Synthesis, biological evaluation and in silico studies of novel N-substituted phthalazine sulfonamide compounds as potent carbonic anhydrase and acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103004

The synthesis, characterization and biological evaluation of a series of novel N-substituted phthalazine sulfonamide (5a-l) are disclosed. Phthalazines which are nitrogen-containing heterocyclic compounds are biologically preferential scaffolds, endowed with versatile pharmacological activity, such as anti-inflammatory, cardiotonic vasorelaxant, anticonvulsant, antihypertensive, antibacterial, anti-cancer action. The compounds were investigated for the inhibition against the cytosolic hCA I, II and AChE. Most screened sulfonamides showed high potency in inhibiting hCA II, widely involved in glaucoma, epilepsy, edema, and other pathologies (K_is in the ranging from 6.32 ± 0.06 to 128.93 ± 23.11 nM). hCA I was inhibited with K_is in the range of 6.80 ± 0.10–85.91 ± 7.57 nM, whereas AChE in the range of 60.79 ± 3.51–249.55 ± 7.89 nM. ADME prediction study of the designed N-substituted phthalazine sulfonamides showed that they are not only with carbonic anhydrase and acetylcholinesterase inhibitory activities but also with appropriate pharmacokinetic, physicochemical parameters and drug-likeness properties. Also, in silico docking studies were investigated the binding modes of selected compounds, to hCA I, II, and AChE.

Full text of DOI:10.1016/j.bioorg.2019.103004

Accepted Manuscript
Synthesis, biological evaluation and in silico studies of novel N-substituted
phthalazine sulfonamide compounds as potent carbonic anhydrase and acetyl-
cholinesterase inhibitors
Cüneyt Türkeş, Mustafa Arslan, Yeliz Demir, Liridon Çoçaj, Arleta Rifati
Nixha, Ş ükrü Beydemir
PII:
S0045-2068(19)30667-4
https://doi.org/10.1016/j.bioorg.2019.103004
103004
DOI:
Article Number:
Reference:
YBIOO 103004
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
27 April 2019
15 May 2019
19 May 2019
Please cite this article as: C. Türkeş, M. Arslan, Y. Demir, L. Çoçaj, A. Rifati Nixha, S. Beydemir, Synthesis,
biological evaluation and in silico studies of novel N-substituted phthalazine sulfonamide compounds as potent
carbonic anhydrase and acetylcholinesterase inhibitors, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/
j.bioorg.2019.103004
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Synthesis, biological evaluation and in silico studies of novel N-substituted phthalazine
sulfonamide compounds as potent carbonic anhydrase and acetylcholinesterase
inhibitors
a, *
b, *
c
d
d
Cüneyt Türkeş , Mustafa Arslan , Yeliz Demir , Liridon Çoçaj , Arleta Rifati Nixha ,
e
Şükrü Beydemir
a
b
c
d
e
Department of Biochemistry, Faculty of Pharmacy, Erzincan Binali Yıldırım University,
2
4100, Erzincan, Turkey
Department of Chemistry, Faculty of Arts and Sciences, Sakarya University, 54187,
Sakarya, Turkey
Department of Pharmacy Services, Nihat Delibalta Göle Vocational High School, Ardahan
University, 75700, Ardahan, Turkey
Department of Chemistry, Faculty of Mathematical and Natural Sciences, University of
Prishtina, 10000, Prishtina, Republic of Kosova
Department of Biochemistry, Faculty of Pharmacy, Anadolu University, 26470, Eskişehir,
Turkey
*
Corresponding author:
E-mail addresses: cuneyt.turkes@erzincan.edu.tr (C.Türkeş), marslan@sakarya.edu.tr
M.Arslan).
(

ABSTRACT
The synthesis, characterization and biological evaluation of a series of novel N-substituted
phthalazine sulfonamide (5a-l) are disclosed. Phthalazines which are nitrogen-containing
heterocyclic compounds are biologically preferential scaffolds, endowed with versatile
pharmacological activity, such as anti-inflammatory, cardiotonic vasorelaxant, anticonvulsant,
antihypertensive, antibacterial, anti-cancer action. The compounds were investigated for the
inhibition against the cytosolic hCA I, II and AChE. Most screened sulfonamides showed
high potency in inhibiting hCA II, widely involved in glaucoma, epilepsy, edema, and other
pathologies (K
with K s in the range of 6.80±0.10-85.91±7.57 nM, whereas AChE in the range of
0.79±3.51-249.55±7.89 nM. ADME prediction study of the designed N-substituted
i
s in the ranging from 6.32±0.06 to 128.93±23.11 nM). hCA I was inhibited
i
6
phthalazine sulfonamides showed that they are not only with carbonic anhydrase and
acetylcholinesterase inhibitory activities but also with appropriate pharmacokinetic,
physicochemical parameters and drug-likeness properties. Also, in silico docking studies were
investigated the binding modes of selected compounds, to hCA I, II, and AChE.
Keywords:
Sulfonamide; phthalazine; carbonic anhydrase; acetylcholinesterase; enzyme inhibitors;
molecular docking

