Antiviral Limonoids Including Khayanolides from ... - ACS Publications

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Antiviral Limonoids Including Khayanolides from the Trang Mangrove Plant Xylocarpus moluccensis Wanshan Li,†,∥ Zhongping Jiang,‡ Li Shen,*,‡ Patchara Pedpradab,§ Torsten Bruhn,⊥ Jun Wu,*,‡ and Gerhard Bringmann*,⊥ †

South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, People’s Republic of China ‡ Marine Drugs Research Center, College of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, People’s Republic of China § Department of Marine Sciences, Faculty of Sciences and Fishery Technology, Rajamangala University of Technology Srivijaya, Trang Campus, Sikao District, Trang Province 92150, Thailand ⊥ Institute of Organic Chemistry, University of Würzburg, Am Hubland, Würzburg 97074, Germany ∥ University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Eight new khayanolides, named thaixylomolins G−N (1−8), two new phragmalins (9 and 10), and two new mexicanolides (11 and 12) were obtained from the seeds of the Trang mangrove plant Xylocarpus moluccensis. The absolute configurations of these limonoids, except for the stereocenter at C-6 of 11 and 12, were assigned by experimental and TDDFT calculated electronic circular dichroism spectra. The khayanolides may be classified into two subclasses, one of which has a C-2 carbonyl and a 3β-acetoxy group, whereas the other possesses a 2β-acetoxy and a C-3 carbonyl function. Khayanolides, rearranged phragmalin-type limonoids, are reported for the first time from plants of the mangrove genus Xylocarpus. The structure of moluccensin J, a known 30ketophragmalin containing a Δ8(14) double bond, was revised to be a khayanolide, named thaixylomolin K. The antiviral activities of the isolates against pandemic influenza A virus (subtype H1N1) were tested by the assay of cytopathic effect inhibition. Three khayanolides, viz., thaixylomolins I, K, and M, exhibited moderate anti-H1N1 activities. The most potent one, thaixylomolin I (IC50 = 77.1 ± 8.7 μM), showed stronger inhibitory activity than that of the positive control, ribavirin (IC50 = 185.9 ± 16.8 μM).

L

of this mangrove genus. Previously, six limonoids, viz., thaixylomolins A−F,16,17 were isolated from a Trang (South Thailand) X. moluccensis. Further investigation of the seeds of the same mangrove yielded 12 new limonoids, including eight khayanolides, two phragmalins, and two mexicanolides. Herein, the isolation, structural identification, and antiviral activities of the new limonoids against influenza A virus (subtype H1N1) are reported.

imonoids are modified tetranortriterpenoids that can be classified based on the cyclization pattern of rings A−D in the triterpenoid backbone. They occur abundantly in the plant families Meliaceae, Rutaceae, and Simaroubaceae. Owing to their broad range of bioactivities and variable frameworks that are created through ring reopening, recyclization, and carbon skeletal rearrangements, limonoids have received renewed interest during the past 10 years. So far, more than 35 types of limonoids with different carbon skeletons have been reported.1,2 The khayanolides are a class of limonoids that are derived from phragmalins via a C-1−C-2 cleavage and subsequent C1−C-30 cyclization and were first characterized from Khaya senegalensis in 1996.3 Thus far, only 18 khayanolides have been reported, all of which were identified from meliaceous plants of the Khaya and Swietenia genera, including K. grandifoliola,4 K. ivorensis,5 K. sengalensis,6−12 and S. mahagoni.13 Plants of the mangrove genus Xylocarpus produce various limonoids, including phragmalins and mexicanolides.14,15 However, khayanolides have not been previously reported from plants © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Thaixylomolin G (1) was obtained as a yellow, amorphous solid. It gave the molecular formula C29H30O11 as established by the (+) HRESIMS ion at m/z 555.1844 ([M + H]+, calcd 555.1861) and 13C NMR data. According to the NMR data of 1 (Tables 1 and 3), nine indices of hydrogen deficiency were due to four carbon−carbon double bonds, two carbonyls, and three ester functionalities, indicating that 1 was hexacyclic. The Received: February 12, 2015

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DOI: 10.1021/acs.jnatprod.5b00151 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

presence of five methyl, three methylene, seven methine, five carbonyl, two oxygenated tertiary, four olefinic quaternary, and three sp3 quaternary carbons in 1 was evidenced by DEPT experiments. The NMR data of 1 were closely related to those of khayseneganin A,12 except for the presence of a 2-O-acetyl, a 30-OH, and a C-11 carbonyl function and the absence of the 6OH group. The HMBC cross-peak from H-2 (δ 5.83 s) to the carbonyl (δC 168.8 qC) carbon of an acetoxy function (Figure 1) suggested the replacement of the 2-OH group in khayseneganin A by an acetoxy function in 1. Similarly, HMBC cross-peaks from the proton of a hydroxy group (δ 5.22 br s) and H-2 (δ 5.83 s) to C-30 (δ 83.3, qC) (Figure 1) placed the hydroxy group at C-30, whereas those from the proton of another hydroxy function (δ 5.32 br s) and H3-19 (δ 1.27 s) to C-1 (δ 85.7, qC) (Figure 1) assigned the hydroxy function at C-1. HMBC cross-peaks from H2-12 [δ 2.04 (d, J = 16.0 Hz), 2.62 (d, J = 16.0 Hz)] to C-11 (δ 193.4) (Figure 1) corroborated the presence of a C-11 carbonyl function, which was conjugated to the Δ8,9 double bond. The presence of an olefinic proton at δ 6.90 (s, H-15) and four conjugated carbons at δ 148.3 (C-8, qC), 146.3 (C-9, qC), 152.4 (C-14, qC), and 120.8 (C-15, CH) revealed the existence of the Δ8,9 and Δ14,15 double bonds, which were confirmed by HMBC cross-peaks between H-15/C-16, H-15/C-8, H3-18/C-14, and H3-19/C-9 (Figure 1). The absence of 6-OH in 1 was supported by the presence of a CH2 group [δH 2.46 (dd, J = 16.0, 10.0 Hz, H-6a), 2.61 (br d, J = 16.0 Hz, H-6b); δC 32.0 (C-6)], of which the protons exhibited 1H−1H COSY correlations to H-5 and HMBC cross-peaks to C-5 and C-7 (Figure 1). Furthermore, the location of a carbonyl function at C-3 and an acetoxy function at C-2 was corroborated by HMBC cross-peaks between H3-28/C-3, H-2/C-8, and H-2/C-30. The relative configuration of 1 was assigned by analysis of the NOE interactions (Figure 2), in which those between H3-18/

