CN108055865B - 用于治疗的新的TNFα结构 - Google Patents

用于治疗的新的TNFα结构

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CN108055865B
CN108055865B CN201580081000.1A CN201580081000A CN108055865B CN 108055865 B CN108055865 B CN 108055865B CN 201580081000 A CN201580081000 A CN 201580081000A CN 108055865 B CN108055865 B CN 108055865B
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tnfr1
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J·P·奥康奈尔
J·R·波特
A·劳森
B·克罗普里安
S·E·拉佩琦
T·J·诺曼
G·J·瓦里楼
T·L·阿拉卡基
A·B·伯金
W·R·皮特
M·D·凯尔米阿诺
D·A·舒伯特
D·J·莱特伍德
R·J·伍顿
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Abstract

公开了一种新的稳定的三聚体TNFα结构，其具有扭曲的对称性，其可以与TNFR1受体结合以减弱其信号，其可以用于治疗和/或预防与可溶性TNFα/TNFR1相互作用有关的疾病。膜结合的TNFα在其通过TNFR2的信号传导能力方面不受影响，因此TNFα的新结构可用于不明显增加感染或恶性风险的治疗。

Description

用于治疗的新的TNFα结构
发明领域
本发明涉及新的稳定的，不对称的三聚体TNFα结构，以及它们在治疗中的用途。本发明进一步涉及三聚体TNFα结构与小分子的复合物，所述小分子可以结合TNFR1受体，但减弱其信号，其可用于治疗和/或预防疾病。本发明进一步涉及不对称三聚体TNFα结构和复合物的晶体，以及从这种晶体获得的3-D结构模型在确定新的三聚体TNFα抑制剂的方法中的用途。
背景技术
肿瘤坏死因子(TNF)超家族是共同调节细胞存活和细胞死亡的主要功能的蛋白质家族。TNF超家族的成员共享共同的核心基序，其由两个反平行的β折叠片(具有反平行的β链)组成，形成“果冻卷”β结构。TNF超家族成员共同的另一个共同特征是形成同源或异源三聚体复合物。TNF超家族成员的这些三聚体形式结合并激活特定的TNF超家族受体。
TNFα是TNF超家族的原型成员-形成对称的同源三聚体。TNFα产生的失调已经牵涉到许多具有重大医学重要性的病理状况。例如，TNFα涉及类风湿性关节炎，炎性肠病(包括克罗恩氏病)，牛皮癣，阿尔茨海默氏病(AD)，帕金森病(PD)，疼痛，癫痫，骨质疏松症，哮喘，系统性红斑狼疮(SLE)硬化症(MS)。TNF超家族的其他成员也涉及病理状况，包括自身免疫性疾病。
TNF超家族成员的常规拮抗剂是大分子的并且通过抑制TNF超家族成员与其受体的结合起作用。常规拮抗剂的实例包括抗-TNFα抗体，特别是单克隆抗体，例如英夫利昔单抗
Figure BDA0001512576410000011
阿达木单抗
Figure BDA0001512576410000012
和赛妥珠单抗
Figure BDA0001512576410000013
或可溶性TNFα受体融合蛋白，例如依那西普
Figure BDA0001512576410000014
这些都抑制可溶性TNFα及其与受体TNFR1(负责炎症)和膜结合TNFα及其与受体TNFR2(参与免疫应答)的相互作用。
发明内容
本发明人已经获得了TNFα的新的，稳定的，不对称的三聚体结构的广泛的结构性理解。该TNFα三聚体通过与TNFR1结合起作用，但不能或不能启动TNFR1下游的信号传导。该TNFα三聚体可以通过在三聚体中心与一类小分子实体(SME)形成复合物而采用该结构。本发明人还对这些化合物，能够诱导不对称TNFα三聚体结构的药效团以及TNFα三聚体内与药效团接触以诱导不对称TNFα三聚体的残基的关键相互作用，获得了广泛的结构理解。这些不对称的TNFα三聚体和复合物可以用于治疗由TNFα介导的病症。本发明人还发现膜结合的TNFα三聚体和复合物不破坏TNFR2下游的信号传导。最后，本发明人已经鉴定了新的不对称TNFα三聚体晶体形式，其可以用于基于结构的合理药物设计方法以确定可以诱导不对称TNFα三聚体的新化合物。
因此，本发明提供了包含SEQ ID NO：36的氨基酸序列或相应序列的蛋白质亚基的不对称TNFα三聚体，其中所述TNFα三聚体当通过X射线晶体学测定时采用构象，其中CαSEQID NO：36的残基12-18，47-50，54-64，76-82，91-97，117-125，129-137和150-156的残基的原子，或对应于SEQ ID NO：36的所有亚基的相应序列所述TNF-α三聚体能够结合TNFR1，但是其中来自所述结合的TNFR1的信号传导被减弱或拮抗，任选地用于方法中的参考结构化合物34.pdb的相同原子的
Figure BDA0001512576410000021
RMSD[均方根偏差]在人体或动物体上进行的治疗。
本文中术语“不对称的TNFα三聚体”，“不对称的TNFα三聚体”或“本发明的TNFα三聚体”可互换使用以表示相同的含义。在此，本发明的TNFα三聚体是非天然存在的(鉴于其不对称构象的性质)。TNFR1受体的“拮抗剂”或“拮抗作用”可以通过使用本文的术语来理解，并且例如广泛地意在表示导致功能性阻止或减少TNFR1信号传导的手段，而不管行动(除非另有说明)。这里“和/或”是指“和或或”。
本发明还提供了包含含有SEQ ID NO：36的氨基酸序列或相应序列的蛋白质亚基的TNFα三聚体和能够与TNFα三聚体结合的化合物的复合物，由此化合物-三聚体复合物结合TNFR1并减弱三聚体通过TNFR1诱导的信号传导，其中通过X射线结晶学测定，化合物包含结合在TNFα三聚体内的药效团，使得其在所有以下残基的范围内：亚基A的Leu57；亚基B的Tyr119；亚基B的Gly121；亚基B的Gly122；C亚基的Tyr59；亚基C的Leu120；和亚基C的Tyr151，并且其中药效团由2个稠合的5-或6-元环(以中心在“R3”和“R2”)，一个环(以R2为中心)与H键受体(“A1”)，并且其还通过连接非氢原子被取代为另外的5-或6-元环(以“R4”为中心)。
还提供了具有空间群P 21 21 21，P 21 21 2或P 1 21 1的TNFα三聚体晶体。此类晶体可用于本发明的TNFα三聚体的结构阐明和比较，或可用于方法用于确定与TNFα三聚体形成复合物的化合物以产生本发明的不对称TNFα三聚体结构。
附图的简要说明
图1显示了式(1)-(64)和(65)的化合物的结构。
图2A显示了影响TNFα与TNF受体结合的测试化合物的筛选结果(MesoscaleDiscovery assay，MSD)。研究了多种测试化合物，计算了TNFα与TNF受体结合的％抑制水平。图2B显示使用该测定的式(3)化合物的剂量响应曲线。图2C显示式(15)化合物的剂量响应曲线。
图3A显示了受体-配体结合测定，证实在式(3)化合物存在下，TNF与TNFR1的胞外结构域(ECD)的结合增强。图4B显示了式(15)的化合物在相同的测定中诱导的增强的结合。
图4(底部曲线)显示了100％水溶液中TNFα的解卷积质谱图。图4(上图)显示了含有10％v/v DMSO的溶液中TNFα的解卷积质谱图。图4(中间曲线)显示了含有10％v/v DMSO和式(3)化合物的溶液中TNFα的解卷积质谱图。
图5显示了含有式(3)化合物的溶液中TNFα的质谱图。
图6显示了尺寸排阻色谱法实验的洗脱曲线的叠加，和随后的(A)与式(3)化合物预温育的TNFα样品然后与TNF-R混合的质谱分析和(B)用TNF-R预温育的TNFα样品，然后与式(3)的化合物混合。
图7显示了(A)TNFα与TNF-R结合的等温量热分析结果和(B)TNFα与TNF-R结合的等温量热分析结果，其中TNFα已经与式(15)的化合物。
图8显示式(3)-三聚体TNFα复合物的晶体结构。
图9显示了按式(3)化合物和式(15)化合物的浓度测量的式(3)化合物和式(15)化合物对人TNFα的中和图。对抗使用L929鼠纤维肉瘤细胞杀伤测定法测得的残余人TNFα浓度(pg/ml)。
图10显示了式(3)化合物的浓度(nM)相对于TNFα处理的人单核细胞中％相对IL-8产量的图。
图11显示在存在(A)TNFα(0.5ng/mL)，(B)IL-1β和TNF-α之前，式(15)化合物的浓度(nM)相对于HEK293细胞中NF-1β(0.5ng/mL)和(C)活化性TNF-R1抗体(300ng/mL)。
图12A显示了使用表面等离子体共振测量的式(3)化合物与TNFα随时间的结合动力学。图12B显示式(15)化合物与TNFα的结合动力学。图12C显示式(39)化合物与TNFα的结合动力学。
图13显示了响应于单独的TNFα或已经用逐渐增加浓度的(A)式(3)化合物或(B)式(15)化合物预孵育的TNFα的嗜中性粒细胞募集水平，并通过腹膜内注射(ip。)。
图14显示单独或在增加浓度的式(3)化合物存在下口服给药时嗜中性粒细胞对TNFα的应答水平。
图15是使用式(3)，(15)和(39)的测试化合物的荧光偏振(FP)测定的结果的图。将测试化合物的浓度对荧光缀合物与TNFα结合的％抑制作图。
图16显示了扭曲的TNFα三聚体结构化合物I-33，35-63的β折叠的Cα原子与发黄的参考结构化合物34的RMSD叠加。
图17显示了畸变的TNFα三聚体结构化合物64的β-折叠的Cα原子与粗化的参考结构化合物34的RMSD叠加。
图18显示说明来自在TNFα三聚体中的特定残基的4以内的64个扭曲的TNFα三聚体结构的配体的百分比的图。
图19显示了化合物1扭曲的TNFα三聚体结构的核心图，突出显示了在64个结构的100％中在配体的4内的所有残基。
图20显示了可能适合本发明的扭曲的TNFα三聚体结构的药效团的一个实例，其显示了三环特征的R2，R3和R4中心的位置以及氢键受体特征A1在R2内的位置环。
图21是显示本文所述化合物(诱导扭曲的可溶性TNFα三聚体结构)对膜TNFα诱导的TNFR2近端和下游信号转导的作用的凝胶；化合物与NS0细胞中过表达的膜TNFα的结合不损害TNFR2特异性膜近端(TRAF-2募集至TNFR2)和下游(全细胞裂解物中pNFκB存在)信号传导。
序列表简述
SEQ ID NO：1显示了CA185_01974.0的LCDR1。
SEQ ID NO：2显示CA185_01974.0的LCDR2。
SEQ ID NO：3显示了CA185_01974.0的LCDR3。
SEQ ID NO：4显示了CA185_01974.0的HCDR1。
SEQ ID NO：5显示了CA185_01974.0的HCDR2。
SEQ ID NO：6显示了CA185_01974.0的HCDR3。
SEQ ID NO：7显示了CA185_01974.0的LCVR的氨基酸序列。
SEQ ID NO：8显示了CA185_01974.0的HCVR的氨基酸序列。
SEQ ID NO：9显示了CA185_01974.0的LCVR的DNA序列。
SEQ ID NO：10显示了CA185_01974.0的HCVR的DNA序列。
SEQ ID NO：11显示了CA185_01974.0的κ轻链的氨基酸序列。
SEQ ID NO：12显示了CA185_01974.0的mIgG1重链的氨基酸序列。
SEQ ID NO：13显示了CA185_01974.0的mFab(无铰链)重链的氨基酸序列。
SEQ ID NO：14显示了CA185_01974.0的κ轻链的DNA序列。
SEQ ID NO：15显示了CA185_01974.0的mIgG1重链的DNA序列。
SEQ ID NO：16显示了CA185_01974.0的mFab(无铰链)重链的DNA序列。
SEQ ID NO：17显示CA185_01979.0的LCDR2。
SEQ ID NO：18显示CA185_01979.0的LCDR3。
SEQ ID NO：19显示了CA185_01979.0的HCDR1。
SEQ ID NO：20显示CA185_01979.0的HCDR2。
SEQ ID NO：21显示CA185_01979.0的HCDR3。
SEQ ID NO：22显示了CA185_01979.0的LCVR的氨基酸序列。
SEQ ID NO：23显示了CA185_01979.0的HCVR的氨基酸序列。
SEQ ID NO：24显示了CA185_01979.0的LCVR的DNA序列。
SEQ ID NO：25显示了CA185_01979.0的HCVR的DNA序列。
SEQ ID NO：26显示了CA185_01979.0的κ轻链的氨基酸序列。
SEQ ID NO：27显示了CA185_01979.0的mIgG1重链的氨基酸序列。
SEQ ID NO：28显示了CA185_01979.0的mFab(无铰链)重链的氨基酸序列。
SEQ ID NO：29显示了CA185_01979.0的κ轻链的DNA序列。
SEQ ID NO：30显示了CA185_01979.0的mIgG1重链的DNA序列。
SEQ ID NO：31显示了CA185_01979.0的mFab(无铰链)重链的DNA序列。
SEQ ID NO：32显示了大鼠TNFα的氨基酸序列。
SEQ ID NO：33显示了小鼠TNFα的氨基酸序列。
SEQ ID NO：34显示人TNFα的氨基酸序列。
SEQ ID NO：35显示了可溶形式的人TNFα的氨基酸序列。
SEQ ID NO：36显示可溶形式的人TNFα的氨基酸序列，但没有起始的“S”(其是SEQID NO：35中的克隆制品)
具体实施方式
在一个实施方案中，提供了包含(或由其组成)SEQ ID NO：36的氨基酸序列或相应序列的蛋白质亚基的不对称TNFα三聚体，其中所述TNFα三聚体当通过X射线测定时采用构象与SEQ ID NO：36的残基12-18，47-50，54-64，76-82，91-97，117-125，129-137和150-156的Cα原子或相应的序列对于参考结构化合物34.pdb的相同原子的
Figure BDA0001512576410000071
RMSD[均方根偏差]内的所有亚基，所述TNF-α三聚体能够结合TNFR1，但是其中来自所述结合的TNFR1的信号传导被减弱或拮抗，任选地用于在人体或动物体上实施的治疗方法中。
本发明的不对称TNFα三聚体或扭曲的TNFα三聚体或TNFα三聚体是TNFα的新型结构，其中对称或apo TNFα三聚体(Eck和Sprang 1989 JBC 264：17595-605)中亚基之间的正常3倍对称轴，被破坏使得三聚体仍然结合TNFR1，但其中来自所述结合的TNFR1的信号传导被减弱或拮抗或被完全抑制。载脂蛋白TNFα三聚体的结构在本领域中是众所周知的，例如来自Protein Data Bank(PDB)的1A8M。
术语“相应序列”表示本发明的TNFα三聚体可以具有任何已知的动物或人TNFα的野生型氨基酸序列，特别是人TNFα，例如SEQ ID NO：36。它可能是可溶性TNFα(sTNFα)或膜结合TNFα或两者。通过金属蛋白酶TNFα转化酶(TACE/ADAM17；虽然其他蛋白酶也可以释放sTNF如ADAM10，ADAM19，基质金属蛋白酶7和蛋白酶)通过蛋白水解切割从膜结合的同源三聚体TNFα(mTNF)释放可溶性同三聚体TNFα3，其可产生相对于TACE切割的sTNFα如SEQ IDNO：36可被1，2，3，4或5个氨基酸延伸或截短的相应的可溶性TNFα序列。可溶性52kDa三聚体sTNF呈三角锥形。由术语mTNF涵盖的人类序列显示在SEQ ID NO：34中，术语sTNF(在SEQ IDNO：34上的TACE的作用的产物)包含的人类序列显示在SEQ ID NO：36中。大鼠和小鼠mTNFα的相应序列分别在SEQ ID NO：32和33中给出。来自其他动物(或人类序列的已知变体)的相应的TNFα序列可以容易地用SEQ ID NO：36序列覆盖，并施用与SEQ ID NO：36相同的氨基酸编号(用于编号TNFα氨基酸酸)。例如，来自各种动物的序列可以在Uniprot数据库(www.uniprot.org)中找到，包括人类序列P01375和Q5STB3。相应的sTNFα序列可以是mTNFα序列的157个氨基酸的C-末端(如SEQ ID NO：36)，或者可以长或短一个，两个或三个氨基酸(大鼠和小鼠序列是156个氨基酸)。相应sTNFα序列可以具有1，2，3，4，5，6，7，8，9，10，11，12，13，14，15，16，17，18，19，20，21，22，23，24，25，26，27，28，29，30，31，32，33，34，35，36，37，38，39或40个氨基酸取代相对于SEQ ID NO：36的氨基酸取代。相应的sTNFα序列在SEQID NO：36的长度上可以与SEQ ID NO：36具有80，90，95，96，97，98或99％的氨基酸序列同一性。
本发明的TNFα三聚体具有通过X射线晶体学(例如通过实施例18和19中详述的方法)测定的共同不对称结构。本发明的TNFα三聚体可通过覆盖残基12-18，47-50，54-64，76-82，91-97，117-125，129-137和150-150的Cα原子的原子坐标来确认。所有亚基的SEQ IDNO：36(β-折叠残基)或相应的序列，并确定它们在参考结构化合物34.pdb的相同原子的
Figure BDA0001512576410000081
RMSD[均方根偏差]内实例如实例19中所述)。
来自任何TNFα序列的β-折叠残基的相应序列可以容易地确定。例如，大鼠和小鼠sTNFα序列是mTNFα的C端156个氨基酸，而不是SEQ ID NO：36的157个氨基酸。它可以容易地确定相应的β折叠的残基为在覆盖，例如，(mTNFα序列SEQ ID NO的：32)大鼠sTNFα序列是残基12-18，47-50，54-64，75-81，90-96，116-124，128-136和149-155。
在本发明的补充数据中给出了64个结构的原子坐标，给出名称为“化合物X”的每个结构等同于术语“化合物X.pdb”，其中X是图1化合物的式(X)1坐在每个三聚体的中心。参考结构Compound34.pdb(64中最平均的结构)的原子坐标因此可以很容易地加载到合适的软件中以便进行覆盖。
在进行覆盖的三聚体亚基链将需要被分配有标签亚基A，B，和C.哪个链A，B或C可通过3之间测量三个距离在x射线晶体学确定结构C确定三个相同残基的α原子-例如每个链中的P117(小鼠sTNFα序列中的P116)；(G121也是合适的)。
三个距离形成一个三角形，在apo TNFα中是等边的，但在本发明的不对称TNFα结构中是扭曲的。最短的距离是BC与AC之间最长的距离(例如AC＝13.8？，AB＝12.3？，BC＝10.2？)。从而通过N/C末端指向您的分子轴向下看，最长的距离定义C，然后A链逆时针旋转，然后B和C再次逆时针旋转。
确定本发明的TNF-α三聚体能够结合TNFR1，但来自所述结合的TNFR1的信号传导被减弱或拮抗，可以通过本文讨论的任何适当方法进行(例如在实施例6，7，8，9，10[Reporter Gene Assay]，12)。
鉴于本发明的TNF-α三聚体的有利性质，它们用于在人体或动物体上实施的治疗方法中。
本发明的TNF-α三聚体可以直接或间接施用于人体或动物体。“间接地”是指已经在动物或人体内的对称TNF-α三聚体可以成为可用于治疗的本发明的不对称TNF-α三聚体。在一个实施方案中，本发明的TNF-α三聚体可通过向动物或人施用本文所述的化合物[例如化合物(1)-(65)]间接施用于人或动物体，引起如本文所述的可用于治疗的本发明复合物的形成。
本发明的TNFα三聚体可以用于治疗和/或预防一种或多种自身免疫病症，炎性病症，神经病症，神经变性病症，疼痛病症，伤害感受性病症或心血管病症。例如，用于治疗和/或预防一种或多种类风湿性关节炎，克罗恩病，牛皮癣，系统性红斑狼疮，阿尔茨海默病，帕金森病和癫痫。
在本文的一个方面，RMSD在0.85，0.8，0.7，0.65，0.6，0.5或0.47内。
在进一步的实施方案中，类似地，提供了通过直接或间接施用于患有自身免疫疾病，炎症性疾病，神经病症，神经变性病症需要的患者包含蛋白质亚基的不对称TNFα三聚体，其包含SEQ ID NO：36的氨基酸序列或相应序列或由其组成，其中所述TNFα三聚体当通过x-射线晶体学测定时采用构象SEQ ID NO：36的残基12-18，47-50，54-64，76-82，91-97，117-125，129-137和150-156的Cα原子，或所有亚基的相应序列在参考结构化合物34.pdb的相同原子的
Figure BDA0001512576410000101
RMSD内，所述TNF-α三聚体能够结合TNFR1，但其中来自所述结合的TNFR1的信号传导被减弱或拮抗。
在上述实施方案的一个方面，本发明的TNFα三聚体的构象可以通过与化合物(1)-(65)中的任何一个形成复合物的TNFα三聚体获得或获得。
在本发明的具体实施方案中：TNFα三聚体拮抗TNFR1受体的信号传导(例如通过本文公开的方法测量)；与对称的TNFα三聚体的稳定性(例如通过本文公开的方法测量)相比，TNFα三聚体具有增加的稳定性，例如稳定性的增加导致TNFα三聚体的热转变中点(Tm)1℃以上，10℃以上，10℃以上20℃以下(例如通过本发明公开的方法测定，TNFα三聚体与TNFR1受体的结合亲和性与结合性相比增加对称TNFα三聚体与TNFR1受体的亲和力(例如通过本文公开的方法测量)；TNFα三聚体通过增加结合率(kon-r)和/或降低与用于结合对称TNFα三聚体与TNFR1受体(例如通过本文公开的方法测量)的kon-r和koff-r值相比，TNF-α的速率(koff-r)TNFR1受体由incr与对称TNFα三聚体与TNFR1受体结合的kon-r值(例如通过本文公开的方法测量)相比，缓解了结合速率(kon-r)。
在另一个实施方案中，如本文进一步描述的，与对称TNFα三聚体与TNFR1受体的KD-r相比，TNFα三聚体与TNFR1受体的KD-r降低，其中：
a)与对称TNFα三聚体与TNFR1受体的KD-r相比，TNFα三聚体与TNFR1受体的KD-r降低至少10倍；
b)TNFα三聚体与TNFR1受体的KD-r值小于10nM。
在进一步的实施方案中，如本文进一步描述的，与对称的TNFα三聚体与TNFR1受体的KD-r相比，TNFα三聚体与TNFR1受体的KD-r降低，其中：
a)与TNFR1受体的对称TNFα三聚体的KD-r相比，TNFα三聚体与TNFR1受体的KD-r降低至少4倍；
b)TNFα三聚体与TNFR1受体的KD-r值小于600pM，例如小于200pM。
在进一步的实施方案中，本发明的TNFα三聚体以下列抗体中的任一种与。KD-ab为1nM或更低的抗体结合：具有序列SEQ ID NO：26的轻链的CA185_1979和序列SEQ ID NO：27；或
CA185_1974，其具有序列SEQ ID NO：11的轻链和SEQ ID NO：12的重链(关于本发明的TNFα三聚体特异性的这些抗体的测量方法和进一步信息参见本文)。通过这种结合，本发明的TNFα三聚体可以说是采用了“稳定的”不对称构象。
本文公开的大量晶体结构已经导致了本发明的TNFα三聚体的结构性理解-特别是清楚地理解了本发明的TNFα三聚体中心的裂缝或空腔或结合位点的性质(参见实施例20和21)。特别地，空腔的形状可以被与本发明的TNFα三聚体形成复合物的化合物占据，以稳定其有利的不对称构象。实施例20描述了总是参与与这样的化合物(例如化合物(1)-(64))接触的TNFα三聚体的残基，并且实施例21描述了可以被包括在化合物内的药效团需要进行这些联系。这种理解容易使技术人员能够使用药效基团作为设计可以使本发明的复合物稳定化的许多不同化合物的基础。
因此在另一个实施方案中，提供了包含蛋白质亚基的TNFα三聚体的复合物，所述蛋白质亚基的TNFα三聚体包含SEQ ID NO：36的氨基酸序列或相应序列或由其组成，所述化合物能够结合TNFα三聚体其中化合物-三聚体复合物与TNFR1结合并减弱或拮抗由三聚体通过TNFR1诱导的信号传导，其中通过X-射线晶体学测定，所述化合物包含结合在TNFα三聚体内的药效团，所有以下残基的4？：亚基A的Leu57；亚基B的Tyr119；亚基B的Gly121；亚基B的Gly122；C亚基的Tyr59；亚基C的Leu120；和亚基C的Tyr151，并且其中药效团由2个稠合的5-或6-元环(以中心在“R3”和“R2”)，一个环(以R2为中心)与H键受体(“A1“)，并且其还通过连接非氢原子被取代为另外的5-或6-元环(以”R4“为中心)。
药效团可具有以下中的一个或多个：R2环为5-或6-元；R3环是5-或6-元；R4环是5-或6-元；R2环是芳香族的；R3环是芳香族的；R4环是芳香族的；R2环是杂芳族的；R3环是杂芳族的；R4环是杂芳族的；共享2个原子的稠合环；连接的非氢原子是碳，氮或氧(其本身可以是连接R2环与R4环的化合物的另一个环的一部分，以形成例如包含三个稠合环的三环化合物-参见化合物48，49，51，52，60，61或63)；A1特征是通过R2环中的氮或氧原子(其可以与TNFα三聚体的亚基C上的Tyr151的侧链氢键结合)。
通常R2是5或6元环。
在一个实施方案中，R2是五元环。在该实施方案的一个方面中，R2是5元芳族环。在该实施方案的另一方面，R2是5元杂芳族环。
在另一个实施方案中，R2是6元环。在该实施方案的一个方面中，R2是6元芳族环。在该实施方案的另一方面，R2是6-元杂芳族环。
芳香族/杂芳香族R2环的π系统合适地与TNFα三聚物的亚基C上的Tyr59的侧链形成CH-π相互作用(合适的是C-β-CH 2基团)。
通常R3是5或6元环。
在一个实施方案中，R3是五元环。在该实施方案的一个方面中，R3是5元芳族环。在该实施方案的另一方面，R3是5元杂芳族环。
在另一个实施方案中，R3是6元环。在该实施方案的一个方面中，R3是6元芳族环。在该实施方案的另一方面，R3是6-元杂芳族环。
合适地，芳族/杂芳族R3环的π体系在TNFα三聚体的亚基C上与Tyr59的侧链的芳族环形成π堆积相互作用。
芳香族/杂芳香族R3环的π系统合适地与TNFα三聚体的亚基A上的Leu57的侧链形成CH-π相互作用(适当地为C-Delta CH2基团)。
通常，R4是5元或6元环。
在一个实施方案中，R4是五元环。在该实施方案的一个方面中，R4是5元芳族环。该实施方案的另一方面，R4是5元杂芳族环。
在另一个实施方案中，R4是6元环。在该实施方案的一个方面中，R4是6元芳族环。该实施方案的另一方面，R4是6-元杂芳族环。
芳香族/杂芳香族R4环的π系统合适地与TNFα三聚体的亚基A上的Leu57的侧链形成CH-π相互作用(适当地为C-Delta CH2基团)。
通常融合的R3和R2环共享2个原子。
通常，连接非氢原子(连接[因此不是R2和R4环的一部分]的单个原子是碳，氮或氧。在第一个实施方案中，连接非氢原子是碳。在第二个实施方案中，连接非氢原子是氮。在第三个实施方案中，连接非氢原子是氧。
通常，A1是R2环中(即部分)的氮或氧原子。在TNFα三聚体的亚基C上，适当地A1与Tyr151的侧链氢键结合。
对于本领域技术人员显而易见的是，药效团表示化合物-三聚体复合物的最小结构和/或化学特征，并且因此该化合物可以包括另外的结构特征。
例如，化合物-三聚体复合物的化合物可以包含如上定义的药效团，其中连接的非氢原子被引入到连接R2环与R4环的另外的环中，从而形成稠合的三环化合物(参见化合物例如48，49，51，52，60，61或63)。
根据下表，药效团可以具有R2，R3和R4环，并且在TNFα三聚体结构内排列A1特征：
Figure BDA0001512576410000141
另一方面，药效团可以具有R1，R2，R3和A1特征之间的一个或多个距离，精确地或在根据下表的值的10％内：
Figure BDA0001512576410000142
在又进一步的方面中，药效团可以在R1，R2，R3和A1特征之间的角度中具有一个或多个角度，精确地或在根据下表的值的10％内：
Figure BDA0001512576410000143
Figure BDA0001512576410000151
有利地，药效团的R3-R2-R4角定义了可能参与本发明的TNFα三聚体的去对称化的香蕉形状。
包含药效团的化合物可以具有20-41个非氢原子，并且例如可以是化合物(1)-(65)中的任何一种。
如本文所述，本发明的复合物可类似地用于在人体或动物体上实施的治疗方法(通过直接[施用复合物]或间接[施用化合物]施用)，或可以直接或在治疗和/或预防一种或多种自身免疫病症，炎性病症，神经病症，神经退行性病症，疼痛病症，伤害感受性病症和/或心血管病症的方法中间接施用有需要的患者；例如在治疗和/或预防类风湿性关节炎，克罗恩氏病，牛皮癣，系统性红斑狼疮，阿尔茨海默病，帕金森病和/或癫痫中的一种或多种中。
如本文所述，本发明的复合物可以类似地包含蛋白质亚基，所述蛋白质亚基包含SEQ ID NO：36的氨基酸序列或相应序列或由其组成。
在一个实施方案中，本发明的复合物包含本发明的TNFα三聚体。
在本发明的具体实施方案中：复合物内的化合物通过TNFR1受体拮抗由TNFα三聚体诱导的信号传导(例如通过本文公开的方法测量)；与不存在该化合物时TNFα三聚体的稳定性(例如通过本文公开的方法测量)相比，该化合物增加了TNFα三聚体的稳定性，例如稳定性的增加导致热转变中点增加(Tm)至少1℃，至少10℃或10℃至20℃；与不存在该化合物时(例如通过本文公开的方法测量的)TNFα三聚体与TNFR1受体的结合亲和力相比，TNFα三聚体对TNFR1受体的结合亲和力增加，例如化合物增加与TNFα三聚体结合的kon-r和koff-r值相比，TNFα三聚体对TNFR1受体的结合亲和力是通过增加开启速率(kon-r)和/或降低关闭速率(koff-r)在不存在化合物的情况下与TNFR1受体结合(如本文所述)；与在不存在化合物的情况下TNFα三聚体与TNFR1受体结合的kon-r值相比，该化合物增加了TNFα三聚体与TNFR1受体的结合亲和力(通过增加kon-r)例如通过本文公开的方法)。
在另一个实施方案中，如本文进一步描述的，与不存在所述化合物时TNFα三聚体与TNFR1受体的KD-r相比，所述化合物将TNFα三聚体的KD-r降低为TNFR1受体，其中：
a)与化合物不存在时TNFα三聚体与TNFR1受体的KD-r相比，化合物使TNFα三聚体的KD-r降低至TNFR1受体至少10倍；
b)化合物存在下TNFα三聚体与TNFR1受体的KD-r值小于10nM。
在另一个实施方案中，如本文进一步描述的，与不存在所述化合物时TNFα三聚体与TNFR1受体的KD-r相比，所述化合物将TNFα三聚体的KD-r降低为TNFR1受体，其中：
a)与化合物不存在时TNFα三聚体与TNFR1受体的KD-r相比，该化合物将TNFα三聚体的KD-r降低至TNFR1受体至少4倍；
b)化合物存在下TNFα三聚体与TNFR1受体的KD-r值小于600pM，例如小于200pM。
在另一个实施方案中，化合物具有500nM或更小的IC50值(例如通过本文公开的方法测量)。
在本发明的另一个实施方案中，复合物与具有1nM或更小的。KD-ab的下列抗体中的任一个结合：具有序列SEQ ID NO：26的轻链和序列SEQ ID NO：27的重链的CA185_1979；或
具有序列SEQ ID NO：11的轻链和序列SEQ ID NO：12的重链(关于这些抗体的测量方法和进一步信息，本发明的复合物和TNFα三聚体是特异性的)的CA185_1974。通过这种结合，可以说本发明的复合物采用“稳定的”不对称构象。
在另一个实施方案中，提供了包含本发明的复合物和药学上可接受的载体的药物组合物。
在本发明的另一方面，本发明人提出的支持本发明的TNFα三聚体，本发明的复合物和本发明的方法的数据可用于治疗，而没有现有TNFα治疗剂的显着副作用。
现有的TNFα抑制剂结合并中和可溶性和膜结合的TNFα(分别为sTNFα和mTNFα)(Nesbitt等，Inflamm Bowel Dis 2007 13：1323-1332)。
已知sTNFα对TNFR1受体具有特异性，对TNFR2受体具有mTNFα(Grell等，Cell 199583：793-802)。
现有的抑制剂在其标签上有黑匣子警告，描述了在严重感染(特别是TB(肺结核)，细菌性败血症和真菌感染)和恶性肿瘤(包括淋巴瘤)中使用的严重后果。
已知对TB(以及李斯特氏菌)的免疫应答是由mTNFα驱动的(Garcia等，Chapter 20p187-201“可溶性和膜TNF和相关配体在分枝杆菌感染中的作用：选择性和非选择性TNF的作用抑制剂在感染期间“，在D.Wallach等(编辑)，Advances in TNF Family Research，Advances in Experimental Medicine and Biology 691，DOI 10.1007/978-1-4419-6612-4_20中)。
选择性抑制sTNFα但不抑制mTNFα的TNFα抑制剂具有减弱实验性关节炎而不抑制对感染的先天免疫性的特征(Zalevsky等，免疫学杂志2007 179：1872-1883)。
实施例22研究了结合本文所述化合物时mTNFα构象如何不影响TNFR2功能；TNFR2近端和下游信号不受损害。
因此，本发明的TNFα三聚体或本发明的复合物可用于在人体或动物体上实施的治疗方法中，其中所述用途不诱导或导致恶化感染和/或恶性肿瘤。例如，感染是TB和/或细菌败血症和/或真菌感染和/或恶性是淋巴瘤。
类似地，本发明人在本发明的方法中提供施用有需要的患者不诱导或导致所述患者中的感染和/或恶性恶化的方法。例如，感染是TB和/或细菌败血症和/或真菌感染和/或恶性是淋巴瘤。
用于鉴定拮抗剂的试验：本发明的TNFα和本发明的复合物
本发明人已经开发了鉴定TNFα拮抗剂的测定法。具体而言，本发明人已经开发了可用于鉴定与TNFα的三聚apo形式结合的化合物的试验，并使这些三聚体稳定在能够结合所需TNF受体(TNFR1)的构象中，从而拮抗信号传导通过所述受体。因此，本发明公开了用于鉴定TNFα拮抗剂的测定法。
具体而言，本文所述的测定法可用于鉴定结合TNFα的三聚apo形式并且形成与TNFR1结合的化合物-三聚体复合物的化合物。
在一个优选的实施方案中，本发明的测定法鉴定结合TNFα的三聚体形式但不结合单体形式的化合物。在特别优选的实施方案中，所述化合物结合并稳定TNFα的三聚体形式，不结合单体形式并且不稳定TNFα的二聚体形式。测试化合物对TNFα三聚体的稳定作用可以通过抑制三聚体之间单体单元交换的测试化合物来实现。
本发明的测定可以包括确定测试化合物是否增强TNFα(本发明的TNFα三聚体和复合物)与其受体的结合，并因此鉴定TNFα拮抗剂。在一个优选的实施方案中，本发明的测定可以包括确定测试化合物是否增强TNFα与TNFR1的结合，并且因此鉴定通过增加TNFα(TNFα)的还原信号传导或非信号传导形式的结合起作用的TNFα拮抗剂三聚体和本发明的复合物)与TNFR1的结合。
根据本发明的用于鉴定TNFα拮抗剂的测定可以包括在使TNFα三聚体的形成不稳定的条件下(例如在DMSO存在下)温育TNFα样品，并测量测试化合物稳定TNFα三聚体形成的程度。或者，根据本发明用于鉴定TNFα拮抗剂的测定可以包括将TNFα与测试化合物结合，并测量本发明的TNFα三聚体或化合物-三聚体复合物与TNFR1的结合程度。
TNFα和TNFR1可以纯化或以混合物存在，例如在培养细胞，组织样品，体液或培养基中。可以开发定性或定量分析，后者可用于确定测试化合物对TNFα的三聚体形式的结合参数(亲和常数和动力学)，以及本发明的TNFα三聚体的结合参数或化合物-三聚体复合物与必需的TNF受体结合。
TNFα的单体，二聚体和三聚体形式的量可以通过测量单体，二聚体和三聚体形式的质量，单体，二聚体和三聚体形式的摩尔量，单体，二聚体和三聚体形式的浓度，以及单体，二聚体和三聚体形式的摩尔浓度。这个数额可以用任何适当的单位给出。例如，单体，二聚体和三聚体形式的浓度可以以pg/ml，ng/ml或μg/ml给出。单体，二聚体和三聚体形式的质量可以以pg，ng或μg给出。
可将感兴趣样品中TNFα的单体，二聚体或三聚体形式的量与另一样品(如对照样品)中TNFα单体，二聚体或三聚体形式的水平进行比较，如本文所述。在这样的方法中，可以评估TNFα的单体，二聚体或三聚体形式的实际量，例如样品中TNFα的单体，二聚体或三聚体形式的质量，摩尔量，浓度或摩尔浓度。可以将TNFα的单体，二聚体或三聚体形式的量与另一个样品中的量进行比较，而不量化TNFα的单体，二聚体或三聚体形式的质量，摩尔量，浓度或摩尔浓度。因此，根据本发明的样品中TNFα的单体，二聚体或三聚体形式的量可以评估为单体，二聚体或三聚体形式的相对量，例如相对质量，相对摩尔量，相对浓度或相对摩尔浓度基于两个或多个样品之间的比较，TNFα的三聚体形式。
在本发明中，可筛选化合物文库以鉴定TNFα拮抗剂(即使用本文公开的测定法)。这样的文库通常包含至少260种化合物。优选地，这样的文库包含至少300，至少500或甚至至少1000个化合物。
基于质谱的测定
本发明人已经发现质谱法可用于鉴定与TNFα的三聚体形式结合的化合物，并使这些三聚体稳定在能够结合TNFR1的构象(本发明的TNFα三聚体或复合物)中。
具体而言，质谱法可用于评估化合物是否稳定TNFα的三聚体形式。
因此，本发明提供了用于鉴定能够结合三聚体TNFα蛋白的化合物的测定法，由此化合物-三聚体复合物(或本发明的TNFα三聚体)结合TNFR1并拮抗受体的信号传导，包括以下步骤鉴定测试化合物与样品中TNFα的三聚体形式的结合，并将该化合物与TNFα的三聚体形式的结合与对照样品的相应值进行比较，该方法包括对含有TNFα的样品进行质谱分析，和该化合物检测TNFα三聚体的量并将样品中TNFα三聚体的量与对照样品比较，并选择能够结合TNFα的三聚体形式的化合物，由此使化合物-三聚体复合物(或TNFα三聚体)结合TNFR1拮抗受体的信号。对照样品可以与待测样品相同，除了它缺乏测试化合物。包含TNFα和化合物的样品可以进一步包含去稳定剂。
在本发明中，试验化合物可以在去稳定剂存在下加入到TNFα溶液中。去稳定剂(也称为离液剂)包括低摩尔浓度(例如1M)尿素，胍或乙腈，这些试剂的高浓度(例如6M或更高)将导致TNFα三聚体的完全解离和组成性TNFα单体的解折叠亚基。去稳定剂优选为DMSO，通常浓度为5％，10％或更高。可以使用质谱分析所得到的溶液。
可以检测到TNFα与结合亲和力低至1mM的测试化合物之间形成的非共价复合物。结合化学计量可以直接从存在或不存在其中结合有多个测试化合物分子的复合物获得。结合亲和力(KD值)可以通过在已知测试化合物浓度下测量TNFα-测试化合物复合物(化合物-三聚体复合物)/TNFα浓度比来确定。
如果测试化合物在不存在测试化合物的情况下与含有TNFα和去稳定剂的样品所观察到的三聚体的量相比增加了三聚体的比例，则其稳定三聚体形式的TNFα。测试化合物可使三聚体的量增加10％，15％，20％，25％，30％，35％，40％，45％，50％，55％，60％，65％，70％，75％与含有TNFα和去稳定剂的样品中存在的三聚体的量相比，在与不存在TNFα的样品中存在的三聚体的量相比，的测试化合物。
与在不存在去稳定剂和测试化合物的情况下对TNFα样品所观察到的相比，测试化合物还可以增加三聚体的量。测试化合物可使三聚体的量增加10％，15％，20％，25％，30％，35％，40％，45％，50％，55％，60％，65％，70％，75％在不存在去稳定剂的情况下，与含有TNFα的样品中存在的三聚体的量相比，所述三聚体的量相对于存在于三聚体中的三聚体的量小于或等于100％，80％，85％，90％，95％，100％，150％，200％，300％，400％和测试化合物。
三聚体稳定在质谱研究中以两种方式被证明。
首先是TNFα三聚体复合物(或本发明的TNFα三聚体)的物理解离，其可以通过在质谱中观察到的单体和三聚体的比率来测量。在我们的研究中没有观察到二聚体。这种分解可能是用于将分子引入质谱仪的高能过程的人造物质。无论如何，它可用于评估测试化合物在雾化和电离过程中稳定三聚复合物的能力，从而减少质谱中观察到的单体的量，单体/三聚体比例用于确定稳定的程度。
其次，在软电离条件下，赋予三聚复合物(或本发明的TNFα三聚体)更少的能量，导致其完整的透射到光谱仪中，从而更密切地反映真正的溶液组成。电喷雾电离过程导致蛋白质多次充电，因为在正离子化模式下，某些氨基酸内的碱性官能团获得正电荷。质谱仪测量质荷比。因此，例如标称52，000DaTNFα三聚体如果携带10次电荷，则其将以m/z比率5，200出现。这种多重带电效应允许质量范围有限的质谱仪用于研究多聚体蛋白质复合物(或本发明的TNFα三聚体)。与光谱仪一起提供的软件允许用户去卷积数据以给出蛋白质的质量，因为如果它只携带单一电荷(即基于其原子组成的真实分子量)，就会出现蛋白质的质量。
在折叠的蛋白质中，许多氨基酸埋藏在核心中，只有暴露在表面上的百分比通常获得6至8个正电荷。没有一个单一的带电物质占主导地位，通常在很小的范围内观察到几种物质(离子)，这些包括所谓的电荷态包络。在另一个极端，在蛋白质完全变性(即展开)的情况下，则暴露更多的氨基酸，并且获得的电荷的典型数量可能高达20，电荷状态包络也包括更大数量的电荷物质现在有更多可用的网站来接受收费。因此，电荷的数量和构成电荷态包膜的带电物质的数量是蛋白质折叠程度的敏感读数。此外，如果折叠的蛋白质可以以表面暴露的氨基酸的相对数量不同的多种构象存在，那么电荷状态包迹的偏移将反映这些差异。
在软纳米电喷雾离子化条件下，对完整折叠的TNFα蛋白质进行质谱研究表明几乎100％的TNFα三聚体被检测到，只有很少的TNFα单体被检测到，而二聚体完全不存在。
在更苛刻的电离条件下，或当向TNFα样品中添加去稳定剂时，观察到单体TNFα的水平升高，伴随三聚体水平的降低。只观察到非常少量的二聚体。
质谱法也可用于确定测试化合物是否结合TNFα的单体，二聚体和三聚体形式。
质谱法也可以用于确定测试化合物与TNFα的化学计量，即有多少分子的测试化合物与TNFα结合。
质谱也可用于确定化合物-TNFα三聚体复合物(或本发明的TNFα三聚体)是否结合TNFR1。
质谱也可以用于测量测试化合物与TNFα结合的速率(“开”速率“kon-c”)和测试化合物从TNFα解离的速率(“关”速率或koff-c)。如本文所用，符号“KD-c”表示测试化合物对TNFα的结合亲和力(解离常数)。KD-c被定义为koff-c/kon-c。测试化合物可具有缓慢的“开”速率，其可通过质谱分析TNFα和化合物-三聚体复合物(或本发明的TNFα三聚体)峰强度以分钟测量。测试化合物的KD-c值可以通过在不同TNFα：化合物-三聚体复合物比率下重复该测量来估计。在一个优选的实施方案中，本发明化合物与TNFα三聚体的结合以快速“开”速率为特征，理想地为约107M-1s-1，具有慢“关”速率，例如通常为10-3s-1，10-4s-1，或者没有可测量的“关”率。
质谱也可以用于确定测试化合物在TNFR1存在下是否结合TNFα。这可能涉及将测试化合物与已经与其受体预温育的TNFα一起温育。然后可以将含有测试化合物和预温育的TNFα和受体的样品根据其分子大小进行分级分离，例如通过分析性凝胶过滤。可以使用质谱法分析所得级分，以确定测试化合物在必需的受体存在下是否与TNFα结合。如果与TNFα结合，化合物将以与TNFα相同的级分洗脱。如果化合物不与TNFα结合，则化合物将以与TNFα不同的部分洗脱。在这种情况下，化合物典型地将在比TNFα更晚的凝胶过滤级分中洗脱。
质谱方法可以包括例如基质辅助激光解吸/电离质谱(MALDI MS)，表面增强激光解吸/电离质谱(SELDI MS)，飞行时间质谱(TOF MS)和液相色谱质谱光谱法(LC MS)。
受体-配体结合测定
常规拮抗剂通过抑制TNFα与其受体的结合起作用。本发明人已经使用受体-配体结合测定来确定测试化合物是否增强本发明的TNFα三聚体或本发明的复合物与其受体的结合。这样的受体-配体结合测定法可用于鉴定通过增加TNFα与TNFR1的还原信号传导或非信号传导形式的结合而起作用的TNFα拮抗剂。
因此，本发明提供了用于鉴定能够结合三聚体蛋白即TNFα的化合物的测定法，由此化合物-三聚体复合物结合TNFR1并拮抗受体的信号传导，包括测量三聚体TNFα与包含测试化合物的样品中的必需受体结合，并将结合于必需受体的三聚体TNFα的水平与对照样品的相应值进行比较，该方法包括进行受体-配体结合测定，其中将TNFα样品和化合物应用于已经结合到表面的TNFR1，并将与TNFR1结合的TNFα三聚体的量与对照样品进行比较，并选择能够结合三聚体形式的TNFα的化合物，由此化合物-三聚体复合物结合TNFR1并拮抗受体的信号传导。对照样品可以与待测样品相同，只是它缺少测试化合物和/或其含有已知化合物。包含TNFα和化合物的样品可以进一步包含去稳定剂。
测试化合物可以加入到含有TNFα和去稳定剂的溶液中。可以将在单独的去稳定剂存在下(在对照样品中)的TNFR1结合水平与在去稳定剂和测试化合物存在下TNFα与其受体的结合水平进行比较。如果测试化合物增加与其受体结合的TNFα的比例，则TNFα与其受体结合的比例与TNFα与其受体结合的水平相比增强了TNFα与其受体的结合，所述TNFα与其受体在不存在测试化合物的情况下观察到含有TNFα和去稳定剂的样品。
受试化合物可以将与其受体结合的TNFα的量增加10％，15％，20％，25％，30％，35％，40％，45％，50％，55％，60％，65％在含有TNFα的样品中TNFα与其受体结合的量相比，在70％，75％，80％，85％，90％，95％，100％，150％，200％，300％，400％没有测试化合物。
本发明的受体-配体结合测定通常需要结合于支持物的TNFα受体。TNFα受体可以直接与载体结合，或者间接地使用接头分子如抗生物素蛋白或抗生蛋白链菌素结合。然后可以通过添加TNFα与去稳定剂的溶液来测定TNFα与其受体的结合水平。去稳定剂(也称为离液剂)包括低摩尔浓度(例如1M)尿素，胍或乙腈，这些试剂的高浓度(例如6M或更高)将导致TNFα三聚体的完全解离和组成性TNFα单体的解折叠亚基。去稳定剂优选为DMSO，通常浓度为5％，10％或更高。
TNFα与其受体的结合通常使用对TNFα特异的抗体并与标记物结合来确定。标记可以是任何可以被检测到的分子。例如，标记可以是放射性同位素(例如125I，32P，35S和3H)，荧光染料(例如荧光素，罗丹明)，酶缀合物等。酶的底物用于定量与表面结合的受体结合的TNFα的量。其他标记包括分子标记，当暴露于某些刺激(例如电)时，其可以被激活以产生光。标记的选择取决于所使用的检测系统。
受体-配体结合测定可以以几种形式进行，包括基于细胞的结合测定，溶液相测定和免疫测定。受体-配体结合反应的固体载体优选含有孔。一般来说，将测试化合物-三聚体复合物(或本发明的TNFα三聚体)与TNFα受体温育一段特定的时间，然后测量与受体结合的化合物-三聚体复合物的量。结合的化合物-三聚体复合物(或本发明的TNFα三聚体)的水平可通过使用显微镜，荧光测定法，闪烁计数器或任何可用的免疫测定法测量标记来计算。
如本文所用，符号“kon-r”表示化合物-三聚体复合物(或本发明的TNFα三聚体)结合TNFα受体时的速率(“开”速率)。如本文所用，符号“koff-r”表示化合物-三聚体复合物(或本发明的TNFα三聚体)从TNFα受体解离时的速率(“关闭”速率)。如本文所用，符号“KD-r”表示化合物-三聚体复合物(或本发明的TNFα)对其受体的结合亲和力(解离常数)。KD-r被定义为koff-r/kon-r。
可使用受体-配体结合测定来测量本发明的化合物-三聚体复合物(或本发明的TNFα三聚体)对TNFR1的结合亲和力。特别地，竞争测定可用于比较本发明的化合物-三聚体复合物(或本发明的TNFα三聚体)对TNFR1的kon-r和koff-r值以及TNFα的kon-r和koff-r值在不存在受试化合物(或apo TNFα)的情况下与其受体结合，并确定本发明的化合物-三聚体复合物(或本发明的TNFα三聚体)与受体结合的KD-r值。
稳定性分析
本发明人已经开发了用于测定测试化合物对TNFα的稳定性(或本发明的TNFα三聚体相对于apoTFAα三聚体的稳定性)的作用的方法。因此，本发明提供了用于鉴定能够结合三聚体TNFα蛋白的化合物的测定法，由此化合物-三聚体复合物结合必需的TNFα受体并调节受体的信号转导，包括测量在包含所述化合物的样品中测定三聚TNFα的浓度，并比较所述三聚TNFα与来自对照样品的相应值的稳定性，所述方法包括进行测定以测定TNFα和所述化合物的样品中TNFα的三聚体形式的Tm，Tm与对照样品的TNFα的三聚体形式，并选择能够结合三聚体形式的TNFα的化合物，由此化合物-三聚体复合物结合TNFα受体并拮抗受体的信号。对照样品可以与待测样品相同，只是它缺少测试化合物和/或其含有已知化合物。包含TNFα和化合物的样品可以进一步包含去稳定剂。
测试化合物可以加入到含有TNFα和去稳定剂的溶液中。在去稳定剂单独存在下(在对照样品中)，TNFα的三聚体形式的稳定性可以与在去稳定剂和测试化合物存在下TNFα的三聚体形式的稳定性进行比较。如果测试化合物增加TNFα的三聚体形式的热转变中点(Tm)，与TNFα的三聚体形式的Tm相比，增加TNFα的三聚体形式的稳定性(对于含有TNFα和去稳定剂的样品测试化合物(或本发明的不对称TNFα三聚体vs.对称TNFα)的缺乏。TNFα的三聚体形式的Tm是50％的生物分子展开的温度。可以使用本领域已知的任何适当的技术，例如使用差示扫描量热法(DSC)或荧光探针热变性分析来测量存在和/或不存在测试化合物的情况下TNFα的三聚形式的Tm。
试验化合物可使TNFα的三聚体形式的Tm增加至少1℃，至少2℃，至少5℃，至少10℃，至少15℃，至少20℃或与不存在测试化合物(或本发明的不对称TNFα三聚体相对于对称TNFα)的含有TNFα的样品中的TNFα的三聚体形式的Tm相比更多。测试化合物优选将TNFα的三聚体形式的Tm提高至少1℃，更优选至少10℃，甚至更优选10℃至20℃。
去稳定剂(也称为离液剂)包括低摩尔浓度(例如1M)尿素，胍或乙腈，这些试剂的高浓度(例如6M或更高)将导致TNFα三聚体的完全解离和组成性TNFα单体的解折叠亚基。去稳定剂优选为DMSO，通常浓度为5％，10％或更高。
等温量热分析
本发明人开发了用于确定测试化合物对TNFα对其受体的结合亲和力(或本发明的不对称TNFα三聚体与对称TNFα三聚体对受体结合的影响)的作用的等温量热法。
因此，本发明提供了用于鉴定能够结合三聚体TNFα的化合物的测定法，其中化合物-三聚体复合物结合TNFR1并拮抗受体的信号传导，包括测定与受体结合的三聚体TNFα水平的步骤包含所述化合物的样品，并将结合于所述受体的三聚体TNFα的水平与对照样品的相应值进行比较(或比较本发明的不对称TNFα三聚体与对称TNFα的结合)，其包括进行等温量热分析以测量结合在化合物存在下TNFα对受体的亲和力；并比较TNFα对受体的结合亲和力和对照样品，并选择能够结合三聚体TNFα的化合物，由此化合物-三聚体复合物结合受体并拮抗受体的信号传导。对照样品可以与待测样品相同，只是它缺少测试化合物和/或其含有已知化合物。
可将TNFα的等分试样依次加入到受体储库中。等分试样的体积可以在任何适当的范围内。等分试样可以是任何合适的体积，例如0.1μl至10μl。在一个优选的实施方案中，等分试样的体积可以是0.5μl，1.0μl或3.0μl。根据注射器的体积，可能会使用更大的体积。
当TNFα三聚体与受体结合时，每次加入TNFα将导致释放或吸收少量热量。通常，当TNFα三聚体与受体结合时，每次加入TNFα将导致释放少量热量。可以使用等温量热法测量放热量，并且该信息用于获得TNFα与其受体的结合亲和力。
该过程可以重复使用含有TNFα和测试化合物的溶液依次加入到受体储库中。优选地，TNFα和测试化合物将是化合物-三聚体复合物(或本发明的不对称TNFα三聚体)的形式。同样，放热量可以用等温量热法测量，并且这个信息用于获得TNFα与其受体的结合亲和力。
可以比较TNFα和化合物-三聚体复合物(或本发明的对称与不对称TNFα三聚体)的结合亲和力，以确定化合物是否增加TNFα与其受体的结合亲和力。
受试化合物可使TNFα与其受体的结合亲和力提高2倍，3倍，4倍，5倍，10倍，20倍，30倍，40倍，50倍，60倍，70倍，80倍，(或本发明的不对称TNFα三聚体相对于对称TNFα三聚体)的TNFα与其受体的结合亲和力的90倍，100倍或更多。
结合亲和力可以以结合亲和力(KD-r)的形式给出，并且可以以任何合适的单位给出，例如μM，nM或pM。KD-r值越小，TNFα与其受体的结合亲和力就越大。
在测试化合物存在下，TNFα与其受体结合的KD-r值可以至少是测试化合物存在下的1.5倍，2倍，3倍，4倍，5倍，10倍，20倍，30倍，40倍，(或本发明的不对称TNFα三聚体与对称的TNFα三聚体的结合)的50倍，60倍，70倍，80倍，90倍，低于TNFα的KD-r值100倍TNFα三聚体)。
