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Exploring Structure-Activity Relationships in Photodynamic Therapy Anticancer Agents Based on Ir(III)-COUPY Conjugates.
Photodynamic therapy holds great promise as a non-invasive
anticancer
tool against drug-resistant cancers. However, highly effective, non-toxic,
and reliable photosensitizers with operability under hypoxic conditions
remain to be developed. Herein, we took the advantageous properties
of COUPY fluorophores and cyclometalated Ir(III) complexes to develop
novel PDT agents based on Ir(III)-COUPY conjugates with the aim of
exploring structure–activity relationships. The structural
modifications carried out within the coumarin scaffold had a strong
impact on the photophysical properties and cellular uptake of the
conjugates. All Ir(III)-COUPY conjugates exhibited high phototoxicity
under green light irradiation, which was attributed to the photogeneration
of ROS, while remaining non-toxic in the dark. Among them, two hit
conjugates showed excellent phototherapeutic indexes in cisplatin-resistant
A2780cis cancer cells, both in normoxia and in hypoxia, suggesting
that photoactive therapy approaches based on the conjugation of far-red/NIR-emitting
COUPY dyes and transition metal complexes could effectively tackle
in vitro acquired resistance to cisplatin.
## Introduction
Introduction Photodynamic therapy (PDT) has gained
great attention in recent
years as a promising cancer treatment modality. 1 , 2 Unlike
conventional approaches based on surgery or chemotherapy, PDT offers
several advantages including low invasiveness, high selectivity and
efficiency, and reduced toxic side effects that usually compromise
patient’s health. Upon light irradiation, the interaction of
a non-toxic photosensitizer (PS) and oxygen triggers local cytotoxicity
through the generation of reactive oxygen species (ROS), which oxidize
biomolecules in cells and lead to irreversible damage on tumor cell
structures (e.g., membranes and organelles), as well as on the vasculature
that deprives the tumor of oxygen and nutrients. 3 In addition, increasing evidences show that PDT can trigger
the activation of the anticancer immune system throughout the body
by immunogenic cell death (ICD). 4 , 5 Depending on the reaction
mechanism, PDT can be classified into two main types (I and II). 6 While type II pathway involves the transformation
of molecular oxygen ( 3 O 2 ) into singlet oxygen
( 1 O 2 ) via an energy transfer process, several
other cytotoxic reactive species, mainly superoxide radicals (O 2 •– ), hydroxyl radicals ( • OH), and hydrogen peroxide, are generated in type I photochemical
pathways via an electron transfer mechanism. 7 Although type II photochemical processes are generally considered
as the main photosensitization mechanism of most of the conventional
PSs, the availability of PDT agents operating at low-oxygen concentrations
in the phototherapeutic window (e.g., 650–800 nm) is highly
desirable to combat deep-seated hypoxic tumors and to avoid toxicity
associated to short wavelengths of light. 8 − 10 Transition
metal complexes have emerged as promising therapeutic
tools in photopharmacology due to several unique properties, including
a wide range of coordination numbers, oxidation states, and geometries. 11 , 12 Among transition metals, Pt(IV), Ru(II), Rh(III), Ir(III), and Os(II)
are very attractive candidates for PDT applications since they tend
to absorb in the visible region of the electromagnetic spectrum and
exhibit relatively high photostability and long luminescence lifetimes
(>100 ns), being an interesting alternative to PSs based on organic
fluorophores on clinical use such as porphyrins or chlorins. 13 − 24 In this context, cyclometalated iridium(III) complexes show excellent
anticancer activities and a great potential to overcome some of the
main drawbacks of conventional platinum-based chemotherapy (i.e.,
resistance and toxic side effects). 25 , 26 Such metal
complexes are likely good candidates for PDT applications as they
combine appealing photophysical and photochemical properties within
a single compound, including large Stokes’ shifts, high luminescent
quantum yield, and high efficient singlet oxygen production upon light
irradiation. Photosensitizers based on cyclometalated Ir(III) complexes
have been used also as photocatalysts in systems for photocatalytic
hydrogen evolution reactions. 27 , 28 However, most reported
cyclometalated iridium(III) complexes are quite cytotoxic in the dark
and only activatable with short wavelengths of light, which compromises
further development of efficient PDT agents. By taking advantage
of the well-established anticancer properties
of transition metal complexes and of the rich and tunable photophysical
and physicochemical properties of small organic chromophores, their
conjugation can be exploited for developing theranostic agents for
imaging-guided PDT. Examples of this strategy include the conjugation
and/or integration of cyclometalated Ir(III) complexes with organic
fluorophores such as boron-dipyrromethene (BODIPY) 29 − 32 porphyrin, 33 xanthene, 34 and rhodamine derivatives. 35 Coumarins, which are also well-known anticancer
scaffolds 36 − 38 and the basis for the development of novel organic
fluorophores, 39 have also been covalently
attached to Ir(III) complexes and used as cyclometalating ligands. 40 − 43 In this context, we have been pioneers in describing a novel class
of PDT agents based on the conjugation of a far red-emitting COUPY
coumarin to a cyclometalated Ir(III) complex (compounds 1a and 2a , respectively, in Figure 1 ). 44 , 45 The resulting Ir(III)-COUPY
conjugate (compound 3a ) was found to be non-cytotoxic
in the dark but highly photocytotoxic after irradiation with visible
light, even under hypoxia, the latter being attributable to the selective
generation of type I superoxide anion radicals. 44 Owing to a strong push–pull character due to the
replacement of the carbonyl group of the lactone in the conventional
coumarin scaffold by N-alkylated cyano(4-pyridine)methylene moieties,
COUPY dyes possess several attractive features for bioimaging applications
such as absorption and emission within the phototherapeutic window,
brightness, high photostability, and large Stokes’ shifts. 46 − 49 In addition, we have recently demonstrated that some COUPY dyes
exhibit effective in vitro anticancer activities upon visible-light
irradiation both under normoxia and hypoxia conditions, while exhibiting
minimal toxicity toward normal cells, which position them as promising
PS candidates for anticancer PDT. 50 Figure 1 (A) Structure
of previously evaluated compounds: COUPY coumarin 1a ,
cyclometalated Ir(III) complex 2a, and the
corresponding Ir(III)-COUPY conjugate 3a . (B) Structure
of the new COUPY coumarins ( 1b–1c ) and Ir(III)-COUPY
conjugates ( 3b–3e ) investigated in this work. Herein, we have synthesized a family of new Ir(III)-COUPY
conjugates
(compounds 3b–3e ) with the aim of exploring structure–activity
relationships (SARs), specially to investigate how the structural
modifications within the COUPY scaffold influence the photophysical,
photochemical, and biological properties of the resulting PSs. As
shown in Figure 1 ,
the newly synthesized Ir(III)-COUPY conjugates combine the highly
potent cyclometalated Ir(III) complex 2a and three COUPY
derivatives ( 1a–1c ), which were connected through
flexible or rigid linkers. The absence of the N , N -diethylamino electron-donating group (EDG) at the 7-position
of the coumarin moiety ( 1b ) could provide insights on
the involvement of this group on the generation of ROS in the resulting
Ir(III)-COUPY conjugate 3b . Moreover, the incorporation
of a julolidine-fused analogue ( 1c ) in Ir(III)-COUPY
conjugate 3c was expected to prevent the twisted intramolecular
charge transfer (TICT) state by incorporating the nitrogen atom into
a system of fused rings, 51 which would
also influence electron charge transfer between the metal center and
the coumarin moiety. Besides investigating the photophysical and photochemical
properties of all of the compounds, the results from cellular uptake
and cyto- and phototoxicity studies in several cancer cells allowed
us to select two Ir(III)-COUPY conjugates ( 3d and 3e ) as promising PSs owing to their excellent phototoxicities
in both normoxic and hypoxic conditions upon green light irradiation
and reduced in vitro dark toxicity.
## Results
Results Synthesis and Characterization of Ir(III)-COUPY Conjugates Synthesis of Ir(III)-COUPY Conjugate 3b As shown in Scheme 1 , a convergent approach was used for the synthesis of Ir(III)-COUPY
conjugate 3b, in which the carboxylic group of the Ir(III)
complex 2b ( Figure 1 ) was linked to the amino group of coumarin 10 through the formation of an amide bond. As previously indicated,
the synthesis of a COUPY derivative lacking the dialkylamino group
at the 7-position of the coumarin skeleton was proposed to explore
the contribution of this EDG to ROS generation when conjugated to
the metal complex. Scheme 1 Synthesis of Ir(III)-COUPY Conjugate 3b and of Control
COUPY Coumarin 1b The synthesis route for COUPY derivative 10 ( Scheme 1 ) involved seven
steps starting with a Pechmann condensation between phenol and ethyl
acetoacetate that afforded the desired coumarin skeleton ( 4 ). 52 Next, coumarin 4 was
reacted with Lawesson’s reagent (LW) to provide thiocoumarin 5 , 53 which was condensed with 4-pyridylacetonitrile
to give compound 6 . Coumarin ester 7 was
obtained with good yield by alkylation of 6 with methyl
bromoacetate. After acid hydrolysis, the carboxyl group of coumarin 8 was reacted with N -Boc-1,3-propanediamine
hydrochloride with the assistance of HATU coupling reagent and DIPEA
to yield coumarin 9 , whose Boc-protecting group was removed
under acidic conditions to give coumarin derivative 10 . Finally, Ir(III)-COUPY conjugate 3b was obtained as
a dark yellow solid after attachment of the Ir(III) complex 2b to the fluorophore via the formation of an amide bond.
All of the compounds depicted in Scheme 1 were purified by silica column chromatography
and fully characterized by high-resolution mass spectrometry (HRMS)
and 1 H and 13 C NMR spectroscopy. Synthesis of Ir(III)-COUPY Conjugate 3c For the synthesis of Ir(III)-COUPY conjugate 3c ( Scheme 2 ), we selected a
julolidine-fused coumarin analogue to red-shift the absorption maximum
with respect to the parent 7-dialkylaminocoumarin. Moreover, as previously
stated, rigidification of the amino group was anticipated to have
an impact on the photophysical and photochemical properties of the
compounds since rotation around the N–C bond is not possible
because of the fusion of the six-membered alkyl rings to the aromatic
ring. The required amino-containing COUPY derivative 16 was synthesized from a commercially available coumarin precursor
following the same procedure as for COUPY 10 . The conjugation
between 16 and the Ir(III) complex 2b afforded
Ir(III)-COUPY conjugate 3c as a dark blue solid after
silica column purification. All the compounds were characterized by 1 H and 13 C NMR spectroscopy and HRMS. Scheme 2 Synthesis
of Ir(III)-COUPY Conjugate 3c and Control
COUPY Coumarin 1c Synthesis of Ir(III)-COUPY Conjugates 3d and 3e With the aim of investigating the effect of the
distance between the COUPY fluorophore and the Ir(III) complex in
the parent Ir(III)-COUPY conjugate 3a on the photophysical
and photochemical properties of the compounds, we designed two analogues
incorporating a longer spacer between both moieties. As shown in Scheme 3 , we selected flexible
( 3d ) and rigid ( 3e ) spacers with the same
number of atoms separating the two moieties. The incorporation of
both linkers was carried out through the formation of an amide bond
between the carboxylic group of each Boc-protected precursor and the
free amino group of coumarin 17 , 37 which afforded COUPY derivatives 18 and 19 containing the flexible and rigid spacers, respectively. Finally,
after acidic Boc removal, the conjugation of coumarins 20 and 21 to the Ir(III) complex 2b provided
Ir(III)-COUPY conjugates 3d and 3e , respectively,
as dark blue solids after silica column purification, which were characterized
by 1 H and 13 C NMR spectroscopy and HRMS. Scheme 3 Synthesis of Ir(III)-COUPY Conjugates 3d and 3e Synthesis of Control COUPY Coumarin 1b and 1c For the synthesis of the corresponding N-methylated
COUPY dyes 1b and 1c to be used as control
compounds, coumarin 6 ( Scheme 1 ) or coumarin 12 ( Scheme 2 ) were reacted with methyl
trifluoromethanesulfonate in DCM at room temperature, yielding the
expected compounds as yellow and dark blue solids, respectively. Photophysical and Photochemical Characterization of the Compounds The photophysical and photochemical properties [absorption and
emission spectra, molar absorption coefficients (ε), fluorescence
(Φ F ) or phosphorescence (Φ P ) quantum
yields, fluorescence (τ F ) or phosphorescent (τ P ) lifetimes, and singlet oxygen quantum yield (Φ Δ )] of the four new Ir(III)-COUPY conjugates ( 3b–3e ) along with the two new coumarins ( 1b–1c ) were
studied in three solvents of different polarities (phosphate-buffered
saline PBS, ACN and DCM), and compared with those of the previously-reported
parent compounds (COUPY dye 1a , Ir(III) complex 2a and Ir(III)-COUPY conjugate 3a ). 44 The UV–vis absorption and emission spectra
are shown in Figures 2 and S1 and S2 , and their photophysical
properties are summarized in Tables S1 and S2 . Figure 2 Comparison of the molar absorptivity (a) and emission spectra (b)
of Ir(III)-COUPY conjugates in ACN. As shown in Table S1 , the structural
modifications carried out within the coumarin scaffold had a strong
impact on the photophysical properties of the compounds. Indeed, the
newly synthesized COUPY dyes showed either a bathochromic shift (about
20–30 nm for coumarin 1c ) or a hypsochromic shift
(about 100–120 nm for coumarin 1b ) in their absorption
and emission maxima compared with those of the parent coumarin 1a (e.g. in ACN, λ abs = 548 nm and λ em = 609 nm for 1a vs λ abs =
446 nm and λ em = 492 nm for 1b , and
λ abs = 572 nm and λ em = 635 nm for 1c ). The replacement of the N , N -dialkylamino group at position 7 of the coumarin skeleton by the
strong electron-donating julolidine-fused analog in coumarin 1c caused the observed red-shifting in the wavelength at the
absorption and emission maxima. On the contrary, the absence of an
EDG at position 7 in coumarin 1b partnering with the
electron-withdrawing cyanomethylenepyridinium moiety had a negative
effect on the spectroscopic properties of the fluorophore since both
absorption and emission maxima were strongly blue-shifted due to the
decreased push–pull character of the chromophore. On the other
hand, the fluorescent quantum yields were slightly lower for coumarin 1c in DCM and PBS, compared with the original COUPY 1a , whereas coumarin 1b was found to be very
weakly fluorescent. For coumarins 1a and 1c , a strong decrease in the fluorescence quantum yield and lifetime
was observed in the more polar solvents, indicating the onset of efficient
intramolecular electron-transfer excited-state deactivation pathways.
Consistent with this, coumarin 1b , lacking the N , N -dialkylamino EDG, showed a less pronounced
solvent dependence. As we had already reported, 44 the Ir(III)
complex 2a showed a strong and long-lived phosphorescence
band around 660 nm, whose intensity and lifetime decreased in the
Ir(III)-COUPY conjugate 3a , suggesting the existence
of competitive excited-state processes. As shown in Table S2 , a similar behavior was observed in the three new
conjugates incorporating a far-red emitting coumarin ( 3c–3e ). Among them, conjugate 3c containing the julolidine-fused
coumarin showed the largest red-shift in the absorption and emission
maxima (∼25–30 nm depending on the solvent), which parallels
the spectroscopic properties of COUPY 1c ( 3c , λ abs = 580 nm and λ em = 647 nm
in ACN). Interestingly, the luminescent quantum yield of 3c was higher than that of the parent conjugate 3a in
DCM, but similar values were obtained in ACN and PBS. As expected,
conjugates 3d and 3e containing the flexible
and the rigid spacer, respectively, showed similar absorption and
emission maxima in all investigated solvents compared to the original
conjugate 3a since they contain the same coumarin (about
λ abs = 555 nm and λ em = 610 nm in
ACN). The luminescence quantum yield values were also similar for
the three conjugates, about 0.1 in DCM and ACN, and below 0.02 in
PBS. The strong decrease in the luminescence quantum yields and lifetimes
in PBS is consistent with the onset of efficient competing intra-
and inter-chromophore electron-transfer processes. In good agreement
with the behavior of COUPY dye 1b , the Ir(III)-COUPY
conjugate 3b practically did not show any fluorescence
in all of the solvents investigated and, as shown in Table S2 , only phosphorescence lifetime values associated
with the Ir(III) complex could be determined and these were shorter
than for 3a . Finally, it is worth noting that the maximum
absorption wavelengths of all the compounds (COUPY dyes and Ir(III)-COUPY
conjugates) in different solvents gradually decrease according to
the following order DCM > ACN > PBS, which agrees with a negative
solvatochromism phenomenon ( Tables S1 and S2 ). Next, we focused on investigating the impact of the structural
modifications on the generation of ROS by Ir(III)-COUPY conjugates.
As we previously reported, 44 the Ir(III)
complex 2a produced singlet oxygen in all of the organic
solvents evaluated but not in PBS as a result of a very efficient
formation of the triplet state due to the heavy-atom effect, while
COUPY 1a did not show significant 1 O 2 quantum yields in any solvent ( Table S1 ). However, the conjugation of Ir(III) complex 2a to
the COUPY fluorophore 1a led to an increase of 1 O 2 quantum yield by one order of magnitude, suggesting
a higher population of COUPY triplet excited states (T 1 ) due to either an enhanced intersystem crossing (ISC) process in
the COUPY moiety of conjugate 3a when directly excited
at 532 nm (as demonstrated by the shorter fluorescence lifetime),
or an efficient triplet–triplet energy transfer process from
the Ir(III) complex to COUPY when the complex is excited at 355 nm
(as demonstrated by its shorter phosphorescence lifetime). In either
case, since the triplet lifetime of COUPY is longer than that of the
Ir(III) complex, localization of the triplet excited-state energy
in the COUPY moiety provides more time for energy transfer to 3 O 2 , thereby favoring the production of 1 O 2 ( Table S2 ). The ability
of all newly synthesized Ir(III)-COUPY conjugates ( 3b–3e ) and control COUPY fluorophores ( 1b–1c ) to produce 1 O 2 was evaluated spectroscopically by the observation
of 1 O 2 emission at 1275 nm upon excitation at
two wavelengths (355 and 532 nm). In the same way as for the original
COUPY derivative 1a , neither 1b nor 1c generated significant amounts of 1 O 2 at either excitation wavelength, indicating that radiative decay
competes efficiently with intersystem crossing to the triplet excited
state, the precursor of singlet oxygen. Interestingly, 1 O 2 emission was observed in all new Ir(III)-COUPY conjugates
in DCM and ACN irrespective of which chromophore was initially excited,
but not in PBS, consistent with the observed decrease in luminescence
quantum yields and lifetimes as a result of efficient electron-transfer
competing processes. Conjugates 3d and 3e containing the same coumarin derivative as the parent compound ( 3a ) showed similar singlet oxygen quantum yields (ca. 0.30–0.40
in DCM upon excitation at both wavelengths) regardless of the spacer
linking both moieties, while the conjugates with new coumarin derivatives, 3b and 3c , exhibited lower values (ca. 0.20 in
DCM). Considering that one of the main features of the parent
Ir(III)-COUPY
conjugate 3a is the generation of superoxide anion radical
(O 2 •– ) in living cells upon irradiation
with visible light, 44 we next investigated
the ability of the new conjugates to produce this specific type-I
ROS in PBS by using a spectroscopic method based on the oxidation
of the non-fluorescent dihydrorhodamine 123 (DHR123) probe by O 2 •– to the corresponding fluorescent
rhodamine 123 derivative. As shown in Figures 3 and S3 , Ir(III)-COUPY
conjugates did not produce any measurable quantity of superoxide anion
radical before irradiation; this result is comparable to that obtained
with DHR123 alone after irradiation, which was used as a negative
control. Surprisingly, under green light irradiation (505 nm), all
of the new conjugates, including the one containing the coumarin fluorophore
lacking the amino group at the 7-position, clearly increased the fluorescence
intensity of DHR123 to a greater extent than the Ir(III) complex 2a and COUPY derivatives 1a–1c , indicating
the generation of superoxide anion radical, consistent with the observed
excited-state electron-transfer processes. Remarkably, all the new
conjugates led to a faster O 2 •– generation rate in comparison to the parent compound 3a . The Ir(III)-COUPY conjugate containing the julolidine-fused system
( 3c ) showed the fastest rates in superoxide generation,
thereby demonstrating the importance of incorporating a strong EDG
in the coumarin scaffold, which not only red-shifts absorption and
emission maxima but also produces more O 2 •– . Interestingly, the Ir(III)-COUPY conjugate 3b reached
the highest maximum emission intensity of superoxide generation after
3 min of irradiation, indicating that the amino group at the 7-position
of the coumarin moiety is not strictly necessary to trigger the generation
of O 2 •– . Overall, the suppression
of singlet oxygen generation in PBS, and the reduction in fluorescence
and phosphorescence lifetimes and quantum yields of Ir(III)-COUPY
conjugates, are consistent with the onset of intra- and inter-chromophore
electron-transfer processes, resulting in the generation of superoxide
radical anion. Figure 3 Detection of ROS in solution. (a) Increase of the fluorescence
spectra emission of DHR123 upon photoirradiation of COUPY coumarin 1a , Ir(III) complex 2a , Ir(III)-COUPY conjugates 3a , 3b, and 3c or without any compound
(DHR 123 alone) at 505 nm in PBS (0.2% DMSO). DHR123 fluorescence
was excited at 500 nm. (b) EPR spectra of Ir(III)-COUPY conjugates 3a–3c trapped by DMPO or TEMP in MeOH in the dark and
after 5 min of irradiation with green light (530 nm and 450 mW cm –2 ). Further evidence of the generation of O 2 •– was obtained by electron paramagnetic
resonance (EPR) using the
DMPO spin trap. As shown in Figure 3 b, the characteristic paramagnetic signal for the DMPO-O 2 •– adduct (peak integral ratio 1:1:1:1)
was observed upon irradiation with 530 nm LED light, which confirmed
that Ir(III)-COUPY conjugates 3a–3c generate O 2 •– regardless of the modification
introduced in the coumarin scaffold. By contrast, no signal was observed
in the absence of light, thereby indicating again that O 2 •– generation is a light-promoted process.
Moreover, EPR studies with TEMP spin trap demonstrated that none of
the Ir(III)-COUPY conjugates was able to photosensitize singlet oxygen
since no signal for the expected TEMP- 1 O 2 adduct
was observed when the mixture was irradiated ( Figure 3 c), which agrees with the results of spectroscopic
studies in polar protic solvents ( Table S2 ). Cellular Uptake and Localization of Ir(III)-COUPY Conjugates The cellular uptake of the conjugates was first investigated using
inductively coupled-plasma mass spectrometry (ICP–MS). After
2 h incubation, the iridium content inside cancer cells treated with
Ir(III)-COUPY conjugates at 10 μM yielded similar results, with
metal accumulations 4- to 9-fold higher than those found in cisplatin-treated
cells ( Figure 4 a).
Intracellular Ir levels varied from 185 ± 40 to 261 ± 25
ng/10 6 cells for Ir(III)-COUPY-treated cells and were comparable
to those incubated with Ir(III) complex 2a (276 ±
12). Strikingly, the amount of metal in cells treated with conjugate 3c doubled that (461 ± 74 ng Ir per million cells), indicating
that the julolidine-fused system of 3c greatly improved
cellular internalization. Figure 4 Cellular uptake and photocytotoxicity of Ir-COUPY
conjugates in
cancer cells. (a) Intracellular accumulation of Ir(III) compounds
and cisplatin in A2780cis cells after 2 h treatment at 10 μM.
Data expressed as mean ± SD from three independent measurements.
(b) Single confocal planes of HeLa cells incubated with the compounds
for 30 min at 37 °C. Upper row: Merge of bright field and fluorescence
images. Lower row: Fluorescence images of the compounds. Scale bar:
20 μm. (c–d) Summary of in vitro photocytotoxicity for
compounds 1a–1c , 2a, and 3a–3e in A2780cis cells after light irradiation (520 nm, 1.5 mW cm –2 , 1 h) under normoxia (21% O 2 ) or hypoxia
(2% O 2 ) represented as log(IC 50 LIGHT ) and phototherapeutic indexes (PI), defined as the ratio of dark
to light IC 50 values. IC 50 , PI values, and their
corresponding SD errors are listed in the Supporting Information ( Table S3 ). To gain more insights on cellular uptake, the compounds
were incubated
with living cancer cells and examined under a confocal microscopy
using yellow light laser (λ ex = 561 nm) excitation.
As shown in Figure 4 b, fluorescence signals from all of the new conjugates containing
a far-red emitting coumarin (e.g. 1a or 1c ), compounds 3c–3e , were appreciated inside cells,
thereby confirming an excellent cellular uptake. By contrast, the
lack of fluorescent emission prevented the cellular uptake of conjugate 3b to be studied by confocal microscopy. In agreement with
ICP–MS results, intracellular fluorescence was slightly higher
in 3c -stained cells than with other conjugates ( Figure 4 a). Overall, the
Ir(III)-COUPY conjugates 3c–3e showed the same
internalization pattern as the parent conjugate 3a , 44 characterized by fluorescent vesicles in the
cytoplasm, which contrasts with that of the unconjugated COUPY coumarins
that typically accumulate in mitochondria (e.g., compound 1a ). 47 Photobiological Studies Having shown that the Ir(III)-COUPY
conjugates can sensitize both type I and II ROS through spectroscopic
techniques and readily internalize into living cells, their photocytotoxicity
was screened in various cancer cell lines. This screening included
two representative melanoma cell lines (female human A375 and male
human SK-MEL-28) and cancer cells with resistance to the clinical
drug cisplatin. For the latter, both sensitive and cisplatin-resistant
ovarian cancer cells (A2780 and A2780cis) as well as HeLa cells, which
also show a degree of chemoresistance to the platinum anticancer drug
in vitro, 54 were used. For the determination
of dark and light cytotoxicity, dose–response curves were assayed
from both conditions to provide the correspondent IC 50 values,
which correspond to the concentration needed for inhibition of cell
growth by 50%. Phototherapeutic index (PI) was calculated as the ratio
of dark to light IC 50 value for each compound. Dark cytotoxicity Except for compound 1c , all the compounds were found non-toxic in the absence of the light
trigger (IC 50 > 100 μM) regardless of the cell
line
( Tables 1 , S3 and S4 ). From this, it was clear that the
julolidine-fused system of 1c impacted on dark cytotoxicity,
rendering dark IC 50 values that oscillated between 5 and
26 μM in the studied cancer cell lines. Noteworthy, conjugate 3c , which also contains 1c as a coumarin moiety
showed no dark cytotoxicity up to 100 μM. Table 1 Photocytotoxicity of the Compounds
toward Cancer Cells in Normoxia a A375 SK-MEL-28 HeLa A2780 A2780cis dark light dark light dark light dark light dark light 1a >100 1.1 ± 0.1 [>89] >100 1.2 ± 0.1 [>86] >100 5.8 ± 0.4 [>17] >100 5.2 ± 0.5 [>19] >100 2.1 ± 0.2 [>48] 1b >100 10 ± 2 [>10] >100 >100 [n.d.] >100 >100 [n.d.] >100 >100
[n.d] >100 >100 [n.d.] 1c 9.9 ± 0.5 0.31 ± 0.02 [32] 12 ± 1 0.2 ± 0.01 [60] 26 ± 4 1.1 ± 0.8 [24] 5.1 ± 0.9 0.09 ± 0.01 [57] 15 ± 2 0.15 ± 0.04 [100] 2a >100 2.1 ± 0.3 [>48] >100 2.8 ± 0.3 [>36] >100 75 ± 6 [>1.3] >100 4 ± 1 [>25] >100 3.5 ± 0.4 [>29] 3a >100 18 ± 3 [>6] >100 1.5 ± 0.1 [>67] >100 8.6 ± 0.7 [>12] >100 1.07 ± 0.07 [>94] >100 0.70 ± 0.06 [>143] 3b >100 78 ± 6 [>1.3] >100 2.1 ± 0.2 [>48] >100 18 ± 2 [>6] >100 7.1 ± 0.1 [>14] >100 61 ± 8 [>1.6] 3c >100 38 ± 5 [>3] >100 >100 [n.d.] >100 45 ± 4 [>2.2] >100 2.1 ± 0.2 [>48] >100 1.04 ± 0.02 [>96] 3d >100 46 ± 4 [>2.2] >100 2.5 ± 0.2 [>40] >100 2.0 ± 0.4 [>50] >100 1.78 ± 0.07 [>56] >100 1.1 ± 0.2 [>91] 3e >100 19 ± 3 [>5] >100 7.6 ± 0.9 [>13] >100 9.3 ± 0.8 [>11] >100 1.9 ± 0.2 [>53] >100 0.93 ± 0.04 [>108] a Cells were treated for 2 h (1 h
of incubation, 1 h of irradiation with green light at 520 nm, 1.5
mW cm –2 ) followed by 48 h incubation in drug-free
medium. Dark analogues were kept in the dark. Data expressed as mean
± SD from three independent experiments. *n.d. = not determined.
PI (phototherapeutic index) is given in brackets; PI defined as IC 50 (dark-non-irradiated cells)/IC 50 (irradiated
cells). Photocytotoxicity in Normoxia First, photoactivation
of conjugates 3a–3e was evaluated in cancer cells,
along with their coumarin precursors 1a–1c and
the parent Ir(III) complex 2a . Light treatments were
applied at doses of 1.5 mW cm –2 using single green
wavelength (520 nm) LED irradiation. Overall, the compounds triggered
cell death upon light exposure at 520 nm in all cancer cell lines
studied, with IC 50 values in the low micromolar range ( Table 1 ). COUPY 1b , which lacks the N , N -dialkylamino
group at position 7, did not display antiproliferative activity after
light irradiation toward the investigated cancer cells, probably due
to its blue-shifted absorption ( Figure S2 ). In contrast, the original COUPY derivative 1a exerted
higher phototoxic action, particularly against melanoma cells, resulting
in more than 86-fold differences in bioactivity upon irradiation ( Table 1 ). The correspondent
conjugate ( 3b ) showed a slight increase in phototoxic
activity compared to 1b . This might be ascribable to
the Ir(III) core of the PS because the Ir(III) complex 2a also presented reasonable inhibitory activity after irradiation.
Nonetheless, the green-light photocytotoxicity of 3b was
lower than that of 3a in all cancer cell lines (light
IC 50 values between 3 and 78 μM compared to 0.7–18
μM). To test whether the poor phototoxicity of 3b was due to a blue-shifted absorption, photoactivation upon blue
light (465 nm) was also assayed. As shown in Table S4 , A2780cis cells dosed with 3b rendered light
IC 50 values that were very similar to those obtained for 3a after blue light irradiation (2.4 and 1.9 μM, respectively).
On the other hand, replacement of the N , N -dialkylamino group of the coumarin by a julolidine-fused system
decreased the phototoxicity of conjugate 3c in A375,
SK-MEL-28 and HeLa cells compared to 3a . Nevertheless,
the behavior of 3c after green light irradiation was
similar to that of 3a in A2780 and A2780cis cancer cells,
with light IC 50 values between 0.7 and 2.1 μM. Phototherapeutic Index To explore the phototherapeutic
potential of the Ir(III)-COUPY conjugates, we performed a SAR analysis
derived from their phototoxic activity in cancer cells. For the identification
of the best performing anticancer PDT agents, PI determination was
used. Noticeably, PIs differed from one cancer cell line to another.
