Novel chelate complexes of Co(II), Ni(II), Cu(II), Pd(II) derived from anti- and syn-isomers of 2-(2-aminothiazole-4-yl)-2-hydroxyiminoacetic acid with pro-/antiproliferative actions on endothelial cells

Article abstract of DOI:10.1016/j.poly.2014.08.034

Anti- and syn-isomers of 2-(2-aminothiazole-4-yl)-2-hydroxyiminoacetic acid (anti-A and syn-A respectively) were used as potential biologically active polydentate complexing agent for Co²⁺, Ni²⁺, Cu²⁺ and Pd²⁺ ions for the synthesis of novel complex compounds with directed angiogenesis-correcting action. It shown that the location of functional groups in ligand molecules, the electronic structure of metal as complexing agent and the synthesis conditions affect the localization of coordination bonds and the structure of formed complexes. The interaction of ethanol solutions of metal salts with anti-A and syn-A in an acidic medium at the component ratios M:L = 1:1 and 1:2 leads to the formation of complexes [Co(anti-A)(H₂O)₃SO₄] (1); [Ni(anti-A)(H₂O)₃SO₄] (2); [Cu(anti-A)₂Cl₂] (3); [Pd(anti-A)₂Cl₂] (4); [Cu(syn-A)₂Cl₂] (5); [Pd(syn-A)Cl₂] (6) with different coordination of ligands. Coordination mode of ligands identified by IR, UV-Vis, XPS and ¹H (¹³C) NMR spectra. The structures of salt of the anti-A and complexes 1, 2, 6 were determined by X-ray diffraction study. Anti-A is coordinated to metal ions in a chelate manner by the nitrogen atom of the hydroxylimino group and the oxygen atom of the deprotonated carboxyl group. In this case, the 2-aminothiazole fragment does not involve in complexation. Syn-A is coordinated to the central metal ion in neutral form through the nitrogen atoms of hydroxyimino group and thiazole ring. All synthesized complex compounds form stable solutions in neutral medium, which makes it possible to use them as potential biologically active substances. Investigation of biological effects for syn-A shows mitogenic and antiapoptotic activity against endotheliocytes, while anti-A causes inhibition of proliferation almost in the entire concentration range. Complex compounds of Cu²⁺ and Pd²⁺ with anti-A and syn-A (3-6) cause an increase of antiproliferative activity of endothelial cells compared with baseline complexing agents. In this case, the activity of complexes with syn-A is superior to activity of analogues with anti-A. The results of cytotoxicity tests revealed a pronounced cytotoxic action for complexes 3 and 4, cytotoxic and proapoptotic activity for 6 and cytostatic effect for 5. For all compounds investigated, a checkpoint from S to G₂ has been established, which may indicate DNA replication disturbance or dysregulation in the endogenous signals of the cell cycle of endotheliocytes.

Full text of DOI:10.1016/j.poly.2014.08.034

Accepted Manuscript
Novel chelate complexes of Co(II), Ni(II), Cu(II), Pd(II) derived from anti- and
syn-isomers of 2-(2-aminothiazole-4-yl)-2-hydroxyiminoacetic acid with pro-/
antiproliferative actions on endothelial cells
S.I. Orysyk, O.O. Zholob, V.V. Bon, V.V. Nikulina, V.V. Orysyk, T.V.
Nikolaienko, L.V. Garmanchuk, Yu.L. Zborovskii, G.M. Tolstanova, N.N.
Khranovskaya, V.I. Pekhnyo, M.V. Vovk
PII:
S0277-5387(14)00556-7
DOI:
http://dx.doi.org/10.1016/j.poly.2014.08.034
POLY 10935
Reference:
To appear in:
Polyhedron
Received Date:
Accepted Date:
12 May 2014
2 August 2014
Please cite this article as: S.I. Orysyk, O.O. Zholob, V.V. Bon, V.V. Nikulina, V.V. Orysyk, T.V. Nikolaienko, L.V.
Garmanchuk, Yu.L. Zborovskii, G.M. Tolstanova, N.N. Khranovskaya, V.I. Pekhnyo, M.V. Vovk, Novel chelate
complexes of Co(II), Ni(II), Cu(II), Pd(II) derived from anti- and syn-isomers of 2-(2-aminothiazole-4-yl)-2-
hydroxyiminoacetic acid with pro-/antiproliferative actions on endothelial cells, Polyhedron (2014), doi: http://
dx.doi.org/10.1016/j.poly.2014.08.034
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1
Novel chelate complexes of Co(II), Ni(II), Cu(II), Pd(II) derived from anti- and
syn-isomers of 2-(2-aminothiazole-4-yl)-2-hydroxyiminoacetic acid with pro-
/antiproliferative actions on endothelial cells
a1
a
b
c
d
S.I. Orysyk , O.O. Zholob , V.V. Bon , V.V. Nikulina , V.V. Orysyk , T.V.
c
c
d
c
Nikolaienko , L.V. Garmanchuk , Yu.L. Zborovskii , G.M. Tolstanova , N.N.
e
a
d
Khranovskaya , V.I. Pekhnyo , M.V. Vovk .
a
V.I. Vernadskii Institute of General and Inorganic Chemistry NAS of Ukraine,
Palladin Ave. 32/34, 03680 Kyiv, Ukraine
b
Department of Inorganic Chemistry, Dresden University of Technology,
Bergstrasse 66, 01069 Dresden, Germany
c
Educational and Scientific Centre "Institute of Biology" of Taras Shevchencko
National University, Glushkov Ave. 2, 03022 Kyiv, Ukraine
d
Institute of Organic Chemistry NAS of Ukraine, Murmanska str. 5, 02660 Kyiv,
Ukraine
e
National Cancer Institute, Lomonosov str., 33/43, 03022 Kyiv, Ukraine
Abstract
Anti- and syn-isomers of 2-(2-aminothiazole-4-yl)-2-hydroxyiminoacetic acid
(
anti-A and syn-A respectively) were used as potential biologically active
2
+
2+
2+
2+
polydentate complexing agent for Co , Ni , Cu and Pd ions for the synthesis
of novel complex compounds with directed angiogenesis-correcting action. It
shown that the location of functional groups in ligand molecules, the electronic
structure of metal as complexing agent and the synthesis conditions affect the
localization of coordination bonds and the structure of formed complexes. The
interaction of ethanol solutions of metal salts with anti-A and syn-A in an acidic
medium at the component ratios M:L=1:1 and 1:2 leads to the formation of
complexes [Co(anti-A)(H O) SO ] (1); [Ni(anti-A)(H O) SO ] (2); [Cu(anti-
2
3
4
2
3
4
A) Cl ] (3); [Pd(anti-A) Cl ] (4); [Cu(syn-A) Cl ] (5); [Pd(syn-A)Cl ] (6) with
2
2
2
2
2
2
2
different coordination of ligands. Coordination mode of ligands identified by IR,
1
13
UV-Vis, XPS and H ( C) NMR spectra. The structures of salt of the anti-A and
complexes 1, 2, 6 were determined by X-ray diffraction study. Anti-A is
coordinated to metal ions in a chelate manner by the nitrogen atom of the
hydroxylimino group and the oxygen atom of the deprotonated carboxyl group. In
this case, the 2-aminothiazole fragment does not involve in complexation. Syn-A is
1
Author for correspondence. Tel.: +38(044)4240511; +38(095)3533288; fax:
+
380(44)4243070
E-mail address: s_oryslend@mail.ru (S.I. Orysyk)

