Structural characterization, photoluminescence, computational studies and bioassay of newly synthesized N-(3-oxo-3-morpholino-1-phenyl-propyl) benzo sulfonamide with multifunctional application

Article abstract of DOI:10.1007/s11164-018-3620-9

N-(3-oxo-3-morpholino-1-phenyl-propyl) benzo sulfonamide (BMS) (C₁₉H₂₂N₂O₄S) has been synthesized and characterized by FT-IR, ¹H and ¹³C NMR, and ESI-mass spectrometric methods. The 2-D NMR

Full text of DOI:10.1007/s11164-018-3620-9

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-018-3620-9
Structural characterization, photoluminescence,
computational studies and bioassay of newly synthesized
N‑(3‑oxo‑3‑morpholino‑1‑phenyl‑propyl) benzo
sulfonamide with multifunctional application
Pamita Awasthi1 · Kirna Devi1
Received: 27 July 2018 / Accepted: 28 September 2018
© Springer Nature B.V. 2018
Abstract
N-(3-oxo-3-morpholino-1-phenyl-propyl) benzo sulfonamide (BMS) (C19H22N2O4S)
1
has been synthesized and characterized by FT-IR, H and 13C NMR, and ESI-mass
spectrometric methods. The 2-D NMR (COSY, HSQC, DEPT and TOCSY spec-
tra) of BMS is recorded to study the correlation between protons and carbons. The
optimized geometry, harmonic vibrational frequencies and infrared intensities are
calculated using B3LYP/6-311++(2d,p) basis set on the Gaussian. 13C and 1H NMR
chemical shifts are calculated using the gauge independent atomic orbital method
with B3LYP/6-311++(2d,p) basis set. The detailed analysis of experimental NMR
chemical shifts as well as IR vibrations is supported by quantum chemical calcula-
tions using the Gaussian approach. The HOMO and LUMO energy calculation and
Mulliken charges were calculated by the DFT approach. The UV–Vis and photolu-
minescence study confrms the light emitting nature of the molecule. BMS showed
better antimicrobial activities against resistant and non-resistant strains. Further, the
anticancer activity of the compound is studied against the PC-3 cancer cell line in
comparison to mitoxantrone. Comparative docking study of BMS with mitoxantrone
and doxorubicin has been carried out on the p53 tumor suppressor-DNA complex.
Efective binding is observed at ARG-248, which is an experimentally proved
mutated site of the p53 protein.
Keywords Sulfonamide · BMS · PL · p53 · Antimicrobial activity · Anticancer
activity
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s1116
4-018-3620-9) contains supplementary material, which is available to authorized users.
*
Pamita Awasthi
pamitawasthi@gmail.com
Kirna Devi
kirnamandi99@gmail.com
1
Department of Chemistry, National Institute of Technology, Hamirpur, Himachal Pradesh, India
Vol.:(0123456789)
1 3

P. Awasthi, K. Devi
Introduction
Molecules with the sulfonamides (R-SO2–NH-R′) (Fig. 1a) feature are proposed to
bear diverse biological properties [1]. Besides showing antibacterial, anticancer,
anti-carbonic anhydrase, diuretic, anti- thyroid, antiviral, anti-diabetic, dermatologi-
cal, antiretroviral, and anticonvulsants activities [2–17], they are showing excellent
anti-juvenile hormone activities against certain Lepidoptera insects [18]. Further
sulfonamide drugs are used in preventing infection in burns; and inhibit the growth
of retroviruses, including HIV. Sulfa drugs are proposed to use for conditions such
as acne, urinary tract infections and also used in treatment of infections caused by
bacteria resistant to other antibiotics.
Very recently –SO2NH– feature have been reported to be a good antitumor agent.
Sulfa drugs AZD5438 are considered to be good CDK2 with an inhibitory property;
arresting cell cycle at G1, M, S and G2 phases. Cell cycle progress is activated by
CDK complexes and cyclins. These CDK complexes and cyclins act as checkpoints
in regulating the transition from one phase to another in the cell cycle. Sulfonamide
groups with diferent functionalities are showing good antitumor activities by inter-
acting with CDK complexes [1]. Further inhibition of the carbonic anhydrase mech-
anism is also shown by the number of molecules bearing the sulfonamide feature.
As per literature review, sulfonamide group bearing compounds, exert their
antitumor action through diferent mechanisms such as disruption of microtubule
assembly, cell cycle arrest in G1 phase, functional suppression of the transcriptional
activator NF-Y and angiogenesis. Mettalloprotease (MMP) inhibitory efects of sul-
fonamides have been evaluated for treatment of arthritis and cancer [2, 3]. Difer-
ent anticancer drugs (EB1089, Tamoxifen), having a –SO2NH– feature, activates
autophagic cell death in breast cancer cells [4]. Two novel sulfonamides, E7070 and
E7010, are potently efective against cancer cells via inhibition of tubulin polym-
erization and proliferation. E7070 are confrmed to be cell-cycle inhibitors and
arrest the cell cycle at the G1 phase [5]. A further E7070 novel sulfonamide anti-
cancer agent is currently in phase II clinical trials for the treatment of solid tumors
21C,
26H
O
S
25C,
28H
19C,
22H
31
R'
B
O
23C,
27H
18C
N
H
15C,
16H,
17H
20C,
24H
40C,41H,42H
O
37C,
38H,
39H
4C,
9H
29
S
R
36
30
N
O
5C,
3C
14
12C,
13H
35
10H
N
A
43C,
44H,
45H
46C,
47H,
48H
H
R = H
6C,
11H
34H
2C,
8H
O
O
1C,
7H
R' = C9H11NO2,
C4H9NO
32
33
(a)
(b)
Fig. 1 a, b Chemical structure of BMS; Patent application No. 201711026145 (Indian)
1 3

