Novel silver(I) complexes of coumarin oxyacetate ligands and their phenanthroline adducts: Biological activity, structural and spectroscopic characterisation

Article abstract of DOI:10.1016/j.jinorgbio.2016.07.010

Novel silver(I) complexes of coumarin oxyacetate ligands and their phenanthroline adducts have been prepared and characterised by microanalytical data and spectroscopic techniques (IR, ¹H, ¹³C NMR, UV–Vis). The crystal structure of one Ag(I) complex was determined by X-ray diffraction analysis. The experimental spectroscopic data have been interpreted on the basis of molecular structure modeling and subsequent spectra simulation with density functional theory method. The binding modes of the coumarins and phenanthroline ligands (monodentate, bidentate, bridging) to Ag(I) have been theoretically modelled and discussed as to the most probable ligand binding in the series of complexes studied. The antimicrobial and antifungal activities have been determined and the complexes were found to have mostly moderate antibacterial activity but some of the phenanthroline adducts were found to have antifungal activity against the clinically important fungus C. albicans, comparable to that of the commercial agents, Amphotericin B and Ketoconazole. Preliminary investigations into the possible mechanism of action of the silver complexes indicated that they did not interact with DNA via nuclease activity or intercalation but the ability to act as a superoxide dismutase mimetic may be related to their antimicrobial activity.

Full text of DOI:10.1016/j.jinorgbio.2016.07.010

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Muhammad Mujahid, Natasha Trendafilova, Agnieszka Foltyn Arfa-Kia,
Georgina Rosair, Kevin Kavanagh, Michael Devereux, Maureen Walsh,
Siobh a´ n McClean, Bernadette S. Creaven, Ivelina Georgieva
PII:
DOI:
Reference:
S0162-0134(16)30211-2
doi: 10.1016/j.jinorgbio.2016.07.010
JIB 10043
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
8 January 2016
5 July 2016
13 July 2016
Please cite this article as: Muhammad Mujahid, Natasha Trendafilova, Agnieszka
Foltyn Arfa-Kia, Georgina Rosair, Kevin Kavanagh, Michael Devereux, Maureen Walsh,
Siobh ´a n McClean, Bernadette S. Creaven, Ivelina Georgieva, Novel ¡!–[INS][S]–¿s¡!–
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Journal of Inorganic Biochemistry (2016), doi: 10.1016/j.jinorgbio.2016.07.010
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ACCEPTED MANUSCRIPT
Novel Silver(I) Complexes Of Coumarin Oxyacetate Ligands And Their Phenanthroline
Adducts: Biological Activity, Structural And Spectroscopic Characterisation.
a
b
a
Muhammad Mujahid, Natasha Trendafilova, Agnieszka Foltyn Arfa-Kia, Georgina
c
d
e
a
a
Rosair, Kevin Kavanagh, Michael Devereux, Maureen Walsh, Siobhán McClean,
a*
b*
Bernadette S. Creaven, Ivelina Georgieva.
a
Centre of Applied Science for Health, Institute of Technology Tallaght, Dublin 24, Ireland.
b
Institute of General and Inorganic Chemistry, Bulgarian Academy of Sciences, 11 Acad. G.
Bonchev Str., Sofia, Bulgaria
c
School of Engineering & Physical Sciences, Heriot-Watt University, Edinburgh, Scotland,
UK EH14 4AS
d
Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland
e
The Centre for Biomimetic and Therapeutic Research (CBTR), College of Sciences and
Health, Dublin Institute of Technology, Camden Row, Dublin 8, Ireland.
Corresponding authors: I. Georgieva, Email: ivelina@svr.igic.bas.bg, Tel: 00-359-2-
9
792592; B. S. Creaven, Email: bernie.creaven@ittdublin.ie , Tel: 00-353-1-4042889.
Abstract: Novel silver(I) complexes of coumarin oxyacetate ligands and their phenanthroline
adducts have been prepared and characterised by microanalytical data and spectroscopic
1
13
techniques (IR, H, C NMR, UV-Vis). The crystal structure of one Ag(I) complex was
determined by X-ray diffraction analysis. The experimental spectroscopic data have been
interpreted on the basis of molecular structure modeling and subsequent spectra simulation
with density functional theory method. The binding modes of the coumarins and
phenanthroline ligands (monodentate, bidenate, bridging) to Ag(I) have been theoretically
modelled and discussed as to the most probable ligand binding in the series of complexes
studied. The antimicrobial and antifungal activities have been determined and the complexes
were found to have mostly moderate antibacterial activity but some of the phenanthroline
adducts were found to have antifungal activity against the clinically important fungus C.
albicans, comparable to that of the commercial agents, Amphotericin B and Ketoconazole.
Preliminary investigations into the possible mechanism of action of the silver complexes
indicated that they did not interact with DNA via nuclease activity or intercalation but the
ability to act as a superoxide dismutase mimetic may be related to their antimicrobial activity.

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Keywords: Coumarin, phenanthroline, Ag(I) complexes, DFT modeling, biological activity,
X-ray structure
1
. Introduction
Several multidrug resistant (MDR) strains of bacteria such as fluoroquinolone- and
carbapenem-resistant Pseudomonas aeruginosa and vancomycin-resistant Staphylococcus
aureus have emerged as a result of the ability of microbes to adapt to continuously changing
environments [1-5]. Treatment of resistant bacterial strains has become a major problem
with rising mortality rates and extended hospitalisation for patients translating into ever
increasing health care costs, with a report in 2015 estimating that cost to the EU to be at 1.5
billion euro annually [6]. Common pathogens such as S. aureus are responsible for harmful
infections including mastitis, upper respiratory complications, bacteraemia, pneumonia, skin
and soft tissue infections. In particular, S. aureus causes a diversity of infections, from skin
infections to disseminated systemic infections leading to organ failure and death [3, 7, 8].
These pathogens can easily be transmitted between patients and have become resistant to
different classes of antibiotic drugs, e.g., some strains of methicillin resistant S. aureus
(
[
MRSA) are often resistant to several antibiotics, including penicillins and cephalosporins
9-11]. In Western Europe a review of intensive care unit infections found that P. aeruginosa
was identified as one of the most common organisms, constituting nearly a third (29%) of all
Gram-negative isolates, and was present in 17% of all positive cultures [12]. In addition, in
2013, the Centre for Disease Control and Prevention published a report outlining the threat
posed by MDR Gram-negative pathogens and highlighted that the world is currently running out
of drugs to treat several Gram-negative infections [13].
The number of incidences of serious fungal infections is also increasing dramatically
and with it a rise in resistance to antifungal treatment and its associated healthcare costs.
Individuals with weakened immune systems are more likely to get fungal infections. These
individuals are either victims of Acquired Immune Deficiency Syndrome (AIDS) or cancer
patients receiving aggressive chemotherapy, or following organ transplantation and long
hospitalisation [14]. Candida albicans is a causal agent of opportunistic oral and genital
infections in humans. It can also form biofilms on the surface of implantable medical devices.
In addition, it is a major pathogen causing systemic fungal infections as well as hospital-
acquired infections and has become an important clinical problem [15]. It is responsible for