在测试化合物存在下TNFα与其受体结合的KD-r值可以是1μM，100nM，10nM，5nM，1nM，100pM，10pM或更少。在一个优选的实施方案中，在测试化合物存在下TNFα与其受体结合的KD-r值是1nM或更低(或本发明的不对称TNFα三聚体与对称TNFα三聚体相比)。
竞争测定
本发明人已经开发了用于鉴定能够与三聚体TNFα结合的化合物的方法，由此化合物-三聚体复合物结合TNFR1并通过研究测试化合物与探针化合物竞争以下物质的能力来拮抗受体的信号传导结合三聚体TNFα。因此，本发明提供了一种测定方法，包括测定测试化合物与探针化合物结合三聚体形式TNFα的竞争性，并将由此观察到的竞争水平与对照样品的相应值进行比较，并选择能够与三聚体TNFα蛋白结合，由此化合物-三聚体复合物结合TNFR1受体并拮抗受体的信号传导。
探针化合物可以包含放射性标记的根据本发明的化合物。可用于本发明探针的放射性核包括氚(3H)，14C，18F，22Na，32P，33P，35S，36Cl，125I，131I和99mTc。
具体而言，竞争测定可以是荧光偏振(FP)测定，其中荧光偏振的程度与荧光分子的旋转弛豫时间相关，并因此与分子大小有关。大分子表现出比小分子更高的偏振度。因此，FP测定可用于测量小荧光配体或探针与更大蛋白质(例如TNFα)的相互作用。偏振度提供了荧光配体的结合/自由比的直接测量。
因此，本发明提供了鉴定能够结合三聚体TNFα蛋白的化合物的方法，由此化合物-三聚体复合物结合TNFR1受体并拮抗受体的信号传导，所述方法包括以下步骤：测量化合物与用于结合TNFα的三聚体形式的探针化合物，将观察到的竞争水平与对照样品的相应值进行比较，并选择能够结合三聚体TNFα蛋白的化合物，由此化合物-三聚体复合物结合TNFR1和其中所述方法包括使用所述化合物和探针化合物进行荧光偏振测定，将所述探针化合物在化合物存在下的极化程度与对照样品中的极化程度进行比较。
测试化合物与探针或配体竞争的能力可使用标准术语(例如半数最大抑制浓度(IC50))来量化。在这种情况下，IC50值代表导致探针与三聚体TNFα结合50％抑制所需的化合物的浓度。测试化合物可具有500nM，400nM，300nM，200nM，100nM，90nM，80nM，70nM，60nM，50nM，40nM，30nM，20nM，10nM，5nM，1nM，100pM或更低的IC50值。优选地，测试化合物具有200nM或更小的IC50值。更优选地，测试化合物具有150nM或更小的IC50值或100nM或更小的IC50值。
如上所述，在本发明中，化合物文库通常进行一种或多种本文所述的测定以鉴定TNFα的拮抗剂。可以包含至少260种化合物，至少300种，至少500种或甚至至少1000种化合物的这样的文库可以使用荧光偏振进行筛选。
当使用荧光偏振筛选化合物文库时，所述方法可以包括如果所述化合物导致特定的IC50值，则选择化合物作为所述TNFα的拮抗剂。例如，如果化合物导致IC50值小于50μM，则可以将化合物鉴定为TNFα的拮抗剂。在一些方面，鉴定其中导致IC50值小于500nM，小于200nM或甚至小于100nM的化合物。
如果来自文库的所有化合物中的化合物具有最低的IC50值，则来自文库的化合物也可以被鉴定为TNFα的拮抗剂。同样地，可以将化合物鉴定为TNFα的拮抗剂，其中它与文库的其他化合物相比具有低的IC50值(即，更好的IC50值)。例如，导致最低IC50值的文库化合物的50％可以被鉴定为拮抗剂。在一些方面，导致最低IC50值的文库化合物的25％或甚至10％可以被鉴定为拮抗剂。
在一个实施方案中，探针化合物包含与荧光配体缀合的根据本发明的化合物。合适地，荧光配体是具有10ns或更少的荧光寿命的荧光染料。合适的荧光染料的典型例子包括荧光素，若丹明，Cy染料(例如Cy2，Cy3，Cy3B，Cy3.5，Cy5，Cy5.5或Cy7)，
Figure BDA0001512576410000301
染料(例如Alexa 350，0405(例如BODIPY FL，BODIPY R6G，BODIPY TMR或BODIPY FL)，或可选地，例如，BODIPY TR)。探针化合物的具体实例在实施例14中描述。
对照样品可以与待测样品相同，只是它缺少测试化合物和/或其含有已知化合物。
包含TNFα和化合物的样品可以进一步包含去稳定剂。去稳定剂(也称为离液剂)包括低摩尔浓度(例如1M)尿素，胍或乙腈，这些试剂的高浓度(例如6M或更高)将导致TNFα三聚体的完全解离和组成性TNFα单体的解折叠亚基。去稳定剂优选为DMSO，通常浓度为5％，10％或更高。
尽管可以使用荧光偏振来鉴定TNFα的拮抗剂，但在本发明的一些方面，这样的拮抗剂可以通过除了荧光偏振之外的任何测定(即通过非荧光偏振的方法)来鉴定。特别地，化合物与三聚体TNFα的结合以及化合物与探针化合物与TNFα的三聚体形式的结合竞争可以通过除荧光偏振以外的任何方法来确定。
通过TNFα受体TNFR1发信号
本发明可涉及鉴定可拮抗(即阻止或减少)TNFα结合的TNFR1的信号传导的化合物的方法。
在一个实施方案中，本发明可以涉及鉴定可以阻止或减少TNFα结合的TNFR1的信号传导的化合物的方法。这种方法可以包括使TNFR1与TNFα和化合物-三聚体复合物两者接触，并检测测试化合物是否通过TNFR1阻止或减少TNFα三聚体信号传导。用化合物-三聚体复合物处理的TNFR1的信号传导量可以与仅用TNFα(或本发明的不对称TNFα三聚体相对于对称的TNFα三聚体)处理的TNFR1的信号传导量相比较。
为了检测信号水平，可以进行测量TNFR1信号传导的下游效应的测定。例如，可以使用L929鼠纤维肉瘤细胞杀伤测定来评估TNFα对细胞死亡的刺激。通过人单核细胞抑制TNFα诱导的IL-8产生也可用于评估测试化合物是否通过其受体抑制TNFα信号传导。
TNFα的拮抗剂
使用本文所述的测定，本发明人已经鉴定了结合三聚体形式的TNFα的测试化合物。这些化合物是分子量为1000Da或更小，750Da或更小，或600Da或更小的小分子实体(SME)。这些化合物稳定了与TNFR1结合的三聚体TNFα的构象，并拮抗了受体的信号。
本发明化合物对TNFα三聚体形式的稳定作用可以通过在存在和不存在该化合物的情况下测量三聚体的热转变中点(Tm)(或类似地本发明的不对称TNFα三聚体与对称TNFα三聚体)。Tm表示50％的生物分子展开时的温度。稳定TNFα三聚体的化合物将增加三聚体的Tm。Tm可以使用本领域已知的任何适当的技术来确定，例如使用差示扫描量热法(DSC)或荧光探针热变性分析。
化合物可以结合在TNFα三聚体(即三聚体的核心)内的中央空间内。
这些化合物可以将TNFα转化为TNFR1拮抗剂。因此这些化合物能够阻断TNFα信号而不必与TNFα和其受体之间的高亲和力相互作用竞争。
通过本发明的方法鉴定的化合物是变构拮抗剂，其与TNFR1受体的天然激动剂结合，即与TNFα的三聚体形式结合，并驱使这些三聚体采用仍与TNFR1结合的构象，并拮抗受体的信号传导。
通过本发明的方法鉴定的化合物可以将天然TNFα激动剂转化成拮抗剂。相反，传统的TNFα拮抗剂与TNFα或TNF受体结合并阻止TNFα与受体结合。
通过本发明的方法鉴定的化合物在其化学式或结构(除了本文提供的除外)方面不受限制，条件是它们与TNFα结合并稳定三聚TNFα的构象(例如本发明的TNFα三聚体)与TNFR1结合并拮抗受体的信号传导。因此可以使用本文所述的测定和方法鉴定通过本发明的方法鉴定的化合物。通过本发明的方法鉴定的化合物可以包含苯并咪唑部分或其电子等排物，例如式(1)-(65)的化合物。
与不存在化合物时TNFα与受体的结合亲和力相比，通过本发明方法鉴定的化合物可增加TNFα(以化合物-三聚体复合物形式)对TNFR1的结合亲和力。
通过本发明的方法鉴定的化合物结合TNFα的三聚体形式。此类化合物可特异性结合一种或多种TNFα的三聚体形式。通过本发明的方法鉴定的化合物可以仅与TNF超家族成员之一(例如TNFα)特异性结合，但不与任何其他TNF超家族成员结合。通过本发明的方法鉴定的化合物还可以特异性结合2，3，4或甚至全部的TNF超家族成员。具体而言，应该理解，化合物结合一种或多种感兴趣的分子，在这种情况下为三聚体形式的TNFα，与任何其他分子(其可能包括TNF超家族的其他成员)没有显着的交叉反应性。交叉反应性可以通过任何合适的方法评估，例如表面等离子体共振。如果化合物与其他分子结合至少5％，10％，15％，20％，那么TNFα三聚体形式的化合物与该特定TNF超家族成员的三聚体形式以外的分子的交叉反应性可以被认为是显着的％，25％，30％，35％，40％，45％，50％，55％，60％，65％，70％，75％，80％，85％，90％或100％结合TNFα的三聚体形式。对TNFα三聚体形式特异的化合物可以以小于90％，85％，80％，75％，70％，65％，60％，55％，50％，45％，40％％，35％，30％，25％或20％的浓度与TNFα的三聚体形式结合。优选地，所述化合物以小于20％，小于15％，小于10％或小于5％，小于2％或小于1％的强度与另一个分子结合，所述强度与TNFα的三聚体形式。
在测试化合物存在下(即以化合物-三聚体复合物形式)，TNFα与其受体结合的KD-r值可以是至少1.5倍，2倍，3倍，4倍，5倍，10倍，20倍，30倍，40倍，50倍，60倍，70倍，80倍，90倍，比TNFα的KD-r值低100倍。在一个优选的实施方案中，用于结合TNFα的化合物-三聚体复合物的KD-r值降低了结合TNFα三聚体的KD-r值的至少1.5倍，优选至少3倍，更优选至少4倍在不存在测试化合物的情况下的TNFR1，即化合物-三聚体复合物对于TNFR1的结合亲和力优选地与结合相比增加至少1.5倍，优选地至少3倍，更优选地至少4倍在不存在测试化合物的情况下TNFα三聚体对TNFR1的亲和力。
与TNFα三聚体单独结合受体的KD-r值相比，化合物-三聚体复合物结合TNFR1的KD-r值的降低可能是由于结合TNFR1的结合率(kon-r)与单独的TNFα三聚体结合的化合物-三聚体复合物，和/或与单独的TNFα三聚体相比降低的释放速率(koff-r)。在一个优选的实施方案中，与单独的TNFα三聚体相比，与受体结合的化合物-三聚体复合物的结合率(kon-r)增加。在另一个实施方案中，与单独的TNFα三聚体相比，与受体结合的化合物-三聚体复合物的解离速率(koff-r)降低。在另一个实施方案中，与受体结合的化合物-三聚体复合物的结合速率(kon-r)增加，与受体结合的化合物-三聚体复合物的解离速率(koff-r)降低单独的TNFα三聚体。与TNFα三聚体与其受体结合的kon-r值相比，化合物-三聚体复合物对TNFR1的kon-r值可以增加至少1.5倍或至少两倍，优选至少三倍化合物-三聚体复合物对TNFR1的化合物的缺乏和/或koff-r值可以降低至少1.2倍，至少1.6倍，至少2倍，更优选至少2.4倍与在不存在所述化合物的情况下与其受体结合的TNFα三聚体的koff-r值相比较。类似的本发明的不对称TNFα三聚体可以与对称的TNFα三聚体相比较。
在一个实施方案中，与TNFα三聚体(kon-c)结合的化合物的结合速率比结合TNFR1的化合物三聚体复合物的结合速率(kon-r)快。在另一个实施方案中，结合TNFR1的化合物-三聚体复合物的解离速率(koff-r)快于化合物与TNFα三聚体结合的解离速率(koff-c)。在另一个实施方案中，与TNFα三聚体(kon-c)结合的化合物的结合速率比结合TNFR1的化合物三聚体复合物的结合速率(kon-r)更快，并且化合物-三聚体复合物与TNFR1(koff-r)的结合比化合物与TNFα三聚体(koff-c)结合的解离速率快。在一个优选的实施方案中，用于结合TNFα三聚体的化合物的KD-c值低于用于结合TNFR1的化合物-三聚体复合物的KD-r值，即化合物对三聚体的亲和力高于化合物三聚体复合物具有受体。
化合物-三聚体复合物和TNFα三聚体与TNFR1的kon-r，koff-r和KD-r值可以使用任何适当的技术，例如表面等离子体共振，质谱和等温量热法来测定，如这里的例子。在测试化合物存在下TNFα与其受体结合的KD-r值可以是1μM，100nM，10nM，5nM，1nM，100pM，10pM或更少。在一个优选的实施方案中，在受试化合物存在下(即在化合物-三聚体复合物中)，TNFα与其受体结合的KD-r值是1nM或更小。在更优选的实施方案中，用于结合TNFR1的化合物-三聚体复合物的KD-r值小于600pM，更优选小于500pM，小于400pM，小于300pM，小于200pM，小于100pM或小于50微米。在最优选的实施方案中，用于结合TNFR1的化合物-三聚体复合物的KD-r值小于200pM。对于本发明的不对称TNFα三聚体与对称TNFα三聚体可以类似地进行。
通过本发明的方法鉴定的化合物可以通过测定来鉴定，所述测定包括测定TNFα样品和化合物中三聚体形式的TNFα的KD-r；比较样品中TNFα的三聚体形式的KD-r与对照样品；并选择本发明的化合物。
当以化合物-三聚体复合物形式的TNFα与受体结合时，通过本发明的方法鉴定的化合物可以完全或部分地抑制通过TNFR1的信号传导。该化合物可以起到减少至少10％，20％，30％，40％，50％，60％，70％，80％，90％或100％的通过TNFR1的信号传导的作用。可通过任何适当的技术来测量信号水平的任何改变，包括通过碱性磷酸酶或萤光素酶测量报告基因活性，使用机器如Cellomics Arrayscan的NF-κB易位，下游效应物的磷酸化，信号分子的募集或细胞死亡。
当化合物-三聚体复合物形式的TNFα与受体结合时，通过本发明方法鉴定的化合物可以拮抗至少一种通过TNFR1的信号传导的下游作用。这些效应在本文中讨论并包括TNFα诱导的IL-8，IL17A/F，IL2和VCAM产生，TNFα诱导的NF-κB活化和嗜中性粒细胞募集。本领域已知标准技术用于测量TNFα的下游效应。通过本发明的方法鉴定的化合物可拮抗至少1，2，3，4，5，10或达到通过TNFR1的信号传导的所有下游效应。
可以使用标准术语例如IC50或半数有效浓度(EC 50)值来量化通过本发明的方法鉴定的化合物的活性。IC50值代表50％抑制特定生物或生物化学功能所需的化合物的浓度。EC50值表示其最大效应的50％所需的化合物的浓度。通过本发明的方法鉴定的化合物可具有500nM，400nM，300nM，200nM，100nM，90nM，80nM，70nM，60nM，50nM，40nM，30nM，20nM，10nM，5nM，1nM，100pM或100nM的IC50或EC 50值减。可以使用任何适当的技术测量IC50和EC 50值，例如可以使用ELISA定量细胞因子的产生。然后可以使用也被称为S形剂量响应模型的标准4-参数逻辑模型生成IC50和EC 50值。
抗体测定
用于本发明分析的抗体是：
·具有序列SEQ ID NO：26的轻链和序列SEQ ID NO：27的重链的CA185_1979；或
·具有序列SEQ ID NO：11的轻链和序列SEQ ID NO：12的重链的CA185_1974。
可以通过例如标准ELISA或Western印迹来测试本发明的抗体与化合物-三聚体复合物的结合。也可以使用ELISA测定来筛选与靶蛋白质显示出阳性反应性的杂交瘤。还可以通过监测抗体与表达靶蛋白的细胞的结合(例如通过流式细胞术)来确定抗体的结合选择性。
本发明的抗体选择性地(或具体地)识别至少一种化合物-三聚体复合物，即化合物-三聚体复合物内的表位(或本发明的不对称TNFα三聚体内的表位)。当抗体或其它化合物以优先或高亲和力与其选择性结合的蛋白质“结合”或“选择性识别”蛋白质时，其与其它蛋白质基本上不结合或以低亲和力结合。本发明抗体对化合物-三聚体复合物靶的选择性可以通过确定抗体是否与其它相关化合物-三聚体复合物结合或是否区分它们来进一步研究
本发明的抗体可以特异性地(或选择性地)与包含三聚体形式的TNFα(或本发明的不对称TNFα三聚体)的化合物-三聚体复合物结合。
通过特异性(或选择性)，将理解抗体结合感兴趣的化合物-三聚体复合物(或本发明的不对称TNFα三聚体)，与任何其它分子(其可包括测试化合物在不存在测试化合物(对称TNFα)的情况下不存在TNFα三聚体或TNFα三聚体。交叉反应性可以通过本文描述的任何合适的方法来评估。如果抗体与另一分子结合至少约5％，10％，15％，20％，25％，则化合物-三聚体复合物的抗体与化合物-三聚体复合物以外的分子的交叉反应性可被认为是显着的％，30％，35％，40％，45％，50％，55％，60％，65％，70％，75％，80％，85％，90％或100％化合物-三聚体复合物。针对化合物-三聚体复合物特异(或选择性)的抗体可以以小于约90％，85％，80％，75％，70％，65％，60％，55％，50％，45％，40％，35％，30％，25％或20％的结合强度的化合物-三聚体复合物。抗体可以以小于约20％，小于约15％，小于约10％或小于约5％，小于约2％或小于约1％与其结合的强度结合至另一个分子化合物-三聚体复合物。与(i)不存在所述化合物的情况下的TNFα的三聚体形式和/或(ii)不存在TNFα三聚体的情况下的化合物(或不对应的TNFα三聚体相对于对称的TNFα三聚体)。
抗体与化合物-三聚体复合物(或本发明的不对称TNFα三聚体)结合的速率在本文中称为“在”速率“kon-ab”和抗体从化合物-三聚体复合物解离的速率(或本发明的不对称TNFα三聚体)在本文中被称为“关闭”速率或koff-ab。如本文所用，符号“。KD-ab”表示抗体对于化合物-三聚体复合物(或本发明的不对称TNFα三聚体)的结合亲和力(解离常数)。。KD-ab定义为koff-ab/kon-ab。抗体可能具有缓慢的“开”速率，可以通过化合物-三聚体复合物的质谱分析和抗体峰强度以分钟测量。通过在不同的抗体：化合物-三聚体复合物比率下重复该测量，可以估计抗体的。KD-ab值。
抗体与化合物-三聚体复合物(或本发明的不对称TNFα三聚体)结合的。KD-ab值可以为至少约1.5倍，2倍，3倍，4倍，5倍，10倍，20倍，30倍，40倍，50倍，60倍，70倍，80倍，90倍，100倍，200倍，300倍或400倍或更低的抗体的。KD-ab值在不存在三聚体TNFα的情况下，化合物不存在时的三聚体TNFα(对称TNFα)和/或抗体与化合物结合的。KD-ab值。结合化合物-三聚体复合物(或本发明的不对称TNFα三聚体)的抗体的。KD-ab值可以降低至少约10倍，至少约100倍，至少约200倍，至少约300乘以测试化合物不存在时与TNFR1受体结合的TNFα三聚体的。KD-ab值，即抗体对化合物-三聚体复合物(或本发明的不对称TNFα三聚体)的结合亲和力通常至少增加(或者在不存在所述化合物的情况下所述抗体对所述三聚体TNFα的结合亲和力)的约10倍，合适地至少约100倍，更合适地至少约200倍，最合适地至少约300倍对称TNFα)和/或在不存在三聚体TNFα的情况下抗体对化合物的结合亲和力。
结合亲和力可以以结合亲和力(。KD-ab)的形式给出，并且可以以任何合适的单位给出，例如μM，nM或pM。。KD-ab值越小，抗体与化合物-三聚体复合物(或本发明的不对称TNFα三聚体)的结合亲和力越大。
抗体与化合物-三聚体复合物(或本发明的不对称TNFα三聚体)结合的抗体的。KD-ab值可以是至少约1.5倍，2倍，3倍，4倍，5倍，10倍，20倍，30倍，40倍，50倍，60倍，70倍，80倍，90倍，100倍或甚至更低的结合三聚体TNFα的抗体的。KD-ab值(或对称的TNFα)和/或在不存在三聚体TNFα的情况下与抗体结合的。KD-ab值。
与不存在该化合物的情况下结合三聚体TNFα的抗体的。KD-ab值(与本发明的不对称TNFα三聚体结合)相比，抗体的。KD-ab值降低或对称TNFα)和/或抗体在不存在三聚体TNFα时与化合物结合的。KD-ab值可能是由于结合化合物-三聚体的抗体的结合速率(kon-ab)增加(对称TNFα)和/或抗体在不存在三聚体TNFα的情况下与化合物结合的情况下，与结合三聚体TNFα的抗体相比，本发明的复合物(或本发明的不对称TNFα三聚体)和/或与不存在化合物(或对称TNFα三聚体)时结合三聚体TNFα的抗体和/或在不存在三聚体的情况下与化合物结合的抗体相比，释放速率(koff-ab)TNFα。
结合于化合物-三聚体复合物(或本发明的不对称TNFα三聚体)的抗体的结合速率(kon-ab)通常与不存在化合物的情况下结合三聚体TNFα的抗体的结合速率(对称的TNFα)和/或在不存在三聚体TNFα的情况下与抗体结合的抗体。结合化合物-三聚体复合物(或本发明的不对称TNFα三聚体)的抗体的解离速率(koff-ab)通常与不存在所述化合物的情况下与三聚体TNFα结合的抗体的解离速率对称的TNFα)和/或在不存在三聚体TNFα的情况下与抗体结合的抗体。最典型地，与化合物-三聚体复合物(或本发明的不对称TNFα三聚体)结合的抗体的结合速率(kon-ab)增加，并且与化合物结合的抗体的解离速率(koff-ab)(或本发明的不对称TNFα三聚体)与在不存在化合物(或对称TNFα)的情况下结合三聚体TNFα的抗体和/或在不存在三聚体的情况下与化合物结合的抗体相比，TNFα。
与本发明的化合物-三聚体复合物(或本发明的不对称TNFα三聚体)结合的抗体的kon-ab值可以增加至少约1.5倍或至少2倍并且典型地至少约3倍在不存在化合物(对称TNFα)的情况下结合三聚体TNFα的抗体的kon-ab值和/或在不存在三聚体TNFα和/或抗体结合的koff-ab值的情况下与化合物结合的抗体与本发明的化合物-三聚体复合物(或本发明的不对称TNFα三聚体)相比，可以降低至少约2倍，至少约10倍，至少约20倍，至少约30倍，至少约40倍，至少约50倍，至少约60倍，至少约70倍，至少约80倍，更合适的是至少约90倍的抗体结合的koff-ab值在不存在化合物(对称TNFα)的情况下与三聚体TNFα和/或与t中化合物结合的抗体他没有三聚TNFα。
kon-ab，koff-ab和。KD-ab值可以使用任何适当的技术来确定，例如表面等离子体共振，质谱和等温量热法。
结合化合物-三聚体复合物(或本发明的不对称TNFα三聚体)的抗体的。KD-ab值可以是1nM，900pM，700pM，500pM，100pM，10pM或更小1pM)。本发明的抗体将理想地以高亲和力结合本发明的化合物-三聚体复合物(或本发明的不对称TNFα三聚体)，例如在皮摩尔范围内。与化合物-三聚体复合物(或本发明的不对称TNFα三聚体)结合的抗体的。KD-ab值可以是1nM或更小，900pM或更小，700pM或更小，500pM或更小，400pM或更小，300pM或更少，200pM或更少，100pM或更少，90pM或更少，80pM或更少，70pM或更少，60pM或更少，50pM或更少，40pM或更少，30pM或更少，20pM或更少，10pM或更少(再次，下降到约1pM)。
抗体可以通过常规方法重组生产。
术语“表位”是被抗体结合的抗原的区域。表位可以被定义为结构或功能。功能表位通常是结构表位的子集，并具有直接有助于相互作用亲和力的那些残基。表位也可以是构象的，即由非线性氨基酸组成。在某些实施方案中，表位可以包括决定簇，所述决定簇是分子的化学活性表面基团，例如氨基酸，糖侧链，磷酰基或磺酰基，并且在某些实施方案中，可以具有特定的三维结构特征，和/或具体的收费特点。
通过使用本领域已知的常规方法，人们可以容易地确定抗体是否与参考抗体结合相同的表位或竞争结合参考抗体。例如，为了确定测试抗体是否与本发明的参考抗体结合相同的表位，允许参考抗体在饱和条件下与蛋白质或肽结合。接下来，评估测试抗体结合蛋白质或肽的能力。如果测试抗体能够在与参考抗体饱和结合后与蛋白质或肽结合，则可以得出结论：测试抗体与参考抗体结合不同的表位。另一方面，如果测试抗体在与参照抗体饱和结合后不能结合蛋白质或肽，那么测试抗体可以结合与本发明参照抗体结合的表位相同的表位。
为了确定抗体是否与参考抗体竞争结合，上述结合方法在两个方向上进行。在第一方向，允许参考抗体在饱和条件下结合蛋白质/肽，然后评估测试抗体与蛋白质/肽分子的结合。在第二方向上，使测试抗体在饱和条件下与蛋白质/肽结合，接着评估参考抗体与蛋白质/肽的结合。如果在两个方向上只有第一个(饱和的)抗体能够与蛋白质/肽结合，则推断测试抗体和参考抗体竞争结合蛋白质/肽。如本领域技术人员将理解的，与参照抗体竞争结合的抗体可能不一定与参照抗体结合相同的表位，但可通过结合重叠或相邻表位来空间阻断参照抗体的结合。
如果两种抗体竞争性抑制(阻断)另一种与抗原的结合，则两种抗体结合相同或重叠的表位。也就是说，如在竞争性结合测定中所测量的，1倍，5倍，10倍，20倍或100倍过量的一种抗体抑制另一种抗体的结合至少50％，75％，90％或甚至99％(参见例如Junghans等，Cancer Res，1990：50：1495-1502)。或者，如果基本上抗原中降低或消除一种抗体的结合的所有氨基酸突变减少或消除另一种抗体的结合，则两种抗体具有相同的表位。如果一些降低或消除一种抗体结合的氨基酸突变降低或消除另一种抗体的结合，则两种抗体具有重叠的表位。
然后可进行额外的常规实验(例如肽突变和结合分析)以确认观察到的测试抗体的结合缺乏是否实际上是由于与参考抗体结合相同的表位，或者如果空间阻断(或另一种现象)是造成缺乏观察结合的原因。可以使用ELISA，RIA，表面等离子体共振，流式细胞术或本领域可用的任何其他定量或定性抗体结合测定来进行这种实验。
本发明的抗体可用于鉴定如本文所述的本发明的化合物(或本发明的复合物或本发明的TNFα三聚体)。本发明的抗体也可以用作靶向参与生物标志物。目标接合生物标志物可用于检测接合，即配体与感兴趣的目标的结合。在目前情况下，本发明的抗体仅结合本发明化合物与TNFα的三聚体形式的复合物。因此，如果本发明的抗体能够与化合物-三聚体复合物结合，则证明配体(化合物)已经结合到感兴趣的靶标(TNFα三聚体)上。如本文所述，可以修饰本发明的抗体以添加可检测标记。因此，可以使用这样的抗体检测本发明的化合物与目标TNFα的接合。
使用本发明的抗体作为靶标接合生物标志物可能在临床或临床前环境中有用，其中样品可以取自根据本发明治疗的受试者。从受试者获得的样品可以用本发明的抗体处理以确定用于治疗受试者的化合物是否与靶标TNFα结合(或者已经在受试者中形成了本发明的不对称TNFα三聚体)。从受试者获得的样品可以是任何合适的组织或流体，例如血液，血浆或尿液。受试者可以是哺乳动物，通常是人。
因此，本发明提供了本发明的抗体作为靶标接合生物标志物用于检测包含三聚体TNFα蛋白质的化合物-三聚体复合物(或已形成本发明的不对称TNFα三聚体)由此化合物-三聚体复合物(或本发明的不对称TNFα三聚体)结合TNFR1，并且拮抗由受试者获得的样品中由三聚体通过受体诱导的信号传导。
类似地，本发明提供了检测化合物与三聚体TNFα的靶向接合的方法，由此化合物-三聚体复合物结合TNFR1并拮抗由三聚体通过受体诱导的信号传导，所述方法包括：
(a)从施用所述化合物的受试者获得样品；
(b)使本发明的抗体与所述样品和对照样品接触，其中所述抗体是可检测的；
(c)确定所述可检测抗体与所述样品和所述对照样品的结合量，
其中所述可检测抗体与所述样品的结合大于所述可检测抗体与所述对照样品的结合指示所述化合物与所述三聚TNFα的靶向接合。
检测抗体和测量抗体与靶结合的量的方法在本领域中是公知的。通常，抗体可以被标记。这种标记包括酶，生物素/链霉抗生物素蛋白，荧光蛋白和荧光染料。
例如，可以通过免疫测定法来测量抗体与靶标的结合。免疫分析包括Western印迹，ELISA，免疫荧光，免疫组织化学和流式细胞术。可以使用任何适当的技术来测量抗体与TNFα的结合。
在上述方法中，将可检测抗体与已施用化合物的受试者的样品的结合与抗体与对照样品的结合进行比较。对照样品可以是任何适当的样品。对照样品通常是“阴性对照”，其代表在不存在化合物的情况下抗体与TNFα的结合。例如，可以在施用化合物之前从患者获得样品。控制还可以基于先前确定的测量，例如，来自不同受试者的多个样品中没有化合物。可以使用约5，10，20，50或100个受试者的测量值来确定对照值。控制可以是平均值，或者是获得的所有值的范围。
实验条件例如来自施用化合物的受试者的样品和对照样品的检测方法是相同的。在这两种情况下抗体也是相同的。
与抗体与对照样品的结合相比，可检测抗体对来自施用化合物的患者的样品的较高结合(增加的结合)表明该化合物与三聚TNFα的靶向结合。换句话说，相对于对照来说，来自施用化合物的患者的样品的等同或较低结合(降低的结合)表明所述化合物没有靶向接合。换句话说，两个金额没有显着差异表示没有目标参与。
技术人员可以容易地确定何时相对于对照存在增加的结合。例如，当控制是数据范围时，可以基于数据的扩展，控制数据与检测到的样本中抗体的检测结合之间的差异以及计算的置信度来确定目标参与。当检测到的样品的结合高于在任何阴性对照中检测到的结合的最大量时，也可能识别目标接合。
如果抗体的结合相对于对照范围的最高量增加约30％或更多，则可以检测目标接合。如果抗体的结合相对于对照范围增加约40％或更多，或约50％或更多，则也可以检测目标接合。当对照是平均值时，这同样适用，或者在施用化合物之前基于来自患者的样品的单一值。相对于对照百分比的增加当然没有上限。
本发明的抗体可用于筛选引发三聚TNFα构象变化的化合物(产生本发明的不对称TNFα三聚体)，其中所述构象变化拮抗TNFR1与三聚TNFα结合的信号传导。
进一步的抗体测定
如本文所述，本发明提供了选择性结合本文所述的至少一种本发明所述的化合物-三聚体复合物(或本发明的不对称TNFα三聚体)的抗体，所述抗体相对于单独与化合物的结合或在不存在所述化合物(或对称的TNFα)。这些抗体可用于鉴定具有相同性质的其他化合物或化合物种类。
因此，本发明提供了用于鉴定本发明化合物的测定法，其包括以下步骤：
a)进行结合测定以测量测试化合物-三聚体复合物与本发明的抗体的结合亲和力；
b)将步骤(a)中测量的结合亲和力与已知以高亲和力结合步骤(a)中提及的抗体的不同化合物-三聚体复合物的结合亲和力；和
c)如果根据步骤(b)中提到的比较考虑其测量的结合亲和力是可接受的，则选择存在于步骤(a)的化合物-三聚体复合物中的化合物。
可以理解，上述步骤(b)中提到的“不同的”化合物-三聚体复合物一般是含有与步骤(a)的化合物-三聚体复合物相同的三聚体的复合物，但是是不同的化合物。该化合物可以是化合物(1)-(65)中的任何一种。
步骤(c)中的“可接受”是指步骤(a)中所述的化合物-三聚体复合物的结合亲和力与步骤(b)中所述的不同化合物-三聚体复合物的结合亲和力大致相当。所述抗体与所述复合物的选择性结合通常相对于所述抗体与TNFα在不存在所述化合物的情况下的结合或不存在TNFα的情况下的所述化合物的结合来测量。
步骤(a)中涉及的化合物-三聚体复合物的结合亲和力通常优于步骤(b)中涉及的不同化合物-三聚体复合物的结合亲和力。适当地，步骤(a)中涉及的化合物-三聚体复合物的结合亲和力相对于步骤(b)中涉及的不同化合物-三聚体复合物的结合亲和力将在10倍，20倍，50倍，100倍，200倍或500倍。
可以使用本发明的抗体来测定化合物文库。文库化合物可以在存在和不存在TNFα的情况下与所述抗体一起温育。仅在TNFα和化合物两者存在下形成与本发明抗体结合的化合物-三聚体复合物的一部分的化合物可能是具有与本文所述化合物相同活性的候选物。然后可以使用本文公开的测定来验证测试化合物是否是如本文所述的化合物。
本发明的一种或多种抗体可以用于测定中。能够结合本发明的任何化合物与TNFα的复合物的通用抗体可以用于本发明的抗体测定中。
本发明的抗体测定可以是能够在短时间内筛选大量测试化合物以鉴定本发明化合物的高通量测定。
TNFα及其受体可以纯化或以混合物存在，例如在培养细胞，组织样品，体液或培养基中。可以开发定性或定量分析，其中后者可用于确定测试化合物对TNFα三聚体形式的结合参数(亲和常数和动力学)，以及化合物-三聚体复合物对TNFR1的结合参数。
包含TNFα和化合物的样品可以进一步包含去稳定剂。去稳定剂(也称为离液剂)包括低摩尔浓度(例如1M)尿素，胍或乙腈，这些试剂的高浓度(例如6M或更高)将导致TNFα三聚体的完全解离和组成性TNFα单体的解折叠亚基。去稳定剂可以是DMSO，通常浓度为5％，10％或更高。
测试化合物可以具有上面讨论的任何/全部性质。
治疗适应症
TNFα是TNF超家族的原型成员。TNFα是介导免疫调节和炎症反应的多效细胞因子。在体内，已知TNFα参与对细菌，寄生虫和病毒感染的应答。具体而言，已知TNFα在类风湿性关节炎(RA)，炎症性肠病(包括克罗恩氏病)，牛皮癣，阿尔茨海默病(AD)，帕金森病(PD)，疼痛，癫痫，骨质疏松症，哮喘，脓毒症，发热，系统性红斑狼疮(SLE)和多发性硬化症(MS)和癌症。也已知TNFα在肌萎缩侧索硬化(ALS)，缺血性中风，免疫复合物介导的肾小球肾炎，狼疮性肾炎(LN)，抗中性粒细胞胞质抗体(ANCA-)相关肾小球肾炎，微小病变，糖尿病性肾病(DN)，急性肾损伤(AKI)，梗阻性尿路病，肾同种异体移植排斥，顺铂诱导的AKI和梗阻性尿路疾病。
可以使用(直接或间接)由本发明的方法或本发明的复合物或本发明的TNFα三聚体鉴定的化合物来治疗，预防或改善可以通过常规方法治疗，预防或改善的任何病症TNFα拮抗剂。通过本发明的方法鉴定的化合物或本发明的复合物或本发明的TNFα三聚体可以单独使用或与常规TNFα拮抗剂组合使用。根据本发明，原则上可以部分或全部由TNFα通过TNF受体的致病性信号传导产生的任何病症得到治疗，预防或改善。TNFα通过TNF受体的致病性信号传导包括通过超过正常生理信号传导的TNF受体的信号传导增加，通过正常启动的TNF受体的信号传导，但是其不能响应正常的生理信号而停止，TNF受体在正常生理范围内，但是由非生理手段启动。
通过本发明方法鉴定的与TNFα(以及本发明的复合物和本发明的TNFα三聚体)相互作用的化合物因此有益于治疗和/或预防各种人类疾病。这些包括自身免疫和炎性疾病；神经和神经退行性疾病；疼痛和伤害性疾病；和心血管疾病。
炎性和自身免疫性疾病包括全身性自身免疫性疾病，自身免疫性内分泌疾病和器官特异性自身免疫性疾病。系统性自身免疫病症包括系统性红斑狼疮(SLE)，牛皮癣，血管炎，多肌炎，硬皮病，多发性硬化症，强直性脊柱炎，类风湿性关节炎和舍格伦综合征。自身免疫性内分泌疾病包括甲状腺炎。器官特异性自身免疫性疾病包括艾迪生病，溶血性或恶性贫血，肾小球性肾炎(包括古德帕斯彻氏综合征)，格雷夫斯病，特发性血小板减少性紫癜，胰岛素依赖性糖尿病，青少年糖尿病，葡萄膜炎，炎性肠病(包括克罗恩氏病和溃疡性结肠炎)，天疱疮，特应性皮炎，自身免疫性肝炎，原发性胆汁性肝硬化，自身免疫性肺炎，自身免疫性心脏炎，重症肌无力，自发性不育，骨质疏松症，哮喘和肌营养不良(包括杜氏肌营养不良)。
神经和神经退行性疾病包括阿尔茨海默病，帕金森病，亨廷顿舞蹈病，中风，肌萎缩性侧索硬化，脊髓损伤，头部创伤，癫痫发作和癫痫。
心血管疾病包括血栓形成，心脏肥大，高血压，心脏不规则收缩(例如在心力衰竭期间)和性功能障碍(包括勃起功能障碍和女性性功能障碍)。
特别地，通过本发明的方法鉴定的化合物或本发明的复合物(或本发明的TNFα三聚体)可用于治疗或预防炎性病症，CNS病症，免疫病症和自身免疫疾病，疼痛，骨质疏松症，发烧和器官移植排斥反应。在一个优选的实施方案中，通过本发明的方法鉴定的化合物或本发明的复合物(或本发明的TNFα三聚体)可用于治疗或预防类风湿性关节炎，炎症性肠病(包括克罗恩氏病)，牛皮癣，阿尔茨海默病帕金森病，癫痫，哮喘，败血症，系统性红斑狼疮，多发性硬化症，哮喘，鼻炎，癌症和骨质疏松症。在另一个优选的实施方案中，通过本发明的方法鉴定的化合物或本发明的复合物(或本发明的TNFα三聚体)可用于治疗或预防类风湿性关节炎(RA)，非特异性炎性关节炎，糜烂性骨病，软骨变性和/或破坏，青少年炎性关节炎，斯蒂尔病(青少年和/或成人发病)，幼年特发性关节炎，少年特发性关节炎(寡关节和多关节形式)，炎性肠病(包括克罗恩病，溃疡性结肠炎，(AD)，白塞病，帕金森病(PD)，肌萎缩性侧索硬化症(ALS)，缺血性中风，疼痛，癫痫，骨质疏松症，骨质减少症，骨质疏松症，慢性病贫血，恶病质，糖尿病，血脂异常，代谢综合征，哮喘，慢性阻塞性气道(或pu(SLE)，多发性硬化症(MS)免疫复合物介导的肾小球肾炎，狼疮性肾炎(LN)，抗中性粒细胞胞质抗体(ANCA-)相关的肾小球肾炎，最小改变疾病，糖尿病性肾病(DN)，急性肾损伤(AKI)，梗阻性尿路病，肾同种异体移植排斥，顺铂诱导的AKI和梗阻性尿路病，眼病(包括糖尿病性视网膜病，糖尿病性黄斑水肿，早产儿视网膜病，年龄相关性黄斑变性，，增生性和/或非增殖性视网膜病，包括新血管形成，视网膜静脉阻塞，各种形式的葡萄膜炎和角膜炎的角膜血管化)，甲状腺炎，纤维化疾病包括各种形式的肝纤维化，各种形式的肺纤维化，系统性硬化症，硬皮病，癌症相关的并发症(包括骨骼并发症，恶病质，d贫血)。
药物组合物，剂量和剂量方案
通过本发明的方法鉴定的化合物和本发明的化合物-三聚体复合物与本发明的TNFα三聚体通常将与药学上可接受的载体一起配制成药物组合物。
如本文所用，“药学上可接受的载体”包括生理上相容的任何和所有溶剂，分散介质，包衣，抗细菌剂和抗真菌剂，等渗剂和吸收延迟剂等。该载体可适用于肠胃外，例如，静脉内，肌肉内，皮内，眼内，腹膜内，皮下，脊髓或其他肠胃外施用途径，例如通过注射或输注。或者，载体可适用于非肠胃外给药，例如局部给药，表皮给药或粘膜给药途径。在一个优选的实施方案中，载体适合于口服给药。取决于施用途径，拮抗剂可以被包被在材料中以保护化合物免于可能使化合物失活的酸和其它自然条件的作用。
本发明的药物组合物可以包含一种或多种药学上可接受的盐。“药学上可接受的盐”是指保留母体化合物的所需生物学活性并且不赋予任何不希望的毒理学作用的盐。这种盐的例子包括酸加成盐和碱加成盐。
优选的药学上可接受的载体包含水性载体或稀释剂可以在本发明的药物组合物中使用的合适的含水载体的例子包括水，缓冲水和盐水。其它载体的实例包括乙醇，多元醇(如甘油，丙二醇，聚乙二醇等)及其合适的混合物，植物油如橄榄油和可注射的有机酯如油酸乙酯。在许多情况下，优选在组合物中包含等渗剂，例如糖，多元醇如甘露醇，山梨糖醇或氯化钠。
治疗组合物通常在制造和储存条件下必须是无菌的和稳定的。该组合物可以配制成适合于高药物浓度的溶液，微乳剂，脂质体或其它有序结构。
本发明的药物组合物可以包含另外的活性成分。
同样在本发明的范围内的是试剂盒，其包含通过本发明的方法鉴定的化合物和本发明的复合物(或本发明的TNFα三聚体)以及使用说明书。试剂盒可以进一步含有一种或多种另外的试剂，如上面讨论的另外的治疗剂或预防剂。
通过本发明的方法鉴定的化合物和本发明的化合物-三聚体复合物(或本发明的TNFα三聚体)或其制剂或组合物可以用于预防性和/或治疗性治疗。
在治疗应用中，以足以治愈，减轻或部分阻滞的量(直接或间接)向已经患有如上所述的病症或病症的受试者施用化合物和化合物-三聚体复合物(或本发明的TNFα三聚体)病症或其一种或多种症状。这种治疗可能导致疾病症状的严重程度降低，或无症状期的频率或持续时间增加。足以实现这一点的量被定义为“治疗有效量”。
在预防性应用中，将制剂以足以预防或减轻病症或其一种或多种症状的后续影响的量施用于处于如上所述的病症或病症风险的受试者。足以实现这一点的量被定义为“预防有效量”。每个目的的有效量将取决于疾病或受伤的严重程度以及受试者的体重和一般状态。
施用对象可以是人类或非人类的动物。术语“非人动物”包括所有的脊椎动物，例如哺乳动物和非哺乳动物，如非人灵长类动物，绵羊，狗，猫，马，牛，鸡，两栖动物，爬行动物等。
通过本发明的方法鉴定的化合物或本发明的化合物-三聚体复合物(或本发明的TNFα三聚体)可以通过一种或多种施用途径，使用本领域已知的多种方法中的一种或多种。如本领域技术人员将理解的，施用的途径和/或模式将取决于期望的结果而变化。本发明的化合物或化合物-三聚体复合物的给药途径的实例包括静脉内，肌肉内，皮内，眼内，腹膜内，皮下，脊髓或其他肠胃外给药途径，例如通过注射或输注。本文使用的短语“肠胃外施用”是指除肠内和局部施用之外的施用模式，通常通过注射。或者，通过本发明的方法鉴定的化合物或本发明的化合物-三聚体复合物(或本发明的TNFα三聚体)可以通过非肠胃外途径施用，例如局部施用，表皮施用或粘膜施用途径。在一个实施方案中，通过本发明的方法鉴定的化合物或本发明的化合物-三聚体复合物(或本发明的TNFα三聚体)用于口服给药。间接施用也可以如本文所述进行。
由本发明的方法鉴定的化合物或本发明的化合物-三聚体复合物(或本发明的TNFα三聚体)的合适剂量可以由熟练的医师确定。本发明药物组合物中活性成分的实际剂量水平可以变化，以获得有效实现对于特定患者，组合物和给药模式的所需治疗反应的活性成分的量，而无需对病人有毒。所选择的剂量水平将取决于各种药物代谢动力学因素，包括所用本发明特定组合物的活性，给药途径，给药时间，所用具体化合物的排泄速率，治疗，与所用特定组合物联合使用的其他药物，化合物和/或材料，所治疗患者的年龄，性别，体重，状况，一般健康状况和既往病史，以及医学领域众所周知的类似因素。
合适的剂量可以是例如约0.01μg/kg至约1000mg/kg体重，典型地约0.1μg/kg至约100mg/kg体重的待治疗患者的范围。例如，合适的剂量可以是每天约1μg/kg至约10mg/kg体重或每天约10μg/kg至约5mg/kg体重。
可以调整剂量方案以提供最佳的期望响应(例如治疗响应)。例如，可以施用单一剂量，可以随着时间的推移施用几个分开的剂量，或者可以根据治疗情况的紧急情况的指示按比例减少或增加剂量。本文使用的剂量单位形式是指适合作为待治疗对象的单位剂量的物理上分立的单位；每个单位含有预定量的活性化合物，其经计算与所需的药物载体结合产生所需的治疗效果。
可以单剂量或多剂量给药。多个剂量可以通过相同或不同的途径和相同或不同的位置施用。或者，剂量可以通过缓释制剂，在这种情况下，需要较少的频率给药。剂量和频率可以根据拮抗剂在患者中的半衰期和期望的治疗持续时间而变化。
如上所述，通过本发明的方法鉴定的化合物或本发明的化合物-三聚体复合物(或本发明的TNFα三聚体)可以与一种或多种其它治疗剂共同施用。例如，其他药剂可以是镇痛剂，麻醉剂，免疫抑制剂或抗炎剂。
两种或更多种药剂的联合给药可以以许多不同的方式实现。两者可以一起施用于单一组合物中，或者可以作为组合疗法的一部分以分开的组合物施用。例如，可以在另一个之前，之后或同时给药。
晶体，使用它们的结构确定以及使用3D模型的方法
本发明人已经发现本发明的TNFα三聚体(或本发明的复合物)可以用适用于X射线衍射的新晶体和三聚体的结构测定进行结晶。利用可获得的结构信息的量，首次本发明允许使用分子技术来鉴定，选择和设计能够结合到在(a)的空腔处的化学实体(包括抑制剂，包括包含本发明的药效团的那些抑制剂)本发明的TNFα三聚体的中心。
因此，TNFα三聚体晶体具有空间群P 21 21 21(最常见)，P 21 21 2或P 1 21 1.所述晶体可根据本领域已知的任何方法制备，例如实施例18(或其中的微小变化)。
在一个实施方案中，晶体包含sTNFα，特别是人sTNFα，例如包含根据SEQ ID NO：35或36的氨基酸残基或由其组成的多肽。
在本发明的实施方案中，晶体可以具有单位晶胞尺寸a＝54埃±1-2埃，b＝81埃±1-2埃，α＝90埃，β＝90°，γ＝90°(最常见)或a＝47.7±1-2，b＝95.8°±1-2°，c＝100.7°±1-2，α＝90°，β＝99.1度，伽马＝90度。
一方面，晶体包含化合物(1)-(64)中的任一种。
晶体可以用于确定其中本发明的复合物或TNFα三聚体的结构，并且所得到的3D模型可以储存在计算机上，并且可以用于确定本发明复合物内相似功能化合物的方法。
因此，进一步提供了一种包括可执行代码的计算机，
a)使用根据化合物1.pdb至化合物64.pdb中的任一种的TNFα三聚体的结构坐标作为不对称TNFα三聚体的三维模型；
b)在三维模型中分析三聚体中心的结合口袋；和
c)在计算机文库中筛选适合所述结合位点的小分子。
在另一个实施方案中，提供鉴定apo TNFα三聚体的潜在抑制剂的方法，其包括以下步骤：
a)使用根据化合物1.pdb至化合物64.pdb中的任何一种的TNFα三聚体的结构坐标来产生不对称TNFα三聚体的三维模型；
b)在三维模型中鉴定三聚体中心的结合口袋的残基；
c)使用结合位点残基产生特定的3-D靶标；
d)使用特定的3-D靶设计或选择潜在的TNFα三聚体抑制剂；
e)获得潜在的抑制剂；和
f)体外使潜在抑制剂与apo TNFα三聚体接触，以确定所述潜在抑制剂与所述apoTNFα三聚体相互作用的能力，
由此相互作用的能力指示所述apo TNFα三聚体的所述潜在抑制剂被确定。
还提供了一种设计结合apo TNFα三聚体的化合物的方法，包括以下步骤：
a)使用根据化合物1.pdb至化合物64.pdb中的任一种的TNFα三聚体的原子坐标来建立TNFα三聚体中心处的结合口袋的三维计算机模型；
b)评估化合物和结合口袋之间的立体化学互补性；
c)通过观察影响蛋白质/化合物缔合的蛋白质或化合物的变化，以迭代方法优化立体化学互补性；和
d)设计优化所述蛋白质/化合物立体化学互补性的化合物。
还提供了用于鉴定与apo TNFα三聚体中心处的结合口袋相互作用的候选抑制剂的方法，其包括以下步骤：
a)获得本发明的晶体；
b)获得步骤a)的晶体的氨基酸的结构坐标；
c)使用步骤b)中产生的氨基酸的结构坐标产生TNFα三聚体的3-D模型，
d)确定来自3-D模型的TNFα三聚体中心处的结合口袋；
e)进行计算机拟合分析以设计或鉴定与结合口袋相互作用的候选抑制剂；和
f)体外设计或鉴定的候选抑制剂与apo TNFα三聚体接触以确定抑制剂对TNFα活性的作用。
还提供了鉴定结合apo TNFα三聚体的化合物的方法，包括以下步骤：
a)使用本发明的晶体获得TNFα三聚体的3-D分子模型；
b)在合理的药物设计方法中使用a)的模型来鉴定可结合于TNFα三聚体中心的候选化合物；和
c)在步骤b)中鉴定的结合候选化合物存在下测定TNFα活性，从而鉴定结合apoTNFα三聚体的化合物。
通常提供本发明晶体(或化合物1.pdb至化合物64.pdb中任何化合物的结构坐标)在鉴定apo TNFα三聚体抑制剂中的用途。
本文描述了测定化合物是否结合本发明的复合物或TNFα的测定法。
根据本发明结合或抑制TNFα结合口袋的化学实体的设计通常涉及两个因素的考虑。首先，实体必须能够在身体和结构上与部分或全部绑定口袋相关联。在该关联中非共价分子相互作用包括氢键，范德华相互作用，疏水相互作用和静电相互作用。
其次，实体必须能够呈现使其能够直接与TNFα结合口袋缔合的构象。虽然实体的某些部分不会直接参与这些关联，但实体的这些部分仍可能影响分子的整体构象。这反过来可能会影响效力。这样的构象要求包括化学实体相对于结合口袋的全部或一部分的总体三维结构和取向，或包含与TNFα结合口袋直接相互作用的几种化学实体的实体的官能团之间的间隔。
可以在其实际合成之前分析化学实体对TNFα结合口袋的潜在抑制或结合作用，并通过使用计算机模拟技术进行测试。如果给定实体的理论结构暗示它与绑定口袋之间的交互和关联不足，那么实体的测试就会被消除。然而，如果计算机建模表明有强烈的相互作用，则可以合成化合物并测试其结合口袋的能力。这可以通过使用本文描述的测定来测试分子抑制TNFα蛋白的能力来实现。以这种方式，可以避免无效化合物的合成。
可通过一系列步骤计算评估TNFα结合口袋的潜在抑制剂，其中筛选化学个体或片段并选择其结合TNFα结合口袋的能力。
本领域技术人员可以使用几种方法之一来筛选化学实体或片段与TNFα结合口袋缔合的能力。该过程可以基于化合物1.pdb至化合物64.pdb中的TNFα结构坐标或定义由机器可读存储器生成的相似形状的其他坐标，通过目视检查例如计算机屏幕上的TNFα结合口袋开始中。然后可以将选定的片段或化学实体定位在该结合口袋内的各种方向或对接。对接可以使用软件来完成，例如QUANTA[Molecular Simulations，Inc.，San Diego，CA；现在是加利福尼亚州圣迭戈的Accelrys的一部分]和SYBYL[Tripos Associates，St.Louis，MO]，随后是具有标准分子力学力场的能量最小化和分子动力学，例如CHARMm[Accelrys，San Diego，CA]和AMBER。
专门的计算机程序也可以帮助选择碎片或化学实体的过程。这些包括：
1.网格[P.J.Goodford，“用于测定生物重要大分子上能量有利的结合位点的计算程序”，J.Med.Chem。28，第849-857页(1985)]。GRID可从英国牛津的牛津大学获得。
2.MCSS[A.Miranker等人，“绑定站点的功能性地图：多重复制同时搜索方法”。Proteins：Structure，Function and Genetics，11，pp.29-34(1991)]。MCSS可得自Molecular Simulations，San Diego，CA。3.AUTODOCK[D.S.Goodsell等人，“AutomatedDocking of Substrates to Proteins by Simulated Annealing”，Proteins：Structure，Function，and Genetics，8，pp.195-202(1990)]。AUTODOCK可从加利福尼亚州La Jolla的Scripps Research Institute获得。
4.DOCK[I.D.Kuntz等人的“A Geometric Approach to Macromolecule-LigandInteractions”，J.Mol.Biol。Biol，161，第269-288页(1982)]。DOCK可从加州大学旧金山分校获得。
5.GLIDE[Schrodinger，Portland，Oregon 97204，USA；Thomas A.Halgren，RobertB.Murphy，Richard A.Friesner，Hege S.Beard，Leah L.Frye，W.Thomas Pollard和JayL.Banks“滑翔：快速准确对接和评分的新方法”。。Enrichment Factors in DatabaseScreening“，J.Med。47(7)，第1750-1759页(2004)]
一旦选择了合适的化学实体或片段，它们可以被组装成单一化合物或化合物的复合物。在显示在计算机屏幕上的三维图像上，相对于TNFα蛋白质的结构坐标，组装之前可以目视检查片段之间的关系。随后，使用QUANTA(R)和DISCOVERY STUDIO(R)[MolecularSimulations，Inc.，San Diego，CA；现在是加利福尼亚州圣地亚哥市Accelrys的一部分]，SYBYL[Tripos Associates，St.Louis，MO]或MAESTRO[Schrodinger，Portland，Oregon97204，USA]或OPENEYE[版权所有(C)1997-2006，OpenEye科学软件，SanteFe，NM 87508，USA]。
有助于本领域技术人员以逐步方式(包括一次片段或化学实体)构建TNFα结合袋的抑制剂的有用程序包括：
1.CAVEAT[P.A.Bartlett等人，“CAVEAT：A Program to Facilitate Structure-Derived Design of Biologically Active Molecules”，Molecular Recognition inChemical and Biological Problems，Special Pub，Royal Chem.Soc。，78，pp.182-196(1989)；G.Lauri和PABartlett，“CAVEAT：a Program to Facilitate the Design ofOrganic Molecules”，J.Computer.Aided Mol.Des。，8，pp.51-66(1994)]。来自加州大学伯克利分校。
2.3D数据库系统，如ISIS(MDL信息系统，San Leandro，CA)。Y.C.Martin在“药物设计中的3D数据库检索”，J.Med.Chem。35，第2145-2154页(1992)。
3.钩[M.B.Eisen等人的“HOOK：寻找能满足大分子结合位点的化学和空间要求的新型分子结构的程序”，蛋白质：结构，基金，遗传学，19，pp.199-221(1994))]。HOOK可从Molecular Simulations，San Diego，CA获得。
抑制性TNFα结合化合物或其它TNFα结合化合物可以设计为整体或“从头”，使用任何一种片段或化学实体一次一步地构建TNFα结合袋的抑制剂空结合口袋或任选地包含已知抑制剂的一些部分或包含本文所述的药效团。有许多从头配体的设计方法，包括：
1.LUDI[H.-J.Bohm，“计算机程序LUDI：酶抑制剂从头设计的新方法”，J.Comp。援助。Molec。Design，6，pp.61-78(1992)]。LUDI可从加利福尼亚州圣地亚哥的分子模拟公司获得；现在Accelrys，圣地亚哥，加利福尼亚州。
2.传奇[Y.Nishibata等，Tetrahedron，47，p。8985(1991)]。LEGEND可从加利福尼亚州圣地亚哥市的分子模拟公司获得；现在Acclerys，圣迭戈，CA.
