Antiviral chlorinated daphnane diterpenoid orthoesters from the bark and wood of Trigonostemon cherrieri

Phytochemistry 84 (2012) 160–168

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Antiviral chlorinated daphnane diterpenoid orthoesters from the bark and wood of Trigonostemon cherrieri Pierre-Marie Allard a, Pieter Leyssen b, Marie-Thérèse Martin a, Mélanie Bourjot a, Vincent Dumontet a, Cécilia Eydoux c, Jean-Claude Guillemot c, Bruno Canard c, Cyril Poullain a, Françoise Guéritte a, Marc Litaudon a,⇑ a b c

Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, LabEx LERMIT, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France Laboratory for Virology and Experimental Chemotherapy, Rega Institute for Medical Research (KU Leuven), Minderbroedersstraat, B3000 Leuven, Belgium Laboratoire d’Architecture et de Fonction des Macromolécules Biologiques (AFMB-AMU-UMR7257), ESIL Case 925, 163, Avenue de Luminy, 13288 Marseille, France

a r t i c l e

i n f o

Article history: Received 30 May 2012 Received in revised form 25 July 2012 Available online 29 August 2012 Keywords: Trigonostemon cherrieri Euphorbiaceae Trigocherrin Trigocherriolide Daphnane Chlorinated diterpenes Antiviral Chikungunya virus Sindbis virus Semliki forest virus Dengue virus

a b s t r a c t The chemical study of the bark and the wood of Trigonostemon cherrieri, a rare endemic plant of New Caledonia, led to the isolation of a series of highly oxygenated daphnane diterpenoid orthoesters (DDO) bearing an uncommon chlorinated moiety: trigocherrins A–F and trigocherriolides A–D. Herein, we describe the isolation and structure elucidation of the DDO (trigocherrins B–F and trigocherriolides A–D). We also report the antiviral activity of trigocherrins A, B and F (1, 2 and 6) and trigocherriolides A, B and C (7–9) against various emerging pathogens: chikungunya virus (CHIKV), Sindbis virus (SINV), Semliki forest virus (SFV) and dengue virus (DENV). Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction In the course of our ongoing program focussing on the characterization of antiviral compounds from New Caledonian plants (Allard et al., 2011), Trigonostemon cherrieri Veillon (Euphorbiaceae), a species collected in the sclerophyllous forest of New Caledonia was selected and chemically investigated. This species is the unique representative of the genus in this island. The genus Trigonostemon comprises about 80 species occurring in tropical Asia, from India and Sri-Lanka to New Guinea (Fan et al., 2010). Numerous structurally interesting compounds such as flavonoidal alkaloids (Kanchanapoom et al., 2002), indole alkaloids (Tan et al., 2010; Zhu et al., 2010), phenanthrenes (Kokpol et al., 1990) and an array of daphnane-type diterpenoids (Chen et al., 2010a; Dong et al., 2011a; Jayasuriya et al., 2000, 2004; Li et al., 2011; Lin et al., 2010; Soonthornchareonnon et al., 2005; Zhang et al., 2010a) have been isolated from this genus. Daphnane-type ⇑ Corresponding author. Tel.: +33 1 69 82 30 85; fax: +33 1 69 07 72 47. E-mail address: [email protected] (M. Litaudon). 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2012.07.023

diterpenoids of Trigonostemon have been shown to possess insecticidal (Jayasuriya et al., 2000, 2004), acaricidal (Soonthornchareonnon et al., 2005), cytotoxic (Chen et al., 2010b; Dong et al., 2011b; Lin et al., 2010) and antiviral (Zhang et al., 2010b) properties. From the species T. cherrieri, we recently reported the isolation and structural characterization of trigocherrin A (1) an unusual chlorinated daphnane diterpenoid orthoester (DDO) possessing a selective and potent antiviral activity against the chikungunya virus replication in cellulo (Allard et al., 2012). Five new analogues of trigocherrin A, named trigocherrins B-F (2–6), and four macrocyclic analogues, named trigocherriolides A–D (7–10), were isolated from the bark and the wood of T. cherrieri. Herein we report the isolation, structure elucidation and antiviral activities observed for these compounds. 2. Results and discussion A total of 1350 EtOAc polyamide cartridge-filtered plant extracts were screened at 50 lg/mL in a dengue polymerase assay using the RNA-dependant RNA polymerase (RdRp) domain of the

P.-M. Allard et al. / Phytochemistry 84 (2012) 160–168

non-structural protein 5 (NS5). A second screen was carried out at 10 lg/mL on 320 selected active extracts, of which the T. cherrieri bark extract was found to significantly inhibit RNA polymerase activity (65% at 10 lg/mL). The dengue polymerase assay was then used to conduct a bioassay-guided purification on a large amount of extract. The air-dried powder of the bark of T. cherrieri (1.2 kg) was extracted with EtOAc to give a crude extract (23 g), which was partitioned between n-hexanes and aqueous MeOH. The aq. MeOH soluble fraction (8 g) was then subjected to C18 flash chromatography, and the active fractions (F11, F13–F15, ca. 90% of inhibition at 10 lg/mL) were then repeatedly purified by preparative, semi-preparative and analytical C18 HPLC to yield trigocherrins A–C (1–3) and trigocherriolides A-D (7–10). Since several of these compounds were isolated in minute quantities, we further investigated chemically the wood of this species, a part of plant that we had collected, stored and preserved at room temperature for several months. Since all the active compounds isolated from the bark were shown to possess one or two chlorine atoms in their structure, a LC/MS-guided purification process was carried out on 6.4 g of crude EtOAc extract (obtained from 900 g of air-dried powdered wood) to isolate the halogenated compounds. This led to further isolation of trigocherrins A and B (1, 2) and trigocherriolide B (8), along with trigocherrins D–F (4–6). Compounds 1–10 were isolated as optically active amorphous white powders. Their spectroscopic data are overall similar and allow to distinguish two groups, trigocherrins A–F (1–6) and the macrocyclic trigocherriolides A–D (7–10). All isolated compounds possessed a common daphnane diterpenoid skeleton substituted by an orthobenzoate moiety at positions 9,12,14; this moiety is characterized by a typical chemical shift at @ C 108.2 ± 0.3 of the quaternary carbon C-10 (Liao et al., 2009). In addition, the systematic presence of a mono or dichlorovinyl group on the five-membered ring A, makes this DDO series extremely original. Indeed, if organohalogens are commonly encountered in marine organisms, they are rarely isolated from higher plants. Furthermore, the dichlorovinyl moiety is particularly scarce in higher plants since it has only been found in the structure of chingazumianine, a probable artifactual alkaloid isolated from the species Corydalis koidzumiana (Chan et al., 2000). The possible implication of a specific type of enzyme (non-heme iron, O2, a-ketoglutarate-dependent halogenase) in the halogenation process of these natural products has been discussed in our previous publication (Allard et al., 2012). The 1H and 13C NMR data of trigocherrins B–F (2–6) and trigocherriolides A–D (7–10) are reported in Tables 1 and 2, respectively, and their respective 1D and 2D NMR spectra are presented in the ‘‘Supplementary data’’ file. Since all compounds share the same DDO backbone, and given that the structure of trigocherrin A (1) has been fully characterized, including its absolute configuration (Allard et al., 2012), only the nature and spatial positions of the substituents on the daphnane core will be discussed in detail. In the NOESY spectra of trigocherrins A and B (1, 2), and trigocherriolide A (7), a set of cross peaks, including a clear correlation between OH-4 and H-8, which indicated a b-orientation of the hydroxy group at C-4, allowed to determine their relative configuration (Figs. 2 and 4). For compounds 3–6 and 8–10, although the signature cross peaks were not clearly visible, the relative configuration of the daphnane skeleton could be assigned as shown since they all have identical correlations to those of 1, 2 and 7, and because they belong to the same chemical series. Compound 2 was obtained as an amorphous powder. The HRESIMS of the [M + Na]+ ion peak at m/z 797.1602 established the molecular formula of 2 as C41H36Cl2O11 (calcd. for C41H36Cl2O11Na 797.1532), thus requiring 23 double-bond equivalents. The 3:2 ratio of [M + H]+ and [M + H + 2]+ confirmed the presence of two chlorine atoms in 2. In accordance with the molecular formula, the 13C NMR data (Table 1) in combination with analysis of the

