A comparison of the 1H and 13C NMR data of 79 with those of 78
indicated that they were almost identical except for the presence of signals
for an aliphatic long-chain acid derivative. This group was determined
through the apperance of one carbonyl ester (C 173.9), fourteen
methylenes (between C 29.2 and 29.7 ppm) and one methyl group (C
14.1) beside the 20 carbons of cassipourol in the 13C NMR spectrum of 79
(Table 3.1). Similarly, it could be seen not only the resonances of 78 but
also the resonances of methyl triplet at H 0.87 (J = 7 Hz), strong broad
singlet at H ca 1.25 and a triplet at H 2.29 (J = 7.5 Hz) in the 1H NMR
of 79. The singlet at 1.25 correlated to the signal of methylene carbon (at
C ca. 29 ppm), while triplet at H 2.29 (J = 7.5 Hz) connected to a shifted
downfield methylene at C 34.4 in the HSQC spectrum. The HR-ESI-MS
exhibited molecular ion peak at m/z 555.5126 [M+Na]+ (calcd for
C36H68NaO2, 555.5117) establishing a molecular formula of C36H68O2 to
compound 79 and the fragment at m/z 318.2965 (C20H39NaO required
318.2899) indicating the presence of a cassipouryl moiety. These data
allowed to identify aliphatic chain as a hexadecanoic acid derivative. The
esterification of this acid with cassipourol on C-15 was suggested by the
chemical shift of C-15 at C 61.19 (shifted downfield, +1.76 ppm due
to este RCOO group). Furthermore, it was also confirmed by the key
HMBC correlations H-14/C-12, C-15; H-15/C-14, C-13, C-1’ (C=O); H-
2’/C-1’, C-3’, C-4’ and H-3’/C-2’, C-4’.
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5.88 (1H, d, J = 2.5 Hz, H-6); 4.59 (1H, d, J
= 8 Hz, H-2); 4.01 (1H, ddd, J = 5.5, 5, 5.5 Hz, H-3); 2.86 (1H, dd, J = 5.5,
5.5 Hz, H-4a); 2.53 (1H, dd, J = 8, 8 Hz, H-4b).
13
C-NMR (125MHz, CD3OD), δ (ppm): 157.8 (C-5); 157.5 (C-7); 156.9
(C-9); 146.2 (C-4’); 146.21 (C-3’); 132.2 (C-1); 120.0 (C-6’); 116.1 (C-2’);
115.2 (C-5’); 100.8 (C-10); 96.3 (C-6); 95.5 (C-8); 82.8 (C-2); 68.8 (C-3);
28.46 (C-4).
● Compound 87 (ponasterone A) white powder, Rf = 0.3
(DCM:MeOH = 4.5:0.5), t
o
nc = 259-260
o
C, HR-ESI-MS m/z = 487,3029
[M+Na]
+
(C27H44O6Na), m/z = 465.3209 [M+H]
+
(C27H45O6), molecular
formula C27H44O6
1
H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 5.83 (1H, d, J = 2 Hz,
H-7); 3.97 (1H, d, J = 2 Hz, H-3); 3.86 (1H, ddd, J = 4, 4.5, 3 Hz, H-2ax);
3.36 (1H, H-22); 3.17 (1H, m, H-9); 2.38 (1H, m, H-5); 2.14 (1H, m, H-
12ax); 2.02 (1H, H-17); 2.00 (1H, H-15α); 1.9 (1H, H-16α); 1.8 (1H, H-
12eq); 1.78 (1H, H-1eq); 1.78 (1H, H-11eq); 1.75 (1H, H-4eq); 1.75 (1H, H-
24a); 1.72 (1H, H-20); 1.60 (1H, H-23a); 1.65 (1H, H-11ax); 1.65 (1H, H-
4ax); 1.53 (1H, H-15β); 1.48 (1H, H-16β); 1.45 (1H, H-24b); 1.43 (1H, H-
1ax); 1.30 (1H, H-23b); 1.20 (3H, s, H-27); 0.98 (3H, s, H-26); 0.97 (3H, s,
H-19); 0.94 (3H, H-21); 0.91 (3H, s, H-18).
13
C-NMR (125MHz, CD3OD), δ (ppm): 206.40 (C-6); 167.94 (C-8);
122.14 (C-7); 85.23 (C-14); 77.96 (C-20); 77.84 (C-22); 68.70 (C-2); 68.51
(C-3); 51.78 (C-5); 50.47 (C-17); 49.62 (C-13); 39.26 (C-10); 37.65 (C-23);
37.37 (C-1); 35.10 (C-9); 32.86 (C-4); 32.52 (C-12); 31.75 (C-15); 30.47 (C-
24); 29.21 (C-25); 24.40 (C-19); 23.40 (C-27); 22.75 (C-26); 21.50 (C-11);
21.50 (C-16); 20.99 (C-21); 18.32 (C-18).
● Compound 88 (20-hydroxyecdysone) colorless solid, Rf = 0.3
(DCM:MeOH = 4:1), [α]
25
D = 58
o
(MeOH, c 0.1), t
o
nc = 244-246
o
C
HR-ESI-MS m/z = 503.2972 [M+Na]
+
(C27H44O7Na), m/z =
481.3151 [M+H]
+
(C27H45O7), molecular formula C27H44O7.
