Improved synthesis of histone deacetylase inhibitors (HDIs) (MS-275 and CI-994) and inhibitory effects of HDIs alone or in combination with RAMBAs or retinoids on growth of human LNCaP prostate cancer cells and tumor xenografts

Article abstract of DOI:10.1016/j.bmc.2007.12.007

We have developed new, simple, and efficient procedures for the synthesis of two promising histone deacetylase inhibitors (HDIs), CI-994, (N-(2-aminophenyl)-4-acetylaminobenzamide), and MS-275 (N-(2-aminophenyl)4-[N-(pyridine-3-yl-methoxycarbonyl)aminomethyl]benzamide) from commercially available acetamidobenzoic acid and 3-(hydroxymethyl)pyridine, respectively. The procedures provide CI-994 and MS-275 in 80% and 72% overall yields, respectively. We found that the combination of four HDIs (CI-994, MS-275, SAHA, and TSA) with retinoids all-trans-retinoic acid (ATRA) or 13-cis-retinoic acid (13-CRA) or our atypical retinoic acid metabolism blocking agents (RAMBAs) 1 (VN/14-1) or 2 (VN/66-1) produced synergistic anti-neoplastic activity on human LNCaP prostate cancer cells. The combination of 2 and SAHA induced G1 and G2/M cell cycle arrest and a decrease in the S phase in LNCaP cells. 2 + SAHA treatment effectively down-regulated cyclin D1 and cdk4, and up-regulated pro-differentiation markers cytokeratins 8/18 and pro-apoptotic Bad and Bax. Following subcutaneous administration, 2, SAHA or 2 + SAHA were well tolerated and caused significant suppression/regression of tumor growth compared with control. These results demonstrate that compound 2 and its combination with SAHA are potentially useful agents that warrant further preclinical development for treatment of prostate cancer.

Full text of DOI:10.1016/j.bmc.2007.12.007

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3352–3360
Improved synthesis of histone deacetylase inhibitors (HDIs)
(MS-275 and CI-994) and inhibitory effects of HDIs alone or
in combination with RAMBAs or retinoids on growth of human
LNCaP prostate cancer cells and tumor xenografts
Lalji K. Gediya,a Aashvini Belosay,a, Aakanksha Khandelwal,a
Puranik Purushottamachara and Vincent C. O. Njara,b,
*
aDepartment of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine,
685 West Baltimore Street, Baltimore, MD 21201-1559, USA
bThe University of Maryland Marlene and Stewart Greenebaum Cancer Center, School of Medicine, Baltimore, MD 21201-1559, USA
Received 20 October 2007; revised 1 December 2007; accepted 4 December 2007
Available online 8 December 2007
Abstract—We have developed new, simple, and efficient procedures for the synthesis of two promising histone deacetylase inhibitors
(HDIs), CI-994, (N-(2-aminophenyl)-4-acetylaminobenzamide), and MS-275 (N-(2-aminophenyl)4-[N-(pyridine-3-yl-methoxycar-
bonyl)aminomethyl]benzamide) from commercially available acetamidobenzoic acid and 3-(hydroxymethyl)pyridine, respectively.
The procedures provide CI-994 and MS-275 in 80% and 72% overall yields, respectively. We found that the combination of four
HDIs (CI-994, MS-275, SAHA, and TSA) with retinoids all-trans-retinoic acid (ATRA) or 13-cis-retinoic acid (13-CRA) or our
atypical retinoic acid metabolism blocking agents (RAMBAs) 1 (VN/14-1) or 2 (VN/66-1) produced synergistic anti-neoplastic activ-
ity on human LNCaP prostate cancer cells. The combination of 2 and SAHA induced G1 and G2/M cell cycle arrest and a decrease
in the S phase in LNCaP cells. 2 + SAHA treatment effectively down-regulated cyclin D1 and cdk4, and up-regulated pro-differen-
tiation markers cytokeratins 8/18 and pro-apoptotic Bad and Bax. Following subcutaneous administration, 2, SAHA or 2 + SAHA
were well tolerated and caused significant suppression/regression of tumor growth compared with control. These results demonstrate
that compound 2 and its combination with SAHA are potentially useful agents that warrant further preclinical development for
treatment of prostate cancer.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
thus, anti-hormonal therapy by surgical or medical sup-
pression of androgen action remains a major treatment
option of the disease.2 Although this treatment may be
initially successful, most tumors eventually recur due
to the expansion of an androgen-refractory population
of PCA cells.3 Metastatic disease that develops even
after potentially curative surgery remains a major clini-
cal challenge. Therapeutic treatments for patients with
metastatic PCA are limited because current chemother-
apeutic and radiotherapeutic regimens are largely inef-
fective.4 Hence, there is urgent need to develop new
therapeutic agents with defined targets to prevent and
treat this disease.
Prostate cancer (PCA) is the most common malignancy
and age-related cause of cancer death worldwide. Apart
from lung cancer, PCA is the most common form of
cancer in men and the second leading cause of death
in American men. In the United States in 2007, an esti-
mated 218,890 new cases of prostate cancer will be diag-
nosed and about 27,050 men will die of this disease.1
The growth of most prostate tumors depends on andro-
gens during the initial stages of tumor development, and
Keywords: Retinoids; RAMBAs; HDIs; Prostate cancer; Anticancer
agents.