2
coordinated to the central metal ion in neutral form through the nitrogen atoms of
hydroxyimino group and thiazole ring. All synthesized complex compounds form
stable solutions in neutral medium, which makes it possible to use them as
potential biologically active substances.
Investigation of biological effects for syn-A shows mitogenic and
antiapoptotic activity against endotheliocytes, while anti-A causes inhibition of
2
+
proliferation almost in the entire concentration range. Complex compounds of Cu
2
+
and Pd with anti-A and syn-A (3–6) cause an increase of antiproliferative activity
of endothelial cells compared with baseline complexing agents. In this case, the
activity of complexes with syn-A is superior to activity of analogues with anti-A.
The results of cytotoxicity tests revealed a pronounced cytotoxic action for
complexes 3 and 4, cytotoxic and proapoptotic activity for 6 and cytostatic effect
for 5. For all compounds investigated, a checkpoint from S to G has been
2
established, which may indicate DNA replication disturbance or dysregulation in
the endogeneous signals of the cell cycle of endotheliocytes.
Keywords: hydroxyiminoacetates, transition metal complexes, X-Ray diffraction
study, pro- and antiproliferative actions, endothelial cell proliferation
1
. Introduction
The development of complex compounds based on biologically active organic
ligands has become a promising trend in pharmaceutical chemistry in the recent
0-15 years. Complex formation prevents hydrolysis of inorganic salts in
1
physiologic media, reduces toxicity and facilitates the penetration of drugs through
cell membranes. Moreover, the pharmacological characteristics of the complexes
are more effective as compared to their initial components [1-4].
Herein we used Anti- and syn-isomers of 2-(2aminothiazole-4-yl)-2-
hydroxyiminoacetic acid (anti-A and syn-A) as potential biologically active
polydentate complexing agents in the synthesis of novel coordination compounds
with Cu(II), Co(II), Ni(II) and Pd(II). These ligands contain biologically active
fragments of 2-iminothiazole and hydroxyiminoacetic acid that are involved in
many biologically active compounds with antibacterial [5, 6] and antiprion [8]
properties, and are used as scaffold in medical chemistry to obtain potential
pharmaceuticals [5-9]. They are inhibitors of interleukin-2-inducible T cell
kinases) [9].
Angiogenesis is known as a multi-step process which consists of remodeling
of extracellular matrix, proliferation and migration of endothelial cells, tube
formation and blood vessels stabilization [10]. It is regulated by a well-

3
orchestrated balance between pro- and anti-angiogenic factors. Dysregulated
angiogenesis is hallmarks of cancer, ischemic diseases and many chronic
inflammatory diseases (e.g., gastrointestinal ulceration, cancer, diabetic
retinopathy, atherosclerosis, rheumatoid arthritis) [10-16]. Preclinical and initial
clinical evidence reveals that normalization of the vascular abnormalities is
emerging as a complementary therapeutic paradigm for cancer and other vascular
disorders, which affect more than half a billion people worldwide [10]. Available
therapeutic approaches to block or increase vascular supply have reached the
clinic, but limited efficacy and resistance pose new challenges
Hypoxia-inducible factor 1 (HIF-1) is a master regulator of hypoxic/ischemic
vascular responses, driving transcriptional activation of many genes encoding
angiogenic cytokines and growth factors involved in vascular reactivity,
angiogenesis, and the mobilization and homing of bone marrow-derived
angiogenic cells. HIF-1 plays the pivotal role in vascular homeostasis and involves
in vascular diseases [17]. HIF-1 activity manipulation is the perspective therapeutic
approach which might overcome the current limitations of pro- and antiangiogenic
medicine. Recently the 2-hydroxyimino-2-phenylacetic acid derivatives are
reported as efficient HIF-1 inhibitors [18].
The high bioactivity of these compounds and other 2-hydroxyiminoacetic acid
derivatives is explained by their ability to form chelate complexes with many
transition metals ions [19, 20]. Polyfunctional 2-hydroxyiminoacetic acids,
coordinated to functional groups of peptide fragments in biological media, are able
to bind metal ions into stable complexes, and act therefore as inhibitors of metal-
containing enzymes and proteins. For instance, a number of amides of 2-
hydroxyiminoalkanecarboxylic acid are inhibitors of zinc-containing enzymes
(histone deacetylases), that increase their high anticancer activity [21].
The effect of copper and palladium complexes on the proliferation of
endothelial cells has been studied. Moreover, the influence of anti/syn isomerism
both on the complexation reactions and biological activity of coordination
compounds has been established.
2
. Materials and methods

4
2
.1. Materials and chemicals
All solvents and reagents were purchased from commercial sources and
directly used without additional purification. Initial reagents Co(NO ) ·6H O
3
2
2
(
20.25% Co), Ni(NO ) ·6H O (20.18% Ni), CuCl ·2H O (37.28% Cu), PdCl
3
2
2
2
2
2
(59% Pd), used for the synthesis of complexes, were purchased from Merck.
2
.2. Preparation of ligands and metal complexes
2
.2.1. 2-(2-Aminothiazole-4-yl)-2-(anti)-hydroxyiminoacetic acid (anti-A) and 2-
(2-aminothiazole-4-yl)-2-(syn)-hydroxyiminoacetic acid (syn-A)
To a solution of NaOH (2.4 g, 0.06 mol) in water (100 mL) was added ethyl
2
-(2-aminothiazole-4-yl)-2-(anti)-hydroxyiminoacetate [22] or ethyl 2-(2-
aminothiazole-4-yl)-2-(syn)-hydroxyiminoacetate [23] (10.76 g, 0.05 mol) with
stirring in an ice bath. The mixture was stirred for 2-3 h at 15-20 C, after that the
solvent was removed in vacuum. The solid residue was dissolved in H О (100
o
2
mL), treated with Norit (1.0 g) and filtered. The glacial acetic acid was added
dropwise to the filtrate with stirring till neutral reaction. The resulting precipitate
was filtered off, washed with water and recrystallized twice from the ethanol.
о
(
Anti-A). Yield: 6.45 g (69 %), m.p. 186–187 С. Anal. Calc. for C H N O S:
5
5
3
3
C, 32.08; H, 2.69; N, 22.45; S, 17.13; Found: C, 31.94; H, 2.82; N, 22.39; S,
1
1
1
7.09%. H NMR (300 MHz; d -DMSO; δ/ppm): 7.30 (br. s, 2Н, NH ); 7.56 (s,
6
2
1
3
H, CHthiazole); ~12.91 (br. s, OН + COOH). C NMR (125.71 MHz; d -DMSO;
6
δ/ppm): 114.48 C(5); 138.04 C(3); 144.01 C(1); 164.85 C(2); 166.82 C(4). IR data,
−1
ν, cm : 3620 (br. m) ν(OH); 3250 (br. m) ν(NH)NH2; 3180 (m) ν(OH)C=N-OH; 2935
m) ν(C-H)thiazole; 2780 (w) (ОН)СООН; 1654 (s) ν(С=О); 1635 (m) ν(C=N)imine
575 (br. m) δ(NH); 1530 (m) ν(C=N)thiazole; 1435 (m) ν(CH−S)thiazole; 1367 (m)
ν(C=C)thiazole; 1220 (w) ν(C−OH); 1021 (m) ν(N−O); 970 (w), 913 (m) δ(С−Н);
23 (m) ν(С−С); 773 (m) δ(С−Н); 641 (w), 620 (w), 556 (m), 484 (w), 423 (m)
(
;
1
8
δ(С−С). To obtain the crystals of anti-A suitable for single crystal X-ray
diffraction study, the compound was crystallized in 0.4M solution of H SO ,
2
4
resulting in the sulfate salt of anti-A. The crystal structure of latter is discussed in
the section of X-ray Diffraction Studies.
о
(Syn-A). Yield: 6.67 g (71 %), m.p. 206–207 С. Anal. Calc. for C H N O S:
5
5
3
3
C, 32.08; H, 2.69; N, 22.45; S, 17.13; Found: C, 31.97; H, 2.76; N, 22.31; S,
1
1
2
7.21%. H NMR (300 MHz; d
6
-DMSO; δ/ppm): 6.84 (s, 1H, CHthiazole) 7.30 (br. s,
1
3
Н, NH ); ~12.43 (br. s, OН + COOH). C NMR (125.71 MHz; d -DMSO;
2
6
δ/ppm): 106.03 C(5); 141.89 C(3); 147.58 C(1); 164.72 C(2); 168.61 C(4). IR data,

