Synthesis and biological activity of nitro heterocycles analogous to megazol, a trypanocidal lead

Article abstract of DOI:10.1021/jm021030a

As part of our efforts to develop new compounds aimed at the therapy of parasitic infections, we synthesized and assayed analogues of a lead compound megazol, 5-(1-methyl-5-nitro-1H-2-imidazolyl)-1,3,4-thiadiazol-2-amine, CAS no. 19622-55-0), in vitro. We first developed a new route for the synthesis of megazol. Subsequently several structural changes were introduced, including substitutions on the two rings of the basic nucleus, replacement of the thiadiazole by an oxadiazole, replacement of the nitroimidazole part by a nitrofurane or a nitrothiophene, and substitutions on the exocyclic nitrogen atom for evaluation of an improved import by the glucose or the purine transporters. Assays of the series of compounds on the protozoan parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani, as either extracellular cells or infected macrophages, indicated that megazol was more active than the derivatives. Megazol was then evaluated on primates infected with Trypanosoma brucei gambiense, including late-stage central nervous system infections in combination with suramin. Full recovery was observed in five monkeys in the study with no relapse of parasitemia within a 2 year follow-up. Because there is a lack of efficacious treatments for sleeping sickness in Africa and Chagas disease in South America, megazol is proposed as a potential alternative. The mutagenicity of this compound is at present being reevaluated, and metabolism is also under investigation prior to possible further developments.

Full text of DOI:10.1021/jm021030a

J . Med. Chem. 2003, 46, 427-440
427
Syn th esis a n d Biologica l Activity of Nitr o Heter ocycles An a logou s to Mega zol, a
Tr yp a n ocid a l Lea d #
Ge´rard Chauvie`re,*,† Bernard Bouteille,‡ Bertin Enanga,‡ Cristina de Albuquerque,§ Simon L. Croft,|
Michel Dumas,‡ and J acques Pe´rie´†
Groupe de Chimie Organique Biologique, Laboratoire de Synthe`se et Physicochimie de Mole´cules d’Inte´reˆt Biologique,
Universite´ Paul Sabatier, UMR CNRS 5068, 31062 Toulouse, France, Faculte´ de Me´decine, Institut d’Epide´miologie
Neurologique et de Neurologie Tropicale (EA 3174), Limoges, France, Faculty of Pharmacy, University of Sao Paulo, Brazil,
and Department of Infections and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, U.K.
Received August 27, 2002
As part of our efforts to develop new compounds aimed at the therapy of parasitic infections,
we synthesized and assayed analogues of a lead compound megazol, 5-(1-methyl-5-nitro-1H-
2-imidazolyl)-1,3,4-thiadiazol-2-amine, CAS no. 19622-55-0), in vitro. We first developed a new
route for the synthesis of megazol. Subsequently several structural changes were introduced,
including substitutions on the two rings of the basic nucleus, replacement of the thiadiazole
by an oxadiazole, replacement of the nitroimidazole part by a nitrofurane or a nitrothiophene,
and substitutions on the exocyclic nitrogen atom for evaluation of an improved import by the
glucose or the purine transporters. Assays of the series of compounds on the protozoan parasites
Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani, as either extracellular
cells or infected macrophages, indicated that megazol was more active than the derivatives.
Megazol was then evaluated on primates infected with Trypanosoma brucei gambiense,
including late-stage central nervous system infections in combination with suramin. Full
recovery was observed in five monkeys in the study with no relapse of parasitemia within a 2
year follow-up. Because there is a lack of efficacious treatments for sleeping sickness in Africa
and Chagas disease in South America, megazol is proposed as a potential alternative. The
mutagenicity of this compound is at present being reevaluated, and metabolism is also under
investigation prior to possible further developments.
In tr od u ction
melarsoprol,8 or where the required amount for an
effective treatment with difluoromethylornithine (DFMO)
is so high (400 g per head) that its use remains
restricted to hospital conditions.9 In this context, we
considered that further investigations on megazol were
justified.
Although nitro heterocycles have been used for a long
time in the treatment of Chagas disease, for example,
nifurtimox and benznidazole,1 or as radiosensitizer in
the case of misonidazole,2 no further important develop-
ments were expected from this class of compounds
because of the mutagenicity risk that they present. This
concern also holds for megazol, a 5-nitroimidazole
bearing a thiadiazole ring compound (Figure 1) that
exhibits a high curative efficacy in mice infected with
different strains of Trypanosoma cruzi3 and also in
Trypanosoma brucei infected rodents.4
However, this limitation should be reconsidered on
the following grounds. First, although mutagenic acti-
vation of megazol is obtained via a nitro reductase
present in the bacteria Salmonella typhimurium used
in the Ames test,5 this does not seem to be the case with
mammalian nitro reductases.6 Second, although the
number of infected people continues to grow by at least
25 000 new cases every year in Africa,7 the therapeutic
arsenal remains largely inadequate, relying on drugs,
either presenting an appreciable toxicity risk such, as
Several goals were pursued in the present work: (i)
to develop a new synthetic route for megazol to obtain
large amounts of compound required for pharmacologi-
cal and toxicological investigations and also for studies
on animals with possible veterinary applications; (ii) to
obtain substituted analogues for studies on the mode
of action in relation to two identified targets, oxygen
metabolism10 and an enzyme of the Krebs cycle, fuma-
rate reductase;11 (iii) to study the drug uptake into the
trypanosome through glucose12 or purine13 transporters
for which glucoconjugates and other modified structures
were synthesized. This paper describes the correspond-
ing syntheses and in vitro biological assays. Preliminary
results of in vivo studies are also given.
Resu lts a n d Discu ssion s
(1) New Syn th esis of Mega zol. This synthesis was
undertaken to increase the poor yield obtained by the
previously developed methods14 (described later in this
work for the synthesis of the 4-methyl analogue of
megazol), which requires a sealed tube reaction. The
purpose was also to find conditions suitable for a large-
scale synthesis.
#
Dedicated to Dr. Benjamin Gilbert, Head of Research at Oswaldo
Cruz Foundation in Rio de J aneiro, Brazil, who interested us in the
study of megazol.
* To whom correspondence should be addressed. PHone: 33(0)5 61
55 68 07. Fax: 33(0)5 61 55 60 11. E -mail: chauvier@cict.fr.
† Universite´ Paul Sabatier.
‡ Institut d’Epide´miologie Neurologique et de Neurologie Tropicale.
§ University of Sao Paulo.
| London School of Hygiene and Tropical Medicine.
10.1021/jm021030a CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/04/2003

428 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
Chauvie`re et al.
F igu r e 1. New procedure for megazol synthesis.
F igu r e 2. Synthesis of 4-methyl-megazol by the cyanamid procedure.
In the first step, the carbanion at position 2 of
(2.a ) Syn th esis of th e 4 Me-Mega zol. This com-
pound was synthesized following the strategy given
below (Figure 2), as in the first approach proposed for
megazol 6a :14 the 4-(5)-Me, 5-(4)-nitroimidazole 7b was
first reacted with dimethyl sulfate, leading to 8b; the
hydroxymethylation at position 2 was then obtained
with formaldehyde in DMSO using a sealed tube at 140
°C over 48 h. Oxidation of the corresponding alcohols
9a ,b into aldehydes 10a ,b was obtained by manganese
dioxide in refluxing toluene; the thiosemicarbazone
11a ,b led then to the 4-substituted compound through
an oxidative cyclization reaction in the presence of ferric
ions.
(2.b) Syn th esis of Oth er 4-Su bstitu ted Mega zol
Der iva tives. A more general strategy (Figure 3) was
developed starting from the dibromo derivative 12.15
After almost quantitative and selective methylation to
13 by action of diazomethane, the more reactive bromine
1-methylimidazole 1 was quantitatively thiomethylated
with dimethyl disulfide, giving 2. 2 was then nitrated
at 70 °C to the corresponding 5-nitroimidazole 3 (Figure
1). Oxidation by hydrogen peroxide leads nearly quan-
titatively to the sulfone 4 where a nucleophilic substitu-
tion by cyanide anion produces the corresponding
carbonitrile 5 in good yield. Finally, a condensation with
thiosemicarbazide in trifluoroacetic acid, further cy-
clization followed by loss of ammonia, and isomerization
lead to megazol 6a .
(2) Syn th esis of Mega zol An a logu es Su bstitu ted
a t P osition 4. These compounds were synthesized to
determine the specificity of the nitro reductase reducing
megazol into the corresponding radical anion and
eventually the importance of the substitution at position
4 to the half-life of the latter.

