Functionalized imidazolium and benzimidazolium salts as paraoxonase 1 inhibitors: Synthesis, characterization and molecular docking studies

Article abstract of DOI:10.1016/j.bmc.2016.02.012

Paraoxonase (PON) is a key enzyme in metabolism of living organisms and decreased activity of PON1 was acknowledged as a risk for atherosclerosis and organophosphate toxicity. The present study describes the synthesis, characterization, PON1 inhibitory properties and molecular docking studies of functionalized imidazolium and benzimidazolium salts (1a-5g). The structures of all compounds were elucidated by IR, NMR, elemental analysis and structures of compounds 2b and 2c were characterized by single-crystal X-ray diffraction. Compound 1c, a coumarin substituted imidazolium salt showed the best inhibitory effect on the activity of PON1 with good IC₅₀ value (6.37 μM). Kinetic investigation was evaluated for this compound and results showed that this compound is competitive inhibitor of PON1 with K_i value of 2.39 μM. Molecular docking studies were also performed for most active compound 1c and one of least active compound 2c in order to determine the probable binding model into active site of PON1 and validation of the experimental results.

Full text of DOI:10.1016/j.bmc.2016.02.012

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Functionalized imidazolium and benzimidazolium salts as
paraoxonase 1 inhibitors: Synthesis, characterization and molecular
docking studies
b
a
c
d,
d
Mert Olgun Karataßs a, , Harun Uslu , Bülent Alıcı , Baßsak Gökçe , Nahit Gencer , Oktay Arslan ,
⇑
⇑
N. Burcu Arslan e, Namık Özdemir f
a Inönü University, Faculty of Arts and Science, Department of Chemistry, 44280 Malatya, Turkey
b Inönü University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 44280 Malatya, Turkey
c Süleyman Demirel University, Faculty of Pharmacy, Department of Biochemistry, 32260 Isparta, Turkey
d Balıkesir University, Faculty of Arts and Science, Department of Chemistry, 10145 Balıkesir, Turkey
e Giresun University, Faculty of Education, Department of Computer Education and Instructional Technology, 28100 Giresun, Turkey
f Ondokuz Mayıs University, Faculty of Arts and Sciences, Department of Physics, 55139 Samsun, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Paraoxonase (PON) is a key enzyme in metabolism of living organisms and decreased activity of PON1
was acknowledged as a risk for atherosclerosis and organophosphate toxicity. The present study
describes the synthesis, characterization, PON1 inhibitory properties and molecular docking studies of
functionalized imidazolium and benzimidazolium salts (1a–5g). The structures of all compounds were
elucidated by IR, NMR, elemental analysis and structures of compounds 2b and 2c were characterized
by single-crystal X-ray diffraction. Compound 1c, a coumarin substituted imidazolium salt showed the
Received 1 January 2016
Revised 3 February 2016
Accepted 8 February 2016
Available online xxxx
Keywords:
Benzimidazolium
Coumarin
Imidazolium
Inhibition
best inhibitory effect on the activity of PON1 with good IC50 value (6.37
evaluated for this compound and results showed that this compound is competitive inhibitor of PON1
with Ki value of 2.39 M. Molecular docking studies were also performed for most active compound 1c
lM). Kinetic investigation was
l
and one of least active compound 2c in order to determine the probable binding model into active site
of PON1 and validation of the experimental results.
Molecular docking
Paraoxonase 1
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
as a risk for atherosclerosis.3 Therefore all factors affecting on
PON1 activity have to be well understood. It is known that almost
Paraoxonase 1 (PON1) is the best studied member of the mam-
malian enzyme family including PON1, PON2 and PON3. PON1 is
the calcium-dependent and high-density lipoprotein (HDL) associ-
ated enzyme that has 355 amino acids with a molecular mass of
43 kDA.1–3 PON1 catalyses the hydrolysis of lactones,4 thiolac-
tones,5 aromatic esters6 and organophosphates,7 but physiological
substrate of this enzyme is still unknown. PON1 is a key enzyme in
metabolism especially for two reasons; (i) it hydrolyses
organophosphates such as serin and soman and protects the ner-
vous system against the neurotoxicity of organophosphates7 and
(ii) it prevents the oxidation of low-density lipoproteins and
reduced levels of oxidized lipids are involved in the initiation of
atherosclerosis so decreased activity of PON 1 was acknowledged
all chemical reactions in metabolism of living organisms are cat-
alyzed by enzymes and they are important drug targets and effects
of bioactive compounds on PON1 activity must also be examined.
Heterocyclic rings are widely found in the structures of large
number of natural products and they are part of various biologi-
cally active compounds so synthesis of heterocyclic compounds
is important target for organic chemists. Therefore, synthesis and
biological evaluation of heterocyclic compounds are still the sub-
ject of intensive research area.8–15 In addition, cyclic structures
are more conformationally restricted than their acyclic analogues
and this fact make them more selective and bioactive.16,17
Azoles and coumarins are well known and widely investigated
class of compounds among the heterocyclic compounds. Imidazole,
triazole and benzimidazole derived compounds have attracted
great interest due to their wide range of pharmacological proper-
ties including anticancer, antitumor, antimicrobial, antiviral, anti-
inflammatory and anticoagulant.8–13 Coumarin derivatives have
been widely investigated owing to their diverse pharmacological
⇑
Corresponding authors. Tel.: +90 4223773855; fax: +90 4223410037 (M.O.K.);
tel.: +90 2666121278; fax: +90 2666121215 (N.G.).
E-mail addresses: mert.karatas@inonu.edu.tr (M.O. Karatasß), ngencer@balikesir.
edu.tr (N. Gencer).
http://dx.doi.org/10.1016/j.bmc.2016.02.012
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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2
M. O. Karataßs et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
R1
N
R1
N
properties such as anticoagulant, anticancer, anti-HIV, antimicro-
bial14,15 and carbonic anhydrase inhibitor.18 As shown, these small
heterocyclic compounds act as highly functional scaffolds and their
vital role in drug design cannot be denied.
DMF, 90 oC, 8h
R2
R2
+
R3CH2Cl
N
N
Cl
R3
In our previous paper, we reported the inhibition of PON1 by
some hydroxy coumarin-benzimidazole hybrid compounds
(doi:10.3109/14756366.2015.1043297)19 In the present study, we
have synthesized nine imidazolium and eight benzimidazolium
derivatives which containing coumarin, triazole, benzimidazole,
benzoxazinone and some aryl or alkyl groups as substituent. The
synthesized compounds were characterized by IR, NMR, elemental
analysis and single-crystal X-ray diffraction. Among the various
activities of these compounds, anti-coagulant effects of coumarin
and benzimidazole derivatives make them more attractive espe-
cially for interactions with PON. Herein, we examined the
in vitro effects of these twenty compounds which were functional-
ized with different substituents. Moreover, in order to determine
the probable binding interactions, molecular docking studies were
performed on PON1 active site.