PCA tumors that arise after anti-hormonal therapy gen-
erally are less differentiated and it is believed that agents
that can induce the cells to differentiate would represent
a new therapeutic strategy.5 Hence, the goal of differen-
tiation therapy is to induce malignant cells to pass the
*
Corresponding author. Tel.: +1 410 706 5885; fax: +1 410 706
0032; e-mail: vnjar001@umaryland.edu
Present address: Department of Food Science and Human Nutrition,
University of Illinois, 905 South Goodwin Avenue, Urbana-Cham-
paign, IL 61801, USA.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.007

L. K. Gediya et al. / Bioorg. Med. Chem. 16 (2008) 3352–3360
3353
block to maturation by allowing them to progress to
more differentiated cell types with less proliferative abil-
ity. Breslow and colleagues6 have led the way in the dis-
covery of agents that inhibit the enzyme histone
deacetylase (HDAC), thereby altering chromatin struc-
ture and changing gene expression patterns. Histone
deacetylase inhibitors (HDIs) are potent differentiating
agents toward a variety of neoplasms, including leuke-
mia, and breast and prostate cancers. Combinations of
HDIs with other known therapies including retinoic
acids (RAs) have been investigated. RAs exert their ef-
fects via a nuclear receptor complex that interacts with
promoters of RA-responsive genes.7 An HDAC subunit
is an integral part of this co-repressor complex, which is
involved in transcriptional silencing in the absence of li-
gand.8 This association provides a rationale for combin-
ing HDIs and RAs/retinoids therapeutically. One of the
early HDIs discovered by Breslow and colleagues is N-
hydroxy-N1-phenylactanediamide, also called suberoy-
lanilide hydroxamic acid (SAHA).9,10 This compound
(trade name: Vorinostatꢁ) was recently (2006) approved
by the U.S. Food and Drug Administration (FDA) for
the treatment of advanced cutaneous T-cell-
lymphoma.11
sponding acid chloride that was coupled with 2-nitroan-
iline in situ to afford N-(21-nitrophenyl)-4-
acetylaminobenzamide in 20.0% yield. This was then
hydrogenated in THF using 10% palladium on activated
charcoal to produce CI-994 (3) in 69.0% yield. The over-
all yield was only 13.8%. Apart from the low yield, this
method is tedious and requires special hydrogenation
conditions.
We have developed a simple and efficient one-step proce-
dure for the synthesis of CI-994 (Scheme 2). The readily
available acetamidobenzoic acid (6) was converted into
its imidazolide derivative by reaction with N, N1-car-
bonyldiimidazole (CDI) in THF at room temperature.
This was further reacted with 1,2-phenylenediamine in
the presence of TFA to obtain CI-994 (3) in 85.0% yield.
The product was recrystallized from THF/methanol to
give pure CI-994 (2) in 80.0% yield.
Another promising HDI N-(2-aminophenyl)4-[N-(pyri-
dine-3-yl-methoxycarbonyl)aminomethyl]benzamide
(MS-275, 4) is currently in several phase I/II clinical tri-
als for various solid tumors and hematological malig-
nancies.21 MS-275 was previously synthesized by
Suzuki et al. via a three-step procedure in 50.96% overall
yield (Scheme 3).22 In addition to the modest overall
yield, this procedure has other disadvantages such as a
tedious method for the preparation of an acid chloride
using oxalyl chloride and also it requires the use of col-
umn chromatography for purification of MS-275.
Recently, we reported on a family of compounds that in-
hibit the P450 enzyme(s) responsible for the metabolism
of all-trans-retinoic acid (ATRA).12 These compounds
also referred to as retinoic acid metabolism blocking
agents (RAMBAs) are able to enhance the antiprolifer-
ative effects of ATRA in breast and prostate cancer cells
in vitro.13 In addition, the RAMBAs were shown to in-
duce differentiation and apoptosis in these cancer cell
lines. However, we also observed that the breast cancer
cell lines were exquisitely more sensitive to the RAM-
BAs.14,15 We also reported recently that combination
of SAHA with either retinoids or RAMBAs resulted
in additive/synergistic PCA (LNCaP and PC-3 cell lines)
growth inhibition in vitro.16
Here again, we have developed a new two-step proce-
dure for preparation of MS-275 as outlined in Scheme
4. Condensation of 3-(hydroxymethyl)pyridine (7) and
4-(aminomethyl)benzoic in the presence of CDI gave
4-[N-(pyridin-3-yl-methoxycarbonyl) aminomethyl]ben-
zoic acid (8) in 91.0% yield. In the previous method
of Suzuki et al., the carboxylic acid derivative 8 was
first converted into acyl chloride hydrochloride by
treatment of oxalyl chloride in toluene and then re-
acted with imidazole to form the acylimidazole inter-
mediate.22 However, we synthesized the imidazolide
of intermediate 8 by treatment with CDI at 55–60 ꢂC
in THF. The imidazolide was then further reacted
in situ with 1,2-phenylenediamine in the presence of
TFA at room temperature to afford MS-275 (4). Fur-
thermore, we developed a simpler process for large
scale purification of crude MS-275 instead of using
conventional column chromatography. Thus, after
the completion of reaction, the solvent was evaporated
and to the concentrate we added the mixture of hex-
ane and water (2:5, v/v) and stirred for 1 h. The result-
ing precipitate was filtered, washed with hexane and
dried. The crude product was further stirred twice in
dichloromethane to remove excess of 1,2-phenylenedi-
amine, filtered, and washed with hexane to give pure
MS-275 in 80.0% yield (>99% as determined by
HPLC). The overall yield for our simple and efficient
production of MS-275 (4) was 72.8%. Finally, the
methods described here for the synthesis of CI-994
and MS-275 are green chemistry methods because of
the greatly increased yields and fewer number of reac-
tion steps.