Structural characterization, photoluminescence,…
[6]. Based upon the cell-cycle analysis study, it is proposed that the E7070 & sul-
fabenzamide with –SO2NH– feature inhibits G0/G1 phase of the cell cycle [8, 9].
NSC158437 with sulfonamide feature have been identifed to interact with the DNA
binding domain of the p53 tumor suppressor-DNA complex and inhibit its function
[19].
Biosynthetic activities are maximum in the G0/G1 phase. All proteins like p53,
p21 and cyclin dependent kinase and nucleotide are made available for DNA repli-
cation/duplication. p53 is proposed to be the most commonly observed genetic fac-
tor in tumor growth as p53 inhibits tumor growth by blocking transformation of an
activated oncogene, or preventing tumor formation. Cell cycle arrest is initiated if
p53 detects DNA damage. Approximately half of cancers involve mutations of one
p53 allele in which the second allele is deleted, resulting in a loss of p53 tumor
suppression properties. Overexpression of p53 results in the arrest of cell cycle dur-
ing G1 phase, therefore, the pathway by which p53 initiates apoptosis is a common
target in cancer-treating drugs. p53 is located in the nucleus of cells throughout the
body and binds directly to the DNA. The core structure of p53 tumor suppressor-
DNA complex has an anti-parallel β-sheet sandwich with four or fve β-strands. If
some of the residues of p53 gets mutated; p53 loses its DNA binding function, and;
therefore, control on DNA goes unchecked and DNA proliferates in uncontrolled
manner, which is the cause of cancers. The two most commonly mutated residues
on p53 tumor suppressor-DNA complex in tumors cells are Arg-248 and Arg-273.
These amino acids (Arg-248, Arg-273 of p53) make direct connection to the DNA
and makes the p53 tumor suppressor-DNA complex. When Arg-248 and Arg-273
get mutated; p53 is unable to bind with DNA or check DNA damage before allowing
the cell to proceed for cell division. This leads to neoplastic growth. The p53 tumor
suppressor-DNA complex structure (Protein ID: 1TUP) study (Fig. 2) showed that
the DNA binding is critical for the biological activity of p53, and mutations at p53
inactivate the functioning. Thus structure suggests the possible targets for design of
compounds to restore activity of mutant p53 proteins found in tumors [19]. Present
study will help in designing molecules with more/additional functional groups for
efective interaction at the binding domain of p53 tumor suppressor-DNA complex.
Further sulfonamide compounds exhibit good activity against some bacterial
strains (Staphylococcus aureus and Escherichia coli) and fungal strains (Candida
albicans) [13]. It is proposed that molecules with benzamide and sulfonamide link-
ages along with a halogen substituent are supposed to be highly potent antibacterial
agents. Synthesized sulfonamides exert their efect by inhibiting the dihydropteroate
synthetase (DHPS) enzyme, which catalyzes folic acid pathways in bacteria. Folic
acid is not synthesized in human cells, however it is taken through the diet, whereas
in bacterial cells, folic acid is synthesized from p-amino benzoic acid (PABA), pteri-
dine and glutamate [16]. Therefore, synthesized sulfonamides compounds compete
with PABA to inhibit the receptor enzyme. Hence, synthesized sulfonamide com-
pounds bind to the active site and block PABA to terminate the synthesis of folic
acid in bacterial cells. Sulfonamides are therefore called bacteriostatic. As per lit-
erature, structure activity relationships between diferent groups on sulfonamides
can afect bioactivity of the molecules like electron withdrawing groups increase the
bioactivity of the compound while electron donating groups lower the efects.
1 3

P. Awasthi, K. Devi
Fig. 2 Structure of p53 tumor
suppressor-DNA complex
(1TUP)
In continuation to our earlier studies we have synthesize a series of com-
pounds having the –SO2NH– feature (Fig. 1a); out of which N-(3-oxo-3-mor-
pholino-1-phenyl-propyl) benzo sulfonamide (BMS) (Fig. 1b) came out to be
a potential antitumor, antibacterial and antifungal agent. Structure of BMS
1
is confrmed by FT-IR, H and 13C NMR, ESI-mass, and 2-D NMR (COSY,
HSQC, TOCSY and DEPT) spectroscopic techniques. Comprehensive antican-
cer, antibacterial, and antifungal study of BMS is reported in this manuscript.
Further, a comparative docking study of BMS, mitoxantrone and doxorubicin
has been carried out with p53 tumor suppressor-DNA complex using Autodock
software. As per literature study, mutated p53 tumor suppressor-DNA complex
leads to deviation in cell cycle progression; hence, DNA multiplications go on
in an uncontrolled way. Interestingly, it has been observed by docking study that
BMS, Mitoxantrone and doxorubicin bind to the mutated site ARG-248 (DNA
binding domain) of p53 tumor suppressor-DNA complex, but does not show any
interaction with DNA. Therefore, the possibility of further blocking the DNA
replication by BMS cannot be denied.
1 3