161

HSQC spectrum revealed 41 carbon signals due to two methyls, two methylenes (one olefinic), 23 methines (5 oxygenated and 16 olefinic), and 14 quaternary carbons (2 ester carbonyls, 5 oxygenated and 7 olefinic). The NMR spectra (Table 1) clearly showed the presence of an isopropenyl [(@ C 138.3, 119.7 and 18.6, C-15, C16 and C-17, respectively), and @ H 5.37 and 5.49, (H2-16), and 1.93, (H-17)], an oxymethylene (@ C 67.0, @ H 4.12 and 5.13, d, 12.2 Hz, CH2-20), two hydroxyl (@ H 3.89, s, and @ H 3.74, d, 9.0 Hz) and two benzoyloxy groups. The position of the substituents was determined by HMBC correlations. (Fig. 2, left) The two hydroxy protons at @ H 3.89 and 3.74 were assigned as 4-OH and 5-OH on the basis of their correlation with C-4 (@ C 82.6) and C-5 (@ C 70.4), respectively. In the HMBC spectrum, correlations from H2-20 and aromatic protons H-300 and H-700 (@ H 8.07) to the carbonyl at @ C 167.0 (C-100 ) suggested that a benzoyloxy group was attached to C-20, whereas correlations from aromatic protons H-3000 and H-7000 (@ H 8.10) to the carbonyl at @ C 165.3 (C-1000 ) confirmed the presence of a second benzoyloxy group, which can be placed at C-13 taking into account the downfield shift of this quaternary carbon when compared with trigocherrin A (1) (@ C 78.8 and 70.4, respectively). The three remaining oxygenated carbons were assigned to C-9 (@ C 74.5), C12 (@ C 76.1) and C-14 (@ C 76.1), and formed a 9,12,14-orthobenzoate pattern as confirmed by HMBC correlations from H-12, H-14, H-30 and H-70 to the typical quaternary C-10 at @ C 108.2. The location of the aforementioned substituents left two double-bond equivalents and two chlorine atoms to dispose on the 5-membered ring of the daphnane core, thus implying the presence of a double bond between C-1 and C-10, and an exocyclic double-bond bearing the two chlorine atoms. Multiple HMBC correlations from H-1 to C2, C-3, C-4, C-9, C-10 and C-19 secured the planar structure of 2. The relative configuration of 2 was established by NOESY experiment (Fig. 2, right). Cross peaks of H-12/H-11, H-11/H-8, H-8/ OH-4, OH-4/OH-5, OH-5/H-20a, H-20b/H-7 and H-7/H-14 indicated that they were all cofacial, and they were randomly assigned as b-oriented. The strong intensity of the H-3/H-5 correlation allowed us to determine H-3 and H-5 as a-oriented. However, as reported in the case of trigochilide A (Chen et al., 2009) a weaker correlation between H-3 and OH-4 was also observed. Correlation of H-14/Me-17 and multiple correlations of H2-16 with H-8, H-11 and H-12 suggested the axial position of the isopropenyl group at C-13. The relative stereochemistry of 2, which was named trigocherrin B, was thus established as depicted in Fig. 2. Compound 3 was given the molecular formula of C38H46Cl2O10 based on the positive ion HR-ESIMS (m/z 733.2513, calcd. 733.2546). In accordance with the molecular formula, 38 carbons were resolved in the 13C NMR spectrum (Table 1), of which 26 were further characterized by HSQC experiment as four methyls, eight methylenes (one olefinic), 14 methines (5 oxygenated and 7 olefinic), along with 12 quaternary carbons (1 ester carbonyl, 5 oxygenated and 6 olefinic). The location of the secondary methyl group at C-11, the isopropenyl group at C-13, the trisubstituted epoxyde at C-6/C-7, the orthobenzoate moiety at C-9/C-12/C-14, as well as the presence of an a,b-unsaturated dichlorovinylic moiety on the five-membered ring of the daphnane skeleton were established by the same key HMBC correlations to those observed for compound 2. Furthermore, 1H–1H COSY and HMBC spectra, in accordance with the molecular formula, suggested the presence of an additional eleven carbons aliphatic side-chain attached to the oxymethine C-3 of the daphnane core, as depicted in Fig. 3. The position of the olefinic methyl 1100 (@ C 12.4) at C-200 was confirmed by the HMBC correlations from H3-1100 to C-200 , C-300 and C-100 . The geometry of the D200 bond was assigned as E on the basis of the NOESY correlation between H3-1100 and H-400 (Fig. 3). Finally, the chemical shifts of C-4, C-5, C-13 and C-20 (@ C 83.2, 72.6, 70.2 and 65.3, respectively) allowed us to place four hydroxy groups at these positions. The relative configuration of compound 3,

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Table 1 1 H and 13C NMR spectroscopic data for trigocherrins B–F (2–6) (in CDCl3, at 600 and 150 MHz, respectively). Position

2

4a

3

5a

6

dC

dH, mult. J (Hz)

dC

dH, mult. J (Hz)

dC

dH, mult. J (Hz)

dC

dH, mult. J (Hz)

dC

dH, mult. J (Hz)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

126.8 140.4 76.4 82.6 70.4 60.3 62.8 34.5 74.5 148.8 35.8 76.1 78.8 76.1 138.3 119.7

127.3 137.1 79.1 83.2 72.6 61.2 62.4 34.8 74.7 148.7 35.8 79.6 70.2 79.2 141.7 115.8

