α-Mercaptoketone based histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.10.058

In an effort to discover novel non-hydroxamic acid histone deacetylase (HDAC) inhibitors, a novel α-mercaptoketone was identified in a high-throughput screen. Lead optimization of the screening hit, led to a number of potent HDAC inhibitors. In particular, α-mercaptoketone 19y (KD5150) exhibited nanomolar in vitro activity and inhibition of tumor growth in vivo.

Full text of DOI:10.1016/j.bmcl.2008.10.058

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6482–6485
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
a-Mercaptoketone based histone deacetylase inhibitors
Paul L. Wash a, Timothy Z. Hoffman a, Brandon M. Wiley a, Céline Bonnefous a, Nicholas D. Smith a,
Michael S. Sertic a, Charles M. Lawrence a, Kent T. Symons b, Phan-Manh Nguyen b, Kevin D. Lustig b,
Xin Guo c, Tami Annable c, Stewart A. Noble a, Jeffrey H. Hager c, Christian A. Hassig b, James W. Malecha a,
*
a Department of Chemistry, Kalypsys, Inc., 10420 Wateridge Circle, San Diego, CA 92121, USA
b Department of Biology, Kalypsys, Inc., 10420 Wateridge Circle, San Diego, CA 92121, USA
c Department of Pharmacology, Kalypsys, Inc., 10420 Wateridge Circle, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to discover novel non-hydroxamic acid histone deacetylase (HDAC) inhibitors, a novel
a
-mer-
Received 29 August 2008
Revised 9 October 2008
Accepted 13 October 2008
Available online 17 October 2008
captoketone was identified in a high-throughput screen. Lead optimization of the screening hit, led to a
number of potent HDAC inhibitors. In particular, -mercaptoketone 19y (KD5150) exhibited nanomolar
in vitro activity and inhibition of tumor growth in vivo.
a
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Histone deacetylase inhibitor
Mercaptoketone
Prodrug
Anti-tumor agent
SAR
Over the past several years, a number of histone deacetylase
(HDAC) inhibitors have entered clinical trials as potential cancer
therapeutics (Fig. 1).1 Recently, Vorinostat 1 was approved for
treatment of cutaneous T-cell lymphoma.2 A common structural
feature of many of these compounds is the presence of a hydroxa-
mic acid which serves to chelate zinc in the active site of the en-
zyme. Hydroxamates, however, have been shown to exhibit
undesirable pharmaceutical properties including poor pharmaco-
kinetics, low solubility, and the potential for chronic toxicities.3,4
We sought to discover a series of HDAC inhibitors devoid of the is-
sues often present in the hydroxamic acid class of compounds by
introducing a novel zinc chelator.
Replacement of the hydroxamate zinc chelating group with
an
a
-mercaptoketone is not without precedent. While our
A high-throughput screen of a 600,000 small molecule library
was carried out in a fluorescence based biochemical assay that uti-
lized the HDAC activity of a partially purified HeLa cell nuclear ex-
tract.5 Disulfide 5 was identified in the screen. Analogous to the
HDAC inhibitor FK228 4 which undergoes a disulfide bond cleav-
age in cells to give the active reduced form6, disulfide 5 is con-
verted to the active
a-mercaptoketone 6 under the reducing
Figure 1. A sample of known HDAC inhibitors.
conditions of our assay (Fig. 2). The proposed binding mode is re-
lated to that of the hydroxamic acids with the
a-mercaptoketone
functioning as a monodentate or bidentate zinc chelating group.7
* Corresponding author. Tel.: +1 858 754 3381; fax: +1 858 754 3301.
E-mail address: jmalecha@kalypsys.com (J.W. Malecha).
Figure 2. Conversion of screening hit to a-mercaptoketone.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.058

P. L. Wash et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6482–6485
6483
Table 1
by, (c) a linker to, (d) a terminal aromatic tail. Our screening hit
contained a novel -mercaptoketone zinc chelating group with a
Cell based HDAC inhibition and cytotoxicity of selected compounds.
a
phenyl spacer, but was devoid of the linker and terminal aromatic
group which have been shown to improve potency in known HDAC
inhibitors. Thus, the lead optimization program focused on the lin-
ker and terminal aromatic units.
