A novel series of histone deacetylase inhibitors incorporating hetero aromatic ring systems as connection units

Article abstract of DOI:10.1016/j.bmcl.2003.07.012

A series of structurally novel HDAC inhibitors, in which a hetero aromatic ring connects the spacer with the hydrophobic group, has been designed and synthesized. These new inhibitors are very potent in in vitro enzymatic assays and display antiproliferation activity against two human cancer cell lines.

Full text of DOI:10.1016/j.bmcl.2003.07.012

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3817–3820
A Novel Series of Histone Deacetylase Inhibitors Incorporating
Hetero Aromatic Ring Systems as Connection Units
Yujia Dai,* Yan Guo, Michael L. Curtin, Junling Li,
Lori J. Pease, Jun Guo, Patrick A. Marcotte, Keith B. Glaser,
Steven K. Davidsen and Michael R. Michaelides
Cancer Research, Abbott Laboratories, Department R47J, Building AP10,
100 Abbott Park Road, Abbott Park, IL 60031, USA
Received11 June 2003; accepted25 July 2003
Abstract—A series of structurally novel HDAC inhibitors, in which a hetero aromatic ring connects the spacer with the hydro-
phobic group, has been designed and synthesized. These new inhibitors are very potent in in vitro enzymatic assays and display
antiproliferation activity against two human cancer cell lines.
# 2003 Elsevier Ltd. All rights reserved.
Reversible acetylation of nuclear histones is essential
for chromatin remodeling and the functional regula-
tion of gene transcription. The balance of acetylation
is controlledandmaintainedby the activities of two
families of enzymes: histone acetyltranferases (HATs)
andhistone edacetylases (HDACs). By altering the
acetylation status of the N-terminal region of histone
proteins packagedwith DNA in the nucleosome, his-
tone deacetylases determine the accessibility of DNA
within chromatin andconsequently influence gene
expression.1 Deregulation of HDAC activity can lead
to the repression of a certain subset of genes in favor
of excessive proliferation andis implicatedin a
number of malignant diseases.2 Identification of
potent HDAC inhibitors offers great opportunities in
the development of therapeutics for the treatment of
cancers.3
A rationale for this structural design is derived from the
X-ray crystal structure of a bacterial HDAC homologue
(HDLP) with boundTSA. 5 It has been suggestedthat the
active site consists of a narrow tubular pocket with a zinc
atom inside. Comparison of the amino acid sequences
aroundthe active site has indicatedthat the structural
features of the active site are well conservedacross all the
HDACs, with exceptions at the solvent-exposedrim of
the pocket. Basedon these observations, it has been rea-
sonedthat modifications of the connection unit and
hydrophobic group, which are assumed to interact with
the entrance area of the catalytic pocket, will provide
opportunities for discovering potent and possibly selec-
tive HDAC inhibitors.6 However, only a limiteddiversity
of connection units, including amide,7 sulfonamide,8
Most of the HDAC inhibitors reportedin the literature
to date generally consist of a zinc-binding group and a
five- or six-carbon hydrophobic spacer attached to a
hydrophobic group via
a connection unit. Sub-
eroylanilide hydroxamic acid (SAHA, 1), a synthetic
small molecule HDAC inhibitor,4 is a well-known
example of this design.
10
ketone9 andether, have been reported. To our knowl-
edge, there have been no reports of utilizing aromatic
heterocycles as connection units in HDAC inhibitors.
*Corresponding author. Fax: +1-847-935-5165; e-mail: yujia.dai@
abbott.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.07.012

