Development of potential antitumor agents. Synthesis and biological evaluation of a new set of sulfonamide derivatives as histone deacetylase inhibitors

Article abstract of DOI:10.1021/jm020377a

A series of sulfonamide hydroxamic acids and anilides have been synthesized and studied as histone deacetylase (HDAC) inhibitors that can induce hyperacetylation of histones in human cancer cells. The inhibition of HDAC activity represents a novel approach for intervening in cell cycle regulation. The lead candidates were screened in a panel of human tumor and normal cell lines. They selectively inhibit proliferation, cause cell cycle blocks, and induce apoptosis in human cancer cells but not in normal cells. The structure-activity relationships, the antiproliferative activity, and the in vivo efficacy are described.

Full text of DOI:10.1021/jm020377a

820
J . Med. Chem. 2003, 46, 820-830
Develop m en t of P oten tia l An titu m or Agen ts. Syn th esis a n d Biologica l
Eva lu a tion of a New Set of Su lfon a m id e Der iva tives a s Histon e Dea cetyla se
In h ibitor s
Giliane Bouchain,† Silvana Leit,† Sylvie Frechette,† Elie Abou Khalil,† Rico Lavoie,†,§ Oscar Moradei,†
Soon Hyung Woo,† Marielle Fournel,‡ Pu T. Yan,‡ Ann Kalita,‡ Marie-Claude Trachy-Bourget,‡ Carole Beaulieu,‡
Zuomei Li,‡ Marie-France Robert,‡ A. Robert MacLeod,‡ J effrey M. Besterman,‡ and Daniel Delorme*,†
Departments of Medicinal Chemistry and Molecular Biology, MethylGene Inc., 7220 Frederick-Banting, Montre´al,
Que´bec H4S 2A1, Canada
Received September 4, 2002
A series of sulfonamide hydroxamic acids and anilides have been synthesized and studied as
histone deacetylase (HDAC) inhibitors that can induce hyperacetylation of histones in human
cancer cells. The inhibition of HDAC activity represents a novel approach for intervening in
cell cycle regulation. The lead candidates were screened in a panel of human tumor and normal
cell lines. They selectively inhibit proliferation, cause cell cycle blocks, and induce apoptosis
in human cancer cells but not in normal cells. The structure-activity relationships, the
antiproliferative activity, and the in vivo efficacy are described.
Ch a r t 1
In tr od u ction
The reversible modification of histones by acetylation,
carried out by histone acetyltransferase (HATs) and
histone deacetylase (HDACs) enzymes, is involved in
chromatin remodeling and plays a crucial role in gene
expression.1
Hyperacetylated histones have been associated with
transcriptionally active genes. The neutralization of
positively charged lysine residues in the N-terminal tails
of core histones weakens the histone-DNA interaction,
which facilitates the accessibility of a variety of factors
to DNA. Conversely, HDACs mediate transcriptional
repression by condensing the structure of chromatin and
restricting the access of transcription factors.2 The
enzymes are involved in many cellular processes, such
as cell cycle progression and differentiation, and the
deregulation is associated with several types of human
cancers. The modulation of HDAC activity might lead
to new therapeutic opportunities against cancer.3
responsible for HDAC inhibition by coordinating to the
zinc ion in the catalytic site. Thus, a need to identify
additional HDAC inhibitors having more druglike struc-
tural features is required to achieve potent in vivo
HDAC inhibitory activity.
To date only a few inhibitors of histone deacetylase
are known. Trichostatin A (TSA)4 (1), a natural product,
and other synthetic compounds such as suberoylanilide
hydroxamic acid (SAHA)5 (2) and analogues,6 oxamfl-
atin7 (3), or MS-2758 (4) have been reported as antitu-
mor agent which inhibits proliferation of tumor cells in
human or animal by inducing terminal differentiation
of tumor cells. Inhibition of HDAC activities in cancer
cells also leads to cell cycle arrest and induction of
apoptosis. The cocrystal structures of the bacterial
HDAC enzyme with TSA and SAHA revealed an active
site consisting of a tubular pocket and a zinc binding
site at the bottom of the pocket.9 It has been shown that
the hydroxamic acid moiety of these compounds is
Recently, the synthesis of a series of sulfonamides
analogues of 5, developed as a new class of potent HDAC
inhibitors, has been published.10 In this study, we
showed that substitution on the sulfonamide or on the
double bond R to the hydroxamic acid function was
insufficient to confere measurable activity, whereas
substitution on the aryl group provided an increase in
HDAC inhibition. Herein, we focused our investigation
on the design, characterization, and structure-activity
relationships of these lead candidates, which were
optimized through variation of the aryl substituents,
sulfonamide functionality, and the zinc-chelating moi-
eties. Poor pharmacokinetic properties associated with
the hydroxamic acid group in matrix metalloproteinases
inhibitors11 prompted us to explore anilide-based sul-
* Corresponding author. Tel.: +1-514-337-3333; Fax: +1-514-337-
0550; e-mail: delormed@methylgene.com.
† Department of Medicinal Chemistry.
‡ Department of Molecular Biology.
§ Present address: Bayer Co., 400 Morgan Lane, West Haven, CT,
06516-4175.
10.1021/jm020377a CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/30/2003

Histone Deacetylase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 821
Sch em e 1a
a
Conditions: (a) Et3N, DCM; (b) NaOMe, MeOH; (c) CH2dCHCO2H, Pd2(dba)3, POT, Et3N, DMF, 100 °C; (d) i. NH2OTHP, EDC,
HOBt, DMF, 50 °C, ii. CSA, MeOH; (e) o-phenylenediamine, BOP, Et3N, DMF, rt, 12 h.
Sch em e 2a
a
Conditions: (a) DCM; (b) NaOH, H2O/THF; (c) EDC, HOBt, NH2OH‚HCl, Et3N.
Sch em e 3a
a
Conditions: (a) (PPh3)4Pd, Cul, propargyl alcohol, pyrrolidine, rt, 20 h, quantitative yield; (b) i. Dess-Martin/MeCN, rt, 16 h; ii. NaClO2,
NaH2PO4, 2-methyl-2-butene, t-BuOH, quantitative yield; (c) o-phenylenediamine, BOP, Et3N, DMF, rt, 12 h.
fonamides in order to develop more pharmaceutically
desirable HDAC inhibitors.
To evaluate the contribution of the sulfonamide
functionality, the reverse sulfonamides 15 and 17 as
well as the urea analogue 21 were synthesized. Com-
pounds 15 and 17, were prepared using routine proce-
dures, starting with the condensation of arylamine 8
with sulfonyl chloride 9. The urea derivative 21 was
readily obtained by reaction of arylisocyanate 18 with
the commercially available intermediate 19, followed by
ester saponification and conversion into the correspond-
ing hydroxamic acid 21.
Ch em istr y
The general synthetic strategies shown in Schemes
1 and 2 were employed for the preparation of the new
hydroxamic acids and anilides analogues. The arylsul-
fonamide 10 was prepared by treating iodoaniline 7 with
an excess of aryl sulfonyl chloride 6 in the presence of
triethylamine, followed by basic methanolysis. Heck
coupling reaction on 10 with acrylic acid in the presence
of Pd2(dba)3 and tri(o-tolyl) phosphine gave the requisite
carboxylic acid 12 quantitatively. Under typical peptide
coupling conditions (EDC/HOBt), 12 was treated with
THP-protected hydroxylamine to form, after removal of
the protecting group under acidic condition, the hydrox-
amic acid 14. In a similar manner, the sulfanilide 16
was obtained by reacting the carboxylic acid with
o-phenylenediamine in the presence of benzotriazol-1-
yloxytris(dimethylamino)phosphonium hexafluorophos-
phate (BOP reagent) and triethylamine.
