Design, synthesis and molecular docking studies of novel piperazine metal complexes as potential antibacterial candidate against MRSA

Article abstract of DOI:10.1016/j.molstruc.2021.130047

In the present study, a series of novel metal piperazine carboxamide analogues 5(a-l) were synthesized and amply characterized by different spectral techniques viz, LC-MS, ¹H-NMR, ¹³C-NMR, and FT-IR. Among these, 5e analogue of piperazine complex showed potent biocidal activity. Further, 5e metal complexes of piperazine copper (II), cobalt (II), zinc (II) and nickel (II) were synthesized and characterized by LC-MS and FT-IR and revealed data showed respectable blends of metal complexes. All the metal complexes of 5e analogue were screened for antibacterial activity. The 5e with copper metal complex showed an excellent antibacterial activity (5Cu). The minimum inhibitory concentration of 5e and copper complex (5cu) against MRSA were found to be 30±0.15 and 20±0.12 μg/mL and 14.5±0.04 and 15±0.08 mm of zone of inhibition respectively compared to drug streptomycin and bacitracin (10 μg/mL). The anti-bacterial potency of the newly synthesized template was also validated by membrane damaging study. The molecular docking study was performed to understand the molecular interaction and binding mode of the compounds on active site of 3VMT and 6FTB protein of MRSA. An in silico docking assisted data strongly correlated to the experimental approach of antibacterial activity against MRSA. The biocompatibility of 5e and 5cu was established and that indicated good compatible at the hemostatic level. The 5e and 5cu analogues were also evaluated for toxicity against L6 cell lines. The eventual outcome of this study shows, the synthesized 5cu complex template can be used for further development to eradicate the MRSA infection.

Full text of DOI:10.1016/j.molstruc.2021.130047

Journal of Molecular Structure 1232 (2021) 130047
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Design, synthesis and molecular docking studies of novel piperazine
metal complexes as potential antibacterial candidate against MRSA
a,∗
b,c
a
a
d
H.S. Nagendra Prasad , A.P. Ananda , S. Najundaswamy , S. Nagashree , L. Mallesha ,
a,∗
e
f
B.P. Dayananda , H.S. Jayanth , P. Mallu
a
Department of Chemistry, Sri Jayachamarajendra College of Engineering, JSS Science and Technology University, Mysuru 570 006, Karnataka, India
Research and Development Centre, Bharathiar University, Coimbatore, 641046, Tamil Nadu, India
b
c
d
e
f
Ganesh Consultancy and Analytical Services, Hebbal Industrial Area, Mysuru, 570016, Karnataka, India
PG Department of Chemistry, JSS College of Arts, Commerce and Science, Mysuru 570025, Karnataka, India
PG Department of Chemistry, Maharani’s college, University of Mysore, Mysuru, Karnataka, India
Department of Microbiology Yuvaraja’s college, University of Mysuru, Mysuru 570005, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, a series of novel metal piperazine carboxamide analogues 5(a-l) were synthesized
and amply characterized by different spectral techniques viz, LC-MS, 1H-NMR, 13 C-NMR, and FT-IR. Among
Received 3 November 2020
Revised 27 January 2021
Accepted 28 January 2021
Available online 3 February 2021
these, 5e analogue of piperazine complex showed potent biocidal activity. Further, 5e metal complexes of
piperazine copper (II), cobalt (II), zinc (II) and nickel (II) were synthesized and characterized by LC-MS
and FT-IR and revealed data showed respectable blends of metal complexes. All the metal complexes of
5e analogue were screened for antibacterial activity. The 5e with copper metal complex showed an excel-
lent antibacterial activity (5Cu). The minimum inhibitory concentration of 5e and copper complex (5cu)
against MRSA were found to be 30± 0.15 and 20± 0.12 μg/mL and 14.5± 0.04 and 15± 0.08 mm of zone
of inhibition respectively compared to drug streptomycin and bacitracin (10 μg/mL). The anti-bacterial
potency of the newly synthesized template was also validated by membrane damaging study. The molec-
ular docking study was performed to understand the molecular interaction and binding mode of the
compounds on active site of 3VMT and 6FTB protein of MRSA. An in silico docking assisted data strongly
correlated to the experimental approach of antibacterial activity against MRSA. The biocompatibility of
Keywords:
Piperazine
Copper complex
MRSA
Molecular docking
Cytotoxicity
5e and 5cu was established and that indicated good compatible at the hemostatic level. The 5e and 5cu
analogues were also evaluated for toxicity against L6 cell lines. The eventual outcome of this study shows,
the synthesized 5cu complex template can be used for further development to eradicate the MRSA infec-
tion.
©
2021 Elsevier B.V. All rights reserved.
1
. Introduction
can be an adventure in the materials of metal in drug design. Cur-
rently, drug designers have shown interest in the utilization and
formation of metal-based drugs as chemotherapeutics agents in
the field of medicinal inorganic chemistry [2]. In a recent investi-
gation, transition metal complexes showed potential biological ac-
tivity [3], which familiarize their coordination sites in the human
body’s function and also in the development of highly functional
novel metal-based complexes, are desired in the pharmaceutical
fields [4].
The transition metal complexes are frequently used in recent
studies to utilize as drugs, to combat several human diseases [1].
The new era started to develop a metal-based transition complex
to achieve a unique therapeutic application in the field of pharma-
cology. These innovations of medicinal inorganic chemistry provide
enhanced active metal complexes. The metal complexes provide
a different approach in the mode of action against living organ-
isms compared to non-metals. The medicinal inorganic chemistry
The molecular architecture heterocyclic piperazine derivatives
have produced a lot of interest due to owing to many rea-
sons, particularly their biological properties as anti-inflammatory,
anti-bacterial, anti-cancer, cardio-protective agents, anti-viral, anti-
tuberculosis, anti-diabetic, and antihistamine profiles [5,6]. The
∗
Corresponding authors.
E-mail addresses: nprasad@jssstuniv.in (H.S.N. Prasad), drmallu66@gmail.com (P.
Mallu).
https://doi.org/10.1016/j.molstruc.2021.130047
0
022-2860/© 2021 Elsevier B.V. All rights reserved.

H.S.N. Prasad, A.P. Ananda, S. Najundaswamy et al.
Journal of Molecular Structure 1232 (2021) 130047
pthalamidepiperazine derivatives have been shown to be highly ac-
tive against drug-resistant strains of Staphylococcus [7]. This syn-
thetic strategy is unique and currently in great demand due to
the use of such multi combination agents in treatments viz. can-
cer, HIV, neurodegenerative diseases, [8,9]. Piperazine itself is a re-
spectable hydrogen-bond acceptor and their metal complex capa-
bility makes it an interesting moiety for supra molecular complex
chemistry [10].
