Structure-activity study to develop cationic lipid-conjugated haloperidol derivatives as a new class of anticancer therapeutics

Article abstract of DOI:10.1021/jm101530j

Haloperidol (HP), a neuroleptic drug, shows high affinity toward-receptors (SR). HP and reduced-HP at higher concentration were known to induce apoptosis in SR-overexpressing carcinomas and melanomas. Herein, we report the development of cationic lipid-conjugated haloperidol as a new class of anticancer therapeutics. In comparison to HP, the C-8 carbon chain analogue (HP-C8) showed significantly high, SR-assisted antiproliferative activity against cancer cells via caspase-3-mediated apoptosis and down-regulation of pAkt. Moreover, melanoma tumor aggressiveness in HP-C8-treated mice was significantly lower than that in HP-treated mice. HP-C8 simultaneously reduced Akt-protein level and increased Bax/Bcl-2 ratio in vascular endothelial cells, thereby indicating a possible protein kinase down-regulatory and apoptosis inducing role in tumor-associated vascular cells. In conclusion, we developed-receptor-targeting cationic lipid-modified HP derivatives as a promising class of anticancer therapeutic that concurrently affects cancer and tumor environment associated angiogenic vascular cells through induction of apoptosis and Akt protein down-regulation.

Full text of DOI:10.1021/jm101530j

ARTICLE
pubs.acs.org/jmc
Structure-Activity Study To Develop Cationic Lipid-Conjugated
Haloperidol Derivatives as a New Class of Anticancer Therapeutics
||
Krishnendu Pal,†, Subrata Kumar Pore,† Sutapa Sinha,‡ Rajiv Janardhanan,§ Debabrata Mukhopadhyay,‡ and
Rajkumar Banerjee*,†
†Division of Lipid Science and Technology, Indian Institute of Chemical Technology, Hyderabad, Andhra Pradesh 500007, India
‡Department of Biochemistry and Molecular Biology and §Department of Radiology, Mayo Clinic Foundation, Rochester,
Minnesota 55902, United States
S Supporting Information
b
ABSTRACT: Haloperidol (HP), a neuroleptic drug, shows high affinity toward σ
receptors (SR). HP and reduced-HP at higher concentration were known to
induce apoptosis in SR-overexpressing carcinomas and melanomas. Herein, we
report the development of cationic lipid-conjugated haloperidol as a new class of
anticancer therapeutics. In comparison to HP, the C-8 carbon chain analogue
(HP-C8) showed significantly high, SR-assisted antiproliferative activity against
cancer cells via caspase-3-mediated apoptosis and down-regulation of pAkt.
Moreover, melanoma tumor aggressiveness in HP-C8-treated mice was signifi-
cantly lower than that in HP-treated mice. HP-C8 simultaneously reduced Akt-
protein level and increased Bax/Bcl-2 ratio in vascular endothelial cells, thereby
indicating a possible protein kinase down-regulatory and apoptosis inducing role
in tumor-associated vascular cells. In conclusion, we developed σ receptor-
targeting cationic lipid-modified HP derivatives as a promising class of anticancer
therapeutic that concurrently affects cancer and tumor environment associated
angiogenic vascular cells through induction of apoptosis and Akt protein down-regulation.
’ INTRODUCTION
potential has never been realized through proper chemical
modifications.
σ receptors, a unique family of membrane-bound orphan
receptor proteins, exist mainly in the central nervous system
(CNS) but are expressed in various peripheral tissues at basal
levels as well.1,2 Moreover, tumor cell lines from diverse origins
including neuroblastoma, glioma, melanoma, and carcinomas of
breast, prostate, and lung overexpress σ receptors.3-9 Interestingly,
σ receptors are more highly expressed in rapidly proliferating
cells and are down-regulated when cells become quiescent.10,11
Their high density in various tumor cell types and particularly in
rapidly proliferating cells makes σ receptors a potential target for
diagnostic imaging and therapeutic agents.8,9
Several studies suggest that σ receptor agonists induce cell
death in various tumor cell lines including prostate and breast
carcinoma and melanoma, with features consistent with
apoptosis.12,13 Haloperidol (Scheme 1) is one of the σ receptor
agonists that has been shown to inhibit cell proliferation and
induce apoptosis in neuroblastomas, melanomas, and mammary
and colon carcinomas in moderate concentrations.12-15 Some
recent studies attributed this cytotoxicity to haloperidol-induced
Akt-sensitive mitochondrial translocation of proapoptotic Bcl-
XS14,15 and disruption of PI3K/PDK1/Akt pathway through
differential subcellular compartmentalization of PI3K and
PDK1.16 However, in spite of the fact that haloperidol shows
significant antiproliferative activity toward cancer cells, its full
Recently we have shown that the targeting ability of haloper-
idol toward σ receptor remains potentially unchanged when the
tertiary OH group of haloperidol is chemically conjugated to
phospholipids with a PEG spacer between.17 This observation
prompted us to investigate whether the apoptosis inducing
ability of haloperidol can be enhanced without hampering the
targeting ability. To this end, we synthesized various haloperidol
derivatives where the tertiary OH group of haloperidol was
conjugated to cationic lipids of varying chain lengths. Twin
carbon chain containing cationic lipids with its natural affinity
toward predominantly negatively charged cell membrane bind
and interact to transfer genes into cells and hence constitute a
distinct class of gene delivery agents.18 We hypothesized that
upon conjugation of twin carbon chain cationic lipid to haloper-
idol, the resultant molecule will maintain its affinity for σ receptor
and thus will not compromise its cellular entry in σ receptor
overexpressing cancer cells. Moreover, the cellular entry may be
further assisted by twin chain cationic lipids conjugated with it.
This way the molecules’ local concentration and availability
inside the cell will be enhanced in comparison to unconjugated
Received: November 30, 2010
Published: March 10, 2011
r
2011 American Chemical Society
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Scheme 1. Structures of Haloperidol (HP), HP-C8, and C8 Control Molecule
haloperidol and might help to induce rapid apoptosis at a given
time of exposure.
HP-C8 Exerts Maximum Toxicity toward Cancer Cells. We
first chose two σ receptor expressing cancer cells, i.e., MCF-7 and
MDA-MB-231, to screen the molecule with maximum toxicity to
cancer cells. From the screening experiments it was clearly
observed that among the four synthesized haloperidol derivatives
HP-C8 has maximum toxicity toward human breast cancer cells,
MCF-7 and MDA-MB-231 cells (Figure 1). In order of toxicity,
HP-C12 is more toxic than HP-C16, but both of them are
significantly less toxic than HP-C8 (p < 0.001) at therapeutic
concentration range. The toxicity of HP-C4 is comparable to that
of haloperidol. The experiment prompted us to select the eight-
carbon chain derivative, HP-C8, for further characterization
experiments.
Our investigation reported herein involves a detailed study of
this new class of cationic haloperidol derivatives. At first, the most
potent derivative was screened by testing against two σ receptor
overexpressing breast cancer cell lines. Then the cytotoxicity of
the selected derivative in four different cancer cell lines and two
normal cell lines was evaluated and compared against that of
unconjugated haloperidol. σ receptor mediated toxicity was
confirmed by siRNA based down-regulation studies. FACS and
further Western blot analyses showed induction of apoptosis via
caspase 3 in cancer cells. Tumor model study and mechanistic
study in vascular endothelial cells highlighted the molecules'
multifunctional role in growth retardation of aggressive tumor.
HP-C8 Is More Toxic toward Cancer Cells Compared to
Normal Cells. σ receptors are basally expressed in almost all
kinds of cells. In this experiment we included a greater number of
cancer cells compared to that in the screening experiment and
also included two cells with noncancer lineage irrespective of
their σ receptor expression status. This experiment was executed
to show HP-C8’s general efficacy as an anticancer molecule. HP-
C8 shows significantly more toxicity (p < 0.001) toward all four
cancer cells tested (MCF-7, MDA-MB-231, ZR-75-1, and
B16F10) compared to either haloperidol or C8 control
(Figure 2). IC50 values of HP-C8 for these cancer cells range
from 2.5 to 3 μM. In normal cells (COS-1 and HEK-293),
however, HP-C8 exhibited more or less similar toxicity compared
to haloperidol or C8 control at concentrations less than 10 μΜ.
IC50 values of HP-C8 for these normal cells vary from 7 to 7.5
μM (data not shown). Therefore, HP-C8 is 2-3 times more
toxic toward cancer cells compared to normal cells. It is inter-
esting to note that toxicities in cancer cells exhibited by individual
treatment of haloperidol or C8-control molecule were signifi-
cantly enhanced when HP-C8, the conjugate of HP and C8
control molecule, was administered. We also treated selected
cancer cells with an equivalent mixture of HP and C8 control
molecule (Figure S1 of Supporting Information). We found that
the mixture had moretoxicity than individual treatmentof eitherof
the components, i.e., HP or C8 control molecule. However, the
toxicity due to the conjugate molecule, i.e., HP-C8, was signifi-
cantly higher than that induced by the mixture of HP and C8. This
clearly indicates that the efficient delivery of HP with the help of
cell surface active cationic lipid certainly induces more HP-
mediated toxicity but is not as efficient as the conjugated molecule.