To access the alkyne analogue outlined in Scheme 3,
the sulfonamide 22 was subjected to a Pd-Cu catalyzed
coupling reaction with propargyl alcohol to give 23
quantitatively. The primary alcohol was oxidized to the
corresponding acid 24 in two steps: Dess-Martin
periodinane oxidation to afford the intermediate alde-
hyde, followed by treatment with sodium chlorite in
buffered aqueous media in the presence of a chlorine
scavenger. The derivatization of 24 into the anilide 25
was performed using standard conditions.

822 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Bouchain et al.
Ta ble 1. HDAC-1 Inhibition and Histone Acetylation Data for Compounds 14, 15, and 21
a
b
Values are means of three separate experiments. Human bladder carcinoma T24 cells treated with escalating doses of compounds
14, 15, and 21. The relative effective concentration (EC, in µM) of a compound to induce the same level of the histone H4 acetylation of
human T24 cells by MS-275 at 1 µM is indicated.
Resu lts a n d Discu ssion
most effective HDAC inhibitors. However, hydroxam-
ates are often found to be biologically labile involving a
limited in vivo efficacy that has prompted additional
efforts toward the discovery of new chelating groups
suitable for use in HDAC templates.11 The benzamide
derivative (MS-275), which diverges structurally from
known HDAC inhibitors, exhibits a decrease in efficacy
in enzymatic assay compared to TSA (IC50 ) 5 nM vs 2
µM). However, MS-275 is still active as an histone
acetylation inhibitor and shows good antitumor activity
against several human tumors in vivo.8 The amine
group of the benzanilide moiety might act as a hydrogen
bond donor or could be involved in other electrostatic
interactions, which are necessary for inhibitory activity.
First, we report the preparation of a series of HDAC
inhibitors incorporating arylsulfonamide hydroxamate
as well as arylurea hydroxamate. The compounds
synthesized and tested in this study for inhibitory
activity on partially purified recombinant human
HDAC-1 and induction of histone acetylation in human
bladder T24 cancer cells are presented in Table 1.12
The SAR studies which focused on the effects of
substitution on the aromatic ring of 5 showed that the
p-methyl and p-tert-butyl analogues (14b, 14c) as well
as substitution with an electron-donating group (e.g.,
p-methoxy 14d ) were equipotent in HDAC-1 inhibitory
activity with the parent compound 14a . Replacement
of the phenyl ring with a polycyclic aromatic system was
well tolerated, with the biphenyl group (14f) slightly
increasing HDAC activity. The sulfonamide functional-
ity was also studied and it appears that similar activity
is observed with the “reverse” sulfonamide derivatives
15a , 15b, and 15c. Since replacement of the sulfona-
mide function with the urea (21) decreased enzymatic
activity and is detrimental for histone acetylation, this
series was not investigated further. All hydroxamates
were more active as HDAC inhibitors as compared to
the corresponding carboxylic acids, probably due to the
enhanced Zn(II) coordinating properties of the hydrox-
amate moiety (bidentate vs monodentate binding). The
use of hydroxamates as the zinc ligand provides the
The hydroxamic acid moiety of 5 has been replaced
by a benzanilide function in compounds 16 and 17,
which were prepared by coupling of the carboxylic acid
derivatives (12 and 13) with 1,2-phenylenediamine in
the usual conditions. Compounds 16 and 17 were
submitted for enzymatic inhibition and induction of
histone acetylation and the IC50 values are provided in
Table 2.
Substitution on the aromatic ring in 16 with electron-
donating groups (16c, 16d ) or replacement of the
aromatic ring with a polycyclic ring (16e-g) was well
tolerated, although these benzamide derivatives exhib-
ited a weaker inhibitory activity in both HDAC-1 and

Histone Deacetylase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 823
Ta ble 2. HDAC-1 Inhibition and Histone Acetylation Data for Compounds 16 and 17
a
b
Values are the means of three separate experiments. Human bladder carcinoma T24 cells treated with escalating doses of compounds
16 and 17. The relative effective concentration (EC, in µM) of a compound to induce the same level of the histone H4 acetylation of
human T24 cells by MS-275 at 1 µM is indicated.
Ta ble 3. HDAC-1 Inhibition Data for Known Inhibitors and Analogues
a
b
Values are means of three separate experiments. The synthetic method7,8 followed a literature procedure. c The synthetic method
was modified by derivatizing the corresponding acids into the hydroxamic acid or the anilide, respectively, using the usual conditions.
histone acetylation than the hydroxamate analogues.
Similar to the hydroxamic acid derivatives, the “reverse”
sulfonamide showed a comparable activity (17a -c).
To compare the effect of the benzamide group with
the hydroxamate, we have compared two known histone
deacetylase inhibitors (Table 3). For this purpose, the
anilide derivative of oxamflatin 26 and the hydroxamate
derivative of MS-275 27 have been synthesized. Sur-
prisingly the oxamflatin derivative, 26, showed a 50-
fold decrease in potency compare to oxamflatin whereas
the hydroxamate analogue, 27, was equipotent to the
parent compound MS-275.
From these results it would appear that the bulkiness
of a phenyl group in the hydroxamate 27 near the zinc
binding site is less tolerated. The compounds are
incorporated into the active-site pocket of the HDAC,
but require a specific length to fit in the narrow, tubelike
pocket. The benzanilide, which might act as a zinc-
chelating moiety, or the amino substituent which is able
to form a hydrogen bond or other electrostatic interac-
tion with key amino acids, seems to be well positioned
at the specific binding site. Although there is no clear
evidence that benzanilide is acting at the zinc active site
and an allosteric binding site cannot be ruled out.
Additional studies are required to understand the exact
mechanism of action of anilide-based HDAC inhibitors.
The evaluation of the distance between the sulfona-
mide and the anilide on enzymatic inhibition has been
accomplished by the synthesis of the compounds re-
ported in Table 4. The replacement of the phenyl ring
with pyridine (16i) or the alkyne (25) were 3- to 4-fold
weaker than the parent compound 16e. Introduction of
a methylene group next to the sulfonamide (16h )
showed a 2-fold lower activity than 16d . This result
suggests that the length between the two functions of
the moiety and the steric parameters would affect the
enzyme inhibitory activity. Lack of an amino group at
the ortho position of the benzamide (28) resulted in an

824 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Ta ble 4. HDAC-1 Inhibition Data for Compounds 16 Analogues
Bouchain et al.
a
Values are means of three separate experiments.
Ta ble 5. Cytotoxic Assay Results of Lead Compounds against Human Tumor and Normal Cells (IC50 in µM)a
cell line
MS-275
TSA
14c
14e
15b
16a
16b
16c
16d
16e
17a
17b
28
tumor type
bladder
colon
T24
HCT116
SW48
A549
H446
Du145
MDAmb231
MCF-7
2.5
0.6
5
8
1
1
3
5
4
0.9
5
4.5
1.5
3.5
3.5
10
2.5
0.9
2.5
3
0.85
2
2
0.2
0.8
2
0.4
0.75
0.7
1.5
1
0.8
0.7
1.5
0.35
1
8
4
10
8
1
2
17
6
12
7
3
3
8
2.5
4
10
2
5
6
8
6
1
7
9
2
3
5
6
>36
15
30
18
8
15
18
29
6.5
3.5
6.5
9.5
2
7
9
11.5
7.5
4
>37
>36
>37
>36
>37
>36
>37
>37
4.5
12
2.5
6.5
5.5
11
lung
prostate
breast
0.8
2.5
0.4
0.7
2
6
7
normal cell
fibroblast
epithelial
MRHF
HMEC
>37
>37
36
32
11
9.5
5
20
3
16
>37
>37
>37
37
>37
>37
>25
>36
34
>41
>37
>37
>36
>37
38
a
b
Values are means of two experiments. The IC50 values represent the drug concentrations producing a 50% decrease in tumor cells
colony formation after 72 h of incubation.
inactive compound, suggesting that aniline is essential
for inhibitory activity in this class of compounds.
We have profiled the more active compounds in
antiproliferative assays and in a p21WAF1/Cip1 promoter
induction assay. The inhibitory potencies (IC50 values)
of compounds 14-17 and 28, against a variety of human
tumor and normal cells, are compared with those of MS-
275 in Table 5. The antiproliferative activities were
determined by MTT assays.
