Structurally Diverse Diterpenoids from Sandwithia ... - ACS Publications

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Cite This: J. Nat. Prod. 2018, 81, 901−912

Structurally Diverse Diterpenoids from Sandwithia guyanensis Simon Remy,† Florent Olivon,† Sandy Desrat,† Florent Blanchard,† Véronique Eparvier,† Pieter Leyssen,§ Johan Neyts,§ Fanny Roussi,† David Touboul,† and Marc Litaudon*,† †

Institut de Chimie des Substances Naturelles, CNRS ICSN, UPR 2301, University of Paris Saclay, 91198 Gif-sur-Yvette, France Laboratory for Virology and Experimental Chemotherapy, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium

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S Supporting Information *

ABSTRACT: Bioassay-guided fractionation of an EtOAc extract of the trunk bark of Sandwithia guyanensis, using a chikungunya virus (CHIKV)-cell-based assay, afforded 17 new diterpenoids 1−17 and the known jatrointelones A and C (18 and 19). The new compounds included two tetranorditerpenoids 1 and 2, a trinorditerpenoid 3, euphoractines P-W (4−11), and euphactine G (13) possessing the rare 5/6/7/3 (4−7), 5/6/6/4 (8−11), and 5/6/8 (13) fused ring skeletons, sikkimenoid E (12), and jatrointelones J-M (14−17) possessing jatropholane and lathyrane carbon skeletons, respectively. Jatrointelones J (14) and M (17) represent the first naturally occurring examples of C-15 nonoxidized lathyrane-type diterpenoids. The structures of the new compounds were elucidated by NMR spectroscopic data analysis. The relative configuration of compound 16 and the absolute configurations of compounds 3−6 and 14 were determined by single-crystal X-ray diffraction analysis. In addition, jatrointelone K (15) was chemically transformed to euphoractine T (8) supporting the biosynthetic relationships between the two types of diterpenoids. Only compound 15 showed a moderate anti-CHIKV activity with an EC50 value of 14 μM. Finally, using a molecular networking-based dereplication strategy, several close analogues of 12-O-tetradecanoylphorbol-13-acetate (TPA), one of the most potent inhibitors of CHIKV replication, were dereplicated.

A

compounds were performed via NMR and MS data analysis. The relative configuration of compound 16 and the absolute configurations of compounds 3−6 and 14 were determined by single-crystal X-ray diffraction analysis. The structures of jatrointelones A (18) and C (19) were elucidated through comparison with literature data.3 The 1H and 13C NMR data of new compounds 1−17 are reported in Tables 1−5. The HRESIMS of compound 1 showed a sodium adduct ion at m/z 325.1422 [M + Na]+ (calcd for 325.1410) consistent with a molecular formula, C18H22O4, indicating eight indices of hydrogen deficiency. Analysis of NMR spectroscopic data indicated that 1 was a phenanthrene-type diterpenoid structurally close to domohinone isolated from Domohinea perrieri.4 Its 13C NMR spectrum accounted for 18 carbon resonances, comprising one carbonyl, eight aromatic/olefinic carbons, two methyl and two methoxy groups, an aliphatic CH and two aliphatic CH2, an oxymethine, and a quaternary carbon (Table 1). However, when compared with domohinone, no ketocarbonyl at C-7 was detected, and compound 1 displayed a methoxy group at C-13 (δC 56.1; δH 3.89) instead of a methyl group in domohinone. The complete assignments of the 1H and 13C NMR spectra of 1 were done by COSY, HSQC, and HMBC NMR experiments as shown in Figure 1. The relative

s part of an ongoing program aiming at the discovery of novel inhibitors of chikungunya virus (CHIKV) replication from plants of the Euphorbiaceae family, the species Sandwithia guyanensis Lanj. was selected for in-depth chemical investigation. The EtOAc extract of the trunk bark showed a significant antiviral activity (EC50 = 7.9 μg/mL). S. guyanensis is an endemic species of the Guyana plateau region, belonging to a genus including only two species.1 S. guyanensis has only been chemically investigated once in 1987, leading to the isolation of two sesquiterpenoids, cyperenol and cyperenoic acid, from the root wood and bark.2

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RESULTS AND DISCUSSION The bioassay-guided fractionation of the EtOAc extract of the trunk bark of S. guyanensis led to the isolation of 19 diterpenoids of six different structural types. The EtOAc extract was first suspended in n-heptane and partitioned with MeCN to yield an MeCN soluble extract. This sample was subjected to a normal-phase flash chromatography, yielding 19 fractions (F1F19). Based on their strong anti-CHIKV activity (0.6 < EC50 < 8 μg/mL) fractions F3-F5, F10, F11, and F13-F15, along with fraction F6 (EC50 > 100 μg/mL) were purified using semipreparative and/or analytical RP-HPLC to yield 17 new compounds 1−17: three degraded diterpenoids 1−3; euphoractines P-W (4−11), sikkimenoid E (12), euphactine G (13), and jatrointelones J-M (14−17); and the known jatrointelones A (18) and C (19). The structural elucidations of these © 2018 American Chemical Society and American Society of Pharmacognosy

Received: December 5, 2017 Published: March 1, 2018 901

DOI: 10.1021/acs.jnatprod.7b01025 J. Nat. Prod. 2018, 81, 901−912

Journal of Natural Products

Article

Table 1. 1H NMR Data for Compounds 1−3 (500 MHz, CDCl3) 1

2

3

position

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

1α 1β 2α 2β 3 5 6α 6β 7α 7β 11 14 15 18 19 20 12-OMe 12-OH 13-OMe 3-OH 13-OH

6.64, d (2.6)

6.62, d (2.6)

4.04, 2.59, 2.13, 1.63, 2.88,

4.04, 2.58, 2.10, 1.60, 2.84,

s m m m m

s ddd (13.0, 3.5, 3.5) dtd (13.0, 3.5, 3.5) dtd (13.0, 12.5, 5.2) m

7.22, s 6.65, s

7.22, s 6.73, s

1.31, s 0.79, s

1.29, s 0.78, s

3.92, s

3.91, s

2.46, 1.97, 2.60, 2.72,

ddd ddd ddd ddd

(13.5, (13.5, (16.0, (16.0,

7.5, 4.3) 10.0, 7.5) 7.5, 4.3) 10.0, 7.5)

1.90, 1.84, 1.80, 2.82, 2.97, 6.90, 7.62,

dd (12.0, 2.5) m ddd (12.0, 12.0, 6.0) ddd (16.5, 12.0, 7.0) ddd (16.5, 5.4, 1.5) s s

1.18, s 1.15, s 1.31, s 10.10, s

3.89, s 3.84, br s

3.85, br s 5.96, br s

Table 2. 1H NMR Data for Compounds 4 (500 MHz, MeOD) and 5−7 (500 MHz, CDCl3) 4 position 1α 1β 2α 3 5 7α 7β 8α 8β 9 11 12 16 17 18 19 20

5

δH (J in Hz) 5.14, d (6.0)

6

δH (J in Hz)

δH (J in Hz)

2.59, dq (6.0, 7.4)

2.80, ddd (19.0, 6.4, 3.1) 2.44, ddd (19.0, 3.1, 2.2) 2.60, qdd (7.5, 6.4, 2.2)

4.53, 1.77, 2.04, 1.79, 1.04, 0.82, 0.56, 4.05, 1.14, 0.87, 1.10, 1.13, 1.37,

4.66, 1.73, 2.10, 1.80, 0.99, 0.77, 0.57, 4.01, 1.26, 0.88, 1.12, 1.13, 1.38,

s m dd (14.4, 7.0) m m m dd (9.4, 9.4) d (9.4) d (7.0) s s s s

7

dd (3.1, 3.1) dd (13.5, 12.5) dd (13.5, 7.0) ddd (14.6, 7.4, 7.0) ddd (14.6, 12.5, 11.0) ddd (11.0, 9.5, 7.4) dd (9.5, 9.5) d (9.5) d (7.5) s s s s

