1-(2-Picolyl)-substituted 1,2,3-triazole as novel chelating ligand for the preparation of ruthenium complexes with potential anticancer activity

Article abstract of DOI:10.1039/c0dt01807d

The 1,4-disubstituted 1,2,3-triazole ligand prepared by click chemistry 1-(2-picolyl)-4-phenyl-1H-1,2,3-triazole (ppt) was investigated as novel chelating ligand for Ru(ii) complexes with potential antitumor activity. The preparation and structural characterization, mainly by NMR spectroscopy in solution and by X-Ray crystallography in the solid state, of four new Ru(ii) complexes is reported: two isomeric Ru-dmso compounds, trans,cis-[RuCl ₂(dmso-S)₂(ppt)] (1) and cis,cis-[RuCl₂(dmso-S) ₂(ppt)] (2), and two half-sandwich Ru-[9]aneS₃ coordination compounds, [Ru([9]aneS₃)(dmso-S)(ppt)][CF ₃SO₃]₂ (3) and [Ru([9]aneS₃)Cl(ppt)] [CF₃SO₃] (4). In all compounds ppt firmly binds to ruthenium in a bidentate fashion through the pyridyl nitrogen atom and the triazole N2, thus forming a puckered six-membered ring. The chemical behavior in aqueous solution of the water-soluble complexes 3 and 4 was studied by UV-Vis and NMR spectroscopy and compared to that of the previously described organometallic analogue [Ru(η⁶-p-cymene)Cl(ppt)][Cl] (5) in view of their potential antitumor activity. Compounds 3-5 were tested also in vitro for cytotoxic activity against two human cancer cell lines, one sensitive and one resistant to cisplatin, in comparison with cisplatin. Compound 4, the one that aquates faster, was found to be more cytotoxic than cisplatin against human lung squamose carcinoma cell line (A-549).

Full text of DOI:10.1039/c0dt01807d

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PAPER
1-(2-Picolyl)-substituted 1,2,3-triazole as novel chelating ligand for the
preparation of ruthenium complexes with potential anticancer activity†
Ioannis Bratsos,*a Damijana Urankar,b Ennio Zangrando,a Petia Genova-Kalou,c Janez Kosˇmrlj,b Enzo Alessioa
and Iztok Turel*b
Received 22nd December 2010, Accepted 24th February 2011
DOI: 10.1039/c0dt01807d
The 1,4-disubstituted 1,2,3-triazole ligand prepared by click chemistry 1-(2-picolyl)-4-phenyl-1H-
1,2,3-triazole (ppt) was investigated as novel chelating ligand for Ru(II) complexes with potential
antitumor activity. The preparation and structural characterization, mainly by NMR spectroscopy in
solution and by X-Ray crystallography in the solid state, of four new Ru(II) complexes is reported: two
isomeric Ru-dmso compounds, trans,cis-[RuCl2(dmso-S)2(ppt)] (1) and cis,cis-[RuCl2(dmso-S)2(ppt)] (2),
and two half-sandwich Ru-[9]aneS3 coordination compounds, [Ru([9]aneS3)(dmso-S)(ppt)][CF3SO3]2 (3)
and [Ru([9]aneS3)Cl(ppt)][CF3SO3] (4). In all compounds ppt firmly binds to ruthenium in a bidentate
fashion through the pyridyl nitrogen atom and the triazole N2, thus forming a puckered six-membered
ring. The chemical behavior in aqueous solution of the water-soluble complexes 3 and 4 was studied by
UV-Vis and NMR spectroscopy and compared to that of the previously described organometallic
6
analogue [Ru(h -p-cymene)Cl(ppt)][Cl] (5) in view of their potential antitumor activity. Compounds 3–5
were tested also in vitro for cytotoxic activity against two human cancer cell lines, one sensitive and one
resistant to cisplatin, in comparison with cisplatin. Compound 4, the one that aquates faster, was found
to be more cytotoxic than cisplatin against human lung squamose carcinoma cell line (A-549).
ind = indazole), in clinical trials.3,4 These two complexes display
Introduction
an activity that is markedly different from that of cisplatin and the
In the development of novel non-platinum metal anticancer drugs
other established platinum anticancer chemotherapeutics: NAMI-
with improved properties, ruthenium compounds occupy a promi-
nent position,1,2 especially after the introduction of two Ru(III)
coordination compounds, NAMI-A ([imH]trans-[RuCl4(dmso-
S)(im)], im = imidazole) and KP1019 ([indH]trans-[RuCl4(ind)2],
A shows negligible cytotoxicity in vitro against cancer cells whereas
it is particularly active against the development and growth of
pulmonary metastases of solid tumors;5 KP1019 is moderately
cytotoxic in vitro but was proved to possess excellent activity
against platinum-resistant colorectal tumors in animal models.6
In recent years, the focus on ruthenium anticancer drugs has
shifted from coordination to organometallic compounds. New
classes of half-sandwich Ru(II)-arene compounds were found to
have promising anticancer activity.7 In particular, compounds
aDipartimento di Scienze Chimiche e Farmaceutiche, Universita` di Trieste,
Via L. Giorgieri 1, 34127, Trieste, Italy. E-mail: jbratsos@units.it
bFaculty of Chemistry and Chemical Technology, University of Ljubljana,
Asˇkercˇeva 5, Ljubljana, Slovenia. E-mail: Iztok.Turel@fkkt.uni-lj.si
cDepartment of Virology, Laboratory of Herpesvirus Infections and Cell
Cultures, National Centre of Infectious and Parasitic Diseases, 44A Stoletov
Street, 1233, Sofia, Bulgaria
6
of the general formula [Ru(h -arene)Cl(en)][PF6] (en = 1,2-
diaminoethane), developed by the group of Sadler,8,9 showed
excellent activity both in vitro against human cancer cells, in-
cluding some platinum-resistant lines, and in vivo against animal
tumor models.10 Extensive structure–activity relationship (SAR)
investigations showed that the anticancer properties of these
complexes - that are believed to be activated through hydrolysis
of the chlorido ligand and to target DNA - are influenced by the
nature of each ligand. In particular, it was found that increasing
the size and hydrophobicity of the arene ligand improves the cell
uptake and the interaction with DNA and, as a consequence,
also the anticancer activity. In addition, the presence of 1,2-
diaminoethane as chelating ligand is very important: replacement
of en with other N–N, N–O or O–O chelating ligands generally
leads to decreased activity.11
† Electronic supplementary information (ESI) available: Tables of X-ray
crystallographic data in CIF format for compounds 1–4; Crystal packing
of 1; 1H NMR spectra of 1 and 2 in CDCl3; 2D homonuclear 1H-1H
COSY and heteronuclear 1H-13C HSQC NMR spectra of 1 in CDCl3; 1H
NMR spectra of 3 and 4 in CD3NO2; 1H-1H COSY NMR spectrum of 3
in CD3NO2; 2D heteronuclear H-13C HSQC and HMBC NMR spectra
1
of 3 in CD3NO2; 2D homonuclear H-1H COSY and heteronuclear H-
1
1
13C HSQC NMR spectrum of 4 in CD3NO2; H NMR spectral changes
1
◦
1
during the aquation of 4 in D2O at 25.0 C; H NMR spectral changes
upon additio◦n of 100mM NaCl to equilibrated solution of 4 and 3a in
1
D2O at 25.0 C; H NMR spectral changes during the aquation of 5 in
D2O; Time evolution of UV-Vis spectra (of 3–5) and UV-Vis difference
spectra (of 4 and 5) in H2O at 25.0 ◦C; Tables with assignments of 1H and
13C resonances (d) for complexes 1–5 in various solvents and for aquation
products 3a and 5a. CCDC reference numbers 805600–805603. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c0dt01807d
5188 | Dalton Trans., 2011, 40, 5188–5199
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6
Intrigued by these results, we reasoned that perhaps the h -
arene fragment of these anticancer half-sandwich organometallic
compounds might be effectively replaced by another neutral
6-electron donor face-capping ligand, while maintaining the
other ligands unchanged. Thus, we developed series of new
half-sandwich Ru(II) coordination compounds of the general
formula [Ru([9]aneS3)Cl(N–N)][CF3SO3], [Ru([9]aneS3)(dmso-S)-
(N–N)][CF3SO3]2,12,13
[Ru([9]aneS3)(dmso-S)(O–O)],14
and
15
[Ru([9]aneN3)(dmso-S)(N–N)][PF6]2 (where N–N = nitrogen
chelating ligand such as en, bpy or substituted bpy; O–O =
chelating dicarboxylate ligand such as oxalate or malonate) in
which the aromatic fragment was replaced either by the tridentate
sulfur macrocycle 1,4,7-trithiacyclononane ([9]aneS3) or by
the corresponding nitrogen macrocycle 1,4,7-triazacyclononane
([9]aneN3). In vitro experiments on selected complexes showed that
only the half-sandwich complex [Ru([9]aneS3)Cl(en)][CF3SO3]
has a moderate cytotoxicity against human and murine cancer cell
lines (IC50 values in the range from 65 to 175 mM), which is ca. one
Fig. 1 Schematic representation of the two isomeric 1,4-disubstituted
1,2,3-triazole chelators A and B.
