Synthesis, in vitro antiprotozoal activity, molecular docking and molecular dynamics studies of some new monocationic guanidinobenzimidazoles

Article abstract of DOI:10.1016/j.ejmech.2021.113545

A series of monocationic new guanidinobenzimidazole derivatives were prepared in a four step process starting from 2-nitro-1,4-phenylendiamine. Their antiparasitic activity against Plasmodium falciparum, Trypanosoma brucei rhodesiense, Trypanosoma cruzi and Leishmania donovani were evaluated in vitro. Two out of 20 tested monocationic compounds (7, 14) showed close activity with reference drug chloroquine against P. Falciparum. To understand the interactions between DNA minor groove and in vitro active compounds (7, 14) molecular docking studies were carried out. Stability and binding energies of DNA-ligand complexes formed by DNA with compounds 7 and 14 were measured by molecular dynamics simulations throughout 200 ns time. Root mean square deviation (RMSD) values of the ligands remained stable below 0.25 mm and root mean square fluctuation (RMSF) values of the active site residues with which it interacted decreased compared to the apo form. All compounds exhibited theoretical absorption, distribution, metabolism and excretion (ADME) profiles conforming to Lipinski's and Ghose's restrictive rules.

Full text of DOI:10.1016/j.ejmech.2021.113545

European Journal of Medicinal Chemistry 221 (2021) 113545
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Synthesis, in vitro antiprotozoal activity, molecular docking and
molecular dynamics studies of some new monocationic
guanidinobenzimidazoles
,
, e
Fatima Doganc a, Ismail Celik b, Gokcen Eren c, Marcel Kaiser d e, Reto Brun d
,
,
*
Hakan Goker a
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, 06100, Tandogan, Ankara, Turkey
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Erciyes University, 38280, Yenidogan, Kayseri, Turkey
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, 06330, Etiler, Ankara, Turkey
d Parasite Chemotherapy Unit, Swiss Tropical and Public Health Institute, Basel, CH, 4002, Switzerland
e University of Basel, Basel, CH, 4001, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of monocationic new guanidinobenzimidazole derivatives were prepared in a four step process
starting from 2-nitro-1,4-phenylendiamine. Their antiparasitic activity against Plasmodium falciparum,
Trypanosoma brucei rhodesiense, Trypanosoma cruzi and Leishmania donovani were evaluated in vitro. Two
out of 20 tested monocationic compounds (7, 14) showed close activity with reference drug chloroquine
against P. Falciparum. To understand the interactions between DNA minor groove and in vitro active
compounds (7, 14) molecular docking studies were carried out. Stability and binding energies of DNA-
ligand complexes formed by DNA with compounds 7 and 14 were measured by molecular dynamics
simulations throughout 200 ns time. Root mean square deviation (RMSD) values of the ligands remained
stable below 0.25 mm and root mean square fluctuation (RMSF) values of the active site residues with
which it interacted decreased compared to the apo form. All compounds exhibited theoretical absorp-
tion, distribution, metabolism and excretion (ADME) profiles conforming to Lipinski's and Ghose's
restrictive rules.
Received 10 March 2021
Received in revised form
7 May 2021
Accepted 8 May 2021
Available online 24 May 2021
Keywords:
Antiparasitic activity
Molecular docking
Molecular dynamics
Monocationic guanidinobenzimidazoles
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
In addition, drugs used in the treatment are highly toxic and have
long treatment periods and significant side effects. Also, cost
Neglected tropical diseases (NTDs) are a diverse group of in-
fectious diseases common in 149 countries especially in tropical
and subtropical regions, affecting more than 1 billion people each
year [1]. Parasitic infections are common even in developing
countries and cause important opportunistic infections, particu-
larly in immunocompromised patients. Among them, malaria,
sleeping sickness (Human Africa Trypanosomiasis, HAT), Chagas
disease and leishmaniasis are responsible for a considerable
amount of human mortality, morbidity and economic hardship
[2,3]. Protozoan parasites are resistant to a large number of drugs.
related problems and lack of oral bioavailability reveal the
requirement for the development of new antiparasitic compounds
[3].
Aromatic amidine derivatives are known as a group of com-
pounds that interact with DNA. These compounds show in vitro
antiprotozoal activity by binding to the DNA minor groove, espe-
cially AT base pairs. These derivatives have been used for years in
the treatment of protozoal diseases. Pentamidine (Fig. 1) is a
compound used clinically in the treatment of sleeping sickness and
antimony-resistant leishmaniasis [4,5]. But, it is not effective when
given orally and various toxic effects such as hypotension, dysgly-
cemia, kidney and liver toxicity have been reported [6].
Until today, based on pentamidine, many similar analogues and
new derivatives with different cationic and heterocyclic structures
have been synthesized and antiprotozoal activities have been
screened. Among these, furamidine has lower toxicity and
Abbreviations: NTDs, Neglected tropical diseases; RMSD, Root mean square
deviation; RMSF, Root mean square fluctuation; TMS, Tetra methyl silane; VMD,
Visual molecular dynamics.
* Corresponding author.
E-mail address: goker@ankara.edu.tr (H. Goker).
https://doi.org/10.1016/j.ejmech.2021.113545
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

F. Doganc, I. Celik, G. Eren et al.
European Journal of Medicinal Chemistry 221 (2021) 113545
Fig. 3. The structures of previously reported monocationic compounds possessing
potent antiprotozoal activity.
Fig. 1. Pentamidine (I), Furamidine (II) and Pafuramidine (III).
remarkably better activity against protozoan parasites than pent-
amidine. Furamidine and methamidoxime prodrug III (Pafur-
amidine) (Fig. 1) performed very potent inhibitory activity against
protozoan parasites [6,7].
how the compounds interacted with DNA and molecular dynamics
simulations were performed to measure the stability and energy of
the DNA-ligand complex formed. Besides, theoretical computa-
tional ADME study of the compounds was carried out.
These compounds that bind to DNA do not directly kill the
parasite, but cause inhibition of DNA-dependent enzymes or direct
inhibition of transcription. Lately, aromatic diguanidine derivatives,
which are bioisosteres of amidines, have also been reported to
show significant antiprotozoal activity [8e10]. Dicationic com-
pounds such as bisguanidine and bis(2-aminoimidazoline) DNA
minor groove binders (Fig. 2) showed in vitro activity against T.b.
rhodesiense and P. falciparum at nanomolar concentrations [11].
Our previous studies have shown that not only dicationic
compounds but also monocationic compounds exhibited good
inhibitory activity against protozoan parasites. Among them,
compounds IV [12] and V-VI [13] (Fig. 3) have significant inhibitory
activity against P. falciparum. In addition, compounds V and VI
show a good inhibitory activity profile against T.b. rhodesiense.
We now report the in vitro antiprotozoal evaluation of new
monocationic guanidinobenzimidazoles against P. falciparum, T.b.
rhodesiense, T.cruzi, L. donovani and their molecular docking studies.
In silico molecular docking analysis was performed to investigate
2. Material and methods
2.1. Chemistry
Compounds 4e23 (Table 1) were prepared using the methods
outlined in Scheme 1. 2-Nitro-1,4-phenylenediamine was con-
verted into HCl salt (1) with 4 M HCl (in dioxane), stirring at room
temperature in ethanol. 1 was reacted with cyanamide to obtain 1-
(4-amino-3-nitrophenyl)guanidine HCl (2). Reduction of 2 with H2/
PdeC afforded 1-(3,4-diaminophenyl)guanidine HCl (3). Cycliza-
tion of 3 with sodium metabisulfite (Na2S2O5) adduct of corre-
sponding
substituted
aldehydes
gave
targeted
guanidinobenzimidazoles (4e23). HCl salts of compounds were
prepared in ethanol with HCl gas.
2.1.1. Experimental
Uncorrected melting points were measured on a Büchi B-540
capillary melting point apparatus. 1H (400 MHz) and 13C (100 MHz)
NMR spectra were recorded employing a Varian Mercury 400 MHz
FT spectrometer, chemical shifts (d) are in ppm relative to TMS, and
coupling constants (J) are reported in Hertz. Mass spectra were
taken on a Waters Micromass ZQ connected with Waters Alliance
HPLC, using ESI (þ) method, with C-18 column. Elemental analyses
were performed by Leco CHNS-932. The compounds reported as
salts frequently analyzed correctly for fractional moles of water
and/or organic solvent of solvation.
Because of the tautomeric effect of the imidazole ring, the 1H
NMR spectra of some compounds was not clear enough, therefore
in order to eliminate the tautomerism compounds were dissolved
in DMSO‑d6, followed by a tiny amount of dry NaH and 2e3 drops of
D2O were added to NMR tubes and stirred well. In case of any
turbidity, the tubes were centrifuged. Clear NMR spectra were
observed as reported in the experimental part.
Fig. 2. Dicationic bisguanidine and bis(2-aminoimidazoline) compounds.
2

