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