Metalloorganic chemistry is becoming an emerging area of re-
search due to the demand for new metal based antibacterial com-
pounds. The serious medical problem of bacterial resistance and
the rate at which it develops has led to increasing levels of resis-
tance to classical antibiotics among Gram-positive organisms such
as Pneumococci, Enterococci, and Staphylococci [11]. Antimicrobial
studies of a novel macro cyclic ligand containing a piperazine moi-
ety and its binuclear metal complexes such as Mn (II), Co (II), Ni
(
II) shows an excellent activity against Salmonella typhi, Bacillus
subtilis, Escherichia coli, Staphylococcus aureus, and Bacillus mega-
terium [12]. In in vitro screening of transition metal complexes of
4
-methylpiperazine-1-carbodithioate and nitrogen based mixed lig-
Fig. 1. Scheme.
and phen against microbial strains, viz. Candida albicans, Escherichia
coli, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococ-
cus faecalis [13]. Copper (II) complexes containing Schiff bases de-
rived from S-benzyldthiocarbazate and saccharinate showed an-
ticancer properties [14]. The complexes were moderately active
against the leukemic cell line (HL-60), but only [Cu (NNS) (sac)]
was found to exhibit strong cytotoxicity against the ovarian can-
cer cell line (Caov3) compared to standard anticancer drug Dox-
orubicin [15]. Complexes of chromium (III) are less cytotoxic than
chromium (VI) to cultured human cells [16]. Platinum metal com-
plexes are amongst the most widely used chemotherapeutic agents
2
2
.1. Synthesis of piperazine ligands
.1.1. 2-(2-(piperazin-1-yl) ethyl) isoindoline-1,3-dione (3)
Equimolar concentrations of isobenzofuran-1, 3-dione (0.5g) (1)
and 2-(piperazine-1-yl)ethamine (0.45g) (2) were refluxed in 25
mL of methanol for 6 hrs and 2-3 drops of glacial acetic acid was
added to the reaction mixture. The completion of the reaction was
confirmed by TLC. The solvent was concentrated in a rotary evap-
orator at 50o C and washed with distilled water. The solid prod-
uct was dried and recrystallized in methanol. The greenish-yellow
product (3) was obtained with good yield (Fig. 1).
[
17]. However, despite the comparative success of the clinically
used platinum-based drugs (cisplatin, carboplatin, and oxaliplatin),
there are significant disadvantages due to its toxicity to healthy tis-
sue and meanwhile cancer cell resistance to the drug. Therefore,
there has been considerable interest in the use of other transition
metals for drug development [18–20].
2
.1.2. Synthesis of 4-(2-(1,3-dioxoisoindolin-2-yl)ethyl)-N-
phenylpiperazine-1-carboxamide
5
(a-l)
A
solution of 2-(2-(piperazin-1-yl)ethyl)isoindoline-1,3-dione
Phthalimide (isoindoline-1,3-dione) has usually been employed
in the design of potential anti-tumor [21], immunomodula-
tory [22], anti-angiogenic [23], anti-microbial [24] and anti-
inflammatory [25] drug candidates.
(
3) (0.5g) in DCM was taken and cooled to 0-5o C in an ice bath.
3
drops of Triethylamine was added to the cold reaction mixture
and stirred for 10 min and then 0.2g of different aromatic carbox-
amide [4(a-l)] were added. The reaction mixture was refluxed for 6
hr (Fig. 1). The progress of the reaction was monitored by TLC. At
the end of the reaction, the solvent was removed under reduced
pressure and re-extracted with ethyl acetate. The obtained organic
layer was washed with 1 % of HCl and Millipore water. The de-
moisturized by passing through anhydrous sodium sulphate and
the compound was dried.
In this study, we have prepared a serious of potent piperazine
phtalamide analogues and further synthesized (Cu, Zn, Co, and
Ni) complexes. All synthesized metal complexes were systemati-
cally characterized. The antibacterial activity was assessed against
MRSA. Also, toxicity assays were carried out against normal cell
lines and piperazine pthalamide copper complexes showed good
blood biocompatibility. All the synthesized analogues validated po-
tency against MRSA by in silico molecular docking proof.
2
.1.3. Synthesis of 4-(2-(1, 3-dioxoisoindolin-2-yl)
2. Materials and methods
ethyl)-N-phenylpiperazine-1-carboxamide metal (Cu, Zn, Co and Ni)
complexes
All chemicals were procured from Sigma Aldrich, Bangalore, In-
The metal complexes of 4-(2-(1,3-dioxoisoindolin-2-yl)ethyl)-
N-phenylpiperazine-1-carboxamide were synthesized as per pro-
tocol shown in Scheme 1. 4-(2-(1,3-dioxoisoindolin-2-yl)ethyl)-N-
phenylpiperazine-1-carboxamide (0.2 g) was dissolved in methanol
and 1.76 g of the metal sulphates such as Cu, Zn, Co and Ni was
dissolved in Millipore water separately and stirred for about 30
min. The catalytic amount of triethylamine was added to the reac-
tion mixture and heated up to 80°C for 15 min, followed by cooling
in a cube of ice, the precipitate was filtered, washed with Millipore
water and dried in an oven (Fig. 1).
dia. Analytical grade solvents and reagents were used for the syn-
thesis. For column chromatography, Silica gel was procured from
Merck Pvt. Ltd., Mumbai, India. The IR spectrum was recorded on
Agilent Cary 630 FTIR spectrometer. All the spectra were run in the
range 400–4000 cm 1 at room temperature. The 1H and 13C NMR
−
spectrum was recorded in CDCl . All chemicals shift are reported
3
in ppm relative to TMS, Mass spectroscopic analysis was performed
in a Water micro TOF QII mass spectrometer. The lyophilized refer-
ence Staphylococcus aureus-96 strain was procured from Microbial
Typing Culture Collection (MTCC), Chandigarh, India, and cultured
in recommended broth as per the revival procedure provided by
MTCC. The required culture medium was purchased from Hi-Media
Laboratories, Mysuru, India.
2.1.3.1. 4-(2-(1,
3-Dioxoisoindolin-2-yl)ethyl)-N-phenylpiperazine-1-
carboxamide (5a). Petroleum ether/ethyl acetate (2:3) v/v solvent
1
system was used in column chromatography. 85% yield. H NMR:
2

H.S.N. Prasad, A.P. Ananda, S. Najundaswamy et al.
Journal of Molecular Structure 1232 (2021) 130047
(
400 MHz, CDCl ) 7.82 (d, 2H,Ar-H, J=6.4Hz) 7.67 (d, 2H, Ar-H,
139.10(Ar-C), 134.30 (Ar-C), 132.50 (Ar-C), 129.25 (Ar-C), 124.30
3
J=7.2Hz) 7.39 (d, 2H, Ar-H, J=8.2Hz) 6.93 (d, 2H,Ar-H, J=8.4Hz)
.85 (t, 1H,Ar-H, J=1.81Hz) 4.21 (s, 1H,N-H) 3.25 (d, 4H,pip-H,
J=7.1Hz) 3.24 (t, 2H,C-H, J=7.5Hz) 2.90 (d, 4H,Pip-H, J=7.2Hz)
(Ar-C), 122.45(Ar-C), 58.78 (Pip-C), 54.52 (Pip-C), 48.27(CH ),
2
6
39.84(CH ). LCMS m/z Calculated for C21H21ClN O : (M-H) 412.87:
2
4
3
found 412.94, IR Vmax(cm 1) 3360(N-H), 1740 (C=O), 1678 (C=C),
1345 (C-N), 754 (C-Cl) Elemental Analysis: Calculated: C, 64.57; H,
6.66; N, 19.85; Experimental: C, 64.56; H, 6.56; N, 19.81.
