The 1,3-diaryltriazenido(p-cymene)ruthenium(II) complexes with a high in vitro anticancer activity

Article abstract of DOI:10.1016/j.jinorgbio.2015.09.005

1,3-Diaryltriazenes (1) were let to react with [RuCl₂(p-cymene)]₂ in the presence of trimethylamine to give neutral 1,3-diaryltriazenido(p-cymene)ruthenium(II) complexes, [RuCl(p-cymene)(ArNNNAr)] (2). The molecular composition of th

Full text of DOI:10.1016/j.jinorgbio.2015.09.005

Journal of Inorganic Biochemistry 153 (2015) 42–48
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
The 1,3-diaryltriazenido(p-cymene)ruthenium(II) complexes with a
☆
high in vitro anticancer activity
Jure Vajs a,1, Ivana Steiner b,1, Anamaria Brozovic b,1, Andrej Pevec a, Andreja Ambriović-Ristov b,
a,
Marija Matković c, Ivo Piantanida c, Damijana Urankar a, Maja Osmak b, , Janez Košmrlj
⁎
⁎
a
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI 1000, Ljubljana, Slovenia
Division of Molecular Biology, Ruđer Bošković Institute, Bijenička cesta 54, HR-10000, Zagreb Croatia
b
c
Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10000 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 May 2015
Received in revised form 26 August 2015
Accepted 9 September 2015
Available online 14 September 2015
1,3-Diaryltriazenes (1) were let to react with [RuCl2(p-cymene)]2 in the presence of trimethylamine to give neu-
tral 1,3-diaryltriazenido(p-cymene)ruthenium(II) complexes, [RuCl(p-cymene)(ArNNNAr)] (2). The molecular
composition of the products 2 was confirmed by NMR spectroscopy and mass spectrometry. The structures of
the selected complexes were confirmed by a single crystal X-ray analysis. All triazenido-ruthenium complexes
were highly cytotoxic against human cervical carcinoma HeLa cells with IC50 below 6 μM, as determined by a
spectrophotometric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) method. The most
active was [RuCl(p-cymene)(ArNNNAr)] (Ar = 4-Cl-3-(CF3)-C6H3) (2g) with IC50 of 0.103 0.006 μM. In com-
parison with the data for the non-coordinated triazenes 1, the triazenido-ruthenium complexes 2 exhibited up to
560-times higher activity. Three selected complexes were highly cytotoxic also against several tumor cell lines:
laryngeal carcinoma HEp-2 cells and their drug-resistant HEp-2 subline (7 T), colorectal carcinoma HCT-116
cells, lung adenocarcinoma H460 cells, and mammary carcinoma MDA-MB-435 cells. The compounds 2g and
[RuCl(p-cymene)(ArNNNAr)] (Ar = 4-I-C6H4) (2j) were similarly cytotoxic against parental and drug-resistant
cells. Time and dose dependent accumulation of the cells in the S phase of the cell cycle was induced by the com-
pound 2g, triggering apoptosis. Our preliminary results indicate triazenido-ruthenium complexes as promising
anticancer drug candidates.
Keywords:
Ruthenium complexes
Triazene
Triazenido
Anticancer activity
© 2015 Elsevier Inc. All rights reserved.
1. Introduction
iron, they are easily reached by the ruthenium compounds accumu-
lating more in the tumor than in the normal cells [12,13]. To date,
The discovery of antitumor properties of cisplatin in 1965 by
Rosenberg and co-workers has been one of the major medicinal
breakthroughs for the metal-based drugs, opening a new field of an-
ticancer research based on metallo-pharmaceuticals [1]. Even
though nowadays cisplatin and its analogues are widely used in a
modern anticancer chemotherapy, these compounds display several
unwanted effects including high toxicity and development of the
drug-resistance [2–4]. Therefore, there is an urge to investigate
new alternative chemotherapeutic agents. Out of the metals that
have been probed for the metal-based anticancer therapies, the ru-
thenium is one of the most promising [5–10]. Besides possessing
rich potential in coordination chemistry [11], its complexes general-
ly show low toxicity and the ability to mimic iron atoms in some
cases. Because the fast dividing cancer cells have higher affinity to
two ruthenium complexes, NAMI-A and KP1019, have entered
human clinical trials [5,9,10].
1,3-Diaryltriazenes have been broadly investigated for their
pharmaceutical potential. In addition to the reports on their antibacteri-
al [14] and antifungal [15] activity, their most widespread use is in the
development of novel anticancer molecules [16–19]. In some cases,
their mode of action is related to the methylation of DNA, leading to a
somatic point mutation represented by C:G → T:A transition in DNA
helix, and finally to the cell death [20].
It has been well accepted that a synergistic effect can arise from
the combination of two pharmacophores into a single compound
[21,22], which prompted us to combine cymene-ruthenium and
1,3-diaryltriazenes entities into the corresponding triazenido-
ruthenium complexes to be screened for a cytotoxic activity against
human cancer cell lines. Surprisingly, although the synthesis and
reactivity of the triazenido complexes with ruthenium [23–27], as
well as osmium [23,25], palladium [28,29], rhodium [30], platinum
[31], aluminium [32], gallium [32], some heavier alkaline earths
[33], and other metals [34] is precedented, to our knowledge no bio-
logical properties of such compounds have been reported to date.
☆
Dedicated to Professor Božo Plesničar on the occasion of his 75th birthday.
Corresponding authors.
⁎
E-mail addresses: Maja.Osmak@irb.hr (M. Osmak), janez.kosmrlj@fkkt.uni-lj.si
(J. Košmrlj).
1
Contributed equally.
http://dx.doi.org/10.1016/j.jinorgbio.2015.09.005
0162-0134/© 2015 Elsevier Inc. All rights reserved.

