Synthesis and electrochemistry of 2-ethenyl and 2-ethanyl derivatives of 5-nitroimidazole and antimicrobial activity against Giardia lamblia

Article abstract of DOI:10.1021/jm900356n

Infections with the diarrheagenic pathogen, Giardia lamblia, are commonly treated with the 5-nitroimidazole (5-NI) metronidazole (Mz), and yet treatment failures and Mz resistance occur. Using a panel of new 2-ethenyl and 2-ethanyl 5-NI derivatives, we fo

Full text of DOI:10.1021/jm900356n

4038
J. Med. Chem. 2009, 52, 4038–4053
Synthesis and Electrochemistry of 2-Ethenyl and 2-Ethanyl Derivatives of 5-Nitroimidazole and
Antimicrobial Activity against Giardia lamblia
Carlos A. Valdez,†,§ Jonathan C. Tripp,†,§ Yukiko Miyamoto,‡ Jaroslaw Kalisiak,† Petr Hruz,‡ Yolanda S. Andersen,‡
Sabrina E. Brown,⊥ Karina Kangas,⊥ Leo V. Arzu,⊥ Barbara J. Davids,‡ Frances D. Gillin,‡ Jacqueline A. Upcroft,|
Peter Upcroft,| Valery V. Fokin,† Diane K. Smith,⊥ K. Barry Sharpless,† and Lars Eckmann*,‡
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037, Departments of Medicine and Pathology, UniVersity of CaliforniasSan Diego, La Jolla, California 92093,
Department of Chemistry and Biochemistry, San Diego State UniVersity, San Diego, California 92182, and The Queensland Institute of
Medical Research, 300 Herston Road, Brisbane, Australia
ReceiVed March 20, 2009
Infections with the diarrheagenic pathogen, Giardia lamblia, are commonly treated with the 5-nitroimidazole
(5-NI) metronidazole (Mz), and yet treatment failures and Mz resistance occur. Using a panel of new 2-ethenyl
and 2-ethanyl 5-NI derivatives, we found that compounds with a saturated bridge between the 5-NI core
and a pendant ring system exhibited only modestly increased antigiardial activity and could not overcome
Mz resistance. By contrast, olefins with a conjugated bridge connecting the core and a substituted phenyl
or heterocyclic ring showed greatly increased antigiardial activity without toxicity, and several overcame
Mz resistance and were more effective than Mz in a murine giardiasis model. Determination of the half-
wave potential of the initial one-electron transfer by cyclic voltammetry revealed that easier redox activation
correlated with greater antigiardial activity and capacity to overcome Mz resistance. These studies show the
potential of combining systematic synthetic approaches with biological and electrochemical evaluations in
developing improved 5-NI drugs.
Introduction
efficacy and safety.8,9 On the basis of the therapeutic utility of
Mz, several other 5-NIsa have been developed more recently.
For example, tinidazole (1, Figure 1) is a N1-position modified
5-NI, which has been approved by the FDA for the treatment
of giardiasis. Not all antigiardial nitro drugs are based on the
5-NI scaffold. Nitazoxanide (2, Figure 1) belongs to an emerging
class of 5-nitrothiazole compounds with potent antigiardial
activity,10 although Giardia lines resistant to Mz are cross-
resistant to nitazoxanide.9 Overall, Mz remains the standard
treatment for giardiasis to date, making the discovery and
development of new therapeutics an important goal in expanding
the arsenal of antibiotics for controlling the infection.
The high specificity of the 5-NIs is due to the requirement
for the prodrug to be reduced to toxic free radical intermediates
by low redox potential reactions present only in anaerobic
microbes.11 These short-lived free radicals cause lethal damage
to the microbe, although their cellular targets are not well-
defined in either eukaryotes or prokaryotes. Energy metabolism
in Giardia, which does not have mitochondria, is fermentative,
and electron transport proceeds in the absence of oxidative
phosphorylation present in organisms with mitochondria. How-
ever, Giardia is microaerotolerant and can reduce O2 and thus
protect the highly oxygen-sensitive, key metabolic enzyme
pyruvate-ferredoxin oxidoreductase and the iron-containing
ferredoxins that activate Mz.12 The oxidoreductase carboxylates
pyruvate and donates electrons to ferredoxin, which in turn
reduces other components in the electron transport chain. ATP
production is linked to pyruvate decarboxylation.13 Reduced
ferredoxin activates the critical nitro group of the prodrug Mz,
leading to formation of toxic free radicals that kill the parasite.
Selective reductive activation in the target microbes, but not
mammalian cells, is generally thought to be critical for the
The anaerobic protozoan parasite, Giardia lamblia, is a major
cause of human diarrheal disease, infecting an estimated 10%
of the world’s population in both endemic and epidemic
fashion.1 In the U.S., Giardia is the most common cause of
waterborne diarrheal disease, with international travelers, hikers,
and children in day care centers at particular risk.2 Infection is
by fecal-oral transmission and is initiated by ingestion of
infectious cysts in contaminated water or through person-to-
person contact. After excystation, flagellated trophozoites
colonize the upper small intestine where they attach to the
epithelial lining but do not invade the mucosa. The duration of
giardial infection is variable, but chronic infection and reinfec-
tion occur commonly.3 Although ∼50% of Giardia infections
are asymptomatic, major symptoms include diarrhea, often
severe and protracted, with malabsorption, dehydration, weight
loss, failure to thrive, cognitive impairment in children, and
chronic fatigue in adults.4,5 These symptoms result from a
combination of intestinal villus atrophy, loss of brush border
microvilli, digestive enzyme deficiencies, and epithelial barrier
dysfunction.6,7
The leading antigiardial agent, metronidazole (Mz, Figure 1),
is a synthetic 5-nitroimidazole (NI) derivative introduced in 1959
by Rhoˆne-Poulenc. In addition to Giardia, Mz is active against
Entamoeba histolytica and Trichomonas Vaginalis, as well as
several clinically important anaerobic bacteria (including Clostrid-
ium difficile, Helicobacter pylori, and Bacteroides fragilis),
making it a highly versatile antibiotic. Mz has a long record of
* To whom correspondence should be addressed. Phone 858-534-0683.
Fax: 858-534-3338. E-mail: leckmann@ucsd.edu.
†
The Scripps Research Institute.
These authors contributed equally to this work.
University of CaliforniasSan Diego.
San Diego State University.
§
‡
⊥
a Abbreviations used: CV, cyclic voltammogram; E1/2, half-wave poten-
tial; Ep, peak potential; Mz, metronidazole; NI, nitroimidazole.
|
The Queensland Institute of Medical Research.
10.1021/jm900356n CCC: $40.75  2009 American Chemical Society
Published on Web 05/29/2009

2-Ethenyl and 2-Ethanyl DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 4039
Figure 1. 5-Nitro heterocyclic compounds active against Giardia.
potency and selectivity of 5-NI drugs.14 However, relatively little
is known about the electrochemical properties of most 5-NIs,
making it difficult to draw detailed conclusions about the
importance of low redox potential for their antimicrobial activity,
selectivity, and mechanisms of action.
by treatment of the crude reaction mixture with a suitable
nucleophile. A more productive functionalization was achieved
by treating olefin 12 with bromine in chloroform to produce
tribromide 33 in 88% yield (Scheme 1b). Additionally, the
double bonds of nitroimidazoles 8 and 12 were dihydroxy-
lated using the acidic Upjohn procedure19 to furnish diol 34
in 28% and diol 35 in 14% yield (Scheme 1c). Subsequent
acetylation of diols 34 and 35 using acetic anhydride
produced compounds 36 and 37 in 66% and 92% yields,
respectively (Scheme 1c).
In spite of the efficacy of 5-NI drugs, treatment failures in
giardiasis occur in up to 20% of cases.14 Clinical resistance of
G. lamblia to Mz and other 5-NIs is proven, and in vitro
resistance can be induced so that parasites grow in physiologi-
cally relevant concentrations of Mz.15 In addition, resistance
has been induced in vitro against all commonly used antigiardial
drugs, including different 5-NIs, furazolidone, albendazole, and
quinacrine,9,14 further demonstrating the need for new com-
pounds to preempt resistance development. Prior studies sug-
gested that modifications in the 2-position of the imidazole ring
can lead to effective 5-NI drugs.16,17 For example, the 2-position
modified 5-NI compound 3 (Figure 1, compound 17 in ref 16)
is very active against G. lamblia and can overcome Mz
resistance.16 Although these 5-NI derivatives were not examined
for their toxicity, in vivo efficacy, or mechanisms of action,
they raised the possibility that 2-position derivatives of 5-NI
may be promising for developing new, potent, and safe drugs
against giardiasis.
Last, we evaluated the complete reduction of the C2-olefin
in the aldol adducts to their saturated counterparts for direct
structure-activity comparisons. Reducing the double bond in
the presence of the nitro group on the imidazole ring proved
difficult. Thus, as expected, hydrogenation of 5 over palladium
or platinum catalysts efficiently hydrogenated the double bond
but also reduced the nitro group to the corresponding amine
38, as outlined in Scheme 2a. We also attempted an alkylation
strategy where dimetridazole 4 was treated with NaOEt and
benzyl bromide but observed no conversion to the desired
product (Scheme 2b). A NiCl2/NaBH4 reducing agent system,20
which is a mild source of hydride, was also tried with olefin 5
but with unsatisfactory results, as only the fully reduced product
38 was again observed (Scheme 2c). We further explored
constructing the imidazole ring independently followed by a
nitration to give the desired product.21 However, the nitration
of known imidazole 39 occurred on the undesired aromatic ring,
forcing us to abandon this approach (Scheme 2d). Ultimately,
we evaluated diimide as the reducing agent. While a more
conventional Cu(II)/hydrazine system22 for generating the
diimide failed to effect the reaction, tosyl hydrazine in pyridine23
selectively reduced the double bond while leaving the nitro
functionality intact (Scheme 2e). This procedure allowed us to
convert the aldol adducts 5, 8, and 12 to their fully saturated
counterparts 40, 41, and 42 in 32%, 18%, and 17% yields,
respectively (Scheme 2e).
In the present study, we synthesized a library of substituted
2-styryl 5-NI compounds and evaluated the significance of the
olefin bridge directly attached to the NI ring by chemically
modifying it by dibromination, dihydroxylation, and reduction.
These new compounds were assessed for their potency against
G. lamblia in vitro and in vivo, their cytotoxicity in human cells,
and their electrochemical properties.
Results and Discussion
Chemistry. We synthesized 2-styryl 5-NI derivatives using
the aldol condensation18 between 1,2-dimethyl-5-nitroimidazole
(dimetridazole, 4, Table 1) and a panel of aryl aldehydes in the
presence of sodium ethoxide in ethanol as shown in Table 1.
By use of this protocol, a diverse array of functionality was
incorporated into the 2-position of the NI core ranging from
electron-rich aromatic systems (Table 1, entries 2-5, 16, and
18) and electron-poor ones (entries 6-15 and 17) to different
heterocyclic scaffolds (entries 23-27). Because certain halogen
substitutions in the aromatic ring can yield potent antimicrobial
compounds,16,17 we systematically explored most of the possible
halogen monosubstitutions around the phenyl ring (entries
6-15). While the yields of these reactions varied widely
(13-77%), sufficient material could be produced for biological
testing after purification by flash chromatography or recrystal-
lization.
In another round of derivatizations, we functionalized the
double bond present in the aldol condensation products. This
allowed us to probe the biological importance of an unsatur-
ated bridge connecting the NI and the phenyl ring in the new
compounds. To this end, we initially attempted to oxidize
the double bond in 5 to the corresponding epoxide 32 using
m-CPBA (Scheme 1a). Although the epoxide was observed
by LCMS, it proved to be difficult to isolate or even capture
Improved Antigiardial Activity and Selectivity of 2-Styryl-
5-NI Derivatives. Prior work had demonstrated that dibro-
minated 5-NI compound 3 (Figure 1), a C2-modified 5-NI,
had increased potency against G. lamblia relative to Mz.16
In the present study, utilizing a panel of isolates representing
assemblages A and B, the two major genetic groupings that
contain all human-infectious Giardia, we confirmed that
compound 3 (Table 2, entry 1) possessed a 40-fold increased
antigiardial activity over Mz (Table 2, entry 10). However,
toxicity analysis in the human cell line, HeLa, indicated that
dibromide 3 was >500-fold more toxic than Mz. To consider
both the potency and toxicity of the new compounds in
comparison to Mz, the in vitro selectivity was determined
by calculating the ratio of the IC50 in HeLa cells to EC50 in
Giardia as an estimate for the therapeutic index. The most
promising candidate compounds for further evaluation will
be ones with an equal or higher in vitro selectivity ratio than
Mz. In the case of dibromide 3, the selectivity ratio was >10-
fold lower than that of Mz (Table 2). Similarly, a close
analogue of 3 bearing a bromine instead of a methyl at the

4040 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Valdez et al.
Table 1. Synthesis of 2-Ethenyl Derivatives of 5-NI by Aldol Condensation
Scheme 1
phenyl ring para to the saturated bridge, compound 33 (Table
2, Entry 2), did not overcome the toxic effects of 3 and had
even lower antimicrobial activity, further reducing the
selectivity ratio. To evaluate whether the toxicity of 3 could