2.10, 2.59, 2.15, 4.55, 4.53, 1.71, 1.95, 1.79, 0.99, 0.74, 0.55, 3.94, 1.20, 0.84, 1.10, 1.10, 1.40,

m m m m s dd (14.2, 12.5) dd (14.2, 7.0) ddd (14.6, 7.5, 7.0) ddd (14.6, 12.5, 11.0) ddd (11.0, 9.6, 7.5) dd (9.6, 9.0) d (9.0) d (7.0) s s s s

δH (J in Hz) 4.82, dd (2.4, 2.0) 2.52, qd (7.5, 2.4) 4.70, 1.71, 2.10, 1.81, 0.97, 0.76, 0.53, 4.02, 1.30, 0.87, 1.10, 1.12, 1.37,

br s dd (13.5, 12.5) dd (13.5, 7.0) dd (14.7, 7.5, 7.0) m m dd (9.4, 9.0) d (9.0) d (7.5) s s s s

resonating at δH 1.31 ppm, a ketocarbonyl (δC 216.8) instead of a conjugated ketocarbonyl in compounds 1 and 2. HMBC correlations from H-14 (δH 7.62) and H-11 (δH 6.90) to the carbonyl carbon C-15 on the one hand, and from the deshielded hydroxy proton at δH 10.10 to C-11, C-12, and C13 on the other hand, indicated that the carboxylic acid and hydroxy groups were attached at C-13 and C-12, respectively. Other HMBC correlations allowed the Me-20 and the ketocarbonyl groups to be located at C-10 and C-3, respectively. The structure and (5S, 10R) absolute configuration of compound 3 was confirmed by X-ray diffraction analysis (Figure 1). The HRESIMS of euphoractine P (4) showed a sodium adduct ion at m/z 371.1840 [M + Na]+ (calcd 349.1829) corresponding to the molecular formula, C20H28O5, indicating

positions of the substituents on the phenanthrene nucleus were determined by NOESY correlations (Figure 1). The HRESIMS data of compound 2 showed a protonated molecular ion at m/z 289.1440 [M + H]+ (calcd 289.1434) corresponding to the molecular formula, C17H20O4. From this formula and its spectroscopic data closely comparable to those of compound 1, it is apparent that compound 2 had a structure similar to 1 but with a hydroxy group attached at C-13 instead of a methoxy group for 1. The molecular formula, C18H22O4, of compound 3 was established by HRESIMS from the protonated molecular ion [M + H]+ at m/z 303.1601 (calcd 303.1591). 1H and 13C NMR data of compound 3 were comparable to those of compounds 1 and 2, but revealed the presence of a carboxylic acid function (δC 173.1), an additional singlet for a third methyl group 902



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Table 3. 1H NMR Data for Compounds 8−11 (500 MHz, CDCl3) position 1α 1β 2 3 5 7α 7β 8α 8β 9 11 12 16 17 18 19 20 5-OAc

8

9

10

11

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

4.76, dd (2.0)

5.20, br d (6.0)

2.54, qd (7.5, 2.0)

2.74, qd (7.5, 6.0)

4.78, 1.57, 1.88, 1.42, 1.54, 1.13, 3.52, 2.09, 1.31, 0.99, 1.11, 1.08, 1.22,

4.75, 1.58, 1.88, 1.42, 1.54, 1.13, 3.52, 2.09, 1.23, 1.00, 1.12, 1.08, 1.19,

br s m m m m m d (8.5) dd (13.0, 8.5) d (7.5) s s s s

br s m m m m m d (8.5) dd (13.0, 8.5) d (7.5) s s s s

2.66, 2.03, 2.17, 4.61, 4.64, 1.55, 1.72, 1.43, 1.51, 1.10, 3.48, 1.97, 1.22, 0.95, 1.10, 1.06, 1.20,

tdd (15.9, 7.4, 2.0) tdd (15.9, 7.4, 2.6) sept (7.4) m br s m brd (13.0) qd (13.0, 4.0) m m d (8.0) dd (13.0, 8.5) d (7.0) s s s s

4.75, dd (2.5, 2.5) 2.48, qd (7.5, 2.5) 6.05, 1.49, 1.59, 1.52, 1.39, 1.14, 3.54, 2.06, 1.28, 1.03, 1.11, 1.07, 1.31, 1.03, 2.17,

br s m m m m m d (8.3) dd (13.0, 8.3) d (7.0) s s s s s s

Table 4. 1H NMR Data for Compounds 12 and 13 (500 MHz, CDCl3) 12 position

δH (J in Hz)

1 2 5 7α 7β 8α 8β 9 11 12α 13 16 17 18 19 20

4.86, d (2.5) 2.45, m 3.24 br d (9.0) 2.54, dd (12.5, 5.5) 2.26, dd (13.0, 12.5) 2.21, m 0.93, m 0.88, m 0.56, dd (9.8, 9.2) 1.41, dd (19.2, 12.5) 2.44 m 1.28, d (7.8) 4.71, d (9.0), 4.04, d (14.4) 1.11, s 0.92, s 1.15, d (6.7)

13 δH (J in Hz) 4.85, 2.57, 4.58, 2.08, 1.19, 1.49, 1.43, 3.34, 5.48, 5.83,

d (2.5) qd (7.5, 2.5) br s dd (15.8, 11.3) dd (15.8, 7.0) m m dd (10.5, 2.5) d (17.0) d (17.0)

1.33, 0.93, 1.26, 1.05, 1.30,

d (7.5) s s s s

Figure 1. Key COSY (bold), HMBC (black arrows), and NOESY (red arrows) correlations of compounds 1 and 2. ORTEP view of compound 3 (bottom).

The HRESIMS of euphoractine Q (5) showed a sodium adduct ion at m/z 355.1886 [M + Na]+ (calcd 355.1880) corresponding to the molecular formula, C20H28O4, indicating one oxygen atom less than the molecular formula of 4. Its 1D and 2D NMR spectroscopic data were similar to those of compound 4, but revealing a methylene group (δC 31.8, δH 2.44, 2.80, 1-CH2) instead of an oxymethine for compound 4. The relative configuration of C-2 was established on the basis of strong NOESY correlations between H-1β at δH 2.44 and Me-16 and Me-17 groups, all oriented above the mean plane of the molecule. The structure and absolute configuration of 5 was confirmed by X-ray diffraction analysis (Figure 2). The HRESIMS of euphoractine R (6) showed a sodium adduct ion at m/z 357.2045 [M + Na]+ (calcd 357.2036) corresponding to the molecular formula, C20H30O4, indicating two more H atoms than 5. Its IR spectrum showed characteristic absorption bands for a carbonyl function (1645 cm−1) and free hydroxy groups (3345 cm−1). When compared

seven indices of hydrogen deficiency. Its 1H and 13C NMR spectra revealed the presence of 20 carbons: five methyl groups, two conjugated ketocarbonyls, five quaternary carbons (two olefinic), six methines (three oxygenated), and two methylene groups. These NMR data supported a tetracyclic structure similar to euphoractine B,5 but with the presence of two conjugated ketocarbonyls (δC 210.1 and 210.9), and the absence of a cinnamoyl moiety. Two spin systems involving protons H-1 and H-2, and from H-7 to H-12 were identified by analysis of 1H−1H correlations observed in the COSY spectrum. The 2D structure of compound 4 was deduced from HMBC correlations and its relative configuration from NOESY correlations. The structure and absolute configuration of 4 was confirmed by X-ray diffraction analysis (Figure 2). It should be noted that, in contrast to euphoractine B, the six- and the seven-membered rings of euphoractine P (4) are transfused. 903