In the hydroxamic series, compounds 14e and 15b
exhibited potent antiproliferative activity against hu-
man cancer cells with IC50 values from 0.2 to 2 µM.
Thus, no difference was observed between the sulfona-
mide 14e and the “reverse” sulfonamide derivative 15b.
Overall, these compounds show selective growth inhibi-
tion of human cancer cells over all types of human
normal cells. In the anilide series, compounds 16a and
16d exhibited weaker but still significant antiprolifera-
tive activity with IC50’s ranging from 1 to 10 µM. A
similar growth inhibition is observed with the “reverse”
sulfonamide derivatives 17a and 17b. It is interesting
that the biphenyl derivative 16e reduced the antipro-
liferative activity of human cancer cells by 2- to 15-fold,
while 28 could not inhibit the growth of any of the
human cancer cell lines. All the sulfonamide derivatives
(16 and 17) displayed selective growth inhibitory activi-
ties against all eight human cancer cell lines compared
to normal human cell lines. Thus, these compounds have
specific antiproliferative activity caused by HDAC in-
hibition and not by their nonspecific toxicity.
pressor.13 Treatment of human HCT116 cancer cells
with compound 16a caused a dose-dependent increase
of p21WAF1/Cip1, while expression of cyclin A and B1 was
downregulated (Figure 1). A time course study showed
that the protein p21WAF1/Cip1 was induced 8 h after
addition of compound 16a (5 µM), reached its maximum
at around 48 h after the treatment and remained
elevated for at least 72 h posttreatment.
The induction of histone acetylation in human bladder
T-24 cancer cells is presented in Figure 2 for three of
the more potent compounds HDAC-1 inhibitors from
Table 2.
Compounds 16a and 16d induced core histone H3 and
H4 acetylation at doses as low as 1 µM in a dose-
dependent manner. Compound 16e was less potent on
induction of histone acetylation in whole cells. Com-
pound 28, which showed no activity in HDAC-1 inhibi-
tion (Table 4), did not induce histone acetylation even
at 25 µM. The ability of these compounds to induce
histone acetylation correlated well with their ability to
inhibit HDAC-1 in vitro.
The effects of compounds 16a and 16d on the cell cycle
were measured by flow cytometric analysis against
human HCT116 cancer cells and HMEC normal cells
after 16 h exposure (Figure 3). The population of G2/
M-phase cells increased in a dose-dependent manner.
Induction of G2/M arrest by these compounds appeared
to be specific to cancer cells, since no induction of cell
cycle arrest was observed in normal cells. Other HDAC
inhibitors including TSA, SAHA, and FR901228 induce
gene expression of p21WAF1/Cip1 and cause G1 and G2/M
cell cycle arrest. In contrast to the G1 arrest, the G2/M
Known HDAC inhibitors were reported to transcrip-
tionally induce p21WAF1/Cip1, a well-known tumor sup-

Histone Deacetylase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 825
F igu r e 1. (A) Protein expression of p21WAF1/Cip1, cyclin A, and cyclin B1 genes in HCT116 cells treated with compound 16a .
Human cancer HCT116 cells were treated with compound 16a at indicated concentrations or DMSO alone for 16 h before whole
cell lysates were harvested and analyzed by Western Blot. (B) Time course of p21WAF1/Cip1 protein induction by compound 16a at
5 µM in human cancer HCT116 cells. In both panels, the expression level of actin was analyzed to reveal protein loading in each
lane.
F igu r e 2. Human T24 cells were treated with compounds 16a , 16d , 16e, and 28 at 0, 1, 5, 25 µM for 16 h. Cells were harvested,
and histones were acid-extracted. Histones were analyzed by SDS-PAGE and immunoblotting with antibodies specific for either
acetylated H4 or acetylated H3 histones. Histones were also stained by Coomassie Blue to reveal the amount of histones loaded
on the blots.
arrest is p21-independent but is associated with sig-
nificant cytotoxicity.5,14
cancer cell cycle progression. This provides selective
inhibition of cancer cell proliferation.
Thus, these observations suggest that compounds 16a
and 16d transcriptionally induced p21WAF1/Cip1 through
acetylation of histones and blocked tumor cell cycle
progression.
Exp er im en ta l Section
Ch em istr y. Nuclear magnetic resonance spectra (1H NMR
and 13C NMR) were measured on Varian 300 MHz. Chemical
shifts (δ) are reported in parts per million (ppm) relative to
TMS peak. MS spectra were obtained on HP1100 instrument.
Elemental analyses were within (0.4% of calculated values.
Flash column chromatography were performed with silica gel
(230-400 mesh). Analytical HPLC were performed on HP1100
instrument equipped with a Zorbax 50 × 4.6 mm C-8 column.
The mobile phases used were A: H2O with 0.01M EDTA +
formic acid to pH ) 5, B: MeOH with 0.05% formic acid, using
a solvent gradient of A/B: 80/20 to 5/95. The analyses were
performed at rt with a constant flow rate of 1 mL/min using a
gradient elution of 0-10 min.
Syn th esis a n d Ch a r a cter iza tion . Compounds 14, 15, 16,
and 17 were essentially prepared according to the same
procedure. The sequence is illustrated for 14a , followed by
analytical data for 14b-j, 15a -c, 16a -i, and 17a -c.
4-(Ben zen esu lfon yla m in o)p h en yl Iod id e (10). To a
solution of 4-iodoaniline 7 (10 g, 45.66 mmol) in CH2Cl2 (150
mL) were added at room-temperature Et3N (15.88 mL, 114.15
mmol) followed by benzenesulfonyl chloride (12.19 mL, 95.89
mmol). After the mixture was stirred 4 h, a saturated sodium
bicarbonate solution was added, and the phases were sepa-
rated. The aqueous layer was extracted with methylene
Some of the HDAC inhibitors emerging from the SAR
studies were evaluated for in vivo antitumor activity
(Table 6). Nude mice bearing established human lung
cancer tumors (A549) or human colon cancer tumors
(HCT116) were used. The compounds were administered
daily intraperitoneally at doses of 40 mg/kg or 50 mg/
kg for 21 days. Compound 14e showed marked antitu-
mor effect against A549 tumors (57% of tumor growth
inhibition), and compounds 16a and 16d suppressed
tumor growth by 55% and 45%, respectively, compared
with mice receiving vehicle alone. The results suggest
that these sulfonamide anilides inhibit cancer cell
growth and may be useful for the treatment of cancer.
In summary, a new series of sulfonamide anilide-
based histone deacetylase inhibitors were discovered.
Our studies demonstrate that sulfonamide anilides
induce hyperacetylation of histones resulting in in-
creased p21WAF1/Cip1 expression and G2/M arrest of

826 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Bouchain et al.
F igu r e 3. Cell cycle analysis of human HCT116 cancer cells and HMEC normal cells treated with compounds 16a and 16d for
16 h. Compound 16a treated cells are shown in upper left (HCT116) and bottom left (HMEC) panels. Compound 16d treated cells
are shown in upper right (HCT116) and bottom right (HMEC) panels. The final concentration of DMSO in cell culture media is
1%. Cells were harvested by trypsinization. Propidium iodide stained DNA contents in fixed cells were analyzed by flow cytometry.
