Chlamydocin-hydroxamic acid analogues as histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmc.2004.08.041

Chlamydocin-hydroxamic acid analogues were designed and synthesized as histone deacetylase (HDAC) inhibitors based on the structure and HDAC inhibitory activity of chlamydocin and trichostatin A. Chlamydocin is a cyclic tetrapeptide containing an epoxyketone moiety in the side chain that makes it an irreversible inhibitor of HDAC. We replaced the epoxyketone moiety of chlamydocin with hydroxamic acid to design potent and reversible inhibitors of HDAC. In addition, a number of amino-cycloalkanecarboxylic acids (Acc) are introduced instead of the simple amino-isobutric acid (Aib) for a variety of the series of chlamydocin analogues. The compounds synthesized were tested for HDAC inhibitory activity and the results showed that many of them are potent inhibitors of HDAC. The replacement of Aib residue of chlamydocin with an aromatic amino acid enhances the in vivo and in vitro inhibitory activity. We have carried out circular dichroism and molecular modeling studies on chlamydocin-hydroxamic acid analogue and compared it with the solution structure of chlamydocin.

Full text of DOI:10.1016/j.bmc.2004.08.041

Bioorganic & Medicinal Chemistry 12 (2004) 5777–5784
Chlamydocin–hydroxamic acid analogues as
histone deacetylase inhibitors
b
a
a
Norikazu Nishino,a,b, Binoy Jose, Ryuzo Shinta, Tamaki Kato,
*
Yasuhiko Komatsub,c and Minoru Yoshidab,d
aGraduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu 808-0196, Japan
bCREST Research Project, Japan Science and Technology Agency, Saitama 332-0012, Japan
cPharmaceuticals and Biotechnology Laboratory, Japan Energy Corporation, Saitama 335-8502, Japan
dRIKEN, Saitama 351-0198, Japan
Received 21 June 2004; revised 26 August 2004; accepted 27 August 2004
Available online 23 September 2004
Abstract—Chlamydocin–hydroxamic acid analogues were designed and synthesized as histone deacetylase (HDAC) inhibitors based
on the structure and HDAC inhibitory activity of chlamydocin and trichostatin A. Chlamydocin is a cyclic tetrapeptide containing
an epoxyketone moiety in the side chain that makes it an irreversible inhibitor of HDAC. We replaced the epoxyketone moiety of
chlamydocin with hydroxamic acid to design potent and reversible inhibitors of HDAC. In addition, a number of amino-cycloal-
kanecarboxylic acids (Acc) are introduced instead of the simple amino-isobutric acid (Aib) for a variety of the series of chlamydocin
analogues. The compounds synthesized were tested for HDAC inhibitory activity and the results showed that many of them are
potent inhibitors of HDAC. The replacement of Aib residue of chlamydocin with an aromatic amino acid enhances the in vivo
and in vitro inhibitory activity. We have carried out circular dichroism and molecular modeling studies on chlamydocin–hydroxamic
acid analogue and compared it with the solution structure of chlamydocin.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
and development of certain human cancers by changing
the expression pattern of various genes. It is therefore
proposed that HDACs are a potential target for the
development of small molecule anticancer agent.
Post-translational modification of proteins by acetyla-
tion and deacetylation of e-amino groups of lysine resi-
dues by histone acetyl transferase (HAT) and histone
deacetylase (HDAC) enzymes is involved in chromatin
remodeling and plays a crucial role in gene expres-
sion.1–4 Modification of the level of histone acetylation
and its consequences have received enormous interest
in recent years and increasing evidence supports their
importance for basic cellular functions such as DNA
replication, transcription, differentiation, and apoptosis.
