Immunomodulatory Biscembranoids and ... - ACS Publications

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Immunomodulatory Biscembranoids and Assignment of Their Relative and Absolute Configurations: Data Set Modulation in the Density Functional Theory/Nuclear Magnetic Resonance Approach Peng Sun,†,‡ Feng-Yuan Cai,†,‡ Gianluigi Lauro,§,‡ Hua Tang,† Li Su,† Hong-Liang Wang,† Huan Huan Li,† Attila Mándi,⊥ Tibor Kurtán,⊥ Raffaele Riccio,§ Giuseppe Bifulco,*,§ and Wen Zhang*,† Downloaded via UNIV AUTONOMA DE COAHUILA on April 8, 2019 at 14:17:45 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Research Centers for Marine Drugs and Pharmaceutical Analysis, School of Pharmacy, Second Military Medical University, 325 Guo-He Road, Shanghai 200433, People’s Republic of China § Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, Fisciano 84084, Italy ⊥ Department of Organic Chemistry, University of Debrecen, POB 400, H-4002 Debrecen, Hungary S Supporting Information *

ABSTRACT: Five new biscembranoids, bistrochelides A−E (3−7), were isolated together with glaucumolides A (1) and B (2) from the soft coral Sarcophyton trocheliophorum. Their structures and absolute configurations were determined by spectroscopic methods, X-ray crystal diffraction, and DFT/ NMR (density functional theory/nuclear magnetic resonance) and TDDFT/ECD (time-dependent density functional theory/electronic circular dichroism) calculations. A new approach is introduced to determine the relative configuration of a stereocenter through the dynamic evaluation of the mean absolute errors (MAEs) between the investigated diastereoisomers, moving from an “extended” to a more diagnostic “restricted” set of atoms. This research leads to the structure revision of glaucumolides A and B. In in vitro immunomodulatory screening, compounds 1 and 4 significantly induced the proliferation of CD3+ T cells, while compounds 1 and 5 significantly increased the CD4+/CD8+ ratio at 3 μM.

B

In the course of an ongoing search for bioactive secondary metabolites from the South China Sea invertebrates,7,10,11 the soft coral S. trocheliophorum was collected off the coast of Xisha, Sansha Province. This species has been reported to be the producer of compounds such as polyhydroxysterols,12,13 sesquiterpenoids,14 cembrane diterpenoids,15−21 and furanones.22,23 This chemical examination on the Et2O extract of S. trocheliophorum led to the isolation of two known compounds glaucumolides A (1) and B (2), the absolute configurations of which were revised, and five new compounds named bistrochelides A−E (3−7) (Figure 1). It is the first report of biscembranoids from the title animal. Herein the isolation, structure elucidation, and bioactivity assessment of these compounds as new members of dimeric cembranoids are reported.

iscembranoids are an interesting family of marine natural products that are featured with two cembranoid diterpenoid moieties connected by a cyclohexene unit.1 The first example of this family, methyl sartortuoate, was isolated from S. tortuosum in 1986.2 Since then, the number of these dimeric cembranoids has rapidly increased. To date, more than 80 members have been discovered. The majority is from soft corals of the genera Sarcophyton (family Alcyoniidae)1,3 with the exception of the sponge Petrosia nigricans.4 The biscembranoids are considered to be constructed by a Diels−Alder cycloaddition between two cembranoid monomers. Normally, the cembranoids with a trisubstituted conjugated Δ21(34)/Δ35(36) butadiene function served as the diene moieties. The dienophiles are cembranoids with a Δ1(2) double bond conjugated by a carbonyl and/or a carboxymethyl ester,1 or cembranoids with a Δ1(2) double bond embedded in an α,β-unsaturated γ-lactone,5,6 or capnosanes with a Δ1(2) double bond embedded in an α,β-unsaturated ε-lactone,7 respectively. The determination of the relative and absolute configuration of biscembranoids has been a challenge due to the high complexity and flexibility of these molecules. The intriguing polycyclic ring systems have attracted great interest as targets for total synthesis.8,9 © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

The workup for extraction and isolation of cembranoids was performed on freshly collected specimens of S. trocheliophorum Received: December 7, 2018

A

DOI: 10.1021/acs.jnatprod.8b01037 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 2. Key NOE correlations of 1.

orientation of both H-4 and H-5 was supported by the NOE cross-peak of H-4/H-5. No coupling constant was observed for H-4/H-5 indicating an ca. 90° dihedral angle between the two protons. The multiplets and coupling constants of H-5 (3JH5,H6 = 11.0, 3.0 Hz) with H2-6 suggested its cis and trans relationships with H-6α and H-6β, respectively. In ring D, the coupling constant between H-21 and H-22 (3JH21,H22 = 10.7 Hz) indicated an anti configuration of the two protons based upon the associated dihedral angle deduced from the Karplus equation.26 An α orientation of H3-39 was deduced from the NOE correlations of H-21/H3-38, H3-38/H-25α, and H-25α/H3-39. The NOE cross-peak of H-22/H-26, H-22/H24β, and H-24β/H-25β suggested a β orientation of H-26, which might explain the absence of an NOE correlation between H-26 and H3-39. Therefore, the relative configuration of 1 was proposed to be (1S*,2R*,3R*,4R*,5S*,21S*,26S*,27R*) in contrast to (1S*,2R*,3S*,4S*,5S*,21S*,26R*,27S*) for glaucumolide A. To confirm these assignments, compound 1 was crystallized and subjected to X-ray crystallographic analysis. The result confirmed the relative configuration and indicated the absolute configuration of 1 as (1S,2R,3R,4R,5S,21S,26S,27R) with a Flack parameter of −0.18 (15) (Figure 3). Therefore,

Figure 1. Structures of glaucumolides A and B (1 and 2) and bistrochelides A−E (3−7).

