Ruthenium(II) trithiacyclononane complexes of 7,3′,4′-trihydroxyflavone, chrysin and tectochrysin: Synthesis, characterisation, and cytotoxic evaluation

Article abstract of DOI:10.1016/j.ica.2019.01.003

This study describes a simple, two-step method for obtaining ruthenium(II) trithiacyclononane ([9]aneS₃) flavonate complexes in yields ranging from 23% to 34%. With 7,3′,4′-trihydroxyflavone (thflv), a neutral complex, [Ru(II)([9]aneS₃)(DMSO)(thflv)] (1), is formed by coordination at the catechol group. With chrysin (chrys) and tectochrysin (tchrys), Ru(II) binds to the chromenone fragment to form cationic complexes that are isolated as [Ru(II)([9]aneS₃)(chrys)(DMSO)]Cl (2) and [Ru(II)([9]aneS₃)(DMSO)(tchrys)]Cl (3). The structure of the complexes is characterised by FT-IR, NMR and ESI⁺-MS. Furthermore, the flavones and the corresponding complexes, 1–3, were investigated regarding their in vitro cytotoxic activity towards four different human tumour cell lines, PC-3 (prostate), MG-63 (osteosarcoma), MCF-7 and MDA-MB-231 (both breast adenocarcinoma).

Full text of DOI:10.1016/j.ica.2019.01.003

Accepted Manuscript
Ruthenium(II) trithiacyclononane complexes of 7,3’,4’-trihydroxyflavone,
chrysin and tectochrysin: synthesis, characterisation, and cytotoxic evaluation
Joana Marques, Artur M.S. Silva, Maria Paula M. Marques, Susana S. Braga
PII:
S0020-1693(18)31298-2
https://doi.org/10.1016/j.ica.2019.01.003
ICA 18726
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 August 2018
23 December 2018
4 January 2019
Please cite this article as: J. Marques, A.M.S. Silva, M.P.M. Marques, S.S. Braga, Ruthenium(II) trithiacyclononane
complexes of 7,3’,4’-trihydroxyflavone, chrysin and tectochrysin: synthesis, characterisation, and cytotoxic
evaluation, Inorganica Chimica Acta (2019), doi: https://doi.org/10.1016/j.ica.2019.01.003
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Ruthenium(II) trithiacyclononane complexes of 7,3’,4’-
trihydroxyflavone, chrysin and tectochrysin: synthesis,
characterisation, and cytotoxic evaluation
Joana Marques,[a,b] Artur M. S. Silva,[a] Maria Paula M. Marques,[b,c] and Susana S. Braga*[a]
[a] QOPNA Research Unit, Department of Chemistry, University of Aveiro, 3810-193
Aveiro, Portugal
[b] “Molecular Physical-Chemistry” R&D Unit, Faculty of Science and Technology,
University of Coimbra, Portugal
[c] Department of Life Sciences, Faculty of Science and Technology, University of Coimbra,
Portugal
* Corresponding author: Susana Santos Braga, sbraga@ua.pt
Acknowledgements
This work was supported by the University of Aveiro, the University of Coimbra and
FCT/MEC (Fundação para a Ciência e a Tecnologia, Ministério da Educação e da Ciência),
through national founds and, where applicable, co-financed by the FEDER (European Fund
for Regional Development) within the PT2020 Partnership Agreement, through the project
FCT UID/QUI/00062/2013 supporting the QOPNA research unit, through support to the
Portuguese NMR Network and through support to the QFM-UC research unit with the
project UID/MULTI/00070/2013. Support through Project ReNATURE - Valorization of the
Natural Endogenous Resources of the Centro Region (Centro 2020, Centro-01-0145-FEDER-
000007) is gracefully acknowledged.
Competing Interests
The authors have no potential conflict of interest to report.
1

Ruthenium(II) trithiacyclononane complexes of 7,3’,4’-
trihydroxyflavone, chrysin and tectochrysin: synthesis,
characterisation, and cytotoxic evaluation
This study describes a simple, two-step method for obtaining ruthenium(II)
trithiacyclononane ([9]aneS3) flavonate complexes in yields of 23 to 43%. With
7,3’,4’-trihydroxyflavone
(thflv),
a
neutral
complex,
[Ru(II)([9]aneS3)(DMSO)(thflv)] (1), is formed by coordination at the catechol
group. With chrysin (chrys) and tectochrysin (tchrys), Ru(II) binds to the
chromenone fragment to form cationic complexes that are isolated as
[Ru(II)([9]aneS3)(chrys)(DMSO)]Cl (2) and [Ru(II)([9]aneS3)(DMSO)(tchrys)]Cl
(3). The structure of the complexes is characterised by FT-IR, NMR and ESI+-
MS. Furthermore, the flavones and the corresponding complexes, 1-3, were
investigated regarding their in vitro cytotoxic activity towards four different
human tumour cell lines, PC-3 (prostate), MG-63 (osteosarcoma), MCF-7 and
MDA-MB-231 (both breast adenocarcinoma).
Keywords: flavones • ruthenium(II) complexes • trithiacyclononane • cytotoxicity •
cancer cell lines
1. Introduction
Flavonoids are polyphenolic natural compounds occurring in a wide variety of edible plants
and known to benefit human health due to their antioxidant and, in a few cases, anti-
inflammatory action [1]. Flavonoids were recently associated with other interesting
medicinal effects, namely protection against fatty liver [2], cardiovascular disease [3] and
with chemopreventive and chemotherapeutic actions. The vast potential of application of
flavonoids has made them subject of intense scientific research. In the year 2017 alone, the
number of publications on the subject was around half a thousand. In the present work, focus
is given to three natural flavonoids: 7,3’,4’-trihydroxyflavone (thflv), 5,7-dihydoxyflavone or
2

chrysin (chrys), and 5-hydroxy-7-methoxyflavone or tectochrysin (tchrys). 7,3’,4’-
trihydroxyflavone is also named 5-deoxyluteolin and it can be found in white clover,
Trifolium repens [4] and in alfalfa, Medicago sativa [5]. It also occurs in Butea monosperma,
a Thailandese fabaceae tree commonly known as ‘flame of the forest’ for its attractive, large,
bright orange-reddish flowers [6]. Its application as an anti-inflammatory compound is under
evaluation [7]. Chrysin occurs naturally in propolis [8], honey [9] and several species of the
Passiflora genus [10], and it exhibits cytotoxic activity against human adenocarcinoma [11]
and prostate cancer cell lines [12, 13]. Methoxyflavones and polymethoxyflavones are also in
the spotlight for their recently discovered growth-inhibitory properties on human colon
cancer cell lines [14, 15]. Tectochrysin (tchrys) is isolated from Kaemperia parviflora, or
Thailand’s black ginger, a herbaceous plant of the Zingiberacea family native to Thailand
[16]. It was shown to induce cell death of colorectal cancer cells by the ROS-mediated
mitochondrial apoptosis pathway [14].
Flavonoids have well-known metal chelating abilities, namely with iron, copper, and
zinc, which may cause their depletion in vivo but may also be used to design new metal drugs
active towards a variety of diseases related to the biological role of metals [17]. For instance,
several aminoflavone complexes with cytotoxic activity are described in the literature,
namely cis-dichlorobis(3-aminoflavone)platinum [18], cis-dichlorobis(3-imino-2-R-O-
flavanone)ruthenium(II) (where R = CH3 or CH2CH3) [19], cis-dichloro(3-nitrosoflavone)(3-
hydroxyiminoflavanone)ruthenium(II) [20], and dichloro(pcym)(6- or 7-amino-
flavone)ruthenium(II), where pcym is η6-p-cymene [21]. Ruthenium complexes with O,O’-
donor ligands are less common due to the lower affinity of this metal ion to bind oxygen,
which makes their preparation more challenging. A few known examples are trans-
bis(DMSO)bis(4’-R-flavone)ruthenium(II), with R = Cl, NO2, OCH3 or N(CH3)2 [22],
bis(2,2’-bipyridine)(flavonolate)ruthenium(II) [23], and two families of organometallic
3

