CN113234026B - 具有b淋巴细胞酪氨酸激酶抑制活性的化合物及其应用 - Google Patents
具有b淋巴细胞酪氨酸激酶抑制活性的化合物及其应用 Download PDFInfo
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- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
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- A—HUMAN NECESSITIES
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明属于医药技术领域,具体提供具有B淋巴细胞酪氨酸激酶抑制活性的化合物及其应用。所述化合物选自九种(T1~T9)化合物中的至少一种。本发明的具有B淋巴细胞酪氨酸激酶抑制活性的化合物,能够有效地选择性抑制BLK活性,可以作为抑制剂或者药物。
Description
技术领域
本发明属于医药技术领域,尤其涉及具有B淋巴细胞酪氨酸激酶抑制活性的化合物及其应用。
背景技术
激酶的作用机理是从高能供体分子(如腺嘌呤核苷三磷酸,英文简称ATP)转移磷酸基团到特定的分子,这一过程称之为磷酸化。蛋白激酶通过磷酸化来改变特定蛋白的活性,从而控制并调节蛋白相关的信号传导和对细胞其他作用。由于蛋白激酶在细胞信号传导的重要作用,利用小分子化合物对于特定激酶的选择性,有助于深入了解细胞的信号传导过程。同时小分子化合物通过调节激酶的活性来控制细胞的功能,使蛋白激酶在临床疾病的治疗中成为良好的药物靶点。
B淋巴细胞酪氨酸激酶(BLK)与SRC、YES、LYN、FYN、LCK、HCK、FGR一并组成SRC激酶家族,属于非受体型蛋白酪氨酸激酶。BLK特异性地表达于B细胞系,也有文献报道,其也在人和小鼠的胰腺β细胞以及小鼠血浆树突细胞中表达。BLK参与BCR信号通路,与B细胞的发育和功能有关。当抗原结合受体,受体交联后,SRC家族蛋白激酶BLK、LYN和FYN激活,激活的SRC家族蛋白激酶BLK、LYN和FYN随即共同参与磷酸化ITAM,ITAM上磷酸化的酪氨酸残基作为停靠位点,招募下游的SYK,定位在膜上的SYK随即被SRC家族蛋白激酶磷酸化激活,活化的SYK磷酸化B细胞连接蛋白,它可以使BCR相关蛋白激酶与一些信号通路桥接起来,导致多种下游分子磷酸化,其中包括BTK激酶等。BCR信号通路参与调节细胞的增殖、分化、凋亡和迁移,并且参与正常B细胞的发育和存活,该通路的活化对于B细胞肿瘤和自身免疫性疾病的发生、发展起着重要的作用,抑制Btk活性而阻断BCR信号通路已经成为多种血液肿瘤治疗的最重要手段。
B细胞的异常激活和自身抗体的分泌是系统性红斑狼疮重要病理特征之一,BCR作为B细胞激活的关键信号通路,通路中的多个成员已成为系统性红斑狼疮的潜在治疗靶点。BLK作为BCR信号通路的潜在负调控因子,其功能的缺失对系统性红斑狼疮的发展的促进作用已被多项研究证实。BLK作为系统性红斑狼疮的风险基因主要来源于研究人员对系统性红斑狼疮病人家族基因的分析,并鉴定了多种BLK风险的突变,体外的实验表明,这些风险突变能造成BLK表达量的下降,失去对IRF5的抑制作用,最终导致IFNβ分泌增多,而促进系统性红斑狼疮的发展。此外,研究人员也通过构建BLK缺失的C57BL/6-lpr/lpr的小鼠模型,证实了BLK缺失导致对系统性红斑狼疮发展的促进作用。2003年,霍普金斯大学医学院的研究人员通过构建BLK(Y495F)激活的转基因小鼠首次揭示了BLK的肿瘤转化作用,提示了BLK是潜在的促瘤基因。2015年,研究人员在从皮肤T细胞淋巴瘤(Cutaneous T-celllymphoma,CTCL)病人分离的原代恶性T细胞中发现了BLK的异常表达,在CTCL中异常表达的BLK处于持续激活的状态,使用siRNA敲低BLK的表达或者利用具有泛Src家族抑制活性的dasatinib和LCKi抑制BLK的激酶活性均能够有效地抑制恶性T细胞的增殖,这一研究首次揭示了BLK参与人类肿瘤的发生。2017,研究人员发现BTK抑制剂ibrutinib对病人来源的急性B细胞型淋巴细胞性白血病(B-cell acute lymphoblastic leukemia,B-ALL)的增殖抑制作用是通过同时靶向BTK和BLK起效的,这一研究提示着BLK参与了某些B细胞肿瘤的发展。除了对B细胞和T细胞肿瘤增殖的促进作用,研究人员发现,BLK表达于恶性胶质瘤细胞系U87,利用shRNA敲低BLK的表达虽然不能抑制U87细胞的增殖,但能够一定程度上抑制U87细胞的迁移,具体参见Samuelson,E.M.,et al.“Reduced B lymphoid kinase(Blk)expression enhances proinflammatory cytokine production and induces nephrosisin C57BL/6-lpr/lpr mice.”PloS one 2014,9(3),e92054-e92054.Jiang,S.H.;et al.“Functional rare and low frequency variants in BLK and BANK1contribute tohuman lupus.”Nat Commun 2019,10(1),2201.Petersen,D.L.;et al.“B-lymphoidtyrosine kinase(Blk)is an oncogene and apotential target for therapy withdasatinib in cutaneous T-cell lymphoma(CTCL).”Leukemia 2014,28(10),2109-12.Kim,E.;et al.“Ibrutinib inhibits pre-BCR(+)B-cell acute lymphoblasticleukemia progression by targeting BTK and BLK.”Blood 2017,129(9),1155-1165.