H-12α, 1-OH/H-2, 30-OH/H-2, H3-19/1-OH, and H3-19/H318 revealed their cofacial relationship and were arbitrarily assigned as α-oriented. The NOE interaction between H-2/ Hpro‑R-29 established the 2α-H and the corresponding 2βacetoxy function. In turn, NOE interactions between H-17/H12β, H-5/H-12β, and H3-28/H-5 indicated the β-orientation for H-5, H-17, and Me-28. Thus, the relative configuration of thaixylomolin G (1) was assigned as depicted. To establish the absolute configuration of thaixylomolin G (1), quantum-chemical electronic circular dichroism (ECD) calculations were performed. Conformational analysis at the B3LYP-D3/def2-TZVP18−22 level for the arbitrarily chosen 1R,2R,4R,5R,10S,13R,17R,30R-configured enantiomer (i.e., following the preliminary α/β denotation) revealed several relevant conformations mainly differing in the orientations of the substituents at C-2 and C-5. The conformation of the main chromophore, i.e., the “northeastern” moiety of the molecule, consisting of the two six-membered rings and the furanyl group, was in all cases identical, except the fact that the dihedral angle at the furanyl axis can have two different values. However, this difference had no impact on the calculated ECD spectrum. Thus, to distinguish between the two possible enantiomers, (1R,2R,4R,5R,10S,13R,17R,30R)-1 and (1S,2S,4S,5S,10R,13S,17S,30S)-1, a TDDFT calculation was performed only for the main conformer using CAM-B3LYP/def2-TZVP(-f).20−23 The calculated and UV-corrected24,25 (UV shift: 25 nm) ECD spectrum of (1R,2R,4R,5R,10S,13R,17R,30R)-1 matched the experimental curve, whereas the one computed for the enantiomer was mirror-image-like (Figure 3). The calculations corroborated the expectation from the exciton chirality rule,26,27 which clearly confirmed that thaixylomolin G (1) was 1R,2R,4R,5R,10S,13R,17R,30R-configured. Thaixylomolin H (2) provided the molecular formula C29H32O10, as determined from the (+) HRESIMS ion at m/ z 541.2063 ([M + H]+, calcd 541.2068) and 13C NMR data. B



Article

Table 1. 1H NMR (500 MHz for 1 and 4 and 400 MHz for 2 and 3) Data for Compounds 1−4 (δ in ppm, J in Hz) 1b

position 2 5 6a 6b 9 11α 11β 12α 12β 15α

5.83 s 2.27 dd (10.0, 4.5) 2.46 dd (16.0, 10.0) 2.61 br d (16.0)

2.04 d (16.0) 2.62 d (16.0) 6.90 s

2c 5.70 s 1.98 m

3c

2.45 m

5.63 s 1.81 br d (7.6) 2.40d

2.45 m

2.47 m

2.05d 2.21m 1.26d 1.26d 6.30 s

2.40d 1.24d 1.69 m 1.15 m 1.24d 3.79 br s

15β

3.79 br s

17 18 19 21 22 23 28 29pro‑S

5.56 1.09 1.27 7.76 6.63 7.64 1.03 2.15

s s s br s br s br s s d (13.5)

29pro‑R

2.57 d (13.5)

7-OMe 2-OAc-2′ 1-OH 30-OH 30-OEt-1″ 2″

3.65 2.12 5.32 5.22

s s br s br s

5.20 s 0.89 s 0.84 s 7.77 br s 6.58 br s 7.69 br s 0.93 s 1.94 d (12.8) 2.34 d (12.8) 3.61 s 2.05 sd 5.45 br s 5.59 br s

5.39 s 0.87 s 0.95 s 7.74 br s 6.54 br s 7.67 br s 0.91 s 1.96 d (12.8) 2.03 d (12.8) 3.57 s 2.20 s 5.32 s 5.39 s

function. Similarly, the NOEs between H3-18/H-12α, H3-19/ H-9, 1-OH/H3 -19, and 30-OH/H-2 revealed their αorientations. In turn, those between H-5/H-12β, H-5/H-11β, H-17/15β, and H-17/H-12β assigned the β-orientation for H-5 and H-17. The relative configuration of 3, named thaixylomolin I, was hence established as depicted. The ECD curve of 3 was not comparable to the spectra of 1 and 2, and thus quantumchemical calculations were performed to unambiguously elucidate the absolute configuration. The computed ECD spectrum of the main conformation of the 1R,2R,4R,5R,9R,10R,13R,17R,30R-configured 3 matched the experimental curve (see the SI, Figure S2). Thus, in addition to the R-configured C-9 stereocenter, 3 had the same absolute configuration as those of compounds 1 and 2. Note the change of the C-10 descriptor in 3 due to the change in the CIP priorities compared to 1 and 2. Thaixylomolin J (4) had the molecular formula C31H38O10 as established by the (+) HRESIMS ion at m/z 593.2351 ([M + Na]+, calcd 593.2357) and 13C NMR data. The NMR data of 4 (Tables 1 and 3) resembled those of 3, but showed that the C30 hydroxy group was replaced by an ethoxy group [δH 3.56 (q, 2H, J = 7.0 Hz, H2-1″), 1.27 (t, 3H, J = 7.0 Hz, H3-2″); δC 61.2 (C-1″), 15.5 (C-2″)]. This was corroborated by HMBC crosspeaks between H2-1″/C-30 and 1H−1H COSY correlations between H2-1″/H3-2″. The relative configuration of 4 was deduced to be identical to that of 3 by analysis of its NOESY spectrum, whereas the absolute configuration of 4 was established to be the same as that of 3 by the similarity of their ECD spectra (see the SI, Figure S2). Thus, the structure of thaixylomolin J (1R,2R,4R,5R,9R,10R,13R,17R,30R-4) was assigned as depicted. Thaixylomolin K (5) gave the molecular formula C29H34O9 as obtained from the (+) HRESIMS ion at m/z 527.2251 ([M + H]+, calcd 527.2276) and 13C NMR data. The NMR data of 5 (Tables 2 and 3) resembled those of 3 (Tables 1 and 3), the difference being the lack of a 30-OH group and the interchange of the C-2 acetoxy and the C-3 carbonyl function in 3. The absence of a 30-OH function was corroborated by the shielded C-30 signal (δ 64.1 in acetone-d6, CH) in 5 and HMBC crosspeaks from H-30 to C-8, C-2, and C-1 (Figure 4). The location of a carbonyl function at C-2 (δ 205.0 in acetone-d6) and an acetoxy function at C-3 (δ 83.2 in acetone-d6) was further corroborated by significant HMBC cross-peaks between H-5/ C-3, H3-28/C-3, H2-29/C-3, H-30/C-9, and H-30/C-14 (Figure 4). The relative configuration of 5 was assigned by NOE interactions. Those between H-5/H-11β and H-17/H12β (Figure 5) demonstrated 5β-H and 17β-H orientations. In turn, those between Hpro‑R-29/H-3, H-15α/H-30, and H-11α/ H-9 established 3α-H, 9α-H, and 30α-H orientations and the corresponding 3β-OAc function (Figure 5). Thus, the relative configuration of 5 was elucidated as depicted. The absolute configuration of 5 was determined by comparing the experimental ECD spectrum of 5 with those of 3 and 4 (see the SI, Figure S3). Owing to the similarities of these spectra, 5 had the same absolute configuration as those of thaixylomolins I and J at the C-13 and C-17 stereocenters, and thus thaixylomolin K (5) was assigned a 1S,3S,4R,5S,9R,10R,13R,17R,30R absolute configuration. Surprisingly, the NMR data of 5 acquired in CDCl3 (Tables 2 and 3) were the same as those of moluccensin J, which was erroneously reported as a phragmalin.28 The proton of a methine group (δH 3.48 s, δC 64.1 in acetone-d6; δH 3.45 s, δC 63.1 in CDCl3), showing crucial HMBC cross-peaks to C-8, C-

