Sulfonamide phenylalanine (SPA) series of analogues as an antibacterial, antifungal, anticancer agents along with p53 tumor suppressor-DNA complex inhibitor–part 1

Article abstract of DOI:10.1080/07391102.2019.1671229

A series of N-[1-benzyl-2-oxo-2-substituted(ethyl)] benzene/p-toluene sulfonamide (K1–K12) are synthesized. Structure of the synthesized analogues has been confirmed by FT-IR, ¹H & ¹³C NMR and ESI-MS spectroscopic techniques. All the

Full text of DOI:10.1080/07391102.2019.1671229

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Sulfonamide phenylalanine (SPA) series of
analogues as an antibacterial, antifungal,
anticancer agents along with p53 tumor
suppressor-DNA complex inhibitor – Part 1
Kirna Devi & Pamita Awasthi
To cite this article: Kirna Devi & Pamita Awasthi (2019): Sulfonamide phenylalanine (SPA)
series of analogues as an antibacterial, antifungal, anticancer agents along with p53 tumor
suppressor-DNA complex inhibitor – Part 1, Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2019.1671229
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Sulfonamide phenylalanine (SPA) series of analogues as an antibacterial,
antifungal, anticancer agents along with p53 tumor suppressor-DNA complex
inhibitor – Part 1
a
a*
Kirna Devi and Pamita Awasthi
a
Department of Chemistry, National Institute of Technology, Hamirpur, H.P. India.
*Corresponding author. Tel. +91-1972-254140.
E-mail address: pamitawasthi@gmail.com
1

Abstract
A series of N-[1-benzyl-2-oxo-2-substituted(ethyl)] benzene/p-toluene sulfonamide (K1–K12)
1
13
are synthesized. Structure of the synthesized analogues has been confirmed by FT-IR, H & C
NMR and ESI-MS spectroscopic techniques. All the synthesized analogues (K1-K12) have also
been examined for their in vitro antibacterial and antifungal activities. Compounds showed good
antibacterial and antifungal activity against standard drug. Anticancer study has been carried out
on three cancer cell lines PC-3, MCF-7 and A549 on two different concentrations (mg/ml and
μg/ml). The K4 sulfonamide analogue showed better anticancer activity amongst all analogues
against PC-3 and A549 cell lines. K4 inhibit G0/G1 phase in cell cycle analysis experiment. All
synthesized molecules (K1-K12) dock at junction p53-DNA and make hydrogen bonded with
residues of p53 protein as per docking study. ADMET predictions of synthesized phenylalanine
sulfonamide analogues (K1-K12) has been done using “Lipinski rule” and it has been observed
that all synthesized analogues did not violate the rule. Electronic, chemical properties and
mulliken atomic charges of analogues were calculated using density functional theory (DFT).
Keywords: phenylalanine sulfonamide; mitoxantrone; streptomycin sulfate; cell cycle; p53.
1
. Introduction
Molecules having sulfonamide (SO NH ) group in their structure have been used as medication
2
2
towards number of diseases. Importance of sulfonamide was realized when sulfonyl amide, a key
analogue of sulfonamide, revealed to be first antibacterial drug. Number of sulfonyl amide
derivatives were synthesized and examined for antibacterial, anticancer, anti-inflammatory,
antidiabetic, carbonic anhydrase inhibitor, diuretic, anti-thyroid, antiviral, dermatological,
antiretroviral, anticonvulsants activities. Some sulfonyl hydrazine are said to have antitumor
effects which prevent malignant cells from developing and lengthening [Shyam et al, 1990].
Chloroquinoxaline sulfonamide (CQS) was the first drug having -SO NH- feature considered to
2
be an anticancer agent. Drug achieved in-vitro toxicity at higher concentration. It has been
observed that drug arrest cell cycle in G0/G1 phase [Branda, Mccormack, and Perlmutter, 1988;
2

Tong et al, 1987]. Later on sulfabenzamide was proposed to effective anticancer agent. Unlike
doxorubicin; sulfabenzamide induced antiproliferative effect due to autophagy. Minimum cell
cycle arrest (G2/M phase) has been observed in T47D cells treated with sulfabenzamide. This is
in contrast to doxorubicin wherein maximum accumulation of cells takes place in S-phase and to
a lesser extent in G2/M phase [Mohammadpur, Safarian, Farahnak, Hasheminasl, and Sheibani,
2
012]. Likewise another analogue, E7070, showed increase in G0/G1 phase and decrease in S-
phase cells population on A549 lung cancer cell line. However, longer exposure would increase
the G2/M fraction in A549 cells [Fukoka et al, 2001].
The structure of E7070 and E7010 (Fig. 1 (a & b)) analogues of sulfonamide is more or less
same but their mechanism of action is different due to change in substituent group attached the
sulfonamide functionality (-SO NH-). E7070 is proposed to show antitumor effect by targeting
2
cell cycle at multiple points (includes both G1/S and G2/S transitions). Further inhibitory effect
on p53 and p21 are considered to be plausible mode of action of E7070 [Yoshino et al, 1992;
Yammato et al, 1998; Supuran 2003; Mohan et al, 2006; Hoyer-Hansen, Bastholm, Mathiasen,
Elling, and Jaattela, 2005; Ozawa et al, 1996; Kesteren et al, 2002; Tsukahara et al, 2001].
Number of analogues of E7070 were synthesized and studied for anti-cancer activity [Mirian et
al. 2011; Badawi, Ali and Ismail, 2008; Yokoi, Kuromitsu and Kawai, 2002]. From cell-cycle
analysis, E7070 and sulfabenzamide proposed to inhibits G0/G1 phase of cell-cycle
[
(
Mohammadpur, Safarian, Farahnak, Hasheminasl, and Sheibani, 2012; Fukuoka et al, 2001].
1R, 2R)-D-threo-2-(N-Myristoylamino)-1-(4'-nitrophenyl)-1,3-propanediol (B13) substituted
with sulfonamide feature along with a C H alkyl chain showed better cytotoxicity in
13
27
comparison to B13. Sulfonamide group along with long alkyl chain increases antitumor activity
and additionally the replacement of phenyl ring by naphthyl ring; significantly enhance the
antitumor effects & hydrophobicity [Lee, Park and Im, 2011].
As per literature study G0/G1 phase have operational effects of p53, p21 and cyclin dependent
kinase along with numerous DNA synthesizers. p53 accomplished to be the most commonly
observed genetic factors in tumor growth. p53 inhibits tumor growth by way of blocking
transformation of an activated oncogene, or stopping tumor formation. When p53 detects
deformed DNA; it immediately signals the cell to start for cell cycle arrest and as the amino acid
residues of p53 genes receive mutations; it lose its DNA binding function. Hence, DNA
multiplication goes unchecked and it proliferates in uncontrolled manner which is proposed to be
3

