Alkamides from Artemisia dracunculus

Alkamides from Artemisia dracunculus

Bouchra Saadali ^a , Driss Boriky ^a , Mohamed Blaghen ^b , Maurice Vanhaelen ^c , Mohammed Talbi ^a, *

a

Laboratoire de Chimie Analytique et Structurale, associe´ au CNRSTP, Faculte´ des Sciences, Ben M’SikBP7960, Casablanca, Morocco

b

Laboratoire de Microbiologie, Biotechnologie et Environnement, Faculte´ des Sciences Aı¨n Chock, Maˆarif BP5366, Casablanca, Morocco

c

Laboratoire de Pharmacognosie et Bromatologie, Institut de Pharmacie de l’Universite´ Libre de Bruxelles, U.L.B. CP205/4, Bd du Triomphe, B-1050 Bruxelles, Belgium

Received 30 May 2001; accepted 6 August 2001

Abstract

From the aerial parts of Artemisia dracunculus, one known alkamide, pellitorine, two new alkamides neopellitorine A and neo- pellitorine B, and one known coumarin herniarine were isolated. Structures were elucidated by means of UV, IR, MS,

¹

H and

¹³

C NMR. These compounds showed insecticidal activity against Sitophilus oryzae and Rhyzopertha dominica at 200 mg/ml concentra- tions. # 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Artemisia dracunculus; Asteraceae; Anthemideae; N-Isobutyl amides; Insecticidal activity; Sitophilus oryzae; Rhyzopertha dominica

1. Introduction

The large genus Artemisia, from the tribe Anthemi- deae, has been the subject of numerous chemical and biological studies (Tan et al., 1998). The species give sesquiterpene lactones, coumarins and acetylenes as the main metabolites (Marco and Barbera, 1990). Prepara- tions from Artemisia plants have a long tradition in adjuvant therapy. In previous papers we reported eudesmanolides from the aerial parts of Artemisia herba alba (Boriky et al., 1996). This paper describes the iso- lation and structural elucidation of three alkamides and one know coumarin from the aerial parts of A. dra- cunculus, and their insecticidal effect against the rice wevil (Sitophilus oryzae) and grain borer (Rhyzopertha dominica). These stored products pests are characterized by their bioecology and the nature of their damage. In fact, these two harmful insects cause the most damage in stocks of corn and barley in Morocco and through out the world.

2. Results and discussion

The petroleum ether extract of aerial parts afforded, after silica gel chromatography pure amide compounds

1–3. Pellitorine 1 is reported for the first time in an Anacyclus species (Dunstan and Garnett, 1895). The IR spectrum of these compounds indicated the presence of amide group (bands at 3300, 3070, 1650 and 1540 cm

¹

) as well as olefinic double bands (1630 and 950 cm

¹

).

This together with the absorption maximum at 257 nm suggested a chromophore of conjugated dienoic acid amides for 1–3. The amide moiety was established by the mass spectrum. The peaks matching [M15]

⁺

, [M43]

⁺

, [M57]

⁺

, [M72]

⁺

and [M100]

⁺

were assigned to a piperidide moiety for 3.

The

¹

H NMR spectra of compounds 1–2 showed characteristic signals at 3.18 (H-1

⁰

), 1.80 (H-2

⁰

), and 0.92(H-3

⁰

,4

⁰

) indicating the isobutylamide moiety (Su and Horvart, 1981; Greger and Hofer, 1987; Bauer et al., 1988, 1989). In addition, the signals of a conjugated diene moiety were apparent ( 5.82(H-2), 7.21 (H-3), 6.24 (H-4) and 6.12(H-5)) in a trans–cis configuration as indicated by the coupling constants of 15 and 11 Hz for each pair of olefinic protons (Greger and Hofer, 1987; Bauer et al., 1988, 1989). The structure of 2 dif- fered from the 1 in the hydrocarbon chain attached to a carbon bearing the conjugated diene moiety, which could be observed from the

¹

H and

¹³

C NMR spectra (Tables 1 and 2). Compound 3 showed different signals in

¹

H NMR spectrum caused by the amine part, three multiplets at 1.57, 1.64 and 1.62that were char- acteristic to the cyclic methylenes and two typical mul- tiplets at 3.49 and 3.61 attached to protons in a

0031-9422/01/$ - see front matter

#

2001 Elsevier Science Ltd. All rights reserved.

P I I : S 0 0 3 1 - 9 4 2 2 ( 0 1 ) 0 0 3 4 7 - 8

Phytochemistry 58 (2001) 1083–1086

www.elsevier.com/locate/phytochem

* Corresponding author. Tel.: +212-270-4671; fax: +212-270-4675.