5
ν, cm−1: 3620 (br. m) ν(OH); 3300 (br. m) ν(NH)NH2; 3150 (br. m) ν(OH)C=N-OH
;
3025 (br. m) ν(C-H)thiazole; 2785 (w) (ОН)СООН; 1665 (s) ν(С=О); 1615 (m)
ν(C=N)imine; 1525 (s) ν(C=N)thiazole; δ(NH)NH2; 1430 (s) ν(CH−S)thiazole; 1350 (br. m)
ν(C=C)thiazole; 1275 (m) ν(C−N); 1164 (m), 1065 (s) ν(C−OH); 1025 (m) ν(N−O);
888 (m) δ(С−Н); 830 (s) ν(С−С); 785 (m), 757 (m) δ(С−Н); 688 (m) δ(N−О); 641
(
w), 620 (w), 567 (m), 477 (w), 428 (br. s) δ(С−С)
2
.2.2. Complex [Co(anti-A)(H O) SO ] (1)
2
3
4
The compound anti-A (0.178 g, 1 mmol) was dissolved in 2M H
2
SO (9 mL),
4
and ethanol (5 mL) was added. To the resulting transparent solution heated to 60 –
o
7
0 C a solution of Co(NO ) ·6H O (0.291 g, 1 mmol) in ethanol (10 mL) was
2
3
2
added with stirring. The reaction mixture was stirred for 75 min under reflux and
cooled down to the room temperature. The pink crystals were formed after 2 days
of staying. The pink crystals are filtered off and dried in a vacuum desiccator over
CaCl2. Yield: 0.29 g (72 %), m.p. ≥ 250 °С (decomp.). Anal. Calc. for
C H CoN O S : C, 15.16; H, 2.80; N, 10.61; S, 16.19; Found: C, 15.32; H, 3.02;
5
11
3
10 2
−1
N, 10.52; S, 16.15%. IR data, ν, cm : 3620 (br. m) intramolecular hydrogen bond;
420 (m) ν(OH)Н2О; 3200 (m) ν(NH)NH2; 3130 (m) ν(OH)C=N-OH; 3005 (br. m) ν(C-
3
H)
; 2755 (m) ν(ОН) related to intramolecular hydrogen bond; 2617 (br. m)
thiazole
+
ν(NH )thiazole; 1640 (s) ν(С=О); 1610 (m) ν(C=N)imine; 1574 (m) ν(C=N)thiazole, δNH
;
1
472 (w) ν(CH−S)
; 1395 (m) ν(C=C)thiazole; 1217 (m) ν(C−OH); 1145 (m);
thiazole
2
4
-
1085 (br. s) ν (F ) SO ; 1060 (m), 983 (w), 963 (w), 870 (m), 785 (m) δ(С−Н);
3
2
2
-
7
00 (w), 632 (w), 590 (m) ν (F ) SO ; 535 (br. w) ν(Co−O); 470 (w), 450 (m)
4
2
4
ν(Co−N)
2
.2.3. Complex [Ni(anti-A)(H O) SO ] (2)
2
3
4
Ni(NO ) ·6H O (0.2907 g, 1 mmol) was dissolved in ethanol (10 mL) at 40–
0 C. To the resulted solution, the solution of anti-A (0.1872 g, 1 mmol),
3
2
2
o
5
dissolved in 2M H SO (9 mL) was added with stirring. The reaction mixture was
2
4
stirred for 45 minutes under reflux and cooled down to the room temperature. The
cyan crystals were formed after 2 days of staying. The turquoise crystals are then
filtered off and dried in a vacuum desiccator over CaCl .Yield: 0.26 g (65 %), m.p.
2
≥
205 °С (decomp.). Anal. Calc. for C H N NiO S : C, 15.17; H, 2.80; N, 10.61;
5
11
3
10 2
−1
S, 16.20; Found: C, 15.25; H, 3.08; N, 10.67; S, 16.18%. IR data, ν, cm : 3620
br. m) intramolecular hydrogen bond; 3400 (br. m) ν(OH)Н2О; 3312 (m) ν(NH)NH2
(
;
3
185 (m) ν(OH)C=N-OH; 3000 (br. m) ν(C-H)
; 2750 (m) ν(ОН) related to
thiazole
+
intramolecular hydrogen bond; 2650 (br. m) ν(NH )thiazole; 1630 (s) ν(С=О); 1620
(
m) ν(C=N)imine; 1560 (m) ν(C=N)thiazole δ(NH); 1485 (m) ν(CH−S)
; 1373 (s)
thiazole
2
-
nν(C=C)thiazole; 1208 (m) ν(C−OH); 1140 (br. s), 1100 (br. s) ν (F ) SO ; 1065 (s),
3
2
4

6
2
-
9
60 (m), 932 (m), 800 (m) δ(С−Н); 730 (br. m), 618 (m) ν (F ) SO ; 555 (m), 505
4
2
4
(m) ν(Ni−O); 438 (m) ν(Ni−N).
2
.2.4. Complex [Cu(anti-A) Cl ] (3)
2
2
CuCl ·2H O (0.0852 g, 0.5 mmol) was dissolved in ethanol (20 mL) at 40–45
2
2
o
C. To the resulted solution a solution of anti-A (0.0936 g, 0.5 mmol), dissolved in
o
ethanol (10 mL) and 2N HCl (5 mL), heated to 40–45 C was added dropwise with
stirring at the same temperature for 45 minutes. After the cooling of resulted dark
olive solution to the room temperature, a dark brown amorphous precipitate was
formed. The precipitate was filtered off after 24 hours, washed with ethanol and
dried in a vacuum desiccator over CaCl . Yield: 0.0572 g (45 %), m.p. ≥ 240 °С
2
(
decomp.). Anal. Calc. for C H Cl CuN O S : C, 23.61; H, 1.98; Cl, 13.94; N,
10
10
2
6
6 2
1
6.52; S, 12.60; Found: C, 23.52; H, 2.05; Cl, 14.06; N, 16.48; S, 12.69%. IR data,
−1
ν, cm : 3620 (br. m) intramolecular hydrogen bond; 3346 (br. m) ν(NH)NH2; 3153
+
(m) ν(OH)C=N-OH; 3020 (m) ν(C-H)thiazole; 2680 (m) ν(NH )thiazole; 1651 (s) ν(С=О);
1610 (m) ν(C=N)imine; 1545 (m) ν(C=N)thiazole; 1400 (m) ν(CH−S)thiazole; 1358 (m)
ν(C=C)thiazole; 1292 (w), 1208 (m) ν(−C−N−); 1071 (m) ν(N−O); 980 (m), 815 (m)
ν(С−С); 787 (s) δ(С−Н); 515 (br. m) ν(Cu−O); 410 (m) ν(Cu−N).
2
.2.5. Complex [Pd(anti-A) Cl ] (4)
2
2
o
PdCl (0.0266 g, 0.15 mmol) was dissolved in 2N HCl (4 mL) at 40–50 C,
2
and ethanol (10 mL) was added. To the resulting light brown solution a hot
solution of anti-A (0.0562 g, 0.3 mmol), dissolved in ethanol (10 mL), and 2N HCl
(2 mL) was added dropwise. The reaction mixture was heated for another 50
minutes. The light yellow precipitate that formed after cooling was filtered off,
washed with ethanol and diethyl ether and dried in a vacuum desiccator over
CaCl . Yield: 0.0494 g (88 %), m.p. ≥ 283 °С (decomp.). Anal. Calc. for
2
C H Cl N O PdS : C, 21.77; H, 1.83; Cl, 12.85; N, 15.23; S, 11.62; Found: C,
1
0
10
2
6
6
2
1
2
1.95; H, 1.88; Cl, 12.05; N, 15.11; S, 11.23%. H NMR, (500 MHz; d -DMSO;
δ/ppm): 7.42 (br. s 2Н, NH ); 7.67 (s 1Н, СН
s 1H, OH). C NMR (125.71 MHz; d -DMSO; δ/ppm): 116.05 C(5); 140.32 C(3);
42.41 C(1); 160.17 C(2); 172.62 C(4). IR data, ν, cm : 3342 (br. m) ν(NH)NH2;
6
+
); 8.17 (br. s 1H, NH ); 9.25 (br.
2
thiazolyl
1
3
6
−1
1
3
+
154 (m) ν(OH)C=N-OH; 2994 (br. m) ν(C-H)thiazole; 2690 (m) ν(NH )thiazole; 1645 (s)
ν(С=О); 1615 (m) ν(C=N)imine; 1580 (m) ν(C=N)thiazole; 1404 (m) ν(CH−S)thiazole
;
1355 (m) ν(C=C)thiazole; 1193 (m) ν(−C−N−); 1075 (m) ν(N−O); 823 (m)ν(С−С),
7
88 (s) δ(С−Н), 530 (br. m) ν(Pd−O), 404 (m) ν(Pd−N).
2
.2.6. Complex [Cu(syn-A) Cl ] (5)
2
2

