Anti-tumor and proapoptotic effect of novel synthetic benzophenone analogues in Ehrlich ascites tumor cells

Article abstract of DOI:10.1016/j.bmc.2005.08.039

A series of substituted benzophenone analogues, (2-aroyl-4-methylphenoxy) acetamides 4a-e, have been synthesized via three-step synthesis sequence beginning with the 2-hydroxybenzophenones 1a-e in excellent yield. 1a-e on reaction with ethyl chloroacetate

Full text of DOI:10.1016/j.bmc.2005.08.039

Bioorganic & Medicinal Chemistry 14 (2006) 435–446
Anti-tumor and proapoptotic effect of novel synthetic
benzophenone analogues in Ehrlich ascites tumor cells
B. T. Prabhakar,a Shaukath Ara Khanum,b K. Jayashree,d Bharathi P. Salimath,a
and S. Shashikanthc,*
aDepartment of Studies in Applied Botany and Biotechnology, University of Mysore, Manasagangotri, Mysore 570 006, India
bDepartment of Chemistry, YuvarajaÕs College, University of Mysore, Mysore 570 005, India
cDepartment of Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India
dDepartment of Pathology, J.S.S. Medical College, Mysore, India
Received 18 July 2005; revised 9 August 2005; accepted 9 August 2005
Available online 7 October 2005
Abstract—A series of substituted benzophenone analogues, (2-aroyl-4-methylphenoxy)acetamides 4a–e, have been synthesized via
three-step synthesis sequence beginning with the 2-hydroxybenzophenones 1a–e in excellent yield. 1a–e on reaction with ethyl chlo-
roacetate afford ethyl (2-aroyl-4-methylphenoxy)acetates 2a–e which on alkaline hydrolysis afforded (2-aroyl-4-methylphenoxy)eth-
anoic acid 3a–e. Compounds 3a–e on condensation with p-chloroaniline furnished benzophenone analogues 4a–e. In the present
report, we investigated the anti-tumor and proapoptotic effect of benzophenones in Ehrlich ascites tumor (EAT) cells. Treatment
of benzophenones in vivo resulted in inhibition of proliferation of EAT cells and ascites formation. Further, we demonstrate that
the induction of apoptosis in EAT cells is mediated through activation of caspase-3. These results suggest a further possible clinical
application of these synthetic compounds as potent anti-tumor and proapoptotic compounds.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
condensation, and nuclear fragments into membrane
enclosed apoptotic bodies.13 The DNA fragmentation
results from activation of caspase-activated endonucle-
ase. DNA fragmentation factor (DFF-40/CAD) is asso-
ciated with the internucleosomal cleavage of DNA
during apoptosis. CAD is normally sequestered in the
cytoplasm via its binding to an inhibitor (ICAD/DFF-
45), which suppresses its endonuclease activity and con-
ceals its nuclear localization signal. During the induc-
tion of apoptosis, ICAD/DFF-45 is cleaved and
inactivated by a caspase-3-like protease, thereby result-
ing in the activation of CAD, its nuclear translocation,
and subsequent oligonucleosomal DNA fragmentation
which is a hallmark of apoptosis.14–16
Apoptosis or programmed cell death has been character-
ized as a fundamental cellular activity occurring under a
wide range of physiological and pathological condi-
tions.1–6 It plays an essential and protective mechanism
against neoplastic development in the organism by elim-
inating genetically damaged cells or excess cells that
have been improperly induced to divide by mitotic stim-
ulus.7–9 Apoptosis results from activation of prepro-
grammed pathway of biochemical events leading to
cell death.10,11 The p53, a tumor suppressor gene, is
implicated in the induction of distinct apoptotic signal-
ing pathways that lead to the activation of aspartate spe-
cific cystiene proteases (caspases) that mediate
apoptosis.12 The activation of apoptotic genes will cul-
minate in morphological and biochemical changes that
include skeletal disruption, membrane blebbing, de-
crease in adhesion and intracellular contacts, chromatin
In this context, it is noteworthy that apoptosis inducing
ability seems to have become a primary factor in consid-
ering the efficacy of chemopreventive agents. There are
several natural and synthetic compounds that a induce
apoptosis both in vivo and in vitro in EAT cells.17–21
Benzophenones are a class of compounds obtained from
natural products22 or by synthetic methods.23 The great
importance of these substances is fundamentally due to
the diverse biological and chemical properties they
Keywords: Benzophenones; EAT cells; Anti-tumor; Apoptosis;
Caspases.
*
Corresponding author. Tel.: +91 0821 2547279; e-mail: skanth1@
rediffmail.com
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.039

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B. T. Prabhakar et al. / Bioorg. Med. Chem. 14 (2006) 435–446
possess. Benzophenones exhibit significant anti-tumor
activity both in vivo and in vitro.24–34 In addition, syn-
thetic benzophenones, such as 2-aminobenzophenone35
and dihydroxy-4-methoxybenzophenone36, have proven
to be antimitotic and anticancer agents, respectively.
Recently, para-methoxy substituted benzophenones were
evaluated as p38a inhibitors with high efficacy and selec-
tivity (IC50: 14 nM).37 Amino- and methoxy-substituted
benzophenones are reported to be potent cytotoxic
agents against a panel of human cancer cell lines includ-
ing multidrug resistant cell lines.24 The tubulin binding
drug combretastatin A-4 exhibits a selective toxicity for
proliferating endothelial cells by induction of apopto-
sis.38 Polyprenylated benzophenone derivatives will in-
duce caspase-mediated apoptosis.31 Encouraged by this
information an attempt was made to achieve new com-
pounds with possible anti-proliferative effect in vivo. In
the present study, we designed and synthesized some nov-
el (2-aroyl-4-methylphenoxy)acetamides 4a–e and veri-
fied their anti-tumor effect and mechanism of inhibition
of proliferation and ascites formation in EAT cells. We
addressed the anti-tumor and proapoptotic effect of com-
pounds 4a–e and relevance of caspase-3. We suggest that
these compounds could be useful for individualization of
therapeutic strategies in the future.
