A novel HDAC inhibitor with a hydroxy-pyrimidine scaffold

Article abstract of DOI:10.1016/j.bmcl.2011.05.098

Histone deacetylases (HDACs) are enzymes involved in many important biological functions. They have been linked to a variety of cancers, psychiatric disorders, and other diseases. Since small molecules can serve as probes to study the relevant biological

Full text of DOI:10.1016/j.bmcl.2011.05.098

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4164–4169
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel HDAC inhibitor with a hydroxy-pyrimidine scaffold
Melissa M. Kemp a, Qiu Wang a,b, Jason H. Fuller a, Nathan West a,c, Nicole M. Martinez a,
Elizabeth M. Morse a,c, Michel Weïwer a, Stuart L. Schreiber a,b,d, James E. Bradner a,c, Angela N. Koehler a,
⇑
a Broad Institute of Harvard and MIT, Cambridge, MA 02142, United States
b Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, United States
c The Dana-Farber Cancer Institute, Division of Hematologic Neoplasia, Boston, MA 02115, United States
d Howard Hughes Medical Institute, Chevy Chase, MD 20815, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Histone deacetylases (HDACs) are enzymes involved in many important biological functions. They have
been linked to a variety of cancers, psychiatric disorders, and other diseases. Since small molecules can
serve as probes to study the relevant biological roles of HDACs, novel scaffolds are necessary to develop
more efficient, selective drug candidates. Screening libraries of molecules may yield structurally diverse
probes that bind these enzymes and modulate their functions in cells. Here we report a small molecule
with a novel hydroxy-pyrimidine scaffold that inhibits multiple HDAC enzymes and modulates acetyla-
tion levels in cells. Analogs were synthesized in an effort to evaluate structure–activity relationships.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 1 March 2011
Revised 24 May 2011
Accepted 25 May 2011
Available online 2 June 2011
Keywords:
Histone deacetylase
Non-selective inhibitor
Hydroxy-pyrimidine
SAR studies
Post-translational modifications on histones are essential for
regulating gene expression.1 A wide variety of chemical modifica-
tions, such as methylation, phosphorylation, and acetylation, can
alter chromatin structure, influencing levels of gene expression.2
numerous diseases, such as inflammation5 and neurosis,6 as well
as regulation of tumor suppressor genes and oncogenes, contribut-
ing to cancer pathogenesis.2,7
Using small molecules to inhibit HDACs has provided promising
clinical results in different models of cancer, such as cutaneous T-
cell lymphoma and neuroblastoma.4,7,8 Among HDAC inhibitors
(HDACi) identified so far, four major classes of structures are fre-
quently studied, including hydoxamates (1), ortho-amino benzam-
ides (2), aliphatic acids (3), and cyclic peptides (4a–b) (Fig. 1).7,9
Most HDACi follow the pharmacophore model of a capping region
that interacts with the surface of the protein, a metal-binding re-
gion that chelates zinc to disrupt the enzymatic activity, and a lin-
ker domain consisting of a hydrophobic spacer that interacts with
the hydrophobic-binding channel (Fig. 1).6b,10
One important modification to histones is N-e-lysine acetylation.
The positive charge of the lysine residues on histone tails allows
the negatively charged DNA to tightly coil around histones, con-
tributing to inhibition of transcription. An increase in lysine side
chain acetylation has the opposite effect, neutralizing the positive
charge and relaxing the DNA from the histone proteins, causing an
increase in the amount of transcriptional activity. This tight bal-
ance of hyperacetylation and hypoacetylation is maintained by
two counteracting families of enzymes, histone acetyltransferases
(HATs) and histone deacetylases (HDACs), respectively.
Since the initial discovery of the first mammalian HDAC,3 17
additional HDACs have been identified. They are divided into sev-
eral classes based on sequence homology to their respective yeast
orthologs.2 The classical zinc-dependent HDACs are divided into
three subclasses, class I (HDAC1, 2, 3, and 8), class II (HDAC4, 5,
6, 7, 9, and 10), and class IV (HDAC11). Class III HDACs, also known
as sirtuins, are zinc-independent and instead require a NAD+ cofac-
tor for deacetylase activity.4 HDACs are present in a variety of
organisms and participate in a myriad of biological functions,
including post-translational modification of various non-histone
substrates. The aberrant activity of HDACs has been connected to
A key motivation for investigating compounds that inhibit
HDACs is to find a compound that selectively targets individual
HDACs or a subclass of HDACs. A few compounds have been found
to selectively target HDACs; however progress has been limited.11
Screening diverse small molecules allows for discovery of structur-
ally novel HDACi that may modulate the proteins through different
mechanisms or with previously unobserved patterns of selectiv-
ity.12 Here we report a structurally novel HDACi, BRD-2577 (7) that
was discovered in a binding assay involving a small-molecule
microarray (SMM) (Fig. 2A). It is interesting to note that the struc-
ture of BRD-2577 is a hydroxy-pyrimidine scaffold, and does not
conform to the four main structural classes of HDACi. We further
explored the structure–activity relationships (SAR) and cellular ef-
fects of this molecule. Recently, Donald and co-workers evaluated
⇑
Corresponding author. Tel.: +1 617 714 7364; fax: +1 617 714 8943.
E-mail address: koehler@broadinstitute.org (A.N. Koehler).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.098

