Investigation of the inhibitory effects of isoindoline-1,3-dion derivatives on hCA-I and hCA-II enzyme activities

Article abstract of DOI:10.1016/j.molstruc.2019.07.070

Inhibition of carbonic anhydrase (CA) has emerged as a promising approach for the treatment of a variety of diseases such as glaucoma, epilepsy, obesity and cancer. As a result, the design of CA inhibitors (CAIs) is an outstanding field of medicinal chemi

Full text of DOI:10.1016/j.molstruc.2019.07.070

Journal of Molecular Structure 1197 (2019) 386e392
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Investigation of the inhibitory effects of isoindoline-1,3-dion
derivatives on hCA-I and hCA-II enzyme activities
,
b
,
,
c
d
Hayrunnisa Nadaroglu a *, Azize Alaylı Gungor b , Ozlem Gundogdu ,
€
Nurhan Horasan Kishali e **, Belgin Kishali f, Mehlika Dilek Altintop f
,
a Ataturk University, Erzurum Vocational School, Department of Food Technology, Erzurum, Turkey
b Department of Nano-Science and Nano-Engineering, Institute of Science and Technology, Ataturk University, 25240 Erzurum, Turkey
c Ataturk University, Erzurum Vocational Collage, Department of Chemical Technology, Erzurum, Turkey
d Ahi Evran University, Kaman Vocational School, Department of Food Technology, Kırsehir, Turkey
e Ataturk University, Faculty of Science, Department of Chemistry, 25240 Erzurum, Turkey
f Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Eskis¸ehir, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of carbonic anhydrase (CA) has emerged as a promising approach for the treatment of a variety
of diseases such as glaucoma, epilepsy, obesity and cancer. As a result, the design of CA inhibitors (CAIs) is
an outstanding field of medicinal chemistry. Due to the therapeutic potential of isoindoline-1,3-diones as
CAIs, herein hCA I and hCA II isozymes were purified from human erythrocytes using affinity chroma-
tography and the inhibitory effects of a series of isoindoline-1,3-diones on hydratase activities of these
isozymes were investigated. Among these compounds, compound 3a was found to be the most effective
Received 20 May 2019
Received in revised form
12 July 2019
Accepted 16 July 2019
Available online 19 July 2019
compound on hCA I with an IC50 value of 7.02
mM, whereas compound 3c was the most potent compound
Keywords:
on hCA II with an IC50 value of 6.39 M. Moreover, molecular docking studies were carried out for all
m
Carbonic anhydrase
Isoindoline-1,3-diones
Molecular docking
compounds and acetazolamide (AAZ), the reference agent, in the active sites of hCA I and hCA II.
Generally, the compounds showed high affinity through salt bridge formation and metal coordination
with Zn2þ ion and
p-stacking interaction with His94 residue. According to in silico Absorption, Distri-
bution, Metabolism and Excretion (ADME) studies, the pharmacokinetic parameters of all compounds
were within the acceptable range.
© 2019 Elsevier B.V. All rights reserved.
1. Introduction
regions, while others catalyze the reaction in the active site without
zinc [3].
Carbonic anhydrases (CAs; EC 4.2.1.1) are ubiquitous zinc-
containing metalloenzymes and they play a pivotal role in all
living metabolism. The CA enzyme is present in 16 isoforms in
mammals [1]. These isoforms are found to be specialized in
different tissues of the organism. Although CAs are involved in
many different reactions, the removal of waste carbon dioxide in
the metabolism is their main task (Fig. 1) [2]. Due to their role in the
conversion of carbon dioxide to bicarbonate, CAs participate in key
biosynthetic reactions such as lipogenesis, gluconeogenesis and
ureagenesis. Some CA isoenzymes contain zinc in their active
The regulation and control of enzyme activity is important for all
living things. There are many regulations that increase the activity
of enzymes, as well as decreasing regulations. In addition, enzyme
activity is regulated by covalent modifications, phosphorylation
and allosteric effects. One of the most important approaches for the
reduction of this type of enzyme activity is the inhibition of CAs
[4,5].
