Synthesis and in vitro antitrypanosomal activity of novel Nifurtimox analogues

Article abstract of DOI:10.1016/j.bmcl.2005.01.002

Eight novel analogues of Nifurtimox, 4-[(5-nitrofurfurylidene)amino]-3- methylthiomorpholine-1,1-dioxide, containing alfa-beta unsaturated amides, were synthesized and evaluated for their in vitro activity against Trypanosoma cruzi epimastigotes. Four der

Full text of DOI:10.1016/j.bmcl.2005.01.002

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1417–1421
Synthesis and in vitro antitrypanosomal activity of novel
Nifurtimox analogues
a
Rocıo Pozas, Javier Carballo, Clementina Castro and Julieta Rubio
a
b
b,
*
´
a
´ ´ ´ ´ ´
Departamento de Quımica Organica, Facultad de Quımica, Universidad Nacional Autonoma de Mexico,
´
C.U. Mexico D.F. 04510, Mexico
b
´
´
Departamento de Medicina Genomica y Toxicologıa Ambiental, Instituto de Investigaciones Biomedicas,
Universidad Nacional Autonoma de Mexico, C.U. Apartado Postal 70228, Mexico DF 04510, Mexico
´
´
´
´
Received 27 April 2004; revised 28 December 2004; accepted 4 January 2005
Available online 25 January 2005
Abstract—Eight novel analogues of Nifurtimox, 4-[(5-nitrofurfurylidene)amino]-3-methylthiomorpholine-1,1-dioxide, containing
alfa–beta unsaturated amides, were synthesized and evaluated for their in vitro activity against Trypanosoma cruzi epimastigotes.
Four derivatives bearing a nitro group at the 5-position of the furan ring were the most active in inhibiting culture growth and pro-
voking cell death, showing trypanocidal activity more than threefold the potency of Nifurtimox, our positive control. Two deriva-
tives lacking a nitro group were less potent than the positive control. Active nitro derivatives very efficiently caused epimastigote
death, which suggests a nitro reduction mechanism of action.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
anti-Trypanosoma cruzi compounds continues to be a
major goal in trypanocidal chemotherapy.
The flagellate Trypanosoma cruzi, the etiological agent
of Chagas disease or American trypanosomiasis, affects
more than 18 million people in Latin America, leading
to approximately 400,000 deaths per year. Since 1909
when this disease was initially characterized1 several ef-
forts were carried out in order to eradicate it, but in spite
of the 75% reduction in the incidence of human cases
observed in the South American countries (i.e., Argen-
tine, Brazil, Chile, Paraguay, Uruguay and Venezuela)
as a consequence of the successful vector control pro-
grams being set up,2 Chagas disease still remains a regio-
nal health problem. Nifurtimox (a 5-nitrofuran
derivative), Benznidazole (a 2-nitroimidazole acetamide)
and Ketoconazole (a piperazine–imidazole–dioxolan),
ameliorate acute T. cruzi infection3 but exert serious side
effects and little or no effect on the chronic phase of the
disease,4 probably due to the inherent characteristics of
the host and the virulence and resistance of the parasite.
Development of safer and more efficient therapeutic
Several authors have reported that T. cruzi is particu-
larly sensitive to compounds that can produce free radi-
cals due to its lack of catalase and glutathione
peroxidase, and proposed that the main mechanism by
which Nifurtimox acts against T. cruzi is through the
intracellular production of superoxide anion.5
Various series of 5-nitrofuran derivatives containing dif-
ferent heterocyclic moieties and alfa–beta unsaturated
amides have been synthesized since their structural
domains were associated with biological activities,
such as trypanocidal, bactericidal, fungicidal and
squistosomicidal.6
2. Chemistry
To prepare compounds 1, 2, 3, 6 and 8, 2-furylacrilic
acid was treated with nitric acid in acetic anhydride,
which led to 5-nitro-2-furylacrilic acid.7 Reaction of
this compound with thionyl chloride in benzene yielded
the corresponding acid chloride, which by reaction with
either isobutylamine, benzylamine, 4-chlorobenzylamine,
Keywords: Trypanosoma cruzi; Antitrypanosomal activity; Nifurtimox
analogues; Antiparasites.
