Fluorescent Imidazo[1,2-a]pyrimidine Compounds as Biocompatible Organic Photosensitizers that Generate Singlet Oxygen: A Potential Tool for Phototheranostics

Article abstract of DOI:10.1002/chem.202004957

Photodynamic therapy has been used to treat a variety of diseases, however, there is continuing search for new biocompatible photosensitizers. Herein, we demonstrate for the first time that imidazo[1,2-a]pyrimidine compounds are able to generate singlet oxygen species and can act as photosensitizers in the intracellular environment. Our results show that this class of compounds absorb and emit in the 400–500 nm region, present low cytotoxicity in the dark, are efficiently uptaken by cells, are fluorescent in intracellular medium, and generate singlet oxygen upon irradiation, killing cancer cells within 2 h at low concentration (2.0 μm). The imidazo[1,2-a]pyrimidine compounds are a potential new tool for phototheranostics, because they can be simultaneously used for fluorescence imaging and photodynamic therapy.

Full text of DOI:10.1002/chem.202004957

Accepted Article
Accepted Article
Title: Fluorescent Imidazo[1,2-a]pyrimidine Compounds as
Biocompatible Organic Photosensitizers that Generate Singlet
Oxygen: A Potential Tool for Phototheranostics
Authors: Maria L. S. O. Lima, Carolyne B. Braga, Tiago B. Becher,
Mikel Odriozola-Gimeno, Miquel Torrent-Sucarrat, Iván Rivilla,
Fernando P. Cossio, Anita J. Marsaioli, and Catia Ornelas
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202004957
Link to VoR: https://doi.org/10.1002/chem.202004957
01/2020

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a
Fluorescent Imidazo[1,2- ]pyrimidine Compounds as
Biocompatible Organic Photosensitizers that Generate Singlet
Oxygen: A Potential Tool for Phototheranostics
Maria L. S. O. Lima,[a]† Carolyne B. Braga,[a] Tiago B. Becher,[a] Mikel Odriozola-Gimeno,[b] Miquel
Torrent-Sucarrat,[b,c] Iván Rivilla,[b] Fernando P. Cossío,[b] Anita J. Marsaioli,[a]* Catia Ornelas[a]*
Abstract: Photodynamic therapy has been used to treat a variety of
diseases, however there is a continuous search for new biocompatible
photosensitizers. Herein, we demonstrate for the first time that
imidazo[1,2- a]pyrimidine compounds are able to generate singlet
oxygen species, and can act as photosensitizers in the intracellular
environment. Our results show that this class of compounds absorbs
and emits in the 400-500 nm region, presents low cytotoxicity in the
dark, is efficiently uptaken by cells, is fluorescent in the intracellular
medium, and generates singlet oxygen upon irradiation killing cancer
cells within 2 h at low concentration (2.0 µM). The imidazo[1,2-
absorbed photons into both fluorescence imaging and
photodynamic activity, can be considered potential tools for
phototheranostics.
Photodynamic therapy (PDT) has been used for the
treatment of a variety of diseases that includes some types of
cancer, bacterial and fungal infections, and skin diseases.[3] PDT,
by definition, refers to the use of visible light to damage or kill a
living tissue in the presence of a photosensitizer and molecular
oxygen.[3b] The three components needed for PDT,
a
photosensitizer, visible light and molecular oxygen, are harmless
individually, but they combine to destroy tumors or other diseased
tissues or to kill bacteria.[4] For PDT to occur, it is crucial to have
the three components combined together near the target tissue.[5]
There are two types of PDT mechanisms: (a) type I involves
electron transfer from the excited photosensitizer to intracellular
molecular oxygen that leads to the formation of several radical
a]pyrimidine compounds are
a
potential new tool for
phototheranostics because they can be simultaneously used for
fluorescence imaging and photodynamic therapy.
Introduction
.-
.
species including superoxide anion (O2 ) and hydroperoxyl (HO2 )
radicals; (b) type II involves energy transfer from the excited
photosensitizer to the ground state of oxygen 3O2 leading to highly
reactive singlet oxygen 1O2.[6] It is difficult to distinguish between
both mechanisms experimentally due to the complexity of the
intracellular environment. However, it is accepted that the
predominant PDT mechanism is type II, and that the singlet
oxygen species destroy the living tissues by reacting directly on
unsaturated lipids, certain amino acids, and on nitrogenous bases
of nucleic acids.[7] Singlet oxygen is highly reactive with a lifetime
of around 40 ns, and a maximum action radius of about 20 nm.[8]
These facts altogether make PDT a very localized, specific and
controllable technique.
Phototheranostics is the combination of diagnosis and
therapeutics in one molecule or macromolecule, triggered by light,
which provides high sensitivity and precision with low toxicity to
healthy tissues.[1] Photoactive compounds can convert the
absorbed photons into signals useful for imaging or therapeutic
activity such as: (i) fluorescence imaging, when the absorbed
photons are reemitted as photons with lower energy; (ii)
photothermal therapy (PTT), when the absorbed photons are
converted into localized heat; (iii) photodynamic activity (PDT),
when the absorbed photons generate reactive oxygen species
(ROS) that cause cellular death.[1-2] Compounds able to convert
PDT has been investigated since the late nineteenth
century; however, only almost one century later, in 1993, the first
PDT agent (Photofrin) was approved for clinical use (for the
treatment of bladder cancer in Canada).[8b, 9] Since then, several
families of compounds have been investigated as potential
photosensitizers[10] that include: porphyrins,[11] phtalocyanines,
tricarbocyanines,[12] BODIPY,[13] perylenes,[14] anthraquinones,
chlorins, terpyridines, RuII complexes[15] and zirconium
complexes.[16]
[a]
[b]
Dr. M. L. S. O. Lima, Dr. C. B. Braga, Dr. T. B. Becher, Prof. A. J.
