Synthesis, Structure, Stability, and Inhibition of Tubulin Polymerization by RuII- p-Cymene Complexes of Trimethoxyaniline-Based Schiff Bases

Article abstract of DOI:10.1021/acs.inorgchem.9b00853

Four trimethoxy- and dimethoxyphenylamine-based Schiff base (L1-L4)-bearing Ru^II-p-cymene complexes (1-4) of the chemical formula [Ru^II(??⁶-p-cymene)(L)(Cl)] were synthesized, isolated in pure form, and structurally characterized using single-crystal X-ray diffraction and other analytical techniques. The complexes showed excellent in vitro antiproliferative activity against various forms of cancer that are difficult to cure, viz., triple negative human metastatic breast carcinoma MDA-MB-231, human pancreatic carcinoma MIA PaCa-2, and hepatocellular carcinoma Hep G2. The ¹H nuclear magnetic resonance data in the presence of 10% dimethylformamide-d₇ or dimethyl sulfoxide-d₆ in phosphate buffer (pD 7.4, containing 4 mM NaCl) showed that the complexes immediately generate the aquated species that is stable for at least 24 h. Electrospray ionization mass spectrometry data showed that they do not bind with guanine nitrogen even in the presence of 5 molar equivalents of 9-EtG, during a period of 24 h. The best complex in the series, 1, exhibits an IC₅₀ of approximately 10-15 μM in the panel of tested cancer cell lines. The complexes do not enhance the production of reactive oxygen species in the cells. Docking studies with a tubulin crystal structure (Protein Data Bank entry 1SAO) revealed that 1 and 3 as well as L1 and L3 have a high affinity for the interface of the α and β tubulin dimer in the colchicine binding site. The immunofluorescence studies showed that 1 and 3 strongly inhibited microtubule network formation in MDA-MB-231 cells after treatment with an IC₂₀ or IC₅₀ dose for 12 h. The cell cycle analysis upon treatment with 1 showed that the complexes inhibit the mitotic phase because the arrest was observed in the G2/M phase. In summary, 1 and 3 are Ru^II half-sandwich complexes that are capable of disrupting a microtubule network in a dose-dependent manner. They depolarize the mitochondria, arrest the cell cycle in the G2/M phase, and kill the cells by an apoptotic pathway.

Full text of DOI:10.1021/acs.inorgchem.9b00853

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Synthesis, Structure, Stability, and Inhibition of Tubulin
Polymerization by RuII−p‑Cymene Complexes of Trimethoxyaniline-
Based Schiff Bases
Sourav Acharya,† Moumita Maji,† Ruturaj,‡ Kallol Purkait,† Arnab Gupta,‡
and Arindam Mukherjee*,†,§
†Department of Chemical Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, Nadia, West Bengal
741246, India
‡Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, Nadia, West Bengal
741246, India
§Center for Advanced Functional Materials (CAFM), Indian Institute of Science Education and Research Kolkata, Mohanpur,
Nadia, West Bengal 741246, India
S
* Supporting Information
ABSTRACT: Four trimethoxy- and dimethoxyphenylamine-
based Schiff base (L1−L4)-bearing RuII−p-cymene complexes
(1−4) of the chemical formula [RuII(η6-p-cymene)(L)(Cl)]
were synthesized, isolated in pure form, and structurally
characterized using single-crystal X-ray diffraction and other
analytical techniques. The complexes showed excellent in vitro
antiproliferative activity against various forms of cancer that are
difficult to cure, viz., triple negative human metastatic breast
carcinoma MDA-MB-231, human pancreatic carcinoma MIA
1
PaCa-2, and hepatocellular carcinoma Hep G2. The H nuclear
magnetic resonance data in the presence of 10% dimethylforma-
mide-d7 or dimethyl sulfoxide-d6 in phosphate buffer (pD 7.4,
containing 4 mM NaCl) showed that the complexes immediately generate the aquated species that is stable for at least 24 h.
Electrospray ionization mass spectrometry data showed that they do not bind with guanine nitrogen even in the presence of 5
molar equivalents of 9-EtG, during a period of 24 h. The best complex in the series, 1, exhibits an IC50 of approximately 10−15
μM in the panel of tested cancer cell lines. The complexes do not enhance the production of reactive oxygen species in the cells.
Docking studies with a tubulin crystal structure (Protein Data Bank entry 1SAO) revealed that 1 and 3 as well as L1 and L3
have a high affinity for the interface of the α and β tubulin dimer in the colchicine binding site. The immunofluorescence studies
showed that 1 and 3 strongly inhibited microtubule network formation in MDA-MB-231 cells after treatment with an IC20 or
IC50 dose for 12 h. The cell cycle analysis upon treatment with 1 showed that the complexes inhibit the mitotic phase because
the arrest was observed in the G2/M phase. In summary, 1 and 3 are RuII half-sandwich complexes that are capable of
disrupting a microtubule network in a dose-dependent manner. They depolarize the mitochondria, arrest the cell cycle in the
G2/M phase, and kill the cells by an apoptotic pathway.
effects.8,16,17 Though their mode of action is not totally
INTRODUCTION
■
understood, activation through reduction (RuIII−RuII) may be
The serendipitous discovery of cisplatin by Rosenberg et al. led
the pathway of activation, and they have both proteins and
to intensive research of metal-based anticancer agents.1,2 Apart
DNA as targets. On the other hand, organometallic half-
from Pt(II/IV), two other metals, Ru and Ga, have shown
sandwich RuII complexes allow versatile modes of interaction
potential, and among them, a few complexes have found their
place in clinical and/or preclinical trials.3−8 Ru complexes have
been found to be excellent candidates for overcoming cisplatin
and flexibility in the structure. Their mechanism of action
varies depending on the attached ligand(s), arene moi-
eties,18−21 and attached halides.22,23 [Ru(η6-C12H10)(en)Cl]-
resistance with weaker side effects showing a different
(PF6) (RM 175), [Ru(η6-p-cymene)(pta)Cl2] (RAPTA-C),
and [Ru(η6-C6H5Me)(pta)Cl2] (RAPTA-T), where en is
ethane-1,2-diamine and pta is 1,3,5-triaza-7-
mechanism of action.9−12 Octahedral RuIII complexes NAMI-
A and NKP-1339 (Figure 1) have undergone clinical
trials.13−15 However, NAMI-A was concluded to be not
effective during a Phase II clinical trial, and NKP1339 has
successfully completed a Phase I clinical trial against non-
small-cell lung carcinoma (NSCLC) with minimum side
Received: March 25, 2019
© XXXX American Chemical Society
A
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Figure 1. RuIII (NKP1339 and NAMI-A) and half-sandwich RuII (RAPTA-C and RM175) anticancer agents, tubulin polymerization inhibitors
(colchicine and combretastatin), and a general representation of the RuII complexes studied herein.
phosphatricyclo[3.3.1.1]decane, belong to the half-sandwich
RuII family of complexes that have undergone preclinical
trials.12,21,24−26 RM 175 with a labile Ru−Cl bond is known as
a DNA targeting molecule. On the other hand, the RAPTA
class of complexes may target various proteins and show
promising anti-metastatic effects in vivo and in vitro.27
the trimethoxyphenyl (TMP) moiety in their chemical
structure. This TMP moiety is present in combretastatine A4
and colchicine and seems to be very crucial for inhibiting
tubulin polymerization.48−51 Keeping that in mind, we have
designed RuII−p-cymene complexes of four different Schiff
bases containing trimethoxy- and dimethoxyaniline as a part of
the ligand system. Docking studies with the ligands suggested
that they may present an orientation that can inhibit tubulin
polymerization by binding to the same active site as colchicine,
during microtubule formation by α and β tubulins. Hence, we
have synthesized four complexes 1−4 with the chemical
formula [RuII(η6-p-cymene)(L)(Cl)] and structurally charac-
terized them using single-crystal X-ray diffraction and other
analytical techniques. These complexes have been analyzed
with respect to their stability in aqueous solutions and
evaluated for their ability to inhibit tubulin polymerization
along with in vitro cytotoxicity in various cancer cell lines.
