DNA interaction, anticancer, antibacterial, ROS and lipid peroxidation studies of quinoxaline based organometallic Re(I) carbonyls

Article abstract of DOI:10.1016/j.molstruc.2021.130529

Hetero mononuclear rhenium(I) complexes (I-V) using ligands (L¹-L⁵) [L¹-L⁵ = 11-((2-chlorobenzylidene)hydrazono)-11H-indeno[1,2-b]quinoxaline (L¹), 8-methyl-11-((4-methyl-benzylidene)hydrazono)-11H-indeno[1,2-b]quinoxaline (L²), 11-((4-bromobenzylidene) hydrazono)-8-nitro-11H-indeno[1,2-b]quinoxaline (L³), 11-((4-bromobenzylidene) hydrazono)-8-chloro-11H-indeno[1,2-b]quinoxaline (L⁴), 8-bromo-11-((4-fluorobenzylidene) hydrazono)-11H-indeno[1,2-b]quinoxaline (L⁵)] were synthesized and characterized by spectroscopic method. All the synthesized compounds have biological importance. DNA interaction studies gave information about the modes of binding and the nucleolytic efficiency of compounds. The binding of the rhenium complexes to Herring sperm DNA (HS DNA) was monitored by UV–visible spectroscopy, viscosity measurements, and molecular docking studies; groove binding was suggested as the most possible mode. The DNA-complexes binding strength was measured in terms of intrinsic binding constants. In vivo and In vitro cytotoxicity against the eukaryotic and prokaryotic cells gave the toxic nature of the synthesized compounds. An antimicrobial study was carried out by estimating MIC (Minimum Inhibitory Concentration) against two Gram-positive (S. aureus, B. subtilis) and three Gram-negative (S. marcescens, P. aeruginosa, E. coli) bacteria. All synthesized complexes are biologically more active than the corresponding ligands. Complexes were having higher MDA and H₂O₂ production than ligands.

Full text of DOI:10.1016/j.molstruc.2021.130529

Journal of Molecular Structure 1240 (2021) 130529
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
DNA interaction, anticancer, antibacterial, ROS and lipid peroxidation
studies of quinoxaline based organometallic Re(I) carbonyls
Reena R. Varmaa, Juhee G. Pandyab, Foram U. Vaidyac, Chandramani Pathakc,
Ravi A. Dabhia, Milan P. Dhaduka, Bhupesh S. Bhatta, Mohan N. Patela,∗
a Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar-388 120, Gujarat, India
b Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar, Gujarat, India
c Department of Cell Biology, School of Biological Sciences and Biotechnology, Indian Institute of Advanced Research, Koba Institutional Area,
Gandhinagar-382007, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Hetero mononuclear rhenium(I) complexes (I-V) using ligands (L1-L5) [L1-L5
=
11-((2-
Article history:
Received 1 November 2020
Revised 12 April 2021
Accepted 19 April 2021
Available online 1 May 2021
chlorobenzylidene)hydrazono)-11H-indeno[1,2-b]quinoxaline
(L1),
8-methyl-11-((4-methyl-
benzylidene)hydrazono)-11H-indeno[1,2-b]quinoxaline (L2), 11-((4-bromobenzylidene) hydrazono)-8-
nitro-11H-indeno[1,2-b]quinoxaline (L3), 11-((4-bromobenzylidene) hydrazono)-8-chloro-11H-indeno[1,2-
b]quinoxaline (L4), 8-bromo-11-((4-fluorobenzylidene) hydrazono)-11H-indeno[1,2-b]quinoxaline (L5)]
were synthesized and characterized by spectroscopic method. All the synthesized compounds have
biological importance. DNA interaction studies gave information about the modes of binding and the
nucleolytic efficiency of compounds. The binding of the rhenium complexes to Herring sperm DNA
(HS DNA) was monitored by UV–visible spectroscopy, viscosity measurements, and molecular docking
studies; groove binding was suggested as the most possible mode. The DNA-complexes binding strength
was measured in terms of intrinsic binding constants. In vivo and In vitro cytotoxicity against the eukary-
otic and prokaryotic cells gave the toxic nature of the synthesized compounds. An antimicrobial study
was carried out by estimating MIC (Minimum Inhibitory Concentration) against two Gram-positive (S.
aureus, B. subtilis) and three Gram-negative (S. marcescens, P. aeruginosa, E. coli) bacteria. All synthesized
complexes are biologically more active than the corresponding ligands. Complexes were having higher
MDA and H2O2 production than ligands.
Keywords:
Organometallic Re(I) carbonyl
Cell proliferation
ROS
DNA
Cytotoxicity
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
conformation differently. Most of the metal complexes exert their
antitumor activity by the metallation of DNA oligonucleotide [13].
The breakthrough discovery of cisplatin has accelerated bioinor-
ganic chemistry research for the discovery of metal-based
chemotherapeutic drugs with a novel mode of action [1]. The
platinum-based anticancer drugs have limitations of side effects
and drug resistance developed among cancer cells [2]. So, many
metal-based compounds have been extensively researched for anti-
cancer activity in search of novel compounds with potent cytotoxic
nature against the cancer cell and fewer side effects [3–12]. The
understanding of biomolecular interaction particularly DNA could
be the most critical thing about the anticancer metallodrug dis-
covery process since the different modes of interaction affect DNA
Quinoxaline intermediates are very significant for the manu-
facturing of pharmaceutically active compounds [14]. Quinoxaline
moiety has very wide biological applications like antibacterial [15],
anticancer [16], antifungal [17], and as photochemically active ma-
terials [18]. Various antibiotics like echinomycine, levomycin, and
actinoleutin contain quinoxaline rings are used for inhibiting gram
+ve bacteria [19]. Many chemical and biological systems are abun-
dant in quinoxaline moiety formed in protein and active for bio-
logical applications [20].
The carbonyl releasing ability of some metal carbonyls has
been utilizing in chemotherapeutic research as an alternative to
platinum-based drugs [21–24]. Also, rhenium metal is very less re-
searched for its biological significance. So, utilizing the extensive
medicinal applications of quinoxaline and CO releasing ability of
metal carbonyls, we synthesized quinoxaline-based rhenium car-
bonyl complexes to explore their diverse biological applications.
∗
Corresponding authors.
E-mail addresses: bhupeshbhatt31@gmail.com (B.S. Bhatt), jeenen@gmail.com
(M.N. Patel).
https://doi.org/10.1016/j.molstruc.2021.130529
0022-2860/© 2021 Elsevier B.V. All rights reserved.

R.R. Varma, J.G. Pandya, F.U. Vaidya et al.
Journal of Molecular Structure 1240 (2021) 130529
The interaction of Re(I) complexes with DNA was studied by UV-
visible spectra, hydrodynamic volume measurement, and molec-
ular modeling. The antibacterial and cytotoxicity studies suggest
higher activity of complexes than ligands. DNA cleavage study was
performed by gel electrophoresis. Synthesized complexes exhibit
a good percentage of the proliferation of human carcinoma cells.
Also, the ability of complexes to produce ROS and lipid peroxida-
tion studies were carried out.
2.3.1. 11-((2-Chlorobenzylidene)hydrazono)-11H-indeno[1,2-b]
quinoxaline (L1)
The synthesis was performed by taking equimolar mix-
ture of 11H-indeno[1,2-b]quinoxalin-11-one (232 mg, 1.0 mmol)
and (2-chlorobenzylidene)hydrazine (155 mg, 1.0 mmol) in
10 mL of methanol and 2 drops of H2SO4. Empirical formula:
C22H13ClN4; Yield: 85%; Melting point: ~192 °C; Molecular weight:
368.82 g/mol; Elemental analysis: Calc. (%): C, 71.64; H, 3.55; N,
15.19; Found (%): C, 71.62; H, 3.53; N, 15.17; Mass (m/z%): 368.80
(100) [M+], 370.80 [M + 2]; 1H NMR (400 MHz, CDCl3) δ/ppm:
ꢀꢀ ꢀꢀ ꢀꢀ ꢀꢀ
8.66 (1H, s, H1’’a), 7.54- 8.66 (12H, m, H2,3,4,5,7,8,9,10,3 ,4 ,5 ,6 ).
