Acaricidal activity of tonka bean extracts. Synthesis and structure-activity relationships of bioactive derivatives

Article abstract of DOI:10.1021/np020563j

The acaricidal effects of tonka bean, Dipterix odorata, extracts were investigated on Dermatophagoides pteronyssinus, the European house dust mite, and compared with benzyl benzoate as a standard acaricidal compound. A cyclohexane extract was the most effective, with an EC₅₀ = 0.075 g/m² after a 24 h period, as compared with benzyl benzoate (0.025 g/m²). Bioassay-guided fractionation of this extract led to the isolation of coumarin (1). Pharmacomodulation of this compound led us to test 20 analogues (2-21), which were either synthesized or purchased.

Full text of DOI:10.1021/np020563j

690
J . Nat. Prod. 2003, 66, 690-692
Aca r icid a l Activity of Ton k a Bea n Extr a cts. Syn th esis a n d Str u ctu r e-Activity
Rela tion sh ip s of Bioa ctive Der iva tives
Christophe Gleye,† Guy Lewin,† Alain Laurens,*,† J ean-Christophe J ullian,† Philippe Loiseau,‡
Christian Bories,‡ and Reynald Hocquemiller†
Laboratoire de Pharmacognosie, UMR 8076 CNRS (BioCIS), Faculte´ de Pharmacie, Universite´ Paris XI,
92296 Chaˆtenay-Malabry Cedex, France, and Laboratoire de Chimiothe´rapie Antiparasitaire, UMR 8076 CNRS (BioCIS),
Faculte´ de Pharmacie, Universite´ Paris XI, 92296 Chaˆtenay-Malabry Cedex, France
Received December 9, 2002
The acaricidal effects of tonka bean, Dipterix odorata, extracts were investigated on Dermatophagoides
pteronyssinus, the European house dust mite, and compared with benzyl benzoate as a standard acaricidal
compound. A cyclohexane extract was the most effective, with an EC50 ) 0.075 g/m2 after a 24 h period,
as compared with benzyl benzoate (0.025 g/m2). Bioassay-guided fractionation of this extract led to the
isolation of coumarin (1). Pharmacomodulation of this compound led us to test 20 analogues (2-21),
which were either synthesized or purchased.
House dust mite allergy has been widely implicated in
the etiology of nonseasonal asthma and atopic disorders,
and this allergy is now considered to be a worldwide
problem.1 The control of insect pests by plants or their bio-
active constituents is being widely investigated. However,
only a few studies deal with acaricidal natural products.
Tonka beans are the dried seeds of Dipteryx odorata
(Aublet) Wild. (Fabaceae). Cultivated in Venezuela, this
tree is used for its fruits to flavor tobacco or in perfumery.
In the course of our systematic search for natural acaricidal
compounds,2,3 the aim of this study was to evaluate the
activity of this species by a simple bioassay on the common
pyroglyphid house dust mite (Dermatophagoides pteron-
yssinus). A cyclohexane extract of tonka beans exhibited a
positive response to the bioassay, and this extract was thus
selected for isolation of active compounds.
The cyclohexane extract was taken to dryness under
reduced pressure to yield a brown gum. This extract
showed a dose-dependent acaricidal effect. The crude
extract was fractionated by column chromatography on
silica gel. Bioassay-guided fractionation by flash chroma-
tography afforded coumarin (1)4 as the active component.
The acaricidal properties of coumarin, a known compound
of tonka beans, were hitherto unknown.5,6 After 24 h, the
EC50 for coumarin (0.032 g/m2) was very similar to that of
benzyl benzoate (0.025 g/m2), a well-known and widely used
acaricidal product.
The coumarin skeleton and benzyl benzoate were then
considered as lead structures in the present study. To
improve the potency and acaricidal profile of these leads,
different modifications were considered. The acaricidal
activities of the synthetic coumarin derivatives are dis-
cussed below.
The derivatives can be classified into three classes: (1)
compounds with a benzo R-pyrone ring (2-8); (2) com-
pounds with a γ-pyrone ring (9-16); (3) compounds deri-
vated from benzyl benzoate (17-21). These compounds
were either obtained from commercial sources or prepared
by synthesis.