The spectrum of activity of a metal-based drug is enhanced
or tuned by incorporation of an organic directing molecule
(ODM) as a ligand, giving researchers an opportunity to target
various organelles or processes, viz., endoplasmic reticu-
lum,28,29 lysosome,30,31 mitochondria,32,33 and tubulin poly-
merization.34−36 Tubulin polymerization involves the assembly
of α and β tubulin hetero-dimers which leads to formation of
the mitotic spindle apparatus that participates in cytoskeleton
formation, intracellular transport, and cell division. Due to
their crucial role in cell division (mitosis), microtubules are a
clear target for anticancer chemotherapeutic drugs.37,38 There
are two major types of microtubule-based anticancer agents.
One group consists of the microtubule destabilizers that inhibit
tubulin polymerization, viz., colchicine, combretastatin A-4,
and vinca alkaloids (Figure 1), while the other type stabilizes
microtubules by promoting tubulin polymerization, viz.,
taxanes.39−41 These complexes inhibit the mitotic phase and
inhibit the cell cycle at the G2/M phase, which ultimately leads
to apoptosis.42
The recent advances showed that formation of Ru and Pt
complexes with tubulin binding agents makes them efficient
inhibitors of tubulin polymerization. Huang et al. showed that
incorporation of different tubulin polymerization inhibitors
into the axial position of Pt(IV) derivatives leads to toxicity
that is higher than those of their corresponding Pt(II)
analogues against cancer cell lines.34,35,43,44 These complexes
showed equal potency toward cisplatin resistant and sensitive
cell lines. Recently, Sadler et al. synthesized RuII arene
derivatives of N-tosyl-1,2-diphenylethane-1,2-diamine, which
were found to accumulate in the cytosol and inhibit tubulin
polymerization.45 Inspired by colchicine and combretastatin,
many new tubulin polymerization inhibitors have been
designed.46,47 Some of them show promising inhibition of
microtubule formation and good antiproliferative activities.
The structure−activity relationship suggests the importance of
EXPERIMENTAL SECTION
■
Materials and Methods. The chemicals were purchased from
multiple commercial sources and used without further purification.
The solvents were distilled and dried using standard procedures, prior
to use. The metal precursor complex [Ru(η6-p-cym)Cl2]2 was
synthesized following a known literature procedure.52 MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (USB) and
all kinds of supplements and assay kits were purchased from Gibco
and used as received. Anti-tubulin antibodies were purchased from
Abcam. All of the solvents used for spectroscopic measurements were
of spectroscopy grade and purchased from Spectrochem, India.
Ultraviolet−visible (UV−vis) spectroscopic measurements were taken
using an Agilent Technologies Cary 300 Bio spectrophotometer. The
FT-IR spectra were recorded using a PerkinElmer SPECTRUM RX I
spectrometer in KBr pellets. The 1H and 13C nuclear magnetic
resonance (NMR) spectra were recorded using a 400 MHz JEOL
ECS or 500 MHz Bruker Avance III spectrometer, at room
temperature (24−27 °C). The chemical shifts (δ) of the relevant
compounds are reported in parts per million. All of the mass spectra
(ESI-HRMS) were recorded in positive electrospray ionization mode
using a Bruker maXis II instrument. Elemental analyses were
performed with a PerkinElmer 2400 series II CHNS/O analyzer.
1
Isolated yields of H NMR pure compounds are reported.
Syntheses. Synthesis of 2-{[(3,4,5-Trimethoxyphenyl)imino]-
methyl}phenol (L1). L1 was synthesized according to the literature
B
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procedure53 with some modifications. 3,4,5-Trimethoxyaniline (5
mmol) and salicylaldehyde (5 mmol) were dissolved in methanol and
refluxed for 12 h in the presence of a catalytic amount of formic acid.
After completion of the reaction, the reaction mixture was
concentrated and kept in an ice bath for 1 h, from which L1
precipitated as a yellow solid. This solid was washed with petroleum
134.7, 121.5, 118.3, 112.9, 101.3, 100.2, 96.9, 85.5, 83.4, 82.4, 80.7,
60.1,55.9, 29.8, 22.1, 21.3, 17.8 (Figure S10). UV−vis [MeOH, λmax
,
nm (ε, M−1 cm−1)]: 294 (8490), 405 (3350). IR (KBr pellets, cm−1):
1608, 1440, 1231 (Figure S17A). ESI-HRMS (MeOH) m/z: (exp)
522.1221 (522.1213) [RuIIC26H30NO4+]. Anal. Calcd for
C26H30ClNO4Ru: C, 56.06; H, 5.43; N, 2.51. Found: C, 56.24; H,
5.41; N, 2.53. Mp: 178 °C.
1
ether and finally dried in a vacuum desiccator. Yield: 82%. H NMR
RuII(p-cym)(L2)Cl (2). Yield: 60%. 1H NMR (500 MHz, DMSO-d6,
298 K) δ: 7.90 (1H, s, CHN), 7.42 (1H, d, J = 1.5 Hz, Ar−H),
7.15−7.10 (3H, m, Ar−H), 7.03 (1H, d, J = 8.5 Hz, Ar−H), 6.72
(1H, d, J = 3.5 Hz, Ar−H), 6.35 (1H, t, J = 7 Hz, Ar−H), 5.4 (1H, d,
J = 6.0 Hz, p-cym-H), 5.34 (1H, d, J = 6.0 Hz, p-cym-H), 5.14 (1H, d,
J = 6.0 Hz, p-cym-H), 4.36 (1H, d, J = 5.5 Hz, p-cym-H), 3.82 (3H, s,
OMe), 3.81 (3H, s, OMe), 2.00 (3H, s, p-cym-Me), 1.10 (3H, d, J =
6.5 Hz, p-cym-isopropyl), 1.07 (3H, d, J = 7.0 Hz, p-cym-isopropyl)
(Figure S11). 13C NMR (125 MHz, DMSO-d6, 298 K) δ: 164.8,
163.8, 152.1, 148.1, 147.3, 135.6, 134.6, 121.5, 118.5, 114.4, 112.9,
111.0, 108.5, 99.9, 97.4, 86.1, 83.0, 82.5, 81.1, 55.7, 55.5, 29.9, 22.2,
21.4, 18.0 (Figure S12). UV−vis [MeOH, λmax, nm (ε, M−1 cm−1)]:
295 (8650), 405 (3350). IR (KBr pellets, cm−1): 1610, 1445, 1233
(Figure S17B). ESI-HRMS (MeOH) m/z: (exp) 492.1161
(400 MHz, CDCl3, 298 K) δ: 13.12 (1H, s, OH), 8.54 (1H, s, CH
N), 7.34−7.19 (2H, m, Ar−H), 6.95 (1H, d, J = 8.3 Hz, Ar−H), 6.88
(1H, t, J = 7.6 Hz, Ar−H), 6.46 (2H, s, Ar−H), 3.84 (6H, s, OMe),
3.80 (3H, s, OMe) (Figure S1). 13C NMR (125 MHz, CDCl3, 298 K)
δ: 161.7, 161.1, 153.8, 137.39, 133.7, 133.4, 132.2, 119.2, 118.8, 117.3,
98.5, 61.0, 56.2 (Figure S2).
