New copper(II) complexes with isoconazole: Synthesis, structures and biological properties

Article abstract of DOI:10.1016/j.poly.2012.10.040

There is an increasing demand for novel metal-based complexes with biologically relevant molecules in technology and medicine. Three new Cu(II) coordination compounds with antifungal agent isoconazole (L), namely mononuclear complexes [CuCl₂(L)₂] (1), and [Cu(O₂CMe) ₂(L)₂]·2H₂O (2) and coordination polymer [Cu(pht)(L)₂]_n (3) (where H₂pht-o-phthalic acid) were synthesized and characterized by IR spectroscopy, thermogravimetric analysis and X-ray crystallography. X-ray analysis showed that in all complexes, the isoconazole is coordinated to Cu(II) centres by a N atom of the imidazole fragment. In complex 1, the square-planar environment of Cu(II) atoms is completed by two N atoms of isoconazole and two chloride ligands, whereas the Cu(II) atoms are coordinated by two N atoms from two isoconazole ligands and two O atoms from the different carboxylate residues: acetate in 2 and phthalate in 3. The formation of an infinite chain through the bridging phthalate ligand is observed in 3. The biosynthetic ability of micromycetes Aspergillus niger CNMN FD 10 in the presence of the prepared complexes 1-3 as well as the antifungal drug isoconazole were studied. Complexes 2 and 3 accelerate the biosynthesis of enzymes (β-glucosidase, xylanase and endoglucanase) by this fungus. Moreover, a simplified and improved method for the preparation of isoconazole nitrate was developed.

Full text of DOI:10.1016/j.poly.2012.10.040

Polyhedron 52 (2013) 106–114
Contents lists available at SciVerse ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
New copper(II) complexes with isoconazole: Synthesis, structures
and biological properties
Galina M. Dulcevscaia a, Victor Ch. Kravtsov a, Fliur Z. Macaev b, Gheorghe G. Duca b, Eugenia P. Stingachi b,
Serghei I. Pogrebnoi b, Veaceslav V. Boldescu b, Steliana F. Clapco c, Janeta P. Tiurina c,
Alexandra A. Deseatnic-Ciloci c, Janusz Lipkowski d, Shi-Xia Liu e, Silvio Decurtins e, Svetlana G. Baca a,
⇑
a Institute of Applied Physics, ASM, Academiei 5, MD2028 Chisinau, Republic of Moldova
b Institute of Chemistry, ASM, Academiei 3, MD2028 Chisinau, Republic of Moldova
c Institute of Microbiology and Biotechnology, ASM, Academiei 1, MD2028 Chisinau, Republic of Moldova
d Institute of Physical Chemistry, Polish Academy of Sciences, 01-224 Warsaw, Poland
e Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 6 November 2012
There is an increasing demand for novel metal-based complexes with biologically relevant molecules in
technology and medicine. Three new Cu(II) coordination compounds with antifungal agent isoconazole
(L), namely mononuclear complexes [CuCl2(L)2] (1), and [Cu(O2CMe)2(L)2]ꢀ2H2O (2) and coordination
polymer [Cu(pht)(L)2]n (3) (where H2pht – o-phthalic acid) were synthesized and characterized by IR
spectroscopy, thermogravimetric analysis and X-ray crystallography. X-ray analysis showed that in all
complexes, the isoconazole is coordinated to Cu(II) centres by a N atom of the imidazole fragment. In
complex 1, the square-planar environment of Cu(II) atoms is completed by two N atoms of isoconazole
and two chloride ligands, whereas the Cu(II) atoms are coordinated by two N atoms from two isoconazole
ligands and two O atoms from the different carboxylate residues: acetate in 2 and phthalate in 3. The for-
mation of an infinite chain through the bridging phthalate ligand is observed in 3. The biosynthetic ability
of micromycetes Aspergillus niger CNMN FD 10 in the presence of the prepared complexes 1–3 as well as
the antifungal drug isoconazole were studied. Complexes 2 and 3 accelerate the biosynthesis of enzymes
(b-glucosidase, xylanase and endoglucanase) by this fungus. Moreover, a simplified and improved
method for the preparation of isoconazole nitrate was developed.
Dedicated to the 100th anniversary of the
award of the Nobel Prize in chemistry to
Professor Alfred Werner.
Keywords:
Isoconazole
Copper(II) complex
Synthesis
X-ray structure
Biological properties
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
dation states, structural diversity and ability to bind a variety of or-
ganic ligands with the possibility to select or develop the organic
Research in the development of new metal-based compounds
with biologically relevant molecules has attracted enormous inter-
est. Such metal coordination complexes have much potential for
design of novel therapeutic and diagnostic agents that target spe-
cific properties and show reduced side effects, avoidance of resis-
tance, improved selectivity and can be used for treating a wide
range of important human diseases; their pharmacological proper-
ties as antiviral, antimicrobial, anti-inflammatory, antihyperlipi-
demic, antitumoral, antidiabetic, diuretic and anticoagulant
remedies were emphasized and widely explored (see some recent
reviews and books, [1]). Moreover, metal coordination compounds
represent an outstanding playground for the creation of the next
generation of realistically useful metal-based drugs and diagnostic
agents. They allow to combine the features of metals which have a
wide range of coordination numbers and geometries, variable oxi-
ligands possessing electron donating or electron withdrawing
groups in an attempt to tune the optimal stability and the biolog-
ical in vitro activity. Furthermore, the coordination compounds
may unite in the same molecule metal ion, biologically relevant li-
gands and some ancillary ligands to create mixed-ligands coordi-
nation species to reach the desired properties [2]. Additional
challenges are the possibility to better understand the mechanism
of action of the small molecules, further evaluation and modula-
tion of the chemical composition and reactivity, and even the
development or improvements in the methods for detection of bio-
logical activity. From the other side, some metal coordination com-
pounds have been recognized as delivery vehicle for CO and NO
molecules in living organisms [3], as powerful probes for DNA-
mediated charge transport [4], and enzyme inhibitors [5]. The sig-
nificant regulatory and biostimulatory effects of coordination com-
pounds in the oriented synthesis of bioactive substances, including
the enzymes (cellulase, protease and amylase), by microorganisms
have also been emphasized [6].
