Structurally simple trichostatin A-like straight chain hydroxamates as potent histone deacetylase inhibitors

Article abstract of DOI:10.1021/jm020154k

A series of new, structurally simple trichostatin A (TSA)-like straight chain hydroxamates were prepared and evaluated for their ability to inhibit partially purified human histone deacetylase 1 (HDAC-1). Some of these compounds such as 8m, 8n, 12, and 15

Full text of DOI:10.1021/jm020154k

J . Med. Chem. 2002, 45, 2877-2885
2877
Str u ctu r a lly Sim p le Tr ich osta tin A-Lik e Str a igh t Ch a in Hyd r oxa m a tes a s
P oten t Histon e Dea cetyla se In h ibitor s
Soon Hyung Woo,*,† Sylvie Frechette,† Elie Abou Khalil,† Giliane Bouchain,† Arkadii Vaisburg,†
Naomy Bernstein,† Oscar Moradei,† Silvana Leit,† Martin Allan,† Marielle Fournel,‡
Marie-Claude Trachy-Bourget,‡ Zuomei Li,‡ 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 April 8, 2002
A series of new, structurally simple trichostatin A (TSA)-like straight chain hydroxamates
were prepared and evaluated for their ability to inhibit partially purified human histone
deacetylase 1 (HDAC-1). Some of these compounds such as 8m , 8n , 12, and 15b exhibited
potent HDAC inhibitory activity with low nanomolar IC50 values, comparable to natural TSA.
These compounds induce hyperacetylation of histones in T24 human cancer cells and
significantly inhibit proliferation in various human cancer cells. They also induce expression
of p21 and cause cell cycle blocks in human cancer cells. In this paper, we describe the synthesis
of these new compounds as well as structure-activity relationship results from enzyme
inhibition and alterations in cellular function.
Ch a r t 1
In tr od u ction
Histone acetylases (HATs) and histone deacetylases
(HDACs) are a group of enzymes that catalyze acetyl-
ation and deacetylation of lysine residues in the N-
terminal tails of core histones.1 HDACs were identified
as transcriptional corepressors by restricting the acces-
sibility of a variety of transcription factors to DNA and
by mediating changes in nucleosome conformation.2
Inhibition of HDAC activity represents a novel approach
for intervening in cell cycle regulation; thus, HDAC
inhibitors could have great therapeutic potential in the
treatment of cell proliferative diseases.3 Several small
molecule inhibitors of HDAC, such as the natural
product trichostatin A (TSA)4 1 (Chart 1), the synthetic
Our synthesis began with the coupling of the readily
available copper reagent 6, derived from ethyl 7-bromo-
heptanoate 5, with aryl and heteroaryl acid chlorides
to afford a variety of aryl ketoesters of type 7. Aryl
ketoesters 7 served as convenient intermediates for
many of the hydroxamates described herein. The Suzuki
coupling of 7 (Ar ) p-Br-phenyl) with a variety of aryl
boronic acids of choice provided various biphenyl deriva-
tives 11. A variety of amino-substituted phenyl keto-
esters 14 could be also accessed by palladium-catalyzed
amination of 7 (Ar ) p-Br-phenyl). Hydrolysis of aryl
ketoesters (7, 11, and 14) and subsequent coupling of
the resulting carboxylic acids with hydroxylamine (1
equiv) under a typical peptide coupling condition (EDC/
HOBt) gave their respective aryl ketone hydroxamates
8, 12, and 15. Hydroxamic acids 9 bearing an oxime
moiety instead of a ketone were prepared in a similar
manner but using an excess of hydroxylamine. Aryl
oxime carboxylic acid 13 could also be prepared by
reacting the corresponding aryl ketone carboxylic acid
with hydroxylamine in pyridine. Wittig reaction of 7
with methyl triphenylphosphonium bromide followed by
the same two step transformation described above
afforded the desired olefinic hydroxamate 10. The
R-methylated hydroxamic acid 16 was prepared from 7
(Ar ) 2-naphthyl) in order to evaluate the steric
tolerance around the Zn-chelating hydroxamate moiety.
compounds suberoylanilide hydroxamic acid (SAHA)5 2,
6
and oxamflatin
3, have been reported to induce
differentiation of several cancer cell lines and to sup-
press cell proliferation.7
As part of our efforts to discover novel HDAC inhibi-
tors,8 we prepared a series of structurally simple TSA-
like straight chain hydroxamate analogues of 4, with
varying chain lengths, aryl substitutions, and aryl-
aliphatic chain connections (e.g., ketone, alkene, oxime,
etc). We now report the synthesis of these new com-
pounds and the resulting structure-activity relation-
ships (SAR) for HDAC inhibition and alterations in
cellular function.
Ch em istr y
To prepare new TSA-like straight chain hydroxamic
acid derivatives of type 4 having various chain lengths,
aryl substitutions, and aryl-aliphatic chain connections,
we chose a general synthetic strategy as outlined in
Scheme 1.
* To whom correspondence should be addressed. Tel.: +1-514-337-
3333. Fax: +1-514-337-0550. E-mail: delormed@methylgene.com.
† Department of Medicinal Chemistry.
‡ Department of Molecular Biology.
10.1021/jm020154k CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/18/2002

2878 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13
Woo et al.
Sch em e 1a
a
Conditions: (a) NaI, acetone, reflux, 83%. (b) (i) Zn, (CH2)2Br2, TMSCl, THF; (ii) LiCl, CuCN, reflux. (c) ArCOCl, THF, 0 °C. (d) 1 N
NaOH, THF-MeOH. (e) EDC, HOBt, NH2OH‚HCl (1.1 equiv), Et3N. (f) EDC, HOBt, NH2OH‚HCl (5 equiv), Et3N. (g) CH3PPh3+Br-,
n-BuLi, THF. (h) ArB(OH)2, Pd(PPh3)4, K2CO3, toluene, 100 °C. (k) (i) HO(CH2)2OH, BF3‚OEt2, HC(OCH3)3, CH2Cl2, 44%; (ii) LDA, CH3I,
THF, 91%; (iii) 1 N HCl, acetone, 50 °C.
Sch em e 2a
a
Conditions: (a) PPh3, toluene, 87%. (b) n-BuLi, THF, and then 2-naphthaldehyde, 34%. (c) 1 N NaOH, THF-MeOH, 60%. (d) EDC,
HOBt, NH2OH‚HCl (1.1 equiv), Et3N, 37%. (e) H2, Pd/C, MeOH, 97%.
Protection of the ketone as a cyclic ketal followed by
R-methylation of the ester group, using methyl iodide
and LDA as a base, furnished the R-methylated ester.
Acidic hydrolysis of the cyclic ketal and hydroxamate
formation as described above allowed the preparation
of hydroxamate 16.
to chelate with the zinc ion and active site residues.10
The aryl portion may act as a cap to bind the molecule
at the rim of the pocket and into an adjacent surface
groove. For rapid SAR development, we first prepared
a series of hydroxamates with different chain lengths
(4-7) between the hydroxamate-carbonyl and aryl
ketone group and evaluated their inhibition using
HDAC-1. Hydroxamate analogues with a chain length
of six carbons were most effective; hydroxamate ana-
logues with either shorter (8a ,b) or longer carbon chain
lengths (8d ) were much less effective in an order of 6 >
7 > 5 > 4 carbon length. The importance of the carbon
chain length in enzyme inhibition can be seen more
drastically in two p-methoxyphenyl hydroxamates, 8e,j,
as well as in two 2-naphthyl hydroxamates, 8f,m .
Analogue 8j with a six carbon chain is a strong inhibitor
of the enzyme with an IC50 of 15 nM, whereas the
analogue with a five carbon chain 8e has 30-fold weaker
inhibitory activity, with an IC50 of 450 nM. These results
suggest that our hydroxamate-based inhibitors with the
optimal six carbon methylene spacer may fit well in the
narrow, tubelike, hydrophobic portion of the active site
and make multiple van der Waals contacts. Thus, like
TSA, these inhibitors probably mimic acetylated lysine,
the natural substrate of core histone proteins.9
Finally, hydroxamates 18 and 19 (Scheme 2) were
readily prepared from ester 17, which in turn was
obtained from a Wittig reaction of 2-naphthylaldehyde
and the phosphonium ylide derived from ethyl 7-bromo-
heptanoate 5. Subsequent catalytic hydrogenation of the
alkene group in 18 gave the 2-naphthyl-alkylhydrox-
amic acid 19.
SAR a n d Biologica l Resu lts
En zym e In h ibition . All compounds were tested in
vitro for the inhibition of recombinant human HDAC-
1.9 We chose HDAC-1 since this isozyme has been
widely implicated in both transcriptional repression and
chromatin remodeling. The IC50 values for some repre-
sentative hydroxamates prepared are shown in Table
1.
Ch a in Len gth . X-ray crystallographic studies using
the HDLP-TSA complex have shown that TSA binds
by inserting its long aliphatic chain into the tubelike
active site pocket, thus allowing the hydroxamate group
Su bstitu tion on th e Ar yl Gr ou p . After the optimal
carbon chain length was established, we next examined

Trichostatin A-Like Straight Chain Hydroxamates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13 2879
Ta ble 1. HDAC-1 Inhibitory Activities
a
Values are means of three separate experiments ( standard deviation (SD).
the effect of substitution on the aryl group. Several
observations arise from examination of IC50 data from
Table 1: (i) an ortho-methoxy substitutent, as in 8i, does
not improve enzyme inhibition relative to the nonsub-
stituted analogue 8c, whereas the corresponding para-
methoxy analogue 8j is 4-5 times more potent than 8c;
(ii) an electron-withdrawing substituent, p-trifluoro-
methyl or p-bromo, has little effect on enzyme inhibitory
activity (cf. 8k ,l as compared to 8c); (iii) the introduction
of an additional hydrophobic aryl group as in 8m ,n and
12 enhances enzyme inhibitory activity significantly.
