CN108464982B - 一种含酰脲结构单元的-5-氰基嘧啶衍生物在制备肿瘤耐药逆转剂中的应用 - Google Patents
一种含酰脲结构单元的-5-氰基嘧啶衍生物在制备肿瘤耐药逆转剂中的应用 Download PDFInfo
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Abstract
本发明公开了一类含酰脲结构单元的‑5‑氰基嘧啶衍生物在制备肿瘤耐药逆转剂中的应用,属于药物化学领域。该类化合物能够对ATP结合盒转运体P‑糖蛋白(ABCB1)介导的耐药性起到良好的逆转作用。该化合物能够在无毒作用下,使ABCB1高表达耐药细胞株SW620/AD300对紫杉醇等ABCB1的转运底物更加敏感,可作为进一步开发的候选或者先导化合物应用于逆转肿瘤耐药的临床辅助治疗药物。
Description
技术领域
本发明涉及药物化学技术领域,特别涉及一种含酰脲结构单元的-5-氰基嘧啶衍生物作为耐药逆转剂的应用。
背景技术
肿瘤是严重危害人类健康的一种疾病,世界范围内因此死亡的人数呈逐年增加的趋势。攻克肿瘤更是世界性的难题,其中一个重要原因就是肿瘤在化疗过程中,均有不同程度的对化疗药物耐药的情况出现,严重影响了治疗的效果。因此,研究肿瘤耐药性产生的分子机制,寻求能够逆转肿瘤耐药性,使其重新对化疗药物敏感的耐药逆转剂是攻克肿瘤的一个重要策略。
临床上大多数肿瘤的耐药情况属于多药耐药(Multidrug resistance,MDR),即肿瘤细胞对某一化疗药物敏感性降低,产生耐受现象后,对未接触过的结构、作用机制、性质不同的其他化疗药物产生了不同程度的交叉耐药现象。多药耐药的产生是一个十分复杂的过程,涉及许多不同的机制,其中研究得最为广泛和深入的是药物的外排作用。细胞的药物外排作用主要由ABC转运蛋白家族完成,肿瘤细胞在发生耐药的过程中,逐步高表达这种转运蛋白,利用ATP水解产生的能量直接将细胞内的药物泵出细胞外,使胞内药物浓度难以达到有效治疗剂量,从而使细胞对化疗药物的敏感性降低,进而产生耐药。
目前发现的人源的ABC家族蛋白有48个,其中研究得最多的是P-糖蛋白(ABCB1,P-gp)、乳腺癌耐药蛋白(ABCG2)和多药耐药蛋白亚家族(ABCC)等。目前已经有文章报道,通过小分子化合物抑制ABC家族蛋白的表达或者功能,可以有效地逆转这种药物泵出过程,使细胞内药物蓄积量增加,达到耐药前的作用效果。因此,耐药逆转剂不仅能够作为研究肿瘤耐药机制的工具化合物,而且可以作为肿瘤耐药的临床辅助治疗药物。
嘧啶类化合物是一类具有非常广泛生物活性的杂环化合物,例如抗病毒、抗菌、抗炎以及抗肿瘤等。Hoff等在2014年报道了一类新型噻吩并嘧啶衍生物(式1),其可作为一种潜在的抗肿瘤成分(Anti-proliferative activity of 2,6-dichloro-9-or 7-(ethoxycarbonylmethyl)-9H-or 7H-purines against several human solid tumourcell lines.Eur.J.Med.Chem.,2014,76,118-124)。另外,脲结构单元作为活性基团,广泛存在于天然和人工合成具有药理活性的化合物中,并广泛应用于抗肿瘤药物的研发。BAY43-9006(式2),一种新型RAF激酶和血管内皮生长因子受体(VEGFR)抑制剂,具有广谱的抗肿瘤活性,例如结肠癌、乳腺癌和非小细胞肺癌体外移植模型中都有明显的抑制作用(Cancer Res.,2004,64,7099-7109)。
发明内容
有鉴于此,本发明目的在于提供一种具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物在制备肿瘤耐药逆转剂中的应用。
本发明提供了一种具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物在制备肿瘤耐药逆转剂中的应用,所述肿瘤耐药逆转剂以P-糖蛋白为靶标;
式I中X为CH或N;
R1为氟、氯、溴、甲基、甲氧基、三氟甲基、硝基或异丙基;
R2为氢、氯、甲基、异丙基、3,4,5-三甲氧基或3,4-二氟。
优选地,当X为CH时,R1为氟、氯、硝基、甲氧基或异丙基,R2为3,4,5-三甲氧基;
当X为N时,R1为甲基、甲氧基、三氟甲基、溴、氯或硝基,R2为氢、甲基、氯、异丙基、3,4,5-三甲氧基或3,4-二氟。
优选地,所述具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物为I-1~1-26中的任意一种,
X为CH时,
I-1 R1=o-OCH3,R2=m,p,m-triOCH3;
I-2 R1=m-Cl,R2=m,p,m-triOCH3;
I-3 R1=m-NO2,R2=m,p,m-triOCH3;
I-4 R1=p-F,R2=m,p,m-triOCH3;
I-5 R1=p-CH(CH3)2,R2=m,p,m-triOCH3;
X为N时,
I-6 R1=p-CH3,R2=p-CH(CH3)2;
I-7 R1=p-OCH3,R2=p-CH(CH3)2;
I-8 R1=m-CH3,R2=p-CH(CH3)2;
I-9 R1=m-CF3,R2=p-CH(CH3)2;
I-10 R1=m-CF3,R2=p-CH3;
I-11 R1=m-CF3,R2=p-Cl;
I-12 R1=m-CF3,R2=m,p-diF;
I-13 R1=m-CF3,R2=m,p,m-triOCH3;
I-14 R1=p-Br,R2=m,p,m-triOCH3;
I-15 R1=o-Cl,R2=m,p,m-triOCH3;
I-16 R1=p-Cl,R2=m,p,m-triOCH3;
I-17 R1=m-Cl,R2=m,p,m-triOCH3;
I-18 R1=m-NO2,R2=m,p,m-triOCH3;
I-19 R1=p-CF3,R2=m,p,m-triOCH3;
I-20 R1=o-OCH3,R2=m,p,m-triOCH3;
I-21 R1=o-Cl,R2=m,p-diF;
I-22 R1=m-Cl,R2=m,p-diF;
I-23 R1=o-OCH3,R2=m,p-diF;
I-24 R1=p-Cl,R2=m,p-diF;
I-25 R1=p-Cl,R2=p-Cl;
I-26 R1=p-CH3,R2=H。
优选地,所述具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物与抗肿瘤药物联用;
所述抗肿瘤药物为P-糖蛋白的底物。
优选地,所述P-糖蛋白的底物为紫杉醇、秋水仙碱、长春新碱或柔红霉素。
优选地,所述联用的方法包括以下步骤:
将具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物在其对耐药细胞株无毒浓度下预孵育后加入抗肿瘤药物,所述预孵育时间为1~8h。
优选地,所述具有抑制细胞耐药性的药物中具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物的浓度为0.1~10μM。
