A novel class for carbonic anhydrases inhibitors and evaluation of their non-zinc binding

Article abstract of DOI:10.1002/ardp.202100188

In this study, 23 different imidazole derivatives were synthesized, and the inhibitory properties of these derivatives against carbonic anhydrase I and II isoenzymes were investigated for the first time. The inhibition concentrations of the imidazole derivatives were found to be in the range of 2.89–115.5 nM. Docking studies examined the binding properties of the imidazole derivatives, and the structure–activity relationship is discussed. Theoretical calculations showed that the binding mode of the imidazole ring was non-zinc binding.

Full text of DOI:10.1002/ardp.202100188

Received: 15 May 2021
Accepted: 17 May 2021
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DOI: 10.1002/ardp.202100188
F U L L P A P E R
A novel class for carbonic anhydrases inhibitors and
evaluation of their non‐zinc binding
Burak Kuzu1 | Meltem Tan1 | İlhami Gülçin2 | Nurettin Menges1
1Pharmaceutical Chemistry Section, Van
Yüzüncü Yil University, Van, Turkey
Abstract
2Department of Chemistry, Faculty of
In this study, 23 different imidazole derivatives were synthesized, and the inhibitory
properties of these derivatives against carbonic anhydrase I and II isoenzymes were
investigated for the first time. The inhibition concentrations of the imidazole deri-
vatives were found to be in the range of 2.89–115.5 nM. Docking studies examined
the binding properties of the imidazole derivatives, and the structure–activity re-
lationship is discussed. Theoretical calculations showed that the binding mode of the
imidazole ring was non‐zinc binding.
Sciences, Atatürk University, Erzurum, Turkey
Correspondence
Nurettin Menges, Pharmaceutical Chemistry
Section, Van Yüzüncü Yil University, 65080
Tuşba, Van, Turkey.
Email: nurettinmenges@yyu.edu.tr
K E Y W O R D S
carbonic anhydrase inhibition, docking, imidazole ring, non‐zinc binding, SAR study
good candidates for CA inhibitors (Figure 1).[18–20] Inhibition mechan-
ism of CAs can be explained as follows: Whereas sulfonamides bind to
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1
INTRODUCTION
Carbonic anhydrases (CAs; also known as carbonate dehydratases,
E.C.4.2.1.1) are metalloenzymes, and 16 α‐CA isozymes or CA‐
related proteins have been described with different catalytic activity,
inhibition profiles, subcellular localization, and tissue distribution in
mammals.[1–14] CA is a pH‐regulatory enzyme and catalyzes carbon
dioxide's hydration to bicarbonate and the corresponding dehydra-
tion of bicarbonate in an acidic medium with the regeneration of
CO2. Many CA isozymes are essential therapeutic targets with the
potential to be inhibited to treat a range of disorders, including os-
teoporosis, cancer, edema, glaucoma, obesity, and epilepsy.[15] Fur-
thermore, inhibition of CAs also has the potential to fight infections
caused by protozoa, fungi, and bacteria.[16]
the Zn2+ ion of the enzyme to release OH− group, the other inhibitors
bind to the active site enzyme via noncovalent bondings in which the
Zn2+ ion is not involved.[21] Different inhibition mechanisms indicate
that inhibitors might bind to the active sites of CAs in different ways,
resulting in either nanomolar inhibition and selectivity of CAIs.
However, in parallel with the studies mentioned above,
significant progress has been made in discovering CAIs that do not
contain sulfonamide groups in the last few years. New CAI class
molecules have emerged as coumarins.[21–24] Coumarin derivatives
have been reported to act as “prodrug inhibitors” and undergo CA‐
mediated hydrolysis of the lactone ring that produces the actual
inhibitor of the 2‐hydroxycinnamic acid class. Through extensive ki-
netics, crystallographic, and synthetic trials, the innovative mechan-
ism of coumarin‐type molecules has been elucidated.[21–24]
According to the mechanism studies in detail, it was found that the
2‐hydroxycinnamic acids formed by coumarin hydrolysis do not
interact with the catalytic metal ion but rather bind in a highly
variable region of the different CA isoforms at the entrance of the
Many of these isoenzymes are essential targets to design different
types of inhibitors with clinical applications. CA inhibitors' (CAIs) main
class is sulfonamides and their bioisostere, sulphamate, and sulphamide
compounds 1–3, 5–7, which Mann and Kellin first discovered.[17]
Besides sulfonamides, new types of molecules such as coumarin 4,
phenol, and cyclopropane‐substituted anisol derivatives 9 have been
The authors have a national patent dealing with the study of synthesized molecules and their carbonic anhydrase inhibition effects
(Turkish Patent Institute; patent number: TR 2017 08596 B).
© 2021 Deutsche Pharmazeutische Gesellschaft
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Arch Pharm. 2021;e2100188.
wileyonlinelibrary.com/journal/ardp
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https://doi.org/10.1002/ardp.202100188

