CN1125079C - 环缩肽 - Google Patents
环缩肽 Download PDFInfo
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- CN1125079C CN1125079C CN96198477A CN96198477A CN1125079C CN 1125079 C CN1125079 C CN 1125079C CN 96198477 A CN96198477 A CN 96198477A CN 96198477 A CN96198477 A CN 96198477A CN 1125079 C CN1125079 C CN 1125079C
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Abstract
式(I)的环缩肽,其中A、B、R1Leu、Leu、C、X和Y如规定的定义,这类环缩肽是粘着分子表达的抑制剂和TNF释放的抑制剂,所以它们适于治疗涉及粘着分子表达水平的升高和/或由TNF介导的炎症和其它疾病。
Description
本发明涉及环缩肽及其作为粘着分子表达的抑制剂的治疗应用。
例如ICAM-1、VCAM-1和E-选择蛋白的细胞粘着分子,是响应包括TNFα、IFNγ、IL1和LPS的促炎介体而在内皮细胞表面以及关于ICAM-1的角质形成细胞表面表达的。相应的反配体,例如LFA-1、VLA-4和SLEX,是在循环血细胞表面表达的。在炎性过程期间白细胞经内皮的迁移,以及血管外的细胞-细胞相互作用,是由于这些粘着分子与其反配体之间的相互作用而被调节的。因此,粘着分子表达的抑制剂有可能治疗许多病态。
环缩肽是环状分子,分子中包括通过肽键连接起来的氨基酸残基和至少一个羟基取代的羧酸残基,该羧酸残基通过它的羟基取代基利用酯键与邻近的酸残基连接。
我们的未决专利申请即公开的国际专利申请WO96/03430中公开了新型的环七缩肽,它们是ICAM-1、VCAM-1和E-选择蛋白表达的抑制剂。我们现在进一步发现了该同一大类化合物的新环七缩肽,包括具有特别理想性能的化合物。
A是乙醇酸(glycolic acid)残基,它任选被甲基、乙基或乙烯基α取代,所述甲基、乙基或乙烯基任选被卤素、烷氧基、任选保护的羟基或氨基、CSNH2、COOR2、乙烯基、-C≡CH或噻唑取代,其中R2是H或低级烷基,任选被烷基、卤素、环烷基、任选取代的噻唑、COOR2或-C≡CH取代,其中R2同上述定义;
B是α-氨基-γ-甲基取代的辛酸残基;
R1是氢或甲基;
X是α-氨基取代的(C2~C14)羧酸残基,以及
Y是α-氨基-或N-甲基-α-氨基取代的(C2~C10)羧酸残基,
条件是:A不是未取代的α-羟基-取代的丁酸残基,条件还有:当A是被乙基α取代的乙醇酸残基时,则该乙基只任选被下列基团取代:氨基、羟基、氯、烷氧基、任选取代的噻唑、任选取代的乙烯基、环丙基、CSNH2或-C≡CH。
在式I中,氨基酸残基的N-端至C-端取向呈顺时针方向,且该缩肽酯键位于残基A和Y之间。当R1是甲基时,则残基R1-Leu和Leu分别是N-甲基亮氨酸残基和亮氨酸残基。
优选的A是乙醇酸残基,它被甲基或乙基α取代,其中的甲基或乙基任选被氨基、羟基、氯、烷氧基、任选取代的噻唑、任选取代的乙烯基、环丙基、CSNH2或-C≡CH取代。
优选的C是式II的N-甲基色氨酸残基,其中R3表示氢、(C1~C4)烷氧基(特别是甲氧基)或烷基,且R4表示氢或卤素。
优选的X是α-氨基取代的(C4~C8)羧酸残基,它任选被β-或γ-(C1~C4)烷基取代。最优选的X是α-氨基-β-或γ-(C1~C4)烷基-(特别是甲基-)取代的辛酸残基或丁酸残基。
优选的Y是N-甲基-α-氨基取代的(C2~C4)羧酸残基,它任选被β-或γ-(C1~C4)烷基取代。最优选的Y是N-甲基丙氨酸残基或N-甲基缬氨酸残基。
本发明包括对应于式I化合物的开链肽或缩肽;例如,通过解离残基Y和A之间的酯键或者解离任何其它邻接的酸残基对之间的酰胺键而得到的开链分子。优选的开链衍生物有式IV和V的化合物,
H-C-X-Y-A-B-R1Leu-Leu.OR7 IV
和
HA-B-R1Leu-Leu-C-X-Y.OR7 V
其中R7表示氢或烷基,如C1-4低级烷基。
其中:
Ap是任选被甲基α取代的乙醇酸残基;
Bp是α-氨基-γ-甲基取代的辛酸残基;
R1p是氢或甲基;
Cp是色氨酸残基或N-甲基色氨酸残基,它任选被N′-(C1~C4)烷氧基取代;
Xp是α-氨基取代的(C2~C14)羧酸残基,以及
Yp是α-氨基-或N-甲基-α-氨基取代的(C2~C10)羧酸残基。
按本发明的另一个实施方案,优选的化合物是式I′p的化合物:
其中Bp、R1p、Cp、Xp和Yp同上述定义,而A′p是α羟基取代的丁酸残基,该残基被式VI的基团γ取代,其中R2表示低级烷基,例如C1-4低级烷基。最优选的R2是甲基或乙基。
式I、IV、V、Ip、I′p和I″p的化合物在后文被称为“本发明的化合物”,该术语还包括呈盐或酯形式以及呈游离态的本发明的所有化合物。
本发明的化合物含不对称原子,因而可呈多种差向异构形式。所有可能的差向异构体及其非对映异构混合物都包括于本发明中。对粘着分子表达活性有抑制作用的差向异构体是优选的。一般地,例如用于本发明的药物应用时,对粘着分子表达活性有抑制作用的差向异构体呈纯形式或基本上纯形式(即没有或基本上没有对粘着分子表达活性缺乏抑制作用的差向异构体),例如包括至少90%的、如至少95%的活性差向异构体(即包括少于10%的、如少于5%的非活性差向异构体)将是优选的。最优选的本发明的化合物具有与下式VII的特别优选的化合物相同的环缩肽环立体化学构象。
特别优选的本发明的化合物是式VIII、IX和X的化合物:
式VIII的化合物已从真菌株F/94-499709的培养物分离出,该真菌株样品是按布达佩斯条约的规定于1995.9.18保藏于the DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen,保藏号为DSM10275。真菌株F/94-499709的特征在下文的实施例1中作了描述。式VIII的化合物是本发明特别的化合物。
菌株F/94-499709的样品也可得自Sandoz.Ltd.CH-4002Basel Switzerland.