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sodium adduct ion at m/z 371.1841 [M + Na]+ (calcd 371.1829) in the HRESIMS data, corresponding to seven indices of hydrogen deficiency. Comparison of the NMR data of compound 8 with those of compound 7 (Tables 2 and 3) revealed that they only differ in the eastern zone of the structure. From the COSY spectrum of 8, two spin systems were assigned from H-1 to H3-16, and from H2-7 to H-12 as depicted in Figure 3. These features together with HMBC

Figure 3. Key COSY (bold), HMBC (black arrows), and NOESY (red arrows) correlations of compound 8.

correlations from H3-18 and H3-19 to C-9, C-10, and C-11 revealed that the structure of compound 8 possessed a 5/6/6/4 fused-ring system similar to that of euphoractine A, a known diterpenoid from Euphorbia micractina.5 The relative configuration of compound 8 was established by NOESY experiment. Cross-peaks between H3-16/H-1, H-1/H3-17, H3-17/H-12, H12/H3-19, indicated the β-orientation of these protons, and αorientation of HO-1. Correlations between H3-18/H-9/H-11/ H-20/H-5 indicated the β-orientation of HO-5 and HO-11 while H-9 and H3-20 were α-oriented. Thus, the structure of euphoractine T (8) was established as shown. The molecular formula, C20H28O5, of euphoractine U (9) was established by HRESIMS data from the sodium adduct ion [M + Na] + at m/z 371.1854 (calcd 371.1829 for C20H28O5Na+). The spectroscopic data showed that 9 was the 2-epimer of 8, with an α-oriented 16-methyl group. The C(1)H/C-(2)H cis-configuration was supported both by a H-1/ H-2 NOESY cross-peak and a larger vicinal coupling constant (3JH‑1/H‑2 = 6.0 Hz) as compared with 8. Additionally, as is the case for compounds 4−6, NOESY cross peak between H-1 and H3-17 suggested their β-orientation, allowing the relative configuration of euphoractine U (9) to be determined as shown in Chart 1. The HRESIMS data of euphoractine V (10) showed a sodium adduct ion [M + Na]+ at m/z 357.2061 (calcd 357.2036), corresponding to the molecular formula, C20H30O4. The analysis of its NMR data revealed a structure comparable to those of compounds 8 and 9 but with a hydroxy group at C3 instead of a carbonyl. The C-(2)H/C-(3)H trans-configuration was supported by analysis of the coupling constants of the protons of the H-1/H-2, H-2/(H3-16)H-3 spin system (J values are indicated in Table 3) and a strong NOESY correlation between H-3 and H3-16. Euphoractine W (11) showed a sodium adduct ion [M + Na]+ at m/z 413.1944 (calcd 413.1935) in the HRESIMS data, corresponding to the molecular formula, C22H30O6Na. The NMR spectroscopic data of compound 11, comparable to those of 8, indicated that it was an acetylated analogue (δC 20.8 and 170.9, δH 2.17) of 8. The location of the acetoxy group at C-5

Figure 2. Key COSY (bold), HMBC (black arrows), and NOESY (red arrows) correlations of compound 7. ORTEP view of the X-ray structures of compounds 4−6.

with 5, the loss of one index of hydrogen deficiency combined with the presence of an additional oxymethine (δC 83.1, δH 4.55) observed in the 13C and HSQC NMR spectra of 6, suggested that one ketocarbonyl function was replaced by a hydroxy group. The HMBC correlation from H3-16 to the oxymethine at δC 83.1 confirmed the substitution of the pentacyclic ring by a hydroxy group at C-3. The structure and absolute configuration of 6 was confirmed by X-ray diffraction analysis (Figure 2). The molecular formula, C 20 H 28 O 5 , was assigned to euphoractine S (7) on the basis of HRESIMS data from the sodium adduct ion [M + Na]+ at m/z 371.1837 (calcd for C20H28O5Na, 371.1829), identical to that of compound 4. 1H and 13C NMR spectroscopic data of compound 7 were closely similar to those of compound 4, but revealed a different coupling constant between H-1 and H-2. Indeed, in compound 7, the C-(1)H/C-(2)H trans-configuration was indicated by the 3 J1,2 coupling constant 2.6 Hz, similar to those reported for lagaspholone A (3J1a/2 = 6.5 Hz, 3J1b/2 = 2.0 Hz) and curcusone A (3J1a/2 = 6.8 Hz, 3J1b/2 = 2.3 Hz).6,7 Additionally, a strong NOESY correlation between H-1 and H3-16 indicated that these protons are cofacial. Key COSY, NOESY, and HMBC correlations are shown in Figure 2. Thus, compound 7, which was assigned a (2S) absolute configuration and is the 2-epimer of euphoractine P (4). The molecular formula of euphoractine T (8) was established as C20H28O5, the same as compound 7, from the 904



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Chart 1. Structures of Compounds 1−19

reported euphactines, neither benzoyl nor cinnamoyl ester moieties were identified. Instead, compound 13 possessed three hydroxy groups at C-1, C-5, and C-9, as indicated by the chemical shifts of the three methines CH-1, CH-5, and CH-9 (δC/δH 73.7/4.85, 70.5/4.58, and 84.6/3.34, respectively). In addition, the 1H and 13C NMR spectra of 13 revealed the presence of two conjugated carbonyls (δC 207.8 and 202.8, C-3 and C-14, respectively) and a trans-disubstituted double bond (δH 5.48 and 5.83, each d, J = 17.0 Hz, C-11C-12). Key COSY, NOESY, and HMBC correlations are shown in Figure 4. The NOESY cross-peaks between H3-16/H-1/H2-17/H-12/ H3-19, arbitrarily β-oriented, indicated the coplanarity of these protons, while correlations between H3-20 and H-11, and, H-9 and H3-18 indicated their α-orientation (Figure 4). It should be noted that in contrast to euphactines A−F,9,10 the six- and eight-membered rings of 13 are trans-fused. Thus, compound 13, named euphactine G, was assigned the structure shown in Chart 1. The HRESIMS data of compound 14 (jatrointelone J) showed a sodium adduct ion [M + Na]+ at m/z 341.2096 (calcd 341.2087), corresponding to the molecular formula C20H30O3. The NMR spectroscopic data of compound 14, similar to those of curculathyrane A and jatrointelone A (18) isolated from Jatropha curcas and J. integerrima, respectively,3,7 revealed that 14 possessed a lathyrane-type skeleton. However, when compared with curculathyrane A, no Δ4(5) double bond nor hydroxy group at C-15 were evident in the NMR spectra of compound 14. The 2D structure of compound 14 was supported by two spin systems observed in the COSY spectrum, from H-1 to H-5 and from H-7 to H3-20 on the one hand, and HMBC correlations from H2-5/H3-17/H2-7 to the oxygenated tertiary carbon C-6 (δC 71.9), from H3-17 to C5 and C-7, from H3-20 to C-12, C-13, and the carbonyl carbon C-14, and from H-15 (δH 3.83) to C-1, C-2, and C-14, on the other hand. NOESY cross-peaks between H-15 (δH 3.83) and H-5b, H-7, H-8b, H-12b, and H-13, indicated the β-orientation of these protons, while cross peaks between H-4 (δH 3.10) and H-7a indicated their α-orientation. The structure and absolute configuration of compound 14 were confirmed by X-ray diffraction analysis (Figure 5). Jatrointelone J (14) is the first

was deduced from the HMBC correlation of H-5 to the ester carbonyl carbon. The relative configuration of compound 11, similar to compound 8, was deduced from NOESY correlations. The HRESIMS data of sikkimenoid E (12) showed a protonated molecular ion [M+H] + at m/z 337.1777, corresponding to the molecular formula, C20H26O3, indicating eight indices of hydrogen deficiency. Its NMR data were closely comparable to those of sikkimenoid A isolated from Euphorbia sikkimensis,8 and possessing a jatropholane-type skeleton. Analysis of 1D and 2D NMR spectroscopic data confirmed that sikkimenoid E (12) was the 1-hydroxylated analogue of sikkimenoid A.8 Key COSY, NOESY, and HMBC correlations are shown in Figure 4. Its relative configuration was elucidated