F. Doganc, I. Celik, G. Eren et al.
European Journal of Medicinal Chemistry 221 (2021) 113545
Table 1
Formulas and in vitro antiprotozoal activities of 4-23.
No
4
Ar
IC50 (
mg/mL) [SI]
P.f.a
T.b.r.a
T.c.a
L.d.a
Cytotoxicity L6 cellsa
4.07
11.6
61.2
>100
>100
5
6
7
1.89
1.17
29.5
90.5
64.5
71.1
>100
42.6
18.2
89.5
>100
0.018 [2360]
21.5 [2]
54.3 [0.8]
52.55 [0.8]
8
9
37.2
72.1
13.6
59.1
38
>100
>100
>100
0.226
43.2
10
1.87
16.1
49.5
>100
51.6
11
12
13
2.67
6.26
0.153
6.04
40.8
23.8
67
>100
>100
>100
>100
97.8
78
62
>100
14
15
0.052 [>1920]
12.5 [>8]
58.6 [>2]
58 [>2]
>100
>100
1.41
11.8
63.6
48.9
16
17
2.72
3.8
8.57
34.3
46.3
40.3
44.6
43.6
0.168
6.03
18
19
0.82
0.57
10.1
12.1
75.1
53.1
56.2
58.6
73.5
40
(continued on next page)
3

F. Doganc, I. Celik, G. Eren et al.
European Journal of Medicinal Chemistry 221 (2021) 113545
Table 1 (continued)
No
Ar
IC50 (mg/mL) [SI]
P.f.a
T.b.r.a
T.c.a
L.d.a
Cytotoxicity L6 cellsa
49.9
20
0.19
11.8
46.1
37.7
21
0.279
16
36.5
44
48.4
22
23
1.14
3.03
5.62
17.3
21.7
64
7.7
46.8
93
90.2
Chl.
Mel.
Bnz.
Mil.
Pod.
0.003
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.003
-
-
-
0.757
-
-
0.555
-
0.006
a
Activities represent the mean of at least two independent experiments; IC50 values used to calculate the average for a given compound were within a factor of two. P.f.:
Plasmodium falciparum NF54, T.b.r.: Trypanosoma brucei rhodesiense STIB900, T.c.: Trypanosoma cruzi Tulahuen C4, L.d.: Leishmania donovani MHOM-ET-67/L82, Chl: Chlo-
roquine, Mel: Melarsoprol, Bnz: Benznidazole, Mil: Miltefosine, Pod: Podophyllotoxin, SI: Selectivity index (IC50 L6 cell/IC50 parasites).
Scheme 1. Synthesis of targeted guanidinobenzimidazoles.
2.1.1.1. 2-Nitro-1,4-phenylenediamine
HCl
(1).
2.1.1.3. 1-(3,4-Diaminophenyl)guanidine HCl (3). The mixture of 2
(0.5 g, 2.16 mmol), PdeC (10%, 0.046 g), tetrahydrofuran (4.2 mL)
and methanol (10 mL) were subjected to hydrogenation using 40
psi of H2 until the end of H2 uptake. The catalyst was filtered on a
bed of Celite, washed with ethanol and concentrated in vacuo.
Powder residue was used for the subsequent steps without crys-
tallization [14]. Yield 93% (0.4 g). Mp: 230e233 ꢀC, MS (ESIþ) m/z:
166 (MþH, 100%).
2-Nitro-1,4-phenylenediamine (2.1 g, 13.7 mmol) was dissolved in
35 mL anhydrous ethanol. 4 M HCl in dioxane (3 mL, 12.3 mmol)
was added and stirred at room temperature for 15 min. Dieth-
ylether (100 mL) was added, the precipitate was collected by
filtration and dried [14]. Yield: 79% (2.57 g), MS (ESIþ) m/z: 154
(MþH, 100%).
2.1.1.2. 1-(4-Amino-3-nitrophenyl)guanidine HCl (2). The mixture of
1 (1.5 g, 7.9 mmol), cyanamide (2.5 g, 59.5 mmol) and water
(0.5 mL) were heated at 60 ꢀC for 1.5 h. It was cooled to room
temperature. The excess of diethylether was added slowly, the
precipitate was collected by filtration and dried [14]. Yield: 73%
(1.09 g). Mp: 170e174 ꢀC, MS (ESIþ)m/z: 196 (MþH, 100%).
2.1.1.4. General synthesis of sodium metabisulfite adduct of
substituted aldehydes. Related substituted aldehydes (30 mmol)
were dissolved in EtOH (100 mL) and sodium metabisulfite (3.2 g)
(in 5 mL of water) was added in portions. The reaction mixture was
stirred vigorously. The mixture was kept in a refrigerator for a
while. The white precipitate was gained by filtration, dried and
4