Over the past 60 years, heterocyclic and aromatic sulfonamides
have been used as CA inhibitors in the treatment of many diseases
such as glaucoma, obesity, epilepsy, cancer, altitude sickness.
Moreover, in the last years CA inhibitors have emerged as anti-
infective agents [6].
* Corresponding author. Ataturk University, Erzurum Vocational School, Depart-
ment of Food Technology, 25240 Erzurum, Turkey.
** Corresponding author.
E-mail addresses: hnisa25@atauni.edu.tr (H. Nadaroglu), nhorasan@atauni.edu.
tr (N.H. Kishali).
In addition to the sulfonamide derivatives such as AAZ, meth-
azolamide, ethoxzolamide, aromatic/heteroaromatic sulfonamide-
based Schiff bases have also been identified as CA inhibitors [6,7].
Isoindoline-1,3-dione is a prominent compound in organic
https://doi.org/10.1016/j.molstruc.2019.07.070
0022-2860/© 2019 Elsevier B.V. All rights reserved.

H. Nadaroglu et al. / Journal of Molecular Structure 1197 (2019) 386e392
387
product was purification with column chromatography. The prod-
uct 3 was crystallized from EtOAc/petroleum ether (Yield: 90e95%)
2.1.1.1. 2,3-Dihydro-2-methyl-1,3-dioxo-1H-isoindole-5-carboxylic
acid (2a). M.p: 232e233 ꢀC (lit: 240 ꢀC) [24], IR (KBr): 3775, 3444,
3185, 1773,1717,1695,1607, 1570,1478, 1449,1378, 1253, 1214,1164.
1H NMR (400 MHz, CDCl3): 8.57 (s, 1H), 8.48 (dd, J ¼ 7.7, 1.5 Hz, 1H),
7.97 (d, J ¼ 7.7 Hz, 1H); 3.23 (s, 3H). 13C NMR (100 MHz, CDCl3):
169.4 (2C), 167.5, 136.5, 136.2, 134.7, 132.8, 125.1, 123.6, 24.5. Anal.
calc. for C10H7NO4 (205.17): C, 58.54; H, 3.44; N, 6.83; found: C,
58.20; H, 3.66; N, 6.41.
2.1.1.2. 2,3-Dihydro-2-ethyl-1,3-dioxo-1H-isoindole-5-carboxylic
acid (2b). M.p: 158e159 ꢀC, IR (KBr): 3772, 3443, 2982, 2947, 1772,
1702, 1441, 1400, 1385, 1308, 1257, 1199, 1077. 1H NMR (400 MHz,
CDCl3): 8.57 (s, 1H), 8.48 (dd, J ¼ 7.7, 1.1 Hz, 1H), 7.97 (d, J ¼ 7.7 Hz,
1H); 3.79 (q, J ¼ 7.3 Hz, 2H), 1.30 (t, J ¼ 7.3 Hz, 3H). 13C NMR
(100 MHz, CDCl3): 169.8 (2C), 167.3, 136.6, 136.2, 134.7, 132.8, 125.1,
123.6, 33.6, 14.1. Anal. calc. for C11H9NO4 (219.20): C, 60.28; H, 4.14;
N, 6.39; found: C, 60.62; H, 4.70, N, 6.48.
Fig. 1. Removal of waste carbon dioxide in the metabolism.
synthesis for the preparation of diverse biologically active mole-
cules [8e12]. Isoindoline-1,3-dione derivatives exhibit various
biological activities, like anticancer [13], antimicrobial [14], anti-
oxidant [15], anti-inflammatory [16] and analgesic [17] activities.
The hydrophobic character of isoindoline-1,3-diones increases their
potential to cross different biological membranes in vivo [18].
In this paper, we synthesized a series of N-substituted isoindo-
line-1,3-diones and investigated their inhibitory effects on hCA I
and hCA II isoenzymes. All compounds were docked to the active
sites of hCA I and hCA II with AAZ to explore their possible binding
modes in the active sites of these enzymes. A computational study
for the prediction of ADME properties of all compounds was also
performed.
