Synthesis and biological evaluation of organoruthenium complexes with azole antifungal agents. First crystal structure of a tioconazole metal complex

Article abstract of DOI:10.1021/om401096y

Nine organoruthenium complexes with azole antifungal agents (L) clotrimazole (ctz), tioconazole (tcz), and miconazole (mcz) with the general formulas [(η⁶-p-cymene)RuCl₂(L)], [(η⁶- p-cymene)RuCl(L)₂]Cl, and [(η⁶-p-cymene)Ru(L) ₃](PF₆)₂ were prepared and characterized by NMR, HRMS, IR, UV-vis, and X-ray crystallography. Herein, we report the first crystal structure of a tioconazole metal complex as well as the structure of the tioconazole ligand itself and the bis-clotrimazole complex as a hexafluorophosphate salt. The complexes possess a pseudooctahedral geometry typical for organoruthenium(II) compounds where half of the coordination sites are occupied by the π-bonded arene ligand p-cymene while the remaining sites are occupied by either the chlorido ligands and/or the azole ligands. The stability of the compounds in dmso solution was studied by NMR spectroscopy. The biological activity of all nine complexes and the ruthenium precursor against the fungus Culvularia lunata was evaluated. The complexes showed antifungal activity at low millimolar concentrations, where the activity decreased with the increasing number of ligands. However at 0.5 mM concentrations all tris-azole complexes statistically significantly reduced the radial growth rate, and also at 0.01 mM concentrations the monoazole complexes showed statistically significant effects. Mcz and its complexes were also tested against the human parasite Schistosoma mansoni and revealed schistocidal activity at 10-100 μg/mL in vitro.

Full text of DOI:10.1021/om401096y

Article
pubs.acs.org/Organometallics
Synthesis and Biological Evaluation of Organoruthenium Complexes
with Azole Antifungal Agents. First Crystal Structure of a Tioconazole
Metal Complex
Jakob Kljun,†,‡ Antony James Scott,§ Tea Lanisnik Rizner,⊥ Jennifer Keiser,∥,# and Iztok Turel*,†,‡
̌
̌
†Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ask
̌ ̌
erceva 5, SI-1000 Ljubljana, Slovenia
‡EN→FIST Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia
§The University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, U.K.
⊥Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia
∥Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, P.O. Box, CH−4002 Basel,
Switzerland
#University of Basel, P.O. Box, CH−4003 Basel, Switzerland
S
* Supporting Information
ABSTRACT: Nine organoruthenium complexes with azole
antifungal agents (L) clotrimazole (ctz), tioconazole (tcz), and
miconazole (mcz) with the general formulas [(η6-p-cymene)-
RuCl2(L)], [(η6-p-cymene)RuCl(L)2]Cl, and [(η6-p-cymene)-
Ru(L)3](PF6)2 were prepared and characterized by NMR,
HRMS, IR, UV−vis, and X-ray crystallography. Herein, we
report the first crystal structure of a tioconazole metal complex
as well as the structure of the tioconazole ligand itself and the
bis-clotrimazole complex as a hexafluorophosphate salt. The
complexes possess a pseudooctahedral geometry typical for
organoruthenium(II) compounds where half of the coordination sites are occupied by the π-bonded arene ligand p-cymene while
the remaining sites are occupied by either the chlorido ligands and/or the azole ligands. The stability of the compounds in dmso
solution was studied by NMR spectroscopy. The biological activity of all nine complexes and the ruthenium precursor against the
fungus Culvularia lunata was evaluated. The complexes showed antifungal activity at low millimolar concentrations, where the
activity decreased with the increasing number of ligands. However at 0.5 mM concentrations all tris-azole complexes statistically
significantly reduced the radial growth rate, and also at 0.01 mM concentrations the monoazole complexes showed statistically
significant effects. Mcz and its complexes were also tested against the human parasite Schistosoma mansoni and revealed
schistocidal activity at 10−100 μg/mL in vitro.
cases the antibacterial potency of the complex is not
significantly altered, while some of the complexes possess
discrete toxicity against certain cancer cell lines and inhibit the
activity of cathepsins, which are enzymes that are involved in
various pathological processes including cancer.3,4 Recently,
two series of novel ruthenium complexes bearing the azole
antifungal agents clotrimazole5 and ketoconazole6 were
prepared by the group of R. A. Sanchez-Delgado, and their
potential as antiparasitic agents against L. major and T. cruzi,
which are the cause of severe ailments such as leishmaniasis and
Chagas disease, was evaluated. It is known that ruthenium
complexes are active against various microorganisms that cause
tropical diseases,7 and also the organoruthenium complex with
ofloxacin, prepared by our group, displayed a modest activity in
such tests.4 It was found by R. A. Sanchez-Delgado that among
INTRODUCTION
■
Azole-type molecules are known sterol biosynthesis inhibitors,
which prevent the growth of certain fungi and parasites by
inhibiting the cytochrome P-450-dependent C-14-α demethy-
lation of lanosterol to ergosterol and cholesterol, respectively
(which are both essential components of their cell mem-
branes).1 Many of them were and some still are in clinical use,
mostly as antifungal agents.
A recurring concept in bioinorganic chemistry is the search
for synergism by combining a metal ion containing moiety and
a bioactive ligand.2 It has been proposed that binding of the
metal ion, even when the active site of the ligand seems thus
blocked, can produce positive effects on the biological activity
through various effects: different modes of action, increased
absorption, higher solubility, and even changed biological
activity. Our group has previously studied the biological
properties of organoruthenium complexes of antibacterial
agents of the quinolone family and found out that in some
Received: November 13, 2013
Published: March 18, 2014
© 2014 American Chemical Society
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Figure 1. The imidazole antifungals used in our study(from left to right) clotrimazole (ctz), tioconazole (tcz), and miconazole (mcz)with the
atom numbering used in the assignment of the NMR peaks.