method (data not shown). In control EAT-bearing mice,
there is a gradual increase in body weight, which from
day six attains an exponential phase of growth. A rise
in body weight of about 7 g over 12 days period of tu-
mor growth was observed. However, when treated with
the compounds 4a–e, about 49.05%, 39.18%, 30.12%,
45.76% and 61.12% decrease in the body weight was ob-
served, respectively, indicating the anti-tumor effect of
compounds 4a–e (Table 1). As shown in Figure 2 com-
pound 4a–e treatment resulted in a dose-dependent inhi-
bition of proliferation of EAT cells. Decrease in tumor
growth also correlates with decrease in ascites volume
of treated mice (Fig. 1). The survivality of EAT cell-
bearing mice (control) halved on the 12th day after
implantation and no animal survived beyond the 14th
day. Compound 4a–e treatment increased the survivali-
ty, where animals were alive beyond the 23rd day. Com-
pound 4e-treated animals survived for 23 days, whereas
compound 4a- and 4d-treated animals survived for 20
days, and of compound 4b- and 4c-treated animals 17
days, correlating with the anti-tumor effect of these com-
pounds. These results showed that compound 4e is the
most effective inhibitor of tumor growth among com-
pounds 4a–d. However, compounds 4a–d did show sig-
nificant inhibition of tumor growth.
All the results are listed in Table 1.
2. Results
2.3. Compounds 4a–e induce apoptosis of EAT cells
in vivo
2.1. Compounds 4a–e inhibit proliferation of EAT cells
in vitro
To elucidate the possible mechanism of compound 4a–e
mediated tumor inhibition, we investigated the effect of
compounds 4a–e on EAT cells. We found that treatment
of compounds 4a–e to EAT cells induced a significant
proportion of cells to undergo apoptosis, as determined
by the flowcytometric analysis. FACS analysis of the
cells indicates the formation of apoptotic bodies and
condensed nuclei that show up in the sub-G1 region.
As shown in Figure 3, when compared to the control
cells, on receiving compound 4a–e treatment some of
the EAT cells undergo apoptosis and are detected in
the sub-G1 area. These results suggested the breakdown
of EAT cells DNA resulting in tumor killing. The obvi-
ous ramifications were the growth arrest of EAT cells.
We examined the effect of compounds 4a–e on the cellular
proliferation of EAT cells by using the MTT assay. Signif-
icant dose-dependent growth inhibition was observed
when cells were treated with compounds 4a–e for 24 h.
Compounds 4a–e showed 50% growth inhibition at
<50 lM (Table 1). These result suggest that compounds
4a–e potently inhibit the proliferation of EAT cells.
2.2. Compounds 4a–e inhibit proliferation EAT cells and
increases the survivality of the animals in vivo
To determine the in vivo effect on cell growth, mouse
mammary carcinoma cell line EAT was treated with
compounds 4a–e (100 mg/kg body weight/ip), during
its growth period, three doses on every other day start-
ing from 6 days after inoculation of tumor cells. Animal
weights were monitored throughout the course of treat-
ment. Compounds were not cytotoxic to the cells as ver-
ified by the MTT assay and trypan blue dye exclusion
2.4. Compounds 4a–e induce nuclear condensation of EAT
cells
An attempt was made to find out whether the inhibition
of proliferation of EAT cells by compounds 4a–e was
Table 1. Effect of compounds 4a–e on proliferation of EAT cells in vitro, tumor growth inhibition, and survivality of the animals in vivo
Control
4a
4b
4c
4d
4e
IC50 value (lM)
Tumor growth inhibition (%)
Survivality of the animals (days)
—
—
14
39.67
49.05
20
38.58
39.18
18
38.22
30.12
18
37.46
45.76
20
42.50
61.12
23
Exponentially growing cells were treated with various concentrations (0–100 lM) of compounds 4a–e for 24 h and were analyzed by the MTT assay
and IC50 values of each compound were determined. The effect of compounds 4a–e on the body weight of the EAT-bearing mice was verified and
percentage of inhibition after treatment was calculated taking 100% growth in the case of untreated mice. Survivality of the untreated mice was 14
days, whereas increased survivality in treated mice was observed. All the experiments contain at least five animals and each experiment was repeated
thrice. Results are mean values (n = 3).

B. T. Prabhakar et al. / Bioorg. Med. Chem. 14 (2006) 435–446
437
Figure 1. Effect of compounds 4a–e on ascites volume. EAT-bearing mice treated with compounds 4a–e were sacrificed after giving each dose and
cells along with ascites fluid were harvested. Ascites volume was recorded. The above results are an average of three individual experiments.
due to induction of apoptosis in EAT cells. During in vivo
studies, cytological analysis of acridine orange/ethidium
bromide stained treated EAT cells (in vivo) showed nucle-
ar condensation, an important feature of apoptosis as
compared to the untreated EAT cells (Fig. 4).
expected, compound 4a–e treatment caused DNA frag-
mentation which then led to the formation of DNA lad-
der in EAT cells. This indicates that the apoptotic role
of compounds 4a–e in EAT cells ruled out the possibility
of it being necrotic to EAT cells (Fig. 6).
2.5. Compounds 4a–e induce externalization of phospha-
tidylserine residues in EAT cells
2.7. Compounds 4a–e modulate p53 expression
For investigating the molecular mechanism of com-
pounds 4a–e induced apoptosis Western blot analysis
of p53 protein was carried out. Result showed an in-
crease in p53 protein levels in treated compared to
untreated. Based on the above results we suggested that
compound 4a–e-mediated apoptosis is p53 dependent
(Fig. 7).
During in vivo studies we verified the flipping of phos-
phatidylserine on the cell membrane by compounds
4a–e, since phosphatidylserine flip-flops onto the cell
surface in the very early stages of apoptosis. Cells under-
going apoptosis were detected using Annexin-V labeled
with fluorescein isothiocyanate. EAT cells treated with
compounds 4a–e clearly showed early apoptotic changes
like annexin binding (greenish red), while control cells
did not show annexin binding (Fig. 5). These results
indicate that compounds 4a–e induce nuclear transloca-
tion of phosphatidylserine to the outer membrane sur-
face in EAT cells.
