Fluorescence, DNA Interaction and Cytotoxicity Studies of 4,5-Dihydro-1H-Pyrazol-1-Yl Moiety Based Os(IV) Compounds: Synthesis, Characterization and Biological Evaluation

Article abstract of DOI:10.1007/s10895-020-02657-1

Osmium(IV) pyrazole compounds and ligands were synthesized and well characterised. Ligands were characterized by heteronuclear NMR spectroscopy (¹H & ¹³C), elemental analysis, IR spectroscopy and liquid crystal mass spectroscopy. Os(

Full text of DOI:10.1007/s10895-020-02657-1

Journal of Fluorescence (2021) 31:349–362
https://doi.org/10.1007/s10895-020-02657-1
ORIGINAL ARTICLE
Fluorescence, DNA Interaction and Cytotoxicity Studies
of 4,5-Dihydro-1H-Pyrazol-1-Yl Moiety Based Os(IV) Compounds:
Synthesis, Characterization and Biological Evaluation
Bharat H. Pursuwani1 & Bhupesh S. Bhatt1 & Foram U. Vaidya2 & Chandramani Pathak2 & Mohan N. Patel1
Received: 28 May 2020 /Accepted: 3 December 2020
/ Published online: 3 January 2021
#
The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Abstract
Osmium(IV) pyrazole compounds and ligands were synthesized and well characterised. Ligands were characterized by
heteronuclear NMR spectroscopy (1H & 13C), elemental analysis, IR spectroscopy and liquid crystal mass spectroscopy.
Os(IV) complexes were characterized by ESI-MS, ICP-OES, IR spectroscopy, conductance measurements, magnetic measure-
ments and electronic spectroscopy. Binding of compounds with HS-DNA were evaluated using viscosity measurements, ab-
sorption titration, fluorescence quenching, and molecular docking, which show effective intercalation mode exhibited by com-
pounds. Binding constant of Os(IV) complexes are found to be 8.1 to 9.2 × 104 M−1. Bacteriostatic and cytotoxic activities were
carried out to evaluate MIC, LC50, and IC50. The compounds have been undergone bacteriostatic screening using three sets of
Gram+ve and two sets of Gram-ve bacteria. MIC of complexes are found to be 72.5–100 μM, whereas that of ligands fall at about
122.5–150 μM.. LC50 count of ligands fall in the range of 16.22–17.28 μg/mL whereas that of complexes of Os(IV) fall in the
range of 4.87–5.87 μg/mL. IC50 of osmium compounds were evaluated using HCT-116 cell line. All the Os(IV) compounds
show moderate IC50.
.
.
.
Keywords Binding Absorption titration Molecular docking HCT-116
Introduction
ions geometrically is equally important [10]. Substitution
on heterocyclic rings enhance biological potentiality of
ligand as well as its metal ion based complex [11]. The
substituents having ability to enhance liphophilicity, sta-
bilize the oxidation state of metal ion based complex and
stabilize the complex making it kinetically inert and ther-
modynamically stable in important aspect of designing
such compounds concluded by structural and activity re-
lationship [12].
DNA has been a primary target of biomolecules to cure
diseases genetically by interaction with base pairs via electro-
static, Vander Waal and hydrophobic forces of attractions.
Complexes with octahedral geometry have been found to be
kinetically more inert since releasing of bioactive ligand on
specific DNA site is of prime importance. Quinoline based
heterocycles have been proved to have good binding affinity
than ciprofloxacin drug [13]. Binding affinity of quinoline
heterocycles are highly stabilized by hydrogen bonding inter-
actions concluded by docking studies [14, 15]. Quinoline class
of heterocycles have been proved to possess greater cytotox-
icity activities when explored to study on wide cancer cell
lines [16, 17].
The field of medicinal inorganic chemistry were explored
widely [1]. Metal complexes with biologically active het-
erocyclic ligands enhance its applications [2]. Design and
synthesis of such compounds having less side effects and
more potentiality has been a primary step towards explor-
ing its application in biological medicinal chemistry
[3–5]. Transition metals have been studied widely since
they often alter and bind with the DNA nucleic bases [6,
7]. The key area of interest for the chemist have been
developing drugs which provide resistance to diseases,
more effective and less toxic or having less side effects
[8, 9]. Design of heterocyclic ligands which fit with metal
* Mohan N. Patel
jeenen@gmail.com
1
Department of Chemistry, Sardar Patel University, Vallabh
Vidyanagar, Gujarat 388 120, India
2
Cell Biology Laboratory, Indian Institute of Advanced Research,
Koba, Gandhinagar, Gujarat 382421, India

