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. Author manuscript; available in PMC: 2012 Jun 3.
Published in final edited form as: Org Lett. 2011 May 11;13(11):2904–2907. doi: 10.1021/ol200889s

Stelleralides A-C, Novel Potent Anti-HIV Daphnane-Type Diterpenoids from Stellera chamaejasme L

Yoshihisa Asada a,*, Aya Sukemori b, Takashi Watanabe c, Kuber J Malla d, Takafumi Yoshikawa b, Wei Li e,*, Kazuo Koike e, Chin-Ho Chen f, Toshiyuki Akiyama g, Keduo Qian g, Kyoko Nakagawa-Goto g, Susan L Morris-Natschke g, Kuo-Hsiung Lee g,h,*
PMCID: PMC3109985  NIHMSID: NIHMS296827  PMID: 21561135

Abstract

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Three novel 1-alkyldaphnane-type diterpenes, stelleralides A–C (4–6), and five known compounds were isolated from the roots of Stellera chamaejasme L. The structures of 4–6 were elucidated by extensive spectroscopic analyses. Several isolated compounds showed potent anti-HIV activity. Compound 4 showed extremely potent anti-HIV activity (EC90 0.40 nM) with the lowest cytotoxicity (IC50 4.3 μM), and appears to be a promising compound for development into anti-AIDS clinical trial candidates.

Stellera chamaejasme L. (Thymelaeaceae) is a toxic perennial herb widespread in northern and southwestern China and Nepal. Its roots have been used in traditional Chinese medicine (TCM) as emulgent and dermatological agents. Previous studies on the chemical constituents in the roots of this plant have identified diterpenoids,1,2 biflavonoids,3,4,5 and lignans6 with antitumor, antimalarial, and antibacterial activities. Highly functionalized daphnane diterpenes from Trigonostemon thyrsoideum also showed inhibitory activity against HIV-1,7 while SJ23B, a jatrophane diterpene from Euphorbia hyberna, displayed strong activity in the nanomolar range against HIV in vitro.8 In addition to the potent antiviral activity of diterpenes, their mechanism of action is also of high interest. For example, prostratin, a tigliane diterpene, interferes with HIV infection by two mechanisms: decreasing the expression of HIV receptors on the surface of healthy cells, and activating the HIV-1 expression of latent HIV hidden in their reservoirs, which is a major stumbling block to the eradication of HIV. The AIDS Research Alliance hoped to complete the final stages of preclinical study on prostratin in 2010 prior to clinical trials.9

During our ongoing chemical studies on Nepalese medicinal plants, we also investigated the chemical constituents of the roots of S. chamaejasme, and isolated eight daphnane-type diterpenes (1–8), including three new compounds, stelleralides A (4), B (5), and C (6).10,11,12 All compounds were screened for anti-HIV activity in MT4 cells.13,14

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A methanol extract of the roots of S. chamaejasme was partitioned between EtOAc and H2O. The EtOAc-soluble fraction was subjected to chromatography on an ODS column, eluting with MeOH and H2O mixtures in different ratios. Separation of the MeOH-eluted fractions using a combination of silica gel column chromatography (CC), ODS CC, and preparative HPLC afforded eight diterpenes, three new (4–6) and five known (1–3, 7 and 8) compounds. The known compounds were identified as gnidimacrin (1),15 pimelea factor P2 (2),1,16 wikstroelide F (3),17,18 simplexin (7),1,19,20 and huratoxin (8),16,21,22 by detailed MS and NMR spectroscopic analysis and comparison with literature data. Wikstroelide F (3) was isolated for the first time from the genus Stellera..

Stelleralide A (4)23 was isolated as a white amorphous powder, [α]D 22 +8.1 (c 0.13, MeOH). Its molecular formula C39H52O12 was determined from the positive-ion HRFABMS data (m/z 735.3328, [M+Na]+). The 1H and 13C NMR spectra of 4 and 1 were quite similar, suggesting that 4 is a 1-alkyldaphnane derivative. Detailed comparison of the NMR data revealed that the resonances for two benzoyl moieties in 1 were replaced by those for one benzoyl moiety and one acetyl moiety in 4. In the HMBC spectrum of 4, correlations were clearly observed between δH 4.92 (H-3) and δC 168.3 (Bz-CO) and between δH 4.11, 4.88 (H2-18) and δC 170.9 (Ac-CO), indicating that the benzoyl and acetyl moieties are attached at C-3 and C-18, respectively. Thus, the structure of 4 was determined as shown in the Chart.

Stelleralide B (5)24 was also isolated as a white amorphous powder, [α]D 23 -17.0 (c 0.12, MeOH), with a molecular formula of C44H54O11 determined from positive-ion HRFABMS (m/z 781.3552, [M+Na]+). The 1H and 13C NMR spectra of 5 resembled those of 1–4, suggesting that 5 is also a 1-alkyldaphnane derivative. A detailed comparison of its 13C NMR spectrum with that of 1 showed that the C-2′ resonance in 1 was shifted upfield to δC 33.4 in 5, indicating that the C-2′ hydroxy group in 1 is replaced by hydrogen in 5. In confirmation, the molecular formula of 5 (C44H54O11) is one oxygen atom lower compared with that of 1 (C44H54O12). Thus, the structure of 5 was determined as shown in the Chart.

