【作者】 赵彤；

【导师】 刘新泳；

【作者基本信息】 山东大学 ， 药物化学， 2022， 博士

【摘要】 痛风(Gout)是一种由于人体嘌呤代谢紊乱所导致的疾病,高尿酸血症(Hyperuricemia)为其病理基础,两者都属于严重危害人类健康的重大代谢性疾病。在过去半个多世纪,痛风呈现爆发性增长及明显年轻化趋势。然而,临床长期使用的抗痛风药物普遍具有活性差及毒副作用大等劣势,且长期用药也造成了不同程度的耐药性。临床长期面临没好药、没新药的窘迫局面。因此,新一代高效安全的抗痛风药物研发是目前新药创制的前沿热点。随着对痛风发病机理的深入探索,研究者发现约90%的痛风患者存在尿酸排泄障碍,其体内的尿酸在肾小球中被各种阴离子转运体大量异常重吸收。尿酸转运蛋白1(Uric acid transporter 1,URAT1)在尿酸重吸收过程中发挥重要作用,使其成为抗痛风药物设计的关键靶标。但现有靶向URAT1的临床药物仍普遍具有活性不足及肝肾毒性高的缺陷。基于目前抗痛风药物的不足及临床迫切需求,本论文以URAT1作为药物设计的靶标,综合运用多种基于配体的药物设计策略对上市药物雷西纳德及前期发现的临床候选药物T7进行了系统的结构修饰。同时通过构建高尿酸血症动物模型,对合成的化合物进行了体内外活性及成药性评价。此外,通过URAT1的同源模建,开展了 URAT1抑制剂的虚拟筛选。具体研究工作分为以下五方面。杂环巯乙酸类URAT1抑制剂的动物体内评价方法建立及吡啶并咪唑或酰基磺酰胺类化合物T7和S3的成药性评价。为了更加有效准确地评价抗痛风活性化合物的动物体内活性,本论文第二章首先通过正交试验法优化、评价并最终获得了可供使用的小鼠高尿酸血症高尿酸血症模型。同时本章也构建了稳定高效的大鼠高尿酸血症高尿酸血症模型以供进一步验证化合物降血尿酸活性。利用上述模型我们对课题组之前发现的苗头化合物T7和S3在体内外活性方面进行了更为深入的研究。结果显示T7和S3在小鼠体内均展示出优良活性,血尿酸下降率是雷西纳德的2.5倍以上。通过梯度剂量下给药,最终确定化合物T7小鼠体内最低起效剂量为1mg/kg,同时两个化合物在大鼠模型中同样展现了突出的降血尿酸活性。体外活性结果显示T7(IC50=1.57μM)的活性是雷西纳德(IC50=7.21 μM)的 4.6 倍。本章也评价了 T7、S3的药代动力学及安全性等成药性指标。结果表明,T7大鼠体内生物利用度为76.3%,小鼠体内最大耐受剂量为500mg/kg,安全性好、成药性佳,值得进一步开发。同时,上述评价也暴露出T7及S3的不足,为后续优化指明了方向。在此基础上,为了获得高效低毒的靶向URAT1抗痛风临床候选药物,我们根据已有雷西纳德类分子的结构特点,对不同区域的关键药效团进行了结构改造,分别开展了以下三部分的工作。基于配体的新型吡啶并咪唑巯基乙酸类抗痛风URAT1抑制剂候选药物TD-3的发现。为了更有效地进行基于配体的化合物设计,本文第三章首先利用前期获得的活性分子及上市药物进行了雷西纳德类分子药效团的构建。结合药效团特征,同时针对杂环巯乙酸类抗痛风药物药效、安全性不足的特点,本章以雷西纳德和候选药物T7为先导化合物,通过优势骨架拼接、电子等排等策略对先导分子中疏水性区域进行了多样性修饰。经Buchwald-Hartwig交叉偶联、环合、亲核取代等反应,本章共合成了 8个子系列共计60个结构全新的URAT1抑制剂。小鼠体内降尿酸活性结果显示26个化合物的动物体内活性均显著超越阳性药物雷西纳德。其中化合物TD-3血尿酸下降率达到96.1%,是本章中活性最优化合物。同时体外活性显示TD-3可显著抑制URAT1,其活性(IC50=1.36μM)是雷西纳德(IC50=5.54 μM)的4.1倍。进一步研究发现TD-3在大鼠体内同样具有突出的降血尿酸活性,小鼠体内最低起效剂量为0.5 mg/kg,显著优于先导化合物T7及雷西纳德。本章最后对TD-3进行了成药性评价,其药代动力学性质良好,绝对生物利用度为59.3%。TD-3小鼠体内最大耐受剂量大于800 mg/kg,且亚急性、组织毒性同样低于先导化合物,安全性显著改善。总之,TD-3可作为候选药物供进一步开发。基于电子等排的新型吡啶并咪唑酰基磺酰胺类抗痛风URAT1抑制剂候选药物TS-2的发现。在本论文第四章中,我们以雷西纳德和候选药物S3作为先导化合物,通过电子等排、分子杂合等策略对硫代侧链阴离子基团进行了多样性改造,设计并合成了两个系列共40个URAT1抑制剂。小鼠体内活性结果显示,两系列中23个化合物具有明显的体内降血尿酸活性,且均优于雷西纳德。其中TS-2在本部分化合物中活性最优,可将模型血尿酸值(1162.00 μM)降至健康空白水平(143.20 μM),血尿酸下降率达到96.8%,是雷西纳德的2倍以上。在体外靶点活性测试中,TS-2抑制URAT1的活性(IC50=0.19 μM)是雷西纳德的29.2倍。动物体内活性结果表明,TS-2在4小时内将大鼠模型血尿酸值(917.80 μM)降至空白血尿酸水平(108.75 μM),血尿酸下降率为91.87%。通过剂量梯度设置发现,TS-2在小鼠体内最低起效剂量为0.25 mg/kg。初步的成药性评价结果显示TS-2药代动力学性质较好。TS-2在小鼠体内的最大耐受剂量大于1000 mg/kg,显著优于雷西纳德,安全性明显提升,值得深入开发。基于骨架跃迁的新型稠环嘧啶类抗痛风URAT1抑制剂先导化合物F-5的发现。第五章在前期研究基础上构建了雷西纳德类分子的3D-QSAR模型。结合模型立体场预测的可修饰区域,本章以雷西纳德及T7为先导化合物,对其核心结构进行了多样性的修饰。通过骨架跃迁等策略,改变母环体积及药效基团的空间排布,设计合成了 9个子系列共计54个全新的URAT1小分子抑制剂。动物体内活性结果显示大部分化合物均表现出强效降血尿酸活性。综合体外活性实验结果,化合物F-5活性最优。其在小鼠体内的降血尿酸下降率为93.2%,抑制URAT1的IC50为2.01 μM,体内外活性均为雷西纳德两倍以上。进一步研究表明,F-5小鼠体内最低起效剂量为0.5 mg/kg,同时具有突出的大鼠体内降尿酸活性。在初步成药性评价实验中,F-5显示出良好的安全性及药代动力学性质,可作为先导化合物进一步研究。基于AlphdFold2的URAT1同源模块构建及URAT1抑制剂的虚拟筛选。由于URAT1为结构复杂的跨膜蛋白,其晶体结构长期未被解析,是制约基于靶标的合理药物设计的“卡脖子”环节。鉴于此,第六章通过AlphdFold2对URAT1进行了同源模建及潜在结合口袋预测。随后利用此模型,对本文中所获得的候选药物的结合模式及药效团特征进行了预测分析,为后续化合物的合理设计提供了依据。同时利用上述模型及结合位点,我们对不同商业小分子化合物库进行了高通量虚拟筛选,综合评价最终获得10个实体小分子。随后通过动物模型对其进行了体内活性筛选。活性结果显示3个小分子在动物体内具有显著的降血尿酸活性,这为后续探索新型URAT1抑制剂提供了基础。总之,本论文针对抗痛风URAT1抑制剂生物活性以及安全性差的问题,综合运用基于配体结构的间接药物设计及计算机辅助药物设计等手段设计合成了三类共计154个全新结构的URAT1抑制剂。通过体内外活性筛选及初步的成药性评价,得到了包括TD-3、TS-2、F-5和T7在内的多个具有重要开发前景的候选药物及先导化合物。此外,本文通过构建URAT1同源模型、药效团模型以及3D-QSAR模型,较为精确地预测和阐明了该类结构的构效关系及其靶点结合模式,分析了该类化合物的药效团特点,为更合理地进行基于靶点高效低毒URAT1抑制剂的设计奠定了基础。更多还原

