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. 2020 Sep 9;11(11):2290–2293. doi: 10.1021/acsmedchemlett.0c00414

Novel Betulinic Acid–Nucleoside Hybrids with Potent Anti-HIV Activity

Qiang Wang †,‡,§, Yujiang Li †, Liyun Zheng §, Xiaowan Huang §, Yanli Wang ‡,*, Chin-Ho Chen ∥, Yung-Yi Cheng ⊥, Susan L Morris-Natschke ⊥, Kuo-Hsiung Lee ⊥,#,*
PMCID: PMC7667853  PMID: 33214842

Abstract

graphic file with name ml0c00414_0004.jpg

Novel betulinic/betulonic acid–nucleoside hybrids were synthesized as possible new anti-HIV agents. Among the synthesized hybrids, two compounds were highly effective against HIV. Compared with AZT and DSB, compounds 10a (IC50 = 0.0078 μM, CC50 = 9.6 μM) and 10b (IC50 = 0.020 μM, CC50 = 23.8 μM) showed more potent or equipotent, respectively, anti-HIV activity but displayed lower cytotoxicity.

Keywords: Betulinic acid, nucleoside, hybrid, click chemistry, anti-HIV activity

Betulinic acid (1) is a natural pentacyclic triterpene with a lupane structure. Studies have demonstrated that 1 displays various beneficial biological activities, including anticancer, antiviral, antibacterial, and anti-inflammatory effects.1−4 Among these bioactivities, anti-HIV activity is a primary interest because of the different anti-HIV mechanisms shown by derivatives of 1 modified at C-3 or C-28. Modification of 1 at C-3 resulted in compounds (e.g., 3) that inhibited HIV-1 maturation,5,6 while modification at C-28 provided compounds (e.g., 4) that inhibited HIV-1 entry by targeting HIV-1 gp-120.7,8 Modification at both C-3 and C-28 resulted in bifunctional derivatives that inhibited HIV-1 entry and maturation.9,10 Additionally, betulonic acid (2), the 3-oxo analogue of 1, exerts potent antitumor activity11−13 (Figure 1).

Figure 1.

Figure 1

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Structures of betulinic acid derivatives.

Furthermore, the low toxicity and good safety index of 1 make it a promising candidate for clinical application as a therapeutic agent.11,14 Unfortunately, the poor solubility of 1 in aqueous solution and many organic solvents is a major obstacle in maximizing its antiviral potency and clinical potential.15 However, some derivatives of 1, including certain ionic derivatives,15 showed improved water solubility and enhanced biological activity compared with unmodified 1.(16−18)

Commonly, nucleoside analogues have good water solubility, and some of them are the backbones of antiretroviral agents used in the treatment of AIDS.19,20 Thus, we postulated that conjugation of 1 with nucleosides might result in better water solubility and greater anti-HIV potency. Here we report the synthesis of novel betulinic/betulonic acid–nucleoside hybrids with a 1,2,3-triazole linking C-2 of the triterpenoid acid with C-3′ or C-4′ of the nucleoside.

The key triterpenoid intermediates 8, 9, and 10 were obtained from the starting material betulin (5) in three steps according to a slightly modified literature method21 (Scheme 1). Ester 7 was obtained from 5 via Jones oxidation followed by esterification using dimethyl carbonate (DMC) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). With DMC and DBU, the esterification reaction was easier and performed under more moderate and safer conditions compared with traditional reagents such as thionyl chloride or diazomethane. Compound 8 was obtained by propargyl α-alkylation of 7 in a KN(SiMe3)2/Et3B system. Then compound 8 was treated with NaBH4 in isopropanol to preferentially give 9; the ratio of α- and β-OH isomers was about 1:1 in methanol but 1:4 in isopropanol. The nuclear Overhauser effect (NOE) between H-3 and H-23 (see Figure S12) indicated an equatorial position (β orientation) for the OH group, and the NOE effects between H-2 and H-24/H-25 (see Figure S12) suggested an axial position (β orientation) for H-2 and thus an equatorial position (α orientation) for the propargyl group. Furthermore, the spin–spin coupling constant between H-3 and H-2 (3JH-2,H-3 = 10.5 Hz, CDCl3) in the 1H NMR spectrum of 10 (see Figure S14) was consistent with axial positions for H-2 and H-3. Demethylation of the sterically hindered ester group in 9 via halogenolysis with LiI in DMF under reflux condition produced 10, the 2-propargyl derivative of 1. Compounds 8, 9, and 10 were coupled separately with different azides (4′-azido-2′-deoxy-2′-fluoro-β-d-arabinocytidine (AFC), 4′-azido-2′-deoxy-2′-fluoro-β-d-arabinouridine (AFU), and AZT) via click chemistry to produce the target compounds 8a–c, 9a–c, and 10a–c, respectively (Scheme 2).

Scheme 1. Synthesis of 2-Propargyl-Substituted Triterpenoid Derivatives.