7
CuCl ·H O (0.0852 g, 0.5 mmol) was dissolved with stirring in ethanol (25
2
2
o
mL) at 40–50 C. To the resulted solution a solution of syn-A (0.1872 g, 1 mmol),
dissolved in 4N HCl (10 mL), and ethanol (20 mL) was added. The reaction
mixture was stirred for 2 h under reflux and cooled down to the room temperature,
resulting in the formation of the dark green amorphous precipitate, which was
filtered off, washed with ethanol and diethyl ether and dried in a vacuum
desiccator over CaCl . Yield: 0.229 g (90 %), m.p. ≥ 215 °С (decomp.). Anal.
2
Calc. for C H Cl CuN O S : C, 23.61; H, 1.98; Cl, 13.94; N, 16.52; S, 12.60;
1
0
10
2
6
6 2
−1
Found: C, 23.55; H, 2.15; Cl, 13.75; N, 16.45; S, 12.53 %. IR data, ν, cm : 3600
br. m) intramolecular hydrogen bond; 3321 (m) ν(OH)COOH; 3235 (m) ν(NH)NH2
070 (br. m) ν(C-H)thiazole; 1650 (s) ν(С=О); 1630 (m) ν(C=N)imine; 1553 (m)
(
3
;
ν(C=N)thiazole; 1440 (m) ν(CH−S)thiazole; 1371 (w), 1326 (m) ν(C=C)thiazole; 1200 (m)
ν(C−N); 1065 (m) ν(N−O); 975 (w); 915 (w); 871 (m), 793 (m) δ(NH); 404 (m)
ν(Cu−N).
2
.2.7. Complex [Pd(syn-A)Cl ] (6)
2
o
PdCl (0.0887 g, 0.5 mmol) was dissolved in 6N HCl (3 mL) at 40–50 C, and
2
ethanol (10 mL) was added. To the resulting transparent light brown solution was
added dropwise a hot solution of syn-A (0.0936 g, 0.5 mmol), dissolved in 4N HCl
(3 mL) and ethanol (7 mL). The reaction mixture was stirred for 45 min under
reflux and cooled down to room temperature. The red transparent solution was
leaved in the dark place for 70 hours. Brown needle-like single crystals were
filtered off, washed with ethanol and diethyl ether and dried in a vacuum
desiccator over CaCl . Yield: 0.101 g (55 %), m.p. ≥ 200 °С (decomp.). Anal.
2
Calc. for C H Cl N O PdS: C, 16.48; H, 1.38; Cl, 19.45; N, 11.53; S, 8.73; Found:
5
5
2
3
3
1
C, 16.55; H, 1.45; Cl, 19.15; N, 11.45; S, 8.73 %. H NMR, (500 MHz; d -DMSO;
6
δ/ppm): 7.36 (s 1Н, СН
C NMR (125.71 MHz; d -DMSO; δ/ppm): 109.04 C(5); 143.08 C(3); 146.91
); 8.64 (br. s 2Н, NH ); 9.28 (br. m 2H, OH, COOH).
thiazole
2
1
3
6
−1
C(1); 160.36 C(2); 172.10 C(4). IR data, ν, cm : 3620 (br. m) intramolecular
hydrogen bond; 3389 (br. m) ν(OH)COOH, 3200 (m) ν(NH)NH2; 3149 (m) ν(OH)C=N-
OH; 2980 (w) ν(C-H)thiazole; 1667 (m) ν(С=О); 1628 (s) ν(C=N)imine; 1540 (m)
ν(C=N)thiazole; 1435 (m) ν(CH−S)thiazole; 1330 (m) ν(C=C)thiazole; 1255 (m), 1212 (m)
ν(C−N); 1177 (m) δ(NH); 1050 (m) ν(N−O); 946 (w); 845 (m), 807 (m), 747 (m)
δ(NH); 395 (m) ν(Pd−N); 318 (m) ν(Pd−Cl).
2
.3. Physical measurements
1
13
The H ( C) NMR spectra were measured on a Varian 300 (300 MHz) and
Bruker Avance DRX-500 (500.00 (125.71) MHz) spectrometers in a d -DMSO
6

8
solution using TMS as internal standard. The IR spectra were recorded on a
-1
Specord M80 in the range 4000–200 cm in KBr pellets. UV-vis spectra in DMF
-3
-1
solutions of anti-А, syn-A and its complexes (10 mol·L ) were recorded on a
-1
Specord M40 spectrophotometer in the range 200–900 nm (50000–11000 cm )
using a 1 mm quartz cell. X-ray photoelectron spectra (XPS) were measured on an
ES-2402 device using Mo Kα radiation. Samples were prepared as thin films from
an acetone suspension on a 10×10 mm aluminum base plate. Elemental analyses
for carbon, hydrogen, nitrogen and sulfur were performed by Carlo Erba Elemental
Analyzer Model 1106. The chlorine was measured by Shoniger method.
2
.3.1. Single crystal X-ray diffraction
Single crystals of anti-A, 1, 2 and 6 were measured on Bruker SMART
APEX2 diffractometer (MoKα radiation, graphite monochromator, λ = 0.71073 Å)
at 173 K in the nitrogen stream. Diffraction images were integrated with SAINT
program [24]. The obtained intensities were corrected for Lorenz, polarization and
absorption using multi-scan technique in SADABS program [25]. All structures
2
were solved by direct methods and refined by full matrix least-square on F using
SHELXTL program package [26]. Hydrogen atoms, connected to O and N were
located from difference Fourier map and refined without constraints. All other H
atoms were put in idealized geometrical positions and refined as a riding model on
the host atom. The detailed experimental data is given in Table 1.
Table 1. X-ray crystallographic experimental data for investigated compounds
2
.4. Cell culture and growth inhibition (IC )
50
Mouse aortic cell line (MAEC were used to determine the
cytotoxic/cytostatic effects of anti-A, syn-A and complexes 3–6. Cells were
incubated with them for 24 and 48 h under normal conditions in 96 well plates.
4
Initial cell concentration was about 5×10 cells/mL in the sample volume of 100
µL. As a culture medium, we used DMEM (Sigma, USA) with 10% FBS (Sigma,
USA), 2 mM L-glutamine, and 40 µg/mL gentamicin. Different concentrations
−6
−4
(
1×10 – 1.25×10 ) of anti-A, syn-A, and 3–6 were added to cell cultures in 100
µL of media after the period of cell adaptation in normal conditions (5 % CO , 100
2
o
%
humidity, 37 C) during 4 h. In order to evaluate cytotoxic/cytostatic effect of
agents MTT-assay was used, that is based on ability dehydrogenases of viable cells
to convert yellow water-soluble MTT into blue crystals of formazan that are
insoluble. The quantity of formazan (determined by the colorimetric method after

