Synthesis and characterization of some new Cu(II), Ni(II) and Zn(II) complexes with salicylidene thiosemicarbazones: Antibacterial, antifungal and in vitro antileukemia activity

Article abstract of DOI:10.3390/molecules18088812

Thirty two new Cu(II), Ni(II) and Zn(II) complexes (1-32) with salicylidene thiosemicarbazones (H₂L¹-H₂L¹⁰) were synthesized. Salicylidene thiosemicarbazones, of general formula (X)N-NH-C(S)-NH(Y), were prepared

Full text of DOI:10.3390/molecules18088812

Molecules 2013, 18, 8812-8836; doi:10.3390/molecules18088812
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Characterization of Some New Cu(II), Ni(II) and
Zn(II) Complexes with Salicylidene Thiosemicarbazones:
Antibacterial, Antifungal and in Vitro Antileukemia Activity
Elena Pahontu 1,*, Valeriu Fala 2, Aurelian Gulea 3, Donald Poirier 4, Victor Tapcov 3 and
Tudor Rosu 5
1
Inorganic Chemistry Department, Faculty of Pharmacy, University of Medicine and
Pharmacy “Carol Davila”, 6 Traian Vuia Street, 020956 Bucharest, Romania
2
Dental Education Department, Moldova State University of Medicine and Pharmacy
“N. Testemitsanu” Chisinau, Republic of Moldova
3
Coordination Chemistry Department, Moldova State University, 60 Mateevici Street,
2009 Chisinau, Republic of Moldova
4
Laboratory of Medicinal Chemistry, CHUQ (CHUL)- Research Center and Université Laval,
2705 Boulevard Laurier, Québec City, QC G1V 4G2 Canada
5
Inorganic Chemistry Department, Faculty of Chemistry, University of Bucharest,
23 Dumbrava Rosie Street, 020462 Bucharest, Romania
* Author to whom correspondence should be addressed; E-Mail: elenaandmihaela@yahoo.com.
Received: 29 May 2013; in revised form: 12 July 2013 / Accepted: 15 July 2013 /
Published: 24 July 2013
Abstract: Thirty two new Cu(II), Ni(II) and Zn(II) complexes (1–32) with salicylidene
thiosemicarbazones (H2L1–H2L10) were synthesized. Salicylidene thiosemicarbazones, of
general formula (X)N-NH-C(S)-NH(Y), were prepared through the condensation
reaction of 2-hydroxybenzaldehyde and its derivatives (X) with thiosemicarbazide or
4-phenylthiosemicarbazide (Y = H, C6H5). The characterization of the new formed
1
compounds was done by H-NMR, 13C-NMR, IR spectroscopy, elemental analysis,
magnetochemical, thermoanalytical and molar conductance measurements. In addition, the
structure of the complex 5 has been determined by X-ray diffraction method. All ligands
and metal complexes were tested as inhibitors of human leukemia (HL-60) cells growth
and antibacterial and antifungal activities.

Molecules 2013, 18
Keywords: copper; nickel; zinc complexes; thiosemicarbazones; antimicrobial activity;
8813
antileukemia
1. Introduction
The design and study of well-arranged metal-containing Schiff bases with ONS – donor atoms is an
interesting field of inorganic and bioinorganic chemistry [1–11]. In-situ one-pot template condensation
reactions lie at the heart of the coordination chemistry. Transition metal complexes have also received
great attention because of their biological interests, including antiviral, anticarcinogenic, antibacterial
and antifungal activities [12–16]. Thiosemicarbazones and their Cu(II) complexes demonstrated potent
cytotoxic activities against a series of murine and human tumor cells in culture [17–19].
In a recent study [20], we have concluded that the in vitro HL-60 leukemia cell growth inhibitory
activity is influenced by the nature and geometric structure of copper complexes. Indeed, copper
complexes containing tridentate ONS Schiff bases as well as salicyliden thiosemicarbazones have been
found as effective inhibitors of cell proliferation. We have started a program directed toward the
synthesis of different classes of anticancer, antibacterial and antifungal agents designed with
complexes of a transition metal and an organic ligand [21–24].
In continuation of this approach, the present paper describes the synthesis, characterisation and in
vitro evaluation of inhibitors of HL-60 cell proliferation, antibacterial and antifungal activity using thirty
two novel Cu(II), Ni(II) and Zn(II) complexes with the salicylidene thiosemicarbazones (H2L1–H2L10),
obtained from the condensation reaction of thiosemicarbazide or 4-phenylthiosemicarbazide with
2-hydroxybenzaldehyde derivatives. All ligands and metal complexes were tested as inhibitors of
human leukemia (HL-60) cell growth. The Cu(II) complexes 21–25, 30 have also been tested for their
in vitro antibacterial activity against Staphylococcus aureus (Wood-46, Smith, 209-P), Staphylococcus
saprophyticus, Streptococcus (group A), Enterococcus faecalis (Gram-positive), Escherichia coli
(O-111), Salmonella typhimurium, Salmonella enteritidis, Klebsiella pneumoniaie, Pseudomonas
aeruginosa, Proteus vulgaris and Proteus mirabilis (Gram-negative) and antifungal activity against
Aspergillus niger, Aspergillus fumigatus, Candida albicans and Penicillium strains.
2. Results and Discussion
2.1. Chemistry
The salicylidene thiosemicarbazones H2L1–H2L10 used in this work were prepared by refluxing (for
30 min.) in ethanol an equimolar amount of aldehyde (salicylaldehyde or its derivatives,
5-chloro-, 5-bromo-, 5-nitro-, 5-methyl- and 3,5-dichlorosalicylaldehyde) and thiosemicarbazide or
4-phenylthiosemicarbazide. The structures of the Schiff bases H2L1–H2L10 were established by IR,
1H-NMR and 13C-NMR spectroscopy.
These Schiff bases were further used for the complexation reaction with Cu2+, Ni2+, Zn2+ metal ions,
using the following salts: CuSO4·5H2O (for complexes 1–7), Cu(NO3)2·3H2O (for 8–14), CuCl2·2H2O
(for 15–30), NiCl2·6H2O (for 31) and ZnCl2 (for 32). To metal salt (10 mmol) dissolved in distilled

Molecules 2013, 18
8814
water was added salicylidene thiosemicarbazone, HL, (10 mmol) dissolved in ethanol. The reaction
mixture was stirred and heated (50–55 °C) for 1.5 h. The precipitate was filtered, washed with ethanol,
ether and dried in air.
The complexes obtained are microcrystalline solids which are stable in air and decompose above
310 °C (Table 1). They are insoluble in organic solvents such as acetone and chloroform but soluble in
DMF and DMSO.
The molar conductance of the soluble complexes in DMF showed values indicating that 1–14
(80–100 ohm−1 cm2 mol−1) are electrolytes and 15–32 (10–20 ohm−1 cm2 mol−1) are non-electrolytes in
nature [25].
The elemental analyses data of Schiff bases (reported in the Experimental section) and their
complexes (Table 1) are in agreement with the proposed composition of the ligands as shown in
Scheme 1 and with the formulas of the complexes as shown in Figure 1a,b.
Scheme 1. General synthesis of organic ligands H2L1−1°.
R1
OH
H
H
N
N
NH
Y
O +
2
- HO
2
R2
S
R1 = H, R2 = H, Y = H (H2L1), R1 = Cl, R2 = Cl, Y = H (H2L6)
R1 = H, R2 = Cl, Y = H (H2L2), R1 = Br, R2 = Br, Y = H (H2L7)
R1 = H, R2 = Br, Y = H (H2L3), R1 = H, R2 = H, Y = C6H5 (H2L8)
R1 = H, R2 = NO2, Y = H (H2L4), R1 = H, R2 = Br, Y = C6H5 (H2L9)
R1 = H, R2 = CH3, Y = H (H2L5), R1 = H, R2 = NO2, Y = C6H5 (H2L10)
Figure 1. (a) General structure of complexes 1–14. (b) General structure of complexes 15–32.
(a)
R1
+
H2O
O
S
Z -
Cu
.
n H2O
NHY
N
N
R2
H

