Isoleucine with secondary sulfonamide functionality as anticancer, antibacterial and antifungal agents

Article abstract of DOI:10.1080/07391102.2021.1893818

Isoleucine substituted analogues with secondary sulfonamide group (I₁–I₆) have been synthesized. Structures of synthesized analogues have been confirmed by Fourier Transform-Infrared Red, Nuclear Magnetic Resonance (¹H and ¹³C) and ESI-MS spectroscopic tools. Cytotoxic screenings of synthesized analogues have been done on MCF-7 (breast), Prostate Cancer-3 (PC-3) and A549 (lung) cancer cell lines. N-(1-isobutyl-2-oxo-2-anilinoethyl) p-toluene sulfonamide (I₅) screened to be better cytotoxic agent on MCF-7 and A549 cell lines whereas N-(1-isobutyl-2-oxo-2-p-chloroanilino ethyl) benzene sulfonamide (I₃) against PC-3 cell line. Cell cycle analysis of N-(1-isobutyl-2-oxo-2-anilinoethyl) p-toluene sulfonamide (I₅) analogue has been carried out on A549 cell line in comparison to control and Vinblastine (standard drug). Complete arrest in G0 and G1 phase along with mild disturbance in S-phase of cell cycle has been observed. The screened analogues (I₁–I₆) also showed good antifungal and antibacterial potential against gram positive as well as gram negative strains. Computer simulation indicated good bioactivity prediction by the ‘Lipinski rule’ and synthesized analogues did not violate this rule. Docking study of isoleucine sulfonamide analogues (I₁–I₆) were carried out to determine the possible interaction sites of the analogues with p53 tumor suppressor-DNA complex and demonstrate that the analogues confirmed binding and inhibition with the most mutated residues of p53. Density functional theory has been used to correlate the electronic and chemical properties of analogues and they were found to be stable and chemically reactive. Thus the results suggest that isoleucine substituted sulfonamide analogues can serve as a structural model for the design of anticancer agents, antibacterial agents as well as antifungal agents with better inhibitory potential. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2021.1893818

Journal of Biomolecular Structure and Dynamics
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Isoleucine with secondary sulfonamide
functionality as anticancer, antibacterial and
antifungal agents
Kirna Devi & Pamita Awasthi
To cite this article: Kirna Devi & Pamita Awasthi (2021): Isoleucine with secondary sulfonamide
functionality as anticancer, antibacterial and antifungal agents, Journal of Biomolecular Structure
and Dynamics, DOI: 10.1080/07391102.2021.1893818
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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2021.1893818
Isoleucine with secondary sulfonamide functionality as anticancer, antibacterial
and antifungal agents
Kirna Devi and Pamita Awasthi
Department of Chemistry, National Institute of Technology, Hamirpur, Himachal Pradesh, India
Communicated by Ramaswamy H. Sarma
ABSTRACT
ARTICLE HISTORY
Isoleucine substituted analogues with secondary sulfonamide group (I
1 6
–I ) have been synthesized.
Received 23 November 2020
Accepted 17 February 2021
Structures of synthesized analogues have been confirmed by Fourier Transform-Infrared Red, Nuclear
1
13
Magnetic Resonance ( H and C) and ESI-MS spectroscopic tools. Cytotoxic screenings of synthesized
analogues have been done on MCF-7 (breast), Prostate Cancer-3 (PC-3) and A549 (lung) cancer cell
KEYWORDS
Antibacterial and antifungal;
bioactivity; cytotoxicity;
density functional theory;
FT-IR; isoleucine amino acid;
Lipinski rule; molecular
docking; sulfonamide
lines. N-(1-isobutyl-2-oxo-2-anilinoethyl) p-toluene sulfonamide (I ) screened to be better cytotoxic
5
agent on MCF-7 and A549 cell lines whereas N-(1-isobutyl-2-oxo-2-p-chloroanilino ethyl) benzene sul-
fonamide (I ) against PC-3 cell line. Cell cycle analysis of N-(1-isobutyl-2-oxo-2-anilinoethyl) p-toluene
3
5
sulfonamide (I ) analogue has been carried out on A549 cell line in comparison to control and
Vinblastine (standard drug). Complete arrest in G0 and G1 phase along with mild disturbance in
S-phase of cell cycle has been observed. The screened analogues (I –I ) also showed good antifungal
1
6
and antibacterial potential against gram positive as well as gram negative strains. Computer simulation
indicated good bioactivity prediction by the ‘Lipinski rule’ and synthesized analogues did not violate
this rule. Docking study of isoleucine sulfonamide analogues (I –I ) were carried out to determine the
1
6
possible interaction sites of the analogues with p53 tumor suppressor-DNA complex and demonstrate
that the analogues confirmed binding and inhibition with the most mutated residues of p53. Density
functional theory has been used to correlate the electronic and chemical properties of analogues and
they were found to be stable and chemically reactive. Thus the results suggest that isoleucine substi-
tuted sulfonamide analogues can serve as a structural model for the design of anticancer agents, anti-
bacterial agents as well as antifungal agents with better inhibitory potential.
1
. Introduction
0.2–2lmol/L with 50% inhibition (Byth et al., 2009).
Imidazo[1,2-a]pyridine derivatives showed tremendous CDK-2
inhibition activity (Babu et al., 2007; Byth et al., 2004). The 4-[2-
Amino-3-cyano-4-(p-tolyl)-5, 6, 7, 8-tetrahydroquinolin-1(4H)-yl]
benzene sulfonamide and 4-[2-Amino-3-cyano-4-(4-hydroxy-
phenyl)-5,6,7,8-tetrahydro-quinolin-1(4H)-yl]benzene sulfonamide
showed anticancer activity at par to standard drug Doxorubicin
The secondary sulfonamides (-SO NH-) is an important structural
2
feature in various biologically active compounds which display
diverse biological activities like-antibacterial (Drews, 2000) anticar-
bonic anhydrase (Supuran & Scozzafava, 2000, 2001) antithyroid
(
Thornber, 1979) and anticancer (Abbate et al., 2004; Ghorab
et al., 2006; Ismail et al., 2006; Rostom, 2006; Supuran et al.,
004). As per literature study, aryl sulfonamide disturbs the cell-
(Al-Said et al., 2010). The derivatives of alkyl/aryl sulfonyl-N, N-
2
diethyl-dithiocarbamates had been successfully evaluated for
anticancer activity and further clinically studied for inhibiting
HIV viral protease (Casini et al., 2002). The phenothiazine deriva-
tives like (Z)-4-(3-Oxo-3-(10H-phenothiazine-2-yl)prop-1-ethyla-
mino)-N-(thiazol-2-yl) benzene sulfonamide and (Z)-4-(3-Oxo-3-
cycle by carrying major damage in G1 phase of cell cycle by
inhibiting carbonic anhydrase activity (Cecchi et al., 2004; Guzel
et al., 2008; Kivela et al., 2005; Supuran, 2008; Supuran et al.,
2003; Supuran & Scozzafava, 2007).
Large number of aromatic and heterocyclic compounds hav-
(10H-phenothiazine-2yl)prop-1-ethylamino)-N-(pyridin-2-yl) ben-
ing -SO NH- feature possess a carbonic anhydrase inhibitory
2
zene sulfonamide showed better anticancer activity at 8.8 lM
concentration than doxorubicin at 9.8lM on T47D (breast can-
property. Among this class of compounds-acetazolamide, meth-
azolamide, ethoxzolamide, dichlorophenamide, etc. have been
used for the last 40years as systemic drugs (Supuran, 1994; cer cell line). Further, cell-cycle analysis of compound (Z)-4-(3-
Supuran & Scozzafava, 2000, 2001). Major side effects associated Oxo-3-(10H-phenothiazine-2-yl) prop-1-enylamino)-N-(thiazol-2-
are fatigue, anorexia paresthesia, etc. and this is due to car- yl)benzene sulfonamide has shown to inhibit G2/M phase and
bonic anhydrase inhibition of non-target cells as well. ADMET study indicated better human intestinal absorption
Compound AZD5438 (Figure 1(e)) showed antiproliferative (Ghorab et al, 2017). Halogenated benzene sulfonamide com-
activity by blocking cell-cycle at G2, M, S and G1 phase at pounds proposed to inhibit HeLa, MDR phenotype of MCF-7/
CONTACT Pamita Awasthi
pamitawasthi@gmail.com
Department of Chemistry, National Institute of Technology, Hamirpur, Himachal Pradesh, India
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2021.1893818.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
K. DEVI AND P. AWASTHI
Figure 1. Structure of the some reported potential sulfonamide derivatives Sulfachloropyridazine (a), bis-arylsulfonamide (b), N-benzoyl-N-(5-bromo/nitropyridin-2-
yl) sulfonamide (c), chloroquinoline sulfonamide (CQS) (d), AZD5438 (e), E7070 compound (f), mitoxantrone (g), basic structures of previous synthesized phenylalan-
ine sulfonamide analogues (K1–K12) [Devi & Awasthi, 2019].
ADR human cancer cell lines (Medina et al., 1999). Number of substituents enhances the antibacterial activity (Basanagouda
quinolone derivatives with sulfonamide group has been pro- et al., 2010). Substituents position along with halogen groups
posed to show excellent anticancer activity whereas pyridine increases the antibacterial activity (Aki-Sener, Bingol, Arpaci,
derivatives show high to weak inhibitory property (Al-Said Yalcin & Altanlar, 2002). The longest carbon chain sulfonamide
et al., 2010; Deininger et al., 2005; Gorre et al., 2001). The diol- molecules are proposed to exhibit greater antifungal activity
containing compounds like 2-(f4-[4-(Acridin-9-ylamino) phenyl- whereas lesser antibacterial activity (Ozbek et al., 2007). The
thio] phenylg (2-hydroxyethyl) amino) ethan-1-ol hydrochloride furan-3-carboxamide analogues containing sulfonamide feature
were most cytotoxic on Chinese hamster lung fibroblasts cells. and benzamidine, hydrazines and imidazoles showed little bio-
Cytotoxic screening against P388 leukemia demonstrated that logical activity (Zanatta et al., 2007). Further incorporation of tri-
same compound to be highly biologically active included azole rings on sulfanilamide resulted into significant increase in
Topoisomerase II inhibition (Chen et al., 2005). Series of N-(p- antibacterial activity (Wang et al., 2010). Further incorporation
Methoxyphenylidene)-imino-4-[(pyridin-2-ylamino) sulfonyl] sul- of -SO -NH feature on azithromycin lower’s the activity of azith-
2
fonamide Schiff’s bases were synthesized and reported to show romycin against some bacterial strains. However, they are
antitumor activity on MCF-7 and HeLa cell lines (Kamel et al., inactive against gram negative, H. influenza and E. coli strains
2010). As per literature study, compounds with sulfonamide fea- (Krajacic et al., 2007).
ture are proposed to arrest G0/G1 phase of cell cycle. DNA syn-
thesis, cyclin-dependent kinase activity along with p53, p21 have incorporated the aromatic ring at the center i.e. R
In our previous reported work (Devi & Awasthi, 2019), we
0
0
activities are the major activities of G0/G1 phase of cell cycle. (Figure 2) along with secondary sulfonamide group and syn-
Mutations at p53 are the cause of cancer and hence the path- thesized series exhibited good anticancer activity on tested
way by which p53 initiate apoptosis of cells is the common tar- cancer cell lines. Some of the compounds having p-chloro-
get of sulfonamide drugs.
substituted aromatic ring did not show any antibacterial
Sulfamethaxazole along with trimethoprim has been very activity but they were showing better antifungal activities. In
extensively used in the treatment of urinary tract infection the present work, we have replaced central aromatic ring
and pneumonia (Brun-Pascaud et al., 1996; Wininger & Fass, with aliphatic side chain i.e. phenylalanine replaced by iso-
0
000
2
002). Number of derivatives of oxa-chromene-sulfonamide leucine while keep R and R same (Figure 2).
showed minimum inhibitory concentration at par to cipro- A series of new secondary sulfonamide feature bearing
floxacin against some of gram positive and gram negative analogues (I –I ) isoleucine amino acid and substituted/
1
6
bacteria except E. coli. Nitro, chloro, bromo and methoxy unsubstituted aromatic aniline groups i.e. (N-(1-isobutyl-2-