of type B.24–31 Thus 1-(2-picolyl)-4-phenyl-1H-1,2,3-triazole (ppt)
was proved to bind in this fashion a variety of metal ions, such as
CoII, NiII, CuII, ZnI, RuII, PdII, AgI, PtII and HgII, forming stable
6-membered chelate rings. Very recently it was proved that ppt,
having three potential coordination sites (i.e. N2 and N3 of the
triazole and the N pyridyl atom) can adopt also other binding
modes including monodentate and bridging bidentate, depending
on the metal center and the experimental conditions.32
6
order of magnitude lower than that of [Ru(h -bip)Cl(en)][PF6]
(bip = biphenyl), i.e. the most cytotoxic among the Ru-arene
analogues, against the same cell lines.12,13 Thus, it emerged that
selected Ru(II)-[9]aneS3 half-sandwich coordination compounds
can have a reasonable antiproliferative activity in vitro against
cancer cell lines and, above all, that the nature of the chelating
ligand is more important than that of the face-capping ligand for
determining such activity. More recently, we also reported that
the conjugation of Ru(II)-[9]aneS3 half-sandwich compounds to
porphyrins leads to enhanced cytotoxicity (in the low micromolar
range), which is further improved upon irradiation with visible
light (photo-toxicity).16
Now, with the aim of expanding the SAR investigation on these
half-sandwich coordination compounds, we are testing other face-
capping and chelating ligands. In this context, we were intrigued
by the possibility of using 1-(2-picolyl)-substituted 1,2,3-triazoles
as a new class of versatile neutral N-N chelators whose physico-
chemical properties can be easily varied.
In this work, we first investigated the reactivity of ppt towards
two common Ru(II)-dmso precursors, namely trans-[RuCl2(dmso-
S)4] and cis,fac-[RuCl2(dmso-S)3(dmso-O)] (from now on simply
trans-[RuCl2(dmso)4] (P1) and cis-[RuCl2(dmso)4] (P2), respec-
tively), and isolated two new isomeric compounds trans,cis-
[RuCl2(dmso-S)2(ppt)] (1), and cis,cis-[RuCl2(dmso-S)2(ppt)] (2),
respectively (Fig. 2). Then, with the aim of evaluating the potential
use of ppt as chelating ligand in Ru-[9]aneS3 compounds and its
influence on in vitro anticancer activity we prepared the two new
half-sandwich complexes [Ru([9]aneS3)(dmso-S)(ppt)][CF3SO3]2
(3) and [Ru([9]aneS3)Cl(ppt)][CF3SO3] (4) (Fig. 2). In all com-
pounds ppt behaves as a chelating ligand through the triazole
N2 and the pyridyl N. The chemical behavior of the water
soluble complexes 3 and 4, and of the previously described
6
organometallic analogue [Ru(h -p-cymene)Cl(ppt)][Cl] (5),24 were
1,4-Disubstituted 1,2,3-triazoles are actively investigated in
diverse areas of chemistry - such as dendrimers, polymers, catalysis,
drug discovery, materials science and bioconjugation - as ready-to-
make linkers between two functional groups.17 By virtue of their
facile and versatile synthetic procedure - the copper(I)-catalyzed
1
studied in aqueous solution by H NMR spectroscopy, whereas
their aquation kinetics were investigated by UV-Vis spectroscopy.
We report also the results of in vitro cytotoxicity tests performed
on complexes 3–5 against two human cancer cell lines (human
larynx carcinoma cell line (HEp-2), and human alveolar squamous
carcinoma epithelial cells (A-549)) in comparison with their
respective precursors, with the free ligand ppt and with cisplatin.
alkyne-azide cycloaddition, known also as “click chemistry”18,19
-
the nature of the functional groups at positions 1 and 4 can be
varied almost at will within a wide range.
In coordination chemistry, appropriately designed 1,4-
disubstituted 1,2,3-triazoles can behave as monodentate ligands
or as chelators towards metal ions, depending on the nature
of the substituents. Bidentate chelation can occur when one of
the substituents bears an appropriate donor atom; in this case,
coordination of the triazole group can involve either the N3 or
N2 atom (chelator A and B, respectively, in Fig. 1), depending
on the position and nature of the pendant coordinating group.
Owing to distinct Lewis basicity of N3 and N2,20 the complexes
arising from these two binding modes can differ greatly in stability.
Conveniently, the stability can be finely tuned by the appropriate
selection of the pendant coordinating group.21–24 Recent reports
demonstrated that when a 2-picolyl group is linked at the N1 atom
the molecule behaves as a highly efficient bidentate ligand through
the triazole N2 and the pyridyl nitrogen atom, i.e. as chelator
Experimental
Materials
The compound [RuCl(h -p-cymene)(ppt)][Cl] (5),24 the precursors
6
trans-[RuCl2(dmso)4] (P1),33 cis-[RuCl2(dmso)4] (P2),33 [Ru([9]-
aneS3)(dmso)3][CF3SO3]2 (P3),34 and [Ru([9]aneS3)Cl(dmso-
S)2][CF3SO3] (P4)34 and the ligand 1-(2-picolyl)-4-phenyl-1H-
1,2,3-triazole (ppt),24 were synthesized according to published
6
procedures. The compounds [RuCl(m-Cl)(h -p-cymene)]2 (P5) and
cis-[PtCl2(NH3)2] (cisplatin) were purchased from Sigma-Aldrich.
All other chemicals were used as purchased without further
purification.
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Fig. 2 Schematic structures of the ligand 1-(2-picolyl)-4-phenyl-1H-1,2,3-triazole (ppt) with numbering scheme used for the NMR characterization,
of the new ruthenium complexes trans,cis-[RuCl2(dmso-S)2(ppt)] (1), cis,cis-[RuCl2(dmso-S)2(ppt)] (2), [Ru([9]aneS3)(dmso-S)(ppt)][CF3SO3]2 (3) and
6
[Ru([9]aneS3)Cl(ppt)][CF3SO3] (4), and of the organometallic analogue [Ru(h -p-cymene)Cl(ppt)][Cl] (5).
Instrumental Methods
dmso-S). dH (500 MHz, CD3NO2) 9.56 (1 H, dd, J 5.9, 1.1 Hz,
C6H), 8.56 (1 H, s, C5¢H), 7.97 (1 H, td, J 7.6, 1.6, C4H), 7.87
(2 H, d, J 7.1, C2¢¢H/C6¢¢H), 7.65 (1 H, d, J 7.6, C3H), 7.59 (1
H, br d, CHa), 7.53–7.40 (4 H, m, C3¢¢H/C5¢¢H/C5H/C4¢¢H),
5.69 (1 H, br d, CHb), 3.78 (3 H, br s, CH3 dmso-S), 3.52 (3 H,
br s, CH3 dmso-S), 3.37 (3 H, br s, CH3 dmso-S), 3.17 (3 H, br s,
CH3 dmso-S). dC (101 MHz, CDCl3) 161.4 (C6H), 153.9 (C2),
149.3 (C4¢), 138.4 (C4H), 129.5 (C4¢¢H), 129.3 (C3¢¢H/C5¢¢H),
128.8 (C1¢¢), 125.8 (C2¢¢H/C6¢¢H), 124.7 (C5¢H), 124.2 (C5H),
124.0 (C3H), 56.3 (CH2 ppt), 46.5 (br, CH3 dmso-S), 44.6 (br,
CH3 dmso-S), 44.2 (br, CH3 dmso-S), 43.7 (br, CH3 dmso-S). dC
(101 MHz, CD3NO2) 161.8 (C6H), 156.3 (C2), 149.9 (C4¢), 140.1
(C4H), 130.4 (C3¢¢H/C5¢¢H), 130.3 (C4¢¢H), 126.9 (C5¢H), 126.8
(C2¢¢H/C6¢¢H), 126.7 (C1¢¢), 125.9 (C3H), 124.5 (C5H), 56.7 (CH2
ppt), 46.4 (br, CH3 dmso-S), 44.6 (br, CH3 dmso-S), 44.5 (br, CH3
dmso-S), 43.8 (br, CH3 dmso-S).