as previously reported.6,24,25 This common procedure on the animal resulted in seven pure compounds (1−7). Compounds 1 and 2 both had a molecular formula of C42H58O8 as determined by HRESIMS (high-resolution electrospray ionization mass spectrometry). They showed identical 1H and 13C NMR data (Table S1, Supporting Information) to those of glaucumolides A and B, two analogues reported recently from the soft coral S. glaucum, suggesting the same structures for the pairs of metabolites, respectively.3 However, detailed analysis of spectroscopic data for 1 and 2 revealed different configurations at C-3, C-4, C-26, and C-27, with respect to glaucumolides A and B, 3 respectively. Exemplified in 1, a different relative configuration was proposed on the basis of NOESY (nuclear Overhauser enhancement spectroscopy) data (Figure 2). In ring C, direct evidence for the NOE (nuclear Overhauser effect) correlation between H-2 and H-21 was hard to identify due to the overlapped resonances in the NOESY spectrum. However, a distinct NOE cross-peak of H-3/H-22 was observed, suggesting a cis relationship between the C-2/C-3 and C-21/ C-22 single bonds, which accordingly showed the cis relationship of H-2/H-21. The NOE correlations of H-21 with H2-14 indicated a (1S*) configuration. In ring B, the NOE correlations of H-3/H-36β, H-2/H-4, and H-2/H-36a suggested a trans relationship between H-2 and H-3, which is in accordance with the absence of NOE correlation of H-2/H3. The coupling constant of H-4 with H-3 (3JH3,H4 = 10.7 Hz) indicated a trans relationship of these two protons. The α

Figure 3. Single-crystal X-ray structure of 1 monohydrate (ORTEP drawing, ellipsoid probability 30%).

compound 1 was designated to be glaucumolide A of which the absolute configuration was revised to (1S,2R,3R,4R,5S,21S,26S,27R). Compound 1 showed different specific rotations ([α]D + 36.0 in MeOH and −4.7 in CHCl3) from that of glaucumolide A ([α]D −207, in CHCl3).3 The unusual [α]D value of glaucumolide A might be attributed to a measurement error or the presence of minor optically active B



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The comparison of measured and calculated ECD spectra corroborated the (1S,2R,3R,4R,5S,21S,26S,27R) absolute configuration of 2 (Figure 5). Compound 2 was thus the same as glaucumolide B3 due to their identical NMR data. The absolute configuration of glaucumolide B should be revised as (1S,2R,3R,4R,5S,21S,26S,27R).

impurities since it was recorded at a very low concentration (c 0.007).3 The established absolute configuration of 1 permitted the application of the TDDFT-ECD (time-dependent density functional theory-electronic circular dichroism) approach to gain information on the solution conformations as references in structure elucidation for the accompanying analogues. ECD calculations were performed for the density functional theory (DFT) reoptimized low-energy conformers obtained by conformational search and molecular dynamics simulations. Specifically, using the MPW1PW91/6-31g(d,p)// MPW1PW91/6-31g(d) level of theory27 (see the Experimental Section) for the prediction of the Boltzmann-weighted ECD spectrum, as successfully employed in previous reports,7,28−31 a good agreement between the experimental and predicted ECD spectra of the (1S,2R,3R,4R,5S,21S,26S,27R)-1 isomer was obtained. This result then indicated (1S,2R,3R,4R,5S,21S,26S,27R) absolute configuration in line with the X-ray results (Figure 4). The low-energy conformers

Figure 5. Comparison of the experimental ECD spectra with the TDDFT-predicted ECD curve of (1S,2R,3R,4R,5S,21S,26S,27R)-2 (MPW1PW91/6-31g(d,p) functional/basis set, optimization at the MPW1PW91/6-31g(d) level, average of seven conformers).

Bistrochelide A (3) was obtained as an amorphous powder. Its molecular formula of C42H58O8 was determined by the HRESIMS ion at m/z 708.4468 [M + NH4]+ (calcd 708.4475), requiring 14 indices of hydrogen deficiency. The IR spectrum displayed absorptions for hydroxy (3465 cm−1), ester carbonyl (1737 cm−1), and conjugated carbonyl (1682 and 1606 cm−1) functionalities. The 13C and distortionless enhancement by polarization transfer (DEPT) NMR spectra revealed the presence of 14 sp2 (4 CO, 4 CCH, 1 CH C) and 28 sp3 carbon atoms (8 CH3, 11 CH2, 4 CH, 3 OCH, 1 C, 1 OC), accounting for nine indices of hydrogen deficiency (Table 1). The remaining indices of hydrogen deficiency were due to the presence of five rings in the molecule. The 1H NMR spectrum displayed resonances of seven methyls, attributable to respectively an isopropyl group, four vinylic methyls, a methyl attached to an oxygenated secondary carbon, and the methyl of an acetoxyl group. Further analyses of the 2D NMR spectra showed that the gross structure of 3 possessed both a five-membered saturated γ-lactone and a seven-membered α,βunsaturated ε-lactone moiety similar to 2. A remarkable difference was observed in the 13C NMR resonance of C-38 which was downfield-shifted from δC 15.9 in 2 to 27.0 in 3, whereas the 13C NMR resonance of C-24 was upfield-shifted from δC 36.3 in 2 to 26.7 in 3. This observation led to the suggestion of a (Z) geometry of the Δ22(23) double bond in 3, which was in good agreement with the distinct NOE correlation between H-22 and H3-38. Compound 3 was thus assigned to be the (22Z) isomer of 2. The absolute configuration of 3 was determined by experimental and calculated TDDFT/ECD spectra. Comparison of calculated and experimental ECD spectra indicated (1S,2R,3R,4R,5S,21S,26S,27R) absolute configuration for 3 (Figures 6 and S3, Supporting Information). Bistrochelide B (4) was obtained as an optically active solid with the same molecular formula as 1, as established by HRESIMS data. The 1H and 13C NMR data of 4 closely

Figure 4. Comparison of the experimental ECD spectrum of 1 measured in MeOH with the TDDFT-predicted ECD curve of (1S,2R,3R,4R,5S,21S,26S,27R)-1 (MPW1PW91/6-31g(d,p) functional/basis set, optimization at the MPW1PW91/6-31g(d) level, average of eight conformers).

identified by DFT shared a high structural similarity with the X-ray geometry especially concerning the left part of the macrocycle, whereas some differences were noticed regarding the conformation of the right macrocycle (see root-mean square deviation (RMSD) values, as reported in Figures S1 and S2, Supporting Information). Overall, ECD calculations accounting for Boltzmann-weighted sampled conformers reproduced the experimental ECD showing a good superposition between measured and predicted curves. Compound 2 possessed the same 2D structure as 1 with the major difference the (11Z) double bond in contrast to the (11E) geometry in 1. The relative configuration of the stereogenetic centers in 2 was deduced to be the same as that of 1 on the basis of NOESY analysis. The experimental ECD spectrum of 2 was quite different from that of 1. In particular, while a negative Cotton effect was observed at 240 nm for 1, a broadened Cotton effect was present in the 230− 290 nm region of the ECD spectrum of 2. In order to clarify this different behavior, the ECD spectrum of 2 was calculated following the same protocol as for 1. Interestingly, the predicted ECD spectrum of 2 showed two broadened Cotton effects at 225 and 270 nm, covering the region of the Cotton effects detected in the experimental spectrum at 230−290 nm. C



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Table 1. 1H and 13C NMR Spectroscopic Data for 3 and 4a 3 no.