2-Ethenyl and 2-Ethanyl DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 4041
Scheme 2
Table 2. Antigiardial Potency and Toxicity of New 5-NI Alkanes
Activity against G. lambliaa
toxicity in HeLa cells,b
pIC50 (mean ( SE)
selectivity ratio,c
IC50/EC50
entry
name
X
Y
R
EC50 (nM)
pEC50 (Mean ( SE)
1
2
3
4
5
6
7
8
9
3
Br
Br
Br
Br
Me
Br
Me
Br
Me
Br
H
65
347
832
447
316
224
209
282
1148
2630
7.19 ( 0.15
6.46 ( 0.15
6.08 ( 0.15
6.35 ( 0.19
6.50 ( 0.19
6.65 ( 0.16
6.68 ( 0.18
6.55 ( 0.25
5.94 ( 0.33
5.58 ( 0.09
4.95 ( 0.06
5.10 ( 0.15
<4.00
174
23
>120
208
>319
436
>474
>357
85
33
34
35
36
37
40
41
42
Mz
OH
OH
OAc
OAc
H
OH
OH
OAc
OAc
H
4.04 ( 0.04
<4.00
4.01 ( 0.01
<4.00
H
H
H
H
Me
Br
<4.00
4.01 ( 0.01
2.22 ( 0.08
10
2291
a Activity against G. lamblia was determined as EC50, the compound concentration that inhibited growth of G. lamblia by 50% over the assay period, and
is expressed as negative log10 value of the EC50 (pEC50). Higher pEC50 values reflect greater potency. Data are mean ( SE of the average pEC50 of four
different G. lamblia isolates (WB/C6, GS/M, 106, and 713). The average pEC50 for each individual isolate is based on the results of three to four separate
experiments. Mean EC50 was calculated from the mean combined pEC50 for all four isolates. b Toxicity against HeLa cells was determined as IC50, the
compound concentration that killed 50% of HeLa cells in the cytotoxicity assays, and is expressed as negative log10 value of the IC50 (pIC50), with higher
values reflecting greater toxicity. Data are the mean ( SE of the results of three separate experiments. c Selectivity ratio, the ratio of IC50 to EC50, reflects
the relative selectivity of compound potency against Giardia compared to toxicity in HeLa cells, with higher ratios indicating greater selectivity for the
parasite.
be attenuated without affecting its potent antigiardial activity,
we synthesized compounds related to dibromide 3 in which
the bromine atoms in the bridge were replaced systematically
with hydroxyl groups (34 and 35, Table 2, entries 3 and 4)
or acetoxy groups (36 and 37, Table 2, entries 5 and 6). These
compounds also had selectivity ratios markedly below Mz,
rendering them significantly less effective than Mz. Finally,
we prepared more direct analogues of dibromide 3, in which
the bromine atoms were replaced with hydrogen atoms
(40-42, Table 2, entries 7-9). Although elimination of the
two bromine atoms in 3 decreased toxicity, it also compro-
mised antigiardial activity and the selectivity ratio. Together,
these results suggest that modifications in the saturated bridge
at C2 of the nitroimidazole core could not overcome toxicity
without markedly reducing antimicrobial activity.
On the basis of the disappointing results obtained with the C2-
saturated bridge analogues, we reasoned that an unsaturated bridge
joining the phenyl ring to the 5-NI core might exert more direct
electronic influence on the 5-NI ring and improve antimicrobial
activity. To explore this idea, we synthesized a series of 5-NI
compounds bearing a substituted styryl unit directly attached to
the C2 position as presented in Table 1. The simplest compound
of this styryl series with a bare phenyl ring, 5 (Table 3, entry 1),
displayed strong antigiardial activity without measurable toxicity,
leading to a high selectivity ratio. Substitutions at the phenyl with
one or several methyl moieties (6-9, Table 3, entries 2-5) did
not improve the activity or selectivity ratio of compound 5.
Similarly, halogen substitutions on the phenyl ring (10-19, Table
3, entries 6-15) also did not significantly improve antigiardial
activity. Toxicity was only very slightly increased by most of these
modifications, with the exceptions of the methyl, chloro, and iodo
substitutions at the meta position of the phenyl ring, which
increased toxicity moderately (7, 14, 18, Table 3, entries 3, 10,
and 14, respectively) but not to the levels of the dibrominated
compounds 3 and 33 (Table 2, entries 1 and 2). Addition of larger
side chains at the phenyl moiety, including OMe (20 and 22, Table
3, entries 16 and 18), OCF3 (21, Table 3, entry 17), and phenyl
(24, Table 3, entry 20), markedly reduced activity against Giardia,
while addition of diethoxymethyl (23, Table 3, entry 19) did not
affect antigiardial activity or toxicity. Beyond substituted styryl
compounds, we performed the aldol condensation reaction between
dimetridazole 4 and a panel of heterocyclic and other aromatic
aldehydes to furnish compounds 25-31 (Table 1, entries 21-27).
Even though these compounds exhibited promisingly low cytotoxic
profiles, their antigiardial activity and selectivity ratio (Table 4,

4042 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Table 3. Antigiardial Potency and Toxicity of New 5-NI Alkenes
Valdez et al.
activity against G. lambliaa
toxicity in HeLa cells,a
pIC50 (mean ( SE)
selectivity ratio,a
IC50/EC50
entry
name
R1
R2
R3
R4
EC50 (nM)
pEC50 (mean ( SE)
1
2
3
4
5
6
7
8
5
6
7
8
9
H
Me
H
H
H
Me
H
H
H
Br
H
H
Cl
H
H
H
I
F
OMe
H
OMe
H
H
H
H
H
Me
Me
H
H
Br
H
H
Cl
Cl
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
39
69
40
162
339
49
47
49
72
22
52
234
60
93
41
155
741
617
52
7.41 ( 0.12
7.16 ( 0.22
7.40 ( 0.16
6.79 ( 0.17
6.47 ( 0.15
7.31 ( 0.20
7.33 ( 0.20
7.31 ( 0.27
7.14 ( 0.15
7.66 ( 0.18
7.28 ( 0.10
6.63 ( 0.22
7.22 ( 0.17
7.03 ( 0.16
7.39 ( 0.15
6.81 ( 0.25
6.13 ( 0.21
6.21 ( 0.27
7.28 ( 0.18
6.07 ( 0.23
<4.00
>2570
1349
776
>617
32
4.03 ( 0.03
4.51 ( 0.24
<4.00
H
Me
Br
H
H
Cl
H
H
Cl
I
H
H
H
H
OMe
H
Ph
4.96 ( 0.12
4.14 ( 0.13
4.06 ( 0.06
4.14 ( 0.10
<4.00
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
1479
1862
1479
>1380
1380
1738
>427
>1660
339
>2455
>646
112
>162
1862
>117
9
10
11
12
13
14
15
16
17
18
19
20
4.52 ( 0.14
4.04 ( 0.05
<4.00
<4.00
4.50 ( 0.23
<4.00
H
<4.00
OCF3
OMe
CH(OEt)2
H
4.08 ( 0.09
<4.00
4.01 ( 0.01
<4.00
851
a Activity against G. lamblia, toxicity in HeLa cells, and selectivity ratios were determined and calculated as described in Table 2.
Table 4. Antigiardial Potency and Toxicity of 5-NI Alkenes with Non-Phenyl Substitutions
a Activity against G. lamblia, toxicity in HeLa cells, and selectivity ratios were determined and calculated as described in Table 2.
entries 1-7) did not surpass that exhibited by the simple phenyl
compound 5 (Table 3, entry 1).
several of the most potent and structurally diverse compounds
in a murine model of G. lamblia infection. Normal adult mice
were infected orally with trophozoites of the GS/M strain of G.
lamblia and left for 2 days to allow full establishment of
infection. Mice were then treated with five doses of the
compounds over a 3-day period, and infectious load was
assessed by counting the number of live trophozoite in the small
intestine. Mz, used as reference drug, caused a ∼1000-fold
reduction in trophozoite numbers, but not parasite eradication,
at a dose of 10 mg/kg, while it had no significant effect at the
lower dose of 2 mg/kg (Figure 2). Six of the seven tested new
Together, these results show that derivatives of 5-NIs
bearing an olefin directly attached to the C2 position of the
imidazole ring have potent antigiardial activity. More im-
portantly, most of the analogues possess low cytoxicity and
excellent selectivity ratios comparable to or exceeding that
of Mz.
Activity of Selected 5-NIs against G. lamblia in a Mu-
rine Infection Model. As a next step toward evaluating the
ultimate clinical potential of the new 5-NI derivatives, we tested

2-Ethenyl and 2-Ethanyl DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 4043
Figure 2. In vivo potency of new 5-NI compounds against giardiasis.
Adult C57BL/6 mice were infected orally with G. lamblia GS/M and
given the indicated compounds at 10 or 2 mg/kg or vehicle only by
oral gavage five times over a 3-day period starting 2 days after infection.
Trophozoite numbers in the small intestine were determined 5 days
after infection. Data are mean ( SE of 3-7 animals. Asterisks designate
a significant decrease (p < 0.05, t test) relative to infection controls
given vehicle only. The gray line depicts the sensitivity of the assay.
The in vitro activies of the respective compounds are taken from Tables
2-4.
Figure 3. Resistance profiles of new 5-NI derivatives. EC50 of all new
compounds were determined for MzR and MzS lines of G. lamblia 713
and 106. The ability to overcome Mz resistance is expressed as the
ratio of EC50 in the MzR line over EC50 in the MzS line and is plotted
for each compound (A, closed circles). Mz (A, open circle) serves as
the reference drug. Compounds were categorized arbitrarily into three
groups to highlight their functional properties. In (B), resistance ratios
for G. lamblia 713 (closed circles) and G. lamblia 106 (open circles)
are plotted against the average antimicrobial activity against four Mz-
sensitive lines for each drug, using the pEC50 values shown in Tables
2-4.
5-NI derivatives were at least as potent as Mz at 10 mg/kg,
with four of them (5, 19, 28, and 29) causing complete clearance
of the infection. One compound, 12, had no significant activity
at either dose. Two of the four compounds, 19 and 28, that
caused complete eradication at 10 mg/kg showed significant
activity at 2 mg/kg, further demonstrating that they were more
potent than Mz (Figure 2). None of the compounds had any
clinically observable untoward effects on the mice during the
3-day treatment period at the higher dose (10 mg/kg).
Further analysis of the in vivo and in vitro data revealed
that the in vitro properties of the compounds only partly
predicted their in vivo behavior. While a positive correlation
existed between in vitro activity and in vivo potency, several
notable exceptions were observed (Figure 2). Compound 12
was the fourth most active in vitro (EC50 of 49 nM, Table 3,
Entry 8) of the seven new 5-NI derivatives tested in vivo,
and yet it showed no activity in vivo. Conversely, compound
28 was only the fifth most active compound in vitro (EC50
of 76 nM, Table 4, entry 4), but it was one of the two most
potent compounds in vivo (Figure 2). Thus, compound 12
relatively underperformed, while compound 28 overper-
formed, in vivo relative to the respective in vitro activity.
Nonetheless, these results indicate that several of the new
5-NI compounds have strong in vivo activity and no apparent
acute toxicity, supporting the potential utility of these
compounds for clinical applications.
These lines exhibit stable Mz resistance with 30- and 6-fold
respective increases in EC50 in the Mz-resistant line relative to
the EC50 in the matched parental line, termed here “Mz
resistance ratio” or “EC50 (MzR)/EC50 (MzS)”, with a lower
resistance ratio indicating a greater ability to overcome Mz re-
sistance. Analysis of the data for the two pairs of G. lamblia
lines revealed that the compounds exhibited a range of resistance
ratios (Figure 3A and Table 5). Most of the ratios of these
compounds were lower than that of Mz for at least one of
the two G. lamblia pairs examined, while compounds 5 and 36
were comparable to and compound 40 higher than Mz (Table
5). Importantly, the correlation of resistance ratios between the
713 line and the 106 line was weak, as a substantial number of
the compounds we tested overcame Mz resistance in only one
line. On the basis of this observation, we divided several of the
compounds into three major resistance groups (Figure 3A and
Table 5). Group A contained compounds that could substantially
overcome Mz resistance in both pairs of G. lamblia lines, while
compounds in groups B and C could overcome resistance in
one of the two pairs but not (or much less so) in the other.
These results provide strong pharmacological evidence for
more than one mechanism of Mz resistance in G. lamblia.
Diversity in antimicrobial resistance mechanism is not surpris-
ing, given that the Giardia lines were derived independently,24,25
and is well recognized for other antimicrobials and microbes.26,27
Moreover, different resistance mechanisms have been proposed
for G. lamblia, including the down-regulation of the critical
pathway required for reductive 5-NI drug activation,13,28,29
although the full extent and relative importance of different
Activity of 2-Position Modified 5-NIs against Mz-Resistant
G. lamblia. Resistance of G. lamblia to Mz has been docu-
mented in the laboratory and threatens to become a serious
clinical problem.9,14 We therefore tested the newly synthesized
5-NIs against two independently derived isogenic pairs of Mz-
resistant isolates of G. lamblia 713 and G. lamblia 106.24,25

