Synthesis and use of dioxime ligands for treatment of leukemia and colon cancer cells

Article abstract of DOI:10.1002/aoc.3752

Two new vicinal dioxime ligands containing thiosemicarbazone units (L¹H₂ and L²H₂) were synthesized and characterized using ¹H NMR, ¹³C NMR, heteronuclear multiple quantum correlation, mass, infrared and UV–visible spectroscopies, elemental analysis and magnetic susceptibility measurements. In addition, homotrinuclear nickel(II), copper(II) and cobalt(II) complexes with a metal-to-ligand ratio of 3:2 for L¹H₂ and L²H₂ were prepared. Synthesis of nickel(II) complex containing a BF₂ ⁺ bridge was carried out using a precursor hydrogen-bridged nickel(II) complex via the template effect. All metal–ligand complexes were tested against two human cancer cell lines (HL-60 and HT-29) for their antiproliferative and apoptotic activities. The results showed that [Co(L²H)₂(H₂O)₂] and [Ni(L²H)₂] exhibited the strongest antiproliferative activity with I_pC₅₀ values ranging between 5 and 10?μM, while [Co(L²H)₂(H₂O)₂] and [Ni(L²H)₂] both induced necrosis of HT-29 cells and 60 and 65% apoptosis in HL-60 cells, respectively.

Full text of DOI:10.1002/aoc.3752

Received: 30 September 2016
DOI 10.1002/aoc.3752
Revised: 3 January 2017
Accepted: 9 January 2017
F U L L PA P E R
Synthesis and use of dioxime ligands for treatment of leukemia
and colon cancer cells
1,2
3
2
Ilknur Babahan
| Ali Özmen | Kadir Aslan
1
Department of Chemistry, Adnan Menderes
University, Faculty of Science, 09010 Aydin,
Turkey
1
Two new vicinal dioxime ligands containing thiosemicarbazone units (L H and
2
2
1
13
L H ) were synthesized and characterized using H NMR, C NMR, heteronuclear
2
2
Department of Chemistry, Morgan State
multiple quantum correlation, mass, infrared and UV–visible spectroscopies, ele-
mental analysis and magnetic susceptibility measurements. In addition,
homotrinuclear nickel(II), copper(II) and cobalt(II) complexes with a metal‐to‐
University, Baltimore, Maryland 21251,
U.S.A.
3
Department of Biology, Adnan Menderes
1
2
University, Faculty of Science, 09010 Aydin,
Turkey
ligand ratio of 3:2 for L H and L H were prepared. Synthesis of nickel(II) complex
2 2
+
containing a BF2 bridge was carried out using a precursor hydrogen‐bridged
nickel(II) complex via the template effect. All metal–ligand complexes were tested
against two human cancer cell lines (HL‐60 and HT‐29) for their antiproliferative
Correspondence
Ilknur Babahan and Ali Özmen, Department
of Chemistry, Adnan Menderes University,
Faculty of Science, 09010, Aydin, Turkey
Email: ilknurbabahan@yahoo.com;
aozmen@adu.edu.tr
2
2
and apoptotic activities. The results showed that [Co(L H) (H O) ] and [Ni(L H) ]
2
2
2
2
exhibited the strongest antiproliferative activity with I
C
p
50 values ranging between 5
2
2
and 10 μM, while [Co(L H) (H O) ] and [Ni(L H) ] both induced necrosis of HT‐
2
2
2
2
29 cells and 60 and 65% apoptosis in HL‐60 cells, respectively.
KEYWORDS
+
2
antiproliferative, apoptosis, BF ‐capped complex, hydrogen‐bridge complex,
thiosemicarbazone, transition metal complexes, vic‐dioximes
1
| INTRODUCTION
In humans, cancer occurs due to multi‐genetic and epige-
netic molecular events that result in systematic failure in
The chemistry of vic‐dioximes and their numerous transition
homeostatic mechanisms governing normal cell proliferation.
Subsequently, the prevention and/or treatment of cancer is
seemingly very difficult using chemical compounds, which
are designed to arrest or reverse carcinogenic changes before
the appearance of malignancy. In the quest to find a cure for
cancer, researchers worldwide are investigating techniques to
facilitate the fast and simultaneous screening of various novel
compounds for their antitumor activities at molecular and
biochemical levels. Although the random screening of vari-
ous substances is time consuming, several anticancer agents
from plants and marine organisms have been discovered
metal complexes has been widely investigated for their poten-
[1–6]
tial use as analytical reagents,
systems (e.g. vitamin B12 analytical reagents)
potential semiconductor materials due to their columnar
as models for biological
[
1–7]
and as
[
8]
stacking properties. The exceptional stability and unique
electronic properties of these complexes can be attributed to
their planar structure stabilized by hydrogen bonding.
Thiosemicarbazones and their derivatives and transition
metal complexes have been demonstrated to show antitumor,
fungicidal, bactericidal and antiviral activity and have also
been used in metal analysis, telecommunications, optical
[
10–12]
recently.
[
9]
computing and information processing applications.
Cell cycle progression, an important regulated biological
event in normal cells, becomes aberrant or deregulated in
transformed and neoplastic cells. In this regard, targeting
deregulated cell cycle progression and its modulation by
various natural and synthetic agents are gaining widespread
Despite the existence of numerous studies of
thiosemicarbazones, and mono‐ and dioximes in the litera-
ture, the derivatives of vic‐dioximes with thiosemicarbazone
side groups are rarely investigated.
Appl Organometal Chem. 2017;e3752.
wileyonlinelibrary.com/journal/aoc
Copyright © 2017 John Wiley & Sons, Ltd.
1 of 10
https://doi.org/10.1002/aoc.3752

2
of 10
BABAHAN ET AL.
1
2
attention for the control of unchecked growth and prolifera-
2.2 | Synthesis of L H (2) and L H (3)
2 2
[10]
tion in cancer cells.
The expanded knowledge of the
A solution of 1a (1 mmol) or a solution of 1b in 30 ml of
absolute ethanol was added dropwise to a solution of 1c
1 mmol) in 10 ml of absolute ethanol over a 30 min period.
The reaction mixture was stirred overnight at room tempera-
ture. After cooling to 0 °C, the pH of the mixture was raised
to 5.0–5.5 by treatment with NaHCO dissolved in 5 ml of
distilled water, and stirring was continued for 1 h. The
solution was poured into 100 ml of cold water with stirring.
