Design and synthesis of CHAP31, trapoxin B and HC-toxin based bicyclic tetrapeptides disulfide as potent histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.06.029

The naturally occurring cyclic depsipeptide, FK228 inhibits histone deacetylase (HDAC) enzymes after reductive cleavage of intra-molecular disulfide bond. One of the sulfhydryl groups produced in the reduction interacts with zinc atom that involved in the

Full text of DOI:10.1016/j.bmc.2014.06.029

Bioorganic & Medicinal Chemistry 22 (2014) 3850–3855
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of CHAP31, trapoxin B and HC-toxin based
bicyclic tetrapeptides disulfide as potent histone deacetylase
inhibitors
Md. Ashraful Hoque a,b, , Md. Nurul Islam a,c, Md. Shahidul Islam a,c, Tamaki Kato a, Norikazu Nishino a,
⇑
Akihiro Ito d, Minoru Yoshida d
a Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu 808-0196, Japan
b Department of Biochemistry and Molecular Biology, Faculty of Science, University of Rajshahi, Rajshahi 6205, Bangladesh
c Department of Chemistry, Faculty of Science, University of Rajshahi, Rajshahi 6205, Bangladesh
d 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:
The naturally occurring cyclic depsipeptide, FK228 inhibits histone deacetylase (HDAC) enzymes after
reductive cleavage of intra-molecular disulfide bond. One of the sulfhydryl groups produced in the reduc-
tion interacts with zinc atom that involved in the catalytic mechanism of type 1 and 2 HDACs such as
HDAC1, HDAC4, and HDAC6. In the present study, we describe the development of CHAP31, trapoxin B
and HC-toxin based cyclic tetrapeptides with intra-molecular disulfide bond as HDAC inhibitors. The
bicyclic tetrapeptides disulfide showed potent HDAC1 and HDAC4 inhibition and p21 promoting activity.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 11 May 2014
Revised 16 June 2014
Accepted 17 June 2014
Available online 25 June 2014
Keywords:
Histone deacetylase inhibitors
Depsipeptide
FK228
Sulfhydryl group
Bicyclic tetrapeptides
1. Introduction
occurring cyclic peptides Trapoxins,11 Cyl-1 and Cyl-2,12–14
WF3161,15 Chlamydocin16 and HC-toxin17–19 may be helpful for
Natural cyclic depsipeptide FK228, one of the two HDAC inhib-
itors approved by the FDA for the treatment of cutaneous T-cell
lymphoma (CTCL), contains intra-molecular disulfide bond.1,2 This
natural product is known to exhibit inhibition of HDAC enzyme
potently after reductive cleavage of the disulfide bond, as the sulf-
hydryl group is a good ligand to zinc atom present in the active site
of class 1 and 2 HDACs.3 Consequently, HDAC inhibitors equipped
with sulfur containing functional groups draw much more
attention of some researchers4–6 for their attractive inhibitory
and specificity profile. Our group reported sulfur containing cyclic
tetrapeptides (SCOPs)7 and bicyclic tetrapeptide disulfide hybrids.8
As a continuation, we reported bicyclic tetrapeptides with intra-
molecular disulfide bridge as potent HDAC inhibitors.9
selective HDAC inhibitors design (Fig. 1).
In the practical use of anticancer drugs, it is desirable that the
drugs exhibit enough stability during the delivery. Some of potent
HDAC inhibitors in vitro often accept modification to lose their
activity in digestive organ and blood. FK228 has an advantage in
the stability of the functional group to be disulfide such as a pro-
drug. In considering the problems in chemical synthesis of FK228
and its analogs,20,21 we challenged the development of cyclic tetra-
peptides bearing disulfide moiety as pro-drug candidates for future
application. For the first time, we reported the successful synthesis
of CHAP31 based cyclic tetrapeptides with intra-molecular disul-
fide bridge9 which has opened a new door to develop novel HDAC
inhibitors. To explore this new type of potent HDAC inhibitors,
here we report the design and synthesis of a series of bicyclic tet-
rapeptide disulfide HDAC inhibitors (1–6) based on different scaf-
folds, for example, CHAP31, trapoxin B, and HC-toxin scaffolds
(Fig. 2).
