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Diarylheptanoids with NO production inhibitory activity from Amomum kravanh
Journal Pre-proofs Diarylheptanoids with NO Production Inhibitory Activity from Amomum kra‐ vanh Jun-Sheng Zhang, Xin-Xin Cao, Jin-Hai Yu, Zhi-Pu Yu, Hua Zhang PII: DOI: Reference:
S0960-894X(20)30096-2 https://doi.org/10.1016/j.bmcl.2020.127026 BMCL 127026
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
18 November 2019 8 February 2020 10 February 2020
Please cite this article as: Zhang, J-S., Cao, X-X., Yu, J-H., Yu, Z-P., Zhang, H., Diarylheptanoids with NO Production Inhibitory Activity from Amomum kravanh, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127026
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© 2020 Elsevier Ltd. All rights reserved.
Diarylheptanoids with NO Production Inhibitory Activity from Amomum kravanh Jun-Sheng Zhang, Xin-Xin Cao, Jin-Hai Yu, Zhi-Pu Yu, Hua Zhang
School of Biological Science and Technology, University of Jinan, Jinan, China
Correspondence Prof. Hua Zhang School of Biological Science and Technology, University of Jinan, Jinan 250022, China Phone: +86-531-89736199 Email: [email protected]
1
Abstract Seven new diarylheptanoids, kravanhols CI (17), along with two known analogues (8 and 9), were isolated from the fruits of Amomum kravanh. The structures of compounds 17 were elucidated by analysis of spectroscopic data, and the absolute configurations of selective ones were determined by time-dependent density functional theory (TD-DFT) based electronic circular dichroism (ECD) calculations. All compounds were evaluated for their inhibitory effects on the nitric oxide (NO) production induced by lipopolysaccharide (LPS) in murine RAW264.7 macrophage cells. Compounds 2, 5, 6 and 9 exhibited moderate inhibitory activity with IC50 values in the range of 17.426.5 M, being more potent than the positive control dexamethasone (IC50 32.5 M).
Key words: Zingiberaceae, Amomum kravanh, diarylheptanoid, ECD calculation, NO production
2
Diarylheptanoids are a group of naturally occurring phenols with the general structure of two aromatic rings linked by a linear seven-carbon chain. These compounds have attracted broad interest from drug discovery field due to their diverse bioactivities, such as anti-inflammatory,1,2 antitumor,3,4 antioxidant,5 antiviral6 and neuroprotective7,8 activities. Curcumin, for instance, has been forwarded to phase II clinical trial for the treatment of pancreatic cancer.9 So far, natural diarylheptanoids have been reported mainly from the plants of Zingiberaceae family. Amomum kravanh (Zingiberaceae) is a tropical plant widely distributed in Thailand, Vietnam and Southern areas of China. The fruits are commonly used as flavoring in dishes and also as a traditional Chinese medicine (TCM) for stomach diseases and digestive disorders. Previous chemical investigations on this plant have revealed the presence of monoterpenoids, diarylheptanoids and diterpenoids, with moderate NO inhibitory activities.1012 In our continuing efforts to explore new bioactive substances from TCM,1315 seven new diarylheptanoids, kravanhols CI (17), along with two known analogues (8 and 9),16 were isolated from the fruits of Amomum kravanh.17 These compounds were structurally characterized mainly by spectroscopic analyses. They were also screened for anti-inflammatory activity by using the lipopolysaccharide (LPS)-induced NO production model in murine RAW264.7 macrophages, and compounds 2, 5, 6 and 9 exhibited moderate inhibitory activity. Herein, details of the isolation, structure elucidation and the NO inhibitory effect of these compounds are described below. O 5'''
HO
OH
D
O
3
3''' 5'
1'''
1'
A
HO
HO
2 1
B
4 5
O
H 6
7
OH
3'
O
O 3''
1''
OH
OH O
1
OH
OH
HO
2
H
OH
O HO
OH 4 R = -OH 5 R = -OH
H
O
O HO
O
H
O OH
O O
6
7
Fig. 1. Structures of compounds 17
O O OH
HO
O
3
OH
3
OH
OH
O
O O
R
O
H
O
O HO
OH
O
H
O
C 5''
HO
OH
Compound 1, a colorless oil, had the molecular formula C28H32O9 as determined by the HRESIMS ion at m/z 535.1942 (calcd for [M Na], 535.1939), indicating 13 degrees of unsaturation. The 1H NMR spectrum of 1 exhibited three typical ABX spin systems at H 6.77 (1H, dd, J = 8.0, 1.8 Hz), 6.61 (1H, d, J = 8.0 Hz) and 6.85 (1H, d, J = 1.8 Hz); 6.61 (1H, dd, J = 8.0, 1.8 Hz), 6.57 (1H, d, J = 8.0 Hz) and 6.77 (1H, d, J = 1.8 Hz); as well as 6.68 (1H, dd, J = 8.0, 1.8 Hz), 6.73 (1H, d, J = 8.0 Hz) and 6.82 (1H, d, J = 1.8 Hz). These data suggested the presence of three 1,3,4-trisubstituted benzene rings. The
13C
NMR spectrum in combination with DEPT135 experiment
resolved 28 carbon resonances attributable to three benzene rings, three methoxyl groups, four oxygenated methines and three methylenes. As 12 of the 13 degrees of unsaturation were consumed by the aforementioned three phenyl rings, the remaining one required the presence of one additional ring in the structure of 1. In the 1H−1H COSY spectrum, a seven-carbon fragment (C-1 to C-7) was first established by the observed correlations (Fig. 2). The aforementioned information implied that compound 1 possessed most structural features of a diarylheptanoid and was quite similar to kravanhol A (8),11 a cometabolite in the current study. In comparison with 8, the major structural changes of 1 were the appearance of an additional trisubstituted benzene ring and a methine (C 54.4, C-2) instead of a methylene in 8. The location of this extra phenyl group was assigned at C-2 by the HMBC correlations from H-2 (H 6.769) and H-6 (H 6.605) to C-2 (C 54.4). The planar structure of 1 was further secured by detailed interpretation of its 2D NMR data (Fig. 2). Particularly, the methoxyl groups were assigned at C-3, C-3 and C-3 by NOE correlations of 3-OMe/H-2, 3-OMe/H-2 and 3-OMe/H-2, respectively. The relative configuration of 1 was established on the basis of NOESY experiment (Fig. 2) and analysis of 1H–1H coupling constants. The NOE correlation of H-1/H-5, together with the large coupling constant between H-1 and H-2 (J = 10.9 Hz), suggested that these protons were axially bonded in the chair-conformational ring B. H-1 and H-5 were then arbitrarily assigned in -orientation while H-2 was in -orientation. Thus, the small coupling constant between H-2 and H-3 (J = 2.6 Hz) indicated that H-3 was equatorially positioned in -orientation. Finally, H-6 was 4
established to be in cis-relationship with H-5 and thus -oriented on the basis of the small J5,6 value (3.1 Hz).18,19
Fig. 2. 1H1H COSY (
), selected HMBC (
) and NOESY (
) correlations
of 1 The absolute configuration of 1 was determined by computational approach of quantum chemistry using Time-Dependent Density Functional Theory (TD-DFT) method.20,21 The established relative configuration of 1 indicated that it possessed either (1S,2S,3S,5R,6S) or (1R,2R,3R,5S,6R) absolute configuration. As shown in Fig. 3, the experimental ECD curve of 1 matched well with the calculated ECD curve of (1R,2R,3R,5S,6R)-enantiomer, confirming the absolute configuration of 1 as shown. The structure of 1 was thus unequivocally assigned and named kravanhol C following kravanhols A and B reported from the same species.11
Fig. 3. Experimental and calculated ECD spectra for 1 5
Table 1 1H NMR data for 17 in CD3OD (600 MHz, in ppm, J in Hz) No.
