Biological evaluation, molecular docking, and sar studies of novel 2-(2,4-dihydroxyphenyl)-1h- benzimidazole analogues

Article abstract of DOI:10.3390/biom9120870

In the present study, new 4-(1H-benzimidazol-2-yl)-benzene-1,3-diols, modified in both rings, have been synthesized and their efficacies as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors have been determined. The modified Ellman’

Full text of DOI:10.3390/biom9120870

Article
Biological Evaluation, Molecular Docking, and SAR
Studies of Novel 2‐(2,4‐Dihydroxyphenyl)‐1H‐
Benzimidazole Analogues
1,
1
2
3
Joanna Matysiak *, Alicja Skrzypek , Monika Karpińska , Kamila Czarnecka ,
3
4
1,2
Paweł Szymański , Marek Bajda and Andrzej Niewiadomy
1
Department of Chemistry, University of Life Sciences in Lublin, Akademicka 15, 20‐950 Lublin, Poland;
alicja.skrzypek@up.lublin.pl (A.S.); andrzej.niewiadomy@up.lublin.pl (A.N.)
Łukasiewicz Research Network—Institute of Industrial Organic Chemistry, Annopol 6,
03‐236 Warsaw, Poland; karpinska@ipo.waw.pl
Department of Pharmaceutical Chemistry, Drug Analyses and Radiopharmacy, Faculty of Pharmacy,
Medical University of Lodz, Muszynskiego 1, 90‐151 Lodz, Poland; kamila.czarnecka@umed.lodz.pl (K.C.);
pawel.szymanski@umed.lodz.pl (P.S.)
2
3
4
Department of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian University Medical
College, Medyczna 9, 30‐688 Cracow, Poland; marek.bajda@uj.edu.pl
* Correspondence: joanna.matysiak@up.lublin.pl
Received: 11 November 2019; Accepted: 10 December 2019; Published: 12 December 2019
Abstract: In the present study, new 4‐(1H‐benzimidazol‐2‐yl)‐benzene‐1,3‐diols, modified in both
rings, have been synthesized and their efficacies as acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE) inhibitors have been determined. The modified Ellman’s
spectrophotometric method was applied for the biological evaluation. The compounds showed
strong (IC₅₀ 80–90 nM) AChE and moderate (IC₅₀ 5–0.2 μM) BuChE inhibition in vitro. Some
compounds were effective toward AChE/BuChE, exhibiting high selectivity ratios versus BuChE,
while the other compounds were active against both enzymes. The structure–activity relationships
were discussed. The compounds inhibited also in vitro self‐induced Aβ(1–42) aggregation and
exhibited antioxidant properties. The docking simulations showed that the benzimidazoles under
consideration interact mainly with the catalytic site of AChE and mimic the binding mode of
tacrine.
Keywords: benzimidazole; acetylcholinesterase; butyrylcholinesterase; inhibitor; beta amyloid;
molecular docking; lipophilicity; SAR
1. Introduction
The therapy of neurodegenerative diseases, including Alzheimer’s disease (AD), is a great
challenge in the modern world [1]. Both acetylcholinesterase (AChE) and butyrylcholinesterase
(BuChE) enzymes are promising molecular targets for AD drug design [2]. Many different
heterocyclic systems are explored in this field including the benzimidazole skeleton [3–6]. The amine
group is an integral part of the structure of numerous designed AChE inhibitors [4].
N‐substituted 2‐aminobenzimidazoles were described as good inhibitors of AChE and BuChE.
The molecular modeling studies revealed that the heterocyclic ring of the active molecule creates
face‐to‐face π–π stacking interactions in the form of a “sandwich” with the indole moiety of the
appropriate amino acids [5]. N‐differently substituted amine derivatives of 2‐arylbenzimidazoles
(compound a, Figure 1) were presented by Alpan et al. [6]. It was found that the compounds
exhibited significant AChE and/or BuChE inhibitory activities (IC50 = 0.6–0.1 μM) and some of them
Biomolecules 2019, 9, 870; doi:10.3390/biom9120870
www.mdpi.com/journal/biomolecules

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were selective against AChE/BuChE. The Mannich bases of 2‐(4‐hydroxyphenyl)benzimidazole
derivatives (e.g., compound b, Figure 1) display moderate to good AChE inhibitory potency with
IC50 ranging from 0.93 to 10 μM as well as moderate BuChE inhibitory activity. The molecular
modeling studies showed that, among others, the 4‐hydroxyl group participated in the crucial
interaction with AChE [7]. Moderate BuChE inhibitors were found in
a group of
2‐[4‐(4‐substitutedpiperazin‐1‐yl)phenyl]benzimidazoles. The most active compounds showed an
inhibitory effect on the self‐mediated amyloid β‐peptide(1–40) (Aβ(1–40)) aggregation [8]. Free of
amine group benzimidazole derivatives were also presented as AChE/BuChE inhibitors. They
include
2‐(chloro/nitro)arylbenzimidazoles
[9]
and
N‐substituted‐2‐aryl‐1H‐benzimidazole‐5‐carboxylates [10]. The highest activity against AChE was
found for 1‐(3‐(1H‐imidazol‐1‐yl)propyl)‐ 2‐(4‐nitrophenyl)‐1H‐benzimidazole‐5‐carboxylate
(compound c, Figure 1) [10]. The other researchers studied benzimidazole salts. A nanomolar
activity was found for N‐propylphthalimide‐ and 4‐vinylbenzyl‐substituted benzimidazole salts
(compounds d and e, Figure 1) [11]. A large group of imidazolium and benzimidazolium oximes
was designed for reactivation of phosphylated human BuChE [12]. A prominent (nanomolar)
activity against AChE/BuChE was found for the pyrazolobenzothiazine‐benzimidazole hybrids
(compound f, Figure 1) [13] and the benzimidazole‐based organophosphorus compounds [14]. Most
of the mentioned compounds are the multitargeted agents: carbonic anhydrase inhibitors [11],
2+
antioxidants [7], interrupters of cell cycle [9], Fe ion chelators [11]. Some of them show anticancer
potency [9,15] or neuroprotective properties [8]. Benzimidazole derivatives are also known as
β‐glucuronidase [16,17], α‐glucosidase [18,19], urease [20], and α‐amylase enzymes inhibitors [21].
O
N
N
N
O2N
N
a
IC50 = 0.13 µM
NO2
NH
N
Cl
OH
N
H
O
b IC50 = 0.93 µM
O
O
N
N
N
NH
N
NH
O
c
e
IC50 = 5.12 µM
N
N
Cl-
N
N
N
IC50 = 0.045 µM
Cl-
N
N
S
O
CH
f IC50 = 0.011 µM
O
3
CH3
d
IC50 = 0.056 µM
Figure 1. Structures of benzimidazole analogs as AChE inhibitors.
Molecular modeling studies for 5‐aryl‐4‐(1,3,4‐thiadiazol‐2‐yl)benzene‐1,3‐diols obtained by us
indicated a network of essential bonds between the ‐OH groups of the benzenediol ring and the
residues of amino acid AChE in the active site which results in a significant inhibitory effect against
AChE [22,23]. Some of them also displayed neuroprotective properties [24]. Therefore, a decision
was made to obtain new benzimidazoles with 2,4‐dihydroxyphenyl moiety as potential
AChE/BuChE inhibitors. Searching for the optimal substitution of the benzenediol ring, the third
group ‐OH or the hydrophobic substituent (‐Cl, ‐Me, ‐Et) was introduced. In the heterocyclic ring,
there were taken into consideration, among others, the substituents ‐OMe and ‐CF3 which intensified
the inhibition activity of 1,3,4‐thiadiazole derivatives.

