Synthesis, crystal structures, spectroscopic, and cytotoxicity study of Cu(II), Co(II), Ni(II) and Pd(II) complexes with 2-anilinomethylidene-5,5-dimethylcyclohexane-1,3-dione

Article abstract of DOI:10.1016/j.poly.2019.114325

The coordination compounds of Cu(II), Co(II), Ni(II) and Pd(II) with 2-anilinomethylidene-5,5-dimethylcyclohexane-1,3-dione (HL) of the composition [ML₂(C₂H₅OH)₂]·nH₂O (M = Co (n = 1), Ni (n = 0)) and

Full text of DOI:10.1016/j.poly.2019.114325

Journal Pre-proofs
Synthesis, crystal structures, spectroscopic, and cytotoxicity study of Cu(II),
Co(II), Ni(II) and Pd(II) complexes with 2-anilinomethylidene-5,5-dimethyl-
cyclohexane-1,3-dione
Julia A. Eremina, Elizaveta V. Lider, Taisiya S. Sukhikh, Ilia V. Eltsov, Natalia
V. Kuratieva, Boris A. Zakharov, Liliya A. Sheludyakova, Lyubov S.
Klyushova, Ekaterina A. Ermakova, Victor V. Dotsenko
PII:
S0277-5387(19)30770-3
DOI:
https://doi.org/10.1016/j.poly.2019.114325
POLY 114325
Reference:
To appear in:
Polyhedron
Received Date:
Revised Date:
Accepted Date:
21 August 2019
18 December 2019
19 December 2019
Please cite this article as: J.A. Eremina, E.V. Lider, T.S. Sukhikh, I.V. Eltsov, N.V. Kuratieva, B.A. Zakharov, L.A.
Sheludyakova, L.S. Klyushova, E.A. Ermakova, V.V. Dotsenko, Synthesis, crystal structures, spectroscopic, and
cytotoxicity study of Cu(II), Co(II), Ni(II) and Pd(II) complexes with 2-anilinomethylidene-5,5-
dimethylcyclohexane-1,3-dione, Polyhedron (2019), doi: https://doi.org/10.1016/j.poly.2019.114325
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Synthesis, crystal structures, spectroscopic, and cytotoxicity study of Cu(II), Co(II), Ni(II)
and Pd(II) complexes with 2-anilinomethylidene-5,5-dimethylcyclohexane-1,3-dione
Julia A. Ereminaa,b,c, Elizaveta V. Lidera,b, Taisiya S. Sukhikh , Ilia V. Eltsov , Natalia V.
a,b
b
a,b
b,d
a,b
c,g
Kuratieva , Boris A. Zakharov , Liliya A. Sheludyakova , Lyubov S. Klyushova , Ekaterina
a,b
e,f
A. Ermakova , Victor V. Dotsenko
a
Nikolaev Institute of Inorganic Chemistry, SB RAS, 3, Acad. Lavrentiev Ave., 630090,
Novosibirsk, Russia
b
Novosibirsk State University, 2, Pirogova Str., 630090, Novosibirsk, Russia
c
Institute of Molecular Biology and Biophysics – subdivision of FRC FTM, 2/12 Timakova str.,
6
30117, Novosibirsk, Russia
d
Boreskov Institute of Catalysis SB RAS, 5, Lavrentieva Str., 630090, Novosibirsk Russia
Kuban State University, 149, Stavropol’skaya str., 350040, Krasnodar, Russia
North Caucasus Federal University, Department of Chemistry, 1a, Pushkin Str., 355009,
e
f
Stavropol, Russia
g
Scientific Institute of Clinical and Experimental Lymphology-Branch of the ICG SB RAS,
6
30060 Novosibirsk, Russia.

E-mail: lisalider@ngs.ru, +7-383-316-51-43, fax +7-383-330-94-89
Abstract:
The coordination compounds of Cu(II), Co(II), Ni(II) and Pd(II) with 2-anilinomethylidene-5,5-
dimethylcyclohexane-1,3-dione (HL) of the composition [ML
2
(C
2
H
5
OH)
2
]·nH O (M = Co
2
(
n=1), Ni (n=0)) and [ML ] (M = Cu, Pd) have been prepared and investigated by elemental
2
analysis, IR-spectroscopy, NMR spectroscopy, powder X-ray diffraction analysis. Single-crystal
X-ray diffraction analysis shows that HL serves as a chelate bidentate ligand coordinated to the
metal ion through the N and O atoms forming a six-membered chelate ring. The coordination
polyhedron of the metal atoms is a square bipyramid [ML
2
(C
2
H
5
OH)
2
] or square planar [ML ].
2

