Half-sandwich iridiumIII complexes with pyrazole-substituted heterocyclic frameworks and their biological applications

Article abstract of DOI:10.1039/c6nj02153k

Low-spin Ir^III organometallic half-sandwich complexes of type [(η⁵-C₅Me₅)Ir(XY)Cl]⁺ (XY = bipyrazoles (4a-4b)/pyrimidin-2-amines (5a-5b)/triazolo[1,5-a]pyrimidines (6a-6b)) have been synthesized and characterized. All the newly synthesized compounds have been evaluated for their DNA binding properties with calf thymus (CT DNA), which revealed enhancement in the binding constant (K_b) of the complexes. The compounds bearing an imidazole substituent proved to be better binders than compounds containing a phenoxy linkage. Molecular docking attests that π-π stacking interactions have been observed between the receptor and the compounds. Furthermore, the observed DNA cleavage potency has been ascribed to a multitarget mechanism of action of these compounds. Intriguingly, the chelation of ligands with Ir^III led to a remarkable enhancement of antibacterial activity against the arbitrarily selected two Gram +ve and three Gram -ve bacterial strains. The complexes of triazolo[1,5-a]pyrimidines proved to be the most cytotoxic compounds towards brine shrimp and S. pombe cells compared to pyrazole-containing heterocyclic frameworks. All complexes showed potent cytotoxicity as compared to the ligands, with IC₅₀ values ranging from 78 to 234 μM toward A549 human lung cancer cells. The potency of the compounds toward these cancer cells was in the order pyrimidin-2-amines > bipyrazoles > triazolopyrimidines.

Full text of DOI:10.1039/c6nj02153k

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Mehta, P. Thakor, V. R. Thakkar, P. Chudasama, J. Patel and M. N. Patel, New J. Chem., 2016, DOI:
1
0.1039/C6NJ02153K.
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DOI: 10.1039/C6NJ02153K
Journal Name
ARTICLE
III
Half-sandwich iridium complexes with pyrazole substituted
heterocyclic frameworks and its biological applications
a
a
b
b
Sanjay B. Gajera, Jugal V. Mehta, Parth Thakor, Vasudev R. Thakkar, Piyushkumar C.
Received 00th January
c
c
a
2
0xx,
Chudasama, Jagdish S. Patel, Mohan N. Patel*
Accepted 00th January
0xx
The low-spin IrIII organometallic half-sandwich complexes of type [(η -C Me )Ir(XY)Cl] , (XY=
5
+
2
5
5
bipyrazoles (4a-4b)/pyrimidin-2-amines (5a-5b)/triazolo[1,5-a]pyrimidines (6a-6b)) motifs have been
synthesized and characterized. All the newly synthesized compounds have been evaluated for DNA
DOI: 10.1039/x0xx00000x
www.rsc.org/
binding properties with calf thymus (CT DNA) revealed enhancement in the binding constant (K ) of
b
complexes. The compounds bearing imidazole substituent are proving a good binder than that of
compounds containing phenoxy linkage. Molecular docking attests 휋-휋 stacking interactions have
been observed between receptor and compounds. Furthermore, the observed DNA cleavage potency
has been ascribed to a multitarget mechanism of action of these compounds. Intriguingly, the
III
chelation of ligands with Ir led to a remarkable enhancement of antibacterial activity against
arbitrarily selected two Gram +ve and three Gram –ve bacterial stains. The complexes of triazolo[1,5-
a]pyrimidines are proved the most cytotoxic compounds and on S. pombe cells compared to pyrazole
incorporated heterocyclic frameworks. All complexes showed potent cytotoxicity as compared to
ligands, with IC50 values ranging from 78 to 234 μM toward A549 human lung cancer cells. Potency of
the compounds toward these cancer cells increased with pyrimidin-2-amines > bipyrazole >
triazolopyrimidine.
8
interaction . The clinical success of cis-platin, carboplatin, and
INTRODUCTION
9, 10
oxaliplatin
have stimulated the search for other transition
The studies of metal chelates, which bind to DNA strand as
reactive models for protein-nucleic acid interaction, provide
metal complexes that possess anticancer activity. New metal-
based anticancer drugs may be able to widen the spectrum of
treatable cancers, reduce toxic side effects, and overcome
platinum resistance. Interest in bio-inorganic chemistry and the
design of complexes as anticancer agents is currently
routes toward rational drug design as well as means to develop
sensitive probes for DNA structure and to get information about
drug design and tools of molecular biology1 . Binding of metal
complexes with DNA has been studied extensively because DNA
is the material of inherence and controls the structure and
, 2
11-14
increasing
. The cyclopentadienyl ligands can provide
hydrophobicity of the faces of the coordination compound
which influences cell uptake and targeting)
metallodrugs are prodrugs, and control over ligand substitution
is vital if the complex is to reach and react with its target site. In
this respect, octahedral low-spin d complexes are attractive for
drug design since they are often kinetically inert. Inertness
increases from the first to second to third row of transition
metals . The lifetime for exchange of an aqua ligand on
3
function of cells . Metal complexes can bind to DNA in a
15-17
(
. Most
noncovalent interaction mode, such as groove binding for large
ligands, electrostatic binding for cations , intercalative mode of
binding for planar ligands, and partial intercalative binding for
incompletely planar ligands . According to the recent reports,
cis-platin is one of the most widely used metal-based antitumor
4
6
5
, 6
7
and anticancer drugs targeting DNA through covalent bonding
18
3+
19, 20
[
Ir(H
O)
2 6
] , is about 300 years
. There are only a limited
number of reported studies on the biological activity of iridium
I
III
a.Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar-388 120,
complexes. Early studies were concerned with Ir and Ir
Gujarat, India.
complexes
21, 22
and more recently, a few studies of
b.
B. R. Doshi School of Bioscience, Sardar Patel University, Vallabh Vidyanagar-
III
23-25
III
organometallic Ir complexes have been reported
. Iridium
3
88120, Gujarat, India
c.
Department of Biological Sciences, P. D. Patel Institute of Applied Sciences,
CHARUSAT, Changa
complexes are generally thought to be too inert to possess high
reactivity. Indeed, the inertness of Ir has allowed the design of
complexes that function as rigid scaffolds and inhibit kinase
III
Electronic Supplementary Information (ESI) available: [Figures S1−S24 contain the,
1
13
H NMR and C NMR (APT) spectrum, S25 is mass spectrum, S26-S33 contains
molecular docking images. Table S34 contain DNA cleavage data, Table S35 contain
anticancer data, Table 36 MIC data and Table 37 contains cytotoxicity data]. See
DOI: 10.1039/x0xx00000x
26
enzymes .
The
biological
activity of
trans-
27
[
IrCl
4
(DMSO)(Im)][ImH] and trans-[IrCl
4
(Im) ][ImH] (ImH =
2
This journal is © The Royal Society of Chemistry 20xx
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ARTICLE
Journal Name
2
8
III
III
13
imidazole) , Ir analogues of the Ru anticancer drugs NAMI-A (7.26 ppm) for chloroform-d
1
.
3
C NMR chemical shifts were
View Article Online
DOI: 10.1039/C6NJ02153K
and the imidazole analogue of the indazole complex KP1019, internally referenced to CDCl
respectively, have been attributed to the kinetic inertness of Ir . and (CD
(77.16 ppm) for chloroform-d
SO (39.52 ppm) for DMSO-d
1
III
3
)
2
6
.
The negatively charged pentamethylcyclopentadienyl (Cp*) is Mass spectrometry
III
an excellent stabilizing ligand for Ir . In the work reported here, Electrospray ionization mass spectra were obtained by infusing
II
II
we apply the designed concepts discovered for Ru and Os
into the mass spectrometer (LC-MS Spectrometer Model Q-ToF
Micro Waters). The mass spectra were recorded with a scan
III
5
+
arene complexes to Ir Cp* complexes [(η -Cp*)Ir(XY)Cl]
containing N,N-bound
bipyrazole/pyrimidin-2- range of m/z 50-1000 for positive ions.
amine/triazolopyrimidine as chelating ligands. Only a few Elemental analysis
iridium complexes containing functionalized Cp* ligands have All biologically evaluated compounds synthesized within this
been reported previously. We have studied the effect of report have been analysed using elemental (CHN) analysis by
functionalization of ligand interaction with DNA, molecular means of combustion. This technique requires the sample to be
docking, anticancer activity on A549, antibacterial and burned in an excess of oxygen and has a variety of traps which
cytotoxicity on S. Pombe cells and brine shrimp lethality assay. collect the combustion products: CO , H O, and NO. These
2
2
Such complexes can be thermodynamically stable and yet masses are then used to help calculate the masses of the
kinetically labile toward substitution reactions and that unknown product. The experimental values are compared with
substituents on chelating ligand can have a dramatic effect on the calculated values of the sample. Thermo finnigan elemental
chemical and biological activities.
analyzer was used to analyse carbon, hydrogen and nitrogen
content of the compounds.
