Molecular design of histone deacetylase inhibitors by aromatic ring shifting in chlamydocin framework

Article abstract of DOI:10.1016/j.bmc.2007.08.041

Chlamydocin, a cyclic tetrapeptide containing aminoisobutyric acid (Aib), l-phenylalanine (l-Phe), d-proline (d-Pro), and a unique amino acid l-2-amino-8-oxo-9,10-epoxydecanoic acid, inhibits the histone deacetylases (HDACs), a class of enzymes, which pla

Full text of DOI:10.1016/j.bmc.2007.08.041

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7830–7839
Molecular design of histone deacetylase inhibitors by aromatic
ring shifting in chlamydocin framework
Gururaj M. Shivashimpi,a Satoshi Amagai,a Tamaki Kato,a Norikazu Nishino,a,*
Satoko Maeda,b Tomonori G. Nishinob and Minoru Yoshidab
aGraduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu 808-0196, Japan
bRIKEN, Saitama 351-0198, Japan
Received 1 July 2007; revised 20 August 2007; accepted 23 August 2007
Available online 26 August 2007
Abstract—Chlamydocin, a cyclic tetrapeptide containing aminoisobutyric acid (Aib), L-phenylalanine (L-Phe), D-proline (D-Pro),
and a unique amino acid L-2-amino-8-oxo-9,10-epoxydecanoic acid, inhibits the histone deacetylases (HDACs), a class of enzymes,
which play important roles in regulation of gene expression. Sulfur containing amino acids can also inhibit potently, so zinc ligand,
such as sulfhydryl group connected with a linker to the so-called capping group, corresponding to cyclic tetrapeptide framework in
case of chlamydocin is supposed to interact with the surface of HDAC molecule. Various changes in amino acid residues in
chlamydocin may afford specific inhibitors toward HDAC paralogs. To find out specific inhibitors, we focused on benzene ring
of L-Phe in chlamydocin framework to shift to various parts of cyclic tetrapeptide. We prepared and introduced several aromatic
amino acids into the cyclic tetrapeptides. By evaluating inhibitory activity of these macrocyclic peptides against HDACs, we could
find potent inhibitors by shifting the aromatic ring to the Aib site.
ꢀ 2007 Published by Elsevier Ltd.
1. Introduction
of the basal transcription machinery or histone acetyl-
transferases.5 These activator complexes, containing
HAT activity, have been shown to induce activation of
transcription. Whereas deacetylation is associated with
condensed chromatin structure resulting in the repres-
sion of gene transcription. Therefore, alteration of equi-
librium in histone acetylation leads to transcriptional
deregulation. This deviation in equilibrium of histone
acetylation either due to HAT mutation or abnormal
recruitment of HDACs has been linked to a number
of malignant diseases.6–9 Inhibition of HDAC enzyme
activity proved to reverse and induce re-expression of
differentiation inducing genes. Therefore, the search
for potent inhibitors possessing adequate selectivity
among the HDAC paralogs demands a considerable
effort to develop therapeutics for the treatment of
epigenetic diseases.
Common cancer therapy techniques, like chemotherapy,
take the advantage of apoptosis to eliminate malignant
cells within tumors. Reversible acetylation and deacety-
lation of the e-amino groups of lysine residues on core
histone tails by histone acetyltransferase (HAT) and his-
tone deacetylase (HDAC) enzymes play an important
role in the epigenetic regulation of gene expression by
altering the chromatin architecture and controlling the
accessibility of transcriptional regulators to DNA and
histones.1–4Modification of the level of histone acetyla-
tion and its consequences have received enormous inter-
est, parallely the growing evidence supports their
importance for basic cellular functions such as DNA
replication, transcription, differentiation, and apoptosis.
Acetylation of lysine residues in N-terminal tails of core
histones reduces the interaction with DNA resulting in
more open chromatin structure. In addition, acetylated
histone tails are specifically recognized and bound by
bromodomain containing proteins such as components
Several structurally unrelated natural and synthetic
compounds have been reported so far as HDAC inhib-
itors (Fig. 1). Among them trichostatin A (TSA, 1),10
depsipeptide FK22811–13 and the cyclic tetrapeptide
family including trapoxin (TPX),14 chlamydocin,15
TAN-1746,16 FR-235222,17 9,10-desepoxy-9-hydroxy-
chlamydocin,18 HC-toxins,19–23 Cyl-1, Cyl-2,24–26 WF-
3161,27 apicidin,28–31 and FR-22549732 are examples of
Keywords: Histone deacetylase; Chlamydocin; Cyclic tetrapeptide;
Inhibitor.
*
Corresponding author. Tel./fax: +81 93 695 6061; e-mail: nishino@
life.kyutech.ac.jp
0968-0896/$ - see front matter ꢀ 2007 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2007.08.041

G. M. Shivashimpi et al. / Bioorg. Med. Chem. 15 (2007) 7830–7839
7831
Figure 1. Structure of natural and synthetic HDAC inhibitors.
naturally occurring HDAC inhibitors. Inhibitors like
suberoylanilide hydroxamic acid (SAHA),33 straight
chain TSA and SAHA analogs,34–36 scriptaid37, and
the benzamide MS-27538,39 have also been designed
and synthesized.
chlamydocin macrocycle by targeting the aromatic ring
of L-Phe residue to shift in different positions. We
shifted the aromatic ring of L-Phe, keeping L-alanine
(L-Ala) at L-Phe position in chlamydocin, to the Aib po-
sition by incorporating 2-aminoindan-2-carboxylic acid
(A2in) (3) and DL-1-aminoindan-1-carboxylic acid
(DL-A1in) (4 and 5) to make rigid aromatic ring. We
then shifted to one of the methyl groups of Aib to make
DL-2-methyl phenylalanine (DL-2MePhe) (6 and 7). Fur-
ther, to shift the aromatic ring to the imino acid posi-
tion, we placed commercially available D-1,2,3,
4-tetrahydro-3-isoquinoline carboxylic acid (D-Tic) (8).
To shift the aromatic ring from cyclic tetrapeptide, we
varied the distance of aromatic ring of L-Phe from cyclic
framework by incorporating several aromatic amino
acids like L-phenylglycine (L-Phg) (9) with no methylene
group from a-carbon, L-2-amino-4-phenylbutanoic acid
(L-Ph4) (10) and L-2-amino-5-phenylpentanoic acid
(L-Ph5) (11) with two and three methylene groups,
respectively, at L-Phe position. To make more flexible
aromatic ring, we incorporated O-benzyl-L-serine
(L-Ser(Bzl)) (12). As reference compounds, we designed
cyclic tetrapeptides containing no aromatic ring by
incorporating L-serine (L-Ser) (13) and L-Ala (14) (not
shown in Fig. 2) at L-Phe position. We designed and
synthesized different cyclic tetrapeptides by introducing
different unusual amino acids. We herein describe the
account on the synthesis of chlamydocin analogs and a
description of the interesting biological results including
circular dichroism studies.