Molecules 2013, 18
8815
Figure 1. Cont.
(b)
M = Cu (15–30), Ni (31), Zn (32); R1 = H (1–19, 30–32), Cl (20), Br (21–29); R2 = H (1, 2, 8, 9,
15, 30–32), CH3 (19), Cl (7, 14, 16, 20), Br (5, 6, 12, 13, 17, 21–29), NO2 (3, 4, 10, 11, 18); Y = H
(1, 3, 5, 7, 8, 10, 12, 14–32), C6H5 (2, 4, 6, 9, 11, 13).
A–Structure
Name
Complex
Py
15–20
H3N
-
21
22
4-MePy
3-MePy
2-MePy
23
24
Etz
Str
25, 30–32
26
Sfc
27
28
Nor
Sdm
29

Molecules 2013, 18
8816
Table 1. Physical and analytical data of the metal complexes 1–32 a.
c
Comp.
No.
µ eff
C, H, N, calc
(found) %
M(3d) d
%
η,
T, C f
dec
Molecular formula
Mr b
IR (cm−1)
e
B.M.
%
H2O (3585, 1575, 920); NH2
(3435, 3420); NH(3335, 3220,
3145); C= N (1605); C-O (1200);
C = S (781);
C: 28.1(28.5);
H: 3.5 (3.0);
C16H24Cu2N6O10S3
[Cu(H2O)(HL1)][Cu(H2O)(HL1)SO4] . 2H2O
18.7
1
2
3
684
2.14
2.07
1.98
65
64
77
460
450
425
N: 12.3(12.5);
S: 14.0 (13.7)
(18.6)
Cu-N (510, 415); Cu-O (470);
Cu-S (450)
C: 41.1(41.4);
H: 3.7 (3.4);
H2O (3580, 1570, 925); NH (3325,
3222, 3143); C = N (1600);
C-0(1195); C = S (780); Cu-N(517,
428); Cu-O (472); Cu-S (445)
H2O (3575, 1570, 922); NH2
(3445, 3425); NH (3330 3230,
3140); C = N (1590); C-O (1195);
C = S (776);
C28H30Cu2N6O9S3
[Cu(H2O)(HL8)][Cu(H2O)][(HL8)SO4] . H2O
15.6
818
774
N: 10.3(10.3);
S: 11.7 (11.6)
(15.8)
C: 24.8 (24.5);
H: 2.8(2.7);
C16H22Cu2N8O14S3
[Cu(H2O)(HL4)][Cu(H2O)(HL4)(SO4) ] . H2O
16.5
N: 14.5 (14.8);
S: 12.4 (12.7)
(16.3)
Cu-N (530, 410);
Cu-O (470); Cu-S (465)
H2O (3580, 1583, 915); NH (3315,
3230, 3138); C = N (1585);
C-O (1197); C = S (779);
Cu-N (525, 430); Cu-O (475);
Cu-S(440)
C: 36.3 (36.5);
H: 3.2 (3.0);
C28H30Cu2N8O14S3
[Cu(H2O)(HL1°)][Cu(H2O)(HL1°)(SO4)].
2H2O
13.8
4
5
926
878
2.09
1.85
72
69
410
450
N: 12.1 (12.4);
S: 10.4 (10.1)
(14.1)
H2O (3565, 1575, 935); NH2
(3445, 3430); NH (3340, 3230,
3137); C = N (1590); C-O (1205);
C = S (780);
C: 21.9 (22.2);
H: 3.3 (3.3);
C16H26Br2Cu2N6O12S3
[Cu(H2O)(HL3)][Cu(H2O)(HL3)(SO4)] . 4H2O
14.6
Br: 18.2 (18.4);
N: 9.6 (9.4);
(14.4)
Cu-N (505, 430);
S: 10.3 (10.5)
Cu-O (485); Cu-S (462)

Molecules 2013, 18
8817
Table 1. Cont.
c
Comp.
No.
µ eff
C, H, N, calc
M(3d) d
%
η,
T, C f
dec
Molecular formula
Mr b
IR (cm−1)
e
B.M.
(found) %
C: 34.4 (34.0);
H: 2.9 (2.7);
Br: 16.4 (16.5);
N: 8.6 (8.4);
S: 9.8 (9.9)
%
H2O (3580, 1565, 930); NH (3330,
3225, 3145); C = N (1585);
C28H28Br2Cu2N6O9S3
[Cu(H2O)(HL9)][Cu(H2O)(HL9)(SO4)] . H2O
13.1
6
976
1.91
56
78
435
(12.8)
C-O (1203); C = S (778); Cu-N
(525, 425); Cu-O (484); Cu-S (465)
H2O (3585, 1575, 920); NH2 (3430,
3430); NH (3335, 3220, 3145);
C = N (1595); C-0(1200);
C = S(785);
C: 26.1 (26.3);
H: 2.7 (2.4);
C16H20Cl2Cu2N6O9S3
[Cu(H2O)(HL2)][Cu(H2O)(HL2)(SO4)] . H2O
17.4
7
735
356
1.79
1.87
Cl: 9.7 (10.0); N:
11.4 (11.5);
430
(17.7)
Cu-N (528, 410);
S: 13.1 (13.3)
Cu-O (482); Cu-S (464)
H2O (3580, 1574, 915); NH2 (3440,
3430); NH (3325, 3230, 3140);
C = N(1600); C-0(1200);
C = S(776);
C: 27.0 (27.3);
H: 3.4 (3.1);
N: 15.7 (15.5);
S: 9.0 (9.4)
C8H12CuN4O6S
[Cu(H2O) (HL1) ]NO3 . H2O
18.0
8
70
390
(18.2)
Cu-N(530, 410);
Cu-O(480); Cu-S(450)
H2O (3576, 1570, 930); NH(3345,
3227, 3146); C = N(1595);
C-O (1198); C = S (787);
Cu-N (525, 430); Cu-O (465);
Cu-S (440)
C: 38.9 (38.4);
H: 3.7 (3.5);
N: 13.0 (13.1);
S: 7.4 (7.2)
C14H16CuN4O6S
[Cu (H2O)(HL8)]NO3 . H2O
14.8
9
432
401
2.12
1.85
54
76
380
325
(14.5)
H2O (3570, 1565, 925); NH2 (3445,
3430); NH (3325, 3215, 3140);
C = N (1598); C-O (1195);
C = S (777); Cu-N (525, 410);
Cu-O (475); Cu-S (440)
C: 23.9 (24.2);
H: 2.7 (2.5);
N: 17.5 (17.1);
S: 8.0 (8.3)
C8H11CuN5O8S
[Cu (H2O)(HL4)]NO3 . H2O
16.0
10
(16.3)

Molecules 2013, 18
8818
Table 1. Cont.
c
Comp.
No.
µ eff
C, H, N, calc
M(3d) d
%
η,
T, C f
dec
Molecular formula
Mr b
IR (cm−1)
e
B.M.
(found) %
%
H2O (3590, 1585, 915); NH(3325,
3225, 3140); C = N(1593);
C-0(1192); C = S(783); Cu-
N(525, 430); Cu-O(480); Cu-
S(455)
C: 33.9 (34.1);
H: 3.4 (3.5);
N: 14.1 (14.2);
S: 6.5 (6.9)
C14H17CuN5O9S
[Cu (H2O)(HL1°)]NO3 . 2H2O
12.9
11
12
13
495
1.94
80
52
65
315
370
360
(12.7)
H2O (3585, 1575, 920);
NH2(3430, 3415); NH(3335,
3220, 3145); C = N(1595); C-
0(1195); C = S(784); Cu-N(525,
425); Cu-O(475);
C: 22.1 (21.8);
H: 2.5 (2.2);
N: 12.9 (13.2);
S: 7.4 (7.5)
C8H11BrCuN4O6S
[Cu (H2O)(HL3)]NO3 . H2O
14.7
435
547
1.80
1.97
(14.5)
Cu-S(460)
C: 30.7 (30.9);
H: 3.4 (3.2);
Br: 14.6 (14.4);
N: 10.2 (9.9);
S: 5.8 (5.7)
H2O (3570, 1565, 925); NH(3330,
3210, 3135); C = N(1590);
C-0(1197); C = S(780); Cu-
N(530, 423); Cu-O(470); Cu-
S(465)
C14H19BrCuN4O8S
[Cu(H2O)(HL9)]NO3 . 3H2O
11.7
(11.5)
H2O (3585, 1575, 920);
NH2(3435, 3425); NH(3335,
3220, 3145); C = N(1605); C-
0(1193); C = S(780);
C: 24.6 (24.6);
H: 2.8 (2.5);
Cl: 9.1 (9.3);
N: 14.3 (14.5);
S: 8.2 (8.5)
C8H11ClCuN4O6S
[Cu(H2O)(HL2)]NO3 . H2O
16.4
14
15
390.5
2.03
1.78
75
71
365
460
(16.1)
Cu-N(515, 430);
Cu-O(490); Cu-S(455)
NH2(3440, 3425); C = N(1590,
1580, 1575, 1310); C-0 (1215);
C-S ( 760); Cu-N(530, 425);
Cu-O(475); Cu-S(460)
C: 46.4 (46.5);
H: 3.6 (3.5);
N: 16.7 (16.5);
S: 9.5 (9.4)
C13H12CuN4OS
[Cu L1Py]
19.0
336
(18.8)

