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Synthesis, Characterization, and Cytotoxicity of Morpholine-Containing Ruthenium(II) p-Cymene Complexes.
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Article
Synthesis, Characterization, and Cytotoxicity of MorpholineContaining Ruthenium(II) p‑Cymene Complexes
Rishav Chatterjee,† Indira Bhattacharya,† Souryadip Roy, Kallol Purkait, Tuhin Subhra Koley,
Arnab Gupta,* and Arindam Mukherjee*
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ABSTRACT: Morpholine motif is an important pharmacophore
and, depending on the molecular design, may localize in cellular
acidic vesicles. To understand the importance of the presence of
pendant morpholine in a metal complex, six bidentate N,O-donor
ligands with or without a pendant morpholine unit and their
corresponding ruthenium(II) p-cymene complexes (1−6) are
synthesized, purified, and structurally characterized by various
analytical methods including X-ray diffraction. Complexes 2−4
crystallized in the P21/c space group, whereas 5 and 6 crystallized
in the P1̅ space group. The solution stability studies using 1H NMR
support instantaneous hydrolysis of the native complexes to form
monoaquated species in a solution of 3:7 (v/v) dimethyl sulfoxided6 and 20 mM phosphate buffer (pH* 7.4, containing 4 mM
NaCl). The monoaquated complexes are stable for at least up to 24 h. The complexes display excellent in vitro antiproliferative
activity (IC50 ca. 1−14 μM) in various cancer cell lines, viz., MDA-MB-231, MiaPaCa2, and Hep-G2. The presence of the pendant
morpholine does not improve the dose efficacy, but rather, with 2-[[(2,6-dimethylphenyl)imino]methyl]phenol (HL1) and its
pendant morpholine analogue (HL3) giving complexes 1 and 3, respectively, the antiproliferative activity was poorer with 3. MDAMB-231 cells treated with the complexes show that the acidic vesicles remain acidic, but the population of acidic vesicles increases or
decreases with time of exposure, as observed from the dispersed red puncta, depending on the complex used. The presence of the
2,6-disubstituted aniline and the naphthyl group seems to improve the antiproliferative dose. The complex treated MDA-MB-231
cells show that cathepsin D, which is otherwise present in the cytosolic lysosomes, translocates to the nucleus as a result of exposure
to the complexes. Irrespective of the presence of a morpholine motif, the complexes do not activate caspase-3 to induce apoptosis
and seem to favor the necrotic pathway of cell killing.
■
INTRODUCTION
Platinum drugs have displayed high cure rates for testicular
cancer and are used to treat various other tumors, viz., prostate,
colon, ovarian, esophageal, bladder, head and neck, and
nonsmall cell lung cancer.1−4 Platinum drugs are also known
for their poor tumor selectivity, serious side effects, and
intrinsic and/or acquired resistance, making them ineffective
for treatment against several tissue types.2,5,6 Thus, in spite of
its clinical success, the deleterious side effects of cisplatin have
led to the search for new anticancer agents with improved
therapeutics. The goal of the modern design by changing the
metal and tuning ligand properties encompasses the reduction
of adverse side effects, activation by external stimuli, and a
focus on targets other than the nucleus to avoid mutagenic
character and improve delivery.7−14 Metal complexes with
potential apart from platinum(II/IV) include gallium(II) and
ruthenium(II/III).15−21 Gallium(III) nitrate has been effective
against lymphoma and bladder cancer.22 Gallium(III)
maltolate and tris(8-quinolinolato)gallium(III) have shown
© XXXX American Chemical Society
efficiency against hepatocellular carcinoma and renal carcinoma, respectively.15,16,22
Ruthenium(II/III) complexes have raised considerable
interest because of the shelf-stability of the complexes in two
oxidation states, the variation of kinetic inertness based on the
oxidation states and geometry, and the scope of the
incorporation of targeting motifs in the ligand without
sacrificing the activity.23−37 Ruthenium(III) complexes act as
prodrugs because of the inert nature of the ruthenium(III)
oxidation state. In a lower oxygen environment (hypoxia) of
tumor cells, ruthenium(III) complexes get reduced to the more
reactive ruthenium(II) complexes, providing enhanced toxicity
Received: May 7, 2021
A
https://doi.org/10.1021/acs.inorgchem.1c01363
Inorg. Chem. XXXX, XXX, XXX−XXX
Inorganic Chemistry
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in tumors.38 The high binding affinity of ruthenium(III) for
the transferrin iron-binding sites offers the possibility of
targeting tumors with high transferrin receptor densities.39−41
Two ruthenium(II/III) complexes, KP1339 and TLD-1433,
are currently undergoing clinical trials as anticancer agents.
KP1339 is a ruthenium(III) complex that has undergone
clinical trials against various solid tumors, including nonsmall
cell lung carcinoma and neuroendocrine tumors.20 TLD-1433
is the first ruthenium complex that has entered clinical trials, as
a photodynamic therapy agent against BCG refractory/
intolerant nonmuscle invasive bladder cancer.17,18 Controlled
multistep synthesis helps to incorporate various ligands of
interest to render optimized ruthenium complexes with
efficient antitumor activity.7,25,31,42−48
In the past few decades, ruthenium(II) half-sandwich
complexes with piano-stool configurations provided researchers the opportunity to design various organic ligands involving
certain targets and then coordinate them to ruthenium(II)
arenes and monitor their activity, selectivity, and kinetic
properties.49−62 Among the various motifs used as pharmacophores, morpholine is found in various anticancer drugs
(Figure 1). The incorporation of morpholine may enhance the
purchased from Spectrochem, India. A PerkinElmer Lambda 35
spectrophotometer was used for UV−vis measurements. The Fourier
transform infrared (FT-IR) spectra were recorded using a
PerkinElmer SPECTRUM RX I spectrometer in KBr pellets. 1H
NMR, 13C NMR, and HMQC spectra were recorded using either a
400 MHz JEOL ECS or 500 MHz Bruker Avance III spectrometer at
room temperature (24−27 °C). All of the 13C NMR spectra reported
are proton-decoupled. The chemical shifts of the relevant compounds
are reported in parts per million (ppm). All of the mass spectrometry
(ESI-MS) spectra were recorded in positive-mode electrospray
ionization using a Bruker maXis II instrument. The reported yields
are of 1H NMR pure compounds.
Synthesis and Characterization. Syntheses of Schiff Base
Ligands. General Procedure I. A literature procedure was adapted for
synthesis of the amine (B2 or B3).78,79 To a methanolic solution of
the desired aniline (B1/B2/B3; 1 mmol) was added the
corresponding aldehyde (1 mmol), and the solution was heated to
reflux for 24 h. After the mixture was cooled, the desired compound
precipitated. The precipitate was filtered, washed with cold methanol
(MeOH) and hexane, and dried under vacuum.
General Procedure II. A literature procedure was adapted for
synthesis of the amine B2.78 To a stirred solution of the respective
aniline (B1/B2; 1 mmol) in MeOH was added the corresponding
aldehyde (1 mmol), and the resulting solution was refluxed for 24 h.
After the mixture was cooled, the solvent was evaporated in a rotary
evaporator, which resulted in a yellow oil. The oil used was further
purified by silica gel column chromatography [mobile phase: 3:2 (v/
v) hexane/ ethyl acetate].
Synthesis of (E)-2-[[(2,6-Dimethylphenyl)imino]methyl]phenol
(HL1). HL1 was synthesized by general procedure II and used
without purification. Yield: 91%, yellow oil. 1H NMR (400 MHz,
CDCl3): δ 8.35 (s, 1H, CHN), 7.45−7.39 (m, 1H, Ar−H), 7.35
(dd, J = 7.6 and 1.6 Hz, 1H, Ar−H), 7.12 (d, J = 7.5 Hz, 2H, Ar−H),
7.09−7.03 (m, 2H), 6.98 (dd, 1H, J = 7.4 and 1.0 Hz, Ar−H), 2.22 (s,
6H) (Figure S1). 13C NMR (100 MHz, CDCl3): δ 166.8, 161.3,
148.2, 133.3, 132.3, 128.6, 128.4, 125.0, 119.1, 118.9, 117.4, 18.6
(Figure S2).
Synthesis of (E)-1-[[(2,6-Dimethylphenyl)imino]methyl]naphthalen-2-ol (HL2). HL2 was synthesized by general procedure
I. Yield: 62%, yellow solid. 1H NMR (500 MHz, CDCl3): δ 9.12 (s,
1H, CHN), 7.99 (d, J = 8.5 Hz, 1H, Ar−H), 7.86 (d, J = 9.1 Hz,
1H, Ar−H), 7.76 (d, J = 8.0 Hz, 1H, Ar−H), 7.51 (t, J = 7.6 Hz, 1H,
Ar−H), 7.35 (t, J = 7.4 Hz, 1H, Ar−H), 7.23 (d, J = 9.1 Hz, 1H, Ar−
H), 7.17 (d, J = 7.4 Hz, 2H, Ar−H), 7.13−7.08 (m, 1H, Ar−H), 2.32
(s, 6H, Ar−CH3) (Figure S3). 13C NMR (125 MHz, CDCl3): δ 168,
161.3, 145.5, 136.2, 133.2, 129.7, 129.3, 128.6, 128.1, 127.4, 125.8,
123.5, 121.4, 118.8, 108.3, 18.6 (Figure S4).
Synthesis of 2-[[[2,6-Dimethyl-4-(morpholinomethyl)phenyl]imino]methyl]phenol (HL3). HL3 was synthesized by general
procedure I. Yield: 79%, light-yellow solid. 1H NMR (500 MHz,
CDCl3): δ 13.08 (s, 1H, OH), 8.34 (s, 1H), 7.40 (t, J = 7.2 Hz, 1H,
CHN), 7.34 (d, J = 7.6 Hz, 1H, Ar−H), 7.05 (d, J = 11.2 Hz, 3H,
Ar−H), 6.95 (t, J = 7.4 Hz, 1H, Ar−H), 3.74 (d, J = 4.0 Hz, 4H, −O−
CH2), 3.45 (s, 2H, Ar−CH2), 2.48 (s, 4H, N−CH2), 2.19 (s, 6H, Ar−
CH3) (Figure S5). 13C NMR (125 MHz, CDCl3): δ 166.7, 161.2,
147.2, 134.2, 133.2, 132.1, 129.2, 128.2, 119.0, 118.8, 117.3, 67.0,
63.1, 53.7, 18.5 (Figure S6).
Synthesis of 2-[[[2,6-Dimethyl-4-(morpholinomethyl)phenyl]imino]methyl]-6-methoxyphenol (HL4). HL4 was synthesized by
general procedure II. Yield: 53%, yellow oil. 1H NMR (500 MHz,
CDCl3): δ 13.52 (s, 1H, O−H), 8.34 (s, 1H, CHN), 7.06 (s, 2H,
Ar−H), 7.01 (d, J = 7.8 Hz, 1H, Ar−H), 6.96 (d, J = 7.6 Hz, 1H, Ar−
H), 6.90 (t, J = 7.7 Hz, 1H, Ar−H), 3.95 (s, 3H, O−CH3), 3.73 (s,
4H, O−CH2), 3.44 (s, 2H, Ar−CH2), 2.47 (s, 4H, N−CH2), 2.19 (s,
6H, Ar−CH3) (Figure S7). 13C NMR (125 MHz, CDCl3): δ 166.8,
151.4, 148.5, 146.9, 134.1, 129.2, 128.3, 123.5, 118.6, 118.5, 114.6,
66.9, 63, 56.1, 53.6, 18.5 (Figure S8).
Synthesis of 1-[[[2,6-Dimethyl-4-(morpholinomethyl)phenyl]imino]methyl]naphthalen-2-ol (HL5). HL5 was synthesized by
general procedure II. Yield: 49%, yellow crystalline solid. 1H NMR
Figure 1. (a) Anticancer ruthenium complexes in clinical trials. (b)
Clinical drugs containing a morpholine moiety.
activity, selectivity, solubility, bioavailability, and pharmacokinetic benefits.63−65 Compounds with morpholine motifs target
various enzymes and receptors including kinases, Amyloid β
peptides, α-glucosidase, serotonin receptors, and histamine
receptors.66−74 Morpholine is also known to help target
lysosomes in cells and acts as the cells’ recycling apparatus.75,76
The work presented here compares the presence and absence
of the morpholine motif in N,O-donor bidentate ligands in a
metal complex, keeping the rest of the environment the same.