Structural characterization, photoluminescence,…
Experimental and theoretical methods
General materials and method
All solvents were distilled and dried before use. Reactions were monitored by
TLC performed on silica gel plates and the product is confrmed as single spot.
Melting points were uncorrected and measured in open capillary tubes on Digital
Melting/Boiling point apparatus (Labtronics). The electron spray positive (ES+)
mass spectra was obtained with a WATERS, MICROMASS Q-TOF micro spec-
trometer with liquid chromatography recorded in HPLC water and methanol.
1H and 13C NMR spectra were recorded in CDCl3 and DMSO on a BRUKER
AVANCE II 400 NMR spectrometer. The IR spectra were recorded on NICOLET
6700 of Thermo scientifc USA using KBr pellets. The spectrum ranges from 500
to 4000 cm−1.
The 2-D NMR experiments were carried out at 298 K. Acquisition parameters
for recording COSY spectra were 2048 (2 K) data points along the t2 dimension
with 32 scans. The 2-D Heteronuclear (13C–1H) HSQC experiment was carried
out at 298 K. Acquisition parameters for recording HSQC spectra were 1024
(1 K) data points along the t2 dimension with 32 scans. Acquisition parameters
for TOCSY spectra were 2048 (2 K) data points along the t2 dimension with 16
scans. Acquisition parameters for dept135 and dept90 spectra were 65 536 (64 k)
data points along the t2 dimension with 256 scans.
Synthesis scheme
The synthetic route for BMS is shown in Scheme 1. A mixture of benzene sul-
fonyl chloride (I) 0.1 mol and phenylalanine (II) 0.1 mol and NaOH solution
1 N was stirred together at 65–70 °C for 4 h. The reaction-mixture was cooled
to 5 °C and treated with concentrated HCl (added slowly to change the pH 6.5)
[20]. The precipitates of benzene sulfonyl phenylalanine (III) were collected by
fltration and washed with distilled water and then dried, the colour of precipitate
was white and yield was 0.89 g with M.P. 130 °C (sharp). Further, the 0.004 mol
of benzene sulfonyl phenylalanine acid was dissolved in dry benzene (10 ml) fol-
lowed by the addition of distilled thionyl chloride (5 ml) to the mixture. The mix-
ture was stirred at room temperature for 20 min and then refuxed for 3 h to result
in the formation of (IV). To the acid chloride of benzene sulfonyl phenylalanine
(IV); morpholine (0.004 mol) was added slowly (drop wise). It was then warmed
up to 45–48 °C and stirred at this temperature for a further 4–6 h. The prod-
uct (V) was kept in ice overnight and then extracted with chloroform and water
for 5–6 times. Product was recrystallized; light yellow colour, yield 0.51 g, M.P.
152 °C (sharp).
1 3

P. Awasthi, K. Devi
O
S
O
NaOH
OH
N
H
OH
S
H2N
Cl
Heat 65-700C
O
O
R
O
O
R
III
II
I
C6H6,
SOCl2
H
N
O
O
O
Cl
S
N
H
S
N
O
N
H
O
-HCl
O
R
O
O
R
IV
V
R = H
Scheme 1 Synthetic route for BMS
Quantum chemical study
DFT calculations were carried out and geometry is optimized at the B3LYP/6-
311++(2d,p) level of theory using Gaussian 03 W program package without any
constraint on the geometry. The optimized structural parameters were used for
vibrational frequencies calculations and NMR chemical shifts at the same level.
1H NMR and 13C NMR isotropic shielding were calculated with the GIAO
method using optimized parameters obtained from the B3LYP/6-311++G(2d,p)
method. 13C isotropic magnetic shielding (IMS) of any carbon atoms (X) was
according to formula, CSX (chemical shift)=IMSTMS–IMSX. The experimental val-
1
ues for H and 13C NMR isotropic chemical shifts for TMS were obtained at 30.84
and 188.1 ppm, respectively [21].
Docking studies
The structures of BMS, Mitoxantrone and Doxorubicin are built using Pymol and
optimization followed by energy minimization with a SPDBV-Swiss-Pdb Viewer.
The p53 protein (protein ID: 1TUP) was downloaded from the RCSB-Protein Data
Bank. The HETATM of 1TUP was removed and optimization followed by energy
minimization with a SPDBV-Swiss-Pdb Viewer. Autodock 4.2 was used for protein
1 3

