CN114380812A - 用于治疗慢性粒细胞白血病的bcr/abl酪氨酸激酶抑制剂 - Google Patents
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Abstract
本发明为用于治疗慢性粒细胞白血病的BCR/ABL酪氨酸激酶抑制剂，公开了抑制BCR/ABL酪氨酸激酶的化合物，该化合物是通过基于已知的BCR/ABL酪氨酸激酶抑制剂，构建药效团模型，对数据库进行虚拟筛选，并对活性最好的化合物进行结构优化，发现优化的化合物能够有效的抑制BCR/ABL酪氨酸激酶的磷酸化，抑制伊马替尼耐药和不耐药的细胞的增殖，并诱导伊马替尼耐药和不耐药的细胞的周期阻滞，自噬和凋亡。本发明公开的化合物作为BCR/ABL酪氨酸激酶抑制剂，为治疗慢性粒细胞白血病提供了新的选择。
Description
技术领域
本发明涉及一种通过计算机虚拟筛选得到的化合物，特别涉及治疗慢性粒细胞白血病的BCR/ABL酪氨酸激酶抑制剂。
背景技术
慢性粒细胞白血病(chronic myeloid leukemia,CML)是一种由造血干细胞转化而来的血液学恶性疾病，该病的标记是Ph染色体，是Nowell和Hungerford在1960年发现的。Ph染色体是9号(abl)和22号(bcr)易位的结果，产生了BCR/ABL融合基因(A newconsistent chromosomal abnormality in chronic myelogenous leukemia identifiedby quinacrine fluorescence and Giemsa staining.Rowley JD.Nature 1973；243:290–3.)。BCR/ABL融合基因导致酪氨酸激酶活性失调，对BCR/ABL酪氨酸激酶活性的解除是CML的主要致病原因(Targeted drugs in chronic myeloid leukemia.leukemia.GoraTybor,J.；Robak,T..Current Medicinal Chemistry 2008,15,3036-51.)，导致细胞增殖，存活和抗凋亡。
伊马替尼是一种能够结合到BCR/ABL激酶区域，并抑制其活性的小分子，是第一代BCR/ABL酪氨酸激酶抑制剂，是CML的一线治疗药物，但是70％CML患者由于耐药性的原因，在疾病晚期会复发。伊马替尼耐药的原因包括BCR/ABL结构域发生点突变，BCR/ABL蛋白扩增，BCR/ABL蛋白高表达和耐药P糖蛋白过表达。伊马替尼耐受的主要原因是BCR/ABL激酶结构域的突变，突变干扰了伊马替尼和BCR/ABL区域的结合，在突变中，T315I突变是最常见的。达沙替尼是第二代酪氨酸激酶抑制剂，已被FDA批准用于治疗CML，但是会有引起罕见疾病的风险—肺动脉高血压。所以寻找新的靶向BCR/ABL酪氨酸激酶的抑制剂，并能够拮抗T315I，这对CML治疗有重要的意义。
发明内容
本发明的目的是提供靶向BCR/ABL酪氨酸激酶的抑制剂，并能够诱导T315I突变细胞凋亡。本发明的技术方案如下：
靶向BCR-ABL酪氨酸激酶的结构通式(Ⅰ)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐：
其中：R1选自氢、甲基、乙基或异丙基；R2选自氢、甲基、乙基或异丙基；
R3：选自
或烷基。
优选的，所述式(Ⅰ)化合物的取代基R3被取代得到如下结构式：
其中R1选自氢、甲基、乙基或异丙基；R2选自氢、甲基、乙基或异丙基；
R4选自甲基、氢、
其中R1选自氢、甲基、乙基或异丙基；R2选自氢、甲基、乙基或异丙基；
R5选自
其中R1选自氢、甲基、乙基或异丙基；R2选自氢、甲基、乙基或异丙基；R6为乙基；
其中R1选自氢、甲基、乙基或异丙基；R2选自氢、甲基、乙基或异丙基；R7选自甲基或
优选的，所述式(Ⅱ)化合物选自：
优选的，所述式(Ⅲ)化合物选自：
优选的，所述式(Ⅳ)化合物选自：
优选的，所述式(Ⅴ)化合物选自：
