Aedes aegypti Larvicidal Sesquiterpene Alkaloids from Maytenus

Article pubs.acs.org/jnp

Aedes aegypti Larvicidal Sesquiterpene Alkaloids from Maytenus oblongata Seindé Touré,† Charlotte Nirma,† Michael Falkowski,‡ Isabelle Dusfour,§ Isabelle Boulogne,‡,⊥ Arnaud Jahn-Oyac,‡ Maïra Coke,∥ Didier Azam,∥ Romain Girod,§ Céline Moriou,† Guillaume Odonne,# Didier Stien,†,¶ Emeline Houel̈ ,*,‡ and Véronique Eparvier*,† †

CNRS, Institut de Chimie des Substances Naturelles, 91198 Gif-sur-Yvette, France CNRS, UMR EcoFoG, AgroParisTech, Cirad, INRA, Université des Antilles, Université de Guyane, 97300 Cayenne, France § Unité de Contrôle et Adaptation des Vecteurs, Institut Pasteur de la Guyane, 97306 Cayenne, France ⊥ UPRES-EA 4358 GlycoMev (Glycobiologie et Matrice Extracellulaire Végétale), Université de Rouen, 76821 Mont-Saint-Aignan, France ∥ Unité Expérimentale d’Ecologie et d’Ecotoxicologie Aquatique, INRA-U3E, 35042 Rennes, France # Laboratoire Ecologie, Evolution, Interactions des Systèmes Amazoniens (LEEISA), CNRS, Université de Guyane, IFREMER, 97300 Cayenne, France ¶ Laboratoire de Biodiversité et Biotechnologies Microbiennes (LBBM), Sorbonne Universités, UPMC Univ. Paris 06, CNRS, Observatoire Océanologique, 66650 Banyuls-sur-Mer, France ‡

S Supporting Information *

ABSTRACT: Four new sesquiterpene alkaloids (1−4) with a β-dihydroagrofuran skeleton and a new triterpenoid (5) were isolated from an ethyl acetate extract of Maytenus oblongata stems. Their structures were elucidated using 1D and 2D NMR spectroscopy as well as MS and ECD experiments. The M. oblongata stem EtOAc extract and the pure compounds isolated were tested for larvicidal activity against Aedes aegypti under laboratory conditions, and compounds 2 and 3 were found to be active.

has therefore become urgent to find novel insecticides or alternative methods for controlling mosquito vectors. Therefore, research on new insecticides, particularly molecules of natural origin, has intensified in recent years. Overall, compounds toxic against adult and immature mosquito stages, insect growth blockers, and excito-repellents are needed urgently.12 Considering this situation, in a constantly evolving epidemiological and regulatory context, the need to discover alternative, environmentally friendly, and safer biopesticides is crucial. Plants are a source of inspiration for new insecticide discovery because they are in constant interaction and have coevolved with herbivores synthesizing complex molecules as a defense, leading to a unique chemical diversity.12,13 In the specific context of the exceptional biodiversity encountered in the Amazonian forest, the biological activities of extracts from 160 plant parts (leaves or aerial parts, inflorescences, fruits, stems, bark, wood, and roots) of 87 species collected in French

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ector-borne diseases account for 17% of the estimated global burden of all infectious diseases.1 Major mosquito species transmitting parasitosis and arbovirosis belong to the genera Aedes, Anopheles, and Culex (Diptera, Culicidae).2 Vector control is the method used most often to circumvent transmission in the absence of vaccines and/or specific treatments for most arboviruses. For this purpose, insecticides have been sprayed for decades, provoking the development of resistance in mosquito populations and resulting in a loss of vector control efficacy.3,4 Moreover, some products have been gradually removed from the market or from the field due to the demonstration of their human and environmental toxicity. A striking example is that of malathion, removed from the European market in 2009 but nevertheless allowed for temporary use in 2014−2015 in French Guiana due to a chikungunya outbreak,5−7 before an abrupt cessation of such spraying occurred following the publication of a monograph published by WHO International Agency for Research on Cancer classifying malathion as probably carcinogenic to humans.8 This concern is even more problematic with the emergence and/or re-emergence of arboviruses such as chikungunya or zika and their consequences.9−11 The situation © 2017 American Chemical Society and American Society of Pharmacognosy

Received: September 16, 2016 Published: February 10, 2017 384

DOI: 10.1021/acs.jnatprod.6b00850 J. Nat. Prod. 2017, 80, 384−390

Journal of Natural Products

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Guiana were evaluated to find new environmentally safe insecticides. During the screening of plant extracts on Aedes aegypti mosquito larvae, it was discovered that an ethyl acetate extract of the stems of Maytenus oblongata Reissek (Celastraceae) exhibited promising larvicidal properties. The family Celastraceae, which comprises approximately 88 genera and 1300 principally pantropical species, has been known for a long time for its insecticidal properties, in particular within the Maytenus genus.14,15 Secondary metabolites reported from Maytenus species are principally alkaloids (maytansinoids) and β-dihydroagarofuran sesquiterpenes and triterpenoids.16−18 Antifeedant and larvicidal β-dihydroagarofurans active against Spodotera spp. were isolated from Maytenus canariensis, M. disticha, M. chiapensis, and M. boaria.19,20

