Low DNA and high BSA binding affinity of cationic ruthenium(II) organometallic featuring pyridine and 2’-hydroxychalcone ligands

Article abstract of DOI:10.1016/j.molstruc.2021.130326

The chiral-at-metal, piano-stool ruthenium(II) racemic organometallic [Ru(cymene)(chalconato)(pyridine)]PF₆ was prepared by a multistep solution synthesis and its molecular and crystal structure was determined by single crystal X-ray diffractio

Full text of DOI:10.1016/j.molstruc.2021.130326

Journal of Molecular Structure 1236 (2021) 130326
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Low DNA and high BSA binding affinity of cationic ruthenium(II)
organometallic featuring pyridine and 2’-hydroxychalcone ligands
Adnan Zahirovic´ a, Suncˇica Rocab, Emira Kahrovic´ a, Aleksandar Višnjevacc,∗
a Laboratory for Inorganic and Bioinorganic Chemistry, Department of Chemistry, Faculty of Science, University of Sarajevo, 71 000 Sarajevo, Bosnia and
Herzegovina
b NMR Centre, Ruder Boškovi´c Institute, 10 000 Zagreb, Croatia
¯
c Laboratory for Chemical and Biological Crystallography, Ruder Boškovi´c Institute, 10 000 Zagreb, Croatia
¯
a r t i c l e i n f o
a b s t r a c t
Article history:
The
chiral-at-metal,
piano-stool
ruthenium(II)
racemic
organometallic
Received 22 December 2020
Revised 4 March 2021
Accepted 15 March 2021
Available online 23 March 2021
[Ru(cymene)(chalconato)(pyridine)]PF6 was prepared by a multistep solution synthesis and its molecular
and crystal structure was determined by single crystal X-ray diffraction. The compound crystallizes
in a centrosymmetric triclinic P-1 space group with two molecules of opposite chirality within the
asymmetric unit. Ruthenium is embedded in an octahedral half-sandwich coordination environment
with four different donors, which generates the chirality of the metal centre. The formation of the
organometallic was monitored ex-situ by infrared spectroscopy. The pyridine coordination to Ru(II) was
particularly analysed to find its characteristic marker bands. The complex was also characterized by
elemental analysis and NMR spectroscopy in solution. Its interaction with CT DNA and bovine serum
albumin (BSA) was investigated by electron spectroscopy and spectrofluorimetry. The organometallic
binds to DNA predominantly by electrostatic forces with low Kb value. On the contrary, its high affinity
for BSA was confirmed by strong fluorescence quenching. The synchronous emission spectra revealed
that the microenvironment of tryptophan is more affected compared to the environment of tyrosine.
Keywords:
ruthenium
chalcone
pyridine
organometallic
X-ray diffraction
infrared
DNA
BSA
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
Z positions are all different, the chirality is generated at the cen-
tral ruthenium atom which opens additional avenues of application
The focus of inorganic chemistry in the 21st century is synthe-
sis – structure – (re)activity correlation as a prerequisite for un-
derstanding chemical dynamics at the molecular level and reach-
ing the point from where desired properties of the new com-
pounds can be controlled. Arising from this concept, ruthenium(II)
organometallic compounds were extensively studied as potential
candidates for the design and development of highly efficient cat-
alysts [1] and drugs [2–4]. The properties of pseudo octahedral
piano-stool organometallics with general formula [(arene)RuIIXYZ]
are controlled through arene/ligand replacement and modification
of the aliphatic or aromatic ligand denticity, as well as by donor
atoms type that occupy three coordination positions designated as
X, Y and Z [5–7]. Due to the steric limitations of many tridentate
ligands, X, Y and Z positions usually belong to one bidentate and
one monodentate ligand, resulting in a vast structural diversity of
these compounds. Moreover, if the donor atoms at the X, Y and
of such chemical edifices as catalysts of enantioselective organic
transformations [8].
Because of their structure, chalcones are prominent building
blocks for cancer [9], malaria [10], tuberculosis [11] and cardio-
vascular diseases [12] drug design. The corresponding ruthenium-
chalcone compounds, on the other hand, exhibited catalytic activ-
ity in hydrogenation of ketones and oxidation of alcohols [13], as
well as in reactivity toward DNA, nuclease activity [14], cytotoxic-
ity [15,16] and antileukemia activity [17] which, combined, greatly
encouraged further investigation of these compounds. The coordi-
nation of heterocycles to ruthenium proved to be crucial in design-
ing the compounds with antitumor activity for which NAMI-A and
KP1019 are probably the best examples [18,19]. Heterocycles are
highly convenient, usually monodentate ligands that allow fine-
tuning of kinetic, electronic, steric or redox properties of coordina-
tion compounds. However, monitoring of heterocycle coordination
to a metal centre in a multistep synthesis can be demanding and
requires time-consuming techniques such as NMR. Fast and widely
available infrared spectroscopy is rarely used to monitor heterocy-
∗
Corresponding author at: Laboratory for Chemical and Biological Crystallogra-
phy, Ruder Boškovic´ Institute, Bijenicˇka cesta 54, HR-10000 Zagreb.
¯
E-mail address: aleksandar.visnjevac@irb.hr (A. Višnjevac).
https://doi.org/10.1016/j.molstruc.2021.130326
0022-2860/© 2021 Elsevier B.V. All rights reserved.