Compound
X
Cell H3a EC50
(lM)
HCT-116 EC50 (lM)
The propensity of thiols to oxidatively dimerize in air or solu-
tion necessitated the use of a prodrug approach. While the disul-
7
8
9
10
11a
12
1
–CH2NH–
–NHC(O)–
–NHC(O)NH–
–NHSO2–
–SO2NH–
–SO2N(CH3)–
SAHA
1.5
67.1
11.2
1.0
0.20
4.3
NT
NT
NT
16.1
8.8
NT
1.6
fide linkage serves as the prodrug for the
a-mercaptoketone in
our screening hit, we chose to incorporate a thioacetate prodrug
in our lead optimization program. The thioacetate prodrug hydro-
lyzes relatively slowly under neutral and acidic conditions, but
undergoes facile enzymatic hydrolysis by esterases, such as those
0.55
NT, not tested.
found in serum, to unmask the a-mercaptoketone.
a
HeLa cell-based histone H3 hyperacetylation assay.
The first series of compounds was prepared with a trifluoro-
methoxyphenyl tail and amine, amide and sulfonamide linkers
(Table 1). Compounds were tested for their pan-HDAC inhibitor
activity by their ability to induce histone H3 hyperacetylation in
a HeLa cell line. Cytotoxicity for the more potent inhibitors was
evaluated against the human colorectal cancer line HCT-116.5
The sulfonamide analogs showed the best HDAC inhibition, but
were not very potent in the cytotoxicity assay. The orientation of
the sulfonamide linkage was very important for activity with sul-
fonamide 11a five times more potent than the related compound
10.
The preferred sulfonamide linker was incorporated into the
next series of molecules targeted at modifications of the tail region.
The general synthetic procedure used to prepare these analogs is
illustrated in Scheme 1. Aniline 16 is prepared in three steps from
6-chloronicotinoyl chloride 13 (dimethylmalonate addition, decar-
boxylation, aminolysis). Aniline 16 or 4-aminoacetophenone 17 is
coupled with sulfonyl chloride 18a-y in pyridine followed by
installation of the thioacetate prodrug (bromination, thioacetate
Table 2
Cell based HDAC inhibition and cytotoxicity of selected compounds.
Compound
R
Cell H3a EC50
(
lM)
HCT-116 EC50 (lM)
11b
11c
l1d
11e
11f
11g
11h
11i
11a
11j
11k
11l
H
2-Cl
3-Cl
4-Cl
0.33
0.85
0.15
0.28
0.42
0.46
0.95
0.79
0.20
2.1
15.4
NT
NT
30.8
NT
NT
3,4-di-Cl
3-F
4-F
3-OCF3
4-OCF3
4-CF3
4-CH3
3-OCH3
4-OCH3
3,4-di-OCH3
NT
NT
8.8
NT
6.5
2.6
0.5
3.2
0.10
0.17
0.05
0.28
addition) to give the corresponding phenyl 11 or pyridyl 19 a-mer-
captoketones (masked as thioacetate prodrug).
11m
11n
Using this chemistry, a variety of substituted phenyl com-
pounds was synthesized to investigate structural modifications of
the terminal aromatic group (Table 2). Ortho substitution de-
creased HDAC inhibitory activity whereas meta or para substitu-
tion was preferred depending on the substituent. Halo
derivatives favored meta substitution, while other analogs favored
para substitution. The 4-trifluoromethyl analog 11j was a poor
HDAC inhibitor, but the 4-methyl 11k and 4-methoxy 11m analogs
were more potent than the unsubstituted compound 11b. In addi-
tion, 11m displayed significant activity in the HCT-116 assay.