2.8. Compound 4a–e treatment induces caspase-3-medi-
ated apoptosis
Delineating the downstream signaling pathway revealed
that compound 4a–e treatment is activating the caspase-
3 protease. In order to verify the involvement of cas-
pase-3 upstream activator of endonuclease, EAT cells
either treated with or without caspase-3 inhibitor were
exposed to the compounds 4a–e. The resultant lanes
(3, 5, 7, 9, and 11) show the DNA fragmentation as a
consequence of increased endonuclease activity and in
lanes (2, 4, 6, 8, and 10) fragmentation was inhibited
2.6. Compounds 4a–e induce degradation of DNA in EAT
cells
Biochemically, apoptosis is characterized by fragmenta-
tion of chromosomal DNA. We further verified the effect
of compounds 4a–e on DNA damage of EAT cells. As

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B. T. Prabhakar et al. / Bioorg. Med. Chem. 14 (2006) 435–446
1800
1600
1400
1800
1600
1800
1600
1400
1200
1000
800
600
400
200
0
1400
1200
1000
800
600
400
200
0
1200
1000
800
600
400
200
0
7th day
9th day
11th day
7th day
9th day
11th day
7th day
9th day
11th day
Number of days
4-a
Number of days
4-b
Number of days
4-c
1800
1600
1400
1200
1000
800
1800
1600
1400
1200
1000
800
600
600
400
400
200
200
0
0
7th day
9th day
11th day
7th day
9th day
11th day
Number of days
4-d
Number of days
4-e
Figure 2. Effect of compounds 4a–e on EAT cell number. EAT-bearing mice treated with compounds 4a–e were sacrificed after giving each dose and
cells along with ascites fluid were harvested. The number of cells per mouse was determined by counting the cells in hemocytometer. The above results
are an average of three individual experiments.
by Ac-DEVD-CHO, a specific inhibitor of caspase-3
enzymes. Lane 1 is control DNA (Fig. 8).
the presence of amide group. The amide moiety is an
important constituent of many biologically significant
compounds. Amide moiety is versatile in organic com-
pounds since all the three atoms in the O–C–N chain
are potentially reactive. These results are partly due to
the delocalization of the p electrons along the O–C–N
chain. This produces partial double-bond character in
the C (O)–N bond. The versatility of the amide group
in forming partial bonds with itself and many other
functional groups is partly responsible for the structural
subtleties of the biologically important proton
derivatives.39,40
2.9. Compounds 4a–e induce translocation of CAD
Since compounds 4a–e activate caspase-3, a death prote-
ase responsible for apoptosis and activation of endonu-
clease in EAT cells, we further verified the coordinate
relationship between caspase-3 and endonuclease in
the cellular system of EAT cells. Western blot analysis
of nuclear extract prepared from EAT cells treated with
compounds 4a–e in vivo showed the presence of CAD,
whereas untreated cells did not show the presence of
CAD (Fig. 9). In apoptotic cells, cleavage of ICAD by
the caspase-3 protease allowed CAD to enter the nucle-
us and degrade DNA. We have demonstrated that the
caspase-activated DNAse (CAD) is translocated from
the cytosol to the nucleus and is responsible for the oli-
gonucleosomal DNA fragmentation. The CAD protein
was identified in the nucleus of compound 4a–e-treated
cell nuclear extract, while CAD was not detected in
the untreated nuclear extract.
To determine whether compound 4a–e treatment inhib-
its tumor growth, effect of compounds 4a–e on EAT cell
growth in vivo was checked. Earlier reports have shown
that benzophenones exhibit significant anti-tumor activ-
ity both in vitro and in vivo in many of the cancer cell
lines.24–34 The synthetic morphiline and thio-morphiline
benzophenones were screened for cytotoxic and anti-tu-
mor activities against p388 murine leukemia and human
lung carcinoma cells.29 Recently, benzophenone ana-
logues with amino and methoxy substitutes at ortho-
and para-positions have been reported as potent cyto-
toxic agents against human cancer cell lines compared
to plenstatin combretastatin A-4 analogue.35 Our results
on in vivo tumor inhibition showed that compounds 4a–
e have potent anti-tumor activity when compared with
3. Discussion
Structurally, (2-aroyl-4-methylphenoxy)acetamides, the
benzophenone analogues 4a–e, exhibit activity due to

B. T. Prabhakar et al. / Bioorg. Med. Chem. 14 (2006) 435–446
439
Figure 3. Analysis of apoptosis in EAT cells by compounds 4a–e. For the determination of cell cycle-phase distribution, EAT cells from tumor-
bearing mice were permeabilized and nuclear DNA was labeled with propidium iodide (PI) using Cycle TEST PLUS DNA reagent kit. Cell cycle
phase distribution of nuclear DNA was determined on FACS, fluorescence detector equipped with 488 nm argon laser light source and 623 nm band
pass filter (linear scale) using CellQuest software (Becton–Dickinson). A total of 10,000 events were acquired and analysis of flowcytometric data was
performed using ModFit software. A histogram of DNA content (x-axis, PI-fluorescence) versus counts (y-axis) has been displayed.
the lead compound. However, we observed that com-
pound 4e with the methoxy group at para-position in
ring B has shown the highest anti-tumor activity of
61.12%, whereas compounds 4a and 4d with the group
at meta- and ortho-positions in ring B, respectively, have
shown moderate decrease in body weight (49% and 45%,
respectively). Compound 4b without substituent in ring
B and 4c with the bromo group at the ortho-position
treatment have shown about 39% and 30% decrease in
body weight, respectively. The results correlated with a
decrease in cell number and ascites volume to consider-
able amount. Treatment of compounds 4a–e also
increased the survivality of the animals. Compound
4e-treated animals were alive for 23 days and the num-
ber of animals whcih survived in this group was 86%,
whereas compound 4a- and 4d-treated animals survived
for 20 days with 70% of the animals surviving. In the
case of compounds 4b and 4c, the percentage of animals
that survived was 58%, whereas the animals were alive
only for 18 days.
Despite variation in the tumor inhibition activity, all the
compounds were able to induce the apoptosis where the
mechanism of tumor growth inhibition was involved.