In 1999 Finnin et al. defined that the aromatic dimethyl
aminophenyl part in TSA plays the role of cap group.40
They also proposed that, natural compounds trapoxin B
and HC-toxins having a cyclic tetrapeptide structure
with hydrophobic groups, such as L-Phe, serve as cap
group. Larger size of cap group may give extensive con-
tacts at the rim of enzyme pocket. There are evidences
which support that the cyclic tetrapeptide framework
has a significant structural role of specific hydrophobic
interaction with the surface of HDAC enzymes.41 Also
in a recent report concerning to the SAR of apicidins29
it is shown, the tryptophan residue in the cyclic tetrapep-
tide is the key constituent for HDACs inhibitory activ-
ity. The cyclic tetrapeptide chlamydocin, originally
isolated from fungus Diheterospora chlamydosphoria is
containing Aib, L-Phe, D-Pro, and L-Aoe, which reacts
and inhibits histone deacetylases.42 Recently we have re-
ported chlamydocin hydroxamic acid analogs,43 and sul-
fur containing chlamydocin analog (2),44 in which the
thiol function is protected as disulfide hybrid (Fig. 1).
Hydrophobicity in capping group of HDAC inhibitors
is crucial in their activity. With the exception of HC-tox-
ins, chlamydocin and other natural or synthetic cyclic
tetrapeptide based inhibitors invariably contain an aro-
matic ring in their macrocyclic cap group. Various
changes of amino acid residues in chlamydocin structure
may afford specific inhibitors toward HDAC paralogs.
In this work, to find out specific inhibitors, we focused
on the benzene ring of L-Phe in chlamydocin frame-
work. In a recent report45 on docking study of TSA
and trapoxin B into the homology model of HDAC1,
it is revealed that aryl group of TSA corresponds to
the cyclic tetrapeptide framework, not to the benzene
ring of L-Phe.
2. Results and discussion
2.1. Chemistry
Our aim was to synthesize potent inhibitors of HDACs
by shifting the benzene ring of L-Phe to Aib position and
proline position of the cyclic tetrapeptide and also var-
ied the distance of benzene ring of L-Phe from cyclic
framework. Synthesis of cyclic tetrapeptide was carried
out according to the general Scheme 1 by the conven-
tional solution phase method, starting from the Z-D-imi-
no acid tert-butyl ester. After the removal of
Z-protection by catalytic hydrogenation, free amine
The cyclic tetrapeptides designed in the present study are
given in Figure 2. We here tried to figure out the role of
aromatic ring and explore its appropriate position in the

7832
G. M. Shivashimpi et al. / Bioorg. Med. Chem. 15 (2007) 7830–7839
Figure 2. Structure of cyclic tetrapeptides containing different aromatic amino acids.
and L-Ala at AA3 position as shown in Scheme 1, to
know the effect of aromatic ring at Aib position. The
cyclic tetrapeptides were synthesized according to the
general method as described above to give the following
compounds, cyclo(-L-Am7(S2Py)-A2in-L-Ala-D-Pro-)
(3), cyclo(-L-Am7(S2Py)-D-A1in-L-Ala-D-Pro-) (4),
cyclo(-L-Am7(S2Py)-L-A1in-L-Ala-D-Pro-) (5), cyclo(-L-
Am7(S2Py)-D-2MePhe-L-Ala-D-Pro-) (6), and cyclo(-L-
Am7(S2Py)-L-2MePhe-L-Ala-D-Pro-) (7). Compounds
4/5 and 6/7 were obtained as diastereomeric mixtures,
which were successfully separated by HPLC. Detailed
HPLC conditions are given in the experimental section.
Next we changed the D-Pro residue of chlamydocin by
D-Tic, to know the effect of aromatic ring at imino acid
position. Using the general method as said above,
we synthesized the following compound cyclo(-L-
Am7(S2Py)-Aib-L-Ala-D-Tic-) (8).
Scheme 1. Synthesis of cyclic tetrapeptides.
was extracted and used for condensation with another
Z-amino acid using DCC/HOBt. Boc-L-2-amino-7-bromo-
heptanoic acid (Ab7)46 was condensed to appropriate tri-
peptide free amine to prepare the linear tetrapeptide.
After the removal of both side protections by treating
with trifluoroacetic acid, cyclization reaction was carried
out with the aid of HATU in DMF (2 mM) with mini-
mum amount of DIEA (2.5 equiv). The yield of cyclic
tetrapeptides was 50–60% after purification by silica
gel chromatography. The cyclic tetrapeptide containing
Ab7 was reacted with potassium thioacetate (Wako,
Japan) to convert the bromide into thioacetate ester.
Further treatment with methylamine in the presence of
2,20-dipyridyl disulfide gave the desired cyclic
tetrapeptides.
To explore the effect of variation in distance between
cyclic framework and benzene ring, we replaced the L-
Phe of chlamydocin at AA3 position, by other unnatural
amino acids, like L-Phg, L-Ph4 amd L-Ph5 and with Aib
at AA2 position. The cyclic tetrapeptides were synthe-
sized according to general method as described above
to obtain following compounds, cyclo(-L-Am7(S2Py)-
Aib-L-Phg-D-Pro-) (9), cyclo(-L-Am7(S2Py)-Aib-L-Ph4-
D-Pro-), (10) and cyclo(-L-Am7(S2Py)-Aib-L-Ph5-D-
Pro-) (11).
Further, we synthesized the cyclic tetrapeptides cyclo(-L-
Am7(S2Py)-Aib-L-Ser(Bzl)-D-Pro-) (12) and cyclo(-L-
Am7(S2Py)-Aib-L-Ser-D-Pro-) (13) by introducing
L-Ser(Bzl) and L-Ser to know the effect of oxygen atom
Initially, we introduced several aromatic Aib analogs
such as A2in, DL-A1in, and DL-2MePhe at AA2 position

G. M. Shivashimpi et al. / Bioorg. Med. Chem. 15 (2007) 7830–7839
7833
in the spacer. Finally we also have synthesized the cyclic
tetrapeptide without having aromatic ring, to obtain cy-
clo(-L-Am7(S2Py)-L-Aib-L-Ala-D-Pro-) (14) as reference
compound.
and HDAC6 enzymes prepared by 293T cells.13 These
compounds containing S–S disulfide were initially as-
sayed for enzyme inhibition; they exhibited less activity
in cell free condition. But in the p21 promoter assay
most of the compounds showed good activity. Maybe
the disulfide bond is reduced in cell medium to give a
sulfhydryl group, to regulate the activity of promoter
gene by inhibiting HDAC enzymes. It is already re-
ported13 that, FK-228 containing an intramolecular
disulfide bridge is a potent HDAC inhibitor in cellular
condition and its activity was found less in the absence
of dithioltritol (DTT) in cell free conditions. The DTT
is known to reduce the disulfide bonds to sulfhydryl
group, which strongly ligates the Zn+2 ion of HDAC en-
zyme. Considering this point, cell free enzyme inhibition
assays were carried out, in presence of 0.1 mM of DTT.