Coumarin-3-carboxylic acid benzyl ester (7), benzyl cou-
malate (8), chromone-2-carboxylic acid benzyl ester (11),
benzyl salicylate (17), 3-hydroxy benzoic acid benzyl ester
(18), 4-hydroxybenzoic acid benzyl ester (19), and benzyl
* To whom correspondence should be addressed. Tel: 331 46835639.
Fax: 331 46835399. E-mail: alain.laurens@cep.u-psud.fr.
† Laboratoire de Pharmacognosie.
‡ Laboratoire de Chimiothe´rapie Antiparasitaire.
10.1021/np020563j CCC: $25.00
© 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/06/2003

Notes
J ournal of Natural Products, 2003, Vol. 66, No. 5 691
Ta ble 1. In Vitro Acaricidal Activities of Compounds 1-21
against D. pteronyssinus (EC50 values after 24 h)
These results could be explained by the occurrence in 17
of a hydrogen bond between the OH and the carbonyl of
the ester group. This hypothesis is supported by the activity
of compound 20, in which a carbonyl substituent adjacent
to the OH allowed a hydrogen bond.
In conclusion, coumarin (1) was isolated by bioassay-
guided fractionation of tonka beans. In terms of acaricidal
activity, this compound with an R-pyrone ring compared
favorably with the known acaricidal benzyl benzoate. On
the basis of these data, γ-pyrones may be worth considering
as potential acaricidal compounds. Structural modifications
on the R-pyrone and γ-pyrone rings showed that the
benzyloxycarbonyl substitution is critical for acaricidal
activity, and substitution by polar hydroxy groups causes
reduction of activity.
tested compound
EC50 (g/m2)
coumarin (1)
6-methylcoumarin (2)
4-hydroxycoumarin (3)
7-hydroxycoumarin (4)
coumarin-6-carboxylic acid benzyl ester (5)
coumarin-5-carboxylic acid benzyl ester (6)
coumarin-3-carboxylic acid benzyl ester (7)
benzyl coumalate (8)
chromone (9)
flavone (10)
chromone-2-carboxylic acid benzyl ester (11)
chromanone (12)
flavanone (13)
dihydrochalcone (14)
flavane (15)
0.032
0.040
>1.50
>1.50
0.495
0.495
0.280
0.495
0.037
0.500
0.495
0.070
0.090
0.110
0.150
0.310
0.025
0.495
0.495
0.242
0.180
0.025
Exp er im en ta l Section
chalcane (16)
benzyl salicylate (17)
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined with a Reichert apparatus and are uncorrected.
1H NMR spectra were obtained with a Bruker AC-200 (at 200
MHz) NMR spectrometer. TLC was performed on precoated
0.25 mm thick Merck plates of silica gel 60 F254. Column
chromatography was carried out on Merck silica gel 60.
3-hydroxybenzoic acid benzyl ester (18)
4-hydroxy benzoic acid benzyl ester (19)
3-formyl-4-hydroxy benzoic acid benzyl ester (20)
benzyltrans cinnamate (21)
benzyl benzoate (reference)
P la n t Ma ter ia l. Tonka beans, D. odorata, were purchased
from Herboristerie Cailleau, 5 Rue Robert d’Arbrissel, BP 69,
49210 Chemille, France, Lot No. 11013.
trans-cinnamate (21) were prepared by esterification of the
commercial carboxylic acids by benzyl alcohol. 3-Formyl-
4-hydroxybenzoic acid benzyl ester (20) was prepared in
two steps from 4-hydroxybenzoic acid: (a) formylation
according to Duff’s process,7 (b) esterification by benzyl
alcohol. Cyclization of 20 into coumarin-6-carboxylic acid
benzyl ester (5) was carried out by a Wittig reaction.8
Coumarin-5-carboxylic acid benzyl ester (6) was prepared
in the same manner from 3-hydroxybenzoic acid. Flavane
(15) and chalcane (16) were obtained by reduction of
flavanone by NaBH3CN/TFA.9 Dihydrochalcone (14) was
prepared by catalytic hydrogenation of flavanone (13). The
acaricidal activities of these coumarin and benzyl benzoate
derivatives are shown in Table 1.
