Received: 10 August 2019 Revised: 14 November 2019 Accepted: 19 November 2019

DOI: 10.1002/aoc.5423

FULL PAPER

Novel 2-formylpyridine 4-allyl-S-

methylisothiosemicarbazone and Zn(II), Cu(II), Ni(II) and
Co(III) complexes: Synthesis, characterization, crystal
structure, antioxidant, antimicrobial and antiproliferative
activity

Greta Balan1 | Olga Burduniuc1 | Irina Usataia2 | Vasilii Graur2 |

Yurii Chumakov3,4 | Peter Petrenko3 | Valentin Gudumac1 | Aurelian Gulea2 |
5
Elena Pahontu

1
State University of Medicine and
Pharmacy “Nicolae Testemiţanu”, 165 New zinc (II), copper (II), nickel (II) and cobalt (III) complexes, [Zn (HL)2]I2
Ştefan cel Mare and Sfânt Street, Chişinau, (1), [Cu (HL)Cl2] (2), [Cu (HL)Br2] (3), [Cu (HL)(H2O)2](ClO4)2 (4),
MD-2004, Republic of Moldova
2
[Ni (HL)2]I2
H2O (5), [Co(L)2]Cl (6), [Co(L)2]NO3 (7), [Co(L)2]I
[Co(L)2](I3)
Laboratory of Advanced Materials in
Biofarmaceutics and Technics, Moldova (8) were obtained with 2-formylpyridine 4-allyl-S-methylisothiosemicarbazone
State University, 60 Mateevici Street, MD (HL). The isothiosemicarbazone ligand was characterized by NMR (1H and
2009, Republic of Moldova 13
C), IR spectroscopy and X-ray diffraction. All the complexes were character-
3
Institute of Applied Physics, Academy of
ized by elemental analysis, IR, UV–Vis, ESI-MS spectroscopy, molar conductiv-
Sciences of Moldova, 5 Academiei Street,
Chişinau, MD 2028, Republic of Moldova ity, magnetic susceptibility measurements. X-ray diffraction analysis on the
4
Gebze Technical University, P.O. Box: monocrystal and powder elucidated the structure of the complexes 1, 5,
141, Gebze, Kocaeli, 41400, Turkey 7 and 8.
5
Inorganic Chemistry Department,
The ligand and the complexes were tested for their antioxidant and antimi-
Faculty of Pharmacy, University of
Medicine and Pharmacy “Carol Davila”, 6 crobial activity against Staphylococcus aureus, Escherichia coli, Klebsiella
Traian Vuia Street, Bucharest, 020956, pneumoniae and Candida albicans. Also, the antiproliferative properties
Romania
of these compounds on human leukemia HL-60, human cervical epithelial
Correspondence HeLa, human epithelial pancreatic adenocarcinoma BxPC-3, human
Dr. Elena Pahontu, Professor, Inorganic muscle rhabdomyosarcoma spindle, large multinucleated RD cells and
Chemistry Department, Faculty of
Pharmacy, University of Medicine and
normal MDCK cells have been investigated. The nickel complex 5 and
Pharmacy “Carol Davila”. Bucharest, cobalt complexes 6, 7 showed promising antiproliferative activity and low
Romania. toxicity.
Email: [email protected]
KEYWORDS
antimicrobial activity, antioxidant activity, antiproliferative activity, complexes, crystal structure

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2019 The Authors. Applied Organometallic Chemistry published by John Wiley & Sons Ltd

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https://doi.org/10.1002/aoc.5423
2 of 17 BALAN ET AL.

1 | INTRODUCTION 2 | EXPERIMENTAL

Malignant tumors, antibiotic resistance are presently a 2.1 | Materials

global health problem urging the development of novel
antitumor and antimicrobial agents.[1–3] Most of the med- All the reagents used were chemically pure. Zn
icines used in medical practice are organic substances. (CH3COO)2
2H2O, CuCl2
2H2O, CuBr2, Cu
Recently, much attention has been paid to transition (ClO4)2
6H2O, Ni (CH3COO)2
4H2O, CoCl2
6H2O, Co
metal complexes as biologically active substances. The (NO3)2
6H2O, Co (CH3COO)2
4H2O (Merck, Darmstadt,
coordination compounds of 3d metals occupy an impor- Germany) were used as supplied. 2-Formylpyridine was
tant place among them, since they have shown promising used as received (Sigma-Aldrich, Munich, Germany).
biological activity.[4–6] N(4)-Allyl-3-thiosemicarbazide was prepared similarly to
The activity of these compounds depends on the the literature procedure[33,34] by reaction of the
structure of molecules and on the arrangement of func- allylisothiocyanate and hydrazine hydrate. The solvents
tional groups. The study of the relationship between the were purified and dried according to standard
chemical structure of substances and their biological procedures.[35]
activity allows for a purposeful synthesis of new
drugs.[7–17]
Thiosemicarbazones are of particular interest among 2.2 | Synthesis of the 2-formylpyridine 4-
a wide range of biologically active substances.[18–23] allyl-S-methylisothiosemicarbazone (HL)
The introduction of alkyl groups on the terminal N4
nitrogen atom and the moiety of 2-formylpyridine and 2-Formylpyridine 4-allyl-S-methylisothioseimicarbazone
their derivatives on the N1 position exert significant bio- (HL) (Scheme 2) was obtained similarly to the method
logical activity.[24] described in the literature.[21,30,36]
Isothiosemicarbazones are of biological interest since Sodium carbonate (1.06 g, 10 mmol) was added to the
thioalkylation leads to a change in the coordination solution of 2-formylpyridine 4-allyl-S-methylisothiosemi-
mode.[25–29] Nevertheless, isothiosemicarbazones have carbazone hydroiodide (3.62 g, 10 mmol). The mixture is
not been studied extensively from a biological point of stirred for 30 min at 50  C. By cooling to room tempera-
view. It is important to study the influence of ture to give a yellow precipitate which was filtered,
thiosemicarbazide's alkylation on the geometry, physico- washed with ethanol and dried in vacuo (Scheme 3).
chemical and biological properties of the corresponding Yellow solid. Yield: 88%; m.p.: 75–77  C; FW:
coordination compounds. 234.321 g/mol; Anal. Calc. for C11H14N4S: C, 56.38; H,
Previously we have described 2-hydroxybenzaldehyde 6.02; N, 23.91; S, 13.68%. Found: C, 56.29; H, 6.14; N,
4-allyl-S-methylisothiosemicarabzone and a series of its 23.84; S, 13.59%.
3d metal coordination compounds.[30] These compounds Main IR peaks (cm−1): ν(N4H) 3010, ν(N2-H) 3180,
exhibit moderate antimicrobial and antifungal activity, ν(C=C allyl) 1639, ν(C=N1) 1614, ν(С = Npyr) 1560, ν
selective anticancer activity which in case of copper com- (CH3–S) 1093, ν(C–S) 643.
plex exceeds the activity of doxorubicine, and promising
antioxidant activity. Since it is known that the substitu-
tion of 2-hydroxybenzaldehyde moiety with
2-formylpyridine moiety leads to a growth of biological
activity of thiosemicarbazones and their coordination
compounds,[28–32] the study of such substitution in case
of isothiosemicarbazones is of interest.
Therefore, the present paper describes the chemical
synthesis, characterization and in vitro biological evalua- S C H E M E 2 Synthesis of 2-formylpyridine 4-allyl-S-
tion of 2-formylpyridine 4-allyl-S-methylisothiosemi- methylisothiosemicarbazone hydroiodide
carbazone (Scheme 1) and its Zn (II), Cu (II), Ni (II) and
Co (III) and coordination compounds.

