Natural Inhibitors of the RhoAâp115 Complex from the Bark of

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

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Natural Inhibitors of the RhoA−p115 Complex from the Bark of Meiogyne baillonii Florent Olivon,† Louis-Feĺ ix Nothias,† Vincent Dumontet,† Pascal Retailleau,† Sylvie Berger,§ Gilles Ferry,§ William Cohen,§ Bruno Pfeiffer,§ Jean A. Boutin,§ Elisabeth Scalbert,§ Fanny Roussi,† and Marc Litaudon*,† †

Institut de Chimie des Substances Naturelles, CNRS-ICSN, UPR 2301, Université Paris-Saclay, 91198, Gif-sur-Yvette, France Institut de Recherches Servier, 78290 Croissy-sur-Seine, France

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§

S Supporting Information *

ABSTRACT: In an effort to find potent natural inhibitors of RhoA and p115 signaling G-proteins, a systematic in vitro evaluation using enzymatic and plasmonic resonance assays was undertaken on 11 317 plant extracts. The screening procedure led to the selection of the New Caledonian endemic species Meiogyne baillonii for a chemical investigation. Using a bioguided isolation procedure, three enediyne-γ-butyrolactones (1−3) and two enediyne-γ-butenolides (4 and 5), named sapranthins H− L, respectively, two enediyne carboxylic acid (6 and 7), two depsidones, stictic acid (8) and baillonic acid (9), aristolactams AIa and AIIa (10 and 11), and two aporphines, dehydroroemerine (12) and noraristolodione (13), were isolated from the ethyl acetate extract of the bark. The structures of the new compounds (1−6, 9, and 11) and their relative configurations were established by NMR spectroscopic analysis and by X-ray diffraction analysis for compound 9. Only stictic acid (8) exhibited a significant inhibiting activity of the RhoA−p115 complex, with an EC50 value of 0.19 ± 0.05 mM. This is the first time that a natural inhibitor of the complex RhoA−p115’s activity was discovered from an HTS performed over a collection of higher plant extracts. Thus, stictic acid (8) could be used as the first reference compound inhibiting the interaction between RhoA and p115.

H

release to allow Rho proteins to bind GTP. Targeting one of these RhoGEFs in order to inhibit RhoA activation is thus of great interest. Among these, Arhgef1 (p115), described to mediate the effect of angiotensin II on vascular tone and blood pressure, was considered as a potential target for the treatment of hypertension.7 A high-throughput screening (HTS) was conducted on a collection of 11 317 EtOAc extracts prepared from various parts of approximately 5200 tropical plant species. The assay aimed at finding extracts able to inhibit the activation of RhoA by p115 and led to the selection of Meiogyne baillonii (Guillaumin) E.C.H van Heusden (Annonaceae). M. baillonii (syn. Uvaria baillonii Guillaumin) is a small tree growing in rain forests of the main island of New Caledonia.10 The genus Meiogyne consists of about 12 species and is distributed

igh blood pressure is an important health concern and a major risk factor for cardiovascular morbidity and mortality. The peptide vasoconstrictor angiotensin II acts directly on vascular smooth muscle, thereby regulating vascular tone by a mechanism involving its binding with the AT1 receptor.1,2 This activation leads to various effects, particularly the activation of RhoA, a small GTP binding protein, and of its downstream signaling cascade.3,4 The inhibition of AT1 reduces the up-regulation of RhoA and Rho-kinase (ROCK) activities in hypertensive rats.5,6 Thus, the RhoA/ROCK pathway has been implicated in numerous diseases of the cardiovascular system.7−9 Therefore, inhibiting RhoA activation could reduce the development of angiotensin-II-dependent hypertension. RhoA is a molecular switch that cycles between an inactive GDP bound form and an active GTPbound one able to activate the ROCK pathway. Rho GTPase activation is controlled by proteins known as Rho guanine nucleotide exchange factors (RhoGEFs). They stimulate GDP © XXXX American Chemical Society and American Society of Pharmacognosy

Received: March 13, 2018

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DOI: 10.1021/acs.jnatprod.8b00209 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

through Southeast Asia from India to New Caledonia.11 Previous studies on species within the genus Meiogyne reported the presence of alkaloids,12 acetylenic butyrolactones,13 and dimeric sesquiterpenoids possessing an unprecedent cis-decalin carbon skeleton.14 No phytochemical study has been carried out on this species. In this paper, the isolation and structural elucidation of three enediyne-γ-butyrolactones (1−3), two enediyne-γ-butenolides (4 and 5), two enediyne carboxylic acids (6 and 7), two depsidones (8 and 9), aristolactams AIa and AIIa (10 and 11), and two aporphines, dehydroroemerine (12) and noraristolodione (13), of which compounds 1−6, 9, and 11 are new, are discussed. These compounds were evaluated for their ability to inhibit the Rho GTPase signaling pathways, particularly RhoA−p115 and RhoA−Net1 complexes.

Figure 1. Bar graph representing sample distribution. The threshold of activity is established at 32% (red bar).

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RESULTS AND DISCUSSION In order to find inhibitors of the RhoA−p115 complex, two bioassays were developed: the first one is an enzymatic assay visualizing the effect of extracts or compounds on the GDP/ GTP exchange capacity of the RhoA complexes, and the second one is based on a surface plasmonic resonance (SPR) approach to detect extracts or compounds able to modulate the association between p115 and RhoA. The first test was used to screen a large number of samples, pure compounds, and extracts, while the SPR assay was used for bioguided fractionation of active extracts. Prior to biological screening of extracts, an in-house library of approximately 115 000 pure compounds was screened at 10 μM using a high-throughput fluorescence polarization assay, but no significantly active hit was detected. We thus turned to an alternative assay involving direct measurement of fluorescence intensity and using a similar approach of guanine nucleotide exchange assay with which the plant extracts collection was screened. One should nevertheless point out that if the approximately 11 000 results were distributed as a Gaussian curve (Figure 1), centered essentially at 0 (no activity, as expected), too many positive results would be found in the first round of the HTS campaign (191 out of 11 316, 1.6%). Among these positive extracts, 136 contained fluorescent compounds interfering with the assay and, thus, leading to many false positive results. A second

round of analysis was performed to separate the fluorescent extracts from the nonfluorescent ones. We also screened the remaining extracts with the initial method of fluorescence polarization but taking into account the increase of the fluorescence emitted over time rather than a change in the fluorescence polarization of the emitted light (Experimental Section). Based on these complementary assays, only 55 of the 11 317 EtOAc extracts were found to show significant activities with more than 32% of inhibition recorded at 10 μg mL−1. An extra round of evaluation was done, and the 15 most active extracts were kept for further studies. Among these extracts, the EtOAc extract of the bark of M. baillonii exhibited significant inhibition of the complex Rhoa−p115 with 97 ± 3% inhibition at 1 mg.mL−1 and an EC50 of 0.4 mg.mL−1, while by using the SPR assay the EC50 value is close to 0.5 mg.mL−1, as indicated by the pink curve in Figure 2. This extract was subjected to a bioassay-guided purification using silica gel chromatography to afford 19 fractions. The active fractions were subjected to multiple normal- and reverse-phase chromatography steps, guided by the measurement of the inhibitory activities of the fraction in the same test. This process led to the isolation of five acetylenic butyrolactones (1−5), two acetylenic fatty acids (6 and 7), two depsidones (8 and 9), and four alkaloids (10−13). B



