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Development of Half-Sandwich Ru, Os, Rh, and Ir Complexes Bearing the Pyridine-2-ylmethanimine Bidentate Ligand Derived from 7-Chloroquinazolin-4(3H)-one with Enhanced Antiproliferative Activity.
Kinesin spindle protein (KSP) inhibitors are one of the
most promising
anticancer agents developed in recent years. Herein, we report the
synthesis of ispinesib-core pyridine derivative conjugates, which
are potent KSP inhibitors, with half-sandwich complexes of ruthenium,
osmium, rhodium, and iridium. Conjugation of 7-chloroquinazolin-4(3H)-one
with the pyridine-2-ylmethylimine group and the organometallic moiety
resulted in up to a 36-fold increased cytotoxicity with IC 50 values in the micromolar and nanomolar range also toward drug-resistant
cells. All studied conjugates increased the percentage of cells in
the G 2 /M phase, simultaneously decreasing the number of
cells in the G 1 /G 0 phase, suggesting mitotic
arrest. Additionally, ruthenium derivatives were able to generate
reactive oxygen species (ROS); however, no significant influence of
the organometallic moiety on KSP inhibition was observed, which suggests
that conjugation of a KSP inhibitor with the organometallic moiety
modulates its mechanism of action.
## Introduction
Introduction Despite the recent development of many
cancer treatments, chemotherapy
remains the primary, and often the only, method used. 1 − 3 Among the numerous anticancer drugs, antimitotic compounds such
as taxanes and Vinca alkaloids are the most important. 2 , 4 Antimitotic agents such as taxanes disrupt the typical microtubule
dynamics, leading to cancer cell death but can also cause many side
effects, such as bleeding, immune system impairment, reduced blood
pressure, and pain in muscles and joints. 5 − 7 Additionally,
the multidrug resistance phenomenon can be observed during chemotherapy,
thus decreasing its efficiency. Therefore, developing new molecules
able to overcome the drawbacks of currently used antimitotic compounds
is still essential. In the last years, low-molecular-weight
inhibitors of the kinesin
spindle protein (KSP) were developed. 5 The
KSP is a member of the motor protein family and plays a crucial role
in spindle pole separation. It is highly active in dividing cells,
while its activity is almost undetectable in nondividing cells. 8 KSP inhibitors disturb the mitosis without direct
microtubule disruption. 8 − 10 Numerous KSP inhibitors have been developed, including
monastrol, 11 dimethylenastron, 8 ispinesib (SB-715992), SB-743921, 8 , 12 litronesib (LY2523355), 13 MK-0731, 14 and filanesib (ARRY-520). 15 , 16 Some of these compounds have been clinically tested in at least
45 phase I/II trials against various types of cancer, 13 with ispinesib 12 , 17 and filanesib 15 , 18 , 19 as the most promising candidates.
Encouraging results of clinical trials of ispinesib use in patients
with metastatic or relapsing squamous cell carcinoma of the head and
neck, with no signs of disease progression or intolerable toxicity,
were observed within 21 days of the first dose; 20 however, up to date, no further phase III studies have
been reported. One of the fruitful methods to develop new anticancer
drug candidates
involves constructing conjugates of active compounds with an organometallic
group. 21 − 23 The most intensively studied organometallic derivatives
include metallocenes 24 (mainly ferrocene
and ruthenocene) and half-sandwich complexes of ruthenium, 25 − 28 osmium, 26 , 29 rhodium, 30 and
iridium. 30 Organometallic compounds have
several advantages over purely organic molecules. The presence of
an organometallic moiety can increase the affinity to the biological
targets by allowing the formation of new hydrophobic or metal–organic
interactions with the protein. Organometallic compounds often have
access to a protein binding site that is inaccessible to organic molecules.
In addition, the presence of a metal atom often increases the ability
of the compound to generate reactive oxygen species (ROS), which can
induce apoptosis. Organometallic conjugates often exhibit stronger
antiproliferative properties than parent compounds and, in many cases,
exhibit additional biological properties. In recent years, many new
organometallic conjugates of antimitotic compounds have been developed,
including derivatives of curcumin, 31 , 32 taxanes, 33 , 34 colchicine, 35 − 37 ethacrynic acid, 38 , 39 paullone, 40 or podophyllotoxin. 41 , 42 The resulting conjugates demonstrate a higher antiproliferative
activity or a new mechanism of action, being highly selective against
tumor cells. Recently, we have reported the synthesis and biological
evaluation
of a series of ferrocenyl 43 and Ru, Os,
Rh, and Ir half-sandwich 44 , 45 conjugates of ispinesib
and its 7-chloroquinazolin-4(3H)-one core. Continuing our study on
novel organometallic antimitotic agents, we designed new half-sandwich
complexes derived from the ispinesib core. Herein, we present the
synthesis, structure, and biological activity studies of novel Ru,
Os, Rh, and Ir half-sandwich complexes bearing the pyridine-2-ylmethanimine
bidentate ligand derived from 7-chloroquinazolin-4(3H)-one ( Figure 1 ). Figure 1 Structure of (a) ispinesib,
(b) its quinazoline-derived Ru, Os,
Rh, and Ir half-sandwich conjugates reported previously, 44 , 45 and (c) compounds studied herein.
## Results and Discussion
Results and Discussion Synthesis The half-sandwich complexes 3a – d and 4a – d were
synthesized in two steps according to Scheme 1 . First, ( R )- and ( S )- 2 imine ligands were generated in situ by
reacting ( R )- and ( S )- 1 with 2 equiv of pyridine-2-carbaldehyde in anhydrous ethanol for
1 h. Next, 0.5 equiv of the proper dimetallic precursor [(cym)MCl 2 ] 2 (M = Ru for 3a and 4a , M = Os for 3b and 4b ) or [(Cp*)MCl 2 ] 2 (M = Rh for 3c and 4c or M = Ir for 3d and 4d ) was added to
the reaction mixture. After 3 h of stirring at RT, the desired complexes 3a – d or 4a – d were isolated as hexafluorophosphate salts in 37–73% yield.
All complexes were fully characterized by 1 H and 13 C{ 1 H} NMR spectroscopy and ESI-MS analyses. The purity
of compounds was confirmed by elemental analysis. Scheme 1 Synthesis of Complexes 3a – 4d It might be expected that a mixture of diastereoisomers
of 3a – d and 4a – d would be formed as the result of the complexation reactions
of enantiomerically pure imines 2 due to the generation
of new chirality on the metal atoms. In the 1 H NMR spectra
of 3a – c and 4a – c , only one main set of peaks was observed in 1 H and 13 C{ 1 H} NMR spectra together with small
amounts (∼15%) of a second species, which can be assigned to
the other diastereoisomers. Yet, for iridium complexes ( 3d and 4d ), we detected much more intensive signals originating
from the second diastereoisomer (ratio of 1:0.4 for 3d and ratio of 1:1 for 4d ). The formation of two diastereoisomers
of the complexes was also confirmed by diffusion-ordered spectroscopy
(DOSY) experiments ( Figures 2 and S1–S3 ). For example,
the DOSY spectra of 3a and ( R )- 1 ( Figure 2 ) confirmed that all 1 H signals observed in the 1 H NMR spectra originate from the molecule(s) showing the same diffusion
coefficient. Figure 2 Overlapped 1 H DOSY spectra of 3a (blue)
and ( R )- 1 (red) in DMSO- d 6 . Notwithstanding, the DOSY experiments confirmed
the presence in
the solution of compounds showing the same diffusion coefficient.
The formation of diastereoisomers of complexes 3a – 4d was confirmed by HPLC-MS analysis ( Figures S9–S16 ). For example, the HPLC-MS analysis
of both ruthenium complexes reveals two peaks at τ 1 = 3.50 and τ 2 = 5.22 min for 3a , with
a ratio of 1:5, with the m / z of
701 assigned to [M 3a -PF6 ] + , and τ 1 = 3.39 and τ 2 = 5.07 min
for 4a , with a ratio of 1:4.3, with the m / z of 701 assigned to [M 4a -PF6 ] + . Likewise, the HPLC-MS analysis of both osmium complexes 3b and 4b confirmed the formation of two diastereoisomers
with the ratio of 1:4 ( Figures S11 and S12 ). In the case of iridium complexes ( 3d and 4d ), the ratio of HPLC peaks is 1:0.4 for 3d and 1:1.4
for 4d , corresponding with the results observed in 1 H NMR. However, for rhodium complexes ( 3c and 4c ), only the main peak at τ 1 = 2.67 min
for 3c and τ 1 = 2.79 min for 4c , with an additional small peak (ratio 1:0.08), and small peaks at
τ 2 = 2.26 min for 3c and τ 2 = 2.40 min for 4c with the m / z of 334 assigned to [M 3c -Cl-PF6 ] 2+ and [M 4c -Cl-PF6 ] 2+ were detected. On the 1 H NMR spectra
of complexes 3a – b and 4a – b at 300 K, aromatic p -cymene
proton signals were broad singlets. Also, no correlation
between aromatic p -cymene protons or proton–carbon
correlations in 1 H– 1 H COSY or 1 H– 13 C HSQC NMR spectra was observed. Therefore,
we performed VT-NMR experiments for 3a and 3b in DMSO- d 6 at various temperatures between
300 and 330 K ( Figures 3 and S4a–d ). An increase in the
temperature of the sample from 300 to 330 K results in a change of
broad singlets at 6.18 and 6.00 ppm into actual doublets and a doublet
at 5.87 ppm, which were assigned to aromatic p -cymene
protons. Additionally, a small set of signals, most likely originating
from the hydrolyzed form of the complex, was observed during the experiment.
The 1 H– 1 H COSY and 1 H– 13 C HSQC spectra allowed observing the expected correlations
between aromatic p -cymene protons and carbon atoms
( Figures S5 and S6 ) at 330 K. However,
those experiments confirmed the partial thermal decomposition of studied
complexes, which impeded the performed 13 C{ 1 H} NMR spectra. Identical results were observed for 4a and 4b (300 and 330 K) ( Figures S7, S8, S45–S47, S59–S61 ). Figure 3 VT-NMR experiments for 3a . 1 H NMR spectra
in DMSO- d 6 (range 6.45–5.35 ppm)
at (a) 300, (b) 310, (c) 320, and (d) 330 K; * denotes the signals
assigned to the solvated compound. It could be expected that ligand 1 may undergo complexation
forming the expected Type I complexes together with two other Type
II and Type III complexes ( Figure 4 ). The formation of Type III complexes was excluded
by MS analysis. In the MS spectra of 3a – 4d , we observed only expected m / z values assigned to monocations [M] + ( Figures S9–S16 ). To further exclude the formation of
Type II complexes, we generated imine 5 in the reaction
of 1 with benzaldehyde ( Scheme 2 ). The obtained imine 5 further
reacted with 0.49 equiv of metal dimers [LMCl 2 ] 2 (M = Rh/Ir, L = Cp* or M = Ru/Os, L = cym) in methanol at RT for
3 h. After the workup, we isolated only previously reported complexes 6a – d bearing 1 as N , N -bidentate ligands in trace yield. As
the formation of imines is reversible, unless coordinated to a metal, 46 imine 5 hydrolyzed in the presence
of a trace of water to amine 1 , which underwent complexation
with [LMCl 2 ] 2 to afford complexes 6a – d . The NMR spectra of the isolated complexes
were identical to those reported previously. 44 Figure 4 Three
possible coordinations of the metal to ligand 2 . Bidentate
coordination of (a) Type 1 and (b) Type II and (c) tridentate
coordination of Type III. Scheme 2 Competitive Complexation of ( S )- 1 and 5 with [LMCl 2 ] 2 X-ray Diffraction Studies Although we obtained complexes
as a mixture of two possible diastereoisomers, the crystallization
of 4a from the dichloromethane/ n -pentane
mixture by slow evaporation in −20 °C allowed to isolate
only one enantiopure isomer 4a S,S Ru . The complex 4a S,S Ru crystallized in the P2 1 space
group and its chiral purity has been confirmed by a low value of the
Flack parameter ( Table S1 ). The imine
( S )- 2 acts as a N , N -bidentate ligand, forming five-membered rings with the
metal ions by coordinating through the iminium and pyridinium nitrogen
( Figure 5 ). Two similar
structures of the complex are present in the unit cell, showing almost
identical ruthenium coordination, varying slightly in the conformation
of the terminal phenyl and i Pr moieties.
In Table 1 , we had
listed bond lengths of the coordination bonds for both forms, which
are typical for such types of complexes. 47 , 48 A more thorough description of the molecular geometry has been presented
in the ESI . Figure 5 Oak Ridge thermal ellipsoid
plot (ORTEP) representation of the
molecular structure of 4a S,S Ru : (a) molecule 4a S,S Ru ″ with
the counterion, (b) molecule 4a S,S Ru ′ with the counterion,
and (c) schematic representation of the ruthenium coordination sphere.
Interatomic distances and angles reported in Table 1 are highlighted in blue. Atomic displacement
parameters are drawn at the 50% probability level. Hydrogen atoms
are represented as fixed-size spheres in panels (a) and (b) and omitted
in panel (c). The cocrystallized disordered solvent molecule has also
been removed for clarity. Table 1 Selected Coordination Bond Lengths
(Å) and Angles (deg) Found in Both Independent Molecules of 4a S,S Ru in Its Crystal Structure bond or angle 4a S,S Ru ′ 4a S,S Ru ″ Ru–Cl 2.396(1) Å 2.383(2) Å Ru–N py 2.082(4) Å 2.096(4) Å Ru–N im 2.131(4) Å 2.119(4) Å Ru−μ [center
of the p-cymene ring] 1.699(2) Å 1.695(2) Å N im –Ru–N py 76.7(2)° 76.7(2)° N im –Ru–Cl 86.4(1)° 85.2(1)° N py –Ru–Cl 85.9(1)° 82.4(1)° N im –Ru−μ 135.55° 134.16° N py –Ru−μ 128.88° 132.27° Cl–Ru−μ 127.11° 125.53° Stability Study For biological studies, compounds are
commonly administered as dimethyl sulfoxide (DMSO) solution to cells
cultured in a specific medium such as Dulbecco’s modified Eagle’s
medium (DMEM). DMEM consists of numerous organic compounds which may
act as ligands for organometallics. Therefore, it is important to
know how the compounds behave in such conditions. The two most prominent
components of DMEM which may coordinate to half-sandwich complexes
are l -cysteine and l -histidine. Both of those amino
acids are present in DMEM at 0.2 mM concentration, so we studied how
the complexes interact with them using UV–vis spectroscopy
and HPLC-MS analysis. The DMSO solutions of complexes were added to
the aqueous solution of l -cysteine or l -histidine
to achieve a complex concentration of 20 μM while keeping the
DMSO concentration at 0.5 vol %. The UV–vis spectra and HPLC-MS
analysis indicate that neither ruthenium 3a nor the osmium
complex 3b reacts with those amino acids within 2 h ( Figures S18–S21, S24 and S25 ). The rhodium
complex 3c slowly reacts with l -cysteine ( Figure S22 ) by increasing the intensity of each
absorbance maximum (λ = 279, 304, 317, 348 nm). HPLC-MS analysis
confirmed the formation of an additional peak at τ = 0.95 min
with m / z 714 assigned to [M-Cl-PF 6 + HCOOH] + ; additionally, the intensity of peaks
corresponding to 3c is lower ( Figure S26 ). A similar effect is observed in the case of l -histidine, with an increase of only one maximum at λ = 278
nm, while the others are almost unchanged ( Figure S23 ). On the other hand, the iridium complex 3d reacts with both l -cysteine and l -histidine ( Figure 6 ) in 40 min. The
intensity of absorbance peaks at λ = 287 and λ = 372 nm
in the presence of cysteine is decreasing, while the intensity of
peaks at λ = 304 and λ = 318 nm is almost intact. HPLC-MS
analysis shows that the intensity of both peaks corresponding to 3d is lower, while the additional peak at τ = 1.09 min
with m / z 804 is assigned to [M-Cl-PF 6 + HCOOH] + for the l -histidine experiment
and at τ = 1.07 min with m / z 804 is assigned to [M-Cl-PF 6 + HCOOH] + for
the l -cysteine experiment ( Figure S27 ). The lack of an isosbestic point on the UV–vis spectra and
HPLC-MS analysis indicate that the reaction does not lead to the dissociation
of ligands 2 and is purely associated with Cl ligand
exchange. Figure 6 UV–vis spectra of 3d in DMSO-water solutions
in the presence of 0.2 mM (a) l -cysteine or (c) l -histidine. The absorbance maxima value changes vs time in the presence
of (b) l -cysteine and (d) l -histidine. Biological Activity Antiproliferative Potential To assess the impact of
conjugating half-sandwich complexes with amines 1 via
an imine-pyridine ligand on biological activity, we examined the antiproliferative
potential of ( R )- and ( S )- 1 and organometallic conjugates 3a – d and 4a – d in selected human
cancer cell lines: alveolar basal epithelial cell adenocarcinoma (A549),
colorectal adenocarcinomas (Colo205 and SW620), colorectal carcinoma
(HCT116), hepatocellular carcinoma (HepG2), and breast adenocarcinoma
(MCF7). The choice of cell lines was dictated by results of previously
published clinical trials on ispinesib. 49 , 50 All complexes
demonstrate an antiproliferative potential in the micromolar or nanomolar
range ( Table 2 , Figures S28 and S30 ). The activity of these compounds
varies significantly depending on the configuration of imine-ligand 2 and the cell line tested. Complexation of the imine derived
from ( R )- 1 by osmium, resulting in complex 3b , leads to an enhanced cytotoxicity toward A549 (2-fold),
HepG2 (3-fold), and MCF7 (3-fold). A similar effect is observed for
Rh 3c and Ir 3d complexes derived from imine
( R )- 2 , characterized by a 2-fold increased
antiproliferative potential toward A549. However, the complexation
of imine ( R )- 2 with ruthenium 3a does not enhance the activity toward studied cell lines.
Nevertheless, the complexation of the imine derived from ( S )- 1 with all metals results in a significantly
increased antiproliferative potential. It is especially evident in
the case of the ruthenium complex 4a (approximately 6-fold
increased activity against MCF7 and Colo205), the osmium complex 4b (increased cytotoxicity against Colo205 (7-fold), HCT116
(10-fold), and MCF7 (9-fold)), and the iridium complex 4d (enhanced activity toward all tested cell lines, ranging from 9-
to 36-fold). Notably, the iridium complex 4d also exhibits
a significantly higher cytotoxicity compared to both ( S )- 1 and the more cytotoxic amine ( R )- 1 (2.6- and 1.6-fold, respectively). Additionally,
within the tested concentration ranges, all of the compounds studied
show no antiproliferative effects on the normal MRC-5 cell line, with
IC 50 values exceeding 100 μM ( Figure S31 ). Table 2 Antiproliferative Activity of ( R )- 1 and ( S )- 1 and Organometallic Complexes 3a – 4d in Human Cancer Cell Lines a IC 50 [μM] compound A549 Colo205 HCT116 HepG2 MCF7 SW620 ( R )- 1 2.21 0.107 0.346 0.566 0.231 0.096 [1.88–2.59] [0.094–0.121] [0.274–0.437] [0.476–0.672] [0.195–0.308] [0.080–0.117] 3a 2.45 1.26 2.88 1.57 0.858 1.22 [2.05–2.93] [1.16–1.38] [2.48–3.43] [1.45–1.70] [0.742–0.988] [1.12–1.33] (0.902) (0.085) (0.120) (0.360) (0.269) (0.079) 3b 1.04 0.448 0.424 0.188 0.073 0.556 [0.983–1.07] [0.412–0.492] [0.388–0.476] [0.174–0.204] [0.068–0.079] [0.514–0.601] (2.12) (0.239) (0.816) (3.01) (3.16) (0.173) 3c 1.16 0.138 0.173 0.689 0.357 0.152 [0.906–1.50] [0.125–0.153] [0.145–0.206] [0.605–0.784] [0.279–0.459] [0.125–0.185] (1.90) (0.775) (2.00) (0.821) (0.647) (0.632) 3d 1.13 0.524 0.476 0.454 0.653 0.198 [0.973–1.32] [0.453–0.606] [0.408–0.550] [0.383–0.539] [0.552–0.767] [0.172–0.226] (1.96) (0.204) (0.727) (1.25) (0.354) (0.485) ( S )- 1 7.05 6.07 8.06 2.40 3.91 2.87 [6.42–7.36] [5.18–7.39] [7.29–8.90] [2.18–2.63] [3.56–4.31] [2.68–3.06] 4a 3.25 0.939 3.76 1.89 0.634 2.91 [2.84–3.73] [0.737–1.20] [3.32–4.27] [1.64–2.17] [0.539–0.743] [2.63–3.21] (2.17) (6.46) (2.14) (1.27) (6.17) (0.986) 4b 2.19 0.904 0.823 1.13 0.438 2.31 [2.00–2.40] [0.825–0.985] [0.658–1.07] [1.04–1.23] [0.398–0.483] [2.18–2.45] (3.22) (6.71) (9.79) (2.12) (8.93) (1.24) 4c 3.79 2.89 2.94 1.15 1.50 2.24 [3.45–4.16] [2.64–3.15] [2.75–3.14] [0.833–1.58] [1.14–1.94] [1.95–2.61] (1.86) (2.10) (2.74) (2.09) (2.61) (1.28) 4d 0.764 0.216 0.222 0.218 0.216 0.139 [0.613–0.954] [0.197–0.235] [0.192–0.254] [0.193–0.244] [0.191–0.244] [0.128–0.150] (9.23) (28.1) (36.31) (11.01) (18.10) (20.65) a Exposure time 72 h; IC 50 values are presented together with the corresponding 95% confidence
intervals (in brackets), n = 3; the activity factors
were calculated as IC 50(1) /IC 50( 3a – 4d ) and are given in parentheses below the confidence
intervals. Next, we evaluated the cytotoxicity of the synthesized
complexes
toward the panel of six multidrug-resistant (MDR) cell lines derived
from SW620 and characterized by the overexpression of various ABC
proteins, namely, ABCG2 (SW620C and SW620Mito), ABCC1 (SW620M and
SW620E), and ABCB1 (SW620D, SW620E, and SW620V) ( Table 3 , Figures S29 and S32 ). Among the series of complexes bearing the ( R )- 2 ligand, only the iridium complex 3d shows a 2.2- and 1.7-fold higher cytotoxicity than the
corresponding amine ( R )- 1 toward SW620C
and SW620D cancer cell lines. The activity of the complexes derived
from the ligand ( S )- 2 is also considerably
higher than that of the compounds containing the ligand ( R )- 2 . The cytotoxicity of both rhodium 4c and iridium 4d complexes is higher than that of amine
( S )- 1 . In the case of 4c , the increase in cytotoxicity is low, with the highest value of
3.7-fold for the SW620Mito line. Nevertheless, the IC 50 values for the iridium complex 4d are 6.1- to 20.6-fold
lower than those for amine ( S )- 1 . Compound 4d also exerts a 2.1- and 2.6-fold higher cytotoxicity than
( R )- 1 against the SW620C and SW620D
lines. Table 3 Antiproliferative Activity of ( R )- 1 and ( S )- 1 and Organometallic Complexes 3a – 4d in Multidrug-Resistant (MDR) Cancer Cell Lines a IC 50 [μM] comp. SW620 SW620C SW620D SW620E SW620M SW620V SW620Mito ( R )- 1 0.096 0.721 1.12 0.835 0.241 0.206 0.261 [0.080–0.117] [0.552–0.942] [0.851–1.47] [0.627–1.11] [0.191–0.305] [0.160–0.267] [0.207–0.329] 3a 1.22 4.38 7.23 5.52 3.45 4.71 4.33 [1.12–1.33] [3.80–5.07] [6.24–8.37] [4.74–6.43] [2.99–3.97] [4.08–5.44] [3.80–4.95] (0.079) (0.165) (0.155) (0.151) (0.070) (0.044) (0.060) 3b 0.556 1.17 3.92 3.52 0.591 1.03 0.748 [0.514–0.601] [1.08–1.27] [3.60–4.27] [3.25–3.83] [0.522–0.667] [0.953–1.12] [0.683–0.815] (0.173) (0.616) (0.286) (0.237) (0.408) (0.200) (0.349) 3c 0.152 0.592 4.18 0.880 1.18 0.210 0.524 [0.125–0.185] [0.526–0.666] [3.13–5.96] [0.657–1.18] [0.998–1.42] [0.175–0.253] [0.418–0.657] (0.632) (1.22) (0.268) (0.949) (0.204) (0.981) (0.498) 3d 0.198 0.335 0.644 1.02 0.247 0.267 0.268 [0.172–0.226] [0.297–0.377] [0.565–0.732] [0.928–1.13] [0.222–0.275] [0.224–0.317] [0.233–0.311] (0.485) (2.15) (1.74) (0.819) (0.976) (0.771) (0.974) ( S )- 1 2.87 3.33 4.15 4.05 3.46 3.44 3.91 [2.68–3.06] [2.94–3.78] [3.65–4.75] [3.57–4.61] [3.03–3.98] [3.00–3.94] [3.46–4.44] 4a 2.91 4.36 6.01 5.46 3.24 5.01 3.68 [2.63–3.21] [3.90–4.88] [5.24–6.88] [4.77–6.26] [2.91–3.61] [4.43–5.58] [3.29–4.10] (0.986) (0.764) (0.690) (0.741) (1.07) (0.687) (1.06) 4b 2.31 4.54 13.5 12.8 2.45 9.30 3.78 [2.18–2.45] [4.15–4.99] [12.3–15.0] [11.7–14.2] [2.25–2.66] [8.52–10.1] [3.48–4.11] (1.24) (0.738) (0.307) (0.316) (1.41) (0.370) (1.03) 4c 2.24 2.97 6.07 6.20 2.25 2.98 1.05 [1.95–2.61] [2.55–3.45] [4.88–7.85] [4.96–8.05] [1.95–2.59] [2.58–3.45] [0.878–1.25] (1.28) (1.12) (0.684) (0.653) (1.54) (1.15) (3.72) 4d 0.139 0.343 0.425 0.663 0.387 0.411 0.365 [0.128–0.150] [0.310–0.379] [0.384–0.472] [0.582–0.754] [0.339–0.443] [0.374–0.451] [0.313–0.428] (20.65) (9.71) (9.76) (6.11) (8.94) (8.37) (10.71) a Exposure time 72 h; IC 50 values are presented together with the corresponding 95% confidence
intervals (in brackets), n = 3; the activity factors
were calculated as IC 50(1) /IC 50( 3a–4d ) and are given in parentheses below the confidence intervals. Cell Cycle Ispinesib leads to the formation of monopolar
mitotic spindles and a blockade of chromosome segregation in cancer
cells. Using flow cytometry, we assessed the cell cycle distribution
in the SW620 and SW620E cells exposed to the studied compounds for
24 and 48 h. Only two complexes, rhodium 3c and iridium 3d , exhibit a significantly different impact on cell cycle
phase distribution. In contrast, all other complexes demonstrate a
pattern similar to the corresponding amines ( R )-
and ( S )- 1 , as shown in Figure 7 and Table S2 . Both complexes, 3c and 3d , decrease
the percentage of cells in the G 1 /G 0 phase and
increase the percentage in the S and G 2 /M phases. All other
compounds exhibit a similar impact on cell phase distribution. Furthermore,
prolonged exposure to the compounds increases the percentage of cells
in the G 2 /M phase, with the most intensive effect observed
for 3c and 3d . These results suggest an
aggravated mitotic arrest in cells treated with the rhodium 3c and iridium 3d complexes. However, none of
the studied compounds affects the cell cycle in SW620E cells, as demonstrated
in Figure S33 . Figure 7 Cell cycle distribution
in SW620 cells: (a) after 24 h and (b)
after 48 h. KSP Inhibitory Activity The mechanism of the anticancer
activity of ispinesib is related to the inhibition of the activity
of the KSP. Thus, we studied the synthesized compounds’ ability
to inhibit KSP activity using the adenosine 5′-triphosphate
(ATP) hydrolysis assay. The inhibitory ability of the KSP is strongly
correlated with the configuration of the organic ligand and the type
of metal coordinated. Only the derivatives bearing an organic ligand
configuration ( R ) exhibit KSP inhibitory activity.