2.1.1.3. 2,3-Dihydro-1,3-dioxo-2-phenyl-1H-isoindole-5-carboxylic
acid (2c). M.p: 259e260 ꢀC (lit: 257e259 ꢀC [25]), IR (KBr): 3445,
3053, 1787, 1720, 1504, 1392, 1296, 1221, 1124, 1104, 1072. 1H NMR
(400 MHz, CD3OD): 8.48 (m, 2H), 8.44 (m, 1H), 7.64e7.33 (m, 5H).
13C NMR (100 MHz, CD3OD): 166.7, 166.6, 166.3, 135.5, 135.0, 132.1,
131.9, 128.6, 128.4, 127.9, 126.68, 123.90, 123.24. Anal. calc. for
C15H9NO4 (267.24): C, 67.42; H, 3.39; N, 5.24; found: C, 67.62; H,
3.21; N, 5.70.
2.1.1.4. Methyl 2,3-dihydro-2-methyl-1,3-dioxo-1H-isoindole-5-
carboxylate (3a). M.p: 122e123 ꢀC, IR (KBr): 3455, 3078, 2956,
1773, 1738, 1717, 1480, 1438, 1383, 1288, 1261, 1188, 1161. 1H NMR
(400 MHz, CDCl3): 8.47 (dd, J ¼ 1.5, 0.7 Hz, 1H), 8.39 (dd, J ¼ 7.7,
1.5 Hz, 1H), 3.98 (s, 3H), 3.21 (s, 3H). 13C NMR (100 MHz, CDCl3):
167.7, 167.7, 165.5, 135.7, 135.6, 132.7, 129.1, 124.5, 123.5, 53.1, 24.5.
Anal. calc. for C11H9NO4 (219.20): C, 60.28; H, 4.14; N, 6.39; found:
C, 60.01; H, 4.15; N, 5.99.
2. Experimental
2.1. Chemistry
All chemicals were commercially available and were used
without purification or after distillation and treatment with drying
agents. Melting points (M.p.) were determined on a capillary
melting apparatus (Buechi 530) and are uncorrected. IR spectra
were obtained from solutions in 0.1 mm cells with a PerkinElmer
spectrophotometer. The 1H (400 MHz) and 13C NMR (100 MHz)
2.1.1.5. Methyl 2,3-dihydro-2-ethyl-1,3-dioxo-1H-isoindole-5-
carboxylate (3b). M.p: 96e97 ꢀC, IR (KBr): 3457, 3115, 2980, 2960,
1773, 1725, 1459, 1431, 1399, 1380, 1330, 1295, 1256, 1197, 1183. 1H
NMR (400 MHz, CDCl3): 8.47 (s, 1H), 8.39 (d, J ¼ 7.8 Hz, 1H), 7.90 (d,
spectra were recorded on Varian and Bruker spectrometers;
d in
ppm. Elemental analyses were performed on a Leco CHNS-932
apparatus. All column chromatography was performed on silica
gel (60-mesh, Merck). Preparative layer chromatography (PLC) is
preparative thin layer chromatography: 1mmof silica gel 60 PF
(Merck) on glass plates.
2.1.1. General procedure for the synthesis of compounds 2a-c and
3a-c
A mixture of compound 1 (1 equiv.), Et3N (3 equiv.), ReNH2 (1.2
equiv.) and toluene (10 mL) was heated at reflux temperature for
24 h. After that, the solvent was evaporated under reduced pressure
and the remaining solid was filtered from a small column. The
product 2 was crystallized from EtOAc/Petroleum ether (Yield:
90e95%).
