In vitro apoptosis-inducing effect and gene expression profiles of mixed ligand Cu(II) complexes derived from 4-acyl pyrazolones on human lung cancer cells

Article abstract of DOI:10.1039/c7ra01025g

In order to study the less understood anti-tumor mechanism of pyrazolone based mixed ligand complexes, four Cu(ii) mixed ligand complexes [Cu(L1)(Phen)NO₃] (1), [Cu(L2)(Bipy)NO₃] (2), [Cu(L2)(Phen)NO₃] (3) and [Cu(L2)(Bipy

Full text of DOI:10.1039/c7ra01025g

RSC Advances
View Article Online
View Journal | View Issue
PAPER
In vitro apoptosis-inducing effect and gene
expression profiles of mixed ligand Cu(II) complexes
derived from 4-acyl pyrazolones on human lung
cancer cells†
Cite this: RSC Adv., 2017, 7, 17107
a
b
c
c
R. N. Jadeja,* K. M. Vyas, K. K. Upadhyay and R. V. Devkar
In order to study the less understood anti-tumor mechanism of pyrazolone based mixed ligand complexes,
four Cu(II) mixed ligand complexes [Cu(L1)(Phen)NO ] (1), [Cu(L2)(Bipy)NO ] (2), [Cu(L2)(Phen)NO ] (3) and
] (4) [L1 ¼ 3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazole-4-carbaldehyde; L2 ¼ 4-
1-naphthoyl)-3-methyl-1-(p-tolyl)-1H-pyrazol-5(4H)-one] have been synthesized and fully characterized.
3
3
3
[Cu(L2)(Bipy)NO
3
(
Single crystal X-ray diffraction analysis of three complexes indicated mononuclear structures exhibiting
square pyramidal geometries. The inhibitory effects of the complexes on the cell population growth of
the human lung carcinoma (A549) and human lung epithelial cell lines (L132) were determined by MTT
assay, DAPI staining and DCFDA staining, which assessed mitochondrial dysfunction, nuclear
condensation and intracellular oxidative stress, respectively. The expression levels of Bax (pro-apoptotic)
and BCl2 (anti-apoptotic) genes were also studied in A549 cells wherein, both genes showed moderate
downregulation after treatment with all the test complexes.
Received 25th January 2017
Accepted 11th March 2017
DOI: 10.1039/c7ra01025g
rsc.li/rsc-advances
being made towards developing novel such agents, with higher
safety margins, possibly targeting novel pathways which exploit
Introduction
4
Cancer is a life threatening group of diseases, in which the particular biology/biochemistry of tumour cells.
deregulated proliferation of cells attacks and disrupts the
Metal complexes have been considered as potential source of
surrounding tissues, leading to dysfunctional metabolic, anticancer drugs for years to overcome toxicity and drug-
1
angiogenetic and immunologic functions. According to World resistance and to achieve higher selectivity and better activity
5
Health Organization (WHO), lung cancer, the most common in comparison with platinum-based therapeutics. Platinum-
cause of cancer-related mortality in humans, was responsible based complexes such as cisplatin, carboplatin and oxaliplatin
2
for 1.56 million deaths in 2012. Surgery, radiotherapy and have been widely used as chemotherapeutic drugs but these
chemotherapy have been widely used to treat lung cancer. drugs always accompany with serious side effects and drug
However, decreasing the mortality and improving the life resistance. Thus, it is very important to develop non-platinum
1,6,7
quality of lung cancer patients remain a huge therapeutic based anticancer drugs with high efficacy and low toxicity.
challenge.
Up to now, many transition-metal based complexes have been
Although a large number of anticancer drugs were launched, investigated in the development of non-platinum-based drugs.
their limited success, the drug resistance problems and severe Amongst all, copper complexes are the best alternative to
side effects indicate that there is an imperative need of novel a group of platinum anticancer drugs with potential application
3
strategies for cancer management. Thus, increasing efforts are for alternative treatments in tumours e.g., (1) copper complexes
with homoscorpionate tridentate tris(pyrazolyl)borate and
auxiliary monodentate phosphine ligands have been reported
a
Department of Chemistry, Faculty of Science, The M. S. University of Baroda, with similar cytotoxicity prole both in cisplatin-sensitive and
8a
Vadodara 390 002, India. E-mail: rajendra_jadeja@yahoo.com; rjadeja-chem@ resistant cell lines. (2) Water-soluble bis(1,10-phenanthroline)
msubaroda.ac.in; Tel: +91-265-2795552 ext 30
octanedioate Cu(II) complexes have shown greater in vivo drug
b
Discipline of Chemistry, School of Basic Sciences, Indian Institute of Technology (IIT)
Indore, Indore 452 017, Madhya Pradesh, India
8b
tolerance compared to cisplatin, etc.
A large number of heterocyclic compounds with ve or six
c
Division of Phytotherapeutics and Metabolic Endocrinology, Faculty of Science,
member rings have been fruitfully incorporated into new drugs
The M.S. University of Baroda, Vadodara 390 002, Gujarat, India
9–11
and therapeutic agents.
Pyrazolones, especially, 4-acyl pyr-
†
Electronic supplementary information (ESI) available: Tables S1–S3 and
Fig. S1–S5. CCDC 1488810–1488812 contain the supplementary crystallographic azolones and its derivatives has been widely studied that forms
data for complexes 1, 3 and 4, respectively. For ESI and crystallographic data in
a key pharmacophore in numerous compounds of biological
CIF or other electronic format see DOI: 10.1039/c7ra01025g
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 17107–17116 | 17107

View Article Online
RSC Advances
Paper
12–22
importance.
Attention has been paid to pyrazole-based
Results and discussion
Synthesis and characterization
metal complexes due to the advantages including stability,
23
easy synthesis, easy alteration, and abundant bio-activity.
24,25
26
These compounds have antibacterial,
cancer activity.
antiviral and anti- The acyl pyrazolone ligands have been synthesized according to
Consequently, the design and preparation of our previously reported procedure.
27,28
32–34
The NMR and mass
novel 4-acyl pyrazolone based metal complexes is still a notable spectra of ligands are given in ESI (Fig. S1 and S2†).