1a-3c
H
N
O
O
N
N
R3;
N
N
S1
S2
S3
Compound no
R1
R2
R3
1a
1b
1c
2a
2b
2c
3a
3b
3c
-CH3
-CH3
-H
S1
S1
S1
S2
S2
S2
S3
S3
S3
-CH3
-H
-CH2CH2CH2CH3
-CH3
-H
-CH3
-CH3
-H
2. Results and discussion
-CH2CH2CH2CH3
-CH3
2.1. Synthesis and spectral characterization of compounds
(1a–5g)
-H
-CH3
-CH3
-H
Compounds C1–3 were synthesized by the procedure described
in literature by Frasinyuk20 as shown in Scheme 1. Synthesis and
structures of imidazolium salts (1a–3c) and benzimidazolium salts
(5a–g) were outlined in Schemes 2 and 3, respectively. These com-
pounds were synthesized by direct quaternization of 1-alkylimida-
zole and 1-alkylbenzimidazole derivatives with different alkyl
chlorides in DMF. All compounds were obtained in good yields
between 54% and 95%. In 1H NMR spectra of imidazolium salts
1a,c, 2a,c and 3a,c, signals of acidic NCHN protons were located
in the range of 9.36–9.92 ppm while these signals were obtained
in the range of 10.34–11.30 ppm for benzimidazolium salts (5a–
g). These signals of acidic hydrogens are in good agreement with
literature.21 For compounds 3a–c, signals of free ANH hydrogens
were located in the range of 13.41–13.49 ppm. Signal of free
ANH hydrogen of compound 5g was located at 11.08 ppm. In IR
spectra of all coumarin derived compounds (C1–3, 1a–c, 5e,f),
sharp carbonyl peaks were observed in the range of 1707–
1723 cmÀ1. Two carbonyl peaks were observed at 1702 and
1685 cmÀ1 for compound 5g. Elemental analysis of all compounds
was also supportive for depicted structures. Hence, all of the syn-
thesized compounds were characterized by 1H NMR, 13C NMR, IR
and elemental analysis in satisfactory manner.
-CH2CH2CH2CH3
Scheme 2. Synthesis and structures of imidazolium salts.
teristics of the two compounds are different, with compounds 2c
ꢀ
and 2b crystallizing in the space groups P21/c and P1, respectively.
In both compounds, the charge is balanced by a ClÀ anion. The
asymmetric unit in the crystal structure of compound 2b contains
the two independent molecules, labelled A and B, and one water
solvent molecule. In the following discussion, parameters for mole-
cule B are quoted in square brackets.
The imidazole ring is planar with an rms deviation of 0.0012 Å
for compound 2c, and 0.0023 Å [0.0034 Å] for compound 2b. The
bonding within the imidazole ring indicates a pattern of delocaliza-
tion that extends from atom N4 to atom N5 through atom C8, the
N4AC8 {1.313(3) Å in compound 2c, 1.346(2) Å [1.3388(19) Å] in
compound 2b} and N5AC8 {1.322(3) Å in compound 2c, 1.323
(2) Å [1.333(2) Å] in compound 2b} distances being significantly
shorter than the N4AC10 {1.376(3) Å in compound 2c, 1.380(2) Å
[1.379(2) Å] in compound 2b} and N5AC9 {1.373(3) Å in com-
pound 2c, 1.371(2) Å [1.380(2) Å] in compound 2b} distances.
The N1AC7 bond lengths of 1.446(3) and 1.440(2) Å [1.445(2) Å]
in compounds 2c and 2b, respectively, show the CAN single bond
character. The N1AC6 and N3AC1 bond lengths in the benzotria-
zole ring are 1.356(3) and 1.378(3) Å in compound 2c, and 1.365
(2) and 1.379(3) Å [1.359(2) and 1.377(3) Å] in compound 2b,
and N1AN2 and N2AN3 bond lengths are 1.359(3) and 1.301
(3) Å in compound 2c, and 1.356(2) and 1.291(2) Å [1.3615(19)
and 1.298(3) Å] in compound 2b, respectively. These shorter
lengths suggest that the CAN and NAN bonds in benzotriazole ring
have part double bond character. In these bond lengths, the N2AN3
bond is relatively shorter, which shows that the N2AN3 distance
has relatively stronger double bond character. All nitrogen atoms
in the benzotriazole ring (N1, N2 and N3) are sp2 hybridized and
occupy positions in the plane of the aromatic moiety. The rms devi-
ation of the benzotriazole ring atoms from their mean plane is
0.0104 Å and 0.0124 Å [0.0089 Å] for compounds 2c and 2b,
respectively. The triazole and benzene rings of the benzotriazole
moiety are inclined to one another at an angle of 0.793(13)° in
2.2. Structural description of the compounds 2b and 2c
The solid-state structures of compounds 2b and 2c have been
determined by single crystal X-ray analysis. The perspective
ORTEP-3 views of the compounds with the adopted atomic num-
bering scheme are depicted in Figures 1 and 2, while selected bond
lengths and angles are given in Table 1. The crystallization charac-
OH
Cl
O
O
O
O
H2SO4 (70%), rt, 24 h
R
+
R
O
Cl
C1, R: 5,7-(CH3)2
;
C2, R: 7,8-(CH3)2
;
C3, R: 6-C(CH3)3
Scheme 1. Synthesis of compounds C1–3.
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M. O. Karataßs et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Cl
H
N
N
N
(i)
(ii)
N
N
N
N
N
N
+
N
N
N
N
N
N
Cl
R4
4
5a- g
O
N
N
N
5a. R4=
5b. R4=
5c. R4=
5d. R4=
CH2CH2CH3
O
O
O
4
4
4
H
5e
5f
5g
. R =
. R =
. R =
O
O
N
O
O
O
O
Scheme 3. Synthesis and structures of benzimidazolium salts. Reagents and conditions: (i) KOH, EtOH, reflux, 24 h, (ii) R4CH2Cl, DMF, 90 °C, 8 h.
Figure 1. A view of compound 2b showing the atom-numbering scheme. Displacement ellipsoids are drawn at the 10% probability level and H atoms are shown as small
spheres of arbitrary radii.
compound 2c, and 1.617(16)° [1.120(11)°] in compound 2b. Fur-
thermore, The mean plane of the benzotriazole system makes a
dihedral angle of 71.536(82)° in compound 2c, and 69.826(52)°
[65.973(59)°] in compound 2b with the imidazole ring. In both
compounds, all the bond lengths and angles are within normal
ranges22 and comparable to those in the related compounds.23–28
derivatives and they showed that 6,7-dihydroxy-3-(4-methylphe-
nyl)-2H-chromen-2-one inhibits PON1 with IC50 value of 3
M.34
In addition, Akbaba et al. reported the interactions of some bromi-
nated compounds and bromophenols with PON1 and compounds
inhibited PON1 with different sensitivities.35
In the present study, the effects of seven-teen imidazole and
benzimidazole derivatives and three simple coumarin on purified
PON1 were investigated. These compounds were synthesized first
time in this study. For evaluating the PON1 activity, all compounds
were subjected to PON1 inhibition assay with paraoxon as sub-
strate. The IC50 values of compounds were given in Table 2. The
results showed that twelve of seventeen imidazolium or benzimi-
dazolium salts inhibit PON1 with different sensitivities while five
compounds show no activity. IC50 values of active compounds
l
2.3. Enzyme inhibition and structure activity relationship
As mentioned in Section 1, inhibition or activation properties of
bioactive compounds on PON1 activity have to be well understood.
According to our literature survey, inhibition effects of some drugs
or metal ions on PON1 activity were reported29–33 but studies
about interactions of novel compounds and PON1 are scarce.
