α-Keto amides as inhibitors of histone deacetylase

Article abstract of DOI:10.1016/S0960-894X(03)00685-1

α-Keto ester and amides were found to be potent inhibitors of histone deacetylase. Nanomolar inhibitors against the isolated enzyme and sub-micromolar inhibitors of cellular proliferation were obtained. The α-keto amide 30 also exhibited significant anti-tumor effects in an in vivo tumor model.

Full text of DOI:10.1016/S0960-894X(03)00685-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3331–3335
ꢀ-Keto Amides as Inhibitors of Histone Deacetylase
Carol K. Wada,* Robin R. Frey, Zhiqin Ji, Michael L. Curtin, Robert B. Garland,
James H. Holms, Junling Li, Lori J. Pease, Jun Guo, Keith B. Glaser,
Patrick A. Marcotte, Paul L. Richardson, Shannon S. Murphy, Jennifer J. Bouska,
Paul Tapang, Terrance J. Magoc, Daniel H. Albert, Steven K. Davidsen
and Michael R. Michaelides
Cancer Research, Abbott Laboratories, Department R47J, Bldg. AP10, 100 Abbott Park Road, Abbott Park, IL 60064, USA
Received 24 February 2003; accepted 13 May 2003
Abstract—a-Keto ester and amides were found to be potent inhibitors of histone deacetylase. Nanomolar inhibitors against the
isolated enzyme and sub-micromolar inhibitors of cellular proliferation were obtained. The a-keto amide 30 also exhibited sig-
nificant anti-tumor effects in an in vivo tumor model.
# 2003 Elsevier Ltd. All rights reserved.
Histone deacetylases (HDACs) catalyze the removal of
acetyl groups from the e-amino groups of lysine residues
at the N-terminus of nucleosomal histones. The altera-
tion of the acetylation status of histone proteins com-
plexed with DNA in the nucleosome determines the
accessibility of DNA within chromatin and conse-
quently influences gene expression.1 Recent studies
indicate that inappropriate recruitment and expression
of HDACs are implicated in several human cancers. The
effects of HDAC inhibitors provide the basis for HDAC
inhibition as a compelling anticancer target. Inhibitors of
HDAC have been reported to induce differentiation of
cancer cells and suppress cell proliferation.2
aromatic ‘cap group’ makes contacts with the pocket
entrance, analogous to the binding of the acetyl group,
lysine side chain, and peptide backbone of the natural
substrate.6
Screening of the Abbott compound library also revealed a
hydroxamic acid-containing HDAC inhibitor (1, Fig. 1).7
Due to the short half-life and low oral bioavailability of
the hydroxamic acids, several substitutes for the hydrox-
amic acid were investigated. Electrophilic ketones, such
as trifluoromethyl ketones and a-keto esters and
amides, are known to be readily hydrated.8 They have
A variety of HDAC inhibitors have been reported in the
literature (Fig. 1). Several are undergoing clinical eval-
uation.2 The majority of these structures have a metal
chelator attached to an aromatic group via a hydro-
phobic linker. The HDAC inhibitors trichostatin A
(TSA) (lit. IC50 3.4 nM)3 and suberoyl anilide hydrox-
amic acid (SAHA) (lit. IC50 10 nM)4 both contain a
hydroxamic acid moiety as the zinc-binding group.
X-ray crystal structures of TSA and SAHA bound to an
HDAC homologue show that the metal-binding group
interacts with the active site zinc, the linker spans the
tube-like portion of the binding pocket, and the
*Corresponding author. Fax: +1-847-935-5165; e-mail: carol.wada@
abbott.com
Figure 1. HDAC inhibitors.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00685-1

