2.2. Structural Determination
The structures of the isolates were determined by the analysis of their spectral data (1H-NMR, 13C-NMR, DEPT, HSQC, HMBC, 1H-1H COSY, ultraviolet, infrared, and mass spectrometry).
The HR-ESI-MS of compound
1 showed a pseudo-molecular ion peak at
m/z 435.1444 [M − H]
−, consistent with the molecular formula of C
25H
24O
7 (Calcd. for C
18H
23O
7, 435.1438), containing 14 degrees of unsaturation. The IR spectrum revealed the absorptions of hydroxyl (3331 cm
−1), and aromatic (1603, 1455 cm
−1) functions. The UV absorption bands at 285 nm suggested that
1 possessed a benzaldehyde or acetophenone functional group. The
13C-NMR and DEPT spectra revealed that compound
1 was categorized into three methoxyl carbons at δ
C 56.1, 56.2, and 56.9; one methylene carbon at δ
C 23.2; 10 methine carbons at δ
C 92.5, 99.1, 107.0, 116.2, 120.3, 126.0, 127.7, 131.2, 131.5, and 138.5; as well as 11 quaternary carbons at δ
C 105.9, 108.2, 118.1, 128.2, 155.6, 161.2, 162.6, 164.1, 164.4, 166.4 and 193.5. Three sections (I–III,
Figure 2) were established from key cross-peaks in the
1H-
1H COSY spectrum (H-5/H-6, H-7/H-8, and H-19/H-20/H-21/H-22). In the HMBC spectrum, on the one hand, the cross-peaks of H-16 (δ
H 3.89) with C-11 (δ
C 166.4), C-12 (δ
C 108.2), C-13 (δ
C 164.4), C-17 (δ
C 128.2), C-18 (δ
C 155.6), and C-22 (δ
C 131.5) and that of H-14 (δ
H 6.20) with C-10 (δ
C 147.7), C-12, C-13, and C-15 (δ
C 145.9) revealed that compound
1 possessed a diphenylmethane structure (
Figure 2). On the other hand, the correlations between H-7 (δ
H 8.02) and C-1 (δ
C 118.1), C-2 (δ
C 161.2), C-6 (δ
C 131.2, and C-9 (δ
C 193.5), as well as between H-3 (δ
H 6.64) with C-1 and C-5 (δ
C 107.0) allowed a cinnamaldehyde unit in
1. Additionally, three methoxy groups were substituted at the C-2, C-4, and C-15 positions due to their respective HMBC correlations. Moreover, the long-range correlations between H-8 and C-9 and C-10 decided the linkage of diphenylmethane and cinnamaldehyde units. The established structure of
1 as above was also consistent with a single-crystal X-ray diffraction analysis showing a perspective drawing, as shown in
Figure 3. Accordingly, the structure of
C-benzylated dihydrochalcone derivative (
1) was determined as (
E)-1-(3,5-dihydroxy-4-(2-hydroxybenzyl)-2-methoxyphenyl)-3-(2,4-dimethoxyphenyl) prop-2-en-1-one and was named thorechalcone A.
