CN117003745A - Gls1/hdac双靶点抑制剂及其合成方法和应用 - Google Patents
Gls1/hdac双靶点抑制剂及其合成方法和应用 Download PDFInfo
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- CN117003745A CN117003745A CN202310946082.8A CN202310946082A CN117003745A CN 117003745 A CN117003745 A CN 117003745A CN 202310946082 A CN202310946082 A CN 202310946082A CN 117003745 A CN117003745 A CN 117003745A
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- thiadiazol
- amino
- phenylacetamido
- piperidin
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Abstract
本发明公开了GLS1/HDAC双靶点抑制剂及其合成方法和应用。一种式I所示的异羟肟酸衍生物或其药学上可接受的盐。式I所示的化合物的HDAC6酶抑制活性达到2.8nM,是阳性对照SAHA(IC50=22nM)的8倍,同时HDAC1酶抑制活性达到29nM,是阳性对照SAHA(IC50=65nM)的2倍,对结直肠癌有明显的抑制效果,能够用于制备治疗结直肠癌的药物。
Description
技术领域
本发明属于小分子化合物领域,涉及GLS1/HDAC双靶点抑制剂及其合成方法和应用。
背景技术
正常细胞主要通过葡萄糖氧化磷酸化作用提供细胞生长的能量,但肿瘤细胞对能量代谢的重编程,其高度依赖谷氨酰胺,表现出“谷氨酰胺成瘾”,这意味着外源性剥夺GLN,可导致肿瘤细胞死亡。
谷氨酰胺是一种条件性必需氨基酸。在正常生理生化条件下,谷氨酰胺是细胞的重要碳源和氮源。细胞外的谷氨酰胺通过细胞膜上的谷氨酰胺转运体SLC1A5进入细胞内,另一部分则通过线粒体膜上的SLC1A5变异体进入线粒体,并在谷氨酰胺酶(GLS)的催化下脱去一个氨基生成谷氨酸,进而在葡萄糖脱氢酶(GDH)或转氨酶的作用下转化为α酮戊二酸,从而参与三羧酸循环。谷氨酸是合成谷胱甘肽的重要前体,还原性谷胱甘肽(GSH)是细胞氧化还原状态的关键调节剂。
谷氨酰胺酶(GLS1)存在两种剪切异构体,KGA和GAC。生理条件下两个无活性的GLS1二聚体同源汇聚成高能活化态的四聚体,该四聚体主要包括两个结合位点——底物催化位点和变构结合位点。DON、重氮丝氨酸和阿西维辛是三种底物位点抑制剂。它们的结构与谷氨酰胺类似,能够和谷氨酰胺竞争性地与GLS1蛋白结合,从而抑制GLS1的生理活性,但是由于其毒性大,选择性低限制了它的开发应用。
2007年第一个变构抑制剂BPTES被发现,在化学结构上,BPTES是一个完全对称的、长链状柔性分子,其对KGA蛋白的抑制IC50为3.3μM,与底物位点抑制剂DON相比,由于BPTES结构上不存在可以和KGA蛋白形成共价结合的基团,因此其特异性更强,安全性更高。BPTES和GLS1共晶复合物结构表明:BPTES占据在GLS1的变构结合位点,通过诱导GLS1产生一定的构象变化,维持其无活性的四聚体构象,阻断其生物学催化功能。目前研究最快的变构位点抑制剂CB839正处于临床1/2期。其结构是将两个苯环分别替换为吡啶环以及三氟甲氧基取代的苯环,共晶复合物显示与CB839类似,两分子的CB839占据GLS1蛋白的变构位点,展现出非常强大的GLS1酶抑制活性以及体外抗肿瘤活性。然而,CB839单用效果非常有限,在临床上大部分以联合给药的方式,在2018年美国癌症研究协会报道了CB839联合了HDAC(组蛋白去乙酰化酶)抑制剂罗米地辛治疗软骨肉瘤有很好的效果,由于这个发现,发明人设想基于HDAC设计出一种双靶点抑制剂。
发明内容
发明人的目的是通过对异羟肟酸的一系列衍生物的研究和筛选,提供一种具有良好的抗肿瘤活性的异羟肟酸衍生物或其药学上可接受的盐,及其制备方法和在制药中的应用。
本发明的另一目的是提供式I所示的异羟肟酸衍生物的制备方法。
本发明的又一目的是提供式I所示的异羟肟酸衍生物的应用。
本发明的目的可通过以下技术方案实现:
一种式I所示的异羟肟酸衍生物或其药学上可接受的盐:
式I结构所示的异羟肟酸衍生物的化学名称:
式I结构所示的异羟肟酸衍生物可与下列酸形成药学上可接受的盐:盐酸、氢溴酸、硫酸、柠檬酸、酒石酸、磷酸、乳酸、乙酸、马来酸或苯磺酸。
式I结构所示的异羟肟酸衍生物的制备方法,包括如下步骤:
(1)N-(5-((1-(5-氨基-1,3,4-噻二唑-2-基)哌啶-4-基)氨基)-1,3,4-噻二唑-2-基)-2-苯乙酰胺与N-(叔丁氧基羰基)-1H-吡唑-4-羧酸在无水溶剂中,在催化剂催化下发生酰胺缩合反应得到4-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-甲酸叔丁酯
(2)4-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-甲酸叔丁酯在三氟乙酸的条件下中,脱去保护基团得到N-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,2,4-噻二唑2-基)-1H-吡唑-4-甲酰胺
(3)N-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,2,4-噻二唑2-基)-1H-吡唑-4-甲酰胺在无水溶剂中,与7-溴庚酸乙酯反应得到7-(4-((5-(4-(5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-基)庚酸乙酯
(4)7-(4-((5-(4-(5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-基)庚酸乙酯在无水溶剂中,与盐酸羟胺以及氢氧化钾反应生成式I结构所示的异羟肟酸衍生物,