Our results on externalization of phosphatidylserine res-
idues, nuclear condensation, formation of apoptotic
bodies, DNA degradation, overexpression of p53, and
caspase-3 inhibition assays showed that compounds
4a–e were inducing caspase-3-mediated apoptosis in
EAT cells. The tumor suppressor gene p53 is regarded
as a key element in maintaining a balance between
growth and cell death in the living system and it plays
an important role in tumor growth inhibition and induc-
tion of apoptosis.41–43 Many compounds upregulate p53

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B. T. Prabhakar et al. / Bioorg. Med. Chem. 14 (2006) 435–446
Figure 4. Changes in the nuclear morphology of EAT cells. Nuclear
staining was performed according to the method of Srinivas et al. EAT
cells either treated or untreated with compounds 4a–e in vivo were
smeared on a glass slide, fixed with methanol/acetic acid (3:1), and air-
dried. The cells were hydrated with PBS and stained with a mixture
(1:1) of acridine orange/ethidium bromide (4 lg/mL) solutions. The
cells were immediately washed with PBS and viewed under a Leitz-
DIAPLAN fluorescent microscope.
Figure 5. Apoptotic morphology of EAT cells upon compounds 4a–e
treatment. EAT cells treated with and without compounds 4a–e in vivo
were harvested and washed with PBS, dropped onto the slide, and air-
dried. Cells were washed with binding buffer (10 mM Hepes, pH 7.5,
containing 140 mM NaCl and 2.5 mM CaCl2) and stained with
Annexin-V detection kit as per the protocol supplied by the manufac-
turer. After 10 min, cells were washed thrice with binding buffer. The
greenish red apoptotic cells were observed using a Leitz-DIAPLAN
fluorescent microscope and photographed.
gene expression both in vivo and in vitro19,44. Our re-
sults showed that compound 4a–e treatment resulted
in increment of p53 protein in EAT cells and it may
be an important molecular mechanism through which
compounds 4a–e inhibit growth of cancer cells.
The caspase family is another evolutionary conserved
gene family that is involved in p53-mediated apoptosis.
Caspases are cysteine proteases that play a critical role
in the execution of apoptosis.45,46 Activation of cas-
pase-3 occurs when p53 is overexpressed in p53 null
Saos-2 cells and BRK cells.47 This suggests that the
apoptotic pathways downstream of p53 may converge
on caspase activation. In fact, activation of p53 induces
caspase-3 activation and nuclear DNA fragmentation in
many cases.47–54 Benzophenone derivatives induce cas-
pase-3-mediated apoptosis both in vitro and in vivo in
many of the human cancer lines.31,55 Activation of cas-
pase-3 was observed in EAT cells treated with com-
pounds 4a–e. In addition, compounds 4a–e induced
activation of caspase-3, which was significantly attenu-
ated in the presence of Ac-DEVD-CHO, suggesting a
caspase-dependent mechanism of cell death by com-
pounds 4a–e. In addition, we have verified the down-
stream activation of caspase-activated DNase upon
Figure 6. In vivo apoptotic effect of compounds 4a–e. Untreated (lane
1) and compound 4a–e-treated EAT cells (lanes 2–6) were used for
extraction of nuclear DNA. DNA was run on 1.5% agarose gel.

B. T. Prabhakar et al. / Bioorg. Med. Chem. 14 (2006) 435–446
441
anti-tumor activity. All the compounds showed proa-
poptotic activity. These effects are likely to be induced
by overexpression of p53, activation of caspase-3, and
translocation of CAD into the nucleus, which is in-
volved in the apoptosis. Such active benzophenone ana-
logues prove to be a potential anti-tumor and apoptosis
inducing factor which could be further developed and
translated into a therapeutic regime for treatment of hu-
man cancer where formation of peritoneal malignant
ascites is a major cause of morbidity and mortality.
Figure 7. Western blot analysis of p53. Nuclear extract from untreated
and compound 4a–e-treated EAT cells in vivo was resolved on 10%
SDS–PAGE. Western blot analysis was performed for p53 using anti-
p53 antibodies. Lane 1, control and lanes 2–6, compound 4a–e treated.
4. Experimental
4.1. Chemistry
Chemicals were purchased from Aldrich Chemical Co.
TLC was performed on aluminum-backed silica plates
with visualization by UV light. Melting points were
determined with a Thomas Hoover capillary melting
point apparatus with a digital thermometer. IR spectra
were recorded in Nujol on an FT-IR Shimadzu 8300
1
spectrometer and H NMR spectra were recorded on a
Bruker 300 MHz spectrometer in CDCl3. Chemical
shifts were recorded in parts per million downfield from
tetramethylsilane. Mass spectra were obtained with a
VG70-70H mass spectrometer and elemental analysis re-
sults are within 0.4% of the calculated value.
The synthesis of the hitherto unreported title com-
pounds 4a–e is as outlined in Scheme 1. Hydroxybenz-
ophenones 1a–e on reaction with ethyl chloroacetate
afford ethyl (2-aroyl-4-methylphenoxy)acetates 2a–e in
excellent yield, which on alkaline hydrolysis afforded
(2-aroyl-4-methylphenoxy)ethanoic acid 3a–e. Com-
pounds 3a–e on condensation with p-chloroaniline in
the presence of boron trifluoride etherate and dry ben-
zene furnished benzophenone analogues, (2-aroyl-4-
methylphenoxy)acetamides 4a–e.43
Figure 8. Effect of caspase-3 inhibitor on compound 4a–e induced
DNA degradation. EAT cells were either untreated or pre-incubated
with 100 lM Ac-DEVD-CHO for 30 min followed by compound 4a–e
treatment for 2 h at 37 ꢁC. Genomic DNA was isolated and verified for
DNA ladder on 1.5% agarose gel, observed under UV-transillumina-
tor, and photographed. Lane 1—control DNA; lanes 3, 5, 7, 9, and
11—compound 4a–e-treated DNA; lanes 2, 4, 6, 8, and 10—compound
4a–e + Ac-DEVD-CHO-treated DNA.
4.1.1. Ethyl[2-(3-chlorobenzoyl)-4-methylphenoxy]acetate
(2a). A mixture of 1a (5 g, 0.02 mol) and ethyl chloroac-
etate (2.4 g, 0.02 mol) in dry acetone (60 mL) and anhy-
drous potassium carbonate (2.8 g, 0.02 mol) was
refluxed for 8 h. Subsequently, the reaction mixture
was cooled and the solvent was removed under reduced
pressure. The residual mass was triturated with ice water
to remove potassium carbonate and extracted with ether
(3 · 50 mL). The ether layer was washed with 10% sodi-
um hydroxide solution (3 · 30 mL), followed by water
(3 · 30 mL), and then dried over anhydrous sodium sul-
fate and evaporated to dryness to yield a crude solid.
