A new simple and high-yield synthesis of suberoylanilide hydroxamic acid and its inhibitory effect alone or in combination with retinoids on proliferation of human prostate cancer cells

Article abstract of DOI:10.1021/jm058214k

We have developed a procedure for the synthesis of TV-hydroxy-N ¹-phenyloctanediamide (suberoylanilide hydroxamic acid (SAHA)), providing the product in 79.8% yield. SAHA is a potent inhibitor of histone deacetylase, induces differentiation and/or apoptosis in certain transformed cells in culture, and suppressed the growth of human prostate cancer LNCaP and PC-3 cell lines. The combination of SAHA with other compounds inhibited cell proliferation of LNCaP cells in additive fashion and resulted in synergistic growth inhibition.

Full text of DOI:10.1021/jm058214k

J. Med. Chem. 2005, 48, 5047-5051
5047
A New Simple and High-Yield Synthesis of Suberoylanilide Hydroxamic Acid
and Its Inhibitory Effect Alone or in Combination with Retinoids on
Proliferation of Human Prostate Cancer Cells
Lalji K. Gediya,† Pankaj Chopra,† Puranik Purushottamachar, Neha Maheshwari, and Vincent C. O. Njar*
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine,
655 West Baltimore Street, Baltimore, Maryland 21201-1559
Received March 28, 2005
We have developed a procedure for the synthesis of N-hydroxy-N1-phenyloctanediamide
(suberoylanilide hydroxamic acid (SAHA)), providing the product in 79.8% yield. SAHA is a
potent inhibitor of histone deacetylase, induces differentiation and/or apoptosis in certain
transformed cells in culture, and suppressed the growth of human prostate cancer LNCaP
and PC-3 cell lines. The combination of SAHA with other compounds inhibited cell proliferation
of LNCaP cells in additive fashion and resulted in synergistic growth inhibition.
Chart 1. Structures of ATRA, 13-CRA, and 4
Introduction
Prostate cancer (PCA) is the most common malig-
nancy 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 U.S. in 2004, an estimated
230 000 new cases of prostate cancer were diagnosed
and about 23 000 men died of this disease.1a During the
period of 1992-1999, the average annual incidence of
PCA among African American men was 59% higher
than among Caucasian men and the average annual
death rate was more than twice that of Caucasian
men.1b
The growth of most prostate tumors depends on
androgens during the initial stages of tumor develop-
ment, and thus, antihormonal therapy, by surgical or
medical suppression of androgen action, remains a
major treatment option of the disease.2 Although this
treatment may be initially successful, most tumors
eventually recur because of the expansion of an androgen-
refractory population of PCA cells.3 Metastatic disease
that develops even after potentially curative surgery
remains a major clinical challenge. Therapeutic treat-
ments for patients with metastatic PCA are limited
because current chemotherapeutic and radiotherapeutic
regimens are largely ineffective.4 Hence, there is urgent
need to develop new therapeutic agents with defined
targets to prevent and treat this disease.
deacetylase (HDAC), thereby altering chromatin struc-
ture and changing gene expression patterns. Histone
deacetylase inhibitors (HDACIs) are potent differentiat-
ing agents toward a variety of neoplasms, including
leukemia and breast and prostate cancers. Combina-
tions of HDACIs with other known therapies including
retinoic acids (RAs) have been investigated. RAs exert
their effects via a nuclear receptor complex that inter-
acts with promoters of RA-responsive genes.7 An HDAC
subunit is an intergral part of this corepressor complex,
which is involved in transcriptional silencing in the
absence of ligand.8 This association provides a rationale
for combining HDACIs and RAs/retinoids therapeuti-
cally. One of the early HDACIs discovered by Breslow
and colleagues is N-hydroxy-N1-phenylactanediamide,
also called suberanilide hydroxamic acid (SAHA).6 This
compound has recently (2004) been approved by the U.S.