4044 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Table 5. Activity of New 5-NIs against Mz-Resistant G. lamblia
Valdez et al.
activity against G. lamblia 713,a
pEC50 (mean ( SE)
activity against G. lamblia 106,a
pEC50 (mean ( SE)
EC50(MzR)/EC50(MzS)b
groupc
A
name
Mz
MzS
MzR
MzS
MzR
G. lamblia 713
G. lamblia 106
5.83 ( 0.08
4.35 ( 0.12
5.40 ( 0.10
4.59 ( 0.08
30.20
6.46
9
6.64 ( 0.23
7.49 ( 0.09
7.44 ( 0.10
7.59 ( 0.01
5.89 ( 0.02
6.90 ( 0.12
6.84 ( 0.10
6.91 ( 0.09
6.69 ( 0.31
7.37 ( 0.38
7.14 ( 0.08
7.51 ( 0.27
6.37 ( 0.24
6.94 ( 0.11
6.59 ( 0.22
7.03 ( 0.10
5.59
3.95
4.06
4.76
2.13
2.73
3.52
3.07
11
15
19
B
C
10
33
7.74 ( 0.10
6.74 ( 0.09
6.93 ( 0.14
6.32 ( 0.51
7.38 ( 0.18
6.67 ( 0.23
6.55 ( 0.43
5.54 ( 0.23
6.40
2.62
6.81
13.67
12
23
24
28
8.09 ( 0.46
7.49 ( 0.09
6.46 ( 0.18
7.29 ( 0.21
7.04 ( 0.39
6.15 ( 0.12
5.34 ( 0.34
5.93 ( 0.02
7.14 ( 0.17
7.19 ( 0.20
6.27 ( 0.27
7.06 ( 0.15
6.70 ( 0.21
6.70 ( 0.14
5.91 ( 0.19
6.60 ( 0.14
11.43
22.00
13.22
22.96
2.78
3.11
2.28
2.84
other
5
7.69 ( 0.10
7.52 ( 0.27
7.69 ( 0.10
7.61 ( 0.61
6.76 ( 0.15
6.99 ( 0.16
6.28 ( 0.20
6.94 ( 0.16
6.31 ( 0.19
5.98 ( 0.06
5.29 ( 0.03
5.30 ( 0.07
7.26 ( 0.24
7.22 ( 0.13
7.45 ( 0.28
7.04 ( 0.03
6.29 ( 0.16
6.76 ( 0.13
6.40 ( 0.14
6.60 ( 0.30
6.44 ( 0.09
6.41 ( 0.06
5.51 ( 0.11
5.59 ( 0.18
25.98
3.77
23.95
42.93
29.51
49.25
7.20
4.22
10.32
4.25
6.00
14.73
13
29
31
36
40
a pEC50 was determined for the respective Mz-sensitive (MzS) and Mz-resistant (MzR) lines of G. lamblia. Data are mean ( SE of three separate experiments.
b The ratio of EC50 in the Mz-resistant line to EC50 in the isogenic Mz-sensitive line (“Mz resistance ratio”) is a measure of the ability of a compound to
overcome Mz resistance in the respective G. lamblia lines. c Group refers to the Mz resistance groups depicted in Figure 3A.
resistance mechanisms remain elusive. In any case, the existence
of group A compounds suggests that it is possible to develop
5-NIs that can markedly overcome at least two forms of Mz
resistance and may be active against other forms of Mz
resistance in G. lamblia.
not readily predictable on the basis of overall antigiardial activity
or structural features.
Electrochemical Properties of 5-NI Alkenes and Alkanes
under Anhydrous Conditions. Since structural features of the
newly synthesized compounds did not provide consistent
explanations for the compounds’ antimicrobial activities, we
began to explore their electrochemical properties, since NIs are
prodrugs that must be activated by reduction in the target
microbes before they exhibit antimicrobial activity. We reasoned
that chemical modification of the molecule with groups that
perturb the electronic properties of the NI ring would affect the
ease with which these molecules undergo reduction. The redox
potential of Mz is more negative than the lowest redox couple
in mammalian cells, which is believed to be the basis for its
high specificity and low toxicity. To determine whether differ-
ences in redox activation might explain the ability of the
different 5-NIs to overcome Mz resistance, we measured the
half-wave potential (E1/2) of the initial one-electron transfer
reaction (0/-1 reduction) for groups A, B, and C compounds
and selected additional compounds. Measurements were done
by cyclic voltammetry in DMSO under anhydrous conditions,
with ferrocene as an internal standard and reference point for
measuring E1/2.
In searching for properties that might explain the resistance
grouping of the compounds, we found that the ability to
overcome Mz resistance was not correlated with the potency
of the respective compounds (Figure 3B). For example, two
group A compounds, 9 and 19, exhibited comparable improve-
ments (i.e., decreases) in resistance ratios in both G. lamblia
pairs, but compound 9 had ∼10-fold lower EC50 against the
respective Mz-sensitive parental lines (Table 5). Moreover,
compounds 11 and 5 had comparable activities against the Mz-
sensitive lines but were markedly different in their resistance
profiles (Table 5). Structural analysis also did not reveal any
clear explanation for the resistance grouping of the compounds,
although some interesting connections could be discerned. For
example, among the 2-styryl 5-NIs, two of the four group A
compounds (11 and 19) possessed a substitution (i.e., Br and
F) at the meta-position in the ring while none of the five group
B or C phenyl-carrying styryl compounds did. The importance
of the substitution position is illustrated by the set of Br-
substituted compounds, 10 (ortho), 11 (meta), and 12 (para).
The m-Br containing compound (11, group A) showed excellent
improvements in resistance ratios for both G. lamblia pairs,
while the o-Br substitution (10, group B) improved the ratio in
the 713 line but not the 106 line, and the p-Br substitution (12,
group C) improved the ratio in the 106 line but less so in the
713 line (Table 5). On the other hand, these structure-activity
relationships did not apply uniformly for other substituents. For
example, while the o-Br and p-Br substituted compounds fell
distinctly into groups B and C, respectively, the same was not
true for the Cl substitutions, where the ortho and para substituted
compounds (13 and 15, respectively) had very similar resistance
profiles (Table 5). Overall, these results demonstrate that several
of the new 5-NIs can markedly, albeit not entirely, overcome
resistance to Mz in G. lamblia. However, the ability to do so is
Representative cyclic voltammograms (CVs) under these
conditions for Mz and one of the new 5-NIs, the 3-thiophene
compound 31, are shown in Figure 4A. From the similarity of
the CVs it can be deduced that both compounds exhibited a
similar electrochemical behavior, corresponding to a reversible
one-electron reduction to the radical anion. The E1/2 of Mz was
-1.561 V under these experimental conditions, which was very
close to the E1/2 of -1.557 V for 1-methyl-5-NI, the unsubsti-
tuted parent compound of all the new 2-substituted N-methyl-
5-NI (Table 6). Most of the new 5-NIs we assayed had more
positive E1/2, indicating that they could be reduced more readily,
whereas six compounds exhibited more negative E1/2 than Mz
and 1-methyl-5-NI (Table 6).
Three of these six compounds had a C2-saturated bridge
between the two ring systems (40-42). This is consistent with

2-Ethenyl and 2-Ethanyl DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 4045
Figure 4. Electrochemistry of new 5-NI compounds. 5-NIs were subjected to cyclic voltammetry in DMSO (A) or pH 7.0 water (C). Representative
scans for Mz and the 3-thiophene compound 31 are shown. Potential is given relative to ferrocene (Fc) in DMSO or a saturated calomel electrode
(SCE) in water. Current was normalized against peak cathodic current for each scan. The arrows indicate the scan direction. A summary of the
electrochemical data is given in Table 6. E1/2 values in DMSO were plotted against Hammett σ values of the 2-alkenylphenyl 5-NI compounds 5,
7, 8, 11, 12, 14, 15, 18, and 19 (B). E1/2 values in DMSO were also plotted against Ep values in water for selected 5-NIs using the data in Table
6 (D). Data in (B) and (D) are the mean ( SE of three to four experiments.
the electron-donating ability of alkyl groups, which add electron
density to the nitroimidazole ring, making reduction more
difficult. Since there is no conjugation connecting the imidazole
ring to the pendant phenyl ring, substitution on the phenyl ring
had no significant effect on the E1/2 (Table 6). The exception to
this notion were the two 5-NI alkanes, 3 and 33, both of which
showed much more positive E1/2 than Mz (Table 6). However,
both compounds had di-Br substitutions in the C2-saturated
bridge and showed more complicated electrochemical behavior
with an irreversible reduction peak in the CV leading into and
overlapping with a reversible wave. The irreversible peak was
likely associated with reductive removal of the Br and the
reversible wave with reduction of the product NI. Interestingly,
both compounds were quite toxic in HeLa cells (Table 2, entries
1 and 2), suggesting that their easier reduction at less negative
redox potentials, or their redox-induced structural change and
probable side chain release, might contribute to toxicity.
chlorines on the phenyl ring, giving it a ∼90 mV more positive
E1/2 than Mz (Table 6).
To gain a better understanding of the observed electrochemi-
cal properties of the different 5-NI compounds, we evaluated
the substituent effects on the redox potentials using Hammett
plots.30-33 In this analysis of aromatic compounds, the E1/2 of
a series of meta- and para-substituted derivatives are plotted
against the Hammett σ values. The latter are derived from the
acid dissociation constants of substituted benzoic acids and
provide a quantitative measurement of the electron-donating/
accepting character of a particular substituent. Figure 4B shows
the Hammett plot for the 2-alkenylphenyl compounds that have
simple meta or para substituents for which good σ values are
available.34 As expected, we observed an approximately linear
correlation with a positive slope, indicating stabilization of
negative charge. This correlation suggests that the C2-olefin
bridge provides an electronic communication between the
pendant phenyl ring and the NI. Dividing the slope of this type
of Hammett plot by the Nernst equation coefficient of 0.0592
V yields a dimensionless parameter that is directly comparable
to the reaction parameter, F, obtained from the slopes of
Hammett plots of other types of reactions.32 This calculation
gives a F of 0.84 for the data in Figure 4B, indicating that the
reduction of these compounds is slightly less sensitive to the
substituent on the phenyl group than the acid dissociation of
benzoic acid with a F value of 1.0. Hammett plots for many
aromatic redox couples give much larger F values, often >2.30-33
The modest value observed in our experiments is consistent with
the substituents being located at some distance from the primary
reduction site, the nitro group of the imidazole core.
All of the other tested compounds with significantly more
positive E1/2 (ranging from 30-90 mV) than Mz and 1-methyl-
5-NI were C2-olefinic derivatives of 5-NI, suggesting that the
conjugation through the unsaturated link between the imidazole
ring and the pendant aromatic ring stabilizes the radical anion
through delocalization and thereby favors reduction of the nitro
group. The compounds substituted with phenyl (5), methylphe-
nyl (6-8), and trimethylphenyl (9) had 30-50 mV more
positive E1/2 than Mz (Table 6). Extended conjugation with
larger aromatic groups (24-26) and/or heterocycles with
electronegative elements (N, O) (28 and 29) made the com-
pounds easier to reduce (50-65 mV more positive E1/2 than
Mz) than the methylphenyl or phenyl derivatives, as did addition
of electron-withdrawing halogen groups on the phenyl ring
(55-90 mV more positive than Mz). The easiest compound to
reduce in this series was 16, which has two electron-withdrawing
Electrochemical Properties of 5-NI Alkenes under Aque-
ous Conditions. We conducted the initial electrochemical
measurements in DMSO because the reduction of the 5-NIs is