Six new ruthenium(II) p-cymene complexes with N,O-donor
Schiff bases with or without pendant morpholine motifs were
synthesized and characterized for this purpose.
■
Article
EXPERIMENTAL SECTION
Materials and Methods. The chemicals and solvents were
purchased from various commercial sources. Unless specifically
mentioned, the chemicals were used as received without further
purification. Solvents were dried following standard procedures.77 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
and the biological assay kits were purchased from Gibco. Solvents
used for spectroscopic measurements were of spectroscopic grade and
B
https://doi.org/10.1021/acs.inorgchem.1c01363
Inorg. Chem. XXXX, XXX, XXX−XXX
Inorganic Chemistry
pubs.acs.org/IC
(400 MHz, CDCl3): δ 9.10 (s, 1H, CHN), 7.97 (d, J = 8.4 Hz, 1H,
Ar−H), 7.83 (d, J = 9.2 Hz, 1H, Ar−H), 7.74 (d, J = 8.0 Hz, 1H, Ar−
H), 7.48 (dt, J = 8.3, 6.8, and 1.2 Hz, 1H, Ar−H), 7.36−7.31 (m, 1H,
Ar−H), 7.16 (d, J = 9.1 Hz, 1H, Ar−H), 7.12 (s, 2H, Ar−H), 3.77−
3.73 (m, 4H, O−CH2), 3.48 (s, 2H, −CH2), 2.52−2.47 (m, 4H, N−
CH2), 2.30 (s, 6H, Ar−CH3) (Figure S9). 13C NMR (100 MHz,
CDCl3): δ 168.3, 161.2, 144.3, 136.1, 134.1, 133.2 129.7, 129.6,
129.4, 128.1, 127.4, 123.5, 121.7, 118.8, 108.4, 67.0, 63.1, 53.7, 18.8
(Figure S10).
Synthesis of (E)-1-[[[2,6-Diisopropyl-4-(morpholinomethyl)phenyl]imino]methyl]naphthalen-2-ol (HL6). HL6 was synthesized
by general procedure I. Yield: 58%, yellow solid. 1H NMR (500 MHz,
CDCl3): δ 15.22 (s, 1H, O−H), 9.07 (d, J = 2.5 Hz, 1H, CHN),
7.99 (d, J = 8.5 Hz, 1H, Ar−H), 7.86 (d, J = 9.1 Hz, 1H, Ar−H), 7.77
(d, J = 8.0 Hz, 1H, Ar−H), 7.49 (t, J = 7.7 Hz, 1H, Ar−H), 7.35 (t, J
= 7.5 Hz, 1H, Ar−H), 7.19 (d, J = 7.6 Hz, 3H, Ar−H), 3.76 (d, J =
3.8 Hz, 4H, O−CH2), 3.56 (s, 2H, Ar−CH2), 3.11 (m, 2H, iPr−CH),
2.51 (s, 4H, N−CH2), 1.24 (d, J = 6.8 Hz, 12H, iPr−CH3) (Figure
S11). 13C NMR (125 MHz, CDCl3): δ 167.7, 161.6, 142.8, 140.2,
135.9, 135.3, 133.2, 129.3, 128.1, 127.4, 124.3, 123.4, 121.5, 118.7,
108.3, 67, 63.4, 53.5, 28.3, 23.7 (Figure S12).
Syntheses of Metal Complexes. General Procedure for the
Synthesis of Metal Complexes 1−6. To a 10 mL stirred methanolic
solution of 0.2 mmol of the respective ligand was added 0.2 mmol of
solid KOH. After 15 min, a 10 mL methanolic solution of 0.1 mmol of
Ru2(p-cym)2Cl4 was added (in the dark), and the resultant solution
was left under stirring for 12 h at 25 °C. The entire solution was
evaporated to dryness, washed multiple times with a minimal amount
of diethyl ether, and purified by column chromatography using neutral
alumina. The mobile phase was 19:1 (v/v) dichloromethane/MeOH.
Ru(p-cym)(L1)Cl (1). Yield: 71%. 1H NMR (400 MHz, DMSO-d6):
δ 7.59 (s, 1H, CHN), 7.24 (m, 2H, Ar−H), 7.21 (m, 1H, Ar−H),
7.09 (t, J = 6.9 Hz, 1H, Ar−H), 7.01 (d, J = 6.4 Hz, 1H, Ar−H), 6.64
(d, J = 8.4 Hz, 1H, Ar−H), 6.31 (t, J = 7.2 Hz, 1H, Ar−H), 5.32 (d, J
= 6.0 Hz, 1H, p-cym−H), 5.14 (d, J = 6.1 Hz, 1H, p-cym−H), 5.06
(d, J = 5.7 Hz, 1H, p-cym−H), 4.26 (d, J = 5.6 Hz, 1H, p-cym−H),
2.54 (m, 1H, p-cym−iPr), 2.45 (s, 3H, Ar−CH3), 2.19 (s, 3H, Ar−
CH3), 1.89 (s, 3H, Ar−CH3), 1.21 (d, J = 6.9 Hz, 6H, p-cym−iPr)
(Figure S13). 13C NMR (125 MHz, DMSO-d6): δ 166.6, 165.9,
154.6, 135.3, 134.5, 131.1, 130.5, 128.6, 128, 126.3, 121, 120.5, 113.1,
102.9, 94.2, 85.8, 84.4, 82.1, 79.1, 30.3, 22, 21.8, 19.2, 18.1, 17 (Figure
S14). IR (KBr pellets, cm−1): 1584 (CHN) (Figure S29). UV−vis
[MeOH; λmax, nm (ε, M−1 cm−1)]: 241 (18620), 292 (5950), 405
(1600), 483 (840) (Figure S28). ESI-HRMS (MeOH). m/z (exp)
460.1214 (460.1281) [Ru II C 25 H 28 NO + ]. Anal. Calcd for
C25H28ClNORu: C, 60.66; H, 5.70; N, 2.83. Found: C, 60.84; H,
5.75; N, 2.79.
Ru(p-cym)(L2)Cl (2). Yield: 78%. 1H NMR (500 MHz, DMSO-d6):
δ 7.99 (s, 1H, CHN), 7.66−7.59 (m, 3H, Ar−H), 7.25 (m, 4H,
Ar−H), 7.12 (t, J = 7.3 Hz, 1H, Ar−H), 6.95 (d, J = 9.2 Hz, 1H, Ar−
H), 5.34 (d, J = 6.1 Hz, 1H, p-cym−H), 5.19 (d, J = 6.0 Hz, 1H, pcym−H), 5.07 (d, J = 5.7 Hz, 1H, p-cym−H), 4.33 (d, J = 5.8 Hz, 1H,
p-cym−H), 2.63−2.59 (m, 1H, p-cym−iPr), 2.17 (s, 3H, Ar−CH3),
1.93 (s, 3H, Ar−CH3), 1.25 (dd, J = 10.4 and 7.0 Hz, 6H, p-cym−iPr).
13
C NMR (125 MHz, DMSO-d6): δ 166.8, 158.4, 155.3, 134.8, 134.4,
131.5, 130.7, 128.5, 128.0, 127.3, 126.1, 125.6, 124.4, 121.6, 119.5,
111.2, 103.1, 94.0, 86.7, 84.7, 82.0, 79.4, 30.3, 22.0, 21.9, 19.1, 17.9,
17.0 (Figures S15−S17). IR (KBr pellets, cm−1): 1582 (CHN)
(Figure S29). UV−vis [MeOH; λmax, nm (ε, M−1 cm−1)]: 254
(34930), 321 (13120), 433 (3370), 485 (1670) (Figure S28). ESIHRMS (MeOH). m/z (exp) 510.1371 (510.1345) [RuIIC29H30NO+].
Anal. Calcd for C29H30ClNORu: C, 63.90; H, 5.55; N, 2.57. Found:
C, 63.77; H, 5.59; N, 2.62.
Ru(p-cym)(L3)Cl (3). Yield: 59%. 1H NMR (500 MHz, DMSO-d6):
δ 7.58 (s, 1H, CHN), 7.20 (s, 2H, Ar−H), 7.09 (t, J = 6.9 Hz, 1H,
Ar−H), 7.01 (d, J = 7.2 Hz, 1H, Ar−H), 6.65 (d, J = 8.4 Hz, 1H, Ar−
H), 6.31 (t, J = 6.9 Hz, 1H, Ar−H), 5.31 (d, J = 5.2 Hz, 1H, p-cym−
H), 5.11 (d, J = 5.3 Hz, 2H, p-cym−H), 4.35 (d, J = 4.1 Hz, 1H, pcym−H), 3.63 (s, 4H, O−CH2), 3.50 (s, 2H, CH2), 2.44 (s, 8H), 2.19
(s, 3H, CH3), 1.90 (s, 3H, CH3), 1.18 (dd, J = 16.8 and 6.7 Hz, 6H, p-
Article
cym−iPr). 13C NMR (125 MHz, DMSO-d6): δ 16.9, 18.0, 19.1, 21.5,
22.2, 30.1, 53.0, 66.1, 79.7, 82.8, 83.7, 84.8, 95.1, 102.3, 113.0, 120.4,
121.0, 128.8, 129.3, 130.3, 130.9, 134.5, 135.2, 165.8, 166.6 (Figures
S18−S20). IR (KBr pellets, cm−1): 1609 (CHN) (Figure S29).
UV−vis [MeOH; λmax, nm (ε, M−1 cm−1)]: 244 (29550), 291 (8130),
403 (2300), 482 (1020) (Figure S28). ESI-HRMS (MeOH). m/z
(exp) 559.1899 (559.1907) [RuIIC30H37N2O2+]. Anal. Calcd for
C30H37ClN2O2Ru: C, 60.65; H, 6.28; N, 4.71. Found: C, 60.42; H,
6.33; N, 4.68.
Ru(p-cym)(L4)Cl (4). Yield: 54%. 1H NMR (400 MHz, DMSO-d6):
δ 7.54 (s, 1H, CHN), 7.17 (s, 2H, Ar−H), 6.70 (d, J = 7.3 Hz, 1H,
Ar−H), 6.64 (d, J = 7.7 Hz, 1H, Ar−H), 6.23 (t, J = 7.6 Hz, 1H, Ar−
H), 5.32 (d, J = 5.7 Hz, 1H, p-cym−H), 5.17 (d, J = 5.5 Hz, 1H, pcym−H), 5.01 (d, J = 5.7 Hz, 1H, p-cym−H), 4.43 (d, J = 5.4 Hz, 1H,
p-cym−H), 3.72 (s, 3H, O−CH3), 3.62−3.59 (m, 4H, O−CH2), 3.48
(s, 2H, CH2), 2.41 (d, J = 10.2 Hz, 8H), 2.17 (s, 3H, Ar−CH3), 1.90
(s, 3H, Ar−CH3), 1.20−1.14 (m, 6H, p-cym−iPr) (Figure S21). 13C
NMR (100 MHz, DMSO-d6): δ 165.6, 158.2, 153.5, 151.3, 135.4,
130.9, 130.3, 129.1, 128.5, 127.0, 120.3, 115.8, 112.1, 102.5, 95.5,
84.4, 83.7, 82.7, 79.9, 66.2, 61.9, 56.0, 29.9, 22.3, 21.5, 19.2, 18.1, 16.8
(Figure S22). IR (KBr pellets, cm−1): 1601 (CHN) (Figure S29).
UV−vis [MeOH; λmax, nm (ε, M−1 cm−1)]: 243 (28900), 304 (8630),
422 (1960), 490 (980) (Figure S28). ESI-HRMS (MeOH). m/z
(exp) 589.2004 (589.1985) [RuIIC31H39N2O3+]. Anal. Calcd for
C31H39ClN2O3Ru: C, 59.65; H, 6.30; N, 4.49. Found: C, 59.46; H,
6.27; N, 4.45.