22
scientic challenge. Pt(II) complexes with the pyrazole based The complexes have been synthesized using methanolic solu-
0
ligands have shown relatively high cytotoxic activity against tion of 4-acyl pyrazolone, 2,2 -bipyridyl/1,10-phenanthrolin and
different cell lines, which is comparable with the activity of Cu(NO ) in 1 : 1 : 1 molar ratio (Schemes 1 and 2). Our primary
3
2
29
cisplatin and much higher than the activity of carboplatin.
aim was to get the ternary mixed ligand Cu(II) complexes with
0
Recently, water soluble Pt(II) complexes bearing acyl pyr- pyrazolone as primary ligand and 2,2 -bipyridyl/1,10-
azolones have been reported with better cytotoxicity compared phenanthrolin as a secondary ligand. The stoichiometry of the
30
to cisplatin. Acylpyrazolone-Cu(II) had been shown to be able reaction was controlled in such a way that no ML2 complex
to induce the apoptosis in KB cells and KBv200 cells via reactive formation takes place with the either of the ligands. All the
31
oxygen species (ROS)-independent mitochondrial pathway.
complexes are coloured and moisture free crystalline solids. The
Following our interest in the design and development of new different spectra of the complexes are given in ESI (Fig. S3–S5†).
mixed-ligand transition metal complexes with good anti-cancer The complexes are insoluble in water and non-polar solvents,
32–34
activity,
four mixed-ligand Cu(II) complexes were synthe- but soluble in MeOH, EtOH, DMF, DMSO, acetonitrile and
sized by the reactions of copper nitrate, different 4-acylpyr- chlorinated solvents. The 4-acyl pyrazolone ligands act as a bi-
azolones and polypyridyls as secondary ligands. The complexes dentate O, O donor towards the Cu(II) core. Molar conduc-
ꢀ
3
ꢁ
were characterized using different analytical and spectroscopic tance values of the complexes in DMF (10 M solution at 25 C)
techniques including single crystal studies. Antitumor activity indicate that the complexes are electrically non-conducting in
35
of these complexes were investigated on A549 lung carcinoma nature. Results of chemical analyses are in good agreement
cell lines by MTT assay, DAPI staining and DCFDA staining, with those of the calculated ones (see Experimental section).
which suggested that the complexes possess good anticancer
property. The expression levels of Bax (pro-apoptotic) and BCl2 1560–1600 cm , which can be assigned to C]N (cyclic). All the
The FT-IR spectra of complexes exhibit the band in the range
ꢀ
1
ꢀ1
(
anti-apoptotic) genes were also studied in A549 cells using all complexes show absorption in the region 3000–2900 cm
,
the synthesized complexes.
which may be due to vC–H. Furthermore, comparison of IR
Scheme 1 Synthesis of complexes 1 & 3.
Scheme 2 Synthesis of complexes 2 & 4.
17108 | RSC Adv., 2017, 7, 17107–17116
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
spectra of the ligands with those of their Cu(II) complexes, Table 1 Summary of crystallographic data of the complexes
indicates that the ligand is coordinated to Cu(II) by two sites,
that is the ligand is bi-dentate. The band due to vC]O is
Complex
1
3
4
completely missing in the spectra of the complexes, suggesting
Chemical formula
C
23
5
H17CuN O
5
34
C H
26CuN
5
O
5
32 5 5
C H25CuN O
enolization of the ligand on complexation. This is supported by Form. weight
506.97
648.15
623.12
ꢁ
the fact that no band for vOH is observed in the infrared spectra a (A)
8.8175(3)
12.8309(4)
19.0229(8)
90.00
102.082(4)
90.00
11.9167(2)
13.7410(2)
18.0275(4)
90.00
100.6935(18)
90.00
11.31171(18)
13.8659(3)
18.2058(3)
90.00
99.5069(17)
90.00
ꢁ
b (A)
of the ligand and its Cu(II) complex. Instead, a band due to vC–O
was observed for the complex, which supports the observation
of its enolization during coordination. This fact suggests that
ꢁ
c (A)
ꢁ
a ( )
ꢁ
b ( )
ꢁ
the ligand remains in the keto form in the solid state, but in g ( )
solution both the keto and enol forms remain in equilibrium.
Deprotonation occurs from the enol form on complexation. In
Z
4
4
4
3
ꢁ
V (A )
2104.50(13)
8325
3238
0.0244
307
Monoclinic
Pꢀ1
1.6000
1.888
1031.3468
23
2900.69(9)
11 617
4818
0.0235
408
Monoclinic
Pꢀ 1
1.484
1.507
1137
23
1.041
0.0488/0.1368
0.0553/0.1427
1488811
2816.31(9)
11 488
4690
0.0209
390
Monoclinic
Pꢀ 1
1.470
1.527
1284
23
0.832
0.0443/0.1740
0.0504/0.1918
1488812
ꢀ
1
Re. Collected
Ind. re.
R(int)
addition, the new band near 580 cm is assigned to Cu–N
ꢀ
1
bond. The new band in the range 410–440 cm is assigned to
Cu–O bond. The wave no. of cyclic C]N does not change in all No. of param.
the complexes, indicates no coordination via C]N cyclic. By Crystal system
Space group
comparing the IR spectra of ligands and metal complexes, it is
ꢀ
3
rcalcd (g cm
)
easy to predict that the ligand binds to the metal ion via O and O
i.e. bi-dentate manner. The IR spectral data of the ligands and
complexes are given in Experimental section.
ꢀ1
Abs coeff, m (cm
F(000)
Temp ( C)
)
ꢁ
2
The visible spectra for the complexes under investigation GOF on F
1.0428
R
R
1
/wR
/wR
2
([I > 2s(I)]
(all data)
0.0546/0.1643
0.0656/0.1826
1488810
were measured in DMF. In DMF, the complexes displayed
absorbance maxima in the 670–720 nm range. For ve-
coordinate Cu(II) complexes, this spectral feature is typical for
Square Planar (SP) or distorted SP geometries, which generally
1
2
CCDC
exhibit a band in the 550–660 nm range (d , dyz / d ); the
geometry around each Cu is SP or distorted SP. In contrast,
Trigonal Bi-pyramidal (TBP) Cu(II) complexes usually show
a maximum at l > 800 nm (dxz, dxꢀy / d ) with a higher energy
z
shoulder. Thus, in DMF, the geometry about the Cu(II) centre in
xz
xꢀy
As theses complexes are pentacoordinated, s values have
been calculated for all complexes. Therefore, we can nd out the
degree of distortion of the polyhedron from square pyramidal to
31–33
trigonal bi-pyramidal according to our previous reports.
