← Back
Rhenium(I) Tricarbonyl Complexes of 1,10-Phenanthroline Derivatives with Unexpectedly High Cytotoxicity.
Eight rhenium(I) tricarbonyl aqua complexes with the
general formula fac -[Re(CO) 3 ( N,N ′-bid)(H 2 O)][NO 3 ] ( 1–8 ), where N,N ′-bid is (2,6-dimethoxypyridyl)imidazo[4,5- f ]1,10-phenanthroline ( L1 ), (indole)imidazo[4,5- f ]1,10-phenanthroline ( L2 ), (5-methoxyindole)-imidazo[4,5- f ]1,10-phenanthroline ( L3 ), (biphenyl)imidazo[4,5- f ]1,10-phenanthroline ( L4 ), (fluorene)imidazo[4,5- f ]1,10-phenanthroline ( L5 ), (benzo[ b ]thiophene)imidazo[4,5- f ]1,10-phenanthroline
( L6 ), (5-bromothiazole)imidazo[4,5- f ]1,10-phenanthroline ( L7 ), and (4,5-dimethylthiophene)imidazo[4,5- f ]1,10-phenanthroline ( L8 ), were synthesized
and characterized using 1 H and 13 C{ 1 H} NMR, FT-IR, UV/Vis absorption spectroscopy, and ESI-mass spectrometry,
and their purity was confirmed by elemental analysis. The stability
of the complexes in aqueous buffer solution (pH 7.4) was confirmed
by UV/Vis spectroscopy. The cytotoxicity of the complexes ( 1–8 ) was then evaluated on prostate cancer cells (PC3), showing a low
nanomolar to low micromolar in vitro cytotoxicity.
Worthy of note, three of the Re(I) tricarbonyl complexes showed very
low (IC 50 = 30–50 nM) cytotoxic activity against
PC3 cells and up to 26-fold selectivity over normal human retinal
pigment epithelial-1 (RPE-1) cells. The cytotoxicity of both complexes 3 and 6 was lowered under hypoxic conditions
in PC3 cells. However, the compounds were still 10 times more active
than cisplatin in these conditions. Additional biological experiments
were then performed on the most selective complexes (complexes 3 and 6 ). Cell fractioning experiments followed
by ICP-MS studies revealed that 3 and 6 accumulate
mostly in the mitochondria and nucleus, respectively. Despite the
respective mitochondrial and nuclear localization of 3 and 6 , 3 did not trigger the apoptosis
pathways for cell killing, whereas 6 can trigger apoptosis
but not as a major pathway. Complex 3 induced a paraptosis
pathway for cell killing while 6 did not induce any of
our other tested pathways, namely, necrosis, paraptosis, and autophagy.
Both complexes 3 and 6 were found to be
involved in mitochondrial dysfunction and downregulated the ATP production
of PC3 cells. To the best of our knowledge, this report presents some
of the most cytotoxic Re(I) carbonyl complexes with exceptionally
low nanomolar cytotoxic activity toward prostate cancer cells, demonstrating
further the future viability of utilizing rhenium in the fight against
cancer.
## Introduction
Introduction Rhenium(I) tricarbonyl complexes are attractive
due to a number
of potential applications. These include but are not limited to photophysics, 1 , 2 artificial photosynthetic materials, 3 − 5 supramolecular systems, 6 − 8 electrocatalysis, 9 , 10 photoinduced catalysis, 9 , 10 and nuclear medicine. 11 , 12 For decades, organometallic
compounds have proven to be auspicious anticancer drug candidates, 13 − 17 and as such, complexes may afford diverse mechanisms of action.
Rhenium organometallic compounds display several innate properties,
such as large Stokes shifts, long-lived emission states, emission
tunability through ligand variation, and high quantum yields. These
characteristics allow simplified elucidation of the mechanism of action
and the cellular distribution of the compounds through emission spectroscopy.
Due to their ability to chelate with metal ions such as N,N′ -donor bidentate ligands, imidazole-phenanthroline derivatives play
a pivotal role in biochemistry, analytical chemistry, and electrochemistry. 18 − 23 Several synthetic polycyclic compounds of the type [M(L,L′) 2 ] n + , where M is a transition metal
ion and L,L′ is an imidazo-phenanthroline-type ligand, have
been reported to successfully interact with DNA. 24 , 25 An essential requirement of cancer treatments is the ability
to
induce cancer cell death while preserving the life of healthy non-cancerous
cells. In recent studies, the use of rhenium(I) tricarbonyl complexes
as possible anticancer agents has shown promising results. 26 − 31 Wang et al . reported rhenium(I) tricarbonyl complexes
with the ability to exert irreversible oxidative stress as well as
glutathione metabolism disorder by means of accumulating in the mitochondria. 32 A rhenium isonitrile complex reported by Wilson et al . induces apoptosis by means of an unfolded protein
response and additionally shows in vitro and in vivo anticancer activity toward ovarian cancer cells. 33 , 34 Furthermore, they reported the scaffold protein NUBP2 that are involved
in Fe–S cluster biogenesis, as a relevant target for a Re(I)
complex (TRIP). 35 Simpson et al. designed rhenium(I) complexes that have the ability to induce cell
death by means of the inhibition of the phosphorylation of Aurora-A
kinase. 36 Delasoie et al . reported a Re(I) tricarbonyl complex that exhibited selectivity
toward HCT-116 cells, 37 while Ye et al . reported a rhenium(I)-based histone deacetylase inhibitor
with the ability to target mitochondria as well as suppress histone
deacetylases activity. 38 The development
of anticancer agents that exhibit numerous mechanisms
of action is of great importance since such compounds may have the
ability to overcome typical obstacles such as ineffectiveness toward
tumors and drug resistance. 39 , 40 A promising strategy
in drug development to increase the potential of anticancer drugs
in exhibiting multiple mechanisms of action is the coordination of
organic molecules with anticancer potential to various metals. With
this in mind and the recent success of imidazo[4,5- f ]1,10-phenanthroline derivatives as prospective anticancer agents
in colorectal and liver cancer, 41 − 43 we designed a small library of
rhenium(I) tricarbonyl complexes ( Figure 1 ) with N,N′ -bidentate
imidazo[4,5- f ]1,10-phenanthroline derivative ligand
systems, with systematic changes with N,S and aromatic motifs on the
backbone, having an appended aqua ligand to evaluate their potentials
as anticancer agents. Rhenium(I) tricarbonyl complexes are usually
kinetically inert, which will help increase the complex half-life
to reach the target. The aqua ligand will further increase the possibility
of target binding. 44 The deprotonation
of the metal bonded labile aqua ligand and DNA binding ability of
the complexes were investigated. Furthermore, we carried out in vitro cytotoxicity studies against prostate cancer and
normal cells to obtain a small structure–activity relationship
(SAR). The cellular localization, cellular uptake pathways, and possible
cell killing pathways of the complexes were also studied. Figure 1 Chemical structures
of rhenium(I) tricarbonyl complexes 1–8 .
## Results and Discussion
Results and Discussion Synthesis and Characterization The procedure reported
by Alberto et al . was utilized for the synthesis
of the rhenium(I) carbonyl complex-starting synthon fac -[NEt 4 ] 2 [Re(CO) 3 (Br) 3 ] (ReAA). 45 The characteristic CO stretching
frequencies of ReAA were observed at 1864 and 1998 cm –1 , respectively. The three Br – ligands were replaced
by labile aqua ligation via the addition of AgNO 3 at pH 2, resulting in the intermediate fac -[Re(CO) 3 (H 2 O) 3 ][NO 3 ]
compound. Water is a good leaving group and can thus be effortlessly
replaced by other ligands. Complexes 1 – 8 were obtained by the addition of the respective N,N′ bidentate
ligands, viz., (2,6-dimethoxypyridyl)imidazo[4,5- f ]1,10-phenanthroline ( L1 ), (indole)imidazo-[4,5- f ]1,10-phenanthroline ( L2 ), (5-methoxyindole)imidazo[4,5- f ]1,10-phenanthroline ( L3) (biphenyl)imidazo[4,5- f ]1,10-phenanthroline ( L4 ), (fluorene)imidazo[4,5- f ]1,10-phenanthroline ( L5 ), (benzo[ b ]thiophene)imidazo[4,5- f ]1,10-phenanthroline
( L6 ), (5-bromothiazole)imidazo[4,5- f ]1,10-phenanthroline ( L7 ), and (4,5-dimethylthiophene)imidazo-[4,5- f ]1,10-phenanthroline ( L8 ), respectively, to
the acidic fac -[Re(CO) 3 (H 2 O) 3 ][NO 3 ] solution, producing the fac -[Re(CO) 3 ( N,N′ -bid)(H 2 O)][NO 3 ]-type compounds. The general synthetic procedure
for 1 – 8 is illustrated in Scheme 1 . All compounds were
characterized using 1 H and 13 C{ 1 H}
NMR, FT-IR, UV/Vis absorption spectroscopy, and ESI-mass spectrometry,
and their purity was confirmed by elemental analysis. Scheme 1 General
Synthetic Procedure for Compounds 1 – 8 X-ray Crystallography fac -[Re(CO) 3 ( L3 )(Br)] ( 3a ) was grown from a
solution of fac -[Re(CO) 3 ( L3 )(H 2 O)][NO 3 ] and NEt 4 Br in DMSO.
The aqua complex did not yield any suitable crystals; however, upon
substitution of the axial aqua ligand with a bromido ligand, crystals
suitable for single crystal crystallography were obtained. The crystallographic
data for 3a is summarized in Table S1 , and the molecular diagram of the crystal structure is given
in Figure 2 . fac -[Re(CO) 3 ( L3 )(Br)] ( 3a ) crystallized in the triclinic P 1̅ space
group, with one molecule and one solvent dimethyl sulfoxide molecule
in the asymmetric unit. The bond distances and angles of 3a are in the range of similar structures and are reported in Table 1 . 46 − 53 Figure 2 Molecular
representation of the crystal structure of fac -[Re I (CO) 3 ( L3 )(Br)]·DMSO
( 3a ). Hydrogen atoms (except two N–H) are omitted
for clarity, and ellipsoids are drawn at the 50% probability level. Table 1 Selected Bond Distances and Angles
(Å, °) of 3a selected bond distances
(Å) selected bond angles
(°) Re1-C3 1.918(6) N1-Re1-N2 75.32(16) Re1-C1 1.905(6) C3-Re1-C2 87.7(2) Re1-C2 1.919(6) C1-Re1-N1 96.03(18) Re1-N2 2.173(4) C2-Re1-Br1 91.79(18) Re1-N1 2.182(4) C1-Re1-Br1 178.37(17) Re1-Br1 2.629(10) C3-Re1-Br1 89.90(16) N1-Re1-Br1 84.32(11) N2-Re1-Br1 83.98(12) A dihedral angle of 14.2(2)°, between the equatorial
plane
(O2–C2–Re1–C3–O3) and the plane through
the imidazo[4,5- f ]1,10-phenanthroline ligand (N1,
N2, C1–C12), is observed and expresses the significant bend
of the (methoxyindole)imidazo[4,5- f ]1,10-phenanthroline
ligand out of the equatorial plane toward the bromido ligand ( Figure S1 ). The solid-state structure of 3a is stabilized by one
intramolecular weak interaction (C21–H21···N4),
two intermolecular hydrogen bonding interactions (N3–H3A···O5,
N5–H5A···Br1), three weak interactions (C11–H11···Br1,
C24–H24···O4, and C25–H25C···O5),
two Y–X···Cg π-interactions (Re1–Br1···Cg1
and S1–O5···Cg2) with X···Cg
distances of 2.947(2) Å and 3.728(5) Å respectively, and
an extensive network (forty nine) of π–π-interactions
with Cg···Cg distances smaller than 3.8 Å ( Tables S2 and S3 and Figure S2 ). An infinite
one-dimensional O–H···O–H chain along
the c axis is observed that is formed by the two
hydrogen bonding interactions (N3–H3A···O5 and
N5–H5A···Br1) ( Figure S3 ). The intra- and intermolecular interactions in the crystal structure
of 3a were quantified and confirmed using Hirshfeld surface
analysis ( Figures S4 and S5 ). From the
fingerprint plots of 3a , it is clear that the H···H
and O···H/H···O interactions contribute
significantly to the packing and overall stabilization of the crystal
structure ( Figure S6 ). Stability Studies of the Complexes The stability of
the complexes in phosphate buffer (pH 7.4) and FBS solution was monitored
by UV/Vis absorption measurements over 6 h. No isosbestic point was
observed during the measurement of UV/Vis spectra of 1–8 in the buffer solution; however, a time-dependent decrease in absorbance
was noticed ( Figures S7–S10 ). This
was due to the poor water solubility, which leads to precipitation
of the complexes in aqueous solution. It is very important to note
that the solubility of the complexes increases significantly in the
presence of FBS, as recently observed with Ru(II) polypyridyl complexes
( Figure S11 ). 54 The stability of complexes 1–8 was also tested
in DMSO solution ( Figures S7–S10 ). Multiple isosbestic points were observed in the DMSO solution
for complexes 6–8 , suggesting a binding with DMSO,
which was not observed for complexes 1–5 . The
binding of complexes 6 and 8 with DMSO was
also confirmed by ESI-MS measurements ( Figure S12 ). In fact, observing the ESI-MS spectra for both complexes 6 and 8 , we noticed a DMSO–complex adduct
([Re I ( L6 / L8 )(CO) 3 (DMSO)] + ) as well as a methanolic adduct ([Re I ( L6 / L8 )(CO) 3 (CH 3 OH)] + ).