Nitro Heterocycles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3 429
F igu r e 3. Synthesis of 4-substituted analogues of megazol.
at the 2 position could be selectively substituted by a
cyano group.16 The key compound 14 then led to
different substituted derivatives. The reaction of thi-
osemicarbazide on 14 in trifluoroacetic acid led to the
formation of the bromo derivative 6c. Nucleophilic
substitution of the bromine atom in 6c allowed the
introduction of two different electron-donating groups
C5H10N and C6H5S. These correspond to soft nucleo-
philes reacting on a soft center. To complete the study,
an electron-withdrawing group CF3 could be introduced
by reacting a soft carbanion synthon of cuprous orga-
nometallic on bromoderivative 14. Further reaction of
thiosemicarbazide led to 6d .
(3.c) Rep la cem en t of th e Nitr oim id a zole P a r t by
a Nitr ofu r a n e or a Nitr oth iop h en e. The correspond-
ing syntheses are given on Figure 6. The starting
materials were aldehydes 21a and 21b, which could be
transformed into the corresponding heterocycles either
by reaction of semicarbazide or thiosemicarbazide with
oxidative cyclization of intermediate 22 or through the
cyano intermediate 24 obtained from the corresponding
oximes 23.
(3.d ) R ep la cem en t of t h e E xt r a cyclic Am in o
Gr ou p . Through a Sandmeyer reaction, this amino
group could be replaced by a chlorine atom (compound
26 in Figure 7). Nucleophilic substitution on the latter
in the presence of sodium methylate produced compound
27.
(4) Ch a n ges in Str u ctu r e in Rela tion sh ip to
P ossible Im p r ovem en t of th e Dr u g Up ta k e. Differ-
ent strategies were considered to decrease the effective
dose of the reference structure by improving the uptake
into the cell.
(4.a ) Str a tegy 1. This was done first by changing the
amphiphilic balance of the reference compound through
the amides 28a -d (Figure 8) where the nature and the
length of the chain were varied. In each case, the
recovery of the drug from the prodrug implied the action
of a peptidase. It has been shown17 that these enzymes
are abundant in the trypanosome cytosol and mi-
crosomes. In this respect also, the sensitivity of the
chain length in the prodrug to peptidases was consid-
ered.
(3) Ch a n ges on th e Tw o Heter ocyclic P a r ts of
th e Refer en ce Mega zol Str u ctu r e. To obtain insight
into the importance of the different parts of the megazol
frame, different syntheses were prepared.
(3.a ) 4-Nitr o Mega zol. This was completed according
to Figure 4 starting from N-methylimidazole 1 trans-
formed into 2-cyano-N-methylimidazole 16 by action of
cyanogen bromide. Further reaction of the thiosemicar-
bazide led to compound 17, the nitration of which
produced the corresponding 4-nitroimidazole 18.
(3.b) Rep la cem en t of th e Th ia d ia zole P a r t by a n
Oxa d ia zole Rin g. This was performed by reaction of
the cyanide ion on sulfone 4 (Figure 5). A subsequent
reaction of semicarbazide on compound 5 as above led
to oxadiazole 19. This route also allowed the straight-
forward synthesis of compound 20, the interest of which
is discussed further in the paper.

430 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
Chauvie`re et al.
F igu r e 4. Synthesis of 4-nitro isomer of megazol.
F igu r e 5. Replacement of thiadiazole ring in megazol by oxadiazole and synthesis of the 2-amino-N-methyl-5-nitroimidazole.
(4.b ) St r a t egy 2. Syn t h esis of a Glu cosa m in o
Con ju ga te. It is known18 that the African trypanosome
relies exclusively on glucose for its energy supply and
that the glucose uptake from the blood is ensured by a
transporter; this transporter has been cloned and over-
expressed.19 A similar transporter also exists in the
South American trypanosome Trypanosoma cruzi.20 To
investigate the possible improvement of megazol uptake
into the cell via these transporters, the glucosamino
conjugate 36 shown in Figure 9 was synthesized. This
reaction involves the following steps: peracetylation of
glucosamine by classical methods, introduction of a
spacer by reaction of succinic anhydride on the latter,
affording the intermediate 33, activation of the acid
function by isobutyl chloroformate, reaction on megazol,
leading to compound 35, and deprotection under cata-
lytic conditions of the acetyl groups on the glucose
moiety by sodium methylate. The overall yield of this
synthesis starting from 29 is 15%.
trypanosomiasis. The purpose was to compare the
activity of megazol to that of the reference compound
suramin and also to determine a possible effect of the
substitution at position 4.
In the second part (Table 2), the representative
compounds were assayed against intracellular parasites
on mouse peritoneal macrophages infected with either
the amastigote form of Trypanosoma cruzi or the
amastigote form of Leishmania infantum. In addition,
the assay was also performed on the bloodstream
trypomastigote form of another strain of Trypanosoma
brucei (strain S427). This gave additional information
on the ability of the corresponding compounds to cross
different membranes.
In the third part (Table 3), all the compounds were
assayed on other strains of the intracellular forms of
Trypanosoma cruzi and Leishmania donovani and the
activities of the synthesized compounds were compared
to the reference drugs sodium stibogluconate for L.
donovani and nifurtimox for Trypanosoma cruzi.
A number of features are shown in Table 1. First, a
similar efficacy of megazol compared to the reference
suramin is observed (twice in order of MEC values).
Second, it is noticeable that substitutions at position 4
(5) In Vitr o Biologica l Assa ys. Three sets of assays
were performed.
In the first part (Table 1) compounds were assayed
in vitro against the bloodstream trypomastigote form
of Trypanosoma brucei, the parasite that causes African

Nitro Heterocycles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3 431
F igu r e 6. Route to analogues of megazol with nitrofurane and nitrothiophene in place of nitroimidazole.
F igu r e 7. Substitutions of the amino group in the thiadiazole ring of megazol.
on the imidazole moiety either, by electron-donating or
withdrawing substituents, reduced or abolished activity.
Third, although compound 28a , the N-acetylated form
of megazol, behaves as a prodrug of this compound, this
was not observed with the trifluoromethyl analogue,
which had no activity.
Finally, the efficacy of the semicarbazone of nitro-
furane 22b, which had the same order of activity as
suramin, and surprisingly the inactivity of the corre-
sponding bicyclic analogue 25b were determined.
Results presented in Tables 2 and 3 show that
megazol appears to be the most active compound against
T. brucei, T. cruzi, Leishmania infantum, and L. dono-