Down-Regulation of σ Receptor Decreases Toxicity of HP-
C8. For this study two cells, MCF-7 (σ receptor overexpressing
’ RESULTS
Synthesis of Haloperidol Derivatives. Since the haloperidol
derivatives differed only in chain lengths, a general procedure was
used to synthesize them (Scheme 2a). At first the amine group of
glycine tert-butyl ester was reacted with different haloalkanes in
the presence of K2CO3 to obtain the respective tertiary amines
(1a-d). This was followed by quaternization with methyl iodide
to give the respective cationic lipid chains with iodide counter-
ions (2a-1d). Then the ester protection of glycine moiety was
removed using strong acid, TFA, to result in cationic lipids
containing a free carboxylic acid group with trifluoroacetate
counterion (3a-d). In a different reaction haloperidol was
conjugated to N-Boc-β-alanine via DCC coupling to form
haloperidol alanine ester (4). Then Boc-protection on the amine
was removed to generate a free amine conjugated to haloperidol
with a three-atom spacer (5). Finally, coupling of this amine with
the carboxylic acid moiety of previously synthesized cationic
lipids was achieved using HATU as a coupling reagent. This was
followed by passing the resultant compound through chloride
ion exchange resin, finally resulting in cationic haloperidol
derivatives with different chain lengths (HP-Cn, where n = 4,
8, 12, and 16).
For the synthesis of C8 control (Scheme 2b), at first diocty-
lamine (7) was synthesized by a nucleophilic reaction between
octan-1-amine and 1-bromooctane. Then dioctylamine was
reacted with 1-bromopropane to obtain a tertiary amine (8)
which on quaternization with methyl iodide and successive
chloride ion exchange with Amberlite IRA-400Cl resin yielded
C8 control molecule 9.
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Scheme 2. Synthesis of Haloperidol Derivatives and C8 Controla
a Reagent and conditions for part a: (i) R-X, K2CO3, ethyl acetate, 70-80 °C; (ii) methyl iodide, dry DCM; (iii) 50% trifluoroacetic acid in dry DCM;
(iv) N-Boc-β-alanine, dicyclohexylcarbodiimide (DCC), N,N-dimethylaminopyridine (DMAP), dry DCM; (v) 10% trifluoroacetic acid in dry DCM,
0 °C; (vi) [dimethylamino(triazolo[4,5-b]pyridine-3-yloxy)methylidene]dimethylazanium hexafluorophosphate (HATU), diisopropylethylamine
(DIPEA), dry DMF; (vii) Amberlite IRA-400Cl resin, methanol. Reagents and conditions for part b: (i) K2CO3, DMSO, 70-80 °C; (ii)
1-bromopropane, K2CO3, ethyl acetate, 70-80 °C; (iii) methyl iodide, dry DCM; (iv) Amberlite IRA-400Cl resin, methanol.
cancer cell) and COS-1 (σ receptor basally expressing noncancer
cell), were chosen. These cells were chosen to pinpoint any σ
receptor-mediated anticancer effects of HP-C8. Hence, if HP-
C8-mediated toxicity is σ-receptor-assisted, then the molecule
should have the least toxicity in noncancer cells, which normally
express the basal level of the σ receptor. They were treated with
either siRNA against σ receptor subtype 1 or universal, control
siRNA, following which the σ receptor levels in treated cells were
respectively compared with the receptor levels of untreated cells
(Figure 3a,b). Down-regulation of σ receptor in MCF-7 cells
with σ receptor siRNA increases cell viability to a significant
extent (p < 0.001) compared to untreated cells or control siRNA
treated cells (Figure 3c). But the recovery in viability in HP-C8-
treated σ-receptor-depleted cells was only moderate (25-30%)
compared to σ-receptor-nondepleted cells. However, in antag-
onism based studies using ditolylguanidine (DTG), a nonspecific
σ receptor antagonist, no such increase was observed (data not
shown). This we presumed was due to greater affinity of HP-C8
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Figure 1. Viability studies of MCF-7 (a) and MDA-MB-231 (b) cells continuously treated with a range of concentrations of haloperidol (O), HP-C4
(9), HP-C8 (2), HP-C12 (0), and HP-C16 (b), respectively, for 48 h. The asterisks (///) denote p < 0.001.
toward σ receptor compared to DTG, leading to irreversible
binding of HP-C8 to σ receptors. On the other hand, by use of
siRNA against σ receptor, the expression level of σ receptors
decreases causing lesser amount of σ receptor-HP-C8 binding
that ultimately leads to increase in cell viability. As expected, with
no visible change in σ receptor level using siRNA in COS-1 cells
there was no change in cellular viability, contrary to what is
witnessed in cancer cells (Figure 3b,d). Here, σ receptor is
expressed at a basal level and further down-regulation does not
make a noticeable difference. In conclusion, the HP-C8-
mediated toxicity in cancer cells is, at least partially, σ-receptor-
dependent.
that HP-C8 treatment truly induced apoptosis specifically in
cancer cells and did not induce apoptosis in normal cells.
Selective Tumor Regression by HP-C8 Compared to Halo-
peridol. To evaluate the tumor regression efficacy of HP-C8
compared to that of haloperidol in vivo, we made a subcutaneous
model of B16F10 tumor in male C57BL6/J mice, divided them
into two groups, and intraperitoneally injected either HP-C8 or
haloperidol (at a dosage of 7.5 mg/kg) suspended in PBS
containing 10% DMSO. The reason behind selection of this
model is that B16F10 cells form rapidly growing tumors and had
been shown to express high levels of σ receptor.20 Figure 5a
shows that a lesser amount of tumor growth is maintained in the
HP-C8 treated group compared to the haloperidol treated group
throughout the experiment. After five injections, the group
treated with HP-C8 showed a significant regression in tumor
growth (P < 0.05) compared to haloperidol treated group.
Figure 5b shows the representative tumors from each group.
After the completion of the experiment tumors from individual
groups were cryosectioned and were imaged for relative density
of apoptotic signs using TUNEL assay. Figure 5c shows that the
tumor section obtained from HP-C8 treated group had more
apoptotic regions compared to the tumor section obtained from
the haloperidol treated group. Therefore, HP-C8 induced apop-
tosis not only in cancer cells in vitro but also in rapidly growing
tumor tissues to result in significant inhibition of tumor growth.
HP-C8 triggers Intrinsic Pathway of Apoptosis and Phos-
pho-Akt Down-Regulation in B16F10 Cells. The tumor model
was made with B16F10, so we chose to see the mechanism of HP-
C8 action in B16F10 cells. HP-C8 was treated with B16F10 in
charcoal stripped serum condition to elucidate the mode of
action of HP-C8 toward B16F10. In the intrinsic pathway of
apoptosis the release of cytochrome c leads to activation of
caspase 9 which finally cleaves procaspase 3 to release effector
protein, activated caspase 3. The release of caspase 3 leads to
apoptosis of cells. However, the release of cytochrome c is
regulated by the Bcl-2 family of proteins. Members of this family
such as Bax causes release of cytochrome c, thus acting as
proapoptotic. But other antiapoptotic members of this family,
such as Bcl-2, bind to pro-apoptotic Bax protein and inactivate
them to release cytochrome c. So a higher value of Bax/Bcl-2
ratio indicates the onset of apoptosis in cells. As can be seen from
Figure 6a, HP-C8 decreased the level of Bcl-2 expression and
increased the expression of Bax in B16F10 cells in comparison to
haloperidol (HP). The overall ratio of Bax/Bcl-2 obtained from
HP-C8 Induces Apoptosis in Cancer Cells but Not in
Normal Cells. The apoptosis inducing effects of the HP-C8
were evaluated by annexin V/propidium iodide (PI) binding.