F. Doganc, I. Celik, G. Eren et al.
European Journal of Medicinal Chemistry 221 (2021) 113545
used for the further steps without purification and characterisation.
30,5’], [H-2’’,6’’: H-3’’,5’’] [H-4’’: H-3’’,5’’]. 13C-NMR & HSQC
d ppm
(DMSO‑d6 þ NaH þ D2O): 159.4, 153.8, 148.15, 143.7, 140.7, 139.4,
138.2, 137.1, 129.5 (CH-3’’,5’’), 127.55 (CH-4’’), 127.35, 126.7 (CH-
2’’,6’’), 126.6, 116.25, 115.5, 109.9 (CH-4). MS (ESIþ) m/z: 328 (MþH,
45%), 164 (100%). Anal Calcd for C20H17N5 . 2HCl . H2O: C, 57.42; H,
5.05; N, 16.74. Found: C, 57.54; H, 4.71; N, 16.78.
2.1.1.5. General synthesis of monocationic guanidinobenzimidazole
derivatives (4e23). The mixture of 3 (1 mmol) and Na2S2O5 adduct
of substituted aldehydes (1.1 mmol) in DMF (1 mL) were heated at
100 ꢀC, for 2 h [15]. The reaction mixture was cooled, poured into
diluted Na2CO3 solution, stirred for a while. The resulting precipi-
tate was collected by filtration washed with water and dried. If the
product was not solid, it was extracted with CH2Cl2: MeOH (95 : 5).
Crystallization or colon chromatography was used for purification.
HCl salts of compounds were made in ethanol with HCl gas.
2.1.1.5.5. 4-(5(6)-Guanidino-1H-benzimidazole-2-yl)benzoic acid
(8). Prepared
from
3
and
Na2S2O5
adduct
of
4-
carboxybenzaldehyde (0.28 g) as described in general method.
The resulting precipitate was crystallised from MeOH, yield 73%,
mp > 300 ꢀC. 1H-NMR
d
ppm (DMSO‑d6 þ NaH þ D2O): 6.46 (dd,1H,
2.1.1.5.1. 1-(2-Phenyl-1H-benzimidazole-5(6)-yl)guanidine
HCl
J ¼ 8.4 & 2 Hz, H-6), 6.98 (d, 1H, J ¼ 2.4 Hz, H-4), 7.36 (d, 1H,
J ¼ 8.8 Hz, H-7), 7.86 (d, 2H, J ¼ 8.4 Hz, 2-phenyl protons), 8.15 (d,
2H, J ¼ 8.4 Hz, 2-phenyl protons). COSY: [H-6: H-7], [H-20,6’: H-
(4). Prepared from 3 and Na2S2O5 adduct of benzaldehyde (0.23 g)
as described in general method. Resulting precipitate was purified
with column chromatography using (CH2Cl2: MeOH: NH4OH 50 :
30: 5) as eluant. Crude product was converted to HCl salt, yield 51%,
30,5’]. 13C-NMR & HSQC
d
ppm (DMSO‑d6 þ NaH þ D2O): 172.7,
160.2, 154.9, 148.2, 143.9, 139.8, 138.8, 137.5, 129.95, 126.4, 116.9 (H-
7), 116.45 (H-6), 110.5 (H-4). MS (ESIþ) m/z: 296 (MþH, 100%). Anal
Calcd for C15H13N5O2 . 3.5H2O: C, 50.25; H, 5.62; N, 19.54. Found: C,
49.85; H, 5.55; N, 19.17.
mp > 300 ꢀC. 1H-NMR
d
ppm (DMSO‑d6 þ NaH þ D2O): 6.91 (dd,1H,
J ¼ 8 & 1.6 Hz, H-6), 7.29 (d,1H, J ¼ 1.6 Hz, H-4), 7.44e7.53 (m, 3H, 2-
phenyl protons), 7.56 (d, 1H, J ¼ 8.8 Hz, H-7), 8.13 (d, 2H, J ¼ 7.6 Hz,
2-phenyl protons). COSY: [H-6: H-7], [H-20,6’: H-30,5’], [H-4’: H-30,
2.1.1.5.6. 1-(2-(4-(Benzyloxy)phenyl)-1H-benzimidazole-5(6)-yl)
guanidine HCl (9). Prepared from 3 and Na2S2O5 adduct of 4-
benzyloxybenzaldehyde (0.35 g) as described in general method.
The resulting precipitate was purified with column chromatog-
raphy using (CH2Cl2: MeOH: NH4OH 50 : 20: 2.5) as eluant. Crude
product converted to HCl salt, yield 48%, mp: 245e247 ꢀC. 1H-NMR
5’]. 13C-NMR & HSQC
d
ppm (DMSO‑d6 þ NaH þ D2O): 155.7, 152.9,
140.2, 138.4, 134.5, 130.6, 130.4, 129.5, 126.9, 120.3 (CH-6), 116.5
(CH-7), 111.2 (CH-4). MS (ESIþ) m/z: 252 (MþH, 100%). Anal Calcd
.
.
for C14H13N5 2HCl 3H2O: C, 44.45; H, 5.59; N, 18.51. Found: C,
44.10; H, 5.44; N, 18.97.
2.1.1.5.2. 1-(2-(4-Chlorophenyl)-1H-benzimidazole-5(6)-yl)gua-
d
ppm (DMSO‑d6 þ NaH þ D2O): 5.13 (s, 1H, CH2), 6.35 (dd, 1H, J ¼ 8
nidine HCl (5). Prepared from
3
and Na2S2O5 adduct of 4-
& 2 Hz, H-6), 6.90 (d, 1H, J ¼ 1.6 Hz, H-4), 6.97 (d, 2H, J ¼ 8.8 Hz, H-
30,50), 7.26 (d, 1H, J ¼ 8.4 Hz, H-7), 7.33e7.36 (m, 1H, H-4’’),
7.40e7.43 (m, 2H, H-3’’,5’’), 7.48 (d, 2H, J ¼ 6.8 Hz, H-2’’,6’’), 8.14 (d,
2H, J ¼ 8.8 Hz, H-20,60). COSY: [H-6: H-7], [H-30,5’: H-20,6’], [3’’,5’’:
2’’,6’’], [H-4’’: H-3’’,5’’]. NOESY: [CH2: H-2’’,6’’(strong) ve H-
chlorobenzaldehyde (0.27 g) as described in general method.
Resulting precipitate was purified with column chromatography
using (CH2Cl2: Isopropanol: NH4OH 50 : 30: 5) as eluant. Crude
product was converted to HCl salt, yield 60%, mp: 175e179 ꢀC. 1H-
NMR
d
ppm (DMSO‑d6 þ NaH þ D2O): 6.38 (dd, 1H, J ¼ 8.8 & 2 Hz,
30,5’(weak)]. 13C-NMR
d
ppm (DMSO‑d6 þ NaH þ D2O): 159.9, 157.7,
H-6), 6.91 (d, 1H, J ¼ 2 Hz, H-4), 7.28 (d, 1H, J ¼ 8.8 Hz, H-7), 7.35 (d,
154.1, 148.05, 143.7, 138.7, 137.8, 131.0, 128.9, 128.3, 128.1, 115.8,
114.9,114.7,109.8, 69.7. MS (ESIþ) m/z: 358 (MþH,100%). Anal Calcd
for C21H19N5O . 2HCl . 1.8H2O: C, 54.50; H, 5.35; N, 15.13. Found: C,
54.64; H, 5.25; N, 15.25.
2H, J ¼ 8.8 Hz, 2-phenyl protons), 8.21 (d, 2H, J ¼ 8.8 Hz, 2-phenyl
protons). 13C-NMR & HSQC
d
ppm (DMSO‑d6 þ NaH þ D2O): 158.6,
153.8, 148.1, 143.6, 139.7, 136.8, 131.1, 128.35, 128.3, 116.35 (CH-7),
115.7 (CH-6), 109.9 (CH-4). MS (ESIþ) m/z: 286 (MþH, 33%), 143
2.1.1.5.7. 1-(2-(2-Phenoxyphenyl)-1H-benzimidazole-5(6)-yl)gua-
nidine HCl (10). Prepared from 3 and Na2S2O5 adduct of 4-
phenoxybenzaldehyde (0.33 g) as described in general method.
The resulting precipitate was purified with column chromatog-
raphy using (CH2Cl2: MeOH: NH4OH 50 : 30: 4) as eluant. Crude
product was converted to HCl salt, yield 78%, mp: 290e293 ꢀC. 1H-
.
.
(100%), 288 (MþHþ2, 11%). Anal Calcd for C14H12ClN5 2HCl
4.5H2O: C, 38.24; H, 5.27; N, 15.92. Found: C, 38.35; H, 4.60; N,
15.98.
2.1.1.5.3. 1-(2-(4-Cyanophenyl)-1H-benzimidazole-5(6)-yl)guani-
dine (6). Prepared from
3
and Na2S2O5 adduct of 4-
cyanobenzaldehyde (0.26 g) as described in general method. The
resulting precipitate was crystallised from MeOH, yield 41%,
NMR
d
ppm (CD3OD): 7.01 (d, 1H, J ¼ 8 Hz), 7.24e7.27 (m, 2H),
7.30e7.35 (m, 1H), 7.40e7.44 (m, 1H), 7.48e7.55 (m, 3H), 7.66e7.71
(m, 1H), 7.820e7.825 (m, 1H), 7.95 (d, 1H, J ¼ 8.8 Hz), 8.18e8.21 (m,
mp > 300 ꢀC. 1H-NMR
d
ppm (DMSO‑d6 þ NaH þ D2O): 6.64 (dd,1H,
J ¼ 8.4 & 2.4 Hz, H-6), 7.10 (d, 1H, J ¼ 1.6 Hz, H-4), 7.44 (d, 1H,
J ¼ 8 Hz, H-7), 7.81 (d, 2H, J ¼ 8.4 Hz, H-30,50), 8.35 (d, 2H, J ¼ 8.4 Hz,
H-20,60). COSY: [H-6: H-7], [H-20,6’: H-30,5’]. 13C-NMR & HSQC &
1H). 13C-NMR
d ppm (CD3OD): 158.75, 158.4, 155.8, 149.0, 136.85,
134.9, 133.5, 131.9, 131.6, 131.5, 127.0, 125.8, 124.85, 122.2, 118.5,
116.6, 113.6, 112.6. MS (ESIþ) m/z: 344 (MþH, 22%), 172 (100%). Anal
.
.
HMBC
d
ppm (DMSO‑d6 þ NaH þ D2O): 155.7 (C-2), 154.3 (C-gua-
Calcd for C20H17N5O 2HCl 1.5H2O: C, 54.18; H, 5.00; N, 15.79.
Found: C, 53.73; H, 4.45; N, 15.88.
nidine), 145.1 (C-3a), 142.8 (C-7a), 139.6 (C-10), 135.3 (C-5), 132.2
(CH-30,50), 126.6 (CH-20,60), 119.4 (CN), 117.35 (CH-6), 116.8 (CH-7),
110.6 (CH-4), 108.9 (C-4’). MS (ESIþ) m/z: 277 (MþH, 100%). Anal
Calcd for C15H12N6 . 2.5H2O . 0.5CH3OH: C, 55.18; H, 5.67; N, 24.91.
Found: C, 55.57; H, 5.55; N, 25.33.
2.1.1.5.8. 1-(2-(3,4-Difluorophenyl)-1H-benzimidazole-5(6)-yl)
guanidine HCl (11). Prepared from 3 and Na2S2O5 adduct of 3,4-
difluorobenzaldehyde (0.27 g) as described in general method.
The resulting precipitate was purified with column chromatog-
raphy using (CH2Cl2: MeOH: NH4OH 50 : 40: 5) as eluant. Crude
product was converted to HCl salt, yield 28%, mp > 300 ꢀC. 1H-NMR
2.1.1.5.4. 1-(2-([1,10-Biphenyl]-4-yl)-1H-benzimidazole-5(6)-yl)
guanidine HCl (7). Prepared from 3 and Na2S2O5 adduct of 4-
phenylbenzaldehyde (0.31 g) as described in general method.
Resulting precipitate was purified with column chromatography
using (CH2Cl2: Isopropanol: NH4OH 50 : 50: 10) as eluant. Crude
product was converted to HCl salt, yield 66%, mp > 300 ꢀC. 1H-NMR
d
ppm (DMSO‑d6 þ NaH þ D2O): 6.39 (dd, 1H, J ¼ 8 & 2 Hz, H-6),
6.91 (d, 1H, J ¼ 1.6 Hz, H-4), 7.28 (d, 1H, J ¼ 8.4 Hz, H-7), 7.28e7.35
(m, 1H, 2-phenyl proton), 7.95e7.99 (d, 1H, 2-phenyl proton),
8.03e8.09 (m,1H, 2-phenyl proton). COSY: [H-6: H-7]. MS (ESIþ) m/
.
.
d
ppm (DMSO‑d6 þ NaH þ D2O): 6.39 (dd, 1H, J ¼ 8.4 & 2 Hz, H-6),
z: 288 (MþH, 82%), 144 (100%). Anal Calcd for C14H11F2N5 2HCl
6.91 (d, 1H, J ¼ 2 Hz, H-4), 7.29 (d, 1H, J ¼ 8.4 Hz, H-7), 7.35 (t, 1H,
J ¼ 7.6 Hz, H-4’’), 7.48 (t, 2H, J ¼ 7.6 Hz, H-3’’,5’’), 7.64 (d, 2H,
J ¼ 8.4 Hz, 2-phenyl protons), 7.72 (d, 2H, J ¼ 6.8 Hz, H-2’’,6’’), 8.30
(d, 2H, J ¼ 8.4 Hz, 2-phenyl protons). COSY: [H-6: H-7], [H-20,6’: H-
CH3OH: C, 45.93; H, 4.36; N, 17.85. Found: C, 45.67; H, 4.32; N, 17.60.
2.1.1.5.9. 1-(2-(2,4-Dimethylphenyl)-1H-benzimidazole-5(6)-yl)
guanidine HCl (12). Prepared from 3 and Na2S2O5 adduct of 2,4-
dimethylbenzaldehyde (0.26 g) as described in general method.
5

