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Discovery of Potent and Selective Inhibitors of Ataxia Telangiectasia Mutated and Rad3 Related (ATR) Protein Kinase as Potential Anticancer Agents ABSTRACT: DNA-damaging agents are among the most frequently used anticancer drugs. However, they provide only modest benefit in mostcancers. Thismay be attributedtoa genomemaintenance network, the DNA damage response (DDR), that recognizes and repairs damaged DNA. ATR is a major regulator of the DDR and an attractive anticancer target. Herein, we describe the discovery of a series of aminopyrazines with potent and selective ATR inhibition. Compound 45 inhibits ATR with a Kiof 6 nM, shows >600-fold selectivity over related kinases ATM or DNA-PK, and blocks ATR signaling in cells with an IC50of 0.42 μM. Using this compound, we show that ATR inhibition markedly enhances death induced by DNA-damaging agents in certain cancers but not normal cells. This differential response between cancer and normal cells highlightsthegreatpotentialforATRinhibitionasanovelmechanismto dramatically increase the efficacy of many established drugs and ionizing radiation. ’INTRODUCTION DNA-damaging agents such as cisplatin, irinotecan, gemcitabine, and ionizing radiation (IR) represent the cornerstone for the treatment of solid tumors. While they can be highly effective in the treatment of certain cancers, for example, testicular cancer,1for the majority of solid tumors, they provide onlymodest benefit. The fact that, intumor cells, proficient processes exist to repair the damaged DNA2-5provides one explanation for the poor response. Important among these processes is the DNAdamageresponse(DDR).6Twophosphoinositol3-kinase-like kinase (PIKK) family members, ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad-3 related), act together as apicalregulatorsofthissignalingpathway.7,8Betweenthem,they actonacomplexnetworkinvolvinghundredsofsubstrates,many of which are shared, to regulate a wide range of critical functions such as cell cycle checkpoint activation and DNA damage repair.Although ATM and ATR are recruited to different DNA strand break structures, it is now known that these structures can be readily interconverted in the cell.8Loss of ATM function is very common in tumors, either through lossof ATM itself or through defects in upstream and downstream signaling.9-12It is believed that such a loss enables the proliferation of incipient cancer cells that carry DNA lesions.13While this may confer a growth advantage on the tumor cells, it is likely to place more reliance on the ATR pathway, for survival following DNA damage. In support of this, some studies have shown that disruption of p53 function, a major substrate for ATM, enhances cell sensitivity to ATR disruption.14,15The exploitation of a potentially synthetic lethal interaction between the ATR and the ATM-p53 pathway provides an attractive opportunity to deliver anticancer drugs that increase the efficacy of established DNA-damaging agents. The benefits of exploiting such synthetic lethal interactions have recently been demonstrated with inhibitors of poly-ADP-ribose polymerase (PARP), an enzyme also involved in DNA repair.16 Cancer cells defective in the breast cancer susceptibility proteins BRCA1/2, which participate in a complementary DNA repair pathway,areacutelysensitivetoinhibitorsofPARP.A number of PARP inhibitors are in clinical trials, and initial results are highly encouraging.17,18 AnumberofpotentandselectiveinhibitorsofthePIKKfamily members ATM (e.g. 1, KU-5593319), DNA-PK(e.g. 2, NU 702620),andtheATRsubstrateChk1(e.g.3,AZD-776221)have been disclosed. In contrast, reported inhibitors of ATR such as 4 (caffeine22) and 5 (schisandrin B23) (Figure 1) are