2.1. Structural Elucidation
5-Olefin phenylpyropene A (
1) was isolated as a yellowish oil. The molecular formula was established as C
32H
36O
9 based on the analysis of
13C-NMR data and HR(+)ESIMS
m/z 565.2448 [M + H]
+ (calcd. for C
32H
36O
9, 565.2432), implying fifteen degrees of unsaturation (
Supplementary Figure S1). The IR spectrum indicated the presence of a carbonyl group (1680 cm
−1) and a benzene ring (3073, 1574 and 1507 cm
−1). UV maxima at 232, 278 and 322 nm supported the long conjugated system containing a benzene ring. The
13C-NMR and DEPT spectra displayed six methyls, four methylenes, ten methines and twelve quaternary carbons. The diagnostic aryl protons at
δH 7.81 (m, 2H) and 7.44 (m, 3H) revealed a monosubstituted benzene ring. Two alkenyl protons at
δH 6.46 (s) and 6.36 (s) indicated there were two trisubstituted double bonds in the molecule. Three methyl group singlets at
δH 2.16, 2.10 and 2.04 showed HMBC correlations with the carbonyl groups at
δC 169.9, 171.1 and 170.4, respectively, confirming there are three acyloxy groups. The other three methyl group singlets at
δH 1.57, 1.25 and 0.87 are connected with the quaternary carbons at
δC 21.2, 24.2 and 13.3, respectively. In the
1H–
1H COSY spectrum, the cross peaks of H-1/H-2, H-2/H-3, H-7/H-8, H-8/H-9, H-2′′(H-6′′)/H-3′′(H-5′′) and H-3′′(H-5′′)/H-4′′ revealed the partial structure of –CHCH
2CH
2–, –CHCH
2CH– and –CHCHCHCHCH– in the molecule (
Figure 2). The key HMBC correlations of H-1/C-10, H-11/C-10, H-15/C-10, H-12/C-3, H-12/C-4, H-12/C-5, H-13/C-4, H-13/C-5, H-13/C-6, H-7/C-6, H-14/C-6, H-12/C-9 and H-15/C-9 deduced the sesquiterpene moiety. The cross peaks of H-13 to C-2′, C-3′ and C-4′, H-5′ to C-4′, C-6′ and C-1′′ in the HMBC spectrum confirmed the connectivity of the sesquiterpene fragment, α-pyrone and the monosubstituted phenyl ring (
Table 1 and
Supplementary Figures S2–S7).
Figure 2.
1H–1H COSY (bold lines) and the main HMBC (arrows) correlations of Compounds 1, 4, 7, 9 and 10.
Figure 2.
1H–1H COSY (bold lines) and the main HMBC (arrows) correlations of Compounds 1, 4, 7, 9 and 10.
Table 1.
1H and 13C-NMR data of 1, 4 and 6 at 400/100 MHz, respectively, δ in ppm.
Table 1.
1H and 13C-NMR data of 1, 4 and 6 at 400/100 MHz, respectively, δ in ppm.
Position | 1 a | 4 a | 6 b |
---|
δC | δH (J in Hz) | δC | δH (J in Hz) | δC | δH (J in Hz) |
---|
1 | 73.3, CH | 4.79, dd (11.6, 4.4) | 73.5, CH | 4.79, dd (11.6, 4.8) | 74.4, CH | 4.79, dd (11.2, 5.6) |
2 | 23.2, CH2 | 1.98, m; 1.75, m | 22.8, CH2 | 1.88, m; 1.70, m | 23.6, CH2 | 1.84, m; 1.88, m |
3 | 35.4, CH2 | 2.08, m; 1.62, m | 36.6, CH2 | 1.83, m; 1.20, ddd (13.2, 13.2, 3.2) | 36.8, CH2 | 1.43, td (12.8, 4.8); 2.15, td (12.8, 4.8) |
4 | 38.7, C | | 36.6, C | | 38.8, C | |