In contrast, all compounds bearing an organic ligand in the (S) configuration
demonstrate no inhibitory activity toward the KSP at a concentration
of 100, 300, and 1000 nM ( Figure 8 ). The reference compound, ispinesib, shows a high
KSP inhibitory activity (KSP residual activity 2.2%) at 100 nM concentration,
while amine ( R )- 1 decreases the KSP
activity to about 35%. While the complexation of ruthenium leads to
the nonactive complex 3a , the other metal complexes 3b – d are able to inhibit KSP activity
with the most active rhodium 3c (47.5%), followed by
iridium 3d (64.0%) and osmium 3b (71.8%)
complexes. Interestingly, the most cytotoxic iridium complexes 3d and 4d are practically deprived of KSP inhibitory
activity. These results suggest the existence of another mechanism
of anticancer activity than the ability to inhibit KSP activity. Figure 8 KSP activity
after being treated with studied compounds at 100,
300, and 1000 nM concentrations. ROS Generation Metal complexes often induce reactive
oxygen species (ROS) generation in cells, 51 which may increase their cytotoxic activity compared to purely organic
molecules. To study the impact of the synthesized compounds on ROS
production, we have measured the ROS generation in SW620 cells by
the dihydrorhodamine 123 (DHR123) oxidation assay ( Figure 9 ). However, there is no correlation
between the antiproliferative potential and the ability of a compound
to generate ROS. Only Ru derivatives ( 3a and 4a ) increase the level of ROS compared to the control or ( R )- and ( S )- 1 , and the level of the
ROS generated by those complexes is virtually the same. In contrast,
the other derivatives do not induce ROS generation. Figure 9 ROS generation in SW620
cells exposure to the studied compounds
(1 μM). Ctrl expressed as 100%, cells in DMEM contained 0.1%
DMSO as the control; verapamil (VER): cells in DMEM contained 0.1%
DMSO and 10 μM VER as an ABC inhibitor to exclude the potential
activity of ABC proteins. Results are presented as mean ± SEM, n = 3. No statistically significant differences were observed
compared to the VER sample, ( R )- 1 or
( S )- 1 ( P < 0.05,
one-way ANOVA followed by the posthoc Tukey test).
## Synthesis
Synthesis The half-sandwich complexes 3a – d and 4a – d were
synthesized in two steps according to Scheme 1 . First, ( R )- and ( S )- 2 imine ligands were generated in situ by
reacting ( R )- and ( S )- 1 with 2 equiv of pyridine-2-carbaldehyde in anhydrous ethanol for
1 h. Next, 0.5 equiv of the proper dimetallic precursor [(cym)MCl 2 ] 2 (M = Ru for 3a and 4a , M = Os for 3b and 4b ) or [(Cp*)MCl 2 ] 2 (M = Rh for 3c and 4c or M = Ir for 3d and 4d ) was added to
the reaction mixture. After 3 h of stirring at RT, the desired complexes 3a – d or 4a – d were isolated as hexafluorophosphate salts in 37–73% yield.
All complexes were fully characterized by 1 H and 13 C{ 1 H} NMR spectroscopy and ESI-MS analyses. The purity
of compounds was confirmed by elemental analysis. Scheme 1 Synthesis of Complexes 3a – 4d It might be expected that a mixture of diastereoisomers
of 3a – d and 4a – d would be formed as the result of the complexation reactions
of enantiomerically pure imines 2 due to the generation
of new chirality on the metal atoms. In the 1 H NMR spectra
of 3a – c and 4a – c , only one main set of peaks was observed in 1 H and 13 C{ 1 H} NMR spectra together with small
amounts (∼15%) of a second species, which can be assigned to
the other diastereoisomers. Yet, for iridium complexes ( 3d and 4d ), we detected much more intensive signals originating
from the second diastereoisomer (ratio of 1:0.4 for 3d and ratio of 1:1 for 4d ). The formation of two diastereoisomers
of the complexes was also confirmed by diffusion-ordered spectroscopy
(DOSY) experiments ( Figures 2 and S1–S3 ). For example,
the DOSY spectra of 3a and ( R )- 1 ( Figure 2 ) confirmed that all 1 H signals observed in the 1 H NMR spectra originate from the molecule(s) showing the same diffusion
coefficient. Figure 2 Overlapped 1 H DOSY spectra of 3a (blue)
and ( R )- 1 (red) in DMSO- d 6 . Notwithstanding, the DOSY experiments confirmed
the presence in
the solution of compounds showing the same diffusion coefficient.
The formation of diastereoisomers of complexes 3a – 4d was confirmed by HPLC-MS analysis ( Figures S9–S16 ). For example, the HPLC-MS analysis
of both ruthenium complexes reveals two peaks at τ 1 = 3.50 and τ 2 = 5.22 min for 3a , with
a ratio of 1:5, with the m / z of
701 assigned to [M 3a -PF6 ] + , and τ 1 = 3.39 and τ 2 = 5.07 min
for 4a , with a ratio of 1:4.3, with the m / z of 701 assigned to [M 4a -PF6 ] + . Likewise, the HPLC-MS analysis of both osmium complexes 3b and 4b confirmed the formation of two diastereoisomers
with the ratio of 1:4 ( Figures S11 and S12 ). In the case of iridium complexes ( 3d and 4d ), the ratio of HPLC peaks is 1:0.4 for 3d and 1:1.4
for 4d , corresponding with the results observed in 1 H NMR. However, for rhodium complexes ( 3c and 4c ), only the main peak at τ 1 = 2.67 min
for 3c and τ 1 = 2.79 min for 4c , with an additional small peak (ratio 1:0.08), and small peaks at
τ 2 = 2.26 min for 3c and τ 2 = 2.40 min for 4c with the m / z of 334 assigned to [M 3c -Cl-PF6 ] 2+ and [M 4c -Cl-PF6 ] 2+ were detected. On the 1 H NMR spectra
of complexes 3a – b and 4a – b at 300 K, aromatic p -cymene
proton signals were broad singlets. Also, no correlation
between aromatic p -cymene protons or proton–carbon
correlations in 1 H– 1 H COSY or 1 H– 13 C HSQC NMR spectra was observed. Therefore,
we performed VT-NMR experiments for 3a and 3b in DMSO- d 6 at various temperatures between
300 and 330 K ( Figures 3 and S4a–d ). An increase in the
temperature of the sample from 300 to 330 K results in a change of
broad singlets at 6.18 and 6.00 ppm into actual doublets and a doublet
at 5.87 ppm, which were assigned to aromatic p -cymene
protons. Additionally, a small set of signals, most likely originating
from the hydrolyzed form of the complex, was observed during the experiment.
The 1 H– 1 H COSY and 1 H– 13 C HSQC spectra allowed observing the expected correlations
between aromatic p -cymene protons and carbon atoms
( Figures S5 and S6 ) at 330 K. However,
those experiments confirmed the partial thermal decomposition of studied
complexes, which impeded the performed 13 C{ 1 H} NMR spectra. Identical results were observed for 4a and 4b (300 and 330 K) ( Figures S7, S8, S45–S47, S59–S61 ). Figure 3 VT-NMR experiments for 3a . 1 H NMR spectra
in DMSO- d 6 (range 6.45–5.35 ppm)
at (a) 300, (b) 310, (c) 320, and (d) 330 K; * denotes the signals
assigned to the solvated compound. It could be expected that ligand 1 may undergo complexation
forming the expected Type I complexes together with two other Type
II and Type III complexes ( Figure 4 ). The formation of Type III complexes was excluded
by MS analysis. In the MS spectra of 3a – 4d , we observed only expected m / z values assigned to monocations [M] + ( Figures S9–S16 ). To further exclude the formation of
Type II complexes, we generated imine 5 in the reaction
of 1 with benzaldehyde ( Scheme 2 ). The obtained imine 5 further
reacted with 0.49 equiv of metal dimers [LMCl 2 ] 2 (M = Rh/Ir, L = Cp* or M = Ru/Os, L = cym) in methanol at RT for
3 h. After the workup, we isolated only previously reported complexes 6a – d bearing 1 as N , N -bidentate ligands in trace yield. As
the formation of imines is reversible, unless coordinated to a metal, 46 imine 5 hydrolyzed in the presence
of a trace of water to amine 1 , which underwent complexation
with [LMCl 2 ] 2 to afford complexes 6a – d . The NMR spectra of the isolated complexes
were identical to those reported previously. 44 Figure 4 Three
possible coordinations of the metal to ligand 2 . Bidentate
coordination of (a) Type 1 and (b) Type II and (c) tridentate
coordination of Type III. Scheme 2 Competitive Complexation of ( S )- 1 and 5 with [LMCl 2 ] 2
## X-ray Diffraction Studies
X-ray Diffraction Studies Although we obtained complexes
as a mixture of two possible diastereoisomers, the crystallization
of 4a from the dichloromethane/ n -pentane
mixture by slow evaporation in −20 °C allowed to isolate
only one enantiopure isomer 4a S,S Ru . The complex 4a S,S Ru crystallized in the P2 1 space
group and its chiral purity has been confirmed by a low value of the
Flack parameter ( Table S1 ). The imine
( S )- 2 acts as a N , N -bidentate ligand, forming five-membered rings with the
metal ions by coordinating through the iminium and pyridinium nitrogen
( Figure 5 ). Two similar
structures of the complex are present in the unit cell, showing almost
identical ruthenium coordination, varying slightly in the conformation
of the terminal phenyl and i Pr moieties.
In Table 1 , we had
listed bond lengths of the coordination bonds for both forms, which
are typical for such types of complexes. 47 , 48 A more thorough description of the molecular geometry has been presented
in the ESI . Figure 5 Oak Ridge thermal ellipsoid
plot (ORTEP) representation of the
molecular structure of 4a S,S Ru : (a) molecule 4a S,S Ru ″ with
the counterion, (b) molecule 4a S,S Ru ′ with the counterion,
and (c) schematic representation of the ruthenium coordination sphere.
Interatomic distances and angles reported in Table 1 are highlighted in blue. Atomic displacement
parameters are drawn at the 50% probability level. Hydrogen atoms
are represented as fixed-size spheres in panels (a) and (b) and omitted
in panel (c). The cocrystallized disordered solvent molecule has also
been removed for clarity. Table 1 Selected Coordination Bond Lengths
(Å) and Angles (deg) Found in Both Independent Molecules of 4a S,S Ru in Its Crystal Structure bond or angle 4a S,S Ru ′ 4a S,S Ru ″ Ru–Cl 2.396(1) Å 2.383(2) Å Ru–N py 2.082(4) Å 2.096(4) Å Ru–N im 2.131(4) Å 2.119(4) Å Ru−μ [center
of the p-cymene ring] 1.699(2) Å 1.695(2) Å N im –Ru–N py 76.7(2)° 76.7(2)° N im –Ru–Cl 86.4(1)° 85.2(1)° N py –Ru–Cl 85.9(1)° 82.4(1)° N im –Ru−μ 135.55° 134.16° N py –Ru−μ 128.88° 132.27° Cl–Ru−μ 127.11° 125.53°
## Stability Study
Stability Study For biological studies, compounds are
commonly administered as dimethyl sulfoxide (DMSO) solution to cells
cultured in a specific medium such as Dulbecco’s modified Eagle’s
medium (DMEM). DMEM consists of numerous organic compounds which may
act as ligands for organometallics. Therefore, it is important to
know how the compounds behave in such conditions. The two most prominent
components of DMEM which may coordinate to half-sandwich complexes
are l -cysteine and l -histidine. Both of those amino
acids are present in DMEM at 0.2 mM concentration, so we studied how
the complexes interact with them using UV–vis spectroscopy
and HPLC-MS analysis. The DMSO solutions of complexes were added to
the aqueous solution of l -cysteine or l -histidine
to achieve a complex concentration of 20 μM while keeping the
DMSO concentration at 0.5 vol %. The UV–vis spectra and HPLC-MS
analysis indicate that neither ruthenium 3a nor the osmium
complex 3b reacts with those amino acids within 2 h ( Figures S18–S21, S24 and S25 ). The rhodium
complex 3c slowly reacts with l -cysteine ( Figure S22 ) by increasing the intensity of each
absorbance maximum (λ = 279, 304, 317, 348 nm). HPLC-MS analysis
confirmed the formation of an additional peak at τ = 0.95 min
with m / z 714 assigned to [M-Cl-PF 6 + HCOOH] + ; additionally, the intensity of peaks
corresponding to 3c is lower ( Figure S26 ). A similar effect is observed in the case of l -histidine, with an increase of only one maximum at λ = 278
nm, while the others are almost unchanged ( Figure S23 ). On the other hand, the iridium complex 3d reacts with both l -cysteine and l -histidine ( Figure 6 ) in 40 min. The
intensity of absorbance peaks at λ = 287 and λ = 372 nm
in the presence of cysteine is decreasing, while the intensity of
peaks at λ = 304 and λ = 318 nm is almost intact. HPLC-MS
analysis shows that the intensity of both peaks corresponding to 3d is lower, while the additional peak at τ = 1.09 min
with m / z 804 is assigned to [M-Cl-PF 6 + HCOOH] + for the l -histidine experiment
and at τ = 1.07 min with m / z 804 is assigned to [M-Cl-PF 6 + HCOOH] + for
the l -cysteine experiment ( Figure S27 ). The lack of an isosbestic point on the UV–vis spectra and
HPLC-MS analysis indicate that the reaction does not lead to the dissociation
of ligands 2 and is purely associated with Cl ligand
exchange. Figure 6 UV–vis spectra of 3d in DMSO-water solutions
in the presence of 0.2 mM (a) l -cysteine or (c) l -histidine. The absorbance maxima value changes vs time in the presence
of (b) l -cysteine and (d) l -histidine.
## Biological Activity
Biological Activity Antiproliferative Potential To assess the impact of
conjugating half-sandwich complexes with amines 1 via
an imine-pyridine ligand on biological activity, we examined the antiproliferative
potential of ( R )- and ( S )- 1 and organometallic conjugates 3a – d and 4a – d in selected human
cancer cell lines: alveolar basal epithelial cell adenocarcinoma (A549),
colorectal adenocarcinomas (Colo205 and SW620), colorectal carcinoma
(HCT116), hepatocellular carcinoma (HepG2), and breast adenocarcinoma
(MCF7). The choice of cell lines was dictated by results of previously
published clinical trials on ispinesib. 49 , 50 All complexes
demonstrate an antiproliferative potential in the micromolar or nanomolar
range ( Table 2 , Figures S28 and S30 ). The activity of these compounds
varies significantly depending on the configuration of imine-ligand 2 and the cell line tested. Complexation of the imine derived
from ( R )- 1 by osmium, resulting in complex 3b , leads to an enhanced cytotoxicity toward A549 (2-fold),
HepG2 (3-fold), and MCF7 (3-fold). A similar effect is observed for
Rh 3c and Ir 3d complexes derived from imine
( R )- 2 , characterized by a 2-fold increased
antiproliferative potential toward A549. However, the complexation
of imine ( R )- 2 with ruthenium 3a does not enhance the activity toward studied cell lines.