vani with no cytotoxicity to macrophages at active
concentrations. Similar results were obtained with the
amide 28a . The enzymatic hydrolysis of this prodrug
seems to occur to a lower extent in Leishmania com-
pared to the two other parasites, since the difference
between 28a and megazol is more pronounced with the
former parasite. Other extra-ring NH2-protected com-
pounds (28b-d ) show no activity likely because the
corresponding protective groups were not hydrolyzed
under the action of intracellular peptidases. Protection
by an acetyl group as in 28a corresponds therefore to
the only possible precursor. However, this protection
does not improve the import of the drug, since the free
compound megazol can cross the two membranes of the
infected macrophages where it was as active (100%
inhibition at 1.5 µM) as against extracellular trypano-
somes (1 µM).
Structural changes on the two heterocycles showed
the following. (a) Replacement of the sulfur atom of
megazol by an oxygen (compound 19) suppresses activ-
ity, and also removing the nitro group (compound 17)

432 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
Chauvie`re et al.
F igu r e 8. Synthesis of the alkylamides of megazol.
F igu r e 9. Synthesis of a glucoconjugate of megazol.
or locating the nitro group in position 4 (compound 18)
makes compounds totally inactive. (b) Some activity
against T. brucei is conserved with sulfone 4, but this
is associated with significant cytotoxicity; other sulfones
have been shown to be active, and their activity has
been related to the inhibition of a cysteine proteinase.21
(c) Surprisingly, the open structure 22b, which combines
a nitrofurane such as in nifurtimox and a carbazide

Nitro Heterocycles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3 433
Ta ble 1. In Vitro Activity on Trypanosoma brucei of Megazol
Analogues
a nitrothiophene in place of a nitroimidazole) is also
inactive. (d) Compound 20 obtained from sulfone 4 has
been prepared to evaluate the activity of the nitroimi-
dazole part of megazol associated with the recognition
motif NH2-CdN- by the purine transporter.13 This
compound was totally inactive, which implies that the
two rings in megazol are required for activity. (e)
Suppressing the extra-cyclic amino group as in com-
pound 27 strongly reduces activity (only active at 25 µM
on T cruzi culture, with 90% inhibition). (f) The inactiv-
ity of the glucosaminoconjugate of megazol 36 suggests
that this molecule is not internalized by the glucose
transporter in T. brucei. Until now, only glucose and
fructose derivatives with minor changes have been
investigated in their affinities for this transporter22 and
little is known for complex molecules.
product
EC50, µM
MEC, µM
suramine
megazol
0.02
0.04
0.50
>41
25
0.8
0.4
6c (megazol-4-Br)
6b (megazol-4-Me)
6d (megazol-4-CF3)
4 (5-NO2-2-SO2Me
22b
3.30
>41
>34
>49
5
8
nd
25b
nd
0.20
nd
>51
1.60
31
28a (megazol(NHCOCH3))
28d (megazol(NHCOCF3))
Ta ble 2. In Vitro Assays on Different Trypanosomatidae of the
Main Analogues of Megazol
% inhibition
25
µM
12.5
µM
6.2
µM
3.1
µM
1.5
µM
The glucose transporter in T. cruzi appears to be less
specific, since an activity is noticed down to 3 µM
concentration for the glucosaminoconjugate of megazol.
product
modela
4
Tb
Tc
Li
27
0
0
0
T
T
0
0
0
0
(6) In Vivo Assa ys w ith Mega zol. (6.a ) Assa ys on
Mice. In vivo, a single injection of megazol at 20 mg
kg-1 cured mice of acute T. brucei brucei AnTat 1.9
infection. A single dose of 80 mg kg-1, given either
intraperitoneally (ip) or per os (po), was effective 48 h
after infection, when parasitemia levels were very high
and infected mice were difficult to cure. This result
confirms that of Winkelmann et al.23 who cured mouse
T. brucei infection with megazol at 25 mg kg-1 admin-
istered PO.
Ct
Tb
Tc
Li
Ct
Tb
Tc
Li
85
29
28
100
99
60
21
100
6a (megazol)
100
99
95
0
0
0
99
99
20
0
80
0
99
99
0
0
60
0
6f (4-SC6H5)
0
0
0
0
Ct
Tb
Tc
Li
40
3
0
22b
100
40
0
100
100
99
0
For subacute T. brucei brucei AnTat 1.1E infections
in mice, megazol or suramin administered alone did not
show any effect on the progression of the disease. In
contrast, a combination treatment of megazol and
suramin proved to be extremely effective. These results
confirm the interest in testing drugs in combinations.
If treatment is delayed until day 21 postinfection,
suramin is totally ineffective, since the central nervous
system (CNS) has become involved, and this drug is
poorly transported across the blood-brain barrier.24 In
such animals, the effect of suramin is to eliminate the
peripheral parasitemia followed by subsequent relapse,
indicating that parasites persist in brain tissue during
the aparasitemic period. Megazol alone, even at the
highest doses (80 mg kg-1 d-1 for 4 consecutive days,
given ip), like suramin, failed to act against the CNS
infection. On day 100 postinjection, mice displayed
several neurological signs: lack of coordination, equi-
librium problems and disorientation with circling move-
ments, altered motor function with difficulty in move-
ment, and sometimes paralysis, somnolence, and rare
convulsive fits. With a combination of suramin (20
mgkg-1 j-1 for 1 day, given ip) and megazol (80 mg kg-1
j-1 for 4 consecutive days, given ip or po), parasitemia
quickly disappeared and all animals treated showed
improvement of their general state from the first week
after treatment. One case of relapse was noted following
each mode of megazol administration. The surviving
animals had no relapses 6 months after treatment. The
results of histological examination of brain tissues
removed at various times were as follows. In untreated
mice, a severe meningitis and a perivasculitis with mild
to strong infiltration of mononuclear cells in the paren-
chymal perivascular spaces were observed. From the
second month after combined chemotherapy, the CNS
Ct
Tb
Tc
Li
Ct
Tb
Tc
Li
Ct
Tb
Tc
Li
Ct
Tb
Tc
Li
0
0
0
0
0
40
0
0
100
95
0
0
0
22c
90
40
0
0
0
89
25a
0
40
4
0
0
0
100
99
28a (NHCOCH3)
100
99
0
0
0
60
0
0
70
99
40
0
99
40
0
0
36
0
20
0
99
0
0
99
0
0
Ct
0
a
Tb ) Trypanosoma brucei; Tc ) Trypanosoma cruzi; Li )
Leishmania infantum; Ct ) cytotoxicity; T ) toxic for macrophage.
Ta ble 3. In Vitro Activity on Intracellular Forms of
Trypanosoma cruzi and Leishmania donovani of Megazol
Analogues
% inhibition
product modela 90 µM 30 µM 10 µM 3 µM ED50 ED50 ref
6a
Tc
Ld
Tc
Ld
Tc
Ld
100
99
41
0
99
20
99
75
3
99
6
0
98
20
10b
6e
11a
93
0
71
0
54
0
3.30
3.50c
a
Tb ) Trypanosoma brucei; Tc ) Trypanosoma cruzi; Ld )
Leishmania donovani. b Reference is sodium stibogluconate. c Ref-
erence is Nifurtimox.
chain, appears as active as megazol on T. brucei, but
the activity of this compound is lower against other
parasites and the cyclic analogue with oxadiazole ring
25b loses activity (see also Table 1). 25c (megazol with