Mono- (1H (400 or 500 MHz), 13C (100.5 MHz)) and bi-
dimensional (1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC) NMR
spectra were recorded on a JEOL Eclipse 400FT or on a Varian
500 spectrometer. 1H chemical shifts in D2O were referenced to the
internal standard 2,2-dimethyl-2,2-silapentane-5-sulfonate (DSS)
at d = 0.00, while in other solvents were referenced to the peak
of residual non-deuterated solvent (d = 7.26 for CDCl3, 4.33
for CD3NO2); 13C chemical shifts were referenced to the peak
of residual non-deuterated solvent (d = 77.2 for CDCl3, 62.8 for
CD3NO2). NMR aquation studies were performed on 2.0 mM
samples of complexes 3–5 in D2O at 25.0 ◦C. Assignments of
1H and 13C resonances (d) of complexes 1–5 in various solvents
and of aquation products are summarized in ESI† (Tables S1
and S2). UV-Vis spectra were obtained on a Jasco V-500 UV-Vis
spectrophotometer equipped with a Peltier temperature controller,
using 1.0 cm path-length quartz cuvettes (3.0 mL). Infrared spectra
(ATR) were recorded on a Perkin-Elmer Spectrum 100 spectrom-
eter (wavelength range: 4000 to 600 cm-1). Elemental analysis was
performed at the Department of Chemistry, University of Udine
(Italy).
cis,cis-[RuCl2(dmso-S)2(ppt)] (2). A 14.6 mg amount of ppt
(0.062 mmol) and a 30.0 mg amount of P2 (0.062 mmol) were
partially dissolved in 5 mL of ethanol and the mixture was refluxed
for 2 h. During this time the solution—obtained upon warming—
turned from yellow to orange and then, after ca. 1 h, the formation
of the product as yellow solid was observed. After cooling, the
product was collected by filtration, washed with ethanol and
diethyl ether and vacuum dried (29.7 mg, 85%). Found: C, 38.0;
H, 4.17; N, 9.74. C18H24Cl2N4O2RuS2 (564.50) requires: C, 38.3;
H, 4.29; N, 9.92. Slow evaporation of the orange filtrate gave
rise to the formation of additional product as orange crystals
suitable for X-ray analysis. Complex 2 is insoluble in water, ethanol
and acetone, while it is soluble in chloroform, dichloromethane
and nitromethane, and slightly soluble in methanol. Selected IR
Synthesis of the complexes
trans,cis-[RuCl2(dmso-S)2(ppt)] (1). Equimolar amounts of P1
(100.0 mg, 0.206 mmol) and ppt (48.7 mg, 0.206 mmol) were
suspended in 5 mL of H2O and the mixture was vigorously stirred
for 24 h at room temperature. During this time, compound 1
precipitated as a yellow solid, and was collected by filtration,
washed with H2O and vacuum dried. (93.9 mg, 81%). Found:
C, 38.6; H, 4.31; N, 9.96. C18H24Cl2N4O2RuS2 (564.50) requires:
C, 38.3; H, 4.29; N, 9.92. Crystals suitable for X-ray analysis
formed spontaneously from the reaction mixture in the absence
of stirring. Complex 1 is insoluble in water, while it is soluble
in acetone, chloroform, dichloromethane and nitromethane, and
slightly soluble in methanol and ethanol. Selected IR nmax/cm-1
3117w, 3086w, 3061w, 1609m, 1486m, 1468m, 1438w, 1412m,
1089s, 763s, 722s and 693s (ppt) and 1069vs (S O(dmso-S)). dH
(500 MHz, CDCl3) 9.67 (1 H, dd, J 5.8, 1.2, C6H), 8.11 (1
H, s, C5¢H), 7.92–7.64 (4 H, m, C4H/CHa/C2¢¢H/C6¢¢H), 7.48–
7.31 (5 H, m, C3¢¢H/C5¢¢H/C4¢¢H/C5H/C3H), 5.26 (1 H, br
d, CHb), 3.86 (3 H, br s, CH3 dmso-S), 3.64 (3 H, br s, CH3
dmso-S), 3.52 (3 H, br s, CH3 dmso-S), 3.19 (3 H, br s, CH3
n
max/cm-1 3116w, 3089w, 2998w, 1610m, 1484m, 1473m, 1422w,
1089s, 767vs, 717m and 694vs (ppt), 1106s and 1082vs (S O(dmso-S)).
dH (500MHz, CDCl3)9.90(1H, d, J 5.8, C6H), 8.16(1H, s, C5¢H),
7.83 (1 H, t, J 7.5, C4H), 7.78 (2 H, d, J 7.6, C2¢¢H/C6¢¢H), 7.52–
7.36 (6 H, m, C3¢¢H/C5¢¢H/C4¢¢H/CHa/C5H/C3H), 5.41 (1 H,
d, J 14.9, CHb), 3.59 (3 H, s, CH3 dmso-S), 3.56 (3 H, s, CH3
dmso-S), 3.35 (3 H, s, CH3 dmso-S), 3.28 (3 H, s, CH3 dmso-S).
dH (500 MHz, CD3NO2) 9.74 (1 H, d, J 5.7, C6H), 8.58 (1 H, s,
C5¢H), 8.02 (1 H, t, J 7.0, C4H), 7.92 (2 H, d, J 7.5, C2¢¢H/C6¢¢H),
7.67 (1 H, d, J 7.3, C3H), 7.56–7.48 (3 H, m, C3¢¢H/C5¢¢H/C5H),
7.45 (1 H, t, J 7.3, C4¢¢H), 7.01 (1 H, d, J 15.3, CHa), 5.87 (1 H,
d, J 15.3, CHb), 3.41 (3 H, s, CH3 dmso-S), 3.41 (3 H, s, CH3
5190 | Dalton Trans., 2011, 40, 5188–5199
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dmso-S), 3.40 (3 H, s, CH3 dmso-S), 3.19 (3 H, s, CH3 dmso-
S). dC (101 MHz, CDCl3) 159.9 (C6H), 151.8 (C2), 148.8 (C4¢),
138.6 (C4H), 129.8 (C4¢¢H), 129.5 (C3¢¢H/C5¢¢H), 128.4 (C1¢¢),
125.6 (C2¢¢H/C6¢¢H), 125.1 (C5¢H), 124.3 (C5H), 124.1 (C3H),
56.1 (CH2 ppt), 45.9 (CH3 dmso-S), 45.8 (CH3 dmso-S), 45.3 (CH3
dmso-S), 44.5 (CH3 dmso-S).
slightly soluble in ethanol and chloroform. UV-Vis lmax (H2O)/nm
248sh, 281, 293 and 400 (e/dm3 mol-1 cm-1 9 796, 7 391, 7 237 and
815). Selected IR nmax/cm-1 3120w, 3089w, 2979w, 1603w, 1474m,
1446m, 1437m, 1415m, 1270vs, 1089m, 766vs, 755m, 723m and
696s (ppt), 1253vs and 1027vs (SO3), 1223s and 1140s (CF3). dH
(500 MHz, D2O) 9.27 (1 H, d, J 5.2, C6H), 8.62 (1 H, s, C5¢H),
8.01 (1 H, t, J 7.7, C4H), 7.84 (2 H, d, J 7.1, C2¢¢H/C6¢¢H), 7.74
(1 H, d, J 7.6, C3H), 7.53 (3 H, t, J 7.6, C3¢¢H/C4¢¢H/C5¢¢H),
7.49 (1 H, m, J 7.1, C5H), 6.36 (1 H, d, J 16.0, CHa ppt), 5.94
(1 H, d, J 16.0, CHb ppt), 3.20–3.11 (1 H, m, CH2 [9]aneS3),
2.96 (2 H, t, J 6.5, CH2 [9]aneS3), 2.87–2.42 (9 H, m, CH2
[9]aneS3). dH (500 MHz, CD3NO2) 9.24 (1 H, d, J 5.3, C6H),
8.52 (1 H, s, C5¢H), 8.02 (1 H, t, J 7.4, C4H), 7.85 (2 H, d,
J 7.4, C2¢¢H/C6¢¢H), 7.74 (1 H, d, J 7.6 Hz, C3H), 7.60–7.38
(4 H, m, C5H/C3¢¢H/C4¢¢H/C5¢¢H), 6.28 (1 H, d, J 15.7, CHa
ppt), 6.10 (1 H, d, J 15.7, CHb ppt), 3.29–3.21 (1 H, m, CH2
[9]aneS3), 3.10–2.90 (5 H, m, CH2 [9]aneS3), 2.88–2.80 (1 H, m,
CH2 [9]aneS3), 2.71–2.62 (1 H, m, CH2 [9]aneS3), 2.59–2.50 (1 H,
m, CH2 [9]aneS3), 2.50–2.38 (3 H, m, CH2 [9]aneS3). dC (101 MHz,
CD3NO2) 158.4 (C6H), 155.1 (C2), 150.2 (C4¢), 140.2 (C4H),
130.7 (C1¢¢), 130.5 (C3¢¢H/C5¢¢H), 130.4 (C4¢¢H), 127.7 (C3H),
127.0 (C2¢¢H/C6¢¢H), 126.8 (C5H), 126.6 (C5¢H), 56.0 (CH2 ppt),
37.9 (CH2 [9]aneS3), 35.1 (CH2 [9]aneS3), 34.9 (CH2 [9]aneS3), 34.8
(CH2 [9]aneS3), 31.7 (CH2 [9]aneS3), 31.4 (CH2 [9]aneS3).