Nevertheless, the complexation of the imine derived from ( S )- 1 with all metals results in a significantly
increased antiproliferative potential. It is especially evident in
the case of the ruthenium complex 4a (approximately 6-fold
increased activity against MCF7 and Colo205), the osmium complex 4b (increased cytotoxicity against Colo205 (7-fold), HCT116
(10-fold), and MCF7 (9-fold)), and the iridium complex 4d (enhanced activity toward all tested cell lines, ranging from 9-
to 36-fold). Notably, the iridium complex 4d also exhibits
a significantly higher cytotoxicity compared to both ( S )- 1 and the more cytotoxic amine ( R )- 1 (2.6- and 1.6-fold, respectively). Additionally,
within the tested concentration ranges, all of the compounds studied
show no antiproliferative effects on the normal MRC-5 cell line, with
IC 50 values exceeding 100 μM ( Figure S31 ). Table 2 Antiproliferative Activity of ( R )- 1 and ( S )- 1 and Organometallic Complexes 3a – 4d in Human Cancer Cell Lines a IC 50 [μM] compound A549 Colo205 HCT116 HepG2 MCF7 SW620 ( R )- 1 2.21 0.107 0.346 0.566 0.231 0.096 [1.88–2.59] [0.094–0.121] [0.274–0.437] [0.476–0.672] [0.195–0.308] [0.080–0.117] 3a 2.45 1.26 2.88 1.57 0.858 1.22 [2.05–2.93] [1.16–1.38] [2.48–3.43] [1.45–1.70] [0.742–0.988] [1.12–1.33] (0.902) (0.085) (0.120) (0.360) (0.269) (0.079) 3b 1.04 0.448 0.424 0.188 0.073 0.556 [0.983–1.07] [0.412–0.492] [0.388–0.476] [0.174–0.204] [0.068–0.079] [0.514–0.601] (2.12) (0.239) (0.816) (3.01) (3.16) (0.173) 3c 1.16 0.138 0.173 0.689 0.357 0.152 [0.906–1.50] [0.125–0.153] [0.145–0.206] [0.605–0.784] [0.279–0.459] [0.125–0.185] (1.90) (0.775) (2.00) (0.821) (0.647) (0.632) 3d 1.13 0.524 0.476 0.454 0.653 0.198 [0.973–1.32] [0.453–0.606] [0.408–0.550] [0.383–0.539] [0.552–0.767] [0.172–0.226] (1.96) (0.204) (0.727) (1.25) (0.354) (0.485) ( S )- 1 7.05 6.07 8.06 2.40 3.91 2.87 [6.42–7.36] [5.18–7.39] [7.29–8.90] [2.18–2.63] [3.56–4.31] [2.68–3.06] 4a 3.25 0.939 3.76 1.89 0.634 2.91 [2.84–3.73] [0.737–1.20] [3.32–4.27] [1.64–2.17] [0.539–0.743] [2.63–3.21] (2.17) (6.46) (2.14) (1.27) (6.17) (0.986) 4b 2.19 0.904 0.823 1.13 0.438 2.31 [2.00–2.40] [0.825–0.985] [0.658–1.07] [1.04–1.23] [0.398–0.483] [2.18–2.45] (3.22) (6.71) (9.79) (2.12) (8.93) (1.24) 4c 3.79 2.89 2.94 1.15 1.50 2.24 [3.45–4.16] [2.64–3.15] [2.75–3.14] [0.833–1.58] [1.14–1.94] [1.95–2.61] (1.86) (2.10) (2.74) (2.09) (2.61) (1.28) 4d 0.764 0.216 0.222 0.218 0.216 0.139 [0.613–0.954] [0.197–0.235] [0.192–0.254] [0.193–0.244] [0.191–0.244] [0.128–0.150] (9.23) (28.1) (36.31) (11.01) (18.10) (20.65) a Exposure time 72 h; IC 50 values are presented together with the corresponding 95% confidence
intervals (in brackets), n = 3; the activity factors
were calculated as IC 50(1) /IC 50( 3a – 4d ) and are given in parentheses below the confidence
intervals. Next, we evaluated the cytotoxicity of the synthesized
complexes
toward the panel of six multidrug-resistant (MDR) cell lines derived
from SW620 and characterized by the overexpression of various ABC
proteins, namely, ABCG2 (SW620C and SW620Mito), ABCC1 (SW620M and
SW620E), and ABCB1 (SW620D, SW620E, and SW620V) ( Table 3 , Figures S29 and S32 ). Among the series of complexes bearing the ( R )- 2 ligand, only the iridium complex 3d shows a 2.2- and 1.7-fold higher cytotoxicity than the
corresponding amine ( R )- 1 toward SW620C
and SW620D cancer cell lines. The activity of the complexes derived
from the ligand ( S )- 2 is also considerably
higher than that of the compounds containing the ligand ( R )- 2 . The cytotoxicity of both rhodium 4c and iridium 4d complexes is higher than that of amine
( S )- 1 . In the case of 4c , the increase in cytotoxicity is low, with the highest value of
3.7-fold for the SW620Mito line. Nevertheless, the IC 50 values for the iridium complex 4d are 6.1- to 20.6-fold
lower than those for amine ( S )- 1 . Compound 4d also exerts a 2.1- and 2.6-fold higher cytotoxicity than
( R )- 1 against the SW620C and SW620D
lines. Table 3 Antiproliferative Activity of ( R )- 1 and ( S )- 1 and Organometallic Complexes 3a – 4d in Multidrug-Resistant (MDR) Cancer Cell Lines a IC 50 [μM] comp. SW620 SW620C SW620D SW620E SW620M SW620V SW620Mito ( R )- 1 0.096 0.721 1.12 0.835 0.241 0.206 0.261 [0.080–0.117] [0.552–0.942] [0.851–1.47] [0.627–1.11] [0.191–0.305] [0.160–0.267] [0.207–0.329] 3a 1.22 4.38 7.23 5.52 3.45 4.71 4.33 [1.12–1.33] [3.80–5.07] [6.24–8.37] [4.74–6.43] [2.99–3.97] [4.08–5.44] [3.80–4.95] (0.079) (0.165) (0.155) (0.151) (0.070) (0.044) (0.060) 3b 0.556 1.17 3.92 3.52 0.591 1.03 0.748 [0.514–0.601] [1.08–1.27] [3.60–4.27] [3.25–3.83] [0.522–0.667] [0.953–1.12] [0.683–0.815] (0.173) (0.616) (0.286) (0.237) (0.408) (0.200) (0.349) 3c 0.152 0.592 4.18 0.880 1.18 0.210 0.524 [0.125–0.185] [0.526–0.666] [3.13–5.96] [0.657–1.18] [0.998–1.42] [0.175–0.253] [0.418–0.657] (0.632) (1.22) (0.268) (0.949) (0.204) (0.981) (0.498) 3d 0.198 0.335 0.644 1.02 0.247 0.267 0.268 [0.172–0.226] [0.297–0.377] [0.565–0.732] [0.928–1.13] [0.222–0.275] [0.224–0.317] [0.233–0.311] (0.485) (2.15) (1.74) (0.819) (0.976) (0.771) (0.974) ( S )- 1 2.87 3.33 4.15 4.05 3.46 3.44 3.91 [2.68–3.06] [2.94–3.78] [3.65–4.75] [3.57–4.61] [3.03–3.98] [3.00–3.94] [3.46–4.44] 4a 2.91 4.36 6.01 5.46 3.24 5.01 3.68 [2.63–3.21] [3.90–4.88] [5.24–6.88] [4.77–6.26] [2.91–3.61] [4.43–5.58] [3.29–4.10] (0.986) (0.764) (0.690) (0.741) (1.07) (0.687) (1.06) 4b 2.31 4.54 13.5 12.8 2.45 9.30 3.78 [2.18–2.45] [4.15–4.99] [12.3–15.0] [11.7–14.2] [2.25–2.66] [8.52–10.1] [3.48–4.11] (1.24) (0.738) (0.307) (0.316) (1.41) (0.370) (1.03) 4c 2.24 2.97 6.07 6.20 2.25 2.98 1.05 [1.95–2.61] [2.55–3.45] [4.88–7.85] [4.96–8.05] [1.95–2.59] [2.58–3.45] [0.878–1.25] (1.28) (1.12) (0.684) (0.653) (1.54) (1.15) (3.72) 4d 0.139 0.343 0.425 0.663 0.387 0.411 0.365 [0.128–0.150] [0.310–0.379] [0.384–0.472] [0.582–0.754] [0.339–0.443] [0.374–0.451] [0.313–0.428] (20.65) (9.71) (9.76) (6.11) (8.94) (8.37) (10.71) a Exposure time 72 h; IC 50 values are presented together with the corresponding 95% confidence
intervals (in brackets), n = 3; the activity factors
were calculated as IC 50(1) /IC 50( 3a–4d ) and are given in parentheses below the confidence intervals. Cell Cycle Ispinesib leads to the formation of monopolar
mitotic spindles and a blockade of chromosome segregation in cancer
cells. Using flow cytometry, we assessed the cell cycle distribution
in the SW620 and SW620E cells exposed to the studied compounds for
24 and 48 h. Only two complexes, rhodium 3c and iridium 3d , exhibit a significantly different impact on cell cycle
phase distribution. In contrast, all other complexes demonstrate a
pattern similar to the corresponding amines ( R )-
and ( S )- 1 , as shown in Figure 7 and Table S2 . Both complexes, 3c and 3d , decrease
the percentage of cells in the G 1 /G 0 phase and
increase the percentage in the S and G 2 /M phases. All other
compounds exhibit a similar impact on cell phase distribution. Furthermore,
prolonged exposure to the compounds increases the percentage of cells
in the G 2 /M phase, with the most intensive effect observed
for 3c and 3d . These results suggest an
aggravated mitotic arrest in cells treated with the rhodium 3c and iridium 3d complexes. However, none of
the studied compounds affects the cell cycle in SW620E cells, as demonstrated
in Figure S33 . Figure 7 Cell cycle distribution
in SW620 cells: (a) after 24 h and (b)
after 48 h.
## Antiproliferative Potential
Antiproliferative Potential To assess the impact of
conjugating half-sandwich complexes with amines 1 via
an imine-pyridine ligand on biological activity, we examined the antiproliferative
potential of ( R )- and ( S )- 1 and organometallic conjugates 3a – d and 4a – d in selected human
cancer cell lines: alveolar basal epithelial cell adenocarcinoma (A549),
colorectal adenocarcinomas (Colo205 and SW620), colorectal carcinoma
(HCT116), hepatocellular carcinoma (HepG2), and breast adenocarcinoma
(MCF7). The choice of cell lines was dictated by results of previously
published clinical trials on ispinesib. 49 , 50 All complexes
demonstrate an antiproliferative potential in the micromolar or nanomolar
range ( Table 2 , Figures S28 and S30 ). The activity of these compounds
varies significantly depending on the configuration of imine-ligand 2 and the cell line tested. Complexation of the imine derived
from ( R )- 1 by osmium, resulting in complex 3b , leads to an enhanced cytotoxicity toward A549 (2-fold),
HepG2 (3-fold), and MCF7 (3-fold). A similar effect is observed for
Rh 3c and Ir 3d complexes derived from imine
( R )- 2 , characterized by a 2-fold increased
antiproliferative potential toward A549. However, the complexation
of imine ( R )- 2 with ruthenium 3a does not enhance the activity toward studied cell lines.
Nevertheless, the complexation of the imine derived from ( S )- 1 with all metals results in a significantly
increased antiproliferative potential. It is especially evident in
the case of the ruthenium complex 4a (approximately 6-fold
increased activity against MCF7 and Colo205), the osmium complex 4b (increased cytotoxicity against Colo205 (7-fold), HCT116
(10-fold), and MCF7 (9-fold)), and the iridium complex 4d (enhanced activity toward all tested cell lines, ranging from 9-
to 36-fold). Notably, the iridium complex 4d also exhibits
a significantly higher cytotoxicity compared to both ( S )- 1 and the more cytotoxic amine ( R )- 1 (2.6- and 1.6-fold, respectively). Additionally,
within the tested concentration ranges, all of the compounds studied
show no antiproliferative effects on the normal MRC-5 cell line, with
IC 50 values exceeding 100 μM ( Figure S31 ). Table 2 Antiproliferative Activity of ( R )- 1 and ( S )- 1 and Organometallic Complexes 3a – 4d in Human Cancer Cell Lines a IC 50 [μM] compound A549 Colo205 HCT116 HepG2 MCF7 SW620 ( R )- 1 2.21 0.107 0.346 0.566 0.231 0.096 [1.88–2.59] [0.094–0.121] [0.274–0.437] [0.476–0.672] [0.195–0.308] [0.080–0.117] 3a 2.45 1.26 2.88 1.57 0.858 1.22 [2.05–2.93] [1.16–1.38] [2.48–3.43] [1.45–1.70] [0.742–0.988] [1.12–1.33] (0.902) (0.085) (0.120) (0.360) (0.269) (0.079) 3b 1.04 0.448 0.424 0.188 0.073 0.556 [0.983–1.07] [0.412–0.492] [0.388–0.476] [0.174–0.204] [0.068–0.079] [0.514–0.601] (2.12) (0.239) (0.816) (3.01) (3.16) (0.173) 3c 1.16 0.138 0.173 0.689 0.357 0.152 [0.906–1.50] [0.125–0.153] [0.145–0.206] [0.605–0.784] [0.279–0.459] [0.125–0.185] (1.90) (0.775) (2.00) (0.821) (0.647) (0.632) 3d 1.13 0.524 0.476 0.454 0.653 0.198 [0.973–1.32] [0.453–0.606] [0.408–0.550] [0.383–0.539] [0.552–0.767] [0.172–0.226] (1.96) (0.204) (0.727) (1.25) (0.354) (0.485) ( S )- 1 7.05 6.07 8.06 2.40 3.91 2.87 [6.42–7.36] [5.18–7.39] [7.29–8.90] [2.18–2.63] [3.56–4.31] [2.68–3.06] 4a 3.25 0.939 3.76 1.89 0.634 2.91 [2.84–3.73] [0.737–1.20] [3.32–4.27] [1.64–2.17] [0.539–0.743] [2.63–3.21] (2.17) (6.46) (2.14) (1.27) (6.17) (0.986) 4b 2.19 0.904 0.823 1.13 0.438 2.31 [2.00–2.40] [0.825–0.985] [0.658–1.07] [1.04–1.23] [0.398–0.483] [2.18–2.45] (3.22) (6.71) (9.79) (2.12) (8.93) (1.24) 4c 3.79 2.89 2.94 1.15 1.50 2.24 [3.45–4.16] [2.64–3.15] [2.75–3.14] [0.833–1.58] [1.14–1.94] [1.95–2.61] (1.86) (2.10) (2.74) (2.09) (2.61) (1.28) 4d 0.764 0.216 0.222 0.218 0.216 0.139 [0.613–0.954] [0.197–0.235] [0.192–0.254] [0.193–0.244] [0.191–0.244] [0.128–0.150] (9.23) (28.1) (36.31) (11.01) (18.10) (20.65) a Exposure time 72 h; IC 50 values are presented together with the corresponding 95% confidence
intervals (in brackets), n = 3; the activity factors
were calculated as IC 50(1) /IC 50( 3a – 4d ) and are given in parentheses below the confidence
intervals. Next, we evaluated the cytotoxicity of the synthesized
complexes
toward the panel of six multidrug-resistant (MDR) cell lines derived
from SW620 and characterized by the overexpression of various ABC
proteins, namely, ABCG2 (SW620C and SW620Mito), ABCC1 (SW620M and
SW620E), and ABCB1 (SW620D, SW620E, and SW620V) ( Table 3 , Figures S29 and S32 ). Among the series of complexes bearing the ( R )- 2 ligand, only the iridium complex 3d shows a 2.2- and 1.7-fold higher cytotoxicity than the
corresponding amine ( R )- 1 toward SW620C
and SW620D cancer cell lines. The activity of the complexes derived
from the ligand ( S )- 2 is also considerably
higher than that of the compounds containing the ligand ( R )- 2 . The cytotoxicity of both rhodium 4c and iridium 4d complexes is higher than that of amine
( S )- 1 . In the case of 4c , the increase in cytotoxicity is low, with the highest value of
3.7-fold for the SW620Mito line. Nevertheless, the IC 50 values for the iridium complex 4d are 6.1- to 20.6-fold
lower than those for amine ( S )- 1 . Compound 4d also exerts a 2.1- and 2.6-fold higher cytotoxicity than
( R )- 1 against the SW620C and SW620D
lines. Table 3 Antiproliferative Activity of ( R )- 1 and ( S )- 1 and Organometallic Complexes 3a – 4d in Multidrug-Resistant (MDR) Cancer Cell Lines a IC 50 [μM] comp. SW620 SW620C SW620D SW620E SW620M SW620V SW620Mito ( R )- 1 0.096 0.721 1.12 0.835 0.241 0.206 0.261 [0.080–0.117] [0.552–0.942] [0.851–1.47] [0.627–1.11] [0.191–0.305] [0.160–0.267] [0.207–0.329] 3a 1.22 4.38 7.23 5.52 3.45 4.71 4.33 [1.12–1.33] [3.80–5.07] [6.24–8.37] [4.74–6.43] [2.99–3.97] [4.08–5.44] [3.80–4.95] (0.079) (0.165) (0.155) (0.151) (0.070) (0.044) (0.060) 3b 0.556 1.17 3.92 3.52 0.591 1.03 0.748 [0.514–0.601] [1.08–1.27] [3.60–4.27] [3.25–3.83] [0.522–0.667] [0.953–1.12] [0.683–0.815] (0.173) (0.616) (0.286) (0.237) (0.408) (0.200) (0.349) 3c 0.152 0.592 4.18 0.880 1.18 0.210 0.524 [0.125–0.185] [0.526–0.666] [3.13–5.96] [0.657–1.18] [0.998–1.42] [0.175–0.253] [0.418–0.657] (0.632) (1.22) (0.268) (0.949) (0.204) (0.981) (0.498) 3d 0.198 0.335 0.644 1.02 0.247 0.267 0.268 [0.172–0.226] [0.297–0.377] [0.565–0.732] [0.928–1.13] [0.222–0.275] [0.224–0.317] [0.233–0.311] (0.485) (2.15) (1.74) (0.819) (0.976) (0.771) (0.974) ( S )- 1 2.87 3.33 4.15 4.05 3.46 3.44 3.91 [2.68–3.06] [2.94–3.78] [3.65–4.75] [3.57–4.61] [3.03–3.98] [3.00–3.94] [3.46–4.44] 4a 2.91 4.36 6.01 5.46 3.24 5.01 3.68 [2.63–3.21] [3.90–4.88] [5.24–6.88] [4.77–6.26] [2.91–3.61] [4.43–5.58] [3.29–4.10] (0.986) (0.764) (0.690) (0.741) (1.07) (0.687) (1.06) 4b 2.31 4.54 13.5 12.8 2.45 9.30 3.78 [2.18–2.45] [4.15–4.99] [12.3–15.0] [11.7–14.2] [2.25–2.66] [8.52–10.1] [3.48–4.11] (1.24) (0.738) (0.307) (0.316) (1.41) (0.370) (1.03) 4c 2.24 2.97 6.07 6.20 2.25 2.98 1.05 [1.95–2.61] [2.55–3.45] [4.88–7.85] [4.96–8.05] [1.95–2.59] [2.58–3.45] [0.878–1.25] (1.28) (1.12) (0.684) (0.653) (1.54) (1.15) (3.72) 4d 0.139 0.343 0.425 0.663 0.387 0.411 0.365 [0.128–0.150] [0.310–0.379] [0.384–0.472] [0.582–0.754] [0.339–0.443] [0.374–0.451] [0.313–0.428] (20.65) (9.71) (9.76) (6.11) (8.94) (8.37) (10.71) a Exposure time 72 h; IC 50 values are presented together with the corresponding 95% confidence
intervals (in brackets), n = 3; the activity factors
were calculated as IC 50(1) /IC 50( 3a–4d ) and are given in parentheses below the confidence intervals.
## Cell Cycle
Cell Cycle Ispinesib leads to the formation of monopolar
mitotic spindles and a blockade of chromosome segregation in cancer
cells. Using flow cytometry, we assessed the cell cycle distribution
in the SW620 and SW620E cells exposed to the studied compounds for
24 and 48 h. Only two complexes, rhodium 3c and iridium 3d , exhibit a significantly different impact on cell cycle
phase distribution. In contrast, all other complexes demonstrate a
pattern similar to the corresponding amines ( R )-
and ( S )- 1 , as shown in Figure 7 and Table S2 . Both complexes, 3c and 3d , decrease
the percentage of cells in the G 1 /G 0 phase and
increase the percentage in the S and G 2 /M phases. All other
compounds exhibit a similar impact on cell phase distribution. Furthermore,
prolonged exposure to the compounds increases the percentage of cells
in the G 2 /M phase, with the most intensive effect observed
for 3c and 3d . These results suggest an
aggravated mitotic arrest in cells treated with the rhodium 3c and iridium 3d complexes. However, none of
the studied compounds affects the cell cycle in SW620E cells, as demonstrated
in Figure S33 . Figure 7 Cell cycle distribution
in SW620 cells: (a) after 24 h and (b)
after 48 h.
## KSP Inhibitory Activity
KSP Inhibitory Activity The mechanism of the anticancer
activity of ispinesib is related to the inhibition of the activity
of the KSP. Thus, we studied the synthesized compounds’ ability
to inhibit KSP activity using the adenosine 5′-triphosphate
(ATP) hydrolysis assay. The inhibitory ability of the KSP is strongly
correlated with the configuration of the organic ligand and the type
of metal coordinated. Only the derivatives bearing an organic ligand
configuration ( R ) exhibit KSP inhibitory activity.
In contrast, all compounds bearing an organic ligand in the (S) configuration
demonstrate no inhibitory activity toward the KSP at a concentration
of 100, 300, and 1000 nM ( Figure 8 ). The reference compound, ispinesib, shows a high
KSP inhibitory activity (KSP residual activity 2.2%) at 100 nM concentration,
while amine ( R )- 1 decreases the KSP
activity to about 35%. While the complexation of ruthenium leads to
the nonactive complex 3a , the other metal complexes 3b – d are able to inhibit KSP activity
with the most active rhodium 3c (47.5%), followed by
iridium 3d (64.0%) and osmium 3b (71.8%)
complexes. Interestingly, the most cytotoxic iridium complexes 3d and 4d are practically deprived of KSP inhibitory
activity. These results suggest the existence of another mechanism
of anticancer activity than the ability to inhibit KSP activity. Figure 8 KSP activity
after being treated with studied compounds at 100,
300, and 1000 nM concentrations.
## ROS Generation
ROS Generation Metal complexes often induce reactive
oxygen species (ROS) generation in cells, 51 which may increase their cytotoxic activity compared to purely organic
molecules. To study the impact of the synthesized compounds on ROS
production, we have measured the ROS generation in SW620 cells by
the dihydrorhodamine 123 (DHR123) oxidation assay ( Figure 9 ). However, there is no correlation
between the antiproliferative potential and the ability of a compound
to generate ROS. Only Ru derivatives ( 3a and 4a ) increase the level of ROS compared to the control or ( R )- and ( S )- 1 , and the level of the
ROS generated by those complexes is virtually the same. In contrast,
the other derivatives do not induce ROS generation. Figure 9 ROS generation in SW620
cells exposure to the studied compounds
(1 μM). Ctrl expressed as 100%, cells in DMEM contained 0.1%
DMSO as the control; verapamil (VER): cells in DMEM contained 0.1%
DMSO and 10 μM VER as an ABC inhibitor to exclude the potential
activity of ABC proteins. Results are presented as mean ± SEM, n = 3. No statistically significant differences were observed
compared to the VER sample, ( R )- 1 or
( S )- 1 ( P < 0.05,
one-way ANOVA followed by the posthoc Tukey test).
## Conclusions
Conclusions We designed and synthesized a series of
organometallic half-sandwich
Ru, Os, Rh, and Ir complexes bearing the pyridine-2-ylmethanimine
bidentate ligand derived from 7-chloroquinazolin-4(3H)-one. We obtained
compounds that exhibited nanomolar IC 50 values, strongly
dependent on the metal center, ligand configuration, and cell type.
All studied molecules, with the most potent rhodium and iridium complexes
derived from ( R )-amine, force the cell cycle arrest
in the G 2 /M phase. Only rhodium and iridium complexes derived
from ( R )-imine possess KSP inhibitory activity, however,
to a lower extent than the corresponding amine. In contrast, all other
complexes were significantly less or even nonactive. The complexation
of imines derived from 1 only for Ru led to compounds
able to do ROS generation. However, there is no clear correlation
between the cytotoxicity, KSP inhibitory activity, impact on the cell
cycle, and ROS generation ability. The results suggest that the complexation
of the imines derived from amines ( R )- and especially
( S )- 1 led to compounds showing different
mechanisms of activity than the organic ligands. Further studies are
planned to determine the mechanism of biological activity of the synthesized
compounds.
## Experimental Section
Experimental Section Materials and Methods All of the reactions were carried
out under an argon atmosphere. All commercially available chemicals
and solvents were of analytical grade and used without further purification.
OsO 4 , RhCl 3 ·xH 2 O, and IrCl 3 ·xH 2 O were purchased from Precious Metals
Online and Sigma-Aldrich. Bis[dichlorido(η 6 - p -cymene)ruthenium(II)] was purchased from Sigma-Aldrich.
