Synthesis and insect antifeedant activity of plumbagin derivatives with the amino acid moiety

Article abstract of DOI:10.1021/jf901760h

A series of plumbagin derivatives (4a-4k) containing an amino acid moiety were synthesized under mild esterification conditions in excellent yields (35%-80%) and screened for their antifeedant activities in tobacco caterpillar (Spodoptera lltura) and cast

Full text of DOI:10.1021/jf901760h

6090 J. Agric. Food Chem. 2009, 57, 6090–6094
DOI:10.1021/jf901760h
Synthesis and Insect Antifeedant Activity of Plumbagin
Derivatives with the Amino Acid Moiety
†
,
T
HONTHULA
S
REELATHA,† ATMAKUR
H
YMAVATHI,† KATRAGADDA
S
URESH
B
ABU
‡
J
OISH
MADHUSUDANA
M
URTHY,‡ USHA
R
ANI
P
ATHIPATI
,†
,
AND
J
ANASWAMY
M
ADHUSUDANA
R
AO*
†Division of Organic Chemistry- I, Natural Product Laboratory, Indian Institute of Chemical Technology,
Tarnaka, Hyderabad 500 607, India, and ‡Biology Division, Indian Institute of Chemical Technology,
Tarnaka, Hyderabad 500 607, India
A series of plumbagin derivatives (4a-4k) containing an amino acid moiety were synthesized under
mild esterification conditions in excellent yields (35%-80%) and screened for their antifeedant
activities in tobacco caterpillar (Spodoptera litura) and castor semilooper (Achaea janata) using a
no-choice laboratory bioassay. The parent compound plumbagin lacked significant activity, but the
analogues were effective in reducing feeding by two insect species. The introduction of an N-acetyl-
L-amino acid side chain to the Michael adduct of plumbagin at the third position of the quinone
moiety significantly increased antifeedant activity. Several of the analogues were also toxic or
caused developmental abnormalities following topical administration.
KEYWORDS: Plumbagin; amino acid esters; Spodoptera litura; Achaea janata; insect antifeedant
INTRODUCTION
which prompted us to synthesize derivatives and screen for
antifeedant activity. In view of the fact that the 1,4-naphthaqui-
none backbone is essential for antifeedant activity, we focused on
the synthesis of the new analogues, by exploiting the C-3 position.
In this context, we have designed analogues of plumbagin by
introducing a peptidyl side chain at the 3-position of plumbagin
and keeping the quinone moiety in tact. This was achieved by the
Michael addition of 5-O-methylplumbagin with ethanolamine
The severe damage caused to the ecology and environment as
well as human health due to the usage of complex chemical
synthetic pesticides necessitated a shift to natural crop protection
and nourishment. Plants produce a diverse range of secondary
metabolites such as terpenoids, alkaloids, polyacetylenes, flavo-
noids, quinones, and sugars as part of their defense mechanism
against insects. Among these classes, naphthaquinones, in parti-
cular, 5-hydroxy-1,4-naphthaquinones such as plumbagin and
juglone, are an important class of natural products that are
extremely useful for the development of potent agrochemicals
in view of their high abundance and relatively nontoxic nature.
A literature precedence revealed that plumbagin isolated from
the Plumbago capensis possesses significant biological activi-
ties such as antimicrobial (1), cytotoxic (2), antimalarial (3),
and antiprotozoal properties (4). Previously, it was illustrated
that the ingestion of the crude aqueous methanol extracts of
P. capensis and its derivatives caused the failure of the molting
cycle in another lepidopteran insect, Bombyx mori (silk worm)(5).
Recently, Tokunaga et al. isolated certain plumbagin analo-
gues from the carnivorous plant, Dionae muscipula Ellis, which
were found to exhibit significant antifeedant activity (6). Further,
they also suggested that the defined substituents on the 1,4-
naphthaquinone backbone can increase the selectivity and pot-
ency of the desired action (7). The naphthaquinone skeletons with
high abundance and favorable structural features form useful
models for the development of potent antifeedants. As part of an
ongoing effort to discover potential leads from Indian medicinal
plants (8, 9), we have isolated large quantities of plumbagin (1),
followed by condensation of a series of N-acetyl-L-amino acids
with the Michael adducts. All of these derivatives along
with plumbagin have been evaluated for their antifeedant, toxic,
and growth regulatory activities against two major agricultural
pests, castor semilooper, Achaea janata, and tobacco caterpillar,
Spodoptera litura.
MATERIALS AND METHODS
General. 1H and 13C spectra were measured on a Bruker 300 MHz
spectrometer using tetramethylsilane as an internal standard. Mass spectra
were recorded on an Agilent LC/MSD trap SL 1100 series spectrometer
with a 70 eV (ESI probe) and the infrared spectra on a Thermo Nicolet
Nexus 670 FTIR spectrometer. Melting points were taken on a Fischer
Scientific melting point apparatus and are uncorrected. The synthetic
compounds were purified by column chromatography using 60-120 mesh
size silica gel (Merck). Thin layer chromatography (TLC) involved the use
of precoated silica gel 60 F254 TLC plates from Merck. The optical
rotations were measured on a Jasco Dip 360 digital polarimeter.
