Synthesis, Characterization, and Cytotoxicity of Morpholine-Containing Ruthenium(II) p-Cymene Complexes

Article abstract of DOI:10.1021/acs.inorgchem.1c01363

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 P1ˉ 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 sulfoxide-d6 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. MDA-MB-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.

Full text of DOI:10.1021/acs.inorgchem.1c01363

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Article
Synthesis, Characterization, and Cytotoxicity of Morpholine-
Containing 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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sı Supporting Information
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 P2 /c space group, whereas 5 and 6 crystallized
1
1
in the P1
̅
space group. The solution stability studies using H NMR
support instantaneous hydrolysis of the native complexes to form
monoaquated species in a solution of 3:7 (v/v) dimethyl sulfoxide-
d6 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 (IC ca. 1−14 μM) in various cancer cell lines, viz., MDA-MB-231, MiaPaCa2, and Hep-G2. The presence of the pendant
5
0
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. MDA-
MB-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
efficiency against hepatocellular carcinoma and renal carcino-
ma, respectively.
■
15,16,22
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.
for their poor tumor selectivity, serious side effects, and
intrinsic and/or acquired resistance, making them ineffective
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
1
−4
Platinum drugs are also known
23−37
2
,5,6
sacrificing the activity.
Ruthenium(III) complexes act as
for treatment against several tissue types.
Thus, in spite of
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
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
Received: May 7, 2021
7
−14
character and improve delivery.
Metal complexes with
potential apart from platinum(II/IV) include gallium(II) and
1
5−21
ruthenium(II/III).
against lymphoma and bladder cancer.
maltolate and tris(8-quinolinolato)gallium(III) have shown
Gallium(III) nitrate has been effective
2
2
Gallium(III)
©
XXXX American Chemical Society
https://doi.org/10.1021/acs.inorgchem.1c01363
A
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
pubs.acs.org/IC
Article
3
8
in tumors. The high binding affinity of ruthenium(III) for
the transferrin iron-binding sites offers the possibility of
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
39−41
targeting tumors with high transferrin receptor densities.
1
PerkinElmer SPECTRUM RX I spectrometer in KBr pellets.
H
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. 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.
multistep synthesis helps to incorporate various ligands of
interest to render optimized ruthenium complexes with
1
3
NMR, C NMR, and HMQC spectra were recorded using either a
00 MHz JEOL ECS or 500 MHz Bruker Avance III spectrometer at
room temperature (24−27 °C). All of the C 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
4
13
20
17,18
1
Controlled
are of H NMR pure compounds.
Synthesis and Characterization. Syntheses of Schiff Base
Ligands. General Procedure I. A literature procedure was adapted for
78,79
7
,25,31,42−48
synthesis of the amine (B2 or B3).
To a methanolic solution of
efficient antitumor activity.
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
In the past few decades, ruthenium(II) half-sandwich
complexes with piano-stool configurations provided research-
ers 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
(
MeOH) and hexane, and dried under vacuum.
General Procedure II. A literature procedure was adapted for
4
9−62
78
properties.
phores, morpholine is found in various anticancer drugs
Figure 1). The incorporation of morpholine may enhance the
Among the various motifs used as pharmaco-
synthesis of the amine B2. 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
1
without purification. Yield: 91%, yellow oil. H NMR (400 MHz,
CDCl ): δ 8.35 (s, 1H, CHN), 7.45−7.39 (m, 1H, Ar−H), 7.35
3
(
dd, J = 7.6 and 1.6 Hz, 1H, Ar−H), 7.12 (d, J = 7.5 Hz, 2H, Ar−H),
7
6
1
.09−7.03 (m, 2H), 6.98 (dd, 1H, J = 7.4 and 1.0 Hz, Ar−H), 2.22 (s,
1
3
H) (Figure S1). C NMR (100 MHz, CDCl ): δ 166.8, 161.3,
3
48.2, 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. H NMR (500 MHz, CDCl ): δ 9.12 (s,
1
3
1
1
H, CHN), 7.99 (d, J = 8.5 Hz, 1H, Ar−H), 7.86 (d, J = 9.1 Hz,
H, 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
1
3
Figure 1. (a) Anticancer ruthenium complexes in clinical trials. (b)
(s, 6H, Ar−CH ) (Figure S3). C NMR (125 MHz, CDCl ): δ 168,
3
3
Clinical drugs containing a morpholine moiety.