Erzengin et al. reported the inhibition effects of some coumarin
were determined in the range of 6.37–3333.30 lM. Among the
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M. O. Karataßs et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
In our previous study, we reported the inhibition of PON1 with
benzimidazole-coumarin hybrid compounds. In the present study,
we aimed to examine the effects of some different substituents to
the inhibitory properties. Here we also examine whether the inhi-
bitory properties of previously reported compounds are specific to
combination of benzimidazole and coumarin or it arises from cou-
marin scaffold or some other substituents. For this purpose, firstly
coumarin compounds C1–3 were investigated. Results showed that
these compounds have good inhibitory properties and IC50 values
were ranged between 21.37 and 40.00 lM. Later, inhibitory prop-
erties of compounds 1a–c were investigated and compounds 1a
and 1c showed good inhibitory activities with IC50 values of
23.70 and 6.37 lM, respectively. In order to observe the effects of
some different heterocyclic cores to the inhibitory activity, benzo-
triazole and benzimidazole moieties were attached to imidazole
core. Interestingly, benzotriazole bearing imidazolium salts
(2a–c) showed no activity. On the other hand, benzimidazole bear-
ing imidazolium salts 3a and 3c showed good inhibitory activities
Figure 2. A view of compound 2c showing the atom-numbering scheme. Displace-
ment ellipsoids are drawn at the 10% probability level and H atoms are shown as
small spheres of arbitrary radii. For clarity, only the major part of the disordered
butyl moiety is shown.
with IC50 values of 19.05 and 28.98 lM, respectively. It was also
observed that, for coumarin and benzimidazole substituted imida-
zolium salts, attaching a methyl group to the 2-position of imida-
zole core decreased activity (IC50 for 1b is 317.31
625.71 M). When benzimidazole derived compounds were per-
used, compound 4 which containing benzimidazole and benzotria-
zole moieties inhibited PON1 activity with IC50 value of 564.13 M.
lM and for 3b
Table 1
l
Selected geometric parameters for compounds 2b and 2c
Parameters
Compound 2c
Compound 2b
l
As seen from Table 2, quaternization of compound 4 with different
substituent changed the inhibitory properties. Compounds 5a and
5b showed lower inhibitory activity than compound 4 with IC50
Molecule A
Molecule B
Bond lengths (Å)
N1AN2
N1AC6
N1AC7
N2AN3
N3AC1
N4AC7
N4AC8
N4AC10
N5AC8
1.359(3)
1.356(3)
1.446(3)
1.301(3)
1.378(3)
1.460(3)
1.313(3)
1.376(3)
1.322(3)
1.373(3)
1.483(4)
1.483(4)
1.336(4)
—
1.356(2)
1.365(2)
1.440(2)
1.291(2)
1.379(3)
1.458(2)
1.346(2)
1.380(2)
1.323(2)
1.371(2)
1.469(2)
—
1.3615(19)
1.359(2)
1.445(2)
1.298(2)
1.377(3)
1.456(2)
1.3388(19)
1.379(2)
1.333(2)
1.380(2)
1.465(2)
—
values of 3333.30 and 2064.20 lM, respectively. In addition,
compounds 5c and 5d showed no activity. So it can be said that,
attaching of butyl, benzyl, trimethoxybenzyl and benzotriazole
moieties to compound 4 decreased inhibitory activities signifi-
cantly. Attaching of 7,8-dimethyl substituted coumarin to the
structure of compound
Compound 5e inhibited PON1 activity with IC50 value 237.50
4
increased the inhibitory activity.
M.
l
N5AC9
Among the benzimidazolium derivatives, compound 5g which
containing benzoxazinone moiety was found out as most active
with IC50 value of 8.76 lM.
According to the results of enzyme inhibition assay the follow-
ing generalizations can be made; (i) all coumarin derived com-
pounds investigated in this study inhibited PON1, (ii) position of
substituents on benzimidazole scaffold is important for inhibitory
activity, benzimidazole derivatives which containing substituent
on the 2-position were found out more active, (iii) benzotriazole
N5AC11A
N5AC11B
C9AC10
C8AC12
1.335(3)
1.472(2)
1.341(2)
1.474(2)
Bond angles (°)
N1AN2AN3
N1AC7AN4
N4AC8AN5
N1AC6AC1
C6AN1AN2
C6AN1AC7
N2AN1AC7
N2AN3AC1
N3AC1AC6
C8AN4AC10
C8AN4AC7
C10AN4AC7
C9AC10AN4
C8AN5AC9
C8AN5AC11A
C9AN5AC11A
C8AN5AC11B
C9AN5AC11B
C10AC9AN5
N4AC8AC12
N5AC8AC12
N5AC11AAC12A
N5AC11BAC12B
108.58(19)
110.83(19)
109.0(2)
104.29(19)
110.33(18)
129.5(2)
120.2(2)
108.23(19)
108.6(2)
108.39(19)
124.9(2)
126.7(2)
107.2(2)
108.4(2)
125.7(4)
125.7(4)
125.0(7)
124.4(7)
107.1(2)
—
108.43(15)
111.50(13)
107.28(14)
103.58(14)
110.67(13)
129.62(14)
119.71(14)
108.80(15)
108.52(15)
108.73(14)
126.19(14)
125.02(15)
106.95(16)
109.55(15)
124.82(17)
125.63(16)
—
108.18(15)
112.24(13)
107.21(13)
104.08(14)
110.51(13)
129.59(13)
119.63(14)
108.66(14)
108.56(15)
109.02(13)
125.63(13)
125.30(13)
107.43(14)
109.64(13)
125.68(15)
124.67(15)
—
Table 2
IC50 values of the synthesized compounds
Compound no
IC50 (lM)
C1
C2
C3
1a
1b
1c
2a
2b
2c
3a
3b
3c
4
31.41
21.37
40.00
23.70
317.31
6.37
NA
NA
NA
19.05
625.71
28.98
564.13
—
—
107.49(15)
127.27(15)
125.45(16)
—
106.71(15)
126.72(15)
126.07(15)
—
—
110.2(3)
110.1(3)
—
—
5a
5b
5c
5d
5e
5f
3333.30
2064.20
NA
tested compounds, 1c was found out as most active compound
with 6.37 M value of IC50. Kinetic investigation was performed
for the compound 1c and results showed that this compound inhi-
bits PON1 activity in a competitive manner with Ki value of
2.39 lM (Table 3, Fig. 3).
l
NA
237.50
627.00
8.76
5g
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M. O. Karataßs et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Table 3
Kinetic and molecular docking data of compounds 1c and 2c
Compound Ki
Vmax
(
l
mol/min) Km
Inhibiton
type
Docking score
(kcal/mole)
Close van der Waals contacts (bond length, (Å))
H-bonds (bond length,
(Å))
no
(l
M)
(lM)
1c
2.39
6.08
—
40.57
Competative À5.4
ALA217 (4.39), GLU218 (3.47), LYS244 (5.47),
TYR248 (5.17)
PHE220 (5.10), HIS246 (4.87), VAl261 (3.70)
HIS246 (2.10)
—
2c
—
—
—
À3.6
y = 40.258x + 168.14
550
500
450
400
350
300
250
200
150
100
50
y = 15.88x + 166.31
y = 6.67x + 164.44
0,004762 mM
0,006667 mM
0
1/[S] mM-1
-25
-20
-15
-10
-5
0
5
10
Figure 3. Inhibition of paraoxonase by compound 1c. The slope of Lineweaver–Burk plots indicates competitive inhibition for paraoxon.
derivatives were found out as inactive against PON1, (iv) in this
study only one benzoxazinone derived compound was investigated
and attaching the benzoxazinone moiety to the structure of com-
pound 4 increased inhibitory activity dramatically.
As mentioned in Section 1, PON1 catalyses the hydrolysis of lac-
tones and in a study it was reported that, besides some lactone
derivatives, dihydrocoumarin was hydrolyzed by PON1 but in the
same study it was also shown that simple coumarin was not
hydrolyzed by PON1.4 As mentioned above, our results revealed
that all coumarin compounds evaluated in this study inhibit
PON1 activity and we suggest that coumarin derivatives are not
suitable substrates for PON1 and they are likely possible inhibitors
of PON1.
