Colaborative Research on Anti-HIV Triterpenoids

在确定白桦酸(1)和二氢白桦酸(9)为抗hiv药物的基础上，我们合成了一系列羽豆烷型三萜酸衍生物，并对其在急性感染的H9细胞中抑制HIV-1复制的活性进行了评价。其中，3- o -（3′，3′-二甲基琥珀基）-白桦酸(3)和3- o -（3′，3′-二甲基琥珀基）-二氢白桦酸（11）均表现出极强的抑制活性，EC_<50>值< 0.00035 muM。它们分别表现出显著的体外治疗指数（TI），分别为>20,000和>14,000。3- o -（3', 3'-二甲基戊二酰）-白桦酸和-二氢白桦酸（12），3- o -二糖酰基-白桦酸和-二氢白桦酸和3- o -戊二酰-白桦酸也能有效抑制HIV的复制，ED_<50>的取值范围为0.01 ~ 0.0023 muM, t.i值为1,017 ~ 2,344。此外，化合物11和12在单核细胞系和外周血单核细胞（PBMCs）中也具有抑制HLV复制的活性。我们的体外实验表明，这些化合物不是HIV- 1逆转录酶的抑制剂，而它们在20 - 40马克杯/毫升的浓度范围内完全抑制合胞体的形成。3-O-（2′，2′-二甲基琥珀酰）-白桦酸(2)也被发现是hiv诱导的膜融合抑制剂，IC_<100>值为20mutg/mL，但其抗hiv活性明显低于上述ED_<50>值为2.7 muM和t.l值为5.9的化合物。这一观察结果表明，除抑制合胞体形成外，这些化合物显示的抗hiv活性可能涉及另一种作用机制。化合物3在MAGI实验中没有活性，这表明它在病毒生命周期的后期（如病毒蛋白合成后）抑制HIV复制。另一方面，齐墩果酸在一些植物中被鉴定为抗hiv的主要成分，包括：Rosa woodsii（叶）、Prosopis glandullosa（叶和细枝）、Phorodendron juniperinum（整株）、Syzygium claviflorum（叶）、Hyptis capitata（整株）和Terniroemia gymnanthera（地上部分）。抑制急性感染H9细胞的HIV- 1复制，EC_<50>值为3.7 muM，抑制H9细胞生长，IC_<50>值为47.8 muM[治疗指数（ti）]。12.8]。结果表明，从木毒和赤藓属植物中分离得到的多酚酸也具有抗hiv的活性（EC_<50> 3.0 muM, t.i 16.6）。熊果酸虽有抗hiv活性（EC_<50> 4.4 muM），但毒性较弱（IC_<50> 14.3 muM, t.i 3.3）。基于这些结果，我们检测了从几种植物中分离的齐墩果酸或多酚酸相关的三萜抗hlv的活性。此外，我们之前已经证明了从S.claviflum叶子中分离的白桦酸衍生物作为抗hiv的原理，表现出极强的抗hiv活性。为此，我们制备齐墩果酸衍生物，并对其抗hiv活性进行了评价。齐墩果酸衍生物中，3- o -（3′，3′-二甲基丁二酰）齐墩果酸抗hiv活性最强，EC_<50>值为0.00086 muM, t.i值为22400。虽然熊果酸对H9细胞有轻微毒性，但其抗hiv活性与齐墩果酸相当。此外，熊果酸的结构与齐墩果酸和白桦酸密切相关，因此被认为是一种潜在的抗hiv药物。因此，对熊果酸进行类似改性，并评价其抗hiv活性。3- o -二甘酰尿醛酸具有较强的抗hiv活性，EC_<50>值为0.31 muM, t.i值为155.5。然而，包括3,3-二甲基琥珀酰衍生物在内的其他衍生物的抗hjv活性不如相应的白桦酸和齐墩果酸衍生物强。少

A series of lupane-type triterpenoic acid derivatives were synthesized and evaluated for their inhibitory activity against HIV-1 replication in acutely infected H9 cells, based on the fact that betulinic acid (1) and dihydrobetulinic acid (9) were identified as anti-HIV agents. Among the derivatives, 3-O-(3 ', 3 '-dimethylsuccinyl)-betulinic acid (3) and 3-O-(3 ', 3 '-dimethyl-succinyl)-dihydrobetulinic acid (11) both demonstrated extremely potent inhibitory activity with EC_<50> values of< 0.00035 muM.They exhibited remarkable in vitro therapeutic index (TI.) values of >20,000 and >14,000, respectively. 3-O-(3', 3'-Dimethylglutaryl)-betulinic acid and -dihydrobetulinic acid(12), 3-O-diglycolyl-betulinic acid and -dihydrobetulinic acid and 3-O-glutaryl- betulinic acid were also potent inhibitors of HIV replication with ED_<50> values ranging from 0.01 to 0.0023 muM and T.I.values from 1,017 to 2,344. In addition, compounds 1 1 and 12 are also active against HLV replica-tion in a monocy … More te cell line and n peripheral blood mononuclear cells (PBMCs). Our in vitro assay indicated that these compounds are not inhibitors on HIV- 1 reverse transcriptase, whereas they inhibited syncytia formation completely in