3.LEAPFROG[可从密苏里州圣路易斯的Tripos Associates获得]。
4.SPROUT[V.Gillet等人，“SPROUT：结构生成程序”，J.Comput。Aided MolDesign，7，pp.127-153(1993)]。SPROUT可从英国利兹大学获得。
5.NEWLEAD(V.Tschinke和N.C.Cohen，“NEWLEAD Program：A New Method forDesign Design for Candidate Structures from Pharmacophoric Hypotheses”，J.Med.Chem。，36，3863-3870(1993))。
根据本发明也可以使用其它分子模拟技术[参见例如NC Cohen等，“MolecularModeling Software and Methods for Medicinal Chemistry，J.Med.Chem。，33，pp.883-894(1990)；参见MA Navia和MA Murcko的“The Use of Structural Information in DrugDesign”，Current Opinions in Structural Biology，2，pp.202-210(1992)；LMBalbes等，“A Perspective of Modern Methods(计算机辅助药物设计)，“计算化学评论”(Review inComputational Chemistry)，第5卷，KBLipkowitz和DBBoyd编辑，VCH，New York，第337-380页(1994)；也参见WCGuida，-Based Drug Design“，Curr.Opin.Struct.Biology，4，pp.777-781(1994)]。
一旦通过上述方法设计或选择了化学实体，则可以通过计算评估来测试和优化该化学实体可以结合TNFα结合口袋的效率。例如，有效的TNFα结合口袋抑制剂必须优选在其结合态和游离态之间表现出较小的能量差异(即结合的小形变能量)。因此，最有效的TNFα结合口袋抑制剂应优选设计成结合变形能不大于约10kcal/mole，更优选不大于7kcal/mole。TNFα结合口袋抑制剂可以以多于一种总体结合能相似的构象与结合口袋相互作用。在这些情况下，结合的变形能量被认为是游离物质的能量与抑制剂结合蛋白质时观察到的构象的平均能量之间的差异。
设计或选择与TNFα结合口袋结合的实体可进一步计算优化，使得在其结合状态下，其优选不会与靶酶和周围水分子发生排斥静电相互作用。这种非互补静电相互作用包括排斥电荷，偶极-偶极和电荷-偶极相互作用。
本领域中可用具体的计算机软件来评估复合变形能量和静电相互作用。设计用于这种用途的程序的例子包括：高斯94，修订C[M.J.Frisch，Gaussian，Inc.，Pittsburgh，PA(C)1995]；AMBER，版本4.1[P.A.Kollman，加利福尼亚大学旧金山分校(C)1995年]；QUANTA/CHARMm[Accelrys，San Diego，CA]；Insight II(R)/Discovery Studio(R)[Accelrys，SanDiego，CA(C)2001，2002]；DelPhi[Accelrys，San Diego，CA(C)2001，2002]；和AMSOL[印第安那大学量子化学计划交流]。这些程序可以用例如硅来实现
Graphics(R)工作站，例如带有“IMPACTTM”图形的INDIG02。其他硬件系统和软件包将为本领域技术人员所知。
本发明所实现的另一种方法是对化学实体或化合物的小分子数据库进行计算筛选，所述化学实体或化合物可以全部或部分结合TNFα结合口袋。在这个筛选中，这些实体对结合口袋的拟合质量可以通过形状互补或通过估计的相互作用能量来判断。C.Mang等人，J.Comp。Chem。，13，第505-524页(1992)]。
根据另一个实施方案，本发明提供了与通过上述方法产生或鉴定的TNFα结合口袋缔合的化合物。
本发明实现的另一种特别有用的药物设计技术是迭代药物设计。迭代药物设计是通过确定和评估连续组的蛋白质/化合物复合物的三维结构来优化蛋白质与化合物之间的缔合的方法。
在迭代药物设计中，获得一系列蛋白质或蛋白质复合物的晶体，然后解析每个晶体的三维结构。这种方法提供了对每种复合物的蛋白质和化合物之间的关联的了解。这是通过选择具有抑制活性的化合物，获得这种新的蛋白质/化合物复合物的晶体，解决复合体的三维结构，并比较新的蛋白质/化合物复合物与先前解决的蛋白质/化合物复合物之间的关联而完成的。通过观察化合物中的变化如何影响蛋白质/化合物结合，可以优化这些结合。为此提供了结构Compound1.pdb-Compound64.pdb。
在一些情况下，迭代药物设计通过形成连续的蛋白质-化合物复合物然后结晶每个新的复合物来进行。或者，将预先形成的蛋白质晶体在抑制剂存在下浸泡，从而形成蛋白质/化合物复合物，并且不需要结晶每个单独的蛋白质/化合物复合物。
以下实施例说明了本发明。
实施例
实施例1-式(1)-(64)&(65)的化合物的合成
实施例1(A)-式(1)-(63)&(65)
Figure BDA0001512576410000571
Figure BDA0001512576410000581
Figure BDA0001512576410000591
实施例1(B)-式(64)的化合物的合成。命名法
化合物是借助于ACD/姓名批(网络版)命名的。12.0或Accelyrs Draw 4.0
缩略语
DCM：二氯甲烷EtOAc：乙酸乙酯
DMF：N，N-二甲基甲酰胺MeOH：甲醇
DMSO：二甲基亚砜SiO 2：二氧化硅
Et2O：乙醚h：小时
THF：四氢呋喃RT：保留时间
室温：MeCN：乙腈
广泛的M：质量
盐水：饱和氯化钠水溶液
HPLC：高效液相色谱
LCMS：液相色谱质谱法
ES+：电喷雾正离子化
TEA：三乙胺
薄层色谱：薄层色谱
分析条件
所有NMR都是在300MHz或400MHz下获得的。
涉及空气或水分敏感试剂的所有反应均在氮气氛下使用干燥的溶剂和玻璃器皿进行。
所有化合物的LCMS数据通过使用下面的方法确定。
方法1：
Waters Acquity-SQD，Waters Acquity UPLC BEH C18，2.1×50mm，1.7μm色谱柱
流动相A：10mM甲酸铵+0.1％氨
流动相B：95％MeCN+5％H 2O+0.1％氨
梯度程序(流速1.0mL/min，柱温40℃)：
时间A％B％
0.00 95 5
0.50 95 5
1.75 5 95
2.00 5 95
2.25 95 5
对于本领域技术人员将显而易见的是，如果使用不同的分析条件，则可以获得LCMS数据的不同保留时间(RT)。
使用Optical Activity PolAAR 2001旋光仪测量旋光度。
中间体1
Figure BDA0001512576410000611
(6-溴-7-氟-2-甲基咪唑并[1，2-a]吡啶-3-基)[2-(二氟甲氧基)苯基]-甲醇-对映体A
外消旋标题化合物按照专利申请WO2014/009295中所述的程序制备。由此制备的外消旋混合物通过手性色谱分离成组分对映异构体，如下所述：
通过在Chiralpak AD上的LC条件下纯化外消旋(6-溴-7-氟-2-甲基咪唑并[1，2-a]吡啶-3-基)[2-(二氟甲氧基)苯基]-甲醇来分离标题化合物(100×500mm×mm，流速300mL/min，30℃，2-PrOH/庚烷1/9，注入浓度为7.5g/L的230mL溶液)。收集第一洗脱对映异构体(RT 27分钟)，蒸发馏分，得到对映异构体A.[α]-12.8°。收集第二洗脱对映异构体(RT50分钟)，蒸发馏分，得到对映异构体B.[α]+12.7°
中间体2
Figure BDA0001512576410000621
3-(三氟甲基)氮杂环丁烷-3-醇
向在冰/盐水浴上冷却至约-5℃的1-boc-3-氮杂环丁酮(11.3g，58.4mmol，)和(三氟甲基)三甲基硅烷(9.22g，64.3mmol)的THF(100mL)逐份加入氟化铯(9.77g，64.3mmol)。将所得混合物在室温下搅拌4小时后进行TLC分析，指示完全消耗起始材料和极性较低的组分。通过加入饱和氯化铵水溶液(100mL)淬灭反应，水相用EtOAc(3×100mL)萃取。将有机相分离，用硫酸钠干燥，过滤，真空除去挥发物，得到粗油状物。将由此获得的油溶于DCM(100mL)中并加入三氟乙酸(40mL)。将混合物在环境温度下搅拌4小时。真空除去挥发物，残余物与甲苯(3×150mL)共沸，得到标题化合物三氟乙酸盐，为棕色固体(15g)。1H NMR(400MHz，d 6DMSO)：δppm9.48(s，2H)，7.95(d，J 0.3Hz，1H)，4.28(d，J 13.1Hz，2H)，4.06(m，2H)。
将由此得到的化合物不经进一步纯化用于随后的反应。
中间体3
Figure BDA0001512576410000622
1-[5-(4，4，5，5-四甲基-1，3，2-二氧硼杂环戊烷-2-基)嘧啶-2-基]-3-(三氟甲基)氮杂环丁烷-3-醇
向中间体2(12g)的乙腈(150mL)溶液中加入TEA(30mL)和2-氯-5-(4，4，5，5-四甲基-1，3，2-二氧杂硼杂环戊烷-2-基)嘧啶(16g)，并将反应在65℃下搅拌18小时。真空除去溶剂，将固体残余物研磨并用蒸馏水洗涤，得到米色固体，高真空干燥，得到标题化合物，为米色固体(18.5g)。1H NMR(300MHz，d6DMSO)：δ/ppm8.53(2H，s)，7.46(1H，s)，4.33-4.31(2H，m)，4.10-4.08(2H，m)，1.29(12H，s)。LCMS(ES+)RT 1.14分钟，346.0(M+H)+。
化合物(64)
Figure BDA0001512576410000631
1-[5-[3-[(S)-[2-(二氟甲氧基)苯基]-羟基-甲基]-7-氟-2-甲基咪唑并[1，2-a]吡啶-6-基]嘧啶-2-基]-3-(三氟甲基)氮杂环丁-3-醇(对映异构体A)
将中间体1(0.7g，2mmol)，中间体3(0.7g，2mmol)，1，1'-双(二苯基膦基)二茂铁-二氯化钯(II)二氯甲烷络合物(36mg，0.044mmol)和2(2mL)在二恶烷(12mL)中的溶液脱气并回流3小时。将冷却的反应混合物用EtOAc稀释，用盐水洗涤两次，将有机层干燥(MgSO 4)并真空浓缩。将残余物进行快速柱层析(SiO 2，0-90％EtOAc/庚烷)，得到标题化合物，为奶油色固体(500mg，50％)。1H NMR(300MHz，DMSO-d6)：δ8.51(m，3H)，7.95(dd，J＝2.3Hz，J26.7Hz，1H)，7.46(m，2H)，7.36(m，2H)，7.12(m，2H)，6.42(d，J 4.4Hz，1H)，6.18(d，J 4.4Hz，1H)，4.35(m，2H)2H)，2.12(s，3H)。LCMS(ES+)RT 1.34分钟，540.0(M+H)+。[α]+39.7°。
实施例2-筛选与TNFα结合的化合物
式(3)和(15)的化合物已经使用以下测定来筛选。
将384孔未包被平板(标准结合)用TNFR胞外域(TNFR-ECD)(10μl，在PBS中1ug/mL)包被过夜的Meso Scale Discovery平板(MSD)。为了确保均匀的分布板以1000rpm离心2分钟。然后将平板密封并在+4℃下储存过夜。
然后在含有0.05％Tween 20(洗涤缓冲液)的50μl磷酸盐缓冲盐水(pH6.5)(PB)中洗板3次，然后用50μl2％BSA封闭。然后将板在室温下在振荡器(600rpm)上孵育2小时。孵育后，将板洗涤(每孔3×50μl洗涤缓冲液)。
在封闭温育期间，将式(3)和(15)的化合物与TNF(R&D Systems)预温育，然后加入到预封闭和洗涤的MSD板中。对于如图2A所示的单点测定法，化合物以100μM的最终浓度(5％最终v/v DMSO)测定。
为了测定EC 50值(图2B和2C)，将式(3)和(15)的化合物在DMSO中进行双倍或三倍稀释，使得当加入到测定中时，测试化合物的最高浓度为50或100μM 5％最终v/v DMSO)。以1：1至4ng/mL TNF(终浓度2ng/ml)的比例加入式(3)和(15)的预稀释化合物，然后在室温下在600rpm摇床上温育1小时。
将10μl式(3)或(15)的化合物与TNFα的预温育的混合物加入制备的MSD板中并在室温下在振荡器上温育1小时。
然后用洗涤缓冲液(每孔3×50μl)洗涤平板。然后将磺基标记的抗-TNF多克隆抗体加入到每个孔中，并将平板在室温下在摇床上再温育1.5小时。
然后洗涤平板(每孔3×50μl洗涤缓冲液)，接着加入50μlMSD Read缓冲液T加表面活性剂(在H2O中稀释1倍)，并在SECTOR Imager 6000上读数。
对于单点测定，使用没有化合物的对照样品计算百分比抑制。
对于EC 50，使用4参数逻辑模型(S形剂量响应模型)通过标准方法计算测定结果。
从图2A可以看出，代表本领域已知的TNFα拮抗剂的标记为“SPD-304”的化合物具有+80％的抑制％值，表明该化合物抑制TNFα与其受体的结合。相比之下，几种测试化合物具有负的％抑制值，表明这些化合物增强了TNFα与TNF受体的结合。
类似地，式(3)(图2B)和式(15)(图2C)的化合物的浓度响应产生阴性抑制曲线。换句话说，TNFα与固定的ECD-TNFR的结合似乎随着化合物浓度的增加而增强。由于这个原因，必须计算EC50(化合物的浓度达到总效应的50％)，而不是IC50。在这种情况下，式(3)化合物的EC 50是4.6μM，式(15)化合物的EC 50是3.7μM。
使用表面等离子体共振的BIA(生物分子相互作用分析)也可用于测量化合物诱导的TNFα与TNF受体的结合增强。为此，使用Biacore A100/4000。在所谓的溶液内竞争/增强测定中，将TNF受体的细胞外结构域(ECD-TNFR)在pH5下固定在1 KRU的水平上到HBS-P缓冲液(10mM HEPES pH7.4，0.15M NaCl，0.005％表面活性剂P20，BIAcore，GE Healthcare)。
将化合物连续稀释两倍，以使测定中最高浓度为20μM。例如，典型的测定可以使用20μM，10μM，5μM，2.5μM，1.25μM，0.625μM，0.312μM，0.156μM，0.078μM，0.039μM的化合物溶液。将化合物与0.5-1nMTNFα混合并平衡至少5小时。每10-15个循环测试对照化合物。使TNFα/化合物混合物在固定化的TNFR上流动3分钟，然后在每个循环之后进行表面再生，其中以30mL/min的流速注射一次30ml的10mM HCL。按照标准程序使用BIAevaluation软件分析背景扣除结合曲线。使用四参数逻辑拟合确定EC50数据。
图3A和图3B分别显示了式(3)和式(15)的化合物的进展曲线。从曲线中减去不存在化合物的情况下TNFα的RU(共振单位)值，所以这些现在仅显示化合物诱导的结合的增加。随着化合物浓度的增加，在更高的RU值下，进展曲线平稳。由此可以通过使用4参数对数拟合模型确定给出50％最大效应的化合物浓度来计算EC 50值。在这些实验中，计算式(3)化合物的EC 50为298nM，式(15)化合物的EC 50为280nM。
可以注意到，EC50显示批间变异性，并且Biacore测定和MSD测定的条件是非常不同的。因此，对于两种测定形式，测得的EC50预计不会相同。
实施例3-化合物3与TNFα结合的质谱分析
质谱通常使用Waters LCT-premier飞行时间质谱仪或Waters SynaptG2 Q-TOF质谱仪进行。使用替代常规光谱仪源的Advion Triversa Nanomate纳流注入装置来引入样品，样品注射是通过具有50μM喷嘴尺寸的“A”系列芯片以100nl/min的标称流速进行的。对Waters LCT-premier飞行时间质谱仪的进一步修改包括一个定制的源冷却装置，允许精确控制源温度，以及一个商业压力调节装置，可精确控制源区的真空条件。这些修饰一起帮助保持天然折叠构象的TNFα三聚体，并促进检测与弱亲和力的测试化合物形成的复合物。典型设置是源温度：10℃，源压力3.74e-3mbar，分析器压力1.54e-6mbar。
使用标准阳离子电喷雾条件产生离子，导致多次给药TNFα。
质谱对蛋白质样品中存在的缓冲盐非常敏感。典型的缓冲盐例如磷酸钾或磷酸钠对离子化具有严重的不利影响。因此，使用Zeba脱盐旋转柱对蛋白质样品进行预处理以除去这些盐，将蛋白质交换成质谱兼容的缓冲液体系，通常为pH 6.8的50mM乙酸铵。
在软离子化条件下，当观察到三聚物的100％透射时，在100％含水环境中的天然条件下，观察到三聚体形式为包含+12，+13和+14离子的电荷状态包膜，加入5％v/v DMSO电荷状态包络转移到较低的am/z(较高的z)，表明如预期的那样，有机共溶剂引起三聚体物质溶液的部分展开，也检测到单体水平的增加。当添加10％v/v DMSO时，仅观察到与单体形式相关的电荷状态包膜，表明该水平的DMSO破坏溶液中三聚体形成。通常，测试化合物以10mMDMSO储备溶液形式存在，使得当它们与溶液中的TNFα一起温育时，最终的DMSO浓度为5％。在软离子化条件下，观察到电荷状态包络不仅与5％DMSO对照谱相比更高的m/z(低z)，而且与在100％水相下获得的谱相比，表明测试化合物能够克服5％DMSO的去稳定作用，并且在天然条件下观察到的稳定性高于和高于稳定性。蛋白质在所述的各种条件下获得的电荷数量的变化证明了这一点。
测量的“开”速率是速率常数kon和测试化合物的浓度的算术乘积，在测试化合物的高浓度下观察到的速率比在低浓度时大。通过在不同测试化合物浓度下通过质谱分析观察到的速率的实验测量允许导出速率常数(kon)的值。在一个典型的实验中，使用来自TNFα储备溶液和测试化合物的Advion Triversa Nanomate机器人以所需浓度制备测试化合物和TNFα三聚体的混合物。然后将样品在数分钟内注入质谱仪，在此期间记录质谱中游离的TNFα和TNFα/测试化合物复合物信号的比例。对于几种不同的测试化合物/TNFα比例重复这一点。
然后使用Graphpad PRISM v.5将针对不同测试化合物/TNFα比率记录的数据拟合至理论单相对数关联曲线以得出kon值。这证实了在Biacore上观察到的低kon值。
将测试化合物制备成在二甲基亚砜(DMSO)中的10mM溶液。因此，有必要确定在不存在测试化合物的情况下DMSO对天然TNFα三聚体的影响。将DMSO加入到TNFα三聚体的水溶液中以得到5％v/v的最终浓度并获得质谱。
在100％水性环境中，即在不存在DMSO的情况下，大部分TNFα以三聚体形式存在，具有显着比例的TNFα单体。在100％水性环境中，观察到TNFα的三聚体形式为包含+12，+13和+14离子的电荷状态包膜(图4，底部迹线)。
添加5％v/v DMSO观察到三聚体TNFα较少。电荷状态包络转移到较低的质量/电荷比(m/z)，表明DMSO引起三聚物的部分展开。在5％v/v DMSO存在下也检测到单体TNFα水平的增加。
当加入10％v/v DMSO时，仅观察到与单体形式相关的电荷状态包膜，表明该水平的DMSO破坏TNFα的三聚体形成(图4，顶部痕迹)。
将式(3)化合物加入到含有TNFα和5％v/v DMSO的溶液中，并获得质谱。在式(3)化合物存在下，发现TNFα存在于5％v/v DMSO溶液中(图4，中间轨迹)。观察到式3结合的TNFα化合物的电荷态包络转移到更高的m/z值(排他性地+12和+11)，揭示了式(3)的化合物不仅克服了DMSO弱的去折叠影响TNFα，而且导致三聚体TNFα复合物的稳定性超过和不存在DMSO时观察到的。
为了解决为了在试验化合物能够结合之前充分减弱三聚体TNFα复合物而存在DMSO的担忧，在100％含水条件下用水溶性化合物重复实验。在不存在与三聚体复合物结合的DMSO化合物时，导致当存在DMSO时观察到更高的m/z比(数据未显示)。这证实了测试化合物不需要DMSO存在以结合TNFα三聚体，并且可以发挥其稳定作用，而不管存在去稳定剂。
测试化合物稳定三聚体形式的TNFα的进一步证据是通过在苛刻的电离条件下分析样品而获得的，所述条件倾向于倾向于将天然三聚体形式分解为单体。当TNFα与式(3)的化合物结合时，在这些条件下检测到的TNFα单体的量显着降低(数据未显示)。这表明测试化合物保护TNFα三聚体免受质谱破坏。
实施例4-TNFα-式(3)化合物的化学计量学复合物
在软电离条件下通过质谱法监测包括式(3)的化合物在内的测试化合物文库与TNFα的孵育。数据显示每个TNFα三聚体作为式(3)化合物的一个分子的结合的化学计量(图5)。未观察到式(3)的化合物与TNFα的单体形式结合。没有证据表明TNFα的二聚体形式稳定。这证实了包括式(3)化合物在内的测试化合物与已知化合物具有不同的作用模式，其稳定了TNFα的二聚体形式。
实施例5-在TNFα三聚体中的单体交换
将TNFα(分别为H3和M3)的人和小鼠同源三聚体一起温育，并通过质谱仪外观杂交异源三聚体监测溶液的等分试样。质谱分析证实天然TNFα三聚体之间的单体交换能够在溶液中发生。交换速度缓慢，并在达到完全平衡之前的4小时内监测(数据未显示)。机制是未知的，虽然是不可能涉及形成二聚体形式，因为没有观察到这些。单体交换可能在纯人和小鼠三聚体之间发生，小鼠和人三聚体的混合仅仅使得这种交换通过质谱可见。
在第二系列实验中，将过量的式(3)化合物与人TNFα一起孵育，然后除去过量的式(3)化合物。质谱分析证实在式(3)化合物和h-TNFα之间形成1:1络合物。现在将小鼠TNFα加入到该样品中，然后在数小时内进行质谱分析。18小时后，样品的组成没有观察到变化。值得注意的是，没有发生单体亚单位交换，没有观察到形成混合异三聚体物质，如MH2和M2H游离或者作为MH2L和M2HL连接。另外，没有证据表明M3L物种的形成，也没有形成未结合的H3物种的证据。这强烈表明，一旦式(3)的化合物与h-TNFα结合，就没有可测量的解离速率。因此，当与h-TNFα预孵育时，式(3)的化合物锁定人三聚体，因此未观察到交叉物种单体亚单位交换。
然后反过来重复实验。将过量的式3化合物与小鼠TNFα一起孵育，然后除去过量的式(3)化合物。质谱分析证实在式(3)化合物和m-TNFα之间形成了1：1络合物。现在将人TNFα加入到该样品中，然后在数小时内进行质谱分析。数据清楚地显示可以发生单体亚单位交换，在游离(MH2和M2H)和连接(MH2L和M2HL)两种状态下观察到混合异三聚体物质的形成。另外有证据表明连接的人同源三聚体(H3L)，未连接的小鼠同源三聚体(M3)和未结合的式(3)(L)的化合物。这表明尽管式(3)化合物与小鼠TNFα同源三聚体之间形成1：1复合物，但存在可测量的解离速率。一旦该复合物(M3L)解离，H3和M3物质之间的单体亚基交换就会进行，释放的配体则能够与存在于溶液中的所有4种三聚物形成复合物。因此，当用m-TNFα预孵育时，式(3)化合物不阻止单体亚基交换，并观察到混合异源三聚体的形成。
然后用式(15)的化合物代替式(3)的化合物重复这两个实验。当式(15)化合物与h-TNFα预温育得到1：1复合物，然后与未连接的m-TNFα混合时的结果与式(3)化合物相同。未观察到单体亚单位交换，18小时后，在溶液中仅观察到H3L和M3物质，证实式(15)的化合物与h-TNFα复合时也没有可测量的解离速率。因此，当用h-TNFα预孵育时，式(15)的化合物锁定人三聚体，因此没有观察到交叉物种单体亚单位交换。
然而，与式(3)化合物相比，当式(15)化合物与m-TNFα预孵育形成1:1复合物，然后与未连接的h-TNFα混合时，未观察到单体亚单位交换18小时内只观察到M3L和H3物质在溶液中。这表明式(15)的化合物在与m-TNFα复合时也没有可测量的解离速率。因此，当用m-TNFα预温育时，式(15)的化合物锁定小鼠三聚体，因此没有观察到交叉物种单体亚单位交换。
这些数据一起表明，虽然式(3)化合物和式(15)化合物对人TNFα具有相似的亲和力，但是该化合物对小鼠TNFα三聚体具有不同的亲和力，式(15)化合物结合更紧密比式(3)的化合物与后者相比。
实施例6-使用TNFα，TNF-R和式(3)的化合物对来自尺寸排阻实验的级分进行质谱分析，
通过液相色谱-质谱(LC-MS)分析来自TNFα，TNF-R和式(3)化合物的混合物的尺寸排阻色谱分离的级分。制备两个样品用于尺寸排阻色谱。在第一个样品中，在向TNF-R中加入化合物-三聚体复合物之前，将式(3)的化合物与TNFα一起预温育。在第二个样品中，将式(3)的化合物加入预先形成的TNFα和TNF-R的复合物中。LC-MS分析显示式(3)化合物与含有两种蛋白质的那些级分相关(图6)，表明不管添加顺序如何，式(3)化合物仍然能够与即即使在TNF-R存在下，式(3)的化合物也与TNFα结合。
实施例7-TNFα和式(15)化合物的等温量热分析-TNFα三聚体复合物结合TNF-R
在ITC缓冲液(50mM HEPES，150mM NaCl，pH7.4)中的TNFα(128μM)与化合物2的DMSO储备液孵育60分钟，得到在5％DMSO中的300mM的最终化合物浓度(测试样品)。将其中DMSO而非化合物添加至TNFα样品的对照样品也温育60分钟(对照)。
温育后，将样品在Nap5大小排阻柱(GE Healthcare)上进行凝胶过滤。在加入500μl进入柱的样品之前，用15ml ITC缓冲液平衡该柱，然后用1ml ITC缓冲液洗脱。该过程从游离化合物和DMSO中分离TNF和化合物结合的TNF。
使用280nm处的吸光度读数来测定测试样品或从NAP 5柱洗脱后的对照中的TNFα浓度，并将样品稀释至64μM的TNFα浓度。
将200μlTNFR1的细胞外结构域(ECD)(10mM)自动加载到AutoITC200(GEHealthcare)的样品池中(使用标准板B方案)。在2个实验中，使用相同的方案将40μl的测试样品或对照自动加载到注射器中。
ITC实验使用在等温线图(图7A和B)上描述的ITC注射方案在25℃下以1000rpm搅拌进行。
使用GE Healthcare ITC在原始4.0软件中的应用收集和分析数据，并使用单站点结合算法计算结果。
计算出在不存在任何测试化合物的情况下与TNF-R结合的TNFα的KD为77nM(图7A)。式(15)化合物存在下与TNF-R结合的TNFα的KD低于量热计的灵敏度范围，因此不能准确计算。然而，热量计具有约1nM的较低灵敏度边界。因此，式(15)化合物存在下与TNF-R结合的TNFα的KD必须是1nM或更低(见图7B)。
实施例8-用本发明化合物中和TNFα
使用Baarsch MJJ等(Immunol Methods 1991；140：15-22)和Galloway CJ等J(Immunol Methods 1991；140：37-43)中公开的方案进行L929中和测定。
简言之，将L929细胞(ECACC，85011425)在含有10％FCS(PAA)，2mM谷氨酰胺(Gibco)，50U/ml青霉素(Gibco)和50μg/ml链霉素(Gibco)的RPMI 1640 GIBCO)。当传代培养时，细胞用10mL不含钙和镁的Dulbecco's磷酸盐缓冲盐水(Gibco)洗涤3次，然后加入3ml胰蛋白酶-EDTA(Gibco)2分钟以从烧瓶中除去细胞。加入培养基中和胰蛋白酶，上下移取细胞以除去任何团块。
使用前一天将L929细胞分开1/2或1/3，再培养24小时。然后如上所述将培养瓶进行胰蛋白酶消化，并在96孔平底平板(Becton Dickinson)的每孔中加入100μl中的2x10 4个细胞。在建立测定之前将平板培养24小时。
从化合物的DMSO储备液中进行连续稀释。典型地，通过从浓缩化合物溶液中双倍稀释产生9点滴定曲线，得到25，12.5，6.25，3.125，1.56，0.78，0.39，0.2，0.1μM的最终测定浓度。
测定培养基与培养基相同，但也含有1μg/ml放线菌素D(Sigma)。将培养基从平板上轻轻摇动，将测定样品加TNFα，标准品和对照以100μl体积加入，一式两份。将平板进一步温育16小时，然后每孔加入10μl5mg/ml甲基噻唑四唑(MTT；Sigma)的培养基溶液4小时。通过加入100μl溶于50％二甲基甲酰胺(DMF；BDH)和50％去离子水的含有20％十二烷基硫酸钠(SDS，BDH)的增溶缓冲液来终止反应。
在37℃孵育过夜以使染料溶解后，在570nm下用Multiskan EX读板仪(Labsystem)在630nm处减去读板。数据使用Genesis软件包进行分析。
式(3)化合物和式(15)化合物均抑制人TNFα的细胞杀伤活性(图9)，表明式(3)化合物和式(15)化合物均能够通过TNF-R抑制人TNFα诱导的信号传导。在这种情况下，式(3)化合物的IC50值为306nM，式(15)化合物的IC50值为125nM。使用式(39)的化合物重复该方案，其还发现通过TNF-R抑制人TNFα诱导的信号传导。因此，式(39)的化合物给出21nM的IC50值。
实施例9-式(3)化合物抑制TNFα诱导的IL-8产生
通过针刺收集来自健康供体的静脉血进入含有钠/肝素的管(BD Biosciences)。通过用Ficoll Paque(Amersham Biosciences)密度梯度离心分离外周血单核细胞(PBMC)。简言之，用RPMI 1640(Gibco)以1：1(v/v)稀释10mL血液并小心地铺在20mL Ficoll Paque上。将细胞在470g离心30分钟(min)，收集PBMC，在RPMI 1640中洗涤一次，并将任何剩余的污染红细胞溶解在红细胞裂解缓冲液(1g/L KHCO 3，8.3g/L NH 4 Cl，0.0372g/L EDTA)。根据制造商的说明，使用CD14+磁性微珠(Miltenyi Biotec)从PBMC中分离单核细胞。简而言之，将PBMC以1x10 7个细胞/ml重悬于含有5％BSA(Sigma)和2mM EDTA(Sigma)的Dulbecco's改良Eagle培养基中。每107个总细胞中25μL的CD14微珠在室温下温育15分钟。使用LS柱(Miltenyi Biotec)进行磁分离。在将细胞/珠混合物应用于柱之前，将柱置于磁场中并用5mL缓冲液洗涤两次。然后将细胞悬浮液在磁场中施加到柱上。单核细胞结合CD14+微珠保留在LS柱上，而剩余的PBMC通过柱子。为了分离单核细胞，然后将柱子(包含保留的细胞)从磁体中取出并置于收集管中。将5mL缓冲液添加到柱中，并通过将注射器柱塞施加到柱的顶部从柱中收集CD14+细胞。收集的细胞在RPMI 1640中洗涤一次。
在96孔圆底平板中，在DMSO中进行11点3倍系列稀释(包括空白)的化合物(原液浓度10mM)。
纯化的单核细胞通过离心(300g，5分钟)洗涤并以1×10 6个细胞/mL的浓度重悬于完全培养基中。将160μL的这种细胞群在含有40μL化合物和TNFα
Figure BDA0001512576410000731
的RPMI 1640或相关对照中一式三份在96孔圆底平板中在37℃温育。
18小时后，将平板离心(300g，5分钟)，收集上清液用于测量细胞因子。
使用来自R&D Systems Ltd.的酶联免疫吸附测定(ELISA)试剂盒，根据制造商的说明在细胞培养上清液中测量人IL-8。用于ELISA的底物是TM Blue(SerologicalsCorporation)。在630nm的波长读取平板，在470nm进行校正。式(3)化合物以浓度依赖性方式抑制TNFα诱导的IL-8产生(图10)，IC50值为454.1nM。
实施例10-式(1)-(64)的化合物对TNFα诱导的NF-κB活化的抑制-报道基因测定
通过TNF-α刺激HEK-293细胞导致NF-kB途径的激活。用于确定TNFα活性的报道细胞系购自Invivogen。HEK-Blue TM CD40L是一种稳定的转染子，表达在与5个NF-kB结合位点融合的IFN-β最小启动子控制下的SEAP(分泌的碱性磷酸酶)。通过TNF-α(0.5ng/ml)，IL-1-β(0.5ng/ml)和活化的抗人TNFR1抗体(300ng/ml)以浓度依赖性方式刺激这些细胞分泌SEAP。
将化合物从10mM DMSO原液(最终测定浓度0.3％)稀释以产生10点3倍连续稀释曲线(30，000nM至2nM终浓度)。将它们与刺激性配体在384孔微量滴定板中混合1小时。将新鲜解冻并洗涤的细胞加入到化合物/刺激混合物中，并进一步温育18小时。使用比色底物Quanti-blue TM(Invivogen)在上清液中测定SEAP活性。
在DMSO对照和最大抑制(通过过量的对照化合物)之间计算化合物稀释的抑制百分数，并使用xlfit(4参数逻辑模型)在活性基质中计算IC50。
将式(15)化合物对TNF-α应答的比活性与对抗屏蔽剂(IL-1β和抗人TNFR1抗体)所观察到的比较进行比较。
式(15)的化合物以浓度依赖性方式抑制TNFα对NF-κB的活化，IC50为113nM(图11A)。相反，式(15)的化合物不抑制IL-1β(图11B)或激活的TNF-R1抗体(图11C)对NF-κB的活化。因此，式(15)的化合物通过TNF-R1特异性地抑制TNFα诱导的信号传导，但对由其他信号传导途径(例如IL-1β)诱导的NF-κB活化没有影响，或者当信号传导的开始(例如通过使用激活的TNF-R1抗体)旁路TNF-R1。
此外，当在报道基因测定中测试时，还发现式(1)-(64)的化合物以浓度依赖性方式抑制TNFα对NF-κB的活化，表现出50μM或更小的IC50值。
实施例11-测定结合TNFα的动力学
表面等离子体共振用于测量式(3)和(15)化合物对TNFα的结合率，解离速率和亲和力(图12A和B)。为了研究的目的，使用Biacore T100/T200。
在HBS-P缓冲液(10mM HEPES pH7.4，0.15M NaCl，0.005％表面活性剂P20，BIAcore，GE Healthcare)中，将TNFα在pH5下固定至5-8KRU的水平。然后用5％DMSO在HBS-P中平衡TNFα至少5小时。将样品从10mM储备液稀释到DMSO匹配的缓冲液中并使其溶解至少5小时。流量为30L/min。
通过从对于式(3)的化合物以25μM的最高浓度和对于式(15)的化合物以1μM开始添加4或5个浓度的化合物并且然后连续地稀释该样品来进行该测定。按照标准程序使用BIAevaluation软件分析背景扣除结合曲线。使用Biacore软件确定结合，亲和力和动力学参数。动力学数据使用levenberg marquardt算法拟合。
实验显示这些化合物非常缓慢地结合固定化的TNFα，如通过式(3)的化合物(图12A)的2.668e3M-1s-1和式(15)的化合物的1.119e3M-1s-1所证明的。(图12B)。它们也具有显着缓慢的解离速率，这似乎是具有这种作用模式的化合物的特征。式(3)化合物的解离速率常数(koff)为9.46e-5s-1，式(15)化合物的解离速率常数(koff)等于2.24e-5s-1。这等同于分解半衰期(t1/2)分别超过2小时和8小时。解离常数(KD)可以由两个常数koff/kon的比值来计算。在该实验中，式(3)的化合物和式(15)的化合物的KD值分别为35nM和2nM。这显着低于实施例3中所示的在Biacore上确定的EC 50，并且可能反映了测定形式的差异。此外，TNFα的形式不同于实施例11的动力学测定，TNFα被固定。
重复该实验以测量式(39)化合物对TNFα的缔合速率，解离速率和亲和力(图12C)。发现式(39)的化合物具有5470M-1s-1的kon，解离速率常数为4.067e-5s-1，KD为7nM。
实施例12-式(3)的化合物和式(15)的化合物在体内拮抗TNFα活性
在另外的研究中，将式(3)和式(15)的化合物与溶解在磷酸盐缓冲盐水(PBS)中的20μMTNFα溶液混合，浓度为2μM，20μM和200μM。因此，每种化合物与TNFα的比例为0.1:1(样品1)，1:1(样品2)和10:1(样品3)。在使用Zeba Spin脱盐柱(Thermo Scientific)进行凝胶过滤之前，将溶液在室温下温育3小时以使化合物结合TNFα。这个过程分离蛋白质结合的化合物和游离化合物。仅含有PBS的对照样品以相同的方式处理以提供用于研究的载体对照。使用Nanodrop(ND-1000)测定洗脱蛋白质的浓度。将TNFα：化合物复合物在PBS中稀释至注射浓度为0.03μg/kg。
对于该研究，通常，除了使用一组5只小鼠的抗人TNFα抗体阳性对照以外，每组包含10只雄性Balb/c小鼠(Charles River)。施用抗体对照小鼠5分钟前(t＝-5)腹膜内(i.p.)注射10mg/kg(100μL)的抗hTNFαα施用i.p.以0.1μg/kg(t＝0)注射PBS或hTNFα。
试验小鼠腹膜内注射。在t＝0时，用100μL凝胶过滤载体(PBS)，hTNFα(0.03μg/kg)或样品1，2和3(化合物与TNFα的比例为0.1:1，1:1和10：1)。
该研究中还包括仅有化合物的小鼠以评估化合物对嗜中性粒细胞募集的作用。
所有小鼠在注射hTNFα(t＝2h)两小时后颈椎脱臼处死，并用3mL FACS缓冲液(500mL含有2g牛血清白蛋白的PBS，6mL HEPES缓冲液和500mL EDTA)灌洗腹腔。抽吸灌洗液并通过如下详述的通过FACS对抗Gr1PE和抗CD45FITC染色细胞来评估嗜中性粒细胞数目。
将100μl来自每个样品的灌洗液分装到FACS管中。使用抗-GR-1PE(BD目录号553128批次#75542)以1:19稀释度和抗-CD45FITC(BD目录号553080批次#80807)在FACS缓冲液中稀释19倍制备FACS鸡尾酒。使用FACS缓冲液以1:10制备Fc区(BD Cat#553142批次#87810)，并在加入抗体混合物之前5分钟将10μL添加至每个样品。每个含有100μL样品的试管中加入10μl抗体混合物。样品在冰上放置20分钟。将1mL FACS裂解液(BD Cat#349202批号29076，在dH2O中以1:10稀释)加入到每个管中，混合并在室温下放置5分钟。然后将1mLFACS缓冲液加入到每个管中，并以400g离心5分钟。然后小心地倒出FACS缓冲液，并将管的尖端在吸水纸上擦拭以使管完全干燥。然后将300μl在FACS缓冲液中稀释的10μl参比珠子溶液(Sigma目录号#P2477批号#116K1612)加入到每个试管中。
使用FACScalibur II和FloJo软件分析样品。
图13显示式(3)(A)化合物和式(15)(B)化合物的结果。单独载体对于嗜中性粒细胞募集的影响可以忽略不计，如同单独使用化合物(在(B)中略高)。来自每个研究的样品1(比例化合物：TNFα0.1：1)与没有化合物时加入TNFα没有显着不同。样品2(1：1)和样品3(10：1)显示显着抑制嗜中性粒细胞募集(分别为86％和85％)。类似地，式(15)化合物的样品2和样品3显示显着抑制嗜中性粒细胞募集(分别为101％和102％)。抗体对照小鼠显示嗜中性粒细胞募集的100％抑制(数据未显示)。
在进一步的实验中，用hTNFα(0.3μg/ml)处理小鼠，口服(p.o.)给药式(3)化合物。
式(3)的化合物在1％甲基纤维素载体中使用covaris机制成悬浮液。
在本研究中，还使用抗人TNFα单克隆抗体(抗hTNFα，UCB)作为阳性对照。
除了接受抗hTNFα的组中使用了十只雄性Balb/c小鼠外，其中使用了4只小鼠。
小鼠以30mg/kg或100mg/kg口服接受100μL任一种载体(1％甲基纤维素)或式(3)化合物。30分钟(t＝-30)或10mg/kg i.p.的抗hTNFα。在注射人TNFα之前5分钟(t＝-5)。在t＝0时，给小鼠注射100μLi.p.的0.03μg/kg的PBS或hTNFα。
所有小鼠在注射hTNFα(t＝2h)两小时后颈椎脱臼处死，腹膜腔被灌洗并且如上所述测量嗜中性粒细胞数目。
式(3)化合物的30mg/kg和100mg/kg的口服给药使TNFα刺激的嗜中性粒细胞募集到腹膜腔中分别减少了49％和79％(图14)。i.p.施用的阳性对照抗体(10mg/kg)注射完全抑制了嗜中性粒细胞的招募。
因此，式(3)的化合物不仅在与TNFα预先混合并通过i.p途径给药时，而且在口服给药时也可以拮抗体内的TNFα活性。
实施例13-通过式(3)和(15)的化合物对TNFα三聚体稳定化的分析
进行荧光探针热变性测定以评估化合物对TNFα热稳定性的影响，作为化合物结合的量度。反应混合物含有5μl30x
Figure BDA0001512576410000782
Orange染料(Invitrogen)和5μlTNFα(1.0mg/ml)，37.5μlPBS，pH 7.4和2.5μl化合物(在DMSO中2mM)。将10μl混合物一式四份分配到384PCR光学孔板中，并在7900HT Fast实时PCR系统(AgilentTechnologies)上运行。PCR系统加热装置设定在20℃至99℃，升温速率为1.1℃/min；通过电荷耦合器件(CCD)监测孔中的荧光变化。将荧光强度增加绘制为温度的函数，并且将Tm计算为该变性曲线的中点(作为拐点确定)(表1)。
通过Tm的增加来表示稳定TNFα。式(3)和(15)的化合物都增加了TNFα的Tm(如表1所示)。因此，式(3)和(15)的化合物都增加了TNFα三聚体的稳定性。
表1显示了存在化合物(3)或(15)时TNFα的热转变中点(Tm)。
Figure BDA0001512576410000781
实施例14-确定式(1)-(64)化合物对荧光缀合物与TNFα结合的作用的荧光偏振测定
化合物(A)的制备
1-(2，5-二甲基苄基)-6-[4-(哌嗪-1-基甲基)苯基]-2-(吡啶-4-基甲基)-1H-苯并咪唑-以下称为“化合物“-通过WO2013/186229(2013年12月19日公开)的实施例499中描述的程序制备。
荧光共轭物的制备
将化合物(A)(27.02mg，0.0538mmol)溶于DMSO(2mL)中。5(-6)将羧基荧光素琥珀酰亚胺酯(24.16mg，0.0510mmol)(Invitrogen目录号：C1311)溶于DMSO(1mL)中，得到亮黄色溶液。两种溶液在室温下混合，混合物变成红色。混合物在室温下搅拌。在混合之后不久，将20μL等分样品移出并在AcOH:H2O的80:20混合物中稀释，以在1200RR-6140LC-MS系统上进行LC-MS分析。色谱图显示保留时间为1.42和1.50分钟的两个密切洗脱峰，质量(M+H)+＝860.8amu，对应于用5-和6-取代的羧基荧光素基团形成的两种产物。保留时间2.21分钟的另一个峰具有对应于化合物(A)的(M+H)+＝502.8amu的质量。未反应的5(-6)羧基荧光素琥珀酰亚胺酯没有观察到峰。三个信号的峰面积分别为22.0％，39.6％和31.4％，表明在该时间点，所需荧光共轭物的两种异构体的转化率为61.6％。进一步将20μL等分试样在数小时后提取，然后搅拌过夜后，如前稀释并进行LC-MS分析。在这些时间点，转换百分比分别为79.8％和88.6％。混合物在UV指导的制备型HPLC系统上纯化。将合并的纯化级分冷冻干燥以除去过量的溶剂。冷冻干燥后，回收橙色固体(23.3mg)，相当于0.027mmol荧光缀合物，对应于反应和制备型HPLC纯化的总产率53％。
抑制荧光缀合物与TNFα的结合
通过在20mM Tris，150mM NaCl，0.05％吐温20中于环境温度下用TNFα预先温育60分钟，在添加之前，以25μM开始，在最终测定浓度的5％DMSO中以10个浓度测试化合物，然后加入的荧光缀合物，并在环境温度下进一步温育20小时。在25μL的总测定体积中，TNFα和荧光缀合物的最终浓度分别为10nM和10nM。在能够检测荧光偏振的平板读数器(例如AnalystHT平板阅读器；或Envision平板阅读器)上读板。在ActivityBase中使用XLfit TM(4参数逻辑模型)计算IC50值。
当在荧光偏振测定中测试时，发现伴随实施例的式(1)-(64)的化合物均显示50μM或更小的IC50值。
在进一步的实验中，从终浓度为5％DMSO的100μM开始，以10个浓度对式(3)化合物进行测试，
在室温下，在20mM Tris，150mM NaCl，0.05％Tween 20中与TNFα温育60分钟，然后加入荧光缀合物，并在环境温度下进一步温育过夜。在总检测体积为25μl时，TNFα和荧光缀合物的最终浓度分别为50nM和10nM。在Analyst HT阅读器上读板。在活性基质中使用xlfit(4参数逻辑模型)计算IC50。
结果如图15所示。式(3)的化合物能够抑制荧光缀合物与TNFα的结合，IC50值为167nM。
使用式(15)和(39)的化合物重复该实验。式(15)的化合物能够抑制荧光缀合物与TNFα的结合，IC50值为102nM。式(39)的化合物能够以20nM的IC50值抑制荧光缀合物与TNFα的结合。
实施例15-抗体衍生物
在用苯并咪唑化合物(15)与人TNFα复合的5只Sprague Dawley大鼠免疫后，将免疫B细胞在96孔板中培养以诱导克隆扩增和抗体分泌(Tickle，S。等，High throughputscreening for high affinity antibodies Journal of Laboratory Automation 200914：303-307)。在基于均质珠粒的FMAT测定中，筛选培养物上清液中与人化合物(15)(与50倍摩尔过量)复合的人TNFα优先结合人源化TNFα。通过使用人TNF受体I-Fc融合蛋白(R&DSystems目录号372-R1)的捕获系统将人TNFα(+/-化合物(15))呈递在珠表面(超级抗生物素蛋白包被的Bangs Beads，目录号CP01N)-050)，与生物素化的抗人Fc(Jackson目录号109-066-098)结合。
证明优先结合TNFα-化合物(15)复合物的抗体被称为“构象选择性”，并被用于克隆。使用荧光焦点法(美国专利7993864/欧洲专利EP1570267B1)从阳性孔中鉴定和分离抗原特异性B细胞，通过逆转录(RT)-PCR从单个细胞中回收特异性抗体可变区基因。
下面显示了两种代表性抗体CA185_01974和CA185_01979的氨基酸序列，其显示了与人和小鼠TNFα+化合物的构象选择性结合：
CA185_01974.0(VR0001837)
轻链可变区(LCVR)SEQ ID NO：7(加下划线的CDR)
DIQMTQSPASLPASPEEIVTITCQASQDIGNWLSWYQQKPGKSPQLLIYGATSLADGVPSRFSASRSGTQYSLKISRLQVEDFGIFYCLQGQSTPYTFGAGTKLELK
重链可变区(HCVR)SEQ ID NO：8(加下划线的CDR)
DVQLVESGGGLVQPGRSLKLSCAASGFTFSAYYMAWVRQAPTKGLEWVASINYDGANTFYRDSVKGRFTVSRDNARSSLYLQMDSLRSEDTATYYCTTEA YGYNSNWFGYWGQGTLVTVSS
CA185_01979.0(VR0001842)
轻链可变区(LCVR)SEQ ID NO：22(加下划线的CDR)
DIQMTQSPASLSASLEEIVTITCQASQDIGNWLSWYQQKPGKSPHLLIYGTTSLADGVPSRFSGSRSGTQYSLKISGLQVADIGIYVCLQAYSTPFTFGSGTKLEIK
重链可变区(HCVR)SEQ ID NO：23(加下划线的CDR)
EVHLVESGPGLVKPSQSLSLTCSVTGYSITNSYWDWIRKFPGNKMEWMGYINYSGSTGYNPSLKSRISISRDTSNNQFFLQLNSITTEDTATYYCARGTYGYNAYHFDYWGRGVMVTVSS
实施例16-高效液相色谱法(HPLC)测定抗体特征
小鼠Fab片段的特异性结合通过使用大小排阻层析(数据未显示)通过CA185_01974和与化合物(15)络合的人TNFα之间的复合物形成来证明。对于0.5x摩尔过量的Fab，主要峰对应于结合的Fab和三聚体化合物复合物(尽管存在小峰表明存在未结合Fab的一些三聚体化合物复合物)。在1.0x摩尔过量的Fab中，存在单独的较高分子量的对应于与三聚体复合物结合的Fab的峰。在Fab的1.5倍和2倍摩尔过量时，存在对应于未结合的Fab的越来越低的分子峰。
因此，化学计量确定为1Fab：1TNFα三聚体，过量的Fab以1.5倍和2倍摩尔过量出现。
还使用尺寸排阻色谱法(数据未显示)研究CA185_01979与化合物(15)络合的人TNFα的结合。至于CA185_01974，化学计量学确定为1Fab：1TNFα三聚体，过量的Fab出现在1.5倍和2倍摩尔过量。
实施例17-测定抗体特性的BIAcore测定
使用BIAcore T200(GE Healthcare)在25℃下进行表面等离子体共振。通过胺偶联化学将抗鼠Fc(Jackson 115-006-071)固定在CM5传感器芯片(GE Healthcare)上，达到约6000个应答单位的捕获水平。使用HBS-EP缓冲液(10mM HEPES pH7.4，0.15M NaCl，3mMEDTA，0.05％(v/v)表面活性剂P20-GE Healthcare)+1％DMSO作为运行缓冲液。使用10μl注射1μg/ml的每种IgG通过固定的抗小鼠Fc来捕获以产生TNFα结合表面。将50nM的人或小鼠TNFα(内部)与2μM化合物在HBS-EP+(1％DMSO)中预温育5小时。
将人或小鼠TNFα+/-测试化合物的3分钟注射以30μl/min的流速通过每个捕获的IgG。以10μl/min的流速通过60s注射40mM HCl x2和30s 5mM NaOH再生表面。按照标准程序，使用T200评估软件(版本1.0)分析双重参考背景扣除的结合曲线。动力学参数由拟合算法确定。
下表2和3中显示了在存在和不存在来自两个化学系列的测试化合物的情况下人和小鼠TNFα的动力学结合数据。
Figure BDA0001512576410000821
Figure BDA0001512576410000831
表2-人TNFα的BIAcore数据
CA185_01974和CA185_01979均显示对化合物扭曲的人TNFα>2log选择性结合，具有来自两个化学系列的代表性测试化合物。
抗体 Mouse TNFα ka(M<sup>-1</sup>s<sup>-1</sup>) kd(s-<sup>1</sup>) KD(M)
CA185_01974 +化合物(39) 6.7x10<sup>4</sup> 4.8x10<sup>-5</sup> 7.1x10<sup>-10</sup>
CA185_01974 +化合物(15) 5.8x10<sup>4</sup> 8.8x10<sup>-5</sup> 1.5x10<sup>-9</sup>
CA185_01974 apo 4.2x10<sup>4</sup> 4.9x10<sup>-3</sup> 1.2x10<sup>-7</sup>
CA185_01979 +化合物(39) 1.9x10<sup>5</sup> 3.5x10<sup>-5</sup> 1.9x10<sup>-10</sup>
CA185_01979 +化合物(15) 1.6x10<sup>5</sup> 6.3x10<sup>-5</sup> 3.8x10<sup>-10</sup>
CA185_01979 apo 7.2x10<sup>4</sup> 2.0x10<sup>-3</sup> 2.7x10<sup>-8</sup>
表3-用小鼠TNFα的BIAcore数据
CA185_01974和CA185_01979均表现出对化合物扭曲的小鼠TNFα>1.5和>2log选择性结合，具有来自两个化学系列的代表性测试化合物。
结论
已经证明抗体CA185_01974和CA185_01989特异性结合TNFα的化合物扭曲状态，并且将是用于检测本发明的TNFα的扭曲三聚体结构的有用的靶标接合生物标志物。
已显示抗体结合TNFα的构象，其由来自不同化学系列的化合物特别稳定。设想这些抗体将成为定义TNFα三聚体的这种以及紧密相关的生物学相关构象的标准，其通过比这里描述的更宽范围的化学系列来稳定。基于所显示的数据，如果CA185_01974或CA185_01989抗体在上述BIAcore测定形式中以优于1nM的KD结合，则认为人TNFα三聚体可以稳定在所述的确定的生物相关构型中。
实施例18-与式(1)-(64)化合物结合的三聚TNFα的晶体结构
可溶形式的人TNFα(VC 2043，UniProt P01375)在大肠杆菌中表达为融合蛋白，并具有最终序列：
SVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL(SEQ ID NO：35)
SEQ ID NO：35的起始“S”是克隆制品，而不是TNF的天然序列的一部分。因此，SEQID NO：35的残基编号从V开始，即V1，R2，S3等。SEQ ID NO：36代表SEQ ID NO：35，但没有该初始“S”残基即
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL(SEQ ID NO：36)
将细胞在富培养基中在37℃下预培养，加入0.1％阿拉伯糖诱导，并使其在25℃下在载体pEMB54中表达过夜。该载体引入可切割的N端His6Smt标签。将细胞裂解并通过Ni-NTA螯合层析进行纯化。用含有咪唑的缓冲液洗脱融合蛋白，并加入蛋白酶切割。通过减去Ni螯合物层析步骤纯化最终切割的TNFα蛋白质以除去融合标签，并通过尺寸排阻色谱进一步纯化以除去剩余的杂质。通常将最终的TNFα产物浓缩至20.0mg/ml并在液氮中快速冷冻。
在10mM HEPES pH7.5，150mM NaCl缓冲液中将纯化的人TNFα(20.0mg/ml，VC2043)稀释至4-7mg/ml，然后在4℃下用0.3-0.5mM化合物(1-2摩尔过量)。通过将0.5μl的复合物与0.5μl的21.44％PEG 3350，100mM Tris，pH9.0(UCB1474433)混合，通过坐滴蒸气扩散使TNFα-化合物复合物结晶；或22.0％PEG 3350，100mM甘氨酸，100mM Tris，pH 8.5(UCB1480595)；或25.6％PEG 3350，100mM Tris，pH8.3，3％木糖醇(UCB5143079)或具有不同沉淀剂浓度的其它制剂或超过100μl的相同结晶溶液的添加剂。其他化合物的其他配方包括8-28％的一系列PEG 3350。其他PEG类添加剂包括PEG 1000(8-18％)或PEG 2000(10-13％)。其他添加剂包括MPD(5-13％)或200mM NaCl。其他缓冲液包括100mM Hepes(pH 7-8)，100mM Tris或bis-Tris(pH 6.7-9.0)。其他非PEG 3350结晶溶液由PEG 2000(14-19％)，100mM Hepes，pH 8，25mM精氨酸；或17％PEG 10000，200mM乙酸铵，100mM双-Tris，pH5.5；或30％PEG 2000MME，100mM乙酸钠，100mM Mes，pH6.5。表4中列出了每种化合物的结晶条件和晶胞的更多细节。
将晶体简单地浸泡在乙二醇中和/或直接在液氮中玻璃化以用于数据收集。
X射线衍射数据从同步加速器源以最常见的波长收集并记录在CCD检测器上。使用XDS软件包可以减少衍射数据(Kabsch，2010a)。与化合物复合的人TNFα(VC 2043)的结构通过使用Phaser的分子置换来解决，输入模型为1TNF。数据被整合到XDS中，并使用XSCALE(Kabsch，2010b)进行缩放。继续使用Coot(Emsley和Cowtan，2004)和Refmac(Murshudov等，1997)的迭代手工建模，直到R和Rfree收敛。使用Coot和MolProbity验证模型质量(Chen等人，2010)。结构的分辨率最经常在2.15左右。
所有64个结构的原子坐标在本发明的补充数据中给出，每个结构都给出了名称“化合物X”或“化合物X.pdb”，其中X是图1中化合物的式(X)每个三聚体的中心。
参考文献：
Kabsch,W.2010a.XDS.Acta Crystallogr.D Biol.Crystallogr.Feb；66(Pt 2):1125-32.PMID:20124692.
Kabsch W.2010b.Integration,scaling,space-group assignment and post-refinement.Acta Crystallogr.D Biol.Crystallogr.Feb；66(Pt 2):133-44.PMID:20124693.
Emsley,P.and Cowtan,K.2004.Coot:model-building tools for moleculargraphics.Acta Crystallogr.D Biol.Crystallogr.Dec；60(Pt 12 Pt 1):2126-32.PMID:15572765.
Murshudov,G.N.,Vagin,A.A.,and Dodson,E.J.1997.Refinement ofmacromolecular structures by the maximum-likelihood method.Acta Crystallogr.DBiol.Crystallogr.May 1；53(Pt 3):240-55.PMID:15299926.
Chen et al.2010.MolProbity:all-atom structure validation formacromolecular crystallography.Acta Crystallogr.D Biol.Crystallogr.Jan；66(Pt1):12-21.PMID:20057044.
表4
Figure BDA0001512576410000861
Figure BDA0001512576410000871
Figure BDA0001512576410000881
Figure BDA0001512576410000891
Figure BDA0001512576410000901
Figure BDA0001512576410000911
Figure BDA0001512576410000921
Figure BDA0001512576410000931
Figure BDA0001512576410000941
Figure BDA0001512576410000951
Figure BDA0001512576410000961
当TNFα与式(3)的化合物预温育时，所得的化合物-三聚体复合物结晶，并且使用X-射线结晶学测定化合物-三聚体TNFα复合物的晶体结构，复合物的晶体结构至图2.2的分辨率)可以在图8中看到。可以在不再对称的三聚体的中间看到该化合物。
更详细地说，当从侧面观察TNFα三聚体的晶体结构时，其大致形状如金字塔/圆锥体。当你向下看三聚体轴，单体末端的N-和C-末端指向你，那么你正在看三聚体的“脂肪”端。在具有化合物的扭曲结构中，A和C亚基之间的裂缝开放，其中，Ab而不受理论约束，Abs结合。
哪一条链是A，B或C可以通过测量三个相同残基的三个C-α原子之间的三个距离来确定-例如，每个链中的P117(G121也是合适的)。
三个距离形成一个三角形，在apo-TNF中是等边的，但是当化合物结合时是扭曲的。最短的距离是BC与AC之间最长的距离(例如AC＝13.8？，AB＝12.3？，BC＝10.2？)。从而通过N/C末端指向您的分子轴向下看，最长的距离定义C，然后A链逆时针旋转，然后B和C再次逆时针旋转。
已经确定，本文所述的化合物与三聚体形式的TNF的结合导致TNF三聚体的构象变化。特别地，当用本文公开的化合物结合时，TNF三聚体呈现变形或扭曲的构象。
例如，当化合物(1)-(64)与人TNFα的可溶性结构域结合时，TNF保留其三聚体结构，但是A和C亚基彼此远离并且A旋转以在这些亚基之间产生裂缝。不受理论束缚，认为这种变形说明了三聚体结合TNFR1的能力，而没有受体的正常活化。
亚基的这种运动也导致化合物结合的三聚体中心处的空腔扩大。化合物(1)-(64)具有20-41个非氢原子。典型地，扭曲的TNFα三聚体结构相对于apo TNFα结构1A8M(使用ThePYMOL Molecular Graphics System，1.7版Schrodinger，LLC；使用命令和设置：
Figure BDA0001512576410000971
dot_solvent＝1，get_area)增加表面积在
Figure BDA0001512576410000972
之间。
实施例19-与式(1)-(63)化合物结合的三聚体TNFα的晶体结构分析，以及确定扭曲的TNFα三聚体结构“化合物64.pdb”在本发明的TNFα三聚体结构内
进行均方根偏差(RMSD)计算以确定扭曲的TNFα三聚体的每个晶体结构到参照结构的距离。
RMSD计算使用程序PyMOL(www.pymol.org)完成。在这个示例版本1.7。被使用。cmd.rms PyMOL命令与定义的原子对一起使用。所用的原子是TNFαβ链中某些残基的主链Cα。使用的残留物编号如下所示：
12-18，47-50，54-64，76-82，91-97，117-125，129-137，150-156
这些残基编号是公开的具有PDB编码1A8M的TNFα结构内的残基，并且是在本发明的扭曲的TNFα三聚体结构内。残基编号也根据SEQ ID NO：36的序列。使用具有TNFα结构的三条链(根据我们的说明书标记为A，B和C)中的每一条中的残基。
命令cmd.rms与以下参数一起使用：
rms＝cmd.rms(“(moving)”，“(target)”，matchmaker＝4，quiet＝0，cycles＝0)
其中“(移动)”是给定TNFα结构中原子的选择，“(靶)”是参考结构内等价原子的选择。
对所有其他结构计算每个结构的RMSD。“代表性结构”或“参考结构”被选择为与所有其他RMSD的总和最低的结构“化合物34”或“化合物34.pdb”。
其余63个结构参照化合物34结构测得的RMSD值以埃为单位测量，显示在下面的表5中。图16显示了具有参考结构的结构的加重；结构紧密围绕参考结构聚集，指示所有64个结构的常见的扭曲的TNFα三聚体排列。
Figure BDA0001512576410000981
Figure BDA0001512576410000991
Figure BDA0001512576410001001
公布为PDB编码1A8M的TNFα三聚体的apo形式的参考结构的RMSD是2.35A。
实施例19b)：确定扭曲的TNFα三聚体结构“化合物64”在本发明的TNFα三聚体结构内
参考结构(化合物34)和查询结构(化合物64)被覆盖(参见具有参考结构的图17)：
1.将两个晶体结构的pdb文件读入PyMol
2.为化合物64(化合物64.pdb)的识别的残基c-α原子创建选择，例如“(移动)”和参考结构(化合物34.pdb)例如“(目标)”晶体结构。在每个结构中应该选择183个原子。
3.使用rms命令计算以埃为单位的均方根偏差(RMSD)。
4.RMSD小于0.9(0.22)，因此化合物64的扭曲的TNFα结构落入本发明的范围内。
PyMol的输入和响应记录：
PyMOL>载入化合物34.pdb
CmdLoad：“化合物34.pdb”加载为“化合物34”。
PyMOL>加载化合物64.pdb
CmdLoad：“化合物64.pdb”加载为“化合物64”。
PyMOL>select移动，化合物64和名称CA和(alt A或alt“\”)和(resi 12-18或resi47-50或resi 54-64或resi 76-82或resi 91-97或resi 117-125或Resi 129-137或Resi150-156)和(链A或链B或链C)
选择器：选择183个原子定义的“移动2”。
PyMOL>选择目标，化合物34和名称CA和(alt A或alt“\”)和(resi 12-18或resi47-50或resi 54-64或resi 76-82或resi 91-97或resi 117-125或Resi 129-137或Resi150-156)和(链A或链B或链C)
选择器：选择183个原子定义的“target2”。
PyMOL>从pymol导入cmd
PyMOL>cmd.rms(“(moving)”，“(target)”，matchmaker＝4，quiet＝0，cycles＝0)
执行：RMS＝0.219(183到183个原子)
实施例19c)：解决与化合物65复合的扭曲的TNFα三聚体结构，并确定该结构在本发明的TNFα三聚体结构内
在10mM HEPES pH7.5，150mM NaCl缓冲液中将纯化的人TNFα(30.0mg/ml，VC2043)(如上所述产生)稀释至4mg/ml，然后在4℃下用0.5mM化合物65(1-2摩尔过量)。通过将0.5μl的复合物与0.5μl的19％PEG2000，15％PEG1000，20mM L-精氨酸，0.1M HEPES，pH8.0在100μl的水中混合，通过坐滴蒸汽扩散使TNFα-化合物65复合物结晶相同的结晶溶液。最初建立后大约2周收集晶体以收集数据。将它们短暂地浸泡在乙二醇中，并直接在液氮中玻璃化以收集数据。结构测定如上所述。统计数据如下表5b所示。
表5b。数据收集和细化统计
Figure BDA0001512576410001021
Figure BDA0001512576410001031
参考结构(化合物34)和查询结构(如以上针对化合物65所测定的)如实施例19b)所述进行重叠。RMSD小于0.9(0.62)，所以化合物65的扭曲的TNFα结构落入本发明的范围内。对于实施例21中描述的结构，化合物65内本发明的药效团在相同的7个TNFα残基的4以内。
实施例19d)-来自其他结构的结果
用其他化合物产生进一步扭曲的TNFα三聚体结构，并如上所述进行分析。与参比结构相比，所有的RMSD都小于0.9埃(最大的是0.85埃。
实施例20-分析畸变的三聚TNFα的晶体结构内的空腔
对抗体/抗原复合物的晶体结构的分析涉及通过在4埃内寻找接触来详细描述互补位/表位界面处的重要相互作用。类似地，对于在TNFα残基和三聚体中心的化合物之间始终产生的那些重要的相互作用，可以分析64个扭曲的三聚体TNFα结构。
Schrodinger程序的大师(
Figure BDA0001512576410001032
Release 2014-4：Maestro，版本10.0，
Figure BDA0001512576410001033
LLC，New York，NY，2014)被用于分析64个扭曲的TNFα三聚体晶体结构。对于每种结构，选择配体周围的4半径，并记录来自位于该半径内的TNFα三聚体的所有残基。
图18显示了说明来自在TNFα三聚体中的特定残基(亚单位特异性)的4以内的64个扭曲的TNFα三聚体结构的配体的百分比的图。观察到7个残基始终在配体的4个之内：来自亚基A的Leu57，来自亚基B的Tyr119，来自亚基B的Gly121，来自亚基B的Gly122，来自亚基C的Leu120和来自亚基C的Tyr151。应注意的是，来自所有三个亚单位总是参与互动；可能是三聚体TNFα结构稳定的原因。
图19显示了化合物1扭曲的TNFα三聚体结构的核心的图片，突出显示化合物1周围的所有上述残基。
实施例21-分析本发明的扭曲的TNFα三聚体结构中心的药效团拟合
使用化合物(1)-(64)的结构和程序阶段(小分子药物发现套件2015-1：小型分子药物发现套件2015-1)，可以描述与所有7个TNFα残基相互作用的配体的药效团，Phase，version 4.2，
Figure BDA0001512576410001041
LLC，New York，NY，2015；Dixon，SL；Smondyrev，AM；Knoll，EH；Rao，SN；Shaw，DE；Friesner，RA，“PHASE：A New Engine for PharmacophorePerception，3D QSAR Model Development and 3D Database Screening。1.Methodologyand Preliminary Results，“J.Comput.Aided Mol.Des。，2006，20，647-671；Dixon，SL；Smondyrev，AM；Rao，SN，”PHASE：A Novel Approach to Pharmacophore Modeling and 3DDatabase Searching，“Chem.Biol.Drug Des。，2006，67，370-372)。
阐明的药效团的特征如下：
1)2个稠合的5-或6-元环(中心在“R3”和“R2”)，一个环(以R2为中心)与H键受体(“A1”)形成氢键亚基C的Tyr151的侧链]，并且还通过连接非氢原子被取代为另外的5-或6-元环(以“R4”为中心)；
2)根据表6，特征可以排列在扭曲的TNFα三聚体结构内；
3)其中一个或多个环可以是芳香族的；
4)其中一个或多个环可以是杂芳族的；
5)稠环共有2个原子；
6)当R3环可以是5或6元时，R2环可以是5或6元，R4环可以是5或6元；
7)连接的非氢原子可以是碳，氮或氧；
8)其中A1可以通过R2环中的氮或氧原子；
9)R2环的pI系统可能与TNFα三聚体亚基C上的Tyr59侧链形成CH-π相互作用；
10)当R3环的pI系统与TNFα三聚体亚基C上的Tyr59侧链形成π堆积相互作用时，
11)当R3环的pI系统与TNFα三聚体的亚基A上的Leu57的侧链形成CH-π相互作用时，
12)R4环的pI系统可能与TNFα三聚体亚基A上Leu57的侧链形成CH-π相互作用；
13)在R1，R2，R3和A1特征之间的一个或多个距离可以根据表7的那些(大约，精确地或在10％之内)的情况下；
14)在R1，R2，R3和A1特征之间的一个或多个角度可以根据表8的那些(大约，精确地或在10％之内)的情况下；限定可能参与TNFα三聚体去对称化的香蕉形状的R3-R2-R4角；
15)如果包含药效团的配体可以具有20-41个非氢原子。
表6
Figure BDA0001512576410001051
表7
Figure BDA0001512576410001052
Figure BDA0001512576410001061
表8
位点1特征 位点2特征 位点3特征 角度(度)
R2 A1 R3 46.6
R2 A1 R4 28.2
R3 A1 R4 63.9
A1 R2 R3 109.2
A1 R2 R4 144.9
R3 R2 R4 89.5
A1 R3 R2 24.2
A1 R3 R4 87.3
R2 R3 R4 66.0
A1 R4 R2 6.9
A1 R4 R3 28.9
R2 R4 R3 24.5
图20显示可能适合本发明的扭曲的TNFα三聚体结构的药效团的说明性实例，其显示三环特征的R2，R3和R4中心的位置以及氢键受体特征A1在R2环。在该实例中，所有的环特征是芳族的(环R2为杂芳族)，环R2与环R4之间的连接原子是碳，环内的特征A1是氮原子，环R2是五元环，环R3和R4是六元的，并且具有根据表7和8的特征之间的距离和角度。
实施例22-TNF小分子对膜TNF-TNFR2信号传导的影响
现有的TNFα抑制剂结合并中和可溶性和膜结合的TNFα(分别为sTNFα和mTNFα)(Nesbitt等，Inflamm Bowel Dis 2007 13：1323-1332)。
已知sTNFα对TNFR1受体具有特异性，对TNFR2受体具有mTNFα(Grell等，Cell 199583：793-802)。
现有的抑制剂在其标签上有黑匣子警告，描述了在严重感染(特别是TB(肺结核)，细菌性败血症和真菌感染)和恶性肿瘤(包括淋巴瘤)中使用的严重后果。
已知对TB(以及李斯特氏菌)的免疫应答是由mTNFα驱动的(Garcia等，Chapter 20p187-201“可溶性和膜TNF和相关配体在分枝杆菌感染中的作用：选择性和非选择性TNF的作用抑制剂在感染期间“，在D.Wallach等(编辑)，Advances in TNF Family Research，Advances in Experimental Medicine and Biology 691，DOI 10.1007/978-1-4419-6612-4_20中)。
选择性抑制sTNFα但不抑制mTNFα的TNFα抑制剂具有减弱实验性关节炎而不抑制对感染的先天免疫性的特征(Zalevsky等，免疫学杂志2007 179：1872-1883)。
本实施例研究本文所述的结合化合物时mTNFα构象如何不影响TNFR2功能；TNFR2近端和下游信号不受损害。
由于在TACE切割位点处的突变，稳定表达不可切割的人膜TNF的鼠B细胞系(NS0-mTNF)被用于触发PHA-L和IL-2扩增的原代人T细胞中的TNFR2信号传导。使用Ficoll梯度离心从最初从全血中分离的PBMC扩增T细胞，并在用PHA-L和IL-2活化和扩增后显示出升高的TNFR2水平。TNFR1也存在于这些细胞上，但是使用UCB小鼠抗人TNFR1抗体5R16阻断。
将NS0-mTNF细胞与10μMNCEs一起培养1小时以使化合物与膜TNF结合。然后将T细胞与NS0-mTNF细胞混合，短暂旋转以使细胞与细胞接触，将细胞孵育5分钟，然后在裂解缓冲液中裂解。分析裂解物的两个近端和下游信号事件，证明TNFR2信号传导。在免疫共沉淀实验中测量了第一次TRAF-2募集到TNFR2受体作为膜近端信号传导的量度。细胞裂解后，使用多克隆山羊抗人TNFR2抗体，然后进行蛋白G琼脂糖珠纯化，从裂解物中分离TNFR2。然后在SDS-PAGE上运行纯化的TNFR2制备物后，在免疫印迹中测量TNFR2相关的TRAF-2。另外，还使用SDS-PAGE和pNFκB特异性免疫印迹对全细胞裂解物取样以检测pNFκB的存在。
使用NS0-wt细胞作为对照，并且在与扩增的人原代T细胞混合时显示对TNFR2受体的非常有限的TRAF-2募集以及低pNFκB信号传导。相比之下，NS0细胞上膜TNF的存在导致TRAF-2的强烈募集和潜在的泛素化，这是由于观察到的梯度作用，表明TNFR2特异性膜近端信号传导。另外，在全细胞裂解物中测量的pNFκB水平增加，显示下游的TNFR2信号传导。化合物的存在并不改变TNFR2特异性的近端和下游信号传导，并且导致TRAF-2类似地招募到TNFR2以及在全细胞裂解物中相似水平的pNFκB。因此，这些结果显示化合物与NS0细胞中过表达的膜TNF的结合不会损害TNFR2特异性近膜(TRAF-2募集至TNFR2)和下游(全细胞裂解物中pNFκB存在)信号传导。
详细的方法部分：
T细胞培养：
·将来自3个不同供体的PBMC在6孔板中在10ml FCS，1％人血清，HEPESGlutaMAX，25U/ml hIL-2(Roche)和2μg/ml hIL-2的5ml RPMI1640中以每孔10x10 6细胞/ml PHA-L(Sigma)生长出T细胞
·3天后，将细胞重新悬浮并在具有与上述相同培养基的75cm2培养瓶中培养，但缺乏PHA-L
·监测培养物的生长情况，每3天更换一次培养基。
NS0-mTNF和NS0-wt细胞的培养
NS0-wt：含10％FCS，NEAA和GlutaMAX的DMEM
·NS0-mTNF：具有10％FCS，NEAA的DMEM(细胞在没有GlutaMAX的情况下培养，因为膜TNF在被表达谷氨酰胺合成酶的载体上引入细胞以产生谷氨酰胺和膜TNF-使用谷氨酰胺缺陷培养基保持选择用于膜TNF表达细胞的细胞。
IP和印迹程序：
·在RPMI+10％FCS+HEPES+GlutMAX+-20μM(化合物15，化合物43或化合物39)或DMSO中制备含有10x10 6 NS0-mTMF细胞的500μl培养基，并在37℃孵育1小时以允许将化合物装载到TNF。这是一个2倍的解决方案
·准备500μl的10x10 6个NS0-wt细胞
·1小时后，在37℃的培养基中加入500μl人类T细胞(50×10^6个细胞/测试)。将T细胞与50μg/ml 5R16抗-TNFR1Fab-PEG在37℃下预温育15分钟以阻断通过TNFR1的信号转导，然后加入到NS0细胞中。混合，短暂旋转使细胞接触并在37℃孵育5分钟，旋转并通过用1ml冰冷的裂解缓冲液重悬沉淀立即裂解并裂解1小时：1％NP-40，150mM NaCl，50mM TrispH8.0，25mM NaF 1mM钒酸盐->10ml该缓冲液加入100μl磷酸酶抑制剂(Sigma P5726-5ml)和100μl磷酸酶抑制剂(Sigma P2850)以及20μl蛋白酶抑制剂混合物(Sigma-P8430)
·在离心机中全速旋转15分钟，并使用上清液。保存一些上清液在印迹中寻找pNFkB
·加入5μg多克隆山羊抗hTNFR2(R&D)并在4℃旋转孵育过夜，第二天早上加入20μlprotG琼脂糖凝胶珠，在旋转的同时再旋转1h
·旋转，用裂解缓冲液洗3次，然后旋转并尽可能多地取出液体，加入SDS-PAGE还原样品缓冲液并煮沸10分钟
·在4-12％Bis-Tris凝胶上加载15μl，并在MOPS缓冲液中在200V下运行55min
·在硝酸纤维素膜上使用iBlot 7min方案进行印迹，并在TBS/0.05％Tween中的5％牛奶中封闭2小时
·在封闭缓冲液中加入第一抗体(抗TRAF-2-1：250 sc-136999-Santa Cruz)，并在5℃下在4℃孵育过夜
·用TBS/0.05％吐温洗涤3次
·在RT下在封闭缓冲液中加入二抗鼠HRP(1：2000)2小时，同时摇动
·用TBS/0.05％吐温洗5次并显色
pNFkB分析：
·在还原条件下，在MOPS缓冲液中的4-12％Bis-Tris凝胶上运行10μl，在200V下1h，并使用7min iBlot程序在硝酸纤维素膜上进行印迹。
·在室温下封闭2小时，同时用含5％牛奶的TBS/0.05％吐温振荡
·加入抗pNFkB抗体1：1000(NEB-3033)和抗GAPDH(1：4000NEB)，并在4℃孵育50mlFalcon on roller
·在封闭缓冲液中用TBS/0.05％Tween-加入的二抗兔-HRP(1：2000)在RT下清洗3次，同时摇动
·用TBS/0.05％吐温洗5次并显色
图21是显示化合物15，43和39与膜TNFα诱导的TNFR2近端和下游信号传导结合的作用的凝胶；化合物与NS0细胞中过表达的膜TNFα的结合不损害TNFR2特异性膜近端(TRAF-2募集至TNFR2)和下游(全细胞裂解物中pNFκB存在)信号传导。
实施例23-Ma等人(2014)和Silvian等人(2011)的化合物和复合物具有与本发明不同的特征
如Ma等人的第12458页所述。(2014)JBC 289：12457-12466，通过虚拟筛选发现C87，试图找到与TNFR1的loop2/domain2的7个氨基酸的肽所占据的空间在与TNFβ的外表面的相互作用中的分子。Ma等人的C87化合物和来自Silvian等人的BIO8898化合物。(2011)ACS化学生物学6：636-647由本发明人测试。
调查结果摘要
·Ma等人描述的Biacore观察。对于C87不能重复。
·没有观察到细胞中TNF特异性抑制的证据。
·另外C87没有观察到绑定的质谱，这是敏感的毫摩尔亲和力。
·广泛的结晶学试验仅产生apo-TNF(没有化合物的TNF)。
·在荧光偏振(FP)测定中，C87在荧光读出的化合物的干扰水平以上没有显着的抑制。
测量TNFα热解链温度稳定性的Thermofluor对C87显示出小的稳定性。
·总之，没有发现C87结合在三聚体中心的证据。绝大多数数据表明与TNFα没有直接的相互作用。BIO8898也被发现不与TNFα结合。
细胞-TNF诱导的HEKNFκB报告基因测定
C87与TNFα预培养1小时，然后加入在NFκB控制下用SEAP稳定转染的HEK-293细胞。为了检测非TNF相关(脱靶)活性，也测试了合适的反筛。将测定温育过夜，然后与对照化合物的100％阻断相比，测量抑制。最大C87浓度是10，000nM，连续稀释3倍。不能检测到不能归因于脱靶活性的抑制作用。
Biacore
使用avi-标签接头固定TNF，并将C87通过芯片。在一个实验中，进行了来自最高浓度10μM的C87的剂量响应。没有观察到结合。在第二个实验中，C87通过芯片的流速降低了。观察到一个小的转变，但整体约束可以忽略不计。Ma等人描述的C87与TNF的结合很可能是基于Y轴上RU值的超化学计量的。在芯片上的标准TNF密度下，该值比单纯1：1结合的预期值高出三十倍。
在另一个实验中，BIO8898在Biacore 4000机器上通过SPR针对CD40L的固定可溶形式和TNFα的可溶形式进行测试。测定了17μM的几何IC50与CD40L的结合，而在该测定中，在TNFα的浓度高达100μM时没有检测到结合。
质谱
在400μM的浓度下没有C87与人TNFα(20μM)结合的证据。一种较低分子量的物质(约473Da似乎在低于5％的占有率时结合)。C87具有503Da的分子量。基于400μM浓度的占有率，预测低分子量物质超过1mM的亲和力。
结晶学
总的来说，将大量努力用于使TNFα结晶C87，包括常规使用本申请中描述的化合物的测试条件。这包括在不同配体浓度，不同蛋白质浓度和不同浸泡时间下进行大量结晶试验。观察到一些晶体，经分析证明是盐或TNF，没有化合物。
荧光偏振(FP)
在针对荧光化合物(探针)进行测定之前，C87与TNFα预温育1小时。通过FP的降低来检测与荧光化合物直接(在相同位点结合)或间接(破坏TNF)的竞争。
抑制曲线的外推产生约100μM的IC50。然而，在抑制剂的最高浓度下观察到荧光猝灭，当在该测定中扣除时抑制C87的作用可忽略不计。
Thermofluor
Thermofluor测量由于化合物稳定或破坏蛋白质而导致的TNFα的解链温度(Tm)的变化。当浓度为500μMC87时，观察到3.8℃的稳定效应，表明弱结合的可能性，这可能不是特异性的。
序列表
SEQ ID NO:1(LCDR1 of 1974)
QASQDIGN
SEQ ID NO:2(LCDR2 of 1974)
GATSLAD
SEQ ID NO:3(LCDR3 of 1974)
LQGQSTPYT
SEQ ID NO:4(HCDR1 of 1974)
AYYMA
SEQ ID NO:5(HCDR2 of 1974)
ASINYDGANTFYRDSVKG
SEQ ID NO:6(HCDR3 of 1974)
EAYGYNSNWFGY
SEQ ID NO:7(LCVR of 1974)
DIQMTQSPASLPASPEEIVTITCQASQDIGNWLSWYQQKPGKSPQLLIYGATSLADGVPSRFSASRSGTQYSLKISRLQVEDFGIFYCLQGQSTPYTFGAGTKLELK
SEQ ID NO:8(HCVR of 1974)
DVQLVESGGGLVQPGRSLKLSCAASGFTFSAYYMAWVRQAPTKGLEWVASINYDGANTFYRDSVKGRFTVSRDNARSSLYLQMDSLRSEDTATYYCTTEAYGYNSNWFGYWGQGTLVTVSS
SEQ ID NO:9(LCVR DNA of 1974)
GACATCCAGATGACCCAGTCTCCTGCCTCCCTGCCTGCATCCCCGGAAGAAATTGTCACCATCACATGCCAGGCAAGCCAGGACATTGGTAATTGGTTATCATGGTATCAGCAGAAACCAGGGAAATCGCCTCAGCTCCTGATCTATGGTGCAACCAGCTTGGCAGATGGGGTCCCATCAAGGTTCAGCGCCAGTAGATCTGGCACACAGTACTCTCTTAAGATCAGCAGACTGCAGGTTGAAGATTTTGGAATCTTTTACTGTCTACAGGGTCAAAGTACTCCGTACACGTTTGGAGCTGGGACCAAGCTGGAACTGAAA
SEQ ID NO:10(HCVR DNA of 1974)