A. Zahirovi´c, S. Roca, E. Kahrovi´c et al.
Journal of Molecular Structure 1236 (2021) 130326
at 600.130 MHz and 300.133 MHz for the 1H, and 150.903 MHz and
75.475 MHz for the 13C nucleus. Chemical shifts, in ppm, are re-
ferred to tetramethylsilane (TMS) as internal standard. Assignment
of 1H and 13C NMR signals was performed using two-dimensional
homo- and heteronuclear correlation experiments with standard
parameters: 1H-1H Correlation Spectroscopy (COSY), 1H-1H Nu-
clear Overhauser Effect Spectroscopy (NOESY), 1H-13C Heteronu-
clear Multiple Quantum Coherence (HMQC) and 1H-13C Heteronu-
clear Multiple Bond Correlation (HMBC).
cle coordination since the marker bands are usually of lower in-
tensity and overlapped by bands of other ligands.
Many ruthenium complexes are extensively investigated as very
promising antitumor agents. The most representative examples in-
clude chlororuthenium(III) coordination complexes featuring het-
erocycles as coligands, such as KP1019 and NAMI-A, and the
organometallic compound of ruthenium(II) with triazaphospho-
adamantane known as RAPTA. These compounds are known to
bind to DNA and proteins which are believed to be their biolog-
ical targets in vivo [20–22], and the investigation of their interac-
tion with new compounds is usually the first step of the biologi-
cal evaluation. Anticancer activity of ruthenium-arene compounds
in vitro can be improved in the presence of proteins. A good ex-
ample is the case of RM175 ([(η6-biph)RuCl(en)]+) whose cyto-
toxicity towards highly-invasive breast MDA-MB-231, human breast
MCF-7 and human epithelial HBL-100 cancer cells is enhanced in
the presence of human serum albumin [23]. Also, ligand substi-
tutions between ruthenium–cymene compounds can control pro-
tein versus DNA targeting and consequently anticancer activity. This
is best illustrated for two ruthenium-cymene compounds: RAED-
C and RAPTA-C. RAED-C ([(cym)RuCl(en)]+) is cytotoxic toward
primary tumours and targets DNA of chromatin, while RAPTA-C
([(cym)RuCl2(PTA)] is relatively non-cytotoxic antimetastatic com-
pound which preferentially forms adducts on the histone pro-
teins [24]. The protein versus DNA binding was also investigated
for many other ruthenium organometallics such as those of NS
donor Schiff bases [25], anthracenyl-appended diazacycloalkanes
[26], substituted pyridylimidazo[1,5-a]pyridines [27], ethylenedi-
amine [28], chloroquine [29], and diimines [30].
Single crystal X-ray data collection was conducted at RT, on
an Oxford diffraction Xcalibur Nova Ruby device. Cu radiation
˚
(λ = 1.54184 A) was employed. Data reduction and cell refine-
ment were carried out using CRYSALIS PRO software [33]. Structure
was solved by direct methods with SIR2014 [34] and refined by a
full matrix least-squares refinement based on F2, with SHELXL [35].
Molecular illustrations were prepared with MERCURY [36]. Calcula-
tions of molecular geometries and crystal packing parameters were
performed with PLATON [37]. All software used is included into the
WinGX package [38]. Hydrogen atoms were included in their geo-
metrically calculated positions and refined according to the riding
model, or (where sustainable throughout the refinement) located
from the difference map and refined freely. Multi-scan absorp-
tion correction procedure was applied [39]. Powder X-ray diffrac-
tion data was collected at RT on Bruker D8 Discover diffractometer
equipped with LYNXEYE XE-T detector, in Bragg-Brentano geom-
etry. Data were collected in 2θ range 5 – 50°. Rietveld structure
refinement was performed in HighScore Xpert Plus program 3.0.
Refinement was carried out by using the split-type pseudo-Voigt
profile function and the polynomial background model. Isotropic
vibration modes were assumed for all atoms. During the refine-
ment, a zero shift, scale factor, half-width parameters, asymmetry
and peak shape parameters were simultaneously refined.
Here we report on the synthesis, chemical characterization
and crystal structure of the racemic chiral-at-metal organometal-
lic compound of ruthenium(II) having 2’-hydroxychalcone ligand
and pyridine as coligand, as well as ex-situ infrared monitoring
of its formation. The interaction of newly prepared ruthenium
organometallic with CT DNA and with bovine serum albumin (BSA)
was investigated.
Interaction of organometallic (f) with DNA and BSA was ob-
served in 10 mM Tris-HCl buffer at pH 7.42 and at RT. Stock so-
lution of organometallic (f) was prepared in methanol at concen-
tration 2 mM. The spectroscopic titration of compound with CT
DNA was carried out by adding 10 μL aliquots of DNA (6.67 mM)
to organometallic solution (2.000 mL, 5 × 10–5 M). Each spectrum
was recorded after 2-minutes equilibration time. The titration of
CT DNA with organometallic (f) was performed by adding 5 μL
aliquots of organometallic (2 mM) to the solution of CT DNA (2.000
mL, 1.67 × 10–4 M). The incubation of organometallic (f) with CT
DNA (1.67 × 10–4 M) was carried out at 2:1 [DNA]/[organometallic]
ratio.
