Molluscicidal activity of synthetic lapachol amino and hydrogenated derivatives

Article abstract of DOI:10.1016/j.bmc.2004.09.043

A series of new amino derivatives and a new partially hydrogenated derivative of the natural naphthoquinone lapachol were found to exhibit molluscicidal activity against Biomphalaria glabrata. A series of new amino derivatives and a new partially hydrogenated derivative of the natural naphthoquinone lapachol were assayed for molluscicidal activity against Biomphalaria glabrata. These derivatives showed low to medium LC₅₀ values, and a 3.1 μg/mL value for the most potent derivative of the series. The toxicity is in agreement with the decrease of polar character of the tested compounds.

Full text of DOI:10.1016/j.bmc.2004.09.043

Bioorganic & Medicinal Chemistry 13 (2005) 193–196
Molluscicidal activity of synthetic lapachol amino and
hydrogenated derivatives
a
a
b
Tania M. S. Silva,a, Celso A. Camara, Ticiano P. Barbosa, Andre Z. Soares,
*
´
Luciana C. da Cunha,b Angelo C. Pintob and Maria D. Vargasc
a
´
ˆ
´
Laboratorio de Tecnologia Farmaceutica, LTF-UFPB, Campus I, Cidade Universitaria, 58051-970 Joa˜o Pessoa, PB, CP 5009, Brazil
b
´ ´
Instituto de Quımica-CT, Bloco A, Universidade Federal do Rio de Janeiro, Cidade Universitaria, 21945-970 Rio de Janeiro,
RJ, Brazil
Instituto de Quımica, Universidade Federal Fluminense, Campus do Valonguinho, Centro, 24020-150 Niteroi, RJ, Brazil
c
´
´
Received 30 August 2004; revised 23 September 2004; accepted 23 September 2004
Available online 13 October 2004
Abstract—A series of new amino derivatives and a new partially hydrogenated derivative of the natural naphthoquinone lapachol
were assayed for molluscicidal activity against Biomphalaria glabrata. These derivatives showed low to medium LC50 values, and a
3.1lg/mL value for the most potent derivative of the series. The toxicity is in agreement with the decrease of polar character of the
tested compounds.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
sp.6 Amino derivatives 2a–h,7 1-aza-anthraquinones
3a–c and a partially hydrogenated derivative of lapachol,
Among human parasitic diseases, schistosomiasis
(sometimes called bilharziasis) ranks second behind ma-
laria in terms of socio-economic and public health
importance in tropical and subtropical areas.1 The dis-
ease is endemic in 74 developing countries, infecting
over 200 million people in rural agricultural and
peri-urban areas. Of these, 20 million suffer severe con-
sequences from the disease and 120 million are sympto-
matic. In many areas, schistosomiasis infects a large
proportion of under-14 children. An estimated 500–
600 million people worldwide are at risk from the dis-
ease.2 In Brazil, the worm Schistosoma mansoni is the
ethyological agent, and it requires the aquatic snail
Biomphalaria glabrata as the major intermediate host
for transmission.3 Molluscicides are of great interest
for the potential focal control of schistosomiasis in ende-
mic countries, and among these compounds of natural
origin, toxic plant saponins have been investigated.4,5
We report herein remarkable molluscicidal activity of
some derivatives of lapachol 1 a natural naphthoqui-
none extracted in 2–5% yield from the bark of Tabebuia
O
OH
1
O
O
R
NHR
a= H
b= CH2CO2H
c= CH2CH2OH
d= CH2CH(OCH3)2
e= CH2CH=CH2
f= CH2Ph
g= CH2CH2CH2CH3
h= CH2CH2Ph
2a-h
O
O
R1
N
R1
3a-c
a= CH2CH(OCH3)2
O
O
b= CH2Ph
c= Ph
OH
O
Keywords: Lapachol; Molluscicide; Naphthoquinones.
*
4
Corresponding author. Tel.: +55 83 2167361; fax: +55 83 2167364;
e-mail: sarmento@ltf.ufpb.br
Figure 1. Derivatives of lapachol investigated.
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.09.043