Ru(p-cym)(L5)Cl (5). Yield: 62%. 1H NMR (400 MHz, DMSO-d6):
δ 7.98 (s, 1H, CHN), 7.62 (m, 3H, Ar−H), 7.26 (t, J = 7.5 Hz, 1H,
Ar−H), 7.19 (d, J = 15.3 Hz, 2H, Ar−H), 7.11 (t, J = 7.4 Hz, 1H, Ar−
H), 6.94 (d, J = 9.2 Hz, 1H, Ar−H), 5.32 (d, J = 6.0 Hz, 1H, p-cym−
H), 5.14−5.09 (m, 2H, p-cym−H), 4.40 (d, J = 5.6 Hz, 1H, p-cym−
H), 3.62 (s, 4H, O−CH2), 3.51 (s, 2H, CH2), 2.54 (m, 1H, pcym−iPr), 2.48 (s, 3H, p-cym−CH3), 2.43 (s, 4H, N−CH2), 2.16 (s,
3H, Ar−CH3), 1.93 (s, 3H, Ar−CH3), 1.22 (t, J = 6.64 Hz, 6H, pcym−iPr) (Figure S23). 13C NMR (100 MHz, DMSO-d6): δ 166.8,
158.5, 154.3, 134.8, 134.3, 129.1,128.5, 128.4, 127.3, 125.6, 124.4,
121.6, 119.5, 111.1, 102.5, 94.8, 85.7, 84.2, 82.7, 80.0, 66.2, 62.0, 30.2,
22.2, 21.8, 19.2, 17.9, 17.0 (Figure S24). IR (KBr pellets, cm−1): 1615
(CHN) (Figure S29). UV−vis [MeOH; λmax, nm (ε, M−1 cm−1)]:
254 (42200), 320 (14480), 422 (3630), 479 (1910) (Figure S28).
ESI-HRMS (MeOH). m/z (exp) 609.2055 (609.1995)
[RuIIC34H39N2O2+]. Anal. Calcd for C34H39ClN2O2Ru: C, 63.39; H,
6.10; N, 4.35. Found: C, 63.47; H, 6.14; N, 4.39.
Ru(p-cym)(L6)Cl (6). Yield: 62%. 1H NMR (500 MHz, DMSO-d6):
δ 7.95 (s, 1H, CHN), 7.63 (dd, J = 19.1 and 8.5 Hz, 2H, Ar−H),
7.47 (d, J = 8.6 Hz, 1H, Ar−H), 7.28 (m, 3H, Ar−H), 7.12 (d, J = 7.4
Hz, 1H, Ar−H), 6.93 (d, J = 9.2 Hz, 1H, Ar−H), 5.40 (d, J = 6.1 Hz,
1H, p-cym−H), 5.34 (d, J = 6.2 Hz, 1H, p-cym−H), 4.97 (d, J = 5.6
Hz, 1H, p-cym−H), 4.36 (d, J = 5.7 Hz, 1H, p-cym−H), 4.08 (m, 1H,
i
Pr), 3.64 (s, 4H, O−CH2), 3.58 (s, 2H, −CH2), 3.01 (m, 1H, iPr),
2.65−2.60 (m, 1H, p-cym−iPr), 2.44 (s, 4H, N−CH2), 1.87 (s, 3H,
Ar−CH3), 1.42 (d, J = 6.8 Hz, 3H, iPr), 1.33 (dd, J = 12.8 and 6.8 Hz,
6H, iPr), 1.24 (d, J = 6.9 Hz, 3H, iPr), 1.06 (d, J = 6.6 Hz, 3H, iPr),
0.80 (d, J = 6.7 Hz, 3H, iPr). 13C NMR (125 MHz, DMSO-d6): δ
167.2, 158.9, 135, 134.1, 128.6, 128.5, 127.6, 127.5, 125.7, 124.3, 124,
121.6, 118.2, 110, 102.1, 95, 86.2, 83, 82.5, 79.3, 66.2, 62.4, 53.1, 30.3,
27.3, 26.6, 26.3, 25.9, 25.8, 22.8, 22.1, 21.2, 17.1 (Figures S25−S27).
IR (KBr pellets, cm−1): 1590 (CHN) (Figure S29). UV−vis
[MeOH; λmax, nm (ε, M−1 cm−1)]: 254 (40460), 322 (3160), 429
(3490), 494 (1730) (Figure S28). ESI-HRMS (MeOH). m/z (exp)
665.2681 (665.2664) [Ru II C 38 H 47 N 2 O 2 + ]. Anal. Calcd for
C38H47ClN2O2Ru: C, 65.17; H, 6.76; N, 4.00. Found: C, 65.32; H,
6.72; N, 4.08.
X-ray Crystallography. Single crystals of complexes 1−6 were
obtained from a methanolic solution of the respective complexes by
slow evaporation at 25 °C. All of the solutions were kept in the dark
during the slow evaporation process to obtain crystals. Good-quality
single crystals suitable for diffraction were mounted over a loop of the
goniometer of a SuperNova, Dual, Cu at zero, Eos diffractometer. The
data of the crystals were collected at 100(1) K to enhance the stability
C
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of the crystal during diffraction and minimize the probability volume
of the thermal ellipsoids. Cu Kα was used as the X-ray source for data
collection due to the nonavailability of Mo Kα. Data reduction was
done using the CrysAlisPro171.37.33c software. The reduced data
were then taken to solve the structure using the ShelXT structure
solution program and consecutively refined with the ShelXL
refinement package using least-squares minimization in Olex2. The
structures were deposited to the CCDC database, and the deposition
numbers are 2081907−2081911.
Solution Stability Study. 1H NMR studies were performed at
different time points at 25 °C for a period of 24 h to estimate the
stability of the complexes in solution. The solution composed of 3:7
(v/v) dimethyl sulfoxide (DMSO)-d6/20 mM phosphate buffer at
pH* 7.4 (pH meter reading without correction for the effects of D on
the glass electrode) and 4 mM NaCl in D2O at 25 °C.
Distribution Coefficient Determination. The standard shakeflask method with an octanol/water mixture following OECD
guidelines80 led to determination of the distribution coefficient (log
Do/w) of the complexes. A known amount of complex was solubilized
with n-octanol presaturated with a 130 mM NaCl solution. The
solution was continuously shaken at 25 °C on an orbital shaker for 12
h, followed by centrifugation of the biphasic solution for 3 min, to
allow complete phase separation. The aliquot from each layer was
measured separately in a UV−vis spectrophotometer with the
appropriate dilution. Each complex was measured in triplicate. The
concentrations of the complexes were calculated from the molar
extinction coefficient, and their ratios provided the distribution
coefficient (log Do/w).
Cell Lines and Culture Condition. The cells were grown in a
100 mm Petri dish with an adherent monolayer and maintained at the
logarithmic phase in a 5% CO2 atmosphere using the appropriate
culture media, supplemented with 10% fetal bovine serum (FBS;
Gibco) and a 1× antibiotic−antimycotic solution. Hep-G2 was grown
in Minimal Essential Medium, while MiaPaCa2 and MDA-MB-231
were cultured in Dulbecco’s Modified Eagle Medium (DMEM) and
in a 1:1 mixture of DMEM with Ham’s F12 nutrient mixture (i.e.,
DMEM/F-12), respectively.
Cell Viability Assay. The effect of the complexes on the growth
inhibition of the tumor cell lines (MDA-MB-231, Hep-G2, and
MiaPaCa2) was assessed with MTT assay. About 6 × 103 cells were
added in each well of a 96-well plate in the appropriate medium (200
μL) and incubated at 37 °C in a 5% CO2 atmosphere. After
incubation for 24 h, the medium was removed, and fresh medium
(200 μL) was added. The compound to be tested was added at
different concentrations in the wells. The compounds were solubilized
in DMSO and added to the respective media such that the
concentration of DMSO in the well was less than 0.2%. Oxaliplatin
was solubilized in N,N-dimethylformamide (DMF) and added to the
respective media such that the concentration of DMF in well was less
than 0.2%. Upon incubation at 37 °C for 72 h, the medium was
removed and renewed with a fresh medium (200 μL) containing 0.1
mg mL−1 MTT. After 3 h of incubation at 37 °C, the medium was
replaced with 200 μL of DMSO. The inhibitory effect on the cells was
calculated by measuring the absorbance of the drug-treated cells to
the untreated ones at 570 nm using a Biotech SYNERGY H1M
microplate reader. Each assay was performed in triplicate. IC50 values
(the drug concentration at which 50% of the cell growth is inhibited)
were calculated by fitting curves with nonlinear regression in
GraphPad Prism 5, version 5.03, by plotting the percent of cell
viability versus the logarithm of the drug concentration in micromolar.
Detection of Apoptosis: PE-Annexin-V/7-AAD Assay. Apoptosis of cells was detected using the PE-Annexin-V and 7-AAD dual
staining apoptosis detection kit (BD Pharmingen) by flow cytometry
according to the manufacturer’s protocol. About 1 × 105 MDA-MB231 cells were seeded in a 6-well plate and incubated for 72 h at 37 °C
in a 5% CO2 atmosphere. Subsequently, the cells were treated with
complexes 3−5 for 8 h with a IC50 solution concentration. Treated
and untreated cells were then harvested with ice-cold 1× phosphatebuffered saline (PBS) containing 0.1 mM ethylenediaminetetraacetic
acid, subsequently washed with cold 1 × PBS twice, and finally
Article
resuspended in an Annexin-V binding buffer. Cells were then
incubated with both PE-Annexin-V and 7-AAD for 15 min in the
dark at 25 °C. Data were analyzed in a BD Biosciences FACS Calibur
flow cytometer within 1 h of sample preparation.
Acridine Orange (AO) Assay. About 5 × 104 MDA-MB-231 cells
were seeded in a 30 mm glass bottom Petri dish and incubated up to
70% confluency at 37 °C in a 5% CO2 atmosphere. Then the medium
was renewed and treated with complexes 2 and 6 at IC50 for 2.5 h.
Then the medium was removed, and the cells were washed with 1×
PBS twice and incubated with AO (2 μM) in the medium for 15 min
at 37 °C in a 5% CO2 atmosphere. AO was removed, and the cells
were washed with 1× PBS twice and visualized under a confocal laser
scanning microscope (Leica TCS SP8). Excitation was at 488 ± 10
nm, and emissions were collected at 520 ± 20 nm (green) and 625 ±
20 nm (red).
Immunofluorescence. About 5 × 104 cells of MDA-MB-231
were seeded in 24-well plates over a glass coverslip and incubated up
to 70% confluency. The cells were treated with complexes 2 and 6 at
IC50 for 6, 12, and 24 h at 37 °C in a 5% CO2 atmosphere. Then
media containing drugs were removed and washed with ice-cold 1×
PBS twice. The cells were fixed with a 2% (v/v) paraformaldehyde
solution for 20 min and quenched with 50 mM ammonium chloride
for 20 min. The cells were washed with 1× PBS twice and blocked
with 1% bovine serum albumin (BSA) and 0.075% saponin in 1× PBS
for 1 h at room temperature. Following blocking, the cells were
washed once with 1× PBS and incubated with the primary antiLAMP1 antibody (antihuman Lamp1, mouse monoclonal antibody,
H4A3, and DSHB) and anti-LAMP2 antibody (DSHB and H4B4)
and mouse monoclonal anti-cathepsin D antibody (BD Biosciences
C47620) in 0.5% BSA for 2 h in a humid chamber at room
temperature. This step was further followed by three washes with 1×
PBS. The cells were incubated with the highly cross-adsorbed
secondary antibody donkey anti-mouse IgG (H+L; Alexa Fluor 568,
Invitrogen A10037) and Phalloidin-iFluor647 (Abcam ab176759) in
0.5% BSA in 1 × PBS for 1 h 30 min at room temperature in a humid
chamber, washed with 1× PBS twice, mounted on slides using 4′,6diamidino-2-phenylindole (DAPI)-containing mounting media (Fluoroshield with DAPI, F6057, Sigma-Aldrich), and imaged under a
confocal laser scanning microscope (Leica TCS SP8). Emission was
collected at 568 nm upon excitation at 552 nm.
Immunoblot Assay. MDA-MB-231 cells were grown in DMEMF12 supplemented with 10% FBS and antibiotics at 37 °C and 5%
CO2. To test the effect of complexes 2 and 6 on lysosomal
degradation, we checked the expression level of the lysosomal
membrane proteins (LAMP1), the autophagy markers (LC3B), and
the level of mTORC1 downstream effectors (phospho-p70S6K/total
p70S6K). The cells were grown up to 70% confluency and treated
with a IC50 dose of complexes 2 and 6 for 2, 6, and 12 h. The cells
were lysed using a RIPA lysis buffer, and the protein samples were
resolved by running through sodium dodecyl−sulfate polyacrylamide
gel electrophoresis. The protein was transferred to a nitrocellulose
membrane and blocked with 5% skim milk in 1× TBST (a mixture of
tris-buffered saline and Tween 20) for 2 h at room temperature. After
blocking, the membrane was incubated with a primary antibody
diluted in 3% skim milk in 1× TBST overnight at 4 °C. Following
primary antibody incubation, the membrane was washed with 1×
TBST and the protein abundance of the respective markers was
detected using a horseradish peroxidase-conjugated secondary
antibody.