1
. Introduction
NitrogenǦ containing heterocyclic compounds have been significant interest because of
their common applicability in the past few decades. These compounds have an essential role
in biological systems as therapeutic agents and found in many of the compounds in medicinal
chemistry [1]. The compounds containing heterocyclic groups such as pyridines, piperazines,
indoles, pyrazoles, thiazolidine-4-ones have received great importance because of their
biological and pharmacological activities [2]. They are widely found in nature, and their
utilization is increasing more and more with each passing day as functional, pharmaceutical,
and agrochemicals materials [3].
Phthalazine derivatives are very important nitrogen-containing heterocyclic compounds
that have variety of pharmacological activity. Heterocycles containing phthalazine moiety
have been reported to possess different pharmacological properties, and they have been
commonly employed as therapeutic agents owing to their anti-inflammatory, cardiotonic
vasorelaxant, anticonvulsant [4], antihypertensive [5], antibacterial [6], anti-cancer features
[
7], and carbonic anhydrase enzyme activity [8]. Several commercially available phthalazine
derivative drugs, for instance, hydralazine, azelastine, budralazine, zaleplon, luminal,
zopolrestat, and MY5445 are used in the treatment of antihypertensive agent for pulmonary
hypertension, allergic rhinitis, vasorelaxation, antihistaminic, insomnia and which are shown
in Scheme 1 [9, 10].
Sulfonamides are an essential compound for the synthesis of bacteriostatic antibiotics.
They are known as sulfa drugs and were used against bacterial infections before discovering
penicillin [11]. The compounds have a wide range of biological activities; for example,
furosemide used as a diuretic, amprenavir used in HIV therapy, sulfathiazole as an

antibacterial agent, and acetazolamide as a carbonic anhydrase inhibitor [12]. However,
sulfonamides are also as effective lactoperoxidase inhibitors [13].
Sulfonamide compounds inhibit carbonic anyhdrase (CA) enzymes in a large scale [14-
1
6], and they have been employed for more than 50 years as agents to reduce blood pressure,
in the treatment of diseases such as now potentially cancer, glaucoma, heart failure, and
epilepsy. Some clinically used sulfonamides such as ethoxzolamide, acetazolamide,
dichlorphenamide, and methazolamide have been employed as systemic CA inhibitors since
the 1950s. [17].
Carbonic anhydrase isoenzymes (CAs) catalyze dehydration and hydration reactions of
carbonic acid (H
2
CO
3
), and carbon dioxide (CO
2
2 3
); in the dehydration reaction H CO is
+
converted to CO
2
, although the hydration reaction produces hydrogen (H ) and bicarbonate
-
(
HCO
3
) ions from CO
2
. [18-22]. This reaction has a crucial role in some vital physiologic
[23]. Because of that, their
modulation is employed for a long time in the treatment of several diseases such as epilepsy
24], edema [25], glaucoma [26]. Recently, inhibitors of CAs have been employed as a novel
functions linked to metabolic pathways involved in CO
2
[
approach for the treatment of the cerebral ischemia [27] different forms of the tumor [28] and
neuropathic pain [29].
Alzheimer’s disease (AD) is a significant problem for old people worldwide. This disease
can affect many aspects of a person’s life [30-32]. Also, there is no cure for AD disease, but
several drugs are employed for the cure [33]. Acetylcholinesterase inhibitors (AChEI) have
been developed as an impact of the cholinergic assumption of cognitive decline [34]. Also,
the effectiveness of these treatments has been investigated in a large number of randomized

controlled tests between cognitive, global, neuropsychiatric domains, and functional [35].
AChEI compounds are employed for the treatment of mild-to-moderate AD. These
compounds inhibit AChE, which is responsible for the separation of acetylcholine (ACh), a
neurotransmitter molecule related to memory function [36].
In this study, a dozen novel N-substituted phthalazine sulfonamide compounds were
synthesized, and their inhibitory effects on the activity of human carbonic anhydrase (hCA) I,
II and acetylcholinesterase (AChE) were evaluated.

2. Results and discussion
2.1. Chemistry
4-Sulfonylamide ester was prepared from 4-Sulfamoylbenzoic acid in ethanol with a
catalytic amount of sulfuric acid by refluxing for 24 hrs. The ester group was converted to the
hydrazide with hydrazine hydrate in ethanol at 70°C for 24 hrs. A mixture of 4-
(
hydrazinecarbonyl)benzen sulfonamide, phthalicanhydride derivatives in acetic acid was
1
13
heated for 8 hours at 100°C. The prepared compounds were characterized by H NMR, C
NMR, IR, and elemental analysis. The proposed compounds (5a-l) were shown in Scheme 2.
1
From the H NMR spectra, sulfanilamide NH
2
and the =CH proton peaks on aromatic
ring resonances at around 7.60 ppm and between 7.00 and 8.00 ppm respectively. From the
1
3
C NMR spectra, the signals of amide and ester carbonyl group are seen at around 165 ppm.
In the infrared spectra of compounds 5a-l, it was possible to observe the absorptions around
-1
-1
3
2
300 cm relating to NH stretchings and absorptions around 1700 cm from carbonyl
moiety stretching. As can be seen in the literature [37], there are two peaks assigned to S=O
as symmetric and asymmetric stretching. The peaks of asymmetric and symmetric stretch are
-1
appeared around 1300 and 1100 cm , respectively.
2.2. Biological evaluation
The novel N-substituted phthalazine sulfonamides obtained here were tested as inhibitors
of pharmacologically and physiologically related the cytosolic hCA I, and II by esterase
assay. Also, it was investigated the inhibition effects on AChE enzyme activity of these
compounds. Their activities were compared to the acetazolamide (AAZ) and tacrine (TAC)
which is standard reference inhibitors for hCA and AChE.