weak and nonselective (Table 1). However, there is a growing interest in ATR as a target for anticancer drugs,24and a number of patent applications have recently been filed that claim ATR inhibitors (from AstraZenecaand Vertex), although limited biochemical or cellular data are disclosed in these documents.25-27The work described here rectifies the shortage of good, well-characterized chemical tools that has hampered characterization of ATR as an oncology target.24Specifically, these compounds will be valuable in helping to define the potential for synthetic lethal interactions with the ATM signaling pathway and the impact of ATR inhibition on normal cells. We report a novel series of 3-amino-6-arylpyrazines that has provided potent and highly selective inhibitors of ATR. Starting from the hit compound 6 (Figure 1), identified from a high-throughput screen (HTS), a combination of structure-activity relationship (SAR) studies and homology modeling led to an understanding of the interactions between the inhibitors and the ATR active site that are critical for both potency and selectivity. Compound 45 represents one of the most potent (Kiappof 6 nM for ATR) and selective members of the series and is used here to illustratetheattractivecellularphenotypethatcanbeachievedby inhibiting ATR。 ’CHEMISTRY Compound 8, with its 6-bromo functional group, provided a late stage intermediate that enabled rapid and versatile modifica-tionofthephenylringatthe6-positionofthepyrazinenucleusin compound 6. The synthetic route for the preparation of ATR inhibitors 6-47 is depicted in Scheme 1. Commercial methyl aminopyrazin-2-carboxylate 53wasbrominatedatthe6-position of the pyrazine nucleus with NBS,28to provide intermediate 54 in high yield. Base hydrolysis of the methyl ester 54 afforded the carboxylic acid 56, which condensed with aniline to give the desired advanced intermediate amide 8.29Diversity of the group at the 6-position of the pyrazine ring was introduced when 8 was subjected to Suzuki cross-coupling30reactions with a range of boronic acids/boronates to generate inhibitors 6 and 10-47. The fully saturated cyclohexane 9 was produced from the cyclohexene derivative 10 by reduction under hydrogenation conditions.31 For alternative amides at the 2-position of the pyrazine core (e.g., cyclohexylamide 48), the chemical sequence described in Scheme 2 was used. A Suzuki cross-coupling reaction be-tween 4-(methylsulfonyl)phenylboronic acid and 54 provided advancedintermediate 57, which in turn was converted toamide 48. An array of bicyclic heteroaryl moieties that could act as phenylamide isosteres was also prepared (benzimidazole, benzoxazole, benzothiazole, and indoles 49-52; Schemes 3 and 4). Early installation of the bicyclic heteroaryl feature preserved the versatility of the 6-position. Thus, treatment of intermediate carboxylic acid 56 with phenylenediamine in DME, in the presence of diethoxy-phosphorylformonitrile, afforded the late stage intermediate benzimidazole 58 in moderate yield.32Subsequent coupling with 4-(methylsulfonyl) phenylboronic acid provided isostere 49. A similar functionalization sequence was adopted for the synthesis of benzoxazole and benzothiazole isosteres 50 and 51. Conversion of the nitrile 59 into late stage intermediatesbenzoxazole60orbenzothiazole61proceededwith 2-aminophenol or 2-aminothiophenol in moderate yield.33-35 Suzuki cross-coupling with 4-(methylsulfonyl)boronic acid gave the required isosteres 50 and 51. Although the chemical sequences described in Scheme 3 for the preparation of isosteres 49-51 have provided the desired compounds,thereversesyntheses(i.e.,functionalizationthrough Suzuki cross-coupling prior to the construction of the isosteric motifs, in a sequence