Figure 2. General formulas of the mono-, bis-, and tris-azole organoruthenium species in which the azole ligands are bonded through the N3
imidazole nitrogen atom. In the case of the bis- and tris-complexes of tcz and mcz the products are mixtures of diastereomers due to the racemic
nature of the ligands.
various complexes the organoruthenium species bearing the
aromatic cymene ligand were the most effective against the two
parasites while presenting no relevant toxicity against human
cells. Nonorganometallic complexes were the least effective,
while substitution of the two chlorido ligands in the
organometallic compounds by bidentate ligands such as bipy,
acac, or en resulted in a slightly lesser activity against the
parasites.
In the last years fungi have become a great global threat to
crops, forests, and animals.8,9 More than 125 million tons of
rice, wheat, maize, potatoes, and soybeans are destroyed by
fungi every year.9 As the use of azole-based agricultural
chemicals has been associated with the increased number of
triazole-resistant fungal species affecting humans8 and azole-
based antifungal drugs used for treatment of human mycoses
have serious side effects,10 new and potent antifungal
compounds with different mechanisms of action are urgently
needed. Synthesis of ruthenium complexes of the azole ligands
was attempted to contribute in this regard.
With this in mind, we have prepared and characterized seven
novel ruthenium complexes using three different imidazole
antifungals (L = clotrimazole (ctz), miconazole (mcz),
tioconazole (tcz); Figure 1) with the general formulas [(η6-p-
cymene)RuCl2(L)], [(η6-p-cymene)RuCl(L)2]Cl, and [(η6-p-
cymene)Ru(L)3](PF6)2 (Figure 2). During our study, complex
[(η6-p-cymene)RuCl2(ctz)], reported by Sanchez-Delgado et
al.,5 and complex [(η6-p-cymene)RuCl2(mcz)], reported by
Gasser et al.,11 were also prepared; however, a different
synthetic procedure was used. Isolation of these complexes
facilitated a comparative study to investigate how different
antifungal ligands and different metal−ligand ratios may
influence the biological properties of the compounds.
On one hand, we wanted to test the antifungal potential of
the novel ruthenium compounds, as we were interested to see if
blocking the N3 atom results in the loss of the antifungal
activity. We selected a model fungal organism, Curvularia
lunata (C. lunata). This darkly pigmented, dematiaceous fungus
is a known plant and human pathogen. The plant diseases
include leaf blight on Sorghum, leaf spot on Canabis sativa, and
seedling blight on Saccharum spp.12 C. lunata is a causative
agent of human diseases in both immunocompromised and
immunocompetent individuals, such as allergic fungal sinusitis,
endocarditis, peritonitis, eumycetoma, keratitis, allergic bron-
chopulmonary disease, endophthalmitis, optic neuropathy, and
disseminated phaeohyphomycosis.13 There are limited ther-
apeutic options for the treatment of these frequently fatal
infections; thus new antimicotics are needed.
On the other hand, due to the promising results of the
ruthenium compounds against parasites causing tropical
diseases, we have decided to perform the tests for the
miconazole complexes (1c, 2c, and 3c) against the human
parasite Schistosoma mansoni (S. mansoni), which causes
schistosomiasis. Schistosomiasis, also known as bilharzia, is
the second most devastating parasitic disease after malaria, and
according to the World Health Organization,14 approximately
200 million people are infected, most of them children. The
drug of choice is praziquantel, but its widespread use may soon
result in resistance. Two vaccines are now in clinical trials,14 but
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mg ctz, (b) 51.9 mg tcz, (c) 55.7 mg mcz) were dissolved in 30 mL of
a MeOH/CHCl3 (1:1) mixture in a round-bottom flask. The color
quickly turned from red to a light orange. The reaction mixture was
stirred until complete dissolution (15−30 min) and then refluxed for
30 min, during which the solution became a deeper orange. The
reaction mixture was cooled to room temperature and stirred for an
additional 4 h, during which time the solution became a deep orange.
The solvent mixture was rotary-evaporated. The orange oily residue
was dissolved in CH2Cl2, the solvent was rotary-evaporated, and the
procedure was repeated again twice to remove traces of CHCl3 or
MeOH. A concentrated CH2Cl2 solution of the complex was then
placed in an ice bath for 10 min, and 15−20 mL of cold n-hexane was
added to produce an orange solid. The product was filtered, washed
twice with 2 mL of n-hexane, left to dry for 5 min on the vacuum
filtration device, and placed in a heater for 3 h at 45 °C (yield: 65 mg,
75% 1a; 64 mg, 68% 1b; 68 mg, 71% 1c).
there are still questions about their effectiveness. Due to these
reasons, the search for new drugs with different mechanisms of
actions for the treatment of fungal and parasitic diseases is very
important.
MATERIALS AND METHODS
■
Synthesis and Characterization. All solvents and reagents,
including the dimeric ruthenium precursor [(η6-p-cymene)Ru(μ-
Cl)Cl]2 (P1), were purchased from Sigma-Aldrich and used as
received.
X-ray diffraction data for all compounds were collected on an
Oxford Diffraction SuperNova diffractometer with Mo microfocus X-
ray source (Kα radiation, λ = 0.71073 Å) with mirror optics and an
Atlas detector. The structures were solved by direct methods
implemented in SIR9215 and refined by a full-matrix least-squares
procedure based on F2 using SHELXL-97.16 All non-hydrogen atoms
were refined anisotropically. The hydrogen atoms were placed at
calculated positions and treated using appropriate riding models. The
programs Mercury17 and Platon18 were used for data analysis and
figure preparation. Additional crystallographic data for the crystal
structures are given in Table S1 and Figures S1 and S2. The crystal
structures of compounds tcz, 1b, and 2a·2CH2Cl2 have been
submitted to the CCDC and have been allocated the deposition
numbers CCDC 971363−5 for tcz, 1b, and 2a, respectively.