complexes, (pcym)(chlorido)(4’-R-3-hydroxyflavonate)ruthenium(II), with R = H, Cl, F [24],
and (pcym)(halogeno)(4’-R-3-hydroxyflavonate)ruthenium(II), with R = H, Cl and halogen =
Br, I [25]. Ruthenium complexes are interesting alternatives to platin compounds for
antitumoral chemotherapy. This is associated with their more versatile ligand-exchange
abilities, in tandem with the octahedral geometry (instead of planar four-coordinated Pt) and
the possibility of existing in two redox states in vivo, (+2 and +3 states) [26]. The complex
indazolium [trans-Ru(III)Cl4bisindazole], commonly known as KP1019 and first described
in the late 1990s [27], attracted interest for its excellent in vitro apoptotic activity against
colorectal cancer [28]. To date, it remains a leading example of transition to the clinic,
having successfully overcome phase I clinical trials and being presently under phase II
studies [29]. Within Ru(II) complexes, emerging families include complexes with facial
arene ligands, namely the RAPTA family (from Ruthenium Arene PTA), based on complex
RAPTA-C, [Ru(II)Cl2(1,3,5-triaza7-phosphatricyclo-[3.3.1.1]decanephosphine)(pcym)] [30].
A few RAPTA complexes have presently completed preclinical trials [31-33]. With the
demonstration that the facial arene ligand is not essential to cytotoxicity and it can be
replaced by other ligands such as the face-capping trithiacyclononane ([9]aneS3) [34], a new
family of cytotoxic Ru(II) complexes emerged [35, 36]. We have developed and tested
several Ru(II)([9]aneS3) complexes, both with aza ligands [37, 38] and with natural ligands
such as glycine [39] and curcumin [40]. Within those with aza-ligands, highlight goes to
[Ru(II)([9]aneS3)Cl(phpz)], where phpz = 5-(2-hydroxyphenyl)-3-[(4-methoxystyryl)-
pyrazole), that has a good cytotoxicity against prostate cancer (PC-3) and breast cancer
(MDA-MB-231) cells [37].
The present paper reports the two-step syntheses of three new Ru(II)
trithiacyclononane ([9]aneS3) complexes with three naturally occurring flavones. In the
particular case of 7,3’,4’-trihydroxyflavone, having a catechol in the B-ring position,
4

coordination occurs via the catechol moiety to form a five-membered ring. This is, to the best
of our knowledge, the first report of ruthenium coordination via the catechol group in large
polyphenolic ligands. The other two complexes feature O,O’-coordination via the chromen-
4-one moiety to Ru(II), thus yielding a six-membered heterocyclic ring containing the Ru(II)
atom. The in vitro cytotoxic activities of these flavonoids are largely unexplored, and for this
reason they were evaluated against four human cancer cell lines, namely prostate cancer (PC-
3), breast cancer (both hormone-dependent (MCF-7) and triple-negative (MDA-MB-63)) and
osteosarcoma (MG-63). The cytotoxicity of the Ru(II) complexes was also assessed,
revealing that metal coordination causes a reduction of the antitumor activity.
Figure 1. Structure and atom labelling of the ligands used in the present work. The available
sites for coordination to Ru(II) are highlighted with dashed lines: a) 7,3’,4’-
trihydroxyflavone binds ruthenium via the catechol; b) chrysin and tectochrysin coordinate in
the positions 4 and 5.
5

2. Experimental
2.1. Chemicals
All the chemicals were used as received from the distributors. 5,7-dihydroxyflavone, also
known as chrysin, was acquired from Alfa Aesar (Karlsruhe, Germany) and 7,3’,4’-
trihydroxyflavone was purchased from the Indofine Chemical Company (Hillsborough,
USA). 5-hydroxy-7-methoxyflavone, with the common name tectochrysin, is not
commercially available, therefore it was prepared as described in 2.3. Tetrabutylammonium
hydroxide (TBAOH) methanol solution (1M) was acquired from Sigma Aldrich (Sintra,
Portugal). [Ru(II)([9]aneS3)Cl2(S-DMSO)] was prepared according to the procedures
described in our previous work [37–42]. Our procedure is adapted from the method described
in 1994 by Landgrafe and Sheldrick [43], replacing the solvent CHCl3 with a non-toxic
solvent, ethanol, and increasing the reaction time to 4h [42].
Solvents were at least of analytical grade and used without further purification.
The cell lines – MCF-7 (estrogen receptor-positive human epithelial breast
adenocarcinoma), MDA-MB-231 (human estrogen receptor-negative epithelial breast
adenocarcinoma), PC-3 (castration-resistant prostate cancer) and MG-63 (osteosarcoma) –
were purchased from the European Collection of Authenticated Cell Cultures (ECACC,
https://www.phe-culturecollections.org.uk/collections/ecacc.aspx)
2.2. Instrumentation
13
1H and C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance
300 spectrometer at 300.13 MHz and 75.47 MHz, respectively, at room temperature. 13C
assignments were made based on 2D heteronuclear single quantum coherence spectroscopy
1
(HSQC, H,13C), and heteronuclear multiple bond correlation (HMBC, delay for long-range
JC/H couplings were optimised for 7 Hz) experiments. The residual protic solvent signal
6

1
(DMSO-d6: H δ 2.50 ppm and 13C δ 39.5 ppm) was used as internal reference. Chemical
shifts are quoted in parts per million (ppm) and the coupling constants (J) in Hertz (Hz).
Fourier-transform infrared (FT-IR) spectra, in the 4000-380 cm-1 range, were
collected as KBr pellets using a Unicam Mattson Mod 7000 FTIR spectrophotometer, by
averaging 64 scans at a maximum resolution of 2 cm-1. In a typical preparation, 2 mg of
sample were mixed in a mortar with 200 mg of KBr (Sigma-Aldrich, ≥ 99%).
Mass spectra were recorded by C. Barros on a Micromass® Q-TOF 2 mass
spectrometer with electrospray ionisation (ESI+-MS), using methanol as the solvent. The m/z
ratios presented in the characterisation data of the sample are monoisotopic, calculated using
12
14
16
32
35
the mass of the most abundant natural isotope of each element (1H, C, N, O, S, Cl
and 102Ru).
Elemental analysis of the newly synthesised complexes was performed by M.
Marques in a TruSpec 630-200-200 CHNS Analyser.
The cell optical density results of the MTT assay were measured using a BioTek
μQuant MQX200 UV-Visible spectrophotometer equipped with the Gen5 software.
2.3. Synthesis of 5-hydroxy-7-methoxyflavone (tchrys)
A solution of chrysin (101.7 mg, 0.40 mmol) and potassium carbonate (165.9 mg, 1.2 mmol)
in refluxing acetone (5 mL) was treated with dimethyl sulphate (41.6 μL, 0.44 mmol). The
mixture was refluxed for one hour to form a yellow precipitate, which was filtered and
subject to column chromatography in silica-gel using as eluent a mixture of 3% methanol in
dichloromethane. Further purification was carried out by recrystallisation in dichloromethane
with 5% methanol over three days. Crystals were washed with ethanol (10 mL) and dried.
Yield: 85 mg (79%).
FT-IR selected bands ν(tilde) = 3340 m (νO–H), 3088 m, 3071 m, 3058 m, 3014 m, 2980 m,
2951 m, 2924 m, 2900 m, 2843 m, 1669 vs (νC=O), 1626 s, 1609 vs (νC2=C3), 1588 vs, 1567 m
7

(νC2=C3–C4), 1495 s, 1453 s, 1436 s, 1423 m, 1352 vs, 1309 m, 1247 m, 1202 vs, 1170 s, 1161
vs, 1121 s, 1103 m, 1041 s, 906 m, 866 s, 850 s, 828 m, 808 s, 721 s, 695 s, 646 s, 566 m,
500 m, 469 m, 390 m.
1H NMR (300.13 MHz, DMSO-d6): δ(ppm) = 12.82 (1H, s, OH-5), 8.13-8.09 (2H, m, H-
2’,6’), 7.66-7.55 (3H, m, H-3’,4’,5’), 7.05 (1H, s, H-3), 6.83 (1H, d, J 2.3 Hz, H-8), 6.41 (1H,
d, J 2.3 Hz, H-6), 3.88 (3H, m, 7-OCH3). Data is in good agrement with literature values
[44].
13C NMR (75.47 MHz, DMSO-d6): δ (ppm) = 182.2 (C-4), 165.4 (C-7), 163.5 (C-2), 161.2
(C-5), 157.4 (C-9), 132.3 (C-4’), 130.6 (C-1’), 129.2 (C-3’,5’), 126.5 (C-2’,6’), 105.4 (C-3),
105.0 (C-10), 98.2 (C-6), 92.9 (C-8), 56.2 (7-OCH3).
2.4. Synthesis of [Ru(II)([9]aneS3)(S-DMSO)(7,3’,4’-trihydroxyflavone)] (1)
A solution of thflv (94.5 mg, 0.35 mmol) in refluxing ethanol (15 mL) was treated with one
equivalent of tetrabutylammonium hydroxide (350 μL, 0.35 mmol). The colour of the
solution changed from light yellow to orange. After 20 minutes, the complex
[Ru([9]aneS3)Cl2(DMSO)] (150 mg, 0.35 mmol) was added and the reaction mixture was left
under reflux for 48 h (Scheme 1). During this time the solution became dark orange and a
dark green precipitate was formed. The hot solution was cannula-filtered and the remaining
solid was washed with ethanol (20 mL), diethyl ether (20 mL) and dried (90 mg, 38% yield).
Elemental analysis for [Ru(C6H12S3)(C15H8O5)(C2H6SO)]·3(H2O) (Mr = 681,8): C, 40.52; H,
4,73 %. Found: C, 40.07; H, 4.28 %.
FT-IR selected bands ν(tilde) = 3443 m (νO–H), 2985 m, 2949 m, 2914 m (all three νC–H),
1624 vs (νCO + νCC (thflv)), 1595 m, 1560 s (both νCC (thflv)), 1490 vs (non-assigned),
1449 m, 1413 m (both δC–H([9]aneS3)), 1305 s, 1257 s, 1160 m (all three δC–H (DMSO)), 1090 s
(νS=O), 721 w (νC–S (DMSO)), 680 w, 650 vw (both νC–S ([9]aneS3)), 490 w, 461 vw (both νRu–
S ([9]aneS3)), 426 w (νRu–S (DMSO)), 397 vw (νRu–O).
8

