Nitroimidazole carboxamides as antiparasitic agents targeting Giardia lamblia, Entamoeba histolytica and Trichomonas vaginalis

Article abstract of DOI:10.1016/j.ejmech.2016.04.064

Diarrhoeal diseases caused by the intestinal parasites Giardia lamblia and Entamoeba histolytica constitute a major global health burden. Nitroimidazoles are first-line drugs for the treatment of giardiasis and amebiasis, with metronidazole 1 being the mo

Full text of DOI:10.1016/j.ejmech.2016.04.064

European Journal of Medicinal Chemistry 120 (2016) 353e362
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Nitroimidazole carboxamides as antiparasitic agents targeting Giardia
lamblia, Entamoeba histolytica and Trichomonas vaginalis
A.M. Jarrad a, A. Debnath b, Y. Miyamoto c, K.A. Hansford a, R. Pelingon a, M.S. Butler a,
,
, *
T. Bains b, T. Karoli a 1, M.A.T. Blaskovich a, L. Eckmann c, M.A. Cooper a
a Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, 4072, Australia
b Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La
Jolla, CA, USA
c Department of Medicine, University of California, San Diego, La Jolla, CA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Diarrhoeal diseases caused by the intestinal parasites Giardia lamblia and Entamoeba histolytica consti-
tute a major global health burden. Nitroimidazoles are first-line drugs for the treatment of giardiasis and
amebiasis, with metronidazole 1 being the most commonly used drug worldwide. However, treatment
failures in giardiasis occur in up to 20% of cases and development of resistance to metronidazole is of
concern. We have re-examined ‘old’ nitroimidazoles as a foundation for the systematic development of
next-generation derivatives. Using this approach, derivatisation of the nitroimidazole carboxamide
scaffold provided improved antiparasitic agents. Thirty-three novel nitroimidazole carboxamides were
synthesised and evaluated for activity against G. lamblia and E. histolytica. Several of the new compounds
exhibited potent activity against G. lamblia strains, including metronidazole-resistant strains of G. lamblia
Received 8 February 2016
Received in revised form
24 April 2016
Accepted 25 April 2016
Available online 27 April 2016
Keywords:
Nitroimidazole
Antiparasitic agent
Giardia lamblia
Entamoeba histolytica
Plasma protein binding
Metabolism
(EC50 ¼ 0.1e2.5
against E. histolytica (EC50 ¼ 1.7e5.1
chomonas vaginalis (EC50 ¼ 0.6e1.4
the intestinal bacterial pathogen Clostridium difficile (0.5e2
new compounds had low toxicity against mammalian kidney and liver cells (CC50 > 100
m
M cf. metronidazole EC50 ¼ 6.1e18
M cf. metronidazole EC50 ¼ 5.0
M cf. metronidazole EC50 ¼ 0.8 M) and moderate activity against
g/mL, cf. metronidazole ¼ 0.5 g/mL). The
M), and
m
M). Other compounds showed improved activity
m
m
M), potent activity against Tri-
m
m
m
m
m
selected antiparasitic hits were assessed for human plasma protein binding and metabolic stability in
liver microsomes to demonstrate their therapeutic potential.
© 2016 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Upon ingestion of G. lamblia cysts, trophozoites emerge from the
cysts and multiply in the lumen of the small intestine, where they
Diarrhoeal diseases caused by intestinal protozoan parasites are
a major global health burden. Two of the most common intestinal
parasites, Giardia lamblia and Entamoeba histolytica, are responsible
for ~280 million and ~50 million annual infections, respectively
[1,2]. Transmission of these parasites occurs by the faecal-oral route
through ingestion of cysts in contaminated water or food, or by
direct person-to-person contact. G. lamblia may also have animal
reservoirs, making the infection a potential zoonotic disease [3].
can attach to the intestinal mucosa. Symptoms of acute giardiasis
include watery diarrhoea, abdominal discomfort, pain and cramps.
Chronic disease can result in malabsorption and failure to thrive in
children [4]. For amebiasis, trophozoites migrate to the large in-
testine and can either reside in the lumen or invade the colonic
mucosa or other extra-intestinal sites, most prominently the liver
[5].
Due to the propensity for spread through contaminated water
and food sources and the low infectious dose of G. lamblia and
E. histolytica cysts [6], the global disease burden is disproportion-
ately shouldered by developing nations in areas with inadequate
sanitation. Protozoan diseases also impact developed nations, often
via travellers visiting regions where disease is endemic. The threat
to developed nations is recognised by the US National Institute of
Allergy and Infectious Diseases as both protozoa are category B
Abbreviation: MtzS, metronidazole sensitive; MtzR, metronidazole resistant;
MIC, minimum inhibition concentration.
* Corresponding author.
E-mail address: m.cooper@uq.edu.au (M.A. Cooper).
Current address: Progen Pharmaceuticals Limited, Darra, Queensland, 4076,
1
Australia.
http://dx.doi.org/10.1016/j.ejmech.2016.04.064
0223-5234/© 2016 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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A.M. Jarrad et al. / European Journal of Medicinal Chemistry 120 (2016) 353e362
bioterrorism threat pathogens [7].
the expanded potential of metronidazole based agents has recently
been demonstrated by modifying metronidazole with a “click
chemistry” approach to generate agents with improved potency
and activity against metronidazole resistant (MtzR) parasites
[15,16].
Metronidazole 1 (Fig. 1) is a generic drug for treatment of a
range of parasitic and anaerobic bacterial infections. For giardiasis,
metronidazole is typically given in 250 mg doses three times a day
for 5e7 days, while amebiasis is treated with a higher 750 mg dose
three times a day for 5e10 days, often followed by treatment with
paromomycin to eradicate cysts from the colon [8,9]. Other 5-
nitroimidazoles, such as tinidazole 2 and ornidazole 3 (Fig. 1)
have improved dosing schedules with only a single 2 g tablet of
either drug for treatment of giardiasis, or 2 g tinidazole once daily
for three days for treatment of amebiasis [8e10]. These agents have
similar adverse effects such as nausea, vomiting and headaches.
Ornidazole is not approved for use in the United States [8e10].
Unfortunately, metronidazole treatment fails in up to 20% of giar-
diasis cases with metronidazole resistance an ever increasing
concern [11,12]. Parasites resistant to metronidazole show cross-
resistance to tinidazole [1]. Furthermore, resistance of
E. histolytica to metronidazole has also been described, as tropho-
zoites can be adapted to grow in the presence of therapeutically
relevant levels of metronidazole [13]. Given the sheer number of
cases of giardiasis and amebiasis, and treatment failures, develop-
ment of alternative treatment options remains an important
priority.
Nitroimidazole carboxamides (Fig. 1) were originally patented
by Merck &. Co. in 1973 for the treatment of infections caused by
Histomonas meleagridis and Trichomonas vaginalis [17].
