Two Novel Lanostane Triterpenoids from Ganoderma Sinense

Abstract

Two novel lanostane-type triterpenes, Ganolactone B and Ganoderiol A triacetate, were isolated from the fruiting bodies of G. sinense, together with six known compounds. The structures of the two new triterpenes were determined as 3β,7β-dihydroxy-11,15-dioxo-lanosta-8-en-24→20 lactone (1) and 3β,24,26-triacetoxy-5α-lanosta-7,9(11)-dien-25-ol (2), respectively, by chemical and spectroscopic means.

Introduction

The fruiting body of the fungus Ganoderma lucidum Karst.(Polyporaceae) has attracted much attention as a well-known traditional Chinese medicine that has been used clinically in China, Japan, and Korea for a long time. Over the past recent two decades, more than 130 highly oxygenated lanostane-type triterpenoids have been isolated from the fruiting bodies, spores and culture mycelia of Ganoderma spp., including common fungal steroids derived from ergosterol. Some of them are known to possess varied bioactive properties. Other Ganoderma spp. have also been used in traditional Chinese, Japanese and Korean medicines, and their pharmacological activities as antitumor [1,2] and antihypertensive agents [3], their use in the treatment of chronic bronchitis and diabetes or their liver protective [4], antioxidant [5] and anti-HIV activity [6] were studied. In this paper, we wish to report the structural elucidation by extensive spectroscopic analyses of two new components isolated together with known six compounds (Figure 1) from the fruiting bodies of G. sinense.

Figure 1.

Figure 1.

Results and Discussion

Dried chipped fruiting bodies of G. sinense were soaked overnight in acetone at room temperature to give an extract which was suspended in water and successively partitioned with petroleum ether, CHCl₃, EtOAc and n-BuOH, respectively. The CHCl₃ layer was chromatographed on silica gel to separate it into six fractions. Some of these were subjected to silica gel column chromatography to give two new triterpene compounds, which we have named Ganolactone B (3β,7β-dihydroxy-11,15-dioxo-lanosta-8-en-24→20 lactone, 1), Ganoderiol A triacetate (3β,24,26-triacetoxy-5α-lanosta-7,9(11)-dien-25-ol, 2), together with six known compounds: Ganoderiol A (3) [7], Ganodermatriol (4), Ganodermanontriol (5), 20(21)-dehydrolucidenic acid (6) [8], Ganoderic acid D (7) [9] and Ganoderic acid A (8) [10].

Ganolactone B (1): colorless needles, m.p. > 300°C, 183°(c=0.3, MeOH). Compound 1 gave a quasi-molecular ion peak at m/z 459 [M+H]⁺ in its positive FABMS spectrum, and was assigned a molecular formula of C₂₇H₃₈O₆, which was confirmed by HRESIMS (C₂₇H₃₈O₆Na, 481.2579; calcd. for C₂₇H₃₈O₆Na, 481.2566) and NMR spectral data (Table 1). The IR spectrum [3433 (br, OH), 1769, 1719, 1655 (C=O), 1207, 1143 and 1065 (C-O and COOC)] indicated the presence of hydroxyl and carbonyl groups. The UV data [(MeOH) λ_max(logε) nm: 255 (0.17)] indicated the presence of an α,β-unsaturated carbonyl group. The ¹H-NMR spectrum of 1 (Table 1), analyzed together with the compound’s HMQC spectrum, exhibited singlet signals (3H each) of six methyl groups at δ_H 1.07, 1.24, 1.36, 1.39, 1.40, 1.44 and two oxygenated methine protons at δ_H 3.46 (1H, dd, J=5.5, 11.0Hz, 3α-H) and 5.19 (1H, t, 8.4Hz, 7α-H). The ¹³C-NMR spectrum displayed signals characteristic of six methyl groups, an oxygen-bearing quaternary carbon at δc 86.49 (s), two hydroxyl-bearing methine carbons at δc 77.59 (d) and 66.70 (d), an α,β-unsaturated C=O at δc 197.98 (s), 158.71 (s), 142.65 (s), a ketone carbon at δc 214.96 (s), and a carboxyl group at δc 176.69 (s). These data suggested a polyoxygenated lanostane-type triterpene with a structure very similar to Ganolactone A, including its chemical configuration at C-20 [11]. Ganolactone B (1) had a signal at δc 86.49 (s). This indicated that linking group was at C-20. The connectivities of 1 were established by interpretation of the HMBC spectrum (Figure 2). In the HMBC experiment, δc 86.49 (s, C-20) showed correlation with two secondary methyl group signals δ_H 2.81, 2.66 (each 1H, H-16), δ_H 2.68, 2.49 (each 1H, H-22) and one methyl signal δ_H 1.39 (3H, H-21). From the above observation and on the basis of all the above results the structure of Ganolactone B was established as 3β,7β-dihydroxy-11,15-dioxo-lanosta-8-en-24→20 lactone.

