Synthesis, anti-bacterial and anti-protozoal activities of amidinobenzimidazole derivatives and their interactions with DNA and RNA

Article abstract of DOI:10.1080/14756366.2018.1484733

Amidinobenzimidazole derivatives connected to 1-aryl-substituted 1,2,3-triazole through phenoxymethylene linkers 7a–7e, 8a–8e, and 9a–9e were designed and synthesised with the aim of evaluating their anti-bacterial and anti-trypanosomal activities and DNA/RNA binding affinity. Results from anti-bacterial evaluations of antibiotic-resistant pathogenic bacteria revealed that both o-chlorophenyl-1,2,3-triazole and N-isopropylamidine moieties in 8c led to strong inhibitory activity against resistant Gram-positive bacteria, particularly the MRSA strain. Furthermore, the non-substituted amidine and phenyl ring in 7a induced a marked anti-bacterial effect, with potency against ESBL-producing Gram-negative E. coli better than those of the antibiotics ceftazidime and ciprofloxacin. UV–Vis and CD spectroscopy, as well as thermal denaturation assays, indicated that compounds 7a and 8c showed also binding affinities towards ctDNA. Anti-trypanosomal evaluations showed that the p-methoxyphenyl-1,2,3-triazole moiety in 7b and 9b enhanced inhibitory activity against T. brucei, with 8b being more potent than nifurtimox, and having minimal toxicity towards mammalian cells.

Full text of DOI:10.1080/14756366.2018.1484733

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis, anti-bacterial and anti-protozoal
activities of amidinobenzimidazole derivatives and
their interactions with DNA and RNA
Andrea Bistrović, Luka Krstulović, Ivana Stolić, Domagoj Drenjančević,
Jasminka Talapko, Martin C. Taylor, John M. Kelly, Miroslav Bajić & Silvana
Raić-Malić
To cite this article: Andrea Bistrović, Luka Krstulović, Ivana Stolić, Domagoj Drenjančević,
Jasminka Talapko, Martin C. Taylor, John M. Kelly, Miroslav Bajić & Silvana Raić-Malić (2018)
Synthesis, anti-bacterial and anti-protozoal activities of amidinobenzimidazole derivatives and
their interactions with DNA and RNA, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1,
1323-1334, DOI: 10.1080/14756366.2018.1484733
To link to this article: https://doi.org/10.1080/14756366.2018.1484733
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 33, NO. 1, 1323–1334
https://doi.org/10.1080/14756366.2018.1484733
RESEARCH PAPER
Synthesis, anti-bacterial and anti-protozoal activities of amidinobenzimidazole
derivatives and their interactions with DNA and RNA
ꢀa
ꢀb
ꢀb
ꢀ
ꢁ
ꢀc,d
Andrea Bistrovic , Luka Krstulovic , Ivana Stolic , Domagoj Drenjancevic , Jasminka Talapkod, Martin C. Taylore,
e
ꢀb
ꢀa
John M. Kelly , Miroslav Bajic and Silvana Raic-Malic
b
aDepartment of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Zagreb, Croatia; Department of
c
Chemistry and Biochemistry, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia; Department of Transfusion Medicine, Osijek
d
University Hospital, Osijek, Croatia; Department of Microbiology and Parasitology, Faculty of Medicine, University of Osijek, Osijek, Croatia;
eDepartment of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, UK
ABSTRACT
ARTICLE HISTORY
Received 25 January 2018
Revised 18 May 2018
Accepted 31 May 2018
Amidinobenzimidazole derivatives connected to 1-aryl-substituted 1,2,3-triazole through phenoxymethy-
lene linkers 7a–7e, 8a–8e, and 9a–9e were designed and synthesised with the aim of evaluating their
anti-bacterial and anti-trypanosomal activities and DNA/RNA binding affinity. Results from anti-bacterial
evaluations of antibiotic-resistant pathogenic bacteria revealed that both o-chlorophenyl-1,2,3-triazole and
N-isopropylamidine moieties in 8c led to strong inhibitory activity against resistant Gram-positive bacteria,
particularly the MRSA strain. Furthermore, the non-substituted amidine and phenyl ring in 7a induced a
marked anti-bacterial effect, with potency against ESBL-producing Gram-negative E. coli better than those
of the antibiotics ceftazidime and ciprofloxacin. UV–Vis and CD spectroscopy, as well as thermal denatur-
ation assays, indicated that compounds 7a and 8c showed also binding affinities towards ctDNA. Anti-
trypanosomal evaluations showed that the p-methoxyphenyl-1,2,3-triazole moiety in 7b and 9b enhanced
inhibitory activity against T. brucei, with 8b being more potent than nifurtimox, and having minimal
toxicity towards mammalian cells.
KEYWORDS
Benzimidazole; 1,2,3-
triazole; resistant bacteria;
antiprotozoal activity;
Trypanosoma brucei; MRSA
Introduction
there are many areas of therapy that might benefit from DNA-
directed intervention, there is currently an urgent need for new
antimicrobials with novel modes of action.
The benzimidazole derivatives, which contain fused heterocyclic
nuclei within their structures, are structural isosteres of purine
bases. This allows them to interact with biopolymers and they,
Antibiotic resistance is a global public threat because of its
effect on health care with prolonged hospitalisations and
increased mortality. The increasing prevalence of hospital and
community-acquired infections caused by multidrug-resistant
(MDR) bacterial pathogens is limiting the options for effective anti-
biotic therapy13,14. Drug-resistant Gram-positive bacterial patho-
gens, including methicillin-resistant Staphylococcus aureus (MRSA)
and vancomycin-resistant enterococci (VRE), have become a serious
clinical problem that impinges on the treatment of various noso-
comial and community-acquired infections15,16. In addition, an
therefore, have diverse biological and clinical applications1–5
.
Much research effort has been aimed at targeting DNA with benzi-
midazole ligands, with the goal of designing agents that have
therapeutic applications5–9. Although RNA is a well-established tar-
get of current antibiotics, designing new compounds that select-
ively recognise RNA has also been a difficult task, particularly
when focused on the treatment of a variety of infections10–12. The
challenge is to produce drug-like molecules with high affinity for
DNA/RNA, while maintaining sufficient sequence selectivity. While increased incidence of MDR Gram-negative bacteria, such as
ꢀ
ꢀ
ꢀ
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Marulicev
CONTACT Silvana Raic-Malic
sraic@fkit.hr
trg 20, HR-10000 Zagreb, Croatia
Supplemental data for this article can be accessed here.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

ꢀ
A. BISTROVIC ET AL.
1324
Figure 1. Representatives of benzimidazoles containing triazole moiety I–V as potential antibacterial agents.
Figure 2. Aromatic amidines and 1,4-diphenyl-1,2,3-triazole amidine VI as anti-HAT agents.
Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumo- triazole-bearing monobenzimidazoles IV and V inhibited growth of
niae, coupled with the lack of novel antibiotics, represents one of Gram-positive bacteria, including two MRSA strains, and displayed
the biggest threats to the control of respiratory and other infec- E. coli DNA topoisomerase I inhibition36.
tions17. In order to overcome these emerging bacterial resistance
The increasingly important role of benzimidazole and triazole
problems, novel anti-bacterial drugs need to be developed. derivatives has been also demonstrated by their in vivo evalua-
Accordingly, in recent years, numerous efforts have focused on tions against Gram-positive37–42 and Gram-negative bacteria43.
discovering novel benzimidazole-based anti-bacterial agents18–24
.
Bis-benzimidazole compound (ridinilazole, SMT-19969)44 recently
The importance of a protonable chemical moiety within anti- entered phase III human clinical trials for the treatment of
bacterial drugs has been investigated in different studies25,26
.
Clostridium difficile.
Besides anti-bacterial activity, benzimidazole containing com-
pounds have shown good anti-protozoal potency45–49. Human
African trypanosomiasis (HAT), also known as sleeping sickness, is
a fatal parasitic disease caused by two subspecies of Trypanosoma
brucei. It has been estimated that over 50 million people are at
risk of infection with HAT in more than 30 African countries, and
there remains a clear need to develop new, safer, and more
affordable agents to combat this fatal infection50. The efficacy of
diarylamidines, such as pentamidine51, berenyl52 and its orally
active prodrug pafuramidine53 (Figure 2), in the treatment of
These have revealed the significant uptake of amidine-containing
DNA ligands into bacteria, and also into the nuclei of eukaryotic
cells27. In addition, the structural features of 1,2,3-triazole also
enable it to mimic different functional groups, justifying its wide
use as a bioisostere for the design of antimicrobial drug ana-
logs28,29. For example 1,4-disubstituted 1,2,3-triazoles are good
Z-amide isosteres, because the C-4 atom can act as an electro-
philic site; the CH bond (in the 5-position) acts as a hydrogen
bond donor, and the lone pair of N-3 electrons acts as a hydrogen
bond acceptor30.
A wide range of pharmacological activities has been attributed protozoal diseases, especially trypanosomiasis, has been known
to the unusual chemical features of azole rings, such as benzimida- for many years.
However, current drugs have problems, such as toxicity, poor
zole and 1,2,3-triazole. These are able to interact in a non-covalent
way with a range of targets, due to the presence of an electron- efficacy, and increasing resistance by the parasites. Although the
rich aromatic system and heteroatoms31,32, and act as promising precise anti-protozoan mechanisms of action of aromatic diami-
moieties for the design of novel scaffolds with anti-bacterial activ- dines have not been fully elucidated, there is considerable
ity. Thus, among the series of [1,2,4-triazolyl]phenyl-substituted evidence that direct interaction with the pathogen genome is
4,6-difluorobenzimidazoles I33, analogues with electronegative sub- important for activity. Recently, a diamidine containing a 1,2,3-tri-
stituents emerged as promising antimicrobials, while 2-thiobenzimi- azole ring as central core was synthesised, which displayed better
dazole with [(1,2,4-triazolyl)ethylthio]phenyl moiety II34 exhibited anti-trypanosomal efficacy than melarsoprol, curing all infected
anti-bacterial properties that were selective for Helicobacter pylori mice54. It was found that incorporation of different hydrophobic
(Figure 1). Benzimidazole–1,2,3-triazole conjugates III with aromatic aromatic head groups linked to the rest of the molecule by an
(p-chlorophenyl and p-fluorophenyl) 4-substituted triazoles exhib- amidine moiety improved both anti-bacterial activity and affinity
ited selective anti-Moraxella catarrhalis activity35. Furthermore, to DNA27.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1325
Figure 3. Design and synthesis of amidinobenzimidazoles connected to 1-aryl-substituted 1,2,3-triazole via phenoxymethylene unit.