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C. K. Wada et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3331–3335
Table 1. Electrophilic ketones
been reported to be inhibitors of lipases9 and serine or
cysteine proteases,10 and trifluoromethyl ketones are
reported to inhibit zinc-dependent enzymes.11 We have
previously reported that trifluoromethyl ketones such as
2 are inhibitors of HDAC.12 However, the ketone was
found to be readily metabolized to the alcohol in vivo.
Other electrophilic ketones were then investigated. It
was found that a-keto esters and amides are potent
inhibitors of HDAC, and a series of these compounds
were synthesized.
Compd
n
X
IC50 (mM)b
1a
1
0.020
The ether-linked compounds (Scheme 1) were prepared
by alkylation of biphenyl-4-ol with a bromo ester of the
appropriate linker length. a-Hydroxylation of the ester
was accomplished by deprotonation using LiHMDS and
treatment with Davis’ oxaziridine.13 Oxidation of the
alcohol with Dess–Martin periodinane gave the a-keto
esters. To prepare the a-keto amides, the a-hydroxy
esters were first treated with the appropriate amine to
give the a-hydroxy amides, which were then oxidized to
the ketones.
2a
1
1
1
0
2
1
1
1
1
1
1
2.9
3.7
3b
4b
0.062
0.082
0.109
>50
0.11
4.9
5b
To vary the aryl group in the amide-linked series the
a-keto amide group was installed first (Scheme 2). The
a-keto ester was prepared via a Horner-Wadsworth-
Emmons olefination14 followed by treatment with CsF.
The ester was then converted to the amide by treatment
with methylamine without concomitant conversion of
the ketone to the imine. The arylamide was then instal-
led using resin-bound coupling reagents, which allowed
the preparation of several arylamides in parallel.
6b
7a
8b
Compounds 15, 16, and 18 were prepared as in Scheme
2 starting with the alkylation of the appropriate aryl
thiol and aryl oxime with O-silyl protected 6-bromo-1-
hexanol. Compound 20 was prepared as in Scheme 2
using N-BOC protected 6-amino-1-hexanol.
9a
10a
11a
12a
>5
>50
>50
aMixture of HDAC1 and HDAC2 from nuclear extraction of K562
erythroleukemia cells.15
bFluorescence-based HDAC assay.16
Scheme 1. (a) Cs2CO3, methyl 7-bromoheptanoate, DMF;
(b) LiHMDS, Davis’ oxaziridine, THF ꢀ78 ꢁC; (c) RNH2, methanol;
(d) Dess–Martin periodinane, CH2Cl2.
Scheme 2. (a) LiOH, H2O, THF; (b) BnBr, NaHCO3, DMF;
(c) Swern; (d) LiCl, DBU, THF; (e) CsF, HOAc, CH3CN; (f) MeNH2(l),
THF, Et3N; (g) ArylNH2, carbodiimide-PS resin, HOAt, DMF.
Scheme 3. (a) NaH, bromoacetaldehyde dimethyl acetal, DMF;
(b) H2SO4, acetone, H2O; (c) KCN, NaHSO3, H2O, THF; (d) HBr(g)
MeOH; (e) RNH2, methanol; (f) Dess–Martin periodinane, CH2Cl2.
,

