Pyridazinone substituted benzenesulfonamides as potent carbonic anhydrase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.12.016

A series of sulfonamide derivatives (2a-l) incorporating substituted pyridazinone moieties were investigated for the inhibition of two human cytosolic carbonic anhydrase isoforms, hCA I and hCA II. All these compounds, together with the clinically used su

Full text of DOI:10.1016/j.bmcl.2015.12.016

Accepted Manuscript
Pyridazinone substituted benzenesulfonamides as potent carbonic anhydrase
inhibitors
Raed Yaseen, Deniz Ekinci, Murat Senturk, Alhamzah Dh. Hameed, Syed
Ovais, Pooja Rathore, Mohammed Samim, Kalim Javed, Claudiu T. Supuran
PII:
S0960-894X(15)30329-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.12.016
BMCL 23377
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 September 2015
3 December 2015
7 December 2015
Please cite this article as: Yaseen, R., Ekinci, D., Senturk, M., Hameed, A.D., Ovais, S., Rathore, P., Samim, M.,
Javed, K., Supuran, C.T., Pyridazinone substituted benzenesulfonamides as potent carbonic anhydrase inhibitors,
Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.12.016
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Pyridazinone substituted benzenesulfonamides as potent carbonic anhydrase inhibitors
Raed Yaseena, Deniz Ekincib, Murat Senturkc,*, Alhamzah Dh. Hameeda, Syed Ovaisa, Pooja
Rathorea, Mohammed Samima, Kalim Javeda,*, Claudiu T. Supurand,*
aDepartment of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New
Delhi 110 062, India.
bOndokuz Mayıs University, Faculty of Agriculture, Department of Agricultural
Biotechnology, 55139, Samsun, Turkey.
cAgri Ibrahim Cecen University, Science and Art Faculty, Chemistry Department, 04100, Agri,
Turkey.
dUniversità degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188,
Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy.
Abstract
A series of sulfonamide derivatives (2a-l) incorporating substituted pyridazinone moieties
were investigated for the inhibition of two human cytosolic carbonic anhydrase isoforms,
hCA I and hCA II. All these compounds, together with the clinically used sulfonamide
acetazolamide were investigated as inhibitors of the physiologically relevant isozymes I and
II. These sulfonamides showed very strong inhibition against all these isoforms with KI’s in
the range of 0.98 to 8.5 nM which makes such molecules possible to be used as leads for
discovery of novel effective CA inhibitors targeting other isoforms with medicinal chemistry
applications.
Keywords: Human carbonic anhydrase, enzyme inhibitors, sulfonamides.
1

The involvement of the metalloenzymes family carbonic anhydrases (carbonate
hydrolyase, EC 4.2.1.1; CA) in various physiological processes have been recognized for a
long period, being shown that the deregulated expression or abnormal performance of the 16
isozymes presently known may have important pathological consequences.1,2 In fact, there are
several human diseases whose pathopysiological characteristics include disbalance in the
conversion between these isonzymes substrates (carbon dioxide and bicarbonate), resulting in
perturbed ion transport, shift in pH, abnormal fluid secretion, etc.1,2 Therefore, it seems
plausible that modulation of CA activity to normal levels either by inhibition or activation
offers interesting therapeutic options.1 Because of their favorable outcomes, sulfonamides
became widely accepted drugs in the treatment of several CA-based diseases, especially as
antiglaucoma agents, diuretics, and antiulcer agents among others.1,2 However, systemic and
even topically administered CA inhibitors regularly showed serious side effects.1-4 It is now
understood that these undesired effects are due to the existence of at least 16 different CA
isoforms,1-5 that are indiscriminately inhibited irrespective of whether they play a real role in
disease or are just coexpressed in the same tissue and elsewhere in the body. Moreover,
certain drugs directed primarily against different CA unrelated targets may also inhibit
activity of CAs. This may be exemplified by the antiinflammatory cyclooxygenase-2-selective
drugs celecoxib and valdecoxib that show nanomolar affinity to several CA isoforms, but are
generally well tolerated and give clinical responses in several disorders.1,2 Thus, it is critically
important to thoroughly characterize the affinity of different isozymes for sulfonamide CAIs,
due to the wide range of applications of such drugs, and also to better understand the side
effects due to inhibition of isozymes, which do not constitute the main target for a certain
disease/application.1-4
Our groups recently investigated the interaction of CA I and II isozymes with several
types of phenols, pyrrole derivatized sulfonamides, dopaminergic bromophenolic compounds
and several of its substituted derivatives, e.g., salicyclates and some of their derivatives.4 Here
we extend these earlier investigations to a series of sulfonamides, some of which are widely
used as prodrug or as drugs. Sulfonamides possess many types of biological activities, and
representatives of this class of pharmacological agents are widely used in clinic as
antibacterial, hypoglycemic, diuretic, anti-hypertensive and antiviral drugs among others.1,2
Recently, a host of structurally novel sulfonamide derivatives have been reported to show
substantial antitumor activity in vitro and/or in vivo.3-4
2