In view of the wide applications of the benzimidazole and General procedure for the synthesis of compounds 3a–e
The reaction mixture of compound 2, Cu(0) (0.8 eq), 1 M CuSO4
(0.3 eq) and the corresponding azide (1.2 eq) was dissolved in
1 ml DMF and a mixture of t-BuOH: H2O ¼ 1: 1 (3 ml). Method A:
The reaction mixture was stirred under microwave irradiation
(300 W) at 80 ꢀC during 1.5 h. Method B: The reaction mixture was
placed in an ultrasonic bath cleaner (1000 W, 35 kHz) at 80 ꢀC for
1.5 h. The solvent was removed under reduced pressure and puri-
fied by column chromatography with CH2Cl2.
1,2,3-triazole moieties in drug development, and encouraged by
the activity profile of both scaffolds35,55, we synthesised molecules
that contained both units attached through a phenoxymethylene
linker as the central core, thereby expanding the electronic envir-
onment of chemical space (Figure 3).
Targeted compounds were designed to contain a non-substi-
tuted amidine, N-isopropylamidine, and imidazoline moiety at the
C-5 position of the benzimidazole core, as the hydrophilic end,
and an aromatic unit at the N-1 position of the 1,2,3-triazole ring,
as the hydrophobic end. It was anticipated that selected 5-amidi-
nobenzimidazoles connected to 1-aryl-substituted 1,2,3-triazole
would exhibit enhanced affinity for DNA/RNA compared to other
aromatic amidines that we have recently studied56,57. Therefore,
interactions between 5-amidinobenzimidazoles 7a–7e, 8a–8e, and
9a–9e and DNA/RNA were assessed and their activities against
Gram-positive, Gram-negative, and antibiotic-resistant, as well as
their trypanocidal properties, were evaluated.
4-((1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyleneoxy)benzal-
dehyde (3b)
Compound 3b was prepared using the above mentioned proced-
ure from 2 (200 mg, 1.15 mmol) and 1-azido-4-methoxybenzene
(2.76 ml, 1.38 mmol) to obtain 3b as white crystals (Method A:
149.3 mg, 42%; Method B: 271.2 mg, 76%; m.p. 127–130 ꢀC) (m.p.
lit.60 ¼ 126–127 ꢀC). 1H NMR (300 MHz, DMSO) d 9.89 (1H, s, CHO),
8.87 (1H, s, H50), 7.90 (2H, d, J ¼ 8.8 Hz, Ph), 7.81 (2H, d, J ¼ 9.1 Hz,
Ph), 7.28 (2H, d, J ¼ 8.7 Hz, Ph), 7.14 (2H, d, J ¼ 9.1 Hz, Ph), 5.36
(2H, s, OCH2), 3.83 (3H, s, OCH3). 13 C NMR (75 MHz, DMSO) d
191.55, 162.99, 159.49, 143.08, 131.96, 130.03, 130.00, 123.25,
122.03, 115.35, 115.03, 61.47, 55.68.
Materials and methods
General
All chemicals and solvents were purchased from Aldrich and
Acros. Pre-coated Merck silica gel 60F-254 plates and Fluka
(0.063–0.2 mm) silica gel using an appropriate solvent system were
employed for thin layer chromatography (TLC) and column chro-
matography, respectively. Melting points were determined using
4-((1–(2-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)benzalde-
hyde (3c)
Compound 3c was prepared using the above mentioned proced-
ure from 2 (200 mg, 1.15 mmol) and 1-azido-2-chlorobenzene
(2.76 ml, 1.38 mmol) to obtain 3c as white solid (Method A:
194.2 mg, 53%; Method B: 231.67 mg, 64%; m.p. ¼ 127–129 ꢀC). 1H
NMR (600 MHz, DMSO) d 9.90 (1H, s, CHO), 8.76 (1H, s, H50), 7.91
(2H, d, J ¼ 8.7 Hz, Ph), 7.79 (1H, dd, J ¼ 8.0, 1.2 Hz, Ph), 7.73 (1H,
dd, J ¼ 7.8, 1.6 Hz, Ph), 7.65 (1H, td, J ¼ 7.8, 1.6 Hz, Ph), 7.60 (1H, td,
J ¼ 7.6, 1.3 Hz, ph), 7.30 (2H, d, J ¼ 8.7 Hz, Ph), 5.40 (s, 2H,
OCH2).13C NMR (151 MHz, DMSO) d 191.84, 163.15, 142.48, 134.54,
132.14, 132.10, 130.84, 130.19, 128.81, 128.74, 128.63, 127.35,
1
Kofler micro hot-stage (Reichert, Vienna, Austria). H and 13 C NMR
spectra were recorded on a Varian Gemini 300 and 600 spectrom-
eters. All NMR spectra were recorded in DMSO-d6 at 298 K. Mass
spectra were recorded on an Agilent 6410 instrument with electro-
spray interface and triple quadrupole analyser. Microwave-assisted
syntheses were carried out in a microwave oven (Milestone start
S) at 80 ꢀC and pressure of 1 bar. The ultrasound-assisted reactions
were performed in a bath cleaner (Bandelin, Sonorex digital 10 P, 115.54, 61.40.
Berlin, Germany) with frequency of 35 kHz and power of 1000 W.
4-((1-((Phenylthio)methyl)-1H-1,2,3-triazol-4-yl)methoxy)benzalde-
hyde (3e)
Compound 3e was prepared using the above mentioned proced-
Experimental procedures for the synthesis of compounds
ure from
2 (200 mg, 1.15 mmol), azidomethyl phenyl sulfide
Amidino-substituted o-phenylenediamines (4–6)58, 4-(prop-2-yny-
loxy)benzaldehyde (2)59, 4–(1,2,3-triazol-4-yl)methoxy)benzaldehyde
(3b)60, 4–(1,2,3-triazol-4-yl)methoxy)benzaldehydes (3a, 3d), and 5-
amidinobenzimidazoles (7a, 7d, 8a, 8d, 9a, and 9d)55 were pre-
pared according to described procedures.
(0.19 ml, 1.38 mmol), Cu (0) (59.8 mg, 0.94 mmol), 1 M CuSO4
(0.24 ml, 0.05 mmol) in DMF (1 ml), t-BuOH: H2O ¼ 1: 1 (4 ml) to
obtain 3e as yellow oil (Method A: 214.3 mg, 57%; Method B:
1
273.6 mg, 73%). H NMR (300 MHz, DMSO) d 9.88 (1H, s, CHO), 8.21
(1H, s, 1H, H50), 7.87 (2H, d, J ¼ 8.8 Hz, Ph), 7.43–7.26 (5H, m, Ph),

ꢀ
A. BISTROVIC ET AL.
1326
7.21 (2H, d, J ¼ 8.7 Hz, Ph), 5.96 (2H, s, 2H, CH2), 5.26 (2H, s, CH2). N-isopropyl-2–(4-((1–(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)
13 C NMR (75 MHz, DMSO) d 191.58, 162.94, 142.57, 132.36, 131.92,
methoxy)phenyl)-1H-benz[d]imidazole-5-carboximidamide dihydro-
130.68, 129.98, 129.43, 127.90, 124.72, 115.36, 61.33, 51.87.
chloride (8b)
Compound 8b was prepared using the above described method
from 3b (200 mg, 0.65 mmol) and 5 (115.2 mg, 0.65 mmol) to
obtain 8b as brown powder (225.8 mg, 58%, m.p. ¼ 188–191 ꢀC).
1H NMR (300 MHz, DMSO) d 9.66 (1H, d, J ¼ 7.7 Hz, NH), 9.51 (1H, s,
NH), 9.08 (1H, s, NH), 8.92 (1H, s, H50), 8.39 (2H, d, J ¼ 8.7 Hz, Ph),
8.08 (1H, s, H4), 7.88 (1H, d, J ¼ 8.5 Hz, H7), 7.83 (2H, d, J ¼ 9.0 Hz,
Ph), 7.69 (1H, d, J ¼ 7.6 Hz, H6), 7.39 (2H, d, J ¼ 8.8 Hz, Ph), 7.15
(2H, d, J ¼ 9.0 Hz, Ph), 5.39 (2H, s, CH2), 4.15–4.02 (1H, m, CH), 3.84
(3H, s, OCH3), 1.32 (6H, d, J ¼ 6.3 Hz, CH3CHCH3).13C NMR
(151 MHz, DMSO) d 162.18, 161.13, 159.50, 152.96, 143.20, 130.02,
129.64, 124.28, 124.21, 123.74, 123.30, 122.03, 115.77, 115.36,
115.05, 61.43, 55.71, 45.25, 21.38. MS (ESI, m/z) 482.1 [M þ H]þ.
Anal. calcd. for C27H27N7O2 ꢁ 2 HCl ꢁ2.6 H2O (Mr ¼ 601.32): C
53.93, H 5.73, N 16.30; found: C 53.62, H 5.71, N 16.39%.
General procedure for the synthesis of compounds 7a–7e, 8a–8e,
and 9a–9e
The reaction mixture of 4-triazolylbenzaldehyde derivatives
(3a–3e), o-phenylenediamine (4, 5, or 6) and 40% NaHSO3 was
dissolved in 15 ml EtOH and stirred under reflux for 6–8 h. After
completion of the reaction NaHSO3 (aq) was filtered and the reac-
tion mixture was evaporated to dryness. Water was added (5 ml)
and the mixture was stirred overnight and filtered. The crude resi-
due was dissolved in HCl saturated MeOH (8–10 ml) and stirred
overnight. Addition of ether resulted in precipitation of products
7a–7e, 8a–8e, and 9a–9e. Solid was collected by filtration, washed
with anhydrous ether, and dried under vacuum.