−
13
2
.62 (t, 2H,C-H, J=7.3Hz). C NMR: (400Hz, CDCl ) 181.19 (C=O),
3
1
68.25(NC=O), 138.13(Ar-C), 134.28 (Ar-C), 132.45(Ar-C), 129.20
Ar-C), 124.21(Ar-C), 122.45(Ar-C), 58.78 (Pip-C), 54.52(Pip-C),
8.27(CH ), 39.84(CH ). LCMS m/z Calculated for C21H22N O :
(
4
2.1.3.6. N-(4-Chlorophenyl)-4-(2-(1,3-dioxoisoindolin-2-yl)
ethyl)
2
2
4
3
M-H) 378.42. Found 378.45, IR Vmax(cm 1) 3360 (N-H), 1740
−
piperazine-1-carboxamide (5f).. Petroleum ether/ethyl acetate (2:3)
(
(
C=O), 1678 (C=C),1345 (C-N), Elemental Analysis: Calculated: C,
v/v solvent system has used in column chromatography.85%
1
6
4.57; H, 6.66; N, 19.85; Experimental: C, 64.56; H, 6.56; N, 19.81
yield. H NMR: (400 MHz, CDCl ) 8.25 (d, 2H,Ar-H, J=8.1Hz) 7.67
3
(
d, 2H, Ar-H, J=7.9Hz) 7.40 (s, 1H, Ar-H) 7.25 (d, 2H,Ar-H, J=7.2Hz)
2
.1.3.2. 4-(2-(1,3-Dioxoisoindolin-2-yl) ethyl)-N-(p-tolyl) piperazine-1-
6.93 (d,1H,Ar-H, J=6.9Hz) 6.85(t, 1H, Ar-H, J=6.9Hz) 6.55 (d, 1H,
carboxamide (5b). Petroleum ether/ethyl acetate (2:3) v/v sol-
Ar-H, J=6.5Hz) 4.21 (s, 1H,N-H) 3.25 (d, 4H,pip-H) 3.24 (t, 2H,C-H,
1
13
vent system was used in column chromatography.80% yield. H
J=7.2Hz) 2.90 (d, 4H,Pip-H, J=7.1Hz) 2.62 (t, 2H,C-H, J=7.3Hz).
C
NMR: (400 MHz, CDCl3) 7.75 (d, 2H,Ar-H, J=6.5Hz) 7.56 (d,
NMR: (400Hz, CDCl ) 180.40 (C=O), 169.21(NC=O), 139.10(Ar-C),
3
2
H, Ar-H, J=7.3Hz) 7.25 (d, 2H, Ar-H, J=7.5Hz) 6.85 (d, 2H,Ar-
134.30 (Ar-C), 132.50(Ar-C), 129.25 (Ar-C), 124.30 (Ar-C), 122.45(Ar-
C), 58.78 (Pip-C), 54.52 (Pip-C), 48.27(CH ), 39.84 (CH ). LCMS
H, J=7.4Hz) 4.25 (s, 1H,N-H) 3.52 (d, 4H,pip-H, J=6.9Hz) 3.40
2
2
(
t, 2H,C-H, J=7.3Hz) 2.65 (d, 4H,Pip-H, J=7.0Hz) 2.45 (t, 2H,C-
m/z Calculated for C21H21ClN O : (M-H) 412.87: found 412.94, IR
4 3
13
−1
H, J=7.2Hz)2.25 (s, 3H,-CH ).
C
NMR: (400Hz, CDCl ) 180.21
Vmax(cm ) 3360(N-H), 1740(C=O), 1678 (C=C),1345(C-N),754(C-
Cl) Elemental Analysis: Calculated: C, 64.57; H, 6.66; N, 19.85;
Experimental: C, 64.56; H, 6.56; N, 19.81.
3
3
(
C=O), 169.10(NC=O), 139.10(Ar-C), 135.20 (Ar-C), 132.50(Ar-C),
1
29.30 (Ar-C), 124.15(Ar-C), 122.50(Ar-C), 59.70 (Pip-C), 55.02(Pip-
C), 48.15(CH ), 39.60(CH ), 25(CH3) LCMS m/z Calculated for
2
2
C22H24N O : (M-H) 392.45: found 392.55, IR Vmax(cm 1) 3350 (N-
−
2.1.3.7. N-(2-Chlorophenyl)-4-(2-(1,3-dioxoisoindolin-2-yl)
ethyl)
4
3
H), 1730 (C=O), 1665 (C=C), 1350 (C-N), Elemental Analysis: Calcu-
lated: C, 64.57; H, 6.66; N, 19.85; Experimental: C, 64.51; H, 6.58;
N, 19.82
piperazine-1-carboxamide (5g). Petroleum ether/ethyl acetate (2:3)
v/v solvent system was used in column chromatography.90%
1
yield. H NMR: (400 MHz, CDCl ) 8.85 (d, 2H,Ar-H, J=7.5Hz) 8.75
3
(
d, 2H, Ar-H, J=7.5Hz) 7.85 (d, 1H, Ar-H, J=7.3Hz) 7.55 (t, 1H,Ar-H,
2
.1.3.3. 4-(2-(1,3-Dioxoisoindolin-2-yl)
ethyl)-N-(4-methoxyphenyl)
J=7.2Hz) 7.15(t,1H,Ar-H, J=7.1Hz) 6.95(d,1H,Ar-H, J=6.9Hz) 4.15 (s,
1H,N-H) 3.20 (d, 4H,pip-H, J=6.8Hz) 3.25 (t, 2H,C-H, J=6.9Hz) 2.95
piperazine-1-carboxami de (5c). Petroleum ether/ethyl acetate (2:3)
13
v/v solvent system was used in column chromatography 89%
(d, 4H,Pip-H, J=7.0Hz) 2.62 (t, 2H,C-H, J=7.2Hz). C NMR: (400Hz,
1
yield. H NMR: (400 MHz, CDCl ) 8.84 (d, 2H,Ar-H, J=6.9Hz) 7.78
CDCl ) 181.19 (C=O), 168.25(NC=O), 138.13(Ar-C), 134.28 (Ar-C),
3
3
(
d, 2H, Ar-H, J=7.3Hz) 7.25 (d, 2H, Ar-H, J=7.5Hz) 6.86 (d, 2H,Ar-H,
132.45(Ar-C), 129.20 (Ar-C), 124.21(Ar-C), 122.45(Ar-C), 58.78 (Pip-
C), 54.52(Pip-C), 48.27(CH ), 39.84 (CH ). LCMS m/z Calculated
J=7.6Hz) 4.20 (s, 1H,N-H) 3.70(s, 3H, O-CH ) 3.15 (d, 4H,pip-H,
3
2
2
for C21H20Cl N O : (M-H) 412.87: found 412.82, IRVmax(cm 1)
−
J=7.2Hz) 3.25 (t, 2H,C-H, J=7.3Hz) 2.95 (d, 4H,Pip-H, J=7.1Hz) 2.70
2 4 3
13
(
(
t, 2H,C-H, J=7.2Hz). C NMR: (400Hz, CDCl ) 180.19 (C=O), 169.30
3360(N-H), 1740(C=O), 1678 (C=C), 1345(C-N), 754(C-Cl) Elemen-
tal Analysis: Calculated: C, 64.57; H, 6.66; N, 19.85; Experimental:
C, 64.56; H, 6.56; N, 19.81.