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several infections in humans including deep mycoses, bloodstream infections, mouth, skin,
vaginal and human immunodeficiency virus (HIV)-related infections as well as Candidiasis
[
16, 17]. Oral infections can be treated with drugs such as Fluconazole and Ketoconazole or
typically with polyene-based Amphotericin B and systemic fungal infections can be treated
with intravenous administration of Amphotericin B or caspofungin. However, azole-resistant
strains of Candida albicans have emerged and these have created problems for
administering azole-based treatments [18]. In addition, some of these drugs, such as
Amphotericin B are nephrotoxic and damage host cells [14]. The increasing incidence of
antifungal resistance in Candida spp. reflects the extensive and escalating use of newer
broad-spectrum antifungal agents and thus presents a serious threat to patients worldwide
[
19, 20].
Generally, silver(I) complexes are known to possess significant antibacterial
activities against Gram-positive and Gram-negative bacteria and have shown excellent
In recent reviews the applications of silver(I)
activity against C. albicans [21-26].
metallodrugs and current silver-based antimicrobial therapeutics which are commercially
available are discussed in detail [27, 28]. More recently the synthesis of silver/amino acid
biopolymer hydrogels as mouldable wound dressings have also been reported [29-31].
Thus
the role of silver(I), acting as a free ion, or in compounds such as sulphurdiazine or as a
hydrogel or nanoparticle has been well established. We have been interested in silver(I)
complexes of coumarins for a number of years and they have shown activity against a
number of antibiotic and antifungal resistant strains. Coumarins are a class of compounds
which occur naturally in many plants and have revealed a diverse array of pharmacological
properties [32]. Previous studies have shown that silver(I) complexes of coumarin-3-
carboxylates and coumarin-4-carboxylates have excellent activity against MRSA and P.
aeruginosa respectively [21, 33, 34]. Exposure of S. aureus to silver nitrate has been shown
to lead to a transient increase in superoxide dismutase (SOD) activity and catalase activity
which may indicate that the silver compound induces oxidative stress in the cell [35]. A more
recent paper has shown that silver enhances antibiotic activity against Gram-negative bacteria
by interfering with cellular processes and sensitizing the cells towards current antibiotics
[
36]. The authors reported that the presence of silver appeared to induce oxidative stress in
treated cells. We have recently reported a series of water-soluble and photo-stable silver(I)
dicarboyxlate complexes with excellent antimicrobial activity and anticancer activity.
Interestingly these complexes appeared to have a different mechanism of action to other

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silver(I) compounds in that they did not induce deoxyribonucleic acid (DNA) damage or
reactive oxygen species (ROS) generation and in fact seemed to act as antioxidants in the
systems studied, significantly reducing superoxide and hydrogen peroxide induced oxidative
stress in the yeast S. cerevisiae [37].
The results of our previous studies have also shown that the mode of antifungal action
for the silver(I) coumarin-based complexes was based on their interference with respiration
in the fungal cells, resulting in reduced ergosterol synthesis and cytochrome production [38].
This mode of action is different to conventional azole and polyene drugs (which bind to
ergosterol or block ergosterol biosynthesis, respectively) and thus they looked like promising
candidates for new antifungal treatment. Subsequent studies on related phenanthroline
adducts of these complexes and other related phenanthroline silver(I) adducts showed that
their mechanism of action was based on disruption of mitochondrial function also [39].
In all of the coumarin complexes previously studied, their solubility was of concern to
us and in many studies the complexes were tested as suspensions. In an attempt to improve
the activity of coumarin carboxylate silver(I) complexes by means of higher solubility and
better pharmacological properties we succeeded in isolating a series of Ag(I) complexes with
coumarin oxyacetate ligands in the 3-, 4-, as well as the 6- and 7-positions on the aromatic
ring. In addition, their 1,10-phenanthroline (phen) adducts were prepared as the solubility
and biological activities of the complexes are also known to increase significantly upon
adduct formation with phen [40-44]. The novel Ag(I) complexes were characterised by
1
13
experimental physico-chemical and spectroscopic (IR, H, C NMR and UV-Vis) studies in
solid state and in solution and by X-ray diffraction analysis. In addition, density functional
theory (DFT) modeling of the compounds studied was performed to support or predict the
metal-ligand binding mode in the coumarin oxyacetate-derived silver(I) complexes and their
phen adducts based on the good agreement between the experimental and calculated
spectroscopic data.
2. Experimental
2
.1. Reagents and instruments
Chemicals and solvents were purchased from Sigma-Aldrich Co. (Dorset, UK) and used
without further purification. Melting points were recorded on a Stuart Scientific SMP-1

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apparatus (up to 300 °C). Thin layer chromatographic (TLC) study was carried out on Silica
gel 60F254 aluminium-backed plates using a pre-saturated elution tank. The compounds were
detected under ultraviolet light and stained using iodine crystals in a closed chamber. Infrared
spectra of solids (as a dispersion in KBr disc) were recorded on a Nicolet Avatar 320 FT-IR
-1
-1
spectrophotometer operating at a resolution of 2 cm in the 4000–400 cm region. The
1
Bruker Biospin 300 and 500 MHz FT-NMR spectrometers were used to record H NMR
1
3
spectra (-5 to 15 ppm with a resolution of 0.18 Hz) and C NMR spectra (-33 to 233 ppm
with a resolution of 2.40 Hz) as solutions in dimethyl sulfoxide (DMSO-d ) using
6
,
tetramethylsilane (TMS) as the internal reference standard. The signal assignments were
made using standard techniques including distortionless enhancement by polarization transfer
(DEPT), homonuclear correlation spectra (COSY) and CH-Shift. Where necessary,
Heteronuclear Single Quantum Correlation (HSQC), Heteronuclear Multiple Bond
Correlation (HMBC) and Nuclear Overhauser effect spectroscopy (NOESY) experiments
were also run to facilitate peak assignments. Microanalytical data were provided by the
Microanalytical Laboratory, Department of Chemistry, University College Dublin, Belfield,
Dublin-4, Ireland, using a Perkin-Elmer CHN analyser. Silver content was determined using
atomic absorption spectroscopy on a Perkin Elmer 460AAS instrument. All samples were
analysed in triplicate. UV-Vis spectra of the complexes and their corresponding ligands were
recorded on a PG Instruments T80 UV/Vis spectrophotometer in DMSO solvent (260-800
nm). The molar conductivity of the complexes and their corresponding ligands was measured
on an EUTOH INSTRUMENTS Cyberscan-500 digital conductometer, using 4 mM solution
of compounds in DMSO at 25 °C. The metal and water contents of the complexes were
determined by using thermogravimetric technique. Thermogravimetric analysis (TGA) was
carried out in the temperature range from 25 to 800 °C in a nitrogen atmosphere using TA
Instrument’s Q50 Thermogravimetric analyser. The experimental conditions were: platinum
weighing pan, nitrogen atmosphere with a 60 ml/min flow rate and a heating rate of 10
°
C/min.
A single crystal was coated in Paratone-N heavy oil then mounted on a Hampton
Research Cryoloop and placed in a cold stream of nitrogen gas (100 K) on a Bruker Nonius
X8 Apex2 CCD diffractometer running the Apex2 software (Bruker 2011). Data collection
consisted of, on average, 3000 frames from a combination of phi and omega scans. After unit
cell determination, intensity data were integrated using SAINT then scaled with TWINABS
[
45] (Ag(I) complex 33 was treated as a 3 component crystal) before Space group
determination with XPREP [46]. The SHELXTL suites of programs [46, 47] were used for