4a 6.07 s 2.06 dd (8.0, 4.0) 2.33 dd (16.5, 4.0) 2.44 dd (16.5, 10.0) 2.40 m 1.75 m 1.41 m 1.41d 1.41d 3.66 br d (19.0) 3.86 dd (19.0, 4.0) 5.21 s 1.01 s 1.03 s 7.47 br s 6.41 br s 7.42 br s 1.05 s 2.11 br s 2.11 br s 3.69 s 2.27s 2.17 s 3.56 q (7.0) 1.27 t (7.0)

a

Measured in CDCl3. bMeasured in acetone-d6. cMeasured in DMSOd6. dThe overlapped signals were assigned with accurate axis calibration of 1H−1H COSY and HSQC spectra.

The similarities between the NMR data of 2 (Tables 1 and 3) and 1 revealed their close structural resemblance. However, the absence of the C-11 carbonyl function in 2 was evidenced by 1 H−1H COSY correlations between H2-11/H2-12 [δ 2.05 (overlapped), 2.21 m, H2-11; δ 1.26 (2H, overlapped, H2-12)], HMBC cross-peaks from H2-11 to C-9, and the shielded C-11 signal (δ 19.2) in 2. The relative configuration of 2 was identical with that of 1 by analysis of its NOESY spectrum. Application of the exciton chirality method26,27 and comparison of the ECD spectrum of 2 (see the SI, Figure S1) with those of 1 and of khayaseneganin A27 showed that thaixylomolin H (2) had the same 1R,2R,4R,5R,10S,13R,17R,30R-absolute configuration as that of thaixylomolin G. The molecular formula of thaixylomolin I (3) was assigned as C29H34O10 from the (+) HRESIMS ion at m/z 543.2220 ([M + H]+, calcd 543.2225) and 13C NMR data, two mass units more than that of 2. The NMR data of 3 (Tables 1 and 3) resembled those of 2, but in 3, the conjugated Δ8,9 and Δ14,15 double bonds of 2 were replaced by a Δ8,14 double bond, which was confirmed by 1H−1H COSY homoallylic couplings between H2-15/H-9 and HMBC cross-peaks between H2-15/C-8, H215/C-14, H3-18/C-14, H-17/C-14, H-2/C-8, and 30-OH/C-8. The NOE association between H-2 and Hpro‑R-29 established the α-orientation for H-2 and the corresponding 2β-acetoxy C



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Table 2. 1H NMR (400 MHz) Data for Compounds 5−8 (δ in ppm, J in Hz) position

5a

3 5 6a 6b 9 11α 11β 12α 12β 15α 15β 17 18 19 21 22 23 28 29pro‑S 29pro‑R 30 7-OMe 3-OAc-2′ 1-OH 30-tigloyl-3″ 4″ 5″ 30-OEt-1″ 2″

4.98 s 2.18 m 2.26 br d (15.6) 2.38 dd (15.6, 11.2) 2.45 m 1.76 m 1.42m 1.38 m 1.54 m 3.44 d (19.6) 3.94 d (19.6) 5.19 s 0.99 s 0.99 s 7.45 br s 6.41 br s 7.40 br s 0.97 s 2.16 d (12.8) 1.98 d (12.8) 3.45 s 3.66 s 2.13 s

5b 4.95 2.25 2.33 2.45 2.48 1.80 1.46 1.34 1.53 3.32 3.85 5.28 0.98 1.01 7.64 6.52 7.56 0.94 2.19 2.01 3.48 3.62 2.05 4.42

6a

s dd (11.2, 2.4) dd (15.6, 2.4) dd (15.6, 11.2) m m m m m br d (19.2) br d (19.2) s s s br s br s br s s d (12.4) d (12.4) s s s s

4.93 s 2.33 br s 2.30 m 2.43 dd (14.8, 10.4) 2.46 m 1.73 m 1.41 m 1.41c 1.53 m 3.79 dd (20.0, 3.2) 3.94 dd (20.0, 3.6) 5.17 s 1.03 s 1.05 s 7.46 br s 6.42 br s 7.41 br s 0.98 s 2.24 d (13.2) 1.78 d (13.2) 3.67 s 2.17 s

7a

8a

5.12 s 2.48 br s 2.44c 2.38c

5.56 s 2.57 t (7.6) 2.41c 2.41c

2.15 m 2.38c 1.52c 1.52c 6.08 s

2.22 dd (12.0, 5.6) 2.46c 1.59 dd (13.0, 4.4) 1.48 m 6.50 s

5.17 1.00 0.99 7.46 6.43 7.42 1.02 2.12 2.07 3.55 3.69 2.15

5.13 1.05 1.03 7.47 6.44 7.42 1.03 2.00 2.69

s s s br s br s br s s d (13.2) d (13.2) s s s

s s s br s br s br s s d (12.8) d (12.8)

3.70 s 2.15 s 6.98 qd (7.2, 1.6) 1.83 d (7.2) 1.84 s

3.51 m, 3.66 m 1.27 t (7.2)

a

Measured in CDCl3. bMeasured in acetone-d6. cThe overlapped signals were assigned with accurate axis calibration of 1H−1H COSY and HSQC spectra.

absence of a Δ8,14 double bond and the existence of the Δ8,9 and Δ14,15 conjugated double bonds. This deduction was verified by HMBC cross-peaks from H-15 to C-8, C-13, C-14, and C-16 and those between H3-18/C-14, H3-19/C-9, and H30/C-9. A comparison of the ECD spectra of compounds 1−6 showed that these curves are clearly dominated by the chromophoric groups in the “northeastern” part of the molecule (the six-membered rings and the furanyl substituent) and thus by the stereocenters at C-13 and C-17. The ECD curve of 7 displayed high similarities to that of 2 (see the SI, Figure S1), proving that both have the same configuration at these stereocenters, and as a consequence, 7 is 1S,3S,4R,5S,10S,13R,17R,30R-configured. The structure of thaixylomolin M (7) was hence elucidated as depicted. Thaixylomolin N (8) provided the molecular formula C34H38O11 as determined from the (+) HRESIMS ion at m/z 645.2310 ([M + Na] +, calcd 645.2306) and 13C NMR data. The NMR data of 8 (Tables 2 and 3) resembled those of 7, the difference being the existence of a tigloyloxy group [δH 6.98 (qd, J = 7.2, 1.6 Hz), 1.83 (d, J = 7.2 Hz), 1.84 s; δC 166.6 qC, 126.9 qC, 141.6 CH, 12.1 CH3, 14.9 CH3], which was confirmed by 1H−1H COSY correlations between H-3″/H3-4″ and significant HMBC cross-peaks between H-3″/C-1″, H3-5″/ C-1″, H3-4″/C-2″, H3-4″/C-3″, and H3-4″/C-1″. Its location at C-30 was supported by the deshielded C-30 signal (δ 92.4 in 8, as compared to δ 64.6 in 7). The relative configuration of 8 was determined to be identical to that of 7 by analysis of its NOESY spectrum, while their identical absolute configurations could be