After the end of the period, the yellow precipitate was
filtered, washed thoroughly with distilled water and dried.
The chemical reaction and molecular structure are shown in
Figure 1. Results of the compositional and spectroscopic
analyses are as follows.
L H . Yield 70%; m.p. 216 °C. FT‐IR (KBr, cm ): 3450
(N─H), 3388 (O─H), 3081 (C─Harom), 2920–2859
C─Haliph), 835 (C═S), 1630 (C═Noxime), 1665
C═Nthisemicar), 997 (N─O). H NMR (DMSO, ppm):
10.28–9.42 s, 2H (OH); 9.08–9.00 s, 2H (NH); 7.36 s, 1H
CH═NOH); 8.54 d, 1H (J:8); 8.29 d, 1H (J:8); 8.04 s, 1H;
7.38 t, 1H (Ar‐H); 2.31 s, 3H (─CH ). C NMR (DMSO,
ppm): 179.81 (─C═S), 148.48 (─HN─C═N─OH), 146.31
(─C─CH═N─OH), 150.43 (─CMe═N─N), 134.55 s,
33.42 s, 134.09 s, 133.90 s, 123.90 s (Ar‐C), 14.44 s
(─CH ). UV–visible (DMSO, λmax, nm): 334, 286 and 251.
FAB MS (m/z): 279 [M − 1] . For C10
280.308 g mol ) calcd (%): 42.85 C, 4.32 H, 29.98 N,
11.44 S; found (%): 42.70 C, 4.45 H, 29.50 N, 11.56 S.
L H . Yield 80%; m.p. 241 °C. FT‐IR (KBr, cm ): 3422
N─H), 3385 (O─H), 3017 (C─Harom), 2963–2882
C─Haliph), 852 (C═S), 1632 (C═Noxime), 1674
C═Nthisemicar), 996 (N─O). H NMR (DMSO, ppm):
molecular basis of tumorigenesis and metastasis, together
with the synthesis of active compounds provide unique
opportunities for discovering new drugs that can target the
abnormal molecular and biochemical signals leading to
(
[
13]
cancer.
3
Thiosemicarbazones and their metal ion complexes have
been investigated for their antitumor activities against various
[
14–16]
cancer cell lines.
The understanding and design of
the chemical structures of the compounds are also important
factors to consider for achieving effective antitumoral
[
17–21]
activity.
Previous studies also reported that
thiosemicarbazones and their metal complexes cause apopto-
tic cell death via cell cycle arrest at G2/M check point and
mitochondrial pathway, which was determined with flow
1
−1
2
[15–21]
cytometric or fluorescence microscopic analysis.
(
(
In this paper, we present the synthesis and characteriza-
1
tion of vic‐dioximes containing thiosemicarbazone units
1
2
(
L H and L H ), new homotrinuclear Ni(II), Cu(II) and
2 2
(
Co(II) complexes of these materials, and a nickel(II) complex
13
+
3
containing a BF2 bridge. Synthesized complexes were tested
against two human cancer cell lines (HL‐60 and HT‐29) for
1
2
their antiproliferative and apoptotic features. L H and L H
2
2
1
ligands possess active groups and are thought to interact with
2
3
cancer cells. Co(II) metal complex of L H ligand was effec-
2
+
2
H O N S
12 2 6
tive on HL‐60 cell line and Ni complex of L H ligand was
2
−
1
(
found quite effective on HL‐60 cell line, where 50% of cells
2
2
were inhibited by [Co(L H) (H O) ] and [Ni(L H) ]. These
2
2
2
2
2
−1
2
results provide initial evidence for future research on pharma-
cological studies on how these ligands and their complexes
can be used for the development of new drugs and their effect
on the cell cycle.
(
(
(
1
1
0.38–9.55 s, 2H (OH); 8.40–8.10 s, 2H (NH); 7.77 s, 1H
(
2
(
(
1
CH═NOH); 8.55 d, 2H (J:8); 7.88 d, 2H (J:8) (Ar‐H);
13
.28 s, 3H (─CH3). C NMR (DMSO, ppm): 180.02
146.77 (─HN─C═N─OH), 145.37
─C─CH═N─OH), 150.44 (─CMe═N─N), 145.75 s,
21.38 s, 123.33 s (Ar‐C), 14.05 s (─CH ). UV–visible
2
2
| EXPERIMENTAL
─C═S),
.1 | Materials and measurements
3
1
(DMSO, λ , nm): 334, 286 and 253. FAB MS (m/z): 279
max
All reagents used were purchased from Merck. H NMR and
+
−1
1
3
[M − 1] . For C H O N S (280.308 g mol ) calcd (%):
10 12 2 6
C NMR spectra (Bruker 400 MHz), Fourier transform
4
2.85 C, 4.32 H, 29.98 N, 11.44 S; found (%): 42.70 C,
infrared (FT‐IR) spectra (Varian 900), melting points (Buchi
SPM‐20) and pH measurements (Orion EA 940 expandable
ion analyzer) were used to elucidate the structures of the
products. The magnetic moments of the complexes were
measured using the Gouy method with a Newport type
D‐104 instrument magnet power supply. Mass spectra were
recorded with a Bruker LC/MS/MS‐8030 triple quadrupole
4
.45 H, 29.50 N, 11.56 S.
mass
spectrometer.
(2E)‐2‐[1‐(Pyridin‐3‐yl)ethylidene]
hydrazinecarbothioamide (Figure 1a), (2E)‐2‐[1‐(pyridin‐4‐
yl)ethylidene]hydrazinecarbothioamide (Figure 1b) and
anti‐chloroglyoxime (Figure 1c) were prepared using previ-
[18,22]
1
2
ously reported methods.
2 2
FIGURE 1 Synthesis of ligands (L H and L H )

BABAHAN ET AL.