Interactions between the rim region of the enzyme and the cap
groups of inhibitors are of great importance to selective inhibitor
design, as this region has less homology between isoforms com-
pared to the active site allowing the cap group to have a significant
impact upon isoform selectivity.10 The cap groups of naturally
After successful synthesis of CHAP31 based bicyclic tetrapep-
tide disulfide,9 to check the effect of hydrophobicity on activity,
first three CHAP31 based (LDLD configuration) bicycle tetrapep-
⇑
Corresponding author.
E-mail address: ashraf_bio2003@yahoo.com (M.A. Hoque).
tides cyclo(-L-Am7(S-)-D-Am7(S-)-L-Ala-D-Pro-) (1), cyclo(-L-Am7
http://dx.doi.org/10.1016/j.bmc.2014.06.029
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

M. A. Hoque et al. / Bioorg. Med. Chem. 22 (2014) 3850–3855
3851
Scheme 1. Synthetic strategy of cyclic tetrapeptides.
occurring cyclic tetrapeptide HDAC inhibitor17–19 and its configur-
aration is LDLD, just reverse arrangement of CHAP31. Thus finally,
the HC-toxin based, cyclo(-L-Am7(S-)-D-Tic-L-Val-D-Am7(S-)-) (6)
(Tic, tetrahydroisoquinoline carboxylic acid) was designed and
synthesized by changing the disulfide bridge position.
2. Results and discussion
2.1. Chemistry
Figure 1. Some natural and synthetic cyclic peptide HDAC inhibitors.
Our aim was to synthesize CHAP31, trapoxin B and HC-toxin
based cyclic tetrapeptide HDAC inhibitors with intra-molecular
disulfide bridge that mimic FK228 (Fig. 2). For that, the building
(S-)-
Phe-
D
-Am7(S-)-
L-Val-D-Pro-) (2) and cyclo(-L-Am7(S-)-D-Am7(S-)-L-
D
-Pro-) (3) (Am7(S-), sulfide of 2-amino-7-mercaptoheptanoic
acid) were designed and synthesized by introducing several hydro-
phobic amino acid residues, for example, alanine, valine and phen-
ylalanine, respectively in the third position of cyclic tetrapeptide
scaffolds (Fig. 2) as this position is suitable for hydrophobic amino
acid for better interaction with surface of enzyme.22 In addition,
we introduced a new amino acid (2-ami no-5-oxy-7-bromohepta-
noic acid, Aob7) expecting the improvement of solubility, activity,
block amino acids Boc-
Ab7), Boc- -2-amino-7-bromoheptanoic acid (Boc-
Boc- -2-amino-5-oxy-7-bromoheptanoic acid (Boc- -Aob7) were
synthesized according to the reported procedure.23
The designed compounds were obtained by synthesizing mono-
cyclic tetrapeptides followed by side chain modification. Synthesis
of cyclic tetra peptides were carried out according to the general
Scheme 1 by the conventional solution phase method. We started
L
-2-amino-7-bromoheptanoic acid (Boc-
L-
D
D-Ab7) and
D
D
and synthetic yield. Thus, we designed and synthesized cyclo(-
Am7(S-)- -Aom7(S-)- -Ala- -Pro-) (4) by introducing -Aob7 in
place of -Ab7 of compound, 1. In the second step, to find out bet-
ter configuration for interacting tightly with surface of the active
pocket of HDACs, cyclo(- -Am7(S-)- -Am7(S-)- -Val- -Pro-) (5)
L-
D
L
D
D
with tert-butyl-protected
N-terninal protected -amino acids (
-Val-OH) using N,N-dicyclohexyl carbodiimide (DCC) and 1-hydro
D
-proline/
D
-Tic and coupled them with
D
L
L
-Ab7/Z- -Ala/Z- -Phe-OH/Z-
L
L
L
L
L
L
D
xybenzotriazole (HOBt) as coupling agents to get protected dipep-
tides. After the removal of N-terminal protection by HCl-dioxane/
catalytic hydrogenation, the free amines were extracted and used
for condensation with other N-terninal protected amino acids
was designed on the basis of trapoxin B scaffold. However, the cyc-
lic tetrapeptide framework (LLLD configuration) of trapoxin affor-
ded rather poor yield in cyclization step than LDLD configuration
and had a solubility problem. HC-toxin is also a potent naturally
(Boc-L/D-Ab7-OH/Boc-D-Aob7-OH) by the same DCC/HOBt method
Figure 2. Structures of designed bicyclic tetrapeptide disulfide HDAC inhibitors.