The Cu(II) and Pd(II) complexes stability in solution have been shown by NMR spectroscopy.
The results of the cell viability study on Hep2 (human larynx carcinoma) cells demonstrates that
the complexes don’t exhibit strong cytotoxic effect against Hep-2 cell line compared to cisplatin
(
IC50 = 9,2 ± 0,5 μM). Palladium(II) complex is the most cytotoxic among all complexes and
shows IC50 value of 68,7 ± 5,3 μM.
Keywords: β-Enaminone, Mononuclear Complexes, Crystal Structures, Cytotoxicity, Hep-2.
1
. INTRODUCTION
β-Enaminones, ···O=C-C=C-NH···, are promising compounds that can be used in synthetic
[
1-3] and medicinal [4-6] chemistry. They can act as synthetic intermediates or building blocks
for the synthesis of various heterocycles such as pyridine, pyrimidine, and pyrrole derivatives. β-
Enaminones are able to form strong intramolecular hydrogen bonds. In addition, they can exist in
various tautomeric forms: enaminone O=C–C=C–NH or iminoenol HO–C=C–C=N [7].
β-Enaminone-containing compounds have been reported to possess anticonvulsant
properties: some of the derivatives have demonstrated potential for anticonvulsant activity on in
vitro and in vivo models [8-13]. Moreover, it has recently been shown that this class of
compounds exhibit histaminergic and uterine relaxant properties [14], anti-inflammatory [15,
1
6], proteasome inhibitory [17] and P-glycoprotein modulating activity [4]. In these works, a
number of enaminones and precursor molecules with potential therapeutic value have been
revealed, but, to date, there are no examples of the β-enaminones that are clinically available.
The complexation ability of enaminones has attracted increasing attention in the last decades
[
18-21]. β-Enamiones are good chelating polydentate ligands that usually have N, O and other
donor atoms depending on the substituents. This class of compounds can differ significantly in
structure, therefore, the formation of both mono- and polynuclear complexes with different
coordination environment is possible. The interest to these compounds is also supported by the
fact that diverse metal complexes of enaminone derivatives can be applied in catalysis [22-23].

Aside from the catalytic properties antibacterial activity of some enaminones and their metal
complexes was studied by T. Mahmud [24] and B. Jeragh [25]. B. Jeragh and co-workers tested
Cu(II), Co(II), Ni(II), Pb(II), Hg(II) and Cd(II) 3-(dimethylamino)-1-(thiophen-2-
yl)/(phenyl)prop-2-en-1-one complexes against strains of bacteria and showed that enaminone
complexes which contain thiophene ring were more effective on microorganisms rather than that
containing aromatic phenyl group. Moreover, S. Mokesch and coworkers synthesized 1,3-
dioxoindan-2-carboxamide-based complexes, investigated their cytotoxic activity against human
cancer cell lines and affinity toward small biomolecules (amino acids, DNA). The benzyl
derivatives showed the highest in vitro activity and promising topoisomerase IIα inhibition [26].
In our work, we report the synthesis and the structure of a series of mononuclear Cu(II),
Co(II), Ni(II) and Pd(II) complexes with the composition [ML
2
(C
2
H
5
OH)
2
]·nH O (M = Co
2
(
n=1), Ni (n=0)) and [ML ] (M = Cu, Pd). The structure of HL was determined for comparison.
2
The Cu(II) and Pd(II) complexes stability in solution was shown by NMR spectroscopy. It was
also demonstrated that complexes don’t exhibit strong cytotoxic effect against Hep-2 (human
larynx carcinoma) cell line compared to cisplatin.
2
2
. MATERIALS AND METHODS
.1. Reagents and Materials
All chemicals used were of reagent grade and were used as purchased without any further
purification. Enamine ligand HL (Scheme 1) was synthesized as described in [27].
O
N
H
H C
3
O
H C
3
Scheme 1. 2-anilinomethylidene-5,5-dimethylcyclohexane-1,3-dione ligand (C15
H
17NO ).
2
2
.2. Methods and Instrumentation