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bands at λmax (log ε) 214 (3.50), 255 (3.61), 269 (3.65), and 285 (3.58) nm for an enediyne-type moiety.13,15 The 1H NMR spectrum of 1 (Table 1) showed the presence of a vinylic group at δH 5.75 (ddt, J = 17.4, 10.1, 6.2 Hz, H-15′), 4.96 (m, H-16′, pro-E), and 5.00 (ddt, J = 17.4, 1.6, 1.6 Hz, H-16′, proZ), a disubstituted double bond at δH 5.48 (H-11′) and δH 6.25 (H-12′), two oxygenated methines at δH 4.74 (H-5) and 5.13 (H-4), and two methyl groups at δH 1.31 (Me-5) and 2.10 (Me-2″). The COSY spectrum displayed two spin systems, from Me-5 to CH2-6′ at δH 2.29, and from the methine H-11′ to the terminal olefinic methylene. The diyne junction between these two moieties was established via the long-range COSY correlation (7JHH) between H-11′ at δH 5.48 and CH2-6′ at δH 2.29 and HMBC correlations from H-5′ (δH 1.50), H-6′ (δH 2.29), H-11′ (δH 5.48), and H-12′ (δH 6.25) to the four acetylenic carbons C-7′ (δC 83.7), C-8′ (δC 65.5), C-9′ (δC 73.2), and C-10′ (δC 74.1). The E configuration of the Δ11′(12′) double bond was confirmed by the large vicinal coupling constant value (J = 15.9 Hz) between H-11′ and H-12′ (δH 6.25). The chemical shifts at δH 4.74 (dq, J = 6.5, 4.9 Hz, H-5), δH 5.13 (dd, J = 4.9, 2.8 Hz, H-4), and δH 2.58 (ddd, J = 8.2, 6.5, 2.8 Hz, H-3) were assigned to the methine protons of the butyrolactone moiety. The trans/cis relationships of protons H3/H-4/H-5 were suggested by the coupling constant values of 2.8 and 4.9 Hz (J3,4 and J4,5, respectively)16 and identical NMR data to those reported for closely related structural analogues.17 The presence of an acetoxy moiety at C-4 was supported by the resonance of a carbonyl function at δC 170.3

Figure 2. Assay of p115 interaction with RhoA in the presence of the EtOAc extract of M. baillonii bark (SD2239). Sensorgrams of p115 interaction with RhoA in the absence (yellow curve) or in the presence of M. baillonii extract. This sample has been evaluated at five different concentrations (0.0625, 0.125, 0.25, 0.5, and 1 mg.mL−1). After being mixed with p115 (4 μM) each extract concentration was injected onto the chip surface, on which RhoA has been captured. The binding level of p115 in the presence of different extract concentrations is then compared to native p115 interaction with RhoA to determine an EC50.

The HRESIMS data of sapranthin H (1) indicated an [M + H]+ ion at m/z 371.2207, which, in conjunction with the 13C NMR spectroscopic data, is consistent with a molecular formula of C23H30O4 (calcd for C23H31O4, 371.2222). The UV spectrum of compound 1 exhibited characteristic absorption

Table 1. NMR Spectroscopic Data (CDCl3) of Compounds 1−3 (300, 75 MHz) 1 position

δC

2 3 4 5 5-Me 1′-a 1′-b 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′-E 16′-Z 17′ 18′-E 18′-Z 1″ 2″

176.7 47.2 75.7 76.9 14.4 28.6a 27.0 28.9a 28.6a 28.3 19.7 83.7 65.5 73.2 74.1 109.3 147.3 32.7 32.8 137.5 115.6

2 δH (J in Hz)

2.58 5.13 4.74 1.31 1.62 1.73 1.50 1.35 1.40 1.50 2.29

ddd (8.2, 6.5, 2.8) dd (4.9, 2.8) qd (6.5, 4.9) d (6.5) m m m m m m t (6.8)

5.48 6.25 2.19 2.13 5.75 4.96 5.00

d (15.9) dt (15.9, 6.7) m m ddt (17.4, 10.1, 6.2) m ddt (17.4, 1.6, 1.6)

δC

3 δH (J in Hz)

176.7 47.2 75.7 76.9 14.4 28.7a 27.1 28.9a 28.7a 28.3 19.8 85.0 65.5 78.6 72.2 108.8 146.9 30.1 33.1 137.8 115.4

2.58 5.14 4.75 1.32 1.62 1.73 1.50 1.37 1.40 1.50 2.31

ddd (8.2, 6.5, 2.9) dd (4.9, 2.9) qd (6.5, 4.9) d (6.5) m m m m m m t (6.8)

5.47 6.01 2.42 2.15 5.79 4.97 5.02

dt (10.8, 1.1) dt (10.8, 7.4) dtd (7.4, 7.4, 1.1) tdm (7.4, 6.5) ddt (17.1, 10.1, 6.5) m ddt (17.1, 1.6, 1.6)

δC 176.8 47.3 75.7 76.8 14.4 28.7a 27.2 28.9a 29.1a 29.3a 29.4a 28.4 19.7 84.0 65.4 73.3 74.0 109.4 147.2 32.7 32.9 137.5 115.6

170.3 20.9

2.10 s

170.3 20.9

2.10 s

170.3 20.9

δH (J in Hz) 2.58 ddd (8.3, 6.5, 2.8) 5.14 dd (4.9, 2.8) 4.75 qd (6.5, 4.9) 1.31 d (6.5) 1.61 m 1.72 m 1.50 m 1.27−1.40 m 1.27−1.40 m 1.27−1.40 m 1.27−1.40 m 1.50 m 2.28 t (6.9)

5.48 6.24 2.19 2.13

d (15.9) dt (15.9, 6.8) m m

5.75 ddt (17.1, 10.3, 6.3) 4.96 dm (10.3) 5.00 ddt (17.1, 1.6, 1.6) 2.09 s

a

Assignments can be interchanged within the same column. C



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Table 2. NMR Spectroscopic Data (CDCl3) of Compounds 4, 5 (500, 125 MHz), and 6 (300, 75 MHz) 4 pos.