1
2
3
4
5
6
7
1
4.93, d (10.9)
4.92, d (10.8)
4.84, d (10.5)
4.40, d (9.8)
4.73, brs
4.33, dd (11.4, 1.7)
4.75 brd (11.9)
2
2.96, dd (10.9, 2.6)
2.95, dd (10.8, 2.2)
2.56, dd (10.5, 9.9)
3.53, dd (9.8, 3.0)
3.57, d (3.5)
2.11, m
1.83, m
1.42, m
1.69, m
3
4.19, m
4.22, m
4.51, ddd (10.9, 9.9, 4.8)
4.11, m
3.98, m
3.93, m
4.25, quintet (2.5)
4
2.13, m
2.14, m
2.26, ddd (13.3, 4.8, 1.7)
1.90, m
1.90, m
2.11, m
1.81, m
1.77, m
1.76, m
1.93, ddd (13.3, 10.9, 6.7)
1.70, m
1.57, m
1.34, m
1.65, m
5
3.99, ddd (11.8, 3.1, 2.3)
3.98, ddd (11.9, 3.4, 2.3)
3.95, ddd (6.7, 6.0, 1.7)
3.85, m
3.80, m
4.10, m
4.55, m
6
3.69, ddd (7.6, 6.5, 3.1)
3.68, ddd (7.6, 6.5, 3.4)
4.16, ddd (7.5, 6.0, 5.4)
1.76, m
1.89, m
3.35, dd (15.4, 7.2)
3.26, dd (15.4, 7.3)
1.67, m
1.69, m
3.04, dd (15.4, 5.3)
2.94, dd (15.4, 5.0)
7
2.89, dd (13.5, 6.5)
2.89, dd (13.5, 6.5)
2.91, dd (13.8, 5.4)
2.60, m
2.69, m
2.72, dd (13.5, 7.6)
2.72, dd (13.5, 7.6)
2.70, dd (13.8, 7.5)
2'
6.85, d (1.8)
6.86, d (1.8)
6.61, m (overlapping)
7.01, d (1.8)
7.07, d (1.7)
6.84, d (1.6)
6.82, brs
5'
6.611, d (8.0)
6.62, d (8.0)
6.60, m (overlapping)
6.78, d (8.1)
6.78, d (8.1)
6.71, brs
6.70, brs
6'
6.766, dd (8.0, 1.8)
6.78, dd (8.1, 1.8)
6.60, m (overlapping)
6.89, dd (8.1, 1.8)
6.85, dd (8.1, 1.7)
6.72, d (1.6)
6.70, brs
2''
6.82, d (1.8)
6.82, d (1.6)
6.90, brs
6.73, d (1.8)
6.73, d (1.8)
7.56, d (1.9)
7.56, d (1.8)
5''
6.73, d (8.0)
6.72, d (8.0)
6.72, brs
6.68, d (8.0)
6.68, d (8.0)
6.85, d (8.3)
6.85, d (8.3)
6''
6.68, dd (8.0, 1.8)
6.68, dd (8.0, 1.6)
6.72, brs
6.60, dd (8.0, 1.8)
6.61, dd (8.0, 1.8)
7.61, dd (8.3, 1.9)
7.61, dd (8.3, 1.8)
2'''
6.769, d (1.8)
6.47, s
6.50, d (1.8)
5'''
6.57, d (8.0)
6'''
6.605, dd (8.0, 1.8)
6.47, s
6.56, dd (8.1, 1.8)
3'-OMe
3.73, s
3.74, s
3.69, s
3.88, s
3.87, s
3.74, s
3.70, s
3''-OMe
3.80, s
3.80, s
3.83, s
3.79, s
3.74, s
3.86, s
3.83, s
3'''-OMe
3.74, s
3.73, s
3.69, s
5'''-OMe
6.63, d (8.1)
3.73, s
6
Compound 2 displayed a quasi-molecular ion peak at m/z 565.2042 [M + Na]+ in the HRESIMS analysis, consistent with a molecular formula of C29H34O10 with 30 mass units more than that of 1. The 1D NMR data (Tables 1 and 2) of 2 were very similar to those of 1 but showed an additional methoxyl unit. In comparison with 1, the NMR data of 2 differed significantly in the D ring, featuring a symmetrical 1,3,4,5-tetrasubstituted benzene ring instead of the 1,3,4-trisubstited benzene ring in 1, which was supported by the observation of a two-proton singlet signal at H 6.47 (2H, s, H-2''' and H-6'''). The assignment of the extra methoxyl group on the D ring and the full structure of 2 were further corroborated by examination of 2D NMR data (Figs. S9-S12, Supplementary data). The absolute configuration of 2 was assigned to be the same as that of 1 by comparison of their ECD spectra, which showed consistent Cotton effects (Fig. S43, Supplementary data). Compound 2 was thus structurally elucidated and named kravanhol D. Compound 3 (kravanhol E) was determined to possess the same planar structure as 1 by HRESIMS and 1D NMR spectra, indicating that they were stereoisomers. The relative configuration of 3 was also established by analyses of NOESY data and 1H– 1H
coupling patterns. Key NOE correlations of H-1/H-3 and H-1/H-6 indicated that
H-1, H-3 and the C-5 sidechain were axially located in the chair conformational B-ring. Thus, H-1 and H-3 were randomly designated in -orientation, while H-5 was equatorially oriented and designated in -orientation. The large values of J1,2 (10.5 Hz) and J2.3 (9.9 Hz) implied that H-2 was axially bonded in -orientation. Moreover, the magnitude of J5,6 (6.0 Hz) in 3 Vs. that (3.1 Hz) in 1 suggested that H-5 and H-6 adopted a trans-relationship in 3. The structure with relative configuration of 3 was thereby established. Compound 4, a colorless oil, had the molecular formula C21H26O7 as determined by the HRESIMS ion at m/z 391.1750 (calcd for [M H], 391.1751). The 1H NMR spectrum of 4 exhibited two typical ABX spin systems at H 6.89 (1H, dd, J = 8.1, 1.8 Hz), 6.78 (1H, d, J = 8.1 Hz) and 7.01 (1H, d, J = 1.8 Hz), as well as 6.60 (1H, dd, J = 8.0, 1.8 Hz), 6.68 (1H, d, J = 8.0 Hz) and 6.73 (1H, d, J = 1.8 Hz). These data suggested the presence of two 1,3,4-trisubstitued benzene rings. The 7
13C
NMR
spectrum in combination with DEPT135 experiment resolved 21 carbon resonances attributable to two benzene rings, two methoxyl groups, four oxygenated methines and three methylenes. The aforementioned NMR data of 4 exhibited high resemblance with those of hedycoropyran C,22 a known diarylheptanoid reported before, and the main differences were attributed to the 3-O-methylation and 6-dehydroxylation in 4. This was supported by the presence of an additional methoxyl group (H 3.79; C 56.3) in 4, together with the replacement of an oxymethine in hedycoropyran C by a methylene (C 38.7) in 4. The planar structure of 4 was further secured by detailed analyses of its 2D NMR data (Fig. 4). The relative configuration of 4 was assigned to be the same as that of hedycoropyran C (except C-6) by comparing their 1D NMR data and NOESY data (Fig. 4). Moreover, the experimental ECD curve of 4 showed negative Cotton effects around 231 and 207 nm, respectively, which matched the calculated ECD spectrum of (1R,2S,3S,5S)-isomer of 4 (Fig. 5A). Thus, the absolute configuration of 4 was assigned as 1R,2S,3S,5S and it was named kravanhol F.
Fig. 4. 1H1H COSY (
), selected HMBC (
) and NOESY (
) correlations
of 4 Compound 5 had the same molecular formula of C21H26O7 as 4 based on the HRESIMS analysis (m/z 391.1754 [M H], calcd 391.1751), indicative of a stereoisomer of the latter. The 1D NMR data (Tables 1 and 2) of 5 showed high similarity to those of 4 with the major differences being attributed to the H-1/H-2 coupling patterns (J1,2 brs in 5 and 9.8 Hz in 4), indicating that 5 was the 2-epimer of 4. Detailed 2D NMR data (Figs. S27-S30, Supplementary data) analyses secured the structure of 5. To assign the absolute configuration of 5, the experimental ECD curve was also compared with the calculated one with a very good match as shown in Fig 5B. Thus, the structure of 5 was assigned as depicted and named kravanhol G. 8
Fig. 5. Experimental and calculated ECD spectra for 4 (A) and 5 (B) Compound 6 displayed a protonated molecular ion at m/z 389.1609 in HRESIMS analysis, consistent with a molecular formula of C21H24O7. The 1D NMR data (Tables 1 and 2) and HSQC spectrum revealed signals for a ketone group (C 199.3), two trisubstituted benzene rings and two methoxyl groups (H 3.87, 3.74; C 56.3, 56.1). The aforementioned spectroscopic information was very similar to that of 1-(3,4-dimethoxyphenyl)-2-((2S,6R)-6-(3,4-dimethoxyphenyl)tetrahydro-2H-pyran-2yl) ethanone, a synthetic analogue reported before,23 except for the presence of an additional oxymethine [H 3.93; C 68.7] in 6 instead of a methylene, together with the absence of two methoxyl groups in the synthetic analogue. Key 1H-1H COSY correlations of H2-2/H-3 and H-3/H2-4 assigned the additional oxymethine at C-3. Further analysis of 2D NMR data (Fig. S44, Supplementary data) led to the proposal of structure of 6, which revealed that 4-OMe and 4-OMe in the above-mentioned synthetic analogue were demethylated in 6. The NOE correlations of H-1/H-3 and H-1/H-5 (Fig. S44, Supplementary data) supported that they were all axially bonded in the B ring and were assigned to be -oriented. Thus, 6 was structurally characterized and given the trivial name kravanhol H. Compound 7 had the same molecular formula as 6, implying that they were structural isomers. The NMR data of 7 were very similar to those of 6 except for the signals for ring B. Detailed 2D NMR analyses permitted the establishment of the planar structure of 7 as shown, being the same as that of 6. In the NOESY spectrum, 9
correlations between H-1 and H-5 together with the large coupling constant between H-1 and H-2 (J = 11.9 Hz) indicated that H-1 and H-5 were axially located in the chair-conformational B-ring and were assigned to be -oriented. Thus, the small coupling constants of H-3 with vicinal protons (J = 2.5 Hz) indicated that it was equatorially positioned and -oriented. Compound 7 was then characterized as the 3-epimer of 6 and was named kravanhol I. Table 2 13C NMR data for 17 in CD3OD (151 MHz, in ppm) No.