Detailed experimental procedure for the preparation
and assay methods were performed according to the re-
ported methods47 which are reproduced in the experi-
mental section. In addition, to know the inhibitory
activity of these compounds in cell based condition,
we also carried out p21 promoter assay according to
the literature.47The results of HDAC inhibitory activity
and the p21 promoter assay of the compounds are
shown in Table 1.
All the synthesized compounds were characterized by
1H NMR and high resolution FAB-MS. The purity
of compounds was determined by HPLC analysis and
all the synthesized cyclic tetrapeptides showed purity
above 97%. The configuration of diastereomers 4/5 con-
taining D-A1 in and L-A1in,43 and 6/7 containing
1
D-2MePhe and L-2MePhe was assigned by H, COSY,
NOESY spectra in CDCl3 and also high resolution
FAB-MS. Difference in chemical shift positions of dia-
stereomers 4 and 5, containing D-A1in and L-A1in was
shown in Figure 3a and b, respectively. In Figure 3a,
the b-proton of D-A1in of compound 4 is appearing
at around 2 ppm as multiplets, and the c-proton is
appearing around 3 ppm. In case of compound 5
(Fig. 3b), these two groups of protons are appearing
at the same position at around 3 ppm. In the NOESY
experiments, we observed NOE of 2.72 and
˚
2.22 Abetween NH- and the b-protons, in compound
4, which corresponds to its D-configuration. We could
not observe any NOE in case of compound 5, which
indicates, it is of L-configuration.
These compounds inhibited HDACs at nanomolar
range. For comparison the inhibitory activity48 of tri-
chostatin A (1) is also shown. Compound 2,44 which
has similar cyclic framework as chlamydocin showing
good activity, is taken as standard for this study. In
compound 3, when Aib is replaced by A2in there was al-
most comparable in vitro activity with 2. Among the
diastereomers 4 and 5, compound 4 with D-A1in found
10 times more active in vitro and in vivo than 5 with
L-A1in. On the contrary among the diastereomers 6
and 7, compound 7 with L-2MePhe was more active
than compound 6 with D-2MePhe. These results indicate
that, in compounds 4 and 7 there is a favorable interac-
tion of benzene ring and the residues at the surface bind-
ing region of HDACs. Naturally occurring cyclic
tetrapeptides rarely contain aromatic iminoacids. There-
fore we replaced the D-Pro residue with D-Tic residue in
compound 8, to study the effect of benzene ring in at
D-Pro position. It showed comparable activity with
compound 2.
2.2. Enzyme inhibition and biological activity
The synthesized cyclic tetrapeptides were assayed for
HDAC inhibitory activity using HDAC1, HDAC4,
Compound 9 with no methylene group on the a-car-
bon, showed less activity compared to 10 and 11 with
two and three methylene groups, respectively, indicat-
ing that increasing the length between cyclic frame-
work and benzene ring will give flexibility and
favorable interaction to the surface binding region of
HDACs. Compound 12 was found more active both
in vitro and in vivo as compared to 13. It further sup-
ports the fact that presence of an aromatic ring has a
positive contribution to the activity of an inhibitor.
The cyclic tetrapeptides 13 and 14 which lack an aro-
matic ring are still retaining the threshold amount of
hydrophobicity to be active. The selectivity of
HDAC1 over HDAC4 is actually insufficient as shown
in Table 1, probably due to the similarity in surface
binding regions of both the enzymes.
Figure 3. Selected region of 1H NMR spectrum of compound 4 (a)
compound 5 (b).

7834
G. M. Shivashimpi et al. / Bioorg. Med. Chem. 15 (2007) 7830–7839
Table 1. HDAC inhibitory activity and p21 promoter assay data
Compound
No
IC50 (nM)
HDAC1/HDAC4a p21 promoter assay EC1000 (nM)
HDAC1 HDAC4 HDAC6
Trichostatin A48
1
2
1.9
3.9
2.0
2.7
2.0
1.8
1.6
2.4
2.8
40
8.8
12
1.0
2.1
1.2
1.1
1.4
2.4
1.6
1.1
1.5
1.9
1.9
2.0
1.6
1.8
190
45
cyclo(-L-Am7(S2Py)-Aib-L-Phe-D-Pro-)44
cyclo(-L-Am7(S2Py)-A2in-L-Ala-D-Pro-)
cyclo(-L-Am7(S2Py)-D-A1in-L-Ala-D-Pro-)
cyclo(-L-Am7(S2Py)-L-A1in-L-Ala-D-Pro-)
cyclo(-L-Am7(S2Py)-D-2MePhe-L-Ala-D-Pro-)
cyclo(-L-Am7(S2Py)-L-2MePhe-L-Ala-D-Pro-)
cyclo(-L-Am7(S2Py)-Aib-L-Ala-D-Tic-)
cyclo(-L-Am7(S2Py)-Aib-L-Phg-D-Pro-)
cyclo(-L-Am7(S2Py)-Aib-L-Ph4-D-Pro-)
cyclo(-L-Am7(S2Py)-Aib-L-Ph5-D-Pro-)
cyclo(-L-Am7(S2Py)-Aib-L-Ser(Bzl)-D-Pro-)
cyclo(-L-Am7(S2Py)-Aib-L-Ser-D-Pro-)
cyclo(-L-Am7(S2Py)-Aib-L-Ala-D-Pro-)
3
140
55
4
5
36
170
3.7
4.7
88
25
70
2.2
4.2
56
33
71
56
29
86
36
43
24
26
43
2000
25600
550
200
7400
320
110
72
6
7
8
9
10
11
12
13
14
6.2
5.3
3.2
6.4
9.4
3.2
2.7
1.6
4.0
5.2
580
750
a Selectivity of the enzymes.
D-imino acid positions. We also varied the spacer length
at L-Phe position. Thus, 11 cyclic tetrapeptides contain-
ing various aromatic amino acids have been successfully
synthesized as the SS-hybrids. Upon p21 promoter assay
we could find out the cyclic peptide 4 with aromatic ring
rigidified tightly at Aib position as potent inhibitor as
mother compound 2. In addition, the data showed that
the aromatic ring at L-Phe position is allowed flexible.
Conformational changes were observed in the diastereo-
mers due to the aromatic ring shift to the Aib site. The
conformations in which the aromatic group and the zinc
ligating spacer are directed to the same side are found to
be more active. This fact leads us to conclude that there
is some kind of hydrophobic interaction of aromatic
ring with the surface binding region of HDAC enzymes.
