CN1219556C - 4-h-1-苯并吡喃-4-酮衍生物作为平滑肌细胞增生抑制剂的用途 - Google Patents
4-h-1-苯并吡喃-4-酮衍生物作为平滑肌细胞增生抑制剂的用途 Download PDFInfo
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Abstract
平滑肌细胞(SMC)增生是血管损伤的许多动物模型中新内膜形成的关键,对人体损伤也是如此。通过基因转移技术的细胞周期抑制作用能够阻止许多动物模型中SMC增生和损伤的形成,虽然这些方法尚不能用于人体疾病治疗。黄酮吡啶酚是近期被认可的、有力的可口服的细胞周期蛋白-依赖性激酶抑制剂。基于血管损伤疾病中SMC增生的作用,我们测试了近期鉴定的、作为细胞周期蛋白-依赖性激酶抑制剂的黄酮吡啶酚对SMC生长的体外和体内作用效果。黄酮吡啶酚(75nmol/L)有力地阻止SMC增生,这是一种与下调细胞周期蛋白-依赖性激酶活性和细胞周期-相关基因表达相联系的作用。我们检测黄酮吡啶酚在大鼠颈动脉损伤模型中体内对SMC增生的效果。气囊损伤后7和14天黄酮吡啶酚(5mg/kg)减少新内膜的大小分别为35%和39%。黄酮吡啶酚是一种治疗SMC-富含的血管损伤的有潜力的药物。4-H-1-苯并吡喃-4-酮衍生物在低剂量水平抑制平滑肌细胞增生。
Description
发明领域
本发明涉及作为平滑肌细胞(SMC)增生抑制剂的4-H-1-苯并吡喃-4-酮衍生物的用途
关于联邦政府提出的研究或开发的说明
本发明以健康国家协会(National Institutes of Health)HL03658和AG15234号批文授权作出。
发明背景
对血管损害的细胞反应——细胞机能障碍、活化、去分化、增生和游走,在临床病案中,例如伴随治疗病人的动脉粥样硬化病的气囊血管成形术(Balloonangioplasty)和斯滕特氏固定模置换术后发生的心瓣再狭窄1,达到了顶点。平滑肌细胞(SMC)增生是血管损害型疾病的一种共同的并且或许是一致的模式,而SMC是新内膜损害的主要细胞构件2,3。重新对抑制SMC增生的关注是与用于治疗冠状动脉疾病(coronary disease)的斯滕特氏固定模的大量使用一起发生的,因为斯滕特氏固定模术心瓣再狭窄几乎完全取决于新内膜的形成和SMC的增生4。据估计,在1997年一年中多达100,000个斯滕特氏固定模术心瓣再狭窄病人需要治疗5;因此,一种容易服用的SMC增生的有效抑制剂就具有重要的临床上和经济上的意义6。
在血管损害模型中抑制SMC增生的努力,不论是通过调节增生反应的细胞介体,还是通过用细胞周期运作直接干涉,都获得新内膜的形成的大量的深入的了解。细胞周期的进程是一种密切受控的过程,由细胞周期蛋白-依赖的激酶(Cdk)和它们的细胞周期蛋白调节亚单位进行正向调节7,由Cdk抑制剂和肿瘤抑制基因如成视网膜细胞瘤蛋白(Rb)和p53进行反向调节8。内源的Cdk抑制剂p21和p27kip1或Rb的基本活性形式的腺病毒调节的过表达(overexpression),在大鼠颈动脉损伤模型中阻断新内膜的形成9-11;类似地,通过竞争性过表达关联DNA结合位点,对转录因子E2F活性的抑制作用也抑制了SMC增生和新内膜形成12。这些研究支持以下的总设想:为了在血管损害的形成中进行介入,细胞周期抑制作用是一个的诱人的目标。
虽然在调节新内膜形成的解剖学机制上基因介入是有助的,但目前,临床上,不适用于人类的血管病治疗是其很大的缺陷。水溶性的、低分子量的具有特定的细胞周期调节作用的,尤其是具有口服活性的化合物,将在试验和将来在临床上获得广泛的使用。近期证实黄酮、黄酮吡啶酚(flavopiridol)是一种有效阻止Cdk2、Cdc2和Cdk4的活性的Cdk抑制剂13-16。同Cdk的其它药物学的抑制剂比较,黄酮吡啶酚以其激酶特性、其可以口服性和其效力,在毫微摩尔浓度下的作用而著称16。这些独一无二的特征产生有利的副作用方面,导致在难治疗的肿瘤治疗的第一阶段临床试验(PhaseI clinical trial)中测试黄酮吡啶酚17。给出这些特性,我们检测黄酮吡啶酚体外和大鼠颈动脉气囊损害后抑制SMC增生的能力。我们成功的发现黄酮吡啶酚是一种有效的和有选择性的细胞周期进程抑制剂,并且在体内和体外它都阻止SMC增生;此外,通过低于其它已知的对人类有毒性作用药物的口服剂量的黄酮吡啶酚,新内膜的形成被有效阻止。
已经令人惊讶的发现,4-H-1-苯并吡喃-4-酮衍生物是合适的SMC增生抑制剂。已经知道4-H-1-苯并吡喃-4-酮衍生物适合用于控制肿瘤。但是,令人惊异的是,根据本发明,作为SMC增生抑制剂,4-H-1-苯并吡喃-4-酮衍生物以低于为控制肿瘤生长必须的剂量水平低的剂量服用,可有效的起作用。
因此,本发明的目的是使用4-H-1-苯并吡喃-4-酮衍生物作为平滑肌增生的抑制剂。
图1、黄酮吡啶酚对HASMC DNA合成的作用。A、在不存在(-)或存在(+)bFGF(10ng/ml)下用指定的黄酮吡啶酚浓度(nmol/L)处理静息的HASMC24小时。作为增生度量的BrdU加入量通过基于ELISA的分析来测定并表达为在bFGF处理不存在下加入量的百分比。*p<0.05,与未处理的细胞比较。p<0.05,与在不存在黄酮吡啶酚下用bFGF处理相比较。B、用bFGF(10ng/ml)、凝血酶(2U/ml)、或在存在或不存在黄酮吡啶酚(75mmol/L)下的载体处理HASMC并测定BrdU的加入量。*p<0.05,与未处理的细胞比较。**p<0.05,与单独用bFGF处理比较。p<0.05,与单独使用凝血酶处理比较。