UV−vis spectra were measured with Perkin-Elmer Lambda 750
UV/vis/NIR spectrometer over a range of 250 to 800 nm (Figures
S3−5). 1H NMR spectra were measured with a Bruker Avance III 500
spectrometer at room temperature. With 1H NMR the spectra
standard was set as trimethylsilane (TMS) with chemical shift at 0
ppm. Infrared spectra (ATR) were recorded on a Perkin-Elmer
Spectrum 100 FTIR spectrometer. The measurements were made in
the range 4000 to 600 cm−1. Elemental analyses were performed on a
Perkin-Elmer 204C microanalyser. HRMS were measured on an
Agilent 6224 Accurate Mass TOF LC mass spectrometer.
Antifungal Activity Test. The antifungal activities of ligands (ctz,
mcz, tcz) and their mono-, bis-, and tris-ruthenium complexes were
tested. Solid malt extract agar pH 6.5 was prepared with 0.5 and 0.01
mM (final concentrations) of azole ligands and their complexes in
dmso or with dmso only. Solutions of azole ligands and their
complexes were freshly prepared and immediately added to cooled
melted malt extract agar. The final concentration of dmso in malt
extract agar was 1%. Petri dishes were centrally inoculated in triplicates
with 5 mm mycelia disks taken from 72 h old agar culture of the
fungus C. lunata. The inoculated plates were then incubated at 26 °C,
and the growth of the inoculated mycelia was measured over the
following week. The diameter of fungal growth was plotted as a
function of time, and the radial growth rate was obtained by linear
regression (GraphPad Prism 5).19
Antischistosomal Activity Test. Adult S. mansoni were collected
from the hepatic portal veins and mesenteric veins of infected NMRI
mice 7−8 weeks postinfection. Freshly harvested schistosomes were
placed in RPMI culture medium supplemented with 5% heat-
inactivated fetal calf serum (iFCS), penicillin (100 U/mL), and
streptomycin (100 μg/m), quickly rinsed, and stored at 37 °C, 5%
CO2 until usage. The miconazole complexes 1−3c were selected for
these tests as model compounds, and stock solutions (10 mg/mL) of
1c, 2c, and 3c as well as the ruthenium precursor P1 were prepared
using dmso. Experiments were conducted in duplicate. Three to five
adult S. mansoni were incubated in 24-flat-bottom-well plates ((BD
Falcon, USA) in the presence of 1−100 μg/mL of the test drugs at 37
°C, 5% CO2. Schistosomes incubated in the presence of blank medium
supplemented with the highest concentration of dmso served as
control. Seventy-two hours post drug exposure schistosomes were
examined phenotypically using a motility scale ranging from 3 (worms
active and healthy) to 1 (worms dead) and an inverse microscope
(Carl Zeiss, Germany, magnification 80×).
[(η6-p-Cymene)RuCl2(ctz)] (1a). 1H NMR (CDCl3, 500.10 MHz): δ
7.83 (t, 1H, J = 1.3 Hz, H2 ctz), 7.45 (dd, 1H, J = 7.9, 1.4 Hz, Hc ctz),
7.38 (dd, 1H, J = 7.5, 1.5 Hz, Hd ctz), 7.35 (dd, 7H, J = 4.2, 2.2 Hz, Ha,
H4 ctz), 7.29 (dd, 1H, J = 7.9, 1.3 Hz, He ctz), 7.15−7.09 (m, 4H, Hb
ctz), 7.01 (dd, 1H, J = 8.0, 1.5 Hz, Hf ctz), 6.82 (t, 1H, J = 1.7 Hz, H5
ctz), 5.31 (d, 2H, J = 6.0 Hz, Ar-H cym), 5.12 (d, 2H, J = 5.9 Hz, Ar-H
cym), 2.86 (dq, 1H, J = 13.8, 7.0 Hz, Ar-CH-(CH3)2, cym), 2.09 (s,
3H, Ar-CH3 cym), 1.19 (d, 6H, J = 6.9 Hz, Ar-CH-(CH3)2 cym) ppm.
Selected IR bands (cm−1, ATR): 3499, 2959, 1445, 1492, 1220, 1042,
1087, 750, 709. UV−vis (λ [nm], ε [L mol−1 cm−1] for c = 1 × 10−3
M, MeOH): 328.5 (1166.0), 406.2 (672.2). HRMS-ESI (m/z): [M −
Cl]+ calcd for C32H31Cl2N2Ru 615.0908, found 615.0906. Anal. Calcd
for C32H31Cl3N2Ru: C, 59.04; H, 4.80; N, 4.30. Found: C, 58.84; H,
4.80; N, 4.32.
[(η6-p-Cymene)RuCl2(tcz)] (1b). 1H NMR (CDCl3, 500.10 MHz): δ
7.87 (s, 1H, H2 tcz), 7.43 (d, 1H, J = 1.9 Hz, Ha tcz), 7.34 (d, 1H, J =
8.4 Hz, Hb tcz), 7.30 (dd, 1H, J = 8.4, 1.9 Hz, Hc tcz), 7.26 (s, 1H,
CDCl, H4 tcz, overlapping with solvent peak), 7.12 (d, 1H, J = 5.7 Hz,
Hd tcz), 6.91 (d, 1H, J = 5.7 Hz, He tcz), 6.84 (t, 1H, J = 1.4 Hz, H5
tcz), 5.39 (dd, 2H, J = 5.3, 2.6 Hz, Ar-H cym), 5.19 (d, 2H, J = 5.9 Hz,
Ar-H cym), 4.93 (dd, 1H, J = 7.5, 2.8 Hz, Hf tcz), 4.40 (d, 1H, J = 11.8
Hz, Hg tcz), 4.27 (d, 1H, J = 11.8 Hz, Hg tcz), 4.10 (dd, 1H, J = 14.5,
2.8 Hz, Hh tcz), 3.98 (dd, 1H, J = 14.5, 7.6 Hz, Hh tcz), 2.96 (hept, 1H,
J = 6.9 Hz, Ar-CH-(CH3)2, cym), 2.14 (s, 3H, Ar-CH3 cym), 1.28 (d,
6H, J = 6.9, 1.7 Hz, Ar-CH-(CH3)2 cym). Selected IR bands (cm−1,
ATR): 3674, 3072, 3039, 2959, 2870, 1525, 1467, 1238, 1382, 1089,
1028, 823, 746, 657. UV−vis (λ [nm], ε [L mol−1 cm−1] for c = 1 ×
10−3 M, MeOH): 327.1 (1070.0), 408.7 (636.6). HRMS-ESI (m/z):
[M − Cl]+ calcd for C26H27Cl4N2ORuS 658.9612, found 658.9620.
