【作者】 陈磊；

【导师】 廖志新；

【作者基本信息】 东南大学 ， 药物化学工程， 2022， 博士

【摘要】 核受体（NR）视黄酸相关孤儿受体C（RORc或RORγ,又称NR1F3）是一种重要的转录因子,它会参与促炎细胞因子白细胞介素（IL）-17的产生和调控。这种受体有三种亚型:RORα、RORβ和RORγ,特别是RORγt（γ的第二异构体,在胸腺高度表达）是人类Th17细胞（T helper cell 17,Th17）分化的主调节因子。由于RORγ的这种作用,使其成为治疗银屑病、类风湿关节炎（RA）、强直性脊柱炎（AS）和葡萄膜炎等自身免疫性疾病药物开发的十分有吸引力的靶点。近年来,已有多种化学结构的小分子RORγt反向激动剂被报道,先后进入临床的候选药物有VTP-43742、GNE-3500、AZD-0284、T0901317和GSK2981278等,其中GSK2981278（化合物01）作为银屑病外用治疗药物进入了II期临床研究。化合物01在酶的结合抑制实验(IC₅₀=10 n M)和RORγt报告基因细胞实验中(IC₅₀=30 n M)分别具有较强的体外RORγt反向激动活性,同时化合物01对咪喹莫特诱导的小鼠皮肤炎症模型中IL-17A细胞因子表达具有较强的抑制作用。尽管化合物01具有优异的生物学特性,但据近年来的报道,该化合物的临床开发已停止,在人肝微粒体（HLM）实验中,化合物01显示出较高的肝清除（CLhep）值和较短的半衰期(HLM,CL_{int（liver）}=1613.9 m L/min/kg,T_1/2=0.8 min),并且亲脂性（Log D=4.10）超出了药物发现最佳的范围（Log D=2.5-3.5）,因而限制了其在临床上的开发应用。GSK2981278良好的生物学活性,引起了本课题组的极大关注。通过合理的结构修饰,在保持强有力的RORγt反向激动剂活性的同时显著提高人体代谢稳定性,不失为一条自身免疫性疾病治疗药物开发的有效途径。本文从GSK2981278（01）出发,通过降低亲脂性和提高LLE（ligand-lipophilicity efficiency）值,设计并合成了4个系列,97个基于2H-色满磺酰胺类衍生物的新型RORγt反向激动剂,开展了相关的化学结构表征及构效关系研究（SAR）。论文对所合成化合物开展了体外酶和细胞活性筛选（化合物HPLC纯度大于95%）,对挑选出的11个活性较好的化合物:a15,a16,c4,c9,c11,c20,c27,d10,d12,d15和d18进行了深入的生物学评价。肝微粒体代谢测试中,c9和d15表现出了良好的代谢稳定性(T_1/2>30 min)。体内研究表明,化合物d15有着作为口服药物的合适的半衰期(T_1/2>30 min)。口服化合物d15对小鼠咪喹莫特皮肤炎症模型中IL6和IL-17A细胞因子表达的抑制作用明显且呈剂量依赖性,并显著降低了皮肤炎症临床症状的发生（PSAI,Psoriasis Area and Severity Index）。进一步的计算机结合模式分析表明化合物d15可以很好地占据RORγt口袋。化合物d15的磺酰基与Ile400相互作用,形成与GSK2981278磺酰基相同的氢键。色满环上的羟基可以通过氢键与Phe377相互作用,就像GSK2981278上的苄醇基团与Phe377和Val376形成氢键一样。这一结果表明,与苄醇相似,d15中色满环并没有失去关键的相互作用。四氢吡喃环上的氧原子可与侧链Arg367形成氢键相互作用。此外,苯乙基基团可以与Trp317（3.2?）紧密接触,导致螺旋12不稳定,从而产生反向激动剂的效果。以上各种数据表明化合物d15具有良好的RORγt反向激动活性和代谢稳定性,可作为治疗Th17驱动的自身免疫性疾病的临床前化合物加以推进。更多还原