2.4. Molecular docking studies
Kinetic evaluation of compound 1c showed that this compound
inhibits PON1 activity in a competitive manner. Therefore molecu-
lar docking studies were performed in order to determine the
probable binding model of compound 1c into active site of PON1.
Moreover, in order to validate the experimental results of com-
pounds and for comparison with 1c, molecular docking studies
were also performed for compound 2c which is one of the inactive
compounds.
Docking scores were obtained using Lamarckian Genetic Algo-
rithm and scoring function of AutoDock 4. After that, interactions
were checked with the aid of ADT and Discovery Studio 4.0 Client.
Docking scores and binding interactions of compounds 1c and 2c
with PON1 (PDB code: 1V04)1 are presented in Table 3. Final
Figure 4. Docking of compound 1c within the active site of PON1 (A) Discovery
Studio 4.0 Client images and (B) ADT images.
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6
M. O. Karataßs et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 5. Docking of compound 2c within the active site of PON1 (A) Discovery Studio 4.0 Client images and (B) ADT images.
images of compounds 1c and 2c for binding interactions are shown
in Figures 4 and 5, respectively.
and some possible side effects of the derivatives of heterocyclic
structures tested in this study.
Molecular docking studies revealed that, compound 1c which is
the most active compound as PON1 inhibitory posses docking
score of À5.4 kcal/mole and forms a hydrogen bond with His-246
(2.10 Å). Moreover 1c in close van der Waals contacts with Ala-
217 (4.39 Å), Glu-218 (3.47 Å), Lys-244 (5.47 Å) and Tyr-248
(5.17 Å). Compound 2c which is an inactive compound exhibits
weaker binding affinity with docking score of À3.6 kcal/mole.
Compound 2c forms no hydrogen bond and in close van der Waals
contact with Phe-220 (5.10 Å), His-246 (4.87 Å) and Val-261
(3.70 Å). These results are in good agreement with experimental
results. Compounds 1c and 2c are bearing same groups in their
structures apart from coumarin or benzotriazole scaffold and
stronger binding affinity of 1c can attributable with coumarin scaf-
fold. Harel et al. reported the crystal structure of recombinant
PON1 which containing two calcium ions and these ions were
shown essential for the catalytic activity and stability of PON1.1
Molecular docking results also suggested that compound 1c inhi-
bits PON1 activity by not interaction with two calcium ions.
4. Experimental
Chemicals and solvents were purchased from Sigma Aldrich,
Merck (Istanbul-Turkey). 6-(chloroacetyl)-2H-1,4-benzoxazine-3
(4H)-one, 1-(chloromethyl)-1H-benzo[d][1,2,3]triazole, 2-(chloro-
methyl)-1H-benzo[d]imidazole were supplied commercially and
used without further purification. Melting points were determined
by Electrothermal-9200 melting point apparatus. The FT-IR spectra
were recorded on an ATR unit in the range of 400–4000 cmÀ1 with
a Perkin Elmer Spectrum 100 Spectrophotometer. The 1H NMR and
13C NMR spectra were recorded using a Bruker FT spectrometer
operating at 300.13 MHz (1H), 75.47 MHz (13C) and Bruker 600
Avance 3 HD spectrometer operating at 600.134 MHz (1H) and
150.918 MHz (13C). Chemical shifts are given in ppm relative to
TMS. NMR multiplicities are abbreviated as follows: s = singlet,
d = doublet, t = triplet, quin = quintet, sex = sextet, m = multiplet
signal. Elemental analyses were performed by a LECO CHNS-932
elemental analyzer at IBTAM (Inonu University Scientific and Tech-
nological Research Center).
3. Conclusion
In summary, nine imidazolium salts and seven benzimidazolium
salts were synthesized in good yields and well characterized. All
compounds were subjected to PON1 inhibition assay with paraoxon
as substrate. Results showed that twelve compounds inhibited
PON1 and IC50 values were determined for active compounds.
Among them, compound 1c was found out as most active and
kinetic evaluation revealed that PON1 is inhibited by 1c in compet-
itive manner. Molecular docking studies were performed for com-
pounds 1c and 2c and results confirmed the data obtained
experimentally. Moreover, according to molecular docking results
1c inhibited PON1 by not interaction with two calcium ions.
Based on the finding in this study, we suggest that coumarins
are potential inhibitory of PON1. In addition, some benzimidazole
or benzoxazinone derived compounds inhibited PON1 effectively.
The derivatives of coumarin, benzimidazole and benzoxazinones
are being investigated as drug candidates for use in future. How-
ever inhibition of PON1 by these compounds reveals that more
carefully and detailed investigations are required about dosage
4.1. General procedure for the preparation of 4-chloro-
methylene substituted coumarin derivatives (C1–3)
4-Chloromethyl-5,7-dimethyl-2H-chromene-2-one (C1), 4-
chloromethyl-7,8-dimethyl-2H-chromene-2-one (C2), 4-chloro-
methyl-6-tertiarybutyl-2H-chromene-2-one (C3) were synthesized
by procedure described in the literature.20 Crude products were
recrystallized from ethanol.
4.1.1. 4-Chloromethyl-5,7-dimethyl-2H-chromene-2-one (C1)
White solid. Yield: 88%, mp: 180–181 °C, Elemental analysis:
Calcd for: C12H11ClO2; C, 64.73; H, 4.98; Found: C, 64.76; H, 4.94;
IR(cmÀ1): 3010, 2949, 1698(AC@O), 1617(AC@CA), 1603, 1550,
1453, 1423, 1389, 1272, 1231, 1161; 1H NMR(300 MHz, DMSO-
d6): 7.11(s, 1H, ArH), 7.04 (s, 1H, ArH), 6.63(s, 1H, AC@CHA),
5.10(s, 2H, ACH2Cl), 2.74(s, 3H, ArCH3), 2.34(s, 3H, ArCH3). 13C
NMR(75 MHz, DMSO-d6): 159.4, 154.9, 152.3, 142.3, 136.1, 129.7,
115.7, 115.6, 110.1, 45.2, 22.3, 20.6.
Please cite this article in press as: Karataßs, M. O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.012

M. O. Karataßs et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
4.1.2. 4-Chloromethyl-7,8-dimethyl-2H-chromene-2-one (C2)
White solid. Yield: 80%, mp: 176–177 °C, Elemental analysis:
Calcd for: C12H11ClO2; C, 64.73; H, 4.98; Found: C, 64.85; H, 4.87;
IR(cmÀ1): 2975, 2938, 1715(AC@O), 1631(AC@CA), 1602, 1563,
1419, 1371, 1281, 1260; 1H NMR(300 MHz, DMSO-d6): 7.57(d,
1H, ArH, J = 8.1 Hz), 7.22(d, 1H, ArH, J = 8.2 Hz), 6.60(s, 1H,
AC@CHA), 4.99(s, 2H, ACH2Cl), 2.36(s, 3H, ArCH3), 2.27(s, 3H,
ArCH3). 13C NMR(75 MHz, DMSO-d6): 159.8, 151.3, 151.0, 141.8,
125.6, 123.9, 121.8, 114.7, 113.8, 41.3, 19.8, 11.2.
(300 MHz, DMSO-d6): 7.71(d, 1H, ANCH@CHNA, J = 8.2 Hz), 7.57
(d, 1H, ANCH@CHNA, J = 8.1 Hz), 7.11(s, 1H, ArH), 7.06(s, 1H,
ArH), 5.89(s, 2H, ANCH2coumarin), 5.37(s, 1H, AC@CHA), 3.77(s,
3H, ANCH3), 2.71(s, 3H, ArCH3), 2.45(s, 3H, ANC(CH3)NA), 2.32(s,
3H, ArCH3). 13C NMR(75 MHz, DMSO-d6): 159.1, 154.5, 151.4,
145.9, 142.6, 136.6, 129.8, 123.2, 121.5, 115.5, 114.4, 110.0, 50.3,
35.1, 23.8, 20.6, 9.2.