Marsaioli, Prof. C. Ornelas
Institute of Chemistry
University of Campinas - Unicamp
Campinas, 13083-861 Sao Paulo, Brazil
E-mail: catiaornelas@catiaornelaslab.com
Dr. M. Odriozola-Gimeno, Dr. M. Torrent-Sucarrat, Dr. I. Rivilla, Prof.
F. P. Cossio
Department of Organic Chemistry I, Centro de Innovación en
Quimica Avanzada (ORFEO-CINQA)
An ideal photosensitizer for PDT should present: chemical
stability, high purity, simple synthesis via high yielding reactions,
effective singlet oxygen 1O2 generation, low cytotoxicity in the
dark, amphiphilic structure, solubility in injectable formulations
and preferential accumulation in diseased tissues.[3c, 17] Another
important aspect to have in consideration when designing a new
photosensitizer is the absorption and emission properties, in
particular, the absorption wavelength. For most diseases located
in internal organs and regions, the photosensitizer must absorb
Universidad del País Vasco/Euskal Herriko Unibertsitatea
(UPV/EHU) and Donostia International Physics Center (DIPC)
P° Manuel Lardizabal 3, E-20018 Donostia/San Sebastián, Spain
Dr. M. Torrent-Sucarrat
[c]
†
Ikerbasque, Basque Foundation for Science, Ma Diaz de Haro 3,
Bilbao 48013, Spain
Present address: M. L. S. O. Lima: Instituto Federal da Bahia, IFBA
- Campus Juazeiro, 48918-900 - Porto Juazeiro, BA, Brasil.
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.202004957
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O
and emit within the near-infrared (NIR) region (700-900 nm).
Whereas, photosensitizers that absorb and emit in the 400-500
nm region have applications in a variety of diseases such as
bladder cancer, cervical intraepithelial neoplasia, oral precancer
lesions, cutaneous T-cell lymphoma and psoriasis. However,
most compounds proposed as photosensitizers for PDT do not
possess all of these characteristics.[3c] Several strategies have
been adopted to improve the performance of photosensitizers that
include chemical modification with solubilizing groups, attachment
of specific targeting groups and vectorization by nanomaterials
through covalent attachment or encapsulation.[18] Nevertheless,
the search for new photosensitizer compounds continues.
Imidazopyridines[19] and imidazopyrimidines[20] are a special
class of fused N-heterocycles containing two or three nitrogen
atoms, respectively, which have a wide variety of pharmaceutical
applications as anti-inflammatory,[21] antitumor,[22] antiviral,[23]
antibacterial,[24] analgesic[25] and anxiolytic.[26]
OH
Br
N
N
NH2
EtOH
reflux, 18h
N
NH2
EtOH
reflux, 18h
N
N
OH
OH
N
N
N
2
1
(H3C)3Si
OSO2CF3
(H3C)3Si
OSO2CF3
18-crown-6, CsF, 1h
18-crown-6, CsF, 1h
MW (120οC, 90W, 40psi)
MW (120οC, 90W, 40psi)
N
N
OH
OH
Recently, the optical properties of some imidazopyridines
and imidazopyrimidines have been studied,[27] and proposed as
biomarkers and biochemical sensors, however, the photophysical
properties of this family of compounds were not fully exploited yet.
Herein, we demonstrate for the first time that imidazo[1,2-
a]pyrimidine compounds are able to generate singlet oxygen 1O2,
are efficiently uptaken by cancer cells, are fluorescent in the
intracellular medium and present photodynamic activity in vitro.
Since these compounds are fluorescent and generate ROS upon
irradiation, they might serve as potential tools for
phototheranostics.
N
N
N
4
0.1 eq. 18-crown-6
K2CO3
trimethylene
bromohydrin
acetone, reflux, 18h
0.1 eq. 18-crown-6
K2CO3
trimethylene
bromohydrin
acetone, reflux, 18h
3
N
N
O
O
N
N
N
OH
OH
6
5
Scheme 1. Synthesis of imidazo[1,2-a]pyrimidine 3 and imidazo[1,2-a]pyridine
4 and their alkylated derivatives 5 and 6, respectively.
Results and Discussion
Synthesis of the Imidazo[1,2-
]pyrimidines
a
]pyridines and Imidazo[1,2-
Photophysical Properties, DFT Calculations and Singlet
Oxygen Generation
a
Substituted imidazo[1,2-a]pyrimidine
1
and imidazo[1,2-
Absorption and emission spectra of compounds 3-6 were
obtained in 5 µmol L-1 solutions in Tris-HCl buffer (100 mmol L-1,
pH 7.0) and the results are shown in Figure 1 and Table 1. All
compounds show one absorption band in the UV region; at 332
nm for the imidazopyrimidine compounds 3 and 5, and at 317 nm
for the imidazopyridine compounds 4 and 6. In the visible region,
all compounds show two major absorption bands; at 423 nm and
445 nm for the imidazopyrimidine compounds 3 and 5, and at 390
and 411 nm for the imidazopyridine compounds 4 and 6.
Fluorescence quantum yields (Φ) were calculated according
to the IUPAC protocol by comparing with the quinine sulfate
standard.[30] Phenolic compounds 3 and 4 showed lower quantum
yields than the respective alkylated derivatives 5 and 6.
The photophysical parameters of the absorption and
emission spectra of compounds 3-6 were computed at B3LYP/6-
311+G(d,p) level of theory, and the calculation data are presented
in Table 2 and Figure 2. In the absorption spectra, one can see a
very good agreement between the theoretical and experimental
values of the absorption and emission wavelengths with average
differences smaller than 18 and 23 nm, respectively.
a]pyridine 2 were obtained employing a synthetic strategy based
on the condensation-elimination reaction between 2-bromo-4’-
hydroxyacetophenone and 2-aminopyrimidine or 2-aminopyridine,
respectively, as the first step. The reaction mechanism involved
in these syntheses was previously described by Hand and
Paudler applied to nitrogenated heterocycles.[28]
The [8+2] cycloadducts 3 and 4 were obtained applying the
methodology proposed by Aginagalde and co-workers, starting
from heterocycles 1 and 2, respectively, and employing 2-
(trimethylsilyl)phenyl triflate as the benzine precursor.[27b] This
method applies microwave radiation as the dielectric thermal
source, making possible the thermally allowed supra-supra
pathway for [π8s+π2s] cycloadditions, according to Woodward-
Hoffman rules.[29] The alkylated alcohols
5 and 6 were
synthesized through Williamson reaction, using 18-crown-6 as
promoter of the Williamson reaction between phenols 3 and 4 and
bromohydrin (Scheme 1). All compounds were fully characterized
1
by H and 13C NMR, IR spectroscopies and mass spectrometry
(Figures SI1-SI24), unambiguously confirming the expected
structures.
