Photodynamic activity and photoantimicrobial chemotherapy studies of ferrocene-substituted 2-thiobarbituric acid

Article abstract of DOI:10.1016/j.bmcl.2021.127922

A ferrocene-substituted thiobarbituric acid (FT) has been synthesized to explore its photophysical properties and photodynamic and photoantimicrobial chemotherapy activities. FT has an intense metal-to-ligand charge transfer (MLCT) band at ca. 575 nm. The

Full text of DOI:10.1016/j.bmcl.2021.127922

Bioorg. Med. Chem. Lett. 40 (2021) 127922
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Photodynamic activity and photoantimicrobial chemotherapy studies of
ferrocene-substituted 2-thiobarbituric acid
,b,
Balaji Babua *, Thivagar Ochappana, Thaslima Asraf Alia, John Mackb, Tebello Nyokongb,
,
*
Mathur Gopalakrishnan Sethuramana
a Department of Chemistry, The Gandhigram Rural Institute – Deemed to be University, Tamil Nadu 624 302, India
b Institute for Nanotechnology Innovation, Department of Chemistry, Rhodes University, Makhanda 6140, South Africa
A R T I C L E I N F O
A B S T R A C T
Keywords:
A ferrocene-substituted thiobarbituric acid (FT) has been synthesized to explore its photophysical properties and
photodynamic and photoantimicrobial chemotherapy activities. FT has an intense metal-to-ligand charge
transfer (MLCT) band at ca. 575 nm. The ferrocene moiety of FT undergoes photooxidation to form a ferrocenium
species which in turn produces hydroxyl radical in an aqueous environment, which was confirmed via the
bleaching reaction of p-nitrosodimethylaniline (RNO). FT exhibits efficient PDT activity against MCF-7 cancer
Ferrocene
Metal to ligand charge transfer
Hydroxyl radical
Photodynamic Therapy
Photo-antimicrobial studies
cells with an IC50 value of 5.6 μM upon irradiation with 595 nm for 30 min with a Thorlabs M595L3 LED (240
mW cmꢀ 2). Photodynamic inactivation of Staphylococcus aureus and Escherichia coli by FT shows significant
activity with log reduction values of 6.62 and 6.16 respectively, under illumination for 60 min at 595 nm. These
results demonstrate that ferrocene-substituted thiobarbituric acids merit further study for developing novel
bioorganometallic PDT agents.
Metallocenes have received considerable research interest in
different fields owing to their interesting physical and chemical prop-
erties.1–3 Ferrocene (Fc), an organometallic compound in which two
mechanistic studies by electron paramagnetic resonance (EPR) spec-
troscopy have shown that ferrocenium salts degrade in an aqueous
environment to undergo a Haber-Weiss-like cycle followed by a Fenton-
type reaction generating highly reactive hydroxyl radicals (•OH), which
exhibit cytotoxic effects on MCF-7 cells.14,15
π
-bonded cyclopentadienyl (
η
5-C5H5) ligands form a sandwich complex
with a Fe(II) ion, is a suitable candidate for a wide range of applications
in catalysis, electroactive materials, sensors, and in medicinal chem-
istry,4–8 due to its facile functionalization and favorable electronic
properties. Incorporation of Fc in various biologically active compounds
has been found to enhance their activity. Chloroquine (CQ) an antima-
larial drug is ineffective against certain strains of Plasmodium falciparum
because of resistance developed by the malarial parasite. Interestingly,
ferroquine (FQ), which is a ferrocenyl analogue of chloroquine, was
found to be active against both CQ-sensitive and CQ-resistant strains of
Plasmodium falciparum.9,10 Ferrocifen is a ferrocene-appended analogue
of tamoxifen, which is highly effective in both hormone-dependent (ER
+) and hormone-independent (ER -) breast cancer, while in contrast,
tamoxifen is active against only hormone-dependent (ER +) breast
cancer.11,12 This enhanced activity was attributed to the redox-active
center of the ferrocene moiety. The ferrocenium (Fc+) ion, which is
the one-electron oxidized species of ferrocene, is known to exhibit an
anti-proliferative effect on certain types of cancer cells.13–15 Detailed
The goal of this study is to develop a ferrocene-substituted thio-
barbituric acid (FT) (Fig. 1a), which is expected to generate the ferro-
cenium moiety upon photoirradiation but remain inactive in the dark.
The rationale for using the thiobarbituric acid is its ability to form
–
–
–
hydrogen bonds through its carbonyl (C O), thiocarbonyl (S O), and
–
NH groups with enzymes and proteins such as Sirtuins (Sirt2) and
nicotinic acetylcholine receptor (nAChR), which can facilitate inter-
nalization of FT into cells.16,17 Conjugating the ferrocene moiety to
thiobarbituric acid forms a donor-
π
-acceptor (D-
π-A) system. The optical
spectra of ferrocene-based D-
π
-A systems usually contain an intense
visible region intramolecular metal-to-ligand charge transfer (MLCT)
band.18–22 The photoinduced charge transfer process leads to the for-
mation of a ferrocenium ion and an anion radical species.23–26 The fer-
rocenium ion is expected to produce hydroxyl radical species, which is
one of the active species in photodynamic therapy (PDT).27,28 During
PDT, a photosensitizer dye enters the S1 state upon photoexcitation,
* Corresponding authors at: Department of Chemistry, The Gandhigram Rural Institute – Deemed to be University, Tamil Nadu 624 302, India.
E-mail addresses: balajibabu1900@gmail.com (B. Babu), mgsethu@gmail.com (M. Gopalakrishnan Sethuraman).
https://doi.org/10.1016/j.bmcl.2021.127922
Received 30 November 2020; Received in revised form 23 February 2021; Accepted 26 February 2021
Available online 8 March 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

