Design and synthesis of novel histone deacetylase inhibitor derived from nuclear localization signal peptide

Article abstract of DOI:10.1016/j.bmcl.2009.10.028

We describe herein the synthesis and characterization of a new class of histone deacetylase (HDAC) inhibitors derived from conjugation of a suberoylanilide hydroxamic acid-like aliphatic-hydroxamate pharmacophore to a nuclear localization signal peptide. We found that these conjugates inhibited the histone deacetylase activities of HDACs 1, 2, 6, and 8 in a manner similar to suberoylanilide hydroxamic acid (SAHA). Notably, compound 7b showed a threefold improvement in HDAC 1/2 inhibition, a threefold increase in HDAC 6 selectivity and a twofold increase in HDAC 8 selectivity when compared to SAHA.

Full text of DOI:10.1016/j.bmcl.2009.10.028

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6588–6590
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of novel histone deacetylase inhibitor derived
from nuclear localization signal peptide
,à
Joshua C. Canzoneri à, Po C. Chen , Adegboyega K. Oyelere
*
School of Chemistry and Biochemistry, Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe herein the synthesis and characterization of a new class of histone deacetylase (HDAC)
inhibitors derived from conjugation of a suberoylanilide hydroxamic acid-like aliphatic-hydroxamate
pharmacophore to a nuclear localization signal peptide. We found that these conjugates inhibited the his-
tone deacetylase activities of HDACs 1, 2, 6, and 8 in a manner similar to suberoylanilide hydroxamic acid
(SAHA). Notably, compound 7b showed a threefold improvement in HDAC 1/2 inhibition, a threefold
increase in HDAC 6 selectivity and a twofold increase in HDAC 8 selectivity when compared to SAHA.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 31 July 2009
Revised 6 October 2009
Accepted 7 October 2009
Available online 13 October 2009
Keywords:
HDAC
NLS
Histone deacetylase inhibition
Histone deacetylase (HDAC) inhibition has been clinically vali-
dated as a therapeutic strategy for cancer treatment with the
FDA approval of suberoylanilide hydroxamic acid (SAHA) for the
treatment of cutaneous T-cell lymphoma.1 HDAC inhibitors
(HDACi) have shown the ability to block angiogenesis and cell
cycling, initiate differentiation and apoptosis. HDACi presumably
derived their biological activities through perturbation of chroma-
tin remodeling and acetylation states of key non-histone pro-
teins.2–7 Most HDACi, including SAHA, non-selectively inhibit the
deacetylase activity of class I/II HDAC enzymes.8,9 This broad HDAC
inhibition is associated with reduced potency and toxic side
effects. Attempts aimed at identifying isoform selective HDACi
have been modestly successful, resulting in very few HDACi that
are only partially isoform selective.10–12
HDACs 1 and 2, the primary targets for the anticancer activity of
HDACi, are exclusively localized in the nucleus.13,14 Thus, the
development of a strategy for nuclear delivery and localization of
HDACi could be an alternative approach to isoform selective
HDACi. Toward this end, we sought novel peptide–HDACi conju-
gates that are capable of crossing both the plasma and nuclear
membrane.15 Most of the nuclear membrane-penetrating peptides
described in the literature are derived from viral sources. Common
examples include the Simian virus nuclear localization peptides
(NLS),15–17 HIV 1 Tat-protein derived peptides,18 and peptides
derived from adenovirus fiber protein. NLS peptides are primarily
utilized by viruses to cross the nuclear membrane, making them
an ideal candidate for HDACi conjugation. Additionally, we rea-
soned that NLS, with lysine-enriched sequences, could also act as
a substrate-mimetic to HDAC by mimicking the N-terminal tail
lysine residues of the core histones. We report here the identifica-
tion of NLS-peptide derived HDACi with anti-HDAC activity and
HDAC isoform selectivities that rival or better that of SAHA.
Our design approach involved conjugation of a SAHA-like ali-
phatic-hydroxamate HDAC inhibition group directly to the NLS
peptide through 1,2,3-triazole moiety.11 This very simple initial de-
sign could facilitate a facile, high yielding synthesis of the proposed
NLS–HDACi conjugates. Accordingly, we prepared compounds 7a–c
having two 1,2,3-triazole rings connecting a NLS-derived peptide to
a HDAC surface recognition group and a hydroxamate zinc binding
group to the surface recognition group through flexible methylene
linkers whose lengths are optimized for HDAC inhibition.11,12
The designed conjugates 7a–c were synthesized through a six-
step synthetic route as shown in Scheme 1. Cu(I)-catalyzed reac-
tion of 4-ethynylaniline with azido esters 1a–c gave cycloadducts
2a–c in excellent yields.11,19 The diazotization of 2a–c by treat-
ment with sodium nitrite followed by exposure of the crude prod-
ucts to sodium azide led to the azido derivatives 3a–c in good
yields. However, a portion of azido derivatives 3 was hydrolyzed
into carboxylic acid giving a mixture of both ester and carboxylic
acid derivatives. To hydrolyze the rest of the ester, lithium hydrox-
ide hydrate was added to the mixture giving a complete conversion
to the azido carboxylic acid derivatives 4a–c in excellent yields.
The reaction of acid 4a–c with O-trityl hydroxylamine gave the
desired O-trityl azido-triazolylhydroxamates 5a–c that were
subsequently coupled to the alkyne-terminated protected NLS
peptide PCS-37689-PI16 to give cycloadducts 6a–c. Exposure of
* Corresponding author. Tel.: +1 404 894 4047; fax: +1 404 894 2291.
E-mail address: aoyelere@gatech.edu (A.K. Oyelere).
Present address: School of Medicinal Chemistry and Pharmacognosy, University
of Illinois at Chicago.
à
These authors contributed equally to the Letter.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.028