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M. A. Hoque et al. / Bioorg. Med. Chem. 22 (2014) 3850–3855
to obtain linear tripeptides. The N-terminals of the tripeptides
were further deprotected by using HCl-dioxane/catalytic hydroge-
nation and coupled them with Boc-protected L-Ab7/L-Val to yield
linear tetrapeptides. After the removal of both side protections
by treating with trifluoroacetic acid (TFA), the linear tetrapeptides
were cyclized under high dilution conditions in dimethylformam-
ide (DMF) using 2-(1H-7-azobenzotriazol-1-yl)-1, 1,3,3-tetrameth-
yluronium hexafluorophosphate (HATU) and diisopropylethyl
amine (DIEA) to give cyclic tetrapeptides after purification by silica
gel column chromatography.
spacer arm of the inhibitor is an important factor for binding with
enzyme and for cell membrane permeability.25 Compound 4 is
much more active than compound 1 against HDAC6. Oxygen pres-
ent in the spacer arm has positive contribution for inhibitory activ-
ity against HDAC6.
Trapoxin B (LLLD configuration) based compound 5 was less
active than CHAP31 (LDLD configuration) based compound 2 both
in vivo and in vitro conditions. These two compounds are different
only in amino acid configuration in the fourth position proving the
superiority of LDLD configuration over LLLD on activity.
In spite of being more hydrophobic, HC-toxin based compound
6 showed less in vitro activity than CHAP31 based compounds
due to shift in disulfide loop and imino acid positions. Thus, it
can be concluded that both hydrophobicity and amino acid
sequence of cyclic tetrapeptide scaffold affect the HDAC inhibi-
tory activity.
The side chain of cyclic tetrapeptides was modified from a
bromo group to a thioacetate group by treating with potassium
thioacetate to yield dithioester compounds. Further treatment
with methylamine in methanol and subsequent oxidation with
iodine yielded the desired cyclic tetrapeptides with intra-molecu-
lar disulfide bridge (Scheme 2). The bicyclic tetrapeptide disulfide
compounds were purified by filtration through a Sephadex LH-20
column.
3. Conclusion
All the synthesized compounds were characterized by 1H NMR
and high resolution FAB-MS. The purity of compounds were deter-
mined by HPLC analysis and showed purity above 98%.
In the present paper, we describe the development of CHAP31,
trapoxin B and HC-toxin based cyclic tetrapeptides disulfide as
potent HDAC inhibitors. Most of the compounds were active in
nano molar range in presence of reducing agent DTT. Among these,
CHAP31 based compound 3 showed the highest activity and spec-
ificity. It can be a promising anti-cancer drug candidate as its activ-
ity and selectivity was comparable with more recently approved
drug, FK228.
2.2. Enzyme inhibition and biological activity
The synthesized compounds were assayed for HDAC inhibitory
activity using HDAC1, HDAC4 and HDAC6 enzymes prepared by
293T cells3 with the aid of 0.1 mM of dithiothreitol (DTT). In addi-
tion, to know the inhibitory activity of these compounds in cell
based condition, we carried out p21 promoter assay according to
the literature.24 The detail experimental procedures for the prepa-
ration and assays of HDAC inhibitory activity and p21 promoter
assay are described in the experimental section of this paper. Tri-
costatin A and FK228 were used as the reference compounds and
the results are summarized in Table 1.