Structural characterization, photoluminescence,…
and ligands preparation. For protein, all hydrogen, including non-polar, Kollaman
charges, Gasteiger charges and AD4 type atoms were added to all atoms. After add-
ing charges; non polar hydrogen atoms were merged. Auto grid was used to gen-
erate grid maps (defned docking area was 52.87×19.01×86.22; with 3-D afnity
grid centered on the binding site of the receptor with 0.375 spacing). The Lamarck-
ian genetic algorithm (LGA) was employed for ligands conformational searching.
Default docking parameters were used with initial populations of 150 randomly
placed individuals, maximum number of 2.5×105 energy evaluations and maximum
number of 2.7×104 generations. Twenty independent docking runs were carried out
for the ligands using the above stated parameters for screening. On the basis of bind-
ing energy (B.E.) and inhibition constant (Ki); the most favorable conformations
were selected [22].
Photoluminescence and UV–Vis studies
Photoluminescence study was performed with an InVia Raman spectrophotometer
(Renishaw) using a He–Cd laser with excitation wavelength at 325 nm in order to
study the fuorescence nature of the compound and absorption spectra by using an
UV/VIS spectrophotometer (Shimadzu).
Cell line and culture
The cytotoxic study of BMS was performed on a human prostate cancer cell line
(PC-3). The cells were grown in the Roswell Park Memorial Institute Medium
(RPMI) containing penicillin G, streptomycin, NaHCO3, RPMI, FBS (fetal bovine
serum 5%), gentamycin and DDW at 37 °C with 90% humidity and 5% CO2. The
BMS was dissolved in dimethyl sulfoxide (DMSO) to prepare the solutions of dif-
ferent concentrations. The selected cell line was treated with diferent concentrations
of BMS to calculate the IC50 value using a MTT assay while control cells received
were mitoxantrone and DMSO [23].
MTT assay
The growth inhibitory efect of BMS on a human prostate cancer cell line (PC-3)
was measured by MTT assay [23]. The cells were seeded in cell culture medium to
get 105 cells/ml, thereafter, 100 µl of cell suspension per well was seeded in tissue
culture plates. The assay was performed on 96 well plates in which cells were treated
with three concentrations of BMS which were incubated for 24 h in CO2 incubator.
Then, 20 µl of freshly prepared MTT solution, 5 mg ml−1 in Phosphate bufer saline
(PBS) was added to each well. The culture plates were stirred at 150 rpm for 5 min
for mixing the MTT into media. The culture plates were incubated again for 4 h
to allow metabolization of MTT. The MTT formazan crystals were resuspended in
100 µl of DMSO and plates were stirred for 20–25 min to dissolve the crystals of
formazan. The absorbance was measured at 570 nm.
1 3

P. Awasthi, K. Devi
Antimicrobial activities
The Staphylococcus aureus (MTCC7443), Pseudomonas aeruginosa (MTCC7837),
Escherichia coli, Candida albicans (MTCC227), Aspergillus fumigatus
(MTCC3007) and Fusarium moniliforme (MTCC6985) strains were used for anti-
microbial studies. Mueller–Hinton agar (MHA) was used as culture medium and
DMSO was used as a solvent control. Streptomycin sulphate and Amphotericin B
were used as standard for antibacterial and antifungal activities. All strains were cul-
tivated for 15 h at 37 °C. The Mueller–Hinton agar (MHA) media was diluted with
water up to 1000 ml and sterilized at 120 °C for 45 min at 15–16 psi pressure in an
autoclave. The media was removed and cooled up to 40–45 °C and placed on Petri
plates (6 mm). The Petri plates were covered and set aside for an hour and then incu-
bated at 37 °C for 48 h. The zones of inhibition were measured in mm [24].
Result and discussion
Molecular geometry
Geometry optimization of molecular structure is achieved by the DFT method using
the Gauss View program, (Gaussian 03 W) (Supporting Information Fig. S1). The
molecule is showing a C1 point group with S confguration. The optimized struc-
tural parameters like interatomic angles are tabulated in (Supporting Information
Table S13). The dipole moment of the compound comes out to be 5.4037 Debye.
Vibrational assignments
The BMS consists of 48 atoms and undergoes 138 modes of vibration (Supporting
Information Fig. S1). All the fundamental vibrations are active in IR absorption.
The FT-IR spectra of the BMS was recorded and calculated in solid phase shown in
(Supporting Information Fig. S2, S3).
The BMS has two benzene rings, a sulfonamide group, an alkyl group, a car-
boxylic group, and morpholine ring. As per literature, the aromatic structure shows
the presence of C–H stretching vibration in the region 3100–3000 cm−1, which is
the characteristic region for the identifcation of C–H stretching vibration [21, 25,
26]. The FT-IR bands observed at 3091.5, 3064.6 and 3031.4 cm−1 are assigned
to C–H stretching vibrations. The scaled vibrations obtained from the B3LYP/6-
311++(2d,p) method shows a good agreement with the recorded spectral data.
The aromatic in-plane C–H bending and out-of-plane bending vibrations normally
occur in the region 1300–1000 cm−1 and 750–1000 cm−1, respectively. These bands
are sharp but have weak to medium intensity as per literature. In-plane C–H bend-
ing vibrational bands with very strong to medium intensity were found at 1232.5,
1256.8, 1277.7, 1298.9 cm−1. The out-of-plane C–H bending vibrations are 949.8,
917.2, 907.9, 870.3, 843.2, 791.3 cm−1 shown in (Supporting Information Fig. S2,
S3). These in-plane and out-of-plane C–H bending vibrations show good agreement
1 3