In continuation of our research in this area, we have dis-
covered improved syntheses of two promising HDIs,17–19
CI-994, (N-(2-aminophenyl)-4-acetylaminobenzamide),
and MS-275 (N-(2-aminophenyl)4-[N-(pyridine-3-yl-
methoxycarbonyl)aminomethyl]benzamide). Further-
more, we assessed the effects of our novel RAMBAs
and retinoids (see Chart 1) in combination with some
HDIs in human prostate cancer model systems in vitro
and in vivo. The molecular effects of compound
2 + SAHA in prostate cancer cells include inhibition of
proliferation, regulation of cell cycle, and induction of
differentiation and apoptosis.
2. Results and discussion
2.1. Chemistry
To the best of our knowledge only one method has been
reported for synthesis of CI-994. This synthesis of CI-
994 by Weiershausen et al.20 is outlined in Scheme 1.
The three-step procedure involves the reaction of oxalyl
chloride with 4-acetamidobenzoic acid to give the corre-

3354
L. K. Gediya et al. / Bioorg. Med. Chem. 16 (2008) 3352–3360
O
O
OH
OH
O
H
N
ATRA
13-CRA
VN/14-1 (1)
N
OH
H
O
N
H
NH
2
N
H
O
N
O
N
VN/66-1 (2)
CI-994 (3)
N
O
H
O
N
H
NH
2
O
N
H
NHOH
N
O
N
O
MS-275 (4)
SAHA (5)
Chart 1. Structures of retinoids, RAMBAs, and HDIs.
NO
2
O
H N
2
H
Cl
H
N
Cl
N
H
N
NO
2
O
O
O
OH
EtOAc, pyridine
(20.0%)
0-5 ºC, DMF
O
O
10% Pd/C,
H2, THF
(69.0%)
H
N
H
N
NH
2
[Overall yield: 0.20 x 0.69 x 100 = 13.8%]
O
O
CI-994
Scheme 1. Previous procedure for synthesis of CI-994 (3).
H
H
N
i. CDI, THF, rt, 1h
N
H
N
NH
2
O
OH
O
NH
2
TFA, rt, 16 h
ii. H N
2
O
O
CI-994, (3)
[Yield = 80.0%]
6
Scheme 2. New synthesis of CI-994 (3).
2.2. Biological studies
of 0.36, 1.0, 7.4, and 5.5 lM, respectively. In contrast,
the retinoids, ATRA and 13-CRA, or RAMBA 1 were
less efficacious, since each of the compounds did not sig-
nificantly inhibit cell growth even at concentrations as
high as 10 lM. However, the combinations of retinoids
or RAMBAs with the HDIs caused dramatic, synergistic
inhibitory effects as shown in Figure 2a–e. Of signifi-
cance is our observation that the combination of excep-
tionally low doses of ATRA (0.1 nM) or 1 (0.1 nM) or 2
(0.1 nM) with MS-275 (0.1 nM) resulted in a growth
inhibition of 65.0%, 67.0%, and 70.0%, respectively
(Fig. 2c–e). As we reported previously,16 treatment with
2.2.1. Effects of retinoids or RAMBAs alone or in
combination with HDIs on LNCaP cell proliferation.
We first studied the effects of retinoids, RAMBAs, and
HDIs as single agents on LNCaP cell viability using
the MTT assay and the IC50 values were determined
from dose–response curves as shown for MS-275
(Fig. 1). The growth inhibitory experiments with the
other compounds gave plots that were essentially the
same as in Figure 1. The HDIs, MS-275, SAHA, and
CI-994, and RAMBA 2 were efficacious with IC50 values

L. K. Gediya et al. / Bioorg. Med. Chem. 16 (2008) 3352–3360
3355
O
O
PhCH3, DMF, rt, 4h
CDI, THF, rt, 1h
DBU, Et3N,
OH
O
N
H
O
N
H
OH
Cl
O
N
N
N
Cl
Cl
O
O
(used without purification)
H N
2
O
OH
O
(91.0%)
i. Imidazole, CDI
THF, rt, 1h
NH
2
(56.0%)
ii. TFA, rt, 15hH, 2N
O
O
N
H
H
N
NH
2
N
O
[Overall yield: 0.91 x 0.56 x 100 = 50.96%]
MS-275
Scheme 3. Previous procedure for synthesis of MS-275 (4).
O
CDI, THF, rt, 1h
DBU, Et3N,
OH
O
N
H
OH
N
N
O
7
8
H N
2
OH
i. CDI, THF, reflux, 1h
O
(91.0%)
NH
2
(80.0%)
ii. TFA, rt, 16hH, 2N
[Overall yield: 0.91 x 0.80 x 100 = 72.8 %]
O
O
N
H
H
N
NH
2
N
O
MS-275 (4)
Scheme 4. New synthesis of MS-275 (4).