Food and Drug Administration for phase II clinical trials
in lymphoma patients.9
Recently, we reported on a family of compounds that
inhibit the P450 enzyme(s) responsible for the metabo-
lism of all-trans retinoic acid (ATRA, Chart 1).10 These
compounds, also referred to as retinoic acid metabolism
blocking agents (RAMBAs), are able to enhance the
antiproliferative effects of ATRA in breast and prostate
cancer cells in vitro. In addition, the RAMBAs were
shown to induce differentiation and apoptosis in these
cancer cell lines. However, we also observed that the
breast cancer cell lines were exquisitely more sensitive
to the RAMBAs.
PCA tumors that arise after antihormonal therapy
generally 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 differentiation therapy is to induce malignant cells
to pass the block to maturation by allowing them to
progress to more differentiated cell types with less
proliferative ability.
Breslow and colleagues6 have led the way in the
discovery of agents that inhibit the enzyme histone
* To whom correspondence should be addressed. Phone:
(410) 706-5885. Fax: (410) 706-0032. E-mail: vnjar001@umaryland.edu.
† These authors contributed equally to this work.
10.1021/jm058214k CCC: $30.25 © 2005 American Chemical Society
Published on Web 06/25/2005

5048 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Scheme 1. Previous Procedures for Syntheses of SAHA
Brief Articles
The present study describes a facile, expeditious, and
high-yield synthesis of SAHA. Furthermore, we assessed
the effects of combinations of RAs or a RAMBA (4) and
SAHA on human LNCaP prostate cancer cells in vitro.
We show that these combinations of agents lead to
enhanced (additive/synergistic) PCA growth inhibition.
synthesis of suberanilic acid, while that described by
Mai et al. was adversely affected by the relatively low
64% yield of the hydroxamate. Although the yield for
the hydroxamate formation step of Stowell et al. was
high (90%), it involved the use of sodium metal, which
is not recommended especially in large-scale prepara-
tions. However, our mild procedure (vide infra) also
resulted in a high yield (90%) of the hydroxamate. We
have developed a two-step procedure that requires
milder reaction conditions and without chromatographic
purification to give SAHA of high purity and high
overall yield of 79.8% (Scheme 2).
The commercially available suberic acid monomethyl
ester (1, 5 g costs $112) was condensed with aniline in
a typical coupling reaction for amide bond formation
using hydroxybenzotriazole (HOBt) and dicyclocarbo-
diimide (DCC) in DMF. The mixture was stirred at room
temperature for 1.5 h. The product was obtained by
diluting the mixture with water, and crude product was
dissolved in petroleum ether and ethyl acetate (1:1) and
then filtered through a short pad of silica gel. Thus,
suberanilic acid methyl ester (2) was obtained in 88.7%
yield. Hydroxylamine was prepared by mixing hydroxy-
lamine hydrochloride with potassium hydroxide in
methanol. The freshly prepared hydroxylamine was
stirred at room temperature with methyl suberanilate
for 1 h. The product was obtained by adding water and
neutralizing with acetic acid to obtain SAHA (3) in
90.0% yield (99.5% purity as determined by HPLC). 2
Results and Discussion
Chemistry. To the best of our knowledge, three
synthetic procedures for the preparation of SAHA have
been reported, and these are presented in Scheme 1.
The first, reported in a patent by Breslow and col-
leagues6 is a one-pot reaction whereby suberoyl chloride
is treated with aniline, hydroxylamine, and aqueous
KOH to give SAHA in low 15-30% yield. In addition to
the low yield, the procedure also suffers from the
formation of a dianilide byproduct that is particularly
difficult to remove, thus requiring tedious column
chromatography purification. In the second procedure
developed by Stowell et al.,11 the dianilide byproduct
was readily separated. However, this three-step proce-
dure starting from suberic acid furnished SAHA in only
35% overall yield. Furthermore, it involves long reaction
times (up to 28 h) and harsh reaction conditions
(185-190 °C). The most recent approach12 is also a
three-step procedure from suberic acid via suberoyl
anhydride, but under mild reaction conditions, it gives
SAHA in 57.8% overall yield.
The overall yield of SAHA via the procedure of Stowell
et al. was adversely affected by the low 41.7% yield for

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 5049
Scheme 2. New Synthesis of 3 (SAHA)
and SAHA were fully characterized by physical and
spectral methods and were consistent with literature
data.5,11,12 The yields of our two-step synthesis of SAHA
are highly reproducible because we have repeated these
reactions at least three times with a standard deviation
of less than 2%. The advantage of our process is its
simplicity and mild reaction conditions and that it does
not require chromatographic purification. In addition,
it gives a higher yield than the alternative procedures.