本发明的第二目的是提供一种药物组合物，其特征在于，所述药物组合物含有治疗有效量的所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐，以及一种或多种药学上可接受的载体，稀释剂或赋形剂。
本发明的第三目的是提供所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐在抑制BCR/ABL酪氨酸激酶中的应用。
本发明的第四目的是提供所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐在拮抗T315I突变中的应用。
本发明的第五目的是提供所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐，在制备预防或治疗白血病药物中的应用。
优选的，所述的白血病为慢性粒细胞白血病。
本发明的有益效果是：经过虚拟筛选，对筛选出的化合物进行细胞毒性实验验证，对活性较好的化合物进行结构优化，再次筛选，对化合物进行生物实验验证，发现化合物能够抑制白血病Imatinib耐药和不耐药细胞的增殖，尤其是以近10倍的效果强于Imatinib抑制BaF3/T315I Imatinib不耐药细胞的增殖。通过流式实验，发现对于K562细胞，化合物诱导细胞周期阻滞在G2期，对于BaF3/WT和BaF3/T315I，化合物诱导细胞周期阻滞在G1期。通过流式实验以及western blot实验，发现化合物能诱导细胞自噬和凋亡，并能抑制BCR/ABL酪氨酸激酶蛋白的表达，有希望成为潜在的BCR/ABL抑制剂，为CML的治疗提供了新的选择。
附图说明
图1：Hypogen药效团中打分值最好的Hypogen1模型，包括一个氢键受体，四个疏水中心。图2：Hiphop药效团中打分值最好的Hiphop1模型，包括两个芳香环中心、三个疏水中心、一个氢键供体、两个氢键受体。
图3：筛选得到的BCR-ABL酪氨酸激酶抑制剂化合物的结构通式。
图4：结构优化的不同浓度的化合物和阳性对照药Imatinib处理K562，BaF3/WT，BaF3/T315I细胞72h的MTT结果。
图5：不同浓度的化合物ZINC21710815作用于K562,BaF3/WT和BaF3/T315I细胞24h对细胞周期的影响。
图6：不同浓度的化合物ZINC21710815作用于K562,BaF3/WT和BaF3/T315I细胞24h对细胞凋亡的影响以及作用于BaF3/WT和BaF3/T315I细胞48h对细胞凋亡蛋白Caspase-3的影响。图7：不同浓度的化合物ZINC21710815作用于BaF3/WT和BaF3/T315I细胞48h对自噬相关蛋白LC3和Beclin1的影响。
图8：不同浓度的化合物ZINC21710815和阳性对照药Imatinib作用于BaF3/WT细胞48h对BCR/ABL靶点及其下游蛋白p-STAT5和p-Crkl的影响。
具体实施方式
以下实施例用于进一步说明本发明的保护范围，但不用来限制本发明的范围。
本发明的目的在于通过计算机筛选出靶向BCR/ABL酪氨酸激酶的抑制剂，并能够诱导T315I突变细胞凋亡。
靶向BCR/ABL酪氨酸激酶的化合物的筛选过程包含以下步骤：(1)收集已知的靶向BCR/ABL酪氨酸激酶的抑制剂，(2)建立药效团模型，(3)验证药效团模型，(4)用药效团模型筛选数据库，(5)进行类药性，ADMET和对接筛选，(6)细胞毒性实验，(7)结构优化，(8)生物实验验证。
本发明药效团模型的构建：收集从文献中获得的已知的靶向BCR/ABL酪氨酸激酶抑制剂，选择药效团特征元素，构建Hypogen和Hiphop药效团。
本发明药效团模型的验证：
挑选出打分值最好的Hypogen药效团模型，收集从文献中获得的已知的靶向BCR/ABL酪氨酸激酶抑制剂作为测试集，验证该药效团模型的预测活性的能力，Fischer随机验证法验证该模型的可靠性，Decoy set方法验证模型的的命中能力。