δC

2 3 4 5 5-Me 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′-E 16′-Z 17′ 18′-E 18′-Z

174.2 134.5 149.1 77.6 19.5 25.4 27.6 28.9a 29.1a 29.3a 29.3a 28.4 19.7 84.0 65.4 73.3 74.1 109.4 147.2 32.7 32.9 137.5 115.6

δH (J in Hz)

6.96 brs 4.97 m 1.39 d (6.8) 2.25 brt (7.9) 1.52 m 1.25−1.40 m 1.25−1.40 m 1.25−1.40 m 1.25−1.40 m 1.50 m 2.29 t (7.0)

5.49 6.25 2.20 2.13

d (15.9) dt (15.9, 6.9) brtd (7.0, 6.9) brtd (7.0, 6.6)

5 δC 174.2 134.3 149.3 77.7 19.5 25.3 27.4 28.8a 28.7a 28.2 19.7 85.1 65.4 78.5 72.1 108.7 147.0 30.1 33.0 137.8 115.4

6

δH (J in Hz)

6.99 4.98 1.39 2.25 1.53 1.33 1.42 1.50 2.32

brs m d (6.8) t (7.5) m m m m t (6.9)

5.47 6.02 2.42 2.15 5.79 4.97 5.02

d (10.8) dt (10.8, 7.5) dt (7.5, 7.3) tdm (7.3, 6.4) ddt (17.0, 10.3, 6.4) m dm (17.0)

pos.

δC

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

179.7 34.3 24.9 29.0a 29.2a 29.2a 29.4a 29.5a 28.5 19.8 84.0 65.4 73.3 74.0 109.4 147.2 32.7 32.9 137.5 115.6

δH (J in Hz) 2.32 t (7.4) 1.60 m 1.25−1.40 m 1.25−1.40 m 1.25−1.40 m 1.25−1.40 m 1.25−1.40 m 1.50 m 2.28 t (6.7)

5.48 6.24 2.19 2.13 5.75 4.97 5.00

d (15.9) dt (15.9, 6.7) m m ddt (17.0, 10.1, 6.3) m m

5.75 ddt (17.2, 10.0, 6.6) 4.97 m 5.00 ddt (17.2, 1.6, 1.6)

a

Assignments can be interchanged within the same column.

(C-1″) observed in the 13C NMR spectrum and an HMBC correlation from H-4 (δ 5.13) to C-1″. The specific rotation of 1 {[α]24D +36 (c 1, CHCl3)} suggested the all-R absolute configuration of C-3, C-4, and C-5 by comparison with data reported for structurally related compounds.16,17 Therefore, compound 1 is (3R,4R,5R)-4-O-acetoxy-3-(hexadeca-11E,15diene-7,9-diynyl)-5-methyltetrahydrofuran-2-one. Sapranthin I (2) was assigned the molecular formula C23H30O4 on the basis of 13C NMR spectroscopic and HRESIMS data. From this formula and its spectroscopic data, closely comparable to those of 1, it is apparent that 2 had a structure similar to 1 but with a coupling constant J11′,12′ of 10.8 Hz for the olefinic protons, indicating a Z-double bond for 2. Taking into account that the sign and magnitude of the specific rotation of 2 {[α]24D +48 (c 0.1, CHCl3)} are comparable to those of compound 1, compound 2 possessed the same absolute configuration. Thus, compound 2 is (3R,4R,5R)-4-O-acetoxy-3-(hexadeca-11Z,15-diene-7,9-diynyl)-5-methyltetrahydrofuran-2-one. Compound 3 was assigned the molecular formula C25H34O4, as supported by HRESIMS, showing an [M + H]+ ion at m/z 399.2519 (calcd for C25H35O4, 399.2535). This molecular formula only differs by 28 mass units from that of 1, suggesting that 3 possessed two additional CH2 groups. From this formula and its spectroscopic data closely comparable to those of compound 1, it is apparent that compound 3 had a structure similar to 1 but with a carbon aliphatic side chain including seven methylene groups instead of the five methylenes for 1. The sign and magnitude of the specific rotation of 3 {[α]24D +36 (c 1, CHCl3)} are identical to those of 1. Compound 3, named sapranthin J, is assigned as (3R,4R,5R)-4-O-acetoxy-3-

(octadeca-13E,17-diene-9,11-diynyl)-5-methyltetrahydrofuran2-one. The HRESIMS data of sapranthin K (4) showed an [M + H]+ ion at m/z 339.2337 (calcd for C23H31O2, 339.2324), which in conjunction with the 13C NMR spectroscopic data is consistent with a molecular formula C23H30O2, indicating nine indices of hydrogen deficiency. The characteristic UV absorption spectrum together with NMR data of compound 4 (Table 2) indicated that compounds 4 and 3 possessed the same aliphatic side chain but a different lactone moiety. Indeed, the analysis of NMR data indicated the absence of the acetoxy group resonances and the presence of a vinylic methine at δC/H 149.1/6.96 (CH-4). In the HMBC spectrum, H-4 is correlated with the quaternary carbon at δC 134.5 (C-3), the methine at δC 77.6 (C-5), and the lactone carbonyl at δC 174.2 (C-2), indicating that compound 4 possesses an α,βunsaturated-γ-lactone moiety. The geometry of the Δ13′(14′) double bond is identical to compound 3. Finally, the (5R) absolute configuration was established by comparison of the specific rotation value of 4 {[α]24D −20 (c 0.1, CHCl3)} with data reported in the literature,18−20 indicating that compound 4 is assigned as (5R)-3-(octadeca-13E,17-diene-9,11-diynyl)-5methylfuran-2-one. The molecular formula, C21H26O2, of sapranthin L (5) was deduced from its HRESIMS ion at m/z 311.2011 [M + H]+ (calcd 311.2011). From this formula and its NMR spectroscopic data, it can be inferred that compound 5 possessed a similar structure to 4 but with an aliphatic side chain including five instead of the seven methylene groups of 4. In addition, a coupling constant J11′,12′ of 10.8 Hz for the olefinic protons indicated a Z-double bond for 2. The value of D