GACGTGCAGCTGGTGGAATCTGGAGGAGGCTTAGTGCAGCCTGGAAGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGTGCCTATTACATGGCCTGGGTCCGCCAGGCTCCAACGAAGGGTCTGGAGTGGGTCGCATCCATTAATTATGATGGTGCTAACACTTTCTATCGCGACTCCGTGAAGGGCCGATTCACTGTCTCCAGAGATAATGCAAGAAGCAGCCTATACCTACAAATGGACAGTCTGAGGTCTGAGGACACGGCCACTTATTACTGTACAACAGAGGCTTACGGATATAACTCAAATTGGTTTGGTTACTGGGGCCAAGGCACTCTGGTCACTGTCTCGAGC
SEQ ID NO:11(1974 LC kappa全长)
DIQMTQSPASLPASPEEIVTITCQASQDIGNWLSWYQQKPGKSPQLLIYGATSLADGVPSRFSASRSGTQYSLKISRLQVEDFGIFYCLQGQSTPYTFGAGTKLELKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
SEQ ID NO:12(1974 HC mIgG1全长)
DVQLVESGGGLVQPGRSLKLSCAASGFTFSAYYMAWVRQAPTKGLEWVASINYDGANTFYRDSVKGRFTVSRDNARSSLYLQMDSLRSEDTATYYCTTEAYGYNSNWFGYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK
SEQ ID NO:13(1974 HC mFabno hinge全长)
DVQLVESGGGLVQPGRSLKLSCAASGFTFSAYYMAWVRQAPTKGLEWVASINYDGANTFYRDSVKGRFTVSRDNARSSLYLQMDSLRSEDTATYYCTTEAYGYNSNWFGYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC
SEQ ID NO:14(1974 LC DNA kappa全长)
GACATCCAGATGACCCAGTCTCCTGCCTCCCTGCCTGCATCCCCGGAAGAAATTGTCACCATCACATGCCAGGCAAGCCAGGACATTGGTAATTGGTTATCATGGTATCAGCAGAAACCAGGGAAATCGCCTCAGCTCCTGATCTATGGTGCAACCAGCTTGGCAGATGGGGTCCCATCAAGGTTCAGCGCCAGTAGATCTGGCACACAGTACTCTCTTAAGATCAGCAGACTGCAGGTTGAAGATTTTGGAATCTTTTACTGTCTACAGGGTCAAAGTACTCCGTACACGTTTGGAGCTGGGACCAAGCTGGAACTGAAACGTACGGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT
SEQ ID NO:15(1974 HC DNA mIgG1全长)
GACGTGCAGCTGGTGGAATCTGGAGGAGGCTTAGTGCAGCCTGGAAGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGTGCCTATTACATGGCCTGGGTCCGCCAGGCTCCAACGAAGGGTCTGGAGTGGGTCGCATCCATTAATTATGATGGTGCTAACACTTTCTATCGCGACTCCGTGAAGGGCCGATTCACTGTCTCCAGAGATAATGCAAGAAGCAGCCTATACCTACAAATGGACAGTCTGAGGTCTGAGGACACGGCCACTTATTACTGTACAACAGAGGCTTACGGATATAACTCAAATTGGTTTGGTTACTGGGGCCAAGGCACTCTGGTCACTGTCTCGAGTGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA
SEQ ID NO:16(1974 HC DNA mFabno hinge全长)
GACGTGCAGCTGGTGGAATCTGGAGGAGGCTTAGTGCAGCCTGGAAGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGTGCCTATTACATGGCCTGGGTCCGCCAGGCTCCAACGAAGGGTCTGGAGTGGGTCGCATCCATTAATTATGATGGTGCTAACACTTTCTATCGCGACTCCGTGAAGGGCCGATTCACTGTCTCCAGAGATAATGCAAGAAGCAGCCTATACCTACAAATGGACAGTCTGAGGTCTGAGGACACGGCCACTTATTACTGTACAACAGAGGCTTACGGATATAACTCAAATTGGTTTGGTTACTGGGGCCAAGGCACTCTGGTCACTGTCTCGAGTGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCGGCTGTCCTGCAATCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT
SEQ ID NO:17(LCDR2 of 1979)
GTTSLAD
SEQ ID NO:18(LCDR3 of 1979)
LQAYSTPFTF
SEQ ID NO:19(HCDR1 of 1979)
NSYWD
SEQ ID NO:20(HCDR2 of 1979)
YINYSGSTGYNPSLKS
SEQ ID NO:21(HCDR3 of 1979)
GTYGYNAYHFDY
SEQ ID NO:22(LCVR of 1979)
DIQMTQSPASLSASLEEIVTITCQASQDIGNWLSWYQQKPGKSPHLLIYGTTSLADGVPSRFSGSRSGTQYSLKISGLQVADIGIYVCLQAYSTPFTFGSGTKLEIK
SEQ ID NO:23(HCVR of 1979)
EVHLVESGPGLVKPSQSLSLTCSVTGYSITNSYWDWIRKFPGNKMEWMGYINYSGSTGYNPSLKSRISISRDTSNNQFFLQLNSITTEDTATYYCARGTYGYNAYHFDYWGRGVMVTVSS
SEQ ID NO:24(LCVR DNA of 1979)
GACATCCAAATGACACAGTCTCCTGCCTCCCTGTCTGCATCTCTGGAAGAAATTGTCACCATTACATGCCAGGCAAGCCAGGACATTGGTAATTGGTTATCATGGTATCAGCAGAAACCAGGGAAATCTCCTCACCTCCTGATCTATGGTACCACCAGCTTGGCAGATGGGGTCCCATCAAGGTTCAGCGGCAGTAGATCTGGTACACAGTATTCTCTTAAGATCAGCGGACTACAGGTTGCAGATATTGGAATCTATGTCTGTCTACAGGCTTATAGTACTCCATTCACGTTCGGCTCAGGGACAAAGCTGGAAATAAAA
SEQ ID NO:25(HCVR DNA of 1979)
GAGGTGCACCTGGTGGAGTCTGGACCTGGCCTTGTGAAACCCTCACAGTCACTCTCCCTCACCTGTTCTGTCACTGGTTACTCCATCACTAATAGTTACTGGGACTGGATCCGGAAGTTCCCAGGAAATAAAATGGAGTGGATGGGATACATAAACTACAGTGGTAGCACTGGCTACAACCCATCTCTCAAAAGTCGAATCTCCATTAGTAGAGACACATCGAACAATCAGTTCTTCCTGCAGCTGAACTCTATAACTACTGAGGACACAGCCACATATTACTGTGCACGAGGGACCTATGGGTATAACGCCTACCACTTTGATTACTGGGGCCGAGGAGTCATGGTCACAGTCTCGAGC
SEQ ID NO:26(1979 LC Kappa全长)
DIQMTQSPASLSASLEEIVTITCQASQDIGNWLSWYQQKPGKSPHLLIYGTTSLADGVPSRFSGSRSGTQYSLKISGLQVADIGIYVCLQAYSTPFTFGSGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
SEQ ID NO:27(1979 HC mIgG1全长)
EVHLVESGPGLVKPSQSLSLTCSVTGYSITNSYWDWIRKFPGNKMEWMGYINYSGSTGYNPSLKSRISISRDTSNNQFFLQLNSITTEDTATYYCARGTYGYNAYHFDYWGRGVMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK
SEQ ID NO:28(1979 HC mFabno hinge全长)
EVHLVESGPGLVKPSQSLSLTCSVTGYSITNSYWDWIRKFPGNKMEWMGYINYSGSTGYNPSLKSRISISRDTSNNQFFLQLNSITTEDTATYYCARGTYGYNAYHFDYWGRGVMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC
SEQ ID NO:29(1979 LC DNA Kappa全长)
GACATCCAAATGACACAGTCTCCTGCCTCCCTGTCTGCATCTCTGGAAGAAATTGTCACCATTACATGCCAGGCAAGCCAGGACATTGGTAATTGGTTATCATGGTATCAGCAGAAACCAGGGAAATCTCCTCACCTCCTGATCTATGGTACCACCAGCTTGGCAGATGGGGTCCCATCAAGGTTCAGCGGCAGTAGATCTGGTACACAGTATTCTCTTAAGATCAGCGGACTACAGGTTGCAGATATTGGAATCTATGTCTGTCTACAGGCTTATAGTACTCCATTCACGTTCGGCTCAGGGACAAAGCTGGAAATAAAACGTACGGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT
SEQ ID NO:30(1979 HC DNA mIgG1全长)
GAGGTGCACCTGGTGGAGTCTGGACCTGGCCTTGTGAAACCCTCACAGTCACTCTCCCTCACCTGTTCTGTCACTGGTTACTCCATCACTAATAGTTACTGGGACTGGATCCGGAAGTTCCCAGGAAATAAAATGGAGTGGATGGGATACATAAACTACAGTGGTAGCACTGGCTACAACCCATCTCTCAAAAGTCGAATCTCCATTAGTAGAGACACATCGAACAATCAGTTCTTCCTGCAGCTGAACTCTATAACTACTGAGGACACAGCCACATATTACTGTGCACGAGGGACCTATGGGTATAACGCCTACCACTTTGATTACTGGGGCCGAGGAGTCATGGTCACAGTCTCGAGTGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA
SEQ ID NO:31(1979 HC DNA mFabno hinge全长)
GAGGTGCACCTGGTGGAGTCTGGACCTGGCCTTGTGAAACCCTCACAGTCACTCTCCCTCACCTGTTCTGTCACTGGTTACTCCATCACTAATAGTTACTGGGACTGGATCCGGAAGTTCCCAGGAAATAAAATGGAGTGGATGGGATACATAAACTACAGTGGTAGCACTGGCTACAACCCATCTCTCAAAAGTCGAATCTCCATTAGTAGAGACACATCGAACAATCAGTTCTTCCTGCAGCTGAACTCTATAACTACTGAGGACACAGCCACATATTACTGTGCACGAGGGACCTATGGGTATAACGCCTACCACTTTGATTACTGGGGCCGAGGAGTCATGGTCACAGTCTCGAGTGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCGGCTGTCCTGCAATCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT
SEQ ID NO:32–Rat TNFα
MSTESMIRDVELAEEALPKKMGGLQNSRRCLCLSLFSFLLVAGATTLFCLLNFGVIGPNKEEKFPNGLPLISSMAQTLTLRSSSQNSSDKPVAHVVANHQAEEQLEWLSQRANALLANGMDLKDNQLVVPADGLYLIYSQVLFKGQGCPDYVLLTHTVSRFAISYQEKVSLLSAIKSPCPKDTPEGAELKPWYEPMYLGGVFQLEKGDLLSAEVNLPKYLDITESGQVYFGVIAL
SEQ ID NO:33–Mouse TNFα
MSTESMIRDVELAEEALPQKMGGFQNSRRCLCLSLFSFLLVAGATTLFCLLNFGVIGPQRDEKFPNGLPLISSMAQTLTLRSSSQNSSDKPVAHVVANHQVEEQLEWLSQRANALLANGMDLKDNQLVVPADGLYLVYSQVLFKGQGCPDYVLLTHTVSRFAISYQEKVNLLSAVKSPCPKDTPEGAELKPWYEPIYLGGVFQLEKGDQLSAEVNLPKYLDFAESGQVYFGVIAL
SEQ ID NO:34–人TNFα
MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL
SEQ ID NO:35–Soluble form of人TNFα
SVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL
SEQ ID NO:36–Soluble form of人TNFα，but lacking the“S”cloning artefactof SEQ ID NO:35
VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL
补充数据：通过晶体的X射线衍射衍生的与化合物(1)-(64)络合的人可溶性TNFα的64个结构的原子结构坐标。这里的结构也称为化合物1.pdb-化合物64.pdb。坐标可以在本专利申请从其中得到的公开的PCT说明书下面或在其中找到。
化合物1
Figure BDA0001512576410001241
Figure BDA0001512576410001251
Figure BDA0001512576410001261
Figure BDA0001512576410001271
Figure BDA0001512576410001281
Figure BDA0001512576410001291
Figure BDA0001512576410001301
Figure BDA0001512576410001311
Figure BDA0001512576410001321
Figure BDA0001512576410001331
Figure BDA0001512576410001341
Figure BDA0001512576410001351
Figure BDA0001512576410001361
Figure BDA0001512576410001371
Figure BDA0001512576410001381
Figure BDA0001512576410001391
Figure BDA0001512576410001401
Figure BDA0001512576410001411
Figure BDA0001512576410001421
Figure BDA0001512576410001431
Figure BDA0001512576410001441
Figure BDA0001512576410001451
Figure BDA0001512576410001461
Figure BDA0001512576410001471
Figure BDA0001512576410001481
Figure BDA0001512576410001491
Figure BDA0001512576410001501
Figure BDA0001512576410001511
Figure BDA0001512576410001521
Figure BDA0001512576410001531
Figure BDA0001512576410001541
Figure BDA0001512576410001551
Figure BDA0001512576410001561
Figure BDA0001512576410001571
Figure BDA0001512576410001581
Figure BDA0001512576410001591
Figure BDA0001512576410001601
Figure BDA0001512576410001611
Figure BDA0001512576410001621
Figure BDA0001512576410001631
Figure BDA0001512576410001641
Figure BDA0001512576410001651
Figure BDA0001512576410001661
Figure BDA0001512576410001671
Figure BDA0001512576410001681
Figure BDA0001512576410001691
Figure BDA0001512576410001701
Figure BDA0001512576410001711
Figure BDA0001512576410001721
Figure BDA0001512576410001731
Figure BDA0001512576410001741
Figure BDA0001512576410001751
Figure BDA0001512576410001761
Figure BDA0001512576410001771
Figure BDA0001512576410001781
Figure BDA0001512576410001791
Figure BDA0001512576410001801
Figure BDA0001512576410001811
Figure BDA0001512576410001821
Figure BDA0001512576410001831
Figure BDA0001512576410001841
Figure BDA0001512576410001851
Figure BDA0001512576410001861
Figure BDA0001512576410001871
Figure BDA0001512576410001881
Figure BDA0001512576410001891
Figure BDA0001512576410001901
Figure BDA0001512576410001911
Figure BDA0001512576410001921
Figure BDA0001512576410001931
Figure BDA0001512576410001941
Figure BDA0001512576410001951
Figure BDA0001512576410001961
Figure BDA0001512576410001971
COMPOUND(2)
Figure BDA0001512576410001972
Figure BDA0001512576410001981
Figure BDA0001512576410001991
Figure BDA0001512576410002001
Figure BDA0001512576410002011
Figure BDA0001512576410002021
Figure BDA0001512576410002031
Figure BDA0001512576410002041
Figure BDA0001512576410002051
Figure BDA0001512576410002061
Figure BDA0001512576410002071
Figure BDA0001512576410002081
Figure BDA0001512576410002091
Figure BDA0001512576410002101
Figure BDA0001512576410002111
Figure BDA0001512576410002121
Figure BDA0001512576410002131
Figure BDA0001512576410002141
Figure BDA0001512576410002151
Figure BDA0001512576410002161
Figure BDA0001512576410002171
Figure BDA0001512576410002181
Figure BDA0001512576410002191
Figure BDA0001512576410002201
Figure BDA0001512576410002211
Figure BDA0001512576410002221
Figure BDA0001512576410002231
Figure BDA0001512576410002241
Figure BDA0001512576410002251
Figure BDA0001512576410002261
Figure BDA0001512576410002271
Figure BDA0001512576410002281
Figure BDA0001512576410002291
Figure BDA0001512576410002301
Figure BDA0001512576410002311
Figure BDA0001512576410002321
Figure BDA0001512576410002331
Figure BDA0001512576410002341
Figure BDA0001512576410002351
Figure BDA0001512576410002361
Figure BDA0001512576410002371
Figure BDA0001512576410002381
Figure BDA0001512576410002391
Figure BDA0001512576410002401
Figure BDA0001512576410002411
Figure BDA0001512576410002421
Figure BDA0001512576410002431
Figure BDA0001512576410002441
Figure BDA0001512576410002451
Figure BDA0001512576410002461
Figure BDA0001512576410002471
Figure BDA0001512576410002481
Figure BDA0001512576410002491
Figure BDA0001512576410002501
Figure BDA0001512576410002511
Figure BDA0001512576410002521
Figure BDA0001512576410002531
Figure BDA0001512576410002541
Figure BDA0001512576410002551
Figure BDA0001512576410002561
Figure BDA0001512576410002571
Figure BDA0001512576410002581
Figure BDA0001512576410002591
Figure BDA0001512576410002601
Figure BDA0001512576410002611
Figure BDA0001512576410002621
Figure BDA0001512576410002631
Figure BDA0001512576410002641
Figure BDA0001512576410002651
Figure BDA0001512576410002661
Figure BDA0001512576410002671
Figure BDA0001512576410002681
Figure BDA0001512576410002691
Figure BDA0001512576410002701
Figure BDA0001512576410002711
COMPOUND(3)
Figure BDA0001512576410002712
Figure BDA0001512576410002721
Figure BDA0001512576410002731
Figure BDA0001512576410002741
Figure BDA0001512576410002751
Figure BDA0001512576410002761
Figure BDA0001512576410002771
Figure BDA0001512576410002781
Figure BDA0001512576410002791
Figure BDA0001512576410002801
Figure BDA0001512576410002811
Figure BDA0001512576410002821
Figure BDA0001512576410002831
Figure BDA0001512576410002841
Figure BDA0001512576410002851
Figure BDA0001512576410002861
Figure BDA0001512576410002871
Figure BDA0001512576410002881
Figure BDA0001512576410002891
Figure BDA0001512576410002901
Figure BDA0001512576410002911
Figure BDA0001512576410002921
Figure BDA0001512576410002931
Figure BDA0001512576410002941
Figure BDA0001512576410002951
Figure BDA0001512576410002961
Figure BDA0001512576410002971
Figure BDA0001512576410002981
Figure BDA0001512576410002991
Figure BDA0001512576410003001
Figure BDA0001512576410003011
Figure BDA0001512576410003021
Figure BDA0001512576410003031
Figure BDA0001512576410003041
Figure BDA0001512576410003051
Figure BDA0001512576410003061
Figure BDA0001512576410003071
Figure BDA0001512576410003081
Figure BDA0001512576410003091
Figure BDA0001512576410003101
Figure BDA0001512576410003111
Figure BDA0001512576410003121
Figure BDA0001512576410003131
Figure BDA0001512576410003141
Figure BDA0001512576410003151
Figure BDA0001512576410003161
Figure BDA0001512576410003171
Figure BDA0001512576410003181
Figure BDA0001512576410003191
Figure BDA0001512576410003201
Figure BDA0001512576410003211
Figure BDA0001512576410003221
Figure BDA0001512576410003231
Figure BDA0001512576410003241
Figure BDA0001512576410003251
Figure BDA0001512576410003261
Figure BDA0001512576410003271
Figure BDA0001512576410003281
Figure BDA0001512576410003291
Figure BDA0001512576410003301
Figure BDA0001512576410003311
Figure BDA0001512576410003321
Figure BDA0001512576410003331
Figure BDA0001512576410003341
Figure BDA0001512576410003351
Figure BDA0001512576410003361
Figure BDA0001512576410003371
Figure BDA0001512576410003381
Figure BDA0001512576410003391
Figure BDA0001512576410003401
Figure BDA0001512576410003411
Figure BDA0001512576410003421
Figure BDA0001512576410003431
Figure BDA0001512576410003441
Figure BDA0001512576410003451
COMPOUND(4)
Figure BDA0001512576410003452
Figure BDA0001512576410003461
Figure BDA0001512576410003471
Figure BDA0001512576410003481
Figure BDA0001512576410003491
Figure BDA0001512576410003501
Figure BDA0001512576410003511
Figure BDA0001512576410003521
Figure BDA0001512576410003531
Figure BDA0001512576410003541
Figure BDA0001512576410003551
Figure BDA0001512576410003561
Figure BDA0001512576410003571
Figure BDA0001512576410003581
Figure BDA0001512576410003591
Figure BDA0001512576410003601
Figure BDA0001512576410003611
Figure BDA0001512576410003621
Figure BDA0001512576410003631
Figure BDA0001512576410003641
Figure BDA0001512576410003651
Figure BDA0001512576410003661
Figure BDA0001512576410003671
Figure BDA0001512576410003681
Figure BDA0001512576410003691
Figure BDA0001512576410003701
Figure BDA0001512576410003711
Figure BDA0001512576410003721
Figure BDA0001512576410003731
Figure BDA0001512576410003741
Figure BDA0001512576410003751
Figure BDA0001512576410003761
Figure BDA0001512576410003771
Figure BDA0001512576410003781
Figure BDA0001512576410003791
Figure BDA0001512576410003801
Figure BDA0001512576410003811
Figure BDA0001512576410003821
Figure BDA0001512576410003831
Figure BDA0001512576410003841
Figure BDA0001512576410003851
Figure BDA0001512576410003861
Figure BDA0001512576410003871
Figure BDA0001512576410003881
Figure BDA0001512576410003891
Figure BDA0001512576410003901
Figure BDA0001512576410003911
Figure BDA0001512576410003921
Figure BDA0001512576410003931
Figure BDA0001512576410003941
Figure BDA0001512576410003951
Figure BDA0001512576410003961
Figure BDA0001512576410003971
Figure BDA0001512576410003981
Figure BDA0001512576410003991
Figure BDA0001512576410004001
Figure BDA0001512576410004011
Figure BDA0001512576410004021
Figure BDA0001512576410004031
Figure BDA0001512576410004041
Figure BDA0001512576410004051
Figure BDA0001512576410004061
Figure BDA0001512576410004071
Figure BDA0001512576410004081
Figure BDA0001512576410004091
Figure BDA0001512576410004101
Figure BDA0001512576410004111
Figure BDA0001512576410004121
Figure BDA0001512576410004131
Figure BDA0001512576410004141
Figure BDA0001512576410004151
Figure BDA0001512576410004161
Figure BDA0001512576410004171
Figure BDA0001512576410004181
Figure BDA0001512576410004191
Figure BDA0001512576410004201
Figure BDA0001512576410004211
Figure BDA0001512576410004221
Figure BDA0001512576410004231
Figure BDA0001512576410004241
Figure BDA0001512576410004251
Figure BDA0001512576410004261
Figure BDA0001512576410004271
Figure BDA0001512576410004281
Figure BDA0001512576410004291
Figure BDA0001512576410004301
Figure BDA0001512576410004311
COMPOUND(5)
Figure BDA0001512576410004312
Figure BDA0001512576410004321
Figure BDA0001512576410004331
Figure BDA0001512576410004341
Figure BDA0001512576410004351
Figure BDA0001512576410004361
Figure BDA0001512576410004371
Figure BDA0001512576410004381
Figure BDA0001512576410004391
Figure BDA0001512576410004401
Figure BDA0001512576410004411
Figure BDA0001512576410004421
Figure BDA0001512576410004431
Figure BDA0001512576410004441
Figure BDA0001512576410004451
Figure BDA0001512576410004461
Figure BDA0001512576410004471
Figure BDA0001512576410004481
Figure BDA0001512576410004491
Figure BDA0001512576410004501
Figure BDA0001512576410004511
Figure BDA0001512576410004521
Figure BDA0001512576410004531
Figure BDA0001512576410004541
Figure BDA0001512576410004551
Figure BDA0001512576410004561
Figure BDA0001512576410004571
Figure BDA0001512576410004581
Figure BDA0001512576410004591
Figure BDA0001512576410004601
Figure BDA0001512576410004611
Figure BDA0001512576410004621
Figure BDA0001512576410004631
Figure BDA0001512576410004641
Figure BDA0001512576410004651
Figure BDA0001512576410004661
Figure BDA0001512576410004671
Figure BDA0001512576410004681
Figure BDA0001512576410004691
Figure BDA0001512576410004701
Figure BDA0001512576410004711
Figure BDA0001512576410004721
Figure BDA0001512576410004731
Figure BDA0001512576410004741
Figure BDA0001512576410004751
Figure BDA0001512576410004761
Figure BDA0001512576410004771
Figure BDA0001512576410004781
Figure BDA0001512576410004791
Figure BDA0001512576410004801
Figure BDA0001512576410004811
Figure BDA0001512576410004821
Figure BDA0001512576410004831
Figure BDA0001512576410004841
Figure BDA0001512576410004851
Figure BDA0001512576410004861
Figure BDA0001512576410004871
Figure BDA0001512576410004881
Figure BDA0001512576410004891
Figure BDA0001512576410004901
Figure BDA0001512576410004911
Figure BDA0001512576410004921
Figure BDA0001512576410004931
Figure BDA0001512576410004941
Figure BDA0001512576410004951
Figure BDA0001512576410004961
Figure BDA0001512576410004971
Figure BDA0001512576410004981
Figure BDA0001512576410004991
Figure BDA0001512576410005001
Figure BDA0001512576410005011
COMPOUND(6)
Figure BDA0001512576410005012
Figure BDA0001512576410005021
Figure BDA0001512576410005031
Figure BDA0001512576410005041
Figure BDA0001512576410005051
Figure BDA0001512576410005061
Figure BDA0001512576410005071
Figure BDA0001512576410005081
Figure BDA0001512576410005091
Figure BDA0001512576410005101
Figure BDA0001512576410005111
Figure BDA0001512576410005121
Figure BDA0001512576410005131
Figure BDA0001512576410005141
Figure BDA0001512576410005151
Figure BDA0001512576410005161
Figure BDA0001512576410005171
Figure BDA0001512576410005181
Figure BDA0001512576410005191
Figure BDA0001512576410005201
Figure BDA0001512576410005211
Figure BDA0001512576410005221
Figure BDA0001512576410005231
Figure BDA0001512576410005241
Figure BDA0001512576410005251
Figure BDA0001512576410005261
Figure BDA0001512576410005271
Figure BDA0001512576410005281
Figure BDA0001512576410005291
Figure BDA0001512576410005301
Figure BDA0001512576410005311
Figure BDA0001512576410005321
Figure BDA0001512576410005331
Figure BDA0001512576410005341
Figure BDA0001512576410005351
Figure BDA0001512576410005361
Figure BDA0001512576410005371
Figure BDA0001512576410005381
Figure BDA0001512576410005391
Figure BDA0001512576410005401
Figure BDA0001512576410005411
Figure BDA0001512576410005421
Figure BDA0001512576410005431
Figure BDA0001512576410005441
Figure BDA0001512576410005451
Figure BDA0001512576410005461
Figure BDA0001512576410005471
Figure BDA0001512576410005481
Figure BDA0001512576410005491
Figure BDA0001512576410005501
Figure BDA0001512576410005511
Figure BDA0001512576410005521
Figure BDA0001512576410005531
Figure BDA0001512576410005541
Figure BDA0001512576410005551
Figure BDA0001512576410005561
Figure BDA0001512576410005571
Figure BDA0001512576410005581
Figure BDA0001512576410005591
Figure BDA0001512576410005601
Figure BDA0001512576410005611
Figure BDA0001512576410005621
Figure BDA0001512576410005631
Figure BDA0001512576410005641
Figure BDA0001512576410005651
Figure BDA0001512576410005661
Figure BDA0001512576410005671
Figure BDA0001512576410005681
Figure BDA0001512576410005691
Figure BDA0001512576410005701
Figure BDA0001512576410005711
Figure BDA0001512576410005721
Figure BDA0001512576410005731
Figure BDA0001512576410005741
Figure BDA0001512576410005751
COMPOUND(7)
Figure BDA0001512576410005752
Figure BDA0001512576410005761
Figure BDA0001512576410005771
Figure BDA0001512576410005781
Figure BDA0001512576410005791
Figure BDA0001512576410005801
Figure BDA0001512576410005811
Figure BDA0001512576410005821
Figure BDA0001512576410005831
Figure BDA0001512576410005841
Figure BDA0001512576410005851
Figure BDA0001512576410005861
Figure BDA0001512576410005871
Figure BDA0001512576410005881
Figure BDA0001512576410005891
Figure BDA0001512576410005901
Figure BDA0001512576410005911
Figure BDA0001512576410005921
Figure BDA0001512576410005931
Figure BDA0001512576410005941
Figure BDA0001512576410005951
Figure BDA0001512576410005961
Figure BDA0001512576410005971
Figure BDA0001512576410005981
Figure BDA0001512576410005991
Figure BDA0001512576410006001
Figure BDA0001512576410006011
Figure BDA0001512576410006021
Figure BDA0001512576410006031
Figure BDA0001512576410006041
Figure BDA0001512576410006051
Figure BDA0001512576410006061
Figure BDA0001512576410006071
Figure BDA0001512576410006081
Figure BDA0001512576410006091
Figure BDA0001512576410006101
Figure BDA0001512576410006111
Figure BDA0001512576410006121
Figure BDA0001512576410006131
Figure BDA0001512576410006141
Figure BDA0001512576410006151
Figure BDA0001512576410006161
Figure BDA0001512576410006171
Figure BDA0001512576410006181
Figure BDA0001512576410006191
Figure BDA0001512576410006201
Figure BDA0001512576410006211
Figure BDA0001512576410006221
Figure BDA0001512576410006231
Figure BDA0001512576410006241
Figure BDA0001512576410006251
Figure BDA0001512576410006261
Figure BDA0001512576410006271
Figure BDA0001512576410006281
Figure BDA0001512576410006291
Figure BDA0001512576410006301
Figure BDA0001512576410006311
Figure BDA0001512576410006321
Figure BDA0001512576410006331
Figure BDA0001512576410006341
Figure BDA0001512576410006351
Figure BDA0001512576410006361
Figure BDA0001512576410006371
Figure BDA0001512576410006381
Figure BDA0001512576410006391
Figure BDA0001512576410006401
Figure BDA0001512576410006411
Figure BDA0001512576410006421
Figure BDA0001512576410006431
Figure BDA0001512576410006441
Figure BDA0001512576410006451
Figure BDA0001512576410006461
Figure BDA0001512576410006471
Figure BDA0001512576410006481
Figure BDA0001512576410006491
COMPOUND(8)
Figure BDA0001512576410006492
Figure BDA0001512576410006501
Figure BDA0001512576410006511
Figure BDA0001512576410006521
Figure BDA0001512576410006531
Figure BDA0001512576410006541
Figure BDA0001512576410006551
Figure BDA0001512576410006561
Figure BDA0001512576410006571
Figure BDA0001512576410006581
Figure BDA0001512576410006591
Figure BDA0001512576410006601
Figure BDA0001512576410006611
Figure BDA0001512576410006621
Figure BDA0001512576410006631
Figure BDA0001512576410006641
Figure BDA0001512576410006651
Figure BDA0001512576410006661
Figure BDA0001512576410006671
Figure BDA0001512576410006681
Figure BDA0001512576410006691
Figure BDA0001512576410006701
Figure BDA0001512576410006711
Figure BDA0001512576410006721
Figure BDA0001512576410006731
Figure BDA0001512576410006741
Figure BDA0001512576410006751
Figure BDA0001512576410006761
Figure BDA0001512576410006771
Figure BDA0001512576410006781
Figure BDA0001512576410006791
Figure BDA0001512576410006801
Figure BDA0001512576410006811
Figure BDA0001512576410006821
Figure BDA0001512576410006831
Figure BDA0001512576410006841
Figure BDA0001512576410006851
Figure BDA0001512576410006861
Figure BDA0001512576410006871
Figure BDA0001512576410006881
Figure BDA0001512576410006891
Figure BDA0001512576410006901
Figure BDA0001512576410006911
Figure BDA0001512576410006921
Figure BDA0001512576410006931
Figure BDA0001512576410006941
Figure BDA0001512576410006951
Figure BDA0001512576410006961
Figure BDA0001512576410006971
Figure BDA0001512576410006981
Figure BDA0001512576410006991
Figure BDA0001512576410007001
Figure BDA0001512576410007011
Figure BDA0001512576410007021
Figure BDA0001512576410007031
Figure BDA0001512576410007041
Figure BDA0001512576410007051
Figure BDA0001512576410007061
Figure BDA0001512576410007071
Figure BDA0001512576410007081
Figure BDA0001512576410007091
Figure BDA0001512576410007101
Figure BDA0001512576410007111
Figure BDA0001512576410007121
Figure BDA0001512576410007131
Figure BDA0001512576410007141
Figure BDA0001512576410007151
Figure BDA0001512576410007161
Figure BDA0001512576410007171
Figure BDA0001512576410007181
Figure BDA0001512576410007191
Figure BDA0001512576410007201
Figure BDA0001512576410007211
Figure BDA0001512576410007221
Figure BDA0001512576410007231
COMPOUND(9)
Figure BDA0001512576410007232
Figure BDA0001512576410007241
Figure BDA0001512576410007251
Figure BDA0001512576410007261
Figure BDA0001512576410007271
Figure BDA0001512576410007281
Figure BDA0001512576410007291
Figure BDA0001512576410007301
Figure BDA0001512576410007311
Figure BDA0001512576410007321
Figure BDA0001512576410007331
Figure BDA0001512576410007341
Figure BDA0001512576410007351
Figure BDA0001512576410007361
Figure BDA0001512576410007371
Figure BDA0001512576410007381
Figure BDA0001512576410007391
Figure BDA0001512576410007401
Figure BDA0001512576410007411
Figure BDA0001512576410007421
Figure BDA0001512576410007431
Figure BDA0001512576410007441
Figure BDA0001512576410007451
Figure BDA0001512576410007461
Figure BDA0001512576410007471
Figure BDA0001512576410007481
Figure BDA0001512576410007491
Figure BDA0001512576410007501
Figure BDA0001512576410007511
Figure BDA0001512576410007521
Figure BDA0001512576410007531
Figure BDA0001512576410007541
Figure BDA0001512576410007551
Figure BDA0001512576410007561
Figure BDA0001512576410007571
Figure BDA0001512576410007581
Figure BDA0001512576410007591
Figure BDA0001512576410007601
Figure BDA0001512576410007611
Figure BDA0001512576410007621
Figure BDA0001512576410007631
Figure BDA0001512576410007641
Figure BDA0001512576410007651
Figure BDA0001512576410007661
Figure BDA0001512576410007671
Figure BDA0001512576410007681
Figure BDA0001512576410007691
Figure BDA0001512576410007701
Figure BDA0001512576410007711
Figure BDA0001512576410007721
Figure BDA0001512576410007731
Figure BDA0001512576410007741
Figure BDA0001512576410007751
Figure BDA0001512576410007761
Figure BDA0001512576410007771
Figure BDA0001512576410007781
Figure BDA0001512576410007791
Figure BDA0001512576410007801
Figure BDA0001512576410007811
Figure BDA0001512576410007821
Figure BDA0001512576410007831
Figure BDA0001512576410007841
Figure BDA0001512576410007851
Figure BDA0001512576410007861
Figure BDA0001512576410007871
Figure BDA0001512576410007881
Figure BDA0001512576410007891
Figure BDA0001512576410007901
Figure BDA0001512576410007911
COMPOUND(10)
Figure BDA0001512576410007912
Figure BDA0001512576410007921
Figure BDA0001512576410007931
Figure BDA0001512576410007941
Figure BDA0001512576410007951
Figure BDA0001512576410007961
Figure BDA0001512576410007971
Figure BDA0001512576410007981
Figure BDA0001512576410007991
Figure BDA0001512576410008001
Figure BDA0001512576410008011
Figure BDA0001512576410008021
Figure BDA0001512576410008031
Figure BDA0001512576410008041
Figure BDA0001512576410008051
Figure BDA0001512576410008061
Figure BDA0001512576410008071
Figure BDA0001512576410008081
Figure BDA0001512576410008091
Figure BDA0001512576410008101
Figure BDA0001512576410008111
Figure BDA0001512576410008121
Figure BDA0001512576410008131
Figure BDA0001512576410008141
Figure BDA0001512576410008151
Figure BDA0001512576410008161
Figure BDA0001512576410008171
Figure BDA0001512576410008181
Figure BDA0001512576410008191
Figure BDA0001512576410008201
Figure BDA0001512576410008211
Figure BDA0001512576410008221
Figure BDA0001512576410008231
Figure BDA0001512576410008241
Figure BDA0001512576410008251
Figure BDA0001512576410008261
Figure BDA0001512576410008271
Figure BDA0001512576410008281
Figure BDA0001512576410008291
Figure BDA0001512576410008301
Figure BDA0001512576410008311
Figure BDA0001512576410008321
Figure BDA0001512576410008331
Figure BDA0001512576410008341
Figure BDA0001512576410008351
Figure BDA0001512576410008361
Figure BDA0001512576410008371
Figure BDA0001512576410008381
Figure BDA0001512576410008391
Figure BDA0001512576410008401
Figure BDA0001512576410008411
Figure BDA0001512576410008421
Figure BDA0001512576410008431
Figure BDA0001512576410008441
Figure BDA0001512576410008451
Figure BDA0001512576410008461
Figure BDA0001512576410008471
Figure BDA0001512576410008481
Figure BDA0001512576410008491
Figure BDA0001512576410008501
Figure BDA0001512576410008511
Figure BDA0001512576410008521
Figure BDA0001512576410008531
Figure BDA0001512576410008541
Figure BDA0001512576410008551
Figure BDA0001512576410008561
Figure BDA0001512576410008571
Figure BDA0001512576410008581
Figure BDA0001512576410008591
Figure BDA0001512576410008601
Figure BDA0001512576410008611
Figure BDA0001512576410008621
Figure BDA0001512576410008631
Figure BDA0001512576410008641
COMPOUND(11)
Figure BDA0001512576410008642
Figure BDA0001512576410008651
Figure BDA0001512576410008661
Figure BDA0001512576410008671
Figure BDA0001512576410008681
Figure BDA0001512576410008691
Figure BDA0001512576410008701
Figure BDA0001512576410008711
Figure BDA0001512576410008721
Figure BDA0001512576410008731
Figure BDA0001512576410008741
Figure BDA0001512576410008751
Figure BDA0001512576410008761
Figure BDA0001512576410008771
Figure BDA0001512576410008781
Figure BDA0001512576410008791
Figure BDA0001512576410008801
Figure BDA0001512576410008811
Figure BDA0001512576410008821
Figure BDA0001512576410008831
Figure BDA0001512576410008841
Figure BDA0001512576410008851
Figure BDA0001512576410008861
Figure BDA0001512576410008871
Figure BDA0001512576410008881
Figure BDA0001512576410008891
Figure BDA0001512576410008901
Figure BDA0001512576410008911
Figure BDA0001512576410008921
Figure BDA0001512576410008931
Figure BDA0001512576410008941
Figure BDA0001512576410008951
Figure BDA0001512576410008961
Figure BDA0001512576410008971
Figure BDA0001512576410008981
Figure BDA0001512576410008991
Figure BDA0001512576410009001
Figure BDA0001512576410009011
Figure BDA0001512576410009021
Figure BDA0001512576410009031
Figure BDA0001512576410009041
Figure BDA0001512576410009051
Figure BDA0001512576410009061
Figure BDA0001512576410009071
Figure BDA0001512576410009081
Figure BDA0001512576410009091
Figure BDA0001512576410009101
Figure BDA0001512576410009111
Figure BDA0001512576410009121
Figure BDA0001512576410009131
Figure BDA0001512576410009141
Figure BDA0001512576410009151
Figure BDA0001512576410009161
Figure BDA0001512576410009171
Figure BDA0001512576410009181
Figure BDA0001512576410009191
Figure BDA0001512576410009201
Figure BDA0001512576410009211
Figure BDA0001512576410009221
Figure BDA0001512576410009231
Figure BDA0001512576410009241
Figure BDA0001512576410009251
Figure BDA0001512576410009261
Figure BDA0001512576410009271
Figure BDA0001512576410009281
Figure BDA0001512576410009291
Figure BDA0001512576410009301
Figure BDA0001512576410009311
Figure BDA0001512576410009321
Figure BDA0001512576410009331
Figure BDA0001512576410009341
Figure BDA0001512576410009351
COMPOUND(12)
Figure BDA0001512576410009352
Figure BDA0001512576410009361
Figure BDA0001512576410009371
Figure BDA0001512576410009381
Figure BDA0001512576410009391
Figure BDA0001512576410009401
Figure BDA0001512576410009411
Figure BDA0001512576410009421
Figure BDA0001512576410009431
Figure BDA0001512576410009441
Figure BDA0001512576410009451
Figure BDA0001512576410009461
Figure BDA0001512576410009471
Figure BDA0001512576410009481
Figure BDA0001512576410009491
Figure BDA0001512576410009501
Figure BDA0001512576410009511
Figure BDA0001512576410009521
Figure BDA0001512576410009531
Figure BDA0001512576410009541
Figure BDA0001512576410009551
Figure BDA0001512576410009561
Figure BDA0001512576410009571
Figure BDA0001512576410009581
Figure BDA0001512576410009591
Figure BDA0001512576410009601
Figure BDA0001512576410009611
Figure BDA0001512576410009621
Figure BDA0001512576410009631
Figure BDA0001512576410009641
Figure BDA0001512576410009651
Figure BDA0001512576410009661
Figure BDA0001512576410009671
Figure BDA0001512576410009681
Figure BDA0001512576410009691
Figure BDA0001512576410009701
Figure BDA0001512576410009711
Figure BDA0001512576410009721
Figure BDA0001512576410009731
Figure BDA0001512576410009741
Figure BDA0001512576410009751
Figure BDA0001512576410009761
Figure BDA0001512576410009771
Figure BDA0001512576410009781
Figure BDA0001512576410009791
Figure BDA0001512576410009801
Figure BDA0001512576410009811
Figure BDA0001512576410009821
Figure BDA0001512576410009831
Figure BDA0001512576410009841
Figure BDA0001512576410009851
Figure BDA0001512576410009861
Figure BDA0001512576410009871
Figure BDA0001512576410009881
Figure BDA0001512576410009891
Figure BDA0001512576410009901
Figure BDA0001512576410009911
Figure BDA0001512576410009921
Figure BDA0001512576410009931
Figure BDA0001512576410009941
Figure BDA0001512576410009951
Figure BDA0001512576410009961
Figure BDA0001512576410009971
Figure BDA0001512576410009981
Figure BDA0001512576410009991
Figure BDA0001512576410010001
Figure BDA0001512576410010011
Figure BDA0001512576410010021
Figure BDA0001512576410010031
Figure BDA0001512576410010041
Figure BDA0001512576410010051
Figure BDA0001512576410010061
Figure BDA0001512576410010071
Figure BDA0001512576410010081
Figure BDA0001512576410010091
Figure BDA0001512576410010101
COMPOUND(13)
Figure BDA0001512576410010102
Figure BDA0001512576410010111
Figure BDA0001512576410010121
Figure BDA0001512576410010131
Figure BDA0001512576410010141
Figure BDA0001512576410010151
Figure BDA0001512576410010161
Figure BDA0001512576410010171
Figure BDA0001512576410010181
Figure BDA0001512576410010191
Figure BDA0001512576410010201
Figure BDA0001512576410010211
Figure BDA0001512576410010221
Figure BDA0001512576410010231
Figure BDA0001512576410010241
Figure BDA0001512576410010251
Figure BDA0001512576410010261
Figure BDA0001512576410010271
Figure BDA0001512576410010281
Figure BDA0001512576410010291
Figure BDA0001512576410010301
Figure BDA0001512576410010311
Figure BDA0001512576410010321
Figure BDA0001512576410010331
Figure BDA0001512576410010341
Figure BDA0001512576410010351
Figure BDA0001512576410010361
Figure BDA0001512576410010371
Figure BDA0001512576410010381
Figure BDA0001512576410010391
Figure BDA0001512576410010401
Figure BDA0001512576410010411
Figure BDA0001512576410010421
Figure BDA0001512576410010431
Figure BDA0001512576410010441
Figure BDA0001512576410010451
Figure BDA0001512576410010461
Figure BDA0001512576410010471
Figure BDA0001512576410010481
Figure BDA0001512576410010491
Figure BDA0001512576410010501
Figure BDA0001512576410010511
Figure BDA0001512576410010521
Figure BDA0001512576410010531
Figure BDA0001512576410010541
Figure BDA0001512576410010551
Figure BDA0001512576410010561
Figure BDA0001512576410010571
Figure BDA0001512576410010581
Figure BDA0001512576410010591
Figure BDA0001512576410010601
Figure BDA0001512576410010611
Figure BDA0001512576410010621
Figure BDA0001512576410010631
Figure BDA0001512576410010641
Figure BDA0001512576410010651
Figure BDA0001512576410010661
Figure BDA0001512576410010671
Figure BDA0001512576410010681
Figure BDA0001512576410010691
Figure BDA0001512576410010701
Figure BDA0001512576410010711
Figure BDA0001512576410010721
Figure BDA0001512576410010731
Figure BDA0001512576410010741
Figure BDA0001512576410010751
Figure BDA0001512576410010761
Figure BDA0001512576410010771
Figure BDA0001512576410010781
Figure BDA0001512576410010791
Figure BDA0001512576410010801
Figure BDA0001512576410010811
COMPOUND(14)
Figure BDA0001512576410010812
Figure BDA0001512576410010821
Figure BDA0001512576410010831
Figure BDA0001512576410010841
Figure BDA0001512576410010851
Figure BDA0001512576410010861
Figure BDA0001512576410010871
Figure BDA0001512576410010881
Figure BDA0001512576410010891
Figure BDA0001512576410010901
Figure BDA0001512576410010911
Figure BDA0001512576410010921
Figure BDA0001512576410010931
Figure BDA0001512576410010941
Figure BDA0001512576410010951
Figure BDA0001512576410010961
Figure BDA0001512576410010971
Figure BDA0001512576410010981
Figure BDA0001512576410010991
Figure BDA0001512576410011001
Figure BDA0001512576410011011
Figure BDA0001512576410011021
Figure BDA0001512576410011031
Figure BDA0001512576410011041
Figure BDA0001512576410011051
Figure BDA0001512576410011061