the main cause of cancers. Approximately half of cancers show mutations of one p53 allele; and
block the p53 tumor suppression properties. Over expression of p53 affects cell cycle arrest
throughout G1 phase. It has been observed that in-presence of sulfabenzamide the anticancer
activity of mutant p53 return to normal and induces autophagical cell death [Mohammadpur,
Safarian, Farahnak, Hasheminasl, and Sheibani, 2012]. Hence the pathway, with the aid of which
p53 initiates apoptosis, is a common target of sulfonamide drugs.
The indolenyl sulfonamides derivatives are proposed to be good antibacterial and antifungal
agents. It is also suggested that these compounds can be good chelating agents. Sulfonamide with
charged substituent group and O & N heteroatom can be effective antibacterial and antifungal
agents. Molecules for multi-therapeutic materials with excessive selectivity have been prepared
[
Cohan, Youssoufi, Jarrahpour and Hadda, 2010]. The cytotoxic activity increases with
substitution in para position of benzene ring. The activity towards Gram-positive and Gram-
negative microorganism is reduced with the aid of larger alkyl substituents. Antibacterial activity
increases when CH group substituted on phenyl ring and decreases with –OCH substitution
3
3
[
Shah, Rivera, and Ashfaq, 2013]. Further analogues of (1H-benzoimidazole) and (1H-
benzoimidazole-1-carbonylphenylsulfamoyl) phenyl acetamide showed better activity against
bacterial strains and no activity towards fungal strains [Kalidhar, Kumar, and Kaur, 2012]. 5-
(bromo/nitropyridin-2-yl)benzamide molecules with benzamide and N-benzoyl-N-(5-
bromopyridin-2-yl) trifluoromethane sulfonamide derivatives; having combination of
benzamides and sulfonamides linkages and halogen substituents; had been proved to be highly
potent antibacterial agents [Rai and Singh, 2011].
Therefore, we designed the synthesis of a series of N-[1-benzyl-2-oxo-2-substituted(ethyl)]
benzene/p-toluene sulfonamide analogues (K1-K12) (Fig. 1 (d)). These K1-K12 analogues bear
sulfonamide and amide functionalities with hydrophobic terminations. They are proposed to be
anticancer, antibacterial and antifungal agent. Structure of new synthesized phenylalanine
1
13
sulfonamide analogues (K1-K12) have been confirmed by FT-IR, H & C NMR and ESI-MS
spectroscopic techniques. Petra Osiris Molinspiration (POM) analysis of synthesized
phenylalanine sulfonamide analogues (K1-K12) has been performed to confirm their drug like
behavior. The anticancer activities of synthesized sulfonamide phenylalanine analogues (K1-
K12) has been studied against PC-3, MCF-7 and A549 cancer cell lines in comparison to
Mitoxantrone (Fig. 1 (c)). Cell-cycle analysis of the best analogue (K4) has been carried out on
4

A549 cell line. The synthesized phenylalanine sulfonamide analogues (K1-K12) analogues
showed good antimicrobial (antibacterial & antifungal) activities towards resistant/non-resistant
strains. The docking study has been carried out with p53 tumor suppressor-DNA complex
(1TUP) using Autodock software. The quantum chemical property of all synthesized
phenylalanine sulfonamide analogues (K1-K12) has been calculated using density functional
theory (DFT).
O
S
O
N
SO NH
N
2
H
HN
MeO
NH
Cl
H NO S
2
2
OH
(
b)
(a)
H
N
O
OH O HN
OH O HN
OH
OH
S
R'
N
H
O
O
R
N
H
R = H, CH3
(d)
(c)
Fig. 1. (a) Structure of E7070 (b) structure of E7010 (C) structure of Mitoxantrone and (d) basic
structure of K1-K12 synthesized series.
2
. Materials and Methods
Solvents had been distilled and dried prior use. Reactions have been monitored on TLC (silica
gel plates) with solvent system of benzene and methanol. Melting points were uncorrected and
measured in open capillary tubes on Digital Melting/Boiling point apparatus. The electron spray
1
positive (ES+) mass spectra were recorded on Waters Micromass Q TOF micro spectrometer. H
1
3
and C NMR spectra were recorded in CDCl and DMSO on a BRUKER AVANCE II 400
3
NMR spectrometer. The records are described as chemical shift, multiplicity (s: singlet, d:
doublet, t: triplet, q: quartet, quin: quintet, m: multiplet, dd: doublet of doublets), coupling
constant in Hz, and the number of protons. The FT-IR spectra were recorded on NICOLET 6700
-1
of Thermo scientific USA using KBr pellets (500 - 4000cm ).
5

2
.1 Procedure for synthesis of K1-K12 analogues
A mixture of benzene/p-toluene sulfonyl chloride (0.1 mole) and phenylalanine (0.1 mole) and
0
NaOH solution (1N) stirred together at 65-70 C for 4 hours. Reaction-mixture were cooled to
0
5
2
C and concentrated HCl was added slowly to change the pH 6.5 [Mahajan, Patial and Sharma,
002]. The precipitates of benzene/p-toluene sulfonyl phenylalanine has been collected and
washed with distilled water and dried. 0.004 mole of benzene/p-toluene sulfonyl phenylalanine
acids dissolved in dry benzene (10 ml). Distilled thionyl chloride (5ml) added to the same
[
Mahajan, Patial and Sharma, 2002]. The mixture was stirred at room temperature for 20 minutes
and refluxed for 3 hours. To the acid chloride of benzene/p-toluene sulfonyl phenylalanine
heterocyclic, aliphatic & aromatic anilines & substituted aromatic aniline (0.004 mole) in
benzene are added drop wise. The temperature of the reaction mixture was raised to 48°C.
Mixture is stirred at this temperature for 4-6 hours. The product was kept in ice overnight and
extracted with chloroform and water. Product was crystallized [Reaction scheme in
supplementary Fig. 1 and characterization in supplementary table 1 and 2].
2
.2. In-silico bioactivity studies
The ADMET predictions of the synthesized analogues K1-K12 has been done by Molinspiration
software (www.molinspiration.com). The physical descriptors like molecular weight, logP,
hydrogen bond acceptors and hydrogen bond donor are calculated. The “Rule of 5” [Lipinski,
Lombardo, Dominy, and Feeney, 2012] properties is a simple set of molecular descriptors used
by Lipinski in formulating (logP<=5, molecular weight <=500, no. of hydrogen bond acceptors
<=10 and no. of hydrogen bond donor <=5) calculated by use of molinspiration software. Total
polar surface area is also calculated by a sum of fragment contributions of the molinspiration
software [Lalitha and Sivakamasundari, 2010]. Molecular volume is acquired by fitting of some
fragment contributions to real 3D volume of molecules [Ertl, Rohde, and Selzer, 2000; Clark,
1
999]. Number of Rotatable Bonds – (nrotb is simple topological parameter defines the
molecular flexibility) shown to be descriptor of oral bioavailability of drugs [Veber et al, 2002]
are also calculated. GPCR ligand, Ion channel modulator, kinase inhibitor, nuclear receptor
6