2. Experimental
2.1. Chemicals
All chemicals used in this study were obtained from com-
mercial sources at highest degree of purity available and used
as received. Dichloro(p-cymene)ruthenium(II) dimer was prepared
according to published procedure [31]. 2’-hydroxychalcone was
Spectrofluorimetric titration of BSA with organometallic (f) was
carried out by adding 10 μL aliquots of organometallic (0.2 mM) to
solution of BSA (2.000 mL, 3.68 × 10–6 M) and recording emission
spectra in 290 – 430 nm range with 279 nm as excitation wave-
length. The synchronous spectra were recorded under the similar
conditions as described above while recording the emission spec-
tra in range 250 – 310 nm with ꢀλ = 15 nm and ꢀλ = 60 nm.
obtained as
a condensation product of benzaldehyde and 2’-
hydroxyacetophenone from Claisen-Schmidt condensation [32].
Deuterated chloroform (CDCl3-d with v/v 0.03% TMS, 99.80% D) for
NMR spectroscopy was purchased from Euriso-Top, France. Highly
polymerized deoxyribonucleic acid sodium salt from calf thymus
(CT DNA), Type I, fibers (A260/A280 > 1.8) was obtained from Sigma.
Bovine serum albumin (BSA, ≥ 98%) was obtained from Sigma as a
lyophilized powder.
2.3. Synthesis
2.2. Physical measurements
Synthesis of chalcone (e)
2’-hydroxyacetophenone (20 mmol, 2.723 g) and benzaldehyde
(20 mmol, 2.123 g) were dissolved in ethanol (40 mL), aqueous so-
lution of sodium hydroxide (86 mmol, 5 M, 17.2 mL) was added
portion-wise and the mixture was vigorously stirred for 24 hours.
The orange solution was acidified to pH 5 using acetic acid. The
resulting yellow chalcone was filtered off, washed with water, and
recrystallized from hot ethanol. Yield: 3.53 g (79%).
Infrared spectra were collected in transmission mode by the
KBr pellet technique using Perkin Elmer BX FTIR. The course of the
reaction was monitored by withdrawing the microliter aliquots of
the reaction mixture and evaporating the solvent after which KBr
pellets were prepared. CHN analyses were performed on a Perkin-
Elmer 2400 Series II CHNS analyser.
The NMR spectra of the chalcone ligand (e) were recorded on
Bruker AV600 spectrometer, and NMR spectra of the organometal-
lic (f) were recorded on Bruker AV300 spectrometer, both with a
5 mm observed RT probe in CDCl3-d. Used spectrometers operate
Synthesis of organometallic [(cym)Ru(chalc)(py)]PF6 (f)
Pyridine (48 μL, 0.60 mmol) was added to
a vigorously
stirred cold suspension of [RuCl2(cym)]2 (184 mg, 0.30 mmol)
in acetone (10 mL) causing an immediate precipitation of or-
2

A. Zahirovi´c, S. Roca, E. Kahrovi´c et al.
Journal of Molecular Structure 1236 (2021) 130326
Scheme 1. Synthesis of 2’-hydroxychalcone (e).
Table 1
ange [(cym)RuCl2(py)]. Stirring was continued for a half hour af-
ter which silver triflate (308 mg, 1.20 mmol) dissolved in acetone
(10 mL) was added portion-wise to the reaction mixture. Approx-
imately a quantitative amount of silver chloride (170 mg) was fil-
tered off through a blue ribbon and the resulting orange filtrate
was mixed with dichloromethane solution (10 mL) of previously
deprotonated chalcone ligand (135 mg, 0.60 mmol) with triethy-
lamine (84 μL, 0.60 mmol). The obtained intensively red solution
was stirred at room temperature for 24 hours and evaporated to
dryness under reduced pressure. The oily product was dissolved in
chloroform (15 mL), transferred to a separating funnel, and washed
with water (2 × 15 mL). The chloroform layer was dried over an-
hydrous sodium sulphate, filtered and evaporated to dryness un-
der reduced pressure. The residue was dissolved in methanol (5
mL), and methanol solution (3 mL) of sodium hexafluorophosphate
(500 mg) was added. The resulting red substance was filtered off,
washed with a water-methanol mixture (1/1, v/v, 2 × 2 mL) and
dried under vacuum over silica. The mother liquor gave single
crystals of (f) as dark red prisms upon an overnight slow evap-
oration at room temperature. Yield: 285 mg (69%). Anal. cal. for
C30H30F6NO2PRu (682.59): C 52.79; H 4.43; N 2.05. Found: C 52.74;
H 4.33; N 2.16%. 1H NMR (300 MHz, CDCl3, 25°C): δ 8.63 (2H, dd,
J = 6.29, 1.26 Hz, H-26 and H-30), 8.18 (1H, d, J = 15.37 Hz, H-
7), 7.80 (2H, dd, J = 7.30, 2.50 Hz, H-2 and H-6), 7.72 (1H, dd, J =
7.54, 1.39, H-28), 7.45–7.37 (7H, m, H-3 and H-5, H-4, H-8, H-11,
H-27 and H-29), 7.25 (1H, ddd, J = 8.52, 6.71, 1.54 Hz, H-13), 6.98
(1H, d, J = 8.66 Hz, H-14), 6.45 (1H, ddd, J = 7.96, 6.57, 0.97 Hz,
H-12), 5.89 (1H, d, J = 6.09 Hz, H-17 or H-19), 5.65 (1H, d, J = 6.09
Hz, H-17 or H-19), 5.58 (1H, d, J = 6.09 Hz, H-16 or H-20), 5.54
(1H, d, J = 6.09 Hz, H-16 or H-20), 2.79 (1H, septet, J = 6.88 Hz,
H-23), 2.07 (3H, s, H-22), 1.34 (3H, d, J = 6.88 Hz, H-24 or H-25),
1.25 (3H, d, J = 6.88 Hz, H-24 or H-25) ppm; 13C NMR (75 MHz,
CDCl3, 25°C): δ 187.2 (1C, C-9), 170.4 (1C, C-15), 152.6 (2C, C-26
and C-30), 147.8 (1C, C-7), 139.0 (1C, C-28), 137.2 (1C, C-13), 134.9
(1C, C-1), 131.5 (1C, C-4), 131.3 (1C, C-11), 129.3 (4C, C-2 and C-6,
C-3 and C-5), 126.2 (2C, C-27 and C-29), 124.7 (1C, C-14), 121.0 (1C,
C-8), 120.4 (1C, C-10), 116.0 (1C, C-12), 102.1 (1C, C-18), 99.6 (1C,
C-21), 85.3 (1C, C-17 or C-19), 84.4 (1C, C-17 or C-19), 81.9 (1C, C-
16 or C-20), 81.0 (1C, C-16 or C-20), 31.0 (1C, C-23), 22.5 (2C, C-24
and C-25), 17.8 (1C, C-22) ppm.
