Design, synthesis, pharmacological evaluation and DNA interaction studies of binuclear Pt(II) complexes with pyrazolo[1,5-a]pyrimidine scaffold

Received: 18 September 2017

DOI: 10.1002/aoc.4222

Revised: 8 November 2017

Accepted: 9 November 2017

F U L L P A P E R

Design, synthesis, pharmacological evaluation and DNA

interaction studies of binuclear Pt(II) complexes with

pyrazolo[1,5‐a]pyrimidine scaffold

Miral V. Lunagariya¹| Khyati P. Thakor¹| Bhargav N. Waghela²| Chadramani Pathak²|

Mohan N. Patel¹

¹Department of Chemistry, Sardar Patel

Substituted pyrazolo[1,5‐a]pyrimidine ligands were synthesized by cyclization,

using 3‐(thiophen‐2‐yl)‐1H‐pyrazol‐5‐amine with substituted enones (3‐phenyl‐

1‐(pyridin‐2‐yl)prop‐2‐en‐1‐one) in presence of KOH and DMF as solvent to form

cyclic aromatic compounds. The substituted pyrazolo[1,5‐a] pyrimidine based

binuclear Pt^IIcomplexes containing neutral tetradentated ligands have general

formula [Pt₂(5a–5f)Cl₄], (where, (5a ‐5f) = pyrazolo[1,5‐a] pyrimidine ligand).

This compounds were characterized by physicochemical and spectroscopic

method like elemental analyses, UV‐Visible, FT‐IR, EDX, TGA, molar conduc-

University, Vallabh Vidyanagar ‐388 120,

Gujarat, India

²Department of Cell Biology, School of

Biological Sciences and Biotechnology,

Indian Institute of Advanced Research,

Koba Institutional Area, Gandhinagar

382007, Gujarat, India

Correspondence

Mohan N. Patel, Department of

Chemistry, Sardar Patel University,

Vallabh Vidyanagar‐388 120, Gujarat,

India.

1

tivity, magnetic susceptibility measurements, mass spectroscopy, H and ¹³C

NMR method. The square planar geometry was predicted by electronic spectral

study. All Pt^IIcompounds were evaluated by antimicrobial assay, in vitro brine

shrimp assay, in vivo cellular level bioassay using S. Pombe cells and anti‐tuber-

culosis study. LC₅₀(50% lethal concentration) values of compounds are observed

between 6.450 ‐ 102.07 μg/mL. UV‐vis absorption titration, competitive displace-

ment assay, molecular docking and viscosity measurement were carried out to

examine the binding type and binding strength of complexes. The binding stud-

ies suggest partial intercalative binding mode of the complexes and the observed

binding constant (K_b) values are found in the order of 6d > 6b > 6c > 6a > 6e > 6 f.

The anti‐proliferative cytotoxicity of the synthesized Pt^IIcomplexes (6a‐6f) were

tested against the HCT‐116 (Human Colorectal Carcinoma) cancer cell line.

Email: jeenen@gmail.com

KEYWORDS

binuclear Pt^IIcomplexes, cellular level bioassay, In vitro antiproliferation cytotoxicity, pyrazolo[1,5‐a]

pyrimidine analogous, thermodynamics parameters

1 | INTRODUCTION

chemistry. Five membered aza‐heterocyclic molecule,

pyrazoles have attained a special status in the eyes of

Incorporation of fused aza‐heterocyclic rings (pyrazole,

pyrimidine and pyrazolo[1,5‐a]pyrimidine) have been

extensively utilized in recent years due to their plethora

of applications in a various domains such as medicinal

chemistry, organometallics, therapeutic and material

chemists and biologists owing to their easy methods of

syntheses and enormous pharmacological applications.

Pyrimidine or m‐diazine moiety is the parent ring system

of a variety of substances which play vital role in biologi-

cal processes. This structural motif is also subset of a large

Appl Organometal Chem. 2018;e4222.

wileyonlinelibrary.com/journal/aoc

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https://doi.org/10.1002/aoc.4222

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LUNAGARIYA ET AL.

number of naturally occurring substances. For example as

vitamins, coenzymes, purines, pterins, nucleotides and

nucleic acids. The properties of pyrimidines are governed

to a large extent by the electron‐attracting properties of

the two nitrogen atoms; each reinforces the electronic

effect of the other in the 2‐, 4‐ and 6‐positions. The

pyrazolo[1,5‐a]pyrimidine motifs are used in medicinal

chemistry dealing with antimicrobial anticancer,

antitumor, anxiolytic, antimicrobial, antibacterial,

antitrichomonal, antischistosomal activities whereas

zaleplon, a hypnotic drug; ocinaplon, an anxiolytic drug;

pyrazophos, a fungicide, herbicides and insecticide are

aspects of pesticide chemistry having a wide range of

enzyme inhibitory activities.^[1–5]

The platinum‐based chemotherapy is currently the

most‐utilised treatment for cancer, one of the most glob-

ally prominent diseases,^[6]since the discovery of anti‐car-

cinogenic activity of cisplatin metal‐based anticancer

drug.^[7–9]Other platinum drugs have been developed

and marketed, such as carboplatin, oxaliplatin nedaplatin,

lobaplatin and heptaplatin, satraplatin and picoplatin.^[10]

While the second generation of carboplatin, and third

generation of oxaplatin, which is more effective and less

toxic than cisplatin but possesses the same spectrum of

antitumor activity used in combination therapy with 5‐

fluorouracil for colon cancer.^[11]

In bioinorganic chemistry, metal complexes have been

used for the treatment of many diseases (cancer, arthritis,

diabetes, alzheimer's, etc.), but their mechanism is not

completely understood in biological systems^[12,13]and

their ability to modulate drug metabolism (hydrolysis)

and target binding through steric and electronic effects

on the substitution mechanism worldwide efforts are

made to develop platinum‐based anticancer agents. The

electron‐rich nature of biomolecular (DNA) and elec-

tron‐deficient metal ions are believed to have strong

interactions, which are thought to be the criteria for the

pharmaceutical activities of the complexes. DNA plays a

central role in replication, transcription and regulation

of genes as an important target of anticancer drugs. The

existence of metal‐binding sites in the DNA structure

makes different types of interaction possible. It has been

reported that the metal complexes can interact with

DNA non‐covalently in the mode of intercalation, groove

binding and electrostatic effect.^[14,15]The insertion of

biologically‐active ligands into metal coordination

compounds have much scope for the design of novel

drugs with improved and targeted activity. Studies on

such complexes indicate that new mechanisms of action

are favourable when combining the bioactivity of the

ligand with the properties inherent to the metal, leading

to the possibility of overcoming the current drug

resistance pathways.

Ligands can be introduced into a system to limit the

adverse effects of excess metal ions. Upon chelation of

ligands to the metal ion, some drastic changes occurred,

which includes increased lipophilicity, stabilizing specific

oxidation states and contributing to substitution inert-

ness. As an alternative, many platinum complexes with

π‐conjugated heterocyclic ligands have been developed

and their anti‐cancer curing properties are investigated.

Precisely designed ligands for metal‐based medicinal

agents can play an integral role in achieving the potential

toxicity of a metallo drug, which can be used for diagnosis

and therapy.

In current work, we have concentrated on following

points: (a) synthesis of ligands (5a‐5f) by two step reaction

employing conventional method and synthesis of target

binuclear Pt^IIcomplexes (6a‐6f) by single step one‐pot

two‐component (b) biological evaluation including DNA

binding studies, DNA nuclease, antibacterial, antituber-

culosis studies, in vitro cytotoxicity against S. pombe cellu-

lar level and brine shrimp lethality assay. In the

framework of biological importance, we intended to

develop new approach for structural diversity of heterocy-

cles incorporating scaffold. The modifications made on

pyrazolo[1,5‐a]pyrimidine centre for probing antibacte-

rial, antitubercular activity includes; (A) thiophene ring

at C‐3 position (B) 4‐substituted phenyl nucleus at C‐8

position and (C) pyridine group on C‐6 position to vali-

date lipophilicity of the target molecules (6a‐6f). All syn-

thesized compounds were evaluated for their biological

applications. Based on the assessment of these inspections

and the persistence of the research work on bioactive het-

erocycles, their design and potential applications are

anticipated.

2 | EXPERIMENTAL

2.1 | Materials and reagents

All the commercial analytical grade reagents, solvents and

chemicals (> 95 % purity) were used without further

purification and purchased from Sigma Aldrich Chemical

Co. (Vadodara, India). K₂PtCl₄salts was purchased from

S. D Fine‐Chem Ltd. (SDFCL). Herring sperm (HS)

DNA, 3‐(thiophen‐2‐yl)‐1H‐pyrazol‐5‐amine, 4‐methoxy

benzaldehyde, 4‐fluoro benzaldehyde, 4‐chloro benzalde-

hyde, 4‐nitro benzaldehyde, 4‐methyl benzaldehyde and

2‐acetyl pyridine were purchased from Sigma Aldrich

Chemical Co. (India). All the bacterial cultures used were

purchased from MTCC, Institute of Microbial Technology

and Chandigarh, India. An Artemia cyst was purchased

from local aquarium store. Nutrient broth (NB), agarose,

ethidium bromide (EtBr), tris‐acetyl‐EDTA (TAE),

bromophenol blue was purchased from Himedia (India).

LUNAGARIYA ET AL.

3 of 25

Thin layer chromatography (TLC) was performed using

Merck aluminium sheets coated with silica gel plates (sil-

ica gel 60 F₂₅₄0.25 mm). Purification by flash chromatog-

raphy was performed using Merck silica gel 60. and

components were visualized by observation under UV

light. GenElute mini Pre Kit for pUC19 DNA isolation

was purchased from Sigma Aldrich (India). S. pombe

Var. Paul Linder 3360 was obtained from IMTECH, Chan-

digarh, India. MilliQ™ (18.2 m℧, Millipore) was used for

the preparation of all deionized water. HPLC grade

DMSO was used to dissolve the platinum compounds.

2.3 | General synthesis of ligands and

complexes

2.3.1 | Claisen‐Schmidt condensation

reaction (3a‐3f)

The α, β unsaturated carbonyl compounds derived by this

method.^[16]Compounds (3a‐3f) were obtained after stir-

ring at room temperature for 2 h a solution containing

(1.241 g, 1 mmol) 1‐(pyridin‐2‐yl)ethan‐1‐one (1a), in

10 ml methanol and KOH (0.673 g, 1.2 mmol, 20 ml)

was added as a catalytic amount Claisen‐Schmidt conden-

sation reaction, with appropriately P‐substituted benzal-

dehyde (‐OCH₃, ‐F, Cl, ‐NO₂, ‐CH₃, ‐H 1 mmol) (2a‐2f)

was added drop wise into the reaction mixture. The reac-

tion mixture was cooled on an ice bath and neutralized

with dilute hydrochloric acid. The crude material

appeared was separated by suction pump and washed

with water‐methanol 50% system (60 ml × 3). The solvent

was removed under reduced pressure. The obtained

material was recrystallized from methanol. The purity of

the products was checked on TLC by using mixture of

(ethylacetate: hexane, 4:1) system. The solvent was

removed under reduced pressure to smoothly afford the

corresponding chalcones as a white solid. The proposed

reaction scheme for the synthesis of chalcone (3a‐3f) is

shown in Scheme 1.

2.2 | Physical measurements

¹H NMR and ¹³C NMR spectra were obtained on a 400

and 100 MHz, Bruker Avance nuclear magnetic reso-

nance spectrometer, either in D₂O (25 °C) or DMSO‐d₆

(35 °C), and TMS as the internal reference. The following

abbreviations apply to splitting patterns: s (singlet), d

(doublet), t (triplet), q (quartet), m (multiplet). The chem-

ical shift (parts per million) of each resonance were

repeated as an approximate midpoint of its multiplicity,

δ (ppm) and the coupling constants (J) in Hz. The

attached proton test in a ¹³C NMR characterisation

showed C, CH, CH₂and CH₃signals unattached to pro-

tons. The APT experiment yields methine (CH) and

methyl (CH₃) signals negative and quaternary (C) and

methylene (CH₂) signals positive. Infrared spectra were

recorded on a FT‐IR ABB Bomen MB‐3000 spectropho-

tometer (Canada) in the range of 4,000 to 400 cm^‐1.

Elemental analysis C, H, N and S of the synthesized

compounds was performed with a model Euro EA ele-

mental analyser. Melting points (⁰C, uncorrected) were

determined in open capillaries on thermoCal10 melting

point apparatus (Analab Scientific Pvt. Ltd, Vadodara,

India). The mass spectra were obtained on a Thermo sci-

entific mass spectrophotometer (USA), using the positive

electrospray ionisation mode. The electronic spectra were

recorded on a UV‐160A UV‐vis spectrophotometer

(Shimadzu, Kyoto, Japan). The magnetic moments were

measured by Gouy's method using mercury

2.4 | General procedure for the

preparation of pyrazolo[1,5‐a]‐pyrimidines

chimeras (5a‐5f)

Lipson and co‐workers reported synthesis of the

pyrazolo[1,5‐a]pyrimidines derivatives method.^[17–19]

Chalcones (3a‐3f) are effective bidentate electrophiles

and have been employed for the synthesis of various bio-

active heterocyclic. To the solution of the appropriate

enones (3a‐3f) (0.50 g, 2.8 mmol) in 10 ml of DMF, 3‐

(thiophen‐2‐yl)‐1H‐pyrazol‐5‐amine

(4a)

(0.33

g,

3.0 mmol) and KOH (0.0056 g, 0.1 mmol) solution were

added, the reaction mixture was refluxed for 30 min.