2–(4-((1–(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-
1H-benz[d]imidazole-5-carboximidamide dihydrochloride (7b)
Compound 7b was prepared using the above described method
from 3b (200 mg, 0.65 mmol) and 4 (87.39 mg, 0.58 mmol) to
N-isopropyl-2–(4-((1–(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methox-
y)phenyl)-1H-benz[d]imidazole-5-carboximidamide
ide (8c)
dihydrochlor-
Compound 8c was prepared using the above described method
from 3c (200 mg, 0.64 mmol) and 5 (101.0 mg, 0.57 mmol) to
1
obtain 7b as white powder (122.7 mg, 53%, m.p. ¼ 195–197 ꢀC). H
NMR (600 MHz, DMSO) d 9.35 (2H, s, NH), 8.94 (2H, s, NH), 8.90
(1H, s, H50), 8.26 (2H d, J ¼ 8.2 Hz, Ph), 8.15 (1H, s, H4), 7.84–7.80
(3H, m, Ph; H7), 7.71 (1H, d, J ¼ 8.0 Hz, H6), 7.34 (2H, d, J ¼ 8.7 Hz,
Ph), 7.15 (2H, d, J ¼ 9.0 Hz, Ph), 5.37 (2H, s, OCH2), 3.84 (3H, s,
OCH3). 13 C NMR (75 MHz, DMSO) d 165.85, 160.58, 159.42, 153.78,
143.21, 140.84, 134.73, 129.97, 129.13, 123.14, 122.69, 121.93,
120.61, 115.55, 114.97, 61.34, 55.63. MS (ESI, m/z) 440.1 [M þ H]þ.
Anal. calcd. for C24H21N7O2 ꢁ 2 HCl ꢁ2.5 H2O (Mr ¼ 557.44): C
51.71, H 5.06, N 17.59; found: C 51.60, H 4.72, N 17.34%.
1
obtain 8c as white powder (105.5 mg, 28%, m.p. ¼ 210–213 ꢀC). H
NMR (300 MHz, DMSO) d 9.58 (1H, d, J ¼ 8.2 Hz, NH), 9.43 (1H, s,
NH), 8.99 (1H, s, NH), 8.78 (1H, s, H50), 8.30 (2H, d, J ¼ 8.6 Hz, Ph),
8.04 (1H, s, H4), 7.87–7.57 (6H, m, H7; H6; Ph), 7.37 (2H, d,
J ¼ 8.4 Hz, Ph), 5.40 (2H, s, OCH2), 4.13–4.00 (1H, m, CH), 1.31 (6H,
d, J ¼ 6.1 Hz, CH3CHCH3). 13 C NMR (151 MHz, DMSO) d 162.41,
160.38, 153.72, 142.46, 134.42, 131.84, 130.61, 128.93, 128.57,
128.55, 128.47, 127.09, 123.10, 127.09, 122.58, 120.24, 116.13,
115.53, 61.14, 45.06, 21.31. MS (ESI, m/z) 486.1 [M þ H]þ. Anal.
calcd. for C26H24ClN7O ꢁ 2 HCl ꢁ2.3 H2O (Mr ¼ 600.33): C 52.02, H
5.14, N 16.33; found: C 52.22, H 5.03, N 16.59%.
2–(4-((1–(2-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-
1H-benz[d]imidazole-5-carboximidamide dihydrochloride (7c)
Compound 7c was prepared using the above described method from
3c (200 mg, 0.64 mmol) and 4 (96.51 mg, 0.64 mmol) to obtain white
powder 7c (210.3 mg, 58%, m.p. ¼ 176–177 ꢀC). 1H NMR (300 MHz,
DMSO) d 9.36 (2H, s, NH), 8.96 (2H, s, NH), 8.77 (1H, s, 1H, H50), 8.28
(2H, d, J ¼ 8.8 Hz, Ph), 8.16 (1H, s, H4), 7.88–7.55 (6H, m, Ph; H5; H6),
7.36 (2H, d, J ¼ 8.9 Hz, Ph), 5.40 (2H, s, OCH2). 13 C NMR (151 MHz,
DMSO) d 165.62, 161.33, 152.72, 142.25, 134.41, 131.84, 131.26,
130.60, 129.84, 128.51, 127.23, 123.76, 123.22, 122.24, 115.90, 115.70,
114.82, 61.23. MS (ESI, m/z) 444.0 [M þ H]þ. Anal. calcd. for
C23H18ClN7O ꢁ 2 HCl ꢁ3 H2O (Mr ¼ 570.86): C 48.39, H 4.59, N 17.17;
found: C 48.11, H 4.47, N 17.38%.
N-isopropyl-2–(4-((1-(phenylthiomethyl)-1H-1,2,3-triazol-4-yl)
methoxy)phenyl)-1H-benz[d]imidazole-5-carboximidamide dihydro-
chloride (8e)
Compound 8e was prepared using the above described method
from 3e (200 mg, 0.61 mmol) and 5 (108.9 mg, 0.61 mmol) to
1
obtain 8e as yellow powder (73.1 mg, 21%, m.p. ¼ 152–154 ꢀC). H
NMR (600 MHz, DMSO) d 9.66 (1H, d, J ¼ 7.5 Hz, NH), 9.51 (1H, s,
NH), 9.09 (1H, s, NH), 8.39 (2H, d, J ¼ 7.9 Hz, Ph), 8.25 (1H, s, H50),
8.08 (1H, s, H4), 7.87 (1H, d, J ¼ 8.3 Hz, H7), 7.68 (1H, d, J ¼ 8.5 Hz,
H6), 7.40 (2H, d, J ¼ 7.5 Hz, Ph), 7.36–7.15 (5H, m, Ph), 5.98 (2H, s,
CH2), 5.29 (2H, s, CH2), 4.24–3.99 (1H, m, CH), 1.31 (6H, d,
J ¼ 6.4 Hz, CH3CHCH3).13C NMR (75 MHz, DMSO) d 162.93, 160.52,
2–(4-((1-((Phenylthio)methyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)- 154.42, 145.37, 143.19, 132.82, 130.98, 129.76, 129.09, 128.18,
124.99, 123.19, 122.71, 122.05, 115.86, 61.55, 52.18, 45.44, 21.75.
MS (ESI, m/z) 498.1 [M þ H]þ. Anal. calcd. for C27H27N7OS ꢁ2 HCl
ꢁ0.3 H2O (Mr ¼ 575.95): C 56.31, H 5.18, N 17.02; found: C 56.33, H
5.37, N 17.28%.
1H-benz[d]imidazole-5-carboximidamide dihydrochloride (7e)
Compound 7e was prepared using the above described method
from 3e (200 mg, 0.64 mmol) and 4 (96.51 mg, 0.64 mmol) to
1
obtain 7e as white powder (324.8 mg, 90%, m.p. ¼ 173–176 ꢀC). H
NMR (300 MHz, DMSO) d 9.46 (2H, s, NH), 9.08 (2H, s, NH), 8.34
(2H, d, J ¼ 8.9 Hz, Ph), 8.25 (1H, s, H50), 8.20 (1H, d, J ¼ 1.1 Hz, H4),
7.89 (2H, t, J ¼ 7.8 Hz, Ph), 7.80 (1H, dd, J ¼ 8.6 Hz, H6), 7.45–7.29
(6H, m, H7; Ph), 5.99 (2H, s, CH2), 5.30 (2H, s, CH2).13C NMR
(75 MHz, DMSO) d 165.70, 161.12, 153.11, 142.62, 139.17, 132.38,
131.84, 130.59, 129.59, 129.38, 127.82, 124.66, 123.55, 122.98,
115.72, 115.30, 61.27, 51.80. MS (ESI, m/z) 456.1 [M þ H]þ. Anal.
5–(4,5-Dihydro-1H-imidazol-2-yl)-2–(4-((1–(4-methoxyphenyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benz[d]imidazole dihydro-
chloride (9b)
Compound 9b was prepared using the above described method
from 3b (200 mg, 0.65 mmol) and 6 (114.9 mg, 0.65 mmol) to
calcd. for C24H21N7OS ꢁ2 HCl ꢁ1.7 H2O (Mr ¼ 559.09): C 51.56, H obtain 9b as yellow powder (279.1 mg, 70%, m.p. ¼ 197–199 ꢀC).
4.76, N 17.54; found: C 51.80, H 4.68, N 17.22%.
1H NMR (300 MHz, DMSO) d 10.68 (2H, s, NH), 8.91 (1H, s, H50),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1327
Table 1. Hypochromic effects (H/%)a, binding constants (log Ks)b and ratios nc
([compound]/[polynucleotide phosphate]) calculated from the UV–Vis titrations
of compounds with ds-DNA/RNA (PBS, I ¼ 0.015 M, and pH ¼ 7).
8.37 (1H, s, H4), 8.32 (2H, d, J ¼ 8.3 Hz, Ph), 7.88 (1H, d, J ¼ 4.3 Hz,
H7), 7.82 (2H, d, J ¼ 8.5 Hz, Ph), 7.35 (2H, d, J ¼ 8.1 Hz, Ph), 7.28
(1H, d, J ¼ 8.3 Hz, H6), 7.15 (2H, d, J ¼ 8.1 Hz, Ph), 5.37 (2H, s,
OCH2), 4.03 (4H, s, CH2CH2), 3.83 (3H, s, OCH3).13C NMR (151 MHz,
DMSO) d 165.49, 160.68, 159.51, 154.27, 143.30, 143.09, 136.43,
131.97 (C4), 130.01 (Ph-q), 129.27 (Ph), 123.21 (C6), 123.00 (C50),
122.04 (Ph), 120.62 (C5), 116.05, 115.63, 115.37, 115.06, 61.38,
55.72, 44.44. MS (ESI, m/z) 466.1 [M þ H]þ. Anal. calcd. for
C26H23N7O2 ꢁ 2 HCl ꢁ0.9 H2O (Mr ¼ 554.65): C 56.30, H 4.87, N
17.68; found: C 56.04, H 4.72, N 17.97%.