The
s values are near 0 for all complexes [0.06 for complex 1, 0.001
for complex 3 and 0.07 for complex 4] revealing square pyra-
midal geometry.
the complexes is closer to SP. These data are consistent with the
36
reported literature for the square pyramidal Cu(II) complexes.
The synthesized complexes are air stable and have higher
The deviation from a perfect TBP as well as SP structure may
be due to the difference in the electro-negativities of all the
different coordinating atoms in the equatorial as well as axial
sites, resulting in different bond lengths and bond angles. A
comparison of the bond lengths of the central metal-donor
atoms shows that the two oxygen donors and two nitrogen
donors are almost similar in the nature of coordination. This
has a net effect that the ve bond lengths are almost similar,
resulting in SP structure. Similarly, the adjacent ve and six
membered chelate rings might also be responsible for distor-
tion from either of the ideal geometries.
thermal stability, which was conrmed by thermo-gravimetric
analysis. Up to 200 C mass loss is not observed, indicating
the absence of lattice water molecule in the complexes. The
complexes slowly started to decompose within the range of 200–
ꢁ
ꢁ
ꢁ
ꢁ
3
3
4
50 C, corresponds to the loss of nitrate molecules. At temp
40 C there is a loss of one ligand molecule and then at temp
50 C the second ligand molecule is decomposed. The nal
product of the thermal decomposition CuO was determined by
elemental analysis.
As shown in Fig. 1, the crystallographically independent Cu(II)
ion in all three complexes is pentacoordinated by three nitrogen
atoms (two bipy-N/phen-N) and three oxygen donors (two
pyrazolone-O and one nitrate-O) in all the three complexes. The
coordination sphere is of the type Cu–N2–N9–O10–O17–O31
Crystal structure description of the complexes
The green block shaped single crystals of three of the
synthesized complexes (1, 3 and 4) suitable for X-ray diffrac-
tion studies, were obtained by slow evaporation in methanol. A
suitable crystal was mounted on a glass bre for data collec-
tion at 298(2) K. The crystal structures of the complexes were
solved by single-crystal XRD in the space group Pꢀ1 of the
monoclinic system. The crystallographic data of these
complexes are listed in Table 1. The important bond lengths
and bond angles of the complexes are listed in Table S1 (ESI†).
The molecular structures of the synthesized complexes
together with the atom-numbering scheme are illustrated
in Fig. 1.
(complex 1)/Cu–N3–N4–O1–O2–O6 (complex 3)/Cu–N2–N3–O4–
O6–O8 (complex 4), exhibiting a geometry close to square pyra-
midal (SP). The coordination geometry of the complexes can best
be described as square pyramidal (SP) with O10, O17, N2 and N9
(complex 1)/O1, O2, N3 and N4 (complex 3)/O4, O6, N2 and N3
(complex 4) forming the plane, and O31 (complex 1)/O6
(complex 3)/O8 (complex 4) occupying axial position, with
a slight deviation at the axial site. The crystal packing of
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 17107–17116 | 17109

View Article Online
RSC Advances
Paper
accessed for their possible cytotoxicity against A549 lung
carcinoma cells and L132 human lung epithelial cells. MTT
produces purple colour formazan in metabolically active cells
wherein, the intensity of colour reects the functional status of
37,38
mitochondria.
In this study, the rationale behind taking
healthy (L132) and cancerous (A549) cell lines was to conrm
specicity of the synthesized complexes against cancer cells.
Results revealed that complexes 1–3 inhibited growth of A549
cells in dose dependent manner (Fig. 2; IC50 4.67, 6 and 8.2 mm,
respectively). Though L132 cells had identical IC50 values,
higher doses did not account for further loss of cell viability
(
Fig. 2). Complex 4 accounted for near normal percentage of
ꢀ1
viable cells (1–10 mm mL ) that prompted us for further
ꢀ1
investigation using a higher dose range (10–100 mm mL ) that
recorded a more pronounced dose dependent cytotoxicity of
A549 cells as compared to L132 cells (IC50 50 vs. 70 mm,
respectively). Hence, it can be tentatively surmised that
complexes 1–4 showed preferential cytotoxicity against A549
cells. These results are prima-facie evidence on selective cyto-
toxicity potential of metal complexes against cancer cells and
therefore qualify them as possible anti-cancer agents.
Mechanism of action
DCFDA staining. Heightened intracellular production of
reactive oxygen species (ROS) accounts for severe damages to
the cell organelles, nucleus and cytoplasmic enzyme
33,39
machinery.
Damage to the mitochondrial membrane and
subsequent leakage of mitochondrial enzymes in the cytoplasm
causes oxidative stress. Hence, prominent uorescence
observed in DCFDA stained cells provide key qualitative
evidence in support of the said hypothesis. In the presence
study, complexes 1–3 and complex 4 accounted for prominent

uorescence in A549 cells at their respective IC50 doses (Fig. 3).
These observations are in agreement with the ndings of MTT
assay and support our claim of mitochondrial damage induce
by complexes 1–4 at the said doses.
DAPI staining. DAPI is a nuclear stain widely used to assess
changes in nuclear morphology and the same was used in our
study. A549 cells treated with complexes 1–4 showed shrunken

uorescent blue nuclei that are characteristic of nuclear
condensation and possible apoptosis (Fig. 4). Further scrutiny
also revealed nuclear fragmentation and aggregation of
Fig. 1 ORTEP views of the (A) complex 1, (B) complex 3 and (C) apoptotic bodies. These observations further corroborate our
complex 4 with displacement ellipsoids drawn at 50%.
results obtained in MTT assay and DCFDA staining.
Gene expression studies. Nuclear condensation induced by
complexes 1–4 in A549 cells prompted further investigation of
complexes 1 and 4 involves weak p–p interactions, which apoptosis. The expression levels of Bax (pro-apoptotic) and BCl2
play a decisive role in cohesion of molecules (Table S2†). C–H–p (anti-apoptotic) genes were studied in A549 cells (Fig. 5). Results
interactions are also present in the crystal structures (Table S3†). revealed that both Bax and BCl2 showed moderate down-
regulation of expression following 1–4 treatments. BCl2/Bax
ratio has been reported as a decisive factor in which a lower
ratio implies towards activation of programmed cell death.
