Discovery and development of 2-aminobenzimidazoles as potent antimalarials

Article abstract of DOI:10.1016/j.ejmech.2021.113518

The emergence of Plasmodium falciparum resistance to frontline antimalarials, including artemisinin combination therapies, highlights the need for new molecules that act via novel mechanisms of action. Herein, we report the design, synthesis and antimalarial activity of a series of 2-aminobenzimidazoles, featuring a phenol moiety that is crucial to the pharmacophore. Two potent molecules exhibited IC₅₀ values against P. falciparum 3D7 strain of 42 ± 4 (3c) and 43 ± 2 nM (3g), and high potency against strains resistant to chloroquine (Dd2), artemisinin (Cam3.II^C580Y) and PfATP4 inhibitors (SJ557733), while demonstrating no cytotoxicity against human cells (HEK293, IC₅₀ > 50 μM). The most potent molecule, possessing a 4,5-dimethyl substituted phenol (3r) displayed an IC₅₀ value of 6.4 ± 0.5 nM against P. falciparum 3D7, representing a 12-fold increase in activity from the parent molecule. The 2-aminobenzimidazoles containing a N¹-substituted phenol represent a new class of molecules that have high potency in vitro against P. falciparum malaria and low cytotoxicity. They possessed attractive pharmaceutical properties, including low molecular weight, high ligand efficiency, high solubility, synthetic tractability and low in vitro clearance in human liver microsomes.

Full text of DOI:10.1016/j.ejmech.2021.113518

European Journal of Medicinal Chemistry 221 (2021) 113518
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery and development of 2-aminobenzimidazoles as potent
antimalarials
,
Shane M. Devine a *, Matthew P. Challis b, Jomo K. Kigotho a, Ghizal Siddiqui b,
,
Amanda De Paoli b, Christopher A. MacRaild a, Vicky M. Avery c d, Darren J. Creek b,
,
, **
Raymond S. Norton a e, Peter J. Scammells a
a Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, 3052, Australia
b Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, 3052, Australia
c Discovery Biology, Griffith University, Nathan, Queensland, 4111, Australia
d School of Environment & Science, Griffith University, Nathan, Queensland, 4111, Australia
e ARC Centre for Fragment-Based Design, Monash University, Parkville, Victoria, 3052, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
The emergence of Plasmodium falciparum resistance to frontline antimalarials, including artemisinin
combination therapies, highlights the need for new molecules that act via novel mechanisms of action.
Herein, we report the design, synthesis and antimalarial activity of a series of 2-aminobenzimidazoles,
featuring a phenol moiety that is crucial to the pharmacophore. Two potent molecules exhibited IC50
values against P. falciparum 3D7 strain of 42 4 (3c) and 43 2 nM (3g), and high potency against strains
resistant to chloroquine (Dd2), artemisinin (Cam3.IIC580Y) and PfATP4 inhibitors (SJ557733), while
Received 17 December 2020
Received in revised form
11 April 2021
Accepted 22 April 2021
Available online 13 May 2021
demonstrating no cytotoxicity against human cells (HEK293, IC50 > 50
possessing a 4,5-dimethyl substituted phenol (3r) displayed an IC50 value of 6.4
m
M). The most potent molecule,
0.5 nM against
Keywords:
Malaria
P. falciparum 3D7, representing a 12-fold increase in activity from the parent molecule. The 2-
aminobenzimidazoles containing a N1-substituted phenol represent a new class of molecules that
have high potency in vitro against P. falciparum malaria and low cytotoxicity. They possessed attractive
pharmaceutical properties, including low molecular weight, high ligand efficiency, high solubility, syn-
thetic tractability and low in vitro clearance in human liver microsomes.
2-Aminobenzimidazole
Plasmodium falciparum
Structure-activity relationships
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
measures, either chemoprophylaxis or active treatment of diag-
nosed disease. Concerted efforts over the last two decades,
Malaria, a parasitic disease that has a major impact on both
human health and economic development, continues to represent a
significant global health challenge, especially to the world's most
vulnerable societies [1]. Malaria is caused by parasites of the genus
Plasmodium, and is transmitted by the female Anopheles mosquito
during a blood-meal. Of the Plasmodium species that infect humans,
Plasmodium falciparum accounts for 90% of deaths. As no highly
effective vaccine is currently available, malaria control involves a
combination of vector control e using insecticide treated bed nets
including implementation of highly efficacious artemisinin com-
bination therapies (ACTs) as the first-line treatment, have resulted
in a steady decline in global malaria mortality, from 736,000 in
2000 to 409,000 deaths in 2019 [1]. During this recent period, a
number of countries have achieved malaria elimination and been
declared malaria-free [1e3]. However, while global malaria inci-
dence has dropped since 2010, the rate of decline has stagnated and
even reversed since 2014 [1].
Alarmingly, resistance to the first- and last-line treatment, ACTs,
is becoming widespread in South-East Asia, with countries in the
Greater Mekong region reporting treatment failure rates of greater
than 10% to at least one of the five ACTs employed in that area [1].
Indeed, Cambodia has seen high failure rates against four of the five
recommended ACTs. This trend raises concern that parasites
resistant to all existing antimalarials will soon spread world-wide
(ITNs) and/or insecticide spraying
e and pharmacological
* Corresponding author.
** Corresponding author.
E-mail addresses: shane.devine@monash.edu (S.M. Devine), peter.scammells@
monash.edu (P.J. Scammells).
https://doi.org/10.1016/j.ejmech.2021.113518
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

S.M. Devine, M.P. Challis, J.K. Kigotho et al.
European Journal of Medicinal Chemistry 221 (2021) 113518
[2e4]. To combat this resistance and to help achieve the ambitious
WHO elimination targets [1], new molecules with novel mecha-
nisms of action are urgently required. Many groups worldwide are
striving to fill this gap, and the Medicines for Malaria Venture
(MMV) supports many of the most promising candidates in clinical
development [2,5]. However, a number of the MMV candidates in
clinical development are based on existing antimalarial chemo-
types, and several early-stage candidates share common mecha-
nisms of action (MoAs). There is a significant risk that molecules
with shared MoA's may well suffer from poor efficacy in the field
owing to cross-resistance. The Open Access Malaria Box [6], which
includes 400 diverse chemotypes triaged from almost 30,000
antimalarial compounds, exhibits many similar MoAs. An analysis
of 90 Malaria Box molecules found that almost half of the tested
compounds demonstrated a similar MoA to existing antimalarials
in clinical usage or development, including inhibition of pyrimidine
biosynthesis and PfATP4 [7,8]. Whilst not surprising, based on the
selection criteria for this specific collection, this does highlight the
need for new chemotypes and potentially new MoAs. Of twenty-
two MMV products in the “Product Development” or “Access”
stages, twelve contain artemisinin (ART) combinations, four
contain sulfadoxine-pyrimethamine and amodiaquine combina-
tions and two are tafenoquine-based therapies. Resistance to
antifolates and 4-aminoquinolines has been known for decades
[2,9] and ACT resistance is continuing to emerge, involving both
ART [10] and its partner drugs [1,11]. The four molecules in the
“Patient Exploratory” phase, ganaplacide [12,13], cipargamin [14],
MMV048 [15,16], and artefenomel [17] (Fig. 1), exhibit different
MoAs, although artefenomel shares some mechanistic features
with ARTs [11]. These molecules are among the most advanced new
agents active against malaria.
Our antimalarial drug discovery program has targeted
P. falciparum apical membrane antigen 1 (AMA1), a protein essen-
tial for parasite invasion and growth [18e20]. This program
employed a fragment-based drug discovery approach to identify,
among other scaffolds, benzimidazoles as an attractive scaffold for
AMA1 inhibition. These benzimidazoles feature 2-phenyl substi-
tution and are exemplified [20] by 1ae1c, which exhibit binding
affinities (KD) for AMA1 ranging from 0.1 to 0.8 mM as measured by
surface plasmon resonance (SPR) (Fig. 2).
A previous report on the antimalarial activity of amino-
azabenzimidazoles described the optimisation of a hit identified in
a HTS-phenotypic screen [21]. The MoA was not investigated and
limited SAR was described, but the structural similarities between
our 2-substituted benzimidazoles and their N1-substituted
Fig. 2. 2-Phenyl-substituted benzimidazoles active against AMA1 [20].
aminoazabenzimidazoles piqued our interest in the possibility of
these acting via AMA1 inhibition. As this was the only report
featuring N1-substituted 2-aminobenzimidazoles, we had an op-
portunity to assess some of the design elements of our 2-
substitution profile with the as-yet-undetermined MoA of the re-
ported aminoazabenzimidazoles [21]. The benzimidazole scaffold
featuring a wide range of substitution is a rich area of ongoing
antimalarial
drug
discovery,
including
N-aryl-2-
aminobenzimidazoles [22], tricyclic pyridobenzimidazoles [23],
1,2,5-trisubstituted benzimidazoles [24] and 2-substituted amide
containing benzimidazoles [25]. Herein, we report our further
investigation of the benzimidazole scaffold and its antimalarial
activity.
2. Results and discussion
2.1. Chemistry
In this current study, we synthesised a range of 1,2-disubstituted
benzimidazoles, predominantly bearing N1-aryl and C2-amino
substituents. Two main synthetic approaches were employed to
prepare the target molecules, namely (i) a one-step copper medi-
ated N1-arylation of 2-aminobenzimidazole or (ii) the cyclisation of
an appropriately substituted o-phenylene diamine with ethox-
ycarbonyl isothiocyanate followed by base hydrolysis.
2.1.1. N1-arylation of 2-aminobenzimidazole
This series of N1-substituted aminobenzimidazoles (ABIs) con-
taining mono-substituted phenyl groups was made via a one-step,
selective
copper-mediated
arylation
reaction
from
2-
aminobenzimidazole and the appropriately substituted hal-
obenzene (Scheme 1). This small initial library featured a variety of
o-, m- and p-substituted benzenes (2ae2k). This reaction, pio-
neered by Ueda et al. [26], utilised CuI, Cs2CO3 and 8-
hydroxyquinoline as a ligand in t-BuOH to preferentially form the
N1-product with no C2-amino substitution being observed. Other
methods of chemoselective N-arylation have been established,
including copper-based approaches using D-glucosamine [27] or
boronic acid [28] methods, as well as nickel based ChaneLam [29]
couplings. Initial attempts to utilise boronic acid chemistry [28]
were explored, but the Ueda et al. method [26] using 8-
hydroxyquinoline gave slightly higher yields in our hands.
This was extended to explore ABIs that featured the o-phenol
substituent in 2a in concert with mono-substitution incorporating
electron-donating (Me, 3ae3d; OMe, 3ee3h) and electron-
withdrawing (F, 3ie3l; Cl, 3me3p) groups installed at the 3-, 4-,
5- and 6-positions of the phenol. Two further analogues in this
series were examined containing dimethylation of the phenol (4,6-
diMe, 3q and 4,5-diMe, 3r). Bioisosteric replacement of a phenol
with other hydrogen bonding motifs such as an indole has been
tolerated by the dopamine D1/D5 receptors [30,31], and a similar
strategy was employed to modify the phenol of 2a. The 3-
substituted compounds (3a, 3e, 3i, 3m) gave the lowest chemical
yields, compared to the other positions substituted owing to the
steric bulk associated with substitution in this position towards the
N-arylation reaction. Ring expansion around the phenolic part of
Fig. 1. The most advanced antimalarial candidates with novel MoAs.
2

S.M. Devine, M.P. Challis, J.K. Kigotho et al.
European Journal of Medicinal Chemistry 221 (2021) 113518
Scheme 1. Synthesis of ABIs 2e6. Reagents and conditions: (a) RePhI or RePhBr, CuI, 8-hydroxyquinoline, Cs2CO3, t-BuOH, 90e110 ꢀC, 16 h, 2e80%.
the molecule gave benzofuran (4a) and indole (4b) via the N-ary-
lation reaction (Scheme 1). The comparable benzothiophene was
attempted and minor amounts were detected by LCMS, but the
desired product could not be isolated. A similar approach was
attempted for the analogous difluoromethyl [32] analogue, but the
N-arylation reaction failed. Replacement of the phenol with
electron-withdrawing groups was attempted, including tri-
fluoromethyl and trifluoromethoxy replacements, as well as halo-
gens including fluoro, chloro, bromo and iodo groups.
Unfortunately, apart from the fluorine replacement (2l), these re-
actions failed suggesting that these functional groups were
incompatible with the scope of the chemoselective N-arylation
reaction. Also, in the formation of 2l, a minor by-product was iso-
lated that corresponded to the O-linked o-fluorobenzene coupling
to 8-hydroxyquinoline, namely 8-(2-fluorophenoxy)quinoline. The
comparable by-product was also detected in reactions aimed at
forming the analogous benzofuran (4a), indole (4b) and attempted
benzothiophene syntheses. Cyclised variants at the 5- and 6-
positions of the phenol with naphthalenol (5a) and quinolinol
(5b) were also synthesised via the N-arylation method. To deter-
mine the effect of changing the phenol to a pyridinol, systematic
nitrogen replacement of a carbon was performed at the four
possible positions on the phenol ring to give 6ae6d. This was also
attempted from 5-iodopyrimidin-4-ol to form the analogous pyr-
imidin-4-ol, but without success. Changing the spatial arrangement
between the amino and phenol moieties in the form of indazole (7)
via the selective N1-arylation depicted in Scheme 1 was also
achieved.
Scheme 2. Synthesis of 2-substituted benzimidazoles 8ae8c. Reagents and condi-
tions: (a) RePhI, CuI, 8-hydroxyquinoline, Cs2CO3, t-BuOH, 100 ꢀC, 16 h, 3e34%.
2.1.3. Linear synthesis to ABIs
A multistep linear sequence utilising nucleophilic aromatic
substitution was employed to access ABIs with variations at the N1-
position (Scheme 3). Analogues 9ae9c were initially accessed from
2.1.2. Replacement of the 2-amino group
Modification of the 2-amino group was probed to elucidate its
importance to activity. The 2-amino group was replaced with a
hydroxyl (8a) or a mercapto group (8b) and featured an o-anilino
group in place of the o-phenol utilising the same N-arylation
method reaction used for the preparation of 2 (Scheme 2). The des-
amino analogue 8c with the o-phenol group was also synthesised
using this approach.
Scheme 3. Synthesis of ABIs 9ae9d, 10. Reagents and conditions: (a) ReNH2, K2CO3,
MeCN, 82 ꢀC, 6 h, 89e93%; (b) H2, 10% Pd/C, EtOH, 25 ꢀC, 2 h, 88e100%; (c) ethox-
ycarbonyl isothiocyanate, EDCI.HCl, Et3N, MeCN, 82 ꢀC, 1 h, 45e88%; (d) 1 M NaOH,
EtOH, 100 ꢀC, 2 h, 40e85%; (e) 2-iodophenol, CuI, 8-hydroxyquinoline, Cs2CO3, t-BuOH,
115 ꢀC, 16 h, 0.3%.
3