ctDNA
polyA-polyU
H/%c log Ks
5.24 0.31
polyC-polyG
Compound H/%c log Ks
n
H/%c log Ks
n
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
7.8
22.1e
36.3
40.4
38.2
41.0
29.7
33.8
41.2
44.2
25.7e
18.9e
11.7
24.6
41.1
6.76 0.27 19.3
–
5.26
13.7
6.44 0.40d
6.27 0.73
–
27.9e
–
–
6.24 0.58 38.9
5.78 0.59 32.5
5.33 0.61 31.7
6.17 0.64 46.5
6.06 0.62f 57.8
5.64 0.69 50.4
5.93 0.47 53.8
6.39 0.36f 47.2
5.21 0.18
5.94 0.11
6.20 0.29
5.87 0.24
5.27 0.23
6.00 0.19
6.13 0.30
5.96 0.30
4.82e
4.96
5.79
5.86
5.05
5.31
6.13
5.96
–
–
–
6.56 0.36
5.89 0.69
6.17 0.4d
6.39 0.39
6.43 0.36
5.54 0.40d
7.20 0.28
6.21 0.40d
5–(4,5-Dihydro-1H-imidazol-2-yl)-2–(4-((1–(2-chlorophenyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benz[d]imidazole dihydro-
chloride (9c)
–
–
–
–
32.7e
–
–
31.6e 5.46 0.30d 5.46e
5.38 0.29 5.34
5.84 0.75 55.5e 5.89 0.30d 5.89
6.21 0.12 31.6 5.36 0.26 5.21
–
–
6.43 0.35 29.3
6.43 0.36
6.59 0.41
4.29 0.4d
Compound 9c was prepared using the above described method
from 3c (200 mg, 0.64 mmol) and 6 (112.9 mg, 0.64 mmol) to
obtain 9c as red powder (115.3 mg, 30%, m.p. ¼ 194–196 ꢀC). 1H
NMR (600 MHz, DMSO) d 10.82 (2H, s, NH), 8.81 (1H, s, H50), 8.45
(1H, s, H4), 8.40 (2H, d, J ¼ 8.6 Hz, Ph), 7.98 (1H, d, J ¼ 8.5 Hz, H7),
7.93 (1H, d, J ¼ 8.3 Hz, H6), 7.81 (1H, dd, J ¼ 8.0, 1.2 Hz, Ph), 7.75
(1H, dd, J ¼ 7.8, 1.6 Hz, Ph), 7.67 (1H, td, J ¼ 7.8, 1.6 Hz, Ph), 7.62
(1H, td, J ¼ 7.7, 1.3 Hz, Ph), 7.40 (2H, d, J ¼ 8.9 Hz, Ph), 5.43 (2H, s,
OCH2), 4.04 (4H, s, CH2CH2).13C NMR (75 MHz, DMSO) 165.20,
161.07, 153.69, 142.40, 138.84, 136.92, 134.44, 131.90, 130.66,
130.15, 129.62, 128.63, 128.52, 127.21, 123.57, 119.46, 116.66,
116.38, 115.69, 61.24, 44.42. MS (ESI, m/z) 470.1 [M þ H]þ. Anal.
calcd. for C25H20ClN7O ꢁ 2 HCl ꢁ1.9 H2O (Mr ¼ 577.08): C 52.03, H
4.51, N 16.99; found: C 52.31, H 4.66, N 16.63%.
aHypochromic effect calculated by Scatchard equation for compounds;
H ¼ (Abs(compound) –Abs(complex))/Abs(compound) ꢁ 100.
bTitration data were processed according to the Scatchard Equation63,64
.
cAccuracy of n 10–30%, consequently logKs values vary in the same order
of magnitude.
dn ¼ fix.
eHypochromic effect calculated from experimental data.
fMixed binding mode and binding constants were calculated in range r ꢃ 0.1.
ꢂ: changes were too small for accurate calculation of binding constants.
increasing ratios was then titrated into the compound buffer solu-
tion (1.48 ꢁ 10ꢂ5 mol dmꢂ3) and the corresponding absorption
spectra were recorded under the same conditions. All data were
graphed and analysed using Origin software version 9.0 (OriginLab
Corporation, Northampton, MA). The stability constants (Ks) and
[bound compound]/[polynucleotide phosphate] ratios (n) were cal-
culated according to the Scatchard Equation63,64. Values for Ks
and n given in Table 1 all have satisfying correlation coeffi-
cients (0.99).
5–(4,5-Dihydro-1H-imidazol-2-yl)-2–(4-((1-(phenylthiomethyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benz[d]imidazole dihydro-
chloride (9e)
Compound 9e was prepared using the above described method
from 3e (200 mg, 0.61 mmol) and 6 (108.9 mg, 0.61 mmol) to
obtain 9e as brown powder (149.4 mg, 41%, m.p. ¼ 162–164 ꢀC).
1H NMR (300 MHz, DMSO) d 10.57 (2H, s, NH), 8.34–8.18 (4H, m,
H50; H4; Ph), 7.85 (2H, s, H7; H6), 7.44–7.21 (7H, m, Ph), 5.97 (2H, s,
CH2), 5.26 (2H, s, CH2), 4.03 (4H, s, CH2CH2).13C NMR (151 MHz,
DMSO) d 165.13, 162.84, 160.80, 153.75, 142.60, 132.38, 131.79,
Thermal melting (Tm)
Tm experiments were conducted with a Perkin Elmer Lambda 25
spectrophotometer in 1 cm quartz cuvettes. The absorbance of the
130.48, 129.86, 129.40, 129.32, 127.72, 124.63, 116.37, 115.52, DNA/RNA-compound complex was monitored at 260 nm as a func-
115.23, 61.17, 51.69, 44.33. MS (ESI, m/z) 482.0 [M þ H]þ. Anal.
tion of temperature. The absorbance of the ligands was subtracted
from every curve, and the absorbance scale was normalised. The
calcd. for C26H23N7OS ꢁ2 HCl ꢁ2.2 H2O (Mr ¼ 594.14): C 52.56, H
4.99, N 16.50; found: C 52.64, H 5.12, N 16.29%.
DTm values were calculated by subtracting Tm of the free nucleic
acid from Tm of the complex. Every reported DTm value was the
average of at least two measurements. The error of DTm is 0.5 ꢀC.
All data were graphed and analysed using Origin software ver-
sion 9.0.
Spectroscopic experiments
Polynucleotides
Polynucleotides were purchased as follows: polyG–polyC,
polyA–polyU (Sigma-Aldrich, St. Louis, MO), calf thymus ctDNA
(Aldrich). Polynucleotides were dissolved in PBS buffer,
I ¼ 0.05 mol dmꢂ3, pH 7.0. The calf thymus ctDNA was additionally
sonicated and filtered through a 0.45 mm filter. The polynucleotide
concentration was spectroscopically determined as the concentra-
tion of nucleobases62.
Circular dichroism (CD)
The CD spectra of DNA/RNA (concentration in cuvette 2 ꢁ 10ꢂ5 M)
were recorded with a JASCO J-800 spectrometer (JASCO UK Ltd.,
Dunmow, United Kingdom) at different ratios r ¼ 0.1, 0.3, 0.5, and
0.7 (r ¼ [compound]/[polynucleotide]) at 25 ꢀC in aqueous buffer
solution (pH ¼ 7, PBS, and I ¼ 0.05 mol dmꢂ3). Titrations were car-
ried out by addition of aliquots of 1 mM stock solutions of the
relevant compound (at increasing ratios) to the buffered poly-
nucleotide (DNA/RNA) solution in a 1 cm quartz cuvette and
scanned over a wavelength range 220–450 nm. All data were
UV–Vis spectroscopy
All UV–Vis absorbance measurements were conducted on a Perkin
Elmer Lambda 25 spectrophotometer (Perkin Elmer, Waltham, MA).
A quartz cell with a 1 cm path length was used for all absorbance
studies. Compound stock solutions were 1 mM. The DNA/RNA at graphed and analysed using Origin software version 9.0.

ꢀ
A. BISTROVIC ET AL.
1328
Scheme 1. Synthesis of 2,5-disubstituted benzimidazoles. (i): propargyl bromide, K2CO3, EtOH, reflux; (ii): corresponding azides, CuSO4, Cu(0), DMF, t-BuOH: H2O ¼ 1: 1,
80 ꢀC; (iii): o-phenylenediamine (4–6), NaHSO3, EtOH, reflux; HCl/MeOH, room temperature.
Biological evaluations
Results and discussion
Chemistry
Anti-bacterial screening
The compounds were evaluated for their in vitro anti-bacterial
activity against Gram-positive bacteria: S. aureus (ATCC 25923),
MRSA, methicillin-sensitive S. aureus (MSSA), E. faecalis, vanco-
mycin-resistant E. faecium (VREF), and Gram-negative bacteria: E.
coli (ATCC 25925), P. aeruginosa (ATCC 27853), A. baumannii (ATCC
19606) and ESBL-producing K. pneumoniae (ATCC 27736). Standard
broth microdilution method as recommended in guidelines of
Clinical and Laboratory Standards Institute61,65,66 was applied and
the minimum inhibitory concentration (MIC) of compounds was
tested. In short, testing was performed in U-bottomed 96-well ster-
ile plastic microdilution trays (Falcon 3077, Becton Dickinson
Labware, Franklin Lakes, NJ) in cation (Ca2þ and Mg2þ) adjusted
Mueller–Hinton broth medium (Becton Dickinson and Co.,
Cockeysville, MD). All testings were performed in triplicate.
1,2,3-Triazole-linked 5-amidinobenzimidazoles 7a–7e, 8a–8e,
and 9a–9e are synthesised as outlined in Scheme 1. 4-
Hydroxybenzaldehyde was propargylated to give 4-(prop-2-ynylox-
y)benzaldehyde (2), which subsequently via the regioselective
Cu(I) catalysed cycloaddition with aromatic azides resulted in
4–(1,2,3-triazol-1-yl)benzaldehyde derivatives (3a–3e) comprising
an N-1-aryl-substituted 1,2,3-triazole subunit. An efficient and
environmentally benign synthetic protocol68, applying microwave
and ultrasound irradiation, was used in the synthesis of 3a–3e.
The efficiency of both ultrasound and microwave conditions were
compared and indicated that ultrasound-assisted syntheses of
3a–3e resulted in higher yields than those of microwave-assisted
reactions. Amidino-substituted 1,2-phenylenediamines (4–6) that
were used for the synthesis of the target 5-amidinobenzimidazoles
7a–7e, 8a–8e, and 9a–9e were synthesised from the correspond-
ing nitrile by the Pinner method58. 4-Amidino 1,2-phenylenedi-
amines (4–6) reacted with the bisulfite adduct of the 4–(1,2,3-
triazol-1-yl)benzaldehyde derivatives (3aꢂe) to produce amidine
(7a–7e), N-isopropylamidine (8a–8e), and imidazoline-substituted
(9a–9e) benzimidazole derivatives69.