F. Doganc, I. Celik, G. Eren et al.
European Journal of Medicinal Chemistry 221 (2021) 113545
The resulting precipitate was purified with column chromatog-
raphy using (CH2Cl2: MeOH: NH4OH 50 : 20: 6) as eluant. Crude
product was converted to HCl salt, yield 54%, mp: 215e220 ꢀC
Crude product was converted to HCl salt, yield 30%, mp:
205e210 ꢀC. 1H-NMR
d
ppm (DMSO‑d6 þ NaH þ D2O): 6.41 (dd, 1H,
J ¼ 8 & 2 Hz, H-6), 6.98 (d, 1H, J ¼ 2 Hz, H-4), 7.37 (d, 1H, J ¼ 8 Hz, H-
7), 7.67 (t, 1H, J ¼ 8 Hz, H-70), 7.97 (d, 1H, J ¼ 8.4 Hz), 8.43 (d, 1H,
J ¼ 6.4 Hz), 8.46 (dd, 1H, J ¼ 7.6 & 1.2 Hz, H-30), 9.22 (s, 1H, H-10),
9.48 (d, 1H, J ¼ 6 Hz, H-40). COSY: [H-6: H-7], [H-3’: H-4’], [H-6’: H-
(bubling). 1H-NMR
d
ppm (DMSO‑d6 þ NaH þ D2O): 2.25 (s, 3H,
CH3), 2.55 (s, 3H, CH3), 6.33 (dd, 1H, J ¼ 8 & 1.6 Hz), 6.90e6.95 (m,
3H), 7.27 (d, 1H, J ¼ 8.4 Hz), 7.73 (d, 1H, J ¼ 7.2 Hz). 13C-NMR
d ppm
(DMSO‑d6 þ NaH þ D2O): 161.4, 154.1, 147.6, 143.35, 138.3, 136.2,
7’], [H-7’: H-8’]. 13C-NMR & HSQC
d
ppm (DMSO‑d6 þ NaH þ D2O):
135.3, 135.0, 131.4, 130.6, 126.1, 115.95, 114.7, 109.9, 22.2, 21.1. MS
159.5, 153.9, 152.6 (CH-10), 148.1, 143.7, 142.5, 139.7, 134.4, 134.0,
131.4 (CH-30), 129.6, 127.7 (CH2-70), 126.6, 121.9 (CH-4’), 116.7 (CH-
7), 115.75 (CH-6), 110.2 (CH-4). MS (ESIþ) m/z: 303 (MþH, 40%), 152
.
.
(ESIþ) m/z: 280 (MþH, 100%). Anal Calcd for C16H17N5 2HCl
0.5C2H5OH: C, 54.40; H, 5.90; N, 18.66. Found: C, 54.05; H, 6.37; N,
18.66.
.
.
(100%). Anal Calcd for C17H14N6 3HCl 4H2O: C, 42.20; H, 5.20; N,
17.37. Found: C, 42.58; H, 4.84; N, 17.73.
2.1.1.5.10. 1-(2-(3,4-Dimetoxyphenyl)-1H-benzimidazole-5(6)-yl)
guanidine HCl (13). Prepared from 3 and Na2S2O5 adduct of 2,4-
dimethoxybenzaldehyde (0.3 g) as described in general method.
The resulting precipitate was crystallised from MeOH. Crude
product was converted to HCl salt, yield 50%, mp: 285e289 ꢀC
2.1.1.5.14. 1-(2-(9H-fluorene-2-yl)-1H-benzimidazole-5(6)-yl)
guanidine HCl (17). Prepared from 3 and Na2S2O5 adduct of fluo-
rene-2-carboxyaldehyde (0.33 g) as described in general method.
The resulting precipitate was purified with column chromatog-
raphy using (CH2Cl2: MeOH: NH4OH 50 : 30: 3) as eluant. Crude
product was converted to HCl salt, yield 14%, mp > 300 ꢀC. 1H-NMR
(bubling). 1H-NMR
d
ppm (DMSO‑d6 þ NaH þ D2O): 3.77 (s, 3H,
CH3), 3.84 (s, 3H, CH3), 6.32 (dd, 1H, J ¼ 8 & 2 Hz, H-6), 6.87 (d, 1H,
J ¼ 2 Hz, H-4), 6.90 (d, 1H, J ¼ 8.4 Hz, H-50), 7.23 (d, 1H, J ¼ 8 Hz, H-
7), 7.76 (dd, 1H, J ¼ 8 & 2 Hz, H-60), 7.90 (d, 1H, J ¼ 2 Hz, H-20). COSY:
d
ppm (DMSO‑d6 þ NaH þ D2O): 4.05 (s, 2H, CH2), 7.21 (dd, 1H,
J ¼ 8.4 & 2 Hz, H-6), 7.35e7.45 (m, 2H), 7.56 (d, 1H, J ¼ 2 Hz), 7.64 (d,
1H, J ¼ 7.2 Hz), 7.74 (d,1H, J ¼ 8 Hz, H-7), 7.97 (d,1H, J ¼ 7.6 Hz), 8.09
(d, 1H, J ¼ 8.4 Hz), 8.20 (d, 1H, J ¼ 8 Hz), 8.36 (s, 1H). COSY: [H-6: H-
[H-6: H-7], [H-5’: H-6’]. 13C-NMR
&
HSQC
d
ppm
(DMSO‑d6 þ NaH þ D2O): 160.0, 153.85, 148.55, 148.1, 147.9, 143.7,
138.9, 131.2, 119.0 (CH-60), 115.7, 114.8 (CH-6), 111.8 (CH-50), 110.7
(CH-2’), 109.6 (CH-4), 55.9 (OCH3), 55.7 (OCH3). MS (ESIþ) m/z: 312
(MþH, 38%), 156 (100%). Anal Calcd for C16H17N5O2 . 2HCl . 2.5H2O:
C, 44.76; H, 5.63; N, 16.77. Found: C, 44.85; H, 4.94; N, 16.35.
2.1.1.5.11. 1-(2-(4-(3,4-Dimetoxyphenoxy)phenyl)-1H-benzimid-
azole-5(6)-yl)guanidine HCl (14). Prepared from 3 and Na2S2O5
adduct of 4-(3,4-Dimethoxyphenoxy)benzaldehyde (0.4 g) as
described in general method. The resulting precipitate was purified
with column chromatography using (CH2Cl2: MeOH: NH4OH 50 :
30: 5) as eluant. Crude product was converted to HCl salt, yield 40%,
7]. 13C-NMR
d
ppm (DMSO‑d6 þ NaH þ D2O): 156.9, 152.2, 146.1,
145.1, 144.9, 140.4, 135.8, 134.15, 131.9, 129.2, 128.1, 127.3, 126.25,
124.7, 123.7, 123.6, 121.7, 116.2, 112.3, 37.3. MS (ESIþ) m/z: 340
(MþH, 82%), 170 (100%). Anal Calcd for C21H17N5 . 2HCl . 1.5H2O: C,
57.41; H, 5.04; N, 15.94. Found: C, 57.30; H, 5.05; N, 16.32.
2.1.1.5.15. 1-(2-(1,4-Benzodioxane-6-yl)-1H-benzimidazole-5(6)-
yl)guanidine HCl (18). Prepared from 3 and Na2S2O5 adduct of 1,4-
benzodioxane-6-carboxyaldehyde (0.3 g) as described in general
method. The resulting precipitate was purified with column chro-
matography using (CH2Cl2: MeOH: NH4OH 50 : 20: 1) as eluant.
Crude product was converted to HCl salt, yield 11%, mp > 300 ꢀC.
mp: 230e234 ꢀC. 1H-NMR
d ppm (CD3OD): 3.79 (s, 3H, 3’’-OCH3),
3.83 (s, 3H, 4’’-OCH3), 6.63 (dd, 1H, J ¼ 8.4 & 1.2 Hz, H-6’’), 6.76 (d,
1H, J ¼ 2.8 Hz, H-2’’), 6.97 (d, 1H, J ¼ 8.4 Hz, H-5’’), 7.06 (d, 2H,
J ¼ 8.4 Hz, H-30,50), 7.14 (dd, 1H, J ¼ 8.4 & 1.6 Hz, H-6), 7.51 (d, 1H,
J ¼ 1.6 Hz, H-4), 7.63 (d, 1H, J ¼ 8.4 Hz, H-7), 8.04 (d, 2H, J ¼ 8.8 Hz,
H-20,60). COSY: [H-6: H-7], [H-20,6’: H-30,5’], [H-5’’: H-6’’]. NOESY:
[3’’-OCH3: H-2’’], [4’’-OCH3: H-5’’]. 13C-NMR & HSQC & HMBC
1H-NMR
d
ppm (DMSO‑d6 þ NaH þ D2O): 4.22 (br.s, 4H, CH2,
overlapped water peak), 6.30 (dd, 1H, J ¼ 8.4 & 2 Hz, H-6), 6.75 (dd,
1H, J ¼ 8.8 Hz, H-80), 6.84 (d, 1H, J ¼ 2 Hz, H-4), 7.20 (d, 1H,
J ¼ 8.4 Hz, H-7), 7.66e7.68 (m, 2H, H-50,70). COSY: [H-6: H-7], [H-7’:
H-8’]. 13C-NMR & HSQC
d
ppm (DMSO‑d6 þ NaH þ D2O): 159.6,
153.8, 148.0, 143.6, 143.1, 142.5, 138.7, 131.8, 120.05, 116.6 (CH-8’),
115.7 (CH-7), 115.3, 114.7 (CH-6), 109.6 (CH-4), 64.5, 64.4. MS (ESIþ)
m/z: 310 (MþH, 65%), 155 (100%). Anal Calcd for C16H15N5O2 . 2HCl .
2H2O: C, 45.94; H, 5.06; N, 16.74. Found: C, 45.54; H, 5.03; N, 17.06.
2.1.1.5.16. 1-(2-(Benzo[b]thiophene-3-yl)-1H-benzimidazole-5(6)-
yl)guanidine HCl (19). Prepared from 3 and Na2S2O5 adduct of
benzo[b]thiophene-3-carboxyaldehyde (0.29 g) as described in
general method. The resulting precipitate was purified with col-
umn chromatography using (CH2Cl2: MeOH: NH4OH 50 : 30: 3) as
eluant. Crude product was converted to HCl salt, yield 35%,
d
ppm (CD3OD): 162.3(C-2), 158.6 (C-guanidine), 154.9 (C-40), 151.8
(C-3’’), 150.95 (C-1’’), 147.7 (C-4’’), 130.7 (C-5), 129.75 (CH-20,60),
124.6 (C-10), 122.3 (CH-6), 118.65 (CH-30,50), 113.95 (CH-5’’), 112.9
(CH-6’’), 106.5 (CH-2’’), 57.0 (4’’-OCH3), 56.6 (3’’-OCH3). MS (ESIþ)
m/z: 404 (MþH, 60%), 202 (100%). Anal Calcd for C22H21N5O3 . 2HCl .
6H2O: C, 45.21; H, 6.03; N, 11.98. Found: C, 45.22; H, 5.13; N, 12.37.
2.1.1.5.12. 1-(2-(Naphthalene-1-yl)-1H-benzimidazole-5(6)-yl)
guanidine (15). Prepared from
3 and Na2S2O5 adduct of 1-
Naphthylbenzaldehyde (0.29 g) as described in general method.
The resulting precipitate was purified with column chromatog-
raphy using (CH2Cl2: MeOH: NH4OH 50 : 30: 5) as eluant, yield 45%,
mp > 300 ꢀC. 1H-NMR
d
ppm (DMSO‑d6 þ NaH þ D2O): 6.39 (dd,1H,
J ¼ 8.4 & 2.4 Hz, H-6), 6.95 (d, 1H, J ¼ 2 Hz, H-4), 7.32e7.44 (m, 3H,
H-7,50,60), 7.91 (d, 1H, J ¼ 8 Hz, H-40), 8.07 (s, 1H, H-20), 9.11 (d, 1H,
J ¼ 7.6 Hz, H-70). COSY: [H-6: H-7], [H-4’: H-5’], [H-5’: H-6’], [H-6’:
mp:
280e286
ꢀC
(bubling).
1H-NMR
d
ppm
(DMSO‑d6 þ NaH þ D2O): 6.41 (dd, 1H, J ¼ 8.4 & 2 Hz, H-6), 6.99 (d,
1H, J ¼ 2 Hz, H-4), 7.37 (d, 1H, J ¼ 8.4 Hz, H-7), 7.45e7.53 (m, 3H),
7.79 (d,1H, J ¼ 8.4 Hz), 7.86e7.89 (m, 1H), 8.15 (dd, 1H, J ¼ 7.2 &
H-7’]. 13C-NMR & HSQC & HMBC
d
ppm (DMSO‑d6 þ NaH þ D2O):
157.5 (C-2), 154.3 (C-guanidine), 147.6 (C-5), 143.3 (C-7a), 140.5 (C-
3'a), 139.3 (C-3a), 138.5 (C-7'a), 134.7 (C-30), 126.7 (CH-70), 124.6
(CH-50,60), 123.7 (CH-20), 122.9 (CH-40), 116.4 (CH-7), 115.6 (CH-6),
110.1 (CH-4). MS (ESIþ) m/z: 308 (MþH, 42%), 154 (100%). Anal
1.2 Hz), 9.51e9.53 (m,1H). COSY: [H-6: H-7]. 13C-NMR
d ppm
(DMSO‑d6 þ NaH þ D2O): 160.7, 153.8, 147.9, 143.65, 139.1, 135.8,
134.1, 131.7, 129.0, 128.0, 127.6, 126.8, 125.8, 125.5, 116.3, 115.1, 109.9.
MS (ESIþ) m/z: 302 (MþH, 52%),151 (100%). Anal Calcd for C18H15N5
.
.
Calcd for C16H13N5S 2HCl 2.5H2O: C, 45.18; H, 4.73; N, 16.46; S,
7.53. Found: C, 45.62; H, 4.002; N, 16.78; S, 7.90.
.
.
4H2O 0.2CH3OH: C, 48.19; H, 4.39; N, 15.43. Found: C, 47.90; H,
4.25; N, 15.13.
2.1.1.5.17. 1-(2-([2,20-Bithiophene]-5-yl)-1H-benzimidazole-5(6)-
yl)guanidine HCl (20). Prepared from 3 and Na2S2O5 adduct of 2,20-
bithiophene-5-carboxyaldehyde (0.33 g) as described in general
method. The resulting precipitate was purified with column chro-
matography using (CH2Cl2: MeOH: NH4OH 60 : 30: 2) as eluant.
Crude product was converted to HCl salt, yield 25%, mp > 300 ꢀC.
2.1.1.5.13. 1-(2-(Isoquinoline-5-yl)-1H-benzimidazole-5(6)-yl)
guanidine HCl (16). Prepared from 3 and Na2S2O5 adduct of iso-
quinoline-5-carboxyaldehyde (0.29 g) as described in general
method. The resulting precipitate was purified with column chro-
matography using (CH2Cl2: MeOH: NH4OH 50 : 30: 5) as eluant.
6