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]-
activity, selectivity, solubility, bioavailability, and pharmacoki-
6
3−65
imino]methyl]phenol (HL3). HL3 was synthesized by general
netic benefits.
Compounds with morpholine motifs target
1
procedure I. Yield: 79%, light-yellow solid. H NMR (500 MHz,
various enzymes and receptors including kinases, Amyloid β
peptides, α-glucosidase, serotonin receptors, and histamine
CDCl ): δ 13.08 (s, 1H, OH), 8.34 (s, 1H), 7.40 (t, J = 7.2 Hz, 1H,
3
CHN), 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−
CH ), 3.45 (s, 2H, Ar−CH ), 2.48 (s, 4H, N−CH ), 2.19 (s, 6H, Ar−
6
6−74
receptors.
Morpholine is also known to help target
75,76
lysosomes in cells and acts as the cells’ recycling apparatus.
2
3
2
2
1
3
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.
CH
) (Figure S5). C NMR (125 MHz, CDCl
3
): δ 166.7, 161.2,
1
6
47.2, 134.2, 133.2, 132.1, 129.2, 128.2, 119.0, 118.8, 117.3, 67.0,
3.1, 53.7, 18.5 (Figure S6).
Synthesis of 2-[[[2,6-Dimethyl-4-(morpholinomethyl)phenyl]-
imino]methyl]-6-methoxyphenol (HL4). HL4 was synthesized by
1
general procedure II. Yield: 53%, yellow oil. H NMR (500 MHz,
CDCl ): δ 13.52 (s, 1H, O−H), 8.34 (s, 1H, CHN), 7.06 (s, 2H,
3
Ar−H), 7.01 (d, J = 7.8 Hz, 1H, Ar−H), 6.96 (d, J = 7.6 Hz, 1H, Ar−
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. 3-
4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
■
H), 6.90 (t, J = 7.7 Hz, 1H, Ar−H), 3.95 (s, 3H, O−CH ), 3.73 (s,
3
4H, O−CH ), 3.44 (s, 2H, Ar−CH ), 2.47 (s, 4H, N−CH ), 2.19 (s,
13
2
2
2
6H, Ar−CH ) (Figure S7). C NMR (125 MHz, CDCl ): δ 166.8,
3
3
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).
77
(
Synthesis of 1-[[[2,6-Dimethyl-4-(morpholinomethyl)phenyl]-
and the biological assay kits were purchased from Gibco. Solvents
used for spectroscopic measurements were of spectroscopic grade and
imino]methyl]naphthalen-2-ol (HL5). HL5 was synthesized by
1
general procedure II. Yield: 49%, yellow crystalline solid. H NMR
B
https://doi.org/10.1021/acs.inorgchem.1c01363
Inorg. Chem. XXXX, XXX, XXX−XXX

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Article
i
13
(
400 MHz, CDCl ): δ 9.10 (s, 1H, CHN), 7.97 (d, J = 8.4 Hz, 1H,
cym− Pr). C NMR (125 MHz, DMSO-d ): δ 16.9, 18.0, 19.1, 21.5,
3
6
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−
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
−
1
S18−S20). IR (KBr pellets, cm ): 1609 (CHN) (Figure S29).