Elemental analysis (C, H, and N) was performed using Euro EA 3000 analyzer. IR
absorption spectra were recorded on SCIMITAR FTS 2000 and VERTEX-80
–
1
−1
spectrophotometers at 4000–100 cm (in fluoridated oil – in the region of 4000–1500 cm , in
–
1
Vaseline oil – 1500–400 см ).
Powder XRD analysis of complexes 1-4 was performed on a Shimadzu XRD-7000
diffractometer (CuKα radiation, Ni filter, 5–50° 2θ range, 0.03° 2θ step, room temperature).
6
The 1D and 2D NMR spectra were recorded in d -acetone and CDCl
3
solutions at room
temperature on a Bruker Avance III 500 FT-spectrometer with working frequencies 500.03,
1
13
15
1
13
1
25.73, 50.67 MHz for H, С, and N nuclei, respectively. The H and С NMR chemical
shifts are reported in ppm of the δ scale and referred to signals of the solvents (2.04 ppm
1
(acetone) and 7.26 ppm (CHCl
3
) for residual protons for H and 29.92 (acetone, CD
3
) and 77.23
13
15
1
ppm (CDCl
3
) for C NMR spectra). N NMR spectra were obtained as projection of 2-D H –
1
5
15
N - correlation. The N NMR chemical shifts are referred to external standard of formamide (δ
(15
N) = 112.5 ppm). 1-D and 2-D (HSQC, HMBC) NMR spectra were used for the signal
assignment.
2
.3. Synthesis and characterization
.3.1. Synthesis of HL
2
Enamine ligand HL was synthesized by modified procedure of Wolfbeis and Ziegler [28] as
follows: a mixture of dimedone (1.40 g, 0.01 mol), freshly distilled aniline (0.9 mL, 0.01 mol)
and triethylorthoformate (2.2 mL, 0.013 mol) was refluxed with vigorous stirring until volatile
products ceased to release (about 3-5 min). Then a mixture was cooled with stirring and diluted
with 20 mL of 50% aq. EtOH. The precipitated product was filtered off and washed with water,
5
1
0% aq. EtOH and petroleum ether to give pure 2-anilinomethylidene-5,5-dimethylcyclohexane-
,3-dione as off-white crystalline solid.
21
Yield (2.2 g): 90%, mp137-138 °C (lit., 141 °C).

1H NMR (500 MHz, CDCl
) δ 1.02 (s, 6H, CH
), 2.35 (s, 2H, 4-CH
), 2.39 (s, 2H, 6-CH ),
3
3
2
2
7
1
.17 (t, 1H, J = 7.1 Hz, p-Ph), 7.20 (d, 2H, J = 8.2 Hz, o-Ph), 7.34 (t, 1H, m-Ph), 8.54 (d, 1H, J =
3.5 Hz, 2-CH), 12.79 (br. d, 1H, NH) .
13C NMR (126 MHz, CDCl
1-C), 118.17 (o-Ph), 126.40 (p-Ph), 129.96 (m-Ph), 138.55 (i-Ph), 150.41 (2-C), 196.05 (3-C),
) δ 28.62 (CH ), 31.13 (5-C), 51.35 (4-C), 51.68 (6-C), 108.94
3
3
(
1
99.81 (7-C).
15N NMR (51 MHz, HCONH
2
) δ 141.0.
2
.3.2. Synthesis of [Cu(L)
2
(C
2
H
5
2 2
OH) ] (1a) and [Cu(L) ] (1b)
A water solution (10 ml) of copper(II) acetate (0.160 g, 0.8 mmol) was added to ethanol
solution (10 ml) of HL (0.390 g, 1.6 mmol) with stirring. Immediately formed green precipitate
was filtered out, washed with ethanol and dried in air. A small amount of light green crystals of
[
Cu(L)
precipitate was recrystallized from CHCl
evaporation. The precipitate was studied by X-ray powder diffraction and identified as [Cu(L)
1b).
Yield (0.41 g): 93%. Elemental analysis for 1b (%): Сalc. for C30
.9; N, 5.1. Found: C, 65.4; H, 5.5; N, 5.0.
2
(C
2
H
5
OH)
2
] (1a) were obtained after a week from mother liquor. The abovementioned
3
and crystals of [Cu(L) ] (1b) were obtained by slow
2
2
]
(
H
32
N
2
O Cu: C, 65.7; H,
4
5
2
.3.3. Synthesis of [Co(L)
An acetone solution (1 ml) of cobalt(II) nitrate (0.087 g, 0.3 mmol) was added to
acetone/ethanol solution (2.5/2.5 ml) of HL (0.073 g, 0.3 mmol) with stirring. Then 1M NaOH
1.5 ml) was added to the mixture. Immediately formed orange precipitate was filtered out,
2
(C
2
H
5
OH)
2
]·H
2
O (2)
(
washed with ethanol and dried in air. Crystals of [Co(L)
from mother liquor by slow evaporation.
2
(C
2
H
5
OH) ] were obtained after a week
2

