SelSA, selenium analogs of SAHA as potent histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.07.068

Cancer treatment and therapy has moved from conventional chemotherapeutics to more mechanism-based targeted approach. Disturbances in the balance of histone acetyltransferase (HAT) and deacetylase (HDAC) leads to a change in cell morphology, cell cycle, differentiation, and carcinogenesis. In particular, HDAC plays an important role in carcinogenesis and therefore it has been a target for cancer therapy. Structurally diverse group of HDAC inhibitors are known. The broadest class of HDAC inhibitor belongs to hydroxamic acid derivatives that have been shown to inhibit both class I and II HDACs. Suberoylanilide hydroxamic acid (SAHA) and Trichostatin A (TSA), which chelate the zinc ions, fall into this group. In particular, SAHA, second generation HDAC inhibitor, is in several cancer clinical trials including solid tumors and hematological malignancy, advanced refractory leukemia, metastatic head and neck cancers, and advanced cancers. To our knowledge, selenium-containing HDAC inhibitors are not reported in the literature. In order to find novel HDAC inhibitors, two selenium based-compounds modeled after SAHA were synthesized. We have compared two selenium-containing compounds; namely, SelSA-1 and SelSA-2 for their inhibitory HDAC activities against SAHA. Both, SelSA-1 and SelSA-2 were potent HDAC inhibitors; SelSA-2 having IC50 values of 8.9 nM whereas SAHA showed HDAC IC₅₀ values of 196 nM. These results provided novel selenium-containing potent HDAC inhibitors.

Full text of DOI:10.1016/j.bmcl.2009.07.068

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2044–2047
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
SelSA, selenium analogs of SAHA as potent histone deacetylase inhibitors
*
Dhimant Desai , Ugur Salli, Kent E. Vrana, Shantu Amin
Department of Pharmacology, H072, The Pennsylvania State Hershey College of Medicine, Hershey, PA 17033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cancer treatment and therapy has moved from conventional chemotherapeutics to more mechanism-
based targeted approach. Disturbances in the balance of histone acetyltransferase (HAT) and deacetylase
(HDAC) leads to a change in cell morphology, cell cycle, differentiation, and carcinogenesis. In particular,
HDAC plays an important role in carcinogenesis and therefore it has been a target for cancer therapy.
Structurally diverse group of HDAC inhibitors are known. The broadest class of HDAC inhibitor belongs
to hydroxamic acid derivatives that have been shown to inhibit both class I and II HDACs. Suberoylanilide
hydroxamic acid (SAHA) and Trichostatin A (TSA), which chelate the zinc ions, fall into this group. In par-
ticular, SAHA, second generation HDAC inhibitor, is in several cancer clinical trials including solid tumors
and hematological malignancy, advanced refractory leukemia, metastatic head and neck cancers, and
advanced cancers. To our knowledge, selenium-containing HDAC inhibitors are not reported in the liter-
ature. In order to find novel HDAC inhibitors, two selenium based-compounds modeled after SAHA were
synthesized. We have compared two selenium-containing compounds; namely, SelSA-1 and SelSA-2 for
their inhibitory HDAC activities against SAHA. Both, SelSA-1 and SelSA-2 were potent HDAC inhibitors;
SelSA-2 having IC50 values of 8.9 nM whereas SAHA showed HDAC IC50 values of 196 nM. These results
provided novel selenium-containing potent HDAC inhibitors.
Received 14 May 2009
Revised 9 July 2009
Accepted 14 July 2009
Available online 17 July 2009
Keywords:
Synthesis
Histone deacetylase inhibitors
Selenium analogs of SAHA
Ó 2010 Published by Elsevier Ltd.