A methyl group at the C-6 position of 1 did not affect
the acaricidal activity (compare 2 with 1), whereas a
hydroxy group at C-4 (3) or C-7 (4) led to a dramatic drop
in activity. A benzyloxycarbonyl group at C-6 (5) or C-5
(6) had 10-fold lower activity, while the same group at C-3
(7) was 2-fold more active than these compounds. The
combination of a coumarin skeleton substituted by a
benzyloxycarbonyl group in these positions lowered the
activity against the pyroglyphid house dust mite. Direct
attachment of this substituent to the R-pyrone ring, as in
benzyl coumalate (8), led to similar activity.
The acaricidal activity of compounds with a γ-pyrone ring
was compared to compounds with an R-pyrone ring. In this
group, chromone (9) showed the strongest activity, quite
similar to coumarin (1). Reduction of the double bond at
C-2/C-3 led to chromanone (12), which was slightly less
active than chromone (9). In this group, it was interesting
to note that flavanone (13), dihydrochalcone (14), flavane
(15), and chalcane (16) preserved this activity, while
flavone (10) was clearly less active.
As for coumarin (1), addition of a benzyloxycarbonyl
group to position 2 of chromone (9) led to a negative effect
on the bioactivity (compared to 9-11). On the other hand,
concerning the benzyl benzoate series, replacement of the
benzoyl by a trans-cinnamoyl group led to a compound (21)
that was 7-fold less active. Finally, substitution of benzyl
benzoate at C-3 or C-4 with a polar hydroxy group led to
greatly reduced activity (compare benzyl benzoate with 18
and 19). However, activity was not reduced when the OH
substituent was at C-2 (compare benzyl benzoate with 17).
E xt r a ct ion a n d Isola t ion of Cou m a r in (1). Powdered
plant material (1 kg) was extracted by C6H12 (cyclohexane) in
a Soxhlet for 24 h. The C6H12 extract was taken to dryness
under reduced pressure to yield a brown gum. When tested
for acaricidal activity, the C6H12-insoluble extract exhibited
activity, with an IC50 value of 0.075 g/m2. The C6H12-insoluble
bioactive extract was subjected to column chromatography
(CC) on silica gel, eluting with C6H12-i-PrOH, 95:5, to give
coumarin (1), identical in all respects (NMR, MS) to a com-
mercial product (Aldrich, France, C8, 557-7).
Ma ter ia ls. Compounds 2, 3, 4, 9, 10, 12, and 13 were
commercially available. Compounds 15 and 16 were prepared
by reduction of 13 using NaBH3CN/TFA.9 Compound 14 was
prepared by catalytic hydrogenation (H2/Pd) of 13. Melting
points and spectroscopic data for compounds 2-4, 9, 10, and
12-16 agreed with those previously reported.9
Gen er a l P r oced u r es for th e Syn th esis of Ben zyl Ester
Der iva tives 7, 8, 11, 17, 18, 19, a n d 21. A mixture of 200
mg of carboxylic acid, 3 mL of benzyl alcohol, and 25 mg of
APTS was refluxed for 15 h. The reaction mixture was
extracted with 200 mL of CH2Cl2, the CH2Cl2 layer was dried
over Na2SO4 and filtered, and the solvent was removed under
reduced pressure. The crude product was purified by column
chromatography (CH2Cl2) over silica gel. Spectroscopic data
agreed with those of commercial products (17, 19, 21) or with
those previously reported in the literature (7,10 8,11 1812).
Ch r om on e-2-ca r boxylic a cid ben zyl ester (11): orange
solid; mp 104-105 °C (from CH2Cl2); 1H NMR (CDCl3) δ 8.20
(1H, dd, J ) 12, 3 Hz, H-5), δ 7.40 (9H, m, H-3, H-6, H-7, H-8,
H-Bn), δ 5.40 (2H, s, CH2-Bn); anal. C 72.93%, H 4.28%,
calcd for C17H12O4, C 72.85%, H 4.32%.