To a magnetically stirred solution of 2 (1 equiv.) in methanol
(10 mL) was cooled to 0 ꢀC. Then SOCl2 (1.2 eq.) was added. The
mixture was stirred refluxed for 18 h. After that, the reaction
mixture was cooled to room temperature and the solvent removed
in evaporator, and The solid was extracted by addition of EtOAc
(2 ꢁ 20 mL) and NH4Cl (20 mL). The organic layer was dried over
Na2SO4 and the solvent was removed in an evaporator. The crude
Scheme 1. The synthetic route for the preparation of isoindoline-1,3-dione derivatives.

388
H. Nadaroglu et al. / Journal of Molecular Structure 1197 (2019) 386e392
Table 1
Purification of hCA I and hCA II from human erythrocytes.
Purification steps
Volume
mL
Activity
(EU/mL)
Total Activity
EU
Protein
Specific Activity
Purification Fold
%
(mg protein/mL)
(EU/mg protein)
Hemolysate
hCA I
hCA II
100
45
35
44.67
59.8
66.8
4467
2691
2338
100
60.2
52.3
1.64*103
0.075
0.033
0.27
797.33
2024.24
e
2953.1
7497.2
Table 2
The results obtained from regression analysis graphs for hCA I and hCA II in the presence of the 1,3-dioxoisoindoline derivatives.
Compound hCA-I Inhibition hCA-II Inhibition
IC50 M) IC50 M)
hCA-I/hCA-II
1.182
(m
r2
Ki (
m
M)
(m
r2
Ki (mM)
16.95
0.9883
12.77 4.33
14.345
0.9971
10.45 1.78
13.04
16.16
0.9990
0.9884
9.56 4.21
8.97
0.9896
0.9923
6.33 1.08
1.454
1.423
10.21 2.26
11.36
8.92 2.66
7.02
0.9974
0.9905
4.13 1.25
9.19
0.9894
0.9914
6.89 2.33
0.538
0.481
12.85
7.21 2.56
26.69
27.23 4.18
10.74
5.76
0.9911
0.9875
8.42 3.33
6.39
6.27
0.9845
0.9925
4.78 1.44
1.68
7.63 1.04
10.45 1.78
0.918
J ¼ 7.7 Hz, 1H), 3.98 (s, 3H), 3.76 (q, J ¼ 7.2 Hz, 1 H), 1.28 (t, J ¼ 7.2 Hz,
3H). 13C NMR (100 MHz, CDCl3): 167.3, 167.2, 165.3, 135.6,
135.5, 135.3, 132.5, 124.2, 123.2, 52.8, 33.2, 13.9. Anal. calc. for
2.1.1.6. Methyl 2,3-dihydro-1,3-dioxo-2-phenyl-1H-isoindole-5-
carboxylic acid (3c). M.p: 210e211 ꢀC, IR (KBr): 3444, 2964, 1776,
1730, 1622, 1597, 1505, 1493, 1458, 1424, 1386, 1353, 1293, 1278,
1217, 1112, 1097. 1H NMR (400 MHz, CDCl3): 8.60 (d, J ¼ 0.7 Hz, 1H),
8.50 (dd, J ¼ 7.7, 1.5 Hz, 1H), 8.04 (d, J ¼ 7.7 Hz, 1H), 7.55e7.43 (m,
C
12H11NO4 (233.22): C, 61.80; H, 4.75; N, 6.01; found: C 62.22; H
4.21; N 6.31.

H. Nadaroglu et al. / Journal of Molecular Structure 1197 (2019) 386e392
389
Fig. 2. Docking poses (A) and interactions (B) of all compounds in the active site of hCA I (PDB code: 1AZM). (Ligand custom carbons are colored as given in the figure and the zinc
atom is displayed in grey sphere).
5H), 4.01 (s, 3H), 1.55 (s, 3H). 13C NMR (100 MHz, CDCl3): 166.7,
166.6, 166.2, 136.2, 136.0, 135.3, 132.5, 129.5, 128.6, 126.7, 125.1,
124.1, 123.2, 53.2. Anal. calc. for C16H11NO4 (281.27): C, 68.33; H,
3.94; N, 4.98; found: C, 68.62, H, 3.81, N, 4.75.
described previously [19].
