摘  要

       结核病（TB）是一种主要由结核分枝杆菌（MTB）引起的传染性疾病，其位列世界十大致死性疾病之一。在新型冠状病毒（COVID-19）大流行之前，TB的死亡率在单一病原体所致感染性疾病中最高。随着耐多药TB（MDR-TB）、广泛耐药TB（XDR-TB）以及全耐药TB（TDR-TB）的出现，以及TB与HIV/AIDS相结合使得TB疫情再度上升，成为全球重大公共卫生问题。尽管具有全新作用机制的抗TB新药贝达喹啉（bedaquiline）、德拉曼尼（delamanid）和普瑞玛尼（pretomanid）分别于2012年、2014年和2019年上市并用于对付MDR-TB，但因存在QT间期延长等副作用，其临床应用受到严格限制。因此，寻找和开发疗效及安全性更高的新型抗TB药势在必行。

       苯并噻嗪酮（BTZs）是一类具有全新作用机制（靶点：DprE1）的抗TB化合物，其代表物BTZ043和macozinone（PBTZ169）目前均处于临床II期试验阶段。近期报道，含有硝基呋喃或1,3,4-噁二唑片段的化合物具有明确的抗MTB活性，并因此受到极大关注。基于此，本论文拟在设计合成新型硝基呋喃-1,3,4-噁二唑杂合体以及PBTZ169衍生物的基础上，通过相关评价，筛选出值得深入研究的苗头化合物。 

本论文包括以下两个课题：

硝基呋喃-1,3,4-噁二唑杂合体的设计、合成与抗结核活性研究

       硝基呋喃类和1,3,4-噁二唑类是近年报道的新型抗TB化合物。本课题通过不同的连接子将这两个片段相连，设计并合成了三个系列的硝基呋喃-1,3,4-噁二唑杂合体，共计28个目标化合物。其中，系列二（连接子为酰胺键）中的代表物2c对MTB-H₃₇Rv和MDR-MTB的活性中等，但其细胞毒性最低，潜在的心脏毒性最小，且口服PK参数均显著优于PBTZ169，值得进一步结构优化。本部分的研究成果已经发表在Bioorg. Med. Chem.，53 (2022) 116529。

PBTZ169衍生物的设计、合成与抗结核活性研究

（一）、结合PBTZ169和利福霉素（RFP/RPT）的结构特点，设计并合成了一系列含有N-（亚甲基）胺基哌嗪片段的PBTZ类化合物（12个）。其中，目标物1a和1e-j表现出与PBTZ169大致相当的抗MTB-H₃₇Rv和MDR-MTB活性（MIC：0.029-0.110 µM），以及可接受的选择性指数（SI: >1100）。此外，化合物1h（即WAP-1902）还显示出优于PBTZ169的PK性质，但存在潜在的心脏毒性，值得进一步结构优化。

（二）、针对上述筛选出的苗头化合物WAP-1902 的潜在心脏毒性（在10 µM时对hERG K⁺的抑制率为88.2%），我们将其结构中的腙键用相对稳定的肼键代替，设计并合成了三个系列含有N-胺基哌嗪片段的PBTZ类化合物（41个）。其中，21个目标物对MTB-H₃₇Rv和MDR-MTB显示优秀的广谱活性（MIC：<0.016 µg/mL）和低细胞毒性（CC₅₀：>64 µg/mL）。尤其是化合物1o 在10 µM时对hERG K⁺的抑制率（22.7%）显著降低，其主要PK参数（C_max，AUC_0-∞，T_1/2）也远优于PBTZ169，值得深入研究。部分研究成果已发表在Eur. J. Med. Chem.，218 (2021) 113398。