4.3.3. 1-(Butyl)-3-((5,7-dimethyl-2H-chromen-2-one-4-yl)
methyl)imidazolium chloride (1c)
4.1.3. 4-Chloromethyl-6-tert-butyl-2H-chromene-2-one (C3)
White solid. Yield: 67%, mp: 134–135 °C, Elemental analysis:
Calcd for: C14H15ClO2; C, 67.07; H, 6.03; Found: C, 67.17; H, 6.12;
IR(cmÀ1): 3074, 2968, 1711(AC@O), 1606(AC@CA), 1569, 1426,
1376, 1261; 1H NMR(300 MHz, DMSO-d6): 7.78–7.36(m, 3H,
ArH), 6.67(s, 1H, AC@CHA), 5.10(s, 2H, ACH2Cl), 1.34(s, 9H, AC
(CH3)3). 13C NMR(75 MHz, DMSO-d6): 159.7, 151.3, 150.8, 146.8,
129.7, 121.4, 116.4, 116.3, 115.3, 41.4, 34.4, 31.0.
White solid. Yield: 59%, mp: 266–268 °C, Elemental analysis:
Calcd for: C19H23ClN2O2; C, 65.79; H, 6.68; N, 8.08; Found: C,
65.70; H, 6.59; N, 8.21; IR(cmÀ1): 3071, 2960, 1709(AC@O), 1618
(AC@CA), 1605, 1559, 1456, 1316, 1157; 1H NMR(300 MHz,
DMSO-d6): 9.49(s, 1H, ANCHNA), 8.01(m, 2H, ANCH@CHNA),
7.16(s, 1H, ArH), 7.10(s, 1H, ArH), 6.19(s, 2H, ANCH2coumarin),
5.44(s, 1H, AC@CHA), 4.28(t, 2H, ANCH2CH2CH2CH3, J = 7.3 Hz),
2.71(s, 3H, ArCH3), 2.37(s, 3H, ArCH3), 1.32(quint, 2H, ANCH2CH2-
CH2CH3, J = 7.4 Hz), 1.29(sex, 2H, ANCH2CH2CH2CH3, J = 7.6 Hz),
0.92(t, 3H, ANCH2CH2CH2CH3, J = 7.4 Hz). 13C NMR(75 MHz,
DMSO-d6): 159.0, 154.4, 152.3, 142.7, 137.3, 136.3, 129.9, 123.3,
123.1, 115.6, 114.3, 110.9, 51.4, 48.9, 31.1, 23.6, 20.6, 18.8, 13.3
4.2. Synthesis of 1-((1H-benzo[d]1,2,3-triazol-1-yl)methyl)-1H-
benzo[d]imidazole (4)
Compound 4 was synthesized by the procedure described in the
literature with some differences in purification step,36 crude pro-
duct was recrystallized from ethanol/diethyl ether. White solid.
Yield: 55%, mp: 191 °C, Elemental analysis: Calcd for: C14H11N5;
C, 67.46; H, 4.45; N, 28.10; Found: C, 67.54; H, 4.36; N, 28.17; IR
(cmÀ1): 3094, 1613, 1501, 1452, 1350, 1279, 1213; 1H NMR
(600 MHz, DMSO-d6): 8.78(s, 1H, ANCHNA), 8.20–7.21(m, 8H,
ArH), 7.37(s, 2H, ANCH2NA). 13C NMR(150 MHz, DMSO-d6):
145.7, 144.7, 143.9, 133.2, 132.5, 128.8, 125.1, 123.7, 123.0,
120.2, 113.3, 110.9, 55.4.
4.3.4. 1-(Methyl)-3-((1H-benzo[d]1,2,3-triazol-1-yl)methyl)
imidazolium chloride (2a)
White solid, Yield: 89%, mp: 217–219 °C, Elemental analysis:
Calcd for: C11H12ClN5; C, 52.91; H, 4.84; N, 28.05; Found: C,
52.94; H, 4.87; N, 28.05; IR(cmÀ1): 3091, 3031, 2947, 2879, 1615,
1593, 1560, 1458, 1308, 1301, 1229, 1220, 1166; 1H NMR
(300 MHz, DMSO-d6): 9.82(s, 1H, ANCHNA), 8.45–7.49(m, 6H,
ArH and ANCH@CHNA), 3.87(s, 3H, ANCH3), 13C NMR(75 MHz,
DMSO-d6): 145.2, 137.6, 132.3, 128.6, 124.9, 124.3, 122.1, 119.5,
110.9, 57.6, 36.1.
4.3. General procedure for the preparation of imidazolium (1a–
3c) and benzimidazolium chlorides (5a–g)
4.3.5. 1-(Methyl)-2-(methyl)-3-((1H-benzo[d]1,2,3-triazol-1-yl)
methyl)imidazolium chloride (2b)
1-Alkylimidazole or compound 4 (2 mmol) were dissolved in
five mililiters of DMF. Two milimoles of corresponding alkyl chlo-
rides was added to this solution and the resulting mixture was
heated for eight hours at 90 °C. Later, the mixture was allowed to
cool to ambient temperature. Ten mililiters of diethyl ether was
added and precipitate was collected by fıltration, The crude pro-
duct was washed with hexane (2 Â 10 mL) and diethyl ether
(2 Â 10 mL) the dried under reduced procedure. The suitable crys-
tals of 2b and 2c for X-ray diffraction analysis was obtained from
recrystallization of 2b and 2c from ethanol/diethyl ether.
White crystal. Yield: 95%, mp: 182–183 °C, Elemental analysis:
Calcd for: C12H14ClN5; C, 54.65; H, 5.35; N, 26.56; Found: C,
54.68; H, 5.37; N, 26.53; IR(cmÀ1): 3086, 3037, 2953, 2944, 2881,
1559, 1466, 1312, 1307, 1231, 1224, 1178; 1H NMR(600 MHz,
DMSO-d6): 8.46(d, 1H, ANCH@CHNA, J = 8.4 Hz), 8.29(d, 1H, ArH,
J = 2.2 Hz), 8.13(d, 1H, ANCH@CHNA, J = 8.4 Hz), 7.78(d, 1H, ArH,
J = 2.2 Hz), 7.69(t, 1H, ArH, J = 6.2 Hz), 7.50(m, 3H, ArH and ANCH2-
triazole), 3.77(s, 3H, ANCH3), 2.87(s, 3H, ANC(CH3)NA). 13C NMR
(150 MHz, DMSO-d6): 146.8, 145.6, 132.9, 129.1, 125.4, 123.4,
121.8, 120.0, 111.4, 57.2, 35.5, 10.5.