−
1
−1
3
.73 (m, 4H, O−CH ), 3.48 (s, 2H, −CH ), 2.52−2.47 (m, 4H, N−
UV−vis [MeOH; λ , nm (ε, M cm )]: 244 (29550), 291 (8130),
2
2
max
1
3
CH ), 2.30 (s, 6H, Ar−CH ) (Figure S9). C NMR (100 MHz,
CDCl ): δ 168.3, 161.2, 144.3, 136.1, 134.1, 133.2 129.7, 129.6,
403 (2300), 482 (1020) (Figure S28). ESI-HRMS (MeOH). m/z
2
3
II
+
(exp) 559.1899 (559.1907) [Ru C H N O ]. Anal. Calcd for
3
30 37 2 2
1
29.4, 128.1, 127.4, 123.5, 121.7, 118.8, 108.4, 67.0, 63.1, 53.7, 18.8
C H ClN O Ru: C, 60.65; H, 6.28; N, 4.71. Found: C, 60.42; H,
30 37 2 2
(
Figure S10).
6.33; N, 4.68.
1
Synthesis of (E)-1-[[[2,6-Diisopropyl-4-(morpholinomethyl)-
Ru(p-cym)(L4)Cl (4). Yield: 54%. H NMR (400 MHz, DMSO-d ):
6
phenyl]imino]methyl]naphthalen-2-ol (HL6). HL6 was synthesized
by general procedure I. Yield: 58%, yellow solid. H NMR (500 MHz,
δ 7.54 (s, 1H, CHN), 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, p-
cym−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−CH ), 3.62−3.59 (m, 4H, O−CH ), 3.48
1
CDCl ): δ 15.22 (s, 1H, O−H), 9.07 (d, J = 2.5 Hz, 1H, CHN),
3
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
3
2
=
7.5 Hz, 1H, Ar−H), 7.19 (d, J = 7.6 Hz, 3H, Ar−H), 3.76 (d, J =
(s, 2H, CH ), 2.41 (d, J = 10.2 Hz, 8H), 2.17 (s, 3H, Ar−CH ), 1.90
2
3
i
i 13
3
2
.8 Hz, 4H, O−CH ), 3.56 (s, 2H, Ar−CH ), 3.11 (m, 2H, Pr−CH),
(s, 3H, Ar−CH ), 1.20−1.14 (m, 6H, p-cym− Pr) (Figure S21). C
2
2
3
i
.51 (s, 4H, N−CH ), 1.24 (d, J = 6.8 Hz, 12H, Pr−CH ) (Figure
NMR (100 MHz, DMSO-d ): δ 165.6, 158.2, 153.5, 151.3, 135.4,
2
3
6
13
S11). C NMR (125 MHz, CDCl ): δ 167.7, 161.6, 142.8, 140.2,
1
1
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
3
35.9, 135.3, 133.2, 129.3, 128.1, 127.4, 124.3, 123.4, 121.5, 118.7,
08.3, 67, 63.4, 53.5, 28.3, 23.7 (Figure S12).
Syntheses of Metal Complexes. General Procedure for the
−
1
(Figure S22). IR (KBr pellets, cm ): 1601 (CHN) (Figure S29).
−
1
−1
UV−vis [MeOH; λ , nm (ε, M cm )]: 243 (28900), 304 (8630),
max
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
Ru (p-cym) Cl 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
422 (1960), 490 (980) (Figure S28). ESI-HRMS (MeOH). m/z
II
+
(exp) 589.2004 (589.1985) [Ru C H N O ]. Anal. Calcd for
3
1
39
2
3
C H ClN O Ru: C, 59.65; H, 6.30; N, 4.49. Found: C, 59.46; H,
3
1
39
2
3
2
2
4
6.27; N, 4.45.
1
Ru(p-cym)(L5)Cl (5). Yield: 62%. H NMR (400 MHz, DMSO-d ):
6
δ 7.98 (s, 1H, CHN), 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−CH ), 3.51 (s, 2H, CH ), 2.54 (m, 1H, p-
alumina. The mobile phase was 19:1 (v/v) dichloromethane/MeOH.