H. meleagridis is a parasite that causes lesions in the cecum and liver
of chickens and turkeys, and is commonly known as turkey black-
head disease [18]. In contrast, T. vaginalis infects the genitourinary
tract in humans causing inflammation and vaginal discharge in
women [19]. The nitroimidazole carboxamides displayed efficacy in
in vivo turkey and mouse models of Histomonas maleagridis and
T. vaginalis [17], respectively, but no substantial antimicrobial
development of this series has since been reported. Given the core
5-nitroimidazole group in the nitroimidazole carboxamides is
similar to metronidazole, we hypothesised that these compounds
could have therapeutic potential against enteric parasites,
including G. lamblia and E. histolytica. In addition, the 20-carbox-
amide substitution provides a convenient handle to optimise
antiparasitic properties. Therefore, we explored the structure ac-
tivity relationships (SAR) of nitroimidazole carboxamides and
conducted preliminary ADME studies to identify improved anti-
parasitic agents with therapeutic potential against G. lamblia and
E. histolytica.
Re-examination of ‘old’ nitroimidazoles is a valuable strategy in
the development of new drugs for treatment of parasitic diseases.
For example, fexinidazole 4 (Fig. 1), initially discovered in the
1980s, has been “rediscovered” and is in clinical development by
the Drugs for Neglected Diseases initiative for treatment of Human
African trypanosomiasis (sleeping sickness) and Chagas disease
[14]. Metronidazole has been in clinical use for over 50 years, but
2. Materials and methods
2.1. Synthesis of 1-methyl-5-nitroimidazoles
The library of 1-methyl-5-nitroimidazole carboxamides 8a-k
was prepared essentially as described by Hoff [17] using the syn-
thetic approach depicted in Scheme 1. Commercially available 1-
methyl-2-hydroxymethyl-5-nitro imidazole 5 was oxidised with
potassium permanganate in acetone to form the potassium
carboxylate salt 6, which was isolated in this form to avoid decar-
boxylation of the free carboxylic acid [17]. The crude carboxylate
salt 6, upon reaction with oxalyl chloride and catalytic DMF, pro-
vided the acid chloride intermediate 7. The library of 5-
nitroimidazole carboxamides 8a-k was then prepared by reacting
the crude acid chloride 7 with the desired primary or secondary
amines in the presence of triethylamine (Scheme 1). Compounds
8a-f and 8i-k are first reported here. Compounds 8g (R ¼ NMe2)
and 8h (R ¼ morpholine), originally reported by Hoff [17], were
prepared for use as comparators due to their activity in in vivo
models of T. vaginalis and H. meleagridis infection. The title com-
pounds 8a-k were all purified by direct or reverse phase chroma-
tography to ꢀ95% purity before biological testing. All compounds
were characterised by 1H and 13C NMR, LCMS and HRMS and
detailed experimental procedures and characterisation data are
provided in the supplementary information.
2.2. Synthesis of 4(5)-nitroimidazoles
To examine the corresponding 4(5)-nitroimidazole carbox-
amide series of 8a-k (i.e no N-methyl substitution) we prepared the
analogous series of novel compounds 12a-k. In addition, four
alternative novel carboxamides 12l-o were prepared, as shown in
Scheme 2. Imidazole-2-carboxylic acid 9 was readily nitrated with
conc. HNO3/H2SO4 to give 4(5)-nitroimidazole carboxylic acid 10.
Carboxamides 12a-o were subsequently prepared by activation of
acid 10 (oxalyl chloride/catalytic DMF or PyBOP/DIPEA) followed by
coupling of the requisite amine. Amidation via intermediate 11 was
the preferred route due to the difficulty of removing the HOBt and
Fig. 1. 5-Nitroimidazoles 1e4 in clinical use or development for treatment of parasitic
diseases, while 8a-k, 12a-o, 13a-g and 14a-e are the nitroimidazole carboxamides
investigated here.

A.M. Jarrad et al. / European Journal of Medicinal Chemistry 120 (2016) 353e362
355
Scheme 1. Synthesis of 1-methyl-5-nitroimidazoles 8a-k. i) KMnO4, acetone, ꢂ5 ꢁC / rt, 85%; ii) oxalyl chloride, cat. DMF, DCM, 0 ꢁC / rt; iii) amine, TEA, DCM, 0 ꢁC / rt, 19e58%.
60 ꢁC. The hydrazide 14e was prepared from 14f via an acid chloride
intermediate and hydrazine. The title compounds 13a-g and 14a-e
were purified by chromatography or recrystallisation and charac-
terised as described for 8a-k.
2.4. Synthesis of des-nitroimidazoles and 4(5)-amino-imidazole
carboxamide
Des-nitro imidazole 17 was prepared by coupling 16 and 4-
fluorobenzylamine with PyBOP and DIPEA (Scheme 4). The
desnitro-imidazole 18 was prepared by alkylation of 19 with 4-
(trifluoromethoxy)benzyl bromide in DMF under basic conditions
achieved with K2CO3 (Scheme 4). The 4(5)-nitroimidazole carbox-
amide 19 was prepared from the intermediate acid chloride formed
with oxalyl chloride and catalytic DMF from 16 (Scheme 4). Amine
20 was prepared by reduction of 12a with Pd/C catalyst at 30 ꢁC, H2,
1 atm in an H-Cube Pro reactor (Scheme 4).
2.5. Antiparasitic assays
2.5.1. Maintenance of G. lamblia, E. histolytica and T. vaginalis
Trophozoites of G. lamblia strains (metronidazole sensitive
(MtzS) line WB and the MtzR line 713-M3 [20,21]), E. histolytica
strain HM1:IMSS and T. vaginalis strain F1623 [15] were axenically
Scheme 2. Synthesis of 4(5)-nitroimidazoles 12a-o. Amide groups a-k are as defined
in Scheme 1. i) HNO3, H2SO4, 80 ꢁC, 54%; ii) oxalyl chloride, cat. DMF, DCM, 0 ꢁC / rt;
iii) amine, PyBOP, DIPEA, 6e75% iv) amine, TEA, DCM, 0 ꢁC / rt, 12e93%.
maintained in TYI-S-33 medium supplemented with penicillin
(100 U/mL) and streptomycin (100 g/mL) [22]. All experiments
m
were performed using trophozoites harvested during the loga-
rithmic phase of growth.
tripyrrolidinophosphine oxide by-products formed during the
PyBOP mediated coupling. The primary amide 12l was prepared by
quenching the acid chloride 11 with concentrated ammonium hy-
droxide solution. The title compounds 12a-o were all purified and
characterised as described for 8a-k.
2.5.2. EC50 assays
Compounds were screened for antiparasitic activity using an
ATP-bioluminescence based assay for cell growth and survival
[23,24]. Briefly, 2.5
sterile water to yield 625
Three-fold serial dilutions were prepared yielding a concentration
range of 0.25e625 M. From this dilution plate, 4 L volumes were
transferred into 96-well microtitre plates followed by addition of
96 L of trophozoites (5000 parasites) to yield a final 8-point
mL of 5 mM stocks were diluted with 17.5
mL
mM working concentration of compounds.