B. Babu et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127922
Fig. 1. The structure of the ferrocene conjugate that was studied in this work and that of the PT control compound; (b) UV–visible absorption spectra of FT and PT in
1:1 DMF/H2O.
which undergoes intersystem crossing to the triplet manifold. The T1
state can either undergo a Type-I process to form reactive oxygen species
(ROS), such as •OH, –•O2, or a Type-II process to transfer its energy to
molecular oxygen to form 1O2 species which are known to be toxic to
surrounding tumor cells.27,28 Photodynamic antimicrobial chemo-
therapy (PACT) follows the same principles of PDT. ROS formed by the
photosensitizer dye cause oxidative damage to cell membrane integrity
causing irreparable biological damage.29,30 The key advantage of PACT
over conventional chemotherapeutic drugs is that it is difficult for mi-
croorganisms to generate resistance against the ROS that are produced.
The synthetic and non-linear optical (NLO) properties of FT have been
reported in the literature,31–33 and also it finds applications in wide
fields,34,35 but its potential application for PDT and PACT studies has not
been explored. The objective of the present work is to synthesize and
characterize FT (Fig. 1a) and to study its PDT activity towards MCF-7
cells and PACT activity against both Gram-positive S. aureus and
Gram-negative E. coli bacteria. PT was used as a control compound to
explore the effect of the ferrocenyl moiety on the PDT and PACT activity,
(Fig. 1a).
FT has a band at 575 nm, which is absent in its precursor. This band
arises from a metal-to-ligand charge-transfer (MLCT) transition from the
ferrocene moiety to thiobarbituric acid. A similar type of charge transfer
spectrum is observed in the literature for ferrocene-based D-π-A conju-
gates.22,26 The MLCT band has a tail of absorbance to ca. 700 nm. This
potentially enables red-light activation in the context of PDT applica-
tions. PT, which lacks a ferrocene moiety, has an absorption band at 378
nm and no absorption bands in the visible region. This demonstrates that
the incorporation of the ferrocene moiety redshifts the MLCT absorption
band. The absorption spectrum of FT measured in 1% DMF/PBS buffers
does not exhibit any change in band morphology but the MLCT band is
red-shifted to 585 nm (Fig. S5).
The structural optimization of FT was performed by density func-
tional theory (DFT) calculations at the B3LYP/6-31G(d) level of theory.
The energy-minimized structure is provided in the Supporting Infor-
mation (Fig. S6). DFT and time-dependent DFT (TD-DFT) studies were
performed to rationalize the electronic structures and photophysical
properties of FT and related compounds. When a comparison is made
between Fc, FT, and Fc-CHO (FA), there is a significant stabilization of
the LUMO and slight stabilization of HOMO (Fig. S7). A calculated
electronic excitation of FT at 592 nm corresponds to the experimentally
observed band at 575 nm. This electronic excitation is associated with
intramolecular charge transfer from the highest occupied molecular
orbital (HOMO) of FT to the lowest unoccupied molecular orbital
(LUMO). The HOMO and LUMO are localized predominantly on the
ferrocenyl and thiobarbituric acid moieties, respectively (Fig. S7).22,26
Thus, visible-light excitation is predicted to transfer an electron from the
ferrocenyl moiety to the thiobarbituric acid which leads to the formation
of a ferrocenium (Fc+) ion with a ferric iron center which can generate
hydroxyl radicals via Fenton-type reactions.14,15,23–26
FT and phenyl-substituted thiobarbituric acid (PT) were synthesized
by condensation of 2-thiobarbituric acid with the corresponding alde-
hyde in ethanol in favorable yield.31,32 The 1H NMR spectrum of FT
contains a peak at 8.25 ppm, which corresponds to the alkene proton.
The signal for the unsubstituted cyclopentadienyl ring (
η
5-C5H5) protons
lies at 4.33 ppm. In contrast, signals of the substituted cyclopentadienyl
ring
η
5-C5H4 protons were observed at 5.04 and 5.39 ppm. The two NH
protons lie at 12.08 and 12.19 ppm (Fig. S1). MALDI-TOF MS analysis of
FT gave a molecular ion peak at 340.02 corresponding to a [M+] species
(Fig. S3).
The electronic absorption spectrum of the ferrocenyl conjugate FT
along with its phenyl analogue (PT) in 1:1 DMF/H2O is shown in Fig. 1b.
For photoirradiation studies, a solution of FT in 1:1 DMF/H2O was
Fig. 2. (a) Changes in the UV–Visible spectra of FT on irradiating with 595 nm light for 0–30 min in 1:1 DMF/H2O; (b) UV–Visible spectra of FT after irradiating for
30 min followed by addition of an excess of 1,10-phenanthroline. (Inset: Pictures of FT in dark, light irradiation for 30 min, light irradiation for 30 min followed by
addition of 1,10-phenanthroline).
2