similar to that described in Scheme 2) have also been successfully utilized and are preferred for exploration of the substitution of the bicyclic isosteres. A sequential derivatization (Scheme 4) of building block 6236 ’RESULTS AND DISCUSSION Compound 6 was identified from a HTS against full-length recombinant ATR. It inhibits ATR with an IC50of 0.62 μM and has good selectivity against ATM and DNAPK (IC50> 8 μM). However, inhibition of ATR activity in cell-based assays, as measured by a reduction in hydroxyurea-induced phosphorylation of H2AX, a direct substrate of ATR37was not observed (IC50> 2.5 μM). Because high-resolution crystallographic data for any member of the PIKK family were unavailable, the related kinase phosphatidylinositol 3-kinase γ (PI3K-γ) was used as a structural template38,39for an ATR homology model to aid inhibitor design. To provide guidance on selectivity, homology models of ATM and DNAPK were also constructed from the same root. The low-resolution structure of DNAPK reported by Sibanda et al.40was used to provide guidance on the overall fold. Although sequence identity between PI3K-γ and PIKK family membersisrelativelylow(e.g.,22%betweenPI3K-γandATRin the kinase domain), a number of important residues are conserved within the active site. These include a salt bridge provided the indole variant 52. This sequence enabled the introduction of the indole motif at the most reactive position (vicinal to amino group), using N-Boc-indol-2-ylboronic ester in a palladium catalyzed cross-coupling reaction; a second Suzuki coupling with 4-(methylsulfonyl)phenylboronic acid, followed by Boc deprotection with TFA, afforded the desired indole containing derivative 52. The homology model with the HTS hit 6 bound to ATR is shown in Figure 2. The positioning of compound 6 in the model active site was guided by a cocomplex structure of a related aminopyrazine in PI3K-γ (not shown). This enabled us to locate critical H-bond interactions between the hinge motif of the protein active site and the core scaffold. Minimization of the co-complex of 6 with ATR predicts H-bond interactions of the ringsp2-nitrogenwiththebackbone-NH ofVal2378(3.01Å  and the exocyclic amine of the amino-pyrazine with the carbonyl of Glu2380 (3.08 Å. The biaryl motif of the inhibitor is also anticipated to contribute to the binding through π-stacking with the indole ring of Trp2379.The hydroxyl of the gatekeeper residue (Tyr2365 in ATR) is located near the amide carbonyl at the 2-position of the pyrazine and appears to be positioned to form a H-bond. A salt bridge between Asp2494 and Lys2327, conserved across the PIKK family,is predicted to be disrupted by the anilinering of inhibitor 6, forcing the side chain of Asp2494 down, thus allowing the aromatic ring to fit under the P loop (Figure 2). This P loop, unusually rich in lipophilic residues when compared with most kinases, appears to fold over the aniline ring at the 2-position (Figure 3) and may also contribute to lipophilic binding. In addition to the interactions described above, the potential interactions between compound 6 and ATM or DNAPK were also examined (described in detail below). This work suggested exploitable differences that should allow selectivity against these related kinases to be engineered. With the objective of increasing potency while preserving selectivity, a first round of exploration was conducted through modification or substitution of the 6-aryl ring (Table 2). As predicted by the homology model, the π-stacking interaction between the 6-aryl in 6 and the Trp2379 makes an important contribution to the potency of the inhibitor. Removal of the phenylring(7and8)resultedina10-foldlossofbindingaffinity, and its complete or partial reduction (cyclohexyl 9 and cyclohex-enyl 10, respectively) led to about 5-fold decrease in potency. These results prompted us to retain an aromatic group at the 6-position of the aminopyrazine core. Exchanging the 6-phenyl group with a pyridine (11) produced 3-fold potency increase. However, inhibition of some Cyp isoforms was also increased (e.g., IC50 cyp3A42 μM for 11 vs IC50 cyp3A4>100 μM for 6), discouraging us from using this motif further. Compounds 13-19 provided information on the effects of substitution at the ortho-position. Introduction of a nitrile group (13) led to a 25-fold improvement in potency against ATR but also increased affinity for ATM and DNAPK. The enhanced potency for ATR and DNAPK can be explained by the possible formation of a new H-bond with a serine residue (Ser2305 in ATRorSer3731inDNAPK).AlthoughATMhasnosuch serine residue, a slight change in conformation of the P loop could, in this case, allow a backbone N-H to participate in an H-bond to the nitrile group. No other substituent gave such affinity en-hancement, although sulfoxide 14 was able to provide a 5-fold improvement. Small group substitutions in the meta-position (compounds20-25) did not lead to significantly increased ATR potency, although selectivity was compromised by polar groups. On the other hand, substitution in the para-position (compounds 26-31) led to the most potent and selective inhibitors in this array. The sulfone 27, and to a lesser extent nitrile 26, provided significant improvements in potency against ATR (25-fold for 27), while retaining >100-fold selectivity against ATM and DNAPK (e.g., for 27, IC50 ATR= 26 nM, IC50 ATM= >8 μM, and IC50DNAPK= 4.4 μM). These results can be attributed to the formation of a putative H-bond with the backbone N-H of the ATR-specific glycine residue (Gly2385), located C-terminal to thehingeregion(Figure3,bluevanderWaalsspheres).InATM, this residue is a proline (Figure 3, red mesh), and no such H-bond can be formed. The equivalent residue in DNAPK is a threonine (Figure 3, yellow mesh), and any chance of a similar H-bond is likely to be blocked by the presence of the threonine side chain. Anilides 6-31 have the potential to form anilines In Vivo and so present a potential toxicological liability. There-fore, the effects of removing the anilide by reduction of the aromatic ring or by providing isosteres of the amide functional group were examined (compounds 48-52). Saturation of the aniline ring (48) resulted in a >100-fold loss in potency. On the other hand, the use of heterocyclic motifs as anilide “isosteres” gave much better results. Benzimidazole 49,41and by shape analogy benzoxazole 50, benzothiazole 51, and indole 52 show similar inhibitory activity to anilide 27 (Table 3) despite the fact only benzimidazole 49 has the potential to form the same H-bonds as the original amide. Neither replacement of the H-bond donor N-H in 27 with an oxygen acceptor in benzox-azole 50 nor removal of the H-bond acceptor carbonyl in indole 52 compromised potency. These results indicate that the proposed H-bond between the anilide carbonyl and the Tyr2365 is not a major contributor to binding, and we concluded that the amide group functions primarily as a linker to position the aromatic group under the P loop. Although these bicyclic heteroaryls led to good ATR inhibition, selectivity against ATM was compromised in all cases. Compounds 49-52 cannot mimic the exact shape of the anilide in27and,asaconsequence,have to bind deeperin the activesite and become more likely to clash with the conserved tyrosine in thebackoftheadenosine-50-triphosphate(ATP)bindingpocket. Inspection of the homology modelsledustohy pothesize that the loss in selectivity may be associated with compounds 49-52 attaining