Chemistry and biology of mercaptoacetamides as novel histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.01.006

A series of mercaptoacetamides were designed and synthesized as novel histone deacetylase inhibitors with the aid of modeling. Their ability to inhibit HDAC activity and their effects on cancer cell growth were investigated. Some compounds exhibit better

Full text of DOI:10.1016/j.bmcl.2005.01.006

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1389–1392
Chemistry and biology of mercaptoacetamides as
novel histone deacetylase inhibitors
Bin Chen,a Pavel A. Petukhov,a Mira Jung,b Alfredo Velena,b Elena Eliseeva,b
Anatoly Dritschilob and Alan P. Kozikowskia,*
aDepartment of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 South Wood Street,
Chicago, IL 60612, USA
bDepartment of Radiation Medicine, Georgetown University, 3970 Reservoir Rd., NW, Washington, DC 20057, USA
Received 22 October 2004; revised 2 January 2005; accepted 6 January 2005
Abstract—A series of mercaptoacetamides were designed and synthesized as novel histone deacetylase inhibitors with the aid of
modeling. Their ability to inhibit HDAC activity and their effects on cancer cell growth were investigated. Some compounds exhibit
better HDAC inhibitory activity than SAHA.
Ó 2005 Elsevier Ltd. All rights reserved.
Histone deacetylases (HDACs) are a family of enzymes
that regulate chromatin remodeling and gene transcrip-
tion. They consequently control critical cellular pro-
cesses, including cell growth, cell cycle regulation,
DNA repair, differentiation, proliferation, and apopto-
sis.1 The post-translational acetylation status of chroma-
tin, which regulates chromatin structure, is determined
by the competing activities of two classes of enzymes,
histone acetyltransferases (HATs) and histone deacetyl-
ases (HDACs). HATs function to acetylate N-terminal
lysine residues in nuclear histones, resulting in the neu-
tralization of the positive charges on the histones and
a more open, transcriptionally active chromatin struc-
ture, while HDACs function to deacetylate and suppress
transcription.2–5 Aberrant acetylation of histone tails
emerging from HAT mutations or abnormal recruit-
ment of HDACs has been linked to carcinogenesis.6–9
HDAC inhibitors have been shown to reverse repression
and to induce reexpression of differentiation inducing
genes.10 It is believed that HDAC inhibitors provide un-
ique opportunities in the discovery of small molecule
therapeutics for the treatment of cancer.
tumor effects (Fig. 1).11 Some of these inhibitors are
currently in phase I/II clinical trials.12 FK228 (or
FR901228), a natural product of a rare family of bicyclic
depsipeptides,13 is currently the only member of the cyc-
lic peptide class under clinical investigation.14 It has
been found that the reduced form of FK228, RedFK
(FK228 + DTT), is more active against HDACs than
FK228 itself.15 The disulfide bond in FK228 is reduced
in cells, releasing a free thiol that can bind to the zinc ion
present at the bottom of a narrow binding pocket in the
HDACs (Fig. 2c). Moreover, Nishino recently reported
that cyclic tetrapeptides bearing a sulfhydryl group po-
tently inhibit histone deacetylases at picomolar concen-
tration levels.16
From this natural product lead, the crystal structure of
HDAC enzyme17 and the key elements of inhibitor–en-
zyme interaction, we designed (Fig. 2) and synthesized
a range of mercaptoacetamides and investigated them
for their ability to inhibit HDAC activity, and for their
effects on cancer cell growth.
The design of mercaptoacetamides has been aided by the
analysis of potential small molecules that may fit into
the binding site of HDAC and their interactions with
the HDAC protein. A homology 3D model of the
HDAC1 protein was built using the HDLP protein
(PDB:1C3S).17,18 The sequence alignment of the
HDAC1 and HDLP proteins were consistent with that
published earlier.17 The homology model of HDAC
A variety of natural and synthetic compounds have been
reported that show HDAC inhibitory activity and anti-
Keywords: Histone deacetylase inhibitor; HDAC; Mercaptoacetamide.
*
Corresponding author. Tel.: +1 312 996 7577; fax: +1 312 996
7107; e-mail: kozikowa@uic.edu
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.006