B. Babu et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127922
Fig. 3. (a) Change in the UV–visible absorption spectra of RNO with irradiation time (0–30 min) in the presence of FT in 1:1 DMF/Water; (b) Plot of the absorption
change of RNO at 440 nm in the presence of FT in the dark (black triangle), light irradiation (red square), light irradiation + t-BuOH (green circle) in 1:1 DMF/H2O.
Fig. 4. (a) Cellular uptake of FT (5 μM) by MCF-7
cells with different time intervals (6, 12, 24, 48 h)
measured by the absorption spectroscopy of lysed
cells. (b) Cytotoxicity of FT against MCF-7 cells after
12 h incubation in the dark followed by photo-
irradiation for 30 min with a Thorlabs M595L3 LED
(240 mW cmꢀ 2). (c) ROS detection in MCF-7 cancer
cells produced by FT (5 μM) kept in the dark and
upon illumination (H2O2 used as a +ve control). (d)
Antimicrobial activities of FT towards S. aureus and
E. coli on photoirradiation for 60 min at 595 nm with
a Thorlabs M595L3 LED (240 mW cmꢀ 2).
irradiated with a 595 nm LED (the same light source used for PDT/PACT
studies) for 30 min to observe the effect of light irradiation, and its
absorption spectra were recorded at regular time intervals (Fig. 2a).
With increased irradiation time, a gradual decrease in the MLCT band
(575 nm) and the band at 370 nm was observed with the appearance of
new bands at 414 and 278 nm. This could be due to the formation of a
ferrocenium (Fc+) ion due to intramolecular charge transfer.25,26 The
color of the solution changes from blue to dark brown after 30 min of
irradiation (inset Fig. 2b). The ferrocenium ions have been shown to
undergo a consecutive chain of reaction in aqueous environments to
cause any change in the absorption spectra over time (Fig. S9). But when
the solution of FT along with RNO is irradiated with 595 nm light, the
absorbance at 440 nm corresponding to RNO decreased gradually with
an increase in irradiation time (Fig. 3a). As expected, the band at 575 nm
for FT also decreased in absorbance with increasing irradiation time.
When the irradiation is performed in the presence of t-butanol as a •OH
quencher39, the rate of decrease of RNO absorbance at 440 nm is
reduced dramatically but still, the band at 575 nm for FT decreased,
which provides evidence for the generation of •OH by FT upon irradi-
ation (Figs. 3b, S9). In contrast, the PT control compound did not cause
any spectral changes with RNO either in the dark or under illumination
(Fig. S10).
•
form OH, Fe2+, and the cyclopentadienyl radical (Cp•).14,15 When an
excess of 1,10-phenanthroline (phen) is added to this irradiated solu-
tion, a red–orange color was immediately observed. The absorption
spectrum of this solution is similar to that of [Fe(Phen)3]2+(Fig. 2b, Fig
S8).36 However, solutions of FT kept in the dark, and PT upon irradia-
tion did not exhibit any apparent change in the visible region spectra.
The intracellular uptake of FT in MCF-7 cells incubated with a 5 μM
solution was measured by absorption spectroscopy of lysed cell extracts
after different time intervals. Fig. 4a shows that FT is internalized
effectively within 6 h, and the highest uptake is observed at 12 h. The
dark toxicity of FT towards MCF-7 cells was tested by MTT assay after
12 h of incubation.40 Since FT is insoluble in water, the addition of FT to
the cell culture required preliminary solubilization in DMSO to form
stock solutions that were then diluted to different concentrations with
DMEM media. The final concentration of DMSO was always < 0.5%.
•
The formation of OH radicals by FT on photo-irradiation is inves-
tigated by using p-nitrosodimethylaniline (RNO) as a hydroxyl radical
quencher.37,38 RNO is highly specific to hydroxyl radicals. The trapping
•
of OH is easily monitored by monitoring its absorbance value at 440
nm. Solutions of the mixture of FT and RNO kept in the dark did not
3