Several etiological pathways like genetic, epigenetic, and cyto-
genetic process play a role in the transformation of normal cells
to cancer cells. Regulation of transcription factors, as well as mod-
ification of chromatin structures (also known as epigenetic regula-
tion) are critical regulatory steps in the cellular response to trauma
and inflammation.1,2 The reversible acetylation of the side chain of
specific histone lysine residues by histone deacetylase (HDACs)
and histone acetyl transferase (HATs) is one of the most widely
studied chromatin modifications.3 The HDACs can be divided into
two families, (1) the Zn+2-dependent HDAC family composed of
Class I, Class IIa/b, and Class IV and (2) Zn+2-independent NAD-
dependent class III enzymes. Class I comprises HDAC1, 2, 3, and
8 which are located in the nuclei of the cells. Class IIa/b contains
HDAC4, 5, 6, 7, 9, and 10 primarily localized to the cytoplasm.
HDAC11 has a conserved domain in the catalytic region of both
Class I and Class II enzymes and it has been grouped to Class IV.
Class III HDACs are NAD+-dependent deacetylase with non-histone
protein as substrate and have been linked to regulation of caloric
utilization of cells.4,5
shown that inhibition of HDAC elicits anticancer effects in several
tumor cells by inhibition of cell growth, and induction of terminal
differentiation in tumor cells. This has led to the development of
HDAC inhibitors for anticancer chemotherapy10 mainly directed at
Zn2+-dependent Class I and II HDACs. Structural–activity relation-
ships (SAR) and reviews of different HDAC inhibitors and analogs
have been previously published.2,11–20 Most of these HDAC inhibi-
tors were designed to have a hydrophobic cap that blocks the en-
trance to the active site, a polar site, and a hydroxamic acid type
zinc-binding active site.15
Hydroxamic acids are the broadest class of inhibitors with high
affinity for HDAC that has been shown to inhibit both Class I and
Class II HDACs. Trichostatin A (TSA) belonging to hydroximates is
one of the first natural product possess HDAC inhibitory activity
and it is widely used as reference compound.21–23 TSA blocks pro-
liferation, inhibits cell growth, decreases differentiation in ovarian
cancer cells, and suppresses growth of pancreatic adenocarcinoma
cells at naonmolar concentrations.24,25 A second generation HDAC
inhibitor, Suberoylanilide hydroxamic acid (SAHA) inhibits secre-
HDAC catalyzesdeacetylation of
e
-aminogroup in lysines located
tion of TNF-a, IL-1b, IL-6, and IFN-c in LPS-induced PBMC cells,
near the N-terminal of core histone proteins.6,7 Specific HDAC activ-
ity results in hypoacetylation that is associated with subsequent
gene silencing, whereas histone hyperacetylation is associated with
unwinding of the DNA and transcriptional activation.8,9 Studies have
inhibits there in vivo production as shown in an LPS-induced ani-
mal model, as well as prevents formation of tumors in mice and
rats.26–28 SAHA (Vorinostat) is under clinical trials in both hemato-
logical and nonhematological malignancies and is approved for
treatment of cutaneous T-cell lymphoma.29,30 Another class of
HDAC inhibitors includes a group of synthetic benzamide deriva-
tives such as MS-275 and CI-994 that are effective inhibitors of
* Corresponding author. Tel.: +1 717 531 6805; fax: +1 717 531 0244.
E-mail addresses: ddesai@psu.edu, dhimant99@hotmail.com (D. Desai).
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.07.068

D. Desai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2044–2047
2045
solid tumors in a murine model, but did not inhibit HDAC directly.31
This class of compounds inhibits both histone deacetylation and
cellular proliferation at the G1-S phase.32 MS-275 and CCI-994
are undergoing clinical trials.33,34 Another class, a cyclic peptide
natural product include Trapoxin, having epoxide group may act
by chemically modifying an active site nucleophile with the epox-
ide group and forming hydrogen bonds through the ketone.35 Trap-
oxin is supposed to trap HDACs through the reaction of the epoxide
moiety with the zinc cation or an amino acid in the binding pock-
et.36–38 FK228 (also referred as depsipeptide) is a natural product
derived from Chromobacterium violaceum, inhibit HDACs at nano-
molar concentrations, and exhibits potent antitumor activity.39
The mechanism of action of FK228 is unknown; however, accord-
ing to one hypothesis, a disulfide bond is reduced inside the cell
or organism and the mercaptobutyenyl residue then fits inside
the HDAC catalytic pocket.35 FK228 is currently undergoing evalu-
ation in clinical trials.40–42 Thus HDACs have been suggested to be
a potential targets for anticancer drug development and many
other non-malignant diseases such as rheumatoid arthritis and
osteoporosis.43 Therefore, demand for new HDAC inhibitors having
strong inhibitory action is increasing. In this communication, we
report the syntheses of two newly developed selenium-based
HDAC inhibitors (namely, SelSA-1 and SelSA-2) and evaluation of
their HDAC activity compare to SAHA and TSA, a known HDAC
inhibitors (Figs. 1–3).