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
3
solvent system. Mass spectra were recorded using electron
spray ionization technique on Waters Micromas Q-TOF
micro instrument.
2
.1. Synthesis of isoleucine substituted sulfonamide
analogues (I –I )
1
6
The synthetic path for the synthesis of N-(1-isobutyl-2-oxo-
substituted-ethyl)benzene/p-toluene sulfonamide (I –I ) ana-
1
6
logues have been shown in Scheme 1. 2-benzenesulfonyla-
mino-3-methyl-pentanoic acid was synthesized by treating
Figure 2. Structures of present synthesized isoleucine sulfonamide ana- isoleucine amino acid (0.1 mol) and benzene sulfonyl chloride
1 6
logues (I –I ).
(
0.1 mol) in NaOH solution (1 N, 200 mL). Mixture was stirred
ꢀ
together at 65–70 C for 4 h. A clear solution was obtained
and cooled to 5 C and treated with concentrated HCl to
make it slightly acidic (pH 6.5–7.0). The I and I acids were
oxo-2-substituted-ethyl) benzene/p-toluene sulfonamide)
have been synthesized (Figure 2). The structures of all syn-
thesized sulfonamide analogues have been studied and con-
ꢀ
1
4
separated out as white crystals. Further I1 and I4 acids
0.004 mol) were dissolved in dry benzene and freshly dis-
1
firmed using spectroscopic techniques i.e. FT-IR, NMR ( H,
(
1
3
C) and ESI-MS spectroscopic tools. Molinspiration software
tilled thionyl chloride (5.0 mL). The mixture was stirred at
room temperature for 20 min followed by refluxing for 3 h.
The solvent was distilled off to give acid chloride of I and I .
The reaction mixture was used for next reaction without
purification. Then aniline and p-chloroaniline (0.004 mol) was
added dropwise to this reaction mixture with constant stir-
ring at 0–5 C and stirred for 6 h at 60–65 C. Reaction com-
pletion was monitored on TLC. Reaction mixture was poured
on ice–water mixture and extracted with chloroform. The
extract was washed with H O. The solvent was distilled off
leaving a solid residue which was recrystallized from chloro-
form to give I –I analogues (Awasthi & Devi, 2018; Devi &
Awasthi, 2019; Mahajan et al., 2002).
calculation indicates the drug like action of synthesized ana-
logues (I –I ). Cytotoxicity of synthesized analogues has been
1
6
1
4
studied against A549, MCF-7 and PC-3 cancer cell lines using
SRB assay. Cell cycle analysis of the best screened out ana-
logue i.e. I on A549 cell line indicates the maximum disturb-
5
ance at G0/G1 phase. The in vitro antibacterial and antifungal
activity of synthesized series has been tested on gram posi-
tive (S. aureus) and gram negative (P. aeruginosa, E. coli) bac-
terial as well as fungal strains (F. moniliforme, A. fumigatus
and C. albicans) using agar well diffusion method. Based on
literature survey and cell cycle study it has been observed
that maximum damage is caused at G0/G1 phase of cell
cycle. Further inhibition of p53 tumor suppressor-DNA com-
ꢀ
ꢀ
2
2
6
plex with -SO -NH feature containing organic compounds
2
have also been proposed in literature. DNA synthesis, cyclin-
dependent kinase activity along with p53, p21 activities are
the major activities of G0/G1 phase of cell cycle. Mutations
at p53 are the cause of cancer and hence the pathway by
which p53 initiate apoptosis of cells is the common target of
sulfonamide drugs. Docking study of all synthesized ana-
logues (I –I ) with p53 tumor suppressor-DNA complex have
2
.1.1. Spectroscopic characterizations
The structures of synthesized isoleucine sulfonamide analogues
1
13
(
I –I ) were solved using spectroscopic techniques i.e. H,
C
1
6
NMR, FT-IR and ESI-MS and the spectra were shown in supple-
mentary material (Supporting information Figures S1(a–f) H-
NMR, S2(a–f) C-NMR, S3(a–f) FT-IR and S4(a–f) ESI-MS).
1
13
1
6
been carried out to check the in silico interaction of the ana-
logues with p53 tumor suppressor-DNA complex. Gaussian
study has been carried out to understand the electronic
behavior and chemical reactivity of the analogues.
2
.1.2. 2-Benzenesulfonylamino-3-methyl-pentanoic acid (I1)
Mol. Wt. 270, Yield 78%, R ¼ 0.45 (benzene: methanol 8:2),
f
ꢀ
ꢁ1
White powder, m.p. 145 C. IR (t cm ): 3548.3 (mOꢁH), 3298.8
mNꢁH), 2967.72, 2886.6 (mCꢁH), 1695.73 (mC¼O), 1617.20,
(
1
1
0
479.26, 1450.3 (mC¼C Ar), 1416.6 (mCꢁH bending), 1163.6,
2
. Materials and methods
1
336.8 (mO¼S¼O), 1091.3 (mCꢁO), 752 (mNꢁH); H-NMR (d CDCl ):
3
.77–0.81 (t, 3H, CH -CH , J ¼ 7.32 Hz) , 0.83–0.85 (d, 3H,
Chemicals used for synthesis were of AR grade (Sigma-
Aldrich). Solvents were distilled before application. Digital
melting/boiling point apparatus was used for the measure-
3
2
-
CH -CH, J ¼ 6.84 Hz) , 1.05–1.41 (m, 2H, -CH -CH ), 1.74–1.80
3
2
3
(m, 1H, -CH-CH ), 3.76–3.80 (d, 1H, -CH-NH, J ¼ 5.12 Hz),
3
ꢀ
5
.90–5.93 (d, 1H, CH-NH-SO , J ¼ 9.36 Hz), 7.80–7.84 (d, Ar-H,
ment of melting points of synthesized analogues ( C uncor-
2
rected). Silica gel plates were used for TLC with benzene and 2H, ortho to SO
methanol (8:2) solvent system at room temperature and spot 2H, meta to SO
were visualized by UV light/or in iodine chamber. NICOLET 1H, para to SO
700 of Thermo scientific USA instrument was used for FT-IR 175.62 (C ¼ O), 139.52 (Ar), 132.99 (Ar), 129.06 (Ar), 128.46
NH group, J ¼ 8.76 Hz), 7.43–7.47 (dd, Ar-H,
2
NH group, J ¼ 7.84 Hz), 7.53–7.55 (dd, Ar-H,
2
13
NH group, J ¼ 5.32 Hz); C NMR (d CDCl
):
3
2
6
ꢁ
1
1
13
spectra using KBr pellets (500–4000 cm ). H and C NMR (Ar), 127.17 (Ar), 125.91 (Ar), 60.20 (-CH-C ¼ O), 37.91 (-CH-
spectra were recorded on Bruker Avance-II 400 NMR spectra CH
), 24.43 (-CH
), 15.35 (-CH
), 11.24 (-CH
); ESI-MS (m/z):
3
2
3
3
using TMS as an internal standard and (CD ) SO-d6 & CDCl ) 271 ¼ [M þ H] , 294 ¼ [M þ Na] , 609 ¼ [2M þ Na þ K] þ
þ
þ
.
3
2
3