1
H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 5.83 (1H, J = 2 Hz, H-
7); 3.97 (1H, d, J = 1.5 Hz, H-3); 3.86 (1H, ddd, J = 3.5, 5, 3.5 Hz, H-2ax);
3.36 (1H, H-22); 3.17 (1H, t, J = 9Hz, H-9); 2.38 (1H, dd, J = 9.5; 5 Hz, H-
5); 2.16 (1H, m, H-12 ax); 2.02 (1H, H-17); 2.00 (1H, H-15α); 1.98 (1H, H-
16α); 1.8 (1H, H-12eq); 1.79 (1H, H-1eq); 1.78 (1H, H-11eq); 1.75 (1H, H-
4eq); 1.75 (1H, H-24a); 1.72 (1H, H-20); 1.67 (1H, H-23a); 1.65 (1H, H-
11ax); 1.65 (1H, H-4ax); 1.59 (1H, H-15β); 1.48 (1H, H-16β); 1.45 (1H, H-
24b); 1.43 (1H, H-1ax); 1.31 (1H, H-23b); 1.22 (3H, s, H-27); 1.22 (3H, s,
H-26); 1.21 (3H, s, H-19); 0.98 (3H, H-21); 0.91 (3H, s, H-18).
7
13
C-NMR (125MHz, CD3OD), δ (ppm): 206.36 (C-6); 167.93 (C-8);
122.12 (C-7); 85.20 (C-14); 78.39 (C-20); 77.88 (C-22); 71.27 (C-25);
68.68 (C-2); 68.49 (C-3); 51.75 (C-5); 50.51 (C-17); 49.50 (C-13); 42.36
(C-24); 39.25 (C-10); 37.36 (C-1); 35.07 (C-9); 32.82 (C-4); 32.50 (C-12);
31.77 (C-15); 29.68 (C-26); 28.99 (C-27); 27.32 (C-23); 24.40 (C-19);
21.49 (C-16); 21.06 (C-21); 18.04 (C-18).
● Compound 84 (lambertic acid) white powder, Rf = 0.5 (n-
hexane:EtOAc = 4:1), [α]
25
D = +58
o
(MeOH, c 0.1), t
o
nc = 129-130
o
C,
molecular formula C20H28O3
1
H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 6.98 (1H, d, J = 8.3
Hz, H-12); 6.52 (1H, d, J = 8.5 Hz, H-11); 3.12-3.14 (1H, m, H-15); 2.94
(1H, dd, J = 14.1, 6.3 Hz, H-7α); 2.74-2.76 (1H, m, H-7β); 2.26-2.28 (1H,
m, H-6α); 2.01(1H, dd, J = 13.8 Hz; H-1α); 1.86 (1H, d, J = 13.4 Hz; H-
3α); 1.71-1.73 (1H, m, H-2α); 1.67-1.69 (1H, m, H-1β); 1.62-1.64 (1H, m,
H-2β); 1.42 (1H, d, J = 13.4 Hz; H-5); 1.38 (1H, d, J = 13.4 Hz; H-6β);
1.34 (3H, d, J = 7 Hz, H-16); 1.33 (3H, d, J = 7 Hz, H-17); 1.18 (3H, s, H-
20); 1.06 (3H, s, H-18); 1.00 (1H, dd, J = 13, 4.3 Hz; H-3β).
13
C-NMR (125MHz, CD3OD), δ (ppm): 181,64 (C-19); 153,38 (C-
12); 147,37 (C-9); 133,61 (C-8); 127,30 (C-14); 127,17 (C-13); 112,58 (C-
11); 54,41 (C-5); 44,87 (C-4); 40,91 (C-6); 39,45 (C-10); 38,83 (C-1);
32,49 (C-3); 29,29 (C-15); 27,73 (C-18); 23,69 (C-20); 23,16 (C-16);
23,12 (C-17); 22,63 (C-7); 21,20 (C-2).
● Compound 79 (cassipouryl hexadecanoat, new compound)
colorless oil, Rf = 0.5 (n-hexane:EtOAc = 5:0.2), m/z: 555.5126 [M+Na]
+
(C36H68NaO2), molecular formula C36H68O2 .
1
H-NMR (500MHz, CDCl3), δ (ppm), J (Hz): 5.33 (1H, t, J = 7.0 Hz,
H-14); 4.58 (2H, d, J = 7.0 Hz; H-15); 2.29 (2H, t, J = 7.5 Hz, H-2’); 2.00
(1H, m, H-12a); 1.69 (3H, br s, H-20); 1.60 (2H, m, H-11); 1.58-1.60 (2H,
m, H-3’); 1,52 (1H, m, H-5); 1.38 (1H, m, H-6); 1.36 (2H, m, H-9); 1.30
(2H, m, H-8); 1.28 (2H, m, H-7); 1.25 (2H, m, H-3); 1.25 (2H, m, H-4);
1.25 (1H, m, H-10a); 1.25 (26H, m, H-4’- H-13’); 1.25 (2H, s, H-14’); 1.25
(2H, br s, H-15’); 1.14 (2H, m, H-2); 1.08 (1H, m, H-10b); 1.08 (1H, m, H-
12b); 0.87 (3H, t, J = 7 Hz; H-16’); 0.86 (3H, s, H-16); 0.85 (3H, s, H-17);
0.845 (3H, d, J = 7; H-18); 0.838 (3H, d, J = 7 Hz; H-19).