Anti-trypanosomal screening and cytotoxicity assays
Bloodstream form T. brucei (strain 221) were cultured in modified
Iscove’s medium, as outlined67 and trypanocidal assays were per-
formed using 96-well microtitre plates. The compound concentra-
tions that inhibited growth by 50% (IC50) and 90% (IC90) were
determined. Parasites were sub-cultured at 2.5 ꢁ 104 mlꢂ1, com-
pounds were added at range of concentrations, and the plates
incubated at 37 ꢀC. Resazurin was added after 48 h, the plates
incubated for a further 16 h, and then read in a Spectramax plate
reader (Molecular Devices Corporation, San Jose, CA). The data
were analysed using GraphPad Prism (GraphPad, La Jolla, CA).
Each drug concentration was tested in triplicate.
Cytotoxicity against mammalian cells was also assessed using
microtitre plates. Briefly, L6 cells (a rat myoblast line) were seeded
at 1 ꢁ 104 mlꢂ1 in 200 ml of growth medium containing different
compound concentrations. The plates were then incubated for 6 d
at 37 ꢀC and 20 ml resazurin added to each well. After a further 8 h
incubation, the fluorescence was determined using a Spectramax
plate reader, as outlined above.
Spectroscopic characterisation of compounds
5-Amidinobenzimidazole derivatives 7a–7e, 8a–8e, and 9a–9e
were synthesised and characterised by UV–Vis spectroscopy.
UV–Vis spectra displayed two absorption maxima at around 260
and 315 nm (Table S1, Supporting Information). Absorbancies of
solutions were proportional to their concentrations up to 1 ꢁ 10ꢂ4
moldmꢂ3, indicating that there is no significant intermolecular
stacking that could give rise to hypochromic effects. Furthermore,
the UV–Vis spectra of 7a–7e, 8a–8e, and 9a–9e revealed negli-
gible temperature dependent changes (25–90 ꢀC) and excellent
reproducibility upon cooling to 25 ꢀC. The results showed that all
evaluated compounds were stable and suitable for further spectro-
scopic and biological investigations.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1329
Table 2. DTm values (ꢀC) of studied ds-polynucleotides upon addition of com-
pounds 7a, 7c–7e, 8a–8e, and 9b–9e at different ratio rb (PBS and pH ¼ 7)a.
Interactions with double-stranded polynucleotides
Spectrophotometric titrations of compounds with ds-DNA/RNA
UV–Vis absorption spectroscopy is simple, widely used and one of
the most effective methods for detecting the interaction of small
molecules with DNA. In general, these interactions and the subse-
quent formation of a new complex leads to changes in UV–Vis
spectra70. Therefore, UV–Vis spectroscopy was applied to investi-
gate the interaction of compounds 7a–7e, 8a–8e, and 9a–9e with
ds-DNA/RNA. UV–Vis titration with ctDNA showed a hypochromic
effect indicating the disappearance of free molecule and the for-
mation of a new compound-DNA species (Figure S1, Supporting
Information). The hypochromic effect (12–44%) was accompanied
by a small bathochromic shift (Dk ¼ 3–9 nm) that was found to ori-
ginate from the stabilisation of DNA secondary structure due to
the interaction with small molecules71.
To assess the sequence selectivity of the compounds, the
experiment was repeated with ds-RNA polynucleotides (polyA-
polyU and polyC-polyG). The addition of polyA-polyU in most cases
led to hypochromic (19–58%) and small bathochromic (2–11 nm)
changes in the visible absorption spectra as a result of complex for-
mation. Absorption spectra obtained by adding aliquots of polyC-
polyG to the compound solutions were recorded until saturation
was achieved. In general, it was observed that addition of polyC-
polyG resulted in a pronounced decrease of UV–Vis absorption
maxima at 300–320 nm (27–50%), followed by small bathochromic
shifts (Dk ¼ 2–7 nm). No further studies were conducted with com-
pounds whose UV–Vis spectra showed minimal changes (DA < 0.08
at r ¼ 1–0.1) during titration with DNA/RNA polynucleotides. It can
be inferred that these compounds interact with polynucleotides
only through a very weak electrostatic and external mode (Table
1). During titration with polyA-polyU, a clear isosbestic point was
observed in UV–Vis spectra of 7a and 9c, pointing to the formation
of one dominant type of complex.
ctDNA
polyA-polyU
Compound
0.3
0.5
0.7
0.1
0.3
0.5
7a
7c
7d
2.46
2.35
3.12
4.84
3.20
3.86
4.69
3.77
3.62
1.74
0.20
2.06
1.74
2.72
1.14
2.53
–
d
1.70
9.05c
1.11
7e
2.19
2.62
2.66
0.39
1.11
12.58c
0.64
15.04c
1.11
8a
8b
3.35
4.03
3.94
3.96
4.25
4.47
0.32
1.17
1.32
1.56
6.38c
0.58
8.49c
1.12
8c
8d
8e
9b
3.40
1.76
2.22
–
3.89
3.32
1.72
–
4.39
3.63
1.71
–
0.76
0.91
0.52
2.49
0.51
0.23
0.72
0.26
2.67
2.92
36.08c
4.27
40.93c
5.53
9c
9d
9e
3.55
3.31
2.87
3.31
3.52
3.47
3.80
2.60
3.60
2.85
1.26
1.11
13.99c
1.42
17.73c
2.06
10.30c
0.47
13.29c
0.47
12.46c
13.89c
aAll values are averaged from at least two measurements. Error in DTm: 0.5 ꢀC.
br ¼ [compound]/[polynucleotide].
cBiphasic melting curve, values for both melting midpoints given when possible.
dNot possible to determine due to the lack of melting midpoint.
Results showed that compounds 8a–8e stabilised ctDNA slightly
better than compounds 7a–7e and 9a–9e. Biphasic curves for
interactions of compounds 7e, 8b, and 9b–9e with polyA-polyU at
higher ratios r indicated additional binding modes. The above-
mentioned compounds have monophasic curves at r ꢄ 0.3, which
together with results on UV–Vis titration confirmed intercalation as
the dominant binding mode, while above that, ratio biphasic
curves indicated agglomeration of compounds along the poly-
nucleotide chains.
The binding constants logKs and the density of the binding
sites n were calculated using Scatchard plot analysis. In addition,
the binding constants Ks for compounds 8b and 8e were calcu-
lated only for titration data taken at the r ꢃ 0.1, because below
that ratio changes in absorption maxima were too small for accur-
ate calculation (DA ꢄ 0.04) (Table 1). The binding constants Ks and
ratios n obtained by processing UV–Vis titration data using the
Scatchard equation are summarised in Table 1.
Circular dichroism (CD) experiments
CD spectroscopy has been extensively employed for the investiga-
tion of small molecule–polynucleotide (DNA/RNA) interactions75,76
.
Binding of achiral small molecules within the chiral DNA/RNA helix
results in an induced CD spectrum (ICD)77,78. The appearance of
ICD bands upon titration (r ¼ 0.1–0.7) at k > 300 nm was used to
estimate the orientation of the chromophore in the ctDNA/RNA
binding site and for the determination of binding mode. ctDNA
features approximately 40% GC and 60% AT base pairs and adopts
a B-helix with a narrow, deep, well-accessible minor groove and a
rather broad, and shallow major groove. The two RNA polymers,
polyA-polyU and polyG-polyC, form a typical A-helix with a broad
minor and narrow major groove. The main difference among ds-
RNAs is the presence of the amino group at N-2 in guanine which
protrudes into the grooves and thereby may influence the affinity
and binding mode of compounds being studied. The addition of
the compounds being investigated resulted in a decrease of the
ds-DNA/RNA CD bands (k ¼ 220–400 nm, S2). Observed changes in
the intensity of CD bands for ds-DNA/RNA indicated the partial
disruption of the polynucleotide helical chirality upon binding of a
small molecule. The addition of the compounds 7a, 7c–7e, 8a–8e,
and 9c–9d in solution with ctDNA generated strong, positive ICD
signals in the range of 300–350 nm (Figures 4 and S2, Supporting
Information). This may arise due to groove binding being the
Thermal denaturation experiments
Thermal melting enables the rapid qualitative evaluation of the
relative binding affinities of the compounds towards selected poly-
nucleotides (Table 2)72,73. The melting temperature (Tm) is defined
as the differences between the melting temperatures of free poly-
nucleotides and their complexes with small non-covalently bound
molecules. The correlation between binding constant and the
increase of Tm was found to be quite complex, because the num-
ber of binding sites, positive charge of compounds, potential
cooperativity, and the affinity for the unfolded polynucleotide
have also to be taken into account74.
Denaturation experiments were carried out at different
amounts of the compounds (r ¼ 0.1, 0.3, 0.5, and 0.7 eq;
r ¼ [compound]/[polynucleotide]) with ctDNA and polyA-polyU.
The results of the denaturation experiments are listed in Table 2.
Generally, results correlated with those of UV–Vis experiments.
Strong non-linear dependence of DTm values on the ratio r was
revealed, suggesting saturation of binding sites at r ¼ 0.5–0.7 (for
7c–7d, 8a–8e, and 9d), r ¼ 0.3–0.5 (for 7a, 9c, and 9e), in good
dominant binding mode for this class of compounds79,80
ICD spectra of polyA-polyU and polyC-polyG with the addition
.
accordance with the calculated values presented in Table 1. of evaluated compounds, except for 7a, 8a–8e, and 9d, showed a

ꢀ
A. BISTROVIC ET AL.
1330
Figure 4. Induced CD spectra of compound 7a (a) and compound 8c (b) with ctDNA (r ¼ 0–0.7).
Figure 5. ICD spectra of RNA polynucleotides with 5-amidinobenzimidazoles with p-methoxyphenyl-1,2,3-triazole unit: compound 7b (a), compound 8b (b), and com-
pound 9b (c).
Table 4. Anti-bacterial activity of selected compounds against antibiotic-resistant
Gram-negative clinical strains.