应注意,按Rule28(4)和(5)EPC的规定,对于DSM10275的样本的获得做了限制。
本发明包括呈分离形式的菌株F/94-499709(DSM10275)及其突变体和衍生物以及由该菌株产生的所有新型环缩肽。
式VIII的化合物及其相关化合物可这样获得,即例如按实施例2中所述那样在营养培养基中培养F/94-499709(DSM10275)或其突变体或衍生物或类似的真菌种,再从培养基中回收这些化合物。
式VIII的化合物的特征给出于实施例3中。
本发明的化合物可通过式XI或XII(下文将描述)或VIII的化合物的衍生作用制备,它包括:
a)-其中A被COOR2取代的式I化合物的制备:将其中A被CN取代的式I的相应化合物与优选是醇的亲核试剂反应,应用合适的碱性或酸性催化剂,优选应用盐酸,反应在优选为醚的有机溶剂中进行,或者
b)-其中A被烷氧基甲基取代的式I化合物的制备:将其中A被CH2-OH取代的式I的相应化合物与例如烷基卤化物或重氮化合物的烷基化化合物反应,可加或不加催化剂,或者
c)-其中A被COOR2取代的式I化合物的制备:应用标准方法酯化其中A被COOH取代的式I的相应化合物,优选通过用例如亚硫酰氯将它转化为酰基氯,再用适当的醇在存在或不存在酸结合剂时进行处理,或者
d)-其中A被CH2OH取代的式I化合物的制备:在有机溶剂中,用金属氢化物或硼氢化物、优选用甲硼烷二甲硫复合物还原其中A被COOR2取代的式I的相应化合物,或者
e)-其中A被任选取代的乙烯基取代的式I化合物的制备:将其中A被CHO取代的式I的相应化合物与维悌希试剂反应,或者
f)-其中A被CH2NH2取代的式I化合物的制备:还原其中A被CH2N3取代的式I的相应化合物,或者
g)-其中A被C≡CH取代的式I化合物的制备:将其中A被CH=CBr2取代的式I的相应化合物进行脱氢反应,或者
h)-其中A被环丙基取代的式I化合物的制备:将其中A被乙烯基取代的式I的相应化合物与重氮甲烷反应,或者
i)-其中A被CSNH2取代的式I化合物的制备:将其中A被CN取代的式I的相应化合物与硫衍生物、优选与二苯基膦连二硫酸反应,例如将该硫化合物的溶液与其中A被CN取代的式I化合物一起回流,或者
Hal-CH2-CO-R6 XIII其中R6同前述定义,Hal表示卤素,或者与式XIII化合物的缩醛反应(该反应可按已知方法进行,例如将式II化合物的溶液在反应条件下为惰性的溶剂中进行反应,例如在二甲基甲酰胺或吡啶中,在优选为60~100℃的较高温度下。终产物可用惯常方法分离和纯化。),或者
k)其中的取代基同前述定义的优选的I′p化合物的制备:将其中A′p表示α-羟基取代的丁酸残基(该丁酸残基被-CHOγ取代)的I′p化合物与烷氧基羰基亚甲基三苯基正膦反应,再分离式I′p的化合物;或者
l)-其中R3表示氢的式I化合物的制备:从其中R3表示OCH3的式I化合物中除去甲氧基,或者
n)-其中R3表示烷基或苄基的式I化合物的制备:将这些基团引入其中R3表示氢的式I化合物,或者
o)-其中R4表示卤素的式I化合物的制备:将其中R4表示氢的式I化合物进行卤化,或者
且需要的话,分离式I的化合物。
在上述a)~i)的优选实施方案中,式I的化合物是其中A是α-羟基丁酸残基的化合物,该丁酸残基适当时被下列基团γ取代:COOR2、CN、烷氧基甲基、CH2OH、COOH、任选取代的乙烯基、CHO、CH2NH2、CH2N3、C≡CH、CH=CBr2、环丙基或乙烯基。
用于制备式I化合物的中间体可这样制备:
(i)制备其中A被-CHO取代的中间体:氧化其中A被-CH2OH取代的式I的相应化合物,以及
(ii)制备其中A被-COOH取代的中间体:在水性醇溶液中,用无机酸(如HCl)或用碱水解其中A被COOAlkyl取代的式I的相应化合物。
用于制备其中A被-CN取代的式I化合物的中间体包括天然化合物。例如式XI和XII的化合物
可从真菌株F92-4471/08的培养物分离而得,该菌株按布达佩斯条约的规定于1993年7月2日在the US Department of Agri-culture,NRRL培养物保藏中心保藏,保藏号为NRRL21123。真菌株F92-4471/08的特性以及化合物XI和XII的分离,在我们的未决专利申请即国际专利申请WO96/03430中作了详细描述。
本发明的化合物也可用化学合成法制备;例如应用惯常的肽合成技术制备。通常在制备这类化合物时最后的步骤是环化反应步骤,其中通过酰胺键或酯键形成反应使线型肽或缩肽进行环化,所述肽或缩肽包括以适当顺序连接在一起的酸残基A、B、R1Leu、Leu、C、X和Y。
因此,本发明包括式I的环缩肽的制备方法,该方法包括将含有以适当顺序连接在一起的酸残基A、B、R1Leu、Leu、C、X和Y的线型肽或缩肽进行环化反应。
本发明的化合物显示药理活性,因而可用作药剂。具体地说,本发明的化合物是细胞粘着分子受激表达的抑制剂,尤其是有关E-选择蛋白的VCAM-1和ICAM-1表达的抑制剂。本发明的化合物也是TNF释放的抑制剂,例如TNFα释放的抑制剂。
在实施例之后描述了可用于检测本发明的化合物对ICAM-1、VCAM-1和E-选择蛋白表达的抑制作用以及对TNFα释放的抑制作用的检验方法。
因此,鉴于它们作为细胞粘着分子表达的抑制剂的活性,这类化合物可用于治疗或预防涉及细胞粘着分子的表达的疾病过程。这些疾病过程包括其中白细胞活化和运输在致病过程中起重要作用的许多后天性和遗传性疾病/失调,最值得注意的是:急性和慢性炎症(例如过敏反应、哮喘、皮炎、牛皮癣、再灌注损伤和败血症性休克),自身免疫病(例如糖尿病、多发性硬化和类风湿性关节炎)以及免疫介导的神经变性(例如获得性免疫缺陷病)。本发明的化合物适用的其它指征包括:转移瘤(例如黑素瘤、骨癌),动脉粥样硬化和同种移植/异种移植排异反应,因为已知对血管粘着分子的抑制可大为改善这些疾病过程的预后。
本发明的化合物对于过度增殖性皮肤病(例如牛皮癣)以及各种恶性肿瘤也有治疗潜力,因为在亚微摩尔浓度下,在基于角质形成细胞以及其它增殖的检查中试验72小时后,可测知它们的抑制活性。
由p24ELISA估测到本发明的化合物在抑制U1单核细胞系中TNFα/IL-6诱导的HIV生产方面有活性,所以这些化合物适用于治疗免疫缺陷病和病毒引起的病,特别是治疗艾滋病。