The cyclic tetrapeptides 13 and 14 which are devoid of
aromatic rings are still retaining their minimal hydro-
phobicity to interact with the surface binding region of
HDACs, thus inhibiting potently. Among the synthe-
sized cyclic peptides, 4 and 12 were found promising,
although they showed insignificant selectivity between
HDAC1 and HDAC4.
Figure 4. CD spectra of cyclic tetrapeptides
7 in methanol.
2, 4,
5,
6,
2.3. Conformational change by aromatic ring shifting
The interesting HDAC activity data of diastereomers 4/
5 and 6/7 forced us to study their conformations. We
have carried out circular dichroism (CD) spectral studies
of these diastereomers (Fig. 4). The CD spectrum of ref-
erence compound 2is similar to chlamydocin49 up to
260 nm. Compounds 4 and 7 showed negative ellipticity
at 220 nm regions, whereas compounds 5 and 6 showed
positive ellipticity. This explains the fact that, com-
pounds 4, 7, and 5, 6 have similar conformations. Fur-
ther 4 and 7 with negative ellipticity are better
inhibitors of HDAC paralogs. We are also carrying
out conformational analysis of these diastereomers by
NMR calculation methods. These results will be pub-
lished elsewhere.
4. Experimental
4.1. General
Unless otherwise noted, all solvents and reagents were of
reagent grade and used without purification. Flash
chromatography was performed using silica gel 60
(230–400 mesh) eluting with solvents as indicated. All
compounds were routinely checked by thin layer
chromatography (TLC) or high performance liquid
chromatography (HPLC). TLC was performed on alu-
minum-backed silica gel plates (Merck DC-Alufolien
Kieselgel 60 F254) with spots visualized by UV light or
charring. Analytical HPLC was performed on a Hitachi
instrument equipped with a chromolith performance
RP-18e column (4.6·50 mm, Merck). The mobile phases
used were A: H2O with 10% CH3CN and 0.1% TFA, B:
CH3CN with 0.1% TFA using a solvent gradient of A
to B over 15 min with a flow rate of 2 ml/min, with
3. Conclusion
In order to develop HDAC inhibitors with good selec-
tivity and potency, we attempted to shift the aromatic
ring of L-Phe in chlamydocin scaffold to Aib and

G. M. Shivashimpi et al. / Bioorg. Med. Chem. 15 (2007) 7830–7839
7835
detection at 220 nm. FAB-mass spectra and high resolu-
tion mass spectra (HR-MS) were measured on a JEOL
JMS-SX 102A instrument. NMR spectra were recorded
on a JEOL JNM A500 MHz spectrometer. Unless
otherwise stated, all NMR spectra were measured in
was solidified by trituration with ether to yield
TFAÆH-L-Ab7-A2in-L-Ala-D-Pro-OH (640 mg, 96%).
HPLC, rt 5.53 min. To a volume of DMF (100 mL)
TFAÆH-L-Ab7-A2in-L-Ala-D-Pro-OH (640 mg, 0.96 mmol),
HATU (548 mg, 1.44 mmol), and DIEA (0.42 mL,
2.4 mmol) were added in five aliquots with 30 min time
interval, while the solution was stirred vigorously. After
the final addition the reaction mixture was allowed to
stir for an additional hour. Completion of the cycliza-
tion reaction was monitored by HPLC and then DMF
was evaporated under reduced pressure. The crude cyc-
lic tetrapeptide was dissolved in ethyl acetate and was
successively washed with 10% citric acid, 4% sodium
bicarbonate, and brine. Finally the ethyl acetate solution
was dried over anhydrous MgSO4 and filtered. After
evaporation of ethyl acetate, the residue was purified
by silica gel chromatography using a mixture of chloro-
form and methanol (99:1 v/v) to yield cyclo(-L-Ab7-
A2in-L-Ala-D-Pro-) (359 mg, 69%) as a white foam
after drying in vacuo. HPLC, rt 7.41 min., HR-FAB
MS, [M+H]+ 533.1745 for C25H34N4O479Br (calcd
533.1685) and 535.1732 for C25H34N4O481Br (calcd
535.1664). The cyclic tetrapeptide cyclo(-L-Ab7-A2in-L-
Ala-D-Pro-) (240 mg, 0.45 mmol) was dissolved in
DMF (2 mL), potassium thioacetate (77 mg, 0.67 mmol)
was added and stirred for 4–5 h. The solvent was evap-
orated and the residue was dissolved in ethyl acetate and
washed with 10% citric acid and brine. The ethyl acetate
solution was dried over anhydrous MgSO4 and evapo-
rated to thioester cyclo(-L-Am7(acetyl)-A2in-L-Ala-D-
Pro-) (212 mg, 89%). HPLC, rt 7.20 min. To a solution
of cyclo(-L-Am7(acetyl)-A2in-L-Ala-D-Pro-) (200 mg,
0.4 mmol) in DMF (2 mL), under argon atmosphere,
2,20-dipyridyldisulfide (176 mg, 0.8 mmol) and 40%
solution of CH3NH2/MeOH (0.22 mL, 2 mmol) were
added and kept stirring for 5 h at room temperature.
After evaporation of DMF residue was dissolved in
ethyl acetate and washed with 10% citric acid, 4%
sodium bicarbonate, brine, and finally dried over MgSO4.
After evaporation of ethyl acetate product was purified
by silica gel chromatography using mixture of chloro-
form and methanol (99:1 v/v) to yield cyclo(-L-Am7
(S2Py)-A2in-L-Ala-D-Pro-) as white foam (145 mg,
61.2%), HPLC, rt 7.78 min, HR-FAB MS, [M+H]+
596.2401 for C30H38O4N5S2 (calcd 596.2287).1H NMR
(500 MHz, CDCl3): dH 1.23 (m, 1H), 1.31 (m, 1H),
1.39 (m, 2H), 1.41 (d, J = 7 Hz, 3H), 1.62 (m, 3H),
1.67 (m, 1H), 1.84 (m, 1H), 1.92 (m, 1H), 2.26 (m,
1H), 2.4 (m, 1H), 2.74 (t, 2H), 2.88 (s, 2H), 2.96 (s,
2H), 3.54 (ddd, J = 10, 10, 8.5 Hz, 1H), 4.02 (m, 1H),
4.19 (ddd, J = 10.0, 9.25, 8.5 Hz, 1H), 4.75 (dd,
J = 2.0, 2.0 Hz, 1H), 6.39 (s, 1H), 7.07 (dd, J = 13.5,
10.0 Hz, 1H), 7.13 (d, J = 10.5 Hz, 1H), 7.17 (m, 2H),
7.21 (m, 2H), 7.34 (d, J = 8 Hz, 1H), 7.52 (d,
J = 10 Hz, 1H).