3 of 10
1
2
.3 | Synthesis of Ni(II), Cu(II) and Co(II)
[Co
3
(L H)
2
.4Cl.(H
2
O)
6
] (2c). Brown; yield 47%; m.p. >
−
1
complexes of ligands (2a–2c, 3a–3c)
300 °C. FT‐IR (KBr, cm ): 3432 (─NH), 3386 (─OH/
H O), 1754 (OH…H), 3056 (C─Harom), 2925–2839
C─Haliph), 815 (C═S), 1661 (C═Nthiosemicar), 1638
C═Noxime), 1014 (N─O), 659 (M─N), 552 (M─S), 450
M─Cl). UV–visible (DMSO, λmax, nm): 590, 459, 355,
73 and 252. FAB MS (m/z): 981 [M − 4] , 792
M − 6H O − 2(C═NOH)] . C H O N Cl S Co
985.301 g mol ) calcd (%): 24.38 C, 3.48 H, 17.06 N,
2
A solution of a metal salt (3 mmol of NiCl ⋅6H O,
CoCl ⋅6H O or CuCl ⋅2H O) in 20 ml of water was
added to 2 mmol of ligand solution (0.560 g of L H
and 0.560 g of L H in 30 ml of ethanol) with stirring.
2
2
(
(
(
2
[
2
2
2
2
1
2
2
2
+
An initial sharp decrease in the pH of the solution from
+
2
20 34 10 12
4
2
3
5.5 to about 3–3.5 was observed. After raising the pH
−1
(
to 5.0–5.5 using 1% aqueous NaOH solution, the reaction
mixture was kept in a hot water bath (60 °C) for 2 h to
complete the precipitation. The precipitated complex
compounds were filtered, washed with water and ethanol,
and dried at room temperature in a vacuum oven.
Structures of complexes are shown in Figure 2. Results
of the compositional and spectroscopic analyses are as
6
.52 S; found (%): 24.68 C, 3.77 H, 17.34 N, 6.87 S.
2
[
Ni (L H) .4Cl] (3a). Brown; yield 58%; m.p. > 300 °C.
3 2
−1
FT‐IR (KBr, cm ): 3394 (─NH), 1775 (OH…H), 3066
(
(
(
C─Harom), 2923–2807 (C─Haliph), 832 (C═S), 1642
C═Nthiosemicar), 1624 (C═Noxime), 1032 (N─O), 642
M─N), 482 (M─S), 445 (M─Cl). H NMR (DMSO, ppm):
1
1
6
2.03–10.21 s, 2H (H─O…H); 7.76–6.79 s, 4H (NH);
follows.
.29 s, 2H (CH═NOH); 7.97 d, 4H (J:8, C ); 6.58 d, 4H
m
1
[
Ni (L H) .4Cl] (2a). Brown; yield 52%; m.p. dec.
3
2
(
J:8, C ); 2.50 s, 6H (─CH ). UV–visible (DMSO, λmax,
o
3
−1
2
3
50 °C. FT‐IR (KBr, cm ): 3422 (─NH), 1743 (OH…H),
080 (C─Harom), 2939–2856 (C─Haliph), 807 (C═S), 1584
nm): 401, 345, 271, 253 and 225. FAB MS (m/z): 875
+
+
[M
−
22
1] , 792 [M
−
2(C═NOH)] . For
(
(
C═Noxime), 1630 (C═Nthiosemicar), 1023 (N─O), 632
M─N), 473 (M─S), 444 (M─Cl). H NMR (DMSO, ppm):
−1
C H O N Cl S Ni (876.490 g mol ) calcd (%): 27.41
20
4
12
4
2
3
1
C, 2.53 H, 19.18 N, 7.21 S; found (%): 27.56 C, 2.45 H,
9.22 N, 7.67 S.
1
6
7
2.15–10.27 s, 2H (H─O…H); 9.93–8.89 s, 4H (NH);
.58 s, 2H (CH═NOH); 8.59 d, 2H (J:8); 8.31 d, 2H (J:8);
1
2
[
Cu (L H) .4Cl] (3b). Brown; yield 43%; m.p. > 300 °C.
3 2
.98 s, 2H; 7.51 t, 2H (Ar‐H); 2.48 s, 6H (─CH ). UV–visi-
−1
3
FT‐IR (KBr, cm ): 3404 (─NH), 1834 (OH…H), 3023
ble (DMSO, λmax, nm): 400, 345, 264, 252 and 235. FAB MS
(
(
(
C─Harom), 2959–2830 (C─Haliph), 824 (C═S), 1625
C═Nthiosemicar), 1590 (C═Noxime), 1019 (N─O), 598
M─N), 519 (M─S), 450 (M─Cl). UV–visible (DMSO, λmax
+
+
(
m/z): 875 [M − 1] , 792 [M − 2(C═NOH)] . For
−1
C H O N Cl S Ni (876.490 g mol ) calcd (%): 27.41
2
0
22
4
12
4
2
3
,
C, 2.53 H, 19.18 N, 7.21 S; found (%): 27.56 C, 2.45 H,
9.22 N, 7.67 S.
nm): 589, 450, 346, 280 and 256. FAB MS (m/z): 890
1
+
+
[M
−
22
1] , 792 [M
−
2(C═NOH)] . For
1
[
Cu (L H) .4Cl] (2b). Brown; yield 40%; m.p. > 300 °C.
−1
3
2
C H O N Cl S Cu (891.048 g mol ) calcd (%): 26.96
C, 2.49 H, 18.86 N, 7.20 S; found (%): 26.45 C, 2.34 H,
1
3
20
4
12
4
2
3
−
1
FT‐IR (KBr, cm ): 3398 (─NH), 1771 (OH…H), 3094
(
(
(
C─Harom), 2925–2859 (C─Haliph), 824 (C═S), 1635
C═Nthiosemicar), 1616 (C═Noxime), 1036 (N─O), 602
8.65 N, 7.56 S.
1
[
Co (L H) .4Cl.(H O) ] (3c). Brown; yield 55%; m.p. >
3 2 2 6
M─N), 519 (M─S), 449 (M─Cl). UV–visible (DMSO, λmax
,
−1
00 °C. FT‐IR (KBr, cm ): 3424 (─NH), 3290 (─OH/
nm): 589, 483, 355, 283 and 246. FAB MS (m/z): 890
H O), 1729 (OH…H), 3064 (C─Harom), 2985–2854
2
+
+
[
M
−
22
1] , 792 [M
−
2(C═NOH)] . For
(
(
(
C─Haliph), 839 (C═S), 1663 (C═Nthiosemicar), 1600
C═Noxime), 1038 (N─O), 615 (M─N), 574 (M─S), 449
M─Cl). UV–visible (DMSO, λmax, nm): 589, 450, 346,
−
1
C H O N Cl S Cu (891.048 g mol ) calcd (%): 26.96
2
0
4
12
4
2
3
C, 2.49 H, 18.86 N, 7.20 S; found (%): 26.45 C, 2.34 H,
8.65 N, 7.56 S.
1
+
2
1
70 and 251. FAB MS (m/z): 981 [M − 4] , 792 [M –
+
− 6H O − 2(C═NOH)] For C H O N Cl S Co
2
20 34 10 12
4
2
3
−
1
(985.301 g mol ) calcd (%): 24.38 C, 3.48 H, 17.06 N,
6
.52 S; found (%): 24.68 C, 3.77 H, 17.34 N, 6.87 S.