13
C-NMR (125MHz, CDCl3), δ (ppm): 173.93 (C-1’); 142.58 (C-13);
118.21 (C-14); 61.19 (C-15); 39.86 (C-12); 39.38 (C-2); 37.44 (C-10);
37.37 (C-4); 37.30 (C-8); 36.64 (C-1); 34.42 (C-2’); 32.80 (C-6); 32.68
(C-9); 31.93 (C-14’); 29.70-29.16 (C4’-C13’); 27.98 (C-5); 25.04 (C-11);
24.80 (C-7); 24.80 (C-3’); 24.47 (C-3); 22.69 (C-16); 22.62 (C-17); 22.71
(C-15’); 19.74 (C-18); 19.71 (C-19); 16.36 (C-20); 14.10 (C-16’).
8
● Compound 85, (+)-spathulenol: colorless oil, Rf = 0.25 (n-
hexane:EtOAc = 5:0.5), [α]
25
D = 5° (CHCl3, c 0.1)
ESI-MS m/z: 221.3 [M+H]
+
, molecular formula C15H24O
1
H-NMR (500 MHz, CDCl3), δ (ppm), J (Hz): 4.69 (1H, s, H-14a);
4.66 (1H, s, H-14b); 2.42 (1H, dd, J = 13.0, 6.0 Hz, H-4a); 2.20 (1H, m, H-
6); 2.06 (1H, d, J = 13.0 Hz, H-4b); 1.77 (1H, m, H-8a); 1.55 (1H, m, H-8b);
1.32 (1H, d, J = 10.5 Hz, H-10); 1.28 (3H, s, H-15); 1.06 (3H, s, H-12); 1.03
(3H, s, H-13); 0.71 (1H, ddd, J = 11.0, 9.5, 6.0 Hz, H-2); 0.47 (1H, dd, J =
11.0, 9.5 Hz, H-1).
13
C-NMR (125MHz, CDCl3), δ (ppm): 153.46 (C-5); 106.26 (C-14);
80.99 (C-9); 54.35 (C-10); 53.41 (C-6); 41.75 (C-8); 38.87 (C-4); 29.92
(C-1); 28.66 (C-12); 27.50 (C-2); 26.72 (C-7); 26.08 (C-15); 24.79 (C-3);
20.27 (C-11); 16.34 (C-13).
● Compound cassipourol: colorless oil, Rf = 0.5 (n-hexane:DCM =
3:1.5), [α]
25
D = + 10.9° (CHCl3, c 0.1), molecular formula C20H38O
1
H-NMR (500MHz, CDCl3), δ (ppm), J (Hz): 5.41 (1H, qt, J = 1.5, 7.0
Hz, H-14); 4.15 (2H, d, J = 7.0 Hz; H-15); 1.99 (1H, m, H-12a); 1.67 (3H, br
s, H-20); 1.52 (1H, m, H-5); 1.51 (2H, m, H-11); 1.40 (1H, m, H-9); 1.38 (1H,
m, H-10a), 1.38 (1H, m, H-6); 1.34 (2H, m, H-8); 1.29 (1H, m, H-10b); 1.28
(1H, m, H-2α); 1.28 (2H, m, H-7); 1.26 (2H, m, H-3); 1.25 (2H, m, H-4); 1.09
(1H, m, H-12b); 1.04 (1H, m, H-2β); 0.86 (3H, s, H-16); 0.853 (3H, d, J = 7,
H-18); 0.85 (3H, s, H-17); 0.846 (3H, d, J = 7 Hz, H-19).
13
C-NMR (125MHz, CDCl3), δ (ppm): 140.30 (C-13); 123.11(C-14);
59.43 (C-15); 39.87 (C-12); 39.38 (C-2); 37.44 (C-10); 37.37 (C-8); 37.37 (C-
4); 36.67 (C-1); 32.70 (C-9); 32.70 (C-6); 27.98 (C-5); 25.14 (C-11); 24.79
(C-3); 24.47 (C-7); 22.71 (C-16); 22.62 (C-17); 19.75 (C-18); 19.71 (C-19);
16.17 (C-20).
● DIH03 the mixture of trans-communic acid (compound 81) and
(compound 82) cis–communic acid, Yellowish oil, molecular formula
C20H30O2
1
H-NMR compound 81 (500MHz, CDCl3), δ (ppm), J (Hz): 6.32 (1H,
dd, J = 17.0, 10.5 Hz, H-14); 5.41 (1H, t, J = 6.0 Hz, H-12); 5.04 (1H, d, J =
17.0 Hz, H-15a); 4.88 (1H, d, J = 10.5 Hz, H-15b); 4.84 (1H, s, H-17a); 4.47
(1H, s, H-17b); 1.75 (3H, s, H-16); 1.25 (3H, s, H-18); 0.65 (3H, s, H-20).