Moreover, a significant amount of the parent complex without the water
([Re I ( L6 / L8 )(CO) 3 ] + ) was detected ( Figure S12 ). We
suggest that the formation of the methanolic adduct is due to the
presence of a high amount of methanol in the ESI-MS sample solution,
replacing either the water molecule or the DMSO. Unfortunately, due
to the poor solubility of complex 7 in methanol, we could
not analyze the DMSO binding using ESI-MS. The stability of
the complexes in DMSO was further confirmed by 1 H NMR;
complexes 3 and 6 were dissolved in DMSO
for 24 h after which it was dried. The 1 H NMR spectrum
of 3 was the same before and after 24 h in DMSO ( Figure S13 ), confirming the results of the UV/Vis
experiments. On the other hand, only 50% of 6 was still
intact after it was dissolved in DMSO for 24 h while 50% was the DMSO
adduct. We confirmed that ∼70% of the aqua complex 6 is still intact after it was dissolved in DMSO for 6 h ( Figure S14 ). Influence of H + Ions – Acid Dissociation Constant
Determination Preliminary cytotoxicity studies indicated
that 3 and 6 showed the best cytotoxicity
and selectivity. For this reason, it was decided to proceed with 3 and 6 with a more detailed investigation, which
also included intracellular accumulation, cellular uptake, mitochondrial
respiration, cell death pathways, and ROS generation. The pH
dependence of 3 and 6 was studied at 25.0
°C from pH 5.5 to 11 in DMF:H 2 O 7:3 ( v/v %) solution. The stability of 3 and 6 in
DMF:H 2 O 7:3 ( v/v %) at pH 5.5 for 24 h
was confirmed; the UV/Vis spectra is provided in the Supporting Information
( Figures S15 and S16 ). Equation 1 describes
the general acid/base behavior of the monoprotic species HA, while eq 2 was utilized for the determination
of the acid dissociation constants of 3 and 6 , where A is the absorbance at the specific pH, A h is
the absorbance of the protonated species, A 0 is the absorbance
of the deprotonated species, and K a1 is
the acid dissociation constant. 1 2 The acid dissociation
constants of 3 and 6 were determined spectrophotometrically
by adjusting the pH of a
2 × 10 –5 M solution of 3 and 6 at wavelengths 433 and 283 nm, respectively, while measuring
the absorbance each time ( Figures S17 and S18 ). The nonlinear fit of this data to eq 2 allowed for determining the p K a1 values of 3 and 6 as 8.95 (±4)
and 9.14 (±2), respectively. This is illustrated in Figure 3 . These values correspond
with those of similar mononuclear deprotonation products and shows
that the complexes are all in the aqua form under physiological conditions. 55 − 57 Egli et a l. 55 determined
the p K a values of fac -[Re(CO) 3 (H 2 O) 3 ] + , yielding
the deprotonation products fac -[Re(CO) 3 (H 2 O) 2 (OH)] and fac -[Re(CO) 3 (H 2 O)(OH) 2 ] − to be
p K a1 = 7.5 ± 0.2 and p K a2 = 9.3 ± 0.3, respectively, while Schutte et al . 56 calculated the p K a of fac -[Re(CO) 3 (Trop)(H 2 O)] (where Trop = tropolonato) to be 8.96 ±
0.02. The p K a values of four rhenium dicarbonyl
complexes ([Re(CO) 2 (N,N)(P)(H 2 O)] + with N,N = 1,10′-phenanthroline and 2,9-dimethyl 1,10-phenanthroline,
P = PTA and DAPTA) were determined and were in the range 9.09 ±
0.03 to 9.28 ± 0.03. Therefore, no considerable difference is
observed in the p K a values in these rhenium(I)
complexes, whether the ligand trans to the aqua ligand
is a carbonyl or phosphine ligand or whether the bidentate ligand
has N,N or O,O atom donors. Figure 3 (a) Plot of absorbance vs pH
for 3 . [Re] = 2 × 10 –5 M, 25 °C,
367 nm, μ
= 1 M (NaClO 4 ). (b) Plot of absorbance vs pH for 6 . [Re] = 2 × 10 –5 M,
25 °C, 332 nm, μ = 1 M (NaClO 4 ). DNA Binding Study A preliminary UV/Vis DNA-complex
titration study indicated that 6 has the highest binding
constant in the series, and later on, we found out that 6 is the most cytotoxic complex in hand. Hence, to check the possible
ways of DNA binding by 6 , we have performed (a) a binding
study with a model nucleobase (guanosine) by 1 HNMR and
ESI-MS for a possibility of covalent binding, (b) an ethidium bromide
displacement assay for intercalation, and (c) a DNA melting assay
to confirm the binding ability. Surprisingly, in the 1 H NMR spectra of guanosine binding kinetics, we only observed a secondary
interaction of 6 with guanosine, despite 6 having a labile neutral water molecule in the Re center ( Figures S19–S22 ). The binding study between
the Re center of complex 6 and N7 of guanosine was monitored
by 1 H NMR in DMF at 37 °C ( Figures S19–S22 ). From the initial time (1 h), we observed a
0.015 ppm downfield shift of the H 8 peak along with the
shift of H b and H c . Considering H a , we did not find any shift during the course of the study. This
would suggest that the shift of the mentioned protons (H b , H c , and H 8 ) is due to some secondary interaction
but not to the binding with N7 of guanosine with the Re center. Moreover,
the amine of guanosine is not taking part in the secondary noncovalent
interaction ( Figure S20 ). When we analyzed
the same solution by ESI-MS, we found a little peak of 6 and a guanosine adduct along with an important amount of free ligand
( Figure S23 ). However, we could not find
any alteration of 1 H NMR peaks of 6 with time
( Figure S19 ) and the peaks were not shifted
in the 1 H NMR spectra studying the stability of 6 ( Figure S23 ). Hence, the formation of
the adduct and the degradation of 6 have occurred only
in the ESI-MS measurement conditions. Therefore, we deduced that the
complex cannot form any covalent bond with DNA via the Re center. The presence of a planar ring system increases
the possibility
of DNA intercalation. Hence, to measure the intercalation ability
of 6 , we performed an ethidium bromide (EB) displacement
assay by fluorescence quenching of the EB-DNA adduct ( Figure S24 ). The apparent binding constant for
intercalation of 6 with ctDNA is in the order of 3.9
(1) × 10 6 M –1 , suggesting the possible
targeting of DNA via intercalation. Finally, the DNA binding properties
of complex 6 was confirmed by measuring DNA melting temperature
( Figure 4 ). We observed
a shift of ctDNA melting temperature toward higher temperature of 6 -bound ctDNA (Δ T m ; 1 ±
0.4 °C and 4 ± 0.1 °C for 6 and EB, respectively,
( Figure 4 )) in comparison
to unbound/normal ctDNA melting temperature. Figure 4 ctDNA melting curve.
Conc. of ctDNA was 40 μM, conc. of EB
and 6 was 30 μM. Temperature: 60–97 °C;
holding time: 1 min, 1 °C/min and the abs was recorded with every
1 °C change in temperature. Distribution Coefficient (log P Values) The distribution coefficient (log P value) of the
complexes was determined using the standard shake flask method. 58 All the complexes showed almost equal distribution
coefficients within 1.5–2.0 ( Figure 5 ), suggesting hydrophobicity. This suggests
that the derivatization on the N–N bidentate ligand of Re complexes
might not be very useful to change the solubility and lipophilicity
of the compounds. Overall, the log P values of our
complexes compared well with other Re tricarbonyl compounds reported
in the literature. 30 , 59 Figure 5 Bar plot representing the log P values of complexes 1–8 . BSA Binding Serum albumin is the most abundant protein
present in the blood and plays an important role in the delivery of
hydrophobic compounds to the cell. Importantly, serum albumin contains
a free thiol from 34-cysteine 60 − 63 and Re complexes have very high binding affinity
for sulfur donors. 64 , 65 BSA has an intrinsic fluorescence
emission maximum at 341 nm upon excitation at 280 nm. 66 , 67 The internal fluorescence quenching was observed upon binding with
our compound. Hence, fluorescence titrations between BSA and different
concentrations of the complex were performed to measure the binding
constant. The bovine serum albumin (BSA) binding study suggests that
all complexes can bind BSA with a moderately high binding constant
in the order of 10 5 M –1 except for complex 7 ( Table 2 , Figures S25–S28 ). A significantly low
binding constant was observed for 7 (0.4 × 10 5 M –1 ). The Stern–Volmer quenching
constant signifies the distance between an inhibitor and the fluorescent
molecules. 68 − 70 The decrease of K sv indicates
a long distance between the inhibitor and fluorescence protein. We
observed the K sv in the order of 10 4 M –1 , suggesting a moderate fitting in the
hydrophobic pocket of BSA. Table 2 Affinity of Complexes 1–8 for BSA a complex Stern–Volmer quenching
constant ( K sv × 10 4 , M –1 ) binding constant ( K a × 10 5 , M –1 ) number of binding sites ( n ) 1 2.41 ± 0.05 2.71 ± 2.91 1.23
± 0.13 2 1.06 ± 0.03 1.10 ± 0.79 1.24
± 0.08 3 2.98 ± 0.15 1.81 ± 1.16 1.18
± 0.07 4 2.95 ± 0.09 1.46 ± 0.06 1.18
± 0.01 5 2.57 ± 0.07 1.04 ± 0.30 1.15
± 0.03 6 2.82 ± 0.07 2.29 ± 0.98 1.23
± 0.05 7 0.83 ± 0.03 0.40 ± 1.16 1.16
± 0.03 8 1.60 ± 0.10 0.94 ± 1.11 1.10
± 0.20 a All plots are available in Figures S25–S28 . Cytotoxicity The in vitro anticancer
activity of the complexes was determined against human prostate cancer
(PC3) and compared with the normal retinal pigment epithelial-1 (RPE-1)
cells. The results are summarized in Table 3 . Among the eight complexes, 4 – 6 showed cytotoxicity in the low nanomolar range
(0.04, 0.03, and 0.05 μM, respectively). Such low values are
scarce in the literature for similar compounds. However, the three
complexes are highly cytotoxic against normal RPE-1 cells as well
(0.12, 0.27, and 0.65 μM, respectively). Despite this, it is
very exciting to notice that almost all complexes are more toxic against
cancer cells than normal cells. Complex 3 is the most
promising by showing a 26-fold selectivity with an IC 50 value of 0.32 μM against PC3 cells. Additionally, it is almost
21 times more cytotoxic than cisplatin against PC3 cells. Interestingly,
it was observed by ICP-MS that the most selective complexes 3 and 6 were localizing in the mitochondria and
nuclei, respectively, suggesting that they have a different mode of
action. In general, we observed that a fused ring or similar derivatization
in the N–N bidentate ligand could be useful to increase cytotoxicity.
Very interestingly, comparing complexes 2 and 3 , the introduction of a methoxy moiety in the indole ring of 2 showed a positive impact on the IC 50 values with
an increase of almost 15-fold in cytotoxicity. Table 3 Cytotoxicity of Complexes 1 – 8 under the Normoxic Conditions in Comparison
to Cisplatin IC 50 (μM) ± S.D. a complex PC3 RPE-1 selectivity b 1 4.6 ± 0.5 0.36 ± 0.02 2 5 ±
3 23 ± 2 5 3 0.32 ± 0.03 8.2 ±
0.4 26 4 0.04 ± 0.01 0.12 ± 0.01 3 5 0.03 ±
0.01 0.27 ± 0.02 9 6 0.05 ± 0.01 0.65 ± 0.03 13 7 1.4 ± 0.40 4.2 ± 0.5 3 8 0.41
± 0.04 1.79 ± 0.04 4 cisplatin 6.9 ± 0.5 36
± 4 5 a S.D. is the standard deviation.
The IC 50 value was determined using the standard Resazurin
assay after 48 h of treatment under normoxic conditions (∼15%
O 2 level). The representative plots are provided in Figures S29–S33 . b Selectivity represents the ratio
between IC 50 against RPE-1 and IC 50 against
PC3 cells. Despite the high cytotoxic activity of complexes against
PC3 cells
in normoxia, a decrease in cytotoxicity (IC 50 , μM:
5.5 ± 1.3 for 3 and 7.0 ± 2.2 for 6 ) was observed for complexes 3 and 6 when
they were evaluated in hypoxic conditions ( Table S4 ). However, a decrease in cytotoxicity of cisplatin (IC 50 , >50 μM) in hypoxic conditions was also observed.
Hence, in hypoxic conditions, despite the decreased cytotoxicity of 3 and 6 against PC3 cells, they were still around
10 times more active than cisplatin. Very importantly, when the compounds
were tested against 3D multicellular PC3 cell spheroids, we observed
a small decrease in IC 50 concentration ( Figure 6 ), indicating that the compounds
are efficient in penetrating the more tumor-like environment of cell
spheroids. Figure 6 IC 50 values represent the cytotoxicity of complexes
against 3D multicellular PC3 cells spheroid (average diameter of 435
μm) after 72 h of incubation with the respective complexes in
normoxic conditions, determined using the AlamarBlue assay. Intracellular Accumulation Study Most of the rhenium(I)
carbonyl complexes reported so far preferentially target mitochondria. 14 , 32 , 59 , 71 In order to investigate the possible cellular target of the complexes,
we measured the accumulation of complexes 3 and 6 in the nuclei and mitochondria using cell fractionation
and ICP-MS and not using the intrinsic luminescent properties of the
Re(I) tricarbonyl complex. In fact, it was reported that different
cellular accumulation results were observed between these methods
since the luminescence properties of this type of metal complex are
environment-dependent and can alter the confocal results. 72 − 74 We found that 3 targets the mitochondria, whereas 6 mostly targets the nuclei and, to a lesser extent, the mitochondria
( Figure 7 ). This observation
is interesting since there are very few examples of Re(I) tricarbonyl
complexes that target the nuclei. Figure 7 Percentage of Re(I) accumulation in the
nucleus and mitochondria
compared to total uptake by PC3 cells. The amount of rhenium in the
isolated nucleus and mitochondria from complex-treated PC3 cells was
quantified using ICP-MS. Cellular Uptake and Uptake Pathways The cellular uptake
pathways of complexes 3 and 6 have been
investigated using known uptake pathways inhibitors, viz., 1 mM tetraethylammonium
chloride (cationic transporter inhibitor), 75 5 μM oligomycin and 50 mM 2-deoxy- d -glucose (metabolic
inhibitors), 76 , 77 and 50 mM ammonium chloride (endocytotic
inhibitor). 78 − 80 The cells were pre-treated with the respective inhibitors
to block the transporter channel. Hence, the uptake of complexes might
get affected if the complex utilizes those transporters. For both
complexes 3 and 6 , a significantly less
amount of complex uptake was observed in the presence of an endocytotic
inhibitor (i.e., ammonium chloride) 78 − 80 and at 4 °C in
comparison to the control ( Figure 8 B,C). Hence, the result suggests that endocytotic and
passive diffusions were majorly involved in complex uptake. Surprisingly,
a significantly high uptake of complex 6 was observed
compared to 3 , which could explain the low nanomolar
cytotoxicity of 6 ( Figure 8 A). Figure 8 (A–C) Amount (ng) of rhenium in PC3 cells upon
2 h of incubation
with either complex 3 or 6 in the presence
and absence of cellular uptake pathways inhibitor. The amount of Re
was quantified by ICP-MS. 1 mM tetraethylammonium chloride (cationic
transporter inhibitor), 5 μM oligomycin and 50 mM 2-deoxy- d -glucose (metabolic inhibitors), and 50 mM ammonium chloride
(endocytotic inhibitor) were used. Mitochondrial Respiration Test Since a significant
amount of the complexes 3 and 6 accumulated
in the mitochondria, the possible influence of the compounds in the
PC3 cells’ oxygen consumption was measured by the Mito stress
test using a Seahorse XF Analyzer. In both cases, a significant amount
of concentration-dependent mitochondrial dysfunction was identified.
Initially, a concentration-dependent lowering of basal respiration
was observed for both complexes 3 and 6 ( Figure 9 and Figure S34 ). Furthermore, a significant drop
in ATP production was identified ( Figure S35 ). Figure 9 Mito stress test profile in PC3 cells measured by a Seahorse XeF4
after 24 h treatment with complexes 3 and 6 and cisplatin in normoxic conditions. OCR stands for oxygen consumption
rate. The experiment was performed using specific electron transport
chain inhibitors, viz. , oligomycin (inhibitor of
ATP synthase (complex V)), FCCP (uncoupling agent), antimycin-A (complex
III inhibitors), and rotenone (complex I inhibitor). Cell Death Pathways and ROS Generation The cell death
pathways induced by 3 and 6 in PC3 cells
were analyzed by measuring cytotoxicity in the presence of specific
cell death pathway inhibitors, viz., 3-methyladenine (autophagy),
Z-VAD-FMK (apoptosis), cycloheximide (paraptosis), and necrostatin-1
(necrosis). A significant amount of increase in the IC 50 value (around 2 times) of complex 3 against paraptosis
inhibitor-treated PC3 cells was observed ( Figure 10 ). Finally, the paraptosis induction in
PC3 cells by complex 3 was confirmed by observing vacuole
formations in the bright field image of PC3 cells treated with compound 3 (SI, Figure S36 ). Of note, similar
kinds of Re complexes are known to induce cell death by paraptosis. 38 , 59 In the case of complex 6 , a very little decrease in
IC 50 dose was noticed when the cells were pre-treated with
an apoptosis inhibitor ( Figure 10 ). Hence, we performed the well-known annexin-V assay
to confirm the apoptosis pathway. As we found a non-apoptosis pathway
for cell killing during IC 50 dose determination of 3 in the presence of an apoptosis inhibitor ( Figure 10 ), complex 3 did
not induce apoptosis, whereas for complex 6 , a concentration-dependent
induction of apoptosis was observed ( Figure 11 ). However, the amount of apoptosis induction
by 6 was very low ( Figure 11 ), suggesting that apoptosis may not be
a major pathway for cell killing. In the case of cisplatin, we observed
an important increase of the IC 50 value in the presence
of both apoptosis and autophagy inhibitors ( Figure 10 ). The observed apoptosis and autophagy
induction by cisplatin agrees with literature reports. 81 Figure 10 IC 50 values of respective complexes against
PC3 cells
in the presence of inhibitors in comparison to the cytotoxicity without
any inhibitor. The inhibitors were pre-incubated for 1 h. The respective
metal complexes were then loaded and incubated for 36 h in normoxic
conditions (details in the Experimental section ). Figure 11 Induction of apoptosis of compound-treated PC3 cells were
measured
by flow cytometry after 24 h compound exposure. The compound-treated
cells were stained dually with Annexin V-PE and PI prior to analysis.
Lower left quadrant (both Annexine V and PI-negative fraction): intact
cells. Lower right quadrant (Annexine V-positive/PI-negative fraction):
early apoptosis. Upper right quadrant (Annexine V-positive/PI-positive
fraction): late apoptosis. Upper left quadrant (Annexine V-negative/PI-positive
fraction): necrosis. Staurosporine, an apoptosis inducer, was used
as positive control. Metal complexes are very well-known to generate
ROS due to the
presence of easily accessible multiple oxidation states. The generation
of ROS inside the PC3 cells treated with the two Re derivatives was
analyzed using a standard ROS detection kit. The result suggested
that the compounds do not play a role in altering the ROS homeostasis
of the cells. In fact, after treatment with complexes 3 and 6 , the ROS level ( Figure S37 ) inside the cells was similar to the control.