6.42, – 4.62, – 5,52, – 3.61, 3.60, – – 2.17, 5.11, – 5.29, – 5.42, 5.52, 1.95,

128.2 136.5 78.1 87.2 74.0 76.2 80.2 34.8 77.5 149.7 35.8 76.6 78.7 77.8 137.4 119.9

6.59, – 6.14, – 3.87, – 4.21, 3.35, – – 2.32, 5.03, – 5.28, – 5.31, 5.37, 1.82,

127.2 136.9 79.5 83.4 72.5 61.2 62.7 34.5 74.8 148.9 35.5 76.3 78.9 76.1 138.1b 119.7

18.6

6.34, – 5.50, – 4.11, – 3.37, 3.33, – – 2.05, 4.28, – 4.47, – 5.23, 4.97, 1.87,

127.0 140.0 76.4 83.2 72.0 60.7 61.7 34.5 74.7 148.4 35.9 76.0 78.8 76.3 138.1 119.8

17

6.35, – 4.61, – 3.87, – 3.43, 3.47, – – 2.11, 5.05, – 5.27, – 5.49, 5.37, 1.93,

6.49, – 5.76, – 4.24, – 3.40, 3.42, – – 2.14, 5.02, – 5.25, – 5.29, 5.38, 1.84,

18 19 20

13.6 118.0 67.0

0

108.2 137.9 125.3 128.0 129.3 128.0 125.3 167.0 130.9 130.0 128.5 133.4 128.5 130.0

1.26, – 5.13, 4.12, – – 7.68, 7.32, 7.32, 7.32, 7.68, – – 8.07, 7.47, 7.59, 7.47, 8.07,

165.3 129.4 129.8 128.3 133.0 128.3 129.8 – –

– – 8.10, 7.43, 7.56, 7.43, 8.10, 3.89, 3.74,

1 20 30 40 50 60 70 100 200 300 400 500 600 700 800 900 1000 1100 1000 2000 3000 4000 5000 6000 7000 4-OH 5-OH

s s d (9.0) s s

q (7.0) br s br s s s s d (7.0) d (12.2) d (12.2)

m m m m m

d (7.8) t (7.8) t (7.8) t (7.8) d (7.8)

18.3 13.6 118.5 65.3 108.3 137.9 125.3 128.2 129.6 128.2 125.3 169.3 126.4 145.3 28.9 28.5 29.5 29.1 31.9 22.6 14.1 12.4

1.21, – 3.78, 3.96, – – 7.75, 7.42, 7.42, 7.42, 7.75, – – 6.83, 2.21, 1.46, 1.26, 1.32, 1.27, 1.31, 0.90, 1.84,

s s br s s s

q (7.0) br s br s s s s d (7.0) d (12.2) d (12.2)

m m m m m

t (7.0) q (7.0) t (7.0) m m m m t (7.0) s

d (7.8) t (7.8) t (7.8) t (7.8) d (7.8) s d (9.0)

18.7 13.6 n.d. 61.6 108.1 n.d. 125.3 127.9 129.4 127.9 125.3 165.8 n.d. 130.1 128.4 134.1 128.4 130.1

1.27, – 3.63, 3.84, – – 7.70, 7.34, 7.34, 7.34, 7.70, – – 8.15, 7.53, 7.67, 7.53, 8.15,

165.2 n.d. 129.8 128.0 133.1 128.0 129.8

– – 8.12, 7.45, 7.58, 7.45, 8.12,

s s s s s

q (7.0) br s br s s s s d (7.0)

18.6

d (7.8) t (7.8) t (7.8) t (7.8) d (7.8)

107.9 137.3 124.8 128.1 129.5 128.1 124.8 167.2 129.0 129.8 128.4 133.5 128.4 129.8

1.32, – 3.73, 4.08, – – 7.62, 7.34, 7.34, 7.34, 7.62, – – 8.06, 7.47, 7.61, 7.47, 8.06,

d (7.8) t (7.8) t (7.8) t (7.8) d (7.8)

164.9 130.5 129.7 128.2 132.7 128.2 129.7

– – 8.07, 7.42, 7.55, 7.42, 8.07,

d (12.2) d (12.2)

m m m m m

13.7 119.6 69.4

s s s s s

m br s br s s s s d (7.0)

18.6

s s s s

q (7.0) br s br s s s s

m m t (7.6) m m

108.1 138.0b 125.3 128.0 129.3 128.0 125.3 167.6 129.0 130.0 128.6 133.7 128.0 130.0

1.30, – 3.71, 4.02, – – 7.69, 7.33, 7.33, 7.33, 7.69, – – 8.06, 7.49, 7.63, 7.49, 8.06,

d (7.8) t (7.8) t (7.8) t (7.8) d (7.8)

m t (7.6) t (7.6) t (7.6) m

165.2 130.9 129.8 128.3 132.9 128.3 129.8

– – 8.10, 7.43, 7.56, 7.43, 8.10,

d (7.8) t (7.8) t (7.8) t (7.8) d (7.8)

d (10.9) d (10.9)

m m m m m

13.7 118.8 65.5

s

d (7.0) d (12.0) d (12.0)

m m m m m

n.d. = not detected. a Assignments of 13C NMR data based on HSQC and HMBC spectra. b Assignments are interchangeable.

named trigocherrin C, could be determined unambiguously, with the exception of the stereocenter C-4, thanks to NOESY correlations similar to those observed for compound 2. A quasi-molecular ion [M + H]+ at m/z 775.1769 for compound 4, and at m/z 775.1785 for compound 6 in the positive HR-ESIMS indicated that they possess an identical molecular formula C41H36Cl2O11 (calcd. 775.1713), and thus are both isomers of 2. Onedimensional NMR spectroscopic data of 2, 4 and 6 are very similar (Table 1), and it could be deduced from the 2D NMR spectra that only the position of the benzoyloxy groups are modified. For compounds 4 and 6, HMBC correlations from H-5 (4), or H-3 (6) and aromatic protons H-300 and H-700 to the carbonyl C-100 allowed to place a benzoyloxy moiety at C-5 (4) or C-3 (6). The chemical shifts of C-13 at @ C 78.8 and 78.9 ppm (4 and 6, respectively) indicated that the second benzoyloxy group is attached at this position, as

it is the case for compound 2, whereas the chemical shifts of C-3, C-4 and C-20 (@ C 76.4, 83.2 and 61.6, respectively) for compound 4, and of C-4, C-5 and C-20 (@ C 83.4, 72.5 and 65.5, respectively) for compound 6, indicated that they all bear hydroxy groups. The relative configuration of compounds 4 and 6, named trigocherrins D and F, could be determined unambiguously, with the exception of the stereocenter C-4, thanks to ROESY correlations similar to those observed for compound 2. From the HR-ESIMS, we deduced the molecular formula C41H38Cl2O12 for compound 5, indicating that it possesses one additional hydroxy group and one proton compared to trigocherrin F (6). The spectroscopic data of 5 are very close to those of 6, with the exception of the chemical shifts of C-6 and C-7 clearly downfield shifted at @ C 76.2, and 80.2, respectively (both around 62.0 ppm in 6), suggesting that the epoxy group have been replaced by two hydroxy