9, C-14, and C-1 (Figure 4), was equivocally assigned as H-30. Then, HMBC cross-peaks from H-30 and H-3 to the carbonyl function (δC 205.0 in acetone-d6; δC 204.2 in CDCl3) (Figure 4) placed it at C-2. Thus, the above results corroborated the key C-1−C-30 linkage in 5, but not the C-1−C-2 linkage. Consequently, moluccensin J28 is a khayanolide, but not a phragmalin. Thaixylomolin L (6) afforded the molecular formula C31H38O10 as established by the (+) HRESIMS ion at m/z 593.2347 ([M + Na]+, calcd 593.2357) and 13C NMR data. The NMR data of 6 (Tables 2 and 3) resembled those of 5, the difference being the presence of an ethoxy group [δH 3.51 m, 3.66 m, 1.27 (t, J = 7.2 Hz); δC 63.3 CH2, 15.7 CH3], its location at C-30 being supported by the significant HMBC cross-peak between H2-1″ and C-30. The relative configuration of 6 was determined to be identical to that of 5 by analysis of its NOESY spectrum. The absolute configuration of 6 was elucidated by the similar ECD spectra of 5 and 6 and by comparison of experimental and calculated ECD spectra (see the SI, Figures S3 and S4). Hence, the structure of thaixylomolin L (6) was assigned the 1R,3S,4R,5S,9R,10S,13R,17R,30S absolute configuration. The change in the C-10 descriptor is due to different CIP priorities as compared to those in 5. Thaixylomolin M (7) gave the molecular formula C29H32O9, as deduced from the (+) HRESIMS ion at m/z 525.2122 ([M + H]+, calcd 525.2119) and 13C NMR data. The NMR data of 7 (Tables 2 and 3) resembled those of 5, the difference being the D



Article

Table 3. 13C NMR (100 MHz for 1−3 and 5−8 and 125 MHz for 4) Data for Compounds 1−8

a

position

1b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 28 29 30 7-OMe 2-OAc-1′ 2′ 3-OAc-1′ 2′ 30-tigloyl-1″ 2″ 3″ 4″ 5″ 30-OEt-1″ 2″

85.7 qC 82.7CH 207.1 qC 52.3 qC 48.5 CH 32.0 CH2 172.4 qC 148.3 qC 146.3 qC 56.2 qC 193.4 qC 46.5 CH2 39.4 qC 152.4 qC 120.8 CH 163.7 qC 78.9 CH 18.4 CH3 16.0 CH3 120.1 qC 141.8 CH 109.9 CH 143.4 CH 15.9 CH3 36.7 CH2 83.3 qC 51.0 CH3 168.8 qC 19.9 CH3

2c 85.6 82.9 208.9 50.9 47.3 31.6 173.3 129.9 157.4 57.9 19.2 29.6 37.1 155.7 111.9 165.2 79.3 15.9 15.0 120.5 141.7 110.2 143.2 17.1 37.2 82.0 51.5 169.2 20.6

qC CH qC qC CH CH2 qC qC qC qC CH2 CH2 qC qC CH qC CH CH3 CH3 qC CH CH CH CH3 CH2 qC CH3 qC CH3

3c 85.8 81.6 203.9 48.6 41.3 33.1 172.5 136.9 44.7 53.1 18.1 30.7 40.6 135.6 34.5 170.7 79.0 14.6 16.6 120.7 141.6 110.4 143.2 14.6 38.3 79.4 51.4 169.6 20.9

qC CH qC qC CH CH2 qC qC CH qC CH2 CH2 qC qC CH2 qC CH CH3 CH3 qC CH CH CH CH3 CH2 qC CH3 qC CH3

4a 86.7 77.3 204.1 48.8 42.1 33.8 172.5 134.4 46.4 53.5 18.9 31.3 41.3 137.9 35.4 170.6 80.6 15.5 17.0 120.3 141.3 110.1 143.0 15.3 40.0 84.8 52.0 169.8 21.2

5a

qC CH qC qC CH CH2 qC qC CH qC CH2 CH2 qC qC CH2 qC CH CH3 CH3 qC CH CH CH CH3 CH2 qC CH3 qC CH3

84.1 204.2 83.1 40.1 40.1 33.7 172.3 134.4 47.5 55.9 19.0 31.4 40.0 132.1 34.9 170.2 80.3 16.4 14.2 120.0 140.7 109.6 142.4 18.7 43.8 63.1 51.2

qC qC CH qC CH CH2 qC qC CH qC CH2 CH2 qC qC CH2 qC CH CH3 CH3 qC CH CH CH CH3 CH2 CH CH3

169.9 qC 20.1 CH3

61.2 CH2 15.5 CH3

5b

6a

7a

8a

qC qC CH qC CH CH2 qC qC CH qC CH2 CH2 qC qC CH2 qC CH CH3 CH3 qC CH CH CH CH3 CH2 CH CH3

84.4qC 204.0qC 83.3CH 40.1qC 39.9CH 34.2CH2 172.9 qC 134.4qC 46.9CH 55.3qC 18.9CH2 31.6CH2 40.8qC 139.0qC 33.1CH2 170.0qC 80.2CH 17.1CH3 15.2CH3 120.5 qC 141.3 CH 110.1 CH 143.0 CH 19.6 CH3 43.7 CH2 92.2 qC 51.8CH3

87.5 qC 203.4 qC 82.6CH 42.0 qC 47.2CH 34.1 CH2 173.2 qC 128.3 qC 159.8 qC 61.4 qC 20.6 CH2 30.4 CH2 37.8 qC 156.1 qC 111.9 CH 166.2 qC 80.4 CH 16.3 CH3 12.4 CH3 120.2 qC 141.1 CH 110.0 CH 142.9 CH 20.3 CH3 42.6 CH2 64.6 CH 51.9 CH3

88.8 qC 198.6 qC 80.0 CH 41.9 qC 46.5CH 34.1 CH2 173.3 qC 125.2 qC 161.8 qC 60.5 qC 20.7 CH2 30.4 CH2 37.8 qC 152.0 qC 113.7 CH 165.6 qC 80.1 CH 16.3 CH3 13.5 CH3 120.4 qC 141.2 CH 110.1 CH 143.0 CH 20.9 CH3 41.4 CH2 92.4 qC 52.0 CH3

169.5 qC 19.7 CH3

170.2 qC 20.6 CH3

170.2 qC 20.5 CH3

169.9 20.6 166.6 126.9 141.6 12.1 14.9

84.3 205.0 83.2 40.6 40.3 33.8 172.7 135.5 48.3 56.5 19.3 31.9 40.2 132.0 34.8 169.1 80.1 16.6 14.1 121.2 141.5 110.2 143.0 18.8 43.6 64.1 50.9

qC CH3 qC qC CH CH3 CH3

63.3CH2 15.7CH3

Measured in CDCl3. bMeasured in acetone-d6. cMeasured in DMSO-d6.