1
CDCl3 solutions with reference to TMS. All H shifts
are given in parts per millions (s = singlet; d = doublet;
t = triplet; m = multiplet). Assignments of proton
resonances were confirmed, when possible, by correlated
spectroscopy. Amino acids were coupled using standard
solution-phase chemistry with dicyclohexyl-carbodiim-
ide (DCC), 2-(1H-benzatriazol-1-yl)-1,1,3,3-tetrameth-
yluronium hexafluorophosphate (HBTU), or O-(7-
azabenzotriazoyl-1-yl)-1,1,3,3-tetramethyluroniumhexa-
fluorophosphate (HATU).
4.1.1. Synthesis of cyclo (-L-Am7(S2Py)-A2in-L-Ala-D-
Pro-) (3). To a cooled solution of H-D-Pro-OtBu (3.5 g,
20 mmol), Z-L-Ala-OH (4.9 g, 22 mmol) and HOBtÆH2O
(3 g, 20 mmol) in dimethylformamide (DMF) (120 mL)
was added DCC (4.9 g, 24 mmol). The mixture was stir-
red at room temperature for 8 h. After completion of the
reaction, DMF was evaporated and the residue was dis-
solved in ethyl acetate and successively washed with 10%
citric acid, 4% sodium bicarbonate, and brine. The ethyl
acetate solution was dried over anhydrous MgSO4 and
concentrated to remain an oily substance, which was
purified by silica gel chromatography using a mixture
of chloroform and methanol (99:1) to yield Z-L-Ala-D-
Pro-OtBu (6.5 g, 90%) as a colorless oil. The protected
dipeptide (1.09 g, 3 mmol) was dissolved in acetic acid
(10 mL). Pd–C (150 mg) was added and the mixture
was stirred under hydrogen atmosphere for 10 h. The
reaction was monitored by TLC and HPLC. After com-
pletion of the reaction Pd–C was filtered off and the ace-
tic acid was evaporated. The residue was dissolved in
ethyl acetate and the organic phase was washed with
2M Na2CO3 solution and dried over anhydrous
Na2CO3. Evaporation of ethyl acetate gave H-L-Ala-D-
Pro-OtBu (685 mg, 95%). To a cooled solution of H-L-
Ala-D-Pro-OtBu (685 mg, 2.8 mmol) and Z-A2in-OH
(1.04 g, 3.6 mmol) in DMF (6 mL) were successively
added HBTU (1.27 g, 3.6 mmol), HOBtÆH2O (429 mg,
2.8 mmol), and DIEA (0.75 ml, 4.2 mmol). The product
Z-A2in-L-Ala-D-Pro-OtBu (785 mg, 75%) was obtained
in the same manner as described earlier as white foam.
The tripeptide Z-A2in-L-Ala-D-Pro-OtBu (1.1 g,
2.1 mmol) was subjected to catalytic hydrogenation with
Pd–C (100 mg) in acetic acid (8 mL). The free amine was
taken into ethyl acetate (25 mL) the aid of 2 M Na2CO3
solution (10 mL). After dried over anhydrous Na2CO3
ethyl acetate solution was evaporated to remain
H-A2in-L-Ala-D-Pro-OtBu (785 mg, 90%). The N-termi-
nal free tripeptide H-A2in-L-Ala-D-Pro-OtBu (785 mg,
1.95 mmol) was coupled with Boc-L-Ab7-OH (758 mg,
2.3 mmol) according to the method described earlier
and the fully protected crude linear tetrapeptide was
purified by silica gel chromatography using a mixture
of chloroform and methanol (99:1 v/v) to yield Boc-L-
Ab7-A2in-L-Ala-D-Pro-OtBu (900 mg, 68%) as a white
foam. Boc-L-Ab7-A2in-L-Ala-D-Pro-OtBu (745 mg,
1 mmol) was dissolved in TFA (3 mL) and left standing
on ice for 3 h. After evaporation of TFA, the residue
4.1.2. Synthesis of cyclo(-L-Am7(S2Py)-D-A1in-L-Ala-D-
Pro-) (4) and cyclo(-L-Am7(S2Py)-L-A1in-L-Ala-D-Pro-)
(5). These compounds were synthesized using DL-A1in
instead of Aib according the method described earlier,
to yield cyclo(-L-Am7(S2Py)-DL-A1in-L-Ala-D-Pro-).
The two distereoisomers were separated by preparative
HPLC [Column: YMC pack, ODS-A (250 · 10 mm),

7836
G. M. Shivashimpi et al. / Bioorg. Med. Chem. 15 (2007) 7830–7839
condition: Isocratic condition from solvent A to B over
20 min with 220 nm detection, Sol. A; 100% H2O with
0.1% TFA and Sol. B; 100% acetonitrile with 0.1%
TFA] to yield cyclo(-L-Am7(S2Py)-D-A1in-L-Ala-D-
Pro-) (60 mg, 23%) HPLC, rt 5.9 min. HR-FAB MS,
[M+H]+ 596.2352 for C30H38O4N5S2 (calcd 596.2287).
1H NMR (500 MHz, CDCl3): dH 1.20 (m, 1H), 1.30
(m, 1H), 1.33 (m, 2H), 1.43 (d, J = 7 Hz, 3H), 1.62 (m,
4H), 1.87 (m, 1H), 1.97 (m, 1H), 2.05 (m, 2H), 2.29
(m, 1H), 2.45 (m, 1H), 2.74 (t, 2H), 2.93 (m, 1H), 2.93
(m, 1H), 3.04 (m, 1H), 3.59 (ddd, J = 10.0, 10.25,
8 Hz, 1H), 3.92 (m, 1H), 4.25 (ddd, J = 9.52, 9.52,
7.5 Hz, 1H), 4.82 (dd, J = 1.5, 1.5 Hz, 1H), 5.18 (m,
1H), 6.46 (s, 1H), 7.09 (d, J = 10.0 Hz, 1H), 7.22 (m,
1H), 7.25 (m, 4H), 7.64 (d, J = 10.0 Hz, 1H), 7.82 (m,
1H), 7.87 (d, J = 7.5 Hz, 1H), 8.51 (d, J = 4.5 Hz, 1H),
and cyclo(-L-Am7(S2Py)-L-A1in-L-Ala-D-Pro-) (70 mg,
33%). HPLC, rt 6.38 min. HR-FAB MS, [M+H]+
596.2367 for C30H38O4N5S2 (calcd 596.2287). 1H
NMR (500 MHz, CDCl3): dH 1.26 (m, 2H), 1.38 (d,
J = 7 Hz, 3H), 1.62 (m, 1H), 1.67 (m, 2H), 1.80 (m,
1H), 1.92 (m, 2H), 2.33 (m, 1H), 2.45 (m, 1H), 2.76 (t,
2H), 2.90 (m, 1H), 2.95 (m, 2H) 3.56 (ddd, J = 10.0,
10.5, 7.5 Hz, 1H) 4.05 (m, 1H), 4.22 (ddd, J = 10.25,
10.0, 7.6 Hz, 1H), 4.77 (dd, J = 2.5, 2.0 Hz, 1H), 5.03
(m, 1H), 6.57 (s, 1H), 7.12 (d, 1H), 7.18 (dd, J = 13.5,
10.0 Hz, 1H), 7.33 (m, 2H), 7.36 (m, 2H), 7.49 (d,
J = 10.5 Hz, 1H), 7.55 (m, 2H), 7.82 (d, J = 8 Hz,
1H), 8.53 (d, J = 4.5 Hz, 1H).