The most potent HDAC inhibitor in the present series
is the p-bromo biphenyl hydroxamate 12 with an IC50
of 2 nM, as compared to the 5 nM IC50 value for natural
TSA. It is noteworthy that none of the parent aryl
ketone carboxylic acids are active but that the biphenyl

2880 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13
Woo et al.
Ta ble 2. Inhibition of Cell Proliferation (MTT Assays) of 8m , 8n , 12, and 15b in Human Normal and Cancer Cellsa
compds
T24
HCT 116
SW48
A549
H446
DU145
MDAmb 231
MCF-7
HMEC
TSA
8m
8n
12
15b
0.15 ( 0.05
17 ( 4
<0.05
0.1 ( 0.01
6.5 ( 1.5
1.5 ( 0.5
3 ( 0.1
0.16 ( 0.04
29 ( 8
0.08 ( 0.02
1.5 ( 0.5
0.65 ( 0.15
0.7 ( 0.2
1 ( 0.1
0.2 ( 0.1
29 ( 8
0.09 ( 0.01
0.95 ( 0.05
0.6 ( 0.2
0.05 ( 0.01
0.8 ( 0.2
0.1 ( 0.01
25 ( 2
2 ( 1
3 ( 0.5
2 ( 1
1.6 ( 0.4
4 ( 1
1.5 ( 0.5
4.5 ( 0.5
0.7 ( 0.2
4 ( 0.1
1 ( 0.1
1 ( 0.1
0.9 ( 0.1
1 ( 0.1
2 ( 1
0.8 ( 0.02
7 ( 1
0.3 ( 0.05
5 ( 1
7 ( 0.5
>20
2.5 ( 0.5
5.5 ( 0.5
a
Substance concentration (µM) required to reduce tumor cell growth by 50%.
F igu r e 2. Induction of p21 expresssion by HDAC Inhibitors.
Human bladder carcinoma T24 cells were treated with 1%
DMSO or HDAC inhibitors in 1% DMSO for 16 h before total
cell lysates were harvested for Western blot analysis using
antibodies specific for p21. The amount of actin protein was
also determined by Western blot as an indication for the
sample loading on each lane of the gel.
F igu r e 1. Histone H3 and H4 acetylation in human bladder
cancer T24 cells induced by compounds 8n , 12, and 15b.
Histones were extracted from T24 cells that had been exposed
to 8n , 12, and 15b at 0, 1, 5, and 25 µM for 16 h and analyzed
by Western blot with antibodies specific for either acetylated
H4 or acetylated H3 histones. The bottom panel (coomassie
staining of the gels) indicates the loading of histones.
We also evaluated the in vitro antiproliferative activi-
ties of all of the above compounds using 3-[4,5-dimeth-
ylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)
assays against eight various human cancer cell lines
(T24, HCT116, SW48, A549, H446, Du145, MDAmb231,
and MCF-7) and a normal human epithelial cell line
(HMEC). All compounds exhibit antiproliferative activi-
ties against human cancer cells with varying degrees
of potency. Among them, compound 12 had the most
potent antiproliferative activities against various hu-
man cancer cells, with IC50 values ranging from 0.05 to
3 µM. In contrast, compound 8m was the weakest on
growth inhibition of cancer cells, with IC50 values
ranging from 1 to 30 µM. Compounds 12, 15b, and TSA
showed selective growth inhibition of human cancer
cells over human normal cells. However, compounds
8m ,n showed no evidence of selective growth inhibition
(Table 2).
Similar to other known HDAC inhibitors, all four of
the above compounds induced p21 protein expression
in T24 human cancer cells. Their abilities to induce p21
correlated well with their abilities to induce histone
acetylation, with the order of potency being 12 > 8n >
15b . 8m (Figure 2). Likewise, compounds 12, 8n , and
15b induced both G1 and G2/M cell cycle arrests at
concentrations equal to or less than 5 µM in human
cancer cells (data not shown).
oxime carboxylic acid 13 has been found to inhibit
HDAC-1, with an IC50 of 200 nM.
Ar yl-Alip h a tic Ch a in Con n ection . To assess the
importance or restriction of an aryl-aliphatic chain
connection in HDAC-1 inhibition, several other func-
tional groups such as an oxime (9), an exo methylene
(10), and an internal alkene (18) were introduced in
place of the ketone. Interestingly, such modifications do
not affect enzyme inhibitory activity (cf. 8m vs 9a , 10,
18, and 8n vs 9b). However, 8-(2-naphthyl)octanoyl
hydroxamic acid (19) or the corresponding secondary
alcohol (20) have lower enzymatic inhibitory activity (cf.
8m vs 19 and 20). These results suggest that an sp2
center at this junction may allow the inhibitor better
interaction at the rim of the active site by forming an
appropriate 110° angle between the adjacent aryl group
and the aliphatic side chain.
Finally, introduction of an R-methyl group adjacent
to the hydroxamate moiety resulted in a considerable
decrease of HDAC inhibitory activity. The R-methylated
derivative 16 (IC50 ) 600 nM) is 120 times less potent
than the corresponding unsubstituted compound 8m
(IC50 ) 5 nM). This result demonstrates that even a
small group may not be tolerable near the zinc binding
site of HDAC-1, and this is in good agreement with the
proposed tubelike narrow active site pocket of HDAC.10a
HDAC In h ibitor s in Cell-Ba sed Assa ys. To con-
firm the ability of our hydroxamates to inhibit HDAC
in cells, we chose 8m ,n , 12, and 15b to evaluate the
induction of histone acetylation in human bladder T24
cancer cells. Three compounds (8n , 12, and 15b) caused
histone hyperacetylation in human cancer cells in a
dose-dependent manner (Figure 1). The ability of these
four compounds to induce histone acetylation in cells
correlated largely with their enzyme inhibitory activities
against HDAC-1 in vitro, with the exception that the
naphthyl derivative 8m induced histone acetylation in
cells weakly. The p-bromobiphenyl compound 12 was
the most effective, being as potent as TSA on induction
of histone acetylation in human cancer T24 cells.
In summary, we have designed and synthesized a
series of structurally simple TSA-like straight chain
hydroxamates. Several of these new compounds were
as potent as TSA in inhibiting HDAC-1 and were
capable of inducing histone acetylation, inducing ex-
pression of p21, and causing cell cycle arrest and
inhibition of cell proliferation. The SAR obtained in this
study may aid in designing nonhydroxamate inhibitors
of HDAC.
Exp er im en ta l Section
Ch em istr y. Nuclear magnetic resonance spectra (1H NMR
and 13C NMR) were recorded on a Varian 300 MHz spectrom-
eter. Chemical shifts (δ) are reported in parts per million (ppm)
relative to solvent peaks. Mass spectra were obtained on a
HP1100 MSD instrument. Elemental analyses were within
(0.4% of calculated values. All reactions were run under a
positive pressure of nitrogen unless otherwise stated. Flash

Trichostatin A-Like Straight Chain Hydroxamates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13 2881
Compounds 8a ,b,d -n were all prepared in manner
similar or identical to that described for compound 8c.
N-Hyd r oxy-5-(ben zoyl)p en ta n a m id e (8a ). 1H NMR (20%
CD3OD in CDCl3): δ 7.86 (d, J ) 6.9 Hz, 2H), 7.59-7.43 (m,
3H), 3.01 (br t, 2H), 2.18 (br t, 2H), 1.72 (m, 4H). 13C NMR
(20% CD3OD in CDCl3): δ 23.24, 24.78, 32.39, 37.79, 127.76,
128.36, 133.00, 136.38, 170.85, 200.82. Anal. (C12H15NO3) C,
H, N.
N-Hyd r oxy-6-(ben zoyl)h exa n a m id e (8b). 1H NMR (20%
CD3OD in CDCl3): δ 7.94 (d, J ) 7.5 Hz, 2H), 7.61-7.40 (m,
3H), 2.98 (t, J ) 6.9 Hz, 2H), 2.15 (t, J ) 6.6 Hz, 2H), 1.80-
1.60 (m, 4H), 1.41 (m, 2H). 13C NMR (20% CD3OD in CDCl3):
δ 23.48, 25.04, 28.37, 32.39, 38.08, 127.87, 128.45, 133.03,
136.57, 171.16, 201.05. Anal. (C13H17NO3) C, H, N.
N-Hyd r oxy-8-(ben zoyl)octa n a m id e (8d ). 1H NMR (20%
CD3OD in CDCl3): δ 7.86 (d, J ) 7.5 Hz, 2H), 7.51-7.35 (m,
3H), 2.89 (t, J ) 6.9 Hz, 2H), 2.02 (t, J ) 6.6 Hz, 2H), 1.70-
1.45 (m, 4H), 1.26 (m, 6H). 13C NMR (20% CD3OD in CDCl3):
δ 23.95, 25.15, 28.59, 28.74, 288.78, 32.55, 38.27, 127.79,
128.36, 132.91, 136.55, 171.27, 201.32. Anal. (C15H21NO3) C,
H, N.