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FIGURE 1 Some of the critical carbonic anhydrase inhibitors
active space cavity. This situation also explains the selectivity of
many isoforms. Moreover, this new class of CA inhibitors brought
the sulfocoumarins found by Zalubovskis et al.[25–27] and the
homocumarins,[28] the homocumarins reported by Ferraroni et al.[29]
Polyamines, such as spermine and its derivatives,[30] and the simple
phenol[31] and many of its derivatives[32–35] inhibit CAs that anchor
to the zinc‐coordinated water molecule. On the contrary,
D'Ambrosio et al.[36] have reported that 2‐benzylsulfinylbenzoic acid
inhibits human CA II (hCA II), showing a binding mode utterly
different from any other class of CA inhibitors investigated so far. The
carboxylated form of the synthesized molecule binds in a pocket si-
tuated out of the enzyme active site. The newest molecule revealed in
the inhibition of CA enzymes was ninhydrin 8 reported by Bouzina et al.
They have shown that ninhydrins coordinate with the Zn(II) ion by one
or two OH moieties in which the molecules most effectively inhibit CA
II and CA VII.[37] D'Ambrosio et al.[38] reported the first structural in-
formation on the CA inhibition mechanism of catechol, showing that
they adopt a peculiar binding mode to the enzyme active site, involving
the zinc‐bound water molecule and the “deep water.” Besides organic
compounds, some of the organometallic complex structures were also
investigated for their inhibition potential against hCAs. Bouchouit
et al.,[39] Alyar and Adem,[40] and Ul‐Hassan et al.[41] reported some
metal complexes with organic structures for hCAs inhibitions. Ghiasi
and her co‐workers also investigated the theoretical modeling of the
complex structure of CA and ligands.[42–44]
with hCA IX/XII and possibly hCA IV[48]; (v) hypoxic tumors, tar-
geting the two isoforms under investigation for tumors, hCA IX and
XII.[49–51] The main reason for the vast amount of research re-
porting recently on the development of inhibitors for CA iso-
enzymes is the hope of developing new drug candidates for the
abovementioned serious diseases. While these research studies are
carried out, it is revealed that the newly discovered ring systems
might target different active regions.
Imidazole ring is a biologically accepted pharmacophore in
medicinal compounds and is also an essential partner of Zn2+
,
responsible for the CA inhibition mechanism. Moreover, the
incorporation of imidazole skeleton has made it a versatile hetero-
cyclic ring possessing a wide spectrum of biological activities such as
antiprotozoal,[52] antibacterial,[53] lipoxygenases (LOXs) inhibiting,[54]
anticancer,[55,56] and antiviral activities.[57]
Although there are only a few examples of inhibitors that do
not have sulfonamide and its bioisosteres, their diffuse localization
in many tissues and organs, and the lack of isozyme selectivity of
the presently available inhibitors, a completely new structural
motif for the inhibition of these enzymes constitutes a valuable
lead for researchers working in the design of CAIs. The studies
described above show that many molecules with different struc-
tures affect the CA isoenzymes. These effects occur as a result of
their settlement in different active regions of the enzyme. This
study aims to reveal a new inhibitor group and determine its ef-
fects on two critical isoenzymes, hCAs I and II. Besides, it aims to
determine the inhibition mechanism through theoretical models.
Thus, it was reported in this study for the first time that it is
possible to use imidazole, which is a critical heterocycle ring, as a
CA enzyme inhibitor. Therefore, in this study, we have reported a
novel class of inhibitors of the metalloenzyme CAs belonging to an
entirely new chemotype, the imidazole derivatives 11–14.[58–61]
Different types of CA isoenzymes have been targeted for dif-
ferent pathogens. These can be briefly summarized as follows: (i)
treatment of idiopathic intracranial hypertension, inhibition with
hCA I–II[45]; (ii) cerebral ischemia/diabetic cerebrovascular pathol-
ogies, inhibition with hCA IX and hCA XII or hCA VA[46]; (iii) tar-
geting the brain/peripheral nervous system, neuropathic pain
isoforms hCA II and VII[47]; (iv) inflammation/arthritis, inhibition

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2
RESULTS AND DISCUSSION
Chemistry
acetazolamide, as shown in Table 1. Furthermore, isozymes CA I
and CA II were inhibited by all of the imidazole derivatives. Imi-
dazole nucleus inhibited CA I and CA II isozymes with promising
concentrations, and we have observed that functional groups al-
tered inhibition constants (Ki) and selectivity, as discussed below
(data extracted from Table 1):
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2.1
Based on the substituted imidazole ring's importance, we synthe-
sized a series of di‐ and trisubstituted imidazole derivatives 11a‐p,
12a, b, 13‐15. The reaction was accomplished by oxidation of acetyl
derivatives 9 to glyoxal 10, formed in situ, followed by treatment
with ammonium acetate (NH4OAc), as depicted in Scheme 1.[59–61]
Further reactions have involved reducing the carbonyl group to
get alcohol derivatives and substitution of the imidazole nitrogen
atom to form N‐substituted imidazole derivatives (Scheme 1). Un-
substituted and substituted phenyl ring, naphthalene, pyrene, and
thiophene rings were incorporated into imidazole skeleton on C‐2
and C‐4 positions. Next, we have substituted the nitrogen atom of
the imidazole ring with allyl bromide in basic conditions, reduced the
carbonyl group of the imidazole ring by NaBH4, and condensed the
carbonyl group by hydroxylamine (Scheme 1). The N‐substituted al-
cohol and oxime‐incorporated imidazole derivatives 12a,b, 13, 14
were formed.
(i) The unsubstituted phenyl ring‐incorporated imidazole 11a
showed an inhibition constant of 2.40 nM for CA I and Ki of
15.22 nM for CA II. Naphthalene ring‐substituted imidazole
derivative 11b inhibited CA I and CA II with inhibition con-
stants of 15.45 and 6.47 nM, respectively. It was shown that
the affinity of CA isozymes could be changed with an extended
ring system. On the contrary, the affinity of α‐naphthalene
11b and β‐naphthalene 11c against CA I and CA II was dif-
ferent. Both derivatives are fully planar, and deviation of
planarity was not observed for both of them. However, we can
say that both phenyl rings of 11c are far away from the imi-
dazole nucleus, and the geometrical difference of 11b gave
higher affinity to CA II and CA I, and surprisingly, we could not
observe this kind of difference for 11c. To determine the limit
of fused aromatic rings incorporated into the imidazole nu-
cleus, we have furnished pyrene ring‐substituted imidazole
molecule 11e, which possesses inhibition constants of 6.21 nM
for CA I and 10.88 nM for CA II. Selectivity in inhibition was
observed in this derivative, as we have observed for 11a.
However, selectivity between 11b and 11e was found to be
the opposite. When the benzene ring located at the C‐4 po-
sition in the structure of molecule 11a was removed (com-
pound 15), it was observed that the activity for both enzymes
decreased considerably. In this case, it has been shown that
the C‐2‐ and C‐4‐substituted benzene ring is required for
optimum activity.
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2.2
Pharmacology/biology
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2.2.1
relationship (SAR)
CA inhibition and structure–activity
Phenol, coumarin 4, and anisole 9 derivatives, which do not carry
sulfonamide groups, have been applied to inhibit CAs with micro-
molar and nanomolar inhibition constant.[18–20] To improve the in-
hibition concentration using an utterly unknown nucleus, we have
synthesized mono‐ and disubstituted imidazole derivatives 11a–p,
12a,b, 13, 14, and 15, whose inhibition data against both catalytically
active human CA isoforms, CAs I–II, are presented in Table 1.
Substituted imidazole derivatives were formed to understand
their effect on enzyme affinity via electronic and steric para-
meters. Therefore, we have incorporated many different func-
tional groups and heterocyclic rings into the imidazole nucleus at
C‐2 and C‐4 positions. Phenyl, α‐naphthalene, β‐naphthalene,
pyrene, substituted phenyl, thiophene, substituted thiophene, al-
cohol, and oxime‐derived imidazole derivatives were investigated
in this study. These compounds, 11a–p, 12a,b, 13, 14, and 15,
inhibited CA I and CA II with inhibition constants of the range of
2.40–115.5 nm for CA I and 1.01–115.5 nm for CA II. There is only
one CA I example with a heterocyclic ring, coumarin, without
sulfonamide group in the literature, and its inhibition concentra-
tions for CA I and CA II are 78 and 59 nM, respectively.[19]
Substituted imidazole derivatives showed excellent affinity to-
ward the hCA isozymes and this study represents the first ex-
ample of this kind of nucleus. An inhibition study was performed
by applying a well‐known standard CA I molecule, acetazolamide,
a clinically used drug. Affinities of imidazole derivatives (except
for 11a–p, and 15) against CA I and CA II were higher than
(ii) After unsubstituted aromatic rings, we have analyzed sub-
stituted phenyl ring‐incorporated imidazole derivatives. The
most minor inhibitor in this study was 11d, which carries two
identical OH groups at the para position of phenyl rings,
compared with the unsubstituted derivatives 11a–c and 11e.
Inhibition constants are 23.64 nM for hCA I and 22.57 nM hCA
II. When we have altered OH groups with OMe group, we have
taken inhibition constants 10.61 nM for hCA I and 6.01 nM for
hCA II (compound 11f in Table 1). It might be stated that the
OH group at para position affects one or more noncovalent
interactions between ligand and enzyme negatively because it
is not only a good hydrogen donor but also an acceptor
functional group. On the contrary, the OMe group positively
changed affinity, as mentioned above, as it is only a hydrogen
acceptor group. The position and number of OMe groups were
also investigated (compounds 11f–h in Table 1). Two OMe
groups at para and meta positions of the phenyl ring increased
inhibitor activity as compared with 11f (compound 11g in
Table 1 and its inhibition constants are 4.91 nM for CA I and
2.10 nM for hCA II). It means that some groups form more

