Z.-S. Wang, et al.
Fitoterapia 134 (2019) 404–410
Table 1
ν
max: 3444, 2924, 2818, 1718, 1571, 1471, 1379, 1292, 1046,
1
−1
HRESIMS at m/z: 684.3619 [M + H]+ (calcd for
H NMR spectral data in CDCl
3
for compounds 1–4 (600 MHz, δ in ppm, J in
772 cm
.
1
Hz).
C
40
H
50
N
3
O
7
, 684.3649). H NMR (600 MHz, CDCl
3
) date, see Table 1;
13
C NMR (150 MHz, CDCl
3
) data, see Table 2.
No.
1
2
3
4
1
2.86 m
2.08 m
2.14 m
1.47 m
1.12 m
1.34 m
1.30 m
1.76 m
1.96 d (7.8)
2.08–2.14 m
2.31 m
1.75 m
2.27 m
1.59 m
3.62 t (4.8)
1.85 m
2.29 m
3.13 dd (9.6)
2.85 m
1.66 m
2.08 m
1.51 m
1.57 m
1.23 m
1.29 m
1.66 m
2.20 m
2.25 m
2.18 m
1.75 m
2.31 m
1.65 m
3.50 m
2.13 m
2.68 m
3.11 dd
2.77–2.87 m
1.37 m
1.75 m
1.14 m
1.50 m
1.35 m
1.34 m
1.71 m
2.01 d (7.6)
2.16 m
1.58 m
1.85 m
2.07 m
2.58–2.62 m
4.80 m
1.82 m
2.35 dd (9.6)
3.14 dd (8.4)
2.83–2.88 m
1.27 m
1.73 m
1.17 m
1.57 m
1.34 m
1.35 m
1.79 m
2.16 m
2.27 m
2.22 m
1.25 m
2.31 m
2.24 m
3.48–3.52 m
1.96 m
2.10 m
3.14–3.17 m
2
.3.2. Brevicatine B (2)
White, amorphous powder; α
2
2
3
3
5
6
6
7
9
1
1
1
1
1
1
1
1
a
b
a
b
20
D
3
= +24.8 (c = 0.40, CHCl ). IR
νmax: 2926, 2855, 2819, 1723, 1687, 1571, 1451, 1292, 1091,
−
1
HRESIMS at m/z: 726.3749 [M + H]+ (calcd for
773 cm
C
42
13
.
1
H
52
N
3
O
8
, 726.3754). H NMR (600 MHz, CDCl
3
) date, see Table 1;
a
b
3
C NMR (150 MHz, CDCl ) data, see Table 2.
2.3.3. Brevicatine C (3)
White, amorphous powder; α
20
0
3
= +28.1 (c = 0.40, CHCl ). IR
D
2a
2b
3
νmax: 3442, 2926, 2817, 1724, 1687, 1571, 1471, 1248, 1090,
−1
HRESIMS at m/z: 712.3607 [M + H]+ (calcd for
7
73 cm
.
1
C
41
H
50
N
3
O
8
, 712.3598). H NMR (600 MHz, CDCl
3
) date, see Table 1;
4
13
5a
5b
6
3
C NMR (150 MHz, CDCl ) data, see Table 2.
2.3.4. Brevicatine D (4)
(
8.4)
20
White, amorphous powder; α
D
3
= +16.3 (c = 0.40, CHCl ). IR
1
7
2.83 m
2.75 m
2.92 br s
3.84 ABq (10.6) 3.83 ABq (10.4)
2.71 br s
νmax: 3441, 2925, 2819, 1688, 1571, 1471, 1291, 1269, 1196,
1
8a
3.80 ABq (10.8) 3.81 ABq
10.4)
3.90 ABq (10.8) 3.90 ABq
10.4)
1.88 m
2.36 m
2.27 m
2.42 m
1.01 t (7.2)
3.20 s
−
1
HRESIMS at m/z: 712.3894 [M + H]+ (calcd for
(
772 cm
C
42
13
.
1
8b
3.93 ABq (10.6) 3.90 ABq (10.4)
1
H
54
N
3
O
7
, 712.3962). H NMR (600 MHz, CDCl
3
) date, see Table 1;
(
3
C NMR (150 MHz, CDCl ) data, see Table 2.
1
1
2
2
2
1
8
8
8
1
1
1
3
9a
9b
1a
1b
1.86 m
2.35 m
2.28 m
2.43 m
0.99 t (7.2)
3.18 s
1.92 m
2.41 m
2.32 m
2.45 m
1.02 t (7.2)
3.19 s
1.90 m
2.37 m
2.29 m
2.43 m
1.01 t (7.2)
3.20 s
3.25 m
2.4. Synthesis
2
2.4.1. 2-methyl-4H-benzo[d][1,3]oxazin-4-one (6)
-OMe
-OCH
-OCH
-OAc
The anthranilic acid 137.1 mg (1.0 mmol) was suspended in acetic
2
2
CH
CH
3
3
anhydride 2 mL. Upon heating all material was dissolved, and the
mixture was refluxed until TLC indicated the reaction was finished
1.06 t (7.8)
1.91 s
4-OAc
2.05 s
(
generally 2 h). Then, the solvent was removed in vacuo to give a solid.