Ta ble 6. In Vivo Antitumor Activity of Compounds 14, 15, 16,
and 17
in methanol (10 mL), and then 10-camphorsulfonic acid (77
mg, 0.33 mmol) was added. The mixture was stirred 2 h at
room temperature, and then the solvents were evaporated
under reduced pressure at room temperature to avoid thermal
decomposition. The crude product was purified by flash chro-
matography using CH2Cl2/MeOH (9/1) as eluent giving com-
pound 14a (116 mg, 55% yield) as an orange solid. 1H NMR
(DMSO-d6) δ 10.69 (s, 1H), 10.54 (s, 1H), 8.98 (bs, 1H), 7.78
(d, J ) 6.9 Hz, 2H), 7.65-7.53 (m, 3H), 7.42 (d, J ) 8.4 Hz,
2H), 7.32 (d, J ) 16.2 Hz, 1H), 7.12 (d, J ) 8.4 Hz, 2H), 6.30
(d, J ) 15.6 Hz, 1H). LRMS (ESI) 319 (MH+).
compd
14e
15b 16a
16d
17a
tumor type
A549 A549 HCT116 HCT116 HCT116
dose ip (mg/kg/day)
tumor growth inhibn (%) 57
50
50
37
40
55
40
45
40
37
chloride, and the combined organic fractions were dried over
MgSO4 then evaporated. The crude mixture was redissolved
in methanol (100 mL) and treated with sodium methoxide (12
g, 228 mmol). The mixture was heated 1 h at 60 °C, cooled to
room temperature, and quenched with 1 N HCl. The mixture
was concentrated under reduced pressure, extracted with
methylene chloride, dried over MgSO4, and concentrated. The
crude material was purified by chromatography on silica gel
using methylene chloride to give the title compound (15.96 g,
97%) as yellow solid. 1H NMR (CDCl3) δ 9.15 (bs, 1H), 7.82 (d,
J ) 8 Hz, 2H), 7.68-7.51 (m, 5H), 7.05 (d, J ) 8 Hz, 2H). Anal.
(C12H10NO2S) C, H, N, S.
N-Hydr oxy-3-[4-(tolu en e-4-su lfon ylam in o)ph en yl]acr yl-
1
a m id e (14b). H NMR (DMSO-d6) δ 10.7 (bs, 1H), 10.45 (bs,
1H), 8.96 (bs, 1H), 7.64 (d, J ) 8.1 Hz, 2H), 7.38 (d, J ) 8.4
Hz, 2H), 7.32-7.29 (m, 3H), 7.09 (d, J ) 8.4 Hz, 2H), 6.29 (d,
J ) 16.2 Hz, 1H), 2.30 (s, 3H). LRMS (ESI) 333 (MH+).
N-H yd r oxy-3-[4-(4-ter t-b u t ylb en zen esu lfon yla m in o)-
p h en yl]a cr yla m id e (14c). 1H NMR (DMSO-d6) δ 10.54 (s,
1H), 7.73 (d, J ) 8.4 Hz, 2H), 7.58 (d, J ) 8.4 Hz, 2H), 7.43 (d,
J ) 8.4 Hz, 2H), 7.32 (d, J ) 15.6 Hz, 1H), 7.15 (d, J ) 8.4 Hz,
2H), 6.30 (d, J ) 15.9 Hz, 1H), 1.25 (s, 9H). LRMS (ESI) 375
(MH+).
3-[4-(Ben zen esu lfon yla m in o)p h en yl]a cr ylic Acid (12).
To a solution of sulfonamide 10 (500 mg, 1.39 mmol) in DMF
(10 mL) were added tris(dibenzylidene acetone)dipalladium (0)
(38 mg, 0.091 mmol), tri-o-tolylphosphine (25 mg, 0.083 mmol),
Et3N (483 µL, 3.48 mmol), and finally acrylic acid (114 µL,
1.67 mmol). The resulting solution was degassed and purged
with nitrogen then heated overnight at 100 °C. The mixture
was filtered through a Celite pad, and then the filtrate was
evaporated. The residue was purified by flash chromatography
using CH2Cl2/MeOH (95/5) as eluent yielding the title com-
N-H yd r oxy-3-[4-(4-m et h oxyb en zen esu lfon yla m in o)-
1
p h en yl]a cr yla m id e (14d ). H NMR (DMSO-d6) δ 10.66 (bs,
1H), 10.37 (bs, 1H), 8.56 (bs, 1H), 7.69 (d, J ) 8.7 Hz, 2H),
7.39 (d, J ) 8.1 Hz, 2H), 7.30 (d, J ) 16.2 Hz, 1H), 7.10-7.03
(m, 4H), 6.27 (d, J ) 15.9 Hz, 1H), 3.77 (s, 3H). LRMS (ESI)
349 (MH+).
N-Hydr oxy-3-[4-(3,4-dim eth oxyben zen esu lfon ylam in o)-
1
1
p h en yl]a cr yla m id e (14e). H NMR (DMSO-d6) δ 10.70 (bs,
pound (415 mg, 99%) as yellow solid. H NMR (acetone-d6) δ
1H), 10.33 (bs, 1H), 8.99 (bs, 1H), 7.44-7.26 (m, 5H), 7.12 (d,
J ) 8.7 Hz, 2H), 7.06 (d, J ) 8.4 Hz, 1H), 6.30 (d, J ) 16.2 Hz,
1H), 3.78 (s, 3H) 3.75 (s, 3H). LRMS (ESI) 379 (MH+).
N-H yd r oxy-3-[4-(b ip h en yl-4-su lfon yla m in o)p h en yl]-
7.88-7.85 (m, 2H), 7.62-7.55 (m, 6H), 7.29 (d, J ) 9 Hz, 2H),
6.41 (d, J ) 16 Hz, 1H). 13C NMR (acetone-d6): 167.69, 144.31,
140.66, 140.44, 133.70, 131.26, 129.94, 129.87, 127.75, 120.73,
118.27.
1
a cr yla m id e (14f). H NMR (DMSO-d6) δ 10.60 (s, 1H), 9.00
N-Hyd r oxy-3-[4-(ben zen esu lfon yla m in o)p h en yl]a cr yl-
a m id e (14a ). To a solution of 12 (200 mg, 0.66 mmol) in DMF
(10 mL) were added 1-(3-dimethylaminopropyl)-3-ethyl-car-
bodiimide hydrochloride (151 mg 0.79 mmol) and hydroxyben-
zotriazole hydrate (134 mg, 0.99 mmol). The mixture was
stirred 20 min at room temperature, and then NH2OTHP (116
mg, 0.99 mmol) was added. The resulting mixture was heated
at 50 °C for 24 h, DMF was removed under reduced pressure,
and the residue was dissolved in methylene chloride and
washed with a saturated solution of NaHCO3. The organic
extract was dried over MgSO4 then condensed. The crude
compound was purified by flash chromatography using hexane/
acetone (7/3) as solvent mixture. The residue was dissolved
(s, 1H), 7.82-7.10 (m, 14H), 6.20 (d, J ) 15.9 Hz, 1H). LRMS
(ESI) 395 (MH+).
N-H yd r oxy-3-[4-(1-n a p h t h ylsu lfon yla m in o)p h en yl]-
a cr yla m id e (14 g). 1H NMR (DMSO-d6) δ 10.94 (bs, 1H),
10.65 (bs, 1H), 8.95 (bs, 1H), 8.73-8.71 (m, 1H), 8.24-8.21
(m, 2H), 8.08-8.05 (m, 1H), 7.74-7.63 (m, 3H), 7.33-7.23 (m,
3H), 7.06-7.04 (m, 2H), 6.24 (d, J ) 15.3 Hz, 1H). LRMS (ESI)
369 (MH+).
N-Hyd r oxy-3-[4-(5-Dim eth yla m in on a p h th a len e-1-su l-
fon yla m in o)p h en yl]a cr yla m id e (14h ). 1H NMR (DMSO-d6)
δ 10.91 (s, 1H), 10.62 (bs, 1H), 8.45 (d, J ) 8.1 Hz, 1H), 8.36
(d, J ) 8.7 Hz, 1H), 8.25 (d, J ) 6.9 Hz, 1H), 7.65-7.59 (m,

Histone Deacetylase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 827
2H), 7.37-7.34 (m, 2H), 7.29-7.23 (m, 2H), 7.06 (d, J ) 8.7
Hz, 2H), 6.25 (d, J ) 15.9 Hz, 1H), 2.80 (s, 6H). LRMS (ESI)
412 (MH+).