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positions of the methyl and methoxy groups on the first aromatic ring were deduced via the correlations from H-5 (δH 6.80) to C-1 (δC 114.3), C-3 (δC 105.3), and C-4 (δC 164.4), from the methyl protons to C-1, C-5 (δC 115.7), and C-6 (δC 145.8), from the methoxy protons to C-4, and from H-3 (δH 7.03) to C-1, C-4, and C-5 (δC 115.7). The position of the substituents of the second aromatic ring, i.e., a carboxylic acid group and a methoxy group, and two oxygenated carbons was established via HMBC correlations from the methoxy protons to C-2′ (δC 157.8), from H-1′ to C-2′, C-3′ (δC 111.1), C-5′ (δC 144.5), C-6′ (δC 126.4), and C-7′ (δC 165.6), and from H3′ to C-1′, C-2′, C-4′ (δC 147.2), and C-5′. The resonance at δC 164.2 (C-7), which suggested the presence of an additional carbonyl ester group, was not yet assigned. Based on these observations and the molecular formula, it could be established that the two aromatic rings are connected by ether and ester bridges, assuming a depsidone skeleton with a central sevenmembered ring. However, at this stage, two structures can be proposed since the connection of the two aromatic rings through an ether bond across either C-2/C-5′ or C-2/C-4′ was uncertain. Baillonic acid (9) was obtained as colorless crystals from acetonitrile. Its structure was unequivocally established by single-crystal X-ray diffraction analysis (Figure 3).

the specific rotation of 5, [α]24D −25 (c 0.1, CHCl3), is similar to that of compound 4, thus defining its structure as (5R)-3(hexadeca-11Z,15-diene-7,9-diynyl)-5-methylfuran-2-one. Sapranthins H−L (1−5) are unstable compounds, as they are oxidized readily in air, turning dark purple, blue, or brown. The HRESIMS data of compound 6 showed an [M + H]+ ion at m/z 301.2193 (calcd for C20H29O2, 301.2168) consistent with the molecular formula C20H28O2 with seven indices of hydrogen deficiency. The UV spectrum exhibited characteristic absorption bands at 214 (3.36), 255 (3.47), 269 (3.53), and 285 (3.52) nm for an enediyne moiety. Its 1H and 13 C NMR data (Table 2) were closely comparable to those reported for octadeca-13E,17-diene-9,11-diynoic acid,21 indicating that both compounds have identical long carbon chains but with nine methylene groups adjacent to the carboxylic acid group for 6, instead of seven methylenes for the known compound 7. Analysis of 1H−1H COSY, HSQC, and HMBC spectra confirmed the assignment of the structure of compound 6 as eicosa-15E,19-diene-11,13-diynoic acid. The HRESIMS of baillonic acid (9) showed an [M − H]− ion peak at m/z 329.0663 (calcd for C17H13O7, 329.0661), consistent with the molecular formula C17H14O7, indicating 11 indices of hydrogen deficiency. The IR spectrum showed a strong absorption band at 1705 cm−1 for a carbonyl function and a large absorption band between 3120 and 2625 cm−1 for a hydroxy group. Its 1H NMR data (Table 3) indicated the Table 3. NMR Spectroscopic Data of Baillonic Acid (9) (500, 125 MHz in Acetone-d6) and Compound 11 (500, 125 MHz in DMSO-d6) 9 pos.

δC

1 2 3 4 4-OMe 5 6 7 8 1′ 2′ 2′-OMe 3′ 4′ 5′ 6′ 7′

114.3 162.2 105.3 164.4 56.3 115.7 145.8 164.2 21.3 113.3 157.8 56.6 111.1 147.2 144.5 126.4 165.6

δH (J in Hz)

7.03 d (2.3) 3.86 s 6.80 d (2.3)

2.46 s 7.21 d (3.0) 3.86 s 7.05 d (3.0)

11 pos.

δC

1 2 3 3-OH 4 4-OMe 4a 5a 5 6 7 7-OH 8 9a 9 10 10a 11 N−H

121.7 111.8 152.0

δH (J in Hz) 7.49 s 10.14 brs

147.8 59.3 120.7 118.7 128.2 114.9 156.6 113.0 136.8 103.4 135.5 121.4 168.6

3.97 s

Figure 3. ORTEP view of baillonic acid (9). 8.90 d (9.0) 7.03 dd (9.0, 2.5)

A second depsidone, stictic acid (8), was isolated as a racemic mixture and identified from the comparison of its observed and reported spectral data.22 Moreover, NMR data and 1D and 2D NMR spectra of compound 8 recorded in DMF-d7 and pyridine-d5 are reported in Figures S3 and S10 (Supporting Information). The present finding is the first example of depsidones being isolated from an Annonaceae species. Only Garcinia species are reported to produce such type of secondary metabolites in higher plants. 23−26 Depsidones are often produced by lichens,27,28 fungi,29,30 and endophytic fungi.31,32 Despite stictic acid (8) being isolated in relatively high yield, an endophytic or lichenic origin cannot be excluded. The known compounds aristolactam AIa (10), dehydroroemerine (12), and noraristolodione (13) were identified by comparison of spectroscopic data (UV, IR, NMR, and MS) with reported data.33−35 Compound 11 was isolated as a reddish, amorphous solid. The HRESIMS data showed an [M + H]+ ion at m/z 282.0765, which in conjunction with the 13 C NMR spectroscopic data established the molecular formula of 11

9.80 s 7.21 d (2.5) 6.91 s

10.67 s

presence of two methoxy groups at δH 3.86 (MeO-4 and MeO2′), an aromatic methyl substituent at δH 2.46 (s, Me-8), and four aromatic resonances at δH 6.80 (d, J = 2.3 Hz, H-5), 7.03 (d, J = 2.3 Hz, H-3), 7.05 (d, J = 3.0 Hz, H-3′), and 7.21 (d, J = 3.0 Hz, H-1′) for two 1,2,3,5-tetrasubstituted benzene rings. The 13C NMR and HSQC spectra confirmed the presence of 17 carbons consisting of three methyl, four sp2 methines, three sp2 quaternary carbons, and seven oxygenated sp2 tertiary carbons. The substitution patterns of the aromatic rings were determined through analysis of the HMBC spectrum. The E