Upon completion of reaction as indicated by TLC plates,

the excess of solvent was removed under reduced pressure

and the reaction mixture was cooled on an ice bath. The

reaction mixture was extracted with ethylacetate (50 ml

× 3), washed thoroughly with water (30 ml × 3), added

brine solution of sodium chloride and dried over sodium

sulphate. The resulting mixture was concentrated under

vacuum. The residue was purified by chromatography

on silica gel system (ethylacetate: hexane, 4:1) to obtain

the pyrazolo[1,5‐a]pyrimidine chimera product. The

proposed reaction scheme for the synthesis of ligands

(5a‐5f) is shown in Scheme 1. ¹H NMR, ¹³C NMR spectra

tetrathiocyanatocobaltate(II) as the calibrate (χ_g

=

16.44×10^–6cgs units at 20 °C) citizen balance. Antibacte-

rial study was carried out by means of laminar airflow

cabinet (Toshiba, Delhi, India). The thermo gram of com-

plexes was recorded with a Mettler Toledo TGA Thermo-

gravimetric analyser. Fluorescence spectroscopy was

carried out by FluoroMax‐4, spectrofluorometer, HORIBA

(Scientific). Conductance measurement was carried out

using conductivity meter model number E‐660A. Photo

quantization of the gel after electrophoresis was done

using AlphaDigiDocTM RT. Version V.4.0.0 PC‐Image

software, CA, USA).

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LUNAGARIYA ET AL.

SCHEME 1 Synthesis of chalcone (3a‐

3f), pyrazolo[1,5‐a] pyrimidine derivatives

ligands (5a–5f) coordinated to Pt^IImetal

ions to give complexes (6a‐6f) and

numbering scheme of atoms

of ligands are shown in supplementary material 1 and 2,

respectively.

136.42 (C_quat.,C₃), 131.83 (CH, C_4”), 128.63 (CH, C_2”’),

128.44 (CH, C_2’, C_6’), 128.0 (CH, C_3”’), 125.38 (C_quat.,

C_1’), 123.55 (CH, C_5”), 121.60 (CH, C_3”), 114.85 (CH, C_3’,

C_5’), 105.97 (CH, C₇), 93.5 (CH, C₄), 55.8 (O‐CH₃). [Total

signal observed = 20: signal of C and CH₂= 8 (Ar‐C =

2, pyridine‐C = 1, thiophene‐C = 1, pyrazolo[1,5‐a]pyrim-

idine‐C = 4), signal of CH and CH₃= 12 (Ar‐CH = 2, pyr-

idine‐CH = 4, thiophene‐CH = 3, pyrazolo[1,5‐a]

pyrimidine‐CH = 2), O‐CH₃= 1]. FT‐IR: (KBr pellet) ν_max

(cm^‐1): 3109 ν_(=C‐H)ar(w), 1604 ν_(C=N)(s), 1458 – 1566 ν_(C‐

2.4.1 | 7‐(4‐Methoxyphenyl)‐5‐(pyridin‐2‐

yl)‐2‐(thiophen‐2‐yl)pyrazolo[1,5‐a] pyrimi-

dine (5a)

It

was

synthesized

using

chalcone

(E)‐3‐(4‐

methoxyphenyl)‐1‐(pyridin‐2‐yl)prop‐2‐en‐1‐one

(3a)

(0.333 g, 2.8 mmol) and 3‐(thiophene‐2‐yl)‐1H‐pyrazol‐5‐

amine (4a) (0.50 g, 3.0 mmol) as described in general pro-

cess. Colour: light yellow crystal, Yield: 82 %, mol. wt.

384.46 g/mol, m.p. 233 °C; Anal. Calc. (%) For

C₂₂H₁₆N₄OS: C, 68.73; H, 4.20; N, 14.57; S, 8.34. Found

(%): C, 68.98; H, 3.92; N, 14.39; S, 8.02. UV‐vis: λ (nm)

(ε, M^‐1cm^‐1): 309 (39,650), 358 (26,830). ¹H NMR

(400 MHz, DMSO‐d₆) δ/ppm: 8.752 (d, 1H, J= 8.0 Hz,

(s), 1257 ν_{(C‐C)alkanes}(s), 1650 ν_{(C=C)conjugated}

H)banding

(w), 1026 ν_{(C‐S‐C)stre. (thiophene}

(s), 709 – 833

alkenes

ring)

ν_{(Ar ‐ H)2 adjacent hydrogen}(s), 586 ν_{(=C‐H)oop band}(s). Mass

(m/z): 385 [M]⁺.

2.4.2 | 7‐(4‐Fluorophenyl)‐5‐(pyridin‐2‐yl)‐

2‐(thiophen‐2 yl)pyrazolo[1,5‐a]pyrimidine

(5b)

H

_2”’), 8.582 (d, 1H, J= 7.6 Hz, H_4”’), 8.365 (d, 2H, J=

3

8.0 Hz, H_2’,6’), 8.160 (s, 1H, H₇), 7.911(t, 1H, J₁= 8.0 Hz,

3

4

J₂= 8.4 Hz, H_3”’), 7.629 (dd, 2H, J₁= 2.4 Hz, J₂=

It was synthesized using chalcone (E)‐3‐(4‐fluorophenyl)‐

1‐(pyridin‐2‐yl)prop‐2‐en‐1‐one (3b) (0.299 g, 2.8 mmol)

and 3‐(thiophene‐2‐yl)‐1H‐pyrazol‐5‐amine (4a) (0.50 g,

3.0 mmol) as described in general process. Colour: yellow

crystal, Yield: 85 %, mol. wt. 372.42 gm/mol, m.p. 223 °C;

Chemical formula: C₂₁H₁₃FN₄S; UV‐vis: λ (nm) (ε, M^‐

3

6.8 Hz, H_3”,6”), 7.396 (t, 1H, J₁= 7.6 Hz, J₂= 8.0 Hz,

H_5”), 7.167 ‐ 7.120 (multiplet, 3H, H_{3’,5’,4”}), 7.008 (s, 1H,

H₄), 3.957 (s, 3H, ‐OCH₃). ¹³C NMR (100 MHz, DMSO‐

d₆) δ/ppm: 165.23 (C_quat.,C₆), 160.63 (C_quat.,O‐CH₃),

155.23 (C_quat.,C_2”), 154.73 (C_quat.,C_5a), 149.98 (CH, C_6”),

cm^‐1): 294 (36,740), 357 (7,960). H NMR (400 MHz,

1

147.51 (C_quat.,C₈), 139.91 (C_quat.,

C

_5”’), 137.19 (CH, C_4”),

LUNAGARIYA ET AL.

5 of 25

DMSO‐d₆) δ/ppm: 8.789 (d, 1H, J= 8.0 Hz, H_2”’), 8.427 (d,

1, thiophene‐C = 1, pyrazolo[1,5‐a]pyrimidine‐C = 4),

signal of CH and CH₃= 11 (Ar‐CH = 2, pyridine‐CH =

4, thiophene‐CH = 3, pyrazolo[1,5‐a]pyrimidine‐CH =

2)]. FT‐IR: (KBr pellet) ν_max(cm^‐1): 3101 ν_(=C‐H)ar(w),

1704 ν_(C=N)(s), 1612 ν_{(C=C)conjugated alkene}(s), 1496 – 1566

ν_{(C‐H)banding}(s), 1218 ν_{(C‐C)alkanes}(s), 1141 ν_{(Ar ‐Cl)}(m),

1080 ν_{(C‐S‐C)stre. (thiophene ring)}(s), 709 – 833 ν_{(Ar‐H)2 adjacent}

1H, J= 8.0 Hz, H_4”’), 8.283 (d, 2H, J= 8.8 Hz, H_2’,6’), 8.127

3

(s, 1H, H₇), 8.079 (t, 1H, J₁= 8.0 Hz, J₂= 8.8 Hz, H_3”’),

7.787 (d, 1H, J= 8.0 Hz, H_3’), 7.763 (d, 1H, J= 7.2 Hz,

3

H_5’), 7.668 (t, 1H, J₁= 5.2 Hz, J₂= 5.6 Hz, H_5”), 7.576

3

4

(dd, 2H, J₁= 8.0 Hz, J₂= 3.6 Hz, H_3”,6”), 7.347 (s, 1H,

3

H₄), 7.212 (t, 1H, J₁= 7.6 Hz, J₂= 7.6 Hz, H_4”). ¹³C

NMR (100 MHz, DMSO‐d₆) δ/ppm: 165.35 (C_quat.,C₆),

162.60 (C_quat.,C_4’), 155.03 (C_quat.,C_2”), 154.43 (C_quat.,

C_5a), 149.18 (CH, C_6”), 147.21 (C_quat.,C₈), 139.44 (C_quat.,

(s), 586 ν_{(=C‐H)oop band}(s). Mass (m/z): 389 [M]⁺,

hydrogen

390 [M+2].

C

_5”’), 137.30 (CH, C_4”), 136.42 (C_quat.,C₃), 131.73 (CH,

C_4”), 130.6 (CH, C_2’, C_6’), 128.44 (CH, C_2”’), 128.0 (CH,

_3”’), 125.20 (C_quat.,C_1’), 123.87 (CH, C_5”), 121.29 (CH,

2.4.4 | 7‐(4‐Nitrophenyl)‐5‐(pyridin‐2‐yl)‐2‐

(thiophen‐2‐yl)pyrazolo[1,5‐a]pyrimidine

(5d)

C

C_3”), 116.85 (CH, C_3’, C_5’), 105.07 (CH, C₇), 93.15 (CH,

C₄). [Total signal observed = 19: signal of C and CH₂=

8 (Ar‐C = 2, pyridine‐C = 1, thiophene‐C = 1,

pyrazolo[1,5‐a]pyrimidine‐C = 4), signal of CH and CH₃

= 11 (Ar‐CH = 2, pyridine‐CH = 4, thiophene‐CH = 3,

pyrazolo[1,5‐a]pyrimidine‐CH = 2)]. FT‐IR: (KBr pellet)

ν_max(cm^‐1): 3102 ν_(=C‐H)ar(w), 1704 ν_(C=N)(s), 1612

It was synthesized using chalcone (E)‐3‐(4‐nitrophenyl)‐1‐

(pyridin‐2‐yl)prop‐2‐en‐1‐one (3d) (0.375 g, 2.8 mmol)

and 3‐(thiophene‐2‐yl)‐1H‐pyrazol‐5‐amine (4a) (0.50 g,

3.0 mmol) as described in general process. Colour: brown

powder, Yield: 88 %, mol. wt. 399.43 gm/mol, m.p. 224 °C;

Anal. Calc. (%) For C₂₁H₁₃N₅O₂S: C, 63.15; H, 3.28; N,

17.53; S, 8.03. Found (%): C 63.09; H, 3.30; N, 17.48; S,

7.95. UV‐Vis: λ (nm) (ε, M^‐1cm^‐1): 294 (32,660), 359

ν_{(C=C)conjugated}

(s), 1496 – 1566 ν_{(C‐H)banding}(s),

alkenes

1218 ν_{(C‐C)alkanes}(s), 1149 ν_(Ar‐F)(s), 1056 ν_{(C‐S‐C)stre. (thio-}

1

(m), 709 – 840 ν_{(Ar‐H)2 adjacent hydrogen}(s), 586

(6,280). H NMR (400 MHz, DMSO‐d₆) δ/ppm: 8.755 (d,

phene ring)

ν_{(=C‐H)oop band}(s). Mass (m/z): 373 [M]⁺.

1H, J= 8.0 Hz, H_2”’), 8.512 (d, 1H, J= 8.0 Hz, H_4”’), 8.329

(d, 2H, J= 9.2 Hz, H_3’,5’), 8.097 (s, 1H, H₇), 8.053 (t, 1H,

³J₁= 7.6 Hz, J₂= 7.6 Hz, H_3”’), 7.778 (d, 1H, J= 7.2 Hz,

3

2.4.3 | 7‐(4‐Chlorophenyl)‐5‐(pyridin‐2‐yl)‐

2‐(thiophen‐2 yl)pyrazolo[1,5‐a] pyrimidine

(5c)

H_2’), 7.651 (d, 1H, J= 7.6 Hz, H_6’) 7.551 (t, 1H, ³J₁=

3

8.0 Hz, J₂= 8.0 Hz, H_5”), 7.506 (dd, 2H, J₁= 9.2 Hz,

⁴J₂= 4.0 Hz, H_3”,6”), 7.311 (s, 1H, H₄), 7.223 (t, 1H, J₁=

3

It was synthesized using chalcone (E)‐3‐(4‐chlorophenyl)‐

1‐(pyridin‐2‐yl)prop‐2‐en‐1‐one (3c) (0.345 g, 2.8 mmol)

and 3‐(thiophene‐2‐yl)‐1H‐pyrazol‐5‐amine (4a) (0.50 g,

3.0 mmol) as described in general process. Colour: yellow

crystal, Yield: 83 %, mol. wt. 388.87 gm/mol, m.p. 226 °C;

Anal. Calc. (%) For C₂₁H₁₃ClN₄S: C, 64.86; H, 3.37; N,

14.41; S, 8.24. Found (%): C, 64.58; H, 2.99; N, 13.97; S,

8.00. UV‐vis: λ (nm) (ε, M^‐1cm^‐1): 283 (40,000), 359

8.4 Hz, J₂= 8.0 Hz, H_4”). ¹³C NMR (100 MHz, DMSO‐

d₆) δ/ppm: 165.48 (C_quat.,C₆), 155.13 (C_quat.,C_2”), 154.79

(C_quat.,C_5a), 149.29 (CH, C_6”), 147.91 (C_quat.,C_4’), 147.50

(C_quat.,C₈), 139.90 (C_quat.,C_5”’), 139.10 (C_quat.,C_1’), 137.20

(CH, C_4”), 136.67 (C_quat.,C₃), 131.90 (CH, C_4”’), 128.60

(CH, C_2”’), 128.00 (CH, C_3”’), 126.20 (CH, C_2’,6’), 123.60

(CH, C_5”), 124.40 (CH, C_3’,5’), 121.37 (CH, C_3”), 105.26

(CH, C₇), 93.37 (CH, C₄). [Total signal observed = 19:

signal of C and CH₂= 8 (Ar‐C = 2, pyridine‐C = 1, thio-

phene‐C = 1, pyrazolo[1,5‐a]pyrimidine‐C = 4), signal of

CH and CH₃= 11 (Ar‐CH = 2, pyridine‐CH = 4, thio-

phene‐CH = 3, pyrazolo[1,5‐a]pyrimidine‐CH = 2)]. FT‐

IR: (KBr pellet) ν_max(cm^‐1): 3116 ν_(=C‐H)ar(w), 1704

ν_(C=N)(s), (w), 1087 ν_{(C‐S‐C)stre. (thiophene ring)}(w), 717 –

840 ν_{(ArH)2 1612 ν(C=C)conjugated alkenes}(s), 1519 ν_{(C‐H)banding}

(s), 1357 ν_(N‐O)nitro(w), 1226 ν_{(C‐C)alkanes}(s), 1142 ν_(Ar‐

1

(12,480). H NMR (400 MHz, DMSO‐d₆) δ/ppm: 8.789

(d, 1H, J= 7.6 Hz, H_2”’), 8.529 (d, 1H, J= 7.6 Hz, H_4”’),

8.342 (d, 2H, J= 6.8 Hz, H_2’,6’), 8.113 (s, 1H, H₇), 8.080

3

(t, 1H, J₁= 9.2 Hz, J₂= 7.6 Hz, H_3”’), 7.790 (d, 1H, J=

8.4 Hz, H_3’), 7.660 (d, 1H, J= 8.0 Hz, H_5’), 7.647 (t, 1H,

3

³J₁= 8.0 Hz, J₂= 8.4 Hz, H_5”), 7.551 (dd, 2H, ³J₁=

4

8.8 Hz, J₂= 4.8 Hz, H_3”,6”), 7.341(s, 1H, H₄), 7.230 (t,

3

1H, J₁= 7.6 Hz, J₂= 8.0 Hz, H_4”). ¹³C NMR (100 MHz,

DMSO‐d₆) δ/ppm: 165.27 (C_quat.,C₆), 155.30 (C_quat.,C_2”),

154.89 (C_quat.,C_5a), 149.20 (CH, C_6”), 147.34 (C_quat.,C₈),

(m), 585 ν_{(=CH)oop band}(s). Mass (m/

NO2) adjacent hydrogen

z): 400 [M]⁺.