图2、黄酮吡啶酚对HASMC增生的作用。单独用bFGF(10ng/ml)(◆)、bFGF和黄酮吡啶酚(75mmo/L)(●)或载体(■)以指定的时间处理静息的HASMC并测量处理后的细胞计数。结果以每孔的细胞计数表示(×103)。
图3、黄酮吡啶酚对在HASMC中的细胞周期蛋白-依赖的激酶活性的作用。用bFGF(10ng/ml)、凝血酶(2U/ml)、或在存在或不存在黄酮吡啶酚(75mmol/L)下的载体处理静息的HASMC,并量化组蛋白H1的磷酰化程度,作为Cdk活性的度量并表达为在不存在bFGF处理下Cdk活性的百分数。*p<0.05,与未处理的细胞比较。**p<0.05,与单独用bFGF处理比较。p<0.05,与单独使用凝血酶处理比较。
图4、细胞周期-相关蛋白通过黄酮吡啶酚的调节作用。静息的HASMC在存在(+)或不存在(-)bFGF(10ng/ml)、凝血酶(2U/ml),和/或黄酮吡啶酚(75nmol/L)处理24小时。细胞溶胞产物的免疫印迹法用特定抗体识别细胞周期蛋白D1(上组)、PCNA(中组),以及磷酰化的(pRb)和过磷酰化(ppRb)的Rb(下组)进行。
图5、黄酮吡啶酚对在HASMC中MAP激酶活性的作用。静息的HASMC在存在(+)或不存在(-)bFGF(10ng/ml)、凝血酶(2U/ml),PD98059(30μmol/L)和/或黄酮吡啶酚(75nmol/L)下处理30分钟。磷酰化Erk1(pErk1)和Erk2(pErk2)的水平通过免疫印迹法用识别两种蛋白的磷酰化特定抗体进行测量(上组)。MAP激酶活性用凝胶中的激酶分析法来测量,使用髓磷脂基的蛋白作为底物(下组)。
图6、用黄酮吡啶酚处理后的HASMC的活力。静息的HASMC用黄酮吡啶酚(75mmo/L)、TNF-□(50ng/ml),或载体以指定的时间处理。细胞存活通过台盼蓝排除法评定。结果表示为存活的细胞对总细胞计数的百分比。
图7、在气囊损害后采用黄酮吡啶酚的大鼠颈动脉新内膜形成的抑制作用。在损害后用或不用黄酮吡啶酚(5mg/kg)治疗5天的组织切片或大鼠颈动脉来测量新内膜/血管中层之比。动脉在损害后在7(n=12)和14(n=12)天进行检查。还给出了每一时段和治疗组在动脉新内膜中PCNA-阳性的核的百分比(±SEM,表示为计数的核的百分比)。*p<0.05,与用载体处理的比较。
图8、大鼠颈动脉的组织切片。切片是动脉损伤后7(组A和B)和14(组C和D)天取样的。示于组A和C中的动脉是通过管饲法用黄酮吡啶酚(5mg/kg)治疗的大鼠的动脉;示于组B和D中的动脉是仅用载体治疗的大鼠的动脉。原始放大率,×100。
图9、大鼠颈动脉气囊损害后Cdk2的表达。切片是动脉损伤后7(组A和B)和14(组C和D)天取样的。示于组A和C中的动脉是通过管饲法用黄酮吡啶酚(5mg/kg)治疗的大鼠的动脉;示于组B和D中的动脉是仅用载体治疗的大鼠的动脉。Cdk2-阳性核,主要位于新内膜,通过碱性磷酸酯酶法用Vector蓝染色。原始放大率,×100。
适用的4-H-1-苯并吡喃是下式的化合物或者是其可药用酸加成盐
式I
式中
R1是氢,1-6碳原子的烷基,芳基-C1-C4-烷基;卤素、羟基或羧基取代的C1-C6-烷基;C3-C6-环烷基,吡啶基,噻吩基,C3-C6-环烷基-C1-C4-烷基,C2-C6-链烯基,C2-C6-链炔基,苯基;卤素、C1-C4-烷基、C1-C4-烷氧基、羟基、羧基、COO-烷基、CONH2、CONH-烷基、CON(烷基)2、硝基、三氟甲基、氨基、C1-C4-烷氨基、二-C1-C4-烷氨基、或苯基一或多取代的苯基;萘,羧基,-CHO-,COO-C1-C4-烷基,伯氨基,烷氨基,芳烷基氨基,二烷基氨基,酰氨基,芳氨基,二芳氨基或-CH2O-C1-C4-烷基;
R2是氢、1-6碳原子的烷基、芳基、硝基、氨基、二-C1-C4-烷氨基、卤素、羟基、烷氧基、-COOH、-COO-C1-C4-烷基、-CHO、-CH2OH或-CH2O-C1-C4-烷基;
R3是氮,C1-C4-烷基;卤素、羟基或羧基取代的C1-C4-烷基;羟基,羧基,硝基,氨基,C1-C6-烷氨基,二-C1-C4-烷氨基,卤素,-O-烷基-C(O)-烷基,-CHO,-CH2OH,-CH2O-C1-C4-烷基或R2N-C(O)-O-,其中R是H、C1-C6-烷基、环烷基;或-O-烷基-C(O)-烷基或芳基;
R4是氢。羟基,C1-C4烷氧基,C1-C4-烷酰氧基,C1-C4-烷氧基羰基,芳氧基,氨基,C1-C4-烷氨基,二-C1-C4-烷氨基,或R’2-N-C(O)-O-,其中R’2是H,C1-C6-烷基,环烷基或芳基;
R5是氢。C1-C6-烷基,芳基-C1-C4-烷基,C3-C8-环烷基,C3-C6-环烷基-C1-C4-烷基,烷基氨基,C1-C4-烷酰基、-C(O)-O-C1-C4-烷基或芳酰基,其中在R1、R2、R3、R4和R5中的芳基是不取代的苯基或由卤素、C1-C4-烷基、C1-C4-烷氧基、羟基、羧基、COO-烷基、CONH2、CONH-烷基、CON(烷基)2、硝基、三氟甲基、氨基、C1-C4-烷氨基、二-C1-C4-烷氨基或苯基一取代或多取代的苯基;
m是0和3之间的整数而n是1。