1
Ru(p-cym)(L1)Cl (1). Yield: 71%. H NMR (400 MHz, DMSO-d ):
6
δ 7.59 (s, 1H, CHN), 7.24 (m, 2H, Ar−H), 7.21 (m, 1H, Ar−H),
2
2
i
7
.09 (t, J = 6.9 Hz, 1H, Ar−H), 7.01 (d, J = 6.4 Hz, 1H, Ar−H), 6.64
cym− Pr), 2.48 (s, 3H, p-cym−CH ), 2.43 (s, 4H, N−CH ), 2.16 (s,
3
2
(
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),
3H, Ar−CH ), 1.93 (s, 3H, Ar−CH ), 1.22 (t, J = 6.64 Hz, 6H, p-
3
3
i
13
=
(
2
cym− Pr) (Figure S23). C NMR (100 MHz, DMSO-d ): δ 166.8,
6
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,
i
.54 (m, 1H, p-cym− Pr), 2.45 (s, 3H, Ar−CH ), 2.19 (s, 3H, Ar−
3
3
i
−1
CH ), 1.89 (s, 3H, Ar−CH ), 1.21 (d, J = 6.9 Hz, 6H, p-cym− Pr)
22.2, 21.8, 19.2, 17.9, 17.0 (Figure S24). IR (KBr pellets, cm ): 1615
3
1
3
−1
−1
(Figure S13). C NMR (125 MHz, DMSO-d ): δ 166.6, 165.9,
(CHN) (Figure S29). UV−vis [MeOH; λmax, nm (ε, M cm )]:
254 (42200), 320 (14480), 422 (3630), 479 (1910) (Figure S28).
ESI-HRMS (MeOH). m/z (exp) 609.2055 (609.1995)
6
1
1
54.6, 135.3, 134.5, 131.1, 130.5, 128.6, 128, 126.3, 121, 120.5, 113.1,
02.9, 94.2, 85.8, 84.4, 82.1, 79.1, 30.3, 22, 21.8, 19.2, 18.1, 17 (Figure
−
1
II
+
S14). IR (KBr pellets, cm ): 1584 (CHN) (Figure S29). UV−vis
[Ru C H N O ]. Anal. Calcd for C H ClN O Ru: C, 63.39; H,
34 39 2 2 34 39 2 2
MeOH; λ , nm (ε, M− cm )]: 241 (18620), 292 (5950), 405
1
−1
[
(
4
6.10; N, 4.35. Found: C, 63.47; H, 6.14; N, 4.39.
Ru(p-cym)(L6)Cl (6). Yield: 62%. H NMR (500 MHz, DMSO-d ):
max
1
1600), 483 (840) (Figure S28). ESI-HRMS (MeOH). m/z (exp)
60.1214 (460.1281) [Ru C H NO ]. Anal. Calcd for
6
II
+
2
5
28
δ 7.95 (s, 1H, CHN), 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,
C H ClNORu: C, 60.66; H, 5.70; N, 2.83. Found: C, 60.84; H,
25
28
5
.75; N, 2.79.
Ru(p-cym)(L2)Cl (2). Yield: 78%. H NMR (500 MHz, DMSO-d ):
1
6
δ 7.99 (s, 1H, CHN), 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, p-
i
i
Pr), 3.64 (s, 4H, O−CH ), 3.58 (s, 2H, −CH ), 3.01 (m, 1H, Pr),
2
2
i
2.65−2.60 (m, 1H, p-cym− Pr), 2.44 (s, 4H, N−CH ), 1.87 (s, 3H,
2
i
cym−H), 5.07 (d, J = 5.7 Hz, 1H, p-cym−H), 4.33 (d, J = 5.8 Hz, 1H,
Ar−CH ), 1.42 (d, J = 6.8 Hz, 3H, Pr), 1.33 (dd, J = 12.8 and 6.8 Hz,
3
i
i
i
i
p-cym−H), 2.63−2.59 (m, 1H, p-cym− Pr), 2.17 (s, 3H, Ar−CH ),
6H, Pr), 1.24 (d, J = 6.9 Hz, 3H, Pr), 1.06 (d, J = 6.6 Hz, 3H, Pr),
3
J
i
i
13
1
.93 (s, 3H, Ar−CH ), 1.25 (dd, = 10.4 and 7.0 Hz, 6H, p-cym− Pr).