2.0 Hz, 1H), 5.10 (m, 1H), 5.95 (s, 1H), 7.14 (m, 2H),
7.24 (m, 4H), 7.75 (d, J = 10 Hz, 1H), 7.85 (m, 1H),
7.92 (d, J = 7.5 Hz, 1H), 8.60 (d, J = 5 Hz, 1H).
4.1.4. Synthesis of cyclo (-L-Am7(S2Py)-Aib-L-Ala-D-
Tic-) (8). Starting from H-D-Tic-OtBu (2.95 g, 10 mmol),
was obtained H-L-Ala-D-Tic-OtBu (1.4 g, 94%) simi-
larly, as explained earlier. The free amine H-L-Ala-D-
Tic-OtBu (1.4 g, 4.6 mmol) was coupled with Z-Aib-OH
(1.14 g, 5 mmol) using DCC (1.13 g, 5.5 mmol) and
HOBtÆH2O (704 mg, 4.6 mmol) in DMF (10 mL) to
yield Z-Aib-L-Ala-D-Tic-OtBu (1.6 g, 70%). Further,
similar reactions were carried out on Z-Aib-L-Ala-D-
Tic-OtBu, according to procedure reported for 3 to yield
finally cyclo(-L-Am7(S2Py)-Aib-L-Ala-D-Tic-) (80 mg
64%). HPLC, rt 6.92 min. HR-FAB MS, [M+H]+
584.2336 for C29H38O4N5S2 (calcd 584.2287). 1H
NMR (500 MHz, CDCl3): dH 1.25 (m, 1H), 1.31 (m,
1H), 1.37 (s, 3H), 1.41 (m, 2H), 1.61 (m, 1H), 1.67 (m,
2H), 1.76 (m, 1H), 1.78 (s, 3H), 2.78 (t, 2H), 2.95 (dd,
J = 8.0, 9.5 Hz, 1H), 3.42 (dd, J = 7.0, 7.0 Hz, 1H),
4.21 (ddd, J = 10.0, 10.0, 7.5 Hz, 1H), 4.30 (d,
J = 15.5 Hz, 1H), 4.97 (d, J = 15.5 Hz, 1H), 5.13 (t,
1H), 5.32 (m, 1H), 6.13 (s, 1H), 6.72 (d, J = 10.0 Hz,
1H), 7.08 (dd, J = 13.5, 10.0 Hz, 1H), 7.14 (d,
J = 7 Hz, 1H), 7.21 (m, 4H), 7.44 (d, J = 10 Hz, 1H),
7.65 (m, 1H), 7.71 (d, J = 8 Hz, 1H), 8.45 (d, J = 5 Hz,
1H).
4.1.5. Synthesis of cyclo (-L-Am7(S2Py)-Aib-L-Phg-D-
Pro-) (9). The N-terminal free H-D-Pro-OtBu (1 g,
5.8 mmol), was coupled with Z-L-Phg-OH (1.82 g,
6.4 mmol) using DCC (1.32 g, 6.4 mmol) and HOBtÆ-
H2O (980 mg, 6.4 mmol) in DMF (15 mL) to yield
Z-L-Phg-D-Pro-OtBu (2.7 g, 99%). The dipeptide Z-L-
Phg-D-Pro-OtBu (2.7 g, 6.2 mmol) was subjected to cat-
alytic hydrogenation with Pd–C (300 mg) in acetic acid
(15 mL). The free amine was taken into ethyl acetate
(50 mL) the aid of 2M Na2CO3 solution (30 mL). After
dried over anhydrous Na2CO3 ethyl acetate solution was
evaporated to remain H-L-Phg-D-Pro-OtBu (1.65 g,
89%). The free amine H-L-Phg-D-Pro-OtBu (1.5 g,
4.8 mmol) was coupled with Z-Aib-OH (1.3 g,
5.5 mmol) in the same manner as described above to
yield Z-Aib-L-Phg-D-Pro-OtBu (2.26 g, 94%). Further,
similar reactions were carried out on Z-Aib-L-Phg-D-
Pro-OtBu, according to procedure reported for 3 to ob-
tain finally cyclo(-L-Am7(S2Py)-Aib-L-Phg-D-Pro-).
Yield (229 mg, 45%), HPLC, rt 7.69 min. HR-FAB
MS, [M+H]+ 584.2347 for C29H37O4N5S2 (calcd
4.1.3. Synthesis of cyclo (-L-Am7(S2Py)-D-2-MePhe-L-
Ala-D-Pro-) (6) and cyclo (-L-Am7(S2Py)-L-2-MePhe-L-
Ala-D-Pro-) (7). These compounds were synthesized
using DL-2MePhe instead of Aib according to the proce-
dure described for 3, to yield cyclo(-L-Am7(S2Py)-DL-
2MePhe-L-Ala-D-Pro-). The two distereoisomers were
separated by preparative HPLC [Column: YMC pack,
ODS-A (250 · 10 mm), condition: Isocratic condition
from solvent A to B over 20 min with 220 nm detection,
Sol. A; 100% H2O with 0.1% TFA and Sol. B; 100% ace-
tonitrile with 0.1% TFA] to yield cyclo(-L-Am7(S2Py)-D-
2MePhe-L-Ala-D-Pro-) (40 mg, 22%) as a white foam.