EXPERIMENTAL SECTION
UV-Vis spectroscopy
UV–160A UV–Vis spectrophotometer was used with 1 cm path
length quartz cuvettes (2.5 mL). Spectra were processed using
Materials and Reagents
All reagents and solvents were purchased commercially and UV-probe software. Experiments were carried out at 298 K
used without further purification unless otherwise noted. The unless otherwise stated.
purity of compounds was assessed by different analytical Synthesis of 5-(1H-imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazole-
1
13
techniques like ESI mass, LC-MS, CHN analysis, H NMR, C NMR 4-carbaldehyde (1a)
and IR spectra. The measured molecular weights of ligands
intermediates) as well as complexes were consistent with
5
-Chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde
mmol), imidazole (1 mmol) and anhydrous potassium carbonate
2 mmol) in dimethyl formamide (10 mL) were charged in a 100
(1
(
expected values which permitted unambiguous identification
and assessment of purity. iridiumIII dimer, guanidine
hydrochloride, 3-amino-1, 2, 4-triazole and potassium tert-
butoxide were purchased from Sigma-Aldrich. Agarose, Luria
broth, ethidium bromide and tris(hydroxymethyl) methylamine
(
mL round bottom flask equipped with a mechanical stirrer and
a condenser. The reaction mixture was heated at 90 ᵒC for 2 h.
The progress of the reaction was monitored by TLC. After the
completion of reaction as confirmed by TLC, the reaction
mixture was poured into 100 mL ice-water, filtered, washed
thoroughly with water, dried and recrystallized from ethanol to
obtain a white solid.
(tris-HCl) were purchased from Hi-media Laboratories Pvt. Ltd.,
India. Culture of pUC19 (MTCC 47), Staphylococcus aureus (S.
aureus) (MTCC–3160), Bacillus subtilis (B. subtilis) (MTCC–
7
193), Serratia marcescens (S. marcescens) (MTCC–7103), Synthesis of 3-methyl-5-phenoxy-1-phenyl-1H-pyrazole-4-
Pseudomonas aeruginosa (P. aeruginosa) (MTCC–1688) and carbaldehyde (1b)
Escherichia coli (E. coli) (MTCC– 433) were purchased from the
5
-Chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde
(1
Institute of Microbial Technology (Chandigarh, India).
mmol), phenol (1 mmol) and anhydrous potassium carbonate (2
mmol) in dimethyl formamide (10 mL) were charged in a 100 mL
round bottom flask equipped with a mechanical stirrer and a
condenser. The reaction mixture was heated at 90 ᵒC for 2 h.
The progress of the reaction was monitored by TLC. After the
completion of reaction as confirmed by TLC, the reaction
mixture was poured in to 100 mL ice-water, filtered, washed
thoroughly with water, dried and recrystallized from ethanol to
obtain a white solid.
Cell lines and culture conditions
The lung carcinoma cell line (A549) was procured from NCCS,
Pune, India. The passage number of 19-25 was used in the
present study. Apart from this the cell line was maintained as a
monolayer in DMEM/F12 (Gibco, Invitrogen, CA, USA)
containing 10% foetal bovine serum (Gibco, Invitrogen, CA,
USA) supplemented with 50 U/mL penicillin, 50 µg/mL
streptomycin at 37˚C in a humidified atmosphere of 5% CO
5% air.
2
/
Synthesis of (E)-3-(5-(1H-imidazol-1-yl)-3-methyl-1-phenyl-1H-
pyrazol-4-yl)-1-(pyridin-2-yl)prop-2-en-1-one (3a)
9
To a mixture of 5-(1H-imidazol-1-yl)-3-methyl-1-phenyl-1H-
pyrazole-4-carbaldehyde (1a) (5.0 mmol), 2- acetyl pyridine (5.0
mmol) and 20% ethanolic NaOH (5 mL) were added. The
reaction mixture was stirred at room temperature until
formation of precipitate. The obtained product was isolated by
Analytical measurements
NMR spectroscopy
1H NMR and 13C NMR spectra were obtained in 5 mm NMR
tubes at 298 K (unless stated otherwise) on Bruker Avance (400
1
MHz) spectrometers. H NMR chemical shifts were internally
2 3 6 3
referenced to (CHD )(CD )SO (2.50 ppm) for DMSO-d and CDCl
2
| J. Name., 2012, 00, 1-3
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Journal Name
ARTICLE
filtration, washed with cold ethanol and recrystallized from 132.88, 137.11, 143.98, 147.15, 147.17, 151.81 (C of aromatic);
View Article Online
DOI: 10.1039/C6NJ02153K
CHCl
Synthesis of (E)-3-(3-methyl-5-phenoxy-1-phenyl-1H-pyrazol-4-yl)-
-(pyridin-2-yl)prop-2-en-1-one (3b)
To a mixture of 3-methyl-5-phenoxy-1-phenyl-1H-pyrazole-4-
carbaldehyde (1b) (5.0 mmol), 2- acetyl pyridine (5.0 mmol) and (E)-3-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-
0% ethanolic NaOH (5 mL) were added. The reaction mixture 1-(pyridin-2-yl)prop-2-en-1-one (3a) (1.0 mmol) and guanidine
3
.
114.50, 121.38, 121.52, 121.63, 123.37, 123.76, 128.85, 130.11,
130.32, 131.55, 136.03, 137.42, 149.55 (CH of aromatic).
4-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-
1
(pyridin-2-yl)pyrimidin-2-amine (pma-N) [5a]
2
was stirred at room temperature until formation of precipitate. hydrochloride (1.0 mmol) were thoroughly mixed in ethanol (5
The obtained product was isolated by filtration, washed with mL) with catalytic amount of potassium tertiary butoxide in a 50
cold ethanol and recrystallized from CHCl
'-(1H-Imidazol-1-yl)-3'-methyl-1',2-diphenyl-5-(pyridin-2-yl)-3,4-
dihydro-1'H,2H-3,4'-bipyrazole (bpy-N) [4a]
3
.
mL round bottom flask. The reaction mixture was refluxed for 6
hrs. After the completion of the reaction as monitored by TLC
(ethyl acetate: hexane: 1:5), the reaction mixture was cooled to
5
(
1
E)-3-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-yl)- room temperature washed with water and dried over Na2SO4.
-(pyridin-2-yl)prop-2-en-1-one (3a) (1.0 mmol) and phenyl The ensuing product upon purification by chromatography
hydrazine (1.0 mmol) were thoroughly mixed in ethanol (5 mL) experiment (silica gel 60–120 mesh, eluent 20%
with catalytic amount of potassium tertiary butoxide in a 50 mL EtOAc/hexanes) gave the desired compound as a white solid
round bottom flask. The reaction mixture was refluxed for 5 hrs. (scheme 1). Yield: 79 %; M.p.: 250 °C; Mol wt.: 394.44 gm/mol;
+
After the completion of the reaction as monitored by TLC (ethyl LC-MS (m/z): 395 (M ); Elemental analysis (%): calc. for
acetate: hexane: 1:5), the reaction mixture was cooled to room
temperature washed with water and dried over Na SO
. The N, 28.45; IR (KBr, ʋmax, cm ): 3309 (-NH
22 18 8
C H N : C, 66.99; H, 4.60; N, 28.41. Found: C, 66.85; H, 4.53;
-1
), 2924 (aromatic ring –
N), 1373 (–CH rocking); 1231 (C-N),
-d ): δ (ppm) 2.379
), 6.940–7.670 (m, 13H, Ar–H);
C{ H} NMR (100 MHz, CHCl -d ): δ (ppm) 12.86 (-CH of
pyrazole); 120.19, 127.67, 128.35, 132.15, 136.57, 136.91,
: C, 72.79; H, 5.20; N, 22.01. Found: C, 72.85; H, 5.15; 137.65, 148.32 (C of aromatic); 119.07, 121.25, 122.69, 123.06,
2
4
2
ensuing product upon purification by chromatography CH stretching), 1581 (–C
experiment (silica gel 60–120 mesh, eluent 20% 763 (-CH bending); H NMR (400 MHz, CHCl
═
3
1
3
1
3 2
EtOAc/hexanes) gave the desired compound as a white solid) (s, 3H, CH ), 5.230 (s, 2H, NH
13
1
(
scheme 1). Yield: 79 %; M.p. 259 °C; Mol wt.: 445.53 gm/mol;
3
1
3
+
LC-MS (m/z): 446 (M ); Elemental analysis (%): calc. for
C
27
H
23
N
7
-
1
125.02, 129.29, 129.91, 136.09, 138.55, 148.38, 149.53 (CH of
aromatic).