5 | 143.7, C | | 50.3, CH | 1.60, dd (12.4, 4.8) | 55.0, CH | 1.66, d (3.6) |
6 | 83.5, C | | 81.9, C | | 86.2, C | |
7 | 77.7, CH | 5.22, d (12.0, 5.2) | 77.5, CH | 5.02, dd (11.6, 4.8) | 77.6, CH | 4.97, dd (12.4, 5.2) |
8 | 24.3, CH2 | 1.81, m; 1.63, m | 24.9, CH2 | 1.78, m; 1.50, m | 29.0, CH2 | 1.60, d (12.4); 1.82, m |
9 | 41.0, CH | 1.73, m | 45.1, CH | 1.66, d (12.0) | 46.2, CH | 1.53, d (3.6) |
10 | 40.5, C | | 40.2, C | | 41.3, C | |
11 | 64.6, CH2 | 3.78, d (12.0); 3.74, d (12.0) | 64.7, CH2 | 3.78, d (12.0); 3.73, d (12.0) | 64.6, CH2 | 3.83, d (11.6); 3.77, d (11.6) |
12 | 24.2, CH3 | 1.25, s | 15.5, CH3 | 1.00, s | 17.9, CH3 | 1.48, s |
13 | 111.4, C | 6.36, s | 16.9, CH2 | 2.57, dd (17.2, 4.8); 2.33, dd (17.2, 12.4) | 60.6, CH | 4.95, d (3.6) |
14 | 21.2, CH3 | 1.57, s | 15.4, CH3 | 1.32, s | 15.9, CH3 | 1.76, s |
15 | 13.3, CH3 | 0.87, s | 13.3, CH3 | 0.86, s | 13.3, CH3 | 0.92, s |
2′ | 162.1, C | | 163.7, C | | 163.5, C | |
3′ | 100.3, C | | 99.8, C | | 100.1, C | |
4′ | 160.2, C | | 162.2, C | | 157.9, C | |
5′ | 97.4, CH | 6.46, s | 99.2, CH | 6.43, s | 147.6, CH | |
6′ | 154.9, C | | 155.9, C | | 128.4, C | |
1′′ | 131.0, C | | | | | |
2′′ | 125.6, CH | 7.81, m | 146.7, CH | 8.99, s | 147.6, CH | 9.07, dd (2.4, 0.8) |
3′′ | 128.9, CH | 7.44, m | 127.3, C | | 127.5, C | |
4′′ | 131.0, CH | 7.44, m | 132.8, CH | 8.09, brd (8.0) | 133.6, CH | 8.22, ddd (8.4, 2.4, 1.6) |
5′′ | 128.9, CH | 7.44, m | 123.6, CH | 7.39, dd (8.0, 4.8) | 124.6, CH | 7.53, ddd (8.4, 4.8, 0.8) |
6′′ | 125.6, CH | 7.81, m | 151.2, CH | 8.66, d (4.8) | 152.3, CH | 8.69, dd (4.8, 1.6) |
1-OCOCH3 | 21.1, CH3 | 2.04, s | 21.1, CH3 | 2.04, s | 21.0, CH3 | 2.00, s |
1-OCOCH3 | 170.0, C | | 170.5, C | | 170.6, C | |
7-OCOCH3 | 21.1, CH3 | 2.16, s | 21.3, CH3 | 2.15, s | | |
7-OCOCH3 | 169.9, C | | 170.1, C | | | |
11-OCOCH3 | 20.8, CH3 | 2.10, s | 20.8, CH3 | 2.11, s | 20.7, CH3 | 1.99, s |
11-OCOCH3 | 171.1, C | | 171.0, C | | 170.8, C | |
13-OH | | | | | | 4.28, brs |
7-OH | | | | | | 4.10, brs |
The relative configuration of
1 was determined by the analysis of the NOESY spectrum. The NOE correlations between H-12/H-14 and H-12/H-15, H-1/H-9, H-7/H-9 and H-7/H-11 were observed. Consequently, H-12, H-14 and H-15 are β-oriented, while H-1, H-7 and H-9 are α-oriented (
Figure 3).
Figure 3.
Key NOESY correlations of Compounds 1, 4 and 7.
Figure 3.
Key NOESY correlations of Compounds 1, 4 and 7.
Compounds
2 and
3 were identified as phenylpyripyropene A and C [
15,
16], by comparing their NMR data to the reference values. The double bond between the C-5 and C-13 positions in
1 was replaced by the saturated carbon-carbon bond in
2. Compounds
1 and
2 have triacyloxy groups at the C-1, C-7 and C-11 positions; however, Compound
3 has only one acyloxy group at the C-1 position (
Supplementary Figures S8–S11).