⇑
Corresponding author. Tel.: +373 22 738154; fax: +373 22 725887.
E-mail address: sbaca_md@yahoo.com (S.G. Baca).
0277-5387/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2012.10.040

G.M. Dulcevscaia et al. / Polyhedron 52 (2013) 106–114
107
Isoconazole nitrate is a well-known antifungal drug and belongs
to a family of N-substituted imidazole derivatives, which showed
their potent activity against a variety of fungal and yeast infections
and have widely been used in the medical treatments of human
and animals [7]. Moreover, the isoconazole nitrate showed a more
powerful antifungal activity compared to other imidazole deriva-
tive drugs against some pathogenic Candida species [8] and has
proven efficacy in treating dermatomycoses [9]. Coordination of
the isoconazole to metal ions may lead to both enhancing its prop-
erties and displaying specific target features. To our knowledge, no
coordination complexes with isoconazole have been reported so
far. As the Cu ion is an essential element for many biological sys-
tems [10] and Cu coordination compounds have recently shown
great promises as therapeutic agents with antifungal and antican-
cer activities [11], three new Cu(II) complexes with the isoconazole
(L) have been synthesized. These include two mononuclear com-
plexes [CuCl2(L)2] (1) and [Cu(O2CMe)2(L)2]ꢀ2H2O (2) and coordina-
tion polymer [Cu(pht)(L)2]n (3) (where H2pht – o-phthalic acid).
Moreover, a simplified and improved method for the synthesis of
the isoconazole nitrate has also been developed. The compounds
were characterized by means of elemental analysis, IR, 1H, and
13C NMR spectroscopy, thermogravimetric and X-ray analyses. Fur-
ther, for studying the influence of these compounds on biosyn-
thetic activity of fungal strain, in particular the production of
hydrolytic enzymes, and revealing important chemical information
that can be implement into more specific structure–activity rela-
tionships, the impact of both the prepared coordination com-
pounds 1–3 and the isoconazole nitrate has been investigated.
tals of 3 revealed non-merohedral twinning and some contamina-
tion of diffraction pattern appeared upon cooling. two
A
components twin sample with component ratio 0.6196/0.3804 at
room temperature has been chosen for the crystal structure inves-
tigation in this case. The structures were solved by direct methods
and refined by full-matrix least squares on weighted F2 values for
all reflections using the SHELX suite of programs [13]. The HKLF5
dataset for refinement of 3 has been generated by the CrysAlisPro,
Agilent Technologies software (Version 1.171.34.49). The non-
hydrogen atoms in all structures were refined with anisotropic dis-
placement parameters. The C-bound H atoms were placed in calcu-
lated positions and were treated in a riding model approximation
with Uiso(H) = 1.2Ueq(C), while the O-bound H atoms of water mol-
ecule in 2 were found from difference Fourier maps and were re-
fined with isotropic displacement parameters Uiso(H) = 1.5Ueq(O).
The Figs. 1 and 2 were produced using the Mercury program
[14]. Crystal data and details of the structure refinement are given
in Table 1.
2.3. Biological testing
To estimate the biological role of the synthesized coordination
compounds 1–3 and isoconazole (L) the fungal strain Aspergillus ni-
ger CNMN FD 10, an active producer of extracellular cellulases and
xylanases, was used. The submerged cultivation of the strain was
carried out in 0.5 L Erlenmeyer flasks on shakers (180–200 rpm)
at 28–30 °C during 6–9 days. The nutrient medium of the following
chosen composition (g/L) was used as a control: beet pulp – 20.0;
wheat bran – 10.0; apple or grape marc – 10.0; KH2PO4 – 1.0;
CaCl2ꢀ2H2O – 0.1; KCl – 0.1; MgSO4ꢀ7H2O – 0.3; NaNO3 – 2.5; FeCl3
– 0.01; pH – 5.5–6.0. The tested compounds were introduced into
the nutritive medium in concentrations of 1, 5 and 10 mg/L. Coor-
dination compounds were added to the sterile cultivation medium
(after medium autoclaving) in the form of solutions prepared as
follows: 100 mg of the compound was dissolved in 20 mL ethylic
alcohol (96%) and was completed to 100 mL with sterile distilled
water.
Activity of cellulosolytic enzymes in culture filtrates was as-
sayed by measuring the amount of reducing sugars released from
the corresponding substrates: 4-nitrophenyl-b-D-glucopyranoside
– for b-glucosidases; Na-carboxymethyl cellulose – for endoglu-
canases; xylan from oat spelt – for xylanases. The determination
of glucose liberated from the substrate was measured calorimetri-
cally using the Somogy–Nelson copper method [15].
2. Experimental
2.1. Materials and physical measurements
All chemicals were purchased from commercial sources and
were used without further purification. All reactions were carried
out under aerobic conditions using commercial grade solvents.
[Cu(Hpht)2(H2O)2] has been synthesized according to previously
reported procedure [12]. Thin-layer chromatography was carried
out on Merck aluminum sheets, silica gel 60 F254. Column chroma-
tography was performed on Fluka silica gel 60, 70–230 mesh. Melt-
ing points were determined on a Boëtius melting point apparatus
(PHMK, VEB Wägetechnik Rapido, Radebeul, Germany) and are
uncorrected. 1H and 13C NMR spectra were acquired on a Bruker
Avance III 400 spectrometer operating at 400.13 MHz for 1H and
100.61 MHz for 13C. Chemical shifts (d) are given in ppm referring
to the signal centre using the solvent peaks for reference: DMSO-d6
2.50/39.52 ppm. The NMR signals were assigned by two-dimen-
sional 1H,1H COSY and 1H,13C correlation spectra (HSQC, HMBC)
using standard pulse sequences. The infrared spectra were re-
corded on a Perkin-Elmer Spectrum 100 FT-IR in the region
4000–650 cmꢁ1 or a Nicolet Avatar 360 FT-IR spectrometer in the
region of 4000 – 400 cmꢁ1 using KBr pellet technique. TGA mea-
surements were carried out with a Mettler Toledo TGA/SDTA 851
2.4. Synthesis of [CuCl2L2] (1)
A solution of CuCl2ꢀ2H2O (0.02 g, 0.12 mmol) in water (1 mL)
was added to
a solution of isoconazole nitrate 15 (0.12 g,
0.25 mmol) in ethanol (4 mL). The dark blue crystals suitable for
X-ray analysis were filtered off next day, washed with ethanol
and dried in air. Yield 0.062 g, 52%. Anal. Calc. for C36H28Cl10CuN4-
O2 (966.71): C, 44.73; H, 2.92; N, 5.79; Found: C, 45.02; H, 3.02; N,
5.78%. IR (KBr, cmꢁ1): 3158 (w), 3136 (m), 3082 (w), 3060 (w),
2890 (w), 1585 (m), 1564 (m), 1527 (m), 1513 (w), 1470 (m),
1436 (s), 1376 (m), 1337 (m), 1277 (w), 1259 (w), 1223 (m),
1196 (m), 1142 (w), 1112 (sh), 1097 (s), 1088 (s), 1074 (sh),
1044 (m), 1034 (sh), 1017 (s), 985 (m), 944 (m), 886 (m), 856
(m), 823 (m), 788 (sh), 778 (s), 759 (m), 746 (s), 725 (m), 696
(w), 656 (s), 639 (m), 623 (s), 562 (m), 520 (w), 497 (w), 454 (w),
432 (w).