此外,鉴于它们作为TNF释放的抑制剂的活性,本发明的化合物适用于预防或治疗由TNF、特别是由TNFα介导的疾病或病理状况,例如炎症,自身免疫病,严重感染,以及包括同种移植和异种移植排异反应的器官或组织移植排异反应,例如用于治疗心、肺、心-肺结合、肝、肾、胰腺、皮肤或角膜移植物的受体,以及预防移植物抗宿主疾病,例如在骨髓移植之后。
本发明的化合物特别适用于治疗、预防或改善自身免疫病和炎症,尤其是病因中包括自身免疫组分的炎症,例如关节炎(如类风湿性关节炎、arthritis chronica progrediente和arthritis deformans)和风湿病。可应用本发明的化合物的具体自身免疫病包括:自身免疫血液病(例如包括溶血性贫血、再生障碍性贫血、纯红细胞性贫血和特发性血小板减少症),全身性红斑狼疮,多软骨炎,硬皮病,韦格纳肉芽肿病,皮肌炎,慢性活性肝炎,重症肌无力,牛皮癣,史蒂文斯-约翰逊综合征,特发性口炎性腹泻,自身免疫炎性肠病(例如包括溃疡性结肠炎和局限性回肠炎),内分泌性眼病,格雷夫斯病,肉样瘤病,多发性硬化,肥大性肝硬变,青少年糖尿病(I型糖尿病),葡萄膜炎(前葡萄膜炎和后葡萄膜炎),干性角膜结膜炎和vernal keratocon-junctivitis,间质性肺纤维变性(interstitiallung fibrosis),牛皮癣性关节炎,以及肾小球性肾炎(伴有或没有肾变病综合征,例如包括特发性肾变病综合征或最小变化的肾病)。
本发明的化合物还适用于治疗、预防或改善哮喘、支气管炎、尘肺、肺气肿,以及其它气道阻塞病或气道炎症。
本发明的化合物适用于治疗由TNF、特别是由TNFα介导的不希望的急性和超急性炎性反应,例如急性感染,如败血症性休克(例如内毒素性休克和成人呼吸窘迫综合征),脑膜炎,肺炎;以及严重烧伤;还适于治疗恶病质或与病态TNF释放相关的、感染、癌或器官功能异常后发作的消耗性综合征,尤其是与艾滋病相关的恶病质,例如与HIV感染相关的或在HIV感染后发作的恶病质。
因此,本发明还包括本发明的化合物的治疗应用,以及含本发明的化合物的治疗组合物。
具体地说,本发明包括治疗或预防涉及粘着分子表达的疾病的方法,这些方法包括给受治疗者施用治疗上或预防上有效量的本发明的化合物。
本发明还包括治疗组合物,它含治疗上有效量的、本发明的化合物。
此外,本发明包括本发明的化合物在制备用于治疗或预防涉及粘着分子表达的疾病的药剂中的应用。
特别地,本发明还提供了系列实施方案:
A.一种方法,它可抑制可溶性TNF、尤其是TNFα的生产,或减轻需要该治疗的受治疗者(例如哺乳动物,尤其是人)的炎症,该方法包括给所述受治疗者施用有效量的、本发明的化合物;或者治疗任何上述疾病的方法,特别是治疗炎症或自身免疫病(例如多发性硬化或类风湿性关节炎)或者减轻一种或多种任何上述病的症状的方法。
B.用作药剂的本发明的化合物,例如作为免疫抑制剂或抗炎剂用于预防或治疗由TNF介导的疾病或病理状况,或者用于预防、改善或治疗上述任何疾病或病况,如自身免疫病或炎症。
C.含有与药物上可接受的稀释剂或载体结合的本发明的化合物的药物组合物,例如作为免疫抑制剂或抗炎剂用于预防或治疗由TNF介导的疾病或病理状况,或者用于预防、改善或治疗上述任何疾病或病况,如自身免疫病或炎症。
D.本发明的化合物在制备用于预防或治疗由TNF介导的疾病或病理状况的药剂(例如作为免疫抑制剂或抗炎剂)方面的应用,或者在制备用于预防、改善或治疗上述任何疾病或病况,如自身免疫病或炎症的药剂方面的应用。
该组合物可作肠胃外的、口服的、气雾剂、吸入剂或局部用药,其中通常包括一种或多种药物上可接受的载体、稀释剂或赋形剂,还可包括例如稳定剂等的添加剂。
应用的这类化合物剂量可根据下列因素变化,即涉及的病况或疾病,是作治疗用还是预防用,尤其是施药方式和途径。不过,要达到满意效果的一般口服剂量是约0.05~约10mg/kg/天,优选是约0.1~约7.5mg/kg/天,更优选是约0.1~约2mg/kg/天,日服一次或分成2~4次的均分剂量。也可肠胃外施药,例如通过静脉滴注或输注,可应用的剂量是约0.01~约5mg/kg/天,优选是约0.05~约1mg/kg/天,更优选是约0.1~约1.0mg/kg/天。
这样,病人口服(p.o.)合适的日剂量是约2.5~约500mg,优选是约5~约250mg,更优选是约5~约100mg;或者静脉注射的日剂量是约0.5~约250mg,优选是约2.5~约125mg,更优选是约2.5~约50mg。
这类化合物可通过任何合适的途径施药,包括经肠地、肠胃外地和局部地或通过吸入器。合适的经肠施药形式有:供饮用的溶液、药片或胶囊。合适的肠胃外形式有注射液或悬浮液。合适的局部施药形式包括乳膏、洗剂等等,这类制剂的浓度范围是0.01-10wt%,优选是0.1~1wt%。口服的适当单位剂量形式可包括1~50mg该化合物,通常是1~10mg。
本发明通过只是阐释性的下述实施例而得以进一步描述,这些实施例参照附图,其中:
图1示出式VIII的化合物的UV光谱;
图2示出式VIII的化合物的IR谱;
图3示出式VIII的化合物的FD-质谱;
图4示出式VIII的化合物的FD-质谱(加有LiI),以及
图5示出式VIII的化合物在CDCl3中的质子NMR谱。
实施例
实施例1:菌株F/94-499709(DSM10275)的鉴定
应用下列培养基来鉴定菌株F/94-499709,其中的培养基组分以去离子水中的“重量/体积”表示,在121℃下加热灭菌20分钟。
MEA:2%麦芽提取物,0.4%酵母提取物,2%琼脂。
在培养皿中的MEA上进行点接种(point-inoculate)再在暗处培养后,菌株F/94-499709表现出如下特性:
生长的最佳温度是24~30℃。培养14天后菌落的直径在24℃下为25~32mm,在27℃下为30~37mm,在33℃下为7~15mm。高于37℃和低于13℃时,菌株F/94-499709一点也未生长。
在27℃的暗处生长的菌落一般是米色至浅黄色,呈颇为扁平至稍微凸起的形状,其中央长出少量的、范围有限的气生菌丝体,它带白色至淡灰色。径向沟纹可变得很明显,当从底部观看时,深灰色中央区可变得突出。在陈化培养物时,菌落中央部分中的气生菌丝体和底物菌丝体可变成暗灰色,而菌落边沿保持米色至浅黄色。