■
RESULTS AND DISCUSSION
Synthesis and Characterization. The N,O-coordinating
ligands HL1−HL6 were prepared by refluxing the respective
amine and aldehyde in MeOH for 24 h. The product was
obtained either by precipitation or by column chromatography
(silica gel 60−120 mesh). The ruthenium(II) p-cymene
complexes 1−6 were synthesized by stirring the ligands with
1 equiv of KOH, followed by the addition of Ru2(p-cym)2Cl4
in MeOH at 25 °C for 12−18 h, as shown in Scheme 1. The
D
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Scheme 1. Synthesis of Ligands HL1−HL6 and Ruthenium Complexes 1−6
Figure 2. ORTEP diagrams of the molecular structures of complexes 2−6. Thermal ellipsoids are drawn at the 50% probability level. All H atoms
are omitted for clarity.
vis spectra suggest that there are π−π* and ligand-to-metal
charge-transfer transitions ca. 290−321, 405−435, and 460−
495 nm, respectively (Figure S28). The IR data show that the
stretching frequency for the imine bonds is 1580−1615 cm−1
for 1−6 (Figure S29).
product was isolated in pure form by column chromatography
in neutral alumina. The complexes presented are new, and all
of them are well-characterized by 1H and 13C NMR, ESIHRMS, FT-IR, and UV−vis analysis. Complexes 2−6 are also
characterized by single-crystal X-ray crystallography. The UV−
E
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Table 1. Selected Bond Lengths (Å) of Complexes 2−6
2
Ru1−Cl1
Ru1−O1
Ru1−N1
Ru1−C20
Ru1−C21
Ru1−C22
Ru1−C23
Ru1−C24
Ru1−C25
3
2.424(2)
2.056(2)
2.100(2)
2.192(3)
2.196(3)
2.179(3)
2.217(3)
2.194(3)
2.185(3)
5
Ru1−Cl1
Ru1−O1
Ru1−N1
Ru1−C25
Ru1−C26
Ru1−C27
Ru1−C28
Ru1−C29
Ru1−C30
Ru1−Cl1
Ru1−O1
Ru1−N1
Ru1−C21
Ru1−C22
Ru1−C23
Ru1−C24
Ru1−C25
Ru1−C26
4
2.418(1)
2.053(2)
2.096(2)
2.199(3)
2.202(3)
2.176(3)
2.236(3)
2.192(3)
2.198(3)
2.432(6)
2.054(2)
2.101(2)
2.215(2)
2.178(2)
2.180(2)
2.202(2)
2.209(2)
2.189(2)
Ru1−Cl1
Ru1−O1
Ru1−N1
Ru1−C22
Ru1−C23
Ru1−C24
Ru1−C25
Ru1−C26
Ru1−C27
6
2.427(1)
2.070(1)
2.110(2)
2.201(2)
2.198(2)
2.179(2)
2.210(2)
2.175(2)
2.194(2)
Ru1−Cl1
Ru1−O1
Ru1−N1
Ru1−C29
Ru1−C30
Ru1−C31
Ru1−C32
Ru1−C33
Ru1−C34
2.418(5)
2.058(1)
2.090(2)
2.215(2)
2.189(2)
2.214(2)
2.201(2)
2.180(2)
2.176(2)
Table 2. Selected Bond Angles (deg) of Complexes 2−6
2
O1−Ru1−Cl1
O1−Ru1−N1
N1−Ru1−Cl1
O1−Ru1−C20
O1−Ru1−C21
O1−Ru1−C22
O1−Ru1−C23
O1−Ru1−C24
O1−Ru1−C25
O1−Ru1−Cl1
O1−Ru1−N1
N1−Ru1−Cl1
O1−Ru1−C25
O1−Ru1−C26
O1−Ru1−C27
O1−Ru1−C28
O1−Ru1−C29
O1−Ru1−C30
3
83.00(6)
86.90(6)
86.90(6)
87.90(10)
112.93(10)
150.57(10)
159.46(10)
121.78(10)
93.29(10)
5
O1−Ru1−Cl1
O1−Ru1−N1
N1−Ru1−Cl1
O1−Ru1−C21
O1−Ru1−C22
O1−Ru1−C23
O1−Ru1−C24
O1−Ru1−C25
O1−Ru1−C26
4
82.92(6)
87.42(9)
85.82(6)
86.13(10)
93.88 (10)
124.50(10)
161.45(10)
146.31(10)
108.95(10)
82.92(6)
87.42(9)
85.82(6)
95.71(8)
127.24(8)
164.19(8)
144.46(8)
108.13(8)
87.71(8)
O1−Ru1−Cl1
O1−Ru1−N1
N1−Ru1−Cl1
O1−Ru1−C29
O1−Ru1−C30
O1−Ru1−C31
O1−Ru1−C32
O1−Ru1−C33
O1−Ru1−C34
X-ray Crystallography. Single crystals of each complex
were obtained from a methanolic solution of the complexes
following a slow evaporation method. Complexes 2−4
crystallized in a monoclinic system with space group P21/c,
whereas complexes 5 and 6 crystallized in a triclinic system
with P1̅ space group. The crystal structure of each complex
showed a chelating N,O coordination bond from the bidentate
Schiff base ligands and monodentate coordination by chloride
(Figure 2). The fourth position was occupied by a p-cymene
ring coordinating with the Ru center in an η6 fashion. Thus, the
RuII centers in these complexes are in a pseudooctahedral
geometry (Figure 2). In complexes 2−4, each unit cell
contained four complexes, whereas for complexes 5 and 6, the
unit cell contained two complexes. Some important bond
distances, angles and crystallographic parameters are listed in
Tables 1−3. The Ru−Cl bond distances of the complexes
range between 2.42 and 2.43 Å, which may be considered
O1−Ru1−Cl1
O1−Ru1−N1
N1−Ru1−Cl1
O1−Ru1−C22
O1−Ru1−C23
O1−Ru1−C24
O1−Ru1−C25
O1−Ru1−C26
O1−Ru1−C27
6
84.31(4)
87.68(6)
85.93(4)
86.25(6)
95.45 (6)
126.97(6)
163.86(6)
144.05(7)
107.31(6)
84.25(5)
86.43(6)
84.60(5)
93.82(7)
88.05(7)
110.04(7)
146.84(7)
162.04(8)
124.28(8)
marginally longer than the Ru−Cl distances of ca. 2.39−2.41 Å
observed for our earlier reported N,N-coordinated complexes.9,81,82 The Ru−O bond distances of the complexes
range between 2.06 and 2.07 Å, and the Ru−C bond lengths of
p-cymene are in the range of 2.18−2.21 Å (Table 2). The
distance between the centroid of the p-cymene ring and RuII in
the 2,6-diisopropylaniline-based N,N-coordinated complex is
ca. 1.70 Å,83 whereas in our complexes, it is ca. 1.68 Å; thus,
the interaction between p-cymene and RuII is stronger in the
N,O-coordinated complexes. In complexes 3−6, weak
intermolecular hydrogen bonding is present, displaying D···A
distances of 3.4−3.8 Å with a ∠D−H···A of 138−162°,
between a RuII-coordinated Cl atom and an aromatic H atom
of the p-cym attached to a RuII from a neighboring complex.
Another intermolecular weak hydrogen-bonding interaction is
present in 3, 5, and 6 among a Ru-coordinated O atom and an
aromatic H atom of p-cym within the range of 3.2−3.4 Å (D···
F
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Table 3. Selected Crystallographic Parameters for Complexes 2−6
empirical formula
radiation
fw/(g/mol)
temp/K
cryst syst
space group
a/Å
b/Å
c/Å
α/deg
β/deg
γ/deg
volume/Å3
Z
ρcalc/(mg/cm3)
μ/mm−1
F(000)
cryst size/mm3
2θ range for data
collection/deg
index ranges
reflns collected
indep reflns
data/restraints/
param
GOF on F2
final R indexes [I ≥
2σ (I)]
final R indexes [all
data]
largest diff
peak/hole/(e /Å3)
2
3
4
5
6
C29H30ClNORu
Cu Kα (λ = 1.54184)
545.06
100.00(10)
monoclinic
P21/c
7.96410(10)
17.0207(3)
17.4922(3)
90
92.9190
90
2368.07(7)
4
1.529
6.561
1120.0
0.3647 × 0.2192 ×
0.0624
7.252−132.336
C30H37ClN2O2Ru
Cu Kα (λ = 1.54184)
594.13
94.8(4)
monoclinic
P21/c
8.8322(3)
14.4477(5)
21.3187(7)
90
98.241(4)
90
2692.28(16)
4
1.466
5.856
1232.0
0.111 × 0.0468 × 0.0213
C31H39ClN2O3Ru
Cu Kα (λ = 1.54184)
624.16
100.00(10)
monoclinic
P21/c
9.5488(2)
14.0931(3)
21.2814(4)
90
92.943(2)
90
2860.11(10)
4
1.450
5.568
1296.0
0.4614 × 0.2865 × 0.1352
C34H39ClN2O2Ru
Cu Kα (λ = 1.54184)
644.19
100.00(10)
triclinic
P1̅
10.7783(5)
10.8455(5)
13.4383(5)
85.531(4)
83.630(4)
69.294(4)
1459.08(12)
2
1.466
5.452
668.0
0.1333 × 0.1333 × 0.0582
C38H47ClN2O2Ru
Cu Kα (λ = 1.54184)
700.326
101(2)
triclinic
P1̅
11.0249(5)
11.7028(5)
13.9028(4)
83.556(3)
89.613(3)
69.622(4)
1669.88(12)
2
1.393
4.808
732.0
0.125 × 0.125 × 0.0480
7.416−132.42
7.526−132.158
6.624−132.266
6.402−132.418
−9 ≤ h ≤ 9, −20 ≤ k ≤
18, −20 ≤ l ≤ 20
41894
4142 [Rint = 0.0472, Rσ =
0.0178]
4142/0/303
−8 ≤ h ≤ 10, −17 ≤ k ≤
11, −24 ≤ l ≤ 25
11310
4695 [Rint = 0.0392, Rσ =
0.0472]
4695/0/330
−10 ≤ h ≤ 11, −16 ≤ k ≤
15, −23 ≤ l ≤ 25
11725
4970 [Rint = 0.0247, Rσ =
0.0274]
4970/0/349
−11 ≤ h ≤ 12, −12 ≤ k ≤
12, −15 ≤ l ≤ 15
10491
5063 [Rint = 0.0282, Rσ =
0.0347]
5063/0/366
−12 ≤ h ≤ 13, −13 ≤ k ≤
13, −16 ≤ l ≤ 16
13122
5716 [Rint = 0.0369, Rσ =
0.0424]
5716/0/404
1.068
R1 = 0.0305, wR2 =
0.0748
R1 = 0.0314, wR2 =
0.0754
0.99/−0.64
1.033
R1 = 0.0334, wR2 =
0.0785
R1 = 0.0393, wR2 =
0.0820
0.89/−0.95
1.063
R1 = 0.0241, wR2 = 0.0611
1.059
R1 = 0.0288, wR2 = 0.0723
1.023
R1 = 0.0270, wR2 = 0.0650
R1 = 0.0250, wR2 = 0.0617
R1 = 0.0310, wR2 = 0.0740
R1 = 0.0301, wR2 = 0.0664
0.37/−0.70
1.47/−0.77
0.42/−0.65
R1 = ∑|Fo| − |Fc||/∑|Fo|. bwR2 = [∑[w(Fo2 − Fc2)2]/∑w(Fo2)2]1/2.
a
A) with a ∠D−H···A of ca. 142−175°. In 4, a weak hydrogenbonding interaction is present among an O atom of methoxy
and an aromatic H atom of p-cym having a distance of ∼3.4 Å
(D···A) with a ∠D−H···A of ca. 143°. In 2, a weak hydrogenbonding interaction is present between the Ru-coordinated Cl
atom and aromatic H atom with a distance of ca. 3.7 Å (D···A)
and a ∠D−H···A of ca. 153−156°. Besides, a weak π···πstacking interaction is also observed in complex 2 (Figure
S30).