F. Doganc, I. Celik, G. Eren et al.
European Journal of Medicinal Chemistry 221 (2021) 113545
1H-NMR
d
ppm (D2O): 6.83 (s, 1H, H-4’’), 6.84 (d, 1H, J ¼ 4 Hz), 6.98
2.3. Computational details
(dd, 1H, J ¼ 3.6 & 1.2 Hz), 7.04 (dd, 1H, J ¼ 8.8 & 2 Hz, H-6), 7.15 (d,
1H, J ¼ 2 Hz, H-4), 7.24 (dd, 1H, J ¼ 5.2 & 1.2 Hz), 7.34 (s, 1H, H-7),
7.37 (d, 1H, J ¼ 4 Hz). COSY: [H-6: H-7], [H-3’: H-4’], [H-3’’: H-4’’],
2.3.1. Molecular docking
The molecular docking studies were carried out using Glide
[H-4’’: H-5’’]. 13C-NMR
d
ppm (D2O): 158.9, 148.45, 146.2, 137.6,
implemented in the Schrodinger Small-Molecule Drug Discovery
€
€
137.15, 135.5, 134.75, 133.9, 131.2, 129.8, 128.5, 127.2, 126.4, 124.9,
Suite (Small-Molecule Drug Discovery Suite 2020-2, Schrodinger,
117.7, 112.9. MS (ESIþ) m/z: 340 (MþH, 100%). Anal Calcd for
LLC, New York, NY, 2020). The compounds which were built via
builder panel in Maestro were subjected to ligand preparation by
.
.
C
16H13N5S2 2HCl 2.5H2O: C, 42.01; H, 4.40; N, 15.31; S, 14.02.
€
€
Found: C, 41.92; H, 3.98; N, 15.22; S, 14.31.
LigPrep (Schrodinger Release 2020-2: LigPrep, Schrodinger, LLC,
New York, NY, 2020) using default conditions. The x-ray crystal
structure of DB819-d(CGCGAATTCGCG)2 complex (PDB code: 3OIE)
[17] was retrieved from the Protein Data Bank. The protein was
prepared using the Protein Preparation Wizard tool. Water mole-
cules and metal ions were deleted and hydrogen atoms were added
followed by the assignment of all atom charges and atom types.
Finally, energy minimization and refinement of the structures were
done up to 0.3 Å RMSD by applying the OPLS3e force field. The
centroid of the x-ray ligand was defined as the grid box. van der
Waals (vdW) radius scaling factor 1.00, partial charge cutoff 0.25,
and OPLS3e force filed were used for receptor grid generation. The
docking protocol was validated by a RMSD value of 1.22 which was
predicted by superimposing the bioactive conformation of x-ray
ligand and its redocked conformation. The compounds prepared by
LigPrep were docked into DNA using the extra-precision (XP)
docking mode of the Glide without any constraints and a 0.75 vdW
radius scaling factor and 0.15 partial charge cutoff [18]. The protocol
facilitates docking by ligand flexibility and generation of multiple
conformers within the rigid receptor. The best conformation for
each compound was chosen based on the lowest XP glide score.
2.1.1.5.18. 1-(2-(5-Phenylthiophene-2-yl)-1H-benzimidazole-
5(6)-yl)guanidine HCl (21). Prepared from 3 and Na2S2O5 adduct of
5-phenylthiophene-2-carboxyaldehyde (0.32 g) as described in
general method. The resulting precipitate was purified with col-
umn chromatography using (CH2Cl2: MeOH: NH4OH 50 : 30: 1) as
eluant. Crude product was converted to HCl salt, yield 41%, mp:
200e204 ꢀC (bubling). 1H-NMR
d
ppm (DMSO‑d6 þ NaH þ D2O):
6.40 (dd, 1H, J ¼ 8.4 & 2 Hz, H-6), 6.86 (d, 1H, J ¼ 2 Hz, H-4),
7.22e7.26 (m, 2H), 7.36e7.40 (m, 3H), 7.54 (d, 1H, J ¼ 4 Hz), 7.64 (d,
1H,
J
¼
6.8 Hz). COSY: [H-6: H-7]. 13C-NMR
d
ppm
(DMSO‑d6 þ NaH þ D2O): 169.0, 156.1, 154.7, 148.1, 143.7, 142.0,
141.9, 140.1, 135.1, 130.1, 128.2, 125.8, 125.3, 124.9, 116.6, 116.5, 110.2.
MS (ESIþ) m/z: 344 (MþH, 61%), 167 (100%). Anal Calcd for
C
18H15N5S . 2HCl . 2.5H2O: C, 47.89; H, 4.91; N, 15.51; S, 7.10. Found:
C, 48.29; H, 4.77; N, 15.90; S, 6.69.
2.1.1.5.19. 1-(2-(5-(4-Chlorophenyl)isoxazole-3-yl)-1H-benzimid-
azole-5(6)-yl)guanidine HCl (22). Prepared from 3 and Na2S2O5
adduct of 5-(4-chlorophenyl)isoxazole-3-carboxyaldehyde (0.34 g)
as described in general method. The resulting precipitate was pu-
rified with column chromatography using (CH2Cl2: MeOH: NH4OH
50 : 20: 2) as eluant. Crude product was converted to HCl salt, yield
34%, mp: 241e246 ꢀC (bubling). 1H-NMR
d
ppm (CD3OD): 7.56 (d,
2.3.2. Molecular dynamics
1H, J ¼ 2 Hz, H-6), 7.58e7.61 (m, 3H), 7.84 (d, 1H, J ¼ 1.6 Hz, H-4),
Molecular dynamics simulations study was performed using
GROMACS 2020.4 [19] version (GROningen MAChine for Chemical
Simulations). The DNA topology file was created using the
Amber99sb-ildn force field [20] and the TIP3P water model was
used. The topology file of compounds 7 and 14 were created via
ACPYPE Server [21] (https://www.bio2byte.be/acpype/) by select-
ing bond charge correction (BCC) charge method and general
Amber force field (GAFF) atom type and net charge þ1. Three
separate system files were created for DNA apo form, DNA-
compound 7 and 14 holo forms. Systems were solvated using
Simple Point Charge water (SPC) 216 and 21 Naþ were added to
equilibrate the systems charges. System energy was minimized, 10
ns duration canonical ensemble (amount of substance (N), pressure
(P) and temperature (T) - NPT) and isothermal-isobaric (amount of
substance (N), volume (V) and equilibrium steps temperature (T) -
NVT) stages were performed, and 200 ns duration molecular dy-
namics simulations were performed. RMSD, RMSF and intermo-
lecular hydrogen bond analyzes were performed. Finally, the
average interaction energy between protein and ligand was calcu-
lated according to short-range Lennard-Jones energy. The results
were monitored using Visual Molecular Dynamics (VMD) [22] and
trajectory graphs were created with QtGrace tools.
7.95e7.97 (m, 3H). COSY: [H-6: H-7], [H-2’’,6’’: H-3’’,5’’]. 13C-NMR
d
ppm (CD3OD): 173.4, 158.4, 152.7, 142.7, 138.75, 135.3, 134.7, 132.9,
130.85, 128.85, 126.3, 126.0, 117.3, 113.1, 100.4. MS (ESIþ) m/z: 353
(MþH, 100%), 355 (MþHþ2, 34%). Anal Calcd for C17H13ClN6O . 2HCl
. 2H2O: C, 44.22; H, 4.14; N, 18.20. Found: C, 43.78; H, 4.12; N, 18.33.
2.1.1.5.20. 1-(2-(4-Methylthiazole-5-yl)-1H-benzimidazole-5(6)-
yl)guanidine HCl (23). Prepared from 3 and Na2S2O5 adduct of 4-
methylthiazole-5-carboxyaldehyde (0.25 g) as described in gen-
eral method. The resulting precipitate was crystallised from EtOH.
Crude product was converted to HCl salt, yield 70%, mp:
265e270 ꢀC (bubling). 1H-NMR
d ppm (CD3OD): 2.82 (s, 3H, -CH3),
7.57 (dd,1H, J ¼ 9.2 & 2 Hz, H-6), 7.83 (d, 1H, J ¼ 1.6 Hz, H-4), 7.95 (d,
1H, J ¼ 9.2 Hz, H-7), 9.45 (s, 1H, H-20). COSY: [H-6: H-7]. 13C-NMR &
HSQC & HMBC d
ppm (CD3OD): 160.3 (CH-20), 159.2 (C-50), 158.4 (C-
guanidine), 145.0 (C-2), 135.2 (C-5), 133.8 (C-3a), 132.2 (C-7a), 126.1
(CH-6), 116.7 (CH-7), 115.4 (C-40), 112.6 (CH-4), 16.9 (40-CH3). MS
.
.
(ESIþ) m/z: 273 (MþH, 100%). Anal Calcd for C12H12N6S 2HCl
4H2O: C, 34.53; H, 5.31; N, 20.13; S, 7.68. Found: C, 34.53; H, 4.41; N,
19.70; S, 8.05.
2.2. Biology
2.3.3. Theoretical ADME calculations
Computational analysis was performed using SwissADME
(http://www.swissadme.ch/) to estimate the physicochemical
properties, pharmacokinetic properties and ADME parameters of
the compounds [23].
2.2.1. Antiprotozoal activity
In vitro assays with P. falciparum NF54, T.b. rhodesiense STIB900,
T. cruzi Tulahuen C4 and L. donovani MHOM-ET-67/L82 were carried
out as previously reported [16].
3. Results and discussion
2.2.2. Cytotoxicity studies
3.1. Antiparasitic activity evaluation
Cytotoxicity was evaluated using cultured L-6 rat myoblast cells
and an Alamar Blue assay as previously reported [16].
All targeted guanidinobenzimidazole compounds 4e23
7