groups, resonances for a ketocarbonyl, two carbonyl esters, three oxygenated quaternary carbons, and one other quaternary carbon were observed in the 13C NMR spectrum of 1. It was proposed that compound 1 has a β-dihydroagarofuran skeleton, based on the sequence of 1H−1H COSY cross-peaks for the H1/H-2/H-3, H-4/H-14, and H-6/H-7 coupling systems and the following HMBC correlations: H-4/C-3, C-5 and C-10; H-7/ C-6, C-8, and C-9; and H-14/C-4 and C-5 (Figure 1). A 2-(3carboxybutanoyl)nicotinic acid moiety was elucidated based on the 2,3-disubstituted pyridine and the −CH2−CH2−C(OH/ CH3)− moieties defined by the HMBC correlations of H-7′/C2′, C-3′, C-9′; H-4′/C-3′; and H-10′/C-8′ C-9′ and the 1H−1H correlations of H-4′/H-5′/H-6′ and H-7′/H-8′. HMBC cross signals H-7/C-13, H-13/C12′, and H-3/C-11′ confirmed the C-3−O−C-11′ and C-13−O−C-12′ linkages. The OAc-1, OAc-2, OAc-6, OAc-9, and OAc-15 groups were determined from the corresponding HMBC correlations of H-1/CO (δC 169.9), H-2/CO (δC 169.0), H-6/CO (δC 169.0), H-9/ CO (δC 166.9), and H-15/CO (δC 169.6) (Figure 1, Table 1). A free hydroxy group was found to be linked to C-9′ from the 13C NMR signal at δC 75.0. It was concluded that compound 1 is an analogue of alatamine without a hydroxy group at C-4.21 The relative configuration of the polyoxygenated β-dihydroagarofuran moiety of 1 was established by analysis of the coupling constants and the ROESY correlations (Figure 2). The determining ROESY correlations were between H-9/H-1 and H-12, H-15/H-6 and H-14, and H-14/H-11 (Figure 2). The electronic circular dichrosim (ECD) spectrum of 1 showed a positive Cotton effect around 230 nm and a negative Cotton effect at 220 nm. On comparison with the absolute configuration of the sesquiterpene part reported in the literature,18 it was determined as 1S, 2R, 3S, 4S, 6S, 7S, and 9R. This new compound was named 4-deoxyalatamine. Compound 2 was isolated as an amorphous, white solid. A molecular formula of C41H45NO17 was determined via HRESITOFMS analysis, which showed a protonated molecular ion peak at m/z 824.2745 [M + H]+ (calcd for C41H46NO17, 824.2766) and corresponding to 16 degrees of unsaturation. The 1H NMR spectrum was similar to the spectrum of compound 1 (Table 1) and suggested that this compound has a β-dihydroagarofuran skeleton. The 1H NMR spectrum of this compound indicated the presence of five protons at δH 7.52, 7.61 (2H) and 7.97 (2H) linked to carbons at δC 135.3, 130.0, and 130.4, respectively. Quaternary carbons at δC 166.3 and 130.9 were also present in this moiety, which was assigned as a benzoyl group. In comparison with 1, compound 2 was found to have one acetyl group less, and the HMBC correlation between H-1 and the quaternary carbon at δC 166.3 enabled the benzoyloxy group to be located at position C-1 (Figure 1). The same patterns of coupling constants and types of ROE correlations were observed as for compound 1 (Figure 2), which allowed the relative configuration for 2 to be confirmed. The ECD spectrum was consistent with the absolute configuration 1S, 2R, 3S, 4S, 6S, 7S, and 9R. This new compound was identified as 1-O-benzoyl-1-deacetyl-4-deoxyalatamine. Compound 3 exhibited a protonated molecular ion peak at m/z 886.2894 [M + H]+ (calcd for C46H48NO17, 886.2922). The NMR data were very similar to those of compounds 1 and 2. The most notable differences with 2 were the disappearance of another acetyl group and the presence of an additional benzoyl group. On the basis of HMBC and ROE experiments, the second benzoyl group was placed at position C-2 (Figure 1,

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RESULTS AND DISCUSSION An M. oblongata stem ethyl acetate extract revealed strong larvicidal activity on both the A. aegypti laboratory reference strain Paea, which is susceptible to all insecticides, and the local field strain Cayenne, which is resistant to pyrethroids and organophosphates. The mortality rates were 95 ± 2% and 83 ± 2%, respectively, after 24 h of exposure at a dose of 100 μg/mL. In comparison, the larval mortality upon exposure to deltamethrin at 50 μg/mL was 100% for the Paea strain but only 26 ± 2% for the Cayenne strain. These results were considered very promising, and therefore bioguided fractionation was performed. Four new sesquiterpene alkaloids (1−4) with a β-dihydroagrofuran skeleton and one new triterpenoid (5) were isolated and characterized structurally.