ESI+-MS (MeOH) m/z (relative intensity %): 522 ([Ru([9]aneS3–CH2CH2)(thflv)]+, 100); 628
([Ru([9]aneS3)(S-DMSO)(thflv)]+, 95).
1H NMR (300.13 MHz, DMSO-d6): δ(ppm) = 10.58 (1H, br s, OH-7), 7.78 (1H, d, J 8.7 Hz,
H-5), 6.90 (1H, dd, J 2.0, 8.4 Hz, H-6'), 6.85 (2H, d, J 2.0 Hz, H-2',8), 6.81 (1H, dd, J 2.0,
8.7 Hz, H-6), 6.35 (1H, d, J 8.4 Hz, H-5'), 6.30 (1H, s, H-3), 3.19 (6H, s, SO(CH3)2), 2.87–
2.58 (12H, m, CH2 of trithiacyclononane).
13C NMR (75.47 MHz, DMSO-d6): δ (ppm) = 176.0 (C-4), 170.1 (C-4'), 165.0 (C-2), 163.5
(C-3'), 162.4 (C-9), 157.5 (C-7), 125.9 (C-5), 116.8 (C-10), 115.7 (C-1'), 114.7 (C-5',6'),
113.9 (C-6), 110.5 (C-2'), 102.1 (C-8), 100.8 (C-3), 43.6 (SO(CH3)2), 34.1, 31.9 and 30.7
(CH2 of trithiacyclononane).
Scheme 1. Synthetic procedure used in the preparation of the complex 1.
2.5. Synthesis of [Ru(II)([9]aneS3)(chrysin)(S-DMSO)]Cl (2)
A solution of 5,7-dihydroxyflavone or chrysin (63.5 mg, 0.25 mmol) in refluxing methanol
(20 mL) was treated with half equivalent of tetrabutylammonium hydroxide (125 μL, 0.125
mmol). The solution turned from light to dark yellow. After 20 minutes, the complex
precursor [Ru([9]aneS3)Cl2(S-DMSO)] (107.5 mg, 0.25 mmol) was added affording a light
orange solution, which was allowed to reflux for 24 h (Scheme 2). The volume of the
solution was reduced to roughly one half and a yellow solid was formed; this solid was
identified as unreacted precursor [Ru([9]aneS3)Cl2(S-DMSO)]. The supernatant was filtered
through a cannula, collected and added with 60 mL of diethyl ether. After storage at 4ºC for
one week, the product [Ru(II)([9]aneS3)(chrys)(S-DMSO)]Cl (2) was isolated as a dark
9

orange precipitate. The product was filtered, washed with cold ethanol (5 mL), diethyl ether
(20 mL) and dried (40 mg, 23% yield).
Elemental analysis for [Ru(C6H12S3)(C15H9O4)(C2H6SO)]Cl·1.6H2O (Mr = 675.0): C, 40.80;
H, 4,50 %. Found: C, 41.00; H, 4.63 %.
FT-IR selected bands ν(tilde) = 3147 m (νO–H), 2992 sh, 2975 m, 2924 m (all three νC–H),
1631 vs (νCO + νCC (chrys)), 1598 s, 1577 s, 1528 vs (all three νCC (chrys)), 1450 m, 1413
m (both δC–H ([9]aneS3)), 1297 w, 1266 m, 1165 s (all three δC–H (DMSO)), 1099 m, 1082 s
(both νS=O), 716 vw (νC–S (DMSO)), 678 w, 653 w (both νC–S ([9]aneS3)), 492 vw, 457 vw
(both νRu–S ([9]aneS3)), 424 m (νRu–S (DMSO)), 400 vw (νRu–O).
ESI+-MS (MeOH) m/z (relative intensity %): 507 ([Ru([9]aneS3–CH2CH2)(chrys)]+, 100);
613 ([Ru([9]aneS3)(chrys)(S-DMSO)]+, 49).
1H NMR (300.13 MHz, DMSO-d6): δ (ppm) = 10.54 (1H, s, OH-7), 8.07-8.02 (2H, m, H-
2',6'), 7.64-7.52 (3H, m, H-3',4',5'), 7.05 (1H, s, H-3), 6.24 (1H, d, J = 2.3 Hz, H-8), 6.11
(1H, d, J = 2.3 Hz, H-6), 2.96 (3H, s, SO(CH3)2), 2.95 (3H, s, SO(CH3)2), 2.93-2.58 (12H, m,
CH2 of trithiacyclononane).
13C NMR (75.47 MHz, DMSO-d6): δ (ppm) = 178.3 (C-4), 168.2 (C-5), 164.3 (C-7), 159.4
(C-2), 158.9 (C-9), 132.0 (C-4'), 130.4 (C-1'), 129.3 (C-3',5'), 126.2 (C-2',6'), 106.7 (C-10),
105.2 (C-3), 103.3 (C-6), 91.1 (C-8), 43.1, 42.4 (SO(CH3)2), 34.6, 34.1, 32.5, 32.4, 29.9 and
29.8 (CH2 of trithiacyclononane).
10

Scheme 2. Synthetic procedure used in the preparation of complexes 2 and 3 (herein
represented in their cationic form).
2.6. Synthesis of [Ru(II)([9]aneS3)(S-DMSO)(tectochrysin)]Cl (3)
A solution of tchrys (18.8 mg, 0.07 mmol) in refluxing methanol (13 mL) was treated with
one equivalent of tetrabutylammonium hydroxide (70 μL, 0.07 mmol). The solution colour
changed from light yellow to fluorescent yellow. After 20 min, the complex
[Ru([9]aneS3)Cl2(S-DMSO)] (30 mg, 0.07 mmol) was added causing the solution to turn
orange. The reaction mixture was left under reflux for 24 h (Scheme 2). After cooling to
room temperature, 20 mL of diethyl ether and 20 mL of dichloromethane were added to the
reaction mixture, but rather than isolating a solid product, an oily product, with dark orange
colour, was obtained. This product was isolated from the solution and recrystallised over one
week in dichloromethane. This allowed isolating a powdered solid that was filtered, washed
with diethyl ether (10 mL) and vacuum-dried (20 mg, 43% yield).
Elemental analysis was not done due to low amount of sample.
FT-IR selected bands ν(tilde) = 3405 m (νO–H), 2988 m, 2961 m, 2932 m, 2910 m (all νC–
H), 1636 vs (νCO + νCC (tcrys)), 1599 s, 1578 m, 1549 s (all three νCC (tcrys)), 1451 m,
1431 m (both δC–H ([9]aneS3)), 1160 m (δC–H (DMSO)), 1099 sh, 1087 m (νS=O), 719 w (νC–S
(DMSO)), 681 w, 657 w (both νC–S ([9]aneS3)), 491 w, 457 vw (νRu–S ([9]aneS3)), 424 w (νRu–S
(DMSO)), 398 vw (νRu–O).
ESI+-MS (MeOH) m/z (relative intensity %): 521 ([Ru([9]aneS3–CH2CH2)(tchrys)]+, 100);
627 ([Ru([9]aneS3)(S-DMSO)(tchrys)]+, 57).
1H NMR (300.13 MHz, DMSO-d6): δ (ppm) = 8.08 (2H, m, H-2',6'), 7.65-7.54 (3H, m, H-
3',4',5'), 7.11 (1H, s, H-3), 6.44 (1H, d, J 2.5 Hz, H-8), 6.26 (1H, d, J 2.5 Hz, H-6), 3.80 (3H,
s, 7-OCH3), 2.97 (6H, s, SO(CH3)2), 2.94-2.66 (12H, m, CH2 of trithiacyclononane).
13C NMR (75.47 MHz, DMSO-d6): δ (ppm) = 178.8 (C-4), 168.4 (C-5), 165.1 (C-7), 159.8
(C-2), 158.7 (C-9), 132.1 (C-4'), 130.2 (C-1'), 129.3 (C-3',5'), 126.3 (C-2',6'), 107.4 (C-10),
11