Evaluation of in vitro cytotoxicity-MTT assay
40
Mitochondrial dysfunctions are a preferred trigger to induce Also, anticancer compounds that induce apoptosis are governed
death in cancer cells by test therapeutants and hence, MTT by BCl2 family of proteins wherein BCl2 suppression is a pro-
41
assay is popular and widely used technique for screening of apoptotic signal. A non-signicant decrements in both, Bax
anti-cancer potential of test compounds. Complexes 1–4 were and BCl2 levels are not in absolute agreement with ndings of
17110 | RSC Adv., 2017, 7, 17107–17116
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
Fig. 2 Effect of metal complexes 1 (A and B), 2 (C and D), 3 (E and F) and 4 (G and H low dose and I and J higher dose) on the cell viability of A549
or L132 cells. Results are expressed as mean ꢂ SD for n ¼ 3 in triplicates. *P < 0.05, **P < 0.01 and ***P < 0.001 as compared to control.
other research groups, but an inhibited BCl2 expression rather imply towards a caspase independent pro-apoptotic signal
than enhanced Bax expression matches with prior reports of induced by compounds 1–4 probably mediated via ‘death’
42
apoptosis of broblasts and breast cancer cells. These ndings receptor that needs further scrutiny.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 17107–17116 | 17111

View Article Online
RSC Advances
Paper
Fig. 3 Effect of test complexes (1–4) on the intracellular oxidative stress in A549 cells (magnification 460ꢃ).
Fig. 4 Effect of test complexes (1–4) on the nuclear condensation in A549 cells (magnification 460ꢃ).
Experimental
Materials
All reagents and solvents were purchased from commercial
sources and were further puried by the standard methods,
wherever necessary. Pyrazolones were obtained from Nutan Dye
Chem. Sachin, Surat. Calcium hydroxide and Cu(NO ) $3H O
3
2
2
were purchased from Sigma-Aldrich. 1,10-Phenanthroline was
0
purchased from LobaChem, Mumbai. 2,2 -Bipyridyl was
purchased from SRL Chem. Formyl chloride and 1-naphthoyl
chloride were obtained from Shiva Pharmachem. Ltd., Baroda
as free gi samples.
Human lung carcinoma (A549) and human lung epithelial
cell line (L132) were obtained from National Centre for Cell
Science, Pune, India. Ethidium bromide (EB), Dulbecco's
Modied Eagle Medium (DMEM), Trypsin Phosphate Versene
Glucose (TPVG) solution trypsin and methylthiazolyldiphenyl-
tetrazolium bromide (MTT) were purchased from HiMedia
Fig. 5 Expression levels of apoptotic genes (Bax and BCl2) in A549
cells. Results are expressed as mean ꢂ SD for n ¼ 3 in triplicates are not
statistically significant.
Conclusions
To conrm the formation of a new series of pyrazolone based Laboratories Pvt. Ltd. (Bombay, India). Fetal bovine serum
mixed-ligand Cu(II) complexes 1–4, analytical, spectral and (FBS) was purchased from Biosera (Ringmer, East Sussex UK)
structural analyses were carried out. The crystal structure of and dimethyl sulfoxide (DMSO) was purchased from the Sisco
three of the synthesized complexes showed that the ligands are Research Laboratories Pvt. Ltd. (Mumbai, India). Rhodamine
0
0
0
coordinated to Cu(II) in a bi-dentate manner. The crystallo- 123, 4 ,6-diamidino-2-phenylindole (DAPI) and 2 ,7 -dichloro-
graphically independent Cu(II) ion is pentacoordinated by two uoresceindiacetate (DCFDA) were purchased from Sigma
nitrogen atoms (two bipy-N/phen-N) and three oxygen donors (Delhi, India). Dulbecco's Modied Eagle Medium (DMEM),
(two pyrazolone-O and one nitrate-O) in all the three complexes Trypsin Phosphate Versene Glucose (TPVG) solution trypsin
exhibiting square pyramidal geometry.
and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were
The synthesized metal complexes were screened for anti- purchased from HiMedia Laboratories Pvt. Ltd. (Bombay,
cancer activities towards human lung carcinoma (A549) and India). Fetal bovine serum (FBS) was purchased from Biosera
human lung epithelial cells (L132). Anti-cancer property of (Ringmer, East Sussex UK) and dimethyl sulfoxide (DMSO)
complexes 1–4 was established through a series of uorescent was purchased from the Sisco Research Laboratories Pvt.
0
staining and gene expression studies. Complexes 1–4 could Ltd. (Mumbai, India). Rhodamine 123, 4 ,6-diamidino-2-
0
0
elevate intracellular oxidative stress, alter nuclear morphology phenylindole (DAPI) and 2 ,7 -dichlorouoresceindiacetate
and induce apoptosis possibly via BCl2 independent pathway. (DCFDA) were purchased from Sigma (Delhi, India).
These results are encouraging and warrant a detailed in vivo
study to establish anti-cancer potential of these complexes. All
Characterization techniques
the results of this study provide guidance for designing struc-
turally modied pyrazolones with specic cytotoxicity towards
human lung cancer cells.
The synthesized compounds were characterized using different
techniques. Elemental analyses (C, H, N) of the synthesized
17112 | RSC Adv., 2017, 7, 17107–17116
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
compounds were performed on a model 2400 Perkin-Elmer acetate. The resultant mixture was reuxed for 4 h. The solid
ꢀ1
elemental analyzer. Infrared spectra (4000–400 cm , KBr discs) green crystalline product obtained was ltered off, washed with
of the samples were recorded on a model RX 1 FT-IR Perkin- hot water followed by methanol and dried. Synthesis of complex
Elmer spectrophotometer. The electronic spectra (in DMF at 1 is summarized in Scheme 1.