1
2
3
4
5
6
7
1
79.5 CH
79.6 CH
80.2 CH
79.3 CH
77.1 CH
78.9 CH
75.1 CH
2
54.4 CH
54.8 CH
59.6 CH
73.7 CH
72.0 CH
43.6 CH2
40.9 CH2
3
70.1 CH
70.1 CH
69.4 CH
69.2 CH
69.4 CH
68.7 CH
65.4 CH
4
36.6 CH2
36.7 CH2
36.8 CH2
39.7 CH2
34.4 CH2
41.8 CH2
39.1 CH2
5
74.3 CH
74.3 CH
76.7 CH
72.0 CH
72.5 CH
74.5 CH
71.3 CH
6
76.2 CH
76.2 CH
75.2 CH
38.7 CH2
38.9 CH2
45.3 CH2
45.6 CH2
7
40.0 CH2
40.0 CH2
41.2 CH2
32.4 CH2
32.2 CH2
199.3 C
199.7 C
1'
134.3 C
134.3 C
134.2 C
133.4 C
133.1 C
135.2 C
135.8 C
2'
112.8 CH
112.9 CH
112.4 CH
112.6 CH
111.3 CH
110.9 CH
110.9 CH
3'
148.2 C
148.3 C
148.6 C
148.7 C
148.5 C
149.0 C
148.9 C
4'
146.6 C
146.7 C
146.7 C
147.2 C
146.2 C
146.9 C
146.7 C
5'
115.4 CH
115.5 CH
115.4 CH
115.7 CH
115.6 CH
115.8 CH
115.7 CH
6'
122.0 CH
122.0 CH
121.3 CH
121.9 CH
119.8 CH
119.7 CH
119.6 CH
1''
131.9 C
131.9 C
131.7 C
135.1 C
134.9 C
130.9 C
131.0 C
2''
114.3 CH
114.3 CH
114.5 CH
113.3 CH
113.2 CH
112.3 CH
112.3 CH
3''
148.7 C
148.7 C
148.7 C
148.7 C
148.5 C
148.7 C
148.7 C
4''
145.8 C
145.8 C
146.0 C
145.4 C
145.1 C
153.4 C
153.3 C
5''
116.0 CH
116.0 CH
115.9 CH
116.0 CH
115.9 CH
115.8 CH
115.7 CH
6''
123.0 CH
123.0 CH
123.0 CH
121.8 CH
121.7 CH
125.1 CH
125.1 CH
1'''
133.5 C
132.7 C
132.7 C
2'''
114.6 CH
108.2 CH
114.4 CH
3'''
148.3 C
148.6 C
148.2 C
4'''
145.7 C
134.8 C
145.9 C
5'''
115.6 CH
148.6 C
116.0 CH
6'''
123.3 CH
108.2 CH
122.2 CH
3'-OMe
56.3 CH3
56.3 CH3
56.3 CH3
56.4 CH3
56.3 CH3
56.3 CH3
56.3 CH3
3''-OMe
56.4 CH3
56.4 CH3
56.4 CH3
56.3 CH3
56.1 CH3
56.3 CH3
56.2 CH3
3'''-OMe
56.3 CH3
56.7 CH3
56.3 CH3
5'''-OMe
56.7 CH3
The known compounds kravanhol A (8) and renealtin A (9)11 were identified by 10
comparison of their NMR data with those in the literature. It is worth noting that the hedycoropyran-type diarylheptanoids are originally derived from the linear diarylheptanoid analogues, and thus it can be comprehended that any configurations of the newly formed chiral centers at C-1 and C-5 are possible, which is also supported by the report of enantiomeric examples in the literature.24 Therefore, special attention should be paid to the future stereochemistry related study of this group of molecules to avoid wrong assignments. Compounds 19 were evaluated for their inhibitory effects on NO production in LPS-activated RAW264.7 macrophages.25 Cell viability was first examined by the MTT method to exclude false positive results caused by the cytotoxicity of the tested compounds.26 As a result, all compounds showed no obvious cytotoxic effects (over 90% cell survival) against RAW264.7 cells at the concentration of 100 M. Subsequent NO inhibition assay27 showed that compounds 2, 5, 6 and 9 could inhibit NO release, with IC50 values ranging from 17.4 to 26.5 M, being more potent than dexamethasone which is a broad-spectrum anti-inflammatory agent (IC50 = 32.5 M) (Table 3). Table 3 IC50 values of the active compounds on LPS-induced NO production in RAW264.7 cellsa Compounds
IC50 (M)
2
23.6 3.3
5
21.8 0.2
6
17.4 0.3
9
26.5 0.8
DXMSb
32.5 3.0
a
Compounds with IC50 > 50 μM were not listed. b Dexamethasone as positive control
Acknowledgements This project was financially supported by Natural Science Foundation of Shandong Province [No. JQ201721], the Young Taishan Scholars Program [No. tsqn20161037], Innovation Team Project of Jinan Science & Technology Bureau [No. 2018GXRC003] and Shandong Talents Team Cultivation Plan of University Preponderant Discipline [No. 10027]. We also thank Prof. Guo-hua Ye for the identification of the plant material. 11
Appendix A. Supplementary data Supplementary data associated with this article can be found online at xx
12
References 1. Matsuda H, Ando S, Kato T, Morikawa T, Yoshikawa M. Inhibitors from the rhizomes of Alpinia officinarum on production of nitric oxide in lipopolysaccharide-activated macrophages and the structural requirements of diarylheptanoids for the activity. Bioorg Med Chem. 2006; 14: 138142. 2. Li J, Zhao F, Li MZ, Chen LX, Qiu F. Diarylheptanoids from the Rhizomes of Curcuma kwangsiensis. J Nat Prod. 2010; 73: 16671671. 3. Lee KS, Li G, Kim SH, Lee CS, Woo MH, Lee SH, Jhang YD, Son JK. Cytotoxic Diarylheptanoids from the Roots of Juglans mandshurica. J Nat Prod. 2002; 65: 17071708. 4. Wohlmuth H, Deseo MA, Brushett DJ, Thompson DR, MacFarlane G, Stevenson LM, Leach DN. Diarylheptanoid from Pleuranthodium racemigerum with in Vitro Prostaglandin E2 Inhibitory and Cytotoxic Activity. J Nat Prod. 2010; 73: 743746. 5. Cerulli A, Lauro G, Masullo M, Cantone V, Olas B, Kontek B, Nazzaro F, Bifulco G, Piacente S. Cyclic Diarylheptanoids from Corylus avellana Green Leafy Covers: Determination of Their Absolute Configurations and Evaluation of Their Antioxidant and Antimicrobial Activities. J Nat Prod. 2017; 80: 17031713. 6. Minassi A, Sánchez-Duffhues G, Collado JA, Muñoz E, Appendino G. Dissecting the Pharmacophore of Curcumin. Which Structural Element Is Critical for Which Action? J Nat Prod. 2013; 76: 11051112. 7. Liu H, Wu ZL, Huang XJ, Peng Y, Huang X, Shi L, Wang Y, Ye WC, Evaluation of Diarylheptanoid–Terpene Adduct Enantiomers from Alpinia officinarum for Neuroprotective Activities. J Nat Prod. 2018; 81: 162170. 8. Jirásek P, Amslinger S, Heilmann J. Synthesis of Natural and Non-natural Curcuminoids and Their Neuroprotective Activity against Glutamate-Induced Oxidative Stress in HT-22 Cells. J Nat Prod. 2014; 77: 22062217. 9. Dhillon N, Aggarwal BB, Newman RA, Wolff RA, Kunnumakkara AB, Abbruzzese JL, Ng CS, Badmaev V, Kurzrock R, Phase II Trial of Curcumin in Patients with Advanced Pancreatic Cancer. Clin Cancer Res. 2008; 14: 4491. 10. Luo JG, Yin H, Kong LY. Monoterpenes from the fruits of Amomum kravanh. J Asian Nat 13
Prod Res. 2014; 16: 471475. 11. Yin H, Luo JG, Kong LY. Diarylheptanoids from the fruits of Amomum kravanh and their inhibitory activities of nitric oxide production. Phytochem Lett. 2013; 6: 403406. 12. Yin H, Luo JG, Kong LY. Tetracyclic diterpenoids with isomerized isospongian skeleton and labdane diterpenoids from the fruits of Amomum kravanh. J Nat Prod. 2013; 76: 237242. 13. Yu JH, Yu ZP, Wang YY, Bao J, Zhu KK, Yuan T, Zhang H. Triterpenoids and triterpenoid saponins from Dipsacus asper and their cytotoxic and antibacterial activities. Phytochemistry. 2019; 162: 241249. 14. Song XQ, Zhang JS, Yu SJ, Yu JH, Zhang H. New octadecanoid derivatives from the seeds of Ipomoea nil. Chin J Nat Med. 2019; 17: 303307. 15. Zhai HJ, Yu JH, Zhang QQ, Liu HS, Zhang JS, Song XQ, Zhang YY, Zhang H. Cytotoxic and antibacterial triterpenoids from the roots of Morinda officinalis var. officinalis. Fitoterapia. 2019; 133: 5661. 16. The air-dried powder of the fruits of A. kravanh (30 kg) was extracted with 95% EtOH at room temperature for four times to yield a crude extract (2600 g). The extract was then suspended in 4.0 L water and partitioned with EtOAc (4.0 L 4). The EtOAc partition (426 g) was subjected to chromatography column (CC) over D101-macroporous absorption resin, eluted with EtOH-H2O (30%, 50%, 65%, 80% and 95%, v/v), to afford five fractions (A, B, C, D and E). Fraction B (67 g) was subjected to an MCI gel column, eluted with MeOH-H2O (40% to 100%, v/v), to give three subfractions (B1–B3). Fraction B1 (31 g) was then separated by silica gel CC, eluted with petroleum ether-EtOAc (10:1 to 1:10, v/v), to produce ten fractions (B1a–B1j). Fraction B1i (410 mg) was subjected to silica gel CC (CH2Cl2/MeOH, 50:1) to give three fractions (B1i1B1i3). Fr. B1i2 (140 mg) was separated by Sephadex LH-20 (CH2Cl2-MeOH, 1:1, v/v), followed by semi-preparative HPLC (45% MeOH-H2O, 3.00 mL/min), to give 8 (5.3 mg, tR = 30 min), 2 (2.4 mg, tR = 33 min), 1 (5.6 mg, tR = 36 min) and 5 (1.8 mg, tR = 38 min). Fr. B1i3 (100 mg) was separated by Sephadex LH-20 (CH2Cl2-MeOH, 1:1, v/v) to give 3 (3.7 mg) and B1i3b (36 mg). B1i3b was further purified by semi-preparative HPLC (30% MeCN-H2O, 3.00 mL/min) to afford 9 (1.3 mg, tR = 10 min), 6 (1.4 mg, tR = 12 min), 7 (2.6 mg, tR = 13 min) and 4 (1.8 mg, tR = 16 min). Kravanhol C (1): Colorless oil; []25D 7.5 (c 0.10, MeOH); UV (MeOH) max (log ) 228 (3.40), 14