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J Fluoresc (2021) 31:349–362
Quinoline nucleus-based complexes have been synthe-
Characterization
sized and studied and their biological assessment have been
proved to be impactful and superior than ligands [18].
Osmium metal ion readily bind with -N, O donor ligands
[19–22]. Osmium metal ion-based complexes have been
explored to in cytotoxic assays and varied cell lines [23].
These have been also in doing interaction with DNA and
adhering its mechanism which puts it into plethora of vast
medicinal synthetic chemistry [24, 25]. Osmium metal ion
complexes often prefer to bind via intercalation mode with
DNA macromolecule Osmium metal ion-based complexes
have been also proved effective in playing role in DNA
binding and cleavage agent [26].
Present report includes, synthesis of six different
substituted pyrazole nucleus-based -N,O donor ligands and
Os(IV) complexes. The compounds have been well charac-
terized and justified using various techniques. All the com-
pounds have undergone cytoxicity assays, bacteriostatic
study, DNA binding and cleavage activities.
General Method for Synthesis of Ligands
2-Chloro-6-methoxyquinoline-3-carbaldehyde have been
synthesized using Vilsmeier–Haack reaction (Scheme 1) [27].
Synthesis and Characterization
of (5-(2-Chloro-6-Methoxyquinolin-3-Yl)-3-(4-Chlorophenyl)
-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone
(L1)
Ligand L1 have been synthesized by Claisen Schmidt conden-
sation reaction between 2-chloro-6-methoxyquinoline-3-
carbaldehyde (2 g, 9 mmol) and 4-chloro acetophenone
(1.39 g, 9 mmol) in presence of potassium hydroxide (0.2 g,
4 mmol) as base. The chalcone formed (2 mmol, 0.42 g) have
been cyclized with (1 mmol, 0.15 g) in presence of potassium
tertiary butoxide (4 mmol, 0.22 g) to form ligand L1. Empirical
formula: C26H19Cl2N3O3, Color: Yellow, yield: 90%, mol. Wt:
492.36 g/mol, m.p.: 285 °C; anal. Cal. (%) for: C, 63.43; H,
3.89, N, 8.53; Found (%), C, 63.22; H, 3.80, N, 8.20; Mass
1
Experimental
m/z(%): 425[M]+. H-NMR (400 MHz, DMSO-d6) δ/ppm:
2.404 (S, 3H, OCH3), 3.248(dd, 1H, H4a, J1 = 5.2 Hz, J2 =
2
3
2
3
Materials and Reagents
5.2 Hz), 4.005(dd, 1H, H4b, J1 = 11.6 Hz, J2 = 11.6 Hz),
6.269(t, 1H, H5), 7.244–8.175(m, 14H, Ar-H), 9.644(s, 1H,
OH) 13C-NMR (100 MHz, DMSO-d6) δ/ppm: 189.65, (C=O,
Phosphorous oxychloride, dimethyl formamide used in reac-
tion were purchased from SDFCL, SD-Fine chemicals ltd. 4-
hydroxide benzhydrazide, potassium tertiary butoxide, osmi-
um tetroxide (Crystals), hydrobromic acid, ammonium bro-
mide was purchased from Sigma Aldrich, Artemia cyst eggs
were purchased from local aquarium. Luria broath, ethidium
bromide and TAE buffer solution were purchased from
Himedia(India). Bacterial culture was borrowed from
MTCC, Chandigarh (India). Herring sperm DNA solution
was purchased from Sigma Aldrich.
C
C
quat); 152.35, (C5, Cquat); 146.35, (C1’, Cquat); 145.06, (C4’,
quat); 142.32, (C2”, Cquat); 132.36, (C3”, Cquat); 132.35, (C2”,
C6”, -CH); 132.05 (C3’, C5’, -CH); 131.43 (C4”, CH); 131.38,
(C5”, CH); 131.37 (C7”, CH); 131.36 (C4a’, Cquat); 131.12,
(C8”, Cquat); 130.88, (C8a’, Cquat); 128.67, (C2”’, C6”, CH);
128.27 (C6”’, Cquat); 126.76, (C3”, C5”, CH); 126.43, (C1”,
C
quat); 126.42, (C4”, Cquat); 55.67, (methoxy, CH3); 44.59 (C4,
CH2); 36.46 (C3, CH). (UV-Vis (DMSO, c = 10−4 mol dm−3):
λmax (ɛ) = 232.00 nm (29,350) mol- 1dm3 cm−1). IR (KBr,
cm−1): 3099 ν(=C-H); 1540 ν(C-N); 1312 ν(C-H); 1666
ν(C=C); 1610 ν(C=O).
Physical Measurement
Synthesis and Characterization
Liquid crystal mass spectroscopy, waters model with
micromass technology were used for mass analysis.
Electronic spectra were taken using UV (160A) spectro-
photometer. Elemental analysis were carried out using
Euro elemental analyzer. Melting point of compounds
were taken using Thermo Cal10. Melting point apparatus.
Magnetic property of complexes were measured using
Guoy’s method. The smeared images of cytotoxicity as-
says were visualized by AlphaDigDoc software
(California). Heteronuclear spectra were taken using
Bruker Avance NMR spectrophotometer (400 MHz).
Infra-red spectra were taken using Shimadzu instrument
(400–4000 cm−1).
of (5-(2-Chloro-6-Methoxyquinolin-3-Yl)-3-(4-Hydroxyphenyl)
-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone
(L2)
It was synthesized using 4-hydroxy acetophenone (9 mmol,
1.22 g) using procedure mentioned in the synthesis of ligand
L1. Empirical formula: C26H20ClN3O4, Color: Yellow, yield:
88%, mol. Wt: 473.91 g/mol, m.p.: 281 °C; anal. Cal. (%) for:
C, 65.90; H, 4.25, N, 8.87; Found (%),C, 65.85; H, 4.12, N,
1
8.80; Mass m/z(%): 473.00 [M]+. H-NMR (400 MHz,
DMSO-d6) δ/ppm: 2.412 (S, 3H, OCH3), 3.260(dd, 1H, H4a,
3
2
2J1 = 5.6 Hz, J2 = 2.8 Hz), 4.017(dd, 1H, H4b, J1 = 7.6 Hz,
3J2 = 12.8 Hz), 6.276(t, 1H, H5), 7.489–8.183(m, 14H, Ar-H),