Evaluation of cell-free and cell-based HDAC inhibitory activity
of the bicyclic tetrapeptides disulfide showed that most of these
compounds were active in nano molar range in presence of reduc-
ing agent DTT. These compounds exhibited poor in vitro activity in
absence of DTT. However, they showed good in vivo activity as the
disulfide bond reduce to active sulfhydryl group inside the cell fol-
lowing the same mechanism reported for FK228.3
In presence of DTT, CHAP31 based bicyclic tetrapeptides disul-
fide (1–3) showed excellent inhibitory activity against HDAC1
and HDAC4. The activity of these compounds increases with
increasing hydrophobicity in the third position of cyclic tetrapep-
tides scaffold. This observation is in agreement with our previous
work.22 Therefore, the third position of the cyclic tetrapeptide
framework demands more attention in designing potent HDAC
inhibitors. Among the CHAP31 based compounds, 3 showed high-
est activity and selectivity, that is, comparable to FK228.
Compounds 1 and 4 have same cyclic tetrapeptide scaffold and
disulfide loop positions. Introduction of oxygen reduced hydropho-
bicity in spacer arm and resulted in less activity of compound 4
compared to its parent compound 1 both in vivo and in vitro
conditions. This result reflects the fact that hydrophobicity of the
4. Experimental
4.1. General
Unless otherwise noted, all solvents and reagents were reagent
grade and used without purification. Flash chromatography was
performed using silica gel 60 (230–400 mesh) eluting with sol-
vents as indicated. All compounds were routinely checked by thin
layer chromatography (TLC) and/or high performance liquid chro-
matography (HPLC). TLC was performed on aluminum-backed sil-
ica gel plates (Merck DC-Alufolien Kieselgel 60 F254) with spots
visualized by UV light. Analytical HPLC was performed on a Hitachi
instrument equipped with a chromolith performance RP-18e col-
umn (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 gradi-
ent of A–B over 15 min with a flow rate of 2 mL/min, with detec-
tion at 220 nm. HR FAB-mass spectra were measured on a JEOL
JMS-SX 102A instrument. NMR spectra were recorded on a JEOL
JNM A500 MHz spectrometer in CDCl3 solutions with reference to
TMS. All 1H shifts are given in parts per million (s = singlet;
d = doublet; t = triplet; m = multiplet). Assignments of proton reso-
nances were confirmed, when possible, by correlated spectroscopy.
Amino acid coupling reactions were performed by standard solu-
tion-phase chemistry using N,N-dicyclohexylcarbodiimide (DCC)
and 1-hydroxybenzotriazol (HOBt). Peptide cyclization was medi-
ated by 2-(1H-7-Azobenzotriazol-1-yl)-1, 1,3,3-tetramethyluroni-
um hexafluorophosphate (HATU) as a coupling reagent.
Scheme 2. Side chain modification of cyclic tetrapeptides.

M. A. Hoque et al. / Bioorg. Med. Chem. 22 (2014) 3850–3855
3853
Table 1
HDAC inhibitory activity and p21 promoter assay data
Compounds
HDAC1
IC50 (nM)
HDAC4
HDAC6/HDAC1a
p21 promoter assay EC1000 (nM)
HDAC6
TSA
23.0
34.0
65.0
3
20.0
FK228 + DTT
1.0
nt
0.625
625
15.7
1
>100
>100
>100
291.0 12
1 + DTT
2
2 + DTT
3
3 + DTT
4
4 + DTT
5
5 + DTT
6
6 + DTT
12.7 0.43
>100,000
8.1 1.0
>100,000
4.9 0.57
>100,000
16.0 0.59
>100,000
13.9 4.3
>100,000
27.0 5.5
10.3 0.85
>100,000
8.3 0.38
>100,000
4.2 0.49
>100,000
11.0 0.49
63,000
1301 77
>100,000
7300 580
>100,000
2319 190
>100,000
320.0 24
>100,000
83.0 4.5
>100,000
590.0 22
102
263
473
20
211.0 31
140.0
9
390.0 37
430.0 28
140.0 12
9.3 0.64
>100,000
37.0 1.0
6
22
a
Selectivity towards HDAC1 over HDAC6; nt: Not tested; The IC50 values were determined as the means SD of the concentrations calculated from at least three
independent dose–response curves.