[Ru([9]aneS3)(dmso-S)(ppt)][CF3SO3]2 (3). Equimolar amo-
unts of P3 (50.0 mg, 0.0614 mmol) and of ppt (14.5 mg,
0.0614 mmol) were partially dissolved in 5 mL of methanol and the
mixture was refluxed for 2h. During this time a yellow solution was
obtained. Rotary evaporation to dryness afforded a pale yellow oil
that was dissolved in acetone (ca. 4 mL). Within 1 day at ambient
temperature the product precipitated as white crystals (plates)
suitable for X-ray analysis that were collected by filtration, washed
with cold acetone and diethyl ether and vacuum dried (41.0 mg,
76%). Found: C, 31.9; H, 3.28; N, 6.12. C24H30F6N4O7RuS6
(893.95) requires: C, 32.2; H, 3.38; N, 6.27. Complex 3 is soluble
in water, methanol and nitromethane, slightly soluble in acetone,
and insoluble in ethanol, chloroform and dichloromethane. UV-
Vis lmax (H2O)/nm 270 and 289 (e/dm3 mol-1 cm-1 12 951 and 10
688). Selected IR nmax/cm-1 3151w, 3017w, 2933w, 1610w, 1486m,
1446m, 1429m, 1271s, 1086m, 775m, 753m, 726m and 683m
(ppt), 1256vs and 1028vs (SO3), 1223s and 1140s (CF3), 1099m
(S O(dmso-S)). dH (500 MHz, D2O) 9.34 (1 H, d, J 5.5, C6H), 8.83
(1 H, s, C5¢H), 8.23 (1 H, t, J 7.7, C4H), 7.95 (1 H, d, J 7.7, C3H),
7.89 (2 H, d, J 6.6, C2¢¢H/C6¢¢H), 7.74 (1 H, t, J 6.4, C5H), 7.54 (3
H, m, C3¢¢H/C4¢¢H/C5¢¢H), 6.32 (1 H, d, J 16.6, CHa ppt), 6.11
(1 H, d, J 16.6, CHb ppt), 3.65–3.25 (6 H, m, CH2 [9]aneS3), 3.54
(3 H, s, CH3 dmso-S), 2.95–2.80 (1 H, m, CH2 [9]aneS3), 2.70–2.40
(5 H, m, CH2 [9]aneS3), 1.99 (3 H, s, CH3 dmso-S). dH (500 MHz,
CD3NO2) 9.48 (1 H, d, J 5.2, C6H), 8.76 (1 H, s, C5¢H), 8.26
(1 H, t, J 7.7, C4H), 7.99 (1 H, d, J 7.5, C3H), 7.88 (2 H, d, J
6.9, C2¢¢H/C6¢¢H), 7.79 (1 H, t, J 6.3, C5H), 7.59–7.42 (3 H, m,
C3¢¢H/C4¢¢H/C5¢¢H), 6.42 (1 H, d, J 16.5, CHa ppt), 6.16 (1 H,
d, J 16.5, CHb ppt), 3.73 (1 H, dd, J 14.0, 4.2, CH2 [9]aneS3),
3.65–3.49 (6 H, m, CH2 [9]aneS3), 3.57 (3 H, s, CH3 dmso-S), 3.42
(2 H, dd, J 27.1, 9.5, CH2 [9]aneS3), 2.93 (1 H, td, J 14.0, 6.0, CH2
[9]aneS3), 2.65 (1 H, td, J 14.0, 6.0, CH2 [9]aneS3), 2.53 (4 H, dd, J
22.4, 9.5, CH2 [9]aneS3), 1.88 (3 H, s, CH3 dmso-S). dC (101 MHz,
CD3NO2) 158.2 (C6H), 154.6 (C2), 152.0 (C4¢), 142.6 (C4H),
131.1 (C4¢¢H), 130.6 (C3¢¢H/C5¢¢H), 130.5 (C3H), 129.6 (C1¢¢),
129.1 (C5¢H), 128.8 (C5H), 127.1 (C2¢¢H/C6¢¢H), 56.0 (CH2 ppt),
47.2 (CH3 dmso-S), 43.1 (CH3 dmso-S), 38.4 (CH2 [9]aneS3), 38.3
(CH2 [9]aneS3), 38.0 (CH2 [9]aneS3), 31.1 (CH2 [9]aneS3), 30.0
(CH2 [9]aneS3), 29.5 (CH2 [9]aneS3).
[Ru(g6-p-cymene)Cl(ppt)][Cl] (5). The preparation and the
NMR characterization (either in CDCl3 as Cl salt or in DMF-
d7 as CF3SO3 salt) have been previously reported.24 Herein, we
report the UV-Vis spectrum in H2O and the assignment of its
1H NMR spectrum in D2O, which are relevant for the current
experiments. UV-Vis lmax (H2O)/nm 246, 267 sh, 286 sh and 401
(e/dm3 mol-1 cm-1 10 788, 8 991, 5 143 and 415). dH (500 MHz,
D2O) 9.06 (1 H, d, J 4.6, C6H), 8.72 (1 H, s, C5¢H), 8.11 (1 H, t, J
7.2, C4H), 7.94–7.72 (3 H, m, C2¢¢H/C6¢¢H/C3H), 7.65 (1 H, t,
J 5.5, C5H), 7.58–7.45 (3 H, m, C3¢¢H/C5¢¢H/C4¢¢H), 6.17–6.08
(2 H, m, CHa/CH Ar), 6.05 (1 H, d, J 5.2, CH Ar), 5.92 (2 H, t,
J 5.9, 2¥CH Ar), 5.68 (1 H, d, J 15.5, CHb), 2.94–2.83 (1 H, m,
CH3CH Ar), 2.08 (3 H, s, CH3 Ar), 1.28 (6 H, t, J 7.7, 2¥CH3CH
Ar).
Crystallographic Measurements
Data collection for crystal structure analysis of 1–4 was carried out
at room temperature on an Enraf Nonius DIP1030H single crystal
˚
diffractometer (Mo-Ka radiation, l = 0.71073 A). Cell refinement,
indexing and scaling of all the data sets were performed using
programs Denzo and Scalepack.35 The structures were solved by
direct methods and subsequent Fourier analyses36 and refined
by the full-matrix least-squares method based on F2 with all
observed reflections.36 Hydrogen atoms were placed at calculated
positions. The structural determination of compound 3 evidenced
[Ru([9]aneS3)Cl(ppt)][CF3SO3] (4). Equimolar amounts of ppt
(19.0 mg, 0.0803 mmol) and of the precursor P4 (50.0 mg,
0.0803 mmol) were partially dissolved in 5 mL of methanol and
the reaction mixture was refluxed for 3h. During this time a yellow
solution was obtained. Rotary concentration to half volume and
saturation by diethyl ether led, within 1 day, to the formation of the
product as a yellow solid that was collected by filtration, washed
with cold acetone and diethyl ether and vacuum dried (43.2 mg,
77%). Found: C, 35.5; H, 3.44; N, 7.84. C21H26ClF3N4O4RuS4
(702.22) requires: C, 35.9; H, 3.44; N, 7.98. Crystals suitable for
X-ray analysis were obtained by slow diffusion of diethyl ether
into a solution of 4 in methanol. Complex 4 is soluble in water,
methanol, acetone, dichloromethane and nitromethane while is
-
a triflate anion (CF3SO3 ) highly disordered that was refined with
isotropic atoms and restrains on its geometry. Consequently the
final R values are higher than those obtained for other compounds
(Table 1). In 4 the DFourier map evidenced a residual interpreted
as a water oxygen (H atoms not located). All the calculations were
performed using the WinGX System, Ver 1.80.05.37 Crystal data
and details of data collections and refinements for the structures
reported are summarized in Table 1.