J. C. Canzoneri et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6588–6590
6589
N
N
N
O
N
N
N
O
O
a
b
N3
N3
H2N
OR
OR
OR
n
n
n
70-90%
3a-c
1a-c
2a-c
a: n = 1, R = Et
b: n = 2, R = Et
c: n = 3, R = Me
a: n = 1, R = Et
a: n = 1, R = Et
b: n = 2, R = Et
c: n = 3, R = Me
b: n = 2, R = Et
c: n = 3, R = Me
c
99%
N
N
N
O
N
N
N
O
d
O
N3
N3
N
H
OH
n
n
73%
4a-c, n = 1-3
5a-c, n = 1-3
e
90%
Boc
HN
Boc
HN
Boc
O
HN
O
O
O
O
O
O
H
N
H
N
H
N
H
N
O
N
H
N
H
N
N
N
O
N
H2N
N
H
N
H
N
H
N
H
N
N
N
N
H
H
O
O
O
O
n
O
O
N
NH
NH
HN
HN
Boc
Pbf
6a-c, n = 1-3
f
100%
H2N
H2N
H2N
O
O
O
O
O
O
O
H
N
H
N
H
H
O
N
H
N
H
N
N
N
N
N
OH
H2N
N
H
N
H
N
H
N
H
N
N
H
N
H
N
O
O
O
O
n
O
O
N
N
NH
HN
H2N
NH2
7a-c, n = 1-3
Scheme 1. Synthesis of NLS–HDACi conjugates. Reagents: (a) 4-ethynylaniline, CuI, DIPEA, THF; (b) NaNO2, NaN3, 17.2% HCl(aq)
(d) o-tritylhydroxylamine, EDCI, HOBT, NMM, DMF; (e) alkyne-terminated NLS peptides, CuI, TBTA, DIPEA, THF/DMF; (f) 90:5:5 TFA/TIPS/phenol.
;
(c) LiOHÁH2O, THF/H2O,
cycloadducts 6a–c to TFA removed all protecting groups yielding
the desired NLS–HDACi conjugates 7a–c in near quantitative
yields.
with a C7 linker, stood out as it showed not only a threefold
improvement in HDAC 1/2 activity when compared to SAHA, but
also a twofold increase in HDAC 8 selectivity and a threefold in-
crease in HDAC 6 selectivity.
Encouraged by the potent anti-HDAC activities of these NLS–
HDACi conjugates, we then evaluated their whole cell anti-prolifer-
ative activities using trypan blue exclusion and MTT assay. Sadly,
none of the conjugates exhibited any appreciable, anti-prolifera-
tive activity in DU-145 or HSC3 cell lines at drug concentrations
The HDAC inhibition activity of 7a–c was tested using a cell free
assay (Fluor de Lys).20 We found that the NLS–HDACi conjugates
display potent HDAC inhibition activities, similar to SAHA, that is
somewhat linker-length dependent against HDAC 1 and 2 from
HeLa cell nuclear extract (Table 1). An increase in the linker-length
from C6 to C7 conferred a better anti-HDAC activity. The NLS–
HDACi conjugates also presented similar isoform selectivity to that
of SAHA with respect to HDAC 6 and 8 (Table 1). Compound 7b,
up to 25
lM in both assays (data not shown). In an attempt to shed
light on the lack of whole cell activity of these conjugates, we