Biological Studies. In a recent study we observed
that some human breast cancer cell lines (MCF-7 and
T-47D) were exquisitely sensitive to ATRA and some of
our novel RAMBAs.10 The apparent lack of sensitivity
of the breast cancer cells (MDA-MB-213) and two
prostate cancer cell lines (LNCaP and PC-3) to ATRA
and our novel RAMBAs may be due to inactivation of
various genes that are essential for retinoid activity.7,8
We then asked whether prostate cancer cells that
display poor antiproliferative responses to ATRA and
our novel RAMBA could be made more sensitive by co-
treatment with SAHA. On the basis of previous evidence
(common signal transduction pathways and possible
modulation at the nuclear receptor levels), we hypoth-
esize that a combination of SAHA and ATRA/RAMBA
may have additive or synergistic inhibitory activity on
prostate cancer cells and tumors.
The dose-response curves of 4 and SAHA showed
that SAHA (IC50 ) 1.0 µM) was a more potent antipro-
liferative agent than 4 (IC50 ) 5.5 µM) against LNCaP
prostate carcinoma cells (Figure 1A,B). To evaluate the
potential additive/synergistic effect of SAHA and 4 on
the growth of LNCaP cells, we determined the effects
of SAHA and 4 alone and in combination. For the
combination, we chose the concentrations of both com-
pounds that gave cell growth inhibition of approximately
50% (Figure 1C). The inhibition of cell growth by this
combination was additive in that its effect was equal to
the sum of the effects of the two compounds separately,
using the Valeriote and Lin analysis.13 Furthermore, we
found that the combination of SAHA (1.0 µM) with
either ATRA (10.0 µM, Figure 2A) or 13-CRA (10.0 µM,
Figure 2B) resulted in additive and synergistic LNCaP
cell growth inhibition, respectively. When evaluated by
ANOVA, the enhanced LNCaP cell growth inhibitions
by the combined agents were in all cases significant (see
Figure 1. Dose-response curves for growth inhibitory action
of 4 (A) and SAHA (B) on LNCaP prostate cancer cells and
growth inhibitory action of 4 (5.0 µM) and SAHA (1.0 µM) (C)
administered alone and in combination on LNCaP prostate
cancer cells. Cells were treated with the compounds for 4 days,
and cell proliferation was measured by MTT assay. Values are
the mean ( SEM from three experiments: (/) P < 0.0001, 4
vs control; (//) P < 0.009, SAHA + 4 vs 4.
Figures 1A and 2B). In all likelihood, these effects of
SAHA and retinoids/RAMBAs would lead to suppression
of tumor growth. Finally, we also found that SAHA
caused a dose-dependent inhibition of PC-3 prostate
cancer cell growth inhibition, with IC50 values of 6.5 µM
(Figure 3). It is most likely that the combination of
SAHA with retinoids/RAMBAs would also be effective
in the hormone-independent PC-3 cell line.
Conclusion
We have described a novel, highly reproducible, and
simple procedure that enabled us to synthesize SAHA
in unprecedented high yield and purity. The biological
data indicate that SAHA is a potent inhibitor of human
prostate cancer cell proliferation. Furthermore, the
observation of synergistic/additive interaction of SAHA
and RAs or a RAMBA on cell growth inhibition suggests
that the combination of retinoids and HDACIs may
show potential in the therapy of prostate cancer and
possibly other cancers. The mechanism(s) that underlies
these observations is currently under investigation.