Synthesis of 2-{[(3,4-Dimethoxyphenyl)imino]methyl}phenol
(L2). L2 was synthesized according to the literature procedure54
with some modifications. 3,4-Dimethoxyaniline (5 mmol) and
salicylaldehyde (5 mmol) were dissolved in methanol and refluxed
for 12 h. After completion of the reaction, the reaction mixture was
evaporated to dryness. Then the solid mass was washed with
petroleum ether, redissolved in diethyl ether, and filtered. The filtrate
was evaporated to dryness and then dried in a vacuum desiccator. The
solid was washed with petroleum ether and finally dried in a vacuum
+
(492.1107) [RuIIC25H28NO3 ]. Anal. Calcd for C25H28ClNO3Ru: C,
1
56.98; H, 5.36; N, 2.66. Found: C, 57.14; H, 5.39; N, 2.68. Mp: 176
desiccator. Yield: 87%. H NMR (400 MHz, DMSO-d6, 298 K) δ:
°C.
13.32 (1H, s, OH), 8.96 (1H, s, CHN), 7.62 (1H, d, J = 8.0 Hz,
Ar−H), 7.38 (1H, m, Ar−H), 7.14 (1H, d, J = 2.2 Hz, Ar−H), 7.01
(4H, m, Ar−H), 3.83 (3H, s, OMe), 3.78 (3H, s, OMe) (Figure S3).
13C NMR (125 MHz, DMSO-d6, 298 K) δ: 161.2, 160.1, 149.3, 148.2,
140.8, 132.7, 132.2, 119.3, 118.9, 116.4, 113.9, 112.0, 105.0, 55.6, 55.6
(Figure S4).
Synthesis of 2-Methoxy-6-{[(3,4,5-trimethoxyphenyl)imino]-
methyl}phenol (L3). 3,4,5-Trimethoxyaniline (5 mmol) and o-
vaniline (5 mmol) were dissolved in methanol and refluxed for 12
h. The solution was then brought to room temperature and
evaporated to dryness. The orange mass was then washed several
times with petroleum benzene, sonicated, and washed with cold
diethyl ether. This solid was further purified by recrystallization from
ethanol. Yield: 95%. 1H NMR (400 MHz, DMSO-d6, 298 K) δ: 13.29
(1H, s, OH), 8.96 (1H, s, CHN), 7.22 (1H, d, J = 7.6 Hz, Ar−H),
7.12 (1H, d, J = 7.6 Hz, Ar−H), 6.91 (1H, t, J = 7.6 Hz, Ar−H), 6.78
(2H, s, Ar−H), 3.84−3.82 (6H, s, OMe), 3.68 (3H, s, OMe) (Figure
S5) 13C NMR (125 MHz, DMSO-d6, 298 K) δ: 162.8, 153.3, 150.5,
147.8, 143.5, 136.6, 123.8, 119.1, 118.5, 115.5, 99.0, 60.1, 55.9, 55.8.
(Figure S6).
1
RuII(p-cym)(L3)Cl (3). Yield: 56%. H NMR (400 MHz, CDCl3,
298 K) δ: 7.77 (1H, s, CHN), 6.97 (2H, d, J = 8.0 Hz, Ar−H), 6.74
(1H, dd, J1 = 7.5 Hz, J2 = 1.5 Hz, Ar−H), 6.59 (1H, dd, J1 = 7.5 Hz, J2
= 1.5 Hz, Ar−H), 6.36 (1H, t, J = 8.4 Hz, Ar−H), 5.36 (1H, d, J = 6.1
Hz, p-cym-H), 5.31 (1H, d, J = 6.0 Hz, p-cym-H), 5.1 (1H, d, J = 6.0
Hz, p-cym-H), 4.4 (1H, d, J = 5.3 Hz, p-cym-H), 3.92 (6H, s, OMe),
3.89 (3H, s, OMe), 3.82 (3H, s, OMe), 2.82 (1H, m, p-cym-
isopropyl-CH), 2.6 (3H, s, p-cym-Me) 1.17 (3H, d, J = 6.8 Hz, p-cym-
isopropyl), 1.12 (3H, d, J = 6.8 Hz, p-cym-isopropyl) (Figure S13).
13C NMR (125 MHz, CDCl3, 298 K) δ: 163.6, 154.4, 153.1, 152.3,
136.5, 126.4, 117.7, 114.8, 113.8, 101.7, 101.5, 98.6, 83.8, 81.0, 80.5,
80.5, 61.1, 56.5, 56.7, 30.5, 22.1, 21.3, 18.9 (Figure S14). IR (KBr
pellets, cm−1): 1608, 1440, 1231. ESI-HRMS (MeOH) m/z: (exp)
+
552.1319 (552.1218) [RuIIC27H32NO5 ]. UV−vis [MeOH, λmax, nm
(ε, M−1 cm−1)]: 272 (10500), 306 (9670), 420 (3180) (Figure
S17C). Anal. Calcd for C27H32ClNO5Ru: C, 55.24; H, 5.49; N, 2.39.
Found: C, 55.04; H, 5.50; N, 2.38. Mp: 180 °C.
RuII(p-cym)(L4)Cl (4). Yield: 56%. 1H NMR (125 MHz, DMSO-d6,
298 K) δ: 7.87 (1H, s, CHN), 7.4 (1H, d, J = 2.0 Hz, Ar−H), 7.12
(1H, m, Ar−H), 7.0 (1H, d, J = 8.5 Hz, Ar−H), 6.76 (2H, d, J = 8.0
Hz, Ar−H), 6.2 (1H, t, J = 7.5 Hz, Ar−H), 5.4 (1H, d, J = 6.0 Hz, p-
cym-H), 5.3 (1H, d, J = 6.0 Hz, p-cym-H), 5.16 (1H, d, J = 6.0 Hz, p-
cym-H), 4.4 (1H, d, J = 6.0 Hz, p-cym-H), 3.82 (3H, s, OMe), 3.81
(3H, s, OMe), 3.7 (3H, s, OMe), 2.8 (3H, s, p-cym-Me), 1.1 (3H, d, J
= 7.0 Hz, p-cym-isopropyl), 1.05 (3H, d, J = 7.0 Hz, p-cym-isopropyl)
(Figure S15). 13C NMR (125 MHz, DMSO-d6, 298 K) δ: 163.5,
156.3, 152.1, 151.9, 148.0, 147.3, 127.1, 118.1, 115.1, 114.4, 111.9,
111.0, 108.5, 99.9, 97.4, 85.9, 82.8, 82.5, 81.1, 55.7, 55.5, 29.8, 22.1,
21.3, 17.9 (Figure S16). UV−vis [MeOH, λmax, nm (ε, M−1 cm−1)]:
275 (10400), 307 (9550), 409 (3050) (Figure S17D). IR (KBr
pellets, cm−1): 1605, 1464, 1183. ESI-HRMS (MeOH) m/z: (exp)
522.1247 (522.1213) [RuIIC26H30NO4+]. Anal. Calcd for
C26H30ClNO4Ru: C, 56.06; H, 5.43; N, 2.51. Found: C, 56.32; H,
5.44; N, 2.49. Mp: 179 °C.
Synthesis of 2-{[(3,4-Dimethoxyphenyl)imino]methyl}phenol
(L4). This compound was synthesized by the same procedure
1
discussed above for L2. Yield: 73%. H NMR (400 MHz, CDCl3,
298 K) δ: 13.72 (1H, s, OH), 8.55 (1H, s, CHN), 6.96−6.90 (2H,
m, Ar−H), 6.83−6.81 (4H, m, Ar−H), 3.86 (6H, s, OMe), 3.84 (3H,
s, OMe) (Figure S7). 13C NMR (125 MHz, CDCl3, 298 K) δ: 160.5,
151.3, 149.6, 148.4, 141.4, 123.5, 119.1, 118.4, 114.5, 112.9, 111.5,
105.1, 56.2, 56.1, 55.9 (Figure S8).