E-mail address: [email protected] (M. Talbi).

position of nitrogen (Greger et al., 1982). Also the pre- sence of an piperidide moiety was supported by the

¹³

C NMR signals at 25.6 (C-2

⁰

), 24.7 (C-3

⁰

), 26.7 (C-4

⁰

), 46.9 (C-1

⁰

) and 43.2(C-5

⁰

). The structure of 3 dif- fered from the 1 in the amine moiety, which could be deduced from the MS-fragmentation pattern [M84]

⁺

instead of [M72]

⁺

and the

¹

H,

¹³

C NMR spectra (Tables 1 and 2).

Compounds 1–3 have never been identified as con- stituents of Artemisia. Compounds 2 and 3 have been isolated for the first time, while compound 1 has pre- viously been found in Anacyclus pyrethrum (Dunstan and Garnett, 1895). According to previous reports (Jacobson, 1954; Bohlmann and Hoffmann, 1983)

unsaturated isobutylamides are well-known from the Asteraceae and for their insecticidal activities (Greger, 1984). They have also been reported from other genera such as Spilanthes (Ramscwak et al., 1999), Echinacea (Bauer et al., 1989), Achillea (Greger et al., 1982; Greger and Hofer, 1989) and Acmella (Martin and Becker, 1985).

Insecticidal tests on Sitophilus oryzae and Rhyzo- pertha dominica revealed that compounds 1–3 were very active, as shown in Fig. 1. Indeed compound 2 showed 100% mortality at a concentration of 200 mg/ml after 3 days on the two insects tested, however, compounds 1 and 3 showed a mortality of about 50 and 80%, respectively, toward R. dominica and S. oryzae at the same concentration after 3 days.

This investigation yielded two purely olefinic alka- mides and an acetylenic alkamide. Two of the three compounds possessed an isobutyl side chain. The dif- ference in insecticidal activity of these compounds could be attributed to their structural difference, especially the acetylenic group contained in compound 2 which showed the best insecticidal activity.

3. Experimental 3.1. General

1

H and

¹³

C NMR spectra were recorded in CDCl

3

on Varian Unity 600 instrument at 600 and 150 MHz, respectively; the chemical shifts are reported in units (ppm) values relative to TMS. IR spectra were recorded as films on a Nicolet 205-FTIR spectrometer. UV spec- tra were determined in MeOH with Shimadzu 265FS spectrophotometer. HR-EI-MS and ES-MS measure- ments were carried out on a Fisons VG Autospec. TLC were carried out on Silica gel F

254

precoated plates (0.25

Table 1

1

H NMR data of compounds

2

and

3

(600 MHz, CDCl

3

, TMS as int.

standard; chemical shifts in values (ppm); coupling constants in Hz in parentheses)

H

2 3

25.81 d (15) 6.25 d (15)

3 7.18 dd (15; 11) 7.23 dd (15; 11)

4 6.20 m 6.17 dd (11; 11)

5 6.06 m 6.05 m

6 2.40 d (3) 2.14 q (7)

7 – 1.42 m

8 – 1.29 m

9 – 1.29 m

10 – 0.89 t (7)

11 1.98 s –

NH 5.55 br s –

1

⁰

3.17 t (6.6) 3.49 m

2

⁰

1.80 m 1.57 m

3

⁰

0.92 d (6.6) 1.64 m

4

⁰

0.92 d (6.6) 1.62 m

5

⁰

– 3.61 m

Table 2

13

C NMR data of compounds

2

and

3

[150 MHz, CDCl

3

, TMS as int.

standard; chemical shifts in values (ppm)]

C

2^a 3^a

1 166.1 165.8

212 3.2118.5

3 138.9a 142.9

4 129.7 128.8

5 140.2a 142.6

6 31.232 .9

7 68.2b 28.5

8 64.9 31.4

9 64.9 22.5

10 65.4b 14.0

11 18.7 –

1

⁰

46.9 46.9b

2

⁰

28.5 25.6a

3

⁰

19.9 24.7a

4

⁰

19.9 26.7a

5

⁰

– 43.2b

a

a,b, interchangeable signals.

Fig. 1. Toxicity of compounds

1–3

on S. oryzae and R. dominica at 200

mg/ml concentration after 3 days. Statistical analysis was done

using ANOVA (P< 10

³

).