N-Hyd r oxy-6-(4-m eth oxyben zoyl)h exa n a m id e (8e). 1H
NMR (20% CD3OD in CDCl3): δ 7.86 (d, J ) 8.7 Hz, 2H), 6.88
(d, J ) 8.7 Hz, 2H), 3.81 (s, 3H), 2.87 (t, J ) 6.6 Hz, 2H), 2.07
(br t, 2H), 1.62 (m, 4H), 1.34 (m, 2H). 13C NMR (20% CD3OD
in CDCl3): δ 23.68, 24.96, 28.32, 32.28, 37.63, 55.14, 113.49,
129.48, 130.13, 163.35, 171.06, 199.81. Anal. (C14H19NO4) C,
H, N.
column chromatography was performed with silica gel 60
(230-400 mesh). Analytical high-performance liquid chroma-
tography (HPLC) was performed on a HP1100 instrument
equipped with a Zorbax (50 mm × 4.6 mm) C-8 column. The
mobile phases used were as follows: A, H2O with 0.01 M
ethylenediaminetetraacetic acid (EDTA) + formic acid to pH
5; B, MeOH with 0.05% formic acid, using a solvent gradient
of A/B 20/80 to 95/5. The analyses were performed at 25 °C
with a constant flow rate of 1 mL/min using a gradient elution
of 0-10 min.
P r ep a r a tion of Or ga n ocop p er Rea gen t (6). To a sus-
pension of zinc foil (3.40 g, 52 mmol) in tetrahydrofuran (THF,
4 mL) was added 1,2-dibromoethane (0.38 g, 2.0 mmol), and
the mixture was heated to 65 °C for 1 min. After it was cooled
to room temperature, chlorotrimethylsilane (0.2 mL, 1.6 mmol)
was added, and the mixture was stirred for 15 min. A solution
of ethyl 7-iodoheptanoate (14.21 g, 50 mmol) in THF (20 mL)
was added slowly at room temperature, and the mixture was
heated to 60 °C for 16 h and then cooled to -10 °C. To this, a
solution of CuCN (3.96 g, 44 mmol) and anhydrous LiCl (3.80
g, 88 mmol) in THF (44 mL) was added via cannula. The
resulting solution was stirred at 0 °C for 10 min and then used
for the coupling reactions with various acid chlorides.
a
Gen er a l P r oced u r e for Con ver sion of 6 to 8. Eth yl
7-ben zoylh ep ta n oa te (7c). To a stirring solution of organo-
copper reagent 6 (2.0 mmol) in THF (4 mL) at -25 °C was
added benzoyl chloride (253 mg, 0.21 mmol), and the reaction
mixture was warmed to 0 °C over 30 min. After it was stirred
for an additional 3 h at 0 °C, the reaction mixture was
quenched with a saturated NH4Cl solution and then extracted
with ethyl acetate. The combined organic extracts were washed
with brine, dried, and concentrated under reduced pressure.
The crude product was purified by flash chromatography (10%
ethyl acetate in hexanes) to afford the title compound 7c (Ar
) phenyl) (376 mg, 80% yield). 1H NMR (CDCl3): δ 7.42-7.97
(m, 5H), 4.12 (q, J ) 6.9 Hz, 2H), 2.97 (t, J ) 7.2 Hz, 2H),
2.30 (t, J ) 7.5 Hz, 2H), 1.74 (m, 2H), 1.65 (m, 2H), 1.39 (m,
4H), 1.25 (t, J ) 6.9 Hz, 3H). 13C NMR (CDCl3): δ 14.19, 24.04,
24.75, 28.90 (2), 34.12, 38.39, 60.11, 127.96, 128.49, 132.84,
136.95, 173.70, 200.29.
N-Hyd r oxy-6-(2-n a p h th oyl)h exa n a m id e (8f). 1H NMR
(20% CD3OD in CDCl3): δ 8.47 (s, 1H), 8.05-7.50 (m, 6H),
3.12 (br t, 2H), 2.15 (br t, 2H), 1.82-1.60 (m, 4H), 1.46 (m,
2H). 13C NMR (20% CD3OD in CDCl3): δ 23.59, 25.02, 28.36,
32.33, 38.06, 123.40, 126.56, 127.46, 128.21, 128.29, 129.29,
129.60, 132.25, 133.78, 135.37, 171.04, 201.04. Anal. (C17H19
NO3) C, H, N.
-
N-Hyd r oxy-7-(2-th iop h en e)ca r bon ylh ep ta n a m id e (8h ).
1H NMR (20% CD3OD in CDCl3): δ 7.76 (d, J ) 2.7 Hz, 1H),
7.68 (d, J ) 4.5 Hz, 1H), 7.16 (m, 1H), 2.92 (d, J ) 7.5 Hz,
2H), 2.10 (t, J ) 7.5 Hz, 2H), 1.74 (m, 2H), 1.64 (m, 2H), 1.36
(m, 4H). Anal. (C12H17NO3S) C, H, N.
N-Hyd r oxy-7-(2-m eth oxyben zoyl)h ep ta n a m id e (8i). 1H
NMR (20% CD3OD in CDCl3): δ 7.81 (dd, J ) 8.1, 1.8 Hz, 1H),
7.47 (m, 1H), 7.02-6.97 (m, 2H), 3.90 (s, 3H), 2.97 (t, J ) 7.2
Hz, 2H), 2.10 (t, J ) 7.2 Hz, 2H), 1.74-1.56 (m, 4H), 1.36 (m,
4H). 13C NMR (20% CD3OD in CDCl3): δ 23.89, 25.05, 28.68,
28.60, 32.50, 43.28, 55.17, 111.35, 120.33, 128.03, 129.69,
133.26, 158.18, 171.18, 204.09. Anal. (C15H21NO4) C, H, N.
N-Hydr oxy-7-(p-an isoyl)h eptan am ide (8j). 1H NMR (20%
CD3OD in CDCl3): δ 7.94 (d, J ) 9.0 Hz, 2H), 6.95 (d, J ) 9.0
Hz, 2H), 3.88 (s, 3H), 2.93 (t, J ) 7.2 Hz, 2H), 2.11 (t, J ) 7.5
Hz, 2H), 1.59-1.79 (m, 4H), 1.37 (m, 4H). 13C NMR (20% CD3-
OD in CDCl3): δ 24.15, 25.04, 28.59 (2), 32.51, 37.88, 55.21,
N-Hyd r oxy-7-ben zoylh ep ta n a m id e (8c). To a stirring
solution of 7c (Ar ) phenyl) (610 mg, 2.3 mmol) in THF (3.5
mL) and MeOH (3.5 mL) at room temperature was added 1 N
NaOH (3.5 mL, 3.5 mmol), and the mixture was stirred for 2
h. After organic solvents were removed, the resulting aqueous
solution was acidified with 1 N HCl and then extracted with
ethyl acetate. The combined organic layers were dried and then
concentrated under reduced pressure to give the crude product.
Purification by flash chromatography (7% methanol in chloro-
form) afforded 7-benzoyl heptanoic acid (511 mg, 94% yield)
as a white solid. 1H NMR (CDCl3): δ 7.96 (d, J ) 6.9 Hz, 2H),
7.41-7.61 (m, 3H), 2.97 (t, J ) 7.5 Hz, 2H), 2.36 (t, J ) 7.5
Hz, 2H), 1.75 (m, 2H), 1.66 (m, 2H), 1.40 (m, 4H). 13C NMR
(CDCl3): δ 24.01, 24.43, 28.80, 28.86, 33.92, 38.37, 127.98,
128.50, 132.88, 136.91, 179.95, 200.44.
113.54, 129.58, 130.20, 163.39, 171.10, 200.06. Anal. (C15H21
NO4) C, H, N.
-
N -H y d r o x y -7-(4-t r iflu o r o m e t h y lb e n zo y l)h e p t a n -
1
a m id e (8k ). H NMR (20% CD3OD in CDCl3): δ 8.07 (d, J )
To a stirring solution of 7-benzoyl heptanoic acid (200 mg,
0.85 mmol) at room temperature in anhydrous dimethyl-
formamide (DMF, 5 mL) was added 1-hydroxybenzotriazole
hydrate (149 mg, 1.11 mmol) followed by 1-(3-dimethylamino-
propyl)-3-ethyl-carbodiimide hydrochloride (191 mg, 1.28 mmol).
After 1 h, hydroxylamine hydrochloride (66 mg, 0.94 mmol)
and Et3N (130 µL, 0.94 mmol) were added, and stirring was
continued at room temperature overnight. The solvent was
removed in vacuo, and the residue was diluted with ethyl
acetate and then was washed with saturated aqueous NaH-
CO3. After it was dried and concentrated, the crude product
was purified by flash chromatography (2-10% methanol in
chloroform) to give 8c (98 mg, 46% yield). 1H NMR (20% CD3-
OD in CDCl3): δ 7.95 (d, J ) 7.2 Hz, 2H), 7.57 (m, 1H), 7.47
(m, 2H), 2.98 (d, J ) 7.5 Hz, 2H), 2.13 (m, 2H), 1.72 (m, 2H),
1.64 (m, 2H), 1.38 (m, 4H). 13C NMR (20% CD3OD in CDCl3):
δ 23.77, 24.98, 28.49 (2), 32.49, 38.13, 127.73, 128.29, 132.86,
136.46, 171.23, 201.16. Anal. (C14H19NO3) C, H, N.