in dry N2 (60 ml minꢁ1) at a heating rate of 10 K minꢁ1
.
2.2. X-ray crystallography
The data for 1, 2 and 3 were collected at 130, 100 and 293 K on
Bruker SMART, Nonius Kappa-CCD, and Xcalibur Oxford Diffraction
diffractometers, respectively, all equipped with a CCD area detec-
tor and employing graphite monochromatized Mo K
a radiation
(k = 0.71073 Å). Final unit cells dimensions were obtained and re-
fined on entire data set. Though many crystallization experiments
have been done, only small and poor diffracting crystals were
available for 2 and 3, which resulted in low resolution data. Crys-
2.5. Synthesis of [Cu(O2CMe)2L2]ꢀ2H2O (2)
A solution of Cu(O2CMe)2ꢀH2O (0.01 g, 0.05 mmol) and isocona-
zole nitrate 15 (0.047 g, 0.1 mmol) in 10 mL of MeOH was refluxed

108
G.M. Dulcevscaia et al. / Polyhedron 52 (2013) 106–114
Fig. 1. Molecular structures of 1 (a) and 2 (b) (ORTEP at 50% level) with an atom labeling scheme. Hydrogen bonds are shown as dotted line.
during 40 min. Dark blue crystals of 2 suitable for X-ray analysis
were filtered off, washed with MeOH and dried in air. Yield
0.03 g, 58%. Anal. Calc. for C40H38Cl8CuN4O8 (1049.93): C, 45.76;
H, 3.65; N, 5.34. Found: C, 45.68; H, 3.97; N, 4.67%. IR (KBr,
cmꢁ1): 3659 (w), 3289 (w), 3195 (w), 3141 (w), 3118 (w), 2931
(w), 2888 (w), 1585 (vs), 1563 (s), 1526 (m), 1467 (m), 1438 (s),
1395 (vs), 1378 (sh), 1336 (m), 1291 (m), 1242 (m), 1218 (m),
1199 (m), 1141 (w), 1107 (s), 1095 (s), 1079 (sh), 1045 (s), 1020
(s), 992 (m), 955 (m), 861 (s), 819 (m), 782 (s), 762 (s), 728 (m),
682 (m), 656 (m).
2.7. Synthesis of isoconazole nitrate
Synthesis of tetrabutylammonium 1H-imidazole (7). A solution of
NaOH (0.4 g, 10 mmol) in 2-propanol (20 mL) was treated with 1H-
imidazole (0.68 g, 10 mmol) followed by refluxing for 20 min.
Then, n-Bu4NBr (3.09 g, 10 mmol) was added at room temperature
and the resulting mixture was refluxing for 10 min. A solid sub-
stance was filtered, solvent was evaporated under vacuum, and
the residue was dried over P2O5 in vacuum desiccator. Yield
3.02 g, 98%. IR (KBr, cmꢁ1): 3101 (w), 2989 (w), 2958 (s), 2874
(m), 1679 (w), 1587 (s), 1491 (m), 1473 (s), 1465 (s), 1379 (m),
882 (s), 738 (s). 1H NMR (DMSO-d6, ppm): d = 0.93 (t, J = 8.0 Hz,
12 H, 4 Me,), 1.26–1.35 (m, 8 H, 4 CH2), 1.53–1.61 (m, 8 H, 4
CH2), 3.18 (t, J = 8.0 Hz, 8 H, 4 CH2-N,), 6.97 (s, 2 H, im), 7.58 (s, 1
H, im). 13C NMR (DMSO-d6, ppm): d = 13.96 (Me x 4), 19.68 (CH2
x 4), 23.56 (CH2 x 4), 58.01 (CH2 x 4), 122.20 (C4 im, C5 im),
135.91 (C2 im).
General procedure for reduction. Procedure for synthesis of halo-
hydrins. NaBH4 (0.21 g, 5.5 mmol) was added (in three portions)
to a solution of the corresponding ketone (5.5 mmol) 4 or 5 in
2-propanol (25 mL) at 3–5 °C. The mixture was stirred at room
temperature for 2 h. Then, the reaction mixture was poured into
water, the solution was extracted with ethyl acetate and dried
over anhydrous Na2SO4. The solvent was distilled. The obtained
light crude product was recrystallized from the appropriate
solvent.
2.6. Synthesis of [Cu(pht)L2]n (3)
To a solution of [Cu(Hpht)2(H2O)2] (0.05 g, 0.12 mmol) in 15 mL
of MeOH 0.12 g of isoconazole nitrate 15 (0.25 mmol) was added.