3
NC=O), 138.40(Ar-C), 135.01 (Ar-C), 132.40(Ar-C), 129.10 (Ar-C),
1
24.15 (Ar-C), 122.50(Ar-C), 59.70 (Pip-C), 55.40(Pip-C), 48.20(CH ),
2
3
9.90(CH ). LCMS m/z Calculated for C24H24N O : (M-H) 408.45:
2 4 4
−
1
found 408.87, IR Vmax(cm ) 3360 (N-H), 1740 (C=O), 1678 (C=C),
2.1.3.8.N-(3,5-Dichlorophenyl)-4-(2-(1,3-dioxoisoindolin-2-
1345 (C-N),1160 (O-CH ) Elemental Analysis: Calculated: C, 64.57;
yl)ethyl)piperazine-1-carboxamide
(5h). Petroleum
ether/ethyl
3
H, 6.66; N, 19.85; Experimental: C, 64.59; H, 6.57; N, 19.83.
acetate (2:3) v/v solvent system was used in column chromatog-
1
raphy. 88% yield. H NMR: (400 MHz, CDCl ) 8.16 (d, 2H,Ar-H,
3
2
.1.3.4. (2-(1,3-Dioxoisoindolin-2-yl)
ethyl)-N-(4-hydroxyphenyl)
J=7.2Hz) 7.95 (d, 2H, Ar-H, J=7.1Hz) 7.39 (s, 1H, Ar-H, J=1.1Hz)
piperazine-1-carboxamide (5d). Petroleum ether/ethyl acetate (2:3)
6.93 (s, 1H,Ar-H) 6.85(s, 1H,Ar-H) 4.20 (s, 1H,N-H) 3.45 (d, 4H,pip-
v/v solvent system was used in column chromatography. 90%
H, J= 6.9Hz) 3.30 (t, 2H,C-H, J=6.9Hz) 2.80 (d, 4H,Pip-H, J=7.1Hz)
1
13
yield. H NMR: (400 MHz, CDCl ) 8.45 (d, 2H,Ar-H, J=7.5Hz) 8.48
2.55 (t, 2H,C-H, J=7.2Hz). C NMR: (400Hz, CDCl ) 180.40 (C=O),
3
3
(
d, 2H, Ar-H, J=7.2Hz) 7.95 (d, 2H, Ar-H, J=7.4Hz) 6.89 (d, 2H,Ar-H,
169.21(NC=O), 139.10(Ar-C), 134.30 (Ar-C), 132.50(Ar-C), 129.25
(Ar-C), 124.30 (Ar-C), 122.45(Ar-C), 58.78 (Pip-C), 54.52 (Pip-C),
48.27(CH ), 39.84 (CH ). LCMS m/z Calculated for C21H20Cl N O :
J=7.5Hz) 5.30(s,1H,OH) 4.19 (s, 1H,N-H) 3.25 (d, 4H,pip-H, J=7.1Hz)
.24 (t, 2H,C-H, J=7.2Hz) 2.90 (d, 4H,Pip-H, J=6.9Hz) 2.62 (t, 2H,C-
3
2
2
2
4
3
13
−1
H, J=7.2Hz). C NMR: (400Hz, CDCl ) 180.50 (C=O), 169.40(NC=O),
(M-H) 447.31: found 447.31, IR Vmax(cm ) 3360(N-H), 1740(C=O),
1678 (C=C), 1345(C-N), 754(C-Cl) Elemental Analysis: Calculated:
C, 64.57; H, 6.66; N, 19.85; Experimental: C, 64.56; H, 6.56; N,
19.81
3
1
39.15(Ar-C), 135.30 (Ar-C), 132.21 (Ar-C), 129.10 (Ar-C), 125.10(Ar-
C), 122.40(Ar-C), 58.70 (Pip-C), 54.50(Pip-C), 49.23(CH ), 39.45
2
(
CH2 . LCMS m/z Calculated for C21H22N O : (M-H) 394.42: found
4 4
94.15, IR Vmax(cm 1) 3355 (N-H), 3400 (OH) 1735 (C=O), 1670
−
3
(
C=C), 1300 (C-N), Elemental Analysis: Calculated: C, 64.57; H,
2.1.3.9. 4-(2-(1,3-Dioxoisoindolin-2-yl)
piperazine-1-carboxamide (5i). Petroleum ether/ethyl acetate
2:3) v/v solvent system was used in column chromatogra-
ethyl)-N-(4-fluorophenyl)
6
.66; N, 19.85; Experimental: C, 64.58; H, 6.53; N, 19.85.
(
1
2
.1.3.5. N-(4-Chlorophenyl)-4-(2-(1,3-dioxoisoindolin-2-yl)
ethyl)
phy. 90% yield. H NMR: (400 MHz, CDCl3) 8.40 (d, 2H,Ar-H,
J=7.0Hz) 7.85(d, 2H, Ar-H, J=7.5Hz) 7.20(d, 2H, Ar-H, J=7.5Hz)
6.60 (d, 2H,Ar-H, J=7.5Hz) 4.15 (s, 1H,N-H) 3.05 (d, 4H,pip-H,
J=6.9Hz) 3.08 (t, 2H,C-H, J=6.9Hz) 2.95 (d, 4H,Pip-H, J=6.8Hz)
piperazine-1-carboxamide (5e). Petroleum ether/ethyl acetate (2:3)
v/v solvent system was used in column chromatography. 95%
1
yield. H NMR: (400 MHz, CDCl ) 8.89 (d, 2H,Ar-H, J=6.9Hz) 7.45
3
13
(
d, 2H, Ar-H, J=8.2Hz) 7.20 (d, 2H, Ar-H, J=8.1Hz) 6.80 (d, 2H,Ar-H,
2.65(t, 2H,C-H, J=6.1Hz). C NMR: (400Hz, CDCl ) 181.10 (C=O),
3
J=7.2Hz) 4.20 (s, 1H,N-H) 3.20 (d, 4H, pip-H, J=7.1Hz) 2.99 (t,
H,C-H, J=6.9Hz) 2.85 (d, 4H,Pip-H, J=6.8Hz) 2.65 (t, 2H,C-H,
169.20(NC=O), 138.14(Ar-C), 134.30 (Ar-C), 132.50(Ar-C), 129.30
(Ar-C), 125.10(Ar-C), 123.50(Ar-C), 59.15 (Pip-C), 54.40(Pip-C),
48.50(CH ), 39.70(CH ). LCMS m/z Calculated for C21H21FN O :
2
13
J=7.1Hz). C NMR: (400Hz, CDCl ) 180.40 (C=O), 169.21(NC=O),
3
2
2
4
3
3

H.S.N. Prasad, A.P. Ananda, S. Najundaswamy et al.
Journal of Molecular Structure 1232 (2021) 130047
−
1
(
M-H) 396.41: found 396.35, IR Vmax(cm ) 3360(N-H), 1740(C=O),
2.1.4.4. Synthesis
of
cobalt
N-(4-chlorophenyl)-4-(2-(1,3-
1
678 (C=C),1345(C-N), 750 (C-F) Elemental Analysis: Calculated: C,
dioxoisoindolin-2-yl) ethyl) piperazine-1-carboxamide complex (5Co).