Recrystallization with ethanol gave 2a in 80% yield.
Compounds 2b–e were synthesized analogously starting
with 1b–e, respectively.
Figure 9. Western blot analysis of CAD. Nuclear extract from
untreated and compounds 4a–e treated EAT cells in vivo was resolved
on 10% SDS–PAGE. Western blot analysis was performed for CAD
using anti-CAD antibodies. Lane 1, control and lanes 2–6, compounds
4a–e treated.
compound 4a–e treatment. Caspase-activated DNase is
associated with inhibitor of CAD (ICAD) in the cytosol.
Upon caspase activation by external stimuli, CAD dis-
sociates from ICAD and translocates to nucleus to dis-
rupt the genetic material.56 Treatment of EAT cells
with compounds 4a–e shows that the activation of
CAD is a downstream target for caspase-3 in EAT cells.
These results indicate the apoptotic signaling pathway
of compounds 4a–e in EAT cells.
Compound 2a: 80%; mp 60–62 ꢁC; IR (Nujol): 1670
1
(C@O), 1735 cmÀ1 (ester C@O); H NMR (CDCl3): d
1.2 (t, J = 7 Hz, 3H, CH3 of ester), 2.3 (s, 3H, CH3),
4.2 (q, J = 6 Hz, 2H, CH2 of ester), 4.45 (s, 2H,
OCH2), 7.2–7.6 (m, 7H, Ar-H); EI-MS: m/z 332.5
(M+, 62), 259.5 (12), 245.5 (12), 221 (100), 193 (06),
139.5 (39), 111.5 (34). Anal. Calcd for C18H17ClO4
Taking together our data compound 4e has very potent
anti-tumor activity that inhibits growth of EAT cells.
However, the compounds 4a–d also showed significant

442
B. T. Prabhakar et al. / Bioorg. Med. Chem. 14 (2006) 435–446
R2
R2
R3
R1
R3
R1
NaOH/H2O
C2H5OH
O
ClCH2COOC2H5
K2CO3/Acetone
O
O
O
OH
H3C
O
H3C
2a-e
1a-e
R2
R2
R3
R1
O
R3
R1
O
p-ClC6H5NH2
O
BF3 etherate
Dry C6H6
NH
Cl
O
O
O
H3C
H3C
OH
4a-e
3a-e
a: R1=R3=H, R2=Cl
b: R1=R2=R3=H
c: R2=R3=H, R1=Br
d: R2=R3=H, R1=Cl
e: R1= R2=H, R3=OCH3
Scheme 1.
(332.5): C, 64.96; H, 5.11; Cl, 10.67. Found: C, 64.94; H,
5.07; Cl, 10.64%.
C19H20O5 (328): C, 69.51; H, 6.09. Found: C, 69.49;
H, 6.05%.
Compound 2b: 79%; mp 61–63 ꢁC; IR (Nujol): 1664
4.1.2.
[2-(3-Chlorobenzoyl)-4-methylphenoxy]ethanoic
1
(C@O), 1760 cmÀ1 (ester C@O); H NMR (CDCl3): d
acid (3a). Compound 2a (2.0 g, 6.0 mmol) was dissolved
in ethanol (10 mL) and treated with a solution of sodi-
um hydroxide (0.6 g, 15 mmol) in water (10 mL). The
mixture was heated under refluxed for 3 h, cooled, and
acidified with 1 N hydrochloric acid. The oily precipitate
was extracted with dichloromethane (3 · 30 mL) and the
solution was washed with water (3 · 25), dried, and
evaporated to give oil. Crystallization from hexane
afforded 3a as a white solid in 75% yield. Compounds
3b–e were synthesized analogously starting with 2b–e,
respectively.
1.2 (t, J = 7 Hz, 3H, CH3 of ester), 2.3 (s, 3H, CH3),
4.1 (q, J = 6 Hz, 2H, CH2 of ester), 4.5 (s, 2H, OCH2),
7.1–7.7 (m, 8H, Ar-H); ms: m/z 298 (M+, 60), 225 (10),
221(100), 211 (10), 193 (05), 105 (36), 77 (31). Anal.
Calcd for C18H18O4 (298): C, 72.48; H, 6.04. Found:
C, 72.46; H, 6.02%.
Compound 2c: 71%; mp 65–67 ꢁC; IR (Nujol): 1665
1
(C@O), 1730 cmÀ1 (ester C@O); H NMR (CDCl3): d
1.21 (t, J = 7 Hz, 3H, CH3 of ester), 2.3 (s, 3H, CH3),
4.22 (q, J = 6 Hz, 2H, CH2 of ester), 4.46 (s, 2H,
OCH2), 7.2–7.6 (m, 7H, Ar-H); EI-MS: m/z 377 (M+,
61), 304 (12), 290 (12), 221 (100), 193 (06), 184 (39),
156 (33). Anal. Calcd for C18H17BrO4 (377): C, 57.29;
H, 4.50; Br, 21.22. Found: C, 57.26; H, 4.53; Br, 21.25%.
Compound 3a: 75%; mp 120–122 ꢁC; IR (Nujol): 1675
(C@O), 1730 (acid C@O), 3400–3500 cmÀ1 (acid OH);
1H NMR (CDCl3): d 2.3 (s, 3H, CH3), 4.46 (s, 2H,
OCH2), 7.2–7.7 (m, 7H, Ar-H), 9.5 (s, 1H, COOH);
MS: m/z 304.5 (M+, 60), 260 (100), 245 (75), 193 (50),
165 (25), 139 (10), 111 (18). Anal. Calcd for C16H13ClO4
(304.5): C, 63.06; H, 4.30; Cl, 11.63. Found: C, 63.04; H,
4.26; Cl, 11.60%.