163

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Table 2 1 H and 13C NMR spectroscopic data for trigocherriolides A–D (7–10) (in CDCl3, respectively at 600 and 150 MHz for 7, 9, and 10, and respectively at 500 and 125 MHz for 8). Position

a

7

8

9

10

dC

dH, mult. J (Hz)

dC

dH, mult. J (Hz)

dC

dH, mult. J (Hz)

dC

dH, mult. J (Hz)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

125.7 139.6 78.5 83.4 71.3 60.9 62.6 34.3 74.5 148.7 34.8 79.4 72.2 79.8 77.1 37.2

125.7 139.7 78.7 83.7 72.0 60.8 62.6 34.7 74.6 149.2 34.7a 79.6 72.1 79.1 75.7 38.6

6.41, – 5.20, – 3.88, – 2.97, 4.43, – – 2.81, 4.28, – 4.72, – 1.84, 1.53, 4.76,

126.2 137.2 78.8 83.5 72.8 60.8 62.5 34.6 74.7 149.9 34.8 79.6 72.1 79.1 75.7 38.4

68.0

6.48, – 5.31, – 3.96, – 3.38, 4.69, – – 2.70, 4.28, – 4.37, – 1.69, 1.49, 1.39,

125.5 139.8 78.5 83.4 70.6 61.2 62.5 34.3 74.6 149.2 34.8 79.5 72.3 79.9 77.3 37.4

17

6.31, – 5.45, – 4.02, – 3.40, 4.72, – – 2.69, 4.27, – 4.37, – 1.70, 1.47, 1.39,

18 19 20

13.8 114.9 65.4

10 20 30 40 50 60 70 100 200 300

108.5 138.2 125.2 128.2 129.4 128.2 125.2 177.8 41.8 35.1

400 –700

800

27.0 27.5 28.9 30.9 38.3

900 1000 1100 1000 2000 3000 4000 5000 6000 7000 4-OH

26.4 24.7 18.8 170.9 111.7 161.8 118.2 136.5 119.6 129.6 –

6.44, s – 5.21, s – 3.94, s – 3.11, s 4.46, br s – – 2.75, q (7.1) 4.29, br s – 4.67, br s – 1.85, dd (15.0, 4.4) 1.55, dd (15.0, 4.8) 4.81, d (12.0) 4.74, d (12.0) 1.23, d (7.0) 6.10, s 3.66, d (12.3) 3.32, d (12.3) – – 7.73, m 7.41, m 7.41, m 7.41, m 7.73, m – 2.46, m 1.67, m 1.35, m 1.68–1.23, m 1.68–1.23, m 1.68–1.23, m 1.68–1.23, m 1.38, m 1.28, m 1.70, m 1.06, d (6.6) 1.17, d (7.1) – – – 7.05, d (8.3) 7.52, ddd (8.3, 7.3, 1.5) 6.95, ddd (8.3, 7.3, 1.5) 7.77, dd (8.3, 1.5) 3.24

23.7 13.8 114.6 64.9 108.5 138.5 125.3 128.2 129.5 128.2 125.3 178.1 41.9 34.8a 26.9 27.5 28.6 29.7 38.0 25.5 24.9 18.6

s s br s s br s

m br s br s d (14.0) d (14.0) s

1.23, d (7.0) 6.07, s 3.85, m 3.78, m – – 7.75, m 7.40, m 7.40, m 7.40, m 7.75, m – 2.43, m 1.67, m 1.36, m 1.72–1.26, m 1.72–1.26, m 1.72–1.26, m 1.72–1.26, m 1.39, m 1.23, m 1.62, m 1.04, d (6.5) 1.19, d (7.0)

68.0 13.8 114.6 65.0 108.5 138.4 125.2 128.2 129.5 128.2 125.2 177.7 41.9 34.9 27.0 27.6 28.7 31.0 38.3 26.3 24.4 18.6 168.3 129.3 129.7 128.7 133.8 128.7 129.7

s s s s br s

m br s br s d (15.0) d (15.0) br s

1.21, d (6,7) 6.04, s 3.54, d (12.1) 3.10, d (12.1) – – 7.73, m 7.40, m 7.40, m 7.40, m 7.73, m – 2.43, m 1.66, m 1.32, m 1.44– 1.14, m 1.44– 1.14, m 1.44– 1.14, m 1.44– 1.14, m 1.36, m 1.30, m 1.75, m 1.06, d (5.9) 1.16, d (6.7) – – 8.04, d (7.2) 7.49, t (7.2) 7.62, t (7.2) 7.49, t (7.2) 8.04, d (7.2)

23.8 13.8 118.1 64.6 108.5 138.4 125.3 128.2 129.5 128.2 125.3 178.1 42.5 35.1 27.0 27.8 28.6 29.7 38.0 25.5 24.8 19.1

s s br s s br s

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

1.21, d (7.0) – 3.89, m 3.78, m – – 7.73, m 7.40, m 7.40, m 7.40, m 7.73, m – 2.49, m 1.69, m 1.37, m 1.66–1.26, m 1.66–1.26, m 1.66–1.26, m 1.66–1.26, m 1.41, m 1.21, m 1.58, m 1.04, d (6.3) 1.23, d (7.0)

Assignments are interchangeable.

groups at these positions. The relative configuration of compound 5, named trigocherrin E, could be determined unambiguously, with the exception of the stereocenter C-4, thanks to ROESY correlations similar to those observed for compounds 2–4 and 6. Compound 7 was obtained as an amorphous powder. The HRESIMS of the [M + Na]+ ion peak at m/z 875.3089 established the molecular formula of 7 as C45H53ClO14 (calcd. for C45H53ClO14Na 875.3022), thus requiring 19 double-bond equivalent. In negative mode, a 3:1 ratio of [M–H] and [M–H + 2] indicated the presence of only one chlorine atom in 7. In accordance with the molecular formula, 45 carbons were resolved in the 13C NMR spectrum (Table 2), of which 32 were further characterized by HSQC experiment as 3 methyls, 9 methylenes, 20 methines (5 oxygenated and 11 olefinic), along with 13 quaternary carbons (2 ester carbonyls, 7 oxygenated and 4 olefinic). The NMR data (Table 2) indicated the presence of three secondary methyls at @ H 1.23, 1.06 and 1.17

(each d, 7.0 Hz), C-18, C-1000 and C-1100 , respectively, and their correlated protons at @ H 2.75 (q, 7.1 Hz, H-11), 1.70 (m, H-900 ) and 2.46 (m, H-200 ). Two AB spin systems for two oxymethylenes were observed at C-17 (@ C 68.0, @ H 4.81 and 4.74, d, 12.0 Hz) and C-20 (@ C 65.4, @ H 3.66 and 3.32, d, 12.3 Hz). Three hydroxyls and signals corresponding to an ortho-hydroxybenzoyl group and a monosubstituted benzene were also observed. The aforementioned data and the quaternary carbon C-10 at @ C 108.5, typical of an orthobenzoate, (Liao et al., 2009) suggested that 7 featured a scaffold of modified DDO. The structure of 7 was established by comprehensive 1D and 2D NMR data analysis. The same HMBC correlations observed for 2 (grey arrows in Fig. 4) allowed us to establish the position of a secondary methyl at C-11, a trisubstituted epoxyde at C-6/C-7, an orthobenzoate at C-9/C-12/C-14 and the general framework of the DDO backbone in 7. Three signals at @ H 3.24, 3.97 and 2.89 were assigned to three hydroxy protons.

