Synthesis, characterization and biological activity of mixed ligands complexes of quinolin-8-ol and substituted chromones with Mn(II), Co(II), Ni(II) and Cu(II) metal ions

Article abstract of DOI:10.1007/s11164-018-3656-x

The mixed ligand complexes were synthesized using quinolin-8-ol and substituted chromone derivative with transition metals like Mn(II), Cu(II), Ni(II) and Co(II). These complexes were characterized using elemental analysis by electron dispersive spectroscopy, Fourier transforms infrared, ultraviolet–visible, proton nuclear magnetic resonance, liquid chromatography coupled with mass spectrometry, electron spin resonance spectra, magnetic susceptibility, powder X-ray diffraction, thermogravimetric analysis and scanning electron microscopy. The complexes were screened by biological activities such as antioxidant activity by 2, 2-Diphenyl-1-picrylhydrazyl, antimicrobial activity by the agar well-diffusion method and anticancer activity by yellow tetrazolium (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide methods. The synthesis of mixed ligand complexes were synthesized by using homoleptic complexes of respective metal complexes of chromones. The FTIR spectra show ν_M–O the ν_M–N frequencies are obtained at 515–645?cm^?1 and 431–496?cm^{? 1} respectively. The NMR spectra of Ni(II) complexes were indicating the complexes are paramagnetic in nature. The ESR spectra of copper complexes shows singlet signal, and they indicate that the copper has 2 + oxidation state in complexes. The complexes showed a well-defined crystal system indicated by powder-X-ray diffraction patterns. The thermogram studies show formation of metal oxides residues at the end product of decomposition of complexes. The scanning electron microscope showed complexes were nanocrystalline in nature. The mixed ligand complex of Ni(II) shows 80.73% antioxidant activity as compared to both the ligands and other metal complexes. The antibacterial activity result obtained clearly showed that all complexes were effective against all the microorganisms of Staphylococcus aures, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginos. The inhibition of the cancerous cell is more the case of Ni (II) complexes as compared with other complexes.

Full text of DOI:10.1007/s11164-018-3656-x

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-018-3656-x
Synthesis, characterization and biological activity of mixed
ligands complexes of quinolin‑8‑ol and substituted
chromones with Mn(II), Co(II), Ni(II) and Cu(II) metal ions
Nitin H. Kolhe · Shridhar S. Jadhav2
1
Received: 1 July 2018 / Accepted: 27 October 2018
©
Springer Nature B.V. 2018
Abstract
The mixed ligand complexes were synthesized using quinolin-8-ol and substituted
chromone derivative with transition metals like Mn(II), Cu(II), Ni(II) and Co(II).
These complexes were characterized using elemental analysis by electron disper-
sive spectroscopy, Fourier transforms infrared, ultraviolet–visible, proton nuclear
magnetic resonance, liquid chromatography coupled with mass spectrometry, elec-
tron spin resonance spectra, magnetic susceptibility, powder X-ray diꢀraction, ther-
mogravimetric analysis and scanning electron microscopy. The complexes were
screened by biological activities such as antioxidant activity by 2, 2-Diphenyl-1-pic-
rylhydrazyl, antimicrobial activity by the agar well-diꢀusion method and anticancer
activity by yellow tetrazolium (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium
bromide methods. The synthesis of mixed ligand complexes were synthesized by
using homoleptic complexes of respective metal complexes of chromones. The
−
1
FTIR spectra show ν
the ν
frequencies are obtained at 515–645 cm and
M–O
M–N
−
1
4
31–496 cm respectively. The NMR spectra of Ni(II) complexes were indicat-
ing the complexes are paramagnetic in nature. The ESR spectra of copper com-
plexes shows singlet signal, and they indicate that the copper has 2+oxidation state
in complexes. The complexes showed a well-deꢁned crystal system indicated by
powder-X-ray diꢀraction patterns. The thermogram studies show formation of metal
oxides residues at the end product of decomposition of complexes. The scanning
electron microscope showed complexes were nanocrystalline in nature. The mixed
ligand complex of Ni(II) shows 80.73% antioxidant activity as compared to both the
ligands and other metal complexes. The antibacterial activity result obtained clearly
showed that all complexes were eꢀective against all the microorganisms of Staphy-
lococcus aures, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginos. The
inhibition of the cancerous cell is more the case of Ni (II) complexes as compared
with other complexes.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s1116
4
-018-3656-x) contains supplementary material, which is available to authorized users.
Extended author information available on the last page of the article
Vol.:(012
1
3456789)
3