δC, type

1 2 3

53.2, C 41.8, CH 84.8, CH

4 5

47.5, CH 72.2, CH

6α 6β 7 8 9α 9β 10 11 12α 12β 13 14α 14β 15 16 17 18 19 20 21 22 23 24α 24β 25α 25β 26 27 28α 28β 29 30 31 32α 32β 33α 33β 34 35 36α 36β 37 38 39 40 -OAc

41.7, CH2 132.2, C 127.8, CH 25.2, CH2 31.4, CH2 162.2, C 125.1, CH 198.6, C 52.4, CH2 25.8, CH 18.5, CH3 25.1, CH3 17.3, CH3 24.7, CH3 179.3, C 48.9, CH 123.1, CH 138.1, C 26.7, CH2 29.7, CH2 67.4, CH 83.6, C 35.0, CH2 27.3, CH2 138.4, CH 135.4, C 37.2, CH2 32.7, CH2 133.8, C 129.3, C 35.0, CH2 21.0, CH3 27.0, CH3 22.7, CH3 167.8, C 170.5, C 21.4, CH3

4

δH (J in Hz) 1.90, d (11.0) 3.95, dd (10.7, 6.2) 1.79, ov 4.80, dd (11.0, 3.0) 2.23, ov 2.15, ov 5.14, 2.19, 2.27, 1.65,

t (7.5) ov m ov

5.97, s

2.98, 2.36, 2.17, 1.08, 1.17, 1.64, 1.87,

d (14.3) d (14.3) ov d (7.3) d (7.3) s s

2.79, d (11.2) 5.23, d (11.2) 1.88, ov 2.75, m 1.86, ov 1.61, m 4.15 d (8.1) 2.21, 2.08, 2.47, 6.09,

ov m m brs

1.98, 3.14, 1.98, 2.59,

ov m ov m

2.18, 1.98, 1.76, 1.82, 1.37,

ov m s s s

2.05, s

δC, type 52.0, C 43.8, CH 85.1, CH 48.1, CH 71.7, CH 43.5, CH2 129.3, C 128.2, CH 27.6, CH2 127.9, CH 129.3, C 47.0, CH2 203.4, C 50.1, CH2 26.2, CH 18.8, CH3 24.3, CH3 17.4, CH3 25.4, CH3 179.1, C 44.8, CH 122.6, CH 138.6, C 36.4, CH2 29.0, CH2 67.9, CH 83.2, C 34.9, CH2 26.4, CH2 135.7, CH 132.0, C 30.2, CH2 29.1, CH2 132.0, C 127.3, C 36.1, CH2 20.4, CH3 16.0, CH3 22.1, CH3 169.0, C 171.1, C 21.5, CH3

δH (J in Hz) 1.68, ov 3.95, dd (11.2, 5.4) 1.95, d (11.2) 4.72, dd (11.2, 2.2) 2.21, ov 2.11, ov 4.97, 2.56, 2.56, 5.63,

t (5.0) ov ov t (8.5)

Figure 6. Comparison of the experimental ECD spectrum of 3 measured in MeOH with the TDDFT-predicted ECD curve of (1S,2R,3R,4R,5S,21S,26S,27R)-3 (MPW1PW91/6-31g(d,p) functional/basis set, optimization at the MPW1PW91/6-31g(d) level, average of 12 conformers).

2.93, d (16.9) 3.06, d (16.9) 2.79, 2.73, 2.19, 1.06, 1.19, 1.66, 1.69,

This assignment was confirmed by the proton sequence from H-8 to H-10 established by the correlation spectroscopy (COSY) experiment and was supported by the presence of an unconjugated ketocarbonyl moiety (δC 203.4, C-13). The (10Z) geometry was assigned by the 13C NMR shift value of the olefinic methyl group (δC 25.4, C-19) and was confirmed by the NOE correlation between H-10 and H3-19.32 The proposed structure of 4 was strongly supported by detailed analysis of 1D and 2D NMR spectra. The relative configurations at the stereogenic centers of 4 were suggested to be the same as those of 1 by NOE data analyses. The absolute configuration of 4 was assigned to be the same as 1, based on the similarity of their experimental ECD spectra (Figure S3, Supporting Information). Bistrochelide C (5) was isolated as an optically active powder. The HRESIMS gave a molecular formula of C42H60O8, which are two atomic mass units more than that of 1. The 1D NMR data of 5 were similar to those of 1 (Tables 2 and 3). A difference was observed for the presence of a methyl doublet (δH 0.92, d, J = 7.1 Hz, H3-19) in the upfield region of the 1H NMR spectrum of 5. The location of the methyl group at C-11 was suggested by the COSY correlation between H3-19 and H-11 and by the heteronuclear multiplebond correlation (HMBC) cross-peaks from H3-19 to C-10 (δC 37.0), C-11 (δC 28.9), and C-12 (δC 48.9). The absence of the Δ11(12) double bond in 5 was in agreement with the presence of an isolated C-13 ketocarbonyl group (δC 208.6). The remaining part of 5 was found to be the same as that of 1 on the basis of detailed analyses of the 1D and 2D NMR data. Compound 5 was then assigned as the 11,12-dihydro derivative of 1. Its NOE pattern was similar to that of 1 except for the C-11 stereogenic center. The relative configuration of C-11 was determined by comparison of the calculated 1H and 13C NMR chemical shifts with experimental values. In particular, the prediction of NMR parameters was performed using the MPW1PW91/6-31g(d,p)//MPW1PW91/6-31g(d) level of theory, as employed in different studies regarding the prediction of the relative configuration of natural compounds.27,33−35 In this context, different investigations were reported to introduce robust parameters for correctly assigning the stereochemical patterns of organic compounds.36,37

d (15.7) d (15.7) m d (7.0) d (7.0) s s

2.98, d (10.6) 5.06, brs 2.11, 2.35, 1.75, 1.36, 4.12,

ov ov m m d (9.2)

2.21, 2.02, 2.45, 5.90,

ov ov ov brs

2.32, 2.56, 2.11, 2.68,

m ov ov m

2.22, 2.01, 1.83, 1.56, 1.29,

m ov s s s

2.09, s

a

In CDCl3, 500 MHz for 1H and 125 MHz for 13C; ov, overlapped signals.

resembled those of 1. A difference was observed for the presence of a Δ10(11) double bond (δC 127.9, C-10; 129.3, C11; δH 5.63, t, J = 8.5 Hz, H-10) in 4 instead of Δ11(12) in 1. D