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SCHEME 1 Synthesis of the imidazole derivatives. DMF, dimethylformamide; TEA, triethylamine
noncovalent bonds with residues of the amino acid inactive
side of isozymes. Two OMe groups on both ortho positions
decreased inhibitor activity in which inhibitor constants are
10.80 nM for hCA I and 14.17 nM for hCA II. Some of the para‐
substituted derivatives 11l–p were also investigated. Electron‐
withdrawing groups attached to the benzene ring decreased
the activities for both isoenzymes.
Density functional theory (DFT) calculation has given an
optimized geometry, because OMe groups at ortho positions of
the phenyl ring show a deviation from planarity at about

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TABLE 1 Inhibition of hCA I and hCA II isoenzymes with
imidazoles 11a–p, 12a,b, 13, 14, and 15
(iii) Besides phenyl ring, the thienyl group‐substituted imidazole 11j
and its brominated analog 11k decreased inhibition constant
with an inhibition constant 4.63 and 9.62 nM and 4.91 and
8.98 nM, respectively. For this type of derivative, it can be said
that there is no extreme effect of bromine at the para position.
Lastly, the substitution of the nitrogen atom of the imidazole
ring with the allyl group increased the inhibition constant
against hCA I (from 4.63 to 8.70 nM) when the inhibition con-
stant against hCA II did not change. It means that a crucial
noncovalent bonding, probably hydrogen bond, was disrupted
by NH substitution.
IC50 (nM)
hCA II
Ki (nM)
hCA I
Compounds
11a
11b
11c
11d
11e
11f
hCA I
5.78
6.30
4.08
6.93
4.95
6.93
3.47
4.08
2.89
4.62
6.30
63.0
86.62
53.3
69.3
77
hCA II
6.93
4.95
5.78
8.66
6.33
5.78
3.85
6.30
4.62
6.93
5.78
115.5
69.3
99
2.40 1.17
15.22 4.36
6.47 0.91
15.45 6.86
10.23 4.21
23.64 1.72
6.21 1.37
11.52 1.97
22.57 7.85
10.88 2.91
6.01 1.08
10.61 2.65
4.91 2.03
(iv) Reduced derivatives showed promising results toward se-
lective inhibition. For example, whereas 11g possesses in-
hibition activity with inhibition constants of 4.91 and
2.10 nM, 12b, reduced analog, decreases the inhibition ac-
tivity against hCA I isozyme with an inhibition constant
ranging from 4.91 to 10.15 nM. On the contrary, 11f shows
inhibition activity with inhibition constants of 10.61 and
6.01 nM in which hCA II can be inhibited selectively. How-
ever, 12a did not show selective inhibition (10.41 and
9.51 nM). There is a significant and sensitive active side in
CA isozymes, which determines inhibition constant when
functional groups are changed. Reduced analogs, 12a and
12b, have alcohol groups, which are significant hydrogen
donor and acceptor units. Consequently, whereas 12a in-
creased inhibition constant against hCA II, 12b increased
inhibition constant against hCA I. With a logical design, a
highly selective CA I analog might be formed by using imi-
dazole derivatives.
11g
11h
11i
2.10 1.03
10.80 4.86
4.43 1.75
14.17 6.69
5.33 1.54
11j
4.63 1.67
9.62 2.62
11k
11l
4.91 1.68
8.98 1.45
158.76 54.34
191.97 108.23
133.36 33.39
142.91 40.93
183.81 69.39
10.41 4.52
10.15 2.11
8.70 2.70
182.06 73.57
208.65 65.63
204.10 138.90
50.76 17.50
146.49 34.78
9.51 1.89
11m
11n
11o
11p
12a
12b
13
69.3
115.5
5.33
3.01
5.77
7.70
86.62
49.50
5.33
6.93
4.62
3.15
115.5
69.30
1.01 0.45
9.59 1.60
14
4.79 2.19
15.97 6.66
133.45 42.64
32.64 13.07
15
280.31 31.06
64.75 14.37
AZAa
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2.3
Molecular docking
Abbreviations: AZA, acetazolamide; CA, carbonic anhydrase.
aAZA was used as a standard inhibitor for all CAs investigated here.
We have desired to observe possible interactions between ligands
and enzymes. Hence, two different enzymes and three different li-
gands, which have significant inhibitor effects, were selected for the
docking process.[63]
44.40° and 70.27° (Figure 2).[62] Therefore, it might be dis-
cussed that planarity might be necessary for the cavity of the
active site of the enzyme and noncovalent bonding with re-
sidues of amino acids at appropriate positions. The substituted
phenyl ring with bromine at para position, 11i, inhibited the
activity of isozymes with inhibition constants of 4.43 nM for
hCA I and 5.33 nM for hCA II (Table 1).
It has been observed that compound 11d has many significant
interactions with CA I enzyme (PDB code: 4WR7) in the docking
study. Some of these are as follows. Compound 11d formed
The carbonyl group of 11f was converted into an oxime by
a reaction between 11f and hydroxylamine under basic con-
ditions resulting in the oxime derivative of imidazole 14.
Surprisingly, 14 possesses more effective potency to inhibit
hCA I with an inhibition constant of 4.79 nM. However, ligand
affinity against hCA II reduced significantly from 6.01 to
15.97 nM. The oxime group is a hydrogen donor and acceptor,
and hydrogen bond accepting or donating might occur in dif-
ferent ways in hCA I and hCA II isoenzymes.
FIGURE 2 Optimized geometry of 11g and 11h