(
i) hCA I is expressed in normal colorectal mucosa and found at the highest level in
erythrocytes [38]. As for hCA I, IC50 values for novel synthesized N-substituted phthalazine
sulfonamide derivatives showed in the ranging from 12.42±0.23 to 32.80±0.49 nM and K
constants in the range of 6.80±0.10-85.91±7.57 nM. It exhibited that K constants order of
synthesized sulfonamides inhibitory strength was 5b (K : 6.80±0.10 nM) > 5h (K : 9.26±0.16
nM) > 5k (K : 9.84±0.13 nM) > 5e (K : 10.35±0.10 nM) > 5c (K : 10.59±0.14 nM) > 5i (K
5.10±0.16 nM) > 5g (K : 16.85±0.20 nM) > 5j (K : 20.01±0.31 nM) > 5d (K : 20.69±0.35
: 85.91±7.57 nM). It is well
i
i
i
i
i
i
i
i
:
1
i
i
i
nM) > 5a (K
i
: 49.31±0.25 nM) > 5l (K
i
: 54.23±4.62 nM) > 5f (K
i
known that incorporation of electron-donating groups resulted in a lessen hCA isoenzymes
activity. Unlike this impact, addition of electron-withdrawing groups in the inhibitor
improved the inhibition impacts. The best inhibitor was 2-(4-sulfamoylbenzoyl)-8-flouro-2,3-
dihydrophthalazine-1,4-dione (5b) (Figs. 1 and 2) when compared to AAZ (K : 434.55±11.91
i
nM), which is used for positive control. Also, the least active was 2-(4-sulfamoylbenzoyl)-6-
carboxy-2,3-dihydrophthalazine-1,4-dione (5f) for hCA I. Compound 5b decreased the
enzyme activity ~13 fold compared to 5f (Tables 1 and 2).
hCA II is a cytosolic enzyme and highly active [39]. As shown in Table 1, the inhibition
profile of the novel synthesized sulfonamides was very active compared to the hCA I. As for
hCA II, the isolated compounds showed IC50 and K
i
values in the low nanomolar range which
constants in the ranging
gave IC50 values in the range of 10.14±0.13-28.57±0.50 nM and K
i
between 6.32±0.06 and 128.93±23.11 nM. The results exhibited that hCA II was effectively
inhibited by novel sulfonamide derivatives compared to hCA I. All studied sulfonamide
derivatives exhibited potent inhibition profile against hCA II. The best inhibitor was 2,8-di(4-
sulfamoylbenzoyl)-1,2,3,4,6,7,8,9-octahydropyridazino[4,5-g]phthalazine-1,4,6,9-tetraone

(
5l) (Figs. 1 and 2), whereas the least effective one 2-(4-sulfamoylbenzoyl)-6-nitro-2,3-
dihydrophthalazine-1,4-dione (5e) for hCA II. On the other hand, AAZ, which is used a
clinical medication had a K constant of 47.60±0.56 nM against hCA II. Compound 5l which
is a more potent inhibitor in comparison with AAZ reduced the hCA II activity approx.7 fold
i
(
Tables 1 and 2).
In literature, it was observed that the sulfonamides inhibit hCA isozymes and the
concentration of these compounds was found in the micromolar to the nanomolar range [40-
2]. The novel ureido benzenesulfonamides incorporating 1,3,5-triazine moieties were
4
investigated as inhibitors of hCA I, II, IX, and XII. All compounds exhibited strong inhibition
effect for hCA isoenzymes [43]. Another study, novel benzenesulfonamide bearing 1,2,3-
triazole linked hydroxy-trifluoromethylpyrazolines were synthesized and tested inhibitory
effects towards hCA I, II, IX, and XII isoforms [44]. 3-aminobenzenesulfonamides
incorporating acylthiourea moieties showed potent inhibitory effect against hCA I, II, IV, and
IX [45].
The results showed that novel synthesized N-substituted phthalazine sulfonamide
derivatives were an excellent inhibitor for hCA isoenzymes. hCA inhibitors have been
employed for the treatment of a great deal of disorder containing epilepsy, glaucoma, cancer,
obesity, and high-altitude sickness [46].
(
ii) AChEIs or anti-cholinesterases inhibit cholinesterase, enhancing the level and length
of ACh action. The variety of use of AChEI compounds is popular in medicine and
agriculture [47]. These compounds traditionally employed for medical aims include
organophosphates and carbamates. In myasthenia gravis and AD diseases in which

cholinergic function is impaired, carbamates/organophosphates offer therapeutic choices
because of high effect as AChEI compounds. The inhibitory effects of the synthesized novel
N-substituted phthalazine sulfonamide compounds had IC50 values in the range of
4
8.69±0.57-221.10±7.14 nM for AChE. These compounds had K constants in ranging
i
between 60.79±3.51 and 249.55±7.89 nM. 2-(4-sulfamoylbenzoyl)-8-flouro-2,3-
dihydrophthalazine-1,4-dione (5b) (Figs. 1 and 2) which is one of the best potent inhibitors in
this study had more effective inhibition profile than that of tacrine (K
standard AChE inhibitor. Additionally, 2-(4-sulfamoylbenzoyl)-7-flouro-2,3-
dihydrophthalazine-1,4-dione (5c) was one of the least effective inhibitors for AChE (Tables
and 2).
i
: 107.96±2.38 nM) as
1
There are many studies about the inhibition of AChE by sulfonamides. For instance, 4-(3-
Substitutedphenyl-5-polymethoxyphenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamides
were synthesized and studied the inhibition effect on AChE enzyme activity. It was found that
i
K constants in ranging from 37.7±14.4 to 89.2±30.2 nM [48]. Another study, novel b-
benzylphenethylamines and their sulfamide derivatives were synthesized and K constants
i
were found in the range of 0.127-2.452 nM [49].
Novel sulfonamide derivatives displayed effective inhibition profile towards AChE.
These compounds might be used as precursor compounds for AD. Because of critical adverse
impacts of the present AChE inhibitors, there is the try to find safer and more effective AChE
inhibitors for the treatment of neurodegenerative disease. Bioassay-guided isolation and
ethnopharmacological approaches have determined as potential in identifying effective AChE
inhibitors.