## Synthesis and Characterization
Synthesis and Characterization The procedure reported
by Alberto et al . was utilized for the synthesis
of the rhenium(I) carbonyl complex-starting synthon fac -[NEt 4 ] 2 [Re(CO) 3 (Br) 3 ] (ReAA). 45 The characteristic CO stretching
frequencies of ReAA were observed at 1864 and 1998 cm –1 , respectively. The three Br – ligands were replaced
by labile aqua ligation via the addition of AgNO 3 at pH 2, resulting in the intermediate fac -[Re(CO) 3 (H 2 O) 3 ][NO 3 ]
compound. Water is a good leaving group and can thus be effortlessly
replaced by other ligands. Complexes 1 – 8 were obtained by the addition of the respective N,N′ bidentate
ligands, viz., (2,6-dimethoxypyridyl)imidazo[4,5- f ]1,10-phenanthroline ( L1 ), (indole)imidazo-[4,5- f ]1,10-phenanthroline ( L2 ), (5-methoxyindole)imidazo[4,5- f ]1,10-phenanthroline ( L3) (biphenyl)imidazo[4,5- f ]1,10-phenanthroline ( L4 ), (fluorene)imidazo[4,5- f ]1,10-phenanthroline ( L5 ), (benzo[ b ]thiophene)imidazo[4,5- f ]1,10-phenanthroline
( L6 ), (5-bromothiazole)imidazo[4,5- f ]1,10-phenanthroline ( L7 ), and (4,5-dimethylthiophene)imidazo-[4,5- f ]1,10-phenanthroline ( L8 ), respectively, to
the acidic fac -[Re(CO) 3 (H 2 O) 3 ][NO 3 ] solution, producing the fac -[Re(CO) 3 ( N,N′ -bid)(H 2 O)][NO 3 ]-type compounds. The general synthetic procedure
for 1 – 8 is illustrated in Scheme 1 . All compounds were
characterized using 1 H and 13 C{ 1 H}
NMR, FT-IR, UV/Vis absorption spectroscopy, and ESI-mass spectrometry,
and their purity was confirmed by elemental analysis. Scheme 1 General
Synthetic Procedure for Compounds 1 – 8
## X-ray Crystallography
X-ray Crystallography fac -[Re(CO) 3 ( L3 )(Br)] ( 3a ) was grown from a
solution of fac -[Re(CO) 3 ( L3 )(H 2 O)][NO 3 ] and NEt 4 Br in DMSO.
The aqua complex did not yield any suitable crystals; however, upon
substitution of the axial aqua ligand with a bromido ligand, crystals
suitable for single crystal crystallography were obtained. The crystallographic
data for 3a is summarized in Table S1 , and the molecular diagram of the crystal structure is given
in Figure 2 . fac -[Re(CO) 3 ( L3 )(Br)] ( 3a ) crystallized in the triclinic P 1̅ space
group, with one molecule and one solvent dimethyl sulfoxide molecule
in the asymmetric unit. The bond distances and angles of 3a are in the range of similar structures and are reported in Table 1 . 46 − 53 Figure 2 Molecular
representation of the crystal structure of fac -[Re I (CO) 3 ( L3 )(Br)]·DMSO
( 3a ). Hydrogen atoms (except two N–H) are omitted
for clarity, and ellipsoids are drawn at the 50% probability level. Table 1 Selected Bond Distances and Angles
(Å, °) of 3a selected bond distances
(Å) selected bond angles
(°) Re1-C3 1.918(6) N1-Re1-N2 75.32(16) Re1-C1 1.905(6) C3-Re1-C2 87.7(2) Re1-C2 1.919(6) C1-Re1-N1 96.03(18) Re1-N2 2.173(4) C2-Re1-Br1 91.79(18) Re1-N1 2.182(4) C1-Re1-Br1 178.37(17) Re1-Br1 2.629(10) C3-Re1-Br1 89.90(16) N1-Re1-Br1 84.32(11) N2-Re1-Br1 83.98(12) A dihedral angle of 14.2(2)°, between the equatorial
plane
(O2–C2–Re1–C3–O3) and the plane through
the imidazo[4,5- f ]1,10-phenanthroline ligand (N1,
N2, C1–C12), is observed and expresses the significant bend
of the (methoxyindole)imidazo[4,5- f ]1,10-phenanthroline
ligand out of the equatorial plane toward the bromido ligand ( Figure S1 ). The solid-state structure of 3a is stabilized by one
intramolecular weak interaction (C21–H21···N4),
two intermolecular hydrogen bonding interactions (N3–H3A···O5,
N5–H5A···Br1), three weak interactions (C11–H11···Br1,
C24–H24···O4, and C25–H25C···O5),
two Y–X···Cg π-interactions (Re1–Br1···Cg1
and S1–O5···Cg2) with X···Cg
distances of 2.947(2) Å and 3.728(5) Å respectively, and
an extensive network (forty nine) of π–π-interactions
with Cg···Cg distances smaller than 3.8 Å ( Tables S2 and S3 and Figure S2 ). An infinite
one-dimensional O–H···O–H chain along
the c axis is observed that is formed by the two
hydrogen bonding interactions (N3–H3A···O5 and
N5–H5A···Br1) ( Figure S3 ). The intra- and intermolecular interactions in the crystal structure
of 3a were quantified and confirmed using Hirshfeld surface
analysis ( Figures S4 and S5 ). From the
fingerprint plots of 3a , it is clear that the H···H
and O···H/H···O interactions contribute
significantly to the packing and overall stabilization of the crystal
structure ( Figure S6 ).
## Stability Studies of the Complexes
Stability Studies of the Complexes The stability of
the complexes in phosphate buffer (pH 7.4) and FBS solution was monitored
by UV/Vis absorption measurements over 6 h. No isosbestic point was
observed during the measurement of UV/Vis spectra of 1–8 in the buffer solution; however, a time-dependent decrease in absorbance
was noticed ( Figures S7–S10 ). This
was due to the poor water solubility, which leads to precipitation
of the complexes in aqueous solution. It is very important to note
that the solubility of the complexes increases significantly in the
presence of FBS, as recently observed with Ru(II) polypyridyl complexes
( Figure S11 ). 54 The stability of complexes 1–8 was also tested
in DMSO solution ( Figures S7–S10 ). Multiple isosbestic points were observed in the DMSO solution
for complexes 6–8 , suggesting a binding with DMSO,
which was not observed for complexes 1–5 . The
binding of complexes 6 and 8 with DMSO was
also confirmed by ESI-MS measurements ( Figure S12 ). In fact, observing the ESI-MS spectra for both complexes 6 and 8 , we noticed a DMSO–complex adduct
([Re I ( L6 / L8 )(CO) 3 (DMSO)] + ) as well as a methanolic adduct ([Re I ( L6 / L8 )(CO) 3 (CH 3 OH)] + ).
Moreover, a significant amount of the parent complex without the water
([Re I ( L6 / L8 )(CO) 3 ] + ) was detected ( Figure S12 ). We
suggest that the formation of the methanolic adduct is due to the
presence of a high amount of methanol in the ESI-MS sample solution,
replacing either the water molecule or the DMSO. Unfortunately, due
to the poor solubility of complex 7 in methanol, we could
not analyze the DMSO binding using ESI-MS. The stability of
the complexes in DMSO was further confirmed by 1 H NMR;
complexes 3 and 6 were dissolved in DMSO
for 24 h after which it was dried. The 1 H NMR spectrum
of 3 was the same before and after 24 h in DMSO ( Figure S13 ), confirming the results of the UV/Vis
experiments. On the other hand, only 50% of 6 was still
intact after it was dissolved in DMSO for 24 h while 50% was the DMSO
adduct. We confirmed that ∼70% of the aqua complex 6 is still intact after it was dissolved in DMSO for 6 h ( Figure S14 ).
## Influence of H
Influence of H + Ions – Acid Dissociation Constant
Determination Preliminary cytotoxicity studies indicated
that 3 and 6 showed the best cytotoxicity
and selectivity. For this reason, it was decided to proceed with 3 and 6 with a more detailed investigation, which
also included intracellular accumulation, cellular uptake, mitochondrial
respiration, cell death pathways, and ROS generation. The pH
dependence of 3 and 6 was studied at 25.0
°C from pH 5.5 to 11 in DMF:H 2 O 7:3 ( v/v %) solution. The stability of 3 and 6 in
DMF:H 2 O 7:3 ( v/v %) at pH 5.5 for 24 h
was confirmed; the UV/Vis spectra is provided in the Supporting Information
( Figures S15 and S16 ). Equation 1 describes
the general acid/base behavior of the monoprotic species HA, while eq 2 was utilized for the determination
of the acid dissociation constants of 3 and 6 , where A is the absorbance at the specific pH, A h is
the absorbance of the protonated species, A 0 is the absorbance
of the deprotonated species, and K a1 is
the acid dissociation constant. 1 2 The acid dissociation
constants of 3 and 6 were determined spectrophotometrically
by adjusting the pH of a
2 × 10 –5 M solution of 3 and 6 at wavelengths 433 and 283 nm, respectively, while measuring
the absorbance each time ( Figures S17 and S18 ). The nonlinear fit of this data to eq 2 allowed for determining the p K a1 values of 3 and 6 as 8.95 (±4)
and 9.14 (±2), respectively. This is illustrated in Figure 3 . These values correspond
with those of similar mononuclear deprotonation products and shows
that the complexes are all in the aqua form under physiological conditions. 55 − 57 Egli et a l. 55 determined
the p K a values of fac -[Re(CO) 3 (H 2 O) 3 ] + , yielding
the deprotonation products fac -[Re(CO) 3 (H 2 O) 2 (OH)] and fac -[Re(CO) 3 (H 2 O)(OH) 2 ] − to be
p K a1 = 7.5 ± 0.2 and p K a2 = 9.3 ± 0.3, respectively, while Schutte et al . 56 calculated the p K a of fac -[Re(CO) 3 (Trop)(H 2 O)] (where Trop = tropolonato) to be 8.96 ±
0.02. The p K a values of four rhenium dicarbonyl
complexes ([Re(CO) 2 (N,N)(P)(H 2 O)] + with N,N = 1,10′-phenanthroline and 2,9-dimethyl 1,10-phenanthroline,
P = PTA and DAPTA) were determined and were in the range 9.09 ±
0.03 to 9.28 ± 0.03. Therefore, no considerable difference is
observed in the p K a values in these rhenium(I)
complexes, whether the ligand trans to the aqua ligand
is a carbonyl or phosphine ligand or whether the bidentate ligand
has N,N or O,O atom donors. Figure 3 (a) Plot of absorbance vs pH
for 3 . [Re] = 2 × 10 –5 M, 25 °C,
367 nm, μ
= 1 M (NaClO 4 ). (b) Plot of absorbance vs pH for 6 . [Re] = 2 × 10 –5 M,
25 °C, 332 nm, μ = 1 M (NaClO 4 ).
## DNA Binding Study
DNA Binding Study A preliminary UV/Vis DNA-complex
titration study indicated that 6 has the highest binding
constant in the series, and later on, we found out that 6 is the most cytotoxic complex in hand. Hence, to check the possible
ways of DNA binding by 6 , we have performed (a) a binding
study with a model nucleobase (guanosine) by 1 HNMR and
ESI-MS for a possibility of covalent binding, (b) an ethidium bromide
displacement assay for intercalation, and (c) a DNA melting assay
to confirm the binding ability. Surprisingly, in the 1 H NMR spectra of guanosine binding kinetics, we only observed a secondary
interaction of 6 with guanosine, despite 6 having a labile neutral water molecule in the Re center ( Figures S19–S22 ). The binding study between
the Re center of complex 6 and N7 of guanosine was monitored
by 1 H NMR in DMF at 37 °C ( Figures S19–S22 ). From the initial time (1 h), we observed a
0.015 ppm downfield shift of the H 8 peak along with the
shift of H b and H c . Considering H a , we did not find any shift during the course of the study. This
would suggest that the shift of the mentioned protons (H b , H c , and H 8 ) is due to some secondary interaction
but not to the binding with N7 of guanosine with the Re center. Moreover,
the amine of guanosine is not taking part in the secondary noncovalent
interaction ( Figure S20 ). When we analyzed
the same solution by ESI-MS, we found a little peak of 6 and a guanosine adduct along with an important amount of free ligand
( Figure S23 ). However, we could not find
any alteration of 1 H NMR peaks of 6 with time
( Figure S19 ) and the peaks were not shifted
in the 1 H NMR spectra studying the stability of 6 ( Figure S23 ). Hence, the formation of
the adduct and the degradation of 6 have occurred only
in the ESI-MS measurement conditions. Therefore, we deduced that the
complex cannot form any covalent bond with DNA via the Re center. The presence of a planar ring system increases
the possibility
of DNA intercalation. Hence, to measure the intercalation ability
of 6 , we performed an ethidium bromide (EB) displacement
assay by fluorescence quenching of the EB-DNA adduct ( Figure S24 ). The apparent binding constant for
intercalation of 6 with ctDNA is in the order of 3.9
(1) × 10 6 M –1 , suggesting the possible
targeting of DNA via intercalation. Finally, the DNA binding properties
of complex 6 was confirmed by measuring DNA melting temperature
( Figure 4 ). We observed
a shift of ctDNA melting temperature toward higher temperature of 6 -bound ctDNA (Δ T m ; 1 ±
0.4 °C and 4 ± 0.1 °C for 6 and EB, respectively,
( Figure 4 )) in comparison
to unbound/normal ctDNA melting temperature. Figure 4 ctDNA melting curve.
Conc. of ctDNA was 40 μM, conc. of EB
and 6 was 30 μM. Temperature: 60–97 °C;
holding time: 1 min, 1 °C/min and the abs was recorded with every
1 °C change in temperature.
## Distribution Coefficient (log
Distribution Coefficient (log P Values) The distribution coefficient (log P value) of the
complexes was determined using the standard shake flask method. 58 All the complexes showed almost equal distribution
coefficients within 1.5–2.0 ( Figure 5 ), suggesting hydrophobicity. This suggests
that the derivatization on the N–N bidentate ligand of Re complexes
might not be very useful to change the solubility and lipophilicity
of the compounds. Overall, the log P values of our
complexes compared well with other Re tricarbonyl compounds reported
in the literature. 30 , 59 Figure 5 Bar plot representing the log P values of complexes 1–8 .
## BSA Binding
BSA Binding Serum albumin is the most abundant protein
present in the blood and plays an important role in the delivery of
hydrophobic compounds to the cell. Importantly, serum albumin contains
a free thiol from 34-cysteine 60 − 63 and Re complexes have very high binding affinity
for sulfur donors. 64 , 65 BSA has an intrinsic fluorescence
emission maximum at 341 nm upon excitation at 280 nm. 66 , 67 The internal fluorescence quenching was observed upon binding with
our compound. Hence, fluorescence titrations between BSA and different
concentrations of the complex were performed to measure the binding
constant. The bovine serum albumin (BSA) binding study suggests that
all complexes can bind BSA with a moderately high binding constant
in the order of 10 5 M –1 except for complex 7 ( Table 2 , Figures S25–S28 ). A significantly low
binding constant was observed for 7 (0.4 × 10 5 M –1 ). The Stern–Volmer quenching
constant signifies the distance between an inhibitor and the fluorescent
molecules. 68 − 70 The decrease of K sv indicates
a long distance between the inhibitor and fluorescence protein. We
observed the K sv in the order of 10 4 M –1 , suggesting a moderate fitting in the
hydrophobic pocket of BSA. Table 2 Affinity of Complexes 1–8 for BSA a complex Stern–Volmer quenching
constant ( K sv × 10 4 , M –1 ) binding constant ( K a × 10 5 , M –1 ) number of binding sites ( n ) 1 2.41 ± 0.05 2.71 ± 2.91 1.23
± 0.13 2 1.06 ± 0.03 1.10 ± 0.79 1.24
± 0.08 3 2.98 ± 0.15 1.81 ± 1.16 1.18
± 0.07 4 2.95 ± 0.09 1.46 ± 0.06 1.18
± 0.01 5 2.57 ± 0.07 1.04 ± 0.30 1.15
± 0.03 6 2.82 ± 0.07 2.29 ± 0.98 1.23
± 0.05 7 0.83 ± 0.03 0.40 ± 1.16 1.16
± 0.03 8 1.60 ± 0.10 0.94 ± 1.11 1.10
± 0.20 a All plots are available in Figures S25–S28 .
## Cytotoxicity
Cytotoxicity The in vitro anticancer
activity of the complexes was determined against human prostate cancer
(PC3) and compared with the normal retinal pigment epithelial-1 (RPE-1)
cells. The results are summarized in Table 3 . Among the eight complexes, 4 – 6 showed cytotoxicity in the low nanomolar range
(0.04, 0.03, and 0.05 μM, respectively). Such low values are
scarce in the literature for similar compounds. However, the three
complexes are highly cytotoxic against normal RPE-1 cells as well
(0.12, 0.27, and 0.65 μM, respectively). Despite this, it is
very exciting to notice that almost all complexes are more toxic against
cancer cells than normal cells. Complex 3 is the most
promising by showing a 26-fold selectivity with an IC 50 value of 0.32 μM against PC3 cells. Additionally, it is almost
21 times more cytotoxic than cisplatin against PC3 cells. Interestingly,
it was observed by ICP-MS that the most selective complexes 3 and 6 were localizing in the mitochondria and
nuclei, respectively, suggesting that they have a different mode of
action. In general, we observed that a fused ring or similar derivatization
in the N–N bidentate ligand could be useful to increase cytotoxicity.