Figure 4. Key COSY (bold), HMBC (black arrows), and NOESY (red arrows) of compounds 12 and 13.

by analysis of NOESY correlation and comparison of chemical shifts and coupling constant values with those of sikkimenoid A.8 NOESY correlations between H-1 and H3-16 and between H-2 and H-5, indicated that the 2-methyl and 1-OH groups were β- and α-oriented, respectively. Compound 13 showed a sodium adduct ion at m/z 371.1832 for [M + Na]+ (calcd for 371.1829) in its HRESIMS data consistent with the molecular formula C20H28O5, the same as compounds 4, 8, and 9. Its NMR spectroscopic data were found to be closely comparable to those of euphactines A−D isolated from E. micractina,9 possessing a 5/6/8 fused-ring skeleton. However, in contrast with the structure of the 905



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example of a naturally occurring C-15 nonoxidized lathyranetype diterpenoid.

Figure 5. ORTEP view of the X-ray structure of compound 14.

The HRESIMS data of jatrointelone K (15), showed a protonated molecular ion [M+H]+ at m/z 331.1933 (calcd for C20H27O3, 331.1904), which in conjunction with the 13C NMR spectroscopic data was consistent with a molecular formula of C20H26O4, indicating eight indices of hydrogen deficiency. Its 1D and 2D NMR data were comparable to those of jatrointelone C (19), isolated from J. integerrima,3 but with the presence of a carbonyl carbon at δC 205.8 (C-3) instead of a hydroxy group in jatrointelone C (19). These data showed similar features with those of 7, 8, 12, and 13, implying the presence of a cyclopentenone moiety hydroxylated at C-1 in 15. These observations were confirmed by HMBC correlations from H3-16 to the oxymethine at δC 75.9 (C-1) and C-3, and from H-1 to C-4, C-14, and C-15. The relative configuration of compound 15 was established from the comparison of NMR data with those of jatrointelone C and NOESY analysis (Figure 6).

Figure 7. Key COSY (bold), HMBC (black arrows), NOESY (red arrows), and ORTEP view of compounds 16.

357.2036) corresponding to the molecular formula, C20H30O4. The NMR spectroscopic data analysis of 17 (Tables 5 and 6) revealed the presence of a lathyrane scaffold similar to those of compounds 14 and 18 characterized by the absence of a Δ4(15) double bond. The spin system from H3-16 to H2-5 (Figure 8) identified from the COSY spectrum, combined with the 3J4/15 coupling constant of 13 Hz provided strong evidence that compound 17 bore a saturated trans-fused cyclopentanone unit. The relative configuration of 17 was determined by a NOESY experiment. However, since the resonances of H-2 and H-4 (δH 2.34 and 2.32, respectively) were overlapped in the 1H NMR spectrum recorded in CDCl3, and in order to remove any ambiguity about the relative configuration of compound 17, additional NMR spectra were recorded in benzene-d6 on a Bruker Avance 600 MHz (Figures S88 to S93, Supporting Information). In the NOESY spectrum, cross-peaks between H3-16/H-1, H-1/H-4, H-4/H-5a, and H-5a/H3-17, indicated that these protons are cofacial, arbitrarily β-oriented, while correlations between H-2/H-15, H-15/H3-20, H3-20/H-11, H11/H-9 and H-9/H3-18, indicated their α-orientation (Figure S93, Supporting Information). It should be noted that the trans configuration of the C4/C15 ring junction of compound 17 is opposite to that of compound 14. Jatrointelone M (17) is the second example of a naturally occurring C-15 nonoxidized lathyrane-type diterpenoid. From a biosynthetic point of view, compounds 1−3 are usually considered as degraded diterpenoids that could be derived from an abietane-type skeleton.11−16 Euphoractanes (4−11) could arise from transannular cyclization of lathyrane derivatives as depicted in Figure 9.17 This hypothesis was supported by the conversion of the lathyrane 15 into euphoractine T (8) in the presence of Lewis acid. Activation of the C-14 carbonyl group by BF3·OEt2 induces the Δ12(13) double bond shift and of the cyclopropane rearrangement into

Figure 6. Key NOESY correlations of 15.

The molecular formula of jatrointelone L (16), was established as C20H28O4 from the protonated molecular ion [M+H]+ observed in the HRESIMS spectrum (m/z 333.2069 calcd for 331.2060), indicating seven indices of hydrogen deficiency, one less than jatrointelone K (15). Its NMR spectroscopic data were comparable to those of curculathyrane B,7 indicating that they shared the same 5,6-oxiranyl tricyclic carbon skeleton with a saturated C-12−C-13 bond. This observation was confirmed by HMBC correlations from H3-20 to two sp3 carbons resonating at δC 26.7 and 44.2 (C-12 and C13, respectively). The relative configuration of compound 16 was established from NOESY correlations and confirmed by Xray analysis (Figure 7). The HRESIMS data of jatrointelone M (17) showed a sodium adduct ion [M + Na]+ at m/z 357.2040 (calcd 906



Article

Table 5. 1H NMR Data for Compounds 14−17 (500 MHz, CDCl3) position 1 2 4 5a 5b 7a 7b 8a 8b 9 11 12a 12b 13 15 16 17 18 19 20 1-OH

14

15

16

17

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

7.07, m 3.10, 2.09, 1.52, 1.42,

ddd (7.5, 2.5, 2.0) dd (15.0, 2.0) dd (15.0, 7.5) m (2H)

1.42, m 0.49, d (11.0) 0.28, dd (9.5, 9.5) 0.59, dd (11.0, 9.5) 1.99, ddd (15.1, 5.6, 1.6) 1.71, ddd (15.1, 12.0, 3.0) 3.14, m 3.83 td (7.5, 2.5, 2.5) 1.78, br s 1.21, s 0.99, s 0.83, s 1.22, d (7.0)

4.69, d (1.5) 2.46, qd (7.5, 1.5)

4.58, d (2.0) 2.46, ddd (14.8, 7.5, 2.0)

3.64, s

3.77, s

1.14, 2.35, 2.19, 1.30, 1.43, 1.65, 6.04,

m dd (13.0, 12.0) m m m dd (11.0, 8.0) d (11.0)

1.31, 2.36, 1.82, 0.98, 0.15, 0.53, 2.08, 1.16, 3.18,

dd (13.5, 13.5) ddd (13.5, 6.0, 1.0) ddd (15.0, 6.0, 1.0) m dd (9.0, 9.0) ddd (10.0, 9.0, 4.8) ddd (15.0, 10.0, 4.5) m m

1.21, 1.26, 1.22, 1.11, 2.02, 4.61,

d (7.5) s s s s br s

1.24, 1.27, 0.99, 0.87, 1.13,

d (7.5) s s s d (7.0)