显微镜检查时未看到芽孢形成结构,于是暂时将菌株F/94-499709称为
不育菌丝体(
mycelium sterilum)。
实施例2:菌株F/94-499709的发酵
下列培养基和方法适用于通过菌株F/94-499709的发酵生产式VIII化合物的方法中。除非另有说明,所有培养基组分以去离子水中的“重量/体积”表示,并在121℃下加热灭菌20分钟。
PCM(预培养和中间培养基):2%麦芽提取物,0.4%酵母提取物,0.1%琼脂。
PRM(生产培养基):2%可溶性淀粉,0.5%酵母提取物,2%葡萄糖,2%玉米浸泡液的浓缩液,0.5%胨,0.2%碳酸钙。
预培养物的生产:将2ml菌株F/94-499709的液氮种子悬浮液融化后,将它们接种入含200ml PCM的500ml锥形瓶,再在24℃下、200RPM的旋转式摇荡器上培养7天。
为了生产原代中间培养物,将各含200ml PCM的14个500ml锥形瓶各用5ml预培养物接种。
继代中间培养物是通过用1.4升原代中间培养物接种两个含PCM的50升发酵罐而生产的。在下列条件下发酵6天:24℃,1升空气/分钟/升培养基,桨式搅拌器转速为150RPM,以及0.5巴的压力。为了生产式VIII的化合物及相关化合物,在含PRM的三个500升发酵罐中,各用13升继代中间培养物接种。发酵条件如下:21℃,1升空气/分钟/升培养基,桨式搅拌器转速为100RPM逐渐加快达150RPM,以及0.5巴的压力。96小时后,收获和合并1500升该发酵产物以回收所需的式VIII的化合物和相关化合物。
实施例3:从菌株F/94-499709中分离式VIII的缩肽
将得自1500升发酵作用的培养液与1700升乙酸乙酯一起在Dispax反应器中均化并强烈搅拌3小时。借助于Westfalia分离器分离有机相。重复该提取步骤,合并有机相后减压蒸发得2745g提取物。用40升甲醇/水混合物(9∶1)和40升环己烷通过三步提取使该提取物脱脂。合并甲醇部份并减压蒸发至干得960g脱脂的提取物。将该提取物在装有于甲醇溶液中15kg Sephadex LH20的柱上进行色谱分离,共得135g含式VIII的缩肽的级分。用该全部135g级分浸渍300g硅胶,再将浸渍了的硅胶加到1.5kg硅胶Merck(0.04~0.063mm)的柱顶,用1升1∶9、2∶8、3∶7、4∶6、5∶5、6∶4、7∶3、8∶2、9∶1的甲基叔丁基醚/环己烷,3升MTBE和3升95∶5的MTBE/甲醇进行色谱分离。收集1升级分并用HPLC和TLC分析。合并级分8和9再蒸发至干。从乙醚中结晶得21.9g纯的式VIII的缩肽。用色谱法在硅胶H Merck(750g)上按前述同样方法进一步纯化母液和级分10~13,得另一批晶状式VIII的环缩肽。从乙醚中纯化后,测得该缩肽的熔点(mp)为143-146℃,旋光度[α]D 20=-233.9°(c=0.908,甲醇)。在VCAM-1细胞ELISA中测得式VIII的缩肽的IC50约为2nM。
分子式:C51H83N7O9(938.2)
在甲醇中的UV光谱:λmax(ε′)=290(5.4),279(5.08),220(38.1),197(55.7)-参见图1
IR(KBr)谱示于图2
MS FAB谱示于图3
MS FAB谱(含LiI)示于图4
在CDCl3中的质子NMR谱示于图5
MTBE=甲基叔丁基醚
VCAM=血管粘着分子
实施例4.式VIII化合物的N1′-脱甲氧基衍生物的合成
把4.9mg式VIII化合物的溶液溶于3ml甲醇,加入8mg钯/活性炭(10%)。在氢气气氛中将该混合物搅拌2小时,通入氩气,过滤并蒸发得无色泡沫状的标题化合物。用薄层色谱法和NMR光谱法分析该化合物,得如下结果。
TLC:硅胶,甲苯/甲醇9/1,Rf=0.28。
1H-NMR(3种构象异构体(conformer)56∶37∶7,以*标记,给出如下特征信号):8.72*(d,J=10Hz,NH);8.08*(s,br,吲哚 NH);6.97*,6.90°(2d,J=2Hz,吲哚H-2);6.34°(d,J=9.5Hz,NH);6.00*(d,J=6.5Hz,NH);5.83′(d,NH);5.32°(ddd,PrLeu α-H);5.12°,5.08*(2q,J=7Hz,乳酸 Me);4.50*(dd,MeLeu α-H);4.10*(ddd,Leu α-H);3.42(q,J=7Hz,MeAla α-H);3.43°,3.19°,3.12*,2.93*,2.53*,2.35°(6s,NMe);1.54*,1.50°(2d,J=7Hz,MeAla α-H),1.38°,1.24*(2d,J=7Hz,乳酸 α-H);0.57*,-0.01*(2d,J=6.5Hz,Me),-0.19*(ddd,Leuβ-H).
实施例5:式VIII化合物的N1′-甲基衍生物的合成
将5mg实施例4的产物于0.5ml干DMF中的溶液与100ml碘代甲烷混合,再加入3mg双(三甲基甲硅烷基)氨基化钠于0.3mlDMF中的溶液。在室温下搅拌该反应混合物达1.5h后,将该混合物倾入0.1M HCl水溶液,用乙酸乙酯提取,再在乙酸乙酯和饱和碳酸氢盐水溶液间分配。用盐水洗涤该有机相,在硫酸钠上干燥后真空蒸发。用色谱法在硅胶上纯化该粗产物(梯度∶甲苯/甲醇=100/0.25~100/2.5)得无色泡沫状标题化合物。用薄层色谱法和NMR谱分析该化合物,得下列结果。
TLC:硅胶,甲苯/甲醇9/1,Rf=0.40。
1H-NMR(2个构象异构体60∶40,以*°标记,给出下列特征信号):8.72*(d,J=10Hz,NH);6.79*,6.74°(2s,吲哚H-2);6.35°(d,J=9.5Hz,NH);5.98*(d,J=6.5Hz,NH);5.32°(ddd,PrLeu α-H);5.12°,5.08*(2q,J=7Hz,乳酸 Me);4.50*(dd,MeLeuα-H);4.06*(ddd,Leu α-H);3.73(s,吲哚 NMe);3.44°,3.19°,3.15*,2.93*,2.53*,2.35°(6s,NMe);1.55*,1.51°(2d,J=7Hz,MeAla α-H),1.39°,1.24*(2d,J=7Hz,乳酸α-H);0.57*,-0.09*(2d,J=6.5Hz,Me),-0.32*(ddd,Leu β-H).