Acetylation of lysine residues of histones results in more
open chromatin structure and therefore activation of
transcription, where as deacetylation of histone is asso-
ciated with a condensed chromatin structure resulting in
the repression of gene transcription. Deregulation of
HAT and HDAC has been implicated in the formation
A number of natural and synthetic HDAC inhibitors
have been reported, and in recent years the importance
of HDAC inhibitors has increased due to their efficacy
against many malignant diseases.5 Several of these
HDAC inhibitors inhibit tumor growth and many of
them are under clinical trials. Trichostatin A, (TSA),6
a
cyclic tetrapeptide family including trapoxins
(TPX),7 chlamydocin,8 HC toxin,9 Cyl-1, Cyl-2,10 WF-
3161,11 apicidin,12,13 and the recent depsipeptide
FK228 (formerly FR901228)14–16 are naturally occur-
ring HDAC inhibitors and inhibitors such as butyrate,17
valproate,18 suberoyl anilide hydroxamic acid,19 ana-
logues of TSA,20–22 and benzamide derivatives of MS-
27523,24 are synthetic compounds. Trichostatin A is a
natural product that contains a hydroxamic acid func-
tional group and is a reversible inhibitor of HDAC.
On the other hand, cyclic tetrapeptides, trapoxin,
chlamydocin, etc. contain an epoxyketone moiety, and
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 ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.08.041

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N. Nishino et al. / Bioorg. Med. Chem. 12 (2004) 5777–5784
naturally occurring cyclic tetrapeptides and our initial
studies showed that these inhibitors have HDAC inhib-
itory activity in nanomolar range (Fig. 1).25,26
In the present study, we proposed to synthesize
chlamydocin–hydroxamic acid analogues as HDAC
inhibitors. During the development of potent inhibitors
of HDAC, we found that amino acid residues in the
cyclic tetrapeptides, and their conformations are very
important for the activity. Therefore we proposed to
compare the activities of several CHAPs with different
amino acid combinations. We herein describe the full
account on synthesis of chlamydocin–hydroxamic acid
analogues and a brief description of the interesting bio-
logical activity. A comparison of the structural features
of chlamydocin and the synthetic chlamydocin–
hydroxamic acid analogues is also described.
Figure 1. The structure of chlamydocin, trichostatin A, and proposed
chlamydocin–hydroxamic acid analogue.
are irreversible inhibitors of HDAC. Chlamydocin was
originally isolated from the fungus Diheterospora chla-
mydosporia, and has been shown to exhibit potent anti-
cancer activity in vitro.8 Chlamydocin belongs to a small
family of hydrophobic cyclic tetrapeptides containing
the unusual amino acid, 2-amino-8-oxo-9,10-epoxy dec-
anoic acid (Aoe), which is essential for their biological
activity. Recently, we have reported a new group of syn-
thetic HDAC inhibitors, CHAPs, by the use of hydroxa-
mic acid as a functional group instead of epoxyketone of
2. Results and discussion
Our aim is to synthesize potent inhibitors of HDACs
based on the natural product chlamydocin. Therefore,
we proposed to synthesize chlamydocin–hydroxamic
acid analogue by replacing the epoxyketone moiety of
chlamydocin by hydroxamic acid. For this, we initially
synthesized chlamydocin–hydroxamic acid analogue
using solution-phase peptide coupling method as de-
picted in Scheme 1. tert-Butyl protected D-Pro was
Scheme 1. Synthesis of cyclic tetrapeptide. Reagents and conditions: (a) HBTU, DIEA, Z-L-Phe; (b) Pd–C, H2; (c) HBTU, DIEA, Z-Aib; (d) HBTU,
DIEA, Boc-L-Asu(OBzl); (e) TFA; (f) HATU, DIEA; (g) NH2OH, BOP, HOBt.

N. Nishino et al. / Bioorg. Med. Chem. 12 (2004) 5777–5784
5779
coupled with Z-L-Phe to give the dipeptide Z-L-Phe-D-
Pro-OtBu. The Z group of dipeptide was deprotected
by catalytic hydrogenation and coupled with Z-Aib.
The Z group of the resulted tripeptide Z-Aib-L-Phe-D-
Pro-OtBu was deprotected by catalytic hydrogenation.
It was then coupled with Boc protected amino suberic
acid benzyl ester, Boc-L-Asu(OBzl) to give Boc-L-
Asu(OBzl)-Aib-L-Phe-D-Pro-OtBu. The C-terminal and
N-terminal protections of the tetrapeptide were removed
by treatment with trifluoroacetic acid (TFA) and the
resulted linear peptide was cyclized in DMF under high
dilution condition using HATU as a coupling reagent to
give cyclo(-L-Asu(OBzl)-Aib-L-Phe-D-Pro-) in 60%
yield. The side chain was deprotected by catalytic hydro-
genation and then coupled with hydroxylamine hydro-
chloride to yield the desired compound 3 and finally
purified using gel filtration.