本发明的化合物有二个不对称中心,一个在含有氮的杂环处,与苯并吡喃的那一部分(C-4’)稠合,另一个在R4-取代的碳原子处(C-3’),这意味着可能有两对旋光异构体。本发明的化合物的定义包含所有可能的立体异构体和它们的混合物。它尤其包括消旋体形式和具有特定活性的分离的旋光异构体。这两种消旋体可以通过物理方法(如分步结晶)拆分。单个旋光异构体可以通过常规的方法从消旋体中得到,例如用旋光酸形成盐然后结晶。
适合用作R1-R5的烷基的例子有,最多6个碳原子,优选最多5个碳原子的直链或支链基团,如甲基、乙基、丙基、异丙基、叔丁基、戊基或异戊基基团。
适合用作R1-R5的取代的烷基基团的例子是,卤烷基(如三氟甲基)、羟烷基(如羟乙基)或羧烷基(如羧乙基)。
有3-6个碳原子的并由R1-R5代表的环烷基的适用的例子有环丙基、环丁基、环戊基或环己基。环烷基烷基的例子是环丙基甲基。
由R1-R5代表的芳烷基的例子有苯基烷基基团,其中的苯基集团是未取代的或由诸如卤素、C1-C4-烷基、C1-C4-烷氧基或硝基或由三氟甲基基团、氨基基团和取代的氨基基团之类的取代基团一取代或多取代的。
由R1-R5代表的芳基的例子有未取代的或由诸如卤素、C1-C4-烷基、C1-C4-烷氧基、羟基、羧基、COO-烷基、CONH2-、CONH-烷基、CON(烷基)2、硝基或三氟甲基、氨基、C1-C4-烷氨基、二-C1-C4-烷氨基、芳族杂环基团(如吡啶基)和多环芳族基团(如萘基)一取代或多取代的苯基基团。
由R1-R5代表的烷基氨基基团的适用的例子是(CH2)n-NR6R7,其中,n是1-3而R6和R7是烷基和如上烷基R1-R5的情况所规定的;此外,R6和R7与氮原子(与它们键连)一起可以是有一个或多个杂原子的杂环。由R6和R7一起与氮原子(与它们键连)所形成的杂环的合适的例子是哌啶、吡咯烷、吗啉、哌嗪或咪唑,它们都可以不取代或由C1-C4-烷基、C1-C4-烷氧基或芳基或由羟基或氨基在一或多个位置上取代。
带有无机或有机酸的本发明的化合物的盐的适用的例子是盐酸盐、氢溴化物、硫酸盐、磷酸盐、乙酸盐、草酸盐、酒石酸盐、柠檬酸盐、马来酸盐或延胡索酸盐。
优选式Ia的化合物
其中,R1是氢、C1-C3-烷基、萘基、苯基;由卤素、C1-C4-烷基、C1-C4-烷氧基、羟基、羧基、COO-烷基、CONH2-、CONH-烷基、CON(烷基)2、硝基、三氟甲基、氨基、C1-C4-烷氨基、二-C1-C4-烷氨基或苯基一取代或多取代的苯基;吡啶基或噻吩基;
R2是氢或C1-C3-烷基;
R5是C1-C3-烷基,C3-C5-环烷基,或C3-C5-环烷基-C1-C4-烷基。
特别优选式Ia的化合物,其中,R1是苯基、噻吩基、吡啶基、氯苯基、二氯苯基、甲苯基、氨基苯基、溴苯基、羟苯基或萘基;
R2是氢而
R5是甲基。
一种特别重要的化合物是(-)-顺式,-5,7-二羟基-2-(2-氯苯基)-8-[4-(3-羟基-1-甲基)-哌啶基]-4H-苯并吡喃-4-酮(黄酮吡啶酚),尤其是呈盐酸盐形式的。
本发明的化合物可以按照US 4 900 727和US 5 284 856的说明制备,这两个专利本文用作参考。上述美国专利的实施例也与本发明相关。
本发明的化合物抑制平滑肌细胞的增生。因此本发明的另一个从属的问题就是抑制平滑肌细胞增生的药物,它含有至少一种如上所定义的式I的化合物或至少一种其可药用酸加成盐,以及使用如上所述的式I的化合物制备具有平滑肌细胞增生抑制作用的药物。
本发明涉及式I的化合物在生产用于抑制平滑肌细胞增生的药物中的应用,其中式I化合物的剂量小于控制肿瘤生长所必须的剂量的70%,
式I
式中,R1是氢,1-6碳原子的烷基,芳基-C1-C4-烷基,由卤素、羟基或羧基取代的C1-C6-烷基,C3-C6-环烷基,吡啶基,噻吩基,C3-C6-环烷基-C1-C4-烷基,C2-C6-链烯基,C2-C6-链炔基,苯基,由卤素、C1-C4-烷基、C1-C4-烷氧基、羟基、羧基、COO-C1-C4-烷基、CONH2、CONH-C1-C4-烷基、CON(C1-C4-烷基)2、硝基、三氟甲基、氨基、C1-C4-烷氨基、二-C1-C4-烷氨基、或苯基一或多取代的苯基,萘基,羧基,-CHO,COO-C1-C4-烷基,伯氨基,C1-C4-烷氨基,芳基C1-C4-烷基氨基,二C1-C4-烷基氨基,C1-C6-酰氨基,芳氨基,二芳氨基或-CH2O-C1-C4-烷基;R2是氢、1-6碳原子的烷基、芳基、硝基、氨基、二-C1-C4-烷氨基、卤素、羟基、C1-C4-烷氧基、-COOH、-COO-C1-C4-烷基、-CHO、-CH2OH或-CH2O-C1-C4-烷基;R3是氢,C1-C4-烷基,由卤素、羟基或羧基取代的C1-C4-烷基,羟基,羧基,硝基,氨基,C1-C4-烷氨基,二-C1-C4-烷氨基,卤素,-O-C1-C4-烷基-C(O)-C1-C4-烷基,-CHO,-CH2OH,-CH2O-C1-C4烷基或(R)2N-C(O)-O-,其中R是H、C1-C6-烷基、C3-C6-环烷基,或芳基;R4是氢,羟基,C1-C4-烷氧基,C1-C4-烷酰氧基,C1-C4-烷氧基羰基,芳氧基,氨基,C1-C4-烷氨基,二-C1-C4-烷氨基,或(R’)2-N-C(O)-O-,其中R’是H、C1-C6-烷基、C3-C6-环烷基或芳基;R5是氢,C1-C6-烷基,芳基-C1-C4-烷基,C3-C6-环烷基,C3-C6-环烷基-C1-C4-烷基,C1-C4-烷基氨基,C1-C4-烷酰基、-C(O)-O-C1-C4-烷基或芳酰基,其中在R1、R2、R3、R4和R5中的芳基是不取代的苯基或由卤素、C1-C4-烷基、C1-C4-烷氧基、羟基、羧基、COO-C1-C4-烷基、CONH2、CONH-C1-C4-烷基、CON(C1-C4-烷基)2、硝基、三氟甲基、氨基、C1-C4-烷氨基、二-C1-C4-烷氨基或苯基一取代或多取代的苯基;和m是0和3之间的整数而n是1。