J. Vajs et al. / Journal of Inorganic Biochemistry 153 (2015) 42–48
43
2. Experimental section
2.3. Reagents and materials
2.1. Physical measurements
Starting materials and solvents for the synthesis of the examined
compounds were used as obtained without further purification from
the commercial sources. 1,3-Diaryltriazenes 1b [14], 1c [14], 1d [18],
1e [14], 1f [39], 1g [39], 1h [14], 1i [14], 1j [39], 1k [40] were prepared
as described earlier. The progress of all reactions was monitored on
Fluka silica-gel TLC-plates (with fluorescence indicator UV254) using
ethyl acetate/petroleum ether (v/v 2:1) as the solvent system. A column
chromatography was performed with Merck silica gel 60 (35–70 μm)
with the solvent mixtures specified in the corresponding experiment.
Melting points were determined on a Kofler micro hot stage appara-
tus and are uncorrected. NMR spectra were recorded at 23 °C with a
Bruker Avance III 500 spectrometer (1H NMR spectra at 500 MHz; 13
C
NMR spectra at 126 MHz). Proton spectra are referenced to TMS as an
internal standard; the carbon chemical shifts are given against the cen-
tral line of the solvent signal (CDCl3 at δ = 77.0 ppm). IR spectra were
obtained with a Bruker ALPHA Platinum ATR spectrometer on a solid
sample support (ATR). High resolution mass spectrometry (HRMS)
analyses were performed using an Agilent 6224 Accurate Mass TOF
LC/MS spectrometer. Elemental analyses (C, H, N) were performed
with Perkin Elmer 2400 Series II CHNS/O Analyser. TG/DSC measure-
ments were performed on a Metter Toledo TGA/DSC1 Instrument.
UV–Vis measurements were performed on UV/vis spectrometer Varian
Cary 100 Bio while the CD experiments were performed on Circular
Dichroism Spectrometer Jasco J-815.
2.4. Synthesis
2.4.1. General procedure for the synthesis of the complexes 2
To a stirred suspension of [RuCl2(η6-p-cymene)]2 (61.2 mg,
0.1 mmol) in methanol (20 mL), 1,3-diaryltriazene 1 (0.2 mmol) and
triethylamine (558.0 μL, 0.4 mmol) were added. The reaction mixture
was stirred for 0.5 h at room temperature. The product was precipitated
by the addition of water (30 mL) and collected by filtration, yielding the
corresponding complex 2.
2.2. X-ray crystal structure determination
In the case of the complexes 2b, 2d and 2k, the products were puri-
fied with a column chromatography using silica gel and petroleum
ether/ethyl acetate (v/v 4:1) as the eluting solvent.
Crystal data and refinement parameters of the compounds 2e and 2h
are listed in Table S1. The X-ray intensity data were collected at room
temperature with an Agilent SuperNova dual source with an Atlas
detector equipped with mirror-monochromated Mo-Kα radiation
(λ = 0.71073 Å) for 2e and with a Nonius Kappa CCD diffractometer
equipped with graphite-monochromated Mo-Kα radiation (λ =
0.71073 Å) for 2h. The data were processed using CRYSALIS PRO [35]
(2e) or DENZO [36] (2h). Both structures were solved by direct methods
using SIR-92 [37] (2e) or SHELXS-97 [38] (2h) and refined against F2 on
all data by a full-matrix least squares procedure with SHELXL-97 [38].
All non-hydrogen atoms were refined anisotropically. All hydrogen
atoms were included in the model at geometrically calculated positions
and refined using a riding model. The fluorine atoms of CF3 substituent
and methine C13 atom in 2e are disordered over two orientations and
were modelled with split positions, with the use of PART instruction.
Selected bond lengths and angles for 2e and 2h are listed in Table 1.
Crystallographic data for structures have been deposited at the
Cambridge Crystallographic Data Centre as supplementary publication
CCDC 1063947 (2e) and 1063948 (2h). Copies of the structures can
be obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
2.4.1.A. Complex 2a. Orange solid; yield 80%; mp 217–219 °C (MeOH/
H2O); IR (cm−1): 3018, 2963, 2865, 1947, 1588, 1478, 1456, 1385,
1369, 1324; 1H NMR (500 MHz, CDCl3): δ (ppm) 1.22 (d, J = 6.9 Hz,
6H), 2.34 (s, 3H), 2.77 (sep, J = 6.9 Hz, 1H), 5.37 (d, J = 5.9 Hz, 2H),
5.78 (d, J = 5.9 Hz, 2H), 7.05–7.11 (m, 2H), 7.28–7.36 (m, 8H); 13C
NMR (126 MHz, CDCl3): δ (ppm) 19.2, 22.7, 31.7, 79.2, 81.4, 102.5,
102.6, 117.0, 124.3, 128.8, 147.0; HRMS (ESI+) for C22H24N3Ru
[M–Cl]+: calcd 432.1014, found 432.1014. Anal. for C22H24ClN3Ru:
C, 56.59; H, 5.18; N, 9.00; found: C, 56.56; H, 4.90; N, 8.98.
2.4.1.B. Complex 2b. Orange solid; yield 88%; mp 168–170 °C (petroleum
ether); IR (cm−1): 3039, 2969, 2101, 2014, 1969, 1935, 1618, 1584,
1475; 1H NMR (500 MHz, CDCl3): δ (ppm) 1.30 (d, J = 6.9 Hz, 6H),
2.32 (s, 3H), 2.97 (sep, J = 6.9 Hz, 1H), 5.27 (d, J = 5.8 Hz, 2H), 5.82
(d, J = 5.8 Hz, 2H), 7.10–7.16 (m, 2H), 7.36–7.42 (m, 2H), 7.49–7.54
(m, 2H); 13C NMR (126 MHz, CDCl3): δ (ppm) 19.3, 22.7, 31.7, 78.5,
82.7 (d, J = 5 Hz), 102.5, 103.0, 119.4 (dq, J1 = 32 Hz, J2 = 11 Hz),
122.6 (q, J = 273 Hz), 123.0 (q, J = 5 Hz), 124.0 (d, J = 4 Hz), 124.9,
136.8 (d, J = 8 Hz), 151.5 (dq, J1 = 258 Hz, J2 = 2 Hz); HRMS (ESI+)
for C24H20F8N3Ru [M–Cl]+: calcd 604.0573, found 604.0575. Anal. for
C24H20ClF8N3Ru: C, 45.12; H, 3.15; N, 6.58; found: C, 45.05; H, 2.78; N,
6.50.
