Toxic effects of natural piperine and its derivatives on epimastigotes and amastigotes of Trypanosoma cruzi

Article abstract of DOI:10.1016/j.bmcl.2004.04.019

We describe herein an evaluation of trypanocidal effects of the natural alkaloid piperine and twelve synthetic derivatives against epimastigote and amastigote forms of the protozoan parasite Trypanosoma cruzi, the causative agent of the incurable human disease, Chagas' disease. The results obtained point to piperine as a suitable template for the development of new drugs with trypanocidal activity.

Full text of DOI:10.1016/j.bmcl.2004.04.019

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3555–3558
Toxic effects of natural piperine and its derivatives on epimastigotes
and amastigotes of Trypanosoma cruzi
a
Tatiana Santana Ribeiro, Leonardo Freire-de-Lima, Jose Osvaldo Previato,
b
b
ꢀ
Lucia Mendonßca-Previato,b Norton Heiseb and Marco Edilson Freire de Limaa,*
a
ꢀ
Universidade Federal Rural do Rio de Janeiro, Instituto de Ci^encias Exatas, Departamento de Quımica, Km 7,
BR 465 CEP: 23.890-000, Seropꢀedica, RJ Brazil
bUniversidade Federal do Rio de Janeiro, Instituto de Biofꢀısica Carlos Chagas Filho, Centro de Ci^encias da Saꢀude. Ilha do Fund~ao,
21.944-970, Rio de Janeiro, RJ Brazil
Received 11 February 2004; revised 7 April 2004; accepted 10 April 2004
Abstract—We describe herein an evaluation of trypanocidal effects of the natural alkaloid piperine and twelve synthetic derivatives
against epimastigote and amastigote forms of the protozoan parasite Trypanosoma cruzi, the causative agent of the incurable human
disease, Chagas’ disease. The results obtained point to piperine as a suitable template for the development of new drugs with
trypanocidal activity.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
is widely used in folk medicine in India, where it origi-
nates.
The alkaloid piperine 1 (Fig. 1) is the main secondary
metabolite in Piper nigrum, occurring mainly in the
fruits.1 Piper nigrum (popularly known as black pepper)
Piperine 1 is very abundant in the plant, being extracted
from the dry fruits with a yield of 3–7%.2 Various
O
O
O
O
N
N
1
2
O
O
O
O
O
O
O
O
N
3
N
O
O
N
4
O
N
O
5
O
O
O
O
O
N
N
7
O
6
Figure 1. Chemical structures of some natural amides isolated from Piper sp.6;7
Keywords: Piperine; Piperine analogues; Piper nigrum; Chagas’ disease; Trypanosoma cruzi.
* Corresponding author. Tel./fax: +55-21-2682-2807; e-mail: marco@ufrrj.br
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.019