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(b) compound 4
(a) compound 3
300
350
400
450
500
550
600
300
350
400
450
500
550
600
Wavelength (nm)
Wavelength (nm)
(d) compound 6
(c) compound 5
300
350
400
450
500
550
600
300
350
400
450
500
550
600
Wavelength
(
nm)
Wavelength (nm)
Figure 1. Excitation (black) and emission (blue) spectra of: (a) 3, (b) 4, (c) 5 and
(d) 6.
Figure 2. Frontier molecular orbitals and their energies (in eV) for compounds
3-6 calculated at B3LYP/6-311+G(d,p) level of theory.
Table 1. Experimental photophysical parameters of compounds 3-6.
a
b
c
Compound
λabs
λexc
λem
Δλd
17
Φf
In Tables 1 and 2, the considered peaks of the lowest-
423
445
462
487
3
445
411
445
411
0.12
energy absorption can be assigned to HOMO
® LUMO
transitions. Figure 2 displays these orbitals and it can be easily
seen that these peaks can be assigned to a π (HOMO) ® π*
(LUMO) transition, where π and π* mainly represent bonding and
antibonding, respectively, interactions of the imidazo[1,2-
a]pyrimidine and imidazo[1,2-a]pyridine units. Moreover, the
calculated absorption spectra (Figure SI25) of these products and
the largest coefficients in the configuration interaction show that
they are mainly dominated by absorption from the three highest
occupied orbitals (HOMO, HOMO-1 and HOMO-2) to the three
lowest unoccupied orbitals (LUMO, LUMO+1 and LUMO+2). In a
similar way, the lowest-energy fluorescence emission wavelength
is dominated by the emission from the lowest unoccupied orbitals
to the highest occupied orbital (π* ® π transition).
390
411
423
446
4
5
6
12
17
12
0.13
0.18
0.18
423
445
462
487
390
411
423
446
aMaximum absorption wavelength that corresponds to the first and second
excited states in H2O at 25°C. bExcitation wavelength in nm. cEmission
wavelength in nm. dStokes shift (λem - λabs). dFluorescence quantum yield in H2O
at 25°C, calculated using quinine sulphate solution standard (1 ppm, in H2SO4
0.5 mol L-1).[30]
These experimental and computational results show that
the presence of the third nitrogen atom on the conjugated system
of the imidazopyrimidine compounds provokes a redshift in the
absorption maxima, when compared to the absorption spectra of
the imidazopyridine compounds. This redshift is due to the
stabilization of the LUMO energies of the imidazopyrimidine that
is more important than the stabilization of the HOMO energies;
i.e., a reduction of the energy HOMO-LUMO gap. It is interesting
to note that the presence of the alkoxy R-OCH2CH2CH2OH group
does not have a significant effect on the absorption spectra.
Conversely, the results displayed in Tables 1 and 2 indicate
that the products with the Ar-OCH2CH2CH2OH group present
higher fluorescence intensities than their equivalents with the Ar-
OH group, which explains the higher fluorescence quantum yields
obtained for the alkylated derivatives 5 and 6.
Table 2. Calculated photophysical parameters of compounds 3-6, computed at
B3LYP/6-311+G(d,p) level.
Com-
pound
a
b
c
b
e
λexc
fexc
λem
fem
ΔEd
3.48
3.69
3.45
3.66
ΔES-T
2.28
2.42
2.26
2.41
405
(3.06)
439
(2.83)
3
0.29
0.25
0.36
0.30
0.23
0.22
0.30
0.28
386
(3.21)
417
(2.97)
4
5
6
408
(3.04)
439
(2.83)
388
(3.19)
418
(2.97)
aAbsorption wavelength for the first excited state, values in nm and eV (in
parenthesis). bOscillator strength. cFluorescence emission wavelength, values
in nm and eV (in parenthesis). dHOMO-LUMO gap in eV. eVertical singlet-triplet
energy gap in eV.
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Cytotoxicity in the dark
The cytotoxicity of compounds 3-6 was evaluated against
HeLa cells (cervical cancer) and MCF-7 cells (breast cancer)
using the CCK-8 assay, and their corresponding IC50 values were
calculated. Compounds 3-5 showed IC50 values in the micromolar
range (Table 3), suggesting that these compounds can have
some potential application as cytotoxic agents against cancer
cells within this concentration range. It is interesting to note that
for the imidazopyrimidine compounds, the IC50 values decreased
upon alkylation of phenolic compound 3 to obtain compound 5.
Table 3. IC50 values (in µM) obtained for imidazopyrimidines and
imidazopyridine compounds 3-6.a
Compound
HeLa cells
MCF-7 cells
3
4
63
28
77
41
5
23
40
6b
>100
>100
aCells were treated for 48 h with the indicated compounds. The half-maximal
inhibitory concentration (IC50) is expressed in µM. Data are presented as the
mean of at least two experiments carried out in triplicate. The IC50 values were
determined by non-linear regression analysis calculated with GraphPad Prism
5.0 software. bCompound 6 presented low solubility in the cell culture medium.