Anal. Calcd for C26H27Cl5N2ORuS: C, 45.00; H, 3.92; N, 4.04. Found:
C, 45.33; H, 4.07; N, 3.97.
[(η6-p-Cymene)RuCl2(mcz)] (1c). 1H NMR (CDCl3, 500.10 MHz):
δ 7.91 (s, 1H, H2 mcz), 7.45 (d, 1H, J = 0.9 Hz, Ha mcz), 7.38 (d, 1H,
J = 4.4 Hz, Hb mcz), 7.36−7.33 (m, 2H, Hc mcz), 7.30 (d, 2H, J = 1.7
Hz, Hd mcz), 7.27 (s, 1H, H4 mcz, overlapping with solvent peak),
6.84 (s, 1H, H5 mcz), 5.39 (d, 2H, J = 5.7 Hz, Ar-H cym), 5.20 (d, 2H,
J = 5.2 Hz, Ar-H cym), 5.02 (dd, 1H, J = 7.4, 2.8 Hz, He mcz), 4.48 (d,
1H, J = 12.3 Hz, Hf mcz), 4.37 (d, 1H, J = 12.3 Hz, Hf mcz), 4.16 (dd,
1H, J = 14.6, 2.8 Hz, Hg mcz), 4.06 (dd, 1H, J = 14.6, 7.4 Hz, Hg mcz),
2.95 (m, 1H, Ar-CH-(CH3)2, cym), 2.15 (s, 3H, Ar-CH3 cym), 1.26 (d
6H, J = 6.9 Hz, Ar-CH-(CH3)2 cym) ppm. Selected IR bands (cm−1,
ATR): 3667, 2971, 2901, 1561, 1471, 1250, 1090, 1044, 864, 789.
UV−vis (λ [nm], ε [L mol−1 cm−1] for c = 1 × 10−3 M, MeOH):
327.5 (1089.3),406.6 (648.6). HRMS-ESI (m/z): [M − Cl]+ calcd for
C28H28Cl5N2ORu 686.9658, found 686.9663. Anal. Calcd for
C28H28Cl6N2ORu: C, 46.56; H, 3.91; N, 3.88. Found: C, 46.30; H,
3.72; N, 4.01.
Bis-azole Complexes with General Formula [(η6-p-Cymene)RuCl-
(L)2]Cl (2a−c). A 40.0 mg (0.065 mmol) amount of P1 and 4.10 equiv
of the azole ligand (0.2678 mmol; (a) 92.4 mg ctz, (b) 103.9 mg tcz,
(c) 111.5 mg mcz) were dissolved in 30 mL of a MeOH/CHCl3 (1:1)
mixture in a round-bottom flask. The color of the reaction mixture
turned from red to a light yellow. The reaction mixture was stirred
until complete dissolution (15−30 min) and then refluxed for 5 h,
Synthetic Procedures. Monoazole Complexes with General
Formula [(η6-p-Cymene)RuCl2(L)] (1a−c). A 40.0 mg (0.065 mmol)
amount of P1 and 2.05 equiv of the azole ligand (0.134 mmol; (a) 46.2
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during which the solution became yellow. The solvent mixture was
rotary-evaporated. The orange oily residue was dissolved in CH2Cl2,
the solvent was rotary-evaporated, and the procedure was repeated
again twice to remove traces of CHCl3 or MeOH. A concentrated
CH2Cl2 solution of the complex was then placed in an ice bath for 10
min, and 15−20 mL of cold n-hexane was added to produce a pale
orange solid. The product was filtered, washed twice with 2 mL of n-
hexane, left to dry for 5 min on the vacuum filtration device, and
placed in a heater for 3 h at 45 °C (yield: 118 mg, 89% 2a; 129 mg,
82% 2b; 115 mg, 77% 2c).
7.03−6.98 (m, 6H, Ar-H ctz), 6.96 (d, 12H, Ar-H ctz), 6.85 (s, 3H. Ar-
H ctz), 6.21 (d, 2H, Ar-H cym), 6.04 (d, 2H, Ar-H cym), 2.01 (dt, 1H,
Ar-CH-(CH3)2, cym), 1.82 (s, 3H, Ar-CH3 cym), 0.95 (d, 6H, Ar-CH-
(CH3)2 cym). Selected IR bands (cm−1, ATR): 2972, 2901, 1447,
1493, 1218, 1087, 1049, 835, 749, 710. UV−vis (λ [nm], ε [L mol−1
cm−1] for c = 1 × 10−3 M, MeOH): 309.4 (948.6), 380.7 (657.9).
HRMS-ESI (m/z): [M + 2]2+ calcd for C76H65Cl3N6Ru 1270.3350,
found 1270.3420. Anal. Calcd for C76H65Cl3F12N6P2Ru: C, 58.52; H,
4.20; N, 5.39. Found: C, 58.63; H, 4.37; N, 5.51.