For instance, A375 melanoma cells, which exhibit the most aggressive
melanoma phenotype, 55 were less sensitive
to Ir(III)-COUPY phototoxicities (PI values not exceeding >6) than
SK-MEL-28 melanoma cells, where PIs reached up to >67 ( Table 1 ). The conjugates
also exerted
mild phototoxic activity in HeLa cancer cells (PIs ranging from 2.2
to >50). The order of potency toward cancer cells generally was 3a > 3c ≈ 3d ≈ 3e > 3b , with small discrepancies in this
trend
depending on the cell line. Nevertheless, the most potent green-light
photoactivation was found in ovarian cancer cells, particularly in
resistant A2780cis cells, where PI values were markedly higher both
for the conjugates containing the parent COUPY dye 1a regardless of the type and length of the spacer, and the one incorporating
the julolidine-fused system (>143 for 3a , >96 for 3c , >91 for 3d, and >108 for 3e ).
Overall, these results led us to initially select 3a and 3c–3e due to their good photocytotoxic profiles, especially
in A2780cis cancer cells. However, since 3c accumulated
in cancer cells to a greater extent than 3a or 3d–3e ( Figure 4 a), we hypothesized that 3c was less efficient
as a PS, as it would require a more intracellular amount of compound
to induce similar phototoxic outcomes. Therefore, 3a was
considered as the best hit Ir(III)-COUPY candidate for green-light
PDT in vitro, followed by 3d and 3e , which
contain the same coumarin and Ir(III) complex moieties. Photocytotoxicity in Hypoxia Since A2780cis cells were
strongly inhibited by Ir(III)-COUPY photosensitizers upon green light
irradiation, this cell line was used for further SARS evaluation.
The phototoxic action of the compounds was assessed toward A2780cis
cells under hypoxic conditions (2% O 2 ) and compared to
the clinically approved PS 5-aminolevulinic acid (5-ALA). In this
second photocytotoxicity testing, dark cytotoxicity was recalculated
using higher concentrations, given that the compounds were deemed
as inactive at 100 μM. Except for 1c , no dark IC 50 values could be determined either in normoxia or hypoxia
up to 250 μM ( Table S3 ), which is
highly desirable for PDT agents. As shown in Table S5 , although none of them inhibited cell growth completely
at 250 μM, some conjugates were relatively toxic at this dose.
For instance, conjugates 3a , 3b, and 3c provided 45–76% of cell killing, while linker-containing
conjugates 3d and 3e barely exhibited dark
cytotoxicity at 250 μM (cell growth inhibition between 12 and
27%). Under hypoxia, poorer PI values were found compared to
those under normoxia, which can be explained in terms of photodynamic
effect restriction by the lack of oxygen ( Figure 4 d and Table S3 ). All the compounds exhibited higher phototherapeutic effects against
A2780cis cells than the protoporphyrin IX (PpIX) precursor 5-ALA ( Table S3 ). The highest PI values in normoxia
were achieved by 3a (>357), followed by the conjugates
containing the longer linkers 3d and 3e (>227
and >269, respectively). However, under hypoxia, the photodynamic
effect of 3a significantly decreased (PI > 66), whereas 3d and 3e yielded comparably higher photocytotoxicity,
with PI values of >131 and >147, respectively. As depicted in Figure 4 d, julolidine-containing
conjugate 3c displayed a similar PI value under normoxia
(>240), but was much less photoactive under hypoxia (PI > 31).
On
the other hand, no obvious photocytotoxicity was observed for 3b upon green light irradiation neither in normoxia nor in
hypoxia (light IC 50 values of 61 and 31 μM, respectively). Photogeneration of ROS Once the photocytotoxicity of
the compounds against cancer cells was demonstrated, the induction
of cellular oxidative stress after irradiation was explored under
both normoxia and hypoxia using the ROS probe 2,7-dichlorodihydrofluorescein
diacetate (DCFH-DA) ( Figure 5 ). In dark conditions, ROS levels from untreated, control
cells remained similar to those treated with COUPY compounds 1a–1c or with iridium(III) complex 2a regardless
of the oxygen tension. Noticeably, conjugates 3a and
its linker-containing derivatives 3d and 3e slightly increased ROS levels under normoxia in the absence of light
( Figure 5 a). Upon 520
nm light application, a strong fluorescence signal from the DCFH-DA
probe was observed in normoxic cells treated with 3a , 3c , 3d, and 3e , indicating efficient
photogeneration of ROS. Although this photogeneration was comparably
lower under hypoxia, differences between dark and light conditions
were still markedly significant under low oxygen environment. Strikingly,
although poor phototoxicity was achieved for 3b in cellular
assays under hypoxia ( Figure 4 d), a slight increase in ROS levels was found after green-light
irradiation in A2780cis cells, these differences being significantly
much larger for the other conjugates ( Figure 5 b). Figure 5 Photoinduced cell death mechanism of Ir-COUPY
conjugates. (a) Relative
ROS levels in A2780cis cells under normoxia (21% O 2 ) and
hypoxia (2% O 2 ) after treatment with the compounds at 10
μM in dark or upon light irradiation (520 nm, 1.5 mW cm –2 , 1 h) as measured with the DCFH-DA probe. Menadione
(Men, 50 μM) was used as a positive control for ROS induction.
Data expressed as mean ± SD from three independent measurements.
(b) Cell death induction in A2780cis after PDT treatment with Ir(III)-COUPY
agents. Data from three independent flow cytometry experiments using
Annexin V/PI dual staining. (c) Change in the relative volume of A2780cis
MCTS over 7 days. PDT treatments were applied on day 3 (dashed line).
Error bars correspond to SD from three replicates. Statistical significance
* p < 0.05, ** p < 0.01, *** p < 0.001 from two-way ANOVA test (a,b) or one-way ANOVA
test (c). Photoinduced Cell Death Mechanism The photogeneration
of ROS by the Ir(III)-COUPY conjugates prompted us to study the cell
death mechanism of these PSs. After 520 nm light irradiation, the
compounds increased the number of detached, non-viable cells as well
as rounded, floating cells. To characterize these morphological changes,
flow cytometry analysis of cell size and cell complexity was performed
using forward scatter and side scatter (FSC vs SSC) plots. Irradiated,
non-treated A2780cis cells grouped into a defined morphological cell
population. However, PDT treatment with Ir(III)-COUPY conjugates induced
a second large population of cell particles with low FSC/SCC ratios
( Figure S4 ). Although 3b did
not produce such changes, 3a , 3c , 3d, and 3e vastly induced these secondary cell
populations that correspond to dead cells with non-viable morphology
( Figure S4 ). To discern the mode of cell
death induced, the Annexin V/Propidium Iodide (AV/PI) labeling method
was used. These assays can detect the translocation of specific phospholipids
during apoptosis in AV + /PI – and AV + /PI + regions and loss of cell membrane integrity
from necrosis in AV – /PI + cells. As depicted
in Figures 5 b and S5 , 3a , 3c , 3d, and 3e photoinduced necrotic cell death, as revealed
by the increase of cells AV – /PI + due
to nuclear staining of PI. 3D Tumor Growth Inhibition Effects To better characterize
the photoinduced anticancer activity of the Ir-COUPY conjugates under
hypoxic conditions, their photocytotoxicity was assayed against 3D
multicellular tumor spheroid (MCTS) culture systems. Cancer cells
growing in MCTS cultures display heterogeneous regions, nutrient and
oxygen gradients, as well as intercellular and cell-extracellular
matrix interactions that mimic the tumor microenvironment. 56 , 57 In addition, 3D MCTS represents a more stringent model for drug
screening since they allow for assay drug penetration, resistance,
and importantly drug response to hypoxic gradients toward the center
of the sphere, which are particularly interesting aspects in the development
of novel PDT agents. 58 , 59 Defined A2780cis MCTS were
uniformly generated with an average radius of ∼350 μm
and an average volume of ∼0.181 mm 3 ( Figure S6 ). These volume values were used to
normalize MCTS growth. After 3 days, MCTS were incubated with Ir(III)-COUPY
conjugates for 4 h and exposed to 1 h of 520 nm light irradiation.
In coherence with what we observed in 2D cellular models under hypoxia,
PDT treatments with conjugates 3a , 3d, and 3e effectively blocked tumoral growth of the A2780cis spheres,
with 3a and 3e producing statistically significant
shrank compared to irradiated controls ( Figures 5 c and S7 ). In
contrast, conjugates 3b and 3c did not show
significant tumor inhibition effects under the same experimental conditions.
## Synthesis and Characterization of Ir(III)-COUPY Conjugates
Synthesis and Characterization of Ir(III)-COUPY Conjugates Synthesis of Ir(III)-COUPY Conjugate 3b As shown in Scheme 1 , a convergent approach was used for the synthesis of Ir(III)-COUPY
conjugate 3b, in which the carboxylic group of the Ir(III)
complex 2b ( Figure 1 ) was linked to the amino group of coumarin 10 through the formation of an amide bond. As previously indicated,
the synthesis of a COUPY derivative lacking the dialkylamino group
at the 7-position of the coumarin skeleton was proposed to explore
the contribution of this EDG to ROS generation when conjugated to
the metal complex. Scheme 1 Synthesis of Ir(III)-COUPY Conjugate 3b and of Control
COUPY Coumarin 1b The synthesis route for COUPY derivative 10 ( Scheme 1 ) involved seven
steps starting with a Pechmann condensation between phenol and ethyl
acetoacetate that afforded the desired coumarin skeleton ( 4 ). 52 Next, coumarin 4 was
reacted with Lawesson’s reagent (LW) to provide thiocoumarin 5 , 53 which was condensed with 4-pyridylacetonitrile
to give compound 6 . Coumarin ester 7 was
obtained with good yield by alkylation of 6 with methyl
bromoacetate. After acid hydrolysis, the carboxyl group of coumarin 8 was reacted with N -Boc-1,3-propanediamine
hydrochloride with the assistance of HATU coupling reagent and DIPEA
to yield coumarin 9 , whose Boc-protecting group was removed
under acidic conditions to give coumarin derivative 10 . Finally, Ir(III)-COUPY conjugate 3b was obtained as
a dark yellow solid after attachment of the Ir(III) complex 2b to the fluorophore via the formation of an amide bond.
All of the compounds depicted in Scheme 1 were purified by silica column chromatography
and fully characterized by high-resolution mass spectrometry (HRMS)
and 1 H and 13 C NMR spectroscopy. Synthesis of Ir(III)-COUPY Conjugate 3c For the synthesis of Ir(III)-COUPY conjugate 3c ( Scheme 2 ), we selected a
julolidine-fused coumarin analogue to red-shift the absorption maximum
with respect to the parent 7-dialkylaminocoumarin. Moreover, as previously
stated, rigidification of the amino group was anticipated to have
an impact on the photophysical and photochemical properties of the
compounds since rotation around the N–C bond is not possible
because of the fusion of the six-membered alkyl rings to the aromatic
ring. The required amino-containing COUPY derivative 16 was synthesized from a commercially available coumarin precursor
following the same procedure as for COUPY 10 . The conjugation
between 16 and the Ir(III) complex 2b afforded
Ir(III)-COUPY conjugate 3c as a dark blue solid after
silica column purification. All the compounds were characterized by 1 H and 13 C NMR spectroscopy and HRMS. Scheme 2 Synthesis
of Ir(III)-COUPY Conjugate 3c and Control
COUPY Coumarin 1c Synthesis of Ir(III)-COUPY Conjugates 3d and 3e With the aim of investigating the effect of the
distance between the COUPY fluorophore and the Ir(III) complex in
the parent Ir(III)-COUPY conjugate 3a on the photophysical
and photochemical properties of the compounds, we designed two analogues
incorporating a longer spacer between both moieties. As shown in Scheme 3 , we selected flexible
( 3d ) and rigid ( 3e ) spacers with the same
number of atoms separating the two moieties. The incorporation of
both linkers was carried out through the formation of an amide bond
between the carboxylic group of each Boc-protected precursor and the
free amino group of coumarin 17 , 37 which afforded COUPY derivatives 18 and 19 containing the flexible and rigid spacers, respectively. Finally,
after acidic Boc removal, the conjugation of coumarins 20 and 21 to the Ir(III) complex 2b provided
Ir(III)-COUPY conjugates 3d and 3e , respectively,
as dark blue solids after silica column purification, which were characterized
by 1 H and 13 C NMR spectroscopy and HRMS. Scheme 3 Synthesis of Ir(III)-COUPY Conjugates 3d and 3e Synthesis of Control COUPY Coumarin 1b and 1c For the synthesis of the corresponding N-methylated
COUPY dyes 1b and 1c to be used as control
compounds, coumarin 6 ( Scheme 1 ) or coumarin 12 ( Scheme 2 ) were reacted with methyl
trifluoromethanesulfonate in DCM at room temperature, yielding the
expected compounds as yellow and dark blue solids, respectively.
## Synthesis of Ir(III)-COUPY Conjugate
Synthesis of Ir(III)-COUPY Conjugate 3b As shown in Scheme 1 , a convergent approach was used for the synthesis of Ir(III)-COUPY
conjugate 3b, in which the carboxylic group of the Ir(III)
complex 2b ( Figure 1 ) was linked to the amino group of coumarin 10 through the formation of an amide bond. As previously indicated,
the synthesis of a COUPY derivative lacking the dialkylamino group
at the 7-position of the coumarin skeleton was proposed to explore
the contribution of this EDG to ROS generation when conjugated to
the metal complex. Scheme 1 Synthesis of Ir(III)-COUPY Conjugate 3b and of Control
COUPY Coumarin 1b The synthesis route for COUPY derivative 10 ( Scheme 1 ) involved seven
steps starting with a Pechmann condensation between phenol and ethyl
acetoacetate that afforded the desired coumarin skeleton ( 4 ). 52 Next, coumarin 4 was
reacted with Lawesson’s reagent (LW) to provide thiocoumarin 5 , 53 which was condensed with 4-pyridylacetonitrile
to give compound 6 . Coumarin ester 7 was
obtained with good yield by alkylation of 6 with methyl
bromoacetate. After acid hydrolysis, the carboxyl group of coumarin 8 was reacted with N -Boc-1,3-propanediamine
hydrochloride with the assistance of HATU coupling reagent and DIPEA
to yield coumarin 9 , whose Boc-protecting group was removed
under acidic conditions to give coumarin derivative 10 . Finally, Ir(III)-COUPY conjugate 3b was obtained as
a dark yellow solid after attachment of the Ir(III) complex 2b to the fluorophore via the formation of an amide bond.
All of the compounds depicted in Scheme 1 were purified by silica column chromatography
and fully characterized by high-resolution mass spectrometry (HRMS)
and 1 H and 13 C NMR spectroscopy.
## Synthesis of Ir(III)-COUPY Conjugate
Synthesis of Ir(III)-COUPY Conjugate 3c For the synthesis of Ir(III)-COUPY conjugate 3c ( Scheme 2 ), we selected a
julolidine-fused coumarin analogue to red-shift the absorption maximum
with respect to the parent 7-dialkylaminocoumarin. Moreover, as previously
stated, rigidification of the amino group was anticipated to have
an impact on the photophysical and photochemical properties of the
compounds since rotation around the N–C bond is not possible
because of the fusion of the six-membered alkyl rings to the aromatic
ring. The required amino-containing COUPY derivative 16 was synthesized from a commercially available coumarin precursor
following the same procedure as for COUPY 10 . The conjugation
between 16 and the Ir(III) complex 2b afforded
Ir(III)-COUPY conjugate 3c as a dark blue solid after
silica column purification. All the compounds were characterized by 1 H and 13 C NMR spectroscopy and HRMS. Scheme 2 Synthesis
of Ir(III)-COUPY Conjugate 3c and Control
COUPY Coumarin 1c
## Synthesis of Ir(III)-COUPY Conjugates
Synthesis of Ir(III)-COUPY Conjugates 3d and 3e With the aim of investigating the effect of the
distance between the COUPY fluorophore and the Ir(III) complex in
the parent Ir(III)-COUPY conjugate 3a on the photophysical
and photochemical properties of the compounds, we designed two analogues
incorporating a longer spacer between both moieties. As shown in Scheme 3 , we selected flexible
( 3d ) and rigid ( 3e ) spacers with the same
number of atoms separating the two moieties. The incorporation of
both linkers was carried out through the formation of an amide bond
between the carboxylic group of each Boc-protected precursor and the
free amino group of coumarin 17 , 37 which afforded COUPY derivatives 18 and 19 containing the flexible and rigid spacers, respectively. Finally,
after acidic Boc removal, the conjugation of coumarins 20 and 21 to the Ir(III) complex 2b provided
Ir(III)-COUPY conjugates 3d and 3e , respectively,
as dark blue solids after silica column purification, which were characterized
by 1 H and 13 C NMR spectroscopy and HRMS. Scheme 3 Synthesis of Ir(III)-COUPY Conjugates 3d and 3e
## Synthesis of Control COUPY Coumarin
Synthesis of Control COUPY Coumarin 1b and 1c For the synthesis of the corresponding N-methylated
COUPY dyes 1b and 1c to be used as control
compounds, coumarin 6 ( Scheme 1 ) or coumarin 12 ( Scheme 2 ) were reacted with methyl
trifluoromethanesulfonate in DCM at room temperature, yielding the
expected compounds as yellow and dark blue solids, respectively.
## Photophysical and Photochemical Characterization of the Compounds
Photophysical and Photochemical Characterization of the Compounds The photophysical and photochemical properties [absorption and
emission spectra, molar absorption coefficients (ε), fluorescence
(Φ F ) or phosphorescence (Φ P ) quantum
yields, fluorescence (τ F ) or phosphorescent (τ P ) lifetimes, and singlet oxygen quantum yield (Φ Δ )] of the four new Ir(III)-COUPY conjugates ( 3b–3e ) along with the two new coumarins ( 1b–1c ) were
studied in three solvents of different polarities (phosphate-buffered
saline PBS, ACN and DCM), and compared with those of the previously-reported
parent compounds (COUPY dye 1a , Ir(III) complex 2a and Ir(III)-COUPY conjugate 3a ). 44 The UV–vis absorption and emission spectra
are shown in Figures 2 and S1 and S2 , and their photophysical
properties are summarized in Tables S1 and S2 . Figure 2 Comparison of the molar absorptivity (a) and emission spectra (b)
of Ir(III)-COUPY conjugates in ACN. As shown in Table S1 , the structural
modifications carried out within the coumarin scaffold had a strong
impact on the photophysical properties of the compounds. Indeed, the
newly synthesized COUPY dyes showed either a bathochromic shift (about
20–30 nm for coumarin 1c ) or a hypsochromic shift
(about 100–120 nm for coumarin 1b ) in their absorption
and emission maxima compared with those of the parent coumarin 1a (e.g. in ACN, λ abs = 548 nm and λ em = 609 nm for 1a vs λ abs =
446 nm and λ em = 492 nm for 1b , and
λ abs = 572 nm and λ em = 635 nm for 1c ). The replacement of the N , N -dialkylamino group at position 7 of the coumarin skeleton by the
strong electron-donating julolidine-fused analog in coumarin 1c caused the observed red-shifting in the wavelength at the
absorption and emission maxima. On the contrary, the absence of an
EDG at position 7 in coumarin 1b partnering with the
electron-withdrawing cyanomethylenepyridinium moiety had a negative
effect on the spectroscopic properties of the fluorophore since both
absorption and emission maxima were strongly blue-shifted due to the
decreased push–pull character of the chromophore. On the other
hand, the fluorescent quantum yields were slightly lower for coumarin 1c in DCM and PBS, compared with the original COUPY 1a , whereas coumarin 1b was found to be very
weakly fluorescent. For coumarins 1a and 1c , a strong decrease in the fluorescence quantum yield and lifetime
was observed in the more polar solvents, indicating the onset of efficient
intramolecular electron-transfer excited-state deactivation pathways.
Consistent with this, coumarin 1b , lacking the N , N -dialkylamino EDG, showed a less pronounced
solvent dependence. As we had already reported, 44 the Ir(III)
complex 2a showed a strong and long-lived phosphorescence
band around 660 nm, whose intensity and lifetime decreased in the
Ir(III)-COUPY conjugate 3a , suggesting the existence
of competitive excited-state processes. As shown in Table S2 , a similar behavior was observed in the three new
conjugates incorporating a far-red emitting coumarin ( 3c–3e ). Among them, conjugate 3c containing the julolidine-fused
coumarin showed the largest red-shift in the absorption and emission
maxima (∼25–30 nm depending on the solvent), which parallels
the spectroscopic properties of COUPY 1c ( 3c , λ abs = 580 nm and λ em = 647 nm
in ACN). Interestingly, the luminescent quantum yield of 3c was higher than that of the parent conjugate 3a in
DCM, but similar values were obtained in ACN and PBS. As expected,
conjugates 3d and 3e containing the flexible
and the rigid spacer, respectively, showed similar absorption and
emission maxima in all investigated solvents compared to the original
conjugate 3a since they contain the same coumarin (about
λ abs = 555 nm and λ em = 610 nm in
ACN). The luminescence quantum yield values were also similar for
the three conjugates, about 0.1 in DCM and ACN, and below 0.02 in
PBS. The strong decrease in the luminescence quantum yields and lifetimes
in PBS is consistent with the onset of efficient competing intra-
and inter-chromophore electron-transfer processes. In good agreement
with the behavior of COUPY dye 1b , the Ir(III)-COUPY
conjugate 3b practically did not show any fluorescence
in all of the solvents investigated and, as shown in Table S2 , only phosphorescence lifetime values associated
with the Ir(III) complex could be determined and these were shorter
than for 3a . Finally, it is worth noting that the maximum
absorption wavelengths of all the compounds (COUPY dyes and Ir(III)-COUPY
conjugates) in different solvents gradually decrease according to
the following order DCM > ACN > PBS, which agrees with a negative
solvatochromism phenomenon ( Tables S1 and S2 ). Next, we focused on investigating the impact of the structural
modifications on the generation of ROS by Ir(III)-COUPY conjugates.
As we previously reported, 44 the Ir(III)
complex 2a produced singlet oxygen in all of the organic
solvents evaluated but not in PBS as a result of a very efficient
formation of the triplet state due to the heavy-atom effect, while
COUPY 1a did not show significant 1 O 2 quantum yields in any solvent ( Table S1 ). However, the conjugation of Ir(III) complex 2a to
the COUPY fluorophore 1a led to an increase of 1 O 2 quantum yield by one order of magnitude, suggesting
a higher population of COUPY triplet excited states (T 1 ) due to either an enhanced intersystem crossing (ISC) process in
the COUPY moiety of conjugate 3a when directly excited
at 532 nm (as demonstrated by the shorter fluorescence lifetime),
or an efficient triplet–triplet energy transfer process from
the Ir(III) complex to COUPY when the complex is excited at 355 nm
(as demonstrated by its shorter phosphorescence lifetime). In either
case, since the triplet lifetime of COUPY is longer than that of the
Ir(III) complex, localization of the triplet excited-state energy
in the COUPY moiety provides more time for energy transfer to 3 O 2 , thereby favoring the production of 1 O 2 ( Table S2 ). The ability
of all newly synthesized Ir(III)-COUPY conjugates ( 3b–3e ) and control COUPY fluorophores ( 1b–1c ) to produce 1 O 2 was evaluated spectroscopically by the observation
of 1 O 2 emission at 1275 nm upon excitation at
two wavelengths (355 and 532 nm). In the same way as for the original
COUPY derivative 1a , neither 1b nor 1c generated significant amounts of 1 O 2 at either excitation wavelength, indicating that radiative decay
competes efficiently with intersystem crossing to the triplet excited
state, the precursor of singlet oxygen. Interestingly, 1 O 2 emission was observed in all new Ir(III)-COUPY conjugates
in DCM and ACN irrespective of which chromophore was initially excited,
but not in PBS, consistent with the observed decrease in luminescence
quantum yields and lifetimes as a result of efficient electron-transfer
competing processes. Conjugates 3d and 3e containing the same coumarin derivative as the parent compound ( 3a ) showed similar singlet oxygen quantum yields (ca. 0.30–0.40
in DCM upon excitation at both wavelengths) regardless of the spacer
linking both moieties, while the conjugates with new coumarin derivatives, 3b and 3c , exhibited lower values (ca. 0.20 in
DCM). Considering that one of the main features of the parent
Ir(III)-COUPY
conjugate 3a is the generation of superoxide anion radical
(O 2 •– ) in living cells upon irradiation
with visible light, 44 we next investigated
the ability of the new conjugates to produce this specific type-I
ROS in PBS by using a spectroscopic method based on the oxidation
of the non-fluorescent dihydrorhodamine 123 (DHR123) probe by O 2 •– to the corresponding fluorescent
rhodamine 123 derivative. As shown in Figures 3 and S3 , Ir(III)-COUPY
conjugates did not produce any measurable quantity of superoxide anion
radical before irradiation; this result is comparable to that obtained
with DHR123 alone after irradiation, which was used as a negative
control. Surprisingly, under green light irradiation (505 nm), all
of the new conjugates, including the one containing the coumarin fluorophore
lacking the amino group at the 7-position, clearly increased the fluorescence
intensity of DHR123 to a greater extent than the Ir(III) complex 2a and COUPY derivatives 1a–1c , indicating
the generation of superoxide anion radical, consistent with the observed
excited-state electron-transfer processes. Remarkably, all the new
conjugates led to a faster O 2 •– generation rate in comparison to the parent compound 3a . The Ir(III)-COUPY conjugate containing the julolidine-fused system
( 3c ) showed the fastest rates in superoxide generation,
thereby demonstrating the importance of incorporating a strong EDG
in the coumarin scaffold, which not only red-shifts absorption and
emission maxima but also produces more O 2 •– . Interestingly, the Ir(III)-COUPY conjugate 3b reached
the highest maximum emission intensity of superoxide generation after
3 min of irradiation, indicating that the amino group at the 7-position
of the coumarin moiety is not strictly necessary to trigger the generation
of O 2 •– . Overall, the suppression
of singlet oxygen generation in PBS, and the reduction in fluorescence
and phosphorescence lifetimes and quantum yields of Ir(III)-COUPY
conjugates, are consistent with the onset of intra- and inter-chromophore
electron-transfer processes, resulting in the generation of superoxide
radical anion. Figure 3 Detection of ROS in solution. (a) Increase of the fluorescence
spectra emission of DHR123 upon photoirradiation of COUPY coumarin 1a , Ir(III) complex 2a , Ir(III)-COUPY conjugates 3a , 3b, and 3c or without any compound
(DHR 123 alone) at 505 nm in PBS (0.2% DMSO). DHR123 fluorescence
was excited at 500 nm. (b) EPR spectra of Ir(III)-COUPY conjugates 3a–3c trapped by DMPO or TEMP in MeOH in the dark and
after 5 min of irradiation with green light (530 nm and 450 mW cm –2 ). Further evidence of the generation of O 2 •– was obtained by electron paramagnetic
resonance (EPR) using the
DMPO spin trap. As shown in Figure 3 b, the characteristic paramagnetic signal for the DMPO-O 2 •– adduct (peak integral ratio 1:1:1:1)
was observed upon irradiation with 530 nm LED light, which confirmed
that Ir(III)-COUPY conjugates 3a–3c generate O 2 •– regardless of the modification
introduced in the coumarin scaffold. By contrast, no signal was observed
in the absence of light, thereby indicating again that O 2 •– generation is a light-promoted process.
Moreover, EPR studies with TEMP spin trap demonstrated that none of
the Ir(III)-COUPY conjugates was able to photosensitize singlet oxygen
since no signal for the expected TEMP- 1 O 2 adduct
was observed when the mixture was irradiated ( Figure 3 c), which agrees with the results of spectroscopic
studies in polar protic solvents ( Table S2 ).
## Cellular Uptake and Localization of Ir(III)-COUPY Conjugates
Cellular Uptake and Localization of Ir(III)-COUPY Conjugates The cellular uptake of the conjugates was first investigated using
inductively coupled-plasma mass spectrometry (ICP–MS). After
2 h incubation, the iridium content inside cancer cells treated with
Ir(III)-COUPY conjugates at 10 μM yielded similar results, with
metal accumulations 4- to 9-fold higher than those found in cisplatin-treated
cells ( Figure 4 a).
Intracellular Ir levels varied from 185 ± 40 to 261 ± 25
ng/10 6 cells for Ir(III)-COUPY-treated cells and were comparable
to those incubated with Ir(III) complex 2a (276 ±
12). Strikingly, the amount of metal in cells treated with conjugate 3c doubled that (461 ± 74 ng Ir per million cells), indicating
that the julolidine-fused system of 3c greatly improved
cellular internalization. Figure 4 Cellular uptake and photocytotoxicity of Ir-COUPY
conjugates in
cancer cells. (a) Intracellular accumulation of Ir(III) compounds
and cisplatin in A2780cis cells after 2 h treatment at 10 μM.
Data expressed as mean ± SD from three independent measurements.
(b) Single confocal planes of HeLa cells incubated with the compounds
for 30 min at 37 °C. Upper row: Merge of bright field and fluorescence
images. Lower row: Fluorescence images of the compounds. Scale bar:
20 μm. (c–d) Summary of in vitro photocytotoxicity for
compounds 1a–1c , 2a, and 3a–3e in A2780cis cells after light irradiation (520 nm, 1.5 mW cm –2 , 1 h) under normoxia (21% O 2 ) or hypoxia
(2% O 2 ) represented as log(IC 50 LIGHT ) and phototherapeutic indexes (PI), defined as the ratio of dark
to light IC 50 values. IC 50 , PI values, and their
corresponding SD errors are listed in the Supporting Information ( Table S3 ). To gain more insights on cellular uptake, the compounds
were incubated
with living cancer cells and examined under a confocal microscopy
using yellow light laser (λ ex = 561 nm) excitation.