Molecules 2013, 18
8819
Table 1. Cont.
c
Comp.
No.
µ eff
C, H, N, calc
M(3d) d
%
η,
T, C f
dec
Molecular formula
Mrb
IR (cm−1)
e
B.M.
(found) %
C: 42.1 (42.0);
H: 3.0 (2.9);
Cl: 9.6 (9.5);
N: 15.1 (15.0);
S: 8.6 (8.4)
%
NH2 (3435, 3420); C = N (1585,
1580, 1570, 1305); C-O (1225);
C-S (750); Cu-N (510, 405);
Cu-O (470); Cu-S (465)
C13H11ClCuN4OS
[Cu L2Py]
17.3
16
370.5
1.78
72
75
440
450
(17.0)
C: 37.6 (37.5);
H: 2.7 (2.5);
Br: 19.3 (19.0);
N: 13.5 (13.3);
S: 7.7 (7.5)
NH2 (3430, 3420); C = N (1585,
1580, 1575, 1300); C-O (1210);
C-S (750); Cu-N (515, 410);
Cu-O (475); Cu-S (465)
C13H11BrCuN4OS
[Cu L3Py]
15.4
17
18
19
415
1.93
(15.5)
C: 40.9 (40.8);
H: 2.9 (2.8);
N: 18.4 (18.2);
S: 8.4 (8.3)
NH2 (3440, 3425); C = N (1585,
1580, 1570, 1315); C-O (1220);
C-S ( 770); Cu-N (525, 410);
Cu-O (470); Cu-S (465)
C13H11CuN5O3S
[Cu L4Py]
16.8
381
350
1.84
1.75
69
70
400
460
(16.7)
C: 48.0 (47.8);
H: 4.0 (3.9);
N: 16.0 (15.9);
S: 9.1 (9.0)
NH2 (3430, 3425); C = N (1590,
1585, 1570, 1315); C-O (1220);
C-S ( 770); Cu-N (520, 415);
Cu-O (470); Cu-S (465)
C14H14CuN4OS
[Cu L5Py]
18.3
(18.0)
C: 38.5 (38.3);
H: 2.5 (2.4);
Cl: 17.5 (17.4);
N: 13.8 (13.6);
S: 7.9 (7.8)
NH2 (3435, 3425); C = N (1585,
1580, 1575, 1305); C-O (1205);
C-S ( 770); Cu-N (515, 430);
Cu-O (485); Cu-S (47 0)
20
21
C13H10Cl2CuN4OS
[Cu L6Py]
15.8
405
468
1.80
1.87
76
78
410
310
(15.7)
C: 20.5 (20.4);
H: 2.6 (2.5);
Br: 34.2 (34.0);
S: 6.8(6.7)
NH2 (3440, 3425); NH (3330, 3215,
3150); C = N (1582, 1585);
C8H12Br2CuN4O3S
[Cu L7(NH3)] ⋅ 2H2O
11.9
(1.7)
C-O (1225); C-S (748); Cu-N (540,
425); Cu-O (490); Cu-S (410);

Molecules 2013, 18
8820
Table 1. Cont.
c
Comp.
No.
µ eff
C, H, N, calc
M(3d) d
%
η,
T, °C f
dec
Molecular formula
Mr b
IR (cm−1)
e
B.M.
(found) %
C: 31.9 (31.8);
H: 2.7 (2.5);
%
NH2 (3435,3430); C = N
(1580,1585); C-O (1225); CNC
(1042); C-S (748); Cu-O (540);
Cu-O (490); Cu-S (410)
C14H14 Br2CuN4O2S
[Cu L7(4-MePy)] ⋅ H2O
12.2
22
23
24
526
1.79
77
76
71
380
390
345
Br: 31.5(31.3);
S: 6.3(6.0)
(12.3)
C: 33.1 (33.0); H:
2.4 (2.2); Br:
31.5(31.4);
NH2 (3440,3425);
C14H12 Br2CuN4OS
[Cu L7(3-MePy)]
12.6
C = N (1580,1585); C-O (1225);
CNC (1042); C-S (748); Cu-O
(540); Cu-O (490); Cu-S (410)
NH2 (3440,3430); C = N
508
508
1.99
1.92
(12.4)
S: 6.3(6.1)
C: 33.1 (32.9); H:
2.4 (2.5); Br:
31.5(31.4);
C14H12 Br2CuN4OS
[Cu L7(2-MePy)]
12.6
(1580,1585); C-O (1225); CNC
(1042); C-S (748); Cu-O (540);
Cu-O (490); Cu-S (410)
(12.3)
S: 6.3(6.0)
C: 30.9 (31.0); H:
2.4 (2.2); Br: 22.9
(22.7); N:
NH2 (3435,3425, 3420, 3410);
C = N (1610, 1600, 1585); SO2
(1320, 1140), C-O (1215); Cu-N
(540, 415); Cu-O (490); Cu-S (440)
C18H17Br2CuN7O3S3
[Cu L7(Etz)]
9.2
25
26
27
699
587
629
1.35
1.28
1.31
81
68
63
470
430
450
(8.6)
14.0(13.9);
S: 13.7(13.5)
C: 28.6 (28.5); H:
2.2 (2.0); Br: 27.3
(27.0); N: 11.9
(12.0);
NH2 (3415,3420,3405,3415); C = N
(1600, 1585); SO2 (1325, 1140);
C-O (1210); Cu-N (525, 410);
Cu-O (475); Cu-S (455)
C14H13Br2CuN5O3S2
[Cu L7(Str)]
10.9
(11.0)
S: 10.9 (10.7)
C: 30.5 (30.3); H
2.4 (2.5); Br: 25.4
(25.3); N: 11.1
(11.0); S: 10.2
(10.0)
NH2 (3420,3415,3415,3405); C = N
(1605, 1590); SO2 (1320, 1145);
C-O (1215); Cu-N (530, 425);
Cu-O (480); Cu-S (465);
C16H15Br2CuN5O4S2
[Cu L7(Sfc)]
10.2
(10.1)