在上述应用中,优选的式I的化合物为式Ia的化合物:
其中,R1是氢,C1-C3-烷基,萘基,苯基,由卤素、C1-C4-烷基、C1-C4-烷氧基、羟基、羧基、COO-C1-C4-烷基、CONH2-、CONH-C1-C4-烷基、CON(C1-C4-烷基)2、硝基、三氟甲基、氨基、C1-C4-烷氨基、二-C1-C4-烷氨基或苯基一取代或多取代的苯基,吡啶基或噻吩基;R2是氢或C1-C3-烷基;R5是C1-C3烷基,C3-C5-环烷基,或C3-C5-环烷基-C1-C4-烷基。
在上述应用中,优选的式Ia的取代基具有以下的意义:R1是苯基、噻吩基、吡啶基、氯苯基、二氯苯基、甲苯基、氨基苯基、溴苯基、羟苯基或萘基;R2是氢而R5是甲基。
本发明的化合物典型的应用是伴随富含平滑肌细胞的血管损害的疾病/紊乱/损伤。一个非常重要的例子是气囊血管术损伤后的损害。另一个重要的应用是防止斯腾特氏植入术后心瓣再狭窄。本发明的化合物可以用于气囊型伤害后损伤的治疗,也可以用于斯滕特氏固定模植入术后的病人的治疗。本发明的化合物还可以用于富含平滑肌细胞的血管损伤的治疗。
本发明的4-H-1-苯并吡喃-4-酮衍生物以技术人员共知的方式使用。对于药品,使用有效剂量的上述活性物质,既可以使用其本身,也可以优选与合适的药物辅剂联合使呈片剂、包衣片剂、胶囊、栓剂、乳剂、悬浮剂或溶液的形式,活性化合物的含量最高达约95%,优选10-75%。
由于其专业知识,技术人员会知道哪一种助剂适合所要求的药物配方。除了片剂的辅剂、或溶剂、凝胶形成剂、栓剂基材和活性物质的其它赋形剂,可以使用例如抗氧化剂、分散剂、乳化剂、消泡剂、矫味剂、防腐剂、助溶剂或着色剂。
活性物质可以口服、肠胃外、静脉注射或直肠给药,优选口服。为了口服,活性物质可以和其它成分一起与适合这一目的的添加剂混合,例如赋形剂、稳定剂、惰性稀释剂,并且可以用传统的方法将它制成合适的服用形式,如片剂、包衣片剂、硬-明胶胶囊,和含水的醇或油的混悬剂或溶液。可以使用的惰性赋形剂的例子是阿拉伯树胶、氧化镁、乳糖、葡萄糖或淀粉,尤其是玉米淀粉。在这一方面,制剂可以制做成干颗粒或湿颗粒。适用的油赋形剂或溶剂的例子是植物油或动物油,例如葵花子油或鱼肝油。
为了皮下或静脉给药,制成活性物质的溶液、悬浮剂或乳剂,如果恰当地使用适合此目的的传统的物质,如助溶剂、乳化剂或其它助剂的话。适用的溶剂有水、生理氯化钠溶液或醇,如乙醇、丙醇或甘油,和其它糖溶液,如葡萄糖溶液或甘露糖醇溶液,或上述各种溶剂的混合物。
4-H-1-苯并吡喃-4-酮衍生物的每日给药的剂量必须适合预期的效力进行选择。4-H-1-苯并吡喃-4-酮衍生物可以按照以下的剂量给药:小于70%,优选小于60%,尤其是小于5%的总药量,这个剂量用于控制各个哺乳动物的肿瘤生长。一个例子是——在裸小鼠异种移植模型中(nude mouse xenograft model)——约5mg/kg体重每天一次的口服剂量。这个剂量是在同一动物模型中抑制肿瘤生长剂量的一半(Drees et al.Clin.Cancer Res.1997;3:273-279)。
4-H-1-苯并吡喃-4-酮衍生物的药动学性质能够使一天几次服用上述化合物或选择缓释的制剂成为必要。
实施例
1.血管损伤的体内大鼠颈动脉损害模型中黄酮吡啶酚抑制平滑肌细胞增生和新内膜的形成。
建立良好的大鼠颈动脉损害模型,其中导管插入损伤后新内膜损害的形成关键取决于SMC增生(Clowes et al.Lab.Ivest.1983;49:327-333,Cloweset.al.Circ.Res.1985;56:139-145)以检测是否黄酮吡啶酚体内诱导SMC的生长抑制,如其体外所为的那样。以每天一次5mg/kg的剂量口服黄酮吡啶酚,从损伤的那天开始并此后服用4天,因为这个时间段覆盖了Cdk2的起始的引发作用和在这个模型中SMC增生的第一波(Circ res.1995;77:445-465,Circ Res.1997;80:418-426)。平均内膜的和血管中层的面积在损伤后7和14天进行量化,而新内膜损害的大小表示为新内膜与血管中层面积之比。治疗组和不治疗组每组12个动物。在7天时比例是,用载体处理的大鼠动脉为1.00±0.05,用黄酮吡啶酚处理的大鼠动脉为0.65±0.04,减少35%。在14天时,用载体处理的大鼠的新内膜/血管中层之比为1.08±0.04,用黄酮吡啶酚处理的大鼠为0.66±0.03,减少38.9%。从统计学上看,在两个时段这些疗效都是显著的(P<0.05)。
方法
材料——黄酮吡啶酚(L86-8275,(-)-顺式,-5,7-二羟基-2-(2-氯苯基)-8-[4-
(3-羟基-1-甲基)哌啶基]-4H-苯并吡喃-4-酮),由Hoechst MarionRoussel,Inc.公司提供,溶于二甲基亚砜作为50mmol/L储备溶液用于细胞培养试验,或溶于水中用于体内试验。碱性成纤维细胞生长因子(bFGF)从Collaborative Biochemical购买,凝血酶从Sigma购买。MEK1抑制剂PD98059从New England Biolabs获得。