S C H E M E 3 Neutralization of 2-formylpyridine 4-allyl-S-

SCHEME 1 Isothiosemicarbazone ligand HL methylisothiosemicarbazone hydroiodide
ANTIMICROBIAL ACTIVITY, ANTIPROLIFERATIVE ACTIVITY, COMPLEXES 3 of 17

Green solid. Yield: 81%; FW: 368.77 g/mol; Anal.

Calc. for C11H14Cl2CuN4S: C, 35.83; H, 3.83; Cl, 19.23;
Cu, 17.23; N, 15.19; S, 8.70; Found: C, 35.77; H, 3.76; Cl,
19.16; Cu, 17.15; N, 15.10; S, 8.62; Main IR peaks (cm−1):
SCHEME 4 The tautomeric forms of the ligand HL ν(N4H) 3104, ν(C=C allyl) 1645, ν(C=N1) 1592, ν(С = Npyr)
1524, ν (CH3–S) 1095, ν(C–S) 646. μeff (292 ± 1 K): 1.48
1st tautomeric form (HL(A) on Scheme 4): 1H NMR μB; χ (CH3OH): 170 Ω−1
cm2
mol−1.
(CDCl3; δ, ppm): 8.44 (s, 1H, CH=N); 8.64 (d, 1H, CH
aromatic); 7.94 (d, 1H, CH aromatic); 7.70 (t, 1H, CH aro- Synthesis of the complex [cu (HL)Br2] 3
matic); 7.25 (t, 1H, CH aromatic); 6.81 (br, 1H, NH); 5.92 The complex 3 was synthesized from CuBr2 (0.224 g;
(m, 1H, CH from allyl moiety); 5.27 (m, 2H, CH2 = C); 1 mmol) and HL (0.234 g; 1 mmol).
3.96 (t, 2H, CH2-N); 2.52 (s, 3H, CH3). 13C NMR (CDCl3; Green solid. Yield: 78%; FW: 457.68 g/mol; Anal.
δ, ppm): 165.15 (C-S); 152.78, 154.32, 149.51, 123.64, Calc. for C11H14Br2CuN4S: C, 28.87; H, 3.08; Br, 34.92;
121.30 (C aromatic); 136.37 (CH=N); 134.38 (CH from Cu, 13.88; N, 12.24; S, 7.01; Found: C, 28.81; H, 3.10; Br,
allyl moiety); 116.63 (CH2=); 45.75 (CH2-N); 12.91 (CH3). 34.85; Cu, 13.79; N, 12.16; S, 6.97; Main IR peaks (cm−1):
2nd tautomeric form (HL(B) on Scheme 4): 1H NMR ν(N4H) 3143, ν(C=C allyl) 1645, ν(C=N1) 1593, ν(С = Npyr)
(CDCl3; δ, ppm): 8.38 (s, 1H, CH=N); 8.60 (d, 1H, CH 1525, ν (CH3–S) 1095, ν(C–S) 648. μeff (292 ± 1 K): 1.37
aromatic); 8.07 (d, 1H, CH aromatic); 7.68 (t, 1H, CH aro- μB; χ (CH3OH): 202 Ω−1
cm2
mol−1.
matic); 7.22 (t, 1H, CH aromatic); 5.99 (m, 1H, CH from
allyl moiety); 5.23 (m, 2H, CH2 = C); 4.56 (br, 1H, NH); Synthesis of the complex [cu (HL)(H2O)2](ClO4)2 4
4.14 (t, 2H, CH2-N); 2.41 (s, 3H, CH3). 13C NMR (CDCl3; The complex 4 was synthesized from Cu (ClO4)2
6H2O
δ, ppm): 163.65 (C-S); 154.84, 153.16, 149.35, 123.43, (0.370 g; 1 mmol) and HL (0.234 g; 1 mmol).
120.78 (C aromatic); 136.11 (CH=N); 134.01 (CH from Green solid. Yield: 75%; FW: 532.80 g/mol; Anal.
allyl moiety); 117.25 (CH2=); 46.13 (CH2-N); 13.18 (CH3). Calc. for C11H18Cl2CuN4O10S: C, 24.80; H, 3.41; Cl, 13.31;
Cu, 11.93; N, 10.52; S, 6.02; Found: C, 24.75; H, 3.35; Cl,
13.27; Cu, 11.88; N, 10.64; S, 6.10. Main IR peaks (cm−1):
2.3 | GENERAL PROCEDURE FOR THE ν(N4H) 3071, ν(C=C allyl) 1637, ν(C=N1) 1593, ν(С = Npyr)
PREPARATION OF THE METAL 1505, ν (CH3–S) 1093, ν(C–S) 667. μeff (292 ± 1 K): 1.80
COMPLEXES 1–8 μB; χ (CH3OH): 207 Ω−1
cm2
mol−1.

Complexes 1–8 were synthesized by combining the ligand Synthesis of the complex [Ni (HL)2]I2
H2O 5
HL with the corresponding metal salts. The complex 5 was synthesized from Ni
(CH3COO)2
4H2O (0.249 g; 1 mmol) and HL
HI (0.724 g;
Synthesis of the complex [Zn (HL)2]I2 1 2 mmol).
To an ethanolic solution (30 ml) of hydroiodide of Brown solid. Yield: 70%; FW: 799.16 g/mol; Anal.
2-formylpyridine 4-allyl-S-methylisothiosemicarbazone Calc. for C22H30I2N8NiOS2: C, 33.06; H, 3.78; I, 31.76; N,
HL
HI (0.724 g, 2 mmol) was added Zn 14.02; Ni, 7.34; S, 8.02; Found: C, 32.98; H, 3.82; I,
(CH3COO)2
2H2O (0.219 g, 1 mmol). The mixture was 31.85; N, 13.96; Ni, 7.28; S, 8.09. Main IR peaks (cm−1):
stirred for 1 hr at 70  C. After filtration a yellow precipi- ν(N4H) 3071, ν(C=C allyl) 1637, ν(C=N1) 1593, ν(С = Npyr)
tate was obtained which was washed with cold ethanol 1524, ν (CH3–S) 1093, ν(C–S) 667. μeff (292 ± 1 K): 2.97
and dried under vacuo. μB; χ (CH3OH): 198 Ω−1
cm2
mol−1.
Yield: 79%; FW: 787.86 g/mol; Anal. Calc. for
C22H28I2N8S2Zn: C, 33.50; H, 3.55; I, 32.22; N, 14.22; S, Synthesis of the complex [Co(L)2]Cl 6
8.14; Zn, 8.30; Found: C, 33.52; H, 3.51; I, 32.16; N, The complex 6 was synthesized from CoCl2
6H2O
14.17; S, 8.11; Zn, 8.24. Main IR peaks (cm−1): ν(N4H) (0.238 g; 1 mmol) and HL (0.468 g; 2 mmol).
3071, ν(C=C allyl) 1637, ν(C=N1) 1593, ν(С = Npyr) 1499, Brown solid. Yield: 82%; FW: 561.01 g/mol; Anal.
ν (CH3–S) 1093, ν(C–S) 667. μeff (292 ± 1 K): 0 μB; χ Calc. for C22H26ClCoN8S2: C, 47.10; H, 4.67; Cl, 6.32; Co,
(CH3OH): 173 Ω−1
cm2
mol−1. 10.50; N, 19.97; S, 11.43; Found: C, 47.02; H, 4.58; Cl,
6.27; Co, 10.45; N, 19.88; S, 11.35. Main IR peaks (cm−1):
Synthesis of the complex [Cu (HL)Cl2] 2 ν(N4H) 3071, ν(C=C allyl) 1637, ν(C=N1) 1593, ν(С = Npyr)
The complex 2 was synthesized using CuCl2
2H2O 1542, ν (CH3–S) 1093, ν(C–S) 667. μeff (292 ± 1 K): 0 μB; χ
(0.171 g; 1 mmol) and HL (0.234 g; 1 mmol). (CH3OH): 85 Ω−1
cm2
mol−1.
4 of 17 BALAN ET AL.

