Mercaptoamide-based non-hydroxamic acid type histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.02.075

Inhibitors of histone deacetylases (HDAC) are emerging as a promising class of anti-cancer agents. A mercaptoamide functionality was designed as a bidentate zinc chelator and incorporated into the hydroxamic acid based SAHA (1) scaffold in order to identify non-hydroxamate compounds as potential inhibitors of histone deacetylases. Two sets of mercaptoamides 2 and 3 with varying spacer length were synthesized and their HDAC inhibitory activity was evaluated. Low micromolar inhibition was observed for mercaptoamides 2e, 3b, and 3d.

Full text of DOI:10.1016/j.bmcl.2005.02.075

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1969–1972
Mercaptoamide-based non-hydroxamic acid type histone
deacetylase inhibitors
Sampath-Kumar Anandan,* John S. Ward, Richard D. Brokx, Mark R. Bray,
Dinesh V. Patel and Xiao-Xi Xiao
Miikana Therapeutics Inc, 6519 Dumbarton Circle, Fremont, CA 94555, USA
Received 4 February 2005; revised 18 February 2005; accepted 24 February 2005
Abstract—Inhibitors of histone deacetylases (HDAC) are emerging as a promising class of anti-cancer agents. A mercaptoamide
functionality was designed as a bidentate zinc chelator and incorporated into the hydroxamic acid based SAHA (1) scaffold in order
to identify non-hydroxamate compounds as potential inhibitors of histone deacetylases. Two sets of mercaptoamides 2 and 3 with
varying spacer length were synthesized and their HDAC inhibitory activity was evaluated. Low micromolar inhibition was observed
for mercaptoamides 2e, 3b, and 3d.
Ó 2005 Elsevier Ltd. All rights reserved.
Inhibitors of histone deacetylases (HDACs) are emerg-
ing as a promising class of anti-cancer agents.1–5 The
Zn2+ binding
head
Spacer
Cap
primary activity of the HDACs is to catalyze the hydro-
lysis of acetyl groups from amino terminal lysine resi-
dues of the nucleosomal core histones.6 HDAC
inhibitors inhibit proliferation of tumor cells by induc-
ing terminal differentiation of tumor cells. Inhibition
of HDAC activities in cancer cells also leads to cell cycle
arrest and induction of apoptosis. Many of these inhibi-
tors have potent anti-tumor effect in vivo and some of
them are currently in phase I/II clinical trials.7,8
O
H
N
OH
N
H
O
SAHA (1)
Figure 1. Structural motifs of HDAC inhibitor SAHA.
Class I and II HDACs are metalloenzymes. The co-crys-
tal structure of the histone deacetylase-like protein
(HDLP) with SAHA9 (1, Fig. 1), the most advanced
hydroxamate compound in clinical trials, reveals an ac-
tive site comprising a tubular pocket with a zinc ion at
the bottom of the pocket. While the hydroxamate head
group of SAHA interacts with the active site zinc ion in
a bidentate fashion, the linker spans the tube-like por-
tion of the binding pocket and the aromatic cap group
makes contact with the pocket entrance. In addition,
hydrogen bonding of the hydroxamic acid with the amino
acid residues of the enzyme, imidazole groups in the his-
tidines (H131, H132), aspartates (D173, D166) along
with active site tyrosine (Y297), provides further ratio-
nale for the potent inhibitory activity of these agents.
The crystal structure of human HDAC8 complexed with
SAHA, reported recently,10,11 also sheds light on such a
catalytic mechanism of the HDACs.
Hydroxamic acid HDAC inhibitors, such as SAHA or-
LAQ824,12 do not discriminate well among the HDAC
subtypes. However, o-aminoanilide non-hydroxamate
SAHA analogs have shown subtype selectivity.13–16 By
contrast, modifications of the cap group of SAHA has
produced selectivity for tubulin acetylation versus his-
tone acetylation.15 Inhibitors that contain hydroxamic
acid as zinc binding group (ZBG) have produced poor
results in clinical trials for other diseases because of poor
pharmacokinetics that may be associated with the
hydroxamic acid functionality.17–20
Keywords: Histone deacetylase inhibitor; Non-hydroxamates;
Mercaptoamides.