Experimental Section
Chemistry. General procedures and techniques were iden-
tical to those previously reported.10 Infrared spectra were
recorded on a Perkin-Elmer 1600 FTIR spectrometer using

5050 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Brief Articles
product, yield 88.7%: mp 64-65 °C; IR (Nujol) 3300, 1725,
1656, 1598, 1533, 1499, 1418, 1329, 1249, 1228, 1197, 1172,
958, 882, 844, 728, 690, 519 cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.52 (d, 2H, J ) 7.5 Hz, 21- and 61-Hs), 7.32 (t, 2H, J )
8.0 Hz, 31- and 51-Hs), 7.17 (brs, 1H, NH), 7.09 (t,1H, J )
7.5 Hz, 41-H), 3.66 (s, 3H, -OCH3), 2.35 (t, 2H, J ) 7.0 Hz,
2- and 7-Hs), 2.31 (t, 2H, J ) 7.5 Hz, 2- and 7-Hs), 1.74
(p, 2H, J ) 7.5 Hz, 3- and 6-Hs), 1.64 (p, 2H, J ) 7.0 Hz,
3- and 6-Hs), 1.38 (m, 4H, 4- and 5-Hs). HRMS calcd 286.1414
(C15H21NO3.Na+), found 286.1411.
Suberyolanilide Hydroxamic Acid (SAHA, 3). Hydroxy-
lamine hydrochloride (59.86 g, 0.861 mol) in methanol
(150 mL) was mixed with KOH (48.26 gm, 0.861 mol) at 40 °C
in methanol (280 mL), cooled to 0 °C, and filtered. The
suberanilic acid methyl ester (12.3 g, 0.0467 mol) was then
added to the filtrate followed by addition (over 30 min) of KOH
(3.93 g, 0.07 mol). The mixture was stirred at room temper-
ature for 1 h. The mixture was added to stirring cold water
(1500 mL), and the pH was adjusted to 7 by adding acetic acid.
The precipitate was filtered off, and the resulting product was
dried in a vacuum oven at 40 °C overnight to yield 10.84 g
(90.0%) of 3 (SAHA): mp 159-160.5 °C; IR (Nujol) 3310, 3267,
2853, 1660, 1614, 1598, 1530, 1464, 1443, 1376, 1316, 976, 761,
1
703, 573 cm-1; H NMR (300 MHz, DMSO-d6) δ 10.32 (s, 1H,
-OH), 9.83 (s, 1H, -NH), 8.64 (s, 1H, -NH), 7.57 (d, 2H, J )
8.5 Hz, 21- and 61-Hs), 7.27 (t, 2H, J ) 7.5 Hz, 31- and 51-Hs),
7.0 (t, 1H, J ) 7.5 Hz, 41-H), 2.28 (t, 2H, J ) 7.5 Hz, 2- and
7-Hs), 1.93 (t, 2H, J ) 7.5 Hz, 2- and 7-Hs), 1.55 (p, 2H, J )
6.5 Hz, 3- and 6-Hs), 1.48 (p, 2H, J ) 6.5 Hz, 3- and 6-Hs),
1.27 (m, 4H, 4- and 5-Hs). HRMS calcd 287.1366 (C14H20N2O3‚
Na+), found 287.1431.
Figure 2. (A) Effects of simultaneous exposure of ATRA
(10.0 µM) and SAHA (1.0 µM) and (B) 13-CRA (10.0 µM) on
human LNCaP prostate cancer cell proliferation. The indicated
concentrations of agents were used alone or in combination.
Values are the mean ( SEM from three experiments. / and
// denote significant difference (P < 0.05) between SAHA or
ATRA and SAHA + ATRA and between SAHA or 13-CRA and
SAHA and 13-CRA, respectively. The results represent the
average and standard deviation of three experiments per-
formed in triplicate.
HPLC Analysis. Chromatographic analysis was achieved
by a reverse-phase HPLC method on a Waters Novapak C18
column (3.9 mm × 150 mm) protected by Waters guard
cartridge packed with pellicle C18 as previously described.
Briefly, the HPLC system used in this study consisted of a
Waters solvent delivery system, a Waters controller (Milford,
MA) coupled to a Waters 717plus autosampler, and a Waters
996 photodiode array detector operating at 240.0 nm. A
multilinear gradient solvent system, (i) 50 mM aqueous
K2PO4 buffer/CH3CN/Et3N (85.975:14:0.025) (100% f 0%) and
(ii) 50 mM aqueous K2PO4 buffer/CH3CN/Et3N (69.975:30:
0.025) (0% f 100%) at a flow rate of 0.75 mL/min, was used.
