2.1. Chemical Structure Elucidation
Compound
1 was isolated as colorless needle, whose molecular formula of C
8H
13O
5N was determined by the positive HRESIMS (
m/
z 204.0870 [M + H]
+, calcd. 204.0866). The IR spectrum indicated the presence of amino (3451, 3368 cm
−1), hydroxyl (3242, 3191 cm
−1), and carbonyl (1635 cm
−1) groups. The
1H NMR spectrum of
1 (
Table 1) indicated protons of one amino at δ
H 6.83 (s, 1H) and 6.30 (s, 1H), three hydroxyls at δ
H 5.94 (s, 1H, exchangeable), 4.94 (s, 1H, exchangeable) and 4.50 (s, 1H, exchangeable), two oxygenated methines at δ
H 4.22 (s, 1H) and 3.72 (s, 1H), one oxygenated methyl at δ
H 3.46 (s, 3H), and one methyl at δ
H 0.83 (s, 3H). The
13C NMR spectrum (
Table 1) revealed the presence of one α,β-unsaturated ketone at δ
C 185.62, 156.36 and 126.93, one methoxyl at δ
C 58.13, one methyl at δ
C 13.39, two oxygenated methines at δ
C 76.99 and 71.55, and one quarternary carbon bearing an oxygen atom at δ
C 77.97. These data presumed compound
1 to be a cyclohexenone with one amino, three hydroxyls, one methoxyl, and one methyl substituents. The assignments of protons attached to their corresponding carbons were readily accomplished by the HSQC technique, which also confirmed the quaternary type of carbons at δ
C 185.62, 156.36, 126.93, and 77.97. In the HMBC spectrum, the correlations from the proton at δ
H 5.94(4-OH) to the carbon at δ
c 156.36 (C-3), from the proton at δ
H 3.46 (2-OCH
3) to the carbon at δ
c 126.93 (C-2), and from the proton at δ
H 4.22 (H-4) to the carbons at δ
c 126.93 (C-2) and 156.36 (C-3), suggested that the –OH at δ
H 5.94 and the –OCH
3 at δ
H 3.46 were respectively attached to C-4 at δ
c 71.55 and C-2 at δ
c 126.93. Simultaneously, the HMBC correlations from the proton at δ
H 4.22 (H-4) to the carbons at δ
c 13.39 (5-CH
3) and 77.97 (C-5), and from the proton at δ
H 4.94 (5-OH) to the carbons at δ
c 71.55 (C-4), 77.97 (C-5) and 13.39 (5-CH
3), indicated that the –OH at δ
H 4.50 and the methyl at δ
c 13.39 were both connected to the same quaternary carbon at δ
c 77.97 (C-5). Furthermore, the position of the –OH at δ
H 4.50 was determined to be at C-6 (δ
c 76.99) by the HMBC correlations from the proton at δ
H 4.50 (6-OH) to the carbons at δ
c 185.62 (C-1), 77.97 (C-5) and 76.99 (C-6), and from the proton at 3.72 (H-6) to the carbons at δ
c 185.62 (C-1), 71.55 (C-4), 77.97 (C-5), and 13.39 (5-CH
3). Thus, the only position left to place the remaining –NH
2 group of compound
1 was at C-3. Therefore, the planar structure of
1 was as shown in
Figure 1. The relative configuration of the three hydroxyls in positions 4, 5, 6 was inferred as
cis-relationship from the NOE correlations between 4-OH (δ
H 4.94) and 5-OH (δ
H 5.94), 4-OH and 6-OH (δ
H 4.50), and 5-OH and 6-OH, as well as no NOE correlations observed from 4-OH, 5-OH and 6-OH to 5-CH
3 (δ
H 0.83) in the NOESY spectrum. So compound
1 was identified as (±)-(4
R*,5
S*,6
S*)-3-amino-4,5,6-trihydroxy-2-methoxy-5-methyl-2-cyclohexen-1-one (4
R*,5
S*,6
S* only represent the relative configuration). Compound
1 gave a zero specific rotation and showed a baseline CD (circular dichroism) curve (Figure S35), indicating a racemic mixture of the enantiomers [
19], which results from the center of chirality (C-4, C-5, and C-6) of the cyclohexenone. This was confirmed also by chiral HPLC analysis, which afforded a doublet peak with cellulose-based stationary phase using
n-hexane/isopropanol (
v/
v 90:10) as mobile phase (Figure S37). Based on the result of the analytical HPLC, they were attempted to separate by preparative chiral HPLC and using three types of chiral columns, but all were unsuccessful due to their small amounts.