4
K. DEVI AND P. AWASTHI
1 6
Scheme 1. Synthetic path for the synthesis of N-(1-isobutyl-2-oxo-2-substituted-ethyl) benzene/p-toluene sulfonamide (I –I ) analogues.
2
.1.3. N-(1-isobutyl-2-oxo-2-anilino-ethyl) benzene sulfonamide (I2) ortho to SO
2
NH group, J¼ 1.52 Hz), 7.59 (s, 1H, NH of aniline);
13
Mol. Wt. 346, Yield 72%, R ¼ 0.51 (benzene: methanol 8:2),
C-NMR (d CDCl ): 168.52 (C ¼ O), 138.98 (Ar), 136.72 (Ar),
f
3
ꢀ
ꢁ1
Light yellow crystals, m.p. 199 C. IR (t cm ): 3302.3 (mNꢁH of 133.16 (Ar), 129.19 (Ar), 128.91 (Ar), 127.26 (Ar), 124.85 (Ar),
aniline), 3249.6 (mNꢁH), 2965.43, 2874.8 (mCꢁH), 1654.98 (mC¼O), 120.13 (Ar), 62.33 (-CH-C ¼ O), 37.79 (-CH-CH ), 24.34 (-CH ),
3
2
þ
1
600.08, 1531.7, 1494.07, 1447.8 (mC¼C Ar), 1403.8 (mCꢁH bend- 15.44 (-CH ), 11.31 (-CH ); ESI-MS (m/z): 347 ¼ [M þ H] ,
3
3
1
þ
þ
ing), 1156.3, 1328.9 (mO¼S¼O), 1092.0 (mCꢁO), 749.2 (mNꢁH); H- 369 ¼ [M þ Na] , 715¼ [2 M þ Na]
NMR (d CDCl ): 0.73–0.76 (t, 3H, CH -CH , J¼ 6.88 Hz), 0.77–0.80
.
3
3
2
(
1
d, 3H, CH -CH, J¼ 7.4 Hz), 0.99–1.51 (m, 2H, -CH -CH ),
3
2
3
.84–1.89 (m, 1H, -CH-CH ), 3.56–3.59 (d, 1H, -CH-NH, 2.1.4. N-(1-isobutyl-2-oxo-2-p-chloroanilino-ethyl) benzene
3
J¼ 5.4 Hz), 5.10–5.12 (d, 1H, CH-NH-SO , J¼ 7.8 Hz), 7.02–7.06
sulfonamide (I3)
d, Ar-H, 2H, para to NH of aniline, J¼ 7.04 Hz), 7.19–7.27 (m, Mol. Wt. 380, Yield 78%, R
Ar-H, 4H, ortho and meta to NH of aniline), 7.38–7.48 (m, Ar-H, Light brown crystals, m.p. 204 C. IR (t cm ): 3301.4 (mNꢁH of
H, meta and para to SO NH group), 7.79–7.81 (d, Ar-H, 2H, aniline), 3253.5 (mNꢁH), 2976.02, 2877.9 (mCꢁH), 1656.9 (mC¼O),
2
(
f
¼ 0.48 (benzene: methanol 8:2),
ꢁ1
ꢀ
3
2