S.M. Devine, M.P. Challis, J.K. Kigotho et al.
European Journal of Medicinal Chemistry 221 (2021) 113518
1-fluoro-2-nitrobenzene and substitution of the respective alkyl or
cycloalkyl amine and then subsequent nitro group reduction. To
avoid using the toxic reagent cyanogen bromide, another method of
benzimidazole ring formation was sought to form the 2-
aminobenzimidazole scaffold. This was achieved via cyclisation of
the respective substituted o-phenylenediamine using ethox-
ycarbonyl isothiocyanate via a modified literature procedure [33],
and the subsequent carbamate was hydrolysed under aqueous
basic conditions to form compounds 9ae9c. An azabenzimidazole
derivative (10) was also pursued to demonstrate the relative effect
of a nitrogen in the typically benzo ring and provide a matched pair
for 2a. This was achieved via the analogous cyclisation of 2,3-
diaminopyridine using ethoxycarbonyl isothiocyanate and subse-
quent carbamate hydrolysis to form the precursor 3H-imidazo[4,5-
b]pyridin-2-amine scaffold (9d). The subsequent N-arylation reac-
tion outlined earlier gave the 2-amino imidazopyridine (10), albeit
in a very low yield.
Scheme 5. Synthesis of Suzuki products 12a and 12b. Reagents and conditions: (a) 3-
bromopyridin-2-amine or 3-bromopyrazin-2-amine, 2-hydroxyphenylboronic acid,
PdCl2(PPh3)2, THF/1 M Na2CO3, 100 ꢀC, 1 h, 19e27%.
antimalarial drugs and from those candidates in the MMV pipeline,
and was the sole ABI featuring an N1-substituted phenol from the
study by Hameed et al. [21] Due to this lack of appreciable AMA1
binding, the remaining molecules 3e12 were not assessed for
AMA1 binding, but solely by in vitro growth inhibition and addi-
tional antimalarial assays.
2.2.1. Structure-activity relationship
To assess the importance of the 2-aminobenzimidazole and N1-
phenol relationship, modifications associated with this moiety
were introduced to determine its relative importance for potency.
The changes undertaken included: 1) phenol substitution, modifi-
cation and replacement; 2) amino modification and replacement;
and 3) the evaluation of divergent scaffolds.
2.1.4. Reductive amination and divergent scaffolds
Modification to the 2-amino group was next explored through
the incorporation of alkyl substituents in order to better under-
stand the tolerance to changes at this position. Initial attempts to
alkylate 2-aminobenzimidazole directly with
1 equivalent of
methyl iodide resulted in inseparable mixtures of 2-aminomethyl
and O-methyl products. Protection of the phenol with TBSCl and
subsequent methylation was also unsuccessful, owing to the highly
labile nature of the O-tert-butylsilyl group, resulting in its cleavage
and O-methylation in situ. Standard reductive amination conditions
were next attempted using 2-aminobenzimidazole and cyclohex-
anone in the presence of AcOH, but only starting material was
recovered. Sørensen et al. [34] noted that standard reductive ami-
nation conditions with 2-aminobenzimidazole and tetralone failed
to produce the expected imine or subsequent amine after reduc-
tion. They modified the reaction to incorporate titanium(IV) iso-
propoxide and successfully achieved imine formation and
subsequent amines after treatment with NaBH(OAc)3. This modi-
fication worked well in our hands and a range of alkyl derivatives
(11ae11i) with both straight and branched chains, as well as cyclic
variants, were made via this 2-step method (Scheme 4).
2.2.2. Phenol substitution, modification and replacement
Compound activity was assessed by an in vitro growth inhibition
assay using asynchronous Pf3D7 culture with 72 h compound
exposure to determine the IC50 (Tables 1 and 2). A negative control
(DMSO, 0.01%) and a positive control (1 mM, 2a) were evaluated
alongside the novel ABIs and associated molecules, and all assays
were carried out in either technical duplicate or triplicate, with at
least three biological replicates for each molecule.
The in vitro IC50 values (activity) of ABIs 2e6, 9 and known an-
timalarials are summarised in Table 1.
The structure-activity relationships (SAR) we have discovered
around this scaffold are striking in that very minor structural
changes result in inactive compounds. Specifically, changing the
potent o-hydroxy (2a) to m-hydroxy (2b), o-amino (2g), o-methoxy
(2d) and o-hydroxymethyl (2k) all resulted in large reductions in
activity (in vitro growth inhibition IC50 > 500 nM). Likewise,
replacement of the hydroxy with a fluorine (2l) resulted in an
inactive compound.
We then investigated retaining the components of the 2-amino
and N1-phenol groups in a divergent scaffold setting. We syn-
thesised the pyridine (12a) and pyrazine (12b) as replacements for
the 2-aminobenzimidazole portion of the molecule via Suzuki
coupling of the respective pyridine and pyrazine with 2-
hydroxyphenylboronic acid (Scheme 5).
However, when the N1-phenol moiety was retained, activity
increases were achieved relative to the parent molecule 2a. The
series 3ae3p, which featured mono-substitution of the phenol
with retention of the 2-amino group, demonstrated remarkable
SAR in our IC50 assay. The 3-substituted compounds (3a, 3e, 3i, 3m)
gave the weakest activity across the four positions substituted. The
4-substituted analogues (3b, 3j and 3n) displayed a range of IC50
values, with the 4-methyl analogue (3b) the most potent at
2.2. Antimalarial activity
Binding to AMA1 was investigated using SPR for compounds
2ae2l [18], but no binding was observed. These compounds were
then assessed for in vitro growth inhibition against the Plasmodium
falciparum 3D7 strain, and one compound, phenol 2a, demon-
strated considerable activity, exhibiting an IC50 of 77 4 nM. The
lack of detectable AMA1 binding implied a distinct MoA for this
compound. ABI 2a is structurally distinct from all existing
56
6 nM. The electron-withdrawing effect of the 4-fluoro (3j;
IC50: 90 11 nM) and 4-chloro (3n; IC50: 162 11 nM) diminished
the activity in comparison. Interestingly, the 4-methoxy analogue
(3f) exhibited an IC50 value of >500 nM, possibly indicating an
unfavourable conformation in the binding site due to its increased
Scheme 4. Synthesis of 2-substituted ABIs 11ae11i. Reagents and conditions: (a) Ti(O-i-Pr)4, RCH ¼ O/RC ¼ O, THF, 25 ꢀC, 5 h; (b) NaBH(OAc)3, THF, 25 ꢀC, 18 h, 8e40%.
4

S.M. Devine, M.P. Challis, J.K. Kigotho et al.
European Journal of Medicinal Chemistry 221 (2021) 113518
Table 1
size when compared to the 4-methyl analogue (3b; IC50
:
IC50 values of ABIs e Phenol substitution, modification and replacement series.
56 6 nM), or an unfavourable dipole-dipole interaction. In gen-
eral, the compounds with electron-donating methyl and methoxy
substituents at the 5-position exhibited the greatest activity, with
the 5-methyl (3c) and 5-methoxy (3g) having IC50 values of 42
4
and 43 2 nM, respectively. These two compounds demonstrate a
1.8-fold increase in activity against P. falciparum 3D7 parasites
compared with 2a.
The electron-withdrawing 5-fluoro (3k; IC50: 188 23 nM) and
5-chloro (3o; IC50: 164 13 nM) derivatives had weaker activity.
The 6-substituted variants had similar activities to each other, of
61 7 nM for 6-methyl (3d) and 55 3 nM for 6-methoxy (3h). The
6-fluoro (3l) and the 6-chloro (3p) showed significant decreases in
their activity, with IC50 values > 500 nM. This large decrease in
activity may be potentially explained by the relative decrease in the
pKa of the phenol with these substituents present. The phenolic pKa
values were therefore examined to assess whether there was any
correlation with the sharp SAR. The electron-withdrawing 6-fluoro
(3l) and 6-chloro (3p) analogues have calculated pKa values 8.70
and 8.28, respectively, compared to the 6-methyl (3d) and 6-
methoxy (3h) compounds with pKa values of 10.46 and 10.17,
respectively. Based on the increased activity of the 5- and 6-methyl
analogues (3c and 3d), dimethylated analogues containing 4,6-
disubstitution (3q) and 4,5-disubstitution (3r) were assessed. The
incorporation of 4,6-dimethyl substitution on the phenol ring (3q)
gave a marked improvement in activity, with an IC50 of 23 2 nM.
This result was amplified when 4,5-dimethyl substitution was
considered, with 4,5-dimethyl (3r) exhibiting an IC50 of
6.4 0.5 nM. These two results establish 3.3-fold (3q) and 12-fold
(3r) increases, respectively, in activity against P. falciparum 3D7
parasites compared with 2a. This demonstrates once again the tight
SAR of the aminobenzimidazole series. Ring expansion of the
phenolic position to a benzofuran (4a) and indole (4b) abolished
activity. Extending this further, naphthalenol (5a) and quinolinol
(5b) were investigated; remarkably, the naphthalenol (5a) had an
Cpd No.
X
R
Y
Z
Q
IC50 (nM)a
CQ
DHA
Pyrimethamine
MQ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
16
2
44
12
2
0.5
4
1
2a
2b
2c
2d
2e
2f
2g
2h
2i
OH
H
H
OMe
H
H
NH2
H
H
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
77
4
6-OH
5-OH
H
6-OMe
5-OMe
H
5-NH2
5-Me
5-CN
H
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
H
H
2j
2k
2l
CH2OH
F
H
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3 m
3n
3o
3p
3q
3r
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
3-Me
4-Me
5-Me
6-Me
3-OMe
4-OMe
5-OMe
6-OMe
3-F
4-F
5-F
6-F
3-Cl
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
135
56
42
9
6
4
7
61
183 15
>500
43
55
2
3
321 29
90 11
188 23
>500
>500
IC50 of 38
1 nM, whereas the quinolinol (5b) was inactive
4-Cl
5-Cl
6-Cl
4,6-diMe
4,5-diMe
162 11
164 13
>500
(>500 nM). The calculated pKa values for 5a and 5b were 9.69 and
9.53, respectively, an insignificant difference compared with the
stark difference in activity, indicating that the phenolic acidity
alone does not account for the activity difference. The four pyr-
idinol compounds (6ae6d) were all inactive and likewise the
phenolic pKa could not account for this loss of activity. Finally, the
inclusion of N1-substitution of ethanol (9a), cyclopentanol (9b) and
cyclohexanol (9c) groups led to IC50 values > 500 nM. Once again,
modification from a phenol (2a) to the aliphatic cyclohexanol (9c)
demonstrated the remarkable SAR changes within this series.
23
2
6.4 0.5
4a
4b
e
e
e
e
O
NH
e
e
e
e
>500
>500
5a
5b
e
e
e
e
e
e
CH
N
e
e
38
1
>500
6a
6b
6c
6d
OH
OH
OH
OH
3-N
4-N
5-N
6-N
e
e
e
e
e
e
e
e
e
e
e
e
>500
>500
>500
>500
2.2.3. Amino modification/replacement and divergent scaffolds
More drastic modifications to the core, including a scaffold
change and relocation of the 2-amino in indazole (7); replacement
(8a, 8b) and removal (8c) of the 2-amino group; inclusion of a 2-
amino imidazopyridine (10); alkylation series of the 2-amino
group (11) and scaffold replacement (12) were assessed in the
same growth inhibition assay format (Table 2).
9a
e
e
e
e
e
e
>500
9b
e
e
>500
9c
e
e
e
e
>500
Investigation of the spatial arrangement between the amino and
phenol in the form of indazole (7) resulted in a complete loss of
activity. Removal of the 2-amino group to give des-amino (8c) with
retention of the N1-substituted o-phenol resulted in a complete loss
of activity, as did modifying the 2-amino group to a hydroxyl (8a) or
mercapto (8b) moiety. The replacement of a carbon with nitrogen
at the 7-position of the benzimidazole to the corresponding aza-
benzimidazole also resulted in an inactive compound (10).
Substitution of the free amine reduced activity, with 11ae11i all
being less potent than the parent 2a. However, these results were
encouraging as earlier attempts to modify the pairing of 2-amino
In vitro growth inhibition assay using asynchronous Pf3D7 culture with 72 h com-
pound exposure (Monash assay).
a
IC50 values are reported as mean SEM (n ꢁ 3).
5

S.M. Devine, M.P. Challis, J.K. Kigotho et al.
European Journal of Medicinal Chemistry 221 (2021) 113518
Table 2
IC50 values of compounds 7, 8, 10e12 e Amino modification/replacement and divergent scaffold series.
Cpd No.
7
X
Y
R
Z
IC50 (nM)a
e
e
e
e
>500
8a
8b
8c
OH
SH
H
NH2
NH2
OH
e
e
e
e
e
e
>500
>500
>500
10
e
e
e
e
>500
11a
11b
11c
11d
11e
11f
e
e
e
e
e
e
e
e
e
e
e
e
Et
Pr
Bu
i-Pr
i-Bu
e
e
e
e
e
e
>500
>500
278 53
>500
356 43
333 68
11g
11h
11i
e
e
e
e
e
e
e
e
e
224 38
>500
>500
12a
12b
e
e
e
e
e
e
CH
N
>500
>500
In vitro growth inhibition assay using asynchronous Pf3D7 culture with 72 h compound exposure (Monash assay).
a
IC50 values are reported as mean SEM (n ꢁ 3).
and N1-phenol motifs resulted in a complete loss of activity. The
most potent analogue (11g; 224 38 nM) was almost 3-fold less
potent compared to 2a. In the straight chain alkyl compounds
(11ae11c), an increase in substituent length resulted in increased
potency. The ethyl- and propyl-containing ABIs (11a and 11b)
exhibited IC50 values > 500 nM, whereas the butyl-containing
pyrimethamine and pyronaridine) with various MoAs to assess
their propensity for cross-resistance (Table 3). ABIs 2a and 3c were
examined for their in vitro anti-parasitic activity against the Dd2
line [35], a CQ-resistant strain with distinct point mutations in the
Pf CQ-resistance transporter (PfCRT) [36e38]. Resistance was
defined as in vitro activity that was 5-fold less potent in the CQ-
resistant line, Dd2, when compared to the CQ-sensitive 3D7 line.
Both of the ABIs, 2a and 3c displayed potent inhibitory activity
against both 3D7 and Dd2 strains, with IC50 values of 22.2 2.1 and
13.5 1.2 nM, respectively, against the drug-sensitive strain 3D7,
compound (11c) had an IC50 value of 278
53 nM. The longer
chain length may interact with hydrophobic residues in the puta-
tive binding site. The additional methyl in the branched chain de-
rivatives (11d, 11e) resulted in increased activity compared to their
straight chain counterparts, but remained less potent than the
parent (2a). The addition of a direct cyclic moiety at the 2-position
amine resulted in cyclohexyl (11f), which was slightly less active
than the cyclohexyl methyl (11g), with IC50 values of 333 68 and
Table 3
Activity of ABIs 2a, 3c and known antimalarials against 3D7 and Dd2 cell lines.
IC50 (nM)a
3D7b
Selectivity Index
Dd2/3D7
224
38 nM, respectively. The addition of a benzyl (11h) or
Dd2
cyclopropyl methyl (11i) abrogated activity.
2a
3c
22.2 2.1
13.5 1.2
0.9 0.1
10.0 1.8
0.4 0.1
148.9 6.5
4.7 1.0
7.4 1.6
97.3 0.8
31.7 1.9
1.3 0.1
87.9 3.5
0.6 0.1
4.4
2.3
1.4
8.8
1.3
0.8
>8000
1.1
The divergent scaffolds represented by 12a and 12b lacked ac-
tivity (IC50 > 500 nM). These results are interesting, as amino-
pyridine (12a) and aminopyrazine (12b) both include the 2-amino
and o-phenol moieties. These results demonstrate the absolute
requirement for the retention of the 2-aminobenzimidazole scaf-
fold in combination with the N1-phenol substitution for potent
antimalarial activity.
Artesunate
CQ
DHA
Puromycin
Pyrimethamine
Pyronaridine
114.4 10.5
>40
m
M
8.3 1.1
In vitro growth inhibition assay using synchronous ring-stage culture with 72 h
compound exposure (Griffith assay). aIC50 values are from two independent bio-
logical replicates, each consisting of two technical repeats each and error is provided
as the standard deviation. b3D7 strain parasites are a different isolate than the
parasites used for other activity assays in Tables 1 and 2
2.2.4. Antimalarial resistance
ABIs were further evaluated alongside known antimalarials
(artesunate,
CQ,
dihydroartemisinin
(DHA),
puromycin,
6