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Table 2. 1H NMR Spectroscopic Data for 5−7 (in CDCl3, 500 MHz, J in Hz)a no. 2 3 4 5 6α 6β 8 9α 9β 10α 10β 11 12α 12β 14α 14β 15 16 17 18 19 21 22 24α 24β 25α 25β 26 28α 28β 29 30 32α 32β 33α 33β 36α 36β 37 38 39 -OAc

5 1.73, 3.91, 2.10, 4.72, 2.32, 2.02, 5.31, 1.85, 1.98, 1.42, 0.87, 2.16, 1.99, 2.17, 2.87, 2.67, 2.22, 1.11, 1.25, 1.65, 0.92, 2.99, 5.12, 2.06, 2.33, 1.69, 1.33, 4.10, 2.21, 2.00, 2.42, 5.90, 2.23, 2.53, 2.16, 2.58, 2.32, 2.06, 1.80, 1.54, 1.28, 2.09,

m dd (11.1, 5.1) ov d (10.5) ov ov t (7.0) m ov m m ov ov ov d (18.3) d (18.3) m d (7.1) d (7.1) s d (5.8) d (10.7) brs ov ov m m d (9.4) ov ov m brs m m ov m ov ov s s s s

7

no.

5

6

7

1.76, ov 4.25, dd (10.3, 6.2) 3.59, dd (10.3, 3.8)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 -OAc

51.9, C 45.4, CH 83.8, CH 49.4, CH 71.6, CH 44.7, CH2 131.3, C 128.7, CH 25.0, CH2 37.0, CH2 28.9, CH 48.9, CH2 208.6, C 53.2, CH2 26.5, CH 18.8, CH3 24.5, CH3 16.6, CH3 21.5, CH3 179.3, C 44.6, CH 122.6, CH 138.7, C 36.3, CH2 28.8, CH2 68.0, CH 83.3, C 35.0, CH2 26.2, CH2 135.6, CH 131.3, C 30.2, CH2 28.9, CH2 132.3, C 128.0, C 36.2, CH2 20.4, CH3 16.0, CH3 22.2, CH3 169.2, C 171.1, C 21.5, CH3

52.4, C 45.3, CH 83.9, CH 49.5, CH 71.7, CH 44.5, CH2 131.6, C 128.7, CH 24.9, CH2 36.8, CH2 28.9, CH 49.3, CH2 208.9, C 52.6, CH2 26.6, CH 18.8, CH3 24.6, CH3 16.6, CH3 21.6, CH3 179.1, C 45.9, CH 123.6, CH 137.7, C 26.7, CH2 29.6, CH2 67.2, CH 83.5, C 34.9, CH2 27.5, CH2 139.2, CH 135.3, C 37.1, CH2 31.8, CH2 133.8, C 130.7, C 36.1, CH2 21.3, CH3 27.0, CH3 22.6, CH3 170.9, C 167.6, C 21.5, CH3

52.0, C 43.1, CH 84.0, CH 60.3, CH 208.7, C 57.2, CH2 126.9, C 130.5, CH 25.3, CH2 36.3, CH2 28.4, CH 48.5, CH2 209.0, C 53.3, CH2 28.7, CH 18.2, CH3 21.4, CH3 15.8, CH3 22.0, CH3 179.0, C 44.9, CH 122.6, CH 138.5, C 36.6, CH2 29.1, CH2 67.7, CH 82.9, C 34.9, CH2 26.7, CH2 135.7, CH 132.0, C 30.3, CH2 29.2, CH2 131.5, C 128.0, C 35.7, CH2 20.3, CH3 16.0, CH3 22.1, CH3 168.8, C

6 1.83, 3.85, 2.07, 4.75, 2.33, 2.04, 5.31, 1.87, 2.05, 1.40, 0.97, 2.11, 2.05, 2.11, 2.88, 2.69, 2.22, 1.12, 1.23, 1.66, 0.94, 2.90, 5.48, 2.22, 2.69, 1.84, 1.61, 4.12, 2.22, 2.02, 2.47, 6.13, 2.00, 3.19, 2.12, 2.49, 2.32, 2.23, 1.81, 1.83, 1.35, 2.08,

ov dd (11.3, 5.0) ov d (9.7) m ov t (7.6) m ov m m ov ov ov d (18.4) d (18.4) ov d (7.2) d (7.2) s d (5.9) d (10.6) d (12.0) ov m ov m d (6.6) ov ov m brs m t (12.2) ov m ov ov s s s s

Table 3. 13C NMR Spectroscopic Data for 5−7 (in CDCl3, 125 MHz)

3.30, 2.74, 5.53, 1.85, 2.01, 2.11, 1.50, 2.40, 2.04, 2.23, 2.94, 2.68, 2.34, 1.02, 1.12, 1.59, 0.98, 2.94, 5.01, 1.98, 2.11, 1.76, 1.35, 4.14, 2.22, 2.05, 2.45, 5.87, 2.34, 2.61, 2.08, 2.74, 2.24, 1.94, 1.78, 1.57, 1.31,

d (10.1) d (9.1) t (7.6) m ov s m m ov ov d (18.0) d (18.0) m d (7.1) d (7.1) s d (6.7) d (11.8) d (10.9) ov ov ov m d (9.2) ov ov m brs m m ov ov ov m s s s

a

ov, overlapped signals.

(11S*)-5] showed a high structural similarity due to the extended cyclic constrained systems (Figure 7). Accordingly, a large set of 13C/1H NMR chemical shift values was similarly

First, for each investigated diastereoisomer, the predicted and experimental NMR chemical shift values are compared atom by atom. In particular, for each atom, it was defined as follows: |Δδ| = |δexp − δcalc|, where δexp (ppm) and δcalc (ppm) are the 13C/1H experimental and calculated chemical shifts, respectively. The mean absolute error (MAE), defined as MAE = ∑(| Δδ |) , is the summation (Σ) of the n computed absolute n error values (|Δδ|), normalized to the number of chemical shifts considered (n), and then it is a positive number. This parameter summarizes, for each investigated diastereoisomer, the similarity between the predicted and experimental chemical shift values. Interestingly, the analysis of the most energetically favored conformers for both possible diastereoisomers [(11R*)-5 and

Figure 7. Superposition between the most energetically favored conformers of (11R*)-5 (gray) and (11S*)-5 (green). E