194
T. M. S. Silva et al. / Bioorg. Med. Chem. 13 (2005) 193–196
Table 1. Molluscicidal activity (lmol/mL) of the lapachol amino
derivatives on B. glabrata (9–16mm, 10 snails per concentration) under
laboratory conditions and 24h exposure
benzoquinone 4 were investigated (Fig. 1). Heterocyclic
quinones and quinones containing nitrogen atoms are
known to exhibit excellent antitumor8 and other biolog-
ical activities.9
Compound
LC10
(lmol/mL)
LC50
(lmol/mL)
LC
90
(lmol/mL)
2a
2b
2c
2d
2e
2f
26.5
86.9
49.3
206.6
72.6
72.1
330.1
102.8
75.9
2. Results and discussion
42.4
25.8
54.9
23.8
Inactivea
13.8
45.2
2.1. Synthesis of the compounds
6.0
44.8
Inactivea
4.0
33.6
Inactivea
23.5
57.6
The amino derivatives 2c–f and 1-aza-anthraquinones
3a–c were synthesized as described previously.7 The
new compounds 2a, 2b, 2g, and 2h were obtained by
nucleophilic displacement of 2-methoxylapachol with
30% ammonium hydroxide solution (45% yield), glycine
in KOH/MeOH10a (80% yield), n-butylamine in metha-
nol (85%) and phenethylamine in methanol (71% yield),
respectively (Scheme 1).
2g
2h
3a
3b
3c
4
32.4
53.8
Inactivea
3.2
66.9
89.0
Inactivea
7.6
101.5
13.6
Inactivea
12.5
a The inactivity corresponds to a value >100lg/mL.
Compound 4 was obtained in high yield (>98%) from the
catalytic hydrogenation of lapachol (5bar) with 10% pal-
ladium on charcoal in acetic acid. Selective hydrogenat-
ing of the prenyl side chain of lapachol was monitored
by 1H NMR spectroscopy. Above 2atm H2 signals
attributed to the hydrogenated quinone started to ap-
pear. The only other related hydrogenation reaction re-
ported to date involves pyrano-quinones derived from
lapachol, not the parent compound itself (Scheme 2).11
LC90 value of 6.18lg/mL (25.5lmol/mL) has been re-
ported.4b In the amino derivatives of lapachol series,
2a–h, the most active compounds were 2e and 2g, whose
activities are superior to that of molluscicide muzigadial
(LC90 = 20–40lmol/mL).12 Compounds 2a (LC50
49.3lmol/mL), 2c (LC50 = 72.6lmol/mL), 2d (LC50
=
=
54.9lmol/mL) and 2h (LC50 = 45.2lmol/mL) presented
activities similar or even superior to that of mukaadial
(LC50 = 80.5lmol/mL).12 The least active compound
of this series was the glycine derivative 2b (LC50
=
2.2. Molluscicidal assays
206.6lmol/mL), and compound 2f was inactive. Among
the 1-aza-anthraquinone derivatives 3a–c, compounds
3a with LC50 = 66.9 and 3b, LC50 = 102.8lmol/mL were
less active than the corresponding amino derivatives of
lapachol, and 3c was inactive (Table 1).
Table 1 summarizes the results of assays performed
against adult snails of B. glabrata, with the LC10,
LC50, and LC90 calculated values. In the bioassays com-
pound 4 presented significant molluscicidal activity
against the adult form of B. glabrata, with
LC90 = 7.6lmol/mL and LC50 = 12.5lmol/mL. This
activity is similar to that presented by molluscicide war-
burganal, with a reported LC50 = 2ppm (8.0lmol/
mL).12 Compound 4, with LC90 = 12.5lmol/mL, is
two times more potent than lapachol 1 for which a
These results indicate a correlation between the hydro-
phobic character of the aminoalkyl side-chain moiety of
the compounds of series 2 and molluscicidal activity,
compounds with the most polar groups generally exhibit-
ing the lowest activities (2b and 2f, Table 1).13 Exceptions
to this trend are compounds 2f, that was inactive and 2c
with a hydroxyl group on the aminoalkyl side chain,
which showed reasonable activity. These compounds
are less active than lapachol 1, and this might be associ-
ated to the more efficient conjugation of the vinylogous
amide present in series 2 and 3 nitrogen compounds,
when compared with the hydroxyl-substituted ones.14
Series 3 of cyclic compounds showed a general loss of
activity when compared with the parent seco-amino com-
pounds of series 2 (except for compound 3b, that is more
potent than compound 2f). In contrast, the partially
hydrogenated lapachol derivative 4, no longer a naphtho-
quinone, but a 2,3,5,6-tetra-substituted benzoquinone, is
two times more potent than lapachol 1. The increase of
activity is probably due to the availability of the new Mi-
chael acceptor from a partially reduced aromatic moiety
arising from the reduction of lapachol 1.13
O
O
NHR
OMe
2a 45%
2b 80%
2g 85%
2h 71%
a, b or c
O
1a
O
Scheme 1. Reagents and conditions: (a) R = H, NH4OH, rt, 24h; (b)
R = CH2CO2H, 10% KOH/MeOH, rt, 12h; (c) R = n-Bu and
CH2CH2Ph, amine, MeOH, rt, 24h.
O
O
OH
OH
a
98%
3. Conclusions
O
O
4
1
The molluscicidal activity of the amino derivatives of
lapachol increased with the increasing lipophilic charac-
Scheme 2. Reagents and conditions: (a) H2, Pd/C 10%, 5.0bar, 2h, 65–
70ꢁC, AcOH.