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
5
Table 1. TGI, LC50 and GI50 value of isoleucine sulfonamide analogues (I
1
–I
6
).
CH -CH , J ¼ 11.04 Hz), 0.84–0.87 (d, 3H, CH -CH,
3
2
3
PC-3
A549
MCF-7
J ¼ 13.96 Hz), 1.49–1.60 (m, 2H, -CH -CH ), 1.89–1.95, (m, 1H,
2
3
-
CH-CH ) 2.30 (s, 3H, Ar-CH ), 3.60–3.64 (d, 1H, -CH-NH,
3 , 3
S. No.
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
J ¼ 5.64 Hz), 5.15–5.17 (d, 1H, CH-NH-SO2, J ¼ 7.9 Hz),
MTX
ADR
2E-03 0.002 3E-05 2E-06 0.008 7E-10 1E-06 4E-04 2E-09
7E-06 0.001 4E-08
0.0
NE
NE
NE
NE
NE
NE
2.0
NE
NE
NE
5E-07 1E-04 0.015 1E-06 7.08–7.12 (d, Ar-H, 2H, para to NH of aniline, J ¼ 6.84 Hz),
3.2E-4 0.001 0.012 3E-04
3.6E-4 2E-03 0.024 0.002
I
1
I
2
I
3
I
4
I
5
I
6
NE
0.07
0.002 0.132
0.06 0.02
1.4E-3
NE
0.132 2E-01
7
7
.29–7.30 (d, Ar-H, 2H, meta to NH of aniline, J ¼ 2.64 Hz),
.31–7.32 (d, Ar-H, 2H, ortho to NH of aniline, J ¼ 2.04 Hz),
0.002 0.003 0.005
NE
NE
NE
NE
NE
NE
11.12
71.31
65.86
NE 2.6E-4 0.003 0.272 4E-05 7.734–7.754 (d, Ar-H, 2H, meta to SO NH group, J ¼ 6.96 Hz),
2
NE
NE
2.5E-4 1E-03 0.034 3.028
7
7
1
.73–7.75 (d, Ar-H, 2H, ortho to SO NH group, J ¼ 8.28 Hz),
2
3.9E-4
NE
NE
7E-05
13
.67 (s, 1H, NH of aniline); C-NMR (d CDCl ): 168.53 (C ¼ O),
3
NE ¼ Nonevaluable data, TGI ¼ concentration of drug causing total inhibition
of cell growth, LC50 ¼ concentration of drug causing 50% cell kill, GI50 ¼ con-
44.19 (Ar), 136.79 (Ar), 135.88 (Ar), 129.78 (Ar), 128.87 (Ar),
centration of drug causing 50% inhibition of cell growth, MTX ¼ Mitoxantrone, 127.36 (Ar), 124.77 (Ar), 120.00 (Ar), 62.36 (-CH-C ¼ O), 37.72
ADR ¼ Adriamycin.
(
(
7
-CH-CH ), 24.38 (-CH ), 21.44 (-CH ), 15.48 (-CH ), 11.31
3 2 3 3
þ
þ
-CH3); ESI-MS (m/z): 361 ¼ [M þ H] , 383 ¼ [M þ Na]
,
1
596.54, 1533.76, 1492.86, 1449.7 (mC¼C Ar), 1400.4 (mCꢁH
þ
43 ¼ [2M þ Na]
.
bending), 1156.9, 1331.0 (mO¼S¼O), 1093.4 (mCꢁO), 771(mNꢁH);
1
H-NMR (d CDCl ): 0.69–0.71 (t, 3H, CH -CH , J ¼ 6.84 Hz),
3
3
2
0
.75–0.79 (d, 3H, CH -CH, J ¼ 7.32 Hz), 0.97–1.47 (m, 2H, -CH - 2.1.7. N-(1-isobutyl-2-oxo-2-p-chloroanilino-ethyl) p-tolu-
3
2
CH ), 1.83–1.89 (m, 1H, -CH-CH ), 3.57–3.60 (d, 1H, -CH-NH,
ene sulfonamide (I
)
6
3
3
Mol. Wt. 394, Yield 78%, R ¼ 0.50 (benzene: methanol 8:2),
J ¼ 5.44 Hz), 5.11–5.13 (d, 1H, CH-NH-SO2, J ¼ 7.68 Hz),
f
ꢀ
ꢁ1
light brown crystals, m.p. ¼ 223 C, IR (t cm ): 3303.4
7
7
7
7
7
7
1
1
.16–7.19 (d, Ar-H, 2H, ortho to NH of aniline, J ¼ 4.36 Hz),
(
1
mNꢁH), 3247.3 (mNꢁH), 2964.2, 2876.1 (mCꢁH), 1659.51 (mC¼O),
.39–7.42 (d, Ar-H, 2H, meta to NH of aniline, J ¼ 6.44 Hz),
595.01, 1529.15, 1493.1, 1446.2 (mC¼C Ar), 1400.6 (mCꢁH bend-
.22–7.25 (d, Ar-H, 2H, meta to SO NH group, J ¼ 2.08 Hz)
2
ing), 1160.0, 1325.7 (mO¼S¼O), 1090.98 (mCꢁO), 818.4 (p-subs. of
ring); H-NMR (d CDCl3): 0.69–0.71 (t, 3H, CH -CH ,
.46–7.49 (d, Ar-H, 1H, para to SO NH group, J ¼ 9.52 Hz),
2
1
.79–7.81 (d, Ar-H, 2H, ortho to SO NH group, J ¼ 5.24 Hz),
3
2
2
1
3
J ¼ 6.84 Hz), 0.76–0.79 (d, 3H, CH -CH, J ¼ 7.36 Hz), 1.00–1.45
.88 (s, 1H, NH of aniline); C-NMR (d CDCl ): 168.68 (C ¼ O),
3
3
(
m, 2H, -CH -CH ), 1.86–1.88 (m, 1H, -CH-CH ), 2.27 (s, 3H, Ar-
39.08 (Ar), 136.86 (Ar), 133.66 (Ar), 129.29 (Ar), 128.98 (Ar),
2 3 3
CH ), 3.52–3.56 (d, 1H, -CH-NH, J ¼ 5.4 Hz), 4.98–5.00 (d, 1H,
27.36 (Ar), 124.95 (Ar), 120.73 (Ar), 62.39 (-CH-C ¼ O), 37.384
3
CH-NH-SO , J ¼ 7.56 Hz), 7.23–7.24 (d, Ar-H, 2H, meta to NH
(
-CH-CH ), 24.64 (-CH ), 15.52 (-CH ), 11.36 (-CH ); ESI-MS (m/
2
3
2
3
þ
3
þ
þ
of aniline, J ¼ 4.68 Hz), 7.19–7.22 (d, Ar-H, 2H, ortho to NH of
z): 381 ¼ [M þ H] , 403 ¼ [M þ Na] , 783 ¼ [2M þ Na]
.
aniline, J ¼ 2.28 Hz), 7.17–7.18 (d, Ar-H, 2H, meta to SO NH
2
group, J ¼ 4.0 Hz), 7.66–7.68 (d, Ar-H, 2H, ortho to SO NH
2
1
3
2
.1.5. 3-Methyl-2-p-tolylsulfonylaminopentanoic acid (I4)
group, J ¼ 8.28 Hz), 7.74 (s, 1H, NH of aniline); C-NMR (d
Mol. Wt. 286, Yield 74%, R ¼ 0.48 (benzene: methanol 8:2),
f
CDCl ): 168.69 (C ¼ O), 144.34 (Ar), 135.72 (Ar), 135.45 (Ar),
3
ꢀ
ꢁ1
White powder, m.p. 160 C, IR (t cm ): 3568.5 (mOꢁH), 3300.5 129.83 (Ar), 129.75 (Ar), 128.88 (Ar), 127.38 (Ar), 121.24 (Ar),
(
mNꢁH), 2968.32, 2886.6 (mCꢁH), 1703.09 (mC¼O), 1922.1 (over- 62.33 (-CH-C ¼ O), 37.54 (-CH-CH ), 24.36 (-CH ), 21.49 (-CH ),
3
2
3
þ
tone bands), 1599.2, 1480.25, 1492.86, 1455.6 (mC¼C Ar), 15.48 (-CH ), 11.31 (-CH ); ESI-MS (m/z): 395 ¼ [M þ H] ,
3
3
þ
þ
1
421.0 (mCꢁH bending), 1135.86, 1331.2 (mO¼S¼O), 1092.4 417 ¼ [M þ Na] , 811 ¼ [2M þ Na]
.
1
(
3
mCꢁO), 808.0 (p-subs. of ring); H-NMR (d CDCl ): 0.77–0.81 (t,
H, CH -CH , J ¼ 7.4 Hz), 0.81–0.85 (d, 3H, CH -CH,
3
3
2
3
2
.2. Anticancer activity
J ¼ 6.84 Hz), 1.03–1.35 (m, 2H, -CH -CH ), 1.71–1.78 (m, 1H,
2
3
-
CH-CH ), 3.74–3.77 (d, 1H, -CH-NH, J ¼ 4.8 Hz), 2.34 (s, 3H,
3
2.2.1. Sulforhodamine B (SRB) assay
SRB assay has been carried out as per standard protocol.
Ar-CH ), 5.08–5.11 (d, 1H, CH-NH-SO , J ¼ 9.72 Hz), 7.19–7.21
3
2
(
d, Ar-H, 2H, meta to SO NH group, J ¼ 8.16 Hz), 7.63–7.65 (d,
2
Cells were inoculated in microtiter plates with different con-
13
ꢁ
7
ꢁ6
ꢁ5
ꢁ4
Ar-H, 2H, ortho to SO NH group, J ¼ 8.32 Hz); C-NMR (d
2
centrations i.e. 10 , 10 , 10 and 10 mg/mL of drugs
CDCl ): 176.30 (C ¼ O), 143.84 (Ar), 136.47 (Ar), 129.59 (Ar), molecules followed by 48 h incubation under standard condi-
3
1
2
2
27.19 (Ar), 59.86 (-CH-C ¼ O), 38.11 (-CH-CH ), 24.40 (-CH ), tions. SRB solution was added to each well after discarding
3
2
1.50 (-CH ), 15.33 (-CH ), 11.29 (-CH ); ESI-MS (m/z): supernatant followed by five times washing of plates and
3
3
3
þ
þ
þ
86 ¼ [M þ H] , 308 ¼ [M þ Na] , 593 ¼ [2M þ Na]
.
then the plates were incubated for 20 min. About 1% acetic
acid was used for the removal of residual dye and absorb-
ance was checked at 540 and 690 nm wavelength.
Percentage growth inhibition of drugs was calculated as: [Ti/
C] ꢂ 100% (Devi & Awasthi, 2019; Skehan et al, 1990; Vichai &
Kirtikara, 2006).
2
.1.6. N-(1-isobutyl-2-oxo-2-anilino-ethyl) p-toluene sul-
fonamide (I5)
Mol. Wt. 360, Yield 67%, R ¼ 0.54 (benzene: methanol 8:2),
f
ꢀ
ꢁ1
off White crystals, m.p. 225 C, IR (t cm ): 3253.2 (mNꢁH),
969.55, 2881.3 (mCꢁH), 1950.19 (overtone bands), 1652.23
2
(
mC¼O), 1599.42, 1536.67, 1493.80, 1438.0 (mC¼C Ar), 1400.9 2.2.2. Cell cycle analysis
(
8
mCꢁH bending), 1157.68, 1328.4 (mO¼S¼O), 1093.6 (mCꢁO), Cell cycle analysis has been carried out as per standard
1
18.8 (p-subs. of ring); H-NMR (d CDCl ): 0.80–0.83 (t, 3H, protocol using Dulbecco’s Modified Eagle Medium for the
3