Compound 1 was obtained as an amorphous, white solid. A molecular formula of C36H43NO17 was determined via HRESITOFMS analysis, which showed a protonated molecular ion at m/z 762.2610 [M + H]+ (calcd for C36H44NO17, 762.2609), corresponding to 16 degrees of unsaturation. The 1 H NMR spectrum suggested the presence of five acetate esters [δH 1.99, 2.01, 2.13, 2.17, and 2.26 (each 3H, s)]. The 1H NMR spectrum of 1 showed also the presence of three other methyl groups at δH 1.40 (d, J = 8.0 Hz), 1.49 (s), and 1.42 (s). The NMR spectroscopic data (Table 1) indicated signals for four methylenes, seven methines, and one 2,3-disubstituted pyridine unit. In addition to the resonances of the aforementioned 385



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Table 1. 13C and 1H NMR Data of Compounds 1−4 in MeOD 1 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ OAc-1 OAc-2 OAc-6

δC,a type 71.7, 69.0, 73.3, 37.1, 91.3, 73.8, 61.7, 196.3, 77.1, 50.7, 84.3, 17.2, 70.9,

CH CH CH CH C CH CH C CH C C CH3 CH2

14.3, CH3 58.9, CH2 161.5, 127.4, 137.7, 120.6, 150.9, 32.1,

C C CH CH CH CH2

40.9, CH2 75.0, 25.4, 173.3, 169.6, 169.9, 19.1, 169.0, 19.4, 169.0, 19.3,

C CH3 C C C CH3 C CH3 C CH3

166.9, 18.5, 169.6, 19.1,

C CH3 C CH3

2 δHb (J in Hz)

5.88 5.30 5.05 2.99

d (2.4) t (2.4) dd (1.26, 2.4) q (8.1)

6.50 s 3.37 s 5.64 s

1.49 3.91 5.63 1.40 4.91 4.59

8.15 7.36 8.63 2.90 3.70 2.23 2.15

(3H, s) d (11.7) d (11.7) d (8.0) d (12.9) d (12.9)

dd (1.6, 7.7) dd (4.8, 7.7) dd (1.6, 4.8) qt (7.2) qt (7.4) m m

1.42 s

δC,a type 73.8, 71.0, 75.3, 38.7, 93.5, 75.7, 62.8, 197.9, 80.6, 63.3, 85.9, 18.7, 70.1,


16.0, CH3 60.9, CH2 163.4, 128.8, 139.7, 122.6, 152.4, 32.1,

C C CH CH CH CH2

40.7, CH2 76.8, 27.0, 175.2, 168.4,

C CH3 C C

3 δHb (J in Hz)

6.13 5.41 5.03 2.94

d (3.2) s s q (7.6)

73.8, 72.1, 75.5, 38.5, 93.5, 75.8, 63.1, 197.9, 80.7, 53.0, 86.4, 18.9, 71.4,

6.42 s 3.35 s 5.63 s

1.45 5.56 3.87 1.38 5.15 4.58

8.09 7.30 8.57 3.64 2.84 2.29 2.13

s d d d d d

δC,a type

(11.6) (11.6) (7.4) (13.1) (13.1)


16.3, CH3 62.1, CH2

dd (1.4, 7.8) dd (4.9, 7.6) dd (1.4, 4.7) qt (7.8) qt (8.0) m m

163.5, 128.8, 139.7, 122.6, 152.6, 32.2,

41.0, CH2 76.9, 27.2, 175.4, 168.5,

1.44 s

C C CH CH CH CH2

C CH3 C C

4 δHb (J in Hz)

6.35 5.70 5.25 3.11

d (3.2) s s q (7.8)

6.53 s 3.37 s 5.77 s

1.56 5.63 3.97 1.48 5.15 4.94

8.18 7.38 8.66 3.74 2.94 2.34 2.23

s d d d d d

(11.5) (11.5) (7.8) (12.6) (12.6)

dd (1.46, 7.7) dd (4.7, 7.8) dd (1.4, 4.7) qt (8.0) qt (8.0) m m

1.47 s

1.99 s

2.26 s 2.17 s

170.7, 21.2, 170.5, 20.9,

C CH3 C CH3

170.9, 19.9, 171.4, 20.9, 166.3, 130.9, 130.4, 130.0, 135.3,

C CH3 C CH3 C C CH CH CH

OAc-15 BzO-1

2.01 s 2.13 s

170.7, C 21.2, CH3

2.13 s

170.9, 16.3, 171.0, 20.6, 166.4, 130.1, 130.9, 130.0, 135.3, 166.5, 130.3, 130.9, 130.2, 135.3,

1.46 s 1.93 s

7.97 d (7.6) 7.61 t (7.6) 7.52 t (7.6)

BzO-2

a

73.8, 71.0, 75.3, 38.7, 93.5, 75.7, 62.8, 74.6, 80.6, 63.3, 85.9, 18.7, 70.1,

CH CH CH CH C CH CH CH CH C C CH3 CH2

16.0, CH3 60.9, CH2 151.2, 129.4, 155.8, 127.1, 152.5, 32.1,

CH C C CH CH CH2

40.7, CH 76.8, 27.0, 175.2, 168.4, 171.8, 21.0,

C CH3 C C C CH3

171.2, 21.2, 171.4, 20.8, 172.0, 21.2, 171.6 21.3

C CH3 C CH3 C CH3

167.6, 130.3, 131.0, 130.0, 135.1,

C C CH CH CH

δHb (J in Hz) 5.80 5.53 5.06 2.84

d (3.6) s s q (7.6)

6.36 2.70 5.68 5.75

s d (2.7) dd (3.4, 9.8) d (9.8)

1.63 5.54 3.88 1.37 5.04 4.61 8.78

s d (11.6) d (11.6) d (8.0) (1H, d, J = 12.7) (1H, d, J = 12.7) s

7.42 8.51 3.54 2.77 2.15

d (5.1) d (5.1) qt (7.3) qt (7.3) m

1.40 s

1.94 s

2.20 s 2.28 s

OAc-8 OAc-9

δC,a type

CH3 C CH3 C C CH CH CH C C CH CH CH

1.47 s 2.01 s

2.18 s 2.11 s 1.98 s 2.24 (3H, s)

7.89 d (7.6) 7.45 t (7.6) 7.62 t (7.6)

8.08 d (7.6) 7.58 t (7.6) 7.72 t (7.6)

8.04 d (7.3) 7.50 t (7.3) 7.63 t (7.3)

Data recorded at 125 MHz. bData recorded at 500 MHz.