Fluorescence microscopy images of the cell culture wells
containing compound 6 clearly showed fluorescent crystals
outside the cells, suggesting that the very low cytotoxicity
obtained for compound 6 is most likely due to the low solubility of
this compound in the cell culture medium precluding the cellular
uptake (Figure SI26). Due to the low solubility of the
imidazopyridine compounds in the cell culture medium, further
work on cell culture was carried only with imidazopyrimidine
compounds 3 and 5.
The cytotoxic activity of alkylated imidazopyrimidine
compound 5 was further explored against PC3 cells (prostate
cancer cells) and PNT2 cells (normal prostate cells), and the
resulting data are depicted in Figure 4. These data suggest that
at concentrations higher than 10 µM compound 5 is more
cytotoxic against cancer cells (PC3) than against healthy cells
(PNT2). This result reveals a potential application of compound 5
as a selective anti-cancer agent (this potential application is
currently under investigation in our laboratory with additional
bioassays).
Figure 3. (a) Lifetime decay curve of compound 5 in methanol at room
temperature upon excitation at 405 nm (R2 = 0.99978). (b) Singlet oxygen
emission spectrum of compound 5 in methanol at room temperature upon
excitation at 445 nm.
Imidazopyrimidine compound 5 was studied using time-
resolved fluorescence spectroscopy and it was found that its
emissive decay occurs in the nanosecond region with
fluorescence lifetime of 7.53 ns (Figure 3a).
a
The singlet excited energy (S1) of the photosensitizer can
be transferred to the triplet excited state (T1) through an
intersystem crossing. The DFT calculations demonstrate that the
vertical singlet-triplet energy gap of 5 (2.26 eV) lies above the
energy necessary to activate the ground state molecular oxygen
(3O2, 0.98 eV) to generate singlet oxygen species (1O2) (Table
1
2).[31] In order to evaluate if this energy transfer occurs, the O2
(b)120
120
phosphorescence was monitored upon irradiation of an oxygen-
rich solution of compound 5. Our results show an intense
emission band around 1250 nm upon excitation of compound 5 at
445 nm (Figure 3b). These data confirm that the
imidazopyrimidine compound 5 generates singlet oxygen species
upon irradiation, therefore, it might be used as a photosensitizer
for photodynamic therapy.
(a)
100
80
60
40
20
0
100
80
60
40
20
0
Control
0.01
0.1
1.0
10
50
Control
0.01
0.1
1.0
10
50
Concentration of imidazopyrimidine 5 (μM)
Concentration of imidazopyrimidine 5 (μM)
Figure 4. Cell viability data obtained for compound 5 against (a) PC3 cells, and
(b) PNT2 cells.
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DAPI
Alexa Fluor
Compound 5
Merged
Figure 5. Confocal fluorescence laser scanning microscopy images of MCF-7 cells incubated with imidazopyrimidine compound 5 (at 2 µM) to observe the cellular
uptake after 6 h and 24 h. Nuclei and cytoskeleton were stained with DAPI (blue) and Alexa Fluor 555-Phalloidin (red), respectively. The green color presented in
the third channel results from compound 5 fluorescence.
Cellular uptake
Photodynamic activity
The cellular internalization of the photosensitizer plays a
The photodynamic activity of compounds 3 and 5 against
MCF-7 cells was investigated by monitoring the cells morphology
variation upon irradiation, during incubation. Exposure of MCF-7
cells to a 469 nm laser during 10 min, in presence of compounds
3 or 5 (at 2 µM) was carried out using a Cytation 5™ cell-imaging
multi-mode reader. After irradiation for 10 min, images were
captured after 1h, 1h30min and 2h. Figure 6 shows the bright field
images merged with fluorescent images obtained with a GFP filter
(excitation wavelength: 469 nm; emission detector 525 nm). The
images demonstrate that both compounds are efficiently uptaken
by the MCF-7 cells within one hour, showing intense green
fluorescence in the cytoplasm. Upon irradiation, the cell
morphology changes dramatically, with surface evaginations,
membrane shrinkage and plasma membrane blebbing. The
plasma membrane deformation developed rapidly, and some
blebs detached from the cell membrane into the medium. These
observations suggest that the photodynamic activity of
compounds 3 and 5 at 2 µM (concentration level much lower than
their IC50) rapidly induces cell death. It is well known that PDT can
induce cells death through three different mechanisms: apoptosis,
necrosis and autophagy, being apoptosis more common when the
photosensitizer is located in the mitochondria.[34] The ability to
activate multiple cell death pathways reduces the probability of
apoptosis resistance development by cancer cells.[35] The cell
death mechanism caused by the imidazopyrimidine compounds
under irradiation is currently under investigation in our laboratory.
crucial role in its therapeutic efficacy.[3b] The cellular response to
photodamage is strongly dependent on the photosensitizer
location, playing
a
significant part in the cell fate.[32]
Photosensitizers are generally located in organelles such as
plasma membrane, lysosomes, mitochondria, Golgi apparatus or
endoplasmatic reticulum.[33]
To evaluate the internalization of imidazopyrimidine
compound 5 in cancer cells, MCF-7 cells were incubated with
compound 5 at 2.0 µM, for 6 h and 24 h. After fixation, the cells
were stained with DAPI (blue, nucleus) and with Alexa Fluor 555-
Phalloidin (red, cytoskeleton), analyzed by confocal fluorescence
laser scanning microscopy, and the obtained images are
displayed in Figure 5.
The images clearly show that compound 5 is efficiently
uptaken by the cells. Within 6 h compound 5 is located in round-
shaped organelles in the cytoplasm, probably lysosomes or
endosomes, showing strong fluorescence. After 24 h, compound
5 can still be found in the round-shaped organelles in the
cytoplasm. Since compound 5 was tested at a concentration level
much lower than its IC50, and the incubation was carried out in the
dark, no significant changes in the cell’s morphology were
observed. These data confirm that compound 5 is not cytotoxic at
2.0 µM in the dark, it is efficiently uptaken by the MCF-7 cells, and
that it shows strong fluorescence in the intracellular medium.