2.3. In silico studies
2
.3.1. ADMET prediction and drug likeness study
ADME (absorption, distribution, metabolism, and excretion)-related pharmacokinetic and
physicochemical parameters of the synthesized N-substituted phthalazine sulfonamides
derivatives were computed. As could be seen from Table 3, the suitability of the novel
compounds was evaluated based on human oral absorption parameters and Lipinski’s rule of
five [50], which is an indication of the drug-likeness of a molecule. According to these
results, all compounds (5a-l) complied with the Lipinski’s rule of five (recommend value is
max. 4). Among the properties and descriptors, molecular weight (MW), dipole moment
(
Dipole), total solvent-accessible volume, van der Waals surface area of polar nitrogen and
oxygen atoms (except for 5l) (PSA), number of property or descriptor values (except for 5l)
#Stars), number of likely metabolic reactions (#Metab), number of non-trivial, non-hindered
rotatable bonds (#Rotor), octanol/water partition coefficient (QPlogPo/w), aqueous solubility
QPlogS) values were calculated within the ranges for drug-like chemical space. On the other
(
(
hand, the predicted Caco-2 cell (QPPCaco), and MDCK cell (QPPMDCK) permeability were
below 25.
2.3.2. Molecular docking study
We performed in silico studies to understand the molecular mechanism underlying
inhibitory activities of N-substituted phthalazine sulfonamides (5a-l). Therefore, compounds
a-l were subjected to the docking analysis against hCA I, II, and AChE receptor proteins. As
5
an initial level of in silico molecular docking computations, the reliability for the Glide SP
docking was estimated by docking the native ligands; 2,3,5,6-tetrafluoro-4-piperidin-1-

ylbenzenesulfonamide (3UG) into the binding site of hCA I, N-[(2Z)-1,3-oxazolidin-2-
ylidene]sulfuric diamide (OVX) into the binding site of hCA II, and donepezil (E20) into the
binding site of AChE. The binding structures of native ligands with receptors were
successfully reproduced yielding the RMSD between the predicted binding pose and the
experimental data pose as 0.295 Å, 0.259 Å, and 0.115 Å for 3UG, OVX, and E20,
respectively.
The hydrogen bonding interactions are between 3UG and OVX and polar regions of the
active sites of hCA I, and II, respectively. The major amino acid residues included in these
regions of the hydrogen bonds are Thr199, and Thr200. Moreover, the critical hydrogen
bonding interaction exists between E20 and hydrophobic part of the binding site of AChE.
Herein, the amino acid residue which is hydrogen bonding interaction is Phe295 (Fig. 3).
In the next step, N-substituted phthalazine sulfonamides (5a-l) and reference inhibitors
(
AAZ and TAC) were docked into the binding pocket. Molecular docking study successfully
generated biologically active conformations of the derivatives (Tables 4, 5, and 6).
Nevertheless, in silico molecular docking results should not be expected to have similar
correlations with the experimental activity, e.g., IC50 values and K constants [51].
i
Compounds 5b and 5l were selected for in silico molecular docking simulations to justify its
activity by investigating its key interactions within the binding sites of hCA I (PDB ID:
4WUQ), hCA II (PDB ID: 4FU5), and AChE (PDB ID: 4EY7) enzymes. The top poses for 5b
and 5l docked within the active site of enzymes were illustrated in Fig. 4.
As a result, the sulfonamide group played the usual role as a zinc-binding moiety for
compound 5b with the hCA I by accepting a coordinate bond with Zn metal and hydrogen

bond from Thr199. Carbonyl group was involved in the interaction within the active site of
hCA I by forming a hydrogen bond with Gln92. Also, the benzene ring was found to have pi-
pi stacking with His200. The subnanomolar inhibitor 5l coordinates the prosthetic Zn ion of
the enzyme through the sulfonamide group of phthalazine, also forms two H-bonds with
Gln92 and Thr199 in the binding site of hCA II. The carbonyl moiety of compound 5b
establishes an H-bond with Phe295 in the active site of AChE, the phthalazine ring is
involved in pi-pi stacking and hydrophobic interactions with Trp286. Second pi-pi stacking
was composed between the benzene ring of the sulfonamide moiety and Phe338. Above
mentioned contacts explained the inhibitory activities for compounds 5b (for the hCA I, and
AChE), and 5l (for the hCA II).