Synthesis of the complex [Co(L)2]NO3 7 2.5 | X-ray Crystallography

The complex 7 was synthesized from Co (NO3)2
6H2O
(0.291 g; 1 mmol) and HL (0.468 g; 2 mmol). The main crystallographic data together with refinement
Brown solid. Yield: 77%; FW: 587.56 g/mol; Anal. details of HL, 1, 5, 7 and 8 compounds are summarized
calc. For C22H26CoN9O3S2 (g
mol−1): C, 44.97; H, 4.46; in Table 1. Data collection and unit cell determination:
Co, 10.03; N, 21.45; S, 10.91; Found: C, 44.86; H, 4.37; Co, CrysAlis PRO CCD (Oxford Diffraction), programs used
9.93; N, 21.56; S, 10.81. Main IR peaks (cm−1): ν(N4H) to solve and refine structures: SHELXS-97 and SHELXL-
3070, ν(C=C allyl) 1637, ν(C=N1) 1601, ν(С = Npyr) 1538, 97.[38,39] All atomic displacements for non-hydrogen
ν (CH3–S) 1097, ν(C–S) 657. μeff (292 ± 1 K): 0 μB; χ atoms were refined using an anisotropic model.
(CH3OH): 97 Ω−1
cm2
mol−1. The structures of 1 and 7 displayed the disorder of the
C2H3 groups which was refined with the help of PART
Synthesis of the complex [Co(L)2]I
[Co(L)2](I3) 8 instruction dividing the disordered atoms into two
The complex 8 was synthesized from Co groups. Selected bond lengths, angles and hydrogen
(CH3COO)2
4H2O (0.249 g; 1 mmol) and HL
HI (0.724 g; bonds are presented in Table 2 and Table 3 respectively.
2 mmol). The geometric parameters were calculated with the help
Brown solid. Yield: 81%; FW: 1558.74 g/mol; Anal. of the PLATON program.[40] Analysis of found X-H…Cg
Calc. for C44H52Co2I4N16S4: C, 33.90; H, 3.36; Co, 7.56; I, (π ring) interactions were performed by the following
32.57; N, 14.38; S, 8.23; Found: C, 33.98; H, 3.41; Co, criteria: H… Cg < 3.0 Å, γ < 30.0 , where γ is the angle
7.48; I, 32.47; N, 14.29; S, 8.31. Main IR peaks (cm−1): between the H… Cg vector and the normal to the aro-
ν(N4H) 3070, ν(C=C allyl) 1640, ν(C=N1) 1600, ν(С = Npyr) matic ring. The hydrogen atoms that are not involved in
1533, ν (CH3–S) 1098, ν(C–S) 656. μeff (292 ± 1 K): 0 μB; χ the hydrogen bonding were omitted from the generation
(CH3OH): 109 Ω−1
cm2
mol−1. of the packing diagrams. Mercury software[41] was used
for visualization of the studied structures.

2.4 | Physical Measurements

2.6 | Biological Studies
Melting points, the determination of metal content, the
chemical elemental analysis for the determination of C, 2.6.1 | Antibacterial and antifungal
H and N, molar conductivity, magnetic susceptibilities, activity
were determined by specific methods.[37] Infrared spectra
of the compounds were recorded on a Bruker ALPHA The antimicrobial activities of the free ligand and com-
FTIR spectrophotometer at room temperature in the plexes were evaluated against three bacterial strains,
range of 4000–400 cm−1. Electronic spectra were recorded Staphylococcus aureus (ATCC 25923), Escherichia coli
using the JascoV-670 spectrophotometer (Tokyo, Japan), (ATCC 25922), Klebsiella pneumoniae (ATCC 13883) and
in diffuse reflectance, using MgO dilution matrices. UV– fungal strain, Candida albicans (ATCC 10231). Determi-
visible absorption spectra in DMSO solutions were mea- nation of the MIC (minimum inhibitory concentration,
sured on Agilent Cary 300 UV–Vis spectrophotometer. μg/ml) and MBC (minimum bactericidal concentration,
The 1H and 13C NMR spectra were recorded on a Bruker μg/ml) was done using the serial dilutions in liquid broth
DRX-400 spectrometer (Billerica, MA, USA), using CDCl3 method.[30,42] The tested substances were dissolved in
as a solvent. The chemical shifts (δ) in ppm were mea- DMSO in concentration of 10 mg/ml. The next dilutions
sured relative to tetramethylsilane (TMS). Electrospray were made using 2% of peptonate bullion. Furacillinum
Ionization Mass Spectrometry (ESI-MS) spectra were col- was used as the standard antibacterial drug while
lected using a Q-TOF instrument supplied by WATERS. nystatine was used as the standard antifungal drug.
Samples were solubilized in methanol at a concentration
of 5 mg. L−1 and were introduced into the spectrometer
via an ACQUITY UPLC WATERS system whilst a Leu- 2.6.2 | Antiproliferative activity
cine Enkephalin solution was co-injected via a micro
pump as internal standard. X-ray powder diffraction Cell cultures
analysis was performed on Panalytical Empyrean diffrac- BxPC-3 (ATCC CRL-1687) and HL-60 (ATCC CCL-240)
tometer system using X-ray tube with copper anode in lines were cultured as monolayer in Roswell Park Memo-
the range of 5–60 degrees 2θ. Similated powder X-ray rial Institute (RPMI) 1640 medium containing L-
diffractograms from single crystal X-ray data were gener- glutamine (2 nM), antibiotics penicillin–streptomycin
ated using Mercury 4 software. (final concentration 100 IU/ml penicillin and 100 μg
ANTIMICROBIAL ACTIVITY, ANTIPROLIFERATIVE ACTIVITY, COMPLEXES 5 of 17

TABLE 1 Crystallographic data, details of data collection and structure refinement parameters for compounds HL, 1 and 5

Compound HL 1 5
Chemical formula C11H15N4S C22H25I2N8S2Zn C44H56I4N16Ni2OS4
M (g mol−1) 234.32 784.79 631.32
Temperature, (K) 293(2) 293(2) 293(2)
Wavelength, (Å) 0.71073 0.71073 0.71073
Crystal system monoclinic triclinic monoclinic
Space group P21/c P-1 P21/c
a (Å) 13.3581(7) 8.6878(8) 8.8120(3)
b (Å) 9.7224(6) 9.2783(8) 17.7401(6)
c (Å) 9.7046(4) 19.1777(16) 20.4101(5)
α( )
0
90 99.670(7) 90
β ( 0) 91.744(4) 93.314(7) 107.177(2)
γ( )
0
90 93.453(8) 90
3
V (Å ) 1259.78(11) 1517.5(2) 3048.31(17)
Dcalc (g cm−3) 1.241 1.718 1.720
μ (mm−1) 0.237 3.006 2.826
F(0 0 0) 500 762 1544
2
Goodness-of-fit on F 1.024 1.027 1.046
Final R1, wR2 [I > 2σ(I)] 0.0548, 0.1205 0.0388, 0.0914 0.0376, 0.0856
R1, wR2 (all data) 0.0972, 0.1406 0.0558, 0.0993 0.0538, 0.0937
−3
Largest difference in peak and hole (e Å ) 0.205, −0.402 0.549, −0.522 0.716, −0.669

Crystallographic data, details of data collection and structure refinement parameters for compounds 7, 8