实施例6:5-[ 8,11-二异丁基-14-(1-甲氧基-1H-吲哚
-3-基甲基)-7,13,19,20-四甲基-5,17-双-(2-甲基-己
基)-3,6,9,12,15,18,21-七氧-1-氧杂-4,7,10,13,16,19-六
氮杂-环二十一烷-2-基]-戊-2-烯酸甲基酯
将185mg式XV(见下文)的化合物和1.26g甲氧基羰基亚甲基三苯基正膦于甲苯中的溶液在室温下搅拌1小时。然后真空蒸发掉溶剂,将残余物在甲醇中的LH-20凝胶过滤柱上进行色谱法纯化,真空蒸发含各级分的产物,进一步用色谱法在硅胶上纯化(洗脱剂∶梯度甲苯/甲醇=99.5/0.5~96/4)得无色固态泡沫体标题产物。将产物从苯中冷冻干燥。1H-NMR(CDCl3,特征信号,3个构象异构体53∶41∶6,以*°′标记):8.67*(d,J=10Hz,C9AA NH);7.87*(d,J=10Hz,C9AA NH);7.80°(d,J=10Hz,NH);7.69°(d,J=10Hz,NH);7.45-7.35(4d,MeMeOTrp H-4′,H-7′);7.23*,7.22°(2m,MeMeOTrp,H-6′);7.4(2m,MeMeOTrp H-5′);7.06*,7.00°(2s,MeMeOTrp H-2′);6.88°(ddd,J=15Hz,J=7Hz,-CH=);6.76*(ddd,J=15Hz,J=7Hz,-CH=);6.24°(d,J=10Hz,Leu NH);6.04*(d,J=6Hz,Leu NH);5.82°,5.78*(2d,J=15Hz,=CH-CO);5.29°(ddd,MeAla a-H);5.07-4.98(m,a-H);4.92(dd,MeMeOTrp a-H);4.86*(ddd,C9AA a-H)4.78*(dd,Leu a-H);4.71(m,a-H);4.48*(dd,MeLeu a-H);4.17*(ddd,Leu a-H);4.06*,4.03′,4.02°(3s,N-OMe);3.75*,3.74°(2s,COOMe);3.43°(s,N-Me);3.20*(s,MeAla NMe);3.17°(s,N-Me);2..92*(s,MeMeOTrp N-Me);2.52*(s,MeLeu N-Me);2.47°(s,N-Me);1.51*,1.48°(2d,J=7Hz,MeAla β-Me);1.04(d,J=6...5Hz,MeLeu Me);0.98-0.84(m);0.63*(d,J=6.6Hz,Leu Me);0.56′,0.39′(2d);0.06*(d,J=6.6Hz,Leu Me);-0.11*(ddd,Leu β-CH).
按与实施例6中所述类似的方法可获得其乙基酯。
式XV的起始原料是已知的(WO96/03430)。在该式中取代基具有下列意义:B′=X′=C′=Y′=在以下实施例7-11中,应用同样的缩写,但下列缩写除外:A″=C″=
实施例7:式IX的化合物
(即式I的化合物,其中A=A″,R8=噻唑-2-基,B=B′,R1=CH3,C=C″,R3=OCH3,
=双键,X=X′,Y=Y′)
将400mg式I化合物(其中A=A″、R8=-CSNH2、B=B′、R1=CH3、C=C″、R3=OCH3、
=双键、X=X′、Y=Y′)和0.5ml氯乙醛(choroacetaldehyde)水合物于8ml干二甲基甲酰胺中的溶液与0.5g4分子筛一起搅拌并加热至60℃达5小时。然后用乙酸乙酯稀释该混合物,过滤后用0.1N HCl萃取。用盐水洗涤该有机相,干燥,再真空蒸发。将粗产物在硅胶上进行色谱法纯化(洗脱剂∶梯度甲苯/甲醇=99.5/0.5~98/2)得固态泡沫体的标题化合物。
将1.1g式XI的化合物(A是α羟基取代的丁酸残基,该残基被-CNγ取代,其中B=B′、R1=CH3、C=C′、R4=OCH3、
=双键、X=X′、Y=Y′)和1.8g二苯基二硫代膦酸(diphenylphospinodithioicacid)于25ml异丙醇中的溶液加热回流达7小时。将该反应混合物真空蒸发,溶于乙酸乙酯,再用碳酸氢钠溶液和盐水萃取。蒸发掉有机层后,将粗产物用色谱法在硅胶上纯化(洗脱剂∶梯度甲苯/甲醇=99.5/0.5~98/2)得无色泡沫体状的起始化合物。
在VCAM-1细胞ELISA中测得实施例4~11的化合物具有类似于式VIII化合物的活性。
1H-NMR谱(CDCl3)实例7. 3个构象异构体46∶51∶3,以*°′标记:8.70*(d,J=10Hz,LeuPr NH);7.89*(d,10Hz,NH);7.83°(d,J=9Hz,NH);7.69,7.66(2d,J=3.3Hz,ok H);7.58(d,J=10Hz,NH);7.53*,7.47°(2dm,J=7Hz,MeTrpOMe H-4′);7.38*,7.37°(2dm,J=8Hz,MeTrpOMeH-7′);7.22*,7.20°(2tm,MeTrpOMe,H-6′);7.17,7.16(2d,J=3.3Hz,噻唑-H);7.09(s,MeTrpOMe H-2′);7.03*,7.00°(2dd,MeTrpOMe H-5′);6.98°(s,MeTrpOMe H-2′);6.18°(d,J=10Hz,Leu NH);6.03*(d,J=7Hz,Leu NH);5.80′(d,J=10Hz,Leu NH);5.30°(ddd,LeuPr a-H);5.11*(dd,羟基丁酸a-H);5.05-4.98(m,a-H);4.94(dd,MeTrpOMe a-H);4.86°(ddd,LeuPra-H);4.73(m,a-H);4.49*(dd,MeLeu a-H);4.23*(ddd,Leu a-H);4.02,4.00(2s,N-OMe);3.84°(m,a-H);3.59-3.40(m);3.38(q,J=7Hz,MeAla a-H);3.33(s,N-Me);3.2-2.85(m);3.21(s,N-Me);3.07(s,N-Me);2.93*(s,MeTrpOMe N-Me);2.53*(s,MeLeu N-Me);2.49(s,N-Me);2.43-2.28(m);2.18(m);1.98(m);1.81(m);1.7-1.1(m);1.48,1.46(2d,J=7Hz,MeAla β-Me);1.06*(d,J=6.5Hz,MeLeu Me);1.00-0.84(m);0.61*(d,J=6.6Hz,Leu Me);0.54′(d,J=6.6Hz,Leu Me);0.35′(d,J=6.6Hz,Leu Me);0.10*(d,J=6.6Hz,Leu Me);-0.12*(ddd,Leu β-CH).8. 