Figure BDA0001512576410011071
Figure BDA0001512576410011081
Figure BDA0001512576410011091
Figure BDA0001512576410011101
Figure BDA0001512576410011111
Figure BDA0001512576410011121
Figure BDA0001512576410011131
Figure BDA0001512576410011141
Figure BDA0001512576410011151
Figure BDA0001512576410011161
Figure BDA0001512576410011171
Figure BDA0001512576410011181
Figure BDA0001512576410011191
Figure BDA0001512576410011201
Figure BDA0001512576410011211
Figure BDA0001512576410011221
Figure BDA0001512576410011231
Figure BDA0001512576410011241
Figure BDA0001512576410011251
Figure BDA0001512576410011261
Figure BDA0001512576410011271
Figure BDA0001512576410011281
Figure BDA0001512576410011291
Figure BDA0001512576410011301
Figure BDA0001512576410011311
Figure BDA0001512576410011321
Figure BDA0001512576410011331
Figure BDA0001512576410011341
Figure BDA0001512576410011351
Figure BDA0001512576410011361
Figure BDA0001512576410011371
Figure BDA0001512576410011381
Figure BDA0001512576410011391
Figure BDA0001512576410011401
Figure BDA0001512576410011411
Figure BDA0001512576410011421
Figure BDA0001512576410011431
Figure BDA0001512576410011441
Figure BDA0001512576410011451
Figure BDA0001512576410011461
Figure BDA0001512576410011471
Figure BDA0001512576410011481
Figure BDA0001512576410011491
Figure BDA0001512576410011501
Figure BDA0001512576410011511
Figure BDA0001512576410011521
Figure BDA0001512576410011531
Figure BDA0001512576410011541
Figure BDA0001512576410011551
Figure BDA0001512576410011561
Figure BDA0001512576410011571
Figure BDA0001512576410011581
COMPOUND(15)
Figure BDA0001512576410011582
Figure BDA0001512576410011591
Figure BDA0001512576410011601
Figure BDA0001512576410011611
Figure BDA0001512576410011621
Figure BDA0001512576410011631
Figure BDA0001512576410011641
Figure BDA0001512576410011651
Figure BDA0001512576410011661
Figure BDA0001512576410011671
Figure BDA0001512576410011681
Figure BDA0001512576410011691
Figure BDA0001512576410011701
Figure BDA0001512576410011711
Figure BDA0001512576410011721
Figure BDA0001512576410011731
Figure BDA0001512576410011741
Figure BDA0001512576410011751
Figure BDA0001512576410011761
Figure BDA0001512576410011771
Figure BDA0001512576410011781
Figure BDA0001512576410011791
Figure BDA0001512576410011801
Figure BDA0001512576410011811
Figure BDA0001512576410011821
Figure BDA0001512576410011831
Figure BDA0001512576410011841
Figure BDA0001512576410011851
Figure BDA0001512576410011861
Figure BDA0001512576410011871
Figure BDA0001512576410011881
Figure BDA0001512576410011891
Figure BDA0001512576410011901
Figure BDA0001512576410011911
Figure BDA0001512576410011921
Figure BDA0001512576410011931
Figure BDA0001512576410011941
Figure BDA0001512576410011951
Figure BDA0001512576410011961
Figure BDA0001512576410011971
Figure BDA0001512576410011981
Figure BDA0001512576410011991
Figure BDA0001512576410012001
Figure BDA0001512576410012011
Figure BDA0001512576410012021
Figure BDA0001512576410012031
Figure BDA0001512576410012041
Figure BDA0001512576410012051
Figure BDA0001512576410012061
Figure BDA0001512576410012071
Figure BDA0001512576410012081
Figure BDA0001512576410012091
Figure BDA0001512576410012101
Figure BDA0001512576410012111
Figure BDA0001512576410012121
Figure BDA0001512576410012131
Figure BDA0001512576410012141
Figure BDA0001512576410012151
Figure BDA0001512576410012161
Figure BDA0001512576410012171
Figure BDA0001512576410012181
Figure BDA0001512576410012191
Figure BDA0001512576410012201
Figure BDA0001512576410012211
Figure BDA0001512576410012221
Figure BDA0001512576410012231
Figure BDA0001512576410012241
Figure BDA0001512576410012251
Figure BDA0001512576410012261
Figure BDA0001512576410012271
Figure BDA0001512576410012281
Figure BDA0001512576410012291
COMPOUND(16)
Figure BDA0001512576410012292
Figure BDA0001512576410012301
Figure BDA0001512576410012311
Figure BDA0001512576410012321
Figure BDA0001512576410012331
Figure BDA0001512576410012341
Figure BDA0001512576410012351
Figure BDA0001512576410012361
Figure BDA0001512576410012371
Figure BDA0001512576410012381
Figure BDA0001512576410012391
Figure BDA0001512576410012401
Figure BDA0001512576410012411
Figure BDA0001512576410012421
Figure BDA0001512576410012431
Figure BDA0001512576410012441
Figure BDA0001512576410012451
Figure BDA0001512576410012461
Figure BDA0001512576410012471
Figure BDA0001512576410012481
Figure BDA0001512576410012491
Figure BDA0001512576410012501
Figure BDA0001512576410012511
Figure BDA0001512576410012521
Figure BDA0001512576410012531
Figure BDA0001512576410012541
Figure BDA0001512576410012551
Figure BDA0001512576410012561
Figure BDA0001512576410012571
Figure BDA0001512576410012581
Figure BDA0001512576410012591
Figure BDA0001512576410012601
Figure BDA0001512576410012611
Figure BDA0001512576410012621
Figure BDA0001512576410012631
Figure BDA0001512576410012641
Figure BDA0001512576410012651
Figure BDA0001512576410012661
Figure BDA0001512576410012671
Figure BDA0001512576410012681
Figure BDA0001512576410012691
Figure BDA0001512576410012701
Figure BDA0001512576410012711
Figure BDA0001512576410012721
Figure BDA0001512576410012731
Figure BDA0001512576410012741
Figure BDA0001512576410012751
Figure BDA0001512576410012761
Figure BDA0001512576410012771
Figure BDA0001512576410012781
Figure BDA0001512576410012791
Figure BDA0001512576410012801
Figure BDA0001512576410012811
Figure BDA0001512576410012821
Figure BDA0001512576410012831
Figure BDA0001512576410012841
Figure BDA0001512576410012851
Figure BDA0001512576410012861
Figure BDA0001512576410012871
Figure BDA0001512576410012881
Figure BDA0001512576410012891
Figure BDA0001512576410012901
Figure BDA0001512576410012911
Figure BDA0001512576410012921
Figure BDA0001512576410012931
Figure BDA0001512576410012941
Figure BDA0001512576410012951
Figure BDA0001512576410012961
Figure BDA0001512576410012971
Figure BDA0001512576410012981
Figure BDA0001512576410012991
Figure BDA0001512576410013001
Figure BDA0001512576410013011
Figure BDA0001512576410013021
Figure BDA0001512576410013031
Figure BDA0001512576410013041
Figure BDA0001512576410013051
Figure BDA0001512576410013061
Figure BDA0001512576410013071
COMPOUND(17)
Figure BDA0001512576410013072
Figure BDA0001512576410013081
Figure BDA0001512576410013091
Figure BDA0001512576410013101
Figure BDA0001512576410013111
Figure BDA0001512576410013121
Figure BDA0001512576410013131
Figure BDA0001512576410013141
Figure BDA0001512576410013151
Figure BDA0001512576410013161
Figure BDA0001512576410013171
Figure BDA0001512576410013181
Figure BDA0001512576410013191
Figure BDA0001512576410013201
Figure BDA0001512576410013211
Figure BDA0001512576410013221
Figure BDA0001512576410013231
Figure BDA0001512576410013241
Figure BDA0001512576410013251
Figure BDA0001512576410013261
Figure BDA0001512576410013271
Figure BDA0001512576410013281
Figure BDA0001512576410013291
Figure BDA0001512576410013301
Figure BDA0001512576410013311
Figure BDA0001512576410013321
Figure BDA0001512576410013331
Figure BDA0001512576410013341
Figure BDA0001512576410013351
Figure BDA0001512576410013361
Figure BDA0001512576410013371
Figure BDA0001512576410013381
Figure BDA0001512576410013391
Figure BDA0001512576410013401
Figure BDA0001512576410013411
Figure BDA0001512576410013421
Figure BDA0001512576410013431
Figure BDA0001512576410013441
Figure BDA0001512576410013451
Figure BDA0001512576410013461
Figure BDA0001512576410013471
Figure BDA0001512576410013481
Figure BDA0001512576410013491
Figure BDA0001512576410013501
Figure BDA0001512576410013511
Figure BDA0001512576410013521
Figure BDA0001512576410013531
Figure BDA0001512576410013541
Figure BDA0001512576410013551
Figure BDA0001512576410013561
Figure BDA0001512576410013571
Figure BDA0001512576410013581
Figure BDA0001512576410013591
Figure BDA0001512576410013601
Figure BDA0001512576410013611
Figure BDA0001512576410013621
Figure BDA0001512576410013631
Figure BDA0001512576410013641
Figure BDA0001512576410013651
Figure BDA0001512576410013661
Figure BDA0001512576410013671
Figure BDA0001512576410013681
Figure BDA0001512576410013691
Figure BDA0001512576410013701
Figure BDA0001512576410013711
Figure BDA0001512576410013721
Figure BDA0001512576410013731
Figure BDA0001512576410013741
Figure BDA0001512576410013751
Figure BDA0001512576410013761
Figure BDA0001512576410013771
Figure BDA0001512576410013781
Figure BDA0001512576410013791
Figure BDA0001512576410013801
Figure BDA0001512576410013811
Figure BDA0001512576410013821
Figure BDA0001512576410013831
COMPOUND(18)
Figure BDA0001512576410013832
Figure BDA0001512576410013841
Figure BDA0001512576410013851
Figure BDA0001512576410013861
Figure BDA0001512576410013871
Figure BDA0001512576410013881
Figure BDA0001512576410013891
Figure BDA0001512576410013901
Figure BDA0001512576410013911
Figure BDA0001512576410013921
Figure BDA0001512576410013931
Figure BDA0001512576410013941
Figure BDA0001512576410013951
Figure BDA0001512576410013961
Figure BDA0001512576410013971
Figure BDA0001512576410013981
Figure BDA0001512576410013991
Figure BDA0001512576410014001
Figure BDA0001512576410014011
Figure BDA0001512576410014021
Figure BDA0001512576410014031
Figure BDA0001512576410014041
Figure BDA0001512576410014051
Figure BDA0001512576410014061
Figure BDA0001512576410014071
Figure BDA0001512576410014081
Figure BDA0001512576410014091
Figure BDA0001512576410014101
Figure BDA0001512576410014111
Figure BDA0001512576410014121
Figure BDA0001512576410014131
Figure BDA0001512576410014141
Figure BDA0001512576410014151
Figure BDA0001512576410014161
Figure BDA0001512576410014171
Figure BDA0001512576410014181
Figure BDA0001512576410014191
Figure BDA0001512576410014201
Figure BDA0001512576410014211
Figure BDA0001512576410014221
Figure BDA0001512576410014231
Figure BDA0001512576410014241
Figure BDA0001512576410014251
Figure BDA0001512576410014261
Figure BDA0001512576410014271
Figure BDA0001512576410014281
Figure BDA0001512576410014291
Figure BDA0001512576410014301
Figure BDA0001512576410014311
Figure BDA0001512576410014321
Figure BDA0001512576410014331
Figure BDA0001512576410014341
Figure BDA0001512576410014351
Figure BDA0001512576410014361
Figure BDA0001512576410014371
Figure BDA0001512576410014381
Figure BDA0001512576410014391
Figure BDA0001512576410014401
Figure BDA0001512576410014411
Figure BDA0001512576410014421
Figure BDA0001512576410014431
Figure BDA0001512576410014441
Figure BDA0001512576410014451
Figure BDA0001512576410014461
Figure BDA0001512576410014471
Figure BDA0001512576410014481
Figure BDA0001512576410014491
Figure BDA0001512576410014501
Figure BDA0001512576410014511
Figure BDA0001512576410014521
Figure BDA0001512576410014531
Figure BDA0001512576410014541
Figure BDA0001512576410014551
Figure BDA0001512576410014561
COMPOUND(19)
Figure BDA0001512576410014562
Figure BDA0001512576410014571
Figure BDA0001512576410014581
Figure BDA0001512576410014591
Figure BDA0001512576410014601
Figure BDA0001512576410014611
Figure BDA0001512576410014621
Figure BDA0001512576410014631
Figure BDA0001512576410014641
Figure BDA0001512576410014651
Figure BDA0001512576410014661
Figure BDA0001512576410014671
Figure BDA0001512576410014681
Figure BDA0001512576410014691
Figure BDA0001512576410014701
Figure BDA0001512576410014711
Figure BDA0001512576410014721
Figure BDA0001512576410014731
Figure BDA0001512576410014741
Figure BDA0001512576410014751
Figure BDA0001512576410014761
Figure BDA0001512576410014771
Figure BDA0001512576410014781
Figure BDA0001512576410014791
Figure BDA0001512576410014801
Figure BDA0001512576410014811
Figure BDA0001512576410014821
Figure BDA0001512576410014831
Figure BDA0001512576410014841
Figure BDA0001512576410014851
Figure BDA0001512576410014861
Figure BDA0001512576410014871
Figure BDA0001512576410014881
Figure BDA0001512576410014891
Figure BDA0001512576410014901
Figure BDA0001512576410014911
Figure BDA0001512576410014921
Figure BDA0001512576410014931
Figure BDA0001512576410014941
Figure BDA0001512576410014951
Figure BDA0001512576410014961
Figure BDA0001512576410014971
Figure BDA0001512576410014981
Figure BDA0001512576410014991
Figure BDA0001512576410015001
Figure BDA0001512576410015011
Figure BDA0001512576410015021
Figure BDA0001512576410015031
Figure BDA0001512576410015041
Figure BDA0001512576410015051
Figure BDA0001512576410015061
Figure BDA0001512576410015071
Figure BDA0001512576410015081
Figure BDA0001512576410015091
Figure BDA0001512576410015101
Figure BDA0001512576410015111
Figure BDA0001512576410015121
Figure BDA0001512576410015131
Figure BDA0001512576410015141
Figure BDA0001512576410015151
Figure BDA0001512576410015161
Figure BDA0001512576410015171
Figure BDA0001512576410015181
Figure BDA0001512576410015191
Figure BDA0001512576410015201
Figure BDA0001512576410015211
Figure BDA0001512576410015221
Figure BDA0001512576410015231
Figure BDA0001512576410015241
Figure BDA0001512576410015251
Figure BDA0001512576410015261
Figure BDA0001512576410015271
Figure BDA0001512576410015281
Figure BDA0001512576410015291
Figure BDA0001512576410015301
COMPOUND(20)
Figure BDA0001512576410015302
Figure BDA0001512576410015311
Figure BDA0001512576410015321
Figure BDA0001512576410015331
Figure BDA0001512576410015341
Figure BDA0001512576410015351
Figure BDA0001512576410015361
Figure BDA0001512576410015371
Figure BDA0001512576410015381
Figure BDA0001512576410015391
Figure BDA0001512576410015401
Figure BDA0001512576410015411
Figure BDA0001512576410015421
Figure BDA0001512576410015431
Figure BDA0001512576410015441
Figure BDA0001512576410015451
Figure BDA0001512576410015461
Figure BDA0001512576410015471
Figure BDA0001512576410015481
Figure BDA0001512576410015491
Figure BDA0001512576410015501
Figure BDA0001512576410015511
Figure BDA0001512576410015521
Figure BDA0001512576410015531
Figure BDA0001512576410015541
Figure BDA0001512576410015551
Figure BDA0001512576410015561
Figure BDA0001512576410015571
Figure BDA0001512576410015581
Figure BDA0001512576410015591
Figure BDA0001512576410015601
Figure BDA0001512576410015611
Figure BDA0001512576410015621
Figure BDA0001512576410015631
Figure BDA0001512576410015641
Figure BDA0001512576410015651
Figure BDA0001512576410015661
Figure BDA0001512576410015671
Figure BDA0001512576410015681
Figure BDA0001512576410015691
Figure BDA0001512576410015701
Figure BDA0001512576410015711
Figure BDA0001512576410015721
Figure BDA0001512576410015731
Figure BDA0001512576410015741
Figure BDA0001512576410015751
Figure BDA0001512576410015761
Figure BDA0001512576410015771
Figure BDA0001512576410015781
Figure BDA0001512576410015791
Figure BDA0001512576410015801
Figure BDA0001512576410015811
Figure BDA0001512576410015821
Figure BDA0001512576410015831
Figure BDA0001512576410015841
Figure BDA0001512576410015851
Figure BDA0001512576410015861
Figure BDA0001512576410015871
Figure BDA0001512576410015881
Figure BDA0001512576410015891
Figure BDA0001512576410015901
Figure BDA0001512576410015911
Figure BDA0001512576410015921
Figure BDA0001512576410015931
Figure BDA0001512576410015941
Figure BDA0001512576410015951
Figure BDA0001512576410015961
Figure BDA0001512576410015971
Figure BDA0001512576410015981
Figure BDA0001512576410015991
Figure BDA0001512576410016001
Figure BDA0001512576410016011
Figure BDA0001512576410016021
Figure BDA0001512576410016031
Figure BDA0001512576410016041
COMPOUND(21)
Figure BDA0001512576410016042
Figure BDA0001512576410016051
Figure BDA0001512576410016061
Figure BDA0001512576410016071
Figure BDA0001512576410016081
Figure BDA0001512576410016091
Figure BDA0001512576410016101
Figure BDA0001512576410016111
Figure BDA0001512576410016121
Figure BDA0001512576410016131
Figure BDA0001512576410016141
Figure BDA0001512576410016151
Figure BDA0001512576410016161
Figure BDA0001512576410016171
Figure BDA0001512576410016181
Figure BDA0001512576410016191
Figure BDA0001512576410016201
Figure BDA0001512576410016211
Figure BDA0001512576410016221
Figure BDA0001512576410016231
Figure BDA0001512576410016241
Figure BDA0001512576410016251
Figure BDA0001512576410016261
Figure BDA0001512576410016271
Figure BDA0001512576410016281
Figure BDA0001512576410016291
Figure BDA0001512576410016301
Figure BDA0001512576410016311
Figure BDA0001512576410016321
Figure BDA0001512576410016331
Figure BDA0001512576410016341
Figure BDA0001512576410016351
Figure BDA0001512576410016361
Figure BDA0001512576410016371
Figure BDA0001512576410016381
Figure BDA0001512576410016391
Figure BDA0001512576410016401
Figure BDA0001512576410016411
Figure BDA0001512576410016421
Figure BDA0001512576410016431
Figure BDA0001512576410016441
Figure BDA0001512576410016451
Figure BDA0001512576410016461
Figure BDA0001512576410016471
Figure BDA0001512576410016481
Figure BDA0001512576410016491
Figure BDA0001512576410016501
Figure BDA0001512576410016511
Figure BDA0001512576410016521
Figure BDA0001512576410016531
Figure BDA0001512576410016541
Figure BDA0001512576410016551
Figure BDA0001512576410016561
Figure BDA0001512576410016571
Figure BDA0001512576410016581
Figure BDA0001512576410016591
Figure BDA0001512576410016601
Figure BDA0001512576410016611
Figure BDA0001512576410016621
Figure BDA0001512576410016631
Figure BDA0001512576410016641
Figure BDA0001512576410016651
Figure BDA0001512576410016661
Figure BDA0001512576410016671
Figure BDA0001512576410016681
Figure BDA0001512576410016691
Figure BDA0001512576410016701
Figure BDA0001512576410016711
Figure BDA0001512576410016721
Figure BDA0001512576410016731
Figure BDA0001512576410016741
Figure BDA0001512576410016751
Figure BDA0001512576410016761
Figure BDA0001512576410016771
Figure BDA0001512576410016781
Figure BDA0001512576410016791
Figure BDA0001512576410016801
COMPOUND(22)
Figure BDA0001512576410016802
Figure BDA0001512576410016811
Figure BDA0001512576410016821
Figure BDA0001512576410016831
Figure BDA0001512576410016841
Figure BDA0001512576410016851
Figure BDA0001512576410016861
Figure BDA0001512576410016871
Figure BDA0001512576410016881
Figure BDA0001512576410016891
Figure BDA0001512576410016901
Figure BDA0001512576410016911
Figure BDA0001512576410016921
Figure BDA0001512576410016931
Figure BDA0001512576410016941
Figure BDA0001512576410016951
Figure BDA0001512576410016961
Figure BDA0001512576410016971
Figure BDA0001512576410016981
Figure BDA0001512576410016991
Figure BDA0001512576410017001
Figure BDA0001512576410017011
Figure BDA0001512576410017021
Figure BDA0001512576410017031
Figure BDA0001512576410017041
Figure BDA0001512576410017051
Figure BDA0001512576410017061
Figure BDA0001512576410017071
Figure BDA0001512576410017081
Figure BDA0001512576410017091
Figure BDA0001512576410017101
Figure BDA0001512576410017111
Figure BDA0001512576410017121
Figure BDA0001512576410017131
Figure BDA0001512576410017141
Figure BDA0001512576410017151
Figure BDA0001512576410017161
Figure BDA0001512576410017171
Figure BDA0001512576410017181
Figure BDA0001512576410017191
Figure BDA0001512576410017201
Figure BDA0001512576410017211
Figure BDA0001512576410017221
Figure BDA0001512576410017231
Figure BDA0001512576410017241
Figure BDA0001512576410017251
Figure BDA0001512576410017261
Figure BDA0001512576410017271
Figure BDA0001512576410017281
Figure BDA0001512576410017291
Figure BDA0001512576410017301
Figure BDA0001512576410017311
Figure BDA0001512576410017321
Figure BDA0001512576410017331
Figure BDA0001512576410017341
Figure BDA0001512576410017351
Figure BDA0001512576410017361
Figure BDA0001512576410017371
Figure BDA0001512576410017381
Figure BDA0001512576410017391
Figure BDA0001512576410017401
Figure BDA0001512576410017411
Figure BDA0001512576410017421
Figure BDA0001512576410017431
Figure BDA0001512576410017441
Figure BDA0001512576410017451
Figure BDA0001512576410017461
Figure BDA0001512576410017471
Figure BDA0001512576410017481
Figure BDA0001512576410017491
Figure BDA0001512576410017501
Figure BDA0001512576410017511
Figure BDA0001512576410017521
Figure BDA0001512576410017531
COMPOUND(23)
Figure BDA0001512576410017532
Figure BDA0001512576410017541
Figure BDA0001512576410017551
Figure BDA0001512576410017561
Figure BDA0001512576410017571
Figure BDA0001512576410017581
Figure BDA0001512576410017591
Figure BDA0001512576410017601
Figure BDA0001512576410017611
Figure BDA0001512576410017621
Figure BDA0001512576410017631
Figure BDA0001512576410017641
Figure BDA0001512576410017651
Figure BDA0001512576410017661
Figure BDA0001512576410017671
Figure BDA0001512576410017681
Figure BDA0001512576410017691
Figure BDA0001512576410017701
Figure BDA0001512576410017711
Figure BDA0001512576410017721
Figure BDA0001512576410017731
Figure BDA0001512576410017741
Figure BDA0001512576410017751
Figure BDA0001512576410017761
Figure BDA0001512576410017771
Figure BDA0001512576410017781
Figure BDA0001512576410017791
Figure BDA0001512576410017801
Figure BDA0001512576410017811
Figure BDA0001512576410017821
Figure BDA0001512576410017831
Figure BDA0001512576410017841
Figure BDA0001512576410017851
Figure BDA0001512576410017861
Figure BDA0001512576410017871
Figure BDA0001512576410017881
Figure BDA0001512576410017891
Figure BDA0001512576410017901
Figure BDA0001512576410017911
Figure BDA0001512576410017921
Figure BDA0001512576410017931
Figure BDA0001512576410017941
Figure BDA0001512576410017951
Figure BDA0001512576410017961
Figure BDA0001512576410017971
Figure BDA0001512576410017981
Figure BDA0001512576410017991
Figure BDA0001512576410018001
Figure BDA0001512576410018011
Figure BDA0001512576410018021
Figure BDA0001512576410018031
Figure BDA0001512576410018041
Figure BDA0001512576410018051
Figure BDA0001512576410018061
Figure BDA0001512576410018071
Figure BDA0001512576410018081
Figure BDA0001512576410018091
Figure BDA0001512576410018101
Figure BDA0001512576410018111
Figure BDA0001512576410018121
Figure BDA0001512576410018131
Figure BDA0001512576410018141
Figure BDA0001512576410018151
Figure BDA0001512576410018161
Figure BDA0001512576410018171
Figure BDA0001512576410018181
Figure BDA0001512576410018191
Figure BDA0001512576410018201
Figure BDA0001512576410018211
Figure BDA0001512576410018221
Figure BDA0001512576410018231
COMPOUND(24)
Figure BDA0001512576410018241
Figure BDA0001512576410018251
Figure BDA0001512576410018261
Figure BDA0001512576410018271
Figure BDA0001512576410018281
Figure BDA0001512576410018291
Figure BDA0001512576410018301
Figure BDA0001512576410018311
Figure BDA0001512576410018321
Figure BDA0001512576410018331
Figure BDA0001512576410018341
Figure BDA0001512576410018351
Figure BDA0001512576410018361
Figure BDA0001512576410018371
Figure BDA0001512576410018381
Figure BDA0001512576410018391
Figure BDA0001512576410018401
Figure BDA0001512576410018411
Figure BDA0001512576410018421
Figure BDA0001512576410018431
Figure BDA0001512576410018441
Figure BDA0001512576410018451
Figure BDA0001512576410018461
Figure BDA0001512576410018471
Figure BDA0001512576410018481
Figure BDA0001512576410018491
Figure BDA0001512576410018501
Figure BDA0001512576410018511
Figure BDA0001512576410018521
Figure BDA0001512576410018531
Figure BDA0001512576410018541
Figure BDA0001512576410018551
Figure BDA0001512576410018561
Figure BDA0001512576410018571
Figure BDA0001512576410018581
Figure BDA0001512576410018591
Figure BDA0001512576410018601
Figure BDA0001512576410018611
Figure BDA0001512576410018621
Figure BDA0001512576410018631
Figure BDA0001512576410018641
Figure BDA0001512576410018651
Figure BDA0001512576410018661
Figure BDA0001512576410018671
Figure BDA0001512576410018681
Figure BDA0001512576410018691
Figure BDA0001512576410018701
Figure BDA0001512576410018711
Figure BDA0001512576410018721
Figure BDA0001512576410018731
Figure BDA0001512576410018741
Figure BDA0001512576410018751
Figure BDA0001512576410018761
Figure BDA0001512576410018771
Figure BDA0001512576410018781
Figure BDA0001512576410018791
Figure BDA0001512576410018801
Figure BDA0001512576410018811
Figure BDA0001512576410018821
Figure BDA0001512576410018831
Figure BDA0001512576410018841
Figure BDA0001512576410018851
Figure BDA0001512576410018861
Figure BDA0001512576410018871
Figure BDA0001512576410018881
Figure BDA0001512576410018891
Figure BDA0001512576410018901
Figure BDA0001512576410018911
Figure BDA0001512576410018921
Figure BDA0001512576410018931
Figure BDA0001512576410018941
Figure BDA0001512576410018951
Figure BDA0001512576410018961
Figure BDA0001512576410018971
Figure BDA0001512576410018981
COMPOUND(25)
Figure BDA0001512576410018982
Figure BDA0001512576410018991
Figure BDA0001512576410019001
Figure BDA0001512576410019011
Figure BDA0001512576410019021
Figure BDA0001512576410019031
Figure BDA0001512576410019041
Figure BDA0001512576410019051
Figure BDA0001512576410019061
Figure BDA0001512576410019071
Figure BDA0001512576410019081
Figure BDA0001512576410019091
Figure BDA0001512576410019101
Figure BDA0001512576410019111
Figure BDA0001512576410019121
Figure BDA0001512576410019131
Figure BDA0001512576410019141
Figure BDA0001512576410019151
Figure BDA0001512576410019161
Figure BDA0001512576410019171
Figure BDA0001512576410019181
Figure BDA0001512576410019191
Figure BDA0001512576410019201
Figure BDA0001512576410019211
Figure BDA0001512576410019221
Figure BDA0001512576410019231
Figure BDA0001512576410019241
Figure BDA0001512576410019251
Figure BDA0001512576410019261
Figure BDA0001512576410019271
Figure BDA0001512576410019281
Figure BDA0001512576410019291
Figure BDA0001512576410019301
Figure BDA0001512576410019311
Figure BDA0001512576410019321
Figure BDA0001512576410019331
Figure BDA0001512576410019341
Figure BDA0001512576410019351
Figure BDA0001512576410019361
Figure BDA0001512576410019371
Figure BDA0001512576410019381
Figure BDA0001512576410019391
Figure BDA0001512576410019401
Figure BDA0001512576410019411
Figure BDA0001512576410019421
Figure BDA0001512576410019431
Figure BDA0001512576410019441
Figure BDA0001512576410019451
Figure BDA0001512576410019461
Figure BDA0001512576410019471
Figure BDA0001512576410019481
Figure BDA0001512576410019491
Figure BDA0001512576410019501
Figure BDA0001512576410019511
Figure BDA0001512576410019521
Figure BDA0001512576410019531
Figure BDA0001512576410019541
Figure BDA0001512576410019551
Figure BDA0001512576410019561
Figure BDA0001512576410019571
Figure BDA0001512576410019581
Figure BDA0001512576410019591
Figure BDA0001512576410019601
Figure BDA0001512576410019611
Figure BDA0001512576410019621
Figure BDA0001512576410019631
Figure BDA0001512576410019641
Figure BDA0001512576410019651
Figure BDA0001512576410019661
Figure BDA0001512576410019671
Figure BDA0001512576410019681
COMPOUND(26)
Figure BDA0001512576410019682
Figure BDA0001512576410019691
Figure BDA0001512576410019701
Figure BDA0001512576410019711
Figure BDA0001512576410019721
Figure BDA0001512576410019731
Figure BDA0001512576410019741
Figure BDA0001512576410019751
Figure BDA0001512576410019761
Figure BDA0001512576410019771
Figure BDA0001512576410019781
Figure BDA0001512576410019791
Figure BDA0001512576410019801
Figure BDA0001512576410019811
Figure BDA0001512576410019821
Figure BDA0001512576410019831
Figure BDA0001512576410019841
Figure BDA0001512576410019851
Figure BDA0001512576410019861
Figure BDA0001512576410019871
Figure BDA0001512576410019881
Figure BDA0001512576410019891
Figure BDA0001512576410019901
Figure BDA0001512576410019911
Figure BDA0001512576410019921
Figure BDA0001512576410019931
Figure BDA0001512576410019941
Figure BDA0001512576410019951
Figure BDA0001512576410019961
Figure BDA0001512576410019971
Figure BDA0001512576410019981
Figure BDA0001512576410019991
Figure BDA0001512576410020001
Figure BDA0001512576410020011
Figure BDA0001512576410020021
Figure BDA0001512576410020031
Figure BDA0001512576410020041
Figure BDA0001512576410020051
Figure BDA0001512576410020061
Figure BDA0001512576410020071
Figure BDA0001512576410020081
Figure BDA0001512576410020091
Figure BDA0001512576410020101
Figure BDA0001512576410020111
Figure BDA0001512576410020121
Figure BDA0001512576410020131
Figure BDA0001512576410020141
Figure BDA0001512576410020151
Figure BDA0001512576410020161
Figure BDA0001512576410020171
Figure BDA0001512576410020181
Figure BDA0001512576410020191
Figure BDA0001512576410020201
Figure BDA0001512576410020211
Figure BDA0001512576410020221
Figure BDA0001512576410020231
Figure BDA0001512576410020241
Figure BDA0001512576410020251
Figure BDA0001512576410020261
Figure BDA0001512576410020271
Figure BDA0001512576410020281
Figure BDA0001512576410020291
Figure BDA0001512576410020301
Figure BDA0001512576410020311
Figure BDA0001512576410020321
Figure BDA0001512576410020331
Figure BDA0001512576410020341
Figure BDA0001512576410020351
Figure BDA0001512576410020361
Figure BDA0001512576410020371
Figure BDA0001512576410020381
Figure BDA0001512576410020391
Figure BDA0001512576410020401
Figure BDA0001512576410020411
Figure BDA0001512576410020421
Figure BDA0001512576410020431
Figure BDA0001512576410020441
Figure BDA0001512576410020451
Figure BDA0001512576410020461
Figure BDA0001512576410020471
COMPOUND(27)
Figure BDA0001512576410020472
Figure BDA0001512576410020481
Figure BDA0001512576410020491
Figure BDA0001512576410020501
Figure BDA0001512576410020511
Figure BDA0001512576410020521
Figure BDA0001512576410020531
Figure BDA0001512576410020541
Figure BDA0001512576410020551
Figure BDA0001512576410020561
Figure BDA0001512576410020571
Figure BDA0001512576410020581
Figure BDA0001512576410020591
Figure BDA0001512576410020601
Figure BDA0001512576410020611
Figure BDA0001512576410020621
Figure BDA0001512576410020631
Figure BDA0001512576410020641
Figure BDA0001512576410020651
Figure BDA0001512576410020661
Figure BDA0001512576410020671
Figure BDA0001512576410020681
Figure BDA0001512576410020691
Figure BDA0001512576410020701
Figure BDA0001512576410020711
Figure BDA0001512576410020721
Figure BDA0001512576410020731
Figure BDA0001512576410020741
Figure BDA0001512576410020751
Figure BDA0001512576410020761
Figure BDA0001512576410020771
Figure BDA0001512576410020781
Figure BDA0001512576410020791
Figure BDA0001512576410020801
Figure BDA0001512576410020811
Figure BDA0001512576410020821
Figure BDA0001512576410020831
Figure BDA0001512576410020841
Figure BDA0001512576410020851
Figure BDA0001512576410020861
Figure BDA0001512576410020871
Figure BDA0001512576410020881
Figure BDA0001512576410020891
Figure BDA0001512576410020901
Figure BDA0001512576410020911
Figure BDA0001512576410020921
Figure BDA0001512576410020931
Figure BDA0001512576410020941
Figure BDA0001512576410020951
Figure BDA0001512576410020961
Figure BDA0001512576410020971
Figure BDA0001512576410020981
Figure BDA0001512576410020991
Figure BDA0001512576410021001
Figure BDA0001512576410021011
Figure BDA0001512576410021021
Figure BDA0001512576410021031
Figure BDA0001512576410021041
Figure BDA0001512576410021051
Figure BDA0001512576410021061
Figure BDA0001512576410021071
Figure BDA0001512576410021081
Figure BDA0001512576410021091
Figure BDA0001512576410021101
Figure BDA0001512576410021111
Figure BDA0001512576410021121
Figure BDA0001512576410021131
Figure BDA0001512576410021141
Figure BDA0001512576410021151
Figure BDA0001512576410021161
Figure BDA0001512576410021171
Figure BDA0001512576410021181
Figure BDA0001512576410021191
Figure BDA0001512576410021201
Figure BDA0001512576410021211
Figure BDA0001512576410021221
Figure BDA0001512576410021231
Figure BDA0001512576410021241
COMPOUND(28)
Figure BDA0001512576410021242
Figure BDA0001512576410021251
Figure BDA0001512576410021261
Figure BDA0001512576410021271
Figure BDA0001512576410021281
Figure BDA0001512576410021291
Figure BDA0001512576410021301
Figure BDA0001512576410021311
Figure BDA0001512576410021321
Figure BDA0001512576410021331
Figure BDA0001512576410021341
Figure BDA0001512576410021351
Figure BDA0001512576410021361
Figure BDA0001512576410021371
Figure BDA0001512576410021381
Figure BDA0001512576410021391
Figure BDA0001512576410021401
Figure BDA0001512576410021411
Figure BDA0001512576410021421
Figure BDA0001512576410021431
Figure BDA0001512576410021441
Figure BDA0001512576410021451
Figure BDA0001512576410021461
Figure BDA0001512576410021471
Figure BDA0001512576410021481
Figure BDA0001512576410021491
Figure BDA0001512576410021501
Figure BDA0001512576410021511
Figure BDA0001512576410021521
Figure BDA0001512576410021531
Figure BDA0001512576410021541
Figure BDA0001512576410021551
Figure BDA0001512576410021561
Figure BDA0001512576410021571
Figure BDA0001512576410021581
Figure BDA0001512576410021591
Figure BDA0001512576410021601
Figure BDA0001512576410021611
Figure BDA0001512576410021621
Figure BDA0001512576410021631
Figure BDA0001512576410021641
Figure BDA0001512576410021651
Figure BDA0001512576410021661
Figure BDA0001512576410021671
Figure BDA0001512576410021681
Figure BDA0001512576410021691
Figure BDA0001512576410021701
Figure BDA0001512576410021711
Figure BDA0001512576410021721
Figure BDA0001512576410021731
Figure BDA0001512576410021741
Figure BDA0001512576410021751
Figure BDA0001512576410021761
Figure BDA0001512576410021771
Figure BDA0001512576410021781
Figure BDA0001512576410021791
Figure BDA0001512576410021801
Figure BDA0001512576410021811
Figure BDA0001512576410021821
Figure BDA0001512576410021831
Figure BDA0001512576410021841
Figure BDA0001512576410021851
Figure BDA0001512576410021861
Figure BDA0001512576410021871
Figure BDA0001512576410021881
Figure BDA0001512576410021891
Figure BDA0001512576410021901
Figure BDA0001512576410021911
Figure BDA0001512576410021921
Figure BDA0001512576410021931
Figure BDA0001512576410021941
Figure BDA0001512576410021951
Figure BDA0001512576410021961
COMPOUND(29)
Figure BDA0001512576410021962
Figure BDA0001512576410021971
Figure BDA0001512576410021981
Figure BDA0001512576410021991
Figure BDA0001512576410022001
Figure BDA0001512576410022011
Figure BDA0001512576410022021
Figure BDA0001512576410022031
Figure BDA0001512576410022041
Figure BDA0001512576410022051
Figure BDA0001512576410022061
Figure BDA0001512576410022071
Figure BDA0001512576410022081
Figure BDA0001512576410022091
Figure BDA0001512576410022101
Figure BDA0001512576410022111
Figure BDA0001512576410022121
Figure BDA0001512576410022131
Figure BDA0001512576410022141
Figure BDA0001512576410022151
Figure BDA0001512576410022161
Figure BDA0001512576410022171
Figure BDA0001512576410022181
Figure BDA0001512576410022191
Figure BDA0001512576410022201
Figure BDA0001512576410022211
Figure BDA0001512576410022221
Figure BDA0001512576410022231
Figure BDA0001512576410022241
Figure BDA0001512576410022251
Figure BDA0001512576410022261
Figure BDA0001512576410022271
Figure BDA0001512576410022281
Figure BDA0001512576410022291
Figure BDA0001512576410022301
Figure BDA0001512576410022311
Figure BDA0001512576410022321
Figure BDA0001512576410022331
Figure BDA0001512576410022341
Figure BDA0001512576410022351
Figure BDA0001512576410022361
Figure BDA0001512576410022371
Figure BDA0001512576410022381
Figure BDA0001512576410022391
Figure BDA0001512576410022401
Figure BDA0001512576410022411
Figure BDA0001512576410022421
Figure BDA0001512576410022431
Figure BDA0001512576410022441
Figure BDA0001512576410022451
Figure BDA0001512576410022461
Figure BDA0001512576410022471
Figure BDA0001512576410022481
Figure BDA0001512576410022491
Figure BDA0001512576410022501
Figure BDA0001512576410022511
Figure BDA0001512576410022521
Figure BDA0001512576410022531
Figure BDA0001512576410022541
Figure BDA0001512576410022551
Figure BDA0001512576410022561
Figure BDA0001512576410022571
Figure BDA0001512576410022581
Figure BDA0001512576410022591
Figure BDA0001512576410022601
Figure BDA0001512576410022611
Figure BDA0001512576410022621
Figure BDA0001512576410022631
Figure BDA0001512576410022641
Figure BDA0001512576410022651
Figure BDA0001512576410022661
Figure BDA0001512576410022671
Figure BDA0001512576410022681
Figure BDA0001512576410022691
Figure BDA0001512576410022701
Figure BDA0001512576410022711
COMPOUND(30)
Figure BDA0001512576410022712
Figure BDA0001512576410022721
Figure BDA0001512576410022731
Figure BDA0001512576410022741
Figure BDA0001512576410022751
Figure BDA0001512576410022761
Figure BDA0001512576410022771
Figure BDA0001512576410022781
Figure BDA0001512576410022791
Figure BDA0001512576410022801
Figure BDA0001512576410022811
Figure BDA0001512576410022821
Figure BDA0001512576410022831
Figure BDA0001512576410022841
Figure BDA0001512576410022851
Figure BDA0001512576410022861
Figure BDA0001512576410022871
Figure BDA0001512576410022881
Figure BDA0001512576410022891
Figure BDA0001512576410022901
Figure BDA0001512576410022911
Figure BDA0001512576410022921
Figure BDA0001512576410022931
Figure BDA0001512576410022941
Figure BDA0001512576410022951
Figure BDA0001512576410022961
Figure BDA0001512576410022971
Figure BDA0001512576410022981
Figure BDA0001512576410022991