Molecules 2013, 18
8821
Table 1. Cont.
c
Comp.
No.
µ eff
C, H, N, calc
M(3d) d
%
η,
T, C f
dec
Molecular formula
Mr b
IR (cm−1)
e
B.M.
(found) %
C: 30.4 (30.2);
H: 2.1 (2.0); Br:
23.9 (24.0); N: 12.5
(12.3); S: 14.3
(14.2)
%
NH2 (3430, 3425, 3415, 3410);
C = N (1610, 1605, 1590); SO2
(1315, 1145); C-O (1210); Cu-N
(530, 420);
C17H14Br2CuN6O3S3
[Cu L7(Nor)]
9.6
28
29
30
670
1.35
69
470
460
500
380
490
(9.5)
Cu-O (480); Cu-S (455);
NH2 (3440, 3430, 3425, 3415);
C = N (1610, 1600, 1595); SO2
(1310, 1150); C-O (1215); Cu-N
(510, 425);
C: 34.6 (34.5); H:
2.7 (2.5); Br: 23.1
(23.0); N: 14.1
C20H19Br2CuN7O3S2
[Cu L7(Sdm)]
9.2
693
541
572
542
1.22
1.45
dia
68
70
80
75
(9.1)
(14.0); S: 9.2 (9.0)
Cu-O (475); Cu-S (450);
NH2 (3435, 3430, 3425, 3415);
C = N (1600, 1595, 1590); SO2
(1310, 1140); C-O (1225); Cu-N
(515, 410);
C: 39.9 (40.0); H:
3.5 (3.4); N: 18.1
(17.9); S: 17.7(17.5)
C18H19CuN7O3S3
[Cu L1(Etz)]
11.8
(11.6)
Cu-O (470); Cu-S (465)
NH2 (3430, 3430, 3420, 3410);
C = N (1605, 1595, 1590); SO2
(1315, 1145); C-O (1220); Cu-N
(525, 415);
C: 37.8 (37.5); H:
4.0 (3.8); N: 17,1
(17.0);
C18H23NiN7O5S3
[Ni L1(Etz)] ⋅ 2H2O
10.3
31
32
(10.2)
S: 16.8 (16.6)
Cu-O (475); Cu-S (460)
NH2 (3430, 3430, 3420, 3415);
C = N (1605, 1595, 1585); SO2
(1315, 1140); C-0 (1215); Cu-N
(525, 425);
C: 39.9 (40.0); H:
3.5 (3.4); N: 18.1
(18.0); S: 17.7
(17.5)
C18H19N7O3S3Zn
[Zn L1(Etz)]
12.0
dia
(11.8)
Cu-O (480); Cu-S (470)
a H2L10, used in the preparation of complexes are reported in Scheme 1. b Mr: relative molecular mass. c µ eff: magnetic moment. d M (3d): metal 3d. e η: yield. f Tdec .:
decomposition temperature.

Molecules 2013, 18
8822
2.1.1. X-ray Structure of [Cu(H2O)(HL3)][Cu(H2O)(HL3)(SO4)]·4H2O (5)
The structure of crystals, obtained from ethanolic solution after recrystallization of (5), has been
determined by means of X-ray analysis and is similar to the structure described in [26].
2.1.2. IR Spectra and Coordination Mode
The tentative assignments of the significant IR spectral bands of H2L1–H2L10 and their Cu(II),
Ni(II) and Zn(II) complexes are presented in Table 1. It has been established that the substituted
salicylaldehyde thiosemicarbazones of complexes 1–14 behave as monodeprotonated tridentate ligands
and are coordinated to the central ions through deprotonated phenolic oxygen atom, azomethinic
nitrogen atom and sulphur atom forming five- and six-membered metalocycles [9,20,21].
The IR spectra of the free ligands shows a broad band at ca. 3600 cm−1 attributed to phenolic group,
δ(OH). This band disappeared from IR spectra of complexes 1–14 [22,23,27]. Moreover, this is
confirmed by the shift of ν(C-O) stretching vibration bands observed in the range of 1250-1240 cm−1
in the spectra of the free ligands, to lower frequency at around 1225–1210 cm−1 in the spectra of the
complexes. This is further confirmed by the presence of the band appearing in the region 500-470 cm−1
assigned to the ν(M-O) frequency [28].
Likewise, the IR spectra of the ligands exhibits a strong band in the range 1620–1610 cm−1
assignable to ν(C = N). In the spectra of the complexes 1–14 this band is shifted to lower frequencies
by ca. 25–15 cm−1 suggesting the coordination of the azomethine nitrogen to the central metal atom.
Also, this coordination is supported of ν(M-N) vibration around 515–540 cm−1 [29].
In the IR spectra of the H2L1–H2L10, the ν(S-H) band at 2570 cm−1 [30–33] was absent, but the
ν(C = S) bands at about 1560 and 822 cm−1 were present. These bands were shifted to lower
wavenumbers in complexes 1–14 and this shift can be assigned to the thiocarbonyl ν(C = S) stretching
and bending modes of vibrations and to the coordination of sulfur atom to metal ion [34–36].
In complexes 15–32, thiosemicarbazones behave as double deprotonated tridentate ligands,
coordinating to the central ion through phenolic oxygen atom, azomethinic nitrogen atom and sulphur
atom forming two five- and six-membered heterocycles. As much, the absorption bands ν(C-OH),
ν(N-NH) and ν(C=S), observed in the spectra of the free thiosemicarbazones, in the range
1245–1240, 1540–1535 and 1125–1120 cm−1, respectively, were shifted to lower frequencies in the
spectra complexes. In the spectra complexes the absorption band ν(C-S) is observed in the range
750–740 cm−1 and the band ν(C-N) is shifted to small frequencies with 35-30 cm−1, being
accompanied by the splitting into two components [27–29].
In the IR spectra of complexes 15–32, an absorption band is observed in the range 1520–1518 cm−1,
conditioned by valence oscillations >C = N-N = C<. This character of IR spectra demonstrates the
thiosemicarbazone enolization in the process of synthesized complexes formation [30–33].
The nitrate complexes 8–14 shows a single band at around 1345-1340 cm−1. It is attributable to
−
ionic NO3 [37].
In compounds 1–14 the absorption bands characteristic to the water molecule from the inner sphere
are observed: ν(H2O) = 3595–3585 cm−1, δ(H2O) = 1590–1585 cm−1, γ(H2O) = 920–915 cm−1,

Molecules 2013, 18
8823
w(H2O) = 640–615 cm−1 due to OH stretching, HOH deformation, H2O rocking and H2O wagging,
respectively [38].
The presence of sulphanilamides in complexes 25–32 is confirmed by the characteristic absorption
bands observed in IR spectra: νas(NH2), νs(NH2): ≈ 3400 cm−1; ν(N-H): 3330 ± 20 cm−1, ν(C-N)(arom)
:
1305 ± 55 cm−1, ν(C = N)(arom) 1580 ± 30 cm−1; νas(SO2), νs(SO2): 1320 ± 20 cm−1, 1100 ± 20 cm−1. It has
been established that the investigated sulphanilamides of the given complexes behave as monodentate
ligands and are coordinated to the central atom through nitrogen atoms and amino groups in the case of
streptocide (Str) and sulphacil (Sfc), thiadiazolic nitrogen atom in the case of ethazole (Etz) and
norsulphazole (Nor) one of the pyrimidinic nitrogen atoms in the case of sulphadimezine (Sdm) [38].
2.1.3. Magnetochemistry
The room temperature magnetic moment of the solid copper (II) complexes 1-24 was found in the
range 1.75–2.00 BM, indicative one unpaired electron per Cu(II) ion [39]. These experimental data
allow us to suppose that in these compounds the spin-spin interaction lacks and probably the
investigated complexes have monomer structure. Also, the magnetic moment values in the range
1.22–1.45 BM for the copper (II) complexes 25–30 are of indicative anti-ferromagnetic spin-spin
interaction through molecular association [40]. Complex 31 is diamagnetic and the central Ni2+ ion is
in a square planar environment [40].
2.1.4. Thermal Decomposition
All complexes studied were investigated by thermogravimetry analysis. The TG thermograms of
complexes 1–14 are characterized by three degradation steps (50–100, 130–170, 310–530 °C). The
weight loss between 50 and 100 °C corresponds to the elimination of water molecules of dehydration
and is an endothermic effect. The second step, also an endothermic effect, corresponds to the
elimination of coordinated water molecules (Table 1). The following effect on DTA curve is
exothermic and corresponds to the complete decomposition (TG, TGD curves) of the organic part of
the complexes.
The TG and TGD curves of the complexes 15–32 are characterized by two steps of weight loss
united (350–480 °C, 480–620 °C) and corresponds to the complete decomposition of the ligands. In
addition, the TG and TGD curves of the complexes 21, 22 and 31 are characterized by a weight loss in
the renge 50–100 °C.
By replacing the sulphate ion from complexes with nitrate ion or by changing the thiosemicarbazide
fragment with 4-phenylthiosemicarbazide fragment, TG and TGD curves show weight loss at lower
temperatures. The final residues were identified by IR spectroscopy as CuO, which provides %Cu
values in the initial samples, by quantitative analyses. They were in agreement wich the theoretical
obtained %Cu values.
2.1.5. NMR Spectra
The NMR spectra of ligands H2L1–H2L10 were recorded in DMSO-d6. The 1H-NMR and 13C-NMR
spectral data are reported along with the possible assignments [41]. All the protons were found to be in