139.91 (C_quat.,

C_5”’), 137.49 (CH, C_4”), 136.87 (C_quat.,C₃),

134.30 (C_quat.,C_4’), 131.90 (CH, C_4”’), 131.10 (C_quat.,C_1’),

129.44 (CH, C_3’,5’), 128.90 (CH, C_2’,6’), 128.60 (CH, C_2”’),

128.00 (CH, C_3”’), 123.29 (CH, C_5”), 121.40 (CH, C_3”),

105.09 (CH, C₇), 93.05 (CH, C₄). [Total signal observed

= 19: signal of C and CH₂= 8 (Ar‐C = 2, pyridine‐C =

2.4.5 | 5‐(Pyridin‐2‐yl)‐2‐(thiophen‐2‐yl)‐7‐

(tolyl)pyrazolo[1,5‐a] pyrimidine (5e)

It was synthesized using chalcone (E)‐1‐(pyridin‐2‐yl)‐3‐

(p‐tolyl)prop‐2‐en‐1‐one (3e) (0.288 g, 2.8 mmol) and 3‐

6 of 25

LUNAGARIYA ET AL.

(thiophene‐2‐yl)‐1H‐pyrazol‐5‐amine (4a) (0.50 g,

3.0 mmol) as described in general process. Colour: yellow

powder, Yield: 81 %, mol. wt. 368.46 gm/mol, m.p. 222 °C;

Anal. Calc. (%) For C₂₂H₁₆N₄S: C, 71.72; H, 4.38; N, 15.21;

S, 8.70. Found (%): C, 72.66; H, 3.89; N, 15.47; S, 8.23. UV‐

(C_quat.,C₈) 131.9 (CH, C_4’”), 140.0 (C_quat.,C_1’), 139.9 (C_quat.,

C_5”’), 137.2 (CH, C_4”), 136.67 (C_quat.,C₃), 129.5 (CH, C_3’,5’),

128.7 (CH, C_4’), 128.6 (CH, C_2”’), 128.0 (CH, C_3”’), 127.5

(CH, C_2’,6’), 123.37 (CH, C_5”), 121.4 (CH, C_3”), 105.26

(CH, C₇), 93.37 (CH, C₄). [Total signal observed = 19: sig-

nal of C and CH₂= 7 (Ar‐C = 1, pyridine‐C = 1, thio-

phene‐C = 1, pyrazolo[1,5‐a]pyrimidine‐C = 4), signal of

CH and CH₃= 12 (Ar‐CH = 3, thiophene‐CH = 3,

pyrazolo[1,5‐a]pyrimidine‐CH = 2, pyridine‐CH = 4)].

FT‐IR: (KBr pellet) ν_max(cm^‐1): 3117 ν_(=C‐H)ar(w), 1717

ν_(C=N)(s), 1612 ν_{(C=C)conjugated alkanes}(s), 1473 ‐ 1566 ν_(C‐

1

vis: λ (nm) (ε, M^‐1cm^‐1): 294.50 (15,830), 358 (3,270). H

NMR (400 MHz, DMSO‐d₆) δ/ppm: 8.779 (d, 1H, J=

8.0 Hz, H_2”’), 8.766 (d, 1H, J= 8.0 Hz, H_4”’), 8.311 (d, 2H,

3

J= 8.0 Hz, H_2’,6’), 8.151 (s, 1H, H₇), 8.078 (t, 1H, J₁=

3

8.4 Hz, J₂= 7.6 Hz, H_3”’), 7.769 (d, 1H, J= 8.0 Hz, H_5’),

3

7.658 (d, 1H, J= 7.6 Hz, H_3’), 7.561 (t, 1H, J₁= 9.2 Hz,

³J₂= 8.8 Hz, H_5”), 7.369 (s, 1H, H₄), 7.361 ‐ 7.196 (multi-

plet, 3H, H_{3”,4”,5”}), 3.916 (s, 3H, ‐CH₃). ¹³C NMR

(100 MHz, DMSO‐d₆) δ/ppm: 165.18 (C_quat.,C₆), 155.30

(C_quat.,C_2”), 154.70 (C_quat.,C_5a), 149.79 (CH, C_6”), 147.50

(s), 1220 ν_{(C‐C)alkanes}(s), 1056 ν_{(C‐S‐C)stre. (thiophene}

H)banding

(m) 717 ‐ 817 ν_{(Ar‐H)2 adjacent hydrogen}(s), 586 ν_(=C‐H)

ring)

(s). Mass (m/z): 355 [M]⁺.

oop band

(C_quat.,C₈) 131.70 (C_quat.,C_4’), 139.90 (C_quat.,C_5”’), 137.20

2.5 | General synthesis of binuclear Pt^II

complexes (6a ‐ 6f)

(CH, C_4”), 136.67 (C_quat.,C₃), 131.90 (CH, C_4”’), 130.00

(C_quat.,C_1’), 129.50 (CH, C_3’,5’), 128.60 (CH, C_2”’), 128.00

(CH, C_3”’), 125.70 (CH, C_2’,6’), 123.60 (CH, C_5”), 121.37

(CH, C_3”), 105.26 (CH, C₇), 93.37 (CH, C₄), 21.50 (‐CH₃).

[Total signal observed = 20: signal of C and CH₂= 8

The pyrazolo[1,5‐a]pyrimidine derived ligands coordinate

with Pt^IImetal ion forming complexes were prepared fol-

lowing published method.^[20,21]The 2 ml aqueous solu-

tion of K₂PtCl₄(0.2 mmol) was mixed with a solution of

ligands, (5a‐5f) (0.1 mmol) respectively 2:1 molar ratio in

methanol (20 ml) and then refluxed for 5‐6 h. After

cooling, the resulting reddish brown slurry was filtered

and the complexes obtained in high yields after filtration

and evaporation of solvents. The products were collected

after washing with methanol twice. The proposed

reaction for the synthesis of complexes (6a‐6f) is shown

(Ar‐C

= 2, pyridine‐C = 1, thiophene‐C = 1,

pyrazolo[1,5‐a]pyrimidine‐C = 4), signal of CH and CH₃

= 12 (Ar‐CH = 2, pyridine‐CH = 4, thiophene‐CH = 3,

pyrazolo[1,5‐a]pyrimidine‐CH = 2, ‐CH3 = 1)]. FT‐IR:

(KBr pellet) ν_max(cm^‐1): 3108 ν_(=C‐H)ar(w), 1708 ν_(C=N)

(s), 1614 ν_{(C=C)conjugated alkenes}(s), 1520 ν_{(C‐H)banding}(s),

1226 ν_{(C‐C)alkanes}(s), 1087 ν_{(C‐S‐C)stre. (thiophene ring)}(w),709

– 840 ν_{(Ar‐H)2 adjacent hydrogen}(m), 586 ν_{(=C‐H)oop band}(s).

Mass (m/z): 369 [M]⁺.

1

in Scheme 1. H NMR, ¹³C NMR spectra of complexes

(6a‐6b) are shown in supplementary material 1 and 2,

respectively.

2.4.6 | 7‐Phenyl‐5‐(pyridin‐2‐yl)‐2‐

(thiophen‐2yl)pyrazolo[1,5‐a] pyrimidine

(5f)

2.6 | Synthesis of [Pt₂(5a)(Cl₄)] (6a)

It was synthesized using chalcone (E)‐3‐phenyl‐1‐

(pyridin‐2‐yl)prop‐2‐en‐1‐one (3f) (0.246 g, 2.8 mmol)

and 3‐(thiophene‐2‐yl)‐1H‐pyrazol‐5‐amine (4a) (0.50 g,

3.0 mmol) as described in general process. Colour: yellow

powder, Yield: 78 %, mol. wt. 354.43 gm/mol, m.p. 228 °C;

Anal. Calc. (%) For C₂₁H₁₄N₄S: C, 71.17; H, 3.98; N, 15.81;

S, 9.05. Found (%): C, 71.89; H, 3.23; N, 15.96; S, 8.90. UV‐

It was synthesized using ligand (5a) (0.036 g, 0.1 mmol)

and K₂PtCl₄salt (0.083 g, 0.2 mmol) as described in gen-

eral process. Colour: brown powder, Yield: 88 %, mol.

wt. 916.43 g/mol, m.p. ≥ 300 °C; Anal. Calc. (%) For

C₂₂H₁₆Cl₄N₄OPt₂S: C, 27.03; H, 1.45; N, 6.11; S, 5.50; Pt,

42.08. Found (%): C, 27.39; H, 1.10; N, 5.78; S, 5.11; Pt,

41.95. Conductance: 25 Ω^‐1cm²mol^‐1. UV‐vis: λ (nm) (ε,

M^‐1cm^‐1): 307 (37,110), 358 (11,620), 472 (1,620). ¹H

NMR (400 MHz, DMSO‐d₆) δ/ppm: 9.591 (d, 1H, J=

7.6 Hz, H_2”’), 8.973 (d, 1H, J= 7.6 Hz, H_4”’), 8.589 (s, 1H,

1

vis: λ (nm) (ε, M^‐1cm^‐1): 294 (35,840), 357 (9,270). H

3

NMR (400 MHz, DMSO‐d₆) δ/ppm: 8.783 (t, 1H, J₁=

3

4.4 Hz, J₂= 4.4 Hz, H_3”’), 8.499 (dd, 2H, J₁= 8.0 Hz,

4

3

J₂= 10.8 Hz, H_4”’), 8.28‐8.205 (multiplet, 1H, H_2”’),

H₇), 8.479 (t, 1H, J₁= 8.0 Hz, J₂= 8.0 Hz, H_3”’), 8.336

(d, 1H, J= 7.6 Hz, H_5’), 8.283 (s, 1H, H₄), 7.878 (t, 1H,

8.161 (d, 1H, J= 10.4 H_4”), 8.126 (s, 1H, H₇), 8.066 (t,

3

1H, J₁= 6.8 Hz, ³J₂= 6.4 HZ, H_5”), 7.663 (d, 2H, J=

³J₁= 7.6 Hz, J₂= 8.0 Hz, H_5”), 7.763 (dd, 2H, ³J₁=

4

5.2 Hz, H_2’,6’), 7.495 (d, 1H, J= 5.6 Hz, H_4”’), 7.587 (dd,

8.0 Hz, J₂= 4.0 Hz, H_3”,6”), 7.651 (d, 1H, J= 7.6 Hz,

3

4

2H, J₁= 8.4 Hz, J₂= 4.4 Hz, H_3’,5’), 7.360 (s, 1H, H₄),

H_3’), 7.259 – 7.295 (multiplet, 3H, H_{4”,2’,6’}), 3.948 (s, 3H,

‐OCH₃). ¹³C NMR (100 MHz, DMSO‐d₆) δ/ppm: 160.64

(C_quat.,C_4’), 150.99 (C_quat.,C₈), 150.15 (C_quat.,C_2”), 144.05

(C_quat.,C_5a), 142.87 (CH, C_6”), 142.30 (CH, C_4”), 139.38

7.213 (t, 1H, J₁= 4.4 Hz, J₂= 5.2 Hz, H_4’). ¹³C NMR

(100 MHz, DMSO‐d₆) δ/ppm: 165.18 (C_quat.,C₆), 155.3

(C_quat.,C_2”), 154.70 (C_quat.,C_5a), 149.79 (CH, C_6”), 147.5

3

LUNAGARIYA ET AL.