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Only the first one can be attributed unambiguously to 4-OH on the basis of its HMBC correlation with carbon C-4 (@ C 83.4). On the basis of HMBC correlations with carbons C-13 (@ C 72.2) and C-15 (@ C 77.1), the second one can be attributed to 13-OH or 15-OH. The chemical shifts of carbons C-5 (@ C 71.3) and C-20 (@ C 65.4) allowed us to place two other hydroxy groups at these positions. In contrast with trigocherrins A–F (1–6), trigocherriolide A (7) bore only one chlorine atom at C-19. HMBC correlations from H-19 (@ H 6.10, s) to C-1, C-2 and C-3 (@ C 125.7, 139.6 and 78.5, respectively) allowed us to place it at a geminal position relative to the chlorine atom. The value of the 1JC–H coupling constant measured for CH-19 (195.2 Hz) is characteristic of a vinyl chloride (Watts and Goldstein, 1966) (see Supplementary data). In the HMBC spectrum, cross peaks from H-3 (@ H 5.21), H2-300 (@ H 1.35 and 1.67) and

Me-1100 to carbonyl C-100 (@ C 177.8) indicated the esterification of the daphnane skeleton at position 3 by an aliphatic substituent. An eleven carbons aliphatic side-chain, attached at the carbonyl ester C100 on one side, and at the quaternary carbon C-15 on the other side, can be constructed with the help of 1H–1H COSY and HMBC experiments. Indeed, COSY correlations (bold lines, Fig. 4), and HMBC correlations from Me-1100 to C-100 , C-200 and C-300 , and from CH2-16 to C-13, C-15, C-17, C-800 , C-900 and C-1000 , allowed us to construct and locate this aliphatic side-chain as depicted in Fig. 4. The second anchor point of the aliphatic side-chain to the daphnane core at C-13 via the oxy-quaternary carbon C-15 was confirmed by HMBC correlations from H2-160 , H-12 and H-14 to C-13. Finally cross peaks from protons H2-17 and H-7000 to the carbonyl ester C-1000 indicated the location of the ortho-hydroxybenzoate at

Fig. 1. Structures of trigocherrins A–F (1–6) and trigocherriolides A–D (7–10).

Fig. 2. Key HMBC (left) and NOESY (right) correlations for 2.

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Table 3 Anti-metabolic and antiviral activities of compounds 1–2 and 6–9 in Vero cells, against CHIKV, SINV and SFV virus, and in a DENV RdRp assay (data in lM). Cpd.

Fig. 3. Selected 1H–1H COSY (bold), HMBC (blue) and NOESY (red) correlations for the aliphatic side-chain of 3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

position C-17. The relative stereochemistry of compound 7 was determined by interpretation of NOESY data. Correlations observed between H-12/H-11, H-11/H-8, H-8/OH-4, H2-20/H-7, H-7/H-8 and H-7/H-14 indicated that these protons had the same orientation that we arbitrary fixed as b, whereas correlations between H-3/ H-5 suggested that they were a-oriented. The clear correlation between H-19 and H-3 indicated that the stereochemistry of the double bond D2 was E. The relative stereochemistry of the macrolactone was not determined due to its high flexibility and the long distance between stereogenic centers C-200 and C-900 with other ones. Compound 7 was named trigocherriolide A. Trigocherriolide B (8) was given the molecular formula of C38H49ClO11 based on the quasimolecular-ion peak at m/z 717.3062 [M + H]+ obtained by HR-ESIMS (calcd. 717.3042). The NMR data of compound 8 were similar to those of compound 7, but the absence of signals for an ortho-hydroxybenzoate moiety, and the presence of an additional tertiary methyl suggested that the latter replaced the former group. The location of this methyl at C-15 was confirmed by correlations from H3-17 to carbons C-13, C-15 and C16 (@ C 72.1, 75.7 and 38.6, respectively) observed in the HMBC spectrum. Other 1H–1H COSY, HMBC and NOESY correlations of trigocherriolide B (8) were similar to trigocherriolide A (7) and allowed to propose the structure depicted in Fig. 1. The HR-ESIMS spectrum of trigocherriolide C (9) revealed a quasimolecular peak [M + H]+ m/z 837.3251 corresponding to the chemical formula C45H53ClO13 (calcd. 837.3253), suggesting the loss of one hydroxy group when compared with compound 7. Indeed, in the 1H NMR spectra of 9, no signal was observed for an ortho-disubstituted aromatic ring, but proton signals for a monosubstituted aromatic ring instead. The location of the benzoyloxy group at C-17 is confirmed by HMBC correlations from H2-17, and H-300 and H-700 to the carbonyl ester C-100 . All other homonuclear (COSY and NOESY) and heteronuclear (HSQC and HMBC) correlations observed for 7 are similar to those of compound 9, thus

1 2 6 7 8 9 Chloroquine 30 -DeoxyGTP

Cellular assay CC50 Vero cells

EC50 (CHIKV)

EC50 (SINV)

EC50 (SFV)

35 ± 8 93 ± 3 23.1 ± 0.6 4.6 ± 0.8 5.3 ± 0.2 10.5 ± 0.1 100 ± 25 n.d.

1.5 ± 0.6 2.6 ± 0.7 3.0 ± 1.2 1.9 ± 0.6 2.5 ± 0.3 3.9 ± 1.0 10 ± 5 n.d.

7.7 ± 1.8 21 ± 3 5.6 ± 0.6 3.2 ± 0.1 3.3 ± 0.2 5.3 ± 1.2 11.0 ± 2.1 n.d.

16 ± 3 45.0 ± 0.6 18 4.0 4.2 5.1 ± 0.4 13.8 ± 2.3 n.d.