N. H. Kolhe, S. S. Jadhav
Keywords Mixed ligand complexes · XRD · SEM · Antioxidant · Antibacterial ·
Anticancer
Introduction
The inorganic co-ordinationated chelate complexes play an important role in biology
[
1]. Most of the organic heterocyclic compounds are biologically active and show
activity as anti-HIV [2], antiviral [3], antibacterial [4], anti-inꢂammatory [5], anti-
coagulant [6], and anticancer [7], etc. The good example of an organic heterocyclic
compound is chromones [8], and quinolin-8-ol. The mixed ligand metal complexes
play an important role in the medicinal ꢁeld. One of the inorganic anticancer drugs
is Cis-DDP [9]. The biological role of transition metal clusters or complexes are
important in living organisms such as manganese [10], metal in the Photosystem-
II, in a plant body while the Mn-Cluster plays an important role in the human body
for the production of dioxygen [11], as well as cobalt in Vitamin B , and copper in
1
2
ceruloplasmin. The chromones are also important for biological purposes in plants
[
12] and for animals in the case of medicinal purposes. The anthocyanidine [13],
contains the chromone nucleus which is responsible for the color of the ꢂower. The
ligands are biologically active. If we form metal complexes using these ligands then,
the activity may be changed. This can be used for the development of new medicine.
Thus by using the idea of formation of active complexes we have prepared the inor-
ganic coordination complexes of transition metals like Mn(II), Co(II), Ni(II), Cu(II)
with quinolin-8-ol and 6-chloro-4-oxo-4H-chromene-3-carbaldehyde.
Literature surveys show that many chemists have synthesized chromone com-
pound but very few of them worked on the chromones metal complexes [14, 15].
Thus in this paper, we have described the synthesis of ligands and their metal com-
plexes. Further, these were characterized by spectral, thermal, magnetic and some
microanalytical methods. The complexes and ligand were tested for their in vitro
antioxidant activity by DPPH assay, and antimicrobial activity against the gram pos-
itive and gram negative strains using agar well diꢀusion method [16], the complexes
were also screened for anticancer activity.
Experiments
Materials and methods
Metal salts like NiCl ·6H O, CuCl ·2H O, CoCl ·6H O, MnCl ·5H O (99.90%,
2
2
2
2
2
2
2
2
Merck) quinolin-8-ol, POCl (99.98%, Merck), dry Pyridine, anhydrous AlCl
3
3
(
(
99.98%, Merck) ethanol, methanol, DMSO, DMF, chloroform and acetic acid
98.99%, Merck) was used as they were for synthesis of ligand and its metal
complexes.
A melting point of the synthesized compounds was determined on digital
Melting Point/boiling Point apparatus EQ-730 equiptronics and is uncorrected.
The IR spectra (KBr Pellets) were measured on an IRAꢃnity-1 shimadzu FTIR
1
3

Synthesis, characterization and biological activity of mixed…
−1
spectrophotometer in the range 4000–400 cm . The electronic spectra for ligands
and complexes were recorded on a UV-1800 Shimadzu spectrophotometer in the
−
3
range 200–800 nm. The electronic spectra of all the complexes were taken at 10 M
concentration in the DMSO and DMF solutions. The magnetic susceptibility was
recorded on the Gouy balance method at room temperature by using Hg[Co(SCN)
]
as the calibrate. The molar conductance of complexes was recorded on an Elico
4
−4
conductivity bridge in DMF 10 M solution using a dip type conductivity cell ꢁtted
with a platinum electrode.
The thermal analysis was measured from room temperature to 9500–1000 °C in
air and some of the complexes in nitrogen atmosphere heated at non-isothermally
with heating rate 8 °C/min. using a TGA-50 Shimadzu thermogravimetric analyser.
1
H NMR spectral measurements were performed on a Brucker Acend™ 500 MHz
spectrometer. The reported chemical shifts were against TMS, DMSO-d and CDCl
6
3
solvents used for ligands and metal complexes analysis at Department of Chemistry,
Savitribai Phule Pune University. ESR spectra were obtained from a JEOL Japan
JES FA200 ESR Spectrometer with X-band at room temperature. The ESR spectra
were obtained from SAIF, IIT Powai University of Mumbai. Elemental analyses of
C, H and N were done on a JEOL C, H, N elemental analyser at the SAIF, Indian
Institute of Technology (IIT-Powai), Mumbai University. LC-MS measurements of
organic ligand were performed on UHPLC-Ultimate-3000, thermo scientiꢁc LC-MS
spectrometer at Department of Chemistry, Savitribai Phule Pune University and
used for characterization.
Synthesis of ligand
The ligand 6-chloro-4-oxo-4H-chromene-3-carbaldehyde was synthesized by the
Vilsmeier-Haack reaction (Scheme 1) by the literature method [17]. The synthesis of
4
chlorophenylacetate 1a was from 1 mol of 4-chlorophenol by reacting with 1 mol
of acetic anhydride in the presence of 5 mL of dry pyridine by the literature method
18].
The obtained product was then run for the Fries reaction by a reported method
to form 1b [19]. The Fries reaction product was further used for the synthesis of
[
6
-chloro-4-oxo-4H-chromene-3-carbaldehyde. In 1b was obtained 25 mL of dimeth-
ylformamide and then 15 mL POCl was added drop wise by maintaining the tem-
3
perature below 20 °C. The whole reaction mixture was stirred with a magnetic
stirrer for about 1 h to get a pink formulating complex [20]. The reaction mixture
was stirred for a few h and then the reaction mixture was kept at room temperature
for overnight. Then the reaction mixture was poured into crushed ice with vigor-
ous stirring. The product was precipitated as a brown solid of 6-chloro-4-oxo-4H-
chromene-3-carbaldehyde 1c, which was ꢁltered and recrystallized from acetic acid.
Synthesis of the complexes of 6‑chloro‑4oxo‑4H‑chromene‑3‑carbaldehyde
The detailed synthesis procedure and synthetic route for homoleptic complexes are
shown in Scheme 2.
1
3