ꢁ
room temperature) in the range of 400–800 nm were recorded on
Green; yield 76.89%, mp
19CuN
>
250 C. Anal. calc. for
a model Perkin Elmer Lambda 35UV-VIS spectrophotometer. LC-
23
C H
5 5
O : MW: 508.97, C (58.28%), H (3.76%), N (13.76%),
MS of the compounds were recorded at SAIF, Panjab University, Cu (12.49%), found: C (58.35%), H (3.56%), N (13.89%), Cu
ꢀ1
Chandigarh. A simultaneous TG/DTA was carried out on a SII (12.67%). IR (KBr, cm ): 1566 (s) (C]N, cyclic), 1610 (m) (C]O),
EXSTAR6000 TG/DTA 6300. The experiments were performed in 585 (s) (Cu–N), 422 (s) (Cu–O); lmax/nm 670 (dxz, dyz / d
2
ꢀy2, square
x
ꢁ
ꢀ1
ꢀ1
2
ꢀ1
N at a heating rate of 10 C min in the temperature range 25– pyramidal); molar conductance (DMF, U cm mol ): 11.00.
2
ꢁ
ꢀ3
7
50 C using a platinum pan. Molar conductivity of 10
solutions of the complexes in DMF was measured at room Cu(NO ) $3H O and 2,2 -bipyridyl. The solid green crystalline
M
[Cu(L1)(Bipy)]NO (2). It was prepared analogously from L1,
3
0
3
2
2
temperature with a model Elico CM 180 digital direct reading product obtained was ltered off, washed with methanol and
deluxe digital conductivity meter. Gravimetric and volumetric dried. Synthesis of complex 2 is summarized in Scheme 2.
ꢁ
analysis were performed for the determination of copper aer
Green; yield 68.99%, mp > 250 C. Anal. calc. for
decomposition of complexes in nitric acid.
C
22
H21CuN
5 5
O : MW: 498.98, C (52.96%), H (4.24%), N (14.04%),
Cu (12.74%), found: C (52.76%), H (4.58%), N (13.98%), Cu
ꢀ1
(
12.83%). IR (KBr, cm ): 1584 (s) (C]N, cyclic), 1519 (m) (C–O),
Preparation of 4-acyl pyrazolones
5
92 (s) (Cu–N), 432 (s) (Cu–O); lmax/nm 673 (d , d / d
2
2
,
xz
ꢀ
yz
x ꢀy
1
2
ꢀ1
3
-Methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazole-4-carbaldehyde square pyramidal); molar conductance (DMF, U cm mol ):
(
L1). 3-Methyl-1-phenyl-1H-pyrazol-5(4H)-one (17.4 g, 0.1 mol) 11.00.
3
was dissolved in hot dioxane (80 cm ) in a ask equipped with
[Cu(L2)(Phen)]NO
3
(3). It was prepared analogously from L2,
a stirrer, separating funnel and reux condenser. Calcium Cu(NO
3
)
2
$3H O and 1,10-phenanthroline. The solid green
2
hydroxide (14.81 g, 0.2 mol) was added to this solution, followed crystalline product obtained was ltered off, washed with
by formyl chloride (6.44 g, 0.1 mol) added drop wise with methanol and dried. Synthesis of complex 3 is summarized in
precaution, as this reaction was exothermic. During this addi- Scheme 1.
ꢁ
tion the whole mass was converted into a thick paste. Aer the
complete addition, the reaction mixture was reuxed for half an
Green; yield 73.45%, mp > 250 C. Anal. calc. for
C H27CuN O : MW: 649.15, C (62.91%), H (4.19%), N (10.79%),
34 5 5
hour and then it was poured into dilute hydrochloric acid (200 Cu (9.79%), found: C (62.60%), H (4.46%), N (10.50%), Cu
3
ꢀ1
cm , 2 M). The coloured crystals (TPMP) thus obtained were (9.80%). IR (KBr, cm ): 1599 (s) (C]N, cyclic), 1487 (m) (C–O),
separated by ltration and recrystallized from an acidied 583 (s) (Cu–N), 412 (s) (Cu–O); lmax/nm 672 (dxz, dyz / d
2
2
,
x
ꢀy
ꢀ1
ꢀ
1
2
methanol–water mixture (HCl : MeOH : H
2
O ¼ 1 : 80 : 19).
square pyramidal); molar conductance (DMF, U cm mol ):
ꢁ
L1 is brown crystalline solid. Yield 82.45% mp 95 C. Anal. 10.00.
calc. for C11
H
10
N
2
O
2
MW: 202.21, C (65.34%), H (4.98%), N
[Cu(L2)(Bipy)]NO
13.85%), found: C (65.76%), H (4.68%), N (13.59%); H NMR Cu(NO $3H
CDCl ): d ppm 2.43 (s, 3H, PZ C–CH
.53 (s, 1H, formyl). IR (KBr, cm ): 1527 (s) (C]N, cyclic), 1636 dried. Synthesis of complex 4 is summarized in Scheme 2.
3
(4). It was prepared analogously from L2,
O and 2,2 -bipyridyl. The solid green crystalline
), 7.28–7.81 (m, 5H, Ph), product obtained was ltered off, washed with methanol and
3
1
0
(
(
3
)
2
2
3
ꢀ1
9
+
ꢁ
(
2
m) (C]O, pyrazolone ring); MS: m/z ¼ 202.14 [C H N O ] ,
Green; yield 72.38%, mp > 250 C. Anal. calc. for
27CuN : MW: 625.13, C (61.48%), H (4.35%), N (11.20%),
-(1-Naphthoyl)-3-methyl-1-(p-tolyl)-1H-pyrazol-5(4H)-one (L2). Cu (10.17%), found: C (61.78%), H (4.54%), N (11.36%), Cu
1
1
11 2 2
+
+
+
01.32 [M ꢀ 1] , 185.29, [C H N O ] , 128.51 [C H N O ] .
C
32
H
5 5
O
1
0
7
2
2
5
6 2 2
4
ꢀ1
It was prepared analogously from 3-methyl-1-(p-tolyl)-1H-pyrazol- (10.89%). IR (KBr, cm ): 1601 (s) (C]N, cyclic), 1472 (m) (C–O),
5
(4H)-one and 1-naphthoyl chloride. The coloured crystals (L2) 581 (s) (Cu–N), 417 (s) (Cu–O); lmax/nm 670 (dxz, dyz / d
x
2
ꢀy
ꢀ1
2
,
ꢀ
1
2
thus obtained were separated by ltration and recrystallized.
square pyramidal); molar conductance (DMF, U cm mol ):
ꢁ
+
+
+
L2 is brown crystalline solid. Yield 71.20% mp 110 C. Anal. 12.00; MS: m/z ¼ 562 [C32
H
27
N
O
2
] , 561 [M ꢀ 1] , 563 [M + 1] .