(11), cyclo(-L-Asu(NHOH)-D-A1in-L-Phe-D-Pro-) (12),
and cyclo(-L-Asu(NHOH)-L-Pro-L-Phe-D-Pro-) (13).
Next we changed the D-Pro residue of chlamydocin to
D-pipecolic acid (D-Pip). This compound was synthe-
sized using the same method reported for compound 3
using DL-Pip instead of D-Pro. After cyclization, the
compound containing D-Pip was purified by column
chromatography and characterized by HPLC and
NMR spectroscopy. Thus, we synthesized compounds,
cyclo(-L-Asu(NHOH)-Aib-L-Phe-D-Pip-) (14), cyclo(-L-
Asu(NHOH)-Acc5-L-Phe-D-Pip-) (15), cyclo(-L-Asu(N-
HOH)-Acc8-L-Phe-D-Pip-) (16), and cyclo(-L-Asu(N-
HOH)-A2in-L-Phe-D-Pip-) (17). We then synthesized
cyclo(-L-Asu(NHOH)-A2in-L-Ile-D-Pip-) (18) by replac-
ing L-Phe of 17 by L-Ile. This compound was synthesized
by using the same method for 17 with L-Ile instead of L-
Phe.
We replaced Aib residue of 3 with many other natural
and nonnatural amino acids to study the effect on the
HDAC inhibitory activity. The amino acids used for
the replacement of Aib are 1-aminocyclopentanecarb-
oxylic acid (Acc5), 1-aminocyclohexanecarboxylic acid
(Acc6), 1-aminocycloheptanecarboxylic acid (Acc7),
1-aminocyclooctanecarboxylic acid (Acc8), 2-aminoin-
dane-2-carboxylic acid (A2in), L-Ala, D-Ala, DL-1-amino-
indane-1-carboxylic acid (DL-A1in), and L-Pro. The
cyclic tetrapeptides chlamydocin–hydroxamic acid ana-
logues were synthesized according to the method
described for compound 3 using the corresponding
amino acids instead of Aib to give the following com-
pounds cyclo(-L-Asu(NHOH)-Acc5-L-Phe-D-Pro-) (4),
cyclo(-L-Asu(NHOH)-Acc6-L-Phe-D-Pro-) (5), cyclo(-L-
Asu(NHOH)-Acc7-L-Phe-D-Pro-) (6), cyclo(-L-Asu(N-
HOH)-Acc8-L-Phe-D-Pro-) (7), cyclo(-L-Asu(NHOH)-
A2in-L-Phe-D-Pro-) (8), cyclo(-L-Asu(NHOH)-L-Ala-L-
Phe-D-Pro-) (9), cyclo(-L-Asu(NHOH)-D-Ala-L-Phe-D-
Pro-) (10), cyclo(-L-Asu(NHOH)-L-A1in-L-Phe-D-Pro-)
To know the effect of retro-enantio-analogues, we
synthesized two compounds, such as cyclo(D-Asu(N-
HOH)-L-Pro-D-Phe-Aib-) (19) and cyclo(-L-Asu(N-
HOH)-L-Pro-D-Phe-Aib-) (20) using the same method
reported for 3 using different amino acids. Finally we
synthesized cyclo(-L-Asu(NHOH)-Aib-L-Phe-L-Pro-)
(21). All the synthesized compounds were characterized
1
by H NMR and high-resolution FAB-MS. The purity
of the compounds was determined by HPLC analysis
and all the synthesized cyclic tetrapeptides showed
purity above 97%.
We evaluated the ability of chlamydocin–hydroxamic
acid analogues to inhibit a partially purified mouse
HDAC in vitro by comparing their IC50s. To evaluate
their cellular activity, we determined the concentra-
tion required for 2-fold increased expression of MHC
class-I molecules in B16/BL6 melenoma cells (C·2).