+
2
.4 | Synthesis of BF2 bridge‐containing
Ni(II) vic‐dioxime complex
A suspension of Ni(II) vic‐dioxime complex [Ni (L H) .4Cl]
1
3
2
(0.5 mmol) in freshly distilled dry acetonitrile (40 ml) was
brought to reflux temperature under nitrogen atmosphere.
Boron trifluoride diethyl ether complex (0.25 ml, 1 mmol)
in acetonitrile (2 ml) was slowly added while the mixture
was stirred. The resulting reaction mixture was completely
FIGURE 2 Suggested structure of metal complexes (2a–2c and 3a–
c)
3

4
of 10
BABAHAN ET AL.
dissolved within 30 min. After this changing, the mixture was
refluxed 30 min further and the solvent was removed to dry-
ness under reduced pressure. Then the residue was dissolved
in acetonitrile (10 ml) and evaporated to dryness. The last
step was repeated twice and the residue was dissolved in
in concentrations of 5, 10, 20 and 40 μM, and 1 μM paclitaxel
and DMSO in the same final concentration of the highest
chemical concentration. Cells were then incubated for 48 h.
At the end of treatment period, MTT (dissolved in phos-
−
1
phate‐buffered saline, final concentration of 0.5 mg ml )
was added to each well and the plate was further incubated
for 4 h. After incubation, medium containing MTT was
carefully aspirated and the formazan crystals produced by
the living cells were dissolved in 100 μl of DMSO. When
dissolved, absorbance of the samples was measured at
560 nm test and 620 nm reference wavelengths using a plate
reader (Thermo Multiskan SPECTRUM, Waltham, MA).
Based on the absorbance data, concentration‐dependent
living cell graphs were prepared for each chemical
(GraphPad 5.0, La Jolla, CA) and effective concentrations
of chemicals were determined. All experiments were done
in triplicate. The average of the triplicate control optical
densities (ODs) was calculated. Then the percentage of cell
density for treated cells was calculated using the following
ratio: (ODcontrol − ODsample)/(ODcontrol − ODblank).
10 ml of acetonitrile and then allowed to cool in a refrigerator
overnight whereupon the product crystallized from the solu-
tion. The resulting product was filtered and recrystallized
from CHCl –hexane (1:1). The last product was filtered,
3
washed with diethyl ether and dried on P O . Structure of
4
10
the complex is shown in Figure 3. Results of the composi-
tional and spectroscopic analyses are as follows.
1
[
Ni (L ) .4Cl.(BF ) ] (2d). Light brown; yield 55%; m.p.
3
2
2 2
−
1
1
2
1
6
45 °C. FT‐IR (KBr, cm ): 3404 (─NH), 3102 (C─Harom),
920–2865 (C─Haliph), 818 (C═S), 1624 (C═Nthiosemicar),
608 (C═Noxime), 1035 (N─O), 1302 (B─O), 1060 (B─F),
53 (M─N), 520 (M─S), 487 (M─Cl). UV–visible (DMSO,
λmax, nm): 400, 345, 314, 260 and 236. FAB MS (m/z): 973
+
+
[
M + 1] , 876 [M − 2BF ] , 792 [M − 2BF –2(C═NOH)]
.
2
2
+
−1
For C H O N Cl B F S Ni (972.099 g mol ) calcd
20 20 4 12 4 2 4 2 3
(%): 24.71 C, 2.07 H, 17.29 N, 6.60 S; found (%): 24.45 C,
2
.32 H, 17.43 N, 6.82 S.
2
.6 | Apoptotic and necrotic cell
determination
2
.5 | Cell culture
Cell apoptosis was determined using Hoechst 33258 and
HL‐60 promyeloic leukemia cell line and HT‐29 human
colorectal adenocarcinoma cell line were purchased from
ATCC. Cells were grown in RPMI‐1640 medium
supplemented with 10% heat inactivated fetal calf serum,
propidium iodide dyes. HL‐60 and HT‐29 cells were seeded
to 48‐well plates, each well containing 0.1 × 10 cells ml
and incubated overnight. An effective 40 μM concentration
of chemicals was added to the wells and incubated at 37 °C
5
−1
1% L‐glutamine and 1% penicillin/streptomycin at 37 °C
in humidified 5% CO atmosphere. After 48 h of incubation,
2
in a humidified atmosphere containing 5% CO . Paclitaxel
2
one plate was taken from the incubator, medium was
removed and the cells were trypsinized. An amount of
was used as positive control.
5
0 μl of trypsinized cells was collected. Hoechst 33258 and
propidium iodide dyes were mixed in a 1:1 ratio by volume
in a dark room and 5 μl of mixture was applied to trypsinized
cells and incubated for 1 h. After incubation, 30 μl of cell
solution was dripped on a slide, examined using a fluorescent
microscope (Olympus BX51) under UV light and DAPI filter
and photographs were taken. Living cells, apoptotic cells and
necrotic cells were counted.
2
.5.1 | Cell viability: MTT proliferation assay
The effects of chemicals on cell proliferation were measured
using colorimetric 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl-
tetrazolium bromide (MTT) assay. An amount of 400 μl of
5
−1
0.1 × 10 cells ml cell stock was placed into each well of
a 48‐well plate and incubated overnight. Stock solutions
of chemicals were prepared by dissolving in
dimethylsulfoxide (DMSO) and diluting with the medium.
After overnight incubation, cells were treated with chemicals
3 | RESULTS AND DISCUSSION
Compounds 1a and 1b were obtained from condensation of
1
‐(pyridin‐3‐yl)ethanone and 1‐(pyridin‐4‐yl)ethanone with
[22]
thiosemicarbazide in the presence of alcohol.