C NMR (125 MHz, DMSO-d ): δ 166.8, 158.4, 155.3, 134.8, 134.4,
31.5, 130.7, 128.5, 128.0, 127.3, 126.1, 125.6, 124.4, 121.6, 119.5,
11.2, 103.1, 94.0, 86.7, 84.7, 82.0, 79.4, 30.3, 22.0, 21.9, 19.1, 17.9,
7.0 (Figures S15−S17). IR (KBr pellets, cm ): 1582 (CHN)
0.80 (d, J = 6.7 Hz, 3H, Pr). C NMR (125 MHz, DMSO-d ): δ
3
6
13
6
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).
1
1
1
−
1
−1
IR (KBr pellets, cm ): 1590 (CHN) (Figure S29). UV−vis
Figure S29). UV−vis [MeOH; λ , nm (ε, M− cm )]: 254
1
−1
−1
−1
(
(
[MeOH; λ , nm (ε, M cm )]: 254 (40460), 322 (3160), 429
max
max
34930), 321 (13120), 433 (3370), 485 (1670) (Figure S28). ESI-
(3490), 494 (1730) (Figure S28). ESI-HRMS (MeOH). m/z (exp)
665.2681 (665.2664) [Ru C H N O ]. Anal. Calcd for
II
+
II
+
HRMS (MeOH). m/z (exp) 510.1371 (510.1345) [Ru C H NO ].
2
9
30
38 47 2 2
Anal. Calcd for C H ClNORu: C, 63.90; H, 5.55; N, 2.57. Found:
C H ClN O Ru: C, 65.17; H, 6.76; N, 4.00. Found: C, 65.32; H,
38 47 2 2
29
30
C, 63.77; H, 5.59; N, 2.62.
Ru(p-cym)(L3)Cl (3). Yield: 59%. H NMR (500 MHz, DMSO-d ):
6.72; N, 4.08.
1
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
6
δ 7.58 (s, 1H, CHN), 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, p-
cym−H), 3.63 (s, 4H, O−CH ), 3.50 (s, 2H, CH ), 2.44 (s, 8H), 2.19
2
2
(
s, 3H, CH ), 1.90 (s, 3H, CH ), 1.18 (dd, J = 16.8 and 6.7 Hz, 6H, p-
3 3
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.
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 × 10 MDA-MB-231 cells
were seeded in a 30 mm glass bottom Petri dish and incubated up to
4
70% confluency at 37 °C in a 5% CO atmosphere. Then the medium
2
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
1
Solution Stability Study. H 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
at 37 °C in a 5% CO atmosphere. AO was removed, and the cells
2
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).
(
v/v) dimethyl sulfoxide (DMSO)-d /20 mM phosphate buffer at
6
pH* 7.4 (pH meter reading without correction for the effects of D on
the glass electrode) and 4 mM NaCl in D O at 25 °C.
2
4
Distribution Coefficient Determination. The standard shake-
flask method with an octanol/water mixture following OECD
Immunofluorescence. About 5 × 10 cells of MDA-MB-231
were seeded in 24-well plates over a glass coverslip and incubated up
80
guidelines 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).
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% CO atmosphere. Then
2
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 anti-
LAMP1 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′,6-
diamidino-2-phenylindole (DAPI)-containing mounting media (Fluo-
roshield 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.
Cell Lines and Culture Condition. The cells were grown in a
00 mm Petri dish with an adherent monolayer and maintained at the
1
logarithmic phase in a 5% CO atmosphere using the appropriate
2
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
3
MiaPaCa2) was assessed with MTT assay. About 6 × 10 cells were
Immunoblot Assay. MDA-MB-231 cells were grown in DMEM-
F12 supplemented with 10% FBS and antibiotics at 37 °C and 5%
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
CO . To test the effect of complexes 2 and 6 on lysosomal
2
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.