281 (3.00) nm; ECD (c 4.9 104 M, MeCN) max () 211 (1.68), 225 (1.19), 242 (1.06), 274 (1.09) nm; 1H and 13C NMR data see Tables 1 and 2; ESIMS m/z 535.1 [M + Na]+; 547.0 [M + Cl]; HRESIMS m/z 535.1942 (calcd for [M Na], 535.1939). Kravanhol D (2): Colorless oil; []25D 12 (c 0.10, MeOH); UV (MeOH) max (log )
227 (3.11),
280 (2.36) nm; ECD (c 3.7 104 M, MeCN) max () 211 (4.37), 225 (0.84) 242 (0.75), 280 (0.56) nm; 1H and 13C NMR data see Tables 1 and 2; ESIMS m/z 565.0 [M + Na]+; 577.1 [M + Cl]; HRESIMS m/z 565.2042 (calcd for [M Na], 565.2044). Kravanhol E (3): Colorless oil; []25D 22 (c 0.10, MeOH); UV (MeOH) max (log ) 229 (3.02), 280 (2.17) nm; ECD (c 3.9 104 M, MeCN) max () 206 (6.81), 237 (1.12), 284 (0.63) nm; 1H
and
13C
NMR data see Tables 1 and 2; ESIMS m/z 535.0 [M + Na]+; 547.0 [M + Cl];
HRESIMS m/z 535.1938 (calcd for [M Na], 535.1939). Kravanhol F (4): Colorless oil; []25D 30 (c 0.10, MeOH); UV (MeOH) max (log ) 228 (3.16), 280 (2.66) nm; ECD (c 5.1 104 M, MeCN) max () 207 (2.75), 231 (2.76) nm; 1H and 13C NMR data see Tables 1 and 2; ESIMS m/z 413.1 [M + Na]+; 425.0 [M + Cl]; HRESIMS m/z 391.1750 (calcd for [M H], 391.1751). Kravanhol G (5): Colorless oil; []25D 35 (c 0.10, MeOH); UV (MeOH) max (log ) 227 (3.29), 281 (2.86) nm; ECD (c 6.4 104 M, MeCN) max () 225 (2.37), 229 (1.48) nm; 1H and 13C NMR data see Tables 1 and 2; ESIMS m/z 413.0 [M + Na]+; 425.0 [M + Cl]; HRESIMS m/z 391.1754 (calcd for [M H], 391.1751). Kravanhol H (6): Colorless oil; []25D 24 (c 0.10, MeOH); UV (MeOH) max (log ) 229 (3.36), 278 (3.05) nm; ECD (c 5.2 104 M, MeCN) max () 204 (2.02), 232 (1.31) nm; 1H and 13C NMR data see Tables 1 and 2; ESIMS m/z 411.1 [M + Na]+; 387.1 [M H], 423.1 [M + Cl]; HRESIMS m/z 389.1609 (calcd for [M H], 389.1595). Kravanhol I (7): Colorless oil; []25D 16.5 (c 0.10, MeOH); UV (MeOH) max (log ) 228 (3.50), 279 (3.20) nm; ECD (c 6.4 104 M, MeCN) max () 206 (0.68), 233 (0.83) nm; 1H and 13C NMR data see Tables 1 and 2; ESIMS m/z 411.0 [M + Na]+; 387.1 [M H], 423.0 [M + Cl]; HRESIMS m/z 389.1598 (calcd for [M H], 389.1595). 17. The fruits of Amomum kravanh Pierre ex Gagnep. were purchased from Kunming ‘Juhuayuan’ herbal market and were collected in Aug 2016 in Xinshuangbanna, Yunnan province, China. The plant materials were authenticated by Prof. Guo-hua Ye from Shandong College of Traditional 15
Chinese Medicine. A voucher specimen has been deposited at School of Biological Science and Technology, University of Jinan (Accession number: npmc-022). 18. Chlipala GE, Tri PH, Hung NV, Krunic A, Shim SH, Soejarto DD, Orjala J. Nhatrangins A and B, Aplysiatoxin-Related Metabolites from the Marine Cyanobacterium Lyngbya majuscula from Vietnam. J Nat Prod. 2010; 73: 784787. 19. Zhu JY, Cheng B, Zheng YJ, Dong Z, Lin SL, Tang GH, Gu Q, Yin S. Enantiomeric neolignans and sesquineolignans from Jatropha integerrima and their absolute configurations. RSC Adv. 2015; 5: 1220212208. 20. Pescitelli G, Bruhn T. Good Computational Practice in the Assignment of Absolute Configurations by TDDFT Calculations of ECD Spectra. Chirality. 2016; 28: 466474. 21. The initial conformations of 1, 4 and 5 were established via the MM2 force field in the ChemDraw_Pro_14.1 software. Conformational searches using mixed torsional/low-mode sampling method with MMFFs in an energy window of 2.58 kcal/mol were carried out by means of the conformational search module in the Maestro 10.2 software. The re-optimization and the following TD-DFT calculations of the re-optimized conformations were all performed with Gaussian 09 at the B3LYP/6-311G(d,p) level in vacuo. Frequency analysis was performed as well to confirm that the re-optimized conformers were at the energy minima. Finally, the SpecDis 1.64 software was used to obtain the Boltzmann-averaged ECD spectra. 22. Lin YS, Lin JH, Chang CC, Lee SS. Tetrahydropyran- and Tetrahydrofuran-Containing Diarylheptanoids from Hedychium coronarium Rhizomes. J Nat Prod. 2015; 78: 181187. 23. Sudarshan K, Aidhen, IS. Synthesis of (+)-Centrolobine and Its Analogues by Using Acyl Anion Chemistry. Eur J Org Chem. 2013; 12: 2298–2302. 24. Dong SH, Nikoli ć D, Simmler C, Qiu F, Breemen RB, Soejarto DD, Pauli GF, Chen SN. Diarylheptanoids from Dioscorea villosa (Wild Yam). J Nat Prod. 2012; 75: 21682177. 25. The RAW264.7 murine macrophage cell line was purchased from the Cell Bank of Beijing Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Beijing, China), and was cultured in Dulbecco’s modified Eagle medium (DMEM) which was supplemented with 10% FBS. The cells were placed at 37 °C in a humidified incubator containing 5% CO2. 26. The cytotoxicity of the isolated compounds toward RAW264.7 cells was determined by MTT 16
assay. RAW264.7 cells were planted in 96-well plates (5 × 103/well) for 24 h. Then they were treated with tested samples which were dissolved in DMSO and diluted in 100 μL DMEM making the final drug concentration 100 μM and 1% DMSO. 1% DMSO served as solvent control, and wells without cells containing only 100 μL DMEM served as blank control. Twenty L solution of MTT was added to each well after 24 h. After incubation for another 4 h, the medium was removed and 100 μL DMSO was added to each well, and then the absorbance (A) was detected at 490 nm using a microplate reader. The inhibition of cell growth was calculated according to the following formula: % Inhibition = [1 (Asample Ablank) / (Asolvent Ablank)] × 100. 27. Nitric oxide release level was assessed by a colorimetric assay based on a diazotization reaction using the Griess reagent system. RAW264.7 cells were planted in 96-well plates (4 × 104/well) for 24 h and then pre-incubated with different concentrations of compounds for 1 h before stimulation with or without LPS (1 g/mL) for 24 h. The NO concentration in culture medium was determined by Griess reagent kit, then the absorbance (A) was measured at 540 nm using a Tecan microplate reader. The inhibition of NO release was calculated according to the following formula: % Inhibition = [1 (Asample Ablank) / (Amodel Ablank)] × 100. The experiments were performed in triplicates, and dexamethasone was used as a positive control.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
17
R2 R1
R4 O R3
HO O
OH O
NO Inhibition 17.4-26.5 M
Amomum kravanh
18
S0960-894X(20)30096-2 https://doi.org/10.1016/j.bmcl.2020.127026 BMCL 127026
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
18 November 2019 8 February 2020 10 February 2020
Please cite this article as: Zhang, J-S., Cao, X-X., Yu, J-H., Yu, Z-P., Zhang, H., Diarylheptanoids with NO Production Inhibitory Activity from Amomum kravanh, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127026
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© 2020 Elsevier Ltd. All rights reserved.