IRυmax(cm 1
−
)
1650(C=C), 1358(C-N), 1700(C=O), 706(C-Cl),
6
4.57; H, 6.66; N, 19.85; Experimental: C, 64.576; H, 6.55; N, 19.83
4
80(Cu-N). LCMS m/z Calculated: (M-H) 470.79: found 470.80
2
.1.3.10.N-(4-Bromophenyl)-4-(2-(1,3-dioxoisoindolin-2-yl)
ethyl)
2
.2. Antibacterial activity against MRSA
piperazine-1-carboxamide (5j). Petroleum ether/ethyl acetate (2:3)
v/v solvent system was used in column chromatography.85%
1
2.2.1. Preparation of bacterial culture and maintenance
yield. H NMR: (400 MHz, CDCl ) 9.15 (d, 2H,Ar-H, J=8.5Hz) 8.60
3
The single purified colony was used, which was obtained from
frozen glass beads of glycerol vial stock (previously culture coated
on glass beads and stored at -18o to -22° C). The glass bead was
inoculated to Brain heart infusion (BHI) broth and incubated at 37o
(
d, 2H, Ar-H, J=7.5Hz) 8.10 (d, 2H, Ar-H, J=7.1Hz) 7.70 (d, 2H,Ar-H,
J=7.2Hz) 4.10 (s, 1H,N-H) 3.10 (d, 4H,pip-H, J=7.1Hz) 3.08 (t,
H,C-H, J=6.9Hz) 2.80 (d, 4H,Pip-H, J=7.1Hz) 2.75 (t, 2H,C-H,
2
13
J=6.9Hz). C NMR: (400Hz, CDCl ) 181.35 (C=O), 168.30(NC=O),
3
6
C for 24 h. The cell density was adjusted to 1 × 10 CFU/mL by
1
38.15 (Ar-C), 134.29 (Ar-C), 132.50(Ar-C), 129.15 (Ar-C), 124.30
using UV-vis spectroscopy at 600 nm [26].
(
Ar-C), 122.50(Ar-C), 59.70 (Pip-C), 54.40 (Pip-C), 49.10(CH ),
2
4
4
0.10(CH ). LCMS m/z Calculated for C21H21BrN O : (M-H)
2 4 3
−
57.32: found 457.25, IR Vmax (cm 1) 3360(N-H), 1740(C=O), 1678
2.2.2. Minimum inhibitory concentration by resazurin assay
The MIC of synthesized metal complexes was evaluated by re-
sazurin assay. 100μL of sterile BHI broth was dispensed in each of
(
C=C),1345(C-N), 760(C-Br) Elemental Analysis: Calculated: C,
6
4.57; H, 6.66; N, 19.85; Experimental: C, 64.56; H, 6.56; N, 19.81
9
6 wells. The different concentrations of metals complex were dis-
solved in dimethyl sulfoxide (DMSO) and mixed with media. To all
the wells 10 μL of resazurin indicator (270 mg in 40 mL sterilized
distilled water) solution was added. Finally, 10 μL of bacterial sus-
pension (5 × 10 cfu/mL) was added to give 5 × 10 cfu/mL. The
broad-spectrum antibiotics Streptomycin and Bacitracin served as
standard bactericidal agents. A combination with all additions, ex-
cept the test compound served as a positive control. The ones with
all solutions without the addition of bacterial culture was a nega-
2
.1.3.11. 4-(2-(1,3-Dioxoisoindolin-2-yl)
ethyl)-N-(4-iodophenyl)
piperazine-1-carboxamide (5k). Petroleum ether/ethyl acetate
2:3) v/v solvent system was used in column chromatogra-
(
6
5
1
phy, 90% yield. H NMR: (400 MHz, CDCl3) 8.85 (d, 2H,Ar-H,
J=8.2Hz) 8.60 (d, 2H, Ar-H, J=7.1Hz) 7.40 (d, 2H, Ar-H, J=7.2Hz)
6
.60 (d, 2H,Ar-H, J=7.1Hz) 4.10 (s, 1H,N-H) 3.15 (d, 4H,pip-H,
J=6.9Hz) 3.10 (t, 2H,C-H, J=6.8Hz) 2.80 (d, 4H,Pip-H, J=6.9Hz)
13
2
.70 (t, 2H,C-H, J=6.8Hz). C NMR: (400Hz, CDCl ) 182.42 (C=O),
3
tive control. The plates were wrapped with thin plastic film and
incubated at 37o C for 24 h [27].
1
68.20(NC=O), 138.40(Ar-C), 134.20 (Ar-C), 132.40(Ar-C), 129.10
Ar-C), 124.20(Ar-C), 122.50(Ar-C), 58.70 (Pip-C), 54.40(Pip-C),
8.25(CH ), 39.80(CH ). LCMS m/z Calculated for C21H21IN O :
(
4
2
2
4
3
2
.2.3. Disc diffusion method
−1
(
M-H) 428.94: found 428.87, IR Vmax(cm ) 3365(N-H), 1745(C=O),
The antibacterial activity of synthesized 5e analogues of piper-
1
670 (C=C),1330 (C-N), 850 (C-I) Elemental Analysis: Calculated: C,
azine and 5cu complex was tested by using disc diffusion method
6
4.57; H, 6.66; N, 19.85; Experimental: C, 64.56; H, 6.56; N, 19.81
in dose dependent manner to susceptibility. The bacterial cultures
7
were prepared from the overnight culture and 1 × 10 CFU/mL
2
.1.3.12. 4-(2-(1,
3-Dioxoisoindolin-2-yl)
ethyl)-N-(4-nitrophenyl)
cells and inoculated on to nutrient agar, and then sterile disc (6
mm) was loaded with different concentration of 5e analogues of
piperazine and 5cu complex. The sterile saline water was used as
negative control and Streptomycin (10μg/disc) as a positive con-
trol. The plates were inverted and incubated at 37°C for 24 h to
examine the zone of inhibition (ZOI) [28].
piperazine-1-carboxamide (5l). Petroleum ether/ethyl acetate (2:3)
v/v solvent system was used in column chromatography. 85%
yield.1H NMR: (400 MHz, CDCl ) 8.80 (d, 2H,Ar-H, J=7.5Hz) 8.60
3
(
d, 2H, Ar-H, J=7.5Hz) 7.40 (d, 2H, Ar-H, J=7.2Hz) 6.90 (d, 2H,Ar-H,
J=7.2Hz) 4.05 (s, 1H,N-H) 3.10 (d, 4H,pip-H, J=7.3Hz) 3.05 (t, 2H,C-
13
H, J=7.1Hz) 2.95 (d, 4H,Pip-H, J=7.3Hz) 2.65 (t, 2H,C-H, J=7.1Hz).
C
NMR: (400Hz, CDCl ) 182.15 (C=O), 168.10(NC=O), 138.14(Ar-C),
3
2.2.4. MRSA membrane damage visualized by SEM
1
34.50 (Ar-C), 132.50(Ar-C), 129.30 (Ar-C), 125.30(Ar-C), 122.50(Ar-
The membrane damage of MRSA was evinced by scanning
electron microscopy (SEM). The freshly grown MRSA culture was
treated with minimum inhibitory concentration of 5Cu for 2 h,
then cells were pelleted by centrifugation (10,000 rpm for 5 min)
at 27°C. For control, MRSA culture was prepared without treat-
ment. The cells were fixed by using glutaraldehyde (2.5%) in PBS,
pelleted and deposited on glass slide followed by stepwise treat-
ment of 30% to 100% ethanol drying. After, 2 days drying under
room temperature. The prepared sample was used for SEM analy-
sis [29].
C), 59.70 (Pip-C), 54.50(Pip-C), 48.25(CH ), 39.85(CH ). LCMS
2
2
m/z Calculated for C21H21N O : (M-H) 428.94: found 428.87, IR
5
5
Vmax (cm-1) 3370(N-H), 1745(C=O), 1670 (C=C), 1340(C-N), 1650
NO ) Elemental Analysis: Calculated: C, 64.57; H, 6.66; N, 19.85;
(
2
Experimental: C, 64.56; H, 6.56; N, 19.81.
2
.1.4.1. Synthesis
of
copper
N-(4-chlorophenyl)-4-(2-(1,3-
dioxoisoindolin-2-yl) ethyl) piperazine 1-carboxamide complex (5Cu).