挑选出打分值最好的Hiphop药效团模型，收集从文献中获得的已知的靶向BCR/ABL酪氨酸激酶抑制剂作为测试集，验证该药效团模型的命中能力。
所述的相关理化性质，包括：
计算化合物的理化性质，包括氢键受体数量(Number of hydrogen bondacceptors，HBA)、氢键供体数量(Number of hydrogen bond donors，HBD)、脂水分配系数(Octanol/water partition coefficient，logP)、分子量(Molecular weight，MW)；
模拟化合物在在有机体内的吸收、分布、代谢、***和毒性等性质，包括预测化合物的肠道吸收性质，水溶性，血脑屏障通透性；
通过对接，预测化合物和受体蛋白的结合能。
实施例1：Hypogen药效团模型的生成
22个已知的BCR/ABL酪氨酸激酶抑制剂被选择作为Hypogen药效团模型的训练集，氢键受体(hydrogen-bond acceptor，HBA)，氢键供体(hydrogen-bond donor，HBD)，疏水中心(hydrophobic)，疏水性芳香环(hydrophobic_aromatic，HA)，正电离子中心(pos_ionizable，PI)药效特征元素被选择生成了10个Hypogen药效团模型。基于最高的相关系数和拟合值，最低的总成本和RMS值，Hypogen1被选择进行下一步分析。采用测试集(testset)，费舍尔(fischer randomization)和诱饵集方法(decoy set)对药效团模型Hypogen1进行验证。Hypogen1药效团能够较好的预测测试集化合物的活性，化合物的预测活性和实际活性之间的相关系数为0.847。费舍尔方法显示在95％置信区间内，结构和活性之间具有统计学相关性。诱饵集方法显示有较高的富集因子(Enrichment factor，10.5)，拟合度(goodness of fit score，0.8022)，表明Hypogen1有较高的效率区分活性分子和非活性分子。
实施例2：Hiphop药效团模型的生成
8个已知的BCR/ABL酪氨酸激酶抑制剂被选择作为Hiphop药效团模型的训练集，氢键受体(hydrogen-bond acceptor，HBA)，氢键供体(hydrogen-bond donor，HBD)，疏水中心(hydrophobic，H)，芳香环(ring_aromatic，RA)，正电离子中心(pos_ionizable，PI)，负电离子中心(neg_ionizable，NI)药效特征元素被选择生成了10个Hiphop药效团模型。根据打分值和特点相似性，10个药效团模型被聚类成两种类型，分别选取两种类型的打分值较高的药效团去匹配训练集的活性化合物和非活性化合物，Hiphop1模型被发现能更好的区别活性和非活性化合物，被选择进行下一步分析。采用测试集对药效团模型Hiphop1进行验证。方法显示有较高的富集因子(Enrichment factor，10.95)，拟合度(goodness of fitscore，0.91)，表明Hypogen1有较高的效率区分活性分子和非活性分子。
实施例3：虚拟筛选的流程如下
分别把已验证的Hypogen1和Hiphop1药效团模型在ZINC Clean Druglike数据库中进行三维搜索，经过Linpinski’s rule of five，ADMET和对接筛选，对两种途径筛选得到的化合物做细胞毒性实验并对活性较好的化合物进行结构优化，并对结构优化的化合物进行再次筛选。两次筛选得到的化合物的通式具有以下结构式：
其中：
(Ⅰ)R1:氢、甲基、乙基、异丙基、
R2:氢、甲基、乙基、异丙基、
R3：通过
和烷基连接侧链。
在结构优化后筛选的化合物中，购买ZINC21710815、ZINC36617889和ZINC20617585，对其做细胞毒性实验。附图4表明了MTT检测的三个化合物对K562，BaF3/WT和BaF3/T315I细胞抑制结果以及化合物ZINC21710815对正常的CCC-HEL-1细胞的毒性作用，其IC50值分别为：