[(η6-p-Cymene)Ru(tcz)3](PF6)2 (3b). 1H NMR (CDCl3, 500.10
MHz): δ 8.14 (d, 3H, H2 tcz), 7.40 (m, 3H, Ar-H tcz), 7.33 (m,
3H, Ar-H tcz), 7.26 (overlapping with CHCl3, s, 6H, Ar-H tcz), 7.10
(m, 3H, Ar-H tcz), 7.02 (m, 3H, Ar-H tcz), 6.81 (m, 3H, Ar-H tcz),
5.68−5.51 (m, 4H, Ar-H cym), 5.06 (d, 3H, Hf tcz), 4.46−4.29 (m,
12H, Hg/h tcz), 2.03 (m, 1H, Ar-CH-(CH3)2, cym), 1.48 (s, 3H, Ar-
CH3 cym), 0.97 (m, 6H, Ar-CH-(CH3)2 cym) ppm. Selected IR bands
(cm−1, ATR): 2870, 1526, 1470, 1245, 1096, 836, 739. UV−vis (λ
[nm], ε [L mol−1 cm−1] for c = 1 × 10−3 M, MeOH): 306.2 (914.8),
[(η6-p-Cymene)RuCl(ctz)2]Cl (2a). 1H NMR (CDCl3, 500.10
MHz): δ 7.89 (s, 2H, H2 ctz), 7.47 (s, 2H, Hc ctz), 7.39 (dd, 2H,
H4 ctz), 7.37 (dd, 2H, Hd ctz), 7.35−7.27 (m, 12H, Ha ctz), 7.22−7.17
(m, 2H, He ctz), 6.98−6.92 (m, 8H, Hb, ctz), 6.89−6.86 (m, 2H, Hf
ctz), 6.85 (t, 2H, H5 ctz), 5.69 (d, 2H, Ar-H cym), 5.50 (d, 2H, Ar-H
cym), 2.47 (sept, 1H, Ar-CH-(CH3)2, cym), 1.81 (s, 3H, Ar-CH3
cym), 1.10 (d, 6H, Ar-CH-(CH3)2 cym) ppm. Selected IR bands
(cm−1, ATR): 3670, 2970, 1446, 1492, 1218, 1087, 836, 749. UV−vis
(λ [nm], ε [L mol−1 cm−1] for c = 1 × 10−3 M, MeOH): 322.2
(953.8), 395.2 (596.7). HRMS-ESI (m/z): [M + 2]+ calcd for
C54H48Cl3N4Ru: 961.1954, found 961.1976. Anal. Calcd for
C54H48Cl4N4Ru: C, 65.13; H, 4.86; N, 5.53. Found: C, 65.35; H,
4,78; N, 5.37.
380.3 (665.4). HRMS-ESI (m/z): [M
+
4]2+ calcd for
C58H53Cl9N6O3RuS3 1397.9523, found 1397.9566. Anal. Calcd for
C58H53Cl9F12N6O3P2RuS3: C, 41.26; H, 3.16; N, 4.98. Found: C,
41.11; H, 3.37; N, 4.79.
[(η6-p-Cymene)Ru(mcz)3](PF6)2 (3c). 1H NMR (CDCl3, 500.10
MHz): δ 8.19−8.11 (m, 3H, H2 mcz), 7.57−7.42 (m, 3H, Ar-H mcz),
7.44−7.27 (m, overlapping with CHCl3, 12H, Ar-H mcz), 6.88−6.78
(m, 3H, Ar-H mcz), 5.98−5.87 (m, 4H, Ar-H cym), 5.64−5.49 (m,
6H, Hd mcz) 5.12 (dt, 3H, Hc mcz), 4.54−4.26 (m, 12H, Hd mcz),
1.96−1.83 (m, 1H, Ar-CH-(CH3)2, cym), 1.39 (s, 3H, Ar-CH3 cym),
0.90−0.86 (m, 6H, Ar-CH-(CH3)2 cym) ppm. Selected IR bands
(cm−1, ATR): 3146, 2974, 2901, 1523, 1473 1246, 1093, 1044, 838,
740. UV−vis (λ [nm], ε [L mol−1 cm−1] for c = 1 × 10−4 M, MeOH):
308.7 (1049.8), 379.9 (778.0). HRMS-ESI (m/z): [M + 6]2+ calcd for
C64H56Cl12N6O3Ru 1483.9631, found 1483.9662. Anal. Calcd for
C64H56Cl12F12N6O3P2Ru: C, 43.34; H, 3.18; N, 4.74. Found: C, 42.99;
H, 3.38; N, 4.61.
[(η6-p-Cymene)RuCl(tcz)2]Cl (2b). 1H NMR (CDCl3, 500.10
MHz): δ 8.05 (d, 1H, H2 tcz), 7.98 (d, 1H, H2 tcz), 7.42−7.34 (m,
3H, Ar-H tcz), 7.28 (m, 1H, Ar-H tcz), 7.25 (m, 4H, Ar-H tcz), 7.10
(m, 2H, Ar-H tcz), 6.94 (m, 1H, Ar-H tcz), 6.80 (m, 2H, Ar-H tcz),
6.66 (d, 1H, Ar-H tcz), 5.58 (m, 2H, Ar-H cym), 5.41 (m, 2H, Ar-H
cym), 4.94 (dd, 2H, Hf tcz), 4.38−4.07 (m, 8H, Hg/h tcz), 2.28 (sept,
1H, Ar-CH-(CH3)2, cym), 1.63 (s, 3H, Ar-CH3 cym), 1.08 (m, 6H, Ar-
CH-(CH3)2 cym) ppm. Selected IR bands (cm−1, ATR): 3140, 2926,
1522, 1469, 1242, 1088, 1032, 835, 738. UV−vis (λ [nm], ε [L mol−1
cm−1] for c = 1 × 10−3 M, MeOH): 320.7 (1009.6), 397.7 (638.3).
HRMS-ESI (m/z): [M + 4]+ calcd for C42H40Cl7N4O2RuS2
1046.9392, found 1046.9418. Anal. Calcd for C42H40Cl8N4O2RuS2:
C, 46.64; H, 3.73; N, 5.18. Found: C, 46.29; H, 3.84; N, 4.98.
