Bicyclic peptides as potent inhibitors of histone deacetylases: Optimization of alkyl loop length

Article abstract of DOI:10.1016/j.bmcl.2009.12.054

Bicyclic tetrapeptide hydroxamic acids were prepared as histone deacetylase (HDAC) inhibitors, and the evaluated inhibitory activity shows that they are potent against HDAC1 and HDAC4. The in vivo activity depends on alkyl loop length.

Full text of DOI:10.1016/j.bmcl.2009.12.054

Bioorganic & Medicinal Chemistry Letters 20 (2010) 997–999
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Bicyclic peptides as potent inhibitors of histone deacetylases:
Optimization of alkyl loop length
b
c
c
Nurul M. Islam a, Tamaki Kato a, Norikazu Nishino a, , Hyun-Jung Kim , Akihiro Ito , Minoru Yoshida
*
a Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu 808-0196, Japan
b BioRunx Co. Ltd, Seoul 110-749, Republic of Korea
c Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Bicyclic tetrapeptide hydroxamic acids were prepared as histone deacetylase (HDAC) inhibitors, and the
evaluated inhibitory activity shows that they are potent against HDAC1 and HDAC4. The in vivo activity
depends on alkyl loop length.
Received 3 November 2009
Revised 8 December 2009
Accepted 11 December 2009
Available online 21 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Bicyclic peptide
Design and synthesis
Ring-closing metathesis
HDAC inhibitor
Optimum loop length
Dynamic acetylation and deacetylation of the
e
-amino groups of
homology between HDAC isoforms compared to the active site,
the modification of the cap group allows to have a significant im-
pact upon isoform selectivity.15 So that, for cap group modification
several cyclic tetrapeptide HDAC inhibitors have been designed
and synthesized.16 On the other hand, synthesis of constrained
peptide by ring closing metathesis (RCM) using ruthenium com-
plexes has been reported.17 On the basis of these reports, we have
previously designed and synthesized a fused bicyclic peptide
(Fig. 1, compound 1) to increase the size and the constraint of ali-
lysines at N-terminal tails of core histones are balanced by histone
acetyl transferase (HAT) and histone deacetylase (HDAC) en-
zymes.1 Imbalance in histone acetylation and deacetylation can
lead to transcriptional deregulation of genes that are involved in
the control of cell cycle progression, differentiation, and/or apopto-
sis. Aberrant histone deacetylation caused by the disrupted HAT
activity or abnormal recruitment of HDACs has been related to
carcenogenesis.2 Inhibition of HDAC enzymatic activity is expected
to induce re-expression of differentiation-inducing genes.
Several natural and synthetic compounds have been reported so
far as HDAC inhibitors. Among them, trichostatin A (TSA),3 depsi-
peptide FK2284 and the cyclic tetrapeptide family including trap-
oxin (TPX),5 chlamydocin,6 HC-toxins7 and apicidin8 are naturally
occurring HDAC inhibitors. As synthetic inhibitors, suberoylanilide
hydroxamic acid (SAHA)9 and the benzamide MS-27510 have been
designed. Recently cyclic tetrapeptide-based HDAC inhibitors,
CHAPs,11 SCOPs12 and ketone-based chlamydocin analogues13 have
also been reported. However, current HDAC inhibitors in clinical
trials are regarded as broad spectrum HDAC inhibitors with mod-
erate anticancer effect. Therefore, it is desirable clinically to devel-
op specific anticancer drugs that are effective for a particular HDAC
that is over expressed in cancer.14 Of various approaches to achieve
this, one is to modify the cap group of the HDAC inhibitors. As the
area surrounding the opening to the binding pocket has less
* Corresponding author.
E-mail address: nishino@life.kyutech.ac.Jp (N. Nishino).
Figure 1. Some selected HDAC inhibitors.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.054

998
N. M. Islam et al. / Bioorg. Med. Chem. Lett. 20 (2010) 997–999
phatic cap group.18 The bicyclic peptide was found to be active in
both cell free and cell based conditions. Moreover, it showed some
selectivity among the HDAC isoforms. These results prompted us to
make further investigation on bicyclic peptide HDAC inhibitors. In
the present study, we design and synthesize a series of bicyclic tet-
rapeptides by changing the length of the aliphatic loop to explore
the effect of the loop length on the activity of the inhibitors. The
sequence and configuration of amino acids in CHAP31 are consid-
ered as the basis for designing the inhibitors. We herein describe
the synthesis of bicyclic tetrapeptides using RCM, and a brief
description of the interesting biological results.
ing with trifluoroacetic acid (TFA), cyclization reaction was carried
out by the aid of N-[(dimetylamino)-1H-1,2,3-triazolo[4,5-b]pyri-
din-1-yl-methylene]-N-methylmethanaminium hexafluorophos-
phate N-oxide (HATU) in dimethylformamide (DMF) under high
dilution conditions with minimum amount of diisopropylethyl
amine (DIEA) (2.5 equiv) to obtain bicyclic tetrapeptides 27–30.