N. H. Kolhe, S. S. Jadhav
Scheme 1 Synthesis of ligand
Ni(II) complex [C H C NiO ] 1
2
0
10 l4
6
The complex 1 was synthesized by adding a methanolic solution of nickel chlo-
ride hexahydrated (0.237 g, 1 mol) to a hot methanolic (10 mL) solution of
6
-chloro-4-oxo-4H-chmene-3-carbaldehyde (0.416 g, 2 mol) with vigorous stir-
ring. Then this solution was reꢂuxed at 80–90 °C for 6.5 h. After 6.5 h the solu-
tion changes its colour to red solution then 2–3 drops of aqueous ammonia was
added and the solution was kept at room temperature for overnight, after which
the orange coloured solid was ꢁltered and was air dried. The weight of product
was 0.427 g, yield 78.20%.
1
3

Synthesis, characterization and biological activity of mixed…
Scheme 2 Synthetic route for homoleptic complexes
Cu(II) complex [C H Cl CuO ] Cl 2 The copper complex 2 was synthesized by
20
14
2
8
2
adding ligand (0.416 g, 2 mmol) to hot methanol and adding the Cu(II) salt a solu-
tion (0.170 g, 1 mmol in 10 mL methanol) into ligand solution drop wise with con-
stant stirring for 10 min. Then the whole mixture was reꢂuxed for 3 h at 70–80 °C.
The little amount of ammonia was added to get a green color solution which was
kept for overnight to form the dark green colour complex. The complex was ꢁltered
1
3

N. H. Kolhe, S. S. Jadhav
through a Buchner funnel, washed with methanol and dried at atmospheric condi-
tion. The practical yield of a complex is 0.438 g, yield 75.85%.
Co(II) complex [C H Cl CoO ] Cl 3
2
0
14
2
8
2
The complex C H Cl CoO ] Cl was synthesized by reacting the mixture of
2
0
14
2
8
2
CoCl ·6H O (0.237 g, 1 mmol) and 6-chloro-4oxo-4H-chomone-3-carbaldehyde
2
2
(
0.416 g, 2 mmol) in hot methanol under N atmosphere and reꢂuxed for 8 h, during
2
which brown product was precipitated. Most of the solvent was evaporated and then
the product was collected, washed with methanol: H O and dried. The weight of
2
product was 0.390 g, yield 67.24%.
Mn(II) complexes [C H Cl MnO ]Cl 4
20
14
2
8
2
The (0.0416 g 2 mmol) 6-chloro-4-oxo-4H-chromene-3-carbaldehyde ligand was
dissolved in hot methanol. To this hot ligand solution 10 mL methanolic solution of
0
.197 g (1 mol) of Mn(II), the salt solution was added drop by drop with continu-
ous stirring while the temperature was maintained at 80–90 °C for 30 min. Then the
whole mixture was reꢂuxed for 12 h and this solution was kept for overnight. Then
the 2–4 drops of cold water solution were added to maintain the pH. The complex
was a precipitate showing brown colour, which was ꢁltered and washed with metha-
nol. The complex was dried at room temperature. The practical weight of the prod-
uct was 0.421 g, yield: 72.71%.
Synthesis of mixed ligand complexes of quinolin‑8‑ol and 6‑chloro‑4‑oxo‑4H‑
chromene‑3‑carbaldehyde with Ni (II), Cu (II) and Co (II) metal ions
The synthetic route for mixed ligand complexes (Scheme 3) and its preparation is
described as follows.
Ni(II) complex [C H Cl NNiO ]Cl 5
2
9
16
2
7
The complex was synthesized by reꢂuxing the complex 1 (0.271 g, 1 mmol and
quinolin-8-ol (0.0725 g, 1 mmol) in hot methanol for 12 h during which the reac-
tion color changes from orange to green. The reaction mixture was evaporated and
dried. The product was puriꢁed by column chromatography. The practical yield was
0
.229 g, % yield 70.67%.
Cu(II) complex [C H Cl CuNO ]Cl 6
2
9
16
2
7
The complex was synthesized by reꢂuxing the anhydrous complex 2 (0.275 g,
mmol) and quinolin-8-ol (0.0725 g, 1 mmol) in hot methanol: acetic acid (1:1, v/v)
for 12 h during which green product was precipitated. The solvent was evaporated
1
1
3

Synthesis, characterization and biological activity of mixed…
Scheme 3 Synthetic route for mixed ligand complexes
and solid was separated. The product was puriꢁed by washing with methanol and
dried. The practical yield was 0.251 g, yield 76.29%.
Co (II) complex [C H Cl CoNO ]Cl 7
29
16
2
7
The complex was synthesized by reꢂuxing the dehydrated complex 3 (0.271 g,
1
mmol) and quinolin-8-ol (0.0725 g, 1 mmol) in hot methanol under N atmosphere
2
for 12 h, during which chocolate brown coloured product was precipitated. The
solvent was evaporated to dryness, and then the product was ꢁltered, washed with
methanol and dried in vacuum. The practical weight of the complex was 0.243 g,
yield 75.00%.
Mn(II) complex [C H Cl MnNO ] Cl 8
29
16
2
7
The complex was synthesized by reꢂuxing the dehydrated complex 4 (0.271 g,
which was dissolved in hot methanol solution with 1 mL acetic acid and quinolin-
8
-ol (0.072 g, 1 mmol) in methanol, then the mixture was reꢂuxed for 6 h then the
pH was maintained by adding ammonia ammonium chloride buꢀer during which a
brown coloured solution was formed. The solvent was evaporated up to a minimum
amount of the solvent remaining and keep for some time until the product appeared.
Then the product was washed with methanol and dried in vacuum. The practical
yield was 0.216 g, yield 66.46%.
1
3