DENV RdRp IC50 12.7 ± 0.2 n.d. n.d. 3.1 ± 0.2 16.0 ± 1.3 n.d. n.d. 0.02

n.d. = not determined. Values are the median ± median absolute deviation calculated from at least 3 independent assays.

defining its planar structure and stereochemistry as depicted in Fig. 1. The HR-ESIMS spectrum in negative mode of trigocherriolide D (10) showed a quasimolecular peak at m/z 749.2465 indicating a chemical formula of C38H48Cl2O11, suggesting that one proton was replaced by a chlorine atom when compared with compound 8. The presence of two chlorine atoms in 10 was confirmed by the 3:2 ratio of the [M + H]+ and [M + H + 2]+ peaks on the HR-ESIMS spectra. All homonuclear (COSY and NOESY) and heteronuclear (HSQC and HMBC) correlations observed for trigocherriolide B (8), with the exception of those concerning H-19, are present in the 2D spectra of 10, allowing us to give its planar structure and stereochemistry as depicted in Fig. 1. The antiviral potential of trigocherrins 1, 2 and 6, and trigocherriolides 7–9 was evaluated in a virus-cell-based assay against three members of the genus Alphavirus (chikungunya virus: CHIKV, Sindbis virus: SINV and Semliki forest virus: SFV), and in an enzymatic assay against the NS5 RNA-dependent RNA polymerase (RdRp) of dengue virus (DENV) of the genus Flavivirus. The observed antiviral properties of the compounds are reported in Table 3. As compared to the reference compound chloroquine, all compounds proved to be more potent, as is apparent from their lower EC50 values. Comparison of the selectivity index (SI calculated as CC50 Vero/EC50 CHIKV, or window for antiviral selectivity) of trigocherrins (1, 2 and 6) (SI = 23, 36 and 8, respectively) versus trigocherriolides (7–9) (SI = 2–3) indicated that trigocherrins are significantly more potent inhibitors of CHIKV replication, some even better than the reference compound chloroquine (SI = 10). Microscopic quality control showed that each of the compounds at least at one concentration perfectly protected the cells from virus-induced CPE (cyto-

Fig. 4. Selected 1H–1H COSY (bold), and key HMBC (left) (in grey common correlations to trigocherrin series, and in blue, additional correlations for compound 7) and NOESY (right) correlations for 7. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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pathic effect) without any significant adverse effects on cell or monolayer morphology (i.e. the treated virus-infected cells perfectly resembled the untreated uninfected cell control condition), confirming a selective inhibitory effect on CHIKV replication. In the same cell line, inhibition of SINV and SFV virus-induced cell death was observed as well, although EC50 values most often were not as low as for CHIKV. Also, upon microscopic quality control, this inhibition was not as pronounced as was the case for CHIKV (i.e. no full inhibition of virus-induced CPE). In the DENV polymerase assay, trigocherrin A (1) and trigocherriolides A and B (7, 8) showed significant inhibitory activity with IC50 values of 12.7, 3.1 and 16 lM, respectively.

3. Conclusion Trigocherrins A–F (1–6) and trigocherriolides A–D (7–10) isolated from the bark and the wood of T. cherrieri represent the first members of an unusual class of vinyl chlorinated daphnane diterpenoids orthoesters. The dichlorovinyl moiety observed in compounds 1–6 and 10 is particularly rare in natural products and, to the best of our knowledge, this is the second time that this functionality has been encountered in higher plants (Chan et al., 2000). The biosynthetic origin of these chlorine atoms is not elucidated yet but we hypothesized the implication of a non-heme iron, O2, a-ketoglutarate-dependent halogenase (Allard et al., 2012). The verification of this hypothesis would require in depth study of the plants genetic material. The results of the biological study indicated that trigocherrins A, B and F (1, 2 and 6) are potent and selective inhibitors of chikungunya virus replication.

4. Experimental 4.1. General experimental procedures Optical rotations were measured at 25 °C on a JASCO P1010 polarimeter. The UV spectra were recorded on a Perkin-Elmer Lambda 5 spectrophotometer. CD spectra were measured at 25 °C on a JASCO J-810 spectropolarimeter. The NMR spectra were recorded on a Bruker 600 MHz instrument (Avance 600) using a 1.7 mm microprobe for compounds 2–7 and 9–10 and on a Bruker 500 MHz instrument (Avance 500) for 8, CDCl3 was used as solvent. HR-ESIMS were run on a Thermoquest TLM LCQ Deca ion-trap spectrometer. Kromasil analytical and preparative C18 columns (250  4.5 mm and 250  21.2 mm; i.d. 5 lm, Thermo) were used for preparative 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. Silica gel 60 (35–70 lm) and analytical TLC plates (Si gel 60 F 254) were purchased from SDS (France). Pre-packed C18 Versapak cartridges (40  75 mm, 70 g) were used for Flash chromatography using a CombiflashCompanion apparatus (Serlabo). LC-ESIMS analyses were performed using a Finnigan Surveyor HPLC system with a Kromasil analytical C18 column (250  4.5 mm; i.d. 5 lm, Thermo) and an ion trap MSn (Finnigan LCQdeca) detector. UPLC-MS analyses were performed using an Acquity Waters UPLC system with BEH column (50  1.0 mm, 1.7 lm) and a LCT Premier XE ESI-TOF detector coupled with a UV-PDA detector (Acquity, Waters). 4.2. Plant material Trigonostemon cherrieri was collected in the Poya region, on the west coast of New Caledonia and identified by one of us (C.P). A voucher specimen (POU-0324) was deposited at the Herbarium

