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ChemistrySelect doi.org/10.1002/slct.202101854

z Biological Chemistry & Chemical Biology

A Practical Method of N-Methylpyrrole Disulfonamides

Synthesis: Computational Studies, Carbonic Anhydrase
Inhibition and Electrochemical DNA Binding Investigations
Mujahid Abas,[a] Yasir Nazir,[a, b] Zaman Ashraf,*[a] Zafar Iqbal,[a] Hussain Raza,[c]
Mubashir Hassan,[d] Erum Jabeen,[a] and Abdul Bais[a]

Heterocyclic compounds bearing sulfonamide moiety have 0.88 μM respectively in comparison to standard acetazolamide
been reported to possess carbonic anhydrase inhibitory (IC50 0.99 � 0.04 μM). The enzyme inhibitory kinetics exhibited
activity. In the present study, a series of novel N-methylpyrrole 3 e a noncompetitive inhibitor with Km and Ki values as
3(a–j) derivatives bearing disulfonamide functional group have 0.34 mM and 18.2 μM respectively. The compounds 3 e and 3 j
been synthesized by following a simple nucleophilic substitu- showed very little cytotoxicity against human keratinocyte
tion reaction route to explore their carbonic anhydrase (HaCaT) with 80 % cell viability and the anticancer activity
inhibitory activity. N-methylpyrrole (1) was converted into N- performed against MCF-7 cell line showed that the compounds
methylpyrrole disulfonyl chloride (2), which upon condensation 3 e and 3 j caused 80 % and 45 % cell death respectively at
with various aliphatic and aromatic amines, yielded the final 125 μM concentrations. Combining the results of DNA binding
products 3(a–j). In silico docking results predicted strong analysis through the UV-Vis spectroscopy (hypochromism),
binding of synthesized compounds in an enzymatic pocket of cyclic voltammetry (current decrease), and fluorescence spec-
human carbonic anhydrase isozyme II (PDB ID 4Q6D). In vitro troscopy (hypochromism in intercalator’s peak); mixed binding
carbonic anhydrase inhibitory assays revealed that analogues mode (intercalation + groove binding) was suggested for 3 e
3 e (1-Methyl-N3,N4-bis(2-(pyridin-2-yl)ethyl)-1H-pyrrole-3,4-di- and intercalation for 3 j with stronger DNA binding of 3 e than
sulfonamide) and 3j (N3,N4,1-trimethyl-1H-pyrrole-3,4-disulfo- 3 j. Based on our results 3 e and 3 j may be proposed to serve
namide) were most potent with IC50 0.38 � 0.01 μM and 0.75 � as a lead structure for designing potentially more active CAIs.

Introduction
gluconeogenesis, and ureagenesis.[3] The family of human
Pyrrole-containing compounds are amongst the most valuable carbonic anhydrase (hCAs) can be classified into different
heterocycles. They possess a wide variety of pharmacological isoforms based on place of origin like cytosol, mitochondria,
applications as antituberculosis, antianalgesics, anticancer, and and saliva. Increased levels of many isoforms of hCAs have
anti-inflammatory agents.[1] The compounds bearing sulphona- been related to various diseases like cancer, glaucoma, and
mide moieties along with pyrrole ring are of special interest for obesity.[4] Cancer is a disease that involves an uncontrolled
possessing attractive carbonic anhydrase inhibitory results. The propagation of cells,-attacks and interrupts the nearby tissues.[5]
carbonic anhydrase (CA) is an enzyme that takes part in a wide According to WHO cancer accounted for 8.2 million deaths in
variety of physiological processes such as buffering of intra and 2012 around the globe.[6]
extracellular pH[2] and biochemical processes as lipogenesis, Despite the existence of a wide variety of anticancer drugs,
there is still a great need for novel drugs for which hCAs have
[a] Dr. M. Abas, Dr. Y. Nazir, Dr. Z. Ashraf, Dr. Z. Iqbal, Dr. E. Jabeen, Dr. A. Bais not developed resistance and are free of serious side effects.
Department of Chemistry, Allama Iqbal Open University, Compounds possessing sulfonamide group (SO2NH2) in their
Islamabad 44000, Pakistan structures are well known to show better CA inhibitory
E-mail: [email protected]
activity.[7] The sulfonamide group includes the crucial nitrogen
[email protected]
[b] Dr. Y. Nazir atom as a zinc binding group which binds with the metal ion in
Faculty of Sciences, Department of Chemistry, a stable tetrahedral geometry in the active site of the enzyme.[8]
University of Sialkot 51300, Pakistan Additionally, active site residues Glu-106 and Thr-199 make
[c] Dr. H. Raza
hydrogen bonds with both NH and oxygen of sulfonamide.
Department of Biological Sciences,
College of Natural Sciences, Moreover, the aromatic/aliphatic heterocycle organic moiety
Kongju National University, binds in an active site of CA.[9] Sulfadrugs include a variety of
Gongju, 314-701, Korea pharmaceutical drugs, comprising of pharmacological repre-
[d] Dr. M. Hassan
sentatives with antitumor, antibacterial,[10] anticarbonic
Institute of Molecular Biology and Biotechnology,
The University of Lahore, Lahore, Pakistan anhydrase,[11] diuretic, hypoglycemic, and protease.[12] Chlorine
Supporting information for this article is available on the WWW under containing sulfadrugs have been reported to possess potent
https://doi.org/10.1002/slct.202101854 anticancer activity[13]. Indole based analogues have the poten-