4
18 2 2
calc. for C22H N O MW: 342.39, C (77.17%), H (5.30%), N
1
(
(
8.18), found: C (77.02%), H (5.45%), N (8.41%); H NMR
Single crystal X-ray structure determination
.28–8.03 (m, 11H, Ph & naphthyl). IR (KBr, cm ): 1554 (s) (C] Crystals having good morphology were chosen for three-
CDCl ): d ppm 1.66 (s, 3H, PZ C–CH ), 2.42 (s, 3H, N-TL C–CH ),
3
3
3
ꢀ
1
7
N, cyclic), 1607 (m) (C]O, pyrazolone ring); MS: m/z ¼ 342.54 dimensional intensity data collection. X-ray intensity data of
+
+
+
[
[
C
C
22
H
H
18
N
N
2
O
O
2
] , 341.22 [M ꢀ 1] , 213.32 [C12
H
10
N
2
O
2
] , 342.54 some of the complexes were collected at room temperature on
+
+
22
18
2
2
] , 127.27 [C
5
H
6
N
2
O
2
] .
X'calibur CCD area-detector diffractometer equipped with
ꢁ
Syntheses of mixed-ligand Cu(II) complexes
Cu(L1)(Phen)]NO (1). To the solution of Cu(NO
graphite monochromated MoKa radiation (l ¼ 0.71073 A). The
[
3
3
)
2
$3H
2
O
crystals used for data collection was of suitable dimensions. The
(1 mmol, 0.241 g) in methanol (5 mL), a solution of L1 (1 mmol, unit cell parameters were determined by least-square rene-
0.202 g) in methanol (10 mL) was added slowly while stirring. To ments of all reections in both cases. All the structures were
this, a solution of 1,10-phenanthroline (1 mmol, 0.198 g) in solved by direct method and rened by full-matrix least squares
methanol (5 mL) was added, followed by small amount of Na- on F2. Data were corrected for Lorentz, polarization and multi-
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 17107–17116 | 17113

View Article Online
RSC Advances
scan absorption correction. The structures were solved by Gene expression studies
Paper
43
44
direct methods using SHELXS97. All non-hydrogen atoms of
the molecule were located in the best E-map. Full-matrix least-
squares renement was carried out using SHELXL97.
Hydrogen atoms were placed at geometrically xed positions
and allowed to ride on the corresponding non-H atoms with
Total RNA was isolated from the A549 cells using TRIzol reagent
(Invitrogen). The quantity and quality of the RNA was assessed
44
by their absorbance at 260 and 280 nm. 1 mg of total RNA was
reverse-transcribed using iScriptcDNA Synthesis Kit (Bio-Rad).
The primer sequences used to amplify the genes were as
ꢁ
C–H ¼ 0.93–0.96 A, and Uiso ¼ 1.5Ueq of the attached C atom for
0
0
follows: forward primer 5 -GGCCCTTTTGCTTCAGGGTT-3 and
methyl H atoms and 1.2Ueq for other H atoms. Atomic scattering
0
0
reverse primer 5 -GAAAAAGGCTCACCGTCGA-3 Bax; forward
factors were taken from International Tables for X-ray Crystal-
0
0
primer 5 -GTCATGTGTGTGGAGAGCGT-3 and reverse primer
45
lography (1992, Vol. C, Tables 4.2.6.8 and 6.1.1.4). An ORTEP
0
0 0
0
and reverse primer 5 -
5
-GGAAACACCTTGACATGCCG-3 BCl2; forward primer 5 -
view of three complexes with atomic atomic labelling is shown
in Fig. 1. The geometry of the molecule has been calculated
0
GCTCTCTGCTCCTCCTGTTC-3
0
CAGTTCCGACTCTTGCCCT-3 GAPDH. Amplication was per-
formed using Thermo Scientic DremTaq green PCR master
mix according to the manufacturer's instructions in a thermal
cycler T100 (Bio-Rad). Data were normalized to the internal
control GAPDH.
46
47
using the soware PLATON and PARST.
Cell culture
Human lung carcinoma (A549) and human lung epithelial cells
5
(
L132) were seeded in T25 asks (1 ꢃ 10 cells per ask) and
ꢁ
Statistical analysis
maintained at 37 C with 5% CO
2
in water jacketed incubator
(Thermo scientic, forma series II 3111, USA). Dulbecco's Data was analyzed for statistical signicance using one way
minimum eagle's medium (DMEM; with 10% FBS and 1% analysis of variance (ANOVA) followed by Bonferroni's multiple
antibiotic-antimycotic solution) was used for cell culture. Cells comparison test and results were expressed as mean ꢂ SEM
were sub-cultured every third day by trypsinization with TPVG using Graph Pad Prism version 3.0 for Windows, Graph Pad
solution. All reagents were ltered through 0.22 m lter Soware, San Diego, California, USA.
purchased from Millipore Biomedical Aids Pvt. Ltd, Pune. Metal
complexes 1–4 were solubilised in DMSO in a way that the total
content of DMSO does not exceed 1%.
Acknowledgements
Financial assistance received from the University Grants
Commission in terms of major research project to RNJ, New
Delhi is gratefully acknowledged. We would like to thank Head,
Department of Chemistry providing necessary facilities. RVD
acknowledges research funding from Gujarat Council on
Science and Technology (GUJCOST/MRP/2014-15/2553) for
conducting biological studies. The help from Dr. Hemant
Mande & Prof. Vivek Gupta in crystallographic data are grate-
fully acknowledged.
Cytotoxicity studies
Cell viability was determined by MTT assay. A549 and L132 cells
3
(
7 ꢃ 10 cells per well) were maintained in 96-well culture plates
as mentioned above for 24 h in absence or presence of metal
complexes. Later, 10 mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide (MTT, 5 mg mL ) was added
to each wells and incubated for 4 h at 37 C. Aer that wells were
washed with Phosphate Buffer Saline. Resulted formazan is
dissolved in 150 mL per well with DMSO and measured at
ꢀ
1
ꢁ
References
540 nm using ELX800 Universal Microplate Reader.
