药学学报

Stage	Type	General structure
Stage 1	Alkyl and haloalkyl phosphate ester
Stage 2	Alkyloxy and haloalkyloxyphosphoramidate
Stage 3	Phosphorodiamidate
Stage 4	Lactyl-derived system
Stage 5	Diaryl phosphate
Stage 6	Aryloxy phosphoramidate

Stage	Type	General structure
Stage 1	Alkyl and haloalkyl phosphate ester
Stage 2	Alkyloxy and haloalkyloxyphosphoramidate
Stage 3	Phosphorodiamidate
Stage 4	Lactyl-derived system
Stage 5	Diaryl phosphate
Stage 6	Aryloxy phosphoramidate

Compd.	EC₅₀/µmol·L^-1
39	0.4	2.5	15.9	1.7	5.6
Remdesivir	3.3	4.7	32	3.7	9.2

Compd.	EC₅₀/µmol·L^-1
39	0.4	2.5	15.9	1.7	5.6
Remdesivir	3.3	4.7	32	3.7	9.2

Compd.	EC₅₀/µmol·L^-1	CC₅₀/µmol·L^-1
37	0.096	0.26
38	0.73	1.44
Hydroxychloroquine	1.74	36.9
Remdesivir	1.52	> 40

Compd.	EC₅₀/µmol·L^-1	CC₅₀/µmol·L^-1
37	0.096	0.26
38	0.73	1.44
Hydroxychloroquine	1.74	36.9
Remdesivir	1.52	> 40

ProTide技术研究进展及其在抗病毒药物研究领域中的应用

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周慧宇 , 朱梅 , 王玉成 ^*

药学学报 | 综述 2023,58(6): 1540-1556

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药学学报 |综述 2023 , 58 (6) : 1540 -1556

ProTide技术研究进展及其在抗病毒药物研究领域中的应用

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周慧宇, 朱梅, 王玉成^*

作者信息

中国医学科学院、北京协和医学院医药生物技术研究所, 北京 100050

通讯作者:

*王玉成, Tel: 86-10-63165263, E-mail: wangyucheng@imb.pumc.edu.cn

Research progress of ProTide technology and its application in the development of antiviral drugs

Hui-yu ZHOU, Mei ZHU, Yu-cheng WANG^*

Affiliations

Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

出版时间: 2023-06-12 doi: 10.16438/j.0513-4870.2022-1210

文章导航

摘要

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ProTide技术是由Christopher McGuigan团队开发的一种前药设计策略, 其特征是将核苷类似物以磷(膦) 酰化的方式引入芳氧基和氨基酸酯, 形成芳氧基磷(膦) 酰胺酯前药。ProTide技术能够使核苷类似物高效透过细胞膜并避开单磷酸化限速步骤, 有效解决了核苷类药物难以被细胞摄取、磷酸化效率低等问题。目前, ProTide技术已广泛应用于抗病毒药物研发领域, 孕育出了包括sofosbuvir在内的多款抗病毒重磅药物以及临床候选药物, 对小分子抗病毒药物的研发进程产生了重要影响。本文对ProTide技术的发展历程及其特点进行了简要介绍, 并概述了近年来ProTide技术在抗病毒药物研究领域的应用情况, 期望为后续抗病毒药物的设计与开发提供参考。

关键词

ProTide / 药物设计 / 前药 / 磷(膦) 酰胺酯 / 抗病毒

Abstract

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ProTide technology is a kind of prodrug design strategy invented by the team of Christopher McGuigan. ProTides are aryloxyphosphoramidates (or aryloxyphosphonamidates) which contain a phosphorus atom combined with an amino acid ester and an aryloxy group. These prodrugs can efficiently cross the cell membrane and escape from the first rate-limiting step of phosphorylation, which afford effective solutions to the drawbacks of current nucleoside analogues. At present, ProTide technology has been extensively applied in the field of antiviral research. It has been successful in providing a number of approved drugs and clinical candidates, such as sofosbuvir and so much more, highlighting the promising future in drug discovery. This review summarizes the brief history and characteristics of ProTide technology, as well as its application in the exploration of antiviral drugs.

Key words

ProTide / drug design / prodrug / phosphoramidate (or phosphonamidate) / antiviral

引用本文

周慧宇, 朱梅, 王玉成. ProTide技术研究进展及其在抗病毒药物研究领域中的应用. 药学学报, 2023 , 58 (6) : 1540 -1556 . DOI: 10.16438/j.0513-4870.2022-1210

Hui-yu ZHOU, Mei ZHU, Yu-cheng WANG. Research progress of ProTide technology and its application in the development of antiviral drugs[J]. Acta Pharmaceutica Sinica, 2023 , 58 (6) : 1540 -1556 . DOI: 10.16438/j.0513-4870.2022-1210

正文

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在社会发展过程中, 病毒对人类的生命健康造成了严重危害。目前已知的致病病毒多达200余种, 引发了多种传染性疾病, 如病毒性肝炎、流行性感冒、麻疹、水痘、艾滋病等^[1]。此外, 2009年H1N1流感疫情、2014年脊髓灰质炎疫情、2016年寨卡病毒疫情、2018年埃博拉疫情、2019年新型冠状病毒疫情以及2022年猴痘疫情等多起国际公共卫生紧急事件也都是由病毒引发。在人类与病毒的斗争中, 抗病毒药物发挥着举足轻重的作用, 因此抗病毒药物的研发与全人类的健康福祉息息相关。