Bis[dichlorido(η 6 - p -cymene)osmium(II)], 52 bis[dichlorido(η 5 -pentamethylcyclopentadienyl)rhodium(III)],
and bis[dichlorido(η 5 -pentamethylcyclopentadienyl)iridium(III)] 53 were synthesized as described previously. ( R )- 1 and ( S )- 1 were synthesized according to a reported procedure. 54 1 H and 13 C{ 1 H} and 1 H– 13 C HSQC NMR spectra were recorded at
294 K on a Bruker Avance III 600 MHz spectrometer at 600.3 MHz for 1 H and at 150.1 MHz for 13 C{ 1 H}. The 1 H and 13 C{ 1 H} chemical shifts were calibrated
based on the residual 1 H and 13 C{ 1 H} solvent peaks, i.e., δ = 3.58 ppm for 1 H and
67.2 ppm for 13 C in THF- d 8 ,
δ = 2.50 ppm for 1 H and 39.5 ppm for 13 C in dmso- d6 and δ = 5.32 ppm for 1 H and 53.8 ppm for 13 C in CD 2 Cl 2 . The UV–vis spectra were recorded at 294 K on a PerkinElmer
Lambda 45 spectrometer. Elemental analyses were performed at the Faculty
of Chemistry, University of Lodz, Poland. The HPLC-MS analysis was
performed using a Shimadzu Nexera XR system equipped with an SPD-M40
and an LCMS-2020 detector on a Phenomenex XB-C18 column (50 ×
4.6 mm, 2.1 mm, 1.7 μm) using a mixture of 55% water with 0.01%
HCOOH (eluent A), 22.5% methanol with 0.01% HCOOH (eluent B), and
22.5% acetonitrile with 0.01% HCOOH (eluent C) with a flow rate of
0.4 mL·min –1 . General Procedure To a solution of ( R )- 1 or ( S )- 1 (1 equiv)
in anhydrous ethanol (12 mL), pyridine-2-carbaldehyde (2 equiv) was
added and the resulting solution was refluxed under argon conditions
for 1 h. Next, the [LMCl 2 ] 2 dimer (M = Rh/Ir,
L = Cp* (Cp* = η 5 -1,2,3,4,5-pentamethylcyclopentadienyl)
or M = Ru/Os, L = cym (cym = η 6 - p -cymene)) (0.49 equiv) was added, the mixture was cooled down to
RT, and stirring was continued for an additional 3 h. The solvent
was evaporated to c.a. 2 mL and methanol (3 mL) and water (10 mL)
were added, followed by a saturated solution of KPF 6 (5
mL). The precipitant was filtrated off, washed with water (3 ×
10 mL), and dried. The products were purified by crystallization from
the methanol/diethyl ether mixture. 3a [(cym)Ru(( R )- 2 )Cl]PF 6 Compound 3a was synthesized
in 69% yield (217 mg) according to the general procedure starting
from 130 mg (0.38 mmol) of ( R )- 1 , 79
mg (70 μL, 0.74 mmol) of pyridine-2-carbaldehyde, and 114 mg
(0.19 mmol) of [(cym)RuCl 2 ] 2 . Elemental analysis
calculated for C 35 H 37 Cl 2 F 6 N 4 OPRu (846.64 g/mol) C 49.65, H 4.41, N 6.62; found C
49.37, H 4.61, N 6.85. HPLC-MS τ 1 = 3.50 min calculated
for C 35 H 37 Cl 2 N 4 ORu + [M-PF 6 ] + m / z = 701.1; found m / z =
701.4, τ 2 = 5.22 min calculated for C 35 H 37 Cl 2 N 4 ORu + [M-PF 6 ] + m / z = 701.1;
found m / z = 701.0. 1 H
NMR (600 MHz, CD 2 Cl 2 ) δ 9.29 (d, J = 5.4 Hz, 1H, CH Ar ), 9.27 (d, J = 5.5 Hz, 0.2H, CH Ar ), 9.21 (s, 0.2H, CH Ar ), 8.45 (s, 1H, CH imine ), 8.33 (d, J =
8.5 Hz, 1H, CH Ar ), 8.24 (d, J = 7.0 Hz,
1H, CH Ar ), 8.20–8.18 (m, 1H, CH Ar ), 8.15–8.11
(m, 0.4H, CH Ar ), 7.92 (d, J = 1.9 Hz,
1H, CH Ar ), 7.80–7.78 (m, 1H, CH Ar ), 7.75–7.72
(m, 0.2H, CH Ar ), 7.63 (d, J = 1.9 Hz,
0.2H, CH Ar ), 7.61 (dd, J = 8.5, 2.0 Hz,
1H, CH Ar ), 7.48–7.44 (m, 0.6H, CH Ar ),
7.38 (t, J = 7.4 Hz, 2H, CH Ar ), 7.32 (t, J = 7.1 Hz, 2H, CH Ar ), 7.09 (d, J = 7.5 Hz, 2H, CH Ar ), 6.09 (d, J = 16.7
Hz, 0.2H, C H 2 Ph), 5.96 (d, J = 6.7 Hz, 0.2H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.92 (br s, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.87 (d, J = 6.0 Hz, 0.2H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.80 (d, J = 17.3 Hz, 1H, C H 2 Ph), 5.71 (br s, 3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.66 (d, J = 6.2 Hz, 0.4H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.48 (d, J = 5.9 Hz, 0.2H, NC H –CH(CH 3 ) 2 ), 5.41 (d, J = 10.3 Hz, 0.2H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.23 (d, J = 16.6 Hz, 0.2H, C H 2 Ph ) , 4.58 (d, J = 10.0 Hz, 1H, H-1′), 4.35 (d, J = 17.3 Hz, 1H, C H 2 Ph), 3.23–3.17 (m, 1H, H-2′), 2.98–2.93
(m, 0.2H, C H (CH 3 ) 2 ),
2.60–2.56 (m, 0.2H, C H (CH 3 ) 2 ), 2.45–2.40 (m, 1H, 4-CH 3 C 6 H 4 C H (CH 3 ) 2 ), 2.38 (s, 0.6H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ),
1.84 (s, 3H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ),
1.21 (d, J = 6.7 Hz, 0.6H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 1.07 (d, J = 6.9 Hz, 0.6H,
4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 1.03–0.99
(m, 7H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 superimposed with
H-3′), 0.89 (d, J = 6.3 Hz, 3H, H-3′),
0.86 (d, J = 7.0 Hz, 3H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.55 (d, J = 6.5 Hz, 0.6H,
4-CH 3 C 6 H 4 CH(C H 3 ) 2 ). 13 C{ 1 H} NMR (151 MHz, CD 2 Cl 2 ) δ 170.7
( C H imine ), 161.5 (C IV ), 156.0 ( C H Ar ), 153.3 (C IV ), 153.0 (C IV ), 147.4 (C IV ), 141.4 (C IV ), 140.3 ( C H Ar ), 136.2 (C IV ), 131.2 ( C H Ar ), 130.3 ( C H Ar ), 129.9 ( C H Ar ), 129.8 ( C H Ar ), 129.2
( C H Ar ), 128.6 ( C H Ar ), 127.1 ( C H Ar ),
127.0 ( C H Ar ), 126.3 ( C H Ar ), 120.2 (C IV ), 46.6 ( C H 2 ), 32.0 (4-CH 3 C 6 H 4 C H(CH 3 ) 2 ), 31.2
(C-2′), 22.9 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 21.9 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 20.4 (C-3′), 19.8 (C-3′), 19.2 (4- C H 3 C 6 H 4 CH(CH 3 ) 2 ). 3b [(cym)Os(( R )- 2 )Cl]PF 6 Compound 3b was synthesized
in 37% yield (128 mg) according to the general procedure starting
from 130 mg (0.38 mmol) of ( R )- 1 , 79
mg (70 μL, 0.74 mmol) of pyridine-2-carbaldehyde, and 148 mg
(0.19 mmol) of [(cym)OsCl 2 ] 2 . Elemental analysis
calculated for C 35 H 37 Cl 2 F 6 N 4 OOsP (935.80 g/mol) C 44.92, H 3.99, N 5.99; found C
44.68, H 3.96, N 6.13. HPLC-MS τ 1 = 4.23 min calculated
for C 35 H 37 Cl 2 N 4 OOs + [M-PF 6 ] + m / z = 791.2; found m / z =
791.3, τ 2 = 7.06 min calculated for C 35 H 37 Cl 2 N 4 OOs + [M-PF 6 ] + m / z = 791.2;
found m / z = 791.2. 1 H
NMR (600 MHz, CD 2 Cl 2 ) δ 9.62 (s, 0.2H,
C H imine ), 9.20 (d, J = 5.5 Hz, 1H, C H Ar ), 8.92 (s,
1H, C H imine ), 8.40 (d, J = 7.4 Hz, 1H, C H Ar ), 8.32 (d, J = 8.5 Hz, 1H, C H Ar ),
8.28 (d, J = 7.8 Hz, 0.2H, C H Ar ), 8.19 (d, J = 8.6 Hz, 0.3H, C H Ar ), 8.16–8.14 (m, 1H, C H Ar ), 8.10–8.09 (m, 0.3H, C H Ar ), 7.90 (d, J = 1.9 Hz,
1H, C H Ar ), 7.74–7.71 (m,
1H, C H Ar ), 7.68–7.66 (m,
0.6H, C H Ar ), 7.61 (dd, J = 8.4, 2.0 Hz, 1H, C H Ar ), 7.47–7.45
(m, 1H, C H Ar ), 7.40 (t, J = 7.5 Hz, 2H, C H Ar ),
7.34 (t, J = 7.4 Hz, 1H, C H Ar ), 7.30–7.27 (m, 1H, C H Ar ), 7.13 (d, J = 7.5 Hz, 2H, C H Ar ), 6.24 (d, J = 5.9 Hz,
0.3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 6.20 (d, J = 5.6
Hz, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 6.06 (d, J = 16.6 Hz, 0.4H, C H 2 Ph), 5.96 (br s, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.93–5.85
(m, 2H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.81 (d, J = 17.0 Hz, 1H, C H 2 Ph), 5.70 (d, J = 5.5 Hz, 0.3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.41 (d, J = 10.3 Hz, 0.2H, H-1′),
5.14 (d, J = 16.5 Hz, 0.3H, C H 2 Ph), 4.80 (d, J = 9.9 Hz, 1H, H-1′), 4.52 (d, J = 13.6 Hz,
1H, C H 2 Ph),
3.16–3.10 (m, 1H, H-2′), 2.99–2.95 (m, 0.25H,
H-2′), 2.91 (s, 0.1H), 2.82 (s, 0.1H), 2.50–2.46 (m,
0.2H), 2.43 (s, 0.7H, 4- CH 3 C 6 H 4 CH(CH 3 ) 2 ), 2.34–2.27
(m, 1H, 4-CH 3 C 6 H 4 C H (CH 3 ) 2 ), 1.90 (s, 3H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ), 1.18 (d, J = 6.6 Hz, 1H,
H-3′), 1.08 (d, J = 6.9 Hz, 0.8H,), 1.00 (d, J = 6.9 Hz, 3H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.97 (d, J = 6.8 Hz, 3H, H-3′),
0.89 (d, J = 6.1 Hz, 3H, H-3′), 0.78 (d, J = 6.9 Hz, 3H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.53 (d, J = 6.5 Hz, 0.7H, H-3′). 13 C{ 1 H} NMR (151 MHz, CD 2 Cl 2 ) δ 172.8 ( C H imine ), 161.5
(C IV ), 155.5 (CH Ar ), 154.7 (C IV ),
152.8 (C IV ), 147.3 (C IV ), 141.5 (C IV ), 140.3 (CH Ar ), 136.1 (C IV ), 131.1 (CH Ar ), 131.1 (C IV ), 129.9 ( C H Ar ), 129.8 ( C H Ar ),
129.3 ( C H Ar ), 128.7 ( C H Ar ), 127.3 ( C H Ar ), 127.1 ( C H Ar ), 126.4
( C H Ar ), 120.2 (C IV ),
83.6 (N C H–CH(CH 3 ) 2 ), 80.8 (4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 76.9 (4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 73.9 (4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 46.9 ( C H 2 Ph), 32.2 (4-CH 3 C 6 H 4 C H(CH 3 ) 2 ), 31.4 (NCH- C H(CH 3 ) 2 ) 23.5 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 21.9 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 20.5 (NCH–CH( C H 3 ) 2 ), 19.8 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 19.1 (4- C H 3 C 6 H 4 CH(CH 3 ) 2 ). 3c [(Cp*)Rh(( R )- 2 )Cl]PF 6 Compound 3c was synthesized
in 69% yield (536 mg) according to the general procedure starting
from 313 mg (0.91 mmol) of ( R )- 1 , 195
mg (174 μL, 1.82 mmol) of pyridine-2-carbaldehyde, and 277 mg
(0.45 mmol) of [Cp*RhCl 2 ] 2 . Elemental analysis
calculated for C 35 H 38 Cl 2 F 6 N 4 OPRh (849.49 g/mol) C 49.49, H 4.51, N 6.60; found C
49.49, H 4.50, N 6.60. HPLC-MS τ 1 = 2.67 min calculated
for C 35 H 38 Cl 2 N 4 ORh + [M-PF 6 ] + m / z = 703.1; found m / z =
703.5, τ 2 = 4.29 min calculated for C 35 H 38 Cl 2 N 4 ORh + [M-PF 6 ] + m / z = 703.1;
found m / z = 703.5. 1 H
NMR (600 MHz, THF- d 8 ) δ 10.76 (s,
0.1H, CH), 9.27 (s, 1H, C H imine ),
8.89 (d, J = 5.4 Hz, 1H, C H Ar ), 8.86 (d, J = 5.5 Hz, 0.1H, C H Ar ), 8.66 (s, 0.1H, CH Ar ), 8.24
(d, J = 7.4 Hz, 1H, C H Ar ), 8.19 (t, J = 7.8 Hz, 1H, C H Ar ), 8.15 (d, J = 8.5 Hz,
1H, C H Ar ), 7.87 (d, J = 1.8 Hz, 0.2H, C H Ar ), 7.84 (t, J = 6.4 Hz, 1H, C H Ar ),
7.73 (d, J = 1.7 Hz, 1H, C H Ar ), 7.56 (dd, J = 8.6, 2.0 Hz, 0.1H,
C H Ar ), 7.47 (dd, J = 8.6, 2.0 Hz, 1H, C H Ar ), 7.39
(t, J = 7.5 Hz, 2H, C H Ar ), 7.32–7.28 (m, 3.5H, C H Ar ), 7.23 (d, J = 7.2 Hz, 0.1H, C H Ar ), 7.20 (d, J = 7.9 Hz,
0.3H, C H Ar ), 5.91 (d, J = 16.9 Hz, 1H, C H 2 Ph), 5.80 (d, J = 17.4 Hz, 0.1H, C H 2 Ph), 5.33 (d, J = 17.1 Hz, 1H, C H 2 Ph), 5.13 (d, J = 8.7 Hz, 1H,
H-1′), 3.08–3.02 (m, 1H, H-2′), 2.39 (s, 0.4H),
1.73 (s, 15H, Cp*-C H 3 ), 1.63 (s, 2H, Cp*-C H 3 ), 1.13 (d, J = 6.7 Hz, 3H, H-3′),
0.95 (d, J = 6.6 Hz, 3H, H-3′). 13 C{ 1 H} NMR (151 MHz, THF- d 8 ) δ 170.4 (C H imine ), 161.5
(C IV ), 157.0 (C IV ), 154.7 (C IV ),
153.6 ( C H Ar ), 148.2 (C IV ), 140.8 ( C H Ar ), 140.7 (C IV ), 137.0 (C IV ), 131.1 ( C H Ar ), 130.7 ( C H Ar ), 129.6 ( C H Ar ), 129.5 ( C H Ar ), 129.3 ( C H Ar ), 128.3
( C H Ar ), 128.3 ( C H Ar ), 127.6 ( C H Ar ),
127.1 ( C H Ar ), 120.4 (C IV ), 98.6 (d, J C–Rh = 7.7 Hz, Cp*),
73.2 (C-1′), 48.2 ( C H 2 Ph),
35.1 (C-2′), 19.9 (C-3′), 18.8 (C-3′), 9.1 (Cp*- C H 3 ). 3d [(Cp*)Ir(( R )- 2 )Cl]PF 6 Compound 3d was synthesized
in 73% yield (593 mg) according to the general procedure starting
from 296 mg (0.87 mmol) of ( R )- 1 , 185
mg (165 μL, 1.73 mmol) of pyridine-2-carbaldehyde, and 338 mg
(0.42 mmol) of [Cp*IrCl 2 ] 2 . Elemental analysis
calculated for C 35 H 38 Cl 2 F 6 IrN 4 OP (938.80 g/mol) C 44.78, H 4.08, N 5.97; found C
44.83, H 4.20, N 5.97. HPLC-MS τ 1 = 3.65 min calculated
for C 35 H 38 Cl 2 N 4 OIr + [M-PF 6 ] + m / z = 793.2; found m / z =
793.4, τ 2 = 7.45 min calculated for C 35 H 38 Cl 2 N 4 OIr + [M-PF 6 ] + m / z = 793.2;
found m / z = 793.6. 1 H
NMR (600 MHz, THF- d 8 ) δ 9.83 (s,
1H, C H imine ), 9.14 (s, 0.4H), 8.86
(d, J = 5.4 Hz, 1H, C H Ar ), 8.84 (d, J = 5.5 Hz, 0.4H, C H Ar ), 8.42 (d, J = 7.6 Hz,
1H, C H Ar ), 8.22 (d, J = 8.5 Hz, 0.6H, C H Ar ), 8.17 (t, J = 7.6 Hz, 1H, C H Ar ),
8.14 (d, J = 8.5 Hz, 1H, C H Ar ), 7.87 (d, J = 2.0 Hz, 0.4H, C H Ar ), 7.84–7.80 (m, 1.5H, C H Ar ), 7.79 (d, J = 2.0 Hz,
1H, C H Ar ), 7.57 (dd, J = 8.6, 2.0 Hz, 0.5H, C H Ar ), 7.47
(dd, J = 8.5, 2.0 Hz, 1H, C H Ar ), 7.38 (t, J = 7.5 Hz, 2H, C H Ar ), 7.32–7.26 (m, 4H, C H Ar ), 7.26–7.22 (m, 1H, C H Ar ), 7.19 (d, J = 7.5 Hz,
1H, C H Ar ), 5.87 (d, J = 17.1 Hz, 1H, C H 2 Ph), 5.78 (d, J = 17.2 Hz, 0.4H), 5.24 (d, J = 17.1 Hz, 1H, C H 2 Ph), 5.15 (d, J = 9.3 Hz, 1H,
H-1′), 3.20–3.13 (m, 1H, H-2′), 2.95–2.89
(m, 0.4H, H-2′), 1.69 (s, 15H, Cp*-C H 3 ), 1.60 (s, 7H, Cp*-C H 3 ), 1.16 (d, J = 6.7 Hz, 3H, H-3′), 0.92 (d, J = 6.6 Hz, 3H, H-3′). 13 C{ 1 H} NMR (151
MHz, THF- d 8 ) δ 172.0 ( C H imine ), 161.3 (C IV ), 157.0 (C IV ), 156.2 (C IV ), 153.1 ( C H Ar ), 148.2 (C IV ), 141.0 ( C H Ar ), 140.8 ( C H Ar ),
136.7 (C IV ), 131.3 ( C H Ar ), 131.2 ( C H Ar ), 129.6 ( C H Ar ), 129.4 ( C H Ar ), 129.3 ( C H Ar ), 128.4
( C H Ar ), 128.3 ( C H Ar ), 128.2 ( C H Ar ),
127.7 ( C H Ar ), 127.0 ( C H Ar ), 120.3 (C IV ), 91.3 (Cp*),
74.8 (C-1′), 48.1 ( C H 2 Ph),
35.9 (C-2′), 19.9 (C-3′), 19.2 (C-3′), 8.80 (Cp*- C H 3 ). 4a [(cym)Ru(( S )- 2 )Cl]PF 6 Compound 4a was synthesized
in 68% yield (215 mg) according to the general procedure starting
from 130 mg (0.38 mmol) of ( S )- 1 , 79
mg (70 μL, 0.74 mmol) of pyridine-2-carbaldehyde, and 114 mg
(0.19 mmol) of [(cym)RuCl 2 ] 2 . Elemental analysis
calculated for C 35 H 37 Cl 2 F 6 N 4 OPRu (846.64 g/mol) C 49.65, H 4.41, N 6.62; found C
49.41, H 4.59, N 6.80. HPLC-MS τ 1 = 3.39 min calculated
for C 35 H 37 Cl 2 N 4 ORu + [M-PF 6 ] + m / z = 701.1; found m / z =
701.3, τ 2 = 5.07 min calculated for C 35 H 37 Cl 2 N 4 ORu + [M-PF 6 ] + m / z = 701.1;
found m / z = 700.8. 1 H
NMR (600 MHz, CD 2 Cl 2 ) δ 9.29 (d, J = 5.4 Hz, 1H, C H Ar ),
9.27 (d, J = 5.6 Hz, 0.2H, C H Ar ), 9.21 (s, 0.2H, C H imine ), 8.45 (s, 1H, C H imine ), 8.33
(d, J = 8.5 Hz, 1H C H Ar ), 8.24 (d, J = 7.1 Hz, 1H, C H Ar ), 8.19 (t, J = 3.8 Hz,
1H, C H Ar ), 8.15–8.11 (m,
0.4H, C H Ar ), 7.92 (d, J = 1.9 Hz, 1H, C H Ar ), 7.80–7.78
(m, 1H, C H Ar ), 7.74–7.72
(m, 0.2H, C H Ar ), 7.63 (d, J = 1.8 Hz, 0.2H, C H Ar ), 7.61 (dd, J = 8.6, 2.0 Hz, 1H, C H Ar ), 7.48–7.44 (m, 0.7H, C H Ar ), 7.41 (d, J = 7.4 Hz, 0.2H, C H Ar ), 7.38 (t, J = 7.4 Hz,
2H, C H Ar ), 7.32 (t, J = 6.8 Hz, 2H, C H Ar ), 7.09 (d, J = 7.5 Hz, 2H, C H Ar ),
6.09 (d, J = 16.9 Hz, 0.2H, C H 2 Ph), 5.96 (d, J = 6.5 Hz, 0.3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.92 (br s, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.87 (d, J = 6.0 Hz, 0.3H,
4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.80 (d, J = 16.8 Hz, 1H,
C H 2 Ph), 5.71
(br s, 3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.66 (d, J = 6.4 Hz, 0.3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.48 (d, J = 5.9 Hz, 0.2H, H-1′), 5.41 (d, J = 10.1
Hz, 0.2H, H-1′), 5.23 (d, J = 16.5 Hz, 0.2H,
C H 2 Ph), 4.58
(d, J = 9.2 Hz, 1H, H-1′), 4.35 (d, J = 14.5 Hz, 1H, C H 2 Ph), 3.23–3.17 (m, 1H, H-2′), 2.98–2.94
(m, 0.2H, H-2′), 2.60–2.55 (m, 0.2H, 4-CH 3 C 6 H 4 C H (CH 3 ) 2 ), 2.45–2.40 (m, 1H, 4-CH 3 C 6 H 4 C H (CH 3 ) 2 ), 2.38 (s, 0.6H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ),
1.84 (s, 3H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ),
1.21 (d, J = 6.6 Hz, 0.8H, H-3′), 1.07 (d, J = 6.9 Hz, 0.7H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 1.03–0.99 (m, 7H, H-3′, superimposed with