13
C-NMR compound 81 (125MHz, CDCl3), δ (ppm): 183.90 (C-19);
147.93 (C-8); 141.61 (C-12); 133.90 (C-14); 133.45 (C-13); 109.91 (C-
15); 107.66 (C-17); 56.43 (C-5); 56.29 (C-9); 44.21 (C-4); 40.37 (C-10);
39.27 (C-6); 38.49 (C-1); 37.93 (C-3); 29.04 (C-18); 25.84 (C-7); 23.31
(C-11); 19.93 (C-2); 12.83 (C-20); 11.84 (C-16).
9
1
H-NMR compound 82 (500MHz, CDCl3), δ (ppm), J (Hz): 6.78
(1H, dd, J = 17.5, 11.0 Hz, H-14); 5.31 (1H, t, J = 6.0 Hz, H-12); 5.18
(1H, d, J = 17.5 Hz, H-15a); 5.08 (1H, d, J = 11.0 Hz, H-15b); 4.84 (1H,
s, H-17a); 4.49 (3H, s, H-17b); 1.77 (3H, s, H-16); 1.25 (3H, s, H-18); 0.65
(3H, s, H-20).
13
C-NMR compound 82 (125MHz, CDCl3), δ (ppm): 183.90 (C-19);
147.93 (C-8); 133.86 (C-14); 131.67 (C-12); 131.56 (C-13); 113.25 (C-
15); 107.80 (C-17); 56.70 (C-9); 56.43 (C-5); 44.21 (C-4); 40.43 (C-10);
39.27 (C-6); 38.52 (C-1); 37.93 (C-3); 29.04 (C-18); 25.84 (C-7); 22.28
(C-11); 19.93 (C-2); 19.72 (C-16); 12.83 (C-20).
● Compound 86 (β-sitosterol): colorless solid, Rf = 0.34 (n-
hexane:DCM = 1:3).
● Compound 83 (pimaric acid): colorless solid, Rf = 0.5 (n-
hexane:EtOAc = 4:1), [α]
20
D = + 87.3
o
(CHCl3, c 0.4), t
o
nc = 217-219
o
C
ESI-MS m/z: 303.6 [M+H]
+
,
molecular formula C20H30O2
1
H-NMR (500MHz, CDCl3), δ (ppm), J (Hz): 5.77 (1H, dd, J = 17.0,
10.5 Hz, H-15); 5.22 (1H, s, H-14); 4.90 (1H, dd, J = 17.0, 1.5 Hz, H-
16a); 4.88 (1H, dd, J = 10.5, 1.5 Hz, H-16b); 1.21 (3H, s, H-19); 1.04 (3H,
s, H-17); 0.84 (3H, s, H-20).
13
C-NMR (125MHz, CDCl3), δ (ppm): 184.92 (C-18); 148.91 (C-
15); 136.63 (C-8); 129.13 (C-14); 110.16 (C-16); 50.58 (C-9); 48.84 (C-5);
47.28 (C-40); 38.30 (C-1); 37.74 (C-10); 37.40 (C-13); 37.05 (C-3); 35.48
(C-7); 34.46 (C-12); 26.04 (C-17); 24.91 (C-6); 18.57 (C-11); 18.16 (C-2);
16.78 (C-19); 15.22 (C-20).
● Compound 80 (12,13-dihydroxylabda-8(17),14-dien-19-oic acid):
colorless oil, Rf = 0.2 (n-hexane:EtOAc = 2:1), molecular formula C20H32O4
1
H-NMR (500MHz, CDCl3), δ (ppm), J (Hz): 5.93 (1H, dd, J = 11,
11 Hz, H-14); 5.34 (1H, d, J = 17 Hz, H-15); 5.21 (1H, dd, J = 10.5, 10.5
Hz, H-15); 4.85 (1H, s, H-17a); 4.44 (1H, s, H-17b); 3.48 (1H, d, J = 10.5
Hz, H-12); 2.4 (1H, m, H-7); 2.18 (1H, d, J = 13 Hz, H-3); 2.03 (1H, m, H-
9); 1.97 (1H, m, H-7); 1.85 (1H, m, H-2α); 1.81-2.0 (2H, m, H-6); 1.79
(1H, m, H-1); 1.6 (1H, m, H-11); 1.53 (1H, m, H-2β); 1.4 (1H, m, H-5);
1.4 (1H, m, H-11); 1.33 (3H, s, H-16); 1.24 (3H, s, H-18); 1.15 (1H, m, H-
1); 1.07 (1H, m, H-3); 0.59 (3H, s, H-20).
13
C-NMR (125MHz, CDCl3), δ (ppm): 183.04 (C-19); 148.36 (C-
19); 140.88 (C-8); 114.57 (C-15); 106.71 (C-17); 75.98 (C-13); 75.84 (C-
12); 56.24 (C-5); 51.65 (C-9); 44.21 (C-4); 40.13 (C-10); 39.03 (C-1);
38.70 (C-7); 37.97 (C-3); 29.02 (C-16); 26.06 (C-6); 24.54 (C-18); 19.87
(C-2); 12.95 (C-20).