As shown in Figure 4 b, fluorescence signals from all of the new conjugates containing
a far-red emitting coumarin (e.g. 1a or 1c ), compounds 3c–3e , were appreciated inside cells,
thereby confirming an excellent cellular uptake. By contrast, the
lack of fluorescent emission prevented the cellular uptake of conjugate 3b to be studied by confocal microscopy. In agreement with
ICP–MS results, intracellular fluorescence was slightly higher
in 3c -stained cells than with other conjugates ( Figure 4 a). Overall, the
Ir(III)-COUPY conjugates 3c–3e showed the same
internalization pattern as the parent conjugate 3a , 44 characterized by fluorescent vesicles in the
cytoplasm, which contrasts with that of the unconjugated COUPY coumarins
that typically accumulate in mitochondria (e.g., compound 1a ). 47
## Photobiological Studies
Photobiological Studies Having shown that the Ir(III)-COUPY
conjugates can sensitize both type I and II ROS through spectroscopic
techniques and readily internalize into living cells, their photocytotoxicity
was screened in various cancer cell lines. This screening included
two representative melanoma cell lines (female human A375 and male
human SK-MEL-28) and cancer cells with resistance to the clinical
drug cisplatin. For the latter, both sensitive and cisplatin-resistant
ovarian cancer cells (A2780 and A2780cis) as well as HeLa cells, which
also show a degree of chemoresistance to the platinum anticancer drug
in vitro, 54 were used. For the determination
of dark and light cytotoxicity, dose–response curves were assayed
from both conditions to provide the correspondent IC 50 values,
which correspond to the concentration needed for inhibition of cell
growth by 50%. Phototherapeutic index (PI) was calculated as the ratio
of dark to light IC 50 value for each compound. Dark cytotoxicity Except for compound 1c , all the compounds were found non-toxic in the absence of the light
trigger (IC 50 > 100 μM) regardless of the cell
line
( Tables 1 , S3 and S4 ). From this, it was clear that the
julolidine-fused system of 1c impacted on dark cytotoxicity,
rendering dark IC 50 values that oscillated between 5 and
26 μM in the studied cancer cell lines. Noteworthy, conjugate 3c , which also contains 1c as a coumarin moiety
showed no dark cytotoxicity up to 100 μM. Table 1 Photocytotoxicity of the Compounds
toward Cancer Cells in Normoxia a A375 SK-MEL-28 HeLa A2780 A2780cis dark light dark light dark light dark light dark light 1a >100 1.1 ± 0.1 [>89] >100 1.2 ± 0.1 [>86] >100 5.8 ± 0.4 [>17] >100 5.2 ± 0.5 [>19] >100 2.1 ± 0.2 [>48] 1b >100 10 ± 2 [>10] >100 >100 [n.d.] >100 >100 [n.d.] >100 >100
[n.d] >100 >100 [n.d.] 1c 9.9 ± 0.5 0.31 ± 0.02 [32] 12 ± 1 0.2 ± 0.01 [60] 26 ± 4 1.1 ± 0.8 [24] 5.1 ± 0.9 0.09 ± 0.01 [57] 15 ± 2 0.15 ± 0.04 [100] 2a >100 2.1 ± 0.3 [>48] >100 2.8 ± 0.3 [>36] >100 75 ± 6 [>1.3] >100 4 ± 1 [>25] >100 3.5 ± 0.4 [>29] 3a >100 18 ± 3 [>6] >100 1.5 ± 0.1 [>67] >100 8.6 ± 0.7 [>12] >100 1.07 ± 0.07 [>94] >100 0.70 ± 0.06 [>143] 3b >100 78 ± 6 [>1.3] >100 2.1 ± 0.2 [>48] >100 18 ± 2 [>6] >100 7.1 ± 0.1 [>14] >100 61 ± 8 [>1.6] 3c >100 38 ± 5 [>3] >100 >100 [n.d.] >100 45 ± 4 [>2.2] >100 2.1 ± 0.2 [>48] >100 1.04 ± 0.02 [>96] 3d >100 46 ± 4 [>2.2] >100 2.5 ± 0.2 [>40] >100 2.0 ± 0.4 [>50] >100 1.78 ± 0.07 [>56] >100 1.1 ± 0.2 [>91] 3e >100 19 ± 3 [>5] >100 7.6 ± 0.9 [>13] >100 9.3 ± 0.8 [>11] >100 1.9 ± 0.2 [>53] >100 0.93 ± 0.04 [>108] a Cells were treated for 2 h (1 h
of incubation, 1 h of irradiation with green light at 520 nm, 1.5
mW cm –2 ) followed by 48 h incubation in drug-free
medium. Dark analogues were kept in the dark. Data expressed as mean
± SD from three independent experiments. *n.d. = not determined.
PI (phototherapeutic index) is given in brackets; PI defined as IC 50 (dark-non-irradiated cells)/IC 50 (irradiated
cells). Photocytotoxicity in Normoxia First, photoactivation
of conjugates 3a–3e was evaluated in cancer cells,
along with their coumarin precursors 1a–1c and
the parent Ir(III) complex 2a . Light treatments were
applied at doses of 1.5 mW cm –2 using single green
wavelength (520 nm) LED irradiation. Overall, the compounds triggered
cell death upon light exposure at 520 nm in all cancer cell lines
studied, with IC 50 values in the low micromolar range ( Table 1 ). COUPY 1b , which lacks the N , N -dialkylamino
group at position 7, did not display antiproliferative activity after
light irradiation toward the investigated cancer cells, probably due
to its blue-shifted absorption ( Figure S2 ). In contrast, the original COUPY derivative 1a exerted
higher phototoxic action, particularly against melanoma cells, resulting
in more than 86-fold differences in bioactivity upon irradiation ( Table 1 ). The correspondent
conjugate ( 3b ) showed a slight increase in phototoxic
activity compared to 1b . This might be ascribable to
the Ir(III) core of the PS because the Ir(III) complex 2a also presented reasonable inhibitory activity after irradiation.
Nonetheless, the green-light photocytotoxicity of 3b was
lower than that of 3a in all cancer cell lines (light
IC 50 values between 3 and 78 μM compared to 0.7–18
μM). To test whether the poor phototoxicity of 3b was due to a blue-shifted absorption, photoactivation upon blue
light (465 nm) was also assayed. As shown in Table S4 , A2780cis cells dosed with 3b rendered light
IC 50 values that were very similar to those obtained for 3a after blue light irradiation (2.4 and 1.9 μM, respectively).
On the other hand, replacement of the N , N -dialkylamino group of the coumarin by a julolidine-fused system
decreased the phototoxicity of conjugate 3c in A375,
SK-MEL-28 and HeLa cells compared to 3a . Nevertheless,
the behavior of 3c after green light irradiation was
similar to that of 3a in A2780 and A2780cis cancer cells,
with light IC 50 values between 0.7 and 2.1 μM. Phototherapeutic Index To explore the phototherapeutic
potential of the Ir(III)-COUPY conjugates, we performed a SAR analysis
derived from their phototoxic activity in cancer cells. For the identification
of the best performing anticancer PDT agents, PI determination was
used. Noticeably, PIs differed from one cancer cell line to another.
For instance, A375 melanoma cells, which exhibit the most aggressive
melanoma phenotype, 55 were less sensitive
to Ir(III)-COUPY phototoxicities (PI values not exceeding >6) than
SK-MEL-28 melanoma cells, where PIs reached up to >67 ( Table 1 ). The conjugates
also exerted
mild phototoxic activity in HeLa cancer cells (PIs ranging from 2.2
to >50). The order of potency toward cancer cells generally was 3a > 3c ≈ 3d ≈ 3e > 3b , with small discrepancies in this
trend
depending on the cell line. Nevertheless, the most potent green-light
photoactivation was found in ovarian cancer cells, particularly in
resistant A2780cis cells, where PI values were markedly higher both
for the conjugates containing the parent COUPY dye 1a regardless of the type and length of the spacer, and the one incorporating
the julolidine-fused system (>143 for 3a , >96 for 3c , >91 for 3d, and >108 for 3e ).
Overall, these results led us to initially select 3a and 3c–3e due to their good photocytotoxic profiles, especially
in A2780cis cancer cells. However, since 3c accumulated
in cancer cells to a greater extent than 3a or 3d–3e ( Figure 4 a), we hypothesized that 3c was less efficient
as a PS, as it would require a more intracellular amount of compound
to induce similar phototoxic outcomes. Therefore, 3a was
considered as the best hit Ir(III)-COUPY candidate for green-light
PDT in vitro, followed by 3d and 3e , which
contain the same coumarin and Ir(III) complex moieties. Photocytotoxicity in Hypoxia Since A2780cis cells were
strongly inhibited by Ir(III)-COUPY photosensitizers upon green light
irradiation, this cell line was used for further SARS evaluation.
The phototoxic action of the compounds was assessed toward A2780cis
cells under hypoxic conditions (2% O 2 ) and compared to
the clinically approved PS 5-aminolevulinic acid (5-ALA). In this
second photocytotoxicity testing, dark cytotoxicity was recalculated
using higher concentrations, given that the compounds were deemed
as inactive at 100 μM. Except for 1c , no dark IC 50 values could be determined either in normoxia or hypoxia
up to 250 μM ( Table S3 ), which is
highly desirable for PDT agents. As shown in Table S5 , although none of them inhibited cell growth completely
at 250 μM, some conjugates were relatively toxic at this dose.
For instance, conjugates 3a , 3b, and 3c provided 45–76% of cell killing, while linker-containing
conjugates 3d and 3e barely exhibited dark
cytotoxicity at 250 μM (cell growth inhibition between 12 and
27%). Under hypoxia, poorer PI values were found compared to
those under normoxia, which can be explained in terms of photodynamic
effect restriction by the lack of oxygen ( Figure 4 d and Table S3 ). All the compounds exhibited higher phototherapeutic effects against
A2780cis cells than the protoporphyrin IX (PpIX) precursor 5-ALA ( Table S3 ). The highest PI values in normoxia
were achieved by 3a (>357), followed by the conjugates
containing the longer linkers 3d and 3e (>227
and >269, respectively). However, under hypoxia, the photodynamic
effect of 3a significantly decreased (PI > 66), whereas 3d and 3e yielded comparably higher photocytotoxicity,
with PI values of >131 and >147, respectively. As depicted in Figure 4 d, julolidine-containing
conjugate 3c displayed a similar PI value under normoxia
(>240), but was much less photoactive under hypoxia (PI > 31).
On
the other hand, no obvious photocytotoxicity was observed for 3b upon green light irradiation neither in normoxia nor in
hypoxia (light IC 50 values of 61 and 31 μM, respectively). Photogeneration of ROS Once the photocytotoxicity of
the compounds against cancer cells was demonstrated, the induction
of cellular oxidative stress after irradiation was explored under
both normoxia and hypoxia using the ROS probe 2,7-dichlorodihydrofluorescein
diacetate (DCFH-DA) ( Figure 5 ). In dark conditions, ROS levels from untreated, control
cells remained similar to those treated with COUPY compounds 1a–1c or with iridium(III) complex 2a regardless
of the oxygen tension. Noticeably, conjugates 3a and
its linker-containing derivatives 3d and 3e slightly increased ROS levels under normoxia in the absence of light
( Figure 5 a). Upon 520
nm light application, a strong fluorescence signal from the DCFH-DA
probe was observed in normoxic cells treated with 3a , 3c , 3d, and 3e , indicating efficient
photogeneration of ROS. Although this photogeneration was comparably
lower under hypoxia, differences between dark and light conditions
were still markedly significant under low oxygen environment. Strikingly,
although poor phototoxicity was achieved for 3b in cellular
assays under hypoxia ( Figure 4 d), a slight increase in ROS levels was found after green-light
irradiation in A2780cis cells, these differences being significantly
much larger for the other conjugates ( Figure 5 b). Figure 5 Photoinduced cell death mechanism of Ir-COUPY
conjugates. (a) Relative
ROS levels in A2780cis cells under normoxia (21% O 2 ) and
hypoxia (2% O 2 ) after treatment with the compounds at 10
μM in dark or upon light irradiation (520 nm, 1.5 mW cm –2 , 1 h) as measured with the DCFH-DA probe. Menadione
(Men, 50 μM) was used as a positive control for ROS induction.
Data expressed as mean ± SD from three independent measurements.
(b) Cell death induction in A2780cis after PDT treatment with Ir(III)-COUPY
agents. Data from three independent flow cytometry experiments using
Annexin V/PI dual staining. (c) Change in the relative volume of A2780cis
MCTS over 7 days. PDT treatments were applied on day 3 (dashed line).
Error bars correspond to SD from three replicates. Statistical significance
* p < 0.05, ** p < 0.01, *** p < 0.001 from two-way ANOVA test (a,b) or one-way ANOVA
test (c). Photoinduced Cell Death Mechanism The photogeneration
of ROS by the Ir(III)-COUPY conjugates prompted us to study the cell
death mechanism of these PSs. After 520 nm light irradiation, the
compounds increased the number of detached, non-viable cells as well
as rounded, floating cells. To characterize these morphological changes,
flow cytometry analysis of cell size and cell complexity was performed
using forward scatter and side scatter (FSC vs SSC) plots. Irradiated,
non-treated A2780cis cells grouped into a defined morphological cell
population. However, PDT treatment with Ir(III)-COUPY conjugates induced
a second large population of cell particles with low FSC/SCC ratios
( Figure S4 ). Although 3b did
not produce such changes, 3a , 3c , 3d, and 3e vastly induced these secondary cell
populations that correspond to dead cells with non-viable morphology
( Figure S4 ). To discern the mode of cell
death induced, the Annexin V/Propidium Iodide (AV/PI) labeling method
was used. These assays can detect the translocation of specific phospholipids
during apoptosis in AV + /PI – and AV + /PI + regions and loss of cell membrane integrity
from necrosis in AV – /PI + cells. As depicted
in Figures 5 b and S5 , 3a , 3c , 3d, and 3e photoinduced necrotic cell death, as revealed
by the increase of cells AV – /PI + due
to nuclear staining of PI. 3D Tumor Growth Inhibition Effects To better characterize
the photoinduced anticancer activity of the Ir-COUPY conjugates under
hypoxic conditions, their photocytotoxicity was assayed against 3D
multicellular tumor spheroid (MCTS) culture systems. Cancer cells
growing in MCTS cultures display heterogeneous regions, nutrient and
oxygen gradients, as well as intercellular and cell-extracellular
matrix interactions that mimic the tumor microenvironment. 56 , 57 In addition, 3D MCTS represents a more stringent model for drug
screening since they allow for assay drug penetration, resistance,
and importantly drug response to hypoxic gradients toward the center
of the sphere, which are particularly interesting aspects in the development
of novel PDT agents. 58 , 59 Defined A2780cis MCTS were
uniformly generated with an average radius of ∼350 μm
and an average volume of ∼0.181 mm 3 ( Figure S6 ). These volume values were used to
normalize MCTS growth. After 3 days, MCTS were incubated with Ir(III)-COUPY
conjugates for 4 h and exposed to 1 h of 520 nm light irradiation.
In coherence with what we observed in 2D cellular models under hypoxia,
PDT treatments with conjugates 3a , 3d, and 3e effectively blocked tumoral growth of the A2780cis spheres,
with 3a and 3e producing statistically significant
shrank compared to irradiated controls ( Figures 5 c and S7 ). In
contrast, conjugates 3b and 3c did not show
significant tumor inhibition effects under the same experimental conditions.
## Dark cytotoxicity
Dark cytotoxicity Except for compound 1c , all the compounds were found non-toxic in the absence of the light
trigger (IC 50 > 100 μM) regardless of the cell
line
( Tables 1 , S3 and S4 ). From this, it was clear that the
julolidine-fused system of 1c impacted on dark cytotoxicity,
rendering dark IC 50 values that oscillated between 5 and
26 μM in the studied cancer cell lines. Noteworthy, conjugate 3c , which also contains 1c as a coumarin moiety
showed no dark cytotoxicity up to 100 μM. Table 1 Photocytotoxicity of the Compounds
toward Cancer Cells in Normoxia a A375 SK-MEL-28 HeLa A2780 A2780cis dark light dark light dark light dark light dark light 1a >100 1.1 ± 0.1 [>89] >100 1.2 ± 0.1 [>86] >100 5.8 ± 0.4 [>17] >100 5.2 ± 0.5 [>19] >100 2.1 ± 0.2 [>48] 1b >100 10 ± 2 [>10] >100 >100 [n.d.] >100 >100 [n.d.] >100 >100
[n.d] >100 >100 [n.d.] 1c 9.9 ± 0.5 0.31 ± 0.02 [32] 12 ± 1 0.2 ± 0.01 [60] 26 ± 4 1.1 ± 0.8 [24] 5.1 ± 0.9 0.09 ± 0.01 [57] 15 ± 2 0.15 ± 0.04 [100] 2a >100 2.1 ± 0.3 [>48] >100 2.8 ± 0.3 [>36] >100 75 ± 6 [>1.3] >100 4 ± 1 [>25] >100 3.5 ± 0.4 [>29] 3a >100 18 ± 3 [>6] >100 1.5 ± 0.1 [>67] >100 8.6 ± 0.7 [>12] >100 1.07 ± 0.07 [>94] >100 0.70 ± 0.06 [>143] 3b >100 78 ± 6 [>1.3] >100 2.1 ± 0.2 [>48] >100 18 ± 2 [>6] >100 7.1 ± 0.1 [>14] >100 61 ± 8 [>1.6] 3c >100 38 ± 5 [>3] >100 >100 [n.d.] >100 45 ± 4 [>2.2] >100 2.1 ± 0.2 [>48] >100 1.04 ± 0.02 [>96] 3d >100 46 ± 4 [>2.2] >100 2.5 ± 0.2 [>40] >100 2.0 ± 0.4 [>50] >100 1.78 ± 0.07 [>56] >100 1.1 ± 0.2 [>91] 3e >100 19 ± 3 [>5] >100 7.6 ± 0.9 [>13] >100 9.3 ± 0.8 [>11] >100 1.9 ± 0.2 [>53] >100 0.93 ± 0.04 [>108] a Cells were treated for 2 h (1 h
of incubation, 1 h of irradiation with green light at 520 nm, 1.5
mW cm –2 ) followed by 48 h incubation in drug-free
medium. Dark analogues were kept in the dark. Data expressed as mean
± SD from three independent experiments. *n.d. = not determined.
PI (phototherapeutic index) is given in brackets; PI defined as IC 50 (dark-non-irradiated cells)/IC 50 (irradiated
cells).
## Photocytotoxicity in Normoxia
Photocytotoxicity in Normoxia First, photoactivation
of conjugates 3a–3e was evaluated in cancer cells,
along with their coumarin precursors 1a–1c and
the parent Ir(III) complex 2a . Light treatments were
applied at doses of 1.5 mW cm –2 using single green
wavelength (520 nm) LED irradiation. Overall, the compounds triggered
cell death upon light exposure at 520 nm in all cancer cell lines
studied, with IC 50 values in the low micromolar range ( Table 1 ). COUPY 1b , which lacks the N , N -dialkylamino
group at position 7, did not display antiproliferative activity after
light irradiation toward the investigated cancer cells, probably due
to its blue-shifted absorption ( Figure S2 ). In contrast, the original COUPY derivative 1a exerted
higher phototoxic action, particularly against melanoma cells, resulting
in more than 86-fold differences in bioactivity upon irradiation ( Table 1 ). The correspondent
conjugate ( 3b ) showed a slight increase in phototoxic
activity compared to 1b . This might be ascribable to
the Ir(III) core of the PS because the Ir(III) complex 2a also presented reasonable inhibitory activity after irradiation.
Nonetheless, the green-light photocytotoxicity of 3b was
lower than that of 3a in all cancer cell lines (light
IC 50 values between 3 and 78 μM compared to 0.7–18
μM). To test whether the poor phototoxicity of 3b was due to a blue-shifted absorption, photoactivation upon blue
light (465 nm) was also assayed. As shown in Table S4 , A2780cis cells dosed with 3b rendered light
IC 50 values that were very similar to those obtained for 3a after blue light irradiation (2.4 and 1.9 μM, respectively).
On the other hand, replacement of the N , N -dialkylamino group of the coumarin by a julolidine-fused system
decreased the phototoxicity of conjugate 3c in A375,
SK-MEL-28 and HeLa cells compared to 3a . Nevertheless,
the behavior of 3c after green light irradiation was
similar to that of 3a in A2780 and A2780cis cancer cells,
with light IC 50 values between 0.7 and 2.1 μM.
## Phototherapeutic Index
Phototherapeutic Index To explore the phototherapeutic
potential of the Ir(III)-COUPY conjugates, we performed a SAR analysis
derived from their phototoxic activity in cancer cells. For the identification
of the best performing anticancer PDT agents, PI determination was
used. Noticeably, PIs differed from one cancer cell line to another.
For instance, A375 melanoma cells, which exhibit the most aggressive
melanoma phenotype, 55 were less sensitive
to Ir(III)-COUPY phototoxicities (PI values not exceeding >6) than
SK-MEL-28 melanoma cells, where PIs reached up to >67 ( Table 1 ). The conjugates
also exerted
mild phototoxic activity in HeLa cancer cells (PIs ranging from 2.2
to >50). The order of potency toward cancer cells generally was 3a > 3c ≈ 3d ≈ 3e > 3b , with small discrepancies in this
trend
depending on the cell line. Nevertheless, the most potent green-light
photoactivation was found in ovarian cancer cells, particularly in
resistant A2780cis cells, where PI values were markedly higher both
for the conjugates containing the parent COUPY dye 1a regardless of the type and length of the spacer, and the one incorporating
the julolidine-fused system (>143 for 3a , >96 for 3c , >91 for 3d, and >108 for 3e ).
Overall, these results led us to initially select 3a and 3c–3e due to their good photocytotoxic profiles, especially
in A2780cis cancer cells. However, since 3c accumulated
in cancer cells to a greater extent than 3a or 3d–3e ( Figure 4 a), we hypothesized that 3c was less efficient
as a PS, as it would require a more intracellular amount of compound
to induce similar phototoxic outcomes. Therefore, 3a was
considered as the best hit Ir(III)-COUPY candidate for green-light
PDT in vitro, followed by 3d and 3e , which
contain the same coumarin and Ir(III) complex moieties.
## Photocytotoxicity in Hypoxia
Photocytotoxicity in Hypoxia Since A2780cis cells were
strongly inhibited by Ir(III)-COUPY photosensitizers upon green light
irradiation, this cell line was used for further SARS evaluation.
The phototoxic action of the compounds was assessed toward A2780cis
cells under hypoxic conditions (2% O 2 ) and compared to
the clinically approved PS 5-aminolevulinic acid (5-ALA). In this
second photocytotoxicity testing, dark cytotoxicity was recalculated
using higher concentrations, given that the compounds were deemed
as inactive at 100 μM. Except for 1c , no dark IC 50 values could be determined either in normoxia or hypoxia
up to 250 μM ( Table S3 ), which is
highly desirable for PDT agents. As shown in Table S5 , although none of them inhibited cell growth completely
at 250 μM, some conjugates were relatively toxic at this dose.
For instance, conjugates 3a , 3b, and 3c provided 45–76% of cell killing, while linker-containing
conjugates 3d and 3e barely exhibited dark
cytotoxicity at 250 μM (cell growth inhibition between 12 and
27%). Under hypoxia, poorer PI values were found compared to
those under normoxia, which can be explained in terms of photodynamic
effect restriction by the lack of oxygen ( Figure 4 d and Table S3 ). All the compounds exhibited higher phototherapeutic effects against
A2780cis cells than the protoporphyrin IX (PpIX) precursor 5-ALA ( Table S3 ). The highest PI values in normoxia
were achieved by 3a (>357), followed by the conjugates
containing the longer linkers 3d and 3e (>227
and >269, respectively). However, under hypoxia, the photodynamic
effect of 3a significantly decreased (PI > 66), whereas 3d and 3e yielded comparably higher photocytotoxicity,
with PI values of >131 and >147, respectively. As depicted in Figure 4 d, julolidine-containing
conjugate 3c displayed a similar PI value under normoxia
(>240), but was much less photoactive under hypoxia (PI > 31).
On
the other hand, no obvious photocytotoxicity was observed for 3b upon green light irradiation neither in normoxia nor in
hypoxia (light IC 50 values of 61 and 31 μM, respectively).
## Photogeneration of ROS
Photogeneration of ROS Once the photocytotoxicity of
the compounds against cancer cells was demonstrated, the induction
of cellular oxidative stress after irradiation was explored under
both normoxia and hypoxia using the ROS probe 2,7-dichlorodihydrofluorescein
diacetate (DCFH-DA) ( Figure 5 ). In dark conditions, ROS levels from untreated, control
cells remained similar to those treated with COUPY compounds 1a–1c or with iridium(III) complex 2a regardless
of the oxygen tension. Noticeably, conjugates 3a and
its linker-containing derivatives 3d and 3e slightly increased ROS levels under normoxia in the absence of light
( Figure 5 a). Upon 520
nm light application, a strong fluorescence signal from the DCFH-DA
probe was observed in normoxic cells treated with 3a , 3c , 3d, and 3e , indicating efficient
photogeneration of ROS. Although this photogeneration was comparably
lower under hypoxia, differences between dark and light conditions
were still markedly significant under low oxygen environment. Strikingly,
although poor phototoxicity was achieved for 3b in cellular
assays under hypoxia ( Figure 4 d), a slight increase in ROS levels was found after green-light
irradiation in A2780cis cells, these differences being significantly
much larger for the other conjugates ( Figure 5 b). Figure 5 Photoinduced cell death mechanism of Ir-COUPY
conjugates. (a) Relative
ROS levels in A2780cis cells under normoxia (21% O 2 ) and
hypoxia (2% O 2 ) after treatment with the compounds at 10
μM in dark or upon light irradiation (520 nm, 1.5 mW cm –2 , 1 h) as measured with the DCFH-DA probe. Menadione
(Men, 50 μM) was used as a positive control for ROS induction.
Data expressed as mean ± SD from three independent measurements.
(b) Cell death induction in A2780cis after PDT treatment with Ir(III)-COUPY
agents. Data from three independent flow cytometry experiments using
Annexin V/PI dual staining. (c) Change in the relative volume of A2780cis
MCTS over 7 days. PDT treatments were applied on day 3 (dashed line).
Error bars correspond to SD from three replicates. Statistical significance
* p < 0.05, ** p < 0.01, *** p < 0.001 from two-way ANOVA test (a,b) or one-way ANOVA
test (c).
## Photoinduced Cell Death Mechanism
Photoinduced Cell Death Mechanism The photogeneration
of ROS by the Ir(III)-COUPY conjugates prompted us to study the cell
death mechanism of these PSs. After 520 nm light irradiation, the
compounds increased the number of detached, non-viable cells as well
as rounded, floating cells. To characterize these morphological changes,
flow cytometry analysis of cell size and cell complexity was performed
using forward scatter and side scatter (FSC vs SSC) plots. Irradiated,
non-treated A2780cis cells grouped into a defined morphological cell
population. However, PDT treatment with Ir(III)-COUPY conjugates induced
a second large population of cell particles with low FSC/SCC ratios
( Figure S4 ). Although 3b did
not produce such changes, 3a , 3c , 3d, and 3e vastly induced these secondary cell
populations that correspond to dead cells with non-viable morphology
( Figure S4 ). To discern the mode of cell
death induced, the Annexin V/Propidium Iodide (AV/PI) labeling method
was used. These assays can detect the translocation of specific phospholipids
during apoptosis in AV + /PI – and AV + /PI + regions and loss of cell membrane integrity
from necrosis in AV – /PI + cells. As depicted
in Figures 5 b and S5 , 3a , 3c , 3d, and 3e photoinduced necrotic cell death, as revealed
by the increase of cells AV – /PI + due
to nuclear staining of PI.
## 3D Tumor Growth Inhibition Effects
3D Tumor Growth Inhibition Effects To better characterize
the photoinduced anticancer activity of the Ir-COUPY conjugates under
hypoxic conditions, their photocytotoxicity was assayed against 3D
multicellular tumor spheroid (MCTS) culture systems. Cancer cells
growing in MCTS cultures display heterogeneous regions, nutrient and
oxygen gradients, as well as intercellular and cell-extracellular
matrix interactions that mimic the tumor microenvironment. 56 , 57 In addition, 3D MCTS represents a more stringent model for drug
screening since they allow for assay drug penetration, resistance,
and importantly drug response to hypoxic gradients toward the center
of the sphere, which are particularly interesting aspects in the development
of novel PDT agents. 58 , 59 Defined A2780cis MCTS were
uniformly generated with an average radius of ∼350 μm
and an average volume of ∼0.181 mm 3 ( Figure S6 ). These volume values were used to
normalize MCTS growth. After 3 days, MCTS were incubated with Ir(III)-COUPY
conjugates for 4 h and exposed to 1 h of 520 nm light irradiation.
In coherence with what we observed in 2D cellular models under hypoxia,
PDT treatments with conjugates 3a , 3d, and 3e effectively blocked tumoral growth of the A2780cis spheres,
with 3a and 3e producing statistically significant
shrank compared to irradiated controls ( Figures 5 c and S7 ). In
contrast, conjugates 3b and 3c did not show
significant tumor inhibition effects under the same experimental conditions.