90
80
70
60
50
40
30
20
10
0
2 (5.0 lM) + SAHA (1.0 lM) resulted in >95.0% growth
inhibition of LNCaP cells (data not shown). We also
found that trichostatin A (TSA), a first generation and
an experimental HDI, in combination with 2 caused a
synergistic inhibitory effect in LNCaP cells (data not
shown). It is pertinent to state here that although we
had previously found that the combination of
2 + SAHA resulted in additive growth inhibition, the ef-
fects observed in the present study with the various com-
binations are clearly synergistic (refer Fig. 2a–e). This
assertion is based on our findings that the inhibition of
cell growth by each of the combinations was greater
than the sums of each of the two compounds separately;
using the Valeriote and Lin analysis.23 These results
clearly show that HDIs can effectively enhance the
growth inhibitory activities of both retinoids and RAM-
BAs. It is well known that although the RAR/RXR
receptors interact with HDACs, the ligands that interact
with either of these proteins, i.e., the retinoids/RAMBAs
and HDIs, have other mechanisms of action, that are
-1
0
1
2
3
4
log MS-275 (nM)
Figure 1. Antiproliferative effect of MS-275. LNCaP cell proliferation
was measured after 6 days of treatment using a MTT assay as
described in Section 4. Data are means (SEM < 10%) of at least three
independent experiments. The experiments with the other compounds
gave dose–response plots that were essentially the same as shown
above.

3356
L. K. Gediya et al. / Bioorg. Med. Chem. 16 (2008) 3352–3360
120
120
100
80
60
40
20
0
a
*, p < 0.0002
b
*, p < 0.0002
100
80
60
40
20
0
*
*
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*, p < 0.0002
*, p < 0.0002
c
d
*
*
120
100
80
60
40
20
0
e
*, p < 0.0002
*
Figure 2. The effect of various HDACIs in combination with retinoids or RAMBAs on LNCaP cell viability as assessed by the MTT assay. (a) The
effect of ATRA, CI-994, and the combination of the two at fixed concentrations. (b) The effect of 13-cis RA and CI-994. (c) The effect of ATRA and
MS-275. (d) The effect of VN/14-1 and MS-275, (d) The effect of VN/66-1 and MS-275. (e) The effect of VN/66-1 and SAHA, *P < 0.0002 (t-test) for
all graphs. Data are means ( SE) of at least three independent experiments.
likely to cause synergistic cell growth inhibitory effects.
It should also be stated that retinoid-specific signaling
alone does not explain the differences in sensitivity of
different cell lines to the different retinoids.24 In addi-
tion, although the HDIs are noted to resensitize certain
genes that are silenced in cancer cells, thereby enhancing
the functional activity of RARs, they also show other
anticancer activities, such as cell cycle arrest and
apoptosis.25
A recent study by Faller and colleagues demonstrated
enhanced (synergistic) suppression of androgen sensitive
LNCaP and CWR22-rv1 cells when retinoids were com-
bined with several HDIs.26 However, Pili and colleagues

L. K. Gediya et al. / Bioorg. Med. Chem. 16 (2008) 3352–3360
3357
reported that phenylbutyrate in combination with 13-
CRA has an additive inhibitory effect in LNCaP cells
in vitro.27 These differences may be due to the pheno-
types/genotypes (number of passages in culture) of the
cell lines utilized.
Cytokeratins 8/18
Cyclin D1
2.2.2. Effects of 2 and SAHA on cell cycle and pro-
differentiation and pro-apoptotic proteins. Treatments of
2 (1 lM) + SAHA (10 nM) on LNCaP cells were used
to further investigate the mechanisms of action. We
elected to use SAHA in these studies because the agent
is currently in clinical use.11 The effects of 2, SAHA or
the combination of both agents on cell cycle profile of
LNCaP cells were examined by flow cytometry as we
have previously described.12,15,28 Compared to control,
2 and SAHA alone induced G1 cell cycle arrest, while
simultaneous treatment of both agents induced both
G1 and G2/M cell cycle arrest with concomitant and sig-
nificant decreases in percentage of cells in S phase (Table
1). The effects of these agents on protein expression of
G1 phase cell cycle regulatory proteins, differentiation,
and apoptosis were also examined in LNCaP cells by
Western blot analysis following treatment for 24 h. As
can be seen in Figure 3, treatment with 2 + SAHA
caused significant up-modulation of cytokeratins 8/18
(4.5-fold), Bad (2.4-fold), Bax (5.4-fold) and down-mod-
ulation of cyclin D1 (>10-fold) and cdk4 (10-fold).
Clearly, this treatment resulted in induction of differen-
tiation, apoptosis, and cell cycle arrest. A lack of cellular
differentiation, uncontrolled cell cycle progression, and
evasion of apoptosis are hallmarks of many human can-
cers.29,30 Therefore, therapeutic agents such as
2 + SAHA that can simultaneously impede cell cycle
progression and promote differentiation and apoptosis
in cancer cells are highly desirable. The cytostatic and
cytotoxic properties of 2 + SAHA in prostate cancer
indicate that the biological mechanisms of action of
these two agents are diverse, thus rendering them more
attractive for preclinical and clinical development for
prostate cancer therapy.