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structure solution and OLEX2 for figures and refinement [48]. All non-hydrogen atoms were
refined anisotropically but significant numbers were subject to restraints. The water
molecules were treated as rigid bodies to find initial suitable H atom positions where close
H∙∙∙H contacts were avoided and hydrogen bonding possibilities maximised and then their
coordinates were fixed (using AFIX 1).
2.2. Syntheses
2.2.1. Synthesis of 2-(2-oxo-2H-chromen-substituted-yl)oxy acetic acid-based ligands (12-22)
Firstly, the ethyl 2-(2-oxo-2H-chromen-substituted-yl)oxy acetate derivatives (1–11) were
prepared by reaction of the appropriate substituted monohydroxy coumarin with ethyl 2-
bromoacetate. The synthetic methodology, together with the analytical and spectroscopic
data is given in Scheme S1, Table S1-S4 and Section S1 in Supplementary material. Then,
the compounds obtained (1–11) were used as precursors for the synthesis of 2-(2-oxo-2H-
chromen-substituted-yl)oxy acetic acids (12-22) (Table 1) using the synthetic methodology
described in Section S2. The analytical and IR spectral data for all the 2-(2-oxo-2H-chromen-
substituted-yl)oxy acetic acid derivatives synthesised are shown in Table S5 (Supplementary
1
13
material). Full H and C NMR spectral data are given in Tables S6 and S7, respectively
1
(
Supplementary material). The atom numbering system used for the assignment of the H and
1
3
C NMR spectra is given in Table 1.
2.2.2. Synthesis of silver(I) complexes of 2-(2-oxo-2H-chromen-substituted-yl)oxy acetic acids
(23-33)
A literature method was adopted for the synthesis of these complexes [21] and full synthetic
details are given in Section S3 in Supplementary material. Selected bond lengths and bond
angles obtained from crystal structure determination of complex 33 are presented in Table 2.
1
13
Full analytical and IR, H, C NMR and UV-vis spectroscopic data are presented in Section
S3, Supplementary material with selected data for representative complexes given in Table
S8 and Tables 3-6.
2
.2.3. Synthesis of 1,10-phenanthroline adducts of silver(I) complexes of 2-(2-oxo-2H-
chromen-substituted-yl)oxy acetic acid-based ligands (34-44)
A literature method was adopted for the synthesis of these adducts [21]. All of the following
reactions were carried out under dark conditions to obtain suitable complexes for structure
elucidation studies. The experimental data recorded for phen adducts (34-44) are given in

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Section S4 in Supplementary material with selected data for representative complexes given in
Tables 3-6.
2.3. Computational details
Molecular structures, vibrational (IR), NMR and absorption spectra calculations of
the model Ag(I) complexes: Ag (C-3oxy-acet) , Ag(C-3oxy-acet)phen , Ag (8acetyl-C-7oxy-
2
2
2
2
acet) and Ag(8acetyl-C-7oxy-acet)phen were carried out with DFT/TDDFT method using
2
2
Gaussian09 program [49]. A bidenate oxylate bridging binding was simulated in Ag (L)
2
2
models as it was found in the X-ray determined structure of [Ag(8acetyl-C-7oxy-acet)] (33).
Following the suggested formula for Ag(I)-phen adducts, a possible monodentate oxylato
bonding of the oxy-acetato coumarin and N,N bidentate bonding of the two phen ligands
were simulated for Ag(C-3oxy-acet)phen and Ag(8acetyl-C-7oxy-acet)phen structures. For
2
2
comparison, the corresponding calculations were performed for the isolated neutral ligands
C-3oxy-acetH (12) and 8acetyl-C-7oxy-acetH (22). Split valence plus polarization basis set
6
-31G(d) was applied for the main group elements: for hydrogen atoms - (4s)/[2s], for carbon
and oxygen atoms - (10s4p1d)/[3s2p1d]. To give a better description of the wave functions in
the intermolecular region and hence to improve the estimation of the Ag(I) – coumarin ligand
interactions, diffuse functions were added to the standard basis set (one s and one p set) for
all O atoms as well as for the two acetoxy carbon, C11 and C12 atoms (see a scheme under
Table 1). The combined basis set is denoted CB1. The computations using CB1 basis set
were carried out with the Lee, Yang, and Parr correlation functional (LYP) [50] combined
with Becke’s three parameters exchange functional (B3) [51]. The adequacy of B3LYP
method for prediction of conformational behaviour, geometry parameters and vibrational
spectra of coumarin derivatives and their metal complexes was proven in our previous
investigations [52, 53]. The Ag(I) ion was calculated with the effective core potential (ECP)
optimized by the Stuttgart–Dresden group [54, 55]. The small quasi-relativistic
pseudopotential, ECP28MWB, was used for Ag(I) in combination with its optimized valence
basis set (8s7p6d)/[6s5p3d]. The small core (RECPs) for the Ag(I) centre considers explicitly
2
6
10
1
10
1
9 valence electrons (4s 4p 4d 5s ), whereas 28 core electrons ([Ar]3d shells) are replaced
by the pseudopotential.
Full geometry optimizations of all the molecular systems were carried out without
symmetry constraint. The minima on the potential energy surfaces were qualified by the
absence of negative eigenvalues in the diagonalized Hessian matrix, giving imaginary normal

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vibrational mode. The projection of the internal coordinates onto each normal mode in terms
of percentage relative weights was computed as implemented in Gaussian09. Each normal
mode is described on the basis of the largest percentage value of the total displacement vector
magnitude. In addition, the vibrational modes have been analysed by visual inspection of the
1
13
modes animated using the ChemCraft program [56]. The H and C NMR chemical shift
calculations of the model compounds 12, 22, 23, 33, 34 were performed in DMSO at
B3LYP/6-31+G(d,p) level. The absolute isotropic magnetic shielding constants ( ) were
i
used to obtain the chemical shifts ( = - ) for C and H atoms by referring to the standard
i
TMS
i
compound, TMS. The UV-Vis absorption spectra of the ligands and their Ag(I) complexes
were simulated in the region 200-400 nm by means of vertical excitation energy calculations
(of singlet excited states) in DMSO using TDDFT method and B3LYP/CB1 level. NMR and
UV–Vis spectra calculations in DMSO were performed for the optimized geometries in the
same solvent. A non-equilibrium implementation of the Polarisable Continuum Model (PCM)
using the integral equation formalism variant (IEFPCM) was applied to simulate the solvent
[
57-59].
2
.4. Antimicrobial studies
The ligands (12-22), Ag(I) complexes (23-33), phenanthroline adducts (34-44) and
commercially available drugs, gentamycin and vancomycin, were screened for their
antimicrobial activity. S. aureus (NCIMB 12702) was obtained from the National Collection
of Industrial, Marine and Food. MRSA strain ATCC4300 and P. aeruginosa strain ATCC
2
7853 were obtained from the American Type Culture Collection. P. aeruginosa were
cultured in Luria Bertani (LB) broth (Sigma) while MRSA was cultured in nutrient broth.
o
Bacteria were stored in broth with 20% glycerol at -80 C and passaged twice on agar plate
(
LB agar, Trypticase Soy agar (TSA) or nutrient agar respectively) before being used in an
experiment. MIC50 were determined by broth microdilution. Stock solutions of each
compound to be tested were prepared in DMSO and serially diluted in triplicate using broth
(100 µl/well) at a concentration range of 0.195–200 µM on microtitre plates, after which the
final concentration of DMSO in the cell suspension was not greater than 1%. Overnight
o
cultures (10 ml) of bacterial strains were inoculated in broth (100 ml) and incubated at 37 C
with agitation, optical density at 600 nm (OD ) was measured and strains were then diluted
6
00
6
to 1 x 10 CFU/ml and seeded at 100 µl/well in microtitre well plates. Bacteria were also