105.3 (C-3), 101.3 (C-6), 90.1 (C-8), 55.6 (7-OCH3), 43.1, 42.5 (SO(CH3)2), 34.7, 34.1, 32.6,
32.3, 30.0 and 29.8 (CH2 of trithiacyclononane).
2.7. Cytotoxicity assays
2.7.1. Preparation of the solutions
All stock solutions were freshly prepared (details described in Table 1). Water soluble
compounds were prepared in phosphate buffered saline (PBS) solution (140.0×10-3 M NaCl,
2.7×10-3 M KCl, 1.5×10-3 M KH2PO4, 8.1×10-3 M Na2HPO4 in Mili-Q water, pH 7.4) and
sterilised by filtration. Poorly water soluble agents were prepared in 50% DMSO in PBS or
in 100% DMSO. Further dilutions of these stock solutions were made with the corresponding
percentages of each solvent, in order to obtain the final concentrations required for the
biological assays (Table 1). Note that the maximal concentration tested was 200 μM for the
compounds prepared in DMSO/PBS (50/50), and 100 μM for those prepared in 100%
DMSO, thus assuring that, in all experiments and for both control and treated cells, the
highest concentrations of DMSO in each well was 0.5% (v/v). Previous studies (which are
not presented) showed that cell viability is not affected upon exposure to 0.5% DMSO, for all
four cell lines studied. Complex 1 was tested to the limit of its solubility in DMSO (18.5
μM).
Table 1. Composition and concentration of the stock solutions and of the highest
concentrations tested for the flavones and complexes 1-3.
Compound
Stock Solution
Highest concentration
7,3',4'-thflv
Complex 1
Chrysin
40 mM, DMSO
3.7 mM, DMSO
100 μM
18.5 μM
100 μM
200 μM
100 μM
200 μM
20 mM, DMSO
Complex 2
Tectochrysin
Complex 3
20 mM, DMSO/PBS
20 mM, DMSO
20 mM, DMSO/PBS
2.7.2. Cell culture
12

Four human cancer cell lines were tested in the present work, representing three types of
cancer: osteosarcoma (MG-63), prostate (PC-3) and breast – both hormone-dependent (MCF-
7) and triple-negative, highly metastatic (MDA-MB-231). The cell lines were cultured as
monolayers and maintained at 37°C, in a humidified incubator under 5%, and sub-cultured
twice a week. Cells were harvested upon addition of trypsin/EDTA (0.05% trypsin/EDTA
solution).
MG-63 cells were grown and maintained in MEM medium supplemented with 10%
inactivated FBS, non-essential amino acids (1 mM), 1% penicillin-streptomycin and sodium
pyruvate (1 mM). PC-3 cells were grown and maintained in RPMI 1640 medium
supplemented with 1,5 g of NaHCO3, 10% inactivated FBS and 1% penicillin-streptomycin.
The MCF-7 cells were maintained in DMEM medium supplemented with 1.5 g of NaHCO3,
10% inactivated FBS and 1% penicillin-streptomycin. The MDA-MB-231 cells were grown
and maintained in RPMI 1640 medium, supplemented with 1.5 g of NaHCO3, 10%
inactivated FBS, non-essential amino acids (1 mM), 1% penicillin-streptomycin and sodium
pyruvate (1 mM).
2.7.3. Toxicity and cell viability assays
Cell viability was assessed by the mitochondrial dehydrogenase activity colorimetric
assay (MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) [45]. Three
independent experiments with triplicates for each drug concentration were performed. Cells
were seeded (MG-63 at 2.5×104 cells/cm2, PC-3 at 1.5×104 cells/cm2, MCF-7 at 3.0×104
cells/cm2 and MDA-MB-231 at 1.5×104 cells/cm2) in 48-well plates (500 μl/well) and
incubated at 37 ºC. After allowing cells to adhere for 24 hours, different concentrations of the
test compounds were added. In the control group the corresponding percentages of solvent,
PBS, DMSO or 50% DMSO in PBS were added. Cisplatin was used as a positive control.
13

The plates were incubated for 72 hours, followed by the addition of MTT solution (5 mg/mL
in PBS) to each well (final concentration 0.5 mg/mL) and incubated for 2 hours. The MTT
containing medium was removed and 200 μl of DMSO was added to each well in order to
dissolve the purple formazan crystals formed by MTT reduction in live viable cells. The
plates were gently homogenised using circular motion at room temperature, to dissolve the
precipitate. The optical density (OD) was then measured at 570 nm.
2.7.4. Statistical analysis
The software GraphPad PRISM 5.00 for Windows (GraphPad Software, San Diego,
California, USA, www.graphpad.com) was used for the analysis of the data from the cell
growth inhibition assays. Results are expressed as a mean and standard deviation obtained
from three independent experiments (n=3), each comprising three replicate measurements
performed for each concentration of each compound tested plus the untreated control.
Statistical analysis was performed by one-way ANOVA followed by the Dunnett's post hoc
test for statistical comparison between the experimental data, p-values less than 0.05 having
been considered as significant. The IC50 values were determined for the compounds which
presented a significant statistical difference relative to the control. The results for each
compound were fitted using nonlinear regression analysis, in sigmoidal dose-response curves
(variable slope).
3. Results and Discussion
3.1. Synthesis of tectochrysin or 5-hydroxy-7-methoxyflavone (tchrys)
Tectochrysin occurs naturally in a variety of plants but it is still not available as a commercial
flavone. This way, for it to be used as a ligand in the ruthenium complexes herein described,
14

it had to be prepared by methylation of chrysin. The reaction is easily achieved using
dimethyl sulphate ((CH3O)2SO2) as the alkylating agent. The product, tectochrysin, was
isolated and purified by crystallisation and its structure was verified by NMR, which is in
good agreement with literature data [44].
3.2. Synthesis of the complexes
The complexes are prepared by a two-step procedure. The flavonol ligand was dissolved in
either methanol or ethanol according to its solubility and treated with one equivalent (or half
equivalent, in the case of chrysin) of base, tetrabutylammonium hydroxide (TBAOH), to
afford the corresponding flavonolate. To the same solution is then added
[Ru([9]aneS3)Cl2(DMSO)] in equimolar amounts. Noteworthy, 7,3’,4’-trihydroxyflavone
(thflv) affords a neutral complex, [Ru(II)([9]aneS3)(DMSO)(thflv)] (1), with a yield of 38%.
The same procedure, using two equivalents of TBAOH (instead of one) also leads to the
formation of the complex 1 with similar yield (37%). Chrysin (chrys) and tectochrysin
(tchrys) afford two monocationic complexes, [Ru(II)([9]aneS3)(chrys)(DMSO)]Cl (2) and
[Ru(II)([9]aneS3)(DMSO)(tchrys)]Cl (3), in moderate yields: 23% for 2 and 43% for 3. For
comparison, the yield reported for [Ru(II)Br(pcym)(3-hydroxyflavonate)] was 47% [25] and
that of [Ru(II)Cl(pcym)(3-hydroxyflavonate)] was 71% [24].
3.3. Infrared spectroscopy
The most relevant FT-IR band frequencies of the powdered complexes, in comparison with
those of the flavones, are presented in the Table 2. Complexation affected mostly the bands
of the flavonoids, in particular those attributed to stretching modes of the ketone group and
the aromatic rings. The assignment of vibrational frequencies of chrysin was carried out by
Sundaraganesan et al., by combining experimental methods and quantum mechanics [46].
The authors explain that the C=O bond in the pyrone ring has a large dipole moment and a
15