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ppm, |Δδ|exp‑(11S*)‑5 = 0.49 ppm, Table S3, Supporting Information). The similarity between the ECD spectra of 5 and 1 (Figure S5, Supporting Information) indicated the absolute configuration of 5 to be (1S,2R,3R,4R,5S,11S,21S,26S,27R). Bistrochelide D (6) was obtained as an amorphous powder and had the same molecular formula as 5. The 1H and 13C NMR spectra of 6 were similar to those of 5. The characteristic methyl doublet (δH 0.94, d, J = 5.9 Hz, H3-19) and the COSY correlation between H3-19 and H-11 indicated the presence of the same Δ11(12) single bond as in 5. Analyses of the 2D NMR data gave the same 2D structure as that of 5. The conspicuously downfield-shifted 13C resonance for C-38 (δC 27.0) and the upfield-shifted one for C-24 (δC 26.7) suggested a (22Z) geometry in 6. This was supported by the NOE correlation between H-22 and H3-38. The relative configuration of 6 was fully assigned by the NOESY analyses except for the C-11 methine. The relative configuration at C-11 was determined by comparison of the calculated 1H and 13C chemical shifts with the measured values, suggesting (11S*) relative configuration, as clearly indicated by the 13C/1H ΔMAE(11R*)‑6/(11S*)‑6/cutoffΔδcalc plots (Figure S6, Tables S4 and S5, Supporting Information, “extended” set of atoms 13 C/1H MAEs = 2.48/0.24 ppm for (11R*)-6 and 2.18/0.19 ppm for (11S*)-6, respectively, 13C/1H ΔMAE(11R*)‑6/(11S*)‑6 = 0.30/0.05 ppm). Also in this case, a difference was detected for the 1H chemical shift values computed for H-11 (δexp = 2.11 ppm, vs δcalc‑(11R*)‑6 = 1.31 ppm, |Δδ|exp‑(11R*)‑6 = 0.80 ppm, vs δcalc‑(11S*)‑6 = 2.35 ppm, |Δδ|exp‑(11S*)‑6 = 0.24 ppm, Table S5, Supporting Information). Compound 6 was defined as the (22Z) isomer of 5, leading to the assignment of its absolute configuration by comparison of experimental and computed ECD data (Figure S7, Supporting Information). Bistrochelide E (7) was obtained as an amorphous powder and had a molecular formula of C40H56O7 on the basis of HRESIMS. Comparison of the NMR spectra of 7 and 5 revealed similar structures. Compound 7 has the same C-11 methine as 5, based on the presence of a methyl doublet (0.98, d, J = 6.7 Hz, H3-19) in the 1H NMR spectrum. The location of the C-5 ketocarbonyl group (δC 208.7) was determined by the HMBC cross-peaks from both H-4 and H2-6 to C-5. The remaining part of 7 was the same as that of 5, which was supported by analyses of the 1D and 2D NMR data. Compound 7 has a C-5 ketocarbonyl group in contrast to a C-5 acetyl group in 5. Further analyses of the NOESY data revealed that 7 had a similar NOE pattern as 5. The comparison of the experimental and calculated 1H and 13C NMR chemical shifts indicated an (11S*) relative configuration for 7, as shown by the 13C/1H ΔMAE(11R*)‑7/(11S*)‑7/ cutoffΔδcalc plots (Figure S8, Tables S6 and S7, Supporting Information; “extended” set of atoms 13C/1H MAEs = 1.95/ 0.26 ppm for (11R*)-7 and 1.79/0.24 ppm for (11S*)-7, respectively, 13C/1H ΔMAE(11R*)‑7/(11S*)‑7 = 0.16/0.02 ppm). Again, for the two diastereoisomers, a large difference was found for the computed shift values for H-11 (δexp = 2.40 ppm, vs δcalc/(11R*)‑7 = 1.22 ppm, |Δδ|exp‑(11R*)‑7 = 1.18 ppm, vs δcalc‑(11S*)‑7 = 2.59 ppm, |Δδ|exp‑(11S*)‑7 = 0.19 ppm, Table S7, Supporting Information). The absolute configuration of 7 was deduced by TDDFT/ ECD calculations. The data showed good agreement between the calculated and experimental ECD curves of (1S,2R,3R,4S,11S,21S,26S,27R)-7 (Figure 8).

predicted for both diastereoisomers (Experimental Section and Tables S2 and S3, Supporting Information), as indicated by the |Δδcalc| parameter (see Computation of NMR Parameters, Experimental Section). In the specific case of compounds (11R*)-5 and (11S*)-5, for the 13 C/ 1 H nuclei, the |Δδcalc‑(11R*)‑5/ (11S*)‑5| parameter is defined as follows: |Δδcalc‑(11R*)‑5/(11S*)‑5| = |δcalc‑(11R*)‑5 − δcalc‑(11S*)‑5|, where, for each atom considered, δcalc‑(11R*)‑5 and δcalc‑(11S*)‑5 are the calculated chemical shift values for (11R*)-5 and (11S*)-5, respectively. Indeed, for two considered isomers of a casestudy compound, this parameter indicates the similarity between the predicted chemical shift values. Starting from these assumptions, the ΔMAE parameter was introduced for the 13C/1H nuclei and is defined as the difference of MAE values (see Experimental Section), respectively, between two diastereoisomers of a case-study compound. Specifically, for the two investigated diastereoisomers of 5, the specific ΔMAE is defined as ΔMAE(11R*)‑5/(11S*)‑5 = MAE(11R*)‑5 − MAE(11S*)‑5, where ΔMAE(11R*)‑5/(11S*)‑5 can be positive or negative, if the MAE of the first considered isomer [(11R*)-5 in this specific case] is higher or lower than the MAE of the second considered isomer [(11S*)-5 in this specific case], respectively. Thus, the variations of the 13C/1H ΔMAE(11R*)‑5/(11S*)‑5 were analyzed by gradually changing the set of atoms used for the comparison, according to the related 13C/1H |Δδcalc‑(11R*)‑5/(11S*)‑5| values. For both nuclei, different cutoffs of |Δδcalc‑(11R*)‑5/(11S*)‑5| values (13C/1H cutoffΔδcalc) were first defined for the selection of the chemical shift data and the subsequent computation of MAE and ΔMAE values, and then 13 C/1H ΔMAE(11R*)‑5/(11S*)‑5 were plotted considering increasing values of 13C/1H cutoffΔδcalc (see Computation of NMR Parameters, Experimental Section). In this way, when 13C/1H cutoffΔδcalc = 0, the “extended” set of atoms was considered for the comparison (namely accounting values related to all the atoms; data related to sp2 carbonyl 13C and 1H bound to heteroatoms were ignored a priori) (Tables S2 and S3, Supporting Information). Increasing the 13C/1H cutoff Δδcalc, we gradually moved to a “restricted” set of atoms, namely accounting only those showing |Δδcalc‑(11R*)‑5/(11S*)‑5| values higher than the cutoffΔδcalc and then selecting the most diagnostic 13C/1H predicted chemical shift data for the comparison. As expected, choosing the “extended” set of atoms (13C/1H cutoffΔδcalc = 0), close 13C/1H MAE were found for both diastereoisomers (13C/1H MAEs = 1.71/0.21 ppm for (11R*)-5 and 1.61/0.20 ppm for (11S*)-5, respectively; 13C/1H ΔMAE(11R*)‑5/(11S*)‑5 = 0.10/0.01 ppm), then indicating the uncertainty in establishing the stereochemical pattern for 5. The 13C/1H ΔMAE(11R*)‑5/(11S*)‑5 values related to increasing 13C/1H cutoffΔδcalc were then analyzed, moving to “restricted” sets of data. Remarkably, the highest |Δδcalc‑(11R*)‑5/(11S*)‑5| were mainly related to the atoms close to the C-11 stereocenter, thus confirming this molecular region as the more diagnostic for determining the relative configuration. The analysis of the plots highlighted a growing tendency of 13C/1H ΔMAE(11R*)‑5/(11S*)‑5 when increasing cutoffΔδcalc, then indicating lower MAEs for (11S*)-5 isomer (Figure S4, Supporting Information). The findings then suggested the (11S*) configuration as the most probable for 5, and these data were corroborated by the close accordance between experimental and calculated 1H chemical shift values related to H-11 for (11S*)-5 (δexp = 2.16 ppm, vs δcalc‑(11R*)‑5 = 1.21 ppm, |Δδ|exp‑(11R*)‑5 = 0.95 ppm, vs δcalc‑(11S*)‑5 = 2.65 F



