Heterocyclic ketones as inhibitors of histone deacetylase

Article abstract of DOI:10.1016/j.bmcl.2003.09.007

Several heterocyclic ketones were investigated as potential inhibitors of histone deacetylase. Nanomolar inhibitors such as 22 and 25 were obtained, the anti-proliferative activity of which were shown to be mediated by HDAC inhibition.

Full text of DOI:10.1016/j.bmcl.2003.09.007

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3909–3913
Heterocyclic Ketones as Inhibitors of Histone Deacetylase
Anil Vasudevan,a,* Zhiqin Ji,b Robin R. Frey,b Carol K. Wada,b Douglas Steinman,b
H. Robin Heyman,b Yan Guo,b Michael L. Curtin,b Jun Guo,b Junling Li,b Lori Pease,b
Keith B. Glaser,b Patrick A. Marcotte,b Jennifer J. Bouska,b Steven K. Davidsenb and
Michael R. Michaelidesb
aMedicinal Chemistry Technologies, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA
bCancer Research, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA
Received 29 July 2003; revised 4 September 2003; accepted 4 September 2003
Abstract—Several heterocyclic ketones were investigated as potential inhibitors of histone deacetylase. Nanomolar inhibitors such
as 22 and 25 were obtained, the anti-proliferative activity of which were shown to be mediated by HDAC inhibition.
# 2003 Published by Elsevier Ltd.
A balance between Histone Deacetylase (HDAC) and
Histone Acetyl Transferase (HAT) determines the
extent of acetylation of chromatin proteins. HDAC-
mediated hypoacetylation increases positive charges at
histone lysine residues and condenses the chromatin
structure, generally leading to transcriptional repres-
sion. Recent studies indicate that HDACs are not only
involved in the regulation of chromatin structure and
gene expression, but also the regulation of cell cycle
progression and tumorigenesis (Fig. 1).1
confirmthat the metal-binding group interacts with the
active site zinc, with the linker spanning the tube-like
portion of the binding pocket and positioning the aro-
matic groups so as to make contact with the pocket
entrance.7
Previous publications have delineated efforts aimed at
optimizing hydroxamic acid containing compounds
such as 1 identified via screening of the Abbott com-
pound collection.8 Recently, non-hydroxamate HDAC
inhibitors containing electrophilic ketones such as tri-
fluoromethyl ketones and a-keto amides, exemplified by
2 and 3 have been reported.9 As a continuation of our
quest for non-hydroxamate HDAC inhibitors, a range
of electrophilic ketones were synthesized and evaluated
in this study, some of which are shown in Table 1. The
synthesis of these compounds was accomplished by one
Several classes of compounds that inhibit the activity of
partially purified HDACs, cause growth arrest of a wide
range of transformed cells in culture and inhibit the
growth of human xenografts in tumor growth studies
have been identified.2 Some of these inhibitors have
been shown to act selectively, altering the transcription
of less than 2% of expressed genes3a and several are
undergoing clinical evaluation.3b Because HDAC is a
zinc metalloenzyme, the majority of inhibitors have a
metal chelating element attached to an aromatic group
via a hydrophobic linker. The HDAC inhibitors tri-
chostatin A (TSA) (lit. IC50 3.4 nM)4 and suberoyl ani-
lide hydroxamic acid (SAHA) (lit. IC50 10 nM)5 both
contain a hydroxamic acid moiety as the zinc-binding
group.6 The X-ray crystal structures of TSA and SAHA
bound to an HDAC homologue have been reported and
*Corresponding author. Tel.: +1-847-938-6594; fax: +1-847-935-
0310; e-mail: anil.vasudevan@abbott.com
Figure 1. HDAC inhibitors.
0960-894X/$ - see front matter # 2003 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2003.09.007

3910
A. Vasudevan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3909–3913
of several methods outlined below. Alkylation of 4-
phenylphenol with bromomethyl heptanoate followed
by saponification afforded the carboxylic acid, which
was then converted to the N-methoxy-N-methyl amide
as shown in Scheme 1. Treatment of this intermediate
with the corresponding lithiated heterocycles afforded
4–7.