[(η6-p-Cymene)RuCl(mcz)2]Cl (2c). 1H NMR (CDCl3, 500 MHz): δ
9.12 (dd, 2H, H2 mcz), 7.61 (d, 2H, Ar-H mcz), 7.55 (dd, 2H, Ar-H
mcz), 7.49−7.16 (m, 10H, Ar-H mcz), 6.72 (dd, 2H, Ar-H mcz),
5.85−5.69 (m, 4H, Ar-H cym), 5.06 (m, 2H, Hd mcz), 4.55−4.03 (m,
8H, He mcz), 2.30−2.15 (m, 1H, Ar-CH-(CH3)2, cym), 1.59 (d, 3H,
Ar-CH3 cym), 1.02 (m, 6H, Ar-CH-(CH3)2 cym) ppm. Selected IR
bands (cm−1, ATR): 3144, 2973, 2901, 1589, 1471, 1244, 1092, 1044,
838, 740. UV−vis (λ [nm], ε [L mol−1 cm−1] for c = 1 × 10−3 M,
MeOH): 321.1 (1011.0), 398.8 (652.3). HRMS-ESI (m/z): [M + 4]+
calcd for C46H42Cl9N4O2Ru 1102.9489, found 1102.9494. Anal. Calcd
for C46H42Cl10N4O2Ru: C, 48.53; H, 3.72; N, 4.92. Found: C, 48.41;
H, 3.97; N, 4.73.
RESULTS AND DISCUSSION
■
Syntheses and Characterization. The three types of
complexes were prepared by fairly similar procedures, in which
the reaction was carried out in a methanol/chloroform mixture,
which offers a high solubility for most organoruthenium
precursors and ligands. For the preparation of monoazole
compounds, the literature offers various synthetic procedures
concerning the choice of solvent (acetone, dichloromethane,
toluene), the reaction conditions, and time (1−16 h).5,20,21 The
group of Sanchez-Delgado reports the synthesis of the
clotrimazole complex 1a as a product of a six-hour reaction
at room temperature in acetone, followed by the precipitation
with Et2O,5 while Gasser et al.11 reported the synthesis of the
monomiconazole complex 1c as a result of a prolonged 20 h
reaction in acetone. We discovered that the use of acetone in
our case was not suitable, since the monoazole compounds
were found to decompose over time, resulting in darker, brown
solutions, which required purification. The decomposition was
avoided by refluxing the MeOH/CHCl3 reaction mixture for 30
min, which jump-started the reaction. The reaction mixture was
then stirred until the completion of the reaction, which was
monitored by TLC. Generally the reactions were complete after
about 2 h, but occasionally additional stirring up to 4 h was
necessary. Moreover, compound 1c was not fully characterized,
as it was reported as unstable in solvents commonly used in
NMR spectroscopy. We have found that some organo-
ruthenium-azole complexes are indeed unstable in solution;
however the compounds can be stabilized for a period of time
by using fresh deuterated solvents and avoiding exposure to
direct sunlight. The photostability of the complexes was not
Tris-azole Complexes with General Formula [(η6-p-Cymene)Ru-
(L)3](PF6)2 (3a−c). A 40.0 mg (0.0663 mmol) amount of P1 and 6.15
equiv of the azole ligand (0.4017 mmol; (a) 138.5 mg ctz, (b) 155.7
mg tcz, (c) 167.2 mg mcz) were dissolved in 30 mL of a MeOH/
CHCl3 (1:1) mixture in a brown round-bottom flask. Soon after
dissolution the color turned from red to yellow. A 68.7 mg (0.272
mmol, 4.10 equiv) amount of AgPF6 was added, and the reaction
mixture was refluxed for 3 h, during which the solution became a very
pale yellow. The solvent was evaporated and the oily residue dissolved
in CH2Cl2. The precipitate AgCl was filtered over Celite, and the
mother solution was evaporated. The dark yellow oily residue was
dissolved in CH2Cl2, the solvent was rotary-evaporated, and the
procedure was repeated again twice to remove traces of CHCl3 or
MeOH. A concentrated CH2Cl2 solution of the complex was then
placed in an ice bath for 10 min, and 15−20 mL of cold n-hexane was
added to produce a yellow solid. The product was filtered with a
vacuum filtration apparatus, washed twice with 2 mL of n-hexane, left
to dry for 5 min on the vacuum filtration device, and placed in a heater
for 3 h at 45 °C (yield: 179 mg, 86% 3a; 181 mg, 80% 3b; 197 mg,
83% 3c).
[(η6-p-Cymene)Ru(ctz)3](PF6)2 (3a). 1H NMR (CDCl3, 500.10
MHz): δ 7.53 (s, 3H, H2 ctz), 7.35−7.20 (m, 27H, Ar-H ctz),
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evaluated; however the photoinduced release of monodentate
nitrogen ligands was observed in the case of structurally similar
(organo)ruthenium complexes22 with a rate and speed that are
strongly dependent on the ruthenium coordination sphere.
Crystallizations were attempted by slowly evaporating the
compound solutions in solvent mixtures containing a
chlorinated solvent, in which the compounds are well soluble
(CH2Cl2, CHCl3), and nonpolar solvents such as n-hexane,
cyclohexane, n-heptane, or toluene. Crystals of compound 1b
were obtained by the slow evaporation of a CH2Cl2/n-hexane
mixture, and herein we report the first crystal structure of a
tioconazole metal complex as well as the crystal structure of the
azole ligand itself.