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treated with a solvent control (0.5% DMSO) in control wells. Following 24 h incubation the
absorbance was read in a microtitre plate reader. Each compound was screened at each
concentration at least in triplicate and three independent experiments were carried out.
In this study, the ligands, their Ag(I) complexes and 1,10-phenanthroline adducts
were tested for anti-Candida activity using a broth microdilution susceptibility protocol
established by the National Committee for Clinical Laboratory Standards (NCCLS),
document M27-A2 with slight modifications. The M27-A2 method was altered by substituting
antibiotic medium 3 (Oxoid Ltd.) for RPMI 1640 medium [60]. All of the test compounds were
prepared in 2% DMSO solution as the compounds were soluble only in DMSO. This solvent
system is frequently used for antifungal testing of drugs. However, complexes 35, 36, 41 and
4
2 did not dissolve well in 2% DMSO and therefore these were tested as suspensions. The
maximum concentration of the DMSO, after serial dilution, in the test wells was 0.5%. The
inhibitory effect of DMSO on the growth of C. albicans was also examined and it was shown
that 0.5% DMSO did not affect the growth of this particular strain (ATCC 10231) of C.
albicans. However, the effect of DMSO concentration on the growth of the Candida species
can be dependent on the strain origin and hence different strains of microbes can have
different tolerances to DMSO. These results indicated that the maximum final concentration
of DMSO that can be used in the assay was ca. 1%. The ligands, complexes and a
commercially available antifungal drug, Amphothericin B were tested against C. albicans
(
ATCC 10231). The screening was carried out according to the broth microdilution
reference method [60].
2
.5. MTT assay
The activity of the ligands and their complexes was assessed using cancer cell lines:
HepG2 cell line, which is a liver-derived cell line, and A498, which is a renal cell line. Testing
was carried out using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay following
9
6 h exposure of the cells [61]. This method is based on the reduction of the tetrazolium salt
MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) into a crystalline blue
formazan product by the cellular oxidoreductases of viable cells. The resultant formazan
crystal formation is proportional to the number of viable cells. The following procedure was
5
used to test the silver complexes and mitoxantrone. Cells were seeded at 4 x 10 cells/ml in
o
9
6-well plates and incubated at 37 C in 5% CO for 96 h. Cells were treated with a range of
2
concentrations of the test compounds, in triplicate, from 0.2 to 200 µM or with a solvent

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control (0.5% DMSO) in complete medium. After 96 h incubation, the cells were assayed by
the addition of one-tenth (20 µl) of the culture volume with MTT (5 mg/ml) in 0.1 M
o
phosphate-buffered saline (PBS), pH 7.4, at 37 C in a humid atmosphere with 5% CO for 4
2
h. The medium was then gently aspirated from test cultures and 100 µl of DMSO was added
to each well. The plates were then shaken for 2 min and the absorbance was read at 550 nm
in a Varioscan plate reader. The IC50 value was defined as the concentration of test
compound required to reduce the absorbance of the MTT–formazan crystals by 50%,
indicating 50% reduction in cellular activity.
2
.6. DNA binding studies
The competitive ethidium bromide (EtBr) displacement studies involved the
preparation of a working solution containing 1 µM calf thymus DNA (CT-DNA) (260=12,824
-1
-1
M(bp) cm ) [bp = base pair] along with 1.26 µM EtBr at neutral pH in N-
tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES) buffer (10 mM TES, 0.10
mM disodium ethylenediamine tetraacetate (Na EDTA), pH = 7). Stock solutions of complex
[
2
3
1 and controls were prepared at 2 mM in 100% DMSO. Two ml of DNA-Et solution were
placed in a 10 mm quartz cuvette (3 ml) and positioned in a temperature controlled (20 °C)
spectrofluorometer. Excitation and emission wavelengths were set to 545 and 595 nm,
respectively. After thermal equilibrium was established the emission and excitation slits were
changed to give a fluorescence reading of 50 arbitrary units with measurements being
recorded over a 20 second interval. An aliquot of complex or drug solution was taken (1-20
µl), added to the cuvette and after equilibration the fluorescence reading was recorded.
Repeated aliquots were added until the fluorescence was 30-40% of the initial control or the
capacity of the 10 mm quartz cuvette (3 ml) to hold more than 1000 µl of sample was
exceeded. Triplicate titrations were performed and the apparent binding constants were
6
-1
calculated using K
= K x 1.26/C where K = 9.50 x 10 M(bp) and 1.26 is the
50
app
e
e
concentration of EtBr in µM [62].
2
.7. Determination of nuclease activity
The nuclease activity of complex 31 and controls was determined by adopting the following
method [62]. Reactions were carried out in a total volume of 20 µl in 0.10 M cacodylate

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buffer (pH = 6) with 50-0.50 µM of complex, which were initially prepared in DMF, then
diluted in buffer, with 1 µl of 0.25 µg/µl pBR322 DNA (Roche). Samples were incubated for 5
h at 37 °C (with and without an added reductant). Quench buffer (3 µl; 0.25% bromophenol
blue, 0.25% xylene cyanole and 30% glycerol) was then added and samples were loaded onto
agarose gel (1%) containing 1.50 µl/100 ml of solution of GelRedTM, 000X).
Electrophoresis was completed at 80 volts for 2 h in 1X TAE buffer (Tris- Acetate-EDTA).
2.8. Determination of SOD Mimetic Ability
The SOD mimetic activities of the metal complex 31 and controls were studied by
following the change in absorbance at  = 550 nm during the reduction of nitroblue
tetrazolium (NBT) to blue formazan in the presence of the tested complex. The assay was
run at pH 7.80 in a cuvette containing 3 ml of the reaction mixture. The reaction mixture
consisted of a double distilled water solution of 50 µM xanthine, 100 µM NBT and 50 mM
PBS. A flux of the superoxide radicals was established by the addition of 20 µl of diluted
xanthine oxidase enzyme to the cuvette. Reduction of NBT by O
was thermostatically
2
.-
controlled for three minutes at 25 °C and resulted in a 0.02/min change in absorbance at  =
.
-
.-
5
50 nm. This corresponded to a flux of 1 µM O /min. After the O flux was established, the
2 2
.
-
quantitative reduction of NBT to blue formazan by O was followed spectrophotometrically
2
at 550 nm, in the presence of the tested complex.
3
. Results and Discussion
3
.1. Synthesis and characterization of silver(I) complexes (23-33) and their phen adducts
(34-44)
Firstly, the ethyl 2-(2-oxo-2H-chromen-substituted-yl)oxy acetate derivatives (1–
1) were prepared by reaction of the appropriate substituted monohydroxy coumarin with
1
ethyl 2-bromoacetate. Then, the compounds obtained were used as precursors for the
synthesis of 2-(2-oxo-2H-chromen-substituted-yl)oxy acetic acids (12-22) (Table 1) by
acid hydrolysis in acetone: water (40:60) mixture (Scheme S1 and Section S2 in
1
13
Supplementary material). The analytical, IR, H and C NMR spectroscopic data for all
ethyl coumarinyloxyacetates (1-11) and coumarin oxyacetic acid-based ligands

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synthesized (12-22) are given in Supplementary material (Tables S1–S4 and S5-S7,
respectively).
The reaction methodology for the synthesis of 2-[(2-oxo-2H-chromen-substituted-
yl)oxy]acetic acid-based silver(I) complexes and its phen adducts is presented in Scheme 1.
The silver(I) complexes (23-33) were synthesized by deprotonation of the 2-(2-oxo-2H-
chromen-substituted-yl)oxy acetic acid-based ligands (12-22) using a stoichiometric amount
of sodium hydrogen carbonate and then adding a stoichiometric amount of silver(I) nitrate.
Further, the silver(I)-phen adducts (34-44) were obtained by stirring of the silver(I)
complexes (23-33) with 1,10-phenanthroline in absolute ethanol.
O
O
O
O
Ag
O
O
1
) NaHCO ,H O
O
OH
3
2
2
) AgNO3
O
O
1
6
,-10-phen, ethanol at
o
0 C
O
N
O
O
N
Ag
N
N
O
Scheme 1. General reaction scheme for the synthesis of 2-[(2-oxo-2H-chromen-3-
yl)oxy]acetic acid-based silver(I) complex and its phen adduct.
All of the synthesized silver(I) complexes and their phen adducts were characterized
by microanalysis and atomic absorption spectroscopy, thermogravimetric analysis,
1
13
conductivity measurements, UV-Vis, IR, H and C NMR spectroscopies, decomposition
temperatures, and in one case (33), by X-ray diffraction analysis.
Microanalytical and atomic absorption spectroscopic data (Table S8) revealed that the
silver(I) complexes 23-33 have a 1:1 metal-to-ligand ratio. The silver(I)-phen adducts 34-44
exhibited a 1:1:2 ratio between metal, ligand and phen, respectively. Complexes 24, 26, 27,
3
1 and 32 also showed the presence of water molecules and the relative amounts of water