4-(3-Methyl-5-phenoxy-1-phenyl-1H-pyrazol-4-yl)-6-(pyridin-2-
yl)pyrimidin-2-amine (pma-O) [5b]
N, 22.05; IR (KBr, ʋmax, cm ): 2924 (aromatic ring –CH
stretching), 1597 (–C N), 1498 (-CH scissoring), 1388 (–CH
rocking); 1246 (C-N), 756 (-CH bending), H NMR (400 MHz,
CHCl -d ): δ (ppm) 2.356 (s, 3H, CH ), 3.434 (dd, 1H, J = 7.2 Hz,
8 Hz, C -H
-H of pyrazoline), 5.240 (dd, 1H, J = 7.2 Hz, 12.8 Hz, C
═
2
3
1
3
1
3
1
C
4
a
of pyrazoline), 3.918 (dd, 1H, J = 13.2 Hz, 18.0 Hz, The synthesis was performed as for 5a using (E)-3-(3-methyl-5-
-H of phenoxy-1-phenyl-1H-pyrazol-4-yl)-1-(pyridin-2-yl)prop-2-en-
4
b
5
1
3
1
pyrazoline), 6.788–8.557 (m, 17H, Ar–H); C{ H} NMR (100 1-one (3b) (1.0 mmol) and guanidine hydrochloride (1.0 mmol).
MHz, CHCl -d ): δ (ppm) 13.13 (-CH of pyrazole), 41.25 (-CH of Yield: 79 %; M.p.: 249 °C; Mol wt.: 420.48 gm/mol; LC-MS (m/z):
pyrazoline), 54.67 (-CH of pyrazoline), 117.03, 131.98, 137.92, 421 (M+); Elemental analysis (%): calc. for C25 O: C, 71.41;
43.58, 147.15, 147.87, 151.71 (C of aromatic); 113.55, , 120.18, H, 4.79; N, 19.99; Found: C, 71.45; H, 4.85; N, 19.95; IR (KBr,
20.59, 120.65, 122.39, 122.86, 127.85, 129.11, 129.34, 130.53, ʋmax, cm-1): 3303 (-NH ), 2924 (aromatic ring –CH stretching),
36.03, 137.41, 149.05 (CH of aromatic). 1574 (–C N), 1381 (–CH rocking), 1202 (-C-N), 1033 (C-O-C),
756 (-CH bending); H NMR (400 MHz, CHCl -d ): δ (ppm) 2.360
(s, 3H, CH ), 5.239 (s, 2H, NH
C{ H} NMR (100 MHz, CHCl
3
1
3
2
20 6
H N
1
1
1
2
═
3
3'-Methyl-5'-phenoxy-1',2-diphenyl-5-(pyridin-2-yl)-3,4-dihydro-
1
3
1
1
'H,2H-3,4'-bipyrazole (bpy-O) [4b]
3
2
), 6.857–7.641 (m, 15H, Ar–H);
13
1
1
The synthesis was performed as for 4a using (E)-3-(3-methyl-5-
phenoxy-1-phenyl-1H-pyrazol-4-yl)-1-(pyridin-2-yl)prop-2-en-
3
-d ): δ (ppm) 13.58 (-CH
3
of
pyrazole); 111.04, 115.26, 115.62, 119.10, 136.41, 138.04,
1
7
-one (3b) (1.0 mmol) and phenyl hydrazine (1.0 mmol). Yield: 145.34, 148.41, 157.22 (C of aromatic); 122.04, 122.70, 122.83,
5 %; M.p.: 249 °C; Mol wt.: 471.56 gm/mol; LC-MS (m/z): 472 123.19, 123.40, 126.64, 129.00, 129.12, 129.67, 129.77, 148.69
(CH of aromatic).
-(5-(1H-Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5-
(pyridin-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidine (tpm-N) [6a]
+
(M ); Elemental analysis (%): calc. for C30
25 5
H N O: C, 76.41; H,
7
5
.34; N, 14.85. Found: C, 76.55; H, 5.43; N, 14.90; IR (KBr, ʋmax
,
-
1
cm ): 2924 (aromatic ring –CH stretching), 1597 (–C
═
N), 1504
(
7
-CH
2
scissoring), 1389 (–CH
3
rocking); 1246 (C-N), 995 (C-O-C) An oven dried 25 mL RB flask was charged with (E)-3-(5-(1H-
): δ (ppm) 2.196 Imidazol-1-yl)-3-methyl-1-phenyl-1H-pyrazol-4-yl)-1-(pyridin-2-
1
71 (-CH bending), H NMR (400 MHz, DMSO-d
6
(
s, 3H, CH
pyrazoline), 4.070 (dd, 1H, J = 13.2 Hz, 18.0 Hz, C
pyrazoline), 5.643 (dd, 1H, J = 6.8 Hz, 13.2 Hz, C
3
), 3.405 (dd, 1H, J = 7.2 Hz, 18.4 Hz, C
4
-H
a
of yl)prop-2-en-1-one (3a) (1.0 mmol), 3- aminotriazole (1.2
4
-H
b
of mmol), KOH (0.1 mmol) and DMF (5 mL). The resulting solution
0
5
-H of was stirred at 110 C for 20 min. On completion, the reaction
1
3
1
pyrazoline), 6.894–8.705 (m, 19H, Ar–H); C{ H} NMR (100 mass was allowed to cool to ambient temperature, diluted with
MHz, DMSO-d ): δ (ppm) 12.33 (-CH of pyrazole), 42.25 (-CH water (20 mL) and extracted into ethyl acetate (2 x 20 mL). The
of pyrazoline), 54.57 (-CH of pyrazoline); 117.03, 124.67, combined organic layers were dried over anhydrous Na SO
and organic solvent was evaporated on a rotatory evaporator.
6
3
2
2
4
,
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DOI: 10.1039/C6NJ02153K
5
5 5
The crude residue was purified by chromatography experiment Synthesis of [(η -C Me )Ir(bpy-O)Cl]Cl [7b]
(
5
silica gel 60–120 mesh, eluent 20% EtOAc/hexanes) affording The synthesis was performed as for 7a using [(η -C Me )-IrCl ]
5
5
2 2
compounds 6a (scheme 1). Yield: 75 %; M.p.: 241 °C; Mol wt.: (0.06 mmol) and bipyrazole (4b) (0.12 mmol). Yield: 65 %; M.p.:
+
+
4
19.45 gm/mol; LC-MS (m/z): 419 (M ); Elemental analysis (%): > 300 °C; Mol wt.: 869.91 gm/mol; MS (ESI, m/z): 834 [M-Cl] ;
-
1
2
-1
17 9
M
calc. for C23H N : C, 65.86; H, 4.09; N, 30.05. Found: C, 65.79; Ω : 72 Ω cm mol ; Elemental analysis (%): calc. for
H, 4.15; N, 30.11; IR (KBr, ʋmax, cm ): 2916 (aromatic ring –CH C H N OCl Ir: C, 55.23; H, 4.63; N, 8.05; Ir, 22.10; Found: C,
-1
40
40
5
2
-1
stretching), 1558 (–C
(
3
═
N), 1381 (–CH
3
rocking); 1203 (C-N), 756 55.20; H, 4.56; N, 8.10; Ir, 22.16; IR (KBr, ʋmax, cm ): 2916
): δ (ppm) 2.348 (s, (aromatic ring –CH stretching), 1597 (–C
N), 1497 (-CH2
1
-CH bending); H NMR (400 MHz, DMSO-d
6
═
1
3
1
H, CH
3
), 7.243–9.310 (m, 14H, Ar–H); C{ H} NMR (100 scissoring), 1381 (–CH rocking); 1249 (C-N), 1026 (C-O-C), 772
3
1
MHz,DMSO-d
6
): δ (ppm) 14.65 (-CH
3
of pyrazole); 110.29, (-CH bending); H NMR (400 MHz, DMSO-d ): δ (ppm) = 1.373
6
1
23.53, 133.69, 137.58, 144.29, 149.91, 153.21, 159.73 (C of (s, 15H, Cp* CH ), 2.251 (s, 3H, CH ), 3.989 (dd, 1H, J = 17.6 Hz,
3 3
aromatic); 109.13, 118.34, 120.89, 121.63, 125.60, 126.02, 30 Hz, C -H of pyrazoline), 4.331 (dd, 1H, J = 14.8 Hz, 30.0 Hz,
4
a
1
27.01, 134.52, 143.13, 150.32, 155.85, 156.84 (CH of C -H of pyrazoline), 5.099 (dd, 1H, J = 11.6 Hz, 23.6 Hz, C -H of
4
b
5
1
3
1
aromatic).