J Fluoresc (2021) 31:349–362
351
Scheme 1 General reaction scheme for synthesis of ligands L1-L6 and complexes C1-C6
9.743(s, 1H, OH) 13C-NMR (100 MHz, DMSO-d6) δ/ppm:
189.70, (C=O, Cquat); 152.40 (C5, Cquat); 146.40 (C1’,
formula: C26H20ClN3O3, Color: Yellow, yield: 82%, mol. Wt:
457.91 g/mol, m.p.: 283 °C; anal. Cal. (%) for: C, 68.20; H,
4.40, N, 9.18; Found (%), C, 68.08; H, 4.30, N, 9.11; Mass
m/z(%):457.00 [M]+. 1H-NMR (400 MHz, DMSO-d6) δ/ppm:
2.418 (S, 3H, OCH3), 3.265(dd, 1H, H4a, 2J1 = 5.6 Hz, 3J2 =
C
quat); 145.11, (C4’, Cquat); 142.37, (C2”, Cquat); 132.41,
(C3”, Cquat); 131.84, (C2’, C6’, CH); 131.83, (C3’, C5’,
CH); 131.53, (C4”, CH); 130.91, (C5”, CH); 130.86, (C7”,
CH); 130.85, (C4a’, Cquat); 129.37, (C8”, CH); 129.13, (C8a’,
8.4 Hz), 4.027(dd, 1H, H4b, J1 = 10.4 Hz, J2 = 9.2 Hz),
6.278(t, 1H, H5), 7.489–8.188(m, 14H, Ar-H), 9.883(s, 1H,
OH) 13C-NMR (100 MHz, DMSO-d6) δ/ppm: 189.75,
(C=O, Cquat); 152.45, (C5, Cquat); 146.45, (C1’, Cquat);
145.16, (C4’, Cquat); 142.42, (C2”, Cquat); 132.46, (C3”,
2
3
Cquat): 126.92 (C6”, Cquat); 126.52 (C2”’, C6”’, CH); 124.01
(C1”, Cquat); 124.84, (C3”’, C5”’); 124.67, (C4”’, Cquat);
54.52, (methoxy, OCH3); 42.84 (C4, CH2); 34.71 (C3, CH).
(UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) = 230.50 nm
(29,350) mol- 1dm3 cm−1). IR (KBr, cm−1): 3067 ν(=C-H);
1521 ν(C-N); 1320 ν(C-H); 1678 ν(C=C); 1625 ν(C=O).
C
quat); 131.89, (C2’, C6’, CH); 131.88, (C3’, C5’, CH);
131.58, (C4”, CH); 130.96, (C5”, CH); 130.91, (C7”, CH);
130.90, (C4a’, Cquat); 129.42, (C8”, CH); 128.18, (C8a’,
C
quat); 126.97, (C2”’, C6”’, CH), 126.57, (C6”, CH);
Synthesis and Characterization
126.06, (C1”, CH); 124.89, (C3”’, C5”’, CH); 124.72,
(C4”’, Cquat); 56.20, (methoxy, OCH3); 42.89, (C4, CH2);
34.46, (C4, CH). (UV-Vis (DMSO, c = 10−4 mol dm−3):
λmax (ɛ) = 238.50 nm (29,350) mol- 1dm3 cm−1). IR (KBr,
cm−1): 3055 ν(=C-H); 1543 ν(C-N); 1335 ν(C-H); 1680
ν(C=C); 1632 ν(C=O).
of (5-(2-Chloro-6-Methoxyquinolin-3-Yl)
-3-Phenyl-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)
Methanone (L3)
It was synthesized using acetophenone (9 mmol, 1.08 g) using
procedure mentioned in the synthesis of ligand L1. Empirical