10
2.3.2. Fokienia hodginsii
Figure 2.4. Schematic diagram showing steps for the isolation of pure
compounds from the twig and leaves of Fokienia hodginsii
● Compound 81 (trans-communic acid) colorless solid, Rf = 0.5 (n-
hexane: EtOAc = 4:1), [α]
25
D = +38
o
(MeOH, c 0.1), t
o
nc = 130-132
o
C,
molecular formula C20H30O2
1
H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 6.34 (1H, dd, J = 17.5,
17 Hz, H-14); 5.40 (1H, t, J = 6.5 Hz, H-12); 5.04 (1H, d, J = 17.5 Hz, H-
15a); 4.88 (1H, d, J = 9.5 Hz; H-15b); 4.85 (1H, br s, H-17a); 4.47 (1H, br s,
H-17b); 1.75 (3H, s, H-16); 1.22 (3H, s, H-18); 0.70 (3H, s, H-20).
13
C-NMR (125MHz, CD3OD), δ (ppm): 181.20 (C-19); 149.55 (C-8);
142.85 (C-14); 134.89 (C-12); 134.55 (C-13); 110.15 (C-15); 107.95 (C-17);
11
57.90 (C-9); 57.43 (C-5); 45.14 (C-4); 41.39 (C-10); 40.62 (C-1); 39.65 (C-3);
39.34 (C-7); 29.62 (C-18); 27.31 (C-6); 24.32 (C-11); 21.20 (C-2); 13.43 (C-
20); 12.02 (C-16).
● Compound 86 (β-sitosterol): colorless solid, Rf = 0.34 (n-
hexane:DCM = 1:3).
● Compound 92 (totarolone): colorless solid, Rf = 0.5 (n-
hexane:EtOAc = 4:1), [α]
25
D = +101.5
o
(MeOH, c 0.1), t
o
nc = 188-189
o
C,
molecular formula C20H28O2.
1
H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 6.95 (1H, d, J = 8.5
Hz, H-11); 6.57 (1H, d, J = 8.5 Hz, H-12); 3.26 (1H, s, H-15); 1.34 (3H, d, J
= 3.5 Hz, H-16); 1.35 (3H, d, J = 3.5 Hz, H-17); 1.28 (3H, s, H-18); 1.16
(3H, s, H-19); 1.14 (3H, s, H-20).
13
C-NMR (125MHz, CD3OD), δ (ppm): 220.56 (C-3); 155.12 (C-
13); 140.11 (C-9); 134.93 (C-8); 132.05 (C-14); 124.81 (C-11); 115.58 (C-
12); 51.52 (C-5); 48.32 (C-4); 39.62 (C-1); 38.39 (C-10); 35.79 (C-2);
30.14 (C-7); 28.85 (C-15); 27.30 (C-18); 25.07 (C-20); 21,68 (C-6); 21,47
(C-19); 20,58 (C-16); 20,51 (C-17).
● Compound 90 (15-nor-labda-8(17),12E-diene-14-carboxaldehyde-
19-oic acid): colorless oil, Rf = 0.5 (n-hexane:EtOAc = 4:1), molecular
formula C19H28O3
1
H-NMR (500MHz, CD3OD): δ (ppm), J (Hz): 9.31 (1H, d, J = 3 Hz; H-
15); 6.56 (1H, ddd, J = 3, 3, 6 Hz; H-12); 4.86 (1H, s, H-17a); 4.44 (1H, s, H-
17b); 1.76 (3H, d, J = 1.1 Hz, H-16); 1.24 (3H, s, H-18); 0.71 (3H, s, H-20).
13
C-NMR (125MHz, CD3OD), δ (ppm): 197.07 (C-14); 181.13 (C-19);
158.69 (C-12); 149.57 (C-8); 140.01 (C-13); 108.03 (C-17); 57.25 (C-5); 57.15
(C-9); 45.15 (C-4); 41.38 (C-10); 40.55 (C-1); 39.42 (C-3); 39.26 (C-7); 29.54
(C-18); 27.24 (C-6); 25.73 (C-11); 21.14 (C-2); 13.37 (C-20); 9.29 (C-16).
● Compound 91 (13-oxo-15,16-dinor-labda-8(17),11E-diene-19-oic
acid): colorless oil, Rf = 0.57 (n-hexane:EtOAc = 4:1), [α]
25
D = +20.5
o
(MeOH, c 0.1), molecular formula C18H26O3
1
H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 7.01 (1H, dd, J = 10.5,
10 Hz, H-11); 6.10 (1H, d, J = 16 Hz, H-12); 4.85 (1H, H-17a); 4.45 (1H, s,
H-17b); 2.6 (1H, dd, J = 10.5 Hz, H-9); 2.47 (1H, m, H-3α); 2.29 (3H, s, H-
16); 2.18 (1H, m, H-7α); 2.04 (1H, m, H-3β); 1.98 (2H, m, H-6); 1.89 (1H, m,
H-2α); 1.48 (1H, m, H-1α); 1.45 (1H, m, H-2β); 1.33 (1H, m, H-5); 1.24 (3H,
s, H-18); 1.09 (1H, m, H-1β); 1.07 (1H, m, H-7β); 0.86 (3H, s, H-20).