1
2.3
0.4
4.5
1
0.07
0.7
0.2
0.5
0
Cdk 4
1
0.1
Bad
Bax
1
1
1.5
1.9
2.4
5.4
2.1
2.8
β-actin
Figure 3. Western blot analysis for various proteins in LNCaP whole
cell lysates after a 24 h treatment with VN/66-1 (1.0 lM), SAHA
(10 nM) or their combination. Densitometry was carried out relative to
loading control (b-actin). Blots are representative of at least three
experiments.
day) or the combination of the two agents. As shown
in Figure 4, growth of tumors was significantly inhibited
with either 2 or SAHA as compared to control (81.6%
and 97.1% inhibition, respectively), and tumors in the
group treated with 2 + SAHA actually regressed by
22% over the duration of the experiment. In addition,
these treatments did not cause any overt toxicity as eval-
uated by weight gain. A recent study by Pili and col-
leagues27 reported that treatment of LNCaP tumor
xenografts with phenylbutyrate (PB) or 13-CRA re-
sulted in modest tumor growth inhibition that was gen-
erally not statistically significant as compared with
control, but that the combination of PB and 13-CRA re-
sulted in a significant additive inhibitory effect (up to
90% growth inhibition as compared with control). Given
the excellent efficacies of 2 or SAHA and their combina-
tion reported in this study, it seems probable that 2 or its
combination with SAHA may be more effective antican-
cer agents. In addition, the anti-tumor efficacies of 2 and
SAHA appear to be superior to the reported efficacy of
SAHA on related androgen-dependent CWR22 prostate
tumors.31 This is the first report of combination of a
RAMBA/retinoid with an HDI that causes LNCaP
tumor regression.
2.2.3. Inhibition of human LNCaP prostate cancer
xenografts by 2 and SAHA in SCID mice. To confirm
our in vitro findings, we evaluated the in vivo anticancer
efficacy of 2 and SAHA in a human LNCaP tumor
xenograft model. LNCaP xenografts were grown in
SCID mice and treated (subcutaneous administration)
with 2 (10 ! 20 mg/kg/day) or SAHA (20 ! 10 mg/kg/
Table 1. Cell cycle analysis of effects of VN/66-1, SAHA or their
combination on LNCaP cells
Cell cycle distribution in LNCaP cellsa
Treatment
G1 (%)
S (%)
G2/M (%)
Control
72.61
89.14
81.90
87.34
27.39
10.86
18.10
4.94
0
VN/66-1 (5 lM)
SAHA (1 lM)
VN/66-1 + SAHA
0
0
7.72
Percentage distribution of cells in each of the cell cycle phase is the
mean obtained from experiments performed in triplicate of at least two
independent experiments, SEM < 10%.
3. Conclusions
a Percent of cells in each phase of the cell cycle after an 18 h treatment
of LNCaP cells with either VN/66-1 (5.0 lM), SAHA (1.0 lM) or
VN/66-1 + SAHA (5.0 + 1.0 lM), respectively.
We have developed new methods that enabled us to syn-
thesize two promising HDIs, CI-994 and MS-275, in
excellent and improved overall yields. In addition, we

3358
L. K. Gediya et al. / Bioorg. Med. Chem. 16 (2008) 3352–3360
1200
1100
1000
900
800
700
600
500
400
300
200
100
0
Control
*, p < 0.005
**, p < 0.001
***, p < 0.0005
*
VN/66-1
**
SAHA
***
VN/66-1 + SAHA
0
5
10
15
20
25
30
Days
DAY 12- SAHA (10 mg/kg/day)
VN/66-1 (20 mg/kg/day)
Figure 4. The effect of VN/66-1 (10 mg/kg), SAHA (20 mg/kg), and the combination (10 + 20 mg/kg, respectively) in a LNCaP xenograft model in
male SCID mice. Doses were switched on day 12 to VN/66-1 (20 mg/kg), SAHA (10 mg/kg), and the combination (20 + 10 mg/kg, respectively) as
indicated by the red arrow. Mice (n = 7) were injected subcutaneously QD, tumors were measured twice a week. Statistical significance was
determined by the t-test. *P < 0.005 and **P < 0.001; ***P < 0.0005 versus control. Not shown on graph: p < 0.05 for VN/66-1 versus SAHA; p < 0.01
for VN/66-1 versus VN/66-1 + SAHA; p < 0.05 for SAHA versus VN/66-1 + SAHA.
have shown that retinoids and RAMBAs interact with
HDIs to cause synergistic inhibition of growth of
LNCaP prostate cancer cells. These studies are the first
to specifically explore the biological mechanisms of ac-
tion of a RAMBA in combination with HDACI. The
combination of 2 and SAHA inhibits the growth of
LNCaP cancer cells by inducing G1 and G2/M cell cycle
arrest and induction of differentiation and apoptosis.
Compound 2, SAHA, and the combination exhibited
potent anti-tumor efficacy in vivo. On the basis of these
impressive results, further preclinical studies are war-
ranted to develop RAMBAs and HDIs such as 2 and
SAHA for prostate cancer treatment.
1H NMR spectra were recorded in CDCl3 and
DMSO-d6 at 500 MHz with Me4Si as an internal
standard using a Varian Inova 500 MHz spectrome-
ter. Melting points (mp) were determined with a
Fischer Johns melting point apparatus and are
uncorrected.