M. M. Kemp et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4164–4169
4165
Figure 1. Pharmacophore model and representative structures of HDACi. Hydroxamic acid: suberoylanilide hydroxamic acid (SAHA) (1), ortho-amino benzamide: MS-275
(2), aliphatic acid: valproic acid (3), and cyclic peptides: largazole (4a) and trapoxin (4b).
Figure 2. (A) Synthesis of BRD-2577 (7). (B) Inhibition profiling of BRD-2577 in triplicate against HDAC1–9. (C) Lineweaver–Burk plot of BRD-2577 at 100
and 1 M (N) for HDAC2.
lM (d), 10 lM (j),
l
the pyrimidine hydroxamic acid moiety and found that their com-
pounds were able to inhibit HDACs, demonstrating the potential
importance of pyrimidines in inhibition of HDACs.13
BRD-2577 was originally identified as a binder to purified
HDAC8 using SMMs.12 To determine whether the binder inhibits
the deacetylase activity of the enzyme, we resynthesized this com-
not interfere with the proteolytic activity of trypsin (Supplemen-
tary data). The mode of inhibition was also evaluated by analyzing
various doses of compound with concentrations of substrate 10
times above and below the Km of HDAC2. The Lineweaver–Burk
plot for BRD-2577 and HDAC2 is shown in Figure 2C. The y-inter-
cept does not vary across concentrations of substrate, suggesting
that BRD-2577 is a competitive inhibitor, and that the inhibitor is
binding to the active site of enzyme.
The structural novelty of BRD-2577 as compared to other HDACi
motivated further studies to gain insight into the molecular fea-
tures that may be responsible for target inhibition. Towards this
end, we investigated whether two major substructures of BRD-
2577, 5 and 6, were independently capable of inhibition. Neither
pound using a nucleophilic substitution reaction between a-chlo-
roketone (5) and mercapto-methylpyrimidine (6) (Fig. 2A) and
evaluated it for HDAC inhibitory effects using a trypsin-coupled
in vitro biochemical assay (Fig. 2B).14 This biochemical assay em-
ploys an acetylated lysine tripeptide substrate amide linked to
aminocoumarin (AMC). When HDACs deacetylate the lysine resi-
due, trypsin is able to cleave the AMC from the substrate, thus
increasing the fluorescent signal.14,15 Small-molecule HDAC inhibi-
tion prevents the deacetylation of the lysine, preventing the gener-
ation of fluorescent signal. The effects of various concentrations of
BRD-2577 were evaluated, and the data were normalized against a
DMSO control. IC50 values were measured by fitting the data to a
non-linear regression model. The IC50 values for BRD-2577 against
the a-chloroketone group (5) nor the pyrimidine ring (6) exhibited
inhibition in the biochemical assay. This may indicate that activity
is not derived from a single biasing element but rather by the
whole skeleton.
Using simple reactions, we synthesized a set of analogs to inves-
tigate the effects of chemical modifications at different positions
(Table 1). The initial SAR study focused on the pyrimidine group
and its substituent groups. The hydroxyl group was necessary for
activity (8–10). The replacement of pyrimidine by a phenyl group
HDAC1–9 ranged from ꢀ11 to 74
lM, suggesting it is a non-selec-
tive inhibitor of HDACs with modest potency. A counter screen
demonstrated that this compound inhibits HDAC activity and does