a concentration range of 20 - 40 mug/mL.However, 3-O-(2', 2'-dimethylsuccinyl)-betulinic acid (2) was also found to be an inhibitor of HIV-induced membrane fusion with IC_<100> value of 20mutg/mL, though it displayed significantly lower anti-HIV activity than foregoing compounds with ED_<50> value of 2.7 muM and T.l. values of 5.9. This observation indicated that another mechanism(s) of action other than inhibition of syncytia formation could be involved in the anti-HIV activity shown by these compounds. Compound 3 was not active in the MAGI assay, suggesting that it inhibit HIV replication at a late stage of the viral life cycle, such as after viral protein synthesis.On the other hand, oleanolic acid was identified as an anti-HIV principle from several plants, including Rosa woodsii (leaves), Prosopis glandulosa (leaves and twigs), Phorodendron juniperinum (whole plant), Syzygium claviflorum (leaves), Hyptis capitata (whole plant), and Terniroemia gymnanthera (aerial part). It inhibited HIV- 1 replication in acutely infected H9 cells with an EC_<50> value of 3.7 muM, and inhibited H9 cell growth with an IC_<50> value of 47.8 muM [therapeutic index (T.I.) 12.8]. Pomolic acid, isolated from R.woodsii and H.capitata, was also identified as an anti-HIV agent (EC_<50> 3.0 muM, T.I.16.6). Although ursolic acid did show anti-HIV activity (EC_<50> 4.4 muM), it was slightly toxic (IC_<50> 14.3 muM, T.I.3.3). Based on these results, we examined anti-HLV the activity of oleanolic acid- or pomolic acid-related triterpenes isolated from several plants. In addition, we previously demonstrated that derivatives of betulinic acid, isolated from the leaves of S.claviflum as an anti-HIV principle, exhibited extreme potent anti-HIV activity. Accordingly, we prepared derivatives of oleanolic acid, and evaluated their anti-HIV activity. Among the oleanolic acid derivatives, 3-O-(3', 3'-dimethylsuccinyl)-oleanolic acid demonstrated most potent anti-HIV activity, with an EC_<50> value of 0.00086 muM and a T.I.value of 22,400.Though ursolic acid is slightly toxic against H9 cells, it shows similar level of anti-HIV acitivity to that of oleanolic acid. In addition, the structure of ursolic acid is closely correlated with oleanolic acid and betulinic acid, hence it is regarded as a potential lead for anti-HIV agent. Therefore, similar modification of ursolic acid, and evaluated their anti-HIV activities. 3-O-diglycoryl-uroslic acid demonstarated relatively potent anti-HIV activity with an EC_<50> value of 0.31 muM and a T.I.value of 155.5. Hoever, anti-HJV activities for other derivatives, including 3,3-dimethysuccinyl derivative, were not potent as corresponding betulinic acid and oleanolic acid derivatives. Less