(
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. Apop-
tosis of cells was detected using the PE-Annexin-V and 7-AAD dual
staining apoptosis detection kit (BD Pharmingen) by flow cytometry
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
5
according to the manufacturer’s protocol. About 1 × 10 MDA-MB-
2
31 cells were seeded in a 6-well plate and incubated for 72 h at 37 °C
in a 5% CO atmosphere. Subsequently, the cells were treated with
2
(
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 Ru (p-cym) Cl
4
complexes 3−5 for 8 h with a IC solution concentration. Treated
5
0
and untreated cells were then harvested with ice-cold 1× phosphate-
buffered saline (PBS) containing 0.1 mM ethylenediaminetetraacetic
acid, subsequently washed with cold 1 × PBS twice, and finally
2
2
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.
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 H and 13C NMR, ESI-
HRMS, FT-IR, and UV−vis analysis. Complexes 2−6 are also
characterized by single-crystal X-ray crystallography. The UV−
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−
for 1−6 (Figure S29).
1
1
E
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Table 1. Selected Bond Lengths (Å) of Complexes 2−6
2
3
4
Ru1−Cl1
Ru1−O1
Ru1−N1
Ru1−C20
Ru1−C21
Ru1−C22
Ru1−C23
Ru1−C24
Ru1−C25
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−C21
Ru1−C22
Ru1−C23
Ru1−C24
Ru1−C25
Ru1−C26
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)
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
2.432(6)
Ru1−Cl1
2.418(5)
Ru1−O1
Ru1−N1
Ru1−C25
Ru1−C26
Ru1−C27
Ru1−C28
Ru1−C29
Ru1−C30
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−O1
Ru1−N1
Ru1−C29
Ru1−C30
Ru1−C31
Ru1−C32
Ru1−C33
Ru1−C34
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
3
4
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
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
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)
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)
O1−Ru1−Cl1
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
84.25(5)
O1−Ru1−N1
N1−Ru1−Cl1
O1−Ru1−C25
O1−Ru1−C26
O1−Ru1−C27
O1−Ru1−C28
O1−Ru1−C29
O1−Ru1−C30
O1−Ru1−N1
N1−Ru1−Cl1
O1−Ru1−C29
O1−Ru1−C30
O1−Ru1−C31
O1−Ru1−C32
O1−Ru1−C33
O1−Ru1−C34
86.43(6)
84.60(5)
93.82(7)
88.05(7)
110.04(7)
146.84(7)
162.04(8)
124.28(8)
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
marginally longer than the Ru−Cl distances of ca. 2.39−2.41 Å
observed for our earlier reported N,N-coordinated com-
9
,81,82
plexes.
The Ru−O bond distances of the complexes
crystallized in a monoclinic system with space group P2 /c,
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 Ru in
1
whereas complexes 5 and 6 crystallized in a triclinic system
II
with P1
̅
space group. The crystal structure of each complex
showed a chelating N,O coordination bond from the bidentate
the 2,6-diisopropylaniline-based N,N-coordinated complex is
83
Schiff base ligands and monodentate coordination by chloride