Encouraged by these results, a series of compounds with a bicyclic
terminal aromatic group was examined (Table 3). In general, these
compounds exhibited even more potent HDAC inhibition than the
simple phenyl substituted derivatives. Indole derivatives 11p and
NT, not tested.
a
HeLa cell-based histone H3 hyperacetylation assay.
work was in progress, an
a-mercaptoketone SAHA analog was
shown to be a potent HDAC inhibitor.8 Mercaptoacetamide9,10
and mercaptoethylamide11 SAHA derivatives have also been
shown to inhibit HDAC activity. However, our screening hit 5
represented a novel lead for the identification of new HDAC
inhibitors; an
structure.
a-mercaptoketone with a non-SAHA like core
Most HDAC inhibitors consist of four structural units1; (a) a zinc
chelating group attached to, (b) a hydrophobic spacer connected
Scheme 1. General synthesis of
a-mercaptoketones. Reagents and conditions: (a) dimethylmalonate, MgCl2, Et3N, toluene, 5 h, 92%; (b) H2O, DMSO, 130 °C, 2 h, 32%; (c) NH3,
120 °C, 10 h, 89%; (d) pyridine, 60 °C, 1 h, 55–90%; (e) i—pyrrolidone hydrotribromide, HBr/AcOH, DMF, 50 °C, 2 h; ii—potassium thioacetate, DMF, rt, 1 h, 45–70%.

6484
P. L. Wash et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6482–6485
Table 3
11r were potent HDAC inhibitors and cytotoxic against the HCT-
116 cell line. Benzimidazole 11u and benzoxazine 11v also induced
histone H3 hyperacetylation, but were less potent in the cytotoxic-
ity assay. Napthyl 11o and 1,4-dioxine 11y were the most potent
HDAC inhibitors. However, 11y demonstrated the best combina-
tion of HDAC inhibition and in vitro cytotoxicity.
Cell based HDAC inhibition and cytotoxicity of selected compounds.
Compound
RING
Cell H3a EC50
0.33
(l
M)
HCT-116 EC50
(lM)
Although the bicyclic compounds were potent HDAC com-
pounds and displayed potent cell killing activity, the initial
in vivo results with these compounds were less encouraging. In
an effort to improve the oral activity, compounds with increased
aqueous solubility were investigated.12 Thus, a series of com-
pounds was prepared which incorporated the optimized sulfon-
amide linker and bicyclic tails, but replaced the phenyl spacer
with a pyridyl group (Table 4). The in vitro data for the pyridyl ser-
ies tracked with the results from the phenyl series with the 1,4-
dioxine 19y and napthyl 19o as the most potent compounds.
Given the in vitro activity of the phenyl and pyridyl dioxins 11y
and 19y, the compounds were tested for their anti-tumor activity
in vivo. To this end, HCT-116 tumor xenograft bearing nude mice
were dosed orally with pyridyl analog 19y and phenyl analog 11y
at 50 mg/kg, once-a-day for 14 days (Fig. 3). Compound 19y exhib-
ited superior anti-tumor efficacy with a T/C value13 of 40% com-
pared to 87% for 11y. At the end of the study, tumors were
excised at 2 and 4 h after the last dose and histone H3 hyperacety-
lation was monitored by Western blotting and LiCor imaging tech-
nology. As depicted in Figure 4, the degree and duration of
11b
15.4
11o
11p
0.029
0.059
2.6
1.5
11q
11r
11s
0.16
1.1
0.6
3.3
0.044
0.047
11t
0.091
4.6
11u
11v
0.079
0.10
1.8
2.6
11w
11x
0.082
0.088
3.2
1.4
11y
0.026
0.4
a
HeLa cell-based histone H3 hyperacetylation assay.
Table 4
Cell based HDAC inhibition and cytotoxicity of selected compounds.
Figure 3. Human colon cancer (HCT-116) xenograft.
Compound
RING
Cell H3a EC50
0.15
(
lM)
HCT-116 EC50
5.3
(lM)
19b
19o
19u
0.042
0.24
4.9
4.9
19v
0.15
9.6
19w
19x
0.095
0.36
6.4
5.5
19y
0.038
1.2
Figure 4. Time course of histone H3 hyperacetylation of end-stage HCT-116
tumors.
a
HeLa cell-based histone H3 hyperacetylation assay.