在目前已上市的抗病毒药物中, 核苷类药物(nucleoside analogues, NAs) 占半数以上。该类药物进入细胞后, 在激酶的作用下经磷酸化依次转化为单磷酸核苷(nucleoside monophosphate, NMP)、二磷酸核苷(nucleoside diphosphate, NDP) 和三磷酸核苷(nucleoside triphosphate, NTP) (图 1), 进而与病毒聚合酶或逆转录酶结合, 掺入病毒DNA或RNA链中, 从而阻断病毒的复制^[2-4]。但由于与天然核苷存在结构上的差异, 激酶对核苷类药物的亲和力十分有限, 单磷酸化过程成为药物代谢过程中的限速步骤, 在很大程度上限制了活性二磷酸及三磷酸核苷的形成, 从而影响了核苷类药物的抗病毒活性^{[5, 6]}。此外, 核苷类似物多为亲水性分子, 主要依靠转运蛋白促进扩散方式跨膜转运, 这导致核苷类药物的膜透过性较差, 难以被细胞摄取^[7]。

为提高核苷类化合物的磷酸化效率、改善生物利用度、增强治疗效果, 磷(膦) 酸酯前药策略在核苷类药物的研发过程中被广泛使用^[8-11]。由英国卡迪夫大学Christopher McGuigan团队首创的ProTide技术(PROdrug + nucleoTIDE) 是目前最成功的前药设计策略之一^{[11, 12]}, 其特征是将核苷类似物以磷(膦) 酰化的方式引入芳氧基和氨基酸酯, 形成芳氧基磷(膦) 酰胺酯, 即ProTide (图 2A)。该前药设计策略能够有效改善母体药物的理化性质, 使其更容易透过细胞膜, 同时也能够提高代谢活化效率, 增强药物的生物利用度和抗病毒活性^{[4, 12-14]}。近年来ProTide技术在抗病毒药物研究领域大放异彩, 目前已有多种ProTide药物应用于临床, 包括丙型肝炎病毒(hepatitis C virus, HCV) 感染治疗领域具有跨时代意义的“重磅炸弹”药物sofosbuvir (1)、人类免疫缺陷病毒(human immunodeficiency virus, HIV) 感染和乙型肝炎病毒(hepatitis B virus, HBV) 感染治疗药物tenofovir alafenamide (2, TAF) 及美国食品和药品管理局(Food and Drug Administration, FDA) 批准的首个新型冠状病毒(SARS-CoV-2) 感染治疗药物remdesivir (3) 等(图 2B)^{[3, 15-18]}。随着ProTide技术的日益成熟, 其优化对象目前已逐步扩展至非核苷类化合物^[19]。ProTide技术的成功开发与应用深刻影响了小分子抗病毒药物的研发进程, 具有极为广阔的发展空间。本文拟对ProTide技术的发展历程及特点进行简要介绍, 并对其在抗病毒药物研发领域的应用进行综述, 以期为后续抗病毒药物的设计与开发提供借鉴和参考。

1 ProTide技术发展历程

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在核苷类药物的研发探索中, 研究者发现磷(膦) 酸酯基团的引入能有效改善核苷类似物磷酸化效率低的问题。但是在生理条件下(pH = 7.0~7.4), 磷(膦) 酸酯片段易失去质子而表现出较强的极性, 药物依旧难以透过细胞膜^{[4, 20]}。因此, 如何将药物更有效地传输到细胞内成为研究者们关注的重点。自20世纪90年代起, Christopher McGuigan团队开始对核苷磷(膦) 酸酯前药进行探索^{[21, 22]}, 逐步建立起了利用芳氧基和氨基酸酯掩蔽磷(膦) 酸基团的ProTide技术, 其发展历程大致可分为6个阶段(表 1)^{[4, 23]}。

Christopher McGuigan团队首先尝试用简单烷氧基及卤代烷氧基对磷酸基团进行掩蔽以提高化合物的亲脂性, 但这些基团在体内难以被水解脱落, 因而化合物的活性并没有得到明显改善^{[21, 22, 24-27]}。在此基础上, 该团队对烷氧基及卤代烷氧基磷酰胺酯前药进行了探索, 并取得了突破性的进展: 氨基酸酯侧链的引入能显著提高母体化合物的抗病毒活性, 其中以L-丙氨酸(L-Ala) 酯效果最佳^[28-30]。于是, 该团队尝试引入两个氨基酸酯侧链形成磷酸二酰胺前药, 但结果却不甚理想^[31]。此外, 用乙醇酸酯或乳酸酯代替氨基酸酯所形成的磷酸酯前药同样效果不佳^[32]。在后续研究中, 该团队将目光投向了芳香基团并制备得到了一系列二芳氧基磷酸酯前药, 此类前药的抗病毒活性与母体化合物相比得到了显著的提高, 这是ProTide技术发展过程中的另一巨大突破^[33-35]。在上述工作的基础上, 该团队用芳氧基和氨基酸酯对磷酸基团进行掩蔽, 获得了具有良好透膜性且抗病毒活性显著的芳氧基磷酰胺酯前药, ProTide技术由此诞生^{[33, 36, 37]}。