However, conversion of aldehyde 9 to the cyanohydrin
followed by treatment with sodium azide, protecting
group removal and Jones oxidation afforded the a-keto
1(H)-tetrazole (Scheme 2).
Treatment of aldehyde 9 (Scheme 3) with 2-lithio het-
erocycles followed by oxidation with manganese dioxide
afforded compounds 10 and 11. The cyanohydrin
obtained fromaldehyde 9 was treated with EtOH/HCl
to afford the iminoether, which when heated with 2-
amino-3-hydroxypyridine, followed by Dess–Martin
Attempts at synthesizing the a-keto tetrazole 8 via alkyl-
ation of BOM-protected 2-lithio tetrazole with 7-chloro-
carbonyl-heptanoic acid methyl ester were unsuccessful.
Table 1. HDAC inhibitory potency of a-keto heterocycles
Compd
X
IC50 (mM)
29
2.9
39
0.11
>50
>50
>50
>50
2.0
Scheme 1. (a) Br(CH2)6COOCH3, Cs2CO3, DMF, 92%; (b) LiOH,
H2O, THF, 95%; (c) HN(CH3)OCH3, DMF, 68%; (d) n-BuLi, THF,
ArH, 0 ꢀC to rt.
4a
5a
6a
7a
Scheme 2. (a) Br(CH2)7OH, Cs2CO3, DMF, 92%; (b) Swern oxdn,
65%; (c) KCN, NaHSO3, THF/H2O, 90%; (d) THP, TsOH, 90%;
(e) NaN3, NH4Cl, DMF, 120 ꢀC, 5 h, 32%; (f) TsOH, CH3OH, 83%;
(g) Jones reagent, 60%.
8b
10a
11a
12a
13b
15b
>50
>50
>50
1.8
1.1
Scheme 3. (a) ArLi, THF, ꢁ78 ꢀC to rt; (b) Dess–Martin periodinane,
CH2Cl2; (c) KCN, NaHSO3, H2O/THF, 90%; (d) AcCl, EtOH, 100%;
(e) 2-amino-3-hydroxypyridine, 2-ethoxyethanol, 130 ꢀC, 40%; (f) 2-
aminoethanol, CH2Cl2; TsOH, CHCl3, 60 ꢀC, 30%.
aRadioactivity-based HDAC assay.13
bFluorescence-based HDAC assay.14

A. Vasudevan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3909–3913
3911
periodinane oxidation afforded the a-keto azabenzox-
azole 12.11 Further, treatment of the iminoether with
2-aminoethanol followed by oxidation afforded the
keto-oxazoline 13.
1,5,7-triazabicyclo[4.4.0]dec-5-ene (PTBD)16 and 16 at
175 ꢀC in acetonitrile using a single-mode microwave unit
to afford compounds such as 18 and 19. [CAUTION:
Heating solvents in a closed-vessel in a microwave to
temperatures above their boiling point should only be
performed in instruments equipped with appropriate
safety features.] Compounds containing an amide,
hydrazide or diacylhydrazine linker (20–28) were syn-
thesized using a suitably protected acid chloride as
shown in Scheme 6. A set of 1,3,4-oxadiazole linked
compounds were also synthesized via a dehydrative
cyclization using the Burgess reagent in a single-mode
microwave.
For the synthesis of the a-keto oxazoles, 2-lithio oxazole
was treated first with ZnCl2 at ꢁ78 ꢀC and then with
CuI at 0 ꢀC, followed by the acid chloride obtained from
carboxylate 14 (Scheme 4).