Table 1
Selected bond lengths (Å) and angles (°) for 2e and 2h.
2e
2h
2.4.1.C. Complex 2c. Yellow solid; yield 74%, mp 162–163 °C (MeOH/
H2O); IR (cm−1): 3359, 3070, 2967, 2033, 1615, 1524, 1485, 1451,
1425, 1375; 1H NMR (500 MHz, CDCl3): δ (ppm) 1.19 (d, J = 6.9 Hz,
6H), 2.14 (s, 3H), 2.75 (sep, J = 6.9 Hz, 1H), 5.30 (d, J = 5.8 Hz, 2H),
5.58 (d, J = 5.8 Hz, 2H), 7.13–7.21 (m, 2H), 7.31–7.38 (m, 2H),
7.43–7.52 (m, 2H); 13C NMR (126 MHz, CDCl3): δ (ppm) 18.9, 22.3,
31.2, 80.2, 82.4, 98.3, 102.9, 114.1 (dq, J1 = 20 Hz, J2 = 3 Hz), 119.2
Ru1–Cl1
Ru1–N1
Ru1–N3
N1–N2
N2–N3
N1–C1
N3–C8
Ru1–N1–N2
Ru1–N1–C1
Ru1–N3–N2
Ru1–N3–C8
Cl1–Ru1–N1
Cl1–Ru1–N3
N1–Ru1–N1i
N1–Ru1–N3
N1–N2–N1i
N1–N2–N3
C1–N1–N2
C8–N3–N2
2.3788(8)
2.1088(18)
2.3942(16)
2.092(4)
2.080(4)
1.323(5)
1.313(5)
1.396(6)
1.409(6)
99.2(3)
140.8(3)
100.2(3)
141.0(3)
85.14(13)
84.87(13)
1.308(2)
1.404(3)
97.50(13)
142.15(14)
(d, J = 22 Hz), 123.1 (qd, J1 = 273 Hz, J2 = 2 Hz), 124.3 (qd, J1
=
32 Hz, J2 = 8 Hz), 128.6 (d, J = 8 Hz), 142.4 (d, J = 3 Hz), 160.0
(d, J = 247 Hz); HRMS (ESI+) for C24H20F8N3Ru [M–Cl]+: calcd
604.0573, found 604.0570. Anal. for C24H20ClF8N3Ru: C, 45.12; H, 3.15;
N, 6.58; found: C, 44.81; H, 2.92; N, 6.67.
83.20(5)
58.87(10)
104.8(2)
58.71(15)
2.4.1.D. Complex 2d. Yellow-green solid; yield 78%; mp 55–57 °C (MeOH/
H2O); IR (cm−1): 2966, 2146, 2064, 2008, 1598, 1568, 1475, 1409, 1364,
1309; 1H NMR (500 MHz, CDCl3): δ (ppm) 1.21 (d, J = 6.9 Hz, 6H),
2.18 (s, 3H), 2.76 (sep, J = 6.9 Hz, 1H), 5.30 (d, J = 5.9 Hz, 2H), 5.59
(d, J = 5.9 Hz, 2H), 7.35–7.46 (m, 4H), 7.60–7.66 (m, 2H); 13C NMR
101.7(4)
119.2(4)
118.6(4)
114.26(17)
Symmetry code: (i) x, −1.5–y, z.

44
J. Vajs et al. / Journal of Inorganic Biochemistry 153 (2015) 42–48
(126 MHz, CDCl3): δ (ppm) 19.0, 22.3, 31.4, 80.3, 82.0, 98.8, 103.2, 123.1
(q, J = 274 Hz), 123.8 (q, J = 32 Hz), 127.1, 127.9, 131.4, 132.4, 144.6;
HRMS (ESI+) for C24H20Cl2F6N3Ru [M–Cl]+: calcd 635.9982, found
635.9978. Anal. for C24H20Cl3F6N3Ru: C, 42.90; H, 3.00; N, 6.25; found:
C, 43.29; H, 2.60; N, 6.18.
2.4.1.J. Complex 2j. Ocher solid; yield 74%; mp 229–231 °C (MeOH/H2O);
IR (cm−1): 2968, 2014, 1892, 1576, 1473, 1408, 1384, 1369, 1288, 1271;
1H NMR (500 MHz, CDCl3): δ (ppm) 1.21 (d, J = 6.9 Hz, 6H), 2.33
(s, 3H), 2.74 (sep, J = 6.9 Hz, 1H), 5.34 (d, J = 5.8 Hz, 2H), 5.75
(d, J = 5.8 Hz, 2H), 7.02–7.10 (m, 4H), 7.57–7.64 (m, 4H); 13C NMR
(126 MHz, CDCl3): δ (ppm) 19.3, 22.7, 31.8, 79.1, 81.4, 88.1, 102.8,
2.4.1.E. Complex 2e. Yellow solid; yield 61%; mp 201–203 °C (MeOH/
H2O); IR (cm−1): 3053, 2967, 2103, 1979, 1898, 1603, 1514, 1477,
1416, 1388; 1H NMR (500 MHz, CDCl3): δ (ppm) 1.30 (d, J = 6.9 Hz,
6H), 2.33 (s, 3H), 2.90 (sep, J = 6.9 Hz, 1H), 5.24 (d, J = 5.9 Hz, 2H),
5.78 (d, J = 5.9 Hz, 2H), 7.17–7.25 (m, 2H), 7.34–7.41 (m, 2H),
7.64–7.70 (m, 2H); 13C NMR (126 MHz, CDCl3): δ (ppm) 19.3, 22.7,
31.8, 78.6, 82.7 (d, J = 5 Hz), 102.9, 103.3, 117.1 (d, J = 21 Hz), 117.7
102.9, 119.0, 137.8, 146.5; HRMS (ESI+) for C22H22I2N3Ru [M–Cl]+
:
calcd 683.8947, found 683.8946. Anal. for C22H22ClI2N3Ru: C, 36.76; H,
3.09; N, 5.85; found: C, 37.01; H, 2.79; N, 5.99.