Table 1 shows the HDAC inhibitory activity of
Table 1. HDAC inhibitory data for reported compounds
No
Compound
IC50 (nM) HDAC1
MHC C·2 (nM)
1
TSA
Chlamydocin
6.02.8
0.15
5.2
2
4.6
3
cyclo(-L-Asu(NHOH)-Aib-L-Phe-D-Pro-)
cyclo(-L-Asu(NHOH)-Acc5-L-Phe-D-Pro-)
cyclo(-L-Asu(NHOH)-Acc6-L-Phe-D-Pro-)
cyclo(-L-Asu(NHOH)-Acc7-L-Phe-D-Pro-)
cyclo(-L-Asu(NHOH)-Acc8-L-Phe-D-Pro-)
cyclo(-L-Asu(NHOH)-A2in-L-Phe-D-Pro-)
cyclo(-L-Asu(NHOH)-L-Ala-L-Phe-D-Pro-)
cyclo(-L-Asu(NHOH)-D-Ala-L-Phe-D-Pro-)
cyclo(-L-Asu(NHOH)-L-A1in-L-Phe-D-Pro-)
cyclo(-L-Asu(NHOH)-D-A1in-L-Phe-D-Pro-)
cyclo(-L-Asu(NHOH)-L-Pro-L-Phe-D-Pro-)
cyclo(-L-Asu(NHOH)-Aib-L-Phe-D-Pip-)
cyclo(-L-Asu(NHOH)-Acc5-L-Phe-D-Pip-)
cyclo(-L-Asu(NHOH)-Acc8-L-Phe-D-Pip-)
cyclo(-L-Asu(NHOH)-A2in-L-Phe-D-Pip-)
cyclo(-L-Asu(NHOH)-A2in-L-Ile-D-Pip-)
cyclo(-D-Asu(NHOH)-L-Pro-D-Phe-Aib-)
cyclo(-L-Asu(NHOH)-L-Pro-D-Phe-Aib-)
cyclo(-L-Asu(NHOH)-Aib-L-Phe-L-Pro-)
33.2
5.67
5.38
2.76
1.88
1.29
325
4
3.02
2.19
2.14
3.99
0.98
1.95
3.23
1.12
1.95
3303
2.98
12.4
2.75
1.2
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
NT
2.69
2.29
NT
NT
37.8
NT
NT
NT
2.68
8.57
31.7
45.7
3950
57,800
717

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N. Nishino et al. / Bioorg. Med. Chem. 12 (2004) 5777–5784
chlamydocin–hydroxamic acid analogue compounds.
These compounds inhibited the HDACs at nanomolar
concentrations. For comparison, the inhibitory activities
of TSA and chlamydocin are also shown. Most of these
chlamydocin–hydroxamic acid analogues are potent
inhibitors of HDAC. The replacement of epoxyketone
moiety of chlamydocin by hydroxamic acid affects the
in vitro and in vivo activities. The activity of chlamydo-
cin–hydroxamic acid analogue
3
is lower than
chlamydocin 2. This fact could be attributed to irrevers-
ible mechanism of 2, though the straightforward com-
parison is difficult. When the amino acid residue Aib is
replaced by Acc5, Acc6, Acc7, and Acc8, the activity in-
creased, and out of these, replacement with Acc7 seems
optimum. The replacement of Aib residue with L-Ala
gives good inhibitory activity in vitro; however, cellular
activity was low. Chlamydocin–hydroxamic analogues
of Aib replaced with aminoindanecarboxylic acids show
very good activity in vitro and in vivo. This high activity
of inhibitors containing an aminoindanecarboxylic acid
residue shows the importance of an aromatic ring for the
activity instead of Aib. This result indicates the presence
of a favorable interaction between the benzene ring and
the residues at the rim of the HDAC binding channel.
Out of these, three compounds 8, 11, and 12, inhibitor
with 2-amino-2-indanecarboxylic acid 8 showed more
potency. On the other hand, the replacement of Aib with
L-Pro in 13 resulted in a significant decrease in the
activity.