8.4 Hz, 2H), 7.74 (d, J ) 8.4 Hz, 2H), 3.02 (t, J ) 7.5 Hz, 2H),
2.12 (t, J ) 7.5 Hz, 2H), 1.75 (m, 2H), 1.65 (m, 2H), 1.40 (m,
4H). 13C NMR (20% CD3OD in CDCl3): δ 23.57, 25.03, 28.51,
28.59, 32.50, 38.50, 123.37 (q, J CF ) 273 z), 125.43 (q, J CF
)
3.7 Hz), 128.18, 134.09 (q, J CF ) 32.6 Hz), 139.32, 171.15,
199.93. Anal. (C15H18F3NO3) C, H, N.
N-Hyd r oxy-7-(4-br om oben zoyl)h ep ta n a m id e (8l). 1H
NMR (20% CD3OD in CDCl3): δ 7.82 (d, J ) 7.5 Hz, 2H), 7.62
(d, J ) 7.5 Hz, 2H), 2.95 (t, J ) 7.2 Hz, 2H), 2.11 (m, 2H),
1.55-1.79 (m, 4H), 1.38 (m, 4H). Anal. (C14H18BrNO3) C, H,
N.
N-Hyd r oxy-7-(2-n a p h th oyl)h ep ta n a m id e (8m ). 1H NMR
(20% CD3OD in CDCl3): δ 8.47 (s, 1H), 7.53-8.01 (m, 6H),
3.10 (t, J ) 7.5 Hz, 2H), 2.12 (m, 2H), 1.78 (m, 2H), 1.66 (m,
2H), 1.42 (m, 4H). 13C NMR (20% CD3OD in CDCl3): δ 24.01,
25.07, 28.62(2), 32.54, 38.27, 123.47, 126.56, 127.48, 128.22,
128.28, 129.03, 129.60, 132.27, 133.86, 135.37, 171.19, 201.18.
Anal. (C18H21NO3) C, H, N.

2882 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13
Woo et al.
N-Hydr oxy-7-[(4-biph en yl)ca r bon yl]h epta n a m ide (8n ).
1H NMR (20% CD3OD in CDCl3): δ 8.03 (d, J ) 7.8 Hz, 2H),
7.70 (d, J ) 7.8 Hz, 2H), 7.64 (d, J ) 7.2 Hz, 2H), 7.55-7.38
(m, 3H), 3.02 (t, J ) 6.9 Hz, 2H), 2.12 (t, J ) 7.2 Hz, 2H), 1.75
(m, 2H), 1.66 (m, 2H), 1.41 (m, 4H). 13C NMR (20% CD3OD in
CDCl3): δ 23.96, 25.06, 28.61(2), 32.54, 38.29, 126.98, 127.02,
128.02, 128.47, 128.71, 135.26, 139.55, 145.66, 171.09, 200.92.
Anal. (C20H23NO3) C, H, N.
7m (Ar ) 2-naphthyl) (82 mg, 0.262 mmol) in THF (4 mL)
was added via cannula. The yellow mixture was stirred over
30 min and quenched with a saturated aqueous solution of
NH4Cl. The solvent was removed by evaporation in vacuo, and
the aqueous residue was then extracted with ethyl acetate.
The combined organic layers were washed with brine, dried
(MgSO4), and concentrated. The crude residue was purified
by flash chromatography (5-8% diethyl ether in hexane) to
afford the title compound (43 mg) in 52% yield. 1H NMR
(CDCl3): δ 7.85-7.78 (m, 4H), 7.58 (dd, J ) 8.8, 1.9 Hz, 1H),
7.48-7.44 (m, 2H), 5.42 (d, J ) 1.1 Hz, 1H), 5.16 (d, J ) 1.4
Hz, 1H), 4.12 (q, J ) 7.1 Hz, 2H), 2.62 (t, J ) 6.9 Hz, 2H),
2.28 (d, J ) 7.4 Hz, 2H), 1.64-1.49 (m, 4H), 1.39-1.33 (m,
4H), 1.25 (t, J ) 7.1 Hz, 3H). LRMS (ESI) ) 311 (MH+).
N-Hyd r oxy-7-[(3-p yr id yl)ca r bon yl]h ep ta n a m id e (8g).
(i) Meth yl-7-[3-(p yr id yl)ca r bon yl]h ep ta n oa te (7g). To a
stirring solution of 3-bromopyridine (1.58 g, 10 mmol) in
anhydrous ethyl ether (100 mL) at -78 °C was added n-
butyllithium (1.6 M in hexane, 7.5 mL), and the resulting
solution was stirred at -78 °C for 10 min. Trimethyltin
chloride (2.39 g, 12 mmol) was added, and the mixture was
stirred at -78 °C for 20 min and then warmed to room
temperature over 1 h. The reaction mixture was diluted with
hexanes (30 mL) and washed with water. The organic phase
was dried and concentrated to provide the crude product,
which was purified by vacuum distillation (125 °C at 15-20
mm Hg) to give 3-trimethylstannyl pyridine (1.93 g, 80% yield).
(ii) N-Hyd r oxy-8-(2-n a p h th yl)n on -8-en a m id e (10). The
above compound was then transformed into 10 in a manner
1
similar to that described for compound 8c. H NMR (CDCl3):
δ 8.22 (bs, 1H), 7.80-7.77 (m, 4H), 7.56 (d, J ) 8.5 Hz, 1H),
7.46-7.44 (m, 2H), 5.39 (s, 1H), 5.13 (s, 1H), 2.59 (t, J ) 6.6
Hz, 2H), 2.05 (bs, 2H), 1.56-1.11 (m, 8H). HRMS: calcd,
297.1729; found, 297.1744.
N -H yd r oxy-7-[4-(4-b r om op h e n yl)b e n zoyl]h e p t a n a -
m id e (12). (i) Eth yl-7-[4-(4-br om op h en yl)ben zoyl]h ep -
ta n oa te (11). To a mixture of 4-bromophenyl boronic acid (1.21
g, 6.0 mmol) and 7l (Ar ) 4-bromophenyl) (2.04 g, 6.0 mmol)
were added toluene (70 mL) and ethanol (3 mL) followed by a
2 M Na2CO3 aqueous solution (6.3 mL, 12.6 mmol). The
resulting mixture was degassed for 10 min, and then, Pd-
(PPh3)4 (0.35 g, 0.3 mmol) was added. After 16 h of reflux, the
mixture was cooled to room temperature, filtered through a
pad of Celite, and rinsed with ethyl acetate. The organic
solution was washed with brine, dried (MgSO4), and concen-
trated. Purification by flash chromatography (10% methanol
in dichloromethane) followed by crystallization from ethanol
and chloroform afforded the ester 11 (Ar ) 4-bromophenyl)
A mixture of 3-trimethylstannyl pyridine prepared above
(1.89 g, 7.81 mmol) and methyl 8-chloro-8-oxooctanoate in
benzene (20 mL) was heated overnight at reflux in the
presence of PdCl2(PPh3)2 (274 mg, 0.39 mmol). After it was
cooled to room temperature, the reaction mixture was parti-
tioned between ethyl acetate and water. The combined organic
layers were dried and concentrated. Purification by flash
chromatography (50% ethyl acetate in hexane) yielded the
desired product 7g (Ar ) 3-pyridyl) (1.15 g, 59% yield) as a
light yellow solid. 1H NMR (CD3OD): δ 9.16 (d, J ) 1.8 Hz,
1H), 8.77 (dd, J ) 4.8, 1.8 Hz, 1H), 8.23 (m, 1H), 7.43 (dd, J )
7.8, 4.8 Hz, 1H), 3.67 (s, 3H), 2.99(t, J ) 7.5 Hz, 2H), 2.32 (t,
J ) 7.5 Hz, 2H), 1.77 (m, 2H), 1.65 (m, 2H), 1.41 (m, 4H). 13C
NMR (CDCl3): δ 23.66, 24.66, 28.78, 28.84, 33.89, 38.67, 51.41,
123.58, 132.11, 135.27, 149.54, 153.31, 174.06, 198.96.
1
(1.30 g, 52% yield). H NMR (20% CD3OD in CDCl3): δ 8.02
(d, J ) 8.7 Hz, 2H), 7.75-7.40 (m, 6H), 4.13 (q, J ) 6.9 Hz,
2H), 3.01 (t, J ) 6.9 Hz, 2H), 2.32 (t, J ) 7.2 Hz, 2H), 1.75 (m,
2H), 1.66 (m, 2H), 1.41 (m, 4H), 1.26 (t, J ) 6.9 Hz, 3H).
(ii) N-H yd r oxy-7-[3-(p yr id yl)ca r b on yl]h ep t a n a m id e
(8g). Compound 8g was prepared from 7g (Ar ) 3-pyridyl) in
an identical manner to that described for compound 8c. 1H
NMR (20% CD3OD in CD3OD): δ 9.12 (d, J ) 1.5 Hz, 1H),
8.74 (dd, J ) 4.8, 1.5 Hz, 1H), 8.30 (m, 1H), 7.50 (dd, J ) 7.8,
4.8 Hz, 1H), 3.02 (t, J ) 7.5 Hz, 2H), 2.12 (t, J ) 7.5 Hz, 2H),
1.75 (m, 2H), 1.65 (m, 2H), 1.40 (m, 4H). 13C NMR (20% CD3-
OD in CD3OD): δ 23.39, 25.00, 28.44, 28.53, 32.48, 38.46,
123.86, 132.17, 135.80, 148.83, 152.61, 171.06, 199.31. Anal.
(C13H18N2O3) C, H, N.