pyrazoline), 7.082–8.921 (m, 19H, Ar–H); C{ H} NMR (100
MHz, DMSO-d ): δ (ppm) 8.73 (Cp*-CH ), 13.32 (-CH of
pyrazole), 42.15 (-CH of pyrazoline), 53.38 (-CH of pyrazoline),
7
[
-(3-Methyl-5-phenoxy-1-phenyl-1H-pyrazol-4-yl)-5-(pyridin-2-yl)-
1,2,4]triazolo[1,5-a]pyrimidine (tpm-O) [6b]
The synthesis was performed as for 6a using (E)-3-(3-methyl-5- 89.98 (Cp*-C); 120.19, 122.69, 123.06, 148.32, 148.38, 149.53,
phenoxy-1-phenyl-1H-pyrazol-4-yl)-1-(pyridin-2-yl)prop-2-en-
150.93, 151.24 (C of aromatic); 119.07, 121.25, 125.02, 127.67,
-one (3b) (1.0 mmol), 3-aminotriazole (1.2 mmol), KOH (0.1 128.35, 129.29, 129.91, 132.15, 136.09, 136.57, 136.91, 137.65,
6
3
3
2
1
mmol) and DMF (5 mL). Yield: 75 %; M.p.: 236 °C; Mol wt.: 138.55 (CH of aromatic).
5
+
Synthesis of [(η -C Me )Ir(pma-N)Cl]Cl [8a]
4
45.49 gm/mol; LC-MS (m/z): 446 (M ); Elemental analysis (%):
calc. for C26
H, 4.15; N, 22.08; IR (KBr, ʋmax, cm ): 2931 (aromatic ring –CH (0.06 mmol) and pyrimidin-2-amine (5a) (0.12 mmol). Yield: 69
5
5
5
19 7 5 5 2 2
H N O: C, 70.10; H, 4.30; N, 22.01; Found: C, 70.05; The synthesis was performed as for 7a using [(η -C Me )-IrCl ]
-1
stretching), 1551 (–C
═
N), 1381 (–CH
3
rocking), 1235 (-C-N), 995 %; M.p.: > 300 °C; Mol wt.: 792.79 gm/mol; MS (ESI, m/z): 757
1
+
-1
2
-1
(C-O-C), 763 (-CH bending); H NMR (400 MHz, DMSO-d
6
): δ [M-Cl] ; Ω
M
: 65 Ω cm mol ; Elemental analysis (%): calc. for
Ir: C, 48.48; H, 4.20; N, 14.13; Ir, 24.25; Found: C,
13
1
(ppm) 2.421 (s, 3H, CH
3
), 7.194–9.211 (m, 16H, Ar–H); C{ H}
32
C H
33
N
8
Cl
2
-1
NMR (100 MHz, DMSO-d
6
): δ (ppm) 12.15 (-CH
10.39, 123.57, 132.15, 133.69, 137.58, 144.16, 149.91, 153.28, NH
59.71 (C of aromatic); 108.18, 117.34, 120.39, 121.63, 124.64, (–CH
25.06, 126.01, 134.52, 143.13, 150.32, 155.85, 156.64 (CH of MHz, DMSO-d
CH ), 5.609 (s, 2H, NH
3
of pyrazole); 48.42; H, 4.26; N, 14.22; Ir, 24.30; IR (KBr, ʋmax, cm ): 3351 (-
1
1
1
2
), 2916 (aromatic ring –CH stretching), 1597 (–C
═
N), 1381
1
3
rocking); 1250 (C-N), 771 (-CH bending); H NMR (400
6
): δ (ppm) = 1.542 (s, 15H, Cp* CH
3
), 2.306 (s, 3H,
), 6.992–8.569 (m, 13H, Ar–H); 13C{ H}
1
aromatic).
3
2
5
Synthesis of [(η -C
5
Me
5
)Ir(bpy-N)Cl]Cl [7a]
Me )-IrCl
(0.06 mmol) and bipyrazole (4a) of pyrazole), 87.69 (Cp*-C); 108.50, 130.64, 137.94, 148.08,
Cl
(15 mL) was stirred for 2 h at ambient 148.98, 155.86, 155.98, 156.77 (C of aromatic); 115.37, 116.01,
6 3 3
NMR (100 MHz, DMSO-d ): δ (ppm) 8.89 (Cp*-CH ), 14.44 (-CH
5
A solution of [(η -C
5
5
2 2
]
(
0.12 mmol) in CH
2
2
temperature. The solution was filtered through Celite. The 122.20, 122.30, 123.87, 124.22, 126.99, 127.59, 129.73, 129.83,
filtrate was evaporated to dryness on a rotary evaporator and 139.55 (CH of aromatic).
5
Synthesis of [(η -C
5
Me
5
)Ir(pma-O)Cl]Cl [8b]
washed with diethyl ether. The product was recrystallized from
CHCl
5
3
/hexane (scheme 1). Yield: 70 %; M.p.: > 300 °C; Mol wt.: The synthesis was performed as for 7a using [(η -C
5
Me
: 69 Ω cm mol (0.06 mmol) and pyrimidin-2-amine (5b) (0.12 mmol). Yield: 71
Cl Ir: C, 52.66; H, %; M.p.: > 300 °C; Mol wt.: 818.82 gm/mol; MS (ESI, m/z): 783
.54; N, 11.62; Ir, 22.78; Found: C, 52.76; H, 4.56; N, 11.66; Ir, [M-Cl] ; Ω
5 2 2
)-IrCl ]
+
-1
2
-
8
;
43.88 gm/mol; MS (ESI, m/z): 809 [M-Cl] ; Ω
M
1
Elemental analysis (%): calc. for C37
H
38
N
7
2
+
-1
2
-1
4
2
1
M
: 66 Ω cm mol ; Elemental analysis (%): calc. for
OCl Ir: C, 51.34; H, 4.31; N, 10.26; Ir, 23.47; Found: C,
3
rocking); 1248 51.44; H, 4.37; N, 10.36; Ir, 23.55; IR (KBr, ʋmax, cm ): 3417 (-
-
1
2.82; IR (KBr, ʋmax, cm ): 2916 (aromatic ring –CH stretching),
597 (–C N), 1499 (-CH scissoring), 1389 (–CH
35
C H
35
N
6
2
-
1
═
2
1
(C-N), 763 (-CH bending); H NMR (400 MHz, DMSO-d
6
): δ (ppm) NH
), 3.984 (dd, 1H, J = (–CH
of pyrazoline), 4.321 (dd, 1H, J = 14.8 Hz, NMR (400 MHz, DMSO-d
of pyrazoline), 5.079 (dd, 1H, J = 11.6 Hz, 23.6 Hz, 2.306 (s, 3H, CH ), 5.622 (s, 2H, NH
-H of pyrazoline), 7.073–8.967 (m, 17H, Ar–H); C{ H} NMR H); C{ H} NMR (100 MHz, DMSO-d
): δ (ppm) 8.65 (Cp*-CH ), 15.08 (-CH of 14.49 (-CH of pyrazole), 89.69 (Cp*-C); 109.50, 130.64, 136.94,
pyrazole), 43.58 (-CH of pyrazoline) 53.53 (-CH of pyrazoline), 148.08, 148.98, 158.06, 159.08, 162.57, 163.98 (C of aromatic);
2
), 2916 (aromatic ring –CH stretching), 1550 (–C
═
N), 1381
1
=
1
3
C
1.370 (s, 15H, Cp* CH
7.6 Hz, 30 Hz, C -H
0.0 Hz, C -H
3
) 2.547 (s, 3H, CH
3
3
rocking), 1196 (-C-N), 1026 (C-O-C), 763 (-CH bending); H
4
a
6
): δ (ppm) = 1.542 (s, 15H, Cp* CH
), 6.732–9.005 (m, 15H, Ar–
): δ (ppm) 9.89 (Cp*-CH ),
3
),
4
b
3
2
13
1
13
1
5
6
3
(
100 MHz, DMSO-d
6
3
3
3
2
9
1
1
0.08 (Cp*-C); 113.55, 120.65, 142.98, 148.03, 149.41, 150.92, 115.37, 116.01, 122.00, 122.30, 123.87, 124.02, 126.99, 127.99,
53.58 (C of aromatic); 117.03, 120.18, 120.59, 122.39, 122.86, 129.73, 129.89, 138.55 (CH of aromatic).