Yield (0.023 g): 24%. Elemental analysis (%): Сalc. for C34
H
46
N
2
7
O Co: C, 62.5; H, 7.1; N,
4
.3. Found: C, 62.6; H, 6.9; N, 4.4.
2
.3.4. Synthesis of [Ni(L)
2
(C
2
H
5
2
OH) ] (3)
A water solution (5 ml) of nickel(II) acetate (0.075 g, 0.3 mmol) was added to ethanol
solution (5 ml) of HL (0.073 g, 0.3 mmol) with stirring. Then 1M NaOH (1.5 ml) was added to
the mixture. Immediately formed light-blue precipitate was filtered out, washed with ethanol and
dried in air. Crystals of [Ni(L)
slow evaporation.
2
(C
2
H
5
OH) ] were obtained after a week from mother liquor by
2
Yield (0.068 g): 71%. Elemental analysis (%): Сalc. for C34
H
44
N
2
6
O Ni: C, 64.3; H, 7.0; N,
4
.4. Found: C, 63.7; H, 6.9; N, 4.35.
2
.3.5. Synthesis of [Pd(L)
2
] (4)
An acetone solution (5 ml) of HL (0.097 g, 0.4 mmol) was added to acetone solution (5 ml)
of palladium(II) acetate (0.045 g, 0.2 mmol) with stirring. During the evaporation on a water
bath a small amount of solid precipitated from the solution. The precipitate was filtered out with
filter paper, but composition of the precipitate could not be established based on the results of
elemental analysis. Orange crystals of [Pd(L) ] suitable for X-ray diffraction analysis were
2
obtained after a week from mother liquor by slow evaporation.
Yield (0.030 g): 25%. Elemental analysis (%): Сalc. for C30
H
32
N
2
O
4
Pd: C, 61.0; H, 5.5; N,
4
7
.7. Found: C, 59.6; H, 5.3; N, 4.6.
1H NMR (500 MHz, CDCl
3
) δ 0.92 (s, 6H, CH
3
), 1.81 (s, 2H, 6-CH
2
), 2.23 (s, 2H, 4-CH ) ,
2
.18 (d, 2H, J = 7.5 Hz, o-Ph), 7.28 (t, 1H, J = 7.3 Hz, p-Ph), 7.33 (t, 2H, m-Ph), 7.99 (s, 1H, 2-
CH).

13C NMR (126 MHz, CDCl
) δ 28.32 (CH ), 30.69 (5-C), 47.47 (6-C), 50.96 (4-C), 111.31
3
3
(1-C), 125.00 (o-Ph), 126.56 (p-Ph), 128.35 (m-Ph), 149.34 (i-Ph), 157.72 (2-C), 187.24 (7-C),
1
96.42 (3-C).
15N NMR (51 MHz, HCONH
2
) δ 192.7.
2
.4. X-ray crystallography
Diffraction data for single-crystals 1a and 2–4 were collected by a Bruker Apex DUO
diffractometer with MoKα radiation (λ = 0.71073 Å) in both cases. The φ- and ω-scan
techniques were employed to measure intensities. Absorption corrections were applied with the
use of the SADABS software (Bruker AXS Inc. (2000-2012). APEX2 (Version 2.0), SAINT
(Version 8.18c), and SADABS (Version 2.11), Bruker Advanced X-ray Solutions. Madison,
Wisconsin, USA). The structures were solved by dual space algorithm (SHELXT [29]) and
refined by the full-matrix least squares technique (SHELXL [30]) in the anisotropic
approximation (except hydrogen atoms). Positions of hydrogen atoms of organic ligands were
geometrically optimized and refined using riding model. H atoms of hydroxyl groups were
localized from the difference electron density map and refined using DFIX restraints for O–H
bonds. The crystallographic data and details of the structure refinements are summarized in
Table S1. Complete structural data were deposited in the CSD with ref codes CCDC 1908039-
1
908041 and 1908044-1908046 [31]. These data can be obtained free of charge from The
Cambridge Crystallographic Data Center at http://www.ccdc.cam.ac.uk/data_request/cif.
A single-crystal X-ray diffraction study for 1b and HL was performed using a STOE IPDS
II diffractometer with MoKα radiation and an image-plate detector. X-AREA (Stoe&Cie, 2013)
was used for data collection; CrysAlis Pro (Rigaku Oxford Diffraction, 2016) – for data
reduction and cell refinement. Crystal structures were solved with SHELXS-2016 [32] and
refined using SHELXL-2017 [30] with ShelXle as a GUI [33]). Atomic parameters for non-
hydrogen atoms were refined in the anisotropic approximation. H-atoms were located in the