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1h30min
1h
2h
Figure 6. Merged images of bright field and fluorescent images captured with GFP filter (excitation wavelength: 469 nm; detector wavelength: 525 nm) of living
MCF-7 cells irradiated in presence of compounds 3 or 5. Two different regions of the 96-well plate is shown for each compound at time points 1h, 1h30 min and 2h.
The wells containing MCF-7 cells exposed to compounds 3
and 5 and irradiation were submitted to the CCK-8 viability assay
after 4h. The results showed no significant values of living cells
confirming the efficacy of the photodynamic activity of the
imidazopyramidine compounds in vitro.
In control experiments with irradiation of MCF-7 cells in
absence of imizadopyrimidines, the cells did not undergo
morphological changes. Compound 6 was also applied to MCF-7
cells and monitored using Cytation 5™, however, images show
that this compound present low solubility on the cell culture
medium, with visible fluorescent crystals outside the cells (Figure
SI26). Since no fluorescence was observed inside the cells the
photodynamic activity of this compound was not evaluated. These
results suggest that the third nitrogen present in the
imidazopyrimidine heterocycles is very important for the solubility
in the cell culture medium, and for the cell penetration process,
when comparing to the imidazopyridine analogs.
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pressure, and the residue was dissolved in a NaOH solution (4 M, 20 mL)
and washed with CHCl3 (4 x 20 mL). The aqueous phase was acidified to
pH 3.0 with HCl solution (6 M), and the resulting solution was kept
refrigerated for 30 minutes. The product was obtained as a precipitated
solid, which was recovered by filtration, dissolution in MeOH and solvent
evaporation under reduced pressure. The product was obtained as a light
red powder in 89% yield. Rf: 0.51 (ethyl acetate/methanol 45:1). Mp: 284-
285 °C. 1H NMR (500 MHz, methanol-d4), δppm: 9.19 (dd, J = 6.7, 1.8 Hz,
1H), 8.97 (dd, J = 4.5, 1.8 Hz, 1H), 8.40 (s, 1H), 7.81 - 7.73 (m, 2H), 7.62
(dd, J = 6.8, 4.4 Hz, 1H), 6.98 (dd, J = 9.1, 2.5 Hz, 2H). 13C NMR (126
MHz, methanol-d4) δppm: 160.3, 156.5, 144.3, 137.4, 137.0, 128.0 (2C),
116.7, 116.1 (2C), 113.9, 107.6. IR (ATR) νmax/cm-1: 3366 and 3217 (O-H),
3081 (C-H), 1603 (C=C), 1538 (C=N), 1370 (C-N), 1172 (C-O). m/z
calculated for C12H9N3O: 211.0746 (100.0%), 212.0779 (13.0%), 212.0716
(1.1%); found (TOF MS EI+): 212.0807 (100.0%), 213.0836 (10.51%).
Conclusion
There is
a
continuous search for new biocompatible
photosensitizers for photodynamic therapy, to treat a variety of
diseases. Moreover, compounds that are able to be used for both
fluorescence imaging and PDT have potential applications in
phototheranostics. Herein, we demonstrate for the first time that
imidazo[1,2-a]pyrimidine compounds generate singlet oxygen
species, and can act as photosensitizers. These compounds are
fluorescent in the intracellular medium, present low cytotoxicity in
the dark, are efficiently uptaken by cancer cells, and kill cancer
cells within 2h at low concentration (2.0 µM) upon irradiation.
Since the imidazo[1,2-a]pyrimidine compounds reported here
absorb and emit in the 400-500 nm region, they have potential
application as photosensitizer for PDT against bladder cancer,
oral precancer lesions, cervical intraepithelial neoplasia,
cutaneous T-cell lymphoma, psoriasis and microbial infections.
Moreover, these compounds can be applied as organic
photosensitizers in environmental applications such as the
phototreatment of polluted waters. This work opened the door to
explore the potential of the imidazopyrimidine compounds as
small organic molecule photosensitizers for phototheranostics.
Our research groups are currently focused on modifying the
substituents groups on the fused heterocycle fragment in order to
obtain related compounds with absorption and emission
wavelength in the near-infrared region (NIR), and in the
investigation of the cell death mechanism(s) promoted by the
imidazopyrimidine compounds upon irradiation.
Synthesis of 4-(imidazo[1,2-a]pyridin-2-yl)phenol, 2. A mixture of 2-
bromo-4’-hydroxy-acetophenone (1 mmol, 0.215 g) and 2-aminopyridine
(1 mmol, 0.094 g) in ethanol (40 mL) was refluxed at 85 °C for 16 h. After
cooling, the solvent was removed under reduced pressure and the residue
was dissolved in a NaOH solution (4 M, 20 mL) and washed with CHCl3 (4
x 20 mL). The aqueous phase was acidified to pH 3.0 with HCl solution (6
M), and the resulting solution was kept refrigerated for 30 minutes. The
product was obtained as a precipitated solid, which was recovered by
filtration, dissolution in MeOH and solvent evaporation under reduced
pressure. The product was obtained as a white powder in 93% yield. Rf:
0.42 (ethyl acetate/hexane 4:1). Mp: 247-278 °C. 1H NMR (500 MHz,
methanol-d4), δppm: 8.77 (dd, J = 6.8, 1.2 Hz, 1H), 8.41 (s, 1H), 8.00 - 7.93
(m, 1H), 7.90 (d, J = 8.9 Hz, 1H), 7.78 - 7.72 (m, 2H), 7.50 (m, 1H), 7.03 -
6.95 (m, 2H). 13C NMR (126 MHz, methanol-d4) δ 161.2, 141.5, 138.1,
134.3, 129.8, 129.0 (2C), 118.5, 118.2, 117.3 (2C), 112.5, 110.6. IR (ATR)
νmax/cm-1: 3469 and 3353 (O-H), 31131 (C-H), 1653 (C=C), 1538 (C=N),
1365 (C-N), 1180 (C-O). m/z Calculated for C13H10N2O: 210.0793
(100.0%), 211.0827 (14.8%); Found: (TOF MS EI+) 211.0854 (100.0%),
212.0910 (11.48%).