ligand, protease inhibitors and enzyme inhibitors properties are calculated [Lagunin,
Stepanchikova, Filimonov, and Poroikov, 2000; Pervez et al, 2010].
2
.3. Antimicrobial studies
The Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans,
Aspergillus fumigatus and Fusarium moniliforme strains has been used for antimicrobial studies.
Mueller-Hinton agar (MHA) has been used as culture medium and DMSO was used as a solvent
control. Streptomycin sulphate and Amphotericin B has been used as a standard for antibacterial
0
and antifungal activities. All strains have been cultivated in for 15 h at 37 C. The Mueller-Hinton
0
agar (MHA) media diluted with water up to 1000 ml and sterilized at 120 C for 45 min at 15-16
0
psi pressure in autoclave. Further media is cooled as much as to 40-45 C and positioned on petri
0
plates (6mm). The petri plates were covered and set aside for an hour and then incubated at 37 C
for 48 hrs. The zones of inhibition have been measured in mm [Barry, Coyle, Thornberry,
Gerlach and Hawkinson, 1979] and results of synthesized analogues are compared with standard
drug Streptomycin sulphate and Amphotericin B.
2
.4. Anticancer activity
2
.4.1. MTT assay [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay against
PC-3 (Prostate cancer cell line)
The synthesized phenylalanine sulfonamide (K1-K12) analogues were examined for cytotoxic
results against human prostate cancer (PC-3) cell line using MTT method and compared with
control. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented
with 10% fetal calf serum, penicillin (100units/ml) and streptomycin (100μg/ml) in a 5% CO2–
humidified atmosphere incubator. The cells were plated in a 96-well plate and treated with
0
different concentrations of the compounds for 24 h in a CO incubator (37 C, 5% CO and 90%
2
2
relative humidity). The cytotoxicity was measured by adding 20μl of freshly prepared MTT
solution (5 mg/mL in PBS, sterile filtered) to every well. Culture plates were gently stirred at150
0
rpm for 5 min and incubated for another 3 h in dark at 37 C to allow metabolization of MTT.
The purple formazan crystals were re-suspended by adding 5mg/ ml in methanol to every well
[
Mosmann, 1983]. The absorbance was examined at 570 nm on spectrophotometer. The cell
7

death was calculated: Cell death = 100 − [(test absorbance/control absorbance) × 100] as
standard protocol.
2
.4.2. SRB (sulforhodamine B) assay against MCF-7 (Human breast cancer cell line) and A549
(Human lung cancer cell line)
The cells have been inoculated into 96 well microtiter plates in 100 µL. After cell inoculation
0
and before the addition of experimental drugs, plates were incubated at 37 C, 5% CO , 95 % air
2
and 100 % relative humidity for 24 hrs. Experimental drugs were dissolved in dimethyl sulfoxide
at 100mg/ml and diluted upto 1mg/ml using water. At the time of drug addition, an aliquot of
frozen concentrate (1mg/ml) was thawed and diluted to 100 μg/ml, 200 μg/ml, 400 μg/ml and
-4
-5
-6
-7
8
00 μg/ml and molar concentrations (10 mg/ml, 10 mg/ml, 10 mg/ml, 10 mg/ml) with
complete medium containing test article. Aliquots of 10 µl of these different drug dilutions were
added to the appropriate microtiter wells having 90 µl of medium.
Further, plates have been incubated under standard conditions for 48 hours. Cells were fixed in
0
situ by the mild addition of 50 µl of cold 30 % (w/v) TCA and incubated for 60 minutes at 4 C.
The supernatant discarded and plates were washed five times with tap water and air dried.
Sulforhodamine B (SRB) solution (50 µl) at 0.4 % (w/v) in 1 % acetic acid added to each of the
wells followed by 20 minutes incubation at room temperature. Residual dye was removed by
washing five times with 1 % acetic acid. The plates were air dried. Absorbance was checked at a
wavelength of 540 nm with 690 nm reference wavelength. Percentage growth inhibition was
calculated as: [Ti/C] x 100 % [Vichai and Kirtikara, 2006; Skehn et al, 1990] as standard
protocol.
2
.4.3. Cell-cycle analysis
Standard protocol on flow cytometery has been followed. Cell culture was trypsinized and
5
washed with phosphate buffered saline (PBS) and incubated at a density of 5×10 cells/well with
5
mL of complete medium in 6 well plates. Cells have been grown in Dulbecco’s Modified Eagle
Medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic-antimitotic.
Test samples dissolved in DMSO (Dimethyl sulfoxide) to make 10 mg/mL stock solution.
Finally operating concentration made by way of further dilution of stock solution in entire
8

medium and several dilutions of the test compounds in 1 mL of whole medium was added. Alone
0
cells (without treatment) are used as control. The plates were then incubated at 37 C for 12 to 24
hours in 5% CO incubator. After Incubation period, each adherent and floating cell have been
2
harvested and centrifuged for 5 min. Pellet was washed with phosphate buffered saline (PBS)
and again centrifuged for 5 min. Further, cell pellets were resuspended in 70% EtOH and fixed
0
for 2h at 4 C. Fixed cells were washed with PBS (14 mL) and centrifuged at 200g for 5 min. Cell
pellets were resuspended in propidium iodide(PI) solution (50μg/mL in 0.1 % sodium citrate, 0.1
%
Triton-X 100 and 20µg/ml RNAs A) for 30 min in dark. Samples were processed using an
Amis flow cytometer. The distribution of cells in each phase of the cell cycle was calculated
using IDEA software.
2
.5. Docking studies
Docking studies of the synthesized analogues were carried out against p53 tumor suppressor-
DNA complex to confirm the site of interaction in G0/G1 phase of cell cycle. Autodock4.2
docking tool (Scripps Research Instituite La Jolla, CA, USA) has been used for docking study.
The 3D structures of ligands were constructed using the Pymol software (www.pymol.com).
Gasteiger charges were assigned followed by merging of non-polar hydrogens. The PDB file of
the structure of p53 protein (1TUP) [Eichhorn, Hiller, Hirschfelder, Frank and Efferth, 2013]
was downloaded from protein data bank (www.rcsb.org). The Kollman charges were added to
each atom and saved as protein.pdbqt. Default values of the parameters were used throughout the
calculations. A grid parameter file (pro.gpf) of the protein was created using Auto Grid Tool.
Calculated output was saved as pro.glg file. Grid points choosen to be 58 × 73 × 65, along the
grid spacing of 0.375 Å. The Lamarckian Genetic Algorithm (LGA) protocol was applied for the
protein fixed: ligand flexible model [Jenwitheesuk and Samudrala, 2003; Morris et al, 1998]. The
LGA protocol includes a local minimization for the given fraction of the population. The LGA
mixes a worldwide search for ligand conformation and orientation to perform energy
minimization. The scoring function is calculated which is a sum of van der Waals, H-bonding
and distance-dependent dielectric electrostatics as well as conformational torsional restriction
entropy and empirical solvation energetics terms of the ligand-protein complex.
9