difference Fourier map and refined using riding model. Platon [34] and Mercury [35] were used
for structure validation and visualization.
2
.5. Cytotoxic activity
Cell viability was evaluated by Hoechst/PI staining by standard method as previously
described [36]. Human larynx carcinoma cell line (Hep2) was grown on 96 wells plate in IMDM
medium in CO
medium solutions of tested compounds dissolved in DMSO (1-4), water (Cu(OAc)
Co(NO ·6H O, Ni(OAc) ·4H O), acetone (Pd(OAc) ) or ethanol (HL) for 48 hours in the range
2
-incubator at 37 °C. After 24 hours cells were treated with diluted with IMDM
2
·H O,
2
3
)
2
2
2
2
2
of concentrations 1-150 μM. Complex 4 is not soluble in DMSO, therefore it was investigated in
the form of suspension in DMSO solution. The final concentration of solvents was less than 1%.
For identification of live and dead cells, treated cells and control cells were stained with a
mixture of fluorescent dyes Hoechst 33342 (Sigma-Aldrich) and propidium iodide (Invitrogen)
for 30 min at 37 °C. IN Cell Analyzer 2200 (GE Healthcare, UK) was used to perform at least
four fields per well automatic imaging in bright-field and fluorescence channels. IN Cell
Investigator image analysis software was used to determine live, apoptotic and dead cells among
the whole population. All data shown are mean of three wells. The quantitative data were
expressed as the mean ± standard deviation (SD).
3
3
. RESULTS AND DISCUSSION
.1. Synthesis
Copper(II), cobalt(II), nickel(II), and palladium(II) complexes were synthesized by the
reaction of ligand HL with Cu(OAc)
respectively. [Pd(L) ] (4) was obtained as the bright-orange crystalline product, while [Cu(L)
1b), [Co(L) (C OH) ]·H O (2) and [Ni(L) (C OH)
orange and light-blue precipitates, respectively. A small amount of light green crystals of
2
·H
2
O, Co(NO
3
)
2
·6H
2
O, Ni(OAc)
2
·4H
2
O and Pd(OAc)
2
2
2
]
(
2
2
H
5
2
2
2
2
H
5
2
] (3) were obtained as light green,

[
Cu(L)
2
(C
2
H
5
OH) ] (1a) were obtained after a week of mother liquor evaporation. The
2
palladium(II) and copper(II) complexes were synthesized with the reagents ratio M: L = 1:2,
while cobalt(II) and nickel(II) complexes with the reagents ratio of 1:1. However, all complexes
prepared reveal metal to ligand ratio of 1:2.
Obtained compounds are soluble in different organic solvents: 1 – ethanol, acetone, DMSO,
CH
2
Cl
2
, 2 – DMSO, CH
2
Cl
2
, 3 – DMSO, 4 – ethanol, acetone, CH
2
Cl .
2
3
.2. Description of crystal structures
According to single-crystal XRD analysis, the complexes 1a, 2 and 3 are isostructural, with
principally the same geometry of the molecules (Fig. S1). Central atoms lie on the inversion
center and coordinate O and N atoms of two enaminonate and two ethanol ligands (Fig. 1a). The
environment of the metals is a square bipyramid with N atoms located in trans-positions. Bond
lengths and angles (Table 1) are close in the compounds, with the exception of M–O(C
2
5
H )
distance being larger by ca. 0.35 Å for the complex 1a as respect to 2 and 3. This difference is
obviously due to strong Jahn-Teller distortion effect for Cu(II) ion, which also manifests itself in
a slightly different orientation of ethanol ligand (Fig. S1). The corresponding distance for 1a is
rather large being longer than typical Cu–O(C
2
5
H ) bonds (of 2.10 – 2.15 Å) found in CSDB (v.
5
.40, February 2019). In fact, the complex 1a loses ethanol ligands upon recrystallization from
chloroform to form 1b. The latter is centrosymmetric and has a square planar coordination
environment of two enaminonates with N atoms at trans-positions (Fig. 1b). τ4-parameter
evaluating the geometry of the coordination polyhedron [37] strictly equals 0, because the
complex is centrosymmetric. This environment is supplemented to square bipyramidal via short
contacts with O atoms of the carbonyl groups of neighboring molecules (Fig. 2a). These contacts
connect the molecules into layers, which are further packed in a direction via van der Waals
interactions into a 3D structure (Fig. 2b). In addition, the compound 1b features greater deviation
of the central atom from enaminonate backbone plane (Fig. S2, Table 1) comparing to 1a, 2 and