Diarylheptanoids with NO Production Inhibitory Activity from Amomum kravanh Jun-Sheng Zhang, Xin-Xin Cao, Jin-Hai Yu, Zhi-Pu Yu, Hua Zhang
School of Biological Science and Technology, University of Jinan, Jinan, China
Correspondence Prof. Hua Zhang School of Biological Science and Technology, University of Jinan, Jinan 250022, China Phone: +86-531-89736199 Email: [email protected]
1
Abstract Seven new diarylheptanoids, kravanhols CI (17), along with two known analogues (8 and 9), were isolated from the fruits of Amomum kravanh. The structures of compounds 17 were elucidated by analysis of spectroscopic data, and the absolute configurations of selective ones were determined by time-dependent density functional theory (TD-DFT) based electronic circular dichroism (ECD) calculations. All compounds were evaluated for their inhibitory effects on the nitric oxide (NO) production induced by lipopolysaccharide (LPS) in murine RAW264.7 macrophage cells. Compounds 2, 5, 6 and 9 exhibited moderate inhibitory activity with IC50 values in the range of 17.426.5 M, being more potent than the positive control dexamethasone (IC50 32.5 M).
Key words: Zingiberaceae, Amomum kravanh, diarylheptanoid, ECD calculation, NO production
2
Diarylheptanoids are a group of naturally occurring phenols with the general structure of two aromatic rings linked by a linear seven-carbon chain. These compounds have attracted broad interest from drug discovery field due to their diverse bioactivities, such as anti-inflammatory,1,2 antitumor,3,4 antioxidant,5 antiviral6 and neuroprotective7,8 activities. Curcumin, for instance, has been forwarded to phase II clinical trial for the treatment of pancreatic cancer.9 So far, natural diarylheptanoids have been reported mainly from the plants of Zingiberaceae family. Amomum kravanh (Zingiberaceae) is a tropical plant widely distributed in Thailand, Vietnam and Southern areas of China. The fruits are commonly used as flavoring in dishes and also as a traditional Chinese medicine (TCM) for stomach diseases and digestive disorders. Previous chemical investigations on this plant have revealed the presence of monoterpenoids, diarylheptanoids and diterpenoids, with moderate NO inhibitory activities.1012 In our continuing efforts to explore new bioactive substances from TCM,1315 seven new diarylheptanoids, kravanhols CI (17), along with two known analogues (8 and 9),16 were isolated from the fruits of Amomum kravanh.17 These compounds were structurally characterized mainly by spectroscopic analyses. They were also screened for anti-inflammatory activity by using the lipopolysaccharide (LPS)-induced NO production model in murine RAW264.7 macrophages, and compounds 2, 5, 6 and 9 exhibited moderate inhibitory activity. Herein, details of the isolation, structure elucidation and the NO inhibitory effect of these compounds are described below. O 5'''
HO
OH
D
O
3
3''' 5'
1'''
1'
A
HO
HO
2 1
B
4 5
O
H 6
7
OH
3'
O
O 3''
1''
OH
OH O
1
OH
OH
HO
2
H
OH
O HO
OH 4 R = -OH 5 R = -OH
H
O
O HO
O
H
O OH
O O
6
7
Fig. 1. Structures of compounds 17
O O OH
HO
O
3
OH
3
OH
OH
O
O O
R
O
H
O
O HO
OH
O
H
O
C 5''
HO
OH
Compound 1, a colorless oil, had the molecular formula C28H32O9 as determined by the HRESIMS ion at m/z 535.1942 (calcd for [M Na], 535.1939), indicating 13 degrees of unsaturation. The 1H NMR spectrum of 1 exhibited three typical ABX spin systems at H 6.77 (1H, dd, J = 8.0, 1.8 Hz), 6.61 (1H, d, J = 8.0 Hz) and 6.85 (1H, d, J = 1.8 Hz); 6.61 (1H, dd, J = 8.0, 1.8 Hz), 6.57 (1H, d, J = 8.0 Hz) and 6.77 (1H, d, J = 1.8 Hz); as well as 6.68 (1H, dd, J = 8.0, 1.8 Hz), 6.73 (1H, d, J = 8.0 Hz) and 6.82 (1H, d, J = 1.8 Hz). These data suggested the presence of three 1,3,4-trisubstituted benzene rings. The
13C
NMR spectrum in combination with DEPT135 experiment
resolved 28 carbon resonances attributable to three benzene rings, three methoxyl groups, four oxygenated methines and three methylenes. As 12 of the 13 degrees of unsaturation were consumed by the aforementioned three phenyl rings, the remaining one required the presence of one additional ring in the structure of 1. In the 1H−1H COSY spectrum, a seven-carbon fragment (C-1 to C-7) was first established by the observed correlations (Fig. 2). The aforementioned information implied that compound 1 possessed most structural features of a diarylheptanoid and was quite similar to kravanhol A (8),11 a cometabolite in the current study. In comparison with 8, the major structural changes of 1 were the appearance of an additional trisubstituted benzene ring and a methine (C 54.4, C-2) instead of a methylene in 8. The location of this extra phenyl group was assigned at C-2 by the HMBC correlations from H-2 (H 6.769) and H-6 (H 6.605) to C-2 (C 54.4). The planar structure of 1 was further secured by detailed interpretation of its 2D NMR data (Fig. 2). Particularly, the methoxyl groups were assigned at C-3, C-3 and C-3 by NOE correlations of 3-OMe/H-2, 3-OMe/H-2 and 3-OMe/H-2, respectively. The relative configuration of 1 was established on the basis of NOESY experiment (Fig. 2) and analysis of 1H–1H coupling constants. The NOE correlation of H-1/H-5, together with the large coupling constant between H-1 and H-2 (J = 10.9 Hz), suggested that these protons were axially bonded in the chair-conformational ring B. H-1 and H-5 were then arbitrarily assigned in -orientation while H-2 was in -orientation. Thus, the small coupling constant between H-2 and H-3 (J = 2.6 Hz) indicated that H-3 was equatorially positioned in -orientation. Finally, H-6 was 4
established to be in cis-relationship with H-5 and thus -oriented on the basis of the small J5,6 value (3.1 Hz).18,19
Fig. 2. 1H1H COSY (
), selected HMBC (
) and NOESY (
) correlations
of 1 The absolute configuration of 1 was determined by computational approach of quantum chemistry using Time-Dependent Density Functional Theory (TD-DFT) method.20,21 The established relative configuration of 1 indicated that it possessed either (1S,2S,3S,5R,6S) or (1R,2R,3R,5S,6R) absolute configuration. As shown in Fig. 3, the experimental ECD curve of 1 matched well with the calculated ECD curve of (1R,2R,3R,5S,6R)-enantiomer, confirming the absolute configuration of 1 as shown. The structure of 1 was thus unequivocally assigned and named kravanhol C following kravanhols A and B reported from the same species.11
Fig. 3. Experimental and calculated ECD spectra for 1 5
Table 1 1H NMR data for 17 in CD3OD (600 MHz, in ppm, J in Hz) No.