strong degree of conjugation, giving rise to two bands at 1612 and 1655 cm-1. Note also that
these bands have a mixed character, comprising ν(CC) and ν(C=O) contributions [46]. The
same observations apply to the bands at 1626 and 1669 cm-1 of tectochrysin. In the
corresponding complexes, 2 and 3, the carbonyl stretch appears as a single band at 1631 and
1636 cm-1, respectively, which is associated with changes in polarisation brought about by
the formation of a six-membered ring with ruthenium (in combination with the deprotonated
C5-O- of ring A). Other reported complexes with O,O’-coordination, including a bis-flavone
ruthenium complex [47] and a curcuminate ruthenium complex [40], exhibited a red shift of
the carbonyl upon metal coordination.
In 7,3’,4’-trihydroxyflavone, a single band is observed for the carbonyl [48], which
appears very slightly blushifted (by only 2 cm-1) in the corresponding ruthenium complex, 1.
This is likely associated with the distinct geometry of coordination, which occurs via the
catechol group and thus has a lower influence on the chromenone group frequencies.
Regarding the backbone of the complexes 1-3, three bands are worth mentioning.
These are the Ru–S stretching band, around 424 cm-1, which helps confirm the presence of
coordinated trithiacyclononane, a band around 397–400 cm-1 that can be tentatively assigned
to the Ru–O stretch, which is expected to occur in the 460–400 cm-1 region [49], and the S=O
stretch of coordinated DMSO that confirms this ligand is retained upon coordination with the
flavones. Note also that the S=O stretching band in complexes 1-3 is found between 1082
and 1099 cm-1 (Table 2), that is, blushifted in regard to free DMSO (at 1055 cm-1), thus
indicating S-coordination; in the case of O-coordination the band would be expected to
appear redshifted to the 862 - 997 cm-1 interval [50].
16

Table 2. Selected FT-IR vibrational frequencies (in cm-1) for 7,3’,4’-trihydoxyflavone (thflv), chrysin (chrys),
tectochrysin (tchrys) and the complexes 1-3.
thflv a
chrys b
tchrys
Description
1
2
3
ν(C=O) + ν(CC)
ν(CC)
1622
1624
1655, 1612
1595, 1577,
1631
1598, 1577,
1528
1669, 1626
1609, 1588,
1636
1599, 1578,
1549
1410
1099, 1087
424
1598, 1568
1595, 1560
1500
—
—
—
—
1567
—
—
—
—
—
—
—
—
1413
1090
426
1413
1082
424
400
δ(CH), [9]aneS3
ν(SO)
ν(Ru–S), DMSO
397
398
ν(Ru–O) c
a 7,3’,4’-trihydroxyflavone group frequencies are assigned according to the report of De Carvalho et al. [48].
b Chrysin group frequencies are assigned according to the work of Sundaraganesan et al. [46].
c The Ru-O stretch is assigned with base on the report of M. Ashok et al. [49].
3.4. Mass spectrometry
The ESI+-MS spectra of the complexes were collected in methanol. The spectrum of the
complex 1 shows two major peaks at m/z 522 and 628, which correspond respectively, to a
fragment and the molecular cation, [Ru([9]aneS3)(DMSO)(thflv)]+. The fragment is
generated by loss of one ethylene unit of the macrocycle along with the coordinated DMSO,
[Ru([9]aneS3–CH2CH2)(thflv)]+. Such type of fragmentation pattern is typical of
ruthenium(II) trithiacyclononane complexes, as demonstrated by the studies developed by the
group of Santana-Marques [51, 52]. The same fragmentation pattern is observed for the
complexes 2 and 3. Besides the peaks corresponding to the molecular cation, found at m/z
613 for 2 and 627 for 3, another intense peak in each spectrum is found respectively at m/z
507 and 521, and attributed to the fragment [Ru([9]aneS3–CH2CH2)(flavonate)]+ (flavonate =
chrys, tchrys) generated by loss of one ethylene of the macrocycle and the labile ligand
DMSO. For all the complexes under study, the isotopic distribution for each set of peaks is
96
99
characteristic of ruthenium-bearing species, with ratios of c.a. 5% for Ru, 14% for Ru,
16% for 100Ru, 30% for 102Ru and 15% for 104Ru. This distribution tallies well with the
theoretical models for the empirical formula of each species, as well as with the reported
experimental data for Ru stable isotopes [53].
17

3.5. NMR
Solution phase NMR determined the structure and geometry of coordination of the
complexes 1-3 and helped confirm the findings resulting from FT-IR data that show the
13
presence of DMSO in the first coordination sphere (1H and C spectra are presented in the
Electronic Supplementary Information).
Complex 1 presents small shifts to lower frequencies for several proton signals of
thflv when compared to the free ligand (Figure 2 and Table 3). Indeed, H-5, H-6 and H-8
shift ca. 0.1 ppm, H-3 shifts 0.3 ppm and H-2’, H-5’ and H-6’ shift ca. 0.5 ppm (refer to
Figure 1 for labeling). The shielding effect is stronger in the protons of ring B due to the
increase of the electronic density in the 3' and 4' positions as a result of the coordination to
Ru(II). In turn, the OH-7 is shifted upfield by 0.19 ppm. The aliphatic region of the spectrum
presents almost no shifts in comparison with the Ru(II) precursor. The methyl groups of the
S-coordinated DMSO appear as a single signal at δ 3.19 ppm and the macrocyclic protons as
a multiplet in the δ 2.87-2.58 ppm range.
Figure 2. 1H NMR spectra in DMSO-d6 of a) thflv and b) complex 1.
18

Table 3. 1H NMR chemical shifts (in ppm) of complex 1 in DMSO-d6,
in comparison with those of the pure flavone ligand.
1H
7,3’,4’-thflv
Complex 1
H-2'
OH-3'
OH-4'
H-5'
H-6'
H-3
7.45 – 7.30 (m)
9.40 (s)
6.85 (d)
n.o.
n.o.
6.35 (d)
6.90 (dd)
6.30 (s)
9.83 (s)
6.91 – 6.87 (m)
7.45 – 7.30 (m)
6.61 (s)
H-5
H-6
OH-7
H-8
CH3-DMSO
7.86 (d)
7.78 (d)
6.81 (dd)
10.58 (br s)
6.85 (d)
3.19 (s)
6.91 – 6.87 (m)
10.77 (s)
6.93 (d)
—
CH2-[9]aneS3
—
2.87 – 2.58 (m)
The DMSO-d6 signal (2.50 ppm) was used as the internal reference.
n.o. – not observed
The low solubility of the complex 1, even in DMSO, has hampered the detection of
aromatic carbons and for this reason the data from 2D NMR experiments (HSQC and HMBC
spectra, presented in the Electronic Supplementary Information) was vital towards
2/3
identification and attribution of attribution of the carbon resonances.
J
C-H
HMBC
correlations allowed identifying the signals of C-2 (H-6’, H-3  C-2), C-4 (H-5, H-3  C-
4), C-7 (H-5, H-6  C-7), C-9 (H-5  C-9), C10 (H-8, H-3  C-10), and C-1’ (H-2’, H-5’
 C-1’). As expected, many 13C resonances of coordinated thflv are shifted in comparison to
the signals of free thflv. The full list of values, for the complexes and the free flavones, is
indicated in the Table 5. We highlight the strong shift observed for the C-3’ and C-4’
resonances (ring B), with values of c.a. 20 ppm that result from the direct coordination of
ring B to Ru(II).
1
Complex 2 features significant shifts in the H NMR signals of the rings A and C of
chrysin, as listed in the Table 4 (atom and ring labelling in Figure 1; spectrum in the Figure
S3 of the Electronic Supplementary Information). The formation of a 6-membered ring
between Ru(II), the deprotonated OH-5 and the 4-carbonyl restores the electronic density of
the C2=C3 unsaturated bond, inducing an anisotropic shift of the H-3 resonance to higher
19