−
IR
υ
max(cm 1
)
1650(C=C), 1358(C-N), 1750(C=O), 706(C-Cl),
4
80(Cu-N). LCMS m/z Calculated: (M-H) 475.41: found 475.42
2
.2.5. Blood biocompatibility
The biocompatibility of synthesized 5e analogues of piperazine
2
2
.1.4.2. Synthesis of zinc N-(4-chlorophenyl)-4-(2-(1,3-dioxoisoindolin-
and 5cu complex were assessed by dispensing different concentra-
tions to freshly collected citrated human blood and incubated at
-yl)
eth
−
-yl)
1610(C=C), 1365(C-N), 1705(C=O), 710(C-Cl),
60(Zn-N). LCMS m/z Calculated: (M-H) 477.24: found 477.25
piperazine-1-carboxamide
complex
(5Zn).
IRυmax(cm 1
5
)
3
7°C for 30 min. The supernatant was collected by centrifuging at
000 rpm for 5 min and the intensity of hemolysis was measured
3
at 545 nm. If samples were highly hemocompatible for HR < 5%,
moderately hemocompatible for HR < 10% and, highly non-hem
compatible for HR > 20% [30].
2
.1.4.3. Synthesis
of
nickel
N-(4-chlorophenyl)-4-(2-(1,3-
dioxoisoindolin-2-yl) ethyl) piperazine-1-carboxamide complex(5Ni).
IRυmax(cm 1)1630(C=C), 1355(C-N), 1710(C=O), 720(C-Cl), 450(Ni-
N). LCMS m/z Calculated: (M-H) 470.55: found 470.50
−
HR=OD treated-OD negative control/OD positive control-OD
negative control × 100.
4

H.S.N. Prasad, A.P. Ananda, S. Najundaswamy et al.
Journal of Molecular Structure 1232 (2021) 130047
2
.3. Molecular docking validation
The co-ordinates of wild-type 3VMT and the 6FTB from Staphy-
lococcus aureus were obtained from the RCSB Protein Data Bank.
The crystal structure was transported and refined by a multistep
procedure through the protein preparation wizard of Maestro 9.3
software, which incorporates energy minimization utilizing OPLS-
2
005 force field, correct bond orders were allocated, hydrogen
˚
atoms were included and the water molecules removed beyond 5A
from hetero atom, formal charges, amide groups of Asn and Gln
were optimized. All amino acid flips were allocated to address ge-
ometry and hydrogen bonds were improved. Utilizing PROPKA, pH
was fixed and optimized to 7.5. Non-hydrogen atoms were mini-
˚
Fig. 2. Pharmacophore model of synthesized compounds 5(a-l).
mized by controlled minimization to default RMSD to 0.3A. Utiliz-
ing Extra-accuracy (XP) docking and scoring each compound were
˚
˚
˚
docking into the receptor grid of radii 30A × 30A × 30A and the
namic property shown in Fig. 2. To explore the depth of the physic-
ochemical behaviour of the synthesized compounds, we designed
a variety of substitutions at thioamide piperazine with a different
type of phenyl group.
docking calculation were judge dependent on the Glide score [31].
2
2
.4. Toxicity of 5e and 5Cu against L6 cell lines
.4.1. Preparation of L6 cell lines and MTT assay
The physicochemical properties of the synthesized compounds
were studied using Osiris data warrior V.4.2.2 [33]. The physico-
chemical parameters such as clogP, clogS, drug-likeness, total sur-
face area, polar surface area, and H-acceptor and H-donor pa-
rameters have been tabulated in Table 1. The acceptability of the
compounds was further evaluated based on Lipinski’s rule of five,
which is essential for rational drug design. Under Lipkinski’s rule
of five [34,35] compound must have molecular weight ≤ 500 Dal-
tons, ≤ 5 hydrogen bond donors, ≤ 10 hydrogen bond acceptors
and logP of ≤ 5.
The monolayer cell culture was trypsinized and the cell count
5
was adjusted to 5.0 × 10 cells/ml using recommended media con-
taining 10% FBS (Fetal Bovine Serum). 100 μL of the diluted cell
suspension (50,000 cells/well) was dispended to 96 well microtiter
plate. After 24h, when a partial monolayer was formed, the su-
pernatant was flicked off, the monolayer was washed once with
medium and 100 μl of different concentrations of test drugs were
mixed with cell line culture. The plate was incubated at 37°C for
2
4 h in 5% CO2 atmosphere. After incubation the test solutions in
The compounds satisfying aforesaid rules are considered as
drug-like. According to this rule, compounds with the number of
violations not more than 1, indicate good bioavailability. Poor in-
gestion and permeation will probably happen when any two of
the previously mentioned rules are neglected. The PSA (polar sur-
face area) of all the combined mixes were inside the worthy range
−
1
the wells were discarded and 100 μl of MTT (5 mg/10 mL of MTT
in PBS) was added to each well. The plates were incubated for 4 h
at 37° C in 5% CO2 atmosphere. The supernatant was removed and
1
00 μl of DMSO was added and the plate was gently shaken to sol-
ubilize the formed formazan. The absorbance was measured using
a microplate reader at a wavelength of 590 nm. The percentage
growth inhibition was calculated based on concentration of test
drug needed to inhibit cell growth by 50% (IC50) values cell line
2
and within the acceptable limit (less 140Ȃ ). The synthesized com-
pounds were within the standards of rules of Lipinski’s parameters
Therefore, the compounds possess the property of drug-likeness.
[
32].
3
.2. Synthesis and characterization of carboxamide derivatives
3. Result and discussion
A series of carboxamide derivatives of 2-(2-(Piperazin-1-yl)
3
.1. Preparation 5(a-l) moiety
ethyl) isoindoline-1,3-dione (3) were synthesized as shown in
the Fig. 1. The structures of synthesized compounds were es-
tablished based on the basis of spectral studies (Fig.S1-S12). The
chemical structure and physical data of synthesized compounds
are given in table S1. The elemental analyses data indicated ad-
mirable results between the experimentally determined values and
the theoretically calculated values within ± 0.4%. Synthesis of (2-
(piperazin-1-yl) ethyl) isoindoline-1,3-dione (3) was single-step re-
The designed molecules were divided into three parts: part A
with phthalimide ring as a hydrophilic unit, part B phthalimide
with only oxygen as electron donor and part C phenyl ring with
substituted to the linker thioamide of piperazine moiety (as hy-
drogen acceptor/donor unit [HAD]) with promising biological activ-
ity to succeed physicochemical, pharmacokinetic and pharmacody-
Table 1
Physicochemical aspects of synthesized compounds.
Sl No
Total MolWeight
eLogP
eLogS
H-Acceptors
H-donors
Total Surface Area
Polar Surface Area
Druglikeness
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
378.431
392.458
408.457
394.430
412.876
412.876
412.876
447.321
396.421
457.327
504.323
423.428
2.2662
2.6101
2.1962
1.9205
2.8722
2.8722
2.8722
3.4782
2.367
-2.585
-2.929
-2.603
-2.289
-3.321
-3.321
-3.321
-4.057
-2.899
-3.419
-3.601
-3.045
7
7
8
8
7
7
7
7
7
7
7
10
1
1
1
2
1
1
1
1
1
1
1
1
284.19
296.45
306.45
290.54
299.61
299.61
299.61
315.03
290.54
302.82
310.47
307.86
72.96
72.96
82.19
93.19
72.96
72.96
72.96
72.96
72.96
72.96
72.96
118.78
7.3716
7.2888
7.2795
7.3378
7.3784
7.3784
7.3784
7.3784
6.0316
5.5816
7.7267
2.2383
g
h
i
j
2.9914
2.7033
0.4181
k
l
5

H.S.N. Prasad, A.P. Ananda, S. Najundaswamy et al.