434 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
Sch em e 1. Bioreduction Routes for Nitro Compounds
Chauvie`re et al.
inflammatory lesions were completely resolved and the
brain appeared histologically normal. These two animals
with relapses presented a degree of inflammatory le-
sions similar to that of untreated animals. A regression
of astrocytosis was also observed that seemed to be
correlated with the presence of inflammatory lesions in
the CNS depending on the time after treatment.25
(6.b) Assa ys on Mon k eys. The efficacy of megazol
was tested at the Centre International de Recherche
Me´dicale (Franceville, Gabon) on vervet monkeys Cer-
copithecus aethiops pygerethrus infected intravenously
with T. b. gambiense MBA (first isolated from a Zairian
patient and kindly donated by D. Le Ray, Institute of
Tropical Medicine Prince Leopold, Antwerpen, Belgique;
unpublished results). The study consisted of two groups
of animals: one group treated in the first stage of the
disease with bloodstream infection only (5 vervets) with
megazol alone (100 mg kg-1 j-1 for 1 day, given po from
18 to 53 days postinfection); one group treated during
the second stage when CNS is involved with trypano-
somes in the CSF (1 vervet, 69 days postinfection) with
megazol (100 mg kg-1 j-1 for 1 day, given po) followed
by suramin (20 mg kg-1 j-1 for 1 day, given ip).
Biological and clinical parameters returned to normal
within 2 weeks following treatment in both groups, and
the posttherapeutic followup of these animals showed
no relapse over 2 years.26
as long as the nitro group is at position 5, since the
isomer 18 with the nitro group at position 4 is totally
inactive. This point had previously been noticed for
other nitroimidazoles when substituted on the imidazole
ring at position 4.8 The decreased activity after substi-
tution on the reference structure also holds when
changes are made on the thiadiazole ring. Substitution
of sulfur by an oxygen abolishes activity; any substitu-
tion on the extra-cyclic amino group has the same
consequence, except when this substitution is acetyl.
Two targets of these nitro compounds were exam-
ined: the detoxification system, since megazol induces
an oxidative stress as shown from ESR experiments,27
and fumarate reductase, an enzyme of the Krebs cycle.11
Because this latter metabolic pathway is absent in T.
brucei,28 the efficacy of megazol via oxidative stress can
be inferred.
The oxidative stress induced by megazol suggests a
first reduction by a nitro reductase (Scheme 1). Several
reductases were assayed in in vitro tests and kcat/KM
values in the range of 2 × 102 to 5 × 103 M-1 s-1 were
found.27 Because the bimolecular rate constant for the
reaction of the nitro radical anion with oxygen, as
determined by pulse radiolysis,29 is about 5 × 106 M-1
s-1, it indicates that the entire process is controlled by
the nitro reductase step. This enzyme has not been
identified yet, but the results suggest that it should be
sensitive to any structural change, since substitution
at position 4 either abolishes or suppresses activity.
Alternatively, the difference in biological efficacy of
4-substituted derivatives may rely on the change in
structure of the compound that no longer would be
planar owing to steric interactions between the sub-
stituent at position 4 and the nitro group. As we showed
previously,30 the most efficient compound in the series,
megazol, has a planar structure, a feature that might
be a requirement for delocalization and consequently
for the optimized half-life of the nitro radical anion.
The loss of activity corresponding to the replacement
of the sulfur atom by an oxygen (megazol and compound
19) cannot be ascribed to a dramatic change in the
Discu ssion
The most salient features of this work are as follows.
First, for the synthesis of megazol, the proposed route
represents a real improvement compared to the previous
synthesis.14 The introduction of a cyano substituent at
position 2 via the sulfone 4 followed by reaction of
thiosemicarbazine on the former and cyclization under
acidic conditions makes this route more convenient than
the previous reaction in a sealed tube. Also, the sup-
pression of contaminant metal avoids environmental
problems.
Second, of the different compounds investigated,
unsubstituted megazol was the most active compound

Nitro Heterocycles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3 435
Ta ble 4. Calculated log P Values of Megazol Analogues
different heterocycles including those without nitro
substituents. The in vivo assays on mice and subse-
quently on monkeys showed that combined therapy of
megazol with suramin was highly effective in the
treatment of experimental T. brucei and T. gambiense
infections with CNS involvement.
The efficacy of treatment of experimental HAT in mice
by a combination of melarsoprol with suramin has been
reported.34 Combination therapy in this case enabled
the treatment of trypanosomiasis with CNS involve-
ment, whereas it has been shown that treatment with
either of the drugs alone is not effective at this stage of
the disease.35 So far, no treatment has been suggested
for treating advanced stages of T. brucei infections when
a high perivascular infiltration of mononuclear cells
invades the CNS associated with neurological disorders
such as those observed in patients with sleeping sick-
ness.36
Y
X
R
log P
megazol
oxadiazol
S
O
S
S
S
S
S
S
H
H
0.58
-1.17
0.56
0.50
5.34
0.38
1.72
0.83
COCH3
COCF3
CO(CH2)12CH3
CF3
C6H5S
(CH2)5N
H
H
H
Therefore, the combined therapy of sleeping sickness
at the CNS infection stage by megazol and suramin as
a nontoxic alternative to previous treatments should be
considered.
In this regard, toxicity and mutagenicity studies are
presently being performed before clinical trials are
considered.
properties of the corresponding radical anion, since ESR
studies indicated a behavior similar to that of megazol.27
But the significant change in calculated log P values (see
Table 4) indicates that the suppression of the biological
activity of this compound may be due to the lack of
import into the cell. It is indeed generally considered
that passive diffusion of compounds into cells is sup-
pressed for compounds with negative log P values.31
The substitution on the amino group indicated that
only the acetylated derivative gave activity, although
lower than that of the reference compound. This implies
that the active form has to bear a free amino group and
that the acetyl group may first have to be hydrolyzed
by cytosolic peptidases. Increasing the lipophilic char-
acter by linking an aliphatic chain (see Table 4) to this
amino group is irrelevant. The inactivity of compounds
28b and 28c indicates that the corresponding alkyl
chains are not enzymatically hydrolyzed. It was alter-
natively considered that this amino group should be free
for recognition of the compound by the purine trans-
porter. Previous work showed that adenine/adenosine
moiety and compounds such as melarsoprol are recog-
nized by their CdN-NH2 frame.13
We showed in another work using tritiated megazol
that this moiety is not necessary, since although uptake
can be via this purine transporter, a large part of the
compound is internalized via passive diffusion.32
As far as the glucose transporter is concerned, al-
though the glucosaminoconjugate of megazol 36 exhibits
a significant affinity for the glucose transporter (Ki )
0.80 mM against KM ) 0.94mM for glucose22), the
glucosamino conjugate showed no activity, possibly
because of lack of uptake. This situation bears some
similarity to results obtained with cancer cells where
glucoconjugates of different anticancer agents exhibit
a strong affinity for the glucose transporter but no
activity on the cell.33
Exp er im en ta l Section
Biologica l Assa ys. Results in Table 1 were determined at
the Institute of Neurologic Epidemiology and Tropical Neurol-
ogy of Limoges Medical Faculty, for Trypanosoma brucei brucei
AnTat 1-9 maintained in cell-free conditions as described
previously4 and in a density of 105 trypanosomes/mL. The
compounds were tested in 10-fold dilutions covering final
concentrations from 50 to 0.01 µM. Each dilution and control
was tested in 16 replicates. The plates were incubated for 48
h (37 °C, CO2 ) 5% in air). Results are expressed as either
50% effective concentration EC50 or 100% minimal effective
concentration (MEC100), the drug concentration that kills 100%
of the population within 48 h.
The results in Table 2 were obtained by Dr. L. Maes at the
J anssen Research Foundation in the Department of Parasitol-
ogy.
For trypomastigotes of Trypanosoma brucei strain S427,
they were derived from axenic culture in HMI-18 medium,
incubated at different drug levels in HMI medium in a 96-
microwell tissue culture plate. Parasite multiplication was
assessed after 3 days of incubation at 37 °C in 5% CO2. Drug
activity was semiquantatively scored as the percent reduction
of parasite multiplication compared to untreated control
cultures. Scoring was performed either microscopically or
colorimetrically.
For trypomastigotes of Trypanosoma cruzi strain Tulahen
CL2, primary mouse peritoneal macrophages were seeded in
96-well microplates at 30 000 cells/well. After 24 h, about
100 000 trypomastigotes of T. cruzi were added per well,
together with 2-fold dilutions of the drug. The cultures were
incubated at 37 °C in 5% CO2 for 4 days. To cope with the
problem of a specific toxicity, noninfected cultures were treated
likewise to enable the calculation of a selectivity index. Drug
activity was semiquantitavely scored as the percent reduction
of the total parasite load (free trypomastigotes and intracel-
lular amastigotes) compared to untreated control cultures.
Scoring was determined microscopically.
The last comment concerning in vitro assays is related
to the differences observed in the T. brucei and T. cruzi
cultures with compounds such as 6f, 36 (Table 2), and
17 (Table 3). Although no efficacy is observed in the
former, an activity was found on T. cruzi cultures. This
result is particularly significant for compound 17 de-
prived of the nitro group. This result could relate to the
observation made by Turrens et al.11 in the identifica-
tion of fumarate reductase as target in T. cruzi for
For amastigotes of Leishmania infantum (MOHM/MA BE/
67), primary mouse peritoneal macrophages were seeded in
16-well Labtek culture slides at 30 000 cells per well. After
24 h, amastigotes of L. infantum (derived from the spleen of
an infected donor animal) were added at an infection ratio of
10/1 together with 2-fold dilutions of the drug. The cultures
were incubated at 37 °C in 5% CO2 for 5 days. Treatment of