N-H yd r oxy-3-[4-(q u in olin e-8-su lfon yla m in o)p h en yl]-
a cr yla m id e (14i). 1H NMR (DMSO-d6) δ 10.63 (s, 1H), 10.36
(bs, 1H), 9.13-9.12 (m, 1H), 8.93 (bs, 1H), 8.51 (d, J ) 8.1 Hz,
1H), 8.40 (d, J ) 7.2 Hz, 1H), 8.28 (d, J ) 8.4 Hz, 1H), 7.75-
7.70 (m, 2H), 7.30-7.20 (m, 3H), 7.09 (d, J ) 8.4 Hz, 2H), 6.21
(d, J ) 15.9 Hz, 1H). LRMS (ESI) 370 (MH+).
N-Hydr oxy-3-[4-(diben zofu r an -3-su lfon ylam in o)ph en yl]-
a cr yla m id e (14j). 1H NMR (DMSO-d6) δ 10.63 (bs, 1H), 10.56
(s, 1H), 8.67 (s, 1H), 8.29 (d, J ) 6.9 Hz, 1H), 7.89-7.85 (m,
2H), 7.75 (d, J ) 8.4 Hz, 1H), 7.59 (t, J ) 7.2 Hz, 1H), 7.47-
7.38 (m, 3H), 7.27 (d, J ) 15.6 Hz, 1H), 7.15 (d, J ) 8.7 Hz,
2H), 6.25 (d, J ) 15.9 Hz, 1H). LRMS (ESI) 409 (MH+).
126.7, 125.2, 122.4, 121.8, 120.8, 119.6, 118.7, 111.9, 110.9,
56.6. HRMS (EI) calcd 453.1358, found 453.1351.
N-(2-Am in op h en yl)-3-[4-(b ip h en yl-4-su lfon yla m in o)-
1
p h en yl]a cr yla m id e (16e). H NMR (DMSO-d6) δ 7.91-7.81
(m, 4H), 7.63-7.58 (m, 5H), 7.48-7.43 (m, 2H), 7.39-7.33 (m,
2H), 7.24 (d, J ) 8.5 Hz, 2H), 6.97 (dd, J ) 9.9, 7.1 Hz, 2H),
6.79 (d, J ) 7.7 Hz, 1H), 6.61 (dd, J ) 7.7, 7.1 Hz, 1H), 5.01
(bs, 2H). 13C NMR (DMSO-d6) δ 162.9,141.9, 141.6, 139.8,
139.2, 137.6, 136.9, 135.8, 128.9, 128.3, 127.4, 127.3, 127.2,
126.3, 126.0, 125.5, 124.8, 123.2, 120.4, 116.2, 115.9. HRMS
(EI) calcd for C27H23N3O3S 469.1460, found 469.1452.
N-(2-Am in op h en yl)-3-[4-(1-n a p h t h ylsu lfon yla m in o)-
1
p h en yl]a cr yla m id e (16f). H NMR (acetone-d6) δ 8.81 (d, J
) 8.4 Hz, 1H), 8.34 (d, J ) 7.2 Hz, 2H), 8.20 (d, J ) 8.1 Hz,
1H), 8.05 (d, J ) 7.5 Hz, 1H), 7.75-7.59 (m, 4H), 7.53-7.41
(m, 3H), 7.23-7.07 (m, 3H), 6.89-6.86 (m, 1H), 6.75 (d, J )
15.3 Hz, 1H). LRMS (ESI) 444 (MH+).
N-H yd r oxy-3-(4-p -t olylsu lfa m oylp h en yl)a cr yla m id e
1
(15a ). H NMR (CD3OD) δ 7.74-7.54 (m, 5H), 7.07-6.96 (m,
N-(2-Am in op h en yl)-3-[4-(d iben zofu r a n -3-su lfon yla m i-
n o)p h en yl]a cr yla m id e (16g). 1H NMR (DMSO-d6) δ 9.66 (s,
1H), 8.72 (s, 1H), 8.33 (d, J ) 8.0 Hz, 1H), 7.94-8.00 (m, 2H),
7.82 (d, J ) 8.0 Hz, 1H), 7.61 (m, 1H), 7.50-7.40 (m, 5H), 7.32
(d, J ) 8.0 Hz, 1H), 7.22 (d, J ) 8.0 Hz, 2H), 7.00 (m, 1H),
6.94 (m, 1H), 6.72 (d, J ) 15.0 Hz, 1H). LRMS (ESI) 484 (MH+).
N-(2-Am in oph en yl)- 3-{4-[(3,4-dim eth oxy-ben zen esu lfo-
n ylam in o)m eth yl]ph en yl}acr ylam ide (16h ). 1H NMR (DM-
SO-d6) δ 8.06 (d, J ) 6.3 Hz, 1H), 7.55 (dd, J ) 8.5, 6.8 Hz,
3H), 7.43-7.32 (m, 4H), 7.12 (d, J ) 8.5 Hz, 1H), 6.97-6.59
(m, 4H), 4.99 (bs, 2H), 4.01 (d, J ) 6.0 Hz, 2H), 3.86 (s, 3H),
3.83 (s, 3H). 13C NMR (DMSO-d6) δ 163.5, 151.8, 148.6, 141.6,
139.4, 133.7, 132.1, 128.1, 127.5, 124.7, 122.7, 120.2, 116.3,
115.9, 111.1, 109.4, 55.8, 55.6, 45.9. HRMS (EI) calcd for
4H), 6.55 (d, J ) 15.7 Hz, 1H), 2.25 (s, 3H). 13C NMR (CD3-
OD) δ 165.4, 141.6, 140.4, 139.5, 136.1, 135.9, 130.6, 129.0,
128.8, 123.1, 121.7, 20.8. HRMS (EI) calcd for C16H16N2O4S
314.0725, found 314.0734.
N-Hyd r oxy-3-[4-(3,4-d im eth oxyp h en ylsu lfa m oyl)p h en -
1
yl]a cr yla m id e (15b). H NMR (DMSO-d6) δ 10.82 (bs, 1H),
9.95 (bs, 1H), 9.12 (bs, 1H), 7.70 (bs, 4H), 7.46 (d, J ) 15.9
Hz, 1H), 6.79 (d, J ) 8.7 Hz, 1H), 6.68 (s, 1H), 6.56-6.51 (m,
2H), 3.65 (s, 3H), 3.62 (s, 3H). LRMS (ESI) 379 (MH+).
N-H yd r oxy-3-[4-(b ip h en yl-4-su lfa m oyl)p h en yl]a cr yl-
1
a m id e (15c). H NMR (CD3OD) δ 10.50 (s, 1H), 9.10 (s, 1H),
7.82 (d, J ) 6.6 Hz, 2H), 7.67 (bs, 2H), 7.65-7.48 (m, 4H),
7.39 (bs, 2H), 7.30 (d, J ) 7.1 Hz, 2H), 7.20 (bs, 2H), 6.56 (d,
J ) 15.7 Hz, 1H). 13C NMR (CD3OD-d4) δ 165.4, 141.6, 141.4,
140.5, 139.5, 139.0, 137.9, 129.8, 129.2, 128.7, 128.6, 128.2,
127.6, 122.7, 121.7. LRMS (ESI) 395 (MH+). HRMS (EI) calcd
for C21H17N2O4S 350.1089, found 350.1095 (M - CO2).
C
24H25N3O5S 467.1515, found 467.1508.
N-(2-Am in op h en yl)-3-[6-(b ip h en yl-4-su lfon yla m in o)-
p yr id in -3-yl]-a cr yla m id e (16i). 1H NMR (CD3OD) δ 8.23 (d,
J ) 1.9 Hz, 1H), 8.03 (bd, J ) 8.5 Hz, 2H), 7.96 (dd, J ) 9.1,
1.9 Hz, 1H), 7.76 (bd, J ) 8.5 Hz, 2H), 7.63 (dd, J ) 8.2, 1.4
Hz, 2H) 7.53 (d, J ) 15.5 Hz, 1H), 7.48-7.36 (m, 3H), 7.29 (d,
J ) 9.1 Hz, 1H), 7.18 (dd, J ) 8.0, 1.4 Hz, 1H), 7.03 (dt, J )
7.8, 1.4 Hz, 1H), 6.86 (d, J ) 7.9, 1.4 Hz, 1H), 6.76 (d, J )
15.6 Hz, 1H), 6.75-6.69 (m, 1H), 4.85 (bs, 4H). 13C (CD3OD) δ
166.4, 154.7, 146.9, 146.2, 143.1, 141.1, 140.6, 138.6, 137.9,
130.1, 129.5, 128.8, 128.5, 128.3, 126.7, 125.6, 125.0, 122.1,
120.8, 119.5, 118.6, 114.9. LRMS (ESI) 471 (MH+).