N. H. Kolhe, S. S. Jadhav
Antioxidant activity
The antioxidant activities of all synthesized complexes were measured by DPPH
assay. The synthesized complexes were dissolved in DMSO with diꢀerent con-
centrations, that is, 20 mg, 40 mg, 60 mg, 80 mg and 100 mg/10 mL. In each con-
centration solution 3 mL of methanolic DPPH solution was added. After 30 min,
the absorbance of the test complexes and ligand was measured at 517 nm using a
UV–Vis spectrophotometer [21]. The standard drug used was ascorbic acid. Free
radical antioxidant activity of DPPH is estimated using the formula,
%DPPH inhibition = Abcontrol − Absample∕Abcontrol × 100,
where, Ab
is the absorbance of DPPH radical+DMSO and Ab
is the
sample
control
absorbance of DPPH radical+sample/standard.
Antimicrobial activity
For this study, the test cultures of bacterial strains Escherichia Coli, Salmonella
typhi, Bacillus subtilis and Staphylococcus aureus were maintained in nutrient
agar slants at 37 °C. The antimicrobial activity of compounds against test bacteria
was determined by the agar well diꢀusion method [22]. The plates containing
2
0 mL of nutrient agar were spread with 100 μL of seeded culture. The wells
were made on these plates with the help of a borer of 9 mm diameter. The test
compounds were loaded (100 μL) into the well as the test sample and DMSO
as negative control. The bacterial plates were incubated in an incubator at 37 °C
for 24 h. Growth was evaluated visually by comparing a particular plate with the
standard antibiotic ciproꢂoxacin.
Anticancer activity by MTT assay
The cytotoxicity was evaluated by MTT [3-4, 5-dimethylthiazol-2-yl)-2,5-diphe-
nyl tetrazolium bromide) according to Mossmann et al. [23]. Hela cell cultures
5
(
5 × 10 cell/ml) were cultured in 96-well ꢂat bottomed microtitreplates and incu-
bated for 48 h. at 37 °C in a humidiꢁed 5% CO incubator. Diꢀerent concen-
2
trations of 25, 50, 75, 100 μL of the synthesized complexes were added to the
wells. The plate was incubated for 48 h. at 37 °C in a humidiꢁed incubator with
5
% CO . MTT (5 mg/ml) was prepared in phosphate buꢀer saline (PBS). MTT
2
(
10 µl) was added to each well and incubated in the dark for 4 h. in CO incuba-
2
tor. The supernatant was removed from the well and the plate was washed three
times with Dulbecco’s formula PBS (pH 7.3). DMSO (100 µl) was added to each
well. The absorbance of the sample was measured at 570 nm after 30 min. The
results of all complexes were compared with standard drug Doxorubicin (10 μM).
1
3

Synthesis, characterization and biological activity of mixed…
Results and discussion
Elemental analysis by electron dispersive X‑ray spectroscopy
Elemental analysis of all complexes was analysed by an EDS/EDX spectrum.
This shows the stoichiometry of complexes matched with the percentage of C, N,
and O. The metal, chloride analysis was also done by EDS spectra; therefore, this
was the great advantage of EDS over C, H, N, O analysis of inorganic coordina-
tion complexes.
The colour, molecular weight, melting point, elemental analysis and conduc-
tivity measurements are illustrated in Table 1 and the EDS spectrum is shown
in Supplementary material Fig. S1. The EDS spectral result match with the cal-
culated % of C, O, N, Cl, and particular metals depends on the complexes, so it
indicates the formation of complexes. The molar conductance measurements of
all the metal complexes 2–8 were found to be electrolytic in nature except for the
complex 1.
Infrared spectra
The most important characteristic frequencies of complexes are shown in Table 2.
The assignments were made by comparison of IR bands of free ligand with its
metal complexes. The IR spectra of both the two ligands behave as bidentate
ligands. The chromone is the O, O donor and neutral ligand, while the quinolin-
8
-ol is the N, O donor and is mononegative ligand.
−
1
The complexes 1–4 show the broadband at 3307–3465 cm is due to ν
and
H2O
the complexes 5–8 show the band is absent because we have used dehydrated com-
plexes for their preparation of mixed ligand-metal complexes. The characteristic
−
1
band of aldhydic carbonyl was found to be 1710 cm in the L2. These frequen-
−1
cies lower by the unit 20–139 cm in the complexes, indicating that the aldehydes
carbonyls donate the pair of electrons toward the metal and forming metal–oxygen
−
1
bond [24]. The ν
frequencies are obtained in the complexes at the 515–645 cm
M–O
and depend on the metal complexes [25]. In the case of the mixed ligand complexes
−
1
5
–8 the ν
frequencies are obtained at 431–496 cm . The L1 donate the electron
M–N
from the C=N atom toward the metal in the case of mixed ligand complexes, in L1
−1
−1
it shows 1540 cm and in the complexes it is lower down by 36 cm and it clearly
indicates a formation bond between ligand and metal in complexes [26].
Electronic spectra of complex and magnetic moments
The electronic spectra (Supplementary material Fig. S2) for the complexes were
scan from 200 to 800 nm on the UV–Vis spectrophotometer. The concentration of
−
3
complexes was in pure DMSO (10 M). The ultraviolet absorption spectrum of
the L1 shows two bands at positions of 275.15 nm and 314.18 nm, and L2 shows
bands at 282.22 nm and 352.73 nm assigned to π → π* and n → π*respectively.
1
3