Figure 1. Estimated active site region in the hCA II-benzenesulfonamide complex X-ray
structure, showing residues participating in recognition of the inhibitor molecule.
In the present study we have purified human CA I and II isoenzymes and examined
the in vitro inhibition effects of some sulfonamide compounds (2a-l) mentioned above on
these enzymes, using the esterase activity.
Sulfonamide type inhibitors binds to CAs, with coordination to the Zn2+ ion from the
enzyme active site by substituting the fourth, non-protein ligand, a water molecule or
hydroxide ion, such as for example acetazolamide (AZA), a clinically used compound since
1954.4,5 The X-ray crystal structure has been extensively used for understanding the inhibition
mechanism of CAIs. For example, for the adduct of hCA II with sulfamide,5 it has been
observed that the compound binds to CA by anchoring its SO2NH- moiety to the zinc ion of
the enzyme active site, through a hydrogen bond as well as through a second hydrogen bond
to the NH amide of Thr199, an amino acid conserved in all α-CAs and critically important for
the catalytic cycle of these enzymes (Figure 1).2-5
The inhibition profile of various CA isozymes with this class of agents is very
variable, with inhibition constants ranging from the millimolar to the submicromolar range.5-7
Thus, it seemed reasonable to us to extend the previous studies,5-10 including in this
investigation a series of Schiff’s bases obtained by condensing formylchromone with
aminosulfonamides.
The synthetic route used to synthesize title compounds (2a-l) is outlined in Scheme 1. The β-
aroylacrylic acids (1a-l) required for the synthesis of pyridazinoneswere obtained by a Friedel
Craft’s acylation through reported methods.11 The cyclization to pyridazinone derivatives
3

bearing a benzenesulfonamide moiety was afforded by the condensation of appropriate β-
aroylacrylic acid and 4-hydrazinobenzenesulfonamide hydrochloride in ethanol in 45-65%
yield. The purity of the compounds was checked by TLC (Silica gel G) which was visualized
by exposing to iodine vapours. The structures of 2a-l were determined on the basis of
elemental analysis and by various spectroscopic methods such as IR, 1H NMR, C NMR and
13
MS. Elemental analysis (C, H, N & S) data were within 0.4% of the theoretical values.
1
Spectral data IR, H NMR, 13C NMR and MS of compounds were found in full agreement
with the proposed structure. IR spectra showed prominent bands for NH2 at 3305-3297 cm-1,
and 3140-3120 cm-1, for cyclic carbonyl at 1662-1638 cm-1, for C=N at 1599-1580 cm-1 and
for SO2N <at 1328-1322 cm-1 and 1156-1133 cm-1. In 1H NMR spectra the aromatic protons
were observed at expected ppms. The signal for SO2NH2 was observed as two-proton singlet
or merged with the signals of aromatic protons in aromatic region.
O
O
Anhydrous AlCl3
O
Ar
Ar
O
+
OH
1, 1, 2, 2 - tetrachloroethane
(
)
1a-l
O
NHNH2HCl
Absolute Ethanol
reflux 48h
O
S
O
NH2
1a, 2a: Ar = Tetralinyl
H
H
3
1b, 2b: Ar = 4-Benzylphenyl
1c, 2c: Ar = 4-Propylphenyl
1d, 2d: Ar = Acenaphthyl
1e, 2e: Ar =Indanyl
4
5
Ar
O
N
N
1f, 2f: Ar = 4-Ethoxyphenyl
1g, 2g: Ar = 4-Phenoxyphenyl
1h, 2h: Ar = 4-Cyclohexyphenyl
1i, 2i: Ar = 4-Iodophenyl
1j, 2j: Ar = 4-Fluorophenyl
1k, 2k: Ar = 4-Bromophenyl
1l, 2l: Ar = 3,4-Difluorophenyl
2"
3"
6"
5"
S
O
(
)
2a-l
O
NH2
Scheme 1. Synthesis of substituted-6-oxopyridazin-1(6H)-yl] benzenesulfonamide (2a-l).
4

The purification of the two CA isozymes was performed with a simple one step
method by a cellulose-benzyl-sulfanylamide affinity column chromatoghrapy. hCA I was
purified, 102.6-fold with a specific activity of 875.12 EUmg-1 and overall yield of 52.42 %,
hCA II was purified, 867.3-fold with a specific activity of 6970 EUmg-1 and overall yield of
63.8 %.2b,5-10 Inhibitory effects of these sulfonamides 2a-l on enzyme activities were tested
under in vitro conditions; KI values were calculated by using the Cheng-Prusoff equations and
are reported in Table 1.
Figure 2. Studied and references molecules.
Table 1. hCA I and II inhibition data with sulfonamides 2a-l and clinically used inhibitors,
and the selectivity ratio hCA I over hCA II.
KI (nM)
Selectivity ratio
hCA I/hCA II
2.39
Compound R
2a
hCA I
hCA II
5.16
2.16
8.50
1.13
1.91
2.90
4.45
2b
0.39
2c
5