4166
M. M. Kemp et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4164–4169
Table 1
SAR on the pyrimidine ring and respective IC50 values
O
R
O
a
HDAC IC50
(lM)
H3C
N
H
Compound
R
1
2
3
4
5
6
7
8
9
O
S
N
CH3
16.4
18.9
15.2
32.5
27.9
1.05)
11.2
1.09)
24.4
23.9
74.1
7
O
(
1.08)
(
1.10)
(
1.05)
(
1.07)
(
(
(
1.07)
(
1.07)
(
1.10)
N
S
H3C
N
H
OH
N
N
N
N
8
9
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
CH3
S
S
N
OCH3
CH3
CH3
N
10
11
12
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
54.2
>100
>100
16.7
>100
>100
88.0
>100
>100
31.9
>100
>100
>100
>100
>100
11.7
CH3
N
S
OH
OH
O
N
CH3
N
O
OH
13
>100
>100
>100
>100
>100
>100
>100
>100
>100
CH3
a
Values are means of >2 experiments.
resulted in loss of activity (11). Interestingly, the activity was re-
tained when the sulfur atom was substituted by oxygen (12), albeit
at a reduced potency and with an altered selectivity pattern. Addi-
tionally, removing nitrogen at the 1-position from 12 (13) led to
loss of activity, demonstrating that both nitrogen atoms are essen-
tial for binding.
the carbonyl group and phenyl group was also examined by syn-
thesizing compounds 31–33. Although most of the modifications
described here led to inactive analogs, several of the compounds
retained inhibition of HDAC6, which is possibly due to its unique
enzymatic structure consisting of two catalytic domains.8
We examined the effects of selected compounds on both his-
Next, we investigated the consequences of chemical modifica-
tion of the opposing end of the structure of BRD-2577 (Table 2).
The removal or the reduction of the carbonyl group (14 and 15,
respectively) resulted in a loss of potency, suggesting the carbonyl
function is indispensable. Similarly, the modifications and the
replacement of the acetamide group with various functionalities
(16–22) resulted in decreased potencies for most of the HDACs,
while the chloride analog (21) retained some inhibition of HDAC1,
2, 4 and 6. The dihydroxy analog (23) was found only to inhibit
HDAC6. Protection of the dihydroxy group via a dioxin (24) led
to a completely inactive compound. The acetamide was also re-
placed with a more rigid oxindole group (25). The role of the phe-
nyl group was further explored. Replacing the phenyl group with a
more hydrophobic thiophene (26) preserved inhibitory activity for
HDAC6. Replacing the aromatic ring with an aliphatic chain (27–
30) was not tolerant to its inhibition activity although 30 retained
some activity for HDAC 1, 2, 3, 5, and 6. The linker region between
tone and a
-tubulin lysine acetylation in cells.16 Tubulin acetylation
is regulated by HDAC617 and many of the analogs inhibited its
activity. HeLa cells were plated in 384-well plates with
2000 cells/well and treated with compounds at various concentra-
tions for 16 h. Acetylation levels were measured using antibodies
that recognize acetylated
a-tubulin or acetylated lysine residues
in post-translationally modified proteins. The fluorescence levels
were measured for each antibody and normalized to the DMSO
control. Treatment with BRD-2577 leads to an increase in both to-
tal lysine and tubulin acetylation (Fig. 3), consistent with our bio-
chemical assay data demonstrating that it inhibits class I and class
II HDACs. Other compounds that inhibited HDAC6 in the enzymatic
assay were also evaluated for their activity in cells but did not
show a significant increase in acetylation level at a concentration
of 20
increases both global lysine and
sistent with the ability to inhibit both class I and class II HDACs.
lM (data not shown). Our positive control compound, SAHA,
a-tubulin acetylation levels, con-

M. M. Kemp et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4164–4169
4167
Table 2
SAR on the phenyl moiety and respective IC50 values
S
N
OH
R
a
N
HDAC IC50
(lM)
CH3
Compound
R
1
2
3
4
5
6
7
8
9
O
14
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
H3C
H3C
N
H
OH
O
15
16
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
13.7
>100
>100
>100
>100
N
H
O
>100
X
X = H
O
X =
X =
17
18
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
10.8
>100
>100
>100
>100
>100
>100
N
CH3
CH3
O
N
H
19
20
21
22
X=OCH3
X=tBu
X=Cl
>100
>100
82.3
>100
>100
67.0
>100
>100
86.4
>100
>100
>100
>100
>100
94.3
>100
>100
60.9
>100
12.9
40.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
X=OH
>100
>100
>100
O
HO
HO
O
23
>100
>100
>100
>100
>100
16.7
>100
>100
>100
O
24
25
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
32.6
>100
>100
>100
>100
>100
>100
O
O
H
N
O
S
O
26
27
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
24.8
>100
>100
>100
>100
>100
>100
O
>100
H3CO
O
28
29
30
>100
>100
77.2
>100
>100
69.3
>100
>100
40.0
>100
>100
>100
>100
>100
48.6
>100
>100
7.2
>100
>100
>100
>100
>100
>100
>100
>100
>100
H3C
O
O
Bu
t
O
EtO
(continued on next page)