352
J Fluoresc (2021) 31:349–362
Synthesis and Characterization
cm−1). IR (KBr, cm−1): 3032 ν(=C-H); 1520 ν(C-N); 1352
ν(C-H); 1653ν(C=C); 1648ν(C=O).
of (5-(2-Chloro-6-Methoxyquinolin-3-Yl)-3-(4-Fluorophenyl)
-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone
(L4)
Synthesis and Characterization of (3-(2-Bromophenyl)
-5-(2-Chloro-6-Methoxyquinolin-3-Yl)
-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone
(L6)
It was synthesized using 4-floro acetophenone (9 mmol,
1.24 g) using procedure mentioned in the synthesis of ligand
L1. Empirical formula: C26H19ClFN3O3, Color: Yellow,
yield: 84%, mol. Wt: 475.90 g/mol, m.p.: 280 °C; Mass
m/z(%):475.00 [M]+. (UV-Vis (DMSO, c = 10−4 mol dm−3):
It was synthesized using 3-bromo acetophenone (9 mmol,
1.79 g) using procedure mentioned in the synthesis of ligand
L1. Empirical formula: C26H19BrClN3O3, Color: Yellow,
yield: 85%, mol. Wt: 536.81 g/mol, m.p.: 280 °C; anal. Cal.
(%) for: C, 58.17; H, 3.57, N, 7.83; Found (%),C, 58.10; H,
3.10, N, 7.70. Mass m/z(%): 536.00 [M]+. 1H-NMR
(400 MHz, DMSO-d6) δ/ppm: 2.438 (S, 3H, OCH3),
λmax (ɛ) = 267.50 nm (29,350) mol- 1dm3 cm−1). H-NMR
1
(400 MHz, DMSO-d6) δ/ppm: 2.422(S, 3H, OCH3),
2
3
3.271(dd, 1H, H4a, J1 = 5.2 Hz, J2 = 4.8 Hz), 4.029(dd,
2
3
1H, H4b, J1 = 11.6 Hz, J2 = 10.4 Hz), 6.286(t, 1H, H5),
7.494–8.195(m, 14H, Ar-H), 9.443(s, 1H, OH) 13C-NMR
(100 MHz, DMSO-d6) δ/ppm: 189.81, (C=O, Cquat); 152.51,
(C5, Cquat); 146.50, (C1’, Cquat); 144.22, (C4’, Cquat); 142.48,
(C2”, Cquat); 132.52, (C3”, Cquat); 131.95, (C2’, C6’, CH);
131.94, (C3’, C5’, CH); 131.64, (C4”, CH); 130.02, (C5”,
CH); 130.97, (C7”, CH); 130.96, (C8”, CH); 129.48, (C2”’,
C6”’); 128.24, (C4a’, Cquat); 126.63, (C8a’, Cquat); 126.03,
(C8a’, Cquat); 125.12, (C1”, Cquat); 124.95, (C3”’, C5”’,
CH); 124.78, (C4”’, CH); 56.43, (Methoxy, OCH3); 42.95,
(C4, CH2); 34.82, (C3, CH). (UV-Vis (DMSO, c =
10−4 mol dm−3): λmax (ɛ) = 239.50 nm (29,350) mol- 1dm3
cm−1). IR (KBr, cm−1): 3046 ν(=C-H); 1533 ν(C-N); 1342
ν(C-H); 1687 ν(C=C); 1646 ν(C=O).
2
3
3.281(dd, 1H, H4a, J1 = 5.2 Hz, J2 = 6.8 Hz), 4.042(dd,
1H, H4b, J1 = 8.4 Hz, J2 = 12.8 Hz), 6.298(t, 1H, H5),
7.519–8.218(m, 14H, Ar-H), 9.748(S, 1H, OH) 13C-NMR
(100 MHz, DMSO-d6) δ/ppm: 189.61, (C=O, Cquat); 152.31,
(C5, Cquat); 146.31, (C1’, Cquat); 145.02, (C2’, Cquat); 142.28,
(C2”, Cquat), 132.32, (C3”, Cquat); 131.75, (C3’, CH); 131.74,
(C4’, CH); 131.44, (C5’, CH); 130.39, (C6’, CH); 130.37,
(C4”, CH); 130.33, (C4a’, Cquat); 129.85, (C5”, CH),
128.61, (C8a’, Cquat), 126.46, (C6”, Cquat); 126.00 (C1”,
2
3
C
quat); 124.49, (C7”, CH), 124.32, (C8”, CH); 124.18,
(C2”’, C6”’, CH); 124.15, (C4”, Cquat); 119.80, (C3”’, C5”’,
CH); 53.12, (Methoxy, OCH3); 42.32, (C4, CH2); 34.19, (C3,
CH). (UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) =
231.50 nm (29,350) mol- 1dm3 cm−1). IR (KBr, cm−1): 3024
ν(=C-H); 1517 ν(C-N); 1361 ν(C-H); 1690 ν(C=C); 1628
ν(C=O).
Synthesis and Characterization of 7(3-(4-Bromophenyl)
-5-(2-Chloro-6-Methoxyquinolin-3-Yl)
-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone
(L5)
Synthesis and Characterization of Metal Salt and
Complexes
It was synthesized using 4-bromo acetophenone (9 mmol,
1.79 g) using procedure mentioned in the synthesis of ligand
L1. Empirical formula: C26H19BrClN3O3, Color: Yellow,
yield: 80%, mol. Wt: 536.81 g/mol, m.p.: 292 °C; anal. Cal.
(%) for: C, 58.17; H, 3.57, N, 7.83; Found (%),C, 58.10; H,
3.45, N, 7.75; Mass m/z(%): 536.00[M]+. 1H-NMR
(400 MHz, DMSO-d6) δ/ppm: 2.435 (S, 3H, OCH3),
Synthesis of Ammonium Hexabromoosmate(IV)
(NH4)2OsBr6 have been synthesized using osmium tetroxide
and hydrobromic acid as starting reactants (Scheme 1) [28,
29].
2
3
3.277(dd, 1H, H4a, J1 = 5.2 Hz, J2 = 5.6 Hz), 4.032(dd,
2
3
1H, H4b, J1 = 8.8 Hz, J2 = 12.4 Hz), 6.288(t, 1H, H5),
7.511–8.210(m, 14H, Ar-H), 9.646(s, 1H, OH). 13C-NMR
(100 MHz, DMSO-d6) δ/ppm: 189.56, (C=O, Cquat); 152.26,
(C5, Cquat); 146.26, (C1’, Cquat); 144.97, (C4’, Cquat); 142.23,
(C2”, Cquat); 132.27, (C3”, Cquat); 131.70, (C2’, C6’, CH);
131.69, (C3’, C5’, CH); 131.39, (C4”, Cquat); 130.77, (C5”,
CH); 130.72, (C7”, CH); 130.71, (C4a’, Cquat); 129.23, (C8”,
CH), 128.99, (C8a’, Cquat); 126.78, (C2”’, C6”’, CH); 126.38,
(C6”, Cquat); 124.87, (C1”, Cquat); 124.70, (C3”’, C5”’, CH);
124.53, (C4”, Cquat); 54.60, (Methoxy, OCH3); 42.70, (C4,
CH2 ); 34.57, (C3, CH). (UV-Vis (DMSO, c =
10−4 mol dm−3): λmax (ɛ) = 230.50 nm (29,350) mol- 1dm3
Synthesis and Characterization
of Tetrabromido(5-(2-Chloro-6-Methoxyquinolin-3-Yl)
-3-(4-Chlorophenyl)-4,5-Dihydro-1H-Pyrazol-1-Yl)
(4-Hydroxyphenyl)Methanone)Osmium(IV) (C1)
Tetrabromido(5-(2-chloro-6-methoxyquinolin-3-yl)-3-(4-
chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(4-
hydroxyphenyl)methanone)osmium(IV) have been syn-
thesized using (NH4)2OsBr6 (0.60 g, 0.085 mmol) and
ligand L1 (0.41 g, 0.085 mmol). Color: Brownish black,
yield: 60%, mol. Wt: 1000.63 g/mol, m.p.: >300 °c. Cal.
(%) for: Os,18.98; Found (%), Os,18.68;. ESI-MS