2.2.2. Hydratase activity assay
CA activity was determined using the Wilbur-Anderson Method.
2.2. Biochemistry
Sepharose 4B, sulphanilamide, L-tyrosine, Tris, Na2SO4, protein
2.2.3. Inhibition assays
assay reagents, and chemicals for electrophoresis were purchased
from Sigma-Aldrich Co. All other chemicals were of analytical grade
and obtained from Merck.
In order to determine the IC50 values for hCA I and hCA II en-
zymes, the compounds (2a-c, 3a-c) were studied at different con-
centrations, keeping the substrate concentration constant on
hydratase activity. The activities of the enzymes in the uninhibited
medium were used as 100% activity. The activity% values were
calculated by measuring the hydratase activities of the enzymes in
the presence of the inhibitors at different concentrations. The IC50
value for each inhibitor was calculated using the plot of Activity%-
[I] plotted [20,21].
2.2.1. Purification of hCA I and hCA II from human erythrocytes
Fresh human blood was obtained from Ataturk University, Blood
Center, kept at 4 ꢀC and used within 2e3 days at most.
Purification of CA isozymes (hCA I and hCA II) from human
erythrocytes was carried out by affinity chromatography as

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H. Nadaroglu et al. / Journal of Molecular Structure 1197 (2019) 386e392
2.3. Molecular docking studies
the reaction of 1,3-dioxo-1,3-dihydroisobenzofuran-5-carboxylic
acid (1) with methyl, ethyl, phenyl amine (2a, 2b, 2c), then con-
verted to the methyl ester derivatives (3a, 3b, 3c) (Scheme 1). The
structures of these compounds were approved on the basis of IR, 1H
NMR, 13C NMR spectral data and elemental analysis. All spectral
data were in good agreement with their structures.
The compounds were docked to the active sites of hCA I and hCA
II. Ligands were set to the physiological pH (pH ¼ 7.4) at the pro-
tonation step and crystal structures of hCA I and hCA II were
retrieved from Protein Data Bank server (PDB codes: 1AZM and
6B59, respectively). In molecular docking simulations: Glide/XP
docking protocols were applied for the prediction of topologies of
3.2. Biochemistry
€
compounds in the active sites of target structures by Schrodinger's
€
Maestro molecular modelling package programme (Schrodinger
hCA I and hCA II isoenzymes were purified in one step using
human blood Sepharose 4B-L-tyrosine-sulphanilamide affinity
€
Release 2016-2: Schrodinger, LLC, New York, NY, USA).
chromatography. hCA I was purified with 2979.1 fold and 60.2%
efficiency with 797.33 specific activity and hCA II was purified with
7497.2 fold and 52.3% efficiency with 797.33 specific activity
(Table 1).
The inhibitory effects on the activity of hCA I and hCA II iso-
enzymes of 1,3-dioxoisoindoline derivatives (2a-c, 3a-c) were
tested in vitro. IC50 values were determined for all 1,3-
dioxoisoindoline derivatives (Table 2). AAZ was used as a stan-
dard inhibitor.
2.4. Prediction of pharmacokinetic parameters
The ADME properties of all compounds were in silico predicted
€
using QikProp module of Schrodinger's Molecular modelling
€
€
package (Schrodinger Release 2016-2: QikProp, Schrodinger, LLC,
New York, NY, 2016).
3. Results and discussion
The following structureeactivity relationships (SAR) were
gathered from the inhibition data reported in Table 2. In order to
determine the relationship between the structures of isoindoline-
1,3-dione derivatives and their hCA-I and hCA-II inhibitory effects,
3.1. Chemistry
In this study, isoindoline-1,3-dione derivatives were obtained by
Fig. 3. Docking poses (A) and interactions (B) of all compounds in the active site of hCA II (PDB code: 6B59). (Ligand custom carbons are colored as given in the figure and the zinc
atom is displayed in grey sphere).