Figure 2. %Inhibition of HDAC in HeLa nuclear extract.
Several structurally diverse HDAC inhibitors have been reported
and many of them belong to the family of hydroxamic acid deriv-
atives.44 Metabolic instability and pharmacokinetic problems such
as glucuronide and sulfate conjugates that could result in short half
life of the drug in biological systems. Therefore, several new non-
hydroxamic HDAC inhibitors have been reported in the literature.9
However, they have a reduced potency compared to hydroxamate
inhibitors. A cyclic peptide HDAC inhibitor FK228 is a potent HDAC
inhibitor having a disulfide bond in the molecule that is reduced in
the cellular environment releasing the free thiol analog as the ac-
tive species.45 Therefore, in a similar manner we hypothesize that
in the cellular environment, the selenium dimer (SelSA-1) and sel-
enocyanide (SelSA-2) will be reduced and free SeH will be release
as the active species that will bind to the acetate group and cause
the potent HDAC inhibitory activities. Based on our hypothesis, we
have synthesized two selenium compounds. Synthesis of SelSA-1
was accomplished as illustrated in Scheme 1.
Figure 3. IC50 value of HDAC inhibitors.
H
O
N
a
NH2
Br
The amino group of aniline was acetylated with the appropriate
acid chloride to give the amide 1 in quantitative yield.46,47 Amide 1
was treated with selenium powder under basic condition in a
biphase system using phase transfer catalyst to give the desired
dimer SelSA-1 in 60% yield.48
Br
Cl
+
O
O
1
H
H
N
N
Se Se
b
O
SelSA-1 (2)
Synthesis of SelSA-2 was accomplished by reacting amide 1
with KSeCN in CH3CN as shown in Scheme 2.49 Acetonitrile was
Scheme 1. Synthesis of SelSA-1. Reagents and conditions: (a) TEA, rt, 18 h, 84%; (b)
KOH, adogen, Se powder, NH2NH2, rt, 60%.
O
H
H
N
O
H
N
OH
N
H
N
N
H
a
SeCN
Br
NH
OH
Me Me
Me
O
NH
Me
O
3
O
1
Trichostatin A (TSA)
SAHA
Scheme 2. Synthesis of SelSA-2. Reagents and conditions: (a) KSeCN, CH3CN, rt,
18 h, 64%.
H
N
H
N
Se - Se
O
O
SelSA-1
the solvent of choice for the reaction to avoid side products as re-
ported earlier with other solvent.50
HeLa cell nuclear extract was used as the source of the HDAC
activity with HDAC1 and HDAC2 being the major contributors.51,52
We found that SelSA-1 and SelSA-2 inhibited HDAC activity
approximately 81% and 95% at 50 nM, respectively. The inhibitory
H
N
SeCN
O
SelSA-2
Figure 1. Structures of HDAC inhibitors.

2046
D. Desai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2044–2047
30. Marks, P. A. Oncogene 2007, 26, 1351.
activity of SelSA-2 was statistically significant higher than TSA
(90%) at the same concentrations (50 nM).53
31. Belien, A.; De Schepper, S.; Floren, W.; Janssens, B.; Marien, A.; King, P.; Van
Dun, J.; Andries, L.; Voeten, J.; Bijnens, L.; Janicot, M.; Arts, J. Mol. Cancer Ther.
2006, 5, 2317.