Figure 1. Selected 1H−1H COSY and HMBC cross-peaks for 1.

deduced from the similar ECD spectra of these molecules (see the SI, Figure S1). Thus, thaixylomolin N (8) is 1R,3S,4R,5S,10S,13R,17R,30S-configured; again different CIP priorities change the descriptors of some of the stereocenters. Compound 9 afforded the molecular formula C31H36O13 as determined by the (+) HRESIMS ion at m/z 639.2051 ([M + Na]+, calcd 639.2048) and 13C NMR data. Its NMR data (Tables 4 and 5) showed high similarity to those of xyloccensin

Figure 2. Selected NOE interactions for 1.

E



Article

Table 4. 1H NMR (400 MHz for 9−11 and 500 MHz for 12) Data for Compounds 9−12 in CDCl3 (δ in ppm, J in Hz) position 3 5 6a 6b 9

Figure 3. Comparison of the experimental ECD spectrum of 1 with TDCAM-B3LYP-calculated spectra of 1 and its enantiomer.

9 5.17 s 2.15 dd (10.0, 2.0) 2.38 d (12.8) 2.41 d (12.8)

10 5.24 s 2.12 m 2.33 d (12.8) 2.36 d (12.8)

2.95 br s 5.56 br s

5.42 br s

2.14 m

2.29 br d (13.5) 1.91 br d (10.0) 1.54 m

1.99 t (14.4)

1.97a

1.89a

11β

2.21 dd (14.4, 4.0)

2.23 dd (14.4, 4.0)

1.89a

3.83 dd (13.2, 4.0) 6.45 s

3.87 dd (13.2, 3.2) 6.60 s

15α 15β 17 18 19 21 22 23 28 29pro‑R 29pro‑S 30 32 7-OMe 1-OH 3-Acyl-2′ 3′ 4′ 6-OAc-2″ 2-OAc-2″

5.93 s 1.48 s 1.31 s 7.62 br s 6.60 br s 7.53 br s 0.75 s 1.80 br d (11.2) 1.85 d (11.2) 4.55 s

5.89 1.47 1.31 7.63 6.60 7.52 0.73 1.74

1.70 s 3.72 s

1.70 s 3.71 s 3.41 2.07 s

2.05 s

s s s br s br s br s s d (11.6)

1.97 d (11.6) 5.34 s

12 4.86 s 3.37 br s

11α

12α 12β

Figure 4. Selected 1H−1H COSY and HMBC cross-peaks for 5.

11

1.15 m 1.89a 3.47 dt 2.4) 3.55 dt 2.4) 5.31 s 1.02 s 1.38 s 7.55 br 6.47 br 7.41 br 1.10 s 1.09 s

(21.6,

1.22 m 2.03 br d (12.0) 6.29 s

(21.6,

s s s

5.14 1.05 1.29 7.52 6.48 7.44 0.87 1.07

s s s br s br s br s s s

2.08 d (13.6) 3.36 d (13.6)

6.28 br d (2.5)

3.78 s

3.73 s

2.20 s

2.73 1.28 1.26 2.18

m d (7.0) d (7.0) s

2.16 s

a

1

The overlapped signals were assigned with accurate axis calibration of H−1H COSY and HSQC spectra.

Compound 10 gave the molecular formula C33H38O14, as deduced from the (+) HRESIMS ion at m/z 681.2151 ([M + Na]+, calcd 681.2154) and 13C NMR data. The NMR data of 10 (Tables 4 and 5) resembled those of 9, the difference being the replacement of the 2-OH function in 9 by an acetoxy group (δH 2.16 s; δC 170.6 qC, 21.9 CH3), the presence of which was corroborated by the deshielded C-2 signal (δ 83.8 in 10, as compared to δ 75.3 in 9). The structure of 10 was thus elucidated as 2-O-acetyl-2-dehydroxy-12-deacetylxyloccensin U with 1R,2S,3S,4R,5S,8R,9S,10R,12S,13S,17S-configuration, as deduced from the similarity of the ECD spectra of 9 and 10 (see the SI, Figure S5). Compound 11 had the molecular formula C29H34O10 on the basis of the (+) HRESIMS ion at m/z 565.2040 ([M + Na]+, calcd 565.2044) and 13C NMR data. The NMR data of 11 (Tables 4 and 5) showed high similarity to those of 2αhydroxymexicanolide,30 the difference being the presence of a 6-acetoxy function (δH 2.20 s, δC 169.6 qC, 20.9 CH3). The HMBC cross-peak between H-6 and the carbonyl carbon of this acetoxy function placed it at C-6. The relative configuration

Figure 5. Selected NOE interactions for 5.

U,29 the difference being the replacement of the 12-acetoxy group in xyloccensin U by a hydroxy function. The shielded C12 signal (δ 66.5 in 9, as compared to δ 69.3 in xyloccensin U) and the absence of 1H and 13C NMR resonances for the 12acetoxy group (δH 1.52 s; δC 20.0 CH3, 170.1 qC in xyloccensin U) in 9 confirmed the above deduction. The relative configuration of 9 was determined to be identical to that of xyloccensin U based on NOE interactions between H-12/H-17, H3-28/H-5, H-17/H-5, H-12/H-5, Hpro‑R-29/H-3, H3-19/H318, and H-11α/H3-18. The structure of 9 was hence identified as 12-deacetylxyloccensin U and is thus closely related to that of andirolide G,27 in which only the C-2 side chain differs. Owing to the nearly identical ECD spectra of andirolide G and 9 (see the SI, Figure S5), the absolute configuration of 9 could be defined as 1R,2S,3S,4R,5S,8R,9S,10R,12S,13S,17S. F



Article

Table 5. 13C NMR (100 MHz for 9 and 10 and 125 MHz for 12) Data for Compounds 9−12 in CDCl3 (δ in ppm) position

9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 28 29 30 31 32 7-OMe 3-Acyl-1′ 2′ 3′ 4′ 6-OAc-1″ 2″ 2-OAc-1″ 2″

