Novel Schiff base copper complexes of quinoline-2 carboxaldehyde as proteasome inhibitors in human prostate cancer cells

Article abstract of DOI:10.1021/jm060712l

We report the synthesis of novel 1:1 Schiff base copper complexes of quinoline-2-carboxaldehyde showing dose-dependent, antiproliferative, and proapoptotic activity in PC-3 and LNCaP prostate cancer cells. We found that quinoline thiosemicarbazone 2 (FPA-

Full text of DOI:10.1021/jm060712l

7
242
J. Med. Chem. 2006, 49, 7242-7246
Novel Schiff Base Copper Complexes of Quinoline-2 Carboxaldehyde as Proteasome Inhibitors
in Human Prostate Cancer Cells
Shreelekha Adsule, Vivek Barve, Di Chen, Fakhara Ahmed, Q. Ping Dou, Subhash Padhye, and Fazlul H. Sarkar*,†
†
‡
§
†
§
†,‡
The PreVention Program and Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State UniVersity, School of Medicine,
9
374 Scott Hall, 540 East Canfield AVenue, Detroit, Michigan 48201, and Department of Chemistry, UniVersity of Pune, Pune 411007, India
ReceiVed June 12, 2006
We report the synthesis of novel 1:1 Schiff base copper complexes of quinoline-2-carboxaldehyde showing
dose-dependent, antiproliferative, and proapoptotic activity in PC-3 and LNCaP prostate cancer cells. We
found that quinoline thiosemicarbazone 2 (FPA-137) was the most potent and inhibited proteosome activity
in intact human prostate cancer PC-3 and LNCaP cells (IC50 of 4 and 3.2 µM, respectively) compared to
clioquinol and pyrrolidine dithiocarbamate (IC50 of 10 and 20 µM), supporting the novelty of 2.
Introduction
copper levels are as high as 2 µg/100 mL (equivalent to 0.3
µM) and in malignant prostates as high as 124 µg/100 mL
The ubiquitin-proteasome pathway has been extensively
1
7
(equivalent to 19.5 µM). The role of copper metal in the
studied in human cancers, which is known to play an important
role in regulating cell proliferation and cell death.1,2 It has been
ubiquitin-proteasome pathway is as yet undetermined. Although
copper alone has been known to inhibit the activity of purified
well-established that through a tight regulation of cell growth
inducers and growth inhibitors, an optimum balance of cell
proliferation and cell death can be achieved. In cancer cells,
however, an altered balance between cell growth inducers and
inhibitors leads to disruption of the cell cycle, causing deregu-
lated growth and inhibition of intrinsic apoptotic cell death
pathways. Thus, proteasome inhibition is a desirable target for
controlling the aberrant growth of tumor cells.
Proteasome inhibitors are known to induce cell death rapidly
and selectively in oncogene-transformed but not normal or
untransformed cells. They are also able to trigger apoptosis in
human cancer cells that are resistant to chemotherapy. Many
natural and synthetic inhibitors of proteasome have been
1
5
proteasome and copper complexes are able to inhibit the
cellular proteasome activity, the exact mechanism remains to
be explored.
The quinoline scaffold having a formyl/acetyl group adjacent
to a heterocyclic nitrogen can be easily appended with phar-
macophores bearing amino groups to yield Schiff base com-
pounds with a wide range of transition metals such as copper,
although their potential of arresting growth of cancer cells
1
8
remained unexplored. In the present study, we describe
synthesis and characterization of four such Schiff bases of
quinoline-2-carboxaldehyde 1, 3, 5, and 7 (FPA 136,138, 140,
and 142) and their copper complexes 2, 4, 6, and 8 (FPA 137,
139, 141, and 143). The cytotoxic effects of quinoline deriva-
tives 1-8 were tested in prostate cancer cell lines PC-3 and
LNCaP, and the apoptotic cell death was measured by histone/
DNA enzyme-linked-immunosorbent assay (ELISA). We further
investigated the in vitro effects of these compounds in LNCaP
cells and observed accumulation of ubiquitinated proteins and
the apoptosis-specific poly ADP-ribose polymerase (PARP)
cleavage after 24 h. On the basis of our results, we propose
that the hydrophilic thiocarbonyl side chain may confer anti-
proliferative activity of quinoline-copper complexes, which
could inhibit the ubiquitin-proteasome pathway in human
cancers.
