Terpenoids and Bibenzyls from Three Argentine Liverworts

Abstract

A new rosane diterpenoid, 3α-hydroxy-5,15-rosadien-11-one (3), was isolated, together with a known rosane diterpenoid, 5,15-rosadiene-3,11-dione (4), and an aromadendrane sesquiterpenoid, ent-cyclocolorenone (5), from the Et₂O extract of an unidentified Argentine liverwort Anastrophyllum species. Moreover, four known sesquiterpene lactones 6–9 and two known bibenzyls 10, 11 were isolated from the Et₂O extracts of Argentine Frullania brasiliensis and Radula voluta, respectively. The structures of compounds 3–11 were determined by the use of NMR techniques.

1. Introduction

Due to their small morphology, liverworts (Hepaticae) are difficult to classify and identify. However, they are a rich source of terpenoids and aromatic compounds, which can be used to evaluate their chemosystematics [1,2]. In our search for new biologically active substances, we continue to study the chemical constituents of liverworts. Many liverworts are endemic to the southern hemisphere, including Oceania and South America. Recently, we have reported the structures of new sesqui- and diterpenoids from New Zealand liverworts [3]. We also reported the isolation of new sesquiterpenoids 1 and 2 from an unidentified Gackstroemia species from New Zealand [4]. During the course of our investigation of the chemical constituents of three Argentine Jungermanniales species (unidentified Anastrophyllum species, Radula voluta and Frullania brasiliensis), we isolated a new rosane diterpenoid 3 and six previously known compounds: Rosane 4, aromadendrane 5, two eudesmanes 6 and 7, two eremophilanes 8 and 9, and two bibenzyls 10 and 11 (Figure 1) and characterized their chemical structures. The chemosystematics of these three species from Argentina are discussed.

Figure 1. Terpenoids and bibenzyls isolated from Argentine liverworts.

Figure 1. Terpenoids and bibenzyls isolated from Argentine liverworts.

2. Results and Discussion

The new rosane diterpenoid 3 was isolated from the ether extract of an unidentified Anastrophyllum species by chromatographic separation (silica gel and Sephadex LH-20), together with ent-cyclocolorenone (5) [5] and 5,15-rosadiene-3,11-dione (4) [6], whose spectral and physical data were identical with those of authentic samples.

The mass spectrum of 3 showed m/z 302 [M]⁺ and its molecular formula, C₂₀H₃₀O₂ (calcd. 302.2245), was confirmed by HR-EIMS. The IR spectrum demonstrated the presence of hydroxy and carbonyl groups. The ¹H-NMR spectrum (Table 1) of 3 showed the signals of terminal vinyl protons (δ 4.94 dd, 4.96 dd, 5.83 dd), an olefinic proton (δ 5.56 d), a proton (δ 3.23 dd) on a carbon bearing a hydroxy group and four tertiary methyls. The ¹³C-NMR spectrum (Table 2) exhibited 20 carbons, and its DEPT spectrum indicated the presence of trisubstituted olefinic carbons (δ 117.0 d, 145.5 s), terminal vinyl carbons (δ 110.2 t, 147.9 d), a carbonyl carbon (δ 214.7) and a methine (δ 77.2) with a hydroxy group, together with four methyls, four methylenes, two methines and three quaternary carbons. Since the ¹³C-NMR (Table 2) data was similar to those of the 5,15-rosadiene-3,11-dione (4) [6], compound 3 was suggested to be a rosane diterpenoid.

Table 1. ¹H-NMR data of 3 (600 MHz, CDCl₃) ^a.

H		H
1	1.01 m	14	1.36 dd (10.2, 2.5) α
	1.97 m		1.76 m β
2	1.69 m	15	5.83 dd (17.3, 10.7)
	1.78 m
3	3.23 dd (11.5, 4.7)b	16	4.94 dd (10.7, 0.8)
			4.96 dd (17.3, 0.8)
6	5.56 d (6.3)	17	0.97 s
7	1.87 m	18	0.99 s
	1.98 m
8	1.77 m	19	1.15 s
10	2.72 m	20	0.98 s
12	1.95 dd (12.6, 2.5) α
	2.73 d (12.6) β

^a Chemical shift values are in δ (ppm); ^b Coupling constants are in Hz.