J Fluoresc (2021) 31:349–362
353
(70 eV): 1002.20, 922.30, 842.39, 762.49, 682.59,
492.59 m/z: Magnetic Moment: 2.70 B.M. (UV-Vis
(DMSO, c = 10−4 mol dm−3): λmax (ɛ) = 284.00 nm
(29,350) mol- 1dm3 cm−1). Conductance: 36 ℧
mol−1 cm2. IR (KBr, cm−1): 3112 ν(=C-H); 1562 ν(C-
N); 1318 ν(C-H); 1680 ν(C=C); 1618 ν(C=O); 556
ν(N-Os); 611 ν(N-Os-Br).
Synthesis and Characterization
of Tetrabromido(3-(4-Bromophenyl)
-5-(2-Chloro-6-Methoxyquinolin-3-Yl)
-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone)
Osmium(IV) (C5)
It was synthesized using (NH4)2OsBr6 (0.60 g, 0.085 mmol)
and ligand L5 (0.45 g, 0.085 mmol). Color: Brownish black,
yield: 60%, mol. Wt: 1046.66 g/mol, m.p.: >300 °c. Cal. (%)
for: Os,18.18; Found (%),Os,18.02; (UV-Vis (DMSO, c =
10−4 mol dm−3): λmax (ɛ) = 225.50 nm (29,350) mol- 1dm3
cm−1). Conductance: 24 ℧ mol−1 cm2. IR (KBr, cm−1): 3047
ν(=C-H); 1538 ν(C-N); 1362 ν(C-H); 1661 ν(C=C); 1653
ν(C=O); 592 ν(N-Os); 627 ν(N-Os-Br).
Synthesis and Characterization of Tetrabromido(of
(5-(2-Chloro-6-Methoxyquinolin-3-Yl)-3-(4-Hydroxyphenyl)
-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone)
Osmium(IV) (C2)
It was synthesized using (NH4)2OsBr6 (0.60 g,
0.085 mmol) and ligand L2 (0.40 g, 0.085 mmol). Color:
Brownish black, yield: 60%, mol. Wt: 983.76 g/mol, m.p.:
>300 °c. Cal. (%) for: Os,19.34; Found (%),Os,19.20;
(UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) =
239.50 nm (29,350) mol- 1dm3 cm−1). Conductance: 32
℧ mol−1 cm2. IR (KBr, cm−1): 3098 ν(=C-H); 1534
ν(C-N); 1342 ν(C-H); 1686 ν(C=C); 1634 ν(C=O); 562
ν(N-Os); 580 ν(N-Os-Br).
Synthesis and Characterization
of Tetrabromido(3-(2-Bromophenyl)
-5-(2-Chloro-6-Methoxyquinolin-3-Yl)
-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)Methanone)
Osmium(IV) (C6)
It was synthesized using (NH4)2OsBr6 (0.60 g, 0.085 mmol)
and ligand L5 (0.45 g, 0.085 mmol). Color: Brownish black,
yield: 60%, mol. Wt: 1046.66 g/mol, m.p.: >300 °c. Cal. (%)
for: Os,18.18; Found (%),Os,17.98; Conductance: 28 ℧
mol−1 cm2. (UV-Vis (DMSO, c = 10−4 mol dm−3): λmax
(ɛ) = 223.00 nm (29,350) mol- 1dm3 cm−1). Conductance: 36
℧ mol-1 cm2. IR (KBr, cm−1): 3044 ν(=C-H); 1526 ν(C-N);
1377 ν(C-H); 1706 ν(C=C); 1634 ν(C=O); 565 ν(N-Os); 610
ν(N-Os-Br).
Synthesis and Characterization
of Tetrabromido(5-(2-Chloro-6-Methoxyquinolin-3-Yl)
-3-Phenyl-4,5-Dihydro-1H-Pyrazol-1-Yl)(4-Hydroxyphenyl)
Methanone)Osmium(IV) (C3)
It was synthesized using (NH4)2OsBr6 (0.60 g, 0.085 mmol)
and ligand L3 (0.38 g, 0.085 mmol). Color: Brownish black,
yield: 60%, mol. Wt: 967.76 g/mol, m.p.: >300 °c. Cal. (%)
for: Os,19.66; Found (%),Os,19.48; (UV-Vis (DMSO, c =
10−4 mol dm−3): λmax (ɛ) = 237.50 nm (29,350) mol- 1dm3
cm−1). Conductance: 28 ℧ mol−1 cm2. IR (KBr, cm−1): 3068
ν(=C-H); 1558 ν(C-N); 1344 ν(C-H); 1710 ν(C=C); 1667
ν(C=O); 575 ν(N-Os); 624 ν(N-Os-Br).
Biological Studies
Hydrodynamic Volume Measurement for Complex to HS-DNA
Interaction
Mode of intercalation between compounds and DNA solution
have been determined with the help of hydrodynamic volume
measurement study. Different concentration of compounds
have been mixed with DNA solution keeping its concentration
at 200 uM. The flow time of incubated solution have been
calculated in triplicate manner keeping it in static environment
at temperature 27 0.5 °C, and phosphate buffer pH 7.2 in
digital viscometric bath [30].
Synthesis and Characterization
of Tetrabromido(5-(2-Chloro-6-Methoxyquinolin-3-Yl)
-3-(4-Fluorophenyl)-4,5-Dihydro-1H-Pyrazol-1-Yl)
(4-Hydroxyphenyl)Methanone)Osmium(IV) (C4)
It was synthesized using (NH4)2OsBr6 (0.60 g,
0.085 mmol) and ligand L4 (0.40 g, 0.085 mmol). Color:
Brownish black, yield: 60%, mol. Wt: 985.75 g/mol, m.p.:
>300 °c. Cal. (%) for: Os,19.30; Found (%),Os,19.13;
(UV-Vis (DMSO, c = 10−4 mol dm−3): λmax (ɛ) =
256.50 nm (29,350) mol- 1dm3 cm−1). Conductance: 26
℧ mol−1 cm2. IR (KBr, cm−1): 3057 ν(=C-H); 1543
ν(C-N); 1348 ν(C-H); 1693 ν(C=C); 1653 ν(C=O); 586
ν(N-Os); 617 ν(N-Os-Br).
Molecular Docking with B-DNA
The PDB files of compounds formatted with the help of
CHIMERA 1.5.1 software have been interacted with B-
DNA PDB file using HEX 6.0 software by activating docking
process [31].