步骤(1)中,所述的N-(5-((1-(5-氨基-1,3,4-噻二唑-2-基)哌啶-4-基)氨基)-1,3,4-噻二唑-2-基)-2-苯乙酰胺与N-(叔丁氧基羰基)-1H-吡唑-4-羧酸在无水溶剂中,室温搅拌8小时反应生成4-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-甲酸叔丁酯;所述的无水溶剂为无水乙腈或无水DMF。所述的N-(5-((1-(5-氨基-1,3,4-噻二唑-2-基)哌啶-4-基)氨基)-1,3,4-噻二唑-2-基)-2-苯乙酰胺和N-(叔丁氧基羰基)-1H-吡唑-4-羧酸的摩尔比为1:1~2。所述的催化剂为HATU;所述的HATU和N-(5-((1-(5-氨基-1,3,4-噻二唑-2-基)哌啶-4-基)氨基)-1,3,4-噻二唑-2-基)-2-苯乙酰胺的摩尔比为1:3。步骤(1)中得到的为4-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-甲酸叔丁酯,经柱层析分离,洗脱液为石油醚∶乙酸乙酯=5∶1(V:V),用于下步反应。
步骤(2)中,所述的4-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-甲酸叔丁酯和三氟乙酸的摩尔比为1:1~10,所述的4-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-甲酸叔丁酯在无水溶剂中,与三氟乙酸室温搅拌12小时,脱去保护基团得到N-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,2,4-噻二唑2-基)-1H-吡唑-4-甲酰胺。所述的无水溶剂为无水二氯甲烷。反应结束后,反应产物减压浓缩,无需进一步纯化,直接用于下步反应。
步骤(3)中,所述的N-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,2,4-噻二唑2-基)-1H-吡唑-4-甲酰胺和7-溴庚酸乙酯的摩尔比为1:1~3,所述的N-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,2,4-噻二唑2-基)-1H-吡唑-4-甲酰胺在无水溶剂中,与7-溴庚酸乙酯回流搅拌24小时,反应生成7-(4-((5-(4-(5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-基)庚酸乙酯。所述的无水溶剂为无水乙醇。反应结束后,反应产物采用柱层析分离,洗脱液为石油醚∶乙酸乙酯=2∶1(V:V)。
步骤(4)中,所述的7-(4-((5-(4-(5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-基)庚酸乙酯和盐酸羟胺的摩尔比为1:1~2,所述的7-(4-((5-(4-(5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-基)庚酸乙酯在无水溶剂中,与盐酸羟胺的氢氧化钾溶液,室温搅拌2小时,反应生成式I结构所示的异羟肟酸衍生物。所述的无水溶剂为无水甲醇。反应结束后,反应产物采用柱层析分离,洗脱液为石油醚∶乙酸乙酯=1∶1(V:V)。
本发明所述的异羟肟酸衍生物或其药学上可接受的盐在制备***的药物中的应用,所述的肿瘤优选结直肠癌或三阴乳腺癌。
一种药物组合物,包含本发明所述的异羟肟酸衍生物或其药学上可接受的盐。
有益效果:
我们一共合成了一系列化合物,并且对这些化合物的生物活性进行了研究,从IV系列化合物的HDAC酶抑制活性可以看出,IV-3相比于IV-1活性有所提升,IV-5相比于IV-4活性有所提升,说明延长R2侧碳链长度有助于提升HDAC酶抑制活性;特别是式I所示的化合物IV-5的HDAC6酶抑制活性达到2.8nM,是阳性对照SAHA(IC50=22nM)的8倍,同时HDAC1酶抑制活性达到29nM,是阳性对照SAHA(IC50=65nM)的2倍,相比于上个系列化合物活性显著提升;化合物IV-5对HDAC6有一定的选择性(HDAC6/HDAC1=10)。通过对式I化合物IV-5进行初步机制学研究发现,该化合物对结直肠癌的抗肿瘤活性与阳性对照相当,并且能够诱导结直肠癌细胞内ROS的生成。
表1
附图说明
图1为采用Western Blot实验检测GLS1/HDAC双抑制剂IV-5在B16F10(图A)和SH-SY5Y(图B)细胞中对于HDAC蛋白底物的乙酰化水平的影响。同阳性对照SAHA一样,化合物IV-5能使乙酰化微管蛋白的比例显著增加,说明化合物在一定程度表明化合物4-5对HDAC有一定抑制活性。
图2为采用细胞内ROS水平检测实验检测异羟肟酸衍生物IV-5对细胞内ROS水平的影响。IV-5处理后的HCT116细胞,其细胞内的ROS水平会显著增加,并且具有浓度依赖性。
图3为采用克隆实验检测异羟肟酸衍生物IV-5对于HCT116细胞集落形成能力的影响。化合物IV-5可以显著抑制HCT116细胞的集落形成,且具有剂量依赖性。
图4为异羟肟酸衍生物IV-5在小鼠体内对结肠癌CT26同种异体移植肿瘤的影响(肿瘤体积)。接种肿瘤时,将CT26细胞(1×107)悬浮于PBS中,并分别在BALB/c小鼠右背侧皮下注射细胞悬液0.1mL。肿瘤体积达到100mm3后,小鼠腹腔单次注射等量药,50mg/kg IV-5、50mg/kg CB839、50mg/kg SAHA或50mg/kg CB839+50mg/kg SAHA,连续28天。肿瘤(A)体外培养图像。第28天肿瘤体积(B)、肿瘤重量(C)、小鼠体重(D)。每2天检测一次肿瘤体积(E)。
图5为异羟肟酸衍生物IV-5在小鼠体内对结肠癌CT26同种异体移植肿瘤的影响(HE染色)。接种肿瘤时,将CT26细胞(1×107)悬浮于PBS中,并分别在BALB/c小鼠右背侧皮下注射细胞悬液0.1mL。肿瘤体积达到100mm3后,小鼠腹腔单次注射等量药,50mg/kg IV-5、50mg/kg CB839、50mg/kg SAHA或50mg/kg CB839+50mg/kg SAHA,连续28天。如图为各组肺、肝、脾、肾、心、肿瘤HE染色。