Figure 2. CD spectra of chlamydocin–hydroxamic acid analogues.
that the synthetic compound and natural product have
similar structural features.27,28 The conformation of
the cyclic peptide framework of chlamydocin is remark-
ably sensitive to solvents. In chloroform, the framework
of chlamydocin exists with bis-c-turn structure with four
transoid amide bonds. On the other hand in DMSO, it
exists as the equilibrium of bis-c-turn structure and b-
turn structure.29 We observed the same behavior for
chlamydocin–hydroxamic acid analogue also. The
molecular modeling study on 3 showed that the exist-
ence of bis-c-turn structure in chloroform (Fig. 3a)
and an equilibrium of bis-c-turn structure and b-turn
structure (Fig. 3b) in DMSO. Similar behavior was ob-
served for compounds 4–8. On the other hand, com-
pounds 9–13 showed only bis-c-turn conformation in
chloroform and also in DMSO indicating that the pres-
ence of Aib is the reason for the two different
conformations.
Most of the naturally occurring cyclic tetrapeptides con-
taining a D-Pro residue, an analogue with D-Pip is also
observed as in trapoxin A, Cyl-2, etc. However, no
chlamydocin analogue with a D-Pip residue was re-
ported. Therefore, we replaced the D-Pro residue of 3
with D-Pip to study the effect of the increase in ring size
on the inhibitory activity. The replacement of D-Pro of
chlamydocin–hydroxamic acid inhibitor to D-Pip,
showed no significant differences in the activity. The
retro-enantio-analogues of chlamydocin 19 and 20
showed lower inhibitory activity than 3. This decrease
in activity indicates the importance of position and
chirality of Pro residue in the cyclic tetrapeptide. The
change from L-Asu to D-Asu affected adversely to the
in vitro and in vivo activity.
CD spectra of some of the chlamydocin–hydroxamic
acid analogues are shown in Figure 2. The CD spectrum
of chlamydocin 2 and 3 are similar up to 260nm. At the
longer wavelength than 260nm, 2 shows a negative
ellipticity due to the chiral ketone in 2,8 which is absent
in 3. Other analogues, except 13, 19, and 21 showed
similar CD spectra as 3 indicating that these compounds
have similar conformations in methanol. Changes in the
Pro position and chirality give different conformations
for 13, 19, and 21.
Conformation analysis of chlamydocin–hydroxamic
acid analogues was studied using H NMR in DMSO-
1
d6 and in CDCl3. Complete assignments of the chemical
shifts were made by using COSY, HOHAHA, and
NOESY spectra. Comparison of the conformations of
hydroxamic acid analogue 3 and chlamydocin 2 showed
Figure 3. Energy minimized structures of the two conformers of
chlamydocin–hydroxamic acid analogue: (a) bis-c-turn and (b) b-turn.

N. Nishino et al. / Bioorg. Med. Chem. 12 (2004) 5777–5784
5781
3. Conclusion
Pro-OtBu (1.40g, 62%). The protected dipeptide
(1.40g, 3.1mmol) was dissolved in acetic acid (5mL).
Pd–C (150mg) was added and the mixture was stirred
under H2 pressure for 10h. After filtration, acetic acid
was evaporated. The residue was dissolved in ethyl
acetate and washed with sodium bicarbonate solution
(4%) and dried over sodium carbonate. Evaporation of
ethyl acetate gave H-L-Phe-D-Pro-OtBu (0.853g,
2.68mmol, 86%), which was coupled with Z-Aib-OH
(954mg and 4.02mmol) according to the method de-
scribed above to yield Z-Aib-L-Phe-D-Pro-OtBu (1.23g,
85%). Z-Aib-L-Phe-D-Pro-OtBu (1.23g, 2mmol) was dis-
solved in acetic acid (5mL) and Pd–C (100mg) was
added. The mixture was stirred under H2 for 10h. After
filtration, acetic acid was evaporated. The residue was
dissolved in ethyl acetate and washed with sodium bicar-
bonate solution (4%) and dried over sodium carbonate.