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KUZU ET AL.
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hydrogen bonding with THR199 and interacted with HIS200, HIS94,
and HIS119 through π–π interaction. On the contrary, the hydrogen
atom of the imidazole ring formed a hydrogen bonding with GLN92.
The binding energy between 11d and CA I was calculated as
−6.4 kcal/mol. In the docking study of molecule 11d with CA II
(5AML), interactions between many essential amino acids in the
active site were observed. The amino acids ASN244, ASN62, ASN67,
and SER197 formed hydrogen bonds with the imidazole ring from
different places. Some unfavorable interactions have also been ob-
served in ligand–enzyme active site interaction. It has been de-
termined that the benzene ring interacts with the Zn265 through
π–cation, and the HIS94 amino acid has π–π interaction with the
imidazole ring. The binding energy for this complexation was found
to be −6.9 kcal/mol. In the docking study of molecule 11i with CA I
(4WR7) enzyme, it was observed that the amino acids HIS119,
HIS94, HIS96, and HIS64 appropriately interacted with the bromine
atom. HIS200 amino acid shows appropriate interactions with imi-
dazole and benzene rings. The binding energy for this docking pro-
cess was −7.2 kcal/mol, and the interactions between molecule 12b
and CA II (5AML) enzyme are as follows: TRP5 and THR199 amino
acids form hydrogen bonding with the oxygen atom of the methoxy
group. While some unfavorable interactions were observed in some
of the water molecules, it was determined that one of the water
molecules revealed hydrogen bonding with the ligand. The binding
energy of this docking was −6.7 kcal/mol (Figure 3 and Table 2).
spectrometer. 1H chemical shifts were referenced to internal stan-
dard tetramethylsilane (δ 0.00 ppm) with solvent CDCl3 or d6‐DMSO.
13C NMR spectra were referenced to the solvent peak at 77 (CDCl3)
and 33 ppm (d6‐DMSO). High‐resolution mass spectra were recorded
using a Thermo Scientific Q Exactive orbitrap detector. Thin‐layer
chromatography (TLC) was carried out on Kieselgel 60.
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting
Information.
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4.1.2
Synthesis of compounds 11a–p[58]
The commercially available acetyl‐substituted aromatic compounds
(1 mmol) were mixed with 4 ml 1,4‐dioxane, and 2.5 mmol SeO2
was added to the solution. The reaction flask was refluxed over-
night, and the completion of the reaction was controlled with the
TLC method. Then after filtration of Se0 particles, 5 mmol am-
monium acetate dissolved in ethanol was added to the reaction
solution at room temperature. The obtained stable molecules were
filtered, washed three times with water (30 ml), and dried in vacuo
over P2O5.
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3
CONCLUSION
Synthesis of compounds 12a and 12b[58]
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In this study, 23 different molecules were synthesized, and their in-
hibitor effects on CA I and CA II enzymes were evaluated using cell‐
free in vitro experiments. Many synthesized molecules gave sound
inhibitor effects than the positive control, acetazolamide molecule.
The SAR was discussed to understand which functional group can
affect the inhibition positively or negatively. Docking studies using two
different enzyme structures gave interaction information. These in-
teractions are favorable, which might be responsible for acceptable
inhibition concentrations. This paper contains the first SAR study for
this new type of CAIs, bringing novel insights regarding the inhibition
mechanism and the structural requirements necessary to be present
in the molecules of such heterocyclic compounds for achieving high
affinity for various CA isoforms.
4.1.3
The obtained imidazole derivatives 11f and 11g (1 mmol) were dis-
solved in methanol, and NaBH4 (2 mmol) was added to the solution
at room temperature. The solution was stirred for 2 h; the reaction
completion was controlled by TLC and the mixture was recrystallized
with water. Moreover, the obtained stable compounds were dried
with P2O5 overnight.
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4.1.4
Synthesis of compound 13
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4
EXPERIMENTAL
Chemistry
The imidazole derivative 11j (1 mmol) was dissolved in dry DMF
(15 ml) (N,N‐dimethylformamide) and NaH (1.6 mmol) was added in
the solution at 0°C. After stirring for 30 min, the allyl bromide
(1.4 mmol) was added to the cooled solution. The reaction comple-
tion was controlled by TLC. The mixture was extracted with ethyl
acetate (10 × 3 ml) and water (30 ml). The organic layer was treated
with anhydrous MgSO4, ethyl acetate was evaporated, and the ob-
tained compound was purified with column chromatography (5:1,
n‐hexane:ethyl acetate).
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4.1
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4.1.1
General information
All solvents and chemicals are commercially available. 1H and 13C
nuclear magnetic resonance (NMR) spectra (see the Supporting In-
formation) were recorded on Varian MR‐400 400 MHz

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FIGURE 3 Docking results for some selected ligands (PDB code: 4WR7 carbonic anhydrase [CA I] and 5AML [CA II]). (a) Docking for 11d
(4WR7), (b) docking for 11d (5AML); (c) docking for 11i (4WR7), and (d) docking for 12b (5AML)
TABLE 2 Binding energies for some docking processes
Compound
11d
Enzyme
hCA I
Binding energy (kcal/mol)
PDB code
4WR7
−6.4
−7.2
−6.7
−6.9
11i
hCA I
4WR7
12b
hCA II
hCA II
5AML
11d
5AML
Abbreviation: hCA, human carbonic anhydrase.