13-Dehydroxylpyripyropene A (
4) was obtained as a white powder. It showed a molecular formula of C
31H
37NO
9 determined by the
13C-NMR data and HR(+)ESIMS peak at
m/
z 568.2549 [M + H]
+ (calcd. for C
31H
37NO
9, 568.2541) (
Supplementary Figure S12). The
13C-NMR and DEPT spectra (
Table 1) displayed six methyls, five methylenes, nine methines and eleven quaternary carbons. By comparing the NMR data with pyripyropene A (
5) (
Supplementary Figures S13–S20) [
17], a quick identification was made revealing that C-13 of
4 was a methylene (
δC 16.9,
δH 2.57, 2.33), corresponding to one methine group (
δC 60.1,
δH 4.99) connected to one hydroxyl group in pyripyropene A (
Figure 2). The relative configuration of
4 was determined on the basis of NOESY data. The NOE correlations of H-12/H-15, H-14/H-15 supported these methyl groups on the β-position of the ring system; while those correlations of H-1/H-9, H-5/H-7, H-5/H-9, H-7/H-9 and H-9/H-11 were used to place these protons on the α-position (
Figure 3).
Compound
6 was elucidated as 7-deacetyl pyripyropene A (
Supplementary Figures S21 and S22).
6 was once obtained by hydrolysis of pyripyropene A with 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) in 80% methanol [
18]. However, this is the first report including the detailed NMR data.
Deacetylsesquiterpene (
7) was isolated as a yellowish oil. The molecular formula was determined as C
15H
24O
3 from the
13C-NMR data and the HR(+)ESIMS peak at
m/
z 275.1603 [M + Na]
+ (calcd. for C
15H
24O
3Na, 275.1618), indicating four degrees of unsaturation (
Supplementary Figure S23). The strong IR absorption at 3443 cm
−1 indicated the presence of the hydroxyl groups. The
13C-NMR and DEPT spectra displayed three methyls, three methylenes, six methines and three quaternary carbons (
Table 2). The quaternary carbon (
δC 132.3) and the methine (
δC 124.4,
δH 5.28) formed a trisubstituted double bond. The quaternary carbon (
δC 147.2) and the methylene (
δC 111.0,
δH 4.94 and 4.72) constructed a terminal double bond. Thus, Compound
7 must be bicyclic to account for the four double bond equivalents required by the molecular formula. The
1H–
1H COSY cross peaks of H-1/H-2, H-4/H-4a, H-4a/H-5, H-5/H-6, H-6/H-7, H-7/H-8 and H-8/H-13 established two partial structures, –CHCH– and –CHCHCHCH
2CH
2CHCH
3 (
Figure 2). The HMBC correlations of H-1/C-8, H-2/C-3, H-5/C-10, H-8/C-8a, H-9/C-3 and H-11/C-10 established the skeleton; three hydroxyl groups were attached to C-1 (
δC 74.6), C-2 (
δC 74.2) and C-8a (
δC 73.8). The NOESY correlations of H-1/H-12, H-7a/H-12, H-6e/H-12, H-2/H-4a, H-4a/H-5, H-4a/8a-OH and H-6a/H-13 revealing 1-OH, 8a-OH, H-5 and H-13 were α-oriented; however, 2-OH was β-oriented (
Figure 3 and
Supplementary Figures S24–S29).
Table 2.
1H and 13C-NMR data of 7, 9 and 10, δ in ppm.
Table 2.
1H and 13C-NMR data of 7, 9 and 10, δ in ppm.