Compound 7 8
Chemical formula C22H24CoN9O3S2 C44H51Co2I4N16S4
−1
M (g mol ) 585.55 1557.71
Temperature, (K) 293(2) 293(2)
Wavelength, (Å) 0.71073 0.71073
Crystal system triclinic triclinic
Space group P-1 P-1
a (Å) 8.8203(4) 9.1984(2)
b (Å) 10.1890(3) 17.7683(7)
c (Å) 14.8156(8) 19.7114(7)
α( )
0
90.362(3) 110.651(4)
β ( 0) 100.013(4) 99.664(3)
γ( )
0
90.763(3) 99.054(3)
3
V (Å ) 1311.02(10) 2888.78(17)
−3
Dcalc (g cm ) 1.483 1.791
μ (mm−1) 0.857 2.902
F(0 0 0) 604 1514
2
Goodness-of-fit on F 1.070 1.044
Final R1, wR2 [I > 2σ(I)] 0.0450, 0.1182 0.0526, 0.1082
R1, wR2 (all data) 0.0579, 0.1274 0.0870, 0.1211
−3
Largest difference in peak and hole (e Å ) 0.356, −0.555 1.351, −1.003
6 of 17 BALAN ET AL.

TABLE 2 Selected bond lengths and angles for HL, 1, 5, 7 and 8

d, Å

Bond lengths HL 1 5 7 8
Zn(1)[Ni(1),Co(1)]-N(1A) 2.113(4) 1.991(3) 1.863(2) 1.868(5) 1.858(5)
Zn(1)[Ni(1),Co(1)]-N(1) 2.119(3) 2.002(3) 1.867(2) 1.878(5) 1.865(5)
Zn(1)[Ni(1),Co(1)]-N(3A) 2.125(4) 2.082(4) 1.919(3) 1.928(5) 1.918(5)
Zn(1)[Ni(1),Co(1)]-N(3) 2.137(4) 2.104(3) 1.923(2) 1.941(5) 1.947(5)
Zn(1)[Ni(1),Co(1)]-N(4A) 2.198(4) 2.131(4) 1.950(3) 1.949(4) 1.946(5)
Zn(1)[Ni(1),Co(1)]-N(4) 2.267(4) 2.110(3) 1.951(2) 1.945(5) 1.962(5)
S(1)-C(1) 1.750(3) 1.757(5) 1.756(4) 1.752(3) 1.744(7) 1.750(7)
S(1)-C(11) 1.785(2) 1.787(6) 1.772(6) 1.777(3) 1.790(9) 1.781(9)
S(1A)-C(1A) 1.752(5) 1.757(4) 1.757(3) 1.756(6) 1.762(6)
S(1A)-C(11A) 1.800(6) 1.785(6) 1.793(4) 1.739(10) 1.769(9)
N(1)-C(5) 1.279(3) 1.266(6) 1.277(5) 1.298(3) 1.289(8) 1.293(8)
N(1)-N(2) 1.383(3) 1.357(5) 1.360(4) 1.352(3) 1.356(7) 1.360(7)
N(2)-C(1) 1.303(3) 1.367(6) 1.382(5) 1.344(4) 1.332(9) 1.334(8)
N(3)-C(1) 1.332(3) 1.279(6) 1.284(5) 1.316(4) 1.356(9) 1.329(8)
N(3)-C(2) 1.436(3) 1.463(6) 1.472(6) 1.464(4) 1.388(9) 1.410(8)
N(4)-C(10) 1.310(6) 1.335(5) 1.335(4) 1.335(7) 1.334(8)
N(4)-C(6) 1.342(5) 1.345(5) 1.359(4) 1.358(8) 1.373(8)
C(2)-C(3) 1.483(9) 1.411(8) 1.491(5) 1.528(12) 1.505(10)
C(3)-C(4) 1.182(9) 1.229(9) 1.303(5) 1.211(14) 1.209(11)
C(5)-C(6) 1.467(7) 1.448(6) 1.444(4) 1.455(9) 1.436(9)
I(1)-I(2) 2.9218(8)
I(2)-I(3) 2.8942(8)
Bond angles ω, deg.
N(1A)-Zn(1)[Ni(1),Co(1)]-N(1) 163.42(15) 173.99(14) 178.93(11) 179.5(3) 177.8(3)
N(1A)-Zn(1)[Ni(1),Co(1)]-N(3A) 73.85(15) 77.16(14) 80.73(11) 80.9(2) 80.8(2)
N(1)-Zn(1)[Ni(1),Co(1)]-N(3A) 115.04(16) 104.12(14) 99.98(11) 99.0(2) 99.1(2)
N(1A)-Zn(1)[Ni(1),Co(1)]-N(3) 120.05(14) 109.02(14) 98.44(10) 99.4(2) 101.0(2)
N(1)-Zn(1)[Ni(1),Co(1)]-N(3) 74.08(14) 76.77(14) 80.76(10) 81.1(2) 81.2(2)
N(3A)-Zn(1)[Ni(1),Co(1)]-N(3) 97.00(16) 96.66(14) 91.51(10) 92.2(2) 90.7(2)
N(1A)-Zn(1)[Ni(1),Co(1)]-N(4A) 73.48(14) 76.85(14) 82.96(11) 82.3(2) 83.0(2)
N(1)-Zn(1)[Ni(1),Co(1)]-N(4A) 97.20(14) 101.80(14) 96.34(11) 97.8(2) 97.1(2)
N(3A)-Zn(1)[Ni(1),Co(1)]-N(4A) 147.25(14) 154.00(14) 163.68(11) 163.1(2) 163.5(2)
N(3)-Zn(1)[Ni(1),Co(1)]-N(4A) 97.29(15) 91.20(13) 91.39(11) 92.0(2) 89.4(2)
N(1A)-Zn(1)[Ni(1),Co(1)]-N(4) 93.21(13) 97.12(13) 98.05(10) 96.6(2) 94.9(2)
N(1)-Zn(1)[Ni(1),Co(1)]-N(4) 72.84(14) 77.03(13) 82.75(10) 82.8(2) 82.9(2)
N(3A)-Zn(1)[Ni(1),Co(1)]-N(4) 93.54(15) 91.74(13) 91.32(10) 90.7(2) 91.3(2)
N(3)-Zn(1)[Ni(1),Co(1)]-N(4) 146.71(14) 153.71(14) 163.51(10) 164.0(2) 164.1(2)
N(4A)-Zn(1)[Ni(1),Co(1)]-N(4) 90.45(13) 92.04(13) 90.44(10) 89.82(19) 93.1(2)
C(1)-S(1)-C(11) 102.2(1) 104.5(3) 104.4(2) 103.63(15) 103.3(4) 102.0(4)
C(1A)-S(1A)-C(11A) 104.9(3) 103.7(3) 102.31(19) 104.4(4) 103.3(4)
I(3)-I(2)-I(1) 178.99(2)
ANTIMICROBIAL ACTIVITY, ANTIPROLIFERATIVE ACTIVITY, COMPLEXES 7 of 17