436 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
Chauvie`re et al.
uninfected control cultures was also included to determine a
selectivity index. Drug activity was semiquantitavely scored
as the percentage reduction of the total parasite load or the
number of infected macrophages in Wright stained prepara-
tions. Scoring was performed microscopically.
Cytotoxicity. MRC-5 cells were seeded in 96-well micro-
titerplates. Drugs were added in 2-fold dilutions. The cultures
were incubated at 37 °C in 5% CO2 for 7 days. Cytotoxicity
was photometrically determined as the percent reduction of
extinction after addition of MTS in treated cultures compared
to untreated control cultures.
(SCH3), 33.64 (NCH3), 133.6 (C4), 151.8 (C2) (C5, no apparent);
mass spectrum (DCI/NH3) m/z 174 (M+ + 1, 100).
1-Meth yl-2-(m eth ylsu lfon yl)-5-n itr o-1H-im id a zole (4).
To 3 (6.2 g,36 mmol) in trifluoroacetic acid (5 mL) was added
hydrogen peroxide (30%,5 mL). The mixture was stirred for 1
h at 40 °C, then cooled and neutralized with saturated
NaHCO3 aqueous solution. After extraction with CH2Cl2 (2 ×
20 mL) and the usual workup, 4 (6.6 g, 90% yield) was
obtained. 1H NMR (CDCl3) δ 3.45 (s, 3H, SO2CH3), 4.28 (s, 3H,
NCH3), 7.93 (s, 1H); 13C NMR (CDCl3) δ 35 (SO2CH3), 42.25
(NCH3), 130.4 (C4), 146.4 (C2) (C5, not apparent); mass
spectrum (DCI/NH3) m/z 206 (M+ + 1, 100), 223 (M+ + 18,
25).
The results in Table 3 were carried out in the Department
of Infectious and Tropical Diseases, London School of Hygiene
and Tropical Medicine.
1-Meth yl-5-n itr o-1H-2-im id a zoleca r bon itr ile (5). Finely
ground potassium cyanide (2.1 g,30 mmol) in DMSO (10 mL)
mixed with 4 (5.4 g, 27 mmol) was stirred at 80 °C for a half-
hour.The mixture was poured into ice-water (50 mL) and
extracted with dichloromethane (3 × 20 mL). The crude solid
obtained after evaporation was purified by flash chromatog-
raphy (petroleum ether/CH2Cl2, 1/1) giving pure 5 (3.28 g, 80%
For Leishmania donovani (MHOM/ET/67/L82), amastigotes,
derived from hamster spleen, were used to infect mouse
intraperitoneal macrophages in Labteck chamber slides. In-
fected macrophages were maintained in the presence of drugs
in the range 90-3 µM in quadruplicate cultures for 7 days at
37 °C. Drug activity was measured from a percentage of
macrophages cleared of amastigotes in treated cultures. Pen-
tostam (sodium stibogluconate) was used as a positive control.
For trypomastigotes of Trypanosoma cruzi (MHOM/BR/OO/
Y) derived from rat, L6 myoblast cultures were used to infect
mouse peritoneal macrophages in medium containing drugs
from 90 to 3 µM for 72 h at 37 °C. Drug activity was
determined from the percent of infected macrophages in
treated cultures. Nifurtimox was used as a positive control.
The standard deviation for all results given in Tables 1-3
is (3%.
yield). 1H NMR (CDCl3) δ 4.19 (s, 3H, NCH3), 8.03 (s, 1H); 13
C
NMR (CDCl3) δ 35.7 (NCH3), 109.2 (CN), 125 (C2), 132 (C4)
(C5, not apparent); IR 2246 cm-1 (CN); mass spectrum (DCI/
NH3) m/z 153 (M+ + 1, 100), 170 (M+ + 18, 40).
5-(1-Meth yl-5-n itr o-1H-2-im id a zolyl)-1,3,4-th ia d ia zol-
2-a m in e (6a ). Mega zol (Or igin a l Syn th esis). Thiosemicar-
bazide (2 g,22 mmol) mixed with 5 (3.28 g,22 mmol) was heated
in trifluoroacetic acid (10 mL) at 60 °C for 15 h. The reaction
medium was poured into ice-water (20 mL) and neutralized
with saturated NaHCO3 aqueous solution. The yellow precipi-
tate formed was filtered on a sintered glass filter, washed with
water (2 × 5 mL), and dried under vacuum to give 6a after
Syn th esis. Melting points were determined on an Electro-
thermal capillary melting point apparatus and were uncor-
rected.
1
recrystallization in acetone (2.5 g, 50% yield). Mp 270 °C; H
1H and 13C NMR spectra were recorded respectively on
Brucker AC80 (80 MHz) and AC200 (50 MHz) spectrometers.
All chemical shifts are reported in ppm relative to TMS as
internal standard. Mass spectra were run on a Nermag R1010
spectrometer at Paul Sabatier University, Toulouse, France.
All chemicals were purchased from Aldrich or Fluka and were
used without additional purification.
DMF and DMSO were dried by stirring over 4 Å molecular
sieves. THF was distilled over sodium in the presence of the
benzophenone ketyl as indicator prior to use.
NMR (DMSO-d6) δ 4.32 (s, 3H, NCH3), 7.8 (broad (b), 2H, NH2),
8.2 (s, 1H); 13C NMR (DMSO-d6) δ 35.00 (s, NCH3), 133.1 (C4),
140.1 (C5), 141.4 (C2), 48.2 (C2*), 169.9 (C5*); mass spectrum
(EI) m/z 227 (M+ + 1, 80). Anal. (C6H6N6O2S) C, H, N.
5-(1,4-Dim eth yl-5-n itr o-1H-2-im id a zolyl)-1,3,4-th ia d ia -
zol-2-a m in e (6b). 11b (2.1 g, 8.8 mmol) and ammonium iron-
(III) sulfate (17.35 g, 36 mmol) were stirred in refluxing water
(200 mL) for 5 h. The solid formed was filtered and washed
with water. 6b was obtained (1.3 g, 60% yield) after recrys-
1
tallization (acetone/DMF, 1:1). Mp 285 °C; H NMR (DMSO-
d6) δ 2.09 (s, 3H, CH3), 4.29 (s, 3H, NCH3), 7.82 (s, 2H, NH2);
13CNMR (DMSO-d6) δ 15.75 (CH3), 35.37 (NCH3), 136.77 (C4),
139.20 (C2), 143.93 (C2*), 148.09 (C5), 169.9 (C5*); mass
spectrum (EI) m/z 241 (M+ + 1). Anal. (C7H8N6O2S) C, H, N.
4-Meth yl-5-n itr o-1H-im id a zole (7b). To a mixture of
anhydrous nitric acid (8 mL) and concentrated sulfuric acid
(8 mL) was added at 0 °C 4-methylimidazole (3.2 g, 38 mmol).
After 30 min at room temperature, the mixture was poured
onto ice. After neutralization with sodium hydroxide, the
yellow precipitate formed was filtered,washed with cold water
(10 mL), recrystallized in water, and dried under vacuum. An
amount of 4.4 g of 7b was obtained (90% yield). Mp 260 °C;
1HNMR (acetone-d6) δ 2.69 (s, 3H, CH3), 7.59 (s, 1H), 10.13
(s, 1H); 13C NMR (acetone-d6) δ 11.6 (CH3), 133.7 (C2), 139.9
(C4) (C5, not apparent); mass spectrum (DCI/NH3) m/z 128
(M+ + 1, 100)
1-Meth yl-5-n itr o-1H-im idazole (8a). 4-Nitroimidazole (5.4
g, 48 mmol) and dimethyl sulfate (5.3 mL) were heated in
refluxing dioxane (20 mL) for 2 h. The solvent was then
evaporated under vacuum, and the oily acidic residue was
neutralized by a NaHCO3 saturated aqueous solution. The
solid formed and the aqueous solution were extracted with
dichloromethane (4 × 20 mL). After the mixture was dried
with magnesium sulfate and after evaporation of the organic
phase, crude 8a was obtained (3.8 g, 62% yield) and used
without further purification. Mp 60 °C; 1H NMR (CDCl3) δ 3.93
(s, 3H, NCH3), 7.5 (s, 1H), 7.87 (s, 1H); 13C NMR (CDCl3) δ
35.2 (NCH3), 133.07 (C4), 141.7 (C2) (C5, not apparent). Anal.
(C4H5N3O2) C, H, N.
Reactions and column chromatographic separations were
followed by thin-layer chromatography using silica gel (with
254 nm fluorescent indicator) on alumina plates.
1-Meth yl-2-(m eth ylsu lfa n yl)-1H-im id a zole (2). Butyl-
lithium 1.6 M (55 mL) was slowly added (10 min) by syringe
through a septum cap to a 1-methylimidazole (6.56 g, 80 mmol)
solution in THF (60 mL) at -30 °C. After 10 min at this
temperature, dimethyl disulfide was added by syringe (9 mL,
90 mmol). The yellow, creamy precipitate formed was mag-
netically agitated for an hour at room temperature. After
vacuum evaporation of THF, water (30 mL) was added to the
residue, which was extracted by dichloromethane (3 × 50 mL).
After the mixture was dried with magnesium sulfate and
evaporation, 2 was obtained as a crude oil (10.5 g, 100% yield)
and nitrated directly without further purification. 1H NMR-
(CDCl3) δ 2.53 (s,3H,SCH3), 3.53 (s,3H,NCH3), 6.84 (d,1H,J )
1.5 Hz), 6.98 (d,1H,J ) 1.5 Hz); 13C NMR (CDCl3) δ 16.1
(SCH3), 32.8 (NCH3), 122.0 (C5), 128.7 (C4), 142.8 (C2); mass
spectrum (DCI/NH3) m/z 129 (M+ + 1, 100), 146 (M+ + 18,
30).
1-Meth yl-2-(m eth ylsu lfa n yl)-5-n itr o-1H-im id a zole (3).
2 (10.5 g, 80 mmol) was added slowly by dropping funnel to
69% nitric acid (30 mL) at 70 °C, and the mixture was
maintained for 1 h. The mixture was then poured in cold water
and neutralized with saturated NaHCO3 aqueous solution. The
yellow precipitate formed was extracted with dichloromethane
(3 × 30 mL). The organic phase dried with magnesium sulfate
gave, after evaporation, a yellow solid purified by flash
chromatograghy on silica gel (CH2Cl2), giving pure 3 (6.2 g,
45% yield). Mp 285 °C; 1H NMR (CDCl3) δ 2.71 (s, 3H, SCH3),
3.85 (s, 3H, NCH3), 7.97 (s, 1H); 13C NMR (CDCl3) δ 14.4
1,4-Dim eth yl-5-n itr o-1H-im id a zole (8b). Starting from
7b (4.4 g, 31 mmol), the same procedure as for 8a was used