图6为异羟肟酸衍生物IV-5在小鼠体内对结肠癌CT26同种异体移植肿瘤的影响(Western Blot)。接种肿瘤时,将CT26细胞(1×107)悬浮于PBS中,并分别在BALB/c小鼠右背侧皮下注射细胞悬液0.1mL。肿瘤体积达到100mm3后,小鼠腹腔单次注射等量药,50mg/kgIV-5、50mg/kg CB839、50mg/kg SAHA或50mg/kg CB839+50mg/kg SAHA,连续28天。WesternBlot法检测各组Ac-α-tublin(A、B)、α-tublin(A、C)、Ac-HistoneH3(A、D)、Histone H3(A、E)的表达。
图7GLS1/HDAC双抑制剂IV-5对CT26移植肿瘤的影响。接种肿瘤时,将CT26细胞(1×107)悬浮于PBS中,并分别在BALB/c小鼠右背侧皮下注射细胞悬液0.1mL。肿瘤体积达到100mm3后,小鼠腹腔单次注射等量药,50mg/kg IV-5、50mg/kg CB839、50mg/kg SAHA或50mg/kg CB839+50mg/kg SAHA,连续28天。流式细胞术检测各组TIL(A、F)、CD4+T(B、G)、CD8+T(C、H)、Activated CD8+T(D、I)、exhaustion CD8+T(E、J)的比例。
图8为异羟肟酸衍生物IV-5在小鼠体内对结肠癌CT26同种异体移植肿瘤的影响(流式细胞术)。接种肿瘤时,将CT26细胞(1×107)悬浮于PBS中,并分别在BALB/c小鼠右背侧皮下注射细胞悬液0.1mL。肿瘤体积达到100mm3后,小鼠腹腔单次注射等量药,50mg/kgIV-5、50mg/kg CB839、50mg/kg SAHA或50mg/kg CB839+50mg/kg SAHA,连续28天。流式细胞术检测各组NK(E)、Proliferating NK(F)、TAM(G)、M1Macrophage(H)、M2Macrophage(I)的比例。
具体实验方式
实施例1
将N-(叔丁氧基羰基)-1H-吡唑-4-羧酸(0.51g,2.41mmol)溶于无水DMF中,加入HATU和DIPEA,室温搅拌30分钟。再向反应体系中加入N-(5-((1-(5-氨基-1,3,4-噻二唑-2-基)哌啶-4-基)氨基)-1,3,4-噻二唑-2-基)-2-苯乙酰胺(1.02g,2.41mmol),室温搅拌8小时,反应完全。将反应液倒入水中,乙酸乙酯萃取,减压浓缩柱层析,得白色固体0.83g,收率56%。m.p.=161-163℃.1H NMR(300MHz,DMSO-d6):δ=12.53(s,1H),12.21(s,1H),9.12(s,1H),8.30(s,1H),7.41-7.25(m,6H),3.82-3.78(m,3H),3.71(s,2H),3.23(t,2H),2.09-2.05(m,2H),1.60(s,9H),1.59-1.47(m,2H)ppm.HRMS(ESI):m/z,calcd for C26H31N10O4S2[M+H]+,611.1966;found:611.1960.
将4-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-甲酸叔丁酯(0.83g,1.36mmol)加入到10mL的三氟乙酸中,室温搅拌12小时,反应完全。减压浓缩除去多余的三氟乙酸,得到灰白色固体0.60g,收率87%。无需进一步纯化,直接用于下步反应。
将N-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,2,4-噻二唑2-基)-1H-吡唑-4-甲酰胺(0.60g,1.17mmol)加入到无水乙醇中,再加入7-溴庚酸乙酯(0.28g,1.17mmol),回流反应24小时,反应完全。减压除去乙醇,加水,乙酸乙酯萃取,减压浓缩柱层析,得白色固体0.68g,收率87%。m.p.=170-172℃.1H NMR(300MHz,DMSO-d6):δ=13.16(s,1H),12.20(s,1H),8.12(s,1H),7.83(s,1H),7.40-7.24(m,6H),4.22(t,J=6.72Hz,2H),4.04-3.71(m,7H),3.25-3.16(m,2H),2.26-2.08(m,2H),1.95(t,J=7.29Hz,2H),1.81-1.77(m,2H),1.55-1.48(m,4H),1.33-1.29(m,4H),1.16-1.13(m,3H)ppm.HRMS(ESI):m/z,calcd for C30H39N10O4S2[M+H]+,667.2592;found:667.2586。
1-(7-(羟基氨基)-7-氧庚基)-N-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-yl)-1,3,4-噻二唑-2-yl)-1H-吡唑-4-羧酰胺
分别将KOH(14.3g,254.36mmol)和盐酸羟胺(12.0g,171.50mmol)加到35mL和60mL甲醇中,搅拌使其溶解,得溶液A,溶液B。将溶液A缓慢的滴加到溶液B中,滴毕,搅拌30min,整个过程控温在5℃以下。抽滤,除去沉淀氯化钾,得到NH2OK甲醇溶液。将7-(4-((5-(4-(5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-基)庚酸乙酯(0.2g,0.37mmol)加到30mL的NH2OK甲醇溶液中,室温搅拌30min,TLC检测原料反应完全,停止反应。减压除去甲醇,然后用1M的盐酸调pH至5-6,析出固体,抽滤,干燥得白色粉末状固体80mg,白色固体,收率56.6%。m.p.=188-191℃.1H NMR(300MHz,DMSO-d6):δ=13.20(s,1H),10.37(s,1H),8.69(s,1H),8.23(s,1H),7.96(s,1H),7.45(d,J=6.87Hz,1H),7.45(m,6H),4.23(t,J=6.72Hz,2H),3.78-3.74(m,5H),3.25-3.18(m,2H),2.11-2.08(m,2H),1.95(t,J=7.29Hz,2H),1.81-1.77(m,2H),1.56-1.52(m,4H),1.33-1.31(m,4H)ppm.HRMS(ESI):m/z,calcd for C28H35N11O4S2[M+H]+,654.2388;found:654.2390.