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J Fluoresc (2021) 31:349–362
Absorption Titration Study of Complex to HS- DNA
Results and Discussion
HS-DNA solution have been dissolved in phosphate buffer
solution keeping pH 7.2 in dimethyl sulfoxide at low temper-
ature of 4 °C. With the fixed concentration of compounds and
varying concentration of DNA solution, titration study have
been carried out [32].
Characterization
1H NMR and 13C-APT Characterization of Compounds
1
All the ligands were well characterized using H-NMR and
13C-APT spectroscopy. In ligand L1, H4a proton represents in
dd pattern at 3.248 δ/ppm whereas its analogues proton H4b
shows same pattern at 4.005 δ/ppm. Further, H3 proton falls at
6.269 δ/ppm as triplet. Aromatic region is justified between
7.244–8.175 δ/ppm. Hydroxy proton and methoxy proton are
obtained as singlet at 9.644 and 2.404 δ/ppm respectively.
Carbon skeleton of ligand L1 were justified using 13C-APT.
Carbonyl carbon shows its presence at 189.65 δ/ppm whereas
methoxy carbon shows its primary carbon peak at 55.67 δ/
Fluorescence Quenching Study
The binding ability and the number of active sites on
biomacromolecules on which compounds are effective have
been determined with the help of fluorescence study.
Depending upon the ability of Os(IV) complexes to displace
EB-DNA, relative change in intensity is observed [33].
1
ppm. H NMR and 13C-APT characterization of all com-
pounds were given in Supplementary Material 1 and 2.
Gel Electrophoresis
LC-MS and ESI-MS Characterization of Compounds
The photolytic cleavage property of Os(IV) complexes have
been determined by gel electrophoresis technique [34].
Compounds were well justified using LC-MS and ESI-MS
spectroscopy. Base peak of ligand L1 is obtained at 358 m/z
as highly intense peak whereas molecular ion peak is obtained
at 492 m/z with lower intensity. Complex C1 were well char-
acterized using ESI-MS spectroscopy. Molecular ion of C1
represents at 1002.20 m/z with [M+], [M + 2], [M + 4],
[M + 6] and [M + 8] peaks whereas ligand detached from mol-
ecule shows its present at 492.59 m/z. Relative isotopic abun-
dance pattern of bromine atoms were reflected at 922.30,
842.39, 762.49 and 682.59 m/z showing quintet, quartet, trip-
let and doublet pattern with relative intensity of 1:3:5:3:1,
1:2:2:1, 1:3:1 and 1:3 respectively. LC-MS of ligands L1-L6,
ESI-MS spectrum and ICP-OES plot of complex C1 are given
in Supplementary Material 3 and 4.
Bacteriostatic Activity
MIC activity have been carried out by growing bacterial cul-
ture in Luria broath solution. The bacterial culture have been
allowed to grow keeping it in orbital shaker at optimum rev-
olution and temperature [35].
In-Vitro Cytotoxicity
The varying concentration of compounds have been treated
with pre-cultured S. Pombe cell’s DNA previously grown in
liquid yeast media to calculate percentage cell viability of
compounds [36].
IR Spectra, Electronic Spectra, Magnetic and Conductance
Measurement of Compounds
IR spectroscopy justify the formation of ligand and metal
complexation. ν(=C-H) stretching band marks its presence
at 3099 cm−1 in ligand L1. Consequently, ν(C-N), ν(C-H),
ν(C=C), and ν(=C-O) stretching bands are observed at
1340, 1312, 1666 and 1610 cm−1, respectively. Upon the
complexation in case of Os(IV) pyrazole compound C1, dis-
turbance in vibrational bands caused marks presence of bands
viz. ν(=C-H), ν(C-N), ν(C-H), ν(C=C), and ν(=C-O) at
3112, 1562, 1318, 1680, and 1618 cm−1, respectively. The
shift in frequency is observed due to the complexation of
osmium metal ion with relative coordinating sites of pyrazole
nucleus ligand L1. The band at 556 cm−1 represents the coor-
dinate covalent bond formed between nitrogen atom of ligand
and osmium metal ion. The complex has octahedral geometry,
In-Vivo Cytotoxicity
Brine shrimp lethality assay have been carried out by using
protocol of Mayer et al., to calculate LC50 values of com-
pounds [37].
Cytotoxicity Using HCT-116 Cell Line
MTT assay have been used to carry out screening of test
compounds using HCT-116 cell line. DMSO have been used
to solubilize the test compounds with different concentration
ranging from 10 to 1000 μg/mL with 1% DMSO as vehicle
control [38].

J Fluoresc (2021) 31:349–362
355
Fig. 1 Plots represent relative viscosity of HS-DNA upon increasing concentration of compounds
consequently the expected ν(N-Os-Br) linkage vibrational
stretching band is observed at 611 cm−1. Electronic spectra
of ligands fall in wavelength of 223.00–284.00 nm whereas
that of Os(IV) complexes shows peaks at 230.50–267.50.
Complex C1 shows bands at 284.00, 339.50 and 458.50 nm
justifying n- π*, π- π* and d-d transition bands accounting for
(t2g)4(eg)0 electronic configuration representing octahedral be-
havior. Magnetic measurement of all the complexes justify
paramagnetic behavior of Os(IV) complexes. All the com-
plexes show non-electrolytic behavior confirmed by conduc-
tance measurements. IR spectrum of all the compounds are
given in Supplementary Material 5. CHNS data of all the
ligands have been given in Supplementary Material 6.
HS-DNA solution were incubated prior to run with the com-
pound’s concentration. The solution was poured into viscom-
eter held in digital viscometric bath in static environment.
Flow time of solution were measured in triplicate manner in
order to get the accurate results. All the compounds show
intercalative mode of binding [39]. Figure shows increase in
viscosity of compounds kept w.r.t. ethidium bromide taken as
reference material. The viscosity measurement plot of com-
pounds is given in Fig. 1.
Molecular Docking
Molecular docking is phenomenon of calculating effective
docking energy upon interaction of molecules with the macro
biomolecules. The molecules were allowed to run in
ChemBio3D software to refine the molecule according to
most stable conformational arrangement. The molecules were
formatted as PDB files and were allowed to interact with B-
DNA in HEX 6.0 software. The molecules and specific sites
of DNA tend to refine, adjust according to conformational
Biological Studies
Viscosity Measurement
Mode of interaction of compounds were well concluded with
the help of hydrodynamic volume measurement study. The
Fig. 2 Docking of Os(IV)
compound C1 with B-DNA