2 ProTide药物的代谢模式及活性影响因素

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与核苷类似物不同, ProTide分子(Ⅰ) 通过被动扩散方式透过细胞膜^{[23, 38]}。进入细胞后, 首先经酯酶[如组织蛋白酶A (CTSA)、羧酸酯酶1 (CES1)] 催化, 发生氨基酸酯酯基的水解, 生成中间体(Ⅱ)。接着, 中间体(Ⅱ) 发生分子内亲核取代反应, 芳基离去, 形成瞬时五元环状中间体(Ⅲ), 继而经水解开环生成磷(膦) 酰胺代谢产物(Ⅳ)。Ⅳ经磷酰胺酶[如三联组氨酸结合蛋白1 (HINT1)] 催化, 发生磷-氮(P-N) 键断裂, 释放出单磷(膦) 酸核苷(Ⅴ)。单磷(膦)酸核苷(Ⅴ) 经两次磷酸化最终形成活性的三磷(膦) 酸核苷(Ⅵ) (图 3)^{[4, 11, 12, 39-44]}。

研究表明, ProTide的活性除与母体化合物有关还受到掩蔽基团性质的影响, 具有较强亲脂性且易于水解的掩蔽基团对化合物的活性更为有利^[45]。其中, 芳香基团是决定ProTide亲脂性的重要因素之一, 以萘氧基和苯氧基取代最为常见^[46]。对于氨基酸酯基团而言, 酯基是影响ProTide亲脂性的又一重要因素, 此外, 酯基也会影响氨基酸酯的水解速度, 通常以短链的伯、仲烷基及苄基为宜^[45]。除酯基外, 氨基酸的性质也会对氨基酸酯的水解速度产生影响, 通常以L-α-氨基酸为最佳选择^{[47, 48]}, 其中L-Ala在已上市及处于临床研究阶段的ProTide药物中最为常见^{[13, 49]}。此外, ProTide分子中五价磷原子的绝对构型(S_p/R_p) 也会影响药物的代谢活化效率^{[48, 50-52]}。因此在实际设计与研发过程中, 为获得理想的ProTide药物, 通常会对芳香基团种类、氨基酸类型及酯基结构、磷原子手性等多种因素进行综合考察^[23]。

3 ProTide技术在抗病毒药物研究领域的应用

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ProTide技术作为一种新颖的前药设计策略受到了研究学者的高度关注并在抗病毒药物研发中得到了广泛应用。近年来, 针对HCV、HIV、HBV等多种病毒开发的ProTide化合物层出不迭, 极大地推动了抗病毒药物的研发进程。ProTide技术在抗病毒药物研究中的主要应用如下。

3.1 ProTide技术在抗丙型肝炎病毒药物研究中的应用

Sofosbuvir (1, 图 2B) 最早由Pharmasset公司开发, 后被Gilead Sciences公司收购, 于2013年12月获得FDA批准用于治疗HCV感染, 是全球首个获批上市的ProTide药物^[15]。Sofosbuvir口服后经肝脏代谢为2ʹ-脱氧-2ʹ-α-氟-β-甲基尿苷-5ʹ-单磷酸, 后经磷酸化形成活性三磷酸代谢物, 从而对HCV NS5B聚合酶产生竞争性抑制作用^[40]。Sofosbuvir是具有S_p构型的单一异构体, 对野生型HCV的抑制活性(90%效应浓度EC₉₀ = 0.42 μmol·L^-1) 约是其R_p异构体(EC₉₀ = 7.5 μmol·L^-1) 的18倍, 对S282T及S96T耐药株的抑制活性(EC_{90 (S282T)}= 7.8 μmol·L^-1, EC_{90 (S96T)}= 0.11 μmol·L^-1) 分别是其R_p异构体(EC_{90 (S282T)} > 100 μmol·L^-1, EC_{90 (S96T)}= 1.3 μmol·L^-1) 的13和12倍。此外, 在原代人肝细胞中, S_p异构体产生三磷酸代谢物的水平比R_p异构体高14%^[50]。临床试验证明, sofosbuvir具有疗效显著、安全性高、耐药屏障高等特点, 可用于1~6型HCV感染的治疗, 其中对2型和3型HCV感染患者的治愈率可高达90%^[53-56]。此外, sofosbuvir也同样适用于HCV合并HIV感染患者以及准备接受肝移植的肝癌患者^[57]。Sofosbuvir的出现在丙肝治疗史上具有里程碑式的意义。

Alfosbuvir (4, SH229, 图 4) 是由南京圣和药业股份有限公司自主研发的国内首个HCV NS5B聚合酶抑制剂。临床前研究结果表明, alfosbuvir的体外抗病毒活性(半数效应浓度EC₅₀ = 10.4~38.4 nmol·L^-1) 是sofosbuvir的2~3倍。两项临床Ⅰ期试验表明, alfosbuvir安全性高且耐受性好, 具有良好的药代动力学性质, 能够与其他抗HCV药物联合使用。一项对124例HCV感染患者进行的临床Ⅱ期试验表明, 96%的受试者在接受12周alfosbuvir (400、600或800 mg·d^-1) 与daclatasvir (60 mg·d^-1) 联合治疗后获得SVR12 (治疗终点后12个月随访时HCV-RNA阴性), 没有观察到与治疗有关的严重不良事件, 没有受试者因不良事件停药^[58]。一项临床Ⅲ期试验结果显示, 326例HCV感染患者在接受12周alfosbuvir (600 mg·d^-1) 与daclatasvir (60 mg·d^-1) 联合治疗后, 98.5%的受试者获得了SVR12, 除1例失访外, 这些受试者在治疗结束第24周均继续获得了持续病毒学应答(SVR24)。用药过程中出现的常见不良反应为高胆固醇血症, 未发生受试者死亡事件^[59]。总体而言, alfosbuvir具有疗效显著且安全性高的特点, 目前正处于申报上市阶段。