Figure BDA0001512576410023001
Figure BDA0001512576410023011
Figure BDA0001512576410023021
Figure BDA0001512576410023031
Figure BDA0001512576410023041
Figure BDA0001512576410023051
Figure BDA0001512576410023061
Figure BDA0001512576410023071
Figure BDA0001512576410023081
Figure BDA0001512576410023091
Figure BDA0001512576410023101
Figure BDA0001512576410023111
Figure BDA0001512576410023121
Figure BDA0001512576410023131
Figure BDA0001512576410023141
Figure BDA0001512576410023151
Figure BDA0001512576410023161
Figure BDA0001512576410023171
Figure BDA0001512576410023181
Figure BDA0001512576410023191
Figure BDA0001512576410023201
Figure BDA0001512576410023211
Figure BDA0001512576410023221
Figure BDA0001512576410023231
Figure BDA0001512576410023241
Figure BDA0001512576410023251
Figure BDA0001512576410023261
Figure BDA0001512576410023271
Figure BDA0001512576410023281
Figure BDA0001512576410023291
Figure BDA0001512576410023301
Figure BDA0001512576410023311
Figure BDA0001512576410023321
Figure BDA0001512576410023331
Figure BDA0001512576410023341
Figure BDA0001512576410023351
Figure BDA0001512576410023361
Figure BDA0001512576410023371
Figure BDA0001512576410023381
Figure BDA0001512576410023391
Figure BDA0001512576410023401
Figure BDA0001512576410023411
Figure BDA0001512576410023421
Figure BDA0001512576410023431
Figure BDA0001512576410023441
Figure BDA0001512576410023451
Figure BDA0001512576410023461
Figure BDA0001512576410023471
Figure BDA0001512576410023481
Figure BDA0001512576410023491
Figure BDA0001512576410023501
Figure BDA0001512576410023511
Figure BDA0001512576410023521
COMPOUND(31)
Figure BDA0001512576410023522
Figure BDA0001512576410023531
Figure BDA0001512576410023541
Figure BDA0001512576410023551
Figure BDA0001512576410023561
Figure BDA0001512576410023571
Figure BDA0001512576410023581
Figure BDA0001512576410023591
Figure BDA0001512576410023601
Figure BDA0001512576410023611
Figure BDA0001512576410023621
Figure BDA0001512576410023631
Figure BDA0001512576410023641
Figure BDA0001512576410023651
Figure BDA0001512576410023661
Figure BDA0001512576410023671
Figure BDA0001512576410023681
Figure BDA0001512576410023691
Figure BDA0001512576410023701
Figure BDA0001512576410023711
Figure BDA0001512576410023721
Figure BDA0001512576410023731
Figure BDA0001512576410023741
Figure BDA0001512576410023751
Figure BDA0001512576410023761
Figure BDA0001512576410023771
Figure BDA0001512576410023781
Figure BDA0001512576410023791
Figure BDA0001512576410023801
Figure BDA0001512576410023811
Figure BDA0001512576410023821
Figure BDA0001512576410023831
Figure BDA0001512576410023841
Figure BDA0001512576410023851
Figure BDA0001512576410023861
Figure BDA0001512576410023871
Figure BDA0001512576410023881
Figure BDA0001512576410023891
Figure BDA0001512576410023901
Figure BDA0001512576410023911
Figure BDA0001512576410023921
Figure BDA0001512576410023931
Figure BDA0001512576410023941
Figure BDA0001512576410023951
Figure BDA0001512576410023961
Figure BDA0001512576410023971
Figure BDA0001512576410023981
Figure BDA0001512576410023991
Figure BDA0001512576410024001
Figure BDA0001512576410024011
Figure BDA0001512576410024021
Figure BDA0001512576410024031
Figure BDA0001512576410024041
Figure BDA0001512576410024051
Figure BDA0001512576410024061
Figure BDA0001512576410024071
Figure BDA0001512576410024081
Figure BDA0001512576410024091
Figure BDA0001512576410024101
Figure BDA0001512576410024111
Figure BDA0001512576410024121
Figure BDA0001512576410024131
Figure BDA0001512576410024141
Figure BDA0001512576410024151
Figure BDA0001512576410024161
Figure BDA0001512576410024171
Figure BDA0001512576410024181
Figure BDA0001512576410024191
Figure BDA0001512576410024201
Figure BDA0001512576410024211
Figure BDA0001512576410024221
Figure BDA0001512576410024231
COMPOUND(32)
Figure BDA0001512576410024232
Figure BDA0001512576410024241
Figure BDA0001512576410024251
Figure BDA0001512576410024261
Figure BDA0001512576410024271
Figure BDA0001512576410024281
Figure BDA0001512576410024291
Figure BDA0001512576410024301
Figure BDA0001512576410024311
Figure BDA0001512576410024321
Figure BDA0001512576410024331
Figure BDA0001512576410024341
Figure BDA0001512576410024351
Figure BDA0001512576410024361
Figure BDA0001512576410024371
Figure BDA0001512576410024381
Figure BDA0001512576410024391
Figure BDA0001512576410024401
Figure BDA0001512576410024411
Figure BDA0001512576410024421
Figure BDA0001512576410024431
Figure BDA0001512576410024441
Figure BDA0001512576410024451
Figure BDA0001512576410024461
Figure BDA0001512576410024471
Figure BDA0001512576410024481
Figure BDA0001512576410024491
Figure BDA0001512576410024501
Figure BDA0001512576410024511
Figure BDA0001512576410024521
Figure BDA0001512576410024531
Figure BDA0001512576410024541
Figure BDA0001512576410024551
Figure BDA0001512576410024561
Figure BDA0001512576410024571
Figure BDA0001512576410024581
Figure BDA0001512576410024591
Figure BDA0001512576410024601
Figure BDA0001512576410024611
Figure BDA0001512576410024621
Figure BDA0001512576410024631
Figure BDA0001512576410024641
Figure BDA0001512576410024651
Figure BDA0001512576410024661
Figure BDA0001512576410024671
Figure BDA0001512576410024681
Figure BDA0001512576410024691
Figure BDA0001512576410024701
Figure BDA0001512576410024711
Figure BDA0001512576410024721
Figure BDA0001512576410024731
Figure BDA0001512576410024741
Figure BDA0001512576410024751
Figure BDA0001512576410024761
Figure BDA0001512576410024771
Figure BDA0001512576410024781
Figure BDA0001512576410024791
Figure BDA0001512576410024801
Figure BDA0001512576410024811
Figure BDA0001512576410024821
Figure BDA0001512576410024831
Figure BDA0001512576410024841
Figure BDA0001512576410024851
Figure BDA0001512576410024861
Figure BDA0001512576410024871
Figure BDA0001512576410024881
Figure BDA0001512576410024891
Figure BDA0001512576410024901
Figure BDA0001512576410024911
Figure BDA0001512576410024921
Figure BDA0001512576410024931
Figure BDA0001512576410024941
Figure BDA0001512576410024951
Figure BDA0001512576410024961
Figure BDA0001512576410024971
Figure BDA0001512576410024981
COMPOUND(33)
Figure BDA0001512576410024982
Figure BDA0001512576410024991
Figure BDA0001512576410025001
Figure BDA0001512576410025011
Figure BDA0001512576410025021
Figure BDA0001512576410025031
Figure BDA0001512576410025041
Figure BDA0001512576410025051
Figure BDA0001512576410025061
Figure BDA0001512576410025071
Figure BDA0001512576410025081
Figure BDA0001512576410025091
Figure BDA0001512576410025101
Figure BDA0001512576410025111
Figure BDA0001512576410025121
Figure BDA0001512576410025131
Figure BDA0001512576410025141
Figure BDA0001512576410025151
Figure BDA0001512576410025161
Figure BDA0001512576410025171
Figure BDA0001512576410025181
Figure BDA0001512576410025191
Figure BDA0001512576410025201
Figure BDA0001512576410025211
Figure BDA0001512576410025221
Figure BDA0001512576410025231
Figure BDA0001512576410025241
Figure BDA0001512576410025251
Figure BDA0001512576410025261
Figure BDA0001512576410025271
Figure BDA0001512576410025281
Figure BDA0001512576410025291
Figure BDA0001512576410025301
Figure BDA0001512576410025311
Figure BDA0001512576410025321
Figure BDA0001512576410025331
Figure BDA0001512576410025341
Figure BDA0001512576410025351
Figure BDA0001512576410025361
Figure BDA0001512576410025371
Figure BDA0001512576410025381
Figure BDA0001512576410025391
Figure BDA0001512576410025401
Figure BDA0001512576410025411
Figure BDA0001512576410025421
Figure BDA0001512576410025431
Figure BDA0001512576410025441
Figure BDA0001512576410025451
Figure BDA0001512576410025461
Figure BDA0001512576410025471
Figure BDA0001512576410025481
Figure BDA0001512576410025491
Figure BDA0001512576410025501
Figure BDA0001512576410025511
Figure BDA0001512576410025521
Figure BDA0001512576410025531
Figure BDA0001512576410025541
Figure BDA0001512576410025551
Figure BDA0001512576410025561
Figure BDA0001512576410025571
Figure BDA0001512576410025581
Figure BDA0001512576410025591
Figure BDA0001512576410025601
Figure BDA0001512576410025611
Figure BDA0001512576410025621
Figure BDA0001512576410025631
Figure BDA0001512576410025641
Figure BDA0001512576410025651
Figure BDA0001512576410025661
Figure BDA0001512576410025671
Figure BDA0001512576410025681
Figure BDA0001512576410025691
Figure BDA0001512576410025701
Figure BDA0001512576410025711
COMPOUND(34)
Figure BDA0001512576410025712
Figure BDA0001512576410025721
Figure BDA0001512576410025731
Figure BDA0001512576410025741
Figure BDA0001512576410025751
Figure BDA0001512576410025761
Figure BDA0001512576410025771
Figure BDA0001512576410025781
Figure BDA0001512576410025791
Figure BDA0001512576410025801
Figure BDA0001512576410025811
Figure BDA0001512576410025821
Figure BDA0001512576410025831
Figure BDA0001512576410025841
Figure BDA0001512576410025851
Figure BDA0001512576410025861
Figure BDA0001512576410025871
Figure BDA0001512576410025881
Figure BDA0001512576410025891
Figure BDA0001512576410025901
Figure BDA0001512576410025911
Figure BDA0001512576410025921
Figure BDA0001512576410025931
Figure BDA0001512576410025941
Figure BDA0001512576410025951
Figure BDA0001512576410025961
Figure BDA0001512576410025971
Figure BDA0001512576410025981
Figure BDA0001512576410025991
Figure BDA0001512576410026001
Figure BDA0001512576410026011
Figure BDA0001512576410026021
Figure BDA0001512576410026031
Figure BDA0001512576410026041
Figure BDA0001512576410026051
Figure BDA0001512576410026061
Figure BDA0001512576410026071
Figure BDA0001512576410026081
Figure BDA0001512576410026091
Figure BDA0001512576410026101
Figure BDA0001512576410026111
Figure BDA0001512576410026121
Figure BDA0001512576410026131
Figure BDA0001512576410026141
Figure BDA0001512576410026151
Figure BDA0001512576410026161
Figure BDA0001512576410026171
Figure BDA0001512576410026181
Figure BDA0001512576410026191
Figure BDA0001512576410026201
Figure BDA0001512576410026211
Figure BDA0001512576410026221
Figure BDA0001512576410026231
Figure BDA0001512576410026241
Figure BDA0001512576410026251
Figure BDA0001512576410026261
Figure BDA0001512576410026271
Figure BDA0001512576410026281
Figure BDA0001512576410026291
Figure BDA0001512576410026301
Figure BDA0001512576410026311
Figure BDA0001512576410026321
Figure BDA0001512576410026331
Figure BDA0001512576410026341
Figure BDA0001512576410026351
Figure BDA0001512576410026361
Figure BDA0001512576410026371
Figure BDA0001512576410026381
Figure BDA0001512576410026391
Figure BDA0001512576410026401
Figure BDA0001512576410026411
Figure BDA0001512576410026421
Figure BDA0001512576410026431
Figure BDA0001512576410026441
Figure BDA0001512576410026451
Figure BDA0001512576410026461
Figure BDA0001512576410026471
Figure BDA0001512576410026481
COMPOUND(35)
Figure BDA0001512576410026482
Figure BDA0001512576410026491
Figure BDA0001512576410026501
Figure BDA0001512576410026511
Figure BDA0001512576410026521
Figure BDA0001512576410026531
Figure BDA0001512576410026541
Figure BDA0001512576410026551
Figure BDA0001512576410026561
Figure BDA0001512576410026571
Figure BDA0001512576410026581
Figure BDA0001512576410026591
Figure BDA0001512576410026601
Figure BDA0001512576410026611
Figure BDA0001512576410026621
Figure BDA0001512576410026631
Figure BDA0001512576410026641
Figure BDA0001512576410026651
Figure BDA0001512576410026661
Figure BDA0001512576410026671
Figure BDA0001512576410026681
Figure BDA0001512576410026691
Figure BDA0001512576410026701
Figure BDA0001512576410026711
Figure BDA0001512576410026721
Figure BDA0001512576410026731
Figure BDA0001512576410026741
Figure BDA0001512576410026751
Figure BDA0001512576410026761
Figure BDA0001512576410026771
Figure BDA0001512576410026781
Figure BDA0001512576410026791
Figure BDA0001512576410026801
Figure BDA0001512576410026811
Figure BDA0001512576410026821
Figure BDA0001512576410026831
Figure BDA0001512576410026841
Figure BDA0001512576410026851
Figure BDA0001512576410026861
Figure BDA0001512576410026871
Figure BDA0001512576410026881
Figure BDA0001512576410026891
Figure BDA0001512576410026901
Figure BDA0001512576410026911
Figure BDA0001512576410026921
Figure BDA0001512576410026931
Figure BDA0001512576410026941
Figure BDA0001512576410026951
Figure BDA0001512576410026961
Figure BDA0001512576410026971
Figure BDA0001512576410026981
Figure BDA0001512576410026991
Figure BDA0001512576410027001
Figure BDA0001512576410027011
Figure BDA0001512576410027021
Figure BDA0001512576410027031
Figure BDA0001512576410027041
Figure BDA0001512576410027051
Figure BDA0001512576410027061
Figure BDA0001512576410027071
Figure BDA0001512576410027081
Figure BDA0001512576410027091
Figure BDA0001512576410027101
Figure BDA0001512576410027111
Figure BDA0001512576410027121
Figure BDA0001512576410027131
Figure BDA0001512576410027141
Figure BDA0001512576410027151
Figure BDA0001512576410027161
Figure BDA0001512576410027171
Figure BDA0001512576410027181
Figure BDA0001512576410027191
Figure BDA0001512576410027201
Figure BDA0001512576410027211
Figure BDA0001512576410027221
Figure BDA0001512576410027231
Figure BDA0001512576410027241
Figure BDA0001512576410027251
Figure BDA0001512576410027261
Figure BDA0001512576410027271
Figure BDA0001512576410027281
COMPOUND(36)
Figure BDA0001512576410027282
Figure BDA0001512576410027291
Figure BDA0001512576410027301
Figure BDA0001512576410027311
Figure BDA0001512576410027321
Figure BDA0001512576410027331
Figure BDA0001512576410027341
Figure BDA0001512576410027351
Figure BDA0001512576410027361
Figure BDA0001512576410027371
Figure BDA0001512576410027381
Figure BDA0001512576410027391
Figure BDA0001512576410027401
Figure BDA0001512576410027411
Figure BDA0001512576410027421
Figure BDA0001512576410027431
Figure BDA0001512576410027441
Figure BDA0001512576410027451
Figure BDA0001512576410027461
Figure BDA0001512576410027471
Figure BDA0001512576410027481
Figure BDA0001512576410027491
Figure BDA0001512576410027501
Figure BDA0001512576410027511
Figure BDA0001512576410027521
Figure BDA0001512576410027531
Figure BDA0001512576410027541
Figure BDA0001512576410027551
Figure BDA0001512576410027561
Figure BDA0001512576410027571
Figure BDA0001512576410027581
Figure BDA0001512576410027591
Figure BDA0001512576410027601
Figure BDA0001512576410027611
Figure BDA0001512576410027621
Figure BDA0001512576410027631
Figure BDA0001512576410027641
Figure BDA0001512576410027651
Figure BDA0001512576410027661
Figure BDA0001512576410027671
Figure BDA0001512576410027681
Figure BDA0001512576410027691
Figure BDA0001512576410027701
Figure BDA0001512576410027711
Figure BDA0001512576410027721
Figure BDA0001512576410027731
Figure BDA0001512576410027741
Figure BDA0001512576410027751
Figure BDA0001512576410027761
Figure BDA0001512576410027771
Figure BDA0001512576410027781
Figure BDA0001512576410027791
Figure BDA0001512576410027801
Figure BDA0001512576410027811
Figure BDA0001512576410027821
Figure BDA0001512576410027831
Figure BDA0001512576410027841
Figure BDA0001512576410027851
Figure BDA0001512576410027861
Figure BDA0001512576410027871
Figure BDA0001512576410027881
Figure BDA0001512576410027891
Figure BDA0001512576410027901
Figure BDA0001512576410027911
Figure BDA0001512576410027921
Figure BDA0001512576410027931
Figure BDA0001512576410027941
Figure BDA0001512576410027951
Figure BDA0001512576410027961
Figure BDA0001512576410027971
Figure BDA0001512576410027981
Figure BDA0001512576410027991
Figure BDA0001512576410028001
Figure BDA0001512576410028011
Figure BDA0001512576410028021
Figure BDA0001512576410028031
COMPOUND(37)
Figure BDA0001512576410028032
Figure BDA0001512576410028041
Figure BDA0001512576410028051
Figure BDA0001512576410028061
Figure BDA0001512576410028071
Figure BDA0001512576410028081
Figure BDA0001512576410028091
Figure BDA0001512576410028101
Figure BDA0001512576410028111
Figure BDA0001512576410028121
Figure BDA0001512576410028131
Figure BDA0001512576410028141
Figure BDA0001512576410028151
Figure BDA0001512576410028161
Figure BDA0001512576410028171
Figure BDA0001512576410028181
Figure BDA0001512576410028191
Figure BDA0001512576410028201
Figure BDA0001512576410028211
Figure BDA0001512576410028221
Figure BDA0001512576410028231
Figure BDA0001512576410028241
Figure BDA0001512576410028251
Figure BDA0001512576410028261
Figure BDA0001512576410028271
Figure BDA0001512576410028281
Figure BDA0001512576410028291
Figure BDA0001512576410028301
Figure BDA0001512576410028311
Figure BDA0001512576410028321
Figure BDA0001512576410028331
Figure BDA0001512576410028341
Figure BDA0001512576410028351
Figure BDA0001512576410028361
Figure BDA0001512576410028371
Figure BDA0001512576410028381
Figure BDA0001512576410028391
Figure BDA0001512576410028401
Figure BDA0001512576410028411
Figure BDA0001512576410028421
Figure BDA0001512576410028431
Figure BDA0001512576410028441
Figure BDA0001512576410028451
Figure BDA0001512576410028461
Figure BDA0001512576410028471
Figure BDA0001512576410028481
Figure BDA0001512576410028491
Figure BDA0001512576410028501
Figure BDA0001512576410028511
Figure BDA0001512576410028521
Figure BDA0001512576410028531
Figure BDA0001512576410028541
Figure BDA0001512576410028551
Figure BDA0001512576410028561
Figure BDA0001512576410028571
Figure BDA0001512576410028581
Figure BDA0001512576410028591
Figure BDA0001512576410028601
Figure BDA0001512576410028611
Figure BDA0001512576410028621
Figure BDA0001512576410028631
Figure BDA0001512576410028641
Figure BDA0001512576410028651
Figure BDA0001512576410028661
Figure BDA0001512576410028671
Figure BDA0001512576410028681
Figure BDA0001512576410028691
Figure BDA0001512576410028701
Figure BDA0001512576410028711
Figure BDA0001512576410028721
Figure BDA0001512576410028731
Figure BDA0001512576410028741
Figure BDA0001512576410028751
Figure BDA0001512576410028761
Figure BDA0001512576410028771
Figure BDA0001512576410028781
Figure BDA0001512576410028791
Figure BDA0001512576410028801
Figure BDA0001512576410028811
COMPOUND(38)
Figure BDA0001512576410028812
Figure BDA0001512576410028821
Figure BDA0001512576410028831
Figure BDA0001512576410028841
Figure BDA0001512576410028851
Figure BDA0001512576410028861
Figure BDA0001512576410028871
Figure BDA0001512576410028881
Figure BDA0001512576410028891
Figure BDA0001512576410028901
Figure BDA0001512576410028911
Figure BDA0001512576410028921
Figure BDA0001512576410028931
Figure BDA0001512576410028941
Figure BDA0001512576410028951
Figure BDA0001512576410028961
Figure BDA0001512576410028971
Figure BDA0001512576410028981
Figure BDA0001512576410028991
Figure BDA0001512576410029001
Figure BDA0001512576410029011
Figure BDA0001512576410029021
Figure BDA0001512576410029031
Figure BDA0001512576410029041
Figure BDA0001512576410029051
Figure BDA0001512576410029061
Figure BDA0001512576410029071
Figure BDA0001512576410029081
Figure BDA0001512576410029091
Figure BDA0001512576410029101
Figure BDA0001512576410029111
Figure BDA0001512576410029121
Figure BDA0001512576410029131
Figure BDA0001512576410029141
Figure BDA0001512576410029151
Figure BDA0001512576410029161
Figure BDA0001512576410029171
Figure BDA0001512576410029181
Figure BDA0001512576410029191
Figure BDA0001512576410029201
Figure BDA0001512576410029211
Figure BDA0001512576410029221
Figure BDA0001512576410029231
Figure BDA0001512576410029241
Figure BDA0001512576410029251
Figure BDA0001512576410029261
Figure BDA0001512576410029271
Figure BDA0001512576410029281
Figure BDA0001512576410029291
Figure BDA0001512576410029301
Figure BDA0001512576410029311
Figure BDA0001512576410029321
Figure BDA0001512576410029331
Figure BDA0001512576410029341
Figure BDA0001512576410029351
Figure BDA0001512576410029361
Figure BDA0001512576410029371
Figure BDA0001512576410029381
Figure BDA0001512576410029391
Figure BDA0001512576410029401
Figure BDA0001512576410029411
Figure BDA0001512576410029421
Figure BDA0001512576410029431
Figure BDA0001512576410029441
Figure BDA0001512576410029451
Figure BDA0001512576410029461
Figure BDA0001512576410029471
Figure BDA0001512576410029481
Figure BDA0001512576410029491
Figure BDA0001512576410029501
Figure BDA0001512576410029511
Figure BDA0001512576410029521
Figure BDA0001512576410029531
COMPOUND(39)
Figure BDA0001512576410029532
Figure BDA0001512576410029541
Figure BDA0001512576410029551
Figure BDA0001512576410029561
Figure BDA0001512576410029571
Figure BDA0001512576410029581
Figure BDA0001512576410029591
Figure BDA0001512576410029601
Figure BDA0001512576410029611
Figure BDA0001512576410029621
Figure BDA0001512576410029631
Figure BDA0001512576410029641
Figure BDA0001512576410029651
Figure BDA0001512576410029661
Figure BDA0001512576410029671
Figure BDA0001512576410029681
Figure BDA0001512576410029691
Figure BDA0001512576410029701
Figure BDA0001512576410029711
Figure BDA0001512576410029721
Figure BDA0001512576410029731
Figure BDA0001512576410029741
Figure BDA0001512576410029751
Figure BDA0001512576410029761
Figure BDA0001512576410029771
Figure BDA0001512576410029781
Figure BDA0001512576410029791
Figure BDA0001512576410029801
Figure BDA0001512576410029811
Figure BDA0001512576410029821
Figure BDA0001512576410029831
Figure BDA0001512576410029841
Figure BDA0001512576410029851
Figure BDA0001512576410029861
Figure BDA0001512576410029871
Figure BDA0001512576410029881
Figure BDA0001512576410029891
Figure BDA0001512576410029901
Figure BDA0001512576410029911
Figure BDA0001512576410029921
Figure BDA0001512576410029931
Figure BDA0001512576410029941
Figure BDA0001512576410029951
Figure BDA0001512576410029961
Figure BDA0001512576410029971
Figure BDA0001512576410029981
Figure BDA0001512576410029991
Figure BDA0001512576410030001
Figure BDA0001512576410030011
Figure BDA0001512576410030021
Figure BDA0001512576410030031
Figure BDA0001512576410030041
Figure BDA0001512576410030051
Figure BDA0001512576410030061
Figure BDA0001512576410030071
Figure BDA0001512576410030081
Figure BDA0001512576410030091
Figure BDA0001512576410030101
Figure BDA0001512576410030111
Figure BDA0001512576410030121
Figure BDA0001512576410030131
Figure BDA0001512576410030141
Figure BDA0001512576410030151
Figure BDA0001512576410030161
Figure BDA0001512576410030171
Figure BDA0001512576410030181
Figure BDA0001512576410030191
Figure BDA0001512576410030201
Figure BDA0001512576410030211
Figure BDA0001512576410030221
Figure BDA0001512576410030231
Figure BDA0001512576410030241
Figure BDA0001512576410030251
Figure BDA0001512576410030261
Figure BDA0001512576410030271
Figure BDA0001512576410030281
Figure BDA0001512576410030291
COMPOUND(40)
Figure BDA0001512576410030301
Figure BDA0001512576410030311
Figure BDA0001512576410030321
Figure BDA0001512576410030331
Figure BDA0001512576410030341
Figure BDA0001512576410030351
Figure BDA0001512576410030361
Figure BDA0001512576410030371
Figure BDA0001512576410030381
Figure BDA0001512576410030391
Figure BDA0001512576410030401
Figure BDA0001512576410030411
Figure BDA0001512576410030421
Figure BDA0001512576410030431
Figure BDA0001512576410030441
Figure BDA0001512576410030451
Figure BDA0001512576410030461
Figure BDA0001512576410030471
Figure BDA0001512576410030481
Figure BDA0001512576410030491
Figure BDA0001512576410030501
Figure BDA0001512576410030511
Figure BDA0001512576410030521
Figure BDA0001512576410030531
Figure BDA0001512576410030541
Figure BDA0001512576410030551
Figure BDA0001512576410030561
Figure BDA0001512576410030571
Figure BDA0001512576410030581
Figure BDA0001512576410030591
Figure BDA0001512576410030601
Figure BDA0001512576410030611
Figure BDA0001512576410030621
Figure BDA0001512576410030631
Figure BDA0001512576410030641
Figure BDA0001512576410030651
Figure BDA0001512576410030661
Figure BDA0001512576410030671
Figure BDA0001512576410030681
Figure BDA0001512576410030691
Figure BDA0001512576410030701
Figure BDA0001512576410030711
Figure BDA0001512576410030721
Figure BDA0001512576410030731
Figure BDA0001512576410030741
Figure BDA0001512576410030751
Figure BDA0001512576410030761
Figure BDA0001512576410030771
Figure BDA0001512576410030781
Figure BDA0001512576410030791
Figure BDA0001512576410030801
Figure BDA0001512576410030811
Figure BDA0001512576410030821
Figure BDA0001512576410030831
Figure BDA0001512576410030841
Figure BDA0001512576410030851
Figure BDA0001512576410030861
Figure BDA0001512576410030871
Figure BDA0001512576410030881
Figure BDA0001512576410030891
Figure BDA0001512576410030901
Figure BDA0001512576410030911
Figure BDA0001512576410030921
Figure BDA0001512576410030931
Figure BDA0001512576410030941
Figure BDA0001512576410030951
Figure BDA0001512576410030961
Figure BDA0001512576410030971
Figure BDA0001512576410030981
Figure BDA0001512576410030991
Figure BDA0001512576410031001
Figure BDA0001512576410031011
Figure BDA0001512576410031021
Figure BDA0001512576410031031
Figure BDA0001512576410031041
Figure BDA0001512576410031051
COMPOUND(41)
Figure BDA0001512576410031052
Figure BDA0001512576410031061
Figure BDA0001512576410031071
Figure BDA0001512576410031081
Figure BDA0001512576410031091
Figure BDA0001512576410031101
Figure BDA0001512576410031111
Figure BDA0001512576410031121
Figure BDA0001512576410031131
Figure BDA0001512576410031141
Figure BDA0001512576410031151
Figure BDA0001512576410031161
Figure BDA0001512576410031171
Figure BDA0001512576410031181
Figure BDA0001512576410031191
Figure BDA0001512576410031201
Figure BDA0001512576410031211
Figure BDA0001512576410031221
Figure BDA0001512576410031231
Figure BDA0001512576410031241
Figure BDA0001512576410031251
Figure BDA0001512576410031261
Figure BDA0001512576410031271
Figure BDA0001512576410031281
Figure BDA0001512576410031291
Figure BDA0001512576410031301
Figure BDA0001512576410031311
Figure BDA0001512576410031321
Figure BDA0001512576410031331
Figure BDA0001512576410031341
Figure BDA0001512576410031351
Figure BDA0001512576410031361
Figure BDA0001512576410031371
Figure BDA0001512576410031381
Figure BDA0001512576410031391
Figure BDA0001512576410031401
Figure BDA0001512576410031411
Figure BDA0001512576410031421
Figure BDA0001512576410031431
Figure BDA0001512576410031441
Figure BDA0001512576410031451
Figure BDA0001512576410031461
Figure BDA0001512576410031471
Figure BDA0001512576410031481
Figure BDA0001512576410031491
Figure BDA0001512576410031501
Figure BDA0001512576410031511
Figure BDA0001512576410031521
Figure BDA0001512576410031531
Figure BDA0001512576410031541
Figure BDA0001512576410031551
Figure BDA0001512576410031561
Figure BDA0001512576410031571
Figure BDA0001512576410031581
Figure BDA0001512576410031591
Figure BDA0001512576410031601
Figure BDA0001512576410031611
Figure BDA0001512576410031621
Figure BDA0001512576410031631
Figure BDA0001512576410031641
Figure BDA0001512576410031651
Figure BDA0001512576410031661
Figure BDA0001512576410031671
Figure BDA0001512576410031681
Figure BDA0001512576410031691
Figure BDA0001512576410031701
Figure BDA0001512576410031711
Figure BDA0001512576410031721
Figure BDA0001512576410031731
Figure BDA0001512576410031741
Figure BDA0001512576410031751
Figure BDA0001512576410031761
Figure BDA0001512576410031771
Figure BDA0001512576410031781
Figure BDA0001512576410031791
Figure BDA0001512576410031801
Figure BDA0001512576410031811
Figure BDA0001512576410031821
Figure BDA0001512576410031831
Figure BDA0001512576410031841
Figure BDA0001512576410031851
Figure BDA0001512576410031861
Figure BDA0001512576410031871
COMPOUND(42)
Figure BDA0001512576410031872
Figure BDA0001512576410031881
Figure BDA0001512576410031891
Figure BDA0001512576410031901
Figure BDA0001512576410031911
Figure BDA0001512576410031921
Figure BDA0001512576410031931
Figure BDA0001512576410031941
Figure BDA0001512576410031951
Figure BDA0001512576410031961
Figure BDA0001512576410031971
Figure BDA0001512576410031981
Figure BDA0001512576410031991
Figure BDA0001512576410032001
Figure BDA0001512576410032011
Figure BDA0001512576410032021
Figure BDA0001512576410032031
Figure BDA0001512576410032041
Figure BDA0001512576410032051
Figure BDA0001512576410032061
Figure BDA0001512576410032071
Figure BDA0001512576410032081
Figure BDA0001512576410032091
Figure BDA0001512576410032101
Figure BDA0001512576410032111
Figure BDA0001512576410032121
Figure BDA0001512576410032131
Figure BDA0001512576410032141
Figure BDA0001512576410032151
Figure BDA0001512576410032161
Figure BDA0001512576410032171
Figure BDA0001512576410032181
Figure BDA0001512576410032191
Figure BDA0001512576410032201
Figure BDA0001512576410032211
Figure BDA0001512576410032221
Figure BDA0001512576410032231
Figure BDA0001512576410032241
Figure BDA0001512576410032251
Figure BDA0001512576410032261
Figure BDA0001512576410032271
Figure BDA0001512576410032281
Figure BDA0001512576410032291
Figure BDA0001512576410032301
Figure BDA0001512576410032311
Figure BDA0001512576410032321
Figure BDA0001512576410032331
Figure BDA0001512576410032341
Figure BDA0001512576410032351
Figure BDA0001512576410032361
Figure BDA0001512576410032371
Figure BDA0001512576410032381
Figure BDA0001512576410032391
Figure BDA0001512576410032401
Figure BDA0001512576410032411
Figure BDA0001512576410032421
Figure BDA0001512576410032431
Figure BDA0001512576410032441
Figure BDA0001512576410032451
Figure BDA0001512576410032461
Figure BDA0001512576410032471
Figure BDA0001512576410032481
Figure BDA0001512576410032491
Figure BDA0001512576410032501
Figure BDA0001512576410032511
Figure BDA0001512576410032521
Figure BDA0001512576410032531
Figure BDA0001512576410032541
Figure BDA0001512576410032551
Figure BDA0001512576410032561
Figure BDA0001512576410032571
Figure BDA0001512576410032581
Figure BDA0001512576410032591
Figure BDA0001512576410032601
Figure BDA0001512576410032611
Figure BDA0001512576410032621
Figure BDA0001512576410032631
Figure BDA0001512576410032641
Figure BDA0001512576410032651
Figure BDA0001512576410032661
Figure BDA0001512576410032671
Figure BDA0001512576410032681
COMPOUND(43)
Figure BDA0001512576410032682
Figure BDA0001512576410032691
Figure BDA0001512576410032701
Figure BDA0001512576410032711
Figure BDA0001512576410032721
Figure BDA0001512576410032731
Figure BDA0001512576410032741
Figure BDA0001512576410032751
Figure BDA0001512576410032761
Figure BDA0001512576410032771
Figure BDA0001512576410032781
Figure BDA0001512576410032791
Figure BDA0001512576410032801
Figure BDA0001512576410032811
Figure BDA0001512576410032821
Figure BDA0001512576410032831
Figure BDA0001512576410032841
Figure BDA0001512576410032851
Figure BDA0001512576410032861
Figure BDA0001512576410032871
Figure BDA0001512576410032881
Figure BDA0001512576410032891
Figure BDA0001512576410032901
Figure BDA0001512576410032911
Figure BDA0001512576410032921
Figure BDA0001512576410032931
Figure BDA0001512576410032941
Figure BDA0001512576410032951
Figure BDA0001512576410032961
Figure BDA0001512576410032971
Figure BDA0001512576410032981
Figure BDA0001512576410032991
Figure BDA0001512576410033001
Figure BDA0001512576410033011
Figure BDA0001512576410033021
Figure BDA0001512576410033031
Figure BDA0001512576410033041
Figure BDA0001512576410033051
Figure BDA0001512576410033061
Figure BDA0001512576410033071
Figure BDA0001512576410033081
Figure BDA0001512576410033091
Figure BDA0001512576410033101
Figure BDA0001512576410033111
Figure BDA0001512576410033121
Figure BDA0001512576410033131
Figure BDA0001512576410033141
Figure BDA0001512576410033151
Figure BDA0001512576410033161
Figure BDA0001512576410033171
Figure BDA0001512576410033181
Figure BDA0001512576410033191
Figure BDA0001512576410033201
Figure BDA0001512576410033211
Figure BDA0001512576410033221
Figure BDA0001512576410033231
Figure BDA0001512576410033241
Figure BDA0001512576410033251
Figure BDA0001512576410033261
Figure BDA0001512576410033271
Figure BDA0001512576410033281
Figure BDA0001512576410033291
Figure BDA0001512576410033301
Figure BDA0001512576410033311
Figure BDA0001512576410033321
Figure BDA0001512576410033331
Figure BDA0001512576410033341
Figure BDA0001512576410033351
Figure BDA0001512576410033361
Figure BDA0001512576410033371
Figure BDA0001512576410033381
Figure BDA0001512576410033391
COMPOUND(44)
Figure BDA0001512576410033392
Figure BDA0001512576410033401
Figure BDA0001512576410033411
Figure BDA0001512576410033421
Figure BDA0001512576410033431
Figure BDA0001512576410033441
Figure BDA0001512576410033451
Figure BDA0001512576410033461
Figure BDA0001512576410033471
Figure BDA0001512576410033481
Figure BDA0001512576410033491
Figure BDA0001512576410033501
Figure BDA0001512576410033511
Figure BDA0001512576410033521
Figure BDA0001512576410033531
Figure BDA0001512576410033541
Figure BDA0001512576410033551
Figure BDA0001512576410033561
Figure BDA0001512576410033571
Figure BDA0001512576410033581
Figure BDA0001512576410033591
Figure BDA0001512576410033601
Figure BDA0001512576410033611
Figure BDA0001512576410033621
Figure BDA0001512576410033631
Figure BDA0001512576410033641
Figure BDA0001512576410033651
Figure BDA0001512576410033661
Figure BDA0001512576410033671
Figure BDA0001512576410033681
Figure BDA0001512576410033691
Figure BDA0001512576410033701
Figure BDA0001512576410033711
Figure BDA0001512576410033721
Figure BDA0001512576410033731
Figure BDA0001512576410033741
Figure BDA0001512576410033751
Figure BDA0001512576410033761
Figure BDA0001512576410033771
Figure BDA0001512576410033781
Figure BDA0001512576410033791
Figure BDA0001512576410033801
Figure BDA0001512576410033811
Figure BDA0001512576410033821
Figure BDA0001512576410033831
Figure BDA0001512576410033841
Figure BDA0001512576410033851
Figure BDA0001512576410033861
Figure BDA0001512576410033871
Figure BDA0001512576410033881
Figure BDA0001512576410033891
Figure BDA0001512576410033901
Figure BDA0001512576410033911
Figure BDA0001512576410033921
Figure BDA0001512576410033931
Figure BDA0001512576410033941
Figure BDA0001512576410033951
Figure BDA0001512576410033961
Figure BDA0001512576410033971
Figure BDA0001512576410033981
Figure BDA0001512576410033991
Figure BDA0001512576410034001
Figure BDA0001512576410034011
Figure BDA0001512576410034021
Figure BDA0001512576410034031
Figure BDA0001512576410034041
Figure BDA0001512576410034051
Figure BDA0001512576410034061
Figure BDA0001512576410034071
Figure BDA0001512576410034081
Figure BDA0001512576410034091
Figure BDA0001512576410034101
Figure BDA0001512576410034111
Figure BDA0001512576410034121
Figure BDA0001512576410034131
Figure BDA0001512576410034141
COMPOUND(45)
Figure BDA0001512576410034142
Figure BDA0001512576410034151
Figure BDA0001512576410034161
Figure BDA0001512576410034171
Figure BDA0001512576410034181
Figure BDA0001512576410034191
Figure BDA0001512576410034201
Figure BDA0001512576410034211
Figure BDA0001512576410034221
Figure BDA0001512576410034231
Figure BDA0001512576410034241
Figure BDA0001512576410034251
Figure BDA0001512576410034261
Figure BDA0001512576410034271
Figure BDA0001512576410034281
Figure BDA0001512576410034291
Figure BDA0001512576410034301
Figure BDA0001512576410034311
Figure BDA0001512576410034321
Figure BDA0001512576410034331
Figure BDA0001512576410034341
Figure BDA0001512576410034351
Figure BDA0001512576410034361
Figure BDA0001512576410034371
Figure BDA0001512576410034381
Figure BDA0001512576410034391
Figure BDA0001512576410034401
Figure BDA0001512576410034411
Figure BDA0001512576410034421
Figure BDA0001512576410034431
Figure BDA0001512576410034441
Figure BDA0001512576410034451
Figure BDA0001512576410034461
Figure BDA0001512576410034471
Figure BDA0001512576410034481
Figure BDA0001512576410034491
Figure BDA0001512576410034501
Figure BDA0001512576410034511
Figure BDA0001512576410034521
Figure BDA0001512576410034531
Figure BDA0001512576410034541
Figure BDA0001512576410034551
Figure BDA0001512576410034561
Figure BDA0001512576410034571
Figure BDA0001512576410034581
Figure BDA0001512576410034591
Figure BDA0001512576410034601
Figure BDA0001512576410034611
Figure BDA0001512576410034621
Figure BDA0001512576410034631
Figure BDA0001512576410034641
Figure BDA0001512576410034651
Figure BDA0001512576410034661
Figure BDA0001512576410034671
Figure BDA0001512576410034681
Figure BDA0001512576410034691
Figure BDA0001512576410034701
Figure BDA0001512576410034711
Figure BDA0001512576410034721
Figure BDA0001512576410034731
Figure BDA0001512576410034741
Figure BDA0001512576410034751
Figure BDA0001512576410034761
Figure BDA0001512576410034771
Figure BDA0001512576410034781
Figure BDA0001512576410034791
Figure BDA0001512576410034801
Figure BDA0001512576410034811
Figure BDA0001512576410034821
Figure BDA0001512576410034831
Figure BDA0001512576410034841
Figure BDA0001512576410034851
Figure BDA0001512576410034861
Figure BDA0001512576410034871
Figure BDA0001512576410034881
Figure BDA0001512576410034891
Figure BDA0001512576410034901
Figure BDA0001512576410034911
COMPOUND(46)
Figure BDA0001512576410034912