发明内容
本发明实施例提供具有B淋巴细胞酪氨酸激酶抑制活性的化合物及其应用,旨在为BLK激酶域的特异性活性抑制提供相应的小分子化合物以及为相关功能研究提供新的工具。
本发明是这样实现的:
一种具有B淋巴细胞酪氨酸激酶抑制活性的化合物,所述化合物选自以下化合物中的至少一种:
相应地,上述具有B淋巴细胞酪氨酸激酶抑制活性的化合物在作为抑制剂的应用。
可选地,所述应用为将所述具有B淋巴细胞酪氨酸激酶抑制活性的化合物作为B淋巴细胞酪氨酸激酶的抑制剂。
以及,上述具有B淋巴细胞酪氨酸激酶抑制活性的化合物作为药物的应用。
可选地,所述应用为将所述具有B淋巴细胞酪氨酸激酶抑制活性的化合物作为B细胞淋巴癌、皮肤T细胞淋巴瘤、自身免疫性疾病、异种免疫性疾病、炎性疾病及其他癌症的的抑制剂。
本发明的有益效果如下:
相对于现有技术,本发明提供的具有B淋巴细胞酪氨酸激酶抑制活性的化合物,能够有效地选择性抑制BLK的活性,因此可以作为抑制剂或者药物,作为BLK的抑制剂时,具有作用快速,且不会影响激酶的蛋白含量,能够克服基因敲除造成的潜在基因补偿效应或者其他信号通路的补偿,此外,还能实现单一地探索BLK激酶域的特异性相关功能,以有效裂解BLK的复杂功能并为BLK生理、病理功能的进一步研究提供有力支撑。而作为药物时可以有效预防及治疗多种癌症、自身免疫性疾病、异种免疫性疾病、炎性疾病等。
附图说明
为了更清楚地说明本发明施例中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍。显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是本发明实施例提供的具有B淋巴细胞酪氨酸激酶抑制活性的化合物对激酶磷酸化程度的影响结果。
具体实施方式
为了使本发明要解决的技术问题、技术方案及有益效果更加清楚明白,以下结合实施例,对本发明做进一步的详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1
一种具有B淋巴细胞酪氨酸激酶抑制活性的化合物的制备方法,包括以下步骤:
(1).将间苯二胺(0.500g,4.62mmol),(Boc)2O(0.92mL,4.02mmol)和三乙胺(1.4mL,9.98mmol)加至已经降温到0℃的1,4-二氧六环和水的混合溶剂体系中(30mL,2:1V/V)。该反应体系在0℃下搅拌1h后,恢复至室温继续搅拌10h。反应液经减压浓缩得到黄色油状物,该油状物被乙酸乙酯溶解后,依次用饱和碳酸氢钠溶液、饱和食盐水洗涤,最终有机相用硫酸镁干燥,过滤并减压浓缩。浓缩物用硅胶柱层析纯化,得到为白色固体(0.48g,产率:58%),即化合物2,整个反应过程如反应方程式一所示。
反应方程式一:
(2).将化合物2(0.352g,1.69mmol)和2-氯-5-硝基嘧啶(0.270g,1.69mmol)先溶于12mL乙腈中。然后在该溶液中加入碳酸钾(0.702g,5.08mmol),整个反应体系在室温下搅拌3h后,将反应溶剂用减压旋蒸去除后,浓缩物用乙酸乙酯溶解后,依次用水、饱和食盐水洗涤。最终有机相用硫酸钠干燥后,减压浓缩,以硅胶柱层析纯化,得到黄色固体(0.50g,产率:89%)为,及化合物3,整个反应过程如反应方程式一所示。
(3).将化合物3(0.500g,1.51mmol)和钯碳(0.16g质量分数:5%)加入一个25mL的两口烧瓶中,在保持缓慢搅拌下10mL甲醇加入该反应体系中,整个反应体系中的空气被氮气替换后,一个充有足够氢气的氢气球被接到该体系上,而后反应体系里面的氮气被气球中的氢气置换(三次)。反应体系在室温下保持搅拌3h后终止反应,反应液用砂芯漏斗滤去残留钯碳得到棕色滤液。滤液浓缩后,以硅胶柱层析纯化,得到黄色固体(0.45g,产率:100%),即为化合物4,整个反应过程如反应方程式一所示。