ASC-21 (5, MIV-802, 图 4) 是由Medivir AB公司研发的一种HCV NS5B聚合酶抑制剂。临床前研究表明, ASC-21对HCV NS5B聚合酶具有较强的抑制作用(半数抑制浓度IC₅₀ = 0.71 μmol·L^-1), 对各基因型HCV的抑制活性(EC₅₀ = 17~58 nmol·L^-1) 普遍优于sofosbuvir (EC₅₀ = 48~210 nmol·L^-1), 且具有良好的安全性(细胞半数毒性浓度CC₅₀ > 200 µmol·L^-1)^[60]。目前, ASC-21正处于临床Ⅰ期试验阶段。

此外, Maiti等^[61]对2′-β-甲基胞苷(6)、2′-β-甲基尿苷(7) 及2ʹ-脱氧-2′-α-氟-β-甲基尿苷(8) 进行前药化修饰, 设计并合成了一系列包含L-Ala、L-天冬氨酸(L-Asp)、亚氨基二乙酸(IDA) 酯片段的ProTide (图 5A), 并对其进行抗HCV活性评价。研究发现, L-Asp ProTide的优化效果显著, 其中化合物7a的抗HCV活性(EC₅₀ = 0.03 µmol·L^-1) 较其母体2′-β-甲基尿苷(7, EC₅₀ = 6.31 µmol·L^-1) 提高了约210倍。构效关系研究表明, L-Asp ProTide的抗病毒活性强弱与L-Asp酯片段的结构有关, 酯基的体积越大、亲脂性越强性则化合物的抗病毒效果越佳, 如正戊酯及异戊酯类化合物的活性(EC₅₀ = 0.03~0.06 µmol·L^-1) 明显优于甲酯类化合物(EC₅₀ = 1.13~3.71 µmol·L^-1)。IDA ProTide未能对母体核苷的抗病毒活性起到改善作用, 这可能是IDA ProTide无法在肝细胞中顺利释放出单磷酸核苷所致。初步的代谢研究表明, L-Asp ProTide可能先在酯酶的催化下发生天冬氨酸酯中β-羧基酯的水解, 而后逐步释放出单磷酸核苷, 最终转化为三磷酸核苷(图 5B)。与L-Ala ProTide相比, L-Asp ProTide的最大优势在于L-Asp具有双羧基, 可以同时引入两种不同的酯片段来调节化合物的理化性质、改善生物利用度、实现靶向给药, 是抗病毒ProTide药物开发的新思路。

Procházková等^[62]设计并合成了5′-氮及5′-硫修饰的2′-β-甲基腺苷ProTide 9和10 (图 6), 但这两个化合物均无抗HCV活性。研究发现, 化合物9和10在细胞内并未经ProTide类似的代谢过程产生单膦酸核苷, 而是直接发生了P-N及P-S键的断裂, 导致活性三膦酸核苷无法顺利生成而使化合物丧失了抗病毒活性, 这为后续ProTide药物的研发提供了重要信息。

3.2 ProTide技术在抗人类免疫缺陷病毒药物研究中的应用

Tenofovir alafenamide (2, TAF, 图 2B) 是由Gilead Sciences公司研发的一种tenofovir (TFV) ProTide, 于2015年11月获得FDA批准用于治疗HIV-1感染^[16]。研究发现, TAF对HIV-1的抑制效果(EC₅₀ = 0.005 µmol·L^-1) 明显强于相应的D-Ala ProTide (EC₅₀ = 10 µmol·L^-1) 和R_p异构体(EC₅₀ = 0.06 µmol·L^-1), 及其母体化合物TFV (EC₅₀ = 5 µmol·L^-1) 和另一种TFV前药tenofovir disoproxil fumarate (TDF, EC₅₀ = 0.05 µmol·L^-1)^[48]。此外, 与TDF相比, TAF具有更强的血浆稳定性, 能够在淋巴组织中特异性积聚, 并在细胞中经CTSA代谢、磷酸化产生具有逆转酶抑制活性的二膦酸代谢物(TFV-DP)^[41], 可以有效改善因血浆高TFV水平而引起的骨密度降低和肾毒性问题^[63]。临床试验结果表明, TAF治疗效果显著, 耐受性好, 在低剂量(25 mg·d^-1) 下即可达到与TDF (300 mg·d^-1) 同样的治疗效果, 具有更高的肾脏及骨骼安全性, 可用于轻度至中度肾损伤HIV-1感染患者的治疗^[64-68]。