Experimental Section
Synthesis of 4-(2,2a1,3-triazacyclopenta[jk]fluoren-1-yl)phenol), 3. 2-
(trimethylsilyl)phenyl triflate (0.3 mmol, 0.101 mL) was added to a mixture
1 (0.2 mmol, 0.042 g), 18-crown-6 (0.6 mmol, 0.159 g) and CsF (0.3 mmol,
0.046 g) in acetonitrile (0.5 mL) and 1,2-dichlorobenzene (1.5 mL). The
reaction mixture was irradiated in a monomode microwave reactor at 120
°C, 90 W and 40 psi. After 1 hour, the resulting mixture was dissolved in
methanol, evaporated and the residue was purified by flash column
chromatography (ethyl acetate/hexane 2:1). The product was obtained as
a yellow powder in 6% yield. Rf: 0.40 (ethyl acetate/hexane 3:1); Mp: 222-
223 °C; 1H NMR (500 MHz, DMSO-d6), δppm: 10.12 (s, 1OH), 9.17 (d, J =
5.0 Hz, 1H), 8.77 (d, J = 8.0 Hz, 1H), 8.60 (d, J = 8.2 Hz, 1H), 8.42 (d, J =
5.0 Hz, 1H), 8.30 (d, J = 8.6 Hz, 2H), 8.03 (t, J = 7.8 Hz, 1H), 7.79 (t, J =
Supporting information contains the excitation and emission spectra of
compounds 1 and 2, all 1H and 13C NMR spectra, IR and mass spectra,
theoretical UV-Vis spectra of compounds 3-6 calculated at B3LYP/6-311+
G(d,p) level, Cartesian coordinates (in Å) of the stationary points in the
ground state optimized at the B3LYP/6-311+G(d,p) level of theory, and
merged image of bright field and fluorescent images captured with GFP
filter of living MCF-7 cells irradiated in presence of compound 6.
General considerations. All reactions were carried out using reagent
grade solvents, and all chemical were purchased from SigmaAldrich and
used without further purification. Flash column chromatography was
performed using normal phase silica gel. The spots of the analytical thin
layer chromatography (TLC) plates were visualized under ultraviolet light
(254 nm and 354 nm). 1H NMR spectra were recorded on Bruker
spectrometers (400 or 500 MHz). Chemical shifts are reported in parts per
million (ppm) relative to the residual solvent peaks. 1H NMR coupling
constants (J) are reported in Hertz (Hz), and multiplicity is indicated as
follows: s (singlet), d (doublet), dd (doublet of doublet), t (triplet), q (quartet)
and m (multiplet). 13C NMR spectra were recorded at 100 or 125 MHz, and
all chemical shift values are reported in ppm on the d scale, with an internal
reference. Cell counting kit 8 (CCK-8) was purchased from Sigma-Aldrich.
DMEM, RPMI-1640 and 0.25% trypsin-EDTA solution were purchased
from Gibco®. Fetal bovine serum (FBS) and Dulbecco modified phosphate
buffered saline (DMPBS, pH 7.4) were purchased from Nutricell Nutrientes
Celulares (Campinas, SP, Brazil). DAPI and Alexa Fluor® 555 Phalloidin
were obtained from Life Technologies.
7.7 Hz, 1H), 7.12 (d, J = 8.6 Hz, 2H). 13C NMR (125 MHz, DMSO-d6), δppm
:
160.1, 150.3, 149.2, 146.2, 134.8, 132.4, 130.7, 130.6, 130.1 (2C), 126.3,
125.9 (2C), 124.8, 121.7, 116.9, 106.0 (2C). IR (ATR) νmax/cm-1: 2881,
2677, 1660, 1603, 1460, 1275, 1244, 1165, 744, 694. m/z Calculated for
C18H11N3O: 285.0902 (100.0%), 286.0936 (19.5 %), 287.0969 (1.8 %),
286.0872 (1.1 %); found (TOF MS EI+): 286.0988 (100.0 %), 287.1016
(17.7 %), 288.1047 (1.1 %).
Synthesis of 4-(benzo[a]imidazo[5,1,2-cd]indolizin-1-yl)phenol, 4. 2-
(trimethylsilyl)phenyl triflate (0.3 mmol, 0.101 mL) was added to a mixture
of 2 (0.2 mmol, 0.042 g), 18-crown-6 (0.6 mmol, 0.159 g) and CsF (0.3
mmol, 0.046 g) in acetonitrile (0.5 mL) and 1,2-dichlorobenzene (1.5 mL).
The reaction mixture was irradiated in a monomode microwave reactor at
120 °C, 90 W and 40 psi. After 1 h, the resulting mixture was dissolved in
methanol, evaporated and the residue was purified by flash column
chromatography (ethyl acetate/hexane 2:1). The product was obtained as
a yellow powder in 6% yield. Rf: 0.67 (ethyl acetate/hexane 3:1). Mp: 232-
233 °C. 1H NMR (500 MHz, DMSO-d6), δppm: 9.99 (s, OH), 8.69 (d, J = 8.0
Hz, 1H), 8.56 (d, J = 8.2 Hz, 1H), 8.42 (d, J = 8.4 Hz, 1H), 8.26 (d, J = 8.3
Synthesis of 4-(imidazo[1,2-a]pyrimidin-2-yl)phenol, 1. A mixture of 2-
bromo-4’-hydroxy-acetophenone (1 mmol, 0.215 g) and 2-
aminopyrimidine (1 mmol, 0.095 g) in ethanol (40 mL) was refluxed at 85
°C for 16 h. After cooling, the solvent was removed under reduced
This article is protected by copyright. All rights reserved.