13
C-NMR (125MHz, CD3OD), δ (ppm): 200.88 (C-13); 180.12 (C-
19); 149.89 (C-8); 148.67 (C-11); 134.58 (C-12); 108.87 (C-17); 61.35 (C-
9); 56.38 (C-5); 45.08 (C-4); 42.13 (C-1); 40.95 (C-10); 39.31 (C-3); 38.24
12
(C-7); 29.42 (C-18); 27.11 (C-14); 26.33 (C-6); 20.86 (C-2); 14.14 (C-20).
● Compound 93 (3β-hydroxytotarol): colorless oil, Rf = 0.55 (n-
hexane:EtOAc = 4:1), [α]
25
D = +29
o
(MeOH, c 0.1),
molecular formula
C20H30O2
1
H-NMR (500MHz, CD3OD),δ (ppm), J (Hz): 6.92 (1H, d, J = 8.5
Hz, H-11); 6.53 (1H, d, J = 8.5 Hz, H-12); 4.1 (1H, d, J = 7 Hz, H-3); 3,24
(1H, dd, J = 5, 5 Hz, H-15); 2.97 (1H, dd, J = 6; 6 Hz, H-7α); 2.71 (1H,
dd, J = 4, 7.5 Hz, H-7β); 2.30 (1H, m, H-1α); 2.03 (1H, s, H-6α); 19.6 (2H,
m, H-2); 1.82 (1H, m, H-6β); 1.42 (1H, m, H-1β); 1.35 (3H, H-17); 1.32
(3H, H-16); 1.29 (1H, m, H-5); 1.17 (3H, s, H-18); 1.07 (3H, s, H-19);
0.88 (3H, s, H-20).
13
C-NMR (125MHz, CD3OD), δ (ppm): 155.32 (C-13); 142.29 (C-9);
134.89 (C-8); 132.07 (C-14); 123.78 (C-11); 115.26 (C-12); 79.58 (C-3);
50.93 (C-5); 39.87 (C-4); 39.27 (C-1); 38.56 (C-10); 30.29 (C-7); 28.96 (C-2);
28.76 (C-19); 25.61 (C-20); 20.56 (C-16&C-17); 20.43 (C-6); 16.08 (C-18).
● Compound 80 (12,13-dihydroxylabda-8(17),14-dien-19-oic acid)
colorless solid, Rf = 0.5 (n-hexane:EtOAc = 4:1), molecular formula
C20H32O4
● Compound 94 [(9S)-drummondol]: colorless oil, Rf = 0.5 (n-
hexane:EtOAc = 4:1), molecular formula C13H20O4
1
H-NMR (500MHz, CD3OD) δ (ppm), J (Hz): 6.19 (1H, dd, J = 16.5,
5.5 Hz, H-8); 6.05 (1H, d, J = 16.5 Hz, H-7); 4.40 (1H, dq, J = 5.2, 6.5 Hz,
H-9); 3.93 (1H, dd, J = 8, 2.5 Hz, H-11α); 3.67 (1H, d, J = 8 Hz, H-11β);
2.68 (1H, dd, J = 18, 2.5 Hz; H-2α); 2.37 (1H, dd, J = 18, 2.5 Hz, H-2β);
2.80 (1H, d, J = 17.5 Hz, H-4α); 2.45 (1H, dd, J = 17.5, 2.5 Hz; H-4β);
1.30 (3H, d, J = 6.5 Hz, H-10); 1.20 (3H, s, H-13); 0.98 (3H, s, H-12).
13
C-NMR (125MHz, CD3OD), δ (ppm): 211.23 (C-3); 140.68 (C-8);
125.66 (C-7); 87.45 (C-5); 82.34 (C-6); 78.39 (C-11); 68.87 (C-9); 53.94
(C-4); 53.23 (C-2); 48.49 (C-1); 23.97 (C-10); 19.17 (C-13); 15.65 (C-12).
● Compound 95 (vomifoliol): colorless oil, Rf = 0.5 (n-
hexane:EtOAc = 4:1), molecular formula C13H20O3
1
H-NMR (500MHz, CD3OD), δ (ppm), J (Hz): 5.90 (1H, d, J = 1 Hz,
H-4); 5.83 (1H, d, J = 15.5 Hz, H-8); 5.79 (1H, d, J = 15.5 Hz, H-7); 4.34
(1H, dq, J = 6.5, 4 Hz, H-9); 2.54 (1H, d, J = 17 Hz, H-2α); 2.18 (1H, d, J =
17 Hz, H-2β); 1.95 (3H, d, J = 1 Hz, H-13); 1.26 (3H, d, J = 7 Hz, H-10);
1.06 (3H, s, H-12); 1.03 (3H, s, H-11).
13
C-NMR (125MHz, CD3OD), δ (ppm): 201.34 (C-3); 167.53 (C-5);
136.91 (C-8); 130.08 (C-7); 127.10 (C-4); 79.97 (C-6); 68.72 (C-9); 50.72
(C-2); 42.43 (C-1); 24.48 (C-11); 23.81 (C-10); 23.47 (C-12); 19.59 (C-13).