Table 1. ¹H-NMR data of 3 (600 MHz, CDCl₃) ^a.

H		H
1	1.01 m	14	1.36 dd (10.2, 2.5) α
	1.97 m		1.76 m β
2	1.69 m	15	5.83 dd (17.3, 10.7)
	1.78 m
3	3.23 dd (11.5, 4.7)b	16	4.94 dd (10.7, 0.8)
			4.96 dd (17.3, 0.8)
6	5.56 d (6.3)	17	0.97 s
7	1.87 m	18	0.99 s
	1.98 m
8	1.77 m	19	1.15 s
10	2.72 m	20	0.98 s
12	1.95 dd (12.6, 2.5) α
	2.73 d (12.6) β

^a Chemical shift values are in δ (ppm); ^b Coupling constants are in Hz.

Table 2. ¹³C-NMR of 3 and 4 (100 MHz, CDCl₃) ^a.

C	3	4 [6]	C	3	4 [6]
1	25.3	25.2	11	214.7	214.4
2	30.3	37.8	12	48.4	48.5
3	77.2	214.6	13	41.6	41.6
4	42.1	51.0	14	38.4	38.4
5	145.5	144.2	15	147.9	147.7
6	117.0	118.2	16	110.2	110.4
7	28.9	29.0	17	23.5	23.5
8	38.3	38.2	18	21.4	22.7
9	48.9	49.4	19	24.4	29.5
10	37.9	38.6	20	12.7	12.0

^a Chemical shift values are in δ (ppm).

Table 2. ¹³C-NMR of 3 and 4 (100 MHz, CDCl₃) ^a.

C	3	4 [6]	C	3	4 [6]
1	25.3	25.2	11	214.7	214.4
2	30.3	37.8	12	48.4	48.5
3	77.2	214.6	13	41.6	41.6
4	42.1	51.0	14	38.4	38.4
5	145.5	144.2	15	147.9	147.7
6	117.0	118.2	16	110.2	110.4
7	28.9	29.0	17	23.5	23.5
8	38.3	38.2	18	21.4	22.7
9	48.9	49.4	19	24.4	29.5
10	37.9	38.6	20	12.7	12.0

^a Chemical shift values are in δ (ppm).

The ¹H-¹H COSY of 3 confirmed three partial segments: (A) -CH(OH)-CH₂-CH₂-CH-, (B) -CH₂-CH-CH₂-CH=C-, and (C) –CH=CH₂. As seen in the HMBC spectrum (Figure 2), the tertiary methyl at Η−17 correlated with the methylene carbon at C-4 in segment B, the terminal vinyl carbon at C-15, the quaternary carbon at C-13 and the isolated methylene carbon at C-12, methylene protons at H-12 of which correlated with the carbonyl carbon at C-11. The other tertiary methyl at H-20 correlated with the methine at C-10 in segment A, the carbonyl carbon, the quaternary carbon at C-9 and the aliphatic methine in segment B. The methyl groups at H-18 and 19 correlated with the methine at C-3 bearing hydroxy group in segment A, an aliphatic quaternary carbon and a trisubstituted olefinic quaternary carbon. On the basis of the above results, the structure of 3 was elucidated to be 3-hydroxy-5,15-rosadien-11-one. The stereochemistry of 3 was clarified by the NOESY spectrum of the m-bromobenzoate derivative 12 of 3. NOE correlations (Figure 3) of 12 were observed between: (i) H-3 and H-1β, H-18; (ii) H-1β and H-20; (iii) H-20 and H-7β, H-12β, H-14β; (iv) H-19 and H-10; (v) H-10 and H-8; (vi) H-8 and H-17. Moreover the CD spectrum of 3 showed a positive Cotton effect (λ_max 297) as the same Cotton effect (λ_max 298) as 4 [6]. Thus, the structure of 3 was shown to be 3α-hydroxy-5,15-rosadien-11-one. However, the absolute configuration of 3 has not yet clarified because the use of only the Cotton effect of the CD spectrum was not able to establish it unequivocally.

Figure 2. ¹H-¹H (bold line) and long range ¹H-¹³C (arrows) correlations of 3.

Figure 2. ¹H-¹H (bold line) and long range ¹H-¹³C (arrows) correlations of 3.

Figure 3. NOE correlations of 12.