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M. M. Kemp et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4164–4169
Table 2 (continued)
S
N
OH
O
R
a
N
HDAC IC50
(lM)
CH3
Compound
R
1
2
3
4
5
6
7
8
9
N
n
H
31
32
33
n = 3
n = 1
n = 5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
a
Values are means of >2 experiments.
Figure 3. Immunofluorescence microscopy of cells treated with BRD-2577. (A) Acetylated lysine and tubulin levels plotted against increasing concentration of BRD-2577. (B)
Representative images of Hoechst (cyan), acetylated lysine (red), acetylated tubulin (green) for DMSO, BRD-2577 (6 M), SAHA (0.6 M), and MS-275 (0.6 M).
l
l
l

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4169
5. Leoni, F.; Zaliani, A.; Bertolini, G.; Porro, G.; Pagani, P.; Pozzi, P.; Dona, G.;
Fossati, G.; Sozzani, S.; Azam, T.; Bufler, P.; Fantuzzi, G.; Goncharov, I.; Kim, S.
H.; Pomerantz, B. J.; Reznikov, L. L.; Siegmund, B.; Dinarello, C. A.; Mascagni, P.
Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 2995.
6. (a) Dompierre, J. P.; Godin, J. D.; Charrin, B. C.; Cordelieres, F. P.; King, S. J.;
Humbert, S.; Saudou, F. J. Neurosci. 2007, 27, 3571; (b) Minucci, S.; Pelicci, P. G.
Nat. Rev. Cancer 2006, 6, 38; (c) Suzuki, T. Chem. Pharm. Bull. (Tokyo) 2009, 57,
897.
MS-275, a control compound, increased global lysine acetylation,
but caused little change in tubulin acetylation, which is consistent
with its selective inhibition of HDAC1–3 versus HDAC6 in our bio-
chemical assay.
In summary, we have identified a novel, pan-selective HDAC
inhibitor from an SMM-based binding screen and demonstrated
inhibitory activity against HDACs using an in vitro biochemical as-
say and cellular acetylation assays. The scaffold does not resemble
previously established pharmacophore models for HDAC inhibi-
tors. This novel HDACi structure may be explored further using
structure-guided approaches to elucidate binding mechanism and
to enhance the potency and selectivity through various structural
modifications, leading towards broad biological and therapeutic
applications.
7. Tan, J.; Cang, S.; Ma, Y.; Petrillo, R. L.; Liu, D. J. Hematol. Oncol. 2010, 3, 5.
8. Marks, P. A.; Xu, W. S. J. Cell. Biochem. 2009, 107, 600.
9. (a) Bowers, A.; West, N.; Taunton, J.; Schreiber, S. L.; Bradner, J. E.; Williams, R.
M. J. Am. Chem. Soc. 2008, 130, 11219; (b) Dokmanovic, M.; Clarke, C.; Marks, P.
A. Mol. Cancer Res. 2007, 5, 981; (c) Miller, T. A.; Witter, D. J.; Belvedere, S. J.
Med. Chem. 2003, 46, 5097; (d) Singh, E. K.; Ravula, S.; Pan, C.-M.; Pan, P.-S.;
Vasko, R. C.; Lapera, S. A.; Weerasinghe, S. V. W.; Pflum, M. K. H.; McAlpine, S. R.
Bioorg. Med. Chem. Lett. 2008, 18, 2549.
10. Sternson, S. M.; Wong, J. C.; Grozinger, C. M.; Schreiber, S. L. Org. Lett. 2001, 3,
4239.
11. (a) Bieliauskas, A. V.; Pflum, M. K. H. Chem. Soc. Rev. 2008, 37, 1402; (b) Butler,
K. V.; Kalin, J.; Brochier, C.; Vistoli, G.; Langley, B.; Kozikowski, A. P. J. Am. Chem.
Soc. 2010, 132, 10842; (c) Chen, Y.; Lopez-Sanchez, M.; Savoy, D. N.; Billadeau,