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Y. Dai et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3817–3820
provide a-bromo ketone 6a. Reaction of 6a with potas-
sium bis(t-butoxycarbonyl)aminide followed by BOC
cleavage gave rise to a-amino ketone 7. Compound 7
was coupledwith aromatic acids to affordthe corre-
sponding amides, which were converted to oxazoles 8
again by reaction with phosphorus pentoxide in reflux-
ing chloroform. Methyl esters 8 were cleavedby sapo-
nification to generate the corresponding acids, which
were then convertedto the hydroxamates 9 as before.
As part of our efforts to search for novel HDAC inhi-
bitors, we investigateda series of new hydroxamates
representedby structure 2, in which the spacer andthe
hydrophobic group are connected via a hetero aromatic
ring system. Structure 2 can be regarded as a product
derived from SAHA through a ‘conformation restric-
tion’ approach. In this paper, we report the synthesis of
these compounds and their structure–activity relation-
ship for HDAC inhibition.
The synthesis of thiazole analogues 12 was achievedby
following the synthetic route shown in Scheme 2. Sube-
ric acidmonomethyl ester was convertedto amide 10 by
treatment with SOCl2 andsubsequently with aqueous
ammonia solution. Lawesson’s Reagent was usedto
transform amide 10 to its corresponding thiamide,
which was reactedwith commercially available amino
ketones to give rise to thiazoles 11 in excellent yields.
Again, the methyl esters 11 were hydrolyzed and con-
verted to hydroxamic acids 12 via reaction of hydroxyl-
amine with the corresponding mixed anhydrides.
The synthesis of 2 startedwith the coupling of a-amino
ketone 3 andsuberic acidmonomethyl ester mediated
by HOBT andEDC ( Scheme 1). Treatment of the
amide 4 with phosphorus pentoxide in refluxing chloro-
form afforded oxazole ester 5 in 84% yield. The ester
was then saponified. The resulting acid was first con-
verted to the corresponding mixed anhydride and then
coupled with hydroxylamine to yield the desired hydro-
xamate 2.
The HDAC inhibitory activity of the synthesizedcom-
pounds was measured using a peptide substrate and a
mixture of HDACs, predominantly HDAC1 and
HDAC2, preparedfrom nuclear extraction of K562
11
erythroleukemia cells followedby partial purification.
We first evaluatedthe effect of replacing the amide in
SAHA (1) with alternate connection moieties (Table 1).
Gratifyingly, the oxazole andthiazole-linkedcompound
2a, 9a and 12a turnedout to be very potent HDAC
inhibitors. With IC50 values of 9.8, 13 and19 nM, 2a,
9a, and 12a are 14-, 11-, and7-foldmore potent,
respectively, than SAHA (1), which exhibiteda 140 nM
activity in the same assay. Compound 13, a reversed
amide analogue of SAHA with an IC50 value of over 1
mM, is a much weaker inhibitor. Replacing the amide
residue of SAHA with a methylene (14) or an ether
linkage (15) also ledto deterioration in HDAC inhibi-
tory activity. Compound 16, which has a benzene ring
insertedin the same location, is more potent than
SAHA, but still less active than 2a, 9a and 12a.
Suberic acidmonomethyl ester also servedas the start-
ing material for the synthesis of oxazoles 9. It was first
convertedto its acidchloride by reacting with oxalyl
chloride and a catalytic amount of DMF, and then
treatedwith diazomethane andconcentratedHBr to
Interestingly, compounds 2a and 9a are equipotent
although they are regioisomers in terms of the nitrogen
atom’s position on the oxazole rings. Compound 12a,
with a thiazole ring as connection unit, also exhibiteda
very comparable activity. This along with the potent
activity of 16 suggests that the position andthe type of
hetero atoms on the five-memberedaromatic ring are
Scheme 1. Synthesis of oxazole hydroxamates: (a) HOBt, EDC,
NMM, DMF; (b) P2O5, CHCl3, reflux; (c) NaOH, MeOH, H2O; (d) i-
PrOCOCl, Et3N, DMF; then 50% aq NH2OH; (e) (COCl)2, cat DMF,
CH2Cl2; (f) CH2N2, HBr, Et2O; (g) (BOC)2NK, DMF, 90 ꢀC; (h) HCl
solution in dioxane; (i) ArCO2H, HOBt, EDC, NMM, DMF.
Scheme 2. Synthesis of thiazole hydroxamates: (a) SOCl2, benzene,
reflux; (b) THF, aq NH4OH; (c) Lawesson’s Reagent, benzene, 70 ꢀC,
1.5 h; (d) ArCOCH2Br, MeOH, rt; (e) NaOH, MeOH, H2O; (f) i-
PrOCOCl, Et3N, DMF; then 50% aq NH2OH.