实施例2
1.实验材料
(1)实验药物
化合物IV5,结构如式I所示
(2)实验试剂
胎牛血清(SH30070.03)(FBS,Hyclone,Logan,UT,USA);青霉素-链霉素(15140148)(Thermo Fisher Scientific,Pittsburgh,PA,USA);RPMI-1640培养基(30-2001)(ATCC,Rockville,MD,USA);胰蛋白酶(15090046)(Gibco,Grand Island,NY,USA);CB839(S7655)、SAHA(S1047)(Selleckchem,Matsonford Road Radnor,PA,USA);HE染色液(KGA224)(KeyGEN Biotech,NanJing,China);PVDF膜(0.45μm)(Millipore,Schwalbach,Germany);StarSignal Western Protein Marker(10-200kDa)(M227-01)(GenStar,Beijing,China);丽春红、吐温20、丙烯酰胺、十二烷基硫酸钠、PMSF(Solon,OH,USA);蛋白印迹膜再生液(ZN1923,Biolab,Beijing,China);蛋白裂解液(RIPA)、1.0mol/LTris HCl(pH6.8)、1.5mol/L Tris HCl(pH8.8)(Beyotime,ShangHai,China);ECL发光液(ThermoFisher Scientific,Pittsburgh,PA,USA);Anti-Ac-α-tubulin antibody(5335)、Anti-α-tubulin antibody(2144)、Anti-Ac-Histone H3 antibody(9649)、Anti-Histone H3antibody(14269)购自CST公司(Cell Signaling Technology,Boston,USA);Anti-β-actinantibody(ab8226)、Goat Anti-Rabbit IgG H&L(HRP)(ab6721)、RabbitAnti-Mouse IgGH&L(HRP)(ab6728)购自Abcam公司(Abcam,Cambridge,UK);流式破膜剂(00-5223-56)(Thermo Fisher Scientific,Pittsburgh,PA,USA);PE-CyTM7 RatAnti-Mouse CD45Antibody(561868)、PerCP-CyTM5.5 RatAnti-Mouse CD3 Molecular Complex Antibody(560527)、FITC RatAnti-Mouse CD4 Antibody(553729)、FITC Rat Anti-Mouse CD8aAntibody(553030)、PE Mouse Anti-Ki-67 Antibody(567719)、PE Rat Anti-Mouse CD223(LAG3)Antibody(552380)、APC Hamster Anti-Mouse CD279(PD-1)Antibody(562671)、APCRat Anti-Mouse F4/80 Antibody(566787)、PerCP-CyTM5.5RatAnti-Mouse I-A/I-E(MHCⅡ)Antibody(562363)、PE Rat Anti-Mouse CD206 Antibody(568273)购自BDBiosciences公司(BD Biosciences,La Jolla,CA,USA);FITC NK1.1 MonoclonalAntibody(PK136)(11-5941-82)、FITC CD11b Monoclonal Antibody(M1/70)(11-0112-41)购自Thermo Fisher Scientific公司(Thermo Fisher Scientific,Pittsburgh,PA,USA)。
(3)实验仪器
FORMA700型超低温冰箱,Thermo公司;YC-300L型药品储存柜,中科美菱低温科技有限责任公司;Direct-Q with pump型超纯水仪,Millipore公司;SW-CJ-2FD型超净化工作台:苏州净化设备有限公司;3K15型低温高速离心机,Sigma公司;BS224型电子天平:北京塞多利斯仪器***有限公司;Forma 3111型水套式CO2培养箱:Thermo Electron company;YXQ-LS-50SⅡ型立式压力蒸汽灭菌器:上海博迅实业医疗设备厂;石蜡包埋机、切片机,德国LEICA公司;奥林巴斯倒置相差显微镜,日本奥林巴斯公司;ZS-MV-IV型小动物麻醉机,北京众实迪创科技发展有限责任公司;Western blotting***(型号:CriterionTM电泳槽,转印槽),Bio-Rad公司;Tanon 6600发光成像工作站,Tanon公司;BeckmanDxFlex流式细胞仪,美国Beckman公司;40μm细胞筛,Corning公司。
(4)实验细胞株
鼠源结肠癌细胞CT26购自American Tissue Culture Collection(ATCC,Rockville,MD,USA)。
(5)细胞培养条件
CT26细胞传代培养,培养条件为含有青霉素(终浓度为100U/mL)、链霉素(终浓度为100μg/mL)以及10%FBS的RPMI-1640培养基,当细胞融合至90%时,弃去旧培养基,用2mLPBS洗涤细胞2次,弃去PBS后加入2mL 0.25%胰蛋白酶-0.02%EDTA混合消化液,置显微镜下观察,约30s,当细胞变圆后迅速加入2mL完全培养基终止消化,轻轻吹打,收集细胞。800rpm,4℃,离心5min,弃去上清,用完全培养基重悬细胞,分瓶培养,隔天换液。
(6)实验动物
BALB/c小小鼠,6周龄;体重18-22g,由常州卡文斯实验动物有限公司提供,动物合格证号:SCXK(苏)2021-0013,饲养于22±2℃的环境中,自由摄食和饮水。
(8)数据分析
数据采用Graphpad Prism 9(Version 9.4.0)进行分析与作图,AdobeIllustrator 2022(Version 2022)进行整理合图。所有数据均以means±SD表示,组间统计学差异采用one-way ANOVA和2way ANOVA检验,P值小于0.05认为有显著性差异。