Table 1.
1H and 13C NMR data of compounds 1 and 2.
Table 1.
1H and 13C NMR data of compounds 1 and 2.
Position | 1 a | 2 a |
---|
δC, mult. | δH | δC, mult. | δH |
---|
1 | 185.62, C | - | 197.95, C | - |
2 | 126.93, C | - | 132.96, C | - |
3 | 156.36, C | - | 157.68, C | - |
4 | 71.55, CH | 4.22 (s) | 81.97, C | - |
5 | 77.97, C | - | 78.15, C | - |
6 | 76.99, CH | 3.72 (s) | - | - |
2-OCH3 | 58.13, CH3 | 3.46 (s) | - | - |
2-OH | - | - | - | 9.00 (s) |
3-NH2 | - | 6.30 (s), 6.83 (s) | - | - |
3-OCH3 | - | - | 58.55, CH3 | 3.99 (s) |
4-OH | - | 5.94 (s) | - | 6.10 (s) |
5-CH3 | 13.39, CH3 | 0.83 (s) | 22.16, CH3 | 1.16 (s) |
5-OH | - | 4.94 (s) | - | 5.63 (s) |
6-OH | - | 4.50 (s) | - | - |
4′ | - | - | 171.13, C | - |
4′-OCH3 | - | - | 52.06, CH3 | 3.59 (s) |
Compound
2 was obtained as colorless needle, and its molecular formula was established as C
9H
12O
7 (
m/
z 255.0471 [M + Na]
+, calcd. 255.0475) on the basis of high-resolution ESIMS measurements, indicating that
2 contained 4 degrees of unsaturation. The IR spectrum indicated the presence of hydroxyl (3434, 3290 cm
−1), ester (1746 cm
−1), and carbonyl (1615 cm
−1) groups. The
1H NMR spectrum of
2 (
Table 1) showed the presence of one methyl at δ
H 1.16 (s, 3H), three hydroxyls at δ
H 9.00 (s, 1H, exchangeable), 6.10 (s, 1H, exchangeable) and 5.63 (s, 1H, exchangeable), and two methoxyls at δ
H 3.99 (s, 3H) and 3.59 (s, 3H). Among them, the methoxyl at δ
H 3.59 (s, 3H) was suggested to be the methoxycarbonyl group revealed by the HMBC correlations between 4′-OCH
3 and C-4′ at δ
C 171.13. The
13C NMR spectrum (
Table 1) showed 9 resolved signals including one α,β-unsaturated ketone at δ
C 197.95, 157.68 and 132.96, one ester carbonyl at δ
C 171.13, two methoxyls at δ
C 58.55 and 52.06, one methyl at δ
C 22.16, and two quaternary carbons at δ
C 81.97 and 78.15. It also showed that 3 of the 4 elements of unsaturation in
2 were due to the carbonyl and α,β-unsaturated ketone groups. The other one degree of unsaturation indicated that
2 contained a monocyclic ring. These NMR data suggested that compound
2 was a cyclopentenone derivative with one methyl, one methoxyl, one methoxycarbonyl, and three hydroxyls. Analysis of the HSQC spectrum provided the assignments of protons attached to their corresponding carbons, which also confirmed what carbons were belonging to the quaternary type. In the HMBC spectrum, the correlations from the hydroxyl proton at δ
H 9.00 (2-OH) to the carbons at δ
c 197.95 (C-1), 132.96 (C-2), and 157.68 (C-3), and from the methoxyl protons at δ
H 3.99 to the carbon at δ
c 157.68 (C-3), suggested that the –OH at δ
H 9.00 and the –OCH
3 at δ
H 3.99 were respectively attached to C-2 at δ
c 132.96 and C-3 at δ
c 157.68. Simultaneously, the HMBC correlations from the hydroxyl proton at δ
H 6.10 (4-OH) to 157.68 (C-3) and 171.13 (C-4′), indicated that the -OH at δ
H 6.10 and the methoxycarbonyl (δ
c 171.13 and 52.06) were both connected to C-4 (δ
c 81.97). Furthermore, the HMBC correlations from the methyl protons at δ
H 1.16 (5-CH
3) to the carbons at δ
c 197.95 (C-1), 81.97 (C-4) and 78.15 (C-5), and from the hydroxyl proton at δ
H 5.63 (5-OH) to the carbons at δ
c 197.95 (C-1), 81.97 (C-4), 78.15 (C-5) and 22.16 (5-CH