Crystallographic data
Structure
[(cym)Ru(chalc)(py)]PF6 (f)
Brutto formula
C30H30F6NO2PRu
Form. weight (gmol–1
Crystal colour, habit
Crystal dim. (mm)
)
682.59
dark red, prism
0.20 × 0.25 × 0.35
1.53
Crystal density (gcm–3
)
Space group
P-1
˚
a (A)
12.7053 (3)
15.0340 (4)
15.7685 (3)
94.879 (2)
90.910 (2)
97.963 (2)
2970.95 (12)
4 (Z’=2)
0.0428
˚
b (A)
˚
c (A)
α (°)
β (°)
γ (°)
3
˚
V (A )
Z
Rint
R
0.0640
σ
μ(CuK ) (mm–1
)
5.37
α
F(000)
1384
θ max (°) data col.
hkl limits of data col.
No. refl. Unique
No. refl. obs. [I>2σ(I)]
Parameters
75.91
-15,15;-18,18:-19,13
12240
10762
777
R1, all
0.0463
R1 [I>2σ(I)]
wR2, all
0.0415
0.1116
wR2 [I>2σ(I)]
S
0.1055
1.103
–3
˚
ρ
max, ρmin (eA
)
0.75; -1.10
The organometallic, chiral-at-metal [(cym)Ru(chalc)(py)]PF6 (f)
was prepared by a multistep solution synthesis and isolated as
racemic mixture (Scheme 2). Complete synthetic procedure was
carried out at normal environmental conditions without the usage
of an inert gas atmosphere. In the first step, the parent ruthenium
dimer [RuCl2(cym)]2 (a) was reacted with one equivalent of pyri-
dine (b) in acetone affording the neutral [RuCl2(cym)(py)] (c). Then
the chlorides were precipitated by the addition of two equivalents
of silver triflate. The chalcone ligand (e) was deprotonated with tri-
ethylamine and reacted with in situ prepared ruthenium-acetone
intermediate (d). The mono cationic [(cym)Ru(chalc)(py)]+ (f) was
separated from Et3NHCF3SO3 by chloroform-water extraction and
precipitated from the organic solvent by the addition of aque-
ous sodium hexafluorophosphate. The reaction steps are shown in
Scheme 2.
In order to check the phase purity, crystalline material, as ob-
tained from the mother liquid, was examined by the X-ray powder
diffraction. Fig. S1 shows Rietveld refinement using the structural
model as determined from single crystal analysis; no additional re-
flections can be noted thus confirming the bulk sample contain
[(cym)Ru(chalc)(py)]PF6 exclusively.
3. Results and discussion
3.2. Molecular and crystal structure
3.1. Synthesis
Experimental data collection details, as well as crystallographic
parameters are given in Table 1, selected torsional angles illus-
trating conformational differences between two molecules of the
asymmetric unit are given in Table S1, while the hydrogen bonds
are listed in Table S2. Fig. 1 depicts molecular structure of (f). The
The base-catalysed condensation of 2’-hydroxyacetophenone
and benzaldehyde in aqueous ethanol gave yellow 2’-
hydroxychalcone as shown in Scheme 1.
3

A. Zahirovi´c, S. Roca, E. Kahrovi´c et al.
Journal of Molecular Structure 1236 (2021) 130326
Scheme 2. Synthesis of [(cym)Ru(chalc)(py)]PF6 (f).
Fig. 1. ORTEP drawing of (f) with atom numbering. Thermal ellipsoids are scaled at 40% probability level for clarity.
overlay of the two molecules of the asymmetric unit is given in
Fig. 2.
Compound (f) crystallizes in centrosymmetric triclinic P-1 space
oxygen. The complexes featuring this type of coordination environ-
ment are also known in literature as half-sandwich complexes [40].