Extraction and Isolation of Plumbagin. The roots of Plumbago
capensis also known as Nila chitramula (10) were collected during August-
September 2005 from Tirumala forest, Tirupati, Andhra Pradesh, India.
A voucher specimen was deposited at the herbarium of Indian Institute
of Chemical Technology, Hyderabad, India. The shade dried roots of
Plumbago capensis were powdered in a pulvarizer (10 kg) and extracted
with chloroform/methanol, 1:1, followed by concentration under reduced
*To whom correspondence should be addressed. Tel: þ91-40-
27193166. Fax: þ91-40-27160512. E-mail: janaswamy@iict.res.in.
pubs.acs.org/JAFC
Published on Web 06/16/2009
© 2009 American Chemical Society

Article
J. Agric. Food Chem., Vol. 57, No. 14, 2009 6091
pressure. The resulting extract was (70 g) chromatographed over silica gel
(60-120 mesh) and eluted with n-hexane/ethyl acetate combinations
of increasing polarity. Plumbagin (12 g) was obtained by elution with
n-hexane/ethyl acetate, 99:1.
130.39, 131.80, 135.60 147.12, 159.62, 169.94, 171.60, 180.80, 183.31.
ESIMS m/z (rel.int.): 374 [Mþ] (C19H22N2O6) (23), 331 [Mþ-COCH3] (4),
304 (11), 260 (63), 248 (47), 234 (79), 220 (9), 203 (100), 175 (33), 147 (11),
99.1 (15).
General Procedure for the Synthesis of Compound 2. To a solution
of plumbagin (1) (0.2 g, 1.0638 mmol) in dichloromethane (10 mL) at room
temperature was added CH3I (1.39 mmol) and freshly prepared Ag2O
(0.05 mmol). The resultant mixture was stirred for 10 h at room
temperature. After completion of the reaction (monitored by TLC), the
reaction mixture was filtered, and then the filtrate was concentrated to
dryness. The residue thus obtained was purified by column chromatogra-
phy with the elution of n-hexane/ethyl acetate, 90:10, to afford 5-O-methyl
plumbagin (2) as yellow needles (11).
2-(8-Methoxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalene-
2-ylamino) ethyl 2-acetamido-3-mercaptopropanoate (4c).
The title compound was prepared by the reaction of compound 3 (0.050 g,
0.13 mmol) with N-acetyl cysteine (0.0426 g, 0.207 mmol). Yield: 42%. mp:
25
119 °C; [R]D þ 6.5° (c1, H2O). IR (KBr) νmax: 3329 (NH), 2923, 1740 (ester
CdO), 1660 (R,β unsaturated CdO), 1575, 1449, 1273, 747 cm-1. 1H NMR
(300 MHz, CDCl3): δ 2.03 (s, 3H, 90-CH3), 2.24 (s, 3H, 2-CH3), 2.32 (d, 2H,
J=7.5 Hz, H-100), 3.84 (dd, 2H, J=6.8 Hz, 14.8 Hz, H-20), 3.98 (s, 3H,
5-OMe), 4.32(dd, 2H, J=6.8 Hz, 14.8 Hz, H-30), 4.73 (t, 1H, J=7.3 Hz, H-60),
7.15 (dd, 1H, J=1.1, 8.5 Hz, H-6), 7.62 (t, 1H, J=7.5 Hz, 8.5 Hz, H-7), 7.75
(dd, 1H, J=1.1 Hz, 7.5 Hz, H-8). 13C NMR (75 MHz, CDCl3,): δ 11.03,
14.12, 22.83, 27.30, 31.37, 51.75, 56.34, 64.35, 115.82, 118.06, 119.16, 131.22,
135.58, 147.24, 159.61, 170.97, 173.04, 180.83, 183.34. ESIMS m/z (rel.int.):
407 [MþþH] (C19H22N2SO6) (23), 373 [Mþ-SH] (8), 315 [373-58] (29), 301
(43), 288 (12), 260 (13), 185 (100), 155 (10), 93 (15).
Procedure for the Synthesis of Compound 3. To a solution of 5-O-
methyl plumbagin (2) (1 mmol) in dry dichloromethane (10 mL) under
nitrogen atmosphere was added dropwise 2-amino ethanol (1.5 mmol) at
room temperature. The resulting orange colored solution was stirred for
1 h at room temperature. The reaction was monitored by TLC, and after
completion, the reaction mixture was concentrated to dryness. The resi-
due was purified by column chromatography using dichloromethane/
methanol (99:1) to yield the Michael adduct (3) as red needles. Yield: 68%.
mp: 126 °C. IR (KBr) νmax: 3312 (NH), 1662 (R,β unsaturated CdO),
1555, 1275, 1038 cm-1. 1H NMR (CDCl3, 300 MHz): δ 2.24 (3H, s, H-2,
-CH3), 3.74 (2H, dd, J=6.8 Hz, 14.8 Hz, H-20), 3.85 (2H, dd, J=6.8 Hz,
14.8 Hz, H-30), 3.98 (3H, s, -OMe), 7.15 (1H, dd, J=1.1 Hz, 8.5 Hz, H-6),
7.62 (1H, t, J=7.5 Hz, 8.5 Hz, H-7), 7.75 (1H, dd, J=1.1 Hz, 7.5 Hz, H-8);
13C NMR (75 M Hz, CDCl3): δ 14.43, 50.95, 56.34, 64.35, 115.55, 118.06,
119.36, 128.65, 135.58, 135.64, 147.24, 161.02, 180.83, 183.34. ESIMS m/z
(rel.int.): 284 [Mþ23] (22), 261 [MþH] (C14H15NO4) (15), 201(100), 175 (7),
147 (34).