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Previously studied 4‐(1H‐benzimidazol‐2‐yl)benzene‐1,3‐diols and their metal complexes
showed significant anticancer and antimicrobial potencies [25–28]. Polyphenol derivatives are
commonly known as antioxidants. Therefore, this is justified to create benzimidazole–resorcinol
conjugates with the additional potential antioxidant properties. It is also known that oxidative stress
has a significant impact on the pathogenesis of neurodegenerative diseases [2,29,30].
The paper presents the synthesis of new 1H‐benzimidazoles functionalized by the resorcinol
moiety and evaluation of their inhibitory activities against cholinesterases, as well as inhibition
mechanism studies, molecular docking, chromatography lipophilicity estimation, and structure–
activity elucidation. The evaluation of in vitro self‐induced Aβ(1–42) aggregation inhibition and
antioxidant properties were also performed.
2. Materials and Methods
2.1. Analytical Studies
Melting points (m.p.) were determined using a BÜCHI B‐540 (Flawil, Switzerland) melting
point apparatus and they were uncorrected. The elemental analysis (C, H, N) was performed on
Perkin‐Elmer 2400. The elemental analysis was within 0.5% of the theoretical value. The purity of
compounds was monitored by reverse‐phase high‐performance liquid chromatography (RP‐HPLC),
RP‐18/MeOH/H2O chromatography. The MS spectra (EI, 70 eV) were recorded using the apparatus
AMD‐604. The IR spectra were registered on the Perkin‐Elmer FT‐IR 1725X spectrophotometer (in
−1
1
13
KBr) in the range of 600–4000 cm . The NMR spectra (1D, H NMR, and C NMR) were recorded in
DMSO‐d6 using a Bruker DRX 500 (Bruker Daltonics, Inc. Billerica, MA, USA). Chemical shifts (,
ppm) were described in relation to tetramethylsilane (TMS) and the coupling constants (J) were
expressed in Hz.
2.2. Synthesis of the Compounds
The general procedure for the synthesis of the compounds is as follows. A mixture of the
corresponding
benzene‐1,2‐diamine
(3
mmol)
and
the
corresponding
sulfinylbis[(2,4‐dihydroxyphenyl)‐ methanethione] (STB) (3 mmol) in MeOH (20 mL) was heated to
reflux for 3–4 h. The hot mixture was filtered. Following, the filtrate was concentrated or left at room
temperature. Finally, the product was crystalized from the MeOH or MeOH/H2O solution.
4‐(1H‐benzimidazol‐2‐yl)‐6‐chlorobenzene‐1,3‐diol (1). Yield: 84%; m.p.: 340–341 °C; EI MS (m/z, %): 260
+
1
(M , 100), 231 (6), 197 (17), 169 (25), 143 (5), 116 (6), 92 (5), 69 (4), 65 (7), 63 (5), 52 (3), 36 (17); H NMR
(500 MHz, DMSO‐d6) δ: 14.12 (s, 1H, NH), 11.46 (s, 1H, C(3)–OH), 10.44 (s, 1H, C(1)–OH), 8.27 (s, 1H,
13
C(5)–H), 7.80 (m, 2H, C(4′,7′)–H), 7.49 (m, 2H, C(5′,6′)–H), 7.03 (s, 1H, C(2)–H) ppm; C NMR (125
MHz, DMSO‐d6) δ: 161.1, 157.3, 152.3, 140.3 (2C), 129.3, 123.2 (2C), 114.8 (2C), 112.6, 111.9, 103.1 ppm;
−1
IR (KBr, cm ): 3281 (OH), 3051 (C–H), 1608 (C=N), 1572 (C=N), 1518 (C=C), 1503 (C=C), 1460, 1403,
1390, 1344, 1308, 1285, 1243, 1229 (C–OH), 1215, 1153, 1098, 1041, 1003, 951, 895, 849, 816, 779, 757,
737, 704. Anal. Calc. for C13H9ClN2O2 (260.68): C, 59.90; H, 3.48; N, 10.75. Found: C, 59.94; H, 3.51; N,
10.68%.
4‐(5,6‐Dichloro‐1H‐benzimidazol‐2‐yl)‐6‐chlorobenzene‐1,3‐diol (2). Yield: 88%; m.p.: 354–355 °C; EI MS
+
(m/z, %): 329 (M , 100), 296 (18), 256 (14), 201 (50), 187 (21), 169 (14), 146 (6), 141 (8), 114 (7), 95 (2), 86
1
(3), 73 (9), 69 (8), 63 (15), 51 (5), 45 (3), 40 (9); H NMR (500 MHz, DMSO‐d6) δ: 11.20 (s, 1H, C(3)–OH),
10.78 (s, 1H, C(1)–OH), 8.11 (s, 1H, C(5)–H), 7.52 (s, 2H, C(4′,7′)–H), 6.72 (s, 1H, C(2)–H) ppm; IR
−1
(KBr, cm ): 3496 (OH), 3354 (NH), 3071 (OH), 1640 (C=N), 1594 (C=N), 1512 (C=C), 1471, 1422, 1399,
1299, 1271, 1211 (C–OH), 1197, 1161, 1089, 1062, 967, 875, 846, 832, 803, 774, 748, 731. Anal. Calc. for
C13H7Cl3N2O2 (329.57): C, 47.38; H, 1.14; N, 8.50. Found: C, 47.29; H, 2.17; N, 8.57%.
4‐[6‐(Trifluoromethyl)‐1H‐benzimidazol‐2‐yl]‐2‐methylbenzene‐1,3‐diol (3). Yield: 72%; m.p.: 263–264 °C;
+
EI MS (m/z, %): 308 (M , 100), 289 (7), 279 (37), 263 (12), 251 (9), 237 (7), 211 (4), 167 (61), 154 (7), 140
1
(5), 65 (5), 39 (4); H NMR (500 MHz, DMSO‐d6) δ: 13.07 (s, 1H, C(3)–OH), 10.54 (s, 1H, C(1)–OH),