Structural characterization, photoluminescence,…
with the literature [21, 25, 26]. The ring stretching vibrations (C=C) are promi-
nent in the spectrum of benzene and these bands are visualized between 1520 and
1650 cm−1 [25, 26]. BMS is having two phenyl rings, where six C=C stretching
vibrations are possible and vibrations are observed at 1584.9 cm−1. The aromatic
C–C stretching vibrations normally occur in the region 1590–1430 cm−1 [21, 25,
26]. In BMS the C–C stretching vibrations are assigned at 1435.0, 1450.6, 1497.0,
1450.6, 1435, 1422 and 1356 cm−1.
The C–S vibrations occur in the region between 700 and 600 cm−1 [26]. In BMS
the C–S vibrations are assigned at 690.2, 656.8, 626.7 cm−1. The O=S=O stretching
vibrations occurs; (i) near 1350–1300 cm−1 and (ii) near 1160–1120 cm−1. In BMS
the O=S=O stretching vibrations are assigned at 1337.2, 1322.9 cm−1 and bend-
ing at 1165.8 cm−1 regions. The vibrations show good agreement within the normal
range [26]. The sulfonamides exhibit S–N stretching vibrations at 910–900 cm−1
[26]. However, in BMS the S–N vibrations are assigned at 907.9 cm−1. A strong
band near 1550 cm−1 and a weaker band near 1250 cm−1 are due to coupling of N–H
in-plane (bending) and C–N (Supporting Information Fig. S2, S3) stretching vibra-
tions. In BMS the N–C vibrations are assigned at 1256.8 cm−1. The C=O stretching
vibrations occur near 1760 cm−1 [26]. In BMS the C=O stretching vibrations are
assigned at 1649.9 cm−1. The shifting to lower frequency could be due to in O=C–N
bonding. The C–O stretching vibration in BMS has been observed in the region
1300–1030 cm−1. The band at 3300–3260 cm−1 is assigned to N–H stretching vibra-
tions. In BMS, due to a secondary N–H group, the N–H vibrations are assigned at
3263.6 cm−1. As per literature, the C–H vibrations occur near 2890 cm−1. However,
in BMS the C–H vibrations are assigned at 2897.7 cm−1.
NMR characterization
Experimental assignments of protons in 1H and 13C NMR spectra of BMS are shown
in (Supporting Information Fig. S4) and along with its theoretically calculated peaks
using the B3LYP/6-311++(2d,p) method confrmed the position of H and C and
(Supporting Information Fig. S5, S6). The phenyl protons resonate as a multiplet at
δ7.12–7.80 ppm. Aliphatic protons resonate as a multiplet at δ2.570–4.33 ppm and
the –NH– proton at the δ5.80–5.82 ppm, respectively.
The aromatic carbons resonate around 110–170 ppm as per literature [26] while
in BMS the aromatic carbons resonate as a multiplet around 127.23–139.8 ppm. C14
(Fig. 1b) of the (C=O) group resonates at the 168.93 ppm. Aliphatic carbons show
the signals in the region of 2–55 ppm as per literature, likewise aliphatic protons of
compound BMS resonate at 41.20–66.07 ppm. The N and O atoms have an electron-
egative property which polarizes the electron distribution in its bonds adjacent to
the carbon atom, hence, it alters the chemical shifts values. The down feld chemical
shift of carbon C40 and C43 attached to the O atom has been observed [26].
The aromatic protons in BMS are of two types, one in which the phenyl group
(ring A) is attached with sulfonamide group and the other in which the phenyl group
(ring B) is attached to a –CH2– group. The fve protons of benzene ring (A) attached
to sulfonamide group were observed as three distinct signals. The two protons ortho
1 3

P. Awasthi, K. Devi
to the sulfonamide group appeared at 7.88 ppm (2H), meta protons were observed
at 7.49 ppm (2H) and the para proton was observed at 7.55 ppm (1H) (Fig. 2). In
the benzene ring (B), which is attached to the methyl group, was observed as two
distinct signals. The two proton ortho to methyl groups appeared at 7.14 ppm (2H)
and the remaining three protons were observed at 7.29 ppm (3H) (Fig. 2). Fur-
ther, in morpholino the ring, the four protons of the two methylene groups ortho
to –N–CO– were observed as two distinct signals at 2.92 and 2.58 ppm and split
up into multiplets. The two axial protons are out of plane while the two equatorial
protons are in plane. The remaining four protons of the morpholino ring ortho to
–O–CH2– group appeared at 3.33 and 3.08 ppm and showed almost the same pattern
as that of four protons attached to the ortho to –N–CO– group. The –NH– protons
appeared as a doublet at 5.77 ppm. The aliphatic protons, i.e., methine and meth-
ylene groups appeared at 2.26 ppm and 4.35 ppm. J values of proton are compiled
in Table 1 and comparatives of experimental and calculated chemical shifts are in
(Supporting Information Table S14).
2‑D NMR characterization
1
The H–1H connectivity’s of BMS are shown in (Supporting Information Fig. S7).
The spectra indicate that neighboring hydrogen’s are coupling with each other. The
protons resonate in the aliphatic region 1.0–5.5 ppm are assigned as H13, H16, H17,
H38, H39, H41, H42, H44, H45, H47 and H48 directly coupled to their respective
bonded protons, i.e., H13, H16, H17, H38, H39, H41, H42, H44, H45, H47 and
H48 (Supporting Information Fig. S7). The aromatic regions protons 7.0–8.8 ppm
are assigned as H7, H8, H9, H10, H11, H22, H24, H26, H27, H28 and H34 are
also directly coupled to their neighboring protons, i.e., H7–8H and 11H, 8H–7H,
9H–10H, 10H–9H and 11H (Supporting Information Fig. S7).
Hydrogen and carbon connectivity is studied using the 1H–13C hetero nuclear sin-
gle-quantum coherence (HSQC) spectra (Supporting Information Fig. S7); tabulated
in (Supporting Information Table S15). The study confrms the structure by con-
nectivity of carbon and to its respective hydrogen and also the stability of the BMS.
Table 1 J connectivity of BMS
Proton
Chemical shift
J (Hz)
–CH2– (dd)
–CH– (td)
–NH– (d)
Ar.H, o– to S=O2 (dd)
m (td)
p-(qd)
O– to –O–CH2 (dd)
p/m
2.2681–2.5918
4.3569–4.2945
5.7701–5.7460
7.8024–7.7809
7.4996–7.4621
7.5778–7.5376
7.1414–7.1182
7.2923–7.2349
5.08, 7.44
5.52, 4.2
9.64
7.12, 3.4
1.36, 5.5
5.08, 8.32, 7.36, 2.6
7.76, 7.24
4.04, 3.04, 2.0
1 3