Article

μM. These results are in line with the minute differences observed in various GEF complexes with RhoA that led to changes in specificities of compounds despite variations of a single amino acid between the various GEF proteins.39 In conclusion, from the EtOAc extract of the bark of M. baillonii, a bioguided fractionation procedure was carried out using both enzymatic and surface plasmonic resonance assays specific for RhoA−p115 complex inhibition, resulting in the isolation of five new acetylenic butyrolactones (1−5) and two known acetylenic fatty acids (6 and 7), a new (9) and a known depsidone (8), a new and a known aristolactam (11 and 10, respectively), and two known aporphine alkaloids (12 and 13). The ability of the isolated compounds to inhibit the activation of RhoA by p115 was evaluated and showed that only stictic acid (8) exhibited a significant activity. This is the first time that a natural inhibitor of the complex RhoA−p115 activity was discovered from an HTS performed over a collection of higher plant extracts. Stictic acid (8) could be used as the first reference compound inhibiting the interaction between RhoA and p115.

as C16H11NO4 (calcd for C16H12NO4, 282.0766), identical to compound 10. The UV spectrum of 11 exhibited characteristic absorption bands at 234, 254, 304, and 314 nm, for a phenanthrene chromophore.36 The IR spectrum showed strong absorption bands at 3220 cm−1 for N−H, at 1714 cm−1 for a carbonyl function, and at 1690 and 1620 cm−1 for an amide group.37,38 The spectroscopic data of compound 11 were similar to those of aristolactam AIa (10), indicating that these compounds possess the same aristolactam skeleton (Table 3). Analysis of 1D and 2D NMR spectra of compounds 10 and 11 indicated that both compounds possess the same substitution pattern for the aromatic ring A and a differently substituted aromatic ring C. Indeed, an ABX system for compound 11 at δH 8.90 (d, J = 9.0 Hz, H-5), 7.03 (dd, J = 9.0, 2.5 Hz, H-6), and 7.21 (d, J = 2.5 Hz, H-8) suggested a 1,2,4trisubstituted aromatic ring C, while compound 10 possesses a 1,2,3-trisubstituted aromatic ring C instead. The C-7 position of the hydroxy group (s, 9.80) was determined through analysis of the HMBC spectrum. A NOESY cross-peak between H-8 and H-9 confirmed the previous observation. Compound 11 is a new aristolactam and is named aristolactam AIIa. Using a Biacore assay, compounds 1−13 were evaluated for their capacity to inhibit the GDP/GTP exchange of the RhoA−p115 complex. Only stictic acid (8) exhibited a significant inhibiting activity on this complex, with an IC50 of 187 ± 51 μM. This is the first time that a natural compound was found to inhibit the RhoA−p115 complex activity. None of the other compounds induced significant inhibition of the RhoaA−p115 complex at a concentration of 200 μM, a concentration at which several of the compounds tested seem to aggregate. In order to check whether the interaction of the isolated compounds with the RhoA−p115 complex had some level of specificity or not, a second assay implying RhoA and Net1, another protein partner among the ∼20 known ones, was designed. However, in this second assay, most of the compounds tend to aggregate at a concentration of 200 μM (Table 4). Only aporphine 12 showed a weak inhibition of the RhoA−Net1 complex, with an IC50 value of 120 ± 31 μM, but the compound tends to aggregate at a concentration of 360

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Table 4. Inhibiting Activities of the RhoA−p115 and RhoA−Net1 Complexes of Compounds 1−13 inhibition complex RhoA−p115 compound

% (at 200 μM)

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

10% 0% 0% nd 0% nd 0% 50.5% 1% 0% nd 21% 0%

IC50 in μM

187 ± 51

RhoA−Net1 % (at 200 μM) nda nd nd nd nd nd nd nd 5% 5% nd 83% 4%

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured at 25 °C on an Anton Paar MCP 300 polarimeter. UV spectra were recorded on a Varian Cary 100 UV−vis spectrophotometer. IR spectra were recorded with a Nicolet FTIR 205 spectrophotometer. NMR spectra were recorded in CDCl3, DMFd7, pyridine-d5, DMSO-d6, and methanol-d4 on a Bruker 600 MHz instrument (Avance 600) using a 1.7 mm microprobe for compound 8, 10, and 12, on a Bruker 500 MHz instrument (Avance 500) for plant extracts, fractions, and compounds 4, 5, 7, 9, 11, and 13, and on a Bruker 300 MHz instrument (Avance 300) for compounds 1, 2, 3, and 6. Chemical shifts (relative to tetramethylsilane) are in ppm, and coupling constants are in Hz. SunFire analytical and preparative C18 columns (250 × 4.6 mm and 250 × 19 mm i.d.; 10 μm Waters) and Kromasil analytical and preparative C18 columns (250 × 4.6 mm and 250 × 21.2 mm i.d.; 5 μm Thermo) were used for HPLC separations using a Dionex system equipped with a sample manager (Gilson 215 liquid handler), a column fluidics organizer, a binary pump (Dionex HPG-3200BX), a UV−vis diode array detector (190−600 nm, Dionex UVD340U), and a PL-ELS 1000 ELSD Polymer Laboratory detector. Some HPLC separations were also performed on a Waters autopurification system equipped with a sample manager (Waters 2767), a column fluidics organizer, a binary pump (Waters 2525), a UV/vis diode-array detector (190−600 nm, Waters 2996), and a PLELS 1000 ELSD Polymer Laboratory detector. SFC analyses were performed with a Thar Waters SFC Investigator II System using a photodiode array detector (Waters 2998), an ELS detector (Waters 2424), and a 4-ethylpyridine column (250 × 4.6 mm, 5 μm). All solvents were purchased from Carlo Erba (France) and SDS (Peypin, France). Silica gel 60 (6−35 μm) was purchased from SDS (Peypin, France), and analytical plates (Si gel 60 F254) were purchased from Merck (France). Prepacked GraceResolv silica cartridges were used for flash chromatography using a Teledyne Isco Combiflash Rf 200i. HRESIMS data were acquired using an Acquity Waters UPLC coupled to a Waters LCT Premier XE mass spectrometer. The UPLC system was equipped with a Waters Acquity PDA dectector. Separation was achieved on a BEHC18 column (1.7 μm, 2.1 mm × 50 mm) at a flow rate of 0.6 mL.min−1. Elution was conducted with a H2O−CH3CN + 0.1% formic acid gradient as follows: 95:5 to 0:100 in 5.5 min. The ionization was carried out using an electrospray ionization source in the positive mode (range 80−1500 m/z). Plant Material. Trunk bark of Meiogyne baillonii (Guillaumin) van Heusden was collected in the Nodela Valley (New Caledonia) during April 2006 by one of the authors (V.D.). The plant was further authenticated by V.D., and a voucher specimen (DUM-0677) has been deposited at the Herbier IRD de Nouméa.