Nitro Heterocycles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3 437
and 8b was obtained (2.65 g, 60% yield). Mp 210 °C; 1H NMR
(CDCl3) δ 2.58 (s, 3H, CH3), 3.94 (s, 3H, NCH3), 7.44 (s, 1H);
13C NMR (CDCl3) δ 16.24 (CH3), 36.2 (NCH3), 139.5 (C2), 143.9
(C5), 145.15 (C4); mass spectrum (DCI/NH3) m/z 142 (M+ + 1,
100).
(C5), 178.3 (CdS); mass spectrum (EI) m/z 243 (M+ + 1, 100).
Anal. (C7H10N6O2S) C, H, N.
2,4-Dibr om o-5-n itr o-1H-im id a zole (12). 12 was prepared
as described in ref 37 by adding bromine to 5-nitroimidazole.
13C NMR (DMSO-d6) δ 126.1 (C4), 120.0 (C2) (C5 not appar-
ent).
(1-Meth yl-5-n itr o-1H-2-im id a zolyl)m eth a n ol (9a ). In a
sealed glass tube, paraformaldehyde (3.6 g, 120 mmol) and 8a
(3.8 g, 28 mmol) in DMSO solution (20 mL) were introduced.
The tube was heated at 140 °C for 48 h. Subsequently the
solution was poured into water (200 mL) and extracted with
ethyl acetate (3 × 50 mL). After the mixture was dried (MgSO4)
and after evaporation of the solvent under vacuum, the solid
was recrystallized (THF/heptane, 1:1) and 9a was isolated
2,4-Dibr om o-1-m eth yl-5-n itr o-1H-im idazole (13). 12 (2.7
g,10 mmol) in ether (20 mL) was added to diazomethane (12
mmol) in ether solution at 20 °C and left for 1 h at room
temperature. After the mixture was dried (magnesium sulfate)
and after evaporation of ether, 13 was obtained (2.7 g, 95%
yield) as a yellow solid that was used without further purifica-
tion. Mp 160 °C; 1H NMR (CDCl3) δ 4.0 (s,CH3); 13C NMR
(CDCl3) δ 37.6 (NCH3), 119.6 (C4), 126.0 (C2), 137.1 (C5); mass
spectrum (DCI/NH3) m/z 284 (M+ + 1, 52), 286 (M+ + 3, 100),
288 (M+ + 5, 50).
4-Br om o-1-m e t h yl-5-n it r o-1H -2-im id a zole ca r b on i-
tr ile (14). 13 (2.85 g, 10 mmol) was mixed with finely ground
potassium cyanide (0.7 g, 11 mmol) in DMSO (10 mL) and
heated for 2 h at 80 °C. The solution was poured into water
(100 mL) and extracted with dichloromethane (3 × 20 mL).
The solid obtained after drying (MgSO4) the organic phase and
after solvent evaporation was purified by flash chromatogra-
phy (ethyl acetate/hexane, 3:1) to give pure 14 (2 g, 85% yield).
Mp 101 °C; IR 2247 cm-1 (CN); 1H NMR (CDCl3) δ 4.18 (s,
NCH3); 13CNMR (CDCl3) δ 37.3 (NCH3), 108.2 (CN), 120.1 (C4),
124.0 (C2), 137.1 (C5); mass spectrum (EI) m/z 230 (M+, 20),
232 (M+ + 2, 20). Anal. (C5H3BrN4O2) C, H, N.
1
(2.63 g, 60% yield). Mp 110 °C; H NMR (DMSO-d6) δ 3.9 (s,
3H, NCH3), 4.5 (d, 2H, CH2, J ) 6 Hz), 5.6 (t, 1H, OH, J ) 6
Hz), 7.9 (s, 1H); 13C NMR (DMSO-d6) δ 33.2 (NCH3), 56 (CH2),
131.4 (C4), 139.1 (C5), 152.3 (C2). Anal. (C5H7N3O3) C, H, N.
(1,4-Dim eth yl-5-n itr o-1H-2-im id a zolyl)m eth a n ol (9b).
Starting with 8b (2.65 g, 18.6 mmol), the same procedure as
for 9a was applied with paraformaldehyde (2.4 g,80 mmol),
and 9b was obtained (2.20 g, 70% yield). Mp 110 °C; 1H NMR
(DMSO-d6) δ 2.4 (s,3H,CH3), 3.87 (s, 3H, NCH3), 4.53 (d, 2H,
CH2, J ) 6 Hz), 5.6 (t, 1H, OH, J ) 6 Hz); 13C NMR (DMSO-
d6) δ 15.53 (CH3), 33.63 (NCH3), 55.57 (CH2OH), 140.6 (C2),
142.6 (C5), 149.93 (C4). Anal. (C6H9N3O3) C, H, N.
1-Meth yl-5-n itr o-1H-2-im id a zolca r ba ld eh yd e (10a ). 9a
(2.6 g,17 mmol) in toluene (50 mL) and activated manganese
dioxide (7.2 g, 85 mmol) were refluxed for 4 h. After filtration,
the mixture was evaporated. The residue was purified by flash
chromatography (CH2Cl2) and gave 10a (2.1 g, 80% yield). Mp
86 °C; 1H NMR (CDCl3) δ 4.4 (s, 3H, NCH3), 8.1 (s,1H), 9.9 (s,
1H, CHO); 13C NMR (CDCl3) δ 34.3 (NCH3), 132.7 (C4), 143.1
(C2), 163.7 (CHO) (C5, not apparent). Anal. (C5H5 N3O3) C,
H, N.
1-Met h yl-5-n it r o-4-(t r iflu or om et h yl)-1H -2-im id a zole-
ca r bon itr ile (15). 15 was synthesized from 14 with “CF3Cu”
generated by the Burton process as previously described.15
5-(4-Br om o-1-m eth yl-5-n itr o-1H-2-im id a zolyl)-1,3,4-th i-
a d ia zol-2-a m in e (6c). 6c was prepared from 14 with thi-
osemicarbazide in trifluoroacetic acid, similar to the prepara-
tion of 6a from 5, described above.
1,4-Dim eth yl-5-n itr o-1H-2-im idazolcar baldeh yde (10b).
9b (2.20 g, 13 mmol) in toluene (50 mL) was refluxed with
manganese dioxide (5.6 g, 65 mmol) for 3 h. After filtration of
the manganese salt, toluene evaporation, and recrystallization
(hexane), 10b was obtained (1.43 g, 80% yield). Mp 115 °C;
1H NMR (CDCl3) δ 2.62 (s, 3H, CH3), 4.3 (s, NCH3), 9.89 (s,
1H, CHO); 13C NMR (acetone-d6) δ 15.93 (CH3), 34.63 (NCH3),
130.04 (C4), 132.2 (C2), 183.65 (CHO) (C5, not apparent). Anal.
(C6H7N3O3) C, H, N.
5-[-1-Met h yl-5-n it r o-4-(t r iflu or om et h yl)-1H -2-im id a -
zolyl]-1,3,4-th ia d ia zol-2-a m in e (6d ). 6d was prepared from
15, similar to preparation described for 6c previously.15
5-(-1-Meth yl-5-n itr o-4-piper idin o-1H-2-im idazolyl)-1,3,4-
th ia d ia zol-2-a m in e (6e). 6e was synthesized from 6c with
piperidine in the presence of sodium methylate, as previously
described.15
5-[-1-Meth yl-5-n itr o-4-(ph en ylsu lfan yl)-1H-2-im idazolyl]-
1,3,4-th ia d ia zol-2-a m in e (6f). 6f was synthesized from 6c
with thiophenol, as previously described.15
1-Meth yl-1H-2-im id a zoleca r bon itr ile (16). 4-N,N-Dim-
ethylaminopyridine (6.1 g, 50 mmol) and cyanogen bromide
(5.3 g, 50 mmol) in DMF (20 mL) under argon at room
temperature generated a yellow precipitate of the cyanoami-
nopyridinium salt to which was added by syringe 1-methylimi-
dazole (1.7 mL, 20 mmol). After 15 h of being stirred, the
mixture was poured into a NaHCO3 saturated solution (100
mL) and extracted with dichloromethane (4 × 25 mL). After
evaporation of the organic phase and flash chromatography
(CH2Cl2), 16 as a brown oil (1.1 g, 50% yield) was obtained.
1H NMR (CDCl3) δ 3.85 (s, 3H, NCH3), 7.06 (d, 1H, H5, J )
1.5 Hz), 7.16 (d, 1H, H4, J ) 1.5 Hz); 13C NMR (CDCl3) δ 34.1
(CH3N), 110.9 (CN), 124.4 (C5), 131.6 (C4) (C2 not apparent);
IR 2235 cm-1 (CN); mass spectrum (EI) m/z 108 (M+ + 1, 20).
5-(1-M e t h y l-1H -2-i m i d a z o ly l)-1,3,4-t h i a d i a z o l-2-
a m in e (17). 16 (0.55 g, 5 mmol) mixed with thiosemicarbazide
(0.5 g, 5 mmol) and heated at 60 °C in trifluoroacetic acid (5
mL) gave 17 (0.55 g, 60% yield) after similar treatment of the
reaction mixture as above for 6a -f . 1H NMR (DMSO-d6) δ
3.96 (s, 3H, NCH3), 6.00 (d, 1H, J ) 1.5 Hz), 7.3 (d, 1H, J )
1.5 Hz); 13C NMR (DMSO-d6) δ 34.7 (NCH3), 124.7 (C5), 128.4
(C4), 137.7 (C2), 149.8 (C2′), 168.2 (C5′); mass spectrum (EI)
m/z 182 (M+ + 1, 20). Anal. (C6H7N5S) C, H, N.
2-[(E)-1-(1-Meth yl-5-n itr o-1H-2-im idazolyl)m eth yliden e]-
1-h yd r a zin eca r both ioa m id e (11a ). 10a (2g, 13 mmol) and
thiosemicarbazide (1.42 g, 15 mmol) were stirred in DMSO
(10 mL) at room temperature for 16 h. The mixture was poured
into water (50 mL) and a yellow precipitate was formed,
filtered, washed with dichloromethane, and recrystallized
(ethyl acetate/acetone, 1:1) to afford 11a (2.6 g, 11 mmol). Mp
240 °C; 1H NMR (DMSO-d6) δ 4.15 (s, 3H, NCH3), 8.11 (s, 1H,
CHdN), 7.75-8.54 (b, 2H, NH2), 8.2 (s, 1H, H4), 11.8 (s, 1H,
NH). In (DMSO-d6/D2O), signals 7.15-8.54 (b, 2H, NH2) and
11.8 (s, 1H, NH) disappear. 13C NMR (DMSO-d6) δ 35.07
(NCH3),133.3 (CHdN), 133.4 (C4), 140.2 (C5), 144.6 (C2), 178.4
(CdS); mass spectrum (EI) m/z 229 (M+ + 1, 80). Anal.
(C6H8N6O2S) C, H, N.
5-(1-Meth yl-5-n itr o-1H-2-im id a zolyl)-1,3,4-th ia d ia zol-
2-a m in e (6a ). Mega zol (14). 11a (2.6 g, 11 mmol) and
ammonium iron(III) sulfate (21g, 44 mmol) in water (200 mL)
were stirred and refluxed for 5 h. The solid formed was filtered,
washed with water, and gave 6a (1.1 g, 40% yield). Analytical
data are given above.
2-[(E)-1-(1,4-Dim et h yl-5-n it r o-1H -2-im id a zolyl)m et h -
ylid en e]-1-h yd r a zin eca r both ioa m id e (11b). The procedure
was similar to that for 11a , using 10b (1.43 g,11 mmol) and
thiosemicarbazide (1.14 g, 12 mmol) in DMSO (10 mL). After
16 h of reaction, hydrolysis, filtration, and recrystallization
(ethanol), 11b was obtained (2.1 g, 80% yield). Mp 255 °C; 1H
NMR (DMSO-d6) δ 2.5 (s, 3H, CH3), 3.35 (b, 2H, NH2), 4.11 (s,
3H, NCH3), 8.08 (s, 1H, CHdN), 11.8 (s, 1H, NH). The 3.35
and 11.8 signals disappear after adding D2O. 13C NMR (DMSO-
d6) δ 15.62 (CH3), 35.45 (NCH3), 133.17 (C4), 142.56 (C2), 143.9
5-(1-Meth yl-4-n itr o-1H-2-im id a zolyl)-1,3,4-th ia d ia zol-
2-a m in e (18). 17 (0.36 g, 2 mmol) was added to a sulfonitric
solution (2 mL of H2SO4 (conc), 2 mL of anhydrous HNO3) at
0 °C. After half an hour, the mixture was poured into ice (5
mL). The yellow precipitate formed was filtered, washed with
water (5 mL), and dried under vacuum, giving 18 (0.2 g, 40%