2. Experimental
13C NMR (100 MHz, CDCl3) δ/ppm: 163.3 (C1"c, Cquat.), 143.2
ꢀ
ꢀ
ꢀ
ꢀ
(C1 a, Cquat.), 153.8 (C5 a, Cquat.), 150.6 (C2 a, Cquat.), 150.0 (C7 a,
2.1. Materials and methods
Cquat.), 161.6 (C1"a, -CH), 143.2 (C1", Cquat.), 139.9 (C2", Cquat.),
ꢀ
ꢀ
138.5 (C10 a, Cquat.), 137.6 (C6 a, Cquat.), 133.74 (C4", −CH), 133.12
(C8, −CH), 132.1 (C3", −CH), 131.0 (C4, −CH), 130.9 (C3, −CH),
133.8 (C5, −CH), 130.0 (C2, −CH), 129.7 (C9, −CH), 129.5 (C6",
−CH), 129.2 (C10, −CH), 129.1 (C5", −CH), 122.6 (C7, −CH). [To-
tal signal observed = 22: signal of C = 9 (phenyl ring-C = 2,
indeno[1,2-b]quinoxaline = 7), signal of CH = 13 (phenyl ring-
CH = 4, indeno[1,2-b]quinoxaline -CH = 8, benzylidene -CH =01)];
All the chemicals and solvents were of the reagent grade, sub-
stituted aldehyde, substituted phenylenediamine were purchased
from Merck Limited (India), ninhydrin was purchased from Thiru-
malai Chemicals Ltd. (TCL), pentacarbonyl chloro rhenium(I) was
purchased from Sigma Aldrich (USA). Luria broth, agarose, nutri-
ent broth, and Luria broth were purchased from Himedia (India).
The culture of two Gram(+ve), i.e. Staphylococcus aureus (S. aureus)
(MTCC-3160) and Bacillus subtilis (MTCC-7193); and three Gram(-
ve), i.e. Serratia marcescens (MTCC-7103), Pseudomonas aeruginosa
(MTCC-1688) and Escherichia coli (MTCC-433), were purchased
from Institute of Microbial Technology (Chandigarh, India). S. cere-
vices Var. Paul Linder 3360 was obtained from IMTECH, Chandigarh
(India). HS DNA was purchased from Sigma Aldrich Chemical Co.
(India).
IR (KBr, 4000–400 cm– 1): 3063 ν(C H)stret., 1596 ν(C=N), 1589
–
–
–
ν(C-H)banding, 1428 ν(C C)stret., 1080 ν(C-N), 810 ν(C Cl)stret.,
702 ν(Ar-H) adjacent hydrogen..
2.3.2. 8-Methyl-11-((4-methylbenzylidene)hydrazono)-11H-indeno
[1,2-b]quinoxaline (L2)
The synthesis was performed by taking equimolar mix-
ture of 8-methyl-11H-indeno[1,2-b]quinoxalin-11-one (246 mg,
1 mmol), (4-methylbenzylidene)hydrazine (134 mg, 1.0 mmol) in
10 mL of methanol and 2 drops of H2SO4. Empirical formula:
C24H18 N4; Yield: 68%; Melting point: ~190 °C; Molecular weight:
362.44 g/mol; Elemental analysis: Calc. (%): C, 79.54; H, 5.01; N,
15.46; Found (%): C, 79.51; H, 5.00; N, 15.44; Mass (m/z%): 362.41
(100) [M+] 1H NMR (400 MHz, CDCl3) δ/ppm: 8.36 (1H, s, H1’’a),
2.2. Physical measurements
The 1H and 13C NMR spectra were recorded on a Bruker Avance
(400 MHz). Infrared spectra were recorded on an FT–IR ABB Bomen
MB 3000 spectrophotometer as KBr pellets in the range 4000–
400 cm−1. C, H, and N elemental analyses were performed with
a Perkin–Elmer 240 elemental analyzer. Molar conductance was
measured using a conductivity meter model no. EQ-660A, Mumbai
(India). Melting points (°C, uncorrected) were determined in open
capillaries on the ThermoCal10 melting point apparatus (Analab
Scientific Pvt. Ltd, India). The electronic spectra were recorded on
a UV–160A UV–Vis spectrophotometer, Shimadzu (Japan). The min-
imum inhibitory concentration (MIC) study was carried out using
the laminar airflow cabinet (Toshiba, Delhi, India). The hydrody-
namic chain length study was carried out by a viscometric mea-
surement bath. Photo quantization of the gel after electrophoresis
was carried out on AlphaDigiDocTM RT. Version V.4.0.0 PC–Image
software. All density functional theory (DFT) calculations were per-
formed using the Gaussian-09 program package.
ꢀꢀ ꢀꢀ ꢀꢀ ꢀꢀ
2.64 (6H,s, 2-CH3), (7.52–8.35 (11H, m, H2,4,5,7,8,9,10,2 ,3 ,5 ,6 ).
13C NMR (100 MHz, CDCl3) δ/ppm: 164.5 (C1"c, Cquat.), 143.1
(C1 a, Cquat.), 153.0 (C2 a, Cquat.), 150.0 (C4 ’, Cquat.), 149.5 (C5 a,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Cquat.), 146.8 (C7 a, Cquat), 161.1 (C1"a, −CH), 143.0 (C3, Cquat.),
ꢀ
ꢀ
139.3 (C1", Cquat.), 138.5 (C10 a, Cquat.), 136.8 (C6 a, Cquat.),
ꢀꢀ
ꢀꢀ
130.3 (C3",5 , −CH), 130.0 (C2",6 , −CH), 129.7 (C4, −CH), 129.0
(C8, −CH), 124.2 (C10, −CH), 122.4 (C9, −CH), 122.2 (C5, −CH),
122.1 (C2, −CH), 120.6 (C7, −CH), 21.7 (-CH3), 21.9 (-CH3). [To-
tal signal observed = 22: signal of C = 10 (phenyl ring-C = 2,
indeno[1,2-b]quinoxaline = 8), signal of CH = 10 (phenyl ring-
CH = 2, indeno[1,2-b]quinoxaline -CH = 7, benzylidene -CH =01,
CH3 = 02)]; IR (KBr, 4000–400 cm– 1): 3063 ν(C H)stret., 1604
–
–
ν(C=N), 1588 ν(C-H)banding, 1488 ν(C C)stret., 1087 ν(C-N), 815
ν(p-substisution), 701 ν(Ar-H) adjacent hydrogen.
2.3.3. 11-((4-Bromobenzylidene)hydrazono)-8-nitro-11H-indeno[1,2-b]
quinoxaline (L3)
2.3. General method for synthesis of quinoxaline based ligands
(L1-L5)
The synthesis was performed by taking equimolar mix-
ture of 8-nitro-11H-indeno[1,2-b]quinoxalin-11-one (277 mg,
1.0 mmol), (4-bromobenzylidene)hydrazine (199 mg, 1.0 mmol)
in 10 mL of methanol and 2 drops of H2SO4. Empirical formula:
C22H12BrN5O2; Yield: 83%; Melting point: ~198 °C; Molec-
ular weight: 458.28 g/mol; Elemental analysis: Calc. (%): C,
57.66; H, 2.64; N, 15.28; Found (%): C, 57.64; H, 2.62; N, 15.25;
Ligands (L1-L5) were obtained from the reaction between
quinoxaline and Schiff base (Scheme 1). These reactions occur in
two steps; the first ninhydrin reacts with different substituted
phenylenediamine using water as solvent and 2 to 4 drops of
H2SO4 as a catalyst to get indeno[1,2-b] quinoxaline derivative
(3a-3e). Second, different substituted aldehyde was reacted with
NH2NH2.H2O using two drops of H2SO4 as a catalyst to get sub-
stituted benzylidenehydrazine (5a-5e). Upon mixing Indeno[1,2-b]
quinoxaline derivatives and benzylidenehydrazine using methanol
as solvent and refluxed at 60 °C, we obtained the ligands (L1-L5).
The 1H and 13C NMR spectra of all synthesized compounds are
shown in supplementary material 1 and 2, respectively.
Mass (m/z%): 458.26 (100) [M+], 460.26 [M
(400 MHz, CDCl3) δ/ppm: 9.28 (1H, s, H1’’a), 7.42- 9.27 (11H, m,
ꢀꢀ ꢀꢀ ꢀꢀ ꢀꢀ
+
2] 1H NMR
H2,4,5,7,8,9,10,2 ,3 ,5 ,6 ). 13C NMR (100 MHz, CDCl3) δ/ppm:
ꢀ
ꢀ
163.6 (C1"c, Cquat.), 160.8 (C6 a, Cquat.), 153.3 (C2 a, Cquat.), 138.2
ꢀ
ꢀ
ꢀ
(C1 a, Cquat.), 149.7 (C5 a, Cquat.), 147.4 (C3, Cquat.), 141.87 (C7 a,
ꢀ
ꢀ
Cquat.), 139.5 (C1", Cquat.), 138.8 (C10 a, Cquat.), 150.0 (C1 ’a,
ꢀꢀ
-CH), 137.5 (C4", Cquat.), 133.9 (C3",5 , −CH), 133.6 (C8, −CH),
2

R.R. Varma, J.G. Pandya, F.U. Vaidya et al.