T. M. S. Silva et al. / Bioorg. Med. Chem. 13 (2005) 193–196
195
ter of the amino alkyl side chain. The partially hydrogen-
ated lapachol derivative 4, obtained from the catalytic
reduction of lapachol 1, showed molluscicidal activity
significantly higher than 1. These findings confirm the
importance of lapachol as an important starting material
for the production of biologically active compounds.
(s, 3H), 3.20 (d, 2H, 6.2Hz), 4.22 (d, 2H, 6.3Hz), 5.01
(m, 1H), 6.76 (t, 1H, 6.3Hz), 7.66 (dt, 1H, 7.0/7.0/
1.6Hz), 7.76 (dt, 1H, 7.0/7.0/1.6Hz), 7.87 (dd, 1H, 7.0/
1.6Hz), 7.91 (dd, 1H, 7.0/1.6Hz); 13C NMR (DMSO-
d6): d 17.96, 22.52, 25.48, 46.06, 115.76, 122.15, 125.38,
125.64, 130.38, 131.79, 132.33, 132.35, 134.49, 146.09,
171.82, 181.31, 182.70. HRMS found: 299.1150. Calcd
for C17H17NO4: 299.1157.
4. Experimental
4.3. Synthesis of 2-butylamino-3-(3-methyl-but-2-enyl)-
[1,4]naphthoquinone (2g)
Melting points are uncorrected and were determined on
an electrically heated metal block apparatus. Column
chromatography was performed on silica-gel G60 (70–
230 mesh, ASTM, Merck), and thin-layer chromatogra-
phy was performed on 0.2mm plates (Merck), visualized
with short wavelength UV light. 1H and 13C NMR spec-
tra wee recorded on a Varian Mercury-200MHz spec-
trometer. Values reported for coupling constants are
first order. High-resolution mass spectra were obtained
by electron impact on a VG Autospec spectrometer. 2-
Methoxy-lapachol 1a was obtained as described
previously.7a
To a methanol solution of 2-methoxylapachol (0.254g,
1mmol) was added 0.146g (2mmol) of n-butylamine,
and the resulting mixture was kept under stirring at
room temperature for 24h. The solvent was removed
by reduced pressure and the resulting deep-brown oil
chromatographed in a silica-gel column with hexane–
CH2Cl2 98:2 to furnish 2g as a red oil (253mg, 85%).
IR (KBr): mmax 3298, 2976, 2914, 2854, 1751, 1682,
1
1590, 1522, 1391, 1364, 1180, and 710cmÀ1; H NMR
(200MHz, CDCl3) d 0.91 (t, 3H, 7.4Hz), 1.39 (m, 2H,
7.0/7.4Hz), 1.58 (m, 2H), 1.67 (d, 3H, 1.2Hz), 1.71 (d,
3H, 1.4Hz), 3.36 (d, 2H, 6.0Hz), 3.49 (m, 2H), 5.08
(dd, 2H, 7.4/1.2Hz), 5.65 (NH, br s), 7.52 (dt, 1H, 7.4/
7.4/1.4Hz), 7.63 (dt, 1H, 7.4/7.4/1.4Hz), 7.94 (dd, 1H,
7.4/1.4Hz) and 8.04 (dd, 1H, 7.4/1.4Hz); 13C NMR
(CDCl3): d 13.66, 18.06, 19.84, 22.94, 25.60, 32.80,
44.73, 114.95, 119.97, 123.31, 131.64, 133.12, 133.40,
133.60, 133.67, 134.27, 145.65, 182.69, 182.91. HRMS
found: 297.1722. Calcd for C19H23NO2: 297.1728.
4.1. Synthesis of 2-amino-3-(3-methyl-but-2-enyl)-
[1,4]naphthoquinone (2a)
A solution of 2-methoxy-lapachol (0.256g, 1mmol) in
10mL of methanol PA was mixed with ammonium
hydroxide 30% solution (10mL), and the solution kept
at room temperature for 24h. The solvent was then
removed by reduced pressure, and the aqueous solu-
tion extracted with dichloromethane (3 · 20mL), the
combined organic extracts were dried with anhydrous
sodium sulfate, and the resulting red-brownish oil chro-
matographed in silica-gel with hexane–dichloromethane
95:5 affording the desired (2a) as red crystals (mp
130–132ꢁC, from hexane–CH2Cl2), in 45% yield. IR
(KBr): mmax 3426, 3315, 3289, 3241, 3064, 2916, 1676,
1625, 1594, 1552, 1437, 1391, 1368, 1277, and
4.4. Synthesis of 2-(3-methyl-but-2-enyl)-3-phenethyl-
amino-[1,4]naphthoquinone (2h)
To a methanol solution of 2-methoxylapachol (0.254g,
1mmol) was added 0.242g (2mmol) of phenethylamine,
and the resulting mixture was kept under stirring at room
temperature for 24h. The solvent was removed by re-
duced pressure and the resulting deep-brown oil chroma-
tographed in a silica-gel column with hexane–CH2Cl2
98:2 to furnish 245mg of red needles (71%), with a mp
1
726cmÀ1; H NMR (200MHz, CDCl3): d 1.71 (s, 3H),
1.80 (s, 3H), 3.27 (d, 2H, J = 7.8Hz), 5.07 (m, 1H),
5.16 (br, NH), 7.67 (dt, 1H, 7.0/7.0/1.4Hz), 7.60 (dt,
1H, 7.0/7.0/1.4Hz), 8.02 (dd, 1H, 7.0/1.4Hz) and 8.10
(dd, 1H, 7.0/1.4Hz); 13C NMR (50MHZ, CDCl3) d
18.1, 23.1, 25.8, 116.4, 120.1, 125.9, 126.5, 126.9,
130.6, 132.1, 133.9, 134.4, 145.5, 181.8, 182.5. HRMS
found: 241.1012. Calcd for C15H15NO2: 241.1102.
80–81ꢁC. IR (KBr): mmax 3290, 2958, 2915, 2834, 1740,
1
1684, 1582, 1533, 1389, 1359, 1180, and 715cmÀ1
;
H
NMR (CDCl3): d 1.67 (s, 3H), 1.67 (s, 3H), 2.91 (t, 2H,
7.2Hz), 3.28 (d, 2H, 7.4Hz), 3.80 (t, 2H, 7.2Hz), 5.11
(ddt, 1H, 7.4/1.4), 5.70 (1H, br s), 7.22 (m, 1H), 7.52
(m, 1H), 7.64 (m, 1H), 7.65 (m, 1H), 7.67 (m, 1H), 8.05
(m, 2H); 13C NMR (CDCl3): d 22.82, 25.48, 25.63,
36.86, 46.00, 115.37, 119.90, 122.87, 125.98, 126.62,
128.58, 131.58, 132.15, 132.86, 133.42, 133.53, 134.11,
137.39, 145.32, 182.60, 182.73. HRMS found: 345.1728.
Calcd for C23H23NO2: 345.1729.
4.2. Synthesis of [3-(3-methyl-but-2-enyl)-1,4-dioxo-1,4-
dihydro-naphthalen-2-ylamino]-acetic acid (2b)
To a solution of 2-methoxy-lapachol 1a (0.256g,
1mmol) and glycine (0.075g, 1mmol) in 20mL of meth-
anol PA was added dropwise a 6.2mL of a KOH 10%
aqueous solution with continuous stirring, and the
resulting solution was stirred for 12h. After TLC inspec-
tion, hydrochloric acid 10% aqueous solution was added
until complete precipitation. The material was collected
4.5. Biological assays
The bioassay was carried out as described by dos Santos
et al.4b by dissolving the sample first in dimethyl sulfox-
ide (DMSO) and then adding dechlorinated water, to
give a solution 0.1% in DMSO.
on a Buchner, washed with distilled water, and recrystal-
¨
lized from methanol, yielding red crystals, with mp
174–175ꢁC. IR (KBr): mmax 3283, 2983, 2921, 2864,
1742, 1677, 1592, 1542, 1394, 1365, 1183, and
Ten adult snails (9–16mm in diameter) were placed
in a beaker, containing 250mL of the molluscicide
1
720cmÀ1; H NMR (DMSO-d6): d 1.62 (s, 3H), 1.70