P r oced u r es for th e Syn th esis of 20, 5, a n d 6. A mixture
of 2.76 g (20 mmol) of 4-hydroxybenzoic acid and 2.8 g of
hexamethylenetetramine (20 mmol) was heated at reflux in
50 mL of trifluoroacetic acid for 6 h. The products were
concentrated and combined with ice water; the resultant
mixture was stirred for 15 min and extracted with EtOAc. The
solvent was washed several times with H2O, dried over Na2-
SO4, then removed under reduced pressure. The crude residue
(400 mg) was added to a stirred solution of APTS (60 mg) in
benzyl alcohol (10 mL) and heated at reflux for 6 h. The
resulting solution was extracted with CH2Cl2, dried over Na2-
SO4, and filtered, and solvent was removed under reduced
pressure. The crude product was then purified with 20%
cyclohexane in CH2Cl2 over silica gel to give 20 (510 mg, 10%).
Reaction of 20 with carbethoxymethylenetriphenylphosphorane

692 J ournal of Natural Products, 2003, Vol. 66, No. 5
Notes
(210 mg) in Et2NPh (30 mL) under reflux was carried out for
90 min. The reaction mixture was diluted with 5% HCl solution
and extracted with Et2O. The residue in CH2Cl2 was chro-
matographed on silica gel. Elution with CH2Cl2 gave 5.
Compound 6 was identically synthesized from 3-hydroxyben-
zoic acid.
3-F or m yl-4-h yd r oxyben zoic a cid ben zyl ester (20):
white waxy solid; 1H NMR (CDCl3) δ 11.40 (1H, s, COH), δ
9.95 (1H, s, OH), δ 8.35 (1H, d, J ) 3 Hz, H-2), δ 8.25 (1H, dd,
J ) 10, 3 Hz, H-6), δ 7.40 (5H, m, H-Bn), δ 7.00 (1H, d, J )
10 Hz, H-5), δ 5.40 (2H, s, CH2-Bn); anal. C 70.22%, H 4.65%,
calcd for C15H12O4, C 70.30%, H 4.72%.
Cou m a r in -6-ca r boxylic a cid ben zyl ester (5): yellow
solid; mp 135-136 °C (from CH2Cl2); 1H NMR (CDCl3) δ 8.25
(2H, m, H-5, H-7), δ 7.70 (1H, d, J ) 12 Hz, H-4), δ 7.40 (6H,
m, H-8, H-Bn), δ 6.50 (1H, d, J ) 12 Hz, H-3), δ 5.40 (2H, s,
CH2-Bn); anal. C 72.78%, H 4.40%, calcd for C17H12O4, C
72.85%, H 4.32%.
Cou m a r in -5-ca r boxylic a cid ben zyl ester (6): yellow
solid; mp 80 °C (from CH2Cl2); 1H NMR (CDCl3) δ 8.90 (1H, d,
J ) 12 Hz, H-4), δ 8.00 (1H, dd, J ) 9, 3 Hz, H-6), δ 7.70 (7H,
m, H-7, H-8, H-Bn), δ 6.50 (1H, d, J ) 12 Hz, H-3); δ 5.40
(2H, s, CH2-Bn); anal. C 72.88%, H 4.26%, calcd for C17H12O4,
C 72.85%, H 4.32%.
Hou se Du st Mites.Dermatophagoides pteronyssinus (Troues-
sart) strain, the European house dust mite, was provided by
Stallerge`ne Laboratories (Antony, France). The strain was
maintained in a culture medium made of 90% Saccharomyces
cerevisiae powder (Organotechnie, La Courneuve, France)
enriched with 10% beard shavings at 25 °C and 75% relative
humidity in darkness.
Biologica l Testin g. Experiments were performed in 24-
well tissue cultures (Costar No. 3524) using the Izri & Rousset
method13 for testing acaricidal compounds on a hard surface,
with minor modifications. The surface of each well was 2 cm2.