2.77
2.89
0.96
2d
3.60
2.34
2.15
2.85
1.67
0.82
2e
2f
1.70
4.87
1.15
0.98
1.48
4.97
2g
2h
3.09
3.49
5.45
3.63
1.86
2.56
3.26
5.19
1.66
1.36
1.67
0.70
2i
2j
2k
2l
F
AZAa
INDa
250
12.0
15.0
20.83
2.06
31.0
Errors in the range of 2-5% of the shown data, from three different assays.
aFrom Ref. 1c.
We report here the inhibitory effects of sulfonamides 2a-l on the eterase activity of
hCA I and II. The sulfonamide CAI acetazolamide AZA and indisulam IDA has been used as
a negative control in our experiments, and for comparison reasons. These compounds were
synthesized and reported by Yaseen, R.14 as potent anti-hyperglycemic agents in glucose fed
hyperglycemic normal rats. Data of Table 1 show the following regarding inhibition of hCA I
and II with compounds 2a-l, AZA, and IND (as standards), by an esterase assay, with p-NPA
(p-nitrophenyl acetate) as substrate: 9b
6

(i) Against the slow cytosolic isozyme hCA I, compound 2b behave as moderate
inhibitor, with KI value in the range of 8.5 nM. It is also interesting to note that derivatives 2c
and 7 were better hCA I inhibitor as compared to other compounds. This might indicate that
hydrophobicity in the pyridazinone moiety is favorable for the inhibition of hCA I.
Acetazolamide AZA had a KI of 250 nM in this assay whereas compounds 2a-l and IND were
more powerful inhibitors than AZA (Table 1).
(ii) A better inhibitory activity has been observed with compounds 2a-l investigated
here for the inhibition of the rapid cytosolic isozyme hCA II (Table 1). Two derivatives, i.e.,
2k and 2l, showed moderate hCA II inhibitory activity with KI-s in the range of 3.26-5.19 nM,
Table 1), whereas the remaining derivatives were quite effective hCA II inhibitors (Table 1).
The best hCA II inhibitor in this series of derivatives was the bulky, pyridazinone substituted
benzenesulfonamide derivative 2h, which has a very low KI value of 0.98 nM, is a better
inhibitor than AZA and IND, a clinically used sulfonamides.
(iii) The rapid human blood cell isozyme (hCA II), ubiquitous in a lot of different
tissues or cells,1,2 is known to possess a high affinity for sulfonamides, we determined the
selectivity ratios of the tested CAIs against human isozyme II over isozyme I (Table 1). It
may be observed that most of the investigated compounds act as more potent hCA II than
hCA I inhibitors, except 2c, 2d, 2f, and 2l, which showed selectivity ratios in the range of
0.39-0.96. Thus, the most hCA I selective inhibitor was compound 2d. The most selective
hCA II over hCA I inhibitors, such as derivatives 2b and 2h, showed selectivity ratios in the
range of 4.45-4.97, which is indeed remarkable. Some other compounds also showed
moderate selectivities, with ratios in the range of 1.36-2.39 (compounds 2b, 2e, 2g, 2i-2l).
Although there are several studies regarding the interactions of sulfonamide
derivatives with carbonic anhydrase isozymes12-13, it is critically important to explore further
classes of potent CAIs in order to detect compounds with a different inhibition profile to find
novel applications for the inhibitors of these widespread enzymes.
Especially, 2c, 2f, and 2g showed good activity against these hCA isozymes, more
effective than the clinical used sulfonamides AZA and IND. Findings of our study indicates
another class of possible CAIs of interest with strong activity, in addition to the well-known
sulfonamides, the phenols/diphenols bearing bulky ortho moieties in their molecules.
Acknowledgments
This study was financed by Agri Ibrahim Cecen University Scientific Research Council,
(project no: Agri BAP-FEF.15.008) for (MS).
7