Kalčic等^[69]用L-酪氨酸(L-Tyr) 衍生物替换苯酚片段对TAF进行了优化改造。采用“一锅法”合成得到了L-Tyr ProTide的非对映异构体混合物, 通过手性高效液相色谱法将混合物拆分为fast-eluting epimer (FEE) 和slow-eluting epimer (SEE), 并利用圆二色谱法、高效液相色谱法等确证SEE为S_P异构体。活性测试结果显示, SEE较FEE对HIV-1具有更强的抑制作用。代谢研究表明, SEE能够在细胞中快速转化为TFV, 且SEE释放的TFV明显多于FEE。在这些L-Tyr ProTide中, 化合物11 (图 7) 的抗病毒活性(EC₅₀ = 0.07 nmol·L^-1) 尤为突出, 较TAF提高了近100倍, 并且化合物11也具有更高的选择性指数, 其SI值(142 857) 较TAF提高了约26倍。对Tyr手性中心的研究进一步证实了L-Tyr结构的重要性, D-Tyr异构体的抗HIV-1活性(EC₅₀ = 0.67 nmol·L^-1) 较化合物11降低了近10倍。稳定性实验表明, 化合物11在pH = 1.5~10范围内较TAF具有更强的化学稳定性, 且化合物11的血浆稳定性也强于TAF。但在肝微粒体细胞中, 化合物11因L-Tyr片段中的酯键易发生断裂而被迅速降解, 这表明化合物11在口服时可能会存在较明显的首过效应。为提高代谢稳定性, 研究者们将L-Tyr片段中的环戊基酯基替换为环戊基-2-氧代乙基得到化合物12 (图 7), 其对HIV-1的EC₅₀值可达6 pmol·L^-1, 且具有更高的选择性指数(SI = 1 533 333)。尽管化合物12的微粒体稳定性较化合物11得到了明显改善, 但仍远不及TAF, 还存在巨大的优化空间。代谢研究表明, 此类ProTide优异的抗病毒活性可能与其能够被细胞高效地摄取并代谢为TFV的独特性质有关, 这为后续高效抗病毒药物的研究提供了参考。

Stavudine (13, 图 8) 是一种核苷类逆转录酶抑制剂, 用于治疗HIV-1感染。Kandil等^[70]对stavudine进行前药化修饰, 设计并合成了一系列ProTide。生物活性评价结果显示, 多氟芳基取代的化合物15和16 (图 8) 对1型和2型HIV均具有明显的抑制作用, 对HIV-1的EC₅₀值分别为0.030和0.037 µmol·L^-1, 对HIV-2的EC₅₀值分别为0.036和0.047 µmol·L^-1, 均高于母体化合物stavudine (图 8, EC_{50 (HIV-1)} = 0.40 µmol·L^-1, EC_{50 (HIV-2)} = 0.45 µmol·L^-1) 和不含氟取代的化合物14 (图 8, EC_{50 (HIV-1)} = 0.21 µmol·L^-1, EC_{50 (HIV-2)}= 0.27 µmol·L^-1), 这可能与多氟芳基取代后化合物的亲脂性增强有关。此外, 化合物14~16在胸苷激酶(TK) 缺陷型细胞中均保持了良好的抗HIV-1活性, EC₅₀值分别为0.32、0.335和0.075 µmol·L^-1, 而stavudine在此类细胞中完全失活, 说明这些化合物对TK介导的磷酸化过程依赖较小, 能有效避开核苷类似物的单磷酸化过程。

此外, 据报道, 抗疱疹病毒药物acyclovir (17, ACV, 图 9A) 经磷酸化后能够对HIV-1逆转录酶产生抑制活性^[71]。受此启发, Derudas等^[72]设计并合成了一系列ACV ProTide, 其中L-Ala ProTide (图 9A) 具有较好的HIV抑制作用(EC_{50 (HIV-1)} = 6.2~17.0 µmol·L^-1, EC_{50 (HIV-2)} = 8.9~42 µmol·L^-1), 但部分化合物表现出了一定的细胞毒性(CC₅₀ ≥ 17 µmol·L^-1)。在该研究基础上, 为获得低毒高效的抗HIV药物, Derudas等^[73]通过分子对接技术使用HIV-1逆转录酶、腺苷酸激酶或鸟苷酸激酶、人DNA聚合酶γ对3 600个无环核苷类似物进行虚拟筛选, 得到了5种具有潜在逆转录酶抑制活性的化合物分子, 并制备了相应的mono-ProTide和bis-ProTide (18~22, 图 9B)。活性结果显示, 绝大多数ProTide具有较好的抗HIV-1活性, 其中化合物22的mono-ProTide活性最佳(23, EC₅₀ = 1.1 µmol·L^-1), 选择性指数为21。但大部分化合物, 尤其是bis-ProTide, 具有较高的细胞毒性(CC₅₀ ≥ 4 µmol·L^-1)。

3.3 ProTide技术在抗乙型肝炎病毒药物研究中的应用

研究发现, TAF (2, 图 2B) 不仅能在淋巴组织中特异性聚积, 也能被肝细胞高效摄取并转化为活性代谢物TFV-DP, 因此除用于治疗HIV-1感染外, TAF同时也可用于HBV感染的治疗^[74]。临床试验表明, HBeAg阴性及阳性HBV感染患者在服用TAF (25 mg·d^-1) 后的治疗效果均与服用TDF (300 mg·d^-1) 相当, 且服用TAF的患者肌酐清除率下降幅度、髋部及脊柱骨密度降低幅度明显更小, 表明TAF具有更高的肾脏及骨骼安全性^{[75, 76]}。2016年11月, FDA正式批准TAF用于HBV感染的治疗^[17]。