of the Botanical and Tropical Ecology Department of the IRD Center, Nouméa, New Caledonia. 4.3. Extraction and isolation Air-dried, grounded bark of T. cherrieri (1.2 kg) was extracted with ethyl acetate (4  1.5 L) at 40 ° C, leading to 23 g of crude EtOAc extract (q = 1.9% w/w). The EtOAc extract (23 g) of the bark was subjected to a liquid/ liquid partition system between n-hexanes/MeOHaq (MeOH:H2O 90:10) (2  750 mL of n-hexanes) leading to the obtention of a non-polar fraction (14.9 g) and a polar fraction (8 g). Half of the polar extract (4.2 g) was subjected to flash chromatography on a C18, Versapak column (40  75 mm, 70 g), and eluted by a H2O:MeOH + 0.1 HCOOH% gradient leading to 20 fractions denoted F1 to F20, combined on the basis of their TLC profiles. The injection of fraction F11 (315 mg) by HPLC on a preparative Kromasil C18 phase using a H2O:ACN + 0.1% HCOOH (60:40 to 0:100 in 30 min, at 15 mL min1) gradient led to 18 fractions, F111 to F1118. The purification of fraction F1118 (5.7 mg), by analytical HPLC (Kromasil C18, isocratic H2O: ACN + 0.1% HCOOH (15:85, 1 mL min1)) (manual collection) led to the isolation of trigocherrin A (1) (1.5 mg). Fraction F13 (605 mg) was subjected to flash chromatography (Versapak C18 column (23  110 mm, 30 g)), leading to 24 fractions denoted F131 to F1324, combined on the basis of their TLC profiles. The purification of the fraction F1313 (6.9 mg) by semi-preparative HPLC (Symetry-Shield C18, H2O:ACN + 0.1% HCOOH (40:60 to 0:80 in 60 min, 7.8 mL min1) allowed the re-isolation of the trigocherrin A (1) (0.6 mg). Purification of fraction F1314 (7.3 mg) under the same conditions allowed the isolation of trigocherrin B (2) (0.6 mg). Purification of fraction F1321 (20.3 mg) by semi-preparative HPLC (Symetry-Shield C18, isocratic H2O:ACN + 0.1% HCOOH (30:70 for 20 min) followed by a gradient (30:70 to 0:100 in 5 min), 7.8 mL min1) led to the isolation of trigocherriolide A (7) (2.3 mg). Separation of fraction F1319 (33.8 mg) by semi-preparative HPLC (Kromasil C18, isocratic H2O:ACN (10:90 at 4.7 mL min1)) led to the obtention of five fractions denoted F13.191 to F13.195. Fraction F13.193 (0.9 mg) is purified by analytical HPLC (Kromasil C18, isocratic H2O:ACN (0:100 to 1 mL min1) to afford trigocherrin C (3) (0.26 mg). Fraction F13.194 corresponds to trigocherriolide B (8) (6.3 mg). The fractions F14 and F15 are combined on the basis of their similar 1H NMR and LC-MS profiles. Combined F14 and F15 (1.25 g) are subject to a flash chromatography (Versapak C18 (23  110 mm, 30 g), gradient H2O:MeOH (80:20 to 30:70 in 30 min and then 30:70 to 0:100 in 120 min, 30 mL min1)) leading to 29 fractions denoted F14/151 to F14/1529, combined on the basis their TLC profiles. After defatting with hexane fraction F14/1518 (34.3 mg) was chromatographed by semi-preparative HPLC (Kromasil C18, isocratic H2O:ACN + 0.1% HCOOH (10:90 to 4.7 mL min1)) to afford 7 fractions denoted F14/15.181 to F14/15.187. Fraction F14/15.183 corresponds to trigocherriolide C (9) (1.99 mg). Fraction F14/15.184 corresponds to trigocherriolide A (7) (3.25 mg), previously isolated. The purification of fraction F14/15.185 (1.6 mg) by analytical HPLC (Kromasil C18, isocratic H2O:ACN + 0.1% HCOOH (10:90 at 1 mL min1) (manual collection)) led to the isolation of trigocherriolide D (10) (0.48 mg). Two EtOAc extractions of the wood of T. cherrieri were prepared. The first extraction was run on 200 g of dry wood (Dionex ASE, 40 °C). The second extraction was run on 700 g of dry wood (40 °C, 100 bar), using a static high-pressure high temperature extractor Zippertex developed in the ICSN Pilot Unit. After comparison of their profile (TLC, 1H NMR and LC-MS) both extract were combined (6.4 g) and then subjected to flash chromatography C18 (Versapak pre-packed cartridge (40  75 mm, 70 g), gradient H2O: MeOH (90:10 to 60:40 in 20 min and then 60:40 to 0:100

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in 100 min, 30 mL min1)) leading to 32 fractions noted F0 1 to F0 32, and combined on the basis of their TLC profiles. The separation of fraction F0 20 (106 mg) by preparative HPLC (Kromasil C18, isocratic H2O:ACN (35:65 at 21 mL min1) led to 14 fractions named F0 201 to F0 2014. Purification of fraction F0 2013 (5.1 mg) by semi-preparative HPLC (Kromasil C18, isocratic H2O:ACN + 0.1% HCOOH (20:80 to 4.7 mL min1) led to the re-isolation of trigocherrin A (7) (1.5 mg). The separation of fraction F0 22 (50 mg) by preparative HPLC (Kromasil C18, isocratic H2O:ACN (15:85 at 21 mL min1) led to 11 fractions named F0 221 to F0 2211. Purification of fraction F0 229 (6.3 mg) by semi-preparative HPLC (Kromasil C18, isocratic H2O:ACN + 0.1% HCOOH (25:75 at 4.7 mL min1)) led to the re-isolation of trigocherrin B (8) (0.4 mg). Separation of fraction F0 24 (100 mg) by preparative HPLC (Kromasil C18, isocratic H2O:ACN (10:90 at 21 mL min1) led to the production of seven fractions named F0 241 to F0 247. Purification of fraction F0 241 (6 mg) by analytical HPLC (Kromasil C18 isocratic H2O:ACN + 0.1% HCOOH (30:70 to 4.7 mL min1) (manual collection)) led to the isolation of trigocherrins D (4) (0.19 mg), E (5) (0.35 mg) and F (6) (0.8 mg), and the re-isolation of trigocherrin B (2). Fraction F0 247 corresponded to the previously isolated trigocherriolide B (8) (1.3 mg). 4.3.1. Trigocherrin B (2) Amorphous powder, [a]D25  105 (c 0.10, MeOH); CD (MeOH, c 1.0  103) kmax (De): 265 (3.7), 210 (1.7) nm; UV (MeOH) kmax (log e) 266 (4.26) nm; IR mmax 3396, 2935, 1719, 1601 cm1; for 1 H and 13C NMR spectroscopic data, see Table 1; HR-ESIMS (pos.) m/z 797.1602 (calcd. for C41H36Cl2O11Na, 797.1532). 4.3.2. Trigocherrin C (3) Amorphous powder, for 1H and 13C NMR spectroscopic data, see Table 1; HR-ESIMS (pos.) m/z 733.2513 (calcd. for C38H47Cl2O10, 733.2546). 4.3.3. Trigocherrin D (4) Amorphous powder, for 1H and 13C NMR spectroscopic data, see Table 1; HR-ESIMS (pos.) m/z 775.1769 (calcd. for C41H37Cl2O11, 775.1713). 4.3.4. Trigocherrin E (5) Amorphous powder, for 1H and 13C NMR spectroscopic data, see Table 1; HR-ESIMS (pos.) m/z 793.1888 (calcd. for C41H39Cl2O12, 793.1819). 4.3.5. Trigocherrin F (6) Amorphous powder, [a]D25  85 (c 0.10, MeOH); CD (MeOH, c 0.8  103) kmax (De): 268 (5.3), 224 (1.8) nm; UV (MeOH) kmax (log e) 266 (4.40) nm; IR mmax 3423, 2928, 1720 cm1; for 1H and 13 C NMR spectroscopic data, see Table 1; HR-ESIMS (pos.) m/z 775.1785 (calcd. for C41H37Cl2O11, 775.1713). 4.3.6. Trigocherriolide A (7) Amorphous powder, [a]D25  9 (c 0.10, MeOH); CD (MeOH, c 1.0  103) kmax (De): 254 (9.1) nm; UV (MeOH) kmax (log e) 303 (3.87), 255 (4.31), 242 (4.34) nm; IR mmax 3459, 2930, 1709, 1674, 1614 cm1; for 1H and 13C NMR spectroscopic data, see Table 2; HR-ESIMS (pos.) m/z 875.3089 (calcd. for C45H53ClO14Na, 875.3021). 4.3.7. Trigocherriolide B (8) Amorphous powder, [a]D25  10 (c 0.10, MeOH); CD (MeOH, c 1.0  103) kmax (De): 253 (9.7), 210 (5.0) nm; UV (MeOH) kmax (log e) 255 (4.41) nm; IR mmax 3459, 2941, 1708 cm1; for 1H and 13 C NMR spectroscopic data, see Table 2; HR-ESIMS (pos.) m/z 717.3062 (calcd. for C38H50ClO11, 717.3042).