References and notes
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Chem.Lett. 2010, 20, 3467; (c) Vullo, D.; Innocenti, A.; Nishimori, I.; Pastorek, J.;
Scozzafava, A.; Pastorekova, S.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 963;
(d) Balaydin, H.T.; Durdagi, S.; Ekinci, D.; Senturk, M.; Goksu, S.; Menzek, A. J. Enzyme
Inhib. Med. Chem. 2012, 27, 467.
2. (a) Supuran, C. T. Carbonic Anhydrases: Catalytic Mechanism, Distribution and
Physiological Roles. In Carbonic Anhydrase-Its Inhibitors and Activators; Supuran, C. T.,
Scozzafava, A., Conway, J., Eds.; CRC: Boca Raton (FL), USA, 2004; pp 1-24; (b) Weber,
A.; Casini, A.; Heine, A.; Kuhn, D.; Supuran, C. T.; Scozzafava, A.; Klebe, G. J. Med.
Chem. 2004, 47, 550; (c) Ekinci, D.; Cavdar, H.; Durdagi, S.; Talaz, O.; Senturk, M.;
Supuran, C.T. Eur. J. Med. Chem. 2012, 49, 68.
3. (a) Sly, W. S.; Hu, P. Y., Annu. Rev. Biochem. 1995, 64, 375; (b) Abdel-Aziz, A.A-M.; El-
Azab, A.S.; Ekinci, D.; Senturk, M.; Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2015, 30,
81; (c) Yerlikaya, E.; Erdogan, O.; Demirdag, R.; Senturk, M.; Kufrevioglu, O.I. Turk. J.
Biochem. 2015, 40, 334.
4. (a) Bayram, E.; Senturk, M.; Kufrevioglu, O. I.; Supuran, C. T. Bioorg. Med. Chem. 2008,
16, 9101; (b) Ekinci, D.; al-Rashida, M.; Abbas, G.; Senturk, M.; Supuran, C. T. J. Enzyme
Inhib. Med. Chem. 2012, 27, 744.; (c) Senturk, M.; Gulcin, I.; Beydemir, S.; Kufrevioglu,
O. I.; Supuran, C. T. Chem. Biol. Drug Des. 2011, 77, 494; (d) Durdagi, S.; Senturk, M.;
Ekinci, D.; Balaydin, H. T.; Goksu, S.; Kufrevioglu, O. I.; Innocenti, A.; Scozzafava, A.;
Supuran, C. T., Bioorg. Med. Chem. 2011, 19, 1381; (e) Balaydin, H.T.; Senturk, M.;
Goksu, S.; Menzek, A. Eur. J. Med. Chem. 2012, 54, 423.
5. Alterio, V.; Vitale, R. M.; Monti, S. M.; Pedone, C.; Scozzafava, A.; Cecchi, A.; De
Simone, G.; Supuran, C. T. J. Am. Chem. Soc. 2006, 128, 8329.
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Ozturk Sarikaya, S.B.; Topal, F.; Senturk, M.; Gulcin, I.; Supuran, C.T. Bioorg. Med.
Chem. Lett. 2011, 21, 4259; (c) Isık, S.; Vullo, D.; Durdagi, S.; Ekinci, D.; Senturk, M.;
Cetin, A.; Senturk, E.; Supuran, C.T. Bioorg. Med. Chem. Lett. 2015, 25, 5636.
7. Nair, S. K.; Ludwig, P. A.; Christianson, D. W., J. Am. Chem. Soc. 1994, 116, 3659.
8. Detailed procedures for enzyme purification can be found in: Firstly, benzoyl chloride was
stirred for four hours at room temperature in CH2Cl2 cellulose. After the spacer arm
cellulose added as a benzyl group and finally diazotized sulfanilamide clamped to the para
position of benzyl group as ligand. The hemolysate was applied to the prepared Cellulose-
8

benzylsulfanylamide affinity column equilibrated with 25 mM Tris-HCl/0.1 M Na2SO4
(pH 8.7). The affinity gel was washed with 25 mM Tris-HCl/22 mM Na2SO4 (pH 8.7). The
human carbonic anhydrase (hCA I and hCA II) isozymes were eluted with 1 M NaCl/25
mM Na2HPO4 (pH 6.3) and 0.1 M CH3COONa/0.5 M NaClO4 (pH 5.6), respectively. All
procedures were performed at 4ºC. (a) Guney, M.; Cavdar, H.; Senturk, M.; Ekinci, D.
Bioorg. Med. Chem. Lett. 2015, 25, 3261; (b) Senturk, E.; Senturk, M.; Ekinci, D. Acta
Physiol. 2015, 215, 99; (c) Ekinci, D.; Senturk, M.; Senturk, E. Acta Physiol. 2015, 215,
99; (d) Fidan, I.; Salmas, R.E.; Arslan, M.; Senturk, M.; Durdagi, S.; Ekinci, D.; Senturk,
E.; Cosgun, S.; Supuran, C.T. Bioorg. Med. Chem. 2015, 23, 7353.
9. (a) Verpoorte, J. A.; Mehta, S.; Edsall, J. T., J. Biol. Chem. 1967, 242, 4221; (b) Cheng, Y.;
Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
10. (a) Bradford, M., Anal. Biochem. 1976, 72, 248; (b) Laemmli, D. K., Nature 1970, 227,
680; (c) Lineweaver, H.; Burk, D., J. Am. Chem. Soc. 1934, 57, 685.
11. (a) Papa, D.; Schwenk, E.; Villani, F.; Klingsberg, E. J. Am. Chem. Soc. 1948, 70, 3356;
(b) Oddy, H.G. J. Am. Chem. Soc. 1923, 45, 2156.
12. (a) Puccetti, L.; Fasolis, G.; Vullo, D.; Chohan, Z. H.; Scozzafava, A.; Supuran, C. T.
Bioorg. Med. Chem. Lett. 2005, 15, 3096; (b) Ekinci, D.; Senturk, M.; Beydemir, S.;
Kufrevioglu, O. I.; Supuran, C. T. Chem. Biol. Drug Des. 2010, 76, 552.
13. (a) Senturk, M.; Talaz, O.; Ekinci, D.; Cavdar, H.; Kufrevioglu, O. I. Bioorg. Med. Chem.
Lett. 2009, 19, 3661; (b) Korkmaz, N.; Obaidi, O.A.; Senturk, M.; Astley, D.; Ekinci, D.;
Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2015, 30, 75; (c) Ceyhun, S. B.; Senturk, M.;
Yerlikaya, E.; Erdogan, O.; Kufrevioglu, O.I.; Ekinci, D. Environ. Toxicol. Pharmacol.
2011, 32, 69.
14. Detailed synthetic procedures for the preparation of all derivatives can be found in:
Melting points were determined in open capillary tubes and are uncorrected. All the
Fourier TransformInfra-Red (FTIR) spectra were recorded on a Bio-rad FTS-135
1
spectrophotometer using KBr pellets; νmax values are given in cm-1. H NMR spectra were
recorded on a BrukerSpectrospin DPX 300 MHz spectrometer using deuterated DMSO as
solvent and tetramethylsilane (TMS) as an internal standard. Chemical shifts are given in δ
(ppm) scale and coupling constants (J values) are expressed in Hz. Mass spectra (MS)
were scanned by affecting FAB ionization JEOL-JMS-DX 303 system, equipped with
direct inlet probe system. The m/z values of the more intense peaks are mentioned. The
purity of the compounds was checked on TLC plate (Silica gel G) in the solvent system
9