*
Corresponding author. Tel.: +52 5 5 56 22 31 59; fax: + 52 5 5 56 22
38 45; e-mail: juruli@servidor.unam.mx
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.002

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R. Pozas et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1417–1421
(CH3CO)2O/HNO3
OH
OH
O2N
Z
Z
stir/-15 to-5 ˚C
O
O
SOCl2/C6H6
Reflux for 4 h
SOCl2/C6H6
Reflux for 4 h
NH2R/C6H6
Cl
O
NHR
Y
Y
Z
stir /r.t
24 h
Z
O
Y = NO2, H
Z= O, S
Scheme 1. General procedure for the chemical synthesis of the eight compounds.
piperonylamine or 4-chlorobenzothiazole, yielded com-
pounds 1, 2, 3, 6 and 8, respectively. Compound 4 was
elaborated through a two-step sequence: (a) reaction
of furylacrilic acid with thionyl chloride and reaction
Table 1. Chemical structures of the synthesized compounds
H
N
R1
R2
Z
O
Comp.
No.
Z
R1
R2
Yield (%) Melting point Name
(ꢁC)
1a
2a
3a
4
O
–NO2
67.5
89.0
85.0
94.0
95.0
88.5
91.3
124–125
N-Isobutyl-3-(5-nitro-2-furyl)-2E-propenamide
N-Benzyl-3-(5-nitro-2-furyl)-2E-propenamide
N-(4-Chlorobenzyl)-3-(5-nitro-2-furyl)-2E-propenamide
N-Benzyl-3-(2-furyl)-2E-propenamide
O
O
O
S
–NO2
–NO2
–H
177–178
163–164
146–147
135–136
147–148
153–154
Cl
5
–H
N-Benzyl-3-(thiophen-2-yl)-2E-propenamide
N-Piperonyl-3-(5-nitro-2-furyl)-2E-propenamide
N-Benzyl-3-(5-nitrothiophen-2-yl)-2E-propenamide
O
O
6a
O
S
–NO2
–NO2
7
Cl
N
S
8
O
–NO2
82.0
186–187
N-(4-Chloro-2-benzothiazole)-3-(5-nitro-2-furyl)-2E-propenamide
a Most biologically potent compounds (see Table 2).

R. Pozas et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1417–1421
1419
with benzylamine; compound 5 was prepared from 3-
thiophen-2-yl-acrylic acid reacted with thionyl chloride
in benzene, followed by treatment with benzylamine;
whereas, compound 7 was synthesized by the reaction
of 3-(5-nitro-thiophen-2-yl) acrylic acid with thionyl
chloride in benzene, followed by treatment with benzyl-
amine.8 These general procedures are shown in Scheme
1.
The structure of these new compounds was confirmed
by IR (recorded in a FTIR Perkin–Elmer 1605 spec-
trometer), RMN (recorded in a Varian 300 MHz 1H
and 750 MHz 13C spectrometer. Chemical shifts are re-
ported in parts per million, relative to tetramethylsilane.
Splitting patterns are designated as s, for singlet, d, dou-
blet and t, triplet), low resolution mass spectra, MS,
were recorded in a JOEL SX-102 A spectrometer in elec-
tron-impact mode and UV spectra were recorded in a
UV/vis Perkin–Elmer Lamda 2 spectrometer.9 Chemical
structure, yield, melting point and name of each com-
pound are described in Table 1.
3. Biological evaluation
Trypanocidal activity was assayed on epimastigote
forms of T. cruzi Y strain, cultured at 28 ꢁC in liver
infusion tryptose medium (LIT), supplemented with
10% heat inactivated (56ꢁ for 30 min) fetal calf serum.
Parasites in logarithmic growth phase (from an initial
culture with 2 · 106 epimastigotes/mL) were incubated
with increasing concentrations of the test compounds
(1, 10 and 100 lg/mL) in dimethylsulfoxide (1% final
concentration) for 24, 48, 72 and 96 h. Parasite viability
and growth response were determined every 24 h in an
inverted microscopy, a Spectronic 20 D+ spectropho-
tometer at 580 nm, and a Neubauer chamber. Morpho-
logy was analyzed in fixed preparations stained with
Giemsa. All assays were carried out in triplicate.