TABLE 3 Hydrogen bonds of HL, 1, 5, 7 and 8 compounds

D-H…A d(D…H), Å d(H…A), Å d(D…A), Å ∠(DHA), deg. Symmetry transformation for H-acceptor.
HL
N(3)-H(3)…N(1) 0.86 2.24 2.5778 103 x,y,z
N(3)-H(3)…N(4) 0.86 2.33 3.0740 145 x,1/2-y,-1/2 + z
C(2)-H(2B)…S(1) 0.97 2.56 2.9694 106 x,y,z
1
N(2)-H(2)…I(2) 0.86 2.88 3.5574 137 -x,1-y,-z
N(2A)-H(2A)…I(1) 0.86 3.01 3.5234 120 x,y,z
C(2A)-H(2AA)… S(1A) 0.97 2.50 2.9834 111 x,y,z
C(2)-H(2C)… S(1) 0.97 2.46 2.9812 113 x,y,z
C(11)-H(11C)…I(2) 0.96 3.05 3.9439 155 -x,1-y,-z
5
N(2)-H(2)…I(2) 0.86 2.95 3.5603 130 1-x,-1/2 + y,1/2-z
N(2A)-H(2A)…I(1) 0.86 2.83 3.5288 140 x,1/2-y,-1/2 + z
C(2A)-H(2AB)…S(1) 0.97 2.63 3.0203 104 x,y,z
C(2)-H(2B)…S(1A) 0.97 2.42 2.9990 118 x,y,z
C(10)-H(10)…I(1) 0.93 2.97 3.8094 151 1-x,-1/2 + y,1/2-z
C(11)-H(11B)…O(1 W) 0.96 2.51 3.4027 154 x,y,z
7
C(2A)-H(2AB)… S(1A) 1.03 2.51 3.0066 109 x,y,z
C(2)-H(2B)… S(1) 0.97 2.55 2.9767 106 x,y,z
C(2)-H(2C)… O(2 N) 0.97 2.43 3.2641 144 x,y,z
C(5)-H(5)… O(2 N) 0.93 2.57 3.1704 123 x,-1 + y,z
C(8A)-H(8A)… O(3 N) 0.93 2.43 3.3140 160 1-x,1-y,-z
C(9)-H(9)… O(1 N) 0.93 2.55 3.4478 161 -x,1-y,-z
C(10)-H(10)… O(3 N) 0.93 2.46 3.1521 131 -x,1-y,-z
C(11A)-H(11F)… O(2 N) 0.96 2.49 3.3783 154 -1 + x,y,z
8
C(2A)-H(2AA)… S(1A) 0.97 2.56 3.0000 108 x,y,z
C(2)-H(2B)… S(1) 0.97 2.55 2.9678 106 x,y,z
C(2B)-H(2BB)… S(1B) 0.97 2.57 2.9972 107 x,y,z
C(9)-H(9)…I(1) 0.93 3.01 3.7550 138 x,y,1 + z
C(2C)-H(2CB)… S(1C) 0.97 2.57 2.9782 105 x,y,z
C(10)-H(10)…I(4) 0.93 3.00 3.7578 140 -x,1-y,1-z
C(11B)-H(11H)…N(2B) 0.96 2.43 2.8036 103 x,y,z

streptomycin/ml) and supplemented with fetal bovine Cells were maintained at 37  C in a 2–5% humidified
serum (FBS) (10% v/v). CO2 atmosphere in the incubator in 75-cm2 culture dis-
Cell lines HeLa (ATCC CCL-2), RD (ATCC CCL-136) hes, and used for experiments between passage 5 and 16.
and MDCK (ATCC CCL-34) were cultured in the The compounds were dissolved at the time of the
Dulbecco's Modified Essential Medium (DMEM) with L- experiments.
glutamine (4 mM), glucose (4.5 g/l), bovine albumin frac-
tion (0,2% v/v), HEPES buffer (20 mM), antibiotics Cell proliferation Resazurin assay
penicillin–streptomycin (final concentration 100 U/ml The number of viable HeLa, BxPC-3, RD and MDCK cells
penicillin and 100 μg streptomycin/ml) and sup- were measured using resazurin sodium salt (MERCK) as
plemented with FBS (10% v/v). a reagent.
8 of 17 BALAN ET AL.

Triplicate cultures of 1
104 cells in 100 μL of stated absorbance is expressed as % inhibition and is calculated
above medium were incubated at 37  C, 2% CO2 in according to the following formula:
96-well microtiter plates. Tested compounds were dis-
solved in dimethyl sulfoxide in order to prepare 1
10−2 M
solutions. After that these solutions were diluted with


Abscontrol – Abssample =Abscontrol
100:
corresponding media, added to each well and incubated
for 24 hr. Subsequently 20 μL resazurin indicator solution
was added to each well and incubated for 4 hr. At last,
the absorbance was read at 570 nm and 600 nm. 3 | RESULTS A ND DISCUSSION
The percentage inhibition was calculated according to
the formula: The ligand HL was synthesized in good yield and charac-
terized by FT-IR, UV–Vis, 1H NMR and 13C NMR spec-
troscopy and X-ray diffraction. The complexes 2–4, 6,
Abs570nm ðsampleÞ− Abs600nm ðsampleÞ 7 were obtained by combining the isothiosemicarbazone
100 − × 100
Abs570nm ðcontrolÞ −Abs600nm ðcontolÞ HL with various metal salts, but complexes 1, 5, 8 were
synthesized using HL
HI and acetates of corresponding
metals. Coordination compounds of copper were synthe-
where Abs570nm and Abs600nm stand for absorbance at sized in 1:1 molar ratio [Cu:HL], zinc, nickel and cobalt
570 and 600 nm. coordination compounds were synthesized similarly, but
The IC50 values were evaluated by statistical software. in 1:2 molar ratio. The synthesis of the complexes is
reproducible and 2-formilpyridine 4-allyl-S-methylisothio
Cell proliferation MTS assay -semicarbazone coordinates as a tridentate ligand
The number of viable HL-60 cells was measured using through the NNN chelating system, which was confirmed
Owen's Reagent (MTS, CAS#138169–43-4) (Cell Titer 96 by the X-ray structure determinations for complexes 1, 5,
Aqueous, Promega, Madison, Wisconsin, USA). Triplicate 7, and 8.
cultures of 1
104 cells in 100 μL of stated above medium The molar conductivity values of the coordination
were incubated at 37  C, 5% CO2 in 96-well microtiter compounds 1–5 are in the range
plates. The 1
10−2 M solutions of the tested compounds 170–207 Ω−1
cm2
mol−1 that indicates that these com-
were obtained by dissolving them in ethanol. After that plexes represent 1:2 electrolytes. The corresponding
these solutions were diluted with culture media and anion (Cl−, Br−, I−, ClO4−) can be either in the outer
added to each well and incubated for 3 days. Following sphere or in the inner sphere as it can be easily
each treatment, 20 μL of Owen's Reagent was added to substituted by the solvent molecule during dissolution
each well and incubated for 4 hr. At last, the plates were process. The data obtained (85–104 Ω−1
cm2
mol−1) for
read at 490 nm using a microplate reader (Molecular cobalt complexes 6–8 demonstrates their electrolotic
Devices, Sunnyvale, CA, USA). nature (1:1).[44]
The elemental analyses data (reported in Experi-
mental Section) confirm the stoichiometry of
2.6.3 | Antioxidant activity the ligand and the corresponding coordination
compounds.
The ABTS free radical-scavenging activity was deter-
mined according to Re et al.[43] A mixture (1:1, v/v) of
1
ABTS (7 mM) and potassium persulfate (4.95 mM) was 3.1 | H-NMR and 13C-NMR Spectra
incubated at 25  C overnight in the dark to prepare the
stock solution. The ABTS•+ solution was diluted with The purity and structure of isothiosemicarbazone HL
acetate-buffered saline (0.02 M, pH 6.5) to give an absor- was determined using 1H and 13C NMR spectroscopy.
bance of 0.7 ± 0.01 at 734 nm. Solutions in DMSO of The alkylation of the sulfur atom is demonstrated
Trolox, HL and complexes 1–8 were prepared. Then, using NMR spectra of 2-formylpyridine 4-allylthio-
20 μl of each dilution was transferred to a 96-well micro- semicarbazone and 4-allyl-S-methylisothiosemi-
titer plate and 180 μl of working solution of ABTS•+ was carbazone. The 13C NMR spectrum of ligand, the char-
dispensed with the dispense module of hybrid reader acteristic peak (179–180 ppm) is not observed for the
(BioTek) and shaken for 15 s. After 30 min of incubation C = S. Is remarkable in the range 12–14 ppm charac-
was measured the decrease in absorbance at 734 nm. teristic peaks of the carbon atom in the methyl group
Blank samples do not contain ABTS•+. The decrease in (Figure S1).
ANTIMICROBIAL ACTIVITY, ANTIPROLIFERATIVE ACTIVITY, COMPLEXES 9 of 17