Compound
2 (7-hydroxy-5,2′,4′-trimethoxyflavanone) was obtained as an amorphous solid with the molecular formula C
18H
20O
7 as analyzed by HRESIMS and had nine degrees of unsaturation. The IR spectrum exhibited absorption bands at 1584 and 1464 cm
−1 for benzyl and strong absorption bands at 3417 cm
−1 for hydroxy together with the UV bands at 230, 250, and 284 nm, which indicated the flavanone skeleton of
2. The
13C-NMR spectrum of
2 showed 18 carbon signals including eight quaternaries (including one conjugated carbonyl carbon at δ
C 187.9), six methines, one methylene, and three methoxyl (δ
C 55.3, 55.6, and 55.7) carbons via the
1H and
13C-NMR and HSQC correlation analyses. The proton signals at δ
H 5.55 (1H, dd,
J = 12.5, 2.5 Hz), 2.95 (1H, dd,
J = 16.5, 12.5 Hz), and 2.67 (1H, dd,
J = 16.5, 2.5 Hz), and the corresponding carbon signals at δ
C 73.0 and 44.0, respectively, were characteristics of the oxygenated methine and methylene groups of a flavanone unit. Moreover, five aromatic protons were evident in the
1H-NMR spectrum, with splitting patterns characteristic of a 1,2,4-trisubstituted aromatic ring (δ
H 6.57, 1H, dd,
J = 8.0, 2.0 Hz; 6.61, 1H, d,
J = 2.0 Hz; and 7.38, 1H, d,
J = 8.0 Hz), and two protons were assigned to H-6 (δ
H 5.95) and H-8 (δ
H 6.06) with w-coupling (
J = 2.5 Hz). The A and B rings of the flavanone framework were established by the HMBC correlation of H-3 with C-2, C-4, C-1′; H-6′ with C-2, C-4′, C-2′; H-6 with C-5, C-7, C-10; and H-8 with C-7, C-9, C-10 (
Figure 2). The chemical shift of C-4 at δ
C 187.9, together with the long-range heteronuclear correlations, indicated the existence of conjugated carbon at C-5 with a methoxyl group, and the other methoxyl groups were substituted at the C-2′ and C-4′ positions of the B ring. The planar structure of
2 was also confirmed by single-crystal X-ray diffraction analysis (
Figure 3). Moreover, the
S configuration of C-2 was deduced based on the CD spectrum of
2 exhibiting negative Cotton effects at 285 nm. Thus, the structure of
2 was elucidated as
S-2-(2,4-dimethoxyphenyl)-7-hydroxy-5-methoxychroman-4-one, possessing the same structure as the reported cerasinone [
15] as assigned only by the
1H-NMR data. However, additional spectroscopic evidence, including the
13C-NMR (
Table 2) and X-ray diffraction data mentioned above were also provided here.
Compound
3 [(2
E)-1-(2,4-Dihydroxy-6-methoxyphenyl)-3-(2,4-dimethoxyphenyl)-2-propen-1-one] was obtained as an amorphous yellow solid C
18H
18O
6; ESIMS
m/z 330 [M − H]
−.
1H-NMR (500 MHz, DMSO) δ
H: 7.89 (d,
J = 16 Hz, 1H); 7.83 (d,
J = 16 Hz, 1H); 7.63 (d,
J = 9 Hz, 1H); 6.65 (d,
J = 2.5 Hz, 1H); 6.62 (dd,
J = 2.5, 8 Hz, 1H); 6.00 (d,
J = 2 Hz, 1H); 5.90 (d,
J = 2.5 Hz, 1H); 3.90 (s, 3H); 3.87 (s, 3H); 3.84 (s, 3H); 3.08 (s, 2H).
13C-NMR (125 MHz, DMSO) δ
C: 191.8, 166.4, 164.6, 162.8, 162.6, 159.9, 137.6, 130.6, 124.8, 116.3, 106.4, 105.1, 98.4, 95.9, 91.6, 55.9, 55.8, 55.5, The data were confirmed by the further comparison of the NMR data with those in the literature [
16].
Compound
4 [(+)-Crotepoxide] was obtained as an amorphous white solid C
18H
18O
8; ESIMS
m/z 362 [M + Na]
+.
1H-NMR (500 MHz, DMSO) δ
H: 7.98 (d,
J = 10.5 Hz, 2H); 7.69 (t,
J = 9.5 Hz, 1H); 7.55 (t,
J = 10 Hz, 1H); 5.74 (d,
J = 2.5 Hz, 1H); 6.62 (dd,
J = 2.5, 8 Hz, 1H); 6.00 (d,
J = 2 Hz, 1H); 5.90 (d,
J = 2.5 Hz, 1H); 3.90 (s, 3H); 3.87 (s, 3H); 3.84 (s, 3H); 3.08 (s, 2H).