【Abstract】 Gout is caused by the disorder of purine metabolism in human body,and hyperuricemia(HUA)is the pathological basis.Both of gout and HUA are metabolic diseases that seriously endanger human health.In the past half century,incidence of gout has explosively increased and showed the increasing trend in younger people.Moreover,the clinically used anti-gout drugs possess disadvantages such as low activity and severe side effects,and long-term usage also leads to drug resistance.It makes clinical treatment facing the dilemma of shortage of effective and new drugs.Therefore,the novel safe and effective drugs are urgently needed for treating gout and hyperuricemia.The studies about pathogenesis of gout showed that abnormal reabsorption of uric acid by anion transporters in the glomerulus resulted in excretion disorder,which existed in more than 90%of hyperuricemia patients.Among the anion transporters,uric acid transporter 1(URAT1)plays an important role in the process of uric acid reabsorption,making it an attractive target for the design of novel hypouricemic drugs.However,the approved uricosuric drugs have the disadvantages of insufficient activity and high hepatorenal toxicity.Considering disadvantages of the clinically used anti-gout drugs and the urgent clinical needs,systematic structural modifications were carried out via the strategies of pharmacophore-based scaffold hopping and bioisosterism with lesinurad and candidate T7 as lead compounds in this study.Meanwhile,a stable mouse hyperuricemia model was well established,with which the activity and druggability of the novel compounds were evaluated.In addition,the virtual screening of URAT1 inhibitors was displayed by establishing URAT1 homology modules.This dissertation is described as the following five parts.Establishment of an in vivo evaluation method for URAT1 inhibitors and druggability evaluation of pyridoimidazole or acylsulfonamide compounds T7 and S3.In order to evaluate the in vivo activity of anti-hyperuricemic compounds more effectively and accurately,a stable acute hyperuricemia model in mice was firstly optimized and evaluated by orthogonal test method.At the same time,a stable and efficient hyperuricemia model in rat and a screening method of target inhibitory activity in vitro for further evaluation of the SUA-reducing activity of the compounds were as well established.Then,the in vitro and in vivo activities of the Lead compounds T7 and S3 which were discovered previously were studied via the models mentioned above.T7 and S3 showed excellent in vivo activity,with decrease ratio(DR)of 2.5 times higher than that of lesinurad.Furthermore,the lowest effective dose of T7 in mice was finally determined to be 1 mg/kg by gradient dose administration,and meanwhile both compounds exhibited excellent SUA-lowering activity in rat models.The in vitro activity results showed that inhibitory activity of T7(IC50=1.57μM)was 4.6-fold stronger than that of lesinurad(IC50=7.21 μM).In this chapter,we also systematically evaluated the druggability of T7 and S3.The bioavailability of T7 in rats was 76.3%,and the maximum tolerated dose was 500 mg/kg.All the results indicated that T7 and S3 were promising anti-gout drug candidates.Meanwhile,T7 and S3 showed several shortcomings through the above evaluations,which compelled us to undertake in-depth optimization.According to the structural characteristics of lesinurad derivatives,the key pharmacophores in different components of the structures were modified to obtain URAT1-targeting drug candidates with high efficiency and low toxicity.The work was described in the following three chapters successively.Discovery of novel