Toluene:Ethyl acetate:Formic acid (5 : 4 : 1,TEF). Elemental analysis was carried out on
CHNS Elementar (Vario EL III).
General procedure for the synthesis of 6-aryl-2-benzenesulfonamide-pyridazinones (2a-
l):
A
mixture of appropriate β-aroylacrylic acid (1a-l) (0.001 mol) and 4-
hydrazinobenzenesulfonamide hydrochloride (0.001 mol) in absolute ethanol (20-30 mL) was
refluxed for 48 h. The solvent ethyl alcohol was removed by distillation method. The solid
residue thus obtained was converted into fine powder, which was stirred with 5% sodium
bicarbonate solution (25 mL). It was filtered, washed with 2% acetic acid and then with water.
It was dried and crystallized from methanol. The intermediatesβ-aroylacrylic acids (1a-l)
required for the synthesis of pyridazinones were prepared through reported procedures.11
4-[6-Oxo-3-(5,6,7,8-tetrahydronaphthalen-2-yl) pyridazin-1(6H)-yl] benzenesulfonamide
(2a): White crystals; yield=61%; m.p. 280-281C
̊
; Rf= 0.45 (TEF); IR νmax (KBr, in cm-1):3304
1
and 3122 (NH2), 1652 (cyclic carbonyl),1582 (C=N), 1331 and 1148(SO2N); H NMR (300
MHz, DMSO-d6, δ): 7.64 (1H, d, J=7.5 Hz, H-6'), 7.18 (1H, d, J=7.2 Hz, H-5'), 7.20 (1H, d,
J=9.9 Hz, H-5), 7.64 (1H, d, J=7.5 Hz, H-2'), 7.89 (2H, d, J=8.4 Hz, H-3", H-5"), 7.96 (2H, d,
J=8.7 Hz, H-2", H-6"), 8.13 (1H, d, J=9.9 Hz, H-4), 7.49 (2H, s, SO2NH2), 1.75 (4H, s, H-3"',
H-4"'), 2.78 (4H, s, H-2"', H-5"'); 13C NMR (75 MHz, DMSO-d6, δ):155.12 (C=N
pyridazinone), 146.21 (C-5 pyridazinone), 124.75 (C-4 pyridazinone), 154.26 (C=O
pyridazinone); ESI-MS: (m/z):381[M+], 382 [M+1], 380[M-1], 379 [M-2];CHNS Analysis for
C20H19N3O3S: Found (Calculated) C, 63.03(63.05%); H, 5.04 (5.05%); N, 10.98 (10.97%); O,
12.58 (12.56%); S 8.40 (8.38%).
4-[3-(4-Benzylphenyl)-6-oxopyridazin-1(6H)-yl] benzenesulfonamide (2b):
White crystals; Yield=65%; m.p. 282-283 C̊
; Rf= 0.48(TEF); IR νmax (KBr, in cm-1):3305 and
3130 (NH2), 1647 (cyclic carbonyl),1582 (C=N), 1580 (C=N), 1335 and 1141(SO2N);1H
NMR (300 MHz, DMSO-d6, δ): 7.19-7.29 (6H, m, H-5, H-2"', H-3"', H-4"', H-5"', H-6"'),
7.36 (2H, d, J=8.1 Hz, H-3', H-5'), 7.49 (2H, s, SO2NH2), 7.84-7.96 (6H, m, H-3", H-5", H-2",
H-6", H-2', H-6'), 8.13 (1H, d, J=9.9 Hz, H-4), 4.005 (2H, s, Ph-CH2-Ph); 13C NMR (75
MHz, DMSO-d6, δ):154.19 (C=N pyridazinone), 147.10 (C-5 pyridazinone), 126.33 (C-4
pyridazinone), 155.12 (C=O pyridazinone);ESI-MS: (m/z):417[M+], 418 [M+1], 416 [M-1],
415 [M-2];CHNS Analysis for C23H19N3O3S: Found (calculated)C, 66.10 (66.12%); H, 4.61
(4.60%); N, 10.12 (10.11%); O, 11.54 (11.52%); S, 7.72 (7.71%).
10