3个构象异构体47∶48∶5,以*°′标记:8.64*(d,J=10Hz,LeuPr NH);7.82(2d,10Hz,NH);7.63°(d,J=10Hz,NH);7.23,7.17,7.08,6.97(4t,芳环H);7.32,7.28(2s,噻唑-H);7.07(s,MeTrpOMe H-2′);6.81(s,MeTrpOMe H-2′);6.22°(d,J=10Hz,Leu NH);6.07*(d,J=7Hz,Leu NH);5.80′(d,J=10Hz,Leu NH);5.29°(ddd,LeuPr a-H);5.20*(dd,MeTrpOMe a-H);5.12°(dd,羟基丁酸 a-H);5.03°(ddd,LeuPr a-H);4.97*(ddd,LeuPr a-H);4.85(m,羟基丁酸+LeuPra-H);4.71°(ddd,Leu a-H);4.52*(dd,MeLeu a-H);4.21*(ddd,Leu a-H);4.02,3.90(2s,N-OMe);3.34(s,N-Me);3.22(s,N-Me);3.02(s,N-Me);2.93(s,N-Me);2.53(s,N-Me);2.48(s,N-Me);1.43,1.42(2d,J=7Hz,MeAla β-Me);1.06*(d,J=6.5Hz,MeLeu Me);0.62*(d,J=6.6Hz,Leu Me);0.10*(d,J=6.6Hz,Leu Me);-0.04*(ddd,Leu β-CH).9. 3个构象异构体42∶52∶6,以*°′标记:8.67*(d,J=10Hz,LeuPr NH);7.83,7.80(2d,10Hz,NH);7.63°(d,J=10Hz,NH);7.55,7.42,7.39,7.36(4d,MeTrpOMe芳环);7.22,7.18,7.05,6.97(4dd,MeTrpOMe H-5′,H-6′);7.06°(s,MeTrpOMe H-2′);6.88*(s,MeTrpOMe H-2′);6.22°(d,J=10Hz,Leu NH);6.02*(d,J=7Hz,Leu NH);5.77′(d,J=10Hz,Leu NH);5.30°(ddd,LeuPr a-H);5.21*(dd,MeTrpOMe a-H);5.0(m,a-H);4.85(m,a-H);4.65(ddd,Leu a-H);4.49*(dd,MeLeu a-H);4.13*(ddd,Leu a-H);4.03,3.98(2s,N-OMe);3.70(m);3.55(m);3.45(q,J=7Hz,MeAla a-H);3.37,3.20,3.11,2.93,2.52,2.43(6s,N-Me);2.73(m,四氢苯并噻唑);1.50,1.48(2d,J=7Hz,MeAla β-Me);1.04*(d,J=6.5Hz,MeLeu Me);0.58*(d,J=6.6Hz,Leu Me);0.50′(d,J=6.6Hz,Leu Me);0.30′(d,J=6.6Hz,Leu Me);0.03*(d,J=6.6Hz,Leu Me);-0.22*(ddd,Leu β-CH).10. 3个构象异构体44∶5 1∶5,以*°′标记:8.66*(d,J=10Hz,LeuPr NH);7.83,7.81(2d,
10Hz,NH);7.63°(d,J=10Hz,NH);7.57*,7.43°,7.38*,7.36°(4d,J=8Hz,吲哚-H);
7.21*,7.18°,7.05,6.97(4t,吲哚-H);7.06*(s,MeTrpOMe H-2′);6.88°(s,MeTrpOMe
H-2′);6.70°,6.69*(2q,J=1Hz,噻唑-H);6.23°(d,J=10Hz,Leu NH);6.05*(d,J=7Hz,
Leu NH);5.80′(d,J=10Hz,Leu NH);5.29°(ddd,LeuPr a-H);5.11°(dd,羟基丁酸
a-H);5.01(m);4.97(dd,a-H);4.85(m,2x a-H);4.69°(ddd,Leu a-H);4.57′(dd,
a-H);4.48*(dd,MeLeu a-H);4.16*(ddd,Leu a-H);4.03,3.98(2s,N-OMe);3.43(q,
J=7Hz,MeAla a-H);3.37,3.20,3.10,2.92,2.52,2.46(6s,N-Me);2.40,2.39(2d,J=1Hz,
Me-噻唑);1.48,1.47(2d,J=7Hz,MeAla β-Me);1.05*(d,J=6.5Hz,MeLeu d-Me);
0.60*(d,J=6.6Hz,Leu d-Me);0.52′,0.33′(2d,J=6.5Hz Leu d-Me);0.05*(d,J=6.6Hz,
Leu d-Me);-0.14*(ddd,Leu β-CH).11. 3个构象异构体47∶50∶3,以*°′标记:8.69*(d,J=10Hz,LeuPr NH);7.79,7.78(2d,
10Hz,NH);7.65°(d,J=10Hz,NH);7.51*,7.41°,7.39*,7.38°(4d,J=8Hz,吲哚-H);
7.23*,7.20°,7.07,7.00(4t,吲哚-H);7.04*(s,MeTrpOMe H-2′);6.85°(s,MeTrpOMe
H-2′);6.71°*(s,噻唑-H);6.25°(d,J=10Hz,Leu NH);6.04*(d,J=7Hz,Leu NH);5.80′
(d,J=10Hz,Leu NH);5.30°(ddd,LeuPr a-H);5.10°(dd,羟基丁酸 a-H);5.02
(m);4.97(dd,a-H);4.85(m,2x a-H);4.72°(ddd,Leu a-H);4.60′(dd,a-H);4.50*(dd,
MeLeu a-H);4.17*(ddd,Leu a-H);4.04,4.00(2s,N-OMe);3.48(q,J=7Hz,MeAla a-H);
3.41,3.21,3.17,2.92,2.53,2.47(6s,N-Me);0.97,0.96(2s,t-Bu- 噻唑);1.50,1.49(2d,
J=7Hz,MeAla β-Me);1.06*(d,J=6.5Hz,MeLeu d-Me);0.62*(d,J=6.6Hz,Leu d-Me);
0.53′,0.35′(2d,J=6.5Hz Leu d-Me);0.07*(d,J=6.6Hz,Leu d-Me);-0.08*(ddd,Leu
β-CH).A)3个构象异构体44∶30∶26,以*°′标记:8.85(d,CSNH2);8.60(d,LeuPr NH);8.17(d,
CSNH2);8.03,8.00(2d,NH);7.88(m,CSNH2);7.6-7.1(芳 m);6.28*(d10Hz,Leu
NH);6.07°(d,7Hz,Leu NH);5.87′(d,9Hz,Leu NH);5.26*(ddd,LeuPr a-H),5.22(dd,
羟基酸 a-H);5.15-4.95,5.08*(dd,羟基酸 a-H);4.83°(ddd,LeuPr a-H);
4.50*(ddd,Leu a-H);4.37*(dd,MeTrpOMe a-H);4.25°(ddd,Leu a-H);4.09,4.05,
4.03*(3s,OMe);3.89(m,a-H);3.65*,3.63′,3.52°(3q,7Hz,MeAla a-H);3.57(m),
3.17,3.16,3.15,3.22,3.20,3.05,2.92,2.55,2.53(9s,N-Me);1.8-1.1;1.05(d,7Hz);
0.99-0.82,0.60°,0.55′,0.23′,0.17°(4d,7Hz,Leu d-Me);-0.15°,-0.17′(ddd,Leu b-CH).
