Hopane-6 α,16 α,22-triol: A New Hopane Triterpenoid from the Lichen Parmotrema sancti-angelii

Abstract

A new compound, namely hopane-6α,16α,22-triol (1), along with 8 known compounds 2 to 9, leucotylin (2), 16β-acetoxyhopane-6α,22-diol (3), 6α-acetoxyhopane-16β,22-diol (4), zeorin (5), 6α-acetoxyhopane-22-ol (6), ergosterol peroxide (7), brassicasterol (8), and atranorin (9), was isolated from the lichen Parmotrema sancti-angelii. The structures of all the isolated compounds 1 to 9 were fully characterized using spectroscopic data, as well as comparison with the previous literature data. Moreover, compounds 2 and 4 were assessed for their in vitro cytotoxicity against 3 human cancer cell lines.

Lichens are symbiotic composites of fungi and algae and/or cyanobacterium which produce various secondary metabolites of interest. ^{1 -3} Most of them are polyphenolic compounds and have many biological activities, including antibiotic, antibacterial, antiviral, anti-inflammatory, analgesic, antipyretic, and antiproliferative activities. ¹ Vietnamese lichens have been studied remarkably for over 3 years with the reports of several new and novel metabolites. ^{4 -9} Parmotrema is a genus of lichen belonging to the family Parmeliaceae with approximately 350 species of foliose lichens and a high level of diversity in the tropical areas of the world. Parmotrema sancti-angelii (Lynge) Hale is a lichen, which has not been fully investigated on biological activity and chemical composition. ¹ In the previous study, we reported 3 new racemic procyanidin analogs from P. sancti-angelii. ⁶ As part of our continuation focusing on the isolation of new chemical constituents from P. sancti-angelii, we report herein the isolation and structural characterization of a new hopane triterpenoid (1) and 8 known compounds 2 to 9, leucotylin (2), ⁹ 16β-acetoxyhopane-6α,22-diol (3), 6α-acetoxyhopane-16β,22-diol (4), ⁸ zeorin (5), ¹⁰ 6α-acetoxyhopane-22-ol (6), ¹¹ ergosterol peroxide (7), ¹² brassicasterol (8), ¹³ and atranorin (9). ¹⁴ The structures of all isolated compounds 1 to 9 (Figure 1) were identified by comparison of their spectroscopic data with those reported in the literature. So far, few hopane-type triterpenes were reported in Parmotrema genus. ¹⁵ Only zeorin (5) was formerly described in Parmotrema austrocetratum. ¹⁶ To the best of our knowledge, compounds 1 to 4 and 6 were first reported in Parmotrema genus. Moreover, the cytotoxic activities of 2 isolated compounds 2 and 4 against liver hepatocellular carcinoma (HepG2), human lung cancer (NCI-H460), and human breast cancer (MCF-7) cell lines were evaluated.

Figure 1

Chemical structures of 1 to 9 from the lichen Parmotrema sancti-angelii.

Compound 1 was isolated as a white amorphous powder, gave the molecular formula C₃₀H₅₂O₃, as deduced by the sodiated molecular ion peak at m/z 483.3829 (calcd. for C₃₀H₅₂O₃ + Na, 483.3814) in high-resolution electrospray ionization (HR-ESI) mass spectrum. Interpretation of the carbon-13 nuclear magnetic resonance (¹³C NMR) and heteronuclear single quantum correlation (HSQC) data of 1 revealed 30 carbon signals, including 8 methyls, 9 methylenes, 7 methines, and 6 quaternary carbons (Table 1). The ¹H NMR spectrum showed 8 singlet methyls (δ_H 0.90, 1.01, 1.04, 1.11, 1.13, 1.15, 1.37, and 1.38) and 2 oxymethines [δ_H 3.89 (ddd, J = 10.7, 10.5, 4.0 Hz, H-6) and 4.64 (m, H-16)]. In the HSQC spectrum, 2 oxymethine proton signals assigned to H-6 (δ_H 3.89, ddd, J = 10.7, 10.5, 4.0 Hz) and H-16 (δ_H 4.64, m) were associated with the corresponding oxymethine carbon signals due to C-6 (δ_C 69.5) and C-16 (δ_C 69.8), respectively. The heteronuclear multiple bond correlation (HMBC) correlations of H-16 to C-14, C-15, and C-18; H-17 to C-16, C-18, C-21, and C-28; and H-21 to C-17, C-18, C-20, and C-22, in accordance with the COSY spin system through H-15/H-16/H-17/H-21 supported the hydroxy group at C-16 (Figure 2a). In addition, hydroxy substitution at C-6 (δ_C 69.5) was also confirmed by HMBC cross-peaks.