Dark blue crystals of 3 suitable for X-ray analysis were filtered
off after a few days, washed with MeOH and dried in air. Yield
0.10 g, 81%. Anal. Calc. for C44H32Cl8CuN4O6 (1059.92): C, 49.86;
H, 3.04; N, 5.29; Found: C, 49.88; H, 3.40; N, 5.05%. IR (KBr,
cmꢁ1): 3136 (w), 3065 (w), 2989 (br.w), 1615 (vs), 1593 (s),
1565 (sh), 1524 (m), 1497 (m), 1467 (m), 1449 (sh), 1437 (sh),
1415 (sh), 1399 (s), 1375 (vs), 1345 (sh), 1278 (w), 1246 (m),
1151 (m), 1096 (s), 1083 (sh), 1041 (m), 1026 (m), 1015 (m), 985
(w), 950 (w), 877 (m), 851 (m), 839 (m), 829 (m), 778 (s), 766
(s), 730 (s), 709 (vs), 653 (m).

G.M. Dulcevscaia et al. / Polyhedron 52 (2013) 106–114
109
Fig. 2. (a) Asymmetric fragment of a polymeric chain in 3; only O4 atom is shown twice to illustrate the coordination surrounding of Cu1 (ORTEP at 30% level). (b) Zigzag like
polymeric chain in 3; two L ligands are represented only by nitrogen atoms N1a and N1b of imidazole fragments. (c) A fragment of the chain with the attached isoconazole
molecules.
2-Chloro-1-(2,4-dichlorophenyl)ethanol (11). Yield 73%. M.p.
53 °C (from acetone). IR (KBr, cmꢁ1): 3600 (w), 3200 (m), 1475
(s), 1100 (m), 815 (s), 750 (m). 1H NMR (DMSO-d6, ppm): 3.32–
3.8 (m, 2 H, 2CH2), 5.0–5.2 (m, 1 H, 1CH), 5.44 (d, J = 10.4 Hz, 1 H,
OH), 7.20 (d, J = 1.94 Hz, 1 H, 6CH), 7.31 (d, J = 1.94 Hz, 1 H, 5CH),
7.60 (s, 1 H, 3CH). 13C NMR (DMSO-d6, ppm): d = 137.20 (2C),
133.20 (5C), 132.14 (6C), 130.01 (3C), 129.20 (4C), 99.12 (6C),
69.14 (7C), 48.7 (8C). Anal. Calc. for C8H7Cl3O (225.5): C, 42.61; H,
3.13; Found: C, 42.62; H, 3.13%.
2CH2), 5.02–5.25 (m, 1H, 1CH), 5.46 (d, J = 10.4 Hz, 1 H, OH), 7.20
(d, J = 1.94 Hz, 1 H, 6CH), 7.31 (d, J = 1.94 Hz, 1H, 5CH), 7.60 (s,
1H, 3CH). Anal. Calc. for C8H7BrCl2O (269.95): C, 35.59; H, 2.61;
Found: C, 35.47; H, 2.76%.
(a) 1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethanol (13). A
solution of halohydrins (1.0 equiv.) and ionic liquid 7 (1.0 equiv.)
in anhydrous MeCN was refluxed for 10–12 h. The reaction has
been monitored by TLC. A 3 volume of solvent was removed under
4
vacuum, the residue poured into water and extracted with ethyl
acetate. The organic phase was dried over anhydrous Na2SO4, dis-
tilled off. The obtained crude product was precipitated in acetone
2-Bromo-1-(2,4-dichlorophenyl)ethanol (12). Yield 57%. M.p. 72–
74 °C (from hexane). 1H NMR (DMSO-d6, ppm): 3.39–4.1 (m, 2 H,

110
G.M. Dulcevscaia et al. / Polyhedron 52 (2013) 106–114
(5C), 71.80
(
12C), 50.99 (4C). Anal. Calc. for C18H15Cl4N3O4
Table 1
Crystallographic data for complexes 1–3.
(479.14): C, 45.12; H, 3.16; N, 8.77; Found C, 45.40; H, 3.15%.
1
2
3
Formula
Crystal system
Space group
a (Å)
b (Å)
c (Å)
C
36H28Cl10CuN4O2
C40H38Cl8CuN4O8
triclinic
P1
C44H32Cl8CuN4O6
monoclinic
P21/n
15.457(6)
13.0982(16)
22.867(4)
90
105.40(3)
90
4463(2)
3. Result and discussion
triclinic
P1
ꢀ
ꢀ
8.6570(16)
9.0619(17)
13.797(3)
107.698(2)
107.840(3)
92.215(3)
971.2(3)
7.8401(8)
8.9852(9)
18.179(2)
99.399(6)
95.422(7)
115.234(9)
1123.4(2)
R1 = 0.0925,
wR2 = 0.2590
3.1. Synthesis of isoconazole nitrate
A general method for the preparation of isoconazole 10 consists
in the initial formation of the 1-(2,4-dichlorophenyl)-2-(1H-imida-
zol-1-yl)ethanol 13 via substitution reaction between 1H-imidaz-
ole and 2-bromo- or 2-chloro-1-(2,4-dichlorophenyl)ethanones
(4, 5) followed the reduction of 1-(2,4-dichlorophenyl)-2-(1H-imi-
dazol-1-yl)ethanone 8 [16–18]. A previous study [19] has shown
that an ionic liquid derivative of 1H-1,2,4-triazole 6 can success-
fully be applied as the synthetic equivalent of the Na (K or Li) salt
of 1,2,4-triazole towards 1-(2,4-dichlorophenyl)-2-(1H-1,2,4-tria-
zol-1-yl)ethanone 9 upon treatment with 2-bromo-1-(2,4-dichlo-
rophenyl)ethanone 4 (Scheme 1). A follow-up study [20–24] on
the preparation of a remarkably promising class of technologically
useful materials called ‘‘task-specific ionic liquids’’ as recyclable re-
agents for ‘‘green chemistry approach’’ has proved the strategic
advantages of using these materials.