6
K. DEVI AND P. AWASTHI
1 6
Figure 3. Percent control growth of isoleucine sulfonamide analogues (I –I ) against human cancer cell lines MCF-7 (a), A549 (b) and PC-3 (c).
1 6
Figure 4. Morphological changes of A549 cells treated with isoleucine sulfonamide analogues (I –I ) with magnification of 100 lm.
ꢀ
ꢀ
growth of cells. Plates were incubated at 37 C for 12–24 h. at 4 C. Further, propidium iodide was added and plates
Phosphate buffer solution was used to wash the plates and were kept for 30 min. IDEA software was used for calculations
then cell plates were resuspended in ethanol solution for 2 h (Devi & Awasthi, 2019).

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
7
1 6
Figure 5. Morphological changes of MCF-7 cells treated with isoleucine sulfonamide analogues (I –I ) with magnification of 100 lm.
2.3. Antibacterial and antifungal assay
to formulate frontier molecular orbitals of analogues (I
–I ) (Devi
1 6
&
Awasthi, 2019). The optimized frontier molecular orbitals of
In vitro antibacterial and antifungal testing of synthesized ana-
logues (I –I ) have been done against gram positive (S. aureus)
and gram negative (P. aeruginosa, E. coli) bacterial as well as
and fungal (F. moniliforme, A. fumigatus and C. albicans) strains
analogues were estimated by Density Functional Theory methods
at B3LYP/6-311þþ(2d,p) level of theory. This method gives bet-
ter results in comparison to Hartree-Fock and Ab-initio methods.
The reactivity descriptors of synthesized isoleucine sulfonamide
analogues (I –I ) were calculated (Raj et al., 2015).
1
6
(Devi & Awasthi, 2019). The Mueller-Hinton agar (MHA) media
1
6
has been used in present study (Barry et al., 1979).
2
.6. In silico bioactivity study
2.4. Docking study
Molinspiration software (www.molinspiration.com) was used for
Autodock 4.2 has been used for docking study of synthesized predictions of drug like behavior of analogues (I –I ) (Devi &
analogues (I –I ) against p53 tumor suppressor-DNA complex Awasthi, 2019). The physical descriptors like miLogP, TPSA,
1
6
1
6
(
1TUP) (Devi & Awasthi, 2019; Eichhorn et al., 2013). Pymol soft- natoms, MW, nON, nOHNH, nviolations, Nrotb were calculated
ware was used to draw the structures of analogues (www. (Lipinski et al., 2012). Molecular volume and total polar surface
pymol.com). Lamarckian Genetic Algorithm protocol was area of the analogues were also estimated (Clark, 1999; Ertl
applied for flexibility (Jenwitheesuk & Samudrala, 2003; Morris et al., 2000; Lalitha & Sivakamasundari, 2010). Further numbers
et al., 1998). Lowest binding energy (DGb) was applied for of rotatable bonds along with other properties were calculated
orientation of final docking (Devi
&
Awasthi, 2019; (Lagunin et al., 2000; Parvez et al., 2010; Veber et al., 2002).
Jenwitheesuk & Samudrala, 2003; Morris et al., 1998).
3. Results and discussion
2.5. Quantum chemical calculation
3.1. Anticancer activity
Gaussian 98 W and Gauss view 05 software tools have been Variation in in vitro cytotoxic activity of synthesized series
used for calculations. Density Functional Theory has been applied (I –I ) has been observed against MCF-7, PC-3 and A549
1
6

8
K. DEVI AND P. AWASTHI
5
Figure 6. Cell-cycles analysis of A549 cells treated with different concentrations of control, vinblastine and I analogue on flow cytometer by staining with propi-
dium iodide.
cancer cell lines. I and I screened out to be potential candi- However, reference drugs Adriamycin and Mitoxantrone have
3
5
dates (Table 1) amongst all the synthesized analogues. shown cytotoxic effect at lower concentrations (Figure 3).