Docking calculations started with an initial population of 150 randomly positioned ligand
conformations. Therefore, in total 100 search attempts (ga_run parameter) done for each ligand.
The maximum number of energy evaluations and generations before the termination of the LGA
6
4
run were 2.5 × 10 and 2.7 × 10 , respectively. For the local search, pseudo-Solis and Wets-
algorithm were applied. Other docking parameters have been set to default values. Final docking
orientation lying within 2 Å of the root-mean square deviation (rmsd) tolerance of each was
represented as the most favorable conformation with low free energy of binding (∆퐺푏)
[Jenwitheesuk and Samudrala, 2003; Morris et al, 1998]. The dock parameter file for the ligand
was saved as lig.dpf. The ligands had been ranked in step with their binding free energy in
kcal/mol and inhibition constant Ki in 휇푀at 298.15 K. Overall free energy of binding (∆퐺푏푖푛푑) is
consists of a sum of free energies measured for the ligand pose.
∆
퐺푏푖푛푑
=
′
Intermolecualr Energy + Internal Energy + torsion Energy − Unbound system senergy
(i)
(Intermolecular Energy = 푎∆퐺푣푑푤 + ꢀ∆퐺푒푙푒 + 푐∆퐺퐻ꢁ푏표푛푑 + ꢂ∆퐺푠표푙)
2
.6. Quantum chemical study
Density Functional Theory (DFT) method has been used to investigate electronic structural
properties of designed ligand (K1-K12) in comparison to Mitoxantrone. The calculation has been
done using DFT protocols. Geometry of the molecules are is optimized at B3LYP/6-311++(2d,p)
level of theory using Gaussian 03W program package without any constraint on the geometry.
Results were visualized by Gauss View 05. The electronic properties; HOMO-LUMO energies
and mulliken charges were calculated. The dipole moment, chemical softness, electrophilicity
index and chemical hardness were calculated and analyzed [Raj, Shoba, Ramalingam and
Periandy, 2015].
3
. Results and Discussion
3
.1. In-silico Bioactivities studies
1
0

All synthesized phenylalanine sulfonamide analogues (K1-K12) were first screened for
bioactivity study (Table 1). As per Lipinski rule, values for good membrane permeability must
be logP ≤5, molecular weight ≤500; range of hydrogen bond acceptors ≤10 and wide array of
hydrogen bond donor ≤5. Physical properties and bioactive properties of the synthesized
phenylalanine sulfonamide analogues are calculated in comparison to E7070. All Sulfonamide
analogues (K1-K12) showed near similar activity for four criteria i.e. GPCR (Guanine-protein
coupled receptor) rate; Ion channel modulator; Kinase inhibitor and Nuclear Receptor Ligand.
All synthesized molecules are arranged in ascending order for GPCR ligand and rated as
K11>K12>K1>K3>K4>K8>K10>K2>K9>E7070, ion channel modulator was in ascending
order and rated as K11>K12>K9>K6=K7>E7070>K2>K5>K10> K8>K4>K3>K1, kinase
inhibitor
was
in
ascending
order
and
rated
as
K8>K1>K12>K11>
K7>K6=K5=K10>K3=K9>K4>K2>E7070 along with nuclear receptor ligand rated as K12>
K11>K6=K7>K5>K9>K2>K10>K3>K4>E7070>K8>K1. This has helped us to have an idea of
probable bioactivity of the synthesized analogues (K1-K12) and kind of interaction with targeted
molecule.
The synthesized phenylalanine sulfonamide analogue does not violate the “rule of 5”. Therefore,
we can say that synthesized phenylalanine sulfonamide analogues (K1-K12) are potential
pharmacophore for antibacterial, antifungals, anticancer, antidiabetic activities and carbonic
anhydrase inhibitor as well. Further, analogues showed good logP value (<5), which is a measure
of the logarithm of octanol/water partition coefficient of organic molecules and measure the
degree of hydrophilicity. High logP value indicates poor intestinal absorption. Synthesized
sulfonamide phenylalanine (K1-K12) analogues having logP values less than five hence
proposed
K12>K7>K11>K6>K10>K3>K4>K9> E7070>K2>K5>K8>K1 order. Total polar surface area
TPSA) is the measure of sum of surfaces of all polar atoms and polar bonds present in the
to
bear
good
membrane
permeability
and
arranged
in
(
molecule like oxygen, nitrogen and attached hydrogen. It predicts drug transport properties
through intestine and blood brain barriers and rated as K1=K8>K2=K9>, K5=K6=K7=K11=K12
>K10=K3=K4>E7070.
1
1

Table 1
Bioactivity score of the synthesized phenylalanine sulfonamide analogues (K1-K12).
*
Data
K1
K2
K3
K4
K5
K6
K7
K8
K9
K10
3.50
K11 K12 E7070
4.05 4.73 2.18
miLogP
TPSA
natoms
MW
0.59
83.47
21
1.99
75.71
26
3.06
66.48
26
2.55
66.48
25
1.90
75.27
22
3.60
75.27
27
4.28
1.03
2.44
75.27 83.47 75.71 66.48 75.27 75.27 122.12
28 22 27 27 28 29 26
305.36 374.46 372.49 358.46 318.40 380.47 414.91 319.3 388.49 386.52 394.50 428.94 385.85
nON
5
2
0
6
6
1
0
6
5
1
0
6
5
1
0
6
5
2
0
6
5
2
0
7
5
2
0
7
5
2
0
6
6
1
0
6
5
1
0
6
5
2
0
7
5
2
0
7
7
4
0
4
nOHNH
nviolations
Nrotb
Volume
GPCR L
ICM
259.92 330.03 337.85 321.05 280.86 335.71 349.25 276.4 346.59 354.41 352.27 365.81 284.52
0.23
-0.14
-0.39
-0.07
0.48
0.13
-0.26
-0.21
-0.23
0.47
0.22
-0.17
-0.24
-0.20
0.51
0.21
-0.19
-0.23
-0.18
0.58
0.16
-0.24
-0.27
-0.27
0.55
0.00
-0.29
-0.27
-0.38
0.31
0.00
-0.29
-0.28
-0.38
0.26
0.19
0.09
0.17
-0.04 -0.04 0.04
-0.21 -0.32 -0.23 -0.35 -0.34 -0.27
-0.40 -0.24 -0.27 -0.30 -0.31 0.30
-0.08 -0.26 -0.22 -0.39 -0.40 -0.16
KI
NRL
PI
0.43
0.40
0.44
0.25 0.20 0.48
EI
0.17
-0.01
0.04
0.02
0.03
-0.06
-0.09
0.09 -0.07 -0.02 -0.12 -0.14 0.30
*
miLogP: logarithm of compound partition coefficient between n-octanol and water, TPSA: total polar surface area, nON: number of
H-bond donor, nOHNH: number of H-bond acceptor, nVio: number of violations, Nrotb: number of rotational bonds; GPCR L =
GPCR ligand, ICM = ion channel modulator, KI = kinase inhibitor, NRL = nuclear receptor ligand, PI = protease inhibitor, EI =
enzyme inhibitor
1
2