Molecules 2013, 18
8824
the expected regions. It was observed that DMSO did not have any coordinating effect on the ligands
or their metal complexes.
2.1.6. Mass Spectra
The FAB mass spectra of Cu(II), Ni(II) and Zn(II) complexes with salicyliden thiosemicarbazones
(H2L1–H2L10) have been recorded (Table 2). The molecular ion [M]+ peaks obtained from Cu(II),
Ni(II) and Zn(II) complexes are as follows: m/z = 274.8 (1), m/z = 319.7 (3), m/z = 309.6 (7),
m/z = 350.9 (9), m/z = 395.6 (11), m/z = 429.8 (13), m/z = 369.8 (16), m/z = 349.3 (19), m/z = 506.8
(22), m/z = 698.2 (25), m/z = 586.1 (26), m/z = 536 (31), m/z = 541.4 (32). The data obtained are in
good agreement with the proposed molecular formula for Cu(II), Ni(II) and Zn(II) complexes. The
FAB mass spectra of these complexes shows peaks assignable to various fragments arising from the
thermal cleavage of the complexes.
Table 2. FAB mass spectral data of Cu(II) Ni(II) and Zn(II) complexes.
Mw
Molecular ion
peak [M]+
Molecular formula
The peaks due to complex fragmentation
(g/mol)
[Cu(H2O)(HL1)][Cu(H2O)(HL1)SO4] .
2H2O (1)
[Cu(H2O)(HL4)][Cu(H2O)(HL4)(SO4)] .
684
774
735
274.8
319.7
309.6
101.2
147.3
136.7
170.3
216.5
206.3
203.4
296.3
287.5
H2O (3)
[Cu(H2O)(HL2)][Cu(H2O)(HL2)(SO4)] .
H2O (7)
[Cu (H2O)(HL8)]NO3 . H2O (9)
[Cu (H2O)(HL1°)]NO3 . 2H2O (11)
[Cu(H2O)(HL9)]NO3 . 3H2O (13)
[Cu L2Py] (16)
432
495
547
370.5
350
526
699
587
572
542
350.9
395.6
429.8
369.8
349.3
506.8
698.2
586.1
536
101.7
147.7
181.2
136.7
132.1
262.3
296.3
284.1
269.5
282.2
171.4
220.2
229.1
207.5
203.3
327.8
357.5
345.6
330.6
344.2
203.8
286.3
295.2
292.1
289.2
403.2
434.4
422.1
401.3
416.1
320.2
372.1
398.8
322.6
318.5
498.8
544.2
532.4
517.8
527.4
[Cu L5Py] (19)
[Cu L7(4-MePy)] ⋅ H2O (22)
[Cu L7(Etz)] (25)
[Cu L7(Str)] (26)
[Ni L1(Etz)] ⋅ 2H2O (31)
[Zn L1(Etz)] (32)
541.4
2.2. Biological Activity
2.2.1. Antiproliferative Activity of Human Leukemia HL-60 Cells
All ligands (Table 3) and their metal complexes (Table 4) were tested as inhibitors of HL-60 cells
proliferation using three concentrations: 0.1, 1.0 and 10 μmol/L. At 0.1 and 1.0 μmol/L the ligands
have unsignificant inhibitor activity, but at 10 μmol/L H2L8 (salicylidene-4-phenylthiosemicarbazone),
H2L9 (5-Br-salicylidene-4-phenylthiosemicarbazone) and H2L1 (5-NO2-salicyliden-4-phenylthio-
semicarbazone) inhibit the cell proliferation (90, 75 and 70%, respectively). So, we can assert that the
presence of phenyl-radical in the Schiff bases composition is important. The same fact is confirmed for
copper complexes, but in the enforced variant. So, copper complexes act selectively in this biological

Molecules 2013, 18
8825
process [23,42–44]. In fact, copper complexes, including inner sphere water and tridentate ONS
ligands, are more active than those containing inner sphere amine, which blocked the metal active
centre. Complexes 1–14 are thus better inhibitors of cell proliferation than complexes 15–30.
If copper is capsulated with amine, the antiproliferative activity change in dependence of
substituents R1 and R2 in the same series Y = H or Y = -C6H5. The following three examples illustrate
our SAR results. If A = Py, Y = H and R1 = H, the antiproliferative activity varies (from 60% to 10%)
depending on R2: H (15) > CH3 (19) > Br (17) > Cl (16) > NO2 (18). If Y = H, R1 = R2 = Br and A - is
variable, the moderate influence of amine nature can be observed depending on the ability of
amine(N)-copper bond force: 25 > 28 > 26 = 27 = 29 > 23 = 24 > 21 > 22. If Y = H, R1 = R2 = H,
A = ethazole and copper ion is replaced by nickel or zinc (31, 32), the antiproliferative activity
dramatically decreases.
Table 3. Schiff bases H2L1–H2L10 and their antiproliferative activity on human leukemia
(HL-60) cells at three concentrations.
Schiff base
(X)N-NH-C(S)-NH(Y)
Inhibition of cell proliferation (%)
X
Y
10 μM
1 μM 0.1μM
R1
OH
R2
R1
R2
H2L1
H2L2
H2L3
H2L4
H2L5
H2L6
H2L7
H2L8
H2L9
H2L10
H
H
Cl
H
H
20
0
10
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
H
H
H
H
Cl
Br
H
H
H
Br
H
5
NO2
CH3
Cl
H
0
H
5
H
10
0
Br
H
H
C6H5
C6H5
C6H5
90
75
70
Br
NO2
SEM < 4% of a single experiment in triplicate.

Molecules 2013, 18
8826
Table 4. Antiproliferative activity of complexes 1–32 on human leukemia (HL-60) cells at
three concentrations.
Structural formula of copper
Inhibition of cell
proliferation (%) b
Structural formula of metal
complexes
Inhibition of cell
proliferation (%)b
complex
Complex a
Complex a
10
1
0.1
10
1
0.1
μM
μM
μM
μM μM μM
R1
H
R2
H
Y
H
R1
H
R2
H
A
1
98
50
0
15
Py
-
35
10
2
3
H
H
H
-C6H5
H
100
90
90
70
0
0
16
17
H
H
Cl
Br
Py
Py
-
-
25
50
5
0
NO2
4
5
H
H
H
H
H
H
H
H
H
H
H
NO2
Br
-C6H5
H
96
95
78
90
90
95
95
0
0
0
0
0
0
0
0
0
0
0
18
19
20
21
22
23
24
25
26
27
28
29
30
31
H
H
NO2
CH3
Cl
Py
Py
-
-
10
55
60
25
20
30
30
60
40
40
55
40
65
5
0
0
6
Br
-C6H5
H
90
Cl
Br
Br
Br
Br
Br
Br
Br
Br
Br
H
Py
-
10
0
7
Cl
95
Br
Br
Br
Br
Br
Br
Br
Br
Br
H
NH3
-
8
H
H
100
4-MePy
3-MePy
2-MePy
Ethazole
Streptocide
Sulfocile
Norsulfosole
Sulfadimizine
Ethazole
Ethazole
Ethazole
-
0
9
H
-C6H5
H
100 100
-
15
5
10
11
12
13
14
NO2
NO2
Br
100
100
98
90
90
95
80
90
-
-C6H5
H
-
15
5
65
65
65
65
60
5
Br
C6H5
H
100
100
5
Cl
5
5
DOX
100 100
30
0
H
H
5
32
H
H
10
5
0
a
b
The molecular formula of complexes are reported in Table 1. SEM < 4% of a single experiment in triplicate.
DOX = Doxorubicine.
2.2.2. Antibacterial Activity
Experimental results obtained from the study of antimicrobial activity (Table 5) demonstrate that
compounds 21–25 and 30 display bacteriostatic and bactericide activity in the concentration range
0.03-4000 µg/mL towards both Gram-positive as well as Gram-negative bacteria. In comparison, the
antimicrobial data characteristic for furacillinum used in medical practice are given. The antimicrobial
activity displayed by the above mentioned compounds is 32–260 times higher towards staphylococcus
and streptococcus than furacillinum and exceeds by 2–260 times her bacteriostatic activity towards the
majority of Gram-negative bacteria. The minimum inhibitory concentration (MIC) and minimum
bactericide concentration (MBC) are influenced by the nature of thiosemicarbazone and amine of the
inner sphere of the coordination compound.