3. Conclusion
In conclusion, we reported a novel N-substituted phthalazine sulfonamide derivatives.
The novel sulfonamides were studied as inhibitors of two pharmacologically and
physiologically relevant isoforms, hCA I, and II, as well as AChE related to AD. All studied
compounds exhibited potent inhibition against these enzymes. K constants were found in the
i
range of 6.32±0.06-128.93±23.11 nM for hCA isoenzymes and 60.79±3.51-249.55±7.89 nM
for AChE. N-substituted phthalazine sulfonamide derivatives might be of interest for further
pharmacologic and medicinal research. Finally, the inhibitory potency of the synthesized N-
substituted phthalazine sulfonamide derivatives was also researched carrying out in silico
molecular docking for compounds 5b and 5l to rationalize the found biological study results.
These derivatives might be of interest for further pharmacologic, and medicinal research.

4
4
4
. Experimental
.1. Chemistry
.1.1. General
Melting points were determined by Yanagimoto micro-melting point apparatus and
uncorrected. IR spectra were measured on a SHIMADZU Prestige-21 (200 VCE)
1
13
spectrometer. H, and C NMR spectra were obtained using VARIAN Infinity Plus in 300
1
13
and 75 Hz, respectively. H and C chemical shifts were referenced to the internal deuterated
solvent. The elemental analysis was carried out with a Leco CHNS-932 instrument. All
chemicals were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany).
4.1.2. General procedure for the preparation of 4-sulfonylamide ester (1)
4-Sulfamoylbenzoic acid (10 mmol) was refluxed for 24 hrs in 50 mL of ethanol and 1.0
mL of sulfuric acid. The solvent was evaporated, and the obtained product was washed with
cold water and dried.
4.1.3. General procedure for the preparation of 4-sulfonylamidebenzohydrazide (2)
4-sulfonylamide ester (10 mmol) and hydrazine hydrate (25 mmol) in ethanol were
refluxed for 24 hrs at 70°C. The reaction mixture was cooled to room temperature, and the
solid was filtered then washed with water and dried.
4.1.4. General procedure for the synthesis of 2-(4-sulfamoylbenzoyl)-2,3-dihydrophthalazine
1,4-dione derivatives (3a-l)

A mixture of 4-(hydrazinecarbonyl)benzen sulfonamide (1 mmol), Phthalic anhydride
derivatives (1 mmol ) in acetic acid (10 ml) was heated for 8 hours at 100°C. After cooling to
room temperature, the product was poured into ice-cold water. Then, filtered and dried. The
1
13
prepared compounds shown in Scheme 2 were characterized by H NMR, C NMR, IR, and
elemental analysis.
4.1.5. 2-(4-sulfamoylbenzoyl)-2,3-dihydrophthalazine-1,4-dione
(5a).
Yield
81%,
-1
recrystallized from ethanol to give as white powder, m.p. 280°C; IR (KBr, υ, cm ): 3350 (N-
1
H), 3220 (NH
MHz, CDCl
2
), 1698 (C=), 1640 (C=C, aromatic), 1355 and 1170 (SO
2
); H NMR (300
3
, δ, ppm) : 11.60(1H, s, NH), 8.10 (2H, d, Ar-H). 8.04 (2H, d, Ar-H), 7.90-7.80
1
3
(
4H, m, Ar-H), 7.60 (2H, s, -NH
2
). C NMR (75 MHz, CDCl
48.3, 136.2, 134.2, 133.8, 130.1, 129.6, 129.3, 126.8, 125.5, 124.7. Anal. Calcd. for
S (%): C, 52.17; H, 3.21; N, 12.17; O, 23.17; S, 9.29. Found: C, 51.38; H, 3.33;
N, 12.33; O, 23.92; S, 9.42.
3
, δ, ppm): 165.9, 165.3, 165.2,
1
15 11 3 5
C H N O
4.1.6. 2-(4-sulfamoylbenzoyl)-8-flouro-2,3-dihydrophthalazine-1,4-dione (5b). Yield 75%,
-1
recrystallized from ethanol to give as white crystals, m.p. 288°C; IR (KBr, υ, cm ): 3350 (N-
1
H), 3250 (NH
MHz, CDCl
m, Ar-H), 7.60 (2H, s, -NH
48.4, 145.2, 137.9, 134.0, 131.8, 130.2, 129.4, 128.6, 126.8, 121.9. Anal. Calcd. for
S (%): C, 49.59; H, 2.77; N, 11.57; F, 5.23; O, 22.02; S, 8.83. Found: C, 49.63;
H, 2.94; N, 11.72; F, 5.30; O, 22.19; S, 8.90.
2
,), 1700 (C=O), 1640 (C=C), 1145 (C-F), 1355 and 1160 (SO
2
); H NMR (300
3
, δ, ppm) : 11.80 (1H,s, NH), 8.12 (2H, d, Ar-H). 8.0 (2H, d, Ar-H), 7.8-8.0(3H,
13
2
3
). C NMR (75 MHz, CDCl , δ, ppm): 165.3, 163.9, 161.3,
1
15 3 5
C H10FN O