3
.2 Antimicrobial and antifungal studies
Molecules with -SO NH- and -NHCOOH- features are proposed to be essential for antimicrobial
2
and antifungal activities. Numbers of drugs are currently in market as an antibacterial and
antifungal agent bearing these features. The antibacterial study of synthesized phenylalanine
sulfonamide analogues (K1-K12) was carried out on Gram-positive bacteria Staphylococcus
aureus, and Gram-negative microorganism Escherichia coli, Pseudomonas aeruginosa and also
on fungal strains like Candida albicans, Aspergillus fumigatus and Fusarium moniliforme (Table
2
& 3). K1-K12 series was also tested against multi-resistant strains Staphylococcus aureus,
Candida albicans and Escherichia coli. The synthesized phenylalanine analogues K2, K3, K4,
K6, K10 and K12 showed better activity over Staphylococcus aureus and Candida albicans
(
resistant strains) but inactive for E.coli (Table 2 & 3). Out of all, K4 showed good activity
against all strains. Structurally K4 differs from rest of analogues in series having pyrrolidine ring
heterocyclic) at on terminal. Due to flexibility in the ring structure; it might raises the
(
hydrophobic character of molecule in comparison to rest and same may be responsible for good
antibacterial action over Streptomycin sulphate. Likewise K10 having piperidine ring at one
terminal showing better zone of inhibition in comparison to Streptomycin sulphate at 10mg/ml
and 1mg/ml concentrations. The K4 analogue showed excellent activity in all examined fungal
strains. K2 and K3 showed moderate activity against Candida albicans while K4 showed better
activity. The K6 and K12 (with chloroaniline ring) showed good activity against Fusarium
moniliforme in comparison to Amphotericin B at all concentrations. Molecules K2, K3 & K4 are
structurally different from rest of the synthesized phenylalanine analogues (K1-K12). They are
having heterocyclic ring at one terminal while rest of them is closing with aromatic/substituted
aromatic ring. Sulfonamides carrying -SO NH - feature which lowers the polarity and alkyl
2
2
group attached with it increases the hydrophobicity which crosses cell wall more easily
Awasthi, Vatsal and Sharma, 2019]. Therefore structure is not the only criteria for antibacterial
[
and antifungal activities but the position of functional group and its orientation is also essential
feature for biological activities.
1
3

Table 2
Antibacterial studies of synthesized phenylalanine sulfonamide analogues (K1-K12).
Species Name Analogues
Zone of inhibition (mm)
Zone of inhibition
Streptomycin
sulphate (mm)
1
0mg/ 1mg 0.1mg/ 0.01mg
ml
16
/ml
ml
13
/ml
12
Pseudomonas
aeruginosa
K4
13.5
20
K10
K4
12
-
12
11.5
-
-
-
-
20
24
Staphylococcus
aureus
Escherichia coli
-
-
-
-
-
Table 3
Antifungal studies of synthesized phenylalanine sulfonamide analogues (K1-K12).
Species Name Analogues
Zone of inhibition (mm)
Zone of inhibition
Amphotericin B (mm)
1
0mg/ml
20
1mg/ml
15
0.1mg/ml 0.01mg/ml
Aspergillus
Fumigatus
Candida
K4
K2
K3
15
10
30
Reduced Reduced
growth growth
Reduced Reduced
Reduced
growth
Reduced
growth
10
Reduced
growth
Reduced
growth
10
20-24
20-24
albicans
growth
20
growth
10
K4
K4
20-24
20
Fusarium
20
10
10
10
moniliforme
K6
K12
10
10
-
24
-
24
-
20
20
20
3
.3 Cytotoxic studies
The synthesized analogues were tested to check their anticancer potentials against different
cancer cell lines.
3
.3.1. MTT assay
1
4

The synthesized synthesized phenylalanine sulfonamide analogues (K1-K12) were screened for
anticancer activity on Human Prostate cancer (PC-3) cell line in comparison with mitoxantrone
at μg/ml concentrations. Cancer cell line was treated with different concentrations for fixed 48
hours. Mitoxantrone is showing cytotoxic effect at lower concentrations. Activity of all the
analogues
(K1-K12)
on
PC-3
cancer
cell
line
is
arranged
as
K4>K10>K2>K1>K5>K11>K6>K8>K9>K12>K7 at μg/ml concentration (Table 4 & Fig. 2).
Interestingly analogue K4 showed good activity in K-series against PC-3 cell lines.
Table 4
IC value on PC-3 cell lines of synthesized phenylalanine sulfonamide analogues (K1-K12).
5
0
Analogues IC (μM)
5
0
K1
K2
K3
K4
K5
K6
K7
K8
K9
K10
K11
K12
MTX
2.92
2.86
3.20
2.73
3.12
3.17
3.32
3.19
3.20
2.79
3.16
3.23
3.24
1
5