2-(8-Methoxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalene-
2-ylamino) ethyl 2-acetamido-4-(methyl thio)butanoate
(4d). The title compound was prepared by the reaction of compound 3
(0.050 g, 0.13 mmol) with N-acetyl methionine (0.039 g, 0.204 mmol).
25
Yield: 68%. mp: 122 °C; [R]D þ25.9° (c 1, H2O). IR (KBr) νmax: 3329
(NH), 2923, 1740 (ester CdO), 1660 (R,β unsaturated CdO), 1575, 1449,
1273, 747 cm-1 1H NMR (300 MHz, CDCl3): δ 1.49 (s, 3H, 400-CH3),
.
1.95-1.97 (2H, m, H-100), 2.03 (s, 3H, 90-CH3), 2.24 (s, 3H, 2-CH3), 2.49
(t, 2H, J=7.2 Hz, H-200), 3.84 (dd, 2H, J=6.8, 14.8 Hz, H-20), 3.98 (s, 3H,
5-OMe), 4.44 (dd, 2H, J=6.8 Hz, 14.8 Hz, H-30), 4.63 (t, 1H, J=7.3 Hz,
H-60), 7.15 (dd, 1H, J=1.1 Hz, 8.5 Hz, H-6), 7.62 (t, 1H, J=7.5 Hz, 8.5 Hz,
H-7), 7.75 (dd, 1H, J=1.1 Hz, 7.5 Hz, H-8). 13C NMR (75 MHz, CDCl3):
δ 11.04, 14.12, 15.49, 22.68, 29.35, 31.37, 44.10, 51.73, 56.36, 64.52, 112.29,
115.93, 119.16, 130.95, 135.60, 147.13, 159.64, 170.12, 171.98, 180.86,
183.35. ESIMS m/z (rel.int.): 435 [MþþH] (C21H26N2SO6) (17), 419 [Mþ-
CH3] (20), 408 [Mþ-CO] (45), 301 (17), 288 (62), 260 (13), 185 (41), 155
(100), 93 (15).
General Procedure for the Synthesis of Derivatives 4a-4k. To a
cooled solution (0 °C) of 3 (1 equiv) in dry dichloromethane (10 mL) under
nitrogen atmosphere was added N,N0-dicyclohexylcarbodimide and (1.5
equiv) and a catalytic amount of 3-hydroxy benzotriazole. N-Acetyl-L-
amino acid (1.2 mmol) was added after stirring for 15 min, and stirring was
continued at room temperature. After completion of the reaction (mon-
itored by TLC), the reaction mixture was filtered to remove the precipi-
tated dicyclohexylurea. The filtrate was evaporated under reduced
pressure, and the residue was purified by silica gel (100-200 mesh) column
chromatography using dichloromethane/methanol (98:2) as eluent to
afford the corresponding plumbagin derivatives (4a-4k). The structures
of the new derivatives were confirmed by the spectral analyses such as
FTIR, 1H NMR, 13C NMR, and mass spectroscopy.
2-(8-Methoxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalene-
2-ylamino) ethyl 2-acetamido-3-methyl Pentanoate (4e). The
title compound was prepared by the reaction of compound 3 (0.050 g, 0.13
mmol) with N-acetyl isoleucine (0.036 g, 0.208 mmol). Yield: 30%. mp: 98
°C; [R]D þ40.0 (c1, H2O). 1H NMR (300 MHz, CDCl3): δ 0.89 (d, 3H, J=
25
5.8 Hz, 100-CH3), 0.90 (t, 3H, J=7.2 Hz, 300-CH3), 1.25-1.28 (2H, m, H-
200), 1.48-1.52 (1H, m, H-100), 2.03 (s, 3H, 90-CH3), 2.24 (3H, s, 2-CH3),
3.84 (dd, 2H, J=6.8 Hz, 14.8 Hz, H-20), 3.98 (s, 3H, -OMe), 4.44 (dd, 2H,
J=6.8 Hz,14.8 Hz, H-30), 4.63 (dd, 1H, J=7.2 Hz, H-60), 7.15 (dd, 1H, J=
1.1, 8.5 Hz, H-6), 7.62 (t, 1H, J=7.5 Hz, 8.5 Hz, H-7), 7.75 (dd, 1H, J=1.1
Hz, 7.5 Hz, H-8). 13C NMR (75 MHz, CDCl3,): δ 11.03, 14.10, 15.42,
22.83, 23.05, 29.35, 30.00, 38.65, 51.73, 56.34, 64.35, 115.90, 118.06,
119.16, 128.59, 135.58, 147.24, 159.61, 170.97, 173.04, 180.83, 183.34.