Table 3. Anti-bacterial activity of selected compounds against antibiotic-resistant
Gram-positive clinical strains.
MIC (mg/ml)
MIC (mg/ml)
E. coli
ESBL
K. pneumoniae
P. aeruginosa
S. aureus
MRSA
S. aureus
MSSA
E. faecium
VRE
Compound
ESBL
ESBL
Compound
7a
7d
7e
9d
4
16
32
128
64
8
16
16
–
128
32
32
128
64
32
8
7a
7b
7c
7d
7e
8a
8b
8c
16
–
16
16
32
8
32
–
32
32
256
256
32
32
9e
–
128
128
64
64
Ceftazidime
Ciprofloxacin
8
>128
1
128
128
32
>128
16
8
16
8d
8e
32
8
64
64
64
64
by UV–Vis and thermal melting methods. Minimal changes of the
intensity of the CD bands of polyC-polyG upon titration with com-
pounds 7a, 8a–8e, and 9d, suggest a non-specific binding mode.
Most probably compounds bind on the outside of the polyC-
polyG polynucleotide. The intensity of negative ICD bands in
polyA-polyU ICD spectra was also observed to be more intense
than those in polyC-polyG spectra obtained with the
same compound.
9a
9b
9c
9d
128
256
8
64
64
4
128
256
128
64
64
1
128
256
64
64
32
9e
Ampicillin
Gentamicin
1
0.25
0.25
0.125
decrease of CD band in the range of 220–300 nm, followed by
appearance of new negative signal at >300 nm (Figures 5 and S2,
Supporting Information). This indicates that intercalation is the
dominant binding mode. Compounds 7b–9b showed higher affin-
ity for dsRNA than ctDNA. While 8b and 9b bound to polyA-
polyU, 7b showed higher affinity for polyC-polyG (Figures 5 and
S2, Supporting Information).
Biological evaluations
In vitro anti-bacterial activity
The in vitro anti-bacterial activity of 5-amidinobenzimidazoles
7a–7e, 8a–8e, and 9a–9e was tested against Gram-positive bac-
teria including S. aureus (ATCC 25923), Enterococcus faecalis (ATCC
ICD spectra of 8b and 9b, in the presence of polyA-polyU,
showed an intense increase of signal above r ¼ 0.3, while ICD 29212), and Gram-negative bacteria including E. coli (ATCC 25925),
spectra of 7b with polyC-polyG showed a weaker intercalation sig- K. pneumoniae (ATCC 700803), P. aeruginosa (ATCC 27853), and
nal above r ¼ 0.5. This is in agreement with the results obtained Acinetobacter baumannii (ATCC 19606). The MICs were determined

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
Table 5. Anti-trypanosomal activitya of compounds 7a–7e, 8a–8e, and 9a–9e against Trypanosoma brucei strain.
1331
N
N
N
R2
N
O
R1
N
H
T. brucei
L6 cells
S.I.c
Compd
7a
R1
R2
IC50 (μM) IC90 (μM) IC50 (μM) L6/Tb IC50
NH2
8.6 0.5 16.2 0.9
-
-
NH2
NH2
OCH3
7b
1.5 0.3 8.1 0.6 302 + 15
200
NH2
NH2
Cl
7c
>15
-
-
-
-
-
-
-
NH2
NH2
7d
12.9 0.2 18.2 1.0
10.2 1.3 15.9 0.4
NH2
NH2
S
7e
NH2
NH2
HN
NH2
HN
NH2
HN
NH2
HN
NH2
HN
8a
8b
8c
8d
8e
9a
9b
9c
9d
7.1 0.7 16.7 1.2
1.1 0.3 3.5 0.2
-
-
OCH3
>300
>270
Cl
>15
-
-
-
-
-
-
12.9 0.2 21.6 0.4
13.5 0.8 22.5 0.3
7.9 0.4 17.2 1.2
-
S
-
H
N
-
N
H
H
N
OCH3
1.6 0.4 7.3 0.3 263 + 15
165
N
H
H
N
Cl
>15
-
-
-
-
-
-
-
N
H
H
N
10.8 0.4
>25
N
H
H
N
S
9e
9.7 0.2 14.0 0.3
N
H
Nifurtimox
-
-
4.4 0.7b
-
aIn vitro activity against bloodstream form T. brucei expressed as the concentration that inhibited growth by 50%
(IC50) and 90% (IC90). Data are the mean of triplicate experiments SEM.
bTaken from Ref. [83].
cSelectivity index.
and compared with those of the antibiotics ceftazidime, ciprofloxa- active against the MSSA strain, although to a lesser extent
cin, ampicillin, and gentamicin (Table S2, Supporting Information).
Generally, compounds showed better activities against
(MIC ¼ 16–128 mg/ml). 8c also displayed modest activity against
VRE-E. faecium. Among other compounds, benzimidazole imidazo-
Gram-positive than Gram-negative bacteria. Only 5-amidinobenzi- line 9c had promising activity against the MRSA strain (MIC ¼ 8 mg/
midazoles 7a, 7d, and 7e proved to be active against three ml). Against the antibiotic-resistant Gram-negative bacteria (Table
Gram-negative strains, particularly amidinobenzimidazole 7d, 4), 5-amidinobenzimidazole 7a, with the N-1-phenyl-1,2,3-triazole,
which has an N-1-benzyl substituent. The type of amidino moiety proved to be the most potent, with IC50 values of 4 mg/ml for E.
in 5-benzimidazole had impact on the anti-bacterial activities, with coli, and 8 mg/ml for K. pneumoniae. However, this compound was
non-substituted amidinobenzimidazoles 7a–7e having the highest only marginally effective against P. aeruginosa. Compounds 7d
overall activities (Table S2, Supporting Information). Compounds and 7e prove to be active against K. pneumoniae (MIC ¼ 16 mg/ml).
that exhibited anti-bacterial activities with MIC <256 mg/ml were Introduction of a methylene (7d and 9d) and sulphide-bridge (7e
evaluated against antibiotic resistant Gram-positive clinical strains, and 9e) between 1,2,3-triazole and the phenyl ring reduced the
such as MRSA, MSSA and VREF (Table 3) and Gram-negative activity against the antibiotic resistant E. coli and K. pneumoniae
clinical strains including extended-spectrum b-lactamase (ESBL)- clinical strains. 7d, 7e, and 9e had slightly greater potency against
P. aeruginosa compared with 7a. Overall, the results indicated that
producing E. coli, K. pneumoniae, and P. aeruginosa (Table 4).
The evaluated compounds had a wide range of activity against the o-chlorophenyl hydrophobic unit, with N-isopropylamidine, as
MRSA, with the 5-N-isopropylamidinobenzimidazoles 8a–8e being the hydrophilic unit, in 8c contributed to anti-bacterial activity,
the most active (MIC ¼ 8–32 mg/mL) (Table 3). 8a–8e were also particularly against the MRSA strain. Importantly, 7a was the most

ꢀ
A. BISTROVIC ET AL.
1332
potent of the compounds against ESBL-producing E. coli, with activity against resistant Gram-positive bacteria, particularly the
higher activity than the reference antibiotics ceftazidime and MRSA strain. On the other hand, non-substituted amidine and
ciprofloxacin.
phenyl rings in 7a contributed to a strong inhibitory effect on an
ESBL-producing E. coli strain, with the potency better than those
of the reference antibiotics ceftazidime and ciprofloxacin
Compounds 7 b, 9a, and 9 b that showed extremely low affinity to
ctDNA had also negligible anti-microbial activity (MIC ꢃ128 mg/ml).
Contrary to this, the 5-N-isopropylamidinobenzimidazole series
8a–8e, which had better binding affinity relative to other ami-
dines, showed some selective activity (MIC ¼ 8–32 mg/ml) against
the MRSA strain. Notably, compound 7a emerged as the most
promising candidate because of its higher potency (MIC ¼ 4 mg/ml)
against ESBL-producing E. coli. It had the highest affinity among
the tested compounds to ctDNA (Tables 1 and 2).
Results of anti-trypanosomal evaluations showed that the o-
chlorophenyl group in 7c–9c had a negative impact on activity,
whereas the p-methoxyphenyl substituent in 7b–9b enhanced
activity, with 8b (IC50 ¼ 1.1 mM and IC90 ¼ 3.5 mM) being more
potent than nifurtimox. In contrast to the observed correlation
between anti-microbial activity and DNA binding, the antiproto-
zoal effects of 8b did not correlate with its DNA affinity. Further
investigations will, therefore, be required to clarify the mechanism
of anti-protozoal activity.
One of our aims was to determine if there was a relationship
between the affinity of compounds towards ds-DNA/RNA and their
antimicrobial activity. UV–Vis and CD spectroscopy, as well as ther-
mal denaturation assays, showed that compounds 7b, 9a, and 9b,
which did not bind to ctDNA, had only marginal anti-microbial
activities (MIC ꢃ128 mg/ml). Conversely, 5-amidinobenzimidazole
7a, which showed the highest affinity to ctDNA, exhibited high
potency against ESBL-producing E. coli, which is in agreement
with previous findings56,81,82
.
Screening of the anti-trypanosomal activity
Results on the in vitro testing against the bloodstream form T. bru-
cei of the 5-amidinobezimidazoles 7a–7e, 8a–8e, and 9a–9e with
1,4-disubstituted 1,2,3-triazole, and nifurtimox as reference drug,
are summarised in Table 5. Similarly to anti-bacterial evaluations,
we investigated how cationic moieties and aromatic substituents
attached to the N-1 of the 1,2,3-triazole ring directly, or through
the methylene and methylenesulphide spacer, influenced, anti-try-
panosomal potencies.