*
Corresponding author. Tel.: +1 510 818 2753; fax: +1 510 818 9955;
e-mail: skumar@miikana.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.075

1970
S.-K. Anandan et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1969–1972
There are non-hydroxamate HDAC inhibitors such as
benzamide, MS-27521 and a depsipeptide, FRK22822
currently in phase I/II clinical trials. Other non-hydroxa-
mate functionalities such as trifluoromethyl ketones,23
ketoamides,24 phosphonates,25 and N-formylhydroxyl-
amine26 are also reported. However, these later com-
pounds have either reduced potency or metabolic
liability. Therefore, there is a clear need to develop
HDAC inhibitors with new non-hydroxamate ZBG to
improve the efficacy, pharmacokinetics and selectivity
of these promising anti-cancer agents.
O
O
O
O
a
R
O
n
N
O
n
H
4b-g (R = Cl, n = 1-6)
4h (R = OH, n = 7)
5
b
O
O
c
O
O
STrt
N
N
n
H
H
N
OH
n
H
7
6
d
The thiol functionality has been shown to be a good
monodentate chelator for zinc-dependent enzymes such
as angiotensin converting enzyme27 and matrix metallo-
proteinases.28 Thiol-based SAHA analogs are reported
to be potent HDAC inhibitors29 in spite of their mono-
dentate zinc binding ability in comparison to SAHAÕs
bidentate hydroxamic acid moiety. Also it has been
shown that the disulfide group in the depsipeptide,
FRK228, undergoes reduction within the cells to release
the zinc binding thiol moiety leading to potent HDAC
inhibition.22 Here we have designed compounds, struc-
turally as close as possible to SAHA, but having the
mercaptoamide replacing the hydroxamate moiety as a
bidentate ZBG and tested them for HDAC inhibitory
activity.
O
O
SH
N
N
n
H
H
2
Scheme 1. Reagents and conditions: (a) aniline, DIEA, DCM, 0–10 °C
for 10 min, rt for 30 min (83–85%) or EDCI, HOBT, DIEA, rt, 9 h
(84–87%); (b) LiOH, EtOH, H2O, rt, 3 h (90–95%); (c) S-trityl
cysteamine, EDC, HOBT, DIEA, DCM, rt, 12 h (45–50%); (d) TFA,
triisopropylsilane, DCM, 1 h (20–25%).
6a–h, which were then coupled with S-trityl protected
cysteamine in presence of EDCI and HOBT to give trityl
protected mercaptoamides 7a–h. The S-trityl protected
cysteamine was prepared from the corresponding cyste-
amine by treating with trityl alcohol and trifluroacetic
acid. Deprotection of the trityl group from 7 with trifluo-
roacetic acid in presence of triisopropylsilane resulted in
the desired mercaptoethylamides 2a–h.
We prepared two sets of compounds possessing an inter-
nal amide or reverse amide attached to the zinc binding
thiol functionality (Fig. 2). Both the mercaptoethyl-
amide in 2 and the mercaptoacetamide in 3 can poten-
tially function as a bidentate Zn2+ chelator. While, the
mercaptoacetamides are recently shown to be a ZBG
in HDAC inhibitors,30–32 we have explored the possibil-
ity of mercatoethylamide as the ZBG in SAHA based
scaffold. We have also varied the distance between the
mercaptoamide head group to the cap group to see its
effect on the HDAC enzyme inhibition. As in SAHA,
a benzamide cap was chosen in compounds 2, but a
quinolinamide cap was incorporated in compounds 3,
since it is shown that incorporation of such a cap have
produced improved HDAC potency.29
Mercaptoacetamides, 3a–d (n = 2–5) were prepared
according to Scheme 2. The amino acids 8a–d (n = 2–
5) were N-protected with the Boc group using di-t-butyl-
dicarbonate in presence of sodium hydroxide and then
coupled with 3-aminoquinoline to obtain amides 10a–d.
Amides 10a–d were then treated with hydrochloric acid
in diethylether to obtain free amines 11a–d, which were
then coupled with S-tritylmercaptoacetic acid to obtain
trityl protected mercaptoacetamides 12a–d in presence
of EDCI and HOBT. Tritylmercaptoacetic acid was pre-
pared from the corresponding mercaptoacetic acid using
trityl alcohol and borontrifluoride etherate. Deprotec-
tion of the trityl group from 12a–d afforded the desired
mercaptoacetamides 3a–d (n = 2–5).