4.3.1. 1-(Methyl)-3–((5,7-dimethyl-2H-chromen-2-one-4-yl)
methyl)imidazolium chloride (1a)
4.3.6. 1-(Butyl)-3-((1H-benzo[d]1,2,3-triazol-1-yl)methyl)
imidazolium chloride (2c)
White solid. Yield: 61%, mp: 279–281 °C, Elemental analysis:
Calcd for: C16H17ClN2O2; C, 63.05; H, 5.62; N, 9.19; Found: C,
63.12; H, 5.72; N, 9.11; IR(cmÀ1): 3070, 2955, 2942, 1707(AC@O),
1614(AC@CA), 1609, 1550, 1466, 1327, 1166; 1H NMR(300 MHz,
White crystal. Yield: 94%, mp: 177–178 °C, Elemental analysis:
Calcd for: C14H18ClN5; C, 57.63; H, 6.22; N, 24.00; Found: C,
57.65; H, 6.25; N, 24.03; IR(cmÀ1): 3165, 3085, 3023, 2950, 2872,
1614, 1592, 1557, 1457, 1434, 1306, 1223, 1165; 1H NMR
(300 MHz, DMSO-d6): 9.92(s, 1H, ANCHNA), 8.41–7.47(m, 6H,
ArH and ANCH@CHNA), 7.43(s, 2H, ANCH2triazole), 4.20(t, 2H,
ANCH2CH2CH2CH3, J = 7.2 Hz), 1.76(quint, 2H, ANCH2CH2CH2CH3,
J = 7.3 Hz), 1.19(sex, 2H, ANCH2CH2CH2CH3, J = 7.6 Hz), 0.86(t, 3H,
ANCH2CH2CH2CH3, J = 7.3 Hz). 13C NMR(75 MHz, DMSO-d6):
145.2, 137.2, 132.3, 128.6, 124.9, 123.1, 122.3, 119.5, 110.9, 57.7,
48.9, 31.1, 18.7, 13.2).
DMSO-d6):
9.36(s,
1H,
ANCHNA),
7.90–7.85(m,
2H,
ANCH@CHNA), 7.16(s, 1H, ArH), 7.10(s, 1H, ArH), 6.18(s, 2H,
ANCH2coumarin), 5.49(s, 1H, AC@CHA), 3.95(s, 3H, ANCH3), 2.72
(s, 3H, ArCH3), 2.37(s, 3H, ArCH3). 13C NMR(75 MHz, DMSO-d6):
159.1, 154.4, 152.4, 142.6, 137.8, 136.3, 129.9, 124.3, 123.0,
115.6, 114.3, 110.8, 51.3, 36.1, 23.6, 20.6.
4.3.2. 1-(Methyl)-2-(methyl)-3-((5,7-dimethyl-2H-chromen-2-
one-4-yl)methyl)imidazolium chloride (1b)
White solid. Yield: 63%, mp: 330–332 °C, Elemental analysis:
Calcd for: C17H19ClN2O2; C, 64.05; H, 6.01; N, 8.79; Found: C,
64.17; H, 6.12; N, 8.91; IR(cmÀ1): 2944, 2935, 1711(AC@O), 1612
(AC@CA), 1606, 1554, 1541, 1460, 1328, 1170; 1H NMR
4.3.7. 1-(Methyl)-3-((1H-benzo[d]imidazol-2-yl)methyl)
imidazolium chloride (3a)
White solid. Yield: 56%, mp: 257–258 °C, Elemental analysis:
Calcd for: C12H13ClN4; C, 57.95; H, 5.27; N, 22.53; Found: C,
57.90; H, 5.20; N, 22.45; IR(cmÀ1): 3055, 2952, 2940, 2856, 2768,
Please cite this article in press as: Karataßs, M. O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.012

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M. O. Karataßs et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
1564, 1533, 1440, 1327, 1270; 1H NMR(300 MHz, DMSO-d6): 13.41
(broad, 1H, ANH), 9.46(s, 1H, ANCHNA), 7.96–7.78(m, 2H,
ANCH@CHNA), 7.57–7.18(m, 4H, ArH), 5.82(s, 2H, ACH2benzimi-
dazole), 3.92(s, 3H, ANCH3), 13C NMR(75 MHz, DMSO-d6): 148.0,
137.5, 123.6, 123.2, 122.2, 44.2, 35.9.
3.76(s, 6H, ArOCH3), 3.63(s, 3H, ArOCH3), 13C NMR(75 MHz,
DMSO-d6): 153.1, 145.2, 143.5, 137.7, 132.4, 130.9, 130.4, 128.8,
128.6, 127.3, 127.1, 124.9, 119.5, 114.3, 114.0, 111.0, 106.6, 59.9,
56.1, 50.4, 30.6.
4.3.13. 1,3-Di((1H-benzo[d]1,2,3-triazol-1-yl)methyl)benzimi-
dazolium chloride (5d)
4.3.8. 1-(Methyl)-2-(methyl)-3-((1H-benzo[d]imidazol-2-yl)
methyl)imidazolium chloride (3b)
White solid. Yield: 73%, mp: 197–198 °C, Elemental analysis:
Calcd for: C21H17ClN8; C, 60.50; H, 4.11; N, 26.88; Found: C,
60.44; H, 4.16; N, 26.76; IR(cmÀ1): 3087, 3061, 1623, 1540, 1509,
1466, 1372, 1344, 1288, 1222, 1209; 1H NMR(600 MHz, DMSO-
d6): 11.30(s, 1H, ANCHNA), 8.47–7.46(m, 12H, ArH), 7.76(s, 4H,
ANCH2triazole), 13C NMR(150 MHz, DMSO-d6): 145.6, 145.1,
132.9, 130.9, 129.2, 128.1, 125.5, 120.0, 114.7, 111.5, 56.7.
White solid. Yield: 54%, mp: 253–255 °C, Elemental analysis:
Calcd for: C13H15ClN4; C, 59.43; H, 5.75; N, 21.32; Found: C,
59.36; H, 5.66; N, 21.38; IR(cmÀ1): 2943, 2891, 2862, 2761, 1590,
1531, 1435, 1385, 1328, 1268; 1H NMR(300 MHz, DMSO-d6):
13.49(broad, 1H, ANH), 7.89(d, 1H, ANCH@CHNA, J = 2.1 Hz),
7.72(d, 1H, ANCH@CHNA, J = 2.1 Hz), 5.77(s, 2H, ANCH2benzimi-
dazole), 3.82(s, 3H, ANCH3), 2.70(s, 3H, ANC(CH3)NA), 13C NMR
(75 MHz, DMSO-d6): 147.8, 145.4, 142.6, 134.2, 122.5, 121.8,
121.6, 118.8, 111.7, 45.1, 34.9, 9.7.
4.3.14. 1-((1H-Benzo[d]1,2,3-triazol-1-yl)methyl)-3-((7,8-dime-
thyl-2H-chromen-2-one-4-yl)methyl)benzimidazolium chloride
(5e)
4.3.9. 1-(Butyl)-3-((1H-benzo[d]imidazol-2-yl)methyl)imidazo-
lium chloride (3c)
White solid. Yield: 78%, mp: 249–251 °C, Elemental analysis:
Calcd for: C26H22ClN5O2; C, 66.17; H, 4.70; N, 14.84; Found: C,
66.26; H, 4.77; N, 14.77; IR(cmÀ1): 2920, 1723, 1608, 1567, 1491,
1443, 1382, 1349, 1262, 1219; 1H NMR(600 MHz, DMSO-d6):
10.34(s, 1H, ANCHNA), 8.38–7.28(m, 10H, ArH), 7.60(s, 2H,
ANCH2triazole), 6.18(s, 2H, ANCH2coumarin), 6.06(s, 1H,
AC@CHA), 2.41(3H, ArCH3), 2.32(s, 3H, ArCH3), 13C NMR
(150 MHz, DMSO-d6): 160.1, 151.6, 149.0, 145.7, 145.0, 142.8,
133.0, 131.8, 131.2, 129.2, 127.9, 126.3, 125.5, 124.6, 122.2,
121.8, 120.0, 115.0, 114.6, 114.5, 111.3, 56.7, 20.4, 11.7.