4046 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Table 6. Electrochemical Properties of Modified 5-NIs
Valdez et al.
mean ( SE
groupa
name
structure
R
E1/2 in DMSO (V vs Fc)b
Ep in water (V vs SCE)b c
,
40
41
42
22
27
20
Mz
I
I
I
IV
IV
IV
phenyl
4-methylphenyl
4-bromophenyl
2,3,4-trimethoxyphenyl
1,3-benzodioxolyl
3-methoxyphenyl
-1.593 ( 0.002
-1.592 ( 0.003
-1.592 ( 0.003
-1.584 ( 0.005
-1.584 ( 0.001
-1.580 ( 0.007
-1.561 ( 0.001
-1.557 ( 0.001
-1.557 ( 0.001
-1.544 ( 0.001
-1.529 ( 0.001
-1.528 ( 0.003
-1.522 ( 0.003
-1.518 ( 0.003
-1.517 ( 0.001
-1.516 ( 0.001
-1.512 ( 0.001
-1.512 ( 0.001
-1.509 ( 0.001
-1.508 ( 0.002
-1.506 ( 0.002
-1.504 ( 0.003
-1.503 ( 0.001
-1.502 ( 0.004
-1.502 ( 0.001
-1.497 ( 0.001
-1.497 ( 0.005
-1.494 ( 0.004
-1.493d ( 0.004
-1.492 ( 0.002
-1.489d ( 0.001
-1.487 ( 0.001
-1.486 ( 0.004
-1.481 ( 0.001
-1.469 ( 0.002
ND
ND
ND
-0.757 ( 0.003
-0.778 ( 0.002
-0.763 ( 0.001
-0.751 ( 0.017
1-methyl-5-NI
ND
ND
ND
ND
34
35
9
30
8
23
5
7
II
II
4-methylphenyl
4-bromophenyl
2,4,6-trimethylphenyl
2-thiopheneyl
4-methylphenyl
4-diethoxymethylphenyl
phenyl
3-methylphenyl
2-methylphenyl
2-biphenyl
2-napthyl
3-thiopheneyl
4-bromophenyl
2-furanyl
4-chlorophenyl
1-napthyl
3-fluorophenyl
1-methylimidazolyl
3-bromophenyl
3-iodophenyl
4-bromophenyl
3-chlorophenyl
4-methylphenyl
4-trifluoromethoxyphenyl
2-chlorophenyl
2-bromophenyl
2,4-dichlorophenyl
A
C
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
III
IV
III
IV
IV
IV
IV
-0.658 ( 0.001
ND
ND
-0.634 ( 0.004
-0.635 ( 0.003
6
-0.621 ( 0.001
C
24
26
31
12
29
15
25
19
28
11
18
33
14
3
ND
ND
-0.595 ( 0.001
C
A
-0.605 ( 0.007
ND
-0.584 ( 0.001
ND
A
C
A
-0.613 ( 0.008
-0.594 ( 0.014
ND
ND
ND
ND
ND
ND
ND
ND
ND
B
21
13
10
16
B
a Group refers to the Mz resistance groups in Figure 3A and Table 5. b Redox activation potential was determined by cyclic voltammetry in DMSO or
water (pH 7.0). Half-wave potential (E1/2) of the one-electron transfer reaction (0/-1 reduction) was determined in DMSO, with ferrocene (Fc) as the
internal reference. Peak potential, Ep, for the irreversible first electron reduction step was determined in water, using a saturated calomel electrode (SCE) as
standard. Data are mean ( SE of three to four separate experiments. c ND, not determined. d The E1/2 values for these compounds are for the reversible wave,
but given the different electrochemical response, these values can not be directly compared to the other E1/2 values.
reversible under these conditions, as shown by the oxidation
peak observed on the return scan in the CVs (Figure 4A). The
resultant E1/2 value (the potential midway between the two peak
values) is close to the thermodynamic redox potential for
reduction to the radical anion and thus provides a fundamental
measure of the ease of adding the first electron to the different
5-NIs. While it is possible that the reduction of the 5-NIs occurs
in a nonaqueous environment inside the microbial target cells
(e.g., in the active site of a redox protein), it is likely that the
reduction occurs in a more aqueous environment. Determination
of the thermodynamic potential for reduction of 5-NIs in water
is confounded by the fact that the electron transfers in aqueous
conditions are irreversible reactions. This is shown by the lack
of an oxidation peak on the return scan in the CVs in aqueous
solution (Figure 4C). The irreversibility is due to the instability
of the radical anion in aqueous solution, which upon protonation
is further reduced at the same potential. Because of this, only
the peak potential (Ep) for the reduction can be derived from
the CVs (Table 6). In contrast to the E1/2, the Ep is strongly
affected by the kinetics of the electron transfer reactions and
any coupled chemical reactions and may not be directly
correlated with the thermodynamic redox potential for the
overall reaction. Furthermore, the kinetic influence means that
variations in the experimental conditions (e.g., differences in
the electrode surface) will affect the Ep, possibly leading to
greater scatter in the data.
For all of these reasons, we chose to rely on the E1/2 values
in DMSO rather than the aqueous Ep values as our primary
indicator of the relative ease of reduction for the different 5-NIs.
However, given that the first step in the reduction in aqueous
solution at physiological pH is likely the same as that of the
overall reaction in DMSO, i.e., one-electron reduction to the
radical anion, we reasoned that the E1/2 values in DMSO should
give us an indication of the relative ease of reduction of the
5-NIs in aqueous solution. This is particularly likely among
compounds of similar structure, for which the difference in
solvation energy between DMSO and water would be expected
to remain fairly constant across the series. In order to test this
hypothesis, we determined the CVs of selected C2-olefinic 5-NI
derivatives in neutral (pH 7.0) aqueous solution and correlated
the data with the E1/2 values obtained under anhydrous conditions
(Table 6). A plot of Ep of the aqueous CVs vs E1/2 for the same
compounds in DMSO showed a strong linear correlation (Figure
4D), supporting the notion that the E1/2 values obtained in

2-Ethenyl and 2-Ethanyl DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 4047
Figure 5. Relationship of redox potential and antigiardial activity and selectivity for new 5-NI compounds. The redox potential in DMSO (E1/2
)
of each new 5-NI was plotted against resistance ratios (A), overall antigiardial activity (B), and selectivity ratio relative to HeLa cells (C). For
compounds with limited aqueous solubility, the data points represent the ratio of EC50 and the highest test concentration (100 µM) and thus the
minimum rather than exact selectivity ratio (closed triangles in C). Mz (open circles) is shown in all panels as the reference drug.
DMSO give a reliable measure of the relative ease of reduction
of the different 5-NIs independent of whether the reduction
occurs in an aqueous or nonaqueous environment in the cell. It
must be noted that the analysis of the true one-electron redox
potential of the new 5-NI compounds in water would likely
require other assay approaches, such as pulse radiolysis, although
the utility of true aqueous one-electron potentials may be limited
if reduction occurs in a purely aqueous environment in the cells
conducive to rapid multielectron transfer.
Importance of Redox Potential of Modified 5-NI for
Antimicrobial Potency and Selectivity. We next examined the
relationship between E1/2 and the ability to overcome Mz
resistance (Figure 5A). In both pairs of Mz-sensitive and Mz-
resistant G. lamblia lines, compounds with improved (i.e.,
decreased) resistance ratios generally had more positive E1/2,
although the importance of this relationship appeared to differ
between the 713 and 106 pairs. In G. lamblia 713, almost all
of the tested compounds showed some ability to overcome
resistance, with the majority exhibiting more positive E1/2 but
a few compounds also showing more negative E1/2 (Figure 5A).
By comparison, in G. lamblia 106, only compounds with more
positive E1/2 than Mz could overcome Mz resistance. This was
not sufficient, however, since only about half of the compounds
with more positive E1/2 had a better (i.e., decreased) resistance
ratio than Mz while the other half exhibited reduced antigiardial
activity compared to Mz in the Mz-resistant line (Figure 5A).
Thus, a decrease in the energy requirements for redox activation,
as reflected in a more positive E1/2, is generally correlated with
the ability of 5-NI to overcome Mz resistance, but such a
decrease is neither necessary nor sufficient.
to Mz, while others (e.g., 21 and 24) had markedly more positive
E1/2 than Mz but only a very modest increase in activity (Tables
2, 3, 4, and 6). Rather surprisingly, comparison of E1/2 and
selectivity ratios revealed little correlation between these
parameters (Figure 5C). Thus, many of the compounds with
more negative E1/2 than Mz showed low selectivity ratios, while
compounds with markedly more positive E1/2 had ratios
comparable to or exceeding that of Mz. These results indicate
that the ease of redox activation is not an important determinant
of the selectivity of 5-NIs in Giardia compared to mammalian
cells, at least as applied to the compounds tested in this study
with their relatively limited E1/2 range.
Conclusions
This study expands on prior work16,17 to demonstrate clearly
that modification of the 2-position of the 5-NI core provides a
promising avenue for improving its antimicrobial potency and
selectivity. All of the new 5-NI derivatives were more potent
than Mz against diverse isolates of G. lamblia, and almost a
third (10 of 35) of these compounds showed 50- to 100-fold
improvement in antigiardial activity. Perhaps more importantly,
a number of the new compounds markedly overcame resistance
to Mz. With the current series of 5-NI derivatives and quantita-
tive approaches, we did not identify compounds that could
overcome Mz resistance completely, as no compound had
identical activity against the Mz-resistant and Mz-sensitive pairs
of isogenic Giardia lines. However, the quantitative analysis
of antigiardial activity revealed that the new compounds
displayed a wide spectrum of activities in the face of Mz
resistance, suggesting that a larger 5-NI library may well contain
compounds whose activity is not compromised by prior Mz
resistance.
A similar conclusion also holds true for the overall antigiardial
activity of the nitro compounds, since we found a positive
correlation between E1/2 and activity (Figure 5B). However,
several compounds (e.g., 20 and 27) with more negative E1/2
than Mz also exhibited increased antigiardial activity compared
Antimicrobial potency and ability to overcome Mz resistance
were poorly if at all correlated. In fact, the compounds with
the most improved Mz resistance ratios had only modest overall

4048 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Valdez et al.
activity against the Mz-sensitive G. lamblia isolates, with pEC50
values in the mid-range of those observed in the entire library.
These results imply that the molecular properties that determine
overall potency are not those that overcome Mz resistance,
although detailed molecular conclusions are impossible to draw
at present, given the lack of mechanistic insights into the relevant
targets of 5-NIs in G. lamblia.
compounds need to be tested rapidly in in vivo models to find
truly effective drugs for treating giardiasis. In doing so, it should
be possible to minimize the negative impact on development
time and resource consumption that follows from the unavoid-
able attrition of compounds that are effective in vitro but exhibit
poor in vivo activity.
Analysis of the electrochemical properties of the new series
of 5-NI compounds revealed fresh insights into the determinants
of antigiardial activity. Compounds with more positive redox
activation potential, which makes them more readily reducible,
generally had greater antimicrobial activity and an improved
capacity to overcome Mz resistance. However, this relationship
was not universal, as several compounds with more negative
redox potentials also had markedly improved antigiardial
profiles, while other compounds with more positive redox
potential did not. Thus, although easier redox activation can
probably explain the improvement in antigiardial activity profile
for many of the compounds, it is neither necessary nor sufficient.
Other yet to be defined molecular properties must therefore also
be important. To our surprise, we did not find a correlation
between ease of redox activation and selectivity, as one might
have expected from the general recognition that the excellent
selectivity of 5-NI compounds for microbial targets over
mammalian cells is based on the differences in the lowest redox
couples present in these systems.11 Our data suggest that, at
least within the relatively negative and narrow E1/2 range of
compounds tested in this study, other determinants beyond ease
of reduction affect toxicity and selectivity of 5-NIs, thus casting
some doubts on the preeminence of the selective redox activation
paradigm in explaining the clinical utility of 5-NI drugs.
Experimental Section
Chemistry. Materials and General Methods. Reagents and
solvents were purchased from commercial sources and were used
as received. Reaction progress was monitored by thin-layer chro-
matography on Merck silica gel 60 F-254 with detection by UV.
Silica gel 60 (Merck 40-63 µm) was used for flash column
chromatography. Melting points are uncorrected and were deter-
mined by use of a Thomas-Hoover Uni-Melt capillary melting point
apparatus. 1H NMR and 13C NMR spectra were recorded with
Bruker DRX-600, Bruker DRX-500, Varian 400, or Bruker AMX-
400 spectrometers using DMSO-d6 or CDCl3. Proton magnetic
resonance (1H NMR) spectra were recorded at 600, 500, or 400
MHz. Data are presented as follows: chemical shift (parts per
million, ppm), multiplicity s ) singlet, d ) doublet, t ) triplet, q
) quartet, quin ) quintet, sep ) septet, m ) multiplet, br ) broad,
coupling constant, J (in hertz), and integration. Carbon magnetic
resonance (13C NMR) spectra were recorded at 150, 125, or 100
MHz. Data for 13C NMR are reported in terms of chemical shifts
(ppm) and coupling constant J (in hertz) for fluorine-containing
compounds. High-resolution mass spectra (HRMS) were recorded
at the mass spectrometry facility at The Scripps Research Institute,
La Jolla, CA. Elemental analyses were performed by Midwest
Microlab, LLC, Indianapolis, IN.
General Procedure for the Aldol Condensation. For the
synthesis of 2-vinyl-5-nitroimidazoles by aldol condensation,
dimetridazole (4, Table 1) was dissolved in EtOH (5.5 mL/mmol
of dimetridazole) and treated with sodium ethoxide (NaOEt,
2.0-2.5 equiv). The resulting amber suspension was vigorously
stirred at ambient temperature for 5-10 min before the addition of
the aryl aldehydes (1.1-1.3 equiv) in small portions or dropwise
using a syringe. The reaction mixture was then heated to 65 °C
overnight, after which the brown reaction mixture was cooled to
ambient temperature, transferred to a separatory funnel, and
partitioned (CH2Cl2//H2O). The organic phase was washed with
brine (NaCl/H2O), dried over anhydrous Na2SO4, and evaporated
in vacuo to generally give a brown residue which was purified by
column chromatography over silica gel.
The 5-NI compounds with a conjugated olefin bridge between
the imidazole core and the pendant ring system generally had
higher antigiardial activity and better selectivity than those with
a saturated alkane connection. In parallel, the energy require-
ments for nitro reduction were lower (i.e., more positive) in
the conjugated compounds and were markedly impacted by the
nature of the functional groups on the distant ring, whereas the
same was not true in the alkanes. The most straightforward
explanation may be that the olefin bridge allows effective
electronic communication between the pendant ring system and
the NI and thereby helps to fine-tune the nitro reducibility by
distant functional groups. It needs to be borne in mind, however,
that the electrochemical behavior of 5-NIs within the microbial
target cells may be more complex than what can be modeled
readily under cyclic voltammetry conditions in DMSO or water.
Inside the target cells, the initial, rate-limiting reductive step
presumably occurs in the presence of electrolytes, proteins,
lipids, and other molecules with varying charges that may affect
the charge distribution and, hence, reducibility and stability of
the 5-NI radicals. In addition, the initial step is followed by a
sequence of multielectron transfers, whose products may interact
differently with microbial target molecules than the radical
formed in the initial one-electron transfer reaction.35
1-Methyl-5-nitro-2-[(E)-2-phenylvinyl]-1H-imidazole (5). Dime-
tridazole (5.0 g, 35.4 mmol) was reacted with benzaldehyde (4.5
g, 42.5 mmol, 1.2 equiv) in the presence of NaOEt (6.03 g, 88.6
mmol, 2.5 equiv). The mixture was purified by column chroma-
tography (14% f 33% EtOAc/hexanes) to give 5 as a bright-yellow
1
solid (1.6 g, 20%). H NMR (500 MHz, CDCl3) δ 8.10 (s, 1H),
7.89 (d, J ) 15.8, 1H), 7.59-7.57 (m, 2H), 7.44-7.37 (m, 3H),
6.90 (d, J ) 15.7, 1H), 4.06 (s, 3H); 13C NMR (125 MHz, CDCl3)
δ 149.8, 139.8, 135.2, 134.2, 129.8, 129.0, 127.5, 111.2, 32.8;
HRMS (ESI+) calcd for C12H12N3O2 [M + H]+ 230.0924, found
230.0931. Anal. (C12H11N3O2) C, H, N.
1-Methyl-2-[(E)-2-(2-methylphenyl)vinyl]-5-nitro-1H-imida-
zole (6). Dimetridazole (1.0 g, 7.1 mmol) was reacted with
2-methylbenzaldehyde (1.02 g, 8.5 mmol, 1.2 equiv) in the presence
of NaOEt (1.2 g, 17.7 mmol, 2.5 equiv). The mixture was purified
by column chromatography (85% CH2Cl2/hexanes f CH2Cl2). The
material obtained in this manner was further purified by a
recrystallization from CH3OH (25 mL) to give 6 as a bright-yellow
The study shows that 5-NI derivatives can be developed that
are more potent than Mz in an animal model of giardiasis and
have no apparent toxic side effects, making them good
candidates for further preclinical development. The data caution,
however, that in vivo activity can differ markedly from what
might be expected on the basis of the observed in vitro activity.
Such differences are most likely accounted for by differential
pharmacokinetics of the respective compounds, which may be
fruitful ground for future studies to develop the clinical potential
of the new 5-NI compounds. Our results also stress that new
1
solid (0.40 g, 23%). H NMR (400 MHz, CDCl3) δ 8.15 (d, J )
15.6, 1H), 8.09 (s, 1H), 7.61 (dd, J ) 7.2, 1.6, 1H), 7.30-7.22 (m,
3H), 6.81 (d, J ) 15.6, 1H), 4.04 (s, 3H), 2.48 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 149.9, 137.4, 137.4, 137.3, 134.2, 134.1,
130.8, 129.5, 126.3, 125.5, 112.3, 32.7, 19.8; HRMS (ESI+) calcd
for C13H14N3O2 [M + H]+ 244.1080, found 244.1076. Anal.
(C13H13N3O2) C, H, N.