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Table 1 Crystallographic data for compounds 1–4
1
2
3
4·H2O
Empirical formula
fw
Crystal system
C18H24Cl2N4O2RuS2
564.50
C18H24Cl2N4O2RuS2
564.50
Monoclinic
P21/c
10.796(3)
C24H30F6N4O7RuS6
893.95
C21H26ClF3N4O4RuS4
720.22
Triclinic
Triclinic
Triclinic
¯
¯
¯
Space group
P1
P1
P1
˚
a/A
8.411(3)
10.009(4)
13.997(4)
104.37(3)
96.23(2)
94.18(2)
1128.6(7)
2
12.590(3)
12.611(3)
12.511(5)
64.09(2)
71.65(3)
85.07(3)
1692.7(9)
2
8.276(3)
13.690(4)
14.262(4)
86.21(3)
73.46(2)
71.34(2)
1467.0(8)
2
˚
b/A
12.813(3)
16.220(4)
˚
c/A
a (◦)
b (◦)
g (◦)
91.19(2)
3
˚
V/A
2243.2(10)
4
1.671
1.146
1144
3.42–28.73
14079
5423
0.0754
2541
267
Z
D
calcd/g cm-3
1.661
1.139
572
2.26–27.08
13115
4678
0.0397
2373
266
1.754
0.915
904
1.71–26.37
15655
6379
0.0516
3177
398
1.630
0.963
728
2.67–27.08
21726
5825
0.0351
2755
343
m(Mo-Ka)/mm-1
F(000)
q range, deg
no. of reflns collcd
no. of indep reflns
Rint
no. of reflns (I > 2s(I))
no. of refined params
goodness-of-fit (F2)
R1, wR2 (I > 2s(I))a
0.839
0.0421, 0.0960
0.503, -0.653
0.838
0.0460, 0.1027
0.813, -0.493
0.911
0.0763, 0.1980
1.097, -0.897
0.860
0.0598, 0.1655
0.760, -0.472
-3
˚
residuals/e A
1
2
a
2
2
2
2 2
R1 = R ꢀFo| - |Fcꢀ/R|Fo|, wR2 = [R w (Fo - Fc ) /Rw (Fo ) ] .
Aquation Kinetics
to 10% fetal bovine serum (FBS), 50 mU/ml penicillin a◦nd
50 mg ml-1 streptomycin. The culture was maintained at 37.0 C
in a humidified CO2 incubator. For routine passages monolayers
were detached using a mixture of 0.05% trypsin (Gibco)/0.02%
ethylendiamino tetraacetic (EDTA).
The aquation kinetics of the water soluble complexes 3–5 were
studied by UV-Vis spectroscopy (0.15 mM samples in distilled
H2O) at 25.0 ◦C. For each complex the wavelength selected for the
kinetic studies corresponded to that of the maximum change in
absorption (derived from the difference spectra). The absorbance
at the selected wavelength was recorded at 60 s intervals and
the absorption/time data for each complex were computer-fitted,
using the program Microcal Origin 5.0, to the first-order rate
equation (eqn (1)), which gave the kH2O value (k) for each aquation
process
Method for determining IC50. Cells were seeded into 96-well
tissue culture plates (Grainer)◦ at a concentration of 2 ¥ 104
cells/well and cultured at 37.0 C in a CO2 atmosphere. On the
24 h cells from confluent monolayers were washed and covered
with media containing the compounds tested in concentrations
0.001 mM, 0.01 mM, 0.1 mM, 1 mM, 10 mM, 100 mM, 250 mM,
500 mM and 800 mM. Cells grown in compound-free medium
served as a control. After 48 h and 72 h the viability of cells was
read by microscopy of unstained cell monolayers and by the MTT-
assay.38 MTT, a tetrazolium dye [3-(4,5-dimethyldiazol-2-yl)-2,5
diphenyl tetrazolium bromide; Thiazolyl blue] (Sigma) was added
to each well in the assay in a volume of 10 ml at a concentration
of 5 mg ml-1. Plates were incubated in the presence of MTT dye
for 3 h. Mitochondrial dehydrogenase activity reduces the yellow
MTT dye to a purple formazan, which is then solubilized with
DMSO:ethanol (1 : 1), and absorbance was read at 570 nm on
an ELISA plate reader. Percentage cell survival was expressed
as: absorbance treated wells/absorbance of control wells. Each
experiment was done in triplicate. IC50 values were determined
from the compound concentrations that induced a 50% reduction
in light absorbance.
A = C0 + C1e-kt
(1)
C0 and C1 are computer-fitted constants, and A is the ab-
sorbance corresponding to time t.
In vitro Studies
Tested Compounds and Precursors. The two new water-
soluble coordination half-sandwich complexes 3 and 4 and the
organometallic analog 5 were selected for in vitro tests in compar-
ison with their precursors P3 and P5, with the free ligand ppt and
with cisplatin.
The compounds under study were first dissolved in PBS (pH 7.4)
to a concentration of 1000 mM and then diluted in cell growth
medium with 2% fetal bovine serum. Eight dilutions of each
compound and precursor (800 mM maximum concentration) were
prepared and immediately tested, with incubation periods of 48 h
or 72 h.
Results and discussion
Cell lines. Human larynx carcinoma cell line (HEp-2) and
human alveolar squamous carcinoma epithelial cells (A-549) were
grown as monolayer culture in DMEM supplemented with 5%
The substituted triazole 1-(2-picolyl)-4-phenyl-1H-1,2,3-triazole
(ppt), recently synthesized via “click chemistry” synthetic
5192 | Dalton Trans., 2011, 40, 5188–5199
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◦
protocols,18,19,24 was used as chelating ligand for Ru(II) complexes
with potential antitumor activity.
˚
Table 2 Selected coordination bond distances (A) and angles ( ) for
compounds 1 and 2
First of all we tested the reactivity of ppt towards two well
known Ru(II)-dmso precursors, namely the isomers trans- and
cis-[RuCl2(dmso)4] (P1 and P2, respectively), in order to verify
its preferred binding mode. It is known that treatment of these
compounds with neutral N–N chelating ligands (e.g. 2,2¢-bipy)
leads mainly to the replacement of two adjacent dmso molecules
and to the formation of either trans,cis-[RuCl2(dmso-S)2(N–N)] or
cis,cis-[RuCl2(dmso-S)2(N–N)] or to mixtures of the two neutral
isomers, depending on the reaction conditions.39
We found that treatment of P1 with an equimolar amount
of ppt in H2O at ambient temperature afforded pure trans,cis-
[RuCl2(dmso-S)2(ppt)] (1) in very good yield. Conversely, a similar
synthetic procedure using P2 as precursor led to the isolation
of mixtures of 1 together with its geometrical isomer cis,cis-
[RuCl2(dmso-S)2(ppt)] (2). Similar mixtures were obtained also
upon treatment of either P1 or P2 with ppt in refluxing water or
methanol. However, we observed that the ratio of the two isomers,
in e.g. methanol, was dependent on the reaction time: whereas
at the beginning compound 1 was the predominant species in
the reaction mixture, upon increasing the time of reflux the ratio
changed in favor of 2. Thus, consistent with what previously found
for similar complexes,40 compound 1 is the kinetic isomer that
isomerizes to the thermodynamically more stable 2 (Scheme 1).
Pure compound 2 was isolated in excellent yield when the reaction
of P2 and ppt was performed in refluxing ethanol, since the product
precipitates spontaneously.
1
2
Ru–N(2)
Ru–N(4)
Ru–S(1)
Ru–S(2)
Ru–Cl(1)
Ru–Cl(2)
N(2)–Ru–N(4)
N(2)–Ru–S(1)
N(2)–Ru–S(2)
N(4)–Ru–S(1)
N(4)–Ru–S(2)
S(1)–Ru–S(2)
N(2)–Ru–Cl(1)
N(4)–Ru–Cl(1)
S(1)–Ru–Cl(1)
S(2)–Ru–Cl(1)
N(2)–Ru–Cl(2)
N(4)–Ru–Cl(2)
S(1)–Ru–Cl(2)
S(2)–Ru–Cl(2)
Cl(1)–Ru–Cl(2)
2.107(4)
2.172(4)
2.274(2)
2.280(2)
2.429(2)
2.397(2)
82.89(16)
93.20(12)
173.94(13)
174.72(12)
91.76(11)
92.31(6)
89.44(12)
94.43(12)
89.07(6)
88.13(6)
90.05(12)
85.26(12)
91.20(6)
92.35(6)
179.43(5)
2.058(4)
2.161(4)
2.276(2)
2.261(2)
2.437(2)
2.422(2)
87.44(16)
93.45(13)
90.36(14)
175.28(12)
88.95(13)
95.68(7)
91.26(14)
88.27(12)
87.07(6)
176.71(6)
177.36(13)
90.22(11)
88.78(6)
90.81(7)
87.44(6)
Fig.
4
ORTEP drawing (35% probability ellipsoids) of complex
Scheme 1
cis,cis-[RuCl2(dmso-S)2(ppt)] (2).