32. Jaboin, J.; Wild, J.; Hamidi, H.; Khanna, C.; Kim, C. J.; Robey, R.; Bates, S. E.;
Thiele, C. J. Cancer Res. 2002, 62, 6108.
33. 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.
34. Prakash, S.; Foster, B. J.; Meyer, M.; Wozniak, A.; Heilbrun, L. K.; Flaherty, L.;
Zalupski, M.; Radulovic, L.; Valdivieso, M.; LoRusso, P. M. Invest. New Drugs
2001, 19, 1.
35. Acharya, M. R.; Sparreboom, A.; Venitz, J.; Figg, W. D. Mol. Pharmacol. 2005, 68,
917.
36. Kijima, M.; Yoshida, M.; Sugita, K.; Horinouchi, S.; Beppu, T. J. Biol. Chem. 1993,
268, 22429.
37. Kosugi, H.; Towatari, M.; Hatano, S.; Kitamura, K.; Kiyoi, H.; Kinoshita, T.;
Tanimoto, M.; Murate, T.; Kawashima, K.; Saito, H.; Naoe, T. Leukemia 1999, 13,
1316.
38. Komatsu, Y.; Tomizaki, K. Y.; Tsukamoto, M.; Kato, T.; Nishino, N.; Sato, S.;
Yamori, T.; Tsuruo, T.; Furumai, R.; Yoshida, M.; Horinouchi, S.; Hayashi, H.
Cancer Res. 2001, 61, 4459.
Both SelSA-2 and TSA showed higher inhibitory activity than
SAHA (77%) at 50 nM which was not different than SelSA-1 at
50 nM. Based on these findings, we assessed the IC50 of SelSA-2,
TSA and SAHA. The IC50 concentrations of SelSA-2, TSA and SAHA
were 8.9, 28.9 and 196 nM, respectively.
In summary, we have developed novel selenium based HDAC
inhibitors and evaluated their inhibitory effect on HDACs. Both
selenium compounds are superior in their inhibitory effect (more
than 20-fold) on HDAC than the known inhibitor, SAHA. Indeed,
SAHA is currently in clinical use for lymphoma and under active
evaluation for other indications.5 However, these selenium-based
small molecules may play an important role in the fight against
cancer. Currently, we are pursuing the structural–activity relation-
ship (SAR) studies with analogs of SelSA as HDAC inhibitors.
39. Piekarz, R.; Bates, S. Curr. Pharm. Des. 2004, 10, 2289.
40. Kwon, H. J.; Kim, M. S.; Kim, M. J.; Nakajima, H.; Kim, K. W. Int. J. Cancer 2002,
97, 290.
Acknowledgments
41. Byrd, J. C.; Marcucci, G.; Parthun, M. R.; Xiao, J. J.; Klisovic, R. B.; Moran, M.; Lin,
T. S.; Liu, S.; Sklenar, A. R.; Davis, M. E.; Lucas, D. M.; Fischer, B.; Shank, R.;
Tejaswi, S. L.; Binkley, P.; Wright, J.; Chan, K. K.; Grever, M. R. Blood 2005, 105,
959.
42. Marshall, J. L.; Rizvi, N.; Kauh, J.; Dahut, W.; Figuera, M.; Kang, M. H.; Figg, W.
D.; Wainer, I.; Chaissang, C.; Li, M. Z.; Hawkins, M. J. J. Exp. Ther. Oncol. 2002, 2,
325.
The authors would like to thank Dr. Jyh-Ming Lin from the Penn
State Hershey Cancer Institute Instrumentation Facility for NMR
spectra and Jenny Dai for performing the MS analysis. This study
was supported by NCI contract N02-CB-56603, and funds from
Penn State Hershey Cancer Institute.
43. Yi, T.; Baek, J. H.; Kim, H. J.; Choi, M. H.; Seo, S. B.; Ryoo, H. M.; Kim, G. S.; Woo,
K. M. Exp. Mol. Med. 2007, 39, 213.
References and notes
44. Vigushin, D. M.; Coombes, R. C. Anticancer Drugs 2002, 13, 1.
45. 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.