N-(2-Am in oph en yl)-3-[4-(tolu en e-4-su lfon ylam in o)ph en -
yl]a cr yla m id e (16a ). To a solution of acid 12a (1.34 g, 4.22
mmol), BOP (2.05 g, 4.64 mmol), and 1,2-phenylenediamine
(502 mg, 4.64 mmol) in DMF (20 mL) was added Et3N (2.34
mL, 16.88 mmol). The mixture was stirred at room tempera-
ture under nitrogen overnight. DMF was removed under
reduced pressure. The crude product was purified by flash
chromatography using hexane/EtOAc (1/1) as eluent giving
1
compound 16a (607 mg, 35%). H NMR (CD3OD) δ 7.68 (d, J
N-(2-Am in oph en yl)- 3-(4-p-tolylsu lfam oyl-ph en yl)-acr yl-
) 8.2 Hz, 2H), 7.55 (d, J ) 15.9 Hz, 1H), 7.47 (d, J ) 8.5 Hz,
2H), 7.30 (d, J ) 8.0 Hz, 2H), 7.19-7.12 (m, 3H), 7.03 (t, J )
7.1 Hz, 1H), 6.86 (d, J ) 8.0 Hz, 1H), 6.75-6.69 (m, 2H), 2.37
(s, 3H). HRMS (EI) calcd for C22H21N3O3S 407.1304, found
407.1293.
1
a m id e (17a ). H NMR (DMSO-d6) δ 7.77 (bs, 4H), 7.57 (d, J
) 15.7 Hz, 1H), 7.35 (d, J ) 6.9 Hz, 1H), 7.03-6.94 (m, 6H),
6.76 (d, J ) 7.1 Hz, 1H), 6.59 (d, J ) 6.9 Hz, 1H), 4.98 (bs,
2H), 2.19 (s, 3H). 13C NMR (DMSO-d6) δ 162.9, 141.6, 139.8,
139.0, 137.6, 134.8, 133.6, 129.6, 128.1, 127.3, 125.9, 125.4,
124.7, 123.2, 120.7, 116.2, 115.9, 20.3. HRMS (EI) calcd for
N-(2-Am in op h en yl)-3-[4-(4-ter t-bu tylben zen esu lfon yl-
a m in o)p h en yl]a cr yla m id e (16b). 1H NMR (acetone-d6) δ
9.25 (bs, 1H), 8.77(bs, 1H), 7.79 (d, J ) 8.5 Hz, 2H), 7.61-
7.57 (m, 3H), 7.53 (d, J ) 8.2 Hz, 2H), 7.34 (d, J ) 8.5 Hz,
1H), 7.30 (d, J ) 8.5 Hz, 2H), 6.96 (t, J ) 8.2 Hz, 1H), 6.85 (d,
J ) 9.3 Hz, 1H), 6.82 (dd, J ) 8.0, 1.4 Hz, 1H), 6.65 (dd, J )
7.7, 1.4 Hz, 1H), 4.63 (bs, 2H), 1.30 (s, 9H).HRMS (EI) calcd
for C25H27N3O3S 449.1773, found 449.1767.
N-(2-Am in oph en yl)-3-[4-(4-m eth oxyph en ylsu lfon ylam i-
n o)p h en yl]a cr yla m id e (16c). 1H NMR (DMSO-d6) δ 7.77 (d,
J ) 8.8 Hz, 2H), 7.51 (d, J ) 8.5 Hz, 2H), 7.45 (d, J ) 16.0 Hz,
1H), 7.34 (d, J ) 8.8 Hz, 1H), 7.18 (d, J ) 8.5 Hz, 2H), 7.11 (d,
J ) 8.8 Hz, 2H), 6.94 (t, J ) 7.4 Hz, 1H), 6.77 (m, 2H), 6.6 (t,
J ) 7.4 Hz, 1H), 4.95 (bs, 1H), 3.83 (s, 3H). 13C NMR (DMSO-
d6) δ 162.5, 141.5, 139.2, 138.8, 130.9, 130.2, 128.9, 128.6,
125.7, 124.7, 119.4, 116.2, 115.9, 114.5, 55.6. LRMS (ESI) 424.5
(MH+). HRMS (EI) calcd for C22H21N3O4S 423.1253, found
423.1235.
C
22H21N3O3S 407.1304, found 407.1301.
N-(2-Am in op h en yl)-3-[4-(3,4-d im et h oxyp h en ylsu lfa -
m oyl)p h en yl]a cr yla m id e (17b). 1H NMR (CD3OD) δ 7.72
(bs, 5H), 7.20 (d, J ) 8.0 Hz, 1H), 7.05 (dd, J ) 7.1, 8.0 Hz,
1H), 6.93 (d, J ) 15.9 Hz, 1H), 6.87 (d, J ) 7.9 Hz, 1H), 6.80
(d, J ) 8.7 Hz, 2H), 6.73 (d, J ) 8.1 Hz, 1H), 6.60 (d, J ) 8.5
Hz, 1H), 3.77 (bs, 3H), 3.74 (bs, 3H). 13C NMR (CD3OD) δ
166.2, 150.7, 148.5, 143.2, 141.7, 140.6, 140.5, 131.9, 129.2,
128.9, 128.4, 126.7, 124.9, 119.5, 118.6, 116.4, 113.2, 108.9,
56.6, 56.4. HRMS (EI) calcd for C23H23N3O5S 453.1358, found
453.1351.
N-(2-Am in op h en yl)- 3-[4-(bip h en yl-4-su lfa m oyl)p h en -
yl]a cr yla m id e (17c). 1H NMR (DMSO-d6) δ 7.91-7.81 (m,
4H), 7.63-7.58 (m, 5H), 7.48-7.43 (m, 2H), 7.39-7.33 (m, 2H),
7.24 (d, J ) 8.5 Hz, 2H), 6.97 (dd, J ) 9.9, 7.1 Hz, 2H), 6.79
(d, J ) 15.9 Hz, 1H), 6.61 (dd, J ) 7.7, 7.1 Hz, 1H), 5.01(bs,
2H). 13C NMR (DMSO) δ 162.9, 141.9, 141.6, 139.8, 139.2,
137.6, 136.9, 135.8, 128.9, 128.3, 127.4, 127.3, 127.2, 126.3,
126.0, 125.5, 124.8, 123.2, 120.4, 116.2, 115.9. HRMS (EI) calcd
for C27H23N3O3S 469.1460, found 469.1452.
N-Hyd r oxy-3-[4-(3-bip h en yl-4-ylu r eid o)p h en yl]a cr yla -
m id e (21). (i) 3-{4-[3-(3,4-Dim eth oxyp h en yl)u r eid o]p h en -
yl}a cr ylic Acid Meth yl Ester (20). To a solution of 4-iso-
cyanato-1,2-dimethoxybenzene 18 (250 mg, 1.4 mmol) in
N-(2-Am in op h en yl)-3-[4-(3,4-d im eth oxyben zen esu lfo-
n yla m in o)p h en yl]a cr yla m id e (16d ). 1H NMR (CD3OD) δ
7.50 (d, J ) 16.0 Hz, 1H), 7.41 (d, J ) 7.9 Hz, 2H), 7.35 (d, J
) 8.2 Hz, 2H), 7.20 (d, J ) 8.2 Hz, 2H), 7.11 (d, J ) 7.9 Hz,
2H), 6.97-6.95 (m, 1H), 6.82 (d, J ) 6.8 Hz, 1H), 6.69-6.65
(m, 2H), 3.81 (bs, 3H), 3.80 (bs, 3H).13C NMR (CD3OD) δ 167.0,
154.4, 150.5, 143.1, 141.9, 141.0, 132.5, 132.3, 129.9, 128.2,

828 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Bouchain et al.
anhydrous dichloromethane (20 mL) was added 3-(4-ami-
nophenyl)acrylic acid methyl ester 19 (248 mg, 1.4 mmol) at
room temperature and under nitrogen. The mixture was
stirred at room temperature for 16 h, quenched with a solution
of ammonium chloride, and extracted with dichloromethane.