Position | 7 a | 9 b | 10 c |
---|
δC, Type | δH, Mult., (J in Hz) | δC, Type | δH, Mult., (J in Hz) | δC, Type | δH, Mult., (J in Hz) |
---|
1 | 74.6, CH | 3.98, d (1.2) | 127.9, CH | 8.92, d (1.5) | 169.1, C | |
2 | 74.2, CH | 4.05, s | 137.4, C | | | |
3 | 132.3, C | | 129.2, CH | 8.22, dd (9.0,1.5) | 78.4, CH | 5.13, ddd (10.0, 6.8, 4.0) |
4 | 124.4, CH | 5.28, s | 121.6, CH | 8.77, d (9.0) | 32.5, CH2 | 3.00, dd (16.4, 4.0); 2.93, dd (16.4, 10.0) |
4a | 38.1, CH | 2.67, s | 127.3, C | | 141.7, C | |
5 | 41.4, CH | 2.38, brd (12.0) | 111.4, C | | 107.1, CH | 6.24, d (2.0) |
6 | 25.9, CH2 | 1.56, m; 1.33, m | 167.1, C | | 163.4, C | |
7 | 30.7, CH2 | 1.48, m; 1.40, m | 117.0, CH | 7.21, s | 101.0, CH | 6.18, d (2.0) |
8 | 31.2, CH | 1.99, m | 159.5, C | | 164.8, C | |
8a | 73.8, C | | 126.3, C | | 100.1, C | |
9 | 20.1, CH3 | 1.78, s | 167.6, C | | 127.0, CH | 5.71, dd (15.2, 6.8) |
10 | 147.2, C | | 195.3, CH | 11.42, brs | 133.3, CH | 6.35, dd (15.2, 10.0) |
11 | 22.4, CH3 | 1.74, brs | 30.1, CH | 3.90, heptet (7.0) | 130.4, CH | 6.10, ddd (15.2, 10.0, 1.2) |
12 | 111.0, CH2 | 4.94, q (1.2 ); 4.72, s | 23.4, CH3 | 1.45, d (7.0) | 131.7, CH | 5.81, dq (15.2, 6.8) |
13 | 15.0, CH3 | 0.93, d (6.8) | 23.4, CH3 | 1.45, d (7.0) | 18.0, CH3 | 1.74, dd (6.8, 1.2) |
1-OH | | 2.00, s | | | | |
2-OH | | 2.00, s | | | | |
2-OCOCH3 | | | | | | |
2-OCOCH3 | | | | | | |
6-OH | | | | 13.42, s | | 11.06, brs |
8-OH | | | | | | 11.06, brs |
8a-OH | | 2.00, s | | | | |
COOH | | | | 10.94, brs | | |
The formula of Compound
8 was identified as C
17H
26O
4, based on the analysis of
13C-NMR data and HREIMS peak at
m/
z 294.1828 [M]
+ (calcd. for C
17H
26O
4, 294.1826). The NMR spectroscopic data of Compound
8 were very similar to those of Compound
7, except that the C-2 hydroxyl group in
7 was replaced by an acetoxy group in
8 (
Supplementary Figures S30 and S31). Fortunately, we obtained a single crystal of
8 from the MeOH solution. In the crystal, the adjacent molecules are interlinked by a pair of strong O1-H...O4 (hydroxyl) and O4-H...O3 (carbonyl) hydrogen bonds to form a zigzag chain running parallel to the b-axis. Neighboring chains of molecules are packed closely together with the hydrophobic methyl groups pointing towards each other. The absolute configuration of
8 was determined as 1
S, 2
R, 4a
R, 5
R, 8
R, 8a
R with the Flack parameter value −0.16(17) by single-crystal X-ray diffraction analysis (
Figure 4 and
Supplementary Crystallographic Information Framework (CIF)) using Cu K
α radiation. Compound
8, named (1
S,2
R,4a
R,5
R,8
R,8a
R)-1,8a-dihydroxy-2-acetoxy- 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,4a,5,6,7,8,8a-octahydronaphthalene, was previously obtained as the degradation product of the natural product CJ-12662, which was obtained from the fermentation broth of
Aspergillus fischeri var.
thermomutatus ATCC 18,618 [
19] and later also isolated from fungi
Neosartorya pseudofischeri and
Eurotium chevalieri [
20,
21]. However, the single-crystal X-ray diffraction data were never reported.
Figure 4.
ORTEP (Oak Ridge Thermal Ellipsoid Plot) drawing of Compound 8.
Figure 4.
ORTEP (Oak Ridge Thermal Ellipsoid Plot) drawing of Compound 8.