4.1.1. N-(2-Aminophenyl)-4-acetylaminobenzamide (3,
CI-994). To the suspension of acetamido benzoic acid
(6, 7.5 g, 0.041 mol) in 75 ml THF was added CDI
(7.5 g, 0.046 mol) portionwise at room temperature.
The reaction mixture was stirred for 1 h to form acylim-
idazole followed by addition of 1,2-phenylenediamine
(36.2 g, 0.335 mol) and TFA (4.19 g, 0.036 mol, 2.8 ml)
and stirring for 16 h. The reaction mixture was then fil-
tered to afford crude CI-994 which was re-crystallized
from THF/methanol to give 9.0 g of CI-994 (3) as white
crystals (yield, 80%). HPLC analysis showed purity
> 99%. Mp 207–208 ꢂC; IR (Nujol): 3289, 1646, 1540,
4. Experimental
4.1. Chemistry
1
General procedures and techniques were identical to
those previously reported.12,16 Infrared spectra were
recorded on a Perkin-Elmer 1600 FT-IR spectrometer
using Nujol paste or KBr pellets. High-resolution
mass spectra (HRMS) were determined on a Bruker
12T APEX-Qe FTICR-MS with an Apollo II ion
source (College of Sciences Major Instrumentation
Cluster, Old Dominion University, Norfolk, VA).
1456, 1297, 739 cmꢀ1; H NMR: d 2.08 (s, 3H, CH3),
4.86 (s, 2H, NH2), 6.59 (s, 1H, Ar-H), 6.78 (d, 1H,
J = 7.5 Hz, Ar-H), 6.96 (s, 1H, Ar-H), 7.16 (d, 1H,
J = 7.5 Hz, Ar-H), 7.69 (d, 2H, J = 7.5, Ar), 7.94 (d,
2H, J = 8.0, Ar-Hs), 9.55 (s, 1H, NH), 10.188 (s, 1H,
NH). HRMS Calcd 269.1164 (C15H15N3O2), found
269.1161. These spectral and analytical data are as pre-
viously reported.20

L. K. Gediya et al. / Bioorg. Med. Chem. 16 (2008) 3352–3360
3359
4.1.2. 4-[N-(Pyridin-3-yl-methoxycarbonyl)amino-
methyl]benzoic acid (8). To a suspension of 1,10-carbon-
yldiimidazole (CDI, 25.6 g, 158 mmol) in THF (120 mL)
was added 3-pyridinemethanol (7, 17.3 g, 158 mmol) in
THF (50 mL) at 10 ꢂC, and the mixture was stirred for
1 h at rt. The resulting solution was added to a suspen-
sion of 4-(aminomethyl)benzoic acid (22.6 g, 158 mmol),
DBU (24.3 g, 158 mmol), and triethylamine (22.2 mL,
158 mmol) in THF (250 mL). After stirring for 5 h at
rt, the mixture was evaporated to remove THF and then
dissolved in water (300 mL). The solution was acidified
with HCl (pH 5) to precipitate a white solid which was
collected by filtration, washed with water (300 mL)
and methanol (50 mL), respectively, and dried to give
pure 8 (41.1 g, 91% yield): mp 207–208 ꢂC; IR (KBr)
3043, 1718, 1568, 1434, 1266, 1108, 1037, 984,
4.2.2. Western immunoblotting. LNCaP cells were trea-
ted for 24 h and harvested thereafter. The cells were
washed with ice-cold DPBS, scraped, processed, and
the supernatant separated and stored at ꢀ80 ꢂC. Wes-
tern blotting was carried out as described previously.32
Antibodies against cytokeratins 8/18 and cyclin D1 were
purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA), and Cdk4, Bad, and Bax were purchased
from Cell Signaling Technology, Inc. (Danvers, MA).
4.2.3. Cell cycle analysis. Cells were plated in T-75 flasks
containing complete RPMI 1640 medium for 24 h. Cells
were serum, starved after washing with phosphate-buf-
fered saline and incubating with RPMI 1640 (minus
phenol red) with 0.2% FBS for 48 h. Under these condi-
tions cells were arrested in the G0/G1 phase as deter-
mined by flow cytometry. Cells were then stimulated
by the addition of complete RMPI 1640 medium con-
taining 10% FBS. Cells were treated with VN/66-1 or
SAHA for various times. Cells were washed with PBS,
trypsinized, resuspended in 10 ml PBS, and counted.
They were then centrifuged (10 min, 2500 rpm at 4 ꢂC),
resuspended in PBS fixed in 70% ice-cold ethanol, and
stored in ꢀ20 ꢂC until staining. Cells were stained for
at least 1 h in the dark with a solution containing
20 lg/ml propidium iodide (Sigma), 0.02 lg/ml RNAse,
and 1% Triton X-100 (Sigma). The DNA content in the
treated and mock-treated groups was measured by flow
cytometry analysis using a FACSort flow cytometer
(Becton–Dickinson, San Jose, CA); 15,000 events were
analyzed for each sample. ModFit LT version 3.1 (Ver-
ity Software House Ind., ME) was used to analyze cell
cycle distribution. The mean of two independent exper-
iments is reported.