9
its dissolution in organic solvents) characterizes the intensity of redox processes in
cells of culture and is an indirect quantitative characteristic of the active biomass.
MTT solution (final concentration is 1 µg/mL) was added to each well before 6 h
of ending of incubation period (48 h). After incubation cells were pelleted by
centrifugation the plates at 1000 rev / min for 2-3 min, than supernatant was
carefully collected and 200 µl of DMSO was added to each well, cells were
resuspended and incubated for 10 min at 37°C, whereupon optical density (λ=570
nm) of formazan solution was measured with multiwell spectrophotometer,
Synergie Biotec (USA). [28]. Cell counts were performed using a tripan blue dye
after 48-hour incubation with agents. The cytotoxic effect was evaluated as percent
of live cells relative to control and characterized by IC index [29], and mitogenic
5
0
effect was evaluated as percentage increase in the number of cells relative to
control.
2
.5. Apoptotic level and cell cycle analysis.
Apoptotic level and the distribution of cells in different phases of the cell
cycle were assessed by flow cytometry [30]. Cells were plated in 6-well plates at a
4
density of 5×10 cells/mL in 5 mL total volume of complete culture medium. Cells
−4
were incubated with anti-A, syn-A, and 3–6 in concentration 3.2×10 M for 48
hours under normal conditions. Cell samples were stained with propidium iodide,
which selectively joins with intercalating places in DNA. Staining of cells by
flyuorohrom dye PI included the following steps: cells after single rinse in 5 ml of
phosphate buffer solution at 1000 rev / min for 10 min, resuspended in 1 ml of
hypotonic lysis buffer (0.1% sodium citrate, 0, 1% Triton X-100, 5 mcg / ml PI),
("Sigma Chemical Co", USA). The proportion of cells in different phases of the
cell cycle was measured by flow cytometry with argon laser (λexcitation=488 µm,
λemission=585/40 µm) (Becton Dickinson, USA) following standard staining. The
samples were analyzed with the help of the Mod Fit LT 3.0 (BDIS, USA) software
[30]. All measurements were conducted in independent triplicates.
3
. Results and discussion
3
.1. Syntheses
The presence of several nucleophilic centers in isomeric hydroxyiminoacetic
acids anti-A and syn-A determines their polydenticity and ability for competitive
coordination. Therefore, the location of functional groups and synthesis conditions

10
affect the localization of coordination bonds and the molecular structure of resulted
complexes.
The compounds 1–6 were synthesized according to the following scheme:
Scheme for the synthesis of complexes
The interaction of ethanol solutions of metal salts with anti-A and syn-A in
the acidic medium at the component ratios M:L 1:1 and 1:2 leads to the formation
of complexes 1–6 with different coordination of ligands. Anti-A is coordinated to
metal ions in a chelate manner by the nitrogen atoms of the hydroxyimino group
and the oxygen atom of the deprotonated carboxyl group. In this case, 2-
aminothiazol fragment does not involve in coordination. In the acidic medium, the
nitrogen atom of the thiazole ring is protonated with subsequent formation of an H-
O-C(O)-C=N-O pseudoheterocycle, based on intramolecular hydrogen bond
(NH…O=C), which promotes the stabilization of the molecular structure. In the
isomorphous compounds 1 and, 2, the central metal ion forms an octahedral
coordination polyhedron, whose vertices are occupied by the nitrogen and oxygen
atoms of ligand, oxygen atoms of three water molecules and sulfate anion
Oppositely, in case of complexes 3 and 4, the metal ion creates a square-planar
coordination geometry. Strong acidic synthesis conditions lead to the formation of
an extra positive charge on the nitrogen atom of thiazole ring due to its protonation
that cause the location of chloride anions in the second coordination sphere
(scheme).
Unlike anti-A, sys-A is coordinated to the metal ion in neutral form by the
nitrogen atoms of the hydroxyimino group and thiazole ring. In this case, the
central metal ion in the complexes 5 and 6 shows square-planar coordination
geometry. Using initial reagent ratio of M:L=1:1, a compound 6 is yielded. The
2
+
Pd ion is surrounded by two nitrogens and two chlorines. It should be noted that
irrespective of the reagent ratio, the analogous copper complex can not be
obtained, since two syn-A molecules completely exchange the chlorine anions in
the starting metal salt, forming the compound 5 (scheme).
3
3
.2. Spectroscopy
.2.1. Infrared spectra
High-frequency region of the IR spectra of anti-A and syn-A isomers is
characterized by absorption bands (ABs), corresponding to the intraligand
-1
hydrogen bond at 3620 сm , stretching vibrations of ν(NH), ν(OH), ν(C–H) and

11
ν(ОН)СООН at 3300-3250 , 3150-3180C=N-OH, 3025-2935νC-H, and 2785-
NH
-
1
2
780(ОН)СООН сm respectively [19, 20, 31–34].
In the frequency range 1700–900 сm , a number of characteristic ABs
correspond to the vibrations ν(С=О) (1665/1654), ν(C=N)imine (1615/1635), δ(NH)
-1
(
(
1575), ν(C=N)thiaziole (1525/1530), ν(CH−S)thiazole (1430/1435), ν(C=C)
1350/1367), ν(C−N) (1275), ν(C−OH) (1164/1190), ν(N−O) (1025/1021). The
position shift of these ABs in the spectra of complexes indicate formation of
coordination bond. The low-frequency region of the IR spectra of the anti-A and
-
1
sys-A compounds (900−200 сm ) contains the following ABs: δ(С−Н) (888/913),
ν(С−С) (830/823), δ(С−Н) (785/773), δ(N−О) (688/641), δ(С−С) (567/556,
4
77/484, 428/423). However, they are less informative in the sense of bond
formation.
The identical IR spectra of the complexes 1 and 2 indicate their isomorphous
nature. In the high-frequency region of the spectra, the ABs of the stretching
vibrations ν(NH), ν(OH), ν(C-H) of the functional groups NH , C=N-OH and C-H
2
-
1
at 3200/3312, 3130/3185 and 3005/3000 сm respectively, Fig. 1. The new ABs in
the spectra at 3420/3400, 2755/2750 and 2617/2650 сm are corresponding to the
-
1
stretching vibrations of coordinated-water O-H bonds, intraligand hydrogen bond
+
and the N-H bond of the protonated nitrogen NH of thiazole ring. The
coordination of anti-A to the metal ion by oxygen atoms of the carboxyl group and
the nitrogen atom of the hydroxyimino group causes a decrease of the frequency of
-
1
the stretching vibrations ν(С=О) and ν(C=N)
on ∆ν=14(24) and 25(15) сm .
imine
2
-
The coordination of SO anions to metal ions confirmed by the presence of ABs
at 1085/1100 and 632/618 сm , which correspond to ν (F ) and ν (F ) of
4
-
1
3
2
4
2
sulfat[35]. In this case, they overlap with vibrations of the N–O bond, which
-1
appeared at 1021 сm in the spectrum of anti-A. In the low-frequency region, the
-1
low-intensity ABs at 535/505 and 450/438 сm correspond to M–O and M–N
bond stretching vibrations [36-38].
Fig. 1. IR spectra of complexes 1−4
The IR spectra of the complexes 3 and 4 are also identical. The ABs at
-
1
3
346/3342, 3153/3154, 3020/2994 сm are caused by (NH)NH2, (OH)
, (C-
at
C=N-OH
+
H)thiazole bond stretches. The presence of a low-intensity AB of ν(NH )
690/2714 сm is common to the spectra of 3 and 4. N–O bond vibrations in the
thiazole
-
1
2
-1
complexes are shifted by ∆ν=54/50 сm to the high-frequency region in
comparison with the spectrum of the ligand. In the low-frequency region ABs of
-
1
the M–O and M–N stretching vibrations at 515/530, 410/404 сm are detected.