The activity was calculated as growth inhibition %
and the data express the mean of three experiments
(Table 2).
4. Discussion and conclusions
In clinical practice, trypanocidal drugs must act upon T.
cruzi infective stages (trypomastigote and amastigote).
Nevertheless, in this study we used the epimastigote
non-infective form, since culture and handle in the lab-
oratory are easier and safer. Moreover, replicative,
non-infective cells have been used in several other stud-
ies for screening natural and synthetic compounds with
potential trypanocidal activity, nitrofurans included.
Agents showing biological activity against T. cruzi epi-
mastigotes have proved to be also efficient against the
infective forms (5).
Trypanocidal activities, reported in Table 2, show that
the most potent derivatives are compounds 6, 1, 2 and
3, all of them containing a 5-nitro furan.

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R. Pozas et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1417–1421
C.; Stoppani, A. O. M.; Paulino, M.; Olea-Azar, C.;
Basombrio, M. A. Eur. J. Med. Chem. 2000, 35, 3443.
4. (a) Castro, J. A.; Dias de Toranzo, E. G. Biomed. Environ.
Sci. 1988, 1, 19; (b) Gorla, N. B.; Ledesma, O. S.; Barbieri,
G. P.; Larripa, I. B. Mut. Res. 1989, 224, 263; (c)
Lack of a nitro group as in derivatives 4 and 5, drasti-
cally decreased trypanocidal response, confirming the
key role played by this portion of the molecule for the
trypanocidal activity. Nevertheless, it is necessary to
point out that the element at the 2-furil position is also
important, since derivatives 7 and 8 also bear the 5-nitro
group and, even they show trypanocidal activity, this is
not as high as that showed by derivatives 1, 2, 3 and 6.
´
Rodrıguez, R. R.; Lane, E.; Chealing, J. J. Inorg. Biochem.
1995, 60, 277.
5. (a) Docampo, R.; Stoppani, A. O. M. Arch. Biochem.
Biophys. 1979, 197(1), 317; (b) Docampo, R.; De Boiso, J.
F.; Stoppani, A. O. M. Experientia 1976, 32, 972; (c)
Boveris, A.; Stoppani, A. O. M. Experientia 1977, 33, 1306;
(d) Boveris, A.; Docampo, R.; Stoppani, A. O. M. Biochem.
The heterocycle in compound 7 is a thiophene, instead
of a furan, and although R2 is the same as in compound
2 its activity was diminished, the only change of O by S
reduced the activity.
´
J. 1980, 188, 643; (e) Denicola, A.; Rubbo, H.; Rodrıguez,
D.; Radi, R. Arch. Biochem. Biophys. 1993, 304, 279; (f)
Rubbo, H.; Denicola, A.; Radi, R. Arch. Biochem. Biophys.
1994, 308, 96; (g) Maya, J. D.; Bollo, S.; Nun˜ez-Vergara, L.
Compounds 2 and 4 have the same R2 substituent, but
compound 4 has not nitro group, therefore its activity
was lower.
´ ´
J.; Squella, J. A.; Repetto, Y.; Morello, A.; Perie, J.;
`
Chauviere, G. Bioch. Pharm. 2003, 65, 999–1006.
6. (a) Freeman, F.; Wilson, P.; Kazan, B. Exp. Parasitol.
1975, 38, 181; (b) Neville, M.; Verge J. Med. Chem. 1997,
20(7), 946; (c) Mester, B.; Elguero, J. Arch. Pharm.
(Weinheim) 1987, 320, 115; (d) Shuyu, W.; Renli, L. Chem.
Abstr. 1986, 105, 21829k; (e) Xiiao-Hui, Z.; Renli, L. Chem.
Abstr. 1980, 93, 14676u; (f) Grivsky, E. Chem. Abstr. 1983,
98, 16431; (g) Kamikawa, T. Chem. Abstr. 1980, 93, 46396g;
(h) Kubo, I.; Matsumoto, T. Experientia 1984, 40, 340; (i)
Abdel-Rahman, M. O. Chem. Abstr. 1969, 70, 114894v.