46. General synthesis information: Melting points were recorded on a Fisher–
Johnson melting point apparatus and are uncorrected. Unless stated otherwise,
1. Alam, H. B.; Rhee, P. Surg. Clin. North. Am. 2007, 87, 55.
2. Bouchain, G.; Leit, S.; Frechette, S.; Khalil, E. A.; Lavoie, R.; Moradei, O.; Woo, S.
H.; Fournel, M.; Yan, P. T.; Kalita, A.; Trachy-Bourget, M. C.; Beaulieu, C.; Li, Z.;
Robert, M. F.; MacLeod, A. R.; Besterman, J. M.; Delorme, D. J. Med. Chem. 2003,
46, 820.
3. Grunstein, M. Nature 1997, 389, 349.
4. Bolden, J. E.; Peart, M. J.; Johnstone, R. W. Nat. Rev. Drug Disc. 2006, 5, 769.
5. Glaser, K. B. Biochem. Pharmacol. 2007, 74, 659.
6. Archer, S. Y.; Hodin, R. A. Curr. Opin. Genet. Dev. 1999, 9, 171.
7. Kouzarides, T. Curr. Opin. Genet. Dev. 1999, 9, 40.
8. Hassig, C. A.; Schreiber, S. L. Curr. Opin. Chem. Biol. 1997, 1, 300.
9. Suzuki, T.; Kouketsu, A.; Matsuura, A.; Kohara, A.; Ninomiya, S.; Kohda, K.;
Miyata, N. Bioorg. Med. Chem. Lett. 2004, 14, 3313.
proton NMR spectra were recorded in CDCl3 using
instrument. The chemical shifts are reported in ppm downfield from TMS.
MS were run on 4000 trap hybrid triple quadrupole/linear ion trap
a Bruker 500 MHz
Q
instrument (Applied Biosystems/MDS Sciex) at the proteomic facility of the
Penn State Hershey Cancer Institute, College of Medicine, Hershey, PA. High-
resolution MS were determined at the Instrument Center, University of Buffalo,
Buffalo, NY. Thin-layer chromatography (TLC) was performed on aluminum-
supported, pre-coated silica gel plates (EM Industries, Gibbstown, NJ). All
starting materials and reagents were obtained from Aldrich Chemical Co.
(Milwaukee, WI) and used without further purification.
10. Johnstone, R. W. Nat. Rev. Drug Disc. 2002, 1, 287.
47. Preparation of 7-bromoheptanoic acid phenylamide (1): A flame-dried round
bottom flask was charged with aniline (1 g, 10.7 mmol) in dry methylene
chloride (20 mL) and triethylamine (2.95 mL, 21.48 mmol). The mixture was
11. Minucci, S.; Pelicci, P. G. Nat. Rev. Cancer 2006, 6, 38.
12. Catley, L.; Weisberg, E.; Tai, Y. T.; Atadja, P.; Remiszewski, S.; Hideshima, T.;
Mitsiades, N.; Shringarpure, R.; LeBlanc, R.; Chauhan, D.; Munshi, N. C.;
Schlossman, R.; Richardson, P.; Griffin, J.; Anderson, K. C. Blood 2003, 102, 2615.
13. Chen, H.; Alam, H. B.; Querol, R. I.; Rhee, P.; Li, Y.; Koustova, E. J. Trauma 2006,
60, 701.
14. Curtin, M. L.; Garland, R. B.; Heyman, 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.
15. Drummond, D. C.; Noble, C. O.; Kirpotin, D. B.; Guo, Z.; Scott, G. K.; Benz, C. C.
Annu. Rev. Pharmacol. Toxicol. 2005, 45, 495.
16. Kouraklis, G.; Theocharis, S. Curr. Med. Chem. Anticancer Agents 2002, 2, 477.
17. Pandolfi, P. P. Cancer Chemother. Pharmacol. 2001, 1, S17.
18. Plumb, J. A.; Steele, N.; Finn, P. W.; Brown, R. Biochem. Soc. Trans. 2004, 32,
1095.