S.M. Devine, M.P. Challis, J.K. Kigotho et al.
European Journal of Medicinal Chemistry 221 (2021) 113518
Table 5
Solubility and metabolic evaluation of ABIs 2a, 3c, 3g, 3q and 3r.
and 97.3 0.8 and 31.7 1.9 nM, respectively, against the drug-
resistant strain Dd2 [35]. The activity against the drug-resistant
strain was therefore lower than against the drug-sensitive strain,
with corresponding selectivity indices (SI) of 4.4 (2a) and 2.3 (3c)
(Table 3). Although, 2a shows minor CQ cross-resistance, this is less
apparent in 3c, suggesting that this issue can be managed with
more advanced compounds in development.
Compounds 2a, 3c, 3g, 3q, 3r and 5a were further examined to
assess drug-sensitivity in field isolates, namely Cam3.IIC580Y (a
Cambodian isolate bearing an ART-resistance conferring mutation
in the propeller domain of the K13 gene), and Cam3.IIrev (in which
the K13 mutation is reverted to wild-type) cell lines [39]. As many
antimalarials have been used in Cambodia and surrounding areas,
these strains also provide the opportunity to study the potential for
cross-resistance to a range of antimalarials. The ABIs exhibited
similar activity against these two Plasmodium lines, with IC50 values
of 264 40 nM and 350 78 nM for 2a, 81 10 nM and 140 3 nM
for 3c, 168 25 nM and 212 10 nM for 3g, 139 10 nM and
133 14 nM for 3q, 63 7 nM and 47 4 nM for 3r, and 114 9 nM
Kinetic solubility (
m
g/
Stability in liver microsomes
Half-life t1/2 (min) In vitro CLint
mL)a
(mL/
min/106 cells)
SolpH 2.0
SolpH 6.5
Mouse
Human
Mouse
Human
2a
3c
3g
3q
3r
>100
>100
>100
>100
>100
>100
25e50
50e100
12.5e25
12.5e25
78
18
6
4
23
>255
162
124
173
129
22
97
271
476
76
<7
11
14
10
13
a
Kinetic solubility was determined by nephelometry.
2.2.5. Preliminary pharmacokinetic studies
ABIs possess favourable drug-like qualities, including low mo-
lecular weight and high aqueous solubility. The ABI core provides
ample opportunities to explore the impact of further functionali-
sation on antimalarial activity without compromising the drug-like
properties. Ligand efficiency (LE) is one method of ranking and
triaging molecules for further development that takes into account
factors other than potency alone. This metric takes into consider-
ation the molecular weight, solubility and potency of a given
and 105
12 nM for 5a, respectively (Table S1). While this is
weaker activity than observed in the laboratory-adapted 3D7 and
Dd2 strains, this may be due to the extended exposure of field
isolates to commonly used antimalarials, leading to greater drug
tolerance compared to the laboratory-adapted strains. Importantly,
similar activity was observed for both of the Cam3.II lines, indi-
cating that the widespread K13 C580Y mutation associated with
ART resistance does not induce resistance to these compounds [40].
However, it is noted that ART resistance is typically determined
through the use of ring stage viability assays [39], and future work
will determine the relevance of ring stage activity to ABI pharma-
cology and investigate any potential for K13-mediated resistance at
the ring stage. CQ, DHA, pyrimethamine and mefloquine were also
examined against these Plasmodium lines and the results were
consistent with previous findings (see Table S1).
molecule and is equal to the
DGbinding/number of heavy atoms
(HAC) [42]. A threshold of 0.3 kcal molꢂ1HACꢂ1 is generally regar-
ded as an excellent starting point for further medicinal chemistry
optimisation. ABIs 3c and 3g have LE values of 0.56 and 0.53 kcal
molꢂ1HACꢂ1, respectively, clearly demonstrating the potential for
elaboration of this class of compounds. The inclusion of a subse-
quent methyl substituent in ABIs 3q and 3r further demonstrates
the attractive prospect of developing this series with LE values of
0.55 and 0.59 kcal molꢂ1HACꢂ1, respectively.
A selection of the most active ABIs were further examined to
determine their physicochemical and metabolic stability charac-
teristics (Table 5). The high aqueous solubility of the ABIs was
demonstrated by kinetic solubility estimation using nephelometry
Apart from the drug resistance mechanisms associated with the
currently used antimalarials, it is necessary to ascertain a resistance
profile against emerging MoAs, such as PfATP4 which is targeted by
a range of chemotypes [8]. The compounds 2a and 3c were there-
fore tested against a parasite strain resistant [41] to the PfATP4
inhibitor SJ557733 and its Dd2 parent strain for comparison
(Table 4). Both compounds showed similar potencies against the
SJ557733-resistant strain and its parent, with IC50 values of 17.5 nM
and 38.2 nM, respectively, for 3c and 46.0 and 102.7 nM, respec-
tively, for 2a (SI ¼ 0.5 for 2a and 3c). In comparison, the PfATP4
inhibitor SJ557733 showed extensive resistance in the Dd2-
SJ557733 resistant strain (SI ¼ >135).
[43] with 2a, 3c, 3g, 3q and 3r exhibiting >100
These five ABIs had solubility of >100 (2a), 50e100 (3g), 25e50 (3c)
and 12.5e25 g/mL (3q and 3r), respectively at pH 6.5. The meta-
mg/mL at pH 2.0.
m
bolic stability of ABIs was assessed by incubation at 1
mM with
either mouse or human liver microsomes at 37 ꢀC at 0.4 mg/mL
protein concentration with the addition of NADPH as co-factor. The
ABIs exhibited a range of half-lives and intrinsic clearance (CLint
)
values, ranging from low to high values across both species tested.
In mice, the CLint was high, with the most potent compounds
against Pf3D7, 3q and 3r, being rapidly metabolised. However, these
same ABIs (3q and 3r) in human liver microsomes fared noticeably
better, with low to intermediate CLint values. Specifically, the most
active ABI in the growth inhibition assay possessing a 4,5-dimethyl
Cytotoxicity evaluation at this early stage of development is
important to determine whether ABIs should be pursued as drug
leads, so ABIs 2a, 3c, 3g, 3q, 3r and 5a were examined against
human embryonic kidney cells (HEK293) to assess their cytotoxic
potential against a human cell line. None of the ABIs examined
substituted phenol (3r) exhibited moderate (76
mL/min/mg protein)
and low (13 L/min/mg protein) intrinsic clearance in mouse and
m
human liver microsomes, respectively. Future work will continue to
assess and address the drug-like properties of the ABIs as well as
investigate them in vivo.
showed cytotoxic effects at 50 mM against HEK293 cells (Table S2).
In fact, the ABIs 2a, 3c, 3g, 3q, 3r and 5a demonstrated >1000-fold
selectivity (HEK293/Pf3D7), demonstrating the safety of the series.
Table 4
3. Conclusions
IC50s of ABIs 2a and 3c against a PfATP4 resistant strain (Dd2-SJ557733).
IC50 (nM)a
Selectivity Index
Novel antimalarial chemical scaffolds that act by new mecha-
nisms of action are required to combat malaria resistance across the
globe. Therefore, new compound classes such as the 2-
aminobenzimidazoles (ABIs) are urgently required. We have
designed, synthesised and functionally evaluated a series of ABIs
and associated molecules (2e12), many of which were produced
via a one-step chemoselective N-arylation reaction. Deletion or
replacement of the 2-amino group abolished antimalarial activity.
Dd2-SJ Parent
Dd2-SJ557733
SJ557733/SJ-Parent
2a
3c
SJ557733
102.7
38.2
58.7
46.0
17.5
>8000
0.5
0.5
>135
In vitro growth inhibition assay using synchronous ring-stage culture with 72 h
compound exposure (Griffith assay). aThe comparisons between ABIs 2a, 3c and
SJ557733 were carried out once.
7

S.M. Devine, M.P. Challis, J.K. Kigotho et al.
European Journal of Medicinal Chemistry 221 (2021) 113518
Extension of the 2-amino group with alkyl, cycloalkyl and benzyl
4.2. General synthetic procedures
modifications (11ae11i), produced via
a titanium-mediated
reductive amination, resulted in weaker activity (224e>500 nM),
while bioisosteric alteration of the phenol to benzofuran (4a) and
indole (4b) scaffolds abolished activity, as did scaffold-hopping
from benzimidazole to pyridine, pyrazine and indazole scaffolds.
These results demonstrate the necessity for retention of the 2-
amino group on the benzimidazole and the N1-phenol arrange-
ment in the ABIs. Substitution of the phenol resulted in active
compounds, with eight molecules (3b, 3c, 3d, 3g, 3h, 3q, 3r, 5a)
possessing greater activity (6.4e61 nM) than the parent 2a (77 nM).
Several molecules were potent against P. falciparum (3D7),
including ABIs featuring 5-methyl (3c), 5-methoxy (3g) and
naphthalenol (5a) moieties, with IC50 values of 42 4, 43 2 and
4.2.1. General procedure for the N-arylation of 2-
aminobenzimidazole [21,26]
To a 2e5 mL microwave vial was added 2-aminobenzimidazole
(150 mg, 1.13 mmol), Cs2CO3 (918 mg, 2.82 mmol, 2.5 eq.), CuI
(21 mg, 0.11 mmol, 0.1 eq.), 8-hydroxyquinoline (25 mg, 0.17 mmol,
0.15 eq.) and the appropriately substituted bromo or iodobenzene
(1.24 mmol, 1.1 eq.) were suspended in t-BuOH (2 mL). The vial was
sealed and heated at 110 ꢀC (for bromobenzenes) or 90 ꢀC (for
iodobenzenes) for 16 h. The cooled mixture was poured into EtOAc/
MeOH (20:1) (30 mL), filtered through Celite™ and evaporated
under reduced pressure. The residue was purified by column
chromatography using DCM/MeOH/NH4OH (89:10:1) to give typi-
cally colourless amorphous solids. This method produced com-
pounds 2ae2l, 3ae3r, 4a, 4b, 5a, 5b, 6ae6d, 7, 8ae8c, 10.
38
1 nM, respectively. Two ABIs featuring 4,6- (3q) and 4,5-
dimethyl substitution (3r) were highly potent as antiparasitic
agents against Pf3D7, with IC50 values of 23 2 and 6.4 0.5 nM,
respectively. The ABIs have favourable pharmaceutical properties,
including low molecular weight, high solubility and ligand effi-
ciency, and are synthetically tractable. Preliminary in vitro meta-
bolism studies of ABIs in human liver microsomes demonstrate
moderate-low half-life and low in vitro intrinsic clearance.
ABIs featuring N1-phenol substitution (5-methyl, 3c) and (5-
methoxy, 3g) were further examined against a range of drug-
resistant parasite lines (Dd2, Cam3.IIC580Y, Cam3.IIrev and Dd2-
SJ557733). As the potency was similar against drug-resistant and
-sensitive lines, this result implies that cross-resistance against
known mechanisms of action is diminished relative to CQ, ART and
SJ557733, which in turn raises the possibility that ABIs act via a
novel mechanism of action. This exciting result warrants further
investigation, including testing against different stages of the par-
asite's lifecycle, in vitro generation of ABI-resistant parasites and
mass spectrometry led -omic approaches to identify the mecha-
nism of action.
4.2.1.1. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)phenol (2a) [21,26].
Compound 2a was formed from 2-iodophenol in 67% yield. 1H NMR
d
10.04 (s, 1H), 7.34 (ddd, J ¼ 8.2, 7.5, 1.7 Hz, 1H), 7.25 (dd, J ¼ 7.8,
1.6 Hz, 1H), 7.18 (d, J ¼ 7.8 Hz, 1H), 7.10 (dd, J ¼ 8.2, 1.2 Hz, 1H), 6.96
(td, J ¼ 7.7, 1.1 Hz, 2H), 6.81 (t, J ¼ 7.5 Hz, 1H), 6.63 (d, J ¼ 7.6 Hz, 1H),
5.96 (s, 2H). 13C NMR
d 154.7, 153.6, 143.1, 135.4, 130.0, 129.2, 121.6,
120.6, 119.7, 118.3, 117.2, 114.8, 107.9. HR-ESMS calcd. for
C
13H12N3Oþ [M þ H] 226.0975, found 226.0981.
4.2.1.2. 3-(2-Amino-1H-benzo[d]imidazole-1-yl)phenol
Compound 2b was formed from 3-iodophenol in 51% yield. 1H NMR
9.96 (s, 1H), 7.39 (t, J ¼ 8.0 Hz, 1H), 7.20 (d, J ¼ 7.8 Hz, 1H),
7.03e6.95 (m,1H), 6.91e6.84 (m, 5H), 6.82 (t, J ¼ 2.1 Hz,1H), 6.22 (s,
2H). 13C NMR
158.7 (C), 154.1 (C), 142.9 (C), 135.9 (C), 134.8 (C),
(2b).
d
d
130.8 (CH), 121.1 (CH), 118.7 (CH), 116.8 (CH), 115.3 (CH), 115.1 (CH),
113.3 (CH), 107.7 (CH). HR-ESMS calcd. for C13H12N3Oþ [M þ H]
226.0975, found 226.0978.
4. Experimental section
4.2.1.3. 4-(2-Amino-1H-benzo[d]imidazole-1-yl)phenol
Compound 2c was formed from 4-iodophenol in 37% yield. 1H NMR
9.85 (s, 1H), 7.26e7.20 (m, 2H), 7.18 (d, J ¼ 7.7 Hz, 1H), 6.99e6.92
(m, 3H), 6.83 (td, J ¼ 7.6,1.0 Hz,1H), 6.75 (dd, J ¼ 7.7, 0.6 Hz,1H), 6.10
(s, 2H). 13C NMR
157.3 (C), 154.6 (C), 142.8 (C), 135.5 (C), 128.2
(2 ꢃ CH), 125.8 (C), 120.9 (CH), 118.5 (CH), 116.5 (2 ꢃ CH), 114.9 (CH),
107.5 (CH). HR-ESMS calcd. for C13H12N3Oþ [M þ H] 226.0975,
found 226.0978.
(2c).
4.1. General methods for chemistry
d
1H, 19F and 13C NMR spectra were recorded on a Bruker Avance
Nanobay III 400 MHz Ultrashield Plus spectrometer at 400.50,
376.85 and 100.72 MHz, respectively, coupled to a BACS 60 auto-
d
matic sample changer at 25 ꢀC. Chemical shifts (
d) are recorded in
parts per million (ppm) by correction with reference to the chem-
ical shift of the solvent (DMSO‑d6 e unless otherwise stated), ac-
cording to the procedure described by Gottlieb et al. [44] Coupling
constants (J) are recorded in Hz, and the significant multiplicities
described by singlet (s), doublet (d), triplet (t), quadruplet (q),
broad (br), multiplet (m), doublet of doublets (dd), and doublet of
triplets (dt). All carbon NMR experiments used the DEPTQ pulse
sequence to determine carbon signals as C, CH, CH2 and CH3. LC-MS
were run to verify reaction outcome and purity using an Agilent
6100 series Single Quad coupled to an Agilent 1200 series HPLC.
The following buffers were used: buffer A, 0.1% formic acid in H2O;
buffer B, 0.1% formic acid in MeCN. The following gradient was used
4.2.1.4. 1-(2-Methoxyphenyl)-1H-benzo[d]imidazole-2-amine (2d)
[45]. Compound 2d was formed from 2-iodoanisole in 2% yield. 1H
NMR
d
7.52 (ddd, J ¼ 8.4, 7.5, 1.7 Hz, 1H), 7.34 (dd, J ¼ 7.7, 1.7 Hz, 1H),
7.29 (dd, J ¼ 8.4, 1.1 Hz, 1H), 7.18 (d, J ¼ 7.5 Hz, 1H), 7.13 (td, J ¼ 7.6,
1.2 Hz, 1H), 6.96 (t, J ¼ 7.2 Hz, 1H), 6.80 (t, J ¼ 7.6 Hz, 1H), 6.58 (dd,
J ¼ 7.7, 0.9 Hz, 1H), 6.03 (s, 2H), 3.74 (s, 3H). 13C NMR
d 155.2 (C),
154.7 (C), 143.1 (C), 135.5 (C), 130.4 (CH), 129.3 (CH), 122.8 (C), 121.1
(CH), 120.7 (CH), 118.4 (CH), 114.8 (CH), 113.2 (CH), 107.6 (CH), 55.6
(CH3). HR-ESMS calcd. for C14H14N3Oþ [M þ H] 240.1131, found
240.1136.
with a Phenomenex Luna 3
mM C8(2) 15 mm ꢃ 4.6 mm column, a
flow rate of 0.5 mL/min and total run time of 12 min: 0e4 min 95%
buffer A and 5% buffer B, 4e7 min 0% buffer A and 100% buffer B,
7e12 min 95% buffer A and 5% buffer B. Mass spectra were acquired
in positive and negative ion mode with a scan range of 0e1000 m/z
at 5 V. UV detection was carried out at 254 nm. All compounds were
of >95% purity. Thin layer chromatography was conducted on
0.2 mm plates using Merck silica gel 60 F254. Column chromatog-
raphy was achieved using Merck silica gel 60 (particle size
4.2.1.5. 1-(3-Methoxyphenyl)-1H-benzo[d]imidazole-2-amine (2e).
Compound 2e was formed from 3-iodoanisole in 56% yield. 1H NMR
d
7.54e7.50 (m, 1H), 7.22 (d, J ¼ 7.4 Hz, 1H), 7.10e6.98 (m, 4H),
6.92e6.84 (m, 2H), 6.28 (s, 2H), 3.83 (s, 3H). 13C NMR
d
160.4 (C),
154.1 (C), 143.0 (C), 136.1 (C), 134.8 (C), 130.8 (CH), 121.2 (CH), 118.7
(CH), 118.5 (CH), 115.1 (CH), 114.2 (CH), 112.0 (CH), 107.8 (CH), 55.4
(CH3). HR-ESMS calcd. for C14H14N3Oþ [M þ H] 240.1131, found
240.1134.
0.063e0.200 mm, 70e230 mesh).
8