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4.3.8. Trigocherriolide C (9) Amorphous powder, [a]D25  65 (c 0.10, MeOH); CD (MeOH, c 1.0  103) kmax (De): 256 (6.2), 210 (3.4) nm; UV (MeOH) kmax (log e) 256 (4.36) nm; for 1H and 13C NMR spectroscopic data, see Table 2; HR-ESIMS (pos.) m/z 837.3251 (calcd. for C45H54ClO13, 837.3253). 4.3.9. Trigocherriolide D (10) Amorphous powder, [a]D25  9 (c 0.10, MeOH); CD (MeOH, c 2.0  103) kmax (De): 265 (9.5), 211 (4.0) nm; UV (MeOH) kmax (log e) 267 (4.27) nm; for 1H and 13C NMR spectroscopic data, see Table 2; HR-ESIMS (neg.) m/z 749.2465 (calcd. for C38H47Cl2O11, 749.2495). 4.4. Enzymatic activity assay of the dengue polymerase Polymerase activity was assayed by monitoring the incorporation of radiolabeled guanosine into a homopolymeric cytosine RNA template, as previously described (Allard et al., 2011). The enzymes were produced and purified as previously described (Selisko et al., 2006). The determination of the IC50 of the pure compounds was performed according to a detailed procedure that was previously described (Allard et al., 2011). IC50 was determined using the following equation: % of enzyme activity = 100/(1 + (I2)/IC50), in which I is the concentration of inhibitor. IC50 was determined from curve-fitting using Kaleidagraph (Synergy Software). For each value, results were obtained using triplicates in a single experiment. 30 -deoxy-GTP was used as the reference. 4.5. Virus-cell-based antiviral assay Throughout the experiments, Vero (African green monkey kidney) cells were used. The following viruses were used: chikungunya virus strain 899, Sindbis virus strain HRsp and Semliki forest virus strain Vietnam. Serial dilutions of extract, fractions or pure compounds, as well as the reference compound chloroquine, were prepared in assay medium [MEM Rega3 (Cat. N°19993013; Invitrogen), 2% FCS (Integro), 5 mL 200 mM L-glutamine, and 5 mL 7.5% sodium bicarbonate] that was added to empty wells of a 96-well microtiter plate (Falcon, BD) on a liquid handling platform (Freedom EVO200, Tecan). Subsequently, 50 lL of a 4x virus dilution in assay medium was added, followed by 50 lL of a cell suspension. This suspension, with a cell density of 25,000 cells/50 lL, was prepared from a Vero cell line subcultured in cell growth medium (MEM Rega3 supplemented with 10% FCS, 5 mL L-glutamine, and 5 mL sodium bicarbonate) at a ratio of 1:4 and grown for 7 days in 150 cm2 tissue culture flasks (Techno Plastic Products). The assay plates were returned to the incubator for 6–7 days (37 °C, 5% CO2, 95–99% relative humidity), a time at which maximal virus-induced cell death or cytopathic effect (CPE) is observed in untreated, infected controls. Subsequently, the assay medium was aspirated, replaced with 75 lL of a 5% MTS (Promega) solution in phenol redfree medium and incubated for 1.5 h. Absorbance was measured at a wavelength of 498 nm (Safire2, Tecan); optical densities (OD values) reached 0.6–0.8 for the untreated, uninfected controls. Raw data were converted to percentage of controls and the EC50 (50% effective concentration or concentration which is calculated to inhibit virus-induced cell death by 50%) and CC50 (50% antimetabolic concentration or concentration which is calculated to inhibit the overall cell metabolism by 50%) were derived from the dose– response curves. All assay conditions producing an antiviral effect exceeding 50% were checked microscopically for minor signs of CPE or adverse effects on the host cell (i.e. altered cell morphology. . .). A compound is only considered to elicit a selective antiviral

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effect on virus replication when, following microscopic quality control, at least at one concentration of compound, no CPE nor any adverse effect is observed (image resembling untreated, uninfected cells). Multiple, independent experiments were performed. Acknowledgments The authors are very grateful to South Province of New Caledonia (Dry Forest Conservation Program), and M. Metzdorf, owner of the parcel of dry forest, who facilitated our field investigations. We are also grateful to ICSN-CNRS for a fellowship (P.-M.A.). We also would like to acknowledge Stijn Delmotte, Tom Bellon, Mieke Flament and Annelies De Ceulaer for their excellent technical assistance in the acquisition of the antiviral data. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2012. 07.023. These data include MOL files and InChiKeys of the most important compounds described in this article. References Allard, P.-M., Dau, E.T.H., Eydoux, C., Guillemot, J.-C., Dumontet, V., Poullain, C., Canard, B., Guéritte, F., Litaudon, M., 2011. Alkylated flavanones from the bark of Cryptocarya chartacea as dengue virus NS5 polymerase inhibitors. J. Nat. Prod. 74, 2446–2453. Allard, P.-M., Martin, M.-T., Tran Huu Dau, M.-E., Leyssen, P., Guéritte, F., Litaudon, M., 2012. Trigocherrin A, the first natural chlorinated daphnane diterpene orthoester from Trigonostemon cherrieri. Org. Lett. 14, 342–345. Chan, S.-C., Chung, M.-I., Yen, M.-H., Lien, G.-H., Lin, C.-N., Chiang, M.Y., 2000. Chingazumianine, a novel dichlorinated alkaloid from Corydalis koidzumiana. Helv. Chim. Acta 83, 2993–2999. Chen, H.-D., He, X.-F., Ai, J., Geng, M.-Y., Yue, J.-M., 2009. Trigochilides A and B, two highly modified daphnane-type diterpenoids from Trigonostemon chinensis. Org. Lett. 11, 4080–4083. Chen, H.-D., Yang, S.-P., He, X.-F., Ai, J., Liu, Z.-K., Liu, H.-B., Geng, M.-Y., Yue, J.-M., 2010a. Trigochinins A–C: Three new daphnane-type diterpenes from Trigonostemon chinensis. Org. Lett. 12, 1168–1171. Chen, H.-D., Yang, S.-P., He, X.-F., Liu, H.-B., Ding, J., Yue, J.-M., 2010b. Trigochinins D–I: Six new daphnane-type diterpenoids from Trigonostemon chinensis. Tetrahedron 66, 5065–5070.

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