H. Nadaroglu et al. / Journal of Molecular Structure 1197 (2019) 386e392
391
Table 3
Docking score (kcal/mol), glide gscore (kcal/mol) and glide emodel (kcal/mol) results of all compounds in the active site of hCA I (PDB code: 1AZM) and hCA II (PDB code: 6B59).
Compound
hCA I
hCA II
Docking score
Glide gscore
Glide emodel
Docking score
Glide gscore
Glide emodel
2a
2b
2c
3a
3b
3c
ꢂ2.91
ꢂ3.48
ꢂ3.28
ꢂ6.99
ꢂ6.34
ꢂ6.32
ꢂ7.24
ꢂ2.91
ꢂ3.48
ꢂ3.28
ꢂ6.99
ꢂ6.34
ꢂ6.32
ꢂ8.23
ꢂ12.02
ꢂ22.30
ꢂ21.47
ꢂ24.47
ꢂ28.03
ꢂ25.71
ꢂ66.57
ꢂ5.46
ꢂ6.14
ꢂ5.62
ꢂ6.09
ꢂ5.29
ꢂ6.38
ꢂ6.87
ꢂ5.46
ꢂ6.14
ꢂ5.62
ꢂ6.09
ꢂ5.29
ꢂ6.38
ꢂ7.87
ꢂ48.21
ꢂ87.74
ꢂ53.39
ꢂ78.98
ꢂ47.31
ꢂ83.59
ꢂ79.31
AAZ
it is necessary to look at these compounds in two aspects. The first
one is N-alkyl/aryl group of the phthalimide ring, namely methyl,
ethyl and phenyl groups. The second one is esters of corresponding
carboxylic acids, for this purpose, two series were prepared for each
group bonded to the nitrogen atom. These are 1,3-dioxoisoindoline-
5-carboxylic acids (2a-c) and their corresponding methyl esters
(3a-c). The compounds inhibited both hCA I and hCA II. This in-
dicates that all synthesized compounds (2a, 2b, 2c, 3a, 3b, 3c) have
groups that can interact with the active site of the enzyme. In
general, the esters were found to be more effective than their
corresponding carboxylic acids. The most effective hCA I inhibitor
in this series was found as compound 3a, whereas the most active
hCA II inhibitor was found as compound 3c. Interestingly, among
carboxylic acid derivatives (2a-c) N-ethyl group enhanced hCA
inhibitory activity, among esters (3a-c) N-ethyl group decreased
hCA inhibitory activity.
As regards to the active site of hCA II, the carboxylates of com-
pounds 2a, 2b and 2c presented hydrogen bonding and salt bridge
formation with Thr199 and Zn301, respectively. However, com-
pounds 3a, 3b and 3c formed metal coordination with Zn301. Be-
sides, the isoindolines of all compounds displayed
interaction with His94 in the active site of hCA II similar to hCA I.
The thiadiazole of AAZ forged a key -stacking interaction with
p-stacking
p
His94, whereas the sulfonamide moiety of AAZ presented hydrogen
bond and metal coordination with Thr199 and Zn301, respectively
(Fig. 3).
In general, Emodel scores are used to compare the different
conformations of the same ligand, whilst docking scores are used
for the comparison of different ligands [22]. The docking score of
compound 3a for hCA I was found as ꢂ6.99 kcal/mol, whereas the
docking score of compound 3c for hCA II was found as ꢂ6.38 kcal/
mol. The docking scores, glide gscores and glide emodel results of
all compounds were given in Table 3.