Free rotation around the C1–C7 i.e. around C31–C37 bonds enables
different orientations of the chalcone phenyl moiety with respect
to the remaining of the complex molecule which may explain the
unusually high Z number (4). Indeed, values of the corresponding
analogous torsion angles differ significantly for two molecules of
group with two molecules of opposite chirality per asymmetric
unit. Pseudo-octahedrally coordinated ruthenium, which sits in a
centre of a piano-stool coordination environment with four dif-
ferent donors is a chiral centre in this molecule and generates
the overall molecular chirality of the complex. While one face of
the pseudo-octahedron around Ru(II) is occupied by the donor p-
cymene phenyl ring, the remaining face comprises three coordi-
nated donor atoms: pyridine nitrogen and two oxygen atoms from
the chalcone moiety: carbonyl oxygen and deprotonated hydroxyl
–
the asymmetric unit (Table S1). One of the PF6 anions (P1) re-
veals large thermal ellipsoids of the fluorine atoms, which sug-
gests multiple orientations (at least two), which, however, are not
crystallographically distinct. Structure of (f) does not feature clas-
sic hydrogen bonds. Nevertheless, a number of intramolecular and
4

A. Zahirovi´c, S. Roca, E. Kahrovi´c et al.
Journal of Molecular Structure 1236 (2021) 130326
Fig. 2. Overlay of two molecules within the asymmetric unit of (f) reveals their opposite chirality and conformational flexibility.
intermolecular C–HˑˑˑO and C–HˑˑˑF interaction are responsible for
a highly complex 3D molecular network characterizing this crystal
structure (Table S2).
species. However, they are unaffected by coordination and based
solely on them it is impossible to conclude whether the pyridine
is present as a coordinated ligand, impurity or solvate. Another
relatively insensitive band in connection with the coordination of
pyridine to metal was found in the region relatively free of vibra-
tions of other ligands is a band assigned to C–H stretching vibra-
tions at 3080 cm–1. This band is somewhat higher positioned with
respect to those arising from other aromatic C–H vibrations (usu-
ally found between 3000 and 3050 cm–1). However, its low inten-
sity makes it relatively insecure for pyridine presence claim. The
band that was found to be the most indicative marker for pyri-
dine coordination to metal centre is a mid-region strong inten-
sity band at 1439 cm–1 arising from pyridine ring deformation (in-
plane C–H wagging). This band does not overlap with bands arising
from cymene or chalcone moieties and has a medium intensity in
the spectrum of a coordination compound. Moreover, it is shifted
to higher wavenumbers upon coordination thus undoubtedly con-
firming the presence of coordinated pyridine. The infrared bands
originating from hexafluorophosphate vibrations are easy to iden-
tify since they are generally of strong intensity. The band at 840
cm–1 arises from P–F bond stretching, while the band at 558 cm–1
is attributed to F2P deformation.
3.3. Infrared spectra
All reactions in applied multistep synthesis (Scheme 2) were
monitored by IR spectroscopy. Particular challenge was to observe
the coordination of pyridine to Ru(II) ion because of extensive
overlapping and lower bands intensity of nonpolar heterocycles in
the IR spectra [41–44]. Recorded infrared spectra of parent com-
pounds (a)–(e), complex (f) and NaPF6 are shown in Fig. S2 with
the selected infrared data summarized in Table S4.
Several weak intensity bands just below 3000 cm–1 arise from
C–H stretching vibrations of cymene saturated methyl and iso-
propyl groups. This is the only region in IR spectra with no overlap
with bands of other ligands and thus can be used to confirm the
presence of cymene moiety. The presence of other ligands in the
first coordination sphere of ruthenium does not significantly affect
the position of these bands. The evidence of chalcone coordina-
tion to Ru(II) is shifting ν(C=O) and ν(C–O(H)) bands upon coor-
dination. Strong absorption band located in the ligand spectrum at
1640 cm–1 is shifted for 16 cm–1 toward lower wavenumbers upon
coordination and is found in the spectrum of complex (f) at 1624
cm–1. This is a direct result of the C=O bond weakening as a re-
sult of chalcone coordination to Ru(II) through carbonyl oxygen. On
the other hand, the band arising from ν(C–O(H)) stretching, found
at 1339 cm–1 in the spectrum of ligand (e), is shifted to higher
wavenumbers upon coordination, as a result of the strengthening
of C–O bond due to the deprotonation of phenolic O–H group and
formation of weaker Ru–O bond.
3.4. NMR spectroscopy
In the recorded 1H and 13C NMR spectra of compounds (e) and
(f) only one set of signals was observed (Figs. 3 and S4–S7). Com-
plexation of hydroxychalcone (e) to ruthenium(II) ion was con-
firmed by difference in the 1H and 13C atom signal chemical shifts
between ligand (e) and complex (f), and by lack of the OH group
signal in the complex (f) proton spectrum.
The bands found in the spectrum (f) at 425 and 661 cm–1 (Fig.
S3), according to Nakamoto’s literature data were attributed to ring
rocking mode and out of plane pyridine vibrations, respectively
[45]. Both bands are weak and found in the low frequency re-
gion, where many otherwise symmetry-forbidden vibrations, are
awakened upon ligand coordination, due to the reduced symme-
try. Two other bands located at 748 and 700 cm–1 in the spec-
trum of pyridine, with moderate and strong intensity, respectively,
arise from C–H out of plane vibrations and are particularly use-
ful for estimation of the pyridine presence in isolated coordination
The biggest changes in 13C chemical shifts were observed for
the C-15 (ꢀδcoord = –6.7 ppm) and C-9 (ꢀδcoord = 6.6 ppm) atom
signals indicating O,O-chelating binding of the chalcone moiety
to the ruthenium(II) ion. The difference in atom signals chemical
shifts after complexation was also noticed for para-cymene: aro-
matic ring H-atoms and H-25 were unshielded (ꢀδcoord = 0.41;
0.06 ppm) and H-21 and H-27 were shielded (ꢀδcoord ≈ –0.09;
–0.03 ppm). Pyridine molecule binds to the Ru(II) ion through
the nitrogen lone electron pair, and the rest of the 1H ring
atom signals were unshielded (ꢀδcoord < 0.06 ppm). Observed in-
5

A. Zahirovi´c, S. Roca, E. Kahrovi´c et al.
Journal of Molecular Structure 1236 (2021) 130326
Fig. 3. 300 MHz 1H NMR spectrum with assignments and numeration scheme of (f).