438 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
Chauvie`re et al.
1
yield). Mp 265 °C; H NMR (DMSO-d6) δ 4.39 (s, 3H, NCH3),
under vacuum to give 25a (275 mg, 65% yield). 1H NMR
(DMSO-d6) δ 7.22 (d, 1H, H4, J ) 5 Hz), 7.72 (d, 1H, H3, J )
5 Hz), 7.77 (b, NH2); 13C NMR (DMSO-d6) δ 111.6 (C4), 114.9
(C3), 144.7 (C5), 147.6 (C2), 151.2 (C′2), 170.0 (C′5); mass
spectrum (EI) m/z 212 (M+, 10). Anal. (C6H4N4O3S) C, H, N.
7.2 (NH2), 8.21 (s,1H); 13C NMR (DMSO-d6) δ 35.0 (NCH3),
133.3 (C5), 140.2 (C4), 142.4 (C2), 153.5 (C2′), 171.5 (C5′); mass
spectrum (EI) m/z 227 (M+ + 1, 10). Anal. (C6H6N6O2S) C, H,
N.
5-(1-Meth yl-5-n itr o-1H-2-im id a zolyl)-1,3,4-oxa d ia zol-2-
a m in e (19). 1-Methyl-2-cyano-5-nitroimidazole 5 (600 mg, 4
mmol) and semicarbazide hydrochloride (500 mg, 4.5 mmol)
were mixed in 10 mL of trifluoroacetic acid and refluxed at 70
°C for 15 h. Then the solution was poured into ice-water (10
mL) and carefully neutralized with NaHCO3 saturated solu-
tion. The yellow solid formed was filtered and washed with
ether and water. After crystallization (ethanol/water, 2:1), 19
was obtained (420 mg, 50% yield). Mp 285 °C; 1H NMR
(DMSO-d6) δ 4.25 (s,NCH3), 7.94 (s,NH2), 8.11 (s,H4); 13C NMR
(DMSO-d6) δ 34.53 (NCH3), 130.65 (C4), 140.05 (C5), 141.34
(C2), 157.0 (C2′), 159.77 (C5′). Anal. (C6H6N6O3) C, H, N.
1-Meth yl-5-n itr o-1H-2-a m in oim id a zole (20). The sulfone
4 (205 mg, 1 mmol) was mixed at -70 °C with gaseous
ammonia condensed in the reaction flask by a Dewar con-
denser. After 2 h, the medium was warmed to room temper-
ature. After ammonia evaporation, the yellow solid formed was
extracted with ethyl acetate (2 × 10 mL), dried (MgSO4),
recovered after solvent evaporation, and purified by flash
chromatography (CH2Cl2/CH3OH, 90:10), and 20 was obtained
5-(5-Nitr o-2-fu r yl)-1,3,4-oxa d ia zol-2-a m in e (25b). 5-Ni-
tro-2-furyl cyanide 24a (700 mg, 5 mmol) and semicarbazide
hydrochloride (560 mg, 5 mmol) were stirred in refluxed
trifluoroacetic acid (20 mL) for 15 h. The cold solution (0 °C)
was then carefully neutralized with
a NaHCO3 aqueous
solution. The orange solid obtained was filtered, washed with
water, and dried in a vacuum, giving 25b (450 mg, 45% yield).
1H NMR (DMSO-d6) δ 6.33 (s, NH2), 7.30 (d, H4), 7.80 (d, H3);
13C NMR (DMSO-d6) δ 111.3 (C3), 114.9 (C4), 133.5 (C2), 140.6
(C5), 150.8 (C′2), 157.0 (C′5); mass spectrum (DCI/NH3) m/z
231 (M+ + 2NH3, 100).
5-(5-Nitr o-2-th ien yl)-1,3,4-th ia d ia zol-2-a m in e (25c). 22c
(1.09 g, 4.4 mmol) mixed in water (50 mL) with ferric chloride
hexahydrate (5.4 g, 20 mmol) was refluxed for 4 h. The yellow
precipitate formed was filtered, washed with water, and dried
under vacuum to give 25c (0.90 g, 85% yield). Mp 245 °C; 13
C
NMR (DMSO-d6) δ 126.4 (C3), 130.6 (C4), 140.5 (C5), 148.6
(C2), 149.7 (C5′), 170.5 (C2′); mass spectrum (DCI/NH3) m/z
229 (M+ + 1, 70), 246 (M+ + 18, 100), 263 (M+ + 34, 40).
2-Ch lor o-5-(1-Met h yl-4-n it r o-1H -2-im id a zolyl)-1,3,4-
th ia d ia zole (26). To megazol (6a ) (230 mg,1 mmol) dissolved
in a mixture of concentrated hydrochloric acid and acetic acid
(1:5) was added copper powder (10 mg) and sodium nitrite (10
mmol). After 30 min of being stirred at 0 °C, the reaction
medium was left at room temperature for 15 h and then poured
into water (20 mL) and extracted with dichloromethane (3 ×
20 mL). After being dried (MgSO4) and after evaporation of
the organic phase, the residue was recrystallized (THF/
1
(60 mg, 40% yield). Mp 205 °C; H NMR (CH3OD) δ 3.71 (s,
3H, NCH3), 7.8 (1H, H4); mass spectrum (DCI/NH3) m/z 143
(M+ + 1, 100).
2-[(E)-1-(5-Nitr o-2-fu r yl)m eth ylid en e]-1-h yd r a zin eca r -
both ioa m id e (22a ). Thiosemicarbazide (455 mg, 5 mmol)
mixed with 5-nitrofurane-2-carboxaldehyde 21a (760 mg, 5
mmol) was stirred for 15 h at room temperature in DMSO (10
mL). After the same workup as that described for 11a , 22a
was obtained (950 mg, 90% yield). 1H NMR (DMSO-d6) δ 7.24
(b, NH), 7.67-7.68 (m, 2H), 7.9 (b, NH2), 11.82 (s, 1H, CHd
N); 13C NMR (DMSO-d6) δ 113.7 (C4), 114.8 (C3), 130.2 (CHd
N), 151.5 (C5), 151.9 (C2), 176.2 (CSNH2); mass spectrum (EI)
m/z 214 (M+). Anal. (C6H6N4O3S) C, H, N.
1
heptane, 1:2) to give 26 (200 mg, 80% yield). Mp 135 °C; H
NMR (DMSO-d6) δ 4.4 (s,NCH3), 8.30 (s,H4); 13C NMR (DMSO-
d6) δ 35.3 (NCH3), 133.0 (C4), 139.3 (C5), 140.9 (C2), 155.8
(C′2), 162.5 (C′5). Anal. (C6H4N5O2SCl) C, H, N.
2-Meth oxy-5-(1-Meth yl-5-n itr o-1H-2-im id a zolyl)-1,3,4-
th ia d ia zole (27). 26 (250 mg, 1 mmol) was stirred with
sodium methylate (210 mg, 4 mmol) in methanol (10 mL) for
4 h at room temperature. After evaporation, the residue was
extracted with ethyl acetate and purified by column chroma-
tography (CH2Cl2/CH3OH, 95:5). 27 was obtained as an oil (130
mg, 55% yield). 1H NMR (CDCl3) δ 4.25 (s, 3H, NCH3), 4.48
(s, 3H, OCH3), 8.00 (1H, H4); 13C NMR (DMSO-d6) δ 35.56
(NCH3), 60.32 (OCH3), 133.0 (C4), 135.78 (C2), 140.9 (C5),
154.6 (C′2), 177.1 (C′5). Anal. (C7H7N5O3S) C, H, N.
N1-[5-(1-Meth yl-5-n itr o-1H-2-im id a zolyl)-1,3,4-th ia d ia -
zol-2-yl]a ceta m id e (28a ). Acetyl chloride (0.8 mL, 10 mmol)
was added, with syringe through a septum cap, to 6a (0.8 g,
3.5 mmol) in THF (20 mL) under nitrogen at room tempera-
ture. After 3 h, the precipitate formed was filtered and
recrystallized (ethanol/acetone, 2:1), giving 28a (0.72 g, 75%
yield). 1H NMR (DMSO-d6) δ 2.24 (s, 3H, COCH3), 4.39 (s, 3H,
NCH3), 8.25 (s, 1H, H4); 13C NMR (DMSO-d6) δ 22.33 (CH3-
CO), 35.23 (CH3N), 133.09 (C4), 140.6 (C2), 141.04 (C5), 153.93
(CONH), 159.74 (C2′), 169.17 (C5′); mass spectrum (EI) m/z
269 (M+ + 1, 10). Anal. (C8H8N6O3S) C, H, N.
N1-[5-(1-Meth yl-5-n itr o-1H-2-im id a zolyl)-1,3,4-th ia d ia -
zol-2-yl]n on a m id e (28b). From 6a (0.225 g,1 mmol) with
nonanoyl chloride (1 mL, 5 mmol) in THF (20 mL), the same
procedure as for 28a was used during 3 h. 28b recrystallized
(ethanol) in 60% yield was obtained. Mp 257 °C; 1H NMR
(DMSO-d6) δ 0.85 (s, 3H, CH3), 1.25 (m, 14H, (CH2)7), 4.39 (s,
3H, NCH3), 8.26 (s, 1H, H4); 13C NMR (DMSO-d6) δ 13.6 (CH3),
21.9-34.8 ((CH2)7), 35.2 (NCH3), 133.1 (C4), 140.5 (C2) (C5
absent), 153.8 (CONH), 159.9 (C′2), 172.2 (C′5); mass spectrum
(DCI/NH3) m/z 367 (M+ + 1, 100), 384 (M+ + 18, 15). Anal.
(C15H22N6O3S) C, H, N.
N1-[5-(1-Meth yl-5-n itr o-1H-2-im id a zolyl)-1,3,4-th ia d ia -
zol-2-yl]tetr a d eca m id e (28c). The same procedure as for 28a
but with 6a (1 mmol) and myristoyl chloride (5 mmol) in THF
gave 28c in 60% yield. Mp 247 °C; 1H NMR (DMSO-d6) δ 0.84
(s, 3H, CH3), 1.23 (m, 24H, (CH2)12), 4.39 (s, 3H, NCH3), 8.27
2-[(E)-1-(5-Nitr o-2-fu r yl)m eth ylid en e]-1-h yd r a zin eca r -
boxa m id e (22b). Semicarbazide hydrochloride (444 mg, 4
mmol) dissolved in DMSO (5 mL) was added to sodium hydride
(110 mg,4 mmol) suspended in DMSO (3 mL). After 5 min
when all hydrogen bubbling had ceased, 5-nitrofurane-2-
carboxaldehyde (570 mg, 4 mmol) diluted in DMSO (2 mL)
was added. After 18 h at room temperature, the brown solution
was poured into water (50 mL) and a dark precipitate was
filtered and purified after dissolution in acetone by flash
chromatography (CH2Cl2/methanol, 90:10) to give 22c (400 mg,
1
55% yield). H NMR (DMSO-d6) δ 6.57 (s, NH), 7.22 (d, H4),
7.77 (d, H3), 10.76 (s, CHdN); 13C NMR (DMSO-d6) δ 112.0
(C4), 115.1 (C3), 127.3 (CHdN), 151.1 (C2), 153.0 (C5), 155.8
(CONH2); mass spectrum (DCI/NH3) m/z 216 (M+ + 18, 100).
2-[(E)-1-(5-Nitr o-2-th ien yl)m eth ylid en e]-1-h yd r a zin e-
ca r both ioa m id e (22c). 21b (0.830 g, 5.3 mmol) and thi-
osemicarbazide (0.48 g, 5.4 mmol) in DMSO (5 mL) were
stirred at room temperature for 18 h. The mixture was poured
into water (20 mL), and the formed precipitate was filtered,
washed with water, and dried under vacuum, leading to 22b
(1.09 g, 90% yield). 1H NMR (DMSO-d6) δ 7.5 (d,1H), 8 (d,-
1H), 11.75 (s,NH2); 13C NMR (DMSO-d6) δ 129 (CdN), 130.2
(C3), 135.2 (C4), 146.6 (C2), 150.6 (C5), 178.1 (CdS). Anal.
(C6H6N4O2S2) C, H, N.
5-Nitr o-2-fu r yl Cya n id e (24a ). Nitrofuroxime 23a , from
Aldrich (2 g, 12 mmol), was refluxed in acetic anhydride (50
mL) at 140 °C for 3 h. After evaporation of the solvent under
vacuum, the oily residue was recrystallized (hexane/ethyl
acetate, 3:1), giving 24a (1.5 g, 90% yield). 1H NMR (CDCl3) δ
7.29 (d, 1H), 7.38 (d, 1H); 13C NMR (CDCl3) δ 109.1 (CN), 111.2
(C3), 123.8 (C4), 127.0 (C2), 144.4 (C5); mass spectrum (EI)
m/z 138 (M+, 35).
5-(5-Nitr o-2-fu r yl)-1,3,4-th ia d ia zol-2-a m in e (25a ). 22a
(425 mg, 2 mmol) mixed in water (20 mL) with ferric chloride
hexahydrate (2.7 g, 10 mmol) was refluxed for 4 h. The yellow
precipitate formed was filtered, washed with water, and dried

