Synthesis of benzologues of Nitazoxanide and Tizoxanide: A comparative study of their in vitro broad-spectrum antiprotozoal activity

Article abstract of DOI:10.1016/j.bmcl.2011.02.100

We have synthesized two new benzologues of Nitazoxanide (NIT) and Tizoxanide (TIZ), using a short synthetic route. Both compounds were tested in vitro against six protozoa (Giardia intestinalis, Trichomonas vaginalis, Entamoeba histolytica, Plasmodium berghei, Leishmania mexicana and Trypanosoma cruzi). Compound 1 (benzologue of NIT) showed broad antiprotozoal effect against all parasites tested, showing IC₅₀'s <5μM. This compound was five-times more active than NIT, and 18-times more potent than metronidazole against G. intestinalis. It was 10-times more active than pentamidine against L. mexicana, and it was sevenfold more potent than benznidazole versus T. cruzi. This compound could be considered as a new broad spectrum antiprotozoal agent.

Full text of DOI:10.1016/j.bmcl.2011.02.100

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3168–3171
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of benzologues of Nitazoxanide and Tizoxanide: A comparative
study of their in vitro broad-spectrum antiprotozoal activity
Gabriel Navarrete-Vazquez a, , Fabiola Chávez-Silva a, Rocío Argotte-Ramos b,
⇑
María del Carmen Rodríguez-Gutiérrez b, Manuel Jesús Chan-Bacab c, Roberto Cedillo-Rivera d,
Rosa Moo-Puc d, Emanuel Hernández-Nuñez a,e
a Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos 62209, Mexico
b Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Morelos 62508, Mexico
c Departamento de Microbiología Ambiental y Biotecnología, Universidad Autónoma de Campeche, Campeche 24030, Mexico
d Unidad de Investigación Médica Yucatán, IMSS Mérida, Yucatán 97000, Mexico
e Facultad de Química, Universidad Autónoma de Yucatán, Mérida, Yucatán 97000, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
We have synthesized two new benzologues of Nitazoxanide (NIT) and Tizoxanide (TIZ), using a short syn-
thetic route. Both compounds were tested in vitro against six protozoa (Giardia intestinalis, Trichomonas
vaginalis, Entamoeba histolytica, Plasmodium berghei, Leishmania mexicana and Trypanosoma cruzi). Com-
pound 1 (benzologue of NIT) showed broad antiprotozoal effect against all parasites tested, showing
Received 19 January 2011
Revised 17 February 2011
Accepted 22 February 2011
Available online 26 February 2011
IC50’s <5lM. This compound was five-times more active than NIT, and 18-times more potent than met-
ronidazole against G. intestinalis. It was 10-times more active than pentamidine against L. mexicana, and it
was sevenfold more potent than benznidazole versus T. cruzi. This compound could be considered as a
new broad spectrum antiprotozoal agent.
Keywords:
Nitazoxanide
Giardia intestinalis
Leishmania mexicana
Benzologue
Ó 2011 Elsevier Ltd. All rights reserved.
Protozoan diseases are a main problem worldwide, affecting
hundreds of millions people and animals.1 The chemotherapy
against diseases such as giardiasis, amoebiasis, trichomoniasis,
leishmaniasis and trypanosomiasis is limited by the existence of
only a few drugs in the market, most of which are of low efficacy,
showing toxic side effects, and frequently lead to the appearance of
resistant strains.2 This reflects the need to continue searching for
new and better antiprotozoal drugs.3
O
H
O
O
O
O
N
N
Plasma
esterase
O N
N
H
O N
N
H
2
2
S
S
NIT (Active)
TIZ (Active)
Nitazoxanide (NIT, AliniaÒ), is a broad-spectrum antiparasitic
compound belonging to a nitroheterocyclic class named thiazo-
lides.4 In humans, NIT is rapidly metabolized to tizoxanide (TIZ),
which is a compound equally effective as the parent drug (Fig. 1).5
Detailed in vitro and in vivo studies have currently been con-
ducted on the efficacy of NIT and other thiazolide drugs against
helminthes, extracellular anaerobic protozoa and bacteria, intra-
cellular parasites and viruses, such hepatitis C and AH1N1.6–9
As a part of our search for basic information about the structural
requirements for new antiprotozoal molecules, we have synthe-
sized two benzologues of NIT and TIZ (Fig. 2). The in vitro antipar-
asitic activities of these compounds on intestinal unicellular
parasites (Giardia intestinalis and Entamoeba histolytica), a urogen-
Figure 1. Metabolism of nitazoxanide (NIT).
Benzologue
X
X
Y
Y
R
O
R
O
O
Benzologue
O
N
N
O2N
O2N
N
H
N
H
S
S
1: R = COCH3
2: R = H
NIT: R = COCH3
TIZ: R = H
⇑
Corresponding author. Tel./fax: +52 777 3297089.
E-mail address: gabriel_navarrete@uaem.mx (G. Navarrete-Vazquez).
Figure 2. Thiazolides used as leads and drug design of compounds 1 and 2 using
vinylogy principle (benzologue).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.100