Syntheses of Metal Complexes (1−4). General Procedure for
the Synthesis of Metal Complexes (1−4). The respective ligand (0.1
mmol) was dissolved in degassed methanol (10 mL) followed by
addition of KOH (0.1 mmol), and then the solution was allowed to
stir for 15 min. Then, a methanolic solution of [Ru(p-cym)Cl2]2 (0.1
mmol) was added in the dark under a nitrogen atmosphere at room
temperature. The resultant solution was stirred at room temperature
for 12 h. The entire solution was then evaporated to dryness, which
gave a yellowish-orange product that was washed several times with
diethyl ether and ultimately purified by column chromatography in
alumina with a 1:1 dichloromethane/acetone mixture.
X-ray Crystallography. Single crystals of a complex (1, 3, or 4)
were obtained by slow evaporation from a methanolic solution at
room temperature. A suitable single crystal was selected and mounted
over a loop of the goniometer of a SuperNova, dual, Cu at zero, Eos
diffractometer. The crystal was kept at 100.00(1) K during data
collection to minimize the probability volume of the ellipsoids. The X-
ray source used to collect data was Mo Kα. Data reduction was
performed in CrysAlisPro171.37.33c, and finally, the structure was
determined with the Superflip55 structure solution program using
Charge Flipping and refined with the ShelXL56 refinement package
using Least Squares minimization in Olex2.57 The CCDC numbers
are 1905422 (1), 1905423 (3), and 1905424 (4).
RuII(p-cym)(L1)Cl (1). Yield: 56%. 1H NMR (400 MHz, DMSO-d6,
298 K) δ: 9.56 (1H, d, J = 5.4 Hz, Ar−H), 8.86 (1H, s, CHN),
8.31−8.25 (2H, m, Ar−H), 7.88 (1H, t, J = 6.0 Hz, Ar−H), 7.16 (2H,
s, Ar−H), 6.06 (1H, d, J = 4.3 Hz, p-cym-H), 5.77 (2H, m, p-cym-H),
5.60 (1H, d, J = 6.1 Hz, p-cym-H), 3.88 (6H, s, OMe), 3.77 (3H, s,
OMe), 2.53 (1H, m, p-cym-isopropyl-CH), 2.17 (3H, s, p-cym-Me),
1.01 (6H, d, J = 6.8 Hz, p-cym-isopropyl) (Figure S9). 13C NMR (125
MHz, DMSO-d6, 298 K) δ: 164.8, 164.0, 154.0, 152.2, 135.8, 135.5,
C
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Solution Stability Study. Stabilities of the complexes (1−3) and
the ligands (L1 and L3) were determined by 1H NMR using DMSO-
d6/DMF-d7 and a 20 mM phosphate buffer mixture (pD 7.4)
containing 4 mM NaCl [1:9 (v/v)] at 25 °C at different time points
up to 24 h. The buffer solution was degassed prior to use.
Distribution Coefficient Determination. Distribution coeffi-
cients of the three complexes in an octanol−water system were
determined using the standard shake-flask method. Each set was
performed in triplicate, and the absorbance was recorded in a UV−vis
spectrophotometer using proper dilution. The concentration of the
substances in each layer was calculated using the respective molar
extinction coefficients of 1−4, and the distribution coefficient values
(log Do/w) were obtained from the ratio.
staining was done by resuspending the cell pellets in a 1× PBS
solution containing PI (55 μg mL−1) and RNaseA (100 μg mL−1).
Cell suspensions were gently mixed and incubated at 37 °C for 0.5 h.
Then samples were analyzed in a BD Biosciences FACS Calibur flow
cytometer.
Detection of Apoptosis: Annexin-V/PE Assay. Apoptotic cells
were 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. A total of 5 × 105 MDA-
MB-231 cells were seeded into a 100 mm sterile tissue culture Petri
dish using 6 mL of DMEM/F-12. Then, cells were incubated at 37 °C
in a 5% carbon dioxide atmosphere for 48 h. Subsequently, the
medium was changed and cells were treated with different
concentrations of drug solutions of 1 and 3 for 24 h. Cells were
then harvested with cold 1× PBS containing 0.1 mM EDTA and
subsequently washed twice with cold 1× PBS and finally resuspended
in Annexin V binding buffer. Cells were then incubated with both
Annexin V-PE 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.
Detection of Mitochondrial Membrane Potential Alteration
by JC-1. Investigation of the change in mitochondrial transmembrane
potential (MMP, ΔΨm) was determined using flow cytometry after
staining live cells with JC-1. A total of 1 × 106 MDA-MB-231 cells
were seeded in a 100 mm Petri dish. After incubation for 48 h,
medium was removed and the cells were treated with complexes 1 and
3 using IC20 and IC50 concentrations for 20 h. Cells were then
harvested by removing the medium and washed with 1× PBS. The
entire washed sample was collected and centrifuged at 2000 rpm for 4
min. The cells were washed twice with 1× PBS and resuspended in
1× PBS supplemented with 10% FBS. The resultant solution was then
incubated with 5 μg mL−1 JC-1 dye for 30 min in the dark. Finally,
after the supernatant had been removed, the cells were suspended in
1× PBS and analyzed in a BD Bioscience FACS Calibur flow
cytometer by measuring the red and green fluorescence intensities.
Caspase 3 Activation Assay. Activation of caspase 3 due to
complex treatment was investigated against MDA-MB-231 using the
caspase 3 colorimetric detection kit (Sigma). The manufacturer’s
protocol was followed throughout the assay. In brief, 5 × 105 MDA-
MB-231 cells were first seeded in a 100 mm sterile tissue culture Petri
dish for 48 h. Cells were then treated with both IC20 and IC50
concentrations of complexes 1 and 3 for 24 h. The release of p-
nitroaniline was monitored over time after caspase 3 substrate
treatment with the cell lysate. The assay was performed following the
96-well plate method, and data were recorded using an enzyme-linked
immunosorbent assay plate reader at 405 nm. A standard curve was
drawn using a known concentration of pNA (p-nitroaniline) to
estimate the amount of pNA released by caspase 3.
Cell Lines and Culture Conditions. Human pancreatic
carcinoma (MIA PaCa-2), triple negative human metastatic breast
adenocarcinoma (MDA-MB-231), and human hepatocellular carci-
noma (Hep G2) cells were obtained from NCCS (Pune, India). The
cells were grown in T-75 flasks as adherent monolayers in a 5%
carbon dioxide atmosphere using a culture medium, supplemented
with 10% fetal bovine serum (GIBCO) and antibiotics (100 units
mL−1 penicillin and 100 μg mL−1 streptomycin). Hep G2 cells were
grown in minimal essential medium (MEM), while MIA PaCa-2 and
MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) and in a 1:1 mixture of DMEM with Ham’s F12
nutrient mixture (i.e., DMEM/F-12), respectively. All cell lines were
maintained in their logarithmic phase of growth before each
experiment and plated when they reached 70% confluency.
Cell Viability Assay. The growth inhibitory effect toward tumor
cell lines (MDA-MB-231, Hep G2, and MIA PaCa-2) was evaluated
with the help of the MTT assay. In brief, 6 × 103 cells per well were
seeded in 96-well microplates in respective media (200 μL) and
incubated at 37 °C in a 5% carbon dioxide atmosphere. In case of
hypoxia, the oxygen level was maintained at 1.5% and cells were
grown under this condition for at least two passages; thereafter, they
were used for the MTT assay as described below. After incubation for
48 h, the medium was renewed with fresh medium (200 μL).