After Bzl deprotection, by catalytic hydrogenation, the carboxyl
group was condensed with hydroxylamine benzyl ester, and finally
Bzl protection was removed by catalytic hydrogenation to obtain
bicyclic tetrapeptide hydroxamic acids 2–5. Reference cyclic tetra-
peptide hydroxamic acid (6) was also synthesized in a similar man-
ner excluding RCM (Fig. 2).
Our synthesis was started from the preparation of the building
block amino acids. Boc-
OH) (7), Boc- -2-amino-7-octenoic acid (Boc-
2-amino-8-nonenoic acid (Boc- -Ae9-OH) (9), Boc-
octenoic acid (Boc- -Ae8-OH) (10) and Boc- -2-amino-8-nonenoic
acid (Boc- -Ae9-OH) (11) were synthesized by the reported proce-
dure.18 Bicyclic tetrapeptides were synthesizedaccording to Scheme
1 by the conventional solution phase method. H-
-Pro-OtBu was
condensed with 7–9 using DCC/HOBt, respectively. Boc protection
was selectively removed by the reported procedure19 using 4 M
HCl/dioxane, and each amine component was condensed with 10
or 11 by the same DCC/HOBt method to obtain linear tripeptides
15–18. The linear tripeptides 19–22 with fused side ring were syn-
L
-2-amino-6-heptenoic acid (Boc-
-Ae8-OH) (8), Boc-
-2-amino-7-
L
-Ae7-
The synthesized compounds (2–6) were assayed for HDAC
inhibitory activity using HDAC1, HDAC4 and HDAC6 prepared from
293T cells. Preparation and assay of HDACs, and p21 promoter as-
say were performed according to the reported methods.13 The re-
sults are summarized in Table 1.
L
L
L-
L
D
D
D
D
All the compounds 2–6 are active in nanomolar range. For com-
parison, the inhibitory activity of tricostatin A is also shown. The
activity toward HDAC1 slightly changes with the difference of
the size of the aliphatic loop. However, the changes in activity to-
ward HDAC4 and HDAC6 are not so remarkable. All the compounds
are specific toward HDAC4 compared with HDAC1 and HDAC6.
They are about two times more active toward HDAC4 than HDAC1,
and about 50 times more active than HDAC6. Most of the currently
available HDAC inhibitors have no or very little specificity toward
HDAC isoforms. Bicyclic tetrapeptides seem to be promising target
for the development of isoform selective inhibitors. Compound 3
showed better selectivity (the ratio of IC50 values: HDAC6/
HDAC4 = 75).
D
thesized by ring closing metathesis between D-Ae8/9 and L-Ae7/8/
9 using Grubb’s first generation Ru catalyst, in dichloromethane
(DCM), followed by catalytic hydrogenation. After selective depro-
tection of 19–22, Boc protected amino suberic acid benzyl ester
[Boc-L-Asu(OBzl)] was incorporated in them to prepare the linear
tetrapeptides 23–26. After removal of both side protections by treat-
All of the bicyclic tetrapeptides except for 2 are excellent in p21
promoter-inducing activity. The variation of HPLC retention time,
which can be used as a parameter for hydrophobicity, could be cor-
related with p21 promoter-inducing activity. A linear increase in
hydrophobicity was observed when loop length was increased
from nine to eleven CH2 groups. However, increase in hydropho-
bicity was not remarkable upon further increase in loop length
(Fig. 3). Similar increasing trend in p21 promoter-inducing activity
was observed (Fig. 3). It seems that the aliphatic loop helped in
penetration through the cell membrane, and resulted in increased
activity up to certain loop length. In our finding, eleven CH2 loop
length is the optimum for p21 promoter-inducing activity, as no
remarkable increase in activity has been observed for further elon-
gation in the loop. Compound 6 was synthesized as a reference
compound to compare the activity between cyclic tetrapeptide
Scheme 1. Synthesis of bicyclic tetrapeptide hydroxamic acids. Reagents and
conditions: (a) DCC, HOBt, DMF, 12 h, 75–80%; (b) 4 M HCl/dioxane, 30 min; (c)
Saturated Na2CO3, 70–80%; (d) Boc-
HOBt, DMF, 12 h, 75–80%; (e) Grubbs‘ first generation catalyst, DCM, 48 h, 50–87%;
(f) AcOH, Pd–C, H2, 12 h, 97–100%; (g) Boc- -Asu(OBzl)-OH, DCC, HOBt, DMF, 12 h,
D-Ae8-OH (10) or Boc-D-Ae9-OH (11), DCC,
L
75–80%; (h) TFA, 3 h, 98–100%; (i) HATU, DIEA, DMF, 4 h, 15–65%; (j) HClÁH2NOBzl,
Figure 2. Bicyclic tetrapeptides and a reference compound synthesized as HDAC
inhibitors.