ESIMS m/z (rel.int.): 417 [MþþH] (C22H28N2O6), 395 (16), 301 (12), 261
(38), 217 (100), 195 (15), 173 (27), 101 (39), 93 (18), 78 (21), 65 (34).
2-(8-Methoxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalene-
2-ylamino) ethyl 2-acetamido-4-methyl Pentanoate (4f). The
title compound was prepared by the reaction of compound 3 (0.050 g, 0.13
mmol) with N-acetyl leucine (0.036 g, 0.208 mmol). Yield: 80%. mp: 96 °C;
2-(8-Methoxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalene-
2-ylamino)ethyl-2-acetamidoacetate (4a). The title compound
was prepared by the reaction of compound 3 (0.050 g, 0.13 mmol) with
N-acetyl-glycine (0.0243 g, 0.207 mmol). Yield: 78%. mp: 105 °C. IR (KBr)
ν
max: 3312 (NH), 1747 (ester CdO), 1662 (R,β unsaturated CdO), 1555,
1275, 1038, 745 cm-1. 1H NMR (300 MHz, CDCl3): δ 2.03 (s, 3H, 90-CH3),
2.24 (s, 3H, 2-CH3), 3.14 (dd, 2H, J=6.8 Hz, 14.8 Hz, H-20), 3.98 (s, 3H,
5-OMe), 3.90 (dd, 1H, J=5.4 Hz, 17.1 Hz, H-60a), 4.03 (dd, 1H, J=5.4 Hz,
17.1 Hz, H-60b), 4.27 (dd, 2H, J=6.8 Hz, 14.8 Hz, H-30), 7.15 (dd, 1H,
J=1.1 Hz, 8.5 Hz, H-6), 7.62 (t, 1H, J=7.5 Hz, 8.5 Hz, H-7), 7.75 (dd, 1H,
J=1.1 Hz, 7.5 Hz, H-8). 13C NMR (75 MHz, CDCl3,): δ 11.04, 14.03, 22.83,
50.95, 56.34, 64.35, 115.93, 118.06, 119.16, 128.80, 135.58, 135.69, 147.24,
159.61, 170.97, 173.04, 180.83, 183.34. ESIMS m/z (rel.int.): 383 [Mþ23],
361 [MþH] (C18H20N2O6) (15), 318 [Mþ-COCH3] (13), 300 [318-H2O] (37),
273 (25), 260 (15), 244 (8) 225 (25), 212 (17), 149 (100), 121 (83), 99 (30).
2-(8-Methoxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalene-
2-ylamino)ethyl2-acetamidopropanoate (4b). The title com-
pound was prepared by the reaction of compound 3 (0.050 g, 0.13 mmol)
with N-acetyl alanine (0.027 g, 0.207 mmol). Yield: 62%. mp: 109 °C;
25
[R]D þ14.5 (c1, H2O). IR (KBr) νmax: 3339 (NH), 2925, 1737(ester CdO),
1657 (R,β unsaturated CdO), 1576, 1452, 1275 cm-1. 1H NMR (300 MHz,
CDCl3,): δ 0.90 (d, 6H, J=9.3 Hz, 300-CH3, 400-CH3), 1.61-1.63 (1H, m, H-
100), 1.78-1.82 (1H, m, H-200), 2.03 (s, 3H, H-90), 2.24 (s, 3H, 2-CH3), 3.84
(dd, 2H, J=6.8 Hz, 14.8 Hz, H-20), 3.98 (s, 3H, -OMe), 4.44 (dd, 2H, dd, J=
6.8 Hz, 14.8 Hz, H-30), 4.63 (t, 1H, J=7.3 Hz, H-60), 7.15 (dd, 1H, J=1.1,
8.5 Hz, H-6), 7.62 (dd, 1H, J=7.5 Hz, 8.5 Hz, H-7), 7.75 (dd, 1H, J=1.1
Hz, 7.5 Hz, H-8). 13C NMR (75 MHz, CDCl3): δ 11.03, 14.10, 22.83, 22.99,
24.88, 31. 24, 42. 07, 41.06, 51.15, 56.34, 64.35, 115. 90, 118.06, 119.16,
128.59, 135.58, 147.24, 159.61, 170.97, 173.04, 180.83, 183.34. ESIMS m/z
(rel.int.): 417 [MþþH] (C22H28N2O6) (18), 395 (16), 301 (12), 261 (38), 217
(100), 195 (15), 173 (27), 101 (39), 93 (18), 78 (21), 65 (34).
20
[R]D þ14.5° (c 1, H2O). IR (KBr) νmax: 3314 (NH), 1742 (ester CdO
stretch), 1665 (R,β unsaturated CdO), 1552, 1270,1034, 742 cm-1 1H
.