ACCEPTED MANUSCRIPT
present was determined by thermogravimetric analysis (Fig. S1, Supplementary material).
Unfortunately the addition of the oxyacetate moiety to the coumarin nucleus did not increase
significantly the solubility of the silver(I) complexes except in the case of complex
[
Ag(8acetyl-C-7oxy-acet)] 33, which was sparingly soluble in water at ambient temperature.
More surprisingly, the formation of the phen adducts, even allowing for the presence of two
additional phen ligands in each silver(I) complex, also did not improve solubility with all of
the coordination compounds being only soluble in DMSO. The observed conductivity values
for the silver(I) complexes and the corresponding phen adducts at 25 °C were in the range 0.43
2
-1
2
-1
to 1.95 Scm mol and at 37 °C were in the range 1.32 to 2.13 Scm mol . These values at both
temperatures were too low to account for any dissociation of the metal complexes and adducts
in DMSO. Hence, these complexes and their phen adducts were regarded as non-electrolyte in
nature and are considered to be similar to other coumarin-based complexes [44, 63-66]. A
number of attempts were made to grow crystals suitable for X-ray diffraction analysis for all
the silver(I) complexes and their phen adducts prepared in this study. Unfortunately, crystals
could not be obtained except for the silver(I) complex [Ag(8acetyl-C-7oxy-acet)] (33).
3
.1.1. Crystal structure of [Ag(8acetyl-C-7oxy-acet)] (33)
Crystal structure data and structure refinement details are given in Supplementary
material (Tables S9-S11). Selected bond lengths and bond angles are presented in Table 2.
The crystal structure, together with the atom numbering scheme and the crystal packing
structure diagram are shown in Fig. 1 and Fig. 2, respectively.
There are two unique silver metal centres in 33 but both possess distorted square
pyramidal coordination geometry with each Ag(I) having five contacts within its coordination
sphere; four Ag-O bonds to four separate coumarin ligands and the fifth contact being to the
second Ag atom with the Ag1-Ag2 separation at 2.749 (15) Å) (see Fig. 1). This distance is
slightly shorter than that in metallic silver (2.88 Å) and significantly less than the van der
Waals radii for silver (3.44 Å), indicating the presence of metal-metal interaction [23]. The
Ag-Ag bond is doubly bridged by two carboxylate groups (O14, O15, O28 and O29) with
Ag(I) to syn-syn carboxylate oxygen atoms, distances ranging from 2.126(9) (Ag1-O28) to
2
.206(9) Å (Ag2-O29). The Ag-carboxylate bonds extend to form a column parallel to the a
axis via longer Ag-O contacts where O29 and O14 make a second longer contact with the
other crystallographically unique Ag atom [Ag2-O14 2.353(8); O29-Ag1 2.384(9) Å]. The
longest and fifth contact to each Ag atom is to the oxygen of the carboxylic group [Ag1-O16
2
.497(11) and Ag2-O2 2.500(10) Å].

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The Ag-carboxylate columns lie in an undulating layer sandwiched between layers of
coumarin ligands. The intra-layer aromatic rings of the coumarin moieties has a centre-to-
centre distance of 3.786(2) Å which is slightly too long for significant π-π interactions. The
layer formed by each coumarin ligand is extended via C-HO hydrogen bonds to another
coumarin ligand related by a centre of inversion. This makes a stack of coumarin-AgO-
coumarin sandwiches parallel to the c axis as shown by the crystal packing in Fig. 2. This
type of polymeric structure, where one silver ion has contacts with four different coumarin
ligands, has been previously proposed by our group for coumarin carboxylate-based silver(I)
complexes [21]. Water molecules build a double column between coumarin ligands forming
O-HO and C-HO hydrogen bonds with themselves and the coumarin ligands.
Crystallographic data for 33 have been deposited at the Cambridge Crystallographic Data
Centre, deposition number CCDC 1437900.
3
.1.2. Experimental and theoretical infrared spectra
For reliable assignment of the observed IR spectrum of the bridged binuclear Ag(I)
complex (33), molecular structure modeling of Ag (8acetyl-C-7oxy-acet) (based on the X-
2
2
ray data) and subsequent vibrational frequencies calculations were performed, Table 3, Fig.
. For the Ag(I) complexes and their phen adducts with unknown crystal and molecular
3
structure, the metal-ligand binding modes and some geometric structure features were
predicted on the basis of interpretation of their experimental and calculated IR spectra. The
experimental and calculated vibrational spectra of selected ligands (12 and 22) and their
Ag(I) complexes (23, 34, and 44) are shown in Figs. S2-S6 (in Supplementary material).
The vibrational analysis of 12, 13 and 22 ligands (representatives of the 2-(2-oxo-2H-
chromen-substituted-yl)oxy acetic acid group) showed that the first intense band in the 1770-
-1
1
670 cm region is due to the acidic carbonyl stretching vibration, (C=O)
and the
COOH
second more intense band is due to the coumarin carbonyl one ((C=O)coum). In some ligands
13, 14, 17) only one intense band was observed since the two bands mentioned above were
(
-1
overlapped. The third intense band at 1673 cm in the IR spectrum of 8acetyl-C-7oxy-acet
ligand (22) was attributed to the (C=O) vibration of the acetyl group, Fig. S2.
Upon complexation there were significant changes in the region associated with
stretching vibrations of the carbonyls most likely as a result of the disappearance of the
carboxylic acid functional group from the region whereas the coumarin carbonyl group
-1
appeared slightly affected (up to 40 cm ). The asym(OCO) and sym(OCO) vibrational

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frequencies, together with the Δ(OCO) values for the carboxylate group of the selected
silver(I) complexes are listed in Table 3. As evidenced by the crystal structure of 33, the
primary ligand bonding type is through carboxylate oxygens to two different Ag(I) centres,
which are involved in oxo-bridge formation, Fig. 1. In addition, the carboxylate oxygens
participate in syn-syn bridge formation with other Ag(I) centre (weaker bonds). The model
calculations of Ag (8acetyl-C-7oxy-acet) and Ag (C-3oxy-acet) species with oxo-bridge
2
2
2
2
simulation reproduce the main vibrational properties, Fig 3 and Fig. S5, respectively. The
-1
calculated Δ(OCO) values (170 – 200 cm ) are in good agreement with the experimental
ones. Hence, the Δ values for the silver(I) compounds (23-33), which were in the range 150
-1
-
205 cm appear typical for bidenate bridging bonding [67] as evidenced by the spectra of
many such complexes including those of 2-(2-oxo-2H-chromen-substituted-yl)oxy acetic
acid ligands in the Ag(I) complexes [68].
The microanalytical data for Ag(I)-phen adduct complexes, which suggested an
empirical formula of Ag(L)phen , indicated that the binding mode of the oxyacetate moiety
2
in these adducts would not be the same as in the phen free Ag(I) complexes. It is likely that in
this case the bonding of the carboxylate is via an unidentate carboxylate bond as it was found
for the comparable Ag(I) adduct complexes formed with 4-coumarin carboxylate and
phenanthroline [33]. The geometry calculations of possible Ag(8acetyl-C-7oxy-acet)phen2
structures confirmed a monomer silver(I) complex with N,N-bidentate binding of two phen
ligands and a monodentate bound coumarin oxyacetate ligand through the carboxylic oxygen,
Fig. S3, in Supplementary material. The vasym(OCO) and vsym(OCO) vibrational frequencies
for the silver(I)-phen adduct 44 were assigned on the basis of IR frequency calculations. The
Δ value was predicted to be larger as compared to the phen free Ag(I) complex in agreement
with the unidentate carboxylate bond (Table 3). The other phen adducts showed similar
spectral behaviour. Unfortunately, we were unable to attain a crystal structure for any of the
-1
phen adducts. The IR region 1740-1640 cm for the silver(I)-phen adducts 34-44 seems
similar to the corresponding silver(I) complexes (23-33) in line with the presence of
uncoordinated coumarin carbonyl group. The IR spectra of silver(I)-phen adducts exhibit
-1
additional bands at ca. 1420, 840 and 725 cm indicating the presence of 1,10-
phenanthroline.
-1
Normally, (Ag-O) stretching vibrations appear in the 280-250 сm frequency region
-1
whereas the (Ag-N) stretching modes are observed in the 448-362 cm IR and Raman
regions [69]. Our vibrational frequency calculations predicted that for the bidentate bridging