Compounds to be studied were added at the appropriate
concentrations. Each concentration was tested in triplicate in the
wells. The compounds to be added were first solubilized in medium
containing DMSO such that the concentration of DMSO in each well
would not exceed 0.2%. The same amount of DMSO was added in the
case of cell-based studies. The incubation was continued for 48 h.
Upon completion of incubation with the compounds, the drug-
containing medium was removed and 200 μL of fresh medium was
added to each well followed by treatment with 20 μL of 1 mg mL−1
MTT in 1× phosphate-buffered saline (PBS) (pH 7.2). After
incubation for 3 h with a MTT solution at 37 °C, medium was
removed and resulting formazan crystals were dissolved in DMSO
(200 μL). The growth inhibition of cells was analyzed by measuring
the absorbance of the drug-treated wells with respect to untreated
ones at 595 nm using a SpectraMax M2e plate reader. IC50 values
(drug concentrations responsible for 50% cell growth inhibition) were
calculated by fitting nonlinear curves in GraphPad Prism 5 version
5.03, using a variable slope model constructed by plotting the cell
viability (percent) versus the log of drug concentration (micromolar)
(Figures S25−S28). IC50 values were calculated by nonlinear four-
parameter curve fitting in a dose−response inhibition−variable slope
model using GraphPad Prism. The data presented here are means of
at least three independent experiments; in a single experiment, each
concentration was assayed in triplicate.
Docking Studies. Molecular modeling was performed using
GOLD (Genetic Optimization for Ligand Docking) Suite (version
5.4.1) software. GOLD generally adopts the genetic algorithm to dock
molecules into protein (macromolecule) active sites. The GOLD
score was used as a search algorithmic function for more effective
complexes. A higher GOLD score fitness value indicates a better
binding interaction in the binding site of the protein. Inhibitor
(colchicine)-bound tubulin protein [Protein Data Bank (PDB) entry
1SAO] was used to dock the complexes. For receptor preparation,
PDB entry 1SAO was first downloaded from the PDB. The protein
was then optimized and energy minimized by applying the OPLS
2005 force field by the protein preparation utility in Maestro Suite
Cell Cycle Arrest. MDA-MB-231 cells (5 × 105 cells per plate)
were grown in a 100 mm sterile cell culture Petri dish suspended in 5
mL of DMEM-F12 under previously described culturing condition.
After 48 h, medium was removed followed by addition of fresh
medium. Appropriate concentrations of the compound solutions were
added and incubated under the same condition described above. After
being exposed to the drug for 24 h, cells were harvested by
trypsinization, centrifuged, and washed twice with cold 1× PBS buffer
(pH 7.2). Cells were again resuspended in 100 μL of cold 1× PBS
buffer and fixed with 70% aqueous ethanol overnight at 4 °C. DNA
2016-1 in Maestro (Schrodinger Suite 2016-1 Protein Preparation
̈
̈
Wizard, Epik, Schrodinger, LLC, New York, NY, 2016; Impact,
̈
̈
Schrodinger, LLC, New York, NY, 2016; Prime, Schrodinger, LLC,
New York, NY, 2016). This optimized protein was used in the GOLD
suite, and the bound inhibitor was extracted. The complexes to be
docked were first optimized using Gaussian 09 software via density
functional theory (DFT) with the B3LYP functional and 6-31G(d)
basis set for C, H, N, and O and sdd basis set for the RuII center and
halides. Orbitals were defined as unrestricted during calculations. The
conductor-like polarizable continuum model (CPCM) was used with
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Scheme 1. Synthesis of Ligands (L1−L4) and Ruthenium Complexes (1−4)
Figure 2. ORTEP diagrams of complexes 1, 3, and 4. Thermal ellipsoids are drawn at the 50% probability level. All hydrogen atoms have been
omitted for the sake of clarity.
KOH, followed by addition of [RuII(η6-p-cym)Cl2]2 in
degassed MeOH at 50 °C for 12 h, as shown in Scheme 1.
The isolated bulk product was obtained by column
water as the solvent. Known inhibitor colchicine, which was extracted
from the active site of 1SAO, was used as is to revalidate the docking
study and to compare the affinity with those of other complexes. The
optimized protein structure was used in the GOLD Docking Wizard
chromatography in neutral alumina. All complexes herein are
to add necessary hydrogen. The active site was defined using the
reported for the first time and well characterized by 1H NMR,
colchicine binding site and within 6 Å of its center. The binding
13C NMR, ESI-HRMS, FT-IR, and UV−vis analysis. The bulk
sphere was large enough to cover the entire active site of the α and β
purity was confirmed by elemental analysis. UV data showed
peaks at approximately 275−295 nm for all of the complexes,
which corresponds to the π−π* transition and a weak peak
around 405−420 nm that corresponds to metal to ligand
charge transfer.
X-ray Crystallography. A suitable single crystal of each
complex was obtained from a methanolic solution by slow
evaporation. Complex 1 crystallizes in a monoclinic system
with space group P21/c, whereas complexes 3 and 4 crystallize
tubulin interface.
Effect on Tubulin Polymerization. A total of 5 × 104 MDA-
MB-231 cells were seeded over glass coverslips (Corning Life
Sciences) for 48 h. Cells were then treated with complexes 1 and 3 at
IC20 and IC50 doses for 12 h. Cisplatin (50 μM) was also treated for
12 h, for use as a negative control, and colchicine (100 nM) was used
as the positive control. After 12 h, media containing drugs were
removed and washed with 1× PBS. Cells were then fixed with 4% (v/
v) paraformaldehyde for 10 min and subsequently quenched with 100
mM glycine. After being washed three times with 1× PBS, cells were
blocked with 3% (w/v) bovine serum albumin (BSA) in PBS
containing 0.1% Tween 20 (PBST) for 20 min at room temperature.
After the BSA had been removed, cells were incubated with the
primary antibody against α-tubulin (anti-α tubulin antibody,
EP1332Y, rabbit monoclonal microtubulin marker) in a 1:400
dilution for 2 h, followed by washing three times with 1× PBST
and 1× PBS. Secondary antibody incubation was done with goat anti-
rabbit IgG H&L (AlexaFluor 488) in 1:1000 dilutions for 2 h in the
dark at room temperature. After being washed with PBST and PBS,
cells were mounted on slides for imaging using the Fluoroshield
mounting medium containing DAPI. The emission of AlexaFluor 488
(λmax = 520 nm) was used to visualize the microtubular network
within the cells. The DAPI enabled the visualization of the nucleus.
All images were taken with a Leica SP8 confocal microscope with a
63× objective.
̅
with a triclinic system in space group P1 (Table S1). The
single-crystal structure of each complex showed a chelating N−
O coordination from the bidentate Schiff base ligand and a
monodentate coordination by a chloride (Figure 2). The
fourth position was occupied by one p-cymene ring that
directly formed an η6 bond with the ruthenium center. In
complex 1, each unit cell contains four complexes, whereas
there are two complex moieties in 3 and 4. Some important
bond angles and bond distances are listed in Tables 1 and 2.
The Ru−C bond lengths of the p-cymene range from ∼2.16 to
∼2.20 Å. The Ru−O bond distances in complexes 1, 3, and 4
are in the range of 2.06−2.08 Å. The Ru−Cl bond distance in
the complexes ranges between ∼2.42 and ∼2.45 Å (Table 1).
The average O−Ru−N, Cl−Ru−N, and O−Ru−Cl bond
angles are ∼88°, ∼84°, and ∼86°, respectively (Table 2).