According to spectroscopic and X-ray evidence (Fig. 3 and
Fig. 4; Tables 2 and 3), in 1 and 2 the ppt ligand firmly binds to
ruthenium in a bidentate fashion - as expected - through the pyridyl
nitrogen atom and the triazole N2, thus forming a 6-membered
chelate ring. Bound ppt adopts a bent conformation, with the
CH2 group that points towards an axial chloride ligand (Cl(1)).
The pyridine and triazole rings form a dihedral angle of 56.0(2)◦
and 42.2(2)◦ in 1 and 2, respectively. The Ru–S (range 2.261(2)–
2.280(2) A) and Ru–Cl (2.397(2)–2.437(2) A) bond distances are
comparable with the values observed in other Ru(II)-dmso-Cl
complexes.12,40,41 The Ru–N(2) bond length is significantly longer in
˚
˚
˚
1 than in 2 (2.107(4) vs. 2.058(4) A) due to the different donor atom
in trans position (dmso-S vs. Cl, respectively). In both isomers the
˚
Ru–N(4) bond distance, with a mean value of 2.167(4) A, is longer
than Ru–N(2). It is worth noting that in 2 the phenyl ring of ppt
is almost coplanar with the triazole ring (dihedral angle 3.8(3)◦,
Table 3) indicating conjugation between them, while the dihedral
angle is significantly wider in 1 (28.8(2)◦). This latter feature favors
a p–p interaction between the phenyl rings and between the pyridyl
˚
rings of symmetry related molecules at 3.660(4) and 3.611(4) A,
respectively, that originates a 1D chain (Fig. S1, ESI†).
The IR spectra of the two isomeric complexes 1 and 2 are very
similar: beside the bands pertaining to the ppt ligand, they show
Fig.
3
ORTEP drawing (35% probability ellipsoids) of complex
trans,cis-[RuCl2(dmso-S)2(ppt)] (1).
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Table 3 Parameters characterizing the conformational geometry of ppt
ligand in compounds 1–4
1
2
3
4
N(1)–C(6)–C(5)/◦
111.6(4)
-59.3(6) 56.8(7)
115.2(5) 112.8(7)
-58.6(10)
-50.6(7) 59.7(11)
112.7(5)
57.0(8)
-60.8(8)
48.7(2)
27.1(1)
33.9(2)
5.5(5)
N(2)–N(1)–C(6)–C(5)/◦
N(4)–C(5)–C(6)–N(1)/◦
pya/triazb/◦
51.3(6)
56.0(2)
42.9(1)
34.2(2)
28.8(2)
1.489(7)
42.2(2)
28.7(1)
30.5(1)
3.8(3)
56.9(4)
32.0(4)
34.8(4)
40.8(3)
py/equator planec/◦
triaz/equator planec/◦
triaz/phenyl/◦
d
˚
d C(6) /A
1.292(8) 1.32(1)
1.327(9)
a mean plane through the pyridine ring. b mean plane through the triazole
ring. c equatorial mean plane through N(2)/N(4)/S(1)/S(2) (in 1, 3, 4) and
N(2)/N(4)//S(1)/Cl(2) in 2. d displacement of C(6) from the equatorial
plane.
strong bands between 1070 and 1105 cm-1 typical of the S
stretching modes of the dmso-S ligands.
O
The 1H NMR spectrum of 1 in CDCl3 (Fig. S2, ESI†) is
characterized by the presence of four relatively broad singlets
in the region typical for dmso-S (d = 3.86, 3.64, 3.52 and 3.20)
which were attributed to the diastereotopic methyl groups of the
two inequivalent dmso-S ligands. The two diastereotopic protons
of the CH2 junction resonate as two broad doublets quite far
apart from one another: one doublet falls at 5.26 ppm whereas
the other is in the aromatic region (d = 7.76). The latter resonance
Fig. 5 1H NMR spectrum of 1 in CDCl3 at various temperatures.
Treatment of the chloride-free Ru(II) precursor [Ru([9]aneS3)-
(dmso)3][CF3SO3]2 (P3) with an equimolar amount of ppt in
refluxing methanol afforded the half-sandwich dmso derivative
[Ru([9]aneS3)(dmso-S)(ppt)][CF3SO3]2 (3) in good yield. Similarly,
treatment of the precursor [Ru([9]aneS3)Cl(dmso-S)2][CF3SO3]
(P4) with ppt led to the replacement of the two adjacent dmso
ligands and to the formation of the monocationic analogue
[Ru([9]aneS3)Cl(ppt)][CF3SO3] (4).
Both compounds were characterized by X-ray structural analy-
sis (Fig. 6 and 7; Tables 3 and 4). In each complex the ruthenium
ion displays a distorted octahedral geometry with the tridentate
[9]aneS3 ligand occupying three positions in facial geometry and
the ppt ligand bound with the same bidentate fashion as found
in 1 and 2, thus forming a 6-membered ring. The coordination
sphere is completed by a dmso molecule bound through S in 3
or by a chloride anion in 4. The dihedral angle formed by the
pyridine and the triazole rings is similar in the two complexes
(ca. 53◦ mean value), whereas a larger difference is found for
the angle measured between the triazole and the phenyl ring:
40.8(3)◦ in 3 and only 5.5(5)◦ in 4. In the lattice of 4 this almost
1
is not distinguishable in the H NMR spectrum since it overlaps
1
with that of C4H, but it is clearly evident in the H-1H COSY
spectrum - where it is coupled with the resonance of the geminal
proton - and in the 1H-13C HSQC in which both proton resonances
are correlated to the same carbon resonance (Fig. S3, ESI†). The
other resonances appear in the aromatic area and are slightly
broadened as well. Broadened peaks were observed also in the 13
C
NMR spectrum, in particular those of the aromatic rings attached
to Ru and of the methyl groups of the dmso ligands. Similar spectra
were also observed in CD3NO2.
The presence of four dmso resonances in the 1H NMR spectrum
of 1 implies that the equatorial plane is not a plane of symmetry for
the complex. The broadening of some resonances suggests that a
motion - that formally exchanges pairwise methyls on each dmso-S
ligand and the diastereotopic CH2 protons - is occurring at a rate
that is relatively slow on the NMR time scale. Most likely this is the
flipping motion of the bent ligand that brings the CH2 junction
in two equivalent limiting positions either above or below the
equatorial plane. Indeed, the resonances of the exchanged protons
become sharp upon lowering the temperature at 273 K, when this
motion becomes slow on the NMR time scale (Fig. 5).
1
In contrast, the H NMR spectrum of complex 2 in CDCl3
at room temperature (Fig. S2, ESI†) displays well resolved sharp
peaks for both the ppt and the inequivalent dmso-S ligands. It is
likely that in 2, owing to the greater bulkiness of one axial ligand
(dmso-S) compared to the other (Cl), the 6-membered ppt chelate
ring is more rigid and basically maintains in solution the same
geometry as that found in the solid state (see above).
Having established that the substituted triazole ppt behaves as a
typical neutral N–N chelating ligand and easily replaces adjacent
dmso ligands in Ru-dmso complexes, we investigated its reactivity
towards the precursors of Ru-[9]aneS3 half-sandwich coordination
compounds.
Fig. 6 ORTEP drawing (35% probability ellipsoids) of the complex cation
of [Ru([9]aneS3)(dmso-S)(ppt)][CF3SO3]2 (3).
5194 | Dalton Trans., 2011, 40, 5188–5199
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◦
˚
Table 4 Selected coordination bond distances (A) and angles ( ) for
compounds 3 and 4
3, X = S(4)
4, X = Cl(1)
Ru–N(2)
Ru–N(4)
Ru–S(1)
Ru–S(2)
Ru–S(3)
Ru–X
N(2)–Ru–N(4)
N(2)–Ru–S(1)
N(2)–Ru–S(2)
N(2)–Ru–S(3)
N(4)–Ru–S(1)
N(4)–Ru–S(2)
N(4)–Ru–S(3)
S(1)–Ru–S(2)
S(1)–Ru–S(3)
S(2)–Ru–S(3)
N(2)–Ru–X
N(4)–Ru–X
S(1)–Ru–X
S(2)–Ru–X
S(3)–Ru–X
2.108(7)
2.170(7)
2.308(2)
2.326(3)
2.374(3)
2.297(3)
85.8(3)
90.97(19)
178.17(19)
95.0(2)
176.5(2)
95.0(2)
91.8(2)
88.27(9)
87.24(9)
86.60(9)
89.8(2)
2.110(5)
2.152(5)
2.283(2)
2.288(2)
2.280(2)
2.441(2)
86.3(2)
90.51(15)
178.53(15)
89.93(14)
175.87(15)
94.78(15)
94.07(14)
88.46(7)
88.54(7)
89.00(7)
94.55(14)
88.70(14)
88.94(7)
86.48(7)
174.88(6)
88.3(2)
92.96(9)
88.54(9)
175.13(9)
Fig. 8 Crystal packing of compound 4: pair of complexes arranged about
a center of symmetry connected by p–p stacking interactions between
phenyl and triazole rings.
strong band at 1099 cm-1 attributed to the S O stretching mode
of the dmso-S molecule.