ATI-2173 (24, 图 10) 是由Antios Therapeutics公司开发的肝靶向clevudine磷酰胺酯前药, 是一种非竞争性、非链终止的聚合酶抑制剂。ATI-2173口服后能被肝细胞高效摄取并转化为活性的三磷酸代谢物, 该活性代谢物通过与HBV聚合酶活性位点结合并使之发生扭曲而阻断病毒DNA的复制^{[77, 78]}。通过采用ProTide技术, ATI-2173避开clevudine的单磷酸化过程, 减少了线粒体胸苷激酶(TK2) 的消耗, 此外, ATI-2173的肝靶向作用也有效降低了clevudine的血浆暴露, 降低了骨骼肌病发生的风险, 具有更好的药代动力学性质和安全性^{[78, 79]}。研究发现, ATI-2173对HBV具有显著的抑制作用(EC₅₀ = 1.31 nmol·L^-1), 对心、肝、肾、骨骼肌、骨髓等无明显毒性, 可与其他抗HBV药物联合使用^[78]。临床Ⅰ期试验结果显示^[80], HBV感染患者在接受为期28天的ATI-2173 (10、25或500 mg·d^-1) 单药治疗后, 体内HBV DNA平均减少2.72~2.78 log₁₀ IU·mL^-1。停药后, 在大部分受试者中仍观察到持续的病毒抑制和共价闭合环状DNA的减少, 1名受试者在停药24周后体内HBV DNA仍低于10 IU·mL^-1, 试验过程中没有观察到与治疗有关的严重不良事件。该试验结果证实ATI-2173能有效减少clevudine全身暴露的风险, 具有良好的抗病毒活性和较高的安全性。目前ATI-2173正处于临床Ⅱ期试验阶段。

此外, Kalčic等^[69]在抗HIV-1药物的探索过程中发现, 部分具有L-Tyr片段的TFV ProTide同样具有良好的抗HBV活性, 其中非对映体混合物25 (图 11) 的抗病毒活性(EC₅₀ = 0.2 nmol·L^-1) 较TAF (EC₅₀ = 5.3 nmol·L^-1) 提高了约26倍, 且具有较高的选择性指数(SI > 250 000), 具有进一步研究的价值。

3.4 ProTide技术在抗疱疹病毒药物研究中的应用

Maiti等^[81]对brivudin (26, BVDU) 及2′-脱氧腺嘌呤核苷类似物(27~30) 进行前药化修饰, 设计并合成了一系列包含L-Ala-Me、IDA-Me及IDA-POM片段的ProTide药物(图 12), 并对其进行抗单纯疱疹病毒(herpes simplex virus, HSV)、抗水痘-带状疱疹病毒(varicella-zoster virus, VZV) 和抗巨细胞病毒(human cytomegalovirus, HCMV) 活性评价。其中化合物31和32对HSV-1和VZV具有较为明显抑制作用, 对HSV-1的EC₅₀值分别为0.3和1.1 µmol·L^-1, 对VZV的EC₅₀值分别为0.21和0.34 µmol·L^-1, 但均不及其母体化合物BVDU (EC_{50 (HSV-1)} = 0.1 µmol·L^-1, EC_{50 (VZV)} = 0.03 µmol·L^-1)。对于2′-脱氧腺嘌呤核苷类似物及其ProTide, 除少部分化合物具有抗VZV活性(EC₅₀ = 2.4~68 µmol·L^-1) 外, 多数化合物基本不具备抗HSV、VZV或HCMV作用。

Wang等^[82]对嘌呤核苷类抗HSV药物vidarabine (33, ARA, 图 13) 进行前药化修饰, 设计并合成了一系列带有不同氨基酸酯片段的ProTide。活性测试结果显示, 这些化合物均具有明显的肝靶向作用和较强的抗HSV-1活性, 其中化合物34和35 (图 13) 活性最佳, EC₅₀值分别为0.52和1.05 µmol·L^-1, 明显优于其母体化合物ARA (EC₅₀ = 10 µmol·L^-1)。

Gemcitabine (36, 图 14) 是一种嘧啶核苷类抗肿瘤药物, 它对HCV、HIV-1、寨卡病毒(zika virus, ZIKV)、甲型流感病毒(influenza A virus, IAV)、SARS-CoV-2等RNA病毒同样具有抑制活性^[83-87]。为增强gemcitabine的抗病毒能力, 降低毒性, Zheng等^[88]在gemcitabine的4′位引入吸电子基团, 设计并合成一系列gemcitabine衍生物及其ProTide。活性测试结果显示, 与gemcitabine (EC_{50 (VZV)} = 0.028 µmol·L^-1, EC_{50 (HCMV)} = 0.074 µmol·L^-1) 和gemcitabine衍生物37 (图 14, EC_{50 (VZV)} = 0.042 µmol·L^-1, EC_{50 (HCMV)} = 0.815 µmol·L^-1) 相比, 化合物38 (图 14) 的抗VZV及抗HCMV活性(EC_{50 (VZV)} = 2.32 µmol·L^-1, EC_{50 (HCMV)} = 15.47 µmol·L^-1) 均有所减弱。但化合物38的细胞毒性(CC₅₀ = 84.35 µmol·L^-1) 较gemcitabine (CC₅₀ = 0.003 6 µmol·L^-1) 和37 (CC₅₀ = 0.11 µmol·L^-1) 更低, 具有更高的选择性。

3.5 ProTide技术在抗新型冠状病毒药物研究中的应用

Remdesivir (3, 图 2B) 是由Gilead Sciences公司研发的一种病毒RNA依赖性RNA聚合酶(RdRp) 抑制剂, 对埃博拉病毒(ebolavirus, EBOV)、SARS冠状病毒(SARS-CoV) 和中东呼吸综合征冠状病毒(MERS-CoV) 等均具有体外抑制活性^[89-92]。2019年新型冠状病毒肺炎疫情暴发后, Wang等^[93]发现remdesivir对SARS-CoV-2具有良好的抑制作用(EC₅₀ = 0.77 μmol·L^-1, SI > 129.87), 结合此前remdesivir在临床试验中表现出的高安全性^[94], remdesivir作为治疗SARS-CoV-2感染的首批候选药物进入临床^[95]。临床试验结果显示, 不同程度的SARS-CoV-2感染患者在接受remdesivir治疗后病情均得到有效改善, 治疗过程中主要存在胃肠道反应、转氨酶水平升高、超敏反应等不良反应^[96]。2020年10月, FDA批准remdesivir用于SARS-CoV-2感染住院患者的治疗^[18]。