White solid. Yield: 67%, mp: 238–239 °C, Elemental analysis:
Calcd for: C15H19ClN4; C, 61.96; H, 6.59; N, 19.27; Found: C,
62.09; H, 6.69; N, 19.24; IR(cmÀ1): 3051, 2959, 2948, 1940, 2866,
2773, 1571, 1544, 1451, 1437, 1331, 1277; 1H NMR(300 MHz,
DMSO-d6): 13.46(broad, 1H, ANH), 9.56(s, 1H, ANCHNA), 8.03–
7.91(m, 2H, ANCH@CHNA), 7.78–7.46(m, 4H, ArH), 6.08(s, 2H,
ANCH2benzimidazole), 4.23(t, 2H, ANCH2CH2CH2CH3, J = 7.2 Hz),
1.83(quint, 2H, ANCH2CH2CH2CH3, J = 7.5 Hz), 1.32(sex, 2H,
ANCH2CH2CH2CH3, J = 7.6 Hz), 0.92(t, 3H, ANCH2CH2CH2CH3,
J = 7.4 Hz), 13C NMR(75 MHz, DMSO-d6): 147.2, 137.6, 133.1,
125.0, 123.3, 122.8, 114.5, 48.8, 44.5, 31.1, 18.8, 13.3.
4.3.15. 1-((1H-Benzo[d]1,2,3-triazol-1-yl)methyl)-3-((6-tert-
butyl-2H-chromen-2-one-4-yl)methyl)benzimidazolium
chloride (5f)
4.3.10. 1-((1H-Benzo[d]1,2,3-triazol-1-yl)methyl)-3-(butyl)-ben-
zimidazolium chloride (5a)
White solid. Yield: 70%, mp: 195–198 °C, Elemental analysis:
Calcd for: C28H26ClN5O2; C, 67.26; H, 5.24; N, 14.01; Found: C,
67.36; H, 5.27; N, 13.90; IR(cmÀ1): 3066, 2918, 1719, 1616, 1602,
1555, 1482, 1470, 1448, 1390, 1340, 1277, 1226; 1H NMR
(300 MHz, DMSO-d6): 10.70(s, 1H, ANCHNA), 8.41–7.44(m, 11H,
ArH), 7.75(s, 2H, ANCH2triazole), 6.36(s, 2H, ANCH2coumarin),
6.09(s, 1H, AC@CHA), 1.27(s, 9H, AArC(CH3)3), 13C NMR
(150 MHz, DMSO-d6): 159.4, 151.0, 148.6, 147.0, 145.2, 144.6,
132.5, 131.4, 130.6, 128.6, 127.5, 127.4, 124.9, 120.6, 119.5,
116.4, 116.1, 114.2, 114.1, 112.8, 110.9, 56.3, 46.9, 34.5, 30.9.
White solid. Yield 92%, mp: 194–196 °C, Elemental analysis:
Calcd for: C18H20ClN5; C, 63.24; H, 5.90; N, 20.49; Found: C,
63.31; H, 5.95; N, 20.33; IR(cmÀ1): 3097, 2962, 2943, 1617, 1505,
1457, 1442, 1366, 1352, 1288, 1217; 1H NMR(300 MHz, DMSO-
d6): 10.93(s, 1H, ANCHNA), 8.57–7.45(m, 8H, ArH), 7.80(s, 2H,
ANCH2triazole), 4.54(t, 2H, ANCH2CH2CH2CH3, J = 7.1 Hz), 1.90
(quint, 2H, ANCH2CH2CH2CH3, J = 7.3 Hz), 1.32(sex, 2H, ANCH2-
CH2CH2CH3, J = 7.6 Hz), 0.90(t, 3H, ANCH2CH2CH2CH3, J = 7.3 Hz),
13C NMR(75 MHz, DMSO-d6): 145.1, 143.4, 132.4, 131.2, 130.3,
128.6, 127.2, 126.9, 124.9, 119.4, 114.1, 113.9, 111.2, 56.0, 46.8,
30.2, 19.0, 13.3.
4.3.16. 1-((1H-Benzo[d]1,2,3-triazol-1-yl)methyl)-3-(6-acetyl-
2H-1,4-benzoxazine-3(4H)-one)benzimidazolium chloride (5g)
White solid. Yield: 54%, mp: 268–270 °C, Elemental analysis:
Calcd for: C24H19ClN6O3; C, 60.70; H, 4.03; N, 17.70; Found: C,
60.61; H, 3.92; N, 17.82; IR(cmÀ1): 3120, 3030, 1702, 1685, 1605,
1566, 1516, 1485, 1384, 1328, 1284; 1H NMR(600 MHz, DMSO-
d6): 11.08(s, 1H, ANH), 10.38(s, 1H, ANCHNA), 8.36–7.19(m,
11H, ArH), 7.86(s, 2H, ANCH2triazole), 6.38(s, 2H, ANCH2benzox-
azinone), 4.75(s, 2H, AC(O)CH2OA), 13C NMR(150 MHz, DMSO-
d6): 189.6, 164.5, 148.8, 145.7, 144.9, 132.8, 132.4, 130.2, 129.3,
128.4, 128.1, 127.8, 127.7, 125.6, 125.3, 120.1, 116.8, 115.8,
114.9, 111.2, 67.2, 56.6, 53.7.
4.3.11. 1-((1H-Benzo[d]1,2,3-triazol-1-yl)methyl)-3-(benzyl)-ben-
zimidazolium chloride (5b)
White solid. Yield: 88%, mp: 193–195 °C, Elemental analysis:
Calcd for: C21H18ClN5; C, 67.11; H, 4.83; N, 18.63; Found: C,
67.20; H, 4.86; N, 18.56; IR(cmÀ1): 3086, 3044, 2944, 1622, 1614,
1512, 1466, 1437, 1380, 1361, 1290, 1218; 1H NMR(300 MHz,
DMSO-d6): 10.70(s, 1H, ANCHNA), 8.38–7.39(m, 13H, ArH), 7.68
(s, 2H, ANCH2triazole), 5.82(s, 2H, ANCH2Ph), 13C NMR(75 MHz,
DMSO-d6): 145.7, 144.1, 134.0, 132.9, 131.4, 131.0, 129.5, 129.3,
129.2, 128.9, 127.9, 127.6, 125.5, 120.1, 114.7, 114.5, 111.3, 56.7,
50.7.
4.4. Single-crystal structure determination
4.3.12. 1-((1H-Benzo[d]1,2,3-triazol-1-yl)methyl)-3-(3,4,5-trime-
thoxybenzyl)-benzimidazolium chloride (5c)
The crystal data of the compounds 2b and 2c were collected on
a STOE IPDS II diffractometer at room temperature (296 K) using
Yellow solid. Yield: 91%, mp: 77–78 °C, Elemental analysis:
Calcd for: C24H24ClN5O3; C, 61.87; H, 5.19; N, 15.03; Found: C,
61.91; H, 5.22; N, 14.91; IR(cmÀ1): 3088, 3051, 3041, 2951, 1619,
1509, 1454, 1447, 1436, 1371, 1353; 1H NMR(300 MHz,
DMSO-d6): 10.99(s, 1H, ANCHNA), 8.32–7.48(m, 8H, ArH), 7.79
(s, 2H, ANCH2triazole), 7.02(s, 2H, ArH), 5.70(s, 2H, ANCH2Ph),
graphite-monochromated Mo
K
a
radiation (k = 0.71073 Å) by
applying the
x
-scan method. The structures were solved by direct
methods using SHELXS-201337 and refined with full-matrix least-
squares calculations on F2 using SHELXL-201437 implemented in
WinGX38 program suit. All of the C-bound H atoms were placed
at calculated positions and included in the refinement in the riding
Please cite this article in press as: Karataßs, M. O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.012

M. O. Karataßs et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
model approximation, fixing the bond lengths at 0.93, 0.97 and
0.96 Å for aromatic CH, CH2 and CH3 atoms, respectively. The water
H atoms in compound 2b were located in a difference Fourier map
and refined isotropically. The displacement parameters of the H
atoms were fixed at Uiso(H) = 1.2Ueq (1.5Ueq for methyl) of their
parent atoms. In compound 2c, the butyl moiety shows positional
disorder and the refined site-occupancy factors of the disordered
parts, viz. C11A–C14A and C11B–C14B, are 0.644(4)/0.356(4)%.