Figure BDA0001512576410034921
Figure BDA0001512576410034931
Figure BDA0001512576410034941
Figure BDA0001512576410034951
Figure BDA0001512576410034961
Figure BDA0001512576410034971
Figure BDA0001512576410034981
Figure BDA0001512576410034991
Figure BDA0001512576410035001
Figure BDA0001512576410035011
Figure BDA0001512576410035021
Figure BDA0001512576410035031
Figure BDA0001512576410035041
Figure BDA0001512576410035051
Figure BDA0001512576410035061
Figure BDA0001512576410035071
Figure BDA0001512576410035081
Figure BDA0001512576410035091
Figure BDA0001512576410035101
Figure BDA0001512576410035111
Figure BDA0001512576410035121
Figure BDA0001512576410035131
Figure BDA0001512576410035141
Figure BDA0001512576410035151
Figure BDA0001512576410035161
Figure BDA0001512576410035171
Figure BDA0001512576410035181
Figure BDA0001512576410035191
Figure BDA0001512576410035201
Figure BDA0001512576410035211
Figure BDA0001512576410035221
Figure BDA0001512576410035231
Figure BDA0001512576410035241
Figure BDA0001512576410035251
Figure BDA0001512576410035261
Figure BDA0001512576410035271
Figure BDA0001512576410035281
Figure BDA0001512576410035291
Figure BDA0001512576410035301
Figure BDA0001512576410035311
Figure BDA0001512576410035321
Figure BDA0001512576410035331
Figure BDA0001512576410035341
Figure BDA0001512576410035351
Figure BDA0001512576410035361
Figure BDA0001512576410035371
Figure BDA0001512576410035381
Figure BDA0001512576410035391
Figure BDA0001512576410035401
Figure BDA0001512576410035411
Figure BDA0001512576410035421
Figure BDA0001512576410035431
Figure BDA0001512576410035441
Figure BDA0001512576410035451
Figure BDA0001512576410035461
Figure BDA0001512576410035471
Figure BDA0001512576410035481
Figure BDA0001512576410035491
Figure BDA0001512576410035501
Figure BDA0001512576410035511
Figure BDA0001512576410035521
Figure BDA0001512576410035531
Figure BDA0001512576410035541
Figure BDA0001512576410035551
Figure BDA0001512576410035561
Figure BDA0001512576410035571
Figure BDA0001512576410035581
Figure BDA0001512576410035591
Figure BDA0001512576410035601
Figure BDA0001512576410035611
Figure BDA0001512576410035621
Figure BDA0001512576410035631
Figure BDA0001512576410035641
Figure BDA0001512576410035651
Figure BDA0001512576410035661
COMPOUND(47)
Figure BDA0001512576410035662
Figure BDA0001512576410035671
Figure BDA0001512576410035681
Figure BDA0001512576410035691
Figure BDA0001512576410035701
Figure BDA0001512576410035711
Figure BDA0001512576410035721
Figure BDA0001512576410035731
Figure BDA0001512576410035741
Figure BDA0001512576410035751
Figure BDA0001512576410035761
Figure BDA0001512576410035771
Figure BDA0001512576410035781
Figure BDA0001512576410035791
Figure BDA0001512576410035801
Figure BDA0001512576410035811
Figure BDA0001512576410035821
Figure BDA0001512576410035831
Figure BDA0001512576410035841
Figure BDA0001512576410035851
Figure BDA0001512576410035861
Figure BDA0001512576410035871
Figure BDA0001512576410035881
Figure BDA0001512576410035891
Figure BDA0001512576410035901
Figure BDA0001512576410035911
Figure BDA0001512576410035921
Figure BDA0001512576410035931
Figure BDA0001512576410035941
Figure BDA0001512576410035951
Figure BDA0001512576410035961
Figure BDA0001512576410035971
Figure BDA0001512576410035981
Figure BDA0001512576410035991
Figure BDA0001512576410036001
Figure BDA0001512576410036011
Figure BDA0001512576410036021
Figure BDA0001512576410036031
Figure BDA0001512576410036041
Figure BDA0001512576410036051
Figure BDA0001512576410036061
Figure BDA0001512576410036071
Figure BDA0001512576410036081
Figure BDA0001512576410036091
Figure BDA0001512576410036101
Figure BDA0001512576410036111
Figure BDA0001512576410036121
Figure BDA0001512576410036131
Figure BDA0001512576410036141
Figure BDA0001512576410036151
Figure BDA0001512576410036161
Figure BDA0001512576410036171
Figure BDA0001512576410036181
Figure BDA0001512576410036191
Figure BDA0001512576410036201
Figure BDA0001512576410036211
Figure BDA0001512576410036221
Figure BDA0001512576410036231
Figure BDA0001512576410036241
Figure BDA0001512576410036251
Figure BDA0001512576410036261
Figure BDA0001512576410036271
Figure BDA0001512576410036281
Figure BDA0001512576410036291
Figure BDA0001512576410036301
Figure BDA0001512576410036311
Figure BDA0001512576410036321
Figure BDA0001512576410036331
Figure BDA0001512576410036341
Figure BDA0001512576410036351
COMPOUND(48)
Figure BDA0001512576410036352
Figure BDA0001512576410036361
Figure BDA0001512576410036371
Figure BDA0001512576410036381
Figure BDA0001512576410036391
Figure BDA0001512576410036401
Figure BDA0001512576410036411
Figure BDA0001512576410036421
Figure BDA0001512576410036431
Figure BDA0001512576410036441
Figure BDA0001512576410036451
Figure BDA0001512576410036461
Figure BDA0001512576410036471
Figure BDA0001512576410036481
Figure BDA0001512576410036491
Figure BDA0001512576410036501
Figure BDA0001512576410036511
Figure BDA0001512576410036521
Figure BDA0001512576410036531
Figure BDA0001512576410036541
Figure BDA0001512576410036551
Figure BDA0001512576410036561
Figure BDA0001512576410036571
Figure BDA0001512576410036581
Figure BDA0001512576410036591
Figure BDA0001512576410036601
Figure BDA0001512576410036611
Figure BDA0001512576410036621
Figure BDA0001512576410036631
Figure BDA0001512576410036641
Figure BDA0001512576410036651
Figure BDA0001512576410036661
Figure BDA0001512576410036671
Figure BDA0001512576410036681
Figure BDA0001512576410036691
Figure BDA0001512576410036701
Figure BDA0001512576410036711
Figure BDA0001512576410036721
Figure BDA0001512576410036731
Figure BDA0001512576410036741
Figure BDA0001512576410036751
Figure BDA0001512576410036761
Figure BDA0001512576410036771
Figure BDA0001512576410036781
Figure BDA0001512576410036791
Figure BDA0001512576410036801
Figure BDA0001512576410036811
Figure BDA0001512576410036821
Figure BDA0001512576410036831
Figure BDA0001512576410036841
Figure BDA0001512576410036851
Figure BDA0001512576410036861
Figure BDA0001512576410036871
Figure BDA0001512576410036881
Figure BDA0001512576410036891
Figure BDA0001512576410036901
Figure BDA0001512576410036911
Figure BDA0001512576410036921
Figure BDA0001512576410036931
Figure BDA0001512576410036941
Figure BDA0001512576410036951
Figure BDA0001512576410036961
Figure BDA0001512576410036971
Figure BDA0001512576410036981
Figure BDA0001512576410036991
Figure BDA0001512576410037001
Figure BDA0001512576410037011
Figure BDA0001512576410037021
Figure BDA0001512576410037031
Figure BDA0001512576410037041
Figure BDA0001512576410037051
Figure BDA0001512576410037061
Figure BDA0001512576410037071
Figure BDA0001512576410037081
Figure BDA0001512576410037091
Figure BDA0001512576410037101
Figure BDA0001512576410037111
Figure BDA0001512576410037121
Figure BDA0001512576410037131
Figure BDA0001512576410037141
Figure BDA0001512576410037151
Figure BDA0001512576410037161
Figure BDA0001512576410037171
Figure BDA0001512576410037181
COMPOUND(49)
Figure BDA0001512576410037182
Figure BDA0001512576410037191
Figure BDA0001512576410037201
Figure BDA0001512576410037211
Figure BDA0001512576410037221
Figure BDA0001512576410037231
Figure BDA0001512576410037241
Figure BDA0001512576410037251
Figure BDA0001512576410037261
Figure BDA0001512576410037271
Figure BDA0001512576410037281
Figure BDA0001512576410037291
Figure BDA0001512576410037301
Figure BDA0001512576410037311
Figure BDA0001512576410037321
Figure BDA0001512576410037331
Figure BDA0001512576410037341
Figure BDA0001512576410037351
Figure BDA0001512576410037361
Figure BDA0001512576410037371
Figure BDA0001512576410037381
Figure BDA0001512576410037391
Figure BDA0001512576410037401
Figure BDA0001512576410037411
Figure BDA0001512576410037421
Figure BDA0001512576410037431
Figure BDA0001512576410037441
Figure BDA0001512576410037451
Figure BDA0001512576410037461
Figure BDA0001512576410037471
Figure BDA0001512576410037481
Figure BDA0001512576410037491
Figure BDA0001512576410037501
Figure BDA0001512576410037511
Figure BDA0001512576410037521
Figure BDA0001512576410037531
Figure BDA0001512576410037541
Figure BDA0001512576410037551
Figure BDA0001512576410037561
Figure BDA0001512576410037571
Figure BDA0001512576410037581
Figure BDA0001512576410037591
Figure BDA0001512576410037601
Figure BDA0001512576410037611
Figure BDA0001512576410037621
Figure BDA0001512576410037631
Figure BDA0001512576410037641
Figure BDA0001512576410037651
Figure BDA0001512576410037661
Figure BDA0001512576410037671
Figure BDA0001512576410037681
Figure BDA0001512576410037691
Figure BDA0001512576410037701
Figure BDA0001512576410037711
Figure BDA0001512576410037721
Figure BDA0001512576410037731
Figure BDA0001512576410037741
Figure BDA0001512576410037751
Figure BDA0001512576410037761
Figure BDA0001512576410037771
Figure BDA0001512576410037781
Figure BDA0001512576410037791
Figure BDA0001512576410037801
Figure BDA0001512576410037811
Figure BDA0001512576410037821
Figure BDA0001512576410037831
Figure BDA0001512576410037841
Figure BDA0001512576410037851
Figure BDA0001512576410037861
Figure BDA0001512576410037871
Figure BDA0001512576410037881
Figure BDA0001512576410037891
Figure BDA0001512576410037901
Figure BDA0001512576410037911
Figure BDA0001512576410037921
Figure BDA0001512576410037931
Figure BDA0001512576410037941
Figure BDA0001512576410037951
Figure BDA0001512576410037961
Figure BDA0001512576410037971
Figure BDA0001512576410037981
COMPOUND(50)
Figure BDA0001512576410037982
Figure BDA0001512576410037991
Figure BDA0001512576410038001
Figure BDA0001512576410038011
Figure BDA0001512576410038021
Figure BDA0001512576410038031
Figure BDA0001512576410038041
Figure BDA0001512576410038051
Figure BDA0001512576410038061
Figure BDA0001512576410038071
Figure BDA0001512576410038081
Figure BDA0001512576410038091
Figure BDA0001512576410038101
Figure BDA0001512576410038111
Figure BDA0001512576410038121
Figure BDA0001512576410038131
Figure BDA0001512576410038141
Figure BDA0001512576410038151
Figure BDA0001512576410038161
Figure BDA0001512576410038171
Figure BDA0001512576410038181
Figure BDA0001512576410038191
Figure BDA0001512576410038201
Figure BDA0001512576410038211
Figure BDA0001512576410038221
Figure BDA0001512576410038231
Figure BDA0001512576410038241
Figure BDA0001512576410038251
Figure BDA0001512576410038261
Figure BDA0001512576410038271
Figure BDA0001512576410038281
Figure BDA0001512576410038291
Figure BDA0001512576410038301
Figure BDA0001512576410038311
Figure BDA0001512576410038321
Figure BDA0001512576410038331
Figure BDA0001512576410038341
Figure BDA0001512576410038351
Figure BDA0001512576410038361
Figure BDA0001512576410038371
Figure BDA0001512576410038381
Figure BDA0001512576410038391
Figure BDA0001512576410038401
Figure BDA0001512576410038411
Figure BDA0001512576410038421
Figure BDA0001512576410038431
Figure BDA0001512576410038441
Figure BDA0001512576410038451
Figure BDA0001512576410038461
Figure BDA0001512576410038471
Figure BDA0001512576410038481
Figure BDA0001512576410038491
Figure BDA0001512576410038501
Figure BDA0001512576410038511
Figure BDA0001512576410038521
Figure BDA0001512576410038531
Figure BDA0001512576410038541
Figure BDA0001512576410038551
Figure BDA0001512576410038561
Figure BDA0001512576410038571
Figure BDA0001512576410038581
Figure BDA0001512576410038591
Figure BDA0001512576410038601
Figure BDA0001512576410038611
Figure BDA0001512576410038621
Figure BDA0001512576410038631
Figure BDA0001512576410038641
Figure BDA0001512576410038651
Figure BDA0001512576410038661
Figure BDA0001512576410038671
Figure BDA0001512576410038681
Figure BDA0001512576410038691
Figure BDA0001512576410038701
Figure BDA0001512576410038711
Figure BDA0001512576410038721
Figure BDA0001512576410038731
Figure BDA0001512576410038741
COMPOUND(51)
Figure BDA0001512576410038742
Figure BDA0001512576410038751
Figure BDA0001512576410038761
Figure BDA0001512576410038771
Figure BDA0001512576410038781
Figure BDA0001512576410038791
Figure BDA0001512576410038801
Figure BDA0001512576410038811
Figure BDA0001512576410038821
Figure BDA0001512576410038831
Figure BDA0001512576410038841
Figure BDA0001512576410038851
Figure BDA0001512576410038861
Figure BDA0001512576410038871
Figure BDA0001512576410038881
Figure BDA0001512576410038891
Figure BDA0001512576410038901
Figure BDA0001512576410038911
Figure BDA0001512576410038921
Figure BDA0001512576410038931
Figure BDA0001512576410038941
Figure BDA0001512576410038951
Figure BDA0001512576410038961
Figure BDA0001512576410038971
Figure BDA0001512576410038981
Figure BDA0001512576410038991
Figure BDA0001512576410039001
Figure BDA0001512576410039011
Figure BDA0001512576410039021
Figure BDA0001512576410039031
Figure BDA0001512576410039041
Figure BDA0001512576410039051
Figure BDA0001512576410039061
Figure BDA0001512576410039071
Figure BDA0001512576410039081
Figure BDA0001512576410039091
Figure BDA0001512576410039101
Figure BDA0001512576410039111
Figure BDA0001512576410039121
Figure BDA0001512576410039131
Figure BDA0001512576410039141
Figure BDA0001512576410039151
Figure BDA0001512576410039161
Figure BDA0001512576410039171
Figure BDA0001512576410039181
Figure BDA0001512576410039191
Figure BDA0001512576410039201
Figure BDA0001512576410039211
Figure BDA0001512576410039221
Figure BDA0001512576410039231
Figure BDA0001512576410039241
Figure BDA0001512576410039251
Figure BDA0001512576410039261
Figure BDA0001512576410039271
Figure BDA0001512576410039281
Figure BDA0001512576410039291
Figure BDA0001512576410039301
Figure BDA0001512576410039311
Figure BDA0001512576410039321
Figure BDA0001512576410039331
Figure BDA0001512576410039341
Figure BDA0001512576410039351
Figure BDA0001512576410039361
Figure BDA0001512576410039371
Figure BDA0001512576410039381
Figure BDA0001512576410039391
Figure BDA0001512576410039401
Figure BDA0001512576410039411
Figure BDA0001512576410039421
Figure BDA0001512576410039431
Figure BDA0001512576410039441
Figure BDA0001512576410039451
Figure BDA0001512576410039461
Figure BDA0001512576410039471
COMPOUND(52)
Figure BDA0001512576410039472
Figure BDA0001512576410039481
Figure BDA0001512576410039491
Figure BDA0001512576410039501
Figure BDA0001512576410039511
Figure BDA0001512576410039521
Figure BDA0001512576410039531
Figure BDA0001512576410039541
Figure BDA0001512576410039551
Figure BDA0001512576410039561
Figure BDA0001512576410039571
Figure BDA0001512576410039581
Figure BDA0001512576410039591
Figure BDA0001512576410039601
Figure BDA0001512576410039611
Figure BDA0001512576410039621
Figure BDA0001512576410039631
Figure BDA0001512576410039641
Figure BDA0001512576410039651
Figure BDA0001512576410039661
Figure BDA0001512576410039671
Figure BDA0001512576410039681
Figure BDA0001512576410039691
Figure BDA0001512576410039701
Figure BDA0001512576410039711
Figure BDA0001512576410039721
Figure BDA0001512576410039731
Figure BDA0001512576410039741
Figure BDA0001512576410039751
Figure BDA0001512576410039761
Figure BDA0001512576410039771
Figure BDA0001512576410039781
Figure BDA0001512576410039791
Figure BDA0001512576410039801
Figure BDA0001512576410039811
Figure BDA0001512576410039821
Figure BDA0001512576410039831
Figure BDA0001512576410039841
Figure BDA0001512576410039851
Figure BDA0001512576410039861
Figure BDA0001512576410039871
Figure BDA0001512576410039881
Figure BDA0001512576410039891
Figure BDA0001512576410039901
Figure BDA0001512576410039911
Figure BDA0001512576410039921
Figure BDA0001512576410039931
Figure BDA0001512576410039941
Figure BDA0001512576410039951
Figure BDA0001512576410039961
Figure BDA0001512576410039971
Figure BDA0001512576410039981
Figure BDA0001512576410039991
Figure BDA0001512576410040001
Figure BDA0001512576410040011
Figure BDA0001512576410040021
Figure BDA0001512576410040031
Figure BDA0001512576410040041
Figure BDA0001512576410040051
Figure BDA0001512576410040061
Figure BDA0001512576410040071
Figure BDA0001512576410040081
Figure BDA0001512576410040091
Figure BDA0001512576410040101
Figure BDA0001512576410040111
Figure BDA0001512576410040121
Figure BDA0001512576410040131
Figure BDA0001512576410040141
Figure BDA0001512576410040151
Figure BDA0001512576410040161
Figure BDA0001512576410040171
Figure BDA0001512576410040181
COMPOUND(53)
Figure BDA0001512576410040182
Figure BDA0001512576410040191
Figure BDA0001512576410040201
Figure BDA0001512576410040211
Figure BDA0001512576410040221
Figure BDA0001512576410040231
Figure BDA0001512576410040241
Figure BDA0001512576410040251
Figure BDA0001512576410040261
Figure BDA0001512576410040271
Figure BDA0001512576410040281
Figure BDA0001512576410040291
Figure BDA0001512576410040301
Figure BDA0001512576410040311
Figure BDA0001512576410040321
Figure BDA0001512576410040331
Figure BDA0001512576410040341
Figure BDA0001512576410040351
Figure BDA0001512576410040361
Figure BDA0001512576410040371
Figure BDA0001512576410040381
Figure BDA0001512576410040391
Figure BDA0001512576410040401
Figure BDA0001512576410040411
Figure BDA0001512576410040421
Figure BDA0001512576410040431
Figure BDA0001512576410040441
Figure BDA0001512576410040451
Figure BDA0001512576410040461
Figure BDA0001512576410040471
Figure BDA0001512576410040481
Figure BDA0001512576410040491
Figure BDA0001512576410040501
Figure BDA0001512576410040511
Figure BDA0001512576410040521
Figure BDA0001512576410040531
Figure BDA0001512576410040541
Figure BDA0001512576410040551
Figure BDA0001512576410040561
Figure BDA0001512576410040571
Figure BDA0001512576410040581
Figure BDA0001512576410040591
Figure BDA0001512576410040601
Figure BDA0001512576410040611
Figure BDA0001512576410040621
Figure BDA0001512576410040631
Figure BDA0001512576410040641
Figure BDA0001512576410040651
Figure BDA0001512576410040661
Figure BDA0001512576410040671
Figure BDA0001512576410040681
Figure BDA0001512576410040691
Figure BDA0001512576410040701
Figure BDA0001512576410040711
Figure BDA0001512576410040721
Figure BDA0001512576410040731
Figure BDA0001512576410040741
Figure BDA0001512576410040751
Figure BDA0001512576410040761
Figure BDA0001512576410040771
Figure BDA0001512576410040781
Figure BDA0001512576410040791
Figure BDA0001512576410040801
Figure BDA0001512576410040811
Figure BDA0001512576410040821
Figure BDA0001512576410040831
Figure BDA0001512576410040841
Figure BDA0001512576410040851
Figure BDA0001512576410040861
Figure BDA0001512576410040871
Figure BDA0001512576410040881
Figure BDA0001512576410040891
Figure BDA0001512576410040901
Figure BDA0001512576410040911
Figure BDA0001512576410040921
Figure BDA0001512576410040931
COMPOUND(54)
Figure BDA0001512576410040932
Figure BDA0001512576410040941
Figure BDA0001512576410040951
Figure BDA0001512576410040961
Figure BDA0001512576410040971
Figure BDA0001512576410040981
Figure BDA0001512576410040991
Figure BDA0001512576410041001
Figure BDA0001512576410041011
Figure BDA0001512576410041021
Figure BDA0001512576410041031
Figure BDA0001512576410041041
Figure BDA0001512576410041051
Figure BDA0001512576410041061
Figure BDA0001512576410041071
Figure BDA0001512576410041081
Figure BDA0001512576410041091
Figure BDA0001512576410041101
Figure BDA0001512576410041111
Figure BDA0001512576410041121
Figure BDA0001512576410041131
Figure BDA0001512576410041141
Figure BDA0001512576410041151
Figure BDA0001512576410041161
Figure BDA0001512576410041171
Figure BDA0001512576410041181
Figure BDA0001512576410041191
Figure BDA0001512576410041201
Figure BDA0001512576410041211
Figure BDA0001512576410041221
Figure BDA0001512576410041231
Figure BDA0001512576410041241
Figure BDA0001512576410041251
Figure BDA0001512576410041261
Figure BDA0001512576410041271
Figure BDA0001512576410041281
Figure BDA0001512576410041291
Figure BDA0001512576410041301
Figure BDA0001512576410041311
Figure BDA0001512576410041321
Figure BDA0001512576410041331
Figure BDA0001512576410041341
Figure BDA0001512576410041351
Figure BDA0001512576410041361
Figure BDA0001512576410041371
Figure BDA0001512576410041381
Figure BDA0001512576410041391
Figure BDA0001512576410041401
Figure BDA0001512576410041411
Figure BDA0001512576410041421
Figure BDA0001512576410041431
Figure BDA0001512576410041441
Figure BDA0001512576410041451
Figure BDA0001512576410041461
Figure BDA0001512576410041471
Figure BDA0001512576410041481
Figure BDA0001512576410041491
Figure BDA0001512576410041501
Figure BDA0001512576410041511
Figure BDA0001512576410041521
Figure BDA0001512576410041531
Figure BDA0001512576410041541
Figure BDA0001512576410041551
Figure BDA0001512576410041561
Figure BDA0001512576410041571
Figure BDA0001512576410041581
Figure BDA0001512576410041591
Figure BDA0001512576410041601
Figure BDA0001512576410041611
Figure BDA0001512576410041621
Figure BDA0001512576410041631
Figure BDA0001512576410041641
Figure BDA0001512576410041651
Figure BDA0001512576410041661
Figure BDA0001512576410041671
Figure BDA0001512576410041681
Figure BDA0001512576410041691
Figure BDA0001512576410041701
Figure BDA0001512576410041711
Figure BDA0001512576410041721
COMPOUND(55)
Figure BDA0001512576410041722
Figure BDA0001512576410041731
Figure BDA0001512576410041741
Figure BDA0001512576410041751
Figure BDA0001512576410041761
Figure BDA0001512576410041771
Figure BDA0001512576410041781
Figure BDA0001512576410041791
Figure BDA0001512576410041801
Figure BDA0001512576410041811
Figure BDA0001512576410041821
Figure BDA0001512576410041831
Figure BDA0001512576410041841
Figure BDA0001512576410041851
Figure BDA0001512576410041861
Figure BDA0001512576410041871
Figure BDA0001512576410041881
Figure BDA0001512576410041891
Figure BDA0001512576410041901
Figure BDA0001512576410041911
Figure BDA0001512576410041921
Figure BDA0001512576410041931
Figure BDA0001512576410041941
Figure BDA0001512576410041951
Figure BDA0001512576410041961
Figure BDA0001512576410041971
Figure BDA0001512576410041981
Figure BDA0001512576410041991
Figure BDA0001512576410042001
Figure BDA0001512576410042011
Figure BDA0001512576410042021
Figure BDA0001512576410042031
Figure BDA0001512576410042041
Figure BDA0001512576410042051
Figure BDA0001512576410042061
Figure BDA0001512576410042071
Figure BDA0001512576410042081
Figure BDA0001512576410042091
Figure BDA0001512576410042101
Figure BDA0001512576410042111
Figure BDA0001512576410042121
Figure BDA0001512576410042131
Figure BDA0001512576410042141
Figure BDA0001512576410042151
Figure BDA0001512576410042161
Figure BDA0001512576410042171
Figure BDA0001512576410042181
Figure BDA0001512576410042191
Figure BDA0001512576410042201
Figure BDA0001512576410042211
Figure BDA0001512576410042221
Figure BDA0001512576410042231
Figure BDA0001512576410042241
Figure BDA0001512576410042251
Figure BDA0001512576410042261
Figure BDA0001512576410042271
Figure BDA0001512576410042281
Figure BDA0001512576410042291
Figure BDA0001512576410042301
Figure BDA0001512576410042311
Figure BDA0001512576410042321
Figure BDA0001512576410042331
Figure BDA0001512576410042341
Figure BDA0001512576410042351
Figure BDA0001512576410042361
Figure BDA0001512576410042371
Figure BDA0001512576410042381
Figure BDA0001512576410042391
Figure BDA0001512576410042401
Figure BDA0001512576410042411
Figure BDA0001512576410042421
Figure BDA0001512576410042431
Figure BDA0001512576410042441
Figure BDA0001512576410042451
Figure BDA0001512576410042461
Figure BDA0001512576410042471
Figure BDA0001512576410042481
Figure BDA0001512576410042491
COMPOUND(56)
Figure BDA0001512576410042492
Figure BDA0001512576410042501
Figure BDA0001512576410042511
Figure BDA0001512576410042521
Figure BDA0001512576410042531
Figure BDA0001512576410042541
Figure BDA0001512576410042551
Figure BDA0001512576410042561
Figure BDA0001512576410042571
Figure BDA0001512576410042581
Figure BDA0001512576410042591
Figure BDA0001512576410042601
Figure BDA0001512576410042611
Figure BDA0001512576410042621
Figure BDA0001512576410042631
Figure BDA0001512576410042641
Figure BDA0001512576410042651
Figure BDA0001512576410042661
Figure BDA0001512576410042671
Figure BDA0001512576410042681
Figure BDA0001512576410042691
Figure BDA0001512576410042701
Figure BDA0001512576410042711
Figure BDA0001512576410042721
Figure BDA0001512576410042731
Figure BDA0001512576410042741
Figure BDA0001512576410042751
Figure BDA0001512576410042761
Figure BDA0001512576410042771
Figure BDA0001512576410042781
Figure BDA0001512576410042791
Figure BDA0001512576410042801
Figure BDA0001512576410042811
Figure BDA0001512576410042821
Figure BDA0001512576410042831
Figure BDA0001512576410042841
Figure BDA0001512576410042851
Figure BDA0001512576410042861
Figure BDA0001512576410042871
Figure BDA0001512576410042881
Figure BDA0001512576410042891
Figure BDA0001512576410042901
Figure BDA0001512576410042911
Figure BDA0001512576410042921
Figure BDA0001512576410042931
Figure BDA0001512576410042941
Figure BDA0001512576410042951
Figure BDA0001512576410042961
Figure BDA0001512576410042971
Figure BDA0001512576410042981
Figure BDA0001512576410042991
Figure BDA0001512576410043001
Figure BDA0001512576410043011
Figure BDA0001512576410043021
Figure BDA0001512576410043031
Figure BDA0001512576410043041
Figure BDA0001512576410043051
Figure BDA0001512576410043061
Figure BDA0001512576410043071
Figure BDA0001512576410043081
Figure BDA0001512576410043091
Figure BDA0001512576410043101
Figure BDA0001512576410043111
Figure BDA0001512576410043121
Figure BDA0001512576410043131
Figure BDA0001512576410043141
Figure BDA0001512576410043151
Figure BDA0001512576410043161
Figure BDA0001512576410043171
Figure BDA0001512576410043181
Figure BDA0001512576410043191
COMPOUND(57)
Figure BDA0001512576410043192
Figure BDA0001512576410043201
Figure BDA0001512576410043211
Figure BDA0001512576410043221
Figure BDA0001512576410043231
Figure BDA0001512576410043241
Figure BDA0001512576410043251
Figure BDA0001512576410043261
Figure BDA0001512576410043271
Figure BDA0001512576410043281
Figure BDA0001512576410043291
Figure BDA0001512576410043301
Figure BDA0001512576410043311
Figure BDA0001512576410043321
Figure BDA0001512576410043331
Figure BDA0001512576410043341
Figure BDA0001512576410043351
Figure BDA0001512576410043361
Figure BDA0001512576410043371
Figure BDA0001512576410043381
Figure BDA0001512576410043391
Figure BDA0001512576410043401
Figure BDA0001512576410043411
Figure BDA0001512576410043421
Figure BDA0001512576410043431
Figure BDA0001512576410043441
Figure BDA0001512576410043451
Figure BDA0001512576410043461
Figure BDA0001512576410043471
Figure BDA0001512576410043481
Figure BDA0001512576410043491
Figure BDA0001512576410043501
Figure BDA0001512576410043511
Figure BDA0001512576410043521
Figure BDA0001512576410043531
Figure BDA0001512576410043541
Figure BDA0001512576410043551
Figure BDA0001512576410043561
Figure BDA0001512576410043571
Figure BDA0001512576410043581
Figure BDA0001512576410043591
Figure BDA0001512576410043601
Figure BDA0001512576410043611
Figure BDA0001512576410043621
Figure BDA0001512576410043631
Figure BDA0001512576410043641
Figure BDA0001512576410043651
Figure BDA0001512576410043661
Figure BDA0001512576410043671
Figure BDA0001512576410043681
Figure BDA0001512576410043691
Figure BDA0001512576410043701
Figure BDA0001512576410043711
Figure BDA0001512576410043721
Figure BDA0001512576410043731
Figure BDA0001512576410043741
Figure BDA0001512576410043751
Figure BDA0001512576410043761
Figure BDA0001512576410043771
Figure BDA0001512576410043781
Figure BDA0001512576410043791
Figure BDA0001512576410043801
Figure BDA0001512576410043811
Figure BDA0001512576410043821
Figure BDA0001512576410043831
Figure BDA0001512576410043841
Figure BDA0001512576410043851
Figure BDA0001512576410043861
Figure BDA0001512576410043871
Figure BDA0001512576410043881
Figure BDA0001512576410043891
Figure BDA0001512576410043901
Figure BDA0001512576410043911
Figure BDA0001512576410043921
Figure BDA0001512576410043931
Figure BDA0001512576410043941
Figure BDA0001512576410043951
Figure BDA0001512576410043961
Figure BDA0001512576410043971
COMPOUND(58)
Figure BDA0001512576410043972
Figure BDA0001512576410043981
Figure BDA0001512576410043991
Figure BDA0001512576410044001
Figure BDA0001512576410044011
Figure BDA0001512576410044021
Figure BDA0001512576410044031
Figure BDA0001512576410044041
Figure BDA0001512576410044051
Figure BDA0001512576410044061
Figure BDA0001512576410044071
Figure BDA0001512576410044081
Figure BDA0001512576410044091
Figure BDA0001512576410044101
Figure BDA0001512576410044111
Figure BDA0001512576410044121
Figure BDA0001512576410044131
Figure BDA0001512576410044141
Figure BDA0001512576410044151
Figure BDA0001512576410044161
Figure BDA0001512576410044171
Figure BDA0001512576410044181
Figure BDA0001512576410044191
Figure BDA0001512576410044201
Figure BDA0001512576410044211
Figure BDA0001512576410044221
Figure BDA0001512576410044231
Figure BDA0001512576410044241
Figure BDA0001512576410044251
Figure BDA0001512576410044261
Figure BDA0001512576410044271
Figure BDA0001512576410044281
Figure BDA0001512576410044291
Figure BDA0001512576410044301
Figure BDA0001512576410044311
Figure BDA0001512576410044321
Figure BDA0001512576410044331
Figure BDA0001512576410044341
Figure BDA0001512576410044351
Figure BDA0001512576410044361
Figure BDA0001512576410044371
Figure BDA0001512576410044381
Figure BDA0001512576410044391
Figure BDA0001512576410044401
Figure BDA0001512576410044411
Figure BDA0001512576410044421
Figure BDA0001512576410044431
Figure BDA0001512576410044441
Figure BDA0001512576410044451
Figure BDA0001512576410044461
Figure BDA0001512576410044471
Figure BDA0001512576410044481
Figure BDA0001512576410044491
Figure BDA0001512576410044501
Figure BDA0001512576410044511
Figure BDA0001512576410044521
Figure BDA0001512576410044531
Figure BDA0001512576410044541
Figure BDA0001512576410044551
Figure BDA0001512576410044561
Figure BDA0001512576410044571
Figure BDA0001512576410044581
Figure BDA0001512576410044591
Figure BDA0001512576410044601
Figure BDA0001512576410044611
Figure BDA0001512576410044621
Figure BDA0001512576410044631
Figure BDA0001512576410044641
Figure BDA0001512576410044651
Figure BDA0001512576410044661
Figure BDA0001512576410044671
Figure BDA0001512576410044681
Figure BDA0001512576410044691
Figure BDA0001512576410044701
Figure BDA0001512576410044711
Figure BDA0001512576410044721
Figure BDA0001512576410044731
Figure BDA0001512576410044741
COMPOUND(59)
Figure BDA0001512576410044742
Figure BDA0001512576410044751
Figure BDA0001512576410044761
Figure BDA0001512576410044771
Figure BDA0001512576410044781
Figure BDA0001512576410044791
Figure BDA0001512576410044801
Figure BDA0001512576410044811
Figure BDA0001512576410044821
Figure BDA0001512576410044831
Figure BDA0001512576410044841
Figure BDA0001512576410044851
Figure BDA0001512576410044861
Figure BDA0001512576410044871
Figure BDA0001512576410044881
Figure BDA0001512576410044891
Figure BDA0001512576410044901
Figure BDA0001512576410044911
Figure BDA0001512576410044921
Figure BDA0001512576410044931
Figure BDA0001512576410044941
Figure BDA0001512576410044951
Figure BDA0001512576410044961
Figure BDA0001512576410044971
Figure BDA0001512576410044981
Figure BDA0001512576410044991
Figure BDA0001512576410045001
Figure BDA0001512576410045011
Figure BDA0001512576410045021
Figure BDA0001512576410045031
Figure BDA0001512576410045041
Figure BDA0001512576410045051
Figure BDA0001512576410045061
Figure BDA0001512576410045071
Figure BDA0001512576410045081
Figure BDA0001512576410045091
Figure BDA0001512576410045101
Figure BDA0001512576410045111
Figure BDA0001512576410045121
Figure BDA0001512576410045131
Figure BDA0001512576410045141
Figure BDA0001512576410045151
Figure BDA0001512576410045161
Figure BDA0001512576410045171
Figure BDA0001512576410045181
Figure BDA0001512576410045191
Figure BDA0001512576410045201
Figure BDA0001512576410045211
Figure BDA0001512576410045221
Figure BDA0001512576410045231
Figure BDA0001512576410045241
Figure BDA0001512576410045251
Figure BDA0001512576410045261
Figure BDA0001512576410045271
Figure BDA0001512576410045281
Figure BDA0001512576410045291
Figure BDA0001512576410045301
Figure BDA0001512576410045311
Figure BDA0001512576410045321
Figure BDA0001512576410045331
Figure BDA0001512576410045341
Figure BDA0001512576410045351
Figure BDA0001512576410045361
Figure BDA0001512576410045371
Figure BDA0001512576410045381
Figure BDA0001512576410045391
Figure BDA0001512576410045401
Figure BDA0001512576410045411
Figure BDA0001512576410045421
Figure BDA0001512576410045431
Figure BDA0001512576410045441
Figure BDA0001512576410045451
Figure BDA0001512576410045461
Figure BDA0001512576410045471
Figure BDA0001512576410045481
Figure BDA0001512576410045491
Figure BDA0001512576410045501
Figure BDA0001512576410045511
Figure BDA0001512576410045521
Figure BDA0001512576410045531
Figure BDA0001512576410045541
COMPOUND(60)
Figure BDA0001512576410045551
Figure BDA0001512576410045561
Figure BDA0001512576410045571
Figure BDA0001512576410045581
Figure BDA0001512576410045591
Figure BDA0001512576410045601
Figure BDA0001512576410045611
Figure BDA0001512576410045621
Figure BDA0001512576410045631
Figure BDA0001512576410045641
Figure BDA0001512576410045651
Figure BDA0001512576410045661
Figure BDA0001512576410045671
Figure BDA0001512576410045681
Figure BDA0001512576410045691
Figure BDA0001512576410045701
Figure BDA0001512576410045711
Figure BDA0001512576410045721
Figure BDA0001512576410045731
Figure BDA0001512576410045741
Figure BDA0001512576410045751
Figure BDA0001512576410045761
Figure BDA0001512576410045771
Figure BDA0001512576410045781
Figure BDA0001512576410045791
Figure BDA0001512576410045801
Figure BDA0001512576410045811
Figure BDA0001512576410045821
Figure BDA0001512576410045831
Figure BDA0001512576410045841
Figure BDA0001512576410045851
Figure BDA0001512576410045861
Figure BDA0001512576410045871
Figure BDA0001512576410045881
Figure BDA0001512576410045891
Figure BDA0001512576410045901
Figure BDA0001512576410045911
Figure BDA0001512576410045921
Figure BDA0001512576410045931
Figure BDA0001512576410045941
Figure BDA0001512576410045951
Figure BDA0001512576410045961
Figure BDA0001512576410045971
Figure BDA0001512576410045981
Figure BDA0001512576410045991
Figure BDA0001512576410046001
Figure BDA0001512576410046011
Figure BDA0001512576410046021
Figure BDA0001512576410046031
Figure BDA0001512576410046041
Figure BDA0001512576410046051
Figure BDA0001512576410046061
Figure BDA0001512576410046071
Figure BDA0001512576410046081
Figure BDA0001512576410046091
Figure BDA0001512576410046101
Figure BDA0001512576410046111
Figure BDA0001512576410046121
Figure BDA0001512576410046131
Figure BDA0001512576410046141
Figure BDA0001512576410046151
Figure BDA0001512576410046161
Figure BDA0001512576410046171
Figure BDA0001512576410046181
Figure BDA0001512576410046191
Figure BDA0001512576410046201
Figure BDA0001512576410046211
Figure BDA0001512576410046221
Figure BDA0001512576410046231
Figure BDA0001512576410046241
Figure BDA0001512576410046251
Figure BDA0001512576410046261
Figure BDA0001512576410046271
Figure BDA0001512576410046281
Figure BDA0001512576410046291
Figure BDA0001512576410046301
Figure BDA0001512576410046311
COMPOUND(61)
Figure BDA0001512576410046312
Figure BDA0001512576410046321
Figure BDA0001512576410046331
Figure BDA0001512576410046341
Figure BDA0001512576410046351
Figure BDA0001512576410046361
Figure BDA0001512576410046371
Figure BDA0001512576410046381
Figure BDA0001512576410046391
Figure BDA0001512576410046401
Figure BDA0001512576410046411
Figure BDA0001512576410046421
Figure BDA0001512576410046431
Figure BDA0001512576410046441
Figure BDA0001512576410046451
Figure BDA0001512576410046461
Figure BDA0001512576410046471
Figure BDA0001512576410046481
Figure BDA0001512576410046491
Figure BDA0001512576410046501
Figure BDA0001512576410046511
Figure BDA0001512576410046521
Figure BDA0001512576410046531
Figure BDA0001512576410046541
Figure BDA0001512576410046551
Figure BDA0001512576410046561
Figure BDA0001512576410046571
Figure BDA0001512576410046581
Figure BDA0001512576410046591
Figure BDA0001512576410046601
Figure BDA0001512576410046611
Figure BDA0001512576410046621
Figure BDA0001512576410046631
Figure BDA0001512576410046641
Figure BDA0001512576410046651
Figure BDA0001512576410046661
Figure BDA0001512576410046671
Figure BDA0001512576410046681
Figure BDA0001512576410046691
Figure BDA0001512576410046701
Figure BDA0001512576410046711
Figure BDA0001512576410046721
Figure BDA0001512576410046731
Figure BDA0001512576410046741
Figure BDA0001512576410046751
Figure BDA0001512576410046761
Figure BDA0001512576410046771
Figure BDA0001512576410046781
Figure BDA0001512576410046791
Figure BDA0001512576410046801
Figure BDA0001512576410046811
Figure BDA0001512576410046821
Figure BDA0001512576410046831
Figure BDA0001512576410046841
Figure BDA0001512576410046851
Figure BDA0001512576410046861