2.4.1.K. Complex 2k. Brown solid; yield 42%; mp 138.1–140.1 °C (diethyl
ether); IR (cm−1): 3068, 2834, 1604, 1580, 1496, 1458, 1440, 1357
1319, 1302; 1H NMR (500 MHz, CDCl3): δ (ppm) 1.23 (d, J = 6.9 Hz,
6H), 2.33 (s, 3H), 2.78 (sep, J = 6.9 Hz, 1H), 3.80 (s, 6H), 5.32 (d, J =
5.8 Hz, 2H), 5.75 (d, J = 5.8 Hz, 2H), 6.81–6.88 (m, 4H), 7.20–7.26
(m, 4H); 13C NMR (126 MHz, CDCl3): δ (ppm) 19.2, 22.7, 31.8, 55.6,
79.1, 81.4, 102.1, 102.3, 114.2, 118.0, 141.4, 156.6; HRMS (ESI+)
for C24H28N3O2Ru [M–Cl]+: calcd 492.1225, found 492.1225. Anal.
for C24H28ClN3O2Ru: C, 54.70; H, 5.36; N, 7.97; found: C, 54.95; H,
5.10; N, 7.97.
(m), 112.9 (m), 123.5 (q, J = 272 Hz), 127.1 (qd, J1 = 33 Hz, J2
=
3 Hz), 135.8 (d, J = 9 Hz), 155.32 (d, J = 254 Hz); HRMS (ESI+)
for C24H20F8N3Ru [M–Cl]+: calcd 604.0573, found 604.0571. Anal.
for C24H20ClF8N3Ru: C, 45.12; H, 3.15; N, 6.58; found: C, 45.02; H, 2.94;
N, 6.49.
2.4.1.F. Complex 2f. Ocher solid; yield 83%; mp 191–193 °C (MeOH/
H2O); IR (cm−1): 3060, 2969, 2872, 2103, 1912, 1594, 1483, 1417,
1384, 1305; 1H NMR (500 MHz, CDCl3): δ (ppm) 1.25 (d, J =
6.9 Hz, 6H), 2.35 (s, 3H), 2.78 (sep, J = 6.9 Hz, 1H), 5.37 (d, J =
5.9 Hz, 2H), 5.78 (d, J = 5.9 Hz, 2H), 7.05–7.18 (m, 4H), 7.31–7.38
(m, 2H); 13C NMR (126 MHz, CDCl3): δ (ppm) 19.3, 22.7, 31.9, 79.1,
81.5, 102.9, 103.1, 116.6, 116.8 (d, J = 14 Hz), 118.6, 121.4 (d, J =
19 Hz), 143.7 (d, J = 3 Hz), 155.5 (d, J = 246 Hz); HRMS (ESI+)
for C22H20Cl2F2N3Ru [M–Cl]+: calcd 536.0046, found 536.0038.
Anal. for C22H20Cl3F2N3Ru: C, 46.21; H, 3.53; N, 7.35; found: C,
46.25; H, 3.23; N, 6.97.
2.5. Biology
2.5.1. Cell culture
Human cervical carcinoma HeLa and laryngeal carcinoma HEp-2
cells were obtained from the cell culture bank (GIBCO/Life Technologies,
Grand Island, NY, USA). The development of the HEp-2 subline resistant
to carboplatin has been published previously [41]. These cells are cross-
resistant to the anticancer drugs cisplatin, transplatin and mitomycine
C, as well as natural compound curcumin [41–43]. Large-cell lung
carcinoma H460 cells, colorectal carcinoma HCT 116 and mammary
carcinoma MDA-MB-435 cells [44] were obtained from American Type
Culture Collection (ATCC; Manassas, VA, USA). All cell lines were
grown as a monolayer culture in Dulbecco's modified Eagle's medium
(DMEM; Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10%
foetal bovine serum (FBS; Sigma-Aldrich) in a humidified atmosphere
of 5% CO2 at 37 °C and were sub-cultured every 3–4 days.
2.4.1.G. Complex 2g. Yellow solid; yield 82%; mp 214–216 °C (diethyl
ether/diisopropyl ether); IR (cm−1): 3037, 2968, 2115, 1576 1474,
1424, 1388, 1366, 1317, 1304; 1H NMR (500 MHz, CDCl3): δ (ppm)
1.25 (d, J = 6.8 Hz, 6H), 2.36 (s, 3H), 2.79 (sep, J = 6.8 Hz, 1H), 5.42
(d, J = 6.0 Hz, 2H), 5.78 (d, J = 6.0 Hz, 2H), 7.40–7.46 (m, 4H),
7.56–7.60 (m, 2H); 13C NMR (126 MHz, CDCl3): δ (ppm) 19.3, 22.7,
31.9, 79.6, 81.4, 102.3, 103.8, 116.5 (q, J = 5 Hz), 120.5, 122.6 (q, J =
274 Hz), 127.6 (q, J = 2 Hz), 128.9 (q, J = 32 Hz), 132.0, 145.2; HRMS
(ESI+) for C24H20Cl2F6N3Ru [M–Cl]+: calcd 635.9982, found 635.9984.
Anal. for C24H20Cl3F6N3Ru: C, 42.90; H, 3.00; N, 6.25; found: C, 43.30;
H, 2.67; N, 6.27.
2.5.2. Cytotoxicity assay
A cytotoxic activity of the triazenido-ruthenium complexes 2 was de-
termined by MTT assay [45], modified as described. The cells were seed-
ed into 96-well tissue culture plates (3000 cells/0.18 mL medium/well).