One of the earliest events of apoptosis is the loss of plasma
membrane integrity, accompanied by translocation of phospha-
tidylserine (PS) from the inner to outer membrane leaflets,
thereby exposing PS to the external environment. The phospho-
lipid binding protein annexin V has high affinity for PS and can
bind to cells with externally exposed PS. Positive staining with
fluorescently labeled annexin V correlates with loss of membrane
polarity but precedes the complete loss of membrane integrity
that accompanies the later stage of cell death resulting from either
apoptosis or necrosis. In contrast, PI can only enter cells after loss
of membrane integrity. Dual staining with annexin V and PI
allows clear discrimination between unaffected cells (annexin V
negative, PI negative, lower left quadrant), early apoptotic cells
(annexin V positive, PI negative, lower right quadrant), late
apoptotic cells (annexin V positive, PI positive, upper right
quadrant), and necrotic cells (annexin V negative, PI positive,
upper left quadrant). A higher amount of dual stained cells in the
upper right quadrant indicates that the cells upon their initiation
in apoptotic phase are in late apoptotic stage and there are
significant losses of plasma membrane integrity that will result in
their eventual death. In this experiment along with MCF-7 we
chose to include B16F10 melanoma cells with which we made
melanoma tumor model later. As can be seen in Figure 4, HP-C8
treatment (5 μM, 24 h) increases the percentage of late apoptotic
cells (with both annexin V and PI positive staining, upper right
quadrant) in MCF-7 and B16F10 cells whereas haloperidol (5
μM, 24 h) has no such effect. In normal cells like COS-1 and
HEK-293 there are no differences between untreated cells and
cells treated with haloperidol or HP-C8. Therefore, it is evident
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Figure 2. Viability studies of MCF-7 (a), MDA-MB-231 (b), ZR-75-1 (c), B16F10 (d), COS-1 (e), and HEK-293 (f) cells continuously treated
with a range of concentrations of haloperidol (white bar), C8 control (gray bar), and HP-C8 (black bar), respectively, for 48 h. The asterisks (///)
denote p < 0.001.
HP-C8-treated cells was almost doubled compared with that
obtained from HP-treated cells. An elevated expression of
cleaved (i.e., activated) caspase 3 was also observed in HP-C8
treated cells, indicating possible cytoplasmic release of cytotoxic
cytochrome c from mitochondria or other proapoptotic factors.
These eventually might have triggered intrinsic pathway of
apoptosis which corroborates with a high level of toxicity to
B16F10 cells as evidenced in viability studies (Figures 1 and 2)
and flow cytometry data (Figure 4). Further, we found that HP-
C8 could trigger the down-regulation of phospho-Akt produc-
tion from Akt. Since a higher ratio of pAkt/Akt is linked with
proproliferative pathways, this kinase down-regulatory property
of HP-C8 might contribute to its anticancer action.
phenomenon to assist the excessive growth and aggressiveness in
B16F10 tumor. Since human umbilical vein endothelial cell
(HUVEC) is a known model of angiogenesis/neovasculature, we
chose to test the effect of HP-C8 on this cell. Figure 6b shows that
HP-C8 clearly has detrimental effect on HUVEC cells. Clearly in
contrast to HP, HP-C8 triggered the formation of cleaved caspase 3
followed by significant reduction of absolute amount of cellular Akt.
As a result no phospho-Akt was formed in cells treated with HP-C8.
The HP-C8-treatment apparently shut down the proproliferative
Akt/pAkt pathway in the HUVEC cell. The up-regulation of caspase
3 in HP-C8-treated cell was further vindicated by the increase in
Bax/Bcl-2 ratio. In conclusion, HP-C8 may have an inhibitory role
in protein kinase B/Akt formation together with a role in triggering
intrinsic pathway of apoptosis in vascular cells. This collective effect
could have provided additional cause of tumor growth retardation
owing to the killing of tumor-associated vascular cells.
Mechanism of Action of HP-C8 in HUVEC Cells. Since
excessive tumor growth is often accompanied by uninterrupted
microangiogenesis and neovasculature formation, we suspected this
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Figure 3. Flow cytometry analysis of σ receptor down-regulation in MCF-7 (a) and COS-1 (b) cells. Blue, green, and red lines represent untreated, σ
receptor siRNA treated, and negative control siRNA treated cells, respectively. Parts c and d represent the viability data of MCF-7 and COS-1 cells
obtained after 48 h following the treatment of 10 μM HP-C8 for 4 h. White, gray, and black bars represent SiRNA untreated, σ receptor siRNA treated,
and negative control siRNA treated cells, respectively. The asterisk (/) denotes p < 0.001.
’ DISCUSSION
the well-known cell-membrane-interacting cationic lipid compo-
nent to accomplish this study.
Development of newer class of drug candidates with multi-
functional role for complicated diseases such as cancer is a
challenging task. There are many existing drug molecules avail-
able with proven and time tested pharmacologic roles. These
molecules are viable candidates for the development of newer
drug molecules. Haloperidol is a well-known antipsychotic drug,
but its usage following proper chemical modification for the
treatment of cancer remained largely unutilized. Modification of
haloperidol should be properly dealt with, since haloperidol is
neurotoxic in nature through the possible down-regulation of
antiapoptotic Bcl-2 or up-regulation of proapoptotic Bcl-XS.21,22
Reduced haloperidol, which triggers cellular calcium release, was
used for anticancer purposes, but this strategy was exploited
limitedly.23 We showed previously the first usage of neuropsy-
chotic drug haloperidol (HP) for selectively targeting σ receptor
overexpressing cancer cells if proper chemical modification could
be accomplished.17 Hypothesizing that better cellular interac-
tion of haloperidol will increase its acceptability as an anticancer
drug for σ receptor expressing cells, we chemically introduced
We find that HP-C8, the eight-numbered-carbon-chain-linked
haloperidol, has the maximum anticancer effect with minimal
toxic effect to noncancer cells (Figures 1 and 2). A 2- to 3-fold
difference in IC50 of HP-C8 in cancer cells with respect to
currently selected normal cells is indeed concerning owing to
narrow therapeutic window. Whether HP-C8 exhibits any spe-
cific antiproliferative effect against these chosen immortalized
noncancer cells is a subject of further scrutiny in future. Further
experiments with freshly isolated primary noncancer cells for
determining actual therapeutic window are currently underway.
However, this study clearly indicates predominant anticancer
efficacy of HP-C8 which is substantiated in later experiments
including in vivo experiments wherein apparently no HP-C8-
induced toxicity is observed in mice during the course of
treatment.
Further we find that this anticancer effect was at least partly
mediated by σ receptor (Figure 3). The prominently higher
percentage of postapoptotic cancer cells than normal cells treated
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Figure 4. Flow cytometric analysis of apoptosis in MCF-7, B16F10, COS-1, and HEK-293 cells costained with FITC-annexin V/propidium iodide (PI).
The horizontal and vertical axes represent FITC-annexin V and PI labeling, respectively. Cells were kept untreated or treated with 5 μM haloperidol or
5 μM HP-C8 for 24 h.
with HP-C8 (Figure 4) prompted us to investigate the pathway
of apoptosis. The significant reduction in tumor aggression by
HP-C8 in comparison to HP (Figure 5) indicated that beyond its
toxic effect to cancer cells, the molecule might have detrimental
effects on tumor-microenvironment-associated vascular en-
dothelial cells. In all these experiments the parent molecule,
HP, seems to have no specific anticancer role. This distinctive
difference in anticancer property is certainly interesting and is
expected to evoke strategies in future to modify pharmacologic
drugs of similar pattern.
which is known to be initiated through the release of signal
factors by mitochondria within the cell. Mitochondria integrate
and propagate intracellular death signals originating from many
factors including those induced by chemotherapeutic drugs.24
This leads cytoplasmic, proapoptotic Bax, Bak, etc. proteins to
bind to outer membrane of mitochondria and signal release of
cytochrome c, which activates initiator protein caspase 9. Acti-
vated caspase 9 cleaves procaspase 3 to produce activated caspase 3,
the effector protein that initiates cellular degradation. In contrast,
Bcl-2, the antiapoptotic protein, down-regulates the release of
cytochrome c.25 Hence, a higher Bax/Bcl-2 ratio indicates the onset
of apoptosis in cells. We found that the Bax/Bcl-2 ratio was nearly
Further Western blot based studies revealed that in B16F10
melanoma cells HP-C8 triggered the intrinsic pathway of apoptosis,
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Figure 5. (a) Tumor growth curve after subcutaneous implantation of B16F10 cells in C57BL6/J mice followed by intraperitoneal injection of
haloperidol (9) or HP-C8 (O) at a dosage of 7.5 mg/kg. The days of injections are indicated by black arrows. Y-axis denotes the size of tumors as tumor
volume in mm3, and X-axis denotes the number of days passed after tumor inoculation. // denotes p < 0.05. (b) Representative samples of B16F10
tumors excised on day 23: (I) tumor from haloperidol treated group and (II) tumor from HP-C8 treated group. (c) Microscopic pictures of 10 μm tumor
sections from haloperidol treated group (upper panel) and HP-C8 treated group (lower panel). Left panels show the tissue architecture in bright field,
and right panels show the apoptotic regions in TUNEL assay. All the images are taken at 10ꢀ magnification. Bar = 2 μm.