In the 1H NMR spectrum of the ligand HL (Figure S5). The electronic spectra in the polycrystal-
(Figure S2), the double peaks of methyl protons in the line state of HL show the intraligand absorption max-
range of 2.41–2.52 ppm are observed.[36,45] Consequently, ima corresponding to π ! π* and n ! π* transitions:
two tautomeric forms in the integral ratio of 1: 0.4 (HL 30,769 and 26,315 cm−1. In the spectra of the com-
(A): HL (B)) are present in the solution. Tautomerism plexes these bands are shifted to lower energies
can be explained by syn/anti isomerism around C=N1 (Table 4).
double bond, and cis/trans isomerism around C=N4 dou- Magnetic moments values for copper complexes 2,
ble bond (Scheme 4).[21,46] 3 are in the range of 1.37–1.48 B.M.These may indicate
their polynuclear structure, because the values are under-
stated in comparison with spin value for one unpaired
3.2 | Infrared Spectra electron 1.73 B.M. Effective magnetic moment of the cop-
per complex 4 is 1.80 B.M., indicate the presence of an
The IR spectra of the complexes (Figure S3) were com- unpaired electron on Cu (II) ion, consequently is a mono-
pared with the ligand (Figure S4) in order to determine mer.[53] The electronic spectra of complexes 2–4 exhibit
the changes that may occur during their formation. It d-d bands at 14,280, 14,490 and 15,380 cm−1, respectively
was observed that three donor nitrogen atoms of the corresponding to the transitions specific to the square-
ligand involved in the coordination to the metal center. pyramidal geometry.[54]
The pyridine υ(С = Npyr) stretching vibration in the The electronic spectrum of the nickel complex 5 shows
free ligand is observed at 1560 cm−1, which is shifted by three spin-allowed bands at 11,290 14,380 and
18–60 cm−1 towards lower wavenumbers in the com- 19,410 cm−1 respectively. These absorption bands may be
plexes, thus indicating coordination of the pyridine nitro- assigned to the 3A2g ! 3T2g(F), 3A2g ! 3T1g(F),
gen atom to the metal ion.[47,48] 3
A2g ! 3T1g(P) transitions respectively, and reflect Ni
A significant change is observed in the area (II) d8 ions in an octahedral geometrical environment.[55]
3010 cm−1, relating to the ν(N4H) stretching vibrations of The magnetic moment value, 3.20 BM corresponds to
the isothiosemicarbazone molecule. It is shifted to the two unpaired electrons per metal ion.
high-frequency region by 60–130 cm−1 in coordination The cobalt complexes 6–8 are diamagnetic, there-
compounds. Such changes in the spectra are the result of fore cobalt ion is in the +3 oxidation state. These com-
participation in the formation of the bond of the plexes show absorption bands in the area 13,980 –
azomethine nitrogen atom. 14,920, 16,390–16,800 and 20,000–22,470 cm−1 that
The intense bands in the spectrum of ligand at 1638 were assigned to the d–d transitions 1A1g ! 3T2g,
and 1585 cm−1 assigned to ν(C=N) and is shifted with up 1
A1g ! 1T1g, 1A1g ! 1T2g, suggesting a octahedral
to 15–27 cm−1,[49–51] in all the complexes, suggesting the geometry.[56]
coordination of the azomethine nitrogen. UV–Vis spectrum of DMSO solution of HL contains
An absorption band is observed at 661 cm−1 in the maximum at 30,581 cm−1 while corresponding spectra of
ligand spectrum that corresponds to ν(C-S), which is not coordination compounds 1–8 contain two maximums in
displaced in the spectra of coordination compounds.[52] the regions 30,959–32,894 cm−1 and 22,123–22,727 cm−1.
Consequently, the sulfur atom is not involved in the coor- It proves that these coordination compounds are stable in
dination of the central ion. DMSO solutions.
The presence of the NO3− anion is demonstrated by
the appearance in the IR spectrum of complex 7 of an
absorption band extended at 1354 cm−1.[30] 3.4 | Mass spectra
The complex 4 exhibits ν (OH) and γ(H2O) bands at
3404 and 1157 cm−1 which are indicative for the coordi- The ESI-MS spectra of the ligand and complexes 1–8
nated water molecules [53] and broad band in the region were recorded to determine molecular species in solution
1100–1000 cm−1 that is characteristic for (Figure S6).
perchlorate ion. The mass spectra of these complexes show peaks
assignable to various fragments arising from the thermal
cleavage of the complexes (Table S1). The molecular ion
3.3 | Electronic Spectra and Magnetic peaks obtained from complexes are as follow:
Studies m/z = 532.1 (1), m/z = 333.9 (2), m/z = 487.1 (3),
m/z = 533.1 (4), m/z = 527.1 (5), m/z = 525.1 (6–8). The
The geometry of the complexes has been deduced from data obtained provide good agreement for the molecular
electronic spectra and magnetic moment data formula of these complexes.
10 of 17 BALAN ET AL.

TABLE 4 Electronic spectral assignments (cm−1) for the complexes 2–8

Compound Transitions d–d (cm−1) μeff (MB) Geometry

[Cu (HL)Cl2] (2) 2
B1g ! B2g B1g ! Eg B1g ! A1g
2 2 2 2 2
1.37 Square-pyramidal
- 14,280 -
[Cu (HL)Br2] (3) 2
B1g ! 2B2g 2B1g ! 2Eg 2B1g ! 2A1g 1.48 Square-pyramidal
- 14,490 -
[Cu (HL)(H2O)2](ClO4)2 (4) 2
B1g ! 2B2g 2B1g ! 2Eg 2B1g ! 2A1g 1.80 Square-pyramidal
- 15,380 -
[Ni (HL)2]I2
H2O (5) 3
A2g(F) ! 3T2g(F) 3A2g(F) ! 3T1g(F) 3.20 Octahedral
3
A2g(F) ! 3T1g(P)
11,290 14,380 19,410
[Co(L)2]Cl (6) 1
A1g ! 3T2g 1A1g ! 1T1g 1A1g ! 1T2g * Octahedral
14,290 16,390 20,000
[Co(L)2]NO3 (7) 1
A1g ! 3T2g 1A1g ! 1T1g 1A1g ! 1T2g * Octahedral
16,390 19,607 22,470
[Co(L)2]I
[Co(L)2](I3) (8) 1
A1g ! 3T2g 1A1g ! 1T1g 1A1g ! 1T2g * Octahedral
13,980 16,806 20,400
*
diamagnetic.