NMR (300 MHz, CDCl3): δ 1.46 (d, 3H, J=6.8 Hz, 60-CH3), 2.03 (s, 3H,
H-90), 2.24 (s, 3H, 2-CH3), 3.87 (dd, 2H, J=6.8 Hz, 14.8 Hz, H-20), 3.98 (s,
3H, 5-OMe), 4.35 (dd, 2H, J=6.8 Hz, 14.8 Hz, H-30), 4.45-4.46 (1H, m, H-
60), 7.15 (dd, 1H, J=1.1, 8.5 Hz, H-6), 7.62 (t, 1H, J=7.5 Hz, 8.5 Hz, H-7),
7.75 (dd, 1H, J=1.1 Hz, 7.5 Hz, H-8). 13C NMR (75 MHz, CDCl3,): δ
11.02, 14.10, 22.83, 50.95, 53.34, 56.34, 64.31, 115.93, 118.08, 119.13,
2-(8-Methoxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalene-
2-ylamino) ethyl 2-acetamido-3-phenyl Propanoate (4g). The
title compound was prepared by the reaction of compound 3 (0.050 g,

6092 J. Agric. Food Chem., Vol. 57, No. 14, 2009
Sreelatha et al.
0.13 mmol) with N-acetyl phenyl alanine (0.054 g, 0.288 mmol). Yield:
mp: 142 °C. IR (KBr) νmax: 3356 (NH), 2925, 2854, 1732 (ester CdO), 1658
25
52%. mp: 132 °C; [R]D þ10.0 (c1, H2O). IR (KBr) νmax: 3327 (NH), 2926,
(R,β unsaturated CdO), 1549, 1460, 1277 cm-1 1H NMR (300 MHz,
.
2854, 1737 (ester CdO), 1662 (R,β unsaturated CdO), 1610, 1576, 1452,
CDCl3): δ 2.00 (s, 3H, 90-CH3), 2.12 (s, 3H, 2-CH3), 3.77 (dd, 2H, J=6.8
Hz, 14.8 Hz, H-20), 3.95 (s, 3H, 5-OCH3), 4.44 (dd, 2H, J=6.8 Hz, 14.8 Hz,
H-30), 7.15 (dd, 1H, J=1.1 Hz, 8.5 Hz, H-6), 7.36 (d, 2H, J=9 Hz, H-300,
H-500), 8.02 (d, 2H, J=9 Hz, H-200, H-600), 7.62 (dd, 1H, J=7.5 Hz, 8.5 Hz,
H-7), 7.75 (dd, 1H, J=1.1 Hz, 7.5 Hz, H-8). 13C NMR (75 MHz, CDCl3):
δ 14.94, 23.14, 44.17, 56.36, 64.15, 114.76, 115.89, 117.62, 119.15 (2C),
128.67, 132.43 (2C), 133.96, 135.55, 149.33, 157.76, 161.75, 167.76, 170.14,
172.08, 180.69, 183.70. ESIMS m/z (rel.int.): 420 [Mþ] (C23H22N2O6) (31),
362 [Mþ-NHCOCH3] (16), 341 (12), 243 (23), 2231(9), 202 (58), 175 (42),
121 (32), 99 (8), 43 (24).
1274 cm-1 1H NMR (300 MHz, CDCl3): δ 2.03 (s,3H, 90-CH3), 2.10
.
(s, 3H, 2-CH3), 3.97 (s,3H, 5-OCH3), 3.18 (d, 2H, J=7.5 Hz, H-100), 3.68
(dd, 2H, J=6.8 Hz, 14.8 Hz, H-20), 4.44 (dd, 2H, J=6.8 Hz, 14.8 Hz, H-30),
4.63 (t, 1H, J=7.5 Hz, H-60), 7.15 (dd, 1H, J=1.1 Hz, 8.5 Hz, H-6), 7.17-
7.20 (5H, m, H-100, Ph), 7.62 (dd, 1H, J=7.5 Hz, 8.5 Hz, H-7), 7.75 (dd, 1H,
J=1.1 Hz, 7.5 Hz, H-8). 13C NMR (75 MHz, CDCl3,): δ 11.41, 14.01,
22.98, 37.87, 44.11, 53.34, 56.34, 64.31, 115.93, 118.08, 119.13, 127.19,
128.59, 129.16, 129.40, 132.47, 135.57, 135.69, 147.12, 159.62, 167.77,
169.94, 171.60, 180.80, 183.31. ESIMS m/z (rel.int.): 451 [MþþH]
(C25H26N2O6) (37), 433 (28), 413 (60), 391 [Mþ-NHCOCH3] (38), 358
[Mþ- CH2Ph] (21), 260 (15), 228 (38), 197 (100), 146 (28), 102 (20), 87 (40).