Journal of Molecular Structure 1240 (2021) 130529
Scheme 1. General scheme for the synthesis of ligands (L1-L5) and complexes (I-V).
ꢀꢀ
ꢀ
ꢀ
133.3 (C9, −CH), 131.0 (C10, −CH), 129.8 (C2",6 , −CH), 127.6
(C5, −CH), 126.6 (C7, −CH), 125.6 (C2, −CH), 123.4 (C4, −CH).
[Total signal observed = 20: signal of C = 10 (phenyl ring-C = 2,
indeno[1,2-b]quinoxaline = 8), signal of CH = 10 (phenyl ring-
CH = 2, indeno[1,2-b]quinoxaline -CH = 7, benzylidene -CH =01)];
Cquat.), 150.3 (C3, Cquat.), 150.0 (C5 a, Cquat.), 146.6 (C7 a, Cquat.),
ꢀ
162.2 (C1"a, -CH), 142.2 (C1", Cquat.), 139.9 (C10 a, Cquat.), 138.2
ꢀ
ꢀꢀ
(C6 a, -CH), 137.3 (C3",5 , Cquat.), 136.7 (C4", Cquat,), 135.9 (C4,
−CH), 134.2 (C8, −CH), 131.0 (C5, −CH), 130.7 (C10, −CH), 130.1
ꢀꢀ
(C9, −CH), 127.7 (C2",6 , −CH), 126.4 (C7, −CH), 125.3 (C2, −CH).
IR (KBr, 4000–400 cm– 1): 3078 ν(C H)stret., 1651 ν(C=N), 1630
[Total signal observed
=
20: signal of
C
=
10 (phenyl ring-
–
–
ν(C-H)banding, 1589 ν(C C)stret., 1527 ν(presence of -NO2), 1040
C = 2, indeno[1,2-b]quinoxaline = 8), signal of CH = 10 (phenyl
ring-CH = 2, indeno[1,2-b]quinoxaline -CH = 7, benzylidene -CH
ν(C-N), 818 ν(p-substisution), 733 ν(Ar-H) adjacent hydrogen, 618
ν(C-Br).
=01)]; IR (KBr, 4000–400 cm– 1): 3032 ν(C H)stret., 1651 ν(C=N),
–
–
1612 ν(C-H)banding, 1597 ν(C C)stret., 1042 ν(C-N), 818 ν(p-
2.3.4. 11-((4-Bromobenzylidene)hydrazono)-8-chloro-11H-indeno[1,2-
b]quinoxaline (L4)
–
substisution), 724 ν(C Cl)stret., 720 ν(Ar-H) adjacent hydrogen,
632 ν(C-Br).
The synthesis was performed by taking equimolar mix-
ture of 8-chloro-11H-indeno[1,2-b]quinoxalin-11-one (267 mg,
1.0 mmol), (4-bromobenzylidene)hydrazine (199 mg, 1.0 mmol)
2.3.5. 8-Bromo-11-((4-fluorobenzylidene)hydrazono)-11H-indeno[1,2-
b]quinoxaline (L5)
in 10 mL of methanol and
2
drops of H2SO4. Empirical for-
The synthesis was performed by taking equimolar mix-
ture of 8-bromo-11H-indeno[1,2-b]quinoxalin-11-one (311 mg,
1.0 mmol), (4-fluorobenzylidene)hydrazine (138 mg, 1.0 mmol)
mula: C22H12BrClN4; Yield: 83%; Melting point: ~194 °C; Molecu-
lar weight: 447.72 g/mol; Elemental analysis: Calc. (%): C, 59.02;
H, 2.70; N, 12.51; Found (%): C, 59.00; H, 2.68; N, 12.49; Mass
(m/z%): 447.70 (100) [M+], 449.70[M + 2], 451.70 [M + 4]; 1H
NMR (400 MHz, CDCl3) δ/ppm: 8.58 (1H, s, H1’’a), 7.32- 8.58
in 10 mL of methanol and
2 drops of H2SO4. Empirical for-
mula: C22H12BrFN4; Yield: 87%; Melting point: ~200 °C; Molecu-
lar weight: 431.27 g/mol; Elemental analysis: Calc. (%): C, 61.27; H,
2.80; N, 12.99; Found (%): -; Mass (m/z%): 450.46 (100) [M+]; 1H
NMR (400 MHz, CDCl3) δ/ppm: 8.41 (1H, s, H1’’a), 7.55- 8.40 (11H,
ꢀꢀ ꢀꢀ ꢀꢀ ꢀꢀ
(11H, m, H2,4,5,7,8,9,10,2 ,3 ,5 ,6 ). 13C NMR (100 MHz, CDCl3)
ꢀ
ꢀ
δ/ppm: 163.9 (C1"c, Cquat.), 142.7 (C1 a, Cquat.), 153.4 (C2 a,
3

R.R. Varma, J.G. Pandya, F.U. Vaidya et al.
Journal of Molecular Structure 1240 (2021) 130529
ꢀꢀ ꢀꢀ ꢀꢀ ꢀꢀ
m, H2,4,5,7,8,9,10,2 ,3 ,5 ,6 ). 13C NMR (100 MHz, CDCl3) δ/ppm:
122.6 (C7, −CH), 22.4 (-CH3), 22.2 (-CH3). [Total signal ob-
served = 24: signal of C = 12 (M-CO = 2, phenyl ring-C = 2,
indeno[1,2-b]quinoxaline = 8), signal of CH = 10 (phenyl ring-
CH = 2, indeno[1,2-b]quinoxaline -CH = 7, benzylidene -CH =01),
ꢀ
ꢀ
164.3 (C4", Cquat.), 162.9 (C1 ’c, Cquat.), 143.4 (C1 a, Cquat.), 151.0
ꢀ
ꢀ
ꢀ
(C5 a, Cquat.), 150.0 (C2 a, Cquat.), 147.1 (C7 a, Cquat.), 154.1 (C1"a,
ꢀ
ꢀ
-CH), 143.1 (C1", Cquat.), 139.9 (C10 a, Cquat.), 138.6 (C6 a, Cquat.),
-CH3 = 02]; IR (KBr, 4000–400 cm– 1): 3063 ν(C H)stret., 1604
–
137.9 (C2, -CH), 137.7 (C3, Cquat,), 135.8 (C8, −CH), 134.7 (C4,
ꢀꢀ
–
ν(C=N), 1588 ν(C-H)banding, 1488 ν(C C)stret., 1087 ν(C-N), 815
−CH), 132.8 (C2",6 , −CH), 130.7 (C5, −CH), 130.0 (C10, −CH),
ꢀꢀ
ν(p-substisution), 701 ν(Ar-H) adjacent hydrogen.
128.2 (C9, −CH), 126.6 (C7, −CH), 125.5 (C3",5 , −CH). [Total signal
observed = 20: signal of C = 10 (phenyl ring-C = 2, indeno[1,2-
b]quinoxaline = 8), signal of CH = 10 (phenyl ring-CH = 2,
indeno[1,2-b]quinoxaline -CH = 7, benzylidene -CH =01)]; IR (KBr,
2.4.3. Synthesis of [Re(CO)3(L3)Cl] (III)
The synthesis was performed by taking equimolar mixture of
Re(CO)5Cl (361 mg, 1.0 mmol) and ligand (L3) (458 mg, 1.0 mmol).
Empirical formula: C25H12BrClN5O5Re; Yield: 85%; Melting point: ~
392 °C; Molecular weight: 763.96 g/mol; Elemental analysis: Calc.
(%): C, 39.30; H, 1.58; N, 9.17; Re, 24.37; Found (%): C, 39.28; H,
4000–400 cm– 1): 3032 ν(C H)stret., 1651 ν(C=N), 1612 ν(C-
–
–
H)banding, 1597 ν(C C)stret., 1042 ν(C-N), 818 ν(p-substisution),
–
722 ν(C Cl)stret., 720 ν(Ar-H) adjacent hydrogen, 635 ν(C-Br)stret.
1.56; N, 9.15; Re,24.35; Conductance: 10.12 ohm−1cm2mol−1 1H
;
2.4. General synthesis of complexes (I-V)
NMR (400 MHz, DMSO-d6) δ/ppm: 9.29 (1H, s, H1’’a), 7.56- 9.29
ꢀꢀ ꢀꢀ ꢀꢀ ꢀꢀ
(11H, m, H2,4,5,7,8,9,10,2 ,3 ,5 ,6 ). 13C NMR (100 MHz, DMSO-d6)
The precursor of [Re(CO)5Cl] in ethanol was refluxed for 10 min
with continuous stirring at ambient conditions. In the solution, the
ethanolic solution of ligand (L1- L5) was poured to get a yellowish-
green solution at the primary stage. The reaction mixture was
refluxed at 60–70 °C temperature for 10 h in inert atmosphere
[25,26]. The completion of the reaction was checked using a silica-
coated TCL plate (ethyl acetate and hexane). The crude was filtered
to remove solid particles. The solvent was removed under reduced
pressure to obtain the brownish-red product (Scheme 1).