## Discussion and Conclusions
Discussion and Conclusions PDT holds great promise
as a non-invasive anticancer tool against
drug-resistant cancers. 60 However, highly
effective, non-toxic, and reliable PSs remain to be developed. Herein,
we took the advantageous properties of both organic fluorophores and
transition metal-based complexes to develop a novel family of PDT
agents. Inspired by our previous encouraging results with this type
of compounds, 44 we synthesized four new
PSs based on the conjugation of COUPY coumarins with far-red/NIR emission
to a cyclometalated Ir(III) complex. The photophysical properties
of the COUPY fluorophores were highly
influenced by the structural modifications carried out within the
coumarin scaffold. In particular, the incorporation of a strong EDG
at position 7 of the coumarin skeleton in the newly synthesized COUPY
dye 1c through fusion of the julolidine system was found
to induce a bathochromic shift in the absorption and emission maxima
compared to the 7- N , N -dialkylamino-containing
parent coumarin 1a . On the other hand, the decreased
push–pull character of the π-delocalized system in COUPY 1b as a consequence of removing the EDG at the 7-position
led to a clear blue-shift in the absorption and emission maxima and
reduced significantly its fluorescence emission. In parallel, the
spectroscopic properties of the conjugates could be also tuned depending
on the COUPY fluorophore attached to the Ir(III) core. For instance,
attachment of julolidine-fused COUPY coumarin 1c to the
Ir(III) complex red-shifted the absorption maximum of the resultant
conjugate 3c compared to 3a , which contains
the parent COUPY dye 1a . In contrast, 3b lost its fluorescence due to the absence of EDG in the coumarin
moiety 1b . This reveals the feasibility of these molecules
as theragnostic agents, which can be tuned to act in different regions
of the visible spectra depending on minimal structural modifications
that do not considerably alter the overall molecular size. In fact,
owing to a strong luminescence, Ir(III)-COUPY conjugates could be
easily tracked inside cancer cells by confocal microscopy. These experiments
revealed that the compounds are highly biocompatible, showing excellent
cellular uptake and forming fluorescent vesicles in the cytosol ( Figure 4 b). It is worth noting
that the julolidine-fused system also increased cell accumulation
of the resulting conjugate 3c ( Figure 4 a), which adds emphasis on how the COUPY
scaffold can act as a handle to modulate not only key photophysical
properties (e.g., absorption and emission within the phototherapeutic
window) but also key biological parameters such as cell entry and
accumulation. Another relevant aspect was that COUPY fluorophores
and their Ir(III) conjugates meet an important requirement for being
considered as promising PDT agents: their minimal toxicity in the
dark ( Tables S3 and S5 ). Only coumarin 1c exhibited dark cytotoxicity in cancer cells, but its correspondent
conjugate ( 3c ) was found to be inactive, thus revealing
that conjugation can be used to diminish undesired dark toxicity of
PSs based on cyclometalated Ir(III) complexes or their derivatives
( Tables 1 and S3 ). The prospect of using Ir(III)-COUPY
photosensitizers was very attractive
since conjugation of COUPY fluorophore to the metal complex increased
by a factor of 10 the 1 O 2 quantum yield of all
the resulting conjugates compared with the corresponding free coumarins
upon excitation at 532 nm ( Tables S1 and S2 ). Compared to the other conjugates, 3a and its spacer-containing
derivatives 3d and 3e had the largest 1 O 2 quantum yields, thereby confirming their capacity
to sensitize highly toxic singlet oxygen after visible light irradiation
in non-aqueous environments, independent of the length and rigidity
of the spacer linking both moieties. In addition, all Ir(III)-COUPY
conjugates were able to promote superoxide (O 2 •– ) generation in PBS through type-I PDT reactions ( Figures 3 and S3 ). Particularly, the julolidine-fused system-containing conjugate
( 3c ) photogenerated O 2 •– with the fastest rates, thereby demonstrating the importance of
incorporating a strong EDG in the coumarin scaffold, which not only
red-shifts absorption and emission maxima but also enhances the production
of superoxide anion radicals. Surprisingly, we also observed that 3b rapidly generated O 2 •– after visible light irradiation, indicating that the amino group
at the 7-position of the coumarin moiety is not strictly necessary
to trigger superoxide generation. The generation of O 2 •– was also corroborated by EPR measurements
using the DMPO spin trap. As anticipated, Ir(III)-COUPY conjugates
with higher quantum yields
for 1 O 2 sensitization gave the largest PI values
in the cancer cell lines studied herein. This preliminary SARS rationale
enabled us to identify 3a , 3d, and 3e as best hit candidates for green light-mediated anticancer
therapy. Interestingly, we detected that A2780cis cells were strongly
inhibited by light-driven treatments, suggesting that in vitro acquired
resistance mechanisms to cisplatin could be tackled by Ir(III)-COUPY-based
photoactive therapy. Local hypoxia within deep-seated tumors
is a serious impediment
for anticancer PDT. 61 Given that Ir(III)-COUPY
PSs possess the ability to generate both O 2 –• and 1 O 2 depending on their microenvironment,
which would overcome the limitation of traditional Type II PDT agents
under low oxygen environments, we tested their photoactivity under
hypoxic conditions. In general, hypoxia decreased the anticancer activity
of the conjugates, which confirmed ROS-generating PDT reactions as
a source of photocytotoxicity ( Figure 4 d). In particular, 3a photocytotoxicity
was greatly reduced under a low oxygen environment. However, the incorporation
of a spacer between the coumarin moiety and the Ir(III) complex enhanced
the phototoxic activity under hypoxia, rendering higher PI values
for 3d and 3e compared to the parent Ir(III)-COUPY
conjugate 3a . These findings suggest that the addition
of rigid or flexible linkers between the metal- and the organic-based
scaffold can be used to improve the phototherapeutic profile of these
PSs, not only increasing the PI under challenging hypoxia condition
but also reducing undesired in vitro dark toxicity ( Figure 4 d and Table S5 ). Besides, we further verified the potential of these
PSs by measuring
intracellular ROS levels in A2780cis cells. In agreement with 1 O 2 and PI values, conjugate 3a along
with 3d and 3e produced higher ROS photogeneration
than other compounds even under low oxygen conditions ( Figure 5 ). It is worth noting that 3c also raised ROS levels that were comparable to those produced
by 3a, 3d, and 3e . However, given that the
intracellular amount of 3c was considerably higher than
that found for the other conjugates, it could be reasoned that 3c is less efficient as PS because a higher amount of compound
is required to catalytically photogenerate similar levels of ROS within
cancer cells. Overall, the massive build-up of ROS after light irradiation
would induce irreversible oxidative damage, triggering necrotic cell
death as a result ( Figure 5 b). Although this cell death mode might be dependent on the
PDT dose applied, i.e., PS concentration and light fluence, the fact
that necrosis was observed following PDT treatments with these Ir(III)-COUPY
conjugates is interesting for two reasons. First, during necrosis,
cytosolic components are released into the extracellular space, which
may elicit acute inflammation and potentiate an antitumor immune response. 62 Second, PDT-induced necrosis could be of particular
interest for the treatment of apoptosis-refractory cancers such as
cisplatin-resistant ovarian cancers. Furthermore, the obtained results
using 3D cell culture models confirmed that this type of PSs exhibits
potent anticancer PDT activity under hypoxic conditions, as conjugates 3a , 3d, and 3e inhibit tumor growth
of MCTS ( Figure 5 c),
which contain hypoxic cellular population in the core of the tumorspheres. In conclusion, we have synthesized four new PDT agents based on
the conjugation between far-red/NIR emitting COUPY fluorophores and
a highly potent cyclometalated Ir(III) complex, with the aim of finding
rational relationships between chemical structure and biological activity.
The structural modifications introduced within the coumarin skeleton
clearly influenced the photophysical and biological properties of
the resulting Ir(III)-COUPY PSs. On the one hand, the conjugates exhibited
appealing features for PDT therapy, including emission in the phototherapeutic
window, excellent cellular uptake, no dark cytotoxicity, and high
photoinduced toxicity under green-light irradiation, which lead to
excellent PI values in several cancer cell lines. On the other hand,
the photoinduced generation of different types of ROS (Type II 1 O 2 and Type I O 2 •– ) for all of the conjugates was demonstrated through spectroscopic
methods, which would facilitate overcoming the limitations of conventional
PSs under low oxygen environments. Remarkably, ROS generation was
also confirmed in cisplatin-resistant A2780cis cancer cells for all
the Ir(III)-COUPY conjugates, in both normoxia and hypoxia. Overall,
three hit conjugates ( 3a , 3d, and 3e ) containing the same COUPY fluorophore were identified
in this SAR study as promising PSs owing to their excellent phototoxicity
in A2780cis cells in normoxia upon green light irradiation (PI = 357.1
for 3a , 227.3 for 3d , and 268.8 for 3e ). Among them, the linker-containing derivatives 3d and 3e are particularly interesting since they exhibited
an enhanced phototoxic activity under hypoxic conditions compared
with the parent Ir(III)-COUPY conjugate 3a (PI = 65.8
for 3a , 131.6 for 3d , and 147.1 for 3e ), as well as reduced undesired in vitro dark toxicity.
Importantly, the massive ROS overproduction during PDT treatments
induced necrotic cell death and effectively blocked tumor growth in
clinically relevant 3D tumoral models. The high anticancer activity
under normoxia and hypoxia conditions and the non-cytotoxicity in
the dark, together with the excellent and tunable photophysical and
photochemical properties of the Ir(III)-COUPY conjugates, make them
useful tools in PDT to deliver efficient ROS-mediated anticancer activity
toward chemo-resistant cancer cells using low doses of clinically-relevant
green light activation.
## Experimental Section
Experimental Section Materials and Methods Unless otherwise stated, common
chemicals and solvents (HPLC grade or reagent grade quality) were
purchased from commercial sources and used without further purification.
Aluminium plates coated with a 0.2 mm thick layer of silica gel 60
F 254 were used for thin-layer chromatography analyses (TLC),
whereas flash column chromatography purification was carried out using
silica gel 60 (230–400 mesh). NMR spectra were recorded at
25 °C in a 400 MHz spectrometer using the deuterated solvent
as an internal deuterium lock. The residual protic signal of CHCl 3 and DMSO was used as a reference in 1 H and 13 C NMR spectra recorded in CDCl 3 and DMSO- d 6 , respectively. Chemical shifts are reported
in part per million (ppm) in the δ scale, coupling constants
in Hz, and multiplicity as follows: s (singlet), d (doublet), t (triplet),
q (quartet), qt (quintuplet), m (multiplet), dd (doublet of doublets),
dt (doublet of triplets), td (triplet of doublets), br (broad signal),
and so forth. Electrospray ionization mass spectra (ESI-MS) were recorded
on an instrument equipped with single quadrupole detector coupled
to an HPLC and high-resolution (HR) ESI-MS on an LC/MS-TOF instrument.
The purity of final compounds was determined by reversed-phase high
performance liquid chromatography (HPLC) analyses on a Jupiter Proteo
C12 column (250 × 4.6 mm, 90 Å, 4 μm, flow rate: 1
mL/min) using linear gradients of 0.1% formic acid in Milli-Q H 2 O (A) and 0.1% formic acid in ACN (B). The HPLC column was
maintained at 25 °C. All final compounds were >95% pure by
this
method. Synthesis of COUPY 1b Synthesis of Compound 4 Phenol (10 g,
106 mmol) and ethyl acetoacetate (13.6 mL, 106 mmol) were mixed and
heated to 100 °C in nitrobenzene (10 mL). In a separate flask,
AlCl 3 (28.1 g, 213 mmol) was dissolved in nitrobenzene
(100 mL) at 0 °C—the AlCl 3 was added in six
portions and left to stir at room temperature for 15–30 min.
Then, the AlCl 3 solution was decanted from excess and added
dropwise to the phenol and ethyl acetoacetate solution over 45 min.
Once the addition was completed, the temperature was increased to
125 °C and the reaction mixture was stirred under reflux for
3 h. The reaction was then cooled to 0 °C and a 1:1 (v/v) mixture
of HCl/H 2 O (100 mL) was added. The mixture was then filtrated,
and the solid was washed with ethyl acetate. The filtrate was transferred
to a separating flask where the aqueous and organic layers were separated:
the organic layer was dried over anhydrous Na 2 SO 4 and filtered. The ethyl acetate was removed by evaporation under
reduced pressure and nitrobenzene by distillation in vacuo. The crude
product was then purified by column chromatography (silica gel, 0–100%
DCM in hexanes) to give 3.79 g (22%) of a brown solid. TLC: R f (DCM) 0.34. 1 H NMR (400 MHz, CDCl 3 ): δ (ppm) 7.61 (1H, dd, J = 8.0, 1.6
Hz), 7.54 (1H, m), 7.32 (2H, m), 6.30 (1H, s), 2.45 (3H, s). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ
(ppm) 160.9, 153.6, 152.5, 131.9, 124.7, 124.3, 120.1, 117.2, 115.2,
18.8. LRMS (ESI-TOF) m / z : [M + H] + calcd for C 10 H 9 O 2 , 161.06;
found, 160.83. Synthesis of Compound 5 Coumarin 4 (4.48 g, 28.0 mmol) was mixed with Lawesson’s Reagent
(5.66 g, 14.0 mmol) in toluene (150 mL) and refluxed for 19 h at 105
°C. The crude was then evaporated under reduced pressure and
purified by column chromatography (silica gel, 0–50% DCM in
hexanes) to obtain 3.80 g of a yellow solid (77% yield). TLC: R f (75:25, DCM/hexanes) 0.88. 1 H NMR
(400 MHz, CDCl 3 ): δ (ppm) 7.65 (1H, dd, J = 8.0, 1.6 Hz), 7.59 (1H, m), 7.49 (1H, m), 7.36 (1H, m), 7.18 (1H,
m), 2.38 (3H, s). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ (ppm) 197.6, 156.2, 144.3, 132.2, 129.1, 125.5,
124.6, 121.6, 117.1, 18.1. HRMS (ESI-TOF) m / z : [M + H] + calcd for C 10 H 9 OS, 177.0369: found, 177.0369. Synthesis of Compound 6 4-Pyridylacetonitrile
hydrochloride (1.71 g, 11.08 mmol) and NaH (3.41 g of a 60% dispersion
in mineral oil, 85.2 mmol) were dried together in a desiccator alongside
compound 5 (1.50 g, 8.52 mmol) in a separate flask. Both
were then dissolved in anhydrous ACN (90 and 30 mL, respectively)
and the first solution passed to the second and the resulting mixture
stirred for 2.5 h at room temperature and under an argon atmosphere.
Afterward, AgNO 3 (3.18 g, 18.74 mmol) was added and the
mixture stirred for 2 h under Ar atmosphere and protected from light.
The crude product was then evaporated under reduced pressure and purified
by column chromatography (silica gel, 50–100% DCM in hexanes
first, then 0–0.65% MeOH in DCM) to give 913 mg (41% yield)
of a brown solid. TLC: R f (DCM/AcOEt 1:1)
0.48. 1 H NMR (400 MHz, CDCl 3 ): δ (ppm)
8.61 (2H, d, J = 5.8 Hz), 7.75 (2H, d, J = 5.8 Hz), 7.51 (2H, m), 7.32 (2H, m), 7.04 (1H, s), 2.42 (3H, s). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ
(ppm) 161.9, 152.1, 150.1, 142.8, 140.1, 131.9, 125.3, 124.6, 121.3,
121.2, 118.9, 118.0, 116.4, 84.8, 18.7. HRMS (ESI-TOF) m / z : [M + H] + calcd for C 17 H 13 N 2 O, 261.1022, found, 261.1026. Synthesis of Compound 1b Methyl trifluoromethanesulfonate
(16 μL, 0.14 mmol) was added to a solution of coumarin 6 (18.1 mg, 0.070 mmol) in DCM (10 mL) under an argon atmosphere.
The mixture was stirred overnight at room temperature and protected
from light. The reaction mixture was evaporated under reduced pressure
and purified by column chromatography (silica gel, 0–10% MeOH
in DCM) to give 16.1 mg of a yellow solid (yield 55%). TLC: R f (10% MeOH in DCM) 0.34. 1 H NMR
(400 MHz, DMSO- d 6 ): δ (ppm) 8.73
(2H, d, J = 8.0 Hz), 8.32 (2H, d, J = 7.6 Hz), 7.92 (2H, m), 7.81 (1H, m), 7.56 (1H, m), 7.30 (1H, d, J = 1.6 Hz), 4.27 (3H, s), 2.62 (3H, d, J = 1.2 Hz). 13 C{ 1 H} NMR (101 Hz, DMSO- d 6 ): δ (ppm) 165.9, 151.6, 150.3, 147.9,
144.5, 133.3, 126.5, 125.6, 122.3, 120.8, 117.4, 117.1, 116.8, 81.3,
46.6, 18.5. HRMS (ESI-TOF) m / z :
[M] + calcd for C 18 H 15 N 2 O, 275.1179; found, 275.1179. Synthesis of Ir(III)-COUPY Conjugate 3b Synthesis of Compound 7 A solution of
methyl bromoacetate (0.9 mL, 9.25 mmol) and coumarin 6 (481 mg, 1.85 mmol) in a 2.5:1 (v/v) mixture of AcOEt and DCM (35
mL) was stirred under reflux at 60 °C for 48 h. The reaction
mixture was then evaporated under reduced pressure and purified by
column chromatography (silica gel, 0–6.5% MeOH in DCM) to give
623 mg (82% yield) of an orange solid. TLC: R f (10% MeOH in DCM) 0.37. 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 8.76 (2H, d, J = 7.4 Hz), 8.41 (2H, d, J = 7.4 Hz), 8.04 (1H,
d, J = 7.6 Hz), 7.95 (1H, dd, J =
8.0, 1.6 Hz), 7.83 (1H, td, J = 8.0, 1.6 Hz), 7.58
(1H, m), 7.35 (1H, s), 5.60 (2H, s), 3.79 (3H, s), 2.65 (3H, s). 13 C{ 1 H} NMR (101 Hz, DMSO- d 6 ): δ (ppm) 167.3, 166.5, 151.7, 151.3, 149.3, 144.9,
133.5, 126.6, 125.6, 122.0, 120.8, 117.4, 117.3, 116.9, 81.6, 58.9,
53.0, 18.6. HRMS (ESI-TOF) m / z :
[M] + calcd for C 20 H 17 N 2 O 3 , 333.1234; found, 333.1243. Synthesis of Compound 8 Compound 7 (214 mg, 0.52 mmol) was refluxed in a solution containing
concentrated HCl (43 mL, 520 mmol) and H 2 O (86 mL, Milli-Q
quality) at 60 °C for 5.5 h. The crude mixture was then evaporated
under reduced pressure to give a red solid, which was used without
further purification in the next step. Analytical HPLC (10 to 100%
B in 30 min, formic acid additive): Rt = 12.5 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 19 H 15 N 2 O 3 , 319.11; found, 319.53. Synthesis of Compound 9 Coumarin 8 (80.4 mg, 0.23 mmol) and HATU (88.8 mg, 0.23 mmol) were
dissolved in anhydrous DMF (8 mL) under an Ar atmosphere. After addition
of DIPEA (40 μL, 0.23 mmol), the reaction mixture was stirred
for 5 min under Ar at room temperature and protected from light. On
the other hand, DIPEA (40 μL, 0.23 mmol) was added to a solution
of N -Boc-1,3-propanediamine hydrochloride (57.3 mg,
0.27 mmol) in anhydrous DMF (5 mL), and the resulting mixture was
combined with the coumarin solution. After addition of DIPEA (40 μL,
0.23 mmol), the reaction mixture was stirred for 2 h at room temperature
under Ar and protected from light. The crude product was evaporated
under reduced pressure and purified by column chromatography (silica
gel, 0–12% MeOH in DCM) to give 30.5 mg of a purple solid (yield:
26%). TLC: R f (10% MeOH in DCM) 0.26. 1 H NMR (400 MHz, DMSO- d 6 ): δ
(ppm) 8.69 (2H, d, J = 7.4 Hz), 8.52 (1H, t, J = 5.6 Hz), 8.36 (2H, d, J = 7.4 Hz),
7.98 (1H, dd, J = 8.4, 1.2 Hz), 7.94 (1H, dd, J = 8.0, 1.2 Hz), 7.82 (1H, td, J = 7.2
Hz, J = 1.6 Hz), 7.57 (1H, td, J = 8.3, 0.8 Hz), 7.33 (1H, d, J = 1.2 Hz), 6.82
(1H, t, J = 5.6 Hz), 5.33 (2H, s), 3.15 (2H, q, J = 6.4 Hz), 2.97 (2H, q, J = 6.4 Hz),
2.64 (3H, d, J = 1.2 Hz), 1.58 (2H, qt, J = 7.0 Hz), 1.37 (9H, s). 13 C{ 1 H} NMR (101
Hz, DMSO- d 6 ): δ (ppm) 166.3, 164.6,
155.6, 151.7, 150.8, 148.7, 145.0, 133.4, 126.6, 125.7, 121.8, 120.8,
117.4, 116.9, 81.5, 77.5, 60.1, 37.5, 36.9, 29.3, 28.2, 18.6. HRMS
(ESI-TOF) m / z : [M] + calcd
for C 27 H 31 N 4 O 4 , 475.2340;
found, 475.2336. Synthesis of Compound 10 A cooled down
solution of hydrochloric acid in dioxane (4 M, 3 mL) was added to
coumarin 9 (10 mg, 0.021 mmol). The reaction mixture
was stirred for 25 min in an ice bath under an Ar atmosphere and protected
from light. After removal of the solvent under reduced pressure, several
co-evaporations from acetonitrile were carried out. The crude product
was used without further purification in the next step since HPLC-MS
analysis revealed that the removal of the Boc group was quantitative.
Analytical HPLC (10 to 100% B in 30 min, formic acid additive): Rt
= 5.5 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 22 H 23 N 4 O 2 , 375.18; found, 375.19. Synthesis of Ir(III)-COUPY Conjugate 3b To a solution of Ir complex 2b (15.6 mg, 14.6 μmol)
and HATU (5.7 mg, 14.6 μmol) in anhydrous DMF (2.5 mL) under
an Ar atmosphere, DIPEA (3 μL, 14.6 μmol) was added and
the mixture stirred for 10 min under Ar at room temperature and protected
from light. After addition of a solution of coumarin 10 (9.8 mg, 21.9 μmol) and DIPEA (15 μL, 73 μmol)
in anhydrous DMF (3 mL), the reaction mixture was stirred for 2.5
h at room temperature under Ar and protected from light. After evaporation
under reduced pressure, the crude was purified by column chromatography
(silica gel, 0–12.5% MeOH in DCM) to give 10.1 mg of a yellow
solid (yield: 47%). TLC: R f (15% MeOH
in DCM) 0.61. 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 14.19 (1H, br s), 13.97 (1H, br s), 8.89
(1H, d, J = 8.7 Hz), 8.72 (3H, m), 8.35 (2H, d, J = 7.4 Hz), 8.28 (1H, d, J = 8.8 Hz),
8.15 (1H, d, J = 8.4 Hz), 8.09 (1H, d, J = 8.8 Hz), 7.95 (2H, m), 7.82 (3H, m), 7.64 (1H, t, J = 7.8 Hz), 7.55 (2H, d, m), 7.47 (1H, d, J = 8.1
Hz), 7.32 (1H, d, J = 1.2 Hz), 7.19 (1H, t, J = 8.5 Hz), 7.10 (2H, m), 7.01 (1H, t, J = 7.5 Hz), 6.89 (1H, t, J = 7.5 Hz), 6.83 (1H,
t, J = 7.7 Hz), 6.76 (2H, t, J =
7.3 Hz), 6.68 (1H, s), 6.31 (1H, d, J = 7.3 Hz),
6.19 (1H, d, J = 7.3 Hz), 5.97 (1H, d, J = 8.3 Hz), 5.71 (1H, d, J = 8.3 Hz), 5.40 (2H,
s), 5.10 (2H, m), 3.20 (4H, m), 2.63 (3H, d, J =
1.1 Hz), 1.69 (4H, m), 1.23 (4H, m), 0.91 (2H, m), 0.65 (3H, t, J = 7.2 Hz). 13 C NMR (101 MHz, DMSO- d 6 ): δ (ppm) 166.3, 165.8, 164.7, 164.4, 163.8, 155.6,
152.4, 151.7, 150.8, 149.8, 148.7, 148.4, 145.0, 143.3, 141.3, 139.5,
139.5, 139.2, 137.9, 134.2, 134.1, 133.4, 133.0, 132.9, 132.3, 132.1,
132.0, 131.3, 130.4, 130.0, 129.6, 129.4, 128.9, 128.9, 128.8, 126.5,
125.7, 124.7, 124.6, 124.4, 123.2, 123.0, 122.4, 121.8, 121.6, 120.8,
120.6, 117.8, 117.4, 117.3, 116.9, 113.4, 112.9, 112.6, 81.5, 60.2,
56.0, 45.9, 37.1, 37.0, 31.9, 29.0, 19.0, 18.6, 13.4. Analytical HPLC
(10 to 100% B in 30 min, formic acid additive): Rt = 12.6 min. HRMS
(ESI-TOF) m / z : [M] 2+ calcd
for C 69 H 58 IrN 11 O 3 , 640.7171;
found, 640.7169. Synthesis of COUPY 1c Synthesis of Compound 11 Coumarin 102
(1.17 g, 4.58 mmol) and Lawesson’s reagent (1.11 g, 2.75 mmol)
were dissolved in toluene (30 mL) and heated at 100 °C for 15
h. After evaporation under reduced pressure, the residue was purified
by column chromatography (silica gel, 50–70% DCM in hexanes)
to give an orange solid (666 mg, 54%): TLC R f (DCM) 0.53. 1 H NMR (400 MHz, CDCl 3 ):
δ (ppm) 7.03 (1H, s), 6.91 (1H, br q, J = 0.8
Hz), 3.29 (4H, q, J = 6.8 Hz), 3.00 (2H, t, J = 6.4 Hz), 2.79 (2H, t, J = 6.4 Hz),
2.27 (3H, d, J = 0.8 Hz), 1.98 (4H, m). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ (ppm) 196.4,
154.5, 146.5, 146.4, 123.4, 121.8, 120.2, 111.2, 106.1, 50.1, 49.8,
28.0, 21.5, 20.7, 20.5, 18.1; HRMS (ESI-TOF) m / z : [M + H] + calcd for C 16 H 18 NOS, 272.1104; found, 272.1108. Synthesis of Compound 12 To a solution
of 4-pyridylacetonitrile hydrochloride (256.4 mg, 1.66 mmol) and NaH
(60% dispersion in mineral oil, 442.2 mg, 11.06 mmol) in dry ACN (50
mL) under an Ar atmosphere and protected from light, a solution of
coumarin 11 (300 mg, 1.11 mmol) in dry ACN (10 mL) was
added. After the mixture was stirred for 2 h at room temperature,
silver nitrate (413.2 mg, 2.43 mmol) was added, and the reaction mixture
was stirred at room temperature for 2 h under an Ar atmosphere and
protected from light. The crude product was evaporated under reduced
pressure and purified by column chromatography (silica gel, 0–3.5%
MeOH in DCM) to give 120.6 mg of an orange solid (yield 31%). TLC: R f (10% MeOH in DCM) 0.67. 1 H NMR
(400 MHz, CDCl 3 ): δ (ppm) 8.54 (2H, m), 7.69 (2H,
m), 6.94 (1H, s), 6.72 (1H, br q, J = 0.8 Hz), 3.27
(4H, m), 2.90 (2H, t, J = 6.5 Hz), 2.78 (2H, t, J = 6.3 Hz), 2.31 (3H, d, J = 0.8 Hz),
2.00 (4H, m). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ (ppm) 163.2, 149.2, 145.4, 144.0, 140.9, 121.4,
120.5, 119.9, 118.4, 111.5, 109.5, 106.0, 80.3, 49.6, 48.9, 27.3,
21.1, 21.0, 20.5, 18.1. HRMS (ESI-TOF) m / z : [M + H] + calcd for C 23 H 22 N 3 O, 356.1757; found, 356.1761. Synthesis of Compound 1c Methyl trifluoromethanesulfonate
(32 μL, 0.28 mmol) was added to a solution of coumarin 12 (49 mg, 0.14 mmol) in DCM (25 mL) under an argon atmosphere.
The mixture was stirred overnight at room temperature and protected
from light. The reaction mixture was evaporated under reduced pressure
and purified by column chromatography (silica gel, 0–6% MeOH
in DCM) to give 47 mg of a red solid (yield 66%). TLC: R f (10% MeOH in DCM) 0.33. 1 H NMR (400 MHz,
DMSO- d 6 ): δ (ppm) 8.60 (2H, d, J = 7.2 Hz), 7.99 (2H, d, J = 7.2 Hz),
7.37 (1H, s), 6.87 (1H, s), 4.13 (3H, s), 3.37 (4H, m), 2.93 (2H,
t, J = 6.4 Hz), 2.81 (2H, t, J =
6.2 Hz), 2.50 (3H, s), 1.96 (2H, m), 1.90 (2H, m). 13 C{ 1 H} NMR (101 Hz, DMSO- d 6 ): δ
(ppm) 166.2, 152.4, 150.4, 148.8, 147.4, 143.5, 122.7, 121.4, 120.1,
118.6, 110.3, 105.0, 77.1, 49.5, 48.7, 45.9, 27.1, 20.8, 20.4, 19.8,
18.5. HRMS (ESI-TOF) m / z : [M] + calcd for C 24 H 24 N 3 O, 370.1912;
found, 370.1914. Synthesis of Ir(III)-COUPY Conjugate 3c Synthesis of Compound 13 Methyl bromoacetate
(52 μL, 0.56 mmol) was added to a solution of coumarin 12 (100 mg, 0.28 mmol) in AcOEt/ACN 1:1 (20 mL). The mixture
was stirred overnight at 50 °C under an Ar atmosphere and protected
from light. The crude product was evaporated under reduced pressure
and purified by column chromatography (silica gel, 0–8% MeOH
in DCM) to give 124 mg of a blue solid (yield, 87%). TLC: R f (10% MeOH in DCM) 0.40. 1 H NMR
(400 MHz, CDCl 3 ): δ (ppm) 8.81 (2H, d, J = 6.8 Hz), 8.01 (2H, d, J = 6.8 Hz), 7.15 (1H,
s), 6.90 (1H, s), 5.80 (2H, s), 3.85 (3H, s), 3.39 (4H, m), 3.05 (2H,
t, J = 6.4 Hz), 2.84 (2H, t, J =
6.4 Hz), 2.49 (3H, s), 2.16 (2H, m), 2.01 (2H, m). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ (ppm) 167.3, 167.2,
152.7, 151.5, 150.8, 148.4, 143.5, 122.5, 122.4, 119.8, 118.5, 111.4,
111.0, 106.0, 79.2, 58.7, 53.6, 50.4, 49.8, 28.1, 21.9, 21.0, 20.3,
19.2. HRMS (ESI-TOF) m / z : [M] + calcd for C 26 H 26 N 3 O 3 , 428.1969; found, 428.1974. Synthesis of Compound 14 A 1:2 (v/v) mixture
of HCl (37%) and Milli-Q water (51 mL) was added to coumarin 13 (98.9 mg, 0.19 mmol). The reaction mixture was stirred
for 5 h at 60 °C under an Ar atmosphere and protected from light.