3.4
3.3
3.2
3.1
3.0
2.9
2.8
2.7
K7
MTX
K12
K3
K8
K9
K6
K11
K5
K1
K2
2
K10
K4
0
4
6
8
10
12
14
Phenylalanine sulfonamide analogues
Fig. 2. Anticancer activity of synthesized phenylalanine sulfonamide analogues (K1-K12) on
PC-3 cell line.
3
.3.2. SRB assay
The synthesized series (K1-K12) have been screened for anticancer activity on Human breast
cancer (MCF-7) and Human lung cancer (A549) cell lines at two concentrations i.e. μg/ml and
molar concentrations along with standard drug Adriamycin and Mitoxantrone. Cancer cell lines
were treated with different concentrations for fixed 48 hours. Mitoxantrone and Adriamycin are
showing cytotoxic effect at lower concentrations.
All synthesized analogues K1-K12 are more or less showing similar activity at all concentrations
(molar and μg/ml) on MCF-7 cell lines and A549 cell line. The activity of synthesized
sulfonamide phenylalanine analogues (K1-K12) on A549 cancer cell line at μg/ml concentration
is arranged as K10>K4>K12>K3>K8> K11>K9>K5>K2>K6>K7 (Fig. 3a) while at molar
concentration the K4 analogue showed good activity amongst all synthesized analogues on A549
were arranged as K4>K10>K1>K3>K2> K9>K8>K11>K12>K6>K7 (Fig. 3b). Activity of all
synthesized analogues against MCF-7 is arranged as K6>K3>K5>K1>K2>K12>K7>K9>K11>
K8>K4 at molar concentration while K7> K2>K5>K6>K12>K9>K3>K1>K10>K4>K8>K11 at
μg/ml concentration ((Table 5 and Fig. 4a, 4b). At both the concentrations molar as well as μg
1
6

molecules are showing more or less same pattern. However, LC , TGI and GI values are quite
5
0
50
different from standard drugs mitoxantrone and adriamycin.
A549 cell line
Molar conc.
A549
conc. g/ml
1
20
140
-4
(b)
10
(
a)
1
10
20
-5
120
10
00
-
6
7
1
1
0
0
4
0
100
-
8
6
4
2
0
0
0
0
0
80
80
60
40
20
0
-20
-20
-40
K1 K2 K3 K4 K5 K6 K7 K8 K9 K10 K11 K12 ADR
Sulfonamide analogues
K1 K2 K3 K4 K5 K6 K7 K8 K9 K11 K12 ADRMTX
Sulfonamide analogues
Fig. 3a. Cytotoxicity measure of synthesized phenylalanine sulfonamide analogues (K1-K12) on
A549 cancer cell line μg/ml; (3b) at molar concentration.
MCF-7
MCF-7
Molar conc.
1
1
1
1
1
80
60
40
20
00
conc. (g/ml)
120
-4
10
(a)
(b)
1
2
40
0
0
-5
100
10
10
10
-
6
80
60
40
20
0
8
0
-7
8
6
4
2
0
0
0
0
0
-20
-
40
60
-
20
40
-
-
K1 K2 K3 K4 K5 K6 K7 K8 K9 K10 K11 K12 ADR
Sulfonamide analogues
K1 K2 K3 K4 K5 K6 K7 K8 K9 K11 K12 MTXADR
Sulfonamide analogues
Fig. 4a. Cytotoxicity measure of synthesized phenylalanine sulfonamide analogues (K1-K12) on
MCF-7 cancer cell line at μg/ml; (4b) at molar concentrations.
1
7

Table 5
LC , TGI and GI of synthesized phenylalanine sulfonamide analogues (K1-K12) on MCF-7
5
0
50
cancer cell line at molar concentrations.
*
*
*
Compounds
K1
LC50
2.385
TGI
GI50
0.009
0.036
0.004
3.106
0.002
0.002
0.043
0.325
0.422
1.888
0.281
1E-06
3E-06
3E-05
7E-05
3E-05
0.002
2E-05
8E-06
4E-05
6E-04
6E-04
0.002
7E-04
2E-09
5E-10
K2
K3
K4
K5
K6
K7
K8
K9
K11
K12
MTX
ADR
NE
0.559
56.50
0.207
0.293
43.7
NE
NE
NE
NE
4E-04
0.023
*
( LC = concentration of drug causing 50% cell kill; TGI = concentration of drug causing total
50
inhibition of cell growth; GI = concentration of drug causing 50% inhibition of cell growth).
5
0
3
.3.3. Cell-cycle analysis
In order to understand the mode of interaction of designed analogues at cellular level; detailed
cell-cycle analysis of best screen analogue K4 has been carried out using flow cytometry. Cell-
cycle analysis of K4 analogue of K series on A549 cell gives interesting results. Literature
studies confirmed that Mitoxantrone arrests G1 and G2 phases of cellular cycle process and
ultimately inhibit the cell growth [Khan, Lal, Kumar and Khan, 2010]. Further Mitoxantrone also
promote the arrest of S-phase of cell cycle. G1 & G2 are the growth phases in cell-cycle analysis
whereas S-phase is a synthetic phase in which DNA replication and DNA synthesis takes place.
Cell undergoes apoptosis upon treatment with Mitoxantrone due to inhibition of cell growth
phases (G1 & G2) in conjuction with inhibiting the DNA-replication/duplication process (S-
phase). Direct assault on cell regulatory protein is suggested and play important role in
controlling the regulation of G2/M and G1 phases. Morphological changes observed in
analogues treated cells included cell shrinkage and extensive detachment of cell from cell culture
substratum. These changes are characteristics of apoptotic cell death in phases of cell-cycle. The
anticancer analogue E7070 having –SO NH- feature is in clinical trials; reduce the S-phase along
2
1
8

with perturbation of cell-cycle in G1 phase; further E7070 block the G1 phase development in
P388 murine leukemia cells [Lee, Park and Im, 2011]. The cell-cycle analysis of E7070 on A549
cell line showed an increase in G0/G1 phase and decrease in S phase fractions for 24 h while
G2/M fraction increases after increased exposure for 48 & 72 h in A549 cells [Kesteren et al,
2
002].
-4
-5
-6
-7
At molar concentration (10 , 10 , 10 and 10 ), K4 analogue showed better effect in
comparison to Mitoxantrone & Adriamycin on A549 and PC-3 in cytotoxic study. Variations are
observed in G0/G1 phases (initial phases) of cell cycle after analyzing data for K4 (Fig. 5).
Hence enhancements of all chemical activities like deliver of proteins, doubling in number of
cell organelles, and so forth has been observed. Biosynthetic activity is high throughout G1
phase which appear to be increased to 5.3% in comparison to control. However activities in G2
phase decrease by ~3.21% i.e. cell accelerating in G2 phase. 2.11% cell is arrested at S phase i.e.
inhibiting the DNA replication mechanism. K4 analogue deregulates the cell cycle and arrest G0
and G1 phase by means of stressful biochemical system along with mild disturbance at S phase.
It arrests 90% growth of cells in G0/G1 phase in comparison to control with 84.7% cent arrest.
Deregulation of cell cycle is the first most important condition for the molecules/drugs to act as
cytotoxic. Therefore, synthesized series (K1-K12) proposed to deregulates the cell cycle at G0
and G1 phase.
1
9