δ/ppm: 188.8 (2M-CO, Cquat.), 185.2 (M-CO, Cquat.), 165.5 (C1"c,
ꢀ
ꢀ
ꢀ
Cquat.), 164.4 (C6 a, Cquat.), 154.6 (C2 a, Cquat.), 139.3 (C1 a, CH),
ꢀ
ꢀ
151.6 (C5 a, Cquat.), 148.5 (C3, Cquat.), 143.3 (C7 a, Cquat.), 140.0
ꢀ
ꢀ
(C1", Cquat.), 139.5 (C10 a, Cquat.), 152.8 (C1 ’a, -CH), 137.8 (C4",
ꢀꢀ
Cquat.), 135.6 (C3",5 , −CH), 135.2 (C8, −CH), 135.1 (C9, −CH),
ꢀꢀ
132.2 (C10, −CH), 130.0 (C2",6 , −CH), 128.3 (C5, −CH), 126.8
(C7, −CH), 126.2 (C2, −CH), 124.7 (C4, −CH). [Total signal ob-
served = 22: signal of C = 12 (M-CO = 2, phenyl ring-C = 2,
indeno[1,2-b]quinoxaline = 8), signal of CH = 10 (phenyl ring-
CH = 2, indeno[1,2-b]quinoxaline -CH = 7, benzylidene -CH =01)];
2.4.1. Synthesis of [Re(CO)3(L1)Cl] (I)
IR (KBr, 4000–400 cm– 1): 3078 ν(C H)stret., 1651 ν(C=N), 1630
–
The synthesis was performed by taking equimolar mixture of
Re(CO)5Cl (361 mg, 1.0 mmol) and ligand (L1) (369 mg, 1.0 mmol).
Empirical formula: C25H13Cl2N4O3Re; Yield: 81%; Melting point:
~392 °C; Molecular weight: 674.51 g/mol; Elemental analysis: Calc.
(%): C, 44.52; H, 1.94; N, 8.31;Re, 27.61; Found (%): C, 44.50; H,
–
ν(C-H)banding, 1589 ν(C C)stret., 1527 ν(presence of -NO2), 1040
ν(C-N), 818 ν(p-substisution), 625 ν(C-Br), 733 ν(Ar-H) adjacent
hydrogen.
2.4.4. Synthesis of [Re(CO)3(L4)Cl] (IV)
1.95; N, 8.28; Re, 27.58; Conductance: 2.97 ohm−1cm2mol−1 1H
;
The synthesis was performed by taking equimolar mixture of
Re(CO)5Cl (361 mg, 1.0 mmol) and ligand (L4) (448 mg, 1.0 mmol).
Empirical formula: C25H12BrCl2N4O3Re; Yield: 79%; Melting point:
~388 °C; Molecular weight: 753.41 g/mol; Elemental analysis:
Calc.(%): C, 39.86; H, 1.61; N, 7.44; Re, 24.72; Found (%): C, 39.84;
NMR (400 MHz, DMSO-d6) δ/ppm: 8.74 (1H, s, H1’’a), 7.61- 8.74
ꢀꢀ ꢀꢀ ꢀꢀ ꢀꢀ
(12H, m, H2,3,4,5,7,8,9,10,3 ,4 ,5 ,6 ). 13C NMR (100 MHz, DMSO-
d6) δ/ppm: 186.3 (2M-CO, Cquat.), 183.3 (M-CO, Cquat.), 165.1
ꢀ
ꢀ
ꢀ
(C1"c, Cquat.), 142.2 (C1 a, Cquat.), 154.5 (C5 a, Cquat.), 152.2 (C2 a,
ꢀ
Cquat.), 151.0 (C7 a, Cquat.), 163.3 (C1"a, -CH), 143.3 (C1", Cquat.),
H, 1.58; N, 7.41; Re,24.68; Conductance: 4.15 ohm−1cm2mol−1 1H
;
ꢀ
ꢀ
140.0 (C2", Cquat.), 139.4 (C10 a, Cquat.), 138.0 (C6 a, Cquat.), 134.1
(C4", −CH), 133.5 (C8, −CH), 132.7 (C3", −CH), 131.5 (C4, −CH),
131.3 (C3, −CH), 131.0 (C5, −CH), 130.6 (C2, −CH), 130.5 (C9, −CH),
130.0 (C6", −CH), 129.9 (C10, −CH), 129.7 (C5", −CH), 123.5 (C7,
−CH). [Total signal observed = 24: signal of C = 11 (M-CO = 2,
phenyl ring-C = 2, indeno[1,2-b]quinoxaline = 7), signal of CH = 13
(phenyl ring-CH = 4, indeno[1,2-b]quinoxaline -CH = 8, benzyli-
NMR (400 MHz, DMSO-d6) δ/ppm: 8.62 (1H, s, H1’’a), 7.25- 8.62
ꢀꢀ ꢀꢀ ꢀꢀ ꢀꢀ
(11H, m, H2,4,5,7,8,9,10,2 ,3 ,5 ,6 ). 13C NMR (100 MHz, DMSO-
d6) δ/ppm: 187.2 (2M-CO, Cquat.), 185.1 (M-CO, Cquat.), 165.4
ꢀ
ꢀ
(C1"c, Cquat.), 144.5 (C1 a, Cquat.), 154.5 (C2 a, Cquat.), 152.7 (C3,
ꢀ
ꢀ
Cquat.), 151.4 (C5 a, Cquat.), 148.6 (C7 a, Cquat.), 163.5 (C1"a, -
ꢀ
ꢀ
CH), 144.2 (C1", Cquat.), 141.4 (C10 a, Cquat.), 139.9 (C6 a, -CH),
ꢀꢀ
138.4 (C3",5 , Cquat.), 138.1 (C4", Cquat,), 136.6 (C4, −CH), 135.2
dene -CH =01)]; IR (KBr, 4000–400 cm– 1): 3063 ν(C H)stret., 1596
–
(C8, −CH), 133.8 (C5, −CH), 133.3 (C10, −CH), 132.2 (C9, −CH),
–
ν(C=N), 1589 ν(C-H)banding, 1428 ν(C C)stret., 1080 ν(C-N), 817
ꢀꢀ
129.6 (C2",6 , −CH), 127.5 (C7, −CH), 126.5 (C2, −CH). [Total sig-
–
ν(C Cl)stret., 702 ν(Ar-H) adjacent hydrogen.
nal observed = 22: signal of C = 12 (M-CO = 2, phenyl ring-
C = 2, indeno[1,2-b]quinoxaline = 8), signal of CH = 10 (phenyl
ring-CH = 2, indeno[1,2-b]quinoxaline -CH = 7, benzylidene -CH
2.4.2. Synthesis of [Re(CO)3(L2)Cl] (II)
=01)]; IR (KBr, 4000–400 cm– 1): 3032 ν(C H)stret., 1651 ν(C=N),
–
The synthesis was performed by taking equimolar mixture of
Re(CO)5Cl (361 mg, 1.0 mmol) and ligand (L2) (362 mg, 1.0 mmol).
Empirical formula: C27H18 ClN4O3Re; Yield: 84%; Melting point:
~388 °C; Molecular weight: 668.12 g/mol; Elemental analysis: Calc.
(%): C, 48.54; H, 2.72; N, 8.39; Re, 27.87; Found (%): C, 48.52;
–
1612 ν(C-H)banding, 1597 ν(C C)stret., 1042 ν(C-N), 818 ν(p-
–
substisution), 725 ν(C Cl)stret., 720 ν(Ar-H) adjacent hydrogen,
640 ν(C-Br).
H, 2.69; N, 8.37; Re, 27.86; Conductance: 5.72 ohm−1cm2mol−1
;
2.4.5. Synthesis of [Re(CO)3(L5)Cl] (V)
1H NMR (400 MHz, DMSO-d6) δ/ppm: 8.34 (1H, s, H1’’a), 2.69
The synthesis was performed by taking equimolar mixture of
Re(CO)5Cl (361 mg, 1.0 mmol) and ligand (L5) (431 mg, 1.0 mmol).