HPLC, rt 6.52 min. HR-FAB MS, [M+H]+ 598.256
for C30H40O4N5S2 (calcd 598.2443). 1H NMR
(500 MHz, CDCl3): dH 1.23 (m, 2H), 1.27 (m, 1H),
1.38 (d, J = 6.5 Hz, 3H), 1.46 (m, 1H), 1.56 (m, 3H),
1.67 (m, 1H), 1.71(s, 3H), 1.81 (m, 1H), 1.92 (m,
1H),2.18 (m, 1H), 2.33 (m, 1H), 2.76 (t, 2H), 2.98 (d,
J = 9 Hz, 1H), 3.12 (d, J = 14 Hz, 1H), 3.53 (ddd, J =
9.75, 7.5, 9.75 Hz, 1H) 3.87 (m, 1H), 4.14 (m, 1H),
4.71 (dd, J = 2.5, 2 Hz, 1H), 5.0 (m, 1H), 6.12 (s, 1H),
7.02 (d, J = 10 Hz), 7.28 (m, 1H), 7.28 (m, 4H), 7.81
(m, 1H), 7.88 (d, J = 8 Hz, 1H), 8.57 (d, J = 5 Hz, 1H),
1
584.2365). H NMR (500 MHz, CDCl3): dH 1.29 (m,
1H), 1.40 (s, 3H), 1.42 (m, 1H), 1.60 (m, 1H), 1.69 (m,
2H), 1.79 (1H, m), 1.85 (S, 3H), 1.88 (m, 1H), 1.99 (m,
2H), 2.32 (m, 1H), 2.43 (m, 1H), 2.77 (t, 2H), 3.74
(ddd, J = 10.0, 10.0, 8.7 Hz, 1H), 4.04 (m, 1H), 4.22
(ddd, J = 11.7, 10.3, 7.0 Hz, 1H), 4.75 (dd, J = 2.5,
2.5 Hz, 1H), 6.05 (s, 1H), 6.15 (d, J = 10.5 Hz, 1H),
7.07 (m, 1H), 7.32 (m, 2H), 7.37 (m, 3H), 7.64 (m,
1H), 7.71 (d, J = 8 Hz, 1H), 8.04 (d, J = 10.5 Hz, 1H),
8.46 (d, J = 4.25 Hz, 1H).
and
cyclo(-L-Am7(S2Py)-L-2MePhe-L-Ala-D-Pro-)
(50 mg, 28%) as a white foam. HPLC, rt 6.78 min.
HR-FAB MS, [M+H]+ 598.2510 for C30H40O4N5S2
(calcd 598.2443). 1H NMR (500 MHz, CDCl3): dH
1.20 (s, 3H), 1.3 (m, 2H), 1.42 (d, J = 7 Hz, 3H), 1.45
(m, 2H), 1.61 (m, 1H), 1.72 (m, 2H), 1.85 (m, 2H),
1.91 (m, 1H), 2.22 (m, 1H), 2.37, 1H), 2.82 (t, 2H),
3.22 (d, J = 13.5 Hz, 1H), 3.57 (ddd, J = 10.0, 10.5,
7.5 Hz, 1H), 3.78 (d, J = 13.5 Hz, 1H) 3.89 (m,1H),
4.18 (dd, J = 8.0, 8.0 Hz, 1H), 4.77 (dd, J = 2.5,
4.1.6. Synthesis of cyclo (-L-Am7(S2Py)-Aib-L-Ph4-D-
Pro-) (10). This compound was synthesized according to

G. M. Shivashimpi et al. / Bioorg. Med. Chem. 15 (2007) 7830–7839
7837
the procedure reported for 9. Yield (326 mg, 68%),
HPLC, rt 7.88 min. HR-FAB MS, [M+H]+ 612.2666
for C31H41O4N5S2 (calcd 612.2678). 1H NMR
(500 MHz, CDCl3): dH 1.28 (m, 1H), 1.33 (s, 3H), 1.41
(m, 2H), 1.61 (m, 1H), 1.72 (s, 3H), 1.79 (m, 3H), 1.83
(m, 2H), 2.05 (m, 1H), 2.19 (m, 1H), 2.37 (m, 1H),
2.64 (m, 2H), 2.77 (t, 2H), 3.31 (ddd, J = 10.5, 10.5,
7 Hz, 1H), 3.74 (m, 1H), 4.17 (ddd, J = 10.5, 10.5,
7.5 Hz, 1H), 4.72 (d, 1H), 4.82 (ddd, J = 9.45, 9.75,
7.65 Hz, 1H), 5.93 (s, 1H), 7.07 (m, 1H), 7.18 (m, 3H),
7.27 (m, 3H), 7.63 (m, 1H), 7.73 (d, J = 8.5 Hz, 1H),
8.47 (d, J = 4.5 Hz, 1H).
for C29H38O4N5S2 (calcd 584.2287). 1H NMR
(500 MHz, CDCl3): dH 1.27 (m, 2H), 1.34 (s, 3H), 1.36
(d, J = Hz, 3H), 1.42 (m, 2H), 1.60 (m, 1H), 1.71 (m,
1H), 1.77 (s, 3H), 1.81 (m, 1H), 1.91 (m, 1H), 2.24 (m,
1H), 2.38 (m, 1H), 2.78 (t, 2H), 3.53 (ddd, J = 16.0,
16.2, 8.9 Hz, 1H), 3.93 (m, 1H), 4.18 (ddd, J = 10.2,
10.5, 7.6 Hz, 1H), 4.73 (dd, J = 2.5, 2.5 Hz, 1H), 5.02
(m, 1H), 5.96 (s, 1H), 7.05 (d, J = 10 Hz, 1H), 7.09 (m,
4H), 7.41 (d, J = 10 Hz, 1H), 7.66 (m, 1H), 7.72 (d,
J = 8.5 Hz, 1H), 8.46 (d, J = 14.5 Hz, 1H).
4.2. Circular dichroism measurement
4.1.7. Synthesis of cyclo (-L-Am7(S2Py)-Aib-L-Ph5-D-
Pro-) (11). This compound was synthesized according to
the procedure reported for 9. Yield (100 mg, 63%),
HPLC, rt 7.96 min. HR-FAB MS, [M+H]+ 626.2798
for C32H43O4N5S2 (calcd 626.2835) 1H NMR
(500 MHz, CDCl3): dH 1.28 (m, 1H), 1.33 (s, 3H), 1.41
(m, 1H), 1.64 (m, 1H), 1.70 (m, 3H), 1.76 (S, 3H), 1.82
(m, 3H), 1.89 (m, 2H), 2.23 (m, 1H), 2.36 (m, 1H),
2.65 (m, 1H), 2.77 (t, 2H), 3.47 (m, 1H), 3.89 (m, 1H),
4.17 (ddd, J = 10.5, 10.3, 7.7 Hz, 1H), 4.71 (dd,
J = 2.0, 1.5 Hz, 1H), 4.85 (ddd, J = 8.75, 10, 4.4), 5.92
(s, 1H), 7.07 (m, 1H), 7.17 (m, 1H), 7.26 (m, 5H), 7.32
(d, J = 8 Hz, 1H)), 7.65 (m, 1H), 8.46 (d, J = 4.0 Hz,
1H).
CD spectra were recorded on a JASCO J-820 spectro-
polarimeter (Tokyo, Japan) using a quartz cell of
1 mm light path length at room temperature. Peptide
solution (0.1 mM) was dissolved in methanol and CD
spectra were recorded in terms of molar ellipticity,
[h]M (deg · cm2 · dmolꢀ1).