Fig. 5. Cell-cycle analysis of K4 on A549 cancer cell line.
3
.4. Docking study
Binding energy (BE) and inhibition constant (Ki) of the synthesized phenylalanine sulfonamide
analogues (K1-K12) are calculated against p53 protein (protein ID: 1TUP). From the cell-cycle
analysis of K4; it is proposed that synthesized phenylalanine sulfonamide analogues (K1-K12)
affect the growth of cancer cells at G0 and G1 phase. In G0 and G1 phase the p53, p21, cyclin
dependent kinase activities are excessive, therefore, detailed binding study of synthesized
analogues with p53 protein has been carried out. It has been observed that majority of mutations
in p53 occurs between 102-292 residues and due to this p53 forgets DNA binding activity. This
kind of p53 activity has been diagnosed in maximum cases of cancers. When residues of p53
undergo mutation; it lacks the ability to check DNA damage before permitting cell to go for cell
division. Hence results in neoplastic growth [Cho, Gorina, Jaffrey and Pavletich, 1994]. The p53
tumor suppressor-DNA complex structural study (Fig. 6) confirmed that DNA binding is critical
for biological activity of p53, and mutations at p53 inactivate the functioning.
The docking study identifies an appropriate pose of ligand inside the binding site of receptor p53,
and also predicts the extent of affinity among ligand (synthesized analogues) and receptor p53.
The search process begins from a random ligand location and orientation inside and outside the
binding sites. By exploring the values of ligand translations, rotations and internal degree of
freedom, it eventually reaches the bound conformation. The energy is estimated as binding free
energy of all conformations of ligand with binding site of receptor p53.
Docking protocol is applied to study binding affinity of sulfonamide analogues (K1-K12) and
mitoxantrone with p53 (1TUP). After creation of docking box, lowest energy conformer of every
single of sulfonamide phenylalanine (K1-K12) analogues is allowed to dock inside the active
sites. Comparative docking analysis of all synthesized sulfonamide analogues (K1-K12) and
mitoxantrone with their common receptor site was carried out. The smaller the value of binding
energy and inhibitory constant, the more effective the compound will be in terms of its
bioactivity. The energy results were ranked according to binding energy, dock energy, torsional
energy and inhibitory concentrations (Ki) (Table 6). The lower value of binding free energy
indicates the greater stability of best docked conformations. Synthesized sulfonamide analogues
(K1-K12) possess lower binding energies upon interaction with 1TUP in comparison to
2
0

mitoxantrone and K4 gives the best fit inside the binding site, followed by analogues K1, K8 and
K12 analogues (Fig. 8).
Vander Waals and electrostatic interactions dominates binding energies between ligand and
protein. Van der Waals terms are associated with shape complementarity among protein and
ligand. Whereas electrostatic interaction are the Coulombic interaction between partial atomic
charges. The atomic charges play a significant role in figuring out binding energy and binding
structure. H-bonds are treated as an aggregate of electrostatic and van der Waals interactions.
Total internal energy also plays important role in binding of complex. The greater is the internal
energy; greater is the probability of intra as well as intermolecular interaction of ligand-for the
protein alone, as well as for the ligand-receptor complex. The synthesized sulfonamide analogues
(K1-K12) having internal energy (-1.86, -1.53, -1.92, -1.77, -1.66, -1.97, -1.94, -2.00, -1.51, -
1
.60, -2.03, -1.44) comparable with MTX (-1.15). There is large difference in the internal energy
of K11 & K8 as compared with other analogues and considered to be least fit. Torsion energy
related with the degree of freedom of ligands. Greater the degree of freedom, greater will be
possibilities of change in the conformations. Therefore internal energy and torsional energy
balanced each other but there is an overall increase in binding energy profile (Fig. 8 (a) and (b)).
Synthesized phenylalanine analogues K1, K6, K7, K8, K11 and K12 showed an increase in
torsion energy. An increase in torsion energy increases the overall binding energy of complex.
Therefore K1 showed the least fit. All synthesized sulfonamides phenylalanine analogues (K1-
K12) show hydrogen bonding interactions with amino acids on the active site of the protein p53
(
Table 6). In addition to H-bonding, π-cation interactions have also been observed. K4 shows
effective interactions with amino acids moieties ARG-248 in comparison to mitoxantrone (Fig.
). The sulfonamide feature is common for all the synthesized sulfonamide analogues (K1-K12);
whereas the substituent group differs at the p-position of ring A (R ) and at the R (Table 7) The
7
1
2
.
change in substituent at R and R changes the conformation in free and bound state which leads
1
2
to torsion angle variations (Fig. 9 (a) and (b)) and (Table 7). High flexibility in main chain angles
i.e. Φ1, Φ2, Φ2”, Φ3, Φ3”, Φ4, Φ4” and Φ5 along with major changes in overall structure has
been observed (Table 7).
Interestingly oxygen atom of SO in K2 analogue makes H-bonds with ARG-248 and oxygen
2
atom of heterocyclic ring (morpholine) makes H-bond with GLN-165 and other interactions with
HIS 168, ASN 239, SER 241 residues of p53. However, mitoxantrone indicates interactions with
2
1

ARG-248, ASN 239 and LEU 137. Maximum inhibition and effective binding interactions is
exhibited by K10 and K2 in comparison to mitoxantrone (Table 6) and binding interactions of
analogues K1-12 with p53 tumor suppressor-DNA complex is confirmed (Fig. 7). K10 makes
hydrogen bond with ARG 248 and MET 243 residues of p53.
Binding activity is based upon the existence of several functional groups present in the molecule.
The polarity of functional group as well as configuration of ligand regulates the relative binding.
Variation in the biological activity among the analogues depends on length and number of
carbon atoms as well as the nature of attached functional groups. Incorporation of
hydrophobicity in main chain and also in side group of main chain lowers the overall binding
energy. The comparative results of anticancer and docking studies of all analogues support each
other and are shown in (Fig. 10).
Fig. 6. Complete structure of p53 (1TUP).
2
2