Figure 6. Western blot analysis. Differential expression of different regulators of apoptosis and proliferation in B16F10 (a) and HUVEC (b) cells treated
with haloperidol (HP) and HP-C8 or kept untreated (UT).
doubled, resulting in concomitant release of more amount of
activated caspase 3 in HP-C8-treated B16F10 cells than in HP-
treated cells (Figure 6a). HP showed an increase in Bax/Bcl-2
ratio compared to untreated control, but it seems that this subtle
increase could not evoke any visible anticancer effect. HP-C8 also
induced apotosis in HUVEC cells, the viable model of cancer
angiogenesis, through prominent up-regulation of Bax/Bcl-2
ratio with concomitant increase in caspase 3 (Figure 6b).
Another important regulator of apoptosis is Akt (protein
kinase B, PKB), a family of serine/threonine kinases. Other activating
kinases phosphorylate and activate this kinase. This prosurviving
factor, i.e., activated Akt (pAkt), exerts many antiapoptotic activities,
such as preventing release of cytochrome c from mitochondria,
inactivating proapoptotic Fas ligand-expressing forkhead tran-
scription factors, phosphorylating and inactivating proapoptotic
factors BAD and pro-caspase-9, etc. Additionally, pAkt also mediates
the activation of endothelial nitric oxide synthase (eNOS), an
important modulator of angiogenesis.26 In addition to the induction
of apoptosis, HP-C8 played a kinase inhibitory role through
down-regulation of the formation of pAkt in B16F10 cancer cells
(Figure 6a). In HUVEC (Figure 6b), HP-C8 treatment inhibited
the expression of Akt protein and hence probably the formation
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of pAkt. Since Akt and pAkt are responsible for the activation of
endothelial nitric oxide synthase (eNOS), an important mod-
ulator of angiogenesis, their absence leads to impaired angiogen-
esis. It remains to be seen if the inhibitory effect on eNOS
activation indeed assists HP-C8 mediated growth inhibition of
highly aggressive B16F10 melanoma tumors. Increase in acti-
vated caspase 3 in HP-C8-treated HUVEC could be partially
vindicated by the increase in Bax/Bcl-2 ratio. However, the
increase in caspase 3 with the complete inhibition of Akt
formation may have a different implication. It is shown recently
that the VEGF induction, which gives protection to Akt protein
from proteasomal degradation, if inhibited, leads to down-
regulation of Akt expression in HUVEC.27 VEGF tyrosine kinase
inhibitor or mTOR inhibitor rapamycin, even in the presence of
VEGF stimulation, leads to a decrease in Akt protein expression
in HUVEC cells, which is followed by the production of cleaved
caspase 3.27 In exact agreement with this published observation,
HP-C8 induces a decrease in Akt expression and also increased
production of cleaved caspase 3. This observation stands with a
marked difference from what is observed when the mother
molecule HP is treated to cells. However, it remains of further
research interest to find if HP-C8 possesses a similar VEGF
tyrosine kinase or mTOR inhibitory role. The data indicate the
onset of apoptosis among the tumor vasculature which ultimately
leads to inhibition of aggressiveness in tumor growth. Together,
our data indicate that HP-C8 induces apoptosis not only in cancer
cells but possibly also in adjoining vascular endothelial cells.
In conclusion, the findings of the present investigation de-
monstrate that structural modification of haloperidol by con-
jugation with a cationic lipid moiety indeed increases its
apoptosis-inducing property in cancer cells without significantly
diminishing its σ receptor targeting ability. Recently, we have
used this strategy to modify estrogen receptor ligand, leading to
the development of a highly potent, selective antibreast cancer
agent.28 Hence, this strategy of chemically modifying antagonists/
agonists of other receptors that are overexpressed in various
types of cancers could gain further importance in the develop-
ment of potential anticancer therapeutics in future.
spectrometry. The final molecules were characterized by 1H NMR and
ESI-HRMS, and the purity was ascertained by HPLC. 1H NMR spectra
were recorded on a Varian FT 200 MHz or Bruker FT 300 MHz
spectrometer. Chemical shifts (δ) are reported in ppm relative to
chloroform resonances (1H: 7.24 ppm for [D]chloroform). Data for
1H NMR are reported as follows: chemical shift (δ in ppm), multiplicity
(s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quadruplet,
m = multiplet), coupling constant (Hz), integration. ESI mass spectra
were obtained using a QStar XL Hybrid QTOF mass spectrometer
(Applied Biosystems). Purities of final products were clarified with a
Varian ProStar HPLC instrument at 210 nm at a flow rate of 1 mL/min
in a Varian Microsorb 100-10 BDS column (4.6 mm ꢀ 250 mm).
Chemistry. Synthesis of tert-Butyl 2-(Dibutylamino)acetate,
1a. Glycine tert-butyl ester (0.45 g, 3.43 mmol) was dissolved in 20 mL
of dry ethyl acetate in a 50 mL round-bottomed flask fitted with a reflux
condenser. Potassium carbonate (1.9 g, 13.77 mmol) and 1-iodobutane
(2.56 g, 13.91 mmol) were added, and the resulting mixture was refluxed
over an oil bath at 70-80 °C for 12 h. Then it was cooled and washed
with water (2 ꢀ 20 mL) and brine (1 ꢀ 20 mL), dried over anhydrous
Na2SO4, and evaporated. Column chromatographic purification of the
residue using 60-120 mesh silica gel and 10% ethyl acetate-hexane (v/v)
as eluent yielded compound 1a as a colorless liquid (0.43 g, 51.4% yield,
1
Rf = 0.6 in 30% ethyl acetate-hexane, v/v). H NMR (300 MHz,
CDCl3): δ = 0.85-0.95 [t, 6H], 1.22-1.42 [m, 8H], 1.45 [s, 9H],
2.50-2.58 [t, 4H], 3.16 [s, 2H]. ESI-MS: m/z = 244 (calcd value for
C14H29O2N = 243.2).
Synthesis of tert-Butyl 2-(Dioctylamino)acetate, 1b. tert-Butyl
2-(dioctylamino)acetate was synthesized following a similar procedure
as for tert-butyl 2-(dibutylamino)acetate (64.2% yield, Rf = 0.65 in 30%
ethyl acetate-hexane, v/v). 1H NMR (300 MHz, CDCl3): δ = 0.84-
0.92 [t, 6H], 1.20-1.34 [m, 20H], 1.36-1.42 [m, 4H,], 1.45 [s, 9H],
2.46-2.56 [t, 4H], 3.16 [s, 2H]. ESI-MS: m/z = 357 (calcd value for
C22H45O2N = 355.3).
Synthesis of tert-Butyl 2-(Didodecylamino)acetate, 1c. tert-
Butyl-2-(didodecylamino)acetate was synthesized following a similar
procedure as for tert-butyl 2-(dibutylamino)acetate (70.1% yield, Rf =
0.65 in 30% ethyl acetate-hexane, v/v). 1H NMR (200 MHz, CDCl3):
δ = 0.82-0.95 [t, 6H], 1.13-1.40 [m, 36H], 1.44 [s, 9H], 1.45-1.65
[m, 4H], 2.45-2.58 [t, 4H], 3.16 [s, 2H]. ESI-MS: m/z = 468 (calcd
value for C30H61O2N = 467.5).
Synthesis of tert-Butyl 2-(Dihexadecylamino)acetate, 1d.
tert-Butyl 2-(dihexadecylamino)acetate was synthesized following a
’ EXPERIMENTAL SECTION
similar procedure as for tert-butyl-2-(dibutylamino)acetate (76.5% yield,
1
Rf = 0.7 in 30% ethyl acetate-hexane, v/v). H NMR (300 MHz,
CDCl3): δ = 0.84-0.92 [t, 6H], 1.20-1.36 [m, 52H], 1.45 [s, 9H],
1.55-1.65 [m, 4H], 2.48-2.56 [t, 4H], 3.16 [s, 2H]. ESI-MS: m/z =
581 (calcd value for C38H77O2N = 579.6).