The cationic complexes 3 and 4 undergo slow hydrolysis of
the monodentate ligand in aqueous solution, and their 1H NMR
spectra in D2O are commented in the next section (see below). The
full assignment of the proton and carbon NMR spectra of 3 and 4
was performed by combination of 1D (1H and 13C) and 2D NMR
(1H-1H COSY, 1H-13C HSQC and 1H-13C HMBC) experiments in
CD3NO2, where both complexes are stable (Fig. S4–S7, ESI†).
Fig. 7 ORTEP drawing (35% probability ellipsoids) of the complex cation
of [Ru([9]aneS3)Cl(ppt)][CF3SO3]·H2O (4).
1
The H NMR spectrum of 3 in CD3NO2 (Fig. S4, ESI†) is
consistent with the molecular structure found in the solid state. The
ppt ligand displays the typical pattern of resonances in the aromatic
region, all downfield shifted compared to the free ligand; the
diastereotopic CH2 protons resonate as two well resolved doublets
flat fragment participates in a double p–p stacking interaction
that involves both rings of symmetry related complexes (distance
1
˚
between centroids of 3.567(5) A, Fig. 8). The Ru–N coordination
(d = 6.42 and 6.16) that are coupled to each other in the H-1H
distances are comparable in the two complexes, while the Ru–
S bond distances of [9]aneS3 are slightly longer in 3 (mean of
COSY spectrum and to the same carbon resonance in the 1H-13C
HSQC spectrum (Fig. S5 and S6, ESI†). In the upfield region,
the methylene groups of [9]aneS3 give a characteristic manifold of
partially overlapping multiplets between 2.40 and 3.80 ppm, and
the two inequivalent methyl groups of the dmso-S ligand resonate
as two well resolved singlets (integrating for 3 H each). Worth
noting, while the methyl resonance at d = 3.57 is in the region
typical for dmso-S, the other one (at d = 1.88) is remarkably shifted
upfield because the methyl falls into the shielding cone of the
adjacent aromatic rings of ppt.13 The 13C NMR spectrum displays
11 resolved resonances between 125.8 and 158.2 ppm, attributed
to the 13 aromatic carbon atoms of the ligand ppt (C2¢¢ and C3¢¢
are equivalent to C6¢¢ and C5¢¢, respectively), and 9 resonances in
the upfield region assigned to the CH2 group of ppt (d = 54.7), to
the two methyl groups of the dmso-S ligand (d = 45.8 and 41.8)
and to the six inequivalent methylene groups of [9]aneS3 (from
37.0 to 28.1 ppm). The assignment of the quaternary carbons (C2,
˚
˚
2.336(3) A) than in 4 (mean 2.284(2) A), probably due to the
bulkier dmso ligand in 3 compared with chloride in 4. It is worth
noting that the Ru–S([9]aneS3) bond distances in 3 and 4 are
˚
slightly shorter (ca. 0.05 A) than in the corresponding precursors
[Ru([9]aneS3)(dmso)3]2+ (P3) and [Ru([9]aneS3)Cl(dmso)2]+ (P4).34
Interestingly, the Ru–Cl and the Ru–N bond lengths in 4 (Table 4)
are significantly longer than those found in the corresponding
6
organometallic compound [Ru(h -p-cymene)Cl(ppt)][Cl] (5): Ru–
Cl = 2.3785(12), Ru–N = 2.082(4) and 2.115(4) A. This feature
˚
suggests the presence of stronger bonds in 5 than in 4, and it
could be relevant for a rationalization of the aquation kinetics (see
below).
Complexes 3 and 4 display very similar IR spectra characterized
by the presence of bands attributed to the ppt ligand and to the
CF3SO3 counter ion. In the spectrum of 3 there is an additional
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C4¢ and C1¢¢) was achieved by the long range 2D heteronuclear
1H-13C HMBC experiments (Fig. S6, ESI†).
The corresponding chloride complex 4 shows similar 1H and 13
C
NMR spectra in CD3NO2 with the obvious absence of the dmso
resonances (Fig. S4 and S7, ESI†).
Chemical behavior in aqueous solution
The chemical behavior of the new water-soluble cationic com-
plexes 3 and 4, and of the previously described organometallic ana-
1
logue 5, was qualitatively investigated by H NMR spectroscopy
in D2O at room temperature.
After dissolution in D2O the 1H NMR spectrum of 3 changed
slowly in time due to dmso dissociation: the two dmso-S singlets
gradually decreased and were eventually replaced (in ca. 24 h) by
the resonance of free DMSO at d = 2.71. The formation of the
aqua species [Ru([9]aneS3)(H2O)(ppt)]2+ (3a, Scheme 2) induced
observable changes also in the resonances of ppt and [9]aneS3; in
particular, the two doublets of the methylene group of ppt (d =
6.32 and 6.11) were replaced by two new doublets (d = 6.40 and
6.00), indicating that the coordination mode of this ligand was
unaffected by the aquation process (Fig. 9).
Fig. 9 1H NMR spectral changes during the aquation of 3 (2.0 mM) in
D2O at 25.0 ◦C. With (*) are indicated selected resonances of the aquated
species 3a (3a: dH (500 MHz, D2O) 9.26 (1 H, d, J 4.8, C6H), 8.71 (1 H, s,
C5¢H), 8.08 (1 H, td, J 7.7, 1.5, C4H), 7.86 (2 H, d, J 7.0, C2¢¢H/C6¢¢H),
7.82 (1 H, d, J 7.8, C3H), 7.62 (1 H, t, J 6.7, C5H), 7.59–7.53 (2 H, m,
C3¢¢H/C5¢¢H), 7.53–7.48 (1 H, m, C4¢¢H), 6.41 (1 H, d, J 16.0, CHa ppt),
6.01 (1 H, d, J 16.0, CHb ppt), 3.22–3.14 (1 H, m, CH2 [9]aneS3), 3.12–3.04
(1 H, m, CH2 [9]aneS3), 3.01–2.70 (4 H, m, CH2 [9]aneS3), 2.70–2.62 (1 H,
m, CH2 [9]aneS3), 2.55–2.41 (4 H, m, CH2 [9]aneS3)).
Scheme 2 Chemical behavior of compounds 3 and 4 in aqueous solution.
A remarkably similar behavior, although faster, was observed
for 4 in aqueous solution. The hydrolytic process involves the
release of the Cl- ion as indicated by the gradual replacement of
the resonances of 4 by those of 3a (Fig. S8, ESI†). At equilibrium,
after ca. 2h, the ratio between 4 and 3a was ca. 1 : 5. Addition
of 100 mM NaCl in the equilibrated solution reversed the ratio
between 4 and 3a from 1 : 5 to 3 : 1 (within ca. 1h, Fig. S9, ESI†).
The chemical behavior of the organometallic compound 5 is
similar to that of the corresponding coordination complex 4. Upon
dissolution in D2O, all the resonances of 5 were slowly replaced by
a new set of resonances with similar pattern which were attributed
The wavelength corresponding to the maximum change in
absorption was selected for the kinetic studies. In each case the
time dependence of the absorbance followed first-order kinetics
(Fig. 10), and yielded the rate constants kH2O listed in Table 5. It
can be seen that the aquation reaction of the chloride derivative
4 is about 5-fold faster than that of the dmso derivative 3. In
addition, the kH2O values are strongly dependent on the nature of
the face-capping ligand: aquation is ca. 10 times faster for the
coordination half-sandwich complex 4 than for the corresponding
6
to the aqua species [Ru(h -p-cymene)(H2O)(ppt)]2+ (5a, Fig. S10,
ESI†). After 1 day the ratio between 5 and 5a was ca. 1 : 1 and
remained unchanged afterwards. Addition of 100 mM NaCl in the
previous solution reversed the equilibrium quantitatively towards
the parent compound 5.
Table 5 Rate constants for the aquation and half-lives at 25.0 ◦C in H2O
for compounds 3–5
Kinetics of aquation
The kinetics of aquation of compounds 3 and 4 were quantitatively
studied by UV-Vis spectroscopy at 25.0 ◦C on 0.15 mM solutions.