5
25.02, 125.44, 129.56, 129.81, 137.85, 139.11, 139.34, 140.53 Synthesis of [(η -C
5
Me
5
)Ir(tpm-N)Cl]Cl [9a]
5
(CH of aromatic).
5 5 2 2
The synthesis was performed as for 7a using [(η -C Me )-IrCl ]
(0.06 mmol) and triazolopyrimidine (6a) (0.12 mmol). Yield: 75
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
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Page 6 of 16
ARTICLE
Journal Name
III
%
; M.p.: > 300 °C; Mol wt.: 817.80 gm/mol; MS (ESI, m/z): 782 ligand (iridium and pyrazole incorporated ligands) files were
View Article Online
DOI: 10.1039/C6NJ02153K
+
-1
2
-1
[
M-Cl] ; Ω
M
: 62 Ω cm mol ; Elemental analysis (%): calc. for prepared using AutoDock Tools. The hetero atoms including
2
Ir: C, 48.47; H, 3.94; N, 15.41; Ir, 23.50; Found: C, water molecules were deleted, polar hydrogen atoms and
C
33
H
32
N
9
Cl
8.56; H, 3.90; N, 15.39; Ir, 23.59; IR (KBr, ʋmax, cm ): 2916 Kollman charges were added to the receptor molecule. All other
N), 1381 (–CH bonds were allowed to be rotatable. In the docking analysis, the
rocking); 1204 (C-N), 764 (-CH bending); H NMR (400 MHz, binding site was assigned across all of the minor and major
DMSO-d ): δ (ppm) = 1.774 (s, 15H, Cp* CH ), 2.483 (s, 3H, CH ), grooves of the DNA molecule, which was enclosed in a box with
.622–9.201 (m, 14H, Ar–H); C{ H} NMR (100 MHz, DMSO-d ): the number of grid points in x × y × z directions, 60 × 74 × 116
δ (ppm) 9.65 (Cp*-CH ), 14.51 (-CH of pyrazole), 91.15 (Cp*-C); and a grid spacing of 0.375 Å. All calculations were performed
12.35, 130.21, 137.43, 143.55, 159.86, 165.22, 165.81, 166.93 on an Intel Core i3 based machine running windows 7 operating
C of aromatic); 113.13, 124.01, 125.90, 127.86, 129.82, 131.23, system. For each of the docking cases, the lowest energy docked
-1
4
(aromatic ring –CH stretching), 1558 (–C
═
3
1
6
3
3
13
1
7
6
3
3
1
(
1
39.55, 141.27, 150.45, 153.27, 153.74, 154.53 (CH of conformation, according to the AutoDock scoring function, was
31
aromatic).
Synthesis of [(η -C
selected as the binding mode . Visualization of the docked
pose was done by using AutoDock 1.5.6 .
5
31
5
Me
5
)Ir(tpm-O)Cl]Cl [9b]
5
The synthesis was performed as for 7a using [(η -C
5 5 2 2
Me )-IrCl ]
Chemical nuclease activity
(0.06 mmol) and triazolopyrimidine (6b) (0.12 mmol). Yield: 73
%
[
C
5
; M.p.: > 300 °C; Mol wt.: 843.83 gm/mol; MS (ESI, m/z): 808 The DNA cleavage study was performed using pUC19 DNA with
+
-1
2
-1
M-Cl] ; Ω
M
: 64 Ω cm mol ; Elemental analysis (%): calc. for synthesized compounds. The samples were incubated for 1 h at
OCl
Ir: C, 51.24; H, 4.06; N, 11.62; Ir, 22.78; Found: C, 37 C. The samples were analysed by 1% agarose gel
1.14; H, 4.00; N, 11.56; Ir, 22.85; IR (KBr, ʋmax, cm ): 2916 electrophoresis [Tris–acetate–ethylenediaminetetraacetic acid,
36
H
34
N
7
2
̊
-1
(
aromatic ring –CH stretching), 1558 (–C
═
N), 1381 (–CH
3
(TAE) buffer, pH 8.0] for 3 h. at 100 mV. The gel was stained with
rocking), 1252 (-C-N), 1026 (C-O-C), 764 (-CH bending); H NMR (0.5 mg mL ) ethidium bromide. The gels were viewed in an
1
-1
(
(
400 MHz, DMSO-d
s, 3H, CH
6
): δ (ppm) = 1.775 (s, 15H, Cp* CH
3
), 2.578 Alpha Innotech Corporation Gel doc system and photographed
), 7.626–9.411 (m, 16H, Ar–H); 13C{ H} NMR (100 using a CCD camera. The cleavage efficiency was measured by
1
3
6 3 3
MHz, DMSO-d ): δ (ppm) 8.86 (Cp*-CH ), 14.21 (-CH
of determining the ability of the complex to convert the
pyrazole), 90.05 (Cp*-C); 114.85, 128.84, 137.59, 141.13, supercoiled DNA (SC) to nicked circular (NC) and linear forms
1
1
1
54.95, 161.13, 161.75, 162.43, 150.70 (C of aromatic); 106.88, (L).
16.09, 121.32, 125.83, 125.96, 126.06, 129.77, 129.95, 130.21,
Antiproliferative study
30.64, 149.96, 153.33 (CH of aromatic).
The 10,000 cells/well were seeded in 96 well culture plates and
allow to grow for 24 h. After 24 h. cells were treated with the
Absorption spectroscopy in the presence of CT-DNA
The absorption spectra were recorded on a Shimadzu UV-2450 various concentrations of synthesized compounds dissolved in
UV-Vis spectrophotometer using cuvettes of 1 cm path length. molecular grade of DMSO to find out appropriate IC50 value of
Absorption spectral titration experiments were performed by the respective compounds. Next day, 10 µL of tetrazolium dye
maintaining a constant concentration of the complex and (MTT) (5 mg/mL) was added to each well and incubated for 3-4
2
varying the nucleic acid concentration. It was achieved by h. in CO incubator at 37˚C. Volume of culture was 100 µL in
dissolving an appropriate amount of the metal complex, DNA each well. After the incubation period, the culture medium was
stock solutions and maintaining the total volume constant. The removed and the purple crystals formed were dissolved in 100
absorbance (A) of the most red-shifted band of each complex µL of molecular grade DMSO. The absorbance was recorded
was recorded after successive additions of CT DNA. The stock with the help of micro plate reader at 570 nm.
solution was prepared by dissolving CT-DNA in a Tris HCl buffer
In vitro antibacterial activity
(pH 7.2) and kept overnight at 4 °C for complete dissolution. The
DNA concentration per nucleotide was determined by The minimal inhibitory concentrations (MIC) were tested
absorption spectroscopy using the molar absorption coefficient against three Gram-negative microorganisms namely
-1
-1
29, 30
(
6600 M cm ) at 260 nm
.
Escherichia coli (MTCC 433), Pseudomonas aeruginosa (MTCC P-
0
9), Serratia marcescens (MTCC 7103) and two Gram-positive
Molecular docking of the complexes with the DNA duplex with
bacteria namely Bacillus subtilis (MTCC 7193) and
Staphylococcus aureus (MTCC 3160). The antibacterial activity
sequence d(CGCGAATTCGCG)
2
AutoDock vina was used for the prediction of binding affinity for the test compounds was performed to determine the
and searching for the optimum binding site together with the bacteriostatic concentration, i.e. minimal inhibitory
AutoDock Tools (ADT) to set up and perform blind docking concentration (MIC). The MIC value was determined by two fold
calculations of all compounds binding to CT-DNA. Since CT-DNA serial dilution technique in triplicates. The lowest compound
used in the experimental work was too large for current concentration inhibiting visible bacterial growth is reported as
computational resources to perform the dock calculation, the MIC32, 33
.