本发明提供了具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物在制备肿瘤耐药逆转剂中的应用,所述肿瘤耐药逆转剂以P-糖蛋白为靶标。该化合物通过作用于耐药机制中关键蛋白P-糖蛋白,使耐药细胞株对临床化疗药重新敏感,以此使化疗药恢复良好的治疗效果。
具体实施方式
本发明提供了一种具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物在制备肿瘤耐药逆转剂中的应用,所述肿瘤耐药逆转剂以P-糖蛋白为靶标,
式I中X为CH或N;
R1为氟、氯、溴、甲基、甲氧基、三氟甲基、硝基或异丙基;
R2为氢、氯、甲基、异丙基、3,4,5-三甲氧基或3,4-二氟。
在本发明中,当X为CH时,R1优选为氟、氯、硝基、甲氧基或异丙基,更优选为氯、硝基或甲氧基,R2优选为3,4,5-三甲氧基;
当X为N时,R1为优选为甲基、甲氧基、三氟甲基、溴、氯或硝基,更优选为甲氧基、三氟甲基或溴,R2优选为氢、甲基、氯、异丙基、3,4,5-三甲氧基或3,4-二氟,更优选为氯、异丙基、3,4,5-三甲氧基或3,4-二氟。
在本发明中,所述具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物优选为I-1~I-26中的一种,更优选为I-2,I-3,I-4,I-5,I-9,I-13,I-14,I-15,I-17或I-18。
X为CH时,
I-1 R1=o-OCH3,R2=m,p,m-triOCH3;
I-2 R1=m-Cl,R2=m,p,m-triOCH3;
I-3 R1=m-NO2,R2=m,p,m-triOCH3;
I-4 R1=p-F,R2=m,p,m-triOCH3;
I-5 R1=p-CH(CH3)2,R2=m,p,m-triOCH3;
X为N时,
I-6 R1=p-CH3,R2=p-CH(CH3)2;
I-7 R1=p-OCH3,R2=p-CH(CH3)2;
I-8 R1=m-CH3,R2=p-CH(CH3)2;
I-9 R1=m-CF3,R2=p-CH(CH3)2;
I-10 R1=m-CF3,R2=p-CH3;
I-11 R1=m-CF3,R2=p-Cl;
I-12 R1=m-CF3,R2=m,p-diF;
I-13 R1=m-CF3,R2=m,p,m-triOCH3;
I-14 R1=p-Br,R2=m,p,m-triOCH3;
I-15 R1=o-Cl,R2=m,p,m-triOCH3;
I-16 R1=p-Cl,R2=m,p,m-triOCH3;
I-17 R1=m-Cl,R2=m,p,m-triOCH3;
I-18 R1=m-NO2,R2=m,p,m-triOCH3;
I-19 R1=p-CF3,R2=m,p,m-triOCH3;
I-20 R1=o-OCH3,R2=m,p,m-triOCH3;
I-21 R1=o-Cl,R2=m,p-diF;
I-22 R1=m-Cl,R2=m,p-diF;
I-23 R1=o-OCH3,R2=m,p-diF;
I-24 R1=p-Cl,R2=m,p-diF;
I-25 R1=p-Cl,R2=p-Cl;
I-26 R1=p-CH3,R2=H。
在本发明中,所述具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物优选与抗肿瘤药物联用;所述抗肿瘤药物为P-糖蛋白的底物。
在本发明中,所述P-糖蛋白的底物优选为紫杉醇、秋水仙碱、长春新碱或柔红霉素,更优选为紫杉醇或秋水仙碱。
在本发明中,所述联用的方法优选包括以下步骤:
将具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物在其对耐药细胞株无毒浓度下预孵育后加入抗肿瘤药物,所述预孵育的时间为1~8h,优选为2~4h。
在本发明中,所述具有抑制细胞耐药性的药物中具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物的浓度优选为0.1~10μM,更优选为2~5μM。在本发明中,当含酰脲结构单元的-5-氰基嘧啶衍生物的浓度为0.1~10μM时,对耐药细胞株无毒。
在本发明中,具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物通过作用于耐药机制中关键蛋白P-糖蛋白,使耐药细胞株对临床化疗药重新敏感,以此使化疗药恢复良好的治疗效果。
在本发明中,所述具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物的制备方法优选采用中国专利CN104402831A中公开的含酰脲结构单元的-5-氰基嘧啶衍生物的制备方法。
为了更好地理解本发明,下面结合实施例进一步阐明本发明的内容,但本发明的内容不仅仅局限于下面的实施例。
实施例1式II所示,R2=m,p,m-triOCH3的衍生物(II-1)的制备
将氰基乙酸乙酯(2.262g,20mmol)与氢氧化钠(1.200g,30mmol)加入乙醇溶液中,回流条件下,反应一段时间,再将硫脲(2.284g,30mmol)和3,4,5-三甲氧基苯甲醛(5.886g,30mmol)加入反应体系中,搅拌反应,TLC跟踪检测。反应结束后,抽滤,重结晶得到纯品。
实施例2式III所示,X=C的衍生物(III-1)的制备
将氯乙酰胺(0.935g,10mmol)分散于溶剂中,在搅拌状态下滴加入草酰氯(1.904g,15mmol),将整个体系转入油浴中加热回流4h,冷却至室温后,滴加苯胺(1.397g,15mmol),搅拌,有固体析出,抽滤,得到固体,分离纯化后得到III-1。产率80%。
实施例3式IV所示,R2=m,p,m-triOCH3,X=C的衍生物(IV-1)的制备
将III-1(6.379g,30mmol)逐滴加入II-1(7.149g,20mmol)的1,4-二氧六环溶液中,加热搅拌反应。用TLC监测反应进程,直至反应完成;未经分离,直接向反应体系中滴加三氯氧磷(4.600g,30mmol),反应完成后,将其倒入冰水中,搅拌,有固体析出,抽滤,得到固体,经柱层析得到纯品化合物IV-1的固体。
Yield 64.5%.Yellow solid.Mp:157–158℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.33(s,1H,NH,D2O exchangeable),10.71(s,1H,NH,D2O exchangeable),8.37–8.21(m,1H,ArH),8.04–7.78(m,4H,ArH),7.68–7.59(m,1H,ArH),7.56–7.37(m,2H,ArH),7.16(ddd,J=7.3,5.0,0.9Hz,1H,ArH),4.34(s,2H,CH2),3.79(d,J=33.4Hz,9H,OCH3),2.26(s,3H,OCH3).13C NMR(100MHz,DMSO-d6,δ,ppm):δ173.87,170.53,168.87,163.10,151.04,150.66,148.25,139.49,134.62,132.82,129.57,129.21,129.07,128.90,120.25,115.19,113.73,102.65,36.54.