19. Remiszewski, S. W. Curr. Opin. Drug Discovery Dev. 2002, 5, 487.
20. Remiszewski, S. W.; Sambucetti, L. C.; Atadja, P.; Bair, K. W.; Cornell, W. D.;
Green, M. A.; Howell, K. L.; Jung, M.; Kwon, P.; Trogani, N.; Walker, H. J. Med.
Chem. 2002, 45, 753.
21. Yoshida, M.; Horinouchi, S.; Beppu, T. BioEssays 1995, 17, 423.
22. Yoshida, M.; Kijima, M.; Akita, M.; Beppu, T. J. Biol. Chem. 1990, 265, 17174.
23. Yoshida, M.; Furumai, R.; Nishiyama, M.; Komatsu, Y.; Nishino, N.; Horinouchi,
S. Cancer Chemother. Pharmacol. 2001, 1, S20.
24. Hoshikawa, Y.; Kwon, H. J.; Yoshida, M.; Horinouchi, S.; Beppu, T. Exp. Cell Res.
1994, 214, 189.
25. Donadelli, M.; Costanzo, C.; Faggioli, L.; Scupoli, M. T.; Moore, P. S.; Bassi, C.;
Scarpa, A.; Palmieri, M. Mol. Carcinog. 2003, 38, 59.
cooled on ice for 10 min. Through
a dropping funnel, a solution of 6-
bromohexanoylchloride (2.3 g, 10.74 mmol) in methylene chloride (10 mL)
was added over a period of 10 min. The mixture was allowed to warm up to
room temperature and stirring was continued for an additional 18 h. The
mixture was poured into water and the organic layer was separated, dried over
MgSO4, filtered, and evaporated to give crude product. Crude product was
purified over a silica gel column using methylene chloride as an eluant to give
bromo amide 1 (2.43 g, 84%).[9] 1H NMR d 1.35 (q, 2H, J = 6.7 Hz), 1.57 (q, 2H,
J = 7.0 Hz), 1.80 (q, 2H, J = 7.5 Hz), 1.95 (q, 2H, J = 7.0 Hz), 2.41 (t, 2H, J = 7.5 Hz),
3.46 (t, 2H, CH2–Br, J = 7.0 Hz), 7.11 (br s, 1H, NH), 7.14 (t, 1H, aromatic,
J = 7.5 Hz), 7.35 (t, 2H, aromatic, J = 7.5 Hz), 7.54 (d, 2H, aromatic, J = 8.0 Hz).
48. Synthesis of bis(5-phenylcarbamoylpentyl) diselenide (SelSA-1) (2): To a stirring
mixture of bromo compound 1 (50 mg, 0.19 mmol) in methylene chloride
(5 mL), selenium powder (25 mg, 0.32 mmol), and water (1 mL) was added.
The mixture was stirred for 5 min and a drop of adogen and 40% KOH solution
were added (1 mL). The mixture was stirred for an additional 5 min and
hydrazine monohydrate (0.5 mL, 10 mmol) was added and the mixture was
stirred overnight at room temperature. The initial black color of the solution
(due to selenium powder) turned yellow after overnight stirring. The organic
layer was separated, dried over MgSO4, filtered, and evaporated to give a crude
product. Column chromatography over silica gel using methylene chloride:
EtOAc (98:2) as eluant gave SelSA-1 (30 mg, 60%); mp 128–130 °C; 1H NMR
(DMSO-d6) d 1.39 (p, 4H, CH2, J = 7.0 Hz), 1.61 (p, 4H, CH2, J = 7.5 Hz), 1.71 (p,
4H, CH2, J = 7.5 Hz), 2.30 (t, 4H, CH2, J = 7.5 Hz), 2.93 (t, 4H, CH2, J = 7.5 Hz),
7.02 (t, 2H, aromatic, J = 7.5 Hz), 7.28 (t, 4H, aromatic, J = 8.0 Hz), 7.59 (d, 4H,
aromatic, J = 8.0 Hz), 9.85 (s, 2H, NH); MS, 563 (M++Na, 100), 540 (M+, 10), 301
(40), 283 (45), 261 (50), 217 (45); HRMS 541.0867 (calculated for C24H32
N2O2Se2, 541.0867).