4.1.7. 2-(4-sulfamoylbenzoyl)-7-flouro-2,3-dihydrophthalazine-1,4-dione (5c). Yield 78%,
-1
recrystallized from ethanol to give as white powder, m.p. 284°C; IR (KBr, υ, cm ): 3320 (N-
1
H), 3220 (NH
CDCl
Ar-H), 7.33 (2H, s, -NH
34.1, 133.1, 133.0, 129.3, 127.8, 127.6, 126.8, 126.4, 123.2. Anal. Calcd. for C15
%): C, 49.59; H, 2.77; N, 11.57; F,5.23; O, 22.02; S, 8.83. Found: C, 49.63; H, 2.85; N,
2
), 1680 (C=O), 1640 (C=C), 1310 and 1165 (SO
2
); H NMR (300 MHz,
3
, δ, ppm) : 11.76 (1H,s, NH), 8.10 (2H, d, Ar-H), 7.90 (2H, d, Ar-H), 7.8-7.5 (3H, m,
13
2
3
). C NMR (75 MHz, CDCl , δ, ppm): 165.3, 164.9, 164.8, 148.3,
1
3 5
H10FN O S
(
11.68; F, 5.28; O, 22.19; S, 8.92.
4.1.8. 2-(4-sulfamoylbenzoyl)-8-nitro-2,3-dihydrophthalazine-1,4-dione (5d). Yield 72%,
-1
recrystallized from ethanol to give as white powder, m.p. 270°C; IR (KBr, υ, cm ): 3325 (N-
1
H, stretch), 3230 (NH
NMR (300 MHz, CDCl
.10 (1H, t, Ar-H), 8.08 (2H, d, Ar-H), 8.0 (2H, d, Ar-H), 7.61 (2H, s, -NH
MHz, CDCl , δ, ppm): 165.3, 164.9, 164.8, 155.9, 148.3, 139.4, 139.3, 134.1, 132.0, 129.4,
26.8, 124.4, 124.2. Anal. Calcd. for C15 S (%): C, 46.16; H, 2.58; N, 14.35; O,
2
), 1698 (C=O), 1650 (C=C), 1540 (NO
2
), 1315 and 1158 (SO
2
); H
3
, δ, ppm) : 11.80 (1H, s, NH), 8.42 (1H, d, Ar-H), 8.40 (1H, d, Ar-H),
13
8
2
). C NMR (75
3
1
2
10 4 7
H N O
8.69; S, 8.21. Found: C, 46.31; H, 2.69; N, 14.49; O, 28.71; S, 8.30.
4.1.9. 2-(4-sulfamoylbenzoyl)-6-nitro-2,3-dihydrophthalazine-1,4-dione (5e). Yield 75%,
-1
recrystallized from ethanol to give as white powder, m.p. 252°C; IR (KBr, υ, cm ): 3540 (N-
1
H), 3370 (NH
2
), 1720 (C=O), 1640 (C=C), 1550 (NO
300 MHz, CDCl
1H, s, Ar-H), 8.10 (2H, d, Ar-H), 7.90 (2H, d, Ar-H), 7.58 (2H, s, -NH
, δ, ppm): 165.3, 164.3, 164.1, 152.7, 148.4, 134.6, 134.0, 131.4, 131.2, 129.4, 126.8,
2
), 1355 and 1170 (SO
2
-NH
2
); H NMR
(
(
3
, δ, ppm) : 11.82 (1H,s, NH), 8.78 (1H, s, Ar-H), 8.60 (1H, s, Ar-H), 7.69
13
2
). C NMR (75 MHz,
CDCl
3

10 4 7
126.3, 119.7. Anal. Calcd. for C15H N O S (%): C, 46.16; H, 2.58; N, 14.35; O, 28.69; S,
8.21. Found: C, 46.33; H, 2.65; N, 14.47; O, 28.82; S, 8.32.
4.1.10. 2-(4-sulfamoylbenzoyl)-6-carboxy-2,3-dihydrophthalazine-1,4-dione (5f). Yield 82%,
-1
recrystallized from ethanol to give as white crystals, m.p. 268°C; IR (KBr, υ, cm ): 3100
(
(
2
carboxylic acid O-H), 3350 (N-H), 3210 (NH ), 1700 (C=O), 1650 (C=C), 1352 and 1155
1
SO
2
); H NMR (300 MHz, CDCl
3
, δ, ppm) : 11.90 (1H,s, NH), 8.20 (2H, d, Ar-H), 8.0 (2H,
1
3
d, Ar-H), 7.81 (1H, s, Ar-H), 7.61 (2H, s, -NH
MHz, CDCl , δ, ppm): 166.3, 165.3, 165.2, 165.1, 148.3, 137.8, 136.9, 134.2, 133.3, 130.6,
29.3, 126.8, 125.2, 124.6. Anal. Calcd. for C16 S (%): C, 49.36; H, 2.85; N, 10.79;
O, 28.77; S, 8.24. Found: C, 49.45; H, 2.97; N, 10.83; O, 28.94; S, 8.32.
2
), 7.71-7.58 (2H, dd, Ar-H). C NMR (75
3
1
11 3 7
H N O
4.1.11. 2-(4-sulfamoylbenzoyl)-5,6,7,8-tetrabromo-2,3-dihydrophthalazine-1,4-dione
(5g).
-
Yield 82%, recrystallized from ethanol to give as white powder, m.p. 292°C; IR (KBr, υ, cm
1
1
)
: 3352 (N-H), 3240 (NH
2 2
), 1700 (C=O), 1665 (C=C), 1355 and 1175 (SO ), 600 (C-Br); H
NMR (300 MHz, CDCl , δ, ppm) : 11.70 (1H,s, NH), 8.18 (2H, d, Ar-H). 8.08 (2H, d, Ar-H),
3
1
3
7
1
2
6
.62 (2H, s, -NH
2
), C NMR (75 Hz, CDCl
3
, δ, ppm): 165.3, 162.0, 161.9, 148.4, 138.4,
Br S (%) C,
34.0, 130.0, 129.7, 129.5, 129.4, 128.8, 126.8, 25.2. Anal. Calcd. for C15
H
7
4
N
3
O
5
:
7.26; H, 1.07; Br, 48.36; N, 6.36; O, 12.10; S, 4.85. Found: C, 27.43; H, 1.15; Br, 48.49; N,
.50; O, 12.25; S, 4.96.
4.1.12. 2-(4-sulfamoylbenzoyl)-5,6,7,8-tetrachloro-2,3-dihydrophthalazine-1,4-dione
(5h).
-
Yield 76%, recrystallized from ethanol to give as white powder, m.p. 286°C; IR (KBr, υ, cm
1
1
)
: 3350 (N-H), 3250 (NH
2 2
), 1700 (C=O), 1655 (C=C), 1348 and 1155 (SO ), 720 (C-Cl); H
NMR (300 MHz, CDCl , δ, ppm) : 11.80 (1H, s, NH), 8.1 (2H, d, Ar-H), 8.01 (2H, d, Ar-H),
3