ligand-based pyridoimidazole mercaptoacetic acid URAT1 inhibitor TD-3 as a hypouricemic drug candidate.In order to carry out ligand-based compound design more efficiently,the lead compounds obtained in the previous work and listed drugs were firstly used to construct the pharmacophore of lesinurad derivatives.Considering the "three-point" pharmacophore and the disadvantages of insufficient efficacy and poor safety of the approved URAT1 inhibitors,the hydrophobic regions of the structures of lesinurad and T7 were modified via scaffold hopping and bioisosterism strategies.Finally,a total of 60 novel URAT1 inhibitors were designed and synthesized via Buchwald Hartwig cross coupling,cyclization and nucleophilic substitution reactions successively.The in vivo activity results in mice showed that 26 compounds possessed robust SUA-lowering activity compared to that of lesinurad.Among them,compound TD-3 showed a decrease rate of 96.1%in the in vivo activity assay,which was the most potent compound in this section.Meanwhile,the in vitro activity results suggested that TD-3 could significantly inhibit URAT1 with IC50 of 1.36 μM,which was 4.1 times higher than that of lesinurad(IC50=5.54 μM).Further activity results show that TD-3 also possessed outstanding SUA-lowering activity in rats,and the minimum effective dose in mice was 0.5 mg/kg,which was significantly better than those of T7 and lesinurad.Finally,in the druggability evaluation experiment,TD-3 achieved excellent pharmacokinetic properties with oral bioavailability of 59.3%.Additionally,TD-3 showed favourable safety profiles.The maximum tolerated dose of TD-3 was 500 mg/kg,and no obvious subacute toxicity was observed in Kunming mice.Overall,all the results indicated that TD-3 could be considered as a promising drug candidate for the treatment of hyperuricemia and gout.Bioisosterism-based discovery of novel pyridzimidazolyl sulfonamide-typed URAT1 inhibitor TS-2 as hypouricemic drug candidate.In this chapter,with drug candidate S3 as starting point,a variety of structural optimization were carried out on the peripheral substituents through bioisosterism and molecular hybridization strategies.Finally,two series of 40 novel URAT1 inhibitors were designed and synthesized.The in vivo activity results showed that 23 compounds in the two series had remarkable in vivo SUA-lowering activity,which were better than that of the control drug lesinurad.Among them,TS-2 showed the best activity.After administration,the blood uric acid level in disease model(SUA=1162.00 μM)was reduced to healthy level(SUA=143.20 μM)rapidly,with the DR of 96.8%.This was more than two times higher than that of lesinurad.In the in vitro activity assay,the URAT1 inhibitory activity of TS-2(IC50=0.19 μM)was 29.2 times higher than that of lesinurad.A more detailed activity study showed that TS-2 reduced the SUA level in the rat model(SUA=917.80μM)to the blank SUA level(SUA=108.75μM)within four hours,with DR of 91.9%.Through the dose gradient setting,it was found that the lowest effective dose of TS-2 in mice was 0.25 mg/kg.The activity of TS-2 was significantly improved compared with that of the lead compounds.Further druggability evaluation showed that TS-2 possessed acceptable pharmacokinetic properties and significantly improved safety properties.The maximum tolerated dose of TS-2 in mice was greater than 1000 mg/kg,which was significantly better than that of lesinurad.In conclusion,TS-2 