Very interestingly, comparing complexes 2 and 3 , the introduction of a methoxy moiety in the indole ring of 2 showed a positive impact on the IC 50 values with
an increase of almost 15-fold in cytotoxicity. Table 3 Cytotoxicity of Complexes 1 – 8 under the Normoxic Conditions in Comparison
to Cisplatin IC 50 (μM) ± S.D. a complex PC3 RPE-1 selectivity b 1 4.6 ± 0.5 0.36 ± 0.02 2 5 ±
3 23 ± 2 5 3 0.32 ± 0.03 8.2 ±
0.4 26 4 0.04 ± 0.01 0.12 ± 0.01 3 5 0.03 ±
0.01 0.27 ± 0.02 9 6 0.05 ± 0.01 0.65 ± 0.03 13 7 1.4 ± 0.40 4.2 ± 0.5 3 8 0.41
± 0.04 1.79 ± 0.04 4 cisplatin 6.9 ± 0.5 36
± 4 5 a S.D. is the standard deviation.
The IC 50 value was determined using the standard Resazurin
assay after 48 h of treatment under normoxic conditions (∼15%
O 2 level). The representative plots are provided in Figures S29–S33 . b Selectivity represents the ratio
between IC 50 against RPE-1 and IC 50 against
PC3 cells. Despite the high cytotoxic activity of complexes against
PC3 cells
in normoxia, a decrease in cytotoxicity (IC 50 , μM:
5.5 ± 1.3 for 3 and 7.0 ± 2.2 for 6 ) was observed for complexes 3 and 6 when
they were evaluated in hypoxic conditions ( Table S4 ). However, a decrease in cytotoxicity of cisplatin (IC 50 , >50 μM) in hypoxic conditions was also observed.
Hence, in hypoxic conditions, despite the decreased cytotoxicity of 3 and 6 against PC3 cells, they were still around
10 times more active than cisplatin. Very importantly, when the compounds
were tested against 3D multicellular PC3 cell spheroids, we observed
a small decrease in IC 50 concentration ( Figure 6 ), indicating that the compounds
are efficient in penetrating the more tumor-like environment of cell
spheroids. Figure 6 IC 50 values represent the cytotoxicity of complexes
against 3D multicellular PC3 cells spheroid (average diameter of 435
μm) after 72 h of incubation with the respective complexes in
normoxic conditions, determined using the AlamarBlue assay.
## Intracellular Accumulation Study
Intracellular Accumulation Study Most of the rhenium(I)
carbonyl complexes reported so far preferentially target mitochondria. 14 , 32 , 59 , 71 In order to investigate the possible cellular target of the complexes,
we measured the accumulation of complexes 3 and 6 in the nuclei and mitochondria using cell fractionation
and ICP-MS and not using the intrinsic luminescent properties of the
Re(I) tricarbonyl complex. In fact, it was reported that different
cellular accumulation results were observed between these methods
since the luminescence properties of this type of metal complex are
environment-dependent and can alter the confocal results. 72 − 74 We found that 3 targets the mitochondria, whereas 6 mostly targets the nuclei and, to a lesser extent, the mitochondria
( Figure 7 ). This observation
is interesting since there are very few examples of Re(I) tricarbonyl
complexes that target the nuclei. Figure 7 Percentage of Re(I) accumulation in the
nucleus and mitochondria
compared to total uptake by PC3 cells. The amount of rhenium in the
isolated nucleus and mitochondria from complex-treated PC3 cells was
quantified using ICP-MS.
## Cellular Uptake and Uptake Pathways
Cellular Uptake and Uptake Pathways The cellular uptake
pathways of complexes 3 and 6 have been
investigated using known uptake pathways inhibitors, viz., 1 mM tetraethylammonium
chloride (cationic transporter inhibitor), 75 5 μM oligomycin and 50 mM 2-deoxy- d -glucose (metabolic
inhibitors), 76 , 77 and 50 mM ammonium chloride (endocytotic
inhibitor). 78 − 80 The cells were pre-treated with the respective inhibitors
to block the transporter channel. Hence, the uptake of complexes might
get affected if the complex utilizes those transporters. For both
complexes 3 and 6 , a significantly less
amount of complex uptake was observed in the presence of an endocytotic
inhibitor (i.e., ammonium chloride) 78 − 80 and at 4 °C in
comparison to the control ( Figure 8 B,C). Hence, the result suggests that endocytotic and
passive diffusions were majorly involved in complex uptake. Surprisingly,
a significantly high uptake of complex 6 was observed
compared to 3 , which could explain the low nanomolar
cytotoxicity of 6 ( Figure 8 A). Figure 8 (A–C) Amount (ng) of rhenium in PC3 cells upon
2 h of incubation
with either complex 3 or 6 in the presence
and absence of cellular uptake pathways inhibitor. The amount of Re
was quantified by ICP-MS. 1 mM tetraethylammonium chloride (cationic
transporter inhibitor), 5 μM oligomycin and 50 mM 2-deoxy- d -glucose (metabolic inhibitors), and 50 mM ammonium chloride
(endocytotic inhibitor) were used.
## Mitochondrial Respiration Test
Mitochondrial Respiration Test Since a significant
amount of the complexes 3 and 6 accumulated
in the mitochondria, the possible influence of the compounds in the
PC3 cells’ oxygen consumption was measured by the Mito stress
test using a Seahorse XF Analyzer. In both cases, a significant amount
of concentration-dependent mitochondrial dysfunction was identified.
Initially, a concentration-dependent lowering of basal respiration
was observed for both complexes 3 and 6 ( Figure 9 and Figure S34 ). Furthermore, a significant drop
in ATP production was identified ( Figure S35 ). Figure 9 Mito stress test profile in PC3 cells measured by a Seahorse XeF4
after 24 h treatment with complexes 3 and 6 and cisplatin in normoxic conditions. OCR stands for oxygen consumption
rate. The experiment was performed using specific electron transport
chain inhibitors, viz. , oligomycin (inhibitor of
ATP synthase (complex V)), FCCP (uncoupling agent), antimycin-A (complex
III inhibitors), and rotenone (complex I inhibitor).
## Cell Death Pathways and ROS Generation
Cell Death Pathways and ROS Generation The cell death
pathways induced by 3 and 6 in PC3 cells
were analyzed by measuring cytotoxicity in the presence of specific
cell death pathway inhibitors, viz., 3-methyladenine (autophagy),
Z-VAD-FMK (apoptosis), cycloheximide (paraptosis), and necrostatin-1
(necrosis). A significant amount of increase in the IC 50 value (around 2 times) of complex 3 against paraptosis
inhibitor-treated PC3 cells was observed ( Figure 10 ). Finally, the paraptosis induction in
PC3 cells by complex 3 was confirmed by observing vacuole
formations in the bright field image of PC3 cells treated with compound 3 (SI, Figure S36 ). Of note, similar
kinds of Re complexes are known to induce cell death by paraptosis. 38 , 59 In the case of complex 6 , a very little decrease in
IC 50 dose was noticed when the cells were pre-treated with
an apoptosis inhibitor ( Figure 10 ). Hence, we performed the well-known annexin-V assay
to confirm the apoptosis pathway. As we found a non-apoptosis pathway
for cell killing during IC 50 dose determination of 3 in the presence of an apoptosis inhibitor ( Figure 10 ), complex 3 did
not induce apoptosis, whereas for complex 6 , a concentration-dependent
induction of apoptosis was observed ( Figure 11 ). However, the amount of apoptosis induction
by 6 was very low ( Figure 11 ), suggesting that apoptosis may not be
a major pathway for cell killing. In the case of cisplatin, we observed
an important increase of the IC 50 value in the presence
of both apoptosis and autophagy inhibitors ( Figure 10 ). The observed apoptosis and autophagy
induction by cisplatin agrees with literature reports. 81 Figure 10 IC 50 values of respective complexes against
PC3 cells
in the presence of inhibitors in comparison to the cytotoxicity without
any inhibitor. The inhibitors were pre-incubated for 1 h. The respective
metal complexes were then loaded and incubated for 36 h in normoxic
conditions (details in the Experimental section ). Figure 11 Induction of apoptosis of compound-treated PC3 cells were
measured
by flow cytometry after 24 h compound exposure. The compound-treated
cells were stained dually with Annexin V-PE and PI prior to analysis.
Lower left quadrant (both Annexine V and PI-negative fraction): intact
cells. Lower right quadrant (Annexine V-positive/PI-negative fraction):
early apoptosis. Upper right quadrant (Annexine V-positive/PI-positive
fraction): late apoptosis. Upper left quadrant (Annexine V-negative/PI-positive
fraction): necrosis. Staurosporine, an apoptosis inducer, was used
as positive control. Metal complexes are very well-known to generate
ROS due to the
presence of easily accessible multiple oxidation states. The generation
of ROS inside the PC3 cells treated with the two Re derivatives was
analyzed using a standard ROS detection kit. The result suggested
that the compounds do not play a role in altering the ROS homeostasis
of the cells. In fact, after treatment with complexes 3 and 6 , the ROS level ( Figure S37 ) inside the cells was similar to the control.
## Conclusions
Conclusions This study reports the synthesis, characterization,
DNA binding
mode, and in vitro cytotoxicity of eight rhenium(I)
tricarbonyl complexes. All complexes showed stability in aqueous solution
and can bind to BSA with binding constants ranging from 10 4 to 10 5 M –1 . The p K a values of 3 and 6 indicate
that these complexes will be in the aqua form under biological conditions.
The Re(I) carbonyl complexes showed exceptionally low nanomolar cytotoxicity
against prostate adenocarcinoma (PC3) and a degree of selectivity
toward cancer cells over normal human retinal pigment epithelial-1
(RPE-1) cells. The most selective complexes 3 and 6 were found to localize in mitochondria and nuclei, respectively.
Moreover, both complexes 3 and 6 downregulated
the mitochondrial ATP production in PC3 cells. The most selective
complex 3 followed a paraptosis pathway for cell killing.
On the other hand, despite the intercalation of complex 6 with DNA and the induction of mitochondrial dysfunction after treatment,
only a very little percentage of cells induced apoptosis. This suggests
that apoptosis is a minor pathway for cell killing. Surprisingly, 6 did not follow any of the other tested cell death pathways,
i.e., necrosis, autophagy, or paraptosis. Very interestingly, this
suggests that the presence of different functional groups on the same
phenanthroline ligand core could induce different cell death mechanisms,
while maintaining a very promising IC 50 range.
## Experimental Section
Experimental Section General Procedures and Instrumentation The IR spectra
were recorded at room temperature on a PerkinElmer BX II IR spectrometer
in the range 4000–370 cm. The liquid-state 1 H and 13 C{ 1 H} NMR spectra were recorded at 25.0 °C
on a 600 MHz Avance II Bruker spectrometer operating at 600 and 151
MHz for 1 H and 13 C, respectively, and dimethyl
sulfoxide- d 6 (DMSO- d 6 ) was used as solvent. The chemical shifts (δ) are reported
in parts per million (ppm); for DMSO- d 6 , the spectra were referenced relative to the solvent peak (2.50
ppm for 1 H and 39.52 ppm for 13 C). Coupling
constants ( J ) are reported in Hz. Electrospray ionization
mass spectrometry (ESI-MS) of 1 – 8 was recorded on a SCIEX 4000 QTRAP hybrid triple quadrupole ion
trap mass spectrometer either in positive or in negative ESI mode
with samples injected as methanol solutions. The ionization voltage
was set at 5500 V in positive mode with a 10 psi curtain gas setting
and 20 psi ionization gas (GS1) setting. The UV/Vis spectra were recorded
in the range of 100–1100 nm at room temperature on a LAMBDA
265 UV/Visible spectrophotometer. The spectrometer utilizes a Xenon
Flash interface. The intensity data was collected on a Bruker X8 ApexII
4 K Kappa CCD diffractometer, equipped with graphite monochromated
Mo K(α) radiation with a wavelength of 0.71073 Å, using
an exposure time of 20 s/frame. A total of 1568 frames were collected
with a frame width of 0.5° with 99.7% completeness accomplished.