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-1
complexes the (Ag-O) frequency should be at ~380 cm . For Ag(I)-phen adducts (where N,
N-bidentate binding of phen ligands and monodentate binding of coumarinyloxy acetic acid
-1
to Ag(I) were proposed) the (Ag-N) frequencies should be at ~ 400 cm and the (Ag-O)
-1
frequency should appear at ~255 cm . However, these bands do not fall into the measured IR
region.
3
.1.3. Experimental and theoretical NMR spectra
The formation of silver(I) complexes and their phen adducts in solution was also
predicted and confirmed by NMR spectroscopy. The good agreement of the experimental
1
13
and calculated H and C NMR data of the Ag(I) complexes 23, 33, 34 (Tables 4 and 5,
respectively [70]) confirms the bridging bidentate carboxylate binding in Ag(I) complexes 23
and 33 (as it is seen from X-ray analysis of 33) and the monodentate coumarin carboxylate
binding in Ag-phen adduct 34. The atom numbering schemes which were used for
1
13
assignment of H and C NMR data of the compounds prepared in this study is given in
1
Table 1. In the H NMR spectra of the silver(I) complexes and their phen adducts, the signal
that corresponded to the carboxylic acid hydrogen at ~13 ppm (for the coumarin ligand) was
absent suggesting that the metal binding is via the deprotonated carboxylate group. For the
1
complexes prepared, the H NMR signals showed upfield chemical shifts for most of the
protons relative to those of the neutral coumarin ligands. The largest upfield shift (0.3 ppm)
1
1
was observed for the methylene protons H , which are the closest to the metal binding site,
1
i.e., carboxylate group. The observed slight upfield shifts (0.1 ppm) of the aromatic H NMR
signals are due to a weakening of the -electron delocalization in the ring as a result of
1
1
electron density withdrawal to Ag(I), Table 4. The degree of upfield shifts for H and the
aromatic hydrogens was greater for the phen adducts compared to the silver(I) complexes
1
a
(
23-33). The H NMR spectra of the phen adduct 34 have additional signals at ca. 9.15 (H ),
b
c
d
7
.95 (H ), 8.70 (H ), 8.15 ppm (H ), which correspond to the phen ligands and the relative
integration confirmed a 1:2 coumarin:phen ratio. All of these signals had shifted downfield
relative to the corresponding signals in the free phen (9.11, 7.79, 8.50, 8.00 ppm) [71].
1
3
Typically, C NMR spectra of 2-(2-oxo-2H-chromen-substituted-yl)oxy acetic acid-
based silver(I) complexes and their phen adducts exhibited downfield chemical shifts relative
to the corresponding ligands (Table 5). The most significant downfield shift observed was for
11
C
being α to the carboxylate group and near to the complexation site. The degree of
1
1
downfield shifts for C was greater for the phen adducts compared to the silver(I) complexes

ACCEPTED MANUSCRIPT
1
3
12
(
23-33). C NMR chemical shifts for C of the carboxylate group, being the coordination
site, was also shifted after complexation as it is observed in the case of substituted coumarin-
-carboxylatosilver(I) complexes [21]. It should be noted that in case of the Ag(I) complexes
3
1
2
12
(
23-33) the C signal is slightly shifted downfield, whereas in their phen adducts the C
signal is shifted upfield (by ~1 ppm) as compared to the free ligand. This result could be
related to the different bonding of carboxylate group - bidentate bridging and monodentate,
respectively. Vinylic carbon and aromatic carbons show a small degree of shielding effect
1
3
upon complexation. In the C NMR spectra of silver(I)-phen adduct 34 the phenanthroline
1
8
23
20
21
22
19
peaks at ca. 151.0 (C ), 142.0 (C ) , 138.0 (C ), 129.0 (C ), 127.0 (C ) and 125.0 (C )
ppm were observed indicating the presence of phenanthroline ligands. Due to the N,N-
1
8
20
19
coordination of phen to Ag(I) the C , C , C signals were downfield shifted relative to free
2
3
phen (149.9, 136.1, 123.2 ppm), whereas the C is upfield shifted (145.5 ppm for phen). The
21
22
C and C signals were not changed in the Ag(I) complexes. The similarity of the coumarin
1
13
and phen signals in the H and C NMR spectra in solution for the Ag(I) complexes (23-33)
on the one hand and for Ag-phen adducts (34-44) on the other hand, indicated that the ligands
would appear in an identical coordination behaviour: bridging bidentate carboxylate binding
and monodentate carboxylate binding of coumarins, respectively and for the phen adducts the
two phen ligands are coordinated to the silver ion through the two nitrogen donors (Tables 4
and 5).
3
.1.4. Experimental and theoretical UV-Vis spectroscopic analysis
The UV-Vis spectra of the silver(I) complexes and their phen adducts were
compared to the electronic spectra of the corresponding ligands. The electronic spectral
data, i.e., the maximum absorption wavelength (λmax in nm) and the molar absorptivity
-1
-1
(
extinction coefficients, M cm ) for all of the complexes and phen adducts along with the
corresponding ligands are given in Table 6. The UV-Vis spectra of selected Ag(I)
complexes are shown in the Fig. S7 (in Supplementary material) and are typical for the
spectra obtained for the other coordination compounds in this series. The observed UV-Vis
spectra of C-3oxy-acetH (12) and 8acetyl-C-7oxy-acet (22) ligands and their Ag(I)
complexes 23, and 33, and the phen adduct 34 were analysed on the basis of
TDDFT/B3LYP/CB1 calculations of the vertical excitation energies in solution (DMSO) for
the model compounds, Table 6. The TDDFT calculations of the model ligands 12 and 22
well reproduce the absorption bands at 310 nm and 280 nm for 12 and at 320, 290 and 240
1
nm for 22 ligand and they are assigned to A(*) excited states (the first band corresponds

ACCEPTED MANUSCRIPT
1
to the 2 A(*) excited state).
The observed absorption bands at 310, 290 and 240 nm for Ag(I) complex 23 are
due to the ligand L()-L(*) electron transitions and they are almost unchanged as
compared to the ligand (12) absorption bands. The corresponding Ag(I)-phen adduct 34,
however, shows a new absorption band at 325 nm assigned to the electron transition from a
mixed orbital, involving Ag(sd ), C-3oxy-acet and phen contributions to phen (*) orbital.
xy
The typical UV-Vis bands of 8acetyl-C-7oxy-acet appear in the spectrum of its
Ag(I) complex 33 and they are assigned to L()-L(*) transitions. The intensity of the band
at 285 nm is strongly increased in the Ag complex. A new band at 265 nm appears for the
Ag(I) complex 33, which is attributed to the transition from L() orbital to the mixed
orbital, with Ag(sd), Ocarbox and Oacet contributions. The good agreement of the experimental
and calculated excitation energies confirmed the suspected structures of the ligands and
their Ag(I) complexes in solution.
In summary, the electronic spectral data of the complexes and phen adducts exhibited
one, two or three bands in the UV region. Typically, the electronic spectra of silver(I)
complexes 23, 26-32 and phen adducts 37-44 exhibited red shifts for at least one of the
absorption bands (345 to 280 nm) while [Ag(C-3oxy-acet)phen ] (34) showed a blue shift to
2
3
05 nm. However, in some cases no change in energy was observed compared to the
corresponding 2-(2-oxo-2H-chromen-substituted-yl)oxy acetic acid ligands e.g., [Ag(C-4oxy-
acet)] (24) and [Ag(6Cl-C-4oxy-acet)] (25). The theoretical analysis of selected Ag(I)
complexes 23, 34 and 33 (along with the ligands 12 and 22) revealed also an appearance of
new absorption bands in their UV-Vis spectra. For phen adducts 34-44, an increase in the
molar absorptivity values was observed. This could be due to the presence of multiple
organic species (ligand and phen). All of the silver(I) complexes 23-33 showed a decrease in
the molar absorptivity values compared to the corresponding 2-(2-oxo-2H-chromen-
substituted-yl)oxy acetic acid ligands. The observed shifts in wavelength (λmax) and molar
absorptivity value may be attributed to the possibility of polarisation of the ligand caused by
the metal-ligand electron interaction [72].
3.2. Determination of antibacterial activities
The MIC values, (the concentration of test compound that inhibits growth by 50%
50
relative to an untreated control) were determined using methicillin resistant Staphylococcus