756 cmꢀ1
;
1H NMR (DMSO-d6) d 4.28 (d, 2H,
J = 5.9 Hz), 5.10 (s, 2H), 7.3–7.5 (m, 3H), 7.7–8.1 (m,
4H), 8.5–8.7 (m, 2H). These spectral and analytical data
are as previously reported.22
4.1.3. N-(2-Aminophenyl)-4-[N-(pyridin-3-yl-methoxycar-
bonyl)aminomethyl]benzamide (4, MS-275). To a suspen-
sion of 8 (5.0 g, 0.017 mol) in THF (100 mL) was added
CDI (3.12 g, 0.019 mol), and the mixture was stirred for
3 h at 60 ꢂC. After formation of acylimidazole the clear
solution was cooled to rt. To this were added 1,2-phen-
ylenediamine (15.11 g, 0.14 mmol) and trifluoroacetic
acid (1.2 mL, 0.015 mol) and then stirred for 16 h. The
reaction mixture was evaporated to remove THF and
the crude product was stirred in a mixture of hexane
and water (2:5, v/v) for 1 h and filtered and dried. The
residue was triturated in dichloromethane twice to af-
ford pure MS-275 (4) as off- white powder 5.25 g, 80%
yield: mp 159–160 ꢂC; IR (KBr) 3295, 1648, 1541,
1
1508, 1457, 1309, 1183, 742 cmꢀ1. H NMR (DMSO-
4.2.4. Animal studies. All animal studies were performed
according to the guidelines approved by the Institution
of Animal Care and Use Committee (IACUC) of the
University of Maryland School of Medicine. Male SCID
mice 4–6 weeks of age were obtained from the National
Cancer Institute (Fredrick, MD). The animals were
housed in a pathogen-free environment under controlled
conditions of light and humidity and received food and
water ad libitum. Subconfluent cells were scraped into
Dulbecco’s phosphate-buffered saline, collected by cen-
trifugation, and resuspended in Matrigel (10 mg/mL)
at 5.0 · 107 cells/ml. Each animal received subcutaneous
inoculations in one site per flank with 100 ll of cell sus-
pension. Animals were randomly grouped with 7 mice
per group. Tumors were measured twice weekly with
calipers, and tumor volume was calculated by the for-
mula (4/3 pr21 ꢁ r2Þ, where r1 is the smaller radius and
r2 is the larger radius. Treatments began when the tu-
d6) d 4.28 (d, 2H, J = 5.9 Hz), 4.86 (s, 2H), 5.10 (s,
2H), 6.60 (t, 1H, J = 7.3 Hz), 6.78 (d, 1H, J = 7 Hz),
6.97 (t, 1H, J = 7 Hz), 7.17 (d, 1H, J = 8 Hz), 7.3–
7.5(m, 3H), 7.78 (d, 1H, J = 8 Hz), 7.93 (d, 2H,
J = 8 Hz), 8.53 (d, 1H, J = 3.7 Hz), 8.59 (s, 1H), 9.61
(s, 1H); HRMS Calcd 376.1560 (C21H20N4O3), found
376.1558. These spectral and analytical data are as pre-
viously reported.22
4.2. Biology
4.2.1. Cell growth inhibition assay (MTT colorimetric
assay). LNCaP cell lines were maintained in RPMI 1640
medium containing 10% fetal bovine serum, 1% penicil-
lin and streptomycin, as the complete culture medium.
Cells (2 · 104) were seeded in 24-well plates and incu-
bated in a 5% CO2 incubator at 37 ꢂC for 1 day. Cul-
tures were treated with various compounds as listed,
alone and in combination on day 2 and 4. Cells were
washed on day 2 and 4 and media were changed. Mito-
chondrial metabolism was measured as a marker for cell
growth by adding 100 ll/well MTT (5 mg/ml in medium)
with 2- h incubation at 37 ꢂC on Day 6. Crystals formed
were dissolved in 500ll of DMSO. The absorbance was
determined using a microplate reader at 560 nm. The
absorbance data were converted into cell proliferation
percentage. Each assay was performed in triplicate.
mors reached
a
measurable size (approximately
100 mm3). VN/66-1 (2) and SAHA were prepared in
sterile conditions as suspensions in 0.3% hydroxypropyl
cellulose (HPC).
4.2.5. Statistical analysis. All experiments were carried
out in at least triplicate and data are expressed as mean-
s SE where applicable. Treatments were compared to
controls using Student’s t-test with either GraphPad
Prism or Sigma Plot. Various treatment groups were

3360
L. K. Gediya et al. / Bioorg. Med. Chem. 16 (2008) 3352–3360
13. Njar, V. C.; Gediya, L.; Purushottamachar, P.; Chopra,
P.; Vasaitis, T. S.; Khandelwal, A.; Mehta, J.; Huynh, C.;
Belosay, A.; Patel, J. Bioorg. Med. Chem. 2006, 14, 4323.
14. Belosay, A.; Brodie, A. M.; Njar, V. C. Cancer Res. 2006,
66, 11485.
compared using the analysis of variance (ANOVA). P
values less than 0.05 were considered to be statistically
significant.
15. Patel, J. B.; Mehta, J.; Belosay, A.; Sabnis, G.; Khandel-
wal, A.; Brodie, A. M.; Soprano, D. R.; Njar, V. C. Br. J.
Cancer 2007, 96, 1204.
16. Gediya, L. K.; Chopra, P.; Purushottamachar, P.;
Maheshwari, N.; Njar, V. C. J. Med. Chem. 2005, 48,
5047.