Phi and omega scans are reported at 100 K. All cell refinements as
well as data reductions were performed using SAINT-Plus software. 82 Absorption corrections were made using the multiscan
technique and the SADABS software package. All structures were solved
with the SIR-97 software package, 83 and
structure refinement was made using SHELXL-2013 84 and WinGX. 85 The molecular graphics
were drawn using DIAMOND. 86 All non-hydrogen
atoms were refined anisotropically. Methyl and aromatic hydrogen atoms
were placed in geometrically idealized positions and constrained to
ride on their parent atoms, with C–H = 0.98 and 0.95 A°
and U iso (H) = 1.5Ueq(C) and 1.2Ueq(C), respectively. The
NH hydrogen atoms were located in the difference Fourier map and freely
refined with U iso (H) = 1.5Ueq(N). The Fc versus Fo plot
proved five reflections to be outliers, and they were removed from
the refinement as systematic errors. Synthesis The starting synthon fac -[NEt 4 ] 2 [Re(CO) 3 (Br) 3 ] (ReAA) and the ligands (2,6-dimethoxypyridyl)imidazo[4,5- f ]1,10-phenanthroline ( L1 ), (indole)imidazo[4,5- f ]1,10-phenanthroline ( L2 ), (5-methoxyindole)imidazo[4,5- f ]1,10-phenanthroline ( L3 ), (biphenyl)imidazo[4,5- f ]1,10-phenanthroline ( L4 ), (fluorene)imidazo[4,5- f ]1,10-phenanthroline ( L5 ), (benzo[ b ]thiophene)imidazo[4,5- f ]1,10-phenanthroline
( L6 ), (5-bromothiazole)imidazo[4,5- f ]1,10-phenanthroline ( L7 ), and (4,5-dimethylthiophene)imidazo[4,5- f ]1,10-phenanthroline ( L8 ) have been synthesized
and characterized as reported previously in the literature. 45 , 50 13 C{ 1 H} NMR was attempted for 1 – 8 ; however, after 18,000 scans on highly concentrated
NMR samples, poor or no peaks were obtained. Due to economic implications,
other characterization methods were relied upon. General Synthetic Procedure for Re(I) Tricarbonyl Complexes ReAA was added to 3 equiv of AgNO 3 solution in 10 mL
of water (at pH 2) and stirred at room temperature for 24 h, which
resulted in a gray/white precipitate of AgBr. The precipitate was
filtered, and 1.1 equiv of the ligand was added to the filtrate. The
solution was stirred at 80 °C for 12 h, and a solid was separated
from the solution. The reaction mixture was filtered and washed three
times with distilled water to yield a solid product. fac -[Re(CO) 3 ( L1 )(H 2 O)][NO 3 ] ( 1 ) 0.206 g (0.267
mmol) ReAA, 0.136 g (0.801 mmol) AgNO 3 , 0.099 g (0.294
mmol) L1 . Yield: 0.0902 g (0.131 mmol, 49%); elemental
analysis for C 23 H 17 N 6 O 9 Re: calculated: C = 39.04%, N = 11.88%, H = 2.42%; found: C = 38.91%,
N = 11.90%, H = 2.44%.; IR (ATR, cm –1 ): ν N-H = 3346, ν O-H = 3064, 2958,
ν C=O = 2028, 1896, ν C=N = 1605,
ν C=N = 1582, 1452, ν C-O =
1323, 1274.; 1 H NMR (400 MHz, DMSO -d 6 ): δ 9.5 (4H, m), 8.6 (1H, d, J = 8.2
Hz), 8.2 (2H, bs), 6.7 (1H, d, J = 8.2 Hz), 4.2 (3H,
s), 4.0 (3H, s) ppm; 13 C{ 1 H} NMR (151 MHz, DMSO -d 6 ): δ 196.9, 164.2, 163.9, 159.8, 159.6,
150.0, 142.5, 142.3, 124.6, 105.1, 104.6, 103.2, 102.9, 54.6, 54.5,
54.4, 54.3 ppm. UV/Vis (DMSO): λ max = 350 nm, ε
= 12,223 M –1 cm –1 . ESI-MS (CH 3 OH, +ve ion mode): m / z 627.9
([M - NO 3 – - H 2 O] + ); calcd, 627.6). fac -[Re(CO) 3 ( L2 )(H 2 O)][NO 3 ] ( 2 ) 0.201 g (0.260
mmol) ReAA, 0.132 g (0.777 mmol) AgNO 3 , 0.096 g (0.286
mmol) L2 . Yield: 0.098 g (0.143 mmol, 55%); elemental
analysis for C 24 H 15 N 6 O 7 Re: calculated: C = 42.04%, N = 12.26%, H = 2.21%; found: C = 41.94%,
N = 12.31%, H = 2.19%; IR (ATR, cm –1 ): ν N-H = 3387, ν O-H = 3074, ν C=O = 2019, 1912, 1886, ν C=N = 1576, ν C=N = 1318; 1 H NMR (400 MHz, DMSO -d 6 ): δ 9.4 (4H, overlying multiplet, singlet, and
doublet), 8.7 (1H, s), 8.3 (2H, overlying singlets), 8.1 (1H, s),
7.6 (1H, s), 7.3 (2H, s) ppm; 13 C{ 1 H} NMR (151
MHz, DMSO -d 6 ): δ 197.0, 143.6, 137.0,
134.3, 127.2, 127.1, 125.5, 123.1, 121.8, 121.1, 112.7 ppm; UV/Vis
(DMSO): λ max = 345 nm, ε = 23,989 M –1 cm –1 . ESI-MS (CH 3 OH, +ve ion mode): m / z 606.2 ([M - NO 3 – - H 2 O] + ); calcd, 605.6). fac -[Re(CO) 3 ( L3 )(H 2 O)][NO 3 ] ( 3 ) 0.20 g (0.260
mmol) ReAA, 0.132 g (0.780 mmol) AgNO 3 , 0.104 g (0.286
mmol) L3 . Yield: 0.0614 g (0.0858 mmol, 33%); elemental
analysis for C 25 H 17 N 6 O 8 Re: calculated: C = 41.96%, N = 11.74%, H = 2.39%; found: C = 41.86%,
N = 11.72%, H = 2.38%; IR (ATR, cm –1 ): ν N-H = 3121, ν O-H = 2921, 2852,
ν C=O = 2028, 1936, 1910, ν C=N =
1603, ν C=N = 1319, ν C-O =
1217. 1 H NMR (600 MHz, DMSO -d 6 ): δ 11.7 (1H, s), 9.3 (2H, broad singlet), 9.2 (2H, singlet),
8.2 (2H, broad singlet), 8.1 (2H, s), 7.5 (1H, d, J = 8.7 Hz), 6.9 (1H, d, J = 8.6 Hz), 3.9 (3H, s)
ppm. 13 C{ 1 H} NMR (151 MHz, DMSO -d 6 ): δ 155.1, 132.1, 126.1, 125.7, 113.3, 113.0,
103.6, 55.9 ppm. UV/Vis (DMSO): λ max = 390 nm, ε
= 19,145 M –1 cm –1 ; ESI-MS (CH 3 OH, +ve ion mode): m / z 636.2
([M - NO 3 – - H 2 O] + ); calcd, 635.6). fac -[Re(CO) 3 ( L4 )(H 2 O)][NO 3 ] ( 4 ) 0.102 g (0.132
mmol) ReAA, 0.067 g (0.397 mmol) AgNO 3 , 0.054 g (0.145
mmol) L4 . Yield: 0.0219 g (0.0303 mmol, 23%); elemental
analysis for C 28 H 18 N 5 O 7 Re: calculated: C = 46.53%, N = 9.69%, H = 2.51%; found: C = 46.23%,
N = 9.71%, H = 2.48%. IR (ATR, cm –1 ): ν O-H = 3091, 3027, ν C=O = 2038, 1993,
1956, ν C=C = 1808, 1775, 1610, ν C=N = 1554, ν C=N = 1339. 1 H NMR (600 MHz,
DMSO -d 6 ): δ 13.9 (1H, s), 9.1 (4H,
s), 8.3 (2H, d, J = 7.3 Hz), 8.0 (2H, broad singlet),
7.9 (2H, d, J = 7.4 Hz), 7.8 (2H, d, J = 7.0 Hz), 7.5 (2H, t, J = 13.8, 6.9 Hz), 7.4 (1H,
t, J = 13.8, 6.9 Hz) ppm. 13 C{ 1 H} NMR (151 MHz, DMSO -d 6 ): δ 151.6,
141.9, 139.6, 129.5, 129.0, 128.5, 127.7, 127.4, 127.2, 125.1 ppm.
UV/Vis (DMSO): λ max = 337 nm, ε = 25,793 M –1 cm –1 . ESI-MS (CH 3 OH,
+ve ion mode): m / z 643.2 ([M - NO 3 – - H 2 O] + ); calcd,
642.6). fac -[Re(CO) 3 ( L5 )(H 2 O)][NO 3 ] ( 5 ) 0.10 g (0.130
mmol) ReAA, 0.066 g (0.39 mmol) AgNO 3 , 0.055 g (0.143 mmol) L5 . Yield: 0.0343 g (0.0469 mmol, 36%); elemental analysis
for C 29 H 18 N 5 O 7 Re: calculated:
C = 47.41%, N = 9.53%, H = 2.47%; found: C = 47.44%, N = 9.50%, H
= 2.43%. IR (ATR, cm –1 ): ν N-H = 3471, ν O-H = 3057, 2902, ν C=O = 2028, 1926, 1905, ν C=N = 1610, ν C=C = 1557, 1476, ν C-H = 1446, ν C=N = 1317. 1 H NMR (600 MHz, DMSO -d 6 ): δ 9.1 (4H, merged singlet), 8.3 (1H, s), 8.2 (1H,
d, J = 7.6 Hz), 8.0 (3H, merged singlet), 7.9 (1H,
d, J = 4.6 Hz), 7.6 (1H, d, J =
7.3 Hz), 7.3 (2H, dt, J = 29.2, 7.3 Hz), 4.0 (2H,
s) ppm. 13 C{ 1 H} NMR (151 MHz, DMSO -d 6 ): δ 152.2, 146.7, 144.1, 144.0, 143.1, 140.7,
139.2, 128.2, 127.8, 127.4, 125.7, 125.6, 125.1, 123.2, 120.9, 120.8,
36.8 ppm. UV/Vis (DMSO): λ max = 342 nm, ε =
29,853 M –1 cm –1 . ESI-MS (CH 3 OH, +ve ion mode): m / z 655.2
([M - NO 3 – - H 2 O] + ); calcd, 654.7). fac -[Re(CO) 3 ( L6 )(H 2 O)][NO 3 ] ( 6 ) 0.199 g (0.258
mmol) ReAA, 0.131 g (0.774 mmol) AgNO 3 , 0.10 g (0.283 mmol) L6 . Yield: 0.105 g (0.149 mmol, 58%); elemental analysis for
C 24 H 14 N 5 O 7 SRe: calculated:
C = 41.02%, N = 9.97%, S = 4.56%, H = 2.01%; found: C = 41.20%, N
= 9.96%, S = 4.60%, H = 2.02%. IR (ATR, cm –1 ): ν N-H = 3397, ν O-H = 3075, ν C=O = 2028, 1920, 1894, ν C=N = 1660, ν C=C = 1581, 1562, ν C=N = 1370, 1304. 1 H NMR (400 MHz, DMSO -d 6 ): δ
9.5 (1H, dd, J = 15.3, 4.7 Hz), 9.3 (1H, broad singlet),
9.1 (2H, overlapping singlets), 8.3 (2H, overlapping singlets), 8.0
(3H, m), 7.5 (2H, overlapping signals) ppm. 13 C{ 1 H} NMR (101 MHz, DMSO -d 6 ): δ 196.9,
152.8, 148.6, 140.2, 140.0, 139.9, 133.2, 132.8, 127.6, 126.6, 126.2,
125.7, 125.5, 125.3, 125.2, 124.9, 124.2, 123.4, 123.2, 123.0 ppm.
UV/Vis (DMSO): λ max = 369 nm, ε = 28,040 M –1 cm –1 . ESI-MS (CH 3 OH,
+ve ion mode): m / z 623.1 ([M - NO 3 – - H 2 O] + ); calcd,
622.6). fac -[Re(CO) 3 ( L7 )(H 2 O)][NO 3 ] ( 7 ) 0.206 g (0.267
mmol) ReAA, 0.136 g (0.801 mmol) AgNO 3 , 0.112 g (0.294
mmol) L7 . Yield: 0.0254 g (0.0347 mmol, 13%); elemental
analysis for C 19 H 10 N 6 O 7 SBrRe: calculated: C = 31.15%, N = 11.47%, S = 4.37%, Br = 10.91%,
H = 1.37%; found: C = 31.25%, N = 11.51%, S = 4.41%, Br = 10.89%,
H = 1.35%. IR (ATR, cm –1 ): ν N-H = 3398, ν O-H = 3059, ν C=O = 2025, 1918, 1893, ν C=N = 1608, ν C=N = 1312, 1254, ν C-Br = 805, ν C-S = 805. 1 H NMR (400 MHz, DMSO -d 6 ): δ 13.9 (1H, s), 11.7 (1H, s), 9.5 (2H, s), 8.2 (3H, overlying
singlets), 7.5 (1H, triplet, J = 16.9, 8.72), 6.9
(1H, doublet, J = 8.7 Hz) ppm. 13 C{ 1 H} NMR (101 MHz, DMSO -d 6 ): δ
197.0, 159.0, 152.4, 145.3, 140.0, 128.1, 124.5 ppm. UV/Vis (DMSO):
λ max = 261 nm, ε = 1494 M –1 cm –1 . ESI-MS (CH 3 OH, +ve ion mode): m / z 671.1 ([M - NO 3 – ] + ); calcd, 670.5). fac -[Re(CO) 3 ( L8 )(H 2 O)][NO 3 ] ( 8 ) 0.203 g (0.264
mmol) ReAA, 0.135 g (0.792 mmol) AgNO 3 , 0.112 g (0.294
mmol) L8 . Yield: 0.0609 g (0.0896 mmol, 34%); elemental
analysis for C 22 H 16 N 5 O 7 SRe: calculated: C = 38.82%, N = 10.29%, S = 4.71%, H = 2.37%; found:
C = 38.85%, N = 10.27%, S = 4.69%, H = 2.38%. IR (ATR, cm –1 ): ν N-H = 3066, ν O-H = 2923, 2865, ν C=O = 2025, 1904, 1894, ν C=N = 1617, ν C=C = 1586, 1545, ν C-S = 806. 1 H NMR (400 MHz, DMSO -d 6 ): δ 14.1 (1H, s), 9.4 (2H, d, J = 4.9 Hz), 9.1 (2H, broad multiplet), 8.1 (2H, broad doublet, J = 19.8 Hz), 7.6 (1H, s), 2.4 (3H, s), 2.2 (3H, s) ppm. 13 C{ 1 H} NMR (101 MHz, DMSO -d 6 ): δ 197.9, 190.0, 151.7, 148.7, 137.0, 134.8, 133.2,
132.9, 130.3, 127.8, 126.9, 13.8, 13.5 ppm. UV/Vis (DMSO): λ max = 341 nm, ε = 6340 M –1 cm –1 . ESI-MS (CH 3 OH, +ve ion mode): m / z 601.1 ([M - NO 3 – - H 2 O] + ); calcd, 600.6). Stability Study The stability of the complexes in DMSO,
aqueous buffer, and FBS solutions was monitored using time-dependent
UV/Vis absorption measurements. Briefly, the concentration of each
complex was maintained at 10 μM except for complex 7 . In fact, due to poor solubility, we used 5 μM concentration
for compound 7 . A 1:20 v/v DMSO:10 mM
phosphate buffer pH 7.4 in the presence of 4 mM NaCl solution of the
complexes was used to evaluate the aqueous solution stability. In
the case of the stability in FBS solution, we prepared the complex
solution in 1:99 v/v DMSO:10 mM phosphate buffer pH 7.4 in the presence
of 10% FBS. The stability of the complexes in solution was monitored
up to 6 h. Determination of Acid Dissociation Constant (p K a ) pH measurements were performed using a Hanna
pH211 Microprocessor pH meter, equipped with a HI 1131 probe, which
was calibrated using standard buffer solutions, with pH = 4.01, 7.00,
and 10.00. A Varian Cary 50 Conc UV/Vis spectrophotometer coupled
to a personal computer capable of performing least squares analyses
on the absorption values vs pH data was utilized
for the kinetic measurements. Microsoft Office Excel (2019) and Scientist
Micromath, version 2.01 were used to fit the collected data to specific
functions. 87 , 88 A circulating water bath system
was used to control and maintain the temperature at 25 °C. The
ionic strength (μ) of all solutions was maintained at 1 M NaClO 4 . The stability of complexes in DMSO solution was also
monitored by ESI-MS measurement. Briefly, 10 μM DMSO solution
of the complexes was incubated for 48 h at 37 °C. Finally, 10
μL of DMSO solution was diluted in 1 mL of methanol and analyzed
by ESI-MS. Distribution Coefficient The distribution coefficient
or lipophilicity (log D ) was determined using a standard
shake flask technique. 58 Briefly, 40 μL
of 1 mM DMSO solution of complex was added to 4 mL of pre-equilibrated
10 mM phosphate buffer (pH 7.4) octanol mixture (1:1 v/v ) and agitated continuously in a vertical shaker for 6 h. Then, each
layer of the mixture was separated by centrifugation and transferred
to new vials. Further, the complex concentration in each layer was
determined by measuring their absorption spectra. Finally, the lipophilicity
was determined using eq 3 below. 3 BSA Binding Study The BSA binding study was performed
by measuring the internal fluorescence quenching of BSA. BSA has an
intrinsic fluorescence emission maxima at 341 nm upon excitation at
280 nm. 66 , 67 Briefly, 20 μM BSA solution in 1×
Tris buffer (pH 7.4) was incubated with different concentrations (0–10
equiv) of complex solution for 15 min at 37 °C. Further, their
fluorescence intensities were recorded at 341 nm upon excitation at
280 nm. Finally, the fluorescence quenching constant and the binding
constant were calculated using the Stern–Volmer equation ( eq 4 ) and the Scatchard equation
( eq 5 ), respectively. 4 5 In the equations, the
fluorescence emission intensity of native protein is denoted as F 0 , the fluorescence emission intensity in the
presence of quencher is denoted as F , [Q] corresponds
to the concentration of quencher for F , K sv is the Stern–Volmer quenching constant, K a is denoted as the binding constant of quencher
and protein, and n is the number of binding sites
per protein molecule. Complexes showed no emission at 341 nm when
excited at 280 nm. DNA Binding Study With Model Nucleobase The DNA binding ability of complex 6 through covalent bond formation was studied in the presence
of a model nucleobase, guanosine, using 1 H NMR and ESI-MS.
Precisely, 1.5 mM DMF- d 7 solution of complex 6 was allowed to react with 3 mM guanosine solution in DMF- d 7 at 37 °C and the binding was monitored
at different intervals of time by recording 1 H NMR spectra
up to 24 h. Finally, after 24 h, 5 μL of DMF solution was diluted
in 1 mL of methanol and analyzed by ESI-MS. Preparation of ctDNA Solution Calf thymus deoxyribonucleic
acid (ctDNA) and tris–HCl buffer solution (pH = 7.4, 1 M) were
purchased from Sigma Aldrich, South Africa, and used without further
purification. The ctDNA stock solution was prepared by stirring a
solution of ctDNA in Tris–HCl buffer for 12 h at room temperature.
The purity of the stock solution was confirmed by the ratio of A 260nm / A 280nm (1.843),
indicating that the solution was free of protein. 45 , 81 , 89 , 91 This solution
was stored at 4 °C between scans and discarded 2 days after preparation.
The molar extinction coefficient value of ctDNA at 260 nm (6600 L
mol –1 cm –1 ) was employed in the
determination of the stock solution concentration. Ethidium Bromide Displacement Assay To check the possibility
of DNA intercalation due to the presence of a planer fused ring ligand
system, we performed the standard ethidium bromide (EB) displacement
assay. Precisely, 10 μM ctDNA solution in 1× Tris·NaCl
buffer was incubated at 37 °C with 3 equiv of EB for 30 min.
After this, 0 to 10 equiv of complex 6 were added to
the solution and incubated for an additional 30 min at 37 °C.
Each solution was excited at 540 nm, and emission was measured from
550 to 800 nm. Finally, the apparent binding constant ( K app ) was measured using eq 6 . 6 where [EB] is the concentration
of EB (30.0 μM) and [C 50 ] is the concentration of
complex when the reduction of fluorescence reach 50%. K EB is the binding constant of EB ( K EB = 1 × 10 7 M –1 for ctDNA). 90 The IC 50 values were calculated from
plots of normalized fluorescence intensity versus complex concentration
( Figure S24B ). DNA Melting The DNA binding ability of complex 6 was confirmed by measuring the DNA melting temperature prior
to complex binding. Briefly, 40 μM ctDNA was incubated with
30 μM of the complex at 37 °C for 15 min. Afterward, the
absorbance of the solution was measured at 260 nm in each °C
increment in temperature within the range from 60–97 °C.