尽管remdesivir获得了FDA的正式批准, 但部分临床试验结果显示, remdesivir对SARS-CoV-2感染患者的治疗效果并不显著^[97-99], 其疗效仍存在诸多争议。研究发现, remdesivir的疗效受限可能与其血浆稳定性差、难以在SARS-CoV-2感染的主要靶器官(如肺) 中积聚、抗病毒效果欠佳等因素有关^[100-103]。针对上述问题, Hu等^[104]在remdesivir的基础上对L-Ala酯片段进行结构修饰, 引入亚油酸结构, 设计并合成了一系列remdesivir衍生物。其中, 化合物39 (图 15) 活性最佳, 它对SARS-CoV-2及其4种突变株的抑制活性(EC₅₀ = 0.4~15.9 μmol·L^-1) 是remdesivir (EC₅₀ = 3.3~32 μmol·L^-1) 的2~7倍(表 2), 且细胞毒性较低(CC₅₀ > 10 µmol·L^-1)。与remdesivir相比, 化合物39具有更强的血浆稳定性和肝微粒体稳定性。静脉注射后, 化合物39在肺部的浓度约是remdesivir的200倍, 其产生的活性三磷酸代谢物的浓度约是remdesivir的5倍, 具有更好的肺靶向性。同时, 化合物39也更容易被肺上皮细胞摄取并活化, 化合物39及其活性代谢物的细胞内总浓度约是remdesivir的16倍。上述结果显示, 化合物39抗SARS-CoV-2效果显著, 具有较大的开发价值。

另外, Zheng等^[88]在对gemcitabine衍生物及其ProTide的探索过程中发现化合物38 (图 14) 对SARS-CoV-2具有明显的抑制作用(EC₅₀ = 0.73 µmol·L^-1), 虽然其活性不及母体化合物37 (EC₅₀ = 0.096 µmol·L^-1), 但较hydroxychloroquine (EC₅₀ = 1.74 µmol·L^-1) 和remdesivir (3, EC₅₀ = 1.52 µmol·L^-1) 活性更佳。但是在抗SARS-CoV-2活性评价过程中发现, 化合物38具有较高的细胞毒性(CC₅₀ = 1.44 µmol·L^-1) (表 3)。

3.6 ProTide技术在抗流感病毒感染药物研究中的应用

Meneghesso等^[105]发现, 尽管三磷酸尿苷衍生物对流感聚合酶表现出了较好的抑制作用, 但尿苷类化合物的抗病毒活性却普遍较弱, 这可能与尿苷类化合物的低磷酸化效率有关。据此, 该研究团队设计并合成了一系列尿苷衍生物及其ProTide以提高尿苷类化合物抗病毒活性和生物利用度。活性测试结果显示, 虽然2ʹ-脱氧-2ʹ-α-氟尿苷40 (图 16) 无抗流感病毒活性(99%效应浓度EC₉₉ > 100 µmol·L^-1), 但其ProTide 41和42 (图 16) 对流感病毒却表现出了中等的抑制作用, EC₉₉分别为49和81 µmol·L^-1。此外, 含萘氧基片段ProTide 41的抗病毒活性强于含苯氧基片段的ProTide 42, 说明增加亲脂性可提高药物的透膜性(计算脂水分配系数ClogP₄₁ = 2.37, ClogP₄₂ = 1.20)。代谢研究表明, 尿苷ProTide能够有效地被CES1水解并缓慢代谢为单磷酸尿苷。与阳性对照ribavirin (EC₉₉ = 8.0 µmol·L^-1) 相比, ProTide 41和42的抗流感病毒效果仍相差甚远, 这可能是其代谢为单磷酸尿苷的过程过于缓慢且不完全, 或是单磷酸尿苷转变为二磷酸尿苷和三磷酸尿苷的效率低下所致。

随后, Meneghesso等^[106]又对鸟苷ProTide进行了探索, 设计并合成了一系列6位修饰的2ʹ-脱氧-2ʹ-α-氟鸟苷衍生物及其ProTide。活性测试结果显示, 6位修饰的2ʹ-脱氧-2ʹ-α-氟鸟苷衍生物43~45 (图 17) 对流感病毒并不具备抑制作用(EC₉₉ > 100 µmol·L^-1)。但是, 6位修饰的2ʹ-脱氧-2ʹ-α-氟鸟苷衍生物ProTide却在不同程度上表现出了抗流感病毒活性, 其中具有萘氧基和L-Ala-Me片段的ProTide 46~48 (图 17) 抗病毒活性最佳(EC₉₉ = 12~15 µmol·L^-1)。分析发现, 亲脂性过高或过低均会对活性产生不利影响, 以ClogP值在2左右为宜。代谢研究表明, 此类ProTide能迅速被CES1水解为磷酰胺代谢产物, 但该代谢产物释放出单磷酸鸟苷的过程则较为缓慢。此外, 2ʹ-脱氧-2ʹ-α-氟鸟苷单磷酸6位上的取代基能够被腺苷脱氨酶代谢并最终转换为2ʹ-脱氧-2ʹ-α-氟鸟苷单磷酸, 这可能是具有不同6位取代基的ProTide表现出了相近抗流感病毒活性的原因, 说明引入合适的6位取代基可将2ʹ-脱氧-2ʹ-α-氟鸟苷衍生物ProTide开发为一种双前药, 从而进一步提高化合物被细胞摄取的能力。