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It is challenging to determine the relative configuration at C11 of bistrochelides C−E. A new approach was introduced in determining the relative configurations by the dynamic evaluation of the MAEs between the investigated diastereoisomers, moving from an “extended” to a more diagnostic “restricted” set of atoms. This would give a methodology reference to the determination of the relative configuration of an alkyl stereocenter in those flexible structures, particularly applicable to compounds featuring a large number of nuclei to be accounted for in the comparison between experimental and calculated chemical shift data. Some of the compounds showed significant T cells proliferation and differentiation activities in the in vitro bioassay. This is the first report of immunoregulatory activity for metabolites of this kind.

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Figure 8. Comparison of the experimental ECD spectra with the TDDFT-predicted curves of (1S,2R,3R,4S,11S,21S,26S,27R)-7 (MPW1PW91/6-31g(d,p) functional/basis set, optimization at the MPW1PW91/6-31g(d) level, average of five conformers).

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured in MeOH with an Autopol IV polarimeter at the sodium D line (589 nm). UV absorption spectra were recorded with a Varian Cary 100 UV/vis spectrophotometer; wavelengths are reported in nm. ECD spectra were recorded with a Jasco-715 and a Jasco-810 spectropolarimeter. Infrared spectra were recorded in thin polymer films on a Nexus 470 FT-IR spectrophotometer (Nicolet); peaks are reported in cm−1. NMR data were acquired at 300 K on a Bruker Avance DRX-500 NMR spectrometer. Chemical shifts are reported relative to the residual CHCl3 signals (δH 7.26; δC 77.0) as an internal standard for 1H and 13C NMR spectra. 1H and 13C NMR assignments were supported by COSY, HMQC, HMBC, and NOESY experiments. The MS and HRMS data were acquired on an Agilent technologies 6224 TOF LC/MS, resolution 5000 equipped with an electron ionization source. An isopropyl alcohol solution of NaI (2 mg/mL) was used as a reference compound. The X-ray diffraction study was carried out on a PANalytical X’Pert Pro diffractometer using CuKα radiation (λ = 1.54178 Å). Semipreparative HPLC was performed on an Agilent 1100 system with a refractive index detector and a YMC Pack ODS-A column (5 μm, 250 × 10 mm). Commercial silica gel (Yantai, 200−300 and 400−600 mesh) was used for column chromatography, and precoated silica gel plates (Yantai, HSGF-254) were used for analytical thin-layer chromatography (TLC). Sephadex LH-20 was used for column chromatography. Spots were detected on TLC under UV or by heating after spraying with an anisaldehyde sulfuric acid reagent. Biological Material. Specimens of S. trocheliophorum, identified by Dr. Xiubao Li of the South China Sea Institute of Oceanology Chinese Academy Sciences, were collected in June 2012 from the coast of Xisha Island in the South China Sea, at a depth of 20 m, and were frozen immediately after collection. A voucher specimen (GE27) is available at the Research Center for Marine Drugs, Second Military Medical University. Extraction and Isolation. The frozen animals (3.2 kg, wet weight) were cut into pieces and extracted exhaustively with acetone at room temperature (5 × 1.5 L). The organic extract was evaporated to afford a residue (61.7 g), which was partitioned between Et2O and H2O. The Et2O solution was concentrated under reduced pressure to give a dark residue (12.2 g). The residue was subjected to silica gel column chromatography eluting with a gradient of acetone in petroleum ether (1:10 to 1:1, v/v), to give 14 fractions (Fr. 1−14). Fr. 7 was fractionated by Sephadex LH-20 (MeOH/CH2Cl2, 1:2) and further purified by RP-HPLC (MeCN/H2O, 74:26, 3.0 mL/min) to give 7 (4.2 mg, tR 34.5 min, yield 0.0001%), 5 (59.6 mg, tR 49.5 min, yield 0.0018%), and 6 (8.8 mg, tR 52.8 min, yield 0.0003%). Fr. 8 was fractionated by Sephadex LH-20 (MeOH/CH2Cl2, 1:2) and then purified by RP-HPLC (MeOH/H2O, 70:30, 3.0 mL/min) to afford 1 (112.8 mg, tR 32.1 min, yield 0.0035%), 2 (194.5 mg, tR 38.1 min, yield 0.0067%), 4 (62.1 mg, tR 43.5 min, yield 0.0019%), and 3 (4.4 mg, tR 45.4 min, yield 0.0001%). Glaucumolide A (1). Colorless crystals (CH3CN/H2O, 5:1); melting point 197−199 °C; Rf 0.35 (CH2Cl2/MeOH 9:1); [α]25D +