3.5 | Structural Characterization of Figure 3b). In crystal packing, it is observed that cobalt
Ligand HL and Complexes 1, 5, 7, 8 complexes 7 are linked to C (2) -H … S (1) HB in
centrosimetric dimers which are bound by the nitrate
The X-ray structures of HL, 1, 5, 7 and 8 are shown on groups in the bridge, forming the 3D hydrogen bonding
the Figures 1–4. networks (Table 2, Figure 4). The crystal structure of
In HL the substituents at the N(2)–C(1) bond are in 8 contains two independent molecules. But only the first
the Е position. The A(S(1)-N(1)-N(2)-N(3)-C(1)-C(5)) core complex forms the hydrogen bonds with triiodide and
is practically planar within 0.028 Å and the dihedral iodine ions through C(9)-H(9)…I(1) and C(10)-H(10)…
angle between given core and pyridine ring is equal to I(4) HB (Table 2, Figure 3c). The C(3)–H … π and C(8)–H
4.1 . However the given molecule is nonplanar because … π interactions were observed for compounds of HL and
the presence of C 3 H5 substituent in the 7 with H … Cg (pyridine) distances 2.91 and 2.71 Å
thiosemicarbazone moiety, the dihedral angle between respectively. All studied compounds are stabilized by
the best planes of A and (С(2)–С(4)) fragment is equal to C-H…S intramolecular HB.
88.8 . In the crystal the ligand HL forms the chains along The performed powder X-ray diffractograms of these
the c-axis where the molecules are linked by N(3)-H… compounds correspond to the simulated ones from single
N(4) (x,1.2-y,-1/2 + z) hydrogen bonds (HB) (Table 2, crystal X-ray data (Figure S7).
Figure 2).
In these complexes, the isothiosemicarbazone coordi-
nates to the metal atom in a tridentate manner using its 3.6 | Biological Activity
the NNN set of donor atoms. They exhibit a distorted
octahedral geometry by the involvement of two ligand 3.6.1 | Antibacterial and antifungal
molecules (Tables 3, 4, Figures 1, 3, 4). The octahedral activity
volumes of Co atoms in 7 and 8 are equal to 9.122 and
9.213 Å3 whereas for Zn and Ni (1, 5) atoms the In a microdilution in vitro assay, ligand and their com-
corresponding values are 12.111, and 11.178 Å3 plexes were tested against Gram-positive (Staphylococcus
respectively. aureus ATCC 25923), Gram-negative (Escherichia coli
In the crystal the complexes of 1 form the hydrogen ATCC 25922, Klebsiella pneumoniae ATCC 13883) bacte-
bonds with iodine atoms (Table 2, Figure 3a) while in the rial strains and fungal strain (Candida albicans ATCC
crystal structure of 5 the iodine atoms I(1) join the com- 10231). Data interpretation was made using furacillinum
plexes into the dimers via (2A)-H…I(1) (x,1/2-y,-1/2 + z) and nystatine as standards drugs.
and C(10)-H…I(1) (1-x,-1/2 + y,1/2-z) HB. In the dimers The results of the antimicrobial activity study
each of complexes are liked by hydrogen bonds with (Table 5) showed that HL and its complexes manifest
another iodine atoms I(2) and water molecules (Table 2, bacteriostatic and bactericidal properties. Activity of
ANTIMICROBIAL ACTIVITY, ANTIPROLIFERATIVE ACTIVITY, COMPLEXES 11 of 17

FIGURE 1 View of HL, 1, 5, 7, 8 compounds with atom numbering. Thermal ellipsoids are drawn at 50% probability level
12 of 17 BALAN ET AL.

F I G U R E 2 The crystal packing of the

ligand HL, showing the chains formation

FIGURE 3 The crystal packing fragments of (a)1, (b) 5, (c) 8

these compounds towards gram-negative bacteria is It has been observed, that the isothiosemicarbazone
lower than towards gram-positive bacteria and fungi. HL and studied coordination compounds manifest
ANTIMICROBIAL ACTIVITY, ANTIPROLIFERATIVE ACTIVITY, COMPLEXES 13 of 17

FIGURE 4 The crystal packing of 7, showing the (a) formation of centrosymmetric dimers and (b) 3D molecular network

TABLE 5 Antibacterial and antifungal activities of ligand HL, HL
HI and complexes 1–4, 6, 7 as MICa/MBCb/MFCc values (μg/ml)

E. coli (G-) K. pneumoniae (G-) S. aureus (G+) C. albicans

Compound MIC MBC MIC MBC MIC MBC MIC MFC

HL 500 500 500 500 0.7 1.5 250 500
HL
HI 250 250 500 1000 1.5 3 500 1000
[Zn (HL)2]I2 (1) 60 120 120 120 0.7 0.7 60 60
[Cu (HL)Cl2] (2) 60 60 60 60 0.7 0.7 30 30
[Cu (HL)Br2] (3) 60 60 120 120 0.7 0.7 30 30
[Cu (HL)(H2O)2](ClO4)2 (4) 60 60 250 250 1.5 1.5 60 60
[Co(L)2]Cl (6) 250 250 120 250 3 3 7 30
[Co(L)2]NO3 (7) 120 250 60 120 0.7 0.7 120 250
Furacillinum 18.5 37.5 75 150 9.3 9.3 - -
Nystatine - - - - - - 80 80

E. coli (Escherichia coli, ATCC 25922); K. pneumoniae (Klebsiella pneumoniae, ATCC 13883); S. Aureus (Staphylococcus aureus, ATCC 25923); C. albicans
(Candida albicans, ATCC 10231).
a
MIC–minimum inhibitory concentration;
b
MBC—minimum bactericide concentration;
c
MFC, minimum fungicidal concentration. G(−): Gram-negative bacteria; G(+): Gram-positive bacteria.

antimicrobial activities within the concentration limits 2-hydroxybenzaldehyde moiety with 2-formylpyridine
0.7–500 μg/ml. moiety leads to a significant intensification of antimicro-
The most vulnerable to the studied substances was bial activity especially towards Gram-positive Staphylo-
Staphylococcus aureus. In this case, the values of mini- coccus aureus.
mum inhibitory and minimum bactericide concentrations The results show that copper complexes 2–4 are the
vary in the range of concentrations 0.7–3 μg/ml, which most active toward gram-negative bacteria Escherichia
indicate the high activity of synthesized compounds. coli. The attention deserve complexes 2 and 7, because its
These values are several times higher than activity of activity towards gram-negative bacteria Klebsiella
furacillinum that is used in medical practice. pneumoniae exceedes the activity of furacillinum. The
The comparison of the activity of HL with the activity cobalt complex 6 exhibits the greatest antifungal activity
of 2-hydroxybenzaldehyde 4-allyl-S-methylisothiosemi- that exceeding the activity of nystatine and other synthe-
carbazone[30] shows that the substitution of sized substances.
14 of 17 BALAN ET AL.