Bis-azole ruthenium complexes were previously reported by
Dyson and Vock in two articles, one with substituted
imidazoles and one with anthracene- or phenoxazine-function-
alized ligands.20,21 These syntheses were carried out in
methanol, 2-propanol, or n-butanol at elevated temperatures
and with 2-fold excess of the ligand, which was needed to be
later removed by extraction using ethyl acetate. In our case the
use of a methanol/chloroform mixture needed only a slight
excess (2−3%) of the ligand and no further purification. In
order to obtain crystals suitable for X-ray diffraction, analogous
compounds containing different counterions were prepared by
the addition of 10−20 molar equiv of the appropriate salt
(NaBF4, KPF6, NaBPh4) to the initial reaction mixture and
subsequent filtration of the precipitated inorganic salts from a
CH2Cl2 solution. In the case of complex 2a, [(η6-p-cymene)-
RuCl(ctz)2](PF6) crystals were obtained by the slow
evaporation of a CH2Cl2/n-hexane solution.
compounds was thus further assessed by elemental analyses and
high-resolution mass spectrometry.
Early studies by Vock et al.21 do not report any issues
concerning the stability of structurally similar complexes in
aqueous media while performing biological assays; however,
Gasser et al.11 recently demonstrated that dissolution of
organoruthenium complexes bearing one monodentate nitro-
gen ligand (with general formula [(η6-p-cymene)Ru(N-ligand)-
Cl2]) can induce the precipitation of the ligand and formation
of the [(η6-p-cymene)Ru(S-dmso)Cl2] species. Interestingly,
parallel cytotoxicity experiments have shown that organo-
ruthenium complexes bearing one nitrogen ligand show
remarkably similar cytotoxic effects to the combination of the
ruthenium-dmso adduct and the free ligand in 1:1 ratio, which
are, however, inactive if applied separately. Since our
compounds have relatively low aqueous solubility and bio-
logical experiments were performed by diluting concentrated
dmso stock solutions with water in order to obtain the desired
concentrations, the stability of the complexes in soliution was
investigated. Our studies confirm that the monoazole
compounds indeed partially dissociate in dmso, resulting in a
mixture of the monoazole- and dmso-ruthenium species. The
bis- and tris-azole complexes however appear stable in dmso
and aqueous solution with a minority (<3%) of decomposition
products appearing after 12 h (Figures S15−18).
Crystal Structures. The crystals of complex 1b were
obtained by slow evaporation of the CH2Cl2/n-hexane (1:1)
solution, which afforded dark yellow needles. So far, this is the
first reported tioconazole metal complex. Complex 2a was
crystallized as a hexafluorophosphate salt. Crystals were
obtained by the slow evaporation of the CH2Cl2/n-hexane
(1:1) solution, which afforded orange needles. Crystals of the
ligand tcz were obtained by the slow evaporation of the
chloroform solution. Crystals of complexes 1b and 2a were
unstable in air due to the presence of volatile solvent molecules
in the crystal structure. In the case of 2a, two dichloromethane
molecules could be located from the Fourier electron density
map, while in the case of 1b the disorder was too severe and the
residual electron density was removed using the SQUEEZE
routine within Platon. The software found a solvent-accessible
void of 310 Å3 and with the electron count of 61, which roughly
corresponds to two dichloromethane molecules.
The synthesis of the tris-azole complexes required the use of
silver salts in order to remove the chlorido ligands from the
reaction mixture, as even the use of 20 molar equiv of the azole
ligands affords the bis-azole complexes as proven by NMR
spectra (Figure 3).
Due to the racemic nature of the tcz and mcz ligands,
complexes 2b, 2c, 3b, and 3c are mixtures of diastereomers/
enantiomers that can be seen in the NMR spectra, as the
multiplets are less defined (Figures S6−14). The purity of the
The crystal structure of ligand tcz (Figure 4) does not
present any strong intermolecular interactions (H-bonds, π-
stacking). However, a weak hydrogen bond between an
aromatic hydrogen on the dichloro-substituted phenyl group
and the thienyl sulfur of the adjacent molecule is observed. This
S···H distance has a value of 2.81(1) Å, which is considerably
smaller than the sum of the van der Waals radii of the two
atoms (3.00 Å). The interaction is further confirmed by the
near coplanarity of the thienyl and dichlorophenyl moieties (φ
= 0.69°) (Figure S1).
The structure of compound 1b (Figure 5) presents a
ruthenium complex with a pseudooctahedral “piano-stool”
geometry with the aromatic p-cymene ligand π-coordinated to
the ruthenium(II) ion. The coordination sphere is completed
with two chlorido ligands and the tioconazole ligand, which is
bound to ruthenium through the imidazole nitrogen atom N3.
The molecules link into dimers about an inversion center by
two pairs of p-cymene C−H···Cl hydrogen bonds with H···Cl
distances of 2.733(1) and 2.716(1) Å, which is considerably less
than the sum of van der Waals radii of the two atoms (2.95 Å).
The distance between the two ruthenium ions in the dimer is
Figure 3. 1H NMR spectra (in CDCl3) in the region pertaining to the
aromatic protons of the ruthenium-bound cymene ligand: (a)
ruthenium precursor P1, (b) compound 1a, (c) compound 2a, (d)
product of the reaction between the precursor P1 and 20 molar equiv
of ctz, (e) compound 3a, synthesized using the silver salt AgPF6.
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Figure 4. Crystal structure of ligand tcz with heteroatom labeling.
Thermal ellipsoids are drawn at the 30% probability level.
Figure 6. Crystal structure of compound 2a with heteroatom labeling.
Thermal ellipsoids are drawn at the 30% probability level. All hydrogen
atoms, the hexafluorophosphate anion, and the cocrystallized solvent
molecules are omitted for clarity.
and their mono-, bis-, and tris-ruthenium complexes on the
fungal growth, we observed more potent antifungal activity for
azole ligands in comparison with their ruthenium complexes
(Table 2; Figure S19). Antifungal activities decreased with the
Table 2. Antifungal Activities of Azole Ligands and Their
Ruthenium Complexes
a
radial growth rate (mm/h)
compound
0.5 mM
0.01 mM
dmso only
0.36
0.01
0.13
0.17
0.16
0.04
0.14
0.15
0.19
0.04
0.09
0.13
0.13
0.40
0.12
0.30
0.33
0.39
0.12
0.17
0.19
0.27
0.09
0.15
0.27
0.27
Figure 5. Crystal structure of compound 1b with heteroatom labeling.
