Z. S. Toh et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
1
0:1) was the only active fraction. The Fr 10 was further purified by
quently, benzoyl chloride (34 mg, 0.0372 mmol) was added and
the mixture was allowed to react overnight at room temperature.
Sephadex LH-20 column (isocratic chloroform/methanol; 1:1) to
obtain dracoflavan B (15 mg). Structural determination of the A
and B type dracoflavan B (Fig. 1) were made by HR-ESI-MS and
Finally, 2 was purified using 250 mm ꢁ 10 mm id., 5
C-18 semi-preparative HPLC column. The products were eluted
using an isocratic eluents with solvent mixture of H O (12.5%)
lM, YMC
1
H NMR, which matches nicely with literature values of the same
2
compound isolated from Daemonorops draco.2
1,24
and methanol (87.5%) at 2.0 mL/min. The A and B type 2 were
collected at 28 and 26 min, respectively. A type 2: White crystals;
UV–Vis kmax = 206 nm and 274 nm; HR-ESI-MS m/z 643.2320 [M
3
.4. Separation of the dracoflavan B diastereomers
+
1
+
35
H] (calculated for C40H O8, 643.2326); H NMR (300 MHz,
Dracoflavan
50 mm ꢁ 10 mm id., 5
B
diastereomers
were
purified
using
acetone) d 8.21 (d, J = 8.5 Hz, 2H, H-18), 7.75 (d, J = 1.5 Hz, 2H,
H-10,14), 7.72 (t, J = 1.3 Hz, 1H, H-16), 7.69 (d, J = 7.0 Hz, 2H,
2
lM, YMC C-18 semi-preparative HPLC col-
0
0
umn. The A and B type dracoflavan B were separated using an iso-
cratic eluents with solvent mixture of H O (25%) and methanol
75%) at 2.0 mL/min. The A and B type dracoflavan B were eluted
H-10 ,14 ), 7.61 (t, J = 7.5 Hz, 2H, H-17), 7.51 – 7.31 (m, 6H, H-
0
0
0
0
2
11,11 ,12,12 ,13,13 ), 6.78 (s, 1H, H-8), 6.20 (s, 1H, H-6 ), 5.07 (dd,
0
(
J = 10.2, 1.6 Hz, 1H, H-2 ), 4.81 (d, J = 3.5 Hz, 1H, H-4), 4.54 (d,
at 46 and 44 min respectively. A type dracoflavan B: white crystal;
J = 5.0 Hz, 1H, H-3-R), 4.30 (dd, J = 5.3, 3.4 Hz, 1H, H-3), 3.79 (s,
ꢀ
0
0
0
UV kmax 208 and 281 nm; HR-ESI-MS m/z 537.1935 [MꢀH] (calcu-
3H, H-5 -OR), 3.59 (s, 3H, H-5), 2.72–2.64 (m, 2H, H-4 a,4 b), 2.29
7, 537.1919); 1H NMR (300 MHz, acetone) d 8.27
–2.21 (m, 1H, H-3 a), 2.09 (s, 3H, H-15), 2.04–1.95 (m, 1H, H-3 b).
0
0
lated for C33
H
29
O
1
(
s, 1H, H-7), 7.74 (d, J = 1.6 Hz, 2H, H-10,14), 7.71 (d, J = 7.6 Hz,
B type 2: H NMR (300 MHz, acetone) d 8.18 (d, J = 8.5 Hz, 2H, H-
0
0
0
0
2
1
4
(
5
H, H-10,14 ), 7.59–7.30 (m, 6H, H-11,11 ,12,12 ,13,13 ), 6.38 (s,
18), 7.77 (d, J = 1.8 Hz, 2H, H-10,14), 7.71 (t, J = 1.3 Hz, 1H, H-16),
0
0
0
0
H, H-8), 6.17 (s, 1H, H-6 ), 5.03 (dd, J = 10.4, 1.8 Hz, 1H, H-2 ),
7.63 (d, J = 7.0 Hz, 2H, H-10 ,14 ), 7.59 (t, J = 7.5 Hz, 2H,, H-17),
0
0
0
.67 (d, J = 3.4 Hz, 1H, H-4), 4.30 (d, J = 5.4 Hz, 1H, H-3-R), 4.16
7.53–7.33 (m, 6H, H-11,11 ,12,12 ,13,13 ), 6.75 (s, 1H, H-8), 6.19
0
0
0
dd, J = 5.3, 3.4 Hz, 1H, H-3), 3.77 (s, 3H, H-5 -OR), 3.54 (s, 3H, H-
(s, 1H, H-6 ), 5.12 (dd, J = 10.5, 2.0 Hz, 1H, H-2 ), 4.86 (d,
J = 3.5 Hz, 1H, H-4), 4.59 (d, J = 5.3 Hz, 1H, H-3-R), 4.35 (dd,
J = 5.3, 3.7 Hz, 1H, H-3), 3.78 (s, 3H, H-5 -OR), 3.42 (s, 3H, H-5),
2.74–2.60 (m, 2H, H-4 a,4 b), 2.33–2.19 (m, 1H, H-3 a), 1.98 (s,
3H, H-15), 1.97–1.87 (m, 1H, H-3 b).