6590
J. C. Canzoneri et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6588–6590
Table 1
Supplementary data
HDAC inhibitory activity and isoform selectivity of SAHA and NLS-linked compounds
HDAC 1/2
IC50 (nM)
HDAC 8
IC50 (nM)
HDAC 8
HDAC 6
IC50 (nM)
HDAC 6
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.10.028.
selectivitya
selectivitya
SAHA
7a
7b
49
36
14
25
2185
3503
1528
1243
45
96
108
50
800
575
716
714
16
16
51
29
References and notes
7c
1. (a) Marks, P. A. Oncogene 2007, 26, 1351; (b) FDA approves vorinostat (Zolinza)
for the treatment of cutaneous manifestations of cutaneous T-cell lymphoma
(CTCL) http://www.fda.gov/AboutFDA/CentersOffices/CDER/ucm094952.htm.
2. Marson, C. M.; Mahadevan, T.; Dines, J.; Segmany, S.; Morrell, J. M.; Alao, J. P.;
Joel, S. P.; Vigushin, D. M.; Coombes, R. C. Bioorg. Med. Chem. Lett. 2007, 17, 136.
3. Moradei, O.; Maroun, C. R.; Paquin, I.; Vaisburg, A. Curr. Med. Chem.: Anti-Cancer
Agents 2005, 5, 529.
4. Hildmann, C.; Riester, D. Appl. Microbiol. Biotechnol. 2007, 75, 487.
5. Marks, P.; Rifkind, R.; Richon, V.; Breslow, R.; Miller, T.; Kelly, W. Nat. Rev.
Cancer 2001, 1, 194.
a
Selectivity is the activity of the HDAC isoform (6 or 8) divided by the activity of
HDAC 1/2.
prepared a BODIPY tagged analog of the NLS (Supplementary
data).21 The syntheses of alkyne BODIPY and NLS-BODIPY are de-
scribed in Supplementary data (Schemes S1 and S2, respectively).
The intracellular localization of the dye labeled NLS was then mon-
itored using fluorescence microscopy at a variety of incubation
times (Supplementary data). It was found that the dye labeled
NLS was not taken up into the nucleus; instead it was sequestered
in the cytosol. This preliminary result may provide an explanation
for the poor whole cell activity of the compounds.
We have described a class of novel NLS-peptide derived HDACi.
All of the compounds demonstrated HDAC inhibition and isoform
selectivity similar to, and in the case of 7b better than, SAHA. De-
spite these promising HDAC inhibitory activities, none of the com-
pounds exhibited whole cell activity at concentrations up to
6. Johnstone, R. W.; Licht, J. D. Cancer Cell 2003, 4, 13.
7. Carey, N.; Thangue, N. Curr. Opin. Pharmacol. 2006, 6, 369.
8. Estiu, G.; Greenberg, E.; Harrison, C.; Kwiatkowski, N.; Mazitschek, R.; Bradner,
J.; Wiest, O. J. Med. Chem. 2008, 51, 2898.
9. Hkan, N.; Jeffers, M.; Kumar, S.; Hackett, C.; Boldog, F.; Khramtsov, N.; Qian, X.;
Mills, E.; Berghs, S.; Carey, N.; Finn, P.; Collins, N.; Tumber, A.; Ritchie, J.; Jensen,
P.; Lichenstein, H.; Sehested, M. Biochem. J. 2008, 409, 581.
10. Miller, T. A.; Witter, D. J.; Belvedere, S. J. Med. Chem. 2003, 46, 5097; 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; (c) Carew, J. S.; Francis, J. G.; Nawrocki, S. T. Cancer
Lett. 2008, 269, 7; (d) Bieliauskas, A. V.; Pflum, M. K. H. Chem. Soc. Rev. 2008, 37,
1402.
11. Chen, P.; Patil, V.; Guerrant, W.; Green, P.; Oyelere, A. Bioorg. Med. Chem. 2008,
16, 4839.
12. Oyelere, A.; Chen, P.; Guerrant, W.; Mwakwari, S.; Hood, R.; Zhnag, Y.; Fan, Y. J.
25 lM. The lack of appreciable whole cell activity of these NLS–
Med. Chem. 2009, 52, 456.
HDACi conjugates may be partly due to their cytosolic entrapment.
Efforts are currently underway in our laboratory to enhance the
nuclear penetration of these conjugates by optimizing their NLS se-
quence linker, and HDAC inhibition moiety.
13. Bolden, J. E.; Part, M. J.; Johnstone, R. W. Nat. Rev. Drug Disc. 2006, 5, 769.
14. Johnstone, R. W. Nat. Rev. Drug Disc. 2002, 1, 287.
15. Tkachenko, A. G.; Xie, H.; Coleman, D.; Glomm, W.; Ryan, J.; Anderson, M. F.;
Franzen, S.; Feldheim, D. L. J. Am. Chem. Soc. 2003, 125, 4700.
16. Oyelere, A. K.; Chen, P. C.; Huang, X.; El-sayed, I. H.; El-sayed, M. A. Bioconjugate
Chem. 2007, 18, 1490.
17. Kalderon, D.; Richardson, W. D.; Markham, A. F.; Smith, A. E. Nature (London)
1984, 311, 33.
Acknowledgments
18. De la Furente, J. M.; Berry, C. C. Bioconjugate Chem. 2005, 16, 1176.
19. (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int.
Ed. 2002, 41, 2596; (b) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem.
2002, 67, 3057.
20. HDAC Fluorimetric Assay/Drug Discovery Kit—AK-500 Manual. Fluorescent
Assay System, BIOMOLÒ International, Plymouth Meeting, PA, 2005.
21. Loudet, A.; Burgess, K. Chem. Rev. 2007, 107, 4891.
This work was financially supported by Georgia Institute of
Technology, by the Blanchard fellowship and by NIH Grant
R01CA131217. J.C.C. and P.C.C. are recipients of the GAANN predoc-
toral fellowship from the Georgia Tech Center for Drug Design,
Development, and Delivery.