2.体外实验
(1)采用细胞培养和抗增殖试验IV-5对人结肠癌HCT116细胞和人乳腺癌MDA-MB-436细胞增殖活性的影响。HCT116细胞在添加10%胎牛血清(FBS)的DMEM中培养,37℃,5%CO2湿度培养箱中培养。用DMEM加10%胎牛血清稀释细胞至100,000细胞/mL。将该细胞悬液以100μL(4000个/孔)的体积接种于96孔板中,孵育24小时。从每孔中除去100μL的培养基后,加入200μL的药物溶液,其中补充了连续稀释的测试化合物或DMSO。然后在37℃、5%CO2下孵育72h,每孔中加入20μL MTT(5mg/mL),孵育4h。小心地除去上清,将甲马甲溶解于150μLDMSO中。测量570nm处的吸光度(OD)。IC50定义为药物浓度对细胞生长的抑制作用达到50%,由剂量-反应曲线确定。实验一式三次。通过生长抑制试验也评估了MDA-MB-436的细胞毒性。接种24小时后,细胞暴露于药物(浓度范围0.5~100μM)中,72h后用MTT法测定。IC50定义为药物浓度对细胞生长的抑制作用达到50%,由剂量-反应曲线确定。实验一式三次。
结果见表3,整体来看,各种化合物的IC50均在微摩尔水平,其中化合物IV-5最优,对结肠癌,三阴性乳腺癌的IC50分别为3μM和7μM,表明化合物IV-5具有一定的体外抗肿瘤细胞增殖活性。
表2
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表3
(2)分子对接研究。为了更好的分析化合物和靶标之间的构效关系,我们选择了代表化合物IV-5进行了分子对接研究。基于已报道的GLS1和HDAC晶体结构,IV-5和GLS1的结合模型显示,IV-5呈现出与阳性对照BPTES相似的构象和结合模式,R1侧酰胺的氮与Leu323形成氢键,R2侧噻二唑上的N以及酰胺键的氮与Phe322和Leu323形成氢键。IV-5和HDAC的结合模型显示,IV-5呈现出与阳性对照SAHA相似的构象和结合模式,末端异羟肟酸的羰基和HDAC蛋白的锌指结构中的Zn2+形成配位作用,末端羰基还与Gly743形成氢键作用。初期对接结果显示代表化合物IV-5能与GLS1和HDAC有较好的结合,证实了化合物改造方向的合理性及代表化合物IV-5作为GLS1和HDAC双靶点小分子抑制剂的潜力。
(3)癌细胞内乙酰化微管蛋白水平检测。化合物预处理后,用RIPA(BeyotimeP0013B)收获B16F10细胞和SH-SY5Y细胞。然后在4℃下以12500rpm离心30分钟,用增强BCA蛋白测定试剂盒(FUDE)定量每个裂解液的浓度。用10%SDS-聚丙烯酰胺凝胶(SDS-PAGE)从总细胞裂解物中分离等效样品,然后转移到PVDF膜(Millipore,USA)上进行蛋白检测。每个膜在室温下封闭2小时,在4℃下孵育一抗过夜。然后每层膜用1x TBST洗涤10min,重复3次,每层膜室温孵育第二抗体1.5h。每个膜在化学发光仪(Tannen 5200)ECL发光液上检测。结果见图1,Westren Blots实验表明化合物IV-5能使乙酰化微管蛋白的比例显著增加,说明化合物在一定程度表明化合物IV-5对HDAC有一定抑制活性。
(4)细胞内ROS水平检测实验。先用无血清培养基稀释阳性对照(Rosup,100mM)到常用工作浓度100μM,对于刺激时间较短的细胞,先装载探针,后用异羟肟酸衍生物IV-5刺激HCT116细胞。检测前一天进行细胞铺板,确保检测时细胞密度达到50~70%。去除细胞培养液,加入适量经合适的缓冲液或无血清培养基稀释到工作浓度的药物,于37℃细胞培养箱内避光孵育,具体诱导时间根据诱导物本身特性,以及细胞类型来决定。按照1:1000用无血清培养基稀释DCFH-DA,使终浓度为10μM。吸去诱导用药物,加入适当体积稀释好的DCFH-DA工作液,以覆盖住细胞为宜。用无血清培养基洗涤细胞1~2次,以充分去除未进入细胞内的DCFH-DA。用激光共聚焦显微镜直接观察。结果见图2,表明异羟肟酸衍生物IV-5处理后的HCT116细胞,其细胞内的ROS水平会显著增加,且呈现剂量或浓度依赖性。
(5)克隆形成实验。HCT116细胞(1000个/孔,体积为2mL,镀于6孔板中,用相应浓度的化合物IV-5(DMSO为对照)处理14天。去除上清液,PBS缓冲液洗涤细胞,用4%多聚甲醛固定液固定。然后用0.1%结晶紫染色30分钟。结果见图3,表明异羟肟酸衍生物IV-5可以显著抑制细胞克隆的形成,且呈现剂量或浓度依赖性。
(6)部分化合物的理化性质研究。对III-4、IV-3、IV-5、BPTES的理化性质进行了探究,结果如表4所示,表明异羟肟酸衍生物IV-5水溶性(0.2mg/mL)得到提高,是阳性对照BPTES(0.05mg/mL)的四倍,水溶性相对于先导BPTES有所改善。
表4
(7)体外血浆稳定性实验。分别在人源和鼠源的血浆中考察了化合物的稳定性,结果如表5所示,发现和孵育2h后的化合物IV-5分别剩余92%和91%,明显优于阳性对照CB839,表明异羟肟酸化合物IV-5在血浆中较为稳定。
表5
3.体内实验
(1)同种异体小鼠结肠癌模型构建
收集培养的CT26细胞,计数,调整使细胞悬液浓度为1.0×107个/mL,于小小鼠右侧腋窝皮下每只接种0.1mL细胞悬液。待肿瘤体积达到100mm3,模型建立成功,开始分组给药。
(2)实验分组
Vehicle组:小鼠CT26结肠癌移植瘤模型建立成功后,腹腔注射等量溶剂,每天一次,连续28天,(N=7);
50mg/kg IV-5组:小鼠CT26结肠癌移植瘤模型建立成功后,腹腔注射50mg/kg的化合物IV-5,每天给药一次,连续给药28天,(N=7);
50mg/kg CB839组:小鼠CT26结肠癌移植瘤模型建立成功后,腹腔注射50mg/kg的化合物CB839,每天给药一次,连续给药28天,(N=7);
50mg/kg SAHA组:小鼠CT26结肠癌移植瘤模型建立成功后,腹腔注射50mg/kg的化合物SAHA,每天给药一次,连续给药28天,(N=7);
50mg/kg CB839+50mg/kg SAHA组:小鼠CT26结肠癌移植瘤模型建立成功后,腹腔注射50mg/kg的化合物CB839和50mg/kg的化合物SAHA,每天给药一次,连续给药28天,(N=7);
给药期间,每隔一天测量小鼠体重和肿瘤体积;给药结束后,处死小鼠并剥离肿瘤,拍摄肿瘤组织,测量肿瘤重量,每组选取2只状态良好的小鼠,剥离心、肝、脾、肺、肾和肿瘤,通过HE染色进行组织病理学评价。Western Blot实验检测肿瘤组织中Ac-α-tublin,α-tublin,Ac-Histone H3,Histone H3蛋白表达水平。