Empirical formula: C25H12BrClFN4O3Re; Yield: 81%; Melting point:
~395 °C; Molecular weight: 736.96 g/mol; Elemental analysis:
Calc. (%): C, 40.75; H, 1.64;N, 7.60; Re, 25.27; Conductance: 18.88
ohm−1cm2mol−1; 1H NMR (400 MHz, DMSO-d6) δ/ppm: 8.65
ꢀꢀ ꢀꢀ ꢀꢀ ꢀꢀ
(6H,s, 2-CH3), 7.61- 8.34 (11H, m, H2,4,5,7,8,9,10,2 ,3 ,5 ,6 ). 13C
NMR (100 MHz, DMSO-d6) δ/ppm: 185.5 (2M-CO, Cquat.), 182.4
ꢀ
(M-CO, Cquat.), 165.4 (C1"c, Cquat.), 144.6 (C1 a, Cquat.), 154.8
ꢀ
ꢀ
ꢀ
ꢀ
(C2 a, Cquat.), 152.8 (C4 ’, CH), 152.3 (C5 a, Cquat.), 148.2 (C7 a,
Cquat.), 163.5 (C1"a, −CH), 144.1 (C3, Cquat.), 140.0 (C1", Cquat.),
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ ꢀꢀ ꢀꢀ ꢀꢀ
140.0 (C10 a, Cquat.), 138.1 (C6 a, Cquat.), 132.4 (C3",5 , −CH),
(1H, s, H1’’a), 8.01- 8.64 (11H, m, H2,4,5,7,8,9,10,2 ,3 ,5 ,6 ). 13C
ꢀꢀ
132.2 (C2",6 , −CH), 130.0 (C4, −CH), 130.0 (C8, −CH), 126.3
NMR (100 MHz, DMSO-d6) δ/ppm: 190.0(2M-CO, Cquat.), 188.1
ꢀ
(C10, −CH), 124.6 (C9, −CH), 124.4 (C5, −CH), 124.3 (C2, −CH),
(M-CO, Cquat.), 166.4 (C4", Cquat.), 164.5 (C1 ’c, Cquat.), 144.8
4

R.R. Varma, J.G. Pandya, F.U. Vaidya et al.
Journal of Molecular Structure 1240 (2021) 130529
ꢀ
ꢀ
ꢀ
ꢀ
(C1 a, Cquat.), 153.8 (C5 a, Cquat.), 152.5 (C2 a, Cquat.), 149.0 (C7 a,
were prepared for the determination of MIC and vaccinated with
five different microorganisms. The solution without turbidity was
noted as the minimum inhibitory concentration after overnight in-
cubation at 37 °C.
ꢀ
Cquat.), 155.6 (C1"a, -CH), 144.3 (C1", Cquat.), 140.0 (C10 a, Cquat.),
ꢀ
139.9 (C6 a, -CH), 138.1 (C2, -CH), 137.9 (C3, Cquat,), 136.8 (C8,
ꢀꢀ
−CH), 135.5 (C4, −CH), 132.8 (C2",6 , −CH), 133.4 (C5, −CH), 131.5
ꢀꢀ
(C10, −CH), 129.1 (C9, −CH), 127.2 (C7, −CH), 126.8 (C3",5 , −CH).
[Total signal observed = 22: signal of C = 12 (M-CO = 2, phenyl
ring-C = 2, indeno[1,2-b]quinoxaline = 8), signal of CH = 10
(phenyl ring-CH = 2, indeno[1,2-b]quinoxaline -CH = 7, benzyli-
2.9. Reactive oxygen species (ROS)
The amount of H2O2 was quantified as described by Loreto
and Velikova [32]. In the process, treated culture centrifuged at
5000 rpm for 5 min, was used for H2O2 determination. The re-
action system consists of 0.5 mL supernatant was added to 0.5 ml
of 10 mM phosphate buffer (pH 7.0) and 1.0 mL of 1 M KI. After
completion of the reaction, absorbance was measured at 390 nm
and results were expressed as μg/mL H2O2 of culture.
dene -CH =01)]; IR (KBr, 4000–400 cm– 1): 3071 ν(C H)stret., 1651
–
–
ν(C=N), 1612 ν(C-H)banding, 1597 ν(C C), 1150 ν(C-F), 1049 ν(C-
N), 818 ν(p-substisution), 741 ν(Ar-H) adjacent hydrogen, 640 ν(C-
Br).
2.5. Antiproliferation study
Stock solutions of 10–100 mg/mL test complexes (I-V) were pre-
pared in dimethyl sulfoxide (DMSO). HCT 116 cells were cultured
in RPMI 1640 medium. The culture media were supplemented
with 10% fetal bovine serum and an antibiotic cocktail contain-
ing penicillin (5 mg/mL), streptomycin (5 mg/mL), and neomycin
(10 mg/mL). The cells were cultured in a humidified atmosphere
of 5% CO2 at 37 °C. The entire study was executed using expo-
nentially growing cells. Twenty-four hours after cell plating, media
was removed and replaced with fresh media containing 10, 25, 50,
100, 500 μg/mL test compounds and DMSO as vehicle control for
the indicated exposure times. The Re(I) tricarbonyl complexes I-V
were tested for their in vitro cytotoxicity against colon carcinoma
(HCT116) cancerous cell lines by the MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) assay. The extent of inhi-
bition was displayed as an IC50 value, which is the concentration
required to inhibit cell growth to half [27].
2.10. Lipid peroxidation
For the measurements of lipid peroxidation in Saccharomyces
cerevisiae, the thiobarbituric acid (TBA) test, which determines
MDA as an end product of lipid peroxidation (Heath and Parker,
1968) [33], was used. We combined 1.0 mL of biological sample
{0.1–2.0 mg of membrane protein or 0.1–0.2 /μmol of lipid phos-
phate} with 2.0 mL of TCA-TBA-HCI and mixed it thoroughly. The
solution was heated for 15 min in a boiling water bath. After cool-
ing, the flocculent precipitates were removed by centrifugation at
1000 rpm for 10 min. The absorbance of the sample was deter-
mined at 535 nm against a blank that contains all the reagents
minus the lipid. The malondialdehyde concentration of the sample
was calculated using an extinction coefficient of 1.56 × 10 ~ M−1
cm−1 [34].
2.6. Cellular level bioassay using S. cerevisiae cells
2.11. DNA interaction
The cellular level bioassay was carried out using S. cerevisiae
cells [28]. The cells were grown in 50 mL yeast extract media in
a 150 mL Erlenmeyer flask. The flask was incubated at 30 °C on a
shaker at 150 rpm till the exponential growth of S. cerevisiae was
obtained (24 to 30 h). The cell culture was treated with different
concentrations (2, 4, 6, 8, 10 mg/mL) of complexes, free ligands,
and DMSO (control), and incubated for 16–18 h. The next day, the
treated cells were collected by centrifugation at 10,000 rpm for
10 min and were dissolved in 500 mL of PBS. The 80 mL of yeast
culture dissolved in PBS and 20 mL of 0.4% trypan blue prepared
in PBS were mixed, and cells were observed in a compound micro-
scope (40X). The dye could enter the dead cell only, so they ap-
peared blue whereas live cells resisted the entry of dye. The num-
ber of dead cells and the number of live cells were counted.
2.11.1. Absorption spectral analysis
The change in spectral behavior of the complex upon interact-
ing with DNA is a tool for determining the interaction of com-
plexes. The extent of the spectral shift of complexes in the pres-
ence of DNA reveals the affinity and nature of binding [35]. In the
experiment, a fixed amount of DNA solution (100 μL) in phosphate
buffer was added to sample cell holding the definite concentration
of the complex solution (20 μmol/L) and reference cell to nullify
the effect of HS DNA, and allowed to incubate for 10 min before
the spectra being recorded. DMSO was also added into the refer-
ence cell as a control to nullify the effect of DMSO [36].
2.11.2. Hydrodynamic volume or viscosity measurement
Viscometric experiments were performed using an Ubbelohde
viscometer, maintained at 25.0 ( 0.5)°C in a thermostatic water
bath. The total system was a 3 mL solution of DNA, buffer, and
the increasing concentration of compounds. The concentration of
DNA was 100 μM and that of metal complex was varied from
5 to 50 μM. The flow time of solutions in phosphate buffer (pH
7.0) was recorded in triplicate for each sample, and an average
flow time was calculated. Data were presented as (η/η0)1/3 versus
[Compound]/[DNA], where η is the viscosity of DNA in the pres-
ence of complex and η0 is the viscosity of DNA alone. The hydro-
dynamic length of DNA generally increases upon partial intercala-
tion while it does not lengthen upon groove binding [37].
2.7. Brine shrimp lethality bioassay
The cytotoxicity of complexes was studied on brine shrimp,
Artemia cysts, according to the reported method [29]. In this ex-
periment, 10 nauplii (larvae) were added to the vials of 24-well
Microtiter plate with a total volume of to 2500 μL per vial [1450
μL sea salt solution + 1000 μL sea salt containing 10 nauplii + 50
μL (complex + DMSO)], [DMSO] <2% (V/V). After 24 h of incuba-
tion, live and dead larvae were counted and the LC50 values were
determined for each complex [30].