1
2
3
4
5
6
7
1
4.93, d (10.9)
4.92, d (10.8)
4.84, d (10.5)
4.40, d (9.8)
4.73, brs
4.33, dd (11.4, 1.7)
4.75 brd (11.9)
2
2.96, dd (10.9, 2.6)
2.95, dd (10.8, 2.2)
2.56, dd (10.5, 9.9)
3.53, dd (9.8, 3.0)
3.57, d (3.5)
2.11, m
1.83, m
1.42, m
1.69, m
3
4.19, m
4.22, m
4.51, ddd (10.9, 9.9, 4.8)
4.11, m
3.98, m
3.93, m
4.25, quintet (2.5)
4
2.13, m
2.14, m
2.26, ddd (13.3, 4.8, 1.7)
1.90, m
1.90, m
2.11, m
1.81, m
1.77, m
1.76, m
1.93, ddd (13.3, 10.9, 6.7)
1.70, m
1.57, m
1.34, m
1.65, m
5
3.99, ddd (11.8, 3.1, 2.3)
3.98, ddd (11.9, 3.4, 2.3)
3.95, ddd (6.7, 6.0, 1.7)
3.85, m
3.80, m
4.10, m
4.55, m
6
3.69, ddd (7.6, 6.5, 3.1)
3.68, ddd (7.6, 6.5, 3.4)
4.16, ddd (7.5, 6.0, 5.4)
1.76, m
1.89, m
3.35, dd (15.4, 7.2)
3.26, dd (15.4, 7.3)
1.67, m
1.69, m
3.04, dd (15.4, 5.3)
2.94, dd (15.4, 5.0)
7
2.89, dd (13.5, 6.5)
2.89, dd (13.5, 6.5)
2.91, dd (13.8, 5.4)
2.60, m
2.69, m
2.72, dd (13.5, 7.6)
2.72, dd (13.5, 7.6)
2.70, dd (13.8, 7.5)
2'
6.85, d (1.8)
6.86, d (1.8)
6.61, m (overlapping)
7.01, d (1.8)
7.07, d (1.7)
6.84, d (1.6)
6.82, brs
5'
6.611, d (8.0)
6.62, d (8.0)
6.60, m (overlapping)
6.78, d (8.1)
6.78, d (8.1)
6.71, brs
6.70, brs
6'
6.766, dd (8.0, 1.8)
6.78, dd (8.1, 1.8)
6.60, m (overlapping)
6.89, dd (8.1, 1.8)
6.85, dd (8.1, 1.7)
6.72, d (1.6)
6.70, brs
2''
6.82, d (1.8)
6.82, d (1.6)
6.90, brs
6.73, d (1.8)
6.73, d (1.8)
7.56, d (1.9)
7.56, d (1.8)
5''
6.73, d (8.0)
6.72, d (8.0)
6.72, brs
6.68, d (8.0)
6.68, d (8.0)
6.85, d (8.3)
6.85, d (8.3)
6''
6.68, dd (8.0, 1.8)
6.68, dd (8.0, 1.6)
6.72, brs
6.60, dd (8.0, 1.8)
6.61, dd (8.0, 1.8)
7.61, dd (8.3, 1.9)
7.61, dd (8.3, 1.8)
2'''
6.769, d (1.8)
6.47, s
6.50, d (1.8)
5'''
6.57, d (8.0)
6'''
6.605, dd (8.0, 1.8)
6.47, s
6.56, dd (8.1, 1.8)
3'-OMe
3.73, s
3.74, s
3.69, s
3.88, s
3.87, s
3.74, s
3.70, s
3''-OMe
3.80, s
3.80, s
3.83, s
3.79, s
3.74, s
3.86, s
3.83, s
3'''-OMe
3.74, s
3.73, s
3.69, s
5'''-OMe
6.63, d (8.1)
3.73, s
6
Compound 2 displayed a quasi-molecular ion peak at m/z 565.2042 [M + Na]+ in the HRESIMS analysis, consistent with a molecular formula of C29H34O10 with 30 mass units more than that of 1. The 1D NMR data (Tables 1 and 2) of 2 were very similar to those of 1 but showed an additional methoxyl unit. In comparison with 1, the NMR data of 2 differed significantly in the D ring, featuring a symmetrical 1,3,4,5-tetrasubstituted benzene ring instead of the 1,3,4-trisubstited benzene ring in 1, which was supported by the observation of a two-proton singlet signal at H 6.47 (2H, s, H-2''' and H-6'''). The assignment of the extra methoxyl group on the D ring and the full structure of 2 were further corroborated by examination of 2D NMR data (Figs. S9-S12, Supplementary data). The absolute configuration of 2 was assigned to be the same as that of 1 by comparison of their ECD spectra, which showed consistent Cotton effects (Fig. S43, Supplementary data). Compound 2 was thus structurally elucidated and named kravanhol D. Compound 3 (kravanhol E) was determined to possess the same planar structure as 1 by HRESIMS and 1D NMR spectra, indicating that they were stereoisomers. The relative configuration of 3 was also established by analyses of NOESY data and 1H– 1H
coupling patterns. Key NOE correlations of H-1/H-3 and H-1/H-6 indicated that
H-1, H-3 and the C-5 sidechain were axially located in the chair conformational B-ring. Thus, H-1 and H-3 were randomly designated in -orientation, while H-5 was equatorially oriented and designated in -orientation. The large values of J1,2 (10.5 Hz) and J2.3 (9.9 Hz) implied that H-2 was axially bonded in -orientation. Moreover, the magnitude of J5,6 (6.0 Hz) in 3 Vs. that (3.1 Hz) in 1 suggested that H-5 and H-6 adopted a trans-relationship in 3. The structure with relative configuration of 3 was thereby established. Compound 4, a colorless oil, had the molecular formula C21H26O7 as determined by the HRESIMS ion at m/z 391.1750 (calcd for [M H], 391.1751). The 1H NMR spectrum of 4 exhibited two typical ABX spin systems at H 6.89 (1H, dd, J = 8.1, 1.8 Hz), 6.78 (1H, d, J = 8.1 Hz) and 7.01 (1H, d, J = 1.8 Hz), as well as 6.60 (1H, dd, J = 8.0, 1.8 Hz), 6.68 (1H, d, J = 8.0 Hz) and 6.73 (1H, d, J = 1.8 Hz). These data suggested the presence of two 1,3,4-trisubstitued benzene rings. The 7
13C
NMR
spectrum in combination with DEPT135 experiment resolved 21 carbon resonances attributable to two benzene rings, two methoxyl groups, four oxygenated methines and three methylenes. The aforementioned NMR data of 4 exhibited high resemblance with those of hedycoropyran C,22 a known diarylheptanoid reported before, and the main differences were attributed to the 3-O-methylation and 6-dehydroxylation in 4. This was supported by the presence of an additional methoxyl group (H 3.79; C 56.3) in 4, together with the replacement of an oxymethine in hedycoropyran C by a methylene (C 38.7) in 4. The planar structure of 4 was further secured by detailed analyses of its 2D NMR data (Fig. 4). The relative configuration of 4 was assigned to be the same as that of hedycoropyran C (except C-6) by comparing their 1D NMR data and NOESY data (Fig. 4). Moreover, the experimental ECD curve of 4 showed negative Cotton effects around 231 and 207 nm, respectively, which matched the calculated ECD spectrum of (1R,2S,3S,5S)-isomer of 4 (Fig. 5A). Thus, the absolute configuration of 4 was assigned as 1R,2S,3S,5S and it was named kravanhol F.
Fig. 4. 1H1H COSY (
), selected HMBC (
) and NOESY (
) correlations
of 4 Compound 5 had the same molecular formula of C21H26O7 as 4 based on the HRESIMS analysis (m/z 391.1754 [M H], calcd 391.1751), indicative of a stereoisomer of the latter. The 1D NMR data (Tables 1 and 2) of 5 showed high similarity to those of 4 with the major differences being attributed to the H-1/H-2 coupling patterns (J1,2 brs in 5 and 9.8 Hz in 4), indicating that 5 was the 2-epimer of 4. Detailed 2D NMR data (Figs. S27-S30, Supplementary data) analyses secured the structure of 5. To assign the absolute configuration of 5, the experimental ECD curve was also compared with the calculated one with a very good match as shown in Fig 5B. Thus, the structure of 5 was assigned as depicted and named kravanhol G. 8
Fig. 5. Experimental and calculated ECD spectra for 4 (A) and 5 (B) Compound 6 displayed a protonated molecular ion at m/z 389.1609 in HRESIMS analysis, consistent with a molecular formula of C21H24O7. The 1D NMR data (Tables 1 and 2) and HSQC spectrum revealed signals for a ketone group (C 199.3), two trisubstituted benzene rings and two methoxyl groups (H 3.87, 3.74; C 56.3, 56.1). The aforementioned spectroscopic information was very similar to that of 1-(3,4-dimethoxyphenyl)-2-((2S,6R)-6-(3,4-dimethoxyphenyl)tetrahydro-2H-pyran-2yl) ethanone, a synthetic analogue reported before,23 except for the presence of an additional oxymethine [H 3.93; C 68.7] in 6 instead of a methylene, together with the absence of two methoxyl groups in the synthetic analogue. Key 1H-1H COSY correlations of H2-2/H-3 and H-3/H2-4 assigned the additional oxymethine at C-3. Further analysis of 2D NMR data (Fig. S44, Supplementary data) led to the proposal of structure of 6, which revealed that 4-OMe and 4-OMe in the above-mentioned synthetic analogue were demethylated in 6. The NOE correlations of H-1/H-3 and H-1/H-5 (Fig. S44, Supplementary data) supported that they were all axially bonded in the B ring and were assigned to be -oriented. Thus, 6 was structurally characterized and given the trivial name kravanhol H. Compound 7 had the same molecular formula as 6, implying that they were structural isomers. The NMR data of 7 were very similar to those of 6 except for the signals for ring B. Detailed 2D NMR analyses permitted the establishment of the planar structure of 7 as shown, being the same as that of 6. In the NOESY spectrum, 9
correlations between H-1 and H-5 together with the large coupling constant between H-1 and H-2 (J = 11.9 Hz) indicated that H-1 and H-5 were axially located in the chair-conformational B-ring and were assigned to be -oriented. Thus, the small coupling constants of H-3 with vicinal protons (J = 2.5 Hz) indicated that it was equatorially positioned and -oriented. Compound 7 was then characterized as the 3-epimer of 6 and was named kravanhol I. Table 2 13C NMR data for 17 in CD3OD (151 MHz, in ppm) No.