1
(a) P. C. McDonald, S. C. Chafe and S. Dedhar, Front. Cell
Dev. Biol., 2016, 4, e27; (b) D. Hanahan and
R. A. Weinberg, Cell, 2011, 144, 646–674; (c) C. T. Supuran,
J. Clin. Oncol., 2012, 3, 98–103; (d) N. M. A. Gawad,
N. H. Amin, M. T. Elsaadi, F. M. M. Mohamed, A. Angeli,
V. De Luca, C. Capasso and C. T. Supuran, Bioorg. Med.
Chem., 2016, 24, 3043–3051.
Determination of reactive oxygen species (DCFDA staining)
A549 cells were cultured as mentioned earlier and aer 18 h of
treatment with metal complexes, cells were incubated with 10
mM 2,7-dichlorodihydrouoroscein diacetate (CM-H2-DCFDA)
ꢁ
at 37 C for 30 min. Control and treated cells were observed
and photographed on EvosFloid cell imaging station (Life
Technologies, USA).
2
(a) B. W. Stewart and C. C. Wild, International Agency for
Research on, O. World Health, World cancer report 2014; (b)
G. Ma, W. Luo, J. Lu, D.-L. Ma, C.-H. Leung, Y. Wang and
X. Chen, Chem.-Biol. Interact., 2016, 253, 1–9.
Determination of nuclear morphology (DAPI staining)
5
A549 cells (5 ꢃ 10 cells per well) were plated in a 6-well plate.
3 (a) C. T. Supuran and A. Scozzafava, Expert Opin. Ther. Pat.,
2004, 14, 25; (b) J. Y. Winum, A. Maresca, F. Carta,
A. Scozzafava and C. T. Supuran, Chem. Commun., 2012,
8177; (c) A. Casini, A. Scozzafava and C. T. Supuran, Expert
Opin. Ther. Pat., 2002, 12, 1307.
Later, cells were treated with or without metal complexes at
ꢁ
3
7 C for 24 h, washed with PBS and xed with 70% ethanol, re-
ꢀ1
washed with PBS and incubated with DAPI (1 mg mL in PBS)
for 10 min. Cells were examined to assess changes in nuclear
morphology (condensed/fragmented nuclei) and photographed
on EvosFloid cell imaging station (Life Technologies, USA).
4 (a) T. Ma, D. A. Fuld, J. R. Rigas, A. E. Hagey, G. B. Gordon,
E. Dmitrovsky and K. H. Dragnev, Chemotherapy, 2012, 58,
17114 | RSC Adv., 2017, 7, 17107–17116
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
3
21; (b) N. Touisni, A. Maresca, P. C. McDonald, Y. Lou,
Y. Li, J. Zhaod, C.-C. He, L. Zhanga, S.-R. Sun and G.-C. Xu,
J. Inorg. Biochem., 2015, 150, 28–37.
23 M. F. Brana, A. Gradillas, A. G. Ovalles, B. Lopez, N. Acero,
F. Llinares and D. M. Mingarro, Bioorg. Med. Chem., 2006,
14, 9–16.
A. Scozzafava, S. Dedhar, J. Y. Winum and C. T. Supuran, J.
Med. Chem., 2011, 54, 8271.
(a) P. F. Liguori, A. Valentini, M. Palma, A. Bellusci,
S. Bernardini, M. Ghedini, M. L. Panno, C. Pettinari,
5
F. Marchetti, A. Crispini and D. Pucci, J. Chem. Soc., Dalton 24 D. Castagnolo, F. Manetti, M. Radi, B. Bechi, M. Pagano,
Trans., 2010, 39, 4205; (b) N. S. Rukk, L. G. Kuzmina,
D. V. Albov, R. S. Shamsiev, S. N. Mudretsova,
A. De Logu, R. Meleddu, M. Saddi and M. Botta, Bioorg.
Med. Chem., 2009, 17, 5716–5721.
G. A. Davydova, V. M. Retivov, P. A. Volkov, 25 A. E. Hassan, A. H. Moustafa, M. M. Tolbah, H. F. Zohdy and
V. V. Kravchenko, G. N. Apryshko, A. N. Streletskii, A. Yu. A. Z. Haikal, Nucleic Acids, 2012, 31, 783–800.
Skryabina, E. A. Mironova and V. V. Zamalyutin, 26 A. Kimata, H. Nakagawa, R. Ohyama, T. Fukuuchi, S. Ohta,
Polyhedron, 2015, 102, 152–162.
T. Suzuki and N. Miyata, J. Med. Chem., 2007, 50, 5053–
6
7
A. M. Evangelou, Crit. Rev. Oncol. Hematol., 2002, 42, 249–
5056.
265.
27 X. H. Wang, X. K. Wang, Y. J. Liang, Z. Shi, J. Y. Zhang,
L. M. Chen and L. W. Fu, Chin. J. Cancer, 2010, 29, 980–
987.
(a) M. Jamshidi, R. Youse, S. M. Nabavizadeh, M. Rashidi,
M. G. Haghighi, A. Niazi and A. A. Moosavi-Movahedi, Int.
J. Biol. Macromol., 2014, 66, 86–96; (b) J. Zhao, L. Zhang, 28 M. H. Norman, L. Liu, M. Lee, N. Xi, I. Fellows,
J. Li, T. Wua, M. Wanga, G. Xu, F. Zhang, L. Liu, J. Yang
N. D. D'Angelo, C. Dominguez, K. Rex, S. F. Bellon,
and S. Sun, Chem.-Biol. Interact., 2015, 231, 1–9.
T. S. Kim and I. Dussault, J. Med. Chem., 2012, 55, 1858–1867.
8
(a) V. Gandin, F. Tisato, A. Dolmella, M. Pellei, C. Santini, 29 (a) E. Budzisz, I.-P. Lorenz, P. Mayer, P. Paneth,
M. Giorgetti, C. Marzano and M. Porchia, J. Med. Chem.,
014, 57, 4745–4760; (b) A. Kellett, M. O'Connor,
M. McCann, O. Howe, A. Casey, P. McCarron, K. Kavanagh,
M. McNamara, S. Kennedy, D. D. May, P. S. Skell,
D. O'Shea and M. Devereux, MedChemComm, 2011, 2, 579–
L. Szatkowski, U. Krajewska, M. Rozalskid and
M. Miernicka, New J. Chem., 2008, 32, 2238–2244; (b)
J. S. Casas, E. E. Castellano, J. Ellena, M. S. Garcıa-
Tasende, M. L. P ´e rez-Parall ´e , A. S ´a nchez, A. S ´a nchez-
Gonz ´a lez, J. Sordo and A. Touceda, J. Inorg. Biochem., 2008,
102, 33–45; (c) E. Budzisz, M. Malecka, B. K. Keppler,
V. B. Arion, G. Andrijewski, U. Krajewska and M. Rozalski,
Eur. J. Inorg. Chem., 2007, 3728–3735.