is suitable for further investigations towards hypouricemic clinical candidates.Scaffold hopping-based discovery of novel fused pyrimidine URAT1 inhibitor F-5 as lead compound.In this chapter,the 3D-QSAR model of lesinurad derivatives was established based on our previous study.Here,combined with the tolerated region predicted by three-dimensional field of this model,multiple modifications were conducted for the core component of lesinurad and T7.The volume of the core ring and the spatial arrangement of the pharmacophore were modified through scaffold hopping and other strategies.And 9 series of URAT1 inhibitors,which contained 54 novel compounds,were finally designed and synthesized.The in vivo activity results showed that most compounds in this chapter exhibited potent SUA-lowering activity.Especially,compound F-5 displayed the best activity whether in vivo or in vitro,with DR of 93.2%and URAT1 inhibitory activity IC50 of 2.01 μM.The in vivo and in vitro activities of F-5 were both more than two times higher than those of lesinurad.Additionally,the lowest effective dose of F-5 was 0.5 mg/kg.At the end of this chapter,the preliminary druggability evaluation showed that F-5 possessed favorable safety and pharmacokinetic properties,and it could be treated as a lead compound for further optimization.AlphdFold2-based establishment of URAT1 homology model and virtual screening of URAT1 inhibitors.Since URAT1 is a transmembrane protein with complex structure,its crystal structure has not been analyzed yet,which restricts the target-based rational drug design.Therefore,in this chapter,the homology model establishment and potential binding pocket prediction of URAT1 were carried out through Alphdfold2.Furthermore,this model was used to predict the binding mode and pharmacophore characteristics of candidate drugs obtained above,which provided a theoretical basis for the rational drug design in subsequent research.A high-throughput virtual screening for different commercial compound libraries was as well conducted via well-established model and predicted binding sites,and several potential URAT1 inhibitors were finally obtained through extensive evaluation.Then the in vivo activity results showed that three small molecules showed obvious SUA-lowering activity in animals,which provided a basis for the follow-up exploration of new URAT1 inhibitors.It is expected that the drug candidates with novel structure,high efficiency and low toxicity would be obtained through further structural optimization.In summary,considering insufficient bioactivity and poor safety of existing URAT1 inhibitors,a total of 154 novel compounds were designed with the combined medicinal chemistry campaign,involving the strategies of molecular hybridization,scaffold hopping,bioisosterism and computer-aided drug design.Subsequently,in vivo activity screening in disease models,in vitro inhibitory activity assay and systematic druggability evaluation were performed,and several candidates were discovered.Among them,TD-3,TS-2,F-5 and T7 showed the outstanding properties.Moreover,the structure-activity relationship,binding mode and pharmacophore characteristics of lesinurad derivatives obtained here were predicted and analyzed through URAT1 homology model,pharmacophore model and 3D-QSAR model.Overall,this study established a solid foundation for the further structure-based design of URAT1 inhibitors with higher efficiency and lower toxicity.更多还原