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Synthesis of compound 14[59]
(100 MHz, DMSO) δ 184.8, 145.3, 142.8, 134.1, 133.5, 133.2, 131.8,
130.9, 130.4, 130.2, 128.5, 128.2, 127.8, 127.3, 126.6, 126.3, 126.2,
125.9, 125.8, 125.4, 125.2, 124.6, 121.7. HRMS (ESI) [M+H]
C24H17N2O :349.1341; found: 349.1344.
|
4.1.5
The synthesized compound 11f (1 mmol) was dissolved in propanol
with heating. Then, 2 mmol NH2OH·HCl and TEA (2 mmol) were
added to this solution. Next, the reaction mixture was refluxed
overnight and controlled by using TLC plate. The reaction solution
was cooled to room temperature and extracted with ethyl acetate
(20 ml × 3) and 30 ml water. The organic layer was dried with MgSO4
and the solvent was evaporated.
[Naphthalen‐2‐yl(4‐(naphthalen‐2‐yl)‐1H‐imidazol‐2‐yl]methanone
(11c)[58]
Light brown solid, M.p: 215–220°C, yield: 72%. Fourier‐transform
infrared (FTIR) attenuated total reflectance (ATR) cm−1: 3275, 3056,
1668, 1631, 1607, 1479, 1463, 1071, 1017, 961, 950, 924, 821. 1H
NMR (400 MHz, DMSO) δ 13.77 (s, 1H, H‐9), 9.45 (s, 1H, H‐8), 8.48
(bs, 1H, Ar–H), 8.26–8.24 (m, 1H, Ar–H), 8.15–8.09 (m, 2H, Ar–H),
8.03 (d, J = 8.0 Hz, 1H, Ar–H), 7.98–7.97 (m, 2H, Ar–H), 7.90
(d, J = 8.5, 1H, Ar–H), 7.70–7.65 (m, 3H, Ar–H), 7.51–7.46 (m, 3H,
Ar–H). 13C NMR (100 MHz, DMSO) δ 181.1, 145.5, 143.4, 135.4,
133.7, 133.5, 132.8, 132.4, 131.6, 130.5, 129.2, 128.7, 128.4, 128.3,
128.1, 127.3, 126.8, 126.3, 126.1, 124.3, 123.3, 119.7.
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4.1.6
Synthesis of compound 15
This molecule was synthesized according to our previous method.[58]
Phenyl(4‐phenyl‐1H‐imidazol‐2‐yl)methanone (11a)[58]
[4‐Hydroxyphenyl(4‐(4‐hydroxyphenyl)‐1H‐imidazol‐2‐yl]methanone
(11d)[59]
Light yellow solid, M.p: 190°C, yield: 68.5%. 1H NMR (400 MHz,
dimethyl sulfoxide [DMSO]) δ 8.48 (d, J = 7.1 Hz, 2H, H‐19 and H‐15),
8.05 (s, 1H, H‐8), 7.92 (d, J = 7.1 Hz, 2H, H‐3 and H‐5), 7.68
(t, J = 7.5 Hz, 1H, H‐17), 7.60–7.58 (m, 2H, H‐16 and H‐18) 7.41
(t, J = 7.5 Hz, 2H, H‐2 and H‐6), 7.29 (t, J = 7.5 Hz, 1H, H‐1). 13C NMR
(100 MHz, DMSO) δ 181.1, 144.7, 141.3, 136.3, 133.6, 132.5, 131.0,
129.2, 128.8, 128.1, 125.6, 120.9.
Yellow solid, M.p: 298–305°C, yield: 56%. FTIR (ATR) cm−1: 3647,
3233, 2980, 2889, 1607, 1579, 1433, 1029, 963, 911, 819, 806. 1H
NMR (400 MHz, DMSO) δ 13.27 (bs, 2H, H‐20, H‐21), 8.59–8.50
(m, AAʹBBʹ system, 2H, Ar–H), 7.77 (s, 1H, H‐8), 7.76–7.73 (m, AAʹBBʹ
system, 2H, Ar–H), 6.92–6.78 (m, AAʹBBʹ system, 4H, Ar–H). 13C
NMR (100 MHz, DMSO) δ 179.2, 162.6, 157.1, 145.1, 143.4, 133.8,
127.7, 126.6, 125.4, 116.7, 115.8, 115.5.
Naphthalen‐1‐yl[4‐(naphthalen‐1‐yl)‐1H‐Imidazol‐2‐yl]
methanone (11b)
Pyren‐1‐yl[4‐(pyren‐4‐yl)‐1H‐imidazol‐2‐yl]methanone (11e)[59]
Yellow solid, M.p: 105–107°C, yield: 61%. 1H NMR (400 MHz,
DMSO) δ 14.0 (s, 1H, N‐H), 8.60 (d, J = 8.4 Hz, 1H, Ar–H), 8.34–8.26
(m, 2H, Ar–H), 8.17 (d, J = 8.2 Hz, 1H, Ar–H), 8.07–8.05 (m, 2H,
Ar–H), 7.95–7.88 (m, 2H, Ar–H), 7.75 (d, J = 6.9 Hz, 1H, Ar–H),
7.69–7.62 (m, 4H, Ar–H), 7.53–7.45 (m, 2H, Ar–H). 13C NMR