1084 B. Saadali et al. / Phytochemistry 58 (2001) 1083–1086

mm). C.C were also performed on Silica gel (Merck Silica gel 60, 70–230 mesh).

3.2. Plant material

Aerial parts of plants A. dracunculus were collected at Sale´, Morocco, in September 1997. Identification of the plant was confirmed by Dr. A. Ouyahya. A voucher specimen is deposited in the herbarium of the DBEV (De´partement de Botanique et Ecologie Ve´ge´tal, Institut Scientifique) in Rabat (Morocco).

3.3. Extraction and isolation

The plant material was air-dried at room temp., finely ground and extracted (100 g), successively with petro- leum ether (pb 40–60

) and CH

2

Cl

2

. The extracts were filtered, and the solvent was concentrated under reduced pressure. One portion (6 g) of the crude petroleum ether extract was fractionated by column chromatography on silica gel [A: hexane–EtOAc (9:1), B: hexane–EtOAc (7:3) and C: hexane–EtOAc (4:6)]. Fraction A (1.06 g) was fractionated by column chromatography on silica gel (hexane–EtOAc, 2:3) to give 1 (36 mg), 2 (17 mg) and 3 (9 mg). Crystallization of the fraction B from CH

2

Cl

2

afforded 476 mg of herniarine 4. This com- pound was isolated from A. dracunculus and identified as described previously (Ahmad and Misra., 1997).

3.4. Deca-2E, 4Z-diynoic acid isobutylamide 1 Yellow oil; UV l

max

nm: 257; IR p

max

cm

¹

: 3300, 3070, 1650, 1540 (–CO–NH–); 2950 (CH–); 1650, 950 (–C¼C–); HR-EI-MS: m/z (%, rel. int.) [M]

⁺

223.1939 (26.5) (calc. for 223.1936), 208.1706 (5.8), 180.1391 (5.7), 166.1236 (6), 151.1123 (100), 124.0763 (2.1), 100.0402(4.3); ES-MS: m/z (%) [2M]

⁺

447.65 (14), 226.42 (4), 225.37 (13), [M+1]

⁺

224.36 (100), 196.18 (4), 177.09 (11), 156.16 (11).

3.5. Undeca-2E, 4Z-dien-7,9-diynoic acid isobutylamide 2 Yellow oil; UV l

max

nm: 257; IR p

max

cm

¹

: 3300, 3070, 1660, 1520 (–CO–NH–); 2960 (CH–); 1620, 990 (–C¼C–); EI-MS: m/z (%, rel. int.) [M]

⁺

229 (33), 214 (10), 186 (7), 172 (12), 157 (100), 129 (31), 127 (38), 57 (43.5); ES-MS: m/z (%) [M]

⁺

459.56 (5), [M+2]

⁺

231.36 (18), [M+1]

⁺

230.31 (100), 226.30 (5), 196.25 (24), 158.18 (12), 156.17 (41);

¹

H and

¹³

C NMR data are presented in Tables 1 and 2.

3.6. Deca-2E, 4Z-dienoic acid piperidide 3 Yellow oil; UV l

max

nm: 257; IR p

max

cm

¹

: 3280, 3080, 1670, 1638 (–CO–N<); 2940 (CH–); 1630, 970 (–C¼C–); EI-MS: m/z (%, re. int.) [M+1]

⁺

236 (4.5), [M]

⁺

235 (17.5), 192 (25.5), 178 (12), 164 (31), 151 (24), 138 (25), 127 (34), 112 (46), 84 (100), 57 (38.5);

¹

H and

13

C NMR data are presented in Tables 1 and 2.

3.7. Insecticidal test

The procedure was similar to the one described by Regnaut–Roger (Hamraoui and Regnault, 1997).

The isolated compounds (each sample at a con- centration 5 mg/ml) were tested for their insecticidal activity, using paper discs (Whatman, Ø=9 mm) impregnated with 30, 40 and 50 ml of the samples from which the solvent was allowed to evaporate at room temperature. Then, the paper disc was introduced into the experimental chamber containing 10 g of wheat and 20 adults insects (each species was tested separately).

The tests were conducted in semi-aerated medium at 25

C and a relative humidity of 70%.

Three replicates were employed for each sample and the mortality was recorded at 24 h, 2, 3 and 4 days.

Acknowledgements

We are grateful to Mr. C. Moulard, to Dr. M. Luh- mer for MS and NMR measurements and, to the

‘‘De´partement des Relations Internationales’’ of U.L.B.

for their financial support.

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