7 of 25

(CH, C_3”’), 137.21 (C_quat.,C₆), 134.48 (CH, C_4”’), 128.85

(CH, C_2’,6’), 125.33 (C_quat.,C_1’), 125.10 (CH, C_5”), 122.09

(CH, C_3”), 121.63 (CH, C₄), 121.22 (CH, C₇), 116.00 (CH,

mol. wt. 920.84 g/mol, m.p. ≥ 300 °C; Anal. Calc. (%)

For C₂₁H₁₃Cl₅FN₄Pt₂S: C, 27.69; H, 1.42; N, 6.08; S, 3.48;

Pt, 42.37. Found (%): C, 28.19; H, 1.49; N, 6.57; S, 3.21;

Pt, 42.18. Conductance: 21 Ω^‐1cm²mol^‐1. UV‐vis: λ

(nm) (ε, M^‐1cm^‐1): 308 (35,420), 358 (9,820), 472.50

C

_2”’), 114.81 (CH, C_3’,5’), 114.62 (C_quat.,C_5”’), 107.06 (C_quat.,

C₃), 55.82 (O‐CH₃). [Total signal observed = 20: signal of

C and CH₂= 8 (Ar‐C = 2, pyridine‐C = 1, thiophene‐C

= 1, pyrazolo[1,5‐a]pyrimidine‐C = 4), signal of CH and

CH₃= 12 (Ar‐CH = 2, thiophene‐CH = 3, pyrazolo[1,5‐

a]pyrimidine‐CH = 2, pyridine‐CH = 4, ‐OCH₃= 1)].

FT‐IR: (KBr pellet) ν_max(cm^‐1): 3078 ν_(=C‐H)ar(w), 1596

ν_(C=N)(s), 1566 ν_{(C=C)conjugated alkenes}(s), 1512 – 1473 ν_(C‐

1

(1,580). H NMR (400 MHz, DMSO‐d₆) δ/ppm: 9.670 (d,

1H, J= 7.2 Hz, H_2”’), 8.951 (d, 1H, J= 7.6 Hz, H_4”’), 8.502

3

4

(dd, 2H, J₁= 8.0 Hz, J₂= 4.0 Hz, H_3”,6”), 8.370 (d, 1H,

J= 8.8 Hz, H_5’), 8.304 (s, 1H, H₇), 7.896 (t, 1H, ³J₁=

3

7.6 Hz, J₂= 7.6 Hz, H_3”’), 7.832 – 7.744 (multiplet, 4H,

3

H

_{3’,2’,6’,5”}), 7.669 (s, 1H, H₄), 7.228 (t, 1H, J₁= 8.8 Hz,

(s), 1249 ν_{(C‐C)alkanes}(s), 1180 ν_{(Ar‐O‐CH3)}(s),

1021 ν_{(C‐S‐C)stre. (thiophene ring)}(s), 717 – 840 ν_{(Ar‐H)2 adjacent}

³J₂= 8.8 Hz, H_4”). ¹³C NMR (100 MHz, DMSO‐d₆) δ/

ppm: 150.91 (C_quat.,C₈), 150.18 (C_quat.,C_2”), 144.15 (C_quat.,

C_5a), 142.89 (CH, C_6”), 142.20 (CH, C_4”), 139.31 (CH, C_3”’),

137.26 (C_quat.,C₆), 134.83 (CH, C_4”’), 134.3(C_quat.,C_4’),

128.56 (CH, C_2’,6’), 131.1 (C_quat.,C_1’), 125.18 (CH, C_5”),

122.89 (CH, C_3”), 121.67 (CH, C₄), 121.20 (CH, C₇),

H)banding

(s), 640 ν_(N‐Pt)(s), 555 ν_(S‐Pt)(s).

hydrogen

2.7 | Synthesis of [Pt₂(5b)(Cl₄)] (6b)

It was synthesized using ligand (5b) (0.037 g, 0.1 mmol)

and K₂PtCl₄salt (0.083 g, 0.2 mmol) as described in gen-

eral process. Colour: brown powder, Yield: 85 %, mol.

wt. 904.39 g/mol, m.p. ≥ 300 °C; Chemical formula:

C₂₁H₁₃Cl₄FN₄Pt₂S. Conductance: 19 Ω^‐1.cm²mol^‐1. UV‐

vis: λ (nm) (ε, M^‐1cm^‐1): 304 (38,020), 357 (10,920),

116.00 (CH, C_2”’), 129.3 (CH, C_3’,5’), 114.68 (C_quat.,C_5”’),

107.39 (C_quat.,C₃). [Total signal observed = 19: signal of

C and CH₂= 8 (Ar‐C = 2, thiophene‐C = 1,

pyrazolo[1,5‐a]pyrimidine‐C = 4, pyridine –C = 1), signal

of CH and CH₃= 11 (Ar‐CH = 2, thiophene‐CH = 3,

pyrazolo[1,5‐a]pyrimidine‐CH = 2, pyridine‐CH = 4)].

FT‐IR: (KBr pellet) ν_max(cm^‐1): 3085 ν_(=C‐H)ar(w), 1596 –

1

471.50 (1,820). H NMR (400 MHz, DMSO‐d₆) δ/ppm:

9.654 (d, 1H, J= 8.0 Hz, H_2”’), 8.964 (d, 1H, J= 8.0 Hz,

1566 ν_(C=N)(m), 1519 – 1473 ν_{(C=C)conjugated}

(s),

alkenes

H

_4”’), 8.694 (s, 1H, H₇), 8.524 ‐ 8.418 (multiplet, 3H,

1396 ν_{(C‐H)banding}(m), 1226 ν_{(C‐C)alkanes}(s), 1149 ν_(Ar‐Cl)

(w), 1010 ν_{(C‐S‐C)stre. (thiophene ring)}(s), 817 – 717 ν_{(Ar ‐H)2}

_{3’,5’,5”}), 8.298 (s, 1H, H₄), 7.937 (t, 1H, ³J₁= 7.6 Hz,

3

J₂= 8.0 Hz, H_3”’), 7.774 (d, 1H, J= 8.0 Hz, H_2’,6’), 7.702

(s), 624 ν_(N‐Pt)(s), 555 ν_(S‐Pt)(s).

adjacent hydrogen

3

4

(d, 1H, J= 8.0 Hz, H_6’), 7.633 (dd, 2H, J₁= 8.0 Hz, J₂=

3

4.8 Hz, H_3”,6”), 7.249 (t, 1H, J₁= 8.0 Hz, J₂= 8.0 Hz,

H_4”). ¹³C NMR (100 MHz, DMSO‐d₆) δ/ppm: 162.91

(C_quat.,C_4’), 150.95 (C_quat.,C₈), 150.18 (C_quat.,C_2”), 144.15

(C_quat.,C_5a), 142.89 (CH, C_6”), 142.20 (CH, C_4”), 139.31

(CH, C_3”’), 137.26 (C_quat.,C₆), 134.43 (CH, C_4”’), 130.56

(CH, C_2’,6’), 128.37 (C_quat.,C_1’), 125.18 (CH, C_5”), 122.19

(CH, C_3”), 121.69 (CH, C₄), 121.20 (CH, C₇), 116.30 (CH,

2.9 | Synthesis of [Pt₂(5d)(Cl₄)] (6d)

It was synthesized using ligand (5d) (0.039 g, 0.1 mmol)

and K₂PtCl₄salt (0.083 g, 0.2 mmol) as described in gen-

eral process. Colour: brown powder, Yield: 90 %, mol.

wt. 931.40 g/mol, m.p. ≥ 300 °C; Anal. Calc. (%) For

C₂₁H₁₃Cl₄N₅O₂Pt₂S: C, 27.08; H, 1.41; N, 7.52; S, 3.44;

Pt, 41.89. Found (%): C, 26.58; H, 1.5; N, 6.83; S, 3.10; Pt,

42.04. Conductance: 22 Ω^‐1cm²mol^‐1. UV‐vis: λ (nm) (ε,

C

_2”’), 116.71 (CH, C_3’,5’), 114.68 (C_quat.,C_5”’), 107.09 (C_quat.,

C₃). [Total signal observed = 19: signal of C and CH₂= 8

(Ar‐C = 2, thiophene‐C = 1, pyrazolo[1,5‐a]pyrimidine‐C

= 4, pyridine –C = 1), signal of CH and CH₃= 11 (Ar‐CH

= 2, thiophene‐CH = 3, pyrazolo[1,5‐a]pyrimidine‐CH =

2, pyridine‐CH = 4)]. FT‐IR: (KBr pellet) ν_max(cm^‐1):

3078 ν_(=C‐H)ar(m), 1604 – 1566 ν_(C=N)(s), 1512 – 1481

ν_{(C=C)conjugated alkenes}(s), 1411 ν_{(C‐H)banding}(m), 1226 ν_(C‐

M^‐1cm^‐1): 305 (34,820), 357 (8,790), 470.50 (1,680). H

1

NMR (400 MHz, DMSO‐d₆) δ/ppm: 8.575 (d, 2H, J=

8.0 Hz, H_{2”’,4”’}), 8.520 (d, 2H, J= 8.4 Hz, H_3’,5’), 8.199 (s,

1H, H₇), 8.115 (d, 1H, J= 8.8 Hz, H_2’), 7.935 (d, 1H, J=

3

4

7.2 Hz, H_6’), 7.789 (dd, 2H, J₁= 8.8 Hz, J₂= 3.2 Hz,

_3”,6”), 7.717 – 7.662 (multiplet, 1H, H_5”), 7.619 (t, 1H,

H

3

(s), 1157 ν_(Ar‐F)(s), 1018 ν_{(C‐S‐C)stre. (thiophene ring)}

³J₁= 7.6 Hz, J₂= 8.0 Hz, H_3”), 7.399 (s, 1H, H₄), 7.242

(t, 1H, ³J₁= 7.2 Hz, ³J₂= 7.6 Hz, H_4”). ¹³C NMR

(100 MHz, DMSO‐d₆) δ/ppm: 150.88 (C_quat.,C₈), 150.18

(C_quat.,C_2”), 147.37 (C_quat.,C_4’), 144.15 (C_quat.,C_5a),

142.89 (CH, C_6”), 142.20 (CH, C_4”), 139.81 (CH, C_2”’),

139.26 (C_quat.,C_1’), 137.1 (C_quat.,C₆), 134.83 (CH, C_4”’),

126.25 (CH, C_2’,6’), 125.18 (CH, C_5”), 122.89 (CH, C_3”),

121.77 (CH, C₄), 121.20 (CH, C₇), 116.00 (CH, C_2”’),

C)alkanes

(w), 725 – 840 ν_{(Ar‐H)2 adjacent hydrogen}(s), 632 ν_(N‐Pt)(s),

570 ν_(S‐Pt)(s).

2.8 | Synthesis of [Pt₂(5c)(Cl₄)] (6c)

It was synthesized using ligand (5c) (0.038 g, 0.1 mmol)

and K₂PtCl₄salt (0.083 g, 0.2 mmol) as described in

general process. Colour: brown powder, Yield: 88 %,

124.4 (CH, C_3’,5’), 114.68 (C_quat.,

C_5”’), 107.39 (C_quat.,C₃).

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1

[Total signal observed = 19: signal of C and CH₂= 8 (Ar‐

C = 2, thiophene‐C = 1, pyrazolo[1,5‐a]pyrimidine‐C = 4,

pyridine –C = 1), signal of CH and CH₃= 11 (Ar‐CH = 2,

thiophene‐CH = 3, pyrazolo[1,5‐a]pyrimidine‐CH = 2,

pyridine‐CH = 4)]. FT‐IR: (KBr pellet) ν_max(cm^‐1): 3078

M^‐1cm^‐1): 306 (37,820), 357 (11,500), 472.50 (1,570). H

3

NMR (400 MHz, DMSO‐d6) δ/ppm: 8.894 (t, 1H, J₁=

3

8.0 Hz, J₂= 8.0 Hz, H_3”’), 8.583 (dd, 2H, J₁= 8.8 Hz,

⁴J₂= 4.0 Hz, H_3”,6”), 8.334 – 8.233 (multiplet, 3H,

H

_{2”’,4”’,5”}), 8.110 (s, 1H, H₇), 7.879 – 7.610 (multiplet, 5H,

3

ν

_(=C‐H)ar(w), 1596 – 1566 ν_(C=N)(s), 1519 – 1473 ν_(C=C)con-

H

_{2’,3’,4’,5’,6’}), 7.329 (s, 1H, H₄), 7.282 (t, 1H, J₁= 8.0 Hz,

3

_{jugated alkenes}(s), 1342 ν_{(C‐H)banding}(s), 1226 ν_{(C‐C)alkanes}(s),

1149 ν_(Ar‐NO2)(w), 1010 ν_{(C‐S‐C)stre (thiophene ring)}(w), 848 –

717 ν_{(Ar‐H)2 adjacent hydrogen}(s), 632 ν_(N‐Pt)(s), 555 ν_(S‐Pt)(s).

J₂= 8.0 Hz, H_4”). ¹³C NMR (100 MHz, DMSO‐d₆) δ/

ppm: 150.98 (C_quat.,C₈), 150.18 (C_quat.,C_2”), 144.50 (C_quat.,

C_5a), 142.89 (CH, C_6”), 142.20 (CH, C_4”), 139.81 (CH,

C_3”’), 137.26 (C_quat.,C₆), 134.16 (CH, C_4”’), 133.03 (C_quat.,

C_1’), 130.05 (CH, C_3’,5’), 127.78 (CH, C_2’,6’), 128.39 (CH,

C_4’), 125.77 (CH, C_5”), 122.9(CH, C_3”), 121.72 (CH, C₇),

2.10 | Synthesis of [Pt₂(5e)(Cl₄)] (6e)

It was synthesized using ligand (5e) (0.0367 g, 0.1 mmol)

and K₂PtCl₄salt (0.083 g, 0.2 mmol) as described in

general process. Colour: brown powder, Yield: 82 %, mol.

wt. 900.43 g/mol, m.p. ≥ 300 °C; Anal. Calc. (%) For

C₂₂H₁₆Cl₄N₄Pt₂S: C, 29.35; H, 1.89; N, 6.92; S, 3.56; Pt,

43.33. Found (%): C, 28.58; H, 2.1; N, 7.09; S, 3.07; Pt,

43.04. Conductance: 18 Ω^‐1cm²mol^‐1. UV‐vis: λ (nm) (ε,

121.19 (CH, C₄), 116.00 (CH, C_2”’), 114.68 (C_quat.,C_5”’),

107.49 (C_quat.,C₃). [Total signal observed = 19: signal of C

and CH₂= 8 (Ar‐C = 2, thiophene‐C = 1, pyrazolo[1,5‐a]

pyrimidine‐C = 4, pyridine –C = 1), signal of CH and

CH₃= 11 (Ar‐CH = 2, thiophene‐CH = 3, pyrazolo[1,5‐a]

pyrimidine‐CH = 2, pyridine‐CH = 4)]. FT‐IR: (KBr pellet)

ν

_max(cm^‐1): 3078 ν_(=C‐H)ar(w), 1558 – 1596 ν_(C=N)(s), 1519 –

1

M^‐1cm^‐1): 307 (36,200), 359 (9,790), 471 (1,790). H NMR

1473 ν_{(C=C)conjugated alkenes}(m), 1350 ν_{(C‐H)banding}(s), 1226

ν_{(C‐C)alkanes}(s), 1033 ν_{(C‐S‐C)stre. (thiophene ring)}(w), 848 –

702 ν_{(Ar‐H)2 adjacent hydrogen}(s), 648 ν_(N‐Pt)(s), 555 ν_(S‐Pt)(s).