4-[6-Oxo-3-(4-propylphenyl) pyridazin-1(6H)-yl] benzenesulfonamide (2c):
White crystals; Yield=53%; m.p. 260-261
C; Rf= 0.46 (TEF); IR νmax (KBr, in cm-1):3297 and
̊
3127 (NH2), 1650(cyclic carbonyl), 1580 (C=N),1338 and 1146(SO2N),1H NMR (300 MHz,
DMSO-d6, δ): 7.21 (1H, d, J=9.9 Hz, H-5), 7.32 (2H, d, J=7.8 Hz, H-3', H-5'), 7.49 (2H, s,
SO2NH2), 7.85 (2H, d, J=7.8 Hz, H-2', H-6'), 7.90 (1H, d, J=7.2 Hz, H-3", H-5"), 7.96 (1H, d,
J=8.7 Hz, H-2", H-6"), 8.15 (1H, d, J=9.6, H-4), 0.90 (3H, t, Ph-CH2CH2CH3), 1.58-1.65
(2H, m, Ph-CH2CH2CH3), 2.61 (2H, t, Ph-CH2CH2CH3); 13C NMR (75 MHz, DMSO-d6,
δ):155.11 (C=N pyridazinone), 145.14 (C-5 pyridazinone), 124.83 (C-4 pyridazinone), 154.15
(C=O pyridazinone);ESI-MS (m/z):369 [M+], 370 [M+1], 368 [M-1], 367 [M-2];CHNS
Analysis for C19H19N3O3S: Found (Calculated) C, 61.74 (61.75%); H, 5.19 (5.20%); N, 11.38
(11.39%); O 12.99 (13.01%); S, 8.71 (8.70%).
4-[3-(1,2-Dihydroacenaphthylen-5-yl)-6-oxo-1,6-dihydropyridazin-1-yl]benzene-1-
sulfonamide (2d):
Light green crystals; Yield=25%; m.p. 250-251 C̊
; Rf =0.54 (TEF); IR νmax (KBr, in cm-
1):3300 and 3131 (NH2), 1662 (cyclic carbonyl) 1595 (C=N),1329 and 1154 (SO2N);1H NMR
(300 MHz, DMSO-d6, δ): 7.11-7.92 (11H, m, aromatic protons of acenaphthyl and N-phenyl,
SO2NH2), 7.25 (1H, d, J=9.6 Hz, H-5), 8.11 (1H, d, J=9.9 Hz, H-4), 3.29 (4H, brs, -CH2-
CH2- ofacenaphthyl); 13C NMR (75 MHz, DMSO-d6, δ):154.51(C=N pyridazinone), 145.23
(C-5 pyridazinone), 124.81 (C-4 pyridazinone), 153.15 (C=O pyridazinone);ESI-MS (m/z):
403 [M+], 404 [M+1], 402 [M-1],401 [M-2];CHNS Analysis for C22H17N3O3S: Found
(Calculated)C, 65.50 (65.51%); H, 4.25 (4.26%); N, 10.42 (10.41%); O, 11.89 (11.88%), S
7.94 (7.95 %).
4-[3-(2,3-Dihydro-1H-inden-5-yl)-6-oxopyridazin-1(6H)-yl] benzenesulfonamide (2e):
Light yellow crystals; Yield=41%; m.p. 286-288 C̊
; Rf =0.55 (TEF); IR νmax (KBr, in cm-
1):3303 and 3120 (NH2); 1655 (cyclic carbonyl), 1590 (C=N),1325 and 1149 (SO2N);1H
NMR (300 MHz, DMSO-d6, δ): 7.19 (1H, d, J=9.6 Hz, H-5), 7.34 (1H, d, J= 8.1 Hz, H-5'),
7.49 (2H, s, SO2NH2), 7.70 (1H, d, J=7.8 Hz, H-6'), 7.79 (1H, s, H-2'), 7.90 (2H, d, J=8.7 Hz,
H-3'', H-5"), 7.96 (2H, d, J=8.7 Hz, H-2", H-6"), 8.14 (1H, d, J=9.9 Hz, H-4), 2.02-2.08 (2H,
m, H-3"'), 2.90 (4H, d, J=4.2 Hz, H-2"', H-4"'); 13C NMR (75 MHz, DMSO-d6,
δ):155.58(C=N pyridazinone), 144.27 (C-5 pyridazinone), 125.81 (C-4 pyridazinone), 152.15
(C=O pyridazinone);ESI-MS (m/z): 367 [M+], 368 [M+1], 366 [M-1], 365 [M-2];CHNS
11