2.8. In vitro antibacterial screening
2.11.3. Molecular docking with DNA sequence d(ACCGACGTCGGT)2
Docking study was measured for Re(I) complexes with deoxyri-
bonucleic acid (DNA). The main purpose of molecular docking is to
identify the binding mode of metal complexes using Hex 8.0 soft-
ware according to the reported procedure [38].
The in vitro antibacterial activity of ligands and complexes was
performed according to the literature procedure [31]. Luria Broth
was used as the bacteria growth medium. Serially diluted concen-
trations of each of test compounds (in DMSO) from 20 to 2800 μM
5

R.R. Varma, J.G. Pandya, F.U. Vaidya et al.
Journal of Molecular Structure 1240 (2021) 130529
2.11.4. Agarose gel electrophoresis
The effect of compounds on the integrity of DNA of S. cerevisiae
cells results of cytotoxicity encouraged us to find out whether the
compounds have any effect on the integrity of DNA. DNA extrac-
tion of S. cerevisiae yeast given in the literature [39]. Gel elec-
trophoresis of plasmid DNA (S. cerevisiae DNA) was carried out in
0.8% agarose gel containing 2.5 μL100mM ethidium bromide (EtBr)
in 50 ml TAE buffer (0.4 gm agarose in 50 ml TAE buffer) submerge
the gel in electrophoresis. Mix the appropriate amount of isolated
DNA with gel and load the mixture (5μL Dye + 3μL sample DNA)
mix it properly then load into the gel. Apply a voltage of 100
Volt, DNA will migrate towards positive anodes, when DNA sam-
ple or dye have migrated a sufficient distance turn off the power
to analyze the gel in UV-light. Image of gel observed in instrument
AlphaDigiDocTM RT. Version V.4.0.0 PC–Image software. The effect
of the complexes (I-V) on the DNA integrity of S. cerevisiae was
carried out by the isolation of DNA from treated and untreated S.
cerevisiae cells and electrophoresed on 1% agarose gel followed by
visualization.
Fig. 1. HOMO (A) and LUMO (B) orbital containing structures of the complex - I.
bounded CO in rhenium(I) metal complexes. Observed frequencies
for metal coordinated CO ligands are lower than free CO ligands
i.e., 2143 cm−1 [43]. CO-stretching frequency data proved that CO
group involves in back-bonding (metal/CO p-back donation into the
p∗ CO orbital), it decreases the bond strength of CO ligands and ul-
timately increases the bond strength of Re-CO bond.
The Ʌm (molar conductance) values of complexes (I-V) were
found in the range of 2.00 – 18.88 ꢀ−1cm2mol−1. It suggests that
complexes (I-V) are non-ionic and chemically neutral.
3. Results and discussion
3.1. Characterization
The electronic spectra of the synthesized ligands (L1-L5) and
complexes (I- V) were recorded at ambient conditions in DMSO.
Two bands appeared in the electronic spectrum: MLCT (metal to
ligand charge transfer) band at 332–400 nm region and LMCT (lig-
and to metal charge transfer) band at 240–330 nm region. The
bands are characteristics of the octahedral environment of ligands
around Re(I) metal ion [44].
The compounds were characterized by 1H NMR, 13C (APT) NMR,
IR, mass, electronic spectra, elemental analysis and conductivity
measurement. The characterization data supports the proposed
structure of Re(I) complexes. We have also performed theoretical
calculations (DFT study) for Re(I) complexes to further support the
proposed structure of Re(I) complexes. The single crystal XRD of
similar type of compounds are reported in the literature [26,40].
The 1H NMR of ligands L1-L5 demonstrate peak at 7.53- 8.65 δ
ppm, 7.52- 8.35 δ ppm, 7.42–9.27 and 7.56- 9.29 δ ppm, 7.32–8.58
δ ppm, and 7.55- 8.40 δ ppm. In the complexes (I-V), these peaks
are shifted to 7.61–8.7 δ ppm, 7.61–8.34 δ ppm, 7.56–9.29 δ ppm,
7.25–8.62 δ ppm, and 8.01–8.64 δ ppm, respectively. In addition to
above data, there is down field shifting occurs for the proton H1’’a
after complexation i.e., 8.65 to 8.74 in L1, 8.36 to 8.34 in L2, 9.28
to 9.29 in L3, 8.58 to 8.62 in L4 and 8.41 to 8.65 in L5. The change
in chemical shift values is observed for two carbon atoms i.e., C-
3.2. Computational analysis
All density functional theory (DFT) calculations were performed
using the Gaussian-09 program package. The DFT/B3LYP (Becke
three-parameter Lee-Yang-Parr) method with LanL2DZ basis set is
used to determine optimized geometry in which bond lengths and
bond angles are calculated. The optimized electronic structure, op-
timized bond length and bond angles of all complexes are shown
in supplementary material 3.
ꢀ
ꢀ
ꢀ
1 ’a and C-1 a, among these two carbon atoms C-1 ’a shows more
ꢀ
3.2.1. Homo-Lumo energy
shifting as compared to C-1 a after complexation. Down field shift-
ing for C-1 ’a are 161.6 to 163.3 in L1, 161.1 to 163.5 in L2, 150.0
to 152.8 in L3, 162.2 to 163.5 in L4 and 154.1 to 155.6 in L5. These
ꢀ
The frontier molecular orbitals (FMO), HOMO, and LUMO play
a significant role in the study of electrical and optical properties.
The HOMO (highest occupied molecular orbital) and LUMO (low-
est unoccupied molecular orbital) give information about the in-
teraction of a molecule with other species. The HOMO signifies the
ability to donate an electron, and the LUMO to accept an elec-
tron. A molecule with a small difference in the orbital energy is
usually more reactive. Among numerous other uses, the energy
gap (Eg) between HOMO and LUMO is used to predict the activ-
ity and intramolecular charge transfer in organic as well as in-
organic molecules with conjugated π bonds. The energy gap be-
tween HOMO-LUMO orbitals of the complexes I - V are 1.8251 eV,
1.8648 eV, 1.7878 eV, 1.8022 eV, and 1.8240 eV, respectively in
the gaseous phase. From Fig. 1, it can be concluded that HOMO
is evenly distributed in the central Re atom through the carbonyl
group whereas LUMO is distributed in the ligand’s moiety. HOMO-
LUMO orbital structures of all the complexes are shown in supple-
mentary material 3.
ꢀ
change in chemical shift for H-1 ’a and more down field shift for
ꢀ
ꢀ
C-1 ’a than C-1 a suggest that coordination occurs from N-20 atom.
13C-APT data of ligand L1-L5 and complexes (I-V) show signals at
126.0–166.0 δ ppm, which are assigned to the aromatic carbon. In
APT spectra, quartet and methylene carbon exhibited normal sig-
nals, while -CH and –CH3 signals are inverted. Signals at 180.0–
195.0 δ ppm value are assigned to carbonyl groups forming a co-
valent bond with the metal ion.
The FT-IR spectra of quinoxaline based ligands (L1-L5) and com-
plexes I -V demonstrate the signal for quinoxaline ring contain
ν(C=N) extending groups at 1600 – 1650 cm−1 range. The signal
at 1500- 1625 cm−1 is assigned for C C stretching, while signals
–
at ~640 cm−1, ~817 cm−1, and ~1150 cm−1 are assigned to C-Br,
–
C Cl and C-F stretching. Each complex indicates (-CO) signal at
around 2030–1860 cm−1, typical of a pseudo-C3v symmetry result-
ing from a facial arrangement of carbonyls [41]. The magnetic mo-
ments (μeff) value of rhenium(I) is found zero, indicate it does not
have unpaired electron (low spin t2g6 eg0 configuration) i.e. all the
rhenium(I) complexes are diamagnetic with +1 oxidation state of
rhenium metal ion. IR spectroscopic data of Re(I) complexes sug-
gest that all three CO in Re(CO)3 have shown its characteristic band
between 1735 and 2021 cm−1 [42], it gives the confirmation of all
3.2.2. Mulliken population analysis
The atomic charge can be determined by Mulliken pop-
ulation analysis (MPA) of the complexes. The bonding abil-
ity of
a molecule depends on the electronic charge on the
atoms. The analysis of complex-I expresses that the atoms Cl28
6

R.R. Varma, J.G. Pandya, F.U. Vaidya et al.
Journal of Molecular Structure 1240 (2021) 130529
Fig. 2. % Cell proliferation of complexes (I-V).