(400 MHz, DMSO‐d₆) δ/ppm: 8.799 (d, 1H, J= 8.0 Hz,

H_2”’), 8.678 (s, 1H, H₇), 8.535 (d, 1H, J= 8.0 Hz, H_4”’), 8.278

(t, 1H, ³J₁= 8.4 Hz, ³J₂= 8.4 Hz, H_3”’), 8.175 – 8.059 (multi-

plet, 2H, H_3’,5’), 7.756 (d, 1H, J= 7.2 Hz, H_2’), 7.654 (d, 1H,

J= 7.2 Hz, H_6’), 7.604 (t, 1H, ³J₁= 7.2 Hz, ³J₂= 7.2 Hz, H_5”),

2.12 | Biological screening of synthesized

binuclear Pt^IIcomplexes

3

4

7.513 (dd, 2H, J₁= 8.0 Hz, J₂= 4.0 Hz, H_3”,6”), 7.317 (s,

1H, H₄), 7.227 (t, 1H, ³J₁= 7.6 Hz, ³J₂= 7.6 Hz, H_4”), 4.173

(s, 3H, ‐CH₃). ¹³C NMR (100 MHz, DMSO‐d₆) δ/ppm:

150.98 (C_quat.,C₈), 150.18 (C_quat.,C_2”), 131.72 (C_quat.,C_4’),

144.50 (C_quat.,C_5a), 142.89 (CH, C_4”), 142.20 (CH, C_6”),

139.81 (CH, C_3”’), 137.26 (C_quat.,C₆), 134.16 (CH, C_4”’),

130.83 (C_quat.,C_1’), 129.66 (CH, C_3’,5’), 125.78 (CH, C_2’,6’),

125.39 (CH, C_5”), 122.77 (CH, C_3”), 121.70 (CH, C₄), 121.22

(CH, C₇), 116.00 (CH, C_2”’), 114.68 (C_quat.,C_5”’), 107.39

(C_quat.,C₃). 21.56 (‐CH₃). [Total signal observed = 20: signal

of C and CH₂= 8 (Ar‐C = 2, thiophene‐C = 1, pyrazolo[1,5‐

a]pyrimidine‐C = 4, pyridine –C = 1), signal of CH and CH₃

= 12 (Ar‐CH = 2, thiophene‐CH = 3, pyrazolo[1,5‐a]pyrim-

idine‐CH = 2, pyridine‐CH = 4, ‐CH₃= 1)]. FT‐IR: (KBr pel-

let) ν_max(cm^‐1): 3078 ν_(=C‐H)ar(w), 1596 ‐1566 ν_(C=N)(s),

1512 ‐1473 ν_{(C=C)conjugated alkenes}(m), 1396 ν_{(C‐H)banding}(m),

1218 ν_{(C‐C)alkanes}(s), 1026 ν_{(C‐S‐C)stre. (thiophene ring)}, (w), 794

– 717 ν_{(Ar‐H)2 adjacent hydrogen}(s), 601 ν_(N‐Pt)(s), 532 ν_(S‐Pt)(s).

2.12.1 | In vitro antibacterial activity

To examine antibacterial activity of compounds under

investigation, we used five test organisms such as two

Gram^(+ve)Bacillus subtilis and Staphylococcus aureus and

three Gram^(–ve)Escherichia coli, Pseudomonas aeruginosa

and Serratia marcescens using the broth dilution

technique. A pre‐culture of bacteria was used to prepare

bacterial culture in Luria Broth. DMSO was used as

medium to acquire preferred concentration of compounds

to check upon microbial strains. The test compounds of

specific concentration was added to the sugar tube

containing Luria Broth solution and sterilized in auto-

clave before the addition of previously cultured bacterial

species. After adding different bacterial species to the

tubes containing specific concentration of test com-

pounds, it was allowed to incubate for 24 h at optimum

temperature. Experiment was repeated till only a faint

turbidity appears in the tube. The lowest concentration,

which showed no visible growth after 24 h subculture

was considered as MIC for each compound.^[22,23]

2.11 | Synthesis of [Pt₂(5f)(Cl₄)] (6f)

It was synthesized using ligand (5f) (0.035 g, 0.1 mmol)

and K₂PtCl₄salt (0.083 g, 0.2 mmol) as described in general

process. Colour: brown powder, Yield: 80 %, mol. wt.

886.40 g/mol, m.p. ≥ 300 °C; Anal. Calc. (%) For

C₂₁H₁₄Cl₄N₄Pt₂S: C, 28.76; H, 1.59; N, 6.32; S, 3.62; Pt,

44.02. Found (%): C, 29.03; H, 1.81; N, 6.01; S, 3.34; Pt,

44.24. Conductance: 20 Ω^‐1cm²mol^‐1. UV‐vis: λ (nm) (ε,

2.13 | Cellular level bioassay using S.

Pombe cells

Cellular level bioassay was carried out using

Schizosaccharomyces Pombe cells, which were grown in

LUNAGARIYA ET AL.

9 of 25

liquid yeast extract media in a 150 ml Erlenmeyer flask

containing 50 ml of yeast extract media. The flask was

incubated at 30 °C on a shaker at 160 rpm until the

exponential growth of S. Pombe was obtained after 24 to

30 h. Then, the cell culture was treated with the different

concentrations (20, 40, 60, 80, 100 μM) of the synthesized

complexes, free ligands, K₂PtCl₄salt and also with

dimethyl sulphoxide as a control and after further allowed

to grow for 16‐18 h. Than the next day, by centrifugation

at 10, 000 rpm for 10 min, the treated cells were collected

and dissolved in 500 μL of PBS (Phosphate Buffered

Saline) 80 μL of yeast culture dissolved in PBS and

20 μL of 0.4% trypan blue prepared in PBS were mixed

and cells were observed with a compound microscope

(40X). The dye could enter dead cells only so they

appeared blue, whereas live cells resisted the entry of

dye. The number of dead cells and number of live cells

were counted in one field. Cell counting was repeated in

two more fields and the average percentage of dead cells

due to the synthesized compounds were calculated.^[24]

2.16 | In vitro antiproliferative cytotoxicity

on human colorectal carcinoma HCT‐116

cells

Antiproliferative cytotoxicity of the synthesized Pt^II

complexes (6a‐6f) was determined by means of the MTT

(3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium

bromide) assay. The HCT‐116 cells were plated at 5000

cells per well in 96‐well culture plates with a culture

medium, incubated for 24 h at 37 °C in a 5% CO₂incuba-

tor. The concentrations of compounds exhibit the vehicle

control (0.5% DMSO), DMSO, 10, 50, 100, 250, 500,

1000 μg/ml. The cells were washed twice with Dulbecco's

Phosphate‐Buffered Saline (DPBS) and incubated with

0.5 mg/ml MTT solution for 4 h at 37 °C. Subsequently,

0.1 mL of SDS‐HCl was added to each well, mixed

carefully and allowed for incubation in the dark for

20 min at 37 °C. Finally, the absorbance of each well

was recorded at 570 nm with a reference wavelength of

650 nm using a multimedia micro plate reader (Spectra

Max M2e, Molecular devices, Sunnyvale, California,

USA). IC₅₀values were calculated from the chart of the

cell proliferation (%) against the compound concentration

(μg/mL). The results are represented as percentage of cell

proliferation in different treatment group. All experi-

ments were carried out in triplicate.^[27]

2.14 | In vitro cytotoxic study using brine

shrimp lethality bioassay (BSLB)

Cytotoxicity assays are widely used by the pharmaceutical

industry to screen for cytotoxicity in compound libraries.

Researchers can look for cytotoxic compounds, if they

are interested in developing a therapeutic that targets rap-

idly dividing cancer cells. In vitro cytotoxicity is a method

by which one can check the toxicity of the compounds on

Brine Shrimp. The experiment was carried out following

the protocol of Mayer et al. A set of 2, 4, 8, 16, 20 μg/ml

in test tube was prepared from a stock solution of

1000 μg/ml of the test compounds. Final volume of in

each test tube was adjusted to 2500 μL (1000 μL sea salt

solution, 450 μL double distilled water, 50 μL DMSO +

complex and remaining 1000 μL was added as 10 nauplii

in a sea salt solution). After 24 h numbers of dead nauplii

were counted and from the survival LC₅₀was

calculated.^[25]

2.17 | DNA interaction studies

2.17.1 | DNA binding study by absorption

analysis

The stock solutions of Pt^IIcomplexes were prepared using

5% DMSO at 4 °C for complete dissolution and used

within 4 days and then diluting them suitably with

phosphate buffer (in MilliQ water) to the required

concentrations for all the experiments.^[4,28]Ratio of UV

absorbance kept at 260 and 280 nm was about 1.89:1 for

HS‐DNA in the buffer, indicating that the HS DNA

sufficiently free from protein. The HS‐DNA concentration

per nucleotide was determined spectrophotometrically by

employing an extinction coefficient of 12858 M^‐1cm^‐1at

260 nm.

2.15 | Mycobacterium tuberculosis (Mtb)

H₃₇Rv strain

2.18 | Viscosity measurements

A primary in vitro antituberculosis activity of the

synthesized binuclear Pt^IIcomplexes were conducted at

concentration of 250 μg/ml against Mycobacterium

tuberculosis H₃₇Rv strain by using Lowenstein ‐ Jensen

medium as described by Rattan ^[26]. The obtained results

of synthesized complex are presented in result and

discussion.

The binding of small molecules to DNA cannot be

determined only by absorption titration method.^[29]

Viscometric measurement is a supporting tool to confirm

the binding mode. The molecule can bind to DNA by

three binding modes as represented below. In electrostatic

and groove binding viscosity of DNA solution remain

unaltered, while in classical intercalative binding viscosity

10 of 25

LUNAGARIYA ET AL.

will rise. Among these interactions, intercalation and

groove binding are the most important DNA‐binding

modes as they invariably lead to cellular degradation.

Herein, we carried out viscosity measurement at 27

0.1 °C using a constant thermometric bath,^[30,31]

results. A competitive interaction of the metal complexes

to HS DNA results in the displacement of the bound

EB.^[34]The quenching constant (K_sq) was calculated using

the linear Stern‐Volmer equation (1):^[35]

I⁰

¼ Ksq r þ 1 OR I₀=I ¼ Ksq½Qꢀ þ 1

(1)

I

2.19 | Molecular modeling study with HS

DNA

where, I₀ and I are the emission intensity of EB‐DNA in

the absence and presence of quencher (complex), K_sqis

the linear Stern‐Volmer quenching constant obtained

from the plot of I₀/I versus [Q] and r is the ratio of total

concentration of complex of DNA or concentration of

quencher [Q].

To determine the strength of the interaction of com-

plexes with DNA, the value of the associative binding con-

stant (K_a) was calculated using the Scatchard

equation 2:^[36]

The most stable configuration was chosen as input for

investigation and the rigid molecular docking study was

performed by using HEX 8.0 software. Pt^IIcomplexes

were taken from their enhanced structure as a molecule

and converted to (Protein Data Bank) .pdb format using

CHIMERA 1.5.1 software. HS‐DNA used in the experi-

mental work was too large for current computational

resources to dock, therefore, the structure of the DNA of

sequence d(ACCGACGTCGGT)2 (1BNA) for groove bind-

ing and an octamer d(GAAGCTTC)2 (1BNA) for interca-

lation^[32](PDB id: 1BNA, a familiar sequence used in

oligodeoxynucleotide study) obtained from the Protein

Data Bank (http://www.rcsb.org/pdb). All calculations

were done using on an Intel CORE i5, 2.5 GHz based

machine running MS Windows 8 64 bit as the operating

system. The by default parameters were used for the

docking calculation with correlation type shape only,

FFT mode at 3D level, grid dimension of 6 with receptor

range 180 and ligand range 180 with twist range 360 and

distance range 40.

log I₀−I=I ¼ logKa þ nlog½Qꢀ

(2)

where, I₀ and I are the fluorescence intensities of the EB‐

DNA in the absence and presence of different concentra-

tions of complexes, respectively and n is the number of

binding. The acting forces between drugs and

biomacromolecules include hydrogen bonds, van der

Waals forces, electrostatic attraction and hydrophobic

interaction, etc. In order to estimate the interaction force

of with all compounds, the standard free energy changes

(ΔG⁰) for the binding process have been calculated using

the Van’t Hoff equation 3:^[37]

2.20 | Competitive displacement assay

ΔG0 ¼ −RTlnKa

(3)

The competitive interaction of all the complexes was

studied by fluorescence quenching analysis in order to

find out whether the compound can partially displace

where, T is the temperature (25 °C, 298 K here), K_ais

associative binding constant and R is gas constant 8.314

Jmol^‐1K^‐1. The negative sign for ΔG means that the spon-

taneous binding process.

ethidium bromide (EB

=

3,8‐diamino‐5‐ethyl‐6‐

phenylphenanthridinium bromide) from DNA‐EB

complex. Ethidium bromide displacement experiment

was done by adding the solution of the complexes to the

phosphate buffer solution (pH, 7.2) up to the value of r

= 3.33 ([DNA]/[Complex]) of pre‐treated EB–DNA mix-

ture ([EB] = 33.3 μM, [DNA] = 10 μM). The changes in

fluorescence intensities of EB and EB bound to DNA were

measured with respect to different concentration of the

complex. The EB has less‐emission intensity in phosphate

buffer solution (pH, 7.2) due to florescence quenching of

free EB by the solvent molecules. In the presence of

DNA, EB exhibits higher intensity due to its partial

intercalative binding mode to DNA. A competitive inter-

action of the metal complexes to HS DNA results in the

displacement of the bound EB and inner filter effect

(IFE) were carried out by equation in literature.^[33]There-

fore, it is needed to eliminate IFE interference with the

2.21 | DNA cleavage assay

The DNA cleavage experiments were performed as

follows: A mixture of TAE buffer (0.04 mol L⁻¹Tris–Ace-

tate, pH 8, 0.001 mol L⁻¹edta), 15 μL reaction mixture

containing 300 μg/ml plasmid DNA in TE buffer (10 mmol

L

⁻¹Tris, 1 mmol L⁻¹edta, pH 8.0) and 200 μmol L⁻¹com-

plex solution. Reactions were allowed to proceed for 3 h at

37 °C in the dark and reactions were settled by addition of

5 μL loading buffer a dye solution (0.25% bromophenol

blue, 40% sucrose, 0.25% xylene cyanol and 200 mmol L

−1

edta). The aliquots were loaded directly on to 1% aga-

rose gel and electrophoresed at 90 V in 1×TAE buffer.