4.06, 2.34, 2.32, 2.08, 1.62, 1.43, 1.43, 1.58, 0.61, 0.99, 1.35, 5.54,

dd (9.4, 9.4) m d (13.0) dd (10.0, 1.9) dd (15.6, 5.5) m m m ddd (13.0, 12.5, 11.0) ddd (12.5, 8.6, 3.4) dd (9.7, 9.3) br d (9.7)

3.59, 1.20, 1.16, 1.11, 1.16, 2.07,

dd (13.0, 9.4) d (7.0) s s s s

be isolated due to concentrations below the limit of detection (0.1 mg) of the ELS detector. In summary, 19 diterpenoids of six different structural types were isolated from the trunk bark extract of S. guyanensis. Among the new compounds (1−17), euphoractins P-W (4− 11) and euphactin G (13) possess the rare 5/6/7/3 (4−7), 5/ 6/6/4 (8−11), and 5/6/8 (13) fused ring skeletons, and sikkimenoid E (12) is only the fifth jatropholane analogue described in nature. Macrocyclic diterpenoids possessing such carbon skeletons were only reported in four Euphorbia species,5,9,10,26,28−30 while jatropholanes, also characterized by a 5/6/7/3 fused ring system, were only isolated in a few Euphorbia and Jatropha species.3,6,8,26,30−32 Lathyrane-type diterpenoids with a 5/11/3-membered ring are more common.33,34 However, it should be noted that all known lathyrane-type diterpenoids are oxidized at C-15, which is not the case of jatrointelone J (14) and jatrointelone M (17). These compounds represent the first examples of naturally occurring C-15 nonoxidized lathyrane-type diterpenoids. Only jatrointelone K (15) exhibited a weak antiviral activity against CHIKV (EC50 = 14 μM, SI = 1.92). The strong antiCHIKV activity of the extract could be explained by the presence of putative bioactive phorbol derivatives in trace quantities. This study exemplifies how the use of tandem mass spectrometry combined with molecular networking analysis can help in explaining unsuccessful bioguided isolation procedure by identifying putative bioactive candidates present at extremely low concentrations in complex mixtures.

a cyclobutane moiety. Enolate addition to the oxirane moiety in aqueous conditions afforded compound 8 with an estimated yield of 80% (based on 1H NMR peak integration, Figure S77, Supporting Information). S. guyanensis was selected for the potent anti-CHIKV activity (EC50 = 7.9 μg/mL) of its bark extract, but only compound 15 (EC50 = 14 μM, SI = 1.92) showed a weak antiviral activity against CHIKV. However, at least two fractions (F14 and F15) exhibited potent anti-CHIKV activities (EC50 < 0.6 μg/mL). In order to identify compounds responsible for the strong antiCHIKV activities initially found in extract and fractions F14 and F15, a molecular network (MN) was built from the LC-MS2 data acquired from the chromatographic fractions F3−F16 (Figure 10A).18 Following the published procedure,19 the relative quantification of the ions detected in each fraction was represented as pie chart diagrams, whose proportions were based on respective areas of the corresponding extracted ion chromatographam area (XIC). The observation of a cluster of MS2 spectra with characteristic acyl loss and diterpenoid backbone fragment ions at m/z 311, 293, and 265, supported that these ions were phorbol derivatives.20 Examination of the MS2 spectra of nodes C, D, E, and F of this cluster showed that these compounds share the same MS2 fragmentation pattern, including neutral losses of various acyl long chains (Figure 10C−F). Moreover when searching for analogues in the GNPS database, the MS2 spectrum of the ion at m/z 595.436 matched with the one of 12-O-tetradecanoylphorbol-13-acetate (TPA), one of the most powerful anti-CHIKV compound yet discovered (EC50 ≈ 3 nM, SI near 2000).21 It is now wellknown that several phorbols and deoxyphorbols possessing long-chain ester moieties located at C-12 or C-13 on the phorbol diterpenoid core were potent inhibitors of CHIKV virus replication.21−27 Because ions displayed in nodes C−F were mainly found in fractions F14 and F15 (Figure 10B), it can be deduced that phorbol derivatives must be responsible for the anti-CHIKV activities initially found in the extract and fractions. Despite significant effort, no phorbol analogue could

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured at 25 °C on an MCP 300 Anton Paar polarimeter at 24 °C, with MeOH used as solvent. UV spectra were recorded on a Varian Cary 100 UV−vis spectrophotometer and measured in a 1 cm quartz cell. NMR spectra were recorded in methanol-d4, CDCl3 or benzene-d6 on Bruker 500 and 600 MHz instruments (Avance 500 and Avance 600). Chemical shifts (relative to TMS) are in ppm, and coupling constants are in Hz. Kromasil analytical and semi preparative 907


908

56.1 56.1

56.1

24.9 13.4

24.9 13.4

12-OMe 13-OMe

115.1 199.4 80.6 43.0 48.0 23.1 30.1 135.8 123.4 148.8 107.1 145.7 157.6 114.8

115.4 199.4 80.6 43.0 47.9 23.2 30.4 135.0 123.7 148.1 107.7 152.0 157.2 111.4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 5-OAc

2

1

position

26.9 21.4 24.5

37.3 34.6 216.8 47.6 50.2 20.2 29.8 126.6 157.5 38.2 114.2 160.1 109.6 131.2 173.1

3 67.4 48.2 210.9 158.2 70.8 51.9 34.0 20.3 28.2 21.0 31.1 71.0 61.0 210.1 149.8 9.1 14.7 29.1 15.4 12.0

4 31.8 41.7 214.1 158 70.9 50.7 32.9 19.6 27.1 20.2 30.2 69.6 59.9 209.3 146 16.2 14.4 28.9 15.4 12.2

5 34.6 41.9 83.1 159.4 71.8 51.2 33.3 19.7 26.9 20.1 30.3 70.2 58.2 209.1 135.4 17.9 14.2 28.9 15.3 13.0

6 72.8 51.1 209.5 156.3 70.6 50.9 32.7 19.4 26.9 20.1 29.7 69.6 59.9 209.5 146.0 13.0 14.2 28.7 15.3 12.0

7 73.4 51.3 210.4 155.7 71.0 48.5 30.5 28.4 37.2 44.1 73.6 45.2 54.5 205.8 147.8 12.9 14.4 28.4 15.3 14.1

8 66.9 46.5 212.2 156.6 70.8 48.2 30.7 21.3 37.3 44.2 73.8 45.4 54.6 205.4 148.4 10.0 14.4 28.4 15.3 14.1

9 34.4 42.3 83.7 160.8 72.3 48.8 30.8 21.6 37.1 44.1 73.7 45.4 53.3 204.5 134.7 17.9 14.4 28.5 15.3 14.8

10 73.2 45.1 204.8 156.7 70.8 47.8 30.6 21.3 37.1 44.2 73.7 45.1 54.9 205.2 145.1 12.2 15.6 28.4 15.3 14.3 170.9 20.8

11 74.2 50.4 207.5 153.7 45.8 153.6 40.2 27.2 26.7 19.7 35.5 47.3 49.4 202.3 157.7 14.3 110.5 28.6 17.0 11.2

12 73.7 51.4 207.8 156.3 70.5 59.4 28.0 29.9 84.6 44.7 141.6 125 53.3 202.8 147.5 12.9 0.93 1.26 1.05 1.3

13 152.1 141.3 210.6 41.6 41.7 71.9 39.8 18.7 26.7 16.1 19.2 27.5 44.2 209.1 60.5 10.4 28.5 28.8 15.0 13.4

14 75.9 48.9 205.8 159.2 60.1 62.8 36.3 18.3 36.6 26.8 28.7 146.7 137.3 195.2 139.1 14.2 16.3 28.3 15.7 11.8