Scheme 3. H-7 and H-25 atom signals chemical shift changes (ꢀδcoord.) in the 1H NMR spectrum before ((a), (e)) and after complexation (f); ꢀδcoord. = δcomplex – δligand.
tramolecular NOE cross peaks between pyridines H-26 and H-30
atoms and H-22, H-23, H-24, H-25 of para-cymene, and H-14 of
chalcone moiety confirmed also the half-sandwich conformation
of (f) in the solution. Coordination effect was observed for H-7
and H-25 atom signals which were unshielded for 0.26 ppm and
0.06 ppm, respectively. It is in accordance with hydrogen interac-
tion found by X-ray, and in the solution NMR it was attributed to
the steric effect of oxygen atoms, O-1 and O-2, which are probably
getting closer to H-7 (O-1) and H-25 (O-2) after breaking ligand
strong hydrogen bond O-1ˑˑˑH–O-2 (δ = 12.81 ppm) when bind-
ing to metal ion (Scheme 3). All other chalcone atom signals in 1H
NMR spectra were shielded (ꢀδcoord = –0.20 – (–0.50) ppm). De-
spite a large signal overlap (7H) in the range 7.45–7.37 ppm, all
complex peaks were assigned by means of the 2D NMR methods
(COSY, NOESY, HMQC, HMBC); Figs. S8–S11.
3.5. Electronic spectra
The electronic spectra of ligands (chalcone and pyridine), par-
ent ruthenium compound and ruthenium organometallic (f) are
shown in Fig. S3. The electronic spectrum of (f) is dominated by
two strong absorption bands, one medium intensity band and one
shoulder in 200 – 600 nm region. The medium intensity band is
located in visible spectrum with maximum near 490 nm and arises
from MLCT transitions [Ru(4dπ)→π∗(ligand)]. The band at 316 nm
in the spectrum of chalcone ligand can be assigned to n→π∗ tran-
sitions. Upon coordination of chalcone to ruthenium this band is
shifted to higher wavelength and is found at 322 nm in the spec-
trum of (f). The bathochromic (red) shift of 6 nm is a consequence
of deprotonation of the chalcone phenolic group and coordination
to Ru(II) [14,46]. Very wide shoulder in spectrum of (f) (220 – 270
6

A. Zahirovi´c, S. Roca, E. Kahrovi´c et al.
Journal of Molecular Structure 1236 (2021) 130326
Fig. 4. Interaction of organometallic (f) with CT DNA: a) spectroscopic titration of (f) with CT DNA; b) behaviour of (f) in buffered aqueous solution; c) graphical determina-
tion of binding constant of (f) with CT DNA.
nm) is the consequence of the superimposed absorptions corre-
sponding to the transitions arising from pyridine (260 nm) and
chalcone ligand (220 nm) [47]. The absorption band at 203 nm
arises from π→π∗ transitions of aromatic cores of all three aro-
matic ligands [48].
separation of the chains. On the contrary, the decrease of the ab-
sorbance at 260 nm indicates shortening of DNA chains due to
electrostatic interactions. The titration experiment (Fig. 5a) shows
that no significant changes in the secondary structure of DNA are
observed within [DNA]/[organometallic] ratio 25 – 3. However, at
[DNA]/[organometallic] = 2, significant decrease of the absorbance
was observed over time (Fig. 5b). The hypochromic effect at DNA
absorption maximum (near 260 nm) is in correlation with confor-
mational change of DNA and indicates shrinkage of helix in length
as a consequence of the electrostatic interaction of (f) with DNA.
This result is in a good correlation with the low Kb value obtained
from spectroscopic titration of organometallic with CT DNA (Fig. 4).
The percentage of the absorbance decrease over time was plotted
in Fig. 5c. The curve clearly shows that the absorbance decrease
is the highest over the first 15 minutes of the reaction, indicating
that the binding of organometallic (f) to DNA is relatively fast.
3.6. Interaction with DNA
The spectroscopic titration of (f) with increasing concentrations
of CT DNA is shown in Fig. 4a. The moderate hypochromism with-
out significant shift of the absorption maximum was observed at
the band positioned near 320 nm. Fig. 4b demonstrates behaviour
of (f) in aqueous solutions over time and indicates that the com-
pound is hydrolytically inert. Thus, all changes in the electronic
spectra of the compound (f) during the spectroscopic titration
(Fig. 4a) can be addressed to interaction of (f) with CT DNA. The
interaction of chalcone ligand (e) with CT DNA also showed mod-
erate hypochromism without observable band shifting (Fig. S12).