The gel was stained with 0.5 mg L⁻¹ethidium bromide

and then bands were visualized by photographed under

LUNAGARIYA ET AL.

11 of 25

UV illuminator. After electrophoresis, the proportion of

DNA in each fraction was estimated quantitatively from

the intensity of the bands using AlphaDigiDoc™ RT.

Version V.4.0.0 PC–Image software. The degree of DNA

cleavage activity was expressed in terms of the percentage

of conversion of the SC‐DNA to OC‐DNA equation

describe in literature.^[38]

3 | RESULTS AND DISCUSSION

3.1 | Magnetic moments, UV‐vis spectral

analysis and molar conductivity (Λ_m)

measurements

FIGURE 2 EDX spectrum of the Pt^IIcomplex 6a

The room temperature magnetic moments show that the

binuclear Pt^IIcomplexes are diamagnetic in nature. The

magnetic susceptibility (μ_eff.) values indicate a pairing of

electron with low‐spin t₂g⁶eg²configuration. Pt^II

complexes have square planer geometry i.e. dsp²

hybridisation. Which can be confirmed by electronic

spectral data. The dz²orbital is in lowest energy state

compare to dx²‐y²orbital energy state for Pt^IIcomplex.

UV‐vis spectra of homoleptic Pt^IIcomplexes (Figure 1)

show three bands in range of 294–472 nm. The highest

wavelength (d–d transition) band is observed at 470–

472 nm region. The bands in the region 357‐360 nm and

294‐309 nm are assigned to MLCT (Metal to ligand charge

transfer) and ILCT (Intra ligands charge transfer)

transitions, respectively.^[39,40]The molar conductivity

(Λ_m) values of Pt^IIcomplexes are observed in the range

of 18 – 25 Ω^‐1cm²mol^‐1, which suggests the non‐electro-

lytic nature of the complexes.

% of C, N, O, S, Pt and Cl as 27.49, 6.34, 2.0, 4.36,

41.93, 16.05, respectively, as estimated with the calcu-

lated values 28.83, 6.11, 1.75, 3.50, 42.58, 15.47, respec-

tively. The elemental analysis data of the synthesized

ligands (5a‐5f) are represented in supplementary mate-

rial 1. ¹H NMR spectra of all synthesized compounds

exhibit a peaks in the range of δ 7.00‐9.00 ppm

assigning aromatic protons. The ¹H NMR spectra of

complexes are shifted to down field compare to corre-

sponding fused heterocyclic pyrazolo[1,5‐a]pyrimidine

1

nucleus based ligands (5a‐5f). H NMR spectra of com-

pounds (5a‐5f and 6a‐6f) are represented in supplemen-

tary material 2 whereas spectral data in experimental

section.

In the ¹³C NMR spectrum, the signals for (‐C, ‐CH)

aromatic carbons appears between 167 and 105 ppm,

respectively. The C4 carbon of ligands (5a‐5f) gave an

intense singlet at about 94.00‐95.00 ppm, it is shifted to

120‐121.50 ppm in the spectra of complexes (6a‐6f)

suggest formation of complex. ¹³C NMR spectra (5a‐5f

and 6a‐6f) are represented in supplementary material 3

whereas spectral data in experimental section.

1

3.2 | EDX, H NMR and ¹³C NMR

Energy dispersive X‐rays analysis (EDX) data (Figure 2)

for the binuclear Pt^IIcomplex 6a confirmed the weight

FIGURE 1 Electronic spectrum of

ligand 5a and Pt^IIcomplex 6a

12 of 25

LUNAGARIYA ET AL.

632‐640 cm^‐1, while thiophene ring of sulphur atom

coordinate with Pt^IIion exhibit the band ⱱ_(Pt‐S)in the

range of 555‐570 cm^‐1.^[41]These bands are shifted to lower

wavenumbers (80‐90 cm^‐1) indicating the coordination of

metal through aza/sulphur in all complexes. The aromatic

(C‐H) band of ligands and complexes are also observed in

the range of 2916‐3130 cm^‐1.^[43]These spectral interpreta-

tions and their assignments are consistent with the

literature data.^[44,45]

3.3 | FT‐IR characterisation

A primary effort was made for the characterization of the

synthesized compounds by FT‐IR spectroscopy. IR spectra

of free N‐aza heterocyclic pyrazolo[1,5‐a] pyrimidine

based compounds, are an illustration in supplementary

material 4 and spectral frequency bands are represented

in experimental section. While pyrazolo[1,5‐a] pyrimidine

derivatives (neutral ‐N,‐S & ‐N,‐N donor tetradentat) of

ligands are capable to coordinate with platinum metal to

form chelating five membered ring in Pt^IIcomplexes.

Infrared spectra of the pyrazolo[1,5‐a] pyrimidine

derivatives ligands exhibit few functional groups i.e. azo

methine (>C=N) group of pyrazolo[1,5‐a]pyrimidine

and pyrimidine moiety, (>C=C<) group of conjugated

aromatic alkene and (C‐S‐C) of thiophene functionality.

The azo methine stretching vibrations ⱱ(C=N), in a N‐

aza heterocyclic pyrazolo[1,5‐a] pyrimidine derivatives

ligands are observed frequency in the range of 1704‐

1710 cm^‐1and in complexes its shifted to lower wavenum-

ber (80‐90 cm^‐1) at 1604‐1566 cm^‐1, which exhibits that

azo methine group coordinated to metal ion by chela-

tion.^[41,42]The band ⱱ(C‐S‐C) of the sulphur containing

heterocyclic five membered ligands are observed in the

3.4 | LC‐MS analysis

Intensity vs mass upon charge ratio spectrum and possible

mass fragmentation pattern of binuclear Pt^IIcomplex (6d)

are illustrated in Figure 3. Mass spectral data of free

ligands (5a‐5f) and platinum(II) complex (6a) are repre-

sented in experimental section. Mass spectral graphs of

ligands (5a‐5f) are represented in supplementary material

5. Complex (6d) shows molecular ion peaks at 928.88 m/z,

930.80 m/z, 932.49 m/z, 934.79 m/z and 936.38 m/z are

assigned to [M], [M+2], [M+4], [M+6] and [M+8],

respectively, due to the presence of four chlorine atom

in the complex 6d. The peak observed at 789.01 m/z value,

is due to the loss of four chlorine atoms. The peak at

399.08 m/z is due to the loss of two platinum metal ions,

which is due to 7‐(4‐nitrophenyl)‐5‐(pyridin‐2‐yl)‐2‐

(thiophen‐2‐yl)pyrazolo[1,5‐a]pyrimidine ligand. The

peak observed at 317.09 m/z value is due to the removal

of the thiophene ring. The peak at 240.06 m/z is due to

the removal of the pyridine ring. The peak at 119.05 m/z

is due to removal of the nitrobenzene ring corresponds

to the pyrazolo[1,5‐a]pyrimidine nucleus.

range of 1080‐1087 cm^‐1and thiophene ring coordinate

,

with Pt^IImetal ion to form complexes, the frequency

appear in range of 1018‐1021 cm^‐1, which indicate the

band of complexes are shifted to (60‐70 cm^‐1) lower

frequency as compared to ligands. N‐aza heterocyclic

pyrazolo[1,5‐a]pyrimidine moiety contain nitrogen atom

coordinates with Pt^IImetal ion forming chelative

compounds, the band ⱱ(Pt‐N) observed in the range of

FIGURE 3 LC‐MS spectrum and fragmentation pattern of Pt^IIcomplex 6d

LUNAGARIYA ET AL.

13 of 25

3.5 | Thermal properties

3.6 | Pharmacology

3.6.1 | In vitro antimicrobial study

Thermogravimetric analysis (TGA) of Pt^IIcomplexes was

carried out under a dinitrogen atmosphere with mass elim-

inate due to heating system of 10 °C per minute from ambi-

ent temperature at 30 ‐ 800 °C, the result of TGA curve

(mass loss% to temperature in ⁰C) of complex (6a) are rep-

resented in supplementary material 6. TG curve is con-

cerned with the change in molecular weight of a

compound with change in temperature. The important

information that can be obtained by TGA, that there is

the amount of weight loss of the compound by heating.

For any complexes, the loss of water molecule from lattice

sphere is observed up to 120 °C, while the coordinated

water molecules directly bonded to platinum metal are

eliminate at higher temperatures in between 120‐180 °C,

but here no mass losses occur up to 200 °C, that indicate

the absence of the lattice or coordinated water molecules

in the complexes. The thermogram of compound [Pt₂(5a)

(Cl₄)] (6a) shows the weight loss occurs in between temper-

ature 230 ‐ 290 °C (weight loss of 15%), which is attributed

to the loss of chlorine molecules, mass loss occurring in

range of temperature 300 ‐ 550 °C (weight loss of 42%) is

due the loss of ligand (5a) and leaving behind metallic

platinum as a residue.^[4]

A comparative study of in vitro antimicrobial screening

indicates that binuclear Pt^IIcomplexes showed greater

activity against Gram^(−ve)and Gram^(+ve)bacteria, when

pyrazolo[1,5‐a]pyrimidine ligand incorporated. The

results concerning MIC values of the compounds and

standard drug such as gatifloxacin (GFLH), ciprofloxacin

(CFLH), levofloxacin (LFLH), norfloxacin (NFLH),

ofloxacin (OFLH), pefloxacin (PFLH) and sparfloxacin

(SFLH) are represented in Figure 4 and supplementary

material 7. Complex (6d) exhibits the excellent potency

amongst all the complexes due to the presence of the

highly electro withdrawing power of the nitro (‐NO₂)

substituted on phenyl ring, its enhance the biological

study.^[46]Additionally, the heights activity of nitro

substituent with complexes are high lipophilic in nature,

that the complexes penetration through cell membrane

due to their increase in lipophilicity. Antibacterial

activities include inhibition of cell wall synthesis,

functions of cellular membrane, protein synthesis,

nucleic acid synthesis and folic acid synthesis in step-

wise manner. MIC values of complexes are increasing

due to chelation. This effect can be explained by

FIGURE 4 Effect of different concentrations of ligands, Pt^IIcomplexes and standard drugs (Gatifloxacin (GFLH), ciprofloxacin (CFLH),

levofloxacin (LFLH), Norfloxacin (NFLH), Ofloxacin (OFLH), Pefloxacin (PFLH) and Sparfloxacin (SFLH) on five different pathogens.

Error bars represent the standard deviation of three repeats

14 of 25

LUNAGARIYA ET AL.

chelation that make possible the ability of a complex to

cross a cell membrane, which can be also explained by

Tweedy's chelation theory.^[47–49]Therefore, we favour

that the toxicity of the complexes can be associated to

the strengths of the metal‐ligands bond, and

furthermore, other factors such as size of the cation,

and receptor sites.

amongst all the compounds due to presence of electron

withdrawing substituents (‐NO₂, ‐F, Cl). Overall

vconclusion is that as concentration of compounds

increases cellular level cytotoxicity also increases. The

percentage viability of compounds are represented in

Figure 6 and cytotoxicity data of the compounds are

shows in supplementary material 8.

3.7 | Cellular level bioassay using S. pombe

cells

3.8 | Effect of platinum(II) complexes (6a‐

6f) on the degradation of DNA of S. pombe

cells

Schizosaccharomyces pombe cells were used to study toxic

effect of compounds in the biological metabolism.^[46,50]A

relative study of cytotoxicity effect of heterocyclic

compounds and their metal complexes indicate that the

metal complexes exhibit better cytotoxicity compare to

The results of in vivo cytotoxicity against eukaryotic cell

stimulated us to discover whether the complexes have

any influence on the degradation of DNA or not. The con-

clusion of the synthesized complexes (6a‐6f), the DNA

degradation of S. pombe cells was approved by the separa-

tion of DNA from treated and untreated S. pombe cells

and also DNA degrade of the S. Pombe cells is visualised

by electrophoresis on 1% agarose gel. DNA destroyed

establish as smear in platinum(II) complexes treated with

S. pombe cells, while in untreated S. pombe cells, entire

band have no smearing. The results of complexes (6a‐6f)

of the degradation of S. Pombe cells DNA represented in

Figure 7. Smearing of S. Pombe cell of DNA concluded

that the damage has followed due to lethal nature of the

compounds. The cellular level cytotoxicity result is in

agreement with that platinum(II) complexes have entered

into the S. Pombe cell and affected the degradation of

DNA. Cisplatin, transplatin and platinum(II) complexes

are more cytotoxic potency as compared to pyrazolo[1,5‐

a] pyrimidine derivatives ligands.

corresponding

pyrazolo[1,5‐a]pyrimidine

ligand.