3
. The complexes 1a, 2 and 3 reveal intermolecular hydrogen bonds (Et)OH···O with
uncoordinated carbonyl group (Tables 1, S2). These interactions connect the molecules into
chains (Fig. 3a). Arrangement of the chains imitates hexagonal packing of rods: each chain is
surrounded by six equivalent ones with symmetry operations ±1/2x, ±1/2y, z and x, ±1+y, z (Fig.
3
b). Interactions between the chains have van der Waals nature.
Similarly to 1b, the compound 4 reveals square planar coordination environment (τ4-
parameter [37] of 0.06) of two enaminonate ligands with N atoms at trans-positions, which is
typical for Pd(II) complexes. It should be noted that in 4, enaminonates are crystallographically
nonequivalent arranging in such a way that Pd atom lies in one plane with the first enaminonate
backbone, and deviates by 0.51 Å from the second enaminonate backbone plane. Contrary to the
compounds 1–3, the structure 4 shows no specific contacts between the molecules. Analysis of
the crystal packing of all complexes reveals similar arrangement patterns of the molecules in the
directions parallel to MN
2
O
2
(L) planes (Figs. S3-S5). In these layers, the dimethylcyclohexane-
1
,3-dione units of adjacent molecules are arranged towards each other. The phenyl units also are
arranged towards each other in all complexes discussed. Considering large distance between the
centers of the phenyl groups (the shortest one is 4.29 Å for 4), this arrangement is caused by
shape-induced van der Waals interaction of the molecules rather than π-π interactions between
the aromatic systems of the ligands. One can conclude that the molecules of the complexes have
oblate shape with specific protrusions and cavities that facilitate packing in the layers close for
all structures 1–4. However, intermolecular separation within the layers can differ considerably
due to the presence of specific intermolecular contacts and coordinated solvent molecules.
Geometry of the molecule of free HL (Fig. 4) resembles that in all complexes. The most
notable difference is that phenyl group is arranged in one plane with enaminone backbone, while
in the complexes, phenyls are inclined with C(H)–C–N–C(H) torsion angle of ca. 55° (Fig. S2,
Table 1). The molecules of HL are arranged in pairs (Fig. S6) similarly to L ligands binded by
the metal in the complexes, but with obviously larger intermolecular separation (N···N distance

is 5.89 Å versus that of 4.06 Å in 4). However, further packing of HL completely differs from
that of the complexes due to diverse arrangement of phenyls.
a)
b)
Fig. 1. Molecular structures of the complexes 1a, 2 and 3, exemplified by the compound 1a
a) and the complexes 1b and 4, exemplified by the compound 4 (b). Hydrogen atoms are
(
omitted for clarity, atomic displacement ellipsoids correspond to 50% probability level.
a)
b)
Fig. 2. Packing diagram of the complex 1b (a, b), showing short contacts Cu–O (dashed).
Hydrogen atoms are omitted for clarity.

a)
b)
Fig. 3. Structure of a chain formed by hydrogen bonding (dashed lines) of the complexes 1a,
2
and 3, exemplified by the compound 1a (a) and crystal packing diagram along c direction.
Hydrogen atoms are omitted except that involved in hydrogen bonding.
Fig. 4. Molecular structure of HL. Hydrogen atoms are omitted for clarity, atomic
displacement ellipsoids correspond to 30% probability level.
Table 1. Selected distances and angles in the complexes 1–4 and HL
*
Compound
M–O(L) , Å
M–O(Et), Å
M–N, Å
OH···O, Å
M-to-
C(H)–C–N–
C(H) torsion,
deg
backbone
plane, Å
1
1
a
1.9569(11)
1.9239(17);
2.4680(14)
2.0058(14)
1.9974(19)
2.7422(19)
0.29
55.8
b
–
–
0.46
47.8
2
.828(3)**
2
3
4
2.0407(18)
2.0145(8)
2.1329(18)
2.1138(9)
–
2.105(2)
2.665(3)
2.6699(12)
–
0.26
55.1
2.0617(10)
2.0393(18);
0.26
55.3
1.9711(16);
0.51; 0.04
56.7; 51.2

1
.9799(16)
2.0303(19)
HL
–
–
–
–
–
1.8
*
O atom belongs to L ligand.
* The distance to O atom of the neighboring molecule
The identity of single crystals of complexes 1–4 and the synthesized polycrystalline phases
was evidenced by powder X-ray diffraction analysis (Fig. S7–11).
*
3
.3. IR spectra
Ligand and complexes are characterized by IR-spectroscopy. Table S3 presents the main
vibrational frequencies in the IR spectra of β-enaminedione derivative and its metal complexes.
The IR spectrum of the ligand shows a low-intensive band of NH group vibrations in the
–
1
–1
range of 3247-3154 cm . The band at 1673 cm is assigned to vibrations of the carbonyl
groups, which are according to structural data involved in the formation of weak intramolecular
hydrogen bonds with the NH-group. As a result of ligand deprotonation the (NH) band
disappears in the spectra of all the complexes. Moreover, shift of (CO) to the low-frequency
region is observed, which indicates the coordination of these groups to the metal ion. The
coordination of solvent molecules in the case of complexes 2 and 3 is indicated by the shift of
-1
the (OH) band to the low-frequency region compared to free ethanol (3358 cm ).Thus, the IR-
spectroscopy data are in good agreement with the structural data.
3
.4. NMR Results
Stability of complexes 1b and 4 in acetone solution was checked by NMR spectroscopy.
6
Due to low solubility in acetone-d , signal assignment isn’t clear: the qualitative comparison of
1H NMR spectra of HL, 1 and 4 is possible only. However, appreciable differences between
spectra of the free ligand and complexes are found clearly. Significant changes in the spectra of