Chemicals and General Procedures. Haloperidol was pur-
chased from Sigma (St. Louis, MO, U.S.). 1-Bromopropane, 1-iodobu-
tane, 1-bromooctane, 1-bromododecane, 1-bromohexadecane, and
octan-1-amine were purchased from Alfa Aesar (Ward Hill, MA). HATU
and glycine tert-butyl ester were purchased from Novabiochem
(Germany). σ receptor siRNA (human) (against subtype 1, catalog
no. SC-42250) was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). This siRNA product is a mixture of three siRNAs with
following sequences: (A) sense, GGAUAUCCAUGCUUAUGUAtt;
antisense, UACAUAAGCAUGGAUAUCCtt; (B) sense, GUCCU-
CACCAUAUGCCUUAtt; antisense, UAAGGCAUAUGGUGAGGACtt;
(C) sense, GGAACGAGAGUAAUUUGAAtt; antisense, UUCAAAU-
UACUCUCGUUCCtt. The universal negative control siRNA (catalog
no. 1022076, sense sequence UUCUCCGAACGUGUCACGUTT,
antisense sequence ACGUGACACGUUCGGAGAATT) was pur-
chased from Qiagen (Valencia, CA). Antibodies were purchased from
Santa Cruz Biotechnology. All other chemicals, reagents, and organic
solvents were purchased either from Sigma (St. Louis, MO, U.S.) or
from S. D. Fine Chemicals Ltd. (Mumbai, India). The reagents were
>95% pure and were used without further purification. All the inter-
mediates in the synthesis were characterized by 1H NMR and/or mass
Synthesis of N-(2-tert-Butoxy-2-oxoethyl)-N-butyl-N-methyl-
butan-1-aminium Iodide, 2a. Compound 1a (0.43 g, 1.76 mmol)
was dissolved in 5 mL of dry DCM in a 25 mL round-bottomed flask, and
excess methyl iodide (3 mL) was added. The reaction mixture was
stirred for 4 h at room temperature. Then it was concentrated and the
residue upon column chromatographic purification using 60-120 mesh
silica gel and 2% methanol-chloroform (v/v) as eluent, yielding
compound 2a as a yellowish gummy liquid (0.61 g, 89.6% yield, Rf =
0.4 in 10% methanol-chloroform, v/v). 1H NMR (300 MHz, CDCl3):
δ = 1.00-1.08 [t, 6H], 1.40-1.51 [m, 4H], 1.53 [s, 9H], 1.75-1.90 [m,
4H], 3.60 [s, 3H], 3.73-3.90 [m, 4H], 4.56 [s, 2H]. ESI-MS: m/z = 258
(calcd value for C15H32O2N = 258.2).
Synthesis of N-(2-tert-Butoxy-2-oxoethyl)-N-methyl-N-
octyloctan-1-aminium Iodide, 2b. Compound 2b was synthesized
following a similar procedure as for compound 2a (86.7% yield, Rf = 0.45
1
in 10% methanol-chloroform, v/v). H NMR (300 MHz, CDCl3):
δ = 0.84-0.92 [t, 6H], 1.20-1.42 [m, 20H], 1.53 [s, 9H], 1.70-1.88
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ARTICLE
[m, 4H], 3.61 [s, 3H], 3.64-3.88 [m, 4H], 4.60 [s, 2H]. ESI-MS: m/z =
371 (calcd value for C23H48O2N = 370.4).
Then 2 mL of trifluoroacetic acid was added dropwise to the solution.
The reaction mixture was further stirred over an ice bath for 4 h, and then
the solution was neutralized using saturated NaHCO3 solution. The
mixture was extracted with DCM (2 ꢀ 15 mL), and the organic layer was
dried with Na2SO4. Evaporation of the organic layer afforded compound
5 as a reddish-yellow gummy material (1.04 g, 90.4% yield, Rf = 0.1 in 5%
methanol-chloroform, v/v, active in ninhydrin charring). Because
compound 5 was obtained as 95% pure (revealed by TLC), it was
directly used for the next step. ESI-MS: m/z = 447 (calcd value for
C24H28O3N2ClF = 446.2).
Synthesis of HP-C4. Compound 3a (0.13 g, 0.41 mmol) was
dissolved in 5 mL of dry DMF in a 25 mL round-bottomed flask and
stirred over an ice bath for 15 min. To this, [dimethylamino(triazolo-
[4,5-b]pyridine-3-yloxy)methylidene]dimethylazanium hexafluoropho-
sphate (HATU) (0.17 g, 0.45 mmol) was added, and stirring was
continued for another 30 min. Then compound 5 (0.27 g, 0.60 mmol)
dissolved in 2 mL of dry DMF was added followed by dropwise addition
of diisopropylethylamine (DIPEA) until the reaction mixture became
slightly basic. The resulting mixture was stirred for 48 h. Then the
reaction mixture was dissolved in 30 mL of DCM, washed with 1 N HCl
(2 ꢀ 30 mL), water (1 ꢀ 30 mL), and brine (1 ꢀ 30 mL), dried with
anhydrous Na2SO4, and evaporated. Column chromatographic purifica-
tion (using >200 mesh silica gel and 2% methanol-chloroform, v/v, as
eluent) of the residue followed by chloride ion exchange chromatogra-
phy (using Amberlite IRA-400Cl resin and methanol as eluent) yielded
HP-C4 as a colorless gummy solid (0.09 g, 32.7% yield, Rf = 0.45 in 10%
methanol-chloroform, v/v). HPLC purity, >98%. 1H NMR (300 MHz,
CDCl3): δ = 0.88-1.00 [t, 6H], 1.15-1.40 [m, 8H], 1.54-1.72 [m,
4H], 2.10-2.25 [m, 2H], 2.30-2.75 [m, 6H], 3.10 [s, 3H], 3.10-3.25
[m, 2H], 3.25-3.50 [m, 8H], 4.23 [s, 2H], 7.00-7.10 [t, 2H], 7.15-
7.30 [m, 4H], 7.85-8.00 [m, 2H]. ESI-HRMS: m/z = 630.3471 (calcd
value for C35H50O4N3ClF = 630.3473).
Synthesis of HP-C8. HP-C8 was synthesized following similar
procedure for the synthesis of HP-C4 (24.3% yield, Rf = 0.5 in 10%
methanol-chloroform, v/v). 1H NMR (300 MHz, CDCl3): δ = 0.83-
0.92 [t, 6H], 1.16-1.38 [m, 20H], 1.56-1.76 [m, 4H], 2.00-2.24 [m,
4H], 2.44-2.57 [m, 2H], 2.60-2.95 [m, 6H], 2.98-3.05 [m, 2H], 3.08
[s, 3H], 3.34-3.53 [m, 8H], 4.30 [s, 2H], 7.04-7.14 [t, 2H], 7.20-7.32
[m, 4H], 7.92-8.02 [m, 2H]. ESI-HRMS: m/z = 742.4746 (calcd value
for C43H66O4N3ClF = 742.4725). HPLC purity, >99%.
Synthesis of HP-C12. HP-C12 was synthesized following similar
procedure for the synthesis of HP-C4 (16.2% yield, Rf = 0.6 in 10%
methanol-chloroform, v/v). 1H NMR (300 MHz, CDCl3): δ = 0.82-
0.95 [t, 6H], 1.17-1.38 [m, 30H], 1.55-1.74 [m, 4H], 1.95-2.26 [m,
4H], 2.45-2.59 [m, 2H], 2.61-2.96 [m, 6H], 2.98-3.05 [m, 2H], 3.07
[s, 3H], 3.34-3.51 [m, 8H], 4.25 [s, 2H], 7.04-7.14 [t, 2H], 7.21-7.30
[m, 4H], 7.91-8.01 [m, 2H]. ESI-HRMS: m/z = 854.6000 (calcd value
for C51H82O4N3ClF = 854.5977). HPLC purity, >98%
Synthesis of N-(2-tert-Butoxy-2-oxoethyl)-N-dodecyl-N-meth-
yldodecan-1-aminium Iodide, 2c. Compound 2c was synthesized
following a similar procedure as for compound 2a (82.8% yield, Rf = 0.55
in 10% methanol-chloroform, v/v). 1H NMR (200 MHz, CDCl3): δ =
0.82-0.95 [t, 6H], 1.15-1.40 [m, 36H], 1.50 [s, 9H], 1.59-1.86 [m,
4H], 3.46-3.63 [m, 4H], 3.65 [s, 3H], 4.33 [s, 2H]. ESI-MS: m/z = 482
(calcd value for C31H64O2N = 482.5).
Synthesis of N-(2-tert-Butoxy-2-oxoethyl)-N-hexadecyl-N-
methylhexadecan-1-aminium Iodide, 2d. Compound 2d was
synthesized following a similar procedure as for compound 2a (80.5%
yield, Rf = 0.6 in 10% methanol-chloroform, v/v). 1H NMR (300 MHz,
CDCl3): δ = 0.84-0.92 [t, 6H], 1.19-1.44 [m, 52H], 1.52 [s, 9H],
1.68-1.88 [m, 4H], 3.61 [s, 3H], 3.64-3.88 [m, 4H], 4.61 [s, 2H]. ESI-
MS: m/z = 595 (calcd value for C39H80O2N = 594.6).