A Cary UV 300 (Agilent Technologies) was used to measure all absorbances.
The experiment was programmed with a 1 °C/min temperature ramp
rate from the temperature range 60–97 °C, and the holding
time was 1 min. Finally, the absorbance of each experiment was normalized
to 0 and 1 and further compared with the melting temperature of native
ctDNA. Cell Lines and Cell Culture Human prostate adenocarcinoma
(PC3) and normal human retinal pigment epithelial-1 (RPE-1) cells
were originally procured from the American Type Culture Collection
(ATCC). The PC3 and RPE-1 cells were maintained in Ham’s F-12K
(Kaighn’s) Medium and DMEM/F-12, GlutaMAX Supplement medium
containing 10% fetal bovine serum (GIBCO) and 1% penicillin–streptomycin
(100 units/mL, GIBCO), respectively. Cytotoxicity The standard Resazurin assay was performed
to determine the cytotoxicity of the synthesized complexes. In particular,
4 × 10 3 cells were seeded in each well of a 96 well
microplate and incubated at 37 °C under normoxic conditions (∼15%
O 2 level). After 24 h of the initial incubation period,
the medium was replenished by a fresh metal complex-containing medium
(100 μL/well) and incubated for an additional 48 h. Initially,
the metal complex was dissolved in DMSO and the target concentration
was achieved by further dilution with the medium. A total of seven
or nine different concentrations of the metal complex solutions from
10 nM to 100 μM were used to observe the dose-dependent cytotoxicity.
The ultimate concentration of DMSO in each well was ≤1%. Finally,
the metal complex-containing medium was carefully aspirated and an
equal volume of Resazurin (0.2 mg/mL)-containing medium was added.
After 4 h of incubation at 37 °C, the fluorescence of the produced
resorufin product (λ ex = 540 nm, λ em = 590 nm) was measured in either a Tecan infinite F200PRO Microplate
Reader or a Cytation 5, Cell Imaging Multi-Mode Reader, BioTek. Finally,
the half-maximal inhibitory concentration (IC 50 ) value
was calculated using the GraphPad Prism 5 software from the plot of
% of cell viability vs log[complex concentration]
after applying nonlinear fitting. Each of the reported IC 50 values is the mean of three individual experiments with standard
deviation, and each concentration was assayed in sextuplicate in each
successive experiment. The cytotoxic inactivation of most selective
complexes 3 and 6 in hypoxic conditions
(2.0% O 2 ) was also measured against PC3 cells. A similar
protocol as stated in normoxia cytotoxicity was followed to determine
their IC 50 in hypoxic conditions, keeping everything unaltered
except the incubation in the presence of 2.0% O 2 . Initially,
at least three consecutive cell passages were maintained in hypoxic
conditions before starting any experiments. Cytotoxicity against 3D Cell Spheroid 1 × 10 4 PC3 cells were seeded in each well of a Corning 96 Well Ultra-Low
Attachment Treated Spheroid Microplate. After 96 h of incubation at
37 °C in normoxic conditions, the average spheroid size observed
was 435 μm. Half of the medium was replaced by an equal volume
of complex-containing (two times of target concentrations) medium.
After 72 h, the cell viability was measured using the AlamarBlue assay.
Finally, the IC 50 values were determined by using GraphPad
Prism 5 software after applying four parameter fitting. Intracellular Accumulation Study The fluorescence property
is highly dependent on pH and the cellular microenvironment. 74 , 91 Hence, we quantified Re in different cellular organelles using ICP-MS
to determine the complex localization in cells. An adequate
number of PC3 cells were seeded in a 150 mm diameter Petri dish using
F12K medium and incubated in normoxic conditions at 37 °C. The
medium from each Petri dish was replenished every 2 days. When the
cell confluency reached around 80–90%, a fresh medium containing
5 μM complexes 3 or 6 was added and
incubated for an additional 1.5 h in normoxic conditions at 37 °C.
Cells were harvested by trypsinization. The medium was removed by
centrifugation, and the cell pellet was washed twice with cold DPBS.
The nucleus and mitochondria of treated cells were then isolated using
the standard kit protocols mentioned below. To determine the total
rhenium uptake, 5 × 10 5 cells were collected and digested
using 100 μL of 70% nitric acid at 60 °C for 12 h. The
acid solution was further diluted using 4900 μL of MilliQ water.
Finally, the rhenium concentration was measured using ICP-MS. Each
experiment was performed in triplicate. Nuclei: The nuclei isolation
kit, Nuclei EZ Prep (NUC101) (Sigma-Aldrich)
was used to isolate the nuclei from the complex-treated cells, and
the standard kit protocol was employed for the isolation process.
The complex-treated cells were exposed to 4 mL of ice-cold Nuclei
EZ lysis buffer and vortexed briefly to lyse the cells. The solution
was settled at 4 °C for another 5 min and centrifuged at 500 g for 5 min at 4 °C. The supernatant was removed carefully.
The cell and nucleus pellets were redissolved using ice-cold Nuclei
EZ lysis buffer, and the same process was repeated to maximize the
nuclei yield. During this isolation step, both the nucleus and cells
were stained with the trypan blue dye prior to counting using a hemocytometer
to obtain the exact number of the nuclei and intact cells. Mitochondria:
The mitochondria isolation kit (MITOISO2) (Sigma-Aldrich)
was used to isolate the mitochondria from the complex-treated cells,
and the standard kit protocol was employed for the isolation procedures.
By following the kit protocol, the complex-treated cells were lysed
using 1× extraction buffer at 4 °C using a Dounce homogenizer.
The exact number of lysed cells was counted using a hemocytometer
after trypan blue staining. The intact cells and other heavier cell
fragments were removed from the solution by centrifugation at 600 g for 10 min at 4 °C. The supernatant was transferred
carefully and centrifuged at 11000 g for 10 min at
4 °C to isolate the mitochondria. Hence, the total amount of
isolated mitochondria corresponds to the total lysed cells. The percentage
of rhenium accumulation in the nucleus and mitochondria was calculated
using the total accumulated rhenium corresponding to 5 × 10 5 cells. Sample Preparation for ICP-MS The samples were digested
in either 100 μL (cell pellet) or 500 μL (nucleus and
mitochondria pellet) of 70% HNO 3 at 60 °C for 12 h.
The acid solution was further diluted (1:50 v/v )
with MilliQ water. Finally, the rhenium concentration in the solution
was analyzed using a high-resolution ICP-MS instrument of ThermoScientific
(Element II, ThermoScientific) at the Institut de Physique du Globe
de Paris, France. Cellular Uptake Pathways 1.5 × 10 6 PC3
cells were seeded in a 6 cm Petri dish and incubated in normoxic conditions
at 37 °C. After 24 h of seeding, the cells were treated with
different cellular uptake pathways inhibitors for 2 h in normoxic
conditions. The inhibitor used here were as follows: 1 mM tetraethylammonium
chloride (cationic transporter inhibitor), 5 μM oligomycin and
50 mM 2-deoxy- d -glucose (metabolic inhibitors), and 50 mM
ammonium chloride (endocytotic inhibitor) as different uptake pathway
inhibitors. Additionally, we pre-incubated the cells at 4 °C
for 2 h to determine the involvement of passive transport on the cellular
uptake process. After 2 h, cells were washed twice with DPBS and 5
μM of complex ( 3 and 6 ) solution was
added. After 2 h of incubation in normoxic conditions, cells were
harvested using trypsinization and counted. 1 × 10 6 cells were separated and digested in 100 μL of conc. HNO 3 . Finally, the acid solution was diluted by addition of 4900
μL of MQ water to keep the acid % within 2. The rhenium concentration
in solution was measured by ICP-MS. Each successive experiment and
control experiments were performed three times to determine the average
value with standard deviation. Mito Stress Test Using Seahorse Apparatus 3 ×
10 4 PC3 cells/well were seeded of a Seahorse XF Cell Culture
Microplate using 80 μL of FBS-supplemented F12K medium and incubated
in normoxic conditions for 24 h at 37 °C. The medium was replaced
by an equal volume of compound ( 3 , 0.4 and 5 μM; 6 , 0.05 and 2 μM; and cisplatin, 7 and 30 μM)-containing
medium. After 24 h of treatment, media were removed, and the treated
cells were washed very carefully with the Seahorse XF medium three
times. Finally, the cells were incubated at 37 °C for 1 h in
a non-CO 2 incubator. The Mito Stress assay was run in a
Seahorse Xfe96 instrument (Agilent) at 37 °C using multiple inhibitors,
viz., ATP synthase inhibitor (oligomycin, 1 μM), proton gradient
and mitochondrial membrane potential collapsing agent (FCCP, 1 μM),
and mitochondrial respiratory complex I and III inhibitors (rotenone,
1 μM and antimycin A, 1 μM, respectively). After the experiment,
the cells were fixed using 4% p -formaldehyde solution
and the nucleus was stained with DAPI. Each well was imaged in a Cytation
5 Cell Imaging Multimode Reader (BioTek) using a 10× objective
lens. Finally, the cells from each image were calculated by using
Gen5 software ( Figure S35 ) and by utilizing
the cell count and the data was normalized against the same cell number. Cell Death Mechanism To evaluate the possible cell
death pathways, we measured the cell viability in the presence of
different cell death pathway inhibitors. 44 , 75 Briefly, 1 × 10 3 PC3 cells/well were seeded in 96-well
plates and incubated for 24 h in normoxic conditions at 37 °C.
The medium was removed and treated with inhibitors for 1 h. Autophagy
inhibitor (3-methyladenine (100 μM)), apoptosis inhibitor (Z-VAD-FMK
(20 μM)), paraptosis inhibitor (cycloheximide (0.1 μM)),
and necrosis inhibitor (necrostatin-1 (60 μM)) were the different
inhibitors used. After 1 h, the inhibitor-containing medium was removed
and washed with 1× DPBS. Then, different concentrations of complex
solution were added and incubated for 36 h in normoxic conditions.
Finally, the cell viability was determined by Resazurin assay (check
the Cytotoxicity section for details) to evaluate the IC 50 values. Annexin-V/PI Assay for Apoptosis Detection 1.5 ×
10 6 PC3 cells were seeded in a 6 cm Petri dish using a
F12K medium and incubated at 37 °C in normoxic conditions. After
24 h of incubation, the medium was replenished with a complex-containing
medium and incubated additionally for 24 h. Two different concentrations
of complexes 3 and 6 were used to determine
the possible apoptosis pathways for cell killing. The cells were then
harvested by trypsinization and washed twice with DPBS. The cells
were further stained with both FITC-tagged Annexin-V (cat. no. 556419,
BD Biosciences) and propidium iodide (cat. no. 51-66211E, BD Biosciences).
Finally, the stained cells were analyzed using a BD LSR II flow cytometry
instrument (BD Biosciences) at Institut Curie, Paris, France. ROS Analysis 1.5 × 10 4 PC3 cells were
seeded in the dark in 96-well plates for 24 h. Four different concentrations
of complexes 3 and 6 were used to treat
the cells for 8 h, and then two different concentrations of H 2 O 2 were used to treat the cells for 30 min (10
and 15 μM). The medium was removed, and 40 μM DCFH-DiOxy-Q
(Kit No. ab238535, Abcam) in 10% FBS containing HEPES was added in
each well followed by incubation at 37 °C for 30 min. The cells
were washed twice with HEPES, and finally fluorescence intensity was
measured (λ ex /λ em ; 485/530 nm) using
a Cytation 5 Cell Imaging Multimode Reader (BioTek). Then, the cells
were fixed using p -formaldehyde and stained using
DAPI. The cells were finally counted in the same way as stated before
and utilized to normalize the data.
## General Procedures and Instrumentation
General Procedures and Instrumentation The IR spectra
were recorded at room temperature on a PerkinElmer BX II IR spectrometer
in the range 4000–370 cm. The liquid-state 1 H and 13 C{ 1 H} NMR spectra were recorded at 25.0 °C
on a 600 MHz Avance II Bruker spectrometer operating at 600 and 151
MHz for 1 H and 13 C, respectively, and dimethyl
sulfoxide- d 6 (DMSO- d 6 ) was used as solvent. The chemical shifts (δ) are reported
in parts per million (ppm); for DMSO- d 6 , the spectra were referenced relative to the solvent peak (2.50
ppm for 1 H and 39.52 ppm for 13 C). Coupling
constants ( J ) are reported in Hz. Electrospray ionization
mass spectrometry (ESI-MS) of 1 – 8 was recorded on a SCIEX 4000 QTRAP hybrid triple quadrupole ion
trap mass spectrometer either in positive or in negative ESI mode
with samples injected as methanol solutions. The ionization voltage
was set at 5500 V in positive mode with a 10 psi curtain gas setting
and 20 psi ionization gas (GS1) setting. The UV/Vis spectra were recorded
in the range of 100–1100 nm at room temperature on a LAMBDA
265 UV/Visible spectrophotometer. The spectrometer utilizes a Xenon
Flash interface. The intensity data was collected on a Bruker X8 ApexII
4 K Kappa CCD diffractometer, equipped with graphite monochromated
Mo K(α) radiation with a wavelength of 0.71073 Å, using
an exposure time of 20 s/frame. A total of 1568 frames were collected
with a frame width of 0.5° with 99.7% completeness accomplished.
Phi and omega scans are reported at 100 K. All cell refinements as
well as data reductions were performed using SAINT-Plus software. 82 Absorption corrections were made using the multiscan
technique and the SADABS software package. All structures were solved
with the SIR-97 software package, 83 and
structure refinement was made using SHELXL-2013 84 and WinGX. 85 The molecular graphics
were drawn using DIAMOND. 86 All non-hydrogen
atoms were refined anisotropically. Methyl and aromatic hydrogen atoms
were placed in geometrically idealized positions and constrained to
ride on their parent atoms, with C–H = 0.98 and 0.95 A°
and U iso (H) = 1.5Ueq(C) and 1.2Ueq(C), respectively. The
NH hydrogen atoms were located in the difference Fourier map and freely
refined with U iso (H) = 1.5Ueq(N). The Fc versus Fo plot
proved five reflections to be outliers, and they were removed from
the refinement as systematic errors.
## Synthesis
Synthesis The starting synthon fac -[NEt 4 ] 2 [Re(CO) 3 (Br) 3 ] (ReAA) and the ligands (2,6-dimethoxypyridyl)imidazo[4,5- f ]1,10-phenanthroline ( L1 ), (indole)imidazo[4,5- f ]1,10-phenanthroline ( L2 ), (5-methoxyindole)imidazo[4,5- f ]1,10-phenanthroline ( L3 ), (biphenyl)imidazo[4,5- f ]1,10-phenanthroline ( L4 ), (fluorene)imidazo[4,5- f ]1,10-phenanthroline ( L5 ), (benzo[ b ]thiophene)imidazo[4,5- f ]1,10-phenanthroline
( L6 ), (5-bromothiazole)imidazo[4,5- f ]1,10-phenanthroline ( L7 ), and (4,5-dimethylthiophene)imidazo[4,5- f ]1,10-phenanthroline ( L8 ) have been synthesized
and characterized as reported previously in the literature. 45 , 50 13 C{ 1 H} NMR was attempted for 1 – 8 ; however, after 18,000 scans on highly concentrated
NMR samples, poor or no peaks were obtained. Due to economic implications,
other characterization methods were relied upon.