Stability in Solution. The solution stability of the
complexes was investigated to understand whether the
complexes dissociate or hydrolyze to monoaquated species in
solution over time. The solution stability was measured at a
physiological pH of 7.4 in a 20 mM phosphate buffer having 4
mM NaCl and mixed with DMSO-d6 in a 7:3 (v/v) ratio. Two
complexes were investigated to represent the solution stability.
These complexes, 3 and 4, hydrolyzed immediately upon
dissolution to form the monoaquated complex releasing the
halide (Figure 3). The immediate hydrolysis was confirmed by
matching the chemical shifts of the aforementioned sample
with a sample incubated with 1 equiv of AgNO3 to precipitate
AgCl and lead to the monoaquated complex. The sample
without added AgNO3 and with AgNO3 showed the exact
same chemical shifts, confirming the immediate hydrolysis
(Figure 3). The hydrolyzed complexes are stable for at least 24
h (time-dependent NMR was done only for a 24 h period). We
also investigated the stability of complex 4 as a representative
in the series for up to 72 h through 1H NMR and found that it
is mostly intact even upon 72 h of incubation (Figure S32).
Complexes 2, 5, and 6 are also hydrolyzed immediately
(Figure S31), but they have poor solubility in the
concentrations (ca. 2 mM) used for NMR and precipitate
after 1 h. In order to understand whether this was due to
coordination with inorganic phosphate, we investigated the
precipitate obtained from the most active complex 6, which
was soluble in MeOH, and studied its ESI-MS, but we found
the monopositive dehalogenated complex [Ru(p-cym)(L6)]+
at m/z 665.2697 (calcd 665.2676) and the dipositive
dehalogenated complex [Ru(p-cym)(HL6)] + at m/z
333.1382 (calcd 333.1374) (Figure S33). The 31P NMR of
the precipitate did not provide any P signal from the
precipitate in CD3OD. One reason could be that the
precipitate does not have a good solubility. The ESI-MS
study of the precipitate did provide a small peak at m/z
850.3422 corresponding to a monomeric ruthenium distribution, but the peak could not be assigned to any phosphate
adduct.
Distribution Coefficient Determination. Lipophilicity is
an important property of a drug for its absorption, distribution,
potency, and elimination. The lipophilicity was determined
G
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Figure 3. Stability of 3 and 4 in a 20 mM phosphate buffer (pH* 7.4) with 4 mM NaCl and 30% DMSO-d6.
with the standard shake-flask method in n-octanol and water as
per the OECD guidelines.80,84 The log D values of complexes
1−6 decreased in the presence of the pendant morpholine
compared to the otherwise same complexes without the
morpholine motif (Figure 4). Thus, the presence of morpho-
carcinoma (MDA-MB-231), human pancreatic carcinoma
(MiaPaCa2), and hepatocellular carcinoma (Hep-G2), all of
which belong to the category of cancers that are relatively
difficult to cure. A comparison of the antiproliferation data
between the complexes with and without the morpholine motif
of the otherwise same ligands, e.g., complexes 1 and 3, shows
that the nonmorpholine-based 1 is more active than the
corresponding pendant morpholine bearing 3 (Table 4). The
difference in the antiproliferative activity minimizes when we
compare 2 and 5. Both 2 and 5 show comparable activity in
MDA-MB-231 and Hep-G2, but 5 is more active in MiaPaCa2.
Table 4. In Vitro Anticancer Activity of Complexes 1−6 in
Various Cancer Cell Lines under Normoxic Conditions
IC50a ± SDb (μM)
Figure 4. Lipophilicity of complexes 1−6 in a 1:1 (v/v) octanol and
water mixture at 25 °C.
line increases the water solubility. The log D continuously
increases for 3−6 because of an increase in the presence of
hydrophobic motifs (viz. naphthyl and isopropyl groups). All
of the complexes exhibit positive log D values in the range of
ca. 0.6−2.2, suggesting that the lipophilicity is well within the
required limits as per Lipinski’s rule of five.
Antiproliferative Activity. The in vitro antiproliferative
activity of complexes 1−6 was tested in three different cell
lines by MTT assay under normoxic conditions. The cell lines
chosen were triple-negative human metastatic breast adeno-
compound
MDA-MB-231
Hep-G2
MiaPaCa2
HL1
1
HL2
2
HL3
3
HL4
4
HL5
5
HL6
6
oxaliplatin
>50
1.9 ± 0.4
>50
1.6 ± 0.3
>100
4.4 ± 0.4
>100
3.0 ± 0.8
>50
1.5 ± 0.3
>50
1.2 ± 0.3
19.2 ± 1.227
ND
5.8 ± 0.9
ND
4.1 ± 0.5
ND
13.9 ± 1.9
ND
9.1 ± 2.2
ND
4.4 ± 0.4
ND
2.8 ± 0.1
9.8 ± 0.3
ND
3.6 ± 0.3
ND
3.1 ± 0.3
ND
8.4 ± 1.6
ND
7.3 ± 2.1
ND
2.0 ± 0.6
ND
2.0 ± 0.2
5.7 ± 0.2
a
MTT assay was performed in normoxic conditions after 72 h of
incubation. bStandard deviation.
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Figure 5. (a) Distribution of LAMP-2 in the MDA-MB-231 cells after incubation with 2 and 6 after 6 and 24 h. (b) Immunofluorescence study of
cathepsin D after incubation with IC50 doses of 2 and 6 for 4 h.
disrupt the lysosomal membrane integrity and breaks them
into smaller vesicles, then the intracellular distribution of
LAMP-1 or LAMP-2 would be different from the control, but
as displayed in Figure 5a, the distribution remains almost
comparable to that of untreated control cells. When the MDAMB-231 cells are incubated with 2 and 6 at IC50 doses for 24 h,
the LAMP-2 population seems not to differ too much to show
a higher dispersion in the cytosol compared to the control. The
above result implies that the lysosomal membrane integrity is
preserved. However, the compound-treated cells show
cathepsin D translocation to the nucleus (Figure 5b). So,
there is a release of cathepsin D from the lysosomes as a result
of treatment of the complexes, and it translocates to the
nucleus. It is well-known that the release of cathepsins in
cytosol may lead to caspase-dependent or -independent cell
killing by apoptosis or necrosis.87
To understand whether the ruthenium(II) complexes (viz. 2
and 6) are affecting the formation and activation of signaling
complexes on the lysosomal membrane, we investigated
expression of the downstream effectors of active mTORC1
in MDA-MB-231 cells treated with 2 and 6. It is known that
the activation of mTORC1 leads to phosphorylation of
ribosomal protein S6 kinase (p70S6K) and the phosphorylated
p70S6K regulates cell growth, proliferation, and protein
synthesis.88,89 The ratio phosphorylated p70S6K to the total
cellular pool of p70S6K is indicative of the mTORC1 activity.
Our results show that the expression is not changed (Figure
S38c) upon treatment with the complexes. Furthermore, we
also investigated whether autophagy is activated by the
Complex 6, which is more hydrophobic (Figure 4), shows the
highest effect (IC50 = ca. 1.2−2.0 μM) in the series (Figures
S34−S37). Complexes 2, 5, and 6 were also investigated in the
nontumorigenic cell line HEK293 to see if they are less toxic to
other cells. The IC50 values obtained are 1.7 ± 0.3, 1.5 ± 0.3,
and 1.4 ± 0.2 μM for 2, 5, and 6, respectively (Figure S37),
suggesting that they are also toxic to noncarcinogenic cells like
cisplatin or oxaliplatin. Notably, most of the complexes require
lower doses than the oxaliplatin in killing the same cancer cells.
Complex 6 is ca. 9 times more effective against the TNBC,
MDA-MB-231, compared to oxaliplatin. It is evident that the
presence of the naphthyl group and diisopropyl substitution in
the aniline is more important in enhancing the antiproliferative
activity and lipophilicity.
Investigation of the lysosomal role in the pathway of
cell killing. The complexes were designed with or without
morpholine as pendant motifs so lysosome membrane
permeabilization was studied. MDA-MB-231 cells were stained
with AO for monitoring the fate of the acidic lysosomal
vesicles. The red puncta for control imply the presence of
acidic vesicles (viz. lysosomes), and after 2.5 h treatment with
complexes 2 and 6, the red dots remained (Figure S38b),
which implies that the lysosomes remained acidic. The
lysosomal membrane has a lipid bilayer that contain
glycoproteins. The most abundant lysosomal membrane
proteins are lysosome-associated membrane proteins 1 and 2
(LAMP-1 and LAMP-2).85,86 The inner lumen of these
proteins is highly glycosylated and protects the lysosomal
membrane from the digestive enzymes. If the complexes
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Figure 6. (a) Expression of LC3B in MDA-MB-231 cell lines after incubation with 2 and 6 for 6, 12, and 24 h. (b) Expression of caspase-3, Bcl-2,
and Bid in MDA-MB-231 cell lines after incubation with 2 and 6 for 6 and 12 h. (c) Tabular representation of the induction of apoptosis and
necrosis by 3−5 in MDA-MB-231 cells. (d) Bar diagram of the induction of apoptosis and necrosis by 3−5 in MDA-MB-231 cells.
complexes by investigating expression of LC3B. During
autophagy, LC3B-I is converted to LC3B-II,90−96 so the
increased ratio of LC3B-II to LC3B-I would indicate
autophagic induction. In our case, the ratio remains the
same, suggesting that autophagy is not initiated (Figure 6a).
Because the complexes are not targeting autophagic cell death,
we monitored the mitochondrial Bcl-2, which is a regulator
protein localized in the outer mitochondrial membrane to
regulate apoptosis.97−99 However, Bcl-2 abundance remains
unaltered in cells treated with the compound (Figure 6b) for
both complexes, suggesting that mitochondria-mediated
apoptosis may not be happening. This was further confirmed
when we investigated caspase-3-mediated apoptosis. The
immunoblot results show that the activation of caspase-3 did
not take place but rather active caspase-3 is absent (Figure 6b)
in cells treated with 2 and 6, suggesting that the cell death is
caspase-independent. The cell death pathway was further
investigated by PE-Annexin-V and 7-AAD double staining
assay in MDA-MB-231 cells for 3−5 using flow cytometry.
Treatments of 8 h with IC50 doses of 3−5 induced ∼13%,
∼10%, and ∼17% late apoptosis and ∼8%, ∼9%, and ∼11%
necrotic/dead cells, respectively (Figures 6c,d and S39), thus
indicating that caspase-independent apoptosis and necrotic cell
death is the probable cell killing pathway for these complexes.
This is a major change in the pathway of action from our
earlier-reported complexes of the same genre.11,81 Complexes
3−5 have the morpholine motif attached to the 2,6disubstituted anilines, but complex 2 does not have the
pendant morpholine but also does not activate caspase-3
(Figure 6b) and translocate cathepsin D to the nucleus (Figure
5b). Thus, irrespective of the presence of the morpholine
motif, the combination of the aldehyde and the disubstituted
anilines renders ruthenium(II) p-cymene complexes that
induce a caspase-independent apoptosis and necrotic pathway
of cell killing.
■
CONCLUSIONS
■
ASSOCIATED CONTENT
The N,O-coordinating 2,6-disubstituted aniline-based Schiff
bases form ruthenium(II) p-cymene complexes that immediately hydrolyze in solution to form monoaquated complexes
stable at pH 7.4 for at least 24 h. The presence or absence of
the pendant morpholine conjugated to the aniline did not
improve the antiproliferative activity, which is evident from the
similarity of the dosage required for complexes 2 and 5. These
complexes do not seem to dismantle the lysosome, but their
presence leads to translocation of cathepsin D, which is not
dependent on the presence of the morpholine in the ligand.
The complexes display high cytotoxicity to the cancer cells.
However, the cytotoxicity is not mediated by autophagy or
caspase-dependent apoptosis, but rather caspase-independent
apoptosis and necrosis are the possible pathways, as is evident
from the immunoblotting and flow cytometry studies.
sı Supporting Information
*
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.1c01363.