868.3035 [M + H]+, calcd for C43H50NO18, 868.3028). Interestingly, the NMR spectroscopic data showed that the carbonyl group at C-1, C-6, C-8, C-9, and C-15 was reduced and confirmed the presence of five acetoxy groups and one benzoyl group at C-2. In 4, the nicotinoyl moiety was also different from the nictotinoyl moiety in compounds 1−3. Here, the pyridine ring was substituted in position C-3′ and C-4′, as

Table 1). The configuration was identical to those at 1 and 2 and was determined by comparison of the ROESY and ECD spectra. This new compound was determined to be 1,2-Odibenzoyl-1,2-deacetyl-4-deoxyalatamine. Like compounds 1−3, compound 4 was assigned as an alkaloid with a β-dihydroagarofuran skeleton. Its molecular formula was C43H49NO18 based on the HRESIMS (m/z 386



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skeleton was determined with one carbonyl and two hydroxy groups at δC 216.0, 73.8, and 75.9 ppm; two oxymethine protons at δH 3.85 (J = 11.0 and 4.4 Hz) and 4.00 (J = 9.2 Hz); and signals for eight methyl singlets (δH 0.73, 0.85, 0.94, 1.00, 1.02, 1.05, 1.16, and 1.21 ppm) (Table 2). However, dissimilarities were observed between the NMR spectrum of 5 and the known friedelane-type compounds and revealed that the hydroxy groups were not in the same position as in the molecules already described. The HMBC correlations between H-2, H-4, and H-23/C-3 and H-1/C-10 and C-5 permitted a carbonyl group to be positioned at C-3. Then, hydroxy groups were placed at positions C-12 and C-16 by analyzing correlations between Me-25/C-8, C-9, and C-10; H-12/C-8, C-27, and C-18; Me-26/C-8, C-13, C-14, and C-15; Me-28/C16, C-17, and C-18; and H-21/C-19, C-20, C-22, C-29, and C30 (Table 2). The relative configuration of 5 was established on the basis of the coupling constants observed and confirmed by a NOESY experiment (Figure 3), which showed correlations of H-10 with H-4, Me-24 with Me-25, Me-25 with H-12 and Me26, Me-27 with H-8 and H-16, H-18 with H-12, and Me-28 with H-12, H-18, and Me-29. This new compound was assigned as 12,16-dihydroxyfriedelan-3-one. The ethyl acetate extract of the stems of M. oblongata exhibited an LD50 value against A. aegypti Paea strain larvae of 74.4 ± 2.5 μg/mL after 24 h exposure and mortality rates of 42.7 ± 6.7%, 69.8 ± 2.5%, and 93.0 ± 0.0% at concentrations of 50, 100, and 1000 μg/mL, respectively, against A. aegypti adult females in an ingestion test model after 72 h of exposure (Figure S3, Supporting Information). Concerning eco- and cytotoxicity, the extract exhibited 45 ± 10% average mortality against C. riparius after 24 h of exposure and 70 ± 26% after 48 h at 75 μg/mL and was shown to be nontoxic against D. magna at the same concentration. The ethyl acetate extract of the stems of M. oblongata also exhibited no cytotoxicity against KB and MCR5 human cell lines at 10 μg/ mL and against Aedes albopictus C6/36 cells at concentrations up to 300 μg/mL. Pure compounds were evaluated for larvicidal activity on the A. aegypti Paea strain. Compounds 2 and 3 showed strong activitiy with LD50 values of 9.4 (95% CI: 6.5−10.0) and 2.7 μM (95% CI: 1.9−2.9), respectively. In parallel, compounds 1 to 5 were also tested on the MRC5 cell line, and no cytotoxicity was observed at 1 and 10 μg/mL (Table 3).

Figure 1. Key 1H−1H COSY (bold line) and HMBC (arrows) correlations for compounds 1−4.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using an Anton Paar MCP 300 polarimeter in a 100-mmlong 350 μL cell. UV spectra were recorded in CHCl3 using a PerkinElmer Lambda 5 spectrophotometer. The ECD spectra were measured at 25 °C on a JASCO J-810 spectropolarimeter. NMR spectra were recorded in CDCl3 on a Bruker 500 MHz NMR spectrometer or a Bruker 600 MHz NMR spectrometer equipped with a 1 mm inverse detection probe. Chemical shifts (δ) are reported as ppm based on the tetramethylsilane signal. High-resolution ESITOFMS measurements were performed using a Waters Acquity UPLC system with a column bypass coupled to a Waters Micromass LCT Premier time-of-flight mass spectrometer equipped with an electrospray interface (ESI). Flash chromatography was performed on a Grace Reveleris system with dual UV and ELSD detection equipped with an 80 g silica column. For UV-based experiments, effluents were monitored at 254 and 280 nm. TLC was conducted on 60 A F254 Merck plates and visualized using UV light and phosphomolybdic acid. Analytical and preparative HPLC work was conducted with a Gilson system equipped with a 322 pumping device, a GX-271 fraction collector, a 171 diode array detector, and a prepELSII detector