The synthesis of mercaptoamides 2 and 3 is outlined in
Schemes 1 and 2 and the compounds prepared for this
study are listed in Table 1. Accordingly, the commer-
cially available acid chlorides 4b–g (n = 1–6) were trea-
ted with aniline in presence of diisopropylethyl amine
(DIEA) to obtain the benzamides 5b–g (n = 1–6). While
the benzamide 5a (n = 0) is available commercially, the
benzamide 5h (n = 7) was obtained by treating the carb-
oxylic acid 4h (n = 7) with aniline in presence of EDCI
and HOBT. The esters of 5a–h were then hydrolyzed
using lithium hydroxide to obtain the carboxylic acids,
The mercaptoamides, 2a–h and 3a–d were tested with an
in vitro assay using a HeLa nuclear extract.33 Inhibition
of HDAC enzyme (IC50) results are summarized in Ta-
ble 1. SAHA was used as a positive control,34 which
had an IC50 value of 0.06 lM in our assays.
The IC50 values reported in Table 1 suggest that the
replacement of hydroxamic acid with mercaptoamide
functionality results in moderately potent HDAC inhibi-
tors. The mercaptoethylamide, 2e or the mercaptoacet-
amide, 3d, whose bidentate Zn2+ binding motif (Fig. 3)
resembles that of SAHA with a similar overall spacer
length, was found to be ꢀ20-fold less potent than
SAHA. Interestingly, the mercaptoethylamide 2e is
O
H
H
H
N
N
N
SH
SH
n
N
n
H
O
O
O
N
2
3
Figure 2. Mercaptoamide based non-hydroxamate HDAC inhibitors.

S.-K. Anandan et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1969–1972
1971
O
O
a
H
N
H2N
Boc
OH
OH
n
n
9
8
b
N
N
O
c
H
O
N
H2N
Boc
N
n
N
H
n
H
10
11
d
N
O
H
N
N
e
O
HS
N
H
N
n
H
O
TrtS
N
n
3
H
O
12
Scheme 2. Reagents and conditions: (a) (Boc)2O, NaOH, t-BuOH, 12 h (82–93%); (b) 3-aminoquinoline, HOBT, EDCI, DIEA, DCM, rt, 12 h (84–
88%); (c) HCl/Et2O, 0 °C, 4 h; (d) S-tritylmercaptoacetic acid, HOBT, EDCI, DIEA, DCM, rt, 12 h (80–85%); (e) TFA, DIEA, DCM, 1 h (41–51%).
Table 1. HDAC inhibition data for mercaptoamides 2 and 3a
series of compounds 2 and 3. As shown by the data in
Table 1, the HDAC inhibition was very much dependent
Entry
Compounds
n
IC50 (lM)
on the chain length with the best activity seen for the
compound with a spacer length of n = 4 (2e), having a
similar spacer distance between the head group to cap
group as in SAHA. Either shorter (n = 1–2) or longer
chain lengths (n = 7) resulted in reduced HDAC inhibi-
tion potency, an observation similar to SAR studies in
the hydroxamate series. Similar conclusion can be
drawn for the mercaptoacetamide series 3. Here again,
it is clear that the optimal distance is n = 3–5. While
the inhibitors with chain length of n = 3–5, 2d–g, had
inhibition in the low micromolar range, it was rather
surprising to see low micromolar inhibiton for 2a with
n = 0, which could be due to the presence of the 1,2-
diketone in 2a that might provide additional bidentate
zinc chelation.
1
2a
2b
2c
2d
2e
2f
0
1
2
3
4
5
6
7
2
3
4
5
2.80
70.0
12.5
3.10
1.50
2.50
2.35
10.2
39.5
0.66
2.80
1.10
2
3
4
5
6
7
2g
2h
3a
3b
3c
3d
8
9
10
11
12
a Average value of two independent experiments.
more potent toward HDAC inhibition compared to the
literature reported mercaptoethylamine 1329 (IC50 > 100
lM) suggesting that the presence of the amide function-
ality in mercatoethylamide 2e enhances HDAC
inhibition.
In summary, replacing a hydroxamic acid with a mer-
captoamide functionality, as in 2e, 3b, and 3d resulted
in HDAC inhibition with IC50 in the low micromolar
range. The mercaptoamide analog 2e seemed to be more
potent than the structurally similar mercaptoamine ana-
log 13 suggesting that the mercaptoethylamine group
We also examined the effect of spacer length between the
cap region and the metal chelating thiol moiety in both
Zn2+
Zn2+
O
O
H
H
N
S-
N
OH
N
N
H
H
O
3d
2e
O
N
SAHA (1)
Zn2+
S-
Zn2+
S-
O
H
N
H
N
N
NH
H
O
O
13
Figure 3. Bidentate chelation of hydroxamic acid in SAHA, mercaptoethylamine 13 and mercaptoamides 2e, 3d with Zn2+ ion.