Biomolecules 2019, 9, 870
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8.17 (d, J = 8.9 Hz, 1H, C(5)–H), 7.85 (s, 1H, C(7′)–H), 7.71 (dd, J = 8.6 and 1.4 Hz, 1H, C(5′)–H), 6.65 (d,
J = 8.5 Hz, 1H, C(4′)–H), 6.59 (d, J = 8.9 Hz, 1H, C(6)–H), 2.00 (s, 3H, Me) ppm; IR (KBr, cm‐1): 3378
(OH, NH), 2924 (CH), 1611 (C=N), 1573 (C=C), 1504 (C=C), 1455, 1377, 1336, 1305, 1267 (C–OH); 1194,
1131, 1111, 1077, 1037, 930, 856, 832, 822, 793, 756, 705. Anal. Calc. for C15H11F3N2O2 (308.26): C, 58.45;
H, 3.60; N, 9.09. Found: C, 58.52; H, 3.58; N, 9.16%.
4‐(7‐Chloro‐5‐(trifluoromethyl)‐1H‐benzimidazol‐2‐yl)‐6‐ethylbenzene‐1,3‐diol (4). Yield: 81%; m.p.: 284–
+
+
+
286 °C; EI MS (m/z, %): 357 (M + 1, 100), 356 (M , 28), 341 (M – Me, 70), 313 (4), 287 (9), 271 (8), 212
1
(5), 181 (46), 164 (16), 149 (3), 77 (5), 69 (11), 45 (2), 39 (3); H NMR (500 MHz, DMSO‐d6) δ: 11.30 (s,
1H, NH), 10.75 (s, 1H, C(3)–OH), 10.04 (s, 1H, C(1)–OH), 7.75 (s, 1H, C(4′)–H), 7.58 (s, 1H, C(5)–H),
7.43 (s, 1H, C(6′)–H), 6.44 (s, 1H, C(2)–H)), 2.46 (q, J = 7.4 Hz, 2H, CH2Me), 1.13 (t, J = 7.4 Hz, 3H,
−1
CH2Me) ppm; IR (KBr, cm ): 3456 (OH), 3402 (OH), 3356 (NH), 2971 (CH), 2939 (CH), 2874 (CH),
1624 (C=N), 1594 (C=C), 1508 (C=C), 1488, 1467, 1346, 1291, 1248 (C–OH), 1146, 1094, 1071, 987, 916,
887, 844, 768, 748. Anal. Calc. for C16H12ClF3N2O2 (356.73): C, 53.87; H, 3.39; N, 7.58. Found: C, 53.94;
H, 3.37; N, 7.62%.
4‐(6‐Methoxy‐1H‐benzimidazol‐2‐yl)‐2‐methylbenzene‐1,3‐diol (5). Yield: 76%; m.p.: 228–229 °C; EI MS
+
(m/z, %): 270 (M , 100), 255 (57), 241 (11), 227 (13), 198 (3), 167 (6), 151 (4), 135 (8), 79 (3), 77 (4), 65 (2),
1
44 (7), 39 (3), 33 (5); H NMR (500 MHz, DMSO‐d6) δ: 13.37 (s, 1H, C(3)–OH), 10.42 (s, 1H, C(1)–OH),
8.15 (d, J = 8.9 Hz, 1H, C(5)–H), 7.38 (d, J = 8.9 Hz, 1H, C(4’)–H), 7.03 (dd, J = 8.9 Hz and 2.8 Hz, 1H,
C(5′)–H), 6.98 (d, J = 2.74 Hz, 1H, C(7′)–H), 6.57 (d, J = 7.9 Hz, 1H, C(6)–H), 3.30 (s, 3H, OMe), 2.00 (s,
13
3H, Me) ppm; C NMR (125 MHz, DMSO‐d6) δ: 161.1, 159.2, 156.9, 156.0, 154.6, 139.5, 138.1, 126.2,
−1
114.8, 111.2, 108.3, 108.8, 102.9, 59.8, 8.2 ppm; IR (KBr, cm ): 3382 (OH), 2937 (CH), 1611 (C=N), 1571
(C=C),1502 (C=C), 1457, 1335, 1319, 1252, 1236 (C–OH), 1183, 1159, 1108, 1091, 1036, 955, 916, 857,
815, 777, 758, 738, 700. Anal. Calc. for C15H14N2O3 (270.16): C, 66.66; H, 5.22; N, 10.36. Found: C, 66.74;
H, 5.24; N, 10.29%.
4‐(6‐Methoxy‐1H‐benzimidazol‐2‐yl)‐6‐chlorobenzene‐1,3‐diol (6). Yield: 73%; m.p.: 219–222 °C; EI MS
+
(m/z, %): 290 (M , 100), 275 (82), 261 (5), 247 (20), 241 (4), 187 (6), 145 (6), 80 (4), 78 (3), 69 (3), 51 (3), 33
1
(6); H NMR (500 MHz, DMSO‐d6) δ: 12.77 (s, 1H, C(3)–OH), 11.40 (s, 1H, C(1)–OH), 8.19 (s, 1H, C(5)–
H), 7.41 (d, J = 8.8 Hz, 1H, C(4′)–H), 7.06 (dd, J = 8.9 and 2.8 Hz, 1H, C(5′)–H), 7.02 (d, J = 2.8 Hz, 1H,
13
C(7′)–H), 6.56 (s, 1H, C(2)–H), 3.27 (s, 3H, CH3) ppm; C NMR (125 MHz, DMSO‐d6) δ: 161.3, 157.1,
−1
156.2, 154.9, 139.1, 134.3, 116.2, 114.7, 111.9, 111.5, 108.2, 104.2, 102.9, 59.8 ppm; IR (KBr, cm ): 3403
(NH), 3198 (OH), 2951 (CH), 1633 (C=N), 1512 (C=C), 1476, 1328, 1228 (C–OH), 1187, 1137, 1109, 1090,
974, 883, 784, 756, 727. Anal. Calc. for C14H11ClN2O3 (290.70): C, 57.84; H, 3.81; N, 9.64. Found: C,
57.92; H, 3.78; N, 9.70%.
4‐(6‐Methoxy‐1H‐benzimidazol‐2‐yl)benzene‐1,2,3‐triol (7) [31].
Ethyl 2‐(2,4‐dihydroxy‐3‐methylphenyl)‐1H‐benzimidazole‐5‐carboxylate (8). Yield: 73%; m.p.: 213–214 °C;
+
1
EI MS (m/z, %): 312 (M , 100), 284 (31), 267 (15), 255 (13), 239 (11), 167 (5), 119 (5), 90 (3), 63 (3); H
NMR (500 MHz, DMSO‐d6) δ: 12.21 (s, 1H, NH), 10.91 (s, 1H, C(2)–OH), 10.06 (s, 1H, C(4)–OH), 7.66
(dd, J = 8.6 and 1.9 Hz, 1H, C(6′)–H), 7.62 (d, J = 8.9 Hz, 1H, C(5)–H), 7.56 (s, 1H, C(4′)–H), 6.79 (d, J =
8.57 Hz, 1H, C(7′)–H), 6.42 (d, J = 8.84 Hz, 1H, C(6)–H), 3.75 (q, J = 7.01 Hz, 2H, CH2Me), 1.82 (s, 3H,
−1
Me), 1.38 (t, J = 7.0 Hz, 3H, CH2Me) ppm; IR (KBr, cm ): 3419 (OH), 3328 (NH), 3204 (OH), 2920
(CH), 1673 (C=O), 1627 (C=N), 1602 (C=C), 1493, 1459, 1394, 1369, 1294, 1263, 1242 (C–OH), 1202,
1155, 1088, 1015, 966, 857, 821, 796, 765, 726. Anal. Calc. for C17H16N2O4 (312.32): C, 65.38; H, 5.16; N,
8.97. Found: C, 65.31; H, 5.19; N, 8.90%.
Ethyl 2‐(5‐ethyl‐2,4‐dihydroxyphenyl)‐1H‐benzimidazole‐5‐carboxylate (9). Yield: 74%; m.p.: 316–317 °C;
+
+
EI MS (m/z, %): 326 (M , 70), 311 (M − Me, 100), 297 (10), 283 (48), 238 (10), 169 (3), 133 (12), 123 (9), 91
1
(4), 44 (4), 36 (5); H NMR (500 MHz, DMSO‐d6) δ: 10.71 (s, 1H, C(2)–OH), 8.33 (s, 1H, C(6)–H), 8.03
(d, J = 8.5 Hz, 1H, C(6′)–H), 7.84 (s, 1H, C(4′)–H), 7.45 (d, J = 8.5 Hz, 1H, C(7′)–H), 6.74 (s, 1H, C(3)–H),
4.38 (q, J = 7.5 Hz, 2H, OCH2Me), 2.50 (q, J = 7.1 Hz, 2H, CH2Me), 1.37 (t, J = 7.1 Hz, 3H, CH2Me), 1.21
−1
(t, J = 7.5 Hz, 3H, OCH2Me) ppm; IR (KBr, cm ): 3425 (NH), 3163 (OH), 2968 (CH),1677 (C=O), 1618