2-Ethenyl and 2-Ethanyl DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 4049
1-Methyl-2-[(E)-2-(3-methylphenyl)vinyl]-5-nitro-1H-imida-
zole (7). Dimetridazole (3.5 g, 24.8 mmol) was reacted with
3-methylbenzaldehyde (3.53 mL, 29.8 mmol, 1.2 equiv) in the
presence of NaOEt (4.2 g, 62 mmol, 2.5 equiv). The mixture was
purified by column chromatography (CH2Cl2 f 10% CH3OH/
d6) δ 8.06 (s, 1H), 7.81 (d, J ) 16.0, 1H), 7.72 (d, J ) 8.4, 2H),
7.61 (d, J ) 8.4, 2H), 7.42 (d, J ) 15.6, 1H), 4.13 (s, 3H); 13C
NMR (100 MHz, DMSO-d6) δ 149.4, 134.7, 134.0, 131.7, 130.8,
129.6, 128.4, 122.5, 114.0, 32.7; HRMS (ESI+) calcd for
C12H11BrN3O2 [M + H]+ 308.0029, found 308.0026. Anal.
(C12H10BrN3O2) C, H, N.
1
CH2Cl2) to give 7 as a yellow solid (2.53 g, 42%). H NMR (400
MHz, CDCl3) δ 8.04 (s, 1H), 7.80 (d, J ) 15.6, 1H), 7.33 (s, 1H),
7.32 (d, J ) 7.2, 1H), 7.27-7.21 (m, 1H), 7.14 (d, J ) 7.6, 1H),
6.82 (d, J ) 15.6, 1H), 3.99 (s, 3H), 2.34 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 149.9, 139.9, 138.6, 135.1, 134.2, 130.7, 128.8,
128.0, 124.7, 110.9, 32.7, 21.3; HRMS (ESI+) calcd for C13H14N3O2
[M + H]+ 244.1080, found 244.1083. Anal. (C13H13N3O2): C, 64.19;
H, 5.39; N, 17.27. Found: C, 64.36; H, 5.50; N, 16.84.
2-[(E)-2-(2-Chlorophenyl)vinyl]-1-methyl-5-nitro-1H-imida-
zole (13). Dimetridazole (1.0 g, 7.1 mmol) was reacted with
2-chlorobenzaldehyde (1.2 g, 8.5 mmol, 1.2 equiv) in the presence
of NaOEt (1.2 g, 17.7 mmol, 2.5 equiv). The mixture was purified
by crystallization from CH3OH/CH2Cl2 (1/2) to give 13 as a bright-
1
yellow solid (0.45 g, 24%). H NMR (500 MHz, CDCl3) δ 8.25
(d, J ) 15.8, 1H), 8.12 (s, 1H), 7.69-7.67 (m, 1H), 7.46-7.43
(m, 1H), 7.34-7.30 (m, 2H), 6.92 (d, J ) 15.7, 1H), 4.07 (s, 3H);
13C NMR (125 MHz, CDCl3) δ 135.7, 134.6, 134.6, 134.9, 133.6,
130.5, 130.3, 127.1, 127.1, 114.1, 32.9; HRMS (ESI+) calcd for
C12H11ClN3O2 [M + H]+ 264.0534, found 264.0539. Anal.
(C12H10ClN3O2) C, H, N.
1-Methyl-2-[(E)-2-(4-methylphenyl)vinyl]-5-nitro-1H-imida-
zole (8). Dimetridazole (7.0 g, 49.6 mmol) was reacted with
4-methylbenzaldehyde (7.0 mL, 59.5 mmol, 1.2 equiv) in the
presence of NaOEt (8.4 g, 124 mmol, 2.5 equiv). The mixture was
purified by column chromatography (CH2Cl2 f 10% CH3OH/
1
CH2Cl2) to give 8 as a yellow solid (2.65 g, 22%). H NMR (400
2-[(E)-2-(3-Chlorophenyl)vinyl]-1-methyl-5-nitro-1H-imida-
zole (14). Dimetridazole (1.0 g, 7.1 mmol) was reacted with
3-chlorobenzaldehyde (1.2 g, 8.5 mmol, 1.2 equiv) in the presence of
NaOEt (1.2 g, 17.7 mmol, 2.5 equiv). The mixture was purified by
crystallization from CH3OH to give 14 as a bright-yellow solid (0.36
g, 19%). 1H NMR (600 MHz, CDCl3) δ 8.09 (s, 1H), δ 7.82 (d, J )
15.7, 1H), 7.57 (s, 1H) 7.43 (td, J ) 4.6, 1.5, 1H), 7.36-7.35 (m,
2H), 6.90 (d, J ) 15.7, 1H), 4.07 (s, 3H); 13C NMR (150 MHz, CDCl3)
δ 149.2, 139.1, 138.1, 137.0, 135.0, 134.1, 130.2, 129.6, 126.8, 126.0,
112.6, 32.8; HRMS (ESI+) calcd for C12H11ClN3O2 [M + H]+
264.0534, found 264.0536. Anal. (C12H10ClN3O2) C, H, N.
MHz, DMSO-d6) δ 8.18 (s, 1H), 7.74 (d, J ) 16.0, 1H), 7.68 (d,
J ) 8.0, 2H), 7.33 (d, J ) 16.0, 1H), 7.24 (d, J ) 8.0, 2H), 4.02
(s, 3H), 2.34 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 149.9,
139.2, 137.9, 134.2, 132.7, 129.4, 127.7, 127.4, 112.0, 32.7, 20.9;
HRMS (ESI+) calcd for C13H14N3O2 [M + H]+ 244.1080, found
244.1074. Anal. (C13H13N3O2): C, 64.19; H, 5.39; N, 17.27. Found:
C, 64.32; H, 4.92; N, 16.85.
1-Methyl-2-[(E)-2-(2,4,6-trimethylphenyl)vinyl]-5-nitro-1H-
imidazole (9). Dimetridazole (1.0 g, 7.1 mmol) was reacted with
2,4,6-trimethylbenzaldehyde (1.36 g, 9.23 mmol, 1.3 equiv) in the
presence of NaOEt (966 mg, 14.2 mmol, 2.0 equiv). The mixture
was purified by column chromatography (CH2Cl2 f 5% CH3OH/
CH2Cl2) to give 9 as a yellow solid (559 mg, 29%). 1H NMR (400
MHz, CDCl3) δ 8.10 (s, 1H), 7.97 (d, J ) 16.0, 1H), 6.93 (s, 2H),
2-[(E)-2-(4-Chlorophenyl)vinyl]-1-methyl-5-nitro-1H-imida-
zole (15). Dimetridazole (1.0 g, 7.1 mmol) was reacted with
4-chlorobenzaldehyde (1.2 g, 8.5 mmol, 1.2 equiv) in the presence
of NaOEt (1.2 g, 17.7 mmol, 2.5 equiv). The mixture was purified
by crystallization from CH3OH/CH2Cl2 (3/1) to give 15 as a bright-
yellow solid (0.27 g, 15%). 1H NMR (500 MHz, CDCl3) δ 8.10 (s,
1H), 7.85 (d, J ) 15.7, 1H), 7.52 (d, J ) 8.5, 2H), 7.40 (d, J )
8.5, 2H), 6.88 (d, J ) 15.8, 1H), 4.07 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 149.5, 138.4, 135.7, 134.2, 133.7, 133.4, 129.3, 128.6,
111.7, 32.8; HRMS (ESI+) calcd for C12H11ClN3O2 [M + H]+
264.0534, found 264.0535. Anal. (C12H10ClN3O2) C, H, N.
2-[(E)-2-(2,4-Dichlorophenyl)vinyl]-1-methyl-5-nitro-1H-imi-
dazole (16). Dimetridazole (1.0 g, 7.1 mmol) was reacted with 2,4-
dichlorobenzaldehyde (1.49 g, 9.23 mmol, 1.3 equiv) in the presence
of NaOEt (966 mg, 14.2 mmol, 2.0 equiv). The mixture was purified
by column chromatography (CH2Cl2 f 10% CH3OH/CH2Cl2) to
6.51 (d, J ) 16.0, 1H), 3.99 (s, 3H), 2.39 (s, 6H), 2.30 (s, 3H); 13
C
NMR (100 MHz, CDCl3) δ 149.8, 139.5, 138.1, 136.5, 134.0, 131.8,
129.9, 129.2, 116.5, 32.7, 21.2, 21.0; HRMS (ESI+) calcd for
C15H18N3O2 [M
(C15H17N3O2): C, 66.40; H, 6.32; N, 15.49. Found: C, 66.87; H,
6.40; N, 15.60.
+
H]+ 272.1393, found 272.1397. Anal.
2-[(E)-2-(2-Bromophenyl)vinyl]-1-methyl-5-nitro-1H-imida-
zole (10). Dimetridazole (1.0 g, 7.1 mmol) was reacted with
2-bromobenzaldehyde (1.6 g, 8.5 mmol, 1.2 equiv) in the presence
of NaOEt (1.2 g, 17.7 mmol, 2.5 equiv). The mixture was purified
by crystallization from CH3OH/CH2Cl2 (1/1) to give 10 as a bright-
1
yellow solid (0.44 g, 20%). H NMR (600 MHz, CDCl3) δ 8.22
1
(d, J ) 15.7, 1H), 8.12 (s, 1H), 7.67 (dd, J ) 7.9, 1.0, 1H), 7.65
(dd, J ) 8.1, 0.8, 1H), 7.37 (t, J ) 7.4, 1H), 7.24 (td, J ) 7.7, 0.7,
1H), 6.87 (d, J ) 15.6, 1H), 4.08 (s, 3H); 13C NMR (100 MHz,
DMSO-d6 + CD2Cl2) δ 149.2, 139.0, 135.6, 134.8, 133.8, 132.9,
130.6, 127.7, 127.6, 124.2, 115.6, 32.7; HRMS (ESI+) calcd for
C12H11BrN3O2 [M + H]+ 308.0029, found 308.0034. Anal.
(C12H10BrN3O2) C, H, N.
give 16 as a yellow solid (316 mg, 15%). H NMR (DMSO-d6) δ
8.20 (s, 1H), 8.19 (d, J ) 8.8, 1H), 8.02 (d, J ) 16.0, 1H), 7.73 (d,
J ) 2.0, 1H), 7.54 (d, J ) 8.8, 1H), 7.53 (d, J ) 16.0, 1H), 4.05
(s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 148.9, 134.3, 134.0,
133.7, 132.0, 131.0, 129.3, 129.2, 128.9, 127.8, 116.6, 32.8; HRMS
(ESI+) calcd for C12H10Cl2N3O2 [M + H]+ 298.0145, found
298.0135. Anal. (C12H9Cl2N3O2): C, 48.34; H, 3.04; N, 14.09.
Found: C, 48.50; H, 3.75; N, 13.87.
2-[(E)-2-(3-Bromophenyl)vinyl]-1-methyl-5-nitro-1H-imida-
zole (11). Dimetridazole (1.5 g, 10.6 mmol) was reacted with
3-bromobenzaldehyde (2.3 g, 12.7 mmol, 1.2 equiv) in the presence
of NaOEt (1.8 g, 26.6 mmol, 2.5 equiv). The mixture was purified
by column chromatography (20% EtOAc/hexanes) to give 11 as a
2-[(E)-2-(2-Iodophenyl)vinyl]-1-methyl-5-nitro-1H-imida-
zole (17). Dimetridazole (1.0 g, 7.1 mmol) was reacted with
2-iodobenzaldehyde (2.0 g, 8.5 mmol, 1.2 equiv) in the presence
of NaOEt (1.2 g, 17.7 mmol, 2.5 equiv). The mixture was purified
by crystallization from CH3OH/CH2Cl2 (1/2) to give 17 as a bright-
orange solid (0.44 g, 20%). 1H NMR (500 MHz, CDCl3) δ 8.14 (s,
1H), 8.08 (d, J ) 15.6, 1H), 7.94 (dd, J ) 7.9, 1.2, 1H), 7.63 (dd,
J ) 7.9, 1.5, 1H), 7.41 (dt, J ) 7.4, 0.6, 1H), 7.07 (dt, J ) 7.7,
1.5, 1H), 6.79 (d, J ) 15.5, 1H), 4.08 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 149.1, 143.1, 140.1, 138.7, 134,3, 130.8, 128.6, 126.8,
114.5, 100.9, 33.0; HRMS (ESI+) calcd for C12H11IN3O2 [M + H]+
355.9891, found 355.9892. Anal. (C12H10IN3O2) C, H, N.
1
bright-yellow solid (0.62 g, 19%). H NMR (600 MHz, CDCl3) δ
8.04 (s, 1H), 7.75 (d, J ) 15.8, 1H), 7.52 (s, 1H), 7.47 (d, J ) 7.7,
1H), 7.42 (d, J ) 7.7, 2H), 6.88 (d, J ) 15.7, 1H), 4.06 (s, 3H);
13C NMR (150 MHz, CDCl3) δ 140.0, 143.1, 138.0, 133.8, 132.5,
130.4, 130.0, 129.7, 126.4, 125.2, 112.3, 32.8; HRMS (ESI+) calcd
for C12H11BrN3O2 [M + H]+ 308.0029, found 308.0034. Anal.
(C12H10BrN3O2): C, 46.78; H, 3.27; N, 13.64. Found: C, 47.65; H,
3.54; N, 13.08.
2-[(E)-2-(4-Bromophenyl)vinyl]-1-methyl-5-nitro-1H-imida-
zole (12). Dimetridazole (5.0 g, 35.4 mmol) was reacted with
4-bromobenzaldehyde (7.8, 42.5 mmol, 1.2 equiv) in the presence
of NaOEt (6.0 g, 88.5 mmol, 2.5 equiv). The mixture was purified
by column chromatography (CH2Cl2 f 10% CH3OH/CH2Cl2) to
give 12 as a yellow solid (7.8 g, 72%). 1H NMR (400 MHz, DMSO-
2-[(E)-2-(3-Iodophenyl)vinyl]-1-methyl-5-nitro-1H-imida-
zole (18). Dimetridazole (1.0 g, 7.1 mmol) was reacted with
3-iodobenzaldehyde (2.0 g, 8.5 mmol, 1.2 equiv) in the presence
of NaOEt (1.2 g, 17.7 mmol, 2.5 equiv). The mixture was purified
by column chromatography (50% CH2Cl2/hexanes f 100% CH2Cl2)
1
to give 18 as a dark-orange solid (0.50 g, 19%). H NMR (600