Figure 2. key HMBC( ) correlations of Ganolactone B (1).

Figure 2. key HMBC( ) correlations of Ganolactone B (1).

Compound 2 gave colorless needles, mp 232~233°C, 80°(c=0.1, C₅D₅N) and a quasi-molecular ion peak at m/z 601 [M+H]⁺ in its positive FABMS spectrum, and was thus assigned a molecular formula of C₃₆H₅₆O₇, which was confirmed by HRESIMS (C₃₆H₅₆O₇Na, 623.3923; calcd. for C₃₆H₅₆O₇Na, 623.3920) and NMR spectral data (Table 1). Its UV spectrum was similar to that of Ganoderiol A [7], indicating the presence a heteroannular diene system in this molecule. The IR spectrum [3492 (br, OH), 1735, 1719 (C=O), 1233, 1152 and 1031 (C-O and COOC)] indicated the presence of hydroxyl and carbonyl groups. The ¹H-NMR spectrum of 2 showed singlets for three acetyl methyl group at δ_H 2.05, 2.07 and 2.10 (3H each). The ¹³C-NMR spectrum of 2 verified the presence of three carbons attached to oxygen at δc 68.45 (t), 76.64 (d) and 80.83 (d), three carbonyl carbons for acetyl moieties at δc 170.64 (s), 170.93 (s), and 171.11 (s), and three acetyl methyl carbons at δc 20.83 (q), 20.98 (q), and 21.29 (q). From these spectral data, the structure of this compound was established as 3β,24,26-triacetoxy-5α-lanosta-7, 9(11)-dien-25-ol, for which we propose the name Ganoderiol A triacetate.

Table 1. ¹H-NMR and ¹³C-NMR data of compounds 1 and 2.

No.	1		2
	δc	δH (J = Hz)	δc	δH (J = Hz)
	C5D5N		CDCl3
1	35.47 t	3.22 m (α) 1.20 m (β)	35.41 t	- -
2	28.59 t	1.89 m (α) 1.89 m (β)	22.77 t	- -
3	77.59 d	3.46 dd(10.96, 5.43) (α)	80.83 d	4.51 dd (12.68, 3.56) (α)
4	49.68 s	-	37.62 s	-
5	49.65 d	1.10 t(16.97, 12.99) (α)	49.26 d	-
6	28.14 t	2.37 m (α) 1.90 m (β)	24.26 t	- -
7	66.70 d	5.19 t(16.97, 12.99) (α)	119.97 d	5.45 m (α)
8	158.71 s	-	142.70 s	-
9	142.65 s	-	146.65 s	-
10	39.29 s	-	37.80 s	-
11	197.98 s	-	116.50 d	5.30 d (4.64)
12	50.94 t	3.09 d(16.88) (α) 2.95 d(16.78) (β)	37.24 t	- -
13	45.78 s	-	43.74 s	-
14	59.03 s	-	50.32 s	-
15	214.96 s	-	25.99 t	-
16	36.53 t	2.81 dd(18.30, 10.17) (α) 2.66 dd(18.30, 10.32) (β)	31.45 t	- -
17	49.65 d	2.65 dd(11.98, 7.26) (α)	50.77 d	-
18	18.64 q	1.40 s	16.92 q	0.54 s
19	19.40 q	1.36 s	22.82 q	0.99 s
20	86.49 s	-	36.45 d	-
21	25.79 q	1.39 s	18.56 q	0.94 d(5.28)
22	27.84 t	2.28 m (α) 2.49 m (β)	32.62 t	- -
23	34.20 t	2.08 m (α) 1.85 m (β)	27.78 t	- -
24	176.69 s	-	76.64 d	4.89 dd(9.80, 1.96)
25	-	-	73.27 s	-
26	-	-	68.45 t	-
27	-	-	20.19 q	1.19 s
28	25.39 q	1.44 s	28.08 q	0.86 s
29	16.47 q	1.24 s	15.66 q	0.86 s
30	28.75 q	1.07 s	25.53 q	0.94 s
OCOMe	-	-	170.64 s	-
	-	-	170.93 s	-
	-	-	171.11 s	-
OCOMe	-	-	20.83 q	2.05(3H, s)
	-	-	20.99 q	2.07(3H, s)
	-	-	21.29 q	2.10(3H, s)

Table 1. ¹H-NMR and ¹³C-NMR data of compounds 1 and 2.