Thermal ellipsoids are drawn at the 30% probability level. Hydrogen
atoms are omitted for clarity.
ctz
1a
2a
3a
5.517(3) Å. The bond lengths and angles are comparable to
those from the previously published clotrimazole analogue
(Table 1).5
mcz
1b
2b
3b
tcz
1c
Compound 2a is a cationic organoruthenium complex in
which two ctz ligands are bonded to the ruthenium ion through
the imidazole nitrogen atom N3. A chlorido ligand and the
aromatic cymene ring complete the coordination sphere around
the metal center with PF6− acting as counterion (Figure 6). The
complex crystallized with two molecules of dichloromethane,
which was used as a solvent in the crystallization process.
Addition of a second clotrimazole ligand did not provoke
drastic changes in the conformation of the molecule since the
bond lengths (Table 1) and angles around the central metal ion
remain virtually unchanged. Moreover, in both complexes’
crystal structures the coordination to the metal ion did not
provoke changes in the bond lengths of the imidazole ring.
Antifungal Activity. The isolated ruthenium complexes
showed antifungal activity at low millimolar concentrations.
When we followed the effects of the individual azole ligands
2c
3c
a
Diameters of fungal colonies were measured daily and plotted as a
function of time; radial growth rate was calculated by linear regression.
increasing number of ligands in these complexes. However, at
0.5 mM concentrations the tris-ruthenium complexes 3a−c still
significantly reduced the radial growth rate by 2.3-, 1.9-, and
2.8-fold, as compared to the control, respectively (Figure S20).
Also at 0.01 mM concentrations a 2.3-, 2.4-, and 2.7-fold
decline in the growth rate was observed for 1a−c, respectively
(Figure S21). Tris-complexes were less potent at 0.01 mM
Table 1. Selected Bond Lengths in the Compounds [(η6-p-cymene)Ru(ctz)Cl2] (1a), [(η6-p-cymene)Ru(tcz)Cl2] (1b), and [(η6-
p-cymene)Ru(ctz)2Cl](PF6) (2a)
1a5
2.098(2)
1b
2.102(4)
2a
Ru−N3
Ru−Cl
Ru−Cymcentroid
2.107(4)/2.111(4)
2.400(2)
2.412(1)/2.399(1)
1.664(2)
2.430(2)/2.421(2)
1.657(2)
1.655(2)
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concentrations, but 3b and 3c still showed a 1.5-fold decrease
in the growth rate.
reported, as the equilibrium between the decomposition
products is reached in about 12 h. The bis-azole complexes
2a−c and the tris-azole complexes 3a−c are on the other hand
stable in dmso solutions, as a minority of decomposition
species (<2%) are observed only after 12 h. All in all, the
ruthenium complexes statistically significantly reduced the
fungal growth rate at 0.5 mM concentration, and the
monoazole complexes also did so at 0.01 mM concentrations.
In azole ruthenium complexes N3 atoms cannot bind to the
heme iron within the active site of lanosterol 14α-demethylase,
which suggests a different mechanism of inhibition. The
miconazole complexes have shown moderately good anti-
schistosomal activity, which decreases with the number of
coordinated azole ligands. However, the gender selectivity
displayed by compound 3c is to the best of our knowledge the
first report of such a phenomenon. Given the interesting
antischistosomal activities of these compounds, further studies
(in vivo studies) should be launched.
The azole antifungal compounds inhibit the fungal growth by
preventing ergosterol biosynthesis via coordination of their N3
atom to the heme iron within the active site of lanosterol 14α-
demethylase.1 In the mono-, bis-, and tris-azole ruthenium
complexes N3 atoms are bound to ruthenium, thus suggesting
that the azole ligand complexes inhibit the fungal growth via
other mechanisms of action, which may not involve lanosterol
14α-demethylase.1
Antischistosomal Activity. No activity was observed
following the incubation of adult S. mansoni with precursor
P1 for 72 h at a 100 μg/mL concentration (Figure 7). On the
ASSOCIATED CONTENT
* Supporting Information
■
S
Supporting Information contains crystallographic data, UV−vis
spectra, 1H NMR spectra, and additional information on
antifungal activity tests. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: iztok.turel@fkkt.uni-lj.si.
■
Figure 7. Activity of compounds 1−3c and the ruthenium precursor
P1 against adult Schistosoma mansoni at 100 μg/mL over 72 h.
Notes
The authors declare no competing financial interest.
other hand the miconazole ligand showed activity at 10 μg/mL
following 24 h of incubation and all worms had died (data not
shown). Sex-specific activity was documented for compound
3c: while female worms incubated with 100 μg/mL compound
3c for 72 h showed only slightly decreased motility, male
worms had died. All worms exposed to 100 μg/mL
concentration of compounds 1c and 2c had died 24−48 h
postincubation. Incubation with lower concentrations (1 and 10
μg/mL) of compounds 1c and 2c resulted in decreased
activities of adult S. mansoni. Worms incubated in the presence
of dmso (controls) remained active and had a healthy
appearance during the entire examination period (Figure 7).
ACKNOWLEDGMENTS
■
We are grateful for the financial support from Project J1-4131
and Program Grants P1-0175 (I.T.) and P1-0170 (T.L.R.) and
the Junior Researcher Grant for J. Kljun, all funded by the
Slovenian Research Agency (ARRS). We thank the EN→FIST
Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, for the
use of the SuperNova diffractometer. A.J.S. is also grateful for
the Erasmus grant, which enabled his 9-month stay in Ljubljana.
J. Keiser is financially supported by the Swiss National Science
Foundation (SNSF; projects nos. PPOOA-114941 and
PPOOP3_135170).
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