3), indicated that the –OH at δ
H 5.63 and the methyl at δ
H 1.16 were both connected to C-5 (δ
c 78.15). Thus, the planar structure of
2 was elucidated as shown in
Figure 1. The relative configuration of the hydroxyls in positions 4 and 5 was inferred as
cis-relationship from the NOE correlations between 4-OH (δ
H 6.10) and 5-OH (δ
H 5.63) in the NOESY spectrum. Therefore, the structure of
2 was elucidated as (±)-(4
S*,5
S*)-2,4,5-trihydroxy-3-methoxy-4-methoxycarbonyl-5-methyl-2-cyclopenten-1-one (4
S*,5
S* only represent the relative configuration). No observable CD cotton effect (Figure S36) and optical rotation of
2 was detected, indicating that it was also a racemic mixture [
19]. This was supported by subsequent HPLC analysis on a chiral cellulose-based phase collumn revealing a symmetrical doublet chromatographic peak by using
n-hexane/isopropanol (
v/
v 95:5) as mobile phase (Figure S38). Unfortunately, resolution of the separation of
2 by preparative chiral HPLC was also unsuccessful.
More detailed experimental information for Chiral HPLC analysis of Compounds
1 and
2 are shown in Figures S37 and S38 at the
Supplementary Information. Natural products are usually biosynthesized in one enantiomer form. Enantiomerically opposite products are less than 1% relative to the overall abundance of natural products, which often result from the action of stereochemically distinct enzymes that can give single and opposite enantiomeric products from achiral substrates [
20]. For examples, (±)-tylopilusin A and (±)-tylopilusin B were two naturalfully substituted 2-cyclopenten-1-one racemates produced by one kind of microorganism, with similar chiral centers as compound
2.
Compound
3 was isolated as yellow powder, and its molecular formula was determined as C
16H
10O
7Cl based on the negative HRESIMS (
m/z 349.0124 [M − H]
−, calcd. 349.0121). The IR spectrum indicated the presence of hydroxyl (3169 cm
−1), ester (1696 cm
−1), carbonyl (1641 cm
−1) and aromatic (1607 cm
−1) groups. The
1H NMR spectrum (
Table 2) exhibited the presence of a 1,2,3,4-tetrasubstituted benzene ring (ring A) at δ
H 7.49 (d, 9 Hz, 1H) and 7.68 (d, 9 Hz, 1H), and a 1,2,3,4,5-pentasubstituted benzene ring (ring B) at δ
H 6.98 (s, 1H), a hydroxyl at δ
H 12.20 (s, 1H), a hydroxymethyl at δ
H 4.65 (d, 5.4 Hz, 2H), and a methoxyl at δ
H 3.85 (s, 3H). The
13C NMR spectrum (
Table 2) showed the presence of 16 carbon signals including one carbonyl at δ
C 180.73, one ester carbonyl at δ
C 167.09, one hydroxymethyl at δ
C 61.03, one methoxyl at δ
C 52.78, one tetrasubstituted benzene ring and one pentasubstituted benzene ring. These NMR data of
3 were closely comparable to those of the known compound
5, fischexanthone [
11]. The only obvious difference between
3 and
5 was ascribed to the substitution of a chlorine atom in ring B of
3. This was proved by the HMBC and HRESIMS spectra. HMBC correlations from H-2 to C-1, 3, 4, 10 and 1′ indicated that the chlorine atom was attached to C-4. So, the structure of
3 was elucidated to be 4-chloro-1,5-dihydroxy-3-hydroxymethyl-6-methoxycarbonyl-xanthen-9-one, and was also named as 4-chlorofischexanthone.
Table 2.
1H and 13C NMR data of compounds 3 and 4, J in Hz.