Evaporation of ethyl acetate yielded H-Aib-L-Phe-D-
Pro-OtBu (0.726g, 79%). H-Aib-L-Phe-D-Pro-OtBu
(0.726g, 1.80mmol) was coupled with Boc-L-Asu-
(OBzl)-OH (1.02g, 2.70mmol) as described earlier to
yield Boc-L-Asu(OBzl)-Aib-L-Phe-D-Pro-OtBu (993mg,
72%). Boc-L-Asu(OBzl)-Aib-L-Phe-D-Pro-OtBu (993
mg, 1.30mmol) was dissolved in TFA (3mL) at 0 ꢁC
and kept for 2h. After evaporation of TFA, the residue
was solidified by titration with ether to yield H-L-
Asu(OBzl)-Aib-L-Phe-D-Pro-OH as TFA salt (737mg,
78%). HPLC, rt 8.66min. To a DMF (400mL) solvent,
We have successfully synthesized several chlamydocin–
hydroxamic acid analogues as HDAC inhibitors. The
in vitro and in vivo HDAC inhibitory activity of the
inhibitors was evaluated and it was found that most of
the inhibitors are potent toward HDAC. The results
indicate that the change of the Aib residue of chlamydo-
cin to aromatic amino acids can increase the inhibitory
activity.
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 solvents as indicated. All
compounds were routinely checked by thin layer
chromatography (TLC) or high-performance liquid
chromatography (HPLC). TLC was performed on
aluminum-backed silica gel plates (Merck DC-Alufolien
Kieselgel 60F 254) with spots visualized by UV light.
Analytical HPLC were performed on
a Hitachi
instrument equipped with a Wako pak C4 column
(4.6mm · 150mm). 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 30min
with detection at 220nm with a flow rate of 1mL/min.
NMR spectra were recorded on a JEOL JNM A500
MHz spectrometer. NMR spectra were measured in
CDCl3 or DMSO-d6 solutions containing TMS as
H-L-Asu(OBzl)-Aib-L-Phe-D-Pro-OHÆTFA
(300mg,
0.42mmol), DIEA/DMF (3.0mL, 1.7mmol), and
HATU (250mg, 0.65mmol) were added in separate five
portions in every 30min with stirring, for the cyclization
reaction. After the reaction, DMF was evaporated
under vacuo, the residue being dissolved in ethyl acetate
and washed with citric acid (10%) solution, sodium
bicarbonate (4%) solution, and brine, respectively. It
was then dried over anhydrous MgSO4 and filtered.
After evaporation of ethyl acetate, the residue was
purified by column chromatography using a mixture of
chloroform and methanol (99:1) to yield cyclo(-L-
Asu(OBzl)-Aib-L-Phe-D-Pro-) (151mg, 61%) HPLC rt
18.1min. cyclo(-L-Asu(OBzl)-Aib-L-Phe-D-Pro-) (151
mg, 0.256mmol) was dissolved in methanol (5mL) and
Pd–C (50mg) was added. The solution was stirred under
H2 for 10h. After the filtration of Pd–C, methanol was
evaporated to yield cyclo(-L-Asu-Aib-L-Phe-D-Pro-)
(124mg, 97%). HPLC, rt 6.32min. The cyclo(-L-Asu-
Aib-L-Phe-D-Pro-) (124mg, 0.248mmol) was dissolved
in DMF (3mL) at 0ꢁC and hydroxylamine hydrochlo-
ride (86mg, 1.24mmol) HOBtÆH2O (57mg, 0.372mmol),
BOP (165mg, 0.372mmol), and triethylamine (0.24mL,
1.74mmol) were added. The mixture was stirred for 2h.
Gel filtration of the reaction mixture using Sephadex
LH-20was followed by lyophilization to yield cyclo
(-L-Asu(NHOH)-Aib-L-Phe-D-Pro-) as white powder
1
reference. All H shifts are given in ppm (s = singlet;
d = doublet; m = multiplet). Assignments of proton
resonances were confirmed, when possible, by selective
homonuclear decoupling experiments or by correlated
spectroscopy. FAB-mass spectra and high-resolution
mass spectra (HRMS) were measured on a JEOL
JMS-SX 102A Instrument. Amino acids were coupled
using standard solution-phase chemistry with
dicyclohexylcarbodimide (DCC), 2-(1H-benzotriazol-1-
yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
(HBTU), or O-(7-azabenzotriazoyl-1-yl)-1,1,3,3-tetra-
methyluronium hexafluorophosphate (HATU) as the
coupling reagent. BOP (benzotriazol-1-yloxytris-
(dimethylamino)-phosphonium hexafluorophosphate)
was used for the reaction with hydroxylamine.