G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3168–3171
3169
ital tract parasite (Trichomonas vaginalis), a red blood cell parasite
(Plasmodium berghei), and kinetoplastid parasites such as Trypano-
soma cruzi and Leishmania mexicana are reported in this letter.
Compounds 1 and 2 were designed on the basis of the structure
of antiprotozoal drug Nitazoxanide (NIT), and its active metabolite,
Tizoxanide (TIZ). The vinylogy principle was used for the drug de-
sign. According to this principle, two substituents X and Y, linked
to aromatic rings in position ortho and para, one in relation to
the other, or separated by a chain of conjugated double bonds or
arenes (benzologue), usually function as being attached directly
one to another (Fig. 2). It implies that biological activity could be
conserved if the electronic communication between X and Y is also
retained.10
The benzamide region of the 1H NMR spectrum contained dis-
placements of the four phenyl protons (H-30 to H-60), ranging from
6.95 to 7.98 ppm, in compounds 1 and 2.
We also observed in both compounds a characteristic ABX pat-
tern for 6-nitrobenzothiazole core: a doublet signal ranging in d
7.87–7.88 ppm, attributable to H-4, with ortho coupling constant
(Jo = 8.9–9.1 Hz); a doublet of doublets signal ranging from 8.28
to 8.31 ppm, assigned to H-5, with ortho and meta coupling con-
stants (Jm = 2.4 and Jo = 9.1 Hz). A second doublet signal in 9.07–
9.10 ppm, with Jm = 2.4 Hz, was assigned to H-7.
The new benzologues 1 and 2 were tested in vitro as antiproto-
zoal agents. Biological assays results against the six protozoa tested
are summarized in Table 1. Comparison was made among new
compounds and the antiprotozoal drugs of choice: metronidazole
and NIT, against G. intestinalis, E. histolytica and T. vaginalis. In order
to compare bioactivities, TIZ, pentamidine and benznidazole were
also tested. In vitro susceptibility assays were performed using a
method previously described.12–14
G. intestinalis (syn. duodenalis, lamblia) is an intestinal protozoan
parasite infecting humans and various other mammalian hosts. It
is one of the most commonly diagnosed protozoal causes of diar-
rhea worldwide. Clinical resistance has been reported for current
chemotherapeutics (metronidazole and albendazole).15 It is inter-
esting to note that compound 1 (benzologue of NIT) was more po-
tent than metronidazole against G. intestinalis, being 18-times
more active than this drug of choice. Compound 1 was also fourfold
more potent than NIT and 2.4-times more active than TIZ. On the
other hand, compound 2 was twofold less active than compound
1, but it was 9-fold more potent than metronidazole, two-times
more active than NIT, and as active as TIZ against G. intestinalis.
Pentamidine (an anti-Pneumocystis, trypanocidal and leishmanicid-
al drug) was as active as metronidazole against this protozoan.
T. vaginalis is the causative agent of trichomoniasis, a common
sexually-transmitted disease in humans.16 Compound 1 showed
nanomolar trichomonicidal potency (IC50 = 842 nM). However, it
was 12-times less active than NIT (IC50 = 68 nM), and three-times
less active than metronidazole and TIZ. Compound 2 was also less
active than the three drugs of choice.
Compounds 1 and 2 were prepared starting from 4-nitroaniline
(3), which was reacted with ammonium thiocyanate and bromine
under reflux, to give 2-amino-6-nitrobenzothiazole (4). This com-
pound was acylated with acetylsalyciloyl chloride, in presence of
triethylamine and catalytic amounts of DMAP, to get compound
1, which was hydrolyzed with lithium hydroxide in a mixture of
THF–H2O 9:1, to obtain compound 2 (Scheme 1). Compounds were
purified by recrystallization. The chemical structures of the synthe-
sized compounds were confirmed on the basis of their spectral
data.11
In the nuclear magnetic resonance spectra (1H NMR; d ppm),
the signals of the respective protons of the compounds were veri-
fied on the basis of their chemical shifts, multiplicities and cou-
pling constants.
NH2
Br2
N
S
NH4SCN
+
NH2
Δ
O2N
O2N
3
4
O
O
O
Cl
TEA, DMAP
The protozoan parasite E. histolytica causes amebic colitis and
amebic liver abscess, diseases that afflict millions of individuals
in developing countries.17 Compounds 1 and 2 showed activity
5
O
against this protozoa in the low micromolar order (IC50’s <9 lM).
However none of them showed more amoebicidal activity com-
pared than NIT, TIZ and metronidazole.
The Leishmania species causes a variety of diseases from self-
healing cutaneous lesions to life-threatening visceral infections.
Clinical manifestations depend on the infecting parasites species.
There is an estimated annual 1.5–2.0 million new cases of leish-
O
O
O H
O
LiOH
N
S
N
S
N
H
THF, H2O
O2N
N
H
O2N
2
1
Scheme 1. Synthesis of benzologues 1 and 2.
Table 1
In vitro antiprotozoal and cytotoxic activities of NIT and TIZ benzologues
R
O
O
N
O2N
N
H
S
Compd
R
IC50
(l
M)
CC50 (lM)
G. intestinalis
T. vaginalis
E. histolytica
L. mexicana
T. cruzi
P. berghei
VERO
1
2
NIT
TIZ
–COCH3
–H
0.297
0.590
1.214
0.716
5.36
0.842
2.147
0.068
0.211
0.290
3.815
18.620
3.515
8.021
0.504
1.229
0.770
11.800
4.270
1.350
>50
6.180
6.190
>50
4.890
>50
18.730
17.470
>50
2.420
2.370
3.890
5.240
>50
683
607
833
388
387
47
Metronidazole
Pentamidine
Benznidazole
4.079
22.58
13.320
>50
>50
34.380
2.942
4.070
14