7. (a) Gilman, H.; Wright, F. G. J. Am. Chem. Soc. 1930, 52,
2550; (b) Abdel-Rahaman, M. O.; Aboul-Enein, M. N.;
Tadros, W. M. J. Chem. U.A.R. 1969, 12(1), 69.
8. Elliott, M.; Farnaham, A. W. Pestic. Sci. 1987, 18, 191.
9. N-Isobutyl-3-(5-nitro-2-furyl)-2E-propenamide (1). Ethyl
acetate (68%) mp 124–125 ꢁC IR m cmÀ1 3270 (N–H),
1664.7 (C@O), 1516.8, 1348.6 (NO2), 979.4 (C@C trans);
1H NMR (300 MHz, DMSO-d6) d 7.4, 6.67 (dd, 2H,
J = 15.38, C@C trans); 7.34,6.69 (dd, 2H, J = 3.7, furane);
6.11 (br s, 1H, –NH); 3.24 (t, 2H, CH–); 1.85 (m, 1H,
–CH–); 0.95 (d, 6H, 2(CH3)). UV (kmax nm) 347.8. MS m/z
238M+.
Compounds 2 and 3 had a very similar biological acti-
vity even though the R2 substituent in compound 3 is
a chlorine; it appears that the addition of this element
had no influence in activity. This also applies for com-
pounds 1 and 2, where 1 has an aliphatic chain, while
2 has an aromatic substituent, and both showed similar
biological activities.
The data show that the nitro derivatives, analogues to
Nifurtimox synthesized by an easy, cheap route, ob-
tained at a high yield, represent useful, novel leads for
the development of new and effective drugs for Ameri-
can Trypanosomiasis treatment. Indeed, these nitro
derivatives appear to be more potent that the commer-
cial analogue Nifurtimox, widely used as trypanocidal
agent.
Since active nitro compounds are highly citotoxic, one
could assume that these kind of molecules perturb physio-
logical cellular functions of the parasite, mainly those
related to its redox system,5 since Trypanosomatides
lack an efficient reduction system; therefore, this class
of free radicals-producing compounds must be effective
to combat the parasite and probably less harmful
against the host, which is entailed with an effective redox
system to detoxicate reactive radicals.
N-Benzyl-3-(5-nitro-2-furyl)-2E-propenamide (2). Ethyl
acetate (89%) mp 177–178 ꢁC. IR m cmÀ1 3250.33 (N–H),
1666.55 (C@O), 1513.09, 1356 (NO2), 969.97 (C@C trans);
1H NMR (300 MHz, DMSO-d6) d 7.44, 6.66 (dd, 2H,
J = 15.5, C@C trans); 7.32, 6.68 (dd, 2H, J = 3.7, furane);
7.31 (m, 5H, aromatic); 6.11 (br s, 1H, –NH); 4.57 (d, 2H,
CH2) UV (kmax nm) 355.65. MS m/z 272M+.
N-(4-Chlorobenzyl)-3-(5-nitro-2-furyl)-2E-propenamide (3).
Ethyl acetate (85%) mp 163–164 ꢁC. IR m cmÀ1 3254.13 (N–
H), 1654.25 (C@O), 1520.7, 1346.38 (NO2), 1H NMR
(300 MHz, DMSO-d6) d 7.37, 6.66 (dd, 2H, J = 15.3, C@C
trans); 7.27, 6.65 (dd, 2H, J = 3.7 furane); 7.26 (m, 4H,
aromatic); 6.55 (br s, 1H, –NH); 4.50 (d, 2H,–CH2) UV
(kmax nm) 347.66 MS m/z 306M+.
References and notes
1. (a) Chagas, C. Mem. Inst. Oswaldo Cruz. 1909, I, 159; (b)
Moncayo, A. Tropical Disease Research Progress 1991–
1992; WHO: Geneva, 1993; pp 67–76.