Intermolecular weak H-bonding interaction between the
metal-coordinated Cl and aromatic hydrogen of a p-cym was
observed for 1 and 3 due to presence of the interacting atoms
within the range of 3.2−3.5 Å (D···A) with a ∠D−H···A of
approximately 140−144°. Another weak H-bond was observed
between the Ru-coordinated O and H atom of an aromatic ring
RESULTS AND DISCUSSION
■
The N−O chelating ligands (L1−L4) were synthesized by
refluxing the respective amines and aldehydes in MeOH using
the literature procedure53,54 with slight modifications. Ru−
arene complexes 1−4 were synthesized in high yields by
stirring the respective ligands with an equivalent amount of
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OMe group with the side-chain −NH2 of Lys254 and the
−CONH2 of Gln11 of the protein. In 3, the m-OMe group is
hydrogen bonded with the carboxylate of the Glu183 residue
(Figure S20). Thus, in the TMP motif-containing complexes,
the salicylaldehyde-based ligand showed better interaction,
leading to a higher affinity of 1 as per the docking studies. The
docking studies show good interaction between the com-
pounds and the tubulin indicating the ability of the compounds
to interact with the protein, but the actual inhibition will
depend on other important factors, viz., the stability of the
molecules in the culture media, the ability to enter cells, and
the flexibility of the molecule inside the protein structure.
Hence, we investigated the stability of the ligands and
complexes in aqueous solutions and probed their lipophilicity.
Stability in an Aqueous Solution. To understand the
fate of the ruthenium complexes under physiological
conditions, we studied the stability of the complexes using
either DMF-d7 or DMSO-d6 with 20 mM phosphate buffer
(pH 7.4, 4 mM NaCl) in a 1:9 (v/v) ratio. Stability studies
showed that complexes 1 and 2 were hydrolyzed within the
first 5 min completely to produce solely aquated species. The
immediate formation of the aquated species was confirmed by
incubating the sample with AgNO3, which yielded the
precipitation of AgCl, but there was no change in the chemical
Table 1. Selected Bond Distances (angstroms) for
Complexes 1, 3, and 4
1
3
4
Ru1−Cl1, 2.4268(6)
Ru1−O1, 2.0590(15)
Ru1−N1, 2.0812(18)
Ru1−C17, 2.192(2)
Ru1−C18, 2.167(2)
Ru1−C19, 2.186(2)
Ru1−C20, 2.195(2)
Ru1−C21, 2.196(2)
Ru1−C22, 2.176(2)
Ru1−Cl1, 2.4453(4)
Ru1−O1, 2.0752(11)
Ru1−N1, 2.0659(13)
Ru1−C18, 2.1867(16)
Ru1−C19, 2.1774(16)
Ru1−C20, 2.1836(16)
Ru1−C21, 2.1898(16)
Ru1−C22, 2.2066(17)
Ru1−C23, 2.1928(16)
Ru1−Cl1, 2.4345(5)
Ru1−O1, 2.0795(15)
Ru1−N1, 2.0807(18)
Ru1−C16, 2.186(2)
Ru1−C17, 2.165(2)
Ru1−C18, 2.159(2)
Ru1−C19, 2.202(2)
Ru1−C20, 2.200(2)
Ru1−C21, 2.204(2)
of p-cym with a ∠D−H···A of approximately 167°. In 4, the H-
bonding interaction distance is 3.55 Å and the ∠D−H···A is
approximately 165° (Figure S18). An oxygen atom of a
methoxy group and the Ru-coordinated O from one complex
form bifurcated H-bonds (∼3.32 Å) with an aromatic
hydrogen of the p-cym ring from another complex with angles
(D−H···A) of 153° and 131°, respectively (Figure S18). A
moderate π···π stacking interaction (∼3.8 Å) between the p-
cymene motifs of two adjacent complex molecules was
observed for complex 3 (Figure S19), whereas for complex
4, a weak π···π stacking was observed between two neighboring
o-vanillin motifs in a unit cell (Figure S19).
Molecular Docking Study. Combretastatin and colchicine
have the same binding site between α and β chains of tubulin
to inhibit their polymerization, thus disrupting the microtubule
network. From the literature reports, it seems that the TMP
motif is a crucial one to inhibit tubulin polymerization and
present in many tubulin polymerization inhibitors, viz.,
combretastatin and colchicine. Among our ligands, the
strongest resemblance to combretastatin and colchicine was
borne by L1 and L3 and their corresponding complexes 1 and
3, containing the TMP motif. Hence, L1, L3, 1, and 3 were
chosen for the docking experiments against α and β tubulin
assembly (PDB entry 1SAO). Colchicine was taken to
compare the docking affinity of the compounds. A higher
GOLD score indicates a better binding interaction of a
molecule with the protein active site. Docking studies with 1
and 3 suggest that they bind to the colchicine binding site of
the α and β tubulin assembly, and GOLD score values (Table
S2) indicate that the binding affinity of 1 (GOLD score of
48.6) is higher than that of 3 (GOLD score of 40.5). On the
other hand, the docking of ligands L1 (GOLD score of 49.9)
and L3 (GOLD score of 52.9) to the same protein structure
displays stronger interactions of the ligands themselves,
indicating their suitability in the design. The noncovalent
interaction of 1 is mainly through hydrogen bonding of the p-
1
shift observed by H NMR. Hence, we concluded that the
complexes may have hydrolyzed very fast. The hydrolyzed
aquated species is stable for at least 24 h (Figure 3 and Figures
Figure 3. Stability kinetics of complexes 1 and 2 in 20 mM phosphate
buffer (pH 7.4) with 4 mM NaCl and 10% DMF-d7. Data for each of
the complexes are separately plotted in Figures S21−S23 for the sake
of clarity.
Table 2. Selected Bond Angles (degrees) for Complexes 1, 3, and 4
1
3
4
O1−Ru1−Cl1, 86.02(5)
O1−Ru1−N1, 87.84(6)
N1−Ru1−Cl1, 84.08(5)
O1−Ru1−C17, 94.90(7)
O1−Ru1−C18, 127.81(8)
O1−Ru1−C19, 163.53(8)
O1−Ru1−C20, 142.05(8)
O1−Ru1−C21, 105.99(8)
O1−Ru1−C22, 86.18(7)
O1−Ru1−Cl1, 85.23(3)
N1−Ru1−O1, 88.37(5)
N1−Ru1−Cl1, 84.10(4)
O1−Ru1−C18, 91.04(5)
O1−Ru1−C19, 121.32(6)
O1−Ru1−C20, 158.90(6)
O1−Ru1−C21, 149.36(5)
O1−Ru1−C22, 111.93(5)
O1−Ru1−C23, 88.20(5)
O1−Ru1−Cl1, 87.80(4)
O1−Ru1−N1, 87.91(6)
N1−Ru1−Cl1, 83.84(5)
N1−Ru1−C16, 124.46(8)
N1−Ru1−C17, 96.45(8)
N1−Ru1−C18, 92.60(7)
N1−Ru1−C19, 115.08(8)
N1−Ru1−C20, 152.10(8)
N1−Ru1−C21, 162.31(8)
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S21 and S22). A similar result was obtained for 3 (Figure S23).
Because the stabilities of the complexes were similar, their
cytotoxicity would be dependent on the amount of interaction
of the complexes with the desired target if the lipophilicities are
similar.
Table 3. In Vitro Anticancer Activity of Complexes 1−4 in
Various Cancer Cell Lines under Normoxic and Hypoxic
Conditions
a
IC50 SD (μM)
The stability of the ligands was also monitored by 1H NMR
using a DMSO-d6/phosphate buffer mixture [3:7 (v/v)]. It was
observed that due to the presence of the hydrolyzable imine
bond both ligands (L1 and L3) were hydrolyzed very quickly
(almost 80% hydrolysis within 12 h) into corresponding amine
and aldehyde (Figure S24). The complexes of the same were
found to be stable as discussed above, suggesting that the
complexation increased the aqueous stability of the ligands.