1
2
3
4
5
6
7
1
79.5 CH
79.6 CH
80.2 CH
79.3 CH
77.1 CH
78.9 CH
75.1 CH
2
54.4 CH
54.8 CH
59.6 CH
73.7 CH
72.0 CH
43.6 CH2
40.9 CH2
3
70.1 CH
70.1 CH
69.4 CH
69.2 CH
69.4 CH
68.7 CH
65.4 CH
4
36.6 CH2
36.7 CH2
36.8 CH2
39.7 CH2
34.4 CH2
41.8 CH2
39.1 CH2
5
74.3 CH
74.3 CH
76.7 CH
72.0 CH
72.5 CH
74.5 CH
71.3 CH
6
76.2 CH
76.2 CH
75.2 CH
38.7 CH2
38.9 CH2
45.3 CH2
45.6 CH2
7
40.0 CH2
40.0 CH2
41.2 CH2
32.4 CH2
32.2 CH2
199.3 C
199.7 C
1'
134.3 C
134.3 C
134.2 C
133.4 C
133.1 C
135.2 C
135.8 C
2'
112.8 CH
112.9 CH
112.4 CH
112.6 CH
111.3 CH
110.9 CH
110.9 CH
3'
148.2 C
148.3 C
148.6 C
148.7 C
148.5 C
149.0 C
148.9 C
4'
146.6 C
146.7 C
146.7 C
147.2 C
146.2 C
146.9 C
146.7 C
5'
115.4 CH
115.5 CH
115.4 CH
115.7 CH
115.6 CH
115.8 CH
115.7 CH
6'
122.0 CH
122.0 CH
121.3 CH
121.9 CH
119.8 CH
119.7 CH
119.6 CH
1''
131.9 C
131.9 C
131.7 C
135.1 C
134.9 C
130.9 C
131.0 C
2''
114.3 CH
114.3 CH
114.5 CH
113.3 CH
113.2 CH
112.3 CH
112.3 CH
3''
148.7 C
148.7 C
148.7 C
148.7 C
148.5 C
148.7 C
148.7 C
4''
145.8 C
145.8 C
146.0 C
145.4 C
145.1 C
153.4 C
153.3 C
5''
116.0 CH
116.0 CH
115.9 CH
116.0 CH
115.9 CH
115.8 CH
115.7 CH
6''
123.0 CH
123.0 CH
123.0 CH
121.8 CH
121.7 CH
125.1 CH
125.1 CH
1'''
133.5 C
132.7 C
132.7 C
2'''
114.6 CH
108.2 CH
114.4 CH
3'''
148.3 C
148.6 C
148.2 C
4'''
145.7 C
134.8 C
145.9 C
5'''
115.6 CH
148.6 C
116.0 CH
6'''
123.3 CH
108.2 CH
122.2 CH
3'-OMe
56.3 CH3
56.3 CH3
56.3 CH3
56.4 CH3
56.3 CH3
56.3 CH3
56.3 CH3
3''-OMe
56.4 CH3
56.4 CH3
56.4 CH3
56.3 CH3
56.1 CH3
56.3 CH3
56.2 CH3
3'''-OMe
56.3 CH3
56.7 CH3
56.3 CH3
5'''-OMe
56.7 CH3
The known compounds kravanhol A (8) and renealtin A (9)11 were identified by 10
comparison of their NMR data with those in the literature. It is worth noting that the hedycoropyran-type diarylheptanoids are originally derived from the linear diarylheptanoid analogues, and thus it can be comprehended that any configurations of the newly formed chiral centers at C-1 and C-5 are possible, which is also supported by the report of enantiomeric examples in the literature.24 Therefore, special attention should be paid to the future stereochemistry related study of this group of molecules to avoid wrong assignments. Compounds 19 were evaluated for their inhibitory effects on NO production in LPS-activated RAW264.7 macrophages.25 Cell viability was first examined by the MTT method to exclude false positive results caused by the cytotoxicity of the tested compounds.26 As a result, all compounds showed no obvious cytotoxic effects (over 90% cell survival) against RAW264.7 cells at the concentration of 100 M. Subsequent NO inhibition assay27 showed that compounds 2, 5, 6 and 9 could inhibit NO release, with IC50 values ranging from 17.4 to 26.5 M, being more potent than dexamethasone which is a broad-spectrum anti-inflammatory agent (IC50 = 32.5 M) (Table 3). Table 3 IC50 values of the active compounds on LPS-induced NO production in RAW264.7 cellsa Compounds
IC50 (M)
2
23.6 3.3
5
21.8 0.2
6
17.4 0.3
9
26.5 0.8
DXMSb
32.5 3.0
a
Compounds with IC50 > 50 μM were not listed. b Dexamethasone as positive control
Acknowledgements This project was financially supported by Natural Science Foundation of Shandong Province [No. JQ201721], the Young Taishan Scholars Program [No. tsqn20161037], Innovation Team Project of Jinan Science & Technology Bureau [No. 2018GXRC003] and Shandong Talents Team Cultivation Plan of University Preponderant Discipline [No. 10027]. We also thank Prof. Guo-hua Ye for the identification of the plant material. 11
Appendix A. Supplementary data Supplementary data associated with this article can be found online at xx
12
References 1. Matsuda H, Ando S, Kato T, Morikawa T, Yoshikawa M. Inhibitors from the rhizomes of Alpinia officinarum on production of nitric oxide in lipopolysaccharide-activated macrophages and the structural requirements of diarylheptanoids for the activity. Bioorg Med Chem. 2006; 14: 138142. 2. Li J, Zhao F, Li MZ, Chen LX, Qiu F. Diarylheptanoids from the Rhizomes of Curcuma kwangsiensis. J Nat Prod. 2010; 73: 16671671. 3. Lee KS, Li G, Kim SH, Lee CS, Woo MH, Lee SH, Jhang YD, Son JK. Cytotoxic Diarylheptanoids from the Roots of Juglans mandshurica. J Nat Prod. 2002; 65: 17071708. 4. Wohlmuth H, Deseo MA, Brushett DJ, Thompson DR, MacFarlane G, Stevenson LM, Leach DN. Diarylheptanoid from Pleuranthodium racemigerum with in Vitro Prostaglandin E2 Inhibitory and Cytotoxic Activity. J Nat Prod. 2010; 73: 743746. 5. Cerulli A, Lauro G, Masullo M, Cantone V, Olas B, Kontek B, Nazzaro F, Bifulco G, Piacente S. Cyclic Diarylheptanoids from Corylus avellana Green Leafy Covers: Determination of Their Absolute Configurations and Evaluation of Their Antioxidant and Antimicrobial Activities. J Nat Prod. 2017; 80: 17031713. 6. Minassi A, Sánchez-Duffhues G, Collado JA, Muñoz E, Appendino G. Dissecting the Pharmacophore of Curcumin. Which Structural Element Is Critical for Which Action? J Nat Prod. 2013; 76: 11051112. 7. Liu H, Wu ZL, Huang XJ, Peng Y, Huang X, Shi L, Wang Y, Ye WC, Evaluation of Diarylheptanoid–Terpene Adduct Enantiomers from Alpinia officinarum for Neuroprotective Activities. J Nat Prod. 2018; 81: 162170. 8. Jirásek P, Amslinger S, Heilmann J. Synthesis of Natural and Non-natural Curcuminoids and Their Neuroprotective Activity against Glutamate-Induced Oxidative Stress in HT-22 Cells. J Nat Prod. 2014; 77: 22062217. 9. Dhillon N, Aggarwal BB, Newman RA, Wolff RA, Kunnumakkara AB, Abbruzzese JL, Ng CS, Badmaev V, Kurzrock R, Phase II Trial of Curcumin in Patients with Advanced Pancreatic Cancer. Clin Cancer Res. 2008; 14: 4491. 10. Luo JG, Yin H, Kong LY. Monoterpenes from the fruits of Amomum kravanh. J Asian Nat 13
Prod Res. 2014; 16: 471475. 11. Yin H, Luo JG, Kong LY. Diarylheptanoids from the fruits of Amomum kravanh and their inhibitory activities of nitric oxide production. Phytochem Lett. 2013; 6: 403406. 12. Yin H, Luo JG, Kong LY. Tetracyclic diterpenoids with isomerized isospongian skeleton and labdane diterpenoids from the fruits of Amomum kravanh. J Nat Prod. 2013; 76: 237242. 13. Yu JH, Yu ZP, Wang YY, Bao J, Zhu KK, Yuan T, Zhang H. Triterpenoids and triterpenoid saponins from Dipsacus asper and their cytotoxic and antibacterial activities. Phytochemistry. 2019; 162: 241249. 14. Song XQ, Zhang JS, Yu SJ, Yu JH, Zhang H. New octadecanoid derivatives from the seeds of Ipomoea nil. Chin J Nat Med. 2019; 17: 303307. 15. Zhai HJ, Yu JH, Zhang QQ, Liu HS, Zhang JS, Song XQ, Zhang YY, Zhang H. Cytotoxic and antibacterial triterpenoids from the roots of Morinda officinalis var. officinalis. Fitoterapia. 2019; 133: 5661. 16. The air-dried powder of the fruits of A. kravanh (30 kg) was extracted with 95% EtOH at room temperature for four times to yield a crude extract (2600 g). The extract was then suspended in 4.0 L water and partitioned with EtOAc (4.0 L 4). The EtOAc partition (426 g) was subjected to chromatography column (CC) over D101-macroporous absorption resin, eluted with EtOH-H2O (30%, 50%, 65%, 80% and 95%, v/v), to afford five fractions (A, B, C, D and E). Fraction B (67 g) was subjected to an MCI gel column, eluted with MeOH-H2O (40% to 100%, v/v), to give three subfractions (B1–B3). Fraction B1 (31 g) was then separated by silica gel CC, eluted with petroleum ether-EtOAc (10:1 to 1:10, v/v), to produce ten fractions (B1a–B1j). Fraction B1i (410 mg) was subjected to silica gel CC (CH2Cl2/MeOH, 50:1) to give three fractions (B1i1B1i3). Fr. B1i2 (140 mg) was separated by Sephadex LH-20 (CH2Cl2-MeOH, 1:1, v/v), followed by semi-preparative HPLC (45% MeOH-H2O, 3.00 mL/min), to give 8 (5.3 mg, tR = 30 min), 2 (2.4 mg, tR = 33 min), 1 (5.6 mg, tR = 36 min) and 5 (1.8 mg, tR = 38 min). Fr. B1i3 (100 mg) was separated by Sephadex LH-20 (CH2Cl2-MeOH, 1:1, v/v) to give 3 (3.7 mg) and B1i3b (36 mg). B1i3b was further purified by semi-preparative HPLC (30% MeCN-H2O, 3.00 mL/min) to afford 9 (1.3 mg, tR = 10 min), 6 (1.4 mg, tR = 12 min), 7 (2.6 mg, tR = 13 min) and 4 (1.8 mg, tR = 16 min). Kravanhol C (1): Colorless oil; []25D 7.5 (c 0.10, MeOH); UV (MeOH) max (log ) 228 (3.40), 14