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
9
5
Figure 7. Morphological changes in the cell cycle analysis of A549 cells treated with (a) control, (b) I analogue, and (c) vinblastine with magnification of 25 lm.
Structurally I and I having common central aliphatic car- -SO NH- group have been proposed to be effective than
1
4
2
0
00
bon chain but differ in R and R ’ para substitution (Figure meta substitution. Further comparative study of some syn-
0
000
2
). Incorporation of p-CH group at R and p-Cl group at R
thesized sulfonamide compounds having NH/NH2 groups
3
enhances the bioactivity. Change in morphological features with chloro and bromo demonstrated the highest anticancer
of cells of A549 and MCF-7 cancer cell lines in the form of activity (Shah et al., 2013). In the I –I synthesized series, we
1
6
cell shrinkage, plasma membrane blebbing, etc, at different have incorporated the aniline and substituted aniline groups
0
00
0
concentrations on 100 lm scale has been observed (Figures at R position along with substituted variation at R (H, CH )
3
4
and 5). All the synthesized analogues inhibited the cell (Figure 2) and fluctuations in the LC50 and GI50 along with
growth but I showed more or less same level of damage to TGI have been observed due to this minor struc-
5
cell and maximum shrinkage of cells (Figures 4 and 5) in tural variation.
both the cancer cell lines in comparison to reference drug. I5
Secondary sulfonamide feature (-SO NH-) is proposed to
2
differs from rest of the analogues only in the position of p- be potential tumor cell growth inhibitor against leukemia,
0
000
CH at R and incorporation of aniline group at R (Figure 2). non-small cell lung, ovarian, melanoma, colon, CNS, renal,
3
As per the literature analysis, para and ortho substitutions to prostate and breast cancer cell lines with GI50 values in

1
0
K. DEVI AND P. AWASTHI
Table 2. Percentage of cell death in different phase of cell cycle after treat-
ment along with provided samples.
1 6
Table 4. Antifungal activity results of isoleucine sulfonamide analogues (I –I )
(mg/mL).
Group/Phase
Control
Replicates
G0/G1 (%)
S (%)
G2/M (%)
Zone of
inhibition (mm)
Amphotericin
B (mm)
Species name
Analogues
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
60.220
64.075
62.630
16.365
12.800
14.755
55.525
51.785
56.770
59.115
56.110
59.530
69.455
68.495
65.890
22.435
19.930
21.400
20.530
22.415
21.015
31.120
34.730
30.125
25.495
27.805
24.285
19.035
19.120
21.405
17.417
16.040
16.045
63.090
64.815
64.310
13.705
13.860
13.495
15.635
16.425
16.480
11.625
12.465
12.850
A. Fumigatus
C. albicans
–
–
10
20
–
I
1
I
3
I
1
I
4
20–24
Vinblastine
–
–
–
F. moniliforme
Maximum reduced growth
Slightly reduced growth
I
5
I
5
I
5
(25 mM)
(50 mM)
(100 mM)
22% cell death in control and Vinblastine. About 17% cell
death of control, 64% cell death by Vinblastine and 16% cells
death caused by I analogue in G2/M phase. In conclusion, I
5
5
analogue arrest G0 and G1 phases along with mild disturb-
ance at S-phase of cell cycle. Therefore, synthesized isoleu-
cine sulfonamide analogues (I –I ) were proposed to arrest
1
6
cell cycle at G0/G1 phase which is similar to our earlier study
on phenylalanine sulfonamide analogues (Figure 1(h) (Devi &
Awasthi, 2019) however S-phase activity in (I –I ) analogues
Table 3. Antibacterial activity results of isoleucine sulfonamide analogues
–I ) (mg/mL).
(I
1 6
1
6
Zone of
inhibition (mm)
increases the cell death by 14%. This variation is due to the
structural difference; as aromatic structures in central part of
analogues is replaced by aliphatic structure (Figures 1(h) and
2). The isoleucine sulfonamide analogues with aliphatic
groups replacing aromatic rings; (Devi & Awasthi, 2019)
could be the reason for the increased/decreased activities of
the G0/G1 and S-phases of cell cycle.
Species
name
10
1
0.1
0.01 Streptomycin
Analogues mg/mL mg/mL mg/mL mg/mL
sulphate
P. aeruginosa
S. aureus
E. coli
I
I
I
1
6
3
15
15
Reduced
growth
–
12
15
12
14
12
12
19
21
24
–
–
–
–
–
1
0–75 nM range (Casini et al., 2002). Further the GI50 value
3
.2. Antibacterial and antifungal assay
for E7070 (Figure 1(f)) has been reported to be 350 nm and
proposed to inhibit the G1/G0 phase of cell cycle.
Synthesized I –I analogues inhibit 50% cell growth in tested
cell lines. However data for LC50 and TGI values were non-
evaluable for A549 cell line which indicates the less toxic
effect of I –I series on A549 cell line (Table 1).
Antibacterial activity of -SO NH- (primary as well as second-
2
ary) feature has been studied extensively (Shah et al., 2013)
and it has been observed that this feature exhibited higher,
moderate and to zero activity against E. coli. Methoxy substi-
tuted sulfonamide compounds showed better activity.
Further it has been proposed that increase in the length at
alkyl chain along with p-substitution of aromatic moiety
enhances the antimicrobial activity.
1
6
1
6
3
.1.1. Cell-cycle analysis
Cell-cycle analysis of one of the representative (I ) has been
carried out on A549 cell line using flow cytometry.
Morphological changes observed in I treated cells are char-
acteristics of apoptotic cell death during cell cycle (Figure 7).
0 000
In the present synthesized series (I –I ), R and R are p-
1 6
5
00
substituted and extension of alkyl chain (R ) has been done.
Antibacterial activity of synthesized analogues (I –I ) have
5
1
6
been carried out against Gram positive (S. aureus) and Gram
negative (E. coli, P. aeruginosa) bacterial as well as fungal
strains (C. albicans, A. fumigatus and F. moniliforme) (Tables 3
and 4). Antibacterial activity has also been checked on multi-
resistant strains i.e. S. aureus and E. coli. Synthesized ana-
logues showed good antibacterial and antifungal activities
on diverse strains (Tables 3 and 4).
E7070 (Figure 1(f)) having -SO NH- blocks G1 phase of cell
2
cycle in P388 murine leukemia cells (Lee et al., 2011). While
treating A549 cell lines with E7070; G0/G1 and G2/M phase
activities increases with decrease in S-phase fractions
(
Kesteren et al., 2002).
Among present synthesized series (I –I ), I analogue
1
6
5
showed better effect against A549 and MCF-7 in cytotoxic
For antibacterial activity the I1 and I6 exhibited zone of
study. Variations have been observed in G0/G1 phases after inhibition (ZOI) of 15 and 12 mm against P. aeruginosa and S.
analyzing data for I5 (Figure 6) along with morphological aureus which is very close to the standard drug Streptomycin
changes in the cells (Figure 7). Percentage cell death in dif- (19 and 21 mm). But all the synthesized analogues (I –I ) were
1
6
ferent phases of cell cycle after treatment with different con- inactive for the E. coli. However for antifungal activity I was
3
centration of samples is tabulated in Table 2. Biosynthetic found to be most effective against C. albicans with ZOI 20 mm
activity is high throughout G1 phase which appear to be which is very close to the standard drug Amphotericin B with
increased to 55% cell death at 25 mM, 59% cell death at ZOI 20–24mm whereas I analogue exhibited 10 mm of ZOI. In
1
5
0 mM and 68% cell death at 100 mM of I in comparison to general, C. albicans has been shown to be resistant to the sul-
5
control which is 62% cell death and 16% cell death for fonamide compounds but present synthesized analogues have
Vinblastine (standard drug). Inhibition of DNA replication shown certain level of antifungal activity. I and I analogues
1
4
mechanism in S-phase increases by 34% in comparison to were found to be least active with slightly reduced growth