细胞培养——人体主动脉的平滑肌细胞(HASMC)从Clonetics获得并如早先所述那样培养18。细胞在5-9传代时使用。在进行试验之前,用含有0.2%胎牛血清的介质于80%融合阻止生长48小时。
细胞增生ELISA——细胞增生用ELISA(Amersham Life Science)测定。HASMC在明胶涂覆的96孔板上生长并使之静息(quiescent)。细胞用10ng/ml的bFGF、2U/ml的凝血酶或载体处理24小时。在生长因子处理之前1小时施用黄酮吡啶酚(75nmol/L)。在最后2小时的处理期间加入5-溴代2’-去氧尿苷(BrdU)最终浓度达到10μmol/L。BrdU的加入量按所说明的测定19。结果表示为从两个独立的实验中每种情形的12个试样的平均±SEM。
细胞计数——生长受抑制的HASMC在6孔板上生长到50%融合,用或不用黄酮吡啶酚(75nmol/L)或bFGF(10ng/ml)处理。处理后相隔一定时间使细胞受胰蛋白酶作用并使用血细胞计数器测定细胞数。
蛋白质印迹分析——静息的HASMC在存在或不存在生长因子和/或黄酮吡啶酚下按所指示的进行处理。蛋白质印迹分析按先前所述实施18。初始的抗体是:多克隆抗人体细胞周期蛋白D1抗体(M-20,Santa Cruz)、单克隆抗人体增生细胞核抗原(PCNA)抗体(PC10,Sigma)、磷酰化-特异p44/42(Erk1/Erk2)MAP激酶单克隆抗体(New England Biolabs),和单克隆抗-Rb抗体(G3-245,Pharmigen),后者识别磷酰化的(pRb)和高磷酰化(ppRb)Rb种。为了免疫印迹研究,试验至少重复三次。
Cdk活性——静息HASMC用兴奋剂和抑制剂处理24小时并按照蛋白质印迹分析法所述制备全部的细胞溶胞产物。激酶测定用组蛋白H1激酶测定试剂盒(Upstate Biotechnology)按照制造商的说明进行。简言之,将用于蛋白激酶C(2μmol/L)和蛋白激酶A(2μmol/L)的10μl肽抑制剂、100μG细胞溶胞产物、10μl测定缓冲液和10μl含有75μmol/L氯化镁、500μmol/L ATP和1μCi/ml [γ-32P]ATP的混合物在微型离心管中混合。在30℃温育10分钟后,用吸移管将等分试样移送到磷酸纤维素纸上。在0.75%的磷酸中洗涤纤维素纸,然后用闪烁计数器(Beckman)测定cpm。结果表示为三个试样的平均±SEM并代表这三个独立的实验。
在凝胶中的激酶测定——静息HASMC用生长因子处理30分钟并按照蛋白质印迹分析法所述制备全部的细胞溶胞产物。在一些实验中,HASMC用30μmol/LPD98059、黄酮吡啶酚或载体预处理60分钟。将等量的蛋白(50μg/管[μg/行])溶于用350μg/ml髓鞘碱性蛋白共聚的聚丙烯酰胺凝胶中。凝胶用[γ-32P]ATP处理并按所描述的进行放射自显影法测定19。
台盼-蓝排除法——在低融合和按照所描述的抑制生长条件下在5-cm皿中生长HASMC。细胞用黄酮吡啶酚(75mmol/L)或肿瘤坏死因子-□(TNF-□;50ng/ml)处理规定的时间。介质去除后,加入0.4%在磷酸盐缓冲的生理盐水中的台盼蓝。5分钟后,在显微镜下计数在皿中的细胞。数出不能存活的蓝色细胞数。
大鼠颈动脉损伤模型——对大鼠颈动脉的损伤基本上按照所描述的要求进行2。成年雄性Sprague-Dawley鼠(400-500g,Zivic-Miller)用腹膜内注射氯胺酮(2mg/kg)和赛拉嗪(4mg/kg)麻醉。然后将左内颈动脉用2F栓子切除术导管插套管。用生理盐水充胀气囊并横向三次抽出气囊以造成扩张和剥脱损伤。右颈动脉不损伤并用作为每只动物的损伤对比。马上进行麻醉苏醒并且此后的4天中给大鼠以盲知的方式通过管饲法服用黄酮吡啶酚(5mg/kg,在水中)或水。所有的大鼠都从手术中生还并且没有涉及按使用的剂量服用药物的毒性的明显迹象。在颈动脉伤害后规定的时间,将大鼠如上述那样麻醉并用4%的在磷酸盐缓冲生理盐水中的低聚甲醛全身灌注-固定(perfusion-fixed)。将右和左颈动脉取下来并通过腔注射4%的低聚甲醛使之膨胀,然后将它们脱水并在4℃于70%的乙醇中保藏。免疫组织化学按照先前所述的进行18,使用单克隆PCNA抗体和多克隆元人体Cdk2抗体(M2-G,Santa Cruz)。
图像分析——除去每一动脉最远侧的和邻近的区域(约500μm)。从每一动脉取间隔500μm的10个中间的横切片(每个8μm)进行分析。按照先前所述固定载物片并用苏木精和曙红着色18。使用Nikon Diaphot 300显微镜和×4倍物镜,使用Hamamatsu C5985摄像机和TCPro 2.14(Coreco.Inc.)将每一横切片拍摄成数字图像。中层的和新内膜区域用NIH图形软件确定。中层的和新内膜的边界由一个载物片观测员(slide reviewer)(A.M.)确定并由第二个观测员以盲知的方式核对。损害大小以新内膜/血管中层之比表示。每一组的结果表达为平均±SEM。在每一组中92%或更多的图像是可解释的(interpretable);其余的受定影的人为作用而不能分析。
统计学分析——当合适的时候,定量的研究数据表达为平均±SEM。对于多个处理数据组,实施由Fisher最低显著差异试验(Fisher’s least significantdifference test)的解析ANOVA。统计学意义在p<0.05时被接受。
结果