4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.89 (d, J = 6.3 Hz, 3H, H-3′), 0.86 (d, J = 7.0 Hz, 3H 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.55
(d, J = 6.5 Hz, 0.7H, H-3′). 13 C{ 1 H} NMR (151 MHz, CD 2 Cl 2 ) δ
170.7 ( C H imine ), 161.5 (C IV ), 156.0 ( C H Ar ), 153.3 (C IV ), 153.0 (C IV ), 147.4 (C IV ), 141.4 (C IV ), 140.3 ( C H Ar ), 136.2 (C IV ), 131.2 ( C H Ar ), 130.3 ( C H Ar ), 129.9 ( C H Ar ), 129.8 ( C H Ar ), 129.2
( C H Ar ), 128.5 ( C H Ar ), 127.1 ( C H Ar ),
127.0 ( C H Ar ), 126.3 ( C H Ar ), 120.2 (C IV ), 82.9 (C-1′),
79.5 (4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 47.9 ( C H 2 Ph), 46.6 ( C H 2 Ph), 32.0 (4-CH 3 C 6 H 4 C H(CH 3 ) 2 ), 31.3 (C-2′),
22.9 (C–H), 22.7 (C–H), 22.6 (C–H), 22.3 (C–H),
21.9 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 20.4 (C-3′), 19.8 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 19.2 (4- C H 3 C 6 H 4 CH(CH 3 ) 2 ). 4b [(cym)Os(( S )- 2 )Cl]PF 6 Compound 4b was synthesized
in 41% yield (144 mg) according to the general procedure starting
from 130 mg (0.38 mmol) of ( S )- 1 , 79
mg (70 μL, 0.74 mmol) of pyridine-2-carbaldehyde, and 149 mg
(0.19 mmol) of [(cym)OsCl 2 ] 2 . Elemental analysis
calculated for C 35 H 37 Cl 2 F 6 N 4 OOsP (935.80 g/mol) C 44.92, H 3.99, N 5.99; found C
44.75, H 4.09, N 5.78. HPLC-MS τ 1 = 4.24 min calculated
for C 35 H 37 Cl 2 N 4 OOs + [M-PF 6 ] + m / z = 791.2; found m / z =
791.4, τ 2 = 7.04 min calculated for C 35 H 37 Cl 2 N 4 OOs + [M-PF 6 ] + m / z = 791.2;
found m / z = 791.6. 1 H
NMR (600 MHz, CD 2 Cl 2 ) δ 9.62 (s, 0.25H,
C H Ar ), 9.20 (d, J = 5.5 Hz, 1H, C H Ar ), 8.91 (s,
1H, C H imine ), 8.40 (d, J = 7.5 Hz, 1H, C H Ar ), 8.31 (d, J = 8.5 Hz, 1H, C H Ar ),
8.27 (d, J = 7.8 Hz, 0.25H, C H Ar ), 8.19 (d, J = 8.5 Hz, 0.25H, C H Ar ), 8.17–8.14 (m, 1H, C H Ar ), 8.11–8.08 (m, 0.25H, C H Ar ), 7.90 (d, J = 1.9 Hz,
1H, C H Ar ), 7.74–7.72 (m,
1H, C H Ar ), 7.68–7.66 (m,
0.5H, C H Ar ), 7.61 (dd, J = 8.2, 2.0 Hz, 1H, C H Ar ), 7.47–7.45
(m, 0.75H, C H Ar ), 7.39 (t, J = 7.5 Hz, 2H, C H Ar ),
7.34 (t, J = 7.4 Hz, 1H, C H Ar ), 7.29 (d, J = 7.5 Hz, 0.5H, C H Ar ), 7.12 (d, J = 7.5 Hz,
2H, C H Ar ), 6.24 (d, J = 6.1 Hz, 0.4H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 6.19 (d, J = 5.5 Hz, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 6.06 (d, J = 16.5 Hz, 0.4H, C H 2 Ph), 5.96
(br s, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.92–5.84 (m, 2H,
4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.81 (d, J = 16.7 Hz, 1H,
C H 2 Ph), 5.69 (d, J = 5.5 Hz, 0.25H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.40 (d, J = 10.4 Hz, 0.25H, NC H –CH(CH 3 ) 2 ), 5.13 (d, J = 16.4 Hz, 0.25H, C H 2 Ph), 4.78 (d, J = 9.9 Hz,
1H, NC H –CH(CH 3 ) 2 ), 4.50 (d, J = 16.2 Hz, 1H, C H 2 Ph), 3.42 (s, 0.1H), 3.16–3.10 (m, 1H, NCH-C H (CH 3 ) 2 ), 3.00–2.95 (m,
0.25H, NCH-C H (CH 3 ) 2 ),
2.43 (s, 1H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ), 2.32–2.27 (m, 1H,
4-CH 3 C 6 H 4 C H (CH 3 ) 2 ), 1.90 (s, 3H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ), 1.35 (d, J = 6.9 Hz, 0.25H), 1.17 (d, J = 6.7 Hz, 1H, NCH–CH(C H 3 ) 2 ), 1.07 (d, J = 6.9 Hz, 1H), 1.00 (d, J = 6.9 Hz, 3H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.96 (d, J = 6.8 Hz, 3H, NCH–CH(C H 3 ) 2 ), 0.89 (d, J =
6.1 Hz, 3H, NCH–CH(C H 3 ) 2 ), 0.77 (d, J =
6.9 Hz, 3H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.51 (d, J = 6.6 Hz, 0.75H, NCH–CH(C H 3 ) 2 ). 13 C{ 1 H}
NMR (151 MHz, CD 2 Cl 2 ) δ 172.4 ( C H imine ), 161.1 (C IV ), 155.2 ( C H Ar ), 154.2 (C IV ), 152.3 (C IV ), 146.9 (C IV ), 141.1 (C IV ), 140.0
( C H Ar ), 135.7 (C IV ),
130.8 ( C H Ar ), 129.5 ( C H Ar ), 129.4 ( C H Ar ), 128.9 ( C H Ar ), 128.2
( C H Ar ), 126.7 ( C H Ar ), 126.0 ( C H Ar ),
119.7 (C IV ), 88.3 (4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 83.2 (C-1′),
46.5 ( C H 2 ), 31.8 (4-CH 3 C 6 H 4 C H(CH 3 ) 2 ), 30.9 (C-2′), 23.1 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 21.4 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 20.1 (C-3′), 19.3
(C-3′), 18.7 (4- C H 3 C 6 H 4 CH(CH 3 ) 2 ). 4c [(Cp*)Rh(( S )- 2 )Cl]PF 6 Compound 4c was synthesized
in 68% yield (535 mg) according to the general procedure starting
from 313 mg (0.91 mmol) of ( S )- 1 , 195
mg (174 μL, 1.82 mmol) of pyridine-2-carbaldehyde, and 276 mg
(0.45 mmol) of [Cp*RhCl 2 ] 2 . Elemental analysis
calculated for C 35 H 38 Cl 2 F 6 N 4 OPRh (849.49 g/mol) C 49.49, H 4.51, N 6.60; found C
49.53, H 4.49, N 6.55. HPLC-MS τ 1 = 2.79 min calculated
for C 35 H 38 Cl 2 N 4 ORh + [M-PF 6 ] + m / z = 703.1; found m / z =
703.5, τ 2 = 4.20 min calculated for C 35 H 38 Cl 2 N 4 ORh + [M-PF 6 ] + m / z = 703.1;
found m / z = 703.5. 1 H
NMR (600 MHz, THF- d 8 ) δ 10.77 (s,
0.1H), 9.27 (s, 1H, C H imine ), 8.90
(d, J = 5.3 Hz, 1H, C H Ar ), 8.86 (d, J = 5.1 Hz, 0.1H, C H Ar ), 8.66 (s, 0.1H), 8.24 (d, J = 7.1 Hz, 1H, C H Ar ), 8.19 (t, J = 7.7 Hz, 1H, C H Ar ),
8.15 (d, J = 8.5 Hz, 1H, C H Ar ), 7.87 (d, J = 1.7 Hz, 0.2H, C H Ar ), 7.84 (t, J = 6.0 Hz,
1H, C H Ar ), 7.73 (d, J = 1.8 Hz, 1H, C H Ar ), 7.56 (dd, J = 8.5, 2.0 Hz, 0.1H, C H Ar ), 7.47 (dd, J = 8.5, 2.0 Hz, 1H, C H Ar ), 7.39 (t, J = 7.4 Hz, 2H, CH Ar ), 7.32–7.28 (m, 3.5H, CH Ar ), 7.23 (d, J = 7.4 Hz, 0.1H, C H Ar ), 7.20 (d, J = 7.7 Hz, 0.3H, C H Ar ) 5.91 (d, J = 16.7 Hz, 1H, C H 2 Ph), 5.80 (d, J = 17.2
Hz, 0.1H, C H 2 Ph), 5.33 (d, J = 17.1 Hz, 1H, C H 2 Ph), 5.13 (d, J = 8.7 Hz, 1H, H-1′),
3.08–3.02 (m, 1H, H-2′), 2.39 (s, 0.5H), 1.73 (s, 15H,
Cp*-C H 3 ),
1.63 (s, 2H, Cp*-C H 3 ), 1.13–1.10 (m, 3H, H-3′ superimposed with the
diethyl ether signal), 0.95 (d, J = 6.6 Hz, 3H, H-3′). 13 C{ 1 H} NMR (151 MHz, THF- d 8 ) δ 170.4 ( C H imine ), 161.5 (C IV ), 154.7 (C IV ), 153.6 ( C H Ar ), 148.2 (C IV ), 140.8 ( C H Ar ), 140.7 (C IV ), 137.0 (C IV ), 131.1 ( C H Ar ), 130.6
( C H Ar ), 129.6 ( C H Ar ), 129.5 ( C H Ar ),
129.3 ( C H Ar ), 128.3 ( C H Ar ), 128.3 ( C H Ar ), 127.6 ( C H Ar ), 127.1
( C H Ar ), 120.4 (C IV ),
98.6 (d, J Rh–C = 7.8 Hz, Cp*), 73.2 (C-1′),
48.2 ( C H 2 Ph), 35.1 (C-2′)
19.9 (C-3′), 18.8 (C-3′), 9.1 (Cp*- C H 3 ). 4d [(Cp*)Ir(( S )- 2 )Cl]PF 6 Compound 4d was synthesized
in 72% yield (413 mg) according to the general procedure starting
from 208 mg (0.61 mmol) of ( S )- 1 , 131
mg (115 μL, 1.22 mmol) of pyridine-2-carbaldehyde, and 237 mg
(0.30 mmol) of [Cp*IrCl 2 ] 2 . Elemental analysis
calculated for C 35 H 38 Cl 2 F 6 IrN 4 OP (938.80 g/mol) C 44.78, H 4.08, N 5.97; found C
44.69, H 4.05, N 5.86. HPLC-MS τ 1 = 3.81 min calculated
for C 35 H 38 Cl 2 N 4 OIr + [M-PF 6 ] + m / z = 793.2; found m / z =
793.6, τ 2 = 7.53 min calculated for C 35 H 38 Cl 2 N 4 OIr + [M-PF 6 ] + m / z = 793.2;
found m / z = 793.5. 1 H
NMR (600 MHz, THF- d 8 ) δ 9.82 (s,
0.7H, CH imine ) 9.14 (s, 1H, CH imine ), 8.87 (d, J = 5.4 Hz, 0.7H, C H Ar ), 8.84 (d, J = 5.4 Hz, 1H, C H Ar ), 8.41 (d, J = 7.7 Hz, 0.7H, C H Ar ), 8.22 (d, J = 8.5 Hz,
1.3H, C H Ar ), 8.18–8.16 (m,
1.4H, C H Ar ), 8.14 (d, J = 8.5 Hz, 1H, C H Ar ), 7.87 (d, J = 1.9 Hz, 1H, C H Ar ),
7.83–7.80 (m, 1.7H, C H Ar ),
7.79 (d, J = 1.9 Hz, 0.7H, C H Ar ), 7.57 (dd, J = 8.6, 1.9 Hz, 1H, C H Ar ), 7.47 (dd, J = 8.5,
2.0 Hz, 0.7H, C H Ar ), 7.38 (t, J = 7.5 Hz, 1.5H, C H Ar ), 7.33–7.28 (m, 4H, C H Ar ), 7.24 (t, J = 7.3 Hz, 1H, C H Ar ), 7.19 (d, J = 7.5 Hz, 2H, C H Ar ), 5.87 (d, J = 17.0 Hz,
0.7H, C H 2 Ph), 5.78 (d, J = 17.1 Hz, 1H, C H 2 Ph), 5.24 (d, J = 17.1 Hz, 0.8H, C H 2 Ph), 5.15 (d, J = 9.3 Hz, 1H, H-1′),
4.74 (br s, 0.7H, C H 2 Ph), 3.19–3.13 (m, 0.7H, H-2′), 2.95–2.87
(m, 1H, H-2′), 1.70 (s, 11.5H, Cp*-C H 3 ), 1.61 (s, 15H, Cp*-CH 3 ), 1.19 (br s, 3H, H-3′) superimposed with 1.16 (d, J = 6.7 Hz, 3H, H-3′), 0.93 (d, J = 6.6 Hz, 2.3H, H-3′). 13 C{ 1 H} NMR
(151 MHz, THF- d 8 ) δ 172.0 ( C H imine ), 161.4 (C IV ), 161.3 (C IV ), 156.9 (C IV ), 156.2 (C IV ), 153.1
( C H Ar ), 152.7 ( C H Ar ), 148.2 (C IV ), 147.9 (C IV ),
141.1 ( C H Ar ), 140.8 ( C H Ar ), 140.6 (C IV ), 136.7 (C IV ), 131.3 ( C H Ar ), 131.2
( C H Ar ), 129.6 ( C H Ar ), 129.4 ( C H Ar ),
129.3 ( C H Ar ), 128.7 ( C H Ar ), 128.4 ( C H Ar ), 128.3 ( C H Ar ), 128.2
( C H Ar ), 127.7 ( C H Ar ), 127.0 ( C H Ar ),
126.7 ( C H Ar ), 121.1 (C IV ), 120.4 (C IV ), 91.4 (Cp*), 91.3 (Cp*), 74.8 (C-1′),
48.1 ( C H 2 Ph), 35.9 (C-2′),
20.2 (C-3′), 19.9 (C-3′), 19.2 (C-3′), 19.1 (C-3′),
8.8 (Cp*- C H 3 ), 8.8 (Cp*- C H 3 ). Stability Studies The stability of 3a – d was studied in the presence of l -cysteine or l -histidine. 3a – d were dissolved
in DMSO and added to 0.2 mM aqueous solution of l -cysteine
or l -histidine to achieve the complex concentration of 20
μM while keeping the DMSO concentration at 0.5 vol %. UV–vis
spectra were recorded over 2 h with 10 min intervals. HPLC-MS analysis
with them using UV–vis spectroscopy and HPLC-MS analysis were
performed on a Phenomenex XB-C18 column (50 × 4.6 mm, 2.1 mm,
1.7 μm) using a mixture of 55% water with 0.01% HCOOH (eluent
A), 22.5% methanol with 0.01% HCOOH (eluent B), and 22.5% acetonitrile
with 0.01% HCOOH (eluent C) with a flow rate of 0.4 mL·min –1 . Cell Lines Cell lines used in this study were purchased
from the American Type Culture Collection via LGC Standards. Human
normal lung fibroblasts (MRC-5), alveolar basal epithelial cell adenocarcinoma
(A549), colorectal adenocarcinoma (Colo205), hepatocellular carcinoma
(HepG2), breast adenocarcinoma (MCF7), and colorectal adenocarcinoma
(SW620) and its MDR variants 55 were cultured
in standard conditions (37 °C, 5% CO 2 , 100% relative
humidity) in high glucose DMEM medium supplemented with GlutaMax,
HEPES (ThermoFisher Scientific) and 10% fetal bovine serum (EURx,
Poland). All cell lines were tested for Mycoplasma contamination using a MycoProbe mycoplasma detection kit (R&D
System). Assaying the Antiproliferative Potential For this purpose,
neutral red uptake assay was performed. 10 4 of cells were
seeded per well of a 96-well plate and left overnight to allow cells
to attach to the surface. Then, the cells were exposed to a desired
concentration of tested compounds. Stock solutions were prepared in
DMSO and were used immediately after preparation. The final concentration
of DMSO was constant and nontoxic (0.1% v/v). After 70 h of culture,
neutral red was added to the final concentration of 1 mM. After 2
h of incubation with the dye, the medium was aspirated and cells were
washed with ice-cold PBS. The dye was released using 100 μL
of the solubilizer (1% acetic acid in 50% ethanol) on an orbital shaker
(10 min). The absorbance at 540 nm was measured using an EnVision
multilabel plate reader (PerkinElmer). The results were presented
as a percentage of control. The IC 90 and IC 50 parameters were calculated using GraphPad Prism v9 software using
the five-parameter nonlinear logistic regression model. Cell Cycle SW620 and SW620E cells lines (vulnerable
and resistant variants, respectively) were seeded in 6-well plates
at a density of 10 5 cells per well. After the time necessary
for the cells to attach to the surface, the cells were treated with
tested compounds at a concentration equal to IC 90 for parent
compounds (15 nM for ( R ) series and 23 nM for ( S ) series). After 24 h, the cells were trypsinized and fixed
with ice-cold 70% v/v ethanol. The cells were stained with 75 μM
propidium iodide with 50 Kunitz units of RNase A in PBS for 30 min
at 37 °C. All samples were analyzed using a LSRII flow cytometer
(Becton Dickinson) at a PE channel (526/26 nm). Cell cycle phase distribution
was determined using a built-in cell cycle module (Watson pragmatic
algorithm) by FlowJo 7.6.1 software. Reactive Oxygen Species Assay Dihydrorhodamine 123
oxidation was used as an indicator of intracellular ROS production.
For this purpose, SW620 cells were seeded in 6-well plates at a density
of 10 5 cells per well. The cells were left overnight (time
needed for them to attach to the surface). Then, 1 μM tested
compounds were added along with 1 μM DHR123. Additionally, since
DHR123 is a substrate of ABCB1 (which may interfere in this assay),
10 μM verapamil, an inhibitor of this protein, was added. The
cells were cultured for an additional 4 h at 37 °C, and then
the cells were harvested by trypsinization, resuspended in a complete
medium, and analyzed using a LSRII flow cytometer (Becton Dickinson)
in a FITC channel (530/30 nm). The results are presented as a percentage
of control (median fluorescence in the presence of DMSO). Kinesin ATPase Inhibition Assay The potential kinesin
modulatory activity of tested compounds was performed using a Kinesin
ATPase end-point biochem kit (Cytoskeleton, Inc.). Compounds were
dissolved in DMSO (the final concentration did not exceed 0.1%). The
experiment was performed according to the manufacturer’s instructions.
One μg of tested kinesin (KSP) was used per reaction. Phosphate
release was measured at the absorbance 650 nm using an EnVision multilabel
plate reader (PerkinElmer).
## Materials and Methods
Materials and Methods All of the reactions were carried
out under an argon atmosphere. All commercially available chemicals
and solvents were of analytical grade and used without further purification.
OsO 4 , RhCl 3 ·xH 2 O, and IrCl 3 ·xH 2 O were purchased from Precious Metals
Online and Sigma-Aldrich. Bis[dichlorido(η 6 - p -cymene)ruthenium(II)] was purchased from Sigma-Aldrich.
Bis[dichlorido(η 6 - p -cymene)osmium(II)], 52 bis[dichlorido(η 5 -pentamethylcyclopentadienyl)rhodium(III)],
and bis[dichlorido(η 5 -pentamethylcyclopentadienyl)iridium(III)] 53 were synthesized as described previously. ( R )- 1 and ( S )- 1 were synthesized according to a reported procedure. 54 1 H and 13 C{ 1 H} and 1 H– 13 C HSQC NMR spectra were recorded at
294 K on a Bruker Avance III 600 MHz spectrometer at 600.3 MHz for 1 H and at 150.1 MHz for 13 C{ 1 H}. The 1 H and 13 C{ 1 H} chemical shifts were calibrated
based on the residual 1 H and 13 C{ 1 H} solvent peaks, i.e., δ = 3.58 ppm for 1 H and
67.2 ppm for 13 C in THF- d 8 ,
δ = 2.50 ppm for 1 H and 39.5 ppm for 13 C in dmso- d6 and δ = 5.32 ppm for 1 H and 53.8 ppm for 13 C in CD 2 Cl 2 . The UV–vis spectra were recorded at 294 K on a PerkinElmer
Lambda 45 spectrometer. Elemental analyses were performed at the Faculty
of Chemistry, University of Lodz, Poland. The HPLC-MS analysis was
performed using a Shimadzu Nexera XR system equipped with an SPD-M40
and an LCMS-2020 detector on a Phenomenex XB-C18 column (50 ×
4.6 mm, 2.1 mm, 1.7 μm) using a mixture of 55% water with 0.01%
HCOOH (eluent A), 22.5% methanol with 0.01% HCOOH (eluent B), and
22.5% acetonitrile with 0.01% HCOOH (eluent C) with a flow rate of
0.4 mL·min –1 .