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
11
1 6
Table 5. B.E, D.E., I.C, T.E and hydrogens bond interactions of isoleucine sulfonamide analogues (I –I ) and mitoxantrone.
S. No.
Analogues
MTX
I.C. Ki (lM)
B.E. (kcal/mol)
T.E. (kcal/mol)
1.79
D.E. (kcal/mol)
11.17
Hydrogen bond interactions
Distances (Å)
Cluster (no.)
12
1
.
928.62nM
–8.23
NH … OH ASN 247
NH … OH LEU 137
NH … NH ASN 239
NH … O oxygen
2.191
2.095
1.948
1.712
Of MTX ring ASN 239
2
3
.
.
I
I
1
670.97
35.0
–4.33
–6.08
2.09
2.09
8.18
9.56
NH … O ¼ C ARG 248
1.837
2.12
2
1
2
2
NH … O S ARG 248
CO … NH of aniline ring ASN 247
NH … O ¼ C MET 243
1.89
4
.
.
I
3
4
21.91
–6.36
–4.77
2.09
2.09
10.96
8.60
1.742
2.052
2.229
1.722
1.949
2.183
2.009
2
3
2
NH … O S ASN 239
5
I
318.17
NH … OH ARG 248
NH … OH ARG 248
6
7
.
.
I
I
5
47.46
37.78
–5.90
–6.03
2.09
2.09
10.43
10.32
NH … O
NH … O
2
S MET 243
3
1
6
2
S ASN 248
NH … O ¼ C MET 243
I.C. ¼ inhibition constant, B.E. ¼ binding energy, D.E. ¼ docking energy, T.E. ¼ torsional energy, MTX ¼ mitoxantrone.
Figure 8. (a) Binding energy profile of isoleucine sulfonamide analogues (I
logues (I –I ) with p53 tumor suppressor-DNA complex.
1 6
–I ); (b) Energy variations i.e. intermolecular, internal and torsional of synthesized ana-
1
6

1
2
K. DEVI AND P. AWASTHI
5 1 6
Figure 9. (a) Hydrogen bond interactions of isoleucine sulfonamide analogue I . (b) Hydrogens bond interactions of isoleucine sulfonamide analogues (I –I ).
against F. moniliforme whereas analogues I , I , I and I did 3.3. Docking study
1
2
5
6
not shown any antifungal activity against the tested strains.
p53 tumor suppressor-DNA complex activities are more
prominent in G0/G1 phase of the cell cycle. The synthesized
analogues (I –I ) arrest the G0/G1 phase of the cell cycle as
Structural variations due to position and type of func-
0
00
000
tional groups at R , R , R (Figure 2) resulted in to the major
variation in the antimicrobial activities. Antibacterial as well
as antifungal activities have been improved by incorporation
of aliphatic structural feature in the analogues. In the present
reported series replacing aromatic ring (Devi & Awasthi,
1
6
per results of cell cycle analysis. The G0/G1 phase consists of
p21, p53 and other DNA synthesis activities. The mutations
in p53 are the major cause of cell proliferation in most of
the cancer types. Therefore, we have carried out docking
study of these analogues (I –I ) with p53 tumor suppressor-
2
019) at the center, which could possibly be the major cause
1
6
of hindrance at the receptor site of the molecule, by aliphatic
chain (Figures 1 (h) and 2) resulted in to better bio-
logical activity.
DNA complex (1TUP) to check the binding affinity of
the analogues.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
13

1
4
K. DEVI AND P. AWASTHI
1 6
Figure 10. (a) Torsional angle variations of isoleucine sulfonamide analogues (I –I ) in free and; (b) docked state.
The binding energy, dock energy, torsional energy and showing H-bonding interactions with amino acids residues
inhibitory concentrations (Ki) are tabulated in Table 5. Lower on the active site of p53 (Table 5). I , I , I and I show effect-
1
2
5
4
value of binding free energy suggested better stability of ive interactions with amino acids moieties ARG-248 (Figure
best docked conformations. I gives best fit inside the bind- 9(a, b)) which is the most mutated residues in cancer cells.
3
ing site followed by I , I , I , I and I analogues (Figure 8). The secondary sulfonamide group is common in all synthe-
2
6
5
4
1
Increase in binding energy upon binding with receptor (p53) sized analogues (I –I ) but they differ from each other by p-
1
6
0
00
000
has been observed however internal and torsional energy substitution at position R of ring A, at the R and R (Table
balances each other (Figure 8(a, b)). Analogues (I –I ) were 6) Torsional angle variations were observed by changing the
1
6
.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
15
Figure 11. Correlation of isoleucine sulfonamide analogues (I
1
–I
6
) for In silico screening (a), In vitro on A549 cell line (b), MCF-7 cell line (c) and PC-3 cell line (d).
Table 7. Frontier molecular orbitals values of isoleucine sulfonamide ana-
logues (I
1 6
–I ).
1
37 and not with MET 243, ARG 243 and ASN 239 residues.
S. No.
E
total (Hartree)
EHOMO (eV) ELUMO (eV) DEHOMO-LUMO gap (eV)
Docking energy as well as binding profile is more or less
same with the earlier study (Devi Awasthi, 2019).
Incorporation of aliphatic group at the center of the ana-
logues (Figure 2) imparts flexibility to the best fit and to
exhibit better interaction with receptor site. Energy wise
change in structural behavior of analogues (I –I ) is slightly
Mitoxantrone
–1517.01
–1220.96
–1432.06
–1891.69
–1260.29
–1471.39
–1931.01
–0.18
–0.22
–0.21
–0.22
–0.22
–0.21
–0.21
–0.12
–0.11
–0.10
–0.11
–0.11
–0.10
–0.10
0.05
0.11
0.11
0.10
0.08
0.10
0.10
&
I
1
I
2
I
3
I
4
I
5
I
6
1
6
better. The anticancer activity and docking study of all the
analogues (I –I ) are compared and shown in Figure 11.
0
00
000
position of substituent at R , R and R in free and docked
state (Figure 10(a, b) and Table 6). Major changes in structure
of analogues with high flexibility at main chain angles i.e.
1 6
3
.4. Quantum chemical calculation
0
0
00
00
U1, U2, U2 , U3, U3 , U4, U4 and U5 have been observed
Table 6). Maximum inhibition and binding interactions have HOMO and LUMO, as frontier molecular orbitals, are consid-
been shown by I , I and I (Table 5 and Figure 9(b)). All the ered to be very important molecular parameter for the stabil-
(
3
2
6
analogues i.e. I , I , I , I , I and I differ in the substitution at ity and chemical reactivity of molecules. Lower HOMO
1
2
3
4
5
6
0
000
R and R position (Figure 2). I2 analogue makes H-bonds energy is noteworthy for participation in chemical interaction
with ARG 248 and ASN 247 residues while I and I make by donating electrons. On the other hand, LUMO energy
3
6
hydrogen bond with MET 243 and ASN 239 residues of p53. determines the ability to accept an electron in a chemical
I5 analogue makes hydrogen bond with MET 243 residues of process. LUMO–HOMO energy gap reflects the chemical
hardness, softness and polarizability of molecule and hence
same can be correlated to its biological activity.
p53 (Figure 9(a)) whereas Mitoxantrone (Figure 1(g)) showed
hydrogen bond interactions with ASN 247, ASN 239 and LEU