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Table 1 Intrinsic binding
constant (Kb) values and
percentage hypochromism shift
values of all the synthesized
compounds
Compound Kb Values × 105 % Hypochromism Compound Kb Values × 105 % Hypochromism
L1
L2
L3
L4
L5
L6
0.136 0.015
0.145 0.011
0.189 0.012
0.170 0.013
0.192 0.014
0.201 0.012
31.12 0.18
11.86 0.17
12.89 0.15
21.23 0.12
24.42 0.18
31.90 0.10
C1
C2
C3
C4
C5
C6
0.822 0.022
0.910 0.020
0.856 0.018
0.811 0.016
0.924 0.014
0.887 0.012
36.20 0.17
20.18 0.18
17.52 0.22
26.24 0.25
32.88 0.21
40.81 0.18
changes and interact with each other by activating docking
process. The effective energy of interaction in which molecule
befit with DNA were counted as its docking energy.. Docking
energy of complexes C1, C2, C3, C4, C5 and C6 are −326.49,
−318.71, −312.94, −292.30, −292.30 and − 292.30 kJ/mol
whereas, their respective ligands show docking interaction
energy of −295.49, −300.25, −289.39, −287.06, −290.79 and
− 293.84 kJ/mol respectively showing effective intercalation
[40, 41]. Docked structure of complex C1 is given in Fig. 2.
Docked images of all the compounds are given in
Supplementary Material 7.
Table 1. shows binding constant and percentage
hypochromism shift of compounds. Absorption titration curve
of complex C1 were represented in Fig. 3. All Os(IV) com-
plexes have better binding constant compared to reported
some Co(III) complexes with different 2-oxo-1,2-
dihydrobenzo[h]quinoline-3-carbaldehyde moiety based li-
gands. While Os(IV) complexes C2, C5 and C6 show selec-
tively better strength compare to Co(III) complex with 2-Oxo-
1,2-dihydro-benzo[h]quinoline-3-carbaldehyde N-
phenylthiosemicarbazone ligand [42].
Fluorescence Quenching Study
Absorption Titration Study of Complex to HS- DNA
Phenomenon of quenching deals with decrease in intensity of
compound in presence of suitable quenchers. Chemically,
bromide ion was used as chemical quenching agent taking
ethidium bromide. Ethidium bromide was used as fluorophore
material since its an effective intercalating agent. Intense
emission bands were observed at 610 nm when allowed to
excite at wavelength of 470 nm due to formation of EB-
DNA bond. Upon addition of varying complex concentration,
the ethidium bromide slowly gets displaced from bonding
with macromolecule resulting in decrease in fluorescence
Absorption titration is UV-visible spectrophotometer-based
activity to calculate binding constant of compounds upon in-
teraction with HS-DNA. Compounds were titrated in absence
and presence of DNA solution. With fixed concentration of
compound and varying concentration of DNA solution, the
experiment was carried out. The relative shifts in DNA treated
and untreated compounds helps in predicting percentage
hypochromism shift. The effective binding constant were cal-
culated with the help of equation given by Wolfe et al.,
Fig. 3 Absorption titration plot of
varying concentration of HS-
DNA with Os(IV) compounds.
Inscribed graph represents change
in absorption

J Fluoresc (2021) 31:349–362
357
Table 2 Stern-Volmer quenching constant (Ksv), binding sites (n), and
binding constant (Kf) values
the bonds formed between its base pairs. DNA converts to
single strand from double stranded structure. The unwinded
helical structure were allowed to move over mobile phase of
gel under specific voltage which stretch the helix more com-
pactly resulting in smeared image when observed under spec-
trophotometer. The RNA remains intact, whereas DNA gets
as smeared forming intense band. Intensity of smearing de-
pends upon the potency of compound at specific concentra-
tion to cleave the macromolecule. Photographed image of
treated compounds with S. Pombe cell’s DNA under gel elec-
trophoresis are represented in Supplementary Material 8.
Complexes
Ksv M−1 ×104
Binding Sites(n)
Kf M−1 ×104
C1
C2
C3
C4
C5
C6
0.521 0.022
0.812 0.021
0.183 0.032
0.522 0.035
0.564 0.028
0.112 0.018
0.9920
0.8693
1.024
0.179 0.023
0.149 0.021
0.107 0.020
0.168 0.018
0.127 0.017
0.211 0.015
0.8898
1.079
0.8692
Bacteriostatic Activity
intensity. Binding constant, effective binding sites and
quenching constant were evaluated from fluorometric titration
and are given in Table 2. The data were in good agreement
with the absorption titration concluding effectiveness of inter-
calation mode of binding. Plots have been represented in
Fig. 4 and mechanism of fluorescence quenching were pre-
sented in Fig. 5. Complexes C2 and C5 have quenching con-
stant greater than reported complexes Pd(II) and their respec-
tive ligand [43].
This activity was carried out using Gram+ve and Gram-ve bac-
teria. All the compounds in different concentration range were
treated with pre-grown bacterial culture. The compounds hav-
ing substituents with electron delocalizing tendency tend to
show better MIC values since they rupture the cell walls of
bacteria there by resisting the growth. The minimal effective
dosage of DMSO solubilized compound which resist the
growth of bacteria is noted as its MIC value. Os(IV) com-
plexes have better bacteriostatic activity compare to their re-
spective ligands. MIC of all the compounds were represented
in Fig. 6. MIC of complexes are found to be 72.5–100 μM,
whereas that of ligands fall at about 122.5–150 μM. MIC of
Os(IV) complexes are better than reported quinoline based
quinazolinone-4-thiazolidinone heterocycles (5a-5 k) [44].
Gel Electrophoresis
Gel electrophoresis is a well-known technique for separation
of charged particles based on difference in polarity to attract
towards opposite poles of gel electrophoresis plate. 1% aga-
rose gel were used in carrying out photolytic cleavage study.
The solutions of compounds were run over casting apparatus
set at optimum voltage required to run the gel. Upon addition
of varying concentration of compounds on the solution of
S. Pombe cell’s DNA. The degradation of DNA were ob-
served resulting in the unwinding of its helix by rupturing
Cellular Level in-Vivo Cytotoxicity
This assay were carried out using S. Pombe cell’s DNA. Cell
culture have been pre-grown 17 h before treatment with the
Fig. 4 Plot of fluorescence
titration whereas, graphs
represent Stern-Volmer equation
parameters to calculate quenching
constant (Ksv), binding sites (n)
and binding constant (Kf)