## General Synthetic Procedure for Re(I) Tricarbonyl Complexes
General Synthetic Procedure for Re(I) Tricarbonyl Complexes ReAA was added to 3 equiv of AgNO 3 solution in 10 mL
of water (at pH 2) and stirred at room temperature for 24 h, which
resulted in a gray/white precipitate of AgBr. The precipitate was
filtered, and 1.1 equiv of the ligand was added to the filtrate. The
solution was stirred at 80 °C for 12 h, and a solid was separated
from the solution. The reaction mixture was filtered and washed three
times with distilled water to yield a solid product.
fac -[Re(CO) 3 ( L1 )(H 2 O)][NO 3 ] ( 1 ) 0.206 g (0.267
mmol) ReAA, 0.136 g (0.801 mmol) AgNO 3 , 0.099 g (0.294
mmol) L1 . Yield: 0.0902 g (0.131 mmol, 49%); elemental
analysis for C 23 H 17 N 6 O 9 Re: calculated: C = 39.04%, N = 11.88%, H = 2.42%; found: C = 38.91%,
N = 11.90%, H = 2.44%.; IR (ATR, cm –1 ): ν N-H = 3346, ν O-H = 3064, 2958,
ν C=O = 2028, 1896, ν C=N = 1605,
ν C=N = 1582, 1452, ν C-O =
1323, 1274.; 1 H NMR (400 MHz, DMSO -d 6 ): δ 9.5 (4H, m), 8.6 (1H, d, J = 8.2
Hz), 8.2 (2H, bs), 6.7 (1H, d, J = 8.2 Hz), 4.2 (3H,
s), 4.0 (3H, s) ppm; 13 C{ 1 H} NMR (151 MHz, DMSO -d 6 ): δ 196.9, 164.2, 163.9, 159.8, 159.6,
150.0, 142.5, 142.3, 124.6, 105.1, 104.6, 103.2, 102.9, 54.6, 54.5,
54.4, 54.3 ppm. UV/Vis (DMSO): λ max = 350 nm, ε
= 12,223 M –1 cm –1 . ESI-MS (CH 3 OH, +ve ion mode): m / z 627.9
([M - NO 3 – - H 2 O] + ); calcd, 627.6).
fac -[Re(CO) 3 ( L2 )(H 2 O)][NO 3 ] ( 2 ) 0.201 g (0.260
mmol) ReAA, 0.132 g (0.777 mmol) AgNO 3 , 0.096 g (0.286
mmol) L2 . Yield: 0.098 g (0.143 mmol, 55%); elemental
analysis for C 24 H 15 N 6 O 7 Re: calculated: C = 42.04%, N = 12.26%, H = 2.21%; found: C = 41.94%,
N = 12.31%, H = 2.19%; IR (ATR, cm –1 ): ν N-H = 3387, ν O-H = 3074, ν C=O = 2019, 1912, 1886, ν C=N = 1576, ν C=N = 1318; 1 H NMR (400 MHz, DMSO -d 6 ): δ 9.4 (4H, overlying multiplet, singlet, and
doublet), 8.7 (1H, s), 8.3 (2H, overlying singlets), 8.1 (1H, s),
7.6 (1H, s), 7.3 (2H, s) ppm; 13 C{ 1 H} NMR (151
MHz, DMSO -d 6 ): δ 197.0, 143.6, 137.0,
134.3, 127.2, 127.1, 125.5, 123.1, 121.8, 121.1, 112.7 ppm; UV/Vis
(DMSO): λ max = 345 nm, ε = 23,989 M –1 cm –1 . ESI-MS (CH 3 OH, +ve ion mode): m / z 606.2 ([M - NO 3 – - H 2 O] + ); calcd, 605.6).
fac -[Re(CO) 3 ( L3 )(H 2 O)][NO 3 ] ( 3 ) 0.20 g (0.260
mmol) ReAA, 0.132 g (0.780 mmol) AgNO 3 , 0.104 g (0.286
mmol) L3 . Yield: 0.0614 g (0.0858 mmol, 33%); elemental
analysis for C 25 H 17 N 6 O 8 Re: calculated: C = 41.96%, N = 11.74%, H = 2.39%; found: C = 41.86%,
N = 11.72%, H = 2.38%; IR (ATR, cm –1 ): ν N-H = 3121, ν O-H = 2921, 2852,
ν C=O = 2028, 1936, 1910, ν C=N =
1603, ν C=N = 1319, ν C-O =
1217. 1 H NMR (600 MHz, DMSO -d 6 ): δ 11.7 (1H, s), 9.3 (2H, broad singlet), 9.2 (2H, singlet),
8.2 (2H, broad singlet), 8.1 (2H, s), 7.5 (1H, d, J = 8.7 Hz), 6.9 (1H, d, J = 8.6 Hz), 3.9 (3H, s)
ppm. 13 C{ 1 H} NMR (151 MHz, DMSO -d 6 ): δ 155.1, 132.1, 126.1, 125.7, 113.3, 113.0,
103.6, 55.9 ppm. UV/Vis (DMSO): λ max = 390 nm, ε
= 19,145 M –1 cm –1 ; ESI-MS (CH 3 OH, +ve ion mode): m / z 636.2
([M - NO 3 – - H 2 O] + ); calcd, 635.6).
fac -[Re(CO) 3 ( L4 )(H 2 O)][NO 3 ] ( 4 ) 0.102 g (0.132
mmol) ReAA, 0.067 g (0.397 mmol) AgNO 3 , 0.054 g (0.145
mmol) L4 . Yield: 0.0219 g (0.0303 mmol, 23%); elemental
analysis for C 28 H 18 N 5 O 7 Re: calculated: C = 46.53%, N = 9.69%, H = 2.51%; found: C = 46.23%,
N = 9.71%, H = 2.48%. IR (ATR, cm –1 ): ν O-H = 3091, 3027, ν C=O = 2038, 1993,
1956, ν C=C = 1808, 1775, 1610, ν C=N = 1554, ν C=N = 1339. 1 H NMR (600 MHz,
DMSO -d 6 ): δ 13.9 (1H, s), 9.1 (4H,
s), 8.3 (2H, d, J = 7.3 Hz), 8.0 (2H, broad singlet),
7.9 (2H, d, J = 7.4 Hz), 7.8 (2H, d, J = 7.0 Hz), 7.5 (2H, t, J = 13.8, 6.9 Hz), 7.4 (1H,
t, J = 13.8, 6.9 Hz) ppm. 13 C{ 1 H} NMR (151 MHz, DMSO -d 6 ): δ 151.6,
141.9, 139.6, 129.5, 129.0, 128.5, 127.7, 127.4, 127.2, 125.1 ppm.
UV/Vis (DMSO): λ max = 337 nm, ε = 25,793 M –1 cm –1 . ESI-MS (CH 3 OH,
+ve ion mode): m / z 643.2 ([M - NO 3 – - H 2 O] + ); calcd,
642.6).
fac -[Re(CO) 3 ( L5 )(H 2 O)][NO 3 ] ( 5 ) 0.10 g (0.130
mmol) ReAA, 0.066 g (0.39 mmol) AgNO 3 , 0.055 g (0.143 mmol) L5 . Yield: 0.0343 g (0.0469 mmol, 36%); elemental analysis
for C 29 H 18 N 5 O 7 Re: calculated:
C = 47.41%, N = 9.53%, H = 2.47%; found: C = 47.44%, N = 9.50%, H
= 2.43%. IR (ATR, cm –1 ): ν N-H = 3471, ν O-H = 3057, 2902, ν C=O = 2028, 1926, 1905, ν C=N = 1610, ν C=C = 1557, 1476, ν C-H = 1446, ν C=N = 1317. 1 H NMR (600 MHz, DMSO -d 6 ): δ 9.1 (4H, merged singlet), 8.3 (1H, s), 8.2 (1H,
d, J = 7.6 Hz), 8.0 (3H, merged singlet), 7.9 (1H,
d, J = 4.6 Hz), 7.6 (1H, d, J =
7.3 Hz), 7.3 (2H, dt, J = 29.2, 7.3 Hz), 4.0 (2H,
s) ppm. 13 C{ 1 H} NMR (151 MHz, DMSO -d 6 ): δ 152.2, 146.7, 144.1, 144.0, 143.1, 140.7,
139.2, 128.2, 127.8, 127.4, 125.7, 125.6, 125.1, 123.2, 120.9, 120.8,
36.8 ppm. UV/Vis (DMSO): λ max = 342 nm, ε =
29,853 M –1 cm –1 . ESI-MS (CH 3 OH, +ve ion mode): m / z 655.2
([M - NO 3 – - H 2 O] + ); calcd, 654.7).
fac -[Re(CO) 3 ( L6 )(H 2 O)][NO 3 ] ( 6 ) 0.199 g (0.258
mmol) ReAA, 0.131 g (0.774 mmol) AgNO 3 , 0.10 g (0.283 mmol) L6 . Yield: 0.105 g (0.149 mmol, 58%); elemental analysis for
C 24 H 14 N 5 O 7 SRe: calculated:
C = 41.02%, N = 9.97%, S = 4.56%, H = 2.01%; found: C = 41.20%, N
= 9.96%, S = 4.60%, H = 2.02%. IR (ATR, cm –1 ): ν N-H = 3397, ν O-H = 3075, ν C=O = 2028, 1920, 1894, ν C=N = 1660, ν C=C = 1581, 1562, ν C=N = 1370, 1304. 1 H NMR (400 MHz, DMSO -d 6 ): δ
9.5 (1H, dd, J = 15.3, 4.7 Hz), 9.3 (1H, broad singlet),
9.1 (2H, overlapping singlets), 8.3 (2H, overlapping singlets), 8.0
(3H, m), 7.5 (2H, overlapping signals) ppm. 13 C{ 1 H} NMR (101 MHz, DMSO -d 6 ): δ 196.9,
152.8, 148.6, 140.2, 140.0, 139.9, 133.2, 132.8, 127.6, 126.6, 126.2,
125.7, 125.5, 125.3, 125.2, 124.9, 124.2, 123.4, 123.2, 123.0 ppm.
UV/Vis (DMSO): λ max = 369 nm, ε = 28,040 M –1 cm –1 . ESI-MS (CH 3 OH,
+ve ion mode): m / z 623.1 ([M - NO 3 – - H 2 O] + ); calcd,
622.6).
fac -[Re(CO) 3 ( L7 )(H 2 O)][NO 3 ] ( 7 ) 0.206 g (0.267
mmol) ReAA, 0.136 g (0.801 mmol) AgNO 3 , 0.112 g (0.294
mmol) L7 . Yield: 0.0254 g (0.0347 mmol, 13%); elemental
analysis for C 19 H 10 N 6 O 7 SBrRe: calculated: C = 31.15%, N = 11.47%, S = 4.37%, Br = 10.91%,
H = 1.37%; found: C = 31.25%, N = 11.51%, S = 4.41%, Br = 10.89%,
H = 1.35%. IR (ATR, cm –1 ): ν N-H = 3398, ν O-H = 3059, ν C=O = 2025, 1918, 1893, ν C=N = 1608, ν C=N = 1312, 1254, ν C-Br = 805, ν C-S = 805. 1 H NMR (400 MHz, DMSO -d 6 ): δ 13.9 (1H, s), 11.7 (1H, s), 9.5 (2H, s), 8.2 (3H, overlying
singlets), 7.5 (1H, triplet, J = 16.9, 8.72), 6.9
(1H, doublet, J = 8.7 Hz) ppm. 13 C{ 1 H} NMR (101 MHz, DMSO -d 6 ): δ
197.0, 159.0, 152.4, 145.3, 140.0, 128.1, 124.5 ppm. UV/Vis (DMSO):
λ max = 261 nm, ε = 1494 M –1 cm –1 . ESI-MS (CH 3 OH, +ve ion mode): m / z 671.1 ([M - NO 3 – ] + ); calcd, 670.5).
fac -[Re(CO) 3 ( L8 )(H 2 O)][NO 3 ] ( 8 ) 0.203 g (0.264
mmol) ReAA, 0.135 g (0.792 mmol) AgNO 3 , 0.112 g (0.294
mmol) L8 . Yield: 0.0609 g (0.0896 mmol, 34%); elemental
analysis for C 22 H 16 N 5 O 7 SRe: calculated: C = 38.82%, N = 10.29%, S = 4.71%, H = 2.37%; found:
C = 38.85%, N = 10.27%, S = 4.69%, H = 2.38%. IR (ATR, cm –1 ): ν N-H = 3066, ν O-H = 2923, 2865, ν C=O = 2025, 1904, 1894, ν C=N = 1617, ν C=C = 1586, 1545, ν C-S = 806. 1 H NMR (400 MHz, DMSO -d 6 ): δ 14.1 (1H, s), 9.4 (2H, d, J = 4.9 Hz), 9.1 (2H, broad multiplet), 8.1 (2H, broad doublet, J = 19.8 Hz), 7.6 (1H, s), 2.4 (3H, s), 2.2 (3H, s) ppm. 13 C{ 1 H} NMR (101 MHz, DMSO -d 6 ): δ 197.9, 190.0, 151.7, 148.7, 137.0, 134.8, 133.2,
132.9, 130.3, 127.8, 126.9, 13.8, 13.5 ppm. UV/Vis (DMSO): λ max = 341 nm, ε = 6340 M –1 cm –1 . ESI-MS (CH 3 OH, +ve ion mode): m / z 601.1 ([M - NO 3 – - H 2 O] + ); calcd, 600.6).
## Stability Study
Stability Study The stability of the complexes in DMSO,
aqueous buffer, and FBS solutions was monitored using time-dependent
UV/Vis absorption measurements. Briefly, the concentration of each
complex was maintained at 10 μM except for complex 7 . In fact, due to poor solubility, we used 5 μM concentration
for compound 7 . A 1:20 v/v DMSO:10 mM
phosphate buffer pH 7.4 in the presence of 4 mM NaCl solution of the
complexes was used to evaluate the aqueous solution stability. In
the case of the stability in FBS solution, we prepared the complex
solution in 1:99 v/v DMSO:10 mM phosphate buffer pH 7.4 in the presence
of 10% FBS. The stability of the complexes in solution was monitored
up to 6 h.
## Determination of Acid Dissociation Constant (p
Determination of Acid Dissociation Constant (p K a ) pH measurements were performed using a Hanna
pH211 Microprocessor pH meter, equipped with a HI 1131 probe, which
was calibrated using standard buffer solutions, with pH = 4.01, 7.00,
and 10.00. A Varian Cary 50 Conc UV/Vis spectrophotometer coupled
to a personal computer capable of performing least squares analyses
on the absorption values vs pH data was utilized
for the kinetic measurements. Microsoft Office Excel (2019) and Scientist
Micromath, version 2.01 were used to fit the collected data to specific
functions. 87 , 88 A circulating water bath system
was used to control and maintain the temperature at 25 °C. The
ionic strength (μ) of all solutions was maintained at 1 M NaClO 4 . The stability of complexes in DMSO solution was also
monitored by ESI-MS measurement. Briefly, 10 μM DMSO solution
of the complexes was incubated for 48 h at 37 °C. Finally, 10
μL of DMSO solution was diluted in 1 mL of methanol and analyzed
by ESI-MS.
## Distribution Coefficient
Distribution Coefficient The distribution coefficient
or lipophilicity (log D ) was determined using a standard
shake flask technique. 58 Briefly, 40 μL
of 1 mM DMSO solution of complex was added to 4 mL of pre-equilibrated
10 mM phosphate buffer (pH 7.4) octanol mixture (1:1 v/v ) and agitated continuously in a vertical shaker for 6 h. Then, each
layer of the mixture was separated by centrifugation and transferred
to new vials. Further, the complex concentration in each layer was
determined by measuring their absorption spectra. Finally, the lipophilicity
was determined using eq 3 below. 3
## BSA Binding Study
BSA Binding Study The BSA binding study was performed
by measuring the internal fluorescence quenching of BSA. BSA has an
intrinsic fluorescence emission maxima at 341 nm upon excitation at
280 nm. 66 , 67 Briefly, 20 μM BSA solution in 1×
Tris buffer (pH 7.4) was incubated with different concentrations (0–10
equiv) of complex solution for 15 min at 37 °C. Further, their
fluorescence intensities were recorded at 341 nm upon excitation at
280 nm. Finally, the fluorescence quenching constant and the binding
constant were calculated using the Stern–Volmer equation ( eq 4 ) and the Scatchard equation
( eq 5 ), respectively. 4 5 In the equations, the
fluorescence emission intensity of native protein is denoted as F 0 , the fluorescence emission intensity in the
presence of quencher is denoted as F , [Q] corresponds
to the concentration of quencher for F , K sv is the Stern–Volmer quenching constant, K a is denoted as the binding constant of quencher
and protein, and n is the number of binding sites
per protein molecule. Complexes showed no emission at 341 nm when
excited at 280 nm.
## DNA Binding Study
DNA Binding Study With Model Nucleobase The DNA binding ability of complex 6 through covalent bond formation was studied in the presence
of a model nucleobase, guanosine, using 1 H NMR and ESI-MS.
Precisely, 1.5 mM DMF- d 7 solution of complex 6 was allowed to react with 3 mM guanosine solution in DMF- d 7 at 37 °C and the binding was monitored
at different intervals of time by recording 1 H NMR spectra
up to 24 h. Finally, after 24 h, 5 μL of DMF solution was diluted
in 1 mL of methanol and analyzed by ESI-MS. Preparation of ctDNA Solution Calf thymus deoxyribonucleic
acid (ctDNA) and tris–HCl buffer solution (pH = 7.4, 1 M) were
purchased from Sigma Aldrich, South Africa, and used without further
purification. The ctDNA stock solution was prepared by stirring a
solution of ctDNA in Tris–HCl buffer for 12 h at room temperature.