13
Chapter 3: RESULTS AND DISCUSSION
3.1. Determination the chemical structure of isolated compounds
from Dacrycarpus imbricatus
13 Compounds have been isolated and determined the structure from
Dacrycarpus imbricatus, including: 3 steroid 86, 87 and 88; 1 flavonoid
89; 1 sugar saccaroza (GB2), 1 secquiterpene 85; 7 diterpene, 3 compound
diterpene labdane 80; 81 and 82, 2 compound diterpene abietane 83, 84
and 2 diterpene monocycle 78 and 79.
● Compound 78 and 79: Cassipourol and cassipouryl
hexadecanoate (new compound)
Compounds 78 and 79 were obtained as colorless semi-liquid
compounds. The molecular formula of 78 was established as C20H38O from
its HR-ESI-MS in combination with
13
C NMR, and DEPT spectroscopic
data. The
13
C NMR and DEPT spectra indicated the presence of 20
14
carbons in this compound including 5xCH3, 9xCH2, 4xCH, 2xCq. These
spectra also revealed the existence of the only one double bond (C 140.3,
123.1) in the structure suggesting compound 78 must be a monocyclic
diterpene. The
l
H NMR showed signals of an olefinic proton as a quartet
triplets at 5.41 (J = 1.5, 7.0 Hz); two oxymethylene protons as a doublet
at H 4.15 (2H, J = 7.0 Hz). Remaining proton signals in the
l
H NMR
spectrum were extremely overlapped, thus the chemical shift assignments
were determined by HSQC experiment. Namely, three methyl singlets at
H 1.67, 0.86, 0.85 correlated with the carbon signals at C 16.2, 22.6,
22.7, respectively in the HSQC spectra. In addtion, it could be assigned
for two methyl doublets at H 0.846; 0.853/C19.7, 19.8; eight methylenes
and three methines between 1.04 and 1.99 (Table 1). A comparison of
the
1
H and
13
C NMR data of 78 with those of cassipourol indicated that
they were identical [45]
A comparison of the
1
H and
13
C NMR data of 79 with those of 78
indicated that they were almost identical except for the presence of signals
for an aliphatic long-chain acid derivative. This group was determined
through the apperance of one carbonyl ester (C 173.9), fourteen
methylenes (between C 29.2 and 29.7 ppm) and one methyl group (C
14.1) beside the 20 carbons of cassipourol in the
13
C NMR spectrum of 79
(Table 3.1). Similarly, it could be seen not only the resonances of 78 but
also the resonances of methyl triplet at H 0.87 (J = 7 Hz), strong broad
singlet at H ca 1.25 and a triplet at H 2.29 (J = 7.5 Hz) in the
1
H NMR
of 79. The singlet at 1.25 correlated to the signal of methylene carbon (at
C ca. 29 ppm), while triplet at H 2.29 (J = 7.5 Hz) connected to a shifted
downfield methylene at C 34.4 in the HSQC spectrum. The HR-ESI-MS
exhibited molecular ion peak at m/z 555.5126 [M+Na]
+
(calcd for
C36H68NaO2, 555.5117) establishing a molecular formula of C36H68O2 to
compound 79 and the fragment at m/z 318.2965 (C20H39NaO required
318.2899) indicating the presence of a cassipouryl moiety. These data
allowed to identify aliphatic chain as a hexadecanoic acid derivative. The
esterification of this acid with cassipourol on C-15 was suggested by the
chemical shift of C-15 at C 61.19 (shifted downfield, +1.76 ppm due
to este RCOO group). Furthermore, it was also confirmed by the key
HMBC correlations H-14/C-12, C-15; H-15/C-14, C-13, C-1’ (C=O); H-
2’/C-1’, C-3’, C-4’ and H-3’/C-2’, C-4’.
15
(CH2)11CH3
O1'
2'
3'
4'
O
1
3 5
6
7
9
19
11
13
14
2
4
8 10
17
18
16
HMBC (H/C ) and NOESY ( )
15
H
Figure 3.4. Selected HMBC and NOESY correlations of compounds 79
The configurations of the chiral centers at C-5, C-6 and three methyl
groups at C-16, C-17, and C-18 of 79 were similar to those of 78 due to
the same phyto origin. This finding was deduced from their almost
identical
13
C NMR values and coupling constants. In addition, it was also
supported by the correlations of Me-16 with H-6 and the Me-17, Me-18
with methylene group H2-7 in the NOESY spectrum. The trans (E
stereochemistry) of the double bond C-13 and C-14 was determined by the
correlation of Me-20 with oxymethylene protons H2-15 (no correlation
between Me-20 with H-14). Consequently, the structure of 79 was
elucidated as cassipouryl hexadecanoate, a new cassipourol dervative.