F. Doganc, I. Celik, G. Eren et al.
European Journal of Medicinal Chemistry 221 (2021) 113545
(Table 1), were tested in vitro in serial drug dilution assays for
antiparasitic activity against P. falciparum, T.b. rhodesiense, T. cruzi
and L. donovani. Cytotoxicity of the synthesized compounds was
determined using the Alamar Blue assay with rat skeletal myoblasts
(L6 cells). Based on the outcomes of this in vitro study these com-
pounds seem to have no cytotoxicity liability. The IC50 values of the
compounds against P. falciparum ranged from 0.018 to 37.15
(Table 1). Best inhibitory activity against P. falciparum were ob-
tained with 7 (IC50 ¼ 0.018 g/mL and SI ¼ 2360) and 14
(IC50 ¼ 0.052 g/mL and SI > 1920) which are possessing biphenyl
mg/mL
m
m
and 4-(3,4-dimethoxyphenoxy)phenyl groups at C-2 position,
respectively. In addition, compounds 13, 17 and 20 also showed
good inhibitory activity with the IC50 values of 0.153, 0.168 and
0.19 mg/mL, respectively. The common feature of these molecules is
that they all possess bulky aromatic rings such as biphenyl, 4-(3,4-
dimethoxy-phenoxy)phenyl, fluorene and bithiophene at the C-2
position of benzimidazole moiety.
The inhibitory effect of the tested molecules against the trypa-
nosomatid parasites was moderate at best. Three compounds, 11
(IC50 ¼ 6.04
mL) exhibited moderate activity against T.b. rhodesiense and one
g/mL) against T. cruzi. None of the
mg/mL), 16 (IC50 ¼ 3.8
mg/mL) and 22 (IC50 ¼ 5.62
mg/
compound, 16 (IC50 ¼ 8.57
m
tested compounds was active against L. donovani.
3.2. Molecular docking studies
To predict the preferred orientation of compounds 7 and 14
inside the DNA, molecular docking studies were performed. The
obtained results indicated that both compounds bound in the mi-
nor groove of the d(CGCGAATTCGCG)2 duplex. The resulting lowest
binding energies for 7 and 14 were found to be ꢁ9.372 kcal/mol
and ꢁ9.764 kcal/mol, respectively. The orientation of 7 allowed to
stack with the center of the DNA minor groove covering almost five
base pairs. The protonated guanidine moiety extended towards
from the groove, leading the NH groups to be closer to DNA
phosphate moiety and favorably interacted with G22 via salt bridge
interaction and H-bonding. The NH moiety of benzimidazole ring
involved in H-bonding with T19 while the aromatic H-bondings
were also observed formed with T7, T8, T19, and T20 base pairs
enhancing the complex stability. In the case of 14, a similar binding
conformation was observed to that of 7, but slightly bended
compared to 7. Compound 14 occupied the region of about six base
pairs in DNA minor groove interacting with the single monomer of
the DNA structure via H-bonding between the guanidine and
carbonyl oxygen of C21 and between the N1 hydrogen of the
benzimidazole ring and the carbonyl oxygen of T19. On the other
hand, the aromatic H-bondings with T7, T8 and C9 carbonyl oxy-
gens through the aromatic hydrogens of phenyl rings were also
observed allowing interaction with the other monomer of the DNA
structure (Fig. 4).
Fig. 4. Predicted binding modes of compounds 7 (A) and 14 (B) in DNA structure (PDB
code: 3OIE). Dashed-yellow lines represent hydrogen bonds and dashed-red lines
represent aromatic hydrogen bonds.
14 with DNA. Trajectory analysis of RMSD, RMSF, intermolecular
hydrogen bond and protein-ligand interaction was performed.
RMSD measurements are the main parameters used to measure
protein-DNA stability and deviations in molecular dynamics sim-
ulations [26]. As shown in Fig. 5a, the RMSD values of the apo form
(3OIE-Apo), compounds 7 (3OIE-LIG7) and 14 (3OIE-LIG14) holo
forms were measured below 0.5 nm 3OIE-Apo, 3OIE-LIG7 and
3OIE-LIG14 gave an average RMSD value of 0.294 nm, 0.334 nm and
0.283 nm, respectively. The interaction of compounds 7 and 14 with
DNA did not cause a significant change in stability. The deviation of
the ligands during the 200 ns simulation period was also analyzed.
As shown in Fig. 5b, compound 7 measured a small deflection of
less than 0.1 nm during 130 ns and an RMSD value of less than 0.15
after 130 ns. Compound 14 showed deviations below 0.15 nm up to
60 ns, RMSD value up to 0.25 nm between 60 and 110 ns and a
fluctuating value below 0.15 nm after 110 ns.
Another important parameter is the RSMF calculation used to
measure the fluctuation and conformational change of macromol-
ecules [27]. As an indicator of the stability and binding strength of
the ligand, it should reduce the fluctuation of the residues with
which it interacts at the active site and increases its stability. RMSF
analysis performed based on residue is given in Fig. 5c for 3OIE-
Apo, 3OIE-LIG7, and 3OIE-LIG14. Besides, in Fig. 6, the 2D diagram
3.3. Molecular dynamics simulations
Molecular dynamics simulations are the basic computational
method used in drug design and development processes to mea-
sure the interaction stability and energy of small molecule lead
compounds with biological macromolecular molecules such as
protein, DNA and RNA [24,25]. To monitor the behavior of the DNA-
ligand complex formed by the protozoal DNA of compounds 7 and
14 obtained by molecular docking under in silico physiological
conditions and to calculate the stability due to time, a molecular
dynamic simulation of 200 ns was performed. Also, ligand-free
DNA was simulated in the environment, conditions and time to
ensure the reliability of the created molecular dynamics system and
to detect the changes caused by the interaction of compounds 7 and
8