The in vitro immunomodulatory activity of these compounds was examined on concanavalin A (Con A)-induced splenocyte activation using flow cytometric analysis.38 An apoptosis analysis of these compounds to splenocytes was first conducted, and the compounds were found to have low cytotoxicity to the tested cells at the concentration of 3 μM (Figure S9, Supporting Information). The percentage of CD3+ cells and the CD4+/CD8+ ratio were analyzed by flow cytometry under these conditions (at 3 μM), using Con A as a positive control (5 μg/mL). It was found that compounds 1 and 4 significantly induced CD3+ T cells proliferation (Figure 9A), which have a potential to restore the reduced T cell

Figure 9. Effects of compounds 1−7 on the proliferation of T cells (*p < 0.05, **p < 0.01).

counts in aged humans. Compounds 1 and 5 significantly increased the CD4+/CD8+ ratio, while 3 decreased the ratio (Figure 9B). This regulation activity correlates to the treatment of dysregulation of the CD4+/CD8+ ratio in autoimmune diseases or viral infections.38 In conclusions, compounds 1−7 were obtained as the first examples of biscembranoids from the soft coral S. trocheliophorum. The chemical diversity was attributed to the presence or absence of double bonds at C-10 or C-11 and the Z/E geometry of the C-11 and C-22 double bonds. The metabolites are presumed to be Diels−Alder adducts of two cembranoid monomers (Figure S10, Supporting Information). Some precursors have been isolated from the Sarcophyton genus such as isosarcophytonolide D,5 sarcophytonolide D,5,39 sarcophytonolide R,17 and (4Z,8S*,9R*,12E,14E)-9-hydroxy1-(prop-1-en-2-yl)-8,12-dimethyloxabicyclo[9.3.2]hexadeca4,12,14-trien-18-one,40 which might partially support the biosynthetic hypothesis. G



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36.0 (c 1.1, MeOH), −4.7 (c 0.1, CHCl3); UV (MeOH) λ max (log ε) 203 (4.04), 242 (3.83) nm; ECD (MeOH, c 4.3 × 10−4) λmax (Δε) 205 (+46.9), 235 (−14.3) nm; ECD (KCl) λmax (Φ) = 205 (+27.7), 236 (−13.9), 375 (+1.4) nm; IR (film) νmax 3469 (OH), 2944, 1737 (CO), 1681, 1676, 1606, 1453 1238 cm−1; 1H and 13C NMR data, Table S1; HRESIMS [M + NH4]+ m/z: 708.4476 (calcd for C42H62NO8, 708.4475). Glaucumolide B (2). White amorphous powder; Rf 0.43 (CH2Cl2/ MeOH 9:1); [α]25D + 18.6 (c 0.7, MeOH), −2.3 (c 1.1, CHCl3); UV (MeOH) λmax (log ε) 203 (4.07), 242 (3.88) nm; ECD (MeOH, c 4.3 × 10−4) λ max (Δε) 203 (+27.3), 233 (−4.6), 255 (−6.6) nm; IR (film) λ max 3464, 2946, 1765, 1737, 1682, 1607, 1439, 1239 cm−1; 1H and 13C NMR data, Table S1; HRESIMS [M + H]+ m/z: 691.4209 (calcd for C42H59O8, 691.4204). Bistrochelide A (3). White amorphous powder; Rf 0.53 (CH2Cl2/ MeOH 9:1); [α]25D −19.2 (c 0.8, MeOH); UV (MeOH) λmax (log ε) 201 (3.77), 244 (3.43) nm; ECD (MeCN, c 4.0 × 10−4) λmax (Δε) 205 (+9.11), 253 (−2.84) nm; IR (film) λmax 3465, 2935, 1959, 1737, 1682, 1606, 1444, 1239 cm−1. 1H and 13C NMR data, Table 1; HRESIMS [M + NH4]+ m/z: 708.4468 (calcd for C42H62NO8, 708.4475). Bistrochelide B (4). White amorphous powder; Rf 0.52 (CH2Cl2/ MeOH 9:1); [α]25D + 26.4 (c 3.0, MeOH); UV (MeOH) λmax (log ε) 204 (4.11), 240 (3.65) nm; ECD (MeOH, c 4.3 × 10−4) λmax (Δε) 207 (+24.1), 233 (−8.9) nm; IR (film) λmax 3419, 2934, 1963, 1736, 1439, 1378, 1240, 1025 cm−1; 1H and 13C NMR data, Table 1; HRESIMS [M + H]+ m/z: 691.4209 (calcd for C42H59O8, 691.4204). Bistrochelide C (5). White amorphous powder; Rf 0.61 (CH2Cl2/ MeOH 9:1); [α]25D + 29.4 (c 1.7, MeOH); UV (MeOH) λmax (log ε) 202 (3.77) nm; ECD (MeOH, c 4.3 × 10−4) λmax (Δε) 205 (+30.3), 231 (−10.7) nm; IR (film) λ max 3451, 2930, 1762, 1735, 1709, 1686, 1453, 1241 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS [M + H]+ m/z: 693.4359 (calcd for C42H61O8, 693.4361). Bistrochelide D (6). White amorphous powder; Rf = 0.67 (CH2Cl2/ MeOH 9:1); [α]25D −8.5 (c 1.1, MeOH); UV (MeOH) λmax (log ε) 201 (3.83) nm; ECD (MeOH, c 2.0 × 10−4) λmax (Δε) 216 (+11.9), 254 (−3.3) nm; IR (film) λmax 3436, 2927, 1759, 1737, 1712, 1687, 1444, 1240 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS [M + NH4]+ m/z: 710.4630 (calcd for C42H64NO8, 710.4626). Bistrochelide E (7). White amorphous powder; Rf 0.40 (CH2Cl2/ MeOH 9:1); [α]25D −18.3 (c 1.4, MeOH); UV (MeOH) λ max (log ε) 202 (3.84) nm; ECD (MeOH, c 5.8 × 10−4) λmax (Δε) 205 (+17.3), 234 (−2.9), 259 (−0.5), 304 (−5.1) nm; IR (film) λmax 3454, 2930, 1766, 1738, 1698, 1454, 1379, 1230 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESIMS [M + Na]+ m/z: 671.3930 (calcd for C40H56NaO7, 671.3918). X-ray Crystallographic Study of 1. A colorless monoclinic crystal of 1 monohydrate (0.29 × 0.21 × 0.13 mm) was obtained by recrystallization from MeCN/H2O:C42H58O8·H2O (Mr = 708.90), monoclinic, space group C2 with a = 18.7168(3) Å, b = 8.9127(2) Å, c = 24.0323(4) Å, β = 103.5320(10)°, V = 3897.71(13) Å3, Z = 4, Dcalcd = 1.208 g/cm3. Intensity data were measured using CuKα radiation (graphite monochromator). A total of 9727 reflections were collected to a maximum 2θ value of 65.99° at 296(2) K. The structure was solved by direct methods (SHELXS-97) and refined using fullmatrix least-squares on F2. All non-hydrogen atoms were given anisotropic thermal parameters; hydrogen atoms were located from difference Fourier maps and refined at idealized positions riding on their parent atoms. The refinement converged at R1 [I > 2σ(I)] = 0.0352, wR2 = 0.0992 for 5187 independent reflections and 460 variables; absolute structure parameter: −0.18(15) (CCDC 1816835 contains the supplementary crystallographic data for this paper. These data are provided free of charge by the Cambridge Crystallographic Data Centre.). Immune Regulation Activity Assay. Splenocytes (2 × 106 cell/ mL) from C57BL/6 mice were incubated with compounds for 24 h using Con A as a positive control (5 μg/mL). The cells were collected and washed with PBS, and then samples were immediately detected by a FACScan flow cytometer (BD, USA) for apoptosis. To investigate the effects of compounds on the differentiation of T cell