frequencies. The H-6 signal appears deshielded, which evidences the decrease in the
electronic density due to the ligand-to-metal sigma (σ) donation. Such effect is known to
attenuate with distance [54] and thus it affects the other protons to a lesser extent. The
aliphatic region of the spectrum presents two singlets at δ 2.95 and 2.96 ppm, attributed to
two non-equivalent methyl groups of an S-coordinated DMSO and a multiplet in the δ 2.93-
2.58 ppm range for the methylene protons of the macrocycle. The shifts of the two DMSO
methyl groups are different from those of the precursor, [Ru(II)([9]aneS3)Cl2(S-DMSO)], and
of those observed for the complex 1, consistent with the different coordination environment
in 2 and the rotational constrains for the DMSO ligands (that result from the presence of the
bulkier flavonate ligand in the coordination sphere). Regarding 13C resonances (Figure S4
and Table 3), chrysin signals were assigned by 2/3JC-H HMBC correlations, namely C-2 (H-6’,
H-2’, H-3  C-2), C-4 (H-3  C-4), C-5 (H-6  C-5), C7 (H-6, H-8 C-7) C-9 (H-8  C-
9), and C-10 (H-8, H-3, H-6  C-10). In complex 2, Ru(II) is coordinated with rings A and
C of chrysin, which is acting, as mentioned, as an electron donor ligand and features
significant changes in its carbon chemical shifts: C-5, C-6 and C-10 appear shifted downfield
by c.a. 6.7, 4.2, and 2.7 ppm, respectively, and C-8 is shifted upfield by 3.1 ppm (regarding
pure chrysin).
Table 4. 1H NMR chemical shifts (in ppm) of complexes 2 and 3 in DMSO-d6, in comparison with those of
the pure flavone ligands.
1H
Chrysin
Complex 2
Tectochrysin
Complex 3
H-2'
H-3'
H-4'
H-5'
H-6'
8.09 - 8.04 (m)
7.64 – 7.53 (m)
7.64 – 7.53 (m)
7.64 – 7.53 (m)
8.09 - 8.04 (m)
6.97 (s)
8.07 – 8.02 (m)
7.64 – 7.52 (m)
7.64 – 7.52 (m)
7.64 – 7.52 (m)
8.07 – 8.02 (m)
7.05 (s)
8.13 – 8.09 (m)
7.66 – 7.55 (m)
7.66 – 7.55 (m)
7.66 – 7.55 (m)
8.13 – 8.09 (m)
7.05 (s)
8.08 (dd)
7.65 – 7.54 (m)
7.65 – 7.54 (m)
7.65 – 7.54 (m)
8.08 (dd)
H-3
7.11 (s)
OH-5
12.83 (s)
n.o.
12.82 (s)
n.o.
H-6
6.22 (d)
6.11 (d)
6.41 (d)
6.26 (d)
OH-7 or 7-OCH3
H-8
10.94 (s)
6.52 (d)
10.54 (s)
6.24 (d)
3.88 (s)
6.83 (d)
3.80 (s)
6.44 (d)
CH3-DMSO
CH2-[9]aneS3
—
—
2.95 (s), 2.96 (s)
2.93 – 2.58 (m)
—
—
2.97 (br s)
2.94 – 2.66 (m)
The DMSO-d6 signal (2.50 ppm) was used as the internal reference.
n.o. – not observed
20

The 1H NMR shifts of complex 3 in DMSO are also presented in the Table 4 (for the
1H spectrum, refer to Figure S7). Given that this complex bears tchrys, a ligand with strong
structural similarities to chrys, its protons present, as expected, chemical shift values similar
those of the complex 2 These confirm coordination of Ru(II) to the deprotonated C5–O- and
the 4-carbonyl of the chromenone. The aliphatic region is also similar to that of 2, with the
twelve methylene protons of [9]aneS3 appearing as a multiplet at δ 2.94-2.66 ppm and two
non-equivalent methyl groups of S-coordinated DMSO appearing collapsed in a single broad
resonance centred at δ 2.97 ppm. Note that non-equivalency is best observed in the 13C
spectrum (Figure S8, Table 5), in which two carbon resonances are observed in the region
13
corresponding to the methyl carbons of coordinated DMSO. The C resonances of C-5 and
C-6 appear shifted downfield by 2.3 and 2.4 ppm, respectively, in regard to free tchrys, as a
result of ligand electron-donating effect. Also noteworthy is the fact that, in 3, the
coordination of Ru(II) to the ketone (C-4) is accompanied by an electronic metal-to-ligand
-backdonation, as evidenced by the shift of the C-4 and C-2 resonances to lower
frequencies.
Table 5. 13C NMR chemical shifts (in ppm) of complexes 1-3 in DMSO-d6, in comparison with those of the
pure flavone ligands.
13C
7,3’,4’-thflv Complex 1
Chrysin
Complex 2
Tectochrysin
Complex 3
C-1'
C-2'
C-3'
C-4'
C-5'
C-6'
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
122.2
113.2
145.7
149.2
116.0
118.6
162.6
104.5
176.3
126.5
114.8
162.6
102.4
157.4
116.2
—
115.7 a
110.5b
163.5 a
170.1 a
114.7 b
114.7 b
165.0 a
100.8 a
176.0 a
125.9 b
113.9 b
157.5 a
102.1 b
162.4 a
116.8 a
—
130.8
126.5
129.2
132.1
129.2
126.5
163.2
105.2
182.0
161.5
99.1
164.5
94.2
157.5
104.0
—
130.4
126.2
129.3
132.0
129.3
126.2
159.4
105.2
178.3
168.2
103.3
164.3
91.1
158.9
106.7
—
130.6
126.5
129.2
132.3
129.2
126.5
163.5
105.4
182.2
161.2
98.2
165.4
92.9
157.4
105.0
56.2
130.2
126.3
129.3
132.1
129.3
126.3
159.8
105.3
178.8
168.4
101.3
165.1
90.1
158.7
107.4
55.6
C-10
O-CH3
CH3 (DMSO)
CH2 ([9]aneS3)
—
—
43.6
34.1, 31.9,
30.7
—
—
43.1, 42.4
34.6, 34.1,
32.5, 32.4,
29.9, 29.8
—
—
43.1, 42.5
34.7, 34.1,
32.6, 32.3,
30.0, 29.8
21

The DMSO-d6 signal (39.5 ppm) was used as the internal reference.
a Projected from HMBC
b Projected from HSQC
3.6. Cytotoxicity assays
The effect of complexes 1 to 3 on the viability of several human cancer cells – prostate
cancer (PC-3), breast cancer (MCF-7 and MDA-MB-231) and osteosarcoma (MG-63) – was
assessed upon a 72 h incubation time. The results were compared with the free ligands –
thflv, chrys and tchrys (Table 6) –, and cisplatin was used as the reference drug.
Table 6. IC50 (μM) values for 7,3’,4’-trihydoxyflavone (thflv), chrysin (chrys),
tectochrysin (tchrys), cisplatin and the complexes 1-3 against four human cancer cell
lines.
Compound
MG-63
PC-3
MCF-7
MDA-MB-231
thflv
complex 1
chrys
complex 2
tchrys
complex 3
cisplatin
38.1
> 18.5 a
41.0
23.2
> 18.5 a
15.7
36.9
> 18.5 a
29.5
24.8
> 18.5 a
17.1
> 200 b
67.9
146.2
32.8
> 200 b
113.5
> 200 b
13.8
180.6
44.8
> 200 b
4.5
> 200 b
6.6
> 200 b
9.5
a 18.5 was the highest concentration tested for this compound due to low solubility in
aqueous medium
b growth inhibition at 200 μM, the highest concentration tested, was lower than 50%.
The results presently obtained confirm the cytotoxic activity of the pure flavonoids,
thflv and chrys having the most interesting IC50 values. Noteworthy is the lower activity
observed for tchrys, a 5-methoxylated derivative of chrys, which demonstrates the relevance
of free hydroxyl groups for the antitumoral activity of flavones. Furthermore, it is worth
highlighting that all the tested flavonoids displayed antineoplastic activity against
osteosarcoma, a kind of cancer with a high incidence in children and teenagers and for which
the currently available chemotherapeutic strategies are still very limited. The compounds´
antitumor efficacy towards the MG-63 cell line, albeit lower than that of the reference drug
22

cisplatin, is within the 38-68 μM range. Overmore, the side effects are expected to be milder
since flavonoids are natural compounds with reported good tolerability. A chrysin glucoside
perfusion was reported to be safely used on mice as a natural hypotensive agent [55] and
intravenous chrysin nanoparticles were shown to control tumour growth in xenograph mice
[56].
Regarding the complexes, 1 could only be tested at a maximal concentration of 18.5
μM, due to poor water solubility (see details in the Experimental section, Table 1). At this
concentration, no inhibition was observed for any of the tested cell lines, and it can then be
considered as inactive. Complexes 2 and 3, in turn, displayed high IC50 values against the
tested cell lines (> 146 μM), revealing a loss of activity as compared to the free ligands.
These results are somewhat unexpected, given the known cytotoxic activity of complexes of
the Ru(II)(trithiacyclononane) family on several of the herein used human tumoral cells,
(MG-63, PC-3 and MDA-MB-231) [37, 38], as well as on a murine cell line (TS/A) [57]. It
may, though, be a phenomenon associated with O,O’–Ru(II) coordination, which was
previously observed for some Ru(II) complexes with curcumin (curc), namely
[Ru(II)Cl(curc)(pcym)])
[58],
[Ru(II)Cl(curc)(hexamethylbenzene)])
[59],
and
[Ru(II)([9]aneS3)(curc)(DMSO)]Cl) [40].
4. Conclusions
The work herein reported describes the first catechol-coordinated ruthenium flavone
complex, [Ru([9]aneS3)(DMSO)(thflv)] (1), as well as two novel complexes
[Ru([9]aneS3)(chrys)(DMSO)]Cl (2) and [Ru([9]aneS3)(DMSO)(tchrys)]Cl (3), with a six-
membered ring formed between Ru and the oxygen atoms at positions 4 and 5 of the
chromenone moiety of each flavone. The complexes 1-3 are obtained in moderate yields of
38, 23 and 43% respectively, using a one-pot generic synthetic procedure that involves an
initial step of deprotonation of the flavone followed by treatment with the ruthenium
23