The data corresponding to the titration experiment are sum-
marized in Table S5 and the binding constant (Kb) of (f) with
CT DNA was determined graphically from the plot [DNA] vs
[DNA]/(εf– εa) as the slope/intercept ratio (Fig. 4c). The bind-
ing constant of (f) with CT DNA was determined as 4.47 × 102
M–1 and is tenfold lower compared to the one measured for
chalcone ligand (e) (4.67 × 103 M–1). The low value of binding
constant indicates that the cationic organometallic (f) predomi-
nantly interacts with CT DNA through electrostatic pairing with
negatively charged phosphate groups. The values of binding con-
stant for groove binding and intercalation are significantly higher,
having values 104 – 106 M–1. Several other cationic ruthenium
organometallics showed electrostatic binding mode to DNA such
as those having ofloxacin [49], ethylenediamine [50] or bis(3,5-
dimethylpyrazolyl) derivatives of benzoic acid [51] as ligands. Also,
some non-arene organometallics of ruthenium(II) such those of 2-
phenylpyridine prefer electrostatic binding mode [52]. On the other
hand, planar π-conjugated aromatic system of chalcone (e) is suit-
able for groove binding or intercalation as it was observed for
some anthraquinone–chalcone hybrids [53] and methoxy substi-
tuted 2’-hydroxychalcones [54].
3.7. Interaction with BSA
The interaction of chalcone ligand (e) and organometallic (f)
with BSA was investigated spectrofluorimetrically utilizing the
spectroscopic titration method. When the excitation of BSA was
carried out at 279 nm, two amino acid residues, tryptophan and
tyrosine, show intrinsic fluorescence with the emission maximum
near 340 nm. Actually, the emission is mostly a consequence of the
fluorescence of two sterically hindered tryptophan residues, Trp-
134 in the first domain and Trp-212 in the second domain of BSA.
Since the intensity and the position of the emission maxima is di-
rectly related to secondary structure of BSA, the changes in the
emission spectrum can be addressed to their interaction with small
molecules.
When the aqueous solution of BSA is titrated with organometal-
lic (f) or chalcone ligand (e), the intrinsic fluorescence is quenched
(Fig. 6a and Fig. S14a). The complexes (f) does not show intrin-
sic florescence in aqueous solution under the same conditions (Fig.
S13). The quenching constant (KSV) can be determined from Stern-
Volmer equation graphically as the slope of [complex] vs I0/I plot
(Fig. 6b and Fig. S14b). The quenching constant of (f) was deter-
mined to equal 2.23 × 105 M–1 indicating that the organometal-
lic strongly quenches BSA fluorescence causing significant changes
in polarity and conformation of tryptophan. The quenching con-
stant of chalcone (e) is slightly lower (1.30 × 105 M–1). The
rate of quenching (kq) was found to be 2.23 × 1013 M–1 s1 for
organometallic (f) and 1.30 × 1013 M–1 s1 for chalcone (e) confirm-
ing that the quenching occurs in both cases through static mecha-
To confirm the electrostatic binding mode of organometallic (f)
with CT DNA, the titration experiment was carried out (Fig. 5a).
The intensity of the absorption near 260 nm in electronic spec-
trum of CT DNA is directly related to DNA helix. The extinction
coefficient of the single stranded DNA is higher (7100 M–1 cm–1
)
as compared to double stranded DNA (6600 M–1 cm-1) and ab-
sorbance increase at 260 nm indicates denaturation of DNA and
7

A. Zahirovi´c, S. Roca, E. Kahrovi´c et al.
Journal of Molecular Structure 1236 (2021) 130326
Fig. 5. Interaction of CT DNA with organometallic (f): a) spectroscopic titration of CT DNA with (f); b) electronic spectra of CT DNA over time during the incubation of CT
DNA and (f) at 2:1 ratio; c) absorbance decrease at CT DNA absorption maximum over time during the incubation of CT DNA and (f).
Fig. 6. Interaction of organometallic (f) with BSA: a) spectrofluorimetric titration of BSA with (f); b) graphical determination of Stern-Volmer constant; c) graphical determi-
nation of binding constant and number of binding sites.
in 1.03 × 104 – 7.77 × 105 M–1 [25]. Also, benzene and cymene
nism, since the maximal value of kq expected for dynamic quench-
ing is 2 × 107 M–1 s–1
.
organometallics of ruthenium(II) with methylhomopiperazine
derived ligands bind moderately strong to BSA (1.7
×
104
The number of binding sites (n) and the binding constant (Kb)
were determined from log[complex] vs log[(I0 – I)/I] plot (Fig. 6c
and Fig. S14c). The number of binding sites was found to be close
to 1, indicating that the organometallic (f) prefers binding with BSA
in 1:1 ratio as many other small molecules. The binding constant
of 1.04 × 106 M–1 demonstrates that organometallic (f) strongly
binds BSA. The binding constant value is comparable to the values
–
4.48 × [26], while half-sandwich Ru(II) arene
104 M–1
)
chloride-complexes of quinolin substituted benzo[d]imidazole,
benzo[d]oxazole and benzo[d]thiazole bind BSA more strongly
with binding constant in 3.63
range [59]. Non-arene ruthenium organometallics of hydrazine
ligands [60], 2-acetyl-5-chlorothiophene thiosemicarbazone
×
105 – 6.52
×
105 M–1
for drugs such as ibuprofen (Kb = 3.6 × 106
M
−1) and diazepam
[61] and binuclear thiosemicarbazone ruthenium complexes
with 5-nitrofuryl pharmacophore [62] bind to BSA with
104 to 105 M–1 binding constants. The binding constant of
[Ru(HL)(CH3CN)(CO)(PPh3)2] with HL= 4-oxo-4H-pyran-2,6-
(Kb = 1.6 × 106
M
−1), which are known to bind albumin very
tightly [55,56]. On the other hand, binding constant of chalcone
(e) with BSA was determined as 1.88 × 104 M−1 and is fiftyfold
lower compared to the one observed for organometallic (f). How-
ever the binding constant value is comparable to fluoro substituted
dicarboxylic acid (Kb = 2.89
×
106 M-1) is comparable to the
binding constant of ruthenium(II) organometallic having chalcone
and pyridine coligands [63].
chalcones ((3.74 – 4.81) × 104
M
−1) [57], but lower compared to
non-substituted chalcone (4.77 × 105
M
−1) [58].