IC50 in μM

120 ± 31

a

nd = not determined. F



Article

X-ray Crystallographic Analysis for 9. All crystallographic data were collected on a Rigaku diffractometer constituted by a MM007 HF copper rotating-anode generator, equipped with Osmic confocal CMF optics, and a Rapid II curved image plate. Orthorhombic space group, P212121, a = 9.176(7) Å, b = 8.194(2) Å, c = 19.269(2) Å, V = 1446.1(12) Å3, Z = 4, Dx = 1.517 mg/m3, μ(Cu Kα) = 1.014 mm−1, and F(000) = 688 e−. Crystal dimensions: 0.49 × 0.45 × 0.29 mm3. A total of 9506 reflections were measured with 2549 independent reflections (Rint = 0.0444). Final R1 = 0.0345, wR2 = 0.0909 for 2045 I > 2σ(I). See Supporting Information for detailed crystallographic data of compound 9, which have been deposited at the Cambridge Crystallographic Data Centre (deposit no. CCDC 953667). Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: + 44-(0)223-336033 or e-mail: [email protected]). Aristolactam AIIa (11): amorphous, orange powder; UV (MeOH) λmax (log ε) 234 (4.61) nm; IR νmax 3224, 2960, 1645, 1210, 1185, 1125, 1118, 806, 730 cm−1; 1H and 13C NMR, see Table 3; HRESIMS m/z 280.0611 [M − H]− (calcd for C16H10NO4, 280.0615). Guanine Nucleotide Exchange Assays: Fluorescence Intensity. Because there were a large number of interferences of fluorescence with the extracts, we used an alternative, more robust method for purification of some extracts. The in vitro nucleotide exchange assays that were used in the present studies are based on the measurement of the increase in fluorescence emitted over time, upon incorporation of free GTP-γNH-BODIPY FL (Invitrogen, ref 35778) into a GDP-loaded His-RhoA F25N (1−180). The capacity of the extracts, or of the purified fractions, to inhibit the GDP/GTP-γNHBODIPY FL exchange reaction catalyzed by the p115 DHPH22 (395−766)-RhoA or the Net1 (149−501)-RhoA complexes was analyzed. The extracts (30 μg mL−1, 5 μL) and 1 μM GEF protein in buffer (20 mM Tris (pH 7,6), 50 mM NaCl, 1 mM MgCl2, 1 mM TCEP, 0.01% IGEPAL, 10% glycerol, 7 nM GTP-Bodipy FL) were mixed at 25 °C during 30 min. The reaction was initiated with the addition of 0.1 μM RhoA in 5 μL. Total fluorescence intensities were measured in kinetics for determination of the initial speed of exchange with an FDSS 6000 spectrophotometer (Hamamatsu) (λex = 480 nm, λem = 540 nm). Guanine Nucleotide Exchange Assays: Polarization of Fluorescence. The in vitro nucleotide exchange assays that were used in the present studies are based on the measurement of the increase in fluorescence emitted over time, upon incorporation of free GTP-γNH BODIPY FL (Invitrogen, ref 35778) into a GDP-loaded His-RhoA F25N (1−180). The capacity of the extracts, or of the purified fractions, to inhibit the GDP/GTP-γNH BODIPY FL exchange reaction catalyzed by the P115 DHPH22 (395−766)− RhoA or the Net1 (149−501)−RhoA complexes was analyzed. In 384-well black plates with clear bottoms (Greiner, ref 781091), the extracts (30 μg/mL, 5 μL/well) and 1 μM GEF protein in 10 μL of reaction buffer (20 mM Tris (pH 7.6), 50 mM NaCl, 1 mM MgCl2, 1 mM TCEP, 0.01% IGEPAL, 10% glycerol) + 7 nM GTP-Bodipy FL were mixed at 25 °C during 30 min. The reaction was initiated with the addition of 0.1 μM RhoA in 5 μL of reaction buffer. Total fluorescence intensities were measured in kinetics every minute after the start of the reaction for determination of the initial speed of exchange with an FDSS 6000 spectrophotometer (Hamamatsu) (λex = 480 nm, λem = 540 nm). The Vi was determined on the first 15 points (15 min), during which the speed of nucleotide exchange was linear. Controls where done to determine Vi max (in the presence of GEF) and Vi min (in the absence of GEF). Z′ and dynamic ranges (Vi max − Vi min) were respectively 0.62 and 42.4 for the P115 assay and 0.67 and 11.4 for the Net1 assay. Surface Plasmon Resonance Measurements. Experiments were performed on a Biacore T200 apparatus (GE Healthcare) at 25 °C in Tris/HCl pH 7.4 50 mM/NaCl 150 mM/MgCl2 1 mM/tris(2carboxyethyl)phosphine (TCEP) 1 mM/P20 0.05%/5% glycerol/2% DMSO (GE Healthcare) as running buffer at a flow rate of 50 μL min−1. Using the biotin capture kit (GE Healthcare), biotinylated RhoA was captured on a CAP sensor chip according to the