26. 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.
49. Synthesis of 5-phenylcarbamoylpentyl selenocyanide (SelSA-2) (3): The starting
material bromo amide 1 (1 g, 3.70 mmol) was dissolved in dry acetonitrile
(15 mL) and the mixture was charged with KSeCN (0.64 g, 4.44 mmol). The
reaction mixture was stirred for 18 h and poured into water. The aqueous layer
was extracted with methylene chloride (3 Â 25 mL). Combined organic layers
were dried over MgSO4, filtered, and evaporated to yield a crude product that
27. Cohen, L. A.; Amin, S.; Marks, P. A.; Rifkind, R. A.; Desai, D.; Richon, V. M.
Anticancer Res. 1999, 19, 4999.
28. Desai, D.; Das, A.; Cohen, L.; El-Bayoumy, K.; Amin, S. Anticancer Res. 2003, 23,
499.
29. Zhang, C.; Richon, V.; Ni, X.; Talpur, R.; Duvic, M. J. Invest. Dermatol. 2005, 125,
1045.

D. Desai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2044–2047
reaction was initiated by adding Color de Lys Substrate (25
2047
was purified by column chromatography using methylene chloride:EtOAc
(99:1) as an eluent to give SelSA-2 (740 mg, 64%), mp 87–88 °C, 1H NMR d 1.60
(q, 2H, J = 7.0 Hz), 1.83 (q, 2H, J = 8.0 Hz), 2.00 (q, 2H, J = 7.5 Hz), 2.42 (t, 2H,
CO–CH2, J = 7.5 Hz), 3.10 (t, 2H, CH2–Se, J = 7.0 Hz), 7.1 (t, 1H, aromatic,
J = 7.5 Hz), 7.17 (br s, 1H, NH), 7.35 (t, 2H, aromatic, J = 7.5H), 7.54 (d, 2H,
aromatic, J = 8.0 Hz); MS m/e 319 (M++Na, 100), 297 (M+, 10), 261 (15), 245 (5),
217 (10), HRMS 297.0489 (calculated for C13H16N2OSe, 297.0501).
50. Jacob, L. A.; Matos, B.; Mostafa, C.; Rodriguez, J.; Tillotson, J. K. Molecules 2004,
9, 622.
l
L) into each well
and samples were incubated at rt for 15 min. Subsequently, the reaction was
stopped by adding 50 L of Color de Lys Developer for additional 30 min at
l
37 °C. Colorimetric changes were measured at 405 nM using a plate reader.
HDAC activity was expressed as percent inhibition based on the relative OD
values of samples. Tests were conducted in triplicate sets. Negative and
positive controls consisted of no HeLa nuclear extract or no inhibitor,
respectively. IC50 of the inhibitor compounds was determined by performing
HDAC assay with increasing concentrations (1, 3.3, 10, 33, 100 and 333 nM) of
the inhibitor compounds (TSA, SAHA and SelSA-2) under the same conditions.
52. Huang, Y.; Tan, M.; Gosink, M.; Wang, K. K.; Sun, Y. Cancer Res. 2002, 62, 2913.
53. Statistical analysis: Data were combined from two separate HDAC assay
51. HDAC assay: HDAC Assay was performed using the colorimetric HDAC
Colorimetric Assay/Drug Discovery Kit (Biomol; Plymouth Meeting, PA)
according to manufacturer’s instructions as reported by Huang et al.52
Briefly, HDAC inhibitors/candidates (10
SelSA-1, and 50 nM SelSA-2) were deposited in a 96-well plate. HeLa cell
nuclear extract (5 L) provided with the kit was added into each well. HDAC
l
L; 50 nM TSA, 500 nM SAHA, 50 nM
experiments and analyzed. Statistical analysis was performed using a
standard one-way ANOVA followed by Student Newman-Keuls post-hoc
ANOVA test. Differences with p <0.05 were considered significant.
l