Compound 2d: 79%; mp 52–54 ꢁC; IR (Nujol): 1672
1
(C@O), 1737 cmÀ1 (ester C@O); H NMR (CDCl3): d
1.21 (t, J = 7 Hz, 3H, CH3 of ester), 2.31 (s, 3H, CH3),
4.21 (q, J = 6 Hz, 2H, CH2 of ester), 4.46 (s, 2H,
OCH2), 7.25–7.7 (m, 7H, Ar-H); EI-MS: m/z 332.5
(M+, 61), 259.5 (11), 245.5 (11), 221 (100), 193 (06),
139.5 (38), 111.5 (33). Anal. Calcd for C18H17ClO4
(332.5): C, 64.96; H, 5.11; Cl, 10.67. Found: C, 64.99;
H, 5.07; Cl, 10.63%.
Compound 3b: 75%; mp 112–115 ꢁC; IR (Nujol): 1655
(C@O), 1733 (acid C@O), 3450–3540 cmÀ1 (acid OH);
1H NMR (CDCl3): d 2.2 (s, 3H, CH3), 4.44 (s, 2H,
OCH2), 6.9–7.55 (m, 8H, Ar-H), 9.2 (s, 1H, COOH);
EI-MS: m/z 270 (M+, 58), 226 (100), 211 (72), 193
(48), 165 (23), 105 (09), 77 (15). Anal. Calcd for
C16H14O4 (270): C, 71.11; H, 5.11. Found: C, 71.13;
H, 5.08%.
Compound 2e: 72%; mp 58–60 ꢁC; IR (Nujol): 1660
1
(C@O), 1730 cmÀ1 (ester C@O); H NMR (CDCl3): d
1.2 (t, J = 7 Hz, 3H, CH3 of ester), 2.25 (s, 3H, CH3),
3.8 (s, 3H, OCH3), 4.2 (q, J = 6 Hz, 2H, CH2 of ester),
4.42 (s, 2H, OCH2), 7.0 (d, J = 8.3 Hz, 2H, Ar-H),
7.15–7.41 (m, 3H, Ar-H), 7.49 (d, J = 8.5 Hz, 2H, Ar-
H); EI-MS: m/z 328 (M+, 59), 255 (09), 241 (09), 221
(100), 193 (05), 135 (35), 107 (29). Anal. Calcd for
Compound 3c: 74%; mp 125–127 ꢁC; IR (Nujol): 1670
(C@O), 1735 (acid C@O), 3410–3510 cmÀ1 (acid OH);
1H NMR (CDCl3): d 2.3 (s, 3H, CH3), 4.45 (s, 2H,
OCH2), 7.1–7.6 (m, 7H, Ar-H), 9.4 (s, 1H, COOH);
EI-MS: m/z 349 (M+, 58), 305 (100), 290 (71), 193

B. T. Prabhakar et al. / Bioorg. Med. Chem. 14 (2006) 435–446
443
(45), 184 (09), 165 (25), 156 (18). Anal. Calcd for
C16H13BrO4 (349): C, 55.01; H, 3.72; Br, 22.92. Found:
C, 55.04; H, 3.75; Br, 22.94%.
Compound 4c: 72%; mp 112–114 ꢁC; IR (Nujol): 1668
(C@O), 1700 (C@O of amide), 3401 cmÀ1 (NH); 1H
NMR (CDCl3): d 2.21 (s, 3H, CH3), 4.41 (s, 2H,
OCH2), 4.75 (br s, 1H, CONH), 6.88–7.25 (m, 7H, Ar-
H), 7.25 (d, J = 8.55 Hz, 2H, Ar-H), 7.59 (d,
J = 8.6 Hz, 2H, Ar-H); EI-MS: m/z 459.5 (M++1, 10),
407 (11), 302.5 (06), 290 (15), 184 (22), 167.5 (100),
156 (24), 111.5 (25). Anal. Calcd for C22H17BrClNO3
(458.5): C, 57.57; H, 3.92; Br, 17.44; Cl, 7.74; N, 3.05.
Found: C, 57.54; H, 3.95; Br, 17.41; Cl, 7.77; N, 3.09%.
Compound 3d: 69%; mp 125–127 ꢁC; IR (Nujol): 1655
(C@O), 1730 (acid C@O), 3410–3555 cmÀ1 (acid OH);
1H NMR (CDCl3): d 2.2 (s, 3H, CH3), 4.45 (s, 2H,
OCH2), 6.9–7.6 (m, 7H, Ar-H), 9.3 (s, 1H, COOH);
EI-MS: m/z 304.5 (M+, 58), 260 (100), 245 (76), 193
(51), 165 (26), 139 (11), 111 (19). Anal. Calcd for
C16H13ClO4 (304.5): C, 63.06; H, 4.30; Cl, 11.63. Found:
C, 63.09; H, 4.27; Cl, 11.65%.
Compound 4d: 72%; mp 96–98 ꢁC; IR (Nujol): 1675
(C@O), 1710 (amide C@O), 3410 cmÀ1 (NH); 1H
NMR (CDCl3): d 2.25 (s, 3H, CH3), 4.46 (s, 2H,
OCH2), 4.85 (br s, 1H, CONH), 6.92–7.28 (m, 7H, Ar-
H), 7.32 (d, J = 8.55 Hz, 2H, Ar-H), 7.63 (d,
J = 8.6 Hz, 2H, Ar-H); EI-MS: m/z 415 (M++1, 09),
397 (10), 302.5 (05), 245.5 (14), 167.5 (100), 139.5 (21),
111.5 (23). Anal. Calcd for C22H17Cl2NO3 (414): C,
63.76; H, 4.10; Cl, 17.14; N, 3.38. Found: C, 63.80; H,
4.15; Cl, 17.01; N, 3.40%.