NMR spectra of ligands (Figures S1−S12) and
complexes (Figures S13−S27), UV−vis spectra of the
complexes (Figure S28), IR spectra of the complexes
(Figure S29), interaction of the crystals in the unit cell
(Figure S30), aquation of 2, 5, and 6 (Figure S31),
aquation of 4 up to 72 h (Figure S32), ESI-MS spectrum
of the precipitate of complex 6 obtained from an NMR
tube during time-dependent stability (Figure S33), MTT
assay (Figures S34−S37), investigation of lysosomal
changes (Figure S38), and investigation of apoptosis
(Figure S39) (PDF)
J
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Accession Codes
R.C. thanks Dr. Sourav Acharya for antiproliferative activity
data of oxaliplatin. A.G. is grateful for an Early Career Research
Award from the Department of Science and Technology,
Government of India (ECR/2015/000220) and a Wellcome
Trust-DBT India Alliance Fellowship (IA/I/16/1/502369).
We thank Tamal Ghosh for helping us with the flow cytometry
analysis studies.
CCDC 2081907−2081911 contain the supplementary crystallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
■
Article
■
AUTHOR INFORMATION
Corresponding Authors
REFERENCES
(1) Kenny, R. G.; Marmion, C. J. Toward Multi-Targeted Platinum
and Ruthenium Drugs-A New Paradigm in Cancer Drug Treatment
Regimens? Chem. Rev. 2019, 119 (2), 1058−1137.
(2) Florea, A.-M.; Buesselberg, D. Cisplatin as an anti-tumor drug:
cellular mechanisms of activity, drug resistance and induced side
effects. Cancers 2011, 3, 1351−1371.
(3) Dilruba, S.; Kalayda, G. V. Platinum-based drugs: past, present
and future. Cancer Chemother. Pharmacol. 2016, 77 (6), 1103−1124.
(4) Johnstone, T. C.; Suntharalingam, K.; Lippard, S. J. The Next
Generation of Platinum Drugs: Targeted Pt(II) Agents, Nanoparticle
Delivery, and Pt(IV) Prodrugs. Chem. Rev. 2016, 116 (5), 3436−
3486.
(5) Zhou, J.; Kang, Y.; Chen, L.; Wang, H.; Liu, J.; Zeng, S.; Yu, L.
The Drug-Resistance Mechanisms of Five Platinum-Based Antitumor
Agents. Front. Pharmacol. 2020, 11, 343.
(6) Oun, R.; Moussa, Y. E.; Wheate, N. J. The side effects of
platinum-based chemotherapy drugs: a review for chemists. Dalton
Trans. 2018, 47 (19), 6645−6653.
(7) Hua, W.; Xu, G.; Zhao, J.; Wang, Z.; Lu, J.; Sun, W.; Gou, S.
DNA-Targeting RuII-Polypyridyl Complex with a Long-Lived
Intraligand Excited State as a Potential Photodynamic Therapy
Agent. Chem. - Eur. J. 2020, 26 (72), 17495−17503.
(8) Crlikova, H.; Kostrhunova, H.; Pracharova, J.; Kozsup, M.; Nagy,
S.; Buglyó, P.; Brabec, V.; Kasparkova, J. Antiproliferative, DNA
binding, and cleavage properties of dinuclear Co(III) complexes
containing the bioactive quinizarin ligand. JBIC, J. Biol. Inorg. Chem.
2020, 25 (2), 339−350.
(9) Mukherjee, A.; Acharya, S.; Purkait, K.; Chakraborty, K.;
Bhattacharjee, A.; Mukherjee, A. Effect of N,N Coordination and RuII
Halide Bond in Enhancing Selective Toxicity of a Tyramine-Based
RuII (p-Cymene) Complex. Inorg. Chem. 2020, 59 (9), 6581−6594.
(10) Maji, M.; Acharya, S.; Maji, S.; Purkait, K.; Gupta, A.;
Mukherjee, A. Differences in Stability, Cytotoxicity, and Mechanism
of Action of Ru(II) and Pt(II) Complexes of a Bidentate N,O Donor
Ligand. Inorg. Chem. 2020, 59 (14), 10262−10274.
(11) Purkait, K.; Ruturaj; Mukherjee, A.; Gupta, A. ATP7B Binds
Ruthenium(II) p-Cymene Half-Sandwich Complexes: Role of Steric
Hindrance and Ru−I Coordination in Rescuing the Sequestration.
Inorg. Chem. 2019, 58 (22), 15659−15670.
(12) Notaro, A.; Jakubaszek, M.; Rotthowe, N.; Maschietto, F.;
Vinck, R.; Felder, P. S.; Goud, B.; Tharaud, M.; Ciofini, I.; Bedioui, F.;
Winter, R. F.; Gasser, G. Increasing the Cytotoxicity of Ru(II)
Polypyridyl Complexes by Tuning the Electronic Structure of Dioxo
Ligands. J. Am. Chem. Soc. 2020, 142 (13), 6066−6084.
(13) Maji, M.; Karmakar, S.; Ruturaj; Gupta, A.; Mukherjee, A.
Oxamusplatin: a cytotoxic Pt(ii) complex of a nitrogen mustard with
resistance to thiol based sequestration displays enhanced selectivity
towards cancer. Dalton Trans. 2020, 49 (8), 2547−2558.
(14) Leal, J.; Santos, L.; Fernández-Aroca, D. M.; Cuevas, J. V.;
Martínez, M. A.; Massaguer, A.; Jalón, F. A.; Ruiz-Hidalgo, M. J.;
Sánchez-Prieto, R.; Rodríguez, A. M.; Castañeda, G.; Durá, G.;
Carrión, M. C.; Barrabés, S.; Manzano, B. R. Effect of the aniline
fragment in Pt(II) and Pt(IV) complexes as anti-proliferative agents.
Standard reduction potential as a more reliable parameter for Pt(IV)
compounds than peak reduction potential. J. Inorg. Biochem. 2021,
218, 111403.
(15) Hummer, A. A.; Bartel, C.; Arion, V. B.; Jakupec, M. A.; MeyerKlaucke, W.; Geraki, T.; Quinn, P. D.; Mijovilovich, A.; Keppler, B.
K.; Rompel, A. X-ray Absorption Spectroscopy of an Investigational
Arindam Mukherjee − Department of Chemical Sciences and
Centre for Advanced Functional Materials, Indian Institute of
Science Education and Research (IISER) Kolkata, Mohanpur
741246, India; orcid.org/0000-0001-9545-8628;
Email: a.mukherjee@iiserkol.ac.in
Arnab Gupta − Department of Biological Sciences, Indian
Institute of Science Education and Research (IISER) Kolkata,
Mohanpur 741246, India; Email: arnab.gupta@
iiserkol.ac.in
Authors
Rishav Chatterjee − Department of Chemical Sciences, Indian
Institute of Science Education and Research (IISER) Kolkata,
Mohanpur 741246, India
Indira Bhattacharya − Department of Biological Sciences,
Indian Institute of Science Education and Research (IISER)
Kolkata, Mohanpur 741246, India
Souryadip Roy − Department of Chemical Sciences, Indian
Institute of Science Education and Research (IISER) Kolkata,
Mohanpur 741246, India
Kallol Purkait − Department of Chemical Sciences, Indian
Institute of Science Education and Research (IISER) Kolkata,
Mohanpur 741246, India
Tuhin Subhra Koley − Department of Chemical Sciences,
Indian Institute of Science Education and Research (IISER)
Kolkata, Mohanpur 741246, India
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.1c01363
Author Contributions
†
Equal contributions.
Author Contributions
R.C. performed the synthesis, characterization, and in vitro
(MTT) screening. I.B. performed immunofluorescence,
immunoblot, and AO assay and contributed to the writing of
the manuscript. S.R. performed 72 h stability tests for complex
4, HEK293 antiproliferative activity, and Annexin-V assay. K.P.
helped in the characterization and in vitro (MTT) screening.
T.S.K. recorded the time-dependent NMR data for the 24 h
stability studies. A.M. contributed to the design and supervision, while A.G. supervised most of the biological assays
including immunofluorescence and Western blot studies.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors earnestly acknowledge funding from CSIR,
Government of India, via 01(2927)/2018/EMR-II and SERB
via EMR/2017/002324 (for A.M.) for the microplate reader.
They also thank IISER Kolkata for infrastructural and financial
support. R.C. and S.R. thank INSPIRE, I.B. thanks CSIR, and
T.S.K. thanks IISER Kolkata for providing research fellowships.
K
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Anticancer Gallium(III) Drug: Interaction with Serum Proteins,
Elemental Distribution Pattern, and Coordination of the Compound
in Tissue. J. Med. Chem. 2012, 55 (11), 5601−5613.
(16) Enyedy, É . A.; Dömötör, O.; Varga, E.; Kiss, T.; Trondl, R.;
Hartinger, C. G.; Keppler, B. K. Comparative solution equilibrium
studies of anticancer gallium(III) complexes of 8-hydroxyquinoline
and hydroxy(thio)pyrone ligands. J. Inorg. Biochem. 2012, 117, 189−
197.
(17) Smithen, D. A.; Yin, H.; Beh, M. H. R.; Hetu, M.; Cameron, T.
S.; McFarland, S. A.; Thompson, A. Synthesis and Photobiological
Activity of Ru(II) Dyads Derived from Pyrrole-2-carboxylate
Thionoesters. Inorg. Chem. 2017, 56 (7), 4121−4132.
(18) Monro, S.; Colón, K. L.; Yin, H.; Roque, J.; Konda, P.; Gujar,
S.; Thummel, R. P.; Lilge, L.; Cameron, C. G.; McFarland, S. A.
Transition Metal Complexes and Photodynamic Therapy from a
Tumor-Centered Approach: Challenges, Opportunities, and Highlights from the Development of TLD1433. Chem. Rev. 2019, 119 (2),
797−828.
(19) Enyedy, É . A.; Dömötör, O.; Bali, K.; Hetényi, A.; Tuccinardi,
T.; Keppler, B. K. Interaction of the anticancer gallium(III) complexes
of 8-hydroxyquinoline and maltol with human serum proteins. JBIC, J.
Biol. Inorg. Chem. 2015, 20 (1), 77−88.
(20) Wernitznig, D.; Kiakos, K.; Del Favero, G.; Harrer, N.; Machat,
H.; Osswald, A.; Jakupec, M. A.; Wernitznig, A.; Sommergruber, W.;
Keppler, B. K. First-in-class ruthenium anticancer drug (KP1339/IT139) induces an immunogenic cell death signature in colorectal
spheroids in vitro†. Metallomics 2019, 11 (6), 1044−1048.
(21) Neuditschko, B.; Legin, A. A.; Baier, D.; Schintlmeister, A.;
Reipert, S.; Wagner, M.; Keppler, B. K.; Berger, W.; Meier-Menches,
S. M.; Gerner, C. Interaction with Ribosomal Proteins Accompanies
Stress Induction of the Anticancer Metallodrug BOLD-100/KP1339
in the Endoplasmic Reticulum. Angew. Chem., Int. Ed. 2021, 60 (10),
5063−5068.
(22) Chitambar, C. R. Gallium Complexes as Anticancer Drugs.
Metal Ions Life Sci. 2018, 18
(23) de Camargo, M. S.; De Grandis, R. A.; da Silva, M. M.; da Silva,
P. B.; Santoni, M. M.; Eismann, C. E.; Menegário, A. A.; Cominetti,
M. R.; Zanelli, C. F.; Pavan, F. R.; Batista, A. A. Determination of in
vitro absorption in Caco-2 monolayers of anticancer Ru(II)-based
complexes acting as dual human topoisomerase and PARP inhibitors.
BioMetals 2019, 32 (1), 89−100.
(24) Lenis-Rojas, O. A.; Robalo, M. P.; Tomaz, A. I.; Fernandes, A.
R.; Roma-Rodrigues, C.; Teixeira, R. G.; Marques, F.; Folgueira, M.;
Yáñez, J.; Gonzalez, A. A.; Salamini-Montemurri, M.; Pech-Puch, D.;
Vázquez-García, D.; Torres, M. L.; Fernández, A.; Fernández, J. J.
Half-Sandwich Ru(p-cymene) Compounds with Diphosphanes: In
Vitro and In Vivo Evaluation As Potential Anticancer Metallodrugs.
Inorg. Chem. 2021, 60 (5), 2914−2930.
(25) Maji, M.; Acharya, S.; Bhattacharya, I.; Gupta, A.; Mukherjee,
A. Effect of an Imidazole-Containing Schiff Base of an Aromatic
Sulfonamide on the Cytotoxic Efficacy of N,N-Coordinated HalfSandwich Ruthenium(II) p-Cymene Complexes. Inorg. Chem. 2021,
60 (7), 4744−4754.