4.1.1. Synthesis of cyclo(-
L
-Am7(S-)-
D
-Am7(S-)-
L
-Ala-
D
-Pro-) (1)
10 min interval with stirring. After completion of the cyclization
reaction, the reaction was quenched by adding a small amount of
acetic acid and DMF was evaporated and the residue was dissolved
in AcOEt. Then solution was washed with 10%) citric acid, 4%
NaHCO3 and brine, respectively. The AcOEt solution was dried over
anhydrous MgSO4 and concentrated to remain an foamy substance,
which was purified by silica gel chromatography using 1% metha-
To a cold solution of Z- -Ala-OH (2.847 g, 12.75 mmol) and H-D-
L
Pro-OtBu (1.82 g, 10.63 mmol) in DMF (22 mL), HOBtÁH2O (1.628 g,
10.63 mmol) and DCC (2.64 g, 12.75 mmol) were added. The mix-
ture was stirred overnight at room temperature. After completion
of the reaction, DMF was removed by evaporation. The residue
was dissolved in ethyl acetate (AcOEt) and filtered. Then it was
washed with 10% citric acid, 4% NaHCO3 and brine, respectively.
The AcOEt solution was dried over anhydrous MgSO4 and concen-
trated to remain an solid substance which was purified by silica gel
chromatography using 1% methanol in chloroform (v/v) to yield
nol in chloroform (v/v) to yield cyclic tetrapeptide, cyclo(-
L-Ab7-D-
Ab7- -Ala- -Pro-) (0.260 g, 62%, HPLC: rt 6.72 min). To a solution of
L
D
cyclic tetrapeptide (0.240 g, 0.41 mmol) in DMF (3 mL), KSAc
(0.137 g, 1.23 mmol) was added and was stirred for 7 h at room
temperature. DMF was evaporated and the residue was dissolved
in AcOEt. Then it was washed with 10% citric acid and brine,
respectively. The AcOEt solution was dried over anhydrous MgSO4
and concentrated to remain crude substance, which was purified
by silica gel chromatography using 1% methanol in chloroform
Z-L-Ala-D
-Pro-OtBu (3.388 g, 85%) as solid. To a solution of pro-
tected dipeptide (0.941 g, 2.50 mmol) in acetic acid (13 mL), Pd-C
(125 mg) was added and the mixture was stirred under hydrogen
atmosphere overnight. The reaction was monitored by TLC and
HPLC. After completion of the reaction, Pd-C was filtered off and
the acetic acid was evaporated. Then the residue was dissolved
in AcOEt and was washed with saturated Na2CO3 solution. The
AcOEt solution was dried over anhydrous Na2CO3 and concentrated
(v/v) to yield cyclo(-
L-Am7(Ac)-D-Am7(Ac)-L-Ala-D-Pro-) (0.200 g,
85%, HPLC: rt 6.50 min) as heavy oil.
The thioester compound (0.145 g, 0.25 mmol) was dissolved in
DMF (3 mL) and passed Ar to remove air. Then methylamine in
methanol (0.255 mL, 2.50 mmol) was added and stirred for 2 h at
room temperature. After completion of the reaction, methylamine
was evaporated and the residue was dissolved in DMF (50). Iodine
in ethanol (0.064 g, 0.25 mmol) was added dropwise and stirred for
30 min at room temperature. After evaporation of DMF, The oxi-
dized peptide was purified by LH-20 gel filtration with DMF as elu-
to remain dipeptide free amine, H-
To a cold solution of Boc- -Ab7-OH (0.694 g, 2.13 mmol) and free
L-Ala-D
-Pro-OtBu (0.517 g, 85%).