K562细胞
ZINC21710815(0.531μM/L)，ZINC36617889(35.878μM/L)，
ZINC20617585(62.813μM/L)。
BaF3/WT细胞
ZINC21710815(0.512μM/L)，ZINC36617889(48.286μM/L)，
ZINC20617585(34.204μM/L)。
BaF3/T315I细胞
ZINC21710815(0.88μM/L)，ZINC36617889(149.629μM/L)，
ZINC20617585(>300μM/L)。
CCC-HEL-1细胞
ZINC21710815(89.587μM/L)
实验结果表明：ZINC21710815对K562细胞，BaF3/WT和BaF3/T315I细胞均有较好抑制活性但对正常的人胚肝二倍体细胞无毒性抑制作用，ZINC21710815作为代表性的化合物进行细胞周期、细胞凋亡、自噬和蛋白质印迹生物实验进行验证。
实施例4：抑制细胞周期的效果
K562细胞以每孔10×105个的密度一式三份的接种于6孔板中，BaF3/WT和BaF3/T315I细胞以每孔5×105个的密度一式三份的接种于6孔板中，每孔加入该化合物(ZINC21710815)，浓度为0，0.1，1，10μM/L孵育24h，收集细胞，并用PBS洗涤细胞，70％的乙醇固定细胞在-20℃至过夜，用PBS洗去固定液。细胞沉淀中加100μLRNase A溶液重悬细胞，37℃水浴30min，再加入400μL PI染色液混匀，4℃避光孵育30min，用流式细胞仪检测。
附图5表明了随着本发明化合物浓度的增大，对于K562细胞，G2期的细胞逐渐增多，该化合物可以诱导K562细胞周期阻滞在G2期；对于BaF3/WT和BaF3/T315I细胞，G1期的细胞逐渐增多，该化合物可以诱导BaF3/WT和BaF3/T315I细胞周期阻滞在G1期。
实施例5：抑制细胞凋亡的效果
K562，BaF3/WT和BaF3/T315I细胞以每孔5×105个的密度一式三份的接种于6孔板中，每孔加入该化合物，浓度为0，0.1，1，10μM/L孵育24h，收集细胞，并用PBS洗涤细胞，用1×Binding Buffer悬浮细胞，离心，弃上清，用1×Binding Buffer重悬细胞，每管加入100μL细胞(1×105个)，加入5μL Annexin V-FITC，室温，避光混匀10min，加入5μL PI避光孵育5min，加入PBS至500μL，1h后用流式细胞仪检测。
附图6表明了随着本发明化合物浓度增大，对于K562，BaF3/WT和BaF3/T315I细胞，凋亡细胞逐渐增多，该化合物可以诱导K562，BaF3/WT和BaF3/T315I细胞凋亡。
实施例6：化合物对凋亡相关蛋白，自噬相关蛋白，BCR/ABL和下游蛋白p-STAT5和p-Crkl表达的效果
低温离心收集经不同浓度的化合物处理48h的白血病细胞，加入细胞裂解液进行裂解。裂解液在低温离心机中14000xg离心15min，上清液待用。测不同浓度的标准品在570nm的吸光度并分别减去空白孔吸光度值，绘制在570nm吸光度对浓度(μg/ml)的标准曲线。使用标准曲线定量待测样品蛋白浓度。待测蛋白样品液加入1×buffer缓冲液，煮沸10min，分装待用。
配制分离胶和浓缩胶，***梳子，加入1×电泳缓冲液，拔去梳子，上样。样品首先在80V恒压下电泳至染料接近分离胶顶端，条带呈平的状态，然后在120V恒压下电泳至染料接近分离胶底部。然后把胶放在1×转膜缓冲液中，按照以下顺序进行转膜操作；白板(正极)-纤维垫-滤纸-PVDF膜-凝胶-滤纸-纤维垫-黑板(负极)，在100V恒压下，4℃条件下转膜。膜在5％脱脂奶粉中室温孵育75min，封闭膜上的非特异性结合，封闭的膜用TBST洗膜10min×3次。膜加入一抗4℃孵育过夜，TBST洗膜10min×3次，加入HRP标记的二抗室温孵育1h，TBST洗膜10min×3次，曝光处理，每个特异性条带的灰度值用ImageJ软件进行分析。