S.M. Devine, M.P. Challis, J.K. Kigotho et al.
European Journal of Medicinal Chemistry 221 (2021) 113518
4.2.1.6. 1-(4-Methoxyphenyl)-1H-benzo[d]imidazole-2-amine
[27,28]. Compound 2f was formed from 4-iodoanisole in 43% yield.
(2f)
J ¼ 13.0 Hz, C), 121.5 (CH), 118.9 (CH), 117.4 (d, J ¼ 19.5 Hz, CH), 115.1
(CH), 107.6 (CH). HR-ESMS calcd. for C13H11FNþ3 [M þ H] 228.0932,
found 228.0935. 8-(2-Fluorophenoxy)quinoline was formed in 1%
1H NMR
d
7.37 (d, J ¼ 8.9 Hz, 2H), 7.20 (d, J ¼ 7.6 Hz, 1H), 7.14 (d,
J ¼ 8.9 Hz, 2H), 6.98 (t, J ¼ 7.1 Hz, 1H), 6.84 (t, J ¼ 7.5 Hz, 1H), 6.77 (d,
yield. 1H NMR
d
8.89 (dd, J ¼ 4.1, 1.7 Hz, 1H), 8.44 (dd, J ¼ 8.4, 1.7 Hz,
J ¼ 7.7 Hz, 1H), 6.15 (s, 2H), 3.84 (s, 3H). 13C NMR
d
158.9 (C), 154.6
1H), 7.81 (dd, J ¼ 8.3, 1.2 Hz, 1H), 7.61 (dd, J ¼ 8.4, 4.2 Hz, 1H),
(C), 142.9 (C), 135.4 (C), 128.3 (2 ꢃ CH), 127.5 (C), 121.0 (CH), 118.6
(CH), 115.2 (2 ꢃ CH), 115.0 (CH), 107.5 (CH), 55.5 (CH3). HR-ESMS
calcd. for C14H14N3Oþ [M þ H] 240.1131, found 240.1135.
7.59e7.55 (m, 1H), 7.44e7.36 (m, 1H), 7.26e7.22 (m, 2H), 7.20e7.09
(m, 2H), 6.92 (td, J ¼ 8.3, 1.9 Hz, 1H). 19F NMR
(min) ¼ 3.33. MS m/z 240.0 (M þ H).
d
ꢂ132.62. (LCMS Rf
4.2.1.7. 1-(2-Aminophenyl)-1H-benzo[d]imidazole-2-amine
(2g).
4.2.1.13. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-3-methylphenol
(3a). Compound 3a was formed in 13% yield. 1H NMR
9.74 (s, 1H),
Compound 2g was formed from 2-iodoaniline in 8% yield. 1H NMR
d
d
7.25e7.16 (m, 2H), 7.03 (dd, J ¼ 7.8, 1.4 Hz, 1H), 6.97 (t, J ¼ 7.2 Hz,
7.23 (t, J ¼ 7.9 Hz, 1H), 7.18 (d, J ¼ 7.8 Hz, 1H), 6.94 (td, J ¼ 7.7, 1.0 Hz,
1H), 6.93 (dd, J ¼ 8.2, 1.3 Hz, 1H), 6.83 (t, J ¼ 7.5 Hz, 1H), 6.70 (td,
J ¼ 7.7, 1.4 Hz, 1H), 6.63 (dd, J ¼ 7.7, 0.9 Hz, 1H), 5.99 (s, 2H), 4.89 (s,
1H), 6.89 (d, J ¼ 8.2 Hz,1H), 6.86 (d, J ¼ 7.5 Hz,1H), 6.79 (t, J ¼ 7.3 Hz,
1H), 6.48 (d, J ¼ 7.6 Hz,1H), 5.93 (s, 2H),1.89 (s, 3H). 13C NMR
d 154.6
2H). 13C NMR
d 154.4 (C), 145.1 (C), 143.4 (C), 134.7 (C), 129.7 (CH),
(C), 152.9 (C), 143.2 (C), 138.1 (C), 134.7 (C), 129.8 (CH), 121.0 (CH),
120.4 (CH), 120.3 (C), 118.2 (CH), 114.7 (CH), 114.3 (CH), 107.4 (CH),
17.2 (CH3). HR-ESMS calcd. for C14H14N3Oþ [M þ H] 240.1131, found
240.1137.
128.7 (CH), 120.8 (CH), 118.9 (C), 118.4 (CH), 116.8 (CH), 116.3 (CH),
115.0 (CH), 107.9 (CH). HR-ESMS calcd. for C13H13Nþ4 [M þ H]
225.1135, found 225.1139.
4.2.1.8. 1-(4-Aminophenyl)-1H-benzo[d]imidazole-2-amine
Compound 2h was formed from 4-iodoaniline in 80% yield. 1H NMR
7.17 (d, J ¼ 7.7 Hz, 1H), 7.04 (d, J ¼ 8.6 Hz, 2H), 6.95 (t, J ¼ 6.9 Hz,
1H), 6.82 (t, J ¼ 7.2 Hz, 1H), 6.73 (ddd, J ¼ 8.7, 7.2, 1.5 Hz, 3H), 6.00 (s,
2H), 5.43 (s, 2H). 13C NMR
154.7 (C), 148.9 (C), 142.8 (C), 135.7 (C),
127.6 (2 ꢃ CH), 122.4 (C), 120.6 (CH), 118.4 (CH), 114.8 (CH), 114.5
(2 ꢃ CH), 107.5 (CH). HR-ESMS calcd. for C13H13Nþ4 [M þ H]
225.1135, found 225.1137.
(2h).
4.2.1.14. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-4-methylphenol
(3b). Compound 3b was formed in 27% yield. 1H NMR
d 9.76 (s, 1H),
d
7.17 (d, J ¼ 7.6 Hz,1H), 7.14 (dd, J ¼ 8.3, 1.7 Hz,1H), 7.05 (d, J ¼ 1.7 Hz,
1H), 7.01e6.92 (m, 2H), 6.81 (td, J ¼ 7.6, 1.0 Hz, 1H), 6.64 (d,
J ¼ 7.3 Hz, 1H), 5.93 (s, 2H), 2.26 (s, 3H). 13C NMR
d 154.6 (C), 151.1
d
(C), 143.0 (C), 135.3 (C), 130.4 (CH), 129.3 (CH), 128.6 (C), 121.2 (C),
120.6 (CH), 118.3 (CH), 117.0 (CH), 114.8 (CH), 108.0 (CH), 19.9 (CH3).
HR-ESMS calcd. for C14H14N3Oþ [M þ H] 240.1131, found 240.1136.
4.2.1.9. 1-(p-Tolyl)-1H-benzo[d]imidazole-2-amine
Compound 2i was formed from 4-iodotoluene in 56% yield. 1H NMR
7.41 (d, J ¼ 8.1 Hz, 2H), 7.34 (d, J ¼ 8.3 Hz, 2H), 7.20 (d, J ¼ 7.7 Hz,
1H), 6.99 (t, J ¼ 6.7 Hz, 1H), 6.88e6.79 (m, 2H), 6.19 (s, 2H), 2.41 (s,
3H). 13C NMR
154.3 (C), 142.9 (C), 137.6 (C), 135.1 (C), 132.4 (C),
130.6 (2 ꢃ CH), 126.5 (2 ꢃ CH), 121.1 (CH), 118.6 (CH), 115.1 (CH),
107.6 (CH), 20.8 (CH3). HR-ESMS calcd. for C14H14Nþ3 [M þ H]
224.1182, found 224.1185.
(2i)
[29].
4.2.1.15. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-5-methylphenol
(3c). Compound 3c was formed in 58% yield. 1H NMR
d 9.90 (s, 1H),
d
7.17 (d, J ¼ 7.7 Hz, 1H), 7.11 (d, J ¼ 7.9 Hz, 1H), 6.94 (t, J ¼ 7.3 Hz, 1H),
6.90 (d, J ¼ 0.7 Hz, 1H), 6.84e6.76 (m, 2H), 6.62 (d, J ¼ 7.6 Hz, 1H),
d
5.93 (s, 2H), 2.32 (s, 3H). 13C NMR
d 154.8 (C), 153.3 (C), 143.1 (C),
139.6 (C), 135.5 (C), 128.9 (CH), 120.5 (2 ꢃ CH), 119.0 (C), 118.2 (CH),
117.6 (CH), 114.8 (CH), 107.9 (CH), 21.0 (CH3). HR-ESMS calcd. for
C
14H14N3Oþ [M þ H] 240.1131, found 240.1133.
4.2.1.10. 4-(2-Amino-1H-benzo[d]imidazole-1-yl)benzonitrile
Compound 2j was formed from 4-iodobenzonitrile in 19% yield. 1H
NMR
8.09 (d, J ¼ 8.6 Hz, 2H), 7.73 (d, J ¼ 8.6 Hz, 2H), 7.24 (d,
J ¼ 7.7 Hz, 1H), 7.04 (t, J ¼ 6.9 Hz, 1H), 6.97e6.86 (m, 2H), 6.45 (s,
2H). 13C NMR
(2 ꢃ CH), 121.8 (CH), 119.0 (CH), 118.5 (C), 115.4 (CH), 110.3 (C), 107.7
(CH). HR-ESMS calcd. for C14H11Nþ4 [M þ H] 235.0978, found
235.0981.
(2j).
4.2.1.16. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-6-methylphenol
(3d). Compound 3d was formed in 32% yield. 1H NMR
9.04 (s, 1H),
d
d
7.25 (ddd, J ¼ 7.4, 1.6, 0.7 Hz, 1H), 7.19 (d, J ¼ 7.6 Hz, 1H), 7.03 (dd,
J ¼ 7.8,1.3 Hz,1H), 6.96 (td, J ¼ 7.6,1.2 Hz,1H), 6.90 (t, J ¼ 7.6 Hz,1H),
6.82 (td, J ¼ 7.6, 1.0 Hz, 1H), 6.62 (d, J ¼ 7.3 Hz, 1H), 6.01 (s, 2H), 2.28
d
153.8 (C), 143.2 (C), 139.4 (C), 134.3 (2 ꢃ CH), 127.4
(s, 3H). 13C NMR
d 154.8 (C), 151.6 (C), 143.2 (C), 135.5 (C), 131.2 (CH),
127.0 (C), 126.5 (CH), 122.1 (C), 120.6 (CH), 119.8 (CH), 118.3 (CH),
114.8 (CH), 107.9 (CH), 16.6 (CH3). HR-ESMS calcd. for C14H14N3Oþ
[M þ H] 240.1131, found 240.1134.
4.2.1.11. (2-(2-Amino-1H-benzo[d]imidazole-1-yl)phenyl)methanol
(2k). Compound 2k was formed from 2-iodobenzyl alcohol in 22%
yield. 1H NMR
d
7.74 (dd, J ¼ 7.6, 0.8 Hz, 1H), 7.58 (td, J ¼ 7.6, 1.2 Hz,
4.2.1.17. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-3-methoxyphenol
(3e). Compound 3e was formed in 27% yield. 1H NMR
d 9.89 (s, 1H),
1H), 7.47 (td, J ¼ 7.6, 1.5 Hz, 1H), 7.29 (dd, J ¼ 7.8, 1.1 Hz, 1H), 7.21 (d,
J ¼ 7.7 Hz, 1H), 6.99 (td, J ¼ 7.6, 1.1 Hz, 1H), 6.82 (td, J ¼ 7.7, 1.0 Hz,
1H), 6.53 (dd, J ¼ 7.7, 0.5 Hz, 1H), 6.11 (s, 2H), 5.23 (t, J ¼ 5.1 Hz, 1H),
4.28 (dd, J ¼ 14.3, 4.9 Hz, 1H), 4.10 (dd, J ¼ 14.2, 4.4 Hz, 1H). 13C NMR
7.28 (t, J ¼ 8.3 Hz, 1H), 7.14 (d, J ¼ 7.8 Hz, 1H), 6.92 (td, J ¼ 7.6, 0.8 Hz,
1H), 6.76 (t, J ¼ 7.5 Hz, 1H), 6.70e6.63 (m, 2H), 6.49 (d, J ¼ 7.5 Hz,
1H), 5.82 (s, 2H), 3.66 (s, 3H). 13C NMR
d 157.0 (C), 155.2 (C), 143.3
(C), 135.2 (C), 134.7 (C), 130.1 (CH), 120.2 (CH), 118.1 (CH), 114.6 (CH),
109.8 (C), 109.0 (CH), 107.7 (CH), 102.9 (CH), 55.7 (CH3). HR-ESMS
calcd. for C14H14N3Oþ2 [M þ H] 256.1081, found 256.1085.
d
154.6 (C), 143.0 (C), 140.7 (C), 135.2 (C), 131.5 (C), 129.3 (CH), 128.6
(CH), 128.2 (CH), 128.1 (CH), 121.0 (CH), 118.6 (CH), 115.0 (CH), 107.5
(CH), 58.5 (CH2). HR-ESMS calcd. for C14H14N3Oþ [M þ H] 240.1131,
found 240.1134.
4.2.1.18. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-4-methoxyphenol
4.2.1.12. 1-(2-Fluorophenyl)-1H-benzo[d]imidazole-2-amine
Compound 2l was formed from 2-fluoroiodobenzene in 15% yield.
1H NMR
7.64e7.48 (m, 3H), 7.42 (td, J ¼ 7.6, 1.5 Hz, 1H), 7.22 (d,
(2l).
(3f). Compound 3f was formed in 55% yield. 1H NMR
d 9.52 (s, 1H),
7.18 (d, J ¼ 7.6 Hz, 1H), 7.02 (d, J ¼ 8.9 Hz, 1H), 6.98e6.92 (m, 2H),
d
6.85e6.80 (m, 2H), 6.68 (d, J ¼ 7.6 Hz, 1H), 6.00 (s, 2H), 3.71 (s, 3H).
J ¼ 7.7 Hz, 1H), 7.01 (td, J ¼ 7.6, 1.2 Hz, 1H), 6.86 (td, J ¼ 7.6, 1.1 Hz,
13C NMR
d 154.5 (C), 152.3 (C), 147.2 (C), 143.0 (C), 135.2 (C), 121.7
1H), 6.68 (d, J ¼ 7.8 Hz, 1H), 6.36 (s, 2H). 19F NMR
d
ꢂ120.67. 13C
(C), 120.6 (CH), 118.3 (CH), 117.7 (CH), 116.0 (CH), 114.8 (CH), 113.8
(CH), 108.1 (CH), 55.5 (CH3). HR-ESMS calcd. for C14H14N3Oþ2 [M þ
H] 256.1081, found 256.1086.
NMR
d
157.7 (d, J ¼ 250.0 Hz, C), 154.5 (C), 143.0 (C), 135.1 (C), 131.2
(d, J ¼ 8.0 Hz, CH), 130.3 (CH), 125.8 (d, J ¼ 3.8 Hz, CH), 122.3 (d,
9