实施例4式I所示,R1=o-OCH3,R2=m,p,m-triOCH3,X=C的衍生物(I-1)的制备
将IV-1(514mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入邻甲氧基苯胺(246mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-1,用乙醇重结晶得到I-1的纯品。Yield 81.2%.Yellowsolid.Mp:231–232℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.01(s,1H,NH,D2Oexchangeable),10.58(s,1H,NH,D2O exchangeable),9.78(s,1H,NH,D2O exchangeable),8.30(s,1H,ArH),7.89(d,J=42.5Hz,2H,ArH),7.58–6.84(m,7H,ArH),4.13(s,2H,CH2),3.79(d,J=33.4Hz,9H,OCH3),2.26(s,3H,OCH3).13C NMR(100MHz,DMSO-d6,δ,ppm)δ172.61,170.58,168.04,160.69,153.13,151.48,150.66,148.66,140.37,139.06,135.25,134.80,131.22,129.33,124.29,120.09,116.62,113.41,106.86,85.28,60.65,56.47,36.06,21.07.HRMS(ESI)calcd for C30H29N6O6S[M+H]+:601.1869,found:601.1866.
实施例5式I所示,R1=m-Cl,R2=m,p,m-triOCH3,X=C的衍生物(I-2)的制备
将IV-1(514mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入间氯苯胺(255mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-2,用乙醇重结晶得到I-2的纯品。Yield 86.0%.Yellowsolid.Mp:184-185℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ10.86(s,1H,D2O exchangeable),10.25(s,1H,D2O exchangeable),9.98(s,1H,D2O exchangeable),7.66(s,1H,ArH),7.56(d,J=8.1Hz,1H,ArH),7.50(d,J=7.9Hz,2H,ArH),7.39(t,J=8.1Hz,1H,ArH),7.32(t,J=7.8Hz,2H,ArH),7.23(d,J=8.1Hz,3H,ArH),7.09(t,J=7.3Hz,1H,ArH),4.16(s,2H,S-CH2),3.84(s,6H,O-CH3),3.75(s,3H,O-CH3).13C NMR(100MHz,DMSO-d6,δ,ppm)δ172.66,170.58,168.19,160.58,153.16,150.88,140.62,139.59,137.91,133.18,131.03,130.46,129.40,129.22,125.12,124.20,123.55,122.46,120.90,120.14,116.45,106.91,85.99,60.64,56.45,35.73.HRMS(ESI)calcd for C29H25ClN6NaO5S[M+Na]+:627.1193,found:627.1193.
实施例6式I所示,R1=m-NO2,R2=m,p,m-triOCH3,X=C的衍生物(I-3)的制备
将IV-1(514mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入间硝基苯胺(276mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-3,用乙醇重结晶得到I-3的纯品。Yield 79.2%.Yellowsolid.Mp:132-134℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ10.80(s,1H,D2O exchangeable),10.26(s,1H,D2O exchangeable),10.18(s,1H,D2O exchangeable),8.51(s,1H,ArH),8.02(dd,J=15.7,5.7Hz,2H,ArH),7.64(t,J=8.2Hz,1H,ArH),7.47(d,J=7.9Hz,2H,ArH),7.32(t,J=7.8Hz,2H,ArH),7.22(d,J=6.7Hz,2H,ArH),7.08(t,J=7.3Hz,1H,ArH),4.16(s,2H,S-CH2),3.84(s,6H,O-CH3),3.75(s,3H,O-CH3).13C NMR(100MHz,DMSO-d6,δ,ppm)δ172.71,170.44,168.24,160.66,153.17,150.79,148.18,140.66,139.37,137.88,130.96,130.17,129.99,129.38,124.18,120.09,119.75,118.28,116.36,106.92,86.23,60.65,56.47,35.88.HRMS(ESI)calcd for C29H26N7O7S[M+H]+:616.1614,found:616.1611.
实施例7式I所示,R1=p-F,R2=m,p,m-triOCH3,X=C的衍生物(I-4)的制备
将IV-1(514mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入对氟苯胺(222mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-4,用乙醇重结晶得到I-4的纯品。Yield 75.0%.Yellowsolid.Mp:208–209℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ10.84(s,1H,D2O exchangeable),10.23(s,1H,D2O exchangeable),9.88(s,1H,D2O exchangeable),7.67–7.43(m,4H,ArH),7.34(dd,J=19.7,12.0Hz,2H,ArH),7.25–7.13(m,4H,ArH),7.09(t,J=7.3Hz,1H),4.11(s,2H,S-CH2),3.84(s,6H,O-CH3),3.75(s,3H,O-CH3).13C NMR(100MHz,DMSO-d6,δ,ppm)δ172.68,170.43,168.04,160.80,153.15,150.88,140.53,137.93,134.14,131.14,129.41,126.50,126.42,124.20,120.15,116.57,115.72,115.50,106.86,85.32,60.64,56.46,35.82.HRMS(ESI)calcd for C29H25FN6NaO5S[M+Na]+:611.1489,found:611.1488.
实施例8式I所示,R1=p-CH(CH3)2,R2=m,p,m-triOCH3,X=C的衍生物(I-5)的制备
将IV-1(514mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入对异丙基苯胺(270mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-5,用乙醇重结晶得到I-5的纯品。Yield 84.5%.Yellowsolid.Mp:151–152℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ10.87(s,1H,D2O exchangeable),10.28(s,1H,D2O exchangeable),9.76(s,1H,D2O exchangeable),7.52(d,J=7.9Hz,2H,ArH),7.44(d,J=8.4Hz,2H,ArH),7.34(dd,J=15.8,8.1Hz,2H,ArH),7.27–7.17(m,4H,ArH),7.09(t,J=7.4Hz,1H,ArH),4.14(s,2H,S-CH2),3.84(s,6H,O-CH3),3.75(s,3H,O-CH3),3.01–2.70(m,1H,-CH(CH3)2),1.19(d,J=6.9Hz,6H,-CH(CH3)2).13C NMR(100MHz,DMSO-d6,δ,ppm)δ172.70,170.54,168.06,160.51,153.14,150.91,145.62,140.50,137.97,135.62,131.21,129.41,126.67,124.18,123.92,120.07,116.64,106.87,85.39,60.63,56.46,35.82,33.42,24.29.HRMS(ESI)calcd for C32H33N6O5S[M+H]+:613.2233,found:613.2231.