Table 1 lists several of the a-keto heterocycles investi-
gated in this study along with the corresponding tri-
fluoromethyl ketone and keto-amide. Initial results
obtained with mono- and bicyclic heterocycles were not
encouraging. However, the a-keto oxazoline 13 and the
corresponding a-keto oxazole analogue 15 displayed
single-digit micromolar potency. The sharp difference in
potency between the a-keto oxazole and a-keto thiazole
was somewhat surprising given their similar s values,12
though a similar trend has been observed in elastase
inhibitors,10b suggesting that subtle differences within
the catalytic site can lead to dramatic changes in
potency. The a-keto tetrazole 8 was equipotent with the
corresponding a-keto acid (data not shown). The lack
of HDAC activity exhibited by the a-keto benzoxazole
and a-keto azabenzoxazole analogues 6 and 12 would
suggest a steric constraint within the active site of
HDAC. As a result, it was decided to focus on the a-
keto oxazole 15 and explore various changes in the ali-
phatic linker as well as the terminal hydrophobic moiety
to improve the HDAC inhibitory potency of this class
of compounds.
The meta phenyl substitution pattern in 18 affords a
slight improvement in potency compared to the para
phenyl substitution in case of the ether-linked com-
pounds, and is equipotent with the 2-naphthyl sub-
stituent (15) in case of the ether and amine-linked
compounds, 17 and 19. The amide linked compounds
tended to afford significantly better inhibitory potency
against HDAC with 22 being 10-fold more potent that
the corresponding ether linked compound 18. In gen-
eral, compounds with a six-atom tether between the
ketone and amide carbonyl were about 5-fold more
potent than compounds with a five-atom tether, con-
sistent with SAR trends observed with trifluoromethyl
The synthesis of several nitrogen and ether linked a-keto
oxazoles was achieved via the modified synthetic route
shown in Scheme 5. Nucleophilic displacement of the
bromine in intermediate 16 with anilines was performed
in a commercial single-mode microwave unit15 at 160ꢀ
in DMF to afford compounds such as 17. A series of
hydrophobic aryl ether substituents were synthesized in
a one-pot reaction of the phenol, polystyrene-supported
Scheme 6. (a) Oxazole, BuLi, THF, ꢁ78 ꢀC; ZnCl2, ꢁ78 ꢀC to rt, CuI,
then acid chloride, 59%; (b) LiOH, THF/H2O, 95%; (c) ArNH2,
EDC, HOBt, 87%; (d) ArCONHNH2, EDC, HOBt, 62%;
(e) (C2H5)3NSO2NHCO2CH3, THF, 150 ꢀC, 15 min, microwave, 38%.
Scheme 4. (a) SOCl2, DMF, reflux, 94%; (b) oxazole, BuLi, THF,
ꢁ78 ꢀC; ZnCl2, ꢁ78 ꢀC to rt, CuI, then acid chloride, 49%.
Scheme 5. (a) Oxazole, BuLi, THF, ꢁ78 ꢀC; ZnCl2, ꢁ78 ꢀC to rt,
CuI, then acid chloride, 59%; (b) Ar-NH2, DMF, 160 ꢀC, 30 min;
(c) PTBD, Ar–OH, acetonitrile, 175 ꢀC, 15 min.
Figure 2. Western blot analysis of the effect of 22 and SAHA on H4
acetylation and p21 induction in MDA435 cells.

3912
A. Vasudevan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3909–3913
ketones and a-keto amides.9 Several potent HDAC
inhibitors were obtained via incorporation of thiazole
containing substituents into the side chain as shown in
25. The hydrazide-linked compound (27) was devoid of
HDAC activity whereas the 1,3,4-oxadiazole (29) linker
afforded a more potent analogue than the corresponding
diacylhydrazine linked analogue (28). The sulfur and
sulfone linked compounds (30 and 31, respectively) were
equipotent with the nitrogen linked compounds, as were
analogues in which the heteroatomlinker was replaced
with an ethylene moiety (32). Reverse amides 33 and 34
with indole substituents in the side chain were also
found to be potent inhibitors of HDAC. Some of the
more potent compounds from this study were found to
have anti-proliferative activity in MDA435 cells in the
micromolar range, with 25 being the most potent com-
pound (IC50 2.3 mM). Figure 2 shows a Western blot
analysis of histone hyperacetylation and p21 induction
in MDA435 cells produced by 22 at 50 mM, consistent
with the notion that the anti-proliferative activity is
mediated by HDAC inhibition (Table 2).17
The anti-proliferative activity of this class of com-
pounds was presumably compromised by their rapid
reduction to the inactive alcohol, as shown in Figure 3.