Table 6
Binding energy (ΔG ), docking energy, Inhibition constant and H-bond interactions of synthesized phenylalanine sulfonamide
b
analogues (K1-K12) and reference compound.
S.
No.
Analogues
Inhibition Binding energy
Docking
energy
(Kcal/mol)
Torsional
energy
(Kcal/mol)
Number
Distances in cluster
(Å)
constant Ki
μM)
(Kcal/mol)
H-bond
interactions
(
1
2
.
.
K-1
K-2
391.78
-4.65
-8.60
2.09
ARG 248 NH…O2S
MET 243 NH…O=C
2.031
1.713
2
2.92
-7.55
-10.87
1.79
HIS 168 NH…O of
morpholine
1.809
4
ASN 239 NH…O=C
SER 241 NH…O2S
2.016
2.203
3
4
5
.
.
.
K-3
K-4
K-5
5.00
3.81
-7.23
-7.39
-6.57
-10.94
-10.95
-10.02
1.79
1.79
1.79
ARG 248 NH…O2S
MET 243 NH…O=C
2.084
1.934
2
10
1
ASN 239 NH…O=C
MET 243 NH…O2S
2.197
1.792
15.26
ARG 273 NH…O2S
ARG 248 CO…NHCH3
2.229
1.936
6
7
8
.
.
.
K-6
K-7
K-8
16.10
6.85
-6.54
-7.05
-5.11
-10.60
-11.07
-9.20
2.09
2.09
2.09
ARG 248 NH…O2S
MET 243 NH…O=C
1.903
1.69
7
6
2
ASN 239 NH…O2S
MET 243 NH…O=C
2.121
1.921
179.67
ARG 248 NH…O=C
ARG 248 NH…O-CH
ASN 239 NH…O2S
1.934
1.948
1.932
2
3

9
.
K-9
14.85
-6.59
-9.89
1.79
ASN 239 NH…O2S
HIS 168 NH…O of
morpholine
2.08
2.205
1
1
0
K-10
K-11
K-12
MTX
1.59
6.79
-7.91
-7.05
-7.31
-8.23
-10.30
-11.17
-10.83
-11.17
1.79
2.09
2.09
1.79
ARG 248 NH…O2S
MET 243 NH…O=C
2.106
1.851
3
2
1
4
1
1
1
1.
2.
3.
ARG 248 NH…O2S
MET 243 NH…O=C
2.057
1.724
4.41
ASN 239 NH…O2S
MET 243 NH…O=C
2.192
1.735
928.62nM
ASN 247 NH…OH
LEU 137 NH…OH
ASN 239 NH…NH
ASN 239 NH…O oxygen
Of MTX ring
1.712
2
4

Table 7
Torsional angles variation of synthesized phenylalanine sulfonamide analogues (K1-K12) before and after docking with p53 tumor
suppressor.
R1
H, CH3
O
O
B
5
'
C
N
C
N
;
;
C
N
4
'











R2
6
R2
2S
1
4
N
3


 5
A
H




C
NH
O
NHCH3
;
; Cl
C
NH
R1
O
S.
Code
Ligands
C S N C (Ф1)
S N C C (Ф2’’)
S N C C ’(Ф2)
C C C ’C ’
N C C ’C ’ (Ф3’’)
N C C R (Ф4)
C ’C C N (Ф4’’)
C C N C (Ф5)
4 5 6 7
1
2
3
4
2
3
4
5
2
3
4
4
5
4
4
5
3
4
4
5
3
4
5
6
4
4
5
6
No.
(Ф3)
R1
H
R2
Free
Docked
Free
Docked
-90.75
Free
Docked
Free
Docked Free
Docked
Free
Docked
-35.78
125.20
Free
159.47
156.69 -114.83
Docked
Free
Docked
1
2
.
.
K1
K2
OH
-110.16
-110.16
19.311 -178.40
122.98 -178.33
61.63 149.35
66.56 80.00
66.58 133.81
-173.44 -154.07 39.49
-173.44 -106.25 36.69
84.12
109.56
167.25
14.49
O
H
-106.11 61.63 133.90
-108.23 61.63 131.70
-62.41
N
H
3
4
5
.
.
.
K3
K4
K5
H
H
H
-110.16
-110.16
-110.16
38.199 -178.33
102.75 -178.33
66.58 96.39
66.58 134.64
66.31 -49.10
-173.44 -143.62 36.69 -161.23 156.69
-41.22
-86.89
96.50
167.25
-62.32
N
H
-93.43
61.63 146.58
-173.44 -105.36 36.12
153.12
-23.43
156.12
159.51
-177.11 -177.11
-179.98 -177.99
N
H
NH CH
80.72
-178.82
-130.55 61.63 109.88
-173.44
71.15
39.59
2
3
2
5

6
7
.
.
K6
K7
H
H
-110.16
-110.16
41.44
-178.82
-151.15 61.63 89.33
-139.17 61.63 101.28
66.31 95.95
66.31 135.59
-173.44 -143.85 39.59
-91.57
2.63
159.51
28.33
-179.98 -177.96
-178.98 -179.98
NH2
Cl
165.31 -178.82
-173.44 -104.13 39.59
-173.44 -143.21 39.49
159.51 122.55
NH2
8
9
.
.
K8
K9
CH3
CH3
OH
-110.16
-110.16
-88.74
-24.30
-178.40
-178.33
177.74
61.63 57.79
66.56 96.78
66.58 -90.13
-16.11
117.61
159.47 103.88
156.69 -122.40
109.56
167.25
-8.80
O
-112.48 61.63 127.49
-115.70 61.63 124.29
-105.11 61.63 135.30
-173.44
29.82
36.69
-62.41
N
H
1
1
0.
1.
K10 CH3
K11 CH3
-110.16
-110.16
143.96 -178.33
66.58 126.69
66.31 90.96
-173.44 -113.33 36.69
-173.44 -148.76 39.59
91.402
178.47
156.69 -148.62
167.25
-62.38
179.99
N
H
51.73
-178.82
159.51
-61.60
-179.98
NH2
Cl
1
2.
K12 CH3
-110.16 -167.65 -178.82
90.69
61.63 -28.81
66.31 96.86
-173.44 -142.92 39.59
35.12
159.51 155.01
-179.98
180.00
NH2
2
6

2
7

Fig. 7. H-bond interactions of synthesized phenylalanine sulfonamide analogues (K1-K12).
2
8

(
a)
-4.5
-5.0
-5.5
-6.0
-6.5
-7.0
-7.5
-8.0
-8.5
K1
K8
K6
K5
K9
K11
K3
K7
K12
K4
K2
K10
Mitoxantrone
0
2
4
6
8
10
12
14
K1-K12
Intermolecular energy
Internal energy
Torsional energy
K7
K11
K1
K9
K12
(
b) 2
K3
K3
K5
K8
K8
K4
K6
K10
Mitoxantrone
K2
0
K12
K2
K9
K10
K5
K7
Mitoxantrone
-
-
-
-
2
4
6
8
K1
K1
K4
K6
K11
K8
K9
K5
K6
K3
K11
K12
K7
-10
K4
K2
K10
Mitoxantrone
0
2
4
6
8
10
12
14
K1-K12
Fig. 8a. Binding energy profile of synthesized phenylalanine sulfonamide analogues (K1-K12);
8b) Intermolecular, internal, and torsional energy (Energy variations) of all (K1-K12) analogues
with p53 tumor suppressor complex.
(
2
9