No.	1		2
	δc	δH (J = Hz)	δc	δH (J = Hz)
	C5D5N		CDCl3
1	35.47 t	3.22 m (α) 1.20 m (β)	35.41 t	- -
2	28.59 t	1.89 m (α) 1.89 m (β)	22.77 t	- -
3	77.59 d	3.46 dd(10.96, 5.43) (α)	80.83 d	4.51 dd (12.68, 3.56) (α)
4	49.68 s	-	37.62 s	-
5	49.65 d	1.10 t(16.97, 12.99) (α)	49.26 d	-
6	28.14 t	2.37 m (α) 1.90 m (β)	24.26 t	- -
7	66.70 d	5.19 t(16.97, 12.99) (α)	119.97 d	5.45 m (α)
8	158.71 s	-	142.70 s	-
9	142.65 s	-	146.65 s	-
10	39.29 s	-	37.80 s	-
11	197.98 s	-	116.50 d	5.30 d (4.64)
12	50.94 t	3.09 d(16.88) (α) 2.95 d(16.78) (β)	37.24 t	- -
13	45.78 s	-	43.74 s	-
14	59.03 s	-	50.32 s	-
15	214.96 s	-	25.99 t	-
16	36.53 t	2.81 dd(18.30, 10.17) (α) 2.66 dd(18.30, 10.32) (β)	31.45 t	- -
17	49.65 d	2.65 dd(11.98, 7.26) (α)	50.77 d	-
18	18.64 q	1.40 s	16.92 q	0.54 s
19	19.40 q	1.36 s	22.82 q	0.99 s
20	86.49 s	-	36.45 d	-
21	25.79 q	1.39 s	18.56 q	0.94 d(5.28)
22	27.84 t	2.28 m (α) 2.49 m (β)	32.62 t	- -
23	34.20 t	2.08 m (α) 1.85 m (β)	27.78 t	- -
24	176.69 s	-	76.64 d	4.89 dd(9.80, 1.96)
25	-	-	73.27 s	-
26	-	-	68.45 t	-
27	-	-	20.19 q	1.19 s
28	25.39 q	1.44 s	28.08 q	0.86 s
29	16.47 q	1.24 s	15.66 q	0.86 s
30	28.75 q	1.07 s	25.53 q	0.94 s
OCOMe	-	-	170.64 s	-
	-	-	170.93 s	-
	-	-	171.11 s	-
OCOMe	-	-	20.83 q	2.05(3H, s)
	-	-	20.99 q	2.07(3H, s)
	-	-	21.29 q	2.10(3H, s)

Experimental

General

Melting points were measured on a XRB-1 micro hot-stage melting point apparatus and are uncorrected. Optical rotations were measured with a DIP-370 automatic polarimeter. UV spectra were measured with a Shimadzu double-beam instrument. IR spectra were obtained on a Bio-Rad FTS-135 infrared spectrophotometer. ¹H-, ¹³C-NMR and 2D-NMR spectra were recorded on a Bruker AM-400 MHz or a DRX-500 spectrometer with TMS as internal standard.

Plant Material

The fruiting bodies of G. sinenes were purchased from the Kunming natural medicine market, Yunnan Province, P. R. China in July 2005. The botanical identification was made by Prof. Yang Zhuliang (Kunming Institute of Botany, The Chinese Academy of Sciences).