Figure 2

(a) Selected HMBC (single-headed arrow curves), COSY (bold lines), and (b) NOESY (double-headed arrow curves) correlations of 1.

Table 1

¹H and ¹³C NMR Data of 1 in CD₃OD.

No.	δH (ppm), J (Hz)	δC (ppm)
1	α 0.85 (m); β 1.62 (m)	41.6
2	α 1.32 (m); β 1.62 (m)	19.6
3	α 1.17 (m); β 1.31 (m)	45.2
4		34.7
5	0.86 (d, 10.7)	62.0
6	3.89 (ddd, 10.7, 10.5, 4.0)	69.5
7	α 1.50 (m); β 1.63 (m)	45.9
8		42.7
9	1.46 (m)	51.2
10		40.4
11	α 1.63 (m); β 1.33 (m)	22.2
12	α 1.61 (m); β 1.34 (m)	24.9
13	1.34 (m)	50.1
14		44.1
15	α 1.74 (m); β 1.58 (m)	41.2
16	4.64 (m)	69.8
17	1.60 (m)	56.9
18		44.5
19	α 1.46 (m); β 1.01 (m)	43.4
20	α 1.74 (m); β 1.62 (m)	27.3
21	2.17 (1H, m)	51.4
22		72.9
23	1.15 (3H, s)	37.3
24	1.02 (3H, s)	22.5
25	0.90 (3H, s)	17.8
26	1.04 (3H, s)	18.9
27	1.37 (3H, s)	17.6
28	1.11 (3H, s)	18.4
29	1.14 (3H, s)	28.7
30	1.38 (3H, s)	31.4

The full analysis of both 1D (¹H and ¹³C) and 2D (COSY, HSQC, and HMBC) NMR spectra showed that compound 1 possesses the same planar structure to those of leucotylin (2) ⁹ but with a slight difference at C-16. The β orientation of H-16 of 1 was evident from its chemical shift value (δ_H 4.64, m), which was different from 2 (δ_H 4.06, ddd, J = 11.5, 9.5, 7.5 Hz, H-16). The relative configuration of 1 was also defined by analysis of its nuclear overhauser effect spectroscopy (NOESY) spectrum (Figure 2b). The NOESY correlation networks of H-16/H-15β/H-17 were cofacial and assigned to the β-orientation. Thus, the structure of 1 was established and named as hopane-6α,16α,22-triol.

Two isolated compounds 2 and 4 were evaluated for their cytotoxicity against HepG2, NCI-H460, and MCF-7 cell lines. Compound 4 showed a moderate activity against HepG2, NCI-H460, and MCF-7 cell lines, with IC₅₀ values of 52.67 ± 2.82, 77.86 ± 5.61, and 47.08 ± 2.64 µg/mL, respectively, while compound 2 displayed no cytotoxicity (IC₅₀ >100 µg/mL) against these cell lines. Camptothecin was used as the reference substance, which exhibited activity against HepG2, NCI-H460, and MCF-7 cell lines (IC₅₀ values of 0.079 ± 0.023, 0.0065 ± 0.003, and 0.036 ± 0.013, respectively).

In conclusion, a new hopane triterpenoid, hopane-6α,16α,22-triol (1), together with 8 known compounds 2 to 9, was isolated from the lichen P. sancti-angelii. Their structures were determined by means of extensive spectroscopic analyses. In addition, 6α-acetoxyhopane-16β,22-diol (4) revealed a moderate activity toward HepG2, NCI-H460, and MCF-7 cell lines.