S.M. Devine, M.P. Challis, J.K. Kigotho et al.
European Journal of Medicinal Chemistry 221 (2021) 113518
4.2.1.19. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-5-methoxyphenol
J ¼ 8.2 Hz, 1H), 7.18e7.02 (m, 4H), 6.95 (t, J ¼ 7.6 Hz, 1H), 6.64 (d,
(3g). Compound 3g was formed in 25% yield. 1H NMR
d
10.01 (s,
J ¼ 7.8 Hz, 1H), 6.12 (s, 2H). 13C NMR
d 156.4 (C), 152.9 (C), 143.0 (C),
1H), 7.16 (d, J ¼ 7.6 Hz, 1H), 7.15 (d, J ¼ 8.6 Hz, 1H), 6.94 (td, J ¼ 7.6,
1.2 Hz, 1H), 6.80 (td, J ¼ 7.6, 1.0 Hz, 1H), 6.63 (d, J ¼ 2.7 Hz, 1H), 6.61
(d, J ¼ 7.5 Hz, 1H), 6.56 (dd, J ¼ 8.7, 2.8 Hz, 1H), 5.91 (s, 2H), 3.78 (s,
133.4 (C),133.0 (C),131.7 (CH),122.0 (CH),120.2 (CH),117.8 (C),116.0
(CH), 115.9 (CH), 113.7 (CH), 108.3 (CH). HR-ESMS calcd. for
C
13H11ClN3Oþ [M þ H] 260.0585, found 260.0588.
3H). 13C NMR
d 160.4 (C), 155.0 (C), 154.7 (C), 142.9 (C), 135.7 (C),
130.0 (CH), 120.5 (CH), 118.23 (CH), 114.67 (CH), 114.5 (C), 107.78
(CH), 105.35 (CH), 102.48 (CH), 55.24 (CH3). HR-ESMS calcd. for
C
4.2.1.26. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-4-chlorophenol
(3n). Compound 3n was formed from 2-bromo-4-chlorophenol in
14H14N3Oþ2 [M þ H] 256.1081, found 256.1086.
46% yield. 1H NMR
d
10.39 (s, 1H), 7.39 (dd, J ¼ 8.7, 2.7 Hz, 1H), 7.35
(d, J ¼ 2.6 Hz,1H), 7.18 (d, J ¼ 7.7 Hz, 1H), 7.10 (d, J ¼ 8.8 Hz, 1H), 6.97
4.2.1.20. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-6-methoxyphenol
(3h). Compound 3h was formed in 45% yield. 1H NMR
9.23 (s, 1H),
(td, J ¼ 7.6, 1.2 Hz, 1H), 6.83 (td, J ¼ 7.6, 1.1 Hz, 1H), 6.65 (dd, J ¼ 7.9,
d
1.2 Hz, 1H), 6.17 (s, 2H). 13C NMR
d 154.6 (C), 153.0 (C), 143.0 (C),
7.18 (d, J ¼ 6.9 Hz, 1H), 7.12 (dd, J ¼ 8.2, 1.2 Hz, 1H), 6.98e6.91 (m,
135.1 (C), 129.9 (CH), 129.1 (CH), 122.7 (C), 122.4 (C), 120.8 (CH),
118.5 (CH), 118.4 (CH), 114.8 (CH), 107.9 (CH). HR-ESMS calcd. for
C
2H), 6.88e6.78 (m, 2H), 6.63 (d, J ¼ 6.6 Hz, 1H), 5.94 (s, 2H), 3.88 (s,
3H). 13C NMR
d
154.7 (C), 149.0 (C), 143.1 (C), 143.0 (C), 135.3 (C),
13H11ClN3Oþ [M þ H] 260.0585, found 260.0582.
121.9 (C), 120.7 (CH), 120.6 (CH), 119.2 (CH), 118.4 (CH), 114.8 (CH),
112.2 (CH), 108.0 (CH), 56.1 (CH3). HR-ESMS calcd. for C14H14N3O2þ
[M þ H] 256.1081, found 256.1085.
4.2.1.27. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-5-chlorophenol
(3o). Compound 3o was formed from 2-bromo-5-chlorophenol in
20% yield. 1H NMR
d
10.53 (s, 1H), 7.29 (d, J ¼ 8.4 Hz, 1H), 7.17 (d,
4.2.1.21. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-3-fluorophenol
J ¼ 7.5 Hz, 1H), 7.10 (d, J ¼ 2.3 Hz, 1H), 7.02 (dd, J ¼ 8.4, 2.3 Hz, 1H),
(3i). Compound 3i was formed in 12% yield. 1H NMR
d 10.46 (s, 1H),
6.96 (td, J ¼ 7.6, 1.2 Hz, 1H), 6.81 (td, J ¼ 7.6, 1.1 Hz, 1H), 6.63 (d,
7.37 (td, J ¼ 8.4, 6.7 Hz, 1H), 7.18 (d, J ¼ 7.7 Hz, 1H), 6.97 (td, J ¼ 7.6,
1.1 Hz, 1H), 6.91 (dd, J ¼ 8.4, 1.1 Hz, 1H), 6.87 (ddd, J ¼ 9.5, 8.5,1.1 Hz,
1H), 6.81 (td, J ¼ 7.6, 1.0 Hz, 1H), 6.58 (d, J ¼ 7.7 Hz, 1H), 6.17 (s, 2H).
J ¼ 7.3 Hz, 1H), 6.08 (s, 2H). 13C NMR
d 154.9 (C), 154.6 (C), 143.1 (C),
135.2 (C), 133.7 (C), 130.9 (CH), 120.9 (C), 120.7 (CH), 119.6 (CH),
118.4 (CH), 117.0 (CH), 114.7 (CH), 107.8 (CH). HR-ESMS calcd. for
19F NMR
d
ꢂ120.53. 13C NMR
d
159.3 (d, J ¼ 247.7 Hz, C), 156.0 (d,
C
13H11ClN3Oþ [M þ H] 260.0585, found 260.0595.
J ¼ 3.9 Hz, C),154.8 (C), 143.4 (C),135.0 (C), 130.6 (d, J ¼ 10.6 Hz, CH),
120.8 (CH), 118.3 (CH), 114.8 (CH), 112.6 (d, J ¼ 2.8 Hz, CH), 109.9 (d,
J ¼ 15.2 Hz, C), 107.5 (CH), 106.4 (d, J ¼ 19.7 Hz, CH). HR-ESMS calcd.
for C13H11FN3Oþ [M þ H] 244.0881, found 244.0887.
4.2.1.28. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-6-chlorophenol
(3p). Compound 3p was formed from 2-bromo-6-chlorophenol in
45% yield. 1H NMR
d
9.89 (s, 1H), 7.53 (dd, J ¼ 8.1, 1.6 Hz, 1H),
7.24e7.17 (m, 2H), 7.01e6.95 (m, 2H), 6.82 (td, J ¼ 7.6, 1.1 Hz, 1H),
4.2.1.22. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-4-fluorophenol
6.62 (d, J ¼ 7.3 Hz, 1H), 6.18 (s, 2H). 13C NMR
d 154.6 (C), 150.3 (C),
(3j). Compound 3j was formed in 27% yield. 1H NMR
d
9.98 (s, 1H),
143.0 (C), 135.2 (C), 130.4 (CH), 128.3 (CH), 123.9 (C), 122.3 (C), 120.9
(CH), 120.2 (CH), 118.5 (CH), 114.7 (CH), 107.8 (CH). HR-ESMS calcd.
for C13H11ClN3Oþ [M þ H] 260.0585, found 260.0593.
7.25e7.15 (m, 3H), 7.08 (dd, J ¼ 8.8, 5.2 Hz, 1H), 6.96 (t, J ¼ 7.2 Hz,
1H), 6.82 (t, J ¼ 7.5 Hz, 1H), 6.67 (d, J ¼ 7.5 Hz, 1H), 6.10 (s, 2H). 19
F
NMR
d
ꢂ124.16. 13C NMR
155.0 (d, J ¼ 235.8 Hz, C), 154.5 (C), 150.3
d
(d, J ¼ 2.3 Hz, C), 143.1 (C), 135.1 (C), 121.9 (d, J ¼ 10.5 Hz, C), 120.8
(CH), 118.4 (CH), 117.7 (d, J ¼ 8.9 Hz, CH), 116.6 (d, J ¼ 22.4 Hz, CH),
116.0 (d, J ¼ 23.7 Hz, CH), 114.8 (CH), 108.0 (CH). HR-ESMS calcd. for
4.2.1.29. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-4,6-
dimethylphenol (3q). Compound 3q was formed from 2-bromo-4,6-
dimethylphenol in 41% yield. 1H NMR
d 8.76 (s, 1H), 7.18 (d,
C
13H11FN3Oþ [M þ H] 244.0881, found 244.0886.
J ¼ 7.7 Hz, 1H), 7.06 (d, J ¼ 2.2 Hz, 1H), 6.95 (td, J ¼ 7.6, 1.2 Hz, 1H),
6.85e6.78 (m, 2H), 6.62 (dd, J ¼ 7.8, 1.1 Hz, 1H), 5.95 (s, 2H), 2.23 (s,
4.2.1.23. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-5-fluorophenol
(3k). Compound 3k was formed in 34% yield. 1H NMR
10.51 (s,
3H), 2.22 (s, 3H). 13C NMR
d 154.8 (C), 149.2 (C), 143.3 (C), 135.5 (C),
d
131.9 (CH), 128.6 (C), 126.7 (C), 126.5 (CH), 121.9 (C), 120.6 (CH),
118.2 (CH), 114.8 (CH), 108.0 (CH), 20.0 (CH3), 16.6 (CH3). HR-ESMS
calcd. for C15H15N3Oþ [M þ H] 254.1288, found 254.1295.
1H), 7.29 (dd, J ¼ 8.7, 6.4 Hz, 1H), 7.17 (d, J ¼ 7.5 Hz, 1H), 6.95 (td,
J ¼ 7.6, 1.2 Hz, 1H), 6.85 (dd, J ¼ 10.5, 2.8 Hz, 1H), 6.83e6.76 (m, 2H),
6.61 (d, J ¼ 7.3 Hz, 1H), 6.03 (s, 2H). 19F NMR
d
ꢂ111.23 (s). 13C NMR
d
163.7 (C), 161.3 (C), 155.5 (d, J ¼ 12.2 Hz, C), 154.8 (C), 143.0 (C),
4.2.1.30. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-4,5-
135.5 (C), 131.0 (d, J ¼ 11.1 Hz, CH), 120.7 (CH), 118.3 (CH), 118.2 (d,
J ¼ 2.9 Hz, C), 114.7 (CH), 107.7 (CH), 106.4 (d, J ¼ 22.7 Hz, CH), 104.1
(d, J ¼ 24.8 Hz, CH). HR-ESMS calcd. for C13H11FN3Oþ [M þ H]
244.0881, found 244.0886.
dimethylphenol (3r). Compound 3r was formed from 2-bromo-4,5-
dimethylphenol in 23% yield. 1H NMR
d
9.66 (s, 1H), 7.16 (dd, J ¼ 7.7,
0.9 Hz,1H), 6.99 (s,1H), 6.94 (td, J ¼ 7.6,1.2 Hz,1H), 6.87 (s,1H), 6.80
(td, J ¼ 7.6, 1.1 Hz, 1H), 6.63 (dd, J ¼ 7.8, 1.1 Hz, 1H), 5.90 (s, 2H), 2.22
(s, 3H), 2.16 (s, 3H). 13C NMR
d 154.7 (C), 151.2 (C), 143.1 (C), 138.0
4.2.1.24. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-6-fluorophenol
(C), 135.5 (C), 129.5 (CH), 127.3 (C), 120.5 (CH), 118.8 (C), 118.2
(2 ꢃ CH), 114.7 (CH), 108.0 (CH), 19.5 (CH3), 18.4 (CH3). HR-ESMS
calcd. for C15H15N3Oþ [M þ H] 254.1288, found 254.1295.
(3l). Compound 3l was formed in 26% yield. 1H NMR
d 10.16 (s, 1H),
7.34 (ddd, J ¼ 10.7, 8.3, 1.5 Hz, 1H), 7.18 (d, J ¼ 7.7 Hz, 1H), 7.10 (dt,
J ¼ 7.9, 1.3 Hz, 1H), 7.01e6.93 (m, 2H), 6.82 (td, J ¼ 7.7, 1.0 Hz, 1H),
6.64 (d, J ¼ 7.6 Hz, 1H), 6.11 (s, 2H). 19F NMR
d
ꢂ132.93 (s). 13C NMR
4.2.1.31. 1-(Benzofuran-7-yl)-1H-benzo[d]imidazole-2-amine (4a).
Compound 4a was formed from 7-bromobenzofuran in 14% yield.
d
154.6 (C), 152.5 (d, J ¼ 240.2 Hz, C), 143.1 (C), 142.2 (d, J ¼ 15.0 Hz,
C), 135.2 (C), 124.8 (d, J ¼ 2.7 Hz, CH), 124.3 (d, J ¼ 4.8 Hz, C) 120.9
(s), 119.1 (d, J ¼ 8.2 Hz, CH), 118.4 (CH), 116.4 (d, J ¼ 18.4 Hz, CH),
114.8 (CH), 107.9 (CH). HR-ESMS calcd. for C13H11FN3Oþ [M þ H]
244.0881, found 244.0887.
1H NMR
d
8.03 (d, J ¼ 2.2 Hz, 1H), 7.84 (dd, J ¼ 7.0, 1.9 Hz, 1H),
7.50e7.41 (m, 2H), 7.25 (d, J ¼ 7.8 Hz, 1H), 7.12 (d, J ¼ 2.2 Hz, 1H),
7.02 (td, J ¼ 7.6, 1.2 Hz, 1H), 6.84 (td, J ¼ 7.6, 1.0 Hz, 1H), 6.61 (d,
J ¼ 7.6 Hz, 1H), 6.36 (s, 2H). 13C NMR
d 154.4 (C), 149.3 (C), 146.8
(CH), 142.8 (C), 134.9 (C), 129.7 (C), 123.9 (CH), 123.5 (CH), 122.1
(CH), 121.3 (CH), 119.0 (C), 118.8 (CH), 115.0 (CH), 107.9 (CH), 107.3
(CH). HR-ESMS calcd. for C15H12N3Oþ [M þ H] 250.0975, found
250.0979.
4.2.1.25. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)-3-chlorophenol
(3m). Compound 3m was formed from 2-bromo-3-chlorophenol in
3% yield. 1H NMR
d
10.64 (s, 1H), 7.42 (t, J ¼ 8.2 Hz, 1H), 7.28 (d,
10

S.M. Devine, M.P. Challis, J.K. Kigotho et al.
European Journal of Medicinal Chemistry 221 (2021) 113518
4.2.1.32. 1-(1H-indol-7-yl)-1H-benzo[d]imidazole-2-amine
(4b).
1.6 Hz, 1H), 7.18 (d, J ¼ 7.7 Hz, 1H), 6.97 (t, J ¼ 7.5 Hz, 1H), 6.84 (t,
J ¼ 7.5 Hz,1H), 6.76 (d, J ¼ 7.7 Hz,1H), 6.37 (t, J ¼ 6.8 Hz,1H), 6.17 (br
Compound 4b was formed from 7-bromoindole in 33% yield. 1H
NMR
d
11.06 (s, 1H), 7.72 (d, J ¼ 7.8 Hz, 1H), 7.30 (t, J ¼ 2.8 Hz, 1H),
s, 2H). 13C NMR
d 159.2 (C), 154.8 (C), 143.3 (C), 140.2 (CH), 136.2
7.25 (d, J ¼ 7.7 Hz, 1H), 7.18 (t, J ¼ 7.6 Hz, 1H), 7.10 (dd, J ¼ 7.4, 0.9 Hz,
(CH), 135.1 (C), 125.1 (C), 120.8 (CH), 118.4 (CH), 114.9 (CH), 108.0
(CH), 105.0 (CH). HR-ESMS calcd. for C12H11N4Oþ [M þ H] 227.0927,
found 227.0935.
1H), 7.00 (t, J ¼ 7.1 Hz, 1H), 6.80 (t, J ¼ 7.6 Hz, 1H), 6.59 (dd, J ¼ 3.0,
1.8 Hz, 1H), 6.52 (d, J ¼ 7.7 Hz, 1H), 6.07 (s, 2H). 13C NMR
d 154.6 (C),
143.4 (C), 135.4 (C), 131.9 (C), 130.2 (C), 126.5 (CH), 120.9 (CH), 120.8
(CH), 120.3 (CH), 119.5 (CH), 118.9 (C), 118.4 (CH), 115.0 (CH), 107.7
(CH), 102.0 (CH). HR-ESMS calcd. for C15H13Nþ4 [M þ H] 249.1135,
found 249.1143.
4.2.1.39. 2-(3-Amino-1H-indazol-1-yl)phenol (7). Compound 7 was
formed from 1H-indazol-3-amine and 2-iodophenol in 18% yield.
1H NMR
d
10.05 (s, 1H), 7.81 (d, J ¼ 8.0 Hz, 1H), 7.39e7.31 (m, 2H),
7.25 (d, J ¼ 8.5 Hz, 1H), 7.19e7.13 (m, 1H), 7.07e7.01 (m, 2H), 6.92
4.2.1.33. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)naphthalen-1-ol
(5a). Compound 5a was formed from 2-bromonaphthalen-1-ol in
(td, J ¼ 7.7, 1.4 Hz, 1H), 5.86 (s, 2H). 13C NMR
d 150.4 (C), 150.2 (C),
140.2 (C), 127.3 (C), 127.2 (CH), 126.9 (CH), 125.0 (CH), 120.7 (CH),
119.4 (CH), 118.6 (CH), 117.0 (CH), 115.7 (C), 110.8 (CH). HR-ESMS
calcd. for C13H12N3Oþ [M þ H] 226.0975, found 226.0979.
9% yield. 1H NMR
d
10.07 (s, 1H), 8.32 (d, J ¼ 7.8 Hz, 1H), 7.95 (d,
J ¼ 7.4 Hz, 1H), 7.64e7.51 (m, 4H), 7.25 (d, J ¼ 8.6 Hz, 1H), 7.22 (d,
J ¼ 7.8 Hz, 1H), 6.99 (t, J ¼ 7.6 Hz, 1H), 6.82 (t, J ¼ 7.5 Hz, 1H), 6.63 (d,
J ¼ 7.6 Hz, 1H), 6.15 (s, 2H). 13C NMR
d
154.6 (C), 149.7 (C), 142.0 (C),
4.2.1.40. 1-(2-Aminophenyl)-1H-benzo[d]imidazole-2-ol
Compound 8a was formed from 2-iodoaniline and 2-
hydroxybenzimidazole in 22% yield. 1H NMR
10.98 (s, 1H), 7.16
(8a).
135.2 (C), 134.3 (C), 127.7 (CH), 127.0 (CH), 126.3 (CH), 126.0 (C),
125.4 (CH), 122.8 (CH), 121.0 (CH), 120.0 (CH), 118.8 (CH), 115.5 (C),
114.4 (CH), 108.0 (CH). HR-ESMS calcd. for C17H14N3Oþ [M þ H]
276.1131, found 276.1139.
d
(ddd, J ¼ 8.2, 7.4, 1.5 Hz, 1H), 7.07e6.98 (m, 3H), 6.93 (td, J ¼ 7.4,
1.9 Hz, 1H), 6.87 (dd, J ¼ 8.1, 1.3 Hz, 1H), 6.65 (td, J ¼ 7.7, 1.4 Hz, 1H),
6.57 (d, J ¼ 7.7, 1H), 4.98 (s, 2H). 13C NMR
d 153.5 (C), 145.5 (C), 130.6
(C), 129.2 (CH), 129.0 (C), 128.9 (CH), 121.2 (CH), 120.5 (CH), 118.8
(C),116.2 (CH),116.1 (CH),108.9 (CH),108.3 (CH). HR-ESMS calcd. for
4.2.1.34. 7-(2-Amino-1H-benzo[d]imidazole-1-yl)quinolin-8-ol (5b).
Compound 5b was formed from 7-bromo-8-hydroxyquinoline in
13% yield. 1H NMR
d
10.48 (s, 1H), 8.95 (dd, J ¼ 3.9, 1.2 Hz, 1H), 8.48
C
13H12N3Oþ [M þ H] 226.0975, found 226.0979.
(dd, J ¼ 8.3, 1.1 Hz, 1H), 7.68 (dd, J ¼ 8.3, 4.2 Hz, 1H), 7.58 (d,
J ¼ 8.7 Hz,1H), 7.49 (d, J ¼ 8.6 Hz,1H), 7.22 (d, J ¼ 7.7 Hz,1H), 6.99 (t,
J ¼ 7.3 Hz, 1H), 6.83 (t, J ¼ 7.4 Hz, 1H), 6.67 (d, J ¼ 7.6 Hz, 1H), 6.16 (s,
4.2.1.41. 1-(2-Aminophenyl)-1H-benzo[d]imidazole-2-thiol
Compound 8b was formed from 2-iodoaniline and 2-
mercaptobenzimidazole in 34% yield. 1H NMR
12.27 (s, 1H), 7.48
(8b).
2H). 13C NMR
d
154.6 (C), 150.1 (C), 148.9 (CH), 142.3 (C), 139.4 (C),
d
136.5 (CH), 135.1 (C), 128.9 (C), 127.5122.8 (CH), 121.1 (CH), 118.9
(CH), 118.3 (CH), 117.6 (C), 114.8 (CH), 108.2 (CH). HR-ESMS calcd. for
C
(dd, J ¼ 6.2, 2.6 Hz, 1H), 7.39 (dd, J ¼ 7.7, 1.5 Hz, 1H), 7.36 (dd, J ¼ 6.2,
2.6 Hz,1H), 7.19 (ddd, J ¼ 8.3, 7.3, 1.6 Hz, 1H), 7.15e7.07 (m, 2H), 6.80
16H13N4Oþ [M þ H] 277.1084, found 277.1085.
(dd, J ¼ 8.2, 1.2 Hz, 1H), 6.61 (td, J ¼ 7.6, 1.3 Hz, 1H), 5.48 (s, 2H). 13
C
NMR d 150.4 (C),142.0 (C),136.6 (CH),133.7 (C),131.3 (CH),131.0 (C),
4.2.1.35. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)pyridin-3-ol (6a).
Compound 6a was formed from 3-hydroxy-2-iodopyridine in 69%
129.3 (CH), 121.6 (CH), 116.7 (CH), 115.3 (CH), 109.8 (CH), 109.7 (C),
109.4(CH). HR-ESMS calcd. for C13H12N3Sþ [M þ H] 242.0746, found
242.0750.
yield. 1H NMR
d
10.60 (br s, 1H), 8.10 (dd, J ¼ 4.6, 1.5 Hz, 1H), 7.52
(dd, J ¼ 8.2, 1.5 Hz, 1H), 7.40 (dd, J ¼ 8.2, 4.6 Hz, 1H), 7.20 (d,
J ¼ 7.6 Hz, 1H), 6.98 (t, J ¼ 7.1 Hz, 1H), 6.83 (t, J ¼ 7.5 Hz, 1H), 6.75 (d,
4.2.1.42. 2-(1H-Benzo[d]imidazole-1-yl)phenol
Compound 8c was formed from 2-iodophenol and benzimidazole
in 4% yield. 1H NMR
10.19 (s, 1H), 8.31 (s, 1H), 7.76e7.71 (m, 1H),
(8c)
[46].
J ¼ 7.6 Hz, 1H), 6.17 (br s, 2H). 13C NMR
d 154.1 (C), 149.1 (C), 142.7
(C), 139.5 (CH), 136.6 (C), 134.1 (C), 125.7 (CH), 125.2 (CH), 120.9
(CH), 118.5 (CH), 114.9 (CH), 109.1 (CH). HR-ESMS calcd. for
d
7.41 (dd, J ¼ 7.8, 1.6 Hz, 1H), 7.36 (ddd, J ¼ 8.2, 7.6, 1.6 Hz, 1H),
C
12H11N4Oþ [M þ H] 227.0927, found 227.0935.
7.28e7.23 (m, 3H), 7.13 (dd, J ¼ 8.2, 1.2 Hz, 1H), 7.01 (td, J ¼ 7.6,
1.3 Hz, 1H). 13C NMR
d 152.1 (C), 144.3 (CH), 143.0 (C), 134.2 (C),
4.2.1.36. 3-(2-Amino-1H-benzo[d]imidazole-1-yl)pyridin-4-ol (6b).
Compound 6b was formed from 3-iodopyridin-4-ol in 28% yield. 1H
129.7 (CH), 127.5 (CH), 122.9 (C), 122.8 (CH), 121.8 (CH), 119.6 (CH),
119.5 (CH), 117.0 (CH), 110.0 (CH). HR-ESMS calcd. for C13H11N2Oþ
[M þ H] 211.0866, found 211.0871.
NMR
d
11.72 (br s, 1H), 8.08 (d, J ¼ 1.5 Hz, 1H), 7.81 (d, J ¼ 7.1 Hz, 1H),
7.17 (d, J ¼ 7.5 Hz, 1H), 6.95 (td, J ¼ 7.6, 1.2 Hz, 1H), 6.83 (td, J ¼ 7.6,
1.1 Hz, 1H), 6.73 (d, J ¼ 7.3 Hz, 1H), 6.40 (d, J ¼ 7.2 Hz, 1H), 6.10 (s,
4.2.2. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)ethan-1-ol
(9a).
2H). 13C NMR
d
173.4 (C), 155.1 (C), 143.1 (C), 138.4 (CH), 137.9 (CH),
1-Fluoro-2-nitrobenzene (250 mg, 1.77 mmol, 1.0 eq.), 2-
aminoethanol hydrochloride (181 mg, 1.86 mmol, 1.05 eq.) and
K2CO3 (514 mg, 3.72 mmol, 2.1 eq.) were heated at reflux in MeCN
(3 mL) at 82 ꢀC for 6 h. The cooled reaction mixture was filtered,
washed with MeCN and the filtrate evaporated at reduced pressure.
The residue was taken up in EtOAc (30 mL), washed with water
(3 ꢃ 10 mL), then saturated NaCl (5 mL). The combined organic
fractions were dried over MgSO4, filtered and the filtrate evapo-
rated at reduced pressure to give 2-((2-nitrophenyl)amino)ethan-
135.3 (C), 123.6 (C), 120.7 (CH), 118.4 (CH), 118.2 (CH), 114.8 (CH),
108.0 (CH). HR-ESMS calcd. for C12H11N4Oþ [M þ H] 227.0927,
found 227.0935.
4.2.1.37. 4-(2-Amino-1H-benzo[d]imidazole-1-yl)pyridin-3-ol (6c).
Compound 6c was formed from 4-iodopyridin-3-ol in 27% yield. 1H
NMR
d
10.74 (br s, 1H), 8.42 (s, 1H), 8.16 (d, J ¼ 5.0 Hz, 1H), 7.34 (d,
J ¼ 5.0 Hz, 1H), 7.20 (d, J ¼ 7.7 Hz, 1H), 6.99 (t, J ¼ 7.4 Hz, 1H), 6.85 (t,
J ¼ 7.4 Hz, 1H), 6.74 (d, J ¼ 7.7 Hz, 1H), 6.29 (s, 2H). 13C NMR
d
154.2
1-ol as a red amorphous solid (286 mg, 89%). 1H NMR
d 8.23 (t,
(C), 150.3 (C), 143.3 (C), 140.8 (CH), 140.5 (CH), 134.4 (C), 128.7 (C),
123.0 (CH), 121.1 (CH), 118.6 (CH), 115.0 (CH), 108.2 (CH). HR-ESMS
calcd. for C12H11N4Oþ [M þ H] 227.0927, found 227.0933.
J ¼ 4.4 Hz, 1H), 8.07 (dd, J ¼ 8.7, 1.5 Hz, 1H), 7.53 (dddd, J ¼ 8.6, 6.9,
1.7, 0.7 Hz, 1H), 7.08 (dd, J ¼ 8.8, 1.2 Hz, 1H), 6.69 (ddd, J ¼ 8.6, 6.9,
1.2 Hz, 1H), 4.98 (t, J ¼ 5.2 Hz, 1H), 3.65 (q, J ¼ 5.4 Hz, 2H), 3.40 (q,
J ¼ 5.5 Hz, 2H).
4.2.1.38. 3-(2-Amino-1H-benzo[d]imidazole-1-yl)pyridin-2-ol (6d).
Compound 6d was formed from 3-iodopyridin-2-ol in 62% yield. 1H
2-((2-Nitrophenyl)amino)ethan-1-ol (286 mg, 1.57 mmol) was
dissolved in EtOH (30 mL) in a three-neck round bottom flask,
purged with N2 and 10% Pd/C (20 mg) added. The stirred mixture
NMR
d
12.30 (br s, 1H), 7.70 (dd, J ¼ 7.0, 1.6 Hz, 1H), 7.59 (dd, J ¼ 6.4,
11