## General Procedure
General Procedure To a solution of ( R )- 1 or ( S )- 1 (1 equiv)
in anhydrous ethanol (12 mL), pyridine-2-carbaldehyde (2 equiv) was
added and the resulting solution was refluxed under argon conditions
for 1 h. Next, the [LMCl 2 ] 2 dimer (M = Rh/Ir,
L = Cp* (Cp* = η 5 -1,2,3,4,5-pentamethylcyclopentadienyl)
or M = Ru/Os, L = cym (cym = η 6 - p -cymene)) (0.49 equiv) was added, the mixture was cooled down to
RT, and stirring was continued for an additional 3 h. The solvent
was evaporated to c.a. 2 mL and methanol (3 mL) and water (10 mL)
were added, followed by a saturated solution of KPF 6 (5
mL). The precipitant was filtrated off, washed with water (3 ×
10 mL), and dried. The products were purified by crystallization from
the methanol/diethyl ether mixture. 3a [(cym)Ru(( R )- 2 )Cl]PF 6 Compound 3a was synthesized
in 69% yield (217 mg) according to the general procedure starting
from 130 mg (0.38 mmol) of ( R )- 1 , 79
mg (70 μL, 0.74 mmol) of pyridine-2-carbaldehyde, and 114 mg
(0.19 mmol) of [(cym)RuCl 2 ] 2 . Elemental analysis
calculated for C 35 H 37 Cl 2 F 6 N 4 OPRu (846.64 g/mol) C 49.65, H 4.41, N 6.62; found C
49.37, H 4.61, N 6.85. HPLC-MS τ 1 = 3.50 min calculated
for C 35 H 37 Cl 2 N 4 ORu + [M-PF 6 ] + m / z = 701.1; found m / z =
701.4, τ 2 = 5.22 min calculated for C 35 H 37 Cl 2 N 4 ORu + [M-PF 6 ] + m / z = 701.1;
found m / z = 701.0. 1 H
NMR (600 MHz, CD 2 Cl 2 ) δ 9.29 (d, J = 5.4 Hz, 1H, CH Ar ), 9.27 (d, J = 5.5 Hz, 0.2H, CH Ar ), 9.21 (s, 0.2H, CH Ar ), 8.45 (s, 1H, CH imine ), 8.33 (d, J =
8.5 Hz, 1H, CH Ar ), 8.24 (d, J = 7.0 Hz,
1H, CH Ar ), 8.20–8.18 (m, 1H, CH Ar ), 8.15–8.11
(m, 0.4H, CH Ar ), 7.92 (d, J = 1.9 Hz,
1H, CH Ar ), 7.80–7.78 (m, 1H, CH Ar ), 7.75–7.72
(m, 0.2H, CH Ar ), 7.63 (d, J = 1.9 Hz,
0.2H, CH Ar ), 7.61 (dd, J = 8.5, 2.0 Hz,
1H, CH Ar ), 7.48–7.44 (m, 0.6H, CH Ar ),
7.38 (t, J = 7.4 Hz, 2H, CH Ar ), 7.32 (t, J = 7.1 Hz, 2H, CH Ar ), 7.09 (d, J = 7.5 Hz, 2H, CH Ar ), 6.09 (d, J = 16.7
Hz, 0.2H, C H 2 Ph), 5.96 (d, J = 6.7 Hz, 0.2H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.92 (br s, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.87 (d, J = 6.0 Hz, 0.2H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.80 (d, J = 17.3 Hz, 1H, C H 2 Ph), 5.71 (br s, 3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.66 (d, J = 6.2 Hz, 0.4H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.48 (d, J = 5.9 Hz, 0.2H, NC H –CH(CH 3 ) 2 ), 5.41 (d, J = 10.3 Hz, 0.2H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.23 (d, J = 16.6 Hz, 0.2H, C H 2 Ph ) , 4.58 (d, J = 10.0 Hz, 1H, H-1′), 4.35 (d, J = 17.3 Hz, 1H, C H 2 Ph), 3.23–3.17 (m, 1H, H-2′), 2.98–2.93
(m, 0.2H, C H (CH 3 ) 2 ),
2.60–2.56 (m, 0.2H, C H (CH 3 ) 2 ), 2.45–2.40 (m, 1H, 4-CH 3 C 6 H 4 C H (CH 3 ) 2 ), 2.38 (s, 0.6H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ),
1.84 (s, 3H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ),
1.21 (d, J = 6.7 Hz, 0.6H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 1.07 (d, J = 6.9 Hz, 0.6H,
4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 1.03–0.99
(m, 7H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 superimposed with
H-3′), 0.89 (d, J = 6.3 Hz, 3H, H-3′),
0.86 (d, J = 7.0 Hz, 3H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.55 (d, J = 6.5 Hz, 0.6H,
4-CH 3 C 6 H 4 CH(C H 3 ) 2 ). 13 C{ 1 H} NMR (151 MHz, CD 2 Cl 2 ) δ 170.7
( C H imine ), 161.5 (C IV ), 156.0 ( C H Ar ), 153.3 (C IV ), 153.0 (C IV ), 147.4 (C IV ), 141.4 (C IV ), 140.3 ( C H Ar ), 136.2 (C IV ), 131.2 ( C H Ar ), 130.3 ( C H Ar ), 129.9 ( C H Ar ), 129.8 ( C H Ar ), 129.2
( C H Ar ), 128.6 ( C H Ar ), 127.1 ( C H Ar ),
127.0 ( C H Ar ), 126.3 ( C H Ar ), 120.2 (C IV ), 46.6 ( C H 2 ), 32.0 (4-CH 3 C 6 H 4 C H(CH 3 ) 2 ), 31.2
(C-2′), 22.9 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 21.9 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 20.4 (C-3′), 19.8 (C-3′), 19.2 (4- C H 3 C 6 H 4 CH(CH 3 ) 2 ). 3b [(cym)Os(( R )- 2 )Cl]PF 6 Compound 3b was synthesized
in 37% yield (128 mg) according to the general procedure starting
from 130 mg (0.38 mmol) of ( R )- 1 , 79
mg (70 μL, 0.74 mmol) of pyridine-2-carbaldehyde, and 148 mg
(0.19 mmol) of [(cym)OsCl 2 ] 2 . Elemental analysis
calculated for C 35 H 37 Cl 2 F 6 N 4 OOsP (935.80 g/mol) C 44.92, H 3.99, N 5.99; found C
44.68, H 3.96, N 6.13. HPLC-MS τ 1 = 4.23 min calculated
for C 35 H 37 Cl 2 N 4 OOs + [M-PF 6 ] + m / z = 791.2; found m / z =
791.3, τ 2 = 7.06 min calculated for C 35 H 37 Cl 2 N 4 OOs + [M-PF 6 ] + m / z = 791.2;
found m / z = 791.2. 1 H
NMR (600 MHz, CD 2 Cl 2 ) δ 9.62 (s, 0.2H,
C H imine ), 9.20 (d, J = 5.5 Hz, 1H, C H Ar ), 8.92 (s,
1H, C H imine ), 8.40 (d, J = 7.4 Hz, 1H, C H Ar ), 8.32 (d, J = 8.5 Hz, 1H, C H Ar ),
8.28 (d, J = 7.8 Hz, 0.2H, C H Ar ), 8.19 (d, J = 8.6 Hz, 0.3H, C H Ar ), 8.16–8.14 (m, 1H, C H Ar ), 8.10–8.09 (m, 0.3H, C H Ar ), 7.90 (d, J = 1.9 Hz,
1H, C H Ar ), 7.74–7.71 (m,
1H, C H Ar ), 7.68–7.66 (m,
0.6H, C H Ar ), 7.61 (dd, J = 8.4, 2.0 Hz, 1H, C H Ar ), 7.47–7.45
(m, 1H, C H Ar ), 7.40 (t, J = 7.5 Hz, 2H, C H Ar ),
7.34 (t, J = 7.4 Hz, 1H, C H Ar ), 7.30–7.27 (m, 1H, C H Ar ), 7.13 (d, J = 7.5 Hz, 2H, C H Ar ), 6.24 (d, J = 5.9 Hz,
0.3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 6.20 (d, J = 5.6
Hz, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 6.06 (d, J = 16.6 Hz, 0.4H, C H 2 Ph), 5.96 (br s, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.93–5.85
(m, 2H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.81 (d, J = 17.0 Hz, 1H, C H 2 Ph), 5.70 (d, J = 5.5 Hz, 0.3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.41 (d, J = 10.3 Hz, 0.2H, H-1′),
5.14 (d, J = 16.5 Hz, 0.3H, C H 2 Ph), 4.80 (d, J = 9.9 Hz, 1H, H-1′), 4.52 (d, J = 13.6 Hz,
1H, C H 2 Ph),
3.16–3.10 (m, 1H, H-2′), 2.99–2.95 (m, 0.25H,
H-2′), 2.91 (s, 0.1H), 2.82 (s, 0.1H), 2.50–2.46 (m,
0.2H), 2.43 (s, 0.7H, 4- CH 3 C 6 H 4 CH(CH 3 ) 2 ), 2.34–2.27
(m, 1H, 4-CH 3 C 6 H 4 C H (CH 3 ) 2 ), 1.90 (s, 3H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ), 1.18 (d, J = 6.6 Hz, 1H,
H-3′), 1.08 (d, J = 6.9 Hz, 0.8H,), 1.00 (d, J = 6.9 Hz, 3H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.97 (d, J = 6.8 Hz, 3H, H-3′),
0.89 (d, J = 6.1 Hz, 3H, H-3′), 0.78 (d, J = 6.9 Hz, 3H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.53 (d, J = 6.5 Hz, 0.7H, H-3′). 13 C{ 1 H} NMR (151 MHz, CD 2 Cl 2 ) δ 172.8 ( C H imine ), 161.5
(C IV ), 155.5 (CH Ar ), 154.7 (C IV ),
152.8 (C IV ), 147.3 (C IV ), 141.5 (C IV ), 140.3 (CH Ar ), 136.1 (C IV ), 131.1 (CH Ar ), 131.1 (C IV ), 129.9 ( C H Ar ), 129.8 ( C H Ar ),
129.3 ( C H Ar ), 128.7 ( C H Ar ), 127.3 ( C H Ar ), 127.1 ( C H Ar ), 126.4
( C H Ar ), 120.2 (C IV ),
83.6 (N C H–CH(CH 3 ) 2 ), 80.8 (4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 76.9 (4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 73.9 (4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 46.9 ( C H 2 Ph), 32.2 (4-CH 3 C 6 H 4 C H(CH 3 ) 2 ), 31.4 (NCH- C H(CH 3 ) 2 ) 23.5 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 21.9 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 20.5 (NCH–CH( C H 3 ) 2 ), 19.8 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 19.1 (4- C H 3 C 6 H 4 CH(CH 3 ) 2 ). 3c [(Cp*)Rh(( R )- 2 )Cl]PF 6 Compound 3c was synthesized
in 69% yield (536 mg) according to the general procedure starting
from 313 mg (0.91 mmol) of ( R )- 1 , 195
mg (174 μL, 1.82 mmol) of pyridine-2-carbaldehyde, and 277 mg
(0.45 mmol) of [Cp*RhCl 2 ] 2 . Elemental analysis
calculated for C 35 H 38 Cl 2 F 6 N 4 OPRh (849.49 g/mol) C 49.49, H 4.51, N 6.60; found C
49.49, H 4.50, N 6.60. HPLC-MS τ 1 = 2.67 min calculated
for C 35 H 38 Cl 2 N 4 ORh + [M-PF 6 ] + m / z = 703.1; found m / z =
703.5, τ 2 = 4.29 min calculated for C 35 H 38 Cl 2 N 4 ORh + [M-PF 6 ] + m / z = 703.1;
found m / z = 703.5. 1 H
NMR (600 MHz, THF- d 8 ) δ 10.76 (s,
0.1H, CH), 9.27 (s, 1H, C H imine ),
8.89 (d, J = 5.4 Hz, 1H, C H Ar ), 8.86 (d, J = 5.5 Hz, 0.1H, C H Ar ), 8.66 (s, 0.1H, CH Ar ), 8.24
(d, J = 7.4 Hz, 1H, C H Ar ), 8.19 (t, J = 7.8 Hz, 1H, C H Ar ), 8.15 (d, J = 8.5 Hz,
1H, C H Ar ), 7.87 (d, J = 1.8 Hz, 0.2H, C H Ar ), 7.84 (t, J = 6.4 Hz, 1H, C H Ar ),
7.73 (d, J = 1.7 Hz, 1H, C H Ar ), 7.56 (dd, J = 8.6, 2.0 Hz, 0.1H,
C H Ar ), 7.47 (dd, J = 8.6, 2.0 Hz, 1H, C H Ar ), 7.39
(t, J = 7.5 Hz, 2H, C H Ar ), 7.32–7.28 (m, 3.5H, C H Ar ), 7.23 (d, J = 7.2 Hz, 0.1H, C H Ar ), 7.20 (d, J = 7.9 Hz,
0.3H, C H Ar ), 5.91 (d, J = 16.9 Hz, 1H, C H 2 Ph), 5.80 (d, J = 17.4 Hz, 0.1H, C H 2 Ph), 5.33 (d, J = 17.1 Hz, 1H, C H 2 Ph), 5.13 (d, J = 8.7 Hz, 1H,
H-1′), 3.08–3.02 (m, 1H, H-2′), 2.39 (s, 0.4H),
1.73 (s, 15H, Cp*-C H 3 ), 1.63 (s, 2H, Cp*-C H 3 ), 1.13 (d, J = 6.7 Hz, 3H, H-3′),
0.95 (d, J = 6.6 Hz, 3H, H-3′). 13 C{ 1 H} NMR (151 MHz, THF- d 8 ) δ 170.4 (C H imine ), 161.5
(C IV ), 157.0 (C IV ), 154.7 (C IV ),
153.6 ( C H Ar ), 148.2 (C IV ), 140.8 ( C H Ar ), 140.7 (C IV ), 137.0 (C IV ), 131.1 ( C H Ar ), 130.7 ( C H Ar ), 129.6 ( C H Ar ), 129.5 ( C H Ar ), 129.3 ( C H Ar ), 128.3
( C H Ar ), 128.3 ( C H Ar ), 127.6 ( C H Ar ),
127.1 ( C H Ar ), 120.4 (C IV ), 98.6 (d, J C–Rh = 7.7 Hz, Cp*),
73.2 (C-1′), 48.2 ( C H 2 Ph),
35.1 (C-2′), 19.9 (C-3′), 18.8 (C-3′), 9.1 (Cp*- C H 3 ). 3d [(Cp*)Ir(( R )- 2 )Cl]PF 6 Compound 3d was synthesized
in 73% yield (593 mg) according to the general procedure starting
from 296 mg (0.87 mmol) of ( R )- 1 , 185
mg (165 μL, 1.73 mmol) of pyridine-2-carbaldehyde, and 338 mg
(0.42 mmol) of [Cp*IrCl 2 ] 2 . Elemental analysis
calculated for C 35 H 38 Cl 2 F 6 IrN 4 OP (938.80 g/mol) C 44.78, H 4.08, N 5.97; found C
44.83, H 4.20, N 5.97. HPLC-MS τ 1 = 3.65 min calculated
for C 35 H 38 Cl 2 N 4 OIr + [M-PF 6 ] + m / z = 793.2; found m / z =
793.4, τ 2 = 7.45 min calculated for C 35 H 38 Cl 2 N 4 OIr + [M-PF 6 ] + m / z = 793.2;
found m / z = 793.6. 1 H
NMR (600 MHz, THF- d 8 ) δ 9.83 (s,
1H, C H imine ), 9.14 (s, 0.4H), 8.86
(d, J = 5.4 Hz, 1H, C H Ar ), 8.84 (d, J = 5.5 Hz, 0.4H, C H Ar ), 8.42 (d, J = 7.6 Hz,
1H, C H Ar ), 8.22 (d, J = 8.5 Hz, 0.6H, C H Ar ), 8.17 (t, J = 7.6 Hz, 1H, C H Ar ),
8.14 (d, J = 8.5 Hz, 1H, C H Ar ), 7.87 (d, J = 2.0 Hz, 0.4H, C H Ar ), 7.84–7.80 (m, 1.5H, C H Ar ), 7.79 (d, J = 2.0 Hz,
1H, C H Ar ), 7.57 (dd, J = 8.6, 2.0 Hz, 0.5H, C H Ar ), 7.47
(dd, J = 8.5, 2.0 Hz, 1H, C H Ar ), 7.38 (t, J = 7.5 Hz, 2H, C H Ar ), 7.32–7.26 (m, 4H, C H Ar ), 7.26–7.22 (m, 1H, C H Ar ), 7.19 (d, J = 7.5 Hz,
1H, C H Ar ), 5.87 (d, J = 17.1 Hz, 1H, C H 2 Ph), 5.78 (d, J = 17.2 Hz, 0.4H), 5.24 (d, J = 17.1 Hz, 1H, C H 2 Ph), 5.15 (d, J = 9.3 Hz, 1H,
H-1′), 3.20–3.13 (m, 1H, H-2′), 2.95–2.89
(m, 0.4H, H-2′), 1.69 (s, 15H, Cp*-C H 3 ), 1.60 (s, 7H, Cp*-C H 3 ), 1.16 (d, J = 6.7 Hz, 3H, H-3′), 0.92 (d, J = 6.6 Hz, 3H, H-3′). 13 C{ 1 H} NMR (151
MHz, THF- d 8 ) δ 172.0 ( C H imine ), 161.3 (C IV ), 157.0 (C IV ), 156.2 (C IV ), 153.1 ( C H Ar ), 148.2 (C IV ), 141.0 ( C H Ar ), 140.8 ( C H Ar ),
136.7 (C IV ), 131.3 ( C H Ar ), 131.2 ( C H Ar ), 129.6 ( C H Ar ), 129.4 ( C H Ar ), 129.3 ( C H Ar ), 128.4
( C H Ar ), 128.3 ( C H Ar ), 128.2 ( C H Ar ),
127.7 ( C H Ar ), 127.0 ( C H Ar ), 120.3 (C IV ), 91.3 (Cp*),
74.8 (C-1′), 48.1 ( C H 2 Ph),
35.9 (C-2′), 19.9 (C-3′), 19.2 (C-3′), 8.80 (Cp*- C H 3 ). 4a [(cym)Ru(( S )- 2 )Cl]PF 6 Compound 4a was synthesized
in 68% yield (215 mg) according to the general procedure starting
from 130 mg (0.38 mmol) of ( S )- 1 , 79
mg (70 μL, 0.74 mmol) of pyridine-2-carbaldehyde, and 114 mg
(0.19 mmol) of [(cym)RuCl 2 ] 2 . Elemental analysis
calculated for C 35 H 37 Cl 2 F 6 N 4 OPRu (846.64 g/mol) C 49.65, H 4.41, N 6.62; found C
49.41, H 4.59, N 6.80. HPLC-MS τ 1 = 3.39 min calculated
for C 35 H 37 Cl 2 N 4 ORu + [M-PF 6 ] + m / z = 701.1; found m / z =
701.3, τ 2 = 5.07 min calculated for C 35 H 37 Cl 2 N 4 ORu + [M-PF 6 ] + m / z = 701.1;
found m / z = 700.8. 1 H
NMR (600 MHz, CD 2 Cl 2 ) δ 9.29 (d, J = 5.4 Hz, 1H, C H Ar ),
9.27 (d, J = 5.6 Hz, 0.2H, C H Ar ), 9.21 (s, 0.2H, C H imine ), 8.45 (s, 1H, C H imine ), 8.33
(d, J = 8.5 Hz, 1H C H Ar ), 8.24 (d, J = 7.1 Hz, 1H, C H Ar ), 8.19 (t, J = 3.8 Hz,
1H, C H Ar ), 8.15–8.11 (m,
0.4H, C H Ar ), 7.92 (d, J = 1.9 Hz, 1H, C H Ar ), 7.80–7.78
(m, 1H, C H Ar ), 7.74–7.72
(m, 0.2H, C H Ar ), 7.63 (d, J = 1.8 Hz, 0.2H, C H Ar ), 7.61 (dd, J = 8.6, 2.0 Hz, 1H, C H Ar ), 7.48–7.44 (m, 0.7H, C H Ar ), 7.41 (d, J = 7.4 Hz, 0.2H, C H Ar ), 7.38 (t, J = 7.4 Hz,
2H, C H Ar ), 7.32 (t, J = 6.8 Hz, 2H, C H Ar ), 7.09 (d, J = 7.5 Hz, 2H, C H Ar ),
6.09 (d, J = 16.9 Hz, 0.2H, C H 2 Ph), 5.96 (d, J = 6.5 Hz, 0.3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.92 (br s, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.87 (d, J = 6.0 Hz, 0.3H,
4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.80 (d, J = 16.8 Hz, 1H,
C H 2 Ph), 5.71
(br s, 3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.66 (d, J = 6.4 Hz, 0.3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.48 (d, J = 5.9 Hz, 0.2H, H-1′), 5.41 (d, J = 10.1
Hz, 0.2H, H-1′), 5.23 (d, J = 16.5 Hz, 0.2H,
C H 2 Ph), 4.58
(d, J = 9.2 Hz, 1H, H-1′), 4.35 (d, J = 14.5 Hz, 1H, C H 2 Ph), 3.23–3.17 (m, 1H, H-2′), 2.98–2.94
(m, 0.2H, H-2′), 2.60–2.55 (m, 0.2H, 4-CH 3 C 6 H 4 C H (CH 3 ) 2 ), 2.45–2.40 (m, 1H, 4-CH 3 C 6 H 4 C H (CH 3 ) 2 ), 2.38 (s, 0.6H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ),
1.84 (s, 3H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ),
1.21 (d, J = 6.6 Hz, 0.8H, H-3′), 1.07 (d, J = 6.9 Hz, 0.7H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 1.03–0.99 (m, 7H, H-3′, superimposed with
4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.89 (d, J = 6.3 Hz, 3H, H-3′), 0.86 (d, J = 7.0 Hz, 3H 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.55
(d, J = 6.5 Hz, 0.7H, H-3′). 13 C{ 1 H} NMR (151 MHz, CD 2 Cl 2 ) δ
170.7 ( C H imine ), 161.5 (C IV ), 156.0 ( C H Ar ), 153.3 (C IV ), 153.0 (C IV ), 147.4 (C IV ), 141.4 (C IV ), 140.3 ( C H Ar ), 136.2 (C IV ), 131.2 ( C H Ar ), 130.3 ( C H Ar ), 129.9 ( C H Ar ), 129.8 ( C H Ar ), 129.2
( C H Ar ), 128.5 ( C H Ar ), 127.1 ( C H Ar ),
127.0 ( C H Ar ), 126.3 ( C H Ar ), 120.2 (C IV ), 82.9 (C-1′),
79.5 (4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 47.9 ( C H 2 Ph), 46.6 ( C H 2 Ph), 32.0 (4-CH 3 C 6 H 4 C H(CH 3 ) 2 ), 31.3 (C-2′),
22.9 (C–H), 22.7 (C–H), 22.6 (C–H), 22.3 (C–H),
21.9 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 20.4 (C-3′), 19.8 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 19.2 (4- C H 3 C 6 H 4 CH(CH 3 ) 2 ). 4b [(cym)Os(( S )- 2 )Cl]PF 6 Compound 4b was synthesized
in 41% yield (144 mg) according to the general procedure starting
from 130 mg (0.38 mmol) of ( S )- 1 , 79
mg (70 μL, 0.74 mmol) of pyridine-2-carbaldehyde, and 149 mg
(0.19 mmol) of [(cym)OsCl 2 ] 2 . Elemental analysis
calculated for C 35 H 37 Cl 2 F 6 N 4 OOsP (935.80 g/mol) C 44.92, H 3.99, N 5.99; found C
44.75, H 4.09, N 5.78. HPLC-MS τ 1 = 4.24 min calculated
for C 35 H 37 Cl 2 N 4 OOs + [M-PF 6 ] + m / z = 791.2; found m / z =
791.4, τ 2 = 7.04 min calculated for C 35 H 37 Cl 2 N 4 OOs + [M-PF 6 ] + m / z = 791.2;
found m / z = 791.6. 1 H
NMR (600 MHz, CD 2 Cl 2 ) δ 9.62 (s, 0.25H,
C H Ar ), 9.20 (d, J = 5.5 Hz, 1H, C H Ar ), 8.91 (s,
1H, C H imine ), 8.40 (d, J = 7.5 Hz, 1H, C H Ar ), 8.31 (d, J = 8.5 Hz, 1H, C H Ar ),
8.27 (d, J = 7.8 Hz, 0.25H, C H Ar ), 8.19 (d, J = 8.5 Hz, 0.25H, C H Ar ), 8.17–8.14 (m, 1H, C H Ar ), 8.11–8.08 (m, 0.25H, C H Ar ), 7.90 (d, J = 1.9 Hz,
1H, C H Ar ), 7.74–7.72 (m,
1H, C H Ar ), 7.68–7.66 (m,
0.5H, C H Ar ), 7.61 (dd, J = 8.2, 2.0 Hz, 1H, C H Ar ), 7.47–7.45
(m, 0.75H, C H Ar ), 7.39 (t, J = 7.5 Hz, 2H, C H Ar ),
7.34 (t, J = 7.4 Hz, 1H, C H Ar ), 7.29 (d, J = 7.5 Hz, 0.5H, C H Ar ), 7.12 (d, J = 7.5 Hz,
2H, C H Ar ), 6.24 (d, J = 6.1 Hz, 0.4H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 6.19 (d, J = 5.5 Hz, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 6.06 (d, J = 16.5 Hz, 0.4H, C H 2 Ph), 5.96
(br s, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.92–5.84 (m, 2H,
4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.81 (d, J = 16.7 Hz, 1H,
C H 2 Ph), 5.69 (d, J = 5.5 Hz, 0.25H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.40 (d, J = 10.4 Hz, 0.25H, NC H –CH(CH 3 ) 2 ), 5.13 (d, J = 16.4 Hz, 0.25H, C H 2 Ph), 4.78 (d, J = 9.9 Hz,
1H, NC H –CH(CH 3 ) 2 ), 4.50 (d, J = 16.2 Hz, 1H, C H 2 Ph), 3.42 (s, 0.1H), 3.16–3.10 (m, 1H, NCH-C H (CH 3 ) 2 ), 3.00–2.95 (m,
0.25H, NCH-C H (CH 3 ) 2 ),
2.43 (s, 1H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ), 2.32–2.27 (m, 1H,
4-CH 3 C 6 H 4 C H (CH 3 ) 2 ), 1.90 (s, 3H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ), 1.35 (d, J = 6.9 Hz, 0.25H), 1.17 (d, J = 6.7 Hz, 1H, NCH–CH(C H 3 ) 2 ), 1.07 (d, J = 6.9 Hz, 1H), 1.00 (d, J = 6.9 Hz, 3H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.96 (d, J = 6.8 Hz, 3H, NCH–CH(C H 3 ) 2 ), 0.89 (d, J =
6.1 Hz, 3H, NCH–CH(C H 3 ) 2 ), 0.77 (d, J =
6.9 Hz, 3H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.51 (d, J = 6.6 Hz, 0.75H, NCH–CH(C H 3 ) 2 ). 13 C{ 1 H}
NMR (151 MHz, CD 2 Cl 2 ) δ 172.4 ( C H imine ), 161.1 (C IV ), 155.2 ( C H Ar ), 154.2 (C IV ), 152.3 (C IV ), 146.9 (C IV ), 141.1 (C IV ), 140.0
( C H Ar ), 135.7 (C IV ),
130.8 ( C H Ar ), 129.5 ( C H Ar ), 129.4 ( C H Ar ), 128.9 ( C H Ar ), 128.2
( C H Ar ), 126.7 ( C H Ar ), 126.0 ( C H Ar ),
119.7 (C IV ), 88.3 (4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 83.2 (C-1′),
46.5 ( C H 2 ), 31.8 (4-CH 3 C 6 H 4 C H(CH 3 ) 2 ), 30.9 (C-2′), 23.1 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 21.4 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 20.1 (C-3′), 19.3
(C-3′), 18.7 (4- C H 3 C 6 H 4 CH(CH 3 ) 2 ). 4c [(Cp*)Rh(( S )- 2 )Cl]PF 6 Compound 4c was synthesized
in 68% yield (535 mg) according to the general procedure starting
from 313 mg (0.91 mmol) of ( S )- 1 , 195
mg (174 μL, 1.82 mmol) of pyridine-2-carbaldehyde, and 276 mg
(0.45 mmol) of [Cp*RhCl 2 ] 2 . Elemental analysis
calculated for C 35 H 38 Cl 2 F 6 N 4 OPRh (849.49 g/mol) C 49.49, H 4.51, N 6.60; found C
49.53, H 4.49, N 6.55. HPLC-MS τ 1 = 2.79 min calculated
for C 35 H 38 Cl 2 N 4 ORh + [M-PF 6 ] + m / z = 703.1; found m / z =
703.5, τ 2 = 4.20 min calculated for C 35 H 38 Cl 2 N 4 ORh + [M-PF 6 ] + m / z = 703.1;
found m / z = 703.5. 1 H
NMR (600 MHz, THF- d 8 ) δ 10.77 (s,
0.1H), 9.27 (s, 1H, C H imine ), 8.90
(d, J = 5.3 Hz, 1H, C H Ar ), 8.86 (d, J = 5.1 Hz, 0.1H, C H Ar ), 8.66 (s, 0.1H), 8.24 (d, J = 7.1 Hz, 1H, C H Ar ), 8.19 (t, J = 7.7 Hz, 1H, C H Ar ),
8.15 (d, J = 8.5 Hz, 1H, C H Ar ), 7.87 (d, J = 1.7 Hz, 0.2H, C H Ar ), 7.84 (t, J = 6.0 Hz,
1H, C H Ar ), 7.73 (d, J = 1.8 Hz, 1H, C H Ar ), 7.56 (dd, J = 8.5, 2.0 Hz, 0.1H, C H Ar ), 7.47 (dd, J = 8.5, 2.0 Hz, 1H, C H Ar ), 7.39 (t, J = 7.4 Hz, 2H, CH Ar ), 7.32–7.28 (m, 3.5H, CH Ar ), 7.23 (d, J = 7.4 Hz, 0.1H, C H Ar ), 7.20 (d, J = 7.7 Hz, 0.3H, C H Ar ) 5.91 (d, J = 16.7 Hz, 1H, C H 2 Ph), 5.80 (d, J = 17.2
Hz, 0.1H, C H 2 Ph), 5.33 (d, J = 17.1 Hz, 1H, C H 2 Ph), 5.13 (d, J = 8.7 Hz, 1H, H-1′),
3.08–3.02 (m, 1H, H-2′), 2.39 (s, 0.5H), 1.73 (s, 15H,
Cp*-C H 3 ),
1.63 (s, 2H, Cp*-C H 3 ), 1.13–1.10 (m, 3H, H-3′ superimposed with the
diethyl ether signal), 0.95 (d, J = 6.6 Hz, 3H, H-3′). 13 C{ 1 H} NMR (151 MHz, THF- d 8 ) δ 170.4 ( C H imine ), 161.5 (C IV ), 154.7 (C IV ), 153.6 ( C H Ar ), 148.2 (C IV ), 140.8 ( C H Ar ), 140.7 (C IV ), 137.0 (C IV ), 131.1 ( C H Ar ), 130.6
( C H Ar ), 129.6 ( C H Ar ), 129.5 ( C H Ar ),
129.3 ( C H Ar ), 128.3 ( C H Ar ), 128.3 ( C H Ar ), 127.6 ( C H Ar ), 127.1
( C H Ar ), 120.4 (C IV ),
98.6 (d, J Rh–C = 7.8 Hz, Cp*), 73.2 (C-1′),
48.2 ( C H 2 Ph), 35.1 (C-2′)
19.9 (C-3′), 18.8 (C-3′), 9.1 (Cp*- C H 3 ). 4d [(Cp*)Ir(( S )- 2 )Cl]PF 6 Compound 4d was synthesized
in 72% yield (413 mg) according to the general procedure starting
from 208 mg (0.61 mmol) of ( S )- 1 , 131
mg (115 μL, 1.22 mmol) of pyridine-2-carbaldehyde, and 237 mg
(0.30 mmol) of [Cp*IrCl 2 ] 2 . Elemental analysis
calculated for C 35 H 38 Cl 2 F 6 IrN 4 OP (938.80 g/mol) C 44.78, H 4.08, N 5.97; found C
44.69, H 4.05, N 5.86. HPLC-MS τ 1 = 3.81 min calculated
for C 35 H 38 Cl 2 N 4 OIr + [M-PF 6 ] + m / z = 793.2; found m / z =
793.6, τ 2 = 7.53 min calculated for C 35 H 38 Cl 2 N 4 OIr + [M-PF 6 ] + m / z = 793.2;
found m / z = 793.5. 1 H
NMR (600 MHz, THF- d 8 ) δ 9.82 (s,
0.7H, CH imine ) 9.14 (s, 1H, CH imine ), 8.87 (d, J = 5.4 Hz, 0.7H, C H Ar ), 8.84 (d, J = 5.4 Hz, 1H, C H Ar ), 8.41 (d, J = 7.7 Hz, 0.7H, C H Ar ), 8.22 (d, J = 8.5 Hz,
1.3H, C H Ar ), 8.18–8.16 (m,
1.4H, C H Ar ), 8.14 (d, J = 8.5 Hz, 1H, C H Ar ), 7.87 (d, J = 1.9 Hz, 1H, C H Ar ),
7.83–7.80 (m, 1.7H, C H Ar ),
7.79 (d, J = 1.9 Hz, 0.7H, C H Ar ), 7.57 (dd, J = 8.6, 1.9 Hz, 1H, C H Ar ), 7.47 (dd, J = 8.5,
2.0 Hz, 0.7H, C H Ar ), 7.38 (t, J = 7.5 Hz, 1.5H, C H Ar ), 7.33–7.28 (m, 4H, C H Ar ), 7.24 (t, J = 7.3 Hz, 1H, C H Ar ), 7.19 (d, J = 7.5 Hz, 2H, C H Ar ), 5.87 (d, J = 17.0 Hz,
0.7H, C H 2 Ph), 5.78 (d, J = 17.1 Hz, 1H, C H 2 Ph), 5.24 (d, J = 17.1 Hz, 0.8H, C H 2 Ph), 5.15 (d, J = 9.3 Hz, 1H, H-1′),
4.74 (br s, 0.7H, C H 2 Ph), 3.19–3.13 (m, 0.7H, H-2′), 2.95–2.87
(m, 1H, H-2′), 1.70 (s, 11.5H, Cp*-C H 3 ), 1.61 (s, 15H, Cp*-CH 3 ), 1.19 (br s, 3H, H-3′) superimposed with 1.16 (d, J = 6.7 Hz, 3H, H-3′), 0.93 (d, J = 6.6 Hz, 2.3H, H-3′). 13 C{ 1 H} NMR
(151 MHz, THF- d 8 ) δ 172.0 ( C H imine ), 161.4 (C IV ), 161.3 (C IV ), 156.9 (C IV ), 156.2 (C IV ), 153.1
( C H Ar ), 152.7 ( C H Ar ), 148.2 (C IV ), 147.9 (C IV ),
141.1 ( C H Ar ), 140.8 ( C H Ar ), 140.6 (C IV ), 136.7 (C IV ), 131.3 ( C H Ar ), 131.2
( C H Ar ), 129.6 ( C H Ar ), 129.4 ( C H Ar ),
129.3 ( C H Ar ), 128.7 ( C H Ar ), 128.4 ( C H Ar ), 128.3 ( C H Ar ), 128.2
( C H Ar ), 127.7 ( C H Ar ), 127.0 ( C H Ar ),
126.7 ( C H Ar ), 121.1 (C IV ), 120.4 (C IV ), 91.4 (Cp*), 91.3 (Cp*), 74.8 (C-1′),
48.1 ( C H 2 Ph), 35.9 (C-2′),
20.2 (C-3′), 19.9 (C-3′), 19.2 (C-3′), 19.1 (C-3′),
8.8 (Cp*- C H 3 ), 8.8 (Cp*- C H 3 ).