0
0
0
), 2.75–2.64 (m, 2H, H-4 a,4 b), 2.39–2.18 (m, 1H, H-3 a), 2.09 (s,
0
1
0
3
H, H-15), 2.03 – 1.95 (m, 1H, H-3 b). B type dracoflavan B:
H
0
0
0
NMR (300 MHz, acetone) d 8.20 (s, 1H, H-7), 7.73 (d, J = 1.6 Hz,
0
0
0
2
H, H-10,14), 7.62 (d, J = 7.6 Hz, 2H, H-10 ,14 ), 7.51–7.29 (m, 6H,
0
0
0
0
H-11,11 ,12,12 ,13,13 ), 6.36 (s, 1H, H-8), 6.16 (s, 1H, H-6 ), 5.11
dd, J = 10.4, 2.2 Hz, 1H, H-2 ), 4.72 (d, J = 3.3 Hz, 1H, H-4), 4.35 (d,
0
(
3.7. Sulfated dracoflavan B, 3
J = 5.3 Hz, 1H, H-3-R), 4.21 (dd, J = 5.2, 3.5 Hz, 1H, H-3), 3.76 (s,
0
0
0
3
2
H, H-5 -OR), 3.37 (s, 3H, H-5), 2.75–2.59 (m, 2H, H-4 a,4 b), 2.32–
Dracoflavan B (15 mg, 0.0279 mmol) was first dissolved in
0
0
.21 (m, 1H, H-3 a), 1.97 (s, 3H, H-15), 1.87–1.80 (m, 1H, H-3 b).
400
by dissolving sodium methoxide (300 mg, 5.55 mmol) in 885
methanol. Thereafter, 30 methanolic solution (8.9
.0558 mmol) was added to the dracoflavan B and the mixture
lL anhyd THF. Next, 30 % methanolic solution was prepared
lL
3
.5. Acylated dracoflavan B, 1
%
lL,
0
Dracoflavan B (30 mg, 0.0557 mmol) was first dissolved in
60 lL pyridine. Next, acetic anhydride (28.4 mg, 0.279 mmol)
was added to dracoflavan B and the mixture was stirred overnight
was stirred at room temperature for 30 min. Subsequently, sulfur
trioxide triethylamine complex (4 mg, 0.0221 mmol) was added
and the mixture was stirred overnight at room temperature.
7
at room temperature. 1 was purified using 250 mm ꢁ 10 mm id.,
Finally, 3 was purified using 250 mm ꢁ 10 mm id., 5
18 semi-preparative HPLC column. The products were eluted using
an isocratic eluents with solvent mixture of H O (20%) and metha-
lM, YMC C-
5
1
H
lM, YMC C-18 semi-preparative HPLC column. The A and B type
were separated using an isocratic eluents with solvent mixture of
2
2
O (20%) and methanol (80%) at 2.0 mL/min. The A and B type 1
nol (80%) at 2 mL/min. Subsequently, 3 were collected at 14 min. A
were collected at 40 and 38 min respectively. A type 1: White crys-
type 3: White crystal; UV kmax 206 and 279 nm; HR-ESI-MS m/z
+
ꢀ
29 10 ,
H O S
617.1487); 1H NMR
tals; UV kmax 211 and 275 nm; HR-ESI-MS m/z 603.1994 [M+Na]
617.1491 [MꢀH] (calculated for C33
(
calculated for C35
H
32NaO8, 603.1989); 1H NMR (300 MHz, ace-
(300 MHz, acetone) d 7.77 (d, J = 1.6 Hz, 2H, H-10, 14), 7.70 (d,
0
0
0
0
tone) d 7.72 (d, J = 1.6 Hz, 2H, H-10,14), 7.67 (d, J = 7.3 Hz, 2H, H-
J = 7.9 Hz, 2H, H-10 , 14 ), 7.49–7.32 (m, 6H, H-11, 11 , 12, 12 , 13,
0
0
0
0
0
0
0
1
8