3
,4
5
developed, which include lactacystin, threonine benzyl ester
macrocycle (TMC95), peptide aldehydes, peptide boronates,
6
7
8
9
10
peptide inyl sulfones, epoxyketones, peptide-R-keto alde-
11
hydes and R-ketoamides, 5-methoxy-1-indanone-3-acetic pep-
12
13
tide derivatives, and bivalent inhibitors. Z-Leu-Leu-Leu-
aldehyde (MG 132) was one of the first synthetic inhibitors to
1
4
be described and used in the inhibition of proteasome. Most
of them have the amide structural motif necessary for specific
recognition by the proteasome site. The major outcome from
the work by Dou et al. has shown that neither 8-hydroxyquino-
line nor copper(II) chloride alone can inhibit proteasome or
induce apoptosis but the copper complex of 8-hydroxyquinoline
can potently and selectively inhibit the chymotrypsin-like
activity of the proteasome in vitro and in vivo. 15 In our earlier
studies, we have shown that certain heterocyclic compounds
such as clioquinol and pyrrolidine dithiocarbamate act as potent
Experimental Section
Chemistry. The novel complexes 1, 3, 5, and 7 were obtained
in satisfactory yields by reacting the parent ligand quinoline-2-
carboxaldehyde with thiosemicarbazone and various hydrazines
such as benzoyl, nicotyl, and salicyl hydrazides in refluxing
methanol. Further, the copper complexes were obtained by heating
antitumor compounds by binding to endogenous copper in
prostate and breast cancer cells.16 We have proposed that highly
elevated levels of copper can be tumor-specific and use of
copper chelators might be one of the useful strategies for cancer
therapies. The levels of copper seem to vary in in vitro cultures
from low to trace levels, whereas in in vivo situations the serum
1
2
equiv of the free ligands and CuCl in methanolic solution at 40
°
C. Overall, the synthetic strategy is summarized in Scheme 1.
Compositional Studies. The analytical and spectroscopic data
on all synthesized ligands and their metal complexes are sum-
marized in Table 1 in Supporting Information, which indicate 1:1
stoichiometry for all copper complexes. The metal complexes are
insoluble in nonpolar organic solvents but dissolve in polar organic
solvent like DMSO. Their molar conductivities in DMSO indicate
the nonelectrolyte nature for these compounds.19 On the basis of
the microanalytical data, it is concluded that the obtained Cu(II)
*
To whom correspondence should be addressed. Phone: 313-576-8327.
Fax: 313-576-8389. E-mail: fsarkar@med.wayne.edu.
†
Department of Pathology, Wayne State University, School of Medicine.
University of Pune.
The Prevention Program and Department of Pathology, Wayne State
‡
§
University, School of Medicine.
1
0.1021/jm060712l CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/01/2006

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7243
Scheme 1. Synthesis of Quinoline-2-carboxaldehyde Derivatives
1
, 3, 5, and 7 and Their Copper Complexes 2, 4, 6, and 8
complexes exist in a monoaligned fashion that requires a ratio of
1
:1:1 for ligand, metal ion, and counterion such as chloride.
Spectroscopy and Magnetism. The electronic spectra of the
Figure 1. (A) Chemical structures of compounds 1 and 2. (B) Plot of
cell growth inhibition in LNCaP cells after treatment with 1 and 2 for
72 h. (C) Plot showing ELISA apoptosis assay in LNCaP cells (control,
10 and 20 µM 1 and 2, respectively) for 72 h.
ligands 1, 3, 5, and 7 in DMSO solvent exhibit intense bands in
the range 300-350 nm, which are assigned n f π* transitions
Table 1 in Supporting Information).20 All copper compounds
(
exhibit a band around 600-700 nm suggestive of square-pyramidal
Table 1. IC50 Values of Compounds 1-8 by MTT Assay in Prostate
geometry around the central metal ion with dx2-y2 as the ground
Cancer Cell Lines PC-3 and LNCaP
state.21 The absorption at 450 nm observed for the compound 2 is
22
ascribed to the S f Cu(II) charge-transfer band, while the
absorption in the region 400-430 nm is due to the oxygen to copper
charge-transfer transition.23
IC50 (µM)
compd
PC-3
LNCaP
1
2
3
4
5
6
7
8
16
5
20
NE
NE
20
20
30
21
7
25
>50
NE
>50
31
35
The observed magnetic moments for the copper complexes are
in the range 1.6-1.7 µ
copper complexes. The slightly lowered magnetic moment (∼1.65
) observed in the case of metal complex 2 is probably due to the
B
, which is characteristic of monomeric
a
a
a
µ
B
covalent nature of the metal-sulfur bond (Table 1 in Supporting
Information).24 The low-temperature EPR spectra of the copper
complexes were recorded in DMSO glass at 77 K. The spectrum
of 2 display four-line copper hyperfine lines and yield a relation g
|
a
NE: not effective.