Table 2.
1H and 13C NMR data of compounds 3 and 4, J in Hz.
Position | 3 a | 4 b |
---|
δC, mult. | δH (J in Hz) | δC, mult. | δH (J in Hz) |
---|
1 | 159.55, C | - | 120.71, C | - |
2 | 108.59, CH | 6.98 (s) | 152.78, C | - |
3 | 150.57, C | - | 119.49, CH | 7.52 (d, 9.6) |
4 | 107.95, C | - | 119.07, CH | 7.38 (d, 8.4) |
5 | 149.15, C | - | 111.29, CH | 7.72 (d, 1.2) |
6 | 117.63, C | - | 135.66, C | - |
7 | 126.08, CH | 7.49 (d, 9.0) | 105.52, CH | 7.41 (d, 1.2) |
8 | 120.74, CH | 7.68 (d, 9.0) | 160.89, C | - |
9 | 180.73, C | - | 175.06, C | - |
10 | 61.03, CH2 | 4.65 (d, 5.4) | 167.74, C | - |
11 | 167.09, C | - | 165.52, C | - |
1′ | 107.11, C | - | 121.40, C | - |
4′ | 150.78, C | - | 149.18, C | - |
5′ | 151.61, C | - | 157.43, C | - |
8′ | 117.57, C | - | 114.18, C | - |
1-OH | - | 12.20 (s) | - | - |
2-OCH3 | - | - | 56.96, CH3 | 3.92 (s) |
5-OH | - | 10.59 (s) | - | - |
8-OCH3 | - | - | 56.79, CH3 | 4.06 (s) |
10-OH | - | 5.71 (s) | - | - |
10-OCH3 | - | - | 53.03, CH3 | 4.09 (s) |
11-OCH3 | 52.78, CH3 | - | 52.83, CH3 | 4.00 (s) |
Compound
4 was obtained as white powder. Its molecular formula of C
19H
16O
8 was determined by HRESIMS (
m/z 395.0732 [M + Na]
+, calcd. 395.0737). The IR spectrum of
4 indicated the presence of ester (1741, 1714 cm
−1), carbonyl (1658 cm
−1) and aromatic (1617, 1586 cm
−1) groups. The
1H NMR spectral data (
Table 2) showed the presence of a 1,3,4,5-tetrasubstituted benzene ring (ring A) at δ
H 7.41 (d, 1.2 Hz, 1H) and 7.72(d, 1.2 Hz, 1H), a 1,2,3,6-tetrasubstituted benzene ring (ring B) at 7.38 (d, 8.4 Hz, 1H) and 7.52 (d, 9.6 Hz, 1H), and four methoxyls at δ
H 4.09 (s, 3H), 4.00 (s, 3H), 3.92 (s, 3H) and 4.06 (s, 3H). The
13C NMR spectrum (
Table 2) revealed the presence of 19 carbon signals including one carbonyl group at δ
C 175.06, two ester carbonyl groups at δ
C 165.52 and 167.74, four methoxyl groups at δ
C 52.83, 53.03, 56.79 and 56.96, and two tetrasubstituted benzene rings. These NMR data of
4 were similar to those of 6-carboxyl-2,8-dihydroxy-1-methoxycarbonyl-xanthen-9-one [
21]. The obvious difference between
4 and 6-carboxyl-2,8-dihydroxy-1-methoxycarbonyl-xanthen-9-one was three methyl groups of the former replaced the three active hydrogen protons of the latter. This was proved by the HMBC spectrum, where correlations from δ
H 3.92 (s, 3H) to C-2 at δ
C 152.78, from δ
H 4.06 (s, 3H) to C-8 at δ
C 160.89, and from δ
H 4.00 (s, 3H) to C-10 at δ
C 167.74 were observed. The positions of all the four substituents were further confirmed to be the same as 6-carboxyl-2,8-dihydroxy-1-methoxycarbonyl-xanthen-9-one by analysis of the HMBC spectrum. Thus, the structure of
4 was elucidated as 2,8-dimethoxy-1,6-dimethoxycarbonyl-xanthen-9-one (
Figure 1).
Figure 2.
Key HMBC and NOESY correlations of compounds 1–4.
Figure 2.
Key HMBC and NOESY correlations of compounds 1–4.