13C-NMR (125 MHz, DMSO) δ
C: 191.8, 166.4, 164.6, 162.8, 162.6, 159.9, 137.6, 130.6, 124.8, 116.3, 106.4, 105.1, 98.4, 95.9, 91.6, 55.9, 55.8, and 55.5. The data were confirmed by the further comparison of the NMR data with those in the literature [
17].
Compound
5 [(−)-1,6-Desoxytingtanoxide] was obtained as an amorphous yellow oil C
23H
20O
6; ESIMS
m/z 392 [M + Na]
+.
1H-NMR (500 MHz, CDCl
3) δ
H: 8.05 (dd,
J = 1, 8.5 Hz, 2H); 7.98 (dd,
J = 1.5, 8 Hz, 2H); 7.58 to 7.53 (m, 2H); 7.42 (t,
J = 10 Hz, 2H); 7.38 (t,
J = 8 Hz, 2H); 6.32 (d,
J = 5.5 Hz, 1H); 6.20 (dd,
J = 5, 10 Hz, 1H); 6.06 (dd,
J= 4.5, 9.5 Hz, 1H); 6.02 (d,
J= 6.5 Hz, 1H); 5.74 (ddd,
J= 6, 4, 1.5 Hz, 1H); 4.94 (d,
J= 3 Hz, 2H), 2.03 (s, 3H).
13C-NMR (125 MHz, CDCl
3) δ
C: 170.5, 166.5, 166.0, 133.6, 133.6, 131.6, 130.3, 130.2, 130.1, 128.8, 128.8, 126.4, 126.0, 125.5, 71.7, 70.1, 65.1, 21.3. The data were confirmed by the further comparison of the NMR data with those in the literature [
18].
Compound
6 (
O-Methoxybenzoyl benzoate) was obtained as an amorphous yellow solid C
15H
14O
3; ESIMS
m/z 242 [M + Na]
+.
1H-NMR (500 MHz, CDCl
3) δ
H: 8.11 (dd,
J = 8.5, 1 Hz, 2H); 7.56 (t,
J = 7.5 Hz, 1H); 7.44 to 7.48 (m, 3H); 7.35 (td,
J = 7.5, 1.5 Hz, 1H); 7.0 (td,
J = 1, 7.5 Hz, 1H); 6.94 (d,
J = 8 Hz, 1H); 5.45 (s, 2H); 3.89 (s, 3H);
13C-NMR (125 MHz, CDCl
3) δ
C: 166.6, 157.5, 132.9, 130.5, 129.7, 129.4, 128.3, 124.5, 120.5, 110.5, 62.2, 55.5. The data were confirmed by the further comparison of the NMR data with those in the literature [
19].
Compound
7 (Sandaracopimaric acid) was obtained as an amorphous yellow solid C
20H
30O
2; ESIMS
m/z 302 [M − H]
−.
1H-NMR (500 MHz, acetone-
d6) δ
H: 5.77 (dd,
J = 14, 9 Hz, 1H); 5.23 (s, 1H); 4.9 (dd,
J = 14.5, 1.5 Hz, 1H); 4.86 (dd,
J = 9, 1Hz, 1H); 2.24 (ddd,
J = 2, 4, 11.5 Hz, 1H); 2.09 (m, 1H); 1.97 (d,
J = 10.5, 2 Hz, 1H); 1.82 (s, 3H); 1.79 (s, 3H); 1.78 (s, 3H); 1.63 (overlap)1.56 (overlap), 1.47 (overlap), 1.44 (dd,
J= 10.5, 3.5 Hz, 1H), 1.36 (ddd,
J = 3, 9, 11 Hz, 1H), 1.28 (m, 1H), 1.19 (s, 3H), 1.16 (td,
J = 3, 11 Hz, 1H), 1.04 (s, 3H), 0.87 (s, 3H).
13C-NMR (125 MHz, acetone-
d6) δ
C: 179.1, 148.7, 136.7, 128.8, 109.7, 50.7, 48.9, 46.7, 38.3, 37.6, 37.2, 36.9, 35.4, 34.4, 25.5, 24.7, 18.4, 18.0, 16.6, and 14.7. The data were confirmed by the further comparison of the NMR data with those in the literature [
20].