2-(8-Methoxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalene-
2-ylamino) ethyl 2-acetamido-3-methyl Butanoate (4h). The
title compound was prepared by the reaction of compound 3 (0.050 g,
0.13 mmol) with N-acetyl valine (0.033 g, 0.207 mmol). Yield: 60%. mp:
Bioassay. Antifeedant activity of the compounds was assessed on
tobacco caterpillar larvae (Spodoptera litura) and castor semilooper
(Achaea janata). The experiments were conductedaccording to the classical
no-choice leaf-disk bioassay described earlier by Akhtar et al. (12). Castor
semilooper, Achaeajanata (L), and thetobacco cutworm, Spodoptera litura
(Fab), were reared on fresh castor leaves (Ricinus communis (L)) grown in
the laboratory at 28 ( 2 °C, relative humidity of 65 ( 5, and a 16:8 light/
dark photo period. Tostudy theantifeedant activity of thetest compounds,
a small circular disk of 5 cm diameter was cut from fresh castor leaves. The
leaf discs were treated on their upper surface with individual concentra-
tions of the compounds, and one leaf disk each was transferred to each
Petri plate of 15 cm diameter containing moist filter paper. Control leaf
discs were treated with the same volume of the solvent only.
In each Petri dish, prestarved healthy third instar larvae of A. janata and
S. litura were introduced to assess antifeedant activity. Progress of the
consumption of the leaf area was measured at 6, 12, and 24 h in both
treated and control leaf disks. Areas of control and treated leaf discs
consumed were measured after 6 h using an AM-300 leaf area meter
(ADC, Bioscientific Limited, England). The antifeedant index was then
calculated as (C - T)/(C þ T) ꢀ 100, where C is the consumption of con-
trol discs, and T is consumption of treated discs (13). For each concentra-
tion, 10 experimental sets were assayed. Each test was replicated three
times. The mean of the 10 sets was taken for each compound, and the
percentage of antifeedant activity with standard deviation was calculated.
Topical Bioassay. Toxicity of the compounds was determined by
topical application to the fourth instar of test insects. The experiments
were conducted according to the topical application method described
earlier by Jamil et al. (14). Four micrograms of compounds was applied
directly to the dorsum of the larva in a 1 μL drop of acetone using a micro
applicator. The controls were treated with the solvent alone. The treated
and control larvae were reared on fresh castor leaves. Mortality was
determined daily until 3 days after treatment. Larvae that lost elasticity
and showed no responses when their tails were pinched with forceps were
regarded as dead. The abnormalities in the form of growth retardation
symptoms were recorded at 4, 14, and 28 days after treatment. On the basis
of three independent trials, each experiment was conducted in four
replicates, leading to 12 replicates with each concentration of compounds.
The mean of 12 replicates was taken for each compound, and the
percentage of mortality with standard error was calculated.
25
99 °C; [R]D þ15.0 (c1, H2O). IR (KBr) νmax: 3356 (NH), 2925, 2854, 1732
(ester CdO), 1658 (R,β unsaturated CdO), 1549, 1460, 1277, 747 cm-1. 1H
NMR (300 MHz, CDCl3): δ 0.91 (d, 3H, J=10.3 Hz, 200-CH3), 0.94 (d, 3H,
J=10.3 Hz, 300-CH3), 2.00 (s, 3H, 90-CH3), 2.12 (s, 3H, 2-CH3), 2.30-2.33
(1H, m, H-100), 3.77 (dd, 2H, J=6.8 Hz, 14.8 Hz, H-20), 3.95 (s, 3H,
5-OCH3), 4.44 (dd, 2H, J=6.8 Hz, 14.8 Hz, H-30), 4.63 (d, 1H, J=7.5 Hz,
H-60), 7.15 (dd, 1H, J=1.1, 8.5 Hz, H-6), 7.62 (t, 1H, J=7.5 Hz, 8.5 Hz,
H-7), 7.75 (dd, 1H, J=1.1 Hz, 7.5 Hz, H-8). 13C NMR (75 MHz, CDCl3,):
δ 14.09, 17.78, 18.95, 23.14, 30.17, 44.17, 56.36, 57.18, 68.15, 115.89,
117.62, 119.15, 123.96, 132.43, 135.56, 147.89, 157.09, 170.14, 172.08,
180.69, 183.70. ESIMS m/z (rel.int.): 402 [Mþ] (C21H26N2O6) (31), 344
[Mþ- NHCOCH3] (16), 301 (12), 288 (23), 260 (9), 217 (100), 202 (58),
175 (42), 99 (8).