(−0.247047), C26 (−0.25944), N19 (−0.182141),
N
(−0.202768)
with rhenium metal is promising in terms of enhancing anticancer
activity.
display highest electronegativity and atoms C4 (0.441342), C5
(0.409261), C21 (0.47035), Re27 (0.411185) display highest elec-
tropositivity. In complex-II, the atoms C7 (−0.332792), C9
(−0.268581), N14 (−0.20396), N20 (−0.205225), Cl30 (−0.25974)
and O32(−0.172988) display highest electronegativity and atoms
C4 (0.439819), C5(0.41042), C17(0.44617), C22(0.444474), C25
(0.454096), Re29 (0.424837) show highest electropositivity. In
complex-III, the atoms C7 (−0.318857), C9 (−0.266415), N14
(−0.206599), N19 (−0.202071), Cl28 (−0.22399), O48 (−0.233999)
show highest electronegativity and atoms C4 (0.439741), C5
(0.419576), C17 (0.347119), C21 (0.452091), Re27 (0.421684) show
highest electropositivity. In complex-IV, the atoms C7 (−0.32735),
C9 (−0.26432), N14 (−0.20268), N19 (−0.2054), C24 (−0.20677),
Cl28 (−0.24086) show highest electronegativity and atoms C4
(0.439861), C5 (0.412316), C13 (0.227544), C21 (0.453369), Re27
(0.422756) show highest electropositivity. In complex-V, the
atoms C7 (−0.329007), C9 (−0.263397), N14 (−0.203263), C17
(−0.301049), N19 (−0.202252), Cl28 (−0.241002), F47 (−0.223174)
show highest electronegativity and atoms C4 (0.440203), C5
(0.411233), C13 (0.221772), C21 (0.458317), C24 (0.347367), Re27
(0.420536) show highest electropositivity. All the values and bar-
chart analysis of Mulliken charges for all complexes are shown in
the supplementary material 3.
3.4. Cellular level bioassay using S. cerevisiae
The cellular level cytotoxicity of ligands L1-L5 and complexes
I-V was carried out by using Saccharomyces Cerevisiae cells in
terms of% viability (Supplementary material 4). The in vitro toxi-
city was found to vary with the concentrations of the synthesized
compounds, chelating ligands environment, and CO ligand directly
linked by a synergic bond with Re(I) metal ion. The increase in
compound concentration resulted in a decrease in% cell viability.
Amongst all ligands, L5 is most potent as it contains the most
electronegative F-atom at p-position to the ancillary ligand, while
L2 is least potent as it contains electron-donating -CH3 group. All
complexes have higher%viability (more potent) than all ligands. At
40.75 μg/mL concentration, complex-V showed maximum cytotox-
icity as it is having fluorine substituted ligand. The increasing order
of%viability of ligands and complexes are V > IV > III > I > II >L5
>L4 >L3 > L1 > L2.
3.5. In vivo brine shrimp lethality bioassay (BSLB)
The BSLB method was used for calculating the%mortality and
toxic nature of synthesized compounds in terms of the death of
the larvae. A plot of the log of the sample’s concentration ver-
sus percentage (%) of mortality showed a linear correlation. The
synthesized ligands have more mortality rate as compared to the
synthesized complexes. The increasing order of mortality rate of
ligands and complexes is L1 (13.94 μg/mL) < L2 (12.12 μg/mL)
< L3 (12.10 μg/mL) < L4 (12.05 μg/mL) < L5 (9.85 μg/mL) <
I (20.21 μg/mL) < II (20.03 μg/mL) < III (18.05 μg/mL) < IV
(16.22 μg/mL) < V (16.06 μg/mL). From the graph, LC50 values
(shown in brackets) of the compounds were calculated, ligand L5 is
most cytotoxic due to the presence of F-atom and L2 has the least
cytotoxicity (due to electron-donating methyl group) amongst lig-
ands. Furthermore, all complexes are more cytotoxic than ligands.
The complex-V is the most potent as it is having the most elec-
tronegative and smallest in size F-atom containing ligand. All com-
pounds have higher cytotoxicity than reported Pd(II) complexes
[45], and [PtCl2(L6)] [46].
3.3. Antiproliferation study
Synthesized complexes (I-V) were tested for cell antiprolifer-
ation activity by MTT assay on the HCT 116 cell line. The% cell
proliferation decreased with an increase in complex concentra-
tion due to inhibition of tumor cells. The decreasing order of IC50
values is cisplatin> oxaliplatin > IV > V > carboplatin > III >
I > II (Fig. 2). Above 500 μg/mL concentration, the solution be-
comes turbid, and coloration occurred. The IC50 values of synthe-
sised complexes (I-V) and standard drugs cisplatin, carboplatin, ox-
aliplatin are >500 μg/mL, >500 μg/mL, 465.2 μg/mL, 60.9 μg/mL,
77.10 μg/mL, 15.49 μg/mL, >111.37 μg/mL, and 22.66 μg/mL, re-
spectively. The results suggest that complex-IV is most cytotoxic,
and we can say that our approach to synthesize metal complexes
having carbon monoxide (CO) and heterocyclic compound chelated
7

R.R. Varma, J.G. Pandya, F.U. Vaidya et al.
Journal of Molecular Structure 1240 (2021) 130529
Fig. 3. % Cell viability of ligands (L1-L5) and complexes (I-V).
3.6. In vitro antibacterial screening
compounds can react with ROS, and thus convert ROS to hydrogen
peroxide. The H2O2 might have contradictory effects on cell prolif-
eration depending on the concentration and cell type, it may cause
an antiproliferative effect [50]. The reaction of metal complexes
with ROS produces higher H2O2 accumulation than ligands that
leads to cell death. So, it can be concluded that metal complexes
are more cytotoxic than ligands as H2O2 accumulation is greater in
treated cells (death cells) than untreated cells. The higher produc-
tion of H2O2 indicates more effectiveness of compounds to inhibit
the production of ROS.
The synthesized heterocyclic ligands (L1-L5) and the complexes
(I-V) were tested against two Gram +ve (S. aureus, B. subtilis) and
three Gram -ve (S. marcescens, P. aeruginosa, E. coli) bacteria. The
data reveals that all complexes have a greater inhibition rate of
bacteria than neutral bidentate ligands (L1-L5) and a metal salt
(Supplementary material 5). For the synthesized complexes, two
factors are applicable, the ligands that are bound to metal ions in
a multidentate fashion, and the nature of the ligand [47]. The MIC
values of the complexes and ligands are observed in the range of
60–90 μM, and 280–320 μM (Supplementary material 5), respec-
tively; which are comparable to the reported rhenium(I) complexes
(MIC = 16–256 μM) [48]. Complex-V and III are the most active
amongst all the compounds. The increasing order of MIC values is
L2 < L1 < L3 < L4 < L5 < II < IV < I < III < V. Bulkiness and elec-
tronegativity affects the MIC values. Amongst all ligands, L2 has a
higher MIC value (least potent) due to presence of electron donat-
ing methyl group and L5 has least MIC value (more potent) due
to presence of most electronegative F-atom to the ancillary ligand.
All metal complexes have lower MIC values (more potent) than lig-
ands. The complex-V has the least MIC value (most potent) due to
the presence of F-atom substituted ligand.
3.8. Lipid peroxidation
Pure lipid suspensions and liposomes can improve metal-based
autoxidation, which produces malondialdehyde that reacts with
lipid tissue to form lipofuscin, thus reducing its concentration in
the cell. Therefore, MDA concentration involves peroxidation in bi-
ological membranes. There are three ways of autoxidation; the first
is free-radical-based oxidation, the second is without free radial
nonenzymatic oxidation and the third is autooxidation in the pres-
ence of enzymes. Lipid contains tissues like fatty acids and excess
cholesterol in the heart that are oxidized by ROS techniques and
decrease its content in the tissue. Production of MDA concentration
increased with time and concentration, it was time- concentration
dependent method. The results state that the MDA content was
higher in complexes than the ligands. So, the toxic nature of com-
plexes was higher than the ligands (Fig. 4) (Supplementary mate-
rial 7). The decreasing order of MDA concentration is V > IV > III
> I > II >L5 >L4 >L3 > L1 > L2. The compounds containing elec-
tronegative fluorine atom as substituent have the highest activity,
while compounds containing electron-donating methyl group pos-
sess the least activity. The ligand L5 stimulates the production of
MDA concentration most effectively, while ligand L2 is the least ef-
fective in the production of MDA concentration. Furthermore, all
complexes exhibit higher MDA concentration than the ligands. The
complex-V is the most effective in stimulating the production of
MDA.