Compound
9 was obtained as a white powder. Its molecular formula was deduced to be C15H14O4 by analysis of the
13C-NMR data and HR(-)ESIMS ion at
m/
z 257.0817 [M − H]
− (calcd. for C
15H
13O
4, 257.0819), which required nine degrees of unsaturation (
Supplementary Figure S32). The IR spectrum revealed the presence of a carboxyl group (3283 and 1671 cm
−1) and an aldehyde group (2812 cm
−1). Two methyl groups at
δH 1.45 (d,
J = 7.0 Hz, 6H) showed
1H–
1H COSY correlations with the methine group at
δH 3.90 (seven peaks,
J = 7.0 Hz) and formed an isopropyl fragment (
Table 2,
Figure 2 and
Supplementary Figures S33–S36). According to the HMBC correlations of H-1/C-2, H-1/C-8a, H-3/C-2 and H-4/C-4a, the protons at
δH 8.92 (d,
J = 1.5 Hz, H-1), 8.22 (dd,
J = 9.0, 1.5 Hz, H-3) and 8.77 (d,
J = 9.0 Hz, H-4) were located at the 1,3,4-position on a phenyl ring. HMBC correlations of H-1/C-2, H-1/C-8a, H-3/C-2, H-4/C-4a, H-4/C-5, H-7/C-5, H-7/C-6, H-11/C-7 and H-11/C-8 indicated the existence of a naphthyl ring, accounting for seven degrees of unsaturation. The remaining carboxyl group (
δC 167.6,
δH 10.94), the aldehyde (
δC 195.3,
δH 11.42), phenolic hydroxyl group (
δH 13.42, and the isopropyl fragment were located on C-2, C-5, C-6 and C-8, respectively, based on the HMBC cross peaks of H-1/C-9, H-3/C-9, H-10/C-6, 6-OH/C-6, 6-OH/C-7 and H-11/C-8. The aldehyde and hydroxyl groups formed an intramolecular hydrogen bond, which accounted for the downfield resonance of the phenolic hydroxyl group. Therefore, the structure of
9 was elucidated as 5-formyl-6-hydroxy-8-isopropyl-2-naphthoic acid.
Compound
10 was isolated as a white powder. The
13C-NMR data and HR(-)ESIMS peak at
m/
z 245.0815 [M − H]
+ (calcd. for C
14H
13O
4, 245.0819), established the molecular formula as C
14H
14O
4 (
Supplementary Figure S37). The
13C-NMR and DEPT spectra revealed fourteen carbon resonances, including one methyl group, one methylene group, seven methine groups and five quaternary carbons. The
1H-NMR spectrum exhibited two meta-aromatic protons at
δH 6.24 (d,
J = 2.0 Hz) and 6.18 (d,
J = 2.0 Hz), indicating the presence of a tetrasubstituted aromatic ring. Two phenolic hydroxyl groups at
δH 11.06 (brs) were connected to C-6 (
δC 163.4) and C-8 (
δC 164.8). Additionally, two pairs of trans-oriented olefinic protons at
δH 5.71 (dd,
J = 15.2, 6.8 Hz), 6.35 (dd,
J = 15.2, 10.0 Hz), 6.10 (ddd,
J = 15.2, 10.0, 1.2 Hz), 5.81 (dq,
J = 15.2, 6.8 Hz), one oxymethine at
δH 5.13 (ddd,
J = 10.0, 6.8, 4.0 Hz), one methylene at
δH 3.00 (dd,
J = 16.4, 4.0 Hz), 2.93 (dd,
J = 16.4, 10.0 Hz) and one methyl group at
δH 1.74 (dd,
J = 6.8, 1.2 Hz) were connected in sequence and formed a pentadienyl group based on their
1H–
1H COSY correlations. The remnant quaternary carbon (
δC 169.1) connected with C-8a (
δC 100.1) and the oxymethine and formed a lactone. The HMBC correlations of H-4/C-4a, H-4/C-8a, H-5/C-6, H-7/C-8 and H-7/C-8a revealed a 3,6,8-trisubstituted isocoumarin ring system (
Table 2,
Figure 2 and
Supplementary Figures S38–S43). The absolute configuration at C-3 remains undetermined. Therefore, Compound
10 was elucidated as 6,8-dihydroxy-3-((1
E,3
E)-penta-1,3- dien-1-yl)isochroman-1-one.
The other known compounds, isochaetominine C (
11) [
22], trichodermamide A (
12) [
23], indolyl-3-acetic acid methyl ester (
13) [
24], 1-acetyl-β-carboline (
14) [
25], 1,2,3,4-tetrahydro- 6-hydroxy-2-methyl-l,3,4-trioxopyrazino[l,2-a]-indole (
15) [
26] and fumiquinazoline F (
16) [
27], were identified by comparing their spectroscopic data with the literature values (
Supplementary Figures S44–S55).