3.3. Molecular docking studies
3.4. In silico ADME studies
Molecular docking studies indicated that compounds 2a-c, 3a-c
showed high affinity in the active sites of hCA I and hCA II when
compared with AAZ, the reference agent (Figs. 2 and 3). The car-
boxylates of compounds 2a, 2b and 2c were engaged with salt
bridge formation and metal coordination with Zn261, whereas the
ester group of compound 3c interacted with Zn261 forming a metal
coordination in the active site of hCA I. The isoindolines of com-
In order to enlighten the biological, pharmaceutical and drug
similarities of these compounds, in silico ADME studies were per-
formed. The results given in Table 4 were found to be within the
acceptable range making these derivatives as promising drug can-
didates for further studies.
pounds 2a, 2b, 2c and 3c displayed
His94 in the active site of hCA I (Fig. 2). On the other hand, com-
pound 3b was found to be capable of forming -stacking interac-
tion with Phe91 due to the isoindoline scaffold. The imide moiety of
compound 3a formed a hydrogen bond with Thr199. The thiadia-
zole of AAZ forged a key p-stacking interaction with His94, whereas
the sulfonamide moiety of AAZ presented hydrogen bond and salt
bridge formation with Thr199 and Zn261, respectively (Fig. 2).
p
-stacking interaction with
4. Conclusion
p
In the present paper, 1,3-dioxoisoindoline derivatives were
found to inhibit hCA I and hCA II at high levels. Accordingly, it has
been found that 1,3-dioxoisoindoline derivatives have the potential
to be used as hCA inhibitors for the development of drugs against
various diseases. Furthermore, molecular docking studies showed
that these compounds presented proper interactions with Zn2þ ion,
Table 4
Predicted ADME properties of compounds 2a-c, 3a-c.
Compound
QPlogPo/w*
QPlogS*
QPlogBB*
QPlogKhsa*
Human Oral Absorption%a
Rule of Fiveb
Rule of Threec
2a
2b
2c
3a
3b
3c
0.46
0.96
1.91
0.46
1.09
2.11
ꢂ1.87
ꢂ2.21
ꢂ3.37
ꢂ2.46
ꢂ2.41
ꢂ3.70
ꢂ1.23
ꢂ1.22
ꢂ1.22
ꢂ0.94
ꢂ0.91
ꢂ0.89
ꢂ0.69
ꢂ0.60
ꢂ0.28
ꢂ0.64
ꢂ0.52
ꢂ0.13
54.92
60.03
66.16
73.67
79.57
86.08
0
0
0
0
0
0
0
0
0
0
0
0
a
QPlogPo/w: Predicted octanol/water partition coefficient (Recommended range: e2.0 to 6.5), QPlogS: Predicted aqueous solubility, log S. S in mol dmꢂ3 is the concen-
tration of the solute in a saturated solution that is in equilibrium with the crystalline solid (Recommended range: e6.5 to 0.5), QPlogBB: brain/blood partition coefficient
(Recommended range: e3 to 1.2), QPlogKhsa: binding to human serum albumin (Recommended range: e1.5 to 1.5), Human Oral Absorption%: Human oral absorption on
0e100% scale (>80% is high, <25% is poor).
b
Rule of Five: Number of violations of Lipinski's rule of five. The rules are: mol_MW (molecular weight of the molecule) < 500, QPlogPo/w (predicted octanol/water partition
coefficient) < 5, donorHB (hydrogen-bond donor atoms) ꢃ 5, accptHB (hydrogen-bond acceptor atoms) ꢃ 10. Compounds that provide these rules are considered as drug-like
molecules.
c
Rule of Three: Number of violations of Jorgensen's rule of three. The three rules are: QPlogS (predicted aqueous solubility) > ꢂ5.7, QPPCaco (predicted apparent Caco-2 cell
permeability in nm/s) > 22 nm/s, # Primary Metabolites < 7. Compounds with fewer (and preferably no) violations of these rules are more likely to be orally available agents
€
€
(Schrodinger Release 2016-2: Schrodinger, LLC, New York, NY, USA).

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His94 and Thr199 residues in the active sites of hCA I and hCA II.
According to in silico ADME studies, the compounds did not violate
Lipinski's rule of five and Jorgensen's rule of three. On the basis of
these findings, these compounds are expected to be potential orally
bioavailable hCA inhibitors.
Acknowledge
All studies related to the kinetics and inhibition studies of hCA I
and hCA II enzyme in this study was financially supported by a the
project numbered FAD-2018-6321 from Research Development
Center of Ataturk University, Turkey.
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