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Scheme 1. Synthesis of N-methyl pyrrole di-sulfonamide derivatives 3(a–j); Reagents and conditions. (i) 78 °C, reflux for 1 h; (ii) Et3N/EtOH, reflux for 10 h.

tial to act as multidrug-resistant bacteria MRSA[14]. The α- Acetazolamide, a standard carbonic anhydrase inhibitor was
fluoroenesulphonamide and α-fluoroenimide revealed the used as a reference (positive control). The pyridyl ring along
possibility to either generate selective/dual hCA IX, XII isoform with methylated substituted carbonic anhydrase inhibitors
confirming the strong potential of these pharmacophores.[15] discussed here are of special interest due to their greater
Benzenesulphonamides, cyclitols and phenolic compounds inhibitory activity. Bioassay results exposed that the main
have shown a potent carbonic anhydrase inhibitory activity reason for inhibitory activity in presence of electron donating
against human and bovine isoenzymes.[16] 2-pyridylethyl and alkyl groups (Table 1). Sulfonamides have
Due to great pharmacological importance of sulfonamides been reported to possess potent human carbonic anhydrase
bearing heterocycles, the current study was aimed to synthe- activity and substitution of 2-pyridylethyl at sulfonamide
size N-methyl pyrrole di-sulfonamides 3(a–j) to explore their moiety resulted in an enhanced inhibitory activity.[18] Moreover,
role as carbonic anhydrase inhibition. The binding potential of compound 3e (1-Methyl-N3,N4-bis(2-(pyridin-2-yl)ethyl)-1H-
target molecules at target protein PDBID 4Q6D was predicted pyrrole-3,4-disulfonamide) bearing 2-pyridylethyl substituted
by molecular docking studies. DNA binding activity was carried sulfonamide showed higher inhibitory activity (IC50 0.38 μM)
out by electrochemical and spectroscopic methods to explore compared to 3 f (1-Methyl-N3,N4-di(pyridin-4-yl)-1H-pyrrole-3,4-
the mechanism of anticancer potential of most potent deriva- disulfonamide) and 3 g (1-Methyl-N3,N4-bis(pyridin-4-ylmethyl)-
tives. Cytotoxicity of these synthesized derivative 3(a–j) was 1H-pyrrole-3,4-disulfonamide) bearing 4-Pyridyl and 4-Picolyl
performed against MCF-7 cell lines and human keratinocyte substituents with IC50 224.86 μM and 326.12 μM respectively.
(HaCaT) cell lines. The lower activity of compounds 3 f and 3 g might be due to
the presence of 4-pyridyl rings in comparison to 2-pyridyl
moieties present in 3 e and 3 j. In the case of compound 3 e the
Results and Discussion presence of 2-pyridyl ring along with CH3 group involved in
inhibition of carbonic anhydrase activity. Derivative 3 e ex-
Synthesis
pressed CA inhibition with IC50 0.38 μM much better than
The title compounds 3(a–j) have been synthesized (Scheme 1) acetazolamide with IC50 0.99 μM used as standard (Table 2).
by following an already reported method with slight
modifications.[17] Briefly, chlorosulphonic acid was added drop-
wise to N-methyl pyrrole (1) in a round bottom flask for 1 h in
1 : 3 at 78 °C using acetone and dry ice bath. The ice-cold
water was added to the reaction mixture to quench the
reaction. Precipitates of N-methyl pyrrole disulphonyl chloride
(2) obtained were recrystallized in aqueous ethanol. Various
Table 1. The % yield, substitution pattern and carbonic anhydrase
organic amines were condensed with N-methyl pyrrole disul- inhibitory activity of derivatives 3(a–j).
phonyl chloride (2) in an equimolar ratio in 10.0 mL of dried
Compound Yield Substitution pattern Carbonic
ethanol and triethylamine as a catalyst in a round bottom flask (%) (R) Anhydrase
and refluxed for 10 h. The progress of the reaction was IC50 � SEM (μM)
monitored by thin-layer chromatography (pet. ether: ethyl
3a 71 H 3.51 � 0.09
acetate in 3 : 1). Upon completion of the reaction, the contents 3b 71 Benzyl 76.49 � 0.52
in the round bottom flask were dropped in ice-cold water and 3c 75 Phenyl 21.61 � 0.44
precipitates of N-methyl pyrrole disulphonamide 3(a–j) ob- 3d 69 Cyclohexyl 1.15 � 0.05
tained were dried and recrystallized in aqueous ethanol. 3e 75 2-Pyridylethyl 0.38 � 0.01
3f 71 4-Pyridyl 224.86 � 0.79
3g 68 4-Picolyl 326.12 � 0.99
3h 71 Biphenyl 97.91 � 0.61
In vitro Bio-evaluation
3i 71 3-Pyridyl 188.12 � 0.79
N-methylpyrrole disulfonamide analogues 3(a–j) were designed 3j 71 n-butyl 0.75 � 0.88
Acetazolamide 0.99 � 0.04
to explore the effects of different substituent groups at
aromatic rings for inhibition of carbonic anhydrase. Each value is expressed as mean � SEM. SEM is the standard error mean.