>
⊥
g > 2.0 characteristic of monomeric copper complex (Table 1
in Supporting Information). A similar feature has been observed
in the spectrum of other copper complexes. Kivelson and Nieman
peak around -1.00 V, which is due to reduction of the azomethine
(CdN) function.30 An additional quasi-reversible peak centered
between -0.55 to -0.75 V is assigned to reduction of aroylhy-
drazone moiety.31 The corresponding copper complexes (2, 4, 6,
and 8) exhibit a reversible Cu(II)/Cu(I) redox couple centered in
the range +0.33 to +0.37 V (Figure 1 in Supporting Information),
indicating a facile reduction. The ipa/ipc ratios for all copper
complexes fall in the range 0.8-0.94, indicating that the copper
redox couples are 75-95% reversible.
have pointed out that compounds having g
while those with g
|
g 2.3 are ionic in nature
|
< 2.3 are covalent in character. The g values
|
for the present series of complexes are higher than g ) 2.0,
revealing the square-pyramidal geometry.25
The infrared spectrum of the parent compound (I) quinoline-2-
-
l
carboxaldehyde has a band at 1709 cm (Table 2 in Supporting
Information) due to the carbonyl group. On the other hand, the
-
l
band at 1583 cm is due to the pyridine nitrogen atom. On
condensation with the thiosemicarbazide side chain, the carbonyl
Biological Results and Discussion
-
l 26
absorption is replaced by a new imine band at 1602 cm , with
Compounds 1-8 shown in the Scheme 1 were tested for their
cell growth inhibitory effect in PC-3 and LNCaP prostate cancer
cell lines. The IC50 values by MTT assay in PC-3 and LNCaP
cells are given in Table 1. Interestingly, the most effective
derivative was found to be 2, the copper complex of the Schiff
base quinoline thiosemicarbazone, which showed high cell kill
in LNCaP and PC-3 cell at IC50 of 5 and 7 µM after 72 h of
treatment. The chemical structures of 1 and 2 are shown in
Figure 1A. A dose-dependent cell growth inhibition of LNCaP
cells after treatment with 1 and its copper complex 2 at 72 h
was observed (Figure 1B).
The isonicotyl hydrazone derivative of quinoline, 5, was
ineffective in all the cell lines tested, whereas the copper
complex 6 showed slight cell growth inhibition at 20 µM in
PC-3 cells. Among the three hydrazone derivatives, none
possessed very good antiproliferative activity in the cancer cell
lines tested except compound 3, which showed slight cytotoxic
activity in LNCaP and PC-3 cells (IC50 of 20 and 25 µM,
respectively). The effects of quinoline derivative 1 and its copper
-
l
two additional bands around 3273 and 3399 cm due to the
2
7
asymmetric and symmetric stretches of the terminal amino group.
The thiocarbonyl absorption in 1 is located at 1116 cm- for this
l
compound, while in case of the hydrazone derivatives (3, 5, and
7
), the stretching NH vibration is observed between 3200 and 3400
-
l
cm . The amide carbonyl frequencies for the compounds can be
-l 28
seen around 1600-1665 m . During metal complexation the Schiff
base ligands (1, 3, 5, and 7) behave as a neutral, tridentate moiety
forming two five-member chelate rings around the central metal
ion through donor atoms. The imino frequency (CdN) and
thiocarbonyl sulfur (CdS) for ligand 1 observed at 1602 and 1116
-
l
cm were found to be shifted to lower wavenumbers upon
-
l
complexation (1522 and 1091 cm ) indicating their involvement
in metal coordination. Similarly, such an observation has been
observed in the case of the ligands (3, 5, and 7), as shown in Table
2
in Supporting Information. The absence of a band around 2600-
-
l
2500 cm in 1 indicates the presence of a thione form for this
29
compound.