Further, in whole blood, representative a-keto oxazoles
15 and 18 were rapidly reduced to the corresponding
alcohol with half-lives of 0.18 and 0.21 h, respectively.
Attempts to modulate this rapid reduction via incor-
poration of a cyclopropyl substituent alpha to the
ketone (35) or substituents on the oxazole moiety (data
not shown) were unsuccessful as these compounds were
inactive against HDAC.
Table 2. Effect of linker variation on HDAC inhibitory activity of a-keto oxazoles
IC50 (mM)b
>10
Compd
n
X
R
IC50 (mM)b
Compd
n
X
R
17b
5
–NH–
0.43
27a
4
–CONHNH–
18b
19b
20b
21b
22b
23b
24b
25b
26b
5
5
5
5
5
4
4
–O–
0.62
0.52
0.32
0.58
0.06
1.50
0.31
0.03
6.40
28a
29a
30b
31b
32b
33b
34b
36a
35a
5
–CONHNHCO–
1.83
0.25
0.54
0.61
0.99
0.09
0.34
>50
>50
–O–
–CONH–
–CONH–
–CONH–
–CONH–
–CONH–
5
5
4
5
4
–S–
–SO2–
–CH¼CH–
–NHCO–
–NHCO–
4
–CONHCH2–
aRadioactivity-based HDAC assay.13
bFluorescence-based HDAC assay.14

A. Vasudevan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3909–3913
3913
nuclear extraction of K562 erythroleukemia cells.11 (b) Saito,
A.; Yamashita, T.; Mariko, Y.; Nosaka, Y.; Tsuchiya, K.;
Ando, T.; Suzuki, T.; Tsuruo, T.; Nakanishi, O. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 4592.
7. Finnin, M. S.; Donigian, J. R.; Cohen, A.; Richon, V. M.;
Rifkind, R. A.; Marks, P. A.; Breslow, R.; Pavletich, N. P.
Nature 1999, 401, 188.
8. Curtin, M. L.; Garland, R. B.; Frey, R. R.; Heyman, H. R.;
Michaelides, M. R.; Li, J.; Pease, L. J.; Glaser, K. B.; Mar-
cotte, P. A.; Davidsen, S. K. Abstracts of Papers, 224th
National Meeting of the American Chemical Society, Boston,
MA, Aug. 18–22, 2002; MEDI 124.
9. (a) Frey, R. R.; Wada, C. K.; Garland, R. B.; Curtin, M. L.;
Michaelides, M. R.; Li, J.; Pease, L. J.; Glaser, K. B.; Mar-
cotte, P. A.; Bouska, J. J.; Murphy, S. S.; Davidsen, S. K.
Bioorg. Med. Chem. Lett. 2002, 12, 3443. (b) Wada, C. K.;
Frey, R. R.; Ji, Z.; Curtin, M. L.; Garland, R. G.; Li, J.;
Pease, L. J.; Guo, J.; Glaser, K. B.; Marcotte, P. A.; Richard-
son, P. L.; Murphy, S. S.; Bouska, J. B.; Tapang, P.; Magoc,
T. J.; Albert, D. H.; Davidsen, S. K.; Michaelides, M. R.
Bioorg. Med. Chem. Lett. 2003, 12, 3331.
10. (a) Tsutsumi, S.; Okonogi, T.; Shibahara, S.; Ohuchi, S.;
Hatsushiba, E.; Patchett, A. A.; Christensen, B. G. J. Med.