Analysis forC19H17N3O3S: Found (Calculated) C, 62.07 (62.09%); H, 4.66 (4.68%); N, 11.48
(11.46%); O, 13.10 (13.08%); S, 8.77 (8.75 %).
4-[3-(4-Ethoxyphenyl)-6-oxopyridazin-1(6H)-yl] benzenesulfonamide (2f):
Light yellow crystals; Yield=55%; m.p. 248-249 C̊
; Rf= 0.46 (TEF);IR νmax (KBr, in cm-
1):3302 and 3121 (NH2); 1644 (cyclic carbonyl), 1584 (C=N),1332 and 1140 (SO2N);1H
NMR (300 MHz, DMSO-d6, δ):7.03 (2H, d, J=8.7 Hz, H-3', H-5'), 7.19 (1H, d, J=9.9 Hz, H-
5), 7.49 (2H, s, SO2NH2), 7.86-7.97 (6H, m, H-2', H-6', H-3", H-5", H-2", H-6"), 8.14 (1H, d,
J=9.9 Hz, H-4), 1.34 (3H, t, Ar-O-CH2-CH3), 4.08 (2H, q, Ar-O-CH2-CH3);13C NMR (75
MHz, DMSO-d6, δ):153.81 (C=N pyridazinone), 144.46(C-5 pyridazinone), 125.13 (C-4
pyridazinone), 155.20 (C=O pyridazinone);ESI-MS (m/z):371[M+], 372[M+1], 370 [M-1],
369[M-2];CHNS Analysis forC18H17N3O4S: Found (Calculated) C, 58.24 (58.25%); H, 4.60
(4.59%); N, 11.29 (11.28%); O, 17.28 (17.27%); S, 8.62 (8.61%).
4-[6-Oxo-3-(4-phenoxyphenyl) pyridazin-1(6H)-yl] benzenesulfonamide (2g):
Light yellow crystals; Yield=58%; m.p. 260-261 C
̊
; Rf= 0.51 (TEF);IR νmax (KBr, in cm-
1
1):3305 and 3132 (NH2); 1647 (cyclic carbonyl), 1580 (C=N),1334 and 1140(SO2N); H
NMR (300 MHz, DMSO-d6, δ):7.07-7.98 (14H, m, H-2', H-6', H-3', H-5', H-3", H-5", H-2",
H-6", H-2"', H-3"', H-4"', H-5"', H-6"', H-5), 8.15 (1H, d, J=9.6 Hz, H-4), 7.49 (2H, s,
SO2NH2); 13C NMR (75 MHz, DMSO-d6):156.02 (C=N pyridazinone), 144.76(C-5
pyridazinone), 125.15 (C-4 pyridazinone), 154.74(C=O pyridazinone);ESI-MS (m/z):419
[M+], 420 [M+1], 418 [M-1], 417 [M-2];CHNS Analysis forC22H17N3O4S: Found
(Calculated)C, 62.96 (62.98%); H, 4.09 (4.11%); N, 10.05 (10.04%); O, 15.30 (15.28%); S
7.68 (7.66%).
4-[3-(4-Cyclohexylphenyl)-6-oxo-1,6-dihydropyridazin-1-yl]benzene-1-sulfonamide (2h):
Yellow crystals; Yield=45%, m.p. 234-235 C̊
; Rf= 0.52 (TEF);IR νmax (KBr, in cm-1):3298 and
3135 (NH2), 1641 (cyclic carbonyl),1599 (C=N), 1328 and 1137(SO2N),1H NMR (300 MHz,
DMSO-d6, δ):7.21 (1H, d, J=9.9 Hz, H-5), 7.35 (2H, d, J=8.1 Hz, H-3', H-5'), 7.48 (2H, s,
SO2NH2), 7.84 (2H, d, J=7.8 Hz, H-2', H-6'), 7.90 (2H, d, J=8.4 Hz, H-3", H-5"), 7.96 (2H, d,
J=7.8 Hz, H-2", H-6"), 8.14 (1H, d, J=9.6 Hz, H-4), 3.1 (1H, s,cyclohexyle ring), 1.31-1.81
(9H, m, cyclohexyl ring);13C NMR (75 MHz, DMSO-d6, δ):154.12 (C=N pyridazinone),
144.96(C-5 pyridazinone), 124.85 (C-4 pyridazinone), 156.24 (C=O pyridazinone);MALDI
(m/z):409 [M+], 410 [M+1], 408 [M-1], 407 [M-2];CHNS Analysis forC22H23N3O3S: Found
12