ACCEPTED MANUSCRIPT
aureus, MRSA strain, ATCC 4300, (Marassas, VA, USA) and P. aeruginosa strain, ATCC
2
7853, as test organisms. Initially, each compound was screened using a single independent
broth microdilution experiment in order to shortlist the most active compounds. Once the
most active compounds were identified, they were tested in triplicate along with a
commercially available antibacterial drug, Vancomycin, according to the broth microdilution
method [60].
The results of this set of screening experiments are given in Table 7 as MIC values
50
in µM. Complexes 35, 36, 41 and 42 were tested as suspensions since they were not soluble
at the final concentration of DMSO. All of the ligands 12-22 were found to be inactive even
at the highest concentration of 100 µM against both bacterial strains. The MIC values of
50
complexes 23-33 against P. aeruginosa ranged from ca. 19 to 36 µM, indicating the
bactericidal potential of these complexes against a resistant Gram-negative strain. Moreover,
they were found to be more active than silver(I) nitrate and Vancomycin (Table 7). On the
other hand, the MIC50 values of these complexes against MRSA were comparable to the
MIC value of silver(I) nitrate but were much higher than that of Vancomycin, (1.0±0.1
5
0
µM). Silver(I) nitrate has a much higher solubility in aqueous systems than the complexes
tested here. The 1,10-phenanthroline adducts 34-44 of these complexes also followed the
same trend with the complexes showing higher activity against the Gram-negative strain and
indeed incorporation of 1,10-phenanthroline molecules into the molecular framework
resulted in significantly increased activity as observed from related previous studies [39, 73-
7
5].
The silver(I) complexes and their 1,10-phenanthroline adducts differ in the
substituents present on the coumarin nucleus but it was not possible to correlate the observed
activity with any physicochemical characteristic since within experimental errors, the
obtained MIC values were found to be comparable with the exception of Ag(8acetyl-C-
5
0
7
oxy-acet) (33). This increase in the effectiveness of 33 could be attributed to its increased
solubility and related bioactivity, induced by the presence of an acetyl group. Overall, the
silver(I) complexes showed selectivity towards Gram-negative bacteria relative to Gram-
positive bacteria.
3.3. Determination of antifungal activities
In this study, the ligands, their metal complexes and 1,10-phenanthroline adducts

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were tested for their anti-Candida activity using a broth microdilution susceptibility protocol
established by the National Committee for Clinical Laboratory Standards (NCCLS),
document M27-A2 with slight modifications. The M27-A2 method was altered by substituting
antibiotic medium 3 (Oxoid Ltd.) for RPMI 1640 medium [60].
All of the test compounds were prepared in 2% DMSO solution as this solvent system
is frequently used for antifungal testing of drugs and the results given in Table 8. Once again,
complexes 35, 36, 41 and 42 were tested as suspensions since they were not soluble at the
final concentration of DMSO. The maximum concentration of the DMSO, after serial
dilution, in the test wells was 0.5%, a concentration which was found to be non-toxic to the
tested cells. The ligands, complexes and a commercially available antifungal drug,
Amphothericin B were tested against C. albicans strain ATCC 10231. The metal-free ligands
were found to be inactive as were silver(I) complexes 24-26, 29, 30, 32 and 33. Low anti-
Candida activity (79 to 97µM) was exhibited by silver(I) complexes 23, 27, 28 and 31.
On the other hand, the adducts formed by these silver(I) complexes with 1,10-
phenanthroline showed significant anti-Candida activity with most showing better activity
than the silver(I) nitrate salt. Complexes 38, 43 and 44 all showed excellent activity (Table 8)
comparable to the benchmark MIC values of the commercial drugs Amphotericin B i.e., 4.3
5
0
µM and Ketoconazole i.e 4.7 µM [75]. It is difficult to suggest a definitive structure-activity
relationship based upon the differences in physicochemical properties of these complexes
given their comparable antimicrobial activities. The improvement in anti-Candida activity
was expected from the 1,10-phenanthroline adducts of silver(I) complexes because such
improvement in activity is well documented [39, 73, 74]. Moreover, this activity could also
be attributed to the presence of three structural moieties, the coumarin-derived ligand and
two 1,10-phenanthroline ligands in one molecular frame, which become biologically active
upon coordinating to a metal centre.
3
.4. Determination of Cytotoxicity
The improved solubility of complex 33, relative to the other silver(I) complexes, did
not result in any increased activity against microbial cells. A number of previous studies on
related complexes had shown that silver(I) complexes of coumarins were active against
immortalised cell lines [76] and it was thought that the increased solubility of compound 33
may give rise to an increase in cytotoxicity against mammalian-derived cells. Thus that series

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of derivatives were screened for their cytotoxicity against two cell lines, HepG2 cell line,
which is a liver-derived cell line, and A498, which is a renal cell line. As can be seen from
Table 9 the cytotoxicities of the complexes were similar to that of silver nitrate (and indeed
comparable to that of the control mitoxantrone) but the most soluble complex again did not
show any significantly different activity in comparison to the other complexes. However the
complexes isolated did have significant cytotoxicity as we have recently identified two series
of related complexes, one series based on hydroxynitro-coumarin complexes of silver(I) [76]
and another series based on silver(I) dicarboxylates [37], both of which had shown
cytotoxicity but whose mechanism of action seemed to be related to their ability to act as
antioxidants, and we were interested to see if that was the case here.
3
.5. DNA binding properties
Complex 31 was chosen for these studies as it had reasonable cytotoxicity against
both mammalian cell lines and was active against the bacterial and fungal cell lines tested.
Many anti-cancer drugs are thought to exert their effect though DNA binding ability and
though there are reports of antimicrobial compounds acting by interfering with DNA
synthesis in a bacterial cell [77]. A recent report has shown that an azolylthioetherquinolone
complex, which had excellent activity against the two bacterial strains used in these studies,
exerted its effect via intercalation into MRSA DNA through a copper ion bridge [78]. Another
paper recently published on bisimidazolium-tagged acridines showed that these compounds
had both anti-bacterial and DNA-binding ability [79].
To confirm whether the silver complex 31, [Ag(3Cl-4Me-C-7oxy-acet)]0.8H O had
2
DNA binding ability, competitive EtBr displacement experiments were conducted with the
complex along with its corresponding ligand, 21, and a known intercalator pentamidine using
CT-DNA over the concentration range of 1 to 1000 μM. Ethidium bound DNA is highly
+
fluorogenic and in the presence of excess ethidium ion (Et ), binding regions within the DNA
polymer become saturated. Thus, during competitive displacement, an exogenous reagent
+
must ‘compete’ at the binding site with Et resulting in a sequential reduction in fluorescence.
As can be seen from Fig. 4, neither the complex, ligand or silver nitrate appears to
bind to DNA via intercalation as no displacement of EtBr was seen. Pentamidine was highly
+
efficient in the displacement of Et bound DNA and, as expected, its apparent binding
5
constant (Kapp = 1.50 x 10 ). The coumarin-derived silver complex which exhibited
cytotoxicity and antimicrobial activity, did not appear to mediate its effect through DNA

