Pyranonaphthoquinones and Naphthoquinones from the Stem Bark of Ventilago harmandiana and Their Anti-HIV-1 Activity

Seven previously undescribed compounds, including five pyranonaphthoquinones (ventilanones L–P) and two naphthoquinones (ventilanones Q and R), along with 15 known compounds were isolated from the stem bark of Ventilago harmandiana (Rhamnaceae). The structures were established by extensive analysis of their spectroscopic data. The absolute configuration of ventilanone L was established from single crystal X-ray crystallographic analysis using Cu Kα radiation and from its electronic circular dichroism data. Anti-HIV-1 activity using a syncytium inhibition assay and the cytotoxic activities of some isolated compounds were evaluated. Compounds 12, 13, 15, and 16 showed activity against syncytium formation with half maximal effective concentration (EC50) values ranging from 9.9 to 47 μM (selectivity index (SI) 2.4–4.5).

T he Ventilago genus belongs to the Rhamnaceae family and is found in tropical and subtropical areas. A number of plants belonging to the Ventilago genus play a role in traditional medicines. For example, Ventilago africana has been used for treatment of dysmenorrhea and as a febrifuge. 1 The root bark of Ventilago madraspatana has been used as a carminative, stomachic, tonic, and stimulant. A mixture of the powdered stem bark of Ventilago madraspatana and sesame seed oil has been applied externally to treat skin diseases and itching. 2 In Taiwan, the stems of Ventilago leiocarpa have been used as a folk medicine to cure rheumatism, hepatitis, and neuralgia. 3 The leaves of Ventilago denticulata are often used as tea products. In Thai folk medicine, the stems of V. denticulata have been used to cure diuresis and arthritis, as well as to reduce cholesterol and sugar contents in blood. 4 Ventilago harmandiana Pierre, called in Thai Khon Tee Dum, is a climber and is endemic to Thailand. V. harmandiana can be found in the limestone mountain areas located in the southern parts of Thailand. In Thai traditional medicine, the water decoction of the heartwood and stem bark of V. harmandiana has been used to treat diabetes, wounds, and chronic inflammation. Previous ethnopharmacological investigation of the MeOH extracts of the heartwood, stem bark, and stemwood of V. harmandiana revealed that they exhibited anti-inflammatory effects in both acute and chronic inflammatory assays. 5 Recently, our research group reported the isolation of ventilanones A−K from the heartwood of V. harmandiana. 6 In our continuing efforts to search for biologically active substances which exhibit cytotoxicity, anti-HIV-1 activity, and anti-inflammatory activity, the MeOH extract of the stem bark of V. harmandiana was investigated and led to the isolation of seven previously undescribed compounds including five pyranonaphthoquinones, ventilanones L−P (1−5), and two naphthoquinones, ventilanones Q and R (6 and 7), together with 15 known compounds (Supporting Information). The structures of these compounds were elucidated using the information from spectroscopic data and by comparison with those of the related analogues previously reported in the literature. The absolute configuration of ventilanone L (1) was established by a combination of single crystal X-ray diffraction analysis using Cu Kα radiation and its electronic circular dichroism (ECD) data. The structures and absolute configuration of ventilanones M− P (2−5) and ventilanone R (7) were established by comparison of their spectroscopic data and ECD patterns with those of ventilanone L (1) and related known compounds. The biological activities of some of the isolated compounds were evaluated in a cytotoxicity assay against a panel of cultured mammalian cancer cell lines, and anti-HIV-1 activity was evaluated using a syncytium inhibition assay. Most of the compounds screened for their cytotoxic activity were found inactive (half maximal inhibitory concentration (IC 50 ) > 20 μM). In the anti-HIV-1 activity, some of the tested compounds exhibited moderate to good inhibitory activity against syncytium formation with half maximal effective concentration (EC 50 ) values in the range 9.9−47 μM (selectivity index (SI) 2.4−4.5). A structure−activity relationship of the pyranonaphthoquinones toward their anti-HIV-1 activity against syncytium formation is also discussed in this article.