Acknowledgments
This research was supported by a grant from the U.S. Na-
tional Institutes of health (NIH) and the National Cancer
Institute (NCI), Grant No.: CA117991 to Vincent C.O.
Njar. We are grateful for the generous support.
17. Acharya, M. R.; Sparreboom, A.; Venitz, J.; Figg, W. D.
Mol. Pharmacol. 2005, 68, 917.
18. Miller, T. A.; Witter, D. J.; Belvedere, S. J. Med. Chem.
2003, 46, 5097.
19. Mork, C. N.; Faller, D. V.; Spanjaard, R. A. Curr. Pharm.
Des. 2005, 11, 1091.
20. Weiershausen, U. S. G. V., K.-O.; Herrmann, W. N-(20-
Aminophenyl)-benzamide derivatives: process for the
preparation thereof and pharmaceutical composition
containing them. USA, 5,137,918, 1992.
21. Hess-Stumpp, H.; Bracker, T. U.; Henderson, D.; Politz,
O. Int. J. Biochem. Cell. Biol. 2007, 39, 1388.
22. Suzuki, T.; Ando, T.; Tsuchiya, K.; Fukazawa, N.; Saito,
A.; Mariko, Y.; Yamashita, T.; Nakanishi, O. J. Med.
Chem. 1999, 42, 3001.
References and notes
1. Jemal, A.; Siegel, R.; Ward, E.; Murray, T.; Xu, J.; Thun,
M. J. CA Cancer J. Clin. 2007, 57, 43.
2. Denmeade, S. R.; Isaacs, J. T. Nat. Rev. Cancer 2002, 2,
389.
3. Barry, M. J.; Fowler, F. J., Jr.; Bin, L.; Oesterling, J. E. J.
Urol. 1997, 158, 488.
4. Feldman, B. J.; Feldman, D. Nat. Rev. Cancer 2001, 1, 34.
5. Sartorelli, A. C. Br. J. Cancer 1985, 52, 293.
6. Marks, P.; Rifkind, R. A.; Richon, V. M.; Breslow, R.;
Miller, T.; Kelly, W. K. Nat. Rev. Cancer 2001, 1, 194.
7. Mangelsdorf, D. J.; Thummel, C.; Beato, M.; Herrlich, P.;
Schutz, G.; Umesono, K.; Blumberg, B.; Kastner, P.;
Mark, M.; Chambon, P.; Evans, R. M. Cell 1995, 83, 835.
8. Nagy, L.; Kao, H. Y.; Chakravarti, D.; Lin, R. J.; Hassig,
C. A.; Ayer, D. E.; Schreiber, S. L.; Evans, R. M. Cell
1997, 89, 373.
23. Valeriote, F.; Lin, H. Cancer Chemother. Rep. 1975, 59,
895.
24. Mongan, N. P.; Gudas, L. J. Differentiation 2007, 75, 853.
25. Richon, V. M. Br. J. Cancer 2006, 95, S2.
26. Gu, J.; Zhao, X.; Spanjaard, R. A.; Chen, T. C.; Flanagan,
J. N.; Boosalis, M.; Perrine, S. P.; Faller, D. V. J. Cancer
Mol. 2006, 2, 25.
9. Richon, V. M.; Webb, Y.; Merger, R.; Sheppard, T.;
Jursic, B.; Ngo, L.; Civoli, F.; Breslow, R.; Rifkind, R. A.;
Marks, P. A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 5705.
10. Kelly, W. K.; Richon, V. M.; O’Connor, O.; Curley, T.;
MacGregor-Curtelli, B.; Tong, W.; Klang, M.; Schwartz,
L.; Richardson, S.; Rosa, E.; Drobnjak, M.; Cordon-
Cordo, C.; Chiao, J. H.; Rifkind, R.; Marks, P. A.; Scher,
H. Clin. Cancer Res. 2003, 9, 3578.
11. Bolden, J. E.; Peart, M. J.; Johnstone, R. W. Nat. Rev.
Drug Discov. 2006, 5, 769.
12. Patel, J. B.; Huynh, C. K.; Handratta, V. D.; Gediya, L.
K.; Brodie, A. M.; Goloubeva, O. G.; Clement, O. O.;
Nanne, I. P.; Soprano, D. R.; Njar, V. C. J. Med. Chem.
2004, 47, 6716.
27. Pili, R.; Kruszewski, M. P.; Hager, B. W.; Lantz, J.;
Carducci, M. A. Cancer Res. 2001, 61, 1477.
28. Huynh, C. K.; Brodie, A. M.; Njar, V. C. Br. J. Cancer
2006, 94, 513.
29. Hanahan, D.; Weinberg, R. A. Cell 2000, 100, 57.
30. Tenen, D. G. Nat. Rev. Cancer 2003, 3, 89.
31. Butler, L. M.; Agus, D. B.; Scher, H. I.; Higgins, B.; Rose,
A.; Cordon-Cardo, C.; Thaler, H. T.; Rifkind, R. A.;
Marks, P. A.; Richon, V. M. Cancer Res. 2000, 60, 5165.
32. Purushottamachar, P.; Khandelwal, A.; Chopra, P.;
Maheshwari, N.; Gediya, L. K.; Vasaitis, T. S.; Bruno,
R. D.; Clement, O. O.; Njar, V. C. Bioorg. Med. Chem.
2007, 15, 3413.