Extraction and Isolation

Dried chipped fruiting bodies of G. sinense (8 kg) were soaked overnight in acetone at room temperature to afford an extract which was suspended in water and successively partitioned with petroleum ether (PE), CHCl₃, EtOAc, and n-BuOH, respectively. The CHCl₃ extracts were concentrated in vacuo to afford a residue (115 g), which was subjected to silica gel column chromatography, using a CHCl₃-Me₂CO gradient (from 100% CHCl₃ to CHCl₃-Me₂CO 1:1) as eluent to separate it into six fractions (Fr. A - F). Fr. D (19.5 g) was subjected repeatedly to silica gel chromatography and eluted stepwise with PE-Me₂CO (from 100% PE to PE-Me₂CO 1:1) to afford compounds 2 (17 mg), 3 (29 mg), 4 (22 mg) and 5 (13 mg), respectively. Fr. E (8.3 g) was rechromatographed on a silica gel column chromatography by elution with PE-Me2CO (5:1 v/v, 3:1, 1:1) to yield 1 (36 mg). Fr. F (4.7g) was rechromatographed on a silica gel column chromatography by elution with PE- Me₂CO (2:1 v/v, 1:2). Further purification of Fr. F-4 (1.7 g) was achieved by a reversed phase C-18 (from MeOH- H₂O 40% to 65%) to give compounds 6 (12 mg), 7 (11 mg) and 8 (9 mg), respectively.

Ganolactone B (1): colorless needles, m.p. > 300°C, 183°(c=0.3, MeOH); positive FABMS m/z 459[M+H]⁺, HRESIMS (calcd. for C₂₇H₃₈O₆: 458.2579); IR(KBr)cm^-1_: 3433 (br, OH), 1769, 1719, 1655 (C=O), 1207, 1143 and 1065 (C-O and COOC); UV (MeOH) λ_max (logε) nm:255(0.17); ¹H- and ¹³C-NMR spectral data, see Table 1.

3β,24,26-Triacetoxy-5α-lanosta-7,9(11)-dien-25-ol (Ganoderiol A triacetate) (2): Colorless needles, mp 232~233°C, 80°(c=0.1, C₅D₅N); positive FABMS m/z 601 [M+H]⁺; HRESIMS (calcd. for C₃₆H₅₆O₇: 600.3920); IR (KBr) cm^-1_: 3492 (br, OH), 1735, 1719 (C=O), 1233, 1152 and 1031 (C-O and COOC); UV (MeOH) λ_max (logε) nm: 243 (0.30); ¹H- and ¹³C-NMR spectral data, see Table 1.

Ganoderiol A (3): colorless needles, mp 232~234°C, +20°(c=0.10, EtOH); HREIMS m/z 474.3740 (calcd for C₃₀H₅₀O₄: 474.3709); UV (EtOH) λ_max (logε) nm: 253 (8058), 244 (962), 253 (6518); IR (KBr) cm^-1: 3350, 2950, 2900, 2850, 1430, 1360, 1060; ¹H-NMR (C₅D₅N) δ_H: 5.55 (1H, m, H-7), 5.40 (1H, m, H-12), 3.83, 3.47 (1H each, d, J=10.6Hz, 26-H), 3.46 (1H, t, J=12.1Hz, C-24), 3.24 (1H, dd, J=10.5 and 5.0 Hz, H-3), 1.11 (3H, s, H-27), 1.01 (3H, s, H-19), 0.98 (3H, s, H-28), 0.92 (3H, d, J=6.2Hz H-21), 0.88 (3H, s, H-29), 0.88 (3H, s, H-30), 0.67 (3H, s, H-18); ¹³C-NMR (C₅D₅N) δc: 36.40 (t, C-1), 28.86 (t, C-2), 78.12 (d, C-3), 39.37 (s, C-4), 49.83 (d, C-5), 23.56 (t, C-6), 120.99 (d, C-7), 143.03 (s, C-8), 146.61 (s, C-9), 37.85 (s, C-10), 116.58 (s, C-11), 38.14 (t, C-12), 44.13 (s,C-13), 50.68 (s, C-14), 28.17 (t, C-15), 31.91 (t, C-16), 51.50 (d, C-17), 16.64 (q, C-18), 23.11 (q, C-19), 37.15 (d, C-20), 19.06 (q, C-21), 34.40 (t, C-22), 28.94 (t, C-23), 77.23 (d, C-24), 74.79 (s, C-25), 69.28 (t, C-26), 20.15 (q,C-27), 28.94 (q, C-28), 16.07 (q, C-29), 25.89 (q, C- 30).