15 77.9 50.7 204.8 162.1 50.2 61.1 40.6 19.6 28.5 18.3 23.4 26.7 44.2 210.9 137.6 14.4 18.6 28.9 15.2 17.8

16

Table 6. 13C NMR Data for Compounds 1, 5, 9, 11−14, 17 (125 MHz, CDCl3), 4 (125 MHz, Methanol-d4), 2, 3, 6−8, 10, 15, 16 (75 MHz, CDCl3) δC in ppm 78.3 51.1 216.7 54.1 31.1 73.0 38.5 21.4 30.9 22.3 25.1 131.5 144 208.0 54.6 12.1 32.2 28.6 15.3 13.0

17

Journal of Natural Products Article



Article

0.1% formic acid, 52:48, in 30 min, 4.7 mL/min) to afford compounds 15 (9.6 mg), 16 (15.0 mg), and 14 (1.8 mg) (9.53, 12.76 and 17.13 min, respectively). F10 (30.9 mg) was subjected to semipreparative HPLC (Kromasil C18, MeCN:H2O + 0.1% formic acid, 35:65, in 15 min, 4.7 mL/min) to afford compound 4 (2.7 mg) (tR = 11.06 min). F11 (40.6 mg) was subjected to semipreparative HPLC (Kromasil C18, MeCN:H2O + 0.1% formic acid, 35:65, in 30 min, 4.7 mL/min) to afford compounds 7 (9.9 mg) and 17 (2.8 mg) (tR = 12.46 and 24.04 min, respectively). F13 (96.4 mg) was subjected to semipreparative HPLC (Kromasil C18, MeCN:H2O + 0.1% formic acid, 35:65, in 30 min, 4.7 mL/min) to afford compound 6 (21.0 mg). F14 (69.9 mg) was subjected to semipreparative HPLC (Kromasil C18, MeCN:H2O + 0.1% formic acid, 25:75 to 45:55, in 30 min, 4.7 mL/min) to afford compounds 9 (2.7 mg), 10 (7.0 mg), and 13 (1.5 mg) (tR = 12.18, 14.11, and 7.49 min, respectively) and F14-1. F14-1 was subjected to analytical HPLC (Kromasil C18, (MeCN:MeOH, 1:1)-H2O + 0.1% formic acid, 55:65, 1.0 mL/min) to afford compound 11 (2.5 mg, tR = 8.52 min). F15 (45.1 mg) was subjected to semipreparative HPLC (Kromasil C18, MeCN:H2O + 0.1% formic acid, 25:75, in 30 min, 4.7 mL/min) to afford compound 8 (6.5 mg, tR = 15.90 min). Compound 1. Amorphous yellowish solid; [α]20 D + 95 (c 0.1, MeOH); UV (CH2Cl2) λmax (log ε) 244 (3.65), 312 (3.83), 351 (3.17) nm; 1H and 13C NMR, see Table 1; HRESIMS m/z 325.1422 [M + Na]+ (calcd 325.1410). Compound 2. Amorphous yellowish solid; [α]20 D + 18 (c 0.1, MeOH); UV (CH2Cl2) λmax (log ε) 244 (3.15), 310 (3.72), 348 (3.74), nm; 1H and 13C NMR, see Table 1; HRESIMS m/z 289.1440 [M+H]+ (calcd 289.1434). Compound 3. Yellowish crystal; [α]20 D + 29 (c 0.1, MeOH); UV (CH2Cl2) λmax (log ε) 237 (3.17), 264 (3.74), 339 (3.76) nm; 1H and 13C NMR, see Table 1; HRESIMS m/z 303.1604 [M + H]+ (calcd 303.1591). X-ray Crystallographic Data of (3) (M = 320.37 g/mol). Monoclinic, space group P21 (no. 4), a = 8.8178(4) Å, b = 7.4089(3) Å, c = 12.7560(9) Å, β = 93.429(7)°, V = 831.86(8) Å3, Z = 2, T = 233 K, μ(CuKα) = 0.758 mm−1, Dcalc = 1.279 g/cm3, 8031 reflections measured (6.942° ≤ 2Θ ≤ 144.722°), 3009 unique (Rint = 0.0536, Rsigma = 0.0703) which were used in all calculations. The final R1 was 0.0433 (I > 2σ(I)) and wR2 was 0.1260 (all data). The absolute configuration of this compound was characterized as (5S, 10R), with an absolute structure parameter, Flack x = −0.17(15) (584 quotients). Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre (deposit no. CCDC 1519648). Copies of the data can be obtained, free of charge, from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/ cif. Euphoractine P (4). White crystals; [α]20 D + 46 (c 0.2, MeOH); UV (CH2Cl2) λmax (log ε) 244 (3.84) nm; 1H and 13C NMR, see Table 3; HRESIMS m/z 371.1840 [M + Na]+ (calcd 371.1829). X-ray Crystallographic Data of (4) (M = 348.42 g/mol). Orthorhombic, space group P212121 (no. 19), a = 6.1491(2) Å, b = 13.1124(4) Å, c = 22.3293(16) Å, V = 1800.40(15) Å3, Z = 4, T = 293 K, μ(CuKα) = 0.741 mm−1, Dcalc = 1.285 g/cm3, 7510 reflections measured (7.82° ≤ 2Θ ≤ 144.11°), 3279 unique (Rint = 0.0432, Rsigma = 0.0748) which were used in all calculations. The final R1 was 0.0608 (I > 2σ(I)) and wR2 was 0.1879 (all data). The absolute configuration of this compound was characterized as (1S, 2R, 5R, 6S, 9S, 11R, 12R,

Figure 8. Key COSY (bold), HMBC (black arrows), and NOESY (red arrows) for compound 17. C18 columns (250 × 4.6 mm and 250 × 10 mm i.d ; 5 μm Thermo Electron) and a Nucleodur PFP analytical column (250 × 4.6 mm i.d ; 5 μm Macherey Nagel) were used for HPLC separations using a Waters autopurification system equipped with a binary pump (Waters 2525), a UV−vis diode array detector (190−600 nm, Waters 2996), and a PL-ELS 1000 ELSD Polymer Laboratory detector. Prepacked GraceResolv silica cartridges were used for flash chromatography using a Teledyne Isco Combiflash Rf 200i. All solvents were purchased from Carlo Erba (France), and analytical plates (Si gel 60 F254) were from Merck (France). Methanol (HPLC grade) was purchased from J. T. Baker and 2-propanol (HPLC grade) from Fisher Scientific (Illkirch, France). Formic acid (purity 98%) was purchased from Fluka (Buchs, Switzerland). Plant Material. Bark of Sandwithia guyanensis was collected by one of us (V. Eparvier) in June 2010 in Saint Elie (French Guyana) and authenticated by Dr. Marie-Françoise Prévost (IRD of French Guyana). A voucher specimen (CAY-3589) has been deposited at IRD Herbarium of French Guyana. Extraction and Isolation. The dried bark (704.2 g) of S. guyanensis were extracted with EtOAc (3 × 500 mL) to give a crude extract (2.2 g) after concentration in vacuo at 40 °C. This extract was dissolved in MeCN (250 mL) and subjected to a liquid/liquid partition using n-heptane (3 × 250 mL) to afford 1.1 g of an MeCN soluble fraction. This residue was subjected to silica gel column chromatography using a gradient of n-heptane/EtOAc/MeOH of increasing polarity (1:0:0 to 0:1:0 to 0:7:3, 40 mL/min) to afford 19 fractions, F1 to F19, according to their TLC profiles. Purification of fraction F3 (55.2 mg) by semipreparative HPLC (Kromasil C18, MeCN:H2O + 0.1% formic acid, 52:48, in 30 min, 4.7 mL/min) afforded five fractions (F3-1 to F3-6). F3-2 was subjected to analytical HPLC (Nucleodur PFP, MeCN:H2O (45:55) + 0.1% formic acid at 1 mL/min), leading to compound 5 (1.5 mg) and compound 3 (1.8 mg) (tR = 9.01 and 12.07 min respectively). F3−3 was subjected to analytical HPLC (Nucleodur PFP, MeCN:H2O (45:55) + 0.1% formic acid at 1 mL/min), to afford compound 19 (3.2 mg) (tR = 12.94 min). F3−5 was subjected to analytical HPLC (Nucleodur PFP, MeCN:H2O (70:30) + 0.1% formic acid at 1 mL/min), leading to compound 12 (0.5 mg) (tR = 6.39 min). F4 (36.1 mg) was subjected to semipreparative HPLC (Kromasil C18, MeCN:H2O + 0.1% formic acid, 52:48, in 30 min, 4.7 mL/min) to afford compound 1 (1.5 mg) (tR = 9.46 min) and compound 18 (1.7 mg) (tR = 11.52 min). F5 (54.2 mg) was subjected to semipreparative HPLC (Kromasil C18, MeCN:H2O + 0.1% formic acid, 40:60, in 30 min, 4.7 mL/min) to afford compound 2 (23.7 mg) (tR = 11.51 min). F6 (36.1 mg) was subjected to semipreparative HPLC (Kromasil C18, MeCN:H2O +