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B. Chen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1389–1392
( )n
N
H O
HN
O
O
O
OH
OH
O
R
N
H
O
N
Trichostatin A (TSA)
NH HN
R1
O
O
O
H
N
Trapoxin A (R = R1 = Benzyl, n = 2)
Trapoxin B (R = R1 = Benzyl, n = 1)
HC-Toxin (R = R1 = Me, n = 1)
N
H
SAHA
O
H O
O
HN
O
O
N
HN
NH
O
S
O
NH HN
O
O
HN
S
O
O
HN
N
OMe
Depsipeptide
FK228
Apicidin
O
Figure 1. Structure of some known HDAC inhibitors.
H
H
H
H
O
H
S
H
Me
H
O
S
O
HN
Zn
Zn
Zn
Zn
HN
O
O
HN
O
O
H
H
H
N
N
N
N
H
O
O
O
O
(a)
(c)
(d)
(b)
Figure 2. Possible mode of interaction of the zinc ion of HDAC with (a) acetylated lysine moiety of histone, (b) hydroxamate group of an HDAC
inhibitor, (c) thiol group from FK228 + DTT, (d) our designed mercaptoacetamide-based inhibitors.
was subjected to relaxation (MD simulations) of the
amino acid side chains followed by final optimization19
using the CHARMm program (C29b1).20,21
mediate 3 was coupled with a carboxylic acid and the tri-
tyl protecting group was removed to provide
mercaptoacetamide 4 (Scheme 1).
All new ligands proposed during the rational drug de-
sign stage were docked to the binding site to ensure that
they would fit into the binding site. The docking, scor-
ing, and ranking were performed using the FlexX and
CScore modules available in Sybyl6.91.22 Most impor-
tantly, it was found that the HDAC binding site can
accommodate head groups as large as thiol and mercap-
toacetamide. The optimal linker according to the com-
puter model of the HDAC binding site was between
four and six CH2 units. The binding mode of the mer-
captoacetamide-based ligands was similar to that found
for SAHA and TSA (Fig. 3). A detailed description of
the computer-aided drug design protocols will be pub-
lished elsewhere.
The mercaptoacetamides of general structure 4 contain-
ing spacers of varying length were synthesized starting
from methyl mercaptoacetate 1. This compound was
protected by tritylation to give ester 2, and reacted in
turn with an alkyldiamine to provide the amine 3. Inter-
Figure 3. Compound 10b docked into the binding site of HDAC1.