黄酮吡啶酚抑制HASMC增生——基于黄酮吡啶酚抑制各种肿瘤细胞增生的能力的曲线,我们检测使用它会阻止原发培养人体SMC的生长的假说。在高浓度黄酮吡啶酚存在下,生长抑制的HAMC用SMC促细胞分裂剂bFGF(10ng/ml)处理24小时,并用基于ELISA的测定来度量增生作用。与未处理的细胞比较,bFGF-处理的细胞的增生增大到5.4-倍(图1A)。用少到50nmol/L黄酮吡啶酚预处理显著地降低了HASMC增生作用(达到3.9倍,p<0.05),在浓度75nmol/L时作用几乎最高。使用胸腺嘧啶核甙摄入作为DNA合成的独立的度量获得相类似的结果(未示出)。
为试验黄酮吡啶酚对SMC增生的通用性,我们检验其对由凝血酶(2U/ml)引起的有丝分裂的作用,凝血酶通过G蛋白联结受体作用,与bFGF比较,它刺激若干受体酪氨酸激酶族。黄酮吡啶酚(75nmol/L)显著的和有力的抑制bFGF-和凝血酶-二者诱导的HASMC增生(分别为5.4-倍对1.8-倍和2.4-倍对0.7-倍,p<0.05,图1B)。我们做了细胞计数以证实,黄酮吡啶酚对在HASMC中细胞周期进程的作用真实反映了在增生作用中的变化。处理后3天bFGF(10ng/ml)诱发3-倍的细胞数增加(图2)。类似的结果在基于ELISA的测定中看到,黄酮吡啶酚(75nmol/L)有效的抑制了bFGF-诱发的增生。
在HASMC中黄酮吡啶酚抑制Cdk活性和细胞周期-相关基因表达——要评价黄酮吡啶酚对细胞周期机制的特定作用,从生长因子-刺激的HASMC中我们测定在细胞溶胞产物中组蛋白H1激酶的活性。组蛋白H1的磷酰化反映Cdc2和Cdk2的活性20。用bFGF和凝血酶处理HASMC,分别导致组蛋白H1激酶活性增加到4.4-倍和3.6倍(图3)。这些细胞周期蛋白-依赖的激酶活性的增加完全被黄酮吡啶酚(75nmol/L)的预处理所阻止。
通过蛋白质印迹分析,我们解决了黄酮吡啶酚是否影响在HASMC中细胞周期-相关的蛋白的生长因子诱发的调节作用。细胞周期蛋白D1是一种G1相细胞周期蛋白,由生长因子的刺激作用上调并且在细胞周期结束后迅速降解21。作为bFGF和凝血酶24小时处理的反应,细胞周期蛋白D1蛋白水平分别被上调到6.3-倍和3.2-倍(图4),一种通过黄酮吡啶酚预处理可以完全阻止的效应。同样,增加的PCNA的表达(主要在S相与DNA复制结合期间合成22),也被黄酮吡啶酚预处理阻止。作为细胞周期-相关蛋白的最终度量,我们使用一种识别磷酰化Rb的抗体检测响应生长因子表达的Rb磷酰化作用。Rb是一种细胞周期调节剂,当Rb处于未磷酰化状态时,它结合或灭活转录因子E2F23,并在体内诱导SMC生长抑制11。磷酰化作用灭活Rb并且可以通过S相发展。Rb磷酰化的分析特别相关是因为Rb是Cdk2和Cdk4体内的目标。凝血酶和bFGF二者都诱发Rb的过磷酰化,一种被黄酮吡啶酚抑制的作用。总括起来,这些结果表明,在与HASMC相关的生长-抑制作用中,黄酮吡啶酚影响在HASMC中G1和S相-相关的细胞周期调节要素的表达和活性。
黄酮吡啶酚对MAP激酶磷酰化作用或活性没有作用——为证实黄酮吡啶酚特异地在细胞周期的水平上起作用,而不是非特异的作用于上游激酶通道上(upstream kinase pathways),我们测量Erk1(p44 MAP激酶)和Erk2(p42 MAP激酶)的磷酰化作用和活性,MAP激酶族的两个成员。我们选择这些激酶是因为它们是直接响应生长刺激发生的转录活动的上游和一些临界促有丝分裂(mitogenic)信号通道的下游24。对MAP激酶的完整响应表明上游致分裂通道也是完整的。我们用单科隆抗体(特定识别磷酰化形态,并进而识别活化形态)测定ERk1和ERk2的磷酰化状态。作为这些试验的对照,我们使用PD98059,一种有力的和有选择性的MAP激酶活性的抑制剂25。用凝血酶和bFGF处HASMC30分钟后检测磷酰化了的Erk1和Erk2的增加量,与未处理的细胞作比较(图5,上组)。用凝血酶和bFGF磷酰化的Erk1和Erk2都被PD98059预处理所阻止,但用黄酮吡啶酚处理的则不同。为了证实这些发现,我们通过在凝胶中的激酶测定法测量Erk1和Erk2活性(图5,下组)。又一次我们发现响应凝血酶和bFGF,Erk1和Erk2活性增加,一种被PD 98059抑制但通过黄酮吡啶酚却不抑制的作用。这些试验,加上图3和4所示的那些一起,证明黄酮吡啶酚对HASMC增生的作用是由于通过不影响上游信号活动来阻止Cdk活性的特定的抑制细胞周期运作(cell cyclemachinery)。
黄酮吡啶酚不降低HASMC的存活力——在其它细胞类型中黄酮吡啶酚活力的早先的报告表明,根据细胞系,黄酮吡啶酚既可以不影响存活力地导致生长的停止,或可以引起编程性细胞死亡16,26-29。因此我们通过在处理后各个时间段测量台盼蓝排除量来评估是否黄酮吡啶酚降低HASMC的存活力。静息的HASMC用黄酮吡啶酚(75mmol/L)、载体或TNF-□(50ng/ml)处理,后者是一种已知在这一细胞类型中引起便出现细胞死亡的细胞因子30。而TNF-□有力地降低HASMC的存活力,导致全部24小时处理的细胞基本上死亡,黄酮吡啶酚没有这种作用(图6)。我们注意到伴随着更高浓度和更长时间的温育,可以发生在黄酮吡啶酚存在下存活力的某些降低(未示出)。但在实验条件下,黄酮吡啶酚主要的是引起生长停止,不影响SMC的存活力。