Molecules 2013, 18
8827
The data concerning the study of antimycotic properties of compounds 22–24 show that they also
display selective bacteriostatic and bactericide activity in the concentration range 9.3–600 μg/mL
towards investigated fungi stems. In order to make a comparison, we also added data regarding the
activity of nystatine, a compound used in medicine at mycoses treatment. The results show that the
synthesized substances have antimycotic activity against most fungi, higher than nystatine activity.
Aspergillus fumigatus is an exception, being less sensible towards mentioned substances. The toxicity
(LD50) of complexes 24 and 30 (some of the most active in this group of substances) is 1,420 mg/kg
and 4,250 mg/kg so it is 8.6–25.5 times lower than that of furacillinum (LD50 = 166.7 mg/kg).
a
b
Table 5. Antimicrobial or antifungal activity (MIC /MBC ) (μg/mL) of some copper
complexes.
Complexes c
Stem
21
0.29
0.29
0.29
0.58
0.58
0.58
0.29
0.29
0.036
0.072
-
22
0.145
0.145
0.145
0.29
0.29
1.16
0.29
0.58
0.009
0.036
-
23
0.145
0.145
0.29
0.29
0.29
1.16
0.145
0.145
1.16
2.33
-
24
0.29
0.29
0.29
0.58
0.29
0.58
0.29
0.29
0.29
0.58
-
25
0.06
0.06
-
30
0.03
0.03
-
Furacillinum Nystatin
Wood-46
Smith
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
9.35
9.35
9.35
9.35
18.7
18.7
9.35
18.7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Staphylococcus
aureus
-
-
209-P
0.06
0.06
0.12
0.24
0.12
0.24
0.06
0.03
0.03
0.03
0.06
0.06
0.06
0.03
Staphylococcus
saprophyticus
Streptococcus( group A)
-
Enterococcus faecalis
Escherichia coli, O-111
Salmonella typhimurium
Salmonella enteritidis
Klebsiella pneumoniae
Pseudomonas aeruginosa
37.5
37.5
18.7
37.5
75
-
-
-
-
0.06 0.097
1.16
37.5
2.33
9.35
2.33
600
9.35
9.35
4.67
4.67
9.35
9.35
1.16
300
18.7
18.7
4.67
1000
4.67
2000
0.29
400
4.67
9.35
0.29
75
15.6
31.2
1.95
62.5
-
15.6
15.6
7.8
31.2
-
150
9.35
9.35
>300
>300
>300
>300
1.16
300
0.29
300
>4000
>4000
-
-
0.58
600
1.95
62.5
1000
>400
0
7.8
15.6
250
250
2000
>4000
1000
1000
2000
4000
Proteus vulgaris
Proteus mirabilis
Aspergillus niger
MIC
MBC
MIC
0.29
2000
2.33
1000
-
1000
>4000
1000
1.16
4000
9.35
1.16
150
0.49
7.8
-
7.8
150
300
150
300
-
-
-
15.6
1.16
>4000
18.7
18.7
-
-
-
-
-
MBC
MIC
>4000 >4000
150
-
-
9.3
-
240
240
MBC
-
150
9.3
-
-

Molecules 2013, 18
8828
Table 5. Cont.
Complexes c
25
Stem
21
-
22
23
300
300
37.5
37.5
37.5
37.5
-
24
30
-
Furacillinum Nystatin
Aspergillus fumigatus
MIC
MBC
MIC
300
300
37.5
37.5
18.7
18.7
-
300
300
-
-
-
-
-
-
-
-
240
240
80
80
80
80
-
-
-
-
Candida albicans
Penicillium
-
37.5
37.5
37.5
37.5
1420
-
-
MBC
MIC
-
-
-
-
-
-
-
MBC
-
-
LD50, mg/kg
-
4250
166.7
a
b
c
MIC – minimum inhibitory concentration. MBC – minimum bactericide concentration. The molecular formula of
complexes are reported in Table 1.
3. Experimental
3.1. Chemistry
All commercially available reagents and chemicals were of analytical- or reagent-grade purity and
used as received. 1H-NMR and 13C-NMR spectra were recorded at room temperature on a Bruker DRX
400 spectrometer in DMSO-d6, using TMS as the internal standard. IR spectra were recorded on a
Specord-M80 spectrophotometer in the 4000–400 cm−1 region using KBr pellets. The chemical
elemental analysis for the determination of C, H, N and Br was done the Carlo-Erba LA-118
microdosimeter. Metal ions were determined following the method described by G. Schwarenbach and
H. Flaschka [45]. The complexes were studied by thermogravimetry (TG), in a current of air, with a
sample heating rate of 1 °C/min, using a SETARAM 92-1600 thermo-balance. Magnetic
measurements were carried out on solid complexes using the Gouy’s method [39].
X-ray diffraction analysis of compound 5 was carried out on a Nonius KappaCCD diffractometer
(MoKα radiation, λ = 0.71069 Å) at room temperature. The structures of complex 5 was solved by the
direct method using SHELXS-86 [46] and SIR-97 [47] software and refined by least squares in the
anisotropic approximation for nonhydrogen atoms (CRYSTALS) [48]. The hydrogen atoms were
refined isotropically. In complex 5, all hydrogen atoms were included in the refinement in
geometrically calculated positions (except for water molecules in which hydrogen atoms were not
located). The C–H and N–H bond lengths varied in the 0.93–0.98 and 0.86–0.89 Å ranges,
respectively. The thermal factors UH were taken to be 1.2–1.5 times as high as the Ueq values of the
carbon and nitrogen atoms.
3.1.1. General Procedures for the Synthesis of the Schiff Bases H2L1–H2L10
A hot solution of salicylaldehyde (10 mmol) in ethanol (20 mL, 50 °C ) was added to a
magnetically stirred solution of H2N-NH-C(S)-NH(Y) (10 mmol), where Y = H, in warm ethanol (20
mL). The mixture was refluxed for 1–2 h. The resulting precipitate was filtered, washed with cold
ethanol, then with diethyl ether, and dried under vacuum. Crystallization from ethanol gave H2L1. The
same method was applied for the synthesis of H2L2–H2L10 by using 2-hydroxybenzaldehyde and its
derivatives (X) with thiosemicarbazide or 4-phenylthiosemicarbazide.