N. H. Kolhe, S. S. Jadhav
1
3

Synthesis, characterization and biological activity of mixed…
Table 2 The characteristic FTIR band of ligands and their metal-complexes
Sr. nos.
Complexes
υO–H/H O
υC–H
υC=O
υC=N
υC–O
υM–N
υM–O
2
L1
L2
1
[C H NO]
3213
–
3074
2974
2869
2981
3200
3009
2819
2810
2698
2816
–
1540
–
–
–
–
1500
1425
1222
1222
1222
1250
1261
1102
1270
1140
–
–
–
–
–
–
431
496
430
421
–
–
9
7
C H ClO
1695
1612
1631
1612
1631
1690
1643
1571
1625
1
0
5
3
[C H Cl NiO ]
3307
3427
3465
3372
–
–
–
–
515
635
505
607
648
579
533
645
2
0
10
4
6
2
[C H Cl CuO ]Cl
2
0
14
2
8
2
3
[C H Cl CoO ] Cl
2
0
14
2
8
2
4
[C H Cl MnO ]Cl
–
2
0
14
2
8
2
5
[C H C2l NNiO ] Cl
1504
1569
1615
1552
2
9
16
2
7
6
[C H Cl CuNO ]Cl
2
9
16
2
7
7
C H Cl CoNO ]Cl
2
9
16
2
7
8
C H Cl MnNO ]Cl
2
9
16
2
7
Absorption spectrum of complexes 1 displays three bands at 264 nm,
3
3
3
08.24 nm and 361 nm, respectively, that corresponds to the A g (F) → T g(F),
2 2
3
3
3
3
A g(F) → T g(F), and A g(F) → T g(P) transitions, which are referred to an
2
1
2
1
octahedral geometry around Ni(II) ion. The mixed ligand complex of nickel com-
−
1
−1
−1
plex 5 exhibits peaks at 28,409.09 cm , 33,670.03 cm and 36,363.04 cm
6
4
4
6
4
4
attributed A g → Eg ( D), A g → T g ( G) to, respectively, and suggests octa-
1
1
2
hedral geometry. The magnetic moment value of complex 5 is found to be 3.24
BM, suggesting octahedral geometry.
Magnetic momentum value of the Cu(II) complex 6 is 2.10 BM, and complex
2
, 2.06 falls within the range observed for octahedral geometry. Further, the elec-
−
1
−1
tronic spectra of Cu(II) complex shows peaks at 27,139.25 cm , 28,762.08 cm ,
−1
2
2
3
2,062.59 cm
due to transition between Eg→ T g indicating octahedral
2
geometry [27] and the colour of the complex is due to d-d transition. Co(II) com-
−1
−1
−1
plexes 3 and 7 show bands at 32,825.63 cm , 37,740.12 cm , 43,425.40 cm
−1
−1
−1
and 14,450 cm , 19,011 cm , 22,323 cm band, respectively, due to the tran-
4
4
4
4
4
4
sitions T g(F)→ T g(F), T g(F)→ A g(F), T g(F)→ T g(P) and magnetic
1
2
1
2
1
1
moment 5.10BM to 4.95BM suggesting octahedral geometry. The mixed ligand
−
1
Mn(II) complex 8 shows three absorption peaks at 14,868 cm expected for
6
4
−1
6
4
A g→ T g(S), at 18,830 cm corresponding to A g→ T g(G) and a broad
1
1
1
2
−1
6
4
band at 21,518–22,688 cm that may be due to A g→ A g, suggesting octahe-
1
1
dral geometry. Further, the octahedral geometry is proposed based on the magnetic
moment. The magnetic moment of the complex is found to be 5.81 BM, and falls
within the range expected for octahedral geometry. While the complex 4 shows two
bands at 18,975.33 cm− and 23,640.64 cm and is due to the d-d transitions.
1
−1
1H NMR spectra of ligand and complex
1
H NMR Spectra of ligand show six indications. The chemical shift (δ) was found to
be 10.37(s), 8.55(s), 8.28(s), 7.70(dd), 7.52(dd), and 2.51(s) and from this data the
ligand was found and it was pure. The PMR of ligand L2 (Supplementary material
Fig. S3) and the complex are shown in Fig. 1.
1
3

N. H. Kolhe, S. S. Jadhav
Fig. 1 NMR spectrum of Ni(II) mixed ligand metal complex 1 (a) and complex 5 (b)
Complex 1 spectrum Fig. 1a and for complex 5 spectrum Fig. 1b show broad
spectra indicating the Ni(II) complexes are paramagnetic in nature. The peak
obtained for complex 5 δ at 3.3.89 (s) and δ at 2.521 are broad singlets so these are
paramagnetic in nature. The aldehydic proton peak was not seen in spectra therefore
it donates the electron toward the metal and formation of complexes. Similarly for
complex 1 shows two signal δs at 3.383 and a second signal δ at 2.516. Both the
peaks were broad it, and they indicate that there was formation of a complex and the
complex was paramagnetic in nature.
LC‑MS spectra of ligand (L2)
Mass spectrum of ligand L2 shows a molecular ion peak [M + H] at m/z 208.88,
which corresponds to its chemical formula. The LC-MS spectrum of ligand is shown
in Supplementary material Fig. S4.
1
3