ACCEPTED MANUSCRIPT
interaction/binding (which can happen through a number of modes) and therefore are likely
to target other sites in the cell other than DNA.
A number of recent papers highlighted the link between antimicrobial activity and
DNA binding and/or nuclease activity suggesting potential mechanisms for antimicrobial
activity that involve binding and/or irreversible cleavage of bacterial nucleic acids [80]. The
nuclease activities of the complex and its corresponding ligand were analysed by monitoring
the unwinding of supercoiled pBR322 plasmid DNA (Form A) to open DNA (Form C) or
linear circular (Form B) over a concentration range of 50-0.50 μM for 5 h without an added
reductant (Figs. 5 and 6).The extent of strand scission was assessed by agarose gel
electrophoresis and compared to copper bisphenanthroline (positive control).
It can be seen from these figures that neither the metal-free ligand or the silver(I)
complex exhibited self-cleaving of supercoiled (SC) DNA (Form A). Copper
bisphenanthroline partially cleaved SC DNA (Form A) to open circular OC (Form C) at 50
μM (lane 7). Moreover, neither complex nor controls cleaved DNA even in the presence of
added reductant (mercaptopropanoic acid) at twice the concentration of the test compounds
(Fig. 6). However, in this case the highest concentrations (50 μM) of copper
bisphenanthroline showed a partial nuclease activity.
3
.6. SOD mimetic activity
Related silver(I) complexes which have shown good antimicrobial activity and
cytotoxicity and which did not show DNA intercalation or nuclease activity did show
potential to reduce oxidative stress in the yeast S. cerevisiae or in cell free models [37, 76].
The SOD mimetic ability of complex 31 was tested for its ability to inhibit the reduction of
NBT to blue formazan by the scavenging of the superoxide radicals, which were generated by
xanthine oxidase during the conversion of xanthine to urate. EC values were considered to
5
0
be the concentration (μM) equivalent to 1 unit of a bovine erythrocyte SOD activity and
therefore the lower the magnitude of EC , the higher will be the SOD mimic activity of the
5
0
complex. The free ligand 21 did not exhibit SOD activity but complex 31 did show SOD
activity (EC = 1.20 ± 0.06 µM), albeit lower than that of native SOD (EC = 0.04 μM), and
5
0
50
indeed also higher than silver(I) nitrate 0.15 ± 0.07 µM. The role of silver(I) complexes in the
redox processes in the cell has been identified by a number of workers in terms or disruption
of mitochondrial function but in more recent times there have been reports where the SOD

ACCEPTED MANUSCRIPT
activity in cells is increased in the presence of colloidal silver and that group posed the
hypothesis that up-regulation of the SOD enzymes was occurring as a result of the presence
of the silver [81]. In another study the effect of silver nanoparticle reduced the antioxidative
biomarkers in rats which were under oxidative stress [82]. The role of silver(I) complexes is
thus under current debate but the activity of the complexes isolated here, which have
antimicrobial, antifungal and cytotoxic effects may have a mechanism of action related to
their ability to act as antioxidants rather than by intercalation or nuclease activity which has
been shown by others in the field as outlined earlier.
4
. Conclusions
A range of novel silver(I) complexes of coumarin-derived oxyacetate ligands and their
,10-phenanthroline adducts were synthesised and characterised by both experimental and
1
computational methods. As only one complex could be isolated in crystalline form the
comparison between the experimental and predicted IR, NMR and UV-Vis data was
necessary to predict and confirm the structure of the other complexes. Polymeric structure
was predicted for the Ag(I) complexes (23-33), where the silver ion has contacts with four
different coumarin ligands and Ag-Ag bond is doubly bridged by two carboxylate groups.
Whereas for their phen adducts (34-44) a monodentate carboxylate binding of the coumarin
ligand, and N,N-bidentate binding of the two phen ligands to the silver ion were proposed.
The complexes showed promising results against the tested bacterial strains and may
have potential as therapeutic agents against infectious diseases. The strain specific inhibitory
response of these complexes, observed during this study, could be attributed to the fact that
their mode of action in Gram-negative bacteria is likely to be different to their mode of action
in Gram-positive bacteria. The anti-Candida results were far more striking with the 1,10-
phenanthroline adducts showing better antifungal activity than the silver(I) salt and were
capable of inhibiting the growth of C. albicans by 50% at a concentration range of ca. 7 to 10
µM. This was comparable to the efficacy of Ketoconazole, a commonly prescribed anti-candida
therapeutic.
The general lack of solubility of the Ag(I) complexes studied may limit their
application to topical infections or as additives in surface protection technologies. Indeed there
are a number of commercially available products such as Foley catheters etc. which are
impregnated with silver(I) additives. However, one of the derivatives isolated had improved
solubility compared to all previous coumarin-based silver(I) complexes but did not show any

ACCEPTED MANUSCRIPT
enhanced antimicrobial activity. Cytotoxicity testing of that series of acetate derivatives against
human-derived cell lines also did not reveal any structure activity relationship (SAR) but did
indicate that the complexes had good activity against the clinically relevant HepG2 and A498
cell lines. Preliminary mechanistic studies using complex 31, [Ag(3Cl-4Me-C-7oxy-
acet)]0.8H O, a complex which had good antimicrobial, antifungal and cytotoxic activity,
2
indicated that the complex did not interact with DNA via intercalation, did not have nuclease
activity but did have SOD mimetic activity. This is consistent with other recent findings on
related complexes which indicated that silver(I) complexes may potentiate their activity
though an antioxidative process which disrupts metabolism of the different cell types studied.
Abbreviations
MDR
AIDS
MRSA
HIV
MultiDrug Resistant
Acquired ImmuneDeficiency Syndrome
methicillin-resistant S. aureus
human immunodeficiency virus
Trypticase Soy agar
TSA
ROS
Reactive oxygen species
DNA
deoxyribonucleic acid
phen
1,10-phenanthroline
SOD
superoxide dismutase
DFT
density functional theory
TDDFT
DMSO
TLC
time dependent density functional theory
dimethyl sulfoxide
Thin layer chromatographic
TGA
thermogravimetric analysis
DEPT
COSY
HSQC
HMBC
distortionless enhancement by polarization transfer
homonuclear correlation spectra
Heteronuclear Single Quantum Correlation
Heteruclear Multiple Bond Correlation

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NOESY
ECP
Nuclear Overhauser effect spectroscopy
effective core potential
TMS
tetramethylsilane
NCCLS
LB
National Committee for Clinical Laboratory Standards
Luria Bertani
NBT
nitrobluetetrazolium
PBS
phosphate-buffered saline
MTT
methylthiazolyldiphenyl-tetrazolium bromide
National Committee for Clinical Laboratory Standards
ethidium bromide
NCCLS
EtBr
PCM
Polarisable Continuum Model
ethidium ion
+
Et
EDTA
CT-DNA
TES
ethylenediaminetetraacetic acid
calf thymus DNA
N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid
supercoiled
SC
DMF
OC
Dimethylformamide
open circular
Acknowledgments
This research was supported by the Technological Sector Research Programme, Strand III
CRS-02 TA01-4 under the European Social Fund and by the Centre of Applied Science for
Health, ITT Dublin funded under PRTLI-5 funding. All DFT calculations were performed on
the MADARA computer cluster of the Bulgarian Academy of Sciences. This work has been
carried out (in part) within the structures of the Facility for Optical Characterisation and
Spectroscopy (now the FOCAS Institute, DIT) funded under The Irish National Development
Plan 2000-2006 with assistance from the European Regional Development Fund.

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