(ii) N-Hyd r oxy-7-[4-(4-br om op h en yl)ben zoyl]h ep ta n a -
m id e (12). The above compound 11 (Ar ) 4-bromophenyl) was
then transformed into 12 in a manner similar to that described
1
for compound 8c. H NMR (DMSO-d6): δ 10.33 (s, 1H), 8.66
(s, 1H), 8.04-7.70 (m, 8H), 3.04 (m, 2H), 1.94 (m, 2H), 1.62
(m, 2H), 1.49 (m, 2H), 1.30 (m, 4H). 13C NMR (DMSO-d6): δ
23.72, 24.98, 28.43 (2), 32.23, 37.90, 121.93, 126.79, 128.64,
129.03, 131.96, 135.78, 138.07, 143.06, 169.06, 199.57.
HRMS: calcd, 385.0677 (M-H2O); found, 385.0685. Anal.
(C20H22BrNO3) C, H, N.
N-H yd r oxy-8-(E)-oxim in o-8-(2-n a p h t h yl)oct a n a m id e
(9a ). Following the procedure described above for compound
8c, but using 5 equiv of NH2OH‚HCl and triethylamine each,
to afford 9a from the corresponding carboxylic acid in 40%
(E)-Oxim in o-8-(4-bip h en yl)octa n oic Acid (13). A mix-
ture of 7-[4-(4-phenyl)benzoyl]heptanoic acid (53 mg, 0.171
mmol) and NH2OH‚HCl (60 mg, 0.854 mmol) was heated at
50 °C in pyridine (0.5 mL) overnight. After excess of pyridine
was removed, the residue was taken up in water (5 mL),
acidified with 1 N HCl (pH ca. 4), and extracted with ethyl
acetate. The combined organic layers were dried and then
concentrated under reduced pressure to give the crude product.
Purification by flash chromatography (10% methanol in chloro-
form) afforded the title compound (50 mg, 89% yield) as a white
1
yield. H NMR (20% CD3OD in CDCl3): δ 8.00 (s, 1H), 7.90-
7.40 (m, 6H), 2.91 (t, J ) 7.2 Hz, 2H), 2.07 (t, J ) 6.9 Hz, 2H),
1.61 (m, 4H), 1.38 (m, 4H). 13C NMR (20% CD3OD in CDCl3):
δ 25.03, 25.46, 25.86, 28.37, 28.92, 32.39, 123.43, 125.38,
125.90, 126.08, 127.15, 127.60, 127.94, 132.79, 133.14, 133.17,
159.01, 171.17. Anal. (C18H22N2O3) C, H, N.
N-H yd r oxy-8-(E)-oxim in o-8-(4-b ip h en yl)oct a n a m id e
(9b). Following the procedure described above for compound
8c, but using 5 equiv of NH2OH‚HCl and triethylamine each,
to afford 9b from the corresponding carboxylic acid in 45%
1
solid. H NMR (20% CD3OD in CDCl3): δ 7.35-7.68 (m, 9H),
2.83 (t, J ) 7.8 Hz, 2H), 2.29 (t, J ) 7.5 Hz, 2H), 1.62 (m, 2H),
1.60 (m, 2H), 1.40 (m, 4H). 13C NMR (20% CD3OD in CDCl3):
δ 24.55, 25.92, 28.58 (2), 29.29, 33.79, 126.52, 126.75, 126.86,
127.31, 128.58, 134.76, 140.17, 141.46, 159.20, 176.54. Anal.
(C20H24N2O3) C, H, N.
1
yield. H NMR (20% CD3OD in CDCl3): δ 7.25-7.67 (m, 9H),
7.36 (m, 3H), 2.82 (t, J ) 7.5 Hz, 2H), 2.06 (br t, J ) 6.9 Hz,
2H), 1.59 (m, 4H), 1.37 (m, 4H). 13C NMR (20% CD3OD in
CDCl3): δ 25.13, 25.78, 25.88, 28.47, 29.00, 32.58, 126.51,
126.72, 126.85, 127.33, 128.59, 134.72, 140.14, 141.47, 159.17,
171.24. Anal. (C18H22N2O3) C, H, N.
N-Hyd r oxy-7-[4-(1-p ip er id in yl)ben zoyl]h ep t a n a m id e
(15a ). (i) E t h yl-7-[4-(1-p ip er id in yl)b en zoyl]h ep t a n oa t e
(14a ). To a mixture of 7l (Ar ) 4-bromophenyl) (1.03 g, 3.02
mmol), Pd2(dba)3 (0.14 g, 0.15 mmol), (()BINAP (0.28 g, 0.45
mmol), and Cs2CO3 (1.37 g, 4.2 mmol) dissolved in dry toluene
(2 mL) was added piperidine (0.9 mL, 9.12 mmol). The mixture
was degassed for 10 min by bubbling with a nitrogen stream
and then heated to 100 °C for 24 h. After it was cooled to room
temperature, the reaction mixture was partitioned between
N-Hyd r oxy-8-(2-n a p h th yl)n on -8-en a m id e (10). (i) Eth yl
8-(2-n a p h th yl)n on -8-en oa te. To a solution of methyltri-
phenylphosphonium bromide (469 mg, 1.31 mmol) in THF (6
mL), precooled to 0 °C under nitrogen, was added n-butyl-
lithium (1.42 M solution in hexanes, 905 µL, 1.28 mmol)
dropwise. The mixture was stirred over 20 min. A solution of

Trichostatin A-Like Straight Chain Hydroxamates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13 2883
ethyl acetate and water. The combined organic layers were
dried (MgSO4) and concentrated. Purification by flash chro-
matography (25% ethyl acetate in hexanes) gave 14a in 40%
7.81 (m, 3H), 7.54 (dd, J ) 8.8, 1.9 Hz, 1H), 7.51-7.45 (m,
2H), 4.12-4.03 (m, 4H), 3.86-3.78 (m, 2H), 2.35 (sext, J )
7.1 Hz, 1H), 1.99-1.94 (m, 2H), 1.60-1.54 (m, 2H), 1.35-1.19
(m, 9H), 1.09 (d, J ) 6.9 Hz, 3H). LRMS (ESI) ) 371 (MH+).
1
yield. H NMR (CDCl3): δ 7.81 (d, J ) 9.0 Hz, 2H), 6.80 (d, J
) 9.0 Hz, 2H), 4.07 (q, J ) 7.2 Hz, 2H), 3.30 (m, 4H), 2.81 (t,
J ) 7.5 Hz, 2H), 2.24 (t, J ) 7.5 Hz, 2H), 1.74-1.56 (m, 10H),
1.20 (m, 4H), 1.24 (t, J ) 7.2 Hz, 3H). 13C NMR (CDCl3): δ
14.23, 24.32, 24.28, 24.81, 25.32, 28.97, 29.07, 34.28, 37.79,
48.58, 60.11, 113.26, 126.40, 130.13, 154.29, 173.77, 198.63.
(iii) Eth yl-2-m eth yl-7-(2-n a p h th oyl)h ep ta n oa te. To a
solution of the previous compound (950 mg, 2.56 mmol) in
acetone (75 mL) was added 1 N HCl (15 mL) until a precipitate
persisted, followed by acetone until a solution was obtained.
The mixture was stirred at room temperature for 1 h and then
at 50 °C for 1 h. The mixture was concentrated in vacuo to 10
mL. The white precipitate formed was filtered and rinsed with
(ii) N-H yd r oxy-7-[4-(1-p ip er id in yl)b en zoyl]h ep t a n a -
m id e (15a ). Following the procedure described above for
compound 8c, the 7-[4-(1-piperidinyl)benzoyl]heptanoic acid
1
water to give the title compound. H NMR (CDCl3): δ 8.47 (s,
1
1H), 8.04-7.96 (m, 2H), 7.91-7.86 (m, 2H), 5.57 (quintd, J )
7.7, 1.9 Hz, 2H), 4.12 (q, J ) 7.1 Hz, 2H), 3.09 (t, J ) 7.4 Hz,
2H), 2.46-2.39 (m, 1H), 1.82-1.51 (m, 4H), 2.45-2.34 (m, 4H),
1.24 (t, J ) 7.1 Hz, 3H), 1.14 (d, J ) 6.9 Hz, 3H). LRMS (ESI)
) 327 (MH+).
was obtained in 98% yield. H NMR (CDCl3): δ 7.84 (d, J )
9.0 Hz, 2H), 6.84 (d, J ) 9.0 Hz, 2H), 3.35 (m, 4H), 2.86 (t, J
) 7.5 Hz, 2H), 2.35 (t, J ) 7.5 Hz, 2H), 1.74-1.56 (m, 10H),
1.20 (m, 4H). 13C NMR (CDCl3): δ 24.25, 24.46, 24.65, 25.24,
28.81, 28.97, 33.92, 37.71, 48.53, 113.24, 126.25, 130.17,
154.27, 179.53, 198.87.
N-Hydr oxy-2-m eth yl-7-(2-n aph th oyl)h eptan am ide (16).
Following a procedure analogous to that described for com-
pound 8c, 2-methyl-7-(2-naphthoyl)heptanoic acid was ob-
tained in 73% yield. 1H NMR (CDCl3): δ 8.47 (s, 1H), 8.03 (dd,
J ) 8.5, 1.6 Hz, 1H), 7.97 (d, J ) 8.0 Hz, 1H), 7.89 (d, J ) 8.5
Hz, 1H), 7.87 (d, J ) 8.0 Hz, 1H), 7.57 (quintd, J ) 7.7, 1.9
Hz, 2H), 3.10 (t, J ) 7.1 Hz, 2H), 2.52-2.46 (m, 1H), 1.83-
1.72 (m, 4H), 1.52-1.42 (m, 4H), 1.19 (d, J ) 6.9 Hz, 3H).