1
3
7
.63 (2H, s, -NH
2
), C NMR (75 MHz, CDCl
3
, δ, ppm) : 165.3, 161.6, 161.4, 148.4, 140.1
Cl S (%)
:
1
33.9, 132.9, 131.3, 130.1, 129.7, 129.4, 126.8 124.1. Anal. Calcd. for C15
H
7
4
N
3
O
5
C, 37.29; H, 1.46; Cl, 29.35; N, 8.70; O, 16.56; S, 6.64. Found: C, 37.38; H, 1.59; Cl, 29.83;
N, 8.92; O, 16.58; S, 6.75.
4.1.13. 2,3-dihydro-2-(4-sulfamoylbenzoyl)naphtha(1,8-de)(1,2)diazepine-1,4-dione
(5i).
-
Yield 76%, recrystallized from ethanol to give as white powder, m.p. 254°C; IR (KBr, υ, cm
1
1
)
: 3320 (N-H,), 3230 (NH
2 2
), 1698 (C=O), 1655 (C=C), 1357 and 1175 (SO ); H NMR (300
MHz, CDCl , δ, ppm) : 11.74 (1H,s, NH), 8.50 (4H, dd, Ar-H), 8.20 (2H, d, Ar-H), 8.02
3
1
3
(
2H, d, Ar-H), 7.82 (2H, t, Ar-H), 7.62 (2H, s, -NH
2
), C NMR (75 MHz, CDCl
65.9, 165.3, 164.5, 148.3, 137.6, 136.2, 134.2, 130.5, 130.1, 129.6, 129.3, 128.9, 128.4,
27.5, 126.8, 124.7, 123.3. Anal. Calcd. for C19 S (%) C, 59.83; H, 3.96; N, 11.02; O,
6.78; S, 8.41. Found: C, 59.97; H, 4.11; N, 11.19; O, 16.90; S, 8.59.
3
, δ, ppm) :
1
1
1
H N
15 3
O
4
:
4.1.14. Octahydro-2-(4-sulfamoylbenzoyl)phthalazine-1,4-dione
(5j).
Yield
70%,
-1
recrystallized from ethanol to give as white powder, m.p. 213°C; IR (KBr, υ, cm ): 3320
1
(
N-H), 3210 (NH
MHz, CDCl
s, -NH ), 1.72-1.84 (2H, t,), 1.30-1.49 (8H, m, -CH
77.3, 177.1, 164.8, 148.1, 134.5, 126.7, 129.2, 40.9, 38.4, 24.1 (2C), 23.9 (2C), Anal. Calcd.
2
), 1698 (C=O), 1610 (C=C, aromatic), 1351 and 1155 (SO
2
); H NMR (300
3
, δ, ppm) : 11.40 (1H, s, NH), 8.05 (2H, d, Ar-H), 7.90 (2H, d, Ar-H), 7.60 (2H,
13
2
2 3
). C NMR (75 MHz, CDCl , δ, ppm):
1
17 3 5
for C15H N O S: C, 51.27; H, 4.88; N, 11.96; O, 22.77; S, 9.13. Found: C, 51.38; H, 5.11; N,
12.11; O, 25.75; S, 9.22.
4.1.15. 2,3,4a,5,8,8a-hexahydro-2-(4-sulfamoylbenzoyl)phthalazine-1,4-dione (5k). Yield
-1
6
6%, recrystallized from ethanol to give as white powder, m.p. 235°C; IR (KBr, υ, cm ):