D
amine (0.517 g, 2.13 mmol) in DMF (5 mL), HOBtÁH2O (0.328 g,
2.13 mmol) and DCC (0.530 g, 2.56 mmol) were added and was
stirred for 5 h on ice-bath. The product Boc-D-Ab7-L-Ala-D-Pro-
OtBu was obtained in a similar manner as described earlier as
white foam (0.880 g, 75%). The protected tripeptide (0.880 g,
1.6 mmol) was dissolved in 4 M HCl/dioxane (4 mL) in ice bath
and was kept for 30 min at room temperature. After completion
of the reaction, HCl/dioxane was removed by evaporation and
the residue was dissolved in AcOEt. The solution was washed with
saturated Na2CO3 solution and dried over anhydrous Na2CO3. The
AcOEt solution was filtered and concentrated to remain free amine
ent. The cyclo(-L-Am7(S-)-D-Am7(S-)-L-Ala-D-Pro-) (1) (0.034 g, 0.0
7mmol, 28%, HPLC: rt 5.33 min) was obtained as solid. HR-FAB-
MS, [M+H]+ 485.2278 for C22H37N4O4S2 (calcd 485.2256). 1H
NMR (500 MHz, CDCl3): dH 7.12 (d, J = 5.5 Hz, 1H), 6.42 (d,
J = 10 Hz, 1H), 6.14 (d, J = 10 Hz, 1H), 4.89–4.98 (m, 1H), 4.71 (d,
J = 7.5 Hz, 1H), 4.41–4.50 (m, 2H), 3.91–4.00 (m, 1H), 3.49–3.56
(m, 1H), 2.58–2.81 (m, 4H), 2.26–2.43 (m, 2H), 2.18 (t, J = 10 Hz,
1H), 1.90–2.00 (m, 1H), 1.74–1.88 (m, 3H), 1.59–1.74 (m, 4H),
1.26–1.54 (m, 12H).
H-
D
-Ab7-
L
-Ala-
D
-Pro-OtBu (0.570 g, 1.27 mmol, 79%) which was
-Ab7-OH (0.412 g, 1.27 mmol) according to
condensed with Boc-
L
the method described earlier and the fully protected crude linear
tetrapeptide was purified by silica gel chromatography using 1%
methanol in chloroform (v/v) to yield Boc-
L
-Ab7-
D
-Ab7-
L
-Ala-
D
-
4.1.2. Synthesis of cyclo(-L-Am7(S-)-D-Am7(S-)-L-Val-D-Pro-) (2)
This compound was synthesized as solid (0.60 g, 1.17 mmol,
45%, HPLC: rt 7.03 min) in a similar manner as described in case
Pro-OtBu (0. 570g, 60%) as white foam. The protected tetrapeptide
(0.555 g, 0.73 mmol) was dissolved in TFA (3 mL) on ice bath and
kept for 3 h at room temperature. After evaporation of TFA, the res-
idue was solidified using ether and petroleum ether to yield TFA
salt of the linear tetrapeptide (0.500 g, 99%). The TFA salt of linear
of compound 1 using Z-L-Val-OH instead of Z-L-Ala-OH for the
synthesis of tripeptide. HR-FAB-MS, [M+H]+ 513.2585 for C24H41-
N4O4S2 (calcd 513.2569). 1H NMR (500 MHz, CDCl3): dH 7.18 (d,
J = 10 Hz, 1H), 6.38 (d, J = 10 Hz, 1H), 6.09 (d, J = 10 Hz, 1H), 4.72
(dd, J = 2, 2 Hz, 1H), 4.47–4.54 (m, 1H), 4.40–4.46 (m, 1H), 4.37
(t, J = 10 Hz, 1H), 3.87–3.94 (m, 1H), 3.47–3.54 (m, 1H), 2.70–2.80
tetrapeptide, TFAÁH-
L-Ab7-D-Ab7-L-Val-D-Pro-OH (0.500 g, 0.72
mmol), HATU (0.410 g, 1.08 mmol) and DIEA (0.313 mL, 1.8 mmol)
were added in separate five portions to DMF (180 mL) in every

3854
M. A. Hoque et al. / Bioorg. Med. Chem. 22 (2014) 3850–3855
(m, 2H), 2.60–2.68 (m, 2H), 2.28–2.41 (m, 2H), 2.08–2.21 (m, 2H),
1.92–2.01 (m, 1H), 1.79–1.89 (m, 2H), 1.70–1.78 (m, 2H), 1.59–1.70
(m, 3H), 1.28–1.53 (m, 9H), 0.98(d, J = 6.5 Hz, 3H), 0.88(d, J = 7 Hz,
3H).
protein A/G plus agarose beads (Santa Cruz Biotechnologies, Inc.).
