M. Grazul et al. / Journal of Inorganic Biochemistry 135 (2014) 68–76
69
DNA fragmentation [17]. This work describes the biological activity of
pyridoxal thiosemicarbazone [18].
dissolved in HCl (100 mL, 0.05 M) and pentano-2,4-dione (2.5 mL,
24.3 mmol) was added dropwise to the stirred mixture. After stirring
for 1.5 h and 3 h incubation at room temperature, a white precipitate
was filtered off, washed with water and dried under reduced pressure.
Yield: 3.320 g (88.0%), and m.p: 95.2–95.9 °C. IR (KBr) ν (cm−1): 3389,
3241 (NH2), 3133 (CH3), 1604, 1574 (C_C, C_N), 1339 (C\NH2),
1029 (N\N), 1099, 880, and 808 (C_S). 1H NMR (400 MHz, DMSO-
d6) δ (ppm): 2.28 (6H, d, \CH3), 6.29 (1H, s, C4\H), and 7.00–7.46
(2H, d, NH2).
Also, many pyrazole complexes coordinated with metal ions are ef-
fective against cancer cells, e.g. pyrazole–rhodium(III) complexes indi-
cate cytostatic activity against HCV29T tumor cells, while their Pd(II)
complexes are active against solid-tumor cell lines and in some cases
exhibit remarkable activity. It has been suggested that their biological
activity depends on the nature of the ligand, the type of the counter
ion used and the configuration of the complex [19,20].
Identification of the mode of action of newly obtained compounds is
essential for designing an effective new drug. It is stated that the appli-
cation of compounds with cytotoxic activity to cells can lead to cell cycle
stop in the G1 (cell cycle phase G1) phase until all induced damages are
fixed or until apoptosis is induced. Necrosis is usually a result of the ex-
position of cells to compounds in high concentrations [21]. Moreover,
these types of compounds can also change the structure of the DNA or
destroy the cytoskeleton [22].
For many years our scientific attention has been focused on the
investigation of chemical compounds with potential anticancer activi-
ty [23,24]. Therefore, in this paper, we present the synthesis as well as
the physico-chemical and biological evaluation of newly obtained
copper(II) complexes of pyrazole derivatives.
2.2.3. Synthesis of dichlorido(1-[amino(thioxo)methyl]-5-hydroxy-3-
phenyl-1H-pyrazole-κN2)copper(II) (3)
A solution of 1-[amino(thioxo)methyl]-5-hydroxy-3-phenyl-1H-
pyrazole (1) (197.4 mg, 0.9 mmol) in ethyl acetate (5 mL) was added
dropwise to a solution of copper(II) chloride dihydrate (153.4 mg,
0.9 mmol) in ethyl acetate (4 mL) and methanol (1 mL) while stirring.
The obtained red-orange precipitate was filtered off, washed with
diethyl ether and dried under reduced pressure. Yield: 164.0 mg
(49.6%), and m.p: 186.1–187.6 °C. IR (KBr) ν (cm−1): 3361 (OH),
3243, 3126 (NH2), 2987 (CH aromat.), 2363 (SH), 1573, 1520 (C_C,
C_N), and 965 (N\N). Anal. Calc. C10H9N3SOCuCl2·3/4H2O (M =
367.229 g/mol) Anal (%): C 32.71, H 2.88, and N 11.44. Found (%): C
32.79, H 2.76, and N 11.25. LSI-MS (m/z): 353(LCuCl+2 ); and 281 (LCu+).
2. Experimental
2.1. General
2.2.4. Synthesis of dichloridobis{1-[amino(thioxo)methyl]-5-hydroxy-3-
phenyl-1H-pyrazole-κN2}copper(II) (4)
The IR spectra were recorded with a Mattson Infinity MI-60 spectro-
photometer in KBr. Melting points were determined using a Buchi Melt-
ing Point B540 apparatus and are uncorrected. Elemental analyses were
obtained in the Microanalytical Laboratory of the Department of
Bioorganic Chemistry (Medical University, Lodz) using a Perkin Elmer
PE 2400 CHNS analyzer. LSI mass spectra (liquid secondary ion) were
recorded on the Finnigan MAT 95 double focusing (BE geometry)
mass spectrometer (Finnigan MAT, Bremen, Germany). Samples were
dissolved in DMSO and 1 μL of 3-nitrobenzylalcohol (NBA) and mixed.
For the ionization, the beam of cesium ions of energy of 13 keV was
used. Spectra were recorded in positive and negative ion mode. Circular
dichroism measurements were performed using a Jasco J-810 spectro-
polarimeter equipped with a Jasco PFD-4255 Peltier temperature con-
troller. UV-visible (UV–vis) spectra were recorded on a Varian Bio 100
UV–vis spectrophotometer at room temperature.