The next day different concentrations of the complexes 2 were added
(0.02 mL) to each well and each concentration was tested in quadrupli-
cate. Following 72 h incubation at 37 °C, the medium was aspirated and
20 μg of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) dye (Sigma-Aldrich) /0.04 mL medium/well was added. Three h
later, formazan crystals were dissolved in DMSO (0.17 mL/well), the
plates were mechanically agitated for 5 min and the optical density at
545 nm was determined on a microtiter plate reader (Awareness
Technology Inc., Palm City, FL, USA). Each experiment was repeated at
least three times. The same protocol was used to determine the cytotoxic
activity of triazenes 1c and 1j.
2.4.1.H. Complex 2h. Red solid; yield 91%; mp 222–224 °C (MeOH/H2O);
IR (cm−1): 3076, 2968, 1608, 1575, 1541, 1525, 1486, 1431, 1371, 1311;
1H NMR (500 MHz, CDCl3): δ (ppm) 1.28 (d, J = 6.9 Hz, 6H), 2.40
(s, 3H), 2.81 (sep, J = 6.9 Hz, 1H), 5.51 (d, J = 6.0 Hz, 2H), 5.83
(d, J = 6.0 Hz, 2H), 7.59–7.63 (m, 2H), 7.70–7.73 (m, 2H), 8.01–8.06
(m, 2H); 13C NMR (126 MHz, CDCl3): δ (ppm) 19.4, 22.7, 32.0, 79.8,
81.5, 102.8, 104.9, 117.0 (q, J = 6 Hz), 120.0, 121.8 (q, J = 274 Hz),
125.7 (q, J = 34 Hz), 127.4, 144.1, 149.5; HRMS (ESI+) for
C24H20F6N5O4Ru [M–Cl]+: calcd 658.0463, found 658.0466. Anal. for
C24H20ClF6N5O4Ru: C, 41.60; H, 2.91; N, 10.11; found: C, 41.88;
H, 2.63; N, 10.04.
2.4.1.I. Complex 2i. Red solid; yield 80%; mp 231–233 °C (MeOH/H2O); IR
(cm−1): 3050, 2970, 2225, 1598, 1567, 1491, 1438, 1382, 1316, 1274;
1H NMR (500 MHz, CDCl3): δ (ppm) 1.26 (d, J = 6.9 Hz, 6H), 2.39
(s, 3H), 2.80 (sep, J = 6.9 Hz, 1H), 5.50 (d, J = 6.0 Hz, 2H), 5.83
(d, J = 6.0 Hz, 2H), 7.54–7.59 (m, 2H), 7.67–7.71 (m, 2H), 7.78–7.83
(m, 2H); 13C NMR (126 MHz, CDCl3): δ (ppm) 19.4, 22.6, 32.0, 79.6,
81.5, 103.0, 104.8, 105.5 (q, J = 2 Hz), 115.6, 115.7 (q, J = 5 Hz),
120.0, 122.1 (q, J = 274 Hz), 134.1 (q, J = 33 Hz), 135.9, 149.3; HRMS
(ESI+) for C26H20F6N5Ru [M–Cl]−: calcd 618.0668, found 618.0661.
Anal. for C26H20ClF6N5Ru: C, 47.82; H, 3.09; N, 10.73; found: C, 47.80;
H, 2.86; N, 10.64.
2.5.3. Cell cycle analysis
Human cervical carcinoma HeLa cells were seeded into 6-well tissue
culture plates (100,000 cells/2 mL medium/well). Next day they were
treated with concentrations of 2g that reduced the cell survival to 60
or 10% (survival fraction 0.6 and 0.1). After 24 and 48 h both adherent
and floating cells were collected, washed with PBS and fixed overnight
in 96% ethanol at −20 °C. Fixed cells were treated with RNase A
(0.1 mg/mL; Sigma-Aldrich) for 1 h at room temperature and afterward
stained with propidium iodide (50 μg/mL; Sigma-Aldrich) for 30 min in
the dark. The DNA content was analysed by a flow cytometry (FACS
Calibur, Becton Dickinson, Mountain View, CA, USA). The data were

J. Vajs et al. / Journal of Inorganic Biochemistry 153 (2015) 42–48
45
analysed with the ModFitLTTM program (Verity Software House Inc.,
Topsham, ME, USA).
as reported in the literature (see: 2.3. Reagents and materials). A subse-
quent reaction of 1 with dinuclear p-cymene complex of ruthenium
[RuCl2(η6-p-cymene)]2 in methanol and in the presence of triethylamine
for 0.5 h afforded the triazenido-ruthenium complexes 2a–j in good to
excellent 61–91% yields (Scheme 1, Table 2). The exception to this was
the 4-methoxyphenyl derivative 2k, obtained in just 42% yield. The drop
in the yield when going from 2a–j to 2k could be explained by the
electron-releasing nature of the 4-methoxyphenyl substituents in the
latter, decreasing the acidity of the triazene and making its reaction
with the ruthenium precursor more sluggish (vide infra). All compounds
2 were isolated as coloured solids, stable in the air and in the solution of
common organic solvents.
2.5.4. Western blot analysis
The western blot analysis was used for the determination of a poly
(ADP-ribose) polymerase (PARP) cleavage as one of the markers for
the apoptosis induction [46]. Upon treatment of the HeLa cells during
24 and 48 h with two concentrations of 2g (IC40 and IC90) the adherent
and floating cells were collected and protein isolation was performed
following the previously used protocol [43]. 30 mg of the total cellular
proteins were loaded onto a 10% SDS polyacrylamide gel and run for
2 h at 35 mA in a Mini-PROTEAN Tetra cell system (Bio-Rad, Hercules,
CA, USA). The separated proteins were transferred onto a 0.2 mm nitro-
cellulose membrane (Schleicher & Schuell, Dassel, Germany; Cat. Nr.