HL and 4 appear in the aromatic area, the signal of NH group at 12.84 ppm disappears, and the
signal of the CH= group shifts to the high-field in the complex spectrum (Fig. 5).
1
3
ppm
8.4
8.2
8.0
7.8
7.6
7.4
ppm
2.5
2.0
1.5
1.0
ppm
1
6
Fig. 5. H NMR spectra of 4 (top), and HL (bottom) in acetone-d .
The NMR spectrum of 1b differs from the HL, showing significant broadening and shift of
the signals due to the presence of paramagnetic ion Cu(II): two broad lines are observed at -8 (ν
3 300 Hz) and 16 ppm (ν ≈ 1 500 Hz). Signals in the typical range of the chemical shifts (0-
2 ppm) are assigned to the species not bonded to Cu(II) directly: acetone, water and ethanol.
½
≈
½
1
Therefore, the coordination of the metal ions with HL preserves in the acetone solution of
compounds (Fig. 6).

7
6
5
4
3
2
1
ppm
x8
2
5
20
15
10
5
0
-5
-10
ppm
1
Fig. 6. H NMR spectra of 1b in CDCl .
3
The solubility of complexes in the chloroform was better than in the acetone, therefore
CDCl
3
solutions were investigated for the characterization and signal assignment of compounds.
1
The compound HL has been described early [27], but H NMR data have been presented only.
Unambiguous assignment of a cyclohexane fragment of the HL is hindered due to
1
3
symmetrical structure of this moiety. Therefore, the C NMR spectrum without CH decoupling
was recorded for the signal assignment (Fig. S12). The coupling constant 3JC,H for trans-isomer
of alkene is always larger than that for the corresponding cis-isomer [38].The signal at 199.81
ppm appears as a quartet with 3JC,H ≈ 6.5 Hz, whereas in contrast, the signal at 196.05 is a poorly
resolved triplet of doublets with 3JC,H ≈ 6.0 Hz and 3JC,H ≈ 2.3 Hz for {4- H; 3- C} and {2- H; 3-
1
13
1
13C} interactions correspondingly. The low-field position of the 13
indicates that the intramolecular H-bond is missing.
C NMR signal for 7-C
1
3
3
In the C NMR spectrum of 4, the signal at 187.24 ppm appears as a quartet with JC,H ≈ 6.3
and the signal at 196.42 is a triplet of doublets with 3JC,H ≈ 6.3 Hz and 3JC,H ≈ 1.8 Hz for {4- H;
1
1
3
1
13
3
- C} and {2- H; 3- C} interactions correspondingly (Fig. S12).
Thus, significant differences appear for the complexes and ligand NMR spectra for both
acetone and chloroform solutions. Signal positions of 4 and HL are shown in the FigureS13.

There are differences between chemical shifts of the complex and the ligand, especially for
nuclei to the chelate ring involved.
1
Signals broadening and paramagnetic shifts are observed in the H NMR spectrum of 1b due
to paramagnetic nature of the Cu(II) ion (Fig. 6). The structure determination and the signal
assignment aren’t possible for this case, but it’s capable to suggest that Cu is bonded with HL as
before.
3
.5. In vitro cytotoxicity study
Ligand HL, its complexes and metal salts (Cu(II), Pd(II), Ni(II) acetates and Co(II) nitrate)
were tested for the cytotoxic activity in vitro against Hep2 (human larynx carcinoma) cell line
using dual staining with Hoechst 33342/propidium iodide. Cisplatin was tested under the same
conditions and used as a comparison. IC50 value was defined as drug concentration that reduces
the number of living cells by 50 %.
Cytotoxicity is not observed after cell treatment for 48 hours with solutions of HL (5-100
μM, Fig. S20b) and metal salts (5-150 μM, graphs are not presented). Copper, cobalt and nickel
complexes shows low toxicity: IC50 values for the [Ni(L)
Co(L) (C OH) ]·H O are higher than 100 μM (Fig. S20c-e).
Palladium(II) complex is the most cytotoxic among all complexes and demonstrates IC50
value of 68,7 ± 5,3 μM. As can be seen in Fig. S20F, treatment of Hep-2 cells for 48 hours with
PdL ] induces cell death already at the 25 μM concentration (13 %). The percentage of
2
(C
2
H
5
OH)
2
], [Cu(L)
2
] and
[
2
2
H
5
2
2
[
2
apoptotic cells is sufficiently high at the maximal concentration tested for this complex (63 %).
However, activity of cisplatin is eight times higher on this cell line (IC50 = 9,2 ± 0,5 μM, Fig.
S20a).
The results shows that palladium(II) coordination compound exhibits significant cytotoxicity
with IC50 values in the micromolar range for Hep2 cells, while the other complexes are less
toxic. Thus, the cytotoxicity of the reported complexes depends on the nature of the metal and