4050 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Valdez et al.
MHz, CDCl3) δ 8.09 (s, 1H), 7.93 (s, 1H), 7.77 (d, J ) 15.7, 1H),
7.70 (d, J ) 7.9, 1H), 7.50 (d, J ) 7.74, 1H), 7.15 (t, J ) 7.8, 1H),
6.87 (d, J ) 15.7, 1H), 4.07 (s, 3H); 13C NMR (150 MHz, CDCl3)
δ 149.2, 139.1, 138.4, 137.8, 137.4, 135.7, 134.1, 130.6, 127.0,
124.5, 94.8, 32.8; HRMS (ESI+) calcd for C12H11IN3O2 [M + H]+
355.9891, found 355.9902. Anal. (C12H10IN3O2): C, 40.58; H, 2.84;
N, 11.83. Found: C, 41.07; H, 2.91; N, 11.12.
32.8, 15.2; HRMS (ESI+) calcd for C17H22N3O4 [M + H]+
332.1605, found 332.1608. Anal. (C17H21N3O4) C, H, N.
2-[(E)-2-Biphenyl-2-ylvinyl]-1-methyl-5-nitro-1H-imidazole (24).
Dimetridazole (1.0 g, 7.1 mmol) was reacted with biphenyl-2-
carbaldehyde (1.67 g, 9.23 mmol, 1.3 equiv) in the presence of
NaOEt (966 mg, 14.2 mmol, 2.0 equiv). The mixture was purified
by column chromatography (CH2Cl2 f 5% CH3OH/CH2Cl2) to give
24 as a yellow solid (455 mg, 21%). 1H NMR (400 MHz, DMSO-
d6) δ 8.13-8.12 (m, 1H), 8.08 (s, 1H), 7.70 (d, J ) 15.6, 1H),
7.51 -7.43 (m, 5H), 7.39-7.32 (m, 4H), 4.02 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 149.7, 141.7, 139.8, 138.9, 135.9, 134.0,
132.9, 130.3, 129.5, 129.2, 128.3, 127.7, 127.4, 126.5, 113.7, 32.7;
HRMS (ESI+) calcd for C18H16N3O2 [M + H]+ 306.1237, found
306.1225. Anal. (C18H15N3O2) C, H, N.
2-[(E)-2-(3-Fluorophenyl)vinyl]-1-methyl-5-nitro-1H-imida-
zole (19). Dimetridazole (1.0 g, 7.1 mmol) was reacted with
3-fluorobenzaldehyde (1.1 g, 8.5 mmol, 1.2 equiv) in the presence
of NaOEt (1.2 g, 17.7 mmol, 2.5 equiv). The mixture was purified
by column chromatography (50% CH2Cl2/hexanes f 100% CH2Cl2)
1
to give 19 as a bright-yellow solid (0.23 g, 13%). H NMR (500
MHz, CDCl3) δ 8.10 (s, 1H), 7.86 (d, J ) 15.7, 1H), 7.40-7.37
(m, 1H), 7.35-7.34 (m, 1H), 7.29 (dt, J ) 10, 2.2, 1H), 7.09 (tdd,
J ) 8.1, 2.6, 1.0, 1H), 6.90 (d, J ) 15.7, 1H), 4.08 (s, 3H); 13C
NMR (150 MHz, CDCl3) δ 163.1 (d, J ) 245.2), 149.2, 139.1,
138.3 (d, J ) 2.9), 137.5 (d, J ) 7.3), 134.1, 130.5 (d, J ) 8.1),
123.6 (d, J ) 2.9), 116.6 (d, J ) 21.5), 113.5 (d, J ) 21.8), 112.5,
32.8; HRMS (ESI+) calcd for C12H11FN3O2 [M + H]+ 248.0830,
found 248.0820. Anal. (C12H10FN3O2) C, H, N.
1-Methyl-2-[(E)-2-(1-naphthyl)vinyl]-5-nitro-1H-imidazole (25).
Dimetridazole (1.0 g, 7.1 mmol) was reacted with 1-naphthaldehyde
(1.25 mL, 9.23 mmol, 1.3 equiv) in the presence of NaOEt (966
mg, 14.2 mmol, 2.0 equiv). The mixture was purified by column
chromatography (CH2Cl2 f 10% CH3OH/CH2Cl2) to give 25 as a
1
yellow solid (554 mg, 28%). H NMR (400 MHz, DMSO-d6) δ
8.59 (d, J ) 15.6, 1H), 8.26 (d, J ) 8.4, 1H), 8.25 (s, 1H), 8.16 (d,
J ) 8.0, 1H), 8.01-7.99 (m, 2H), 7.66-7.59 (m, 3H), 7.49 (d, J
) 15.6, 1H), 4.08 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 149.7,
134.1, 133.7, 133.3, 132.0, 130.6, 129.7, 128.7, 127.0, 126.1, 125.8,
125.6, 124.6, 128.8, 115.5, 32.8; HRMS (ESI+) calcd for
2-[(E)-2-(3-Methoxyphenyl)vinyl]-1-methyl-5-nitro-1H-imida-
zole (20). Dimetridazole (1.0 g, 7.1 mmol) was reacted with
3-methoxybenzaldehyde (1.13 mL, 9.23 mmol, 1.3 equiv) in the
presence of NaOEt (966 mg, 14.2 mmol, 2.0 equiv). The mixture
was purified by column chromatography (CH2Cl2 f 5% CH3OH/
C16H14N3O2 [M
(C16H13N3O2) C, H, N.
+
H]+ 280.1080, found 280.1085. Anal.
1
CH2Cl2) to give 20 as a yellow solid (588 mg, 32%). H NMR
(400 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.70 (d, J ) 16.0, 1H), 7.35
(d, J ) 16.0, 1H), 7.34 (br s, 1H), 7.29 (d, J ) 5.2, 1H), 6.93-6.90
(m, 1H), 3.99 (s, 3H), 3.77 (s, 3H); 13C NMR (100 MHz, DMSO-
d6) δ 159.6, 149.7, 139.0, 137.9, 136.8, 134.1, 129.8, 120.5, 115.3,
113.3, 112.4, 55.2, 32.8; HRMS (ESI+) calcd for C13H14N3O3 [M
+ H]+ 260.1030, found 260.1029. Anal. (C13H13N3O3) C, H, N.
1-Methyl-5-nitro-2-{(E)-2-[4-(trifluoromethoxy)phenyl]vinyl}-
1H-imidazole (21). Dimetridazole (1.0 g, 7.1 mmol) was reacted
with 4-(trifluoromethoxy)benzaldehyde (1.33 mL, 9.23 mmol, 1.3
equiv) in the presence of NaOEt (966 mg, 14.2 mmol, 2.0 equiv).
The mixture was purified by column chromatography (CH2Cl2 f
10% CH3OH/CH2Cl2) to give 21 as a yellow solid (311 mg, 14%).
1H NMR (400 MHz, DMSO-d6) δ 8.13 (s, 1H), 7.88 (d, J ) 8.8,
2H), 7.73 (d, J ) 15.6, 1H), 7.38 (d, J ) 15.6, 2H), 7.36 (d, J )
8.8, 1H), 3.98 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 149.3,
148.6, 148.5, 148.6, 148.6, 142.0, 139.1, 136.1, 134.8, 134.0, 129.5,
128.0, 121.2, 120.6, 118.7, 114.3, 32.8; HRMS (ESI+) calcd for
C13H11F3N3O3 [M + H]+ 314.0747, found 314.0752. Anal.
(C13H10F3N3O3): C, 49.85; H, 3.22; N, 13.42. Found: C, 49.92; H,
3.60; N, 13.88.
1-Methyl-2-[(E)-2-(2-naphthyl)vinyl]-5-nitro-1H-imidazole (26).
Dimetridazole (1.0 g, 7.1 mmol) was reacted with 2-naphthaldehyde
(1.44 g, 9.23 mmol, 1.3 equiv) in the presence of NaOEt (966 g,
14.2 mmol, 2.0 equiv). The mixture was purified by column
chromatography (CH2Cl2 f 10% CH3OH/CH2Cl2) to give 26 as a
1
yellow solid (385 mg, 19%). H NMR (400 MHz, DMSO-d6) δ
8.22 (s, 1H), 8.21 (br s, 1H), 8.07 (dd, J ) 8.8, 1.6, 1H), 7.98-7.92
(m, 4H), 7.56-7.52 (m, 3H), 4.08 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 149.8, 137.8, 134.2, 133.3, 133.1, 133.0, 128.6, 128.3,
128.2, 127.6, 126.9, 126.8, 126.6, 123.9, 113.5, 32.8; HRMS (ESI+)
calcd for C16H14N3O2 [M + H]+ 280.1080, found 280.1081. Anal.
(C16H13N3O2): C, 68.81; H, 4.69; N, 15.05. Found: C, 68.21; H,
4.78; N, 14.95.
2-[(E)-2-(3a,7a-Dihydro-1,3-benzodioxol-5-yl)vinyl]-1-methyl-
5-nitro-1H-imidazole (27). Dimetridazole (1.0 g, 7.1 mmol) was
reacted with piperonal (1.39 g, 9.23 mmol, 1.3 equiv) in the
presence of NaOEt (966 mg, 14.2 mmol, 2.0 equiv). The mixture
was purified by column chromatography (CH2Cl2 f 10% CH3OH/
1
CH2Cl2) to give 27 as a yellow solid (600 mg, 31%). H NMR
(400 MHz, DMSO-d6) δ 8.17 (s, 1H), 7.70 (d, J ) 15.6, 1H), 7.57
(d, J ) 1.6, 1H), 7.25 (d, J ) 15.6, 1H), 7.21 (dd, J ) 8.0, 1.6z,
1H), 6.96 (d, J ) 8.0, 1H), 6.09 (s, 2H), 4.01 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 150.1, 148.4, 148.0, 137.9, 134.3, 130.0,
124.0, 111.0, 108.4, 106.0, 101.4, 89.3, 32.7; HRMS (ESI+) calcd
for C13H12N3O4 [M + H]+ 274.0822, found 274.0823. Anal.
(C13H11N3O4) C, H, N.
1-Methyl-5-nitro-2-[(E)-2-(2,3,4-trimethoxyphenyl)vinyl]-1H-
imidazole (22). Dimetridazole (1.0 g, 7.1 mmol) was reacted with
2,3,4-trimethoxybenzaldehyde (1.67 g, 8.52 mmol, 1.2 equiv) in
the presence of NaOEt (1.2 g, 17.8 mmol, 2.5 equiv). The mixture
was purified by column chromatography (CH2Cl2 f 10% CH3OH/
1
CH2Cl2) to give 22 as a yellow solid (362 mg, 16%). H NMR
(400 MHz, CDCl3) δ 8.06 (s, 1H), 7.94 (d, J ) 16.0, 1H), 7.23 (d,
J ) 8.8, 1H), 6.96 (d, J ) 16.0, 1H), 6.68 (d, J ) 8.8, 1H), 4.00
(s, 3H), 3.93 (s, 3H), 3.88 (s, 3H), 3.87 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 155.1, 152.9, 150.7, 142.4, 138.7, 135.2, 134.4,
123.4, 122.2, 110.6, 107.5, 61.1, 60.8, 56.0, 32.6; HRMS (ESI+)
calcd for C15H18N3O5 [M + H]+ 320.1241, found 320.1240. Anal.
(C15H17N3O5) C, H, N.
1-Methyl-2-[(E)-2-(1-methyl-1H-imidazol-2-yl)vinyl]-5-nitro-
1H-imidazole (28). Dimetridazole (1.06 g, 7.5 mmol) was reacted
with 1-methyl-1H-imidazole-2-carbaldehyde (1.0 g, 9.1 mmol, 1.2
equiv) in the presence of NaOEt (1.27 g, 18.8 mmol, 2.5 equiv).
The mixture was purified by column chromatography (CH2Cl2 f
10% CH3OH/CH2Cl2) to give 28 as a yellow solid (1.36 g, 77%).
1H NMR (400 MHz, DMSO-d6) δ 8.20 (s, 1H), 7.64 (d, J ) 15.2,
1H), 7.33 (d, J ) 15.2, 1H), 7.31 (s, 1H), 7.07 (s, 1H), 3.99 (s,
3H), 3.78 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 149.3, 143.4,
134.1, 129.2, 124.0, 123.0, 113.6, 32.7, 32.4; HRMS (ESI+) calcd
for C10H12N5O2 [M + H]+ 234.0985, found 234.0986. Anal.
(C10H11N5O2) C, H, N.
2-[(E)-2-(2-Furyl)vinyl]-1-methyl-5-nitro-1H-imidazole (29).
Dimetridazole (1.0 g, 7.1 mmol) was reacted with furan-2-
carbaldehyde (0.65 mL, 7.81 mmol, 1.1 equiv) in the presence of
NaOEt (1.2 g, 17.8 mmol, 2.5 equiv). The mixture was purified by
column chromatography (CH2Cl2 f 10% CH3OH/CH2Cl2) to give
29 as a yellow solid (825 mg, 53%). 1H NMR (400 MHz, DMSO-
2-{(E)-2-[4-(Diethoxymethyl)phenyl]vinyl}-1-methyl-5-nitro-
1H-imidazole (23). Dimetridazole (20 g, 142 mmol) was reacted
with 4-(diethoxymethyl)benzaldehyde (34.5 mL, 170 mmol, 1.2
equiv) in the presence of NaOEt (24 g, 354 mmol, 2.5 equiv). The
mixture was purified by column chromatography (CH2Cl2 f 10%
1
CH3OH/CH2Cl2) to give 23 as a yellow solid (5.67 g, 24%). H
NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 7.87 (d, J ) 16.0, 1H),
7.56 (d, J ) 8.0, 2H), 7.51 (d, J ) 8.0, 2H), 6.89 (d, J ) 16.0,
1H), 5.52 (s, 1H), 4.05 (s, 3H), 3.65-3.53 (m, 4H, 2 × OCH2CH3),
1.25 (t, J ) 7.2, 6H, 2 × OCH2CH3); 13C NMR (100 MHz, CDCl3)
δ 149.8, 140.9, 139.4, 135.2, 134.2, 127.3, 111.3, 101.0, 89.8, 61.1,