DCC, HOBt, TEA, DMF, 60–80%; (k) Pd–BaSO4, AcOH, H2, 70–80%.

N. M. Islam et al. / Bioorg. Med. Chem. Lett. 20 (2010) 997–999
999
Table 1
HDAC inhibitory activity and p21 promoter activity data for bicyclic tetrapeptide hydroxamic acids and reference compounds
Compounds
Loop size
HPLC20 tR (min)
IC50 (nM)
HDAC4
p21 promoter assay, EC1000 (nM)
HDAC1
HDAC6
Tricostatin A
—
—
23
9.1
9.1
11
13
25
44
65
20
2
3
4
5
6
–(CH2)9–
4.74
6.03
7.39
7.52
7.51
5.4
5.5
4.5
5.0
12
330
410
280
240
340
92
7.2
2.6
2.0
13
–(CH2)10
–(CH2)11
–(CH2)12
—
–
–
–
Lehrmann, H.; Polesskaya, A.; Harel-Bellan, A. Cell Mol. Life Sci. 2001, 58, 728–
736.
3. Yoshida, M.; Kijima, M.; Akita, M.; Beppu, T. J. Biol. Chem. 1990, 265, 17174–
17179.
4. Ueda, H.; Nakajima, H.; Hori, Y.; Fujita, T.; Nishimura, M.; Goto, T.; Okuhara, M.
J. Antibiot. 1994, 47, 301–310.
5. Kijima, M.; Yoshida, M.; Sugita, K.; Horinouchi, S.; Beppu, T. J. Biol. Chem. 1993,
268, 22429–22435.
6. De Schepper, S.; Bruwiere, H.; Verhulst, T.; Steller, U.; Andries, L.;
Wouters, W.; Janicot, M.; Arts, J.; Van Heusden, J. J. Pharmacol. Exp. Ther.
2003, 304, 881–888.
7. Pope, M. R.; Ciuffetti, L. M.; Knoche, H. W.; McCrery, D.; Daly, J. M.; Dunkle, L. D.
Biochemistry 1983, 22, 3502–3506.
8. Darkin-Rattray, S. J.; Gurnett, A. M.; Myers, R. W.; Dulski, P. M.;
Crumley, T. M.; Allocco, J. J.; Cannova, C.; Meinke, P. T.; Colletti, S. L.;
Bednarek, M. A.; Singh, S. B.; Goetz, M. A.; Dombrowski, A. W.;
Polishook, J. D.; Schmatz, D. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,
13143–13147.
9. 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, 3003–3007.
10. Suzuki, T.; Ando, T.; Tsuchiya, K.; Fukazawa, N.; Saito, A.; Mariko, Y.;
Yamanashi, T.; Nakanishi, O. J. Med. Chem. 1999, 42, 3001–3003.
11. Komatsu, Y.; Tomizaki, K.; Tsukamoto, M.; Kato, T.; Nishino, N.; Sato, S.;
Yamori, T.; Tsuruo, T.; Furumai, R.; Yoshida, M.; Horinouchi, S.; Hayashi, H.
Cancer Res. 2001, 61, 4459–4466.
12. Nishino, N.; Jose, B.; Okamura, S.; Ebisusaki, S.; Kato, T.; Sumida, Y.; Yoshida, M.
Org. Lett. 2003, 5, 5079–5082.
13. Bhuiyan, M. P. I.; Kato, T.; Okauchi, T.; Nishino, N.; Maeda, S.; Nishino, T. G.;
Yoshida, M. Bioorg. Med. Chem. 2006, 14, 3438–3446.