subtypes, splenocytes were incubated with compounds for 24 h using Con A as a positive control. The cells were collected and stained with PE-CD3, FITC-CD4, and Percp/cy5.5-CD8, respectively. The percentage of CD3+T, CD4+T, and CD8+T cells was analyzed by flow cytometry.41,42 The values are presented as means ± SD (n = 3). *P < 0.05 and **P < 0.01 versus blank treatment. ECD Calculations. 3D starting conformers of compounds 1−3 and (11S*)-7 were produced using Monte Carlo Multiple Minimum (MCMM), Low Mode Conformational Search (LMCS), and molecular dynamics. The selected geometries were optimized at the quantum mechanical (QM) level by using the MPW1PW91 functional and the 6-31G(d) basis set. QM calculations were performed on the selected conformers at the TDDFT (NStates = 40) MPW1PW91/6-31g(d,p) level. QM calculations were performed using Gaussian software.43−45 The final ECD spectra were computed considering the influence of each conformer on the total Boltzmann distribution taking into account the relative energies and were graphically plotted using SpecDis software.46 Computation of NMR Parameters. 3D starting models of compounds 5−7 were built by Maestro 10.247 and optimized by MacroModel 10.248 with the Optimized Potentials for Liquid Simulations (OPLS) force field49 and the Polak-Ribier conjugate gradient algorithm (PRCG, maximum derivative less than 0.001 kcal/ mol). Conformational search rounds for the compounds were performed using MacroModel 10.247,48 at the empirical molecular mechanics (MM) level. Specifically, MCMM and LMCS methods were first employed in order to explore the conformational space. Furthermore, rounds of molecular dynamics simulations were performed at 450, 600, 700, and 750 K, with a time step of 2.0 fs, an equilibration time of 0.1 ns, and a simulation time of 10 ns. All the produced conformers were collected and analyzed in order to discard the redundant ones. Specifically, the nonredundant conformers were selected by using the “Redundant Conformer Elimination” module of Macromodel 10.247 excluding those differing more than 12.5 kJ/mol (3.0 kcal/mol) from the most energetically favored conformation and setting a 0.1 Å RMSD minimum cutoff for saving structures. The following reported QM calculations were performed using Gaussian 09 software.43 The conformers were geometry optimized at the quantum mechanical (QM) level by using the MPW1PW91 functional and the 6-31G(d) basis set. After this step, the new geometries were visually inspected in order to filter out further possible redundant conformers. Finally, the conformers were used for the subsequent computation of the 13C and 1H NMR chemical shifts, using the MPW1PW91 functional and the 6-31G(d,p) basis set. The final 13C NMR and 1H NMR chemical shift data were computed considering the influence of each conformer on the total Boltzmann distribution and taking into account the relative energies. Calibrations of calculated 13C and 1H chemical shifts were performed following the multistandard approach (MSTD).50,51 The sp2 13C and 1H NMR chemical shifts were computed using 2-methyl-2-butene, 2,3dimethyl-2-butene, and methyl acrylate as reference compounds,50,51 while tetramethylsilane (TMS) was used for computing sp3 13C and 1 H chemical shift data (Tables S2−S7, Supporting Information). Experimental and calculated 13C and 1H NMR chemical shifts were compared computing the Δδ parameter (see Tables S2−S7, Supporting Information): |Δδ| = |δexp − δcalc|, where δexp (ppm) and δcalc (ppm) are the 13C/1H experimental and calculated chemical shifts, respectively. The comparison of the calculated 13C/1H chemical shift values for the diastereoisomeric pairs of 5 and 6 was performed introducing the |Δδcalc| parameter. For two possible isomers of a case-study compound (isomer A and isomer B), each considered atom was defined: |Δδcalc| = |δcalc‑isomerA − δcalc‑isomerB|. The MAEs for all the possible diastereoisomers (Tables 2 and 4 and Tables S2−S7, Supporting Information) were computed: MAE = ∑(| Δδ |) , defined as the summation (Σ) of the n computed absolute n error values (|Δδ|), normalized to the number of chemical shifts considered (n). For two possible isomers of a case-study compound H



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(isomers A and B), the ΔMAE parameter was defined (Results and Discussion): ΔMAE = MAEisomer A − MAEisomer B.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b01037. HRESIMS and NMR spectra for compounds 1−7, crystallographic data for 1, scaled calculated NMR chemical shifts |Δδ|(13C), |Δδcalc| (13C), and MAE values 5−7, with Cartesian coordinates of the optimized geometries for the conformers of 5−7 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Peng Sun: 0000-0002-9508-2904 Gianluigi Lauro: 0000-0001-5065-9717 Huan Huan Li: 0000-0001-7678-9819 Attila Mándi: 0000-0002-7867-7084 Tibor Kurtán: 0000-0002-8831-8499 Raffaele Riccio: 0000-0001-5073-5513 Giuseppe Bifulco: 0000-0002-1788-5170 Wen Zhang: 0000-0002-5747-4413 Author Contributions ‡

P.S., F.-Y.C., and G.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research work was financially supported by the Natural Science Foundation of China (U1405227, 41576157, 81573342, 81622044, 81820108030, 18ZR1449600). The research of the Hungarian authors was supported by the EU and cofinanced by the European Regional Development Fund under Project GINOP-2.3.2-15-2016-00008. G. B. acknowledges the financial support of MIUR Italy PRIN 2017 project (2017A95NCJ).

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