The organic layer was washed with water and brine and then
dried over MgSO4. The solvent was evaporated, and the
residue was purified by flash chromatography on silica gel
using CH2Cl2/MeOH (9/1) to give the desired product 20 in 75%
7.12 (d, J ) 8.7 Hz, 2H), 7.58 (d, J ) 7.5 Hz, 2H), 7.50-7.20
(m, 5H), 7.39 (d, J ) 7.5 Hz, 2H), 6.22 (bs, 2H), 4.37 (s, 2H).
13C NMR (acetone-d6) δ 146.4, 139.5, 139.2, 138.7, 133.3, 129.7,
129.2, 128.4, 128.1, 127.8, 120.8, 119.6, 89.5, 84.1, 50.9. LRMS
(ESI) 364 (MH+), 386 (M + Na).
(iii) Bip h en yl-4-su lfon ic Acid [4-(3-oxop r op -1-yn yl)-
p h en yl]a m id e. To a solution of alcohol 23 (1.37 g, 3.77 mmol)
in dry acetonitrile was added Dess-Martin reagent (1.87 g,
4.41 mmol) under nitrogen and the mixture stirred at room
temperature in the dark for 4 h. The reaction mixture was
diluted with ethyl acetate and the oxidant quenched with a
solution of Na2S2O3 (4.8 g) in water (25 mL). The organic layer
was washed (sat. NaHCO3), dried (MgSO4), filtered, and
concentrated. Upon purification by flash chromatography on
silica gel (elution 50% EtOAc in hexane), a yellow crystalline
solid (930 mg, 68% yield) corresponding to the aldehyde was
obtained. 1H NMR (acetone-d6) δ 9.64 (bs, 1H), 9.34 (s, 1H),
7.98 (d, J ) 8.7 Hz, 2H), 7.70 (d, J ) 8.7 Hz, 2H), 7.53-7.50
(m, 2H), 7.49 (d, J ) 8.7 Hz, 2H), 7.40 (d, J ) 8.7 Hz, 2H),
7.39-7.27 (m, 3H). 13C NMR (acetone-d6) δ 177.4, 146.1, 141.5,
139.2, 138.7, 135.2, 129.6, 129.1, 128.3, 128.1, 127.6, 119.8,
114.8, 94.5, 89.2. HRMS (EI) calcd for C21H15NO3S: 361.0773,
found: 361.0771.
1
yield (377 mg). H NMR: (CD3OD) δ 7.60-7.40 (m, 4H), 7.20
(s, 1H), 7.00-6.82 (m, 3H), 6.43 (d, J ) 16.0 Hz, 1H), 3.99 (s,
3H), 3.80 (s, 3H), 3.75 (s, 3H). LRMS (ESI) 358 (MH+).
(ii) N-Hydr oxy-3-[4-(3-biph en yl-4-ylu r eido)ph en yl]acr yl-
a m id e (21). To a solution of compound 20 (786 mg, 2 mmol)
in methanol (10 mL) was added at room temperature a 5%
sodium hydroxide solution (10 mL). The mixture was stirred
at room temperature for 4 h. The solvent was evaporated, and
the residue was diluted with water. The mixture was washed
with dichloromethane, acidified with 10% HCl, extracted with
ethyl acetate, and then dried over MgSO4. The solvent was
evaporated to give the product in 73% yield (554 mg). LRMS
(ESI) 341(M - H).
To a solution of carboxylic acid (316 mg, 1 mmol) in DMF
(10 mL) were added 1-(3-dimethylaminopropyl)-3-ethylcarbo-
diimide hydrochloride (248 mg 1.3 mmol) and hydroxybenzo-
triazole hydrate (162 mg, 1.2 mmol). The mixture was stirred
at room temperature for 2 h. Hydroxylamine hydrochloride
(348 mg, 5 mmol) was added followed by triethylamine (696
µL, 5 mmol). After the mixture was stirred at room-temper-
ature overnight, the solvent was evaporated. Water was added,
and the mixture was extracted with EtOAc. The organic layer
was washed with saturated solution of NaHCO3 and brine,
dried over MgSO4, and evaporated to dryness. The residue was
purified by column chromatography on silica gel using CH2-
(iv) [4-(Bip h en yl-4-su lfon yla m in o)p h en yl]p r op yn oic
Acid (24). A solution of aldehyde precursor (1.03 g, 2.85 mmol)
in tert-butyl alcohol/dichloromethane (3/1, 60 mL) was treated
with 2-methyl-2-butene (15 mL) followed by a freshly prepared
solution of NaH2PO4 (2.53 g, 18.3 mmol) and NaClO2 (2.53 g,
28 mmol) in water (24 mL), which was added dropwise during
12 min. The mixture was allowed to react for 17 h at room
temperature, diluted with ethyl acetate, and extracted with
saturated NaHCO3 and the organic layer discarded. The
aqueous layer then was acidified to pH ) 2 with solid KHSO4
and extracted with EtOAc, and the organic phase was dried
with MgSO4 and concentrated, to afford the acid 24 (1.075 g,
100% yield). 1H NMR (acetone-d6) δ 9.51 (bs, 1H), 7.92 (d, J )
8.8 Hz, 2H), 7.78 (d, J ) 8.8 Hz, 2H), 7.69 (d, J ) 8.7 Hz, 2H),
7.51 (d, J ) 8.7 Hz, 2H), 7.46-7.31 (m, 5H). 13C NMR (acetone-
d6) δ 154.4, 146.4, 141.2, 139.7, 139.2, 134.9, 129.9, 129.8,
129.3, 128.5, 128.4, 128.0, 120.3, 115.5, 85.7. HRMS (EI) calcd
for C21H16NO4S: 378.0800, found: 378.0809.
1
Cl2/MeOH (9/1) to give 21 in 35% yield. H NMR (CD3OD) δ
7.62-7.40 (m, 4H), 7.20 (s, 1H), 7.03-6.80 (m, 3H), 6.45 (d, J
) 16 Hz, 1H), 3.80 (s, 3H), 3.75 (s, 3H). LRMS (ESI) 358 (MH+).
N-(2-Am in op h en yl)- 3-[4-(bip h en yl-4-su lfon yla m in o)-
p h en yl]p r op yn a m id e (25). (i) 4-(4-Bip h en ylsu lfon yla m i-
n o)p h en yl Iod id e (22). A solution of biphenylsulfonyl chlo-
ride (3.35 g, 13.26 mmol) and 4-iodoaniline (7) (1.79 g, 8.15
mmol) in a mixture of acetonitrile/dichloromethane/pyridine
(4/2/1, 35 mL) stirred at 0 °C under nitrogen was treated with
triethylamine (4 mL) and slowly warmed to 10 °C during 16
h. Aqueous saturated sodium bicarbonate solution was then
added and the mixture stirred at room temperature for 3 h,
extracted with ethyl acetate, dried (MgSO4), filtered, and
concentrated. The crude residue was suspended in dry metha-
nol (100 mL), warmed to 60 °C, treated with 4.4 M NaOMe
solution in methanol (41 mmol), and stirred at the same
temperature for 4 h. After being cooled to 0 °C, 2 N HCl was
carefully added until pH ) 2 and extracted with ethyl acetate,
and the combined organic layers were washed (sat. NaHCO3),
dried, and concentrated. The crude was purified through
chromatographic column on silica gel (elution 50% to 100%
ether in hexanes) to afford 2.96 g (6.8 mmol, 83% yield) of
(v) N-(2-Am in op h en yl)- 3-[4-(bip h en yl-4-su lfon yla m i-
n o)p h en yl]p r op yn a m id e (25). A mixture of acid 24 (442 mg,
1.17 mmol) and BOP (728 mg, 1.65 mmol) was dissolved in
DMF (12 mL) and treated with triethylamine (0.7 mL, 5
mmol), followed by 1,2-phenylenediamine (295 mg, 2.73 mmol).