1
6
K. DEVI AND P. AWASTHI
1 6
Figure 12. Frontier Molecular Orbitals (FMO’s) of isoleucine sulfonamide analogues (I –I ).
Table 8. Reactivity parameter of isoleucine sulfonamide analogues (I
1 6
–I ).
and Figure 12). Further, reactivity parameters are also show-
S. No. I.P.
E.A. D.P. (Debye) E.N. (v) C.S. ( n) C.H. (g) E.I. (x) ing similar pattern (Table 8). Hence electronically synthesized
I
1
I
2
I
3
I
4
I
5
I
6
–0.22 –0.11
–0.21 –0.10
–0.22 –0.11
–0.22 –0.11
–0.21 –0.10
–0.21 –0.10
2.95
1.75
3.62
3.36
3.31
2.38
0.17
0.16
0.16
0.16
0.15
0.16
8.77
9.25
9.17
8.77
10.10
9.17
0.05
0.05
0.05
0.05
0.04
0.05
0.25
0.24
0.24
0.24
0.25
0.24
analogues as well as Mitoxantrone are behaving more or less
in similar pattern as per present study.
3
.5. In silico bioactivity study
E.A. ¼ electron affinity, D.P. ¼ dipole moment, E.N. ¼ electron negativity,
C.S. ¼ chemical softness, C.H. ¼ chemical hardness, I.P. ¼ ionization potential,
E.I. ¼ electrophilicity index.
E7070, with secondary sulfonamide feature, (Figure 1(f)) is
used in phase II clinical trials. As per Lipinski rule, logP
should be ꢃ5 (measure of degree of hydrophilicity and loga-
Table 9. Bioactivity scores of isoleucine sulfonamide analogues (I
erence compound.
–I
1 6
) and ref- rithm of water/octanol partition coefficient); hydrogen bond
donor should be ꢃ5; hydrogen bond acceptors should
ꢄData
I
I
2
I
3
I
I
5
I
6
E7070
1
4
be ꢃ10 and molecular weight should be ꢃ500. We have
miLogP
TPSA
natoms
MW
0.41
83.47
18
3.42
75.27
24
4.10
75.27
25
0.86
83.47
19
3.87
75.27
25
4.55
75.27
26
2.18
122.12
26
compared the potential drug like behavior of designed and
synthesized analogues (I –I ) with E7070 using computational
1
6
271.34 346.45 380.90 285.3 360.48 394.92 385.85 tool (Table 9). Synthesized isoleucine sulfonamide analogues
nON
5
2
0
6
5
2
0
7
5
2
0
7
5
2
0
6
5
2
0
7
5
2
0
7
7
4
0
4
(
I –I ) were in agreement to this criterion. However, variation
1 6
nOHNH
nviolations
Nrotb
Volume
GPCR L
ICM
KI
NRL
PI
in logP value, number of hydrogen bond donor and acceptor
(nON and nOHNH) has been observed. Proposed molecules
238.46 314.25 327.79 255.0 330.81 344.35 284.52
(
I –I ) are more flexible than E7070 upon comparing the
1 6
–0.15
–0.25
–0.78
–0.41
0.41
–0.17
–0.41
–0.45
–0.55
0.30
–0.17
–0.40
–0.45
–0.56
0.25
–0.15
–0.33
–0.75
–0.36
0.36
–0.21
–0.46
–0.48
–0.56
0.24
–0.20
–0.45
–0.48
–0.56
0.19
0.04
–0.27
0.30
–0.16
0.48
rotatable bond (nrotb) property. Some variation has been
observed in kinase and protease inhibitors (KI and PI) includ-
ing enzyme inhibitors and nuclear receptors (EI and NRL).
EI
0.03
–0.13
–0.16
–0.03
–0.19
–0.21
0.30
ꢄ
miLogP: logarithm of molecules partition coefficient between water and n-
octanol; TPSA: total polar surface area, nON: number of Hydrogen bond donor,
nOHNH: number of Hydrogen bond acceptor, nVio: number of violations,
Nrotb: number of rotational bonds; GPCR L ¼ Guanine Protein-Coupled
4
. Conclusion
Number of substituted secondary sulfonamide analogues
Receptor ligand, ICM ¼ ion channel modulator, KI ¼ kinase inhibitor, have been reported in literature and proposed to be effect-
NRL ¼ nuclear receptor ligand, PI ¼ protease inhibitor, EI ¼ enzyme inhibitor.
ive anticancer agents. These classes of analogues are pro-
posed to be potential carbonic anhydrase inhibitors and
No key difference has been observed in the energy profile affect the nucleotide synthesis. However exact mechanism of
of synthesized series I –I upon comparing frontier molecular action is not proposed till date but majority of the analogues
1
6
orbital analysis with standard drug Mitoxantrone (Table 7 with -SO NH- feature showed inhibition action against
2

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
17
leukemia, ovarian, colon, prostate and breast cancer cell lines
with proposed GI50 values in the range of 10–75 nm.
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of secondary sulfonamide analogues (I –I ) with diverse sub-
1
6
stituted features. Structure of synthesized analogues (I –I )
1
6
1
13
was conferred by IR, NMR ( H, C) and ESI-MS tools. All syn-
thesized analogues showed anticancer activity on PC-3
(
Prostrate), MCF-7 (Breast) and A549 (Lung) cancer cell lines.
I5 and I3 showed better anticancer activity among all. Cell
cycle analysis of the I analogue on A549 cell line indicates
5
the complete arrest in G0/G1 phase along with mild disturb-
ance in S-phase. Interestingly, minor change in cell cycle
inhibition has been observed upon comparing the cell cycle
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5
fonamide feature (Devi & Awasthi, 2019). Replacing aromatic
feature with aliphatic one (I ) changes the course of inter-
5
10(6), 885–889. https://doi.org/10.1128/JCM.10.6.885-889.1979
action with receptor site and hence over all mechanism of
interaction. Screened analogues also showed good antibac-
terial and antifungal potential against gram positive as well
as gram negative strains. In order to co-relate the structural
and biological data, we have carried out docking and
Gaussian study of synthesized analogues I –I series in com-
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1
6
parison to mitoxantrone and E7070. In silico bioactivity study
indicates that proposed analogues are bioactive, however it
required detail experimentation and same will be reported
subsequently. As per present study synthesized isoleucine
sulfonamide analogues (I –I ) are proposed to be potential
1
6
anticancer, antibacterial and antifungal agents.
Acknowledgements
1
13
Thanks to SAIF lab, PU Chandigarh for H-NMR, C-NMR, ESI-MS, and
FT-IR spectroscopic experiments. Authors thank Anti-Cancer Drug screen-
ing facility (ACDSF) at ACTREC, Tata Memorial Centre, Navi Mumbai for
cytotoxic study. Thanks to Pharmacology and Toxicology lab (CSIR-IHBT),
Palampur, Himachal Pradesh for cell-cycle analysis. We are thankful to
Dr. Sunita Siwach, Ubiquitous Analytical Techniques Lab, CSIR-CSIO
Chandigarh for antibacterial and antifungal studies. This research work is
also filed as Indian patent with Application no. 202011017638. We are
thankful the Director, National Institute of Technology, Hamirpur (H.P)
India for extending all laboratory and administrative facilities.
(
2004). Imidazo[1,2- b ]pyridazines: A potent and selective class of
cyclin-dependent kinase inhibitors. Bioorganic & Medicinal Chemistry
Letters, 14(9), 2249–2252. https://doi.org/10.1016/j.bmcl.2004.02.008
Casini, A., Scozzafava, A., Mastrolorenzo, A., & Supuran, T. C. (2002).
Sulfonamides and sulfonylated derivatives as anticancer agents.
Current Cancer Drug Targets, 2(1), 55–75. https://doi.org/10.2174/
1
568009023334060
Cecchi, A., Winum, J., Innocenti, A., Vullo, D., Montero, J., Scozzafava, A.,
Supuran, C. T. (2004). Carbonic anhydrase inhibitors: Synthesis and
&
Disclosure statement
inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes
I, II, and IX with sulfonamides derived from 4-isothiocyanato-benzola-
mide. Bioorganic & Medicinal Chemistry Letters, 14(23), 5775–5780.
https://doi.org/10.1016/j.bmcl.2004.09.062
No potential conflict of interest was reported by the authors.
Chen, K. M., Sun, Y. W., Tang, Y. W., Sun, Z. Y., & Kwon, C. H. (2005). Synthesis
and antitumor activity of sulfur-containing 9-anilinoacridines. Molecular
Pharmaceutics, 2(2), 118–128. https://doi.org/10.1021/mp049913g
Clark, D. E. (1999). Rapid calculation of polar molecular surface area and
its application to the prediction of transport phenomena. 2.
Prediction of blood-brain barrier penetration. Journal of
Pharmaceutical Sciences, 88(8), 815–821. https://doi.org/10.1021/
js980402t10430548
Funding
This work is financially supported by UGC-RGNF, New Delhi, India (F1-
1
7.1/2014-15/RGNF-2014-15-ST-HIM-84222/(SAIII/website)).
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