Synthesis, characterization and biological activity of mixed…
ESR spectra of Cu(II) complexes 2 and 6
ESR spectra of Cu(II) complexes 2 and 6 (Fig. 2) was recorded at room tempera-
ture in the polycrystalline state, on X-band at frequency of 9107.60 MH under
Z
the magnetic ꢁeld strength of 298.303 mT. The spectrum of complex 2 shows
one intense absorption in the high ꢁeld and is isotropic due to tumbling of the
molecule.
The g values of the complex are of g value 2.322 which is more than the free
ion g value (2.0023) indicating that the unpaired electron in the ground state of
2
2
Cu(II) that is in dx − y . While the ESR spectrum of complex 6 shows superhy-
perꢁne lines. The signal shows the triplet is due to the Cu nucleus coupled with
the nitrogen of the ligand quinolin-8-ol and gives a triplet intense line. So that it
indicates that only one molecule of the quinolin-8-ol binds to the copper metal
and forms a Cu–N bond. It also indicates that the unpaired electron delocalised
through the structures [28].
X‑ray diꢀraction study
The powder XRD pattern (Supplementary material Fig. S5 (a) and (b) of all
the homoleptic and heteroleptic complexes were recorded over the 2θ = 20º–80º
range. The all metal complexes including mixed ligand complexes exhibit a crys-
talline nature with sharp peaks. This XRD study is also one of the pieces of evi-
dence about formation of metal-ligand complexes. The XRD unit cell parameter
such as crystal nature, bravis lattices, diꢀraction angles, cell length, volume, etc.,
are illustrated in Table 3. The XRD peak at the bottom side is broad in all the
complexes to indicate smaller particle size may be in nanometers.
Average size of these complexes was calculated by Scherer’s formula [29].
0
.94ꢀ
D =
ꢁ
cos ꢂ
where λ is wavelength of X-ray radiation, β is the full width at half maximum of dif-
fraction line and θ is the diꢀraction angle.
The average crystallite sizes of all the complexes were found to be in nanom-
eter scale. Therefore, we have taken the SEM pictograph. The XRD pattern gives
the information about the crystal system and the complexes shows a diꢀerent
crystal nature with diꢀerent bravis lattices.
Thermogravimetric analysis of complexes
Thermogravimetric studies (TG) for the complexes were carried out from room tem-
perature to 1000 °C. The stages of decomposition, temperature range, and decompo-
sition products as well as the observed mass loss percentages of all complexes are
studied. The thermogram of all the complexes is shown in Fig. 3(a) and (b).
1
3

N. H. Kolhe, S. S. Jadhav
Fig. 2 ESR Spectra of Cu(II) complexes 2 and 6
1
3

Synthesis, characterization and biological activity of mixed…
1
3

N. H. Kolhe, S. S. Jadhav
(
a)
25
20
15
10
5
0
1
2
3
4
0
100
200
300
400
500
600
700
800
900 1000
Temperature°C
14
(
b)
5
6
7
8
1
2
0
8
6
4
2
0
1
0
100
200
300
400
500
600
700
800
900 1000
Temperature (°C)
Fig. 3 Thermogram of complexes 1–4 (a) and 5–8 (b)
The TG of complex 1 shows single step decomposition between the tempera-
tures 356–382.70 °C with % wt. loss 82.78%, which indicate that organic moiety
and chloride ions are evaporated and the remaining NiO as a product was formed.
The complex 2 decomposes into two steps. In the ꢁrst step for the H Cl O group
4
2
2
there was the loss at temperature 255 °C and in the second step the C H ClO there
1
0
5
3
1
3

Synthesis, characterization and biological activity of mixed…
was the loss at temperature 480.67 °C leaving the CuO as residue [30]. The com-
plex 3 shows one step decomposition in the thermogram. In these steps both the
coordinated water molecules and chromones were lost between the temperatures
1
50–643.82, and the % wt. loss was found to 83.61%; this is a match with calcu-
lated % wt. loss and ꢁnally the formation of cobalt oxide as a product. The thermo-
graph of Mn(II) complex 4 of a homoleptic complex showed the vertical curve at
temperatures 272–342 °C with loss of an organic part of the complexes and forma-
tion of black residue of MnO . Complex 5 showed total % weight loss was found
2
to be 84.582% that matches with calculated wt. loss for 85.64% at temperatures
1
75–650 °C. The complex 5 was decomposed at temperatures 366–568 °C with loss
of both the ligands.
The mixed ligand complexes 6–8 are more stable as compared with their homo-
leptic complexes. The decomposition occurs in one step. The ꢁrst step was due to
the loss of the ligands chromone and quinolin-8-ol simultaneously and remaining is
the metal oxide residue that depends on the metal complexes. All data of the com-
plexes were imputed into Table 4.
Scanning electron microscopy
The surface morphology and the grain size of the metal complexes have been illus-
trated by the scanning electron micrograph. The SEM micrograph of all the metal
complexes is shown in Fig. 4.
Table 4 Thermogravimetric analysis of complexes
Sr. no.
Complexes
Temperature (°C)
% Weight loss
Decomposition assignment
–C H Cl O
found (calculated)
1
2
[C H Cl NiO ]
200–382
82.00 (80.00)
18.00 (20.00)
22.16 (23.49)
43.27 (45.01)
2
0
10
4
6
20 10
4
6
3
82–650
57.37–255
00–689
NiO
–H Cl O
4
[C H Cl CuO ]Cl
2
0
14
2
8
2
4
2
3
–C H ClO
10 5 3
CuO
–C H Cl O
3
4
5
6
7
8
[C H Cl CoO ] Cl
209–643
272–342.15
366–568
82.179 (83.61)
2
0
14
2
8
2
20 14
4
8
1
8.12 (16.97)
89.69 (89.25)
0.12 (11.54)
83.52 (85.64)
6.50 (14.66)
84.95 (85.64)
5.05 (14.35)
Co O4
3
[C H Cl MnO ]Cl
-C H Cl O
20 14 4 8
2
0
14
2
8
2
1
MnO2
–C H Cl NO
[C H Cl NNiO ] Cl
2
9
16
2
7
29 16
2
7
7
7
7
1
NiO
–C H Cl NO
[C H Cl CuNO ]Cl
527.10–650.41
150–443
2
9
16
2
7
29 16
2
1
CuO
–C H Cl NO
C H Cl CoNO ]Cl
80.46 (83.85)
19.33 (16.15)
69.46 (70.77)
2
9
16
2
7
29 16
2
5
45–669.47
CoO
–C H Cl NO
[C H Cl NNiO ] Cl
388.50–570
2
9
16
2
7
29 16
2
3
0.20 (25.78)
MnO2
1
3