Sensitive and selective cells can interact with the

compounds and can be easily monitored by trypan blue

dye. Trypan blue dye did not penetrate to living S.pombe

cells because of its cell membrane cannot be destroyed,

while it can penetrate to dead cell of S. pombe as a result

blue colour under microscope is observed (Figure 5). The

toxicity is found to be differ with the type of substituent

present in the synthesized compounds. After 17‐20 h of

the treatment, many of the S. pombe cells are destroyed

due to the toxic nature of the compound.^[47]Cytotoxicity

activity of all complexes are higher than their respective

free ligands and are comparable with cisplatin and

transplatin.^[51]Complexes (6d, 6b, 6c) are the most active

3.9 | In vitro cytotoxicity bioassay

Synthesized compounds were screen for their cytotoxicity

using the protocol of Meyer et al. ^[50,52]This cytotoxic pro-

cess is inexpensive, less time consuming and consistent.

LC₅₀value is calculated from the plot of the log concen-

tration of compounds vs LC₅₀values. Antilog of the

obtained value at 50% fatality gives the value of LC₅₀.

From the graph, the LC₅₀values of the synthesized com-

pounds are calculated and they are found in the range of

6.450 ‐ 102.07 μg/ml. Complex (6b) is more potent com-

pare to other complexes and corresponding ligands

(Figure 8). The order of potency of compounds is

cis‐platin > 6d > 6b > 6c > 6a > 6e > 6f > trans‐platin

> pyrazolo[1,5‐a] pyrimidine derivatives ligands.^[46]Par-

ticularly, the complexes 6b, 6d and 6c exhibit high

potency due to their high electro–withdrawing substitu-

tion and less sterically hindered on the ligand. The LC₅₀

FIGURE 5 Effect of compounds on S. pombe cells. Dead cells are

blue colour, whereas live cells are transparent

LUNAGARIYA ET AL.

15 of 25

FIGURE 6 The percentage viability of compounds at different concentrations with error uncertainty in the value 5%

FIGURE 7 Lane 1‐5 represent the DNA damage of the S. pombe cells by the treatment of platinum(II) complexes (6a‐6f) in the range of (20

– 100 μM) while lane untreated represents S. pombe cells as the control and another lane DMSO solvent + DNA + S. Pombe cells to study

genotoxicity

value of all synthesized compounds are comparable to

standard anticancer agent cis‐platin and transplatin.

heterocyclic ligands. The compounds which showed

higher inhibition against M. tuberculosis H₃₇Rv, are fur-

ther study for their MICs.

3.10 | Mycobacterium tuberculosis (Mtb)

H₃₇Rv strain

3.11 | In vitro antiproliferative cytotoxicity

on human colorectal carcinoma HCT‐116

cells

In vitro antituberculosis activity of the synthesized Pt^II

complex (6d) have been screened against Mycobacterium

tuberculosis H₃₇Rv strain. The complex (6d) possess excel-

lent activity (12.5 μg/ml) against M. tuberculosis H₃₇Rv

strain due to presence of nitro substituent on fused

The positive results observed in DNA binding and DNA

cleavage studies have directed us to examine the cell

growth inhibition of the platinum(II) complexes. To

16 of 25

LUNAGARIYA ET AL.

growth (%) is calculated. The IC₅₀(i.e., the concentration

producing 50% inhibition of growth) is determined and

expressed in μg/ml concentration (Figure 9). The IC₅₀

values of the platinum(II) complexes (6b, 6c and 6d)

exhibit a certain inhibitory effect on HCT‐116 cancer cell

line, while platinum(II) complexes (6a, 6e and 6f) are

found to be practically ineffective. Pt^IIcomplexes (6b ‐F,

6c ‐Cl and 6d ‐NO₂) containing electron withdrawing

functional group are greater in vitro cytotoxicity than the

complexes (6a –OCH₃, 6e –CH₃and 6f ‐H) containing

electron releasing group. Furthermore, the IC₅₀values

of the Pt^IIcomplexes (6b, 6c and 6d) are comparable with

standard drug such as cisplatin (51.64 μM)^[53]

,

FIGURE 8 Plot of LC₅₀values of different compounds in μg L⁻¹

using brine shrimp to determine the cytotoxic effects. Error bars

represent the standard deviation of three replicates 5%

carboplatin (273.05 μM) and oxaliplatin (57.04 μM).^[27]

IC₅₀value of the complexes 6b, 6c and 6d are compara-

ble to the standard drugs cisplatin and oxaliplatin. Com-

plexes (6d –NO₂and 6b –F) are the most cytotoxic

against HCT‐116 cancer cell line as compared to other

complexes. To found the effect of DMSO on the cells’

viability, control solutions with the respective DMSO

content are also screened and the cytotoxic effect of

DMSO is very low. The IC₅₀value of the Pt^IIcomplexes

are found in increasing order of 6d (182.93 μM) > 6b

(320.36 μM) > 6c (325.52 μM) > 6a (344.73 μM) > 6e

(674.15 μM) > 6f (> 1000 μM) > DMSO.

investigate the potential of the complexes as antitumor

agents, In vitro antiproliferative assay of the synthesized

platinum(II) complexes (6a‐6f) against Human Colorectal

Carcinoma HCT‐116 cell line were evaluated by MTT

assay. The concentration of platinum(II) complexes are

taken in the range of 10‐1000 μg/ml. The viability of cells

in the presence of the tested compounds is compared to

that observed in control cultures and the inhibition of

FIGURE 9 Effect of synthesized Pt^IIcomplexes conjugate on cell proliferation. Determination of IC₅₀values of Pt^IIcomplexes (6a–6f)

conjugate in HCT 116 cells. HCT 116 cells were treated with the various concentrations control (100% DMSO), vehicle control (0.5%

DMSO), 10, 50, 100, 250, 500, 1000 μg/mL) of Pt^IIcomplexes (6a–6f) conjugate for 24 h. MTT assay determined inhibition of HCT 116 cells.

The obtained values were plotted and the IC₅₀values were analysed the data with t‐test using Sigmaplot 12.0. Note: Error bars represent

standard deviation of three replicates

LUNAGARIYA ET AL.

17 of 25

cytotoxicity as compared to other complexes (6e, 6a,

6f).^[39]It is observed that presence of electron withdraw-

ing substituents (‐F, ‐Cl, ‐NO₂) in complexes (6b, 6c, 6d)

increased antimicrobial activity. The pyrazolo[1,5‐a]

pyrimidine nucleus with nitro substituent at para‐posi-

tion on phenyl moiety at C‐8 position, exhibits moderate

antituberculosis activity against Mycobacterium tuberculo-

sis H₃₇Rv strain as compared to isoniazid and rifampicin

drugs. The existence of electron releasing group (‐OCH₃,

‐CH₃and ‐H) at para‐position in phenyl moiety decreased

antibacterial potency and also decrease toxicity against S.

pombe cells and brine shrimp lethality bioassay.

3.12 | Structure–activity relationship

(SAR)

The biological activity is significantly affected by intro-

ducing six‐different (‐OCH₃, ‐F, ‐Cl, ‐NO₂, ‐CH₃, ‐H)

substituted phenyl ring at C‐8 position, thiophene ring

at C‐3 position and pyridine ring at C‐6 position on

pyrazolo[1,5‐a]pyrimidine scaffold (Figure 10). It is

observed that the highly electron withdrawing nitro sub-

stituent in phenyl moiety at C‐8 position in pyrazolo[1,5‐

a]pyrimidine core (compound 6d) exhibit higher antitu-

berculosis assay against Mycobacterium tuberculosis

H₃₇Rv strain as compared other compounds (6a, 6c, 6b,

6e, 6f) and also enhanced antibacterial activity against

Gram^(‐ve)bacterial species. The replacement of electron

donating substituents with methoxy group (6a), methyl

group (6e) and hydrogen group (6f) at para‐position on

phenyl moiety decreases the antibacterial activity against

P. aeruginosa and E.coli, while the electron withdrawing

chloro group (6c) and nitro (6d) and fluoro (6b) group

exhibit highest activity against S. marcescens and B.

subtilis, respectively. The complexes (6b, 6c, 6d) show

excellent cytotoxicity against S. pombe cells, in vitro brine

shrimp cytotoxicity and HCT‐116 cancer cell line

3.13 | DNA interactions studies

3.13.1 | By electronic absorption titration

Quantitative binding strength of the complexes have

been carried out by electronic absorption titration

(Figure 11). Electron rich (Lewis bases) biomolecule

compound is bound to the electron deficient Pt^IIcom-

plexes. Electronic absorption titration technique gives

good evidence of the complex‐DNA interaction and also

measured for binding mode and binding strength of the

FIGURE 10 Structure–activity relationships for inhibitory concentration (IC₅₀) and lethal concentration (LC₅₀) values of the synthesized

compounds

18 of 25

LUNAGARIYA ET AL.

FIGURE 11 Absorption spectral changes upon addition of HS DNA to the solution of Pt^IIcomplex (6d) after incubating it for 10 minutes at

five different temperature (a) at 298 K (b) at 301 K (c) at 303 K (d) at 308 K (e) at 313 K in phosphate buffer (Na₂HPO₄/NaH₂PO₄, pH = 7.2).

Inset: Plot of [DNA]/ (εa‐εf) vs. [DNA]

complex‐DNA adduct. The overall binding constants,

(K_b) of the complexes to HS DNA, are quantitatively

determined by observing the changes in the absorption

intensity of the spectral bands by successive addition of

HS DNA at different temperatures as represented in

Figure 10. There are mainly two binding modes for the

drug or complex‐DNA interaction. (I) covalently binding

(ii) non‐covalently binding mode. In covalent binding

mode, complexes that undergoes fast substitution reaction

take place that means the labile group of Pt^IIcompound is

replaced by a N7 position of the sugar. The Pt^IIcomplexes,

along with most drugs tend to interact with DNA non‐

covalently through three selective modes: intercalative,

electrostatic (binding to the phosphate group) and groove

binding.^[54]The interaction affinity of the complex‐DNA

led to a decrease in hypochromism with a slight

bathochromic shift at 2‐4 nm.^[55,56]This spectral study

suggests stacking interaction between the aromatic conju-

gated cyclic compound and DNA base pairs.^[57]

DNA binding ability. It is lower than the K_bvalue of the clas-

sical intercalator (EB) ethidium bromide (7.16 × 10⁵M⁻¹

)

and have higher binding affinity compared to the cisplatin

(5.73 × 10⁴M^‐1),^[58]oxaliplatin (5.3 × 10³M^‐1)^[59]and

carboplatin (0.33 × 10³M^‐1).^[60]The K_bvalues are found in

the order of 6d > 6b > 6c > 6a > 6e > 6 f. The decrease

absorption intensity and thoroughly increase wavelength

suggest that the complex‐DNA interaction via partial

intercalative mode of binding. Complex (6d) shows the

highest binding affinity as compared to the other complexes

and ligands due to the existence of a nitro group.

To get additional information of complex‐DNA inter-

actions, the thermal parameters like the change in

enthalpy (ΔH°), change in free energy (ΔG°), and change

in entropy (ΔS°) changes are calculated by using the Van't

Hoff equation 4 and 5.^[61]The K_band thermal parameters

are represented in Table 1.

ΔH^°ΔS^°

RT

¼ −

þ

R

The calculated binding constant (K_b) of the Pt^IIcom-

plexes with DNA are found in the range of 0.23‐6.74 ×

10⁵M^‐1indicating that the complexes show an effective

ln Kb

(4)

(5)

ΔG^°¼ ΔH0−TΔS^°

LUNAGARIYA ET AL.

19 of 25

TABLE 1 The binding constant (K_b) and thermodynamic parameters (ΔH⁰, ΔS⁰ and ΔG⁰) for the interaction of Pt^IIcomplexes with DNA at

five different temperatures

λ_max(nm)

Δλ

(nm)

red

shift

K_b×

10⁵

ΔH⁰

(kJ

ΔS⁰ (J

T

(K)

(M^‐

Hyper.

(%)

mol^‐

ΔG⁰ (J

1

Compounds

6a

Bound free

)

K^‐1)

mol^‐1)

308 305

265 264

308 306

310 306

310 312

3

1

2

4

2

298

301

303

308

313

3.82

1.64

1.06

0.51

0.47

21.56

19.12

23.63

18.02

27.83

‐106.7

‐158.8

‐73.15

‐141.4

‐89.20

‐106.4

‐254.059

‐422.784

‐136.998

‐360.645

‐192.161

‐250.401

‐31,033.80

‐30,271.62

‐29,763.50

‐28,493.21

‐27,222.91

6b

6c

6d

6e

6f

299 297

284 280

308 306

299 298

306 302

2

4

2

1

4

298

301

303

308

313

4.11

3.67

3.19

0.49

0.28

39.27

19.78

37.46

21.95

32.91

‐32,882.73

‐31,614.38

‐30,768.82

‐28,654.90

‐26,540.98

265 264

269 266

306 305

266 265

270 268

1

3

1

2

298

301

303

308

313

5.23

3.35

2.33

1.91

1.14

18.34

19.30

20.98

17.59

18.34

‐32,323.63

‐31,912.64

‐31,638.64

‐30,953.65

‐30,268.66

266 265

263 261

303 299

297 296

286 275

1

2

4

1

9

298

301

303

308

313

6.74

6.29

2.93

2.58

0.41

17.78

20.12

19.35

18.34

42.55

‐33,949.02

‐32,867.09

‐32,145.80

‐30,342.57

‐28,539.35

310 306

308 306

313 308

307 304

332 327

4

2

5

3

5

298

301

303

308

313

4.79

2.45

2.12

0.93

0.87

21.87

23.23

17.33

20.46

19.40

‐31,936.78

‐31,360.30

‐30,975.98

‐30,015.17

‐29,054.36

265 264

300 298

306 305

307 301

295 293

1

2

1

5

2

298

301

303

308

313

3.86

2.27

2.19

0.76

0.53

16.70

17.29

18.12

18.39

20.68

‐31,832.94

‐31,081.74

‐30,580.94

‐29,328.93

‐28,076.93

Hyper.(%) = [(A_free‐ A_bound)/A_free] × 100%,

K_b= Intrinsic DNA binding constant determined from the UV‐vis absorption spectral titration,

T = Temperature in kelvin (K),

ΔH = Change in enthalpy,

ΔS = Change in entropy,

ΔG = Gibb's free energy,

Δλ = Difference between bound wavelength and free wavelength.

where, K_bis at the corresponding temperature (T), and

value of gas constant (R) is 8.314 Jmol^‐1K^‐1. The Van't

Hoff plots for the interaction are shown in Figure 12.