This solvent system was adapted with modifications from that
reported by Kelley et al.14 The retention time was 13.44 min
for SAHA. Mobile phase ii was used for suberanilic acid methyl
ester, and the retention time was 15.59 min. The HPLC
analysis was performed at ambient temperature, and data
acquisition and management were achieved with a Waters
millennium chromatography manager.
Figure 3. Effects of SAHA on human prostate cancer cell
(PC-3) proliferation.
Cell Growth Inhibition Assay (MTT Colorimetric As-
say). LNCAP cell lines were maintained in RPMI 1640
medium containing 10% fetal bovine serum, 1% penicillin, and
streptomycin, as the complete culture medium. The 2 × 104
cells were seeded in 24-well plates and incubated in a 5% CO2
incubator at 37 °C for 1 day. Cultures were treated with
various compounds as listed, alone and in combination on days
2 and 4. Cells were washed on day 2, and media were changed.
Mitochondrial metabolism was measured as a marker for cell
growth by adding 100 µL/well MTT (5 mg/mL in medium) with
2 h of incubation at 37 °C on day 6. Crystals formed were
dissolved in 500 µL of DMSO. The absorbance was determined
using a microplate reader at 560 nm. The absorbance data
were converted into a cell proliferation percentage. Each assay
was performed in triplicate.
PC-3 cell lines were maintained in RPMI 1640 medium
containing 10% fetal bovine serum, 1% penicillin, and strep-
tomycin, as the complete culture medium. The 2 × 104 cells
were seeded in 24-well plates and incubated in a 5% CO2
incubator at 37 °C for 1 day. Cultures were treated with
various compounds as listed, alone and in combination on days
2 and 4. Cells were washed on day 2, and media were changed.
Mitochondrial metabolism was measured as a marker for cell
growth by adding 100 µL/well MTT (5 mg/mL in medium) with
Nujol paste. High-resolution mass spectra (HRMS) were
determined on a 3-Tesla Finnigan FTMS-2000 FT mass
spectrometer, ESI mode (Department of Chemistry, The Ohio
State University). 1H NMR Spectra were performed in CDCl3
and DMSO-d6 at 500 MHz with Me4Si as an internal standard
using a Varian Inova 500 MHz spectrometer. As a criterion of
purity for key target compounds, we have provided (see
Supporting Information) high-resolution mass spectral data
with HPLC chromatographic data indicating compound ho-
mogeneity. Melting points (mp) were determined with a
Fischer Johns melting point apparatus and are uncorrected.
Suberanilic Acid Methyl Ester (2). Suberic acid mono-
methyl ester (1, 10 g, 0.0531 mol), 1-hydroxybenzotriazole
(8.61 g, 0.0637 mol), and aniline (5.93 g, 0.0637 mol) were
dissolved in DMF (60 mL) at room temperature. Dicyclohexy-
lcarbodiimide (DCC) (13.14 gm, 0.0637 mol) was added, and
the mixture was stirred at room temperature for 1.5 h. The
precipitate of dicyclohexylurea was filtered off and was washed
with 5 mL DMF. The filtrate was added in cold stirring water
(900 mL). The precipitate was filtered and dried under
vacuum. The crude product was placed on a short pad of silica
gel and eluted with petroleum ether and ethyl acetate (1:1).
The solvent was removed under vacuum to give 12.4 g of

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2 h of incubation at 37 °C on day 6. The crystals formed were
dissolved in 500 µL of DMSO. The absorbance was determined
using a microplate reader at 560 nm. The absorbance data
were converted into a cell proliferation percentage. Each assay
was performed in triplicate.
Acknowledgment. This research was supported by
grants from the U.S. Department of Defense under the
Peer Review Medical Research Program (Grant
W81XWH-04-1-0101), the U.S. Army Medical Research
and Material Command (Grant DAM D17-01-1549), and
TEDCO to Vincent C. O. Njar. We thank Dr. Gary
Strahan, Department of Pharmaceutical Sciences, School
of Pharmacy, University of Maryland, Baltimore (UMB),
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1
for some of H NMR data.
Supporting Information Available: HPLC chromato-
grams and high-resolution mass spectral data for 2 and SAHA.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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