Cyclic Voltammetry. Under the experimental conditions, the
Schiff base ligands (1, 3, 5, and 7) exhibit an irreversible reduction

7
244 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Brief Articles
Figure 3. Inhibition of proteosome activity and apoptosis inhibition
in prostate cancer LNCaP cells treated with DMSO for control, CuCl
2
alone, 1 (10 µM), solution mixtures of 1 and CuCl (10 µM), and 2 (5
2
and 10 µM). (A) Western blot analysis for accumulation of ubiquitinated
proteins as an indicator for proteosome inhibition. Treatment of LNCaP
2
cells with (1) a solution mixture of 1 with CuCl and (2) copper complex
2
alone results in ubiquitinated protein accumulation, suggesting
proteosome inhibition. (B) Western blot analysis for cleavage of PARP
as an indication of apoptosis. Treatment with 1 alone, solution mixture
of 1 and CuCl , and copper complex 2 results in cleavage of PARP,
2
indicating that these complexes are capable of inducing apoptosis.
Figure 2. Inorganic copper complex mediated proteasome inhibition
in vitro: (A) 20S purified proteasome; (B) LNCaP whole-cell extract
after treatment with CuCl alone, 1, solution mixture of 1 with CuCl ,
2 2
and 2.
complexed with metals such as copper, it leads to stable
proteasome inhibitory molecules, as indicated by the cyclic
2+
voltammetric studies and the scan rate dependence of the Cu /
1
+
Cu couple rather than physical mixtures of known quinones
1
6
complex 2 were evaluated by ELISA for the detection of
apoptotic cell death in LNCaP cells after 72 h of treatment.
The IC50 concentrations of quinoline were correlated with
the inhibition of cell growth because measured derivatives
showed increased apoptosis (Figure 1C) by the MTT assay in
all the cell lines tested. Our observations suggest that apoptosis
ELISA assay could be useful for drug development and
chemosensitivity assessment because it can distinguish clinically
useful anticancer compounds from toxic compounds with a very
potent IC50 (as in the case of 3), and thus, it is a sensitive assay
for detecting apoptosis induced by novel antitumor compounds.
From these observations we concluded that the thiol side chain
substitution at the C2 position of the quinoline moiety upon
copper complexation leads to generation of a highly potent
antiproliferative compound. Previously, it had been reported that
the thioester adduct, lactathione, leads to generation of an active
form within Jurkat cells, which leads to proteasome inactiva-
tion.32 It has been shown by Kikuchi and others that inhibition
of proteasomal chymotrypsin-like activity is associated with
induction of apoptosis in tumor cells.33
and copper. Ligand 1 shows an irreversible peak at -1.00 V
(Figure 1A in Supporting Information) and a peak due to the
aroylhydrazone side chain around -0.55 to -0.75 V, whereas
its copper complex 2 has a reversible redox peak centered at
+0.33 to +0.37 V (Figure 1B in Supporting Information),
2+
1+
indicating scan rate dependence of the Cu /Cu couple (Figure
1C in Supporting Information).
Owing to the inhibitory effect on PC-3 and LNCaP cell
proliferation, copper complex 2 and its ligand 1 were selected
for further investigation to test the in vitro effects of these
compounds along with the inorganic copper metal alone in
LNCaP cells. When LNCaP cells were treated with DMSO
(vehicle control), copper salt alone, no accumulation of ubiq-
uitinated proteins was observed. However, when the solution
mixture of ligand 1 and copper (1:1) was tested on LNCaP cells,
after 24 h, a moderate level of ubiquitinated proteins was seen
compared to the ligand 1 or copper salt alone. A significant
accumulation of ubiquitinated proteins (Figure 3A) was observed
with the Schiff base copper complex 2 at 10 µM compared to
a lower concentration (5 µM) of 2, DMSO control, and the
mixture of ligand 1 and copper (1:1). The accumulation of
ubiquitinated protein was comparable between the Schiff base
copper complex 2 and the physical mixture of 1 and CuCl2,
indicating that quinolines upon metal complexation yield potent
molecules capable of inhibiting proteosome activity. Moreover,
we observed that 2 shows the apoptosis-specific PARP cleavage,
which was apparent within 24 h in LNCaP cells (Figure 3B).
These results suggest that Schiff base copper compounds 2 can
inhibit the proteasome and induce apoptosis in tumor cells.