S.M. Devine, M.P. Challis, J.K. Kigotho et al.
European Journal of Medicinal Chemistry 221 (2021) 113518
was purged and cycled three times from vacuum to H2. The mixture
was stirred at 25 ꢀC for 2 h, at which time water was added (1 mL).
The mixture was filtered through Celite™, washed with 20:1 EtOH/
water (20 mL) and evaporated under reduced pressure to give 2-
((2-aminophenyl)amino)ethan-1-ol as an orange amorphous solid
1.6 Hz, 1H), 4.67 (d, J ¼ 4.0 Hz, 1H), 4.47 (s, 2H), 4.14 (d, J ¼ 5.2 Hz,
1H), 3.89 (dq, J ¼ 6.2, 3.3 Hz, 1H), 3.39 (ddd, J ¼ 9.4, 4.7, 2.6 Hz, 1H),
2.18e2.03 (m, 1H), 1.80 (dddd, J ¼ 12.3, 8.9, 7.2, 5.5 Hz, 1H),
1.76e1.62 (m, 2H), 1.54e1.36 (m, 2H).
(1S,2S)-2-((2-Aminophenyl)amino)cyclopent-1-ol (303 mg,
1.58 mmol, 1.0 eq.), was dissolved in MeCN (12 mL) with Et3N
(239 mg, 100%). 1H NMR
d 6.57e6.53 (m, 1H), 6.52e6.47 (m, 1H),
6.45e6.39 (m, 2H), 4.67 (t, J ¼ 5.5 Hz, 1H), 4.42 (s, 2H), 4.33 (t,
J ¼ 5.7 Hz, 1H), 3.60 (q, J ¼ 5.8 Hz, 2H), 3.08 (q, J ¼ 5.9 Hz, 2H).
2-((2-Aminophenyl)amino)ethan-1-ol (239 mg, 1.57 mmol, 1.0
(220
thiocyanate (196
lowed by EDCI.HCl (363 mg, 1.89 mmol, 1.2 eq.) and additional Et3N
(330 L, 2.36 mmol, 1.5 eq.). The mixture was heated at reflux at
m
L, 1.58 mmol, 1.0 eq.). To this solution, ethoxycarbonyl iso-
mL, 1.73 mmol, 1.1 eq.) was added dropwise, fol-
eq.), was dissolved in MeCN (12 mL) with Et3N (218
1.0 eq.). To this solution, ethoxycarbonyl isothiocyanate (195
1.72 mmol, 1.1 eq.) was added dropwise, followed by EDCI.HCl
mL, 1.57 mmol,
m
mL,
82 ꢀC for 1 h, whereby a precipitate formed. The cooled reaction
mixture was filtered and washed with a small volume of MeCN to
give ethyl (1-((1S,2S)-2-hydroxycyclopentyl)-1H-benzo[d]imid-
azole-2-yl)carbamate as a white solid (211 mg, 46%). 1H NMR
(361 mg, 1.88 mmol, 1.2 eq.) and additional Et3N (328
mL,
2.36 mmol, 1.5 eq.). The mixture was heated at reflux at 82 ꢀC for
1 h, whereby a precipitate formed. The cooled reaction mixture was
filtered and washed with a small volume of MeCN to give ethyl (1-
(2-hydroxyethyl)-1H-benzo[d]imidazole-2-yl)carbamate as a white
d
12.09 (s, 1H), 7.47e7.43 (m, 1H), 7.40e7.36 (m, 1H), 7.18e7.12 (m,
2H), 4.99 (d, J ¼ 5.2 Hz, 1H), 4.79 (q, J ¼ 9.1 Hz, 1H), 4.71e4.63 (m,
1H), 4.05 (q, J ¼ 7.1 Hz, 2H), 2.19e2.01 (m, 2H), 2.00e1.71 (m, 3H),
1.59 (dq, J ¼ 12.4, 8.2 Hz, 1H), 1.22 (t, J ¼ 7.1 Hz, 3H).
solid (175 mg, 45%). 1H NMR
d 12.04 (s, 1H), 7.43e7.38 (m, 2H),
7.19e7.10 (m, 2H), 4.92 (s, 1H), 4.10e4.02 (m, 4H), 3.68 (q, J ¼ 7.1 Hz,
Ethyl
(1-((1S,2S)-2-hydroxycyclopentyl)-1H-benzo[d]imid-
2H), 1.22 (t, J ¼ 7.1 Hz, 3H).
azole-2-yl)carbamate (180 mg, 0.62 mmol) was dissolved in EtOH
(2 mL) and 1 M NaOH (2 mL) in a sealed tube and the mixture
heated at 100 ꢀC for 3 h. The resultant cooled solution was poured
onto ice/water (50 mL) and the pH adjusted to 8 with HCl. The
solution was extracted with EtOAc (3 ꢃ 10 mL). The combined
organic fractions were washed with saturated NaCl (5 mL), dried
over MgSO4, filtered and the filtrate evaporated at reduced pressure
to give a colourless semi-solid. The residue was purified by column
chromatography using DCM/MeOH/NH4OH (89:10:1) to give
(1S,2S)-2-(2-amino-1H-benzo[d]imidazole-1-yl)cyclopentan-1-ol
Ethyl (1-(2-hydroxyethyl)-1H-benzo[d]imidazole-2-yl)carba-
mate (170 mg, 0.68 mmol) was dissolved in EtOH (2 mL) and 1 M
NaOH (2 mL) in a sealed tube and the mixture heated at 100 ꢀC for
3 h. The resultant cooled solution was poured onto ice/water
(50 mL) and the pH adjusted to 8 with HCl. The solution was
extracted with EtOAc (3 ꢃ 10 mL). The combined organic fractions
were washed with saturated NaCl (5 mL), dried over MgSO4, filtered
and the filtrate evaporated at reduced pressure to give a yellow
semi-solid. The residue was purified by column chromatography
using DCM/MeOH/NH4OH (89:10:1) to give 2-(2-amino-1H-benzo
9b (104 mg, 77%). 1H NMR
d
7.16e7.11 (m, 2H), 6.92 (td, J ¼ 7.7,
[d]-imidazole-1-yl)ethan-1-ol 9a (48 mg, 40%). 1H NMR
(d
1.1 Hz, 1H), 6.83 (td, J ¼ 7.7, 1.2 Hz, 1H), 6.20 (s, 2H), 5.02 (d,
J ¼ 5.2 Hz, 1H), 4.55 (qd, J ¼ 7.4, 5.1 Hz, 1H), 4.40 (td, J ¼ 9.4, 7.6 Hz,
1H), 2.12e1.95 (m, 3H), 1.92e1.72 (m, 2H), 1.58 (dq, J ¼ 12.6, 8.0 Hz,
7.16e7.09 (m, 2H), 6.91 (td, J ¼ 7.5, 1.4 Hz, 1H), 6.86 (td, J ¼ 7.5,
1.3 Hz, 1H), 6.22 (s, 2H), 4.95 (t, J ¼ 5.2 Hz, 1H), 4.01 (t, J ¼ 5.6 Hz,
2H), 3.65 (q, J ¼ 5.5 Hz, 2H). 13C NMR
d
155.2 (C), 142.7 (C), 134.8 (C),
1H). 13C NMR
d 155.4 (C), 143.3 (C), 132.4 (C), 120.0 (CH), 117.7 (CH),
120.1 (CH), 117.9 (CH), 114.7 (CH), 107.7 (CH), 59.6 (CH2), 44.3 (CH2).
115.1 (CH), 109.1 (CH), 72.5 (CH), 62.0 (CH), 33.0 (CH2), 26.0 (CH2),
20.2 (CH2). HR-ESMS calcd. for C12H16N3Oþ [M þ H] 218.1288, found
218.1278.
HR-ESMS calcd. for C9H12N3Oþ [M þ H] 178.0975, found 178.0979.
4.2.3. 2-(2-Amino-1H-benzo[d]imidazole-1-yl)cyclopentan-1-ol
(9b)
4.2.4. (1S,2S)-2-(2-amino-1H-benzo[d]imidazole-1-yl)cyclohexan-
1-ol (9c)
1-Fluoro-2-nitrobenzene (250 mg, 1.77 mmol, 1.0 eq.), (1S,2S)-
trans-2-aminocyclopentanol hydrochloride (256 mg, 1.86 mmol,
1.05 eq.) and K2CO3 (514 mg, 3.72 mmol, 2.1 eq.) were heated at
reflux in MeCN (3 mL) for 6 h. The cooled reaction mixture was
filtered, washed with MeCN and the filtrate evaporated at reduced
pressure. The residue was taken up in EtOAc (30 mL), washed with
water (3 ꢃ 10 mL), then saturated NaCl (5 mL). The combined
organic fractions were dried over MgSO4, filtered and the filtrate
evaporated at reduced pressure to give (1S,2S)-2-((2-nitrophenyl)
1-Fluoro-2-nitrobenzene (250 mg, 1.77 mmol, 1.0 eq.), (1S,2S)-
trans-2-aminocyclohexanol hydrochloride (282 mg,1.86 mmol,1.05
eq.) and K2CO3 (514 mg, 3.72 mmol, 2.1 eq.) were heated at reflux in
MeCN (3 mL) for 6 h. The cooled reaction mixture was filtered,
washed with MeCN and the filtrate evaporated at reduced pressure.
The residue was taken up in EtOAc (30 mL), washed with water
(3 ꢃ 10 mL), then saturated NaCl (5 mL). The combined organic
fractions were dried over MgSO4, filtered and the filtrate evapo-
rated at reduced pressure to give (1S,2S)-2-((2-nitrophenyl)amino)
amino)cyclopent-1-ol as a red oil (368 mg, 93%). 1H NMR
d 8.06 (dd,
J ¼ 8.6,1.6 Hz,1H), 7.95 (d, J ¼ 6.5 Hz,1H), 7.55 (dddd, J ¼ 7.5, 6.9,1.7,
0.6 Hz,1H), 7.18 (dd, J ¼ 8.9,1.2 Hz,1H), 6.70 (ddd, J ¼ 8.4, 6.9,1.2 Hz,
1H), 5.09 (d, J ¼ 4.3 Hz, 1H), 3.96 (dq, J ¼ 6.2, 4.5 Hz, 1H), 3.73 (ddd,
J ¼ 11.2, 8.4, 5.3 Hz,1H), 2.24 (dtd, J ¼ 13.2, 7.9, 5.6 Hz,1H), 1.86 (ddt,
J ¼ 12.4, 8.5, 6.3 Hz,1H),1.80e1.63 (m, 2H),1.51 (dddd, J ¼ 25.8,12.8,
8.5, 5.8 Hz, 2H).
cyclohexan-1-ol as a red oil (386 mg, 92%). 1H NMR
d 8.13 (d,
J ¼ 6.0 Hz, 1H), 8.04 (dd, J ¼ 8.7, 1.6 Hz, 1H), 7.53e7.44 (m, 1H), 7.16
(dd, J ¼ 8.9, 1.2 Hz, 1H), 6.64 (ddd, J ¼ 8.6, 6.8, 1.2 Hz, 1H), 4.97 (d,
J ¼ 5.0 Hz, 1H), 3.47e3.38 (m, 2H), 2.06e1.99 (m, 1H), 1.92e1.86 (m,
1H), 1.70e1.56 (m, 2H), 1.41e1.20 (m, 4H).
(1S,2S)-2-((2-Nitrophenyl)amino)cyclohexan-1-ol (354 mg,
1.50 mmol) was dissolved in EtOH (30 mL) in a three-neck round
bottom flask, purged with N2 and 10% Pd/C (20 mg) added. The
stirred mixture was purged and cycled three times from vacuum to
H2. The mixture was stirred at 25 ꢀC for 2 h, at which time water
was added (1 mL). The mixture was filtered through Celite™,
washed with 20:1 EtOH/water (20 mL) and evaporated under
reduced pressure to give (1S,2S)-2-((2-aminophenyl)amino)cyclo-
(1S,2S)-2-((2-Nitrophenyl)amino)cyclopent-1-ol
(364
mg,
1.64 mmol) was dissolved in EtOH (30 mL) in a three-neck round
bottom flask, purged with N2 and 10% Pd/C (20 mg) added. The
stirred mixture was purged and cycled three times from vacuum to
H2. The mixture was stirred at 25 ꢀC for 2 h, at which time water
was added (1 mL). The mixture was filtered through Celite™,
washed with 20:1 EtOH/water (20 mL) and evaporated under
reduced pressure to give (1S,2S)-2-((2-aminophenyl)amino)cyclo-
pentan-1-ol as a light brown oil (272 mg, 88%). 1H NMR
d 6.59e6.52
pent-1-ol as a light brown oil (314 mg, 100%). 1H NMR
J ¼ 7.6, 1.6 Hz, 2H), 6.48 (td, J ¼ 7.5, 1.6 Hz, 1H), 6.39 (td, J ¼ 7.3,
d
6.52 (td,
(m, 1H), 6.52e6.43 (m, 2H), 6.47e6.36 (m, 1H), 4.63 (d, J ¼ 4.7 Hz,
1H), 4.44 (s, 2H), 4.02 (d, J ¼ 5.7 Hz, 1H), 3.38 (dt, J ¼ 8.8, 4.6 Hz,1H),
12