N-Benzyl-3-(2-furyl)-2E-propenamide (4). Ethyl acetate
(94%) mp146–147 ꢁC IR m cmÀ1 3257.64 (N–H), 1654.85
1
(C@O), 974.18 (C@C trans); H NMR (300 MHz, DMSO-
2. TDR news, UNDP/WORLD BANK/WHO Special Pro-
gramme for Research and Training in Tropical Diseases
(TDR), No. 56 June 1998, No. 59 June 1999, No. 60
October 1999, No. 62 June 2000.
d6) d 7.46, 6.32 (dd, 2H, J = 15.35, C@C trans); 7.29 (m,
5H, aromatic); 7.30, 6.54, 6.41 (3d, 3H, furane); 6.0 (br s,
1H, –NH); 4.54 (d, 2H,–CH2) UV (kmax nm) 398.31 MS m/z
227 M+.
3. (a) Brener, Z.; Cancado, J. R.; Galvao, L. M.; da Luz, Z.
M. P.; Filardi, L. S. D.; Pereira, M. E. S.; Santos, L. M. S.;
Cancado, C. D. Mem. Do Inst. Oswaldo Cruz. 1993, 88, 149;
(b) Jernigan, J.; Pearson, R. Curr. Opin. Infect. Diseases
N-Benzyl-3-(2-thiol)-2E-propenamide (5). Ethyl acetate
(95%) mp 135–136 ꢁC IR m cmÀ1 3247.48 (N–H), 1648.16
1
(C@O), 973.62 (C@C trans) H NMR (300 MHz, DMSO-
d6) d 7.46, 6.32 (dd, 2H, J = 16.15, C@C trans); 7.29 (m,
5H, aromatic); 7.75, 7.25, 7.10 (3d, 3H, thiophene);6.18 (br
s, 1H, –NH); 4.55 (d, 2H, –CH2) UV (kmax nm) 306.37 MS
m/z 243 M+.
´
1993, 6, 794; (c) Cerecetto, H.; Di Maio, R.; Gonzalez, M.;
Risso, M.; Saenz, P.; Seoane, G.; Denicola, A.; Peluffo, G.;
Quijano, C.; Olea-Azar, C. J. Med. Chem. 1999, 42, 1941;
´
(d) Cerecetto, H.; Di Maio, R.; Gonzalez, M.; Risso, M.;
Sagrera, G.; Seoane, G.; Denicola, A.; Peluffo, G.; Quijano,
N-Piperonil-3-(5-nitro-2-furyl)-2E-propenamide (6). Ethyl
acetate (88.5%) mp 147–148 ꢁC IR m cmÀ1 3273.31 (N–H),

R. Pozas et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1417–1421
1421
1653.41 (C@O), 1511.88, 1343.51 (NO2), 1H NMR
(300 MHz, DMSO-d6) d 7.37, 6.66 (dd, 2H, J = 15.3,
C@C trans); 7.27, 6.65 (m, 2H, J = 3.7 furane); 7.45, 6.80
(dd, 2H, J = 3.0 aromatic); and 6.10 (s, 1H, aromatic); 6.55
(br s, 1H, –NH); 5.95 (s, 2H, –CH2); 4.45 (d, 2H, –CH2);
UV (kmax nm) 348.72 MS m/z 316 M+.
N-Benzyl-3-(5-nitro-2-thiol)-2E-propenamide (7). Ethyl
acetate (91.3%) mp 153–154 ꢁC IR m cmÀ1 3257.18 (N–H),
1664.91 (C@O), 1532.17, 1341.00 (NO2); 1H NMR
(300 MHz, DMSO-d6) d 7.46, 6.45 (dd, 2H, J = 15.3,
C@C trans); 7.80, 7.05 (m, 2H, J = 3.4 thiol); 7.28 (m,
5H, aromatic); 6.10 (br s, 1H, –NH); 4.50 (d, 2H, –CH2)
UV (kmax nm) 357.74 MS m/z 288 M+.
N-(4-Chloro-2-benzothiazole)-3-(5-nitro-2-furyl)-2E-prop-
enamide (8). Ethyl acetate (82%) mp 186–187 ꢁC. IR m cmÀ1
3466.57 (N–H), 1646.00 (C@O), 1536.57, 1348.37 (NO2),
1H NMR (300 MHz, DMSO-d6) d 7.46, 6.45 (dd, 2H,
J = 16.3, C@C trans); 7.30 (m, 3H, aromatic); 7.40, 6.95 (m,
2H, J = 3.7 furane); 6.05 (br s, 1H, –NH); UV (kmax nm)
266.76 MS m/z 349 M+.