a slightly different position in the active site relative to 27 (clockwise rotation illustrated in Figure 4). In this binding mode, compounds 49-52 avoid a clash with the tyrosine gate-keeper residue and also appear to alleviate a potential clash between the para-sulfone and Pro2775 in ATM that is observed for 27 (the equivalent residue is Gly2385 in ATR and Thr3809 in DNAPK). In addition, the ATM homology model predicts that an arginine residue (Arg2691) may also contribute to the enhanced potency through a H-bond with the sulfone in compounds 49-52. Because the anilide27remainedthemostpotentandselective compound, it was chosen as the platform from which to further understand the interaction of the sulfone group. SARs between compoundsin Table2 and the ATR homology model(Figure3)suggested that para-H-bond acceptors were important and that an optimal interaction with the protein could be formed when the H-bond acceptor is positioned below the plane of the 6-phenyl ring. A set of para-amides (32-38) (Table 4) that have a range of in vacuo dihedral angles between the carbonyl and the plane of the6-phenylwereusedtoassessthishypothesis.The piperidine amide 37, with a natural dihedral angle of ～60?, was the most potentin this series.This is consistent with the ATR homology model, which suggests that the dihedral angle in the co-complex is～65? for an optimal H-bond with Gly2385. For other amide analogues, the decreased potency can be explained by rotational energy barriers to the optimal ～65? dihedral angle: for example, the primary amide 32, which is conjugated with the phenyl ring, has to overcome a 6 kcal/mol barrier, which leads to a 4-fold reduction in potency(IC50of110nMascomparedtoanIC50of26nMfor37).In the case of sulfone 27, the improved potency is attributed to a low rotation alenergy barrier (<1 kcal/mol) to positiona sulfone oxygen at the optimal angle and distance for a H-bond with the -NH- of Gly2385. While the use of amides offers advantages in terms of the provision of chemical diversity, the requirement for favorable dihedral angles for good H-bond interactions limits the possibilities.Wethereforecontinuedtoexplore theutility ofsulfone 27. Substituting the methyl-sulfone itself with both lipophilic (40-44) and polar groups (45-47) led to compounds with potency improvements of up to 4-fold, while retaining selectivity versus DNAPK and ATM. In addition, polar substitution (45-47) provided an enhancementin cell potency that may have been due toim proved physical properties.Compound45,forexample,has an enzyme potency of 12 nM and inhibits ATR-mediated phosphorylation of H2AX in cells with an IC50of 0.42 μM; in contrast,compound42,with as imilarenzyme potency of14nM, inhibits ATR in cells with an IC50of 2 μM. Besides its attractive combination of potency, selectivity, and cellular activity, compound 45 has good “druglike” properties that include aqueous solubility, lipophilicity (cLogP 3.0), and a profilein Caco 2 study that suggests good passive diffusionacross membranes with minimal efflux liability (A-B = 17.10-6cm/s, and B-A = 23.10-6cm/s). It was therefore selected for further biological evaluation. |
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RXMCDM: 你不是误人吗？ 2014-04-18 23:36:57
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RXMCDM: 你不是误人吗？ 2014-04-18 23:36:57
RXMCDM: 金币+2, 幸苦你了！ 2014-04-20 00:05:08