Experimental

General

NMR, NMR, Bruker Avance III (500 MHz for ¹H NMR and 125 MHz for ¹³C NMR) and Varian Mercury-400 Plus NMR; HR-ESI-MS, HR-ESI-MS Bruker micrOTOF Q-II; IR, Bruker Tensor-27 infrared spectrophotometer; Thin-layer chromatography was carried out on precoated silica gel 60 F₂₅₄ or silica gel 60 RP-18 F₂₅₄S (Merck, Germany) and spots were visualized by spraying with 30% H₂SO₄ solution followed by heating. Gravity column chromatography was performed with silica gel 60 (0.040-0.063 mm, Himedia, India).

Lichen Material

P armotrema sancti-angelii was collected from the bark of tea trees Camellia sinensis at Bao Loc city, Lam Dong province, Vietnam (07/2013-09/2013) and the scientific name was identified by Dr Harrie J.M. Sipman, Botanic Garden and Botany Museum Berlin-Dahlem, Freie University, Berlin, Germany. A voucher specimen (No US-B021) was deposited in the herbarium of the Department of Organic Chemistry, University of Science, Ho Chi Minh City, Vietnam.

Extraction and Isolation

The clean, air-dried, and ground material (950 g) was macerated with acetone at room temperature (3× 10 L) and the filtrated solution was evaporated under reduced pressure to afford acetone crude extract (110.4 g). This crude extract was applied on silica gel (Merck Art 7730) column chromatography (CC), eluted with the mobile phase n-hexane-ethyl acetate (9:1) to afford a major fraction (HE, 6.1 g). This fraction was applied to silica gel CC (n-hexane-ethyl acetate [9:1]) to give 3 fractions (HE1-HE3). Fraction HE1 (3.3 g) was fractionated by silica gel CC to give 4 subfractions (HE1.1-HE1.4) using chloroform as the eluent. Subfraction HE1.3 was applied to preparative thin-layer chromatography (PTLC) using chloroform–methanol (100:4) to afford 1 (1.2 mg), 3 (1.1 mg), and 4 (5.2 mg). Subfraction HE1.4 was rechromatographed using n-hexane-ethyl acetate (9:1) to afford 6 (6.8 mg), 8 (3.9 mg), and 9 (495.8 mg). Fraction HE2 was applied to silica gel CC using n-hexane-ethyl acetate-acetic acid (9:1:0.05) to give 5 subfractions (H2.1-H2.5). Subfraction H2.1 was subjected to silica gel CC and eluted with the increasing of polarity mobile phase by mixing hexane, CH₂Cl₂, EtOAc, and MeOH, to yield 5 (99.2 mg) and 7 (3.8 mg). Finally, subfraction H2.5 was applied to PTLC, eluted with the solvent system of n-hexane-ethyl acetate-methanol (7:3:0.4) to give 2 (4.9 mg).

Cytotoxic Assays

Compounds 2 and 4 were subjected to cytotoxic evaluation against HepG2, MCF-7, and NCI-H460 cell lines using camptothecin as the positive control. ¹⁷

Hopane-6α,16α,22-triol (1)

White amorphous powder.

[α]²⁵ _D: +348.0 (c 0.1, MeOH).

IR (KBr): 3385, 2945, 1382, 1159, 1040, 1024, 947 cm⁻¹.

¹H and ¹³C NMR: Table 1

HRESIMS: m/z [M + Na]⁺ calcd for C₃₀H₅₂O₃ + Na: 483.3814; found: 483.3829

Supplemental Material

Supporting Information – Supplemental material for Hopane-6α,16α,22-triol: A New Hopane Triterpenoid from the Lichen Parmotrema sancti-angelii

Supplemental material, Supporting Information, for Hopane-6α,16α,22-triol: A New Hopane Triterpenoid from the Lichen Parmotrema sancti-angelii by Jirapast Sichaem, Huu-Hung Nguyen, and Thuc-Huy Duong in Natural Product Communications