(3)肿瘤体积测量
小鼠结肠癌模型造模成功后,每隔一天监测并记录各组小鼠的肿瘤体积,肿瘤体积(tumor volume,TV)的计算公式为:TV=1/2×a×b2,其中a、b分别表示长宽。
(4)HE染色
将制好的心、肝、脾、肺、肾和肿瘤组织石蜡切片置于电热恒温干燥箱中,60℃烘烤3小时;将干燥的石蜡切片进行常规二甲苯脱蜡,下行梯度乙醇水化,蒸馏水洗;苏木素染核2min,盐酸酒精分化数秒,水洗返蓝;伊红染液染片1min,水洗冲掉残留染液;切片经梯度酒精脱水干燥,二甲苯透明,中性树胶封片;使用相差显微镜随机选择一个视野使用400×倍视野下拍照。
(5)Western Blot
按照动物实验分组,用预冷得PBS洗涤组织两次,每100mg压缩体积的组织样本加入1mL添加了PMSF的RIPA,使用电动匀浆仪充***解后,4℃12000g离心5min,立即吸取上清至一预冷的Eppendorf管中,即为抽提得到的细胞蛋白,冻存于-80℃备用。通过BCA法进行蛋白定量,最后加入5×loading buffer沸水浴10min,样品制备完成,可以-20℃保存。
根据待测蛋白分子量的不同,配制10%或12%的分离胶和5%的压缩胶,,灌制SDS-PAGE凝胶。加入适量提前预冷的1×电泳缓冲液后,将之前生物标记的样品或细胞内总蛋白提取物加入泳道中(预染蛋白Marker和样品)。80V稳压电泳约30min,待样品进入分离胶后,调整电压为120V继续电泳。待目的条带到达合适位置时(参照预染蛋白Marker的位置),结束电泳。比照凝胶大小剪PVDF膜,置于甲醇中活化1min,随后放入转膜缓冲液中浸泡,滤纸一并放入转膜缓冲液中浸泡15min。按照PVDF膜≥凝胶≥滤纸的原则制作转膜“三明治”,确保去除气泡后开始恒压转膜。转完成后,用丽春红s染液染膜5min,随后TBST清洗2次,观察膜上的蛋白。
将膜用TBS从下向上浸湿后,移至含有封闭液(5%BSA)的平皿中,室温下脱色摇床上摇动封闭1h,以封闭PVDF膜的免疫球蛋白结合位点。用TBST将膜上的残余液体洗干净,然后用封口机将膜置于自封袋中,封好三边后,加入用TBST稀释至适当浓度的一抗(具体一抗及稀释比见表3),尽可能排出气泡,封闭袋口,4℃过夜。剪开自封袋,用TBST洗膜3次,每次10min。然后将膜置于封闭袋中,加入适量适当浓度的二抗(具体二抗及稀释比见表6),封闭袋口,室温下孵育1h。剪开自封袋,用TBST洗膜3次,每次10min。等体积混合化学发光试剂A液和B液,将膜蛋白面向下与此混合液充分接触;5min后,用Tanon 6600发光成像工作站进行检测,同一个PVDF膜需多次曝光时,采用strip液(蛋白印迹膜再生液)于室温摇动洗涤45min后,用TBST洗涤3次,再从封闭步骤重新开始,后续步骤同前;蛋白表达量使用ImagePro Plus 6.0软件对光密度值进行分析,蛋白相对表达量计算为目的蛋白灰度值/内参蛋白灰度值。
表6
抗体 | 稀释(应用程序) |
Ac-α-tubulin | 1:1000(WB) |
α-tubulin | 1:1000(WB) |
Ac-Histone H3 | 1:1000(WB) |
Histone H3 | 1:1000(WB) |
β-actin | 1μg/mL |
Rabbit Anti-Mouse IgG H&L(HRP) | 1:10000(WB) |
Goat Anti-Rabbit IgG H&L(HRP) | 1:10000(WB) |
(6)流式细胞术检测肿瘤免疫细胞相关指标
1)样品处理:治疗结束后,处死小鼠,收集各组小鼠肿瘤组织,浸泡于PBS中。研磨,收集过40μm细胞筛,800g离心5min,最后用PBS调整成适量浓度的细胞悬液备用。
2)TIL肿瘤浸润淋巴细胞
PBS洗涤两次后用200μL PBS重悬细胞,当细胞呈悬液状态时装入流式管中,流式管标号,分别于标号的流式管中加入PE-CyTM7Rat Anti-Mouse CD45 Antibody,充分混匀,室温下避光孵育30min。然后加入500μL PBS洗涤2次除去未标记上的抗体,最后用400μLPBS重悬细胞,将细胞悬液转移至流式细胞仪专用上机管中上机检测。
3)CD4+
PBS洗涤两次后用200μL PBS重悬细胞,当细胞呈悬液状态时装入流式管中,流式管标号,分别于标号的流式管中加入PE-CyTM7 Rat Anti-Mouse CD45 Antibody、PerCP-CyTM5.5 Rat Anti-Mouse CD3 Molecular Complex Antibody、FITC Rat Anti-Mouse CD4Antibody,充分混匀,室温下避光孵育30min。然后加入500μL PBS洗涤2次,除去未标记上的抗体,最后用400μL PBS重悬细胞,将细胞悬液转移至流式细胞仪专用上机管中上机检测。
4)CD8+
PBS洗涤两次后用200μL PBS重悬细胞,当细胞呈悬液状态时装入流式管中,流式管标号,分别于标号的流式管中加入PE-CyTM7 Rat Anti-Mouse CD45 Antibody、PerCP-CyTM5.5 Rat Anti-Mouse CD3 Molecular Complex Antibody、FITC Rat Anti-Mouse CD8aAntibody,充分混匀,室温下避光孵育30min。然后加入500μL PBS洗涤2次除去未标记上的抗体,最后用400μL PBS重悬细胞,将细胞悬液转移至流式细胞仪专用上机管中上机检测。
5)Activated CD8+
PBS洗涤两次后用200μL PBS重悬细胞,当细胞呈悬液状态时装入流式管中,流式管标号,分别于标号的流式管中加入PE-CyTM7 Rat Anti-Mouse CD45 Antibody、PerCP-CyTM5.5 Rat Anti-Mouse CD3 Molecular Complex Antibody、FITC Rat Anti-Mouse CD8aAntibody、PE Mouse Anti-Ki-67 Antibody,充分混匀,室温下避光孵育30min。PBS洗涤两次后用200μL PBS重悬细胞,1500rpm离心5min,弃掉上清,加入100μL破膜剂,室温放置20min,洗涤液洗涤一次加入FITC Mouse Anti-Ki-67 antibody,4℃避光放置30min。然后加入500μL PBS洗涤2次除去未标记上的抗体,最后用400μL PBS重悬细胞,将细胞悬液转移至流式细胞仪专用上机管中上机检测。