84.1 75.3 85.3 44.4 40.5 33.7 173.9 83.7 87.2 47.3 34.3 66.5 44.8 154.3 123.3 163.5 78.8 13.0 15.6 121.3 142.4 109.6 144.9 14.6 39.2 78.1 119.5 16.5 52.4 169.8 21.7

10 qC qC CH qC CH CH2 qC qC qC qC CH2 CH qC qC CH qC CH CH3 CH3 qC CH CH CH CH3 CH2 CH qC CH3 CH3 qC CH3

84.1 83.8 84.9 44.6 39.9 33.7 173.8 83.7 86.2 48.1 34.6 66.6 44.8 153.9 123.7 163.6 78.8 13.0 15.5 121.4 142.4 109.6 144.8 14.4 39.8 74.2 119.7 16.5 52.3 169.2 21.7

qC qC CH qC CH CH2 qC qC qC qC CH2 CH qC qC CH qC CH CH3 CH3 qC CH CH CH CH3 CH2 CH qC CH3 CH3 qC CH3

11 212.5 80.1 207.6 49.2 43.4 72.5 170.6 123.4 51.5 53.2 18.4 28.2 38.3 136.0 32.8 169.3 80.3 17.5 17.0 120.2 141.6 109.9 143.0 20.9 20.2 44.5

qC qC qC qC CH CH qC qC CH qC CH2 CH2 qC qC CH2 qC CH CH3 CH3 qC CH CH CH CH3 CH3 CH2

53.5CH3

169.6 qC 20.9 CH3

configuration of 12 was determined by ECD calculations, and only the simulated curve of 2R,3S,5S,9S,10R,13R,17R-12 showed an acceptable match with the experimental spectrum (see SI, Figure S7). The structure of 12 was thus defined as 6O-acetyl-6-dehydroxymoluccensin T. Among the eight khayanolides, compounds 4 and 6 contain an allylic C-30 ethoxy group, which might be derived from EtOH that was used for the extraction of plant material. Thus, 4 could have been derived from alcohol. However, a similar precursor of 6 has not been obtained. In order to clarify the origin of the C-30 ethoxy group, compound 3 was kept in EtOH at room temperature for a week. However, UPLC-MS analysis confirmed that the C-30 ethoxy group in 4 was not derived from EtOH (see the SI, Figures S8−10). Since plant anaerobic respiration may produce ethanol, the ethoxy groups in 4 and 6 might be derived via this source.32,33 Antiviral activities against the pandemic influenza A virus (subtype H1N1) were tested by the assay of cytopathic effect (CPE) inhibition.34,35 Three khayanolides, viz., thaixylomolins I (3), K (5), and M (7), exhibited moderate anti-H1N1 activities, with IC50 values of 77.1 ± 8.7, 113.5 ± 8.6, and 121.5 ± 1.5 μM, respectively, and all three compounds showed stronger inhibitory activity than that of the positive control, ribavirin (IC50 = 185.9 ± 16.8 μM). Thaixylomolins G−N are eight new khayanolides that can be classified into two subclasses, of which one has a C-2 carbonyl and a 3β-acetoxy group, whereas the second group possesses a 2β-acetoxy and a C-3 carbonyl function. To date, only 18 khayanolides have been reported.4−13 Thaixylomolin G is the first khayanolide that contains a C-11 carbonyl group in conjugation with two double bonds, viz., Δ8,9 and Δ14,15. Thaixylomolins I−L are the first khayanolides containing a Δ8,14 double bond, whereas thaixylomolins H, M, and N are rare khayanolides possessing two conjugated double bonds, viz., Δ8,9 and Δ14,15. In conclusion, khayanolides have been identified for the first time from plants of the mangrove genus Xylocarpus. Thaixylomolins I, K, and M showed stronger anti-H1N1 activities than that of ribavirin.

12 211.4 77.6 86.3 40.2 43.7 71.9 170.4 134.4 55.0 52.9 22.3 33.1 37.6 160.1 113.6 164.6 79.7 22.5 15.2 120.0 141.5 110.2 143.3 21.4 23.0 133.2

qC qC CH qC CH CH qC qC CH qC CH2 CH2 qC qC CH qC CH CH3 CH3 qC CH CH CH CH3 CH3 CH

53.1 175.8 34.4 18.9 19.3 169.6 20.9

CH3 qC CH CH3 CH3 qC CH3

■

170.6 qC 21.9 CH3

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined on an MCP500 modular circular polarimeter (Anton Paar GmbH). UV spectra were recorded on a GENESYS 10S UV−vis spectrophotometer (Thermo Scientific), and HRESIMS were obtained on an LC-DAD-ESIMS system in the positive-ion mode. NMR spectra were measured on a Bruker spectrometer (AV-400 or AV-500). Preparative HPLC was performed on a Waters 2535 pump equipped with a 2998 photodiode array detector and YMC C18 reversed-phased columns (250 × 10 mm i.d., 5 μm). Silica gel (100−200 mesh) (Qingdao Mar. Chem. Ind. Co. Ltd.) and C18 reversed-phased silica gel (50 μm, YMC) were employed for column chromatography. Plant Material. From the Thai mangrove swamps of Trang Province, seeds of Xylocarpus moluccensis were collected in December 2012 and June 2013. Identification of the mangrove was done by one of the authors (J.W.). Voucher specimens (ThaiXM-02 and ThaiXM03) were deposited in the Marine Drugs Research Center, College of Pharmacy, Jinan University. Extraction and Isolation. The air-dried seeds (1.0 kg, ThaiXM02) were powdered and extracted at normal atmospheric temperature with 95% (v/v) EtOH (5 × 5 L) to afford an extract (310.0 g), which was partitioned with EtOAc and H2O to afford the EtOAc portion (66.0 g). The EtOAc fraction was further subjected to a silica gel column (120 × 10 cm i.d.), eluted with a gradient mixture of CHCl3/ MeOH (100:0 to 5:1), to afford 156 fractions. Fractions 51−75 (3.9 g)

was elucidated by NOE interactions except for the C-6 stereocenter. In addition, this center should have no effect on the ECD spectrum, so that only the absolute configurations of the other centers were defined using quantum-chemical ECD calculations. The ECD spectrum computed for the main conformer of 2R,5S,9S,10R,13R,17R-11 showed a very good match with the experimental ECD curve (see the SI, Figure S6); thus, the structure of 11 was assigned as 6-O-acetyl-2αhydroxymexicanolide. Compound 12 gave the molecular formula C33H40O11 according to the (+) HRESIMS ion at m/z 635.2463 ([M + Na]+, calcd 635.2463) and 13C NMR data. The NMR data of 12 (Tables 4 and 5) resembled those of moluccensin T,31 the difference being the replacement of the 6-OH function in moluccensin T by an acetoxy group (δH 2.18 s; δC 169.6 qC, 20.9 CH). The HMBC cross-peak between H-6 and the carbonyl (δC 169.6 qC) carbon of the acetoxy group verified the above deduction. The relative configuration of 12 was determined to be identical to that of moluccensin T by analysis of its NOESY spectrum; however, the configuration at C-6 was again not assignable. In analogy to 11, the absolute G