2.3. Synthesis of 4-nitroimidazoles
m
m
The novel 4-nitroimidazoles 13a-g and 14a-c were synthesised
from the respective 1H-imidazole carboxamides 12g, 12l, 12m and
12p by alkylation with benzyl or alkyl halides under basic condi-
tions (K2CO3) (Scheme 3). The hydroxamic acid 14d was prepared
by treatment of the ester 14c with hydroxylamine in methanol at
m
concentration range spanning 0.01e25
mM. Assay plates were
incubated for 24e48 h at 37 ꢁC in the GasPak™ EZ Anaerobe Gas
Generating Pouch Systems (VWR, West Chester, PA) to maintain

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A.M. Jarrad et al. / European Journal of Medicinal Chemistry 120 (2016) 353e362
Scheme 3. Synthesis of 4-nitroimidazoles 13a-g and 14a-f. i) benzyl or alkyl bromide, K2CO3, DMF, rt / m
W 80 ꢁC, 7e98%; ii) HNO3, H2SO4, 60 ꢁC, 64%; iii) NH2OH, MeOH, 60 ꢁC,
37%; iv) 1 M NaOH, THF: MeOH (1:1), rt, 37%; v) oxalyl chloride, cat. DMF, DCM, 0 ꢁC / rt; vi) NH2NH2ꢄH2O, DCM, 0 ꢁC, 65%.
2.6. MIC assays
Clostridium difficile strains (630, ATCC BAA-1382 and NAP1/027,
ATCC BAA-1803) were maintained as previously described [16]. The
minimum inhibition concentration (MIC) was determined accord-
ing to the CLSI Methods with modifications in broth and inoculum
for C. difficile [16,25,26]. Briefly, compounds and control antibiotics
were serially diluted two-fold in 96-well plates (Non-binding sur-
face, Corning). The plates were placed in a Coy anaerobic chamber
(5% H2, 10% CO2, 85% N2) overnight to reduce the medium. C. difficile
bacteria from BHIS(TA) agar plates were cultured anaerobically in
BHIS at 37 ꢁC overnight. A sample of culture was then diluted 40-
fold in BHIS broth and incubated at 37 ꢁC for approximately 4.5 h.
The resultant mid-log phase culture (OD600 ¼ 0.4e0.6) was diluted
to a final concentration of ~1 ꢃ 106 CFU/mL, then 50
mL was added
to each well of the compound-containing 96-well plates, yielding a
final cell concentration of 5 ꢃ 105 CFU/mL and final volume of
100 mL with 3% maximum DMSO concentration. Compound con-
centration ranged from 64 to 0.03 mg/mL. An antibiotic standard, a
positive growth control (no compound) and a sterility control (no
bacteria) were included on each 96 well plate. Plates were covered
and incubated at 37 ꢁC for 24 h. MICs for each strain were deter-
mined as the lowest concentration without visible growth. Vari-
ance between replicates was typically within one 2-fold dilution.
Median MICs are reported with a range given when the median MIC
was between two tested concentrations.
Scheme 4. Synthesis of desnitro-imidazoles 17 and 18 and amine 20. i) 4-
fluorobenzylamine, PyBOP, DIPEA, DCM, rt, 67% ii) oxalyl chloride, cat. DMF,
0
ꢁC / rt; iii) conc. NH4OH, 0 ꢁC / rt; 73%; iv) 4-(trifluoromethoxy)benzyl bromide,
K2CO3, DMF, rt, 58%; v) MeOH, Pd/C, H-cube, 1 atm, 30 ꢁC, 74%.
2.7. Cytotoxicity
anaerobic condition throughout the incubation period. Viable cell
numbers were determined in triplicate using the CellTiter-Glo
Luminescent Cell Viability Assay [23].
Human HEK293 and HepG2 cells were seeded at 3000 and
5000 cells per well in 384-well plates, respectively. Cells were
cultured in Dulbecco's modified Eagle's medium with 10% FBS for

A.M. Jarrad et al. / European Journal of Medicinal Chemistry 120 (2016) 353e362
357
24 h at 37 ꢁC, 5% CO2. A dilution series of compounds was added,
with the highest concentration of 100 M. The final concentration
of DMSO in culture media was 0.5%, which showed no effect on cell
growth. After 24 h incubation with the compounds, 5 M resazurin
of the filtered sample and the concentration of unfiltered sample.
All samples were tested in triplicate with sulfamethoxazole as a
control. LC/MS/MS parameters are detailed in the supplementary
information (Supplementary Table 1).
m
m
was added into each well and incubated at 37 ꢁC for 2 h. As a
negative control, 1% Triton X-100 was added into the culture media
to lyse all of the cells. The fluorescence intensity was read using
Polarstar Omega with excitation/emission 560/590 nm. Data were
analysed with GraphPad Prism 6 software (La Jolla, California USA)
to calculate CC50 values.
2.11. Metabolic stability
Metabolic stability studies were performed with human liver
microsomes (HMMC-PL, Lot# PL050B-B, Thermo Fisher Scientific
USA) with test compound (5 mM) degradation monitored by LC/MS/
MS analysis. Stock solutions (2.5 mM) of the test compounds were
prepared in DMSO. The reaction mixture containing test compound
2.8. Correlation of compound properties with activity
(5
mM) and liver microsomes (1 mg/mL) in 100 mM potassium
A correlation matrix between compound activity and physico-
chemical properties was calculated using Excel correlation analysis
(Supplementary Table 2). AlogP, logD, MW, logS and tPSA were
calculated from the 2D structure of the compounds, using Pipeline
Pilot (Accelrys, Version 9.1.0.13). Antimicrobial activity was
expressed as -log10 values of MIC or EC50, using average MIC (mol
phosphate buffer pH 7.4 was preincubated at 37 ꢁC (1 mL volume).
The reaction was initiated by addition of NADPH solution (cofactor)
in potassium phosphate buffer (final concentration 1 mM). Aliquots
(180
60 and 120 min) and quenched by adding ice-cold stop solution
(540 L) comprising 0.5 M carbutamide internal standard in
m
L) from the reaction mixture were withdrawn (t ¼ 0, 10, 30,
m
m
L
ꢂ1) of MtzS C. difficile ATCC BAA-1382 and ATCC BAA-1803 strains
acetonitrile: methanol: formic acid (1: 1: 0.001). Reaction samples
were incubated at 4 ꢁC for 30 min, centrifuged at 14,000 ꢃ g for
8 min and the clear supernatant was transferred to a vial for LC/MS/
MS analysis. The percentage of compound metabolised at different
times was calculated as a percentage of the levels at the start of
incubation (t ¼ 0 min sample). Matrix blank was also prepared as a
control. All samples were tested in triplicate except for the control
samples (without NADPH), matrix blank and verapamil standard
(time points ¼ 0,10 and 30 min). LC/MS/MS parameters are detailed
in the supplementary information (Supplementary Table 1).
and EC50 (mol Lꢂ1) against G. lamblia WB strain, E. histolytica
HM1:1MSS strain and T. vaginalis F1623 strain. Correlations of
determination (R2) between compound activities and logD, MW or
logS were determined by linear regression analysis in GraphPad
Prism
6 software (La Jolla, California USA) (Supplementary
Figs. 1e3).