实施例9式I所示,R1=p-CH3,R2=p-CH(CH3)2,X=N的衍生物(I-6)的制备
采取实施例1同样的方法制备II-2,R2=p-CH(CH3)2;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-2。
将IV-2(467mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入对甲基苯胺(214mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-6,用乙醇重结晶得到I-6的纯品。Yield 78.5%.Yellowsolid.Mp:216–217℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.04(s,1H,NH,D2Oexchangeable),10.63(s,1H,NH,D2O exchangeable),9.77(s,1H,NH,D2O exchangeable),8.30(s,1H,ArH),8.00(s,1H,ArH),7.93–7.68(m,3H,ArH),7.36(s,4H,ArH),7.13(d,J=6.1Hz,3H,ArH),4.10(s,2H,CH2),2.94(s,1H,CH),2.26(s,3H,CH3),1.21(d,J=6.0Hz,6H,CH3).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.77,170.77,168.18,160.72,152.43,151.42,150.72,148.70,139.03,135.29,134.74,133.78,129.33,129.26,126.91,124.24,120.09,116.52,114.64,113.41,85.03,36.02,33.86,24.04,21.06.HRMS(ESI)calcd forC29H28N7O2S[M+H]+:538.2025,found:538.2023.
实施例10式I所示,R1=p-OCH3,R2=p-CH(CH3)2,X=N的衍生物(I-7)的制备
采取实施例1同样的方法制备II-2,R2=p-CH(CH3)2;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-2。
将IV-2(467mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入对甲氧基苯胺(246mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-7,用乙醇重结晶得到I-7的纯品。Yield 86.7%.Yellowsolid.Mp:175–176℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.05(s,1H,NH,D2Oexchangeable),10.66(s,1H,NH,D2O exchangeable),9.72(s,1H,NH,D2O exchangeable),8.30(d,J=4.2Hz,1H,ArH),7.99(d,J=7.2Hz,1H,ArH),7.91–7.68(m,2H,ArH),7.36(dd,J=10.3,8.8Hz,4H,ArH),6.89(d,J=8.9Hz,1H,ArH),6.65(dd,J=34.8,8.7Hz,2H,ArH),4.07(s,2H,CH2),3.73(s,3H,OCH3),2.94(dt,J=13.7,6.8Hz,1H,CH),1.20(d,J=6.9Hz,6H,CH3).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.73,170.84,168.11,160.78,157.17,152.41,151.36,150.72,148.70,139.05,133.79,130.56,129.23,126.90,125.98,120.14,116.48,114.98,114.02,113.46,84.74,55.54,35.95,33.85,24.03.HRMS(ESI)calcd forC29H28N7O3S[M+H]+:554.1974,found:554.1972.
实施例11式I所示,R1=m-CH3,R2=p-CH(CH3)2,X=N的衍生物(I-8)的制备
采取实施例1同样的方法制备II-2,R2=p-CH(CH3)2;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-2。
将IV-2(467mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入间甲基苯胺(214mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-8,用乙醇重结晶得到I-8的纯品。Yield 81.0%.Yellowsolid.Mp:156–157℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.06(s,1H,NH,D2Oexchangeable),10.63(s,1H,NH,D2O exchangeable),9.78(s,1H,NH,D2O exchangeable),8.30(d,J=4.7Hz,1H,ArH),7.99(d,J=7.1Hz,1H,ArH),7.90–7.70(m,3H,ArH),7.49–7.09(m,6H,ArH),6.97(d,J=7.5Hz,1H,ArH),4.10(s,2H,CH2),2.94(dt,J=13.7,6.9Hz,1H,CH),2.27(s,3H,CH3),1.21(d,J=6.9Hz,6H,CH3).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.69,170.90,168.24,160.72,152.47,151.37,150.69,148.69,139.04,138.23,137.83,133.75,129.27,128.68,126.91,126.26,124.75,121.43,120.11,116.46,113.44,85.23,35.89,33.85,24.03,21.44.HRMS(ESI)calcd for C29H28N7O2S[M+H]+:538.2025,found:538.2022.
实施例12式I所示,R1=m-CF3,R2=p-CH(CH3)2,X=N的衍生物(I-9)的制备
采取实施例1同样的方法制备II-2,R2=p-CH(CH3)2;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-2。
将IV-2(467mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入间三氟甲基苯胺(322mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-13,用乙醇重结晶得到I-13的纯品。Yield 88.6%.Yellowsolid.Mp:194–195℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.08(s,1H,NH,D2Oexchangeable),10.63(s,1H,NH,D2O exchangeable),10.10(s,1H,NH,D2O exchangeable),8.29(d,J=4.0Hz,1H,ArH),8.06–7.89(m,3H,ArH),7.89–7.77(m,2H,ArH),7.60(t,J=7.8Hz,1H,ArH),7.54–7.40(m,1H,ArH),7.35(d,J=8.3Hz,2H,ArH),7.14(dd,J=6.9,5.3Hz,1H,ArH),4.14(s,2H,CH2),2.94(dt,J=13.7,6.9Hz,1H,CH),1.21(d,J=6.9Hz,6H,CH3).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.74,170.91,168.36,160.69,152.61,151.35,150.67,148.67,138.99,138.96,133.63,129.98,129.31,127.66,126.95,125.84,123.13,121.54,120.33,120.10,116.26,113.45,85.89,35.91,33.85,24.01.HRMS(ESI)calcd forC29H25F3N7O2S[M+H]+:592.1743,found:592.1741.