Extraction and Isolation. The dried bark (dry wt, 1400 g) was extracted with EtOAc (3 × 10 L) at 40 °C using a static highpressure/high-temperature Zippertex extractor developed in the ICSN Pilot Unit. The EtOAc extract was concentrated in vacuo at 40 °C to yield 10.9 g of residue. This extract was subjected to silica gel column chromatography (6−35 μm) by using a gradient of n-heptane/ acetone/MeOH (1:0:0 to 0:8:2) of increasing polarity to afford 19 fractions (F1 to F19), according to their TLC profiles. F5 (71 mg) was further purified using a preparative TLC plate with n-heptane/ CH2Cl2 (6:4) to give 12 (1.1 mg). F7 (1.44 g) was subjected to silica gel column chromatography using a gradient of n-heptane/acetone/ MeOH (75:25:0 to 0:8:2) of increasing polarity to afford 12 fractions (F7-1 to F7-12), according to their TLC profiles. F7-4 (311.4 mg) was purified by preparative HPLC (SunFire C18, MeCN/H2O, 75:25, + 0.1% formic acid at 20 mL min−1) to afford a mixture F7-4-1 and compounds 5 (2 mg) and 3 (7.3 mg) (tR: 19.0 and 33.5 min, respectively). F7-4-1 (14 mg) was further separated by semipreparative HPLC (Sunfire C18, (MeCN/MeOH, 1:1)/H2O, 67:33, at 4 mL.min−1), leading to compound 4 (2.6 mg) (tR: 44.5 min). Purification of fraction F8 (161 mg) by preparative HPLC (SunFire C18, MeCN/H2O, 65:35, + 0.1% formic acid, 20 mL min−1) afforded compounds 1 (10.2 mg) and 2 (5.3 mg) (tR: 47.0 and 43.0 min, respectively). Fraction F9 (413 mg) was subjected to preparative HPLC (SunFire C18, MeCN/H2O, 70:30, + 0.1% formic acid at 20 mL min−1) to yield a mixture of F9-1 and compound 6 (13 mg) (tR: 31.0 min). F9-1 (8.6 mg) was further purified by semipreparative HPLC (SunFire C18, MeCN/H2O, 60:40, + 0.1% formic acid to 100:0 at 4 mL min−1) to afford compound 7 (1.6 mg) (tR: 25.5 min). F14 (233 mg) was subjected to silica gel column chromatography by using a gradient of n-heptane/acetone/MeOH (1:0:0 to 0:8:2) to afford three fractions (F14-1 to F14-3), from which F14-3 (150 mg) was further subjected to preparative HPLC (Kromasil C18, MeCN/H2O, 35:65, + 0.1% formic acid to 50:50 in 23 min at 20 mL min−1), affording a mixture of F14-3-1 and compounds 9 (2.5 mg), 11 (2.8 mg), and 13 (1.6 mg). F14-3-1 (7 mg) was later purified by semipreparative SFC, using a 4-ethylpyridine column and a gradient of CO2 and MeOH as cosolvent (MeOH 30% to 35% in 10 min), leading to compound 10 (1.0 mg). F15 (254.5 mg) was subjected to preparative HPLC (SunFire C18, MeCN/H2O, 30:70, + 0.1% formic acid to 50:50 at 20 mL min−1) to afford compound 8 (53 mg). Sapranthin H (1): yellowish oil; [α]24D +36 (c 1, CHCl3); UV (CHCl3) λmax (log ε) 214 (3.50), 255 (3.61), 269 (3.65), 285 (3.58) nm; 1H and 13C NMR, see Table 1; HRESIMS m/z 371.2207 [M + H]+ (calcd for C23H31O4, 371.2222). Sapranthin I (2): yellowish oil; [α]24D +48 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 214 (3.46), 255 (3.61), 269 (3.65), 285 (3.60) nm; 1H and 13C NMR, see Table 1; HRESIMS m/z 371.2217 [M + H]+ (calcd for C23H31O4, 371.2222). Sapranthin J (3): yellowish oil; [α]24D +36 (c 1, CHCl3); UV (CHCl3) λmax (log ε) 214 (3.45), 255 (3.60), 269 (3.66), 285 (3.60) nm; 1H and 13C NMR, see Table 1; HRESIMS m/z 399.2519 [M + H]+ (calcd for C25H35O4, 399.2535). Sapranthin K (4): yellowish oil; [α]24D −20 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 214 (3.50), 255 (3.54), 269 (3.61), 285 (3.54) nm; 1H and 13C NMR, see Table 2; HRESIMS m/z 339.2337 [M + H]+ (calcd for C23H31O2, 339.2324). Sapranthin L (5): yellowish oil; [α]24D −25 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 214 (3.47), 255 (3.56), 269 (3.66), 285 (3.51) nm; 1H and 13C NMR, see Table 2; HRESIMS m/z 311.2011 [M + H]+ (calcd for C21H26O2, 311.2011). Eicosa-15E,19-diene-11,13-diynoic acid (6): yellowish oil; [α]24D 0 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 214 (3.36), 255 (3.47), 269 (3.53), 285 (3.52) nm; 1H and 13C NMR, see Table 2; HRESIMS m/z 301.2193 [M + H]+ (calcd for C20H29O2, 301.2168). Stictic acid (8): see NMR Table S3, Supporting Information (spectra recorded in DMF-d7 and pyridine-d5). Baillonic acid (9): amorphous, white powder; [α]24D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 216 (3.35) nm; IR νmax 3396, 2935, 1719, 1601 cm−1; 1H and 13C NMR, see Table 3; HRESIMS m/z 329.0663 [M − H]− (calcd for C17H13O7, 329.0661). G



Article

manufacturer’s instructions. The immobilization levels were around 250 to 300 U. A control surface was prepared with the same protocol with biotinylated Arf1 (17−181), a small G protein unrelated to RhoA. After the capture of the biotinylated protein, both flow cell surfaces were saturated with two injections of EZ-Link-Amine-PEG2Biotin (Pierce, #21346) at 100 μM, for 60 s each at 30 μL.min−1 with a pause of 180 s in-between). Solutions are obtained from a 1:1 v/v mix of the compound solution in 10% DMSO buffer with the protein solution in a 0% DMSO buffer, resulting in a 5% DMSO mix. The target protein was His-p115DH-PH (395−766; MW 43 475.3 Da) or Net1 (149−501; MW 42 163 Da). Binding is monitored by MultiCycle Kinetics (High Performance). Injection runs were for 120 s at a 30 μL min−1 flow rate, and dissociation runs for 120 s at a 30 μL min−1 flow rate, both at 25 °C. An extra wash after injection was done with a solution of 50% DMSO. Between each run, the surface was fully regenerated with a mixture of 8 M guanidine hydrochloride and 1 M NaOH (3:1 v/v) (GE Healthcare). Carryover and solvent correction were applied. Binding of the compound to the target protein occurs free in solution. Between each compound concentration p115 or Net1 was injected alone (100% of binding). For each compound concentration inhibition percent is determined from the 100% binding.