12
Since the OH group of the carboxyl fragment of syn-A does not involved in
the formation of coordination bond to the metal ion in the complexes 5 and 6, the
position of the AB of the carboxyl group does not change. In this case, the ABs of
-1
the thiazole ring are shifted by ∆ν=50 сm , and the (N−O) vibration frequency is
-
1
-1
higher by ∆ν=40/25 сm . The ABs at 404/395 and 310 сm assigned to M–N and
M–Cl bond stretches.
3
.2.2. UV-vis spectra
The electronic absorption spectrum of the aqueous solution of anti-A consists
-1
-1
-1
of broad ABs at 45200 сm (221 nm), 39700 сm (251 nm) and 34700 сm (288
nm). The decomposition of the spectrum into Gaussians gave four peaks at 45200,
-1
3
9700, 34700 and 31800 сm , which assigned to the intraligand π→π* (C=N)
electron transitions of thiazole ring, π→π* (C=N) of hydroxyiminogroup and
π→π*/n→π* (C=О) of carboxyl group [19, 20, 31, 34, 38, 39] (Fig. 2a).
The UV-vis spectrum of the aqueous solution of syn-A contains of two broad
-1
-1
ABs at 44250 сm (226 nm) and 38300 сm (261 nm). The decomposition of the
spectrum into Gaussians revealed four peaks at 44250, 38300, 35000 and 32200
-
1
сm (Fig. 2b), which correspond to the intraligand electron transitions π→π*
(
(
C=N) of thiazol ring, π→π* (C=N) of hydroxyiminogroup and π→π*/n→π*
C=О) of carboxyl group [19, 20, 31, 34, 38, 39].
Fig 2. UV-vis spectra of aqueous solutions of anti-A (a) and syn-A (b) with
decomposition into Gaussian components
The spectra of the anti-A and syn-A isomers differ in the position of the ABs
of intraligand electron transitions, which are explained by different orientation of
chromophore groups relative to hydroxyiminogroup (C=N-OH). The frequency of
the electron transitions π→π* of thiazole ring and (C=N) hydroxyiminigroup of
-
1
anti-A is shifted by ∆ν=+950, +1400 сm to the high-frequency region in
comparison with syn-A. Oppositely, the position of the Gaussians corresponding to
the electron transitions π→π*/n→π* (C=O) of anti-A is shifted by ∆ν=-300, -400
-
1
сm to the low-frequency region.
The UV-vis spectra of aqueous solutions of the complexes 1 and 2 are similar.
They differ only in the position of the ABs, which correspond to ligand-to-metal
-
1
-1
charge transfer transitions (LMСTT), at 28100 сm (complex 1) and 28300 сm
-1
(
complex 2). In the range of 45000–38000 сm , the ABs of the π→π* (C=C, C=N)
electron transitions of thiazole ring and (C=N)imine at 44580, 42970, 39400 сm are
detected. In this case, the bathochromic shift by ∆ν=-(620-300) сm of these bands
-1
-1

13
relative to pristine anti-A is observed. It could be explained by protonation of the
nitrogen atom of the thiazole rings and coordination of the hydroxyiminogroup to
the metal ion. The ABs that assigned to the π→π* transitions of C=O are also
-1
-1
shifted by ∆ν=+70 сm and -250 сm , which is probably caused by the formation
-1
of a metallocycle. The ABs of d-d transitions appears at 19500, 15100 сm in 1
-
1
and 21800, 15000 сm in 2.
The spectra of the aqueous solutions of the complexes 3 and 4 are also
similar, which indicates the same coordination mode of the ligand to the central
-1
metal ion (Table 2). The LMCTTs of these complexes appears at 28900 сm (for
-
1
-1
-1
3
) and 28720 сm (for 4) and d-d transitions at 16750 сm and 24700 см
respectively.
-1
Table 2. UV-Vis spectra of synthesized compounds (ν, cm )
The UV-vis spectra of the aqueous solutions of the complexes 5 and 6 are
similar. The ABs of electron transitions π→π* of thiazole ring and (C=N) are
imine
-
1
-1
at 44720, 43150 and 36160 сm in the complex 5 and at 44700, 43000, 36160 сm
in the complex 6. They undergo a hypsochromic shift by ∆ν=+470/450 and 1160
сm relative to the ABs of starting syn-A, which indicates their coordination to the
-
1
central metal ion. Besides, in the spectra of the complexes there are LMCTTs at
-
1
-1
2
2
9100 and 28800 сm . The position of the d-d transitions at 16700 сm in 5 and
-1
4400 сm in 6 is typical of the square-planar shape of the coordination unit.
1 13
.3.3. H ( C) NMR and X-ray photoelectron spectra
3
1
The H NMR spectra of the compounds anti-A and syn-A contain a broadened
singlet of amine group at 7.30 ppm, and the oxime and carboxyl groups appear as
one broadened signal in a range of 12.40-13.0 ppm due to protons exchange. It
should be noted that the positions of the singlets of the 5-Н protons of the thiazole
ring in the spectra of anti-A and syn-A differ noticeably (anti-A, 7.56 ppm; syn-A,
6
.84 ppm), Fig. 3 a, b. An analogous dependence was noted for the corresponding
1
3
ethyl ethers of anti-A and syn-A [40]. The C NMR spectra of the anti-A and syn-
A contains five carbon signals, the position of which differ from each other by (δ =
8
.45 (C5), 3.85 (C3), 3.57 (C1), 0.13 (C2), 1.79 (C4) ppm.
1
Fig. 3. H NMR spectra of anti-A (a) and syn-A (b)
1
In the H NMR spectrum of the complex 6, the signals from the 5-Н protons
of the thiazole ring and amine group undergo a shift of ∆δ=+0.52 and +1.34 ppm,

14
respectively, to the downfield in comparison with starting syn-A, which is due to
the coordination of the thiazole ring to the metal ion. In this case, the broadened
singlet of oxime and carboxyl group protons shifts by ∆δ=-3.15 ppm to the upfield,
which is associated with the formation of intraligand hydrogen bonds and
coordination of the oxime nitrogen atom to the palladium ion.
In contrast to the compound 6, the signals from the 5-Н proton of the thiazole
1
ring and amine group in the H NMR spectrum of the complex 4 shift slightly by
∆
δ=+0.01 and +0.23 ppm to the downfield, indicating a lack of coordination of the
thiazole ring to the palladium ion. Besides, in the spectrum of the complex there is
+
a broadened singlet at 8.17 ppm, which corresponds to the proton NH at the
nitrogen atom of the thiazole ring, Fig 4. The protonation of the thiazole ring is
also confirmed by the IR spectra of this complex and by the X-ray analysis of the
complexes 1 and 2, in which coordination of anti-A is carried in a similar manner.
1
Fig. 4. H NMR spectrum of compound 4
13
Carbon signals of C(1), C(2) in the C NMR spectra of complexes 4, 6 are
shifted upfield by (δ = -1.6/-0.67 and -4.68/-4.36 ppm, whereas carbon signals of
C(3), C(4), C(5) are shifted downfield relative to the starting isomers by (δ =
2
.28/1.19, 5.8/3.94, 1.57/3.01 ppm, due to their coordination to the central metal
ion, Fig. 5.
1
3
Fig. 5. C NMR spectra of anti-A, syn-A and compounds 4, 6
It is known that coordination of the ligand to the central metal ion can be
determined by X-ray photoelectron spectra. Therefore, we used XPS spectroscopy
for studying the coordination mode of anti-A and syn-A to palladium (complexes
4
, 6) and copper (complexes 3, 5) ions.
Analysis of the spectra showed that the N1s line of anti-A and syn-A consists
of three components with Ebond = 400.10/399.71, 400.95/400.70, 401.71/401.50 eV,
which correspond to the three non-equivalent nitrogen atoms NH , C=N and
2
thiazole
C=Nimine, Fig. 6. In the spectra of complexes 4, 6 Ebond of these groups increased by
.02/0.06 (NH ), 0.19/0.37 (C=Nthiazole) and 0.81/0.91 (C=Nimine) eV. Their values
0
2
are 400.12/399.77, 401.14/401.07, 402.52/402.41 eV. Based on this it is evident
that Ebond of (C=Nthiazole) and (C=Nimine) are greatest in complex 6, while in complex
4
only most increased the value of Ebond (C=Nimine). This indicates to polytypic
coordination of anti-A and syn-A to the metal ions according to the proposed
synthesis scheme.