2-(8-Methoxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalene-
2-ylamino) ethyl 2-acetamido-2-(4-hydroxy phenyl) Acetate
(4i). The title compound was prepared by the reaction of compound 3
(0.050 g, 0.13 mmol) with N-acetyl tyrosine (0.046 g, 0.206 mmol). Yield:
25
40%. mp: 151 °C. [R]D -12.0 (c 1, H2O). IR (KBr) νmax: 3356 (NH),
2925, 2854, 1732 (ester CdO), 1658 (R,β unsaturated CdO), 1549, 1460,
1277 cm-1. 1H NMR (300 MHz, CDCl3): δ 1.98 (s, 3H, 90-CH3), 2.12 (s,
3H, 2-CH3), 2.86 (dd, 1H, J=5.0 Hz, 13.2 Hz, H-100a), 3.15 (dd, 1H, J=5.0
Hz, 13.2 Hz, H-100b), 3.77 (dd, 2H, J=6.8 Hz, 14.8 Hz, H-20), 3.95 (s, 3H, 5-
OCH3), 4.44 (t, 2H, J=6.8 Hz, 14.8 Hz, H-30), 4.60 (t, 1H, J=7.5 Hz, H-60),
6.63 (d, 2H, J=10.2 Hz, H-300, H-700), 7.15 (dd, 1H, J=1.1, 8.5 Hz, H-6),
7.18 (d, 2H, J=10.2 Hz, H-400, H-600) 7.62 (t, 1H, J=7.5 Hz, 8.5 Hz, H-7),
7.75 (dd, 1H, J=1.1 Hz, 7.5 Hz, H-8). 13C NMR (75 MHz, CDCl3,):
δ 14.06, 22.94, 37.17, 44.17, 53.36, 57.18, 68.15, 115.14, 115.89, 117.62,
119.15, 127.96 (2C), 128.65(2C), 131.40, 133.67, 135.18, 147.89, 158.82,
159.62, 170.14, 172.08, 180.33, 183.14. ESIMS m/z (rel.int.): 466 [Mþ]
(C25H26N2O7) (15), 408 [Mþ- NHCOCH3] (21), 315 (49), 301 (12), 288
(23), 260 (9), 217 (100), 202 (58), 175 (42), 99 (8).
2-(8-Methoxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalene-
2-ylamino)ethyl-4-acetamido Butanoate (4j). The title com-
pound was prepared by the reaction of compound 3 (0.050 g, 0.13 mmol)
with N-acetyl-4-amino butyric acid (0.030 g, 0.206 mmol) .Yield: 35%. mp:
101 °C. IR (KBr) νmax: 3356 (NH), 2925, 2854, 1732 (ester CdO), 1658
Data Analysis. Antifeedant indices were calculated using five different
concentrations of each compound, and data was subjected to probit
analysis (15) to determine the ED50 value representing the concentrations
that caused 50% feeding deterrence along with the 95% confidence
intervals. Results from the topical and growth regulatory bioassays were
analyzed by one way ANOVA. Post hoc testing was carried out using the
Tukey test. A significant level of 0.05 was used for all statistical tests.
(R,β unsaturated CdO), 1549, 1460, 1277 cm-1 1H NMR (300 MHz,
.
CDCl3): δ 1.83 (dd, 2H, J=6.8 Hz, 13.4 Hz, H-70), 1.98 (s, 3H, 110-CH3),
2.16 (3H, s, 2-CH3), 2.76 (t, 2H, J=8.5 Hz, H-60), 3.26 (dd, 2H, J=7.0 Hz,
13.5 Hz, H-80), 3.77 (dd, 2H, J=5.4 Hz, J=10.7 Hz, H-20), 3.95 (s, 3H, 5-
OCH3), 4.44 (t, 2H, J=6.8 Hz, H-30), 4.26 (dd, 2H, J=5.4 Hz, J=10.7 Hz,
H-30), 7.16 (dd, 1H, J=1.1, 8.5 Hz, H-6), 7.62 (t, 1H, J=7.5 Hz, 8.5 Hz,
H-7), 7.75 (dd, 1H, J=1.1 Hz, 7.5 Hz, H-8). 13C NMR (75 MHz, CDCl3,):
δ 14.33, 17.78, 18.95, 23.14, 30.17, 44.17, 56.36, 57.18, 68.15, 115.89,
117.62, 119.15, 123.96, 132.43, 147.32, 167.76, 170.14, 172.08, 180.69,
183.70. ESIMS m/z (rel.int.): 388 [Mþ] (C20H24N2O6) (7), 330 [Mþ-
NHCOCH3] (23), 301 (8), 288 (23), 260 (9), 217 (13), 202 (58), 169 (100),
97 (8).
RESULTS AND DISCUSSION
Figure 1 outlines the general procedure used to synthesize
plumbagin analogues, which were prepared in good overall yields
via a three-step procedure. Initially, plumbagin was converted to its
methyl ether 2 in 85% yield through a reaction with MeI in the
presence of Ag2O in dry dichloromethane. Michael addition of
5-O-methyl plumbagin with ethanolamine afforded the Michael
adduct 3. Thus, condensation of the Michael adducts with the
2-(8-Methoxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalene-
2-ylamino)-ethyl-4-acetamido Benzoate (4k). The title com-
pound was prepared by the reaction of compound 3 (0.050 g, 0.13 mmol)
with N-acetyl para-amino benzoic acid (0.037 g, 0.206 mmol). Yield: 58%.
N-acetyl-
dimide and 3-hydroxy benzotriazole yielded the targeted products.
L
-amino acids in the presence of N,N0-dicyclohexylcarbo-

Article
J. Agric. Food Chem., Vol. 57, No. 14, 2009 6093
Figure 1. Synthesis of plumbagin derivatives.