Determination of Distribution Coefficients. Lipophi-
licity is considered to be an indication of understanding the
cellular uptake capability of a compound. Hence, lipophilicity
is an important parameter while studying cytotoxic efficacy.
The distribution coefficient values for ligands (L1−L4) as well
as complexes 1−4 in octanol/water systems were determined.
The values of log D for the ligands vary from 2.5 to 4, showing
a wide spectrum of lipophilicity and indicating the low water
solubility of the ligands. Complexation increased the water
solubility and decreased the log D to approximately 0.18−0.4
(Figure 4). The log D values of new pharmacophores in the
MDA-MB-
231
MDA-MB-231
(hypo)
MIA PaCa-2
Hep G2
1
2
3
4
11.4 1.9
11.5 0.4
16.9 0.3
18.6 1.7
37.2 2.5
12.5 0.8
ND
9.8 1.2
11.6 0.3
14.8 1.1
19.8 1.4
31.8 4.8
14.5 1.0
16.6 0.8
17.1 1.3
24.1 3.3
14.3 0.8
c
12.5 1.8
c
ND
b
CDDP
37.8 2.3
a
IC50 SD values are determined by the MTT assay in normoxia
(∼15% O2) and hypoxia (1.5% O2). SD, standard deviation. The
statistical significance (P) of the data ranges from >0.001 to <0.05.
b
c
CDDP, cisplatin. Not determined. See the Experimental Section for
full details.
receptors (estrogen, progesterone, and HER2). Thus, the
treatment of metastatic MDA-MB-231 almost solely depends
on chemotherapy and has a high relapse rate with existing
drugs. The MIA PaCa-2 cell line was chosen because it is
known to be an aggressive pancreatic cancer with high
tumorigenicity59 and does not express a detectable amount
of carcino embryonic antigen, thus making detection difficult.60
The in vitro studies showed that the ligands are nontoxic but
complexation increased their solubility and toxicity. The
docking studies (Figure S20) did, however, suggest that the
ligands should be toxic but did not take into account the
stability of the ligands in solution. The stability of the free
ligands is poor in a buffer solution as depicted by ∼80%
degradation of L1 and L3, to corresponding amine and
aldehyde, during a 12 h incubation period (Figure S24). The
ligands dissociate to their respective amine and aldehyde
(Figure S24). In addition, due to the high lipophilicity of the
ligands, it is also possible that most of the intact ligands are
stuck at the cell membrane. The metal complexation imparts
stability to the ligand and increases the hydrophilicity, allowing
the complexes to enter the cell cytoplasm thus enhancing their
cytotoxicity.
IC50 values of all four complexes range between 10 and 25
μM (Table 3). Complex 1 is the most toxic and is
approximately 3 times more potent than cisplatin (a drug
used in treatment of TNBC)61 against MDA-MB-231 and MIA
PaCa-2 under the same testing conditions. When 1 and 3 were
tested in the MDA-MB-231 cell line under hypoxic conditions
to check if there was any deactivation under hypoxia, which is
known to happen for many drugs,62,63 it was found that the
cytotoxicity remained unaltered. The anticancer activities of
the salicylaldimino derivatives are higher than those of o-
vanillinaldimino analogues as is evident from Table 3 on going
from 1 to 4.
Figure 4. Lipophilicity of the ligands (L1−L4) and their
corresponding metal complexes (1−4) in a 1:1 (v/v) octanol/water
mixture at 37 °C.
Comprehensive Medicinal Chemistry database range from
−0.4 to 5.6.58 It is encouraging to find that the log D values for
the ligands and complexes 1−4 are within the range of new
pharmacophores as shown by the database. In addition, the log
D values are quite similar, suggesting that the activity would be
mostly dependent only on the interaction of the complexes
with their targets due to the variation mostly in the number of
methoxy groups and the overall geometry of the complex.
In Vitro Cytotoxicity. Cytotoxicities of all four ligands
(L1−L4) and metal complexes (1−4) were tested in three
different cancer cell lines under normoxic conditions by the
MTT assay. The cell lines chosen for this purpose were human
pancreatic carcinoma (MIA PaCa-2), triple negative human
metastatic breast adenocarcinoma (MDA-MB-231), and
hepatocellular carcinoma (Hep G2). Data obtained are listed
in Table 3. Triple negative breast cancer, pancreatic carcinoma,
and liver carcinoma are difficult to cure; hence, these cell lines
have been chosen. In particular, triple negative breast cancer
MDA-MB-231 is one of the most difficult to treat and has a
poor response rate due to absence of three main targeting
Effect on Tubulin Polymerization. The in vitro toxicity
profile showed that complex 1 is the most toxic among all of
the complexes and 3 is less toxic than 1, which also corelates
with the docking study. However, there is an extra methoxy
group present in 3 compared to 1 that hence makes it more
resemble the combretastatin structure. Therefore, 1 and 3 were
selected to evaluate their effect on the disruption of the
microtubule network in the MDA-MB-231 cells. Colchicine
was taken as a positive control because it is known to inhibit
tubulin polymerization, whereas cisplatin was taken as a
negative control because it is a DNA cross-linking agent. With
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Figure 5. Immunofluorescence study of the disruption of the microtubule network in MDA-MB-231 cells after incubation for 12 h in the presence
of (A) 0.1% DMSO, (B) colchicine (100 nM), (C) cisplatin (50 μM), (D) 1 (IC50), and (E) 3 (IC50). Microtubules were visualized with a
monoclonal anti-α-tubulin antibody (green) interacting with a secondary antibody tagged with AlexFluor 488 (λem = 520 nm). The nucleus was
stained with DAPI (blue). Images were acquired with a Leica SP8 confocal microscope with a 63× objective. The scale bar represents 20 μm.
regard to cisplatin, we would like the readers to note that there
are some discrete reports indicating the potency of cisplatin to
interact with tubulin and disrupt polymerization at significantly
high dosages, although no cellular studies have been reported.
However, in our cellular studies when we used 50 μM CDDP
and treated MDA-MB-231 cells with them for 12 h, we did not
observe any disruption of the microtubule formation as
evidenced from Figure 5C.
The control cells without drug treatment exhibited a normal
filamentous microtubule array (green fluorescence) (Figure
5A) and so did the cisplatin-treated cells (Figure 5C).
However, the effect of cisplatin on the nucleus could be
observed in some portions (Figure S29). The cells treated with
Figure 6. Quantification of the inhibition of the microtubule network
colchicine (positive control) exhibited microtubule network
in MDA-MB-231 cells.
damage (Figure 5B). When cells were treated with complexes
1 and 3 at IC50 doses (Figure 5D,E), they exhibited disruption
of the microtubule networks leading to deformation similar to
that observed for colchicine. Even at only IC20 dosages of 1
and 3, the disruption of the microtubule network was evident
(Figure S29). Thus, 1 and 3 induced disruption of the
microtubule in a dose-dependent manner, indicating tubulin
polymerization was inhibited by these complexes in MDA-MB-
231 cells.
The inhibition effect became more apparent when the data
were quantified (Figure 6). It was done by averaging the
perimeter of the microtubule network for at least 10 treated
cells versus the same number of untreated cells from a control.
The relative perimeter (i.e., treated vs control) was plotted,
and better dose responses of 1 over 3 were observed.