2
structure of the DNA with sequence d(CGCGAATTCGCG) (PDB
id: 1BNA, a familiar sequence) obtained from the Protein Data
Bank (www.rcsb.org/pdb). The receptor (CT-DNA) and the
In vitro cytotoxicity bioassay
6
| J. Name., 2012, 00, 1-3
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Page 7 of 16
PNl ee aws eJ do ou rnn oa tl ao df j uC sht emm ai rs gt ir ny s
Journal Name
ARTICLE
5
III
to form piano stool iridium
View Article Online
The cytotoxicity assay was performed on brine shrimp nauplii [{(η -C
5
Me
5
4
)Ir(μ-Cl)Cl}
2
]
1
1
DOI: 10.1039/C6NJ02153K
using Meyer method. The lethal concentrations of compounds complexes . In the H NMR spectra of complexes, the protons
resulting in 50% mortality (LC50) of the brine shrimp from the 24 due to pro-chiral methylene protons resonated toward
h and the dose–response data were transformed into a straight downfield side by difference of δ 0.5-1.5 ppm similarly the chiral
line by means of a trend line fit linear regression analysis; the C–H proton of pyrazoline and aromatic proton in complexes,
LC50 was determine from the best-fit line obtained by plotting % resonated towards downfield side by difference of δ upto 1.0
mortality vs. concentration. All data has been collected from at and 0.5 ppm, respectively. It may be ascribed to the
least three independent experiments and the LC50 determined coordination of ligands to metal centre. The protons due to Cp*-
34-36
using OriginPro 8 software as previously described
.
CH
almost
complexes, which indicates a rather small change in the
3
in complexes displayed an upfield shift and resonated at
∼
1.48 ppm with respect to the metal precursor
Cellular level cytotoxicity
Due to eukaryotic and fairly big size characteristics of S. pombe, electronic environment about the metal centre. 13C NMR
it is become an important tool to study cell biology. Cytotoxicity spectral data of compounds further supported the formation of
of synthesized compounds was tested using bioassay on S. complexes and the proposed formulations (Supplementary
37
42-47
pombe cells at the cellular level as previously described .
material 1)
. The ESI-MS of complexes have been acquired to
understand the relative composition and stability of the
complexes. Notably these displayed the most abundant peaks
for complex (7a) at m/z 808.37 [M]+ and 810.43 [M+2]+
RESULTS AND DISCUSSION
Molecular design
(Supplementary material 2).
As shown in Fig. 1, the typical active N−N functionality
embedded in pyridyl−pyrazole, pyridyl-pyrimidine and pyridyl-
DNA binding by absorption spectral studies
triazolopyrimidine will serve as a basic core ligand with a readily There are a number of ways in which small molecules can
available metalation site. The designed molecule was interact with DNA. For example, groove binding, which is
synthesized using developed strategy reported earlier38-40. This
primarily an electrostatic interaction between a cationic
incorporated imidazole and phenol features an installation of molecule and the negative phosphate backbone of DNA. In
different biological application.
covalent binding, a molecule coordinates to one of the base
pairs of DNA (cis-platin covalently binds to DNA), and
intercalation, resulting from insertion of the molecule or part of
the molecule between the base pairs of DNA. Intercalation
typically requires an extended flat aromatic group to
48
accomplish this type of DNA interaction . Because of the
intense photophysical properties of metal complexes, a great
deal of research has been conducted. In beginning with the
initial studies by Barton and Turro in the mid-1980s4
9,
50
,
associated with the development of ruthenium(II) complexes as
DNA probes and phototherapeutics. To this end, we evaluated
III
the interaction of iridium complexes with CT DNA by
spectroscopic means51-53. The DNA binding experiment was
performed in Tris-HCl buffer using DMSO solution of all
compounds. The DNA concentration per nucleotide was
determined by absorption spectroscopy using the molar
-1
-1
absorption coefficient 6600 M cm at 260 and 280 nm
A260/A280 of 1.9, indicating that the DNA was sufficiently free of
Fig. 1 Modifications of the piano stool complexes.
5
4
0
protein . The stock solutions were stored at 4 C and used
within 4 days. Absorption titrations were carried out by varying
the concentration of CT-DNA from 0 to 500 µL and with constant
concentration of the compound (125 µL). In order to eliminate
the absorbance of DNA itself; a reference cell containing DNA
alone was prepared at the same concentration to which
increments of the DNA stock solution were added. The binding
of the ligands/complexes to DNA led to decrease in the
Synthesis and general aspects
1
In the H NMR spectra of bipyrazoles, the pro-chiral methylene
protons of pyrazoline appeared as two distinct doublets of a
doublet, thereby indicating that both the protons are
magnetically non-equivalent and diastereotopic. The chiral C–H
proton of pyrazoline appeared as doublets of a doublet. In H
2
NMR of pyrimidin-2-amines, –NH protons resonates as singlet
1
absorption of the UV-Vis spectrum, indicating that the
휋*
at around δ 5.5-5.7 ppm. The aromatic protons resonate as
orbital of the compounds can couple with the
DNA base pairs, resulting in the decrease in the
휋
orbital of the
multiples at around δ 6.00–8.00 ppm. The complexes of the
휋– 휋* transition
5
type [(η -C
5
Me
5
)Ir(L)]Cl (where L = bipyrazole; 7a-7b/pyrimidin-
energy and causing a bathochromic shift. The binding constants
2
-amine; 8a-8b/triazolopyrimidine; 9a-9b) were prepared by
(K
b
) of the ligands/complexes to CT-DNA were determined by
addition of the chelating ligands to dichloromethane solution of
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Page 8 of 16
ARTICLE
Journal Name
monitoring the changes in the absorbance with increasing
concentration of CT-DNA (Fig.2).
View Article Online
DOI: 10.1039/C6NJ02153K
5
5
-1
5
Fig. 2 (A) Absorption spectra of [(η -C
5
Me
5
)Ir(bpy-N)Cl]Cl (7a) (K
b
= 1.13 ± 0.125 ×10 M ) with inset plot of [DNA]/(εa–εf) vs. [DNA]. Fig. 2 (B) Absorption spectra of [(η -C
5
Me
5
)Ir(bpy-
4
-1
O)Cl]Cl (7b) (K
b
= 9.20 ± 0.049×10 M ) with inset plot of [DNA]/(εa–εf) vs. [DNA].
The absorption data were analysed to evaluate the intrinsic the presence of an extended
binding constant (K ) determined from the spectroscopic imidazole ring and planarity of the molecule. The binding
titration data using the following equation.
DNA]/(ε - ε ) = [DNA]/(ε - ε ) + 1/K
The ‘apparent’ extinction coefficient (ε
calculating absorbance of compounds. The terms ε
correspond to the extinction coefficients of free (unbound) and calf thymus DNA to compounds show a decrease in absorbance
the fully bound ligands/complexes. The binding constant (K of the * band, which indicates that the binding of ligands
was calculated using a plot inset (Fig. 2) of [DNA]/(ε - ε ) vs. and complexes with DNA are due to a strong interaction. The
DNA] from the ratio of slope to intercept . In the present binding constant (K ) values of the complexes are observed in
study, the heteroatom present in the ligands/complexes the following order 8a > 9a > 7a > 8b > 7b > 9b. Table 1 shows
휋-system of the aromatic
b
constant (K
b
) of 7a, 8a and 9a with CT-DNA are found to be 1.13
5
5
5
5
-1
[
a
f
b
f
b
(ε
b
- ε
f
)
± 0.12 ×10 , 1.51 ± 0.076 ×10 and 1.16 ± 0.085 ×10 M ,
a
) was obtained by respectively. Which are found greater than rest of the
and ε complexes due to the presence of imidazole ring. Addition of
f
b
b
)
휋–휋
a
f
5
4
[
b
55
aromatic moiety can easily bind DNA base pairs . The the static binding constant of ligands and complexes with CT-
interaction of the complexes 7a and 7b with increase in calf DNA.
thymus DNA is shown in Fig. 2. In the absorption spectrum,
ligands and complexes exhibit more intense absorption bands,
which are attributed to strong stacking interaction between the
planar extended
휋-system, facilitating
a
non-covalent
interaction of the compounds with the base pair of the DNA
5
6, 57
molecule
. While increasing the concentration of the calf
thymus DNA, the absorption band of the compounds is affected,
this results in hypochromism and bathochromic (red) shift. The
synthesized complexes 7a, 8a and 9a exhibit 25%, 32% and 30%
of hypochromism, respectively and 3 nm, 5 nm and 5 nm of
bathochromic shift, respectively, upon addition of DNA. The
extent of hypochromism gives a measure of the strength of the
intercalative binding. Results show that the compounds with
imidazole substituent are better DNA binder as compared to the
compounds having phenoxy substituent. The presence of the
imidazole ring in the 7a, 8a and 9a is responsible for more
hypochromism, indicating that the extent of interaction of
compounds contain imidazole ring are stronger than that of
others containing phenoxy substituent. The complexes 7a, 8a
and 9a exhibit strong hydrophobic CT-DNA interaction due to
8
| J. Name., 2012, 00, 1-3
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Page 9 of 16
PNl ee aws eJ do ou rnn oa tl ao df j uC sht emm ai rs gt ir ny s
Journal Name
ARTICLE
Table 1: Absorption spectral properties of ligands and IrIII complexes bound to CT DNA
presented with standard deviation for three independent experiments.