Structural characterization, photoluminescence,…
TOCSY (Total correlation spectroscopy)
TOCSY spectra of BMS exhibit several interproton contacts shown in (Sup-
porting Information Fig. S8) and connectivity among the protons are tabulated
in (Supporting Information Table S16). The protons H7, H8, H9, H10, and H11
of benzene ring (ring A) followed by –SO2NH– group are showing intercon-
nected peaks but are not showing any cross peaks to the other protons of BMS
due to a –SO2– group, which is a highly electronegative group and stabilizes the
–O=C–NH– bond. Likewise, all the protons of ring B and heterocyclic morpho-
line ring resonate independently and did not show any cross-connectivity (Sup-
porting Information Table S16). Further cross-connectivity of 13H (12C); 34H
(30N); 16 and 17H (15C) confrms the structure of BMS.
It can be presumed that due to fexibility of the chain 12C–15C–18C, ring B
is not stable; hence, it rotates by 3600 around the 12C–15C bond. Further, due
to the possibility of boat confrmation of the heterocyclic ring, the cross-connec-
tivity between the protons of ring B and protons of morpholine ring could not be
observed (Supporting Information Table S16).
DEPT (distortionless enhancement by polarization transfer)
The dept90 spectra give peaks for –CH– and methylene and dept135 for –CH– and
methylene carbon which give negative peaks. The dept90 spectra show the peaks
for all carbons that are part of the methine (CH) group while in the dept135 spec-
tra, the methylene carbon shows inverted peaks for carbons C15, C46, C37, C40
and C43 and methine and methyl carbons show positive peaks, and the results are
shown in the (Supporting Information Fig. S9) confrms the 13C NMR and also
the stability of the BMS.
ESI‑Mass spectroscopy
The structure of the BMS was further confrmed by recording an electron spray
mass spectrum. The molecular formula of the BMS is C19H22N2O4S showing
a molecular ion peak m/z 375.2 [M + H]+ (Supporting Information Fig. S10).
The dimerized adduct of sodium or potassium molecular ion peak is at 397
[M + Na]+. The dimer molecular ion peak with sodium adduct is observed at m/z
771 [2M + Na]+. The diferent fragment ion peaks, base peaks, and molecular ion
peaks support the stoichiometric formulation of BMS, and the results are tabu-
lated in (Supporting Information Table S17).
Molecular ions themselves act as a base peak; hence, the molecule is consid-
ered to be stable. After going through the fragmentation pattern, it is observed
that –C=O– is the weakest area in the molecule and separated with stable
fragments.
1 3

P. Awasthi, K. Devi
Fig. 3 Frontier molecular orbital (HOMO–LUMO) of BMS
Frontier molecular orbital analysis
Highest occupied molecular orbital (HOMO) and lowest occupied molecular orbital
(LUMO) energy gap are calculated and shown in Fig. 3. “HOMO” level is defned
Table 2 Calculated
DFT/B3LYP/6311++(2d,p)
BMS
Mitoxantrone
energies values of BMS and
mitoxantrone
Etotal (Hartree)
EHOMO (eV)
ELUMO (eV)
−1516.57
−0.183
−0.149
0.034
−1517.016
−0.181
−0.126
0.055
ΔEHOMO−LUMO gap (eV)
EHOMO−1 (eV)
ELUMO+1 (eV)
ΔEHOMO−1−LUMO+1 gap (eV)
−0.202
−0.122
0.080
−0.026
+0.020
0.006
Chemical hardness η (eV)
Electronegativity χ (eV)
Chemical softness ξ (eV)
Electrophilicity index ω (eV)
Dipole moment (Debye)
0.017
−0.166
29.41
0.8088
5.4035
0.0275
−0.1535
18.18
0.4283
6.89
1 3