281 (3.00) nm; ECD (c 4.9 104 M, MeCN) max () 211 (1.68), 225 (1.19), 242 (1.06), 274 (1.09) nm; 1H and 13C NMR data see Tables 1 and 2; ESIMS m/z 535.1 [M + Na]+; 547.0 [M + Cl]; HRESIMS m/z 535.1942 (calcd for [M Na], 535.1939). Kravanhol D (2): Colorless oil; []25D 12 (c 0.10, MeOH); UV (MeOH) max (log )
227 (3.11),
280 (2.36) nm; ECD (c 3.7 104 M, MeCN) max () 211 (4.37), 225 (0.84) 242 (0.75), 280 (0.56) nm; 1H and 13C NMR data see Tables 1 and 2; ESIMS m/z 565.0 [M + Na]+; 577.1 [M + Cl]; HRESIMS m/z 565.2042 (calcd for [M Na], 565.2044). Kravanhol E (3): Colorless oil; []25D 22 (c 0.10, MeOH); UV (MeOH) max (log ) 229 (3.02), 280 (2.17) nm; ECD (c 3.9 104 M, MeCN) max () 206 (6.81), 237 (1.12), 284 (0.63) nm; 1H
and
13C
NMR data see Tables 1 and 2; ESIMS m/z 535.0 [M + Na]+; 547.0 [M + Cl];
HRESIMS m/z 535.1938 (calcd for [M Na], 535.1939). Kravanhol F (4): Colorless oil; []25D 30 (c 0.10, MeOH); UV (MeOH) max (log ) 228 (3.16), 280 (2.66) nm; ECD (c 5.1 104 M, MeCN) max () 207 (2.75), 231 (2.76) nm; 1H and 13C NMR data see Tables 1 and 2; ESIMS m/z 413.1 [M + Na]+; 425.0 [M + Cl]; HRESIMS m/z 391.1750 (calcd for [M H], 391.1751). Kravanhol G (5): Colorless oil; []25D 35 (c 0.10, MeOH); UV (MeOH) max (log ) 227 (3.29), 281 (2.86) nm; ECD (c 6.4 104 M, MeCN) max () 225 (2.37), 229 (1.48) nm; 1H and 13C NMR data see Tables 1 and 2; ESIMS m/z 413.0 [M + Na]+; 425.0 [M + Cl]; HRESIMS m/z 391.1754 (calcd for [M H], 391.1751). Kravanhol H (6): Colorless oil; []25D 24 (c 0.10, MeOH); UV (MeOH) max (log ) 229 (3.36), 278 (3.05) nm; ECD (c 5.2 104 M, MeCN) max () 204 (2.02), 232 (1.31) nm; 1H and 13C NMR data see Tables 1 and 2; ESIMS m/z 411.1 [M + Na]+; 387.1 [M H], 423.1 [M + Cl]; HRESIMS m/z 389.1609 (calcd for [M H], 389.1595). Kravanhol I (7): Colorless oil; []25D 16.5 (c 0.10, MeOH); UV (MeOH) max (log ) 228 (3.50), 279 (3.20) nm; ECD (c 6.4 104 M, MeCN) max () 206 (0.68), 233 (0.83) nm; 1H and 13C NMR data see Tables 1 and 2; ESIMS m/z 411.0 [M + Na]+; 387.1 [M H], 423.0 [M + Cl]; HRESIMS m/z 389.1598 (calcd for [M H], 389.1595). 17. The fruits of Amomum kravanh Pierre ex Gagnep. were purchased from Kunming ‘Juhuayuan’ herbal market and were collected in Aug 2016 in Xinshuangbanna, Yunnan province, China. The plant materials were authenticated by Prof. Guo-hua Ye from Shandong College of Traditional 15
Chinese Medicine. A voucher specimen has been deposited at School of Biological Science and Technology, University of Jinan (Accession number: npmc-022). 18. Chlipala GE, Tri PH, Hung NV, Krunic A, Shim SH, Soejarto DD, Orjala J. Nhatrangins A and B, Aplysiatoxin-Related Metabolites from the Marine Cyanobacterium Lyngbya majuscula from Vietnam. J Nat Prod. 2010; 73: 784787. 19. Zhu JY, Cheng B, Zheng YJ, Dong Z, Lin SL, Tang GH, Gu Q, Yin S. Enantiomeric neolignans and sesquineolignans from Jatropha integerrima and their absolute configurations. RSC Adv. 2015; 5: 1220212208. 20. Pescitelli G, Bruhn T. Good Computational Practice in the Assignment of Absolute Configurations by TDDFT Calculations of ECD Spectra. Chirality. 2016; 28: 466474. 21. The initial conformations of 1, 4 and 5 were established via the MM2 force field in the ChemDraw_Pro_14.1 software. Conformational searches using mixed torsional/low-mode sampling method with MMFFs in an energy window of 2.58 kcal/mol were carried out by means of the conformational search module in the Maestro 10.2 software. The re-optimization and the following TD-DFT calculations of the re-optimized conformations were all performed with Gaussian 09 at the B3LYP/6-311G(d,p) level in vacuo. Frequency analysis was performed as well to confirm that the re-optimized conformers were at the energy minima. Finally, the SpecDis 1.64 software was used to obtain the Boltzmann-averaged ECD spectra. 22. Lin YS, Lin JH, Chang CC, Lee SS. Tetrahydropyran- and Tetrahydrofuran-Containing Diarylheptanoids from Hedychium coronarium Rhizomes. J Nat Prod. 2015; 78: 181187. 23. Sudarshan K, Aidhen, IS. Synthesis of (+)-Centrolobine and Its Analogues by Using Acyl Anion Chemistry. Eur J Org Chem. 2013; 12: 2298–2302. 24. Dong SH, Nikoli ć D, Simmler C, Qiu F, Breemen RB, Soejarto DD, Pauli GF, Chen SN. Diarylheptanoids from Dioscorea villosa (Wild Yam). J Nat Prod. 2012; 75: 21682177. 25. The RAW264.7 murine macrophage cell line was purchased from the Cell Bank of Beijing Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Beijing, China), and was cultured in Dulbecco’s modified Eagle medium (DMEM) which was supplemented with 10% FBS. The cells were placed at 37 °C in a humidified incubator containing 5% CO2. 26. The cytotoxicity of the isolated compounds toward RAW264.7 cells was determined by MTT 16
assay. RAW264.7 cells were planted in 96-well plates (5 × 103/well) for 24 h. Then they were treated with tested samples which were dissolved in DMSO and diluted in 100 μL DMEM making the final drug concentration 100 μM and 1% DMSO. 1% DMSO served as solvent control, and wells without cells containing only 100 μL DMEM served as blank control. Twenty L solution of MTT was added to each well after 24 h. After incubation for another 4 h, the medium was removed and 100 μL DMSO was added to each well, and then the absorbance (A) was detected at 490 nm using a microplate reader. The inhibition of cell growth was calculated according to the following formula: % Inhibition = [1 (Asample Ablank) / (Asolvent Ablank)] × 100. 27. Nitric oxide release level was assessed by a colorimetric assay based on a diazotization reaction using the Griess reagent system. RAW264.7 cells were planted in 96-well plates (4 × 104/well) for 24 h and then pre-incubated with different concentrations of compounds for 1 h before stimulation with or without LPS (1 g/mL) for 24 h. The NO concentration in culture medium was determined by Griess reagent kit, then the absorbance (A) was measured at 540 nm using a Tecan microplate reader. The inhibition of NO release was calculated according to the following formula: % Inhibition = [1 (Asample Ablank) / (Amodel Ablank)] × 100. The experiments were performed in triplicates, and dexamethasone was used as a positive control.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
17
R2 R1
R4 O R3
HO O
OH O
NO Inhibition 17.4-26.5 M
Amomum kravanh
18