6)Exhausted CD8+
PBS洗涤两次后用200μL PBS重悬细胞,当细胞呈悬液状态时装入流式管中,流式管标号,分别于标号的流式管中加入PE-CyTM7Rat Anti-Mouse CD45 Antibody、PerCP-CyTM5.5 Rat Anti-Mouse CD3 Molecular Complex Antibody、FITC Rat Anti-Mouse CD8aAntibody、PE Rat Anti-Mouse CD223(LAG3)Antibody、APC Hamster Anti-Mouse CD279(PD-1)Antibody充分混匀,室温下避光孵育30min。然后加入500μL PBS洗涤2次除去未标记上的抗体,最后用400μL PBS重悬细胞,将细胞悬液转移至流式细胞仪专用上机管中上机检测。
7)NK
PBS洗涤两次后用200μL PBS重悬细胞,当细胞呈悬液状态时装入流式管中,流式管标号,分别于标号的流式管中加入PE-CyTM7Rat Anti-Mouse CD45 Antibody、PerCP-CyTM5.5 Rat Anti-Mouse CD3 Molecular Complex Antibody、FITC NK1.1 MonoclonalAntibody充分混匀,室温下避光孵育30min。然后加入500μLPBS洗涤2次除去未标记上的抗体,最后用400μL PBS重悬细胞,将细胞悬液转移至流式细胞仪专用上机管中上机检测。
8)Proliferating NK
PBS洗涤两次后用200μL PBS重悬细胞,当细胞呈悬液状态时装入流式管中,流式管标号,分别于标号的流式管中加入PE-CyTM7 Rat Anti-Mouse CD45 Antibody、PerCP-CyTM5.5 Rat Anti-Mouse CD3 Molecular Complex Antibody、FITC NK1.1 MonoclonalAntibody、PE Mouse Anti-Ki-67 Antibody充分混匀,室温下避光孵育30min。PBS洗涤两次后用200μL PBS重悬细胞,1500rpm离心5min,弃掉上清,加入100μL破膜剂,室温放置20min,洗涤液洗涤一次加入Anti-Mouse Ki-67 Monoclonal Antibody APC,4℃避光放置30min。然后加入500μL PBS洗涤2次除去未标记上的抗体,最后用400μL PBS重悬细胞,将细胞悬液转移至流式细胞仪专用上机管中上机检测。
9)TAM肿瘤相关巨噬细胞
PBS洗涤两次后用200μLPBS重悬细胞,当细胞呈悬液状态时装入流式管中,流式管标号,分别于标号的流式管中加入PE-CyTM7 Rat Anti-Mouse CD45 Antibody、APC RatAnti-Mouse F4/80 Antibody,充分混匀,室温下避光孵育30min。PBS洗涤两次后用200μLPBS重悬细胞,1500rpm离心5min,弃掉上清,加入100μL破膜剂,室温放置20min,洗涤液洗涤一次加入FITC CD11b Monoclonal Antibody,4℃避光放置30min。然后加入500μL PBS洗涤2次除去未标记上的抗体,最后用400μL PBS重悬细胞,将细胞悬液转移至流式细胞仪专用上机管中上机检测。
10)M1、M2型巨噬细胞
PBS洗涤两次后用200μL PBS重悬细胞,当细胞呈悬液状态时装入流式管中,流式管标号,分别于标号的流式管中加入PE-CyTM7 Rat Anti-Mouse CD45 Antibody、APC RatAnti-Mouse F4/80 Antibody、PE Rat Anti-Mouse CD206 Antibody充分混匀,室温下避光孵育30min。PBS洗涤两次后用200μL PBS重悬细胞,1500rpm离心5min,弃掉上清,加入100μL破膜剂,室温放置20min,洗涤液洗涤一次加入FITC CD11b Monoclonal Antibody、PerCP-CyTM5.5 Rat Anti-Mouse I-A/I-E(MHCⅡ)Antibody,4℃避光放置30min。然后加入500μLPBS洗涤2次除去未标记上的抗体,最后用400μL PBS重悬细胞,将细胞悬液转移至流式细胞仪专用上机管中上机检测。
结果见图4-图7,图4表明与Vehicle组相比,50mg/kg IV-5组、50mg/kg CB839组、50mg/kg SAHA组和50mg/kg CB839+50mg/kg SAHA组小鼠28天和最后一天的移植瘤体积均减小,移植瘤重量减轻;其中50mg/kg IV-5组最为显著。
图5表明,与Vehicle组相比,50mg/kg IV-5组、50mg/kg CB839组、50mg/kg SAHA组和50mg/kg CB839+50mg/kg SAHA组小鼠心肝脾肺肾HE均无明显变化;与Vehicle组相比,50mg/kg IV-5组、50mg/kg CB839组、50mg/kg SAHA组和50mg/kg CB839+50mg/kg SAHA组小鼠移植瘤组织肿瘤细胞数量减少,肿瘤细胞坏死增多,出现炎性细胞浸润现象。
图6表明,与Vehicle组相比,50mg/kg IV-5组、50mg/kg CB839组、50mg/kg SAHA组和50mg/kg CB839+50mg/kg SAHA组小鼠瘤组织中α-tublin、Histone H3的表达水平无明显变化,50mg/kg IV-5组、50mg/kg SAHA组和50mg/kg CB839+50mg/kg SAHA组小鼠瘤组织中Ac-α-tublin、Ac-Histone H3的表达水平均上升。
图7表明,与Vehicle组相比,50mg/kg IV-5组、50mg/kg CB839组和50mg/kg CB839+50mg/kg SAHA组小鼠瘤组织中TIL细胞、CD4+T细胞、CD8+T细胞和Activated CD8+T细胞的比例均不同程度的上升,Exhausted CD8+T细胞的比例呈不同程度的下降,其中50mg/kgIV-5组最为显著。50mg/kg SAHA组中TIL细胞的占比无明显变化,CD4+T细胞、CD8+T细胞和Activated CD8+T细胞的占比均升高,Exhausted CD8+T细胞的比例降低。