3a [(cym)Ru(( R )- 2 )Cl]PF 6 Compound 3a was synthesized
in 69% yield (217 mg) according to the general procedure starting
from 130 mg (0.38 mmol) of ( R )- 1 , 79
mg (70 μL, 0.74 mmol) of pyridine-2-carbaldehyde, and 114 mg
(0.19 mmol) of [(cym)RuCl 2 ] 2 . Elemental analysis
calculated for C 35 H 37 Cl 2 F 6 N 4 OPRu (846.64 g/mol) C 49.65, H 4.41, N 6.62; found C
49.37, H 4.61, N 6.85. HPLC-MS τ 1 = 3.50 min calculated
for C 35 H 37 Cl 2 N 4 ORu + [M-PF 6 ] + m / z = 701.1; found m / z =
701.4, τ 2 = 5.22 min calculated for C 35 H 37 Cl 2 N 4 ORu + [M-PF 6 ] + m / z = 701.1;
found m / z = 701.0. 1 H
NMR (600 MHz, CD 2 Cl 2 ) δ 9.29 (d, J = 5.4 Hz, 1H, CH Ar ), 9.27 (d, J = 5.5 Hz, 0.2H, CH Ar ), 9.21 (s, 0.2H, CH Ar ), 8.45 (s, 1H, CH imine ), 8.33 (d, J =
8.5 Hz, 1H, CH Ar ), 8.24 (d, J = 7.0 Hz,
1H, CH Ar ), 8.20–8.18 (m, 1H, CH Ar ), 8.15–8.11
(m, 0.4H, CH Ar ), 7.92 (d, J = 1.9 Hz,
1H, CH Ar ), 7.80–7.78 (m, 1H, CH Ar ), 7.75–7.72
(m, 0.2H, CH Ar ), 7.63 (d, J = 1.9 Hz,
0.2H, CH Ar ), 7.61 (dd, J = 8.5, 2.0 Hz,
1H, CH Ar ), 7.48–7.44 (m, 0.6H, CH Ar ),
7.38 (t, J = 7.4 Hz, 2H, CH Ar ), 7.32 (t, J = 7.1 Hz, 2H, CH Ar ), 7.09 (d, J = 7.5 Hz, 2H, CH Ar ), 6.09 (d, J = 16.7
Hz, 0.2H, C H 2 Ph), 5.96 (d, J = 6.7 Hz, 0.2H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.92 (br s, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.87 (d, J = 6.0 Hz, 0.2H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.80 (d, J = 17.3 Hz, 1H, C H 2 Ph), 5.71 (br s, 3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.66 (d, J = 6.2 Hz, 0.4H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.48 (d, J = 5.9 Hz, 0.2H, NC H –CH(CH 3 ) 2 ), 5.41 (d, J = 10.3 Hz, 0.2H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.23 (d, J = 16.6 Hz, 0.2H, C H 2 Ph ) , 4.58 (d, J = 10.0 Hz, 1H, H-1′), 4.35 (d, J = 17.3 Hz, 1H, C H 2 Ph), 3.23–3.17 (m, 1H, H-2′), 2.98–2.93
(m, 0.2H, C H (CH 3 ) 2 ),
2.60–2.56 (m, 0.2H, C H (CH 3 ) 2 ), 2.45–2.40 (m, 1H, 4-CH 3 C 6 H 4 C H (CH 3 ) 2 ), 2.38 (s, 0.6H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ),
1.84 (s, 3H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ),
1.21 (d, J = 6.7 Hz, 0.6H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 1.07 (d, J = 6.9 Hz, 0.6H,
4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 1.03–0.99
(m, 7H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 superimposed with
H-3′), 0.89 (d, J = 6.3 Hz, 3H, H-3′),
0.86 (d, J = 7.0 Hz, 3H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.55 (d, J = 6.5 Hz, 0.6H,
4-CH 3 C 6 H 4 CH(C H 3 ) 2 ). 13 C{ 1 H} NMR (151 MHz, CD 2 Cl 2 ) δ 170.7
( C H imine ), 161.5 (C IV ), 156.0 ( C H Ar ), 153.3 (C IV ), 153.0 (C IV ), 147.4 (C IV ), 141.4 (C IV ), 140.3 ( C H Ar ), 136.2 (C IV ), 131.2 ( C H Ar ), 130.3 ( C H Ar ), 129.9 ( C H Ar ), 129.8 ( C H Ar ), 129.2
( C H Ar ), 128.6 ( C H Ar ), 127.1 ( C H Ar ),
127.0 ( C H Ar ), 126.3 ( C H Ar ), 120.2 (C IV ), 46.6 ( C H 2 ), 32.0 (4-CH 3 C 6 H 4 C H(CH 3 ) 2 ), 31.2
(C-2′), 22.9 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 21.9 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 20.4 (C-3′), 19.8 (C-3′), 19.2 (4- C H 3 C 6 H 4 CH(CH 3 ) 2 ).
3b [(cym)Os(( R )- 2 )Cl]PF 6 Compound 3b was synthesized
in 37% yield (128 mg) according to the general procedure starting
from 130 mg (0.38 mmol) of ( R )- 1 , 79
mg (70 μL, 0.74 mmol) of pyridine-2-carbaldehyde, and 148 mg
(0.19 mmol) of [(cym)OsCl 2 ] 2 . Elemental analysis
calculated for C 35 H 37 Cl 2 F 6 N 4 OOsP (935.80 g/mol) C 44.92, H 3.99, N 5.99; found C
44.68, H 3.96, N 6.13. HPLC-MS τ 1 = 4.23 min calculated
for C 35 H 37 Cl 2 N 4 OOs + [M-PF 6 ] + m / z = 791.2; found m / z =
791.3, τ 2 = 7.06 min calculated for C 35 H 37 Cl 2 N 4 OOs + [M-PF 6 ] + m / z = 791.2;
found m / z = 791.2. 1 H
NMR (600 MHz, CD 2 Cl 2 ) δ 9.62 (s, 0.2H,
C H imine ), 9.20 (d, J = 5.5 Hz, 1H, C H Ar ), 8.92 (s,
1H, C H imine ), 8.40 (d, J = 7.4 Hz, 1H, C H Ar ), 8.32 (d, J = 8.5 Hz, 1H, C H Ar ),
8.28 (d, J = 7.8 Hz, 0.2H, C H Ar ), 8.19 (d, J = 8.6 Hz, 0.3H, C H Ar ), 8.16–8.14 (m, 1H, C H Ar ), 8.10–8.09 (m, 0.3H, C H Ar ), 7.90 (d, J = 1.9 Hz,
1H, C H Ar ), 7.74–7.71 (m,
1H, C H Ar ), 7.68–7.66 (m,
0.6H, C H Ar ), 7.61 (dd, J = 8.4, 2.0 Hz, 1H, C H Ar ), 7.47–7.45
(m, 1H, C H Ar ), 7.40 (t, J = 7.5 Hz, 2H, C H Ar ),
7.34 (t, J = 7.4 Hz, 1H, C H Ar ), 7.30–7.27 (m, 1H, C H Ar ), 7.13 (d, J = 7.5 Hz, 2H, C H Ar ), 6.24 (d, J = 5.9 Hz,
0.3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 6.20 (d, J = 5.6
Hz, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 6.06 (d, J = 16.6 Hz, 0.4H, C H 2 Ph), 5.96 (br s, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.93–5.85
(m, 2H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.81 (d, J = 17.0 Hz, 1H, C H 2 Ph), 5.70 (d, J = 5.5 Hz, 0.3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.41 (d, J = 10.3 Hz, 0.2H, H-1′),
5.14 (d, J = 16.5 Hz, 0.3H, C H 2 Ph), 4.80 (d, J = 9.9 Hz, 1H, H-1′), 4.52 (d, J = 13.6 Hz,
1H, C H 2 Ph),
3.16–3.10 (m, 1H, H-2′), 2.99–2.95 (m, 0.25H,
H-2′), 2.91 (s, 0.1H), 2.82 (s, 0.1H), 2.50–2.46 (m,
0.2H), 2.43 (s, 0.7H, 4- CH 3 C 6 H 4 CH(CH 3 ) 2 ), 2.34–2.27
(m, 1H, 4-CH 3 C 6 H 4 C H (CH 3 ) 2 ), 1.90 (s, 3H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ), 1.18 (d, J = 6.6 Hz, 1H,
H-3′), 1.08 (d, J = 6.9 Hz, 0.8H,), 1.00 (d, J = 6.9 Hz, 3H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.97 (d, J = 6.8 Hz, 3H, H-3′),
0.89 (d, J = 6.1 Hz, 3H, H-3′), 0.78 (d, J = 6.9 Hz, 3H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.53 (d, J = 6.5 Hz, 0.7H, H-3′). 13 C{ 1 H} NMR (151 MHz, CD 2 Cl 2 ) δ 172.8 ( C H imine ), 161.5
(C IV ), 155.5 (CH Ar ), 154.7 (C IV ),
152.8 (C IV ), 147.3 (C IV ), 141.5 (C IV ), 140.3 (CH Ar ), 136.1 (C IV ), 131.1 (CH Ar ), 131.1 (C IV ), 129.9 ( C H Ar ), 129.8 ( C H Ar ),
129.3 ( C H Ar ), 128.7 ( C H Ar ), 127.3 ( C H Ar ), 127.1 ( C H Ar ), 126.4
( C H Ar ), 120.2 (C IV ),
83.6 (N C H–CH(CH 3 ) 2 ), 80.8 (4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 76.9 (4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 73.9 (4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 46.9 ( C H 2 Ph), 32.2 (4-CH 3 C 6 H 4 C H(CH 3 ) 2 ), 31.4 (NCH- C H(CH 3 ) 2 ) 23.5 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 21.9 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 20.5 (NCH–CH( C H 3 ) 2 ), 19.8 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 19.1 (4- C H 3 C 6 H 4 CH(CH 3 ) 2 ).
3c [(Cp*)Rh(( R )- 2 )Cl]PF 6 Compound 3c was synthesized
in 69% yield (536 mg) according to the general procedure starting
from 313 mg (0.91 mmol) of ( R )- 1 , 195
mg (174 μL, 1.82 mmol) of pyridine-2-carbaldehyde, and 277 mg
(0.45 mmol) of [Cp*RhCl 2 ] 2 . Elemental analysis
calculated for C 35 H 38 Cl 2 F 6 N 4 OPRh (849.49 g/mol) C 49.49, H 4.51, N 6.60; found C
49.49, H 4.50, N 6.60. HPLC-MS τ 1 = 2.67 min calculated
for C 35 H 38 Cl 2 N 4 ORh + [M-PF 6 ] + m / z = 703.1; found m / z =
703.5, τ 2 = 4.29 min calculated for C 35 H 38 Cl 2 N 4 ORh + [M-PF 6 ] + m / z = 703.1;
found m / z = 703.5. 1 H
NMR (600 MHz, THF- d 8 ) δ 10.76 (s,
0.1H, CH), 9.27 (s, 1H, C H imine ),
8.89 (d, J = 5.4 Hz, 1H, C H Ar ), 8.86 (d, J = 5.5 Hz, 0.1H, C H Ar ), 8.66 (s, 0.1H, CH Ar ), 8.24
(d, J = 7.4 Hz, 1H, C H Ar ), 8.19 (t, J = 7.8 Hz, 1H, C H Ar ), 8.15 (d, J = 8.5 Hz,
1H, C H Ar ), 7.87 (d, J = 1.8 Hz, 0.2H, C H Ar ), 7.84 (t, J = 6.4 Hz, 1H, C H Ar ),
7.73 (d, J = 1.7 Hz, 1H, C H Ar ), 7.56 (dd, J = 8.6, 2.0 Hz, 0.1H,
C H Ar ), 7.47 (dd, J = 8.6, 2.0 Hz, 1H, C H Ar ), 7.39
(t, J = 7.5 Hz, 2H, C H Ar ), 7.32–7.28 (m, 3.5H, C H Ar ), 7.23 (d, J = 7.2 Hz, 0.1H, C H Ar ), 7.20 (d, J = 7.9 Hz,
0.3H, C H Ar ), 5.91 (d, J = 16.9 Hz, 1H, C H 2 Ph), 5.80 (d, J = 17.4 Hz, 0.1H, C H 2 Ph), 5.33 (d, J = 17.1 Hz, 1H, C H 2 Ph), 5.13 (d, J = 8.7 Hz, 1H,
H-1′), 3.08–3.02 (m, 1H, H-2′), 2.39 (s, 0.4H),
1.73 (s, 15H, Cp*-C H 3 ), 1.63 (s, 2H, Cp*-C H 3 ), 1.13 (d, J = 6.7 Hz, 3H, H-3′),
0.95 (d, J = 6.6 Hz, 3H, H-3′). 13 C{ 1 H} NMR (151 MHz, THF- d 8 ) δ 170.4 (C H imine ), 161.5
(C IV ), 157.0 (C IV ), 154.7 (C IV ),
153.6 ( C H Ar ), 148.2 (C IV ), 140.8 ( C H Ar ), 140.7 (C IV ), 137.0 (C IV ), 131.1 ( C H Ar ), 130.7 ( C H Ar ), 129.6 ( C H Ar ), 129.5 ( C H Ar ), 129.3 ( C H Ar ), 128.3
( C H Ar ), 128.3 ( C H Ar ), 127.6 ( C H Ar ),
127.1 ( C H Ar ), 120.4 (C IV ), 98.6 (d, J C–Rh = 7.7 Hz, Cp*),
73.2 (C-1′), 48.2 ( C H 2 Ph),
35.1 (C-2′), 19.9 (C-3′), 18.8 (C-3′), 9.1 (Cp*- C H 3 ).
3d [(Cp*)Ir(( R )- 2 )Cl]PF 6 Compound 3d was synthesized
in 73% yield (593 mg) according to the general procedure starting
from 296 mg (0.87 mmol) of ( R )- 1 , 185
mg (165 μL, 1.73 mmol) of pyridine-2-carbaldehyde, and 338 mg
(0.42 mmol) of [Cp*IrCl 2 ] 2 . Elemental analysis
calculated for C 35 H 38 Cl 2 F 6 IrN 4 OP (938.80 g/mol) C 44.78, H 4.08, N 5.97; found C
44.83, H 4.20, N 5.97. HPLC-MS τ 1 = 3.65 min calculated
for C 35 H 38 Cl 2 N 4 OIr + [M-PF 6 ] + m / z = 793.2; found m / z =
793.4, τ 2 = 7.45 min calculated for C 35 H 38 Cl 2 N 4 OIr + [M-PF 6 ] + m / z = 793.2;
found m / z = 793.6. 1 H
NMR (600 MHz, THF- d 8 ) δ 9.83 (s,
1H, C H imine ), 9.14 (s, 0.4H), 8.86
(d, J = 5.4 Hz, 1H, C H Ar ), 8.84 (d, J = 5.5 Hz, 0.4H, C H Ar ), 8.42 (d, J = 7.6 Hz,
1H, C H Ar ), 8.22 (d, J = 8.5 Hz, 0.6H, C H Ar ), 8.17 (t, J = 7.6 Hz, 1H, C H Ar ),
8.14 (d, J = 8.5 Hz, 1H, C H Ar ), 7.87 (d, J = 2.0 Hz, 0.4H, C H Ar ), 7.84–7.80 (m, 1.5H, C H Ar ), 7.79 (d, J = 2.0 Hz,
1H, C H Ar ), 7.57 (dd, J = 8.6, 2.0 Hz, 0.5H, C H Ar ), 7.47
(dd, J = 8.5, 2.0 Hz, 1H, C H Ar ), 7.38 (t, J = 7.5 Hz, 2H, C H Ar ), 7.32–7.26 (m, 4H, C H Ar ), 7.26–7.22 (m, 1H, C H Ar ), 7.19 (d, J = 7.5 Hz,
1H, C H Ar ), 5.87 (d, J = 17.1 Hz, 1H, C H 2 Ph), 5.78 (d, J = 17.2 Hz, 0.4H), 5.24 (d, J = 17.1 Hz, 1H, C H 2 Ph), 5.15 (d, J = 9.3 Hz, 1H,
H-1′), 3.20–3.13 (m, 1H, H-2′), 2.95–2.89
(m, 0.4H, H-2′), 1.69 (s, 15H, Cp*-C H 3 ), 1.60 (s, 7H, Cp*-C H 3 ), 1.16 (d, J = 6.7 Hz, 3H, H-3′), 0.92 (d, J = 6.6 Hz, 3H, H-3′). 13 C{ 1 H} NMR (151
MHz, THF- d 8 ) δ 172.0 ( C H imine ), 161.3 (C IV ), 157.0 (C IV ), 156.2 (C IV ), 153.1 ( C H Ar ), 148.2 (C IV ), 141.0 ( C H Ar ), 140.8 ( C H Ar ),
136.7 (C IV ), 131.3 ( C H Ar ), 131.2 ( C H Ar ), 129.6 ( C H Ar ), 129.4 ( C H Ar ), 129.3 ( C H Ar ), 128.4
( C H Ar ), 128.3 ( C H Ar ), 128.2 ( C H Ar ),
127.7 ( C H Ar ), 127.0 ( C H Ar ), 120.3 (C IV ), 91.3 (Cp*),
74.8 (C-1′), 48.1 ( C H 2 Ph),
35.9 (C-2′), 19.9 (C-3′), 19.2 (C-3′), 8.80 (Cp*- C H 3 ).