Compound 3 was obtained as pale yellow rods with mp 116−117°C (crystallized from CH 2 Cl 2 −hexane). Its molecular formula, C 18 H 22 O 6 , was determined by HRESIMS, which showed a sodium adduct ion peak at m/z 357.1309 [M + Na] + . A comparison of its 1 H and 13 C NMR experimental data (Tables 1 and 2, respectively) with those of compound 2 suggested that these compounds have related structures with the only difference being the resonance of a phenol proton at δ H 6.75 (s) at C-7 was replaced by the resonance of methoxy protons at δ H 3.95 (s, 3H). This assumption was supported by the HMBC correlation between H 3 -7 (δ H 3.95) and C-7 (δ C 157.8). On the basis of the NOESY correlations, compound 3 shares the same relative configurations as those of 1 and 2. The absolute configuration of 3 was confirmed by comparison of its experimental ECD data with those of 1 and 2 ( Figure 3). From the aforementioned data, compound 3 was identified as (1R,3S,4aR,10aS)-7,8-dimethoxy-9-hydroxy-1,3,6-trimethyl-3,4,4a,10a-tetrahydro-1H-naphtho[2,3-c]pyran-5,10-dione and was named ventilanone N.  (Tables 1  and 2, respectively) closely resembled those of ventilanone A (8). 6 The key difference was the shift of a methoxy resonance at δ H 4.03 (OCH 3 -8) in compound 8 to a new methoxy group at δ H 3.90 (OCH 3 -7)/δ C 60.0 (OCH 3 -7) in compound 4. The HMBC data shown in Figure 1 supported the assignment of the location of the methoxy group at C-7 [δ H 3.90 (H 3 -7) to δ C 149.7 (C-7)]. The complete assignment of each 1 H and 13 C signal (see Tables 1 and 2, respectively) was deduced by 2D NMR data (HMQC and HMBC). The NOESY correlations between H-1 and H-3 as well as between H 3 -1 and H 3 -3 ( Figure 2) suggested the cis-relationship of CH 3 -1 and CH 3 -3. Therefore, the relative configuration of compound 4 was concluded to be as shown. The ECD spectrum of compound 4 ( Figure 5) was similar to that of compound 8, whose absolute configuration was previously established by means of X-ray crystallographic analysis of its p-bromobenzenesulfonate derivative, with the ECD data suggesting the (1R,3S) absolute configuration. 7 On the basis of the spectroscopic data of 4 above and the comparison of its spectroscopic data with those of 8, compound 4 was assigned as (1R,3S)-7,9-dimethoxy-8-hydroxy-1,3,6-trimethyl-3,4-dihydro-1H-naphtho[2,3-c]pyran-5,10-dione and was named ventilanone O.
The 4a,10a-dihydropyranonaphthoquinones are a unique class of compounds and are commonly found in fungi and related microorganisms. 25−27 Some of the 4a,10a-dihydropyranonaphthoquinones possessed potent biological activities, e.g., cytotoxicity and anti-HIV-1 and antibacterial activities. 28−31 Dihydrofusarubin, a 4a,10a-dihydropyranonaphthoquinone derivative, is a secondary metabolite obtained from various species of Fusarium and exhibited antifungal activity. 20 From our exhaustive search (SciFinder Scholar database), this work is the first report on the isolation of 4a,10adihydropyranonaphthoquinone derivatives from plants in the Ventilago genus.
Compound 6 was obtained as orange rods with mp 146− 147°C (crystallized from CH 2 Cl 2 −hexane). Its molecular formula, C 15 H 14 O 6 , was established from HRESIMS data which displayed a sodium adduct ion peak at m/z 313.0691 [M + Na] + . 1 H and 13 C NMR spectroscopic data of 6 ( Table  3) indicated the presence of an aromatic proton at δ H 7.45 (s, H-5), two methyl groups at δ H 2.30 (s, CH 3 -6) and 2.56 (s, CH 3 -10), and two methoxy groups at δ H 4.10 (s, OCH 3 -3) and 4.13 (s, OCH 3 -2). According to the 1 H− 13 C correlations in the   (Figure 1). Complete assignments of 1 H and 13 C NMR data of 6 as shown in Table 3 were deduced by HMBC data ( Figure  1). Additionally, the NOESY experiment ( Figure 2) that showed correlations of H-5/H 3 -6 and H 3 -6/H 3 -10 led to the assignment of the location of a methyl group at C-6 and an acetyl group at C-7. On the basis of the aforementioned data and by comparison of the spectroscopic data of 6 with those of the previously reported compounds, compound 6 was identified as 7-acetyl-2,3-dimethoxy-8-hydroxy-6-methylnaphthalene-1,4-dione and was named ventilanone O.