Journal of Molecular Structure 1232 (2021) 130047
Fig. 3. FTIR Spectra of synthesized complexes.
Fig. 5. XRD of complex 5Cu.
action between commercially available phthalic anhydride (1) and
range of 4000-400 cm 1. The ligand 5e shows absorption bands
−
2
-(piperazine-1-yl) ethamine(1equi) (2). The spectral analysis of
−
1
−1
synthesized compound (3) using FT-IR spectra range at 3350 cm
at 1740 cm
due to the presence of C=O stretch, the absorp-
−1
indicates the presence of N-H group at the piperazine ring. The
tion bands at 1500cm
are assigned to the aromatic C=C stretch
−1
−1
−1
band at 1258 cm
and 770 cm
is due to the stretching vibra-
and the appearance of the band at 1260 cm
is due to the
tion C-N and -CH2 rocking respectively. The peak at 1780 cm 1 in-
dicates the formation of the keto group (C=O) in the synthesized
compound (3). The 1H-NMR spectra analysis of the compound (3)
shows doublet peak at 7.3 indicating the presence aromatic pro-
ton and singlet peak at 4.2 shows the presence of NH group in the
synthesized compound. In the mass spectra of compound 3, the
observed molecular ion peak at m/z 259.30 is in agreement with
−
stretching of vibration C-N. The appearance of absorption bands
−
1 is
at 751-795 cm
due to -CH2 Rocking and absorption band at
−1
3253-3326 cm
is due to the N-H stretch in carboxamide 5(a-
†
l) derivatives (ESI ). N-(4-Chlorophenyl)-4-(2-(1,3-dioxoisoindolin-
2-yl)ethyl)piperazine-1-carboxamide complexes, 5Cu, 5Zn, 5Ni and
5Co were synthesized (Fig. 1). Elemental analyses data of theo-
retically calculated values and experimentally determined values
were within the limits of permissible error. The absence of NH ab-
sorption bands in the IR spectra confirmed the synthesized com-
plexes shown in Fig 2A. 5 Cu complexes attributed the appear-
the molecular formula C14 H17 N O of compound 3. Twelve ((2-
3
2
(
piperazin-1-yl) ethyl) isoindoline-1,3-dione derivatives were syn-
thesized by using (2-(piperazin-1-yl) ethyl) isoindoline-1,3-dione
and isocyanates, as shown in the Fig. 1. All synthesized com-
pounds were characterized by different spectral techniques. The
FT-IR spectra of all synthesized compounds were recorded in the
−1
ance of a medium to strong absorption band at 3200 cm
due
to the stretching vibration of Ar-H bond formation in the syn-
thesized complex as shown in Fig. 3. The appearance of band at
Fig. 4. Mass spectra of complex 5Cu.
6

H.S.N. Prasad, A.P. Ananda, S. Najundaswamy et al.
Journal of Molecular Structure 1232 (2021) 130047
1
750 cm 1 and 1610 cm
−
−1
are due to the bending of vibration
Table 2
C=O and stretching vibration of C=C bonds [36,37]. The compound
Structures of synthesized compounds 5(a-l) and metal com-
plexes with MIC value against MRSA compared with stan-
dards Streptomycin and Bacitracin.
−1
gives a C-Cl band in the region of 706 cm . Regarding the lig-
and to metal vibrations, only the stretching vibration of the Cu-N
bonds could tentatively be assigned in the IR spectrum, as a band
at 480 cm 1 [38]. The FT-IR spectra of 5Zn are changes in inte-
gral intensities and bandwidths, especially of the bands originating
from NH stretching vibrations didn’t show in products. The peak
−
−1
appearance at 1360 cm
indicates the presence of C-N bond, re-
garding ligand to metal vibrations, the stretching vibration of the
Zn-N bond at 490 cm 1. Elemental analyses data of theoretically
−
calculated values and experimentally determined values lie within
±
0.4% (Fig. S13). The theoretical molecular weight of 5 Cu com-
plexes is 475.41 and the molecular ion peak appears at 475.52,
showing that clear agreement with theoretical data (Fig. 4). Mean
particle size of the same complex can be measured from the pow-
der XRD result (Fig. 5) using Debye Scherrer equation [39] which is
0.03 nm and observed 2θ values are also in good agreement with
literature [40]. The molecular ion peak of the synthesized com-
pounds confirmed its formula weight, which is the same as the
calculated molecular ion value. The stability constant of the for-
mation of metal complexes is used to measure interaction strength
of reagents. From this cycle, metal particle and ligand communica-
tion shaped the two kinds of metal edifices, one is supra molecular
buildings known as host-visitor edifices and the other is anion-
containing edifices. In the arrangement it gives and figures the
necessary data about the centralization of metal structures. The
9
stability of complex 5 Cu is 1.2 × 10 (Fig. S14).
3
.3. Antibacterial activity of Piperazine metal complexes against
MRSA
The synthesized 5(a-l) analogues were assessed for MIC by re-
sazurin assay. The parental structural piperazine and phthalim-
ide 5(a-l) were tested against MRSA. The 5e and 5Cu metal com-
plex showed remarkable MIC value of 30± 0.15μg/mL and 20± 0.12
μg/mL, respectively confirmed with a wide spectrum antibiotic
drug Streptomycin (10.2± 0.02) and Bacitracin (10.0± 0.06) in μg/mL
(
Table 2).
The metal complexes of 5e such as 5Cu, 5Zn, 5Ni, and 5Co were
screened for antibacterial activity against MRSA. Among these, 5Cu
metal complex showed prominent activity. The antibacterial activ-
ity of 5e and 5Cu were also assessed by disc diffusion in dose-
dependent manner. The assay was validated with drug strepto-
mycin and bacitracin shows 14.5± 0.04 and 15± 0.08 mm of zone
of inhibition respectively (Fig. 6A). The 5e analogues of piper-
azine showed a moderate level of inhibition of zone in radius
(
ZIO) 4.51± 0.02, 4.92± 0.01, 5.20± 0.02, 6.11± 0.03, 6.80± 0.02 and
7
.40± 0.03mm (Fig. 6B). The 5Cu metal-based complex showed
an enhanced antibacterial activity against MRSA in inhibition of
zone in radius (ZIO) 5.89± 0.03, 5.98± 0.02, 6.20± 0.02, 6.95± 0.01,
7.80± 0.03 and 8.25± 0.03mm (Fig. 6C). The revealed data confirms
that the synthetic piperazine derivative showed significant results
due to its structure and chemical moieties in the compound. The
analogue 5Cu showed potent nature due to its electronegative
chlorine group present in the para position of the aromatic ring
and phenyl ring with substituted to the linker carboxamide of
piperazine moiety with copper metal exhibited promising biolog-
ical activity to govern physicochemical, pharmacokinetic and phar-
macodynamic property.
3
.4. Molecular docking for protein Glycosyltransferase and PDB ids
are 3VMT and 6FTB
Molecular docking is a current method in computational sci-
ence to significantly explore ligand assertion and it has provoked
7

H.S.N. Prasad, A.P. Ananda, S. Najundaswamy et al.