Reaction of ketones 4 or 5 with 1 equiv. of ionic liquid 7 in boil-
ing MeCN afforded the substituted imidazole 8 with 50% and 70%
yields, respectively. On the other side, the ketones 4 and 5 were
transformed into the racemic halohydrins 11 and 12 by the reac-
tion with NaBH4 in 2-propanol. The addition of 2-chloro-1-(2,4-
dichlorophenyl)ethanol 11 or 2-bromo-1-(2,4-dichlorophe-
nyl)ethanol 12 to ionic liquid 7 gave directly 13 with 68% and
40% yields, respectively. Finally, the 2,6-dichlorobenzyl ester 10
was prepared via treatment of alcohol 13 and 1,3-dichloro-2-(chlo-
romethyl)benzene 14 with 1.25 equiv. of NaH in refluxing 1,4-
dioxane for 1.5 h. Isoconazole 10 can easily be transformed into
the corresponding salt 15 upon treatment with an excess of
HNO3 in acetone at room temperature with a nearly quantitative
yield. Our methodology offers an easy and alternative way for
the procedure developed by others [16,17].
a
(°)
b (°)
c
(°)
U (Å3)
R indices
[I > 2r(I)]
R1 = 0.0299
wR2 = 0.0798
R1 = 0.0794
wR2 = 0.2624
as a white solid. Yield 68% from 11. Yield 40% from 12. M.p. 142–
143 °C. IR (KBr, cmꢁ1): 1512 (m), 1079 (s), 845 (s), 733 (m). 1H
NMR (DMSO-d6, ppm): d = 4.0–4.06 (m, 2 H, 2CH2), 5.06–5.08 (m,
1 H, 1CH), 4.16 (d, J = 10.8 Hz, 1 H, OH), 6.03 (d, J = 4 Hz, 1 H, 4CH
of the imidazole ring), 6.83 (d, J = 4 Hz, 1 H, 5CH of the imidazole
ring), 7.03 (s, 1 H, 2CH), 7.42–7.46 (m, 2 H, 5CH, 6CH). 7.59 (s, 1
H, 3CH). 13C NMR (DMSO-d6, ppm): d = 137.14 (6C), 136.20 (1C),
132.99 (10C), 130.11 (7C), 130.01 (2C), 129.68 (8C), 128.04 (11C),
126.12 (9C), 118.44 (3C) 63.88 (5C); 53.22 (4C). Anal. Calc. for C11-
H10Cl2N2O (257.12): C, 51.38; H, 3.92; N, 10.89; Found C, 51.39;
H, 3.92; N, 10.90%.
(b) NaBH4 (0.21 g, 5.5 mmol) was added (in three portions) to a
solution of the ketone (1.4 g, 5.5 mmol) 8 in 2-propanol (25 mL) at
3–5 °C. The mixture was stirred at room temperature for 2 h (mon-
itored by TLC). Then, 50% of solvent was removed under reduced
pressure and 3% solution of HCl (3 mL) was added. The reaction
was neutralized with sodium carbonate and poured into water.
The mixture was extracted with ethyl acetate and the organic
phase was dried over anhydrous Na2SO4. The solvent was removed
under reduced pressure on a rotary evaporator to yield a viscous
gum, which was recrystallized in acetone to give 13. Yield:
1.19 g, 85%.
1-[2-(2,6-Dichlorobenzyloxy)-(2,6-dichlorophenyl)ethyl]-1H-imid-
azole (10) and its salt (15). To a suspension of 60% dispersion of NaH
in mineral oil (0.25 g, 7.5 mmol) in dry 1,4-dioxane (15 mL) at
room temperature under a nitrogen atmosphere was added drop-
wise a solution of 13 (1.28 g, 5 mmol) in 1,4-dioxane (20 mL).
The reaction mixture was refluxed for 1 h. 1,3-Dichloro-2-(chloro-
methyl)benzene 14 (1 g, 5 mmol) was added dropwise and re-
fluxed for 2.5 h. Solvent was removed in vacuum, and after that
the reaction was quenched with water (100 mL). The aqueous solu-
tion was extracted with ethyl acetate (3x50 mL), the organic
phases were combined, dried over anhydrous Na2SO4, and filtered.
The solvent was removed by distillation under reduced pressure,
giving a crude reaction mixture that was purified followed by crys-
tallization from acetone to give 1.45 g of ester 10. Yield: 70%. M.p.
115–116 °C.
A solution of base 10 (1.2 g, 2.8 mmol) in acetone (5 mL) was
treated with concentrated (86%) HNO3 (5 mL) followed by stirring
for 12 h. The white solid 15 was filtered and dried over P2O5 in vac-
uum desiccator. Yield 1.35 g, 98%. M.p. 203–204 °C. IR (KBr, cmꢁ1):
1381 (w), 1380 (w), 1510 (m), 1150 (m), 810 (s), 720 (s). 1H NMR
(DMSO-d6, ppm): d = 4.40–4.50 (m, 2 H, 1CH), 4.62 (d, J = 10.8 Hz, 2
H, -OCH2), 5.12–5.20 (m, 1 H, 2CH), 7.30–7.42 (m, 3 H, phenyl of 2-
(2,6-dichlorobenzyloxy)), 7.48–7.59 (m, 2 H, 5CH, 6CH), 7.60 (s, 2 H,
4CH, 5CH of the imidazole ring), 7.72 (s, 1 H, 3CH) 9.04 (s, 1 H, 2CH
of the imidazole ring), 14.6 (s, 1 H, HNO3). 13C NMR (DMSO-d6,
ppm): d = 138.10 (1C), 137.79 (13C), 135.35 (6C), 133.12 (14C, 7C),
131.88 (10C), 131.22 (2C), 130.00 (16C), 129.41 (7C), 128.21 (8C),
128.16 (15C, 18C) 127.13 (11C), 125.18 (9C), 118.29 (3C), 74.31
3.2. Synthesis of Cu(II) complexes and preliminary characterization
The reaction of Cu(II) chloride dihydrate with isoconazole ni-
trate 15 in water:ethanol (1:4) solution at room temperature leads
to the crystalline product with the formula [CuCl2(L)2] (1) in ca.
52% yield. Using Cu(II) acetate hydrate or Cu(II) phthalate dihy-
drate in the reaction with isoconazole nitrate 15 in methanol re-
sults in [Cu(O2CMe)2(L)2]ꢀ2H2O (2) and [Cu(pht)(L)2]n (3) in ca.
58% and 81% yield, respectively. The materials 1–3 were character-
ized by elemental analysis, IR data, and thermogravimetric and sin-
gle-crystal X-ray diffraction analyses to confirm the homogeneity
of the obtained solids.