Figure 2. Key 1H−1H ROE correlations for compounds 1 and 4.

shown in the 1H NMR spectrum. For example, the protons α to the nitrogen resonated at δH 8.51 (d, J = 5.1 Hz, H-6′) and 8.78 (s, H-2′). The structure of the nicotinic part was confirmed by comparison with the literature (Figure 1).22 The configuration 1S, 2R, 3S, 4S, 6S, 7S, 8R, and 9R was determined by analysis of the 1H NMR coupling constants and by a ROESY experiment (Figure 2) and from the ECD spectrum. This new compound was assigned as 4-deoxyisowilfordine. Compound 5 was isolated as a white powder and showed an exact mass of 458.3760 Da in its HRESIMS, which accounted for a molecular formula of C30H50O3 and was indicative of six sites of unsaturation. By comparison of the 1H and 13C NMR spectra with the literature,23−25 it was proposed that 5 is of the friedelane pentacyclic structural type. The presence on this 387



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Table 2. 13C and 1H NMR Data and 2D Correlations of Compound 5 in MeOD 5 position

δC,a type

1

23.6, CH2

2

42.2, CH2

3 4 5 6

a

216.0, 59.1, 43.3, 42.3,

C CH C CH2

7

19.5, CH2

8 9 10 11

54.6, 39.2, 60.4, 48.0,

CH C CH CH2

12 13 14 15

73.8, 46.5, 42.2, 45.7,

CH C C CH2

16 17 18 19

75.9, 37.8, 47.7, 39.6,

CH C CH CH2

20 21

29.2, C 33.1, CH2

22

37.4, CH2

23 24 25 26 27 28 29 30

7.3, 15.1, 20.2, 22.2, 13.7, 26.3, 31.1, 36.0,

CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

δHb (J in Hz)

COSY

HMBC

a: 1.74 m b: 1.99 m a: 2.31 m b: 2.41 m

1b, 2a, 2b 1a, 2a, 2b 1a, 1b, 2b, 4 1a, 1b, 2a, 4

2, 2, 1, 1,

2.39 m

2a, 2b, 23, 24

2, 3, 23, 24

eq: 1.39 m ax: 1.78 brd (12.5) eq: 1.48 m ax: 1.57 brd (12.5) 1.44 m

6ax, 7eq, 7ax 6eq, 7eq, 7ax 6eq, 6ax 6eq, 6ax, 8 8ax, 26

5, 5, 5, 5, 9,

7, 24 7, 24 6 6 14, 25, 26

1.67 dd (12.5, 2.2) eq: 1.62 dd (12.5, 4.4) ax: 1.32 m 3.85 dd (11.0, 4.4)

1a, 1b, 25 11ax, 12 11eq, 12 11eq, 11ax

1, 8, 8, 8,

4, 5, 8, 9, 24, 25 9, 12, 13, 25 9, 12, 13, 25 18, 27

eq: 1.34 m ax: 1.85 br dd (14.7, 9.5) 4.00 t (9.2)

15ax, 16 15eq, 16 15eq, 15ax

8, 14, 16, 17, 26 8, 14, 16, 17, 26 15, 22, 28

2.03 dd (13.4, 4.4) eq: 2.13 dd (13.9, 4.4) ax: 1.44 m

19eq, 19ax, 27 18, 19ax 18, 19eq, 29

12, 13, 16, 17, 19, 27, 28 17, 18, 20, 21, 29 17, 18, 20, 21, 29

a: 1.32 m b: 1.40 m a: 1.40 m b: 1.50 m 0.85 d (6.6) 0.73 s 1.00 s 1.16 s 1.02 s 1.21 s 1.05 s 0.94 s

5, 5, 3, 3,

10 10 4, 10 4, 10

19, 20, 22, 29, 19, 20, 22, 29, 17, 18, 20, 22, 17, 18, 20, 22, 3, 4, 5 4, 5, 6, 10 8, 9, 10, 11 8, 13, 14, 15 12, 13, 14, 18 16, 17, 18 19, 20, 21, 30 19, 20, 21, 29

22b 22a 4 4 10 18 19ax, 30 29

30 30 28 28

Data recorded at 150 MHz. bData recorded at 600 MHz.

Table 3. Larvicidal Activities and Cytotoxicity of Compounds 1−5 bioassay larvicidal activity (PAEA strain, LD50, μM) MRC5 cell mortality (10 μg/mL) MRC5 cell mortality (1 μg/mL)a a

1

2

3

9.4 ± 2

2.7 ± 1

38 ± 2

15 ± 2

21 ± 1

17 ± 1

48 ± 2

24 ± 2

15 ± 1

18 ± 1

15 ± 1

16 ± 2

>10

4 >10

5 >10

Positive control: docetaxel showing 0.0005% mortality at 1 μg/mL.

electrospray nebulizer. Columns used for these experiments included a Phenomenex Luna C18 5 μm 4.6 × 250 mm analytical column and a Phenomenex Luna C18 5 μm 21.2 × 250 mm preparative column. The flow rate was set to 1 or 17 mL/min, respectively, using a linear gradient of H2O mixed with an increasing proportion of CH3CN or