ca. 1.70 Å, whereas in our complexes, it is ca. 1.68 Å; thus,
II
(
Figure 2). The fourth position was occupied by a p-cymene
the interaction between p-cymene and Ru is stronger in the
6
ring coordinating with the Ru center in an η fashion. Thus, the
Ru centers in these complexes are in a pseudooctahedral
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°,
II
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
II
between a Ru -coordinated Cl atom and an aromatic H atom
II
of the p-cym attached to a Ru 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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Article
Table 3. Selected Crystallographic Parameters for Complexes 2−6
2
3
4
5
6
empirical formula
radiation
fw/(g/mol)
temp/K
cryst syst
space group
a/Å
b/Å
c/Å
α/deg
β/deg
C H ClNORu
Cu Kα (λ = 1.54184)
545.06
100.00(10)
monoclinic
C H ClN O Ru
Cu Kα (λ = 1.54184)
594.13
94.8(4)
monoclinic
C H ClN O Ru
Cu Kα (λ = 1.54184)
624.16
100.00(10)
monoclinic
C H ClN O Ru
Cu Kα (λ = 1.54184)
644.19
100.00(10)
triclinic
C H ClN O Ru
38 47 2 2
2
9
30
30 37
2
2
31 39
2
3
34 39
2
2
Cu Kα (λ = 1.54184)
700.326
101(2)
triclinic
P1
̅
11.0249(5)
11.7028(5)
13.9028(4)
83.556(3)
89.613(3)
69.622(4)
1669.88(12)
2
P2 /c
P2 /c
P2 /c
P1
̅
1
1
1
7.96410(10)
17.0207(3)
17.4922(3)
90
92.9190
90
2368.07(7)
4
1.529
8.8322(3)
14.4477(5)
21.3187(7)
90
98.241(4)
90
2692.28(16)
4
1.466
9.5488(2)
14.0931(3)
21.2814(4)
90
92.943(2)
90
2860.11(10)
4
1.450
10.7783(5)
10.8455(5)
13.4383(5)
85.531(4)
83.630(4)
69.294(4)
1459.08(12)
2
γ/deg
volume/Å3
Z
3
ρcalc/(mg/cm )
1.466
5.452
1.393
4.808
μ/mm−
1
6.561
5.856
5.568
F(000)
cryst size/mm3
1120.0
1232.0
1296.0
668.0
732.0
0.3647 × 0.2192 ×
0.111 × 0.0468 × 0.0213 0.4614 × 0.2865 × 0.1352 0.1333 × 0.1333 × 0.0582 0.125 × 0.125 × 0.0480
0
.0624
2
θ range for data
7.252−132.336
7.416−132.42 7.526−132.158 6.624−132.266 6.402−132.418
collection/deg
index ranges
−9 ≤ h ≤ 9, −20 ≤ k ≤ −8 ≤ h ≤ 10, −17 ≤ k ≤ −10 ≤ h ≤ 11, −16 ≤ k ≤ −11 ≤ h ≤ 12, −12 ≤ k ≤ −12 ≤ h ≤ 13, −13 ≤ k ≤
8, −20 ≤ l ≤ 20 11, −24 ≤ l ≤ 25 15, −23 ≤ l ≤ 25 12, −15 ≤ l ≤ 15 13, −16 ≤ l ≤ 16
1
reflns collected
indep reflns
41894 11310 11725 10491 13122
4142 [R = 0.0472, R = 4695 [R = 0.0392, R = 4970 [R = 0.0247, R = 5063 [R = 0.0282, R = 5716 [R = 0.0369, R =
int
σ
int
σ
int
σ
int
σ
int
σ
0
.0178]
0.0472]
0.0274]
0.0347]
0.0424]
data/restraints/
param
GOF on F2
4142/0/303
4695/0/330
4970/0/349
5063/0/366
5716/0/404
1.068
1.033
1.063
1.059
1.023
final R indexes [I ≥ R = 0.0305, wR =
R = 0.0334, wR =
R = 0.0241, wR = 0.0611 R = 0.0288, wR = 0.0723 R = 0.0270, wR = 0.0650
1 2 1 2 1 2
1
2
1
2
2σ (I)]
0.0748
0.0785
final R indexes [all
data]
R = 0.0314, wR =
R = 0.0393, wR =
R = 0.0250, wR = 0.0617 R = 0.0310, wR = 0.0740 R = 0.0301, wR = 0.0664
1
2
1
2
1
2
1
2
1
2
0.0754
0.0820
largest diff
0.99/−0.64
0.89/−0.95
0.37/−0.70
1.47/−0.77
0.42/−0.65
3
peak/hole/(e /Å )
a
R = ∑|F | − |F ||/∑|F |. wR = [∑[w(F − F ) ]/∑w(F ) ]1/2.