For comparison, compound 5 was also studied under the same
conditions. The UV-Vis spectra of all compounds show significant
time-dependent changes in the region 200–500 nm (Fig. 10, and
Fig. S11 and S12 in ESI†) with clean isosbestic points, suggesting
the occurrence of a single hydrolytic process (i.e. conversion of
the parent dmso/Cl complex to the corresponding aqua species)
consistent with the NMR observations.
a
Com-
lmin
Isosbestic
a
pound lmax [nm] [nm]
points [nm] kH2O [10-5 s-1] (t1/2
) H2O [min]
3
4
5
319
273
243
266, 289 297
337, 411 240, 302
213, 273 233, 253
8.43 0.01
40.27 0.01 28.7 0.01
4.10 0.04 281.9 2.7
137.2 0.2
a Wavelengths obtained from the time evolution difference electronic
absorption spectra. In bold are indicated the wavelengths selected for the
kinetic studies.
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Fig. 10 Left: Time evolution of UV-Vis difference spectra during the aquation of complex 3 (DA = At - A0, where At = absorbance at time t and A0 =
absorbance at t = 2 min (i.e. the time at which the first spectrum was recorded)); Right: Time-dependence of the absorbance at selected wavelengths for
the aquation of the complexes 3–5 in H2O at 25.0 ◦C (Full lines represent computer fits giving the first-order rate constants for the aquation, listed in
Table 5).
Table 6 Cytotoxic activity expressed as IC50 (mM SD) of all tested
compounds at 48 h and 72 h on the human cancer cell lines HEp-2 and
A-549
In contrast to chloride-derivative 4, the dmso-derivative 3
exhibits no activity towards both tested cell lines indicating that
the leaving group, and as consequence the aquation rate, plays an
important role on the activity of such complexes.
IC50, mM SD
HEp-2
48h
A-549
48h
Conclusions
Compound
72h
72h
We described here the preparation and structural characterization,
mainly by NMR spectroscopy in solution and by X-ray crystal-
lography in the solid state, of four new Ru(II) complexes bearing
a 1,4-disubstituted 1,2,3-triazole, i.e. 1-(2-picolyl)-4-phenyl-1H-
1,2,3-triazole (ppt) as chelating ligand: two isomeric neutral Ru-
dmso complexes, trans,cis-[RuCl2(dmso-S)2(ppt)] (1) and cis,cis-
[RuCl2(dmso-S)2(ppt)] (2), and two half-sandwich Ru-[9]aneS3
coordination compounds, [Ru([9]aneS3)(dmso-S)(ppt)][CF3SO3]2
(3) and [Ru([9]aneS3)Cl(ppt)][CF3SO3] (4). In all four compounds
ppt is bound to Ru through the N2 of the azole ring and the
pyridyl nitrogen atom forming a stable 6-membered ring. The
original aim of this work was to evaluate the preferential binding
mode of ppt on octahedral Ru complexes and to assess how
it influences the antiproliferative activity of half-sandwich Ru
coordination compounds, thus developing further the structure–
activity relationship investigation in this series of structurally
similar compounds.
ppt
150 19
280 18
> 300
> 300
> 300
150 16
> 300
> 300
> 300
> 300
100 14
80 10
100 32
100
> 300
250 13
80
125
210 12
105 10
8
3
> 300
100
P3
4
5
P5
Cisplatin
6
60 17
200 16
170 12
3
6
94
9
7.5 11
200
7
organometallic compound 5. The aquation process is also more
pronounced for 4 than for 5. These observations are in agreement
with the finding that, in solid state, the Ru–Cl bond is weaker in 4
than in 5.
In vitro experiments
The cytotoxic effects of compounds 3–5 were evaluated after 48
and 72 h of exposure on two human cancer cell lines: the cisplatin-
sensitive human larynx carcinoma cell line (HEp-2) and the
cisplatin-resistant human alveolar squamous carcinoma epithelial
cells (A-549).42 The tested compounds exhibited cytotoxic effects
that enabled the construction of concentration-response curves
and calculation of the IC50 values (Table 6).
The results show that, in general, the antiproliferative activity
of the tested ruthenium compounds is lower than that of cisplatin
and basically they are not cytotoxic against the specific cells. Often
the activity of ppt or of the precursors is higher than that of
the half-sandwich derivatives. Notable exception is compound 4
that, on the A-549 cell line shows an IC50 value almost one-order
of magnitude lower than that of cisplatin. Conversely, 4 does
not show any activity on HEp-2 cells, indicating a remarkable
selectivity towards the cisplatin-resistant cell line.
The two neutral Ru-dmso complexes 1 and 2 are insoluble in
water, and therefore no further biological investigation was per-
formed. On the other hand, the cationic half-sandwich complexes
3 and 4 are fairly soluble in water. Hence, in view of their potential
antitumor activity, their chemical behavior in aqueous solution
was studied by UV-Vis and NMR spectroscopy and compared
to that of the previously described organometallic analogue
6
[Ru(h -p-cymene)Cl(ppt)][Cl] (5). We found that the aquation rate
of such complexes, and consequently their potential biological
activity, depends on the nature of the face-capping ligand (Cl is
released faster from the coordination compound 4 than from the
organometallic analogue 5) and of the leaving group (Cl > dmso-
S). Compounds 3–5 were tested also in vitro for antiproliferative
activity against two human cancer cell lines in comparison with
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the precursors [Ru([9]aneS3)(dmso)3][CF3SO3]2 (P3) and [RuCl(m-
Cl)(h -p-cymene)]2 (P5), the free ligand (ppt) and cisplatin. All
of hydrated ruthenium chloride. Fondazione CRTrieste is also
gratefully acknowledged for the munificent donation of a Varian
500 NMR spectrometer to the Department of Chemical Sciences.
Financial support from the Slovenian Research Agency (ARRS)
through projects J1-0200-0103-008, P1-0230-0103 and EN→FIST
Centre of Excellence, Dunajska 156, SI-1000 Ljubljana is grate-
fully acknowledged. We thank Dr A. Bergamo (Callerio Foun-
dation Onlus, Trieste, Italy) for her critical comments on the
manuscript, as well as Dr Radostina Alexandrova (Bulgarian
Academy of Sciences, Sofia, Bulgaria) for her contribution to the
biological experiments.
6
tested compounds were found to be less cytotoxic than cisplatin
with the exception of 4, the compound that hydrolyses fastest,
against cisplatin-resistant human lung squamose carcinoma cells
(A-549). The selective cytotoxic activity found for 4 against A-549
cells is particularly interesting: lung cancer is the most common
cause of cancer-related death and squamous cell carcinoma is the
second most common type of lung cancer.
So far, only half-sandwich Ru-[9]aneS3 complexes capable of
hydrolyzing the monodentate ligand at a reasonable rate and of
acting as hydrogen bond donors through the chelating ligand, like
[Ru([9]aneS3)Cl(en)][CF3SO3] and [Ru([9]aneS3)Cl(dach)][PF6]
(where dach = 1,2-diaminocyclohexane), were found to have some
antiproliferative activity in vitro.12,13,15 However, these compounds
were less active compared to their organometallic analogues.
Here we show that the nature of the face-capping ligand is
not the most important feature for activity: the cytotoxicity of
the organometallic compound 5 towards the two tested cancer
cell lines is comparable or lower than that of the analogous
coordination compound 4. Most likely the rate of aquation plays
an important role in differentiating these two structurally similar
compounds. In addition, we showed here that introduction of a
chelating ligand that is unable of making hydrogen bonds, such
as ppt, not necessarily induces the total loss of antiproliferative
activity. Possibly, also the nature of the pendant group in position
4 influences the activity. For example, the phenyl ring in ppt is
expected to impart hydrophobicity to the complex that improves
its membrane permeability, and might be involved in additional
stacking interaction (e.g. intercalation) with DNA bases. In the
future, we aim to prepare by click reaction new 1-(2-picolyl)-
1,2,3-triazole ligands bearing more advanced pendant functional
groups in position 4 that might improve the properties of their
half-sandwich complexes.
The compounds described in this manuscript, despite their lim-
ited cytotoxic properties in vitro, may hold promise for therapeutic
purposes in vivo. Indeed, the Ru(III) compound NAMI-A, that is
currently being tested on patients in a phase 1–2 study, was found
to be poorly cytotoxic in vitro but has an effect on metastasis
formation in vivo. Nowadays, it is becoming increasingly evident
that the classical cytotoxicity tests are often insufficient models for
drug screening, since they lack essential components of the in vivo
environment, such as the extracellular matrix components (ECM).
In this respect, some organo-ruthenium half-sandwich complexes
belonging to the so-called RAPTA family developed by the group
of Dyson,43 although poorly toxic in classical proliferation assays
(probably because they interact with ECM components),44,45 were
found to be effective against metastasis formation and as inhibitors
of cell invasion pathways. Therefore, in this context, our new
complexes might be of interest for further evaluation in biological
systems.
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