Structural characterization, photoluminescence,…
as the one which essentially shows an electron donation behavior while “LUMO”
shows an electron acceptor behavior. A primary postulation is that the reaction
occurs with the highest possible interaction between the HOMO of one moiety with
the LUMO on another one. The interaction between them is a major reason for most
of the reactions. From DFT calculations, the chemical reactivity and kinetic stability
can be related to the energy gap (ΔΕ EHOMO−ELUMO). DFT calculation gives infor-
mation about reactivity parameters Table 2 and is calculated as follows:
(
)
ꢀ(electronegativity) = −1∕2 ELUMO + EHOMO
,
(1)
(2)
(3)
(4)
(5)
(
)
ꢀ(potential) = −ꢁ = 1∕2 ELUMO + EHOMO
,
(
)
ꢀ(hardness) = 1∕2 ELUMO − EHOMO
,
S(softness) = 1∕2ꢀ,
ꢀ(electrophilicity) = ꢁ2∕2ꢂ.
The impact of η and σ is illustrated in the estimation of both reactivity and stability.
Reactivity of synthesized analogue BMS is compared with known anticancer drug
MTX in terms of transition from HOMO to LUMO. It is well explained that organic
molecules with diverse functionality and an intense Π-system are highly reactive.
And the reactivity can be easily explained on the basis of transitions from HOMO to
LUMO. And this intramolecular electronic transition plays a vital role in its action.
The lower the gap between HOMO and LUMO, the higher would be the reactivity
and molecules are at ease to form intramolecular charge transfer complexes.
Upon comparing the HOMO–LUMO energy separation of MTX and BMS,
which comes out to be 0.055 eV and 0.034 eV respectively; BMS is chemically
reactive up to the level of MTX. All the parameters of chemical hardness, electro-
negativity, chemical softness, electrophilicity index [27] and dipole moment of the
BMS as well as MTX are calculated using B3LYP/6-311++(2d,p) on the Gaussian
and tabulated in Table 2.
Mulliken atomic charges
The Mulliken charges are directly related to the vibrational properties of a mole-
cule and quantify how the electronic structure changes under atomic displacement;
it is therefore related to the chemical bonds present in the molecule. The Mulliken
charges of BMS and mitoxantrone are computed by the B3LYP/6-311++(2d,p)
basis set shown in (Supporting Information Fig. S11). The sulfur atoms have posi-
tive values due to their electron donating ability. On the other hand, hydrogen atoms
and most of carbon atoms displayed negative values because of their electron accept-
ing ability. The negative charge on oxygen and nitrogen and the positive charge on
hydrogen atoms attached to that carbon confrms the charge transfer in the mole-
cules. The carbon atom which is attached to the oxygen and nitrogen atoms bears
positive charge due to the electron withdrawing nature of O/N and the other carbon
1 3

P. Awasthi, K. Devi
Fig. 4 UV–Vis and PL spectra of BMS
atoms are positively charged which confrms the charge transfer and the chemical
reactivity of the molecules.
UV–Vis and photoluminescence (PL) spectroscopy
The UV–Vis and PL spectra of BMS, Fig. 4, show a broad and prominent emission
peak in the visible region. The peaks in UV regions give absorbance at 262 and
273 nm. The peaks in PL gives four emission peaks in the visible region centered
at 598, 838, 894 and 1073 nm. The shifting of peaks to higher wavelength shows
that the compound is highly luminescent and has application in the area of photody-
namic therapy.
Biological activities
MTT assay
The IC50 value of BMS came out to be 2.86 µM; this shows good activity over the
tested prostate cancer cell line, better than the known anticancer drug Mitoxantrone
(3.24 µM); and is shown in (Supporting Information Fig. S12). These preliminary
results are promising, and the BMS is an active anticancer agent. Compounds bear-
ing sulfonamide features are proposed to stop the growth of cells in the G1 phase of
the cell cycle by inhibiting the synthesis of protein (p53, p21, CDKs) and nucleotide
which are needed for preparation of DNA.
Antimicrobial activities
The BMS showed good activities against Staphylococcus aureus, Pseudomonas
aeruginosa, Candida albicans, Aspergillus fumigatus and Fusarium moniliforme,
and the results are compiled in Table 3. It also showed good activities against
1 3

Structural characterization, photoluminescence,…
Table 3 Antimicrobial activities of BMS and standard drugs
Antibacterial strains
Zone of
Antifungal strains
Zone of inhibition (mm)
inhibition(mm)
BMS
Streptomy-
BMS
Amphotericin B
cin Sulphate
Staphylococcus aureus
Pseudomonas aeruginosa
Escherichia coli
11
16
–
20
20
16
Candida albicans
Aspergillus fumigatus
Fusarium moniliforme
22
14
16
30
20–24
20
resistant strains to sulfonamide, i.e., Candida albicans, Staphylococcus aureus.
The zone of inhibitions for Streptomycin is 20 mm and for BMS are 16 mm
against Pseudomonas aeruginosa and 11 mm against Staphylococcus aureus and
also no activity against E. coli. From all the bacterial and fungal strains, BMS
showed good activity against Candida albicans and Pseudomonas aeruginosa.
From this study the BMS showed good antifungal activity in comparison to the
antibacterial activity.
Docking studies
As per literature review, the majority of mutations at the p53 tumor suppressor-
DNA complex occurs in between 102 and 292 residues and, due to this, it results
in loss of DNA binding [19]. This kind of p53 activity has been identifed in
most of the tumors. Interestingly, the oxygen atom (O32) of SO2 in BMS forms
hydrogen bonds with ARG-248 and the oxygen atom (O36) of the morpholine
ring forms a hydrogen bond with GLN-165 in the DNA binding domain of the
p53 tumor suppressor-DNA complex. However, mitoxantrone and doxorubicin
show interactions with ARG-248 only. Maximum inhibition and efective binding
interactions are shown by BMS in comparison to mitoxantrone and doxorubicin
and are tabulated in Table 4 and H-bonds interactions of BMS with p53 tumor
suppressor-DNA complex shown in (Fig. 5a, b).
Table 4 Binding energy and inhibition constant obtained from docking studies
Compounds
Binding energy
Inhibition con-
stant (Ki) (μM)
Binding interactions
(B.E.) (kcal/mol)
BMS
−6.51
16.91
ARG248 NH…O2S
GLN165 NH…O oxygen of morpholine ring
ARG248 NH…O oxygen of MTX ring
ARG248 NH…OH
Mitoxantrone
Doxorubicin
−4.50
−5.38
495.34
113.56
1 3