Pathways of Cell Killing. The compounds under
investigation were half-sandwich complexes of RuII, many of
which exhibit excellent interactions with DNA bases. Hence, in
spite of the tubulin network disrupting ability, these complexes
were probed for their ability to bind 9-ethyl guanine, a model
nucleobase, using ESI-MS. Complexes were treated with 5
molar equivalents of 9-EtG in a 1:9 (v/v) mixture of MeOH
and 5 mM phosphate buffer (pH 7.4, containing 4 mM NaCl)
and studied in ESI-MS. The ESI-MS data did not show any
binding even after incubation for 24 h (Figures S30−S37). In
general, any 9-EtG adduct formation is prominently found in
the resultant spectrum for this p-cymene class of RuII
complexes, as reported previously.23 These indicate that
DNA may not be the prime target for these classes of
compounds; rather, they may noncovalently interact with the
interface of the α and β tubulin during the mitotic phase, at the
colchicine binding site.
Certain half-sandwich RuII−p-cymene complexes are also
known to enhance ROS production or concentration, which
actively helps apoptosis by inducing oxidative stress in cells.
ROS may be generated by these RuII−p-cymene complexes
due to their ability to disrupt the intracellular redox balance by
catalytic conversion of intracellular NADH to NAD+.30,64
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Complexes 1 and 3 were treated with NADH [1:9 (v/v)
MeOH and water containing 4 mM NaCl], and the absorption
at λmax values of 260 and 340 nm was monitored for 6 h
(Figure S38). There was almost no change observed, and it
was rather similar to the control experiment. This suggested
that the complexes were not converting NADH to NAD+. To
investigate, if there was any other process involved in the
enhancement of ROS by the complexes, we performed an in
vitro cellular assay using IC20 and IC50 dosages of 1 and 3 in
the MDA-MB-231 cell line. After being incubated for 12 h, the
cells were stained by DCFHDA and analyzed by flow
cytometry for the ROS population. The data showed that
there is no increase in the ROS level upon treatment,
suggesting that these complexes do not enhance ROS
production in cells (Figure S39).
The studies with 1 and 3 showed that the complexes
inhibited tubulin polymerization and did not enhance ROS
production. If the disruption of the microtubule network is the
pathway of cell killing, then the cell cycle arrest should occur in
the mitotic phase. Complex 1 was thus probed for cell cycle
arrest in the MDA-MB-231 cell line by flow cytometry using a
propidium iodide assay with two concentrations (8 and 12
μM). It was found that with increasing concentrations of 1, the
population of cells arrested at the G2/M phase increased
(Figure 7A and Figure S40). This further supported the idea
and ∼70% apoptosis at IC20 and IC50 dosages, respectively
(compared to 7% in control) (Figure 7B and Figure S41). The
late apoptotic stage cells become stained with both Annexin-V
and 7-AAD; the early apoptotic population becomes stained
with only Annexin-V, and dead cells are stained with only 7-
AAD, thus allowing the detection.
It is important to know if the apoptosis is induced through
extrinsic or intrinsic pathways or both are involved. The
intrinsic pathway is regulated via mitochondria and may be
monitored by the change in the mitochondrial membrane
potential (MMP, ΔΨm) using the fluorescent dye JC-1 using
flow cytometry. JC-1 dye in aggregated form emits red
fluorescence at a λmax of 590 nm, but when the mitochondrial
membrane depolarizes, it stabilizes the monomeric form of JC-
1 that emits a green fluorescence at a λmax of 550 nm. MDA-
MB-231 cells treated with the IC20 or IC50 concentration of 1
and 3 for 18 h showed an increase in the green fluorescence
intensity in a dose-dependent manner, suggesting the intrinsic
pathway may be mostly involved in apoptosis (Figure 7C and
Figure S42). Apoptosis was also further confirmed by the
release of caspase 3. The mitochondrial membrane potential
change leads to the release of cytochrome c, which activates
caspase 9 (initiator caspase) that ultimately cascades to activate
caspase 3 (effector caspase), leading to apoptotic cell death
after proteolytic cleavage of a series of cellular targets. MDA-
MB-231 cells treated with IC20 and IC50 concentrations of 1
and 3 showed release of free pNA in a dose-dependent manner
(Figure 7D), from the active caspase 3 substrate (Ac-DEVD-
pNA) thus further emphasizing the apoptotic pathway.
CONCLUSION
■
The N−O chelating half-sandwich RuII−p-cymene complexes
containing di- and trimethoxyphenyl motifs showed inhibition
of tubulin polymerization, thereby disrupting the microtubule
network in triple negative metastatic breast cancer MDA-MB-
231. The complexes exhibited aquation at physiological pH
(pH 7.4); the trimethoxyphenyl-containing aquated RuII−p-
cymene complexes do not bind to the guanine N7, unlike
DNA-targeting drugs like cisplatin, nor do they enhance ROS.
Complex 1 that is most efficient in microtubule network
disruption during the mitotic phase is also the most cytotoxic
complex in the series and arrests the cell cycle in the G2/M
phase, suggesting microtubule network disruption is the major
pathway of cytotoxicity. The inhibition described above led to
depolarization of the mitochondria, activation of caspase 3, and
finally apoptosis.
Figure 7. In vitro mechanistic studies of the apoptotic pathway of the
complexes. (A) Cell cycle arrest in the G2/M phase by 1 in a dose-
dependent manner (8 and 12 μM) in the MDA-MB-231 cell line. (B)
Induction of apoptosis by complexes 1 and 3 in the MDA-MB-231
cell line in a dose-dependent manner. (C) Influence on the change in
the mitochondrial membrane potential of MDA-MB-231 cells treated
with complexes 1 and 3 at their respective IC20 and IC50
concentrations for 18 h and stained with JC-1 (5 μg mL−1) analyzed
by flow cytometry. (D) Colorimetric determination showing caspase
3 activation in the MDA-MB-231 cell line with 1 and 3 at two
different concentrations for 24 h.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.9b00853.
NMR spectra of ligands (Figures S1−S8) and complexes
(Figures S9−S16), UV−vis spectra of the complexes
(Figure S17), different crystallization parameters and
interactions (Table S1 and Figures S18 and S19),
docking studies with tubulin protein 1SAO (Figure S20
and Table S2), hydrolysis of the complexes and ligands
in buffer (Figures S21−S24), MTT assay (Figures S25−
S28), microtubule disruption (Figure S29), binding with
9EtG of the complexes by ESI-MS (Figures S30−S37),
and different pathways of cell killing by the complexes,
that the cytotoxicity of the complexes is mediated via
disruption of the microtubule network in the mitotic phase.
Both 1 and 3 induced apoptosis as per the Annexin-V/PE and
7AAD double-staining assay in MDA-MB-231 cells. The IC50
as well as the sub-IC50 dose showed that cell killing occurs
through apoptosis. 1 induced ∼60% and ∼74% of apoptosis at
IC20 and IC50 dosages, respectively, whereas 3 induced ∼63%
I
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Inorganic Chemistry
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: a.mukherjee@iiserkol.ac.in.
ORCID
Arindam Mukherjee: 0000-0001-9545-8628
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors earnestly acknowledge CSIR, Government of
India, via Project 01(2927)/18/EMR-II. The authors also
thank IISER Kolkata for infrastructural and financial support.
S.A. thanks UGC. M.M. thanks CSIR. Ruturaj thanks IISER
Kolkata. K.P. thanks UGC for research fellowships. A.G. is
thankful for the Early Career research Award from SERB, DST,
Government of India (ECR/2015/000220), and a Wellcome
Trust-DBT India Alliance Fellowship (IA/I/16/1/502369).
The authors thank Dr. Supratim Dutta for the microplate
reader facility. The authors sincerely thank Mr. Tamal Ghosh
for helping with flow cytometry analysis studies.
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