N. H. Kolhe, S. S. Jadhav
Fig. 4 SEM micrographs of all metal complexes
The complex 1 shows stone shape nanoparticles with the presence of a well in the
stone, while the complex 2 does not give ꢁne nanoparticles. The complexes 3 and 4
show spherical shape and aggumlation type shape. The formation of the metal complex
has the reduction of phase separation and softness of the surface [31]. The SEM image
shows that there was a change in the morphology of complexes to mixed ligand metal
complexes [32].
SEM micrograph taken in the three diꢀerent scales 5 μm, 10 μm, 20 μm. From the
SEM analysis all complexes have been shown to be nanocrystalline in nature. The
mixed ligand complexes 5–8 shows pod, rough rock, spherical ball and the ꢂower type
shapes, respectively and the nanocrystalline sizes are 10.15, 8.80, 9.27, 13.45 nm.
While for the complexes 1–4 the grain size was found to be deep well, rock, ball, and
stone like morphology, respectively, and the sizes for complexes are 8.52, 5.87, 5.07,
7
.67 nm with the diꢀerent morphology obtained for the diꢀerent complexes. It also
clearly indicates that the morphology was also changing to changes for the homoleptic
complexes that are changes into the heteroleptic complexes.
1
3

Synthesis, characterization and biological activity of mixed…
Fig. 4 (continued)
Biological activity
The biological activity of ligand and their metal complexes were screened for anti-
oxidant activity by DPPH assay, antibacterial activity and anticancer activity by
MTT assay methods.
Antioxidant activity
Free radicals are generated by enzymatic and non-enzymatic processes and if the
colours of these free radicals disappear, it is understood that the compounds have the
ability to scavenge free radicals [33].The antioxidant activity of ligand and synthe-
sized complexes have been evaluated for DPPH radical scavenging assay against the
ascorbic acid that was used as a reference drug at various concentrations. The per-
centage of scavenging activity and the IC value are elaborated in Table 5 and the
5
0
graph was plotted % S.A. versus concentration (mg) in Fig. 5(a) and (b).
The mixed ligand complex 5 of Ni(II) shows 80.73% scavenging activity as com-
pared to both the ligands and other metal complexes. While these Ni(II) complexes
show IC at 40.60 mg. The complexes of Cu(II) show 48.27 percent scavenging
5
0
activity and the IC value is more at 103. 93 mg. The complex of cobalt shows %
5
0
S.A. 66. 62% and IC was 57.12 mg, Mn(II) complex shows % S.A. 45.62% and
5
0
1
3

N. H. Kolhe, S. S. Jadhav
Table 5 Percent scavenging
activity and inhibition
Comp.
% S.A.
IC50 (mg/mL)
concentration of ligands and
their metal complexes
L1
31.45
77.91
78.57
34.44
65.50
45.77
80.73
48.27
66.62
45.62
71.77
164.88
50.45
43.27
251.76
57.67
119.78
40.61
103.93
57.12
109.04
L2
1
2
3
4
5
6
7
8
Ascorbic acid
36.10
IC 109.04 mg. In the case of all these complexes the Ni(II) complex shows very
5
0
good scavenging activity at the least concentration that is below 50 mg. The L1
and L2 show 31.45% and 77.91% scavenging activity as compared with L1, the L2
shows highest % S.A. The chromone has an IC value that was 50.45 mg and qui-
5
0
nolin-8-ol shows 164.88 mg. Thus chromone ligand and Ni(II) complexes showed
good antioxidant complexes [34].
Antimicrobial activity
In antimicrobial activity of the ligands 6-chloro-3-formyl chromone, N,O donor
and their mixed ligand complexes with Mn(II), Co(II), Ni(II) and Cu(II) complexes
against gram positive S. aureus, B. subtilis and gram negative bacteria like E. coli,
P. aeruginos was carried out. Only the mixed ligands synthesized complexes were
dissolved in a minimum quantity of DMSO and adjusted to make up the volume to
get 100 µg/mL, 200 µg/mL, 300 µg/mL, 400 µg/mL, 500 µg/mL concentrations. The
zone of inhibitions was observed and measured to compare with ciproꢂoxacin as
standard drug. The result obtained clearly showed that all complexes were eꢀective
against the entire microorganisms of S. aures, B. subtilis, E. coli, and P. aeruginos-
was. The zone of inhibition was described in Supplementary material Table T1 and
Fig. S6. The Complexes 6 and 7 show more activity as compared with complexes 5
and 8. The antimicrobial activity goes on to increase with increased concentration
in all the complexes [35]. Many researchers found that the less active compound
become more active upon complexion [36]. This is due to the decrease in polarity by
forming a chelate complex [2].
Anticancer activity by MTT assay
The eꢀect of diꢀerent complexes was checked on the cancerous cell line. The
result of the cytotoxicity assay was expressed in terms of percentage inhibition of
1
3