Data collection: X-AREA [39], cell refinement: X-AREA, data reduc-
tion: X-RED32.39 Crystal data, data collection and structure refine-
ment details are summarized in Table 4. The general-purpose
crystallographic tool PLATON40 was used for the structure analysis
and presentation of the results. ORTEP-3 was used to generate the
molecular graphics.41
CCDC 929882 and 1005944 contain the supplementary crystal-
lographic data (excluding structure factors) for the compounds
reported in this article. These data can be obtained free of charge
via http://www.ccdc.cam.ac.uk/cgi-bin/catreq.cgi [or from the
Cambridge Crystallographic Data Centre (CCDC), 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44(0)1223-336033; e-mail: data_re-
quest@ccdc.cam.ac.uk].
at 412 nm. The molar extinction coefficient of p-nitrophenol
= 17,100 MÀ1 cmÀ1at pH 8) was used to calculate enzyme activ-
(e
ity. The reaction was followed for 2 min at 37 °C by monitoring the
appearance of p-nitrophenol at 412 nm in automated recording
spectrophotometer (Biotek, Winooski, VT). 2 mM of final substrate
concentration was used during enzyme assay, and all measure-
ments were taken in duplicate and corrected for the non-enzy-
matic hydrolysis.
4.5.1. Purification of paraoxonase from human serum by
hydrophobic interaction chromatography
Human serum was isolated from 40 ml fresh human blood and
put into a dry tube. The blood samples were centrifuged at
3000 rpm for 15 min and the serum was removed. Firstly, serum
paraoxonase was isolated by ammonium sulphate precipitation
(60–80%). The precipitate was collected by centrifugation at
15,000 rpm for 20 min, and re-dissolved in 100 mM Tris–HCl buffer
(pH 8.0). Then, we synthesized the hydrophobic gel, including
Sepharose 4B, L-tyrosine and 1-napthylamine, for the purification
of human serum paraoxonase.43 The purified enzyme had aspecific
activity of 11.76 U/mg. The column was equilibrated with 0.1 M of
a Na2HPO4 buffer (pH 8.00) including 1 M ammonium sulphate.
The paraoxonase was eluted with an ammonium sulphate gradient
using 0.1 M Na2HPO4 buffer with and without ammonium sulphate
(pH 8.00). The purified PON1 enzyme was stored in the presence of
2 mM calcium chloride in order to maintain activity.
4.5. Paraoxonase enzyme assay
Paraoxonase enzyme activity towards paraoxon was quantified
spectrophotometrically by the method described by Gan et al.42
The enzyme assay was based on the estimating of p-nitrophenol
4.5.2. Total protein determination
The absorbance at 280 nm was used to monitor the protein in
the column effluents and ammonium sulphate precipitation. Quan-
titative protein determination was achieved by absorbance mea-
surements at 595 nm according to Bradford,44 with bovine serum
albumin as a standard.
Table 4
Crystal data and structure refinement parameters for compounds 2b and 2c
Parameters
Compound 2b
Compound 2c
CCDC deposition no.
Color/shape
1005944
1003894
Colorless/plate
Colorless/prism
Chemical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
2(C12H14N5)+Á2ClÀÁH2O C14H18N5+ÁClÀ
4.5.3. SDS polyacrylamide gel electrophoresis
545.48
296
291.78
296
0.71073 Mo K
Monoclinic
P21/c (No. 14)
SDS polyacrylamide gel electrophoresis was performed after
purification of the enzyme. It was carried out in 10% and 3% acry-
lamide concentration for the running and stacking gel, respec-
tively, containing 0.1% SDS according to Laemmli.45 A 20 mg
sample was applied to the electrophoresis medium. Gel was
stained overnight in 0.1% Coomassie Brilliant Blue R-250 in 50%
methanol and 10% acetic acid, then destained by frequently chang-
ing the same solvent, without dye. The electrophoretic pattern was
photographed with the system of produce as an image of the gel.
0.71073 Mo K
Triclinic
P1 (No. 2)
a
a
ꢀ
Unit cell parameters
a, b, c (Å)
8.1818(6), 13.1643(9), 8.3066(5), 19.2956
14.1659(10) (9), 9.7770(7)
102.865(5), 106.491(6), 90, 99.164(5), 90
104.493(5)
1343.70(17)
2
1.348
a
, b,
c
(°)
Volume (Å3)
Z
1547.06(16)
4
1.253
Dcalc (g/cm3)
4.5.4. In vitro kinetic studies and calculation of IC50 and Ki
values
l
(mmÀ1
)
0.280
0.245
Absorption correction
Tmin, Tmax
Integration
0.864, 0.962
572
Integration
0.889, 0.980
616
For the inhibition studies of synthesized compounds, different
concentrations of compounds were added to the reaction medium.
PON1 activity with compounds was assayed by following the
hydration of paraoxon. Activity percentage values of PON for five
different concentrations of each compounds were determined by
regression analysis using the Microsoft Office 2000 Excel. PON1
enzyme activity without a synthesized compound was considered
as 100% activity. The inhibitor concentration causing up to 50%
inhibition (IC50 values) for compounds were determined from the
graphs. So as to determinate Ki, Km and Vmax values of the PON1
enzyme using paraoxon as a substrate was measured at eight dif-
ferent substrate concentrations (0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 and
1.2 mM) at pH 8.00 and 37 °C in presence of two different cou-
F000
Crystal size (mm3)
0.27 Â 0.48 Â 0.71
0.08 Â 0.39 Â 0.60
Diffractometer/measurement STOE IPDS II/
method
Index ranges
x
scan
STOE IPDS II/x scan
À10 6 h 6 10,
À10 6 h 6 10,
À25 6 k 6 25,
À12 6 l 6 9
À17 6 k 6 17,
À18 6 l 6 18
h range for data collection (°) 1.68 6 h 6 28.03
2.11 6 h 6 27.55
10564
Reflections collected
Independent/observed
reflections
18208
6480/4720
3559/2175
Rint
0.062
0.049
Refinement method
Full-matrix least-
squares on F2
6480/0/346
1.061
R1 = 0.0426,
wR2 = 0.1117
R1 = 0.0613,
wR2 = 0.1186
0.23, À0.27
Full-matrix least-
squares on F2
3559/113/218
1.006
R1 = 0.0509,
wR2 = 0.1145
R1 = 0.0952,
wR2 = 0.1291
0.28, À0.23
Data/restraints/parameters
Goodness-of-fit on F2
marin derivative concentrations of compound 1c (6.7
4.7 M) as knowing highest and lowest final concentrations. Km
and Vmax values were calculated 40.57 M, 6.08 mol/min respec-
lM and
Final R indices [I >2
r(I)]
l
l
l
R indices (all data)
tively through Lineweaver–Burke graphs. The coumarin compound
numbered 1c inhibited PON1 enzyme activity in a competitive
D
qmax, D
qmin (e/Å3)
inhibition type with Ki of 2.39
lM value. As a result of in vitro
Please cite this article in press as: Karataßs, M. O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.012

10
M. O. Karataßs et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
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The authors declare no competing financial interest.
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Please cite this article in press as: Karataßs, M. O.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.012