After the cleared supernatant had been incubated for 1 h at 4 °C
with 4 lg of an anti-FLAG M2 antibody (Sigma–Aldrich Inc.) for
HDAC1, HDAC4 and HDAC6, the agarose beads were washed three
times with lysis buffer and once with histone deacetylase buffer
consisting of 20 mM Tris–HCl (pH 8.0), 150 mM NaCl, and 10%
glycerol. The bound proteins were released from the immune com-
4.1.3. Synthesis of cyclo(-L-Am7(S-)-D-Am7(S-)-L-Phe-D-Pro-) (3)
This compound was synthesized as solid (0.083 g, 0.15 mmol,
28%, HPLC: rt 7.22 min) in a similar manner as described in case
plex by incubation for 1 h at 4 °C with 40
(Sigma–Aldrich Inc.) in histone deacetylase buffer (200
supernatant was collected by centrifugation. For the enzyme assay,
10 L of the enzyme fraction was added to 1 L of fluorescent sub-
strate (2 mM Ac-KGLGK(Ac)-MCA) and 9 L of histone deacetylase
buffer, and the mixture was incubated at 37 °C for 30 min. The
reaction was stopped by the addition of 30 L of trypsin (20 mg
lg of the FLAG peptide
of compound 1 using Z-
L
-Phe-OH instead of Z-
L
-Ala-OH for the syn-
lL). The
thesis of tripeptide. HR-FAB-MS, [M+H]+ 561.2546 for C28H41N4O4-
S2 (calcd 561.2569). 1H NMR (500 MHz, CDCl3): dH 7.21 (m, 5H, Ph),
l
l
7.15 (d, J = 10 Hz, 1H,
L
-Am7NH), 6.56 (d, J = 10 Hz, 1H, PhNH), 6.10
l
a
a-
(d, J = 10 Hz, 1H, -Am7NH), 5.08 (m, 1H, CH Ph), 4.63 (m, 1H, CH
D
a
aD
L
-Am7), 4.46 (m, 2H, CH Pro, CH -Am7), 3.83 and 3.12 (two m,
l
2H, CH2dPro), 3.20 and 2.92 (two m, 2H, CH2bPh), 2.75 (m, 2H, CH2-
/mL) and incubated at 37 °C for 15 min. The released amino methyl
coumarin (AMC) was measured using a fluorescence plate reader.
The 50% inhibitory concentrations (IC50) were determined as the
means with SD calculated from at least three independent dose
response curves.
xL
xD
-Am7), 2.65 (m, 2H, CH2 -Am7), 2.32 and 1.71 (two m, 2H, CH2-
bpro), 2.21 and 1.71 (two m, 2H, CH2Pro), 2.21 and 1.47 (two m, 2H,
c
CHb2
L D-Am7), 1.71 and 1.37 (two m, 2H, CH2-
-Am7), 1.47 (m, 2H, CH2b
eL
-Am7), 1.71 (m, 6H), 1.47 and 1.37 (two m, 2H, CH2d
D
-Am7), 1.37
cL
(m, 2H, CH2 -Am7).