2-Ethenyl and 2-Ethanyl DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 4051
d6) δ 8.18 (s, 1H), 7.83 (d, J ) 1.6, 1H), 7.58 (d, J ) 15.6, 1H),
7.00 (d, J ) 15.6, 1H), 6.90 (d, J ) 3.2, 1H), 6.64 (dd, J ) 3.2,
2.0, 1H), 3.98 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 151.4, 149.5,
144.9, 139.0, 134.2, 124.8, 113.6, 112.7, 110.3, 32.6; HRMS (ESI+)
calcd for C10H10N3O3 [M + H]+ 220.0717, found 220.0720. Anal.
(C10H9N3O3) C, H, N.
1-(4-Bromophenyl)-2-(1-methyl-5-nitro-1H-imidazol-2-yl)-
ethane-1,2-diol (35). Compound 12 (1.42 g, 4.62 mmol) was made
into a suspension in 1:1 t-BuOH/H2O (60 mL). Citric acid (1.78 g,
9.25 mmol, 2.0 equiv) was added in one portion followed by
potassium osmate dihydrate (3.0 mg, 0.0092 mmol, 0.2 mol %).
To the black suspension was added N-methylmorpholine N-oxide
(NMO, 0.52 mL, 5.08 mmol, 1.1 equiv) dropwise at ambient
temperature, and the resulting suspension was stirred vigorously
overnight. The mixture was filtered through Celite, and the brown
filtrate was transferred to a separatory funnel and partitioned
(CH2Cl2/H2O). The organic phase was washed with brine (2 × 100
mL), dried over Na2SO4, and evaporated in vacuo to give a brown
residue. The residue was purified by column chromatography
(CH2Cl2 f 5% MeOH/CH2Cl2) to give diol 35 as a white foam
1-Methyl-5-nitro-2-[(E)-2-(2-thienyl)vinyl]-1H-imidazole (30).
Dimetridazole (1.0 g, 7.1 mmol) was reacted with thiophene-2-
carbaldehyde (0.86 mL, 9.23 mmol, 1.3 equiv) in the presence of
NaOEt (966 mg, 14.2 mmol, 2.0 equiv). The mixture was purified
by column chromatography (CH2Cl2 f 10% CH3OH/CH2Cl2) to
1
give 30 as a yellow solid (384 mg, 23%). H NMR (400 MHz,
DMSO-d6) δ 8.18 (s, 1H), 7.90 (d, J ) 1.6, 1H), 7.78 (d, J ) 15.6,
1H), 7.70 (dd, J ) 5.2, 1.2, 1H), 7.63 (dd, J ) 4.8, 2.8, 1H), 7.21
(d, J ) 15.6, 1H), 4.00 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
150.0, 138.9, 138.7, 134.2, 132.1, 127.4, 125.6, 112.6, 32.7; HRMS
(ESI+) calcd for C10H10N3O2S [M + H]+ 236.0488, found 236.0491.
Anal. (C10H9N3O2S): C, 51.05; H, 3.86; N, 17.86. Found: C, 51.48;
H, 3.35; N, 17.57.
1
(225 mg, 14%). H NMR (400 MHz, DMSO-d6) δ 7.96 (s, 1H),
7.43 (d, J ) 8.4, 2H), 7.21 (d, J ) 8.4, 2H), 6.07 (d, J ) 4.8, 1H),
5.81 (d, J ) 4.8, 1H), 4.93-4.91 (m, 1H), 4.80-4.78 (m, 1H),
3.85 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 152.1, 140.9, 138.9,
131.6, 130.5, 129.1, 120.2, 74.1, 71.5, 33.4; HRMS (ESI+) calcd
for C12H13BrN3O4 [M + H]+ 342.0084, found 342.0086. Anal.
(C12H12BrN3O4): C, 42.12; H, 3.54; N, 12.28. Found: C, 41.84; H,
3.77; N, 12.47.
1-Methyl-5-nitro-2-[(E)-2-(3-thienyl)vinyl]-1H-imidazole (31).
Dimetridazole (1.0 g, 7.1 mmol) was reacted with thiophene-3-
carbaldehyde (0.81 mL, 9.23 mmol, 1.3 equiv) in the presence of
NaOEt (966 mg, 14.2 mmol, 2.0 equiv). The mixture was purified
by column chromatography (CH2Cl2 f 10% CH3OH/CH2Cl2) to
1-(1-Methyl-5-nitro-1H-imidazol-2-yl)-2-(4-methylphenyl)-
ethane-1,2-diyl Diacetate (36). Diol 34 (90 mg, 0.325 mmol) was
dissolved in anhydrous pyridine (0.5 mL), and the solution was
cooled to 0 °C. Acetic anhydride (132.6 mg, 122 µL, 1.3 mmol,
4.0 equiv) was added dropwise via syringe, and the resulting
solution was stirred at ambient temperature for 3 h. The mixture
was transferred to a separatory funnel and partitioned (CH2Cl2/
H2O). The organic phase was washed with 1.0 N HCl (2 × 50
mL), brine (2 × 50 mL), dried over Na2SO4, and evaporated in
vacuo to give a yellow oil. The residue was purified by column
chromatography (hexanes f 40% EtOAc/hexanes) to provide
1
give 31 as a yellow solid (367 mg, 22%). H NMR (400 MHz,
DMSO-d6) δ 8.18 (s, 1H), 7.91 (d, 15.6, 1H), 7.67 (d, J ) 5.2,
1H), 7.52 (d, J ) 3.6, 1H), 7.15 (dd, J ) 5.2, 3.6, 1H), 7.03 (d, J
) 15.6, 1H), 3.99 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 149.5,
140.3, 138.9, 134.2, 130.8, 130.1, 128.5, 128.3, 111.6, 32.7; HRMS
(ESI+) calcd for C10H10N3O2S [M + H]+ 236.0488, found 236.0493.
Anal. (C10H9N3O2S): C, 51.05; H, 3.86; N, 17.86. Found: C, 50.90;
H, 3.54; N, 17.67.
2-[1,2-Dibromo-2-(4-bromophenyl)ethyl]-1-methyl-5-nitro-
1H-imidazole (33). Compound 12 (2.05 g, 6.7 mmol) was dissolved
in anhydrous CHCl3 (120 mL), and the pale-yellow solution was
cooled to 0 °C. Bromine (0.37 mL, 1.12 g, 7.03 mmol, 1.05 equiv)
was added via syringe at 0 °C, and the resulting orange solution
was stirred at ambient temperature for 4 h. The mixture was
transferred to a separatory funnel and partitioned (CH2Cl2//H2O).
The organic phase was washed with Na2S2O3 (3 × 100 mL), brine
(NaCl/H2O, 2 × 50 mL), dried over Na2SO4, and evaporated in
vacuo to give a pale-yellow solid. The residue was purified by
column chromatography (hexanes f 30% EtOAc/hexanes) to give
33 as an off-white solid (2.73 g, 88%). 1H NMR (400 MHz, DMSO-
d6) δ 8.20 (s, 1H), 7.74 (d, J ) 8.4, 2H), 7.61 (d, J ) 8.4, 2H),
6.49 (d, J ) 11.2, 1H), 6.03 (d, J ) 11.6, 1H), 4.01 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ 149.1, 139.1, 138.4, 132.3, 131.5, 130.7,
122.3, 51.7, 42.0, 33.3; HRMS (ESI+) calcd for C12H11Br3N3O2
[M + H]+ 465.8296, found 465.8394. Anal. (C12H10Br3N3O2) C,
H, N.
1-(1-Methyl-5-nitro-1H-imidazol-2-yl)-2-(4-methylphenyl)-
ethane-1,2-diol (34). Compound 8 (0.69 g, 2.85 mmol) was made
into a suspension in t-BuOH/H2O (1:1, 37 mL). Citric acid (1.10
g, 5.7 mmol, 2.0 equiv) was added in one portion followed by
potassium osmate dihydrate (2.1 mg, 0.0057 mmol, 0.2 mol %).
To the black suspension was added N-methylmorpholine N-oxide
(NMO, 0.32 mL, 1.1 equiv) dropwise at ambient temperature, and
the resulting suspension was stirred vigorously overnight. The
mixture was filtered through Celite, and the brown filtrate was
transferred to a separatory funnel and partitioned (CH2Cl2/H2O).
The organic phase was washed with brine (2 × 100 mL), dried
over Na2SO4, and evaporated in vacuo to give a brown residue.
Crude product was purified by column chromatography (EtOAc)
to give 34 as a yellowish solid (220 mg, 28%). 1H NMR (400 MHz,
CDCl3 + CD3OD) δ 7.86 (s, 1H), 7.01 (s, 4H), 4.92 (d, J ) 6.9,
1H), 4.64 (d, J ) 6.9, 1H), 3.49 (s, 3H), 3.35 (br s, 2H), 2.23 (s,
3H); 13C NMR (100 MHz, CDCl3 + CD3OD) δ 151.4, 138.0, 135.8,
131.3, 129.0, 129.0, 126.2, 75.6, 70.9, 32.8, 20.9; HRMS (ESI+)
calcd for C13H16N3O4 [M + H]+ 278.1135, found 278.1137. Anal.
(C13H15N3O4): C, 56.31; H, 5.45; N, 15.15. Found: C, 56.41; H,
5.44; N, 15.11.
1
acetylated adduct 36 as a yellowish wax (74 mg, 66%). H NMR
(400 MHz, CDCl3) δ 7.93 (s, 1H), 7.11-7.04 (m, 4H), 6.36 (d, J
) 9.4, 1H), 5.93 (d, J ) 9.4, 1H), 3.62 (s, 3H), 2.28 (s, 3H), 2.13
(s, 3H), 2.11 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 170.4, 169.2,
147.0, 139.3, 138.5, 132.5, 131.7, 129.4, 127.0, 74.7, 68.6, 32.7,
21.6, 21.0, 20.6; HRMS (ESI+) calcd for C17H19N3O6Na [M + Na]+
384.1166, found 384.1172. Anal. (C17H19N3O6): C, 56.51; H, 5.30;
N, 11.63. Found: C, 53.32; H, 5.13; N, 10.22.
1-(1-Methyl-5-nitro-1H-imidazol-2-yl)-2-(4-bromophenyl)-
ethane-1,2-diyl Diacetate (37). Diol 35 (200 mg, 0.58 mmol) was
dissolved in anhydrous pyridine (10 mL), and the solution was
cooled to 0 °C. Acetic anhydride (0.14 mL, 1.45 mmol, 2.5 equiv)
was added dropwise via syringe, and the resulting solution was
stirred at ambient temperature for 3 h. The mixture was transferred
to a separatory funnel and partitioned (CH2Cl2//H2O). The organic
phase was washed with 1.0 N HCl (2 × 50 mL), brine (2 × 50
mL), dried over Na2SO4, and evaporated in vacuo to give a yellow
oil. The residue was purified by column chromatography (hexanes
f 40% EtOAc/hexanes) to provide acetylated adduct 37 as a
1
colorless oil (227 mg, 92%). H NMR (400 MHz, CDCl3) δ 7.93
(s, 1H), 7.41 (d, J ) 8.4, 2H), 7.13 (d, J ) 8.4, 2H), 6.39 (d, J )
9.2, 1H), 5.96 (d, J ) 9.2, 1H), 3.73 (s, 3H), 2.13 (s, 3H), 2.11 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 170.3, 169.1, 146.4, 143.9,
133.9, 132.4, 132.0, 129.0, 123.5, 74.1, 68.0, 32.9, 20.9, 20.6;
HRMS (ESI+) calcd for C16H17BrN3O6 [M + H]+ 426.0295, found
426.0300. Anal. (C16H16BrN3O6) C, H, N.
1-Methyl-5-nitro-2-(2-phenylethyl)-1H-imidazole (40). Com-
pound 5 (1.0 g, 4.37 mmol), p-toluenesulfonhydrazine (4.06 g, 21.82
mmol, 5.0 equiv), and K2CO3 (3.02 g, 21.82 mmol, 5.0 equiv) in
pyridine (30 mL) were stirred at 120 °C for 14 h. Purification of
the mixture was carried out by preparatory thin layer chromatog-
raphy on silica gel, eluting with 70% EtOAc/hexanes to furnish 40
1
as a white solid (0.324 g, 32%). H NMR (600 MHz, CDCl3) δ
8.00 (s, 1H), 7.35-7.29 (m, 2H), 7.25 (tt, J ) 7.2, 1.7, 1H), 7.14
(dd, J ) 7.0, 1.3, 2H), 3.26 (s, 3H), 3.13 (t, J ) 7.3, 2H), 3.04 (t,
J ) 8.0, 2H); 13C NMR (150 MHz, CDCl3) δ 152.6, 139.8, 138.8,
132.6, 128.8, 128.3, 126.8, 33.6, 32.7, 29.7; HRMS (ESI+) calcd
for C12H14N3O2 [M + H]+ 232.1080, found 232.1083. Anal.