Figure 3. Key 1H−1H NOE correlations for compound 5. 388



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MeOH. Both solvents were modified with 0.1% formic acid. All solvents were HPLC grade. Potato dextrose agar was purchased from Fluka Analytical. Plant Material. Maytenus oblongata stems were collected on the Couy River near Kourou (22N, east 302167, north 558167), French Guiana, in March 2012. This species is not a protected plant and can be collected without restriction at this location. A herbarium voucher (GO726) was deposited in Cayenne Herbarium (CAY). Extraction and Isolation. Plant samples were dried (room temperature, 10% air relative humidity) and finely powdered. Stems (320 g) were extracted at room temperature by maceration using 3 × 2 L of EtOAc; the residue was subsequently extracted using the same volume of EtOAc and, finally, with methanol, providing 4.4 and 8.5 g of extract, respectively, after evaporation of the solvents. A portion of the EtOAc extract (1.0 g) was purified by flash chromatography on C18 silica gel with a linear gradient of H2O−CH3CN (1:0 to 0:1 in 25 min, flow rate 40 mL/min). Six fractions were gathered based on their TLC profiles. The larvicidal activity was found to be concentrated in fractions 4 (131.5 mg) and 5 (67.0 mg). Upon further fractionation using preparative HPLC with H2O−CH3CN (40:60 to 0:100 in 20 min, flow rate 21 mL/min), fraction 4 afforded compounds 1 (2.1 mg, 0.000 65%, tR 8.42 min) and 2 (17.8 mg, 0.000 55%, tR 15.79 min). Compounds 3 (9.5 mg, 0.000 68%, tR 10.10 min), 4 (1.0 mg, 0.000 31%, tR 7.13 min), and 5 (2.2 mg, 0.000 68%, tR 15.19 min) were isolated from fraction 5. 4-Deoxyalatamine (1): white powder; [α]25D +10.0 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 270 (3.4) nm; 1H and 13C NMR data, see Table 1; HRESITOFMS m/z 762.2572 [M + H]+ (calcd for C36H44NO17, 762.2604). 1-O-Benzoyl-1-deacetyl-4-deoxyalatamine (2): white powder; [α]25D +24.0 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 269 (3.5) nm; 1H and 13C NMR data, see Table 1; HRESITOFMS m/z 824.2767 [M + H]+ (calcd for C41H46NO17, 824.2770). 1,2-O-Dibenzoyl-1,2-deacetyl-4-deoxyalatamine (3): white powder; [α]25D +74.0 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 269 (3.6) nm; 1H and 13C NMR data, see Table 1; HRESITOFMS m/z 886.2897 [M + H]+ (calcd for C46H48NO1, 886.2922). 4-Deoxyisowilfordine (4): white powder; [α]25D +38.0 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 271 (2.7) nm; 1H and 13C NMR data, see Table 1; HRESITOFMS m/z 868.3035 [M + H]+ (calcd for C43H50NO18, 868.3028). 12,16-Dihydroxyfriedelan-3-one (5): white powder; [α]25D +6.0 (c 0.5, MeOH); 1H and 13C NMR data, see Table 2; HRESITOFMS m/z 503.3743 [M + FA − H]− (calcd for C31H51O5, 503.3737). Evaluation of Larvicidal Activity. Insect Collection and Rearing. Two Aedes aegypti strains were used for testing the extracts and compounds. The laboratory strain Paea is susceptible to all insecticides. This strain, collected in French Polynesia, has been maintained for a decade in an insectary at the Institut Pasteur de la Guyane, Cayenne, French Guiana. The Cayenne strain is resistant to both pyrethroid and organophosphate insecticides and is a first generation (F1) strain from wild-caught mosquito larvae (F0). Mosquito rearing was performed under natural conditions: 28 ± 2 °C, 80 ± 20% RH, and 12 h ± 20 min over the year. A. aegypti eggs were preserved on dried paper strips at ambient temperature. Hatching was produced by dropping these strips in water and placing them in a vacuum pump for at least 40 min. Larvae were fed with yeast pellets and late third- or early fourth-instar larvae were used in the tests. All plant crude extracts and fractions were investigated using the WHO procedure for testing mosquito larvicides.26 The larvicidal activity of pure compounds was evaluated using a tube assay.27 Cup Assay. For each bioassay, 25 larvae of each strain were transferred to cups containing 99 mL of distilled water. For the determination of mortality rates, four cups, representing a total of 100 larvae, were used. The plant crude extract and fractions were tested at 100 and 10 ppm, respectively. For LD50 calculations, four cups per concentration (100 larvae) were used and seven concentrations from 10 to 150 μg/mL of each test material diluted in ethanol were tested in order to generate a dose−response curve. A 1 mL amount of the control insecticide at the desired concentration was added to each cup.