C. K. Wada et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3331–3335
3333
Table 2. Varying the hydrophobic linker and linking group
Table 3. Varying the aromatic group of amide-linked compounds
Compd
R
IC50 PLF-HT1080 PLF-MDA435
(nM)a
Compd
Regiochem
X
Linker
IC50 (nM)
(mM)
(mM)
6a
para
para
para
para
para
para
para
para
meta
meta
O
O
O
S
SO2
–(CH2)6–
–(CH2)4OCH2–
–(CH2)4CꢂC–
–(CH2)6–
114
>10,000
>1000
526
13b
14b
15b
16b
17b
18b
19b
20b
21b
21
22
23
24
25
26
27
28
29
30
31c
32
3.7
338
9.4
6.9
ndb
17
14
nd
–(CH2)6–
143
241
853
201
21
3.7
SO2NH–(CH
CH¼NO
2)6–
–(CH2)5–
–(CH2)6–
2)6–
NHC(O)
C(O)NH–(CH
NHC(O)
–(CH2)6–
22
aMixture of HDAC1 and HDAC2 from nuclear extraction of K562
erythroleukemia cells.15
bFluorescence-based HDAC assay.16
1041
9.5
35
39
Compounds 13 and 14 were synthesized via the cyano-
hydrin as shown for 13 in Scheme 3. The cyanohydrin
was converted to the a-hydroxy ester then carried on to
the a-keto amide as described in Scheme 1. All com-
pounds were characterized by 1HNMR, MS, and
combustion analysis.
2.9
4.0
4.5
14
18
3.4
Table 1 lists several of the electrophilic ketones which
were investigated. The a-keto ester 4 and the a-keto
amide 8 were found to be potent inhibitors of HDAC.
Interestingly, only the methyl ester and methyl amide
derivatives were active. The corresponding acid, pri-
mary amide, ethyl amide, and dimethyl amide were all
inactive. X-ray crystal structures of an HDAC homo-
logue show a water pocket near the active site6 which
could perhaps be accessed by extending off the methyl
amide. However, attaching groups such as a dimethyl-
amine (11) or carboxylic acid (12) led to a loss of
HDAC activity. The structure activity relationship of
the a-keto esters and amides correlates with the hydrox-
amates with regard to linker length with six carbons
being optimal, indicating a similar binding mode.
133
11
13
1.2
3.1
>50
5.8
2.0
1.4
>50
2.7
9.1
4.3
0.67
3.1
0.12
0.16
As with the trifluoromethyl ketones, the a-keto amides
are readily metabolized to the alcohol in vivo. The
a-keto esters are also subject to hydrolysis. In an effort
to find more a metabolically stable a-keto amide and
increase potency, changes in the hydrophobic linker and
the group (X) attaching the aryl moiety were investi-
gated (Table 2). A heteroatom (13) was added to the
linker. It was hoped that the electron-withdrawing oxy-
gen group would increase the hydration state of the
ketone and therefore influence its metabolism. However,
it was found to exist primarily as the enol form which
was inactive. Insertion of an alkyne linker (14) also led
to a loss of activity. para-Sufide, sulfone, sulfonamide,
oxime, and amide (15–19) were slightly less potent,
while the meta-amide linked compounds 20 and 21
increased potency dramatically.
aFluorescence-based HDAC assay.16
bnd, not determined.
cTrifluoroacetic acid salt.
We then investigated the effect of different aryl groups in
place of the biphenyl of 21. The cellular activities of these
compounds are shown as IC50 values for inhibition of
Figure 2. Western blot analysis the effect of 30 and SAHA on H4
acetyliation and p21 induction in MDA435 cells.

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C. K. Wada et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3331–3335
administered intraperitoneal (ip), at 30 and 100 mg/kg,
every other day (q2d). Significant inhibition of tumor
growth to 2 g relative to control was achieved. How-
ever, some evidence of toxicity (rough coats, weight
loss, lethality) was observed in the 100 mg dose group.
The a-keto amide 30 was observed to have a short half-
life in vivo, in cell culture, and in whole blood due to its
rapid reduction to the inactive a-hydroxy amide. Over-
all exposure was low with only transient concentrations
of inhibitor above the cellular proliferation IC50 value
(Fig. 4). Despite the poor pharmacokinetics of the
a-keto amides, they exhibit significant anti-tumor effects
in xenograft models, suggesting that transient exposure
to HDAC inhibitors may be sufficient for anti-tumor
effects.
Alpha-keto esters and amides have been shown to be
potent inhibitors of HDAC. By altering the linker and
aryl group, nanomolar HDAC inhibitors, some with
single digit to sub-micromolar IC50 values against cel-
lular proliferation, were obtained. Significant anti-
tumor effects were observed in an in vivo tumor model
with a-keto amide 30 despite the short half-life of this
compound. In conclusion, it has been shown that alter-
natives to the hydroxamate zinc-binding group can lead
to potent, single-digit nanomolar, inhibitors of HDAC.
Figure 3. Effects of 30 on tumor growth in an HT1080 mouse tumor
model (n=10 per dose group). Percent inhibition of growth to 2 g
relative to control is in parentheses of legend.
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