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T. S. Ribeiro et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3555–3558
biological activities have been attributed to piperine
including insecticidal3 and nematocidal activity,4 and
inhibition of liver metabolism.5 Other natural amides
have been isolated from Piper species. Some are shown
in Figure 1.6;7
antiparasitic activity of curcumin (isolated from
Curcuma longa) and its derivatives on Leishmania ama-
zonensis.14
We began our studies with the isolation of natural pip-
erine 1 from the powdered dry fruits of Piper nigrum
using the methodology described by Ikan,2 and obtained
the amide in a very pure form in 7% yield. The isolated
product showed physical and spectrometric data iden-
tical to that reported in the literature.2;15
The easy access to plant material (which may be
acquired from different sources), the abundance of the
natural product as well as the ease of extraction make
piperine a useful starting material for the preparation of
potentially bioactive compounds.
Firstly, piperine was tested against T. cruzi epimastig-
otes. It showed a dose-dependent toxicity, with IC50 of
7.36 lM. This preliminary result encouraged us to pre-
pare a series of derivatives in order to determine the
chemical features present in piperine responsible for the
trypanocidal activity. We have carried out the satura-
tion of the 2,4-diene moiety as well as some chemical
transformations on the amide function and have also
prepared amides with different length side chains.
Kapil described the results of an investigation of pip-
erine activity against promastigote forms of Leishmania
donovani.8 More recently, Raay and co-workers
described encouraging data obtained in vivo testing
hamsters infected with L. donovani with piperine inter-
calated into mannose-coated liposomes.9 Based on these
results, we investigated the activity of this alkaloid and
of a series of derivatives on Trypanosoma cruzi, another
important protozoan parasite and the cause of Chagas’
disease (American trypanosomiasis) in humans, an
incurable infectious disease responsible for 21,000
deaths and 200,000 new cases annually in 15 Southern
Cone countries.10
Scheme 1 shows the chemical transformations carried
out on 1, affording derivatives 11–20. The saturated
derivative 11 was prepared from 1 by catalytic hydro-
genation of the conjugated double bonds.16 The piperic
acid 13 was obtained from 1 in excellent yield through
basic hydrolysis.2 Acid 13 was used as precursor of a
series of derivatives, the ester 14 and the amides 15–19,
through the intermediacy of the acyl chloride (13a)
prepared by treatment of 13 with oxalyl chloride,17 and
addition of the relevant alcohol or amine. The allylic
amine 12 was obtained as a single product by reduction
of 1 with diisobutylaluminum hydride.18
The most common treatment for this disease involves
two drugs, nifurtimox 8 and benznidazole 9 (Fig. 2),
which are active only during the acute and short-term
chronic phase. Benznidazole is now the only drug still
available since the production of nifurtimox was stop-
ped. Unfortunately, narrow therapeutic windows,
strong side effects, and variable drug susceptibilities
among T. cruzi strains result in low clinical efficacies for
these nitro derivatives.11 Thus, it is important to study
and develop new compounds with antitrypanosomal
activity, which may possess enhanced antiparasitic
activity associated with low toxicity. Natural products
have long been used as templates for the development of
new molecules, which may be useful against parasitic
diseases (e.g., quinine 10, Fig. 2, the antimalarial iso-
lated from Cinchona officinalis).12;13
The derivatives of the cinnamic series (Scheme 2) were
obtained through classical Knoevenagel’s methodology
employing piperonal 20 as starting material.19 The cin-