Figure 3. NOE correlations of 12.

Frullania brasiliensis contains sesquiterpene lactones [7]. We reexamined the same species for the purpose of potentially isolating new compounds. A chromatographic separation of the ether extract of Frullania brasiliensis resulted in the isolation of two eudesmanes, (+)-frullanolide (6), which causes potent allergenic contact dermatitis [8], and nepalensolide A (7) [9], and two eremophilanes, 5-epi-dilatanolide A (8) [7] and 5-epi-dilatanolide B (9) [7].

Two bibenzyl compounds, 3,5-dihydroxy-2-(3-methyl-2-butenyl)bibenzyl (10) [10,11] and 2-geranyl-3,5-dihydroxybibenzyl (11) [12], were chromatographically isolated from the ether extract of Radula voluta.

Fusicoccane and sphenolobane diterpenoids are common chemical components isolated from Anastrophyllum species, e.g., from A. minutum [13], A. aurztum [14] and A. donnianum [15]. These diterpenoids are very characteristic for Anastrophyllum genus belonging to the Jungermanniaceae. However, neither sphenolobane nor fusicoccane were isolated from the present unidentified Anastrophyllum species. Those isolates included naturally rare rosane diterpenoid and aromadendrane sesquiterpenoid. Therefore, the unidentified Anastrophyllum species may be from a chemically different taxon from the other Anastrophyllum species.

Eudesmane and eremophilane sesquiterpene lactones have been isolated from F. brasiliensis (Frullaniaceae) [7] and are considered the most important chemical markers of the Frullaniaceae [1,2,16]. The present F. brasiliensis also contained the same eudesmanolides and eremophilanolides as those reported.

A number of bibenzyls and prenyl bibenzyls were isolated from European, New Zealand, Ecuador and Japanese Radula species (Radulaceae) [1,2,17]. R. voluta also produced bibenzyls 10 and 11, which are ubiquitous components in the Radula species. The Radula including R. voluta is chemically very isolated from the other liverworts examined so far, since the presence of terpenoids is very rare.

3. Experimental

3.1. General

¹H and ¹³C-NMR: 200, 400 and 600 MHz (¹-NMR) and 100, 150 MHz (¹³C-NMR). Chemical shift values were expressed in δ (ppm) downfield from tetramethylsilane as an internal standard (¹H-NMR) and δ 77.03 (ppm) from CDCl₃ as a standard (¹³C-NMR). TLC: Visualized under UV (254 nm) light and by spraying with 10% H₂SO₄ or Godin reagent [18] followed by heating at 120–130 °C. MeOH-CH₂Cl₂ (1:1) was used for Sephadex LH-20. [α]_D: CHCl₃. Radula voluta Tayl. ex Gott., Lindenb. & Nees, Frullania brasiliensis Raddi and unidentified Anastrophyllum species were collected in Argentina in 2005 and identified by Prof. Dr. S. R. Gradstein (University of Göttingen, Germany). The voucher specimen was deposited at the Institute of Pharmacognosy, Tokushima Bunri University.

3.2. Extraction and Isolation

The dry material (14.7 g) of the unidentified Anastrophyllum species was ground and extracted with Et₂O. The crude extract (471.4 mg) was divided into 10 fractions by column chromatography on silica gel (n-hexane-EtOAc gradient). Fraction 4 gave ent-cyclocolorenone (5) ([α]_D +376.9° c 1.45; 15.6 mg) and 5,15-rosadiene-3,11-dione (4) (6 mg) by rechromatography on silica gel (n-hexane-EtOAc 17:3, CH₂Cl₂-Et₂O 49:1). 3α-Hydroxy-5,15-rosadien-11-one (3) (4.5 mg) was purified from Fraction 6 by Sephadex LH-20 and silica gel (n-hexane-EtOAc 19:1).

The crude Et₂O extract (1.9 g) of F. brasiliensis (90.8 g) was chromatographed on silica gel (n-hexane-EtOAc gradient) to give six fractions. Fraction 3 was rechromatographed on Sephadex LH-20, silica gel (n-hexane-EtOAc or n-hexane-Et₂O), MPLC (Si-60, toluene) and prep. HPLC (Cosmosil 5SL-II, n-hexane-Et₂O 98:2) to give (+)-frullanolide (6) (59.3 mg) and nepalensolide A (7) (15.1 mg). 5-Epi-dilatanolide A (8) (13.9 mg) and 5-epi-dilatanolide B (9) (5.4 mg) were isolated from Fraction 5 by a combination of reverse phase silica gel (CH₃CN), MPLC (Si-60, n-hexane-Et₂O 4:1) and prep. HPLC (UK-silica, n-hexane-EtOAc 9:1).