B. Babu et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127922
provided direct evidence for the generation of intracellular ROS upon
light irradiation. FT was found to have significant PACT activity against
S. aureus and E. coli with high log reduction values. The results
demonstrate that FT has the potential to be developed further as a
photosensitizer dye for PDT and PACT. Further studies are in progress to
Table 1
Photophysical data of FT in 1:1 DMF/H2O and its cytotoxicity data in MCF-7
cells and PACT data on S. aureus and E. coli.
FT
λmax/nm (
IC50 M)
ε
× 103 Mꢀ 1cmꢀ 1
)
370 (15.9), 575 (3.7)
> 100
(
μ
Dark a
Light b
S. aureus
E. coli
structurally modify ferrocene-based D-π-A conjugates to improve their
5.6 (±0.9)
6.62 (20)
aqueous solubility so they can be further evaluated in PDT studies.
Log redc (MBC /
μ
M)d
6.16 (30)
a
12 h incubation in MCF-7 cells in the dark, b12 h incubation in MCF-7 cells in
Declaration of Competing Interest
the dark followed by exposure to a Thorlabs M595L3 LED (240 mW cmꢀ 2) for 30
min, cLog reduction values for the photoinactivation effects of FT (15 and 25
μM
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
for S. aureus and E. coli, respectively) after 60 min irradiation at 595 nm with a
Thorlabs M595L3 LED (240 mW cmꢀ 2), d Minimum bactericidal concentration
(MBC).
Acknowledgements
Upon photoirradiation with a 595 nm LED, MCF-7 cells incubated with
FT exhibited significant cytotoxicity effects with an IC50 value (con-
Theoretical calculations were carried out at the Centre for High
Performance Computing in Cape Town. Thanks are due to the author-
ities of GRI-DTBU for encouragement and UGC-SAP for financial
support.
centration required for 50% inhibition of cell proliferation) of 5.6 μM
(Fig. 4b, Table 1). FT treated cells that were kept in the dark exhibited no
significant cytotoxicity (IC50 > 100 M). Fig. 4b contains the dos-
μ
e–response curves for FT in the dark and under illumination. Control
experiments with PT exhibited no cytotoxicity in the dark or when
irradiated (data not shown). The 2′,7′-dichlorofluorescein diacetate
(DCFDA) assay was performed to provide direct evidence for intracel-
lular ROS formation.41 MCF-7 cells treated with FT exhibited a signifi-
cant increase in 2ʹ,7ʹ-dichlorofluorescein fluorescence intensity when
irradiated with light when a comparison is made to the response of the
dark treated cells (Fig. 4c). When irradiation was carried out in the
presence of N-acetyl cysteine (NAC), a ROS scavenger, the fluorescence
intensity decreased, which indicates the involvement of reactive oxygen
species.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.127922.
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