4.3. The p21 promoter assay
4.1.4. Synthesis of cyclo(-L-Am7(S-)-D-Aom7(S-)-L-Ala-D-Pro-) (4)
This compound was synthesized as solid (0.084 g, 0.17 mmol,
34%, HPLC: rt 4.92 min). in a similar manner as described in case
of compound 1 using Boc-D-Aob7-OH instead of Boc-D-Ab7-OH
The human wild-type p21 promoter luciferase fusion plasmid,
WWP-Luc, was a kind gift from Dr. B. Vogelstein. A luciferase
reporter plasmid (pGW-FL) was constructed by cloning the
2.4 kb genomic fragment containing the transcription start site
into HindIII and SmaI sites of the pGL3-Basic plasmid (Promega
Co., Madison, WI). Mv1Lu (mink lung epitherial cell line) cells
were transfected with the pGW-FL and a phagemid expressing
neomycin/kanamycin resistance gene (pBK-CMV, Stratagene, La
Jolla, CA) with the Lipofectamine reagent (Life Technology, Rock-
ville, MD USA). After the transfected cells had been selected by
for the synthesis of tetrapeptide. HR-FAB-MS, [M+H]+ 487.2045
for C21H35N4O5S2 (calcd 487.2049). 1H NMR (500 MHz, CDCl3): dH
7.12 (d, J = 10 Hz, 1H), 6.57 (d, J = 10 Hz, 1H), 6.45 (d, J = 10 Hz,
1H), 4.90–4.97 (m, 1H), 4.68–4.74(m, 2H), 4.39–4.46 (m, 1H),
3.91–3.97 (m, 1H), 3.50–3.68 (m, 5H), 2.82–2.94 (m, 2H), 2.74–
2.80 (m, 1H), 2.64–2.73 (m, 1H), 2.36–2.43 (m, 1H), 2.23–2.34
(m, 1H), 2.10–2.19 (m, 1H), 1.90–2.05 (m, 4H), 1.68–1.87 (m,
2H), 1.47–1.64(m, 3H), 1.31–1.45 (m, 5H).
400 lg/mL Geneticin (G418, Life Technology), colonies formed
were isolated. One of the clones was selected and named
MFLL-9. MFLL-9 expressed a low level of luciferase, of which
activity was enhanced by TSA in a dose-dependent manner.
MFLL-9 cells (1 Â 105) cultured in a 96-well multi-well plate for
6 h were incubated for 18 h in the medium containing various
concentrations of drugs. The luciferase activity of each cell lysate
was measured with a LucLite luciferase Reporter Gene Assay Kit
(Packard Instrument Co., Meriden, CT) and recorded with a Lumi-
nescencer-JNR luminometer (ATTO, Tokyo, Japan). Data were nor-
malized to the protein concentration in cell lysates. Concent
rations at which a drug induces the luciferase activity 10-fold
higher than the basal level are presented as the 1000% effective
concentration (EC1000).
4.1.5. Synthesis of cyclo(-L-Am7(S-)-L-Am7(S-)-L-Val-D-Pro-) (5)
This compound was synthesized as solid (0.04 g, 0.08 mmol,
31%, HPLC: rt 6.58 min) in a similar manner as described in case
of compound 2 using Boc-L-Ab7-OH instead of Boc-D-Ab7-OH for
the synthesis of tripeptide. HR-FAB-MS, [M+H]+ 513.2558 for C24-
H41N4O4S2 (calcd 513.2569).
4.1.6. Synthesis of cyclo(-
This compound was synthesized as solid (0.017 g, 0.03 mmol,
23%, HPLC: rt 7.90 min) from H-
-Tic-OtBu by stepwise elongating
to tetrapeptide, Boc- -Val- -Ab7- -Ab7-
-Tic-OtBu followed by
L-Am7(S-)-D-Tic-L-Val-D-Am7(S-)-) (6)
D
L
D
L
D
cyclization and side chain modification by same method as
described in above. HR-FAB-MS, [M+H]+ 575.2608 for C29H43N4O4-
S2 (calcd 575.2726). 1H NMR (500 MHz, CDCl3): dH 7.14–7.25
(m, 1H), 6.75 (d, J = 10 Hz, 1H), 6.53 (d, J = 10 Hz, 1H), 6.20 (d,
J = 10 Hz, 1H), 5.16–5.25 (m, 2H), 4.93–4.96 (m, 1H), 4.49–4.65
(m, 2H), 3.85 (t, J = 10, Hz, 1H), 3.39 (dd, J = 6, 6 Hz, 1H), 2.97
(dd, J = 7.5, 7.5 Hz, 1H), 2.63–2.84 (m, 4H), 2.10–2.21 (m, 2H),
2.01–2.10 (m, 1H), 1.59–1.78(m, 6H), 1.39–1.59 (m, 11H), 0.95 (d,
J = 7 Hz, 3H), 0.89 (d, J = 6 Hz, 3H).
Acknowledgments
This study was supported by Kitakyushu Foundation for the
Advancement of Industry, Science and Technology (FAIS).
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