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谷歌翻译的，哈哈 可以参考参考！！ 的共济失调毛细血管扩张症突变和RAD3相关（ATR）蛋白激酶的强效选择性抑制剂的发现作为潜在抗癌药物摘要：DNA损伤剂是最常用的抗癌药物之一。然而，它们仅提供适度的好处mostcancers。Thismay是attributedtoa genomemaintenance网络，在DNA损伤反应（DDR），识别和修复受损的DNA。ATR是DDR和一个有吸引力的抗癌目标的主要调节剂。本文中，我们描述了一系列氨基吡嗪与有效和选择性抑制ATR的发现。化合物45抑制ATR与Kiof 6纳米，显示> 600倍的选择性超过相关激酶ATM或DNA-PK，并阻止ATR信号在细胞与IC50of 0.42微米。使用该化合物，我们表明，ATR抑制显着增强了死亡诱导在某些癌症的DNA损伤剂，但不能正常细胞。这种癌症和正常细胞之间的差别响应highlightsthegreatpotentialforATRinhibitionasanovelmechanismto显着增加的许多已建立的药物和电离辐射的功效。'引言DNA损伤剂，例如顺铂，伊立替康，吉西他滨，和电离辐射（IR）所代表的基石用于实体瘤的治疗。虽然它们可以用于治疗某些癌症，例如，睾丸癌，1对于大多数实体肿瘤的高度有效的，它们提供onlymodest好处。即，intumor细胞，精通工艺存在修复损坏的DNA2-5provides一种解释为穷人响应的事实。这些过程中重要的是DNAdamageresponse（DDR）.6 Twophosphoinositol3-激酶样激酶（PIKK）家族成员，ATM（共济失调毛细血管扩张症突变）和ATR（ATM和抗辐射3相关），一起行动起来为apicalregulatorsofthissignalingpathway.7，8Betweenthem，他们actonacomplexnetworkinvolvinghundredsofsubstrates，其中有许多是共享的，调节范围广的关键功能，如细 胞周期检查点的激活和DNA损伤repair.Although ATM和ATR被招募到不同的DNA链断裂的结构，它是目前已知这些结构可以很容易地互变中的ATM功能的cell.8Loss是在肿瘤很常见的，无论是通过lossof ATM本身或通过在上游和下游signaling.9-12It的缺陷被认为是这样的损失使初期的癌细胞进行DNA损伤的增殖。 13While这可能赋予肿瘤细胞生长优势，它很可能放置在ATR途径更加依赖，对于生存下列DNA损伤。为支持这项工作，一些研究显示p53功能的破坏，对ATM的主要底物，促进细胞的敏感性ATR disruption.14，在ATR和ATM-p53通路间潜在的合成致死的互动第十五开发提供了一个有吸引力的机会来传递抗癌药物，增加既定的DNA损伤剂的有效性。利用这样合成致死相互作用的好处最近已证明与聚-ADP-核糖聚合酶（PARP）抑制剂，酶也参与DNA repair.16癌细胞有缺陷的乳腺癌易感蛋白BRCA1 / 2，其中参与互补DNA修复途径，PARP抑制剂的areacutelysensitivetoinhibitorsofPARP.A数量正在临床试验，并初步结果是高度encouraging.17，18 AnumberofpotentandselectiveinhibitorsofthePIKKfamily成员自动柜员机（如1，KU-5593319），DNA-PK（例如2，NU 702620） ，andtheATRsubstrateChk1（例3，AZD¬776221）披露。相反，报告的ATR抑制剂，例如4（caffeine22）和5（五味子B23）（图1）是弱和非选择性（表1）。然而，在ATR作为抗癌药物，24and申请了多项专利的最近已提出了索赔的ATR抑制剂（从AstraZenecaand顶点），虽然有限的生物化学或细胞的数据披露这些documents.25 ¬一个目标一个越来越大的兴趣这里所描述第二十七纠正工作中的阻碍了ATR的特征为肿瘤target.24Specifically好，良好的特点化学工具短缺，这些化合物将是有价值的，帮助确定潜在的与ATM合成致死相互作用信号通路和ATR抑制对正常细胞的影响。我们报道了一系列新的3 -氨基-6 -arylpyrazines已经提供ATR的强有力的和高度选择性的抑制剂。从命中化合物6（图1），从一个高通量筛选（HTS）中标识的起始，结构-活性关系（SAR）研究和同源建模的组合导致了抑制剂和ATR活性之间的相互作用的理解网站是为效力和选择性的关键。化合物45是最有效的（Kiappof 6纳米为ATR）中的一个和该系列的选择性部件，在这里是用来illustratetheattractivecellularphenotypethatcanbeachievedby抑制ATR。“化学化合物8，其 -溴官能团，提供了一种后期中间那个启用快速和通用的modifica¬tionofthephenylringatthe6-positionofthepyrazinenucleusin化合物6。为ATR抑制剂6-47的制备合成路线路线1中被描绘。商业甲基aminopyrazin -2 -羧酸吡嗪核用NBS的 53wasbrominatedatthe6 -位，28to提供中间体54在高收益率。甲基酯54，得到羧酸56，其与苯胺缩合，得到该组所希望的高级中间体酰胺8.29Diversity在吡嗪环的6 -位上的基水解时引入8进行Suzuki交叉coupling30reactions与一系列的硼酸/硼酸盐生成抑制剂6和10-47。完全饱和的环己烷9是从环己烯衍生物10生成由下氢化conditions.31减少对于替代酰胺的吡嗪核心（例如，环己基48）的2 -位，在方案2中所述的化学序列被使用。甲铃木交叉偶联反应，之间为4 -（甲基磺酰基）苯基硼酸和54提供advancedintermediate 57，这反过来又被转换toamide 48的二环杂芳基部分，可以作为苯基酰胺等排物还制备（苯并咪唑，苯并恶唑的数组，苯并噻唑，吲哚和49-52;方案3和4）。初安装的双环杂芳基的功能的保留的6位的通用性。因此，治疗中间体羧酸56与苯二胺在二甲醚，在二乙氧基phosphorylformonitrile的存在，所提供的后期中间体苯并咪唑58在温和yield.32Subsequent耦合用的4 -（甲基磺酰基）苯基硼酸，提供电子等排体49类似的官能化序列。为苯并恶唑和苯并噻唑的合成采用等排物的腈59的50和51转换成后期 intermediatesbenzoxazole60orbenzothiazole61proceededwith 2 -氨基苯酚或2 -氨基苯硫酚在温和yield.33-35 Suzuki交叉偶联用4 -（甲基磺酰基）苯基硼酸得到所需的排物50和51，虽然在方案3中的化学序列描述为电子等排物的49-51提供了所要求的化合物，thereversesyntheses（制剂即functionalizationthrough Suzuki交叉偶联之前，电子等排基序的结构，在一个序列中类似于在方案2中描述的）也已成功地利用和优选为双环电子等排体的取代的探索。积木6236'结果与讨论化合物6的顺序衍生（计划4）被确定从对全长重组ATR高温超导。它能抑制ATR与IC50of 0.62微米，并具有针对ATM和DNAPK良好的选择性（IC50> 8微米）。然而，在基于细胞的测定抑制ATR活性，如通过在H2AX的羟基脲诱导的磷酸化的减少测定ATR37was的直接底物未观察到（IC 50> 2.5微米）。因为高分辨率晶体学数据的PIKK家族的任何成员都不可用，则相关激酶的磷脂酰肌醇3 -激酶γ（PI3K-γ）被用作结构template38，39for的ATR的同源性模型，以帮助抑制剂的设计。为了提供选择性的指导，还建造了本是同根ATM和DNAPK的同源性模型。DNAPK的低分辨率结构报告由西班达等al.40was用来对整体折提供指导。虽然PI3K-γ和PIKK家族membersisrelativelylow（例如，22％betweenPI3K-γandATRin激酶结构域）之间的序列同一性，一些重要的残基的活性位点中是保守的。这些包括一个盐桥中提供的吲哚变种52。该序列使能引入吲哚基序中的反应性最强的位置（邻位，以氨基基团），用N-BOC-吲哚-2 -基硼酸酯在钯催化交叉偶联反应; 第二Suzuki偶联与4 -（甲基磺酰基）苯基硼酸，随后进行Boc脱保护，用TFA，得到含有衍生52所需的吲哚与高温超导的同源性模型击中6绑定到ATR示于图2的化合物6的定位。模型中的活性位点是由在PI3K-γ相关的氨基吡嗪的cocomplex结构导向（未示出）。这使我们能够找到的蛋白质活性位点的铰链主题和核心支架之间关键的氢键相互作用。最小化6共同配合物与ATR的预测的氢键相互作用 ringsp2¬nitrogenwiththebackbone-NH ofVal2378（3.01Å和的环外胺 的氨基吡嗪与Glu2380的羰基（3.08埃。抑制剂的联芳基序还预期有助于通过π-堆积与Trp2379的吲哚环的结合。网守残基（Tyr2365在ATR）的羟基靠近酰胺羰基的吡嗪的2位，并且看起来似乎定位于形成氢键。Asp2494和Lys2327之间的盐桥，横跨PIKK家族保守的，被预测为通过的抑制剂6 anilinering被打乱，迫使Asp2494的侧链下来，从而使芳族环，以适应在P下循环（图2）。这个P环，异常丰富的亲脂性残基时，与大多数激酶相比，显得倍，在苯胺环的2 -位上（图3），也可以向亲脂性绑定。