实施例13式I所示,R1=m-CF3,R2=p-CH3,X=N的衍生物(I-10)的制备
采取实施例1同样的方法制备II-3,R2=p-CH3;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-3。
将IV-3(439mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入间三氟甲基苯胺(322mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-10,用乙醇重结晶得到I-10的纯品。Yield 72.3%.Yellowsolid.Mp:181–182℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.09(s,1H,NH,D2Oexchangeable),10.62(s,1H,NH,D2O exchangeable),10.12(s,1H,NH,D2O exchangeable),8.28(d,J=4.4Hz,1H,ArH),7.95(dd,J=18.1,8.7Hz,3H,ArH),7.82(dd,J=20.8,8.0Hz,3H,ArH),7.60(t,J=7.8Hz,1H,ArH),7.49(d,J=7.7Hz,1H,ArH),7.30(d,J=8.0Hz,2H,ArH),7.19–7.04(m,1H,ArH),4.14(s,2H,CH2),2.37(s,3H,Ar-CH3).13C NMR(100MHz,DMSO-d6,δ,ppm)δ172.72,170.90,168.39,151.30,150.65,148.65,142.03,139.00,138.94,133.21,129.96,129.56,129.17,127.67,126.15,125.84,123.13,121.53,120.33,120.10,116.25,113.47,85.87.HRMS(ESI)calcd for C27H21F3N7O2S[M+H]+:564.1430,found:564.1428.
实施例14式I所示,R1=m-CF3,R2=p-Cl,X=N的衍生物(I-11)的制备
采取实施例1同样的方法制备II-4,R2=p-Cl;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-4。
将IV-4(459mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入间三氟甲基苯胺(322mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-11,用乙醇重结晶得到I-11的纯品。Yield 87.5%.Yellowsolid.Mp:202–203℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.07(s,1H,NH,D2Oexchangeable),10.58(s,1H,NH,D2O exchangeable),10.18(s,1H,NH,D2O exchangeable),8.28(s,1H,ArH),8.12–7.69(m,6H,ArH),7.69–7.34(m,4H,ArH),7.13(s,1H,ArH),4.14(s,2H,CH2).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.92,170.83,167.37,160.54,151.27,150.63,148.63,138.99,138.82,136.72,134.81,131.02,129.97,129.52,129.09,127.73,125.81,123.11,121.69,120.41,120.10,115.93,113.46,86.33,35.91.HRMS(ESI)calcdfor C26H18ClF3N7O2S[M+H]+:584.0883,found:584.0882.
实施例15式I所示,R1=m-CF3,R2=m,p-diF,X=N的衍生物(I-12)的制备
采取实施例1同样的方法制备II-5,R2=m,p-diF;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-5。
将IV-5(461mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入间三氟甲基苯胺(322mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-12,用乙醇重结晶得到I-12的纯品。Yield 86.7%.Yellowsolid.Mp:134–135℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.08(s,1H,NH,D2Oexchangeable),10.58(s,1H,NH,D2O exchangeable),10.22(s,1H,NH,D2O exchangeable),8.27(d,J=4.4Hz,1H,ArH),8.04–7.72(m,6H,ArH),7.56(dt,J=27.0,8.0Hz,3H,ArH),7.25–7.03(m,1H,ArH),4.15(s,2H,CH2).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.94,170.83,166.29,160.45,151.26,150.56,148.59,138.97,138.75,133.40,130.00,129.84,127.78,126.68,125.80,123.10,121.76,120.42,120.08,118.71,118.52,118.37,118.19,115.82,113.46,86.53,35.92.HRMS(ESI)calcd for C26H17F5N7O2S[M+H]+:586.1085,found:586.1086
实施例16式I所示,R1=m-CF3,R2=m,p,m-triOCH3,X=N的衍生物(I-13)的制备
采取实施例1同样的方法制备II-6,R2=m,p,m-triOCH3;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-6。
将IV-6(515mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入间三氟甲基苯胺(322mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-13,用乙醇重结晶得到I-13的纯品。Yield 72.0%.Yellowsolid.Mp:145–146℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.18(s,1H,NH,D2Oexchangeable),10.59(s,1H,NH,D2O exchangeable),10.14(s,1H,NH,D2O exchangeable),8.27(s,1H,ArH),8.07–7.73(m,4H,ArH),7.70–7.39(m,2H,ArH),7.16(d,J=38.9Hz,3H,ArH),4.21(s,2H,CH2),3.83(s,6H,OCH3),3.74(s,3H,OCH3).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.64,170.62,168.16,160.64,153.15,151.31,150.63,148.58,140.64,138.97,138.93,131.01,130.02,129.82,129.50,127.67,125.83,123.12,121.54,120.30,120.03,116.39,113.42,106.94,86.05,60.65,56.49,36.03.HRMS(ESI)calcd for C29H25F3N7O5S[M+H]+:640.1590,found:640.1588.
实施例17式I所示,R1=p-Br,R2=m,p,m-triOCH3,X=N的衍生物(I-14)的制备
采取实施例1同样的方法制备II-6,R2=m,p,m-triOCH3;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-6。
将IV-6(515mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入对溴苯胺(344mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-14,用乙醇重结晶得到I-14的纯品。Yield 75.0%.Yellowsolid.Mp:216–217℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.08(s,1H,NH,D2Oexchangeable),10.58(s,1H,NH,D2O exchangeable),9.92(s,1H,NH,D2O exchangeable),8.29(d,J=4.3Hz,1H,ArH),7.95(d,J=7.2Hz,1H,ArH),7.83(t,J=7.2Hz,1H,ArH),7.62–7.40(m,4H,ArH),7.20(s,2H,ArH),7.17–7.07(m,1H,ArH),4.18(s,2H,CH2),3.83(s,6H,OCH3),3.75(s,3H,OCH3).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.67,170.64,168.13,160.52,153.15,151.30,150.64,148.62,140.60,139.04,137.41,131.73,131.07,125.92,120.11,117.54,116.49,113.50,106.94,85.86,60.66,56.49,36.10.HRMS(ESI)calcd forC28H25BrN7O5S[M+H]+:650.0821,found:650.0820.
实施例18式I所示,R1=o-Cl,R2=m,p,m-triOCH3,X=N的衍生物(I-15)的制备
采取实施例1同样的方法制备II-6,R2=m,p,m-triOCH3;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-6。
将IV-6(515mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入邻氯苯胺(255mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-15,用乙醇重结晶得到I-15的纯品。Yield 87.5%.Yellowsolid.Mp:206–207℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.17(s,1H,NH,D2Oexchangeable),10.60(s,1H,NH,D2O exchangeable),9.97(s,1H,NH,D2O exchangeable),8.28(d,J=4.1Hz,1H,ArH),8.03–7.71(m,2H,ArH),7.75–7.45(m,2H,ArH),7.39(t,J=8.1Hz,1H,ArH),7.30–7.04(m,4H,ArH),4.21(s,2H,CH2),3.83(s,6H,OCH3),3.75(s,3H,OCH3).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.64,170.63,168.14,160.61,153.15,151.34,150.67,148.60,140.65,139.59,138.99,133.15,131.02,130.46,125.08,123.54,122.50,120.04,116.43,113.46,106.96,85.97,60.66,56.50,36.04.HRMS(ESI)calcd forC28H25ClN7O5S[M+H]+:606.1326,found:606.1324.