       通过以上研究工作，共合成81个目标化合物。其中，80个化合物未见文献报道。目标化合物经¹H NMR、¹³C NMR和MS确证，部分化合物还经过HRMS确证。

Abstract

Tuberculosis (TB), an infectious disease mainly caused by Mycobacterium tuberculosis (MTB), is one of the world's top 10 lethal diseases. Before the pandemic of the novel coronavirus (COVID-19), TB was the disease with the highest mortality among those infectious diseases caused by single pathogen. The rising incidence of TB due to the emergence of multi-drug resistant TB (MDR-TB), extensively drug-resistant TB (XDR-TB), and totally drug-resistant TB (TDR-TB), as well as the association of TB with HIV/AIDS, presents a global health problem. Thankfully, bedaquiline, delamanid and pretomanid were marketed to treat MDR-TB in 2012, 2014 and 2019, respectively, but their clinical use has been severely limited due to some adverse events, such as QT interval prolongation. Therefore, there is a great demand to develop more effective and safer anti-TB drugs with novel mechanisms of action.

Benzothiazinones (BTZs) are a novel class of anti-TB agents targeting DprE1, and two representatives BTZ043 and macozinone (PBTZ169) are in Phase II clinical trials at present, for the treatment of both susceptible TB and MDR-TB. On the other hand, compounds containing a nitrofuran or an 1,3,4-oxadiazole moiety have garnered great interest recently, because of their promising potence against MTB. Based on this, it was intended to do further structural modifications on PBTZ169, and combine nitrofuran with 1,3,4-oxadiazole moieties into hybrids in this work. Our primary objective was to find new agents with potent anti-MTB activity and facilitate the further research of these compounds.

This paper specifically includes the following two aspects:

Design, synthesis and antitubercular activity of nitrofuran-1,3,4-oxadiazole

hybrids

Recently, nitrofuran- and 1,3,4-oxadiazole-based compounds have been identified as novel antitubercular agents. Three series of nitrofuran-1,3,4-oxadiazole hybrids through different linkers, were designed and synthesized. Our results revealed that compound 2c in the second series with an amide bond as the linker, showed moderate in vitro activity against both MTB H37Rv strain and MDR-MTB 16883 clinical isolate, low cytotoxicity, low inhibition of hERG and good oral PK, indicating its promising potential to be a lead for further structural modifications. Relevant findings have been published at Bioorg. Med. Chem., 53 (2022) 116529.

2. Design, synthesis and antitubercular activity of PBTZ169 derivatives

1) Based on the structural features of PBTZ169 and rifampicin/rifapentine, a series of novel benzothiazinone derivatives containing a N-((methylene)amino) piperazine moiety were designed and synthesized. Target compounds 1a and 1e-j showed excellent in vitro activity against both drug-sensitive MTB strain H37Rv and two MDR-MTB clinical isolates (MIC: 0.029-0.110 µM) roughly comparable to PBTZ169, and accepted selective index (SI: >1100). Compound 1h had good safety and PK profiles, suggesting its promising potential to be lead compound for further modifications.

2)  Aiming at reducing hERG cardiac toxicity （inhibition rate: 88.2% at 10 µM） of WAP-1902 discovered in our lab, a series of novel benzothiazinone derivatives containing a N-(amino) piperazine moiety, were designed and synthesized, by replacement of the unstable hydrazone bond with the corresponding hydrazine one. Twenty-one compounds exhibited excellent in vitro activity against both drug-sensitive MTB H37Rv and MDR-MTB strains (MIC: <0.016 µg/mL), and low cytotoxicity (CC₅₀: >64 µg/mL). Especially compound 1o displayed low hERG cardiac toxicity (inhibition rate: 22.7% at 10 µM), and much greater PK parameters(C_max, AUC_0-∞, T_1/2) than PBTZ169, deserving further research. Relevant research findings have also been presented in Eur. J. Med. Chem., 2021, 218:113398.

Through the above research works, a total of 81 target compounds were synthesized. Among them, 80 compounds have not been reported in the literature. The target compounds were confirmed by ¹H NMR, ¹³C NMR and MS, and some compounds were also confirmed by HRMS.