2.9. Microscopy
The effect of compounds on G. lamblia WB growth and survival
was examined by light microscopy. Briefly, stock compounds were
diluted in DMSO (100%) to give 400 ꢃ final concentration of com-
3. Results and discussion
pound. An aliquot of 2.5
m
L of working stock was added to each well
3.1. Biological activity of 1-methyl-5-nitroimidazole carboxamides
of a 24 well tissue culture clear bottom plate (Corning, 3524), fol-
lowed by trophozoites (1 mL, 50,000 parasites/mL) to yield a final
concentration of 3 ꢃ EC50. Metronidazole (3 ꢃ EC50) served as a
positive control. Media only wells were included as a sterility
control, and vehicle only (0.25% DMSO) was included as a control
for growth. Assay plates were incubated for 48 h at 37 ꢁC in the
GasPak™ EZ Anaerobe Gas Generating Pouch Systems (VWR, West
Chester, PA) to maintain anaerobic conditions throughout the in-
cubation period. The assays were performed in triplicate (3 wells/
treatment). Growth inhibition was visualised by phase contrast
microscopy (200 ꢃ magnification) (Carl Zeiss).
The antiparasitic activity of the 1-methyl-5-nitroimidazole car-
boxamides 8a-k against G. lamblia and E. histolytica was first
assessed (Table 1). All of the 5-nitroimidazoles were active against
MtzS G. lamblia WB, with EC50 values ranging from 1.6
(cf. metronidazole EC50 ¼ 6.1 M). Compounds 8f (R ¼ NHCH2(2-
pyridinyl)) and 8h (R ¼ morpholine) were the most active de-
M) with ~4-fold greater potency than
mM to 4.9 mM
m
rivatives (EC50 ¼ 1.6
m
metronidazole, suggesting that polar groups conferred superior
activity. The aromatic substituted benzyl amides (8a-d) and 8e
(R
¼
NHCH2CH2(4-Me-Ph))
all
had
similar
activity
(EC50 ¼ 2.8e3.5
mM), demonstrating that carboxamide groups with
2.10. Plasma protein binding
different aromatic substitution patterns are tolerated at the 20-
imidazole ring position. The aliphatic cyclic secondary amides of 8j
(R ¼ NH-cyclopropyl) and 8k (R ¼ NH-cyclohexyl) were also both
well tolerated, as were the tertiary amide groups of 8g (R ¼ NMe2)
and 8i (R ¼ pyrrolidine). Encouragingly, a number of compounds
were also active against MtzR G. lamblia 713M3 (Table 1). For
example, 8c (R ¼ NHCH2(3-OCF3-Ph), 8d (R ¼ NHCHMe-(4-F-Ph)),
8f (R ¼ NCH2(2-pyridinyl)), 8i (R ¼ pyrrolidine) and 8k (R ¼ NH-
cyclohexyl) were 3e12-fold more active than metronidazole
Plasma Protein Binding (PPB) was performed using an Ultrafil-
tration method [27,28]. Fresh frozen human plasma was pooled
from 0 Positive (Product Number 2799882) and 0 Negative (Product
number 5398256) blood from the R & D division of the Australian
Red Cross Blood Services (Brisbane). Stock solutions (2.5 mM) of the
test compounds were prepared in DMSO. Test compounds (5
were incubated in 100% human plasma at 37 ꢁC for 30 min (1 mL
volume). For unfiltered samples, an aliquot (50 L) was removed,
diluted with PBS (50 L) and quenched with ice-cold precipitating
solution comprising 0.5 M carbutamide MS internal standard in
mM)
m
against this MtzR G. lamblia strain (EC50 1.5e5.1
dazole EC50 ¼ 18 M).
Carboxamides 8a-k generally exhibited moderate activity
against E. histolytica with a wide range of EC50 values from 3.7 M to
22
M (Table 1). Compound 8k (R ¼ NH-cyclohexyl) was the most
potent (EC50 ¼ 3.7 M) with similar activity to metronidazole
(EC50 ¼ 5.0 M), a notable difference in SAR compared to the
mM, cf. metroni-
m
m
m
acetonitrile: methanol: formic acid (1: 1: 0.001). Samples were
incubated at 4 ꢁC for 30 min, then centrifuged at 14,000 ꢃ g for
8 min, with the clear supernatant transferred to a vial for LC/MS/MS
m
m
m
analysis. For filtered samples, the plasma sample (250
filtered using Amicon Ultra-0.5 Centrifugal Filter Devices 30K
L) was
mL) was
m
preference for greater polarity against G. lamblia. However, the
majority of the series, including the smaller lipophilic cyclopropyl
amide 8j, were 2e4-fold less potent than metronidazole, leaving
room for further optimisation.
NMWL at 14,000 ꢃ g for 7 min and then an aliquot (50
m
processed as described for unfiltered samples. The fraction of un-
bound compound was calculated by determining the concentration

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A.M. Jarrad et al. / European Journal of Medicinal Chemistry 120 (2016) 353e362
Table 1
Activity of 1-methyl-5-nitroimidazole carboxamides and 4(5)-nitroimidazole carboxamides against G. lamblia, E. histolytica, T. vaginalis and C. difficile.
EC50
(
m
M) (pEC50 SE)
MIC (
m
g/mL) CC50
(
m
M) (pCC50 SE) S.Ia (CC50/EC50
)
G. lamblia
E. histolytica
T. vaginalis
C. difficileb
HEK
293
Hep
G2
WB
713M3
HM1:IMSS
F1623
630
No.