namic acid 21 prepared in this way was used to prepare
amides 2 and 22. Piperamide 2 is a natural product
isolated by Loder et al. from Piper novae hollandie.20
Preparation of amide 3, an analogue with a seven car-
bon extended side chain, involved the synthetic route
shown in Scheme 3. This amide, named piperettine, is a
natural product isolated from Piper nigrum and Piper
aurantiacum.4;15 Amide 3 was prepared from 13 in five
steps, employing an Emmos–Horner reaction of alde-
hyde 24 with triethylphosphonoacetate,21 which affor-
ded the ester 25 stereoselectively, in good yield with
E-configuration at D2;3. The reaction sequence from acid
26 to analogue 3 was similar to that employed for other
amides (Schemes 1 and 2). This synthetic path afforded
piperettine 3 in six steps from piperine 1, in 26% overall
yield.
This work is part of a research program aiming at the
use of abundant natural products in the synthesis of
new molecules with potential application as antipara-
sitic drugs. We previously reported the evaluation of
CH3
O
N
N
N
O2N
N
N
H
O
SO2
NO2
9
8
H
The structures of all derivatives prepared in this work
(Schemes 1–3) were confirmed by IR, MS, H, and 13C
NMR spectral data. The assignments are based on 2D
NMR experiments and comparison with literature val-
ues2–5 and are consistent with the structures described.
H
H
1
HO
N
CH3O
N
10
Evaluation of the antiparasitic activity of the natural
product and its derivatives was carried by screening
Figure 2. Structures of nifurtimox 8, benznidazole 9 and quinine 10.

T. S. Ribeiro et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3555–3558
3557
O
O
N
a
11
O
O
O
O
O
N
N
b
12
1
O
c
O
O
O
O
Cl
d
O
OH
13a
e
13
O
O
O
R
O
17. R= N[i-CH(CH3)2]2
18. R= Morpholyl
14. R= OC6H11
15. R= NHCH2C6H5
19. R= N-Methylpiperazyl
HN
OCH3
16. R=
OCH3
Scheme 1. Chemical transformations on piperine 1. Reagents and conditions: (a) ethyl acetate, Pd/C, H2, 2 h (90%); (b) DIBAL-H, toluene, )10 °C,
30 min (80%); (c) KOH, ethanol, reflux, 24 h; then HCl (aq), 0 °C (90%); (d) (COCl)2, 25 °C, 30 min (100%); (e) CH2Cl2, alcohol, or amine, 0 °C, 1 h
(70–90%).
compounds were also evaluated for their toxicity against
noninfected murine peritoneal macrophages and their
ability to reduce the number of intracellular amastigotes
in in vitro infected cells.22 The results are presented in
Table 1. The IC50 values obtained for the compounds
tested were compared with those for benznidazole 9
(Fig. 2), the drug of choice for the treatment of Chagas’
disease.
CO2H
O
O
CHO
b
O
O
a
21
20
2
O
O
O
N
X
X= CH2
22 X= O
Neither benznidazole nor piperine or any of the syn-
thesized derivatives interfered with the cellular viability
of noninfected macrophages up to the concentration of
20 lM as judged by measurements of Trypan blue
exclusion and phagocytosis (not shown). As expected,
the IC50 values for benznidazole are similar to those
described previously for epimastigotes and intracellular
amastigotes.26;27 Piperine proved to be a potent T. cruzi
inhibitor, being more toxic to intracellular amastigotes
Scheme 2. Preparation of cinnamic analogues of piperine. Reagents
and conditions: (a) malonic acid, pyridine, piperidine (cat.), reflux 3 h;
then HCl (aq), 0 °C (85%); (b); (c) (COCl)2, 25 °C, 30 min (100%); then
CH2Cl2, piperidine, or morpholine, 0 °C, 1 h (90%).
their effects on the proliferation of T. cruzi Y-strain
epimastigotes grown in axenic culture.22 The most active
O
O
O
a
OH
b
O
OH
23
13
O
O
O
O
d
OCH2CH3
O
c
O
O
H
25
24
O
O
O
O
O
O
N
OH
e
3
26
Scheme 3. Preparation of piperettine 3 from piperine 1. Reagents and conditions: (a) DIBAL-H, THF, )10 °C, 1 h (60%); (b) MnO2, THF, reflux, 3 h
(93%); (c) Triethylphosphonoacetate, THF, NaH, )15 °C, 1 h (90%); (d) KOH, CH3CH2OH, 25 °C; then HCl (aq), 0 °C (90%); (e) (COCl)2, 25 °C,
30 min; then CH2Cl2, piperidine, 0 °C, 1 h (90%).