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ChemistrySelect doi.org/10.1002/slct.202101854

Table 2. Carbonic anhydrase inhibitory kinetics analysis of the derivative Table 3. Glide docking score of docking complexes 3(a–j).
3 e.
Compound Docking score (PDB ID 4Q6D)
Concentration Vmax Km (μM) Inhibition Type Ki kcal/mol
(μM) (ΔA /min) (μM)
3a 3.028
5
0.00 1.26061 × 10 0.34 3b 3.387
6
0.19 7.73751 × 10 0.34 Non-Competitive 0.87 3c 3.273
6
0.38 7.12121 × 10 0.34 3d 2.794
6
0.76 5.27241 × 10 0.34 3e 5.154
3f 3.135
3g 3.244
3h 2.829
3i 4.436
3j 2.638
Kinetic Study Acetazolamide 3.211
Kinetic studies were carried to find out the inhibitory mecha-
nism of synthesized compounds 3(a–j) against carbonic
anhydrase. Based on IC50 values, 3 e was selected as the most Computational Studies
active derivative to explore its mechanism of inhibition. The
Molecular Docking of Synthesized Compounds 3(a–j)
enzyme inhibitory kinetics was determined by the Lineweaver-
Burk plot of 1/V vs 1/[S] using different concentrations of 3 e Molecular docking approach is significant approach to examine
which produced a series of straight-lines intersecting in the the binding conformations of ligands within the active site of
second-quadrant. It has been further investigated that 1/Vmax target proteins.[19] The newly synthesized ligands 3(a–j) were
increased while Km remains the same by increasing concen- docked against human carbonic anhydrase isozyme II to predict
tration of 3 e predicting non-competitive inhibition of carbonic their conformational positions and interactions with active site
anhydrase by interrupting enzyme-substrate complex (ESI). residues. The docked complexes were examined based on glide
Secondary plots of concentration vs slope of compound 3 e, dock energy values (kcal/mol) (Table 3) and bonding interac-
expressed EI dissociation constants Ki 18.2 μM and Michealis- tion pattern (hydrogen/hydrophobic) with the active site
Menten constant Km 0.59 mM have been presented by secon- residues.
dary plot intercept vs conc. of derivative 3e (Figure 1). Thus,
compound 3 e exhibited noncompetitive mode of binding with
Ligand-Binding Analysis of Human Carbonic Anhydrase
carbonic anhydrase enzyme.
Isozyme II Docked Complexes
Each inhibitor has a different interaction with active site
residues of protein due to its different substitution pattern. The
reference inhibitor acetazolamide interacts CA II catalytic site
with thiadiazole ring closer to zinc (301) ion forming π-π