15
Fig. 6. N1s lines of anti-A, syn-A and complexes 4, 6 in X-ray photoelectron
spectra
In the XPS spectra of complexes 3, 5, the values of component N1s are
400.15/399.81, 401.32/400.93, 402.59/402.38 eV. Similar to palladium complexes,
in compounds 3, 5 the values of N1s increased by 0.05/0.10 (NH ), 0.37/0.23
2
(C=Nthiazole) and 0.88/0.88 (C=Nimine) eV, indicating the similar coordination of
anti-A and syn-A to copper ions.
The value Ebond Pd3d and Pd3d in the complexes 4, 6 are 340.11/345.35
5/2
3/2
and 339.70/344.87 eV, while the Ebond Cu 2p are 932.70 and 933,10 eV in copper
3/2
complexes 3, 5, indicating the divalent state of metal ions, Fig.7.
Fig. 7. Pd3d , Pd3d and Cu2p lines in XPS
5/2
3/2
3/2
3
.3. X-ray diffraction study
3
.3.1. anti-A
A sulfate salt of the anti-A was crystallized from the 0.4M solution of H SO .
2
4
The compound crystallized in a monoclinic crystal system (space group P2 /n)
1
with two organic cations, sulfate anion and lattice water molecule in the
asymmetric unit (Fig. 8). Thiazole rings in both molecules are planar. The mean
deviation from least square plane amount to 0.003 and 0.008 Å for
C1/N1/C2/C3/S2 and C6/N4/C7/C8/S3 correspondingly. 2-Hydroxyiminoacetate
groups are found in anti-conformation in both molecules that confirms by torsion
angles C5-C4-N2-O5 = -176.72 ° and C10-C9-N5-O8 = -174.82°. The carboxylate
groups in both molecules are protonated showing values of C-O bond lengths
characteristic for the presence of pronounced single and double bonds (See Table
S1, ESI). The whole molecules have a non planar structure with most significant
deviation from between thiazole and 2-hydroxyiminoacetate groups. The dihedral
angles between C1/N1C2/C3/S2 and C4/C5/O7/O6/N2/O5 planes amount to
15.74°. The second molecule has a twice higher divergence of 31.95° between
C6/N4/C7/C8/S3 and C9/C10/O9/O10/N5/O8 planes. In both molecules, the
hydrogen atoms were found on thiazole nitrogens from difference Fourier map,
confirming the cationic nature of species. Mentioned hydrogen atoms involved in
intramolecular H-bonds (Table SX, ESI) with carboxylate C=O groups, forming
pseudoheterocycles N1/C2/C4/C5/O7/H1 and N4/C7/C9/C10/O9/H4. In the crystal
structure all structural units are connected in a complicated 3D Framework of
intermolecular H-bons (See Table S2, ESI).

16
Fig. 8. Molecular structure of sulfate salt of the anti-A. Dashed lines indicate
hydrogen bonds
3
.3.2. [Co(anti-A)(H O) SO ] (1) and [Ni(anti-A)(H O) SO ] (2)
2
3
4
2
3
4
Compounds 1 and 2 crystallized in the same space group P2 /n. Similar unit
1
cell parameters and composition revealed the isostructural nature of the
compounds. The crystallographic independent part contains one molecule of
complex (Fig. 9). The metal atoms have a nearly regular MNO (M – Ni, Co)
5
octahedral coordination geometry with typical M—O and M—N bond lengths
(Table S3, S5, ESI).
Fig. 9. Molecular structure of complexes 1 and 2 with labeling scheme. Dashed
lines indicate hydrogen bonds
The coordination environment of the central atom consists of O5 and N1 from
the ligand molecule, coordinated in the bidentate chelate manner. Additional four
oxygen atoms originate from coordinated sulfate anion and three water molecules.
Analyzing the torsion angles C1-C2-O1-O7 (-177.43° for 1 and -176.76° for 2) one
can conclude about the anti-conformation of the 2-hydroxyiminoacetate group in
both cases. The metallarings M1-N1-C2-C1-O5 are planar with deviation from
least square plane of 0.0212 Å for 1 and 0.0247 Å for 2. Remarkably that
chelatation to the metal center also influenced the geometry of the ligand molecule.
Thus, decreasing of dihedral angle between thiazole and 2-hydroxyiminoacetate
groups to 6.39(14)° for 1 and 6.55(14)° for 2 is pointed on planar configuration of
the coordinated ligand molecule. Intramolecular hydrogen bonds N2—H2N…O6,
O7—H7O…O2 and O8—H82O…O3 are additionally stabilizing the molecule of
complex. As in the case of anti-A, the complex 3D Framework of hydrogen bonds
is found in the crystal structure (Table S4 and S6, ESI).
3
.3.3. [Pd(syn-A)Cl ] (6)
2
Using PdCl in the synthesis leads to formation of complex 6 that crystallized
2
in orthorhombic symmetry (space group Pna2 ). The asymmetric unit contains one
1
molecule of complex and lattice water molecule (Fig. 10). The central Pd atom
revealed slightly distorted square-planar PdCl N coordination geometry (Table
2
2
S7, ESI) with mean deviation from least-square plane of 0.0104 Å. In contrast to
Co(II) and Ni(II) complexes, a five membered planar metallaring
Pd1/N1/C1/C3/N2 is formed due to the strong affinity of Pd(II) to nitrogen. This
forces the switch of ligand conformation from anti- to syn-. This evidenced by
C2/C1/N1/O1 torsion angle of 0.49°. The molecule of complex is not planar

17
because of dihedral angle between O2—C2—O3 carboxylate and remaining
moiety of 34.9(1)°. Besides, the compound is additionally stabilized by
intramolecular H-bonds O1—H1O—Cl2 and N3—H32N—Cl1 (Table S8, ESI). In
the crystal structure, the 3D framework of H-bonds is found.
Fig. 10. Molecular structure of 6 with labeling scheme. Dashed lines indicate
hydrogen bonds
3
.4. Cell culture tests
Our study showed that anti-A and syn-A demonstrated multidirectional
effects on endothelial cells. Syn-A showed mitogenic effect by stimulating dose-
-6
-4
dependently proliferation of cells in the concentration range 8х10 –1.25х10 М
average effect was 23.7%±7.2% vs. control). At the same concentration range,
anti-A slightly inhibited proliferation of endothelial cells (Fig. 11 a, b), but had
(
-
4
opposite effect in concentration 3.20х10 М.
Cytofluorometric analysis revealed that syn-A increased 1.8-fold (p<0.05)
cells population of the proliferative pool (G /M+S) vs. control and anti-A effects
2
(
Table 3). This effect was associated with increase number of cells both in G /M
2
(
p<0.05) and S (p<0.001) phases of cell cycle and no change in G /G cell
0
1
population. Anti-A increased number of cells in S phase (p<0.01), decreased - in
G /M (p<0.05), but didn’t affect number of cells in the proliferative pool (G /M+S)
2
2
as well as in G /G phases of cell cycle.
0
1
In spite of multidirectional effect of tested compounds on cells proliferative
-4
activity, both anti-A and syn-A (3.20х10 М) induced anti-apoptotic effect vs.
control. Anti-A and syn-A have reduced the percentage of apoptotic cells in 3- and
2
.6-fold (p<0.01), respectively (Fig. 12).
Fig. 11. The influence of anti-A with compounds 3, 4 (a) and syn-A with
complexes 5, 6 (b) on the endothelial cells proliferative activity. MTT–test
Fig. 12. The influence of anti-A, syn-A and complexes 3-6 on the level of
endothelial cells apoptotic index: ^ p<0.05 – vs. control, * p<0.01 – vs. control
Table 3. Cells distribution in phases of cell cycle under the influence of tested anti-
A, syn-A and complexes 3-6
The complexation of anti-A and syn-A with transition metals changed their
effectiveness. Complex 3 and 4 had more profound anti-proliferative effect vs.
anti-A. However, complex 4 was more effective in comparison with complex 3