X
R
MtzS
MtzR
MtzS
MtzS
MtzS
0.5
1
Metronidazole
6.1 (5.21 0.05) 18 (4.74 0.02) 5.0 (5.30 0.03) 0.8 (6.1 0.07)
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>16
>29
>33
>32
>34
>36
>63
>42
>63
>34
>29
>20
8a
8b
8c
8d
8e
8f
8g
8h
8i
Me NHCH2(4-F-Ph)
Me NHCH2(4-OCF3-Ph)
Me NHCH2(3-OCF3-Ph)
Me NHCHMe(4-F-Ph)
Me NHCH2CH2(4-Me-Ph) 2.8 (5.55 0.03) >20 (<4.70)
Me NHCH2(2-pyridinyl)
Me N(Me)2
Me morpholine
Me pyrrolidine
Me NH-cyclopropyl
Me NH-cyclohexyl
3.5 (5.46 0.03) 13 (4.89 0.11) 9.7 (5.01 0.01) 5.2 (5.28 0.11) >64
3.0 (5.52 0.03) 8.8 (5.06 0.29) 14 (4.85 0.02) 13 (4.88 0.01) 32
3.1 (5.51 0.02) 1.5 (5.84 0.69) 17 (4.77 0.02) 8.1 (5.09 0.09) 32e64
2.9 (5.54 0.03) 1.9 (5.73 0.39) 10 (5.00 0.03) 3.8 (5.42 0.24) 64
13 (4.89 0.03) 8.1 (5.09 0.16) >64
1.6 (5.80 0.03) 4.1 (5.39 0.29) 14 (4.85 0.14) 1.4 (5.84 0.09) 64
2.4 (5.62 0.03) >20 (<4.70) 14 (4.85 0.03) 1.3 (5.89 0.30) 32
1.6 (5.80 0.03) 11 (4.95 0.20) 14 (4.85 0.02) 0.6 (6.24 0.34) 32
2.9 (5.54 0.04) 4.0 (5.40 0.33) 22 (4.66 0.02) 1.7 (5.76 0.17) 64
3.4 (5.47 0.04) 8.6 (5.06 0.23) 12 (4.92 0.03) 1.1 (5.96 0.30) >64
4.9 (5.31 0.04) 5.1 (5.30 0.20) 3.7 (5.43 0.03) 11 (4.97 0.14) >64
8j
8k
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NHCH2(4-F-Ph)
0.5 (6.28 0.10) 2.4 (5.63 0.35) 3.6 (5.44 0.06) 2.3 (5.63 0.17)
0.2 (6.61 0.03) 2.5 (5.60 0.38) 4.5 (5.35 0.06) 2.2 (5.65 0.23) 16
0.2 (6.70 0.03) 15 (4.83 0.11) 3.6 (5.44 0.01) 1.9 (5.72 0.17) 16
4
>100 >100
>100 93 (4.03 0.07) >500/465
>250
NHCH2(4-OCF3-Ph)
NHCH2(3-OCF3-Ph)
NHCHMe(4-F-Ph)
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>500
>250
>1000
>14
>12
>11
>29
>20
>167
N/A
0.4 (6.40 0.6)
1.3 (5.90 0.31) 1.7 (5.77 0.04) 0.6 (6.23 0.14)
8
8
2
2
2
2
1
8
1
NHCH2CH2(4-Me-Ph) 0.1 (7.00 0.04) 1.4 (5.87 0.39) 2.1 (5.68 0.04) 1.2 (5.92 0.16)
NHCH2(2-pyridinyl)
N(Me)2
morpholine
pyrrolidine
NH-cyclopropyl
NH-cyclohexyl
NH2
7.2 (5.14 0.03) >20 (<4.70)
8.3 (5.08 0.02) >20 (<4.70)
8.8 (5.06 0.02) >20 (<4.70)
15 (4.82 0.02) 7.1 (5.15 0.11)
2.7 (5.57 0.03) 2.4 (5.63 0.11)
5.1 (5.29 0.02) 2.9 (5.54 0.05)
3.4 (5.47 0.02) 13 (4.88 0.11) 4.3 (5.37 0.02) 1.7 (5.77 0.11)
5.0 (5.30 0.03) >20 (<4.70) 5.3 (5.28 0.02) 4.4 (5.36 0.05)
0.6 (6.22 0.03) 5.5 (5.26 0.26) 2.8 (5.55 0.04) 2.4 (5.61 0.10)
>50 (<4.3) >20 (<4.70) 13 (4.88 0.03) >20 (<4.70)
9.9 (5.00 0.02) >20 (<4.70)
>50 (<4.3) >20 (<4.70)
24 (4.62 0.05) >20 (<4.70)
NHMe
NHCH2CH2OH
NMeCH2CH2OH
6.1 (5.21 0.02) 8.5 (5.07 0.15) 0.5e1
>10
N/A
>4
>50 (<4.3)
>50 (<4.3)
>20 (<4.70)
>20 (<4.70)
8
2
a
Selectivity Index: average cytotoxicity against HEK293 and HepG2 cell lines/G. lamblia WB activity(CC50/EC50).
Similar results were obtained for C. difficile NAP/027 strain (Supplementary Table 3). EC50 minimum n ¼ 3 EC50, pEC50 SE, MIC median of n ¼ 4, CC50 n ¼ 3, pCC50 SE.
b
For comparison, we next tested the new derivatives against
parasitic and bacterial proposed mechanisms of activation of the
nitroimidazole carboxamides, or possibly differences in cellular
uptake.
The compound series 8a-k was not cytotoxic against human
kidney or liver cell lines at the highest concentration tested
T. vaginalis, which was the original indication for nitroimidazole
carboxamides [17]. Several of the compounds had potent activity
against T. vaginalis, with EC50 values that were similar to metroni-
dazole (EC50 ¼ 0.6e1.4
Notably, the smaller, more polar and nonaromatic side chains of
8g-j were favourable, with activities from 0.6 to 1.7 M, which was
mM cf. metronidazole EC50 ¼ 0.8
mM).
(CC50 > 100
mM), so the calculated minimal selectivity indices
m
ranged from >16 to >63 (Table 1).
consistent with 8g-h having activity in an in vivo mouse model of
T. vaginalis infection [17]. Similarly, an ionisable aromatic group of
3.2. Biological activity of 4(5)-nitroimidazoles
compound 8f (R
(EC50 ¼ 1.4 M). In contrast the non-polar, aromatic benzyl (8a-d)
and phenethyl (8e) substituents resulted in comparatively weak
activity against T. vaginalis (EC50 ¼ 3.8e13 M).
¼
CH2-(2-pyridinyl)) was beneficial
m
The promising activity of the 1-methyl-5-nitrocarboxamide se-
ries as antiparasitic agents led us to explore the nitroimidazole
carboxamide scaffold further to understand the SAR and determine
whether the activity could be improved. Hoff [17] previously re-
ported the influence of alkyl and hydroxyl alkyl groups at the 10-
imidazole nitrogen position on antiparasitic activity but did not
describe the corresponding compound series in which the 10-
imidazole nitrogen lacks substitution. Hence we generated an
analogous series of 4(5)-nitroimidazole carboxamides (12a-k) with
the unsubstituted 1-position and matched carboxamide R groups
to 8a-k, and tested them for antiparasitic and antimicrobial activity.
Compared to the 1-methyl-5-nitro series, the presence of an acidic
imidazole proton in the 4(5)-nitro series permits ring tautomerism,
which may alter the reduction potential of the nitro group.