Phenyl, p-methoxyphenyl, o-chlorophenyl, benzyl and (phenyl-
thio)methyl substituents had a significant negative impact on IC50
values of anti-trypanosomal activity in the following order: p-
OCH3 > Ph > PhSCH2 ꢅ Bn > o-Cl. Except for 7c–9c, all compounds
were active against T. brucei with IC50 values ranging from 1.1 to
13.5 mM. Interestingly, the o-chlorophenyl substituent in 7c–9c
caused the loss of anti-trypanosomal activity (IC50 > 15 mM). The
presence of the p-methoxyphenyl substituent in 7b–9b led to
enhanced anti-trypanosomal potency, with the 5-N-isopropylamidi-
nobenzimidazole analogue 8b being the most promising com-
pound (IC50 ¼ 1.1 mM, IC90 ¼ 3.5 mM), which is 4-fold more potent
than nifurtimox. UV–Vis titrations and thermal denaturation assays
suggested that 7b–9b have low affinity to ctDNA (Table 1) indicat-
ing that DNA is not the primary target for their anti-trypanosomal
activity. Cytotoxicity assays against the rat myoblast cell line L6,
revealed negligible activity, with three-figure selectivity index
(Table 5).
The promising anti-bacterial activity of compounds 7a and 8c
and the anti-trypanosomal potency of compound 8b suggest that
further structural optimisation of the 1,2,3-triazole-linked 5-amidi-
nobenzimidazole class could enhance the potential anti-HAT and
anti-bacterial activity against resistant pathogenic microorganisms.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
We greatly appreciate the financial support of the Croatian
Science Foundation (project No. IP-2013–11-5596) and University
of Osijek, Faculty of Medicine [research grant VIF2016-MEFOS-27].
References
Conclusions
1. Akhtar W, Khan MF, Verma G, et al. Therapeutic evolution of
benzimidazole derivatives in the last quinquennial period.
Eur J Med Chem 2017;126:705–53.
2. Akhtar J, Khan AA, Ali Z, et al. Structure-activity relationship
(SAR) study and design strategies of nitrogen-containing
heterocyclic moieties for their anticancer activities. Eur J
Med Chem 2017;125:143–89.
3. Yadav G, Ganguly S. Structure activity relationship (SAR)
study of benzimidazole scaffold for different biological activ-
ities: a mini-review. Eur J Med Chem 2015;97:419–43.
4. Akhtar MJ, Siddiqui AA, Khan AA, et al. Design, synthesis,
docking and QSAR study of substituted benzimidazole linked
oxadiazole as cytotoxic agents, EGFR and erbB2 receptor
inhibitors. Eur J Med Chem 2017;126:853–69.
5. Bhattacharya S, Chaudhuri P. Medical implications of benzimi-
dazole derivatives as drugs designed for targeting DNA and
DNA associated processes. Curr Med Chem 2008;15:1762–77.
6. Sontakke VA, Lawande PP, Kate AN, et al. Antiproliferative
activity of bicyclic benzimidazole nucleosides: synthesis,
DNA-binding and cell cycle analysis. Org Biomol Chem
2016;14:4136–45.
The 1,2,3-triazole-linked 5-amidinobenzimidazoles 7a–7e, 8a–8e,
and 9a–9e were synthesised by a Cu(I)catalysed 1,3-dipolar cyclo-
addition reaction applying microwave and ultrasound irradiation,
with subsequent formation of a benzimidazole moiety by oxidative
coupling of o-phenylenediamines with benzaldehydes. It was
found that the 7c–9c, 7d–9d, and 7e–9e sets of compounds non-
covalently bound to ds-DNA/RNA. The small bathochromic shifts
in UV–Vis titration spectra upon addition of ctDNA, modest ther-
mal stabilisation effects, and strong positive ICD bands in CD titra-
tion experiments supported minor groove binding as the
dominant binding mode of these compounds. Conversely, the
appearance of negative ICD bands in CD titration experiments
with polyA-polyU and polyC-polyG, and density of binding sites
obtained from UV–Vis titrations, identified intercalation as the pre-
dominant binding mode.
Furthermore, SARs showed that the type of aromatic substitu-
ents at N-1 of 1,2,3-triazole had profound effects on anti-bacterial
and anti-protozoal activities. Thus, results of anti-bacterial evalua-
tions revealed that o-chlorophenyl-1,2,3-triazole and N-isopropyla-
midine moieties in 8c had a considerable impact on inhibitory

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1333
7. Hegde M, Kumar KSS, Thomas E, et al. A novel benzimidazole 28. Bonandi E, Christodoulou MS, Fumagalli G, et al. The 1,2,3-
derivative binds to the DNA minor groove and induces apop-
triazole ring as a bioisostere in medicinal chemistry. Drug
tosis in leukemic cells. RSC Adv 2015;5:93194–208.
Discov Today 2017;22:1572–81.
8. Singla P, Luxami V, Paul K. Triazine–benzimidazole conju- 29. Dheer D, Singh V, Shankar R. Medicinal attributes of 1,2,3-tri-
gates: synthesis, spectroscopic and molecular modelling azoles: current developments. Bioorg Chem 2017;71:30–54.
studies for interaction with calf thymus DNA. RSC Adv 30. Tiwari VK, Mishra BB, Mishra KB, et al. Cu-catalyzed click
2016;6:14741–50.
reaction in carbohydrate chemistry. Chem Rev 2016;
9. Chaudhuri P, Ganguly B, Bhattacharya S. An experimental
116:3086–240.
and computational analysis on the differential role of the 31. DeSimone RW, Currie KS, Mitchell SA, et al. Privileged struc-
positional isomers of symmetric bis-2-(pyridyl)-1H-benzimida-
tures: applications in drug discovery. Comb Chem High
zoles as DNA binding agents. J Org Chem 2007;72:1912–23.
Throughput Screening 2004;7:473–93.
10. Thomas JR, Hergenrother PJ. Targeting RNA with small mole- 32. Gaba M, Singh S, Mohan C. Benzimidazole: an emerging
cules. Chem Rev 2008;108:1171–224.
scaffold for analgesic and anti-inflammatory agents. Eur J
Med Chem 2014;76:494–505.
33. Jadhav GR, Shaikh MU, Kale RP, et al. SAR study of clubbed
[1,2,4]-triazolyl with fluorobenzimidazoles as antimicrobial
and antituberculosis agents. Eur J Med Chem 2009;44:
2930–5.
11. Guan L, Disney MD. Recent advances in developing small
molecules targeting RNA. ACS Chem Biol 2012;7:73–86.
12. Zheng Y, Cai X, Bradley JE. MicroRNAs in parasites and para-
site infection. RNA Biol 2013;10:371–9.
13. Cheng G, Dai M, Ahmed S, et al. Antimicrobial drugs in
€
fighting against antimicrobial resistance. Front Microbiol 34. Kuhler TC, Swanson M, Christenson B, et al. Novel structures
2016;7:470.
derived from 2-[[(2-pyridyl)methyl]thio]-1H-benzimidazole as
anti-Helicobacter pylori agents, Part 1. J Med Chem 2002;
45:4282–99.
14. Cassir N, Rolain JM, Brouqui P. A new strategy to fight anti-
microbial resistance: the revival of old antibiotics. Front
Microbiol 2014;5:551.
15. Yang SW, Pan J, Yang C, et al. Benzimidazole analogs as
WTA biosynthesis inhibitors targeting methicillin resistant
Staphylococcus aureus. Bioorg Med Chem Lett 2016;26:
4743–7.
16. Hu L, Kully ML, Boykin DW, Abood N. Optimization of the
central linker of dicationic bis-benzimidazole anti-MRSA and
anti-VRE agents. Bioorg Med Chem Lett 2009;19:3374–7.
ꢁ
ꢁ ꢀ
ꢀ
35. Maracic S, Kraljevic TG, Paljetak HC, et al. 1,2,3-Triazole
pharmacophore-based benzofused nitrogen/sulfur hetero-
cycles with potential anti-Moraxella catarrhalis activity.
Bioorg Med Chem 2015;23:7448–63.
36. Ranjan N, Story S, Fulcrand G, et al. Selective inhibition of
Escherichia coli RNA and DNA topoisomerase I by Hoechst
33258 derived mono- and bisbenzimidazoles. J Med Chem
2017;60:4904–22.
17. Rodrigo-Troyano A, Sibila O. The respiratory threat posed by 37. Grillot AL, Tiran AL, Shannon D, et al. Second-generation
multidrug resistant Gram-negative bacteria. Respirology
2017;22:1288–99.
18. Ansari KF, Lal C. Synthesis and evaluation of some new ben-
antibacterial benzimidazole ureas: discovery of a preclinical
candidate with reduced metabolic liability. J Med Chem
2014;57:8792–816.
zimidazole derivatives as potential antimicrobial agents. Eur 38. Janupally R, Jeankumar VU, Bobesh KA, et al. Structure-
J Med Chem 2009;44:2294–9.
19. Sharma D, Narasimhan B, Kumar P, Jalbout A. Synthesis and
QSAR evaluation of 2-(substituted phenyl)-1H-benzimida-
guided design and development of novel benzimidazole
class of compounds targeting DNA gyrase B enzyme of
Staphylococcus aureus. Bioorg Med Chem 2014;22:5970–87.
zoles and [2-(substituted phenyl)-benzimidazol-1-yl]-pyridin- 39. Lam T, Hilgers MT, Cunningham ML, et al. Structure-based
3-yl-methanones. Eur J Med Chem 2009;44:1119–27.
20. Ates-Alagoz Z. Antimicrobial activities of 1-H-benzimidazole-
based molecules. Curr Top Med Chem 2016;16:2953–62.
design of new dihydrofolate reductase antibacterial agents:
7-(benzimidazol-1-yl)-2,4-diaminoquinazolines. J Med Chem
2014;57:651–68.
21. Song D, Ma S. Recent development of benzimidazole-con- 40. Whalen KL, Chau AC, Spies MA. In silico Optimization of a
taining antibacterial agents. Chem Med Chem 2016;11:
646–59.
22. Desai N, Trivedi A, Pandit U, et al. Hybrid bioactive hetero-
Fragment-Based Hit Yields Biologically Active, High-Efficiency
Inhibitors for Glutamate Racemase. ChemMedChem 2013;8:
1681–1689.
cycles as potential antimicrobial agents: a review. Mini Rev 41. Pan W, Fan M, Wu H, et al. A new small molecule inhibits
Med Chem 2016;16:1500–26.
Streptococcus mutans biofilms in vitro and in vivo. J Appl
23. El-Gohary NS, Shaaban MI. Synthesis and biological evalu-
Microbiol 2015;119:1403–11.
ation of a new series of benzimidazole derivatives as anti- 42. Luo G, Spellberg B, Gebremariam T, et al. Combination ther-
microbial, antiquorum-sensing and antitumor agents. Eur J
Med Chem 2017;131:255–62.
apy with iron chelation and vancomycin in treating murine
staphylococcemia. Eur J Clin Microbiol Infect Dis 2014;33:
845–51.