10.1002/chem.202004957
Chemistry - A European Journal
FULL PAPER
Hz, 2H), 8.14 - 8.06 (m, 2H), 7.91 (t, J = 7.5 Hz, 1H), 7.73 (t, J = 7.5 Hz,
1H), 7.09 (d, J = 8.6 Hz, 2H). 13C NMR (125 MHz, DMSO-d6), δppm: 159.3,
146.9, 139.5, 131.0, 130.1, 130.0, 128.4, 127.3, 125.5, 125.3 (2C), 124.4
(2C), 121.1, 119.5, 116,7, 113.0 (2C), 109.4. IR (ATR) νmax/cm-1: 3058,
2594, 1601, 1492, 1470, 1275, 1248, 1020, 749. m/z Calculated for
C19H12N2O: 284.0950 (100.0%); found (TOF MS EI+): 285.1038 (100.0 %),
286.1064 (18.54 %).
In Vitro Cellular Uptake Study. Confocal laser scanning microscopy
(CLSM) was used to visualize cellular uptake of compounds. MCF-7 cells
were seeded into 6-well plates at a density of 3 × 105 cells/well in 3 mL of
RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin.
After culturing for 24 h at 37 °C in a humidified atmosphere with 5% CO2,
the cells were further incubated for 6 h with compound 5 at 1 µM. The cells
were washed three times with PBS, fixed with 4% p-formaldehyde for 15
min at room temperature, and washed again with PBS. Then, the cells
were permeabilized with 0.2% Triton X-100 for 5 min, washed with PBS,
and incubated with DAPI to label the nuclei and with Alexa Fluor 555
Phalloidin for staining the cytoskeleton, according to the standard protocol
provided by the supplier. The slides containing the cells were rinsed once
with PBS and mounted with antifading solution. Finally, the fluorescence
images were obtained using an Upright LSM780-NLO Zeiss confocal laser
scanning microscope equipped with 405, 458, 488, 514, 543, 561 and 633
nm laser excitation sources.
Synthesis
of
3-(4-(2,2a1,3-triazacyclopenta[jk]fluoren
-1-
yl)phenoxy)propan-1-ol, 5. A mixture of compound 3 (1 mmol), K2CO3 (3
mmol, 0.415 g), trimethylene bromohydrin (1 mmol, 0.214 mL) and 18-
crown-6 (0.1 mmol, 2.65 x 10-5 g) in acetone (40 mL) was refluxed for 16
h. After cooling, the solution was filtered (to remove the K2CO3), and the
solvent was removed under reduced pressure. The obtained crude product
were purified by flash column chromatography (ethyl acetate/hexane 2:1).
The product was obtained as a yellow powder in 60% yield. Rf: 0.24 (ethyl
acetate/hexane 3:1). Mp: 149-150 °C. 1H NMR (400 MHz, CDCl3-d), δppm
:
9.13 (d, J = 4.0 Hz, 1H), 8.37 (d, J = 6.4 Hz, 2H), 8.34 (d, J = 6.4 Hz, 2H),
7.93 (d, J = 4.0 Hz, 1H), 7.82 (t, J = 5.6 Hz, 1H), 7.57 (t, J = 6.0 Hz, 1H),
7.14 (d, J = 6.8 Hz, 2H), 4.29 (t, J = 4.8 Hz, 2H), 3.98 (, J = 4.4 Hz, 2H),
3.30 (s, OH), 2.18 (q, J = 4.8 Hz, 2H). 13C NMR (125 MHz, DMSO-d6),
δppm: 160.3, 149.1, 149.05, 145.6, 134.5, 131.9, 130.2, 129.8, 129.6, 125.8
(2C), 125.7, 125.5 (2C), 121.1, 117.4, 115.4, 105.6 (2C), 64.9, 57.2, 32.1.
IR (ATR) νmax/cm-1: 3224, 1605, 1252, 1168, 827, 742. m/z calculated for
C21H17N3O2: 343.1354 (100.0 %); found (TOF MS EI+): 344.1407
(100.0%), 344.1396 (28.10 %).
Cell Viability Assay. Cells were seeded into 96-well plates (7.5 × 103
cells/well) containing 200 μL of complete cell culture medium and
incubated for 24 h at 37 °C in a humidified atmosphere with 5% CO2. Then,
the cells were treated with different concentrations of compounds 3-6 for
further 48 h. After incubation, the growth medium was removed, the cells
were washed twice with PBS, and the cell viability was evaluated using the
Cell Counting Kit-8 (CCK-8) according to the manufacturer instructions.
The absorbance of the solution in each well was measured at 450 nm in a
microplate reader (FlashScan 530 Analitic Jena). The negative control was
designed as 100% of dehydrogenase activity, and cell viability was
expressed as a percentage of the untreated cells.
Synthesis
of
3-(4-(benzo[a]imidazo[5,1,2-cd]indolizin-1-
yl)phenoxy)propan-1-ol, 6. A mixture of compound 4 (1 mmol), K2CO3 (3
mmol, 0.415 g), trimethylene bromohydrin (1 mmol, 0.214 mL) and 18-
crown-6 (0.1 mmol, 2.65 x 10-5 g) in acetone (40 mL) was refluxed for 16
h. After cooling, the solution was filtered (to remove the K2CO3), and the
solvent removed under reduced pressure. The obtained crude products
were purified by flash column chromatography (ethyl acetate/hexane 2:1).
The product was obtained as a yellow powder in 65% yield. to yield the
corresponding pure products. Rf: 0.36 (ethyl acetate/hexane 3:1). Mp:
251-252 °C. 1H NMR (400 MHz, methanol-d4), δppm: 8.37 (d, J = 8.0 Hz,
1H), 8.26 (d, J = 8.0 Hz, 1H), 8.13 (m, 3H), 7.97 (t, J = 8.4 Hz, 1H), 7.92
(d, J = 7.6 Hz, 1H), 7.71 (t, J = 6.0 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.16
(d, J = 8.8 Hz, 2H), 4.21 (t, J = 6.0 Hz, 2H), 3.83 (t, J = 6.4 Hz, 2H), 2.09
(q, J = 6.4 Hz, 2H). The OH signal is next to the solvent signal in the NMR
spectrum. 13C NMR (100 MHz, methanol-d4), δppm: 161.8, 147.0, 140.6,
132.6, 131.8, 130.9, 130.6, 130.1, 128.7, 127.6, 126.3 (2C), 124.5 (2C),
121.7, 121.1, 116.3, 113.0 (2C), 110.3, 66.1, 59.7, 33.6. IR (ATR) νmax/cm-
1: 3199, 2923, 1603, 1496, 1248, 737. m/z Calculated for C22H18N2O2:
342.1368 (100.0 %); found (TOF MS EI+): 343.1455 (100.0%), 344.1481
(22.92 %).