NBA083C001EA) by using the same Bio-Rad system optimised for a pro-
tein transfer with a Mini Trans-Blot® Module (Bio-Rad) and buffer
consisting of 25 mM Tris/HCl, 86 mM glycine and 20% methanol. To
avoid a nonspecific binding, the membrane was incubated in blocking
buffer (5% non-fat dry milk, 0.1% Tween 20 in 1 × TBS) for one h at
room temperature. The incubation with a primary antibody recognizing
cleaved anti-PARP fragment (mouse monoclonal Cat. Nr. 9544, Cell
Signalling, Beverly, MA, USA) was performed overnight at 4 °C (diluted
1:1000). After washing with 0.1% Tween-20 in 1 × TBS and incubation
with the corresponding horseradish peroxidase-coupled secondary
anti-rabbit antibody (Santa Cruz Biotechnology, Solna Sweden) (diluted
1:10,000) for 2 h at room temperature, the proteins were visualized
with an Amersham ECL Western Blotting Detection Reagent (GE
Healthcare Life Sciences, Little Chalfont, UK; Cat. Nr. RPN2106) accord-
ing to the manufacturer's protocol. All membranes were incubated
with the anti-extracellular-signal-regulated kinases 1/2 (anti-ERK1/2)
(Santa Cruz Biotechnology, USA; Cat. Nr. sc-153) antibody to confirm
equal protein loading. ERK1/2 were used as loading controls since no
changes in total ERK1 and ERK2 expression was detected upon exposure
of the cells to the different drugs [47,48].
With the exception of 1a, 1j, and 1k, the triazenes that had
trifluoromethyl substituent in the structure were selected for this
investigation. This was based on our previous observation that CF3
substituent has the strongest influence on an antiproliferative activity
of 1,3-diaryltriazenes as well as some of N-acyl derivatives to the
tumor cells [18]. In the line with the fact that the fluorinated compounds
could be more biologically active as their non-fluorinated analogues
[51] is also our recent observation that CF3 group has a distinct place
in an anti-mycobacterial activity of 1,3-diaryltriazenes [14].
The structures of the complexes 2 were confirmed by the 1H and 13
C
NMR spectroscopy, a high-resolution time-of-flight mass spectrometry
with an atmospheric pressure electrospray ionization (ESI HRMS) and
microanalysis. It has been previously reported that in the carbon NMR
spectra of 1,3-diaryltriazenes the resonances are broadened to the base-
line [14,39], which could be accounted for by a dynamic triazene-
triazene tautomeric exchange process that proceeds at the 13C NMR
time-scale. In contrast, the 13C NMR resonances of the triazenido ligands
in the compounds 2 show well defined line-shape. For both Ar groups in
the triazenido ligand and the p-cymene ligand, one set of resonances
were observed in the 1H and 13C NMR spectra indicating the symmetry
of the complex 2. The copies of the 1H and 13C NMR spectra are shown in
the Supplementary Information. In ESI HRMS spectra the compounds 2
(measured in H2O/CH3CN) were characterized by the presence of [M–
Cl]+, whereas the ions for [M + H]+ and [M–Cl + CH3CN]+ could also
be observed. The complexes 2 were also characterized by determining
melting points. The melting of the crystals is accompanied by a decom-
position as evident from the TG/DSC measurements for the selected
complexes 2e, f, g (Fig. S1, Supplementary Information).
2.5.5. Determination of DNA binding
The calf thymus ct-DNA was purchased from Sigma-Aldrich, dis-
solved in Na-cacodylate buffer, I = 0.05 M, pH = 7, additionally sonicat-
ed, and the solution was filtered through a 0.45 mm filter. The
polynucleotide concentration was determined spectroscopically as the
concentration of phosphates by ε260 nm = 6600 M−1 cm−1. Circular di-
chroism (CD) experiments were performed in quartz cuvette of 1 cm
path length, by collecting CD spectrum of c(ct-DNA) = 2.86 × 10−6 M
and subsequently adding small portions of compound to obtain ratios
r[compound]/[ct-DNA] = 0.3, 0.5 and 0.7. The CD spectra of the mix-
tures were collected after incubation times of 1 and 10 min to check
any kinetic effect.
Thermal denaturation curves for the ct-DNA and its complexes with
the studied compounds were determined in Na-cacodylate buffer, I =
0.05 M, pH = 7 by following the absorption change at 260 nm as a func-
tion of temperature, as previously described [49,50]. The absorbance of
the compound was subtracted from each curve and the absorbance
scale was normalized. The measured Tm values are the midpoints
of the transition curves determined from the maximum of the first
derivative and checked graphically by the tangent method. The ΔTm
values were calculated subtracting Tm of the free nucleic acid from Tm
of the complex. Every ΔTm value here reported was an average of at
least two measurements. The error in ΔTm is 0.5 °C.
3.2. Crystal structures
The crystals, suitable for an X-ray analysis were obtained by a slow
diffusion of petroleum ether into a solution of 2e and 2h in ethyl acetate.
The structures of both compounds are displayed in Figs. 1 and 2, respec-
tively. The structures of 2e and 2h exhibit a piano-stool geometry. The
two nitrogen atoms of the triazenido bidentate chelating ligand, η6-p-
cymene ligand and chloride ion fulfil the coordination sphere around
the ruthenium atom in both complexes. The compound 2e crystalizes
in orthorhombic Pnma space group with imposed mirror plane through
Ru1, Cl1 and N2 atoms (Fig. 1) while 2h crystalizes in monoclinic P21/n
3. Results and discussion
3.1. Synthesis and characterization
The preparation of the 1,3-diaryltriazenes 1 was accomplished by a
treatment of the appropriately substituted anilines with either sodium
nitrite in hydrochloric acid or with isopentyl nitrite in dichloromethane
Scheme 1. Preparation of triazenido-ruthenium complexes 2.