Article

were combined and further separated on an RP-18 column (60 × 4 cm i.d.), eluted with a gradient mixture of acetone/H2O (50:50 to 100:0), to afford 77 subfractions, among which the combination of subfractions 8−13 in acetone afforded mixed crystals. Further purification of the resulting crystals by preparative HPLC (MeCN/ H2O, 35:65) gave compounds 1 (5.2 mg), 2 (6.3 mg), and 3 (6.1 mg), whereas HPLC (MeCN/H2O, 35:65 or 40:60) purification of the remaining mother liquor yielded compounds 5 (47.5 mg) and 7 (18.2 mg). The combination of fractions 27−36 (21.0 g) and further separation on an RP-18 column (100 × 5 cm i.d.), eluted with a gradient mixture of acetone/H2O (50:50 to 100:0), afforded 63 subfractions. The fifth subfraction was purified by preparative HPLC (MeCN/H2O, 35:65) to give compounds 9 (7.3 mg) and 10 (20.0 mg), whereas HPLC (MeOH/H2O, 48:52) purification of subfraction 15 yielded compounds 4 (7.9 mg), 6 (1.8 mg), and 8 (8.5 mg). The air-dried seeds (10.0 kg, ThaiXM-03) were powdered and extracted at room temperature with 95% (v/v) EtOH (5 × 20 L) to afford the resulting extract (680.0 g), which was partitioned with EtOAc and H2O to afford an EtOAc portion (296.0 g). The EtOAc portion was subjected to a silica gel column (120 × 10 cm i.d.), eluted with a gradient mixture of CHCl3/MeOH (100:0 to 5:1), to afford 160 fractions. The combination of fractions 26−28 (31.6 g) and further separation on an RP-18 column (100 × 5 cm i.d.), eluted with a gradient mixture of acetone/H2O (50:50 to 100:0), yielded 57 subfractions, among which the combination of subfractions 6−16 and further HPLC (MeCN/MeOH/H2O, 27:20:53) purification afforded compound 11 (2.8 mg), whereas HPLC (MeCN/MeOH/H2O, 28:20:52) purification of the 23rd subfraction gave compound 12 (32.4 mg). Thaixylomolin G (1): yellow, amorphous solid; [α]25 D = +163 (c 0.1, acetone); UV (MeCN) λmax (log ε) 199 (3.59), 297 (2.90) nm; ECD (c 0.18 mM, MeCN) λmax (Δε) 218 (+2.6), 246 (−3.8), 302 (+14.3) nm; 1H and 13C NMR data (Tables 1 and 3); HRESIMS [M + H]+ m/ z 555.1844 (calcd for C29H31O11, 555.1861). Thaixylomolin H (2): yellow, amorphous solid; [α]25 D = +106 (c 0.1, acetone); UV (MeCN) λmax (log ε) 198 (2.89), 282 (2.60) nm; ECD (c 0.19 mM, MeCN) λmax (Δε) 195 (+4.0), 226 (+2.5), 256 (−1.8), 289 (+5.1) nm; 1H and 13C NMR data (Tables 1 and 3); HRESIMS [M + H]+ m/z 541.2063 (calcd for C29H33O10, 541.2068). Thaixylomolin I (3): white, amorphous solid; [α]25 D = −8 (c 0.03, acetone); UV (MeCN) λmax (log ε) 201 (1.52) nm; ECD (c 0.46 mM, MeCN) λmax (Δε) 217 (−5.5), 277 (+0.7) nm; 1H and 13C NMR data (Tables 1 and 3); HRESIMS [M + H]+ m/z 543.2220 (calcd for C29H35O10, 543.2225). Thaixylomolin J (4): white, amorphous solid; [α]25 D = +42 (c 0.2, acetone); UV (MeCN) λmax (log ε) 208 (1.24) nm; ECD (c 0.44 mM, MeCN) λmax (Δε) 222 (−3.8), 281 (+0.4) nm; 1H and 13C NMR data (Tables 1 and 3); HRESIMS [M + Na]+ m/z 593.2351 (calcd for C31H38NaO10, 593.2357). Thaixylomolin K (5): white, amorphous solid; [α]25 D = +15 (c 0.1, acetone); UV (MeCN) λmax (log ε) 197 (2.56) nm; ECD (c 0.48 mM, MeCN) λmax (Δε) 205 (+3.8), 232 (−8.6), 337 (+2.7) nm; 1H and 13 C NMR data (Tables 2 and 3); HRESIMS [M + H]+ m/z 527.2251 (calcd for C29H35O9, 527.2276). Thaixylomolin L (6): white, amorphous solid; [α]25 D = −9 (c 0.2, acetone); UV (MeCN) λmax (log ε) 204 (2.16) nm; ECD (c 0.44 mM, MeCN) λmax (Δε) 206 (+1.6), 232 (−5.2), 336 (+1.7) nm; 1H and 13 C NMR data (Tables 2 and 3); HRESIMS [M + Na]+ m/z 593.2347 (calcd for C31H38NaO10, 593.2357). Thaixylomolin M (7): yellow, amorphous solid; [α]25 D = +161 (c 0.2, acetone); UV (MeCN) λmax (log ε) 197 (2.37), 290 (2.46) nm; ECD (c 0.19 mM, MeCN) λmax (Δε) 208 (+5.9), 258 (−0.6), 292 (+6.4) nm; 1H and 13C NMR data (Tables 2 and 3); HRESIMS [M + H]+ m/ z 525.2122 (calcd for C29H33O9, 525.2119). Thaixylomolin N (8): yellow, amorphous solid; [α]25 D = −16 (c 0.03, acetone); UV (MeCN) λmax (log ε) 216 (3.12), 282 (2.43) nm; ECD (c 0.16 mM, MeCN) λmax (Δε) 207 (+4.7), 255 (−0.3), 286 (+4.4), 319 (−3.3) nm; 1H and 13C NMR data (Tables 2 and 3); HRESIMS [M + Na]+ m/z 645.2310 (calcd for C34H38NaO11, 645.2306).