Compound 3e: 70%; mp 130–132 ꢁC; IR (Nujol): 1660
(C@O), 1738 (acid C@O), 3470–3575 cmÀ1 (acid OH);
1H NMR (CDCl3): d 2.25 (s, 3H, CH3), 3.8 (s, 3H,
OCH3), 4.42 (s, 2H, OCH2), 7.0 (d, J = 8.3 Hz, 2H,
Ar-H), 7.15–7.45 (m, 3H, Ar-H), 7.5 (d, J = 8.5 Hz,
2H, Ar-H), 9.1 (s, 1H, COOH); EI-MS: m/z 300 (M+,
55), 256 (100), 241 (70), 193 (45), 165 (21), 135 (08),
107 (14). Anal. Calcd for C17H16O5 (300): C, 68.0; H,
5.33. Found: C, 68.03; H, 5.35%.
Compound 4e: 72%; mp 119–121 ꢁC; IR (Nujol): 1665
(C@O), 1710 (C@O of amide), 3410 cmÀ1 (NH); 1H
NMR (CDCl3): d 2.32 (s, 3H, CH3), 3.78 (s, 3H,
OCH3), 4.39 (s, 2H, OCH2), 4.79 (br s, 1H, CONH),
6.87 (d, J = 8.3 Hz, 2H, Ar-H), 6.9–7.21 (m, 3H, Ar-
H), 7.25 (d, J = 8.55 Hz, 2H, Ar-H), 7.58 (d,
J = 8.6 Hz, 2H, Ar-H), 7.59 (d, J = 8.5 Hz, 2H, Ar-H);
EI-MS: m/z 410.5 (M++1, 09), 392.5 (10), 302.5 (06),
241 (05), 167.5 (100), 135 (22), 111.5 (25), 107 (17). Anal.
Calcd for C23H20ClNO4 (409.5): C, 67.39; H, 4.88; Cl,
8.66; N, 3.41. Found: C, 67.43; H, 4.91; Cl, 8.63; N,
3.44%.
4.1.3. [2-(3-Chlorobenzoyl)-4-methylphenoxy]-N-(4-chlor-
ophenyl)-acetamide (4a). A mixture of 3a (1.0 g,
3.2 mmol), p-chloroaniline (0.51 g, 4.0 mmol), and bor-
on trifluoride etherate (0.83 g, 6.0 mmol) in dry ben-
zene (15 mL) was refluxed for 6 h. The refluxing
solvent was dried by circulation over anhydrous sodi-
um sulfate in a SoxhletÕs apparatus. The reaction mix-
ture was washed with 10% aqueous sodium hydroxide
(3 · 20 mL) and 10% hydrochloric acid (3 · 20 mL),
and then with water (3 · 25 mL). After being dried
with anhydrous sodium sulfate, the organic layer was
concentrated under reduced pressure. The amide was
purified by crystallization from ethanol. Further purifi-
cation was carried out by chromatography on silica gel
using hexane and ethanol (7:2) as eluent to give 4a as a
white solid in 70% yield. Compounds 4b–e were syn-
thesized by analogous procedures using 3b–e,
respectively.
4.2. Biology
Swiss albino mice were obtained from Central Animal
Facility, Department of Zoology, University of Mysore,
Mysore, India. MTT assay kit was procured from
Merck. Annexin-V apoptosis detection kit was procured
from Sigma, USA. ACDEVD-CHO caspase-3 inhibitor,
anti-p53, and anti-caspase DNase antibodies were pro-
cured from Santa Cruz Biotechnology, USA. All other
reagents were of highest analytical grade.
Compound 4a: 72%; mp 127–129 ꢁC; IR (Nujol): 1670
(C@O), 1705 (amide C@O), 3405 cmÀ1 (NH); 1H
NMR (CDCl3): d 2.23 (s, 3H, CH3), 4.45 (s, 2H,
OCH2), 4.82 (br s, 1H, CONH), 6.9–7.26 (m, 7H, Ar-
H), 7.3 (d, J = 8.55 Hz, 2H, Ar-H), 7.61 (d, J = 8.6
Hz, 2H, Ar-H); EI-MS: m/z 415 (M++1, 10), 397 (11),
302.5 (06), 245.5 (15), 167.5 (100), 139.5 (22), 111.5
(24). Anal. Calcd for C22H17Cl2NO3 (414): C, 63.76;
H, 4.10; Cl, 17.14; N, 3.38. Found: C, 63.73; H, 4.14;
Cl, 17.11; N, 3.40%.
4.2.1. Cell culture and in vitro compound 4a–e treatment.
EAT cells were grown in NCTC-135 medium with 10%
FCS and penicillin/streptomycin (1 mg/mL). After the
cells were 80% confluent, they were treated with various
concentrations of compounds 4a–e (0–100 lM) for var-
ious time periods (0–24 h). Then, the cells were trypsin-
ized and collected before being used for further
experiments.
Compound 4b: 71%; mp 105–107 ꢁC; IR (Nujol): 1665
(C@O), 1701 (C@O of amide), 3402 cmÀ1 (NH); 1H
NMR (CDCl3): d 2.2 (s, 3H, CH3), 4.4 (s, 2H, OCH2),
4.72 (br s, 1H, CONH), 6.8–7.2 (m, 8H, Ar-H), 7.22
(d, J = 8.55 Hz, 2H, Ar-H), 7.52 (d, J = 8.6 Hz, 2H,
Ar-H); EI-MS: m/z 379.5 (M++1, 10), 361.5 (11), 302.5
(06), 210.5 (15), 167.5 (100), 105 (22), 77 (25). Anal.
Calcd for C22H18ClNO3 (379.5): C, 69.57; H, 4.78; Cl,
9.33; N, 3.69. Found: C, 69.54; H, 4.75; Cl, 9.36; N,
3.64%.
4.2.2. Cytotoxicity assay. In vitro growth inhibitory ef-
fect of compounds 4a–e on EAT cells was determined
by measuring the absorbance of 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazoliumbromide (MTT, MERCK)
dye for living cells as per the manufacturerÕs instruc-
tions. Briefly, EAT cells were seeded in 96-well microti-
ter plates containing NCTC medium. After exposure to
compounds 4a–e for 24 h in a dose-dependent way for

444
B. T. Prabhakar et al. / Bioorg. Med. Chem. 14 (2006) 435–446
various time periods (0–24 h), 50 lL of MTT solution
(2 mg/mL in PBS) was added to each well, and plates
were incubated for an additional 4 h at 37 ꢁC. The
MTT solution in medium was then aspirated off. To dis-
solve the formazan crystals formed in viable cells,
200 lL DMSO was added to each well before measuring
the absorbance at 570 nm.
ar DNA was labeled with propidium iodide (PI) using
Cycle TEST PLUS DNA reagent kit. Cell cycle-phase
distribution of nuclear DNA was determined on FACS,
fluorescence detector equipped with 488 nm argon laser
light source and 623 nm band pass filter (linear scale)
using CellQuest software (Becton–Dickinson). A total
of 10,000 events were acquired and analysis of flowcy-
tometric data was performed using ModFit software.