F. Doganc, I. Celik, G. Eren et al.
European Journal of Medicinal Chemistry 221 (2021) 113545
Fig. 5. (a) DNA-root mean square deviation (RMSD), (b) Ligand-RMSD and (c) root mean square fluctuation (RMSF), analysis of the apo form (3OIE-Apo), compounds 7 (3OIE-LIG7)
and 14 (3OIE-LIG14) holo forms of protozoal DNA throughout 200 ns.
of the DNA-ligand interactions obtained with Glide XP at the DNA
active site of compounds 7 and 14 is presented. When the RMSF
graph and the 2D DNA-ligand interaction diagram are evaluated
together, the base pairs T19 and G22 of B chain in which they form
hydrogen bonds show less fluctuation than the apo form and it is
understood to make it more stable. Compound 14, on the other
hand, gave lower RMSF values compared to the apo form in T19 and
C21 base pairs of B chain which are involved in hydrogen bonding
was formed and the DNA became more stable.
The presence and number of hydrogen bonds in protein-ligand
or DNA-ligand interactions could indicate that the ligand will
interact more with the macromolecule and form a more stable
complex [28]. Therefore, the time-dependent number and change
of hydrogen bonds were analyzed. As shown in Fig. 7a, during the
200 ns simulation, compound 7 often formed two hydrogen bonds,
sometimes three hydrogen bonds and occasionally four hydrogen
bonds. As shown in Fig. 7b, compound 14 frequently formed four to
five hydrogen bonds. Besides hydrogen bond analysis, the Lennard-
Jones DNA-ligand interaction energy was measured over time.
Lennard-Jones energy is an energy measurement method that is
widely used in molecular dynamics simulations to calculate the
interaction energies of molecules that are not directly bonded but
are standing together [29]. As given in Fig. 7c, the 3OIE-LIG7
complex produced approximately ꢁ150 kJ molꢁ1 to 175 kJ molꢁ1
,
while 3OIE-LIG14 produced ꢁ175 kJ molꢁ1 to ꢁ250 kJ molꢁ1 DNA
ligand binding energy, respectively. ꢁ167.75 kJ molꢁ1 and
-220.671 kJ molꢁ1 average short-range Lennard-Jones gave energy
values.
3.4. Computational ADME estimations
Many drug molecule candidates do not have suitable
9

Fig. 6. 2D Schematic diagram describing the DNA-ligand interactions obtained with Glide XP at the DNA active site of (A) x-ray ligand, compounds (B) 7 and (C) 14 (PDB ID: 3OIE). (D) The interaction fingerprint matrix that identifying
the binding patterns of compounds with DNA (PDB ID: 3OIE).

F. Doganc, I. Celik, G. Eren et al.
European Journal of Medicinal Chemistry 221 (2021) 113545
Fig. 7. (a, b) Intermolecular H bonds number between protozoal DNA and compounds 7e14, (c) the short-range Lennard-Jones DNA-ligand interaction energy for 200 ns (PDB ID:
3OIE).
pharmacokinetic properties, although they show active properties
during phase studies, they fail to succeed. For this reason, it is
beneficial to calculate some computational and predictable pa-
rameters. Molecular weight, number of rotatable bonds, hydrogen
acceptors, hydrogen donors, molar refractivity, and octanol/water
partition coefficient were calculated. Lipinski and Ghose were
evaluated according to the restrictive rules. According to Lipinski's
rule of 5, MW ꢂ 500, MLogP ꢂ4.15, N or O ꢂ 10 and NH or OH ꢂ 5
4. Conclusions
This study indicated that 2-(aryl-substituted-1H-benzimid-
azole-5(6)-yl)guanidine derivatives show a good inhibitory activity
profile against P. falciparum. Especially, two of them (7, 14) showed
close activity against P. falciparum compared to reference drug
chloroquine. It is apparent that substitution of C-2 position with
bulky aromatic rings is critical for optimal anti-malarial activity.
These compounds also showed moderate activity against T.b. rho-
desiense as compared to the reference drug melarsoprol. None of
the tested compounds exhibited a good activity against T. cruzi or
L. donovani. Molecular docking studies for compounds 7 and 14
supported the biological data indicating that these compounds are
interacting DNA through minor groove binding. According to the
molecular dynamics study, compounds 7 and 14 remain stable
during 200 ns simulation, form 4 to 5 hydrogen bonds and average
short-range Lennard-Jones DNA-ligand energy of ꢁ167.75 kJ molꢁ1
and -220.67 kJ molꢁ1 form, respectively. In vivo studies of
[30],
according
to
Ghose's
rule,
160 ꢂ MW ꢂ 480, ꢁ0.4 ꢂ WLogP ꢂ 5.6, 40 ꢂ MR ꢂ 130 and
20 ꢂ atoms ꢂ 70 [31]. Accordingly, as seen in Table 2, all of the
compounds comply with Lipinski's and Ghose's rules. All calculated
parameters are given in supporting data. According to theoretical
calculations, the ADME parameters of the compounds are within
suitable limits.
Table 2
Calculated and evaluated SwissADME parameters.
Comp. MW
#Rotatable bonds #H-bond acceptors #H-bond donors MLogP WLogP MR
GI absorption Lipinski #violations Ghose #violations
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
251.29
285.73
276.30
327.38
295.30
357.41
343.38
287.27
279.34
311.34
403.43
301.35
302.33
339.39
309.32
307.37
339.44
333.41
352.78
272.33
3
3
3
4
4
6
5
3
3
5
7
3
3
3
3
3
4
4
4
3
2
2
3
2
4
3
3
4
2
4
5
2
3
2
4
2
2
2
4
3
4
4
4
4
5
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
1.92
2.45
1.29
3.15
1.31
2.83
2.88
2.72
2.43
1.35
2.27
2.73
1.68
3.38
1.35
2.55
2.28
2.73
2.20
0.61
2.34
3.00
2.22
4.01
2.04
3.77
4.14
3.46
2.96
2.36
4.15
3.50
2.89
3.92
2.12
3.56
4.13
4.07
3.65
2.11
77.13
82.14
81.85
102.57 High
84.09 High
108.11 High
103.65 High
77.05
87.07
90.12
High
High
High
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
High
High
High
116.63 High
94.64
92.43
High
High
105.58 High
88.00
92.52
98.32
100.45 High
97.64
77.77
High
Low
High
High
High
MW: Molecular weight. #Rotatable bonds: Number of rotatable bonds. #H-bond acceptors: Number of hydrogen acceptors. #H-bond donors: Number of hydrogen donors.
MLogP: Topological method - WLogP atomistic method octanol/water partition coefficient. MR: Molar refractivity. GI: Gastrointestinal absorption.
11