PKF 285-916(噻唑)
生物活性
在分析中测试本发明的化合物的活性以测定其细胞毒性和对于ICAM-1、VCAM-1和E-选择蛋白表达、细胞增殖的抑制作用,以及对于TNF释放的抑制作用和相应的细胞毒性分析。
分析方法如下:
HaCaT细胞,即自发转化的、具有正常角质形成细胞的高度保守性表型分化特性的非致瘤性人角质形成细胞系(Boukamp等,1988,J.Cell Biol.106,761-771),被用作细胞增殖分析和ICAM-1细胞Elisa。
A.ICAM-1细胞-ELISA分析
I.
角质形成细胞ICAM-1细胞Elisa
用于测定对ICAM-1表达的抑制作用的ICAM-1细胞Elisa基本上如Winiski和Foster描述的(1992,J.Invest.Dermatol.,99,48-52)那样。将HaCaT细胞接种于96孔微量滴定板(2×104个细胞/孔,于下列培养基中:DMEM,其中含5%FCS,100U/ml青霉素,100mg/ml链霉素,2mM谷氨酰胺,1mM丙酮酸钠),待长到铺满后在新鲜的试验培养基(如上述培养基,但其中含0.5%而不是5%FCS)中培养约24小时,其中加有或不加IFN-γ/TNF-α刺激培养基(试验培养基+1000U/ml IFN-γ/3ng/ml TNF-α),二者都分为存在或不存在试验化合物的情况。然后洗去培养基,用1%多聚甲醛固定该细胞单层。将该单层与饱和量的初级(小鼠抗ICAM-1单克隆)抗体和次级(山羊抗小鼠过氧化物酶缀合)抗体一起培养。随后的过氧化物酶反应中应用3-氨基-9-乙基咔唑(AEC)作底物,生成不溶性的有色产物,它容易用标准的微量滴定板读出器测定。
II.
细胞毒性的测定
当完成测定ICAM-1的AEC反应之后,用PBS(200mL)漂洗该HaCaT单层,从板上倾掉PBS,然后将板在毛巾纸顶部轻拍至干以除去过剩的液体。用湿纸巾轻轻地擦干微量滴定板底面,然后再用干纸巾擦,在492nm处读取吸光度。在这些单层变干之前,往每个孔中加入0.1ml0.1%结晶紫于PBS中的溶液(先滤过0.2mm滤器)。接着在室温下将这些板培养10分钟,用PBS充分洗涤5次,按前述方法除去过剩的流体,再在这些单层变干之前又一次在492nm处读取吸光度。将染色前后的光密度相减得因为结晶紫染色而产生的值,从而与孔中存在的细胞单层量相关联。应用这些值来校正AEC值。
B.内皮细胞VCAM-1、ICAM-1和E-选择蛋白细胞-Elisa
分析
该分析基于96孔细胞Elisa方法,其中应用人微血管内皮细胞系HMEC-1和人脐静脉内皮细胞(HUVEC)。用试验化合物对细胞预处理4小时,然后用TNFα刺激6-16小时,再用多聚甲醛固定,随后用间接的免疫过氧化物酶染色法估测VCAM-1、ICAM-1或E-选择蛋白表达。细胞毒性效果的测定是这样进行的,即通过计数暴露于试验物质后的相对细胞数(吉姆萨核染液),与对照孔(只有溶剂和培养基)比较。如果化合物表现为≥50%VCAM-1、I-CAM-1或E-选择蛋白抑制而细胞损失<25%,则将它们记为阳性。
方法学
I.
细胞系:VCAM-1和ICAM-1分析中应用无限增殖化(SV-40病毒大T抗原)人微血管内皮细胞系(HMEC-1;Ades等,J.Invest.Dermatol.99:683-690,1992)。HMEC-1细胞组成性地表达低水平的、被炎症介质上调的ICAM-1。然而,它们只在细胞因子刺激之后表达VCAM-1。进行了剂量-响应实验和时间过程实验以测定诱导VCAM-1和ICAM-1表达的最佳条件。
II.
生长条件:使HMEC-1细胞在标准条件(37℃,5%CO2)下生长于T-75烧瓶(Nunc)中,生长密度为1.5×106个细胞/ml培养基(CM=内皮细胞基本培养基[EBM;Clonetics],补加有10%FCS、10ng/ml人EGF(Boehringer)、1mg/ml氢化可的松(Sigma#0888)、2.2g/l NaHCO3、15mM Hepes、0.11g/l丙酮酸钠、4mM谷氨酰胺、100U/ml青霉素和100mg/ml链霉素)。在适度胰蛋白酶消化(0.25%胰蛋白酶+0.1%EDTA,达8min)和重悬浮之后,每2-3天以1∶3分割比再接种细胞。
III.