After removal of the major part of the solvent, several coevaporations
from acetonitrile were carried out. The crude product was used without
further purification in the next step. Analytical HPLC (10 to 100%
B in 30 min, formic acid additive): Rt = 16.6 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 25 H 24 N 3 O 3 , 414.18; found, 413.81. Synthesis of Compound 15 Coumarin 14 (87.5 mg, 0.19 mmol) and HATU (76 mg, 0.19 mmol) were dissolved
in anhydrous DMF (8 mL) under an Ar atmosphere. After addition of
DIPEA (70 μL, 0.39 mmol), the reaction mixture was stirred for
5 min under Ar at room temperature and protected from light. On the
other hand, DIPEA (35 μL, 0.19 mmol) was added to a solution
of N -Boc-1,3-propanediamine hydrochloride (62 mg,
0.29 mmol) in anhydrous DMF (5 mL), and the resulting mixture was
combined with the coumarin solution. After addition of DIPEA (70 μL,
0.39 mmol), the reaction mixture was stirred for 2 h at room temperature
under Ar and protected from light. The crude product was evaporated
under reduced pressure and purified by column chromatography (silica
gel, 0–12.0% MeOH in DCM) to give 29 mg of a purple solid (yield:
24%). TLC: R f (10% MeOH in DCM) 0.59. 1 H NMR (400 MHz, DMSO- d 6 ): δ
(ppm) 8.69 (1H, br s), 8.55 (2H, d, J = 6.8 Hz),
7.96 (2H, d, J = 6.6 Hz), 7.35 (1H, s), 6.84 (2H,
m), 5.19 (2H, s), 3.37 (4H, m), 3.13 (2H, q, J =
6.6 Hz), 2.95 (4H, m), 2.80 (2H, m), 2.49 (3H, s), 1.92 (4H, m), 1.57
(2H, quint, J = 6.9 Hz), 1.37 (9H, s). 13 C NMR (101 MHz, DMSO- d 6 ): δ (ppm)
166.2, 164.8, 155.6, 152.8, 150.5, 149.3, 147.6, 143.9, 122.7, 121.7,
119.3, 118.5, 110.5, 109.8, 104.9, 77.5, 77.5, 59.5, 49.6, 48.7, 40.2,
37.5, 36.8, 31.3, 29.3, 29.0, 28.2, 27.1, 22.1, 20.8, 20.4, 19.8,
18.5, 14.0. HRMS (ESI-TOF) m / z :
[M] + calcd for C 33 H 40 N 5 O 4 , 570.3075; found, 570.3079. Synthesis of Compound 16 A cooled down
solution of hydrochloric acid in dioxane (4 M, 8 mL) was added to
coumarin 15 (19 mg, 0.031 mmol). The reaction mixture
was stirred for 25 min in an ice bath under an Ar atmosphere and protected
from light. After removal of the solvent, several co-evaporations
from acetonitrile were carried out. The crude product was used without
further purification since HPLC-MS analysis revealed that the removal
of the Boc group was quantitative. Analytical HPLC (10 to 100% B in
30 min, formic acid additive): Rt = 10.6 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 28 H 32 N 5 O 2 , 470.26; found, 470.11. Synthesis of Ir(III)-COUPY Conjugate 3c To a solution of Ir complex 2b (30 mg, 28.1 μmol)
and HATU (11.0 mg, 28.1 μmol) in anhydrous DMF (5 mL) under
an Ar atmosphere, DIPEA (10 μL, 56.1 μmol) was added and
the mixture stirred for 10 min under Ar at room temperature and protected
from light. After addition of a solution of coumarin 16 (16.9 mg, 31.2 μmol) and DIPEA (25 μL, 140.3 μmol)
in anhydrous DMF (6 mL), the reaction mixture was stirred for 2.5
h at room temperature under Ar and protected from light. After evaporation
under reduced pressure, the crude was purified by column chromatography
(silica gel, 0–12% MeOH in DCM) to give 14.5 mg of a dark blue
solid (yield: 33%). TLC: R f (10% MeOH
in DCM) 0.47. 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 13.99 (2H, br s), 8.88 (1H, d, J = 8.8 Hz), 8.69 (1H, d, J = 9.0 Hz), 8.66 (1H,
m), 8.55 (2H, d, J = 7.0 Hz), 8.30 (1H, d, J = 9.0 Hz), 8.15 (1H, d, J = 8.3 Hz),
8.08 (1H, d, J = 8.9 Hz), 7.99 (2H, d, J = 7.2 Hz), 7.93 (1H, d, J = 7.7 Hz), 7.83 (2H,
m), 7.72 (1H, m), 7.63 (1H, t, J = 7.8 Hz), 7.51
(1H, d, J = 8.1 Hz), 7.45 (1H, d, J = 8.1 Hz), 7.40 (1H, s), 7.19 (1H, t, J = 8.7 Hz),
7.09 (3H, m), 7.00 (1H, t, J = 7.4 Hz), 6.90 (2H,
m), 6.81 (1H, t, J = 7.5 Hz), 6.73 (2H, m), 6.68
(1H, s), 6.30 (1H, d, J = 7.6 Hz), 6.19 (1H, d, J = 7.6 Hz), 5.95 (1H, d, J = 8.2 Hz),
5.70 (1H, d, J = 8.2 Hz), 5.21 (2H, s), 5.10 (2H,
m), 3.19 (4H, sext, J = 7.2 Hz), 2.92 (2H, t, J = 6.1 Hz), 2.81 (2H, t, J = 6.0 Hz),
1.91 (4H, m), 1.68 (5H, m), 1.23 (4H, m), 0.94 (4H, m), 0.66 (3H,
t, J = 7.2 Hz). 13 C NMR (101 MHz, DMSO- d 6 ): δ (ppm) 165.8, 164.9, 164.0, 155.6,
152.9, 152.4, 150.6, 149.8, 149.4, 148.5, 147.6, 143.9, 141.2, 139.5,
137.9, 132.4, 132.1, 132.0, 131.2, 130.2, 129.9, 129.3, 129.0, 128.9,
128.7, 124.7, 124.5, 124.2, 124.2, 123.0, 122.9, 122.8, 122.7, 122.3,
121.7, 121.6, 120.5, 119.4, 118.5, 117.7, 113.4, 113.3, 113.1, 113.0,
112.8, 112.6, 110.6, 105.0, 77.4, 67.5, 59.5, 49.6, 48.7, 45.9, 37.0,
31.9, 29.0, 27.1, 20.8, 20.4, 19.8, 19.0, 18.8, 18.5, 13.4. HRMS (ESI-TOF) m / z : [M] 2+ calcd for C 75 H 67 IrN 12 O 3 , 688.2539; found, 688.2545.
Analytical HPLC (10 to 100% B in 30 min, formic acid additive): Rt
= 15.5 min. Synthesis of Ir(III)-COUPY Conjugate 3d Synthesis of Compound 18 To a solution
of Boc-6-aminohexanoic acid (42.8 mg, 0.19 mmol) and HATU (72.5 mg,
0.19 mmol) in anhydrous DMF (6 mL) under an Ar atmosphere, DIPEA (65
μL, 0.37 mmol) was added and the mixture stirred for 10 min
under Ar at room temperature and protected from light. After addition
of a solution of coumarin 17 (32 mg, 0.062 mmol) and
DIPEA (54 μL, 0.31 mmol) in anhydrous DMF (6 mL), the reaction
mixture was stirred for 2.5 h at room temperature under Ar and protected
from light. After evaporation under reduced pressure, the crude was
purified by column chromatography (silica gel, 0–12% MeOH in
DCM) to give 19.7 mg of a pink solid (yield: 49%). TLC: R f (10% MeOH in DCM) 0.43. 1 H NMR (400 MHz,
DMSO- d 6 ): δ (ppm) 8.59 (1H, br s),
8.53 (2H, d, J = 6.2 Hz), 8.16 (2H, d, J = 6.4 Hz), 7.81 (1H, br s), 7.72 (1H, d, J = 9.0
Hz), 6.99 (3H, m), 6.74 (1H, br s), 5.24 (2H, s), 3.55 (4H, m), 3.10
(4H, m), 2.86 (2H, m), 2.55 (3H, s), 2.03 (2H, m), 1.58 (2H, m), 1.46
(2H, m), 1.36 (11H, m), 1.18 (8H, m). 13 C NMR (101 MHz,
DMSO- d 6 ): δ (ppm) 172.1, 166.8,
164.8, 155.6, 154.9, 152.8, 152.0, 149.2, 144.2, 127.0, 120.1, 118.2,
111.9, 110.5, 110.4, 96.4, 78.1, 77.3, 59.6, 44.2, 36.9, 36.1, 35.4,
29.3, 29.0, 28.3, 26.0, 25.1, 18.4, 12.4. HRMS (ESI-TOF) m / z : [M] + calcd for C 37 H 51 N 6 O 5 , 659.3915; found, 659.3928. Synthesis of Compound 20 A cooled down
solution of hydrochloric acid in dioxane (4 M, 6 mL) was added to
coumarin 18 (16.1 mg, 0.023 mmol). The reaction mixture
was stirred for 20 min in an ice bath under an Ar atmosphere and protected
from light. After removal of the solvent, several co-evaporations
from acetonitrile were carried out. The crude product was used without
further purification since HPLC-MS analysis revealed that the removal
of the Boc group was quantitative. Analytical HPLC (10 to 100% B in
30 min, formic acid additive): Rt = 8.5 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 32 H 43 N 6 O 3 , 559.34; found, 559.49. Synthesis of Ir(III)-COUPY Conjugate 3d To a solution of Ir complex 2b (20.6 mg, 19.3 μmol)
and HATU (7.4 mg, 19.3 μmol) in anhydrous DMF (4 mL) under an
Ar atmosphere, DIPEA (7 μL, 38.6 μmol) was added and the
mixture stirred for 10 min under Ar at room temperature and protected
from light. After addition of a solution of coumarin 20 (14.6 mg, 23.2 μmol) and DIPEA (17 μL, 96.4 μmol)
in anhydrous DMF (5 mL), the reaction mixture was stirred for 2.5
h at room temperature under Ar and protected from light. After evaporation
under reduced pressure, the crude was purified by column chromatography
(silica gel, 0–12% MeOH in DCM) to give 27.9 mg of a purple
solid (yield: 68%). 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 14.16 (2H, br s), 8.88 (1H, d, J = 8.7 Hz), 8.69 (1H, d, J = 8.9 Hz),
8.65 (1H, t, J = 5.5 Hz), 8.53 (2H, d, J = 7.5 Hz), 8.29 (1H, d, J = 9.0 Hz), 8.15 (3H,
m), 8.07 (1H, d, J = 8.9 Hz), 7.96 (1H, d, J = 7.5 Hz), 7.92 (1H, t, J = 5.6 Hz),
7.87 (1H, d, J = 7.6 Hz), 7.83 (1H, dd, J = 8.8 Hz, J = 1.5 Hz), 7.70 (1H, d, J = 9.2 Hz), 7.63 (1H, t, J = 8.0 Hz), 7.53 (2H,
m), 7.46 (1H, d, J = 8.1 Hz), 7.19 (1H, t, J = 8.8 Hz), 7.00 (8H, m), 6.81 (1H, td, J = 7.6 Hz, J = 1.3 Hz), 6.74 (2H, m), 6.64 (1H,
d, J = 1.1 Hz), 6.30 (1H, d, J =
7.3 Hz), 6.19 (1H, d, J = 7.4 Hz), 5.95 (1H, d, J = 8.3 Hz), 5.70 (1H, d, J = 8.2 Hz),
5.25 (2H, m), 5.09 (2H, m), 3.53 (4H, q, J = 6.9
Hz), 3.11 (6H, m), 2.53 (2H, d, J = 0.8 Hz), 2.09
(2H, t, J = 7.4 Hz), 1.71 (2H, m), 1.51 (6H, m),
1.25 (3H, m), 1.15 (6H, t, J = 7.0 Hz), 0.99 (2H,
m), 0.65 (3H, t, J = 7.2 Hz). 13 C NMR
(101 MHz, DMSO- d 6 ): δ (ppm) 172.1,
166.8, 165.5, 164.8, 164.6, 164.0, 155.6, 154.9, 152.8, 152.4, 152.0,
149.8, 149.2, 148.5, 144.2, 143.4, 141.2, 139.6, 139.5, 139.3, 137.8,
134.5, 134.3, 132.4, 132.1, 132.0, 131.2, 130.2, 129.8, 129.0, 128.8,
128.8, 127.0, 124.8, 124.5, 124.3, 123.0, 123.0, 122.2, 121.6, 120.5,
120.1, 118.2, 117.5, 113.3, 113.1, 112.8, 112.6, 111.9, 110.5, 110.4,
96.4, 78.1, 59.6, 45.9, 44.2, 36.9, 36.2, 35.4, 31.9, 29.0, 26.2,
25.1, 19.0, 18.4, 13.4, 12.4. HRMS (ESI-TOF) m / z : [M] 2+ calcd for C 79 H 78 IrN 13 O 4 , 732.7959; found, 732.7973. Analytical
HPLC (10 to 100% B in 30 min, formic acid additive): Rt = 13.7 min. Synthesis of Ir(III)-COUPY Conjugate 3e Synthesis of Compound 19 To a solution
of trans -4- N -Boc-aminomethyl-cyclohexanecarboxylic
acid (56 mg, 0.22 mmol) and HATU (85.3 mg, 0.22 mmol) in anhydrous
DMF (6 mL) under an Ar atmosphere, DIPEA (77 μL, 0.44 mmol)
was added and the mixture stirred for 10 min under Ar at room temperature
and protected from light. After addition of a solution of coumarin 17 (37.6 mg, 0.073 mmol) and DIPEA (64 μL, 0.36 mmol)
in anhydrous DMF (6 mL), the reaction mixture was stirred for 2.5
h at room temperature under Ar and protected from light. After evaporation
under reduced pressure, the crude was purified by column chromatography
(silica gel, 0–11% MeOH in DCM) to give 35.4 mg of a pink solid
(yield: 68%). TLC: R f (10% MeOH in DCM)
0.38. 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 8.60 (1H, m), 8.53 (2H, d, J =
7.2 Hz), 8.16 (2H, d, J = 7.2 Hz), 7.73 (1H, m),
7.72 (1H, d, J = 9.2 Hz), 7.02 (1H, m), 6.98 (1H,
dd, J = 9.2, 2.4 Hz), 6.93 (1H, s), 6.78 (1H, t, J = 6.0 Hz), 5.24 (2H, s), 3.56 (4H, q, J = 7.2 Hz), 3.13 (2H, q, J = 6.4 Hz), 3.07 (2H,
q, J = 6.0 Hz), 2.75 (2H, t, J =
6.4 Hz), 2.55 (3H, s), 2.00 (1H, m), 1.68 (4H, m), 1.57 (2H, qt, J = 6.8 Hz), 1.36 (9H, s), 1.26 (4H, m), 1.18 (6H, t, J = 7.2 Hz), 0.84 (2H, m). 13 C{ 1 H}
NMR (101 Hz, DMSO- d 6 ): δ (ppm) 175.2,
166.8, 164.8, 155.7, 154.9, 152.8, 152.0, 149.2, 144.2, 127.0, 120.1,
118.2, 111.9, 110.5, 110.4, 96.4, 78.1, 77.3, 59.6, 46.1, 44.2, 44.1,
37.4, 36.8, 36.0, 29.5, 29.0, 28.8, 28.3, 18.4, 12.4. HRMS (ESI-TOF) m / z : [M] + calcd for C 39 H 53 N 6 O 5 , 685.4072; found, 685.4072. Synthesis of Compound 21 A cooled down
solution of hydrochloric acid in dioxane (4 M, 11 mL) was added to
coumarin 19 (28 mg, 0.039 mmol). The reaction mixture
was stirred for 20 min in an ice bath under an Ar atmosphere and protected
from light. After removal of the solvent, several co-evaporations
from acetonitrile were carried out. The crude product was used without
further purification since HPLC-MS analysis revealed that the removal
of the Boc group was quantitative. Analytical HPLC (10 to 100% B in
30 min, formic acid additive): Rt = 8.6 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 34 H 45 N 6 O 3 , 585.35; found, 585.31. Synthesis of Ir(III)-COUPY Conjugate 3e To a solution of Ir complex 2b (34.6 mg, 32.3 μmol)
and HATU (12.7 mg, 32.3 μmol) in anhydrous DMF (5 mL) under
an Ar atmosphere, DIPEA (12 μL, 64.7 μmol) was added and
the mixture stirred for 10 min under Ar at room temperature and protected
from light. After addition of a solution of coumarin 21 (25.5 mg, 38.8 μmol) and DIPEA (29 μL, 161.7 μmol)
in anhydrous DMF (6 mL), the reaction mixture was stirred for 2.5
h at room temperature under Ar and protected from light. After evaporation
under reduced pressure, the crude was purified by column chromatography
(silica gel, 0–12% MeOH in DCM) to give 34.7 mg of a purple
solid (yield: 58%). 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 14.40 (2H, br s), 8.89 (1H, d, J = 8.8 Hz), 8.70 (2H, m), 8.54 (2H, d, J = 7.5 Hz), 8.28 (1H, d, J = 9.0 Hz), 8.15 (3H,
m), 8.07 (1H, d, J = 8.9 Hz), 7.97 (1H, d, J = 6.9 Hz), 7.91 (1H, d, J = 7.6 Hz),
7.82 (2H, m), 7.70 (1H, d, J = 9.2 Hz), 7.62 (2H,
m), 7.52 (1H, d, J = 8.1 Hz), 7.46 (1H, d, J = 8.1 Hz), 7.19 (1H, t, J = 8.8 Hz),
7.12 (2H, q, J = 7.9 Hz), 6.95 (6H, m), 6.80 (1H,
t, J = 7.6 Hz), 6.75 (2H, t, J =
7.4 Hz), 6.63 (1H, s), 6.30 (1H, d, J = 7.2 Hz),
6.19 (1H, d, J = 7.4 Hz), 5.96 (1H, d, J = 8.3 Hz), 5.73 (1H, d, J = 8.2 Hz), 5.26 (2H,
s), 5.10 (2H, m), 3.54 (4H, q, J = 6.9 Hz), 3.07
(7H, m), 2.53 (2H, s), 2.07 (1H, m), 1.73 (6H, m), 1.58 (2H, qt, J = 6.9 Hz), 1.35 (3H, m), 1.16 (6H, t, J = 7.0 Hz), 0.92 (4H, m), 0.63 (3H, t, J = 7.2 Hz). 13 C NMR (101 MHz, DMSO- d 6 ): δ
(ppm) 175.2, 166.8, 165.8, 164.8, 164.5, 163.8, 155.6, 154.9, 152.8,
152.3, 152.0, 149.8, 149.2, 148.5, 144.2, 143.4, 141.3, 139.5, 139.2,
137.8, 134.5, 134.3, 134.2, 133.2, 133.1, 132.3, 132.3, 132.0, 131.2,
130.3, 129.9, 129.4, 128.9, 128.9, 128.8, 127.6, 127.0, 124.8, 124.6,
124.3, 123.1, 123.0, 122.9, 122.2, 121.6, 120.6, 120.1, 119.3, 118.2,
117.5, 113.3, 113.0, 112.9, 112.5, 111.9, 110.5, 110.4, 96.5, 78.1,
59.6, 45.9, 45.3, 44.2, 44.1, 37.1, 36.9, 36.0, 31.9, 29.7, 29.0,
28.9, 19.0, 18.4, 13.4, 12.4. HRMS (ESI-TOF) m / z : [M] 2+ calcd for C 81 H 80 IrN 13 O 4 , 745.8037; found, 745.8047. Analytical
HPLC (10 to 100% B in 30 min, formic acid additive): Rt = 15.8 min. Photophysical and Photochemical Characterization For
photophysical measurements, all solvents used were spectroscopic grade.
Absorption spectra were recorded in a Varian Cary 500 UV/vis/NIR or
Varian Cary 6000i spectrophotometer at room temperature. Molar absorption
coefficients (ε) were determined by direct application of the
Beer–Lambert law, using solutions of the compounds in each
solvent with concentrations ranging from 10 –6 to
10 –5 M. Emission spectra were registered in a Photon
Technology International (PTI) fluorimeter or in a Horiba Fluoromax-4
spectrofluorometer. Fluorescence quantum yields (Φ F ) were measured by the comparative method using cresyl violet in
ethanol (CV; Φ F;ref = 0.54 ± 0.03) as reference. 63 Then, optically matched solutions of the compounds
and CV were excited and the fluorescence spectra were recorded. The
absorbance of sample and reference solutions was set below 0.1 at
the excitation wavelength, and Φ F was calculated
using the following eq 1 1 where area sample and area ref are the integrated fluorescence for the sample and the reference
and η sample and η ref are the refractive
index of sample and reference solutions respectively. The uncertainty
in the experimental value of Φ F has been estimated
to be approximately 10%. Phosphorescence quantum yields (Φ P ) of the iridium complex were determined analogously to Φ F using argon-saturated meso-tetra-5,10,15,20-phenylporphine
as reference (Φ F = 0.11) in toluene. 64 Singlet oxygen generation was studied by time-resolved
near-infrared
phosphorescence by means of a customized setup. Briefly, a pulsed
Nd:YAG laser (FTSS355-Q, Crystal Laser, Berlin, Germany) working at
a 1 or 10 kHz repetition rate at 355 nm (0.5 μJ per pulse) or
532 nm (1.2 μJ per pulse) was used to excite the sample. A 1064
nm rugate notch filter (Edmund Optics) and an uncoated SKG-5 filter
(CVI Laser Corporation) were placed in the laser path to remove any
NIR emission. The light emitted by the sample was filtered with a
1000 nm long-pass filter (Edmund Optics) and later by a narrow bandpass
filter at 1275 nm (BK-1270-70-B, bk Interferenzoptik). A thermoelectric-cooled
NIR-sensitive photomultiplier tube assembly (H9170-45, Hamamatsu Photonics,
Hamamatsu, Japan) was used as a detector. Photon counting was achieved
with a multichannel scaler (NanoHarp 250, PicoQuant). The time dependence
of the 1 O 2 phosphorescence with the signal intensity S ( t ) is described by eq 2 , in which τ T and τ Δ are the lifetimes of the photosensitizer triplet state
and of 1 O 2 respectively, and S 0 is a preexponential parameter proportional to Φ Δ . 2 The Φ Δ values
of the different samples were
obtained by comparing S 0 values of optically
matched samples and using an appropriate reference by means of eq 3 . 3 The same setup was used to monitor
the phosphorescence of the complex
and the conjugate, except that the red-sensitive Hamamatsu H5783 photosensor
module was used for detection. Superoxide Anion Radical Generation and Characterization Using
DHR123 All compounds (10 μM) were prepared in PBS (0.2%
DMSO). To this solution, DHR123 was added so that its final concentration
was 10 μM. Then, the samples were irradiated in 1.0 × 0.5
cm cuvette by green light (505 nm centered LED) for indicated time
intervals. Immediately, the fluorescence spectra were collected by
using a Photon Technology International (PTI) fluorimeter. The excitation
wavelength was set to 500 nm, the excitation and emission slit widths
were 2 nm, and the integration time was set to 1 s. EPR Studies EPR measurements were carried out with
a Bruker Elexys 580E spectrometer working in X-band at room temperature.
Microwave frequency was 9.68 GHz. The modulation amplitude was 0.12
mT. Both the modulation and the microwave power were selected in such
a way that no distortion or saturation of the signal was produced.
A LED source was used for the irradiation of the sample (530 nm, 450
mW cm –2 ). The spin traps 2,2,6,6-tetramethylpiperidine
(TEMP, 60 mM) and 5,5-dimethyl-1-pyrroline- N -oxide
(DMPO, 360 mM) were dissolved in MeOH. In a typical experiment, 20
μL of TEMP or DMPO solutions was added to an Eppendorf tube
containing the compounds ( 3a–3c ) in solid form,
and the resulting solution was diluted with 280 μL of MeOH (final
concentration of conjugates 250 μM). The samples were loaded
into capillary tubes, which were placed in the EPR to register the
spectrum in dark conditions. Then, the same samples were irradiated
for 5 min and the EPR spectrum was registered again. Cell Culture and Cell Lines A375, HeLa, and SK-MEL-28
cells were cultured in Dulbecco’s modified Eagle medium (DMEM)
supplemented with 10% FBS, l -glutamine, and 1% penicillin–streptomycin.
A2780 and A2780cis cells were grown in RPMI-1640 cell medium supplemented
with 10% fetal bovine serum (FBS) and 2 mM l -glutamine. Acquired
resistance to cisplatin in the A2780cis cell line was maintained by
adding cisplatin 1 μM to culture medium every second passage.
Cells were cultured in a humidified incubator at 310 K with 5% CO 2 atmosphere and subcultured two or three times a week with
appropriate densities and were confirmed to be mycoplasma-free using
a standard Hoechst DNA staining method. Fluorescence Imaging HeLa Cells were maintained in
DMEM containing low glucose (1 g/L) and supplemented with 10% fetal
bovine serum (FBS), 50 U/mL penicillin–streptomycin, and 2
mM l -glutamine. For cellular uptake experiments and posterior
observation under the microscope, cells were seeded on glass bottom
dishes (P35G-1.5-14-C, Mattek). 24 h after cell seeding, cells were
incubated at 37 °C for 30 min with Ir(III)-COUPY conjugates 3a–3e (5 μM) in supplemented DMEM. Then, cells
were washed three times with Dulbecco’s phosphate-buffered
saline to remove the excess of the compounds and kept in low glucose
DMEM without phenol red supplemented with 10 mM Hepes for fluorescence
imaging. All microscopy observations were performed using a
Zeiss LSM 880 confocal microscope equipped with a 561 nm laser. The
microscope was also equipped with a heating insert (P Lab-Tek S, Pecon)
to keep cells at 37 °C. Cells were observed using a 63×
1.4 oil immersion objective. The compounds were excited using the
561 nm laser and emission detected from 570 to 670 nm. Image analysis
was performed using Fiji. 65 All images
are colorized using the Fire lookup table from Fiji. Cellular Accumulation by ICP–MS Briefly, A2780cis
cells were seeded onto a 12-well plate (3 × 10 5 cells/well).
Treatments with tested agents for 2 h were applied at 10 μM.
Cisplatin was included for comparative purposes. Cells were then trypsinized,
and pellets were counted. Samples were then digested with 30% HNO 3 suprapur acid (Sigma-Aldrich) and subjected to Inductively
Coupled Plasma Mass Spectrometry analysis in Agilent 7900 ICP–MS. 99 Ru, 101 Ru, 194 Pt, and 195 Pt isotopes were measured. Three independent experiments were performed
( n = 2 replicates). Photo- and Cytotoxic Activity Determination A2780,
A2780cis, HeLa, A375, and SK-MEL-28 cells were maintained at logarithmic
growth-phase and cultured in 96-well plates at a density of 5000 cells/well
in complete medium for 24 h at 310 K, 5% CO 2 in a humidified
incubator. For hypoxia experiments, a hypoxia condition was set up
by Tissue Culture Service at University of Murcia using nitrogen (N 2 ) to displace O 2 down to a minimum of 2% in a Forma
Steri-Cycle i160 incubator (Thermo Fisher Scientific). Serial dilutions
of the compounds were added at the final concentrations in the range
of 0–100 μM in a final volume of 100 μL per well.
A light-based treatment schedule was performed as follows: 1 h incubation
with the compounds in the dark, followed by 1 h incubation under irradiation
conditions by placing the Photoreactor EXPO-LED from Luzchem (Canada)
fitted with green lamps (final light intensity applied of 1.5 mW/cm 2 at λ max = 520 nm) inside the CO 2 incubator. All the cell culture plates subjected to light irradiation
included untreated controls to verify that cell viability was not
affected by light. Then, treatment-containing media was removed, and
fresh media was added for a 48 h cell recovery period; the temperature
throughout the experiment remained at 310 K. Dark control samples
were placed in the same dark conditions and then kept incubated for
1 h in the dark in the humidified CO 2 incubator. Medium
was then aspirated by suction, cells were loaded with 50 μL
of MTT solution (1 mg/mL) for additional 4 h and then removed, and
50 μL of DMSO was added to solubilize the purple formazan crystals
formed in active cells. The absorbance was measured at 570 nm using
a microplate reader (FLUOstar Omega), and the IC 50 values
were calculated based on the inhibitory rate curves using the following
equation 4 where I represents the percentage
inhibition of viability observed, I max is the maximal inhibitory effect, IC 50 is the concentration
that inhibits 50% of maximal growth, C is the concentration
of the treatment, and n is the slope of the semi-logarithmic
dose–response sigmoidal curves. The non-linear fitting was
performed using SigmaPlot 14.0 software. All experiments were performed
in three independent studies with n = 3 replicates
per concentration level. For a detailed phototoxicity procedure, ref ( 43 ) includes technical explanations. ROS Photogeneration in A2780cis Cells ROS levels were
determined using the 2′-7′dichlorofluorescein diacetate
(DCFH-DA) probe. A2780cis cells were seeded onto 96-well black plates
at 2 × 10 4 cells/well for 24 h in a humidified CO 2 incubator either in normoxia (21% O 2 ) or hypoxia
(2% O 2 ). Tested compounds were then administered in cell
media for the allowed time. Treatments were then removed, and cells
were stained with 10 μM of DCFH-DA for 0.5 h. After staining,
cells were washed with PBS twice and irradiated for 1 h with LED source
light from Luzchem photoreactor (Canada) fitted with green lamps (final
light intensity applied of 1.5 mW/cm 2 at λ max = 520 nm). Fluorescence readings were performed in FLUOstar Omega
(λ exc = 488 nm and λ em = 530 ±
30 nm). Non-irradiated plates were used for dark conditions, whereas
treated, unstained cells were used to subtract basal fluorescence
of compounds and correct fluorescence readings. Unstained cells served
as blank. Three independent experiments were performed with n = 3 replicates. Cell Death Induction Assays Cell death induction was
evaluated using the Annexin V-FITC/Propidium iodide (AV/PI) dual staining
method. Briefly, A2780cis cells were seeded in 12-well plates at a
density of 2 × 10 5 cells/well and incubated overnight
at 310 K. Tested compounds (10 μM) were added, following the
described treatment schedule (1 h incubation + 1 h irradiation with
520 nm light). Irradiated, non-treated cells served as the control
group. After 24 h of the drug-free recovery period, cells were harvested
by trypsinization, washed with PBS and centrifuged, and the pellets
were resuspended in 200 μL of a binding buffer as instructed
by the manufacturer (Cayman). The resuspended cell solution was left
at room temperature in the dark for 15 min before analysis by flow
cytometry (FACSCalibur BecktonDickinson; 10 4 events acquired
per sample). Cells were visualized using λ exc = 488
channels and registered at 525 nm and 620 nm for Annexin V and propidium
iodide in FL1 and FL2 channels, respectively. Cell populations were
classified as follows: AV - /PI - (viable cells);
AV - /PI + (necrotic cells); AV + /PI - and AV + /PI + (apoptotic cells). Alternatively,
A2780cis cells were subjected to flow cytometry after phototreatments
and morphological changes were analyzed using FSC and SSC contour
plots. Data were analyzed using FlowJo software. Three independent
experiments were performed. 3D Multicellular Tumor Spheroid Growth Inhibition Assays Briefly, a single suspension of A2780cis cells at a density of 5
× 10 3 cells/well in complete RPMI medium was dispatched
onto 96-well Corning microplates with ultralow attachment surface
coating. The plates were covered and transferred to an incubator at
310 K with a 5% CO 2 atmosphere. After 3 days post-seeding,
uniform MCTSs were formed, which was confirmed using an inverted Zeiss
AXIO observer 7 microscope. Cell media of MCTSs was changed by replacing
50% with fresh cell media and allowed to grow for 3 days. On day 3,
MTCS was incubated with tested agents (10 μM) for 4 h and then
irradiated with 520 nm light for 1 h. Treatments were then carefully
removed, and fresh media was added. The integrity, radius, size, and
volume of the MCTSs were monitored using a DMi1 inverted phase contrast
microscope (Leica Microsystems) over 7 days. The radius of the tumorspheres
was measured using Fiji software, and the volume was calculated using
the following equation: V = 4/3π r 3 .
## Materials and Methods
Materials and Methods Unless otherwise stated, common
chemicals and solvents (HPLC grade or reagent grade quality) were
purchased from commercial sources and used without further purification.
Aluminium plates coated with a 0.2 mm thick layer of silica gel 60
F 254 were used for thin-layer chromatography analyses (TLC),
whereas flash column chromatography purification was carried out using
silica gel 60 (230–400 mesh). NMR spectra were recorded at
25 °C in a 400 MHz spectrometer using the deuterated solvent
as an internal deuterium lock. The residual protic signal of CHCl 3 and DMSO was used as a reference in 1 H and 13 C NMR spectra recorded in CDCl 3 and DMSO- d 6 , respectively. Chemical shifts are reported
in part per million (ppm) in the δ scale, coupling constants
in Hz, and multiplicity as follows: s (singlet), d (doublet), t (triplet),
q (quartet), qt (quintuplet), m (multiplet), dd (doublet of doublets),
dt (doublet of triplets), td (triplet of doublets), br (broad signal),
and so forth. Electrospray ionization mass spectra (ESI-MS) were recorded
on an instrument equipped with single quadrupole detector coupled
to an HPLC and high-resolution (HR) ESI-MS on an LC/MS-TOF instrument.
The purity of final compounds was determined by reversed-phase high
performance liquid chromatography (HPLC) analyses on a Jupiter Proteo
C12 column (250 × 4.6 mm, 90 Å, 4 μm, flow rate: 1
mL/min) using linear gradients of 0.1% formic acid in Milli-Q H 2 O (A) and 0.1% formic acid in ACN (B). The HPLC column was
maintained at 25 °C. All final compounds were >95% pure by
this
method.
## Synthesis of COUPY
Synthesis of COUPY 1b Synthesis of Compound 4 Phenol (10 g,
106 mmol) and ethyl acetoacetate (13.6 mL, 106 mmol) were mixed and
heated to 100 °C in nitrobenzene (10 mL). In a separate flask,
AlCl 3 (28.1 g, 213 mmol) was dissolved in nitrobenzene
(100 mL) at 0 °C—the AlCl 3 was added in six
portions and left to stir at room temperature for 15–30 min.