The purity of the stock solution was confirmed by the ratio of A 260nm / A 280nm (1.843),
indicating that the solution was free of protein. 45 , 81 , 89 , 91 This solution
was stored at 4 °C between scans and discarded 2 days after preparation.
The molar extinction coefficient value of ctDNA at 260 nm (6600 L
mol –1 cm –1 ) was employed in the
determination of the stock solution concentration. Ethidium Bromide Displacement Assay To check the possibility
of DNA intercalation due to the presence of a planer fused ring ligand
system, we performed the standard ethidium bromide (EB) displacement
assay. Precisely, 10 μM ctDNA solution in 1× Tris·NaCl
buffer was incubated at 37 °C with 3 equiv of EB for 30 min.
After this, 0 to 10 equiv of complex 6 were added to
the solution and incubated for an additional 30 min at 37 °C.
Each solution was excited at 540 nm, and emission was measured from
550 to 800 nm. Finally, the apparent binding constant ( K app ) was measured using eq 6 . 6 where [EB] is the concentration
of EB (30.0 μM) and [C 50 ] is the concentration of
complex when the reduction of fluorescence reach 50%. K EB is the binding constant of EB ( K EB = 1 × 10 7 M –1 for ctDNA). 90 The IC 50 values were calculated from
plots of normalized fluorescence intensity versus complex concentration
( Figure S24B ). DNA Melting The DNA binding ability of complex 6 was confirmed by measuring the DNA melting temperature prior
to complex binding. Briefly, 40 μM ctDNA was incubated with
30 μM of the complex at 37 °C for 15 min. Afterward, the
absorbance of the solution was measured at 260 nm in each °C
increment in temperature within the range from 60–97 °C.
A Cary UV 300 (Agilent Technologies) was used to measure all absorbances.
The experiment was programmed with a 1 °C/min temperature ramp
rate from the temperature range 60–97 °C, and the holding
time was 1 min. Finally, the absorbance of each experiment was normalized
to 0 and 1 and further compared with the melting temperature of native
ctDNA.
## With Model Nucleobase
With Model Nucleobase The DNA binding ability of complex 6 through covalent bond formation was studied in the presence
of a model nucleobase, guanosine, using 1 H NMR and ESI-MS.
Precisely, 1.5 mM DMF- d 7 solution of complex 6 was allowed to react with 3 mM guanosine solution in DMF- d 7 at 37 °C and the binding was monitored
at different intervals of time by recording 1 H NMR spectra
up to 24 h. Finally, after 24 h, 5 μL of DMF solution was diluted
in 1 mL of methanol and analyzed by ESI-MS.
## Preparation of ctDNA Solution
Preparation of ctDNA Solution Calf thymus deoxyribonucleic
acid (ctDNA) and tris–HCl buffer solution (pH = 7.4, 1 M) were
purchased from Sigma Aldrich, South Africa, and used without further
purification. The ctDNA stock solution was prepared by stirring a
solution of ctDNA in Tris–HCl buffer for 12 h at room temperature.
The purity of the stock solution was confirmed by the ratio of A 260nm / A 280nm (1.843),
indicating that the solution was free of protein. 45 , 81 , 89 , 91 This solution
was stored at 4 °C between scans and discarded 2 days after preparation.
The molar extinction coefficient value of ctDNA at 260 nm (6600 L
mol –1 cm –1 ) was employed in the
determination of the stock solution concentration.
## Ethidium Bromide Displacement Assay
Ethidium Bromide Displacement Assay To check the possibility
of DNA intercalation due to the presence of a planer fused ring ligand
system, we performed the standard ethidium bromide (EB) displacement
assay. Precisely, 10 μM ctDNA solution in 1× Tris·NaCl
buffer was incubated at 37 °C with 3 equiv of EB for 30 min.
After this, 0 to 10 equiv of complex 6 were added to
the solution and incubated for an additional 30 min at 37 °C.
Each solution was excited at 540 nm, and emission was measured from
550 to 800 nm. Finally, the apparent binding constant ( K app ) was measured using eq 6 . 6 where [EB] is the concentration
of EB (30.0 μM) and [C 50 ] is the concentration of
complex when the reduction of fluorescence reach 50%. K EB is the binding constant of EB ( K EB = 1 × 10 7 M –1 for ctDNA). 90 The IC 50 values were calculated from
plots of normalized fluorescence intensity versus complex concentration
( Figure S24B ).
## DNA Melting
DNA Melting The DNA binding ability of complex 6 was confirmed by measuring the DNA melting temperature prior
to complex binding. Briefly, 40 μM ctDNA was incubated with
30 μM of the complex at 37 °C for 15 min. Afterward, the
absorbance of the solution was measured at 260 nm in each °C
increment in temperature within the range from 60–97 °C.
A Cary UV 300 (Agilent Technologies) was used to measure all absorbances.
The experiment was programmed with a 1 °C/min temperature ramp
rate from the temperature range 60–97 °C, and the holding
time was 1 min. Finally, the absorbance of each experiment was normalized
to 0 and 1 and further compared with the melting temperature of native
ctDNA.
## Cell Lines and Cell Culture
Cell Lines and Cell Culture Human prostate adenocarcinoma
(PC3) and normal human retinal pigment epithelial-1 (RPE-1) cells
were originally procured from the American Type Culture Collection
(ATCC). The PC3 and RPE-1 cells were maintained in Ham’s F-12K
(Kaighn’s) Medium and DMEM/F-12, GlutaMAX Supplement medium
containing 10% fetal bovine serum (GIBCO) and 1% penicillin–streptomycin
(100 units/mL, GIBCO), respectively.
## Cytotoxicity
Cytotoxicity The standard Resazurin assay was performed
to determine the cytotoxicity of the synthesized complexes. In particular,
4 × 10 3 cells were seeded in each well of a 96 well
microplate and incubated at 37 °C under normoxic conditions (∼15%
O 2 level). After 24 h of the initial incubation period,
the medium was replenished by a fresh metal complex-containing medium
(100 μL/well) and incubated for an additional 48 h. Initially,
the metal complex was dissolved in DMSO and the target concentration
was achieved by further dilution with the medium. A total of seven
or nine different concentrations of the metal complex solutions from
10 nM to 100 μM were used to observe the dose-dependent cytotoxicity.
The ultimate concentration of DMSO in each well was ≤1%. Finally,
the metal complex-containing medium was carefully aspirated and an
equal volume of Resazurin (0.2 mg/mL)-containing medium was added.
After 4 h of incubation at 37 °C, the fluorescence of the produced
resorufin product (λ ex = 540 nm, λ em = 590 nm) was measured in either a Tecan infinite F200PRO Microplate
Reader or a Cytation 5, Cell Imaging Multi-Mode Reader, BioTek. Finally,
the half-maximal inhibitory concentration (IC 50 ) value
was calculated using the GraphPad Prism 5 software from the plot of
% of cell viability vs log[complex concentration]
after applying nonlinear fitting. Each of the reported IC 50 values is the mean of three individual experiments with standard
deviation, and each concentration was assayed in sextuplicate in each
successive experiment. The cytotoxic inactivation of most selective
complexes 3 and 6 in hypoxic conditions
(2.0% O 2 ) was also measured against PC3 cells. A similar
protocol as stated in normoxia cytotoxicity was followed to determine
their IC 50 in hypoxic conditions, keeping everything unaltered
except the incubation in the presence of 2.0% O 2 . Initially,
at least three consecutive cell passages were maintained in hypoxic
conditions before starting any experiments.
## Cytotoxicity against 3D Cell Spheroid
Cytotoxicity against 3D Cell Spheroid 1 × 10 4 PC3 cells were seeded in each well of a Corning 96 Well Ultra-Low
Attachment Treated Spheroid Microplate. After 96 h of incubation at
37 °C in normoxic conditions, the average spheroid size observed
was 435 μm. Half of the medium was replaced by an equal volume
of complex-containing (two times of target concentrations) medium.
After 72 h, the cell viability was measured using the AlamarBlue assay.
Finally, the IC 50 values were determined by using GraphPad
Prism 5 software after applying four parameter fitting.
## Intracellular Accumulation Study
Intracellular Accumulation Study The fluorescence property
is highly dependent on pH and the cellular microenvironment. 74 , 91 Hence, we quantified Re in different cellular organelles using ICP-MS
to determine the complex localization in cells. An adequate
number of PC3 cells were seeded in a 150 mm diameter Petri dish using
F12K medium and incubated in normoxic conditions at 37 °C. The
medium from each Petri dish was replenished every 2 days. When the
cell confluency reached around 80–90%, a fresh medium containing
5 μM complexes 3 or 6 was added and
incubated for an additional 1.5 h in normoxic conditions at 37 °C.
Cells were harvested by trypsinization. The medium was removed by
centrifugation, and the cell pellet was washed twice with cold DPBS.
The nucleus and mitochondria of treated cells were then isolated using
the standard kit protocols mentioned below. To determine the total
rhenium uptake, 5 × 10 5 cells were collected and digested
using 100 μL of 70% nitric acid at 60 °C for 12 h. The
acid solution was further diluted using 4900 μL of MilliQ water.
Finally, the rhenium concentration was measured using ICP-MS. Each
experiment was performed in triplicate. Nuclei: The nuclei isolation
kit, Nuclei EZ Prep (NUC101) (Sigma-Aldrich)
was used to isolate the nuclei from the complex-treated cells, and
the standard kit protocol was employed for the isolation process.
The complex-treated cells were exposed to 4 mL of ice-cold Nuclei
EZ lysis buffer and vortexed briefly to lyse the cells. The solution
was settled at 4 °C for another 5 min and centrifuged at 500 g for 5 min at 4 °C. The supernatant was removed carefully.
The cell and nucleus pellets were redissolved using ice-cold Nuclei
EZ lysis buffer, and the same process was repeated to maximize the
nuclei yield. During this isolation step, both the nucleus and cells
were stained with the trypan blue dye prior to counting using a hemocytometer
to obtain the exact number of the nuclei and intact cells. Mitochondria:
The mitochondria isolation kit (MITOISO2) (Sigma-Aldrich)
was used to isolate the mitochondria from the complex-treated cells,
and the standard kit protocol was employed for the isolation procedures.
By following the kit protocol, the complex-treated cells were lysed
using 1× extraction buffer at 4 °C using a Dounce homogenizer.
The exact number of lysed cells was counted using a hemocytometer
after trypan blue staining. The intact cells and other heavier cell
fragments were removed from the solution by centrifugation at 600 g for 10 min at 4 °C. The supernatant was transferred
carefully and centrifuged at 11000 g for 10 min at
4 °C to isolate the mitochondria. Hence, the total amount of
isolated mitochondria corresponds to the total lysed cells. The percentage
of rhenium accumulation in the nucleus and mitochondria was calculated
using the total accumulated rhenium corresponding to 5 × 10 5 cells.
## Sample Preparation for ICP-MS
Sample Preparation for ICP-MS The samples were digested
in either 100 μL (cell pellet) or 500 μL (nucleus and
mitochondria pellet) of 70% HNO 3 at 60 °C for 12 h.
The acid solution was further diluted (1:50 v/v )
with MilliQ water. Finally, the rhenium concentration in the solution
was analyzed using a high-resolution ICP-MS instrument of ThermoScientific
(Element II, ThermoScientific) at the Institut de Physique du Globe
de Paris, France.
## Cellular Uptake Pathways
Cellular Uptake Pathways 1.5 × 10 6 PC3
cells were seeded in a 6 cm Petri dish and incubated in normoxic conditions
at 37 °C. After 24 h of seeding, the cells were treated with
different cellular uptake pathways inhibitors for 2 h in normoxic
conditions. The inhibitor used here were as follows: 1 mM tetraethylammonium
chloride (cationic transporter inhibitor), 5 μM oligomycin and
50 mM 2-deoxy- d -glucose (metabolic inhibitors), and 50 mM
ammonium chloride (endocytotic inhibitor) as different uptake pathway
inhibitors. Additionally, we pre-incubated the cells at 4 °C
for 2 h to determine the involvement of passive transport on the cellular
uptake process. After 2 h, cells were washed twice with DPBS and 5
μM of complex ( 3 and 6 ) solution was
added. After 2 h of incubation in normoxic conditions, cells were
harvested using trypsinization and counted. 1 × 10 6 cells were separated and digested in 100 μL of conc. HNO 3 . Finally, the acid solution was diluted by addition of 4900
μL of MQ water to keep the acid % within 2. The rhenium concentration
in solution was measured by ICP-MS. Each successive experiment and
control experiments were performed three times to determine the average
value with standard deviation.
## Mito Stress Test Using Seahorse Apparatus
Mito Stress Test Using Seahorse Apparatus 3 ×
10 4 PC3 cells/well were seeded of a Seahorse XF Cell Culture
Microplate using 80 μL of FBS-supplemented F12K medium and incubated
in normoxic conditions for 24 h at 37 °C. The medium was replaced
by an equal volume of compound ( 3 , 0.4 and 5 μM; 6 , 0.05 and 2 μM; and cisplatin, 7 and 30 μM)-containing
medium. After 24 h of treatment, media were removed, and the treated
cells were washed very carefully with the Seahorse XF medium three
times. Finally, the cells were incubated at 37 °C for 1 h in
a non-CO 2 incubator. The Mito Stress assay was run in a
Seahorse Xfe96 instrument (Agilent) at 37 °C using multiple inhibitors,
viz., ATP synthase inhibitor (oligomycin, 1 μM), proton gradient
and mitochondrial membrane potential collapsing agent (FCCP, 1 μM),
and mitochondrial respiratory complex I and III inhibitors (rotenone,
1 μM and antimycin A, 1 μM, respectively). After the experiment,
the cells were fixed using 4% p -formaldehyde solution
and the nucleus was stained with DAPI. Each well was imaged in a Cytation
5 Cell Imaging Multimode Reader (BioTek) using a 10× objective
lens. Finally, the cells from each image were calculated by using
Gen5 software ( Figure S35 ) and by utilizing
the cell count and the data was normalized against the same cell number.
## Cell Death Mechanism
Cell Death Mechanism To evaluate the possible cell
death pathways, we measured the cell viability in the presence of
different cell death pathway inhibitors. 44 , 75 Briefly, 1 × 10 3 PC3 cells/well were seeded in 96-well
plates and incubated for 24 h in normoxic conditions at 37 °C.
The medium was removed and treated with inhibitors for 1 h. Autophagy
inhibitor (3-methyladenine (100 μM)), apoptosis inhibitor (Z-VAD-FMK
(20 μM)), paraptosis inhibitor (cycloheximide (0.1 μM)),
and necrosis inhibitor (necrostatin-1 (60 μM)) were the different
inhibitors used. After 1 h, the inhibitor-containing medium was removed
and washed with 1× DPBS. Then, different concentrations of complex
solution were added and incubated for 36 h in normoxic conditions.
Finally, the cell viability was determined by Resazurin assay (check
the Cytotoxicity section for details) to evaluate the IC 50 values.
## Annexin-V/PI Assay for Apoptosis Detection
Annexin-V/PI Assay for Apoptosis Detection 1.5 ×
10 6 PC3 cells were seeded in a 6 cm Petri dish using a
F12K medium and incubated at 37 °C in normoxic conditions. After
24 h of incubation, the medium was replenished with a complex-containing
medium and incubated additionally for 24 h. Two different concentrations
of complexes 3 and 6 were used to determine
the possible apoptosis pathways for cell killing. The cells were then
harvested by trypsinization and washed twice with DPBS. The cells
were further stained with both FITC-tagged Annexin-V (cat. no. 556419,
BD Biosciences) and propidium iodide (cat. no. 51-66211E, BD Biosciences).
Finally, the stained cells were analyzed using a BD LSR II flow cytometry
instrument (BD Biosciences) at Institut Curie, Paris, France.
## ROS Analysis
ROS Analysis 1.5 × 10 4 PC3 cells were
seeded in the dark in 96-well plates for 24 h. Four different concentrations
of complexes 3 and 6 were used to treat
the cells for 8 h, and then two different concentrations of H 2 O 2 were used to treat the cells for 30 min (10
and 15 μM). The medium was removed, and 40 μM DCFH-DiOxy-Q
(Kit No. ab238535, Abcam) in 10% FBS containing HEPES was added in
each well followed by incubation at 37 °C for 30 min. The cells
were washed twice with HEPES, and finally fluorescence intensity was
measured (λ ex /λ em ; 485/530 nm) using
a Cytation 5 Cell Imaging Multimode Reader (BioTek). Then, the cells
were fixed using p -formaldehyde and stained using
DAPI. The cells were finally counted in the same way as stated before
and utilized to normalize the data.