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(C=N), 1569 (C=C), 1511 (C=C), 1466, 1408, 1373, 1296, 1245 (C–OH), 1149, 1094, 1014, 951, 837, 765,
742. Anal. Calc. for C18H18N2O4 (326.35): C, 66.25; H, 5.56; N, 8.58. Found: C, 66.31; H, 5.55; N, 8.51%.
Ethyl 2‐(2,4,6‐trihydroxyphenyl)‐1H‐benzimidazole‐5‐carboxylate (10). Yield: 63%; m.p.: 300–301 °C; EI
+
MS (m/z, %): 314 (M , 36), 286 (9), 269 (9), 250 (10), 230 (21), 222 (22), 206 (6), 194 (13), 185 (6), 177 (18),
149 (8), 126 (100), 111 (7), 97 (8), 90 (5), 85 (22), 80 (13), 69 (23), 63 (10), 52 (12), 44 (27), 39 (10), 36 (7);
1
H NMR (500 MHz, DMSO‐d6) δ: heteroatoms protons are illegible; 8.02 (m, 2H, CH), 7.65 (m, 1H,
CH), 5.91 (s, 1H, CH), 5.89 (s, 1H, CH), 4.35 (q, J = 7.0 Hz, 2H, CH2Me), 1.35 (t, J = 7.1 Hz, 3H, CH2Me)
−1
ppm; IR (KBr, cm ): 3435 (OH, NH), 1667 (C=O), 1615 (C=N), 1517 (C=C), 1478, 1390, 1366, 1289, 1244
(C–OH), 1189, 1156, 1028, 934, 823, 766, 729. Anal. Calc. for C16H14N2O5 (314.29): C, 61.14; H, 4.49; N,
8.91. Found: C, 61.08; H, 4.52; N, 8.85%.
2‐(2,4‐Dihydroxy‐3‐methylphenyl)‐1H‐benzimidazole‐6‐carbonitrile (11). Yield: 76%; m.p.: 338–339 °C; EI
+
1
MS (m/z, %): 265 (M , 100), 236 (32), 220 (10), 208 (10), 194 (6), 181 (3), 168 (3), 117 (4), 90 (4), 36 (4); H
NMR (500 MHz, DMSO‐d6) δ: 10.07 (s, 1H, C(1)–OH), 8.11 (s, 1H, C(7′)–H), 7.75 (m, 2H, CH), 7.65
(dd, J = 8.3 and 1.3 Hz, 1H, C(5′)–H), 6.57 (d, J = 8.7 Hz, 1H, C(6)–H), 2.07 (s, 3H, Me) ppm; IR (KBr,
−1
cm ): 3369 (NH), 3134 (OH), 2837 (CH), 1659 (C=N), 1605 (C=C), 1570 (C=C), 1474, 1413, 1369, 1317,
1273, 1235 (C–OH), 1213, 1087, 981, 874, 819, 756, 710. Anal. Calc. for C15H11N3O2 (265.27): C, 67.92; H,
4.18; N, 15.84. Found: C, 68.01; H, 4.20; N, 15.77%.
2‐(5‐Ethyl‐2,4‐dihydroxyphenyl)‐1H‐benzimidazole‐6‐carbonitrile (12). Yield: 84%; m.p.: 284–285 °C; EI
+
+
MS (m/z, %): 279 (M , 40), 264 (M − Me, 100); 236 (5), 207 (4), 194 (7), 181 (3), 117 (3), 90 (3), 40 (2), 36
1
(3); H NMR (500 MHz, DMSO‐d6) δ: heteroatoms protons are illegible; 8.18 (s, 1H, C(7′)–H), 7.89 (s,
1H, C(6)–H), 7.84 (d, J = 8.3 Hz, 1H, C(4′)–H), 7.77 (m, 1H, C(5′)–H), 6.66 (s, 1H, C(3)–H), 2.45 (q, J =
7.5 Hz, 2H, CH2Me), 1.20 (t, J = 7.5 Hz, 3H, CH2Me) ppm. Anal. Calc. for C16H13N3O2 (279.29): C,
68.81; H, 4.69; N, 15.05. Found: C, 68.72; H, 4.71; N, 14.98%.
4‐(6‐Nitro‐1H‐benzimidazol‐2‐yl)benzene‐1,3‐diol (13) [31].
2‐Methyl‐4‐(6‐nitro‐1H‐benzimidazol‐2‐yl)benzene‐1,3‐diol (14). Yield: 74%; m.p.: 213–215 °C; EI MS
+
(m/z, %): 285 (M , 100), 256 (14), 239 (55), 210 (16), 167 (13), 151 (11), 119 (5), 90 (8), 77 (6), 63 (11), 52
1
(5), 39 (5), 33 (3); H NMR (500 MHz, DMSO‐d6) δ: 12.15 (s, 1 H, NH), 10.99 (s, 1H, C(3)–OH), 10.15 (s,
1H, C(1)–OH), 7.98 (dd, J = 9.1 Hz and 2.6 Hz, 1H, C(5′)–H), 7.92 (d, J = 2.6 Hz, 1H, C(7′)–H), 7.65 (d, J
= 8.6 Hz, 1H, C(5)–H), 6.82 (d, J = 9.1 Hz, 1H, C(4′)–H), 6.47 (d, J = 8.8 Hz, 1H, C(6)–H), 2.02 (s, 3H,
13
Me) ppm; C NMR (125 MHz, DMSO‐d6) δ: 195.1, 158.3, 151.3, 135.3, 133.2, 126.2, 124.8, 123.4, 122.6,
−1
117.1, 111.3, 110.1, 107.4, 8.4 ppm; IR (KBr, cm ): 3426 (NH), 3340 (OH), 3222 (OH), 2855 (CH), 1641
(C=N), 1604 (C=C), 1514 (C=C), 1499, 1462, 1428, 1382, 1382, 1307, 1262 (C–OH), 1203, 1153, 1080, 887.
Anal. Calc. for C14H11N3O4 (285.25): C, 58.95; H, 3.89; N, 14.73. Found: C, 59.03; H, 3.91; N, 14.68%.
2.3. RP‐18 HPLC Chromatography
The Eurosil Bioselect C18 (5 μm, 300 × 4.6 mm) column was used as the stationary phase. The
mobile phase consisted of different volume mixtures of methanol and 20 mM acetate buffer as the
aqueous phase to give pH 4.0. The organic modifier concentration ranged from 0.30 to 0.75,
3
−1
depending on the structure of the compound at 0.05 (0.1) intervals. The flow rate was 0.5 cm ∙min at
room temperature. Measurements were made at 292 nm. The retention time of an unretained solute
(t0) was determined by the injection of a small amount of acetone dissolved in water. All compounds
showed the regular retention behavior in the function of methanol concentration (v/v). This is
described by the Soczewiński–Wachtmeister dependence:
log k = log kw + S (% organic modifier)
(1)
in which log kw is the retention factor of a solute with pure water as the mobile phase (intercept) and
S is the regression curve (slope) [32,33]. The log k values were calculated as log k = log (tR − t0)/t0,
where tR is the retention time of a solute and t0 is the retention time of an unretained solute.
2.4. In Vitro AChE and BuChE Inhibition Assay