Subse-
quently,
(2E)‐N‐[(1Z,2E)‐N‐hydroxy‐2‐(hydroxyimino)
ethanimidoyl]‐2‐[1‐(pyridin‐3‐yl)ethylidene]
hydrazine
1
carbothioamide (2, L H ) and (2E)‐N‐[(1Z,2E)‐N‐hydroxyl‐
2
2
‐(hydroxylimino)ethanimidoyl]‐2‐[1‐(pyridin‐4‐yl)ethyli-
2
FIGURE 3 Suggested structure of 2d, the boron‐containing
dene] hydazine carbothioamide (3, L H ) were prepared from
1a and 1b with 1c in the presence of alcohol. Then, Ni(II),
2
1
[18]
[Ni
3
(L )
2
.4Cl] complex (X: N; Y: CH)

BABAHAN ET AL.
5 of 10
1
Cu(II) and Co(II) complexes of the ligands were prepared in
information; FT‐IR spectrum of [Ni (L H) .4Cl]), in which
3
2
ethanol using MCl ⋅xH O as metal salts. M(II)–dioxime
M is Ni(II), Cu(II) or Co(II).2H O, exhibited C═N
and
2
2
2
oxime
1
complex of the type [M (L ) .4Cl.(BF ) ] (M(II) = Ni(II))
C═Nthiosemicar absorption bands at 1584–1638 and
3
2
2 2
[19]
+
−1
was synthesized using a standard procedure.
BF2 bridge
1625–1674 cm , respectively. These bands suggest that the
containing 3‐acetylpyridine thiosemicarbazone glyoxime
complex was synthesized by adding boron trifluoride diethyl
ether complex to a refluxing acetonitrile solution containing
ligands are N,N′ coordinated with the metal ion in correspon-
dence with the proposed structures in Figure 2. The shifting
of ν(C═N)oxime to lower wavenumbers indicates that nitrogen
atom is coordinated to metal ion after the complexation
(Table 1).
1
the appropriate metal–dioxime complex ([Ni (L H) .4Cl]).
3
2
The metal complexes were characterized using mass FT‐IR
and UV–visible spectroscopies, elemental analysis and mag-
netic susceptibility measurements. Attempts to grow X‐ray‐
quality single crystals were unsuccessful.
N─H stretching vibrations of the ligands shift to lower
−1
frequencies in the 3450–3420 cm range suggesting that
nitrogen atom of the oxime group is coordinated to metal
ions. Therefore, O─H stretching vibrations of coordinate
H O and O─H in the complexes probably occur in the same
2
3
.1 | FT‐IR and mass spectra
range as N─H stretching vibrations. The disappearance of
functional groups relating to δOH for compounds 2a–2c
FT‐IR data of the ligands and their complexes are given in
Table 1. The FT‐IR spectra of compounds L H and L H
exhibit ─NH (3450 cm for L H and 3422 cm for
L H ), ─OH (3388 cm for L H and 3385 cm for L H ),
1
2
−1
and 3a–3c at 3388–3385 cm indicates that intramolecular
2
2
−
1
1
−1
hydrogen bonds form with a peak between 1729 and
2
2
−1
1
−1
2
−1
[22–28]
1834 cm for the complexes.
2
2
1
2
−
1
for L H and 1632 cm−1 for
─
C═Noxime (1630 cm
Coordination of sulfur with the metal ion would result in
the displacement of electrons towards the latter, thus resulting
in the weakening of the (C═S) bond. Hence, on complexation
2
2
−1
1
−1
L H ) and ─NO (997 cm for L H and 996 cm for
L H ) stretching vibrations.
2
2
2
[18,19,23]
2
[
18,22]
1
Disappearance of O─H vibrations of the oxime groups
in the FT‐IR spectra of the complexes indicates that oxime
protons are separated upon complex formation and bonded
to the metals. This result is supported by the shifting of
N─O vibrations to frequencies higher than those of free
(C═S) stretching vibrations should decrease.
Therefore,
on complexation the bands at 835–852 cm− are shifted to
lower wavenumbers and the intensity of the bands is also
reduced. All these changes on complexation confidently pre-
clude any unambiguous confirmation of the metal–sulfur
bond. The FT‐IR spectral bands for both the ligands are
unchanged in the complexes, but show additional new bands
[
4,17,22–25]
ligands.
. FAB mass spectral analysis indicated
1
molecular ion m/z ratios of 279 for L H and 279 for
L H , in agreement with the proposed structures
2
2
−1
with medium to weak intensity in the 473–659 cm region,
2
(
Figure 1).
which are assigned tentatively to ν(M─N) and ν(M─S), and
[
18,22]
The FT‐IR spectra of KBr pellets containing the 3‐acetyl‐
are in agreement with previously reported values.
Coor-
and 4‐acetylpyridine thiosemicarbazone glyoxime complexes
dination of the metal ion with sulfur and nitrogen atoms in
the complexes is supported by the appearance of new bands
of the general formula M (LH) (Figure S1b in supporting
3
2
TABLE 1 Characteristic FT‐IR bands (cm− , KBr) of ligands and their metal complexes
1
a
b
–
NH
–Oh/H
2
O
C═S
…H… C═N
C═N
N–O
B–O
B–F
M–N
M–S
M–Cl
1
L H
2
3450 b
3422
3388 b
—
835 m
—
1665 s 1630 s
997 m
—
—
—
—
—
—
—
—
—
—
—
1
[
[
[
[
Ni
3
(L H)
2
.4Cl]
807 m 1720 w 1630 S 1584 s 1023 m
824 m 1771 w 1635 s 1616 s 1036 m
815 m 1754 w 1661 s 1638 s 1014 m
630 m 473 m 444 m
602 m 519 m 449 m
659 m 552 m 450 m
1
Cu
Co
3
(L H)
2
.4Cl]
3398 b
3432 b
3404 b
3422 b
3394 b
3404 b
3420 b
—
1
3
(L H)
2
.4Cl.(H
2
O)
6
]
3386 b
—
1
Ni
3
(L )
2
.4Cl.(BF
2
)
2
]
818 m
852 m
—
—
1624 s 1608 s 1035 m 1302 m 1060 m 653 m 520 m 487 m
2
L H
2
2
3385 b
—
1674 s 1632 s
996 m
—
—
—
—
—
—
—
—
—
—
—
[
[
[
Ni
3
(L H)
2
.4Cl]
832 m 1775 w 1642 s 1624 s 1032 m
824 m 1834 w 1625 s 1590 s 1019 m
839 m 1729 w 1663 s 1600 s 1038 m
642 m 482 m 445 m
598 m 519 m 450 m
615 m 574 m 449 m
2
Cu
3
(L H)
2
2
.4Cl]
.4Cl.(H
—
2
Co
3
(L H)
2
O)
6
]
3290 b
b: broad; s: strong; m: medium; w: weak.
a
Thiosemicarbazone moiety.
b
Oxime moiety.