S.M. Devine, M.P. Challis, J.K. Kigotho et al.
European Journal of Medicinal Chemistry 221 (2021) 113518
2.92 (tt, J ¼ 9.4, 4.5 Hz, 1H), 2.08e2.00 (m, 1H), 1.94e1.86 (m, 1H),
1.68e1.57 (m, 2H), 1.36e1.20 (m, 3H), 1.10e0.96 (m, 1H).
(1S,2S)-2-((2-Aminophenyl)amino)cyclohexan-1-ol (270 mg,
1.31 mmol, 1.0 eq.) was dissolved in MeCN (15 mL) with Et3N
amine (9d) (200 mg, 61%) for a total yield of 80%. This was used
without further purification. 1H NMR
d
11.06 (s,1H), 7.85 (dd, J ¼ 5.1,
1.5 Hz, 1H), 7.33 (dd, J ¼ 7.6, 1.5 Hz, 1H), 6.82 (dd, J ¼ 7.6, 5.1 Hz, 1H),
6.49 (s, 2H). Using the general procedure for N-arylation from 2-
iodophenol formed 10 in 0.3% yield, with 3H-imidazo[4,5-b]pyr-
(182
thiocyanate (163
lowed by EDCI.HCl (301 mg, 1.57 mmol, 1.2 eq.) and additional Et3N
(274 L, 1.96 mmol, 1.5 eq.). The mixture was heated at reflux for
mL, 1.31 mmol, 1.0 eq.). To this solution, ethoxycarbonyl iso-
mL, 1.44 mmol, 1.1 eq.) was added dropwise, fol-
idin-2-amine recovered (80%). 1H NMR (CD3OD):
d
7.82 (dd, J ¼ 5.1,
1.1 Hz, 1H), 7.59 (dd, J ¼ 7.8, 1.4 Hz, 1H), 7.41 (ddd, J ¼ 8.3, 7.4, 1.7 Hz,
1H), 7.33 (dd, J ¼ 7.8, 1.6 Hz, 1H), 7.13e7.08 (m, 2H), 7.05 (td, J ¼ 7.6,
1.3 Hz, 1H). HR-ESMS calcd. for C12H11N4Oþ [M þ H] 227.0927,
found 227.0930.
m
1 h, whereby a precipitate formed. The cooled reaction mixture was
filtered and washed with a small volume of MeCN to give ethyl (1-
((1S,2S)-2-hydroxycyclohexyl)-1H-benzo[d]imidazole-2-yl)carba-
mate as a white solid (270 mg, 68%). 1H NMR
d
12.05 (s, 1H), 7.53 (s,
4.2.6. General procedure for synthesis of C2-amino substituted
1H), 7.47e7.40 (m, 1H), 7.17e7.07 (m, 2H), 4.75 (d, J ¼ 4.9 Hz, 1H),
4.46e4.15 (m, 2H), 4.05 (q, J ¼ 7.0 Hz, 2H), 2.26e2.11 (m, 1H),
2.04e1.96 (m, 1H), 1.79e1.66 (m, 3H), 1.49e1.26 (m, 3H), 1.23 (t,
J ¼ 7.1 Hz, 3H). Ethyl (1-((1S,2S)-2-hydroxycyclohexyl)-1H-benzo[d]
imidazole-2-yl)carbamate (260 mg, 0.86 mmol) was dissolved in
EtOH (2 mL) and 1 M NaOH (2 mL) in a sealed tube and the mixture
heated at 100 ꢀC for 3 h. The resultant cooled solution was poured
onto ice/water (50 mL) and the pH adjusted to 8 with HCl. The
solution was extracted with EtOAc (3 ꢃ 10 mL). The combined
organic fractions were washed with saturated NaCl (5 mL), dried
over MgSO4, filtered and the filtrate evaporated at reduced pressure
to give a colourless semi-solid. The residue was purified by column
chromatography using DCM/MeOH/NH4OH (89:10:1) to give
(1S,2S)-2-(2-amino-1H-benzo[d]imidazole-1-yl)cyclohexan-1-ol
analogues (11ae11i)
Titanium (IV) isopropoxide (329 mL, 1.11 mmol) and the appro-
priate aldehyde or ketone (0.58 mmol) was stirred in dry THF
(1 mL) for 1 h. These were added dropwise to 2a (100 mg,
0.44 mmol) in dry THF (1 mL) and stirred at 25 ꢀC for 5 h. To the
intermediate imine, NaBH(OAc)3 (188 mg, 0.89 mmol) was added
directly and the reaction left to stir at 25 ꢀC for 18 h. The reaction
was quenched with saturated NaHCO3 (20 mL) and extracted with
DCM (3 ꢃ 20 mL). The organic layer was then washed with water
(10 mL) followed by saturated sodium chloride (10 mL) and evap-
orated under reduced pressure. The residue was purified by column
chromatography using DCM/MeOH/NH4OH (89:10:1) to give com-
pounds 11ae11i.
9c (168 mg, 85%). 1H NMR
d
7.30 (d, J ¼ 7.8 Hz, 1H), 7.11 (dd, J ¼ 7.8,
4.2.6.1. 2-(2-(Ethylamino)-1H-benzo[d]imidazole-1-yl)phenol (11a).
1.2 Hz, 1H), 6.89 (td, J ¼ 7.5, 1.1 Hz, 1H), 6.80 (td, J ¼ 7.6, 1.3 Hz, 1H),
6.10 (s, 2H), 4.67 (d, J ¼ 5.8 Hz, 1H), 4.07 (tt, J ¼ 9.5, 5.0 Hz, 1H), 3.90
(ddd, J ¼ 12.3, 10.0, 4.0 Hz, 1H), 2.13 (qd, J ¼ 12.7, 3.8 Hz, 1H),
2.04e1.97 (m, 1H), 1.78e1.65 (m, 3H), 1.49e1.30 (m, 3H). 13C NMR
Compound 11a was formed in 37% yield. 1H NMR
d 9.98 (s, 1H), 7.35
(ddd, J ¼ 8.2, 7.5, 1.7 Hz, 1H), 7.28e7.19 (m, 2H), 7.10 (dd, J ¼ 8.2,
1.2 Hz, 1H), 7.01e6.91 (m, 2H), 6.81 (td, J ¼ 7.6, 1.1 Hz, 1H), 6.60 (d,
J ¼ 7.3 Hz, 1H), 5.90 (t, J ¼ 5.7 Hz, 1H), 3.39e3.26 (m, 2H), 1.14 (t,
d
155.1 (C), 143.1 (C), 132.8 (C), 119.7 (CH), 117.6 (CH), 114.8 (CH),
J ¼ 7.1 Hz, 3H). 13C NMR
d 155.0 (C), 154.3 (C), 143.5 (C), 136.2 (C),
109.8 (CH), 68.1 (CH), 59.9 (CH), 35.4 (CH2), 29.1 (CH2), 25.0 (CH2),
23.9 (CH2). HR-ESMS calcd. for C13H18N3Oþ [M þ H] 232.1444,
found 232.1450.
130.6 (CH), 130.0 (CH), 121.7 (C), 121.0 (CH), 120.3 (CH), 118.9 (CH),
117.7 (CH), 115.3 (CH), 108.1 (CH), 37.7 (CH2), 15.6 (CH3). HR-ESMS
calcd. for C15H16N3Oþ [M þ H] 254.1288, found 254.1291.
4.2.5. 2-(2-Amino-3H-imidazo[4,5-b]pyridin-3-yl)phenol (10)
2,3-Diaminopyridine (1.00 g) was first purified by adding MeCN
(50 mL) and stirring at 25 ꢀC for 2 h. The mixture was filtered to
remove a black solid, washed with MeCN and the filtrate evapo-
rated at reduced pressure to give 639 mg of 2,3-diaminopyridine as
a fawn solid available for further reaction. 2,3-Diaminopyridine
(639 mg, 5.86 mmol, 1.0 eq.) was dissolved in MeCN (30 mL) and
4.2.6.2. 2-(2-(Propyl)-1H-benzo[d]imidazole-1-yl)phenol
(11b).
Compound 11b was formed in 25% yield. 1H NMR
d
10.00 (s, 1H),
7.35 (ddd, J ¼ 8.2, 7.4, 1.7 Hz, 1H), 7.27e7.19 (m, 2H), 7.10 (dd, J ¼ 8.2,
1.2 Hz, 1H), 7.02e6.91 (m, 2H), 6.80 (td, J ¼ 7.6, 1.1 Hz, 1H), 6.60 (dd,
J ¼ 7.6, 0.5 Hz, 1H), 5.94 (t, J ¼ 5.5 Hz, 1H), 3.26 (dd, J ¼ 14.6, 5.5 Hz,
2H), 1.58 (dd, J ¼ 14.6, 7.4 Hz, 2H), 0.86 (t, J ¼ 7.4 Hz, 3H). 13C NMR
(CDCl3)
d 154.9 (C), 153.7 (C), 141.3 (C), 134.5 (C), 131.2 (CH), 128.9
to this solution was added Et3N (816
m
L, 5.86 mmol, 1.0 eq.).
(CH), 122.1 (CH) 120.7 (C), 120.6 (CH), 120.3 (2 ꢃ CH), 115.2 (CH),
Ethoxycarbonyl isothiocyanate (728 L, 6.44 mmol, 1.1 eq.) was
m
109.1 (CH), 44.3 (CH2), 22.6 (CH2), 11.2 (CH3). HR-ESMS calcd. for
added dropwise, whereby the reaction warmed and a precipitate
quickly formed. The mixture was stirred at 25 ꢀC for 10 min, before
EDCI.HCl (1.35 g, 7.03 mmol, 1.2 eq.) and additional Et3N (1.22 mL,
8.78 mmol, 1.5 eq.) were added. The mixture was heated at reflux at
82 ꢀC for 1 h. The cooled reaction mixture was filtered and washed
with a small volume of MeCN to give ethyl (3H-imidazo[4,5-b]
C
16H18N3Oþ [M þ H] 268.1444, found 268.1448.
4.2.6.3. 2-(2-(Butylamino)-1H-benzo[d]imidazole-1-yl)phenol (11c).
Compound 11c was formed in 25% yield. 1H NMR
9.99 (s, 1H), 7.35
d
(ddd, J ¼ 8.2, 7.4, 1.7 Hz, 1H), 7.26e7.18 (m, 2H), 7.10 (dd, J ¼ 8.2,
1.2 Hz, 1H), 6.94 (dd, J ¼ 7.4, 1.2 Hz, 1H), 6.95 (td, J ¼ 7.6, 1.2 Hz, 1H),
6.80 (td, J ¼ 7.6,1.2 Hz,1H), 6.60 (d, J ¼ 7.6 Hz,1H), 5.89 (t, J ¼ 5.6 Hz,
1H), 3.34e3.26 (m, 2H) 1.55 (tt, J ¼ 14.4, 7.0 Hz, 2H), 1.31 (tq,
pyridin-2-yl)carbamate (1.06 g, 88%). 1H NMR
d 11.90 (s, 1H), 11.49
(s, 1H), 8.16 (dd, J ¼ 4.9, 1.6 Hz, 1H), 7.72 (dd, J ¼ 7.8, 1.6 Hz, 1H), 7.07
(dd, J ¼ 7.8, 4.9 Hz,1H), 4.23 (q, J ¼ 7.1 Hz, 2H),1.28 (t, J ¼ 7.1 Hz, 3H).
J ¼ 14.4, 7.4 Hz, 2H), 0.88 (t, J ¼ 7.4 Hz, 3H). 13C NMR
d 154.6 (C),
Ethyl
(3H-imidazo[4,5-b]pyridin-2-yl)carbamate
(500
mg,
153.8 (C), 143.0 (C), 135.7 (C), 130.1 (CH), 129.5 (CH), 121.3 (C) 120.5
(CH), 119.8 (CH), 118.4 (CH), 117.3 (CH), 114.8 (CH), 107.6 (CH), 42.1
(CH2), 31.4 (CH2), 19.5 (CH2), 13.9 (CH3). HR-ESMS calcd. for
2.43 mmol, 1.0 eq.) was taken up in MeOH (2 mL) and 1 M NaOH
(2 mL) in a sealed tube and the mixture heated at 100 ꢀC for 2 h. The
resultant cooled solution was poured onto ice/water (50 mL) and
the pH adjusted to 8 with HCl. The resultant precipitate was filtered
and washed with water to give 3H-imidazo[4,5-b]pyridin-2-amine
(9d) (63 mg, 19%). The filtrate was washed with EtOAc (2 ꢃ 10 mL)
to remove trace amounts of starting material and the aqueous
fraction evaporated at reduced pressure. The residue was taken up
in MeCN (50 mL), stirred for 1 h, filtered and the filtrate evaporated
at reduced pressure to give further 3H-imidazo[4,5-b]pyridin-2-
C
17H20N3Oþ [M þ H] 282.1601, found 282.1559.
4.2.6.4. 2-(2-(Isopropylamino)-1H-benzo[d]imidazole-1-yl)phenol
(11d). Compound 11d was formed in 8% yield. 1H NMR
10.05 (s,
d
1H), 7.35 (td, J ¼ 8.2, 1.6 Hz, 1H), 7.24 (d, J ¼ 7.8 Hz, 1H), 7.24 (d,
J ¼ 7.8 Hz, 1H) 7.11 (dd, J ¼ 8.2, 1.2 Hz, 1H), 6.98 (td, J ¼ 15.2, 7.6 Hz,
1H), 6.97 (td, J ¼ 15.1, 7.6 Hz, 2H), 6.82 (td, J ¼ 7.7,1.0 Hz,1H), 6.62 (d,
J ¼ 7.6 Hz, 1H), 5.57 (d, J ¼ 8.0 Hz, 1H), 4.09 (dh, J ¼ 8.0, 6.5 Hz, 1H),
13