D. D.; Dow, G. S.; Kozikowski, A. P. J. Med. Chem. 2008, 51, 3437; (d) Kozikowski,
A. P.; Tapadar, S.; Luchini, D. N.; Kim, K. H.; Billadeau, D. D. J. Med. Chem. 2008,
51, 4370; (e) Mai, A.; Massa, S.; Pezzi, R.; Simeoni, S.; Rotili, D.; Nebbioso, A.;
Scognamiglio, A.; Altucci, L.; Loidl, P.; Brosch, G. J. Med. Chem. 2005, 48, 3344;
(f) Matsuyama, A.; Shimazu, T.; Sumida, Y.; Saito, A.; Yoshimatsu, Y.;
Seigneurin-Berny, D.; Osada, H.; Komatsu, Y.; Nishino, N.; Khochbin, S.;
Horinouchi, S.; Yoshida, M. EMBO J. 2002, 21, 6820; (g) Paris, M.; Porcelloni,
M.; Binaschi, M.; Fattori, D. J. Med. Chem. 2008, 51, 1505; (h) Suzuki, T.;
Matsuura, A.; Kouketsu, A.; Hisakawa, S.; Nakagawa, H.; Miyata, N. Bioorg. Med.
Chem. 2005, 13, 4332; (i) Tang, W.; Luo, T.; Greenberg, E. F.; Bradner, J. E.;
Schreiber, S. L. Bioorg. Med. Chem. Lett. 2011, 21, 2601; (j) Wang, D.-F.; Helquist,
P.; Wiech, N. L.; Wiest, O. J. Med. Chem. 2005, 48, 6936.
12. Vegas, A. J.; Fuller, J. H.; Koehler, A. N. Chem. Soc. Rev. 2008, 37, 1385.
13. (a) Donald, A. D. G.; Clark, V. L.; Patel, S.; Day, F. A.; Rowlands, M. G.; Wibata, J.;
Stimson, L.; Hardcastle, A.; Eccles, S. A.; McNamara, D.; Needham, L. A.;
Raynaud, F. I.; Aherne, W.; Moffat, D. F. Bioorg. Med. Chem. Lett. 2010, 20, 6657;
(b) Moffat, D.; Patel, S.; Day, F.; Belfield, A.; Donald, A.; Rowlands, M.; Wibawa,
J.; Brotherton, D.; Stimson, L.; Clark, V.; Owen, J.; Bawden, L.; Box, G.; Bone, E.;
Mortenson, P.; Hardcastle, A.; van Meurs, S.; Eccles, S.; Raynaud, F.; Aherne, W.
J. Med. Chem. 2010, 53, 8663.
Acknowledgments
This research was supported in part by a grant from the Na-
tional Institute of General Medical Sciences (NIGMS 38627 to
S.L.S.) and in part with Federal funds from the National Cancer
Institute’s Initiative for Chemical Genetics (ICG), under Contract
No. N01-CO-12400. The content of this publication does not neces-
sarily reflect the views or policies of the Department of Health and
Human Services, nor does the mention of trade names, commercial
products or organizations imply endorsement by the US Govern-
ment. S.L.S is an investigator with the Howard Hughes Medical
Institute.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.05.098.
14. Bradner, J. E.; West, N.; Grachan, M. L.; Greenberg, E. F.; Haggarty, S. J.;
Warnow, T.; Mazitschek, R. Nat. Chem. Biol. 2010, 6, 238.
15. Wegener, D.; Wirsching, F.; Riester, D.; Schwienhorst, A. Chem. Biol. 2003, 10,
61.
References and notes
16. (a) Haggarty, S. J.; Koeller, K. M.; Wong, J. C.; Grozinger, C. M.; Schreiber, S. L.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4389; (b) Wong, J. C.; Hong, R.; Schreiber,
S. L. J. Am. Chem. Soc. 2003, 125, 5586.
17. Hubbert, C.; Guardiola, A.; Shao, R.; Kawaguchi, Y.; Ito, A.; Nixon, A.; Yoshida,
M.; Wang, X. F.; Yao, T. P. Nature 2002, 417, 455.
1. Cole, P. A. Nat. Chem. Biol. 2008, 4, 590.
2. de Ruijter, A. J.; van Gennip, A. H.; Caron, H. N.; Kemp, S.; van Kuilenburg, A. B.
Biochem. J. 2003, 370, 737.
3. Taunton, J.; Hassig, C. A.; Schreiber, S. L. Science 1996, 272, 408.
4. Witt, O.; Deubzer, H. E.; Milde, T.; Oehme, I. Cancer Lett. 2009, 277, 8.