实施例19式I所示,R1=p-Cl,R2=m,p,m-triOCH3,X=N的衍生物(I-16)的制备
采取实施例1同样的方法制备II-6,R2=m,p,m-triOCH3;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-6。
将IV-6(515mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入对氯苯胺(255mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-16,用乙醇重结晶得到I-16的纯品。Yield 80.0%.Yellowsolid.Mp:128–129℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.07(s,1H,NH,D2Oexchangeable),10.57(s,1H,NH,D2O exchangeable),9.93(s,1H,NH,D2O exchangeable),8.29(s,1H,ArH),8.09–7.72(m,2H,ArH),7.55(d,J=7.9Hz,2H,ArH),7.39(d,J=8.0Hz,2H,ArH),7.17(d,J=26.0Hz,3H,ArH),4.17(s,2H,CH2),3.83(s,6H,OCH3),3.75(s,2H,OCH3).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.67,170.63,168.13,160.59,153.15,151.29,150.64,148.62,140.59,139.04,136.94,131.08,129.37,128.81,125.66,120.11,116.50,113.48,106.92,85.78,60.66,56.48,36.12.HRMS(ESI)calcd for C28H25ClN7O5S[M+H]+:606.1326,found:606.1324.
实施例20式I所示,R1=m-Cl,R2=m,p,m-triOCH3,X=N的衍生物(I-17)的制备
采取实施例1同样的方法制备II-6,R2=m,p,m-triOCH3;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-6。
将IV-6(515mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入间氯苯胺(255mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-17,用乙醇重结晶得到I-17的纯品。Yield 76.5%.Yellowsolid.Mp:194–195℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.17(s,1H,NH,D2Oexchangeable),10.60(s,1H,NH,D2O exchangeable),9.97(s,1H,NH,D2O exchangeable),8.28(d,J=4.1Hz,1H,ArH),8.03–7.71(m,2H,ArH),7.75–7.45(m,2H,ArH),7.39(t,J=8.1Hz,1H,ArH),7.30–7.04(m,4H,ArH),4.21(s,2H,CH2),3.83(s,6H,OCH3),3.75(s,3H,OCH3).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.64,170.63,168.14,160.61,153.15,151.34,150.67,148.60,140.65,139.59,138.99,133.15,131.02,130.46,125.08,123.54,122.50,120.04,116.43,113.46,106.96,85.97,60.66,56.50,36.04.HRMS(ESI)calcd forC28H24ClN7NaO5S[M+H]+:628.1146,found:628.1147.
实施例21式I所示,R1=m-NO2,R2=m,p,m-triOCH3,X=N的衍生物(I-18)的制备
采取实施例1同样的方法制备II-6,R2=m,p,m-triOCH3;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-6。
将IV-6(515mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入间硝基苯胺(276mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-18,用乙醇重结晶得到I-18的纯品。Yield 79.0%.Yellowsolid.Mp:213–214℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.12(s,1H,NH,D2Oexchangeable),10.52(s,1H,NH,D2O exchangeable),10.28(s,1H,NH,D2O exchangeable),8.48(s,1H,ArH),8.27(s,1H,ArH),7.91(ddd,J=36.8,22.0,7.6Hz,4H,ArH),7.64(t,J=7.9Hz,1H,ArH),7.17(d,J=43.4Hz,3H,ArH),4.21(s,2H,CH2),3.79(d,J=35.6Hz,9H,OCH3).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.71,170.46,168.19,160.67,153.15,151.30,150.59,148.56,148.10,140.63,139.36,138.96,130.96,130.18,130.06,120.01,119.70,118.24,116.37,113.37,106.92,86.18,60.66,56.49,36.22.HRMS(ESI)calcd forC28H25N8O7S[M+H]+:617.1567,found:617.1564.
实施例22式I所示,R1=p-CF3,R2=m,p,m-triOCH3,X=N的衍生物(I-19)的制备
采取实施例1同样的方法制备II-6,R2=m,p,m-triOCH3;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-6。
将IV-6(515mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入对三氟甲基苯胺(322mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-19,用乙醇重结晶得到I-19的纯品。Yield 87.5%.Yellowsolid.Mp:201–202℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.10(s,1H,NH,D2Oexchangeable),10.57(s,1H,NH,D2O exchangeable),10.14(s,1H,NH,D2O exchangeable),8.29(d,J=4.2Hz,1H,ArH),7.82(ddd,J=46.5,37.2,8.6Hz,6H,ArH),7.37–7.05(m,3H,ArH),4.21(s,2H,CH2),3.84(s,6H,OCH3),3.75(s,3H,OCH3).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.78,170.56,168.31,160.56,153.17,151.27,150.61,148.63,143.55,141.92,140.66,139.02,130.98,126.10,123.41,120.12,116.43,113.42,106.97,86.47,60.67,56.49,36.34.HRMS(ESI)calcd for C29H25F3N7O5S[M+H]+:640.1590,found:640.1603.
实施例23式I所示,R1=o-OCH3,R2=m,p,m-triOCH3,X=N的衍生物(I-20)的制备
采取实施例1同样的方法制备II-6,R2=m,p,m-triOCH3;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-6。
将IV-6(515mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入邻甲氧基苯胺(246mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-20,用乙醇重结晶得到I-20的纯品。Yield 74.2%,Yellowsolid.Mp:204-205℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.12(s,1H,NH,D2Oexchangeable),10.52(s,1H,NH,D2O exchangeable),10.28(s,1H,NH,D2O exchangeable),8.48(s,1H,ArH),8.27(s,1H,ArH),7.91(ddd,J=36.8,22.0,7.6Hz,4H,ArH),7.64(t,J=7.9Hz,1H,ArH),7.17(d,J=43.4Hz,3H,ArH),4.21(s,2H,CH2),3.79(d,J=35.6Hz,9H,OCH3).13C NMR(100MHz,DMSO-d6,δ,ppm)δ172.71,170.46,168.19,160.67,153.15,151.30,150.59,148.56,148.10,140.63,139.36,138.96,130.96,130.18,130.06,120.01,119.70,118.24,116.37,113.37,106.92,86.18,60.66,56.49,36.22.HRMS(ESI)calcd forC29H28N7O6S[M+H]+:602.1822,found:602.1820.