precursor, [Ru([9]aneS3)Cl2(DMSO)]. The unequivocal confirmation of the geometry of
complexes 1-3 was achieved by use of both solution and solid-state characterisation
techniques, namely FT-IR, 1-D and 2-D NMR and ESI+-MS.
The cytotoxic activity against several human cancer cells was studied for the flavones
as well as their complexes. The free ligands showed good inhibitory action on the various
tested cell lines (prostate cancer, breast cancer, both hormone-dependent and triple-negative)
and, more importantly, osteosarcoma (a strongly chemoresistant, low prognosis type of
cancer). Anti-osteosarcoma action has been reported for other flavones and their derivatives,
in particular for baicalin [60], nobiletin [61], luteolin [62], and wogonin [63], but it is, to the
best of our knowledge, unprecedented for chrysin, tectochrysin and 7,3’,4’-trihydroxyflavone
(also called 5-deoxyluteolin). In turn, complexes 1 to 3 displayed no antitumor activity
against the MG-63 osteosarcoma cell line. Particularly for complexes 2 and 3, tested at
concentrations up to 200 μM, a very reduced activity was obtained. Complex 2, comprising
chrys as the ligand, prompts a decreased viability of PC-3 prostate cancer and MDA-MB-231
breast cancer cells, but with high IC50 values (146.2 and 180.6 μM, respectively). As for
complex 3, with tchrys as the ligand, an IC50 > 200 μM was determined, which is in line with
the higher IC50 values observed for free tchrys in these cell lines. Hence, the low activity of
the metal complexes may be somewhat associated with the properties of the ligand, and
partly caused by solubility issues that do not allow the complexes to achieve their cellular
targets in suitable amounts. Future directions towards the improvement of these structures
should thus contemplate an improved cellular uptake and increased water solubility, either by
incorporating a solubilising group as PTA (1,3,5-triaza-7-phosphaadamantane) in their
coordination sphere or by loading them into adequate carriers such as cyclodextrins and
liposomes.
24

References
A. N. Panche, A. D. Diwan, S. R. Chandra, J. Nutr. Sci. 5 (2016) e47.
1.
2.
3.
4.
5.
6.
7.
8.
doi:10.1017/jns.2016.41
B. Van De Wier, G. H. Koek, A. Bast, G. R. M. M. Haenen, Crit. Rev. Food Sci.
Nutr. 57 (2017) 834–855. doi:10.1080/10408398.2014.952399
X. M. Liu, Y. J. Liu, Y. Huang, H. J. Yu, S. Yuan, B. W. Tang, P. G. Wang, Q. Q.
He, Mol. Nutr. Food Res. 61 (2017) article 1601003. doi:10.1002/mnfr.201601003
A. L. Livingston, E. M. Bickoff, J. Pharm. Sci. 53 (1964) 1557.
doi:10.1002/jps.2600531237
E. M. Bickoff, S. C. Witt, E. M. Bickoff, J. Pharm. Sci. 54 (1965) 1555.
doi:10.1002/jps.2600541042
R. Chokchaisiri, C. Suaisom, S. Sriphota, A. Chindaduang, T. Chuprajob, A.
Suksamrarn, Chem. Pharm. Bull. 57 (2009) 428–432. doi:10.1248/cpb.57.428
C. Selvam, S. M. Jachak, K. K. Bhutani, Phytother. Res. 18 (2004) 582–584.
doi:10.1002/ptr.1492
M. Barbaric, K. Miskovic, M. Bojic, M. B. Loncar, A. Smolcic-Bubalo, Z. Debeljak,
M.
Medic-Saric,
J.
Ethnopharmacol.
135
(2011)
772772–778.
doi:10.1016/j.jep.2011.04.015.
9.
M. I. Isla, A. Craig, R. Ordoñez, C. Zampini, J. Sayago, E. Bedascarrasbure, A.
Alvarez, V. Salomón, L. Malnonado, LWT Food Sci. Technol. 11 (2011) 1922–1930.
doi:10.1016/j.lwt.2011.04.003
10.
M. Ozarowski, A. Piasecka, A. Paszel-Jaworska, D.S.d.A. Chaves, A. Romaniuk, M.
Rybczynska, A. Gryszczynska, A. Sawikowska, P. Kachlicki, P.L. Mikolajczak, A.
Seremak-Mrozikiewicz, A. Klejewski, B. Thiem, Rev. Bras. Farmacogn. 28 (2018)
179–191. doi:10.1016/j.bjp.2018.01.006
11.
12.
M. Cardénas, M. Marder, V. C. Blank L. P. Roguin, Bioorg. Med. Chem. 14 (2006)
2966–2971. doi:10.1016/j.bmc.2005.12.021.
K. Hu, W. Wang, H. Cheng, S. Pan, J. Ren, Med. Chem. Res. 20 (2011) 838–846.
doi:10.1007/s00044-010-9395-1
25

13.
14.
15.
16.
17.
18.
19.
20.
S. Samarghandian, J. T. Afshari, S. Davoodi, Clinics 66 (2011) 1073–1079.
doi:10.1590/S1807-59322011000600026
M. Bhardwaj , N.-H. Kim , S. Paul, R. Jakhar, J. Han, S. C. Kang, Plos One 11 (2016)
e0154525. doi:10.1371/journal.pone.0154525
P. Qiu, P. Dong, H. Guan, S. Li, C. T. Ho, M. H. Pan, D. J. McClements, H. Xiao,
Mol. Nutr. Food Res. 54 (2010) S244–S252. doi:10.1002/mnfr.200900605.
P. Sawasdee, C. Sabphon, D. Sitthiwongwanit, U. Kokpol, Phytother Res. 23 (2009)
1792–1794. doi:10.1002/ptr.2858
M. Grazul, E. Budzisz, Coord. Chem. Rev. 253 (2009) 2588–2598.
doi:10.1016/j.ccr.2009.06.015.
E. Ciesielska, K. Studzian, E. Zyner, J. Ochocki, L. Szmigiero, Cell. Mol. Biol. Lett. 5
(2000) 441–450.
M. M. Kasprzak, L. Szmigiero, E. Zyner, J. Ochocki, J. Inorg. Biochem. 105 (2011)
518–524. doi:10.1016/j.jinorgbio.2010.12.013.
M. Kasprzak, M. Fabijańska, L. Chȩcińska, K. Studzian, M. Markowicz-Piasecka, J.
Sikora, E. Mikiciuk-Olasik, J. Ochocki, Inorg. Chim. Acta 457 (2017) 69–80.
doi:10.1016/j.ica.2016.11.021
21.
22.
23.
24.
25.
A. Pastuszko, K. Niewinna, M. Czyz, A. J. Zwiak, M. Ma1ecka, E. Budzisz, J.
Organomet. Chem. 745-746 (2013) 64–70. doi:10.1016/j.jorganchem.2013.07.020
A. K. Singh, G. Saxena, Sahabjada, M. Arshad, Spectrochim. Acta A: Mol. Biomol.
Spec. 180 (2017) 97–104. doi:10.1016/j.saa.2017.02.056
X. Han, K. K. Klausmeyer, P. J. Farmer, Inorg. Chem. 55 (2016) 7320-7322.
doi:10.1021/acs.inorgchem.6b00852
S. L. Saraf, T. J. Fish, A. D. Benninghoff, A. A. Buelt, R. C. Smith, L. M. Berreau,
Organometallics 33, (2014) 6341–6351. doi:10.1021/om5006337
A. Kurzwernhart, W. Kandioller, É. A. Enyedy, M. Novak, M. A. Jakupec, B. K.
Keppler, C. G. Hartinger, Dalton Trans. 42 (2013) 6193–6202.
doi:10.1039/C2DT32206D
26.
A. Bergamo, C. Gaiddon, J. H. M. Schellens, J. H. Beijnen, G. Sava, J. Inorg.
Biochem. 106 (2012) 90–99. doi:10.1016/j.jinorgbio.2011.09.030
26