Figure BDA0001512576410046871
Figure BDA0001512576410046881
Figure BDA0001512576410046891
Figure BDA0001512576410046901
Figure BDA0001512576410046911
Figure BDA0001512576410046921
Figure BDA0001512576410046931
Figure BDA0001512576410046941
Figure BDA0001512576410046951
Figure BDA0001512576410046961
Figure BDA0001512576410046971
Figure BDA0001512576410046981
Figure BDA0001512576410046991
Figure BDA0001512576410047001
Figure BDA0001512576410047011
Figure BDA0001512576410047021
Figure BDA0001512576410047031
Figure BDA0001512576410047041
Figure BDA0001512576410047051
COMPOUND(62)
Figure BDA0001512576410047052
Figure BDA0001512576410047061
Figure BDA0001512576410047071
Figure BDA0001512576410047081
Figure BDA0001512576410047091
Figure BDA0001512576410047101
Figure BDA0001512576410047111
Figure BDA0001512576410047121
Figure BDA0001512576410047131
Figure BDA0001512576410047141
Figure BDA0001512576410047151
Figure BDA0001512576410047161
Figure BDA0001512576410047171
Figure BDA0001512576410047181
Figure BDA0001512576410047191
Figure BDA0001512576410047201
Figure BDA0001512576410047211
Figure BDA0001512576410047221
Figure BDA0001512576410047231
Figure BDA0001512576410047241
Figure BDA0001512576410047251
Figure BDA0001512576410047261
Figure BDA0001512576410047271
Figure BDA0001512576410047281
Figure BDA0001512576410047291
Figure BDA0001512576410047301
Figure BDA0001512576410047311
Figure BDA0001512576410047321
Figure BDA0001512576410047331
Figure BDA0001512576410047341
Figure BDA0001512576410047351
Figure BDA0001512576410047361
Figure BDA0001512576410047371
Figure BDA0001512576410047381
Figure BDA0001512576410047391
Figure BDA0001512576410047401
Figure BDA0001512576410047411
Figure BDA0001512576410047421
Figure BDA0001512576410047431
Figure BDA0001512576410047441
Figure BDA0001512576410047451
Figure BDA0001512576410047461
Figure BDA0001512576410047471
Figure BDA0001512576410047481
Figure BDA0001512576410047491
Figure BDA0001512576410047501
Figure BDA0001512576410047511
Figure BDA0001512576410047521
Figure BDA0001512576410047531
Figure BDA0001512576410047541
Figure BDA0001512576410047551
Figure BDA0001512576410047561
Figure BDA0001512576410047571
Figure BDA0001512576410047581
Figure BDA0001512576410047591
Figure BDA0001512576410047601
Figure BDA0001512576410047611
Figure BDA0001512576410047621
Figure BDA0001512576410047631
Figure BDA0001512576410047641
Figure BDA0001512576410047651
Figure BDA0001512576410047661
Figure BDA0001512576410047671
Figure BDA0001512576410047681
Figure BDA0001512576410047691
Figure BDA0001512576410047701
Figure BDA0001512576410047711
Figure BDA0001512576410047721
Figure BDA0001512576410047731
Figure BDA0001512576410047741
Figure BDA0001512576410047751
Figure BDA0001512576410047761
Figure BDA0001512576410047771
Figure BDA0001512576410047781
Figure BDA0001512576410047791
Figure BDA0001512576410047801
Figure BDA0001512576410047811
Figure BDA0001512576410047821
Figure BDA0001512576410047831
COMPOUND(63)
Figure BDA0001512576410047832
Figure BDA0001512576410047841
Figure BDA0001512576410047851
Figure BDA0001512576410047861
Figure BDA0001512576410047871
Figure BDA0001512576410047881
Figure BDA0001512576410047891
Figure BDA0001512576410047901
Figure BDA0001512576410047911
Figure BDA0001512576410047921
Figure BDA0001512576410047931
Figure BDA0001512576410047941
Figure BDA0001512576410047951
Figure BDA0001512576410047961
Figure BDA0001512576410047971
Figure BDA0001512576410047981
Figure BDA0001512576410047991
Figure BDA0001512576410048001
Figure BDA0001512576410048011
Figure BDA0001512576410048021
Figure BDA0001512576410048031
Figure BDA0001512576410048041
Figure BDA0001512576410048051
Figure BDA0001512576410048061
Figure BDA0001512576410048071
Figure BDA0001512576410048081
Figure BDA0001512576410048091
Figure BDA0001512576410048101
Figure BDA0001512576410048111
Figure BDA0001512576410048121
Figure BDA0001512576410048131
Figure BDA0001512576410048141
Figure BDA0001512576410048151
Figure BDA0001512576410048161
Figure BDA0001512576410048171
Figure BDA0001512576410048181
Figure BDA0001512576410048191
Figure BDA0001512576410048201
Figure BDA0001512576410048211
Figure BDA0001512576410048221
Figure BDA0001512576410048231
Figure BDA0001512576410048241
Figure BDA0001512576410048251
Figure BDA0001512576410048261
Figure BDA0001512576410048271
Figure BDA0001512576410048281
Figure BDA0001512576410048291
Figure BDA0001512576410048301
Figure BDA0001512576410048311
Figure BDA0001512576410048321
Figure BDA0001512576410048331
Figure BDA0001512576410048341
Figure BDA0001512576410048351
Figure BDA0001512576410048361
Figure BDA0001512576410048371
Figure BDA0001512576410048381
Figure BDA0001512576410048391
Figure BDA0001512576410048401
Figure BDA0001512576410048411
Figure BDA0001512576410048421
Figure BDA0001512576410048431
Figure BDA0001512576410048441
Figure BDA0001512576410048451
Figure BDA0001512576410048461
Figure BDA0001512576410048471
Figure BDA0001512576410048481
Figure BDA0001512576410048491
Figure BDA0001512576410048501
Figure BDA0001512576410048511
Figure BDA0001512576410048521
Figure BDA0001512576410048531
Figure BDA0001512576410048541
Figure BDA0001512576410048551
Figure BDA0001512576410048561
COMPOUND(64)
Figure BDA0001512576410048562
Figure BDA0001512576410048571
Figure BDA0001512576410048581
Figure BDA0001512576410048591
Figure BDA0001512576410048601
Figure BDA0001512576410048611
Figure BDA0001512576410048621
Figure BDA0001512576410048631
Figure BDA0001512576410048641
Figure BDA0001512576410048651
Figure BDA0001512576410048661
Figure BDA0001512576410048671
Figure BDA0001512576410048681
Figure BDA0001512576410048691
Figure BDA0001512576410048701
Figure BDA0001512576410048711
Figure BDA0001512576410048721
Figure BDA0001512576410048731
Figure BDA0001512576410048741
Figure BDA0001512576410048751
Figure BDA0001512576410048761
Figure BDA0001512576410048771
Figure BDA0001512576410048781
Figure BDA0001512576410048791
Figure BDA0001512576410048801
Figure BDA0001512576410048811
Figure BDA0001512576410048821
Figure BDA0001512576410048831
Figure BDA0001512576410048841
Figure BDA0001512576410048851
Figure BDA0001512576410048861
Figure BDA0001512576410048871
Figure BDA0001512576410048881
Figure BDA0001512576410048891
Figure BDA0001512576410048901
Figure BDA0001512576410048911
Figure BDA0001512576410048921
Figure BDA0001512576410048931
Figure BDA0001512576410048941
Figure BDA0001512576410048951
Figure BDA0001512576410048961
Figure BDA0001512576410048971
Figure BDA0001512576410048981
Figure BDA0001512576410048991
Figure BDA0001512576410049001
Figure BDA0001512576410049011
Figure BDA0001512576410049021
Figure BDA0001512576410049031
Figure BDA0001512576410049041
Figure BDA0001512576410049051
Figure BDA0001512576410049061
Figure BDA0001512576410049071
Figure BDA0001512576410049081
Figure BDA0001512576410049091
Figure BDA0001512576410049101
Figure BDA0001512576410049111
Figure BDA0001512576410049121
Figure BDA0001512576410049131
Figure BDA0001512576410049141
Figure BDA0001512576410049151
Figure BDA0001512576410049161
Figure BDA0001512576410049171
Figure BDA0001512576410049181
Figure BDA0001512576410049191
Figure BDA0001512576410049201
Figure BDA0001512576410049211
Figure BDA0001512576410049221
Figure BDA0001512576410049231
Figure BDA0001512576410049241
Figure BDA0001512576410049251
Figure BDA0001512576410049261
Figure BDA0001512576410049271
Figure BDA0001512576410049281
Figure BDA0001512576410049291
Figure BDA0001512576410049301
序列表
<110> UCB Biopharma SPRL
<120> 用于治疗的新的TNFA结构
<130> N405790WO
<150> GB 1418776.9
<151> 2014-10-22
<150> GB 1418774.4
<151> 2014-10-22
<150> GB 1418773.6
<151> 2014-10-22
<150> GB 1418781.9
<151> 2014-10-22
<150> GB 1510758.4
<151> 2015-06-18
<160> 36
<170> PatentIn version 3.5
<210> 1
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> LCDR1 of 1974/1979
<400> 1
Gln Ala Ser Gln Asp Ile Gly Asn
1 5
<210> 2
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> LCDR2 of 1974
<400> 2
Gly Ala Thr Ser Leu Ala Asp
1 5
<210> 3
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> LCDR3 of 1974
<400> 3
Leu Gln Gly Gln Ser Thr Pro Tyr Thr
1 5
<210> 4
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> HCDR1 of 1974
<400> 4
Ala Tyr Tyr Met Ala
1 5
<210> 5
<211> 18
<212> PRT
<213> 人工序列
<220>
<223> HCDR2 of 1974
<400> 5
Ala Ser Ile Asn Tyr Asp Gly Ala Asn Thr Phe Tyr Arg Asp Ser Val
1 5 10 15
Lys Gly
<210> 6
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> HCDR3 of 1974
<400> 6
Glu Ala Tyr Gly Tyr Asn Ser Asn Trp Phe Gly Tyr
1 5 10
<210> 7
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> LCVR of 1974
<400> 7
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Pro Ala Ser Pro Glu
1 5 10 15
Glu Ile Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Asn Trp
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile
35 40 45
Tyr Gly Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Ala
50 55 60
Ser Arg Ser Gly Thr Gln Tyr Ser Leu Lys Ile Ser Arg Leu Gln Val
65 70 75 80
Glu Asp Phe Gly Ile Phe Tyr Cys Leu Gln Gly Gln Ser Thr Pro Tyr
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 8
<211> 121
<212> PRT
<213> 人工序列
<220>
<223> HCVR of 1974
<400> 8
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr
20 25 30
Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Asn Tyr Asp Gly Ala Asn Thr Phe Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Arg Ser Ser Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Thr Thr Glu Ala Tyr Gly Tyr Asn Ser Asn Trp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 9
<211> 321
<212> DNA
<213> 人工序列
<220>
<223> LCVR DNA of 1974
<400> 9
gacatccaga tgacccagtc tcctgcctcc ctgcctgcat ccccggaaga aattgtcacc 60
atcacatgcc aggcaagcca ggacattggt aattggttat catggtatca gcagaaacca 120
gggaaatcgc ctcagctcct gatctatggt gcaaccagct tggcagatgg ggtcccatca 180
aggttcagcg ccagtagatc tggcacacag tactctctta agatcagcag actgcaggtt 240
gaagattttg gaatctttta ctgtctacag ggtcaaagta ctccgtacac gtttggagct 300
gggaccaagc tggaactgaa a 321
<210> 10
<211> 363
<212> DNA
<213> 人工序列
<220>
<223> HCVR DNA of 1974
<400> 10
gacgtgcagc tggtggaatc tggaggaggc ttagtgcagc ctggaaggtc cctgaaactc 60
tcctgtgcag cctcaggatt cactttcagt gcctattaca tggcctgggt ccgccaggct 120
ccaacgaagg gtctggagtg ggtcgcatcc attaattatg atggtgctaa cactttctat 180
cgcgactccg tgaagggccg attcactgtc tccagagata atgcaagaag cagcctatac 240
ctacaaatgg acagtctgag gtctgaggac acggccactt attactgtac aacagaggct 300
tacggatata actcaaattg gtttggttac tggggccaag gcactctggt cactgtctcg 360
agc 363
<210> 11
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> 1974 LC kappa full
<400> 11
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Pro Ala Ser Pro Glu
1 5 10 15
Glu Ile Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Asn Trp
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile
35 40 45
Tyr Gly Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Ala
50 55 60
Ser Arg Ser Gly Thr Gln Tyr Ser Leu Lys Ile Ser Arg Leu Gln Val
65 70 75 80
Glu Asp Phe Gly Ile Phe Tyr Cys Leu Gln Gly Gln Ser Thr Pro Tyr
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
<210> 12
<211> 445
<212> PRT
<213> 人工序列
<220>
<223> 1974 HC mIgG1 full
<400> 12
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr
20 25 30
Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Asn Tyr Asp Gly Ala Asn Thr Phe Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Arg Ser Ser Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Thr Thr Glu Ala Tyr Gly Tyr Asn Ser Asn Trp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser
115 120 125
Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val
130 135 140
Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro
180 185 190
Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro
195 200 205
Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly
210 215 220
Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys
245 250 255
Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln
260 265 270
Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln
275 280 285
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu
290 295 300
Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg
305 310 315 320
Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro
340 345 350
Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr
355 360 365
Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln
370 375 380
Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly
385 390 395 400
Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu
405 410 415
Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn
420 425 430
His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
435 440 445
<210> 13
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 1974 HC mFabno hinge full
<400> 13
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr
20 25 30
Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Asn Tyr Asp Gly Ala Asn Thr Phe Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Arg Ser Ser Leu Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Thr Thr Glu Ala Tyr Gly Tyr Asn Ser Asn Trp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser
115 120 125
Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val
130 135 140
Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro
180 185 190
Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro
195 200 205
Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys
210 215 220
<210> 14
<211> 642
<212> DNA
<213> 人工序列
<220>
<223> 1974 LC DNA kappa full
<400> 14
gacatccaga tgacccagtc tcctgcctcc ctgcctgcat ccccggaaga aattgtcacc 60
atcacatgcc aggcaagcca ggacattggt aattggttat catggtatca gcagaaacca 120
gggaaatcgc ctcagctcct gatctatggt gcaaccagct tggcagatgg ggtcccatca 180
aggttcagcg ccagtagatc tggcacacag tactctctta agatcagcag actgcaggtt 240
gaagattttg gaatctttta ctgtctacag ggtcaaagta ctccgtacac gtttggagct 300
gggaccaagc tggaactgaa acgtacggat gctgcaccaa ctgtatccat cttcccacca 360
tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 420
cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 480
aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg 540
ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca 600
tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gt 642
<210> 15
<211> 1335
<212> DNA
<213> 人工序列
<220>
<223> 1974 HC DNA mIgG1 full
<400> 15
gacgtgcagc tggtggaatc tggaggaggc ttagtgcagc ctggaaggtc cctgaaactc 60
tcctgtgcag cctcaggatt cactttcagt gcctattaca tggcctgggt ccgccaggct 120
ccaacgaagg gtctggagtg ggtcgcatcc attaattatg atggtgctaa cactttctat 180
cgcgactccg tgaagggccg attcactgtc tccagagata atgcaagaag cagcctatac 240
ctacaaatgg acagtctgag gtctgaggac acggccactt attactgtac aacagaggct 300
tacggatata actcaaattg gtttggttac tggggccaag gcactctggt cactgtctcg 360
agtgccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact 420
aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg 480
acctggaact ctggatccct gtccagcggt gtgcacacct tcccagctgt cctgcagtct 540
gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc 600
gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc 660
agggattgtg gttgtaagcc ttgcatatgt acagtcccag aagtatcatc tgtcttcatc 720
ttccccccaa agcccaagga tgtgctcacc attactctga ctcctaaggt cacgtgtgtt 780
gtggtagaca tcagcaagga tgatcccgag gtccagttca gctggtttgt agatgatgtg 840
gaggtgcaca cagctcagac gcaaccccgg gaggagcagt tcaacagcac tttccgctca 900
gtcagtgaac ttcccatcat gcaccaggac tggctcaatg gcaaggagtt caaatgcagg 960
gtcaacagtg cagctttccc tgcccccatc gagaaaacca tctccaaaac caaaggcaga 1020
ccgaaggctc cacaggtgta caccattcca cctcccaagg agcagatggc caaggataaa 1080
gtcagtctga cctgcatgat aacagacttc ttccctgaag acattactgt ggagtggcag 1140
tggaatgggc agccagcgga gaactacaag aacactcagc ccatcatgga cacagatggc 1200
tcttacttcg tctacagcaa gctcaatgtg cagaagagca actgggaggc aggaaatact 1260
ttcacctgct ctgtgttaca tgagggcctg cacaaccacc atactgagaa gagcctctcc 1320
cactctcctg gtaaa 1335
<210> 16
<211> 669
<212> DNA
<213> 人工序列
<220>
<223> 1974 HC DNA mFabno hinge full
<400> 16
gacgtgcagc tggtggaatc tggaggaggc ttagtgcagc ctggaaggtc cctgaaactc 60
tcctgtgcag cctcaggatt cactttcagt gcctattaca tggcctgggt ccgccaggct 120
ccaacgaagg gtctggagtg ggtcgcatcc attaattatg atggtgctaa cactttctat 180
cgcgactccg tgaagggccg attcactgtc tccagagata atgcaagaag cagcctatac 240
ctacaaatgg acagtctgag gtctgaggac acggccactt attactgtac aacagaggct 300
tacggatata actcaaattg gtttggttac tggggccaag gcactctggt cactgtctcg 360
agtgccaaaa cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact 420
aactccatgg tgaccctggg atgcctggtc aagggctatt tccctgagcc agtgacagtg 480
acctggaact ctggatccct gtccagcggt gtgcacacct tcccggctgt cctgcaatct 540
gacctctaca ctctgagcag ctcagtgact gtcccctcca gcacctggcc cagcgagacc 600
gtcacctgca acgttgccca cccggccagc agcaccaagg tggacaagaa aattgtgccc 660
agggattgt 669
<210> 17
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> LCDR2 of 1979
<400> 17
Gly Thr Thr Ser Leu Ala Asp
1 5
<210> 18
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> LCDR3 of 1979
<400> 18
Leu Gln Ala Tyr Ser Thr Pro Phe Thr Phe
1 5 10
<210> 19
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> HCDR1 of 1979
<400> 19
Asn Ser Tyr Trp Asp
1 5
<210> 20
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> HCDR2 of 1979
<400> 20
Tyr Ile Asn Tyr Ser Gly Ser Thr Gly Tyr Asn Pro Ser Leu Lys Ser
1 5 10 15
<210> 21
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> HCDR3 of 1979
<400> 21
Gly Thr Tyr Gly Tyr Asn Ala Tyr His Phe Asp Tyr
1 5 10
<210> 22
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> LCVR 1979
<400> 22
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Glu
1 5 10 15
Glu Ile Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Asn Trp
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro His Leu Leu Ile
35 40 45
Tyr Gly Thr Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Gln Tyr Ser Leu Lys Ile Ser Gly Leu Gln Val
65 70 75 80
Ala Asp Ile Gly Ile Tyr Val Cys Leu Gln Ala Tyr Ser Thr Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 23
<211> 120
<212> PRT
<213> 人工序列
<220>
<223> HCVR of 1979
<400> 23
Glu Val His Leu Val Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Asn Ser
20 25 30
Tyr Trp Asp Trp Ile Arg Lys Phe Pro Gly Asn Lys Met Glu Trp Met
35 40 45
Gly Tyr Ile Asn Tyr Ser Gly Ser Thr Gly Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Ser Arg Asp Thr Ser Asn Asn Gln Phe Phe Leu
65 70 75 80
Gln Leu Asn Ser Ile Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg Gly Thr Tyr Gly Tyr Asn Ala Tyr His Phe Asp Tyr Trp Gly Arg
100 105 110
Gly Val Met Val Thr Val Ser Ser
115 120
<210> 24
<211> 321
<212> DNA
<213> 人工序列
<220>
<223> LCVR DNA of 1979
<400> 24
gacatccaaa tgacacagtc tcctgcctcc ctgtctgcat ctctggaaga aattgtcacc 60
attacatgcc aggcaagcca ggacattggt aattggttat catggtatca gcagaaacca 120
gggaaatctc ctcacctcct gatctatggt accaccagct tggcagatgg ggtcccatca 180
aggttcagcg gcagtagatc tggtacacag tattctctta agatcagcgg actacaggtt 240
gcagatattg gaatctatgt ctgtctacag gcttatagta ctccattcac gttcggctca 300
gggacaaagc tggaaataaa a 321
<210> 25
<211> 360
<212> DNA
<213> 人工序列
<220>
<223> HCVR DNA of 1979
<400> 25
gaggtgcacc tggtggagtc tggacctggc cttgtgaaac cctcacagtc actctccctc 60
acctgttctg tcactggtta ctccatcact aatagttact gggactggat ccggaagttc 120
ccaggaaata aaatggagtg gatgggatac ataaactaca gtggtagcac tggctacaac 180
ccatctctca aaagtcgaat ctccattagt agagacacat cgaacaatca gttcttcctg 240
cagctgaact ctataactac tgaggacaca gccacatatt actgtgcacg agggacctat 300
gggtataacg cctaccactt tgattactgg ggccgaggag tcatggtcac agtctcgagc 360
<210> 26
<211> 214
<212> PRT
<213> 人工序列
<220>
<223> 1979 LC Kappa full
<400> 26
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Glu
1 5 10 15
Glu Ile Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Asn Trp
20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro His Leu Leu Ile
35 40 45
Tyr Gly Thr Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Gln Tyr Ser Leu Lys Ile Ser Gly Leu Gln Val
65 70 75 80
Ala Asp Ile Gly Ile Tyr Val Cys Leu Gln Ala Tyr Ser Thr Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
<210> 27
<211> 444
<212> PRT
<213> 人工序列
<220>
<223> 1979 HC mIgG1 full
<400> 27
Glu Val His Leu Val Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Asn Ser
20 25 30
Tyr Trp Asp Trp Ile Arg Lys Phe Pro Gly Asn Lys Met Glu Trp Met
35 40 45
Gly Tyr Ile Asn Tyr Ser Gly Ser Thr Gly Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Ser Arg Asp Thr Ser Asn Asn Gln Phe Phe Leu
65 70 75 80
Gln Leu Asn Ser Ile Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg Gly Thr Tyr Gly Tyr Asn Ala Tyr His Phe Asp Tyr Trp Gly Arg
100 105 110
Gly Val Met Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val
115 120 125
Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr
130 135 140
Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr
145 150 155 160
Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser
180 185 190
Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala
195 200 205
Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys
210 215 220
Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val
245 250 255
Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe
260 265 270
Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro
290 295 300
Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val
305 310 315 320
Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335
Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys
340 345 350
Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp
355 360 365
Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro
370 375 380
Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser
385 390 395 400
Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala
405 410 415
Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His
420 425 430
His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
435 440
<210> 28
<211> 222
<212> PRT
<213> 人工序列
<220>
<223> 1979 HC mFabno hinge full
<400> 28
Glu Val His Leu Val Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Asn Ser
20 25 30
Tyr Trp Asp Trp Ile Arg Lys Phe Pro Gly Asn Lys Met Glu Trp Met
35 40 45
Gly Tyr Ile Asn Tyr Ser Gly Ser Thr Gly Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Ser Arg Asp Thr Ser Asn Asn Gln Phe Phe Leu
65 70 75 80
Gln Leu Asn Ser Ile Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg Gly Thr Tyr Gly Tyr Asn Ala Tyr His Phe Asp Tyr Trp Gly Arg
100 105 110
Gly Val Met Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val
115 120 125
Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr
130 135 140
Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr
145 150 155 160
Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser
180 185 190
Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala
195 200 205
Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys
210 215 220
<210> 29
<211> 642
<212> DNA
<213> 人工序列
<220>
<223> 1979 LC DNA Kappa full
<400> 29
gacatccaaa tgacacagtc tcctgcctcc ctgtctgcat ctctggaaga aattgtcacc 60
attacatgcc aggcaagcca ggacattggt aattggttat catggtatca gcagaaacca 120
gggaaatctc ctcacctcct gatctatggt accaccagct tggcagatgg ggtcccatca 180
aggttcagcg gcagtagatc tggtacacag tattctctta agatcagcgg actacaggtt 240
gcagatattg gaatctatgt ctgtctacag gcttatagta ctccattcac gttcggctca 300
gggacaaagc tggaaataaa acgtacggat gctgcaccaa ctgtatccat cttcccacca 360
tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 420
cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 480
aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg 540
ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca 600
tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gt 642
<210> 30
<211> 1332
<212> DNA
<213> 人工序列
<220>
<223> 1979 HC DNA mIgG1 full
<400> 30
gaggtgcacc tggtggagtc tggacctggc cttgtgaaac cctcacagtc actctccctc 60
acctgttctg tcactggtta ctccatcact aatagttact gggactggat ccggaagttc 120
ccaggaaata aaatggagtg gatgggatac ataaactaca gtggtagcac tggctacaac 180
ccatctctca aaagtcgaat ctccattagt agagacacat cgaacaatca gttcttcctg 240
cagctgaact ctataactac tgaggacaca gccacatatt actgtgcacg agggacctat 300
gggtataacg cctaccactt tgattactgg ggccgaggag tcatggtcac agtctcgagt 360
gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 420
tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc 480
tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 540
ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc 600
acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg 660
gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 720
cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 780
gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 840
gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 900
agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 960
aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 1020
aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 1080
agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 1140
aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 1200
tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 1260
acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 1320
tctcctggta aa 1332
<210> 31
<211> 666
<212> DNA
<213> 人工序列
<220>
<223> 1979 HC DNA mFabno hinge full
<400> 31
gaggtgcacc tggtggagtc tggacctggc cttgtgaaac cctcacagtc actctccctc 60
acctgttctg tcactggtta ctccatcact aatagttact gggactggat ccggaagttc 120
ccaggaaata aaatggagtg gatgggatac ataaactaca gtggtagcac tggctacaac 180
ccatctctca aaagtcgaat ctccattagt agagacacat cgaacaatca gttcttcctg 240
cagctgaact ctataactac tgaggacaca gccacatatt actgtgcacg agggacctat 300
gggtataacg cctaccactt tgattactgg ggccgaggag tcatggtcac agtctcgagt 360
gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 420
tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc 480
tggaactctg gatccctgtc cagcggtgtg cacaccttcc cggctgtcct gcaatctgac 540
ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc 600
acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg 660
gattgt 666
<210> 32
<211> 235
<212> PRT
<213> 褐家鼠
<400> 32
Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala
1 5 10 15
Leu Pro Lys Lys Met Gly Gly Leu Gln Asn Ser Arg Arg Cys Leu Cys
20 25 30
Leu Ser Leu Phe Ser Phe Leu Leu Val Ala Gly Ala Thr Thr Leu Phe
35 40 45
Cys Leu Leu Asn Phe Gly Val Ile Gly Pro Asn Lys Glu Glu Lys Phe
50 55 60
Pro Asn Gly Leu Pro Leu Ile Ser Ser Met Ala Gln Thr Leu Thr Leu
65 70 75 80
Arg Ser Ser Ser Gln Asn Ser Ser Asp Lys Pro Val Ala His Val Val
85 90 95
Ala Asn His Gln Ala Glu Glu Gln Leu Glu Trp Leu Ser Gln Arg Ala
100 105 110
Asn Ala Leu Leu Ala Asn Gly Met Asp Leu Lys Asp Asn Gln Leu Val
115 120 125
Val Pro Ala Asp Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys
130 135 140
Gly Gln Gly Cys Pro Asp Tyr Val Leu Leu Thr His Thr Val Ser Arg
145 150 155 160
Phe Ala Ile Ser Tyr Gln Glu Lys Val Ser Leu Leu Ser Ala Ile Lys
165 170 175
Ser Pro Cys Pro Lys Asp Thr Pro Glu Gly Ala Glu Leu Lys Pro Trp
180 185 190
Tyr Glu Pro Met Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp
195 200 205
Leu Leu Ser Ala Glu Val Asn Leu Pro Lys Tyr Leu Asp Ile Thr Glu
210 215 220
Ser Gly Gln Val Tyr Phe Gly Val Ile Ala Leu
225 230 235
<210> 33
<211> 235
<212> PRT
<213> 小家鼠
<400> 33
Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala
1 5 10 15
Leu Pro Gln Lys Met Gly Gly Phe Gln Asn Ser Arg Arg Cys Leu Cys
20 25 30
Leu Ser Leu Phe Ser Phe Leu Leu Val Ala Gly Ala Thr Thr Leu Phe
35 40 45
Cys Leu Leu Asn Phe Gly Val Ile Gly Pro Gln Arg Asp Glu Lys Phe
50 55 60
Pro Asn Gly Leu Pro Leu Ile Ser Ser Met Ala Gln Thr Leu Thr Leu
65 70 75 80
Arg Ser Ser Ser Gln Asn Ser Ser Asp Lys Pro Val Ala His Val Val
85 90 95
Ala Asn His Gln Val Glu Glu Gln Leu Glu Trp Leu Ser Gln Arg Ala
100 105 110
Asn Ala Leu Leu Ala Asn Gly Met Asp Leu Lys Asp Asn Gln Leu Val
115 120 125
Val Pro Ala Asp Gly Leu Tyr Leu Val Tyr Ser Gln Val Leu Phe Lys
130 135 140
Gly Gln Gly Cys Pro Asp Tyr Val Leu Leu Thr His Thr Val Ser Arg
145 150 155 160
Phe Ala Ile Ser Tyr Gln Glu Lys Val Asn Leu Leu Ser Ala Val Lys
165 170 175
Ser Pro Cys Pro Lys Asp Thr Pro Glu Gly Ala Glu Leu Lys Pro Trp
180 185 190
Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp
195 200 205
Gln Leu Ser Ala Glu Val Asn Leu Pro Lys Tyr Leu Asp Phe Ala Glu
210 215 220
Ser Gly Gln Val Tyr Phe Gly Val Ile Ala Leu
225 230 235
<210> 34
<211> 233
<212> PRT
<213> 智人
<400> 34
Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala
1 5 10 15
Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe
20 25 30
Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe
35 40 45
Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro
50 55 60
Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser
65 70 75 80
Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro
85 90 95
Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu
100 105 110
Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser
115 120 125
Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly
130 135 140
Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala
145 150 155 160
Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro
165 170 175
Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu
180 185 190
Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu
195 200 205
Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly
210 215 220
Gln Val Tyr Phe Gly Ile Ile Ala Leu
225 230
<210> 35
<211> 158
<212> PRT
<213> 智人
<400> 35
Ser Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His
1 5 10 15
Val Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg
20 25 30
Arg Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln
35 40 45
Leu Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu
50 55 60
Phe Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr
65 70 75 80
Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser
85 90 95
Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala
100 105 110
Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu
115 120 125
Lys Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp
130 135 140
Phe Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu
145 150 155
<210> 36
<211> 157
<212> PRT
<213> 智人
<400> 36
Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val
1 5 10 15
Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg
20 25 30
Ala Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu
35 40 45
Val Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe
50 55 60
Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile
65 70 75 80
Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala
85 90 95
Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys
100 105 110
Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys
115 120 125
Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe
130 135 140
Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu
145 150 155

Claims (67)

1.包含SEQ ID NO：36的氨基酸序列或在SEQ ID NO：36的长度上与SEQ ID NO:36具有90、95、96、97、98或99％氨基酸序列同一性的相应序列的蛋白质亚基的不对称TNFα三聚体在制备用于治疗人体或动物体的药物中的用途，其中所述TNFα三聚体采用的构象为，当通过X射线晶体学测定时，对于所有亚基而言，SEQ ID NO：36的残基12-18、47-50、54-64、76-82、91-97、117-125、129-137和150-156或者相应序列的相应残基的Cα原子在参考结构化合物34.pdb的相同原子的
Figure FDA0002940744420000011
RMSD之内，所述TNF-α三聚体能够结合TNFR1，但其中来自所述结合的TNFR1的信号传导被减弱或拮抗，其中所述药物用于治疗和/或预防一种或多种以下疾病：类风湿性关节炎，克罗恩氏病，牛皮癣，系统性红斑狼疮，阿尔茨海默氏病，帕金森病和癫痫。
2.权利要求1的用途，其中RMSD在
Figure FDA0002940744420000012
之内。
3.权利要求1的用途，其中RMSD在
Figure FDA0002940744420000013
之内。
4.权利要求1的用途，其中RMSD在
Figure FDA0002940744420000014
之内。
5.权利要求1的用途，其中RMSD在
Figure FDA0002940744420000015
之内。
6.权利要求1的用途，其中RMSD在
Figure FDA0002940744420000016
之内。
7.权利要求1的用途，其中RMSD在
Figure FDA0002940744420000017
之内。
8.权利要求1的用途，其中RMSD在
Figure FDA0002940744420000018
之内。
9.权利要求1的用途，其中所述蛋白质亚基包含SEQ ID NO：36的氨基酸序列或由其组成，任选地其中所述药物用于治疗人体。
10.权利要求1的用途，其中所述构象能够通过TNFα三聚体与化合物(1)-(64)中的任何一种形成复合物来获得，其中化合物(1)-(64)具有下述结构：
Figure FDA0002940744420000021
Figure FDA0002940744420000031
Figure FDA0002940744420000041
Figure FDA0002940744420000051
Figure FDA0002940744420000061
Figure FDA0002940744420000071
Figure FDA0002940744420000081
Figure FDA0002940744420000091
Figure FDA0002940744420000101
Figure FDA0002940744420000111
Figure FDA0002940744420000121
Figure FDA0002940744420000131
Figure FDA0002940744420000141
11.复合物在制备用于治疗人体或动物体的药物中的用途，所述复合物包含蛋白质亚基的TNFα三聚体和能够结合所述TNFα三聚体的化合物，其中所述蛋白质亚基包含SEQ IDNO：36的氨基酸序列或在SEQ ID NO：36的长度上与SEQ ID NO:36具有90、95、96、97、98或99％氨基酸序列同一性的相应序列，其中所述化合物-三聚体复合物结合TNFR1并减弱所述三聚体通过TNFR1诱导的信号传导，其中当通过X射线晶体学测定时，所述化合物包含结合在TNFα三聚体内的药效团，使得所述药效团在SEQ ID NO：36的所有以下残基或者所述相应序列的相应残基的
Figure FDA0002940744420000142
之内：亚基A的Leu57；亚基B的Tyr119；亚基B的Gly121；亚基B的Gly122；亚基C的Tyr59；亚基C的Leu120；和亚基C的Tyr151；并且其中所述药效团由2个稠合的中心在“R3”和“R2”的5-或6-元环、中心在R2的一个环与H键受体“A1”，并且其还通过连接非氢原子被取代为另外的中心在“R4”的5-或6-元环，其中所述药物用于治疗和/或预防一种或多种以下疾病：类风湿性关节炎，克罗恩氏病，牛皮癣，系统性红斑狼疮，阿尔茨海默氏病，帕金森病和癫痫。
12.权利要求11的用途，其中R2环是5元或6元。
13.权利要求11的用途，其中R3环是5元或6元的。
14.权利要求11的用途，其中R4环是5元或6元的。
15.权利要求11的用途，其中R2环是芳族的。
16.权利要求11的用途，其中R3环是芳族的。
17.权利要求11的用途，其中R4环是芳族的。
18.权利要求11的用途，其中R2环是杂芳族的。
19.权利要求11的用途，其中R3环是杂芳族的。
20.权利要求11的用途，其中R4环是杂芳族的。
21.权利要求11的用途，其中稠合环共有2个原子。
22.权利要求11的用途，其中连接性非氢原子是碳、氮或氧。
23.权利要求11的用途，其中A1特征是通过R2环中的氮或氧原子。
24.权利要求11的用途，其中A1特征与所述TNFα三聚体的亚基C上的Tyr151的侧链氢键结合。
25.权利要求11的用途，其中所述R2、R3和R4环以及A1特征根据下表排列在所述TNFα三聚体结构内：
Figure FDA0002940744420000151
Figure FDA0002940744420000161
26.权利要求11的用途，其中所述R2，R3，R4和A1特征之间的一个或多个距离是根据下表中的值或在该值的10％内
Figure FDA0002940744420000162
27.权利要求11的用途，其中所述R2，R3，R4和A1特征之间的一个或多个角度是根据下表中的值或在该值的10％内
Figure FDA0002940744420000163
Figure FDA0002940744420000171
28.权利要求11的用途，其中所述R3-R2-R4角限定涉及所述TNFα三聚体的去对称化的香蕉形状。
29.权利要求11的用途，其中包含药效团的化合物具有20-41个非氢原子。
30.权利要求11的用途，其中所述化合物是化合物(1)-(64)中的任何一种。
31.权利要求11的用途，其中所述药物用于治疗人体。
32.权利要求11的用途，其中所述蛋白质亚基包含SEQ ID NO：36的氨基酸序列或由其组成。
33.权利要求11的用途，其中所述复合物中的TNFα三聚体是如权利要求1所定义的TNFα三聚体。
34.权利要求11的用途，其中所述化合物拮抗TNFα三聚体通过TNFR1受体诱导的信号传导。
35.权利要求11的用途，其中所述化合物与不存在所述化合物时所述TNFα三聚体的稳定性相比增加所述TNFα三聚体的稳定性。
36.权利要求35的用途，其中稳定性的增加导致TNFα三聚体的热转变中点升高至少1℃。
37.权利要求35的用途，其中稳定性的增加导致TNFα三聚体的热转变中点升高至少10℃。
38.权利要求35的用途，其中稳定性的增加导致TNFα三聚体的热转变中点升高10℃至20℃。
39.权利要求11的用途，其中所述化合物与不存在所述化合物的情况下所述TNFα三聚体对所述TNFR1受体的结合亲和力相比，增加所述TNFα三聚体对所述TNFR1受体的结合亲和力。
40.权利要求39的用途，其中所述化合物通过相较于在不存在所述化合物的情况下TNFα三聚体与TNFR1受体结合的缔合速率和解离速率值增加缔合速率和/或降低解离速率来增加所述TNFα三聚体与TNFR1受体的结合亲和力。
41.权利要求40的用途，其中所述化合物通过相较于在不存在所述化合物的情况下TNFα三聚体与TNFR1受体结合的缔合速率值增加缔合速率来增加所述TNFα三聚体与TNFR1受体的结合亲和力。
42.权利要求39的用途，其中与不存在所述化合物的情况下TNFα三聚体对TNFR1受体的KD-r相比，所述化合物降低所述TNFα三聚体对TNFR1受体的KD-r，其中：
a)与不存在所述化合物的情况下所述TNFα三聚体对TNFR1受体的KD-r相比，所述化合物降低所述TNFα三聚体对TNFR1受体的KD-r至少10倍；
b)在所述化合物的存在下TNFα三聚体对TNFR1受体的KD-r值小于10nM。
43.权利要求39的用途，其中与不存在所述化合物的情况下TNFα三聚体对TNFR1受体的KD-r相比，所述化合物降低所述TNFα三聚体对TNFR1受体的KD-r，其中：
a)与不存在所述化合物的情况下所述TNFα三聚体对TNFR1受体的KD-r相比，所述化合物降低所述TNFα三聚体对TNFR1受体的KD-r至少4倍；
b)在所述化合物的存在下TNFα三聚体对TNFR1受体的KD-r值小于600pM。
44.权利要求43的用途，其中在所述化合物存在下所述TNFα三聚体对所述TNFR1受体的KD-r值小于200pM。
45.权利要求11的用途，其中所述化合物具有500nM或更低的IC50值。
46.权利要求11的用途，其中所述复合物以1nM或更低的KD-ab结合以下抗体中的任一种：
具有序列SEQ ID NO：26的轻链和序列SEQ ID NO：27的重链的CA185_1979；或
具有序列SEQ ID NO：11的轻链和序列SEQ ID NO：12的重链的CA185_1974。
47.权利要求1的用途，其中TNFα三聚体拮抗TNFR1受体的信号传导。
48.权利要求1的用途，其中所述TNFα三聚体与对称TNFα三聚体的稳定性相比具有增加的稳定性。
49.权利要求48的用途，其中稳定性增加导致TNFα三聚体的热转变中点升高至少1℃。
50.权利要求48的用途，其中稳定性增加导致TNFα三聚体的热转变中点升高至少10℃。
51.权利要求48的用途，其中稳定性增加导致TNFα三聚体的热转变中点升高10℃至20℃。
52.权利要求1的用途，其中与对称TNFα三聚体与TNFR1的结合亲和力相比，所述TNFα三聚体对TNFR1受体的结合亲和力增加。
53.权利要求52的用途，其中通过相较于对称TNFα三聚体与TNFR1受体结合的缔合速率和解离速率值而言增加其缔合速率和/或降低其解离速率，所述TNFα三聚体对TNFR1受体的结合亲和力增加。
54.权利要求52的用途，其中通过相较于对称TNFα三聚体与TNFR1受体结合的缔合速率值而言增加其缔合速率，所述TNFα三聚体对TNFR1受体的结合亲和力增加。
55.权利要求52的用途，其中与对称TNFα三聚体对TNFR1受体的KD-r相比，所述TNFα三聚体对TNFR1受体的KD-r降低，其中：
a)与对称TNFα三聚体对TNFR1受体的KD-r相比，所述TNFα三聚体对TNFR1受体的KD-r降低至少10倍；
b)所述TNFα三聚体对TNFR1受体的KD-r值小于10nM。
56.权利要求52的用途，其中与对称TNFα三聚体对TNFR1受体的KD-r相比，所述TNFα三聚体对TNFR1受体的KD-r降低，其中：
a)与对称TNFα三聚体对TNFR1受体的KD-r相比，所述TNFα三聚体对TNFR1受体的KD-r降低至少4倍；
b)所述TNFα三聚体对TNFR1受体的KD-r值小于600pM。
57.权利要求56的用途，其中所述TNFα三聚体与TNFR1受体的KD-r值小于200pM。
58.权利要求1的用途，其中所述TNFα三聚体以1nM或更低的KD-ab结合以下抗体中的任一种：
具有序列SEQ ID NO：26的轻链和序列SEQ ID NO：27的重链的CA185_1979；或
具有序列SEQ ID NO：11的轻链和序列SEQ ID NO：12的重链的CA185_1974。
59.权利要求1或7的用途，其中TNFα三聚体是可溶性TNFα、膜结合TNFα或可溶性TNFα和膜结合TNFα。
60.权利要求1或11的用途，其中所述TNFα三聚体包含SEQ ID NO：36的蛋白质序列或由其组成。
61.权利要求1或11的用途，其中所述用途不诱导或导致恶化感染和/或恶性肿瘤。
62.权利要求61的用途，其中所述感染是TB和/或细菌败血症和/或真菌感染。
63.权利要求61的用途，其中所述恶性肿瘤是淋巴瘤。
64.一种包括可执行代码的计算机，其用于：
a)使用化合物1.pdb至化合物64.pdb中的任一种的TNFα三聚体的结构坐标作为不对称TNFα三聚体的三维模型；
b)在三维模型中分析三聚体中心的结合口袋；和
c)在计算机文库中筛选适合所述结合位点的小分子。
65.鉴定apo TNFα三聚体的潜在抑制剂的方法，包括以下步骤：
a)使用化合物1.pdb至化合物64.pdb中的任何一种的TNFα三聚体的结构坐标来产生不对称TNFα三聚体的三维模型；
b)在三维模型中鉴定三聚体中心的结合口袋的残基；
c)使用结合位点残基产生特定的3-D靶标；
d)使用特定的3-D靶设计或选择潜在的TNFα三聚体抑制剂；
e)获得潜在的抑制剂；和
f)体外使潜在抑制剂与apo TNFα三聚体接触，以确定所述潜在抑制剂与所述apo TNFα三聚体相互作用的能力，
由此相互作用的能力指示所述apo TNFα三聚体的所述潜在抑制剂被确定。
66.一种设计结合apo TNFα三聚体的化合物的方法，包括以下步骤：
a)使用化合物1.pdb至化合物64.pdb中的任一种的TNFα三聚体的原子坐标来建立TNFα三聚体中心处的结合口袋的三维计算机模型；
b)评估化合物和结合口袋之间的立体化学互补性；
c)通过观察影响蛋白质/化合物缔合的蛋白质或化合物的变化，以迭代方法优化立体化学互补性；和
d)设计优化所述蛋白质/化合物立体化学互补性的化合物。
67.化合物1.pdb至化合物64.pdb中任何化合物的结构坐标在鉴定apo TNFα三聚体抑制剂中的用途。
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