After 17 h at room temperature, the mixture was diluted with
ethyl acetate and washed with aqueous 5% KHSO4/brine then
saturated NaHCO3. The organic layer was dried (Na2SO4),
filtered, and concentrated. The crude mixture was purified by
flash chromatography on silica gel (elution 50% to 100% EtOAc
in hexane), to give 330 mg of 25 (0.69 mmol; 59% yield). 1H
NMR (acetone-d6) δ 9.43 (bs, 1H), 8.02 (d, J ) 8.5 Hz, 2H),
7.93 (d, J ) 8.5 Hz, 2H), 7.90 (d, J ) 8.5 Hz, 2H), 7.65 (d, J )
8.5 Hz, 2H), 7.47-7.34 (m, 7H), 7.21-7.17 (m, 2H). 13C NMR
(acetone-d6) δ 167.2, 158.6, 146.3, 141.3, 140.9, 139.8, 139.5,
134.2, 131.0, 129.9, 129.8, 129.3, 128.7, 128.6, 128.4, 128.0,
126.8, 125.1, 122.7, 103.6, 99.1. HRMS (EI) calcd for
1
sulfonamide 22. H NMR (CDCl3) δ 7.85 (d, J ) 8.7 Hz, 2H),
7.66 (d, J ) 8.7 Hz, 2H), 7.58-7.56 (m, 2H), 7.55 (d, J ) 8.7
Hz, 2H), 7.46-7.43 (m, 3H), 6.90 (d, J ) 8.7 Hz, 2H). 13C NMR
(CDCl3) δ 146.1, 138.9, 138.4, 137.2, 136.3, 129.9, 129.0, 128.6,
127.8, 127.7, 127.3, 123.2. LRMS (ESI): 436 (MH+), 458 (M +
Na).
C
27H21N3O3S: 467.1304, found: 467.1307.
5-(3-Ben zen esu lfon yla m in op h en yl)-p en t -2-en -4-yn o-
ic Acid (2-Am in op h en yl)a m id e (26). 1H NMR (CDCl3) δ
7.76 (d, J ) 7.5 Hz, 2H), 7.51 (t, J ) 7.5 Hz, 1H), 7.41 (t, J )
7.5 Hz, 1H), 7.29-6.95 (m, 7H), 6.86 (d, J ) 15.6 Hz, 1H), 6.73
(d, J ) 8.6 Hz, 2H), 6.50 (d, J ) 15.6 Hz, 1H). 13C NMR (CDCl3)
δ 163.61, 140.45, 138.97, 137.26, 132.79, 132.28, 129.17,
128.87, 128.04, 127.24, 126.80, 125.29, 125.22, 123.81, 123.13,
121.90, 121.54, 119.38, 117.95, 96.19, 86.99. LRMS (ESI) 418
(MH+).
(ii) Bip h en yl-4-su lfon ic Acid [4-(3-H yd r oxyp r op -1-
yn yl)p h en yl]a m id e (23). A mixture of iodosulfonamide 22
(2.63 g, 6.05 mmol), CuI (228 mg, 1.19 mmol), and Pd(PPh3)4
(376 mg, 0.325 mmol) was dissolved in degassed pyrrolidine
(22 mL) at room temperature, under nitrogen and at dark.
After the mixture was stirred 8 min, propargyl alcohol (1.2
mL, 20.5 mmol) was added and the mixture stirred for 20 h,
then poured on saturated NH4Cl, and extracted with ethyl
acetate. The organic layer was dried (Na2SO4), filtered,
concentrated, and purified by column chromatography on silica
gel (elution 5% MeOH in DCM) to give 23 as a yelow solid
(2.2 g, 100%). 1H NMR (acetone-d6) δ 7.89 (d, J ) 8.1 Hz, 2H),
(4-Hyd r oxyca r ba m oylben zyl)ca r ba m ic Acid P yr id in -
1
3-ylm eth yl Ester (27). H NMR (DMSO-d6) δ 8.57 (bs, 1H),
8.52-8.50 (m, 1H), 7.94-7.86 (m, 2H), 7.76 (bd, J ) 8.1 Hz,
1H), 7.69-7.65 (m, 1H), 7.42-7.37 (m, 1H), 7.30-7.25 (m, 2H),
5.70 (s, 2H), 4.23 (d, J ) 8.7 Hz, 2H). LRMS (ESI) 302 (MH+).

Histone Deacetylase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 829
N-P h en yl-3-[4-(biph en yl-4-su lfon ylam in o)ph en yl]acr yl-
a m id e (28). 1H NMR (CDCl3) δ 7.80 (d, J ) 8.5 Hz, 2H), 7.65-
7.55 (m, 5H), 7.50-7.30 (m, 6H), 7.10 (d, J ) 8.8 Hz, 3H), 6.90
(d, J ) 8.8 Hz, 3H), 6.45 (bd, J ) 15.0 Hz, 1H). LRMS (ESI)
455 (MH+).
Ack n ow led gm en t. The authors would like to thank
Isabelle Dupont and Eric Chan for cloning and expres-
sion of the HDAC enzyme.
Su p p or tin g In for m a tion Ava ila ble: HPLC purity data
on final compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
Biology. P r od u ction of Recom bin a n t HDAC1 En zym e.
Human HDAC1 cDNA was generated by RT-PCR reactions
using primers complementary to the 5′ and 3′ coding sequence
of human HDAC1 gene (Genbank accession number U50079).
Error-free HDAC1 cDNA were inserted either into pcDNA3.1
vector (Invitrogen) or into the pBlueBAC (Invitrogen) vector,
both with Flag epitope tagged at the C-terminus of the protein.
Insect High Five cells (Invitrogen) were used to produce
recombinant HDAC1. HDAC1 recombinant enzymes were
partially purified by a Q-Sepharose column (Pharmacia)
followed by purification using a column with anti-Flag M2
affinity gels (Sigma), according to manufacturer’s instructions.
HDAC En zym e Assa y. [3H]-Labeled acetylated histones
were prepared in J urkat-T cells as described12 and used as
HDAC enzyme substrates. Assay was performed as in the
literature.12 Briefly, small molecule inhibitors diluted in DMSO
at various concentrations were preincubated with recombinant
HDAC1 enzyme for 30 min at 4 °C in buffer containing 40 mM
Tris-Cl, pH 7.6, 20 mM EDTA, and 50% glycerol. At 37 °C,
[3H]-labeled acetylated histones were added into the reaction
mixture. Incubation time for HDAC1 enzyme reaction was 10
min. Reaction was stopped, and released [3H] acetic acid was
extracted and quantified by scintillation counting. The 50%
inhibitory concentrations (IC50) for inhibitors were determined
by analyzing dose-response inhibition curves.
Wester n Blot An a lysis. Whole cell extracts or acid ex-
tracted histones prepared from inhibitor-treated cells were
analyzed by SDS-PAGE. Proteins were transferred on the
PVDF membrane and probed with various primary antibodies.
Primary antibodies were ordered from Santa Cruz Biotech-
nology except for acetylated H4 or acetylated H3 antibodies
(Upstate Biotechnology), or antibodies against p21WAF1/Cip1
(Transduction Laborotories). Horseradish peroxidase-conju-
gated secondary antibodies (Sigma) were used and the en-
hanced chemiluminescence (ECL, Amersham) was followed for
detection.
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