Figure 1. Inhibition of carbonic anhydrase by compound 3 e. Concentration of 3 e used were 0.00, 0.19, 0.38 and 0.76 μM, respectively. Substrate p-nitrophenyl
acetate concentrations were 0.125, 0.25, 0.5, 1 and 2 mM, respectively.

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interaction with side-chain residue His94. The 3e was selected PSA, and molar volume 389.96 A3 values which significantly
as the best inhibitor against target protein giving the highest justified its drug-like behaviour with a drug like model score
binding energy ( 5.154 kcal/mol) as compared to reference 0.51. In addition, its molecular weight (449.12 g/mol) was also
acetazolamide ( 3.211 kcal/mol) (Table 4). Therefore, 3 e- better than other drugs (Table 4).
docked complex was visualized to check their binding pattern
and conformational position within the active site of protein
Cytotoxicity Studies
4Q6D. The ligand-protein binding analyses revealed that two
terminal pyridine rings of 3 e interact through π-π stacking The cytotoxic effects of the synthesized derivatives have been
with active site His94 closer to Zn (301) ion and His4 checked against the human keratinocyte cell line (HaCaT). The
respectively. Moreover, the rigid structure of 3 e is preventing it results expressed that most of the target molecules are non-
from entering the active site, predicting its non-competitive toxic against HaCaT cells, and the effects are produced in a
nature (Figure 2). The residue that played a vital role in dose-dependent pattern (Figure 3). The cytotoxicity assay
stabilizing the protein inhibitor complex is His94 and these exhibited more than 80 % cell viability for human keratinocyte
findings are in accordance with the literature.[20] (HaCaT) cell line by derivative 3 e and 3 j at 125 μM concen-
tration. 100 % DMSO was used as a positive control.
Chemo-informatics and Lipinski’s Rule
Anticancer Activity
The predicted chemo-informatics properties such as molecular
weight, number of hydrogen bond acceptors, number of Anticancer activity of the target molecules 3(a–j) was per-
hydrogen bond donors, polarizability, solubility, molar volume, formed against breast cancer cell lines MCF-7 and results
and polar surface area were computationally evaluated. The showed that most of the tested compounds including 3 e and
literature study established a standard value for molar molec- 3 j possess good anticancer activity. The results assured that
ular weight (160–480), the number of atoms (20–70), polar- compound 3 e causes 80 % cell death while 3 j causes 45 %
izability � 5, and solubility � 4.[21] Results revealed that pre- cancer cell death at a concentration of 125 μM (Figure 4). The
dicted values of 3 e are much better than other compounds. cytotoxic effects produced by derivative 3 e and 3 j against
Moreover, Lipinski’s rule of five (RO5) about specific chemistry human keratinocyte (HaCaT) at 125 μM concentration were
or structural features found in 3 e is followed in this drug. The quite smaller which revealed that these derivatives are safer for
computational results predicted that 3 e possesses 8 HBA (� healthy cells compared to cancer cells.
10), 2 HBD (� 5), 0.72 LogP (� 5), 2.37 mol/L LogS, 106.69 A2

Table 4. Chemo-informatics, Lipinski’s rule of 5 (RO5), and drug-likeness behavior of drugs 3(a–j).

Compound M.F M.Wt � 500 HBA � 10 HBD � 5 LogP � 5 LogS � 4(mol/ PSA � 140 Vol Drug likeness RO5
L) (A2) (A3) Score > 0