Synthesis of N-(Carboxymethy)-N-butyl-N-methylbutan-
1-aminium Trifluoroacetate, 3a. Compound 2a (0.61 g, 1.58
mmol) was dissolved in 2 mL of dry DCM in a 25 mL round-bottomed
flask and stirred over an ice bath for 15 min. Then 2 mL of trifluoroacetic
acid was added dropwise to the solution. The reaction mixture was
stirred over an ice bath for a further 15 min and then for 4 h at room
temperature. DCM and excess trifluoroacetic acid were chased by
nitrogen flushing. The residue was purified by flush column chroma-
tography using 60-120 mesh silica gel and 8% methanol-chloroform
(v/v) as eluent to obtain compound 3a as a colorless gummy liquid (0.33
g, 66.4% yield, Rf = 0.3 in 10% methanol-chloroform, v/v). ESI-MS:
m/z = 202 (calcd value for C11H24O2N = 202.2)
Synthesis of N-(Carboxymethyl)-N-methyl-N-octyloctan-
1-aminium Trifluoroacetate, 3b. Compound 3b was synthesized
following a similar procedure as for compound 3a (71.9% yield, Rf = 0.4
in 10% methanol-chloroform, v/v). ESI-MS: m/z = 315 (calcd value for
C19H40O2N = 314.3).
Synthesis of N-(Carboxymethyl)-N-dodecyl-N-methyldo-
decan-1-aminium Trifluoroacetate, 3c. Compound 3c was
synthesized following a similar procedure as for compound 3a (72.5%
yield, Rf = 0.5 in 10% methanol-chloroform, v/v). ESI-MS: m/z = 426
(calcd value for C27H56O2N = 426.4).
Synthesis of N-(Carboxymethyl)-N-hexadecyl-N-methyl-
hexadecan-1-aminium Trifluoroacetate, 3d. Compound 3d
was synthesized following a similar procedure as for compound 3a
(78.4% yield, Rf = 0.55 in 10% methanol-chloroform, v/v). ESI-MS:
m/z = 539 (calcd value for C35H72O2N = 538.6).
Synthesis of N-Boc-β-alanine-Haloperidol Conjugate, 4.
A mixture of N-Boc-β-alanine (0.91 g, 4.81 mmol), haloperidol (1.50 g,
3.99 mmol), and N,N-dimethylaminopyridine (DMAP) (0.24 g, 1.97
mmol) was dissolved in 10 mL of dry DCM in a 50 mL round-bottom
flask and stirred in ice for half an hour. To the mixture, DCC (0.99 g, 4.8
mmol) dissolved in 5 mL of dry DCM was added, and the sample was
stirred in an ice bath for 1 h. The reaction mixture was further stirred for
24 h at room temperature. Then the reaction mixture was filtered. The
filtrate was washed with water (2 ꢀ 30 mL) and brine (1 ꢀ 30 mL),
dried with anhydrous Na2SO4, and evaporated. Column chromato-
graphic purification of the residue using 60-120 mesh silica gel and 1%
methanol-chloroform (v/v) as eluent yielded compound 4 as a reddish-
yellow gummy material (1.41 g, 64.6% yield, Rf = 0.6 in 5% methanol-
chloroform, v/v). 1H NMR (200 MHz, CDCl3): δ = 1.43 [s, 9H], 1.75-
2.05 [m, 4H], 2.15-2.55 [m, 8H], 2.65-2.80 [m, 2H], 2.85-3.00 [t,
2H], 3.2-3.35 [m, 2H], 4.70-4.90 [m, 1H], 7.05-7.35 [m, 6H], 7.90-
8.05 [m, 2H]. ESI-MS: m/z = 547 (calcd value for C29H36O5N2ClF =
546.2).
Synthesis of HP-C16. HP-C16 was synthesized following similar
procedure for the synthesis of HP-C4 (13% yield, Rf = 0.65 in 10%
methanol-chloroform, v/v). 1H NMR (200 MHz, CDCl3): δ = 0.78-
0.93 [t, 6H], 1.12-1.41 [m, 52H], 1.53-1.77 [m, 4H], 1.96-2.27 [m,
4H], 2.39-2.54 [m, 2H], 2.61-2.85 [m, 6H], 2.90-3.05 [m, 2H], 3.06
[s, 3H], 3.30-3.55 [m, 8H], 4.34 [s, 2H], 7.04-7.16 [t, 2H], 7.23-7.33
[m, 4H], 7.91-8.03 [m, 2H]. ESI-MS: m/z = 966.7216 (calcd value for
C59H98O4N3ClF = 966.7229). HPLC purity, >99%.
Synthesis of Dioctylamine, 7. A mixture of octan-1-amine (2 g,
15.5 mmol), 1-bromooctane (2.98 g, 15.5 mmol), and anhydrous
potassium carbonate (2.34 g, 18.01 mmol) in 4 mL of DMSO was kept
under stirring for 12 h at 80 °C. The reaction mixture was taken in 50 mL
of chloroform, washed with water (3 ꢀ 50 mL), dried over anhydrous
Na2SO4, and evaporated. Column chromatographic purification of the
residue using 60-120 mesh silica gel and 1% methanol-chloroform (v/v)
as eluent afforded compound 7 as a white solid (1.3 g, 30.8% yield,
Synthesis of β-Alanine-Haloperidol Conjugate, 5. Com-
pound 4 (1.41 g, 2.58 mmol) was dissolved in 18 mL of dry DCM in a
50 mL round-bottomed flask and stirred over an ice bath for 15 min.
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Rf = 0.55 in 10% methanol-chloroform, v/v). 1HNMR(200MHz, CDCl3):
δ = 0.9 [t, 6H], 1.05-1.45 [m, 20H], 1.6-1.85 [m, 4H], 2.8 [t, 4H].
Synthesis of N-Octyl-N-propyloctan-1-amine, 8. In a 50 mL
round-bottomed flask a mixture of compound 7 (0.5 g, 2.07 mmol),
1-bromopropane (0.381 g, 3.1 mmol), and anhydrous potassium
carbonate (0.55 g, 4.14 mmol) in 10 mL of ethyl acetate was kept under
stirring for 24 h at 70 °C. Then the solvent was evaporated. Column
chromatographic purification of the residue using 60-120 mesh silica
gel and 10% ethyl acetate in hexane (v/v) as the eluent afforded
compound 8 as a white solid (0.55 g, 77.8% yield, Rf = 0.6 in 30% ethyl
acetate-hexane, v/v). 1H NMR (200 MHz, CDCl3): δ = 0.9 [t, 9H],
1.05-1.45 [m, 20H], 1.4-1.6 [m, 6H], 2.2-2.4 [m, 6H].
Synthesis of C8 Control, 9. Compound 8 (0.5 g, 1.46 mmol) was
dissolved in 5 mL of dry DCM and treated with excess methyl iodide
(3 mL) and stirred for 4 h at room temperature. Then the reaction
mixture was concentrated. Column chromatographic purification of the
residue (using 100-200 mesh silica gel and 1% methanol-chloroform
(v/v) as eluent) followed by chloride ion exchange chromatography
(using Amberlite IRA-400Cl resin and methanol as eluent) yielded the
C8 control molecule (9) as a light yellow gummy solid (0.22 g, 40%
yield, Rf = 0.3 in 10% methanol-chloroform, v/v). 1H NMR (200 MHz,
CDCl3): δ = 0.9 [t, 6H], 1-1.1 [t, 3H], 1.2-1.5 [m, 20H], 1.65-1.9
[m, 6H], 3.5-3.6 [m, 6H], 3.4 [s, 3H]. ESI-HRMS: m/z = 298.3473
(calcd value for C20H44N = 298.3473). HPLC purity >98%.
Biology. Preparation of Samples and Sample Treatment. The
compounds were dissolved in cell culture grade DMSO to get a primary
stock. The stock is progressively diluted with DMSO to get secondary
stocks. Finally, the working concentrations of the derivatives were
obtained by adding the secondary DMSO stocks in 10% fetal bovine
serum containing cell culture medium. The amount of DMSO in working
solutions never exceeded more than 1% with respect to the serum
containing culture medium. For the viability studies an amount of 100 μL
of cell culture solutions containing respective concentrations of compounds
was added to each well of 96-well plates. For quantification of apoptosis
studies an amount of 1.5 mL of culture medium containing respective
concentration of compounds was added to each well of 6-well plates.
Cell Culture. MCF-7, MDA-MB-231, ZR-75-1, B16F10, COS-1, and
HEK-293 cells were purchased from the National Center for Cell
Sciences (Pune, India) and were mycoplasma free. Cells were cultured
in RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine
serum (GIBCO), 50 μg/mL penicillin, 50 μg/mL streptomycin, and 100
μg/mL kanamycin at 37 °C in a humidified atmosphere of 5% CO2 in
air. Cultures of 85-90% confluency were used for all of the experiments.
The cells were trypsinized, counted, and seeded in 96-well plates for
viability studies, in 6-well plates for quantification of apoptosis studies,
and in 100 ꢀ 20 mm2 tissue culture Petri dish for Western blot studies.