b
2
2
2
2 2
1
o
c
o
2
o
c
o
A) with a ∠D−H···A of ca. 142−175°. In 4, a weak hydrogen-
bonding interaction is present among an O atom of methoxy
and an aromatic H atom of p-cym having a distance of ∼3.4 Å
h (time-dependent NMR was done only for a 24 h period). We
also investigated the stability of complex 4 as a representative
1
in the series for up to 72 h through H NMR and found that it
(
D···A) with a ∠D−H···A of ca. 143°. In 2, a weak hydrogen-
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
bonding 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···π-
stacking 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
+
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
3
1
mM NaCl and mixed with DMSO-d in a 7:3 (v/v) ratio. Two
333.1382 (calcd 333.1374) (Figure S33). The P NMR of
the precipitate did not provide any P signal from the
6
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
precipitate in CD OD. One reason could be that the
3
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 distribu-
tion, 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
with a sample incubated with 1 equiv of AgNO to precipitate
3
AgCl and lead to the monoaquated complex. The sample
without added AgNO and with AgNO showed the exact
3
3
same chemical shifts, confirming the immediate hydrolysis
Figure 3). The hydrolyzed complexes are stable for at least 24
(
G
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Article
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
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.
80,84
per the OECD guidelines.
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-
Table 4. In Vitro Anticancer Activity of Complexes 1−6 in
Various Cancer Cell Lines under Normoxic Conditions
a
b
IC50 ± SD (μM)
compound
MDA-MB-231
Hep-G2
MiaPaCa2
HL1
>50
ND
ND
1
HL2
1.9 ± 0.4
>50
5.8 ± 0.9
ND
3.6 ± 0.3
ND
Figure 4. Lipophilicity of complexes 1−6 in a 1:1 (v/v) octanol and
water mixture at 25 °C.
2
HL3
1.6 ± 0.3
>100
4.1 ± 0.5
ND
3.1 ± 0.3
ND
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-
3
HL4
4
HL5
5
HL6
6
4.4 ± 0.4
>100
3.0 ± 0.8
>50
1.5 ± 0.3
>50
1.2 ± 0.3
13.9 ± 1.9
ND
9.1 ± 2.2
ND
4.4 ± 0.4
ND
2.8 ± 0.1
9.8 ± 0.3
8.4 ± 1.6
ND
7.3 ± 2.1
ND
2.0 ± 0.6
ND
2.0 ± 0.2
5.7 ± 0.2
27
oxaliplatin
19.2 ± 1.2
a
MTT assay was performed in normoxic conditions after 72 h of
incubation. Standard deviation.
b
H
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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.
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.
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 MDA-
MB-231 cells are incubated with 2 and 6 at IC doses for 24 h,
5
0
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
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
87
killing by apoptosis or necrosis.
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
88,89
synthesis.
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
85,86
(
LAMP-1 and LAMP-2).
The inner lumen of these
proteins is highly glycosylated and protects the lysosomal
membrane from the digestive enzymes. If the complexes
I
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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
CONCLUSIONS
■
90−96
autophagy, LC3B-I is converted to LC3B-II,
so the
The N,O-coordinating 2,6-disubstituted aniline-based Schiff
bases form ruthenium(II) p-cymene complexes that immedi-
ately 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.
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
9
7−99
regulate apoptosis.
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%,
ASSOCIATED CONTENT
■
∼
10%, and ∼17% late apoptosis and ∼8%, ∼9%, and ∼11%
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.1c01363.
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
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
11,81
earlier-reported complexes of the same genre.
Complexes
3
−5 have the morpholine motif attached to the 2,6-
(
(
Figure S29), interaction of the crystals in the unit cell
Figure S30), aquation of 2, 5, and 6 (Figure S31),
disubstituted anilines, but complex 2 does not have the
pendant morpholine but also does not activate caspase-3
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
(
5
Figure 6b) and translocate cathepsin D to the nucleus (Figure
b). 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.
(
Figure S39) (PDF)
J
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Article
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 crys-
tallographic 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.
AUTHOR INFORMATION
Corresponding Authors
Arindam Mukherjee − Department of Chemical Sciences and
Centre for Advanced Functional Materials, Indian Institute of
Science Education and Research (IISER) Kolkata, Mohanpur
■
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