formation of Pd(II) complex with nontoxic reagents (HL and Pd(OAc) ) leads to the appearance
2
of cytotoxic activity. The fact that synthesized compounds possess non-toxic profile can be used
in the future in drug development based on enaminone moiety. For example, many non-toxic,
copper chelate complexes with small molecular mass are essential for the de novo synthesis of
metalloelement-dependent enzymes required for oxygen utilization and prevention of oxygen
superoxide accumulation [39].
4
. CONCLUSIONS
In summary, mononuclear copper(II), nickel(II), cobalt(II) and palladium(II) complexes with
-anilinomethylidene-5,5-dimethylcyclohexane-1,3-dione have been prepared and characterized
2
both in solution and solid state. According to single-crystal XRD analysis, the complexes 1a and
reveal square planar coordination environment of two enaminonate ligands with N atoms at
trans-positions, while the environment of 1a, 2 and 3 is supplemented to square bipyramidal by
coordinated C OH molecules. It should be stressed that the complexes with the composition
] (M = Co (n=1), Cu and Ni (n=0)) reveal intermolecular hydrogen bonds
Et)OH···O=C with uncoordinated carbonyl group of HL. Interestingly, these interactions have
4
2
H
5
[
(
ML
2
(C
2
H
5
OH)
2
van der Waals nature and connect the molecules into chains imitating hexagonal packing of rods.
The NMR studies have shown appreciable differences between spectra of the free ligand and
complexes: significant changes appear in the aromatic area, the signal of NH group at 12.84 ppm
disappears from the ligand spectrum, and the signal of the CH= group shifts to the high-field in
the complex spectrum. We can conclude from the above results that the coordination of the
copper(II) and palladium(II) ions to HL is preserved in the acetone solution of the compounds.
The cytotoxicity study on Hep-2 cell line has revealed that all compounds obtained are almost an
order of magnitude less active than clinically approved drug cisplatin. In particular, the most
cytotoxic palladium(II) complex shows IC50 value of 68,7 ± 5,3 μM. The results presented
contribute to the coordination chemistry of β-enaminones and could be useful for a better

understanding of structure – cytotoxic activity relationships for the metal complexes of β-
enaminones.
Acknowledgements
This work was supported by the Russian Science Foundation (Project № 18-73-00294).
Prof. Dotsenko V.V. is grateful for financial support by the Russian Ministry of Education and
Science (State Assignment to Higher Education Institutions, project no. 4.5547.2017/8.9).
Dr. Zakharov B.A. is grateful to the Ministry of Science and Higher Education of the Russian
Federation (project АААА-А19-119020890025-3).
The authors thank Anna P. Zubareva and Valentina V. Ankudovich for the elemental analysis,
Nina I. Alferova for the IR spectroscopy data, and Ilya V. Korolkov for the X-ray phase analysis
data. This work involved the use of equipment from the «Proteomic analysis» Center of the
Institute of Molecular Biology and Biophysics of Federal State Budget Scientific Institution
“Federal Research Centre of Fundamental and Translational Medicine” (IMBB FRC FTM),
Novosibirsk, Russia.
Conflicts of interest. There are no conflicts to declare.

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Graphical abstract
Reaction of Cu(II), Co(II), Ni(II) and Pd(II) salts with 2-anilinomethylidene-5,5-
dimethylcyclohexane-1,3-dione produced the coordination compounds of the
composition [ML (C H OH) ] (M = Cu, Co, Ni) and [ML ] (M = Cu, Pd), whose
2
2
5
2
2
crystal structures were determined. The cytotoxic properties of the complexes have

been investigated in vitro on Hep-2 cell line and the Pd(II) compound showed the
highest cytotoxicity.
40.
Credit Author Statement
Julia A. Eremina: Investigation, Writing – Original Draft; Elizaveta V. Lider: Supervision,
Writing - Review & Editing; Taisiya S. Sukhikh: Investigation; Ilia V. Eltsov: Investigation;
Natalia V. Kuratieva: Investigation; Boris A. Zakharov: Investigation; Liliya A. Sheludyakova:
Investigation; Lyubov S. Klyushova: Investigation; Ekaterina A. Ermakova: Investigation;
Victor V. Dotsenko: Conceptualization
41.