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Salicylidene thiosemicarbazone (H2L1). Yield: 75%. Anal. Calc. (%) for C8H9N3OS (195 g/mol): C,
49.23; H, 4.61; N, 21.53; S, 16.41. Found: C, 49.40; H, 4.52; N, 21.35; S,16.28. IR (cm−1, KBr): 3600
1
(m, OH), 3058 (m, NH), 1560 (s, C=S), 1586 (w, C=N), 1535 (m, NNH), 822 (m, C=S). H-NMR
(DMSO-d6, δ, ppm): 11.39 (s, 1H, NNH); 9.88 (s, 1H, OH); 8.37 (s, 1H, HC=N); 7.93, 7.91 (2s,
13
1H+1H, NH2); 8.20, 7.21, 6.85, 6.80 (m, 4H, benzene). C-NMR (DMSO-d6, δ, ppm): 177.6 (C=S);
156.4 (HC=N); 139.6 (C-OH); 116.0, 131.1 120.4, 126.7, 118.9 (benzene).
5-Chlorosalicylidene thiosemicarbazone (H2L2). Yield: 70%. Anal. Calc. (%) for C8H8ClN3OS
(229.5 g/mol): C, 41.83; H, 3.48; N, 18.30; S, 13.94. Found: C, 42.26; H, 3.34; N, 18.15; S, 13.79. IR
(cm−1, KBr): 3600 (m, OH), 3058 (m, NH), 1565 (s, C=S), 1585 (w, C=N), 1535 (m, NNH), 820 (m,
1
C=S). H-NMR (DMSO-d6, δ, ppm): 11.44 (s, 1H, NNH); 10.21 (s, 1H, OH); 8.30 (s, 1H, HC=N);
13
8.16, 8.11 (2s, 1H+1H, NH2); 8.10, 7.21, 6.86 , (m, 3H, benzene). C-NMR (DMSO-d6, δ, ppm):
177.8 (C=S); 155.1 (HC=N); 137.7 (C-OH); 117.7, 132.6, 130.4, 122.4, 123.5, 126.5 (benzene).
5-Bromosalicylidene thiosemicarbazone (H2L3). Yield: 71%. Anal. Calc. (%) for C8H8 BrN3OS
(274 g/mol): C, 35.03; H, 2.91; N, 15.32; Br, 29.19; S, 11.67. Found: C, 34.89; H, 2.78; N, 15.25; Br,
28.91; S, 11.45. IR (cm−1, KBr): 3600 (m, OH), 3055 (m, NH), 1562 (s, C=S), 1584 (w, C=N), 1535
(m, NNH), 823 (m, C=S). 1H-NMR (DMSO-d6, δ, ppm): 11.42 (s, 1H, NNH); 10.23 (s, 1H, OH); 8.29
13
(s, 1H, HC=N); 8.21, 8.17 (2s, 1H+1H, NH2); 8.21, 7.32, 6.81 (m, 3H, benzene). C-NMR (DMSO-
d6, δ, ppm): 177.8 (C=S); 155.6 (HC=N); 137.2 (C-OH); 118.2, 133.2, 111.2, 128.3, 122.9 (benzene).
5-Nitrosalicylidene thiosemicarbazone (H2L4). Yield: 62%. Anal. Calc. (%) for C8H8N4O3S
(240 g/mol): C, 40.00; H, 3.33; N, 23.33; S, 13.33. Found: C, 40.46; H, 3.12; N, 23.16; S, 13.10. IR
(cm−1, KBr): 3600 (m, OH), 3058 (m, NH), 1559 (s, C=S), 1586 (w, C=N), 1535 (m, NNH), 821 (m,
1
C=S). H-NMR (DMSO-d6, δ, ppm): 11.53 (s, 1H, NNH); 11.55 (s, 1H, OH); 8.37 (s, 1H, HC=N;
8.29, 8.24 (2s 1H+1H, NH2); 8.86, 78.11, 7.04 (m, 3H, benzene). 13C-NMR (DMSO-d6, δ, ppm): 178.0
(C=S); 161.9 (HC=N); 136.8 (C-OH); 116.5, 126.3, 140.3, 122.2, 121.4 (benzene).
5-Methylsalicylidene thiosemicarbazone (H2L5). Yield: 68%. Anal. Calc. (%) for C9H11N3OS
(209 g/mol): C, 51.67; H, 5.26; N, 20.09; S, 15.31. Found: C, 52.02; H, 5.00; N, 19.83; S, 15.04. IR
(cm−1, KBr): 3600 (m, OH), 3058 (m, NH), 1558 (s, C=S), 1583 (w, C=N), 1535 (m, NNH), 824 (m,
C=S). 1H-NMR (DMSO-d6,, δ, ppm): 11.50 (s, 1H, NNH); 9.85 (s, 1H, OH); 8.31 (s, 1H, HC=N); 8.02,
8.07 (2s 1H+1H, NH2); 7.22, 6.85, 6.62 (m, 3H, benzene); 2.30 (s, 3H, CH3). 13C-NMR (DMSO-d6, δ,
ppm): 178.2 (C=S); 153.4 (HC=N); 140.3 (C-OH); 116.0, 133.4, 130.8, 130.5, 118.0 (benzene); 20.9
(CH3).
3,5-Dichlorosalicylidene thiosemicarbazone (H2L6). Yield: 75%. Anal. Calc. (%) for C8H7 Cl2N3OS
(264 g/mol): C, 36.36; H, 2.65; N, 15.90; Cl, 26.89; S, 12.12. Found: C, 36.53; H, 2.48; N, 15.73; Cl,
26.57; S, 11.98. IR (cm−1, KBr): 3600 (m, OH), 3058 (m, NH), 1558 (s, C=S), 1587 (w, C=N), 1535
1
(m, NNH), 822 (m, C=S). H-NMR (DMSO-d6, δ, ppm): 11.48 (s, 1H, NNH); 10.3 (s, 1H, OH); 8.35
13
(s, 1H, HC=N); 7.98, 7.93 (2s 1H+1H, NH2); 7.28, 7.13, (m, 2H, benzene). C-NMR (DMSO-d6, δ,
ppm): 177.9 (C=S); 154.0 (HC=N); 140.5 (C-OH); 123.0, 133.1, 126.9, 127.1, 121.5 (benzene).

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3,5-Dibromosalicylidene thiosemicarbazone (H2L7). Yield: 72%. Anal. Calc. (%) for C8H7Br2N3OS
(353 g/mol): C, 27.19; H, 1.98; N, 11.89; Br, 45.32; S, 9.06. Found: C, 27.40; H, 1.78; N, 11.68; Br,
45.03; S, 8.83. IR (cm−1, KBr): 3650 (m, OH), 3058 (m, NH), 1560 (s, C=S), 1586 (w, C=N), 1535 (m,
NNH), 819 (m, C=S). 1H-NMR (DMSO-d6, δ, ppm): 11.45 (s, 1H, NNH); 10.55 (s, 1H, OH); 8.29 (s,
13
1H, HC=N); 8.10, 8.01 (2s, 2H, NH2); 8.20, 7.56 (d, 2H, benzene). C-NMR (DMSO-d6, δ, ppm):
178.5 (C=S); 155.4 (HC=N); 150.2 (C-OH); 118.1, 137.5, 111.2, 130.8, 123.0 (benzene).
Salicylidene-4-phenylnthiosemicarbazone (H2L8). Yield: 58%. Anal. Calc. (%) for C14H13N3OS
(271 g/mol): C, 61.99; H, 4.79; N, 15.49, S, 11.80. Found: C, 62.27; H, 4.58; N, 15.28; S, 11.73. IR
(cm−1, KBr): 3600 (m, OH), 3060 (m, NH), 1565 (s, C=S), 1586 (w, C=N), 1535 (m, NNH), 823 (m,
1
C=S). H-NMR (DMSO-d6, δ, ppm): 11.78 (s, 1H, NNH); 9.98 (s, 1H, OH); 8.50 (s, 1H, HC=N);
10.06 (1s 1H, NH-C6H5); 8.10, 7.22, 6.90, 6.88 (m, 4H, benzene-OH); 7.38, 7.34, 7.34, 7.25, 7.25 (m,
13
5H-benzene-NH). C-NMR (DMSO-d6, δ, ppm): 177.2 (C=S); 157.1 (HC=N); 140.5 (C-OH); 116.5,
131.8, 120.7, 128.5, 118.4 (benzene-OH); 139.6 (C-NH); 127.5, 127.5, 126.1, 126.1, 127.7 (benzene-NH).
5-Bromosalicylidene-4-phenylthiosemicarbazone (H2L9). Yield: 70%. Anal. Calc. (%) for
C14H12BrN3OS (350 g/mol): C, 48.00; H, 3.42; N, 12.00; Br, 22.85; S, 9.14. Found: C, 48.39; H, 3.25;
N, 11.80; Br, 22.63; S, 9.00. IR (cm−1, KBr): 3600 (m, OH), 3060 (m, NH), 1565 (s, C=S), 1586 (w,
1
C=N), 1535 (m, NNH), 822 (m, C=S). H-NMR (DMSO-d6, δ, ppm): 11.82 (s, 1H, NNH); 10.32 (s,
1H, OH); 8.42 (s, 1H, HC=N); 10.20 (1s 1H, NH-C6H5); 8.35, 7.35, 6.85 (m, 3H, benzene-OH); 7.38,
7.39, 7.52, 7.51, 7.24 (m, 5H-benzene-NH). 13C-NMR (DMSO-d6, δ, ppm): 176.6 (C=S); 156.2
(HC=N); 139.7 (C-OH); 118.6, 138.4, 111.6, 133.9, 123.2 (benzene-OH); 139.4 (C-NH); 128.5, 128.5,
126.9, 126.9, 125.9 (benzene-NH).
5-Nitrosalicylidene-4-phenylthiosemicarbazone (H2L10). Yield: 76%. Anal. Calc. (%) for C14H12N4O3S
(316 g/mol): C, 53.16; H, 3.79; N, 17.72; S, 10.12. Found: C, 53.34; H, 3.57; N, 17.58; S, 9.97. IR
(cm−1, KBr): 3600 (m, OH), 3060 (m, NH), 1565 (s, C=S), 1586 (w, C=N), 1535 (m, NNH), 822 (m,
1
C=S). H-NMR (DMSO-d6, δ, ppm): 11.91 (s, 1H, NNH); 11.67 (s, 1H, OH); 8.49 (s, 1H, HC=N);
10.35 (1s 1H, NH-C6H5); 8.98, 8.15, 7.06 (m, 3H, benzene-OH); 7.37, 7.39, 7.31, 7.52, 7.21 (m,
13
5H-benzene-NH). C-NMR (DMSO-d6, δ, ppm): 176.6 (C=S); 156.2 (HC=N); 139.7 (C-OH); 118.6,
138.4, 111.6, 133.9, 123.2 (benzene-OH); 139.4 (C-NH); 128.5, 128.5, 126.9, 126.9, 125.9 (benzene-NH).
3.1.2. General Procedure for the Preparation of Complexes 1–32
Synthesis of compound 1. 30 mL of ethanolic solution, which contains 10 mmol of salicyliden
thiosemicarbazone is mixed with 10 mmol of CuSO4·5H2O, dissolved in 20 mL of distilled water. The
reaction mixture is heated (50–55 °C) and stirred continuously for 1.5 h. The green colored solid,
which separated on cooling, was filtered, washed with ethanol, diethyl ether and dried in air. Method
for the synthesis of compound 1 is similar to that of work [26] but were modified working conditions.
Synthesis of Compounds 2–14. This compounds have been synthesized according to the above
described procedure, using CuSO4·5H2O or Cu(NO3)2·3H2O and salicyliden thiosemicarbazone,
5-chloro-, 5-bromo-, 5-nitro- salicyliden thiosemicarbazones or 5-bromo-, 5-nitro-salicyliden-4-
phenylthiosemicarbazones, in 1:1 molar ratio.