Copper complexes have been reported to mediate cell death
via oxidative stress through generation of hydrogen peroxide.3
To determine whether the Schiff base quinoline derivative 1
and its copper complex 2 generated H2O2 in LNCaP cells during
induction of apoptosis or acted via a different mechanism, we
carried out an H2O2 assay using the Amplex Red hydrogen
peroxide kit (Molecular Probes). The LNCaP cells were treated
with three concentrations of 1 and 2 (4, 7, and 10 µM) for 24
h followed by measurement of H2O2 at various time points from
1 to 6 h. The absorbance values were normalized to control
To investigate the effects of these novel quinoline derivatives
1, 3, 5, and 7 and their copper complexes, we also carried out
in vitro assays using 20S purified proteasome and intact LNCaP
cells to assess the effects of the Schiff base copper complexes
with CuCl2 as control. Our results using 20S proteasome
confirmed previous findings by Dou et al.15 where CuCl2 salt
alone inhibited proteasome activity at an IC50 of 3.5 µM
compared to the copper complex 2 and its ligand 1, which
showed an IC50 higher than 10 µM in inhibiting 20S proteasome
(Figure 2A). However, when we tested the effects of the copper
4,35
complex 2 and its ligand 1 with copper salt alone, our results
in intact LNCaP cells revealed that the copper complex 2 and
mixture of 1 and copper (IC50 of 3.2 and 4 µM, respectively)
are more potent in inhibiting proteasome-chymotrypsin-like
activity compared to 1 and CuCl2 alone (IC50 g 10 µM), as
shown in Figure 2B.
These observations show that the presence of a cytotoxic
pharmacophore at position C2 in the parent quinoline creates a
far more potent anticancer agent. When these ligands are further

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7245
and plotted as absorbance at 560 nm versus concentration in
µM. The Schiff base quinoline derivative and its copper complex
did not generate high levels of H2O2 (Figure 2A in Supporting
Information). The standard H2O2 curve was plotted as shown
in Figure 2B in Supporting Information. At 10 µM, copper
derivative 2 shows an almost 2-fold lowered absorbance than
(8) Adams, J.; Behnke, M.; Chen, S.; Cruikshank, A. A.; Dick, L. R.;
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10) Elofsson, M.; Splittgerber, U.; Myung, J.; Mohan, R.; Crews, C. M.
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10 µM H2O2 as indicated by the plot in Figure 2A in Supporting
Information. Thus, induction of apoptosis by these compounds
in LNCaP cells is not through oxidative stress but through
inhibition of the proteasome-ubiquitin pathway, resulting in
the induction of apoptosis in LNCaP prostate cancer cells as
shown by our results.
822.
11) Lynas, J. F.; Harriott, P.; Healy, A.; Mckarvey, M. A.; Walker, B.
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Conclusions
Nitrogen heterocyclic compounds have been used widely in
the pharmaceutical industry, medicine, and agriculture for their
biological activity because of their antimicrobial, antipyretic,
anti-inflammatory, and anticancer properties. We have described
the synthesis and structural characterization of novel copper
quinoline-2-carboxaldehyde complexes. These synthesized Schiff
base compounds are different with respect to their various
functional groups attached to the quinoline ligand. The cytotoxic
activity is also affected by the nature of the side chains at
position C2, and our in vitro findings show that these compounds
have high antiproliferative activity against prostate cancer cell
lines PC-3 and LNCaP. Furthermore, these compounds are
capable of inducing apoptosis in prostate cancer cells without
oxidative stress, as indicated by the H2O2 assay. The highest
cytotoxic activity was observed for the copper complex 2 that
inhibited chymotrypsin-like proteasome activity in intact prostate
LNCaP cancer cells. Our study strongly suggests that the
strategies adopted in modifying the parent ligand with introduc-
tion of cytotoxic thiocarbonyl side chains enhance the antitumor
property with subsequent lowering of IC50 values. Furthermore,
the Schiff base copper complex 2 can inhibit the proteasome
and induce apoptosis as shown by PARP cleavage in prostate
tumor LNCaP cells. In conclusion, the present work indicates
that introduction of a thiocarbonyl group at the C2 position in
the quinoline moiety upon copper complexation leads to
generation of a potent anticancer agent that can be used for
targeting the ubiquitin-proteasome pathway for the treatment
of prostate cancer.
2
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Supporting Information Available: Experimental details for
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analysis results for all new compounds. This material is available
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Brief Articles
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34) Chen, S. H.; Liu, S. H.; Liang, Y. C.; Lin, J. K.; Lin-Shiau, S. Y.
Death signaling pathway induced by pyrrolidine dithiocarbamate-
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