196
T. M. S. Silva et al. / Bioorg. Med. Chem. 13 (2005) 193–196
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suspension at four appropriate concentrations. Each test
concentration was set in duplicate. Snails were exposed
to the potential molluscicide for 24h at room tempera-
ture and were kept under normal diurnal lighting. After
24h, the suspension was decanted; the snails were
washed with water and offered lettuce leaves as food.
The tested snails were then left in water for another
24h, and at the end of this period were examined to
assess mortality. Snails were considered dead if they
either remained motionless or did not respond to the
presence of food, or if the shell looked discolored. In
order to verify the snailÕs susceptibility, two control sets
were used: one with cupric carbonate at 50ppm and the
other containing 0.1% DMSO dechlorinated water. The
collected data were computerized, and the LC10, LC50,
and LC90 values determined by performing a probit
analysis.4b
Acknowledgements
The authors thank CNPq, CAPES, FAPERJ, IM-
SEAR-CNPq, and PIBIC-UFPb for scholarships and
financial support and A. J. de Almeida and J. T. F. do
Nascimento (Chemistry Institute-UNICAMP) for the
HRM spectra.
11. Ferreira, V. F.; Pinto, A. V.; Pinto, M. C. F. R.; Silva, M.
M. An. Acad. Bras. Ci. 1987, 59, 329.
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