附图6表明了随着本发明化合物浓度的升高，凋亡相关蛋白Cleaved Caspase3的表达量逐渐增加，该化合物可能通过凋亡相关蛋白caspase3诱导BaF3/WT和BaF3/T315I细胞凋亡。
附图7表明了随着本发明化合物浓度的升高，自噬相关蛋白LC3-Ⅱ和Beclin1的表达量逐渐增加，该化合物可能通过自噬相关蛋白LC3和Beclin1诱导BaF3/WT和BaF3/T315I细胞自噬。
附图8表明了在本发明化合物的作用下，对BCR-ABL酪氨酸激酶和下游靶蛋白p-STAT5和p-Crkl的表达有抑制作用，该化合物可以抑制BCR/ABL酪氨酸激酶及其下游靶蛋白p-STAT5和p-Crkl磷酸化。
本发明的化合物适用于慢性粒细胞白血病，尤其是化合物ZINC21710815能够强烈的抑制K562，BaF3/WT和BaF3/T315I细胞的增殖，诱导K562，BaF3/WT和BaF3/T315I细胞周期阻滞，诱导K562，BaF3/WT和BaF3/T315I细胞凋亡，抑制BaF3/WT和BaF3/T315I细胞凋亡相关蛋白和自噬相关蛋白的表达，抑制BCR-ABL酪氨酸激酶及其下游蛋白的磷酸化。
Claims (11)
1.一种靶向BCR-ABL酪氨酸激酶的结构通式(Ⅰ)所示的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐：
其中：R1选自氢、甲基、乙基或异丙基；R2选自氢、甲基、乙基或异丙基；
R3：选自
或烷基。
2.如权利要求1所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐，其特征在于，所述式(Ⅰ)化合物的取代基R3被取代得到如下结构式：
其中R1选自氢、甲基、乙基或异丙基；R2选自氢、甲基、乙基或异丙基；
R4选自甲基、氢、
其中R1:选自氢、甲基、乙基或异丙基；R2选自氢、甲基、乙基或异丙基；
R5选自
其中R1选自氢、甲基、乙基或异丙基；R2选自氢、甲基、乙基或异丙基；R6为乙基；
其中R1选自氢、甲基、乙基或异丙基；R2选自氢、甲基、乙基或异丙基；R7选自甲基或
3.如权利要求2所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐，其特征在于，所述式(Ⅱ)化合物选自：
4.如权利要求2所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐，其特征在于，所述式(Ⅲ)化合物选自：
5.如权利要求2所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐，其特征在于，所述式(Ⅳ)化合物选自：
6.如权利要求2所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐，其特征在于，所述式(Ⅴ)化合物选自：
7.一种药物组合物，其特征在于，所述药物组合物含有治疗有效量的如权利要求1-6任一项所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐，以及一种或多种药学上可接受的载体，稀释剂或赋形剂。
8.如权利要求1-6任一项所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐在抑制BCR/ABL酪氨酸激酶中的应用。
9.如权利要求1-6任一项所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐在拮抗T315I突变中的应用。
10.如权利要求1-6任一项所述的化合物或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体及其混合物形式，及其可药用盐，在制备预防或治疗白血病药物中的应用。
11.如权利要求10所述的应用，其特征在于，所述的白血病为慢性粒细胞白血病。
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