6
of 10
BABAHAN ET AL.
35
−
at 473–659 cm which can be assigned to ν(M─N) and
1
58
Ni(II) complex (fragments are based on Ni and Cl) shows
[
23]
+
ν(M─S) vibrations.
In addition, ν(M─Cl) is assigned in
molecular ion peaks at m/z 973 [M] (calcd 973), 876
[M − 2BF ] (calcd 876) and 792 [M − 2BF –2(C═NOH)]
(calcd 792), confirming the formation of the desired com-
the 444–487 cm− region.
1
[18,24]
+
2
2
+
The mass spectra of 2c and 3c (Figure S2 in supporting
5
9
35
information; fragments are based on Co and Cl for
pound (Figure 3).
1
[Co (L H) .4Cl.(H O) ]) show molecular ion peaks at m/z
The possibility of thione–thiol tautomerism (H─N─C═S,
C═N─SH; Figure 4) in the ligands was determined by the
3
2
2
6
+
+
9
81 [M − 4] (calcd 981.301), 927 [M − 4 − 3H O]
2
+
−1
(calcd 927.255), 906 [M − 7 − 4H O] (calcd 906.240),
presence of characteristics thiol bands near 2500 cm in
2
+
8
−
1
74 [M – 3 − 6H O] (calcd 874.209), 832 [M − 6H O
(─NOH) − OH] (calcd 832. 227) and 792 [M –
− 6H O − 2(C═NOH)] (calcd 792.175), confirming
the FT‐IR spectra. The observation of strong ν(S─H) absorp-
2
2
+
−1
1
−1
2
tion bands at 2548 cm for L H and 2554 cm for L H in
2 2
+
the FT‐IR spectra of the ligands also suggests that they are in
[20,21,29]
2
the formation of the desired compounds (Figure 1). The
loss of six water molecules, one proton and after the break-
ing of two of the M─NC═OH bonds is a general feature of
the prepared Co(II) complexes and gives the characteristic
peak at m/z 792.
the thiol tautomeric form in the solid state.
3.2 | Magnetic susceptibility
Trinuclear nickel complexes show magnetic moments in the
range 4.50–4.52 BM indicating that the metal centers have
different environments and geometries. It can be concluded
from these data that the nickel atom bonded to the oxime
group is diamagnetic and has a square‐planar geometry,
whereas the other two nickel atoms, each of which is bonded
to the thiosemicarbazone side of the mononuclear nickel
complex with a simple ligand such as Cl─ and SCN─, are
The mass spectrum of [Ni (LH) .4Cl] (fragments are
3
2
58
35
based on Ni and Cl) shows molecular ion peaks at m/z
8
7
+
75 [M − 1] (calcd 875) and 792 [M − 2(C═NOH)] + (calcd
92), confirming the formation of the desired compound
(Figure 1). The mass spectrum of [Cu (LH) .4Cl] (fragments
3 2
6
3
35
are based on Cu and Cl) shows molecular ion peaks at m/
+
+
z 890 [M − 1] (calcd 890) and 807 [M − 2(C═NOH)]
calcd 807), also confirming the formation of the desired
[
30]
(
paramagnetic and have tetrahedral geometry.
compound (Figure 2). For the complexes the highest intensity
The room temperature magnetic moment measurements
peak at m/z 792 for Ni(II) and Co(II) complexes, and at m/z
show that the Co(II) complexes are paramagnetic with mag-
1
8
07 for Cu(II) complexes can be assigned to the removal of
netic susceptibility of 3.43 BM for L H and 3.45 BM for
2
+
2
the ─C═NOH groups, resulting in a [M − 2(C═NOH)] spe-
cies with the highest peak at m/z 792 for Ni(II) and Co(II)
complexes, and at m/z 807 for Cu(II) complexes. The MS‐
determined metal‐to‐ligand ratio is 3:2 for complexes 2a–2c
and 3a–3c. Elemental analysis data also indicate that the
L H per Co(II). The Cu(II) complexes also are found to be
paramagnetic with μ = 1.49 BM for L H and 1.53 BM
for L H per Cu(II) ion. Though these are somewhat low
2
1
eff
2
2
2
values the magnetic moments, there is likely some diamagne-
tism being contributed from the ligand ultimately decreasing
the total paramagnetism of the complexes. It has previously
desired compounds are synthesized.
In the FT‐IR spectrum of the BF ‐capped Ni(II) com-
plex, a shift for the C═N frequencies (1624 to 1608 cm )
+
2
2+
2+
been reported that bridged Cu and Co complexes of
−
1
some vic‐dioximes exhibit moderate to weak antiferromag-
[
31–33]
indicates coordination through the N atoms. The shift
netic interactions.
+
2
1
2
observed in the BF ‐bridged complex is due to the strong
For [Co (L H) .4Cl.(H O) ] and [Co (L H) .4Cl.
3 2 2 6 3 2
electron‐withdrawing effect of the boron linked groups incor-
(H O) ], coordinated H O molecules are identified by a
2 6 2
[17]
−1
porated into the dioxime complex.
The broad band at
743 cm , assigned to the O─H─O bending vibrations, dis-
appears upon insertion of BF groups with the simultaneous
broad OH absorption around 3386–3290 cm , with constant
−
1
[32]
1
intensities after heating above 110 °C for 24 h.
2
−
1
appearance of peaks at 1302 and 1060 cm for the B─O
and B─F resonances, respectively.