P. L. Wash et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6482–6485
6485
5. Hassig, C. A.; Symons, K. T.; Guo, X.; Nguyen, P.; Annable, T.; Wash, P. L.; Payne,
J. E.; Jenkins, D. A.; Bonnefous, C.; Trotter, C.; Wang, Y.; Anzola, J. V.; Milkova, E.
L.; Hoffman, T. Z.; Dozier, S. J.; Wiley, B. M.; Saven, A.; Malecha, J. W.; Davis, R.
L.; Shiau, A. K.; Noble, S. A.; Rao, T. S.; Smith, N. D.; Hager, J. H. Mol. Cancer Ther.
2008, 7, 1054.
6. Furumai, R.; Matsuyama, A.; Kobashi, N.; Lee, K. H.; Nishiyama, M.; Nakajima,
H.; Tanaka, A.; Komatsu, Y.; Nishino, N.; Yoshida, M.; Horinouchi, S. Cancer Res.
2002, 62, 4916.
7. Vanommeslaeghe, K.; Loverix, S.; Geerlings, P.; Tourwe, D. Bioorg. Med. Chem.
2005, 13, 6070.
8. Gu, W.; Nusinzon, I.; Smith, R. D.; Horvath, C. M.; Silverman, R. B. Bioorg. Med.
Chem. 2006, 14, 3320.
9. Suzuki, T.; Matsuura, A.; Kouketsu, A.; Nakagawa, H. M.; Miyata, N. Bioorg. Med.
Chem. Lett. 2005, 15, 331.
10. Chen, B.; Petukhov, P. A.; Jung, M.; Velena, A.; Eliseeva, E.; Dritschilo, A.;
Kozikowski, A. P. Bioorg. Med. Chem. Lett. 2005, 15, 1389.
11. Anandan, S. K.; Ward, J. S.; Brokx, R. D.; Bray, M. R.; Patel, D. V.; Xiao, X. X.
Bioorg. Med. Chem. Lett. 2005, 15, 1969.
induction of hyperacetylation paralleled the anti-tumor efficacy.
Robust histone H3 acetylation was observed with 19y at both 2
and 4 h post-single dose. In contrast, 11y treatment led to transient
H3 hyperacetylation as evidenced by significant biomarker induc-
tion only at 2 h. Thus, the incorporation of the pyridyl spacer in
19y effects improved target modulation and, as a result, improved
anti-tumor activity.
A novel series of potent HDAC inhibitors incorporating an
a-
mercaptoketone has been identified. Based on superior in vivo
activity, 19y was advanced for further study and designated
KD5150. These results support the continued investigation of the
KD5150 series of
a-mercaptoketone HDAC inhibitors as potential
cancer therapeutics.5,12,14
12. Additional efforts at improving the aqueous solubility in the series are
detailed in the following paper: Smith, N. D. et al., Bioorg. Med. Chem.
Lett.
References and notes
1. Paris, G.; Porcelloni, M.; Binaschi, M.; Fattori, D. J. Med. Chem. 2008, 51, 1505.
2. Marks, P. A. Oncogene 2007, 26, 1351.
3. Vassiliou, S.; Mucha, A.; Cuniasse, P.; Georgiadis, D.; Lucet-Levannier, K.; Beau,
F.; Kannan, R.; Murphy, G.; Knaeuper, V.; Rio, M. C.; Basset, P.; Yiotakis, A.; Dive,
V. J. Med. Chem. 1999, 42, 2610.
13. T/C: (treated final volume À treated initial volume)/(control final
volume À control initial volume) Â 100.
A T/C value of 0 equals tumor
stasis.
14. Feng, R.; Ma, H.; Hassig, C. A.; Payne, J. E.; Smith, N. D.; Mapara, M. Y.; Hager, J.
H.; Lentzsch, S. Mol. Cancer Ther. 2008, 7, 1494.
4. Suzuki, T.; Miyata, N. Curr. Med. Chem. 2005, 12, 2867.