Photodynamic activity. Cells irradiated and imaged using a Cytation™
5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc., Winooski,
VT) configured with a GFP light cube. The microscope uses a combination
of LED light sources in conjunction with band pass filters to provide the
appropriate wavelength of light. The GFP light cube uses a 469 nm
excitation filter and a 525 nm emission filter. Installed objectives include
4x, 10x, and 20x magnifications. Irradiation and imaging ware carried out
in living cells using 96-well plates, at 37 °C in a humidified atmosphere with
5% CO2. After 30 min of incubation with compounds 3 or 5, cells were
irradiated with GFP laser (100 mW) for 10 min, and images were captured
at different time points: 1 h, 1h30 min and 2h.
Computational Calculations. The geometries for all studied systems in
the ground state have been fully optimized in the gas phase using the
B3LYP functional[36] and the 6-311+G(d,p) basis set.[37] The harmonic
vibrational frequencies were also calculated at the same level of theory in
order to verify that all the stationary points are minima of their potential
energy surface. The time-dependent (TD) DFT methodology[38] was
applied to compute the absorption and emission spectra at the ground and
the first singlet excited states, respectively, with the B3LYP/6-311+G(d,p)
level. All calculations were performed using the Gaussian09 program
package.[39]
Photophysical characterization. Excitation and emission spectra were
recorded with a 2300 EnSpireTM Multimodal Reader (Perkin Elmer).
Fluorescence quantum yields were obtained in a Spectrofluorometer
(Varian, Cary Eclipse model), using 1 cm quartz cell, and were calculated
ST
using quinine sulphate
(1 ppm, in H2SO4 0.5 M; ΦST = 0.54) as
reference, according to IUPAC protocol.[30] All the excitation and emission
spectra of compounds 3-6 were previously optimized under the same
conditions used for the fluorescence quantum yields experiments.
Acknowledgements
Cell Culture. The human MCF-7 (breast cancer), PC3 (prostate cancer)
and PNT2 (normal prostate) cells were cultured in RPMI-1640
supplemented with 10% FBS and 1% penicillin‒streptomycin. HeLa
(human cervical cancer) cells were cultured in DMEM (Dulbecco’s
Modified Eagle Medium) with high glucose supplemented with 10% FBS
and 1% penicillin-streptomycin. All cells were incubated at 37 °C in a
humidified atmosphere with 5% CO2. The cell culture medium was
changed every 2 days, and cells were passaged at 80-90% confluency
using 0.25% trypsin-0.02% EDTA solution.
The authors gratefully acknowledge Sao Paulo research
Foundation – FAPESP (grant #2018/02093-0 for C.O.; fellowship
#2017/06146-8 for C.B.B.), National Council for Scientific and
Technological Development – CNPq (grant #140741/2013-5 for
A.J.M.; productivity award for C.O. #307403/2018-1), Petrobras-
ANP (grant #140741/2013-5 for A.J.M.), and Coordination for the
Improvement of Higher Education Personnel – CAPES (Ph.D.
scholarship for T.B.B.; CAPES/PDSE #010181/2014-08 for
This article is protected by copyright. All rights reserved.

10.1002/chem.202004957
Chemistry - A European Journal
FULL PAPER
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Yim, S. Kim, B. Ryu, K. M. K. Swamy, G. Kim, N. Kwon, C. Y. Kim, S.
Park, J. Yoon, Angew. Chem. Int. Ed. 2020, 59, 8957-8962; c) Z.
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M.L.S.O.L.). This study was financed in part by the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior – Brasil
(CAPES), finance code 001. The group at Department of Organic
Chemistry I acknowledges the financial support provided by the
Ministerio de Economía y Competitividad (MINECO) of Spain and
FEDER (projects CTQ2016-80375-P and Red de Excelencia
Consolider
CTQ2014-51912-REDC),
and
the
Basque
Government (GV/EJ, grant IT-324-07).
AUTHOR INFORMATION
ORCID
Maria L. S. O. Lima: 0000-0002-7824-8108
Carolyne B. Braga: 0000-0003-1800-386X
Miquel Torrent-Sucarrat: 0000-0003-2689-0278
Fernando P. Cossío: 0000-0001-8668-536X
Anita J. Marsaioli: 0000-0002-7894-6942
Catia Ornelas: 000-0001-8020-9776
[14] Y. Cai, D. Ni, W. Cheng, C. Ji, Y. Wang, K. Müllen, Z. Su, Y. Liu,
C. Chen, M. Yin, Angew. Chem. Int. Ed. 2020, 59, 14014-14018.
[15] a) S. Chakrabortty, B. K. Agrawalla, A. Stumper, N. M. Vegi, S.
Fischer, C. Reichardt, M. Kögler, B. Dietzek, M. Feuring-Buske, C.
Buske, S. Rau, T. Weil, J. Am. Chem. Soc. 2017, 139, 2512-2519; b)
J. Karges, T. Yempala, M. Tharaud, D. Gibson, G. Gasser, Angew.
Chem. Int. Ed. 2020, 59, 7069-7075.
Keywords: imidazopyrimidine • singlet oxygen • organic
synthesis •photosensitizer • photodynamic therapy
[16] Y. Zhang, T. S. Lee, J. L. Petersen, C. Milsmann, J. Am. Chem.
Soc. 2018, 140, 5934-5947.
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