实施例24式I所示,R1=o-Cl,R2=m,p-diF,X=N的衍生物(I-21)的制备
采取实施例1同样的方法制备II-5,R2=m,p-diF;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-5。
将IV-5(461mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入邻氯苯胺(255mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-21,用乙醇重结晶得到I-21的纯品。Yield 75.5%.Yellowsolid.Mp:106–107℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ10.88(s,1H,NH,D2Oexchangeable),10.56(s,1H,NH,D2O exchangeable),10.05(s,1H,NH,D2O exchangeable),8.29(d,J=4.3Hz,1H,ArH),8.08–7.68(m,4H,ArH),7.63–7.20(m,5H,ArH),7.20–7.02(m,1H,ArH),3.99(s,2H,CH2).13C NMR(100MHz,DMSO-d6,δ,ppm):δ173.06,170.44,166.02,161.18,151.35,150.56,148.67,139.01,134.91,133.39,131.39,130.08,129.66,129.03,128.03,126.73,120.07,118.73,118.54,118.37,118.19,115.85,113.44,85.21,35.96.HRMS(ESI)calcd for C25H17ClF2N7O2S[M+H]+:552.0821,found:552.0818.
实施例25式I所示,R1=m-Cl,R2=m,p-diF,X=N的衍生物(I-22)的制备
采取实施例1同样的方法制备II-5,R2=m,p-diF;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-5。
将IV-5(461mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入间氯苯胺(255mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-22,用乙醇重结晶得到I-22的纯品。Yield 85.0%.Yellowsolid.Mp:192–193℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.08(s,1H,NH,D2Oexchangeable),10.60(s,1H,NH,D2O exchangeable),10.08(s,1H,NH,D2O exchangeable),8.28(d,J=4.4Hz,1H,ArH),8.05–7.69(m,4H,ArH),7.67–7.48(m,3H,ArH),7.38(t,J=8.1Hz,1H,ArH),7.28–7.03(m,2H,ArH),4.15(s,2H,CH2).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.95,170.83,166.28,160.42,151.29,150.60,148.62,139.41,138.98,133.16,130.45,126.72,126.68,125.27,123.70,122.64,120.09,118.71,118.53,118.36,118.18,115.86,113.48,86.42,35.93.HRMS(ESI)calcd for C25H17ClF2N7O2S[M+H]+:552.0821,found:552.0820.
实施例26式I所示,R1=o-OCH3,R2=m,p-diF,X=N的衍生物(I-23)的制备
采取实施例1同样的方法制备II-5,R2=m,p-diF;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-5。
将IV-5(461mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入邻甲氧基苯胺(246mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-23,用乙醇重结晶得到I-23的纯品。Yield 78.5%.Yellowsolid.Mp:198–199℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.00(s,1H,NH,D2Oexchangeable),10.57(s,1H,NH,D2O exchangeable),9.32(s,1H,NH,D2O exchangeable),8.28(d,J=4.3Hz,1H,ArH),8.09–7.69(m,4H,ArH),7.68–7.44(m,2H,ArH),7.28–6.82(m,4H,ArH),4.08(s,2H,CH2),3.81(s,3H,OCH3).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.96,170.66,165.54,160.57,153.08,151.33,150.59,148.63,139.00,133.46,127.61,126.56,126.11,126.05,120.69,120.06,118.64,118.45,118.36,118.18,115.99,113.45,112.14,100.00,85.53,56.19,36.02.HRMS(ESI)calcd for C26H20F2N7O3S[M+H]+:548.1316,found:548.1314.
实施例27式I所示,R1=p-Cl,R2=m,p-diF,X=N的衍生物(I-24)的制备
采取实施例1同样的方法制备II-5,R2=m,p-diF;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-5。
将IV-5(461mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入对氯苯胺(255mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-24,用乙醇重结晶得到I-24的纯品。Yield 80.9%.Yellowsolid.Mp:213–214℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.09(s,1H,NH,D2Oexchangeable),10.58(s,1H,NH,D2O exchangeable),10.03(s,1H,NH,D2O exchangeable),8.28(d,J=4.1Hz,1H,ArH),8.09–7.71(m,4H,ArH),7.71–7.45(m,3H,ArH),7.39(d,J=8.8Hz,2H,ArH),7.20–7.00(m,1H,ArH),4.15(s,2H,CH2).13C NMR(100MHz,DMSO-d6,δ,ppm):δ173.01,170.71,166.20,160.37,151.29,150.60,148.55,139.01,136.77,133.41,129.53,128.80,126.66,125.73,120.08,118.69,118.51,118.35,118.17,115.93,113.51,86.15,36.09.HRMS(ESI)calcd for C25H17ClF2N7O2S[M+H]+:552.0821,found:552.0819.
实施例28式I所示,R1=p-Cl,R2=p-Cl,X=N的衍生物(I-25)的制备
采取实施例1同样的方法制备II-4,R2=p-Cl;采取实施例2同样的方法用氨基吡啶替代苯胺制备III-2,X=N;采取实施例3同样的方法制备IV-4。
将IV-4(459mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入对氯苯胺(255mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-25,用乙醇重结晶得到I-25的纯品。Yield 74.5%.Yellowsolid.Mp:212–213℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.07(s,1H,NH,D2Oexchangeable),10.57(s,1H,NH,D2O exchangeable),9.99(s,1H,NH,D2O exchangeable),8.29(d,J=4.1Hz,1H,ArH),8.10–7.71(m,4H,ArH),7.56(dd,J=8.7,7.2Hz,4H,ArH),7.39(d,J=8.8Hz,2H,ArH),7.13(dd,J=6.9,5.3Hz,1H,ArH),4.14(s,2H,CH2).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.99,170.71,167.31,160.46,151.31,150.66,148.63,139.02,136.84,136.66,134.86,131.01,129.47,129.08,128.80,125.68,120.11,116.05,113.51,85.98,36.08.HRMS(ESI)calcd for C25H18Cl2N7O2S[M+H]+:550.0620,found:550.0617.