Chem. 1994, 37, 3492. (b) Edwards, P. E.; Wolanin, D. J.;
Andisik, D. W.; Davis, M. W. J. Med. Chem. 1995, 38, 76. (c)
Dragovich, P. S.; Zhou, R.; Webber, S. E.; Prins, T. J.; Kwok,
A. K.; Okano, K.; Fuhrman, S. A.; Zalman, L. S.; Mal-
donado, F. C.; Brown, M. A.; Meador, J. W.; Patick, A. K.;
Ford, C. E.; Brothers, M. A.; Binford, S. L.; Matthews, D. A.;
Ferre, R. A.; Worland, S. T. Bioorg. Med. Chem. 2000, 10, 45.
11. Boger, D. L.; Sato, H.; Lerner, A. E.; Hendrick, M. P.;
Fecik, R. A.; Miyauchi, H.; Wilkie, G. D.; Austin, B. J.;
Patricelli, M. P.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 5044.
Figure 3. Metabolic fate of 18 in the presence of MDA435 cells with
concomitant formation of 36.
In summary, a series of novel, potent a-keto oxazoles
were identified as HDAC inhibitors in this study.
Efforts to mitigate the metabolic liability of these elec-
trophilic carbonyl containing compounds will be the
focus of future communications.
References and Notes
1. Review: Johnstone, R. W. Nat. Rev. Drug Discov. 2002, 1,
287.
2. (a) Massa, S.; Mai, A.; Sbardella, G.; Esposito, M.; Ragno,
R.; Loidl, P.; Brosh, G. J. Med. Chem. 2001, 13, 2069. (b)
Jung, M.; Brosch, G.; Kolle, D.; Scherf, H.; Gerhauser, C.;
Loidl, P. J. Med. Chem. 1999, 42, 4669. (c) 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.; Delorme, D. Bioorg. Med. Chem. Lett.
2001, 11, 2847.
12. Chan, A. W. E.; Golec, J. M. C. Bioorg. Med. Chem. 1996,
10, 1673.
13. The given IC50 value was determined using a mixture of
HDAC1 and HDAC2 prepared fromnuclear extraction of
K562 erythroleukemia cells. This protocol is described in
Yoshida, M.; Kijima, M.; Akita, M.; Beppu, T. J. Biol. Chem.
1990, 265, 17174.
14. See ref 15 in Dai, Y.; Guo, Y.; Michaelides, M. R.; Guo, J.;
Pease, L. J.; Li, J.; Marcotte, P. A.; Glaser, K. B.; Davidsen,
S. K. Bioorg. Med. Chem. Lett. 2003, 13, 1897.
15. All microwave reactions were performed in a CEM Dis-
cover1 unit equipped with a pressure load cell and an IR
sensor to measure the temperature.
16. Xu, W.; Mohan, R.; Morrissey, M. M. Tetrahedron Lett.
1997, 38, 7337.
3. (a) Van, L. C.; Emiliani, S.; Verdin, E. Gene Expr. 1996, 5,
245. (b) Kelly, W. K.; O’Connor, O. A.; Marks, P. A. Expert
Opin. Investig. Drugs 2002, 11, 1695.
4. (a) IC50 value determined using immunoprecipitated
HDAC1. Tsuji, N.; Kobayashi, M.; Nagashima, K.; Waki-
saka, Y.; Koizumi, K. J. Antibiotics 1976, 29, 1. (b) Yoshida,
M.; Nomura, S.; Beppu, T. Cancer Res. 1987, 47, 3688.
5. IC50 value determined using immunoprecipitated HDAC1.
Richon, V. M.; Emiliani, S.; Verdin, E.; Webb, Y.; Breslow,
R.; Rifkind, R. A.; Marks, P. A. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 3003.
17. See reference 20 in Curtin, M. L.; Garland, R. B.; Hey-
man, H. R.; Frey, R. R.; Michaelides, M. R.; Li, J.; Pease,
L. J.; Glaser, K. B.; Marcotte, P. A.; Davidsen, S. K. Bioorg.
Med. Chem. Lett. 2002, 12, 2919.
6. Non-hydroxamate inhibitors of HDAC such as MS 27-275
have been reported. (a) MS 27-275 IC50 2.0 mM determined
using a mixture of HDAC1 and HDAC2 prepared from