(26) Maji, M.; Bhattacharya, I.; Acharya, S.; Chakraborty, M. P.;
Gupta, A.; Mukherjee, A. Hypoxia Active Platinum(IV) Prodrugs of
Orotic Acid Selective to Liver Cancer Cells. Inorg. Chem. 2021, 60
(7), 4342−4346.
(27) Acharya, S.; Maji, M.; Chakraborty, M. P.; Bhattacharya, I.;
Das, R.; Gupta, A.; Mukherjee, A. Disruption of the Microtubule
Network and Inhibition of VEGFR2 Phosphorylation by Cytotoxic
N,O-Coordinated Pt(II) and Ru(II) Complexes of Trimethoxy
Aniline-Based Schiff Bases. Inorg. Chem. 2021, 60 (5), 3418−3430.
(28) Acharya, S.; Maji, M.; Ruturaj; Purkait, K.; Gupta, A.;
Mukherjee, A. Synthesis, Structure, Stability, and Inhibition of
Tubulin Polymerization by RuII−p-Cymene Complexes of Trimethoxyaniline-Based Schiff Bases. Inorg. Chem. 2019, 58 (14), 9213−
9224.
(29) Ilmi, R.; Tseriotou, E.; Stylianou, P.; Christou, Y. A.; Ttofi, I.;
Dietis, N.; Pitris, C.; Odysseos, A. D.; Georgiades, S. N. A Novel
Article
Conjugate of Bis[((4-bromophenyl)amino)quinazoline], a EGFR-TK
Ligand, with a Fluorescent Ru(II)-Bipyridine Complex Exhibits
Specific Subcellular Localization in Mitochondria. Mol. Pharmaceutics
2019, 16 (10), 4260−4273.
(30) Sonkar, C.; Malviya, N.; Ranjan, R.; Pakhira, S.;
Mukhopadhyay, S. Mechanistic Insight for Targeting Biomolecules
by Ruthenium(II) NSAID Complexes. ACS Appl. Bio Mater. 2020, 3
(7), 4600−4612.
(31) Mondal, A.; Sen, U.; Roy, N.; Muthukumar, V.; Sahoo, S. K.;
Bose, B.; Paira, P. DNA targeting half sandwich Ru(ii)-p-cymeneN∧N complexes as cancer cell imaging and terminating agents:
influence of regioisomers in cytotoxicity. Dalton Trans. 2021, 50 (3),
979−997.
(32) Qiao, L.; Liu, J.; Han, Y.; Wei, F.; Liao, X.; Zhang, C.; Xie, L.;
Ji, L.; Chao, H. Rational design of a lysosome-targeting and nearinfrared absorbing Ru(ii)−BODIPY conjugate for photodynamic
therapy. Chem. Commun. 2021, 57 (14), 1790−1793.
(33) Zhang, C.; Guan, R.; Liao, X.; Ouyang, C.; Rees, T. W.; Liu, J.;
Chen, Y.; Ji, L.; Chao, H. A mitochondria-targeting dinuclear Ir−Ru
complex as a synergistic photoactivated chemotherapy and photodynamic therapy agent against cisplatin-resistant tumour cells. Chem.
Commun. 2019, 55 (83), 12547−12550.
(34) Acharya, S.; Ghosh, S.; Maji, M.; Parambil, A. R. U.; Singh, S.;
Mukherjee, A. Inhibition of 3D colon cancer stem cell spheroids by
cytotoxic RuII-p-cymene complexes of mesalazine derivatives. Chem.
Commun. 2020, 56 (40), 5421−5424.
(35) Liu, R.; Yuan, C.; Feng, Y.; Qian, J.; Huang, X.; Chen, Q.;
Zhou, S.; Ding, Y.; Zhai, B.; Mei, W.; Yao, L. Microwave-assisted
synthesis of ruthenium(ii) complexes containing levofloxacin-induced
G2/M phase arrest by triggering DNA damage. RSC Adv. 2021, 11
(8), 4444−4453.
(36) Steel, T. R.; Walsh, F.; Wieczorek-Błauż, A.; Hanif, M.;
Hartinger, C. G. Monodentately-coordinated bioactive moieties in
multimodal half-sandwich organoruthenium anticancer agents. Coord.
Chem. Rev. 2021, 439, 213890.
(37) Liu, Z.; Li, J.; Kong, D.; Tian, M.; Zhao, Y.; Xu, Z.; Gao, W.;
Zhou, Y. Dual Functional Half-Sandwich Ru(II) Complexes:
Lysosome-Targeting Probes and Anticancer Agents. Eur. J. Inorg.
Chem. 2019, 2019 (2), 287−294.
(38) Hartinger, C. G.; Zorbas-Seifried, S.; Jakupec, M. A.; Kynast, B.;
Zorbas, H.; Keppler, B. K. From bench to bedside − preclinical and
early clinical development of the anticancer agent indazolium trans[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A). J.
Inorg. Biochem. 2006, 100 (5), 891−904.
(39) Motswainyana, W. M.; Ajibade, P. A. Anticancer activities of
mononuclear ruthenium (II) coordination complexes. Adv. Chem.
2015, 2015, 1.
(40) Lin, K.; Zhao, Z.-Z.; Bo, H.-B.; Hao, X.-J.; Wang, J.-Q.
Applications of ruthenium complex in tumor diagnosis and therapy.
Front. Pharmacol. 2018, 9, 1323.
(41) Kostova, I. Ruthenium complexes as anticancer agents. Curr.
Med. Chem. 2006, 13 (9), 1085−1107.
(42) Wang, Y.; Jin, J.; Shu, L.; Li, T.; Lu, S.; Subarkhan, M. K. M.;
Chen, C.; Wang, H. New Organometallic Ruthenium(II) Compounds
Synergistically Show Cytotoxic, Antimetastatic and Antiangiogenic
Activities for the Treatment of Metastatic Cancer. Chem. - Eur. J.
2020, 26 (66), 15170−15182.
(43) Travassos, I. O.; Mello-Andrade, F.; Caldeira, R. P.; Pires, W.
C.; da Silva, P. F. F.; Correa, R. S.; Teixeira, T.; Martins-Oliveira, A.;
Batista, A. A.; de Silveira-Lacerda, E. P. Ruthenium (II)/allopurinol
complex inhibits breast cancer progression via multiple targets. JBIC,
J. Biol. Inorg. Chem. 2021, 26, 385.
(44) Ribeiro, G. H.; Guedes, A. P. M.; de Oliveira, T. D.; de Correia,
C. R. S. T. b.; Colina-Vegas, L.; Lima, M. A.; Nóbrega, J. A.;
Cominetti, M. R.; Rocha, F. V.; Ferreira, A. G.; Castellano, E. E.;
Teixeira, F. R.; Batista, A. A. Ruthenium(II) Phosphine/Mercapto
Complexes: Their in Vitro Cytotoxicity Evaluation and Actions as
Inhibitors of Topoisomerase and Proteasome Acting as Possible
L
https://doi.org/10.1021/acs.inorgchem.1c01363
Inorg. Chem. XXXX, XXX, XXX−XXX
Inorganic Chemistry
pubs.acs.org/IC
Triggers of Cell Death Induction. Inorg. Chem. 2020, 59 (20),
15004−15018.
(45) Karmakar, J.; Nandy, P.; Das, S.; Bhattacharya, D.; Karmakar,
P.; Bhattacharya, S. Utilization of Guanidine-Based Ancillary Ligands
in Arene−Ruthenium Complexes for Selective Cytotoxicity. ACS
Omega 2021, 6 (12), 8226−8238.
(46) Jin, Z.; Qi, S.; Guo, X.; Jian, Y.; Hou, Y.; Li, C.; Wang, X.;
Zhou, Q. The modification of a pyrene group makes a Ru(ii) complex
versatile. Chem. Commun. 2021, 57 (26), 3259−3262.
(47) Wise, D. E.; Gamble, A. J.; Arkawazi, S. W.; Walton, P. H.;
Galan, M. C.; O’Hagan, M. P.; Hogg, K. G.; Marrison, J. L.; O’Toole,
P. J.; Sparkes, H. A.; Lynam, J. M.; Pringle, P. G. Cytotoxic (cis,cis1,3,5-triaminocyclohexane)ruthenium(ii)-diphosphine complexes;
evidence for covalent binding and intercalation with DNA. Dalton
Trans. 2020, 49 (43), 15219−15230.
(48) Pereira, S. A. P.; Bobbink, F. D.; Dyson, P. J.; Saraiva, M. L. M.
F. S. Automatic evaluation of cyclooxygenase 2 inhibition induced by
metal-based anticancer compounds. J. Inorg. Biochem. 2021, 218,
111399.
(49) Du, E.; Hu, X.; Roy, S.; Wang, P.; Deasy, K.; Mochizuki, T.;
Zhang, Y. Taurine-modified Ru (II)-complex targets cancerous brain
cells for photodynamic therapy. Chem. Commun. 2017, 53 (44),
6033−6036.
(50) Du, Q.; Guo, L.; Ge, X.; Zhao, L.; Tian, Z.; Liu, X.; Zhang, F.;
Liu, Z. Serendipitous synthesis of five-coordinated half-sandwich
aminoimine iridium (iii) and ruthenium (ii) complexes and their
application as potent anticancer agents. Inorg. Chem. 2019, 58 (9),
5956−5965.
(51) Li, J.; Tian, Z.; Ge, X.; Xu, Z.; Feng, Y.; Liu, Z. Design,
synthesis, and evaluation of fluorine and Naphthyridine−Based halfsandwich organoiridium/ruthenium complexes with bioimaging and
anticancer activity. Eur. J. Med. Chem. 2019, 163, 830−839.
(52) Li, J.; Tian, Z.; Xu, Z.; Zhang, S.; Feng, Y.; Zhang, L.; Liu, Z.
Highly potent half-sandwich iridium and ruthenium complexes as
lysosome-targeted imaging and anticancer agents. Dalton Trans. 2018,
47 (44), 15772−15782.
(53) Ma, W.; Guo, L.; Tian, Z.; Zhang, S.; He, X.; Li, J.; Yang, Y.;
Liu, Z. Rhodamine-modified fluorescent half-sandwich iridium and
ruthenium complexes: potential application as bioimaging and
anticancer agents. Dalton Trans. 2019, 48 (15), 4788−4793.
(54) Ma, W.; Zhang, S.; Tian, Z.; Xu, Z.; Zhang, Y.; Xia, X.; Chen,
X.; Liu, Z. Potential anticancer agent for selective damage to
mitochondria or lysosomes: Naphthalimide-modified fluorescent
biomarker half-sandwich iridium (III) and ruthenium (II) complexes.
Eur. J. Med. Chem. 2019, 181, 111599.
(55) Mitrović, A.; Kljun, J.; Sosič, I.; Gobec, S.; Turel, I.; Kos, J.
Clioquinol−ruthenium complex impairs tumour cell invasion by
inhibiting cathepsin B activity. Dalton Trans. 2016, 45 (42), 16913−
16921.
(56) Mitrović, A.; Kljun, J.; Sosič, I.; Uršič, M.; Meden, A.; Gobec,
S.; Kos, J.; Turel, I. Organoruthenated nitroxoline derivatives impair
tumor cell invasion through inhibition of cathepsin B activity. Inorg.
Chem. 2019, 58 (18), 12334−12347.
(57) Paitandi, R. P.; Sharma, V.; Singh, V. D.; Dwivedi, B. K.;
Mobin, S. M.; Pandey, D. S. Pyrazole appended quinoline-BODIPY
based arene ruthenium complexes: their anticancer activity and
potential applications in cellular imaging. Dalton Trans. 2018, 47 (48),
17500−17514.
(58) Thota, S.; Rodrigues, D. A.; Crans, D. C.; Barreiro, E. J. Ru (II)
compounds: next-generation anticancer metallotherapeutics? J. Med.
Chem. 2018, 61 (14), 5805−5821.
(59) Xie, Y.; Zhang, S.; Ge, X.; Ma, W.; He, X.; Zhao, Y.; Ye, J.;
Zhang, H.; Wang, A.; Liu, Z. Lysosomal-targeted anticancer halfsandwich iridium (III) complexes modified with lonidamine amide
derivatives. Appl. Organomet. Chem. 2020, 34 (5), e5589.