The IR spectrum of the free isoconazole ligand shows medium
intensity bands at 1582 and 1561 cmꢁ1 due to C@N bond stretch-
ing of aromatic rings. In 1, these bands are slightly shifted to higher
values of 1585 and 1564 cmꢁ1, indicating the coordination of a
imidazole nitrogen atom in the Cu(II) complex. In this region, the
IR spectra of compounds 2 and 3 exhibit intense bands at 1615–
1563 cm–1 due to the asymmetric stretching vibrations of the coor-
dinated carboxylate groups of the acetate and phthalate ligands,
that overlap the
m(C@N) stretching vibrations of the imidazole
fragment of the isoconazole ligand. The symmetric stretching
vibrations of coordinated carboxylate groups are observed in the
region 1415–1374 cm–1. IR spectra of all complexes 1–3 also show
absorptions due to C-H bond stretching of aromatic rings in the
range 3289–2817 cm–1. The presence of solvate water molecules

G.M. Dulcevscaia et al. / Polyhedron 52 (2013) 106–114
111
O
X
N
O
N
Hal
Cl
Cl
N
Cl
Cl
O
Cl
Cl
8
9
4
X=C (70%)
X=N
N
Hal=Br
5 Hal=Cl
X
N
n-Bu4N
Cl
Cl
N
NaBH4
10
6
7
X=N
X=C (98%)
NaBH4
Cl
Cl
14
Cl
NaH,
OH
Cl
Cl
N
N
Cl
OH
NO3
Hal
then HNO3
NH
O
N
Cl
Cl
Cl
11
12
Hal=Cl (73%)
Hal=Br (57%)
Cl
15 (
Overall 69%)
Cl
13
11
)
(68 % from
(40 % from
(85 % from
12
)
8
)
Scheme 1. The synthesis of isoconazole nitrate.
in 2 caused the appearance of absorption bands with maxima at
3659 cm–1
Table 2
.
Selected bond lengths [Å] and angles [°] in 1–3.
Thermogravimetric analyses for all complexes were performed
in a nitrogen atmosphere in the temperature range of 25–800 °C.
The TGA data show that complex 1 is stable up to approximately
185 °C and then it starts to decompose in one step with total
weight loss of 65.9% to the final products (observed 34.1%). It is
accompanied by an endothermic effect at 196 °C. Coordination
polymer 3 is also stable up to approximately 200 °C and then the
decomposition of the organic ligands takes place in two unidenti-
fied steps with total weight loss of 70.0% to the final products (ob-
served 30.0%). It is accompanied by an endothermic effect at
219 °C. For complex 2, the loss of solvate water molecules with a
weight loss of 4.25% (calculated 3.43%) was observed before
200 °C followed by the decomposition of organic part in two
unidentified steps to final products (observed 29.2%).
1
2
3
Cu1–N1 (N1a)
Cu1–N1b
2.006(1)
2.2564(5)
89.90(4)
1.964(9)
1.953(8)
89.1(3)
1.972(16)
1.990(12)
1.989(15)
1.936(17)
88.4(7)
93.1(8)
90.6(6)
88.6(6)
163.3(6)
177.4(7)
Cu1–Cl5 (O2)
Cu1–O4#1
N1(N1a)–Cu1–Cl5(O2)
N1a–Cu1–O4#1
N1b–Cu1–O2
N1b–Cu1–O4#1
N1a–Cu1–N1b
O2–Cu1–O4#1
Symmetry transformations used to generate equivalent atoms: #1 ꢁx + 1/2, y + 1/2,
ꢁz + 3/2.
3.3. X-ray analysis
tion of the donor atoms from the coordination plane is less than
0.165 Å and results in some tetrahedral distortion of the coordina-
tion plane. In contrast to 1 and 2, the isoconazole molecules in 3
reside on the same side of the copper(II) coordination plane and
the phthalate ligands are situated on opposite sides of this plane.
They serve as exo-bidentate O,O0-ligands and coordinate via car-
boxylic groups to neighboring Cu(II) atoms, linking them to a zig-
X-ray crystallographic studies revealed that 1 and 2 contain cen-
trosymmetric mononuclear [CuCl2(L)2] and [Cu(O2CMe)2(L)2]ꢀ2H2O
complexes, respectively. Each Cu(II) atom in both structures resides
on a crystallographic center of symmetry and coordinates in trans-
positions to two isoconazole molecules via a nitrogen atom of a
imidazole fragment and two chlorine atoms in 1 or two oxygen
atoms from different acetate groups in 2 resulting in a square-pla-
nar coordination geometry (Fig. 1). The imidazole fragments form
with the coordination plane of the copper atom dihedral angles of
18.31° in 1 and 13.82° in 2. This difference in dihedral angles as well
as the longer Cu1–N1 bond length of 2.006(1) Å in 1 compared with
1.964(9) Å in 2, Table 2, is related with the different nature of the
ligands cis–situated with respect to the two imidazole fragments.
Solvate water molecules are hydrogen bonded to acetate groups
zag-like polymeric chain along the crystallographic
b axis,
Fig. 2b. Repeating units in the chain are symmetry related by 2-fold
screw axis. Three dihedral angles between the planes of carboxylic
groups and benzene fragment are very similar and possess values
in the narrow range 59.2–60.8°. Isoconazole molecules attached
to copper(II) atoms decorate the polymeric chain, as shown in
Fig. 2c.
The analysis of torsion angles reveals a similarity in the confor-
mation of isoconazole molecules in 1–3. In 1 and 2, isoconazole
molecules differ only by reverse orientation of an imidazole frag-
ment with respect to other parts of the molecule; the torsion angle
C1–N2–C4–C5 equals 91.58° and –115.3°, respectively, Table 3. The
structural conformations of the crystallographically independent
isoconazole molecules a and b in 3 are rather similar and close to
that found in 2. The largest difference in the conformation of the
isoconazole molecule in 1 and 2 compared to 3 is related with
the torsion angle C5–O1–C12–C13, which is anti in 1 and 2, and
in
2 [O1W–H. . .O3 = 2.795(12) Å, O1W–H = 0.92(2) Å, H. . .O3 =
1.87(2) Å, \O1W–H. . .O3 = 178(16)°].