Compound 7 was obtained as pale yellow needles with mp 139−140°C (crystallized from CH 2 Cl 2 −hexane). Its molecular formula was established as C 16 H 18 O 6 from HRESIMS data which displayed a sodium adduct peak at m/z 329.1002 [M + Na] + ]. The NMR spectroscopic data of 7 are similar to those of 6 ( Table 3). The differences included the disappearance of a carbonyl resonance at δ C 180.9 (C-4) in 6 but the existence of an additional methyl group at δ H 1.66 (s, CH 3 -4)/δ C 32.6 (CH 3 -4) found in 7. This methyl group was located at C-4 according to its HMBC correlations with an aromatic proton at  Journal of Natural Products pubs.acs.org/jnp Article δ H 7.05 (H-5) and an oxygenated nonhydroxygenated carbon at δ C 71.7 (C-4). This assignment was also confirmed by the NOESY correlations of H 3 -4 to the nearby aromatic proton, H-5 ( Figure 2). The lack of an optical rotation and the flat ECD spectrum indicated that 7 was a racemic mixture. Subsequently, the enantiomers of 7 were resolved approximately in a ratio of 1:1 by HPLC using a Chiralpak OD-H column. The determination of absolute configurations was performed by comparison of specific rotation and ECD data with those of the previously reported derivatives (Supporting Information). 34−36 Thus, compound 7 was determined to be 7acetyl-4,5-dihydroxy-2,3-dimethoxy-4,7-dimethylnaphthalen-1-(1H)-one and was named ventilanone R. Some isolated pyranonaphthoquinone and naphthoquinone derivatives have been evaluated for their cytotoxic effects and anti-HIV-1 activities. In this study, the anti-HIV-1 activity was studied using a syncytium inhibitory assay (Table 4). In the present work, in order to focus on the active compounds, the EC 50 maximum cutoff was set at 50 μM. Compounds 12, 13, and 15 along with a dimeric 16 showed moderate to good inhibitory activities in the syncytium reduction assay with EC 50 values in the range 9.9−47 μM (SI 2.4−4.5) while 4a,10adihydropyranonaphthoquinone derivatives are inactive (EC 50 > 50 μM). Compound 15 was the most active in the syncytium reduction assay with an EC 50 value of 9.9 μM (SI 4.5). Almost all the compounds screened for cytotoxicity against a panel of cultured mammalian cancer cell lines did not show cytotoxicity (IC 50 > 20 μM) (see Table S1).
Based on the preliminary screening results on anti-HIV-1 activities of pyranonaphthoquinones, the structure−activity relationship can be discussed. The presence of the unsaturation bond between C-4a and C-10a in the structure of a pyranonaphthoquinone is important for the anti-HIV-1 activity in the syncytium reduction assay, while the position and number of oxygenated groups on the naphthoquinone moiety at C-7, C-8, and C-9 had no effect. Additionally, the hydroxy group at C-1 of the pyran ring found in 15 had a significant effect and enhanced inhibitory activity against syncytium formation. ■ EXPERIMENTAL SECTION General Experimental Procedures. Melting points (uncorrected) were measured on an Electrothermal 9100 melting point apparatus. Optical rotations were measured on a JASCO DIP-370 digital polarimeter by using a 50 mm microcell (1 mL). The UV spectra were recorded on a JASCO V-530 spectrometer. The ECD spectra were measured on a JASCO J-815 spectropolarimeter by using 10 and 50 mm microcells. The IR spectra were recorded on a PerkinElmer System 2000 FTIR spectrophotometer. The NMR spectra were recorded on a Bruker DPX 300, Bruker Ascend 400, or Jeol NMR 400 MHz spectrometer with tetramethylsilane (TMS) as an internal reference. HRESIMS spectra were measured on a Bruker micro TOF spectrometer. EIMS spectra were measured on a Thermo Finnigan Polaris Q mass spectrometer at 70 eV (probe Extraction and Isolation. The air-dried stem bark of V. harmandiana (9.7 kg) was ground and macerated with MeOH (33 L × 5 days × 3 times) at room temperature, followed by filtration. The MeOH was evaporated under reduced pressure and the residual water was removed by freeze-drying to afford the MeOH extract (770