Furthermore, the small structural change will influence other
physicochemical parameters (e.g. polar surface area and logS)
which is likely to be reflected in different SAR profiles between the
two series.
m
Since nitroimidazole carboxamides contain a 5-nitroimidazole
warhead similar to metronidazole, a common treatment for in-
fections caused by both parasites and anaerobic bacteria, we also
determined the antimicrobial activity of 8a-k against the anaerobic
bacterium C. difficile. C. difficile is an anaerobic Gram-positive bac-
terium that infects the colon and causes inflammation and diar-
rhoea [29], similar to symptoms of G. lamblia and E. histolytica
infection. Surprisingly, none of the 1-methyl-5-nitroimidazole
carboxamides 8a-k had significant activity against the 630 or
NAP1/027 strains of C. difficile (MIC ꢀ 32
m
g/mL) whereas metro-
nidazole was quite potent (MIC
¼
0.5 g/mL) (Table 1,
m
Supplementary Table 3). Therefore this compound series, contain-
ing the 1-methyl-5-nitroimidazole core, exhibited greater selec-
tivity toward anaerobic protozoan parasites compared to the
anaerobic bacteria C. difficile, suggesting differences between the

A.M. Jarrad et al. / European Journal of Medicinal Chemistry 120 (2016) 353e362
359
In general the 4(5)-nitroimidazole carboxamides 12a-k exhibi-
ted improved activity against G. lamblia and E. histolytica relative to
their 5-nitroimidazole counterparts 8a-k (Table 1). For G. lamblia,
the aromatic benzyl amides 12a-d, phenethyl 12e and cyclohexyl
vehicle control (which produced a confluent cell layer under the
test conditions). The morphology of G. lamblia treated with 12a was
altered, while the morphology of the metronidazole-treated cells
remained similar to the vehicle control (Fig. 2). A prior study by
Tejman-Yarden et al. reported that metronidazole slowed the rate
of oscillation of the Giardia flagella, while auranofin, a compound
with a proposed different mode of action, caused cell blebbing [24].
The different morphology of the G. lamblia treated with the 4(5)-
nitroimidazole carboxamide 12a may indicate an additional mode
of action compared to metronidazole.
12k groups were very potent (EC50 ¼ 0.1e0.6
m
M). In contrast 12f
NMe2) and 12h
(R NHeCH2(2-pyridinyl)), 12g (R
¼
¼
(R ¼ morpholine) were 3.5e5.5-fold less active than the respective
1-methyl analogues 8f, 8g and 8h. Compounds 12i (R ¼ pyrrolidine)
and 12j (R
(EC50 ¼ 3.4 and 5
¼
NH-cyclopropyl) maintained similar activity
m
M, respectively) to the 1-methyl analogues 8i
and 8j. A number of compounds with substituted benzyl groups
(12a-b and 12d) and the phenethyl derivative 12e also displayed
3.2.2. Influence of physicochemical properties on compound
activity in the 4(5)-nitromidazole compound series
good activity against MtzR G. lamblia (EC50 ꢅ 2.5
mM).
For E. histolytica, the 4(5)-nitromidazole carboxamides were
We observed improved activity profiles of 4(5)-nitroimidazoles
relative to the corresponding analogue in the 5-nitroimidazole
series against G. lamblia, E. histolytica and C. difficile, but not
T. vaginalis. In addition, the 4(5)-nitroimidazoles with the most
potent activity against G. lamblia differed significantly from the
compounds with the most potent activity against C. difficile. To
better understand the relationship between biological activity and
physicochemical properties, the correlation coefficients (r) were
determined between a range of calculated compound properties
(AlogP, logD, molecular weight, logS and topological polar surface
area) and biological activity against the different organisms
(Supplementary Table 2). G. lamblia activity was positively corre-
lated with AlogP (r ¼ 0.94), logD (r ¼ 0.93) and MW (r ¼ 0.82). A
negative correlation with LogS (r ¼ ꢂ0.92) was also observed, while
there was no meaningful relationship with tPSA (r ¼ 0.06). Nearly
identical results were obtained with logP and logD values as only
12f (R ¼ NHCH2(2-pyridinyl)) contained an ionisable group. Mod-
erate to weak correlations were observed between E. histolytica or
T. vaginalis activity and compound properties (AlogP, logD, MW,
logS and tPSA). In contrast, C. difficile activity was positively
correlated with LogS (r ¼ 0.72), negatively correlated with AlogP
(r ¼ ꢂ0.72), logD (r ¼ ꢂ0.72) and MW (r ¼ ꢂ0.75) and poorly
correlated with tPSA (r ¼ ꢂ0.23), supporting the qualitative ob-
servations made from examination of the SAR.
To quantify the extent that the variability in activity against each
organism was dependent on logD, MW and logS, the coefficient of
determination (R2) was next calculated (Fig. 3, Supplementary
Figs. 1e3). This analysis supported the correlation between
G. lamblia activity and logD, MW and logS properties of the com-
pounds (R2 ranged from 0.67 to 0.86) (Fig. 3, Supplementary
Figs. 1e3). No correlation was found for E. histolytica and
T. vaginalis activity and compound properties (R2 ranged from 0.15
to 0.28) (Fig. 3, Supplementary Figs. 1e3). In contrast, a weak cor-
relation between C. difficile activity and logD, MW and logS was
observed (R2 ranged from 0.47 to 0.56) (Fig. 3, Supplementary
Figs. 1e3), demonstrating greater variability in the data that was
not accounted for by changes to logD, MW or logS.
overall more potent than the 1-methyl series, with activities
ranging from 1.7 to 15
mM for compounds 12a-k compared to
3.7e22 M for the 8a-k series. Several compounds in the 12a-k
m
series (12d (R ¼ NHCHMe-(4-F-Ph)), 12e (R ¼ NHCH2CH2(4-Me-
Ph)), 12g (R ¼ NMe2) and 12k (R ¼ NH-cyclohexyl)) were 2e3-fold
more potent than metronidazole, while all of the other compounds
had similar activity to metronidazole, except for the pyridine 12f
that was the least potent compound (EC50 ¼ 15
mM).
In contrast to their improved activity against G. lamblia and
E. histolytica, compounds 12a-k were not overall more active than
8a-k against T. vaginalis (Table 1). The SAR was relatively flat: the
trend for improved potency with more polar substituents seen with
series 8a-k disappeared. The most potent compound was 12d
(R ¼ NHCHMe(4-F-Ph)) with EC50 ¼ 0.6
benzyl compounds 12a-12c, 12e and the pyrrolidine 12i had similar
M), but were generally 2e3 fold less
mM. The other aromatic
activity (EC50 ¼ 1.2e2.3
m
potent than 12d. Interestingly, the absence of N-substitution on the
imidazole ring for 12a-k also greatly improved activity against both
the 630 and NAP1/027 strains of C. difficile (MIC ¼ 0.5e16
mg/mL),
whereas the 1-methyl-5-nitro series were all essentially inactive
(ꢀ32
mg/mL) (Table 1, Supplementary Table 3). Small lipophilic and
polar 20-carboxamide substituents were preferred in the case of
C. difficile. For example, 12j (R ¼ NH-cyclopropyl) was the most
active derivative against C. difficile (MIC ¼ 1
m
g/mL), although less
active than metronidazole (MIC ¼ 0.5
dimethyl, morpholine and pyrrolidine derivatives) had MIC ¼ 2
mL. In contrast, the aromatic benzyl 12a-d, phenethyl 12e and
g/mL). To
m
g/mL), while 12f-i (pyridine,
mg/
cyclohexyl 12k compounds were less active (MIC ¼ 4e16
m
further understand this preference for activity against C. difficile,
additional small, polar amides 12l-o were synthesised. These
included 12l (R ¼ NH2), 12m (R ¼ NHMe) and two compounds
inspired from the side chain of metronidazole: 12n
(R ¼ NHCH2CH2OH) and 12o (R ¼ NMeCH2CH2OH). Compounds
12l-m and 12o gave results that supported the previous trend
observed against C. difficile (MIC ¼ 0.5e2
m
g/mL), while 12n
(R ¼ NHCH2CH2OH) was less active (MIC ¼ 8e16
mg/mL). These
additional compounds 12l-o had weak to no activity against the
parasites.