As it was mentioned above, the fluorescence of the BSA arises
from tryptophan and tyrosine. The distinction between the emis-
sion of these two amino acid residues can be made by synchronous
spectroscopy, when the difference between the emission and ex-
citation wavelength is fixed (ꢀλ = λem – λex). If the ꢀλ is set
to 15 nm, the emission corresponds to tyrosine residues, while
The binding constant of ruthenium(II) organometallic having
chalcone and pyridine coligand to BSA is somewhat larger as
compared to many other ruthenium-arene organometallics. The
cyclopentadienylruthenium(II) compounds of acetophenone-4(N)-
substituted thiosemicarbazone binding BSA with binding constant
8

A. Zahirovi´c, S. Roca, E. Kahrovi´c et al.
Journal of Molecular Structure 1236 (2021) 130326
Fig. 7. Synchronous emission spectra of BSA in presence of increasing concentrations of (f) at: a) ꢀλ = 15 nm; b) ꢀλ = 60 nm.
the tryptophan emission is observed when ꢀλ = 60 nm [64].
The synchronous emission spectra of BSA in the presence of in-
creasing concentrations of organometallic (f) and chalcone (e) are
shown in Fig. 7 and Fig. S15, respectively. Both compounds induce
emission decrease at both bands indicating that the microenviron-
ment around tryptophan and tyrosine of BSA changes when the
organometallic (f) or chalcone (e) are present. Under the same ex-
perimental conditions, the larger emission decrease at tryptophan
emission maximum is observed in presence of complex (f) com-
pared to ligand (e). Moreover, this decrease in presence of com-
plex (f) is followed with small 1 nm hypochromic shift indicating
that the binding of organometallic (f) to BSA disturbs tryptophan
environment more than the one of tyrosine.
comparable to the one of commercial drugs such as ibuprofen
and diazepam, and is fiftyfold higher as compared to parent chal-
cone ligand. The synchronous spectroscopy confirmed that the mi-
croenvironment around tryptophan is more affected as compared
to the one of tyrosine. The results encourage further testing of
[(cym)Ru(py)(chalc)]PF6 for potential anticancer activity, especially
due to the fact that it shows strong protein and low DNA binding
affinity, which can be used is design of novel non-cytotoxic an-
timetastatic ruthenium organometallics. The higher affinity of our
organometallic to BSA as compared to DNA can be associated with
more hydrophobic nature of BSA compared to DNA.
Author statement
The higher affinity of (f) for BSA compared to DNA can be at-
tributed to cationic, non-planar and hydrophobic nature of molec-
ular ion of organometallic (f). The cationic nature dictates the elec-
trostatic binding mode of (f) with DNA, while non-planar pseudo
octahedral piano-stool conformation does not facilitate intercala-
tion. The absence of heteroatoms, beyond those included in coor-
dination to Ru, suitable for H-bond formation or other weak inter-
actions of complex with DNA results in poor affinity of the com-
plex to DNA and leaves electrostatic binding as the only possibility
for interaction (f) with DNA. On the other hand, cationic nature of
(f) facilitates binding with BSA but it is not decisive factor as in
the case of DNA since BSA abounds with many surface available
hydrophobic pockets [65,66] which accommodate hydrophobic in-
teraction with lipophilic aromatic motives of organometallic (f).
Aleksandar Višnjevac: X-ray structure determination, conceptu-
alization, manuscript preparation
Suncˇica Roca: NMR measurements and interpretation
Adnan Zahirovic´: Syntheses of the compounds, physico-
chemical measurements and interpretation (with the exception of
NMR and single crystal X-ray studies), interaction with BSA and
DNA assays
Emira Kahrovic´: conceptualization, manuscript preparation
Supplement
CCDC 2041048 contain the supplementary crystallographic data.
These data can be obtained free of charge via http://www.ccdc.
cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallo-
graphic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax:
(+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.
4. Conclusions
The solution synthesis afforded cationic racemic chiral-at-metal
organometallic ruthenium(II) compound fully characterized in the
solid state and in the solution. The ruthenium(II) is embed-
ded in pseudo-octahedral piano-stool conformation coordinated by
cymene ligand in a η6-manner, bidentate monobasic chalcone lig-
and through phenolic and carbonyl oxygen atoms and the nitro-
gen atom of pyridine. The ex-situ monitoring of the pyridine co-
ordination to Ru(II) by infrared spectroscopy revealed that band
arising from pyridine ring deformation is the most useful marker
for pyridine coordination. The interaction of organometallic with
CT DNA revealed that synthesized compound only weakly binds
to DNA by electrostatic interaction with negatively charged phos-
phate backbone and has lower affinity to DNA compared to par-
ent chalcone ligand. The emission spectroscopy showed that the
intrinsic fluorescence of BSA is strongly quenched in the pres-
ence of ruthenium organometallic and chalcone ligand. The high
value of [(cym)Ru(py)(chalc)]PF6 binding constant with BSA is
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgements
The authors gratefully acknowledge the contribution of Dr. Jas-
minka Popovic´ for the PXRD analysis.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.130326.
9

A. Zahirovi´c, S. Roca, E. Kahrovi´c et al.
Journal of Molecular Structure 1236 (2021) 130326
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