The structure of 3 reveals a [Cu(pht)(L)2]n chain coordination
polymer. The coordination of Cu(II) atom is square-planar and real-
ized by two nitrogen atoms from two symmetry independent
monodentate
L
ligands [Cu1–N1a = 1.972(16) Å and Cu1–
N1b = 1.990(12) Å] and oxygen atoms from two pht2ꢁ ligands
[Cu–O2 = 1.989(15) and Cu–O4 = 1.936(17) Å], Fig. 2a. The devia-

112
G.M. Dulcevscaia et al. / Polyhedron 52 (2013) 106–114
Table 3
Table 5
Selected torsion angles [°] in the isoconazole molecules in 1–3.
The influence of 1–3 and isoconazole nitrate (L) on xylanase biosynthesis by
Aspergillus niger CNMN FD 10.
1
2
3a
3b
Compounds
Concentration (mg/L)
Enzyme activity (U/mL)
C1–N2–C4–C5
N2–C4–C5–C6
C4–C5–C6–C7
N2–C4–C5–O1
C4–C5–O1–C12
C5–O1–C12–C13
91.58(18)
ꢁ115.3(12) ꢁ100.4(18) ꢁ102(2)
ꢁ174.32(13) ꢁ179.6(10) ꢁ170.0(15) ꢁ168.7(13)
6 days
7 days
8 days
9 days
79.31(18)
64.76(16)
78.0(14)
55.7(13)
88.1(19)
63(2)
78.5(18)
61(2)
1
1
5
10
1
5
10
1
5
10
1
5
11.42
7.01
5.62
27.41
23.39
11.78
38.00
35.43
26.76
39.09
31.99
24.44
15.05
20.67
18.68
28.29
22.12
12.87
12.15
21.47
27.99
19.58
21.51
20.16
18.31
15.77
10.33
8.16
19.00
15.23
8.41
24.14
24.66
22.19
26.11
24.17
22.77
8.26
ꢁ167.42(13) ꢁ154.0(10) ꢁ157.5(17) ꢁ159.1(13)
169.26(13)
169.9(11)
ꢁ92.9(14)
59(2)
71(2)
59.6(14)
77.9(7)
2
34.85
27.09
18.61
32.82
30.46
10.44
10.34
5.26
O1–C12–C13–C14 ꢁ85.57(18)
C7–C6–C5–O1
C6–C5–O1–C12
ꢁ164.77(14) ꢁ160.2(11) ꢁ144.8(14) ꢁ151.2(12)
76.32(16)
83.7(13)
75(2)
71.5(19)
3
L
adopts gauche conformation in 3. The opposite sign of O1–C12–
C13–C14 torsion angles in 1 and 2 with regard to 3 is also worth
to be mentioned. Crystal packing diagrams of 1–3 (Figs. S1–S3)
do not reveal any specific intermolecular contacts in addition to
van der Waals interactions.
7.11
0.00
33.29
10
0
1.63
22.55
Control
38.73
The maximum accumulation of enzymes has been highlight in bold.
3.4. Biological studies
Table 6
The influence of 1–3 and isoconazole nitrate (L) concentrations in cultivation culture
on endoglucanase biosynthesis by Aspergillus niger CNMN FD 10.
Biological activity of complex compounds can be explained by
the presence in their composition as a complexing metal atoms
of trace elements Co, Cu, Fe, Mn, Zn, Ni, and Mo, etc. Oligoelements
interact with proteins, enzymes, vitamins or hormones and ac-
tively take part in the regulation of important biochemical pro-
cesses in living organisms [25]. Copper is one of the most
abundant metallic elements in biological systems [7]. In enzymol-
ogy, copper is somewhat similar to iron in that it function in a ser-
ies of oxidases, oxygenases and low-molecular-weight electron
transfer proteins that are reminiscent of ferredoxins. Furthermore,
one class of superoxide dismutases contains copper, as well as
polyphenoloxidases and tyrosinases. Copper is a bioactive metal
required for growth of microorganisms since it is a cofactor for
numerous enzymes. It is known that in the presence of copper
the iron absorption is facilitated as well as its incorporation into
the cytochromes [26–28]. From the foregoing, it is apparent that
studies on the biological properties of the synthesized copper coor-
dination compounds are very promising as a theoretical and a
practical point of view.
Compounds
Concentration (mg/L)
Enzyme activity (U/mL)
6 days
7 days
8 days
9 days
1
1
5
10
1
5
10
1
5
10
1
5
3.76
3.41
2.46
5.36
4.67
3.22
5.42
5.07
3.24
3.24
3.09
2.87
3.76
3.94
3.34
3.28
5.09
4.89
4.46
5.11
4.89
4.49
3.44
3.44
1.74
3.84
2.37
2.18
1.47
4.31
3.03
3.20
4.87
4.12
2.27
2.06
2.01
1.63
5.40
2.58
2.48
2.28
3.41
3.55
2.92
3.28
3.38
3.09
2.31
1.64
1.40
3.73
2
3
L
10
0
Control
The maximum accumulation of enzymes has been highlight in bold.
cule, as well as synergistic action of two or more components,
additionally to the coordination compounds 1–3 the free isocona-
zole ligand has also been screened. The results are presented in Ta-
bles 4–6. The data show that the addition of both the free
isoconazole ligand and coordination compound 1, in which Cu(II)
ions are coordinated to isoconazole and Clꢁ groups, to the cultiva-
tion medium of micromycete A. niger CNMN FD 10 causes a pro-
nounced inhibitory effect on the enzymatic activity of all types of
hydrolases (b-glucosidase, endoglucanase and xylanase) as com-
pared to the control (Figs. 3–5). The inhibitory effect increases with
increasing concentration of 1. It may be due to the presence of
chloride atoms in the ligand, as well as in the coordination sphere
of the metal in complex 1. Chloride ions and some of their com-
pounds are some of the strongest antimicrobial remedies.
In the case of addition of coordination compounds 2 and 3, in
which chloride ions coordinated to metal were replaced by carbox-
ylic groups, with a concentration of 1.0 mg/L into nutritive medium
of A. niger CNMN FD 10, a positive effect was observed. The maxi-
mum accumulation of the enzymes was detected 24–48 h earlier
and it was significantly higher than in the control. Thus, the max-
imum accumulation of b-glucosidases in the control was 2.35 U/
mL on the 7th day of cultivation and 2.36 U/mL (2), and 2.38 U/
mL (3) on the 6th day of cultivation (Fig. 3).