The negative values of change in Gibb's free energy

(ΔG°) indicate a spontaneous interaction of the complex‐

DNA, while the positive values of the change in enthalpy

(ΔH°) and change in entropy (ΔS°) indicate that the bind-

ing between the complex‐DNA is mainly hydrophobic in

nature.^[39,62]Furthermore, the K_bdecrease with the

increasing temperature of the complexes. The K_bvalues

are calculated at different temperature, such as 298, 301,

303, 308 and 313 K. The binding mode of the complexes

are further conformed by viscosity measurements and

fluorescence quenching analysis.

3.14 | Viscosity measurement

Pt^IIcomplexes are coordinated with N7 position of base

pairs (A = T and G≡C) on DNA through intercalation,

partial intercalation, electrostatic interaction and groove

20 of 25

LUNAGARIYA ET AL.

3.15 | Molecular docking with DNA

Molecular docking study played important roles in the

molecular recognition of nucleoside and nucleotide in

the rational design of new chemotherapeutic drugs.

Docking study of complex‐DNA performed in order to

predict the binding site along with desired orientation of

the molecules inside the DNA.^[34]Docking structure of

the complex (6d) is illustrate in Figure 14 and other

docking structure of Pt^IIcomplexes and ligands are illus-

trate in supplementary material 9. The binding energies

of docking structure of ligands (5a‐5f) and complexes

(6a‐6f) are found to be ‐257.45 (5a), ‐271.89 (5b), ‐275.50

(5c), ‐265.96 (5d), ‐256.06 (5e), ‐268.07 (5f), ‐287.88(6a),

‐275.07 (6b), ‐238.75 (6c), ‐280.51 (6d), ‐291.06 (6e), ‐

275.12 (6f) kJ mol⁻¹, respectively. The DNA binding

energy of the compounds is negative, which with an

increase the binding affinity of complex‐DNA. The molec-

ular docking study is also confirmed by electronic absorp-

tion titration, fluorescence quenching analysis and

viscosity measurements studies that the complex‐DNA

interaction take place through the partial intercalative

mode of DNA binding.

FIGURE 12 The Van't Hoff plot of Pt^IIcomplex (6d) at different

temperatures

binding. The relative viscosity curve of complexes are

represented in Figure 13. The plot of relative viscosity is

a (η/η⁰)^1/3vs the ratio of the decrease concentration of

the complex‐DNA solutions. Increasing concentrations

of complexes (6a‐6f), relative viscosity of the HS‐DNA

solutions steadily increases, suggests the intercalative

binding mode. Complex 6d shows increase in relative vis-

cosity due to presence of electron withdrawing (‐NO₂)

functionality. Increase in viscosity values shows affinity

of compounds to bind with DNA, which follows the order

6d > 6b > 6c > 6a > 6e > 6 f. Results conclude that com-

plexes show partial intercalation binding with DNA base

pairs, which is consistent with our interpretation based

on binding constants.

3.16 | Competitive displacement assay

For confirmation of partial intercalative mechanism, fluo-

rescence quenching study was performed at ambient tem-

perature. The complexes are not fluorescent in addition of

the HS‐DNA. Therefore, binding assay of complex‐DNA

cannot carried out directly through emission spectra.

The planar phenanthridine ring of EB can intercalate

between adjacent DNA base pairs. This intercalative

mechanism suggests the intense fluorescence emission

band at 610 nm and excitation wavelength at 480 nm

due to fluorescence quenching and EB‐DNA interaction,

respectively. Within this context, the compound which

can intercalate to DNA equally or more strongly than

EB (typical intercalator), is added into a solution of the

EB–DNA compound.^[63]The addition of complexes (6a‐

6f) at diverse r‐ values resulted in a significant gradually

decrease of the intensity of the emission band of the

DNA–EB system at 610 nm indicating the competition

of the Pt^IIcomplexes with EB in binding to DNA. The

observed quenching of DNA–EB fluorescence by the com-

pounds proposes that the complexes can significantly dis-

place to ethidium bromide (EB) from the DNA–EB

system, thus the illuminating the partial interaction with

HS‐DNA by the intercalative mode.^[64,65]The linear

Stern–Volmer plots of DNA–EB for the complexes show

that the quenching of EB–DNA by the complexes is in bet-

ter agreement (r = 3.33) with the linear Stern–Volmer

quenching equation proving that the addition of EB from

FIGURE 13 The effect of increasing amounts of complexes on

the relative viscosity of HS‐DNA at 27 ( 0.1) ⁰C in phosphate

buffer at pH = 7.2. Error bars represent the standard deviation of

three replicates

LUNAGARIYA ET AL.

21 of 25

EB–DNA by each compound result in a decrease in the

fluorescence intensity.^[66,67]This is related to binding con-

stant (K_a), linear Stern‐Volmer quenching constants (K_sq),

standard change in Gibb's free energy (ΔG⁰) and number

of compound binds with DNA (binding site, n) are repre-

sented in Table 2. The K_avalues calculated from

Scatchard plots indicating that complex (6c) in

Figure 15, the binding constant (K_a) and linear Stern‐

Volmer quenching constants (K_sq) values of Pt^IIcom-

plexes are found in range of 1.20 × 10³– 4.20 × 10⁵and

3.3 × 10²– 6.0 × 10³, respectively. The results of fluores-

cence quenching data and graphs of complexes (6a‐6f)

are represented in supplementary material 10. The values

of n is observed around 1.0 indicates the 1: 1 molar ratio

between the complexes and HS‐DNA.

3.17 | Gel electrophoresis

Pt^IIcomplexes are in good correlation with the DNA

cleavage because some of the anticancer agents approved

for clinical use cause cell death by damaging DNA.^[9,68]

Cleavage activity on plasmid pUC19 DNA induced by

synthesized Pt^IIcomplexes can be observed using DNA

electrophoresis. The DNA cleavage affinity of the

complexes could follow by two pathway that effectively

cleave of DNA molecule either with hydrolytic or oxida-

tive pathways due to the importance of DNA nuclease in

biological applications. (i) Breaking of phosphodiester

moiety to further sub units can be illustrated by hydrolytic

DNA cleavage study. In hydrolytic cleavage, the ability of

complexes to bring about supercoil (SC) form of DNA to

the open‐circular (OC).^[69,70](ii) Abstracting proton of

sugar (adenosine, guanine, thymine, cytosine) might lead

to oxidation DNA nuclease.^[71]There has been extensive

interest in DNA nuclease process that are activated by

metal complexes. Gel electrophoresis is an appropriate

technique to evaluate the cleavage of DNA by metal based

drugs, to determine the factors affecting the nucleolytic

TABLE 2 Linear stern‐Volmer quenching constants (K_sq), num-

ber of binding sites (n), change in standard free energy (ΔG⁰) and

associative binding constant (K_a) of Pt^IIcomplexes (6a‐6f) with HS‐

DNA

Complexes

K_sq(M^‐1)

4.6 × 10³

K_a(M^‐1)

1.05 × 10⁵

n

ΔG(J mol^‐1)

‐28524.10

6a

1.290

6b

6c

6d

6e

6f

3.9 × 10³

3.3 × 10²

6.0 × 10³

3.6 × 10³

1.3 × 10³

1.07 × 10⁵

3.71 × 10⁴

1.47 × 10⁵

0.91 × 10⁴

1.40 × 10³

1.295

1.011

1.32

‐28,540.65

‐26,071.03

‐29,490.49

‐22,605.68

‐18,261.11

FIGURE 14 Molecular docking of ligand (5d) and complex (6d)

with the DNA duplex (VDW spheres) of sequence

d(ACCGACGTCGGT)₂. The complex and ligand are docked into the

DNA base pairs (1 BNA) using hex 8.0 software

1.11

1.02

22 of 25

LUNAGARIYA ET AL.

FIGURE 15 Fluorescence emission spectra of EB binds to HS‐DNA in the presence of complex (6c). [EB] = 33.3 μM, [DNA] = 10 μM;

[complex] = (i) 3.33, (ii) 6.66, (iii) 10, (iv) 13.33, (v) 16.66, (vi) 20, (vii) 23.33, (viii) 26.66, (iX) 30, (X) 3.33 μM; λ_ex= 480 nm. The arrows

show the intensity changes upon increasing the concentrations of complex. Inset graph: Plots of I₀/I versus [Q], with • for the experimental

data points and the full line for the linear fitting of the data. Comparative plot of log[I₀‐I/I] versus log[complex] for the titration of HS‐DNA EB

system with complexes (6c) in (Na₂HPO₄/NaH₂PO₄, pH = 7.2) phosphate buffer medium

efficiency of a compound and to compare the nucleolytic

properties of different compounds.^[72]

Results of % cleavage ability of the complexes are

represented in Figure 16. And data of % cleavage of Pt^II

complexes are illustrate in supplementary material 11.

(Figure 17) is due to the change in binding affinity of the

Pt^IIcomplexes to DNA. The similar behavior of Pt^IIcom-

plexes with plasmid DNA is shown by reported

compounds of the types [Pt(barb‐κN)₂(bpy‐κN,N′)],^[73]

[Pt(2,2′‐bipyridyl‐5,5′‐dicarboxylic

acid)Cl₂·2H₂O],^[74]

[Pt(Fc‐bpa)Cl]Cl.^[75]The DNA cleavage efficiency of the

complexes is in order of cisplatin > 6d > 6b > 6c > 6a >

FIGURE 16 Photogenic view of the cleavage of pUC19 DNA

(300 μg cm⁻³) with a series of compounds using 1% agarose gel

containing 0.5 μg cm⁻³EtBr. All reactions were incubated in TE

buffer (pH 8) at a final volume of 15 mm³for 3 h at 37 °C. Lane 1.

DNA control, lane 2. K₂PtCl₄, lane 3. Cisplatin, lane 4. Transplatin,

lane 5. (6a), lane 6. (6b), lane 7. (6c), lane 8. (6d), lane 9. (6e), lane

10. (6f)

FIGURE 17 Plot of DNA cleavage data by agarose gel

electrophoresis of different compounds using pUC19 DNA. Error

bars represent the standard deviation of three replicates 5%

LUNAGARIYA ET AL.

23 of 25

6e > 6f > transplatin. The different DNA‐cleavage

efficiency of the complexes, metal salt, transplatin and

cisplatin is due to the change in binding affinity of the

complexes to DNA and the structural dissimilarities of

ligands.

gratefully acknowledge the University Grants Commis-

sion, New Delhi, India for meritorious fellowships

awarded to them during year 2014‐2018 and ‘BSR UGC

One Time Grant’, vide UGC letter no. F.19‐119/

2014(BSR).

DECLARATION OF INTEREST

4 | CONCLUSION

The authors report no conflicts of interest. The authors

alone are responsible for the content and writing of the

paper.

In this article, a series of new pyrazolo[1,5‐a] pyrimidine

nuclease based ligands (5a ‐5f) and their Pt^IIcomplexes

(6a – 6f) were synthesized and characterized. And also

we report the biological study of homoleptic Pt^II

complexes (6a‐6f). The complexes 6b, 6c and 6d displayed

greater antibacterial activity, brine shrimp cytotoxicity

and cellular level cytotoxicity against S. pombe cells

compared to the complexes 6a, 6e, 6 f. Nitro substituted

compounds 6d may be recognized as the most biologically

active compound showing an interesting dual antituber-

culosis and antibacterial profile. All Pt^IIcomplexes exhibit

good antibacterial activity with low MIC values against S.

aureus, B.substilus and S. marcescens. DNA binding of all

Pt^IIcomplexes observed via partial intercalative mode that

confirmed by bathochromism and hypochromism of band

in UV‐vis absorption titration spectroscopy, increase in

relative viscosity of DNA and partially decrease in fluores-

cence emission intensity. Complex (6d) containing nitro

group is binds more strongly than the other complexes.

Presence of nitro group act as not only chemical isosteres

for the oxygen atoms in the heterocyclic base of thymi-

dine, but also participate in ‘strong’ O–H bonding as a

result it shows greater DNA binding study and antimicro-

bial activity, than other complexes. All the complexes and

cisplatin drug show strong in vitro cytotoxic, as well as

cellular level bioassay than transplatin. Pt^IIcomplexes

(6b, 6c and 6d) exhibited significant cytotoxicity against

HTC‐116 cancer cells and comparable to clinically used

metallodrugs such as cisplatin, carboplatin and

oxaliplatin. Presence of electron withdrawing group in

complex 6d, 6b and 6c, improves its biological property.

The preliminary studies encourage for carrying out

further in vivo enzymatic behaviours experiments.

ORCID

Mohan N. Patel http://orcid.org/0000-0001-9016-9245

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How to cite this article: Lunagariya MV, Thakor

KP, Waghela BN, Pathak C, Patel MN. Design,

synthesis, pharmacological evaluation and DNA

interaction studies of binuclear Pt(II) complexes

with pyrazolo[1,5‐a]pyrimidine scaffold. Appl

Organometal Chem. 2018;e4222. https://doi.org/

10.1002/aoc.4222

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Buyukgungor, Dalton Trans. 2015, 44, 6880.

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Design, synthesis, pharmacological evaluation and DNA interaction studies of binuclear Pt(II) complexes with pyrazolo[1,5-a]pyrimidine scaffold

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