3a C15H15NO3 257.11 3 1 2.68 2.84 43.86 262.08 0.29 Yes

3b C19H21N3O4S2 419.10 6 2 2.15 2.76 87.93 361.85 0.09 Yes
3c C17H17N3O4S2 391.07 4 2 1.96 2.66 85.71 324.90 0.63 Yes
3d C17H29N3O4S2 403.16 6 2 2.54 2.58 87.80 380.30 0.07 Yes
3e C19H23N5O4S2 449.12 8 2 0.72 2.37 106.69 389.96 0.51 Yes
3f C15H15N5O4S2 393.06 6 2 0.20 2.06 104.56 315.50 0.12 Yes
3g C17H19N5O4S2 421.09 8 2 0.35 1.94 106.78 352.44 0.32 Yes
3h C29H25N3O4S2 543.13 4 2 5.79 6.12 85.16 476.63 0.27 No
3i C15H15N5O4S2 393.06 6 2 0.40 1.85 104.74 315.84 0.20 Yes
3j C7H13N3O4S2 267.03 6 2 0.93 1.62 86.71 212.92 0.29 Yes
Acetazolamide C4H6N4O3S2 221.99 7 3 0.01 1.95 97.42 167.21 0.11 Yes

M.F: Molecular Formula, M.Wt: Molecular Weight, HBA: H-Bond Acceptor, HBD: H-Bond Donor, LogP: Lippophilicity of partition coefficient, LogS:
Lippophilicity of water, PSA: Polar Surface Area, Vol: Volume, RO5: Lipinski’s Rule of 5.

Figure 2. Illustration of the docking pose of (a) reference, acetazolamide (b) surface and stick representation of 3 e and (c) ball and stick representation of 3 e in
the active site of 4Q6D. The inhibitor binds in the active site closer to the His94. Amino acid residues forming the binding pocket of the protein 4Q6D are
labeled in stick representation. Zn2 + ion is shown in grey and labelled.

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Figure 3. The effects of derivatives 3(a–j) on cell viability of HaCaT cells.

Figure 4. The anticancer activity of derivative 3(a–j) against MCF-7 cells.

+ 0.120 eV for 3 j. The frontier orbital energies predicted that

DFT Calculations
both 3 e and 3 j may be involved in any charge transfer process.
The DFT computed charge distribution in 3 e revealed that The isodensity of both HOMO and LUMO orbitals were
both –S atoms attached to exhibited electropositive behavior concentrated over terminal ethyl pyridiyl substituent moities in
but are surrounded tetrahedrally with two slightly 3 e Figure S2 ((a) & (c)) but the HOMO and LUMO were more
electronegative O, one electronegative C, and one suffi- concentrated over the pyrrole ring in 3 j. In the 3D optimized
ciently electronegative N atom which may shield structures of both molecules, there are regions of planarity and
electropositive S from interacting with negative charges of nonplannarity. Their planner regions can be involved in
DNA phosphate backbone Figure S1(a). A similar shield of intercalation while non-planner part may or may not be able to
electronegative atoms was present around electropositive S fit in the groove of DNA (Figure S3). Therefore, based on DFT
atoms in 3 j Figure S1(b). In the rest of the structures of both calculations, it can be predicted that if any or both of these
molecules, atoms are the least electropositive. So, the charge molecules would have interacted with DNA they will be doing
distribution predicts that there is the least or no chance of so through intercalation/groove binding/ mix mode of binding.
electrostatic interactions of any of the molecules with DNA.
The HOMO and LUMO of 3 e and 3 j are given in Figure S2.
The energy of HOMO of 3 e was 0.331 eV and that of LUMO
orbital was 0.106 eV. EHOMO was 0.378 eV and ELUMO was

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DNA Binding Investigations intercalation. The DNA binding was further investigated
through fluorescence spectroscopy where the addition of 3 e
The UV-Vis spectrum of DNA exhibited a single peak with λmax resulted in a slight decrease in fluorescence intensity of NR-
at 255 nm and absorbance 0.926. When 20 μM 3 e was added DNA at 256 nm. Combing the results from all three techniques
to the solution, the DNA peak was shifted towards higher λmax. the mix mode of binding (intercalation + groove binding) can
The incremental addition of 3 e resulted in more redshift with a be assigned to 3 e (Figure 5).
decrease in absorbance. The redshift with a decrease in The UV-Vis spectra of DNA exhibited a decrease in peak
absorption is an indicator of the intercalation mode of binding. absorption along with a redshift when 3 j was added
When the vice versa was performed by adding different incrementally. When DNA was added to 3 j and differential
concentrations of DNA to 3 e and recording differential spectra, spectra of 3 j were recorded, a red shift was observed with
the peak absorbance was found to decrease without any shift hypochromism. Such bathochromic shifts in UV-Vis spectra are
in λmax. The absorbance decrease without a shift in absorption labeled as intercalation. The cyclic voltammogram of 3 j was
maxima indicates groove binding. The DNA interaction was associated with a reversible peak (Epo/Epr = 0.185/0.139 V)
further evaluated through cyclic voltammetry where the coupled with irreversible oxidation peak at 0.743 V. the
titration of 3 e with DNA was recorded. The cyclic voltammo- addition of DNA resulted in a decrease in peak current and Ep
gram of DNA was associated with the reversible redox peak of oxidation peak at 0.743 showed a significant positive shift.
with Epo/Epr = 0.230/0.191 V coupled with an irreversible oxida- This complemented the intercalation of 3 j into DNA. The
tion peak at 0.730 V. The incremental addition of DNA to the intercalation was further confirmed through fluorescence
solution of 3 e increased peak current with a shift in Ep of analysis where 3 j was able to replace NR intercalated into DNA
oxidation peak at 0.230 V. thus electrochemical behaviors and hence significantly redesigning the fluorescence intensities
indicated mix mode of binding involving groove binding and of NR-DNA (Figure 6).