1
3
350 (N-H), 3240 (NH
MHz, CDCl , δ, ppm) : 11.74 (1H,s, NH), 8.02 (2H, d, Ar-H), 7.80 (2H, d, Ar-H), 7.59 (2H, s,
NH ), 5.90-5.99 (2H, q, =CH, cyclohexenyl), 2.08-2.12 (2H, t, -CH, cyclohexenyl), 1.86-
2 2
), 1698 (C=O), 1610 (C=C), 1355 and 1160 (SO ); H NMR (300
3
-
2
1
3
1
1
4
.92 (4H, t, -CH
2
). C NMR (75 MHz, CDCl
3
, δ, ppm): 177.3, 177.0, 165.3, 148.3, 136.7,
S: C, 51.57; H,
31.7, 129.4, 127.6, 125.6, 41.4, 38.7, 27.2, 27.0. Anal. Calcd. for C15
15 3 5
H N O
.33; N, 12.03; O, 22.90; S, 9.18. Found: C, 51.62; H, 4.41; N, 12.15; O, 23.05; S, 9.28.
4
1
2
1
.1.16. 2,8-di(4-sulfamoylbenzoyl)-1,2,3,4,6,7,8,9-octahydropyridazino[4,5-g]phthalazine-
,4,6,9-tetraone (5l). Yield 71%, recrystallized from ethanol to give as white powder, m.p.
-1
98°C; IR (KBr, υ, cm ): 3330 (N-H), 3230 (NH
2
), 1700 (C=O), 1626 (C=C, aromatic),
1
315 and 1165 (SO
2
); H NMR (300 MHz, CDCl
3
, δ, ppm) : 11.70 (1H,s, NH), 8.33 (2H, s,
13
Ar-H), 8.20 (2H, d, Ar-H), 8.00 (2H, d, Ar-H), 7.58 (4H, s, -NH
CDCl , δ, ppm): 165.1, 165.0, 162.4, 148.0, 136.1, 135.2, 132.4,132.2, 129.2, 128.2, 127.9.
Anal. Calcd. for C24 (%) C, 47.06; H, 2.63; N, 13.72; O, 26.12; S, 10.47. Found:
C, 47.32; H, 2.79; N, 13.95; O, 26.32; S, 10.52.
2
). C NMR (75 MHz,
3
H
16
N
6
O
10
S
2
:
4.2. Biological studies
4.2.1.Purification of CA and activity assay
Carbonic anhydrase isoenzymes (I, and II) were purified from human erythrocytes in a
shortly using Sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography which is a
rapid and straightforward chromatographic technique. The amount of the protein was
determined according to the Bradford method which is a simple and quick analytical
procedure at 595 nm wavelength, quantitatively as described previously [52, 53]. Bovine
serum albumin (BSA) was used as a standard protein for this method [54, 55]. Enzyme purity

and subunit composition were controlled with Laemmli’s SDS-PAGE procedure (3-8%) [56,
7]. PageRuler prestained protein ladder was used as the reference protein standard, a mixture
5
of proteins ranging from 10 to 180 kDa [58, 59] conforming to our previous studies [60, 61].
The reaction mix (approx. 1 mL) consisted of p-nitrophenyl acetate (3 mM) as a substrate, p-
acetoxynitrobenzene, purified enzyme, and Tris-sulfate buffer (50 mM, pH 7.4). The enzyme
activity measurements were achieved with three times for each sample by spectrophotometer
3
-1
-1
at 25°C. Enzyme unit was calculated using the absorption coefficient (ε=5.4x10 M cm ) of
p-nitrophenol at 348 nm [62] by using our earlier reported methods [63, 64]. The inhibitory
activities of the N-substituted phthalazine sulfonamide compounds (5a-l) were determined on
the esterase activity of hCA I, and II isoenzymes. The activities were assayed in the presence
of different inhibitor concentrations. The experiments were repeated in triplicate for each drug
concentration used. IC50 values and K
65, 66]. The inhibition type for each of the compounds was determined using Lineweaver-
Burk curves [67, 68].
i
constants were computed as in our previous assays
[
4
.2.2.AChE activity assay
Acetylcholinesterase activity measurement was carried out according to Ellman’s method
69] by the 1.4 mL quartz cuvette at room temperature. The reaction mixture contained 10
[
mM of acetylthiocholine iodide (AChI), 2-(acetylthio)-N,N,N-trimethylethanaminium iodide,
as substrate and 5 mM 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) in 1 mM Tris/HCl (pH
8.0) containing 5 mM EDTA as described previously [70]. AChE assay was depended on the
-1
-1
performed of DTNB (ε=13,6 M cm ) at 412 nm, spectrophotometrically according to our
previous study [71]. The inhibition effects on AChE of these compounds (5a-l) were
determined by Activity%-[Phthalazine] curves. The IC50 values were obtained from

Activity% versus the inhibitors. Various concentrations of the phthalazines were utilized for
the determination of K constants and inhibition types as described in a previous study [72].
i
4
.3. In silico studies
.3.1.ADMET study
4
The physicochemical and pharmacokinetic properties (i.e., ADMET prediction and drug-
likeness) of the N-substituted phthalazine sulfonamide compounds (5a-l) were predicted using
the QikProp [73, 74] and Molecular Properties Panel of Maestro [75].
4.3.2.Molecular docking study
Protein Preparation Wizard [76] was used to pre-process the crystal structures of the hCA
I, II, and AChE in complex with the ligands 2,3,5,6-tetrafluoro-4-piperidin-1-
ylbenzenesulfonamide (PDB ID: 4WUQ, 1.75 Å), N-[(2Z)-1,3-oxazolidin-2-ylidene]sulfuric
diamide (PDB ID: 4FU5, 0.98 Å), and donepezil (PDB ID: 4EY7, 2.35 Å), respectively. The
N-substituted phthalazine sulfonamide derivatives were prepared with the LigPrep module
[
77, 78] as in our previous studies [79]. Protonation states at pH 7.0±2.0 and tautomers were
generated and minimizationed using OPLS3e force field [80, 81]. The ligands were docked to
the protein structure using the standard precision (SP) mode [82-84].
4.4. Statistical studies
Analysis of the data and drawing of graphs were implemented using GraphPad Prism6
and Systat SigmaPlot12 software. The results were shown as mean±standard deviation (95%
confidence intervals). Differences between data sets were considered statistically significant
when the p-value was less than 0.05.

Acknowledgments
This work was supported by The Research Fund of Erzincan Binali Yıldırım University (grant
number FBA-2017-501) and The Research Fund of Sakarya University (grant number 2016-
02-04-018).

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