在血管损伤的大鼠颈动脉损伤模型中体内黄酮吡啶酚抑制平滑肌细胞增生和新内膜的形成——我们利用良好建立的大鼠颈动脉模型(其中导管插入损伤后新内膜损害的形成关键地取决于SMC增生2,3),以检验黄酮吡啶酚是否体内引起SMC生长停止,如同它的体外所为。我们以每天一次5mg/kg的剂量口服给药,从损伤的当天开始并持续4天,因为这个时间段覆盖了在这个模型中Cdk2的起始引发作用和SMC增生的第一波31,32。平均内膜的和血管中层的面积在损伤后7和14天进行量化,而新内膜损害的大小表示为新内膜与血管中层面积之比。治疗组和不治疗组每组12个动物。在7天时新内膜/血管中层之比是,用载体处理的大鼠为1.00±0.05,用黄酮吡啶酚处理的大鼠为0.65±0.04,减少35.0%(图7)。在14天时,用载体处理的大鼠的新内膜/血管中层之比为1.08±0.04,用黄酮吡啶酚处理的大鼠为0.66±0.03,减少38.9%。从统计学上看,在两个时段这些疗效都是显著的(P<0.05)。各个动脉切片示于图8中。
为了直接表现黄酮吡啶酚抑制平滑肌细胞增生作用,我们将切片染色以用于每一动脉代表性区域的PCNA表达并确定新内膜中PCNA-阳性的核的百分比。第7天,未处理大鼠的受损动脉31.1±7.2%的核是PCNA-阳性,而黄酮吡啶酚处理的大鼠的受损颈动脉仅11.8±1.5%的核PCNA-阳性(图7;p<0.05)。第14天,受损动脉被处理的10.4±2.0%新内膜细胞出现PCNA-阳性核,而受损动脉未处理的仅4.2±0.5%新内膜细胞出现PCNA-阳性核(p<0.05)。(PCNA-阳性核在未损害动脉中很少见到,不论处理与否。)同样,与未处理的大鼠动脉比较(组B和组D),Cdk2-阳性细胞在黄酮吡啶酚-处理的大鼠的新内膜中很少见(图9,组A和组C),在损害后第7和14天都如此。
讨论
在本研究中,我们检测了新Cdk抑制剂、黄酮吡啶酚——已知的Cdk最有力的和特定的抑制剂,是否为体内抑制SMC增生的合适的候选,尤其是对于所设定的血管损伤而言。先前治疗上尝试针对细胞周期运作来治疗血管疾病治疗,已经为本研究提供了逻辑依据9-12;但为此目的所使用的方法却依赖抑制细胞周期过程的基因转移技术。当前,巨大的障碍阻止这些技术的临床应用33。在我们进行研究期间,有报道CTV-313——一种新近鉴定的也有Cdk抑制性能的化合物(但需在毫摩尔浓度级),也可以抑制新内膜的形成;但要求CVT-313在损伤时滴注到颈动脉中以产生这种作用34。作为比对,我们显示黄酮吡啶酚,当口服时,能够以可与其它临床相关药剂相比较的程度,有力地抑制新内膜增生11,35,,36。在血管损伤的动物模型中显示活性的药剂中黄酮吡啶酚的口服活性使其确实非比寻常。它的选择性、效力和服用的方便性,使得黄酮吡啶酚成为检验人体血管损害中体内细胞周期抑制作用的治疗益处的优秀候选者。
我们选择口服黄酮吡啶酚的浓度(5mg/kg)是裸小鼠异种移植模型中抑制肿瘤生长的浓度的一半27。值得注意的是,75nnmol/L的黄酮吡啶酚浓度在我们的研究中产生SMC增生的几乎完全的抑制作用,而以低于难治疗癌的第一阶段人体研究中的毒性阈值的剂量,中位值血清浓度达到425nmol/L17。我们的研究结果建议,比用于瘤形成的剂量低很多的细胞周期抑制剂剂量,就可以在给定的血管疾病如心瓣再狭窄中有效果,伴随的好处是增加了可忍性。
虽然我们已经指出,黄酮吡啶酚导致培养中的HASMC生长停止而不影响存活,并且显示在黄酮吡啶酚体内处理后新内膜的形成减少,我们不能确定细胞周期的停止是在颈动脉损伤中降低新内膜生成的唯一的因素。黄酮吡啶酚可以在引发或不引发编程性细胞死亡的(取决于被观测的细胞类型)条件下导致生长停止29。有趣的是,黄酮吡啶酚抑制在终末分化的PC12细胞中的编程性细胞死亡,但对于正在增生的未分化的PC12细胞它诱发编程性细胞死亡28。虽然我们的体内试验是摹拟在损伤前SMC的表型的条件下进行的,仍有可能SMC会在损伤后对黄酮吡啶酚作出不同的反应并且甚至会引发编程性细胞死亡。当在血管损害中的编程性细胞死亡的功用不清楚时,在SMC中的Fas配体的表达引发编程性细胞死亡并且在家兔的气囊型损害后阻止新内膜的生成37,我们提议如果黄酮吡啶酚确实体内引发SMC的编程性细胞死亡,像在增生PC12细胞中所做的那样,则在新内膜生成的背景中这可以是一种适于健康的现象。其它机制对黄酮吡啶酚对损害形成的作用也可以起一份作用。例如,反义寡脱氧核甙酸-介导的细胞周期抑制作用改善了家兔静脉移植物的内皮功能38。针对黄酮吡啶酚对SMC体内的作用的机制,除了生长停止的外,需更做进一步研究。
给出了证明大鼠颈动脉损伤后在损害形成中SMC增生的作用2,3,注意到以下所述是有趣的:尽管黄酮吡啶酚的体外作用显著,在我们体内试验的条件下其抑制新内膜生成的能力(虽然量大)是更适度的。对这种观察现象我们考虑了几种解释。停止黄酮吡啶酚后不太可能发生SMC增生加速,因为自从损伤后增生指标的差异持续了14天之久(图7和9)。似乎有道理的是,即使没有大量的SMC增生,损伤形成的其它因素,如SMC的迁移和细胞外基质的产生,仍然对损伤的形成起作用,或者每天一次的黄酮吡啶酚的给药量在这个模型中不足以完全阻止增生。近期的数据表明,假定后面的假说可能是正确的话,黄酮吡啶酚的生物学半衰期短至2.5小时;进一步的研究可能会确定一种更有效的给药方案39。