Table 1. Antifeedant Effect of Plumbagin and Their Derivatives 4a-k against
Achaea janata and Spodoptera litura by the Leaf Disc Method
Table 2. Toxicity Effects of Plumbagin and Their Derivatives 4a-4k against
Achaea janata and Spodoptera litura by a Topical Bioassay
LC50 (95% FLa) μg/cm2
mean (%) toxicity ( SEa
compound
4a
4b
A. janata
S. litura
A. janata
S. litura
69.51(35.02-92.45)
340.63(290.88-509.14)
>500
36.87(32.33-41.14)
>500
>500
4a
4b
100.0 ( 0.0a
þþ
þþ
þþ
4c
4d
4c
4d
þþ
100 ( 0.0
þþ
100.0 ( 0.0a
þþ
37.37(34.14-40.47)
292.00(256.89-392.71)
>500
48.47(16.77-77.36)
>500
4e
4e
þþ
4f
4g
466.28(335.42-2260.61)
105.93(94.21-119.48)
>500
4f
4g
þþ
74.2 ( 0.4
þþ
51.96(43.32-58.03)
104.59(65.71-134.12)
199.45(179.52-229.41)
>500
23.4 ( 0.4
32.6 ( 0.3
24.6 ( 0.3
þþ
4h
4i
4h
4i
84.5 ( 0.2
þþ
461(298.04-4105.42)
247.59(210.62-348.87)
117.25 (79.03-170.46)
335.52(246.41-701.22)
18.89 (16.77- 29.08)
4j
4j
þþ
4k
268.88(238.17-327.93)
252.52(222.81-959.92)
21.30(17.70-24.80)
4k
þþ
63.8 ( 0.2
þþ
plumbagin
azadirachtin
plumbagin
azadirachtin
þþ
93.6 ( 1.2a
84.0 ( 0.5
a Fiducial limits.
a Percentage mean toxicity ( SE values are for doses tested at 4 μg/ insect for
compounds 4a-4k and plumbagin 0.1 μg/insect for azadirachtin. þþ: moderately
active (less than 20%).
With the 11 derivatives of plumbagin in hand, we next
examined their antifeedant activities by using the conventional
no-choice disk method. The results are summarized in Table 1.
It is interesting to note that compared to the parent compound
plumbagin, most of the derivatives synthesized displayed higher
antifeedancy. Among the test compounds, plumbagin with gly-
cine (4a), methionine (4d), valine (4h), and with the phenylalanine
side chain (4g) displayed good antifeedancy against A. janata and
S. litura. However, the introduction of the methionine (4d) side
chain into plumbagin significantly enhanced activity against
S. litura larvae. It is important to note that the derivatives 4c,
4f, and 4j were totally inactive against S. litura larvae (Table 1).
In the topical bioassay, (4a) and (4d) were extremely toxic and
produced 100% lethal activity in A. janata. In an analogous
study, these derivatives did not show mortality against S. litura,
but the treated larvae experienced prolonged molting and adult
deformities with undeveloped wings swollen, abdomen, shor-
tened and partial development of antennae (Table 2). Similar
results were observed with 4i and 4f. The mortality rate in
plumbagin derivatives with cysteine (4c), valine (4h), and the
para-amino benzoic acid side chain (4k) against S. litura was high
in comparison to that of A. janata. Plumbagin derivative 4k
caused abnormalities in the emerging adults by affecting their
wing formation. Almost all of the abnormal adults with crumpled
wings died shortly without mating and egg laying. The phenyl-
alanine ester (4g) showed moderate toxic effects against A. janata,
and the molting process of the survived larvae was prevented or
was not carried out to completion. The defects intensified with the
sequence of molts into the later instars. Insects went through
normal apolysis but failed to complete ecdysis, resulting in larval
pupal intermediates with exuvia adhering to the head capsule.
The retention of the cuticular skin disrupts the excretory and
locomotory functions and leads to death. It is possible that the
compound acts as a chitin synthesis inhibitor, which needs
verification. This indicates that the treatment of this derivative
resulted in irreversible damage to physiological processes essen-
tial to the development of A. janata. Overall, these results imply
that the mode of perception as well as the structure-activity
relationship of the plumbagin analogues differs considerably
between the insect species examined in this study. However, some
of these compounds were highly promising as toxicants as well as
antifeedants against the two prominent agricultural pests, and

6094 J. Agric. Food Chem., Vol. 57, No. 14, 2009
Sreelatha et al.
future evaluations of the susceptibility of other pest species is
likely to yield better pest management compounds.
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It is intriguing that, though the test insects belong to the same
order, i.e, Lepidoptera, the biological activity of the derivatives
differed. The reason perhaps is the genetic variability of each
genera. Both insects belong to different genera as well as different
feeding habitats. S. litura is a polyphagous pest that can feed on
any plant, and A. janata being monophagous has restricted
feeding on a limited species of plants.
It is interesting tonote that plumbagin was neither effective as a
toxicant nor produced any growth inhibition in both test insects
at the dosage employed. It is possible that the activity of the
compound differed with the type of insect or that the dosage
employed was too low to obtain visible effects. However, a dosage
of 0.1% of plumbagin employed in the present investiga-
tion produced a moderate percentage of antifeedant activity
against the third instar larvae of S. litura (26%) and A. janata
(35.69%).
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results observed, glycine could be identified as the best possible
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basic amino acids will throw more light on the efficacy of these
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Received March 31, 2009. Revised manuscript received May 28, 2009.
Accepted May 28, 2009.