To summarise, while activity against G. lamblia was improved by
increasing logD, MW and decreasing logS, this trend was not
apparent for E. histolytica or T. vaginalis. In contrast, activity against
C. difficile was weakly improved with compounds with lower logD,
MW and greater logS.
The majority of the 4(5)-imidazole series 12a-o were not cyto-
toxic at the highest concentration tested (CC50 > 100 mM) against
mammalian liver or kidney cell lines. The only compound found to
show cytotoxicity was 12b (R ¼ NHCH2(4-OCF3-Ph)) against the
HepG2 liver cell line (CC50 ¼ 93
m
M), but the selectivity index
3.3. Biological activity of 4-nitroimidazoles
(SI ¼ 465) relative to G. lamblia activity remained excellent.
Given the potent activity of the 4(5)-nitroimidazoles relative to
the 1-methyl-5-nitroimidazoles, we were interested to determine
the activity of 4-nitroimidazole carboxamide analogues, since
research by Trunz et al. showed that 4-nitroimidazoles can have
potent antiparasitic activity [30]. We therefore prepared a series of
4-nitroimidazole carboxamides 13a-g. Since polar substituents
were favourable for activity against G. lamblia in the 5-
3.2.1. Phenotypic effect of 4(5)-nitroimidazole 12a on G. lamblia
Microscopy was used to visually examine the impact of one of
the most potent compounds, the 4(5)-nitroimidazole 12a
(R ¼ NHCH2(4-F-Ph)), on G. lamblia trophozoites. Parasite cell
growth was similarly inhibited by treatment with 3 ꢃ EC50 of either
metronidazole (18 mM) or compound 12a (1.5 mM) relative to the

360
A.M. Jarrad et al. / European Journal of Medicinal Chemistry 120 (2016) 353e362
Fig. 2. Compound 12a inhibited Giardia lamblia trophozoites growth A) vehicle (DMSO), B) metronidazole (3 ꢃ EC50 ¼ 18
m
M) and C) 12a (3 ꢃ EC50 ¼ 1.5
mM).
Fig. 3. Activity vs logD of active 4(5)-nitroimidazoles. Compounds were classified as active against G. lamblia and E. histolytica with EC50 < 50
mM and were considered active against
T. vaginalis with EC50 < 20 g/mL). Metronidazole (black symbols) is shown for comparison.
mM. All 4(5)-nitroimidazoles were classified as active against C. difficile (MIC ꢅ 16
m
nitroimidazole series (though not the 4(5)-nitroimidazole series),
compounds were synthesised with the 20-substituent as a primary
carboxamide group with the 10-ring position substituted with
benzyl, phenethyl, heteroaromatic pyridine, cyclopropyl and
cyclohexyl groups. The compounds were found to have selective
activity against G. lamblia (Table 2). Several of these compounds,
Table 2
In vitro activity of 1-substituted 4-nitroimidazoles against G. lamblia and E. histolytica.
EC50
(m
M) (pEC50 SE)
MIC (
m
g/mL)
CC50
(
m
M) (pCC50 SE)
S.Ia (CC50/EC50
)
G. lamblia
E. histolytica
C. difficile
HEK293
HepG2
No.
X
R
WB
HM1:IMSS
630
1
Metronidazole
CH2(4-F-Ph)
6.1 (5.21 0.05)
>50 (<4.3)
4.1 (5.39 0.03)
8.4 (5.08 0.05)
>50 (<4.3)
27 (4.57 0.07)
>50 (<4.3)
5.0 (5.30 0.06)
5.0 (5.30 0.03)
>50 (<4.3)
>25 (<4.6)
>50 (<4.3)
>50 (<4.3)
>50 (<4.3)
>50 (<4.3)
>50 (<4.3)
0.5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>16
N/A
>22
>12
N/A
>3.7
N/A
>20
13a
13b
13c
13d
13e
13f
13g
-NH2
-NH2
-NH2
-NH2
-NH2
-NH2
-NH2
>64
>64
>64
>64
>64
>64
>64
CH2(4-OCF3-Ph)
CH2(3-OCF3-Ph)
CH2CH2(4-Me-Ph)
CH2(2-pyridinyl)
CH2-cyclopropyl
CH2-cyclohexyl
14a
14b
14c
14d
14e
CH2(4-OCF3-Ph)
CH2(4-OCF3-Ph)
CH2(4-OCF3-Ph)
CH2(4-OCF3-Ph)
CH2(4-OCF3-Ph)
-NHMe
-NMe2
-OEt
-NHOH
-NHNH2
3.4 (5.47 0.04)
2.7 (5.57 0.04)
6.0 (5.22 0.05)
5.1 (5.29 0.04)
7.5 (5.12 0.05)
30 (4.52 0.03)
19 (4.72 0.04)
51 (4.29 0.03)
10 (5.00 0.03)
45 (4.35 0.03)
64e>64
64
>64
16
>100
>100
>100
36 (4.44 0.06)
>100
>100
>100
>100
>100
>100
>29
>37
>17
7/>20
>13
>64
a
Selectivity Index: average cytotoxicity of HEK293 and HepG2 cell lines/G. lamblia WB activity (CC50/EC50). EC50 minimum n ¼ 3 EC50 (pEC50 SE), MIC median of n ¼ 4, CC50
n ¼ 3 (pCC50 SE).

A.M. Jarrad et al. / European Journal of Medicinal Chemistry 120 (2016) 353e362
361
including 13b (R ¼ CH2(4-OCF3-Ph)), 13c (R ¼ CH2(3-OCF3-Ph)), and
13g (R ¼ CH2(cyclohexyl)) displayed activities similar to metroni-
exposure of G. lamblia parasites to the drug in the intestinal tract
after the initial absorption period continues to occur by biliary
excretion and enterohepatic circulation [34]. Oral absorption is also
necessary for treatment of invasive amebiasis, underlying the
importance of adequate absorption of nitro drugs for in vivo effi-
cacy. To delineate preliminary ADME characteristics of the new
nitroimidazole carboxamide compounds, we determined their
plasma protein binding and microsome stability, as these proper-
ties are likely to influence compound half-life and free drug avail-
able at the sites of infection.