The biological activity of coordination compounds 1–3 towards
the biosynthesis of enzymes (b-glucosidase, xylanase and endoglu-
canase) by the fungal strain A. niger CNMN FD 10 has been tested.
Because of the evaluation and explanation of the biological activity
of a coordinative compound requires knowledge of the contribu-
tion of the metal ion, the ligand and of the integral complex mole-
Table 4
The influence of 1–3 and isoconazole nitrate (L) concentrations in cultivation culture
on b-glucosidase biosynthesis by Aspergillus niger CNMN FD 10.
Compounds
Concentration (mg/L)
Enzyme activity (U/mL)
6 days
7 days
8 days
9 days
1
1
5
10
1
5
10
1
5
10
1
5
1.62
0.56
0.36
2.38
2.31
0.23
2.36
2.25
0.44
0.22
0.14
0.11
2.09
1.87
0.92
0.36
2.20
2.05
0.15
2.05
2.02
0.89
1.82
0.75
0.61
2.35
1.30
0.51
0.38
1.58
1.71
0.81
1.69
1.63
1.42
1.54
0.54
0.20
1.75
0.29
0.30
0.28
0.75
0.72
0.47
0.20
0.56
0.35
0.06
0.05
0.06
0.72
2
3
L
10
0
Maximum xylanases biosynthesis in control (38.73 U/mL) was
observed on the 8th day of cultivation, and in optimized media –
39.09 U/mL (2) and 38.00 U/mL (3) on the 7th day of cultivation,
Control
The maximum accumulation of enzymes has been highlight in bold.

G.M. Dulcevscaia et al. / Polyhedron 52 (2013) 106–114
113
Thus, this study showed that Cu(II) coordination compounds 2
and 3 in the selected optimal concentration (1.0 mg/L) can be used
to accelerate by 24–48 h the biosynthesis of b-glucosidase, xylan-
ase and endoglucanase from micromycetes A. niger CNMN FD 10,
which is economically profitable, since it significantly reduces en-
ergy costs for industrial production of the mentioned enzymes. The
reduction of technologic cycles presents significance for biotech-
nology, offering several economic advantages – increasing the
amount of product per unit of time, reduces energy consumption
that contributes to increasing technological profitability and
reducing of final product cost. It is worth to mention that enzymes
play the most crucial roles as biocatalysts in organic synthesis
where they show greater specificities than more conventional
forms of organic reactions; in chemical industry they were used
for the synthesis of specific chemicals and polymers as well as in
developing and producing key pharmaceutical ingredients and
new therapeutic agents. [30]. Moreover, enzymes have contributed
greatly in the development of more environmentally adapted
household and industrial detergents, in textile industry, in oil
and gas drilling, in the production of biopolymers and fuel ethanol,
in paper processing, and in the food industry [31].
Fig. 3. b-Glucosidase activities obtained with the concentration of compounds of
1.0 mg/L in nutritive medium on the 6th, 7th, 8th and 9th day of cultivation.
4. Conclusions
The reaction of copper(II) salts with an antifungal agent isocon-
azole nitrate (L), for which a simplified and improved method of
synthesis was developed, affords three new Cu(II) coordination
compounds, namely mononuclear complexes [CuCl2(L)2] (1) and
[Cu(O2CMe)2(L)2]ꢀ2H2O (2), and one-dimensional coordination
polymer [Cu(pht)(L)2]n (3). Single-crystal X-ray diffraction analysis
showed that in all complexes the isoconazole is coordinated to
Cu(II) centres by a N atom of the imidazole fragment. The forma-
tion of an infinite zigzag chain through the bridging phthalate li-
gand was observed in 3. The investigation of the biosynthetic
ability of micromycetes A. niger CNMN FD 10 in the presence of
the prepared coordination compounds 1–3 as well as the anti-
fungal drug isoconazole showed that complexes 2 and 3 accelerate
the biosynthesis of enzymes (b-glucosidase, xylanase and endoglu-
canase) by this fungus and their regulatory and biostimulatory ef-
fect on the biosynthesis of enzymes can be explored. Both complex
1 and isoconazole nitrate displayed pronounced inhibitory effect
on the enzymatic activity. The potent biological activity of this
family of Cu–isoconazole compounds stresses the need for
improvements of their solubility properties and for more studies
comprising the interaction with other biologically relevant struc-
tures. The synthesis of other metal complexes with isoconazole
and the investigation of their biological properties are under way.
Fig. 4. b-Xylanase activities obtained with the concentration of compounds of
1.0 mg/L in nutritive medium on the 6th, 7th, 8th and 9th day of cultivation.
Acknowledgments
Fig. 5. b-Endoglucanase activities obtained with the concentration of compounds of
1.0 mg/L in nutritive medium on the 6th, 7th, 8th and 9th day of cultivation.
This study was supported by the Supreme Council for Science
and Technological Development of R. Moldova (Project
09.836.05.02A) and the Swiss National Science Foundation
(SCOPES IZ73Z0_127925).
which is 24 h earlier (Fig. 4). Endogluconase’s maximum accumu-
lation in the control was 5.4 U/mL (on 8th day of cultivation), but
in optimized media it was 5.42 U/mL (complex 2) and 5.36 U/mL
(complex 3) on 6th day of cultivation, reducing the biological cycle
by 48 h (Fig. 5). However, increasing the concentration of coordina-
tion compounds up to 10.0 mg/L causes a significant reduction in
enzymatic activity of all studied components of the enzymatic
complex. Perhaps this phenomenon is caused by the toxicity of
tested compounds. The results correlate with literature data and
show that toxicity is a function of concentration of the substance.
Metals are characterized by a very narrow boundary between the
concentrations necessary for vital activity of organisms and those
which are toxic to them [29].
Appendix A. Supplementary data
CCDC 869079 (1), 869077 (2), and 869078 (3) contain the crys-
tallographic data for 1–3. 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, Cam-
bridge CB2 1EZ, UK; fax: +44 1223 336 033; or e-mail: depos-
it@ccdc.cam.ac.uk. Supplementary data associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.poly.2012.10.040.

114
G.M. Dulcevscaia et al. / Polyhedron 52 (2013) 106–114
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