4052 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Valdez et al.
(C12H13N3O2): C, 62.33; H, 5.67; N, 18.17. Found: C, 62.71; H,
5.77; N, 17.57.
volume. Final compound concentrations ranged from 100 µM
to 2 nM. Cells were cultured for 2 days, and viable cell numbers
were determined with a dye reduction assay. For this, Alamar-
Blue reagent (AbD Serotec) was added at 5 µL/well, and cultures
were incubated for 4 h, after which fluorescence was assayed in
a microplate reader. The 50% inhibitory concentration (IC50),
the drug concentration that caused 50% loss of viability in the
HeLa cultures compared to parallel cultures without added drugs,
was determined by graphic extrapolation of the concentration-
response curves. Toxicity is expressed as negative log10 value
of the IC50 (pIC50), with results calculated as the mean ( SE of
three to four independent experiments.
Murine Giardia Infections. Adult C57BL/6 mice were obtained
from The Jackson Laboratory (Bar Harbor, ME). For infections,
trophozoites of G. lamblia GS/M were grown to mid-logarithmic
growth phase and given by oral gavage at 107/mouse in a 0.2 mL
volume in TYI-S-33 growth medium. After 2 days (to allow
establishment of infection), mice were given a test compound in
1% methylcellulose in PBS by oral gavage five times over a 3-day
period. Controls received only methylcellulose/PBS. At day 5, the
entire small intestine was removed and opened longitudinally,
placed into 5 mL PBS, and cooled on ice for 10 min. After vigorous
shaking to detach trophozoites, live G. lamblia were counted in a
hemocytometer with a phase-contrast microscope. All animal studies
were reviewed and approved by the UCSD Institutional Animal
Care and Use Committee.
Electrochemical Measurements. E1/2 values were measured by
cyclic voltammetry, using a CH Instruments model 600c digital
potentiostat with its standard software package. All measure-
ments were conducted under Ar gas in a jacketed, one-
compartment cell with a Pt wire counter electrode. For the
nonaqueous CVs, a Au disk working electrode (∼1 mm diameter)
was used along with a Ag wire pseudoreference electrode. For
the aqueous CVs, a glass carbon working electrode (∼2 mm
diameter) was used with a SCE reference electrode. Immediately
before use, the working electrode was polished with 0.25 µm
diamond polishing paste, rinsed thoroughly with deionized water,
polished with 0.05 µm alumina paste, rinsed again with deionized
water, then rinsed with acetone and dried with a tissue. The
electrolyte solution used for the nonaqueous CVs was 0.10 M
NBu4PF6 in DMSO. Prior to use, the solid electrolyte was
recrystallized three times from 95% ethanol, dried overnight in
vacuum at 100 °C, and stored in a desiccator. The solvent,
anhydrous grade DMSO (Aldrich, sure-seal), was passed through
a short column of activated alumina directly into the cell.
Ferrocene was added as an internal potential reference. For the
aqueous CVs, the electrolyte solution was 0.1 M KCl in pH 7.0
phosphate buffer (0.1 M ionic strength). For all experiments,
oxygen was removed by bubbling Ar gas through the solution
prior to making measurements. The temperature was controlled
by using a circulating water bath to run 25 °C water through
the outer cell jacket. In most experiments, 5 µL of a 100 mM
stock solution of the 5-NI compounds in DMSO was added to
3 mL of electrolyte solution in the cell to give a measurement
concentration of 0.17 mM. CVs were recorded at 100 mV/s.
1-Methyl-2-[2-(4-methylphenyl)ethyl]-5-nitro-1H-imidazole
(41). Compound 8 (0.9 g, 3.75 mmol), p-toluenesulfonhydrazine
(3.49 g, 18.8 mmol, 5.0 equiv), and K2CO3 (2.59 g, 18.8 mmol,
5.0 equiv) in anhydrous pyridine (30 mL) were stirred at 120 °C
for 14 h. The mixture was cooled to ambient temperature,
transferred to a separatory funnel, and partitioned (CH2Cl2/H2O).
The organic phase was washed with brine (NaCl/H2O, 2 × 50 mL),
NaHCO3/H2O (2 × 50 mL), dried over Na2SO4, and evaporated in
vacuo to give a brown residue. The residue was purified by flash
chromatography (CH2Cl2 f 5% MeOH/CH2Cl2) to give 41 as a
1
pale-yellow solid (166 mg, 18%). H NMR (400 MHz, CDCl3) δ
7.98 (s, 1H), 7.10 (d, J ) 8.0, 2H), 7.03 (d, J ) 8.0, 2H), 3.66 (s,
3H), 3.10-3.06 (m, 2H), 3.04-2.99 (m, 2H), 2.33 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ 152.7, 136.6, 136.2, 132.5, 129.3, 128.1,
127.9, 33.0, 32.7, 29.6, 20.9; HRMS (ESI+) calcd for C13H16N3O2
[M + H]+ 246.1237, found 246.1239. Anal. (C13H15N3O2): C, 63.66;
H, 6.16; N, 17.13. Found: C, 63.15; H, 6.54; N, 17.65.
2-[2-(4-Bromophenyl)ethyl]-1-methyl-5-nitro-1H-imidazole (42).
Compound 12 (0.9 g, 2.95 mmol)), p-toluenesulfonhydrazine (2.73
g, 14.7 mmol, 5.0 equiv), and K2CO3 (2.03 g, 14.7 mmol, 5.0 equiv)
in anhydrous pyridine (30 mL) were stirred at 120 °C for 14 h.
The mixture was transferred to a separatory funnel and partitioned
(CH2Cl2/H2O). The organic phase was washed with 1.0 N HCl (3
× 100 mL), dried over Na2SO4, and evaporated in vacuo to give a
brown residue which was purified by flash chromatography (hexanes
f 40% EtOAc/hexanes) to provide 42 as an off-white solid (151
1
mg, 17%). H NMR (400 MHz, DMSO-d6) δ 8.03 (s, 1H), 7.47
(d, J ) 8.4, 2H), 7.23 (d, J ) 8.4, 2H), 3.78 (s, 3H), 3.07-3.04
(m, 2H), 3.03-2.98 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 152.8,
139.8, 138.7, 132.2, 131.0, 130.7, 119.1, 32.7, 31.3, 28.0; HRMS
(ESI+) calcd for C12H13BrN3O2 [M + H]+ 310.0186, found
310.0189. Anal. (C12H12BrN3O2) C, H, N.
Antimicrobial Assays. G. lamblia C6/WB (ATCC 50803),
GS/M (ATCC 50580), BRIS/87/HEPU/713 (713),24 BRIS/83/
HEPU/106 (106)25 were used as Mz-sensitive lines. The respective
isogenic lines, 713-M3 and 106-21D10, were employed as Mz-
resistant lines.24,25 C6/WB, 713, and 106 belong to G. lamblia
assemblage A, while GS/M belongs to assemblage B. All lines were
grown in TYI-S-33 medium supplemented with 10% bovine serum
and 1 mg/mL bovine bile (Sigma) under anaerobic conditions.36
The Mz-resistant lines were routinely cultured with 50 µM Mz but
were grown without Mz for 2-3 days before experiments. Stocks
of the test compounds (10 mM in DMSO) were diluted in PBS to
75 µM, and 1:3 serial dilutions were made in 40 µL TYI-S-33
medium in 96-well plates. Trophozoites were harvested in the mid-
logarithmic growth phase and were added to the wells in a 10 µL
volume at 1000-2000 cells/well (total volume 50 µL/well). Final
compound concentrations ranged from 20 µM to 0.4 nM, covering
a 5 log10 range. Appropriately diluted solvent alone was used as a
control. Cultures were grown for 2 days at 37 °C under anaerobic
conditions (AnaeroPack-Anaero system, Remel). After incubation,
cell growth and viability were determined with an ATP assay by
adding 50 µL/well BacTiter-Glo microbial cell viability assay
reagent (Promega) and measuring the luminescent signal in a
microplate reader. The 50% effective concentration (EC50), the drug
concentration that inhibits G. lamblia growth by 50% compared to
parallel cultures without added drugs, was determined by graphic
extrapolation of the concentration-response curves. Antigiardial
potency is expressed as negative log10 value of the EC50 (pEC50),
with results calculated as mean ( SE of three to four independent
experiments. Initial studies showed that the results of the ATP
assays correlated well with direct microscopic evaluation.
Acknowledgment. This work was supported by NIH U01
Cooperative Research Agreement AI75527 and the UCSD
Digestive Diseases Research Development Center (DK80506).
J.C.T. was supported by an NIH postdoctoral fellowship under
the Ruth L. Kirschstein National Research Service Award
program. P.H. was supported by a fellowship from the Swiss
National Science Foundation (SSMBS; PASMA 114623). K.K.
and L.V.A. received financial support from the CSU-LSAMP
program funded by National Science Foundation, Grant HRD-
0331537. We thank Taryn K. Fernandes, Cameron A. Collins,
and M. Naomi King for help with the cyclic voltammetry
studies.
Cytotoxicity Assays. The human epithelial cell line HeLa
(ATCC CCL-2) was maintained in DMEM, supplemented with
10% FBS, 5 mM HEPES, 50 U/mL penicillin, and 50 µg/mL
streptomycin, at 37 °C in 5% CO2/95% air. Compound stocks
in DMSO were diluted in DMEM to a concentration of 375 µM.
Serial 1:3 dilutions were made in DMEM in 96-well plates (40
µL/well), and 2000 HeLa cells were added per well in a 10 µL

2-Ethenyl and 2-Ethanyl DeriVatiVes
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