LRMS (ESI) ) 299 (MH+).
Following the procedure described above for compound 8c,
compound 15a was obtained in 45% yield. 1H NMR (20% CD3-
OD in CDCl3): δ 7.71 (d, J ) 8.7 Hz, 2H), 6.73 (d, J ) 8.7 Hz,
2H), 3.23 (m, 4H), 2.74 (t, J ) 7.2 Hz, 2H), 1.98 (t, J ) 6.6 Hz,
2H), 1.54 (m, 10H), 1.24 (m, 4H). 13C NMR (20% CD3OD in
CDCl3): δ 24.00, 24.50 24.99(2), 28.49, 28.58, 32.43, 37.49,
48.23, 112.96, 125.55, 130.11, 154.25, 171.15, 199.72. Anal.
(C19H28N2O3) C, H, N.
Following a procedure analogous to that described for
compound 8c, compound 16 was obtained in 65% yield. 1H
NMR (CDCl3): δ 8.47 (s, 1H), 8.02 (d, J ) 8.8 Hz, 1H), 7.97
(d, J ) 8.8 Hz, 1H), 7.91-7.86 (m, 2H), 7.63-7.55 (m, 2H),
3.18-3.02 (m, 2H), 2.31-2.17 (m, 1H), 1.73-1.694 (m, 2H),
1.50-1.28 (m, 4H), 1.11-1.07 (m, 3H). HRMS: calcd, 313.1678;
found, 313.1685.
(E/Z)-Eth yl 8-(2-n a p h th yl)-7-octen oa te (17). To a solu-
tion of ethyl 7-bromoheptanoate 5 (10.0 g, 42.17 mmol) in
toluene (280 mL) was added triphenylphosphine (11.06 g, 42.17
mmol). The solution was refluxed over 24 h under nitrogen
and then cooled to room temperature. The supernatant was
transferred to a flask and an excess of triphenylphosphine
(5.53 g, 21.09 mmol) was added. The solution was refluxed over
3 days and then cooled to room temperature without stirring
to favor sedimentation. The supernatant was again removed
by decantation, and the resulting colorless oil was dried under
high vacuum affording (carbethoxyhexyl)triphenylphosphonium
bromide (18.24 g) in 87% yield. 1H NMR (CDCl3): δ 7.96-
7.62 (m, 9H), 7.37-7.08 (m, 6H), 4.11-4.00 (m, 2H), 3.86-
3.70 (m, 2H), 2.37-2.32 (m, 4H), 2.28-2.19 (m, 2H), 1.72-
1.46 (m, 6H), 1.31-1.25 (m, 2H), 1.22-1.15 (m, 3H). LRMS
(ESI) ) 419 (phosphonium ion).
To a suspension of the phosphonium salt (18.14 g, 36.32
mmol) in THF (300 mL) cooled to 0 °C was added n-
butyllithium (1.13 M solution in hexanes, 35.4 mL, 39.95
mmol) dropwise. The resulting solution was stirred at 0 °C
over 30 min, and a solution of 2-naphthaldehyde (5.67 g, 36.32
mmol) in THF (60 mL) was transferred dropwise via cannula.
The mixture was stirred overnight at room temperature. The
reaction was quenched with saturated aqueous NH4Cl and
extracted with diethyl ether. The combined organic layers were
successively washed with water and brine, dried over magne-
sium sulfate, and then concentrated in vacuo. The crude
residue was purified by flash chromatography (6-8% ethyl
acetate in hexane) to afford 17 in 34% yield as a mixture of E
and Z isomers in ca. a 1:2 ratio. 1H NMR (CDCl3): δ 7.83-
7.75 (m, 3H), 7.71 (s, 0.5H), 7.67 (s, 0.5H), 7.57 (dd, J ) 8.5,
1.9 Hz, 0.5H), 7.49-7.38 (m, 2.5H), 6.57 (d, J ) 11.5 Hz, 0.5H),
6.54 (d, J ) 15.9 Hz, 0.5H), 6.34 (dt, J ) 15.7, 7.1 Hz, 0.5H),
5.73 (dt, J ) 11.8, 7.1 Hz, 0.5H), 4.17-4.10 (m, 2H), 2.42 (qd),
J ) 7.4, 1.9 Hz, 1H), 2.34-2.24 (m, 3H), 1.73-1.31 (m, 6H),
1.28-1.22 (m, 3H). LRMS (ESI) ) 297 (MH+).
N-Hyd r oxy-7-[4-(1-(4-p h en ylp ip er a zin yl))ben zoyl]h ep -
ta n a m id e (15b). Following the procedure described above for
compound 15a but using the 1-phenyl-piperidine, the com-
pound 15b was obtained in 12% yield over three steps. 1H
NMR (20% CD3OD in CDCl3): δ 7.79 (d, J ) 8.7 Hz, 2H), 7.19
(m, 2H), 6.89-6.78 (m, 5H), 3.41 (m, 4H), 3.24 (m, 4H), 2.78
(t, J ) 7.5 Hz, 2H), 2.00 (t, J ) 7.5 Hz, 2H), 1.66-1.46 (m,
4H), 1.26 (m, 4H). 13C NMR (20% CD3OD in CDCl3): δ 24.40,
25.03, 28.61 (2), 32.48, 37.65, 47.06, 48.96, 113.35, 116.24,
120.22, 126.89, 128.98, 130.06, 150.65, 153.89, 171.11, 199.86.
Anal. (C24H31N3O3) C, H, N.
N-Hydr oxy-2-m eth yl-7-(2-n aph th oyl)h eptan am ide (16).
(i) Eth yl-8-(2-n a p h th yl)-8-[2-(1,3-d ioxolyl)]octa n oa te. To
a solution of 7m (Ar ) 2-naphthyl) (4.52 g, 14.47 mmol) in
dichloromethane (140 mL) was added ethylene glycol (8.07 mL,
144.7 mmol) followed by boron trifluoride etherate (3.67 mL,
28.94 mmol). The mixture was stirred for 1 h at room
temperature. Then, trimethylorthoformate (2.73 mL, 21.71
mmol) was added and the mixture was stirred overnight. The
reaction was quenched with a saturated aqueous solution of
sodium bicarbonate, and the layers were separated. The
aqueous layer was extracted with dichloromethane, and the
combined organic layers were washed with brine, dried
(MgSO4), and concentrated. The crude residue was purified
by flash chromatography (15-20% ethyl acetate/hexane) to
afford the title compound in 44% yield. 1H NMR (CDCl3): δ
7.91 (s, 1H), 7.87-7.81 (m, 3H), 7.54 (dd, J ) 8.5, 1.4 Hz, 1H),
7.49-7.46 (m, 2H), 4.12-4.03 (m, 4H), 3.82-3.78 (m, 2H), 2.23
(t, J ) 7.4 Hz, 2H), 1.99-1.94 (m, 2H), 1.59-1.54 (m, 2H).
LRMS (ESI) ) 357 (MH+).
(ii) Eth yl-2-m eth yl-8-(2-n a p h th yl)-8-[2-(1,3-d ioxolyl)]-
octa n oa te. To a solution of diisopropylamine (550 µL, 3.93
mmol) in THF (40 mL) cooled to 0 °C under nitrogen was added
dropwise n-butyllithium (1.2 M solution in hexanes, 3.12 mL,
3.65 mmol). The mixture was stirred at 0 °C for 20 min and
then cooled to -78 °C. A cooled (-78 °C) solution of the above
ester (1.0 g, 2.81 mmol) in THF (15 mL) was transferred via
cannula. The mixture was stirred for 1 h at -78 °C. Then, a
cooled (-78 °C) solution of iodomethane (349 µL, 5.61 mmol)
in THF (15 mL) was transferred via cannula. The resulting
mixture was stirred at -78 °C for 30 min, quenched with
saturated aqueous NH4Cl, and then stirred for 4 h at room
temperature. The mixture was concentrated in vacuo, and the
aqueous residue was extracted with diethyl ether. The organic
layer was washed with brine, dried (MgSO4), and concentrated.
The crude residue was purified by flash chromatography (10%
ethyl acetate in hexane) to afford the title compound (950 mg)
in 91% yield. 1H NMR (300 MHz, CDCl3): δ 7.91 (s, 1H), 7.87-
(E)-N-Hyd r oxy-8-(2-n a p h th yl)-7-octen a m id e (18). Fol-
lowing a procedure analogous to that described for compound
8c, (E)-8-(2-naphthyl)-7-octenoic acid was obtained in 60%
yield. 1H NMR (CDCl3): δ 7.79-7.75 (m, 3H), 7.67 (s, 1H), 7.57
(dd, J ) 8.5, 1.7 Hz, 1H), 7.47-7.38 (m, 2H), 6.54 (d, J ) 15.7

2884 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13
Woo et al.
Hz, 1H), 6.34 (dt, J ) 9.0, 6.6 Hz, 1H), 2.38 (t, J ) 7.4 Hz,
2H), 2.28 (q, J ) 6.6 Hz, 1H), 1.74-1.64 (m, 2H), 1.57-1.40
(m, 4H). LRMS (ESI) ) 269 (MH+).