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Dark brown solid, M.p: 145–149°C, yield; 72%. FTIR (ATR) cm−1
:
(2,6‐Dimethoxyphenyl)[4‐(2,6‐dimethoxyphenyl)‐1H‐imidazol‐2‐
yl]methanone (11h)
3039, 1622, 1593, 1537, 1506, 1446, 1069, 991, 959, 921, 872, 840.
1H NMR (400 MHz, DMSO) δ 8.87 (d, J = 9.3 Hz, 1H, Ar–H), 8.80
(s, 1H, Ar–H), 8.63–8.61 (m, 1H, Ar–H), 8.52 (d, J = 9.3 Hz, 1H, Ar–H),
8.44 (s, 1H, Ar–H), 8.39 (d, J = 9.7 Hz, 2H, Ar–H), 8.35–8.33 (m, 1H,
Ar–H), 8.32–8.31 (m, 1H, Ar–H), 8.30–8.29 (m, 1H, Ar–H), 8.28 (bs,
1H, Ar–H), 8.25–8.23 (m, 1H, Ar–H), 8.21–8.20 (m, 1H, Ar–H),
8.19–8.17 (m, 1H, Ar–H), 8.15–8.12 (m, 2H, Ar–H), 8.13–8.11 (m, 1H,
Ar–H), 8.08 (bs, 1H, Ar–H), 8.03–8.01 (m, 1H, Ar–H). 13C NMR
(100 MHz, DMSO) δ 185.5, 146.7, 133.3, 132.1, 131.4, 131.1, 130.8,
130.7, 130.5, 129.8, 129.7 129.5, 129.3, 128.0, 127.9, 127.8, 127.7,
127.6, 127.2, 126.8, 126.5 125.7, 125.4, 125.3, 125.0, 124.8, 124.4,
124.3, 124.2, 124.0. HRMS (ESI) [M+H] C36H21N2O: 497.1654;
found: 497.1649.
Light yellow solid, M.p: 239–241°C, yield: 30%. FTIR (ATR) cm−1
:
3426, 3096, 3006, 2980, 2951, 1641, 1592, 1486, 1472, 1431, 950,
937, 899. 1H NMR (400 MHz, CDCI3) δ 11.64 (bs, 1 H, H‐9), 7.93
(s, 1H, H‐8), 7.34 (t, J17,18 = 8.4 Hz, 1H, H‐17), 7.26 (t, J1,6 = 8.4 Hz,
1H, H‐1), 6.69 (d,
J18,17 = 8.4 Hz, 2H, H‐18, H‐16), 6.63
(d, J6,1 = 8.4 Hz, 2H, H‐6 and H‐2), 3.97 (s, 6H, OMe) 3.75 (s, 6H,
OMe). 13C NMR (100 MHz, CDCI3) δ 184.6, 157.9, 157.5, 143.9,
134.3, 131.2, 129.4, 129.2, 117.0, 106.4, 104.4, 104.2, 56.02, 56.01.
HRMS (ES) [M+H]; m/z C20H20N2O5: 368.1372; found: 368.1389.
(4‐Methoxyphenyl)[4‐(4‐methoxyphenyl)‐1H‐imidazol‐2‐
yl]methanone (11f)[59]
(4‐Bromophenyl)[4‐(4‐bromophenyl)‐1H‐imidazol‐2‐yl]methanone
(11i)[59]
Yellow solid, M.p: 204–206°C, yield: 87.5%. FTIR (ATR) cm−1: 3264,
3095, 2980, 2840, 1608, 1595, 1564, 1511, 1026, 972, 957, 903,
864. 1H NMR (400 MHz, DMSO) δ 8.50–8.48 (m, AAʹBBʹ system, 2H,
Ar–H), 7.94 (s, 1H, H‐8), 7.87–7.86 (m, AAʹBBʹ system, 2H, Ar–H),
7.13–7.11 (m, AAʹBBʹ system, 2H, Ar–H), 7.00–6.68 (m, AAʹBBʹ sys-
tem, 2H, H‐2, H‐6), 3.87 (s, 3H, OMe), 3.78 (s, 3H, OMe). 13C NMR
(100 MHz, DMSO) δ 179.1, 163.9, 159.5, 144.1, 133.5, 128.8, 127.2,
124.2, 124.1, 119.8, 114.7, 114.3, 56.1, 55.6.
Yellow solid, M.p: 298–305°C, yield: 56%. FTIR (ATR) cm−1: 3266,
3094, 1612, 1580, 1557, 1483, 1010, 902, 869, 830, 766. 1H NMR
(400 MHz, DMSO) δ 13.71 (bs, 1H, H‐9), 8.50–8.48 (m, AAʹBBʹ sys-
tem, 2H, Ar–H), 8.13 (bs, 1H, H‐8), 7.87–7.85 (m, AAʹBBʹ system, 2H,
Ar–H), 7.81–7.79 (m, AAʹBBʹ system, 2H, Ar–H), 7.60–7.59
(m, AAʹBBʹ system, 2H, Ar–H). 13C NMR (100 MHz, DMSO) δ
180.1, 144.9, 142.3, 135.3, 133.2, 133.0, 132.0, 131.9, 127.9, 127.3,
120.6, 119.9.
(3,4‐Dimethoxyphenyl)[4‐(3,4‐dimethoxyphenyl)‐1H‐imidazol‐2‐
yl]methanone (11g)[59]
Thiophen‐2‐yl[4‐(thiophen‐2‐yl)‐1H‐imidazol‐2‐yl]methanone
(11j)[60]
Brown‐yellow mixture solid, M.p: 120–125°C, yield: 52%. FTIR (ATR)
cm−1: 3217, 3086, 2980, 2836, 1609, 1588, 1567, 1511, 1017, 948,
894, 856, 846. 1H NMR (400 MHz, DMSO) δ 8.35 (d, J = 8.3 Hz, 1H,
H‐19), 7.86 (bs, 1H, H‐9), 7.52 (s, 2H, H‐5 and H‐15), 7.52 (s, 1H, H‐8),
7.12 (d, J = 8.3 Hz, 1H, H‐3), 6.97 (d, J = 8.3 Hz, 1H, H‐2), 3.86 (s, 3H,
Orange solid, M.p: 195–200°C, yield: 55%. FTIR (ATR) cm−1: 3235,
3092, 1644, 1594, 1506, 1473, 1051, 997, 915, 889, 870. 1H NMR
(400 MHz, DMSO) δ 13.67 (bs, 1 H, H‐8), 8.66 (dd, J17,16 = 3.8,
J17,15 = 1.1 Hz, 1H, H‐17), 8.10 (dd, J2,3 = 4.5, J2,4 = 1.1 Hz, 1H, H‐2),
7.92 (s, 1H, H‐7), 7.46 (dd, J4,3 = 3.5, J4,2 = 1.1 Hz, 1H, H‐4), 7.45 (dd,
OMe), 3.85 (s, 3H, OMe), 3.81 (s, 3H, OMe), 3.75 (s, 3H, OMe).13
C
NMR (100 MHz, DMSO) δ 179.1, 153.5, 149.4, 148.7, 148.5, 145.4,
128.9, 125.9, 117.9, 113.7, 112.4, 111.2, 109.1, 56.1, 55.9, 55.8, 55.7.
J15,16 = 5.0, J15,17 = 1.1 Hz, 1H, H‐15), 7.32 (dd, J16,15 = 5.0,