1972
S.-K. Anandan et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1969–1972
17. Pikul, S.; Ohleer, N. E.; Ciszewski, G.; Laueersweiler, M.
C.; Almstead, N. G.; De, B.; Mieling, G. E. J. Med. Chem.
2001, 44, 2499–2502.
18. Michaelides, M. R.; Dellaria, J. F.; Gong, J.; Holms, J. H.;
Davidson, S. K. Bioorg. Med. Chem. Lett. 2001, 11, 1553.
19. Peng, S. X.; Strojnowski, M. J.; Hu, J. K.; Smith, B. J.;
Eichhold, T. H.; Wehmeyer, K. R.; Pikel, S.; Almstead, N.
G. J. Chromatogr. B 1999, 724, 181.
adjacent to a carbonyl moiety could have similar biden-
tate chelating geometry with the zinc ion (Fig. 3) as in
SAHA. The reduced potency for 2e or 3d compared to
SAHA would suggest that the zinc chelation might be
weaker with either the mercaptoethylamide or the mer-
captoacetamide. Further SAR studies are in progress
to improve the HDAC potency, physiological properties
and overall pharmacological behavior of this class of
inhibitors over the hydroxamate class.
20. Sugihara, K.; Kitamura, S.; Ohta, S.; Tatsumi, K.
Xenobiotica 2000, 30, 457.
21. Suzuki, T.; Ando, T.; Tsuchiya, K.; Fukazawa, N.; Saito,
A. J. Med. Chem. 1999, 42, 3001.
References and notes
22. Furumai, R.; Matsuyama, A.; Kohashi, N.; Lee, K.-H.;
Nishiyama, M.; Nakajima, H.; Tanaka, A.; Komatsu, Y.;
Nishino, N.; Yoshida, M.; Horinouchi, S. Cancer Res.
2002, 62, 4916.
23. Frey, R. R.; Wada, C. K.; Garland, R. B.; Curtin, M. L.;
Michaelides, M. R. Bioorg. Med. Chem. Lett. 2002, 12,
3443.
24. Frey, R. R.; Wada, C. K.; Garland, R. B.; Curtin, M. L.;
Michaelides, M. R. Bioorg. Med. Chem. Lett. 2003, 13,
3331.
1. Marks, P. A.; Rifkind, R. A.; Richon, V. M.; Breslow, R.;
Miller, T.; Kelly, W. K. Nat. Rev. Cancer 2001, 1, 194.
2. Kelly, W. K.; OÕConnor, O. A.; Marks, P. A. Expert Opin.
Invest. Drugs 2002, 11, 1695.
3. Marks, P. A.; Richon, V. M.; Breslow, R.; Rifkind, R. A.
Curr. Opin. Oncol. 2001, 13, 477.
4. Marks, P. A.; Rifkind, R. A.; Richon, V. M.; Breslow, R.
Clin. Cancer Res. 2001, 7, 759.
25. Kapustin, G. V.; Fejer, G.; Gronlund, J. L.; McCafferty,
D. G.; Seto, E.; Etzkorn, F. A. Org. Lett. 2003, 5, 3053.
26. Wu, T. Y. H.; Hassig, C.; Wu, Y.; Ding, S.; Shultz, P. G.
Bioorg. Med. Chem. Lett. 2004, 14, 449.
5. Meinke, P. T.; Liberator, P. Curr. Med. Chem. 2001, 8,
211.
6. Grozinger, C. M.; Schreiber, S. L. Chem. Biol. 2002, 9, 3.
7. Jung, M. Curr. Med. Chem. 2001, 8, 1505.
27. Ondetti, M. A.; Rubin, B.; Cushman, D. W. Science 1977,
196, 2735.
28. Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A. J.
Chem. Rev. 1999, 99, 2735.
8. Curtin, M. L. Expert Opin. Ther. Pat. 2002, 12, 1375.
9. 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.
29. Suzuki, T.; Kouketsu, A.; Matsuura, A.; Kohara, A.;
Ninomiya, S.; Kohda, K.; Miyata, N. Bioorg. Med. Chem.