(60) Yang, Y.; Guo, L.; Tian, Z.; Gong, Y.; Zheng, H.; Zhang, S.; Xu,
Z.; Ge, X.; Liu, Z. Novel and versatile imine-N-heterocyclic carbene
half-sandwich iridium (III) complexes as lysosome-targeted anticancer
agents. Inorg. Chem. 2018, 57 (17), 11087−11098.
Article
(61) Yang, Y.; Guo, L.; Tian, Z.; Liu, X.; Gong, Y.; Zheng, H.; Ge,
X.; Liu, Z. Imine-N-Heterocyclic Carbenes as Versatile Ligands in
Ruthenium (II) p-Cymene Anticancer Complexes: A Structure−
Activity Relationship Study. Chem. - Asian J. 2018, 13 (19), 2923−
2933.
(62) Zeng, L.; Gupta, P.; Chen, Y.; Wang, E.; Ji, L.; Chao, H.; Chen,
Z.-S. The development of anticancer ruthenium (II) complexes: from
single molecule compounds to nanomaterials. Chem. Soc. Rev. 2017,
46 (19), 5771−5804.
(63) Kumari, A.; Singh, R. K. Morpholine as ubiquitous
pharmacophore in medicinal chemistry: deep insight into the
structure-activity relationship (SAR). Bioorg. Chem. 2020, 96, 103578.
(64) Kourounakis, A. P.; Xanthopoulos, D.; Tzara, A. Morpholine as
a privileged structure: a review on the medicinal chemistry and
pharmacological activity of morpholine containing bioactive molecules. Med. Res. Rev. 2020, 40 (2), 709−752.
(65) Tzara, A.; Xanthopoulos, D.; Kourounakis, A. P. Morpholine As
a Scaffold in Medicinal Chemistry: An Update on Synthetic Strategies.
ChemMedChem 2020, 15, 392.
(66) Walker, E. H.; Pacold, M. E.; Perisic, O.; Stephens, L.;
Hawkins, P. T.; Wymann, M. P.; Williams, R. L. Structural
determinants of phosphoinositide 3-kinase inhibition by wortmannin,
LY294002, quercetin, myricetin, and staurosporine. Mol. Cell 2000, 6
(4), 909−919.
(67) Knight, Z. A.; Chiang, G. G.; Alaimo, P. J.; Kenski, D. M.; Ho,
C. B.; Coan, K.; Abraham, R. T.; Shokat, K. M. Isoform-specific
phosphoinositide 3-kinase inhibitors from an arylmorpholine scaffold.
Bioorg. Med. Chem. 2004, 12 (17), 4749−4759.
(68) Vlahos, C. J.; Matter, W. F.; Hui, K. Y.; Brown, R. F. A specific
inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 1994,
269 (7), 5241−5248.
(69) Zask, A.; Kaplan, J.; Verheijen, J. C.; Richard, D. J.; Curran, K.;
Brooijmans, N.; Bennett, E. M.; Toral-Barza, L.; Hollander, I.; AyralKaloustian, S.; Yu, K. Morpholine derivatives greatly enhance the
selectivity of mammalian target of rapamycin (mTOR) inhibitors. J.
Med. Chem. 2009, 52 (24), 7942−7945.
(70) Chresta, C. M.; Davies, B. R.; Hickson, I.; Harding, T.;
Cosulich, S.; Critchlow, S. E.; Vincent, J. P.; Ellston, R.; Jones, D.;
Sini, P.; et al. AZD8055 is a potent, selective, and orally bioavailable
ATP-competitive mammalian target of rapamycin kinase inhibitor
with in vitro and in vivo antitumor activity. Cancer Res. 2010, 70 (1),
288−298.
(71) Zheng, J.; Xin, Y.; Zhang, J.; Subramanian, R.; Murray, B. P.;
Whitney, J. A.; Warr, M. R.; Ling, J.; Moorehead, L.; Kwan, E.;
Hemenway, J.; Smith, B. J.; Silverman, J. A. Pharmacokinetics and
Disposition of Momelotinib Revealed a Disproportionate Human
MetaboliteResolution for Clinical Development. Drug Metab.
Dispos. 2018, 46 (3), 237−247.
(72) Liu, D.; Xu, Y.; Feng, Y.; Liu, H.; Shen, X.; Chen, K.; Ma, J.;
Jiang, H. Inhibitor discovery targeting the intermediate structure of βamyloid peptide on the conformational transition pathway:
implications in the aggregation mechanism of β-amyloid peptide.
Biochemistry 2006, 45 (36), 10963−10972.
(73) Yun, C.-H.; Boggon, T. J.; Li, Y.; Woo, M. S.; Greulich, H.;
Meyerson, M.; Eck, M. J. Structures of lung cancer-derived EGFR
mutants and inhibitor complexes: mechanism of activation and
insights into differential inhibitor sensitivity. Cancer Cell 2007, 11 (3),
217−227.
(74) Barker, A. J.; Gibson, K. H.; Grundy, W.; Godfrey, A. A.;
Barlow, J. J.; Healy, M. P.; Woodburn, J. R.; Ashton, S. E.; Curry, B. J.;
Scarlett, L.; Henthorn, L.; Richards, L. Studies leading to the
identification of ZD1839 (Iressa): an orally active, selective epidermal
growth factor receptor tyrosine kinase inhibitor targeted to the
treatment of cancer. Bioorg. Med. Chem. Lett. 2001, 11 (14), 1911−
1914.
(75) Falcone, S.; Cocucci, E.; Podini, P.; Kirchhausen, T.; Clementi,
E.; Meldolesi, J. Macropinocytosis: regulated coordination of
M
https://doi.org/10.1021/acs.inorgchem.1c01363
Inorg. Chem. XXXX, XXX, XXX−XXX
Inorganic Chemistry
pubs.acs.org/IC
endocytic and exocytic membrane traffic events. J. Cell Sci. 2006, 119
(22), 4758−4769.
(76) Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter,
P. Molecular Biology of the Cell, 4th ed.; Garland Science, 2002.
(77) Armarego, W. L. F.; Chai, C. In Purification of Laboratory
Chemicals, 7th ed.; Armarego, W. L. F., Chai, C., Eds.; ButterworthHeinemann: Boston, 2013; pp 71−90.
(78) Sashuk, V.; Schoeps, D.; Plenio, H. Fluorophore tagged crosscoupling catalysts. Chem. Commun. 2009, 7, 770−772.
(79) Oliveira, K. M.; Honorato, J.; Gonçalves, G. R.; Cominetti, M.
R.; Batista, A. A.; Correa, R. S. Ru(ii)/diclofenac-based complexes:
DNA, BSA interaction and their anticancer evaluation against lung
and breast tumor cells. Dalton Trans. 2020, 49 (36), 12643−12652.
(80) OECD. Test No. 107: Partition Coefficient (n-octanol/water):
Shake Flask Method, 1995.
(81) Purkait, K.; Chatterjee, S.; Karmakar, S.; Mukherjee, A.
Alteration of steric hindrance modulates glutathione resistance and
cytotoxicity of three structurally related RuII-p-cymene complexes.
Dalton Trans. 2016, 45 (20), 8541−8555.
(82) Bhattacharyya, S.; Purkait, K.; Mukherjee, A. Ruthenium(II) pcymene complexes of a benzimidazole-based ligand capable of
VEGFR2 inhibition: hydrolysis, reactivity and cytotoxicity studies.
Dalton Trans. 2017, 46 (26), 8539−8554.
(83) Ramos, T. S.; Luz, D. M.; Nascimento, R. D.; Silva, A. K.; Lião,
L. M.; Miranda, V. M.; Deflon, V. M.; de Araujo, M. P.; Ueno, L. T.;
Machado, F. B. C.; Dinelli, L. R.; Bogado, A. L. Ruthenium-cymene
containing pyridine-derived aldiimine ligands: Synthesis, characterization and application in the transfer hydrogenation of aryl ketones
and kinetics studies. J. Organomet. Chem. 2019, 892, 51−65.
(84) Wenlock, M. C.; Potter, T.; Barton, P.; Austin, R. P. A method
for measuring the lipophilicity of compounds in mixtures of 10. J.
Biomol. Screening 2011, 16 (3), 348−355.
(85) Chen, J. W.; Murphy, T. L.; Willingham, M. C.; Pastan, I.;
August, J. T. Identification of two lysosomal membrane glycoproteins.
J. Cell Biol. 1985, 101 (1), 85−95.
(86) Kundra, R.; Kornfeld, S. Asparagine-linked oligosaccharides
protect Lamp-1 and Lamp-2 from intracellular proteolysis. J. Biol.
Chem. 1999, 274 (43), 31039−31046.
(87) Boya, P.; Kroemer, G. Lysosomal membrane permeabilization
in cell death. Oncogene 2008, 27 (50), 6434−6451.
(88) Xiao, L.; Wang, Y. C.; Li, W. S.; Du, Y. The role of mTOR and
phospho-p70S6K in pathogenesis and progression of gastric
carcinomas: an immunohistochemical study on tissue microarray. J.
Exp. Clin. Cancer Res. 2009, 28 (1), 152.
(89) Liu, H.; Huang, B.; Xue, S.; U, K. P.; Tsang, L. L.; Zhang, X.;
Li, G.; Jiang, X. Functional crosstalk between mTORC1/p70S6K
pathway and heterochromatin organization in stress-induced
senescence of MSCs. Stem Cell Res. Ther. 2020, 11 (1), 279.
(90) Satyavarapu, E. M.; Das, R.; Mandal, C.; Mukhopadhyay, A.;
Mandal, C. Autophagy-independent induction of LC3B through
oxidative stress reveals its non-canonical role in anoikis of ovarian
cancer cells. Cell Death Dis. 2018, 9 (10), 934.
(91) Wu, J.; Dang, Y.; Su, W.; Liu, C.; Ma, H.; Shan, Y.; Pei, Y.;
Wan, B.; Guo, J.; Yu, L. Molecular cloning and characterization of rat
LC3A and LC3BTwo novel markers of autophagosome. Biochem.
Biophys. Res. Commun. 2006, 339 (1), 437−442.
(92) Tanida, I.; Ueno, T.; Kominami, E. Human Light Chain 3/
MAP1LC3B Is Cleaved at Its Carboxyl-terminal Met121 to Expose
Gly120 for Lipidation and Targeting to Autophagosomal Membranes*. J. Biol. Chem. 2004, 279 (46), 47704−47710.
(93) Kabeya, Y.; Mizushima, N.; Yamamoto, A.; Oshitani-Okamoto,
S.; Ohsumi, Y.; Yoshimori, T. LC3, GABARAP and GATE16 localize
to autophagosomal membrane depending on form-II formation. J. Cell
Sci. 2004, 117, 2805−2812.
(94) Kabeya, Y. LC3, a mammalian homolog of yeast Apg8p, is
localized in autophagosome membranes after processing. EMBO J.
2000, 19 (21), 5720.
(95) He, H.; Dang, Y.; Dai, F.; Guo, Z.; Wu, J.; She, X.; Pei, Y.;
Chen, Y.; Ling, W.; Wu, C.; Zhao, S.; Liu, J. O.; Yu, L. Post-
Article
translational Modifications of Three Members of the Human
MAP1LC3 Family and Detection of a Novel Type of Modification
for MAP1LC3B*. J. Biol. Chem. 2003, 278 (31), 29278−29287.
(96) Ichimura, Y.; Kirisako, T.; Takao, T.; Satomi, Y.; Shimonishi,
Y.; Ishihara, N.; Mizushima, N.; Tanida, I.; Kominami, E.; Ohsumi,
M.; Noda, T.; Ohsumi, Y. A ubiquitin-like system mediates protein
lipidation. Nature 2000, 408 (6811), 488−492.
(97) Cleary, M. L.; Smith, S. D.; Sklar, J. Cloning and structural
analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin
transcript resulting from the t(14;18) translocation. Cell 1986, 47 (1),
19−28.
(98) Tsujimoto, Y.; Finger, L.; Yunis, J.; Nowell, P.; Croce, C.
Cloning of the chromosome breakpoint of neoplastic B cells with the
t(14;18) chromosome translocation. Science 1984, 226 (4678),
1097−1099.
(99) Kelly, G. L.; Strasser, A. Toward Targeting Antiapoptotic MCL1 for Cancer Therapy. Annu. Rev. Cancer Biol. 2020, 4 (1), 299−313.
N
https://doi.org/10.1021/acs.inorgchem.1c01363
Inorg. Chem. XXXX, XXX, XXX−XXX