3.3 | NMR spectra
[17]
In the FT‐IR spectrum
1
of the boron‐containing metal complex, bands at 3404 and
In the H NMR spectra (data summarized in Table 2), the
035 cm− correspond to N─H and N─O stretching vibra-
1
proton resonances appear as two peaks, low intensity
1
1
tions, respectively. The mass spectrum of the BF ‐bridged
singlets, at 10.28 and 9.42 ppm for L H and 10.38 and
2
2
FIGURE 4 Thione (I) and thiol (II and
1
III) forms of ligands (X: N; Y: CH for L H
2
;
2
2
X: CH; Y: N for L H )

BABAHAN ET AL.
1
7 of 10
13
a,b
TABLE 2 H NMR and C NMR spectral data (δ, ppm) of ligands in DMSO‐d
6
1
H NMR spectra of ligands and Ni(II) complexes
c
c
–
OH
10.28–9.42 s, 2H 9.08–9.00 s, 2H 8.54 d, 1H; 8.29 d, 1H; 8.04 s, 1H; 7.38 t, 1H 7.36 s, 1H
10.38–9.55 s, 2H 8.40–8.10 s, 2H 8.55 d, 2H; 7.88 d, 2H 7.77 s, 1H
.4Cl] 12.15–10.27 s, 2H 9.93–8.89 s, 4H 8.59 d, 2H; 8.31 d, 2H; 7.98 s, 2H; 7.51 t, 2H 6.58 s, 2H
NH
Ar–H
CH═Noh
CH
3
1
L H
2
2
2.31 s, 3H
2.28 s, 3H
2.48 s, 6H
2.50 s, 6H
2
L H
1
[
[
Ni
3
(L H)
2
2
2
Ni
3
(L H)
.4Cl] 12.03–10.21 s, 2H 7.76–6.79 s, 4H
7.97 d, 4H; 6.58 d, 4H
6.29 s, 2H
13C NMR spectra of ligands
C═S
HNC═Noh
148.48
HC═Noh
146.31
MeC═NNH
150.43
Ar–C
CH
3
1
L H
2
2
179.81
180.02
134.55–123.90 14.44
145.75–123.33 14.05
2
L H
146.77
145.37
150.44
a
4
Chemical shifts are reported in ppm relative to SiMe at 30 °C; s: singlet; d: doublet.
b
6
In DMSO‐d .
c
Disappears on D
2
O exchange.
2
9
.55 ppm for L H , respectively. These two D O‐exchange-
2
2
able singlets correspond to two non‐equivalent OH protons,
which also indicate the anti configuration of the ─OH rela-
[
19,23,30,33]
tive to each other.
The chemical shifts related to
NH protons are observed at 9.08 and 9.00 ppm as a singlet
1
2
for L H , at 8.40 and 8.10 ppm as a singlet for L H ,
2
2
respectively, and disappear with D O exchange. Also, the
2
chemical shifts assigned to CH═N─ protons are observed
1
2
at 7.36 ppm for L H and 7.77 ppm for L H as a singlet.
2
2
1
The H NMR spectra of the diamagnetic metal complexes
Ni (L H) .4Cl] and [Ni (L H) .4Cl] are characterized by
3 2 3 2
1
2
[
the absence of the ═N─OH signals for the ligands and the
existence of intramolecular D O‐exchangeable H‐bridge
2
protons is evident from new signals at low field,
1
1
2.15 ppm for [Ni (L H) .4Cl] and 12.03 ppm for
3
2
2
[
Ni (L H) .4Cl]. Consequently, one can conclude that both
d metal ions are coordinated with the dioximato donor sites
in square‐planar geometry.
3 2
8
[34]
The carbon resonances of
oxime groups are observed at 148.48 and 146.31 ppm for
1
2
L H , and 146.77 and 145.37 ppm for L H .
2
2
These non‐equivalent carbon atoms, particularly, are
hydroxyimino carbon atoms, also confirming the anti struc-
2
FIGURE 5 Geometric isomers of Ni(II), Cu(II) and Co(II).2H O
complexes with ligands
1
2
[35]
ture of L H2 and L H .
There is a singlet peak at
2
1
2
1
2.15 ppm for L H and 12.03 ppm for L H in place of
2 2
1
the OH functional group in the H NMR spectra of the
square‐planar complexes (2a and 3a) indicating the existence
of intramolecular complexes that have only one signal at ca
alternative chemical environments will show two O─H⋅⋅⋅O
bridge protons for the cis form but only one for the trans
[
36]
1
12.00 ppm confirming the trans form of the complexes.
form. The observed H NMR spectra of the chelate structures
It is known that Ni(II), Cu(II) and Co(II) metal complexes
of dimethylglyoxime have five‐membered hydrogen bonds
indicate that the metal ion coordinates to the nitrogen atoms
of the dioxime groups.
A hydrogen atom separates each of the two oxime groups,
and thus a hydrogen bridge is formed. Oxime groups possess
[
37]
(O─H─O), which can be easily observed by the deuterium‐
exchange method. The complexes are expected to form trans
and cis isomers, which can be attributed to the asymmetry in
the ligand (Figure 5). The H NMR spectra of the complexes
stronger hydrogen‐bonding capabilities than alcohols,
1
[38]
phenols or carboxylic acids,
in which intermolecular
can be used to determine the isomer formed, since the
hydrogen bonding of moderate strength and directionality

8
of 10
BABAHAN ET AL.
[
39]
links
molecules to form supramolecular structures; these
spectra of Co(II) complexes in DMSO exhibit broad
2
2
directional noncovalent intermolecular interactions have
received considerable attention.
bands between 589 and 590 nm assigned to E → T
g
2g
[33,36]
[36]
Here are observed two
transitions, characteristic for octahedral geometry.
The
O…H─O peaks, as expected for metal complexes with
structures such as those in Figures 2–4.
Cu(II) complexes show a broad band at 589 nm, which
2
2
[35,40,41]
can be assigned to a B1g → A transition.
This,
1g
together with the measured μeff values, suggests square‐
planar geometry. The weak d–d transitions of square‐pla-
3
.4 | UV–visible spectra
[
35]
nar Ni(II) complexes result in peaks at 400 nm.
The spectra of the ligands and metal complexes contain
five absorption bands between 225 and 590 nm in DMSO
(
data not shown for the sake of brevity). In the electronic
3.5 | Antiproliferative activity
spectra of the ligands and their Ni(II), Cu(II) and Co(II)
complexes and the BF ‐briged Ni(II) complex, the wide
HL‐60 and HT‐29 cells were seeded to 48‐well plates and
treated with increasing concentrations of test chemicals. Cell
proliferation was calculated using MTT data and from these
results I C values were determined. The results are shown
2
range of bands seems to be due to both π → π*,
n → π* and d–d transitions of C═N and charge transfer
transition arising from p electron interactions between
metal and ligand, which involves either a metal‐to‐ligand
p
50
in Figure 6 and in the supporting information (Figure S3).