46
J. Vajs et al. / Journal of Inorganic Biochemistry 153 (2015) 42–48
Table 2
A preparation of the triazenide complexes 2 and the key of the aromatic substituents
(Scheme 1).
Triazene 1
Ar−
Complex 2
Yielda
80
1a
2a
1b
1c
1d
1e
2b
2c
2d
2e
88
74
78
61
Fig. 2. ORTEP plot of 2h with thermal ellipsoids at 30% probability level. Hydrogen atoms
are omitted for clarity.
1f
2f
83
82
91
80
interactions could be observed in the crystal packing of 2e and 2h. The
non-covalent C4–H4 · · · Cl1a [a: −x + 1, y + 1/2, −z + 2] and
C12–H12C · · · Cl1b [b: x–1/2, −y–3/2, −z + 3/2] intermolecular con-
tacts in 2e connect the molecules into 3D supramolecular network
(Fig. S2, Supplementary Information). In 2h the molecules weakly
interact each other through the C5–H5 · · · Cl1a [a: −x + 1, −y,
−z], C19–H19 · · · F4b [b: −x + 1/2, −y + 1/2, z + 1/2], C17–
H17 · · · O3c [c: x–1/2, −y + 1/2, z + 1/2] and C21–H21C · · · O4d
[d: −x + 2, −y, −z] (Fig. S3, Supplementary Information) forming
3D supramolecular array. This 3D network in 2h is further stabilized
by the non-covalent C7–F1 · · · π interaction of 2.97 Å to one of the
aromatic rings (C8–C13) in the crystal structure.
1g
1h
1i
2g
2h
2i
1j
2j
74
42
1k
2k
a
Percent yield of the isolated pure product.
with the whole molecule in the asymmetric unit (Fig. 2). The Ru–N1
bond distances are from 2.080(4) to 2.109(2) Å and the bite angles
N–Ru–N are from 101.7(4)° to 104.8(2)° in both complexes. All bond
lengths and angles in the bidentate chelating system are within the
range of the values described in literature for the related triazenido-
ruthenium(II) complexes [23,25,27]. No significant hydrogen-bonding
3.3. Cancer cell growth inhibition
A cytotoxic effect of the compounds 2 was first evaluated on a
human cervical carcinoma HeLa cells, the cell model system that we
found previously to be suitable for the screening of the new compounds
[18,52]. As evident from Table 3, all compounds strongly inhibited the
growth of the HeLa cells with IC50 below 6 μM. The compound 2g was
the most cytotoxic with IC50 of 0.103 0.006 (after 72 h incubation),
which is over 2000-times higher in comparison to the ruthenium
precursor [RuCl2(η6-p-cymene)]2 (IC50 = 212.97 2.14 [53]).
In general, the cytotoxicity of the triazenido-ruthenium complexes 2
was significantly increased as compared to the parental triazenes [16,
18], confirming the occurrence of a synergistic effect. This is evident
by comparison the data for the complexes 2c, 2d, 2g and 2j with those
for the uncoordinated triazenes 1c, 1d, 1g and 1j, respectively
(Table 3). The most pronounced difference was observed for the
Table 3
An antiproliferative activity of the compounds 2 towards the HeLa cells (IC50 μM SD).
IC50 value after 72 h incubation.
2
IC50 SD
1
IC50
2a
2b
2c
2d
2e
2f
2g
2h
2i
1.543 0.060
0.633 0.038
0.230 0.053
5.923 0.178
0.603 0.015
0.763 0.099
0.103 0.006
0.280 0.035
0.770 0.086
0.203 0.025
2.290 0.184
1c
1d
10.86 0.18
62.2a,b
1g
1j
58.38 2.96a
9.63 0.06
2j
2k
a
Data from the literature [18].
b
Fig. 1. ORTEP plot of 2e with thermal ellipsoids at 30% probability level. Hydrogen atoms
are omitted for clarity. Symmetry code: (i) (i) x, −1.5–y, z.
The highest concentration that could be evaluated, but with no influence on the cell
survival.

J. Vajs et al. / Journal of Inorganic Biochemistry 153 (2015) 42–48
47
Table 4
An antiproliferative activity of the selected ruthenium-triazenide complexes 2c, 2g and 2j
towards various tumor cell lines (IC50 μM + SD). IC50 value after 72 h incubation.
Cell linea
Compound
2c
2g
2j
HeLa
HEp-2
7 T
HCT 116
H460
0.230 0.053
0.226 0.002
0.330 0.037
0.249 0.010
0.300 0.033
0.297 0.015
0.103 0.006
0.108 0.015
0.109 0.006
0.106 0.009
0.117 0.004
0.123 0.018
0.203 0.025
0.180 0.015
0.212 0.020
0.180 0.001
0.208 0.018
0.329 0.174
Fig. 3. A cleavage of PARP in HeLa cells upon 24 and 48 h treatment with two doses of 2g
which reduced the cell survival to 60% or 10% (IC40 and IC90). Control cells were collected
24 and 48 h after the beginning of the treatment. ERK1/2 was used as a loading control.
Precision Plus Protein™ All Blue Prestained Protein Standards (Bio-Rad, Cat. Nr.
1610373) is indicated in the figure. Representative data of three independent experiments
are presented. Densitometric data are shown. Non-treated cells were set as 1.
MDA-MB-435
a
HeLa = human cervical carcinoma cells; HEp-2 = laryngeal carcinoma cells; 7 T =
drug-resistant HEp-2 subline; HCT-116 = colorectal carcinoma cells, H460 = lung
adenocarcinoma cells; MDA-MB-435 = mammary carcinoma cells.
triazene 1g, the activity of which increased upon a coordination to the
ruthenium into 2g by cca. 560-times.
with a lower dose and a shorter period of time, i.e. 24 h, the cleavage of
PARP was observed.