除了交互如上所述，化合物6和ATM或DNAPK之间的潜在相互作用，也进行（在下面详细描述）。这项工作表明利用的差异，应该允许对这些相关激酶选择性进行设计。随着越来越多的效力，同时保持选择性的目的，第一轮的探索通过修改或6 -芳基环（表2）的置换进行。作为预测的同源性模型，将6 -芳基在6和Trp2379之间的π-堆积相互作用使得该抑制剂的效力的一个重要贡献。除去苯基环（7and8）resultedina10-foldlossofbindingaffinity，并且其完全或部分还原（环己基9和环己烯基10，分别）导致了在效力大约5倍的降低。这些结果促使我们保留芳族基团氨基吡嗪芯的6 -位上。交换-6 -苯基与吡啶（11），得到3倍的效力增加。然而，抑制一些噻庚啶亚型也增加（例如，IC50 cyp3A42微米为11比CYP3A4的IC50> 100μM6），进一步使用这个主题劝阻我们。化合物13-19的邻位上提供替代的影响的信息。腈基（13）的引入导致了25倍的改善的效力针对ATR而且对于ATM和DNAPK亲和力增加。增强的效力为ATR和DNAPK可以通过用一个丝氨酸残基（Ser2305在ATRorSer3731inDNAPK），可能会形成新的氢键来解释。AlthoughATMhasnosuch丝氨酸残基，在P环的构象的轻微变化可能，在此情况下，允许骨干新罕布什尔州参加一个氢键的腈基。没有其他取代基得到烯hancement这样的亲和力，尽管亚砜14能够提供一个5倍的改善。小群的替换在间位（compounds20-25）并没有导致显著增加ATR效力，虽然选择性是由极性基团破坏。在另一方面，取代在对位（化合物26-31）导致了在这个阵列中的最有效的和选择性的抑制剂。砜27，并在较小程度上腈26，设置在效力针对ATR显著改善（25倍为27），同时保留了对ATM和DNAPK（例如，> 100倍的选择性为27，IC 50的ATR = 26纳米， IC50 ATM => 8μM，并 IC50DNAPK = 4.4微米）。这些结果可以归因于一个假定的氢键与ATR特定的甘氨酸残基的主链NH（Gly2385）的形成中，位于C-末端thehingeregion（图3，bluevanderWaalsspheres）。InATM，该残基是脯氨酸（图3，红色网格），并没有这样的氢键可以形成。在DNAPK等价残基是苏氨酸（图3，黄色网格），和一个类似氢键的任何机会很可能被封锁的苏氨酸侧链的存在。酰替苯胺6-31必须形成苯胺在体内，因此存在潜在的毒性责任的潜力。目前，脱颖而出，通过还原芳环或通过提供的酰胺官能团的电子等排物除去N-酰苯胺的影响进行了检查（化合物48-52）。苯胺环（48）的饱和导致效力一个> 100倍的损失。另一方面，使用杂环基序的N-酰苯胺如“等排物”给更好的结果。苯并咪唑49,41和由形状类似于苯并恶唑50，苯并噻唑51，和吲哚52显示出类似的抑制活性，以苯胺27（表3）尽管只苯并咪唑49具有形成同一氢键作为原酰胺的潜在的事实。无论是更换新的氢键供体的NH在27与氧气受体benzox唑50，也不去除吲哚52效力受损的氢键受体羰基。这些结果表明，苯胺羰基和Tyr2365之间的拟议氢键是不是一个主要因素结合，我们的结论是，酰胺基团的功能主要是作为一个连接到P循环下放置芳香基团。虽然这些双环杂芳基导致良好的ATR的抑制作用，抑制ATM选择性损害在所有情况下。化合物49-52不能模仿苯胺in27and，asaconsequence的确切形状，具有结合deeperin的activesite，变得更容易碰在thebackoftheadenosine-50-三磷酸（ATP）bindingpocket保守的酪氨酸。检查，在选择性的损失可以与化合物49-52中获得的活性位点相对的一个稍微不同的位置，以27（图4中所示的顺时针方向转动）相关的同源性modelsledustohy pothesize的。在这种结合模式，化合物49-52避免冲突与酪氨酸守门员残留，似乎也减轻是观察27帕拉砜和Pro2775在ATM之间的潜在冲突（相当于残留物Gly2385在ATR和在DNAPK Thr3809）。另外，在ATM同源性模型预测，一个精氨酸残基（Arg2691）也可通过氢键与化合物49-52砜有助于增强的效力。因为anilide27remainedthemostpotentandselective化合物，它被选为从中进一步了解砜基的相互作用的平台。增值compoundsin表2和ATR的同源性模型（图3）之间的建议，对-氢键受体是重要的，且当氢键受体定 位在6 -苯基环平面的下面可以形成与蛋白质的最佳交互。一组对-酰胺（32¬38）（表4），其具有在真空中二面角The6广告-phenylwereusedtoassessthishypothesis.The哌啶酰胺37的羰基与平面，具有～60的自然二面角之间的范围内？，是最potentin这series.This与ATR的同源性模型，这表明在共同复杂的二面角为～65一致？一个最佳的氢键与Gly2385。对于其他酰胺类似物的效价降低可以通过旋转能垒到最佳～65来解释？二面角：例如，伯酰胺32，其结合有苯环，具有克服一个6千卡/摩尔的屏障，这导致了4倍降低效力（IC50of110nMascomparedtoanIC50of26nMfor37）在砜27的情况下，。改进的效力归因于低旋转alenergy屏障（<1千卡/摩尔），以位置a砜氧在最佳角度和距离的H-键合的-NH-的Gly2385。虽然使用酰胺的酒店在提供化学多样性方面的优势，有利的二面角为好氢键相互作用的要求限制了 possibilities.Wethereforecontinuedtoexplore theutility ofsulfone 27代的甲基砜本身都是具有亲脂性（40-44 ）和极性基团（45-47）导致化合物具有高达4倍效力的改进，同时保留了选择性与DNAPK和ATM。此外，极性取代（45-47）提供了一种enhancementin细胞效力可能是由于toim证明物理properties.Compound45，forexample，具有12纳米的一种酶的效力和抑制H2AX在细胞ATR-介导的磷酸化与IC50of 0.42微米; 相比之下，compound42，以作为imilarenzyme效力of14nM，抑制ATR细胞与IC50of 2微米。除了 其诱人的效力，选择性和细胞活动的组合，化合物45具有良好的“druglike”的属性，包括水溶性，脂溶性（疏水参数3.0），和一个profilein的 Caco 2的研究，结果显示，好的被动diffusionacross膜以最小的外排责任（AB = 17.10～6公分/秒，和BA = 23.10～6公分/秒）。因此，它被选择用于进一步的生物评估。小群的替换在间位（compounds20-25）并没有导致显著增加ATR效力，虽然选择性是由极性基团破坏。在另一方面，取代在对位（化合物26-31）导致了在这个阵列中的最有效的和选择性的抑制剂。砜27，并在较小程度上腈26，设置在效力针对ATR显著改善（25倍为27），同时保留了对ATM和DNAPK（例如，>100倍的选择性为27，IC 50的ATR = 26纳米， IC50 ATM => 8μM，并IC50DNAPK = 4.4微米）。这些结果可以归因于一个假定的氢键与ATR特定的甘氨酸残基的主链NH（Gly2385）的形成中，位于C-末端thehingeregion（图3，bluevanderWaalsspheres）。InATM，该残基是脯氨酸（图3，红色网格），并没有这样的氢键可以形成。在DNAPK等价残基是苏氨酸（图3，黄色网格），和一个类似氢键的任何机会很可能被封锁的苏氨酸侧链的存在。酰替苯胺6-31必须形成苯胺在体内，因此存在潜在的毒性责任的潜力。目前，脱颖而出，通过还原芳环或通过提供的酰胺官能团的电子等排物除去N-酰苯胺的影响进行了检查（化合物48-52）。苯胺环（48）的饱和导致效力一个> 100倍的损失。另一方面，使用杂环基序的N-酰苯胺如“等排物”给更好的结果。苯并咪唑49,41和由形状类似于苯并恶唑50，苯并噻唑51，和吲哚52显示出类似的抑制活性，以苯胺27（表3）尽管只苯并咪唑49具有形成同一氢键作为原酰胺的潜在的事实。无论是更换新的氢键供体的NH在27与氧气受体benzox唑50，也不去除吲哚52效力受损的氢键受体羰基。这些结果表明，苯胺羰基和Tyr2365之间的拟议氢键是不是一个主要因素结合，我们的结论是，酰胺基团的功能主要是作为一个连接到P循环下放置芳香基团。虽然这些双环杂芳基导致良好的ATR的抑制作用，抑制ATM选择性损害在所有情况下。化合物49-52不能模仿苯胺in27and，asaconsequence的确切形状，具有结合deeperin的activesite，变得更容易碰在thebackoftheadenosine-50-三磷酸（ATP）bindingpocket保守的酪氨酸。