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J16,17 = 3.8 Hz, 1H, H‐16), 7.09 (dd, J3,2 = 4.5, J3,5 = 3.5 Hz, 1H, H‐3).
13C NMR (100 MHz, DMSO) δ 173.0, 144.0, 141.1, 138.7, 137.6,
136.9, 136.6, 129.0, 128.3, 125.0, 123.4, 118.5.
(m, AAʹBBʹ system, 2H, Ar–H). 13C NMR (100 MHz, d6‐DMSO)
δ = 179.8, 144.9, 142.2, 138.6, 134.9, 132.9, 132.8, 132.1, 132.0,
129.1, 128.9, 127.0, 119.8.
(5‐Bromothiophen‐2‐yl)[4‐(5‐bromothiophen‐2‐yl)‐1H‐imidazol‐2‐
yl]methanone (11k)
(4‐Nitrophenyl)[4‐(4‐nitrophenyl)‐1H‐imidazol‐2‐yl]methanone
(11n)[59]
Brown solid, M.p: 170–171°C, yield: 92%. 1H NMR (400 MHz,
DMSO) δ 8.25 (bs, 1H, Ar–H), 7.93 (bs, 1H, Ar–H), 7.42 (d, J = 4.1 Hz,
1H, Ar–H), 7.30 (bs, 1H, Ar–H), 7.19 (d, J = 3.7 Hz, 1H, Ar–H). 13C
NMR (100 MHz, DMSO) δ 170.8, 143.2, 141.1, 136.2, 131.7, 131.1,
123.9, 123.7, 109.9. HRMS (ESI) [M+H] C12H7Br2N2OS2: 416.8367;
found: 416.8372.
Dark brown solid, M.p: 280–282°C, yield: 88%. 1H NMR (400 MHz,
d6‐DMSO) δ = 13.99 (bs, 1H, –NH), 8.71–8.64 (m, AAʹBBʹ system, 2H,
Ar–H), 8.40–8.33 (m, 3H, Ar–H), 8.26–8.20 (m, AAʹBBʹ system, 2H,
Ar–H), 8.17–8.10 (m, AAʹBBʹ system, 2H, Ar–H). 13C NMR (100 MHz,
d6‐DMSO) δ = 180.1, 150.2, 146.6, 145.3, 141.5, 141.2, 132.4, 126.0,
124.6, 123.8.
(4‐Fluorophenyl)[4‐(4‐fluorophenyl)‐1H‐imidazol‐2‐yl]methanone
(11l)[59]
[4‐(Trifluoromethyl)phenyl][4‐(4‐(trifluoromethyl)phenyl)‐1H‐
imidazol‐2‐yl]methanone (11o)[64]
Dark yellow solid, M.p: 224–226°C, yield: 92%. 1H NMR (400 MHz,
d6‐DMSO) δ = 13.64 (bs, 1H, –NH), 8.71–8.67 (m, AAʹBBʹ system, 2H,
Ar–H), 8.06 (d, J = 2.5 Hz, 1H, Ar–H), 7.96–7.93 (m, AAʹBBʹ system,
2H, Ar–H), 7.44–7.39 (m, AAʹBBʹ system, 2H, Ar–H), 7.26–7.22
(m, AAʹBBʹ system, 2H, Ar–H). 13C NMR (100 MHz, d6‐DMSO)
δ = 179.5, 166.5 (d, J = 252.3 Hz), 161.9 (d, J = 243.5 Hz), 144.9,
142.5, 134.1 (d, J = 9.4 Hz), 132.9 (d, J = 2.9 Hz), 130.6 (d, J = 3.0 Hz),
127.2 (d, J = 8.1 Hz), 119.0, 116.0 (d, J = 6.3 Hz), 115.8 (d, J = 6.6 Hz).
Dark yellow solid, M.p: 199–200°C, yield: 82%. 1H NMR (400 MHz,
d6‐DMSO) δ = 13.91 (bs, 1H, –NH), 8.69–8.64 (m, AAʹBBʹ system, 2H,
Ar–H), 8.30 (s, 1H, Ar–H), 8.15–8.11 (m, AAʹBBʹ system, 2H, Ar–H),
7.98–7.92 (m, AAʹBBʹ system, 2H, Ar–H), 7.77–7.72 (m, AAʹBBʹ sys-
tem, 2H, Ar–H). 13C NMR (100 MHz, d6‐DMSO) δ = 180.4, 145.1,
142.0, 139.7, 137.9, 132.8 (d, J = 32.0 Hz), 131.8, 130.4, 127.8
(d, J = 31.7 Hz), 126.0 (d, J = 3.6 Hz), 125.8, 125.7 (d, J = 3.7 Hz),
125.2, 121.2.
(4‐Chlorophenyl)[4‐(4‐chlorophenyl)‐1H‐imidazol‐2‐yl]methanone
(11m)[59]
4‐[2‐(4‐Cyanobenzoyl)‐1H‐imidazol‐4‐yl]benzonitrile (11p)[65]
Light brown solid, M.p: 189–191°C, yield: 90%. 1H NMR (400 MHz,
d6‐DMSO) δ = 13.72 (bs, 1H, –NH), 8.60–8.56 (m, AAʹBBʹ system, 2H,
Ar–H), 8.13 (d, J = 2.5 Hz, 1H, Ar–H), 7.94–7.91 (m, AAʹBBʹ system,
2H, Ar–H), 7.67–7.64 (m, AAʹBBʹ system, 2H, Ar–H), 7.49–7.44
Brown solid, M.p: 269–270°C, yield: 74%. 1H NMR (400 MHz, d6‐
DMSO) δ = 8.62–8.57 (m, AAʹBBʹ system, 2H, Ar–H), 8.23 (s, 1H,
Ar–H), 8.06–8.02 (m, AAʹBBʹ system, 2H, Ar–H), 8.01–7.98
(m, AAʹBBʹ system, 2H, Ar–H), 7.83–7.80 (m, AAʹBBʹ system, 2H,

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Ar–H). 13C NMR (100 MHz, d6‐DMSO) δ = 180.0, 145.5, 141.2, 139.7,
1H, Ar–H), 7.36 (dd, J = 1.2, 3.5 Hz, 1H, Ar–H), 7.33 (s, 1H, Ar–H),
7.27–7.25 (m, 1H, Ar–H), 7.19 (dd, J = 3.9, 4.9 Hz, 1H, Ar–H), 7.07
(dd, J = 3.5, 5.1 Hz, 1H, Ar–H), 6.13–6.02 (m, 1H, –CH), 5.30–5.25
(m, 1H, –CH2), 5.22–5.17 (m, 1H, –CH2), 5.13 (dt, J = 1.5, 5.7 Hz, 2H,
–CH2). 13C NMR (100 MHz, CDCl3) δ 174.7, 141.9, 141.2, 137.2,
136.9, 136.4, 135.7, 132.9, 127.9, 127.6, 124.4, 122.9, 120.8, 118.4,
51.1. HRMS (ESI) [M+H] C15H13N2OS2: 301.0469; found: 301.0462.
138.0, 133.1, 133.0, 132.6, 131.5, 125.8, 119.5, 118.7, 115.4, 109.8.
(4‐Methoxyphenyl)[4‐(4‐methoxyphenyl)‐1H‐imidazol‐2‐yl]methanol
(12f)[59]
(Z/E)‐(4‐Methoxyphenyl)[4‐(4‐methoxyphenyl)‐1H‐imidazol‐2‐
yl]methanone oxime (14)[61]
Light brown solid, M.p: 180–181°C, yield: 87%. 1H NMR (400 MHz,
DMSO) δ 11.91 (s, 1H, H‐9), 7.63 (d, J = 8.8 Hz, 2H, Ar–H), 7.35
(d, J = 8.7 Hz, 3H, Ar–H), 6.89–6.85 (m, 4H, Ar–H), 6.10
(d, J13,12 = 4.0 Hz, 1H, H‐13), 5.70 (d, J12,13 = 4.0, 1H, H‐12), 3.72
(s, 3H, OMe), 3.70 (s, 3H, OMe). 13C NMR (100 MHz, DMSO) δ 158.8,
158.1, 135.7, 128.1, 125.9, 114.2, 113.8, 69.7, 55.5, 55.4. HRMS (ESI)
[M+H] C18H19N2O3:311.1396; found (M+H)+: 311.1397.
Light brown viscous, yield: 89.2%. 1H NMR (400 MHz, DMSO) δ
12.29 (s, 1H, N–H), 11.30 (s, 1H, O–H), 7.73–7.65 (m, 1H, Ar–H),
7.63–7.60 (m, 2H, Ar–H), 7.55–7.53 (m, 1H, Ar–H), 7.51–7.47 (m, 1H,
Ar–H), 6.99–6.94 (m, 2H, Ar–H), 6.92–6.90 (m, 1H, Ar–H), 6.87–6.85
(3,4‐Dimethoxyphenyl)[4‐(3,4‐dimethoxyphenyl)‐1H‐imidazol‐2‐
yl]methanol (12b)[59]
(m, 1H, Ar–H), 3.80–3.78 (m, 3H, OMe), 3.76–3.72 (m, 3H, OMe). 13
C
NMR (100 MHz, DMSO) δ 160.1, 159.8, 158.3, 147.9, 143.8, 141.2,
132.0, 127.7, 126.0, 123.9, 114.6, 114.4, 114.2, 113.6, 113.5, 113.2,
55.6, 55.5. HRMS (ESI) [M+H] C18H18N3O3: 324.1348; found [M
+H]+: 324.1350.
(1H‐Imidazol‐5‐yl)(phenyl)methanone (15)[59]
Light pink solid, M.p: 210–211°C, yield: 75%. FTIR (ATR) cm−1: 3288,
3113, 3008, 2954, 2904, 1591, 1532, 1511, 1451, 1320, 944, 871,
854. 1H NMR (400 MHz, d6‐DMSO) δ 11.96 (bs, 1H, N–H), 7.35 (bs,
1H, Ar–H), 7.31 (d, J = 2.0 Hz, 1H, Ar–H), 7.24 (dd, J = 2.0, 8.3 Hz, 1H,
Ar–H), 7.14 (d, J = 1.6 Hz, 1H, Ar–H), 6.92–6.88 (m, 3H, Ar–H), 6.13
(d, J = 3.4 Hz, 1H, OH), 5.70 (d, J = 3.4 Hz, 1H, CH) 3.78 (s, 3H, OMe),
3.75 (s, 3H, OMe), 3.73 (s, 3H, OMe), 3.71 (s, 3H, OMe). 13C NMR
(100 MHz, d6‐DMSO) δ 151.2, 149.2, 148.9, 148.4, 147.7, 136.1,
119.0, 116.8, 112.4, 111.8, 110.7, 108.6, 69.9, 56.0, 55.9, 55.8, 55.7.
HRMS (ESI) [M+H] C20H23N3O5: 371. 1607; found: 371.1600.
Brown solid, M.p: 153–155°C, yield: 65%. 1H NMR (400 MHz, CDCl3)
δ = 11.33 (bs, 1H, –NH), 8.59–8.56 (m, 2H, Ar–H), 7.63–7.59 (m, 1H,
Ar–H), 7.53–7.49 (m, 2H, Ar–H), 7.41 (bs, 1H, Ar–H), 7.27 (bs, 1H,
Ar–H). 13C NMR (100 MHz, CDCl3) δ = 182.2, 145.2, 135.6, 133.4,
131.8, 131.0, 128.3, 120.3.
|
Biological activity
[1‐Allyl‐4‐(thiophen‐2‐yl)‐1H‐imidazol‐2‐yl](thiophen‐2‐
yl)methanone (13)
4.2
To investigate the inhibitory effects of imidazole derivatives on hCA I
and hCA II from human erythrocytes, they were purified via a simple
single‐step sepharose‐4B‐L‐tyrosine‐sulfanilamide affinity gel chro-
matography. For this purpose, the human erythrocyte samples were
centrifuged at 13,000 rpm for 25 min. Then the solution was filtered
to remove the precipitate. Both hCA isoenzymes were isolated from
the serum, where the pH was adjusted to 8.7 by adding solid Tris.
The affinity column was equilibrated by buffer solution (25 mM
Tris‐HCl/0.1 M Na2SO4) at pH 8.7. The serum was loaded to affinity
Yellow solid, M.p: 165–167°C, yield: 95%. 1H NMR (400 MHz,
CDCl3) δ 8.58 (dd, J = 1.2, 3.9 Hz, 1H, Ar–H), 7.73 (dd, J = 1.2, 4.9 Hz,