Y. Dai et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3817–3820
3819
Table 1. HDAC inhibition of hydroxamic acids
position (2b: 2.1 nM; 12b: 2.5 nM). However, incorpor-
ating a CF3O at the same position causeda drop in their
activity (9c: 89 nM and 12f: 74 nM). The position of
substitution seems to have little impact on activity. This
is exemplifiedby 12g (5.8 nM) and 12h (4.3 nM). Their
activities are comparable to those of 12c and 12b,
respectively. Introduction of a second pendant phenyl
ring did not have an effect on potency (2d, 9d and 12d).
However, replacement of the phenyl with a naphthyl
group ledto a 4-foldincrease in potency ( 12i).
11
11
CompdX
HDAC
CompdX
HDAC
IC50, nM
IC50, nM
2a
9a
12a
1
9.8
13
14
15
16
1010
560
330
58
The compounds in Table 2 were also evaluatedfor
antiproliferative activity against human HT1080 fibro-
sarcoma andhuman MDA435 breast carcinoma cell
lines. Most of these compounds displayed a significant
inhibitory effect against the growth of both cell lines,
comparable to the activity shown by SAHA (HT1080:
2.4 mM; MDA435: 1.9 mM).
13
19
CH2
O
140
Encouragedby the results from these hyrdoxamate
HDAC inhibitors, we also appliedthe hetero aromatic
connection units to keto amides, a new class of HDAC
inhibitors discovered recently, in which a keto amide
moiety functions as the zinc-binding group.13
not important for retaining enzymatic potency. It is
unclear what role the hetero aromatic systems may play
in the interaction between the inhibitors andHDAC
enzymes. However, the results in Table 1 indicate that:
(1) there is no critical hydrogen bond interaction
between the connection units andthe enzymes, and(2) a
flat or sp2 configuration of the connection unit is crucial
for potent HDAC inhibition. These observations are
consistent with a recent report9b that hydroxamates
with an olefinic moiety as the connection unit exhibited
potent HDAC activity.
The strategy for the synthesis of compounds 17–20
(Table 3) is representedby the preparation of com-
pound 20 in Scheme 3. Reaction of a-bromo ketone 6b
with thiobenzamide afforded thiazole 21, which was
then reduced to aldehyde 22 using DIABAL-H. The
aldehyde was treated with Horner–Wadsworth–
Emmons reagent 23 to give rise to enol ether 24.
Removal of the silyl group with CsF generatedthe cor-
responding a-keto ester, which was finally convertedto
the amide 20 by treatment with methylamine. Exhibit-
ing double-digit nanomolar IC50 values, compounds 17–
20 are much more potent than compound 25,13 in which
an amide linker served as connection unit.
The influence on HDAC activity of substitution at the
terminal phenyl ring in 2a, 9a and 12a was then exam-
ined( Table 2). Not surprisingly, similar trends are
observedacross the oxazole andthiazole series. Intro-
duction of a bromide at the p-positions of the phenyl
rings in 2a, 9a and 12a enhancedtheir activity to 2.7, 4.4
and8.5 nM, respectively. Improvedpotency was also
seen when a methoxy group was introduced to the same
In summary, we have synthesizeda series of structurally
novel andpotent HDAC inhibitors, in which a five-
memberedhetero aromatic ring connects the spacer to
the hydrophobic group. These compounds are sig-
Table 2. HDAC inhibition of hydroxamic acids
11
11
CompdR
HDAC
HT108012
MDA43512
IC50 (nM) proliferation proliferation
CompdR
HDAC
HT108012
MDA43512
IC50 (nM) proliferation proliferation
IC50 (mM)
IC50 (mM)
IC50 (mM)
IC50 (mM)
2a
2b
2c
2d
9a
9b
9c
9d
9e
H
9.8
2.1
2.7
6.8
13
4.4
89
8.41
0.31
7.7
0.66
23%@50
27
5.14
15.3
76%
2.2
2.52
6.7
0.95
12a
12b
12c
12d
12e
12f
12g
12h
12i
H
19
2.5
8.5
11
42
74
5.8
4.3
4.3
5.07
3.63
5.68
1.32
99.7%
2.97
8.11
1.95
0.85
p-CH3O
p-Br
p-(p-ClC6H4)
H
p-Br
p-CF3O
p-Ph
p-F
p-CH3O
p-Br
p-Ph
p-CF3
p-CF3O
6.12%@50
97%
20.4
2.39
100%@50
10.1
2.0
2.04
m-Br
2-CH3O-6-CH3O
b-Naphthyl
98.6%@50
2.91
0.85
12
23
98.7
7.94
2.39

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Y. Dai et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3817–3820
Table 3. HDAC inhibition of keto amides
References and Notes
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Scheme 3. Synthesis of keto amide 20: (a) PhCSNH2, MeOH, rt, 78%;
(b) DIBAL-H, CH2Cl2, À78 ꢀC; (c) LiCl, DBU, THF, 95%; (d) CsF,
HOAc, CH3CN, 64%; (e) CH3NH2, Et3N, THF, rt, 25%.
11. The given IC50 values of HDAC inhibition were deter-
mined using the procedure described by note 15 in ref 7a.
12. The given IC50 values of cell growth inhibition were
determined using the procedure described by note 19 in ref 7b.
13. Wada, C. K.; Frey, R. R.; Ji, Z.; Curtin, M. L.; Garland,
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J. J.; Tapang, P.; Magoc, T. J.; Albert, D. H.; Davidsen, S. K.;
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nificantly more potent than SAHA in the HDAC enzy-
matic assay andexhibit significant antiproliferative
activity against the growth of human HT1080 fibro-
sarcoma andhuman MDA435 breast carcinoma cell
lines. We have shown that these hetero aromatic ring
systems provide new opportunities for the identification
of potent andpossibly selective HDAC inhibitors as
potential antitumor agents.