检查，在选择性的损失可以与化合物49-52中获得的活性位点相对的一个稍微不同的位置，以27（图4中所示的顺时针方向转动）相关的同源性 modelsledustohy pothesize的。在这种结合模式，化合物49-52避免冲突与酪氨酸守门员残留，似乎也减轻是观察27帕拉砜和Pro2775在ATM之间的潜在冲突（相当于残留物Gly2385在ATR和在 DNAPK Thr3809）。另外，在ATM同源性模型预测，一个精氨酸残基（Arg2691）也可通过氢键与化合物49-52砜有助于增强的效力。因为anilide27remainedthemostpotentandselective化合物，它被选为从中进一步了解砜基的相互作用的平台。增值compoundsin表2和ATR的同源性模型（图3）之间的建议，对 -氢键受体是重要的，且当氢键受体定位在6 -苯基环平面的下面可以形成与蛋白质的最佳交互。一组对 -酰胺（32-38）（表4），其具有在真空中二面角The6广告¬phenylwereusedtoassessthishypothesis.The哌啶酰胺37的羰基与平面，具有～60的自然二面角之间的范围内？，是最potentin这series.This与ATR的同源性模型，这表明在共同复杂的二面角为～65一致？一个最佳的氢键与Gly2385。对于其他酰胺类似物的效价降低可以通过旋转能垒到最佳～65来解释？二面角：例如，伯酰胺32，其结合有苯环，具有克服一个6千卡/摩尔的屏障，这导致了4倍降低效力（IC50of110nMascomparedtoanIC50of26nMfor37）在砜27的情况下，。改进的效力归因于低旋转alenergy屏障（<1千卡/摩尔），以位置a砜氧在最佳角度和距离的H-键合的¬NH-的Gly2385。虽然使用酰胺的酒店在提供化学多样性方面的优势，有利的二面角为好氢键相互作用的要求限制了possibilities.Wethereforecontinuedtoexplore theutility ofsulfone 27代的甲基砜本身都是具有亲脂性（40-44 ）和极性基团（45-47）导致化合物具有高达4倍效力的改进，同时保留了选择性与DNAPK和ATM。此外，极性取代（45-47）提供了一种enhancementin细胞效力可能是由于toim证明物理properties.Compound45，forexample，具有12纳米的一种酶的效力和抑制H2AX在细胞ATR-介导的磷酸化与IC50of 0.42微米; 相比之下，compound42，以作为imilarenzyme效力of14nM，抑制ATR细胞与IC50of 2微米。除了其诱人的效力，选择性和细胞活动的组合，化合物45具有良好的“druglike”的属性，包括水溶性，脂溶性（疏水参数3.0），和一个profilein的Caco 2的研究，结果显示，好的被动diffusionacross膜以最小的外排责任（AB = 17.10～6公分/秒，和BA = 23.10～6公分/秒）。因此，它被选择用于进一步的生物评估。小群的替换在间位（compounds20-25）并没有导致显著增加ATR效力，虽然选择性是由极性基团破坏。在另一方面，取代在对位（化合物26-31）导致了在这个阵列中的最有效的和选择性的抑制剂。砜27，并在较小程度上腈26，设置在效力针对ATR显著改善（25倍为27），同时保留了对ATM和DNAPK（例如，> 100倍的选择性为27，IC 50的ATR = 26纳米， IC50 ATM => 8μM，并IC50DNAPK = 4.4微米）。这些结果可以归因于一个假定的氢键与ATR特定的甘氨酸残基的主链NH（Gly2385）的形成中，位于C-末端thehingeregion（图3，bluevanderWaalsspheres）。InATM，该残基是脯氨酸（图3，红色网格），并没有这样的氢键可以形成。在DNAPK等价残基是苏氨酸（图3，黄色网 格），和一个类似氢键的任何机会很可能被封锁的苏氨酸侧链的存在。酰替苯胺6-31必须形成苯胺在体内，因此存在潜在的毒性责任的潜力。目前，脱颖而出，通过还原芳环或通过提供的酰胺官能团的电子等排物除去N-酰苯胺的影响进行了检查（化合物48-52）。苯胺环（48）的饱和导致效力一个> 100倍的损失。另一方面，使用杂环基序的N-酰苯胺如“等排物”给更好的结果。苯并咪唑49,41和由形状类似于苯并恶唑50，苯并噻唑51，和吲哚52显示出类似的抑制活性，以苯胺27（表3）尽管只苯并咪唑49具有形成同一氢键作为原酰胺的潜在的事实。无论是更换新的氢键供体的NH在27与氧气受体benzox唑50，也不去除吲哚52效力受损的氢键受体羰基。这些结果表明，苯胺羰基和Tyr2365之间的拟议氢键是不是一个主要因素结合，我们的结论是，酰胺基团的功能主要是作为一个连接到P循环下放置芳香基团。虽然这些双环杂芳基导致良好的ATR的抑制作用，抑制ATM选择性损害在所有情况下。化合物49-52不能模仿苯胺in27and，asaconsequence的确切形状，具有结合deeperin的activesite，变得更容易碰在thebackoftheadenosine-50-三磷酸（ATP）bindingpocket保守的酪氨酸。检查，在选择性的损失可以与化合物49-52中获得的活性位点相对的一个稍微不同的位置，以27（图4中所示的顺时针方向转动）相关的同源性 modelsledustohy pothesize的。在这种结合模式，化合物49-52避免冲突与酪氨酸守门员残留，似乎也减轻是观察27帕拉砜和Pro2775在ATM之间的潜在冲突（相当于残留物Gly2385在ATR和在 DNAPK Thr3809）。另外，在ATM同源性模型预测，一个精氨酸残基（Arg2691）也可通过氢键与化合物49-52砜有助于增强的效力。因为anilide27remainedthemostpotentandselective化合物，它被选为从中进一步了解砜基的相互作用的平台。增值compoundsin表2和ATR的同源性模型（图3）之间的建议，对 -氢键受体是重要的，且当氢键受体定位在6 -苯基环平面的下面可以形成与蛋白质的最佳交互。一组对 -酰胺（32-38）（表4），其具有在真空中二面角The6广告¬phenylwereusedtoassessthishypothesis.The哌啶酰胺37的羰基与平面，具有～60的自然二面角之间的范围内？，是最potentin这series.This与ATR的同源性模型，这表明在共同复杂的二面角为～65一致？一个最佳的氢键与Gly2385。对于其他酰胺类似物的效价降低可以通过旋转能垒到最佳～65来解释？二面角：例如，伯酰胺32，其结合有苯环，具有克服一个6千卡/摩尔的屏障，这导致了4倍降低效力（IC50of110nMascomparedtoanIC50of26nMfor37）在砜27的情况下，。改进的效力归因于低旋转alenergy屏障（<1千卡/摩尔），以位置a砜氧在最佳角度和距离的H-键合的¬NH-的Gly2385。虽然使用酰胺的酒店在提供化学多样性方面的优势，有利的二面角为好氢键相互 作用的要求限制了possibilities.Wethereforecontinuedtoexplore theutility ofsulfone 27代的甲基砜本身都是具有亲脂性（40-44 ）和极性基团（45-47）导致化合物具有高达4倍效力的改进，同时保留了选择性与DNAPK和ATM。此外，极性取代（45-47）提供了一种enhancementin细胞效力可能是由于toim证明物理properties.Compound45，forexample，具有12纳米的一种酶的效力和抑制H2AX在细胞ATR-介导的磷酸化与IC50of 0.42微米; 相比之下，compound42，以作为imilarenzyme效力of14nM，抑制ATR细胞与IC50of 2微米。除了其诱人的效力，选择性和细胞活动的组合，化合物45具有良好的“druglike”的属性，包括水溶性，脂溶性（疏水参数3.0），和一个profilein的Caco 2的研究，结果显示，好的被动diffusionacross膜以最小的外排责任（AB = 17.10～6公分/秒，和BA = 23.10～6公分/秒）。因此，它被选择用于进一步的生物评估。 |
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2楼2014-04-18 23:35:01
18766177254
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3楼2014-04-19 08:19:51