4.1.1. Synthesis of cyclo(-L-Asu(NHOH)-Aib-L-Phe-D-
Pro-) (3). To a solution of Z-L-Phe-OH (2.25g,
7.5mmol) and H-D-Pro-OtBu (0.862g, 5.0mmol) in
DMF (10mL) HOBtÆH2O (766mg, 5.0mmol), HBTU
(2.84g, 7.5mmol), and triethylamine (1.75mL,
12.5mmol) were added and the mixture was stirred for
2h. DMF was evaporated and the residue was dissolved
in ethyl acetate and washed with citric acid solution
(10%), sodium bicarbonate solution (4%), and brine,
respectively, and then dried over anhydrous MgSO4.
After evaporation of ethyl acetate, the residue was puri-
fied by silica gel chromatography using a mixture of
chloroform and methanol (99:1) to yield Z-L-Phe-D-
1
(95mg, 74%) HPLC, rt 14.3min, H NMR (500MHz,
CDCl3): d 7.56 (d, 1H), 7.11–7.29 (m, 7H), 6.31 (d,
1H), 5.12 (m, 1H), 4.68 (d, 1H), 4.22 (d, 1H), 3.86 (m,
1H), 3.16 (m, 2H), 2.94 (m, 3H), 2.31 (m, 1H), 2.13
(m, 2H), 1.77–1.25 (m, 16H); CD (methanol) k ([h]M)
243 (À31,000), 228 (12,000), 210 (À15,000) nm; HR-
FAB MS [M+H]+ 516.2819 for C26H38O6N5 (calcd
516.2822).

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N. Nishino et al. / Bioorg. Med. Chem. 12 (2004) 5777–5784
4.1.2. Synthesis of cyclo(-L-Asu(NHOH)-Acc5-L-Phe-D-
Pro-) (4). This compound was synthesized according to
the procedure reported for 3 using Acc5 instead of Aib.
1H), 7.06–7.35 (m, 9H), 4.92 (m, 1H), 4.74 (d, 1H),
4.27 (m, 1H), 3.40–3.58 (m, 2H), 2.80–3.35 (m, 6H),
2.20(m, 2H), 1.10–1.95 (m, 12H); Conformer 2, 8.75
(br, 1H), 8.65 (br, 1H), 7.06–7.35 (m, 10H), 4.98 (d,
1H), 4.58 (m, 1H), 4.08 (m, 1H), 3.40–3.58 (m, 2H),
2.80–3.35 (m, 6H), 2.20 (m, 2H), 1.10–1.95 (m, 12H);
HR FAB-MS [M+H]+ 590.2970 for C32H40O6N5 (calcd
590.2979).
1
HPLC, rt 15.9min, H NMR (500MHz, DMSO-d6) d:
Conformer 1, 8.36 (s, 1H), 7.63 (d, 1H), 7.17–7.28 (m,
5H), 7.00 (d, 1H), 4.95 (m, 1H), 4.74 (m, 1H), 4.26 (m,
1H), 3.05–3.35 (m, 2H), 2.89 (m, 2H), 1.24–2.27 (m,
22H); Conformer 2, 8.43 (d, 1H), 7.96 (d, 1H), 7.17–
7.28 (m, 5H), 6.85 (d, 1H), 4.84 (m, 1H), 4.55 (m, 1H),
4.18 (m, 1H), 3.05–3.35 (m, 2H), 2.89 (m, 2H), 1.24–
2.27 (m, 22H); HR FAB-MS [M+H]+ 542.2982 for
C28H40O6N5 (calcd 542.2979).
4.1.7. Synthesis of cyclo(-L-Asu(NHOH)-L-Ala-L-Phe-D-
Pro-) (9). This compound was synthesized according to
the procedure reported for 3 using L-Ala instead of Aib.