Following a procedure analogous to that described for
compound 8c, 18 was obtained in 37% yield. 1H NMR (CD3-
OD): δ 7.79-7.74 (m, 3H), 7.67 (s, 1H), 7.60 (d, J ) 8.8 Hz,
1H), 7.45-7.36 (m, 2H), 6.56 (d, J ) 15.7 Hz, 1H), 6.38 (dt, J
) 15.7, 6.9 Hz, 1H), 2.28 (q, J ) 6.3 Hz, 2H), 2.11 (t, J ) 7.4
Hz, 2H), 1.72-1.62 (m, 2H), 1.58-1.50 (m, 2H), 1.46-1.40 (m,
2H). HRMS: calcd, 283.1572; found, 283.1567.
N-Hyd r oxy-8-(2-n a p h th yl)octa n a m id e (19). To a solu-
tion of 18 (30 mg, 0.106 mmol) in methanol (2 mL) was added
a catalytic amount of 10% palladuim on charcoal. The mixture
was degassed, placed under a hydrogen atmosphere, and
stirred for 15 min. The mixture was filtered through a pad of
Celite and rinsed with methanol. The filtrate was concentrated
in vacuo to afford 19 (29.1 mg) in 97% yield. 1H NMR (CD3-
OD): δ 7.80-7.74 (m, 3H), 7.60 (s, 1H), 7.44-7.37 (m, 2H),
7.33 (dd, J ) 8.2, 1.6 Hz, 1H), 2.77 (t, J ) 7.4 Hz, 2H), 2.07 (t,
J ) 7.1 Hz, 2H), 1.71-1.64 (m, 2H), 1.62-1.57 (m, 2H), 1.37-
1.28 (m, 6H). HRMS: calcd, 285.1729; found, 285.1727.
(MgSO4), and concentrated, yielding 6-tert-butyldimethylsilyl-
oxy-6-(2-naphthyl) hexanal as a crude oil in 91% yield (1.05
g). 1H NMR (CDCl3): δ 9.73 (t, J ) 1.9 Hz, 1H), 7.83-7.78 (m,
3H), 7.69 (s, 1H), 7.49-7.43 (m, 3H), 4.80 (t, J ) 6.9 Hz, 1H),
2.39 (td, J ) 7.1, 1.6 Hz, 2H), 1.81-1.55 (m, 4H), 1.43-1.25
(m, 2H) 0.90 (s, 9H), 0.04 (s, 3H), - 0.15 (s, 3H). LRMS (ESI)
) 225 (M+1-TBSO).
To a suspension of sodium hydride (60% in mineral oil, 177
mg, 4.42 mmol) in THF (25 mL) at room temperature under
nitrogen was added dropwise methyl diethylphosphonoacetate
(811 µL, 4.42 mmol). The resulting solution was stirred for 10
min while cooling to 0 °C. A solution of the crude aldehyde
(1.05 g, 2.94 mmol) in THF (10 mL) was transferred via
cannula. The resulting solution was stirred for 15 min at 0 °C
and quenched with a saturated aqueous solution of NH4Cl.
THF was removed in vacuo, and the aqueous residue was
partitioned between diethyl ether and water. The aqueous
layer was extracted with diethyl ether, and the combined
organic layers were washed with brine, dried (MgSO4), and
concentrated. The crude residue was purified by flash chro-
matography (5% ethyl acetate/hexane) to afford the title
compound in 63% yield (1.30 g). 1H NMR (CDCl3): δ 7.83-
7.78 (m, 3H), 7.69 (s, 1H), 7.49-7.41 (m, 3H), 6.94 (dt, J )
15.4, 7.1 Hz, 1H), 5.79 (dt, J ) 15.7, 1.1 Hz, 1H), 4.79 (dd, J
) 7.1, 5.2 Hz, 1H), 3.71 (s, 3H), 2.16 (q, J ) 7.4 Hz, 2H), 1.81-
1.62 (m, 2H), 1.48-1.26 (m, 4H), 0.90 (s, 9H), 0.04 (s, 3H),
-0.15 (s, 3H). LRMS (ESI) ) 413 (MH+).
8-t-Bu t yld im et h ylsilyloxy-8-(2-n a p h t h yl)-2-oct en oic
Acid . Following a procedure analogous to that described for
compound 8c, the title compound was obtained in 100% yield.
1H NMR (CDCl3): δ 7.83-7.78 (m, 3H), 7.72 (s, 1H), 7.48-
7.40 (m, 3H), 6.74 (dt, J ) 15.4, 7.1 Hz, 1H), 5.78 (d, J ) 15.4
Hz, 1H), 4.88-4.84 (m, 1H), 2.17-2.11 (m, 2H), 1.82-1.69 (m,
2H), 1.47-1.28 (m, 4H), 0.89 (s, 9H), 0.06 (s, 3H), -0.16 (s,
3H). LRMS (ESI) ) 421 (M++Na).
Syn th esis of N-Hyd r oxy-8-h yd r oxy-8-(2-n a p h th yl)-2-
octen a m id e (20). O-(t-Bu tyld im eth ylsilyl)-1-(2-n a p h th yl)-
6-h ep ten ol. A flame-dried round-bottomed flask was charged
with magnesium turnings (1.23 g, 50.75 mmol). THF (70 mL)
followed by 1,2-dibromoethane (381 mg, 2.03 mmol) were
added under nitrogen. A solution of 6-bromohexene (8.28 g,
50.75 mmol) in THF (30 mL) was added via cannula, and the
mixture was refluxed overnight. A solution of 2-naphthalde-
hyde (6.10 g, 39.04 mmol) in THF (30 mL) was transferred
via cannula to the Grignard reagent precooled to -78 °C, and
the mixture was slowly warmed to 0 °C over 3 h. After it was
stirred at that temperature for an additional 2 h, the reaction
mixture was quenched with saturated aqueous NH4Cl and
extracted with diethyl ether. The combined organic layers were
washed with brine, dried over magnesium sulfate, and con-
centrated in vacuo. The residue was purified by flash chro-
matography (10-15% ethyl acetate in hexane) to afford 1-(2-
N-Hyd r oxy-8-t-b u t yld im et h ylsilyloxy-8-(2-n a p h t h yl)-
2-octen a m id e. Following a procedure analogous to that
described for compound 8c, the title compound was obtained
1
1
in 44% yield. H NMR (CD3OD): δ 7.83-7.72 (m, 4H), 7.48-
naphthyl)-6-heptenol in 89% yield (8.33 g). H NMR (CDCl3):
7.40 (m, 3H), 6.77 (dt, J ) 15.1, 7.1 Hz, 1H), 5.75 (d, J ) 15.4
Hz, 1H), 4.91-4.79 (m, 1H), 2.18-2.14 (m, 2H), 1.79-1.68 (m,
2H), 1.48-1.39 (m, 2H), 1.28-1.21 (m, 2H), 0.89 (s, 9H), 0.06
(s, 3H), -0.16 (s, 3H). LRMS (ESI) ) 436 (M++Na).
δ 7.85-7.78 (m, 4H), 7.51-7.44 (m, 3H), 5.85-5.72 (m, 1H),
5.02-4.91 (m, 2H), 4.87-4.82 (m, 2H), 2.08-2.01 (m, 2H),
1.94-1.80 (m, 2H), 1.53-1.23(m, 4H). LRMS (ESI) ) 223
(MH+-H2O).
N-Hyd r oxy-8-h yd r oxy-8-(2-n a p h th yl)-2-octen a m id e. To
a solution of the silyl ether (300 mg, 0.725 mmol) in THF (3
mL) at room temperature was added 1 N HCl (3 mL). The
mixture was stirred for 3 h at 30 °C. The aqueous layer was
extracted with ethyl acetate, and the combined organic layers
were washed with brine, dried (MgSO4), and concentrated. The
crude residue was purified by flash chromatography (75-100%
ethyl acetate in hexane) to afford the title compound in 33%
To a solution of the above alcohol (1.0 g, 4.16 mmol) in
dichloromethane (40 mL) at room temperature under
a
nitrogen atmosphere were successively added tert-butyldim-
ethylsilyl chloride (815 mg, 5.41 mmol), imidazole (340 mg,
4.99 mmol), and a catalytic amount of 2,6-(dimethylamino)-
pyridine. The mixture was stirred for 1 h, and then, triethy-
lamine (696 µL, 4.99 mmol) was added. After it was stirred
for 2 days, the reaction was quenched with water, and the
aqueous layer was extracted with dichloromethane. The
combined organic layers were washed successively with 1 N
HCl, saturated aqueous sodium bicarbonate, then brine, dried
(MgSO4), and concentrated. The crude residue was purified
by flash chromatography (hexanes) to afford the title com-
pound in 84% yield (1.24 g). 1H NMR (CDCl3): δ 7.83-7.79
(m, 3H), 7.70 (s, 1H), 7.47-7.40 (m, 3H), 5.78 (ddt, J ) 17.0,
10.4, 6.6 Hz, 1H), 5.85-5.71 (m, 2H), 4.79 (dd, J ) 6.9, 4.9
Hz, 1H), 2.04-1.98 (m, 2H), 1.80-1.54 (m, 2H), 0.90 (s, 9H),
0.04 (s, 3H), - 0.14 (s, 3H). LRMS (ESI) ) 223 (MH+-TBSO).