27.
W. Peti, T. Pieper, M. Sommer, B. K. Keppler, G. Giester, Eur. J. Inorg. Chem. 1999
(1999) 1551–1555. doi:10.1002/(SICI)1099-0682(199909)1999:9<1551::AID-
EJIC1551>3.0.CO;2-7
28.
29.
S. Kapitza, M. A. Jakupec, M. Uhl, B. K. Keppler, B. Marian, Cancer Lett. 226
(2005) 115–121. doi:10.1016/j.canlet.2005.01.002
F. Lentz, A. Drescher, A. Lindauer, M. Henke, R. A. Hilger, C. G. Hartinger, M. E.
Scheulen, C. Dittrich, B. K. Keppler, U. Jaehde, Anticancer Drugs 20 (2009) 97–103.
doi:10.1097/CAD.0b013e328322fbc5
30.
31.
32.
33.
C. Scolaro, C. G. Hartinger, C. S. Allardyce, B. K. Keppler, P. J. Dyson, J. Inorg.
Biochem. 102 (2008) 1743–1748. doi:10.1016/j.jinorgbio.2008.05.004
B. S. Murray, M. V. Babak, C. G. Hartinger, P. J. Dyson, Coord. Chem. Rev. 306
(2016) 86–114. doi:10.1016/j.ccr.2015.06.014
C. S. Allardyce, P. J. Dyson, Dalton Trans. 45 (2016) 3201–3209.
doi:10.1039/c5dt03919c
R. E. Morris, R. E. Aird, P. S. Murdoch, H. M. Chen, J. Cummings, N. D. Hughes, S.
Parsons, A. Parkin, G. Boyd, D. I. Jodrell, P. J. Sadler, J. Med. Chem. 44 (2001)
3616–3621. doi:10.1021/jm010051m
34.
35.
36.
B. Serli, E. Zangrando, T. Gianferrara, C. Scolaro, P. J. Dyson, A. Bergamo, E.
Alessio, Eur. J. Inorg. Chem. 2005 (2005) 3423–3434. doi:10.1002/ejic.200500210
A. Rilak, I. Bratsos, E. Zangrando, J. Kljun, I. Turel, Ž. D. Bugarčić, E. Alessio,
Inorg. Chem. 53 (2014) 6113–6126. doi:10.1021/ic5005215
T. Gianferrara, A. Bergamo, I. Bratsos, B. Milani, C. Spagnul, G. Sava, E. Alessio, J.
Med. Chem. 53 (2010) 4678–4690. doi:10.1021/jm1002588
37.
38.
J. Marques, V. L. M. Silva, A. M. S. Silva, M. P. M. Marques, S. S. Braga. Complex
Metals 1 (2014) 7–12. doi:10.1080/2164232X.2013.873992
J. Marques, J. A. Fernandes, F. A. A. Paz, M. P. M. Marques, S. S. Braga. J. Coord.
Chem. 65 (2012) 2489–2499. doi:10.1080/00958972.2012.696624
39.
J. Marques, T. M. Santos, M. P. M. Marques, S. S. Braga. Dalton Trans. (2009)
9812–9819. doi:10.1039/b915839a
27

40.
41.
42.
M. C. Henriques, M. A.F. Faustino, A.M. S. Silva, J. Felgueiras, M. Fardilha, S. S.
Braga, J. Coord. Chem. 70 (2017) 2393–2408. doi:10.1080/00958972.2017.1336232
J. Marques, T. M. Braga, F. A. A. Paz, T. M. Santos, M. F. S. Lopes. Biometals 22
(2009) 541–556. doi:10.1007/s10534-009-9211-x
S. S. Braga, J. Marques, E. Heister, C. V. Diogo, P. J. Oliveira, F. A. A. Paz, T. M.
Santos, M. P. M. Marques. Biometals 27 (2014) 507–525. doi:10.1007/s10534-014-
9725-8
43.
44.
C. Landgrafe, W. S. Sheldrick, J. Chem. Soc., Dalton Trans. 0 (1994) 1885–1893.
doi:10.1039/DT9940001885
M. Hasan, Q. U. Ahmed, S. Z. M. Soad, J. Latip, M. Taher, T. M. F. Syafiq, M. N.
Sarian, A. M. Alhassan, Z. A. Zakaria, BMC Compl. Altern. Med. 17 (2017) article
431. doi:10.1186/s12906-017-1929-3
45.
46.
T. Mosmann. J. Immunol. Methods 65 (1983) 55–63. doi:10.1016/0022-
1759(83)90303-4
N. Sundaraganesan, G. Mariappan, S. Manoharan, Spectrochim. Acta A 87 (2012) 67–
76. doi:10.1016/j.saa.2012.06.011
47.
48.
R. Gaur, L. Mishra, RSC Adv. 3 (2013) 12210–12219. doi:10.1039/C3RA41451E
M. G. de Carvalho, M. S. R. Gomes, M. C. C. de Oliveira, C. J. da Silva, A. G. de
Carvalho, Braz. J. Farmacogn. 21 (2011) 397–401. doi:10.1590/S0102-
695X2011005000037
49.
M. Ashok, A.V.S.S. Prasad, V. Ravinder. J. Braz. Chem. Soc. 18 (2007) 1492–1499.
doi:10.1590/S0103-50532007000800007
50.
51.
M. Calligaris. Coord. Chem. Rev. 248 (2004) 351–375. doi:10.1016/j.ccr.2004.02.005
M. G. O. Santana-Marques, F. M. L. Amado, A. J. F. Correia, M. Lucena, J.
Madureira, B. J. Goodfellow, V. Felix, T. M. Santos. J. Mass Spectrom. 36 (2001)
529–537. doi:10.1002/jms.152
52.
53.
R. A. Izquierdo, J. Madureira, C. I. V. Ramos, M. G. O. Santana-Marques, T. M.
Santos. Int. J. Mass Spectrom. 301 (2011) 143–150. doi:10.1016/j.ijms.2010.08.007
T. Hopp, M. Fischer-Gödde, T. Kleine, J. Anal. At. Spectrom. 31 (2016) 1515–1526.
doi:10.1039/C6JA00041J
28

54.
55.
56.
57.
58.
A. M. S. Silva, J. A. S. Cavaleiro, G. Tarrago, C. Marzin, J. Heterocycl. Chem. 31
(1994) 97–103. doi:10.1002/jhet.5570310118
K. Cherkaoui-Tangi, M. Lachkar, M. Wibo, N. Morel, A. H. Gilani, B.
Lyoussi, Phytother. Res. 22 (2008) 356–361. doi:10.1002/ptr.2322
K. M. Kim, H. K. Lim, S. H. Shim, J. Jung, Int. J. Nanomed. 12 (2017) 1917–1925.
doi:10.2147/IJN.S132043
B. Serli, E. Zangrando, P. J. Dyson, T. Gianferrara, A. Bergamo, C. Scolaro, E.
Alessio. Eur. J. Inorg. Chem. (2005) 3423–3434. doi:10.1002/ejic.200500210.
F. Caruso, M. Rossi, A. Benson, C. Opazo, D. Freedman, E. Monti, M. B. Gariboldi,
J. Shaulky, F. Marchetti, R. Pettinari, C. Pettinari. J. Med. Chem. 55 (2012) 1072–
1081. doi:10.1021/jm200912j
59.
L. Bonfili, R. Pettinari, M. Cuccioloni, V. Cecarini, M. Mozzicafreddo, M. Angeletti,
G. Lupidi, F. Marchetti, C. Pettinari, A. M. Eleuteri. ChemMedChem. 7, (2012)
2010–2020. doi:10.1002/cmdc.201200341
60.
61.
62.
63.
Y. Wang, H. Wang, R. Zhou, W. Zhong, S. Lu, Z. Ma, Y. Chai, Anti-Canc. Drugs 28
(2017) 581–587. doi:10.1097/CAD.0000000000000495
H.-L. Cheng, M.-J. Hsieh, J.-S. Yang, C.-W. Lin, K.-H. Lue, K.-H. Lu, S.-F. Yang,
Oncotarget 7 (2016) 35208–35223. doi:10.18632/oncotarget.9106
Y. Wang, D. Kong, X. Wang, X. Dong, Y. Tao, H. Gong, Iran. J. Pharm. Res. 14
(2015) 531–538.
C. Lin, C. Kuo, M. Lee, K. Lai, J. Lin, J. Yang, C. Yu, C. Lu, J. Chiang, F. Chueh, J.
Chung, Int. J. Oncol. 39 (2011) 217–224. doi:10.3892/ijo.2011.1027
29