12-Deacetylxyloccensin U (9): white, amorphous solid; [α]25 D = +23 (c 0.1, acetone); UV (MeCN) λmax (log ε) 201 (2.23) nm; ECD (c 0.16 mM, MeCN) λmax (Δε) 207 (+6.5), 271 (+1.2) nm; 1H and 13C NMR data (Tables 4 and 5); HRESIMS [M + Na]+ m/z 639.2051 (calcd for C31H36NaO13, 639.2048). 2-O-Acetyl-2-dehydroxy-12-deacetylxyloccensin U (10): white, amorphous solid; [α]25 D = +37 (c 0.1, acetone); UV (MeCN) λmax (log ε) 214 (1.87) nm; ECD (c 0.15 mM, MeCN) λmax (Δε) 202 (+ 5.2), 273 (+ 0.8) nm; 1H and 13C NMR data (Tables 4 and 5); HRESIMS [M + Na]+ m/z 681.2151 (calcd for C33H38NaO14, 681.2154). 6-O-Acetyl-2α-hydroxymexicanolide (11): white, amorphous solid; [α]25 D = −44 (c 0.1, acetone); UV (MeCN) λmax (log ε) 198 (2.50) nm; ECD (c 0.18 mM, MeCN) λmax (Δε) 204 (+2.7), 301 (−0.3) nm; 1 H and 13C NMR data (Tables 4 and 5); HRESIMS [M + Na]+ m/z 565.2040 (calcd for C29H34NaO10, 565.2044). 6-O-Acetyl-6-dehydroxymoluccensin T (12): yellow, amorphous solid; [α]25 D = +108 (c 0.1, acetone); UV (MeCN) λmax (log ε) 196 (2.12), 275 (1.45) nm; ECD (c 0.16 mM, MeCN) λmax (Δε) 198 (+3.5), 222 (+0.1), 292 (+8.1) nm; 1H and 13C NMR data (Tables 4 and 5); HRESIMS [M + Na]+ m/z 635.2463 (calcd for C33H40NaO11, 635.2463). Computational Details. All conformational analyses and the TDDFT calculations have been done with ORCA36,37 using the chain of spheres approximation.38,39 For the structure optimizations B3LYPD3/def2-SVP20−22 (for 1 def2-TZVP22 was used) has been applied, while the TDDFT computations were done with CAM-B3LYP/def2TZVP(-f).20−23,40 The ECD spectra of the limonoids shown here are dominated by the conformation of the “northeastern” part of the molecules and thus by the C-13 and C-17 stereocenters. These two six-membered rings and the furanyl group are structurally quite rigid, and only two different conformers were found, in which only the dihedral angle of the furanyl axis differed. However, both conformations showed nearly identical simulated ECD spectra. In addition, the different conformations of the “southern”moieties of the compounds showed a negligible effect on the ECD; thus only the ECD curves of the main configurations were calculated and compared with the experimental spectra. The relative configuration of the stereocenter at C-6 of compounds 11 and 12 could not be elucidated by NOE correlations due to the conformational flexibility in this part of the molecule. However, this stereocenter has no significant effect on the ECD spectrum because of lacking nearby chromophores and because of the dominance of the “northeastern” chromophore to the overall spectrum of the liminoids. Thus, we arbitrarily chose the Rconfiguration for this stereogenic center based on biosynthetic reasons. For comparison of experimental and calculated spectra, SpecDis25 was applied using the following UV shifts24 and σ values for the computed curves: (1) 25 nm at 0.3 eV; (3) 10 nm at 0.4 eV; (4) 15 nm at 0.4 eV; (6) 30 nm at 0.4 eV; (11) 15 nm at 0.4 eV; and (12) 5 nm at 0.4 eV.

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ASSOCIATED CONTENT

S Supporting Information *

NMR and HRESIMS spectra for compounds 1−12, CD spectra for compounds 2−12, and quantum-chemical ECD calculations for compounds 3, 4, 6, 11, and 12. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00151.

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AUTHOR INFORMATION

Corresponding Authors

*(L. Shen) Tel: +86-20-85222050. Fax: +86-20-85224766. Email: [email protected]. *(J. Wu) Tel: +86-20-85222377. Fax: +86-20-85224766. Email: [email protected]. *(G. Bringmann) Tel: +49 931-31-85323. Fax: +49 931-3184750. E-mail: [email protected]. H



Article

Notes

(26) Berova, N.; Polavarapu, P. L.; Nakanishi, K.; Woody, R. W., Eds. Comprehensive Chiroptical Spectroscopy; John Wiley & Sons, Inc.: Hoboken, 2012. (27) Tanaka, Y.; Yamada, T.; In, Y.; Muraoka, O.; Kajimoto, T.; Tanaka, R. Tetrahedron 2011, 67, 782−792. (28) Pudhom, K.; Sommit, D.; Nuclear, P.; Ngamrojanavanich, N. J. Nat. Prod. 2010, 73, 263−266. (29) Wu, J.; Xiao, Q.; Zhang, S.; Li, X. Tetrahedron 2005, 61, 8382− 8389. (30) Govindachari, T. R.; Kumari, G. N. K. Phytochemistry 1998, 47, 1423−1425. (31) Li, J.; Li, M. Y.; Feng, G.; Wu, J. J. Nat. Prod. 2012, 75, 1277− 1283. (32) Williams, C. J.; Yavitt, J. B. Soil Sci. Soc. Am. J. 2010, 74, 317− 325. (33) Gibbs, J.; Greenway, H. Funct. Plant Biol. 2003, 30, 1−47. (34) Grassauer, A.; Weinmuellner, R.; Meier, C.; Pretsch, A.; Prieschl-Grassauer, E.; Unger, H. Virol. J. 2008, 5, 107. (35) Fan, Y. Q.; Wang, Y.; Liu, P. P.; Fu, P.; Zhu, T. H.; Wang, W.; Zhu, W. M. J. Nat. Prod. 2013, 76, 1328−1336. (36) Neese, F. WIREs Comput. Mol. Sci. 2012, 2, 73−78. (37) Neese, F.; Wennmohs, F.; Becker, U.; Bykov, D.; Ganyushin, D.; Hansen, A.; Izsák, R.; Liakos, D. G.; Kollmar, C.; Kossmann, S.; Pantazis, D. A.; Petrenko, T.; Reimann, C.; Riplinger, C.; Roemelt, M.; Sandhöfer, B.; Schapiro, I.; Sivalingam, K.; Wezisla, B. ORCA, version 3.0.3 ed.; MPI CEC: Mühlheim a.d.R., Germany, 2014. (38) Izsak, R.; Neese, F. J. Chem. Phys. 2011, 135, 144105. (39) Petrenko, T.; Kossmann, S.; Neese, F. J. Chem. Phys. 2011, 134, 054116. (40) Ekström, U.; Visscher, L.; Bast, R.; Thorvaldsen, A. J.; Ruud, K. J. Chem. Theory Comput. 2010, 6, 1971−1980.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the NSFC (81302693, 31170331, and 81125022), the Cultivation and Innovation Fund of Scientific Research of Jinan University Youth Fund (21613332), Pearl River S&T Nova Program of Guangzhou (201506010023), the Science and Technology Planning Project of Guangdong Province (2013B051000057), the Guangdong Key Science and Technology Special Project (2011A080403020), the Special Financial Fund of Innovative Development of Marine Economic Demonstration Project (GD2012-D01-001), and the DFG (SFB 630).

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