The cells were allowed to adhere overnight before they were used for
experiments. HUVEC cells (Lonza Inc.) were cultured in company
recommended medium, EGM (endothelial growth medium)-2 Bullet
Kit (Lonza Inc.), and were maintained as described above. HUVEC cell-
based experiments were done in second and third cellular passages only.
Cytotoxicity Studies. Cytotoxicities of the compounds were evaluated
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) reduction assay. Briefly, cells were seeded at a density of 5000
cells/well in a 96-well plate usually 18-24 h before experiment.
Solutions containing respective concentrations of compounds were
added to triplicate wells. In the screening studies derivatives of haloper-
idol were continuously treated to the cells for 48 h. In other studies,
cytotoxic cationic haloperidol derivative HP-C8 along with haloperidol
and C8 control was continuously treated for 48 h. Following the
termination of experiment cells were washed and promptly assayed
for viability using MTT. Results were expressed as percent viability =
{[A540(treated cells) - background]/[A540(untreated cells) - back-
ground]} ꢀ 100.
σ Receptor Down-Regulation by siRNA Delivery. siRNA based σ
receptor down-regulation was performed following a similar procedure
described earlier.19 In brief, MCF-7 or COS-1 cells were seeded at a
density of 2 ꢀ 105 cells per well in a 6-well plate usually between 18 and
24 h before transfection. Then 5 pmol of σ receptor siRNA (subtype 1)
(Santa Cruz Biotechnology) or 5 pmol of negative control siRNA
(Qiagen) (diluted to 50 μL with serum free RPMI) was complexed
with 0.5 μL of Lipofectamine 2000 (Invitrogen) (diluted to 50 μL with
serum free RPMI) for 15 min. An amount of 900 μL of serum free RPMI
was added to the resulting complex. Cells were washed with phos-
phate-buffered saline (PBS) and then treated with respective com-
plexes. After 4 h of incubation, cells were harvested, counted, and
again seeded at a density of 10 000 cells per well in 96-well plates for
cytotoxicity assay. After 16-18 h cells were kept untreated or treated
with 10 μM HP-C8 for 4 h. After 4 h of treatment, the cells were
washed and kept in the presence of fresh cell culture medium for 48 h.
The cells were then assayed for viability using MTT as described
earlier.
Flow Cytometric Analysis. The remaining cells after seeding from the
above experiment were used to evaluate the extent of down-regulation of
σ receptor by flow cytometry. They were replated, and after 24 h they
were harvested, washed, fixed with 2% formaldehyde, and kept at 4 °C
for 2 h. Then they were collected by centrifugation, washed, and treated
with polyclonal antibody raised against full-length σ receptor of human
origin in rabbit (Santa Cruz Biotechnology) at 1:100 dilution (in PBS
containing 2% fetal bovine serum). After incubating at 4 °C for 2 h, cells
were collected by centrifugation, washed, and treated with FITC
conjugated goat-antirabbit secondary antibody (Santa Cruz Biotechnology)
at 1:200 dilution (in PBS containing 2% fetal bovine serum) and
incubated at 4 °C for 1 h in the dark. Finally cells were analyzed using
a flow cytometer (FACS Canto II, Becton Dickinson, San Jose, CA, U.S.)
and data were analyzed with FCS Express V3 software. A minimum of
10 000 events were gated per sample.
Quantification of Apoptosis Studies. The annexin V-FITC-labeled
apoptosis detection kit (Sigma) was used to detect and quantify
apoptosis by flow cytometry per manufacturer’s instructions. In brief,
cells (2 ꢀ 105 cells/well) were seeded in six-well plates and cultured
overnight in 10% serum medium. After 16-18 h cells were kept
untreated or treated with 5 μM haloperidol or 5 μM HP-C8 for 24 h.
Then the cells were harvested and collected by centrifugation for 5 min
at 1000g. Cells were then resuspended at a density of 1 ꢀ 106 cells/mL in
1ꢀ binding buffer and stained simultaneously with FITC-labeled
annexin V (25 ng/mL) and propidium iodide (50 ng/mL). Cells were
analyzed using a flow cytometer (FACS Canto II), and data were
analyzed with FCS Express V3 software. A minimum of 10 000 events
were gated per sample.
Preclinical Studies Using C57BL6/J Mice. Male C57BL6/J mice were
obtained from CCMB (Hyderabad, India). The protocols for animal
experimentations were approved through Institutional Animal Ethical
Committee. Male C57BL6/J mice, 6-8 weeks old, were subcutaneously
inoculated with 2 ꢀ 105 B16F10 cells in the lower right abdomen. Eleven
days following cancer cell implantation, mice were grouped as per
treatment. The groups were as follows: (i) group treated with haloper-
idol (four mice) and (ii) group treated with HP-C8 (four mice). For
each treatment group an amount of 7.5 mg/kg respective compounds
suspended in PBS containing 10% DMSO was injected. Five intraper-
itoneal injections with a gap of 2-3 days were administered. Tumors
were measured three times a week. The tumor sizes were expressed in
volume (mm3) and calculated using the formula (0.5ab2), where a is the
longest dimension and b is the shortest dimension of the tumors.
Experiment was terminated when the average tumor volume of the
haloperidol treated group reached ∼4000 mm3.
TUNEL Assay. After completion of the above experiment one mouse
from each group was sacrificed for detection of apoptosis in tumor tissues.
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The tumors were frozen in Jung tissue freeze medium (Leica Microsystem,
Germany) followed by cryosectioning of 10 μm thin sections using Leica
CM1850 cryostat (Germany). The sections were fixed and TUNEL
assayed using DeadEnd fluorometric TUNEL assay kit (Promega,
Madison, WI) per manufacturer’s protocol. Images were acquired in
Nikon TE2000E microscope at 10ꢀ magnification.
’ REFERENCES
(1) Wolfe, S. A., Jr.; Culp, S. G.; De Souza, E. B. Sigma-receptors in
endocrine organs: identification, characterization, and autoradiographic
localization in rat pituitary, adrenal, testis, and ovary. Endocrinology
1989, 124, 1160–1172.
(2) Hellewell, S. B.; Bruce, A.; Feinstein, G.; Orringer, J.; Williams,
W.; Bowen, W. D. Rat liver and kidney contain high densities of sigma 1
and sigma 2 receptors: characterization by ligand binding and photo-
affinity labeling. Eur. J. Pharmacol. 1994, 268, 9–18.
(3) Vilner, B. J.; John, C. S.; Bowen, W. D. Sigma-1 and sigma-2
receptors are expressed in a wide variety of human and rodent tumor cell
lines. Cancer Res. 1995, 55, 408–13.
(4) John, C. S.; Bowen, W. D.; Saga, T.; Kinuya, S.; Vilner, B. J.;
Baumgold, J.; Paik, C. H.; Reba, R. C.; Neumann, R. D.; Varma, V. M.;
McAfee, J. G. A malignant melanoma imaging agent: synthesis, char-
acterization, in vitro binding and biodistribution of iodine-125-(2-
piperidinylaminoethyl)4-iodobenzamide. J. Nucl. Med. 1993, 34, 2169–
2175.
(5) John, C. S.; Vilner, B. J.; Bowen, W. D. Synthesis and character-
ization of [125I]-N-(N-benzylpiperidin-4-yl)-4-iodobenzamide, a new
sigma receptor radiopharmaceutical: high affinity binding to MCF-7
breast tumor cells. J. Med. Chem. 1994, 37, 1737–1739.
(6) John, C. S.; Vilner, B. J.; Gulden, M. E.; Efange, S. M.; Langason,
R. B.; Moody, T. W.; Bowen, W. D. Synthesis and pharmacological
characterization of 4-[125I]-N-(N-benzylpiperidine-4-yl)-4-iodobenz-
mide: a high affinity sigma receptor ligand for potential imaging of
breast cancer. Cancer Res. 1995, 55, 3022–3027.
(7) John, C. S.; Gulden, M. E.; Vilner, B. J.; Bowen, W. D. Synthesis,
in vitro validation and in vivo pharmacokinetics of [125I]N-[2-(4-
iodophenyl)ethyl]-N-methyl-2-(1-piperidinyl)ethylamine: a high-affi-
nity ligand for imaging sigma receptor positive tumors. Nucl. Med. Biol.
1996, 23, 761–766.
Western Blot. For this study, both B16F10 and HUVEC cells were
treated with HP or HP-C8 (10 μM) or kept untreated for 24 h in
charcoal stripped serum condition (RPMI for B16F10 and EBM
solution with bullet kit (Lonza) except the recommended serum).