4a [(cym)Ru(( S )- 2 )Cl]PF 6 Compound 4a was synthesized
in 68% yield (215 mg) according to the general procedure starting
from 130 mg (0.38 mmol) of ( S )- 1 , 79
mg (70 μL, 0.74 mmol) of pyridine-2-carbaldehyde, and 114 mg
(0.19 mmol) of [(cym)RuCl 2 ] 2 . Elemental analysis
calculated for C 35 H 37 Cl 2 F 6 N 4 OPRu (846.64 g/mol) C 49.65, H 4.41, N 6.62; found C
49.41, H 4.59, N 6.80. HPLC-MS τ 1 = 3.39 min calculated
for C 35 H 37 Cl 2 N 4 ORu + [M-PF 6 ] + m / z = 701.1; found m / z =
701.3, τ 2 = 5.07 min calculated for C 35 H 37 Cl 2 N 4 ORu + [M-PF 6 ] + m / z = 701.1;
found m / z = 700.8. 1 H
NMR (600 MHz, CD 2 Cl 2 ) δ 9.29 (d, J = 5.4 Hz, 1H, C H Ar ),
9.27 (d, J = 5.6 Hz, 0.2H, C H Ar ), 9.21 (s, 0.2H, C H imine ), 8.45 (s, 1H, C H imine ), 8.33
(d, J = 8.5 Hz, 1H C H Ar ), 8.24 (d, J = 7.1 Hz, 1H, C H Ar ), 8.19 (t, J = 3.8 Hz,
1H, C H Ar ), 8.15–8.11 (m,
0.4H, C H Ar ), 7.92 (d, J = 1.9 Hz, 1H, C H Ar ), 7.80–7.78
(m, 1H, C H Ar ), 7.74–7.72
(m, 0.2H, C H Ar ), 7.63 (d, J = 1.8 Hz, 0.2H, C H Ar ), 7.61 (dd, J = 8.6, 2.0 Hz, 1H, C H Ar ), 7.48–7.44 (m, 0.7H, C H Ar ), 7.41 (d, J = 7.4 Hz, 0.2H, C H Ar ), 7.38 (t, J = 7.4 Hz,
2H, C H Ar ), 7.32 (t, J = 6.8 Hz, 2H, C H Ar ), 7.09 (d, J = 7.5 Hz, 2H, C H Ar ),
6.09 (d, J = 16.9 Hz, 0.2H, C H 2 Ph), 5.96 (d, J = 6.5 Hz, 0.3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.92 (br s, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.87 (d, J = 6.0 Hz, 0.3H,
4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.80 (d, J = 16.8 Hz, 1H,
C H 2 Ph), 5.71
(br s, 3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.66 (d, J = 6.4 Hz, 0.3H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.48 (d, J = 5.9 Hz, 0.2H, H-1′), 5.41 (d, J = 10.1
Hz, 0.2H, H-1′), 5.23 (d, J = 16.5 Hz, 0.2H,
C H 2 Ph), 4.58
(d, J = 9.2 Hz, 1H, H-1′), 4.35 (d, J = 14.5 Hz, 1H, C H 2 Ph), 3.23–3.17 (m, 1H, H-2′), 2.98–2.94
(m, 0.2H, H-2′), 2.60–2.55 (m, 0.2H, 4-CH 3 C 6 H 4 C H (CH 3 ) 2 ), 2.45–2.40 (m, 1H, 4-CH 3 C 6 H 4 C H (CH 3 ) 2 ), 2.38 (s, 0.6H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ),
1.84 (s, 3H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ),
1.21 (d, J = 6.6 Hz, 0.8H, H-3′), 1.07 (d, J = 6.9 Hz, 0.7H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 1.03–0.99 (m, 7H, H-3′, superimposed with
4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.89 (d, J = 6.3 Hz, 3H, H-3′), 0.86 (d, J = 7.0 Hz, 3H 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.55
(d, J = 6.5 Hz, 0.7H, H-3′). 13 C{ 1 H} NMR (151 MHz, CD 2 Cl 2 ) δ
170.7 ( C H imine ), 161.5 (C IV ), 156.0 ( C H Ar ), 153.3 (C IV ), 153.0 (C IV ), 147.4 (C IV ), 141.4 (C IV ), 140.3 ( C H Ar ), 136.2 (C IV ), 131.2 ( C H Ar ), 130.3 ( C H Ar ), 129.9 ( C H Ar ), 129.8 ( C H Ar ), 129.2
( C H Ar ), 128.5 ( C H Ar ), 127.1 ( C H Ar ),
127.0 ( C H Ar ), 126.3 ( C H Ar ), 120.2 (C IV ), 82.9 (C-1′),
79.5 (4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 47.9 ( C H 2 Ph), 46.6 ( C H 2 Ph), 32.0 (4-CH 3 C 6 H 4 C H(CH 3 ) 2 ), 31.3 (C-2′),
22.9 (C–H), 22.7 (C–H), 22.6 (C–H), 22.3 (C–H),
21.9 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 20.4 (C-3′), 19.8 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 19.2 (4- C H 3 C 6 H 4 CH(CH 3 ) 2 ).
4b [(cym)Os(( S )- 2 )Cl]PF 6 Compound 4b was synthesized
in 41% yield (144 mg) according to the general procedure starting
from 130 mg (0.38 mmol) of ( S )- 1 , 79
mg (70 μL, 0.74 mmol) of pyridine-2-carbaldehyde, and 149 mg
(0.19 mmol) of [(cym)OsCl 2 ] 2 . Elemental analysis
calculated for C 35 H 37 Cl 2 F 6 N 4 OOsP (935.80 g/mol) C 44.92, H 3.99, N 5.99; found C
44.75, H 4.09, N 5.78. HPLC-MS τ 1 = 4.24 min calculated
for C 35 H 37 Cl 2 N 4 OOs + [M-PF 6 ] + m / z = 791.2; found m / z =
791.4, τ 2 = 7.04 min calculated for C 35 H 37 Cl 2 N 4 OOs + [M-PF 6 ] + m / z = 791.2;
found m / z = 791.6. 1 H
NMR (600 MHz, CD 2 Cl 2 ) δ 9.62 (s, 0.25H,
C H Ar ), 9.20 (d, J = 5.5 Hz, 1H, C H Ar ), 8.91 (s,
1H, C H imine ), 8.40 (d, J = 7.5 Hz, 1H, C H Ar ), 8.31 (d, J = 8.5 Hz, 1H, C H Ar ),
8.27 (d, J = 7.8 Hz, 0.25H, C H Ar ), 8.19 (d, J = 8.5 Hz, 0.25H, C H Ar ), 8.17–8.14 (m, 1H, C H Ar ), 8.11–8.08 (m, 0.25H, C H Ar ), 7.90 (d, J = 1.9 Hz,
1H, C H Ar ), 7.74–7.72 (m,
1H, C H Ar ), 7.68–7.66 (m,
0.5H, C H Ar ), 7.61 (dd, J = 8.2, 2.0 Hz, 1H, C H Ar ), 7.47–7.45
(m, 0.75H, C H Ar ), 7.39 (t, J = 7.5 Hz, 2H, C H Ar ),
7.34 (t, J = 7.4 Hz, 1H, C H Ar ), 7.29 (d, J = 7.5 Hz, 0.5H, C H Ar ), 7.12 (d, J = 7.5 Hz,
2H, C H Ar ), 6.24 (d, J = 6.1 Hz, 0.4H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 6.19 (d, J = 5.5 Hz, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 6.06 (d, J = 16.5 Hz, 0.4H, C H 2 Ph), 5.96
(br s, 1H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.92–5.84 (m, 2H,
4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.81 (d, J = 16.7 Hz, 1H,
C H 2 Ph), 5.69 (d, J = 5.5 Hz, 0.25H, 4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 5.40 (d, J = 10.4 Hz, 0.25H, NC H –CH(CH 3 ) 2 ), 5.13 (d, J = 16.4 Hz, 0.25H, C H 2 Ph), 4.78 (d, J = 9.9 Hz,
1H, NC H –CH(CH 3 ) 2 ), 4.50 (d, J = 16.2 Hz, 1H, C H 2 Ph), 3.42 (s, 0.1H), 3.16–3.10 (m, 1H, NCH-C H (CH 3 ) 2 ), 3.00–2.95 (m,
0.25H, NCH-C H (CH 3 ) 2 ),
2.43 (s, 1H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ), 2.32–2.27 (m, 1H,
4-CH 3 C 6 H 4 C H (CH 3 ) 2 ), 1.90 (s, 3H, 4-C H 3 C 6 H 4 CH(CH 3 ) 2 ), 1.35 (d, J = 6.9 Hz, 0.25H), 1.17 (d, J = 6.7 Hz, 1H, NCH–CH(C H 3 ) 2 ), 1.07 (d, J = 6.9 Hz, 1H), 1.00 (d, J = 6.9 Hz, 3H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.96 (d, J = 6.8 Hz, 3H, NCH–CH(C H 3 ) 2 ), 0.89 (d, J =
6.1 Hz, 3H, NCH–CH(C H 3 ) 2 ), 0.77 (d, J =
6.9 Hz, 3H, 4-CH 3 C 6 H 4 CH(C H 3 ) 2 ), 0.51 (d, J = 6.6 Hz, 0.75H, NCH–CH(C H 3 ) 2 ). 13 C{ 1 H}
NMR (151 MHz, CD 2 Cl 2 ) δ 172.4 ( C H imine ), 161.1 (C IV ), 155.2 ( C H Ar ), 154.2 (C IV ), 152.3 (C IV ), 146.9 (C IV ), 141.1 (C IV ), 140.0
( C H Ar ), 135.7 (C IV ),
130.8 ( C H Ar ), 129.5 ( C H Ar ), 129.4 ( C H Ar ), 128.9 ( C H Ar ), 128.2
( C H Ar ), 126.7 ( C H Ar ), 126.0 ( C H Ar ),
119.7 (C IV ), 88.3 (4-CH 3 C 6 H 4 CH(CH 3 ) 2 ), 83.2 (C-1′),
46.5 ( C H 2 ), 31.8 (4-CH 3 C 6 H 4 C H(CH 3 ) 2 ), 30.9 (C-2′), 23.1 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 21.4 (4-CH 3 C 6 H 4 CH( C H 3 ) 2 ), 20.1 (C-3′), 19.3
(C-3′), 18.7 (4- C H 3 C 6 H 4 CH(CH 3 ) 2 ).
4c [(Cp*)Rh(( S )- 2 )Cl]PF 6 Compound 4c was synthesized
in 68% yield (535 mg) according to the general procedure starting
from 313 mg (0.91 mmol) of ( S )- 1 , 195
mg (174 μL, 1.82 mmol) of pyridine-2-carbaldehyde, and 276 mg
(0.45 mmol) of [Cp*RhCl 2 ] 2 . Elemental analysis
calculated for C 35 H 38 Cl 2 F 6 N 4 OPRh (849.49 g/mol) C 49.49, H 4.51, N 6.60; found C
49.53, H 4.49, N 6.55. HPLC-MS τ 1 = 2.79 min calculated
for C 35 H 38 Cl 2 N 4 ORh + [M-PF 6 ] + m / z = 703.1; found m / z =
703.5, τ 2 = 4.20 min calculated for C 35 H 38 Cl 2 N 4 ORh + [M-PF 6 ] + m / z = 703.1;
found m / z = 703.5. 1 H
NMR (600 MHz, THF- d 8 ) δ 10.77 (s,
0.1H), 9.27 (s, 1H, C H imine ), 8.90
(d, J = 5.3 Hz, 1H, C H Ar ), 8.86 (d, J = 5.1 Hz, 0.1H, C H Ar ), 8.66 (s, 0.1H), 8.24 (d, J = 7.1 Hz, 1H, C H Ar ), 8.19 (t, J = 7.7 Hz, 1H, C H Ar ),
8.15 (d, J = 8.5 Hz, 1H, C H Ar ), 7.87 (d, J = 1.7 Hz, 0.2H, C H Ar ), 7.84 (t, J = 6.0 Hz,
1H, C H Ar ), 7.73 (d, J = 1.8 Hz, 1H, C H Ar ), 7.56 (dd, J = 8.5, 2.0 Hz, 0.1H, C H Ar ), 7.47 (dd, J = 8.5, 2.0 Hz, 1H, C H Ar ), 7.39 (t, J = 7.4 Hz, 2H, CH Ar ), 7.32–7.28 (m, 3.5H, CH Ar ), 7.23 (d, J = 7.4 Hz, 0.1H, C H Ar ), 7.20 (d, J = 7.7 Hz, 0.3H, C H Ar ) 5.91 (d, J = 16.7 Hz, 1H, C H 2 Ph), 5.80 (d, J = 17.2
Hz, 0.1H, C H 2 Ph), 5.33 (d, J = 17.1 Hz, 1H, C H 2 Ph), 5.13 (d, J = 8.7 Hz, 1H, H-1′),
3.08–3.02 (m, 1H, H-2′), 2.39 (s, 0.5H), 1.73 (s, 15H,
Cp*-C H 3 ),
1.63 (s, 2H, Cp*-C H 3 ), 1.13–1.10 (m, 3H, H-3′ superimposed with the
diethyl ether signal), 0.95 (d, J = 6.6 Hz, 3H, H-3′). 13 C{ 1 H} NMR (151 MHz, THF- d 8 ) δ 170.4 ( C H imine ), 161.5 (C IV ), 154.7 (C IV ), 153.6 ( C H Ar ), 148.2 (C IV ), 140.8 ( C H Ar ), 140.7 (C IV ), 137.0 (C IV ), 131.1 ( C H Ar ), 130.6
( C H Ar ), 129.6 ( C H Ar ), 129.5 ( C H Ar ),
129.3 ( C H Ar ), 128.3 ( C H Ar ), 128.3 ( C H Ar ), 127.6 ( C H Ar ), 127.1
( C H Ar ), 120.4 (C IV ),
98.6 (d, J Rh–C = 7.8 Hz, Cp*), 73.2 (C-1′),
48.2 ( C H 2 Ph), 35.1 (C-2′)
19.9 (C-3′), 18.8 (C-3′), 9.1 (Cp*- C H 3 ).
4d [(Cp*)Ir(( S )- 2 )Cl]PF 6 Compound 4d was synthesized
in 72% yield (413 mg) according to the general procedure starting
from 208 mg (0.61 mmol) of ( S )- 1 , 131
mg (115 μL, 1.22 mmol) of pyridine-2-carbaldehyde, and 237 mg
(0.30 mmol) of [Cp*IrCl 2 ] 2 . Elemental analysis
calculated for C 35 H 38 Cl 2 F 6 IrN 4 OP (938.80 g/mol) C 44.78, H 4.08, N 5.97; found C
44.69, H 4.05, N 5.86. HPLC-MS τ 1 = 3.81 min calculated
for C 35 H 38 Cl 2 N 4 OIr + [M-PF 6 ] + m / z = 793.2; found m / z =
793.6, τ 2 = 7.53 min calculated for C 35 H 38 Cl 2 N 4 OIr + [M-PF 6 ] + m / z = 793.2;
found m / z = 793.5. 1 H
NMR (600 MHz, THF- d 8 ) δ 9.82 (s,
0.7H, CH imine ) 9.14 (s, 1H, CH imine ), 8.87 (d, J = 5.4 Hz, 0.7H, C H Ar ), 8.84 (d, J = 5.4 Hz, 1H, C H Ar ), 8.41 (d, J = 7.7 Hz, 0.7H, C H Ar ), 8.22 (d, J = 8.5 Hz,
1.3H, C H Ar ), 8.18–8.16 (m,
1.4H, C H Ar ), 8.14 (d, J = 8.5 Hz, 1H, C H Ar ), 7.87 (d, J = 1.9 Hz, 1H, C H Ar ),
7.83–7.80 (m, 1.7H, C H Ar ),
7.79 (d, J = 1.9 Hz, 0.7H, C H Ar ), 7.57 (dd, J = 8.6, 1.9 Hz, 1H, C H Ar ), 7.47 (dd, J = 8.5,
2.0 Hz, 0.7H, C H Ar ), 7.38 (t, J = 7.5 Hz, 1.5H, C H Ar ), 7.33–7.28 (m, 4H, C H Ar ), 7.24 (t, J = 7.3 Hz, 1H, C H Ar ), 7.19 (d, J = 7.5 Hz, 2H, C H Ar ), 5.87 (d, J = 17.0 Hz,
0.7H, C H 2 Ph), 5.78 (d, J = 17.1 Hz, 1H, C H 2 Ph), 5.24 (d, J = 17.1 Hz, 0.8H, C H 2 Ph), 5.15 (d, J = 9.3 Hz, 1H, H-1′),
4.74 (br s, 0.7H, C H 2 Ph), 3.19–3.13 (m, 0.7H, H-2′), 2.95–2.87
(m, 1H, H-2′), 1.70 (s, 11.5H, Cp*-C H 3 ), 1.61 (s, 15H, Cp*-CH 3 ), 1.19 (br s, 3H, H-3′) superimposed with 1.16 (d, J = 6.7 Hz, 3H, H-3′), 0.93 (d, J = 6.6 Hz, 2.3H, H-3′). 13 C{ 1 H} NMR
(151 MHz, THF- d 8 ) δ 172.0 ( C H imine ), 161.4 (C IV ), 161.3 (C IV ), 156.9 (C IV ), 156.2 (C IV ), 153.1
( C H Ar ), 152.7 ( C H Ar ), 148.2 (C IV ), 147.9 (C IV ),
141.1 ( C H Ar ), 140.8 ( C H Ar ), 140.6 (C IV ), 136.7 (C IV ), 131.3 ( C H Ar ), 131.2
( C H Ar ), 129.6 ( C H Ar ), 129.4 ( C H Ar ),
129.3 ( C H Ar ), 128.7 ( C H Ar ), 128.4 ( C H Ar ), 128.3 ( C H Ar ), 128.2
( C H Ar ), 127.7 ( C H Ar ), 127.0 ( C H Ar ),
126.7 ( C H Ar ), 121.1 (C IV ), 120.4 (C IV ), 91.4 (Cp*), 91.3 (Cp*), 74.8 (C-1′),
48.1 ( C H 2 Ph), 35.9 (C-2′),
20.2 (C-3′), 19.9 (C-3′), 19.2 (C-3′), 19.1 (C-3′),
8.8 (Cp*- C H 3 ), 8.8 (Cp*- C H 3 ).
## Stability Studies
Stability Studies The stability of 3a – d was studied in the presence of l -cysteine or l -histidine. 3a – d were dissolved
in DMSO and added to 0.2 mM aqueous solution of l -cysteine
or l -histidine to achieve the complex concentration of 20
μM while keeping the DMSO concentration at 0.5 vol %. UV–vis
spectra were recorded over 2 h with 10 min intervals. HPLC-MS analysis
with them using UV–vis spectroscopy and HPLC-MS analysis were
performed on a Phenomenex XB-C18 column (50 × 4.6 mm, 2.1 mm,
1.7 μm) using a mixture of 55% water with 0.01% HCOOH (eluent
A), 22.5% methanol with 0.01% HCOOH (eluent B), and 22.5% acetonitrile
with 0.01% HCOOH (eluent C) with a flow rate of 0.4 mL·min –1 .
## Cell Lines
Cell Lines Cell lines used in this study were purchased
from the American Type Culture Collection via LGC Standards. Human
normal lung fibroblasts (MRC-5), alveolar basal epithelial cell adenocarcinoma
(A549), colorectal adenocarcinoma (Colo205), hepatocellular carcinoma
(HepG2), breast adenocarcinoma (MCF7), and colorectal adenocarcinoma
(SW620) and its MDR variants 55 were cultured
in standard conditions (37 °C, 5% CO 2 , 100% relative
humidity) in high glucose DMEM medium supplemented with GlutaMax,
HEPES (ThermoFisher Scientific) and 10% fetal bovine serum (EURx,
Poland). All cell lines were tested for Mycoplasma contamination using a MycoProbe mycoplasma detection kit (R&D
System).
## Assaying the Antiproliferative Potential
Assaying the Antiproliferative Potential For this purpose,
neutral red uptake assay was performed. 10 4 of cells were
seeded per well of a 96-well plate and left overnight to allow cells
to attach to the surface. Then, the cells were exposed to a desired
concentration of tested compounds. Stock solutions were prepared in
DMSO and were used immediately after preparation. The final concentration
of DMSO was constant and nontoxic (0.1% v/v). After 70 h of culture,
neutral red was added to the final concentration of 1 mM. After 2
h of incubation with the dye, the medium was aspirated and cells were
washed with ice-cold PBS. The dye was released using 100 μL
of the solubilizer (1% acetic acid in 50% ethanol) on an orbital shaker
(10 min). The absorbance at 540 nm was measured using an EnVision
multilabel plate reader (PerkinElmer). The results were presented
as a percentage of control. The IC 90 and IC 50 parameters were calculated using GraphPad Prism v9 software using
the five-parameter nonlinear logistic regression model.
## Cell Cycle
Cell Cycle SW620 and SW620E cells lines (vulnerable
and resistant variants, respectively) were seeded in 6-well plates
at a density of 10 5 cells per well. After the time necessary
for the cells to attach to the surface, the cells were treated with
tested compounds at a concentration equal to IC 90 for parent
compounds (15 nM for ( R ) series and 23 nM for ( S ) series). After 24 h, the cells were trypsinized and fixed
with ice-cold 70% v/v ethanol. The cells were stained with 75 μM
propidium iodide with 50 Kunitz units of RNase A in PBS for 30 min
at 37 °C. All samples were analyzed using a LSRII flow cytometer
(Becton Dickinson) at a PE channel (526/26 nm). Cell cycle phase distribution
was determined using a built-in cell cycle module (Watson pragmatic
algorithm) by FlowJo 7.6.1 software.
## Reactive Oxygen Species Assay
Reactive Oxygen Species Assay Dihydrorhodamine 123
oxidation was used as an indicator of intracellular ROS production.
For this purpose, SW620 cells were seeded in 6-well plates at a density
of 10 5 cells per well. The cells were left overnight (time
needed for them to attach to the surface). Then, 1 μM tested
compounds were added along with 1 μM DHR123. Additionally, since
DHR123 is a substrate of ABCB1 (which may interfere in this assay),
10 μM verapamil, an inhibitor of this protein, was added. The
cells were cultured for an additional 4 h at 37 °C, and then
the cells were harvested by trypsinization, resuspended in a complete
medium, and analyzed using a LSRII flow cytometer (Becton Dickinson)
in a FITC channel (530/30 nm). The results are presented as a percentage
of control (median fluorescence in the presence of DMSO).
## Kinesin ATPase Inhibition Assay
Kinesin ATPase Inhibition Assay The potential kinesin
modulatory activity of tested compounds was performed using a Kinesin
ATPase end-point biochem kit (Cytoskeleton, Inc.). Compounds were
dissolved in DMSO (the final concentration did not exceed 0.1%). The
experiment was performed according to the manufacturer’s instructions.
One μg of tested kinesin (KSP) was used per reaction. Phosphate
release was measured at the absorbance 650 nm using an EnVision multilabel
plate reader (PerkinElmer).