14. Kahnberg, P.; Lucke, A. J.; Glenn, M. P.; Boyle, G. M.; Tyndall, J. D. A.; Parsons, P.
G.; Fairlie, D. P. J. Med. Chem. 2006, 49, 7611–7622.
15. Nielsen, T. K.; Hildmann, C.; Dickmanns, A.; Schwienhorst, A.; Ficner, R. J. Mol.
Biol. 2005, 354, 107–120.
16. (a) Shivashimpi, G. M.; Amagai, S.; Kato, T.; Nishino, N.; Maeda, S.; Nishino, T.
G.; Yoshida, M. Bioorg. Med. Chem. 2007, 15, 7830–7839; (b) Nishino, N.; Jose,
B.; Shinta, R.; Kato, T.; Komatsu, Y.; Yoshida, M. Bioorg. Med. Chem. 2004, 12,
5777–5784; (c) Nishino, N.; Yoshikawa, D.; Watanabe, L. A.; Kato, T.; Jose, B.;
Komatsu, Y.; Sumida, Y.; Yoshida, M. Bioorg. Med. Chem. Lett. 2004, 14, 2427–
2431.
17. (a) Wels, B.; Kruijtzer, J. A. W.; Nijenhuis, W. A. J.; Gispen, W. H.; Adan, R. A. H.;
Liskamp, R. M. J. Bioorg. Med. Chem. 2005, 13, 4221–4227; (b) Mollica, A.;
Guardiani, G.; Davi, P.; Ma, S.; Prreca, F.; Lai, J.; Mannina, L.; Sobolev, A. P.;
Hruby, V. J. J. Med. Chem. 2007, 50, 3138–3142; (c) Deshmukh, P. H.; Schultz-
Fademrecht, C.; Procopiou, P. A.; Vigushin, D. A.; Coombes, C.; Barett, A. G. M.
Adv. Synth. Catal. 2007, 349, 175–183.
Figure 3. Change in (d) hydrophobicity, and (s) activity in cell based test with loop
length of bicyclic tetrapeptides (2–5).
and bicyclic tetrapeptide. Compound 3 corresponds to the fused
ring one of compound 6. The improvement in activity and selectiv-
ity in both cell free and cell based conditions of compound 3 over
compound 6 reflects the importance of closed ring.
In summary, in order to find novel and potent non-aromatic
HDAC inhibitors, we designed and synthesized CHAP31-based
bicyclic tetrapeptide hydroxamic acids by changing the aliphatic
loop length. These inhibitors show potent HDAC inhibitory activity
in vivo and in vitro. They also show some selectivity among the
HDAC isoforms. The aliphatic loop length is important, and eleven
CH2 loop is the optimum for in vivo activity. These results further
confirm the hypothesis that modification of the cap group of HDAC
inhibitors can lead to potent HDAC inhibitors, which may have po-
tential as anticancer agents. We are also carrying out conforma-
tional analysis of these inhibitors by NMR calculation methods.
These results will be published elsewhere.
References and notes
18. Nishino, N.; Shivashimpi, G. M.; Soni, P. B.; Bhuiyan, M. P. I.; Kato, T.; Maeda, S.;
Nishino, T. G.; Yoshida, M. Bioorg. Med. Chem. 2008, 16, 437–445.
19. Han, G.; Tamaki, M.; Hruby, V. J. J. Peptide Res. 2001, 58, 338–341.
1. (a) Hassig, C. A.; Schreiber, S. L. Curr. Opin. Chem. Biol. 1997, 1, 300–308; (b)
Grozinger, C. M.; Schreiber, S. L. Chem. Biol. 2002, 9, 3–16; (c) Yoshida, M.;
Matsuayama, A.; Komatsu, Y.; Nishino, N. Curr. Med. Chem. 2003, 10, 2351–
2358.
2. (a) Urnov, F. D.; Wolffe, A. P. Emerg. Ther. Targets 2000, 4, 665–685; (b)
Mahlknecht, U.; Hoelzer, D. Mol. Med. 2000, 6, 623–644; (c) Timmermann, S.;
20. HPLC analysis was performed on
a Hitachi instrument equipped with a
Chromolith performance RP-18e column (4.6 Â 100 mm, Merck). The mobile
phases used were A: H2O with 0.1% TFA, B: CH3CN with 0.1% TFA using a
solvent gradient of A–B over 15 min with a flow rate of 2 mL/min, with
detection at 220 nm.