Following treatment whole cell lysates from both the cells were
prepared as follows: Cells were lysed with cold radioimmunopreci-
pitation assay buffer (RIPA, Boston BioProducts, MA, U.S.) (50 mM
Tris-HCl, pH 7.4, 0.15 M NaCl, 1% Nonidet P-40, 0.5% sodium
deoxycholate, and 0.1% SDS), which was premixed with 1ꢀ Halt
phosphatase inhibitor cocktail, 1ꢀ protease inhibitor cocktail
(Thermo Scientific, IL, U.S.), and 2 mM sodium vanadate. Respective
cell lysates were run in SDS-PAGE gradient gel, and then the bands
were transferred to PVDF membrane. Antibodies such as caspase 3,
Bax, Akt1/2/3, and pAkt1/2/3 (Santa Cruz), Bcl-2 (Cell Signaling
Technology, Beverly, MA), and anti-β-actin (Sigma Chemical Com-
pany, St. Louis, MO) followed by horseradish peroxidase conjugated
secondary antibody (Santa Cruz Biotechnology) and the SuperSignal
West Pico substrate (Pierce Biotechnology) were used for detection
of protein bands.
Statistical Analysis. Data were expressed as the mean ( SD and
statistically analyzed by the two-tailed, unpaired, Student’s t-test using
Microsoft Excel (Seattle, WA). For animal studies, scores were con-
sidered significant when p < 0.05. For in vitro studies, scores were
considered significant when p < 0.01.
(8) John, C. S.; Vilner, B. J.; Geyer, B. C.; Moody, T.; Bowen, W. D.
Targeting sigma receptor-binding benzamides as in vivo diagnostic and
therapeutic agents for human prostate tumors. Cancer Res. 1999,
59, 4578–4583.
(9) John, C. S.; Bowen, W. D.; Fisher, S. J.; Lim, B. B.; Geyer, B. C.;
Vilner, B. J.; Wahl, R. L. Synthesis, in vitro pharmacologic characteriza-
tion, and preclinical evaluation of N-[2-(10-piperidinyl)ethyl]-
3-[125I]iodo-4-methoxybenzamide (P[125I]MBA) for imaging breast
cancer. Nucl. Med. Biol. 1999, 26 (4), 377–382.
’ ASSOCIATED CONTENT
S
Supporting Information. Detailed spectral characteriza-
b
tion data and HPLC purity data of all final molecules; Figure S1
depicting toxicity comparison of HP-C8 with mixture of HP and
C8-molecule in B16F10 cell. This material is available free of
charge via the Internet at http://pubs.acs.org.
(10) Wheeler, K. T.; Wang, L. M.; Wallen, C. A.; Childers, S. R.;
Cline, J. M.; Keng, P. C.; Mach, R. H. Sigma-2 receptors as a biomarker
of proliferation in solid tumours. Br. J. Cancer 2000, 82, 1223–1232.
(11) Zamora, P. O.; Moody, T. W.; John, C. S. Increased binding to
sigma sites of N-[10(2-piperidinyl)ethyl)-4-[I-125]-iodobenzamide (I-125-
PAB) with onset of tumor cell proliferation. Life Sci. 1998, 63, 1611–1618.
(12) Crawford, K. W.; Bowen, W. D. Sigma-2 receptor agonists
activate a novel apoptotic pathway and potentiate antineoplastic drugs in
breast tumor cell lines. Cancer Res. 2002, 62, 313–322.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 91-40-27191476. Fax: 91-40-27193370. E-mail: rkbanerjee@
yahoo.com and banerjee@iict.res.in.
Present Addresses
Department of Biochemistry and Molecular Biology, Mayo
Clinic Foundation, Rochester, Minnesota 55902, U.S.
(13) Brent, P. J.; Pang, G. T. Sigma binding site ligands inhibit cell
proliferation in mammary and colon carcinoma cell lines and melanoma
cells in culture. Eur. J. Pharmacol. 1995, 278, 151–160.
’ ACKNOWLEDGMENT
(14) Wei, Z.; Mousseau, D. D.; Dai, Y.; Cao, X.; Li, X. M. Haloper-
idol induces apoptosis via the sigma2 receptor system and Bcl-XS.
Pharmacogenomics J. 2006, 6, 279–288.
(15) Nordenberg, J.; Perlmutter, I.; Lavie, G.; Beery, E.; Uziel, O.;
Morgenstern, C.; Fenig, E.; Weizman, A. Anti-proliferative activity of
haloperidol in B16 mouse and human SK-MEL-28 melanoma cell lines.
Int. J. Oncol. 2005, 27, 1097–1103.
K.P. and S.K.P thank CSIR, Government of India, for their
doctoral fellowship. R.B. thanks Department of Science and
Technology, Government of India, for research support (Grant
SR/S1/OC-64/2008).
’ ABBREVIATIONS USED
(16) Dai, Y.; Wei, Z.; Sephton, C. F.; Zhang, D.; Anderson, D. H.;
Mousseau, D. D. Haloperidol induces the nuclear translocation of
phosphatidylinositol 30-kinase to disrupt Akt phosphorylation in PC12
cells. J. Psychiatry Neurosci. 2007, 32, 323–330.
(17) Mukherjee, A.; Prasad, T. K.; Rao, N. M.; Banerjee, R. Haloper-
idol-associated stealth liposomes: a potent carrier for delivering genes to
human breast cancer cells. J. Biol. Chem. 2005, 280, 15619–15627.
HP, haloperidol; PI, propidium iodide; PI3K, phosphatidyli-
nositol 3-kinase; Akt, protein kinase B; HUVEC, human umbilical
cord vascular endothelial cell; FITC, fluorescein isothiocyanate;
TUNEL, terminal deoxynucleotide transferase dUTP nick end
labeling; Bcl-2, B-cell lymphoma 2; BAX, Bcl-2-associated X
protein
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Journal of Medicinal Chemistry
ARTICLE
(18) Karmali, P. P.; Chaudhuri, A. Cationic liposomes as non-viral
carriers of gene medicines: resolved issues, open questions, and future
promises. Med. Res. Rev. 2007, 27, 696–722.
(19) Mukherjee, A.; Narayan, K. P.; Pal, K.; Kumar, J. M.; Rangaraj,
N.; Kalivendi, S. V.; Banerjee, R. Selective cancer targeting via aberrant
behavior of cancer cell-associated glucocorticoid receptor. Mol. Ther.
2009, 17, 623–631.
(20) Pham, T. Q.; Berghofer, P.; Liu, X.; Greguric, I.; Dikic, B.;
Ballantyne, P.; Mattner, F.; Nguyen, V.; Loc’h, V.; Katsifis, A. Prepara-
tion and biologic evaluation of a novel radioiodinated benzylpiperazine,
123I-MEL037, for malignant melanoma. J. Nucl. Med. 2007, 48, 1348–
1356.
(21) Kim, N. R.; Park, S. W.; Lee, J. G.; Kim, Y. H. Protective effects
of olanzapine and haloperidol on serum withdrawal-induced apoptosis
in SH-SY5Y cells. Prog. Neuropsychopharmacol. Biol. Psychiatry 2008,
32, 633–642.
(22) Wei, Z.; Qi, J.; Dai, Y.; Bowen, W. D.; Mousseau, D. D.
Haloperidol disrupts Akt signalling to reveal a phosphorylation-depen-
dent regulation of pro-apoptotic Bcl-XS function. Cell. Signallling 2009,
21, 161–168.
(23) Brent, P. J.; Pang, G.; Little, G.; Dosen, P. J.; Van Helden, D. F.
The sigma receptor ligand, reduced haloperidol, induces apoptosis and
increases intracellular-free calcium levels [Ca2þ] in colon and mammary
adenocarcinoma cells. Biochem. Biophys. Res. Commun. 1996, 219, 219–
226.
(24) Kim, R.; Emi, M.; Tanabe, K. Role of mitochondria as the
gardens of cell death. Cancer Chemother. Pharmacol. 2006, 57, 545–553.
(25) Gross, A.; McDonnell, J. M.; Korsmeyer, S. J. BCL-2 family
members and the mitochondria in apoptosis. Genes Dev. 1999, 13, 1899–
1911.
(26) Testa, J. R.; Bellacosa, A. Akt plays a central role in tumorigen-
esis. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 10983–10985.
(27) Riesterer, O.; Zingg, D.; Hummerjohann, J.; Bodis, S.; Pruschy,
M. Degradation of PKB/Akt protein by inhibition of the VEGF
receptor/mTOR pathway in endothelial cells. Oncogene 2004,
23, 4624–4635.
(28) Sinha, S.; Roy, S.; Reddy, B. S.; Pal, K.; Sudhakar, G.; Iyer, S.;
Dutta, S.; Wang, E.; Vohra, P. K.; Roy, K. R.; Reddanna, P.; Mukho-
padhyay, D.; Banerjee, R. A lipid modified estrogen derivative that treats
breast cancer independent of estrogen receptor expression through
simultaneous induction of autophagy and apoptosis. Mol. Cancer Res.
[Online early access]. DOI: 10.1158/1541-7786.MCR-10-0526. Pub-
lished Online: February 2, 2011.
2390
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