A histogram of DNA content (x-axis, PI-fluorescence)
versus counts (y-axis) has been displayed.
4.2.3. Animals and in vivo tumor growth. Six- to eight-
week-old female mice were acclimated for 1 week while
caged in groups of five. Mice were housed and fed a diet
of animal chow and water ad libitum throughout the
experiment. All experiments were approved by the Insti-
tutional Animal Care and Use Committee of the Univer-
sity of Mysore, Mysore, India. The EAT cell lines were
maintained by in vivo passage. Swiss albino mice bear-
ing EAT were sacrificed and the tumors were resected
under aseptic conditions. A suspension of the resected
tumor cell was made in 0.9% normal saline. Approxi-
mately 5 · 106 cells/mouse (300 lL of suspension) were
injected into dorsa of the mice (weighing 30 g) intra-
peritoneally. These cells grow in mice peritoneum form-
ing an ascites tumor with massive abdominal swelling.
The animals showed a dramatic increase in the body
weight over the growth period and the animals suc-
cumbed to the tumor burden 14–16 days after implanta-
tion. The number of cells increased over the 10 days of
growth with formation of 6–7 mL of ascites fluid.
4.2.7. Trypan blue assay. EAT cells were treated with dif-
ferent concentrations of compounds 4a–e for various
time periods (0–8 h). Cell viability was assessed by mix-
ing aliquots of cell suspension with 0.4% trypan blue.
Cells that picked up the dye were considered to be dead.
4.2.8. Acridine orange/ethidium bromide staining. Nucle-
ar staining was performed according to the method of
Srinivas et al.57 EAT cells either treated or untreated
with compounds 4a–e in vivo were smeared on a glass
slide and fixed with methanol/acetic acid (3:1), and air-
dried. The cells were hydrated with PBS and stained
with a mixture (1:1) of acridine orange/ethidium bro-
mide (4 lg/mL) solutions. The cells were immediately
washed with PBS and viewed under a Leitz-DIAPLAN
fluorescent microscope.
4.2.9. Annexin-V staining. EAT cells treated with and
without compounds 4a–e in vivo were harvested and
washed with PBS, dropped onto the slide, and air-dried.
Cells were washed with binding buffer (10 mM Hepes,
pH 7.5, containing 140 mM NaCl and 2.5 mM CaCl2)
and stained with Annexin-V detection kit as per the pro-
tocol supplied by the manufacturer. After 10 min cells
were washed thrice with binding buffer. The greenish
red apoptotic cells were observed using a Leitz-DIA-
PLAN fluorescent microscope and photographed.
4.2.4. In vivo compound 4a–e treatment. To determine
whether compounds 4a–e inhibits tumor growth in vivo,
compounds 4a–e (100 mg/kg body weight/ip) were inject-
ed into the EAT-bearing mice intraperitoneally using a 26
gauge needle on every alternate day starting 6 days of tu-
mor implantation and growth of the tumor was moni-
tored by taking the body weight of the animals every
day. Control mice were injected with 0.2 mL saline (ip)
on every alternate day. Each treatment consisted of at
least five mice and each experiment was repeated thrice.
After giving each dose (compounds + 200 lL of 0.9% sal-
ine), animals were sacrificed and the EAT cells along with
ascites fluid were harvested. The set of animals was used
to study the survivality after treatment until their death.
4.2.10. DNA isolation. EAT cells were collected from
mice treated with or without compounds 4a–e in vivo
and DNA was isolated using the phenol–chloroform
method. In brief, cells were lysed in a buffer containing
50 mM Tris–HCl, pH 8.0, and 0.5% SDS, and incubated
for 30 min at 37 ꢁC. The cell lysate was subjected to 8 M
potassium acetate precipitation and left for 1 h at 4 ꢁC.
The supernatant was subjected to phenol/chloroform/
isoamyl alcohol (25:24:1) extraction and once to chloro-
form extraction. DNA was precipitated by adding 1:2
volumes of ice-cold ethanol. The precipitated DNA
was dissolved in 50 lL TE buffer (pH 8.0). The DNA
was digested with 20 lg/mL RNase at 37 ꢁC for 1 h.
The DNA was quantitated and equal concentration of
DNA (50 lg) was resolved on 1.5% agarose gel, viewed
under UV light, and documented using Uvp-BioDoc-
ItTM system.
4.2.5. Isolation of EAT cells from mice peritoneal cavity.
The EAT cells were isolated from the peritoneal cavity
of tumor-bearing mice (control or treated). Two to three
milliliters of sterile PBS was injected into the peritoneal
cavity of the mice and the peritoneal fluid containing the
tumor cells was withdrawn, collected in sterile petri dish-
es, and incubated at 37 ꢁC for 2 h. The cells of macro-
phage lineage adhered to the bottom of the petri
dishes. The non-adherent population was aspirated out
gently and washed repeatedly with PBS. EAT cells were
then separated from other non-adherent contaminating
cells by fluorescence-activated cell sorter (FACS). More-
over, viability of these cells was assessed and was found
to be >95% by trypan blue dye exclusion. The viable
EAT cells were processed for further experiments.
4.2.11. Caspase-3 inhibition assay. EAT cells were har-
vested and washed with PBS and suspended in HBSS
buffer, pH 7.4. The cells were preincubated with or with-
out caspase-3 inhibitor AC-DEVD CHO (100 lM) at
37 ꢁC for 1 h. Subsequently, the cells were treated with
compounds 4a–e and incubated for 2 h at 37 ꢁC. DNA
4.2.6. Detection of apoptosis by flowcytometry. For the
determination of cell cycle-phase distribution, EAT cells
from tumor-bearing mice were permeabilized and nucle-

B. T. Prabhakar et al. / Bioorg. Med. Chem. 14 (2006) 435–446
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The authors express their sincere gratitude to the Uni-
versity of Mysore, Mysore, for the laboratory facility.
P.B.T. is indebted to the Department of Atomic Energy,
Government of India, Mumbai, for the award of project
fellow and S.A.K. is indebted to the UGC for the award
of teacherÕs fellowship.
33. Hou, A.; Fukai, T.; Shimazaki, M.; Sakgami, H.; Sun, H.;
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