The dry material (690 mg) of R. voluta was ground and extracted with Et₂O. The crude extract (79 mg) was chromatographed on Sephadex LH-20 and silica gel to give 3,5-dihydroxy-2-(3-methyl-2-butenyl)bibenzyl (10) (17.6 mg) and 2-geranyl-3,5-dihydroxybibenzyl (11) (3.1 mg).

3α-Hydroxy-5,15-rosadien-11-one (3): [α]_D +71.3° (c 1.31); CD (EtOH): Δε_297nm +1.70, Δε_211nm −0.51 (c = 9.77 × 10⁻⁴); FTIR ν_max cm⁻¹: 3438, 1704; ¹H-NMR see Table 1; and ¹³C-NMR see Table 2; HR-EIMS: calcd for C₂₀H₃₀O₂: 302.2245. Found: 302.2253; EIMS m/z (rel. int.): 302[M]⁺(10), 284(100), 269(47), 241(76), 226(11), 211(11), 201(11), 187(47), 173(47), 171(46), 159(23), 151(34), 145(21), 134(27), 119(26), 105(29), 91(25), 79(113), 67(11), 55(13), 41(15).

m-Bromobenzoate 12:To compound 3 (3.5 mg) in dry CH₂Cl₂ (2 mL) was added m-bromobenzoic acid (5 mg), DCC (4 mg) and DMAP (2 mg) and the solution was stirred at r.t. overnight. The reaction mixture was filtered and chromatographed on silica gel (n-hexane-EtOAc 19:1) to yield m-bromobenzoate 12 (4.2 mg). ¹H-NMR (600 MHz, CDCl₃): δ 8.17 (1H, t, J = 1.9 Hz), 7.98 (1H, dd, J = 8.0, 1.1 Hz), 7.69 (1H, ddd, J = 8.0, 1.9, 1.1 Hz), 7.33 (1H, t, J = 8.0 Hz), 2.05 (1H, m, H-1α), 1.16 (1H, dd, J = 12.9, 4.1 Hz, H-1β), 1.82-1.95 (3H, m, H-2, H-2, H-7α), 4.73 (1H, dd, J = 11.3, 4.7 Hz, H-3), 5.62 (1H, d, J = 6.3 Hz, H-6), 1.99 (1H, m, H-7β), 1.80 (1H, m, H-8), 2.81 (1H, brd, J = 12.4 Hz, H-10), 1.97 (1H, dd, J = 12.6, 2.2 Hz, H-12α), 2.74 (1H, d, J = 12.6 Hz, H-12β), 1.38 (1H, dd, J = 9.9, 2.5 Hz, H-8α), 1.77 (1H, d, J = 12.4 Hz, H-14β), 5.84 (1H, dd, J = 17.3, 10.7 Hz, H-15), 4.95 (1H, dd, J = 10.7, 0.8 Hz, H-16), 4.97 (1H, dd, J = 17.3, 0.8 Hz, H-16), 0.99 (3H, s, H-17), 1.22 (3H, s, H-18), 1.10 (3H, s, H-19), 1.02 (3H, s, H-20); ¹³C-NMR (100 MHz, CDCl₃): δ 214.6, 164.8, 147.9, 144.5, 135.8, 132.8, 132.6, 130.0, 128.2, 122.5, 117.9, 110.2, 80.0, 49.0, 48.4, 41.6, 41.2, 38.4, 38.3, 37.9, 28.9, 26.7, 25.0, 24.6, 23.5, 23.1, 12.6.

4. Conclusions

A new rosane diterpenoid 3 was isolated from the unidentified Argentine liverwort Anastrophyllum species, together with a known rosanediterpenoid 4 and an aromadendranesesquiterpenoid 5. The known bibenzyls 10 and 11 and sesquiterpene lactones 6–9 were isolated from Argentine Radula voluta and Frullania brasiliensis.