Then, the AlCl 3 solution was decanted from excess and added
dropwise to the phenol and ethyl acetoacetate solution over 45 min.
Once the addition was completed, the temperature was increased to
125 °C and the reaction mixture was stirred under reflux for
3 h. The reaction was then cooled to 0 °C and a 1:1 (v/v) mixture
of HCl/H 2 O (100 mL) was added. The mixture was then filtrated,
and the solid was washed with ethyl acetate. The filtrate was transferred
to a separating flask where the aqueous and organic layers were separated:
the organic layer was dried over anhydrous Na 2 SO 4 and filtered. The ethyl acetate was removed by evaporation under
reduced pressure and nitrobenzene by distillation in vacuo. The crude
product was then purified by column chromatography (silica gel, 0–100%
DCM in hexanes) to give 3.79 g (22%) of a brown solid. TLC: R f (DCM) 0.34. 1 H NMR (400 MHz, CDCl 3 ): δ (ppm) 7.61 (1H, dd, J = 8.0, 1.6
Hz), 7.54 (1H, m), 7.32 (2H, m), 6.30 (1H, s), 2.45 (3H, s). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ
(ppm) 160.9, 153.6, 152.5, 131.9, 124.7, 124.3, 120.1, 117.2, 115.2,
18.8. LRMS (ESI-TOF) m / z : [M + H] + calcd for C 10 H 9 O 2 , 161.06;
found, 160.83. Synthesis of Compound 5 Coumarin 4 (4.48 g, 28.0 mmol) was mixed with Lawesson’s Reagent
(5.66 g, 14.0 mmol) in toluene (150 mL) and refluxed for 19 h at 105
°C. The crude was then evaporated under reduced pressure and
purified by column chromatography (silica gel, 0–50% DCM in
hexanes) to obtain 3.80 g of a yellow solid (77% yield). TLC: R f (75:25, DCM/hexanes) 0.88. 1 H NMR
(400 MHz, CDCl 3 ): δ (ppm) 7.65 (1H, dd, J = 8.0, 1.6 Hz), 7.59 (1H, m), 7.49 (1H, m), 7.36 (1H, m), 7.18 (1H,
m), 2.38 (3H, s). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ (ppm) 197.6, 156.2, 144.3, 132.2, 129.1, 125.5,
124.6, 121.6, 117.1, 18.1. HRMS (ESI-TOF) m / z : [M + H] + calcd for C 10 H 9 OS, 177.0369: found, 177.0369. Synthesis of Compound 6 4-Pyridylacetonitrile
hydrochloride (1.71 g, 11.08 mmol) and NaH (3.41 g of a 60% dispersion
in mineral oil, 85.2 mmol) were dried together in a desiccator alongside
compound 5 (1.50 g, 8.52 mmol) in a separate flask. Both
were then dissolved in anhydrous ACN (90 and 30 mL, respectively)
and the first solution passed to the second and the resulting mixture
stirred for 2.5 h at room temperature and under an argon atmosphere.
Afterward, AgNO 3 (3.18 g, 18.74 mmol) was added and the
mixture stirred for 2 h under Ar atmosphere and protected from light.
The crude product was then evaporated under reduced pressure and purified
by column chromatography (silica gel, 50–100% DCM in hexanes
first, then 0–0.65% MeOH in DCM) to give 913 mg (41% yield)
of a brown solid. TLC: R f (DCM/AcOEt 1:1)
0.48. 1 H NMR (400 MHz, CDCl 3 ): δ (ppm)
8.61 (2H, d, J = 5.8 Hz), 7.75 (2H, d, J = 5.8 Hz), 7.51 (2H, m), 7.32 (2H, m), 7.04 (1H, s), 2.42 (3H, s). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ
(ppm) 161.9, 152.1, 150.1, 142.8, 140.1, 131.9, 125.3, 124.6, 121.3,
121.2, 118.9, 118.0, 116.4, 84.8, 18.7. HRMS (ESI-TOF) m / z : [M + H] + calcd for C 17 H 13 N 2 O, 261.1022, found, 261.1026. Synthesis of Compound 1b Methyl trifluoromethanesulfonate
(16 μL, 0.14 mmol) was added to a solution of coumarin 6 (18.1 mg, 0.070 mmol) in DCM (10 mL) under an argon atmosphere.
The mixture was stirred overnight at room temperature and protected
from light. The reaction mixture was evaporated under reduced pressure
and purified by column chromatography (silica gel, 0–10% MeOH
in DCM) to give 16.1 mg of a yellow solid (yield 55%). TLC: R f (10% MeOH in DCM) 0.34. 1 H NMR
(400 MHz, DMSO- d 6 ): δ (ppm) 8.73
(2H, d, J = 8.0 Hz), 8.32 (2H, d, J = 7.6 Hz), 7.92 (2H, m), 7.81 (1H, m), 7.56 (1H, m), 7.30 (1H, d, J = 1.6 Hz), 4.27 (3H, s), 2.62 (3H, d, J = 1.2 Hz). 13 C{ 1 H} NMR (101 Hz, DMSO- d 6 ): δ (ppm) 165.9, 151.6, 150.3, 147.9,
144.5, 133.3, 126.5, 125.6, 122.3, 120.8, 117.4, 117.1, 116.8, 81.3,
46.6, 18.5. HRMS (ESI-TOF) m / z :
[M] + calcd for C 18 H 15 N 2 O, 275.1179; found, 275.1179.
## Synthesis of Compound
Synthesis of Compound 4 Phenol (10 g,
106 mmol) and ethyl acetoacetate (13.6 mL, 106 mmol) were mixed and
heated to 100 °C in nitrobenzene (10 mL). In a separate flask,
AlCl 3 (28.1 g, 213 mmol) was dissolved in nitrobenzene
(100 mL) at 0 °C—the AlCl 3 was added in six
portions and left to stir at room temperature for 15–30 min.
Then, the AlCl 3 solution was decanted from excess and added
dropwise to the phenol and ethyl acetoacetate solution over 45 min.
Once the addition was completed, the temperature was increased to
125 °C and the reaction mixture was stirred under reflux for
3 h. The reaction was then cooled to 0 °C and a 1:1 (v/v) mixture
of HCl/H 2 O (100 mL) was added. The mixture was then filtrated,
and the solid was washed with ethyl acetate. The filtrate was transferred
to a separating flask where the aqueous and organic layers were separated:
the organic layer was dried over anhydrous Na 2 SO 4 and filtered. The ethyl acetate was removed by evaporation under
reduced pressure and nitrobenzene by distillation in vacuo. The crude
product was then purified by column chromatography (silica gel, 0–100%
DCM in hexanes) to give 3.79 g (22%) of a brown solid. TLC: R f (DCM) 0.34. 1 H NMR (400 MHz, CDCl 3 ): δ (ppm) 7.61 (1H, dd, J = 8.0, 1.6
Hz), 7.54 (1H, m), 7.32 (2H, m), 6.30 (1H, s), 2.45 (3H, s). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ
(ppm) 160.9, 153.6, 152.5, 131.9, 124.7, 124.3, 120.1, 117.2, 115.2,
18.8. LRMS (ESI-TOF) m / z : [M + H] + calcd for C 10 H 9 O 2 , 161.06;
found, 160.83.
## Synthesis of Compound
Synthesis of Compound 5 Coumarin 4 (4.48 g, 28.0 mmol) was mixed with Lawesson’s Reagent
(5.66 g, 14.0 mmol) in toluene (150 mL) and refluxed for 19 h at 105
°C. The crude was then evaporated under reduced pressure and
purified by column chromatography (silica gel, 0–50% DCM in
hexanes) to obtain 3.80 g of a yellow solid (77% yield). TLC: R f (75:25, DCM/hexanes) 0.88. 1 H NMR
(400 MHz, CDCl 3 ): δ (ppm) 7.65 (1H, dd, J = 8.0, 1.6 Hz), 7.59 (1H, m), 7.49 (1H, m), 7.36 (1H, m), 7.18 (1H,
m), 2.38 (3H, s). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ (ppm) 197.6, 156.2, 144.3, 132.2, 129.1, 125.5,
124.6, 121.6, 117.1, 18.1. HRMS (ESI-TOF) m / z : [M + H] + calcd for C 10 H 9 OS, 177.0369: found, 177.0369.
## Synthesis of Compound
Synthesis of Compound 6 4-Pyridylacetonitrile
hydrochloride (1.71 g, 11.08 mmol) and NaH (3.41 g of a 60% dispersion
in mineral oil, 85.2 mmol) were dried together in a desiccator alongside
compound 5 (1.50 g, 8.52 mmol) in a separate flask. Both
were then dissolved in anhydrous ACN (90 and 30 mL, respectively)
and the first solution passed to the second and the resulting mixture
stirred for 2.5 h at room temperature and under an argon atmosphere.
Afterward, AgNO 3 (3.18 g, 18.74 mmol) was added and the
mixture stirred for 2 h under Ar atmosphere and protected from light.
The crude product was then evaporated under reduced pressure and purified
by column chromatography (silica gel, 50–100% DCM in hexanes
first, then 0–0.65% MeOH in DCM) to give 913 mg (41% yield)
of a brown solid. TLC: R f (DCM/AcOEt 1:1)
0.48. 1 H NMR (400 MHz, CDCl 3 ): δ (ppm)
8.61 (2H, d, J = 5.8 Hz), 7.75 (2H, d, J = 5.8 Hz), 7.51 (2H, m), 7.32 (2H, m), 7.04 (1H, s), 2.42 (3H, s). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ
(ppm) 161.9, 152.1, 150.1, 142.8, 140.1, 131.9, 125.3, 124.6, 121.3,
121.2, 118.9, 118.0, 116.4, 84.8, 18.7. HRMS (ESI-TOF) m / z : [M + H] + calcd for C 17 H 13 N 2 O, 261.1022, found, 261.1026.
## Synthesis of Compound
Synthesis of Compound 1b Methyl trifluoromethanesulfonate
(16 μL, 0.14 mmol) was added to a solution of coumarin 6 (18.1 mg, 0.070 mmol) in DCM (10 mL) under an argon atmosphere.
The mixture was stirred overnight at room temperature and protected
from light. The reaction mixture was evaporated under reduced pressure
and purified by column chromatography (silica gel, 0–10% MeOH
in DCM) to give 16.1 mg of a yellow solid (yield 55%). TLC: R f (10% MeOH in DCM) 0.34. 1 H NMR
(400 MHz, DMSO- d 6 ): δ (ppm) 8.73
(2H, d, J = 8.0 Hz), 8.32 (2H, d, J = 7.6 Hz), 7.92 (2H, m), 7.81 (1H, m), 7.56 (1H, m), 7.30 (1H, d, J = 1.6 Hz), 4.27 (3H, s), 2.62 (3H, d, J = 1.2 Hz). 13 C{ 1 H} NMR (101 Hz, DMSO- d 6 ): δ (ppm) 165.9, 151.6, 150.3, 147.9,
144.5, 133.3, 126.5, 125.6, 122.3, 120.8, 117.4, 117.1, 116.8, 81.3,
46.6, 18.5. HRMS (ESI-TOF) m / z :
[M] + calcd for C 18 H 15 N 2 O, 275.1179; found, 275.1179.
## Synthesis of Ir(III)-COUPY Conjugate
Synthesis of Ir(III)-COUPY Conjugate 3b Synthesis of Compound 7 A solution of
methyl bromoacetate (0.9 mL, 9.25 mmol) and coumarin 6 (481 mg, 1.85 mmol) in a 2.5:1 (v/v) mixture of AcOEt and DCM (35
mL) was stirred under reflux at 60 °C for 48 h. The reaction
mixture was then evaporated under reduced pressure and purified by
column chromatography (silica gel, 0–6.5% MeOH in DCM) to give
623 mg (82% yield) of an orange solid. TLC: R f (10% MeOH in DCM) 0.37. 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 8.76 (2H, d, J = 7.4 Hz), 8.41 (2H, d, J = 7.4 Hz), 8.04 (1H,
d, J = 7.6 Hz), 7.95 (1H, dd, J =
8.0, 1.6 Hz), 7.83 (1H, td, J = 8.0, 1.6 Hz), 7.58
(1H, m), 7.35 (1H, s), 5.60 (2H, s), 3.79 (3H, s), 2.65 (3H, s). 13 C{ 1 H} NMR (101 Hz, DMSO- d 6 ): δ (ppm) 167.3, 166.5, 151.7, 151.3, 149.3, 144.9,
133.5, 126.6, 125.6, 122.0, 120.8, 117.4, 117.3, 116.9, 81.6, 58.9,
53.0, 18.6. HRMS (ESI-TOF) m / z :
[M] + calcd for C 20 H 17 N 2 O 3 , 333.1234; found, 333.1243. Synthesis of Compound 8 Compound 7 (214 mg, 0.52 mmol) was refluxed in a solution containing
concentrated HCl (43 mL, 520 mmol) and H 2 O (86 mL, Milli-Q
quality) at 60 °C for 5.5 h. The crude mixture was then evaporated
under reduced pressure to give a red solid, which was used without
further purification in the next step. Analytical HPLC (10 to 100%
B in 30 min, formic acid additive): Rt = 12.5 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 19 H 15 N 2 O 3 , 319.11; found, 319.53. Synthesis of Compound 9 Coumarin 8 (80.4 mg, 0.23 mmol) and HATU (88.8 mg, 0.23 mmol) were
dissolved in anhydrous DMF (8 mL) under an Ar atmosphere. After addition
of DIPEA (40 μL, 0.23 mmol), the reaction mixture was stirred
for 5 min under Ar at room temperature and protected from light. On
the other hand, DIPEA (40 μL, 0.23 mmol) was added to a solution
of N -Boc-1,3-propanediamine hydrochloride (57.3 mg,
0.27 mmol) in anhydrous DMF (5 mL), and the resulting mixture was
combined with the coumarin solution. After addition of DIPEA (40 μL,
0.23 mmol), the reaction mixture was stirred for 2 h at room temperature
under Ar and protected from light. The crude product was evaporated
under reduced pressure and purified by column chromatography (silica
gel, 0–12% MeOH in DCM) to give 30.5 mg of a purple solid (yield:
26%). TLC: R f (10% MeOH in DCM) 0.26. 1 H NMR (400 MHz, DMSO- d 6 ): δ
(ppm) 8.69 (2H, d, J = 7.4 Hz), 8.52 (1H, t, J = 5.6 Hz), 8.36 (2H, d, J = 7.4 Hz),
7.98 (1H, dd, J = 8.4, 1.2 Hz), 7.94 (1H, dd, J = 8.0, 1.2 Hz), 7.82 (1H, td, J = 7.2
Hz, J = 1.6 Hz), 7.57 (1H, td, J = 8.3, 0.8 Hz), 7.33 (1H, d, J = 1.2 Hz), 6.82
(1H, t, J = 5.6 Hz), 5.33 (2H, s), 3.15 (2H, q, J = 6.4 Hz), 2.97 (2H, q, J = 6.4 Hz),
2.64 (3H, d, J = 1.2 Hz), 1.58 (2H, qt, J = 7.0 Hz), 1.37 (9H, s). 13 C{ 1 H} NMR (101
Hz, DMSO- d 6 ): δ (ppm) 166.3, 164.6,
155.6, 151.7, 150.8, 148.7, 145.0, 133.4, 126.6, 125.7, 121.8, 120.8,
117.4, 116.9, 81.5, 77.5, 60.1, 37.5, 36.9, 29.3, 28.2, 18.6. HRMS
(ESI-TOF) m / z : [M] + calcd
for C 27 H 31 N 4 O 4 , 475.2340;
found, 475.2336. Synthesis of Compound 10 A cooled down
solution of hydrochloric acid in dioxane (4 M, 3 mL) was added to
coumarin 9 (10 mg, 0.021 mmol). The reaction mixture
was stirred for 25 min in an ice bath under an Ar atmosphere and protected
from light. After removal of the solvent under reduced pressure, several
co-evaporations from acetonitrile were carried out. The crude product
was used without further purification in the next step since HPLC-MS
analysis revealed that the removal of the Boc group was quantitative.
Analytical HPLC (10 to 100% B in 30 min, formic acid additive): Rt
= 5.5 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 22 H 23 N 4 O 2 , 375.18; found, 375.19. Synthesis of Ir(III)-COUPY Conjugate 3b To a solution of Ir complex 2b (15.6 mg, 14.6 μmol)
and HATU (5.7 mg, 14.6 μmol) in anhydrous DMF (2.5 mL) under
an Ar atmosphere, DIPEA (3 μL, 14.6 μmol) was added and
the mixture stirred for 10 min under Ar at room temperature and protected
from light. After addition of a solution of coumarin 10 (9.8 mg, 21.9 μmol) and DIPEA (15 μL, 73 μmol)
in anhydrous DMF (3 mL), the reaction mixture was stirred for 2.5
h at room temperature under Ar and protected from light. After evaporation
under reduced pressure, the crude was purified by column chromatography
(silica gel, 0–12.5% MeOH in DCM) to give 10.1 mg of a yellow
solid (yield: 47%). TLC: R f (15% MeOH
in DCM) 0.61. 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 14.19 (1H, br s), 13.97 (1H, br s), 8.89
(1H, d, J = 8.7 Hz), 8.72 (3H, m), 8.35 (2H, d, J = 7.4 Hz), 8.28 (1H, d, J = 8.8 Hz),
8.15 (1H, d, J = 8.4 Hz), 8.09 (1H, d, J = 8.8 Hz), 7.95 (2H, m), 7.82 (3H, m), 7.64 (1H, t, J = 7.8 Hz), 7.55 (2H, d, m), 7.47 (1H, d, J = 8.1
Hz), 7.32 (1H, d, J = 1.2 Hz), 7.19 (1H, t, J = 8.5 Hz), 7.10 (2H, m), 7.01 (1H, t, J = 7.5 Hz), 6.89 (1H, t, J = 7.5 Hz), 6.83 (1H,
t, J = 7.7 Hz), 6.76 (2H, t, J =
7.3 Hz), 6.68 (1H, s), 6.31 (1H, d, J = 7.3 Hz),
6.19 (1H, d, J = 7.3 Hz), 5.97 (1H, d, J = 8.3 Hz), 5.71 (1H, d, J = 8.3 Hz), 5.40 (2H,
s), 5.10 (2H, m), 3.20 (4H, m), 2.63 (3H, d, J =
1.1 Hz), 1.69 (4H, m), 1.23 (4H, m), 0.91 (2H, m), 0.65 (3H, t, J = 7.2 Hz). 13 C NMR (101 MHz, DMSO- d 6 ): δ (ppm) 166.3, 165.8, 164.7, 164.4, 163.8, 155.6,
152.4, 151.7, 150.8, 149.8, 148.7, 148.4, 145.0, 143.3, 141.3, 139.5,
139.5, 139.2, 137.9, 134.2, 134.1, 133.4, 133.0, 132.9, 132.3, 132.1,
132.0, 131.3, 130.4, 130.0, 129.6, 129.4, 128.9, 128.9, 128.8, 126.5,
125.7, 124.7, 124.6, 124.4, 123.2, 123.0, 122.4, 121.8, 121.6, 120.8,
120.6, 117.8, 117.4, 117.3, 116.9, 113.4, 112.9, 112.6, 81.5, 60.2,
56.0, 45.9, 37.1, 37.0, 31.9, 29.0, 19.0, 18.6, 13.4. Analytical HPLC
(10 to 100% B in 30 min, formic acid additive): Rt = 12.6 min. HRMS
(ESI-TOF) m / z : [M] 2+ calcd
for C 69 H 58 IrN 11 O 3 , 640.7171;
found, 640.7169.
## Synthesis of Compound
Synthesis of Compound 7 A solution of
methyl bromoacetate (0.9 mL, 9.25 mmol) and coumarin 6 (481 mg, 1.85 mmol) in a 2.5:1 (v/v) mixture of AcOEt and DCM (35
mL) was stirred under reflux at 60 °C for 48 h. The reaction
mixture was then evaporated under reduced pressure and purified by
column chromatography (silica gel, 0–6.5% MeOH in DCM) to give
623 mg (82% yield) of an orange solid. TLC: R f (10% MeOH in DCM) 0.37. 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 8.76 (2H, d, J = 7.4 Hz), 8.41 (2H, d, J = 7.4 Hz), 8.04 (1H,
d, J = 7.6 Hz), 7.95 (1H, dd, J =
8.0, 1.6 Hz), 7.83 (1H, td, J = 8.0, 1.6 Hz), 7.58
(1H, m), 7.35 (1H, s), 5.60 (2H, s), 3.79 (3H, s), 2.65 (3H, s). 13 C{ 1 H} NMR (101 Hz, DMSO- d 6 ): δ (ppm) 167.3, 166.5, 151.7, 151.3, 149.3, 144.9,
133.5, 126.6, 125.6, 122.0, 120.8, 117.4, 117.3, 116.9, 81.6, 58.9,
53.0, 18.6. HRMS (ESI-TOF) m / z :
[M] + calcd for C 20 H 17 N 2 O 3 , 333.1234; found, 333.1243.
## Synthesis of Compound
Synthesis of Compound 8 Compound 7 (214 mg, 0.52 mmol) was refluxed in a solution containing
concentrated HCl (43 mL, 520 mmol) and H 2 O (86 mL, Milli-Q
quality) at 60 °C for 5.5 h. The crude mixture was then evaporated
under reduced pressure to give a red solid, which was used without
further purification in the next step. Analytical HPLC (10 to 100%
B in 30 min, formic acid additive): Rt = 12.5 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 19 H 15 N 2 O 3 , 319.11; found, 319.53.
## Synthesis of Compound
Synthesis of Compound 9 Coumarin 8 (80.4 mg, 0.23 mmol) and HATU (88.8 mg, 0.23 mmol) were
dissolved in anhydrous DMF (8 mL) under an Ar atmosphere. After addition
of DIPEA (40 μL, 0.23 mmol), the reaction mixture was stirred
for 5 min under Ar at room temperature and protected from light. On
the other hand, DIPEA (40 μL, 0.23 mmol) was added to a solution
of N -Boc-1,3-propanediamine hydrochloride (57.3 mg,
0.27 mmol) in anhydrous DMF (5 mL), and the resulting mixture was
combined with the coumarin solution. After addition of DIPEA (40 μL,
0.23 mmol), the reaction mixture was stirred for 2 h at room temperature
under Ar and protected from light. The crude product was evaporated
under reduced pressure and purified by column chromatography (silica
gel, 0–12% MeOH in DCM) to give 30.5 mg of a purple solid (yield:
26%). TLC: R f (10% MeOH in DCM) 0.26. 1 H NMR (400 MHz, DMSO- d 6 ): δ
(ppm) 8.69 (2H, d, J = 7.4 Hz), 8.52 (1H, t, J = 5.6 Hz), 8.36 (2H, d, J = 7.4 Hz),
7.98 (1H, dd, J = 8.4, 1.2 Hz), 7.94 (1H, dd, J = 8.0, 1.2 Hz), 7.82 (1H, td, J = 7.2
Hz, J = 1.6 Hz), 7.57 (1H, td, J = 8.3, 0.8 Hz), 7.33 (1H, d, J = 1.2 Hz), 6.82
(1H, t, J = 5.6 Hz), 5.33 (2H, s), 3.15 (2H, q, J = 6.4 Hz), 2.97 (2H, q, J = 6.4 Hz),
2.64 (3H, d, J = 1.2 Hz), 1.58 (2H, qt, J = 7.0 Hz), 1.37 (9H, s). 13 C{ 1 H} NMR (101
Hz, DMSO- d 6 ): δ (ppm) 166.3, 164.6,
155.6, 151.7, 150.8, 148.7, 145.0, 133.4, 126.6, 125.7, 121.8, 120.8,
117.4, 116.9, 81.5, 77.5, 60.1, 37.5, 36.9, 29.3, 28.2, 18.6. HRMS
(ESI-TOF) m / z : [M] + calcd
for C 27 H 31 N 4 O 4 , 475.2340;
found, 475.2336.
## Synthesis of Compound
Synthesis of Compound 10 A cooled down
solution of hydrochloric acid in dioxane (4 M, 3 mL) was added to
coumarin 9 (10 mg, 0.021 mmol). The reaction mixture
was stirred for 25 min in an ice bath under an Ar atmosphere and protected
from light. After removal of the solvent under reduced pressure, several
co-evaporations from acetonitrile were carried out. The crude product
was used without further purification in the next step since HPLC-MS
analysis revealed that the removal of the Boc group was quantitative.
Analytical HPLC (10 to 100% B in 30 min, formic acid additive): Rt
= 5.5 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 22 H 23 N 4 O 2 , 375.18; found, 375.19.
## Synthesis of Ir(III)-COUPY Conjugate
Synthesis of Ir(III)-COUPY Conjugate 3b To a solution of Ir complex 2b (15.6 mg, 14.6 μmol)
and HATU (5.7 mg, 14.6 μmol) in anhydrous DMF (2.5 mL) under
an Ar atmosphere, DIPEA (3 μL, 14.6 μmol) was added and
the mixture stirred for 10 min under Ar at room temperature and protected
from light. After addition of a solution of coumarin 10 (9.8 mg, 21.9 μmol) and DIPEA (15 μL, 73 μmol)
in anhydrous DMF (3 mL), the reaction mixture was stirred for 2.5
h at room temperature under Ar and protected from light. After evaporation
under reduced pressure, the crude was purified by column chromatography
(silica gel, 0–12.5% MeOH in DCM) to give 10.1 mg of a yellow
solid (yield: 47%). TLC: R f (15% MeOH
in DCM) 0.61. 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 14.19 (1H, br s), 13.97 (1H, br s), 8.89
(1H, d, J = 8.7 Hz), 8.72 (3H, m), 8.35 (2H, d, J = 7.4 Hz), 8.28 (1H, d, J = 8.8 Hz),
8.15 (1H, d, J = 8.4 Hz), 8.09 (1H, d, J = 8.8 Hz), 7.95 (2H, m), 7.82 (3H, m), 7.64 (1H, t, J = 7.8 Hz), 7.55 (2H, d, m), 7.47 (1H, d, J = 8.1
Hz), 7.32 (1H, d, J = 1.2 Hz), 7.19 (1H, t, J = 8.5 Hz), 7.10 (2H, m), 7.01 (1H, t, J = 7.5 Hz), 6.89 (1H, t, J = 7.5 Hz), 6.83 (1H,
t, J = 7.7 Hz), 6.76 (2H, t, J =
7.3 Hz), 6.68 (1H, s), 6.31 (1H, d, J = 7.3 Hz),
6.19 (1H, d, J = 7.3 Hz), 5.97 (1H, d, J = 8.3 Hz), 5.71 (1H, d, J = 8.3 Hz), 5.40 (2H,
s), 5.10 (2H, m), 3.20 (4H, m), 2.63 (3H, d, J =
1.1 Hz), 1.69 (4H, m), 1.23 (4H, m), 0.91 (2H, m), 0.65 (3H, t, J = 7.2 Hz). 13 C NMR (101 MHz, DMSO- d 6 ): δ (ppm) 166.3, 165.8, 164.7, 164.4, 163.8, 155.6,
152.4, 151.7, 150.8, 149.8, 148.7, 148.4, 145.0, 143.3, 141.3, 139.5,
139.5, 139.2, 137.9, 134.2, 134.1, 133.4, 133.0, 132.9, 132.3, 132.1,
132.0, 131.3, 130.4, 130.0, 129.6, 129.4, 128.9, 128.9, 128.8, 126.5,
125.7, 124.7, 124.6, 124.4, 123.2, 123.0, 122.4, 121.8, 121.6, 120.8,
120.6, 117.8, 117.4, 117.3, 116.9, 113.4, 112.9, 112.6, 81.5, 60.2,
56.0, 45.9, 37.1, 37.0, 31.9, 29.0, 19.0, 18.6, 13.4. Analytical HPLC
(10 to 100% B in 30 min, formic acid additive): Rt = 12.6 min. HRMS
(ESI-TOF) m / z : [M] 2+ calcd
for C 69 H 58 IrN 11 O 3 , 640.7171;
found, 640.7169.
## Synthesis of COUPY
Synthesis of COUPY 1c Synthesis of Compound 11 Coumarin 102
(1.17 g, 4.58 mmol) and Lawesson’s reagent (1.11 g, 2.75 mmol)
were dissolved in toluene (30 mL) and heated at 100 °C for 15
h. After evaporation under reduced pressure, the residue was purified
by column chromatography (silica gel, 50–70% DCM in hexanes)
to give an orange solid (666 mg, 54%): TLC R f (DCM) 0.53. 1 H NMR (400 MHz, CDCl 3 ):
δ (ppm) 7.03 (1H, s), 6.91 (1H, br q, J = 0.8
Hz), 3.29 (4H, q, J = 6.8 Hz), 3.00 (2H, t, J = 6.4 Hz), 2.79 (2H, t, J = 6.4 Hz),
2.27 (3H, d, J = 0.8 Hz), 1.98 (4H, m). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ (ppm) 196.4,
154.5, 146.5, 146.4, 123.4, 121.8, 120.2, 111.2, 106.1, 50.1, 49.8,
28.0, 21.5, 20.7, 20.5, 18.1; HRMS (ESI-TOF) m / z : [M + H] + calcd for C 16 H 18 NOS, 272.1104; found, 272.1108. Synthesis of Compound 12 To a solution
of 4-pyridylacetonitrile hydrochloride (256.4 mg, 1.66 mmol) and NaH
(60% dispersion in mineral oil, 442.2 mg, 11.06 mmol) in dry ACN (50
mL) under an Ar atmosphere and protected from light, a solution of
coumarin 11 (300 mg, 1.11 mmol) in dry ACN (10 mL) was
added. After the mixture was stirred for 2 h at room temperature,
silver nitrate (413.2 mg, 2.43 mmol) was added, and the reaction mixture
was stirred at room temperature for 2 h under an Ar atmosphere and
protected from light. The crude product was evaporated under reduced
pressure and purified by column chromatography (silica gel, 0–3.5%
MeOH in DCM) to give 120.6 mg of an orange solid (yield 31%). TLC: R f (10% MeOH in DCM) 0.67. 1 H NMR
(400 MHz, CDCl 3 ): δ (ppm) 8.54 (2H, m), 7.69 (2H,
m), 6.94 (1H, s), 6.72 (1H, br q, J = 0.8 Hz), 3.27
(4H, m), 2.90 (2H, t, J = 6.5 Hz), 2.78 (2H, t, J = 6.3 Hz), 2.31 (3H, d, J = 0.8 Hz),
2.00 (4H, m). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ (ppm) 163.2, 149.2, 145.4, 144.0, 140.9, 121.4,
120.5, 119.9, 118.4, 111.5, 109.5, 106.0, 80.3, 49.6, 48.9, 27.3,
21.1, 21.0, 20.5, 18.1. HRMS (ESI-TOF) m / z : [M + H] + calcd for C 23 H 22 N 3 O, 356.1757; found, 356.1761. Synthesis of Compound 1c Methyl trifluoromethanesulfonate
(32 μL, 0.28 mmol) was added to a solution of coumarin 12 (49 mg, 0.14 mmol) in DCM (25 mL) under an argon atmosphere.