VCAM-1和ICAM-1细胞-Elisa
将96孔平底微量滴定板预涂上牛纤连蛋白(FN;Sigma#F1141),然后以2×104个细胞/孔的密度接种在EBM生长培养基中并培养一夜。第二天,将该培养基(CM)先用200ml/孔的EBM分析培养基(CM中补加5%而不是10%FCS)置换,随后用180ml含下列3组物质之一的培养基置换:(1)适当浓度的试验化合物,(2)相应浓度的溶剂/甲醇萃取的培养基,或(3)只有EBM分析培养基,并在37℃下培养4hr。每个96孔分析都重复一次。再加入20ml浓细胞因子溶液(2000U/ml TNFα)刺激这些细胞,接着在37℃下培养16hr。
然后将该细胞单层用含1%多聚甲醛的EBM培养基洗涤,室温(RT)下在2%的多聚甲醛中固定15min,再用PBS漂洗数次。从细胞中除去PBS,将该单层在含10%正常山羊血清(NGS)的PBS中培养30min。用100ml/孔的抗VCAM-1或ICAM-1单克隆抗体置换该NGS溶液,再在4℃下培养一夜。然后除去该mAb溶液,用PBS将这些细胞漂洗数次,接着在RT下与含10%NGS的PBS一起培养30-60min。除去该NGS溶液,添加100ml辣根过氧化物酶缀合的山羊F(Ab′)2抗小鼠IgG抗体(Tago;在含5%NGS的PBS中稀释至1∶500),在RT下将这些板培养1hr。然后除去次级抗体,用PBS漂洗细胞,再用150ml/孔新制备并过滤了的AEC溶液(3-氨基-9-乙基咔唑;Sigma)代替PBS,接着将这些板在RT下培养45-60min。除去过氧化物酶底物,用PBS漂洗细胞。在微量滴定板读出器上读取550nm处的AEC吸光度并用690nm处的“空白”或参比值修正。
IV.
E-选择蛋白分析:用新分离的HUVEC进行该E-选择蛋白分析,基本上按VCAM-1或ICAM-1分析中所述方法,但采用更短时间的TNFα刺激(6-8小时)。
V.
细胞毒性的测定(基于核染色剂的细胞损失):
用95%乙醇置换PBS达20min(每10min变更一次)使内皮细胞脱色,其间用显微镜检法来控制。然后在蒸馏水(Aquadest)中漂洗这些细胞,在RT下用33%Giemsa溶液于Aquadest中覆盖该细胞单层达5min。再用Aquadest洗涤这些孔,随后风干至少15min。应用显微镜检法来检查只有核被染色,而胞质基本上没染色。在微量滴定板读出器上读取550nm处的Giemsa吸光度值,并以690nm处的“空白”值(没有细胞的几排孔)修正。
VI.
数据评估:组成性VCAM-1或E-选择蛋白表达的AEC值(未刺激的对照孔)基本上等于同种型配对的对照mAb的值并代表本底染色剂(background stain)。在每个96孔板中,从每个细胞因子刺激的组(EBM和溶剂对照物,以及试验物质)的平均AEC值减去平均固有值,所得值表示上调的ICAM-1和诱导型VCAM-1或E-选择蛋白细胞粘着分子(CAM)表达(称为AEC-CAM)。然后将每个AEC-CAM值除以相应的平均Giemsa值,由所得值可估测对给定的细胞密度、基于核数的CAM相对表达水平(称为AEC:Giemsa比)。
AEC(刺激的)-AEC(未刺激的)=AEC-CAM
AEC-CAM/Giemsa=AEC∶Giemsa比
因此,“实际的”CAM IC50值是通过将试验物质的AEC∶Giemsa值与刺激的对照物(EBM,溶剂)的值进行比较而测定的。然后将这些值相对于只有Giemsa的IC50值进行分析。严格的判据可确定CAM抑制作用对细胞毒性(Giemsa)曲线是否表明所希望的“真正”成功。
C.HaCaT细胞增殖分析
将HaCaT细胞在其中补加有下列物质的DMEM(Gibco#074-02100)中培养:2.2g/l NaHCO3、0.11g/l丙酮酸钠、15mM Hepes、5%胎牛血清(FCS)、青霉素(100U/ml)、链霉素(100mg/ml)以及谷氨酰胺(将最终浓度增大4mM)。为进行该增殖分析,采用胰蛋白酶消化使细胞脱附,悬浮于新鲜培养基中,再接种入96孔微量滴定板,最终密度为4000个细胞/0.2ml/孔。24小时(第0天)后,用含梯度浓度的试验化合物的新鲜培养基置换该培养基。在37℃/5%CO2下培养3天后,通过比色分析法测定与溶剂对照物相比细胞增殖的程度,该分析法中应用染料sulforhodamine B测定相对细胞量(Ske-han等,1990,J.Natl.Cancer Inst.82,1107-1112)。“起始细胞数”是通过测定第0天相对细胞量而测定的。将结果表示为%抑制=100-%对照吸光度(其中溶剂对照物=100%),它代表3次测定的平均值±标准偏差。用半对数法描绘剂量-响应曲线,再通过线性内插法确定半最大抑制所需的浓度(IC50)。以“起始细胞数”表示没有细胞净损失时的最大抑制,它通常为90~98%。
D.TNF释放的抑制
I.
单核细胞的制备:应用来自健康志愿者的外周血,采用Hansell等的ficoll-hypaque密度分离法(J.Imm.Methods(1991)145:105.);使用的细胞浓度为于RPMI1640+10%FCS中105个细胞/孔。先用系列稀释的试验化合物在37℃下培养细胞达30分钟,随后加入IFNγ(100U/ml)和LPS(5mg/ml),再进一步培养3小时。然后在1400RPM下离心10min终止培养。应用商购的ELISA(In-notest hTNFα,得自Innogenetics N.V.,Zwijnaarde,比利时)测定上清液中的TNFα。在0~10mM的浓度下测试这些化合物。例举的式I化合物,尤其是式Ip、I′p、I″p、VII、IX和X的优选化合物,在该分析中抑制TNF释放的IC50从约5至高达约nM。
II.细胞毒性
对THP1细胞(5×104个/孔)测定细胞毒性,在IFNγ(100U/ml)和LPS(5mg/ml)存在下,分别存在和不存在试验化合物时,于37℃下将这些细胞培养24小时。采用比色读出器(MTT)估测活细胞和死细胞百分数,如Mosman在J.Imm.Methods(1983)65:55中所述,它是通过测定活细胞中的线粒体脱氢酶而进行分析的。该分析中测知本发明的优选化合物一般具有较低的细胞毒性,例如IC50从约100至约1000nM。
Claims (6)
1.式Ip的化合物:
其中:
Ap是任选被H或甲基或乙基α取代的乙醇酸残基;
Bp是α-氨基-γ-甲基取代的辛酸残基;
R1p是氢或甲基;
Cp是色氨酸残基或N-甲基色氨酸残基,它任选被N′-(C1~C4)烷氧基取代;
Xp是α-氨基取代的(C2~C14)羧酸残基;以及
Yp是α-氨基-或N-甲基-α-氨基取代的(C2~C10)羧酸残基。
5.以DSM10275保藏的真菌株F/94-499709。
6.权利要求1中所述的式Ip的化合物在制备具有治疗作用的药物中的应用。
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