图8表明,与Vehicle组相比,50mg/kg IV-5组、50mg/kg CB839组和50mg/kg CB839+50mg/kg SAHA组小鼠瘤组织NK细胞、Proliferating NK细胞、TAM细胞和M1 Macrophage细胞的比例均不同程度的上升,M2 Macrophage细胞的比例呈不同程度的下降,其中50mg/kgIV-5组最为显著。
Claims (10)
1.一种异羟肟酸衍生物或其药学上可接受的盐,其特征在于,所述的异羟肟酸衍生物结构如式I所示:
2.根据权利要求1所述的GLS1/HDAC双靶点抑制剂或其药学上可接受的盐,其特征在于,式I所示的异羟肟酸衍生物可与下列酸形成药学上可接受的盐:盐酸、氢溴酸、硫酸、柠檬酸、酒石酸、磷酸、乳酸、乙酸、马来酸或苯磺酸。
3.权利要求1所述的式I结构所示的异羟肟酸衍生物的制备方法,其特征在于,包括如下步骤:
4.根据权利要求3所述的制备方法,其特征在于,包括如下步骤:
(1)N-(5-((1-(5-氨基-1,3,4-噻二唑-2-基)哌啶-4-基)氨基)-1,3,4-噻二唑-2-基)-2-苯乙酰胺与N-(叔丁氧基羰基)-1H-吡唑-4-羧酸在无水溶剂中,在催化剂催化下发生酰胺缩合反应得到4-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-甲酸叔丁酯
(2)4-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-甲酸叔丁酯在三氟乙酸的条件下中,脱去保护基团得到N-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,2,4-噻二唑2-基)-1H-吡唑-4-甲酰胺
(3)N-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,2,4-噻二唑2-基)-1H-吡唑-4-甲酰胺在无水溶剂中,与7-溴庚酸乙酯反应得到7-(4-((5-(4-(5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-基)庚酸乙酯
(4)7-(4-((5-(4-(5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-基)庚酸乙酯在无水溶剂中,与盐酸羟胺以及氢氧化钾反应生成式I结构所示的异羟肟酸衍生物,
5.根据权利要求4所述的制备方法,其特征在于,步骤(1)中,所述的N-(5-((1-(5-氨基-1,3,4-噻二唑-2-基)哌啶-4-基)氨基)-1,3,4-噻二唑-2-基)-2-苯乙酰胺与N-(叔丁氧基羰基)-1H-吡唑-4-羧酸在无水溶剂中,室温搅拌8小时反应生成4-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-甲酸叔丁酯;所述的无水溶剂为无水乙腈或无水DMF;所述的N-(5-((1-(5-氨基-1,3,4-噻二唑-2-基)哌啶-4-基)氨基)-1,3,4-噻二唑-2-基)-2-苯乙酰胺和N-(叔丁氧基羰基)-1H-吡唑-4-羧酸的摩尔比为1:1~2;所述的催化剂为HATU;所述的HATU和N-(5-((1-(5-氨基-1,3,4-噻二唑-2-基)哌啶-4-基)氨基)-1,3,4-噻二唑-2-基)-2-苯乙酰胺的摩尔比为1:3;步骤(1)中得到的为4-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-甲酸叔丁酯,经柱层析分离,洗脱液为石油醚∶乙酸乙酯=5∶1(V:V),用于下步反应。
6.根据权利要求4所述的制备方法,其特征在于,步骤(2)中,所述的4-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-甲酸叔丁酯和三氟乙酸的摩尔比为1:1~10,所述的4-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-甲酸叔丁酯在无水溶剂中,与三氟乙酸室温搅拌12小时,脱去保护基团得到N-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,2,4-噻二唑2-基)-1H-吡唑-4-甲酰胺;所述的无水溶剂为无水二氯甲烷;反应结束后,反应产物减压浓缩,无需进一步纯化,直接用于下步反应。
7.根据权利要求4所述的制备方法,其特征在于,步骤(3)中,所述的N-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,2,4-噻二唑2-基)-1H-吡唑-4-甲酰胺和7-溴庚酸乙酯的摩尔比为1:1~3,所述的N-(5-(4-((5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,2,4-噻二唑2-基)-1H-吡唑-4-甲酰胺在无水溶剂中,与7-溴庚酸乙酯回流搅拌24小时,反应生成7-(4-((5-(4-(5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-基)庚酸乙酯;所述的无水溶剂为无水乙醇;反应结束后,反应产物采用柱层析分离,洗脱液为石油醚∶乙酸乙酯=2∶1(V:V)。
8.根据权利要求4所述的制备方法,其特征在于,步骤(4)中,所述的7-(4-((5-(4-(5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-基)庚酸乙酯和盐酸羟胺的摩尔比为1:1~2,所述的7-(4-((5-(4-(5-(2-苯基乙酰胺基)-1,3,4-噻二唑-2-基)氨基)哌啶-1-基)-1,3,4-噻二唑2-基)氨基甲酰基)-1H-吡唑-1-基)庚酸乙酯在无水溶剂中,与盐酸羟胺的氢氧化钾溶液,室温搅拌2小时,反应生成式I结构所示的异羟肟酸衍生物;所述的无水溶剂为无水甲醇。反应结束后,反应产物采用柱层析分离,洗脱液为石油醚∶乙酸乙酯=1∶1(V:V)。
9.权利要求1所述的异羟肟酸衍生物或其药学上可接受的盐在制备***的药物中的应用,所述的肿瘤优选结直肠癌或三阴乳腺癌。
10.一种药物组合物,其特征在于包含权利要求1所述的异羟肟酸衍生物或其药学上可接受的盐。
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