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Acetylcholinesterase (AChE, E.C. 3.1.1.7, from human erythrocytes), butyrylcholinesterase
(BuChE, E.C. 3.1.1.8, from equine serum), acetylthiocholine iodide (ATCh), butylthiocholine iodide
(BTCh), 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB), donepezil, and neostigmine bromide were
purchased from Sigma‐Aldrich (Steinheim, Germany). The inhibitory activities against AChE and
BuChE of the prepared compounds were performed by means of the method previously developed
by Ellman et al. using neostigmine and donepezil as the reference compound [34]. The details were
described previously [22].
2.5. Kinetic Characterization of AChE and BuChE Inhibition
The kinetic analysis was evaluated according to the Ellman’s method [34]. Plots of 1/velocity
versus the reciprocal substrate concentration were constructed at different final concentrations of the
ATC iodide in the range of 100–10 μM. The studies were carried out using the final concentrations of
1 inhibitor: (AChE: 0.25, 1.0 μM; BuChE: 0.25, 1.2 μM) and 5 (AChE: 0.1, 1.0 μM; BuChE: 0.25, 0.55
μM) and without it. The tested medium contained DTNB solution (0.4 mg/mL), AChE solution (2
U/mL) or BuChE solution (4 U/mL), solution of the examined compound, and different
concentrations of the ATC iodide substrate solution. Absorbance was detected at 412 nm at room
temperature after 7 min. The obtained data were calculated and presented on the Lineweaver–Burk
plot. The determined the Michaelis‐Menten constant (KM) and the maximum velocity of the enzyme
(υmax) values were used to define the type of enzyme inhibition.
2.6. Determination of the Inhibitory Effect on the Self‐Mediated Aβ(1–42) Aggregation
Amyloid β‐peptide, (1–42) (Aβ42), was purchased from Aldrich. Aβ42 was diluted in the
phosphate buffer (pH 8.0) to obtain a 125.8 μM solution. The compounds under study were first
dissolved in a small amount of methanol, and then further diluted in the phosphate buffer to the
final concentration of 5–160 μM. To study the Aβ42 aggregation inhibition, the fluorescence
emission of thioflavin T (ThT) was followed with curcumin as the reference compound [35,36]. At
first, the Aβ42 (10 μL) samples and the tested compounds (10 μL) were diluted with the phosphate
buffer (60 μL) and then they were incubated for 24 h at 37 °C without stirring. As for the control, a
sample of the peptide was incubated without the inhibitor. The tests were carried out in black,
flat‐bottom, 96‐well plates. Then 20 μL of 2.5 μM ThT in 50 mM glycine–NaOH (pH 8.5) was added
and incubated for 5 min. Fluorescence was monitored with the excitation at 446 nm and the emission
at 490 nm. A time scan of fluorescence was performed and the intensity values reached the plateau
(300 s). The percent inhibition was calculated from:
I% = 100 − (IFI/IF0 * 100)
(2)
in which IFI and IF0 correspond to the fluorescence intensities, in the presence and absence of the test
compounds, respectively, minus the fluorescence intensities due to the blanks. The reported values
were obtained as the mean ± SD of triplicate of three independent experiments.
2.7. In Vitro Antioxidant Activity Assay
Antioxidant activity of each compound and standards (rutin) was assessed based on the radical
scavenging effect of stable DPPH free radical [37]. An amount of 10 μL of each tested compound or
standard series of different concentrations (from 0.0 to 100 μM) was added to 9 mL of methanol
solution of 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) (100 μM). After incubation at 37 °C for 30 min,
decrease in the absorbance of each solution was measured at 520 nm. The absorbance of the blank
sample containing the same amount of DMSO and DPPH solution was also prepared and measured.
All experiments were conducted in triplicate. The scavenging potential was compared with a solvent
control (0% radical scavenging) and the standard compound. Radical scavenging activity (%AA)
was calculated by the following formula:
(%AA) = [(A0 – A1)/A0] × 100%
(3)