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Fig. 5 Fluorescence quenching observed with Os(IV) compound
test compounds. Varying concentration of compounds have
been treated with the cell culture keeping it in conical flask in
laminar air flow cabinet in presence of FV light. The solutions
were kept for 24 h overnight before taking readings. The treat-
ed solutions were centrifuged to collect the culture. The col-
lected culture was kept in incubation with tryptan blue dye and
dead cells were counted under digital microscope. The dye
allows dead cells to penetrate into it whereas it resists live
cells. Percentage cell viability of Os(IV) complexes were
found better than that of respective ligands. Table 3 shows
percentage cell viability of compounds. Percentage cell via-
bility of Os(IV) complexes is more effective compare to re-
ported oxovanadium(IV) complexes [45].
importance. Nauplii hatched from brine shrimp eggs have been
treated with different concentration of compounds. The death of
larvae is noted as lethality result. It is concluded that with increase
in concentration of compounds, percentage mortality was in-
creased. Cytotoxic value of ligands and Os(IV) complexes are
respectively. Os(IV) complexes possess better LC50 than synthe-
sized ligands. LC50 count of ligands fall in the range of 16.22–
17.28 μg/mL whereas that of Os(IV) fall in the range of 4.87–
5.87 μg/mL. LC50 of Os(IV) compounds show better value com-
pare to reported oxovanadium (IV) complexes with 2-thiophene
carboxylic acid hydrazide ligand [46]. LC50 of compounds are
represented in Fig. 7.
Cytotoxicity Using HCT-116 Cell Line
In-Vitro Cytotoxicity against Artemia Cyst Lethality Bioassay
Cytotoxicity of complexes were screened over HCT-116 cell
line. Figure 8 Show plot of percentage proliferation vs. con-
centration (μg/mL) with different concentration of test
Meyer et al.’s protocol was implemented to carry out the assay.
This is primary assay to judge molecule for the pharmaceutical
Fig. 6 MIC values of compounds
MIC(μM)
in terms of μM upon exposure to
series of Gram(+ve) and
Gram(−ve) bacteria
180
160
140
120
100
80
60
40
20
0
C1
C2
C3
C4
C5
C6
Compounds
Gram(+ve) B. subꢀlis
Gram(-ve) P. aeruginosa Gram(-ve) E. coli
L1
L2
L3
L4
L5
L6
Gram(+ve) S. Aureus
Gram(-ve) S. marcescens

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359
Table 3 Percentage viability on
S. Pobme cell’s DNA presented
with standard deviation for three
independent experiment
Ligands
Percentage viability per concentration
(μg/mL)
L1
L 2
L 3
L 4
L 5
L 6
2
42 1.5 35 2.2 37 2.5 44 2.4 41 2.5 39 1.5
40 1.6 31 2.0 34 1.4 42 2.2 38 1.5 36 1.2
38 1.5 29 1.8 33 1.6 40 1.4 36 1.0 34 1.8
36 2.0 27 1.6 29 1.8 38 1.6 34 1.2 31 1.6
34 2.1 24 1.5 26 2.0 35 1.8 32 1.3 29 2.0
4
6
8
10
Complexes
Percentage viability per concentration
C1
C 2
C 3
C 4
C 5
C 6
(μg/mL)
2
36 1.6 29 1.4 31 1.2 38 1.7 35 1.6 33 2.2
32 1.5 26 1.6 28 1.5 34 1.7 31 1.5 29 2.1
30 1.5 24 1.8 26 1.8 32 1.8 29 1.5 27 2.0
28 2.0 21 2.1 24 2.0 29 2.0 26 1.5 25 1.8
26 2.0 19 2.5 20 2.5 28 2.0 25 2.0 22 2.2
4
6
8
10
compounds taken with respect to vehicle and control media.
Complexes C1, C2, C4 and C5 have IC50 of 423, 752, 520 and
666 μg/mL while others show moderate impact. IC50 of
Os(IV) C1 dominate over reported Re(I) with 5-(4-
Chlorophenyl)-1-(4-nitrophenyl)-3- (thiophen-2-yl)-4,5-
dihydro-1H-pyrazole ligand [47]. Complex C1 represents least
IC50 amongst the complexes since it possess chlorine func-
tionality which put an inductive effect in dominance.
biological screening. All the compounds show intercala-
tion mode of binding when interacted with HS-DNA jus-
tified with docking energy and DNA interaction study.
Docking energy of ligands fall at about −287.06 to
−300.25 kJ/mol. Whereas, complexes fall at about
−292.30 to −326.49 kJ/mol showing effective intercala-
tion. Binding constant of Os(IV) complexes are found to
be 8.1 to 9.2 × 104 M−1.All Os(IV) complexes have better
binding constant compared to reported Co(III) complexes
w i t h d i f f e r e n t s u b s t i t u t e d 2 - o x o - 1 , 2 -
dihydrobenzo[h]quinoline-3-carbaldehyde moiety based
ligands. Binding constant have been further justified with
fluorescence quenching study. Quenching constant of
complexes fall at about 1.1 to 8.1 × 103 M−1. Complexes
C2 and C5 have quenching constant better than reported
Pd(II) complexes. MIC of complexes are found to be
72.5–100 μM, whereas that of ligands fall at about
122.5–150 μM. MIC of Os(IV) complexes are better than
Conclusion
Various pyrazole nucleus-based ligands and their Os(IV)
complexes were synthesized and well characterized using
different spectroscopic techniques viz. 1H-NMR, 13C-
APT, LC-MS, ESI-MS, ICP-OES, IR spectroscopy, elec-
tronic spectroscopy, conductance and magnetic measure-
ments. The synthesized compounds were subjected for
Fig. 7 Bar graph represents LC50
of osmium(IV) compounds and
ligands

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J Fluoresc (2021) 31:349–362
Fig. 8 Plots of % cell proliferation vs. concentration represents IC50 of Os(IV) compounds
reported quinoline based quinazolinone-4-thiazolidinone
heterocycles. Percentage cell viability of Os(IV) com-
plexes is more effective compare to reported
oxovanadium(IV) complexes. LC50 of Os(IV) compounds
show better value compare to reported oxovanadium (IV)
complexes with 2-thiophene carboxylic acid hydrazide li-
gand. Os(IV) complexes showed intense and impactful
smearing of S. Pombe cell’s DNA when allowed to run
over gel electrophoresis. IC50 of Os(IV) C1 dominate over
reported Re(I) with 5-(4-chlorophenyl)-1-(4-nitrophenyl)-
3- (thiophen-2-yl)-4,5-dihydro-1H-pyrazole ligand.
Compliance with Ethical Standards
Competing Interests Not applicable
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