0 ,14 ), 7.51–7.31 (m, 6H, H-11,11 ,12,12 ,13,13 ), 6.59 (s, 1H, H-
13 ), 7.13 (s, 1H, H-8), 6.15 (s, 1H, H-6 ), 5.03 (dd, J = 10.3, 1.7 Hz,
0
0
0
), 6.18 (s, 1H, H-6 ), 5.05 (dd, J = 10.2, 1.9 Hz, 1H, H-2 ), 4.76 (d,
1H, H-2 ), 4.71 (d, J = 3.7 Hz, 1H, H-4), 4.19 (d, J = 5.0 Hz, 1H, H-3-
0
J = 3.5 Hz, 1H, H-4), 4.52 (d, J = 5.1 Hz, 1H, H-3-R), 4.27 (dd,
R), 3.76 (s, 1H, H-5 -OR), 3.53 (s, 3H, H-5), 2.73–2.63 (m, 2H, H-
0
0
0
0
J = 5.0, 3.5 Hz, 1H, H-3), 3.77 (s, 3H, H-5 -OR), 3.55 (s, 3H, H-5),
4 a, 4 b), 2.28–2.20 (m, 1H, H-3 a), 2.09 (s, 3H, H-15), 2.00–1.95
(m, 1H, H-3 b). B type 3: H NMR (300 MHz, acetone) d 7.75 (d,
J = 1.4 Hz, 2H, H-10, 14), 7.62 (d, J = 7.3 Hz, 2H, H-10 , 14 ), 7.49–
7.32 (m, 6H, H-11, 11 , 12, 12 , 13, 13 ), 7.10 (s, 1H, H-8), 6.14 (s,
1H, H-6 ), 5.10 (dd, J = 10.6, 2 Hz, 1H, H-2 ), 4.76 (d, J = 3.3 Hz, 1H,
H-4), 4.24 (d, J = 5.3 Hz, 1H, H-3-R), 3.75 (s, 1H, H-5 -OR), 3.35 (s,
3H, H-5), 2.73–2.63 (m, 2H, H-4 a, 4 b), 2.28–2.20 (m, 1H, H-3 a),
2.07 (s, 3H, H-15), 1.89–1.81 (m, 1H, H-3 b).
0
0
0
1
2
.72–2.61 (m, 2H, H-4 a,4 b), 2.28 (s, 3H, H-16), 2.25–2.19 (m,
0
0
0
0
1
H, H-3 a), 2.02 (s, 3H, H-15), 2.02–1.93 (m, 1H, H-3 b). B type 1:
H NMR (300 MHz, acetone) d 7.73 (d, J = 1.6 Hz, 2H, H-10,14),
.62 (d, J = 7.1 Hz, 2H, H-10 ,14 ), 7.51 – 7.33 (m, 6H, H-
1,11 ,12,12 ,13,13 ), 6.57 (s, 1H, H-8), 6.17 (s, 1H, H-6 ), 5.10 (dd,
J = 10.5, 2.1 Hz, 1H, H-2 ), 4.81 (d, J = 3.4 Hz, 1H, H-4), 4.57 (d,
J = 5.1 Hz, 1H, H-3-R), 4.31 (dd, J = 5.0, 3.5 Hz, 1H, H-3), 3.77 (s,
H, H-5 -OR), 3.38 (s, 3H, H-5), 2.78–2.54 (m, 2H, H-4 a,4 b), 2.26
1
0
0
0
0
0
0
0
7
1
0
0
0
0
0
0
0
0
0
0
0
0
0
3
(
1
0
s, 3H, H-16), 2.27–2.19 (m, 1H, H-3 a), 1.92 (s, 3H, H-15), 1.89–
3.8. Acetyl-protected galloylated dracoflavan B, 4
0
.76 (m, 1H, H-3 b).
The hydroxyl group of gallic acid (1 g, 5.88 mmol) was pro-
tected by reacting with acetic anhydride (3.4 mL, 35.28 mmol) in
3
.6. Benzoylated dracoflavan B, 2
the presence of catalytic amount of sulfuric acid (32 lL) at 75 °C
Dracoflavan B (20 mg, 0.0372 mmol) was dissolved in 1 mL
for 1 min. Subsequently, a clear yellow solution was obtained
and cooled to room temperature. Next, 10 mL of water was added
and stirred at room temperature for 2.5 h to obtain a white crystal
product. Later, the acetyl-protected gallic acid was collected using
dichloromethane. Thereafter, triethylamine (TEA) (24 mg,
.0372 mmol) was added to dracoflavan B and the deprotonation
reaction was performed at room temperature for 30 min. Subse-
0