Stelleralide C (6)25 was isolated as a white amorphous powder, [α]D 23 +14.0 (c 0.12, MeOH). Its molecular formula C37H46O11 was determined from the positive-ion HRFABMS data (m/z 689.2932, [M+Na]+). The 1H and 13C NMR spectra of 6 showed resonances characteristic of the alkyl part of 1-alkyldaphnane derivatives, including a quaternary carbon resonance at δC 120.7 (C-1′). However, large differences were observed between the A ring backbone resonances of 6 and those of 1–5. Namely, in the 13C NMR spectrum, 6 showed resonance for an acetal carbon at δC 112.1 and a lactone carbonyl carbon at δC 175.7. In addition, a singlet methyl at δH 1.96 assignable to H-19 was observed in the 1H NMR spectrum of 6. The acetal carbon resonance at δC 112.1 was assigned to C-2, because HMBC correlations (Figure 1) with protons at δH 3.15 (H-1), 3.49 (H-10) and 1.96 (H-19) were clearly observed. Similarly, the lactone carbonyl resonance at δC 175.7 was assigned to C-3 based on the HMBC correlations (Figure 1) with protons at δH 5.42 (H-5) and 3.49 (H-10). In addition, the presence of a β-oriented 2,4-epoxide moiety was deduced from spectroscopic evidence, in which resonances assignable to H-2 and H-4 were not observed in 1H NMR spectrum and the resonance for H-5α (δH 5.42) was shifted to lower field as compared with those of 1–5, due to the anisotropic effect of the adjacent carbonyl group attached to C-4. In the ROESY spectrum (Figure 2) of 6, the presence of correlations of δH 1.96 (H3-19) with δH 1.10 (H3-10′) and 3.15 (H-1), and the lack of a correlation between δH 1.96 (H3-19) and 1.42 (H-9′), indicated a β-orientation of 19-methyl moiety. Furthermore, the correlations between H3-10′/H-1 and H-10/H-9′ in the ROESY spectrum indicated a 9′S configuration in 6.

Figure 1.

Figure 1

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Key HMBC and 1H-1H correlations of 6.

Figure 2.

Figure 2

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Key correlations in ROESY spectrum of 6.

The isolated compounds (1–8) were evaluated for anti-HIV activity against NL4-3 in MT4 cells, as well as cytotoxicity. The data (EC90 and IC50 values, respectively) are listed in Table 1. New compound 4 and the known compound 1 were the most potent compounds, with anti-HIV EC90 values of less than 1 nM (0.40 and 0.41 nM, respectively), where EC90 was the compound concentration that inhibited HIV-1 replication by 90%. Structurally, the two compounds differed only in the C-18 substituent (OAc in 4 and OBz in 1). Compound 4 (IC50 4.3 μM) was less cytotoxic than 1 (IC50 2.8 μM). Compounds 2, 3, and 5 showed lower, but significant anti-HIV potency with EC90 values between 1.4 and 1.7 nM. All three compounds are unsubstituted at C-2′, while the more potent 1 and 4 contain an OH group at this position. Unlike in the other compounds, the long C-1′ alkyl chain of 7 and 8 does not form a macrocyclic ring at C-1, and these two compounds were less potent. The least potent compound was 6, which contains a 2,4-epoxide moiety and lactone ring.

Table 1.

Anti-HIV Activity of 1–8.

no. anti-HIV (NL4-3) EC90 (μM) cytotoxicity (MT4) IC50 (μM)
1 0.00041 2.8
2 0.0014 3.2
3 0.0017 7.3
4 0.00040 4.3
5 0.0016 14.5
6 0.091 3.4
7 0.0080 22.5
8 0.0058 9.4
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In conclusion, eight 1-alkyldaphnane type diterpenes, including three new compounds, stelleralides A–C, were isolated from the roots of Stellera chamaejasme. Two compounds, new 4 and known 1, exhibited extremely potent anti-HIV EC90 values of less than 1 nM. The structural difference between these two and several less potent compounds was the presence of a C-2′ OH group. Synthesis of structurally modified analogs will be pursued to establish structure-activity relationship (SAR) correlations and optimize these lead compounds as anti-AIDS clinical trial candidates..

The daphnane diterpenes described in this study share some structural similarity to other diterpenes, such as prostratin (12-deoxyphorbol-13-acetate), DPP (12-deoxyphorbol-13-phenylacetate), and ingenol derivatives. Prostratin has been well documented for its anti-HIV-1 activity.26,27,28 DPP is a tigliane diterpene, similar to prostratin. DPP exhibited more potent anti-HIV-1 activity than prostratin.29 In addition to the tigliane diterpenes, ingenol derivatives were reported to have anti-HIV-1 activity comparable to that of DPP.30,31 The anti-HIV-1 activity of these compounds was, at least in part, due to their ability to activate protein kinase C and downregulate HIV-1 receptors, CD4 and chemokine receptors.26,28,30 The mechanism of action of the daphnane diterpenes reported here is currently under investigation. However, due to their structural similarity to other phorboids, it is likely that activation protein kinase C and down regulation of HIV-1 cellular receptors might be responsible for their potent anti-HIV-1 activity.

Supplementary Material

1_si_001

Acknowledgments

This investigation was supported by NIH Grant AI-033066 from the National Institute of Allergy and Infectious Diseases (NIAID) awarded to K.H.L. This study was also supported in part by the Department of Health Clinical Trial and Research Center of Excellence (DOH100-TD-B-111-004).

Footnotes

Supporting Information Available: Tables of 1H, 13C, and HMBC NMR spectroscopic data for 4–6. Copies of 1H and 13C spectra for 1–8. Copies of 2D NMR spectra and MS data for 4–6. This material is available free of charge via the Internet at http://pubs.acs.org.

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Associated Data

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Supplementary Materials

1_si_001
NIHMS296827-supplement-1_si_001.pdf (5.7MB, pdf)

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