Biomolecules 2019, 9, 870
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in which (A0) is the absorbance of the blank sample and (A1) is the absorbance of the tested
compound (t = 30 min). Concentration of each compound required for scavenging 50% of DPPH
(IC50) was also determined [38].
2.8. Computational Methods
The partition coefficient (log P) values were predicted using Crippen′s fragmentation by the CS
ChemProp module from ChemDraw Ultra 12 (CambridgeSoft, Cambridge, MA, USA) according to
the fragmentation method introduced by Crippen [39]. The polar surface area (tPSA) was calculated
by the atom‐based method [40].
2.9. Molecular Modeling Studies
The spatial structures of compounds 1, 5, and 7 were created by Corina online (Molecular
Networks) and later prepared by Sybyl 8.0 (Tripos). All atom and bond types were checked,
necessary hydrogen atoms were added, and finally, the Gasteiger–Marsili charges were assigned.
The pKa values for the compounds and the ionization states corresponding to the physiological
conditions (pH 7.4) were assessed by Marvin (ChemAxon). The compounds were docked to
acetylcholinesterase from the 1ACJ crystal structure. Before docking with GoldSuite (CCDC), the
enzyme structure was prepared. All histidine residues were protonated at Nε, the hydrogen atoms
were added, and some water molecules (616, 634, 643) were retained. The binding site was defined
as all amino acid residues within the radius of 12 Å from the reference compound, tacrine. A
standard set of genetic algorithms was applied. The population size was equal to 100, and the
number of operations 100,000. As a result, 10 conformations for each compound were obtained. The
GoldScore function was used for the selection of the best poses. The results were visualized by
PyMOL 0.99 (DeLano Scientific LLC, Palo Alto, CA, USA).
3. Results and Discussion
3.1. Chemistry
The target benzimidazole derivatives (Scheme 1) have been obtained in a one‐step procedure
using benzene‐1,2‐diamines with sulfinylbis[(2,4‐dihydroxyphenyl)methanethione] (STB) or its
analogs substituted by ‐Me, ‐Et, ‐Cl, or ‐OH on the resorcinol moiety in the reaction. These
compounds are the agents cyclizing and introducing resorcinol (or modified one) into final
products. They were obtained according to the previously described way [41]. The synthesis
procedure was conducted for 2–4 h and the compounds were obtained in a good yield (60–87%). The
product purity was evaluated by RP‐HPLC (C‐18, methanol–water mobile phase). The synthetic
scheme and structures of benzimidazoles are presented in Scheme 1.

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R1
X
HO
NH2
NH2
X
N
STB
OH
R2

MeOH, 3 h
Y
N
H
Y
Z
R3
1 - 14
Z
STB
:
1. X, Y, Z: H
2
R1, R3: H, R2: Cl
R1, R3: H, R2: Cl
R1: Me, R2, R3: H
R1, R3: H, R2: Et
R1: Me, R2, R3: H
R1, R3: H, R2: Cl
R1: OH, R2, R3: H
R1 = Me, OH
. X, Y: Cl, Z: H
R2 = Me, Et, Cl
R3 = OH
3. X: H, Y: CF3, Z: H
. X: CF3, Y: H, Z: Cl
5. X: H, Y: OMe, Z: H
. X: H, Y: OMe, Z: H
4
6
R2
R2
7. X: H, Y: OMe, Z: H
HO
R1
R3 R3
OH
. X: EtOC(=O), Y, Z: H R1: Me, R2, R3: H
. X: EtOC(=O), Y, Z: H R1, R3: H, R2: Et
8
9
O
S
10. X: EtOC(=O), Y, Z: H R1, R2: H, R3: OH
R1
R1: Me, R2, R3: H
R1, R3: H, R2: Et
R1, R2, R3: H
11
. X: H, Y: CN, Z: H
12. X: H, Y: CN, Z: H
. X: H, Y: NO2, Z: H
14. X: H, Y: NO2, Z: H
OH
S
S
OH
13
R1: Me, R2, R3: H
Scheme 1. Synthesis of 2‐(2,4‐dihydroxyphenyl)‐1H‐benzimidazoles.
1
13
The structures of the compounds were confirmed by mass spectrometry, H NMR, and
C
NMR, as well as by IR spectroscopy. The obtained data are consistent with those for the other
derivatives [25,42].
3.2. Inhibitory Potency Against AChE and BuChE
All obtained compounds have been assessed as the AChE and BuChE inhibitors. Their potency
was determined using the modified Ellman’s method and expressed as the half of maximal
inhibitory concentration, IC50 [34]. Neostigmine and donepezil were applied as the reference agents.
The results are collected in Table 1.
The results show that the potency of compounds is varied and some of them are characterized
by good to moderate inhibitory activities. The IC50 values toward AChE and BuChE ranged from 76
to 98 nM or from 0.117 to 18.711 μM, respectively, for the most active derivatives. All considered
compounds showed greater activity toward AChE than BuChE. Some of them were highly selective
for AChE with respect to BuChE (compounds 4 and 6), whereas compound 5 was a strong inhibitor
of both cholineserases (IC50 = 86 nM and 117 nM). Differences in the inhibitory potency toward these
enzymes result from some structural differences between the hydrophobic gorges of the active
center of both enzymes [43].
The studied phenol–benzimidazole conjugates with the additional substituent including the
modified amine group on the phenolic moiety showed a lower activity compared with the
considered polyphenols and the highest activity was found for 5‐nitroderivative [6,7]. This suggests
that the introduction of the second hydroxyl group into the phenyl ring enhances an inhibitory
potency of the compound against AChE.
Table 1. In vitro inhibitory concentration of the compounds against AChE and BuChE (IC50, μM) and
selectivity of the studied compounds.
1
2
AChE
BuChE
Selectivity
for AChE
Compound
3
IC50 [μM]
0.076 ± 0.002
0.541 ± 0.036
0.098 ± 0.005
0.118 ± 0.009
IC50 [μM]
4.24 ± 0.205
22.69 ± 0.078
14.022 ± 0.111
46.025 ± 0.104
1
2
3
4
55.8
41.9
143.1
390.0