While stirring a solution of 1-[amino(thioxo)methyl]-5-hydroxy-3-
phenyl-1H-pyrazole (1) (131.6 mg, 0.6 mmol) in ethyl acetate (5 mL),
a solution of copper(II) chloride dihydrate (51.1 mg, 0.3 mmol) in
ethyl acetate (4 mL) and methanol (1 mL) was added dropwise. Next,
the mixture was refluxed and stirred for 2 h at 45 °C. The obtained
black precipitate was filtered off, washed with ethyl acetate and diethyl
ether and dried under reduced pressure. Yield: 87.9 mg (51.2%), and
m.p: 160.2–160.9 °C. IR (KBr) ν (cm−1): 3423 (OH), 3152, 3128
(NH2), 3028 (CH, aromat.), 1606, 1523 (C_C, C_N), 1316 (C\NH2),
1001 (N\N), 867, and 808 (C_S). Anal. Calc. C20H18N6S2O2CuCl2
(M = 572.984 g/mol) Anal (%): C 41.92, H 3.17, and N 14.67. Found
(%): C 41.79, H 3.31, and N 14.27. LSI-MS (m/z): 574 (L2CuCl2+
+
1H); 538 (L2CuCl+); and 219 (L).
2.2.5. Synthesis of dichlorido(1-[amino(thioxo)methyl]-3,5-dimethyl-1H-
pyrazole-κN2)copper(II) (5)
In all experiments, each compound was dissolved in 10 μL of DMSO
and diluted with bidistilled water or Tris–HCl NaCl buffer in order to cal-
culate its concentration. The final percentage of DMSO was 0.1% at most.
Compound 5 was synthesized according to the literature with minor
modifications [29]. Copper(II) chloride dehydrate (85.2 mg, 0.5 mmol)
in ethyl acetate (4 mL) and methanol (1 mL) was added dropwise to a
solution of 1-[amino(thioxo)methyl]-3,5-dimethyl-1H-pyrazole (2)
(77.6 mg, 0.5 mmol) in ethyl acetate (5 mL) while stirring. The reaction
mixture was stirred for 24 h. Obtained gray-green precipitate was fil-
tered off, washed with ethyl acetate and diethyl ether and dried under
reduced pressure. Yield: 128.0 mg (88.4%), and m.p: 164.8–166.2 °C.
IR (KBr) ν (cm−1): 3296, 3106 (NH2), 1621, 1588 (C_C, C_N), 1346
(C\NH2), 999 (N\N), and 847 (C_S). Anal. Calc. C6H9N3SCuCl2
(M = 289.675 g/mol) Anal (%): C 24.88, H 3.13, and N 14.51.
Found (%): C 24.72, H 3.14, and N 14.48. LSI-MS (m/z): 289 (LCuCl+2 );
254 (LCuCl+); and 155 (L).
2.2. Synthesis of compounds
2.2.1. Synthesis of 1-[amino(thioxo)methyl]-5-hydroxy-3-phenyl-1H-
pyrazole (1)
The synthesis of ligand 1 was slightly modified from methods pub-
lished previously [25,26]. Hydrazinecarbothioamide (2.37 g, 26 mmol)
was dissolved in a mixture of ethanol (20 mL) and HCl (1 mL). Next
ethyl benzoylacetate was added (4.5 mL, 26 mmol). The obtained mix-
ture was refluxed for 1 h under an argon atmosphere. After cooling, the
white precipitate was filtered off, washed with water and dried under
reduced pressure. Yield: 3.85 g (67.5%), m.p: 159.1–160.3 °C. IR (KBr)
ν (cm−1): 3290, 3159, 3113 (NH2), 3095 (CH aromat.), 1651 (C_O),
1584, 1558 (C_C, C_N), 1320 (C\NH2), 1026 (N\N), 1102, 883,
and 798 (C_S). 1H NMR (270 MHz, DMSO-d6) δ (ppm): 6.18 (1H,
s, C4-H), 7.40–8.04 (5H, m, arom.), 9.50–10.40 (2H, s, NH2), and
11.75–12.65 (1H, s, OH/NH).
2.3. The stability of compounds in aqueous solution
The stability of the metal(II) complexes in water/DMSO solution at
concentration 10 μM was assessed by UV–vis spectrophotometric anal-
ysis in the range of 200–800 nm. Spectra were recorded over a period of
24 h using a Scanning Kinetics program. The timetable of all measure-
ments is presented in Table S1. The obtained UV–vis spectra were com-
pared to each other.
2.2.2. Synthesis of 1-[amino(thioxo)methyl]-3,5-dimethyl-1H-pyrazole (2)
Ligand 2 was synthesized according to a known procedure with slight
modifications [27,28]. Hydrazinecarbothioamide (2.5 g, 27.4 mmol) was