Figure 5. UV-Vis spectra of (a) DNA in variable concentrations of 3 e (b) 3 e (20 mM) in variable concentrations of DNA (c) cyclic voltammograms of 3 e in
variable concentrations of DNA at 0.1 V/s (d) fluorescence spectra of NR-DNA in variable concentration of 3 e in 50 % methanol-PBS (pH-7.4).

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Figure 6. Cyclic voltammograms of 3 j (20 mM) in 50 % methanol-PBS (pH-7.4) at 0.1 V/s vs. SCE with variable concentrations of DNA.

Therefore, combining UV-Vis spectroscopy, cyclic voltam-

Conclusion
metry, and fluorescence spectroscopy assisted by DFT predic-
tions, a mix mode of binding (intercalation + groove binding) A series of novel sulfonamide-based N-methylpyrrole deriva-
was suggested for 3 e while 3 j was able to intercalate into tives 3(a–j) with various hydrophilic and hydrophobic moieties
DNA. The binding strength was calculated from all four were synthesized in good yield (68-75 %) to find their role in CA
techniques is given in Table 5. The values of binding constants inhibition. All compounds exhibited carbonic anhydrase inhib-
are higher for 3 e as compared with 3 j. This reveals that 3 e can itory activity depending upon substitution pattern but ana-
bind more strongly with DNA than 3 j. This complements our logues 3 e bearing 2-pyridylethyl moiety and 3 j containing
IC50 values for their anti-cancerous activity against MCF-7 cell butyl substituent showed excellent carbonic anhydrase inhib-
lines where 3 e exhibited less IC50 than 3 j. ition with IC50 0.38 μM and 0.75 μM respectively better than
reference acetazolamide with IC50 0.99 μM in a noncompetitive
manner. Employing an in silico model of the protein-ligand
complex, the molecular origin of selectivity of compound 3 e
towards the hCA II isoform (PDB ID 4Q6D) was investigated and
identified the regions that likely contribute to the observed
selectivity of compound 3 e towards hCA II. The most important
Table 5. UV-Vis, cyclic voltammetry, and fluorescence spectroscopic studies
interaction was the π-π type association of the pyridine rings
of synthesized compounds 3(a–j) to intercalate into DNA.
of compound 3 e with the His94 and His4. The in vitro studies
a
Kb × 103 M 1
UV-Vis Spectroscopy Fluorescence Cyclic revealed that compounds 3 e and 3 j displayed a prominent
Compound DNA Spectroscopy voltammetry
variable variable
cytotoxic effect selectively toward MCF-7 cancer cells and did
not exhibit toxicity to the non-cancerous HaCaT cells at 125 μM
3e 9.84 9.80 6.30 9.7 concentration. The mechanism of anticancer potential was
3j 8.79 8.81 4.26 7.98
further investigated by DNA binding studies through electro-
[a] Kb was calculated from cyclic voltammetry using first oxidation peak. chemical methods. The UV-Vis spectroscopy, cyclic voltamme-

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ChemistrySelect doi.org/10.1002/slct.202101854

try, and fluorescence spectroscopy revealed that the 3 j [6] J.-Y. Winum, A. Maresca, F. Carta, A. Scozzafava, C. T. Supuran, Chem.
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