3170
G. Navarrete-Vazquez et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3168–3171
maniasis, from which approximately 500,000 belong to the visceral
form, which is potentially fatal.18
anide and the lack of nitro reduction as part of its mechanism of
action, make it an important exception.23,24
In vitro antileishmanial assay was carried out using a method
In conclusion, we have synthesized and screened the in vitro
antiprotozoal activities of two new NIT and TIZ benzologues. This
study demonstrated that the insertion of benzene ring between
the NIT pharmacophore region (nitro and thiazole), have generated
a new antiprotozoal scaffold. Compound 1 (benzologue of NIT),
previously described.12,18 Compound 1 had an excellent activity
against this kinetoplastid protozoa (IC50 = 1.350 lM). It was 4.5-
times more active than NIT and TIZ. The last two drugs were pre-
viously reported by our group as potential new antikinetoplastid
parasite compounds.18 Pentamidine (second-line antileishmanial
drug) was used as positive control. In this research, compound 1
was almost 10-times more potent than pentamidine against prom-
astigotes of L. mexicana. Compound 2 was inactive against this
protozoan.
American trypanosomiasis or Chagas’ disease, caused by its eti-
ological agent T. cruzi, is still one of the major causes of morbidity
and mortality due to cardiovascular diseases in Latin America.18
In vitro trypanocidal assay was performed using a technique
previously reported.12,18 Compound 1 was four-times more active
than NIT and TIZ, which were twofold more potent than benzni-
dazole (first-line antichagasic drug). Compound 1 was seven-times
more active than benznidazole against epimastigotes of T. cruzi.
Compound 2 did not show activity in this assay.
Malaria continues to be a major health challenge in most trop-
ical and many subtropical regions. It is caused by protozoan para-
sites of the genus Plasmodium. The rodent malaria parasite, P.
berghei, is a useful model to screen new antimalarial drugs.19 Cul-
tured schizonts of P. berghei were used to assess antimalarial activ-
ity of compounds and were prepared following the protocol
described previously.12,20,21 We evaluated the antiplasmodial
activity of compounds 1 and 2, which were as active as pentami-
dine (a known plasmocidal drug). NIT and TIZ showed micromolar
showed high bioactivity against all parasites tested (IC50’s <5 lM)
and low cytotoxic effect. The obtained results are very promising
since compound 1 exhibited higher bioactivity than NIT, TIZ, ben-
znidazole and pentamidine especially towards G. intestinalis, L.
mexicana, T. cruzi and P. berghei. Compound 1 could be considered
as a new broad spectrum antiprotozoal agent. Further optimization
of new series and pharmacokinetic characterization of both com-