The mixture was stirred overnight at room temperature and protected
from light. The reaction mixture was evaporated under reduced pressure
and purified by column chromatography (silica gel, 0–6% MeOH
in DCM) to give 47 mg of a red solid (yield 66%). TLC: R f (10% MeOH in DCM) 0.33. 1 H NMR (400 MHz,
DMSO- d 6 ): δ (ppm) 8.60 (2H, d, J = 7.2 Hz), 7.99 (2H, d, J = 7.2 Hz),
7.37 (1H, s), 6.87 (1H, s), 4.13 (3H, s), 3.37 (4H, m), 2.93 (2H,
t, J = 6.4 Hz), 2.81 (2H, t, J =
6.2 Hz), 2.50 (3H, s), 1.96 (2H, m), 1.90 (2H, m). 13 C{ 1 H} NMR (101 Hz, DMSO- d 6 ): δ
(ppm) 166.2, 152.4, 150.4, 148.8, 147.4, 143.5, 122.7, 121.4, 120.1,
118.6, 110.3, 105.0, 77.1, 49.5, 48.7, 45.9, 27.1, 20.8, 20.4, 19.8,
18.5. HRMS (ESI-TOF) m / z : [M] + calcd for C 24 H 24 N 3 O, 370.1912;
found, 370.1914.
## Synthesis of Compound
Synthesis of Compound 11 Coumarin 102
(1.17 g, 4.58 mmol) and Lawesson’s reagent (1.11 g, 2.75 mmol)
were dissolved in toluene (30 mL) and heated at 100 °C for 15
h. After evaporation under reduced pressure, the residue was purified
by column chromatography (silica gel, 50–70% DCM in hexanes)
to give an orange solid (666 mg, 54%): TLC R f (DCM) 0.53. 1 H NMR (400 MHz, CDCl 3 ):
δ (ppm) 7.03 (1H, s), 6.91 (1H, br q, J = 0.8
Hz), 3.29 (4H, q, J = 6.8 Hz), 3.00 (2H, t, J = 6.4 Hz), 2.79 (2H, t, J = 6.4 Hz),
2.27 (3H, d, J = 0.8 Hz), 1.98 (4H, m). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ (ppm) 196.4,
154.5, 146.5, 146.4, 123.4, 121.8, 120.2, 111.2, 106.1, 50.1, 49.8,
28.0, 21.5, 20.7, 20.5, 18.1; HRMS (ESI-TOF) m / z : [M + H] + calcd for C 16 H 18 NOS, 272.1104; found, 272.1108.
## Synthesis of Compound
Synthesis of Compound 12 To a solution
of 4-pyridylacetonitrile hydrochloride (256.4 mg, 1.66 mmol) and NaH
(60% dispersion in mineral oil, 442.2 mg, 11.06 mmol) in dry ACN (50
mL) under an Ar atmosphere and protected from light, a solution of
coumarin 11 (300 mg, 1.11 mmol) in dry ACN (10 mL) was
added. After the mixture was stirred for 2 h at room temperature,
silver nitrate (413.2 mg, 2.43 mmol) was added, and the reaction mixture
was stirred at room temperature for 2 h under an Ar atmosphere and
protected from light. The crude product was evaporated under reduced
pressure and purified by column chromatography (silica gel, 0–3.5%
MeOH in DCM) to give 120.6 mg of an orange solid (yield 31%). TLC: R f (10% MeOH in DCM) 0.67. 1 H NMR
(400 MHz, CDCl 3 ): δ (ppm) 8.54 (2H, m), 7.69 (2H,
m), 6.94 (1H, s), 6.72 (1H, br q, J = 0.8 Hz), 3.27
(4H, m), 2.90 (2H, t, J = 6.5 Hz), 2.78 (2H, t, J = 6.3 Hz), 2.31 (3H, d, J = 0.8 Hz),
2.00 (4H, m). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ (ppm) 163.2, 149.2, 145.4, 144.0, 140.9, 121.4,
120.5, 119.9, 118.4, 111.5, 109.5, 106.0, 80.3, 49.6, 48.9, 27.3,
21.1, 21.0, 20.5, 18.1. HRMS (ESI-TOF) m / z : [M + H] + calcd for C 23 H 22 N 3 O, 356.1757; found, 356.1761.
## Synthesis of Compound
Synthesis of Compound 1c Methyl trifluoromethanesulfonate
(32 μL, 0.28 mmol) was added to a solution of coumarin 12 (49 mg, 0.14 mmol) in DCM (25 mL) under an argon atmosphere.
The mixture was stirred overnight at room temperature and protected
from light. The reaction mixture was evaporated under reduced pressure
and purified by column chromatography (silica gel, 0–6% MeOH
in DCM) to give 47 mg of a red solid (yield 66%). TLC: R f (10% MeOH in DCM) 0.33. 1 H NMR (400 MHz,
DMSO- d 6 ): δ (ppm) 8.60 (2H, d, J = 7.2 Hz), 7.99 (2H, d, J = 7.2 Hz),
7.37 (1H, s), 6.87 (1H, s), 4.13 (3H, s), 3.37 (4H, m), 2.93 (2H,
t, J = 6.4 Hz), 2.81 (2H, t, J =
6.2 Hz), 2.50 (3H, s), 1.96 (2H, m), 1.90 (2H, m). 13 C{ 1 H} NMR (101 Hz, DMSO- d 6 ): δ
(ppm) 166.2, 152.4, 150.4, 148.8, 147.4, 143.5, 122.7, 121.4, 120.1,
118.6, 110.3, 105.0, 77.1, 49.5, 48.7, 45.9, 27.1, 20.8, 20.4, 19.8,
18.5. HRMS (ESI-TOF) m / z : [M] + calcd for C 24 H 24 N 3 O, 370.1912;
found, 370.1914.
## Synthesis of Ir(III)-COUPY Conjugate
Synthesis of Ir(III)-COUPY Conjugate 3c Synthesis of Compound 13 Methyl bromoacetate
(52 μL, 0.56 mmol) was added to a solution of coumarin 12 (100 mg, 0.28 mmol) in AcOEt/ACN 1:1 (20 mL). The mixture
was stirred overnight at 50 °C under an Ar atmosphere and protected
from light. The crude product was evaporated under reduced pressure
and purified by column chromatography (silica gel, 0–8% MeOH
in DCM) to give 124 mg of a blue solid (yield, 87%). TLC: R f (10% MeOH in DCM) 0.40. 1 H NMR
(400 MHz, CDCl 3 ): δ (ppm) 8.81 (2H, d, J = 6.8 Hz), 8.01 (2H, d, J = 6.8 Hz), 7.15 (1H,
s), 6.90 (1H, s), 5.80 (2H, s), 3.85 (3H, s), 3.39 (4H, m), 3.05 (2H,
t, J = 6.4 Hz), 2.84 (2H, t, J =
6.4 Hz), 2.49 (3H, s), 2.16 (2H, m), 2.01 (2H, m). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ (ppm) 167.3, 167.2,
152.7, 151.5, 150.8, 148.4, 143.5, 122.5, 122.4, 119.8, 118.5, 111.4,
111.0, 106.0, 79.2, 58.7, 53.6, 50.4, 49.8, 28.1, 21.9, 21.0, 20.3,
19.2. HRMS (ESI-TOF) m / z : [M] + calcd for C 26 H 26 N 3 O 3 , 428.1969; found, 428.1974. Synthesis of Compound 14 A 1:2 (v/v) mixture
of HCl (37%) and Milli-Q water (51 mL) was added to coumarin 13 (98.9 mg, 0.19 mmol). The reaction mixture was stirred
for 5 h at 60 °C under an Ar atmosphere and protected from light.
After removal of the major part of the solvent, several coevaporations
from acetonitrile were carried out. The crude product was used without
further purification in the next step. Analytical HPLC (10 to 100%
B in 30 min, formic acid additive): Rt = 16.6 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 25 H 24 N 3 O 3 , 414.18; found, 413.81. Synthesis of Compound 15 Coumarin 14 (87.5 mg, 0.19 mmol) and HATU (76 mg, 0.19 mmol) were dissolved
in anhydrous DMF (8 mL) under an Ar atmosphere. After addition of
DIPEA (70 μL, 0.39 mmol), the reaction mixture was stirred for
5 min under Ar at room temperature and protected from light. On the
other hand, DIPEA (35 μL, 0.19 mmol) was added to a solution
of N -Boc-1,3-propanediamine hydrochloride (62 mg,
0.29 mmol) in anhydrous DMF (5 mL), and the resulting mixture was
combined with the coumarin solution. After addition of DIPEA (70 μL,
0.39 mmol), the reaction mixture was stirred for 2 h at room temperature
under Ar and protected from light. The crude product was evaporated
under reduced pressure and purified by column chromatography (silica
gel, 0–12.0% MeOH in DCM) to give 29 mg of a purple solid (yield:
24%). TLC: R f (10% MeOH in DCM) 0.59. 1 H NMR (400 MHz, DMSO- d 6 ): δ
(ppm) 8.69 (1H, br s), 8.55 (2H, d, J = 6.8 Hz),
7.96 (2H, d, J = 6.6 Hz), 7.35 (1H, s), 6.84 (2H,
m), 5.19 (2H, s), 3.37 (4H, m), 3.13 (2H, q, J =
6.6 Hz), 2.95 (4H, m), 2.80 (2H, m), 2.49 (3H, s), 1.92 (4H, m), 1.57
(2H, quint, J = 6.9 Hz), 1.37 (9H, s). 13 C NMR (101 MHz, DMSO- d 6 ): δ (ppm)
166.2, 164.8, 155.6, 152.8, 150.5, 149.3, 147.6, 143.9, 122.7, 121.7,
119.3, 118.5, 110.5, 109.8, 104.9, 77.5, 77.5, 59.5, 49.6, 48.7, 40.2,
37.5, 36.8, 31.3, 29.3, 29.0, 28.2, 27.1, 22.1, 20.8, 20.4, 19.8,
18.5, 14.0. HRMS (ESI-TOF) m / z :
[M] + calcd for C 33 H 40 N 5 O 4 , 570.3075; found, 570.3079. Synthesis of Compound 16 A cooled down
solution of hydrochloric acid in dioxane (4 M, 8 mL) was added to
coumarin 15 (19 mg, 0.031 mmol). The reaction mixture
was stirred for 25 min in an ice bath under an Ar atmosphere and protected
from light. After removal of the solvent, several co-evaporations
from acetonitrile were carried out. The crude product was used without
further purification since HPLC-MS analysis revealed that the removal
of the Boc group was quantitative. Analytical HPLC (10 to 100% B in
30 min, formic acid additive): Rt = 10.6 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 28 H 32 N 5 O 2 , 470.26; found, 470.11. Synthesis of Ir(III)-COUPY Conjugate 3c To a solution of Ir complex 2b (30 mg, 28.1 μmol)
and HATU (11.0 mg, 28.1 μmol) in anhydrous DMF (5 mL) under
an Ar atmosphere, DIPEA (10 μL, 56.1 μmol) was added and
the mixture stirred for 10 min under Ar at room temperature and protected
from light. After addition of a solution of coumarin 16 (16.9 mg, 31.2 μmol) and DIPEA (25 μL, 140.3 μmol)
in anhydrous DMF (6 mL), the reaction mixture was stirred for 2.5
h at room temperature under Ar and protected from light. After evaporation
under reduced pressure, the crude was purified by column chromatography
(silica gel, 0–12% MeOH in DCM) to give 14.5 mg of a dark blue
solid (yield: 33%). TLC: R f (10% MeOH
in DCM) 0.47. 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 13.99 (2H, br s), 8.88 (1H, d, J = 8.8 Hz), 8.69 (1H, d, J = 9.0 Hz), 8.66 (1H,
m), 8.55 (2H, d, J = 7.0 Hz), 8.30 (1H, d, J = 9.0 Hz), 8.15 (1H, d, J = 8.3 Hz),
8.08 (1H, d, J = 8.9 Hz), 7.99 (2H, d, J = 7.2 Hz), 7.93 (1H, d, J = 7.7 Hz), 7.83 (2H,
m), 7.72 (1H, m), 7.63 (1H, t, J = 7.8 Hz), 7.51
(1H, d, J = 8.1 Hz), 7.45 (1H, d, J = 8.1 Hz), 7.40 (1H, s), 7.19 (1H, t, J = 8.7 Hz),
7.09 (3H, m), 7.00 (1H, t, J = 7.4 Hz), 6.90 (2H,
m), 6.81 (1H, t, J = 7.5 Hz), 6.73 (2H, m), 6.68
(1H, s), 6.30 (1H, d, J = 7.6 Hz), 6.19 (1H, d, J = 7.6 Hz), 5.95 (1H, d, J = 8.2 Hz),
5.70 (1H, d, J = 8.2 Hz), 5.21 (2H, s), 5.10 (2H,
m), 3.19 (4H, sext, J = 7.2 Hz), 2.92 (2H, t, J = 6.1 Hz), 2.81 (2H, t, J = 6.0 Hz),
1.91 (4H, m), 1.68 (5H, m), 1.23 (4H, m), 0.94 (4H, m), 0.66 (3H,
t, J = 7.2 Hz). 13 C NMR (101 MHz, DMSO- d 6 ): δ (ppm) 165.8, 164.9, 164.0, 155.6,
152.9, 152.4, 150.6, 149.8, 149.4, 148.5, 147.6, 143.9, 141.2, 139.5,
137.9, 132.4, 132.1, 132.0, 131.2, 130.2, 129.9, 129.3, 129.0, 128.9,
128.7, 124.7, 124.5, 124.2, 124.2, 123.0, 122.9, 122.8, 122.7, 122.3,
121.7, 121.6, 120.5, 119.4, 118.5, 117.7, 113.4, 113.3, 113.1, 113.0,
112.8, 112.6, 110.6, 105.0, 77.4, 67.5, 59.5, 49.6, 48.7, 45.9, 37.0,
31.9, 29.0, 27.1, 20.8, 20.4, 19.8, 19.0, 18.8, 18.5, 13.4. HRMS (ESI-TOF) m / z : [M] 2+ calcd for C 75 H 67 IrN 12 O 3 , 688.2539; found, 688.2545.
Analytical HPLC (10 to 100% B in 30 min, formic acid additive): Rt
= 15.5 min.
## Synthesis of Compound
Synthesis of Compound 13 Methyl bromoacetate
(52 μL, 0.56 mmol) was added to a solution of coumarin 12 (100 mg, 0.28 mmol) in AcOEt/ACN 1:1 (20 mL). The mixture
was stirred overnight at 50 °C under an Ar atmosphere and protected
from light. The crude product was evaporated under reduced pressure
and purified by column chromatography (silica gel, 0–8% MeOH
in DCM) to give 124 mg of a blue solid (yield, 87%). TLC: R f (10% MeOH in DCM) 0.40. 1 H NMR
(400 MHz, CDCl 3 ): δ (ppm) 8.81 (2H, d, J = 6.8 Hz), 8.01 (2H, d, J = 6.8 Hz), 7.15 (1H,
s), 6.90 (1H, s), 5.80 (2H, s), 3.85 (3H, s), 3.39 (4H, m), 3.05 (2H,
t, J = 6.4 Hz), 2.84 (2H, t, J =
6.4 Hz), 2.49 (3H, s), 2.16 (2H, m), 2.01 (2H, m). 13 C{ 1 H} NMR (101 Hz, CDCl 3 ): δ (ppm) 167.3, 167.2,
152.7, 151.5, 150.8, 148.4, 143.5, 122.5, 122.4, 119.8, 118.5, 111.4,
111.0, 106.0, 79.2, 58.7, 53.6, 50.4, 49.8, 28.1, 21.9, 21.0, 20.3,
19.2. HRMS (ESI-TOF) m / z : [M] + calcd for C 26 H 26 N 3 O 3 , 428.1969; found, 428.1974.
## Synthesis of Compound
Synthesis of Compound 14 A 1:2 (v/v) mixture
of HCl (37%) and Milli-Q water (51 mL) was added to coumarin 13 (98.9 mg, 0.19 mmol). The reaction mixture was stirred
for 5 h at 60 °C under an Ar atmosphere and protected from light.
After removal of the major part of the solvent, several coevaporations
from acetonitrile were carried out. The crude product was used without
further purification in the next step. Analytical HPLC (10 to 100%
B in 30 min, formic acid additive): Rt = 16.6 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 25 H 24 N 3 O 3 , 414.18; found, 413.81.
## Synthesis of Compound
Synthesis of Compound 15 Coumarin 14 (87.5 mg, 0.19 mmol) and HATU (76 mg, 0.19 mmol) were dissolved
in anhydrous DMF (8 mL) under an Ar atmosphere. After addition of
DIPEA (70 μL, 0.39 mmol), the reaction mixture was stirred for
5 min under Ar at room temperature and protected from light. On the
other hand, DIPEA (35 μL, 0.19 mmol) was added to a solution
of N -Boc-1,3-propanediamine hydrochloride (62 mg,
0.29 mmol) in anhydrous DMF (5 mL), and the resulting mixture was
combined with the coumarin solution. After addition of DIPEA (70 μL,
0.39 mmol), the reaction mixture was stirred for 2 h at room temperature
under Ar and protected from light. The crude product was evaporated
under reduced pressure and purified by column chromatography (silica
gel, 0–12.0% MeOH in DCM) to give 29 mg of a purple solid (yield:
24%). TLC: R f (10% MeOH in DCM) 0.59. 1 H NMR (400 MHz, DMSO- d 6 ): δ
(ppm) 8.69 (1H, br s), 8.55 (2H, d, J = 6.8 Hz),
7.96 (2H, d, J = 6.6 Hz), 7.35 (1H, s), 6.84 (2H,
m), 5.19 (2H, s), 3.37 (4H, m), 3.13 (2H, q, J =
6.6 Hz), 2.95 (4H, m), 2.80 (2H, m), 2.49 (3H, s), 1.92 (4H, m), 1.57
(2H, quint, J = 6.9 Hz), 1.37 (9H, s). 13 C NMR (101 MHz, DMSO- d 6 ): δ (ppm)
166.2, 164.8, 155.6, 152.8, 150.5, 149.3, 147.6, 143.9, 122.7, 121.7,
119.3, 118.5, 110.5, 109.8, 104.9, 77.5, 77.5, 59.5, 49.6, 48.7, 40.2,
37.5, 36.8, 31.3, 29.3, 29.0, 28.2, 27.1, 22.1, 20.8, 20.4, 19.8,
18.5, 14.0. HRMS (ESI-TOF) m / z :
[M] + calcd for C 33 H 40 N 5 O 4 , 570.3075; found, 570.3079.
## Synthesis of Compound
Synthesis of Compound 16 A cooled down
solution of hydrochloric acid in dioxane (4 M, 8 mL) was added to
coumarin 15 (19 mg, 0.031 mmol). The reaction mixture
was stirred for 25 min in an ice bath under an Ar atmosphere and protected
from light. After removal of the solvent, several co-evaporations
from acetonitrile were carried out. The crude product was used without
further purification since HPLC-MS analysis revealed that the removal
of the Boc group was quantitative. Analytical HPLC (10 to 100% B in
30 min, formic acid additive): Rt = 10.6 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 28 H 32 N 5 O 2 , 470.26; found, 470.11.
## Synthesis of Ir(III)-COUPY Conjugate
Synthesis of Ir(III)-COUPY Conjugate 3c To a solution of Ir complex 2b (30 mg, 28.1 μmol)
and HATU (11.0 mg, 28.1 μmol) in anhydrous DMF (5 mL) under
an Ar atmosphere, DIPEA (10 μL, 56.1 μmol) was added and
the mixture stirred for 10 min under Ar at room temperature and protected
from light. After addition of a solution of coumarin 16 (16.9 mg, 31.2 μmol) and DIPEA (25 μL, 140.3 μmol)
in anhydrous DMF (6 mL), the reaction mixture was stirred for 2.5
h at room temperature under Ar and protected from light. After evaporation
under reduced pressure, the crude was purified by column chromatography
(silica gel, 0–12% MeOH in DCM) to give 14.5 mg of a dark blue
solid (yield: 33%). TLC: R f (10% MeOH
in DCM) 0.47. 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 13.99 (2H, br s), 8.88 (1H, d, J = 8.8 Hz), 8.69 (1H, d, J = 9.0 Hz), 8.66 (1H,
m), 8.55 (2H, d, J = 7.0 Hz), 8.30 (1H, d, J = 9.0 Hz), 8.15 (1H, d, J = 8.3 Hz),
8.08 (1H, d, J = 8.9 Hz), 7.99 (2H, d, J = 7.2 Hz), 7.93 (1H, d, J = 7.7 Hz), 7.83 (2H,
m), 7.72 (1H, m), 7.63 (1H, t, J = 7.8 Hz), 7.51
(1H, d, J = 8.1 Hz), 7.45 (1H, d, J = 8.1 Hz), 7.40 (1H, s), 7.19 (1H, t, J = 8.7 Hz),
7.09 (3H, m), 7.00 (1H, t, J = 7.4 Hz), 6.90 (2H,
m), 6.81 (1H, t, J = 7.5 Hz), 6.73 (2H, m), 6.68
(1H, s), 6.30 (1H, d, J = 7.6 Hz), 6.19 (1H, d, J = 7.6 Hz), 5.95 (1H, d, J = 8.2 Hz),
5.70 (1H, d, J = 8.2 Hz), 5.21 (2H, s), 5.10 (2H,
m), 3.19 (4H, sext, J = 7.2 Hz), 2.92 (2H, t, J = 6.1 Hz), 2.81 (2H, t, J = 6.0 Hz),
1.91 (4H, m), 1.68 (5H, m), 1.23 (4H, m), 0.94 (4H, m), 0.66 (3H,
t, J = 7.2 Hz). 13 C NMR (101 MHz, DMSO- d 6 ): δ (ppm) 165.8, 164.9, 164.0, 155.6,
152.9, 152.4, 150.6, 149.8, 149.4, 148.5, 147.6, 143.9, 141.2, 139.5,
137.9, 132.4, 132.1, 132.0, 131.2, 130.2, 129.9, 129.3, 129.0, 128.9,
128.7, 124.7, 124.5, 124.2, 124.2, 123.0, 122.9, 122.8, 122.7, 122.3,
121.7, 121.6, 120.5, 119.4, 118.5, 117.7, 113.4, 113.3, 113.1, 113.0,
112.8, 112.6, 110.6, 105.0, 77.4, 67.5, 59.5, 49.6, 48.7, 45.9, 37.0,
31.9, 29.0, 27.1, 20.8, 20.4, 19.8, 19.0, 18.8, 18.5, 13.4. HRMS (ESI-TOF) m / z : [M] 2+ calcd for C 75 H 67 IrN 12 O 3 , 688.2539; found, 688.2545.
Analytical HPLC (10 to 100% B in 30 min, formic acid additive): Rt
= 15.5 min.
## Synthesis of Ir(III)-COUPY Conjugate
Synthesis of Ir(III)-COUPY Conjugate 3d Synthesis of Compound 18 To a solution
of Boc-6-aminohexanoic acid (42.8 mg, 0.19 mmol) and HATU (72.5 mg,
0.19 mmol) in anhydrous DMF (6 mL) under an Ar atmosphere, DIPEA (65
μL, 0.37 mmol) was added and the mixture stirred for 10 min
under Ar at room temperature and protected from light. After addition
of a solution of coumarin 17 (32 mg, 0.062 mmol) and
DIPEA (54 μL, 0.31 mmol) in anhydrous DMF (6 mL), the reaction
mixture was stirred for 2.5 h at room temperature under Ar and protected
from light. After evaporation under reduced pressure, the crude was
purified by column chromatography (silica gel, 0–12% MeOH in
DCM) to give 19.7 mg of a pink solid (yield: 49%). TLC: R f (10% MeOH in DCM) 0.43. 1 H NMR (400 MHz,
DMSO- d 6 ): δ (ppm) 8.59 (1H, br s),
8.53 (2H, d, J = 6.2 Hz), 8.16 (2H, d, J = 6.4 Hz), 7.81 (1H, br s), 7.72 (1H, d, J = 9.0
Hz), 6.99 (3H, m), 6.74 (1H, br s), 5.24 (2H, s), 3.55 (4H, m), 3.10
(4H, m), 2.86 (2H, m), 2.55 (3H, s), 2.03 (2H, m), 1.58 (2H, m), 1.46
(2H, m), 1.36 (11H, m), 1.18 (8H, m). 13 C NMR (101 MHz,
DMSO- d 6 ): δ (ppm) 172.1, 166.8,
164.8, 155.6, 154.9, 152.8, 152.0, 149.2, 144.2, 127.0, 120.1, 118.2,
111.9, 110.5, 110.4, 96.4, 78.1, 77.3, 59.6, 44.2, 36.9, 36.1, 35.4,
29.3, 29.0, 28.3, 26.0, 25.1, 18.4, 12.4. HRMS (ESI-TOF) m / z : [M] + calcd for C 37 H 51 N 6 O 5 , 659.3915; found, 659.3928. Synthesis of Compound 20 A cooled down
solution of hydrochloric acid in dioxane (4 M, 6 mL) was added to
coumarin 18 (16.1 mg, 0.023 mmol). The reaction mixture
was stirred for 20 min in an ice bath under an Ar atmosphere and protected
from light. After removal of the solvent, several co-evaporations
from acetonitrile were carried out. The crude product was used without
further purification since HPLC-MS analysis revealed that the removal
of the Boc group was quantitative. Analytical HPLC (10 to 100% B in
30 min, formic acid additive): Rt = 8.5 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 32 H 43 N 6 O 3 , 559.34; found, 559.49. Synthesis of Ir(III)-COUPY Conjugate 3d To a solution of Ir complex 2b (20.6 mg, 19.3 μmol)
and HATU (7.4 mg, 19.3 μmol) in anhydrous DMF (4 mL) under an
Ar atmosphere, DIPEA (7 μL, 38.6 μmol) was added and the
mixture stirred for 10 min under Ar at room temperature and protected
from light. After addition of a solution of coumarin 20 (14.6 mg, 23.2 μmol) and DIPEA (17 μL, 96.4 μmol)
in anhydrous DMF (5 mL), the reaction mixture was stirred for 2.5
h at room temperature under Ar and protected from light. After evaporation
under reduced pressure, the crude was purified by column chromatography
(silica gel, 0–12% MeOH in DCM) to give 27.9 mg of a purple
solid (yield: 68%). 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 14.16 (2H, br s), 8.88 (1H, d, J = 8.7 Hz), 8.69 (1H, d, J = 8.9 Hz),
8.65 (1H, t, J = 5.5 Hz), 8.53 (2H, d, J = 7.5 Hz), 8.29 (1H, d, J = 9.0 Hz), 8.15 (3H,
m), 8.07 (1H, d, J = 8.9 Hz), 7.96 (1H, d, J = 7.5 Hz), 7.92 (1H, t, J = 5.6 Hz),
7.87 (1H, d, J = 7.6 Hz), 7.83 (1H, dd, J = 8.8 Hz, J = 1.5 Hz), 7.70 (1H, d, J = 9.2 Hz), 7.63 (1H, t, J = 8.0 Hz), 7.53 (2H,
m), 7.46 (1H, d, J = 8.1 Hz), 7.19 (1H, t, J = 8.8 Hz), 7.00 (8H, m), 6.81 (1H, td, J = 7.6 Hz, J = 1.3 Hz), 6.74 (2H, m), 6.64 (1H,
d, J = 1.1 Hz), 6.30 (1H, d, J =
7.3 Hz), 6.19 (1H, d, J = 7.4 Hz), 5.95 (1H, d, J = 8.3 Hz), 5.70 (1H, d, J = 8.2 Hz),
5.25 (2H, m), 5.09 (2H, m), 3.53 (4H, q, J = 6.9
Hz), 3.11 (6H, m), 2.53 (2H, d, J = 0.8 Hz), 2.09
(2H, t, J = 7.4 Hz), 1.71 (2H, m), 1.51 (6H, m),
1.25 (3H, m), 1.15 (6H, t, J = 7.0 Hz), 0.99 (2H,
m), 0.65 (3H, t, J = 7.2 Hz). 13 C NMR
(101 MHz, DMSO- d 6 ): δ (ppm) 172.1,
166.8, 165.5, 164.8, 164.6, 164.0, 155.6, 154.9, 152.8, 152.4, 152.0,
149.8, 149.2, 148.5, 144.2, 143.4, 141.2, 139.6, 139.5, 139.3, 137.8,
134.5, 134.3, 132.4, 132.1, 132.0, 131.2, 130.2, 129.8, 129.0, 128.8,
128.8, 127.0, 124.8, 124.5, 124.3, 123.0, 123.0, 122.2, 121.6, 120.5,
120.1, 118.2, 117.5, 113.3, 113.1, 112.8, 112.6, 111.9, 110.5, 110.4,
96.4, 78.1, 59.6, 45.9, 44.2, 36.9, 36.2, 35.4, 31.9, 29.0, 26.2,
25.1, 19.0, 18.4, 13.4, 12.4. HRMS (ESI-TOF) m / z : [M] 2+ calcd for C 79 H 78 IrN 13 O 4 , 732.7959; found, 732.7973. Analytical
HPLC (10 to 100% B in 30 min, formic acid additive): Rt = 13.7 min.
## Synthesis of Compound
Synthesis of Compound 18 To a solution
of Boc-6-aminohexanoic acid (42.8 mg, 0.19 mmol) and HATU (72.5 mg,
0.19 mmol) in anhydrous DMF (6 mL) under an Ar atmosphere, DIPEA (65
μL, 0.37 mmol) was added and the mixture stirred for 10 min
under Ar at room temperature and protected from light. After addition
of a solution of coumarin 17 (32 mg, 0.062 mmol) and
DIPEA (54 μL, 0.31 mmol) in anhydrous DMF (6 mL), the reaction
mixture was stirred for 2.5 h at room temperature under Ar and protected
from light. After evaporation under reduced pressure, the crude was
purified by column chromatography (silica gel, 0–12% MeOH in
DCM) to give 19.7 mg of a pink solid (yield: 49%). TLC: R f (10% MeOH in DCM) 0.43. 1 H NMR (400 MHz,
DMSO- d 6 ): δ (ppm) 8.59 (1H, br s),
8.53 (2H, d, J = 6.2 Hz), 8.16 (2H, d, J = 6.4 Hz), 7.81 (1H, br s), 7.72 (1H, d, J = 9.0
Hz), 6.99 (3H, m), 6.74 (1H, br s), 5.24 (2H, s), 3.55 (4H, m), 3.10
(4H, m), 2.86 (2H, m), 2.55 (3H, s), 2.03 (2H, m), 1.58 (2H, m), 1.46
(2H, m), 1.36 (11H, m), 1.18 (8H, m). 13 C NMR (101 MHz,
DMSO- d 6 ): δ (ppm) 172.1, 166.8,
164.8, 155.6, 154.9, 152.8, 152.0, 149.2, 144.2, 127.0, 120.1, 118.2,
111.9, 110.5, 110.4, 96.4, 78.1, 77.3, 59.6, 44.2, 36.9, 36.1, 35.4,
29.3, 29.0, 28.3, 26.0, 25.1, 18.4, 12.4. HRMS (ESI-TOF) m / z : [M] + calcd for C 37 H 51 N 6 O 5 , 659.3915; found, 659.3928.