Nitro Heterocycles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3 439
(s, 1H, H4); mass spectrum (DCI/NH3) m/z: 437 (M+ + 1, 100),
454 (M+ + 18,5). Anal. (C20H32N6O3S) C, H, N.
4.37 (s, 3H, NCH3), 4.70-5.30 (m, 2H, H3, H4), 5.67-5.76 (m,
1H, H2), 7.98 (d, 1H, J ) 7 Hz, H1), 8.26 (s, 1H, C4); 13C NMR
(DMSO-d6) δ 20.21-20.36 (CH3COO), 20.53 (CH2), 33.99.34.46
(CH2CH2), 35.13 (NCH3), 51.77 (C2′), 61.39 (C6′), 67.99 (C5′),
71.48 (C5′), 71.48 (C3′), 72.07 (C4′), 91.64 (C1′), 132.95 (C4),
140.46 (C5), 141.09 (C2), 153.62 (C2*), 159.77 (C5*), 166.59-
169.75 (OOCCH3), 171.46-171.74 (CONH); mass spectrum
(DCI/NH3) m/z 670 (M+ + 1, 20). Anal. (C25H31N7O13S) C, H,
N.
N1-[5-(1-Meth yl-5-n itr o-1H-2-im id a zolyl)-1,3,4-th ia d ia -
zol-2-yl]-2,2,2,tr iflu or oa ceta m id e (28d ). To 6a (226 mg,1
mmol) suspended in THF (5 mL) was added trifluoroacetic
anhydride (2 mL), Instantaneously the suspension was dis-
solved. After 5 min the THF was evaporated under vacuum.
The oily residue neutralized with an aqueous solution of
NaHCO3 gave a yellow precipitate of 28d , which was washed
with water and dried under vacuum (300 mg, 90% yield). 13C
N1-[5-(1-Meth yl-5-n itr o-1H-2-im id a zolyl)-1,3,4-th ia d ia -
zol-2-yl]-N5-[2,4,5-tr ih yd r oxy-6-(h yd r oxym eth yl)tetr a h y-
d r o-2H-3-p yr a n yl]p en ta n ed ia m id e (36). Sodium methylate
(0.22 mmol) was mixed and stirred with 35 (70 mg, 0.1 mmol)
dissolved in methanol (10 mL) at room temperature for 2 h.
Deprotection was monitered by TLC (AcOEt). The mixture was
treated with acid Dowex 50WA resin. After methanol evapora-
tion under vacuum, compound 36 was recovered and recrystal-
1
NMR (DMSO-d6) δ 35.0 (NCH3), 117.0 (q, CF3, J CF ) 186 Hz),
132.9 (C4), 140.5 (C2) (C5 absent), 141 (C2′), 167.7 (CO), 170
(C5′); mass spectrum (DCI/NH3) m/z 323 (M+ + 1, 66), 340
(M+ + 18, 52). Anal. (C8H5F3N6O3S) C, H, N.
6-(Hyd r oxym eth yl)-3-[4-m eth oxyben zylid en ea m in o]-
t et r a h yd r op yr a n -2,4,5-t r iol (30). Paramethoxybenzalde-
hyde (0.136 g, 1 mmol) was added by syringe to a glucose
amine hydrochloride 29 (0.215 g, 1 mmol) in NaOH solution
(20 mL, 20 mmol) at 0 °C. After the mixture was stirred for
12 h at room temperature, 30 was obtained as a white solid
1
lized in ethanol (60% yield). H NMR (DMSO-d6) δ 1.83-2.17
(m, 6H, CH2CH2CH2), 3.09-4.10 (m, 5H, H3, H4, H5, H6), 4.38
(s, 3H, NCH3), 4.95 (m, 1H, H2), 7.62 (d, 1H, 8 Hz, H1), 8.24
(s, 1H, C4). 13C NMR (DMSO-d6) 20.57 (CH2), 34.14 (COCH2),
35.19 (NCH3), 54.16 (C2′), 61.04 (C6′), 70.40 (C3′), 70.73 (C4′),
71.98 (C5′), 90.51 (C1′), 133.07 (C4), 140.58 (C5), 141.01 (C2),
153.66 (C2*), 159.67 (C5*), 171.62 (CONH), 171.76 (CONH);
mass spectrum, FAB (matrix, 3-nitrobenzyl alcohol/DMSO) m/z
502 (M+ + 1).
1
(60% yield). H NMR (D2O) δ 3.84 (s, 3H, OCH3), 6.7-6.9 (m,
4H, phenyl), 8.23 (s, 1H, HCdN).
6-[(Acetyloxy)m eth yl]-3-[4-m eth oxyben zyliden eam in o]-
tetr a h yd r o-2,4,5-tr ia cetyloxyp yr a n e (31). 30 (0.298 g, 1
mmol) in pyridine (10 mL) in an ice bath was mixed with acetic
anhydride (0.3 mL, 5 mmol) and stirred for 48 h at room
temperature. After azeotropic solvent evaporation with tolu-
ene, 31 obtained in 80% yield was used directly without further
Ack n ow led gm en t. The Pierre FABRE Foundation
is greatly acknowledged for supporting the toxicity and
mutagenicity studies. Financial support from Ministe`re
des Affaires Etrange`res, from WHO/TDR and GDR
CNRS/DRET is also greatly appreciated. Dr. A. J .
Georges, Dr. P. Millet, Dr. F. Deloron, and Dr. G.
Dubreuil from the International Center of Medical
Research of Franceville (Gabon) are greatly acknowl-
edged for their contributions to this work. S.L.C.
received support from the WHO/TDR program.
1
purification. H NMR (D2O) δ 1.84-2.06 (m, 12H, CH3COO),
3.8 (s, 3H, OCH3), 4.01-4.31 (m, 3H, H5, H6), 5.09-5.40 (m,
3H, H2, H3, H4), 5.90 (d, 1H, J ) 7 Hz, H1), 6.81-7.68 (d,
4H, phenyl), 8.13 (s, 1H, CHdN); 13C NMR (D2O) δ 20.5-20.6
(CH3COO), 55.43 (CH3O), 61.86 (C6′), 68.06 (C2′), 72.7-73.2
(C3′,4′,5′), 91.2 (C1′), 114.1, 128.3, 130.3, 162.3 (aromatic
carbons), 164.3 (CHdN), 168.8-170.7 (CH3COO); IR (KBr/
cm-1) 2918 (CH3), 1750-1740 (COO).
2,5-Di(a cetyloxy)-6-[(a cetyloxy)m eth yl]-3-a m m on iotet-
r a h yd r o-4-p yr a n yl Acet a t e Ch lor id e (32). Concentrated
hydrochloric acid was carefully added in an acetone solution
of 31 kept at 0 °C in an ice bath to reach pH 5.The precipitate
Refer en ces
1
formed was filtered and washed with acetone (yield 90%). H
(1) De Castro, S. L. The challenge of Chagas’ disease chemo-
therapy: An update of drugs assayed against Trypanosoma
cruzi. Acta Trop. 1993, 53, 83-98.
(2) Chapman, J . D.; Lee, J .; Meekes, B. E. Cellular reduction of
nitroimidazole drugs potential for selective chemotherapy and
diagnosis for hypoxic cells. Int. J . Radiat. Oncol., Biol., Phys.
1989, 16, 911-917.
NMR (D2O) δ 2.06-2.25 (m, 12H, CH3COO), 5.95 (d, 1H, J )
8 Hz, H1); 13C NMR (D2O) δ 22.6-22.7(CH3COO), 54.0 (C2′),
64.0 (C6′), 70.5 (C3′), 73.4 (C4′), 74.7 (C5′), 92.9 (C1′), 175.7-
176.1 (CH3COO).
5-Oxo-5-({2,4,5-tr i(a cetyloxy)-6-[(a cetyloxy)m eth yl]tet-
r a h yd r o-2H-3-p yr a n yl}a m in o)p en ta n oic a cid (33). Glu-
taric anhydride (0.30 g, 2 mmol) and 32 (0.384 g, 1 mmol) in
pyridine (10 mL) with 4-dimethylaminopyridine (0.04 g, 1
mmol) were stirred for 48 h at room temperature. After
evaporation of pyridine in the presence of toluene and chro-
matography (CH2Cl2/CH3OH, 95:5), 33 was obtained (80%
yield). 1H NMR (CDCl3) δ 1.71-2.10 (m, 12H, CH3COO), 2.16-
2.34 (m, 6H, (CH2)3), 3.71-4.41 (m, 3H, H5, H6), 4.80-5.30
(m, 2H, H3, H4), 5.60-5.70 (m, 1H, H2), 6.65 (d, 1H, J ) 7
Hz, H1); 13C NMR (CD3OD) δ 20.5-20.6 (CH3COO), 32.57-
35.O ((CH2)3), 52.44 (C2′), 61.69 (C6′), 66.3 (C5′), 72.5 (C3′C4′),
92.3 (C1′), 169.6-171.4 (CH3COO), 173.3 (CONH), 177.4
(COO); IR (KBr/cm-1) 3365 (COOH), 2968 (CH3), 1750 (COO).
3-Meth ylpen tan oic-4-oxo-4-({2,4,5-(acetyloxy)-6-[(acety-
l o x y ) m e t h y l ] t e t r a h y d r o -2 H -3 -p y r a n y l }a m i n o ) -
p en ta n oic An h yd r id e (34). N-Methylmorpholine (0.12 mL,
1.1 mmol) and isobutyl chloroformate (0.15 mL, 1.1 mmol) at
-40 °C were mixed with 33 (1 mmol) under argon in THF (10
mL) for 10 min, and 34 formed in situ was not isolated.
2,5-Di(a ce t yloxy)-6-[(a ce t yloxy)m e t h yl]-3-[(5-{[5-(1-
m eth yl-5-n itr o-1H-2im id a zolyl)-1,3,4-th ia d ia zol-2-yl]a m i-
n o}-5-oxop en ta n oyl)a m in o]tetr a h yd r o-2H-4-p yr a n yl Ac-
eta te (35). Megazol (225 mg, 1 mmol) was added to the above
mixture containing 34 and kept at ambient temperature for 2
h. After evaporation under vacuum and flash chromatography
(CH2Cl2/CH3OH, 95:5), 35 was isolated (230 mg, yield 25%).
1H NMR (DMSO-d6) δ 1.92-2.04 (m, 12H, CH3COO), 1.6-2.11
(m, 6H, CH2CH2CH2), 3.27 (NH), 3.90-4.10 (m, 3H, H5, H6),
(3) Filardi, L. S.; Brener, Z. A nitroimidazole-thiadiazole derivative
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