pounds are in progress in our laboratory.
Acknowledgments
This work was supported in part by internal funds from Facul-
tad de Farmacia, UAEM. We are in debt to Johnatan Cauich Chan
from the UADY, for his technical assistance during antiprotozoal
susceptibility assays.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.100.
References and notes
activity (IC50 = 3.890 and 5.240
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good potency (IC50 = 4.070 M) in this assay. To the best of our
knowledge, this is the first study reporting the in vitro activity of
NIT, TIZ and benznidazole, against P. berghei.
lM) against schizonts develop-
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11. 2-{[(6-Nitro-1,3-benzothiazol-2-yl)amino]carbonyl}phenyl
acetate
(1).
Recrystallized from ethanol. Yield: 73%, mp: 300–303.5 °C. 1H NMR
(400 MHz, DMSO) d: 2.25 (s, 3H, CH3), 7.03 (t, 1H, H-5, J = 7.5 Hz), 7.08 (d,
1H, H-30, J = 8.2 Hz), 7.52 (td, 1H, H-40, J = 1.7, J = 8.2 Hz), 7.88 (d, 1H, H-4,
J = 8.9 Hz), 7.98 (dd, 1H, H-60, J = 1.7, J = 8.2 Hz), 8.31 (dd, 1H, H-5, J = 2.4,
J = 8.9 Hz), 9.10 (d, 1H, H-7, J = 2.4 Hz), 12.76 (br s, 1H, NH) ppm. 13C NMR
(100 MHz, DMSO) d: 22.9 (CH3), 117.3 (C-30), 119.1 (C-7), 120.6 (C-4), 121.8 (C-
5), 122.0 (C-10), 130.6 (C-6), 132.2 (C-40, 135.0 (C-60), 135.0 (C-7a), 142.9 (C-50),
143.1 (C-20), 143.1 (C-3a), 153.5 (C-2), 163.5 (NHC@O), 170.2 ((OC@O) ppm;
MS/FAB+: m/z 358 (M+H+). HRMS (FAB+): m/z 358.0497 [M+H]+ (calcd for
16H11N3O5SH+
358.0498).
2-Hydroxy-N-(6-nitro-1,3-benzothiazol-2-yl)
Although nitro compound-containing drugs and analogues are
frequently avoid in a drug discovery program due to mutagenic
and potentially toxic side effects, the metabolic stability of nitazox-
C
benzamide (2). Recrystallized from ethanol. Yield: 81%, mp: 317–320.9 °C. 1H
NMR (400 MHz, DMSO) d: 6.95 (t, 1H, H-5, J = 7.9 Hz), 7.08 (d, 1H, H-30,
J = 7.9 Hz), 7.51 (td, 1H, H-40, J = 1.7, J = 8.5 Hz), 7.87 (d, 1H, H-4, J = 9.2 Hz),
7.97 (dd, 1H, H-60, J = 1.7, J = 7.9 Hz), 8.28 (dd, 1H, H-5, J = 2.5, J = 8.9 Hz), 9.07
(d, 1H, H-7, J = 2.4 Hz), 12.76 (br s, 1H, NH) ppm. 13C NMR (100 MHz, DMSO) d:
117.3 (C-30), 119.0 (C-7), 120.6 (C-4), 121.8 (C-5), 122.0 (C-10), 130.6 (C-6),
132.2 (C-40), 135.0 (C-60), 135.0 (C-7a), 142.9 (C-50), 143.1 (C-3a), 153.5 (C-2),
157.5 (C-20), 163.5 (NHC@O) ppm; MS/FAB+: m/z 316 (M+H+). HRMS (FAB+): m/
z 316.0356 [M+H]+ (calcd for C14H12N3O4SH+ 316.0392).
Table 2
Selectivity indexes of 1, 2, NIT and TIZ
Compd G.
intestinalis
T.
E.
L.
T.
P.
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vaginalis
histolytica mexicana cruzi
berghei
Selectivity index (SI = CC50/IC50
)
1
2
NIT
TIZ
2300
1029
686
811
283
12250
1839
194
76
1653
316
506
ND
135
63
140
ND
44
282
256
214
74
542
22

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