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Synthesis of Compound 15. To CuCl2·2H2O (10 mmol) dissolved in 20 mL ethanol was added
salicyliden thiosemicarbazone (10 mmol) dissolved in 15 mL hot ethanol.The mixture was stirred
continuously (1 h) and then pyridine alcoholic solution is added till pH=7.5–8. The dark green
microcrystals was filtered, washed with ethanol, diethyl ether and dried in air.
Synthesis of Compound 16–32. This compounds have been synthesized according to the above
described procedure, using as initial substances CuCl2·2H2O, thiosemicarbazones H2L2−7 and ethanolic
solution of pyridine, 2-, 3-, 4-picoline, streptocide (Str), sulphacil (Sfc), norsulphazol (Nor), ethazol
(Etz) or sulphadimezine (Sdm), in 1:1:1 molar ratio. The elemental analysis confirms the molecular
formula. The physical and analytical data are presented in Table 1.
3.2. Cytotoxicity Assay
3.2.1. Preparation of Test Solutions
Stock solutions of the investigated compounds (H2L1–H2L10) and copper complexes 1–30 were
prepared in dimethylsulfoxide (DMSO) at a concentration of 10 mM and diluted with nutrient medium
to various working concentrations. DMSO was used instead of ethanol due to solubility problems.
3.2.2. Cell Culture
Human promyelocytic leukemia cells HL-60 (ATCC, Rockville, MD, USA) were routinely grown
in suspension in 90% RPMI-1640 (Sigma, Saint Louis, USA) containing L-glutamine (2 nM),
antibiotics (100 IU penicillin/mL, 100 µg streptomycin/mL) and supplemented with 10% (v/v) foetal
bovine serum (FBS), in a 5% CO2 humidified atmosphere at 37 °C. Cells were currently maintained in
continuous exponential growth with twice a week dilution of the cells in culture medium [9].
3.2.3. Cell Proliferation Assay
The cell proliferation assay was performed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Cell Titer 96 Aqueous, Promega,
Madison, Wi, USA), which allowed us to measure the number of viable cells. In brief, triplicate
cultures of 1 x 104 cells in a total of 100 µL medium in 96-well microtiter plates (Becton Dickinson
and Company, Lincoln Park, NJ, USA) were incubated at 37 °C, 5% CO2. Compounds were dissolved
in DMSO to prepare the stock solution of 1 × 10−2 M. These compounds were diluted at the
appropriate concentration (1 or 10 µM) with culture media, added to each well and incubated for
3 days. Following each treatment, 20 µL MTS was added to each well and incubated for 4 h. MTS is
converted to water-soluble coloured formazan by dehydrogenase enzymes present in metabolically
active cells. Subsequently, the plates were read at 490 nm using a microplate reader (Molecular
Devices, Sunnyvale, CA). The results were reported as the percentage of cell proliferation inhibition
compared to the control (basal cell proliferation=100%).
3.3. Antibacterial Activity

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The antibacterial activity of complexes and also of their prototype Furaciline has been determined
under liquid nutritive environment [2% of peptonate bullion (pH 7.0)] using successive dilutions
method [36–38]. Staphylococcus aureus (Wood-46, Smith, 209-P), Staphylococcus saprophyticus,
Streptococcus faecalis, Escherihia coli (O-111), Salmonella typhimurium, Salmonella enteritidis,
Klebsiella pneumoniaie, Pseudomonas aeruginosa, Proteus vulgaris and Proteus mirabilis standard
stems were used as reference culture for in vitro experiments. The dissolution of studied substances in
dimethylformamide, microorganisms cultivation, suspension obtaining, determination of minimal
inhibition concentration (MIC) and minimal bactericide concentration (MBC) have been carried out
according to the method previously reported [39].
3.4. Antifungal Bioassay
Antimycotic properties of the complexes were investigated in vitro on laboratory stems: Aspergillus
niger, Aspergillus fumigatusi, Candida albicans and Penicillium. The activity has been determined in
liquid Sabouroud nutritive environment (pH 6.8). The inoculates were prepared from fungi stems
which were harvested during 3–7 days. Their concentration in suspension is (2–4) × 106 colonies form
unities in milliliter. Sowings for levures and micelles were incubated at 37 °C during 7 and 14 days,
respectively.
4. Conclusions
Ten new salicyliden thiosemicarbazones ligands and their corresponding Cu(II), Ni(II) and Zn(II)
complexes have been synthesized and characterized. The molecular structure of the complex 5 has
been determined by single crystal X-ray diffraction method. The IR, 1H-NMR and 13C-NMR data were
successfully used to elucidate the formation of the salicyliden thiosemicarbazones ligands. All ligands
and their metal complexes were tested as inhibitors of HL-60 cells proliferation. The ligands have
unsignificant inhibitor activity at 0.1 and 1.0 μmol/L, but at 10 μmol/L H2L8, H2L9 and H2L10 inhibit
the cell proliferation. The copper complexes, including inner sphere water and tridentate ONS ligands,
are more active than those containing inner sphere amine, which blocked the metal active centre. The
most indicative criteria for future synthesis of biological active coordination compounds from the
viewpoint of the inhibition of HL-60 cell proliferation, antibacterial and antifungal activity: use of
copper (II) complexes and presence of sulphur atom in the tridentate organic ligand.
Supplementary Data
CCDC 623449 contain the supplementary crystallographic data for C16H8Br2Cu2N6O12S3 (5).
These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or
from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK;
fax: (+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.

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Acknowledgements
The authors wish to thank the CHUQ (CHUL) Research Center at Québec City (Canada) and State
University of Medicine and Pharmacy of Chisinau (Moldova) for their help in carrying out biological
studies. The authors thank the Academy of Sciences of Moldova (Institute of Chemistry), for their help
in carrying out C, H, N and Br elemental analyses. Also authors thank the FQRNT grant funding
project 115825 for financial support.
Conflict of Interest
The authors declare no conflict of interest.
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