Scheme 1

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Reagents and conditions: (a) CrO3, H2SO4, acetone, 0 °C, 6 h, 59%; (b) DMC, DBU, reflux, 24 h, 72%; (c) KN(SiMe3)2, Et3B, propargyl bromide, DME, rt, N2, 6 h, 85%; (d) NaBH4, i-PrOH, rt, 51%; (e) LiI, DMF, reflux, N2, 24 h, 78%.

Scheme 2. Synthesis of Triterpenoid–Nucleoside Hybrids via Click Chemistry.

Scheme 2

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The solubilities of 1 and 8b in methanol were determined with the help of quantitative 1H NMR analysis. The results showed that 8b had better solubility than 1; the calculated solubilities of 1 and 8b in methanol were 4.9 and 52.6 mg/mL (10.7 and 66.3 mmol/L), respectively. Details of the experiment are shown on page S9 in the Supporting Information.

Compounds 8a–c, 9a–c, and 10a–c were evaluated in an anti-HIV (wild-type) replication assay,22 and the in vitro anti-HIV activity results are listed in Table 1. Cytotoxicity was evaluated using the MTT assay.22

Table 1. Anti-HIV Activities of Compounds 8a–10c.

compound IC50 (μM)a CC50 (MT4 cells, μM)b SIc
8a 0.097 ± 0.028 >25.2 >260
8b –d –d –
8c –d –d –
9a 0.048 ± 0.018 >25.2 >525
9b –d –d –
9c >1.3 >1.3 –
10a 0.0078 ± 0.0020 9.6 ± 0.82 1231
10b 0.020 ± 0.005 23.8 ± 1.79 1190
10c >1.3 >1.3 –
3 0.021 ± 0.007 >6.84 >326
AFC 0.000262 ± 0.000084 0.09957 ± 0.00863 380
AFU 0.219 ± 0.060 >3.48 >16
AZT 0.018 ± 0.006 >0.37 >20.6
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a

IC50 is the 50% HIV inhibitory concentration. The results are presented as mean ± standard deviation (SD) of triplicate experiments.

b

CC50 is the 50% cytotoxic concentration. The results are presented as mean ± SD of triplicate experiments.

c

Selectivity index, defined as SI = CC50/IC50.

d

No selective anti-HIV activity (SI < 5).

As shown in Table 1, none of the three hybrids conjugated with AZT (8c, 9c, 10c) displayed significant anti-HIV activity, one of the three hybrids conjugated with AFU (10b) showed anti-HIV activity, and all three hybrids conjugated with AFC (8a, 9a, 10a) exerted potent anti-HIV activity. Compared with the positive controls AZT and 3, compounds 10a and 10b showed greater and comparable potency, respectively, as well as lower cytotoxicity. Although compound 10b (the hybrid of 1 and AFU) was less potent than 10a (the hybrid of 1 and AFC), it was also less cytotoxic, resulting in similar selectivity index (SI) values (10a, 1231; 10b, 1190). These values were higher than the SI of 380 for AFC, the most potent compound, which exhibits nanomolar anti-HIV activity. Moreover, the three triterpenoid–AFC hybrids had the following order of potency: 10a (3β-OH, 17-COOH) > 9a (3β-OH, 17-COOMe) > 8a (3-oxo, 17-COOMe). Also, comparison of the structural differences between 9a–c and 10a–c shows that the betulinic acid scaffold with a 17-COOH is preferred over its methyl ester analogues.

In summary, we synthesized a novel class of betulinic/betulonic acid–nucleoside hybrids. From this class, we identified two analogues, 10a and 10b, with highly potent anti-HIV activity. Although the hybrid compounds were less potent than the nucleoside (AFC) itself, they were also less cytotoxic and perhaps would be safer therapeutic agents. Mechanism studies, further optimization of activity, and determination of efficacy in animal models will be reported in the future.

Acknowledgments

This work was supported by the Science and Technology Research Project of Henan Province (192102310407), the Basal Research Fund of Henan Academy of Sciences (200602007), and the Medical Science and Technology Research Project of Henan Province (LHGJ20190829). Grant support from the National Institute of Allergy and Infectious Diseases, U.S. National Institutes of Health (AI033066 to K.-H.L.) is also acknowledged.

Glossary

Abbreviations

AFC

4′-azido-2′-deoxy-2′-fluoro-β-d-arabinocytidine

AFU

4′-azido-2′-deoxy-2′-fluoro-β-d-arabinouridine

CC50

50% cytotoxic concentration

DBU

1,8-diazabicyclo[5.4.0]undec-7-ene

DMC

dimethyl carbonate

IC50

50% HIV inhibitory concentration

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00414.

  • Biological assays, synthetic methods, solubility determination, chemical characterization of final products, 1H and 13C NMR spectra, and HRMS spectra (PDF)

The authors declare no competing financial interest.

Supplementary Material

ml0c00414_si_001.pdf (4.3MB, pdf)

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Supplementary Materials

ml0c00414_si_001.pdf (4.3MB, pdf)

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