18
(
(
(
fig. 11 a). Complex 3 slightly increased number of cells in the proliferative pool
G /M+S) (1.1-fold, p<0.05), which was associated with increase - in S phase
2
p<0.05) and no change in G /M vs. control. The number of cells in G /G phase
2
0
1
was decreased by complex 3 vs. control, anti-A and complex 4 effects. Influence of
complex 4 on cell cycle phase was similar to effect of anti-A (Table 3).
Redistribution of cells in cell cycle phases described for anti-A, complexes 3 and
4
, may result in cells entrance in postsynthetic checkpoint and autophagic death
[41].
The incubation of endothelial cells with complex 3 led to reduced apoptotic
cells percent. Complex 4 didn’t affect apoptotic level of cells vs. control, but
increased apoptotic cells percent vs. anti-A (Fig. 12).
We could not detect cytotoxic effect for anty-A, while both complexes (3 and
-4
-5
4
) had cytotoxic effect (ІС = 1.33х10 М for 3 and ІС =2.5х10 М for 4).
5
0
50
Complexes of metal with syn-A (5 and 6) induced opposite effect in
comparison to syn-A: compound 6 did not affect cells percent in G /G phase vs.
0
1
control, but increase vs. syn-A effect. Redistribution in G /M and S was the most
2
pronounced relatively to control and compound syn-A. The population of cells in S
phase exceeded control and syn-A effects, in 10- and 1.2-fold, respectively (Table
3
). It was associated with 3.2- and 10-fold (p<0.01) increase number or apoptotic
cells vs. control and syn-A effects, respectively (Fig. 8). The similar effects were
described for compounds that modify activity of p53 and can lead to checkpoint of
cells conversion in G and initiation of apoptosis [42]. Complex 5 had slight
2
cytostatic effect (1.1-fold) vs. control and complex 6, and more profound (1.8-fold,
p<0.01) vs. syn-A.
4
. Conclusions
2
+
2+
2+
We showed that anti-A and syn-A coordinate bidentately to Co , Ni , Cu
2
+
and Pd ions yielding chelate-type complexes. Anti-A coordinates to the central
metal ion by the oxygen atom of the deprotonated carboxyl group and the nitrogen
atom of the oxime group. Oppositely, syn-A coordinates to the central metal ions
by two nitrogen atoms of the oxim group and thiazole ring. The molecular
structures of synthesized complexes were stabilized by intramolecular hydrogen
bonds, forming so-called pseudoheterocycles. All complex compounds form stable
solutions in neutral medium, which makes it possible to use them as potential
biologically active substances.
When studying the biological effect of the compounds anti-A and syn-A on
the main cell target (endotheliocytes), it has been found that syn-A exhibits

19
mitogenic and antiapoptotic effect, whereas anti-A causes inhibition of
proliferation almost in all tested diapason.
Under the influence of complex compounds on endothelial cells, an increase
in the antiproliferative activity of the complexes 3−6 in comparison with starting
complexing agents is observed. Thus the activity of the syn-A-based complexes is
higher than that of the analogous complexes with anti-A. The complex compounds
3
and 4 show pronounced cytotoxic action, and 6 has cytotoxic and proapoptotic
properties. For the complex 5, cytostatic effect has been established. The “arrest”
of conversion from S to G was detected for all tested compounds that can be
2
evidence of malfunction in DNA replication or deregulation in endogenic signals
of cell cycle in endotheliocytes associated with hypoxia and further HIF-1
activation. Profile of tested compounds influence on endothelial cells denotes its
potential utilization as pharmacological angiogenesis directed drug.
Acknowledgements
Prof. Ludmila Garmanchuk is thankful to the Dr I. Goot, University of London for
the kindly providing of Mouse aortic cell line.
Appendix A. Supplementary data
The crystal structures of compounds anti-A and 1, 2 and 6 have been
submitted to the CCDC and have been allocated the deposition numbers CCDC
987450-987453 for compounds anti-A, 1, 2 and 6, respectively. These data can be
obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the
Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ,
UK; fax: (+44) 1223-336-033). Supplementary data associated with this article can
be found, in the online version, at doi:
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22
Table 1
X-ray crystallographic experimental data for investigated compounds
Compound
anti-A
1
2
6
Брутто-формула
C
10
H
14
N
6
O
11
S
3
C
5
H
11CoN
3
O
10
S
2
C
5
H
11
N
3
NiO10
S
2
C
5
H
7
Cl
382.50
Orthorhombic,
Pna2
a = 7.2574(14)
b = 13.6007(4) b = 12.233(2)
2 3 4
N O PdS
Молекулярна маса
490.45
Monoclinic,
P2 /n
396.21
Monoclinic, P2
396.00
/n Monoclinic,
P2 /n
a = 6.7770(2)
Crystal system, space
group
Unit cell dimensions (Å) a = 7.0929(13) a = 6.8185(3)
1
1
1
1
b = 21.166(4)
c = 12.230(3)
β = 104.906(10) β = 102.961(4)
b = 13.6661(8)
c = 13.8939(9)
c = 13.8496(4)
β = 102.798(2)
1244.83(6)
c = 12.487(3)
3
Volume (Å )
1774.3(6)
1261.68(12)
1108.6(4)
4, 2.292
Z, Calculated density
4, 1.836
4, 2.086
4, 2.113
3
(
g/cm )
-
1
µ, mm
0.495
1.751
1.954
2.345
F(000)
1008
804
808
744
θ range for data
collection (º)
1.92 to 26.38
2.12 to 26.43
2.13 to 26.53
2.33 to 26.42
Limiting indices
-8 ≤ h ≤ 8
-8 ≤ h ≤ 8
0 ≤ k ≤ 17
0 ≤ l ≤ 17
2704
-8 ≤ h ≤ 8
-8 ≤ h ≤ 9
-
-
21 ≤ k ≤ 26
15 ≤ l ≤ 9
-17 ≤ k ≤ 14
-17 ≤ l ≤17
9883/2580
-15 ≤ k ≤ 15
-13 ≤ l ≤ 15
7900/1912
Reflections
8866/3595
collected/unique
Data/parameters (I >
[R(int)=0.0282]
3595/307
[R(int) =0.0341] [R(int) = 0.0651]
2704/231
1.087
2580/220
1912/163
4
σ(I))
2
Goodness-of-fit on F
1.082
1.038
1.091
R [I > 4σ(I)], Rw (all
data)
0.0340, 0.1050 0.0340, 0.0933
0.0336, 0.0865 0.0365, 0.0830
Largest diff. peak and
hole (eÅ )
0.308 and -
0.407
0.318 and -0.511 0.391 and -0.483 0.875 and -0.649
-3

23
-1
Table 2. UV-Vis spectra of synthesized compounds (ν, cm )
compounds π→π*
π→π* π→π*
(C=N)th (C=N)imine
iazole
π→π*/n→π* LMCTT
(C=О)
d-d
thiazole
ring
anti-A
syn-A
1
45200
44250
39700
38300
34700
35000
34770
31800
32200
31400
−
−
−
−
sh 44580, 39400
2970
28100
19500,
15100
4
2
3
4
5
6
sh 44580, 39400
2970
47900,
2000
sh 44500, 37400
2800
sh 44720, 39500
3150
44700,
3000
34450
35200
35250
36160
36160
31225
33500
33000
32100
32500
28300
28900
28720
30800
28800
21800,
15000
16750
4
39500
4
24700
16700
24400
4
4
39300
4
Sh, shoulder absorption band; LMCTT, Ligand-to-Metal Charge Transfer Transition
Table 3.
Cells distribution in phases of cell cycle under the influence of tested anti-A, syn-A
and complexes 3-6
compounds
G /G
0
G /M
2
S
G /M+S
2
1
Control
anti-A
syn-A
67.39±1.2
29.07±2.16
3.54±0.01
32.61±2.17
32.95±2.06
[a]
[b]
67.05±2.06
19.24±1.02
36.29±2.09
13.71±1.04
22.07±0.74
16.49±1.34
17.26±1.65
[
b],[c]
[a]
[b]
[b]
[b]
[b]
41.64±1.02
62.40±1.06
58.36±2.83
[
a],[c]
[a],[c]
3
4
5
6
21.12±1.16
37.61±2.52
31.34±2.45
[
b]
68.66±1.32
14.08±0.80
18.64±1.00
6.36±0.07
[
a],[d]
[a],[d]
[a],[d]
[a],[d]
72.07±0.34
9.29±0.33
27.93±1.33
34.61±0.95
[
d]
[b],[d]
[b],[d]
[d]
65.09±0.22
28.25±2.18
[
a] p<0.05; [b] p<0.01 – vs. control; [c] p<0.05 vs. anti-A; [d] p<0.05 – vs. syn A

Figures

Figures

Figures

Figures

Figures

Figures