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5
6
7
0.086 ± 0.021
0.092 ± 0.002
>500
0.117 ± 0.041
18.711 ± 0.173
>500
1.4
203.4
‐
8
9
10
11
12
13
14
0.184 ± 0.022
0.962 ± 0.023
>500
>500
>500
1.487 ± 0.118
1.003 ± 0.196
0.284 ± 0.014
0.052 ± 0.003
0.020 ± 0.008
25.352 ± 0.232
43.855 ± 0.381
>500
>500
>500
106.557 ± 0.201
88.363 ± 0.155
31.024 ± 0.133
0.068 ± 0.005
7.520 ± 0.200
137.8
45.6
‐
‐
‐
71.7
88.1
109.2
1.3
376
4
bib‐1,3‐diol
neostigmine
donepezil
1 IC50—50% inhibitory concentration (means ± SD of three independent experiments) of AChE.
IC50—50% inhibitory concentration (means ± SD of three experiments) of BuChE. 3 Selectivity for
2
4
AChE
=
IC50(BuChE)/IC50(AChE).
4‐(1H‐benzimidazol‐2‐yl)benzene‐1,3‐diol was previously
described [27].
3.3. Kinetic Studies
The mechanism of AChE inhibition was investigated for inhibitors 1 and 5 as the most valuable
compounds. The Lineweaver–Burk plots present 1/velocity versus 1/substrate concentrations in the
range of 100–10 μM for compounds 5 (0.1, 1.0 μM) and 1 (0.25, 1.0 μM) and without inhibitors (0 μM)
(Figure 2). Based on the Lineweaver–Burk plots analysis, the mixed type of inhibition was proven
because the lines cross the same point of the coordinate system. As follows from the studies, 1 and 5
showed a different relationship: increased K
M
and decreased υmax values with higher inhibitor
concentrations (Figure 2, Table 2). The mechanism of BuChE inhibition was also investigated for
compounds 1 and 5 and the tests showed the same relationships as in the case of AChE (Figure 3,
Table 3).
1

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5
Figure 2. Lineweaver–Burk reciprocal plots for AChE inhibition by compounds 1 and 5.
Table 2. The K and υmax values at different concentrations of the inhibitor toward AChE.
M
υ
max
Compound/Concentration [μM]
KM [μM]
−1
[A min ]
Compound 1
1.0
0.25
0
46.41
33.16
28.45
105.26
135.14
153.85
Compound 5
1.0
0.15
0
487.09
147.05
70.33
120.48
175.44
454.55
1

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5
Figure 3. Lineweaver–Burk reciprocal plots for BuChE inhibition by compounds 1 and 5.
Table 3. The K
M
and υmax values at different concentrations of the inhibitor toward BuChE.
−1
Compound/Concentration [μM]
K
M
[μM]
υ
max [A min ]
Compound 1
1.2
0.25
0
73.53
64.92
58.14
163.93
322.58
526.32
Compound 5
0.55
0.25
0
209.21
154.08
110.74
178.57
196.08
222.22
3.4. Inhibition of Self‐induced Aβ Aggregation
Nowadays, the amyloid hypothesis has become the dominating paradigm for understanding
AD [44]. Aβ(1–42) is the most amyloidogenic Aβ fragment found in the AD plaques. To determine
the amyloid Aβ(1–42) aggregation inhibition of the strongest AChE inhibitors (1, 5, 6), thioflavin‐T
(ThT) assay was performed with curcumin as the reference compound. Interestingly, all the
synthesized compounds presented moderate to good potencies on the self‐induced Aβ(1–42)
aggregation. The data in Figure 4 show that the compounds at 5 μM inhibited Aβ(1–42)
self‐aggregation in the range from 14% to 22% and at 160 μM in the range from 71% to 77%. They
indicate that new compounds are found to be similar or more potent than curcumin in inhibiting the
self‐induced Aβ42 aggregation at higher concentrations. Surprisingly, at lower concentrations, the
tested compounds are less potent than the reference compound. The most effective compound is 6,
followed by 5 and 1; their IC50 values were 27.04 ± 2.29, 33.07 ± 2.07, and 48.00 ± 3.43 μM, respectively
(Table 4).

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100
1
5
6
curcumin
80
60
40
20
0
160
80
40
20
10
5
compounds concentrations (µM)
Figure 4. Inhibition of self‐aggregation of Aβ(1–42) by compounds 1, 5, and 6. The thioflavin‐T fluorescence
method was used and the measurements were carried out in the presence of test compounds. The mean ± SD
values from three independent experiments are shown.
3.5. Antioxidant Potential
Reactive oxygen species are a causal factor in the process of aging and the cumulative damage
has been associated with AD [45]. In this study, the DPPH radical scavenging activity of the newly
synthesized compounds was estimated using the Brand‐Williams method. The data showed that all
tested compounds demonstrated the dose‐dependent DPPH inhibition activity (Table 4). Compound
1 exhibited the lowest DPPH radical scavenging activity (0.641 ± 0.03 μM). On the contrary, in
compound 8 (IC50(Aβ(1–42)) = 0.125 ± 0.06 μM), the radical scavenging properties were at a level similar
to the used standard.
3.6. Molecular Modeling Studies
Three benzimidazole derivatives were taken for the molecular docking studies to present their
binding mode within the enzyme and to explain the differences in the activity. Two of them were
active (1, 5) while one was inactive (7). The crystal structure of AChE from the electric ray in a
complex with the reference inhibitor, tacrine, was used for docking (PDB code: 1ACJ). The docking
procedure was fully validated and described previously [46].
All compounds presented a similar arrangement within the active site of AChE, interacting
mainly with the catalytic site (Figure 5). They mimicked the binding mode of tacrine that is a
mixed‐type, reversible inhibitor of cholinesterase that binds close to the catalytically active serine in
the active site of the enzyme [47]. The benzimidazole ring interacted by π–π stacking with the side
chains of Phe330 and Trp84. The ‐NH fragment of this heterocyclic system created a hydrogen bond
with the carbonyl group of His440. The methoxy group (compound 5) was not engaged in any
specific interactions and it seems that it was not necessary for the enzyme inhibition. The substituted
phenyl moiety created π–π interactions with Trp84.
Table 4. In vitro inhibition of the self‐induced aggregation IC50(Aβ(1–42)) and the radical scavenging
effect (DPPH free radical) IC50(DPPH) by the synthesized compounds.
1
1
Compound
1
IC50(Aβ(1–42)) [μM]
IC50(DPPH) [μM]
0.641 ± 0.03
48.00 ± 3.43
2
2
‐
0.213 ± 0.08