Ganodermatriol (4): white powder, mp 180~190°C; +9°(c=0.04, EtOH); HREIMS m/z 456.3612 (calcd for C₃₀H₄₈O₃: 456.3605); UV (EtOH) λ_max (logε) nm: 237 (4740), 245 (5400), 253 (3650); ¹H-NMR (C₅D₅N) δ_H: 5.56 (1H, J=7.5 , H-24), 5.48 (1H, m, H-7), 5.32 (1H brd, J=6.2Hz, H-11), 4.33 (2H, s, H-27), 4.21 (2H, s, H-26), 3.24 (3H, dd, J=10.6 and 4.8 Hz H-3), 1.01 (3H, s, H-19), 0.98 (3H, s, H-28), 0.91 (3H, d , J=6.2Hz, H-21), 0.88 (3H, s, H-29), 0.88 (3H, s, H-30), 0.67 (3H, s, H-18); ¹³C-NMR (C₅D₅N) δc: 36.42 (t, C-1), 28.74 (t, C-2), 78.12 (d, C-3), 39.37 (s, C-4), 49.84 (d, C-5), 23.57 (t, C-6), 121.06 (d,C-7), 142.98 (s, C-8), 146.64 (s, C-9), 37.86 (s, C-10), 116.54 (s, C-11), 38.11 (t, C-12), 44.13 (s,C-13), 50.69 (s, C-14), 28.16 (t, C-15), 31.90 (t,C-16), 51.24 (d, C-17), 16.65 (q, C-18), 23.10 (q, C-19), 36.42 (d, C-20), 18.62 (q, C-21), 36.91 (t, C-22), 24.66 (t, C-23), 127.74 (d, C-24), 140.75 (s, C-25), 65.51 (t, C-26), 58.59 (q,C-27), 28.86 (q, C-28), 16.02 (q, C-29), 25.87 (q, C- 30).

Ganodermanontriol (5): colorless prisms, mp 168~170°C; +41°(c=0.20, MeOH); HREIMS m/z 472.3540 (calcd. for C₃₀H₄₈O₄: 472.3550); ¹H-NMR (C₅D₅N) δ_H: 5.51 (1H, d, J=6.2Hz, H-7), 5.39 (1H, d, J=5.9Hz, H-11), 3.83, 3.48 (1H each, d, J=11.3Hz, 26-H), 3.46 (1H, t, J=11.0Hz, C-24), 1.20 (3H, s, H-19), 1.13 (3H, s, H-28), 1.11 (3H, s, H-27), 1.09 (3H, s, H-29), 0.92 (3H, d, J=6.2Hz, H-21), 0.88 (3H, s, H-30), 0.60 (3H, s, H-18); ¹³C-NMR (C₅D₅N) δc: 36.86 (t, C-1), 34.39 (t, C-2), 215.18 (s, C-3), 47.52 (s, C-4), 51.11 (d, C-5), 23.92 (t, C-6), 120.43 (d,C-7), 143.14 (s, C-8), 146.97 (s, C-9), 37.13 (s, C-10), 117.69 (s, C-11), 37.50 (t, C-12), 44.08 (s,C-13), 50.60 (s, C-14), 31.39 (t, C-15), 28.14 (t, C-16), 51.11 (d, C-17), 16.06 (q, C-18), 22.11 (q, C-19), 36.55 (d, C-20), 19.06 (q, C-21), 34.39 (t, C-22), 28.94 (t, C-23), 77.24 (d, C-24), 74.79 (s, C-25), 69.28 (t, C-26), 20.14 (q,C-27), 25.66 (q, C-28), 22.39 (q, C-29), 25.66 (q, C- 30).

20(21)-Dehydrolucidenic acid A (6): colorless needles, mp 135-137°C; +69.9°(c 0.20, CHCl₃); HREIMS m/z456.2512 (calcd. for C₂₇H₃₆O₆ [M], 456.2511); UV (MeOH) λ_max (logε) nm: 253 (3.78); IR (KBr) cm^-1: 3445, 1735, 1702, 1659, 897; ¹H-NMR (CHCl₃) δ_H: 5.06 (1H, s, H-21), 4.91 (1H, s, H-21), 1.39 (3H, s, H-30), 1.25 (3H, s, H-19), 1.13 (3H, s, H-28), 1.10 (3H, s, H-29), 0.89 (3H, s, H-19); ¹³C-NMR (CHCl₃) δc: 35.64 (t, C-1), 34.27 (t, C-2), 216.57 (s, C-3), 46.78 (s, C-4),49.07 (d, C-5), 27.66 (t, C-6), 66.34 (d,C-7), 157.75 (s, C-8), 141.15 (s, C-9), 38.31 (s, C-10), 197.49 (s, C-11), 49.07 (t, C-12), 45.26 (s,C-13), 58.82 (s, C-14), 217.67 (s, C-15), 38.64 (t,C-16), 46.24 (d, C-17), 18.82 (q, C-18), 18.14 (q, C-19), 143.91 (s, C-20), 112.35 (t, C-21), 31.28 (t, C-22), 32.26 (t, C-23), 176.88 (s, C-24), 27.03(q, C-28), 20.78(q, C-29), 24.55(q, 30).