Figure 9. Biosynthetic hypothesis of euphoractane 8 formation from lathyrane 15. 909



Article

Figure 10. (A) Molecular network of fractions F3-F16 of S. guyanensis bark extract. (B) Cluster of phorbol analogues. Relative quantification of each ion within the fractions are represented as a XIC area-dependent pie-chart drawing. (C) MS2 spectrum of node C matching as a TPA analogue when comparing to GNPS spectral libraries. (D, E, and F) MS2 spectra of nodes D, E, and F from cluster (B) showing characteristic phorbol backbone fragment ions, and losses of different fatty acyl chains. and 13C NMR, see Table 3; HRESIMS m/z 371.1837 [M + Na]+ (calcd 371.1829). Euphoractine T (8). Amorphous white solid; [α]20 D + 26 (c 0.2, MeOH); UV (CH2Cl2) λmax (log ε) 252 (3.84) nm; 1H and 13C NMR, see Table 5; HRESIMS m/z 371.1841 [M + Na]+ (calcd 371.1829). Euphoractine U (9). Amorphous white solid; [α]20 D − 27 (c 0.2, MeOH); UV (CH2Cl2) λmax (log ε) 245 (3.77) nm; 1H and 13C NMR, see Table 5; HRESIMS m/z 371.1854 [M + Na]+ (calcd 371.1829). Euphoractine V (10). Amorphous white solid; [α]20 D + 40 (c 0.4, MeOH); UV (CH2Cl2) λmax (log ε) 245 (3.78) nm; 1H and 13C NMR, see Table 5; HRESIMS m/z 357.2061 [M + Na]+ (calcd 357.2036). Euphoractine W (11). Amorphous white solid; [α]20 D − 0.67 (c 0.1, MeOH); UV (CH2Cl2) λmax (log ε) 245 (3.82) nm; 1H and 13C NMR, see Table 5; HRESIMS m/z 413.1977 [M + Na]+ (calcd 413.1935). Sikkimenoid E (12). Amorphous white solid; [α]20 D + 12 (c 0.1, MeOH); UV (CH2Cl2) λmax (log ε) 252 (3.27) nm; 1H and 13C NMR, see Table 5; HRESIMS m/z 337.1777 [M + Na]+ (calcd 337.1774). Euphactin G (13). Amorphous white solid; [α]20 D + 111 (c 0.1, MeOH); UV (CH2Cl2) λmax (log ε) 271 (3.75) nm; 1H and 13C NMR, see Table 5; HRESIMS m/z 371.1894 [M + Na]+ (calcd 371.1829). Jatrointelone J (14). White crystal; [α]20 D + 95 (c 0.1, CH2Cl2); UV (CH2Cl2) λmax (log ε) 238 (3.79); 1H and 13C NMR, see Table 6; HRESIMS m/z 659.4291 [2M + Na]+ (calcd 659.4282). X-ray Crystallographic Data of (14) (M = 334.44 g/mol). monoclinic, space group P21 (no. 4), a = 13.2449(19) Å, b = 10.3812(14) Å, c = 14.7800(17) Å, β = 93.224(7)°, V = 2029.0(5) Å3, Z = 4, T = 293 K, μ(Cu Kα) = 0.598 mm−1, Dcalc = 1.095 g/cm3, 21132 reflections measured (6.684° ≤ 2Θ ≤ 147.946°), 7502 unique (Rint = 0.1059, Rsigma = 0.2245) which were used in all calculations. The final R1 was 0.1103 (I > 2σ(I)) and wR2 was 0.3781 (all data). The absolute configuration of this compound was characterized as (4R, 6S,

13R), with an absolute structure parameter, Flack x = 0.24(15) (546 quotients). Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre (deposit no. CCDC 1519649). Euphoractine Q (5). White crystal; [α]20 D + 88 (c 0.1, MeOH); UV (CH2Cl2) λmax (log ε) 254 (3.82), 231 (3.65) nm; 1H and 13C NMR, see Table 3; HRESIMS m/z 355,1886 [M + Na]+ (calcd 355.1880). X-ray Crystallographic Data of (5) (M = 331.41 g/mol). Monoclinic, space group P21 (no. 4), a = 8.7269(4) Å, b = 8.1173(5) Å, c = 13.0392(9) Å, β = 94.329(7)°, V = 921.05(10) Å3, Z = 2, T = 293 K, μ(CuKα) = 0.658 mm−1, Dcalc = 1.199 g/cm3, 13743 reflections measured (6.798° ≤ 2Θ ≤ 144.82°), 3456 unique (Rint = 0.0482, Rsigma = 0.0706) which were used in all calculations. The final R1 was 0.0513 (I > 2σ(I)) and wR2 was 0.1403 (all data). The absolute configuration of this compound was characterized as (2S, 6R, 7R, 8R, 10S, 13S, 14R), with an absolute structure parameter, Flack x = 0.07(15) (492 quotients). Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre (deposit no. CCDC 1519647). Euphoractine R (6). White crystal; [α]20 D + 22 (c 0.1, MeOH); UV (CH2Cl2) λmax (log ε) 266 (3.82), 239 (3.70) nm; IR υmax 3345, 1645 cm−1; 1H and 13C NMR, see Table 4; HRESIMS m/z 357.2021 [M + Na]+ (calcd 357.2036). X-ray Crystallographic Data of (6) (M = 700.92 g/mol). Orthorhombic, space group C2221 (no. 20), a = 7.8136(4) Å, b = 22.8068(11) Å, c = 65.613(5) Å, V = 11692.5(12) Å3, Z = 12, T = 293 K, μ(CuKα) = 0.664 mm−1, Dcalc = 1.195 g/cm3, 50476 reflections measured (5.388° ≤ 2Θ ≤ 144.694°), 11178 unique (Rint = 0.1031, Rsigma = 0.1391) which were used in all calculations. The final R1 was 0.0795 (I > 2σ(I)) and wR2 was 0.1907 (all data). The absolute configuration of this compound was characterized as (2S, 3R, 5R, 6S, 9S, 11R, 12R, 13R), with an absolute structure parameter, Flack x = −0.04(16) (1017 quotients). Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre (deposit no. CCDC 1519644). Euphoractine S (7). Amorphous white solid; [α]20 D + 23 (c 0.1, MeOH); UV (CH2Cl2) λmax (log ε) 265 (3.79), 237 (3.71) nm; 1H 910