Table 3.1.:
13
C- and
1
H NMR data for compounds 78 and 79
(125/500 MHz, CDCl3, ppm)
a
C 78 79
C H C H HMBC (HC)
1 36.7 - 36.6 - -
2 39.4 1.28 m, 1.04 m 39.4 1.14 m, 2H C-3; C-4; C-16; C-17
3 24.8 1,26 m 24.5 1.25 m C-2
4 37.4 1,25 m 37.4
d
* 1.25 m C-5; C-18
5 28.0 1.52 m 28.0 1.52 m C-3;
6 32.7 1.38 m 32.8 1.38 m C-4; C-7; C-18
7 24.5 1.28 m 24.8 1.28 m C-6; C-8,
8 37.4 1.34 m 37.3
d
* 1.30* m C-7; C-19
9 32.7 1.40 m 32.7 1.36 m C-8
10 37.4 1.38 m, 1.29 m 37.4 1.25 m, 1.08m C-8; C-9; C-19
11 25.1 1.51 m 25.0 1.60 m C-10; C-12
12 39.9 1.99 m, 1.09 m 39.9 2.00 m, 1.08 m C-20; C-11;
C-10; C-14; C-13
13 140.3 - 142.6 - -
14 123.1 5.41 qt (1.5.
7.0)
118.2 5.33 t (7.0) C-20; C-12; C-15 (w)
15 59.4 4.15 d (7.0) 61.2 4.58 d (7.0) C-14; C-13; C-1’ (C=O);
16 22.7
b
0.86 s 22.7
e
0.86 s* C-1; C-2; C-17
17 22.6
b
0.85 s 22.6
e
0.85 s* C-1; C-2; C-16;
18 19.8
c
0.853b d (7) 19.7
f
0.845 d (7) C-5
16
19 19.7
c
0.846 d (7) 19.7
f
0.838 d (7) C-8; C-9
20 16.2 1.67 br s 16.4 1.69 br s C-12; C-14
C-1’ - - 173.9 - -
2’ - - 34.4 2.29 t (7.5) C-1’, C-3’, C-4’
3’ - - 24.8 1.58-1.60 m C-2’, C-4’
4’-13’ - - 29.7 -
29.2
1.25 m, 26 H *
14’ - - 31.9 1.25 s *
15’ - - 22.7 1.25* br s *
16’ - - 14.1 0.87 t (7) *
a
Assignments made on the basis of HSQC and HMBC spectroscopic
data and in comparison with the literature values [45]
b-h
values having the same superscript in the respective columns may
be interchanged.
* Overlap signals (of C1’-C16’= hexadecanoate).
Figure 3.5. HR-ESI-MS spectrum of compound 79
17
Figure 3.6.
1
H-NMR spectrum of compound 79 (CDCl3, 500MHz)
Figure 3.7.
13
C-NMR spectrum of compound 79 (CDCl3, 125 MHz)
18
Figure 3.8. HSQC spectrum of compound 79
Figure 3.8. HSQC spectrum of compound 79
19
Figure 3.8. HSQC spectrum of compound 79
Figure 3.9. HMBC spectrum of compound 79
20
Figure 3.10. COSY spectrum of compound 79
Figure 3.11. NOESY spectrum of compound 79
3.2. Determination the structure of isolated compounds from
Fokienia hodginsii
9 Compounds have been isolated and determined the structure from
Fokienia hodginsii, including: 1 steroid 86; 6 diterpene, 2 compound
21
abietane diterpene 92, 93, 2 compound labdane diterpene 81, 80, 2 nor-
labdane 90, 91; 2 megastigmen 94 and 95.
● Compound 92: Totarolone
The
1
H NMR spectrum of 92 revealed two aromatic ortho-coupled
protons at δH 6.95 (1H, d, J = 8.5 Hz, H-11); 6.58 (1H, d, J = 8.5 Hz, H-
12), three singlet methyl groups at δH 1.29 (3H, s, H-20); 1.17 (3H, s, H-
19); 1.14 (3H, s, H-18), an isopropyl group attached to a benzene ring by
two doublet methyl signals at δH 1.34, 1.33 (each 3H, J = 6.5 Hz) and one
methine proton at δH 3.27 (1H, br s, H-15), and aliphatic protons in the
range from δH 1.73 to 3.03 ppm. Its
13
C-NMR and DEPT spectra showed
signals of 20 carbon atoms including a ketone group, 5xCH3, 4xCH2,
4xCH, and 6xCq. The presence of an aromatic ring was confirmed by two
methine signals at δC 124.81 (C-11), 115.58 (C-12); one quaternary carbon
bonded to an oxygen atom at δC 155.12 (C-13) and three other quaternary
carbons at δC 140.11 (C-9), 134.93 (C-8), 132.05 (C-14). The HMBC
correlations observed between H-16, H-17 and C-14; H-11 and the C-13
indicated that the isopropyl group was at C-14, and the phenolic hydroxyl
group at C-13. Thus, the structure of 92 was concluded to be 13-hydroxy-
8,11,13-totaratrien-3-one (3-oxototarol, 3-ketototarol). This compound
showed the strong activity against some gram-positive and gram-negative
bacteria as well as against some human pathogenic fungi [87-91].
Figure 3.58.
1
H-NMR spectrum of compound 92 (500MHz, CD3OD)
22
Figure 3.59.
13
C-NMR spectrum of compound 92 (125 MHz, CD3OD)
3.3. Biological activities of some isolated compounds
Some of the compounds of Dacrycarpus imbricatus and Fokienia
hodginsii: pimaric acid (83), lambetic acid (84), 12,13-dihydroxylabda-8(17),14-
dien-19-oic acid (80), trans–communic acid (81), 15-nor-labda-8(17),12E-
diene-14-carboxaldehyde-19-oic a
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