4.2.1. HDACs preparation and enzyme activity assay. In
a 100-mm dish, 293T cells (1–2 · 106) were grown for
24 h and transiently transfected with 10 lg each of the
vector pcDNA3-HDAC1 for human HDAC1,
pcDNA3-HDAC4 for human HDAC4, or pcDNA3-
mHDA2/HDAC6 for mouse HDAC6, using the Lipo-
fectAMINE2000 reagent (Invitrogen). After successive
cultivation in DMEM for 24 h, the cells were washed
with PBS and lysed by sonication in lysis buffer contain-
ing 50 mM Tris–HCl (pH 7.5), 120 mM NaCl, 5 mM
EDTA, and 0.5% NP40. The soluble fraction collected
by microcentrifugation was precleared by incubation
with protein A/G plus agarose beads (Santa Cruz Bio-
technologies, 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 deace-
tylase buffer consisting of 20 mM Tris–HCl (pH 8.0),
150 mM NaCl, and 10% glycerol. The bound proteins
were released from the immune complex by incubation
for 1 h at 4 ꢁC with 40 lg of the FLAG peptide (Sig-
ma–Aldrich Inc.) in histone deacetylase buffer
(200 lL). The supernatant was collected by centrifuga-
tion. For the enzyme assay, 10 lL of the enzyme frac-
tion was added to 1 lL of fluorescent substrate (2 mM
Ac-KGLGK(Ac)-MCA) and 9 lL of histone deacetyl-
ase buffer, and the mixture was incubated at 37 ꢁC for
30 min. The reaction was stopped by the addition of
30 lL of tripsin (20 mg/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 deter-
mined as the means with SD calculated from at least
three independent dose–response curves.
4.1.8. Synthesis of cyclo (-L-Am7(S2Py)-Aib-L-Ser(Bzl)-
D-Pro-) (12). This compound was synthesized according
to the procedure reported for 9. Yield (86 mg, 60%),
HPLC, rt 7.61 min. HR-FAB MS, [M+H]+ 628.2598
for C30H39O5N5S2 (calcd 628.2627) 1H NMR
(500 MHz, CDCl3): dH 1.27 (m, 1H), 1.34 (s, 3H), 1.41
(m, 1H), 1.60 (m, 2H), 1.71 (m, 1H), 1.74 (S, 3H), 1.79
(m, 1H), 1.85 (m, 1H), 1.92 (m, 1H), 2.23 (m, 1H),
2.37 (m, 1H), 2.77 (t, 2H), 3.56 (m, 2H), 3.85 (m, 1H),
3.93 (t, 1H), 4.13 (ddd, J = 10.0, 10.0, 8.0 Hz, 1H),
4.55 (dd, J = 12, 12.5), 4.75 (dd, J = 2.0, 2.0 Hz, 1H),
5.12 (m, 1H), 5.98 (s, 1H), 7.07 (m, 1H), 7.32 (m, 5H),
7.65 (m, 1H), 7.71 (d, J = 10 Hz, 1H), 8.44 (d,
J = 4.25 Hz, 1H).
4.1.9. Synthesis of cyclo (-L-Am7(S2Py)-Aib-L-Ser-D-
Pro-) (13). This compound was synthesized according to
the procedure reported for 9. Yield (86 mg, 60%),
HPLC, rt 4.98 min. HR-FAB MS, [M+H]+ 538.2205
for C24H35O5N5S2 (calcd 538.2158). 1H NMR
(500 MHz, CDCl3): dH 1.29 (m, 1H), 1.35 (s, 3H), 1.42
(m, 1H), 1.60 (m, 2H), 1.70 (m, 1H), 1.79 (S, 3H), 1.84
(m, 2H), 1.94 (m, 1H), 2.25 (m, 1H), 2.39 (m, 1H),
2.77 (t, 2H), 3.37 (dd, J = 3.0, 3.35 Hz, 1H, 3.58 (ddd,
J = 10.3, 10.3, 7.5 Hz, 1H), 3.87 (m, 2H), 3.95 (m,
2H), 4.19 (ddd, J = 10.0, 10.0, 7.3 Hz, 1H), 4.76 (d,
J = 6.5 Hz, 1H), 4.95 (m, 1H), 6.05 (s, 1H), 6.92 (d,
J = 6.5 Hz, 1H), 7.08 (m, 1H), 7.65 (m, 1H), 7.72 (d,
J = 8.25 Hz, 1H), 7.78 (d, J = 10.0 Hz, 1H), 8.46 (d,
J = 3.5 Hz, 1H).
4.2.2. The p21 promoter assay. 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-Ba-
sic plasmid (Promega Co., Madison, WI). Mv1Lu (mink
lung epithelial cell line) cells were transfected with the
pGW-FL and a phagemid expressing neomycin/kanamycin
4.1.10. Synthesis of cyclo (-L-Am7(S2Py)-Aib-L-Ala-D-
Pro-) (14). This compound was synthesized according to
the procedure reported for 3. Yield (50 mg, 30%),
HPLC, rt 5.98 min. HR-FAB MS, [M+H]+ 584.2336

7838
G. M. Shivashimpi et al. / Bioorg. Med. Chem. 15 (2007) 7830–7839
18. Gupta, S.; Peiser, G.; Nakajima, T.; Hwang, Y.-S.
Tetrahedron Lett. 1994, 35, 6009–6012.
19. Liesch, J. M.; Sweeley, C. C.; Staffeld, G. D.; Anderson,
M. S.; Weber, D. J.; Scheffer, R. P. Tetrahedron 1982, 38,
45–48.
20. Pope, M. R.; Ciuffetti, L. M.; Knoche, H. W.; McCrery,
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3502–3506.
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M. R.; Ciuffetti, L. M.; Knoche, H. W.; Daly, J. M.;
Dunkle, L. D. Tetrahedron Lett. 1982, 23, 5381–5384.
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Biol. Chem. 1973, 37, 955–956.
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 400 lg/mL Geneticin (G418, Life Technol-
ogy), colonies formed were isolated. One of the clones
was selected and named MFLL-9. MFLL-9 expressed
a low level of luciferase, whose activity was enhanced
by TSA in a dose-dependent manner. MFLL-9 cells
(1 · 105) cultured in a 96-well multi-well plate for 6 h
were incubated for 18 h in the medium containing vari-
ous 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 Luminescencer-
JNR luminometer (ATTO, Tokyo, Japan). Data were
normalized to the protein concentration in cell lysates.
Concentrations at which a drug induces the luciferase
activity 10-fold higher than the basal level are presented
as the 1000% effective concentration 1000% (EC1000).
The human wild-type p21 promoter luciferase fusion
plasmid, WWP-Luc, was a kind gift from Dr. B.
Vogelstein.
26. Hirota, A.; Suzuki, A.; Tamura, S. Agric. Biol. Chem.
1973, 37, 1185–1189.
27. Umehara, K.; Nakahara, K.; Kiyoto, S.; Iwami, M.;
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Acknowledgment
This study was supported by the Program for Promo-
tion of Fundamental Studies in Health Sciences of the
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