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(5) Kataoka, C.; Egashira, K.; Inoue, S.; Takemoto, M.; Ni, W.; Koyanagi, M.; Kitamoto, S.; Usui, M.; Kaibuchi, K.; Shimokawa, H.; Takeshita, A. Hypertension 2002, 39, 245−250. (6) Moriki, N.; Ito, M.; Seko, T.; Kureishi, Y.; Okamoto, R.; Nakakuki, T.; Kongo, M.; Isaka, N.; Kaibuchi, K.; Nakano, T. Hypertens. Res. 2004, 27, 263−270. (7) Guilluy, C.; Brégeon, J.; Toumaniantz, G.; Rolli-Derkinderen, M.; Retailleau, K.; Loufrani, L.; Henrion, D.; Scalbert, E.; Bril, A.; Torres, R. M.; Offermanns, S.; Pacaud, P.; Loirand, G. Nat. Med. 2010, 16, 183−190. (8) Uehata, M.; Ishizaki, T.; Satoh, H.; Ono, T.; Kawahara, T.; Morishita, T.; Tamakawa, H.; Yamagami, K.; Inui, J.; Maekawa, M.; Narumiya, S. Nature 1997, 389, 990−994. (9) Loirand, G.; Guérin, P.; Pacaud, P. Circ. Res. 2006, 98, 322−334. (10) Van Heusden, E. C. H. Bulletin du Muséum National d’Histoire Naturelle, 4ème Série 1996, 18, 75−83. (11) http://www.theplantlist.org/1.1/browse/A/Annonaceae/ Meiogyne/. (12) Tadić, D.; Cassels, B. K.; Leboeuf, M.; Cavé, A. Phytochemistry 1987, 26, 537−541. (13) Bousserouel, H.; Awang, K.; Guéritte, F.; Litaudon, M. Phytochem. Lett. 2012, 5, 29−32. (14) Litaudon, M.; Bousserouel, H.; Awang, K.; Nosjean, O.; Martin, M.-T.; Dau, M. E. T. H.; Hadi, H. A.; Boutin, J. A.; Sévenet, T.; Guéritte, F. J. Nat. Prod. 2009, 72, 480−483. (15) Etse, J. T.; Waterman, P. G. Phytochemistry 1986, 25, 1903− 1905. (16) Harcken, C.; Brückner, R.; Rank, E. Chem. - Eur. J. 1998, 4, 2342−2352. (17) Ravi, B.; Wells, R. Aust. J. Chem. 1982, 35, 105−112. (18) Guo, Y.-W.; Gavagnin, M.; Mollo, E.; Trivellone, E.; Cimino, G. J. Nat. Prod. 1999, 62, 1194−1196. (19) Lorenzo, M.; Brito, I.; Cueto, M.; D’Croz, L.; Darias, J. Org. Lett. 2006, 8, 5001−5004. (20) Marshall, J. A.; Wolf, M. A.; Wallace, E. M. J. Org. Chem. 1997, 62, 367−371. (21) Zgoda, J. R.; Freyer, A. J.; Killmer, L. B.; Porter, J. R. J. Nat. Prod. 2001, 64, 1348−1349. (22) Elix, J.; Adler, M.; Wardlaw, J. Aust. J. Chem. 1996, 49, 1175− 1178. (23) Ito, C.; Itoigawa, M.; Mishina, Y.; Tomiyasu, H.; Litaudon, M.; Cosson, J.-P.; Mukainaka, T.; Tokuda, H.; Nishino, H.; Furukawa, H. J. Nat. Prod. 2001, 64, 147−150. (24) Ito, C.; Miyamoto, Y.; Nakayama, M.; Kawai, Y.; Rao, K. S.; Furukawa, H. Chem. Pharm. Bull. 1997, 45, 1403−1413. (25) Permana, D.; Lajis, N. H.; Mackeen, M. M.; Ali, A. M.; Aimi, N.; Kitajima, M.; Takayama, H. J. Nat. Prod. 2001, 64, 976−979. (26) Xu, Y.-J.; Chiang, P.-Y.; Lai, Y.-H.; Vittal, J. J.; Wu, X.-H.; Tan, B. K. H.; Imiyabir, Z.; Goh, S.-H. J. Nat. Prod. 2000, 63, 1361−1363. (27) Elix, J. A.; Wardlaw, J. H. Aust. J. Chem. 2000, 53, 815−818. (28) Millot, M.; Tomasi, S.; Articus, K.; Rouaud, I.; Bernard, A.; Boustie, J. J. Nat. Prod. 2007, 70, 316−318. (29) Khumkomkhet, P.; Kanokmedhakul, S.; Kanokmedhakul, K.; Hahnvajanawong, C.; Soytong, K. J. Nat. Prod. 2009, 72, 1487−1491. (30) Stodola, F. H.; Vesonder, R. F.; Fennell, D. I.; Weisleder, D. Phytochemistry 1972, 11, 2107−2108. (31) Lang, G.; Cole, A. L. J.; Blunt, J. W.; Robinson, W. T.; Munro, M. H. G. J. Nat. Prod. 2007, 70, 310−311. (32) Zhang, W.; Xu, L.; Yang, L.; Huang, Y.; Li, S.; Shen, Y. Fitoterapia 2014, 96, 146−151. (33) Priestap, H. A. Phytochemistry 1985, 24, 849−852. (34) Guinaudeau, H.; Leboeuf, M.; Debray, M.; Cavé, A.; Paris, R. R. Planta Med. 1975, 27, 304−318. (35) Chia, Y.-C.; Chang, F.-R.; Teng, C.-M.; Wu, Y.-C. J. Nat. Prod. 2000, 63, 1160−1163. (36) Tabopda, T. K.; Ngoupayo, J.; Liu, J.; Mitaine-Offer, A.-C.; Tanoli, S. A. K.; Khan, S. N.; Ali, M. S.; Ngadjui, B. T.; Tsamo, E.; Lacaille-Dubois, M.-A.; Luu, B. Phytochemistry 2008, 69, 1726−1731.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00209. HRESIMS data, 1H, 13C, and 2D NMR spectra for 1−6, 8, 9, and 11; NMR shifts of compound 8 recorded in DMF-d7 and pyridine-d5; ORTEP view and a detailed discussion on X-ray structure determination for compound 9 (PDF) Corresponding crystallographic information file (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: marc. [email protected]. ORCID

Vincent Dumontet: 0000-0002-1770-6566 Fanny Roussi: 0000-0002-5941-9901 Marc Litaudon: 0000-0002-0877-8234 Author Contributions

F.O. and L.-F.N. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to South Province of New Caledonia, which has facilitated our field investigation (permit collect number 6024-4255/DRN/ENV). This work has benefited from an “Investissement d’Avenir” grant managed by Agence Nationale de la Recherche (CEBA, ANR-10-LABX-25-01).

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REFERENCES

(1) Staessen, J. A.; Wang, J.; Bianchi, G.; Birkenhäger, W. H. Lancet 2003, 361, 1629−1641. (2) de Gasparo, M.; Catt, K. J.; Inagami, T.; Wright, J. W.; Unger, T. Pharmacol. Rev. 2000, 52, 415−472. (3) Kimura, K.; Eguchi, S. Am. J. Physiol. Cell Physiol. 2009, 297, C1059−C1061. (4) Wirth, A. Biochim. Biophys. Acta, Mol. Basis Dis. 2010, 1802, 1276−1284. H



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