Binding to human plasma proteins was measured for several
4(5)- and 5-nitroimidazole carboxamide matched pairs, including
8a and 12a (R ¼ NHCH2(4-F-Ph)), 8k and 12k ((R ¼ NH-cyclohexyl))
and 8h and 12h (R ¼ morpholine) (Table 3). Plasma protein binding
varied depending on the 10- and 20-substituents. The 4-F-benzyla-
mide (8a and 12a) and cyclohexylamide (8k and 12k) imidazoles
were highly bound to plasma proteins (ꢀ94%) regardless of the 10-
substituent (H or Me), with the plasma protein binding for 10-H
derivatives slightly greater in each instance. In contrast, the mor-
pholine group of 8h ameliorated plasma protein binding (9%
bound) for the 5-nitroimidazole but the 4(5)- matched pair 12h
displayed high plasma protein binding, while metronidazole was
almost completely unbound (<5% bound). The contrast in plasma
protein binding between 8h and 12h could be explained by the
acidic nature of the imidazole NeH bond observed in the proton
dazole (EC50 ¼ 4.1e8.4
mM cf. metronidazole EC50 ¼ 6.1
mM), while
being non-cytotoxic to human liver or kidney cell lines
(CC50 > 100 M). Interestingly, none of the 13a-f series had activity
m
against E. histolytica or C. difficile.
To determine the relative influence of the 20 position on the
potency and selectivity for G. lamblia, we next modified the 20 po-
sition to methyl amide 14a, dimethyl amide 14b, ethyl ester 14c,
hydroxamide 14d and hydrazide 14e while maintaining the 10 ring
position with the preferred CH2(4-OCF3-Ph) group. Compounds 14a
(R ¼ NHMe) and 14b (R ¼ NMe2) were the most active against
G. lamblia (EC50 ¼ 3.4 and 2.7
m
M, respectively), slightly more
M) and metroni-
potent than the primary amide 13b (EC50 ¼ 4.1
m
dazole (EC50 ¼ 6.1
mM). Compounds 14c (R ¼ COOEt), 14d
(R ¼ NHOH) and 14e (R ¼ NHNH2) had similar or slightly reduced
activity relative to 13b (R ¼ NH2). Therefore different 20 sub-
stituents were tolerated for activity against G. lamblia. Although
compound series 14 displayed improved activity compared to
compound series 13 against E. histolytica (EC50 ¼ 10e45
mM
vs >50 g/mL vs >64 g/mL),
m
M) and C. difficile (MIC ¼ 16 to ꢀ64
m
m
the overall activity profile of both series remained considerably
inferior to metronidazole (Table 2, Supplementary Table 4) and
compounds within series 8 and 12. These results demonstrate that
G. lamblia is selectively sensitive to 4-nitromidazoles, suggesting
differences in the nitro-reduction activation and/or uptake of 4-
nitroimidazoles compared to E. histolytica and C. difficile.
NMR (NH
d ~14.3 ppm) and tendency for plasma proteins such as
human serum albumin to bind anionic compounds [27]. While both
metronidazole and tinidazole are mostly unbound to plasma pro-
teins [35], tizoxanide, the active metabolite of the prodrug nita-
zoxanide, is highly plasma protein bound [36]. The influence of
plasma protein binding on the free drug concentration at the site of
infection is also related to other complex factors, including meta-
bolism, distribution and half-life, and further in vivo efficacy ex-
periments are necessary to determine the impact of high plasma
protein binding on in vivo efficacy in this series [37].
The metabolic stability of a compound influences the concen-
tration of drug available in the circulation for treatment of invasive
amebiasis, and for prolonged exposure of G. lamblia to drug treat-
ment via enterohepatic recirculation pathways. Therefore, we
measured the human liver microsome stability for the 4(5)- and 5-
nitroimidazoles matched pairs 8a and 12a (R ¼ NHCH2(4-F-Ph))
and 8h and 12h (R ¼ morpholine) as these compounds showed
good potency and a range of plasma protein binding. All of the
compounds were metabolically stable after 2 h incubation with
human liver microsomes (Table 3). This result was comparable to
metronidazole, suggesting that the compounds have favourable
metabolic stability and that different 20 substituents were tolerated.
3.4. Desnitro and amine derivatives
5-Nitroimidazole antimicrobial agents are pro-drugs that are
activated by reduction of the nitro group to reactive intermediates
that cause cellular damage [31]. The reduction step is catalysed by
organism specific oxidoreductase enzymes, confounding target
based drug design and enzymatic assays as approaches to drug
development. In G. lamblia, the enzymes pyruvate ferredoxin
oxidoreductase, nitroreductase 1 and thioredoxin reductase 1 have
been implicated in the reductive activation of metronidazole [32].
Since the nitro group is key to the mode of action of metronidazole,
we sought to establish whether this functional group is also
important for the activity of these nitroimidazole carboxamides,
which are thought to act by similar mechanisms as metronidazole.
Thus, we prepared desnitro analogues 17 and 18 and the reduced
amine derivative 20 (Scheme 4). As hypothesised, all three com-
pounds displayed no discernable activity against parasites or
C. difficile, supporting the importance of the nitro group in the
mode of action of nitroimidazole carboxamides (Supplementary
Table 5).
4. Conclusion
3.5. Plasma protein binding and microsome stability
New nitroimidazole carboxamides were identified with activity
Metronidazole is essentially 100% orally absorbed [33], yet
against the pathogenic parasites G. lamblia, including
a
Table 3
Human plasma protein binding and microsome stability for selected compounds.
No.
X
R
G. lamblia EC50
(
m
M)
Plasma protein binding (% bound)
Microsome stability (% remaining at 2 h)
1
8a
12a
8h
12h
8k
Metronidazole
6.1
3.5
0.5
1.6
8.8
4.9
0.6
<5
95
>99
9
78
94
99
100
88
94
Me
H
Me
H
NHCH2(4-F-Ph)
NHCH2(4-F-Ph)
morpholine
97
morpholine
100
N.D
N.D
Me
H
NH-cyclohexyl
NH-cyclohexyl
12k
Plasma protein binding sulfamethoxazole control ¼ 68% bound (consistent with literature 66% bound [38]); microsome stability verapamil control ¼ 25% remaining at 2 h
(consistent with literature [39]).

362
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Acknowledgement
We thank Madison Hoffman, Andrew Barnett and Alexander
Booy for their contribution to chemical syntheses during under-
graduate research projects and to Johnny Huang and Geraldine
Kaeslin for assisting with the cytotoxicity assays. This research was
supported by a UQ CIEF grant, NHMRC grant AP1063214, and NIH
grants AI114671 and DE020607. AMJ was supported by an Austra-
lian Postgraduate Award (APA), QLD Government Smart Futures
PhD scholarship and IMB scholarship, TK, RP, MATB and MSB by a
Wellcome Trust Seeding Drug Discovery Award (094977/Z/10/Z)
and Wellcome Trust Strategic Award (066648 CO-ADD), and MAC
by NHMRC Fellowships (AF511105 and APP1059354).
Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.04.064.
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