Article

9S, 11R, 13R, 15S), with an absolute structure parameter, Flack x = −0.1(3) (485 quotients). Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre (deposit no. CCDC 1519645). Jatrointelone K (15). amorphous white solid; [α]20 D − 6 (c 0.1, MeOH); UV (CH2Cl2) λmax (log ε) 281 (3.55), 240 (3.77) nm; 1H and 13C NMR, see Table 6; HRESIMS m/z 331.1949 [M + H]+ (calcd 331.1904). Jatrointelone L (16). orange crystals; [α]20 D + 9 (c 0.1, MeOH); UV (CH2Cl2) λmax (log ε) 245 (3.80), nm; 1H and 13C NMR, see Table 6; HRESIMS m/z 333.2069 [M + H]+ (calcd 333.2060). X-ray Crystallographic Data of (16) (M = 332.42 g/mol). Monoclinic, space group P21 (no. 4), a = 5.8124(12) Å, b = 33.429(7) Å, c = 10.239(2) Å, β = 106.297(7)°, V = 1909.6(7) Å3, Z = 4, T = 293 K, μ(CuKα) = 0.635 mm−1, Dcalc = 1.156 g/cm3, 15444 reflections measured (10.442° ≤ 2Θ ≤ 136.502°), 6658 unique (Rint = 0.0747, Rsigma = 0.1779) which were used in all calculations. The final R1 was 0.0883 (I > 2σ(I)) and wR2 was 0.2924 (all data). The relative configuration of this compound was confirmed by X-ray diffraction analysis, but unfortunately, the absolute configuration could not be established. Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre (deposit no. CCDC 1583803). Jatrointelone M (17). amorphous white solid; [α]20 D + 88 (c 0.1, MeOH); UV (CH2Cl2) λmax (log ε) 258 (3.56) nm; 1H and 13C NMR, see Table 1; HRESIMS m/z 357.2040 [M + Na]+ (calcd 357.2036). Conversion of Compound 15 into Compound 8. To a solution of 15 (1 mg) in CDCl3 (400 μL) was added BF3·OEt2 (10 equiv, 4 μL). After 4 h at room temperature, a drop of water was added and the mixture was dried over MgSO4, filtered, and concentrated under reduced pressure. 1H NMR anaysis was performed in CDCl3 at 500 MHz. Antiviral Assays. The anti-Chikungunya virus bioassays were conducted according to a protocol previously described, using chloroquine as a positive control.24 Data Dependent LC ESI HRMS2 Analysis. LC analyses were performed with a Dionex Ultimate 3000 RSLC system equipped with an Accucore C18 column (2.1 × 100 mm; 2.6 μm, ThermoScientific). Elution was conducted with a mobile phase consisting of water (A) and MeCN (B), following an isocratic step at 20% B during 5 min, a gradient 20 to 100% B in 20 min, then maintaining 100% B for 4 min at a flow rate of 350 μL.min 1. The column oven was set at 30 °C and the injection volume at 5 μL. LC ESI HRMS2 analyses were achieved by coupling the LC system to a hybrid quadrupole time-of-flight mass spectrometer Agilent 6540 (Agilent Technologies, Massy, France) equipped with an ESI dual source, operating in positive ion mode. Source parameters were set as follows: capillary temperature at 325 °C, source voltage at 3500 V, sheath gas flow rate at 7 L.min−1, nebulizer pressure at 30 psi, drying gas flow rate at 10 L·min−1, drying gas temperature at 350 °C, stealth gas temperature at 350 °C, skimmer voltage at 45 V, fragmentor voltage at 150 V, and nozzle voltage at 500 V. MS scans were operated in full scan mode from m/z 100 to 1000 (100 ms scan time) with a mass resolution of 20 000 at m/z 922. MS1 scan was followed by MS2 scans of the five most intense ions above an absolute threshold of 3000 counts. Selected parent ions were fragmented at a fixed collision energy value of 35 eV and an isolation window of 1.3 amu. Calibration solution, containing two internal reference masses (purine, C5H4N4, m/z 121.0509, and HP 921 [hexakis (1H,1H,3H tetrafluoropentoxy)phosphazene], C18H18O6N3P3F24, m/z 922.0098), routinely led to mass accuracy below 2 ppm. MS data acquisitions were performed using MassHunter Workstation software (Agilent Technologies, Massy, France). MZmine 2 Data Preprocessing Parameters. Raw files were directly processed using the MZmine 2.28. The mass detection was realized keeping the noise level at 0. The chromatogram building was achieved using a minimum time span of 0.1 min, minimum height of 5000 and m/z tolerance of 0.006 (or 20 ppm). The local minimum search deconvolution algorithm was used with the following settings:

chromatographic threshold = 1%, minimum retention time range = 0.3 min, minimum relative height = 1%, minimum absolute height = 5000, minimum ratio of peak top/edge = 2, peak duration range = 0.1 − 2.0 min. Chromatograms were deisotoped using the isotopic peaks grouper algorithm with an m/z tolerance of 0.006 (or 20 ppm) and an RT tolerance of 0.5 min. Peak alignment was performed using the RANSAC aligner method (m/z tolerance at 0.006 (or 20 ppm), absolute tR tolerance 1.5 min, absolute RT tolerance after correction of 0.5 min, and a threshold value of 1). The peak list was gap filled with the peak finder module (intensity tolerance at 50%, m/z tolerance at 0.006 (or 20 ppm) and absolute RT tolerance of 0.5 min). Molecular Network Analysis. The molecular network was created using the online workflow at Global Natural Products Social molecular networking (GNPS) (http://gnps.ucsd.edu). The data was filtered by removing all MS/MS peaks within ± 17 Da of the precursor m/z. MS2 spectra were window filtered by choosing only the top 6 peaks in the ± 50 Da window throughout the spectrum. A network was created where edges were filtered to have a cosine score above 0.7 and more than six matched peaks. Further edges between two nodes were kept in the network if and only if each of the nodes appeared in each other’s respective top 10 most similar nodes. The spectra in the network were searched against GNPS′ spectral libraries. The library spectra were filtered in the same manner as the input data. All matches kept between network spectra and library spectra were required to have a score above 0.4 and at least four matched peaks. Analog search was enabled against the library with a maximum mass shift of 100 Da. Finally, MNs were visualized using Cytoscape 3.4.0 software. All data files used during this study were deposited in the GNPS MassIVE data repository and are publicly available (MassIVE ID: MSV000081542).

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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.7b01025. NMR spectra of all new compounds (PDF) X-ray data of compound 3 (CIF) X-ray data of compound 4 (CIF) X-ray data of compound 5 (CIF) X-ray data of compound 6 (CIF) X-ray data of compound 14(CIF) X-ray data of compound 16 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: + 33 1 69 82 30 85. Fax: + 33 1 69 07 72 47. E-mail: [email protected]. ORCID

Véronique Eparvier: 0000-0002-2954-0866 Fanny Roussi: 0000-0002-5941-9901 David Touboul: 0000-0003-2751-774X Marc Litaudon: 0000-0002-0877-8234 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by an “Investissement d’Avenir” grant managed by Agence Nationale de la Recherche (CEBA, ANR10-LABX-25-01). The authors are grateful to Caroline Collard, Nick Verstraeten and Charlotte Vanderheydt from the “Laboratory for Virology and Experimental Chemotherapy” at the “Rega Institute for Medical Research” in Leuven, for the evaluation of antiviral activity. We are also grateful to Jean911



Article

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François Gallard from ICSN for his help in carrying out NMR experiments.

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