实施例29式I所示,R1=p-CH3,R2=H,X=N的衍生物(I-26)的制备
采取实施例1同样的方法制备II-7,R2=H;采取实施例2同样的方法,用氨基吡啶替代苯胺,制备III-2,X=N;采取实施例3同样的方法制备IV-7。
将IV-7(425mg,1mmol)加入50ml圆底烧瓶中,加入20ml乙醇,溶解后,加热搅拌下加入对甲基苯胺(214mg,2mmol),加热回流反应,TLC跟踪监测。待反应完成后,冷凝,析出固体,抽滤即得到化合物I-26,用乙醇重结晶得到I-26的纯品。Yield 70.5%.Yellowsolid.Mp:206–207℃.1H NMR(400MHz,DMSO-d6,δ,ppm)δ11.03(s,1H,NH,D2Oexchangeable),10.63(s,1H,NH,D2O exchangeable),9.81(s,1H,NH,D2O exchangeable),8.30(d,J=4.1Hz,1H,ArH),8.09–7.77(m,4H,ArH),7.54(dt,J=32.2,7.3Hz,3H,ArH),7.36(d,J=8.3Hz,2H,ArH),7.14(dd,J=10.5,4.9Hz,3H,ArH),4.10(s,2H,CH2),2.25(s,3H,CH3).13C NMR(100MHz,DMSO-d6,δ,ppm):δ172.85,170.73,168.42,160.65,151.38,150.70,148.70,139.07,136.21,135.24,134.80,131.68,129.33,129.13,128.94,124.28,120.11,116.35,113.44,85.43,36.01,21.06.HRMS(ESI)calcd for C26H22N7O2S[M+H]+:496.1556,found:496.1553.
实施例33上述化合物的逆转耐药的活性评价
1、实验方法
取对数生长期的细胞,消化计数后,用DMEM高糖培养基(Dulbecco’s ModifiedEagle Media high glucose)调整细胞密度,以5000~6000个细胞/孔接种至96孔板中,每孔100μL。培养24h后,待细胞恢复至正常贴壁形态,弃去培养基,每孔加入160μL空白培养基后,加入20μL浓度为20μM的实施例中制备得到的化合物,预孵育4h之后,加入20μL不同浓度的紫杉醇(PTX),PTX的浓度分别为(196.83,65.61,21.87,7.29,2.43,0.81,0.27,0.09,0.03,0.01μM)。最终使每孔中培养基总体积为200μL,化合物的终浓度为2μM(此浓度对耐药细胞株SW620/AD300为无毒浓度,细胞存活率均为90%以上),PTX的终浓度为(19.683,6.561,2.187,0.729,0.243,0.081,0.027,0.009,0.003,0.001μM。每个浓度设6个复孔,另设空白对照组(含细胞,含完全培养基,不含化合物)。继续作用72h后,每孔加入20μLMTT
(3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐),培养4h后,吸去液体,加入150μL的DMSO,振荡均匀,酶标仪490nm处检测吸光度值。试验结果采用SPSS软件计算IC50值和相关系数。
用计算所得IC50值得出在耐药细胞株SW620/AD300中,无毒浓度的化合物的对临床抗肿瘤药物PTX的耐药逆转倍数。
2、实验结果
实验结果如下表1所示。
表1上述化合物的逆转耐药活性评价
实验结果表明,耐药细胞株SW620/AD300在含酰脲结构单元的-5-氰基嘧啶衍生物的无毒浓度2μM作用下,对临床药物PTX的敏感性得到了明显的增强。说明此类衍生物有明显的逆转耐药的能力,而且,其中化合物I-2,I-3,I-4,I-5,I-9,I-13,I-14,I-15,I-17,I-18的逆转活性明显优于ABCB1抑制剂Verapamil,可作为进一步开发的候选或者先导化合物,应用于制备耐药逆转剂。
实施例34
按5×106个细胞每只将对数期耐药细胞SW620/AD300接种于体重18-20g,周龄5-6周的雌性BABL/c裸鼠腋下。观察测量,当移植瘤长至100mm3时,将老鼠分为以下四组:①对照组,0.2mL/kg/day,腹腔注射生理盐水;②PTX单用组,5mg/kg/3day,腹腔注射;③化合物单用组,20mg/kg/day,口服灌胃;④化合物和PTX联用组(化合物,20mg/kg/day,腹腔注射;PTX,5mg/kg/3day,腹腔注射)。在持续给药的21天内,每三天测量记录裸鼠的体重和瘤体积。最后一天给药结束后,处死裸鼠,将移植瘤剥离称重记录。
最终结果表明,在整个过程中裸鼠的体重没有明显变化,说明化合物对裸鼠没有毒性;瘤体积的增长,联用组明显要慢于对照组以及两个单用组;最终的瘤重数据显示,化合物单用组的瘤重与对照组相比没有明显变化,PTX单用组的瘤重降低约18.87%,而化合物和PTX联用组的瘤重相对于对照组,下降了50.94%。因此,该化合物能在无毒的情况下,明显逆转耐药细胞株对PTX的敏感性,使PTX发挥更好的抗肿瘤效果。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (4)
1.一种具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物在制备结肠癌肿瘤耐药逆转剂中的应用,所述肿瘤耐药逆转剂以P-糖蛋白为靶标;所述抗肿瘤药物为P-糖蛋白的底物,其中抗肿瘤药物为紫杉醇;
所述具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物为I-2~1-5、I-9、I-13~I-15、I-17和I-18中的任意一种,
X为CH时,
I-2 R1=m-Cl,R2=m,p,m-triOCH3;
I-3 R1=m-NO2,R2=m,p,m-triOCH3;
I-4 R1=p-F,R2=m,p,m-triOCH3;
I-5 R1=p-CH(CH3)2,R2=m,p,m-triOCH3;
x为N时,
I-9 R1=m-CF3,R2=p-CH(CH3)2;
I-13 R1=m-CF3,R2=m,p,m-triOCH3;
I-14 R1=p-Br,R2=m,p,m-triOCH3;
I-15 R1=o-Cl,R2=m,p,m-triOCH3;
I-17 R1=m-Cl,R2=m,p,m-triOCH3;
I-18 R1=m-NO2,R2=m,p,m-triOCH3。
2.根据权利要求1所述的应用,其特征在于,所述具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物与抗肿瘤药物联用;
所述抗肿瘤药物为P-糖蛋白的底物。
3.根据权利要求2所述的应用,其特征在于,所述联用的方法包括以下步骤:
将具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物在其对耐药细胞株无毒浓度下预孵育后加入抗肿瘤药物,所述预孵育时间为1~8h。
4.根据权利要求3所述的应用,其特征在于,所述具有式I所示结构的含酰脲结构单元的-5-氰基嘧啶衍生物的浓度为0.1~10μM。
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