The activity of this series of complexes exceeds the 3.6.2 | Antiproliferative activity
activity of corresponding complexes of
2-hydroxybenzaldehyde 4-allyl-S-methylisothiosemicar- The compounds have been tested for antiproliferative
bazone.[30] The activity of copper (2–4) and cobalt (6–7) activity on the following cancer cells: human leukemia
complexes is 2–8 times higher towards gram-negative (HL-60), human cervical epithelial (HeLa), human epi-
Escherichia coli than the activity of corresponding copper thelial pancreatic adenocarcinoma (BxPC-3), human
and cobalt complexes of 2-hydroxybenzaldehyde 4-allyl- muscle rhabdomyosarcoma spindle and large
S-methylisothiosemicarbazone. Therefore, the the substi- multinucleated (RD) cells.
tution of 2-hydroxybenzaldehyde moiety with The isothiosemicarbazone and six coordination
2-formylpyridine moiety leads to a significant growth of compounds were tested as inhibitors of HL-60 cells
antimicrobial activity of coordination compounds proliferation. These human promyelocytic leukemia
towards gram-negative microorganisms. cells were incubated for three days in the presence of
The present experimental results show that these synthesized compounds (ligand and complexes)
complexes display the potential application in the and the number of viable cells was measured using
antibacterial and antifungal arias. the MTS assay. The results are expressed as the per-
centage of cell growth inhibition at three
T A B L E 6 Antiproliferative activity of ligand and metal concentrations.
complexes on human leukaemia HL-60 cells at three The HL, HL
HI and zinc complex 1 do not manifest
concentrations and IC50 values inhibitor activity in the concentration range 10–0.1 μM.
Inhibition of cell Copper, nickel and cobalt complexes with this ligand pos-
proliferation (%)a sess anticancer activity with IC50 values 0.4–6.3 μM
IC50,
(Table 6). The nature of the metal ion, the number and
Compound 10 μM 1 μM 0.1 μM μM
nature of the donor atoms, the stereochemistry of the
HL 0 0 0 >100 coordination polyhedral, seem to influence cellular
HL
HI 0 0 0 >100 proliferation.
[Zn (HL)2]I2 (1) 0 0 0 >100 The copper complex 3 manifests the highest activity
[Cu (HL)Cl2] (2) 100 30.1 0 3.3 in comparison to all studied coordination compounds
[Cu (HL)Br2] (3) 99.5 96.0 0 0.4
with this ligand and approaches to the activity of doxoru-
bicin, which is used in medicine. The activity of the stud-
[Cu (HL)(H2O)2] 100 7.6 5.0 5.2
ied complexes practically disappears at 0.1 μM
(ClO4)2 (4)
concentration.
[Ni (HL)2]I2
H2O (5) 94.7 5.7 1.1 5.5
The search for novel biologically active substances
[Co(L)2]NO3 (7) 77.7 10.0 4.1 6.3 with more selectivity and lower toxicity continues to be
Doxorubicin 99 98 15 0.2 an area of intensive investigation. So that, the activity of
a
SEM < ±4% of a single experiment in triplicate. The IC50 values were these compounds was tested on normal MDCK cells.
calculate using statistical software. Experimental data indicate that the ligand HL does not

TABLE 7 Antiproliferative activity of ligand and metal complexes on MDCK cells at three concentrations and IC50 values

Inhibition of cell proliferation (%)a

Compound 100 μM 10 μM 1 μM 0.1 μM IC50, μM

HL 0 0 0 0 >100
[Zn (HL)2]I2 (1) 96.7 0 0 0 68
[Cu (HL)Cl2] (2) 100 79.5 2.4 0 5.0
[Cu (HL)Br2] (3) 100 100 7.3 0 1.5
[Ni (HL)2]I2
H2O (5) 57.4 0 0 0 95
[Co(L)2]Cl (6) 45.8 0 0 0 >100
[Co(L)2]NO3 (7) 23.8 0 0 0 >100
[Co(L)2]I
[Co(L)2](I3) (8) 31.0 0 0 0 >100
Doxorubicin - 56.0 25.1 19.1 7.1
a
SEM < ±4% of a single experiment in triplicate.
ANTIMICROBIAL ACTIVITY, ANTIPROLIFERATIVE ACTIVITY, COMPLEXES 15 of 17

TABLE 8 IC50 values of ligand and metal complexes towards MDCK, HL-60, HeLa, BxPC-3 and RD cells

MDCK HL-60 HeLa BxPC-3 RD

Compound IC50, μM IC50, μM SIa IC50, μM SIa IC50, μM SIa IC50, μM SIa
HL >100 >100 - >100 - >100 - 16.1 >6.2
[Zn (HL)2]I2 (1) 68 >100 - >100 - >100 - 10.9 6.24
[Cu (HL)Cl2] (2) 5.0 3.3 1.52 7.92 0.63 1.08 4.63 1.16 4.31
[Cu (HL)Br2] (3) 1.5 0.4 3.75 1.25 1.20 0.36 4.17 0.68 2.21
[Ni (HL)2]I2
H2O (5) 95 5.5 17.3 54.0 1.76 51.6 1.84 14.1 6.74
[Co(L)2]Cl (6) >100 - - 2.1 >47 48.2 >2.1 15.9 6.29
[Co(L)2]NO3 (7) >100 6.3 >16 >100 - >100 - 57.0 >1.7
[Co(L)2]I
[Co(L)2](I3) (8) >100 - - >100 - >100 - >100 -
Doxorubicin 7.1 0.2 35.5 10.0 0.71 3.7 1.92 16.2 0.44
50 ðMDCK Þ
a
SI – selectivity index SI ¼ ICIC
50 ðcancer cellÞ
.

inhibit proliferation of these normal cells (Table 7). 4 | CONCLUSIONS

Cobalt coordination compounds also do not affect the
growth and multiplication of MDCK cells. Physico-chemical analyses confirmed the composition
By performing primary screening on several cancer and structures of the newly obtained complexes. The
cells, the selectivity of antiproliferative activity was coordination ability of the isothiosemicarbazone HL
determined for some of the synthesized complexes has been proved in complexation reaction with Zn (II),
(Table 8). Ni (II), Cu (II) and Co (III) ions. The structure of the
The isothiosemicarbazone HL selectively inhibits HL and complexes 1, 5, 7 and 8 has been elucidated
only RD cells. The copper complex 3 is the most active by single-crystal X-ray diffraction analysis. These stud-
towards all studied cancer cells, but it also affects prolif- ies demonstrate the nonplanar configuration of the
eration of normal cells, so it is not enough selective ligand by the presence of the C3H5 radical in the iso-
(SI (selectivity index) = 1.2–4.2). Copper complexes 2, thiosemicarbazone structure.
3 have selectivity indexes towards BxPC-3 and RD cells Depending on the metal salt used, the iso-
that exceed corresponding SI values of doxorubicin in thiosemicarbazone act as ligand tridentate through
2.2–9.8 times. Cobalt complex 6 selectively inhibits prolif- deprotonated N4 nitrogen atom (when we used
eration of HeLa cells (SI > 47) and practically does not CoCl2
6H2O, Co (NO3)2
6H2O, Co (CH3COO)2
4H2O),
influence the growth of normal cells. Its SI value towards nondeprotonated N4 (when we used Zn
this cancer cell is more than 60 times higher than the (CH3COO)2
2H2O, CuCl2
2H2O, CuBr2, Cu
corresponding SI value of doxorubicin that is used in (ClO4)2
6H2O, Ni (CH3COO)2
4H2O), N1 and pyridine
medical practice. nitrogen atoms.
Copper coordination compound 3 is more active than The antimicrobial activity of the ligand and its
corresponding copper complex of 2-hydroxybenzal- complexes was evaluated on a series of standard
dehyde 4-allyl-S-methylisothiosemicarbazone[30] but it is strains of Staphylococcus aureus, Escherichia coli,
less selective at the same time. So the subtituition of Klebsiella pneumoniae and Candida albicans and antip-
2-hydroxybenzaldehyde moiety with 2-formylpyridine roliferative activity was investigated using HL-60,
moiety leads to a decrease of selectivity. HeLa, BxPC-3, RD cancer cell lines and normal
MDCK cell.
The quantitative antimicrobial activity test results
3.6.3 | Antioxidant activity proved that the complexes have specific antimicrobial
activity, depending on the microbial species tested, in the
The antioxidant activity of the compounds was deter- range of concentration 0.7–500 μg/ml.
mined by the ABTS• + method. Experimental data rev- The screening results showed that the synthesized
ealed that ligand HL and complexes 1–8 show moderate substances inhibit proliferation of the studied cancer cells
antioxidant activity against ABTS•+ with an at concentrations 100–0.1 μM. The study of the influence
IC50 ≥ 100 μM. of synthesized compounds on healthy MDCK cells
16 of 17 BALAN ET AL.

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