Adequate dilutions (30 µL) of the compounds or crude extracts
to be tested in absolute EtOH were applied on the bottom of
the wells and allowed to dry for 24 h before the experiment.
Concentrations of crude extracts and tested drugs were
expressed in g/m2. Control wells received only EtOH or crude
extract solvent. To prevent the mites from escaping, the upper
rims of the wells were covered with “Glue Pelton” (Rhoˆne-
Poulenc J ardin, Lyon, France). Treatments and blanks were
executed in triplicate and repeated after one week. Benzyl
benzoate, one of the most potent acaricidal compounds for
human and domestic use (Sigma-Aldrich, Saint Quentin
Fallavier, France), was used as the reference compound. One
hundred mites (3.5 mg of culture medium) were added in each
well, and the mortality was observed with a stereobinocular
microscope after a 24 h incubation period at 25 °C, 75% relative
humidity in darkness. Mites that did not move when prodded
with a fine brush were considered as dead. The observed
mortality in treated wells was corrected for the mortality
observed in control wells according to Abbott’s formula;
concentrations were determined by means of linear regression
analysis. For the determination of the activity of pure com-
pounds, seven 2-fold dilutions in triplicate were tested, ranging
from 0.01 to 0.640 g/m2. Student’s t-tests were used for
comparison between treated and untreated groups of house
dust mites. A P value e 0.05 was considered to represent a
significant difference.
Ack n ow led gm en t. This research was sponsored by the
“Direction de la Recherche et des EÄ tudes Doctorales (DRED)”,
through a biennal contract with the “Re´seau de Recherche
Pharmacochimie”. The authors are grateful to O. Ruth from
Lavandin de Grignan for his support.
Refer en ces a n d Notes
(1) Platts-Mills, T.; Vervloet, D.; Thomas, W.; Aalberse, R.; Chapman,
N. J . Allergy Clin. Fundam. 1997, 100, S1-S24.
(2) Akendengue, B.; Ngou-Milama, E.; Bourobou-Bourobou, H.; Essouma,
J .; Roblot, F.; Gleye, C.; Laurens, A.; Hocquemiller, R.; Loiseau, P.;
Bories, C. Phytother. Res., accepted.
(3) Raynaud, S.; Fourneau, C.; Laurens, A.; Hocquemiller, R.; Loiseau,
P.; Bories, C. Planta Med. 2000, 66, 173-175.
(4) Buckingham, J . Dictionary of Natural Products; Chapman & Hall:
London, 1992; Vol 1, p 631.
(5) Hoult, J . R. S.; Paya, M. Gen. Pharmacol. 1996, 27, 713-722.
(6) Estevez-Braun, A.; Gonzalez, A. G. Nat. Prod. Rep. 1997, 14, 465-
475.
(7) Smith, W. E. J . Org. Chem. 1972, 37, 3972-3973.
(8) Harayama, T.; Nakatsuka, K.; Nishioka, H.; Murakami, K.;
Hayashida, N.; Ishii, H. Chem. Pharm. Bull. 1994, 42, 2170-2173.
(9) Lewin, G.; Bert, M. Dauguet, J . C.; Dolley, J .; Le Menez, P.; Schaeffer,
C.; Guinamant, J . L.; Volland, J . P. Bull. Soc. Chim. Fr. 1991, 128,
939-944.
(10) Orita, M.; Yamamoto, S.; Katayama, N.; Aoki, M.; Takayama, K.;
Yamagiwa, Y.; Seki, N.; Suzuki, H.; Kurihara, H.; Sakashita, H.;
Takeuchi, M.; Fujita, S.; Yamada, T.; Tanaka, A. J . Med. Chem. 2001,
44, 540-547.
(11) J avaheripour, H.; Neckers, D. C. J . Org. Chem. 1977, 42, 1844-1850.
(12) Arnoldi, A.; Grasso, S.; Meinardi, G.; Merlini, L. Eur. J . Med. Chem.
1988, 23, 149-154.
(13) Izri, M.; Rousset, J . J . Rev. Fr. Allergol. 1995, 36, 507-509.
NP020563J