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5
33.07 ± 2.07
27.04 ± 2.29
0.627 ± 0.11
0.362 ± 0.01
0.125 ± 0.06
0.209 ± 0.02
0.087 ± 0.04
6
2
8
‐
2
12
‐
2
rutin
‐
1 IC50: 50% inhibitory concentration (means ± SD of three experiments). 2 Experiment was not conducted.
The hydroxyl group in position 4 was always bound to the carboxyl group of Glu199 by the
hydrogen bond, whereas the hydroxyl group in position 2 usually created the H bond with the water
molecule. Comparing two active compounds with an inactive one, it could be noticed that they
differed significantly from one another as far as lipophilicity is concerned. Introduction of the third
hydroxyl group in position 3 (compound 7, log P = 1.57) led to a decrease of lipophilic properties in
comparison with the compounds containing an extra methyl group (chlorine atom (compound 1, log
P = 2.64 or compound 5, log P = 2.45)). The detailed binding mode of compound 5 is presented in
Figure 6.
The lower log P value (lower lipophilicity) resulted in reduction of hydrophobic and aromatic
interactions, especially those of the phenyl moiety, and as a consequence, a lack of the activity. The
other compound 10 with the third ‐OH group on the phenyl ring is also inactive. This observation is
consistent with the results of our previous studies which confirmed an important role of lipophilic
properties in binding to AChE [48]. Structure–activity relationship (SAR) analysis shows also an
important role of lipophilicity and the polar surface area on inhibitory potency against
cholinesterases. The docking simulation of benzimidazole–phenol conjugates with the additional
tertiary amine group and resorcinol‐1,3,4‐thiadiazole hybrids proved that the compounds bound
mainly with the catalytic site and confirmed an important role of hydroxyl groups in the interactions
with an enzyme [7,22].
3.7. SAR Studies
To find the structure–activity relationships, some quantities of the compounds were estimated
(Table 5). They included the lipophilicity parameter log k
silico log P, the topological polar surface area (tPSA), and the molar refractivity (MR) (Table 5). The
log k and S parameters obtained by the extrapolation technique are commonly used as lipophilicity
descriptors [32,49,50].
w
obtained by RP‐HPLC and calculated in
w

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Figure 5. Comparison of the binding modes of selected derivatives (1, 5, and 7) and tacrine. The compounds are
in the colors: 1—pink, 5—green, 7—blue, and tacrine—yellow. The red spheres represent the water molecules.
Figure 6. The binding mode of compound 5 within the active site of AChE. Hydrogen bonds are
shown as red dashes, the distance between the donor and the acceptor for each hydrogen bond is
given in Å. The red spheres represent water molecules.
To obtain better results, the previously described compound 4‐(1H‐benzimidazol‐2‐yl)‐benzene
‐1,3‐diol (bib‐1,3‐diol) as a parent compound was included in the biological screening (Tables 1 and
5) [26]. The structure–activity elucidation shows that the presence of the third ‐OH group in the
benzenediol moiety (compounds 7 and 10) or ‐NO
benzimidazole ring decreases significantly the inhibitory potency of the compounds against both
enzymes. Introduction of the hydrophobic substituent (‐Cl, ‐Me, ‐CF ) as well as ‐OMe on the
2
(13, 14), particularly ‐CN (11, 12) on the
3
heterocyclic ring or ‐Cl in the aryl moiety enhances biological activity of analogs in comparison to
the unsubstituted bib‐1,3‐diol. The activity of compounds with the ethyl ester group is moderate (8–
10) and it deteriorates with the addition of the hydroxyl substituent.
Table 5. Chromatographic parameters: log k
w
(intercept) and −S (slope) of the linear dependences
obtained by RP 18 HPLC, log P values, molar refractivity (MR), topological polar surface area (tPSA).
2
2
3
−1
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
log k
w
−S
n
6
5
6
6
6
6
6
5
6
6
6
5
4
5
5
r
log P
2.64
3.76
3.49
4.47
2.45
2.54
1.57
2.73
3.15
1.85
2.61
3.02
2.52
2.94
2.09
tPSA [Å ] MR [cm ∙mol ]
3.602
5.460
5.720
4.237
4.591
4.808
3.589
2.566
3.067
2.088
3.590
3.360
2.475
2.674
1.732
3.896
5.770
6.299
4.635
5.179
5.390
3.870
4.097
4.160
3.237
3.867
4.854
4.655
4.596
2.979
0.992
0.985
0.992
0.992
0.994
0.993
0.996
0.875
0.999
0.979
0.996
0.975
0.999
0.981
0.968
64.85
64.85
64.85
64.85
74.08
74.08
94,31
91.15
91.15
111.38
88.64
88.64
116.66
116.66
64.85
67.86
77.07
75.66
84.87
76.40
75.11
72.32
86.20
90.80
82.12
75.25
79.85
1
‐
‐
1
bib‐1,3‐diol
63.26
1 Parameter was not calculated.

Biomolecules 2019, 9, 870
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On the other hand, taking into account the values of the calculated descriptors (Table 5), the low
tPSA as well as MR favor the activity. Small particles with a low polar surface area are preferred.
However, the most active compounds are characterized by a diverse lipophilicity expressed by both
log P and log kw. It seems that not the log P value of compounds but the location of the polar region
in the molecule is important. That confirms a lack of activity for the compounds with the third polar
substituent in the aryl ring (IC50 > 500 μM, compounds 7 and 10).
4. Conclusions
In summary, a series of new benzimidazoles functionalized by 2,4‐dihydroxyphenyl moiety has
been obtained and evaluated as AChE and BuChE inhibitors. All compounds were more active
against AChE than BuChE. Analogs 1 and 5 are the strongest inhibitors in the series (IC50 = 76 and 86
nM, respectively). At the same time, compound 5 is highly active against BuChE (IC50 = 117 nM). The
kinetic studies suggest the mixed type of inhibition for both enzymes. Besides the ability to inhibit
AChE and BuChE, the most active derivative also inhibited in vitro self‐induced Aβ(1–42)
aggregation and exhibited antioxidant properties. The compounds under consideration have the
properties expected for potential drugs used in AD therapy.
The docking simulation exhibited that modeled compounds 1 and 5 create a lot of interactions
with the catalytic site of AChE and they mimic the binding mode of tacrine, which confirms their
high inhibitory potency. The force of binding with the enzyme and the activity depend on the
lipophilicity of the compounds, molar refractivity, and topological polar surface area. Introduction
of the third hydroxyl group into the phenyl ring leads to reduction of lipophilic properties (increase
of tPSA) as well as of hydrophobic and aromatic interactions of enzymes, particularly those of the
phenyl moiety, and as a consequence, a lack of activity is observed. The presence of the additional
lipophilic substituent of both rings enhances the anticholinesterase potency of analogs compared
with the parent compound.
Author Contributions: Conceptualization, J.M.; Investigation, A.S., M.K., and K.C.; Methodology, J.M.,
Supervision, A.N.; Visualization, M.B; Writing—original draft, J.M., A.S., P.S., and M.B.; Writing—review &
editing, J. M.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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