P. Awasthi, K. Devi
Fig. 5 a Docked structure of BMS bound to p53 tumor suppressor-DNA complex (1TUP); b insightful
view of hydrogen bonds interactions of BMS
Conclusion
BMS (C19H22N2O4S) is designed and synthesized using conventional methods of
synthetic organic chemistry. The objective was to study the multifunctional appli-
cations (anticancer, antibacterial and antifungal) of the synthesized BMS mole-
cule. Structure of the BMS was confrmed by FT-IR, 1H and 13C NMR, 2-D NMR
and mass spectroscopic tools. As per literature sulfonamide shows a S–N vibra-
tional band between 910 and 900 cm−1 [26]. Likewise in BMS, the S(29)–N(30)
vibrational band appears at 907.9 cm−1 (Supporting Information Fig. S2, S3). The
shifting of the C(14)=O(33) bond to lower wave number, i.e., 1649.9 cm−1 and
168.9 ppm in 13C NMR (Supporting Information Fig. 4) confrms the bonding of
–C(14)=O(33)– to (3N5) of morpholine ring. The connectivity of H(13) to H(34) of
C(12)–N(30) bond in COSY spectra (Supporting Information Fig. S7) and connec-
tivity of C(12) atom to its proton, i.e., H(13) in HSQC spectra (Supporting Informa-
tion Fig. S7) confrms the stability of BMS structure. Further
–O(33)=C(14)–N(35)– stable fragmentation in ESI-Mass (Supporting Information
Fig. S10) confrms the attachment of the morpholine ring in BMS. Structure was
further validated by computational tools. Frontier molecular orbital theory and
Mulliken atomic charges of the BMS and MTX using B3LYP/6-311++(2d,p) on
the Gaussian has been carried out. Study indicates that the molecule is chemi-
cally reactive and participates in intramolecular charge transfer complexes. The
UV–Vis and photo luminescent study showed that the compound is highly lumi-
nescent; hence, it can have wider application in the area of photodynamic
therapy.
1 3

Structural characterization, photoluminescence,…
The antibacterial and antifungal activities are good over resistant and non-resist-
ant bacterial and fungal strains. As per literature, the available sulfonamide com-
pounds are resistant to Candida albicans, Staphylococcus aureus, E. coli, Shigella,
Streptomycin pyogenes, Streptomycin viridans, anaerobes, Gonococci, and Pnemo-
cocci strains. BMS showed good activities over two resistant strains, i.e., Candida
albicans, Staphylococcus aureus and no activity was observed in E. coli. The stand-
ard drugs used in antibacterial and antifungal assays were Straptomycin sulphate
and Amphotericin B in comparison to BMS. The –SO2NH– feature along with the
morpholino entity in the BMS molecule is responsible for their good antibacterial
activity over Straptomycin sulphate and antifungal activity over Amphotericin B due
to competitive binding with PABA on the receptor enzyme DHPS [16] of the bacte-
rial cell.
Cytotoxic study indicates that BMS has comparable anticancer activities against
the PC-3 cancer cell line in comparison to standard drug MTX. In reference to lit-
erature study, the binding mode of MTX is proposed to be an intercalative mode
of interaction with DNA. Interestingly, in the present random docking study; BMS,
Mitoxantrone and doxorubicin show binding with the ARG248 site of the p53 tumor
suppressor-DNA complex but no interactions with DNA.
Therefore, it is concluded that BMS inhibits or interacts with the most mutated
residues, i.e., ARG-248 and GLN-165 and blocks p53 function as per the docking
study and the same is confrmed by cell cycle analysis (unpublished results). Car-
bonic anhydrase inhibition and anti-diabetic studies along with detailed synthesis of
a series of analogues will be reported subsequently.
Acknowledgements The authors thank University Grants Commission-Rajiv Gandhi National Fel-
lowship for ST students, New Delhi, India (F1-17.1/2014-15/RGNF-2014-15-ST-HIM-84222/(SAIII/
Website)) for giving the fnancial assistance. We would like to thank Director, NIT Hamirpur for pro-
viding infrastructure facilities. The facilities provided by the NMR Centre and computational lab, IIT
Roorkee, Uttarakhand are highly acknowledged. Authors are thankful to Prof. Ritu Barthwal Department
of Biotechnology IIT Roorkee for her NMR and computational discussion. The paper is the original
research of the authors, and the work is also being fled as a patent with Indian Patent Application No.
201711026145.
Compliance with ethical standards
Confict of interests There is no confict to declare.
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