Negative-control treatment was performed for each test with 1 mL of ethanol. Larval mortality was recorded at 24 h after exposure. Tube Assay. Fifty larvae of the sensitive Paea strain were transferred to test tubes (75 × 12 × 0.8−1.0), soda rimLess, containing approximately 2.97 mL of distilled water. Ten tubes per concentration (10 × 5 larvae) and five concentrations from 1 to 10 μg/mL of each compound diluted in ethanol were used. A solution of 30 μL of the insecticide at the desired concentration was added to each tube. Control treatments were performed for each test with 30 μL of ethanol. Larval mortality was recorded at 24 h after exposure. Statistical Analysis. Initially, Abbott’s formula28 was applied to mortalities if mortality in the control was between 5% and 20%. The test was invalidated if mortality in the control was greater than 20%. Lethal doses were obtained by a probit regression under a general linearized model [BioRssay 6.1. script29 in R software version 3.2.0 (https://www.r-project.org/)]. Evaluation of Adulticidal Activity. Sixty 3−5-day adult females of A. aegypti Paea strain were selected for this test. The ingestion bioassay30 consisted of impregnating a cotton disk with 2 mL of the insecticide solution, prepared with sucrose (10% v/v) and trypan blue (0.02% v/v). Trypan blue was used to control the ingestion of the product by the mosquitoes. Three concentrations were used (30, 300, and 1000 μg/mL). Cypermethrin (0.5 μg/mL) was used as a positive control, and sucrose mixed with trypan blue was the negative control. Mortality was evaluated at 24, 48, and 72 h. Each test was performed with 20 females in triplicate (n = 60). A chi square test was applied to test the significance of the response relative to the negative control. Mosquito blood feeding was performed using Swiss mice from Charles River, France. Experimental authorization was obtained under the agreement number B973-02-01 delivered by “Préfecture de Guyane” and renewed on June 6, 2015. The specific experimental project was reapproved by the Ethical Committee CETEA Institut Pasteur (number 89), report number 2015-0010, issued on May 18, 2015. Cytotoxicity Assays. Cytotoxicity assays were conducted with KB (nasopharyngeal epidermoid carcinoma) and MRC5 (normal lung tissue of a 14-week-old male fetus) cell lines using the procedure described by Tempête et al.;31 docetaxel was used as positive control. For the evaluation of cytotoxicity against Aedes albopictus C6/36 cells, the protocol was adapted from a published procedure.32−34 Each well of a 24-well plate was filled with 720 μL of a cellular suspension (age 1 week, concentration 105−107 cells/mL). The plate was then incubated for 24 h. After incubation, the supernatant was eliminated, and 400 μL of test solution in L15 Leibovitz culture medium was added to obtain final concentrations of 50, 100, and 300 μg/mL. Plates were incubated with the test material for 24, 48, or 72 h at room temperature. After incubation, 40 μL of a 5 mg/mL MTT solution was added to each well. A new incubation period of 1 h was then observed, and the supernatants were eliminated and replaced by 400 μL of DMSO to dissolve formazan crystals formed in the cells. After homogenization, 100 μL of each well was transferred to a 96-well plate, and 100 μL of DMSO was added to each well. The absorbance was eventually read at 570 nm. The cytotoxic effect was evaluated by comparing the percentage of living cells treated with the test material versus living cells treated with the solvent alone. The following formula was used: mortality rate = (absorbance of the control − absorbance of the sample)/absorbance of the control × 100. Nonparametric analyses were performed with a Kruskal−Wallis test, and multiple comparisons were performed with the Dunn method and Bonferroni correction. Inhibition concentrations values (IC50) and 95% confidence intervals were calculated with logistic regression via probit analysis. Ecotoxicological Assessment on Nontarget Species, Daphnia magna and Chironomus riparius. The guidelines of the “Immediate Immobilization Test” (OECD 202) for Daphnia magna and the “Immediate Immobilization Test” (OECD No. 235) for Chironomus riparius were followed. The extract was tested only at the LC50 value defined from A. aegypti sensitivity (75 μg/mL). Three conditions were tested: control, control/solvent, and LC50, with four replicates per condition. The physicochemical measurements (pH, dissolved oxygen, conductivity) were performed with measuring devices (sensors). The remaining measures (chlorine, nitrites, nitrates, 389



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phosphates) were performed with aquarium strips. Photoperiod and temperature were recorded using a “templight” recorder throughout the test period, from clutch incubation until the end of the exposure.