3.7. Reactive oxygen species (ROS)
The lethal effect of synthesized ligands (L1-L5) and complexes
(I- V) on growth inhibition of Saccharomyces cerevisiae was evalu-
ated by quantification of H2O2, as ROS. Here we found that treated
cells were showing greater production of H2O2, as compared to un-
treated cells. The H2O2 concentration was increasing with increas-
ing the test sample concentration (Supplementary material 6). The
decreasing order of H2O2 accumulation is as V > IV > III > I > II
> L5 >L4 >L3 > L1 > L2 (Fig. 3). The results state that complexes
show a higher toxic effect than ligands, and the ROS generation
rate depends upon compounds concentration and functionality on
compounds. This was further confirmed by observing their effects
on the DNA integrity of Saccharomyces cerevisiae as higher H2O2
causes DNA damage. Metabolic processes in cells produce reactive
oxygen species (ROS) i.e., oxygen-derived m•olecules such as hydro-
gen peroxide (H2O2), superoxide anion (O2 −), and hydroxyl radi-
cal (•OH) [49]. The presence of reactive oxygen species in the cell
is very harmful to the DNA and leads to cancer. The synthesized
3.9. DNA interactions
3.9.1. Absorption titration
The binding mode and binding constant (Kb) of the complexes
toward DNA gives an idea about the strength of interaction, which
8

R.R. Varma, J.G. Pandya, F.U. Vaidya et al.
Journal of Molecular Structure 1240 (2021) 130529
Fig. 4. H2O2 accumulation of ligands (L1-L5) and complexes (I-V).
Fig. 5. MDA production in S. cerevisiae yeast using ligands (L1-L5) and complex (I-V).
can be obtained by studying UV–vis. absorption spectra. The suc-
cessive addition of DNA (100 μL) to the fixed concentration of
complexes at 10 min break time resulted in a decrease in absorp-
tion (hypochromism) and a small redshift. It suggests that all com-
plexes (I-V) bind to DNA through major or minor groove results
[35]. The binding constant (Kb) values were estimated from the
plot of [DNA]/(εa-εf) versus [DNA] concentration, the slope to in-
tercept ratio value gives binding constant Kb value (Fig. 5). The ab-
sorption spectral changes were monitored at around 240–330 nm
for the investigation of the DNA binding mode and strength. The
decreasing order of binding constant (shown in brackets) is V
DNA. While the effect was less pronounced in the case of synthe-
sized compounds, which showed a slight increase in viscosity of
DNA similar to minor groove binder netropsin [54,55]. These show
that compounds are more likely to have DNA groove binding mode
[44,55].
3.9.3. Molecular docking with DNA sequence d(ACCGACGTCGGT)2
Molecular docking study gives the idea of the orientation of
ligands and complexes inside the DNA groove [56,57]. The com-
plexes (I- V) and ligands (L1-L5) are shown by the ball and stick
and DNA base pairs are shown by the licorice model using Hex
8.0 software. The structure of ligands and complexes were drawn
in .CDX format using ChemBioDraw Ultra 14.0, then converted to
PDB format using Chem3D (Cambridge Soft). For docking studies,
the structural coordinates of DNA were obtained from the pro-
tein data bank (pdb id: 423D) [58]. Re(I) complexes bind to base-
pair A–T, C–G, G–C, A–T (B-DNA) at minor grooves of the DNA
(Fig. 6) (Supplementary material 9). Docking energies of ligands
(L1-L5) and complexes (I-V) are −268.84 kJ/mol, −270.95 kJ/mol,
−281.54 kJ/mol, −277.15 kJ/mol, −277.65 kJ/mol, −283.75 kJ/mol,
−280.82 kJ/mol, −302.12 kJ/mol, −288.47 kJ/mol, −290.23 kJ/mol,
respectively. Complex III shows higher negative binding energy
(−302.12 KJ/mol) than the other complexes and ligands. The as-
cending order of energy is L1 < L2 < L4 < L5 < L3 < II < I < IV <
V < III.
(2.6 × 105
M
−1) > IV (2.3 × 105
M
−1) > II (1.9 × 105
M
−1) > I
M
−1) >
(1.8 × 105
M
−1) > III (1.1 × 105
M
−1) > L4 (0.9 × 105
L3 (0.8 × 105
M
−1) > L1 (0.6 × 105
M
−1) > L2 (0.4 × 105 M−1
)
> L5 (0.3 × 105
M
−1). All complexes show higher binding con-
stant values than the ligands. Complex-V containing electronega-
tive fluorine atom as a substituent to the ancillary ligand exhibit
the highest binding strength towards DNA. The synthesized com-
pounds possess higher binding strength than reported Re(I) com-
plexes (Kb = 7.95 ( 1.89) × 104
M
−1) [42]. The Kb value of all
complexes are comparable with the reported rhodium(III) and irid-
ium(III) complexes (Kb = 1.94 × 105 –3.12 × 105
M
−1) [51].
3.9.2. Hydrodynamic volume or viscosity measurement
Viscosity measurements of DNA solution were carried out by
varying the concentration of compounds to get an idea of the bind-
ing mode. Groove binding causes less pronounced or only a minor
change in the viscosity [52]. The experimental values were plot-
ted as (η/η0)1/3 v/s [complex]/[DNA] and [ligand]/[DNA] (Supple-
mentary material 8). The ability to decrease the (η/η0)1/3 of com-
3.9.4. Effect of compounds on the DNA of S. cerevisiae cells
The Agarose gel electrophoresis technique was used to check
the DNA cleavage efficiency of ligands (L1-L5) and complexes (I-
V) [59]. The electrophoretic separation of S. cerevisiae DNA upon
treatment with compounds under aerobic conditions suggests that
complexes (I-V) have much higher binding efficacy to the S. cere-
visiae DNA than the quinoxaline based ligands (L1-L5). Ligands
show lesser smearing as compared to the complexes (Fig. 7). It
suggests that DNA cleavage efficiency complexes are higher than
plexes is in order V > IV > I > II > III > L2 > L3 > L1 > L5
>
L4. Complexes show a higher (η/η0)1/3 value than ligands, which
was also supported by reported compounds [53]. The complex-V
has the highest (η/η0)1/3 value. The EB (ethidium bromide) shows
the intercalation mode of binding, which increase the viscosity of
9

R.R. Varma, J.G. Pandya, F.U. Vaidya et al.
Journal of Molecular Structure 1240 (2021) 130529
Fig. 6. UV–visible spectra of (a) ligand (L1) and (b) complex-I.
Fig. 7. Molecular docking of ligand (L1) and complex (I) by the ball and stick model and DNA by VDW sphere.
Fig. 8. Electrophoretic cleavage of S. cerevisiae DNA using ligands (L1-L5) and complexes (I-V).
10

R.R. Varma, J.G. Pandya, F.U. Vaidya et al.
Journal of Molecular Structure 1240 (2021) 130529
the ligands. Complexes-V, I, and II show a better cleavage effect
of DNA, complex-III show moderate cleavage effect of DNA and
complex-IV shows lesser cleavage effect on DNA.
DST-PURSE Sardar Patel University, Vallabh Vidyanagar for LC-MS
analysis.
Supplementary materials
4. Conclusions
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.130529.
A series of substituted quinoxaline based organometallic rhe-
nium(I) complexes were synthesized and well-characterized, in
search of new organometallic complexes that can show better an-
tibacterial, cytotoxicity, reactive oxygen species production, lipid
peroxidation, genotoxicity, DNA binding, and DNA cleavage activ-
ities. The synthetic approach was carried out by pentacarbonyl
chloro rhenium(I) as the starting material for preparing the novel
neutral organometallic complexes having octahedral structure and
diamagnetic. 1H NMR and 13C NMR data concluded that coor-
dination occurs through N-20 atom and resultant complex have
six membered chelate ring. The DFT calculations were performed
for estimation of bond length and bond angle data, theoretically.
The Mulliken charge analysis of complexes by DFT studies was
performed for the evaluation of atomic charge in complexes. The
HOMO-LUMO molecular energy diagram and energy gap determi-
nation by DFT calculation show the charge distribution in com-
plexes. DNA binding study was carried out by absorption titration,
viscosity measurement, and molecular modeling. Binding constant
(Kb) values of complexes were higher than the ligands, it shows
the higher binding capability of complexes with DNA i.e., groove
binding. There was a minor change in the viscosity in complexes
than the ethidium bromide suggests DNA groove binding of com-
plexes. In molecular modeling, docking energies of complexes were
found higher than the ligands. The presence of a more electroneg-
ative environment improves the antibacterial activity as suggested
by MIC data. Complex-IV was observed most antiproliferative than
all synthesized compounds, cisplatin, oxaliplatin, and carboplatin.
All the complexes showed well in vitro cytotoxic, cellular level
bioassay, reactive oxygen species production, and lipid peroxida-
tion compared to ligands. The ability to produce reactive oxygen
species and lipid peroxidation may be the reason for the good an-
tiproliferative activity of complexes.
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