S.M. Devine, M.P. Challis, J.K. Kigotho et al.
European Journal of Medicinal Chemistry 221 (2021) 113518
1.18 (d, J ¼ 6.5 Hz, 6H). 13C NMR
d
153.9 (C),153.6 (C),142.8 (C),135.5
bromopyridin-2-amine (150 mg, 0.87 mmol, 1.0 eq.), 2-
hydroxyphenylboronic acid (179 mg, 1.30 mmol, 1.5 eq.) and
PdCl2(PPh3)2 (61 mg, 0.087 mmol, 0.1 eq.) and the mixture was
heated at 100 ꢀC for 1 h. The cooled reaction mixture was diluted
with EtOAc (3 mL) and the organic layer filtered through cotton
wool. The filtrate was evaporated and purified by column chro-
matography using an eluted with EtOAc/Et3N (99:1) to give (12a)
(C), 130.1 (CH), 129.4 (CH), 121.3 (C), 120.6 (CH), 119.9 (CH), 118.5
(CH), 117.3 (CH), 114.9 (CH), 107.8 (CH), 44.4 (CH), 22.6 (CH3), 22.5
(CH3). HR-ESMS calcd. for C16H18N3Oþ [M þ H] 268.1444, found
268.1449.
4.2.6.5. 2-(2-(Isobutylamino)-1H-benzo[d]imidazole-1-yl)phenol
(11e). Compound 11e was formed in 23% yield. 1H NMR
d
10.02 (s,
(31 mg, 19%) as an orange amorphous solid. 1H NMR
d 9.68 (s, 1H),
1H), 7.35 (ddd, J ¼ 8.2, 7.4, 1.6 Hz, 1H), 7.27e7.19 (m, 2H), 7.11 (dd,
J ¼ 8.2, 1.2 Hz, 1H), 6.97 (m, 2H), 6.80 (td, J ¼ 7.6, 1.2 Hz, 1H), 6.60 (d,
J ¼ 7.6 Hz, 1H), 5.97 (app t, 1H), 3.10 (ddd, J ¼ 20.2, 11.1, 6.4 Hz, 2H),
7.94 (dd, J ¼ 4.9, 1.7 Hz, 1H), 7.27 (dd, J ¼ 7.3, 1.7 Hz, 1H), 7.20 (td,
J ¼ 8.1, 1.7 Hz, 1H), 7.10 (dd, J ¼ 7.5, 1.6 Hz, 1H), 6.95 (d, J ¼ 8.1 Hz,
1H), 6.88 (td, J ¼ 7.4, 1.0 Hz, 1H), 6.64 (dd, J ¼ 7.3, 5.0 Hz, 1H), 5.24 (s,
1.99 (dp, J ¼ 13.6, 6.8 Hz, 1H), 0.86 (dd, J ¼ 6.8 Hz, 2.0 Hz, 6H). 13
C
2H). 13C NMR
d 157.0 (C), 154.3 (C), 146.6 (CH), 138.2 (CH), 131.2
NMR
d
154.6 (C), 153.9 (C), 142.8 (C), 135.6 (C), 130.1 (CH), 129.5
(CH), 128.9 (CH), 124.9 (C), 119.5 (CH), 118.9 (C), 115.9 (CH), 112.8
(CH). HR-ESMS calcd. for C11H11N2Oþ [M þ H] 187.0866, found
187.0871.
(CH), 121.2 (C), 120.6 (CH), 119.9 (CH), 118.4 (CH), 117.3 (CH), 114.7
(CH), 107.6 (CH), 49.9 (CH2), 27.4 (CH), 20.1 (CH3), 20.0 (CH3). HR-
ESMS calcd. for C17H20N3Oþ [M þ H] 282.1601, found 282.1609.
4.2.8. 2-(3-Aminopyrazin-2-yl)phenol (12b). To a degassed biphasic
solution of THF (1.5 mL) and 1 M Na2CO3 (0.5 mL), was added 3-
bromopyrazin-2-amine (150 mg, 0.86 mmol, 1.0 eq.), 2-
hydroxyphenylboronic acid (178 mg, 1.30 mmol, 1.5 eq.) and
PdCl2(PPh3)2 (61 mg, 0.087 mmol, 0.1 eq.) and the mixture was
heated at 100 ꢀC for 1 h. The cooled reaction mixture was diluted
with EtOAc (3 mL) and the organic layer filtered through cotton
wool. The filtrate was evaporated and purified by column chro-
matography and eluted with petroleum spirits/EtOAc (4:1) to give
4.2.6.6. 2-(2-(Cyclohexylamino)-1H-benzo[d]imidazole-1-yl)phenol
(11f). Compound 11f was formed in 20% yield. 1H NMR
d 10.10 (s,
1H), 7.35 (td, J ¼ 8.2, 1.6 Hz, 1H), 7.28e7.21 (m, 2H), 7.11 (dd, J ¼ 8.2,
1.2 Hz, 1H), 7.03e6.91 (m, 2H), 6.82 (td, J ¼ 7.6, 0.9 Hz, 1H), 6.32 (d,
J ¼ 7.6 Hz, 1H), 5.56 (d, J ¼ 7.6 Hz, 1H), 3.84e3.62 (m, 1H), 1.94e1.88
(m, 2H), 1.77e1.63 (m, 2H), 1.58 (d, J ¼ 12.7 Hz, 1H), 1.40e1.19 (m,
4H), 1.15e1.02 (m, 1H). 13C NMR
d 153.8 (C), 153.6 (C), 142.6 (C),
135.4 (C), 130.0 (CH), 129.3 (CH), 121.3 (C), 120.7 (CH), 119.9 (CH),
118.6 (CH), 117.3 (CH), 114.8 (CH), 107.8 (CH), 51.6 (CH), 32.6 (CH2),
32.6 (CH2), 25.4 (CH2), 25.0 (CH2), 25.0 (CH2). HR-ESMS calcd. for
(12b) (43 mg, 27%) as an orange amorphous solid. 1H NMR
d 10.12
(s, 1H), 7.94 (d, J ¼ 2.7 Hz, 1H), 7.85 (d, J ¼ 2.7 Hz, 1H), 7.31e7.24 (m,
C
19H22N3Oþ [M þ H] 308.1757, found 308.1753.
2H), 6.96 (d, J ¼ 7.7 Hz, 1H), 6.91 (td, J ¼ 7.4, 1.1 Hz, 1H), 5.79 (s, 2H).
13C NMR
d 154.7 (C), 153.7 (C), 140.8 (CH), 139.3 (C), 131.9 (CH), 131.0
4.2.6.7. 2-(2-((Cyclohexylmethyl)amino)-1H-benzo[d]imidazole-1-
(CH),129.9 (CH),124.3 (C),119.4 (CH),116.1 (CH). HR-ESMS calcd. for
C
yl)phenol (11g). Compound 11g was formed in 39% yield. 1H NMR
10H10N3Oþ [M þ H] 188.0818, found 188.0822.
d
10.03 (s, 1H), 7.35 (ddd, J ¼ 8.2, 7.6, 1.7 Hz, 1H), 7.27e7.20 (m, 2H),
7.11 (dd, J ¼ 8.2, 1.2 Hz, 1H), 6.98 (td, J ¼ 7.5, 1.2 Hz), 6.94 (td,
J ¼ 7.7 Hz,1.1 Hz), 6.80 (td, J ¼ 7.6,1.0 Hz,1H), 6.60 (d, J ¼ 7.4 Hz,1H),
5.93 (t, J ¼ 4.8 Hz, 1H), 3.14 (qt, J ¼ 12.9, 6.3 Hz, 2H), 1.87e1.54 (m,
4.3. Biology
Compound preparation and storage. A 10 mM stock solution in
6H), 1.28e1.02 (m, 3H), 0.97e0.77 (m, 2H). 13C NMR
d
154.6 (C),
DMSO (Sigma D2650) was prepared for each compound, which was
153.8 (C), 135.6 (C), 130.1 (CH), 129.5 (CH), 121.2 (C), 120.5 (CH),
119.8 (CH), 118.4 (CH), 117.3 (CH), 114.7 (CH), 107.6 (CH), 48.6 (CH2),
36.8 (CH), 30.4 (CH2), 30.3 (CH2), 26.2 (CH2), 25.5 (CH2), 25.4 (CH2).
HR-ESMS calcd. for C20H24N3Oþ [M þ H] 322.1914, found 322.1919.
stored at ꢂ20 ꢀC until use.
4.3.1. IC50 determination (Monash assay)
P. falciparum strains 3D7, ART resistant (Cam3.IIC580Y) and ART
sensitive (Cam3.IIrev) [39] were cultured continuously as previously
described [47]. In-vitro growth inhibition IC50 assays were used to
determine the concentration of drug at which 50% of parasites were
killed. Asynchronous cultures were adjusted to 0.2% parasitaemia
4.2.6.8. 2-(2-(Benzylamino)-1H-benzo[d]imidazole-1-yl)phenol
(11h). Compound 11h was formed in 40% yield. 1H NMR
d 10.15 (s,
1H), 7.40e7.34 (m, 3H), 7.32e7.25 (m, 3H), 7.23e7.17 (m, 2H), 7.13
(dd, J ¼ 8.2, 1.2 Hz, 1H), 7.00 (td, J ¼ 7.6, 1.3 Hz, 1H), 6.96 (td, J ¼ 7.6,
1.2 Hz, 1H), 6.84 (td, J ¼ 7.6, 1.1 Hz, 1H), 6.68 (t, J ¼ 6.0 Hz, 1H), 6.64
and 4% haematocrit. Relevant compounds were made up to 2 mM
(twice the desired highest concentration) in complete RPMI media
(Merck/Sigma Aldrich R6504, supplemented with 25 mM HEPES,
0.5% (wt/vol) Albumax II, 0.37 mM hypoxanthine and 2.0 g/l sodium
bicarbonate). A serial dilution was performed from this concen-
tration in order to produce a range of test concentrations. IC50 as-
says were performed in 96-well plates in either duplicate or
triplicate with three biological replicates performed for each
compound. The negative control used was 0.01% DMSO while the
(d, J ¼ 7.7 Hz, 1H), 4.61e4.47 (m, 2H). 13C NMR
d 154.6 (C), 154.0 (C),
142.6 (C), 140.6 (C), 135.9 (C), 130.3 (CH), 129.7 (CH), 128.1 (2 ꢃ CH),
127.0 (2 ꢃ CH), 126.5 (CH), 121.1 (C), 120.7 (CH), 119.9 (CH), 118.7
(CH), 117.3 (CH), 115.0 (CH), 107.8 (CH), 45.5 (CH2). HR-ESMS calcd.
for C20H18N3Oþ [M þ H] 316.1444, found 316.1446.
4.2.6.9. 2-(2-((Cyclopropylmethyl)amino)-1H-benzo[d]imidazole-1-
yl)phenol (11i). Compound 11i was formed in 25% yield. 1H NMR
positive control was 1 mM of 2a. Adjusted culture (100 mL) was
d
10.02 (s, 1H), 7.36 (td, J ¼ 8.2, 1.6 Hz, 1H), 7.30e7.19 (m, 2H), 7.12
added to each well and incubated for 72 h at 37 ꢀC under a defined
atmosphere (95% N2, 4% CO2, 1% O2). Following addition of culture,
the final haematocrit was 2% and the final starting concentration of
(dd, J ¼ 8.2, 0.9 Hz, 1H), 7.05e6.91 (m, 2H), 6.82 (t, J ¼ 7.4 Hz, 1H),
6.62 (d, J ¼ 7.6 Hz, 1H), 5.95 (t, J ¼ 5.4 Hz,1H), 3.20 (t, J ¼ 6.1 Hz, 2H),
1.21e1.08 (m, 1H), 0.38 (dd, J ¼ 6.5, 1.6 Hz, 2H), 0.23 (dd, J ¼ 6.0,
drug was 1
removed from each well and replaced with 140
After media replacement, 100 L of lysis buffer (20 mM Tris, 5 mM
EDTA, 0.008% w/v Saponin and 0.08% w/v Triton X) with SYBR
Green dye (0.1 L/1 mL; Life Technologies) was added and wells
mM. Following the 72 h incubation, 140
mL of media was
4.2 Hz, 2H). 13C NMR
d
154.7 (C), 153.7 (C), 135.7 (C), 130.1 (CH),
mL of fresh media.
129.4 (CH), 121.3 (C), 120.5 (CH), 119.9 (CH), 118.4 (CH), 117.3 (CH),
114.9 (CH), 107.7 (CH), 46.9 (CH2), 11.2 (CH), 3.3 (CH2), 3.3 (CH2). HR-
ESMS calcd. for C17H18N3Oþ [M þ H] 280.1444, found 280.1448.
m
m
were thoroughly mixed. The plate was incubated at room temper-
ature in the dark for 2 h before fluorescence detection using a
Novostar V plate reader (BMG Labtech) with excitation and
4.2.7. 2-(2-Aminopyridin-3-yl)phenol (12a). To a degassed biphasic
solution of THF (1.5 mL) and 1 M Na2CO3 (0.5 mL), was added 3-
14

S.M. Devine, M.P. Challis, J.K. Kigotho et al.
European Journal of Medicinal Chemistry 221 (2021) 113518
emission wavelengths of 485 and 530 nm, respectively. Analysis of
the data was performed on GraphPad Prism (San Diego, Calif)
software. The data were normalised to the positive and negative
controls and plotted against the logarithm of the concentration of
drug, which was then used to calculate the IC50 values through
fitting a 4-parameter variable slope.
4.3.5. Estimation of kinetic solubility using nephelometry
Compounds in DMSO were spiked into either pH 6.5 phosphate
buffer or 0.01 M HCl (pH~2.0) with the final DMSO concentration
being 1%. After 30 min had elapsed, the samples were then
analyzed via nephelometry to determine a solubility range [43].
4.3.6. In vitro metabolic stability
The metabolic stability assay was performed by incubating each
compound (1 mM) in human or mouse liver microsomes (Sekisui
4.3.2. Resistant cell line antimalarial activity assay (Griffith assay)
CQ, ART, puromycin, DHA, pyronaridine, pyrimethamine and
SJ557733 were used as reference compounds. Stock solutions were
prepared at 2.5 mM (DHA, artesunate) or 10 mM (all other refer-
ence compounds) in DMSO, apart from pyronaridine and CQ, which
XenoTech) at 37 ꢀC and a protein concentration of 0.4 mg/mL. The
metabolic reaction was initiated by the addition of an NADPH-
regenerating system and quenched at various time points over a
60 min incubation period by the addition of acetonitrile containing
diazepam as internal standard. Control samples (containing no
NADPH) were included (and quenched at 2, 30, and 60 min) to
monitor for potential degradation in the absence of cofactor.
were dissolved in water. Puromycin (5 mM) and 0.4% DMSO were
used as positive and negative controls, respectively. The experi-
mental compounds and the reference antimalarials were diluted
1:25 in water, then 1:10 in the final assay plates, to give a final assay
top concentration of 40 mM. The final DMSO concentration for all
compounds in the assay was 0.4%. All the compounds were tested
in full dose-response using three concentrations per log dose (22
Author contribution
The manuscript was written through contributions of all au-
thors, and all authors have given approval to the final version of the
manuscript.
points, final concentration range 40
imental compounds and 40 Me0.01 nM for the reference anti-
malarials (10 Me0.003 nM for DHA and artesunate). P. falciparum
mMe0.004 nM) for the exper-
m
m
parasite strains Dd2-SJ557733 resistant and Dd2-SJ557733 parent
were grown in RPMI 1640 supplemented with 25 mM HEPES, 5% AB
human male serum, 2.5 mg/mL Albumax II, and 0.37 mM hypo-
xanthine. Parasites were subjected to two rounds of sorbitol syn-
chronisation before undergoing compound treatment. Ring-stage
parasites were exposed to the experimental compounds in 384-
wells imaging microplates (PerkinElmer CellCarrier), as previ-
ously described [48]. Plates were incubated for 72 h at 37 ꢀC, 90%
N2, 5% CO2, 5% O2, then the parasites were stained with 2-(4-
amidinophenyl)-1H-indole-6-carboxamidine (DAPI), and imaged
using an Opera QEHS micro-plate confocal imaging system (Per-
kinElmer). Images were analyzed as previously described [48].
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
The authors would like to thank Ben Capuano for productive
discussions on this project, Jason Dang for obtaining HRMS data
and the Australian Red Cross Blood Service for the provision of
human red blood cells. This work was supported in part by
Australian National Health and Medical Research Council (NHMRC)
Project (1098884 & 1163235) and Synergy grants (1185354). R. S. N.
and D. J. C. acknowledge fellowship support from the NHMRC. The
Centre for Drug Candidate Optimisation (CDCO, Monash University)
is acknowledged for conducting the ADME studies. The CDCO is
partially supported by the Monash University Technology Research
Platform network and Therapeutic Innovation Australia (TIA)
through the Australian Government National Collaborative
Research Infrastructure Strategy (NCRIS) program.
4.3.3. Cytotoxicity assay (MTT assay e Monash)
Human embryonic kidney (HEK293) cells were maintained in
complete DMEM medium (DMEM containing 2 mM GlutaMAX,
supplemented with 10% FBS and antibiotics). 24 h prior to com-
pound exposure, HEK293 cells were seeded (8 ꢃ 104 cells/well) into
flat bottom 96-well plates (Corning) and incubated at 37 ꢀC, 5% CO2.
Serial dilutions of test compounds ranging from 100 mMe391 nM
were incubated at standard conditions for 24 h. After compound
exposure, the media was removed from the wells and replaced with
an equal volume of MTT media (complete DMEM medium con-
taining 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT,
Sigma)). Plates were incubated for an additional 3e4 h at standard
conditions, after which MTT media was removed and an equal
volume of DMSO added to dissolve formazan crystals. The total
absorbance was measured at 570 nm (EnSight plate reader, Perki-
nElmer). Raw data was blank adjusted, normalised to DMSO control
to obtain % viability data. Data is representative of at least 3 bio-
logical replicates with at least 2 technical repeats.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113518.
Abbreviations
ABI
aminobenzimidazole
artemisinin combination therapy
apical membrane antigen 1
artemisinin
ACT
AMA1
ART
CQ
4.3.4. Data analysis
Raw data were normalised using the in-plate positive and
negative controls to obtain normalised % inhibition data, which
were then used to calculate IC50 values, through a 4-parameter
logistic curve fitting in GraphPad Prism. The experiments against
3D7 and Dd2 cells were carried out in two independent biological
replicates, each consisting of two technical repeats each. The
comparison between 3D7, Dd2 and the SJ-resistant/parent strains
were carried out once.
chloroquine
DHA
dihydroartemisinin
EDCI.HCl 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride
MMV
MoA
SAR
Medicines for Malaria Venture
mechanism of action
structure-activity relationship
surface plasmon resonance
SPR
15

S.M. Devine, M.P. Challis, J.K. Kigotho et al.
European Journal of Medicinal Chemistry 221 (2021) 113518
identifies pan-active N-Aryl-3-trifluoromethyl pyrido[1,2-a]benzimidazoles
which are efficacious in an in vivo mouse model of malaria, J. Med. Chem. 62
(2019) 1022e1035, https://doi.org/10.1021/acs.jmedchem.8b01769.
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