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T. S. Ribeiro et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3555–3558
Table 1. Growth inhibition of T. cruzi (epimastigotes and amastigotes)
for benznidazole, piperine and its derivatives
References and notes
1. Semler, U.; Gross, G. G. Phytochemistry 1988, 27, 1566.
2. Ikan, R. In Natural Products: A Laboratory Guide, 2nd
ed.; Academic Press: 1991; pp 233–238.
3. de Paula, V. F.; Barbosa, L. C. D.; Demuner, A. J.; Pilo-
Veloso, D.; Picanco, M. C. Pest Manag. Sci. 2000, 56, 168.
4. Kiuchi, F.; Nakamura, N.; Tsuda, Y.; Kondo, K.;
Yoshimura, H. Chem. Pharm. Bull. 1988, 36, 1452.
5. Koul, S.; Koul, J. L.; Taneja, S. C.; Dhar, K. L.; Jamwal,
D. S.; Singh, K.; Reen, R. K.; Sing, J. Bioorg. Med. Chem.
2000, 8, 251.
6. Parmar, V. S.; Jain, S. C.; Bisht, K. S.; Jain, R.; Taneja, P.;
Jha, A.; Tyagi, O. D.; Prasad, A. K.; Wengel, J.; Olsen, C.
E.; Boll, P. M. Phytochemistry 1997, 46, 597.
7. Tsukamoto, S.; Cha, B.-C.; Ohta, T. Tetrahedron 2002, 58,
1667.
8. Kapil, A. Planta Med. 1993, 59, 474.
9. Raay, B.; Medda, S.; Mukhopadhyay, S.; Basu, M. K.
Indian J. Biochem. Biophys. 1999, 36, 248.
Compound
Epimastigotes IC50 (lM) Amastigotes IC50 (lM)
1
7.36
>96.52
10.67
4.91
NTa
2
3
7.40
2.58
11.52
9.63
NT
9
2.20
19.41
17.49
11
12
13
14
15
16
17
18
19
22
>114.67
>83.33
>81.43
>65.61
14.85
NT
NT
NT
7.77
5.71
NT
56.13
>83.33
>95.78
NT
a NT: Not tested.
10. Moncayo, A. Mem. Inst. Oswaldo Cruz 2003, 98, 577.
11. Coura, J. R.; DeCastro, S. L. Mem. Inst. Oswaldo Cruz
2002, 97, 3.
12. Cordell, G. A. Phytochemistry 2000, 463.
13. Mann, J. In Murder, Magic, and Medicine; Oxford
University Press: England, 1992; pp 82–96.
14. Gomes, D. C. F.; Alegrio, L. V.; Lima, M. E. F.; Leon, L. L.;
ꢀ
Araujo, C. A. C. Arzneim.-Forsch./Drug Res. 2002, 52, 120.
15. Araujo-Junior, J. X.; Da-Cunha, E. V. L.; Chaves, M. C.
than epimastigotes. The apparently higher potency on
amastigotes may be explained by its preferential uptake
by tissue culture cells or by a greater capacity of intra-
cellular amastigotes to fluid-phase pinocytosis.28 Among
the derivatives prepared, acid 13 and ester 14 did not
show activity at the maximum dose on epimastigotes,
evidence for the need for a nitrogen-containing function
for activity. Amides 2, 15, 16, 19, and 22 were also
shown to be inactive against epimastigotes at the dosage
tested. Derivatives 2, 13–16, 19, and 22 were not eval-
uated against amastigotes. The loss of toxicity observed
for amide 2 clearly demonstrates the importance of the
extended carbon side chain in the original molecule,
corroborated by the maintenance of toxicity by piper-
ettine 3 against epimastigotes and amastigotes (Table 1).
Removal of the double bonds (derivative 11) did not
interfere significantly with activity, suggesting that
conjugation is not essential for trypanocidal activity.
Surprisingly, changing the piperidine moiety of the
natural product for diisopropyl (17) or morpholyl
groups (18) produces loss of activity on epimastigotes
but does not interfere significantly with toxicity against
intracellular amastigotes. Finally, reduction of the car-
bonylamide group of piperine gave the allylic amine 12,
which retained significant toxic effects against the par-
asites, showing that the carbonyl group is not important
for the toxic effect. In conclusion, we have synthesized a
series of piperine derivatives, which behave as potent
inhibitors of the proliferation of T. cruzi parasites, and
which may be considered suitable template compounds
for the design of new and more potent drugs for the
treatment of Chagas’ disease.
O.; Gray, A. I. Phytochemistry 1997, 44, 559.
ꢀ
16. Hudlicky, M. Reductions in Organic Chemistry; John
Wiley & Sons: New York, 1986; pp 1–3.
17. Szmuszkovice, J. J. Org. Chem. 1964, 29, 843.
18. Berardi, F.; Loiodice, F.; Frachiolla, G.; Calabufo, N. A.;
Perrone, R.; Tortorella, V. J. Med. Chem. 2003, 46, 2117.
19. Silva, E. F.; Canto-Cavalheiro, M. M.; Braz, V. R.; Cysne-
Finkelstein, L.; Leon, L. L.; Echevarria, A. Eur. J. Med.
Chem. 2002, 37, 979.
20. Loder, J. W.; Moorhous, A.; Russell, G. B. Austr. J.
Chem. 1969, 22, 1531.
21. Marianoff, B. E.; Reitz, A. B. Chem. Rev. 1989, 89, 863.
22. Drug screening. Biological assays on Y-strain epimasti-
~
gotes (Fundacßao Oswaldo Cruz, RJ, Brazil) were per-
formed as previously described.23 Benznidazole 9 (Roche,
Rio de Janeiro, Brazil), piperine 1 and its derivatives
(Table 1) were stored as 10 mg mLÀ1 stock solutions in
dimethylsulfoxide (DMSO), and were serially diluted (1:2)
in medium before use (the concentrations of 0.76, 1.56,
3.125, 6.25, 12.5, and 25 lg mLÀ1 were used) in triplicate.
Experiments on the intracellular form of the parasite were
conducted on T. cruzi-infected (Y-strain) macrophages as
described before.23 The 50% inhibitory concentration
(IC50) values were determined by linear regression analysis.24
For cellular viability tests, peritoneal mouse macrophages
were treated with the indicated concentrations of com-
pounds for 72 h as described elsewhere.25
23. Saraiva, V. B.; Gibaldi, D.; Previato, J. O.; Mendoncßa-
Previato, L.; Bozza, M. T.; Freire-de-Lima, C. G.; Heise,
N. Antimicrob. Agents Chemother. 2002, 46, 3472.
24. Lux, H.; Heise, N.; Klenner, T.; Hart, D. T.; Opperdoes,
F. R. Mol. Biochem. Parasitol. 2000, 111, 1.
25. Delorenzi, J. C.; Freire-de-Lima, L.; Gattass, C. R.; Costa,
D. A.; He, L.; Kuehne, M. E.; Saraiva, E. M. B.
Antimicrob. Agents Chemother. 2002, 46, 2111.
Acknowledgements
Financial support from FAPERJ and fellowships from
Capes to T. S. Ribeiro and from CNPq to L. Freire-de-
Lima are gratefully acknowledged. We also thank Dr.
Christopher Jones (Laboratory for Molecular Structure,
NIBSC, Herts, UK) for critical reading of the manu-
script.
26. Neal, R. A.; Bueren, J. Trans. R. Soc. Trop. Med. Hyg.
1988, 82, 709.
ꢀ
27. Martınez-Dıaz, R. A.; Escario, J. A.; Nogal-Ruiz, J. J.;
Gomez-Barrio, A. Mem. Inst. Oswaldo Cruz 2001, 96, 53.
ꢀ
ꢀ
28. Soares, M. J.; de Souza, W. Parasitol. Res. 1991, 77, 461.