4-OMe
6-OMe
’
3.38 s
3.28 s
8.24 d (7.8)
3.39 s
3.29 s
8.17 d
3.38 s
3.33 s
8.24 t (7.8)
The crude material was recrystallized from hexane and ethyl acetate.
The product was collected by filtration and washed with hexane.
Yield = 90.3%, m.p. 82–83 °C. H NMR (DMSO-d
J = 7.6 Hz, 1H), 7.48 (td, J = 1.5 and 7.6 Hz, 1H), 7.20–7.16 (m, 2H),
3.21 s
8.25 d (7.8)
1
(
7.8)
6
, 400 MHz) δ 7.86 (d,
4’
5’
6’
7.60 d (7.2)
7.71 d (7.8)
7.25 t (7.8)
7.67 t (7.8)
7.75 t (7.2)
7.20 d
7.70 t (7.8)
7.77 d (7.2)
7.27 d (7.8)
7.72 d (7.8)
7.76 t (7.8)
7.28 d (7.8)
2
.15 (s, 3H).
(
7.8)
8.10 d
7.8)
7.63 d
8.4)
2
.4.2. 2-(2-Methyl-4-oxoquinazolin-3-yl) benzoic acid (7)
2-Methyl-4H-benzo[d][1,3]oxazin-4-one (6) 161.2 mg (1.0 mmol)
5
”
”
8.20 d (7.8)
7.45 t (7.8)
8.20 d (7.8)
7.47 t (7.2)
8.20 d (7.8)
7.46 t (7.2)
(
6
and anthranilic acid 164.1 mg (1.2 mmol) were dissolved in glacial
acetic acid 5 mL. The solution was refluxed until TLC indicated the
reaction was finished (about 8 h). After cooling to room temperature,
the mixture was concentrated in vacuo to give a solid. Then, the crude
material was recrystallized and the product 7 was collected by filtration
and washed with ethyl acetate. Yield = 75.5%, m.p. 256–258 °C.
NMR (DMSO-d
(
7
8
”
”
7.75 t (7.8)
7.69 t (7.8)
7.54 t (7.8)
7.70 d
(
7.73 t (7.8)
7.64 t (7.8)
7.70 t (7.8)
7.62 t (7.8)
7.8)
2”-OMe
2.21 s
2.21 s
2.21 s
2.21 s
1
H
6
, 400 MHz) δ 13.11 (s, 1H), 8.10 (m, 2H), 7.82 (m, 2H),
Sichuan Province, P.R. China, where a voucher specimen (No.
ZN361520160821) was deposited.
7
8
.67 (m, 2H), 7.57 (dd, J = 0.7 and 7.5 Hz, 1H), 7.50 (td, J = 0.9 and
.3 Hz, 1H), 2.11 (s, 3H).
2.3. Extraction and isolation
2.4.3. Brevicatine A (1)
A solution of the scaconine (82.3 mg, 0.194 mmol), 2-(2-methyl-4-
oxoquinazolin-3-yl) benzoic acid (65.1 mg, 0.232 mmol), EDC·HCl
56.4 mg, 0.293 mmol), and DMAP (19.1 mg, 0.156 mmol) was added
in dichloromethane (10 mL), and stirred at room temperature for 10 h.
The solvent was removed by distillation under reduced pressure and
concentrated to give a crude yellow solid. The crude material was se-
parated by flash column chromatography using 7% methanol/di-
chloromethane as eluent to yield the desired product 1 as a white solid
The powdered roots of A. brevicalcaratum (2.1 kg) were extracted
with 95% EtOH (3 × 10 L) at room temperature for 3 days. The crude
extract (300 g) was suspended in H O, the pH was adjusted to 3.0 using
0% HCl solution, and the suspension was extracted with petroleum
(
2
1
ether (4 × 2 L) and EtOAc (4 × 2 L), successively. Then, the pH of the
aqueous layer was adjusted to 9 using an aqueous ammonia solution
and then extracted with CH
40 g). By repeated column chromatography, the alkaloid extract was
separated to afford compounds 1 (78 mg), 2 (56 mg), 3 (63 mg) and 4
37 mg) (Scheme S1, Supporting information).
2 2
Cl (4 × 2 L) to obtain the alkaloid extract
(
(
100.2 mg, 76.7%).
(
2
.4.4. Brevicatine B (2)
A solution of compound 1 (36.2 mg, 0.052 mmol) and TsOH
2
.3.1. Brevicatine A (1)
White amorphous powder; α
(20.1 mg, 0.12 mmol) in acetic anhydrate (4 mL) was heated to reflux
for 4 h. The acetic anhydrate was removed by simple distillation, and
2
0
D
= −10.6 (c = 0.40, CHCl
3
). IR
405