Molecular docking of the complexes with the DNA duplex with
View Article Online
DOI: 10.1039/C6NJ02153K
sequence d(CGCGAATTCGCG)2
In addition to the wet lab activity, we have also performed the
computational work (molecular docking) for all synthesized
compounds (Supplementary material 4). Molecular docking of
the complexes with the DNA duplex with sequence
Binding
Compo Wavele Shif
%
b
constant (K )
unds
ngth
t
Hypochromism
5
-1
×
10 (M )
4
a
375
379
265
268
314
316
367
368
399
380
398
409
2
2
2
3
2
3
3
4
5
5
5
4
12±1.5
11±0.6
12±1.2
13±1.0
16±0.6
15±0.6
25±1.0
22±1.0
32±6.1
24±1.2
30±1.0
17±1.2
0.35±0.027
0.14±0.023
0.43±0.020
0.26±0.008
0.58±0.012
0.48±0.008
1.13±0.125
0.92±0.049
1.51±0.076
0.94±0.016
1.16±0.085
0.51±0.013
2
d(ACCGACGTCGGT) . The molecular docking technique has
4b
played important roles in understanding the drug–DNA
interactions for the rational drug design and discovery, as well
as in the mechanistic study by placing a small molecule in the
binding site of the target specific region of the DNA mainly in a
5a
5b
6a
5
8
non-covalent fashion. Docking was performed to preliminarily
predict the binding affinity and the chosen binding site along
with the sterically acceptable conformations. It is generally
accepted that the lower the binding free energy, the more
potent the binding affinity is between the receptor (DNA) and
6b
7a
7b
8a
‘
‘the ligand’’ molecules. Amongst all possible conformation, a
8b
structure having the most optimal energy was chosen for the
final binding orientation analysis (supplementary material 3).
9a
9b
Fig. 3: Most probable 2D/3D model of docking pose to active site of compounds with CT-DNA
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-
1
Table 2: Molecular docking results of in terms of glide docking score (kcal mol )
Compounds
Glide docking
score
-6.7
Compounds
Glide docking
score
-8.1
4a
7a
7b
8a
8b
9a
9b
4
5
b
a
-7.0
-7.2
-7.5
-7.3
-8.0
-7.2
-8.2
-8.0
5b
6a
6b
-7.4
-8.2
The docked pose with minimum energy clearly showed that
minor groove mode played a predominant role in the
interaction. As can be seen from the Docking score,59, 60 listed in
Table 2. The negative values of the binding free energy of
Fig. 4: Cleavage of SC pUC19 DNA with series of ligands and complexes (50 μM) using 1%
agarose gel containing 0.5 μg/mL ethidium bromide. All reactions were incubated in TE
buffer (pH 8) in a final volume of 20 μL, for 24 h. at 37 ᵒC. Lane 1, DNA control; Lane 2,
docked complexes suggest that the compounds reasonably bind DNA+dimer; Lane 3-4, DNA+(4a-4b); Lane 5-6, DNA+(5a-5b); Lane 7-8, DNA+(6a-6b);
Lane 9-10, DNA+(7a-7b); Lane 11-12, DNA+(8a-8b); Lane 13-14, DNA+(9a-9b);
Abbreviations: N = nicked; S = supercoiled; L= linear.
to the DNA. The complex 8b stacked between DG24 and DG22
of chain A and DC3 and DA5 of chain B. The complex 9b stacked
between DG12 of chain A and DG14, DC15 and DA17 of chain B.
The complexes 8b and 9b are having a good interaction with the
receptor (Fig. 3). From the resulting docked structures it is clear
that compounds fit well into the minor groove of the targeted
DNA and G−C rich region, stabilized by van der Waals
interactions and hydrophobic contacts.
9
0
SC
L
80
7
0
NC
Chemical nuclease activity
60
50
The activity of the complexes as chemical nucleases was studied
using supercoiled pUC19 DNA in 1% agarose gel at 100 V for 2
h. The efficiency of the complexes was compared with that of
the ligands and dimer under the same conditions. The DNA
4
3
0
0
6
1
cleavage can occur by hydrolytic and oxidative pathways , in
which hydrolytic DNA cleavage involves cleavage of
phosphodiester bond to generate fragments and converting
supercoil (SC) form of DNA to open-circular (OC) form and last
20
1
0
0
6
2
in linear (L) form , is being used for identifying the percentage
of cleavage as a function of concentration of nuclease. Oxidative
DNA cleavage involves either oxidation of the deoxyribose
moiety by abstraction of sugar hydrogen or oxidation of
Fig. 5: Percentage cleavage of SC, NC and linear form of DNA presented with standard
deviation for three independent experiments.
6
3
nucleobases .
The principle of this method is that molecules migrate in the gel
as a function of their mass, charge and shape. The supercoiled
DNA, migrating faster than open circular molecules of the same
mass and charge. The native DNA remains in the supercoiled
(SC) form. Single strand cleavage results in so called nicked (NC)
or open circular (OC) form of DNA whereas the double-strand
cleavage results in linear form of DNA. The migration rate during
agarose gel electrophoresis depends on both size (base pairs)
and conformation, with smaller or condensed DNA migrating
faster than larger or unfolded DNA. SC has a tightly packed
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conformation and therefore migrates faster through agarose
gels than linear DNA (intermediate migration) or open circle
DNA (slowest migration) . Results show that all complexes are
better DNA cleaving agents as compared to ligands and salt as
well. Complexes 7a and 7b induce almost degradation of DNA,
View Article Online
DOI: 10.1039/C6NJ02153K
64
9
0% and 86%, respectively. The pertinent cleavage data are
presented in supplementary material 4.
Antiproliferative activity
The antiproliferative activity of ligands and complexes in A549
(
(
lung) cancer cells have been shown in Fig. 6. The ligand 5b
phenoxy substituted pyrimidin-2-amine) shows excellent
anticancer activity than other synthesized ligands. All the
synthesized complexes are found better anticancer agents as
compared to ligands (supplementary material 5). The
complexes having ether linkage are more potent than
complexes having N –linkage. The complexes of pyrimidin-2- Fig. 6: Cell viability assay presented in percentage with standard deviation for three
amine i.e. 8a and 8b exhibit better activity as compared to independent experiments.
complexes of bipyrazole and triazolopyrimidine. This proves
that compounds of pyrimidin-2-amine are most potent than In vitro antibacterial activity
pyrazole or fused triazolopyrimidine. The anticancer activity of
complexes are in order of 7a < 9a < 9b < 8a < 7b < 8b.
The antimicrobial activities of all newly synthesized bipyrazole,
pyrimidin-2-amine and triazolopyrimidines derivatives were
Synthesized complexes exhibit better anticancer activity than
tested against arbitrarily selected two Gram positive and three
previously
reported
half-sandwich
Gram negative bacterial strains. When tested for antibacterial
activity, cyclopentadienyl dimer has been found to be inactive.
Thus, oxidative stress by iridium seems to enhance antibacterial
potency but is not the only component determining antibiotic
activity. The aim was to incorporate pyrazole on iridium to
check the antibiotic activity. So, we synthesized pyrazole
incorporatedbipyrazoles/pyrimidin-2-amines/
III
pentamethylcyclopentadienyl iridium complexes (IC50 = 1-89
66
μM ) and iridium-Cp* chloride picolinamide complexes (IC50
=
67
3
0-190 μM ).
triazolopyrimidines. When tested for antibacterial activity of 6a
and 6b, they exhibit good results compared to rest with MIC
values in the range of 248–267 µM (Fig.7). These results suggest
that neither iridium dimer nor pyrazole based ligands are found
active as an individual, but the coordination of both is
indispensable for antibacterial activity demonstrating that the
organometallic moiety alone is not sufficient for antibiotic
activity but needs the pyrazole based backbone. A satisfactory
reason for this increase in antibacterial activity may be
3
0
considered in the light of Overtone's concept and chelation
theory.
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7
6
5
4
3
2
1
00
00
00
00
00
00
00
0
Salt
4a
4b
5a
B.subtilis
5b
6a
6b
7a
7b
8a
8b
9a
E.coli
9b
S.aureus
S.marcescens
P.aeruginosa
Fig. 7: The antimicrobial activity results in μM presented with standard deviation for three independent experiments.