Synthesis, characterization and biological activity of mixed…
100
L1
L2
1
(
a)
90
80
70
60
50
40
30
20
2
3
4
20
40
60
80
100
Concentration (mg)
100
5
(
b)
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
6
7
8
20
40
60
80
100
Concentration (mg)
Fig. 5 Antioxidant activity of ligands L1, L2 and complexes 1–4 (a) and 5–8 (b)
cancerous cells. The control standard drug Doxorubicin (10 Μm) vial and remain-
ing vials contained chemically synthesized metal–ligand complexes with variable
concentration.
The MTT assay was referred to for checking the anticancer activity of the com-
plexes 1–8. The anticancer activity of the complexes was evaluated against the
1
3

N. H. Kolhe, S. S. Jadhav
50
48
46
44
42
40
38
36
34
32
30
28
26
L1
L2
1
2
3
4
5
6
7
8
25
50
75
100
125
150
Concentration (µg /ml)
Fig. 6 Anticancer activity of ligands and complexes
Hela-cell line. All the complexes 1–8 showed good anticancer activity against
the HeLa cell line shown in Fig. 6. Figure 6 was plotted with % cell inhibition
versus concentration in µg/mL of complexes. It clearly indicates that the concen-
tration goes on increasing as the % cell inhibition increases. The complexes 1 and
5
showed excellent activity against the other complexes. The complex 5 shows
6.66% cell inhibition at 100 µg/mL. While 1 shows 45.40% inhibition. These
4
complexes also show good activity at lower concentration of 35.42% and 40.06%
at 25ug/mL concentration. As followed by complexes 1 and 5 complexes 2 and
6
, these showed 42.08% and 41.38%, respectively, at the highest concentration.
The complex 8 shows variable activity with diꢀerent concentration, at highest
concentration the complex 8 showed 43.65% cell inhibition. At lowest concentra-
tion, this complex showed very low cell inhibition 28.05%, as compared with L1,
L2 and all remaining complexes. The complexes of Co(II) 3, 7 showed 40.15,
4
1.66% cell deaths. The Mn(II) complex 4 showed moderate activity, the activ-
ity of homoleptic complex 4 showed good with diꢀerent concentration, but in
case of complex 8 it was diꢀerent. From Fig. 6, this indicates that the concentra-
tion increases with increasing anticancer activity in all the complexes except for
the complex 8. Overall complexes like 1, 5, 2, 6, and 7 show good activity and
the other 3, 4, 8 displayed moderate activity (Supplementary material Table T2).
The result indicates that mixed ligand metal complexes showed good activity
as compared with ligands and homoleptic complexes. This is may be due to the
change in stereochemistry, structure-relationship activity and induction of activ-
ity through the ligand into the metal complexes [37].
1
3

Synthesis, characterization and biological activity of mixed…
Conclusion
We have synthesized heteroleptic complexes from the homoleptic complexes with
good yield. Most of the complexes were more biologically active than the ligands
molecules. The bonding mode of L1 was via phenolic oxygen and nitrogen donor
atoms, while the L2 was bound to metals ions through two carbonyl oxygen atoms.
The bonding modes of ligand have been elucidated by FTIR, ESR spectra. The
geometry of all the chelated complexes were octahedral in nature as was indicated
by electronic and magnetic studies. In the case of antioxidant and anticancer activi-
ties of the heteroleptic Ni(II) complex; it showed higher activity than the other
complexes and the ligands. This has been due to the ligand inducing its activity by
forming the chelate complex due to change in its stereochemistry, potential. The
antimicrobial activity of complexes was more as compared with ligands, especially
heteroleptic complexes that showed higher activity.
Acknowledgements The authors are very grateful to Principal Dr. Muktaja Mathkari, MES, Abasaheb
Garware College, Pune and Principal Dr.B.H.Zaware, New Arts Commerce and Science College Ahmed-
nagar for support and providing a laboratory facility. Authors are thankful to HOD, Prof. R.C.Chikate,
MES, Abasaheb Garware College Pune for encouragement. Authors are thankful for BCUD, Savitribai
Phule Pune University for providing funds under a minor research project (Project No. 14SCI000295).
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Aꢀliations
Nitin H. Kolhe · Shridhar S. Jadhav2
1
*
Nitin H. Kolhe
kolhenitin18@gmail.com
1
2
Department of Chemistry and Research Centre, MES, Abasaheb Garware College, Pune,
Aꢃliated to Savitribai Phule Pune University, Pune 411004, India
Department of Chemistry and Research Centre, New Arts, Commerce and Science College,
Ahmednagar 414401, India
1
3