From the triazenido-ruthenium complexes 2 we selected three high-
ly active representatives 2c, 2g and 2j, and examined their cytotoxicity
against laryngeal carcinoma HEp-2 cells and their drug-resistant 7 T
subline, colorectal carcinoma HCT-116 cells, lung adenocarcinoma
H460 cells, and mammary carcinoma MDA-MB-435 cells. As evident
from Table 4, the compound 2g which was mostly cytotoxic for the
Hela cells, strongly inhibited the growth of other tested cell lines as
well. Modest differences in sensitivity between different cell lines
were observed for 2c and 2j. The ratio in IC50 between the most sensitive
and the most resistant tumor cell line was lower than 2 (for 2j). For 2g
similar cytotoxicity against all cells examined was determined. In com-
parison to the other tumor cell lines, the H460 and MDA-MB-435 cells
were more resistant to the selected complexes. It is important to note
that the compounds 2g and 2j were similarly cytotoxic to the parental
laryngeal carcinoma HEp-2 cells and to their drug-resistant 7 T subline,
whereas the growth inhibitory effect of 2c was more expressed on the
parental than on the resistant 7 T cells.
3.6. DNA as possible target
To examine whether DNA is the target for the most cytotoxic com-
pounds 2c, 2g and 2j, as well as the corresponding uncoordinated
triazenes 1c, 1g and 1j, respectively, we studied their interaction with
the double stranded DNA, namely calf thymus ct-DNA. All compounds
and their metal complexes show an aggregation at rather low concen-
trations (starting from 4 to 9 μM). The study of interactions of the
compounds with the ds-DNA (calf thymus; ct-DNA) was performed in
the concentration range with linear absorbance-to-concentration
dependence, i.e. the concentration in which the biological effect of com-
plexes was determined (the IC50
value of all three selected complexes
was below 0.35 μM). In a thermal denaturation experiment these com-
pounds were mixed with the ct-DNA in a ratio r[compound]/[ct-DNA] = 0.3
(pH 7, c(compound) b3.5 μM), at which any DNA binding mode should
give a measurable change in DNA melting point transition. However,
none of the studied molecules stabilized the ct-DNA against thermal
denaturation, indicating that at the biologically relevant conditions
(pH 7, c(compound) = 10 μM) they do not form a stable non-covalent
complex with the double stranded DNA (Fig. S4, Supplementary
Information).
Furthermore, an addition of the ct-DNA caused changes in the UV–Vis
spectra (Fig. S5, Supplementary Information) that could be assigned
to the aggregation process (Fig. S4, Supplementary Information) particu-
larly due to the noticeable increase of the baseline N450 nm, at which
compounds do not absorb light. In the circular dichroism (CD) experi-
ments CD bands of DNA (230–290 nm) did not change significantly
upon the addition of any compound studied, which suggests that the
DNA secondary structure is not affected by a small molecule binding
[54,55]. Moreover, none of the studied compounds showed any specific
CD band N300 nm upon mixing with DNA (Fig. S4, Supplementary
Information), which usually appears for the achiral small molecules
upon uniform binding to the DNA [56–58].
3.4. Influence on the cell cycle
To get some insight into the molecular mechanism involved in the
cytotoxicity of the triazenido-ruthenium complexes, the influence of
2g on the progression of HeLa cells was monitored. This effect was
investigated by a flow cytometry after 24 h and 48 h of the treatment
with two concentrations that in MTT assay reduced the survival of the
cells to 60 or 10% (survival fraction 0.6 and 0.1, respectively).
As shown in Table 5, the high dose of the compound 2g caused a
time and a dose dependent accumulation of the cells in the S phase of
the cell cycle. It was accompanied by an increase of the cells present
in the subG1 fraction, which represents the apoptotic cells.
3.5. Induction of an apoptosis
To further confirm that the compound 2g indeed induces apoptosis,
we explored the cleavage of the apoptosis marker PARP following the
treatment of HeLa cells. As shown in Fig. 3., the compound 2g induced
a dose and time dependent cleavage of PARP. Even upon the treatment
To conclude, thermal denaturation and CD and UV–Vis experiments
suggest a non-specific aggregation of some compounds along DNA close
to 0.1 mM concentrations of dye, with no significant impact on DNA
properties (melting point, CD spectrum). Metal complexes did not
show any interaction with DNA. Thus, the studied compounds do not
show any biologically relevant interaction with ds-DNA.
Table 5
Effects of the compound 2g on the cell cycle of HeLa cells.a
Survival
fraction
24 h
48 h
G0/G1
S
G2M
subG1
G0/G1
S
G2M
subG1
4. Conclusions
0
0.6
0.1
58.58
61.91
29.68
20.54
24.43
58.73
20.88
13.66
11.59
7.33
10.06
14.53
60.39
57.44
31.37
27.79
34.77
55.52
11.82
7.79
13.11
5.97
6.24
22.16
Eleven new triazenido-ruthenium complexes were easily prepared
by combining 1,3-diaryltriazenes with [RuCl2(η6-p-cymene)]2 in the
presence of trimethylamine. All complexes exhibited high cytotoxic ac-
tivity against several human carcinoma cells as well as against the drug-
resistant subline. A selected complex 2g induced time and dose
a
HeLa cells were treated for 24 h and 48 h with 2g, stained with propidium iodide and
analysed by flow cytometry. A cell cycle distribution was assessed as described in the
Experimental section.

48
J. Vajs et al. / Journal of Inorganic Biochemistry 153 (2015) 42–48
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We acknowledge a financial support from the Ministry of Science,
Education and Sport of the Republic of Croatia (Projects 098-0982913-
2748, 098-0982913-2850 and 098-0982914-2918) and the Ministry of
Education, Science and Sport, Republic of Slovenia, the Slovenian
Research Agency (Grant P1-0230; Young Researcher Grant to J.V., and
Grant P1-0175). This work was also partially supported through the
infrastructure of the EN-FIST Centre of Excellence, Ljubljana, Slovenia.
A financial support from the Joint Projects BI-HR/12-13-028 and BI-
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Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jinorgbio.2015.09.005
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