## Synthesis of Compound
Synthesis of Compound 20 A cooled down
solution of hydrochloric acid in dioxane (4 M, 6 mL) was added to
coumarin 18 (16.1 mg, 0.023 mmol). The reaction mixture
was stirred for 20 min in an ice bath under an Ar atmosphere and protected
from light. After removal of the solvent, several co-evaporations
from acetonitrile were carried out. The crude product was used without
further purification since HPLC-MS analysis revealed that the removal
of the Boc group was quantitative. Analytical HPLC (10 to 100% B in
30 min, formic acid additive): Rt = 8.5 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 32 H 43 N 6 O 3 , 559.34; found, 559.49.
## Synthesis of Ir(III)-COUPY Conjugate
Synthesis of Ir(III)-COUPY Conjugate 3d To a solution of Ir complex 2b (20.6 mg, 19.3 μmol)
and HATU (7.4 mg, 19.3 μmol) in anhydrous DMF (4 mL) under an
Ar atmosphere, DIPEA (7 μL, 38.6 μmol) was added and the
mixture stirred for 10 min under Ar at room temperature and protected
from light. After addition of a solution of coumarin 20 (14.6 mg, 23.2 μmol) and DIPEA (17 μL, 96.4 μmol)
in anhydrous DMF (5 mL), the reaction mixture was stirred for 2.5
h at room temperature under Ar and protected from light. After evaporation
under reduced pressure, the crude was purified by column chromatography
(silica gel, 0–12% MeOH in DCM) to give 27.9 mg of a purple
solid (yield: 68%). 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 14.16 (2H, br s), 8.88 (1H, d, J = 8.7 Hz), 8.69 (1H, d, J = 8.9 Hz),
8.65 (1H, t, J = 5.5 Hz), 8.53 (2H, d, J = 7.5 Hz), 8.29 (1H, d, J = 9.0 Hz), 8.15 (3H,
m), 8.07 (1H, d, J = 8.9 Hz), 7.96 (1H, d, J = 7.5 Hz), 7.92 (1H, t, J = 5.6 Hz),
7.87 (1H, d, J = 7.6 Hz), 7.83 (1H, dd, J = 8.8 Hz, J = 1.5 Hz), 7.70 (1H, d, J = 9.2 Hz), 7.63 (1H, t, J = 8.0 Hz), 7.53 (2H,
m), 7.46 (1H, d, J = 8.1 Hz), 7.19 (1H, t, J = 8.8 Hz), 7.00 (8H, m), 6.81 (1H, td, J = 7.6 Hz, J = 1.3 Hz), 6.74 (2H, m), 6.64 (1H,
d, J = 1.1 Hz), 6.30 (1H, d, J =
7.3 Hz), 6.19 (1H, d, J = 7.4 Hz), 5.95 (1H, d, J = 8.3 Hz), 5.70 (1H, d, J = 8.2 Hz),
5.25 (2H, m), 5.09 (2H, m), 3.53 (4H, q, J = 6.9
Hz), 3.11 (6H, m), 2.53 (2H, d, J = 0.8 Hz), 2.09
(2H, t, J = 7.4 Hz), 1.71 (2H, m), 1.51 (6H, m),
1.25 (3H, m), 1.15 (6H, t, J = 7.0 Hz), 0.99 (2H,
m), 0.65 (3H, t, J = 7.2 Hz). 13 C NMR
(101 MHz, DMSO- d 6 ): δ (ppm) 172.1,
166.8, 165.5, 164.8, 164.6, 164.0, 155.6, 154.9, 152.8, 152.4, 152.0,
149.8, 149.2, 148.5, 144.2, 143.4, 141.2, 139.6, 139.5, 139.3, 137.8,
134.5, 134.3, 132.4, 132.1, 132.0, 131.2, 130.2, 129.8, 129.0, 128.8,
128.8, 127.0, 124.8, 124.5, 124.3, 123.0, 123.0, 122.2, 121.6, 120.5,
120.1, 118.2, 117.5, 113.3, 113.1, 112.8, 112.6, 111.9, 110.5, 110.4,
96.4, 78.1, 59.6, 45.9, 44.2, 36.9, 36.2, 35.4, 31.9, 29.0, 26.2,
25.1, 19.0, 18.4, 13.4, 12.4. HRMS (ESI-TOF) m / z : [M] 2+ calcd for C 79 H 78 IrN 13 O 4 , 732.7959; found, 732.7973. Analytical
HPLC (10 to 100% B in 30 min, formic acid additive): Rt = 13.7 min.
## Synthesis of Ir(III)-COUPY Conjugate
Synthesis of Ir(III)-COUPY Conjugate 3e Synthesis of Compound 19 To a solution
of trans -4- N -Boc-aminomethyl-cyclohexanecarboxylic
acid (56 mg, 0.22 mmol) and HATU (85.3 mg, 0.22 mmol) in anhydrous
DMF (6 mL) under an Ar atmosphere, DIPEA (77 μL, 0.44 mmol)
was added and the mixture stirred for 10 min under Ar at room temperature
and protected from light. After addition of a solution of coumarin 17 (37.6 mg, 0.073 mmol) and DIPEA (64 μL, 0.36 mmol)
in anhydrous DMF (6 mL), the reaction mixture was stirred for 2.5
h at room temperature under Ar and protected from light. After evaporation
under reduced pressure, the crude was purified by column chromatography
(silica gel, 0–11% MeOH in DCM) to give 35.4 mg of a pink solid
(yield: 68%). TLC: R f (10% MeOH in DCM)
0.38. 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 8.60 (1H, m), 8.53 (2H, d, J =
7.2 Hz), 8.16 (2H, d, J = 7.2 Hz), 7.73 (1H, m),
7.72 (1H, d, J = 9.2 Hz), 7.02 (1H, m), 6.98 (1H,
dd, J = 9.2, 2.4 Hz), 6.93 (1H, s), 6.78 (1H, t, J = 6.0 Hz), 5.24 (2H, s), 3.56 (4H, q, J = 7.2 Hz), 3.13 (2H, q, J = 6.4 Hz), 3.07 (2H,
q, J = 6.0 Hz), 2.75 (2H, t, J =
6.4 Hz), 2.55 (3H, s), 2.00 (1H, m), 1.68 (4H, m), 1.57 (2H, qt, J = 6.8 Hz), 1.36 (9H, s), 1.26 (4H, m), 1.18 (6H, t, J = 7.2 Hz), 0.84 (2H, m). 13 C{ 1 H}
NMR (101 Hz, DMSO- d 6 ): δ (ppm) 175.2,
166.8, 164.8, 155.7, 154.9, 152.8, 152.0, 149.2, 144.2, 127.0, 120.1,
118.2, 111.9, 110.5, 110.4, 96.4, 78.1, 77.3, 59.6, 46.1, 44.2, 44.1,
37.4, 36.8, 36.0, 29.5, 29.0, 28.8, 28.3, 18.4, 12.4. HRMS (ESI-TOF) m / z : [M] + calcd for C 39 H 53 N 6 O 5 , 685.4072; found, 685.4072. Synthesis of Compound 21 A cooled down
solution of hydrochloric acid in dioxane (4 M, 11 mL) was added to
coumarin 19 (28 mg, 0.039 mmol). The reaction mixture
was stirred for 20 min in an ice bath under an Ar atmosphere and protected
from light. After removal of the solvent, several co-evaporations
from acetonitrile were carried out. The crude product was used without
further purification since HPLC-MS analysis revealed that the removal
of the Boc group was quantitative. Analytical HPLC (10 to 100% B in
30 min, formic acid additive): Rt = 8.6 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 34 H 45 N 6 O 3 , 585.35; found, 585.31. Synthesis of Ir(III)-COUPY Conjugate 3e To a solution of Ir complex 2b (34.6 mg, 32.3 μmol)
and HATU (12.7 mg, 32.3 μmol) in anhydrous DMF (5 mL) under
an Ar atmosphere, DIPEA (12 μL, 64.7 μmol) was added and
the mixture stirred for 10 min under Ar at room temperature and protected
from light. After addition of a solution of coumarin 21 (25.5 mg, 38.8 μmol) and DIPEA (29 μL, 161.7 μmol)
in anhydrous DMF (6 mL), the reaction mixture was stirred for 2.5
h at room temperature under Ar and protected from light. After evaporation
under reduced pressure, the crude was purified by column chromatography
(silica gel, 0–12% MeOH in DCM) to give 34.7 mg of a purple
solid (yield: 58%). 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 14.40 (2H, br s), 8.89 (1H, d, J = 8.8 Hz), 8.70 (2H, m), 8.54 (2H, d, J = 7.5 Hz), 8.28 (1H, d, J = 9.0 Hz), 8.15 (3H,
m), 8.07 (1H, d, J = 8.9 Hz), 7.97 (1H, d, J = 6.9 Hz), 7.91 (1H, d, J = 7.6 Hz),
7.82 (2H, m), 7.70 (1H, d, J = 9.2 Hz), 7.62 (2H,
m), 7.52 (1H, d, J = 8.1 Hz), 7.46 (1H, d, J = 8.1 Hz), 7.19 (1H, t, J = 8.8 Hz),
7.12 (2H, q, J = 7.9 Hz), 6.95 (6H, m), 6.80 (1H,
t, J = 7.6 Hz), 6.75 (2H, t, J =
7.4 Hz), 6.63 (1H, s), 6.30 (1H, d, J = 7.2 Hz),
6.19 (1H, d, J = 7.4 Hz), 5.96 (1H, d, J = 8.3 Hz), 5.73 (1H, d, J = 8.2 Hz), 5.26 (2H,
s), 5.10 (2H, m), 3.54 (4H, q, J = 6.9 Hz), 3.07
(7H, m), 2.53 (2H, s), 2.07 (1H, m), 1.73 (6H, m), 1.58 (2H, qt, J = 6.9 Hz), 1.35 (3H, m), 1.16 (6H, t, J = 7.0 Hz), 0.92 (4H, m), 0.63 (3H, t, J = 7.2 Hz). 13 C NMR (101 MHz, DMSO- d 6 ): δ
(ppm) 175.2, 166.8, 165.8, 164.8, 164.5, 163.8, 155.6, 154.9, 152.8,
152.3, 152.0, 149.8, 149.2, 148.5, 144.2, 143.4, 141.3, 139.5, 139.2,
137.8, 134.5, 134.3, 134.2, 133.2, 133.1, 132.3, 132.3, 132.0, 131.2,
130.3, 129.9, 129.4, 128.9, 128.9, 128.8, 127.6, 127.0, 124.8, 124.6,
124.3, 123.1, 123.0, 122.9, 122.2, 121.6, 120.6, 120.1, 119.3, 118.2,
117.5, 113.3, 113.0, 112.9, 112.5, 111.9, 110.5, 110.4, 96.5, 78.1,
59.6, 45.9, 45.3, 44.2, 44.1, 37.1, 36.9, 36.0, 31.9, 29.7, 29.0,
28.9, 19.0, 18.4, 13.4, 12.4. HRMS (ESI-TOF) m / z : [M] 2+ calcd for C 81 H 80 IrN 13 O 4 , 745.8037; found, 745.8047. Analytical
HPLC (10 to 100% B in 30 min, formic acid additive): Rt = 15.8 min.
## Synthesis of Compound
Synthesis of Compound 19 To a solution
of trans -4- N -Boc-aminomethyl-cyclohexanecarboxylic
acid (56 mg, 0.22 mmol) and HATU (85.3 mg, 0.22 mmol) in anhydrous
DMF (6 mL) under an Ar atmosphere, DIPEA (77 μL, 0.44 mmol)
was added and the mixture stirred for 10 min under Ar at room temperature
and protected from light. After addition of a solution of coumarin 17 (37.6 mg, 0.073 mmol) and DIPEA (64 μL, 0.36 mmol)
in anhydrous DMF (6 mL), the reaction mixture was stirred for 2.5
h at room temperature under Ar and protected from light. After evaporation
under reduced pressure, the crude was purified by column chromatography
(silica gel, 0–11% MeOH in DCM) to give 35.4 mg of a pink solid
(yield: 68%). TLC: R f (10% MeOH in DCM)
0.38. 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 8.60 (1H, m), 8.53 (2H, d, J =
7.2 Hz), 8.16 (2H, d, J = 7.2 Hz), 7.73 (1H, m),
7.72 (1H, d, J = 9.2 Hz), 7.02 (1H, m), 6.98 (1H,
dd, J = 9.2, 2.4 Hz), 6.93 (1H, s), 6.78 (1H, t, J = 6.0 Hz), 5.24 (2H, s), 3.56 (4H, q, J = 7.2 Hz), 3.13 (2H, q, J = 6.4 Hz), 3.07 (2H,
q, J = 6.0 Hz), 2.75 (2H, t, J =
6.4 Hz), 2.55 (3H, s), 2.00 (1H, m), 1.68 (4H, m), 1.57 (2H, qt, J = 6.8 Hz), 1.36 (9H, s), 1.26 (4H, m), 1.18 (6H, t, J = 7.2 Hz), 0.84 (2H, m). 13 C{ 1 H}
NMR (101 Hz, DMSO- d 6 ): δ (ppm) 175.2,
166.8, 164.8, 155.7, 154.9, 152.8, 152.0, 149.2, 144.2, 127.0, 120.1,
118.2, 111.9, 110.5, 110.4, 96.4, 78.1, 77.3, 59.6, 46.1, 44.2, 44.1,
37.4, 36.8, 36.0, 29.5, 29.0, 28.8, 28.3, 18.4, 12.4. HRMS (ESI-TOF) m / z : [M] + calcd for C 39 H 53 N 6 O 5 , 685.4072; found, 685.4072.
## Synthesis of Compound
Synthesis of Compound 21 A cooled down
solution of hydrochloric acid in dioxane (4 M, 11 mL) was added to
coumarin 19 (28 mg, 0.039 mmol). The reaction mixture
was stirred for 20 min in an ice bath under an Ar atmosphere and protected
from light. After removal of the solvent, several co-evaporations
from acetonitrile were carried out. The crude product was used without
further purification since HPLC-MS analysis revealed that the removal
of the Boc group was quantitative. Analytical HPLC (10 to 100% B in
30 min, formic acid additive): Rt = 8.6 min. LRMS (ESI-TOF) m / z : [M] + calcd for C 34 H 45 N 6 O 3 , 585.35; found, 585.31.
## Synthesis of Ir(III)-COUPY Conjugate
Synthesis of Ir(III)-COUPY Conjugate 3e To a solution of Ir complex 2b (34.6 mg, 32.3 μmol)
and HATU (12.7 mg, 32.3 μmol) in anhydrous DMF (5 mL) under
an Ar atmosphere, DIPEA (12 μL, 64.7 μmol) was added and
the mixture stirred for 10 min under Ar at room temperature and protected
from light. After addition of a solution of coumarin 21 (25.5 mg, 38.8 μmol) and DIPEA (29 μL, 161.7 μmol)
in anhydrous DMF (6 mL), the reaction mixture was stirred for 2.5
h at room temperature under Ar and protected from light. After evaporation
under reduced pressure, the crude was purified by column chromatography
(silica gel, 0–12% MeOH in DCM) to give 34.7 mg of a purple
solid (yield: 58%). 1 H NMR (400 MHz, DMSO- d 6 ): δ (ppm) 14.40 (2H, br s), 8.89 (1H, d, J = 8.8 Hz), 8.70 (2H, m), 8.54 (2H, d, J = 7.5 Hz), 8.28 (1H, d, J = 9.0 Hz), 8.15 (3H,
m), 8.07 (1H, d, J = 8.9 Hz), 7.97 (1H, d, J = 6.9 Hz), 7.91 (1H, d, J = 7.6 Hz),
7.82 (2H, m), 7.70 (1H, d, J = 9.2 Hz), 7.62 (2H,
m), 7.52 (1H, d, J = 8.1 Hz), 7.46 (1H, d, J = 8.1 Hz), 7.19 (1H, t, J = 8.8 Hz),
7.12 (2H, q, J = 7.9 Hz), 6.95 (6H, m), 6.80 (1H,
t, J = 7.6 Hz), 6.75 (2H, t, J =
7.4 Hz), 6.63 (1H, s), 6.30 (1H, d, J = 7.2 Hz),
6.19 (1H, d, J = 7.4 Hz), 5.96 (1H, d, J = 8.3 Hz), 5.73 (1H, d, J = 8.2 Hz), 5.26 (2H,
s), 5.10 (2H, m), 3.54 (4H, q, J = 6.9 Hz), 3.07
(7H, m), 2.53 (2H, s), 2.07 (1H, m), 1.73 (6H, m), 1.58 (2H, qt, J = 6.9 Hz), 1.35 (3H, m), 1.16 (6H, t, J = 7.0 Hz), 0.92 (4H, m), 0.63 (3H, t, J = 7.2 Hz). 13 C NMR (101 MHz, DMSO- d 6 ): δ
(ppm) 175.2, 166.8, 165.8, 164.8, 164.5, 163.8, 155.6, 154.9, 152.8,
152.3, 152.0, 149.8, 149.2, 148.5, 144.2, 143.4, 141.3, 139.5, 139.2,
137.8, 134.5, 134.3, 134.2, 133.2, 133.1, 132.3, 132.3, 132.0, 131.2,
130.3, 129.9, 129.4, 128.9, 128.9, 128.8, 127.6, 127.0, 124.8, 124.6,
124.3, 123.1, 123.0, 122.9, 122.2, 121.6, 120.6, 120.1, 119.3, 118.2,
117.5, 113.3, 113.0, 112.9, 112.5, 111.9, 110.5, 110.4, 96.5, 78.1,
59.6, 45.9, 45.3, 44.2, 44.1, 37.1, 36.9, 36.0, 31.9, 29.7, 29.0,
28.9, 19.0, 18.4, 13.4, 12.4. HRMS (ESI-TOF) m / z : [M] 2+ calcd for C 81 H 80 IrN 13 O 4 , 745.8037; found, 745.8047. Analytical
HPLC (10 to 100% B in 30 min, formic acid additive): Rt = 15.8 min.
## Photophysical and Photochemical Characterization
Photophysical and Photochemical Characterization For
photophysical measurements, all solvents used were spectroscopic grade.
Absorption spectra were recorded in a Varian Cary 500 UV/vis/NIR or
Varian Cary 6000i spectrophotometer at room temperature. Molar absorption
coefficients (ε) were determined by direct application of the
Beer–Lambert law, using solutions of the compounds in each
solvent with concentrations ranging from 10 –6 to
10 –5 M. Emission spectra were registered in a Photon
Technology International (PTI) fluorimeter or in a Horiba Fluoromax-4
spectrofluorometer. Fluorescence quantum yields (Φ F ) were measured by the comparative method using cresyl violet in
ethanol (CV; Φ F;ref = 0.54 ± 0.03) as reference. 63 Then, optically matched solutions of the compounds
and CV were excited and the fluorescence spectra were recorded. The
absorbance of sample and reference solutions was set below 0.1 at
the excitation wavelength, and Φ F was calculated
using the following eq 1 1 where area sample and area ref are the integrated fluorescence for the sample and the reference
and η sample and η ref are the refractive
index of sample and reference solutions respectively. The uncertainty
in the experimental value of Φ F has been estimated
to be approximately 10%. Phosphorescence quantum yields (Φ P ) of the iridium complex were determined analogously to Φ F using argon-saturated meso-tetra-5,10,15,20-phenylporphine
as reference (Φ F = 0.11) in toluene. 64 Singlet oxygen generation was studied by time-resolved
near-infrared
phosphorescence by means of a customized setup. Briefly, a pulsed
Nd:YAG laser (FTSS355-Q, Crystal Laser, Berlin, Germany) working at
a 1 or 10 kHz repetition rate at 355 nm (0.5 μJ per pulse) or
532 nm (1.2 μJ per pulse) was used to excite the sample. A 1064
nm rugate notch filter (Edmund Optics) and an uncoated SKG-5 filter
(CVI Laser Corporation) were placed in the laser path to remove any
NIR emission. The light emitted by the sample was filtered with a
1000 nm long-pass filter (Edmund Optics) and later by a narrow bandpass
filter at 1275 nm (BK-1270-70-B, bk Interferenzoptik). A thermoelectric-cooled
NIR-sensitive photomultiplier tube assembly (H9170-45, Hamamatsu Photonics,
Hamamatsu, Japan) was used as a detector. Photon counting was achieved
with a multichannel scaler (NanoHarp 250, PicoQuant). The time dependence
of the 1 O 2 phosphorescence with the signal intensity S ( t ) is described by eq 2 , in which τ T and τ Δ are the lifetimes of the photosensitizer triplet state
and of 1 O 2 respectively, and S 0 is a preexponential parameter proportional to Φ Δ . 2 The Φ Δ values
of the different samples were
obtained by comparing S 0 values of optically
matched samples and using an appropriate reference by means of eq 3 . 3 The same setup was used to monitor
the phosphorescence of the complex
and the conjugate, except that the red-sensitive Hamamatsu H5783 photosensor
module was used for detection.
## Superoxide Anion Radical Generation and Characterization Using
DHR123
Superoxide Anion Radical Generation and Characterization Using
DHR123 All compounds (10 μM) were prepared in PBS (0.2%
DMSO). To this solution, DHR123 was added so that its final concentration
was 10 μM. Then, the samples were irradiated in 1.0 × 0.5
cm cuvette by green light (505 nm centered LED) for indicated time
intervals. Immediately, the fluorescence spectra were collected by
using a Photon Technology International (PTI) fluorimeter. The excitation
wavelength was set to 500 nm, the excitation and emission slit widths
were 2 nm, and the integration time was set to 1 s.
## EPR Studies
EPR Studies EPR measurements were carried out with
a Bruker Elexys 580E spectrometer working in X-band at room temperature.
Microwave frequency was 9.68 GHz. The modulation amplitude was 0.12
mT. Both the modulation and the microwave power were selected in such
a way that no distortion or saturation of the signal was produced.
A LED source was used for the irradiation of the sample (530 nm, 450
mW cm –2 ). The spin traps 2,2,6,6-tetramethylpiperidine
(TEMP, 60 mM) and 5,5-dimethyl-1-pyrroline- N -oxide
(DMPO, 360 mM) were dissolved in MeOH. In a typical experiment, 20
μL of TEMP or DMPO solutions was added to an Eppendorf tube
containing the compounds ( 3a–3c ) in solid form,
and the resulting solution was diluted with 280 μL of MeOH (final
concentration of conjugates 250 μM). The samples were loaded
into capillary tubes, which were placed in the EPR to register the
spectrum in dark conditions. Then, the same samples were irradiated
for 5 min and the EPR spectrum was registered again.
## Cell Culture and Cell Lines
Cell Culture and Cell Lines A375, HeLa, and SK-MEL-28
cells were cultured in Dulbecco’s modified Eagle medium (DMEM)
supplemented with 10% FBS, l -glutamine, and 1% penicillin–streptomycin.
A2780 and A2780cis cells were grown in RPMI-1640 cell medium supplemented
with 10% fetal bovine serum (FBS) and 2 mM l -glutamine. Acquired
resistance to cisplatin in the A2780cis cell line was maintained by
adding cisplatin 1 μM to culture medium every second passage.
Cells were cultured in a humidified incubator at 310 K with 5% CO 2 atmosphere and subcultured two or three times a week with
appropriate densities and were confirmed to be mycoplasma-free using
a standard Hoechst DNA staining method.
## Fluorescence Imaging
Fluorescence Imaging HeLa Cells were maintained in
DMEM containing low glucose (1 g/L) and supplemented with 10% fetal
bovine serum (FBS), 50 U/mL penicillin–streptomycin, and 2
mM l -glutamine. For cellular uptake experiments and posterior
observation under the microscope, cells were seeded on glass bottom
dishes (P35G-1.5-14-C, Mattek). 24 h after cell seeding, cells were
incubated at 37 °C for 30 min with Ir(III)-COUPY conjugates 3a–3e (5 μM) in supplemented DMEM. Then, cells
were washed three times with Dulbecco’s phosphate-buffered
saline to remove the excess of the compounds and kept in low glucose
DMEM without phenol red supplemented with 10 mM Hepes for fluorescence
imaging. All microscopy observations were performed using a
Zeiss LSM 880 confocal microscope equipped with a 561 nm laser. The
microscope was also equipped with a heating insert (P Lab-Tek S, Pecon)
to keep cells at 37 °C. Cells were observed using a 63×
1.4 oil immersion objective. The compounds were excited using the
561 nm laser and emission detected from 570 to 670 nm. Image analysis
was performed using Fiji. 65 All images
are colorized using the Fire lookup table from Fiji.
## Cellular Accumulation by ICP–MS
Cellular Accumulation by ICP–MS Briefly, A2780cis
cells were seeded onto a 12-well plate (3 × 10 5 cells/well).
Treatments with tested agents for 2 h were applied at 10 μM.
Cisplatin was included for comparative purposes. Cells were then trypsinized,
and pellets were counted. Samples were then digested with 30% HNO 3 suprapur acid (Sigma-Aldrich) and subjected to Inductively
Coupled Plasma Mass Spectrometry analysis in Agilent 7900 ICP–MS. 99 Ru, 101 Ru, 194 Pt, and 195 Pt isotopes were measured. Three independent experiments were performed
( n = 2 replicates).
## Photo- and Cytotoxic Activity Determination
Photo- and Cytotoxic Activity Determination A2780,
A2780cis, HeLa, A375, and SK-MEL-28 cells were maintained at logarithmic
growth-phase and cultured in 96-well plates at a density of 5000 cells/well
in complete medium for 24 h at 310 K, 5% CO 2 in a humidified
incubator. For hypoxia experiments, a hypoxia condition was set up
by Tissue Culture Service at University of Murcia using nitrogen (N 2 ) to displace O 2 down to a minimum of 2% in a Forma
Steri-Cycle i160 incubator (Thermo Fisher Scientific). Serial dilutions
of the compounds were added at the final concentrations in the range
of 0–100 μM in a final volume of 100 μL per well.
A light-based treatment schedule was performed as follows: 1 h incubation
with the compounds in the dark, followed by 1 h incubation under irradiation
conditions by placing the Photoreactor EXPO-LED from Luzchem (Canada)
fitted with green lamps (final light intensity applied of 1.5 mW/cm 2 at λ max = 520 nm) inside the CO 2 incubator. All the cell culture plates subjected to light irradiation
included untreated controls to verify that cell viability was not
affected by light. Then, treatment-containing media was removed, and
fresh media was added for a 48 h cell recovery period; the temperature
throughout the experiment remained at 310 K. Dark control samples
were placed in the same dark conditions and then kept incubated for
1 h in the dark in the humidified CO 2 incubator. Medium
was then aspirated by suction, cells were loaded with 50 μL
of MTT solution (1 mg/mL) for additional 4 h and then removed, and
50 μL of DMSO was added to solubilize the purple formazan crystals
formed in active cells. The absorbance was measured at 570 nm using
a microplate reader (FLUOstar Omega), and the IC 50 values
were calculated based on the inhibitory rate curves using the following
equation 4 where I represents the percentage
inhibition of viability observed, I max is the maximal inhibitory effect, IC 50 is the concentration
that inhibits 50% of maximal growth, C is the concentration
of the treatment, and n is the slope of the semi-logarithmic
dose–response sigmoidal curves. The non-linear fitting was
performed using SigmaPlot 14.0 software. All experiments were performed
in three independent studies with n = 3 replicates
per concentration level. For a detailed phototoxicity procedure, ref ( 43 ) includes technical explanations.
## ROS Photogeneration in A2780cis Cells
ROS Photogeneration in A2780cis Cells ROS levels were
determined using the 2′-7′dichlorofluorescein diacetate
(DCFH-DA) probe. A2780cis cells were seeded onto 96-well black plates
at 2 × 10 4 cells/well for 24 h in a humidified CO 2 incubator either in normoxia (21% O 2 ) or hypoxia
(2% O 2 ). Tested compounds were then administered in cell
media for the allowed time. Treatments were then removed, and cells
were stained with 10 μM of DCFH-DA for 0.5 h. After staining,
cells were washed with PBS twice and irradiated for 1 h with LED source
light from Luzchem photoreactor (Canada) fitted with green lamps (final
light intensity applied of 1.5 mW/cm 2 at λ max = 520 nm). Fluorescence readings were performed in FLUOstar Omega
(λ exc = 488 nm and λ em = 530 ±
30 nm). Non-irradiated plates were used for dark conditions, whereas
treated, unstained cells were used to subtract basal fluorescence
of compounds and correct fluorescence readings. Unstained cells served
as blank. Three independent experiments were performed with n = 3 replicates.
## Cell Death Induction Assays
Cell Death Induction Assays Cell death induction was
evaluated using the Annexin V-FITC/Propidium iodide (AV/PI) dual staining
method. Briefly, A2780cis cells were seeded in 12-well plates at a
density of 2 × 10 5 cells/well and incubated overnight
at 310 K. Tested compounds (10 μM) were added, following the
described treatment schedule (1 h incubation + 1 h irradiation with
520 nm light). Irradiated, non-treated cells served as the control
group. After 24 h of the drug-free recovery period, cells were harvested
by trypsinization, washed with PBS and centrifuged, and the pellets
were resuspended in 200 μL of a binding buffer as instructed
by the manufacturer (Cayman). The resuspended cell solution was left
at room temperature in the dark for 15 min before analysis by flow
cytometry (FACSCalibur BecktonDickinson; 10 4 events acquired
per sample). Cells were visualized using λ exc = 488
channels and registered at 525 nm and 620 nm for Annexin V and propidium
iodide in FL1 and FL2 channels, respectively. Cell populations were
classified as follows: AV - /PI - (viable cells);
AV - /PI + (necrotic cells); AV + /PI - and AV + /PI + (apoptotic cells). Alternatively,
A2780cis cells were subjected to flow cytometry after phototreatments
and morphological changes were analyzed using FSC and SSC contour
plots. Data were analyzed using FlowJo software. Three independent
experiments were performed.
## 3D Multicellular Tumor Spheroid Growth Inhibition Assays
3D Multicellular Tumor Spheroid Growth Inhibition Assays Briefly, a single suspension of A2780cis cells at a density of 5
× 10 3 cells/well in complete RPMI medium was dispatched
onto 96-well Corning microplates with ultralow attachment surface
coating. The plates were covered and transferred to an incubator at
310 K with a 5% CO 2 atmosphere. After 3 days post-seeding,
uniform MCTSs were formed, which was confirmed using an inverted Zeiss
AXIO observer 7 microscope. Cell media of MCTSs was changed by replacing
50% with fresh cell media and allowed to grow for 3 days. On day 3,
MTCS was incubated with tested agents (10 μM) for 4 h and then
irradiated with 520 nm light for 1 h. Treatments were then carefully
removed, and fresh media was added. The integrity, radius, size, and
volume of the MCTSs were monitored using a DMi1 inverted phase contrast
microscope (Leica Microsystems) over 7 days. The radius of the tumorspheres
was measured using Fiji software, and the volume was calculated using
the following equation: V = 4/3π r 3 .