【Abstract】 The nuclear receptor(NR)retinoic acid receptor-related orphan receptor C(RORc or RORγ,also known as NR1F3)is an important transcription factor involved in the production and regulation of the pro-inflammatory cytokine interleukin(IL)-17.There are three subtypes known as RORα,RORβ,and RORγ.In particular,RORγt(the second isoform of γ which is highly restricted to expression in the thymus)is a master regulator for the differentiation of human Th17 cells.It has been identified as the master regulator of function and differentiation of IL-17 cells,making it an attractive drug-target for the treatment of autoimmune diseases: psoriasis,rheumatoid arthritis(RA),ankylosing spondylitis(AS),and uveitis.On the basis of the influence of RORγ over multiple inflammatory pathways,it has been proposed that RORγ is a potentially valuable molecular target for the treatment of inflammatory diseases.Indeed,a diverse range of chemical structures has been reported as RORγt inhibitors by many research groups.Recent clinical candidates from these efforts include VTP-43742,GNE-3500,AZD-0284,T0901317,GSK2981278,etc.Among them,GSK2981278(Compound 01)was developed in a Phase II study for the topical treatment of psoriasis.Compound 01 has potent in vitro RORγt inverse agonistic activity in the binding inhibitory assay(IC50 = 10 n M)and in a RORγt reporter gene assay(IC50 = 30 n M),respectively.Compound 01 exerted robust inhibition of IL-17 A cytokine expression in a mouse imiquimod-induced skin inflammation model.But recently,clinical development of this compound was halted.Despite the attractive biological profiles for target potency,compound 01 displays high hepatic clearance(CLhep)values incubated with human liver microsomes(HLM,CLint(liver)=1613.9 m L/min/kg,T1/2 = 0.8 min)and has lipophilicity(Log D = 4.10)outside the typical optimal range for drug discovery(approximate Log D average of 2.5-3.5),thereby limiting its clinical application.The good biological activity of GSK2981278 has attracted the great attention of our research group.Through reasonable structural modification,RORγt inverse agonist can maintain strong activity and significantly improve metabolic stability,which is aneffective way to develop drugs for the treatment of autoimmune diseases.Starting from the reported GSK2981278(01),we engineered structural modifications to design and synthesize novel RORγt inverse agonists that reduced the lipophilicity and improvement of LLE values.A total of 97 compounds based on 2H-chromone sulfonamide derivatives in 4 series were synthesized.The chemical structure characterization and structure-activity relationship were studied.Through in vitro enzyme and cell activity screening,we selected 11 compounds with good activity: a15,a16,c4,c9,c11,c20,c27,d10,d12,d15,and d18(HPLC purity of each target compound was greater than 95%)for subsequent biological evaluation.In the liver microsomal metabolism test,c9 and d15 showed good metabolic stability(T1/2 > 30 min).In vivo efficacy test of imiquimod skin inflammation model in mice showed that it was superior to GSK2981278.In vivo studies showed that compound d15 had an appropriate half-life(T1/2 > 30 min)for oral use.Compound d15 exhibited a robust and dose-dependent inhibition of IL6 and IL-17 A cytokine expression and significantly reduced the development of skin inflammation clinical symptoms in a mouse imiquimod-induced skin inflammation model.d15 significantly reduces the occurrence of clinical symptoms of skin inflammation(PSAI,Psoriasis Area and Severity Index).Further analysis of the binding mode revealed that d15 can well occupy the RORγt pocket.The sulfonyl group of the compound d15 can interact with Ile400 by forming hydrogen bonds as same as sulfonyl group of GSK2981278.The hydroxy group on the chromone ring can interact with Phe377 by a hydrogen bond similar to the benzyl alcohol group on GSK2981278,forming a hydrogen bond with Phe377 and Val376.This result suggested that we did not lose the key interaction by replacing the benzyl alcohol with a chroman ring.The oxygen atoms of the tetrahydropyran ring can interact with side chain Arg367 by forming hydrogen bonds.In addition,phenyl ethyl can make close contact with Trp317(3.2 ?),which leads to the destabilization of helix 12 in the agonist position.The final compound d15 was shown good inverse excitatory activity and metabolicstability of RORγt.Therefore,based on the above experimental data,compound d15 was selected as a preclinical compound for the treatment of TH17-driven autoimmune diseases.更多还原