3.7 ProTide技术在抗登革热病毒药物研究中的应用

AT-752 (49, 图 18) 是由Atea Pharmaceuticals公司开发的RdRp抑制剂, 是一种双前药, 对2型和3型登革热病毒(dengue virus, DENV) 均具有良好的抑制作用, EC₅₀值分别为0.48和0.77 µmol·L^-1, 且无明显的细胞毒性(CC₅₀ > 170 µmol·L^-1)。AT-752的代谢过程如图 18所示, AT-752通过在外周血单个核细胞(peripheral blood mononuclear cell, PBMC) 中形成活性的三磷酸代谢物AT-9010 (50) 而发挥竞争性抑制作用。体内活性测试结果显示, AT-752可有效降低登革热病毒感染小鼠病毒血症的发病率并提高感染小鼠的存活率^[107]。有关AT-752在健康受试者中安全性、耐受性和药代动力学性质的临床Ⅰ期试验于2021年11月完成, 结果尚未公布, 目前AT-752处于临床Ⅱ期试验阶段。

Okon等^[108]通过引入色胺和2-甲基硫代乙基对2′-β-甲基鸟苷进行磷酰胺酯前药化修饰获得了一种结构新颖的ProTide化合物51 (图 19)。与经典ProTide不同, 该化合物无需酶的启动激活即可通过化学裂解产生磷酰胺核苷, 而后经HINT1催化发生P-N键断裂, 释放出单磷酸核苷并最终产生活性的三磷酸代谢物(图 19), 具有全身给药的潜力。活性测试结果显示, ProTide 51对DENV-2的抑制效果(EC₅₀ = 1.59 µmol·L^-1) 是其母体核苷(EC₅₀ = 8.14 µmol·L^-1) 的5倍, 且无明显的细胞毒性(CC₅₀ > 200 µmol·L^-1), 具有深入开发的价值。

3.8 ProTide技术在其他抗病毒药物研究中的应用

Pileggi等^[109]利用交叉复分解反应合成了一系列烯基嘧啶衍生物ProTide并评价了其抗DNA及RNA病毒活性。其中, 烯基胸腺嘧啶ProTide 52 (图 20) 对VZV、HCMV、辛德比斯病毒(sindbis virus, SINV)、柯萨奇病毒B4 (coxsackie virus B4)、庞特托罗病毒(punta toro virus, PTV) 和黄热病毒(yellow fever virus, YFV) 均表现出了一定的抑制作用(EC₅₀ = 20~58 µmol·L^-1), 且无明显细胞毒性(CC₅₀ > 100 µmol·L^-1), 具有潜在的广谱抗病毒能力。

4 总结与展望

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由Christopher McGuigan团队开创的ProTide技术深刻影响了小分子药物的研发进程, 尤其为小分子抗病毒药物的研究带来了突破性的进展。ProTide技术不仅能够实现对现有抗病毒药物的活性优化, 还能将惰性分子转变为活性抗病毒药物, 这为新型抗病毒药物的设计与开发, 特别是“老药新用”的发掘提供了思路, 具有极其重要的借鉴意义。目前, ProTide技术在抗丙型肝炎病毒、人类免疫缺陷病毒、乙型肝炎病毒、疱疹病毒、新型冠状病毒、流感病毒等多种抗病毒药物的研发中取得了令人瞩目的成果。随着该项技术的不断发展, ProTide的结构类型日益丰富, 各种功能化磷酸掩蔽基团的引入为调节药物的理化性质、改善生物利用度、提高抗病毒活性提供了更为多样的选择。此外, ProTide技术的应用范围日益也扩大, 其优化对象逐步从核苷类化合物扩展至非核苷类化合物。但是, 与其他前药策略类似, 前体药物的细胞外激活仍是ProTide技术面临的一大挑战, 它会在一定程度上降低药物的生物活性并引起毒副作用, 因此提高ProTide药物的稳定性和靶向性将是未来研发的重要方向。总体而言, ProTide技术的应用为抗病毒药物的研究提供了高效便利的手段, 也为应对当下和未来病毒性疾病的威胁提供了强大助力, 具有广阔的发展空间和良好的发展前景。

作者贡献: 周慧宇负责文献检索和文章撰写; 朱梅负责文章内容核对和修改; 王玉成负责文章选题、指导和审校。

利益冲突: 所有作者均声明没有利益冲突。

基金

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国家自然科学基金资助项目(82204207)

参考文献

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文献

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2023年第58卷第6期

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doi: 10.16438/j.0513-4870.2022-1210

接收时间：2022-11-14
首发时间：2025-11-21
出版时间：2023-06-12

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收稿日期：2022-11-14
修回日期：2022-12-07

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国家自然科学基金资助项目(82204207)

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中国医学科学院、北京协和医学院医药生物技术研究所, 北京 100050

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*王玉成, Tel: 86-10-63165263, E-mail: wangyucheng@imb.pumc.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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