Lett. 2004, 13, 3331.
30. Suzuki, T.; Nagano, Y.; Kouketsu, A.; Matsuura, A.;
Maruyama, S.; Kurotaki, M.; Nakagawa, H.; Miyata, N.
J. Med. Chem. 2005, 48, 1019.
10. John, R.; Somoza, J. R.; Skene, R. J.; Katz, B. A.; Mol,
C.; Ho, J. D.; Jennings, A. J.; Luong, C.; Arvai, A.;
Buggy, J. J.; Chi, E.; Tang, J.; Sang, B.; Verner, E.;
Wynands, R.; Leahy, E. M.; Dougan, D. R.; Snell, G.;
Navre, M.; Knuth, M. W.; Swanson, R. V.; McRee, D. E.;
Tari, S. W. Structure 2004, 12, 1325.
31. Suzuki, T.; Matsuura, A.; Kouketsu, A.; Nakagawa, H.;
Miyata, N. Bioorg. Med. Chem. Lett. 2005, 15, 331.
32. Chen, B.; Petukhov, P. A.; Jung, M.; Velena, A.; Eliseeva,
E.; Dritschilo, A.; Kozikowski, A. P. Bioorg. Med. Chem.
Lett. 2005, 15, 1389.
11. Vannini, A.; Volpari, C.; Filocamo, G.; Casavola, E. C.;
Brunetti, M.; Renzoni, D.; Chakravarty, P.; Paolini, C.;
Francesco, R. D.; Gallinari, P.; Steinkuhler, C.; Marco, S.
¨
F. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15064.
12. Martin, M. S.; Mou, Y.; Perez, L. B.; Sharma, S.; Smith,
T.; Sorensen, E.; Taplin, F.; Trogani, N.; Versace, R.;
Walker, H.; Weltchek-Engler, S.; Wood, A.; Wu, A.;
Atadja, P.; Remiszewski, S. W.; Sambucetti, L. C.; Bair,
K. W.; Bontempo, J.; Cesarz, D.; Chandramouli, N.;
Chen, R.; Cheung, M.; Cornell-Kennon, S.; Dean, K.;
Diamantidis, G.; France, D.; Green, M. A.; Howell, K. L.;
Kashi, R.; Kwon, P.; Lassota, P. J. Med. Chem. 2003, 46,
4609.
13. Matsuyama, A.; Yoshimatsu, Y.; Shimazu, T.; Sumida,
Y.; Osada, H.; Komatsu, Y.; Nishino, N.; Khochbin, S.;
Horinouchi, S.; Yoshida, M. EMBO J. 2002, 21,
6820.
14. 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.
33. In vitro fluorescent histone deacetylase assay: HDAC
inhibition assay was performed using the HDAC fluores-
cent activity assay kit (Biomol Research Laboratories,
Plymouth Meeting, PA). HeLa cell nuclear extract, which
contains a number of HDAC isozymes and other nuclear
factors, was used as the source of HDAC activity. The
final substrate concentration in the assay mixture was
50 lM. The reaction was allowed to proceed for 10 min at
room temperature before stopping the reaction. Test
compounds were prepared as 20 mM stock solutions in
DMSO (Molecular Biology grade, Sigma–Aldrich Co., St.
Louis, MO) and stored at À70 °C. DMSO had no
significant effect on the activity of this assay at concen-
trations up to 5%; the final DMSO concentration in the
wells will be no more than 2%. Assays were performed in
white polystyrene 96-well half-area assay plates (Corning,
NY) and measured on a Wallac 1420 fluorescent plate
reader (Wallac Oy, Turku, Finland) with an excitation
wavelength of 355 nm, an emission wavelength of 460 nm,
and a 1 s signal averaging time.
15. Wong, J. C.; Hong, R.; Schreiber, S. L. J. Am. Chem. Soc.
2003, 125, 5586.
16. Glaser, K. B.; Li, J.; Pease, L. J.; Staver, M. J.; Marcotte,
P. A.; Guo, J.; Frey, R. R.; Garland, R. B.; Heyman, H.
R.; Wada, C. K.; Vasudevan, A.; Michaelides, M. R.;
Davidsen, S. K.; Curtin, M. L. Biochem. Biophys. Res.
Commun. 2004, 325, 683.
34. Mai, A.; Esposito, M.; Sbardella, G.; Massa, S. A. Org.
Prep. Proced. Int. 2001, 33, 391.