2
584.
9
1
1
A. Kumar and R. Kumar, Int. Res. J. Pharm., 2011, 2, 11–12.
0 N. B. Patel and F. M. Shaikh, Sci. Pharm., 2010, 78, 753–765.
1 (a) S. Bala, S. Kamboj and A. Kumar, J. Pharm. Res., 2010, 3, 30 S. A. De Pascali, D. Migoni, M. Monari, C. Pettinari,
2
993–2997; (b) A. Abbas, S. Murtaza, M. N. Tahir, S. Shamim,
F. Marchetti, A. Muscella and F. P. Fanizzi, Eur. J. Inorg.
Chem., 2014, 1249–1259.
31 X. H. Wang, D. Z. Jia, Y. J. Liang, S. L. Yan, Y. Ding,
L. M. Chen, Z. Shi, M. S. Zeng, G. F. Liu and L. W. Fu,
Cancer Lett., 2007, 249, 256–270.
M. Sirajuddin, U. A. Rana, K. Naseem and H. Raque, J. Mol.
Struct., 2016, 1117, 269–275.
2 N. Raman, A. Kulandaisamy, A. Shunmugasundaram and
1
K. Jeyasubr-amanian, Transition Met. Chem., 2001, 26, 131–
1
35.
32 K. M. Vyas, R. V. Devkar, A. Prajapati and R. N. Jadeja, Chem.-
Biol. Interact., 2015, 240, 250–266.
1
1
3 T. Yoshikuni, J. Mol. Catal. A: Chem., 1999, 148, 285–288.
4 B. A. Uzoukwu, P. U. Adiukwu, S. S. Al-Juaid, P. B. Hitchcock 33 K. M. Vyas, R. N. Jadeja, D. Patel, R. V. Devkar and
and J. D. Smith, Inorg. Chim. Acta, 1996, 250, 173–176. V. K. Gupta, Polyhedron, 2014, 80, 20–33.
5 Z. Y. Yang, R. D. Yang, F. S. Li and K. B. Yu, Polyhedron, 2000, 34 K. M. Vyas, R. N. Jadeja, D. Patel, R. V. Devkar and
9, 2599–2604. V. K. Gupta, Polyhedron, 2013, 65, 262–274.
6 W. F. Yang, S. G. Yuan, Y. B. Xu, Y. H. Xiao and K. M. Fang, J. 35 W. J. Geary, Coord. Chem. Rev., 1971, 7, 81–122.
1
1
1
1
Radioanal. Nucl. Chem., 2003, 256, 149–152.
36 S. S. Massoud, F. A. Mautner, M. A. M. Abu-Youssef and
7 P. Chiba, W. Holzer, M. Landau, G. Bechmann, K. Lorenz,
N. M. Shuaib, Polyhedron, 1999, 18, 2061–2067.
B. Plagens, M. Hitzler, E. Richter and G. Ecker, J. Med. 37 D. Madamwar, D. K. Patel, S. N. Desai, K. K. Upadhyay and
Chem., 1998, 41, 4001–4011. R. V. Devkar, EXCLI J., 2015, 14, 527–539.
8 G. C. Xu, L. Zhang, L. Liu, G. F. Liu and D. Z. Jia, Polyhedron, 38 M. C. Thounaojam, R. N. Jadeja, M. Valodkar, P. S. Nagar,
1
1
2008, 27, 12–24.
R. V. Devkar and S. Thakore, Food Chem. Toxicol., 2011,
49(11), 2990–2996.
9 X. H. Wang, D. Z. Jia, Y. J. Liang, S. L. Yan, Y. Ding,
L. M. Chen, Z. Shi, M. S. Zeng, G. F. Liu and L. W. Fu, 39 H. Kim, G. R. Lee, J. Kim, J. Y. Baek, Y. J. Jo, S. E. Hong and
Cancer Lett., 2007, 249, 256–270. H. M. Kim, Free Radical Biol. Med., 2016, 91, 264–274.
0 R. N. Jadeja, M. Chhatrola and V. K. Gupta, Polyhedron, 2013, 40 T. W. Sedlak, Z. N. Oltvai, E. Yang, K. Wang, L. H. Boise,
2
2
2
6
3, 117–126.
1 K. M. Vyas, R. N. Jadeja, J. Patel and R. V. Gupta, Polyhedron,
013, 65, 262–274.
C. B. Thompson and S. J. Korsmeyer, Proc. Natl. Acad. Sci.
U. S. A., 1995, 92(17), 7834–7838.
41 J. M. Adams and S. Cory, Science, 1998, 281, 1322–1326.
2
2 (a) B. Shaabani, A. A. Khandar, M. Dusek, M. Pojarova and 42 B. Rostkowska-Nadolska, M. Fraczek, W. Gawron and
K. Mahmoudi, Inorg. Chim. Acta, 2013, 394, 563–568; (b) M. Latocha, Acta Biochim. Pol., 2009, 56, 235–242.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 17107–17116 | 17115

View Article Online
RSC Advances
Paper
4
4
3 Bruker, SMART (Version 5.631), SAINT (Version 6.45) and 45 C. K. Johnson, ORTEP II; Report ORNL-5138, Oak Ridge
SADABS (Version 2.05), Bruker AXS Inc., Madison,
Wisconsin, USA, 2003.
4 G. M. Sheldrick, SHELXS97 and SHELXL97, University of
Gottingen, Germany, 1997.
National Laboratory, Oak Ridge, TN, 1976.
46 A. L. Spek, PLATON for Windows. September 1999 Version,
University of Utrecht, Netherlands, 1999.
47 M. Nardelli, J. Appl. Crystallogr., 1995, 28, 659.
17116 | RSC Adv., 2017, 7, 17107–17116
This journal is © The Royal Society of Chemistry 2017