Me t h yl-8-t -b u t yld im e t h ylsilyloxy-8-(2-n a p h t h yl)-2-
octen oa te. To a solution of the alkene (1.05 g, 3.23 mmol) in
tert-butyl alcohol (50 mL) were successively added water (10
mL), sodium bicarbonate (2.71 g, 32.3 mmol), sodium periodate
(4.15 g, 19.38 mmol), and osmium tetroxide (8 mg, 0.032
mmol). The mixture was stirred for 3 h and then quenched
with a 10% aqueous solution of sodium thiosulfate. After 30
min, the white precipitate was filtered off and rinsed with
diethyl ether. The filtrate was concentrated in vacuo. The
residue was partitioned between diethyl ether and water. The
aqueous layer was extracted with diethyl ether. The combined
organic layers were washed successively with a 10% aqueous
solution of sodium thiosulfate, water, and then brine, dried
1
yield (77 mg). H NMR (CD3OD): δ 7.87-7.71 (m, 4H), 7.52-
7.40 (m, 3H), 6.77 (dt, J ) 15.1, 6.9 Hz, 1H), 5.75 (d, J ) 15.1
Hz, 1H), 4.76 (t, J ) 6.9 Hz, 1H), 2.17-2.10 (m, 2H), 1.84-
1.77 (m, 2H), 1.49-1.27 (m, 4H) 1.88-1.75 (m, 4H). LRMS
(ESI) ) 282 (M+-OH), 322 (M++Na).
N-Hyd r oxy-8-h yd r oxy-8-(2-n a p h th yl)octa n a m id e (20).
To a solution of the alkene (20 mg, 0.067 mmol) in methanol
(670 µL) was added sodium borohydride (2.5 mg, 0.067 mmol).
The mixture was stirred for 15 min, and the solvent was
evaporated. The residue was partitioned between ethyl acetate
and water. The organic layer was dried (MgSO4) and concen-
trated. Purification by flash chromatography (6% methanol in
1
dichloromethane) afforded 20 in 46% yield (9.1 mg). H NMR
(CD3OD): δ 7.84-7.76 (m, 4H), 7.50-7.40 (m, 3H), 4.75 (t, J
) 6.6 Hz, 1H), 2.05 (t, J ) 7.4 Hz, 2H), 1.86-1.76 (m, 2H),
1.62-1.55 (m, 2H), 1.43-1.23 (m, 6H). HRMS: calcd, 283.1572
(M-H2O); found, 283.1581.
Biological Assays. P r odu ction of Recom bin an t HDAC1
En zym e. Human HDAC-1 cDNA was generated by RT-
polymerase chain reaction (PCR) reactions using primers
complementary to the 5′ and 3′ coding sequence of human
HDAC1 gene (Genbank accession number U50079). Error-free
HDAC1 cDNA was inserted either into the pcDNA3.1 vector

Trichostatin A-Like Straight Chain Hydroxamates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 13 2885
(2) (a) Kouzarides, T. Histone acetylases and deacetylases in cell
proliferation. Curr. Opin. Genet. Dev. 1999, 9, 40-48. (b) Review
Strahl, B. D.; Allis, C. D. Nature 2000, 403, 41-45.
(3) Liu, R. J .; Nagy, L.; Inoue, S.; Shao, W.; Miller, W. H.; Evans,
R. M. Role of the histone deacetylase complex in acute promy-
elocytic leukaemia. Nature 1998, 391, 811-814.
(4) (a) Beppu, T.; Suginami-ku, H.; Iwamoto; Yoshida, M. U.S.
Patent 4,690,918, 1987. (b) Yoshida, M.; Kijima, M.; Akita, M.;
Beppu, T. Potent and specific inhibition of mammalian histone
deacetylase both in vivo and in vitro by trichostatin A. J . Biol.
Chem. 1990, 265, 17174-17179. (c) Fleming, I.; Iqbal, J .; Krebs,
E. P. The total synthesis of (()-trichostatin A. Tetrahedron 1983,
39, 841-846. (d) Mori, K.; Koseki, K. Synthesis of trichostatin
A, a potent differentiation inducer of friend leukemic cells, and
its antipode. Tetrahedron 1988, 44, 6013-6020.
(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 described9 and used as
HDAC enzyme substrates. The assay was performed as in the
literature.9 Briefly, small molecule inhibitors diluted in di-
methyl sulfoxide (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. The incubation time for
the HDAC1 enzyme reaction was 10 min. The reaction was
stopped, and released [3H] acetic acid was extracted and
quantified by scintillation counting. The 50% inhibitory con-
centrations (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 sodium dodecyl sulfate polyacrylamide gel elec-
trophoresis (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
enhanced chemiluminescence (ECL, Amersham) was followed
for detection.
(5) (a) Richon, V. M.; Emiliani, S.; Verdin, E.; Webb, Y.; Breslow,
R.; Rifkind, R. A.; Marks, P. A. A class of hybrid polar inducers
of transformed cell differentiation inhibits histone deacetylases.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3003-3007. (b) Butler,
L. M.; Agus, D. B.; Scher, H. I.; Higgins, B.; Rose, A.; Cordon-
Cardo, C.; Thaler, H. T.; Rifkind, R. A.; Marks, P.; Richon, V.
M. Suberoylanilide hydroxamic acid, an inhibitor of histone
deacetylase, suppresses the growth of prostate cancer cells in
vitro and in vivo. Cancer Res. 2000, 60, 5165-5170. (c) Huang,
L.; Pardee, A. B. Suberoylanilide hydroxamic acid as a potential
therapeutic agent for human breast cancer treatment. Mol. Med.
2000, 6, 849-866.
(6) Kim, Y. B.; Lee, K. H.; Sugita, K.; Yoshida, M.; Horinouchi, S.
Oncogene 1999, 18, 2461.
(7) (a) J ung, M.; Brosch, G.; Ko¨lle, D.; Scherf, H.; Gerha¨user, C.;
Loidl, P. Amide analogues of trichostatin A as inhibitors of
histone deacetylase and inducers of terminal cell differentiation.
J . Med. Chem. 1999, 42, 4669-4679. (b) Review Meinke, P. T.;
Liberator, P. Histone deacetylase: a target for antiproliferative
and antiprotozoal agents. Curr. Med. Chem. 2000, 8, 211-235.
(c) Marks, P. A.; Rifkind, R. A.; Richon, V. M.; Breslow, R.; Miller,
T.; Kely, W. K. Histone deacetylases and cancer: causes and
therapies. Nat. Rev. Cancer 2001, 1, 194-202. (d) Remiszewski,
S. W.; Sambucetti, L. C.; Atadja, P.; Bair, K. W.; Cornell, W. D.;
Green, M. A.; Howell, K. L.; J ung, M.; Kwon, P.; Trogani, N.;
Walker, H. Inhibitors of human histone deacetylase: synthesis
and enzyme and cellular activity of straight chain hydroxamates.
J . Med. Chem. 2002, 4, 753-757. (e) Massa, S.; Mai, A.;
Sbardella, G.; Esposito, M.; Ragno, R.; Loidl, P.; Brosch, G. 3-(4-
Aroyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamides, a new class of
synthetic histone deacetylases inhibitors. J . Med. Chem. 2001,
13, 2069-2072.
(8) (a) Lavoie, R.; Bouchain, G.; Frechette, S.; Woo, S. H.; Abou-
Khalil, E.; Leit, S.; Fournel, M.; Yan, P. T.; Trachy-Bourget, M.
C.; Beaulieu, C.; Li, Z.; Besterman, J .; Delorme, D. Design and
synthesis of a novel class of histone deacetylase inhibitors.
Bioorg. Med. Chem. Lett. 2001, 11, 2847-2850. (b) Delorme, D.;
Woo, S. H.; Vaisburg, A. WO 0170675. (c) Delorme, D.; Ruel, R.;
Lavoie, R.; Thibault, C.; Abou-Khalil, E. WO 0138322, 2001.
(9) Carmen, A. A.; Rundlett, S. E.; Grunstein, M. HDA1 and HDA3
are components of a yeast histone deacetylase (HDA) complex.
J . Biol. Chem. 1996, 271, 15837-15844.
MTT Assa y. Compounds at various concentrations were
added to cells plated in 96 well plates. Cells were incubated
for 72 h at 37 °C in 5% CO2 incubator. MTT (Sigma) was added
at a final concentration of 0.5 mg/mL and incubated with the
cells for 4 h before an equal volume of solubilization buffer
(50% N,N-dimethylformamide, 20% SDS, pH 4.7) was added
onto cultured cells. After overnight incubation, solubilized dye
was quantified by colorimetric reading at 570 nM using a
reference at 630 nM. OD values were converted to cell numbers
according to a standard growth curve of the relevant cell line.
The concentration that reduces cell numbers to 50% of those
of DMSO-treated cells is determined as MTT IC50
.
Ack n ow led gm en t. We thank C. Bonfils and Y. Hou
for cloning of the HDAC enzyme, M. F. Robert and I.
Dupont for expression of the HDAC enzyme, and P. T.
Yan, Ann Kalita, and Carole Beaulieu for biological
assays.
(10) (a) Finnin, M. S.; Donigian, J . R.; Cohen, A.; Richon, V. M.;
Rifkind, R. A.; Marks, P. A.; Breslow, R.; Pavietich, N. P.
Structures of a histone deacetylase homologue bound to the TSA
and SAHA inhibitors. Nature 1999, 401, 188-193. (b) Pavietich,
N. P.; Finnin, M. S.; Donigian, J . R.; Richon, V. M.; Rifkind, R.
A.; Marks, P. A.; Breslow, R. U.S. 0024700, 2000.
Refer en ces
(1) Hassig, C. A.; Schreiber, S. L. Nuclear histone acetylases and
deacetylases and transcriptional regulation: HATs off to HDACs.
Curr. Opin. Chem. Biol. 1997, 1, 300-308.
J M020154K