Journal of Molecular Structure 1232 (2021) 130047
Fig. 6. Antimicrobial activity 5Cu and 5e against MRSA. (A) MRSA tested with standard drug bacitracin μg/disc (10.42 ± 0.01) and streptomycin μg/disc (12.12 ± 0.02) ZOI
in mm, (B) 5e metal complex and (C) 5Cu analogue.
Table 3
Molecular docking scores of synthesized analogues, complex and antibiotic streptomycin against 3VMT and 6FTB protein.
PDB id
3VMT
6FTB
Compounds
DockingScore XP Gscore Glide Energy(kcal/mol) Glide Emodel(kcal/mol) Docking Score XP Gscore Glide Energy(kcal/mol) Glide Emodel(kcal/mol)
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
-3.025
-5.324
-4.291
-5.055
-5.918
-5.276
-3.506
-4.561
-5.369
-4.092
-5.098
-3.085
-3.777
-5.745
-4.712
-5.476
-6.339
-5.697
-4.246
-4.109
-3.881
-3.008
-3.313
-3.825
-10.828
-43.448
-54.29
-57.543
-68.712
-65.601
-68.013
-69.874
-57.318
-56.827
-57.025
-50.96
-4.624
-5.44
-5.364
-6.18
-37.185
-46.014
-40.21
-54.35
-65.245
-62.976
-58.101
-63.824
-53.655
-57.486
-57.65
-50.735
-48.466
-56.239
-47.677
-40.179
-45.062
-47.53
-4.18
-4.601
-5.132
-7.022
-5.517
-4.48
-4.486
-6.658
-4.253
-3.741
-5.093
-6.254
-5.889
-5.44
-44.699
-49.385
-39.924
-41.355
-32.367
-42.826
-40.534
-47.301
-42.813
-55.213
g
h
i
-5.833
-5.925
-6.629
-6.55
-53.259
-59.468
-62.767
-57.947
-69.461
j
-47.557
-36.896
-45.994
-59.987
-56.843
-51.134
-64.073
-77.870
k
l
-2.145
-7.879
-6.807
-7.517
Streptomycin -10.656
Fig. 7. Moleculardocking proof for 3VMT(A), 6 FTB (B) and standard streptomycin (C, D) proteins of MRSA intern best pose at active sites of targets by 5e and streptomycin
respectively.
8

H.S.N. Prasad, A.P. Ananda, S. Najundaswamy et al.
Journal of Molecular Structure 1232 (2021) 130047
huge headways in prescription disclosure and plan in the
field of medicinal science [41]. In silico docking procedure re-
searches the coupling mode and inclination of a little particle
inside the coupling site of the receptor target protein. The docked
ligands were situated by their coupling affection in ligand receptor
structures.
In the present study, molecular docking was performed on a set
of test compounds, viz. piperazine carboxamide analogues 5(a-l)
against two lipid II-bound glycosyltransferase proteins of MRSA like
3VMT and 6FTB (PDB ids). This lipid is an important component in
the cell wall synthesis. The experimental data strongly indicated
that piperazine carboxamide analogues 5(a-l) show inhibitory ac-
tion in the control of MRSA growth by in vitro antibacterial stud-
ies. To validate this, the in silico molecular docking approach was
studied to give a more scientific way of justification to the drug in-
teraction for target perilous superbug MRSA. The molecular dock-
ing was implemented to evaluate the behaviour at biological inter-
faces by choosing S. aureus proteins such as 3VMT and 6FTB from
RCSB Protein Data Bank. The scores due to docking for the tested
ligands determined that all the prepared admixtures possessed po-
tential for interaction with one or more amino acids in the active
site (binding pocket) of the receptor. The compound 5e was the
most potent inhibitor of the receptor. In the docking, the bind-
ing of 5e was supported by strong interactions such as hydrogen,
Vander Waals and hydrophobic with possible amino acid residues
Fig. 8. Antibacterial activity of 5Cu was evidenced by cell membrane damage of
MRSA. The MRSA was treated with MIC value of 5Cu and without treated MRSA as
control.
10.656 kcal/mol and -7.879 kcal/mol, respectively. The binding site
of analogue 5e in the active site of 3VMT and 6FTB interaction 3D
were represented respectively (Fig. 7A and 7B) and 2D structure of
all synthesized ligands tabulated in supplementary file (Table S2).
The compound 5e of protein 3VMT demonstrated hydrogen bond,
(π-π) stacking and π cation interaction associations with ARG117
in compound 5e and indicated that other than hydrogen interac-
tions, hydrophobic interaction plays a role with the protein 6FTB.
The involvement of the chloro, sulphur and the nitrogen groups
in electrostatic forces of attraction enhancing the docking scores
of these analogues was predicted compared to the standard strep-
(
Table 3). The ligand piperazine having phthalic anhydride, chloro
groups (5e), contributed the highest docking score -5.918 kcal/mol
with 3VMT protein and significant score of -6.688 kcal/mol with
6
FTB protein compared to the standard antibiotic streptomycin -
Fig. 9. Toxicity of 5e and 5Cu. A. L6 cell lines, B and C. Toxic effect of 5e and 5Cu and D. % of inhibition with different from 10–320 μg/Ml.
9

H.S.N. Prasad, A.P. Ananda, S. Najundaswamy et al.
Journal of Molecular Structure 1232 (2021) 130047
Fig. 10. Biocompatible nature of 5e and 5Cu. A. and B. Biocompatibility of 5e and 5Cu with different from 20–500 μ/mL. C. Graphical representation of % hemolysis rate.
tomycin, which interacted strongly with 3MVT through MET 99,
CYS248, GLU 102, ARG 117 for hydrogen interaction, and with 6FTB
GLN136, THR133, GLY131, LEU112, VAL154 and LEU 157 showed rel-
evant significant anticipated interaction compared with the potent
role of synthesized analogue 5e. In this study, the 5e has good
docking score compared to other analogues and also this theoreti-
cal data strongly correlated to our experimental results.
cant changes in structural membrane with respect to control. The
treated MRSA showed some pores, breaks and cracks compared to
smooth control surface of (untreated) by SEM (Fig. 8) [42]. The re-
sult showed bactericidal activity is due to this metal complex bind-
ing to membrane or membrane-bound factors, which leads to cel-
lular content leakage, cell death and indication of protect from the
adverse inflammatory conditions that can be induced during MRSA
infection in the host.
3
.5. Membrane damage
3
.6. Toxic effect of piperazine copper complexes-5e and 5Cu
In the series of metal piperazine carboxamide analogues, 5 (Cu)
complexwas chosen due its potent activity against superbug MRSA
to visualize the cell membrane damage. The 5Cu showed signifi-
The toxicity of metal depends on intermediate metabolism.
Some metals like lead, mercury, and thallium can gather in the mi-
10

H.S.N. Prasad, A.P. Ananda, S. Najundaswamy et al.
Journal of Molecular Structure 1232 (2021) 130047
tochondrial membrane and inhibit oxidative phosphorylation both
invitro and invivo. In this scenario, these accumulated metals dis-
rupt heme synthesis and cause damage to heme-containing en-
zymes. However, toxicities from copper metals are witnessed as
not toxic as compared to lead, mercury, and arsenic poisoning. In
this consequence, toxicity is one of the pre-requisite criteria for
drug design. The piperazine complex 5e and 5Cu metal complex
revealed low toxicity to the L6 cell line (Fig. 9A–9D).
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Declaration of Competing Interest
The authors declare no competing financial interest.
Acknowledgements
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