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(11) Ioos, S.; Mallet, H. P.; Leparc Goffart, I.; Gauthier, V.; Cardoso, T.; Herida, M. Med. Mal. Infect. 2014, 44, 302−307. (12) Isman, M. B.; Grieneisen, M. L. Trends Plant Sci. 2014, 19, 140− 145. (13) Fine, P. V. A.; Metz, M. R.; Lokvam, J.; Mesones, I.; Zuniga, J. M. A.; Lamarre, G. P. A.; Pilco, M. V.; Baraloto, C. Ecology 2013, 94, 1764−1775. (14) Gonzales, A. G.; Bazzocchi, I. L.; Moujir, L.; Jiménez, I. A. Stud. Nat. Prod. Chem. 2000, 23, 649−738. (15) Deepa, M. A.; Narmatha Bai, V. Int. J. Bot. 2010, 6, 220−227. (16) Madrigal, R. V.; Zwilowski, B. W.; Smith, C. R., Jr. J. Chem. Ecol. 1985, 11, 501−506. (17) Shirota, O.; Morita, H.; Takeya, K.; Itokawa, H. J. Nat. Prod. 1994, 57, 1675−1681. (18) Gutiérrez-Nicolàs, F.; Obertir, J. C.; Ravelo, A. R.; EstévezBraun, A. J. Nat. Prod. 2014, 77, 1853−1863. (19) Gao, J. M.; Wu, W. J.; Zhang, J. W.; Konishi, Y. Nat. Prod. Rep. 2007, 24, 1153−1189. (20) Gonzales, A. G.; Jiménez, I. A.; Ravelo, A. G.; Bazzocchi, I. L. Tetrahedron 1993, 49, 6637−6644. (21) Yamada, K.; Shizuri, Y.; Hirata, Y. Tetrahedron 1978, 34, 1915− 1920. (22) Ya, L.; Strunz, G. M.; Calhoun, L. A. Phytochemistry 1991, 30, 719−721. (23) Guo, Z. H.; Huang, J.; Wan, G. S.; Huo, X. L.; Gao, H. Y. J. Nat. Med. 2013, 67, 844−849. (24) Minh, T. T.; Anh, N. T.; Thang, V. D.; Sung, T. V. Z. Naturforsch., B: J. Chem. Sci. 2010, 65, 1284−1288. (25) Gunatilaka, A. A. L.; Nanayakkara, N. P. D.; Sultanbawa, M. U. S. Tetrahedron Lett. 1979, 20, 1727−1730. (26) World Health Organization. Guidelines for Laboratory and Field Testing of Mosquito Larvicides. http://apps.who.int/iris/bitstream/ 10665/69101/1/WHO_CDS_WHOPES_GCDPP_2005.13.pdf (accessed August 2016). (27) Falkowski, M.; Jahn-Oyac, O.; Ferrero, E.; Issaly, J.; Eparvier, V.; Rodrigues, A. M. S.; Stien, D.; Houël, E.; Dusfour, I. J. Am. Mosq. Control Assoc. 2016, 32, in press. (28) Abbott, W. J. Econ. Entomol. 1925, 18, 265−267. (29) Milesi, P.; Pocquet, N.; Labbé, P. N. Engl. J. Med. 2014, 371, 887−889. (30) Stell, F. M.; Roe, R. M.; Arellano, C.; Kennedy, L.; Thornton, H.; Saavedra-Rodriguez, K.; Wesson, D. M.; Black, W. C.; Apperson, C. S. Med. Vet. Entomol. 2013, 27, 284−297. (31) Tempête, C.; Werner, G.; Favre, F. Eur. J. Med. Chem. 1995, 30, 647−650. (32) Carrasco-Pozo, C.; Pastene, E.; Vergara, C.; Zapata, M.; Sandoval, C.; Gotteland, M. Biochim. Biophys. Acta, Gen. Subj. 2012, 1820, 2052−2061. (33) Gieseler, A.; Schultze, A. T.; Kupsch, K.; Haroon, M. F.; Wolf, G.; Siemen, D.; Kreutzmann, P. Biochem. Pharmacol. 2009, 77, 888− 896. (34) Vidau, C.; Brunet, J. L.; Badiou, A.; Belzunces, L. P. Toxicol. In Vitro 2009, 23, 589−597.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00850. NMR spectra of compounds 1−5 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel (E. Houël): +594 594 29 31 34. Fax: +594 594 29 51 32. E-mail: [email protected]. *Tel (V. Eparvier): +33 169 82 36 79. Fax: +33 169 07 72 47. E-mail: [email protected]. ORCID

Véronique Eparvier: 0000-0002-2954-0866 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was part of the Laboratory of Excellence “Centre de la Biodiversité Amazonienne” [Labex CEBA (CEBA, ref ANR-10-LABX-25-01)] and of the STRonGer consortium (Institut Pasteur de la Guyane). This work was part of the INSECTICIDES project funded by Europe (ERDF OP, PRESAGE No. 31220), French Guiana Regional Council, and the Air Liquide Foundation. This work benefited from an “Investissement d’Avenir” grant managed by Agence Nationale de la Recherche (Infrastructure Nationale en Biologie Santé “ANAEE-France” ANR-11-INBS-0001) through the use of the U3E INRA1036 PEARL platform.

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REFERENCES

(1) World Health Organization, Media Center. Vector-borne Diseases. http://www.who.int/mediacentre/factsheets/fs387/en/ (accessed February 2016). (2) McGraw, E. A.; O’Neill, S. L. Nat. Rev. Microbiol. 2013, 11, 181− 193. (3) David, J.-P.; Ismail, H. M.; Chandor-Proust, A.; Paine, M. J. I. Philos. Trans. R. Soc., B 2013, 368, 20120429. (4) Sokhna, C.; Ndiath, M. O.; Rogier, C. Clin. Microbiol. Infect. 2013, 19, 902−907. (5) EC - European Commission 2007/565/EC. http://eur-lex. europa.eu/legal-content/EN/TXT/?uri=CELEX%3A32007D0565 (accessed July 2016). (6) MEEDDAT - Ministère de l’Ecologie, de l’Energie, du Développement Durable et de l’Aménagement du Territoire. www. legifrance.gouv.fr/affichTexte.do?cidTexte=JORFTEXT000019375714 (accessed July 2016). (7) JORF (Journal Officiel de la République Française) no. 0186 du 13 août 2014 page 13530 texte no. 42. http://www.legifrance.gouv.fr/ affichTexte.do?cidTexte=JORFTEXT000029358124 (accessed August 2016). (8) Guyton, K. Z.; Loomis, D.; Grosse, Y.; El Ghissassi, F.; Benbrahim-Tallaa, L.; Scoccianti, C.; Mattock, H.; Straif, K. Lancet Oncol. 2015, 16, 490−491. (9) Leparc-Goffart, I.; Nougairede, A.; Cassadou, S.; Prat, C.; de Lamballerie, X. Lancet 2014, 383, 514. (10) Musso, D.; Cao-Lormeau, V. M.; Gubler, D. J. Lancet 2015, 386, 243−244. 390


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