(4).将化合物5(1.06g,1eq)悬浮于50mL干燥的二氯甲烷中,加入重蒸的三乙胺(1.46mL,1.5eq),冰浴冷却。同时将Fmoc-Osu(3.07g,1.3eq)溶于20mL干燥的二氯甲烷中,冰浴冷却后,用注射器缓慢打入IV-1的二氯甲烷的悬液中,缓慢恢复室温,反应4-6h。TLC监测反应结束后,减压除去易挥发的组分,用乙酸乙酯和1N HCl萃取,有机相用饱和食盐水洗,并用无水硫酸钠干燥。浓缩,重结晶得到目标产物,白色固体2.68g,产率80%,即化合物6,整个反应过程如反应方程式二所示。
反应方程式二:
(5).将化合物6(2.19g,1.1eq),化合物4(1.61g,1eq),HATU(4.06g,2eq)溶于25mL干燥的DMF中,搅拌溶解,加入DIEA(1.76mL,2eq),室温搅拌反应过夜。TLC监测反应结束后,减压尽量除去DMF,乙酸乙酯和饱和碳酸钠溶液萃取,有机相用饱和食盐水洗,并用无水硫酸钠干燥。浓缩,柱层析得浅黄色固体(2.58g,产率74%),即化合物7,整个反应过程如反应方程式二所示。
(6).将化合物7(2.58g)溶于含有体积比为50%吗啡啉的DMF溶液,室温搅拌过夜。TLC监测反应结束后,减压尽量除去DMF,乙酸乙酯和饱和碳酸钠溶液萃取,有机相用饱和食盐水洗,并用无水硫酸钠干燥。浓缩,柱层析得白色固体(1.45g,产率85%),即化合物8。
(7).将化合物8(50mg,1eq),4’-氟-[1,1’-联苯基]-3-羧酸(27mg,1.1eq),HATU(87mg,2eq)溶于1mL干燥的DMF中,加入DIEA(38uL,2eq),反应过夜。TLC监测反应结束后,减压除去易挥发组分,用乙酸乙酯和饱和碳酸钠溶液萃取,有机相用饱和食盐水洗,无水硫酸钠干燥。浓缩,柱层析得到产物74mg;其溶液4mL二氯甲烷中,加入2mL TFA,室温反应2h。减压除去有机溶剂和多余的TFA,用乙酸乙酯和饱和碳酸钠溶液萃取,有机相用饱和食盐水洗,无水硫酸钠干燥。浓缩,重结晶得淡黄色固体42mg,即化合物9,两步总产率66%。
(8).将化合物9(0.094g),Boc保护的甘氨酸9(0.032g)和HATU(0.101g)溶于3mLDMF中,在保持缓慢搅拌下加入二异丙基乙胺(44μL)。反应液在室温下保持搅拌2h后,溶剂用减压旋蒸去除后,残留物溶于乙酸乙酯,然后依次用饱和碳酸氢钠溶液和饱和食盐水洗涤。最终的有机相以无水硫酸镁干燥后,过滤,减压浓缩,以硅胶柱层析纯化;接着将得到的化合物分散于2mL二氯甲烷中,在保持搅拌下将2mL三氟乙酸慢慢的滴入反应体系中。最终的反应体系在室温下保持搅拌1h后,减压浓缩得到固状物。该残留物用乙酸乙酯溶解后依次用10%氢氧化钠溶液和饱和食盐水洗涤。最终有机相以无水硫酸镁干燥后,减压浓缩,真空干燥过夜后得到的黄色固体(0.052g)分散于THF和水的混合溶剂中(2mL,1:1V/V),然后加入二异丙基乙胺(18μL,0.11mmol)。在保持缓慢搅拌下,丙烯酰氯(14μL,0.18mmol)缓慢的滴入反应体系中。反应液在室温下搅拌2h后,减压浓缩,残留物用乙酸乙酯溶解后依次用饱和碳酸氢钠溶液、饱和食盐水洗涤。最终有机相以无水硫酸镁干燥后减压浓缩,浓缩物用硅胶柱层析纯化得产物T2(50mg,白色固体),具体如反应方程式二所示,多步总产量为44%。
实施例2
一种具有B淋巴细胞酪氨酸激酶抑制活性的化合物的制备方法,反应生成化合物T1。其制备方法与实施例1相同,所不同的是化合物9反应生成化合物T1的过程中,添加的反应物不同,而是根据生成的化合物T1的结构添加反应物,其中,化合物T1的结构式如下:
实施例3
一种具有B淋巴细胞酪氨酸激酶抑制活性的化合物的制备方法,反应生成化合物T3。其制备方法与实施例1相同,所不同的是化合物9反应生成化合物T3的过程中,添加的反应物根据生成的化合物T3的结构进行添加,其中,化合物T3的结构式如下:
实施例4
一种具有B淋巴细胞酪氨酸激酶抑制活性的化合物的制备方法,反应生成化合物T4。其制备方法与实施例1相同,所不同的是化合物9反应生成化合物T4的过程中,添加的反应物根据生成的化合物T4的结构进行添加,其中,化合物T4的结构式如下:
实施例5
一种具有B淋巴细胞酪氨酸激酶抑制活性的化合物的制备方法,反应生成化合物T5。其制备方法与实施例1相同,所不同的是化合物9反应生成化合物T5的过程中,添加的反应物根据生成的化合物T5的结构进行添加,其中,化合物T5的结构式如下:
实施例6
一种具有B淋巴细胞酪氨酸激酶抑制活性的化合物的制备方法,反应生成化合物T6。其制备方法与实施例1相同,所不同的是化合物9反应生成化合物T6的过程中,添加的反应物根据生成的化合物T6的结构进行添加,其中,化合物T6的结构式如下:
实施例7
一种具有B淋巴细胞酪氨酸激酶抑制活性的化合物的制备方法,包括以下步骤:
(1).将化合物10(1.28g)(化合物10与化合物4的制备方法相同),2-甲基-5-硝基苯甲酸(0.72g)和HATU(1.89g)溶于10mL DMF,滴加DIEA(1.2mL),室温反应过夜。TLC监测反应结束后,减压除去易挥发组分,用乙酸乙酯和饱和碳酸钠溶液萃取,有机相用饱和食盐水洗,无水硫酸钠干燥。浓缩,柱层析得到黄色固体(1.37g,75%),即化合物11,具体反应过程如反应方程式三所示。其中,化合物10的获得类似化合物4的合成步骤。
反应方程式三:
(2).将化合物11(0.082g)溶于2mL TFA和DCM中,室温搅拌2h,真空浓缩得到的黄色固体容易2mL DMF,接着加入DIEA(0.104mL),丙烯酸(0.027g)和HATU(0.114g),室温搅拌过夜。TLC监测反应结束后,减压除去易挥发组分,用乙酸乙酯和饱和碳酸钠溶液萃取,有机相用饱和食盐水洗,无水硫酸钠干燥。浓缩,柱层析得到化合物12,0.042g,产率56%。具体反应过程如反应方程式三所示。
(3).将化合物12(0.06g),SnCl2·2H2O(0.176g)溶解于5mL乙醇中,回流2h。TCL检测反应结束后,带冷却至室温,用乙酸乙酯和饱和碳酸钠溶液萃取,有机相用饱和食盐水洗,无水硫酸钠干燥。浓缩,柱层析得到的黄色固体(0.087g),以及6-异喹啉甲酸(0.044g),HATU(0.174g)溶于2mL DMF中,接着滴加DIEA(0.16mL),室温反应过夜。TLC监测反应结束后,减压除去易挥发组分,用乙酸乙酯和饱和碳酸钠溶液萃取,有机相用饱和食盐水洗,无水硫酸钠干燥。浓缩,TLC制备板分离以及HPLC分离得到白色固体(0.049g,产率65%),即化合物T7。具体反应过程如反应方程式三所示。
实施例8
一种具有B淋巴细胞酪氨酸激酶抑制活性的化合物的制备方法,反应生成化合物T8。其制备方法与实施例7相同,所不同的是化合物12反应生成化合物T8的过程中,添加的反应物根据生成的化合物T8的结构进行添加,其中,化合物T8的结构式如下:
实施例9
一种具有B淋巴细胞酪氨酸激酶抑制活性的化合物的制备方法,反应生成化合物T9。其制备方法与实施例7相同,所不同的是化合物12反应生成化合物T9的过程中,添加的反应物根据生成的化合物T9的结构进行添加,其中,化合物T9的结构式如下:
性能测试:
对实施例1~9得到的化合物进行了BLK的体外抑制活性性能验证分析,具体验证过程如下:
在重组激酶活性实验中,使用如下所述的方法测定实施例1~9所得化合物对BLK的半抑制浓度IC50。
使用时间分辨荧光共振能量转移(time-resolved fluorescence resonanceenergy transfer)(TR-FRET)方法测定BLK激酶活性。使用384孔测定板,在10μL反应体积中进行测定。将激酶、抑制剂、ATP(在激酶的Km)和1μM肽底物(生物素-AVLESEEELYSSARQ-NH2)在反应缓冲液(pH 7.4)中孵育1h,所述反应缓冲液由20mM Tris、50mM NaCl、MgCl2(5-25mM,取决于激酶)、MnCl2(0-10mM)、1mM DTT、0.1mM EDTA、0.01%牛血清白蛋白、0.005%吐温-20和10%DMSO组成。通过加入5μL的1×Lance缓冲液(Perkin-Elmer)中的1.2当量的EDTA(相对于二价阳离子)猝灭反应物。在5μL体积中加入链霉亲和素-APC(Perkin-Elmer)和Eu标记的p-Tyr100抗体(Perkin-Elmer)的1×Lance缓冲液,混合物温育1h。使用多模式读板仪(multimode plate reader),330nm的激发波长(λEx)及615nm和665nm的检测波长(λEm)下测量TR-FRET信号。通过665nm与615nm下的荧光比确定活性。对每一种化合物,测定了不同浓度化合物下的酶活性。阴性对照反应在缺少抑制剂情况下进行(做六个一式两份),并且用两个无酶对照来确定基线荧光水平,使用软件拟合获得IC50。
在BLK的体外抑制活性分析中,测定了本发明的实施例1~9的化合物的IC50值,并且记录在表1中,按照IC50值所处区间给出IC50值,其中“+++”代表IC50<50nM;“++”代表50nM<IC50<500nM;“+”代表500nM<IC50<5000nM。
表1
采用一系列浓度梯度(10nM、30nM、100nM、300nM、1000nM)的化合物(T9)处理Ramos细胞1小时后,收集细胞,用蛋白免疫印迹的方法检测化合物对激酶磷酸化程度的影响,结果如图1所示。其中,p-SYK-352、p-SYK-525/6、p-BTK-551分别表示BLK激酶下游底物SYK激酶和BTK激酶被检测的具体磷酸化位点,以二甲亚砜(DMSO)作为阴性对照。
从图1可知,该化合物能有效抑制p-SYK及p-BTK-551的磷酸化。由于化合物T1~T8与化合物T9具有类似的结构,也具有类似的性能。
综上可见,本发明设计合成的具有B淋巴细胞酪氨酸激酶抑制活性的化合物,能够有效地选择性抑制BLK活性,因此可以做为B淋巴细胞酪氨酸激酶的抑制剂。提供了能够潜在的治疗B细胞淋巴癌、皮肤T细胞淋巴瘤、自身免疫性疾病、异种免疫性疾病、炎性疾病及其他癌症的药物;同时也为科研领域提供了进一步研究BLK生理、病理功能的小分子化学工具。
由此也可以得出,本发明实施例提供的具有B淋巴细胞酪氨酸激酶抑制活性的化合物也可以作为药物,以用于治疗癌症等病症。具体地,作为B细胞淋巴癌、皮肤T细胞淋巴瘤(CTCL)、自身免疫性疾病、异种免疫性疾病、炎性疾病及其他癌症的药物。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (2)
1.一种具有B淋巴细胞酪氨酸激酶抑制活性的化合物,其特征在于,所述化合物选自以下六种化合物中的至少一种:
2.如权利要求1所述的具有B淋巴细胞酪氨酸激酶抑制活性的化合物在制备p-SYK-352、p-SYK-525/6、p-BTK-551激酶抑制剂的应用。
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