F. Doganc, I. Celik, G. Eren et al.
European Journal of Medicinal Chemistry 221 (2021) 113545
Opin. Ther. Pat. 19 (10) (2009) 1417e1448, https://doi.org/10.1517/
compounds 7 and 14 are in progress, to test efficacy in an animal
model of infection and to establish pharmacokinetic profiles.
13543770903216675.
€
[12] C. Karaaslan, M. Kaiser, R. Brun, H. Goker, Synthesis and potent antiprotozoal
activity of mono/di amidino 2-anilinobenzimidazoles versus Plasmodium fal-
ciparum and Trypanosoma brucei rhodesiense, Bioorg. Med. Chem. 24 (2016)
4038e4044, https://doi.org/10.1016/j.bmc.2016.06.047.
Declaration of competing interest
€
[13] M. Alp, H. Goker, R. Brun, S. Yıldız, Synthesis and antiparasitic and antifungal
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.
evaluation
of
2’-arylsubstituted-1H,1’H-[2,5’]bisbenzimidazolyl-5-
carboxamidines, Eur. J. Med. Chem. 44 (2009) 2002e2008, https://doi.org/
10.1016/j.ejmech.2008.10.003.
[14] T.J. Church, N.S. Cutshall, A.R. Gangloff, T.E. Jenkins, M.S. Linsell, J. Litvak,
K.D. Rice, J.R. Spencer, V.R. Wang, Compounds and Compositions for Treating
Diseases Associated with Serine Protease, Particularly Tryptase, Activity, 1998.
WO 98/45275.
Acknowledgements
[15] H.F. Ridley, R.G.W. Spickett, G.M. Timmis, A new synthesis of benzimidazoles
This work was supported by Ankara University Scientific
Research Fund (BAP-17L0237006). Central Laboratory of Pharmacy
Faculty of Ankara University provided support for acquisition of the
NMR, Mass spectrometer and elemental analyzer used in this work.
All molecular dynamics simulations reported were performed uti-
lizing TUBITAK (The Scientific and Technological Research Council
of Turkey), ULAKBIM (Turkish Academic Network and Information
Centre), High Performance and Grid Computing Centre (TRUBA
resources).
and aza-analogs, J. Heterocycl. Chem.
10.1002/jhet.5570020424.
2 (1965) 453e456, https://doi.org/
[16] F.A. Bernal, M. Kaiser, B. Wünsch, T.J. Schmidt, Structure-activity relationships
of cinnamate ester analogues as potent antiprotozoal agents, ChemMedChem
15 (1) (2020) 68e78, https://doi.org/10.1002/cmdc.201900544.
[17] Y. Chai, M. Munde, A. Kumar, L. Mickelson, S. Lin, N.H. Campbell, M. Banerjee,
S. Akay, Z. Liu, A.A. Farahat, R. Nhili, S. Depauw, M.H. David-Cordonnier,
S. Neidle, W.D. Wilson, D.W. Boykin, Structure-dependent binding of aryli-
midamides to the DNA minor groove, Chembiochem 15 (2014) 68e79,
https://doi.org/10.1002/cbic.201300622.
[18] R.A. Friesner, R.B. Murphy, M.P. Repasky, L.L. Frye, J.R. Greenwood,
T.A. Halgren, P.C. Sanschagrin, D.T. Mainz, Extra precision glide: docking and
scoring incorporating a model of hydrophobic enclosure for protein-ligand
complexes, J. Med. Chem. 49 (2006) 6177e6196, https://doi.org/10.1021/
jm051256o.
Appendix A. Supplementary data
[19] M.J. Abraham, T. Murtola, R. Schulz, S. Pall, J.C. Smith, B. Hess, E. Lindahl,
GROMACS: High performance molecular simulations through multi-level
parallelism from laptops to supercomputers, SoftwareX 1 (2015) 19e25,
https://doi.org/10.1016/j.softx.2015.06.001.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113545.
[20] K. Lindorff-Larsen, S. Piana, K. Palmo, P. Maragakis, J.L. Klepeis, R.O. Dror,
D.E. Shaw, Improved side-chain torsion potentials for the Amber ff99SB
protein force field, Proteins Struct. Funct. Bioinf. 78 (8) (2010) 1950e1958,
https://doi.org/10.1002/prot.22711.
[21] A.W.S. da Silva, W.F. Vranken, ACPYPE-Antechamber python parser interface,
BMC Res. Notes 5 (1) (2012) 1e8, https://doi.org/10.1186/1756-0500-5-367.
[22] W. Humphrey, A. Dalke, K. Schulten, VMD: visual molecular dynamics, J. Mol.
Graph. 14 (1) (1996) 33e38, https://doi.org/10.1016/0263-7855(96)00018-5.
[23] A. Daina, O. Michielin, V. Zoete, SwissADME: a free web tool to evaluate
pharmacokinetics, drug-likeness and medicinal chemistry friendliness of
References
[1] WHO neglected tropical diseases. https://www.who.int/neglected_diseases/
diseases/en/, 2019. (Accessed 1 October 2019) accessed.
[2] M.N.C. Soeiro, E.M. de Souza, C.E. Stephens, D.W. Boykin, Aromatic diamidines
as antiparasitic agents, Expet Opin. Invest. Drugs 14 (8) (2005) 957e972,
https://doi.org/10.1517/13543784.14.8.957.
[3] M.S. Bartlett, T.D. Edlind, C.H. Lee, R. Dean, S.F. Queener, M.M. Shaw,
J.W. Smith, Albendazole inhibits Pneumocystis carinii proliferation in inocu-
lated immunosuppressed mice, Antimicrob. Agents Chemother. 38 (8) (1994)
1834e1837, https://doi.org/10.1128/AAC.38.8.1834.
small molecules, Sci. Rep.
srep42717.
7 (1) (2017) 1e13, https://doi.org/10.1038/
[4] M.A. Ismail, A. Batista-Parra, Y. Miao, W.D. Wilson, T. Wenzler, R. Brun,
D.W. Boykin, Dicationic near-linear biphenyl benzimidazole derivatives as
DNA-targeted antiprotozoal agents, Bioorg. Med. Chem. 13 (2005)
6718e6726, https://doi.org/10.1016/j.bmc.2005.07.024.
[24] H. Zhao, A. Caflisch, Molecular dynamics in drug design, Eur. J. Med. Chem. 91
(2015) 4e14, https://doi.org/10.1016/j.ejmech.2014.08.004.
ꢀ
ꢀ
[25] P. Sledz, A. Caflisch, Protein structure-based drug design: from docking to
molecular dynamics, Curr. Opin. Struct. Biol. 48 (2018) 93e102, https://
doi.org/10.1016/j.sbi.2017.10.010.
€
[5] H. Goker, D.W. Boykin, S. Yıldız, Synthesis and potent antimicrobial activity of
some novel 2-phenyl or methyl-4H-1-benzopyran-4-ones carrying amidino-
benzimidazoles, Bioorg. Med. Chem. 13 (2005) 1707e1714, https://doi.org/
10.1016/j.bmc.2004.12.006.
[26] K. Sargsyan, C. Grauffel, C. Lim, How molecular size impacts RMSD applica-
tions in molecular dynamics simulations, J. Chem. Theor. Comput. 13 (4)
(2017) 1518e1524, https://doi.org/10.1021/acs.jctc.7b00028.
[27] L. Martínez, Automatic identification of mobile and rigid substructures in
molecular dynamics simulations and fractional structural fluctuation analysis,
[6] D.W. Boykin, Antimicrobial activity of the DNA minor groove binders fur-
amidine and analogs, J. Braz. Chem. Soc. 13 (6) (2002) 763e771, https://
doi.org/10.1590/S0103-50532002000600006.
[7] S. Mallena, M.P.H. Lee, C. Bailly, S. Neidle, A. Kumar, D.W. Boykin, W.D. Wilson,
Thiophene-based diamidine forms a “super” AT binding minor groove agent,
J. Am. Chem. Soc. 126 (2004) 13659e13669, https://doi.org/10.1021/
ja048175m.
[8] C.E. Stephens, R. Brun, M.M. Salem, K.A. Werbovetz, F. Tanious, W.D. Wilson,
D.W. Boykin, The activity of diguanidino and ‘reversed’ diamidino 2,5-
diarylfurans versus Trypanosoma cruzi and Leishmania donovani, Bioorg.
Med. Chem. Lett 13 (2003) 2065e2069, https://doi.org/10.1016/S0960-
894X(03)00319-6.
[9] R.K. Arafa, R. Brun, T. Wenzler, F.A. Tanious, W.D. Wilson, C.E. Stephens,
D.W. Boykin, Synthesis, DNA affinity, and antiprotozoal activity of fused ring
dicationic compounds and their prodrugs, J. Med. Chem. 48 (2005)
5480e5488, https://doi.org/10.1021/jm058190h.
PloS
One
10
(3)
(2015),
e0119264,
https://doi.org/10.1371/
journal.pone.0119264.
[28] H. Wang, Z. Gao, P. Song, B. Hu, J. Wang, M. Cheng, Molecular dynamics
simulation and QM/MM calculation reveal the selectivity mechanism of type I
1/2 kinase inhibitors: the effect of intramolecular H-bonds and conforma-
tional restriction for improved selectivity, Phys. Chem. Chem. Phys. 21 (43)
(2019) 24147e24164, https://doi.org/10.1039/c9cp04353e.
[29] T. Steinbrecher, D.L. Mobley, D.A. Case, Nonlinear scaling schemes for
Lennard-Jones interactions in free energy calculations, J. Chem. Phys. 127 (21)
(2007) 214108, https://doi.org/10.1063/1.2799191.
[30] C.A. Lipinski, Lead- and drug-like compounds: the rule-of-five revolution,
Drug Discov. Today Technol. 1 (4) (2004) 337e341, https://doi.org/10.1016/
j.ddtec.2004.11.007.
[31] A.K. Ghose, V.N. Viswanadhan, J.J. Wendoloski, A knowledge-based approach
in designing combinatorial or medicinal chemistry libraries for drug discov-
ery. 1. A qualitative and quantitative characterization of known drug data-
bases, J. Comb. Chem. 1 (1) (1999) 55e68, https://doi.org/10.1021/cc9800071.
[10] P.G. Baraldi, A. Bovero, F. Fruttarolo, D. Preti, M.A. Tabrizi, M.G. Pavani,
R. Romagnoli, DNA minor groove binders as potential antitumor and anti-
microbial agents, Med. Res. Rev. 24 (4) (2004) 475e528, https://doi.org/
10.1002/med.20000.
[11] F. Saczewski, L. Balewski, Biological activities of guanidine compounds, Expert
12