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gel and washed with buffer solution (25 mM Tris–HCl/22 mM
Na2SO4) at pH 8.7. The hCA I isoenzyme was eluted by buffer
solution (1.0 M NaCl/0.25 M sodium phosphate) at pH 6.3. On the
contrary, hCA II isoenzyme was eluted by another buffer solution
(0.1 M sodium acetate/0.5 M NaClO4) at pH 5.6. Both isoenzymes
were taken from the column in fractions of 2 ml. All works were
realized at 4°C. The hCA isoenzymes activity was measured by
following procedure; the change at absorbance a specific wavelenght
(348 nm) of p‐nitrophenyl acetate (PNA) to p‐nitrophenolate ion over
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
AUTHOR CONTRIBUTIONS
Burak Kuzu and Meltem Tan Uygun conducted the synthesis of
compounds and evaluation of spectral data. İlhami Gulcin completed
in vitro experiments and extracted results. Nurettin Menges
designed the experiments, evaluated spectral data, and wrote the
manuscript.
3 min at room temperature (25°C) using
a spectrophotometer
(UV–Vis spectrophotometer; Thermo Fisher Scientific) according to
the method of Verpoorte et al., as described previously. There were
0.4 ml Tris‐SO4 buffer (0.05 M, pH 7.4), 0.3 ml of 3 mM PNA, 0.2 ml of
H2O, and 0.1 ml of enzyme solution in a test tube content of this
reaction.[66]
ORCID
Nurettin Menges
http://orcid.org/0000-0002-5990-6275
REFERENCES
[1] I. Nishimori, T. Minakuchi, S. Onishi, D. Vullo, A. Cecchi,
A. Scozzafava, C. T. Supuran, Bioorg. Med. Chem. 2007, 15, 7229.
[2] A. Yıldırım, U. Atmaca, A. Keskin, M. Topal, M. Çelik, İ. Gülçin,
C. T. Supuran, Bioorg. Med. Chem. 2015, 23, 2598.
|
4.3
Docking studies
[3] D. Vullo, M. Franchi, E. Gallori, J. Antel, A. Scozzafava,
C. T. Supuran, J. Med. Chem. 2004, 47, 1272.
[4] İ. Gulci̧ n, B. Trofimov, R. Kaya, P. Taslimi, L. Sobenina, E. Schmidt,
The protein molecule was obtained from the Protein Data Bank (PDB
code: 4WR7 and 5AML). For docking studies, Autodock 4.2 program
was utilized. The macromolecule was reconstructed by discovering the
ligand molecules and water molecules by the Discovery Studio 2016
program. Again with the same program, the amino acids in the active
region were determined. Enzyme and ligand molecules were pre-
pared, and the AutoDock 4.2 program prepared a grid map.[63] The
search grid of 4WR7 was identified as center X: 13.845, Y: 34.568,
Z: 18.971 coordinates for 4WR7 with dimension size_x: 60, size_y:
60, and size_z: 60, and X: −4.923, Y: 3.828, Z: 15.223 coordinates
for 5AML with dimension size_x: 60, size_y: 60, and size_z: 60. The
docking parameters are set to default, and the population size is
set to 300 and docking is performed. The enzyme ligand con-
formation of the smallest binding energy from 10 different con-
formations was created for each molecule obtained. This complex
was then studied with Discovery Studio and the 2D diagram of the
interaction with amino acids in the ligand‐binding region.
O. Petrova, S. Malysheva, N. Gusarova, V. Farzaliyev, A. Sujayev,
S. Alwasel, C. T. Supuran, Bioorg. Chem. 2020, 103, 104171.
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4.4
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SUPPORTING INFORMATION
Additional Supporting Information may be found online in the sup-
porting information tab for this article.
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How to cite this article: B. Kuzu, M. T. Uygun, İ. Gülçin, N.
Menges, A novel class for carbonic anhydrases inhibitors and
evaluation of their non‐zinc binding. Arch. Pharm. 2021,
e2100188. https://doi.org/10.1002/ardp.202100188.