Ganoderic acid D (7): colorless needles, mp 201~203°C: +185°(c=0.10, EtOH); HREIMS m/z 530.2875 (calcd. for C₃₀H₄₂O₈: 530.2881); UV (EtOH) λ_max (logε) nm: 253 (8800); IR (KBr) cm^-1: 3410, 3400~2500 (br), 1730, 1720, 1660; ¹H-NMR (C₅D₅N) δ_H: 5.16 (1H, dd, J=8.4 and 8.4Hz, H-7), 4.69 (1H, s, H-12), 1.47 (3H, s, H-28), 1.43 (3H, s, H-19), 1.41 (3H, d, J=6.7Hz, C-21), 1.36 (3H, d, J=7.3Hz, C-27), 1.22 (3H, s, H-18), 1.15 (3H, s, H-30), 1.07 (3H, s, H-29); ¹³C-NMR (C₅D₅N) δc: 35.47 (t, C-1), 34.59 (t, C-2), 216.64 (s, C-3), 46.93 (s, C-4), 49.34 (d, C-5), 28.94 (t, C-6), 65.72 (d, C-7), 159.83 (s, C-8), 140.90 (s, C-9), 38.23 (s, C-10), 201.07 (s, C-11), 79.26 (d, C-12), 51.78 (s, C-13), 59.91 (s, C-14), 215.26 (s, C-15), 39.24 (t,C-16), 46.93 (d, C-17), 13.03 (q, C-18), 18.37 (q, C-19), 29.09 (d, C-20), 21.89 (q, C-21), 49.02 (t, C-22), 209.04 (s, C-23), 46.93 (t, C-24), 46.93 (d, C-25), 178.26 (s, C-26), 17.66 (q,C-27), 24.01 (q, C-28), 21.14 (q, C-29), 26.60 (q, C- 30).

Ganoderic acid A (8): colorless needles, mp 233~236°C; +150°(c=0.13, CHCl₃); HREIMS m/z 516.3070 (calcd. for C₃₀H₄₂O₈: 516.3087); UV (EtOH) λ_max (logε) nm: 254 (3.70); IR (KBr) cm^-1: 3400, 2700~2300 (br), 1700, 1655, 1270, 1000; ¹H-NMR (C₅D₅N) δ_H: 5.23 (1H, dd, J=9.2 and 7.8Hz, H-15), 4.94 (1H, dd, J=9.7 and 7.6Hz, H-7), 1.51 (3H, s, H-28), 1.41 (3H, s, H-19), 1.33 (3H, d, J=7.3Hz, C-27), 1.15 (3H, s, H-30), 1.11 (3H, s, H-29), 1.07 (3H, s, H-18), 0.95 (3H, d, J=6.2Hz, C-21); ¹³C-NMR (C₅D₅N) δc: 36.07 (t, C-1), 34.63 (t, C-2), 216.11 (s, C-3), 47.10 (s, C-4), 49.04 (d, C-5), 29.69 (t, C-6), 68.76 (d,C-7), 161.51 (s, C-8), 139.98 (s, C-9), 38.35 (s, C-10), 199.70 (s, C-11), 52.44 (t, C-12), 46.78 (s, C-13), 54.76 (s, C-14), 72.22 (d, C-15), 36.96 (t, C-16), 48.68 (d, C-17), 17.54 (q, C-18), 19.58 (q, C-19), 33.10 (d, C-20), 19.77 (q, C-21), 49.97 (t, C-22), 208.95 (s, C-23), 47.15 (t, C-24), 35.60 (d, C-25), 176.23 (s, C-26), 17.65 (q,C-27), 20.29 (q, C-28), 20.87 (q, C-29), 27.26 (q, C- 30).