According to Overtone's concept of cell permeability, the lipid All the synthesized compounds were screened for their
membrane that surrounds cell favours the passage of only lipid cytotoxicity (brine shrimp bioassay) using the protocol of Meyer
35
soluble materials. As a consequence of that the liposolubility is et al . Brine shrimp lethality bioassay is a development in the
an important factor which controls bacteriostatic activity. On assay procedure of bioactive compound, which indicates
III
chelation, the polarity of the Ir ion will be lowered to a greater cytotoxicity as well as a wide range of pharmacological activities
extent due to the overlap of the ligand orbital and partial of the compounds. Results for the lethality were noted in terms
III
sharing of the positive charge of the Ir ion with donor groups. of deaths of larvae. The mortality rate of brine shrimp nauplii
Further, it increases the delocalization of
휋- electrons over was found to increase with increasing the concentration of
whole chelate ring and enhances lipophilicity of the complexes. complexes. A plot of the log of sample's concentration versus
This increased lipophilicity, enhances the penetration of the percentage of mortality showed a linear correlation. From the
complexes into lipid membranes and blocks the metal binding graph, the LC50 values of the compounds were calculated and it
sites in bacterial enzymes. The complexes also disturb the is observed from the results (Fig.8) that 6a and 6b proved to be
respiratory processes of the cell and thus block the synthesis of good cytotoxic agents (5.0 ± 0.032 μM and 5.34 ± 0.059 μM,
proteins, which restricts further growth of the organism. The respectively) as compared to all other ligands.
antibacterial activity was evaluated by minimal inhibitory
concentration (MIC) values. The pertinent MIC data are
presented in supplementary material 6. The purely organic
intermediate compounds were inactive at some extent whilst
upon chelation they proved better antimicrobial agents (70-125
µM). It is worth mentioning that compounds showed lower
activity against Gram positive bacteria. This might be due to the
presence of the outer membrane of Gram positive bacteria,
which hinders access of many compounds to the cytoplasmic
membrane where complexes exert its activity.
Cytotoxicity on brine shrimp
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DOI: 10.1039/C6NJ02153K
9
8
7
6
5
4
3
2
1
0
4a 7a 4b 7b 5a 8a 5b 8b 6a 9a 6b 9b
Fig. 8: Influence of the compounds on the brine shrimp lethality bioassay presented with
standard deviation for three independent experiments.
Fig. 9: Effect of compounds on S. pombe cells, dead cells are seen dark whereas live cells
are seen transparent.
It indicates that the triazolopyrimidines are more cytotoxic than
that of pyrazolines and pyrimidin-2-amine. The compounds
containing imidazole nucleus are more potent than that of
phenoxy nucleus substituted at 5th position on 1-phenyl-3-
methyl-pyrazole. The order of potency of pyrazole incorporated
The toxicity was found to vary with the type of substituent,
principal moieties and concentrations of the synthesized
compounds. General observation is that as concentration of
compounds increases the cytotoxicity is also increases . After
treatment, many of the S. pombe cells are killed due to toxic
nature of the compound. From Table 3, we observed that the
37
compounds is 5b < 5a < 8b < 4b < 4a < 7b < 8a < 7a < 6b < 6a <
III
9
b < 9a. The tested Ir complexes have strong cytotoxic activity
but this examination is a primary one and further tests are
required to investigate its actual mechanism of cytotoxicity and
its probable effects on higher animal model and on cancer cell
line. The pertinent cytotoxic data are presented in
supplementary material 7.
III
cytotoxicity activity of Ir complexes found better than that of
respective free ligands. The order of potency of compounds is
complexes > triazolopyrimidines > pyrazolines > pyrimidin-2-
amines. The compounds bearing imidazole ring show better
results than rest of all. 6a is more potent amongst all ligands and
complex 9a is most toxic amongst all synthesized compounds.
Cytotoxicity on S. pombe
In the present study, S. pombe cells have been used to study Table 3: Percentage viability on S.pombe cells presented with standard deviation for
three independent experiments.
cytotoxic effect of compounds at a cellular level. S. pombe cells
has become an important tool to study cell biology due to its
Compounds
% Viability per concentration
eukaryotic and fairly big size characteristics. A comparative
study of cellular level cytotoxicity values of the free ligands and
their complexes indicate that the metal complexes show better
activity against S. pombe cells compared to the pyrazole
incorporated ligands. Cell death caused by toxicity of the
chemically synthesized compounds could be easily monitored
by vital staining (Fig.9).
2
µM
81
83
82
85
79
81
65
67
66
69
63
65
4 µM
76
78
77
80
74
76
60
62
62
64
57
60
6 µM
70
71
72
74
68
70
54
56
58
58
52
54
8 µM
65
67
66
69
63
65
49
51
53
53
47`
49
10 µM
61
62
63
65
59
61
45
47
49
49
43
45
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
CONCLUSION
In conclusion, the biological and medicinal chemistry of iridiumIII
complexes has been little explored previously, perhaps because
6
III
it is often assumed that low-spin 5d Ir complexes are highly
kinetically inert. Our data show that this is not always the case.
III
The cyclopentadienyl ligands, while stabilizing Ir , can confer
kinetic labile ligands such as chloride. Moreover, pyrazole based
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Journal Name
heterocyclic frameworks can have a major effect on the 11. C. G. Hartinger and P. J. Dyson, Chem. Soc. Rev., 2009, 38, 391-
View Article Online
5
+
DOI: 10.1039/C6NJ02153K
chemical and biological behaviour of [(η -Cp*)Ir(XY)Cl]
401.
2177.
complexes. This appears to be the first time that 12. R. H. Fish and G. Jaouen, Organometallics, 2003, 22, 2166-
bipyrazole/pyrimidin-2-amine/triazolopyrimidine ligands have
been used in iridium complexes. In general, the introduction 13. Y. K. Yan, M. Melchart, A. Habtemariam and P. J. Sadler, Chem.
of pyrazole based ligands to iridiumIII enhances its activity
III
Commun., 2005, 4764-4776.
against brine shrimp and S. pombe. The complexes of 14. G. Suss-Fink, Dalton Transactions, 2010, 39, 1673-1688.
triazolo[1,5-a]pyrimidines are proven to be most cytotoxic. The 15. B. T. Loughrey, P. C. Healy, P. G. Parsons and M. L. Williams,
chelating ligands and metal complexes appear to determine the
Inorg. Chem., 2008, 47, 8589-8591.
selectivity of nucleobases binding. The complexes containing 16. M. G. Mendoza-Ferri, C. G. Hartinger, M. A. Mendoza, M.
imidazole in N, N-chelating ligands stack strongly between the
base pairs of CT DNA, in contrast the complexes containing
phenoxy shows poor binding. Furthermore, the complexes 7b
and 8b are found excellent anticancer agent as compared to
Groessl, A. E. Egger, R. E. Eichinger, J. B. Mangrum, N. P.
Farrell, M. Maruszak, P. J. Bednarski, F. Klein, M. A. Jakupec,
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presence of phenoxy substituent on pyrazole ring as compared
to imidazole substituent. In addition IrIII complexes show
Chen, P. J. Sadler and D. I. Jodrell, Br. J. Cancer, 2002, 86, 1652-
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III
be applied to Ir complexes to achieve potent biological activity. 20. L. Helm and A. E. Merbach, Coord. Chem. Rev., 1999, 187, 151-
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181.
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ACKNOWLEDGMENTS
7
533.
2
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4. C. G. Hartinger, Angew. Chem. Int. Ed., 2010, 49, 8304-8305.
5. F. Shao and J. K. Barton, J. Am. Chem. Soc., 2007, 129, 14733-
This work was supported by UGC, New Delhi for providing BSR-
one time grant fellowship and UGC-BSR JRF/SRF 5/100. The
authors thank the Head, Department of Chemistry, Sardar Patel
University for providing research facilities and CISST, Vallabh
Vidyanagar for LC-MS. Authors are thankful to P. D. Patel
Institute of applied sciences for providing cell culture facilities.
1
4738.
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New Journal of Chemistry
Page 16 of 16
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DOI: 10.1039/C6NJ02153K
Table of Contents Graphic:
IrIII
complexes
Ligands
Ir salt
N
N
N
N
N
N
Ir
NH2
Y
Cl
X
N
N
Ph
Table of Contents synopsis:
Enhancement in the biological function i.e., DNA binding, molecular docking,
Antiproliferative and DNA cleavage of metal complexes as compared to free ligands.