B. Chen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1389–1392
1391
O
O
( )n
H2N
NH2
Ph3COH
TFA
TrtS
HS
OMe
OMe
2
1
O
O
O
EDCI
NH2
RCOOH
TFA
Et3SiH
TrtS
SH
SH
( )n
( )n
4
N
H
R
N
N
H
H
3
O
O
TFA
Et3SiH
RNCO
R
( )n
N
H
N
H
N
H
5
Scheme 1.
The preparation of mercaptoacetamides of structure 5
containing a urea spacer was brought about by reaction
of amine 3 with an isocyanate, followed by the deprotec-
tion (Scheme 1).
Table 1. HDAC inhibitory activity of mercaptoacetamides
Compound
50% HDAC activity
inhibition (lM)
4a R = p-Me2NPh, n = 1
4b R = p-Me2NPh, n = 2
4c R = p-Me2NPh, n = 3
4d R = p-Me2NPh, n = 4
4e R = p-Biphenyl, n = 1
4f R = p-Biphenyl, n = 2
4g R = p-Biphenyl, n = 3
4h R = Ph, n = 3
0.80
4.70
0.20
0.45
0.80
13.0
0.55
1.1
Mercaptoacetamides of structure 10 were synthesized
from mercaptoacetic acid 6 by trityl protection and con-
densation with an amino ester to afford 8. Next, base
hydrolysis of compound 8, amide formation in the pres-
ence of EDCI, and deprotection of the trityl group gave
mercaptoacetamide 10 (Scheme 2).
4i R = Ph, n = 4
4j R = HSCH2, n = 4
0.90
10.0
0.20
0.25
0.40
The in vitro HDAC inhibitory activity of these com-
pounds was determined by using fluor-Lys as the sub-
strate (BIOMOL). These data are displayed as 50%
HDAC activity inhibition values in Table 1. Both TSA
and SAHA were used as positive controls. From these
data, it is apparent that the activity does show some
dependence on chain length, with n = 3 or 4 being best,23
and amide linkers being better than urea linkers. Substi-
tution of the five methylene spacer with a p-xylylene unit
as in 4k gives comparable HDAC inhibitory activity. As
observed previously, the optimum activity is achieved
for R = p-dimethylaminophenyl in comparison to biphe-
nyl, phenyl, or mercaptomethyl bearing ligands.
4k R = p-Me2NPh, (CH2)n = p-Ph
4l R = 8-Quinolinyl, n = 3
4m R = 3-Quinolinyl, n = 3
5a R = p-Me2NPh, n = 3
5b R = p-Me2NBn, n = 4
5c R = Ph, n = 3
1.7
5.0
0.75
0.63
1.0
5d R = Ph, n = 4
5e R = Bn, n = 3
10a R = p-Me2NPh, n = 3
10b R = 8-Quinolinyl, n = 3
10c R = 3-Quinolinyl, n = 3
10d R = 6-Quinolinyl, n = 3
10e R = Ph, n = 3
0.60
0.044
0.048
0.90
0.30
TSA
SAHA
0.0035
0.080
Compounds 10a–e represent the reverse amide analogs
of 4. While 10a, e, and 4c have comparable activities,
10b and c are particularly potent, and they show a clear
dependence on the site of attachment of the thiol bear-
ing appendage to the quinoline ring system. The aro-
matic cap of these HDAC inhibitors may thus be able
to have a better interaction with the outside rim of the
gorge region of the HDACs.
To test the biological effects of these ligands, cytotoxi-
cities were then determined following 24 h exposure of
human cancer cell lines, including cervix carcinoma
(HeLa), prostate cancer (PC-3), breast cancer (MCF-7)
and squamous carcinoma (SQ-20B), to four compounds
(Table 2). The IC50 values of these compounds range
from 30 to 130 lM. as shown in Table 2. Compound
10b shows potent cytotoxicity in these cancer cells.
TsOH
O
O
( )n
H2N
COOBn
Ph3COH
TFA
HS
TrtS
OH
OH
EDCI, Et3N
6
7
O
RNH2
EDCI
5% NaOH
MeOH
Table 2. Antiproliferative activities of mercaptoacetamides
BnO
STrt
STrt
( )n
N
H
O
Compound
IC50 (lM) SD
SQ-20B MCF-7
8
HeLa
PC-3
O
O
H
H
N
TFA
Et3SiH
N
SH
4c
4g
108 0.020 100 0.093 125 0.090 110 0.081
80 0.018 130 0.043 110 0.044 100 0.056
( )n
( )n
R
N
H
R
N
H
O
O
9
10
10b
10c
30 0.031 31 0.024
94 0.016 100 0.043
42 0.035
80 0.020
33 0.021
50 0.026
Scheme 2.

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B. Chen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1389–1392
14. Sandor, V.; Bakke, S.; Robey, R. W.; Kang, M. H.;
Blagosklonny, M. V.; Bender, J.; Brooks, R.; Piekarz, R.
L.; Tucker, E.; Figg, W. D.; Chan, K. K.; Goldspiel, B.;
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As is evident from the present work, some of these read-
ily prepared mercaptoacetamides are potent HDAC
inhibitors that show promising activity in inhibiting cel-
lular proliferation. Issues relating to isoform selectivity
as well as chemosensitization using these ligands are
now under study.
16. Nishino, N.; Jose, B.; Okamura, S.; Ebisusaki, S.; Kato,
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Acknowledgments
We are pleased to acknowledge funding from Genether-
apy Pharmaceuticals Inc, Springfield, VA.
18. In the course of preparation of this paper, the X-ray
structure of human HDAC8 was published. Somoza, J.
R.; Skene, R. J.; Katz, B. A.; Mol, C.; Ho, J. D.; Jennings,
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