(Calculated)C, 64.48 (64.50%); H, 5.68 (5.69%); N, 10.28 (10.29%); O, 11.74 (11.75%); S
7.85 (7.86%).
4-[3-(4-Iodophenyl)-6-oxopyridazin-1(6H)-yl]benzenesulfonamide (2i):
Light brown crystals; Yield=31%; 269-270 C̊
; Rf= 0.5 (TEF);IR νmax (KBr, in cm-1):3301 and
3140 (NH2), 1638 (cyclic carbonyl), 1591 (C=N),1326 and 1133(SO2N); 1H NMR (300 MHz,
DMSO-d6, δ): 7.23 (1H, d, J=9.9 Hz, H-5), 7.48 (2H, s, SO2NH2), 7.52-7.98 (8H, m, H-2', H-
3', H-5', H-6', H-2'', H-3", H-5", H-6"), 8.16 (1H, d, J=9.9 Hz, H-4); 13C NMR (75 MHz,
DMSO-d6, δ):156.11 (C=N pyridazinone), 144.91 (C-5 pyridazinone), 125.16 (C-4
pyridazinone), 155.18 (C=O pyridazinone);ESI-MS (m/z):453 [M+], 454 [M+1], 452[M-1],
451 [M-2];CHNS Analysis for C16H12IN3O3S: Found (Calculated) C, 42.41 (42.40%); H, 2.68
(2.67%); I, 28.01 (28.00%); N, 9.26(9.27%); O, 10.60 (10.59%); S, 7.06 (7.07%).
4-[3-(4-Fluorophenyl)-6-oxopyridazin-1(6H)-yl] benzenesulfonamide (2j):
Dark yellow crystals; Yield=45%; m.p. 288-289 ̊
C; Rf = 0.44(TEF);IR νmax (KBr, in cm-
1):3299 and 3130 (NH2); 1659 (cyclic carbonyl), 1599 (C=N),1328 and 1156 (SO2N);1H
NMR (300 MHz, DMSO-d6, δ):7.23 (1H, d, J=9.9 Hz, H-5), 7.36 (2H, d, J=8.7 Hz, H-2', H-
6'), 7.50 (2H, s, SO2NH2), 7.93-8.03 (6H, m, H-3', H-5', H-3", H-5", H-2', H-6"), 8.17 (1H, d,
J=9.9 Hz, H-4); 13C NMR (75 MHz, DMSO-d6, δ):157.09 (C=N pyridazinone), 146.13 (C-5
pyridazinone), 125.11 (C-4 pyridazinone), 156.17 (C=O pyridazinone);ESI-MS
(m/z):345[M+], 346 [M+1], 344[M-1], 343 [M-2]; CHNS Analysis forC16H12FN3O3S: Found
(Calculated) C, 55.65 (55.67%); H, 3.53 (3.51%); F, 5.54 (5.52%); N, 12.19 (12.18%); O,
13.88 (13.87%); S; 9.27 (9.26%).
4-[3-(4-Bromophenyl)-6-oxopyridazin-1(6H)-yl] benzenesulfonamide (2k):
Light yellow crystals; Yield=35%; m.p. 278-279 ̊
C; Rf= 0.4 (TEF);IR νmax (KBr, in cm-
1):3301 and 3135 (NH2), 1641 (cyclic carbonyl),1588 (C=N)1328 and 1137 (SO2N); 1H NMR
(300 MHz, DMSO-d6, δ): 7.24 (1H, d, J=9.9 Hz, H-5), 7.49 (2H, s, SO2NH2), 7.71 (2H, d,
J=8.4 Hz, H-2', H-6'), 7.93 (6H, m, H-2", H-3", H-5", H-6", H-3', H-5'), 8.18 (1H, d, J=9.9
Hz, H-4); 13C NMR (75 MHz, DMSO-d6, δ):154.34 (C=N pyridazinone), 144.87 (C-5
pyridazinone), 124.96 (C-4 pyridazinone), 156.35 (C=O pyridazinone); ESI-MS (m/z):406
[M+], 408 [M+2], 405 [M-1], 404 [M-2]; CHNS Analysis for C16H12BrN3O3S: Found
(Calculated) C, 47.31 (47.32%); H, 2.97 (2.95%); Br, 19.71 (19.70%); N, 10.37 (10.35%), O,
11.82 (11.80%); S 7.89 (7.90%).
13

4-[3-(3, 4-Difluorophenyl)-6-oxopyridazin-1(6H)-yl] benzenesulfonamide (2l):
White crystals; Yield=37%; m.p. 270-271 C
; Rf = 0.46; (TEF);IR νmax (KBr, in cm-1):3298
̊
and 3121 (NH2), 1657 (cyclic carbonyl), 1589 (C=N),1322 and 1152 (SO2N);1H NMR (300
MHz, DMSO-d6, δ): 7.25 (1H, d,J=9.6 Hz, H-5), 7.49 (1H, s, SO2NH2), 7.58 (1H, d, J=9.6
Hz, H-5'), 7.83 (1H, d, J=5.7 Hz, H-2'), 7.92 (2H, d,J=9 Hz, H-3", H-5"), 7.97 (2H, d, J=8.7
Hz, H-2", H-6"), 8.004-8.039 (1H, m, H-6'), 8.19 (1H, d, J=9.9 Hz, H-4); 13C NMR (75 MHz,
DMSO-d6, δ):154.41 (C=N pyridazinone), 143.77 (C-5 pyridazinone), 125.20 (C-4
pyridazinone), 154.13 (C=O pyridazinone); ESI-MS (m/z):363[M+], 364 [M+1], 362[M-1],
361 [M-2]; CHNS Analysis for C16H11F2N3O3S: Found (Calculated): C, 52.87 (52.86%); H,
3.12 (3.11%); F, 10.46 (10.45%); N, 11.56 (11.55%); O, 13.19 (13.20%); S, 8.81 (8.83%).
14

Pyridazinone substituted benzenesulfonamides as potent carbonic anhydrase inhibitors
Raed Yaseena, Deniz Ekincib, Murat Senturkc,*, Alhamzah Dh. Hameeda, Syed Ovaisa, Pooja
Rathorea, Mohammed Samima, Kalim Javeda,*, Claudiu T. Supurand,*
Hydrophobic part
of active site
Hydrophilic
part of active site
R
O S
O
N
Thr 199
H
NH-
O
H
Zn2+
O-
His 94
His 119
O
His 96
Glu 106
15