我们的研究结果显示,在公认的血管疾病的小动物模型中,黄酮吡啶酚能够抑制SMC增生,并且进而抑制新内膜的形成。必须指出的是,在人体血管损伤中SMC增生的抑制的关联性是有争议的,并且会依据损伤的性质和增生观察的时间而不同。在人体动脉粥样化斑切除术标本中SMC的增生指数非常低40,虽然这些标本可能不反映在损伤发展中更早期、更关键阶段的增生变化。此外,动脉的改造不同于新内膜生长可以解释在人体血管成形术后高比率的腔阻塞(luminalobstruction)41。比较起来,在伴有斯滕特氏固定模术后心瓣再狭窄的人体损伤的动脉粥样化斑切除术标本中,SMC中有丝分裂活性的指数高的多(25%PCNA-阳性),与SMC增生的已承认的作用一致,但在这一过程中,与改造术不同4。因为斯滕特氏固定模置换术和斯滕特氏固定模术心瓣再狭窄的临床问题增多,所以需要阻止SMC增生和新内膜的形成的有效的方法。由于黄酮吡啶酚是一种具有特定Cdk-抑制活性的有力的、可以口服的药剂,而且由于黄酮吡啶酚对人体的安全剂量是已知的,因此可以考虑将黄酮吡啶酚作为预防人体斯滕特氏固定模术心瓣再狭窄的候选药物。
方法和结果:使用培养的人体主动脉平滑肌细胞,我们发现浓度低达75nmol/L的黄酮吡啶酚,对碱性成纤维细胞生长因子-引起的和凝血酶-引起的增生,产生近乎完全的抑制作用。在这一剂量上,黄酮吡啶酚抑制细胞周期蛋白-依赖的激酶活性,如由组蛋白H1磷酰化作用所检测的那样,但对MAP激酶的活性没有作用。细胞周期-相关蛋白细胞周期蛋白D1、增生细胞核抗原,以及磷酰化的视网膜胚细胞瘤蛋白的诱导作用也被黄酮吡啶酚阻止。黄酮吡啶酚对细胞的活力没有影响。为试验口服时黄酮吡啶酚在体内是否有相似的活性,我们检查气囊损害后大鼠颈动脉的新内膜的形成。在损伤后,通过管饲法服用黄酮吡啶酚(5mg/kg),新内膜面积在7天和14天,分别减少35%和39%。
结论:黄酮吡啶酚在体外和体内抑制SMC生长。它可以口服并且对细胞周期蛋白-依赖的激酶的选择性使它在SMC-富含的血管损害的治疗中成为一种有潜力的药物。
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Claims (9)
1.式I的化合物在生产用于抑制平滑肌细胞增生的药物中的应用,其中式I化合物的剂量小于控制肿瘤生长所必须的剂量的70%,
式中
R1是氢,1-6碳原子的烷基,芳基-C1-C4-烷基,由卤素、羟基或羧基取代的C1-C6-烷基,C3-C6-环烷基,吡啶基,噻吩基,C3-C6-环烷基-C1-C4-烷基,C2-C6-链烯基,C2-C6-链炔基,苯基,由卤素、C1-C4-烷基、C1-C4-烷氧基、羟基、羧基、COO-C1-C4-烷基、CONH2、CONH-C1-C4-烷基、CON(C1-C4-烷基)2、硝基、三氟甲基、氨基、C1-C4-烷氨基、二-C1-C4-烷氨基、或苯基一或多取代的苯基,萘基,羧基,-CHO,COO-C1-C4-烷基,伯氨基,C1-C4-烷氨基,芳基C1-C4-烷基氨基,二C1-C4-烷基氨基,C1-C6-酰氨基,芳氨基,二芳氨基或-CH2O-C1-C4-烷基;
R2是氢、1-6碳原子的烷基、芳基、硝基、氨基、二-C1-C4-烷氨基、卤素、羟基、C1-C4-烷氧基、-COOH、-COO-C1-C4-烷基、-CHO、-CH2OH或-CH2O-C1-C4-烷基;
R3是氢,C1-C4-烷基,由卤素、羟基或羧基取代的C1-C4-烷基,羟基,羧基,硝基,氨基,C1-C4-烷氨基,二-C1-C4-烷氨基,卤素,-O-C1-C4-烷基-C(O)-C1-C4-烷基,-CHO,-CH2OH,-CH2O-C1-C4-烷基或(R)2N-C(O)-O-,其中R是H、C1-C6-烷基、C3-C6-环烷基,或芳基;
R4是氢,羟基,C1-C4-烷氧基,C1-C4-烷酰氧基,C1-C4-烷氧基羰基,芳氧基,氨基,C1-C4-烷氨基,二-C1-C4-烷氨基,或(R’)2-N-C(O)-O-,其中R’是H、C1-C6-烷基、C3-C6-环烷基或芳基;
R5是氢,C1-C6-烷基,芳基-C1-C4-烷基,C3-C6-环烷基,C3-C6-环烷基-C1-C4-烷基,C1-C4-烷基氨基,C1-C4-烷酰基、-C(O)-O-C1-C4-烷基或芳酰基,
其中在R1、R2、R3、R4和R5中的芳基是不取代的苯基或由卤素、C1-C4-烷基、C1-C4-烷氧基、羟基、羧基、COO-C1-C4-烷基、CONH2、CONH-C1-C4-烷基、CON(C1-C4-烷基)2、硝基、三氟甲基、氨基、C1-C4-烷氨基、二-C1-C4-烷氨基或苯基一取代或多取代的苯基;
m是0和3之间的整数而n是1。
3.权利要求2的应用,其中式Ia的取代基具有以下的意义
R1是苯基、噻吩基、吡啶基、氯苯基、二氯苯基、甲苯基、氨基苯基、溴苯基、羟苯基或萘基;
R2是氢而
R5是甲基。
4.权利要求1的应用,其中的化合物是(-)-顺式,-5,7-二羟基-2-(2-氯苯基)-8-[4-(3-羟基-1-甲基)-哌啶基]-4H-苯并吡喃-4-酮。
5.权利要求1-4中任一项所说的应用,其中的药物用于富含平滑肌细胞的血管损伤治疗。
6.权利要求1-4中任一项所说的应用,其中的药物用于治疗气囊型伤害后损伤的治疗。
7.权利要求1-4中任一项所说的应用,其中的药物用于斯滕特氏固定模植入术后的病人的治疗。
8.根据权利要求1所说的应用,其中式I化合物的剂量小于控制肿瘤生长所必须的剂量的60%。
9.根据权利要求8所说的应用,其中式I化合物的剂量小于控制肿瘤生长所必须的剂量的50%。
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