ꢀ
ꢀ
24. Klimesova V, Kocı J, Pour M, et al. Synthesis and preliminary
evaluation of benzimidazole derivatives as antimicrobial 43. Nimesh H, Sur S, Sinha D, et al. Synthesis and biological
agents. Eur J Med Chem 2002;37:409–18.
evaluation of novel bisbenzimidazoles as Escherichia coli
topoisomerase IA inhibitors and potential antibacterial
agents. J Med Chem 2014;57:5238–57.
25. Gibney KA, Sovadinova I, Lopez AI, et al. Poly(ethylene
imine)s as antimicrobial agents with selective activity.
Macromol Biosci 2012;12:1279–89.
26. Sharma V, Chitranshi N, Agarwal AK. Significance and bio-
logical importance of pyrimidine in the microbial world. Int J
Med Chem 2014;2014:202784.
44. Mann J, Taylor PW, Dorgan CR, et al. The discovery of a
novel antibiotic for the treatment of Clostridium difficile
infections: a story of an effective academic–industrial part-
nership. MedChemComm 2015;6:1420–6.
27. Anthony NG, Breen D, Clarke J, et al. Antimicrobial lexitrop- 45. Farahat AA, Bennett-Vaughn C, Mineva EM, et al. Synthesis,
sins containing amide, amidine, and alkene linking groups.
DNA binding and antitrypanosomal activity of benzimidazole
J Med Chem 2007;50:6116–25.
analogues of DAPI. Bioorg Med Chem Lett 2016;26:5907–10.

ꢀ
A. BISTROVIC ET AL.
1334
€
46. Karaaslan C, Kaiser M, Brun R, Goker H. Synthesis and potent 64. McGhee JD, von Hippel PH. Theoretical aspects of DNA-pro-
tein interactions: co-operative and non-co-operative binding
of large ligands to a one-dimensional homogeneous lattice.
J Mol Biol 1974 1974;86:469–89.
antiprotozoal activity of mono/di amidino 2-anilinobenzimi-
dazoles versus Plasmodium falciparum and Trypanosoma
brucei rhodesiense. Bioorg Med Chem 2016;24:4038–44.
65. Clinical and Laboratory Standards Institute. Methods for dilu-
tion antimicrobial susceptibility tests for bacteria that grow
aerobically. M7-A7, Wayne (PA): CLSI; 2006.
66. Clinical and Laboratory Standards Institute. Performance
standards for antimicrobial susceptibility testing. M100-S17,
Wayne (PA): CLSI; 2007.
47. Ismail MA, Batista-Parra A, Miao Y, et al. Dicationic near-lin-
ear biphenyl benzimidazole derivatives as DNA-targeted
antiprotozoal agents. Bioorg Med Chem 2005;13:6718–26.
48. Rice CA, Colon BL, Alp M, et al. Bis-benzimidazole hits
against Naegleria fowleri discovered with new high-through-
put screens. Antimicrob Agents Chemother 2015;59:2037–44.
67. Taylor MC, Lewis MD, Fortes Francisco A, et al. The
Trypanosoma cruzi vitamin C dependent peroxidase confers
protection against oxidative stress but is not a determinant
of virulence. PLoS Negl Trop Dis 2015;9:e0003707.
€
49. Alp M, Goker H, Brun R, Yildiz S. Synthesis and antiparasitic
and antifungal evaluation of 20-arylsubstituted-1H,10H-
[2,5']bisbenzimidazolyl-5-carboxamidines. Eur J Med Chem
2009;44:2002–8.
68. Kappe CO, Van der Eycken E. Click chemistry under non-clas-
sical reaction conditions. Chem Soc Rev 2010;39:1280–90.
69. Ohemeng KA, Nguyen VN. Ortho -Mcneil Pharmaceutical. 2-
substituted phenylbenzimidazole antibacterial agents, US.
Patent Application 08/924,558, 1999, WO1999011627A1.
70. Rehman SU, Sarwar T, Husain MA, et al. Studying non-cova-
lent drug–DNA interactions. Arch Biochem Biophys
2015;576:49–60.
71. Arjmand F, Parveen S, Afzal M, et al. Molecular drug design,
synthesis and crystal structure determination of Cu II–Sn IV
heterobimetallic core: DNA binding and cleavage studies.
Eur J Med Chem 2012;49:141–50.
72. Banerjee M, Farahat AA, Kumar A, et al. Synthesis, DNA bind-
ing and antileishmanial activity of low molecular weight bis-
arylimidamides. Eur J Med Chem 2012;55:449–54.
73. Wilson WD, Tanious FA, Fernandez-Saiz M, Rigl CT.
Evaluation of drug-nucleic acid interactions by thermal melt-
ing curves. Methods Mol Biol 1997;90:219–40.
74. Mergny JL, Lacroix L. Analysis of thermal melting curves.
Oligonucleotides 2003;13:515–37.
75. Husain MA, Yaseen Z, Rehman SU, et al. Naproxen interca-
lates with DNA and causes photocleavage through ROS gen-
eration. Febs J 2013;280:6569–80.
76. Rodger A. Circular dichroism and linear dichroism.
Encyclopedia of Analytical Chemistry. John Hoboken (NJ):
Wiley & Sons, Ltd. 2014.
77. Liu Y, Kumar A, Boykin DW, Wilson WD. Sequence and
length dependent thermodynamic differences in heterocyclic
diamidine interactions at AT base pairs in the DNA minor
groove. Biophys Chem 2007;131:1–14.
78. O'Sullivan P, Rozas I. Understanding the guanidine-like cat-
ionic moiety for optimal binding into the DNA minor groove
. ChemMedChem 2014;9:2065–73.
79. Nagle PS, Quinn SJ, Kelly JM, et al. Understanding the DNA
binding of novel non-symmetrical guanidinium/2-aminoimi-
dazolinium derivatives. Org Biomol Chem 2010;8:5558–67.
80. Nguyen B, Tardy C, Bailly C, et al. Influence of compound
structure on affinity, sequence selectivity, and mode of bind-
ing to DNA for unfused aromatic dications related to furami-
dine. Biopolymers 2002;63:281–97.
81. Dyatkina NB, Roberts CD, Keicher JD, et al. Minor groove
DNA binders as antimicrobial agents. 1. Pyrrole tetraamides
are potent antibacterials against vancomycin resistant
Enteroccoci and methicillin resistant Staphylococcus aureus.
J Med Chem 2002;45:805–17.
82. Khalaf AI. Minor groove binders: some recent research in
drug development. Curr Trends Med Chem 2009;6:53–63.
83. Havrylyuk D, Zimenkovsky B, Karpenko O, et al. Synthesis of
pyrazoline–thiazolidinone hybrids with trypanocidal activity.
Eur J Med Chem 2014;85:245–54.
50. Sola I, Artigas A, Taylor MC, et al. Synthesis and biological
evaluation of N-cyanoalkyl-, N-aminoalkyl-, and N-guanidi-
noalkyl-substituted 4-aminoquinoline derivatives as potent,
selective, brain permeable antitrypanosomal agents. Bioorg
Med Chem 2016;24:5162–71.
51. Pai S, Alam AS, Kapoor JN. Pentamidine salts useful in the
treatment of pneumocystis carinii pneumonia, US. Patent
Application US/07383, 243. US5084480 A, Fujisawa USA Inc.,
1992.
52. Jensch H4. 4'-Diamidino-diazoaminobenzene, a new agent in
the treatment of trypanosomiasis and babesiasis. Arzneimitt
Forsch 1955;5:634–635.
53. Ansede JH, Anbazhagan M, Brun R, et al. As indicators of in
vivo efficacy in a mouse model of Trypanosoma brucei rho-
desiense infection. J Med Chem 2004;47:4335–8.
54. Bakunov SA, Bakunova SM, Wenzler T, et al. Synthesis and
antiprotozoal activity of cationic 1,4-diphenyl-1H-1,2,3-tria-
zoles. J Med Chem 2010;53:254–72.
ꢀ
ꢀ
55. Bistrovic A, Krstulovic L, Harej A, et al. Design, synthesis
and biological evaluation of novel benzimidazole amidines
as potent multi-target inhibitors for the treatment of non-
small cell lung cancer. Eur J Med Chem 2018;143:1616–34.
2017
ꢁ
ꢀ
ꢀ
56. Stolic I, Paljetak HC, Peric M, et al. Synthesis and structur-
e–activity relationship of amidine derivatives of 3,4-ethylene-
dioxythiophene as novel antibacterial agents. Eur J Med
Chem 2015;90:68–81.
ꢀ
ꢀ
ꢀ
57. Krstulovic L, Stolic I, Jukic M, et al. New quinoline-arylami-
dine hybrids: synthesis, DNA/RNA binding and antitumor
activity. Eur J Med Chem 2017;137:196–210.
ꢁ
ꢀ
58. Hranjec M, Starcevic K, Zamola B, et al. New amidino-benzi-
midazolyl derivatives of tylosin and desmycosin. J Antibiot
2002;55:308–14.
59. Chandrika NT, Shrestha SK, Ngo HX, Garneau-Tsodikova S.
Synthesis and investigation of novel benzimidazole deriva-
tives as antifungal agents. Bioorg Med Chem 2016;24:
3680–6.
60. Kumar R, Arora J, Prasad AK, et al. Synthesis and antimicro-
bial activity of pyrimidine chalcones. Med Chem Res
2013;22:5624–32.
61. Harbeck RJ, McCarter YS, Ortez JH, et al. Manual of anti-
microbial susceptibility testing, Washington: American
Society for Microbiology; 2005.
ꢀ
ꢁ
ꢀ
62. Stolic I, Miskovic K, Magdaleno A, et al. Effect of 3,4-ethyle-
nedioxy-extension of thiophene core on the DNA/RNA bind-
ing properties and biological activity of bisbenzimidazole
amidines. Bioorg Med Chem 2009;17:2544–54.
63. Scatchard G. The attractions of proteins for small molecules
and ions. Ann NY Acad Sci 1949;51:660–72.