[18]
or ligand‐to‐metal electron transfer.
The absorption
The explanations for each test chemical are given in the
2
bands below 286 nm in DMSO are practically identical
and may be attributed to π → π* transitions in the aro-
matic ring or azomethine (─C═N) groups. The absorption
bands observed within the range 314–355 nm in DMSO
are most probably due to the n → π* transition of imine
figure captions. Our results indicate that the L H ligand is
2
effective on cancer cells in terms of cell death.
The test chemicals are more effective on HL‐60 cell line
but the effect differs depending on the metal. Figure 6 shows
2
that Co metal complexes of the L H ligand are effective in
2
[
18]
group corresponding to the ligands or complexes.
The
applied minimum concentration on HL‐60 cell line and the
absorption bands at 400–459 nm are assigned to metal‐
to‐ligand charge transfer or ligand‐to‐metal charge transfer
1
1
[35,40,41]
and A1g
→
B1g transitions.
The electronic
(a)
(
a)
(b)
(b)
2
FIGURE
6
(a) [Co(L H)
2
(H
2
O)
2
]
applied in increasing
2
concentrations by comparing with positive control paclitaxel on HL‐60
and HT‐29 cell lines. It was tested between 5 and 40 μM concentrations
and found to be effective on HL‐60 cell line in applied minimum
2 2 2
FIGURE 7 (a) [Co(L H) (H O) ] applied in effective concentration
by comparing with positive control paclitaxel on HL‐60 and HT‐29
cell lines. It was found to be more effective on HL‐60 cell line and
concentration. The I
p
C
50 value was nearly 5 μM. Solvent: DMSO. (b)
triggered apoptosis in more than 60% of cells. In HT‐29 cell line it
2
2
[Ni(L H)
2
] applied in increasing concentrations by comparing with
caused necrotic cell death in low levels. (b) [Ni(L H)
2
] applied in
positive control paclitaxel on HL‐60 and HT‐29 cell lines. It was tested
between 5 and 40 μM concentrations and found to be more effective on
effective concentration by comparing with positive control paclitaxel on
HL‐60 and HT‐29 cell lines. It showed nearly the same rate of cell death
in both cell lines. It triggered apoptosis at 65% level in HL‐60 cell line
but in HT‐29 cell line there were apoptotic and necrotic cells together
HL‐60 cell line in applied minimum concentration. The I
nearly 5 μM. Solvent: DMSO
p
C50 value was

BABAHAN ET AL.
9 of 10
2
I C value was ca 5 μM (Figure 6a). Ni complex of the L H2
necrotic cell death is observed with low levels of Co metal
complex for HT‐29 cell line. The level of apoptosis is only
at 65% in HL‐60 cell line and apoptosis and necrosis in
HT‐29 (Figure 8b).
p
50
ligand is found to be quite effective on HL‐60 cell line. There
is 50% cell inhibition even in applied minimum concentration
Figure 6b). These results also show that [Co(L H) (H O) ]
and [Ni(L H) ] complexes containing the L H ligand dis-
2
(
2
2
2
2
2
2
2
play strong inhibitory effect on HL‐60 cell line.
4
| CONCLUSIONS
1
2
3
.6 | Cell death assay
We prepared two new vic‐dioxime ligands (L H and L H )
2
2
containing 3‐acetyl‐ or 4‐acetylpyridine thiosemicarbazone
moieties and their novel homotrinuclear complexes (Ni(II),
Cu(II), Co(II) and BF ‐briged Ni(II)). Reaction of L H and
L H with metal(II) salts yielded homotrinuclear complexes
corresponding to the general formula [M (LH) ] (M: Ni(II),
Cu(II) or Co(II).2H O). The hydrogen‐bridged Ni(II) was
converted into its BF ‐capped analogue by the reaction with
BF ⋅Et O. Thiosemicarbazone dioxime complexes of Ni(II)
were stabilized by replacing the bridging protons of the vic‐
dioxime with BF and removing acidic protons from the O
binding sites. Our results show the coordinating ability of
the vic‐dioximes together with the thiosemicarbazone moie-
ties, and provide synthetic strategies for the preparation of
Chemical‐induced cell apoptosis was determined using
Hoechst 33258 and propidium iodide dyes and the results
are shown in Figures 7 and 8 and in the supporting informa-
tion (Figure S4). This method allows one to distinguish
between apoptosis and necrosis.
according to their morphology and the integrity of their cell
membranes, which can easily be seen upon propidium iodide
1
2
2
2
2
[42]
3
2
Cells were judged
2
+
2
3
2
staining.
2
Ni complex of the L H ligand results in a rate of cell
2
2
death for both cell lines similar to that of the Co metal coun-
terpart (Figure S5b): Co metal complex of the L H ligand is
found to be effective on HL‐60 cell line and triggers apopto-
sis (Figure 8a) in more than 60% of cells (Figure S5a), while
2
2
2
mixed ligand complexes. Our results also indicate that L H2
ligands are effective on cancer cells in terms of cell death.
2
Co metal complex of the L H ligand was found effective
2
in applied minimum concentration on HL‐60 cell line and
2
the I C value was ca 5 μM. Ni complex of the L H ligand
p
50
2
was found to be effective on HL‐60 cell line, where 50% of
cells were inhibited by 5 μM of the ligand. Co and Ni metal
2
complexes of the L H ligand triggered greater than 60 and
2
6
5% apoptosis in HL‐60, respectively, and apoptosis and
necrosis in HT‐29 cell line.
Overall, we conclude that [Co(L H) (H O) ] and
2
2
2
2
2
[Ni(L H) ] show the best effect in terms of cell death among
2
the metal–ligand complexes reported in this study. These
results afford a path for further research on pharmacological
studies on how these ligands and their complexes can be used
for developing new drugs and how they affect the cell cycle.
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FIGURE 8 (a) [Co(L H)
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How to cite this article: Babahan I, Özmen A, Aslan
K. Synthesis and use of dioxime ligands for treatment
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