1
HPLC, rt 12.1min, H NMR (500MHz, DMSO-d6): d
4.1.3. Synthesis of cyclo(-L-Asu(NHOH)-Acc6-L-Phe-D-
Pro-) (5). This compound was synthesized according to
the procedure reported for 3 using Acc6 instead of Aib.
10.34 (s, 1H), 8.67 (s, 1H), 8.16 (br, 1H), 7.90 (m, 1H),
7.72 (m, 1H), 7.19–7.38 (m, 5H), 5.05 (m, 1H), 4.62
(m, 1H), 4.10(m, 1H), 3.81 (m, 1H), 2.6–03.20(m,
4H), 0.96–2.18 (m, 17H); HR FAB-MS [M+H]+
502.2642 for C25H36O6N5 (calcd 502.2666).
1
HPLC, rt 16.9min, H NMR (500MHz, DMSO-d6) d:
Conformer 1, 7.99 (br, 1H), 7.56 (d, 1H), 7.18–7.31
(m, 5H), 7.07 (d, 1H), 4.97 (m, 1H), 4.73 (m, 1H), 4.35
(m, 1H), 3.30–3.60 (m, 2H), 2.82–3.10 (m, 2H), 1.10–
2.25 (m, 24H); Conformer 2, 8.41 (d, 1H), 7.48 (br,
1H), 7.18–7.31 (m, 5H), 6.81 (d, 1H), 4.80(m, 1H),
4.55 (m, 1H), 4.26 (m, 1H), 3.30–3.60 (m, 2H), 2.82–
4.1.8. Synthesis of cyclo(-L-Asu(NHOH)-D-Ala-L-Phe-D-
Pro-) (10). This compound was synthesized according to
the procedure reported for 3 using D-Ala instead of Aib.
1
HPLC, rt 13.9min, H NMR (500MHz, DMSO-d6): d
3.10(m, 2H), 1.1–02.25 (m, 24H); CD (methanol)
k
10.31 (br, 1H), 8.98 (br, 1H), 8.53 (m, 1H), 7.15–7.32
(m, 7H), 4.92 (m, 1H), 4.68 (m, 1H), 4.41 (m, 1H),
4.21 (m, 1H), 3.67 (m, 1H), 3.30(m, 1H), 3.20 (m,
1H), 2.88 (m, 1H), 1.07–2.18 (m, 17H); HR FAB-MS
[M+H]+ 502.2658 for C25H36N5O6 (calcd 502.2666).
([h]M) 241 (À30,000), 227 (19,000), 210 (À28,000) nm;
HR-FAB MS [M+H]+ 556.3156 for C29H42O6N5 (calcd
556.3135).
4.1.4. Synthesis of cyclo(-L-Asu(NHOH)-Acc7-L-Phe-D-
Pro-) (6). This compound was synthesized according to
the procedure reported for 3 using Acc7 instead of Aib.
4.1.9. Synthesis of cyclo(-L-Asu(NHOH)-L-A1in-L-Phe-
D-Pro-) (11) and cyclo(-L-Asu(NHOH)-D-A1in-L-Phe-D-
Pro-) (12). These compounds were synthesized using
DL-A1in instead of Aib for 3 to yield Boc-L-Asu-
(OBzl)-DL-A1in-L-Phe-D-Pro-OtBu. Then Boc-L-Asu-
(OBzl)-DL-A1in-L-Phe-D-Pro-OtBu (889mg and 1.00
mmol) was deprotected using TFA to yield H-L-A-
su(OBzl)-DL-A1in-L-Phe-D-Pro-OH-TFA 717mg (90%).
It was cyclized according to the method described earlier
and purified by column chromatography using a
mixture of chloroform and methanol (9:1) to yield
cyclo(-L-Asu(OBzl)-L-A1in-L-Phe-D-Pro-) (77mg, 26%)
(HPLC, rt 16.00min) and cyclo(-L-Asu(OBzl)-D-A1in-
L-Phe-D-Pro-) (225mg, 75%) HPLC, rt 17.04min.
cyclo(-L-Asu(OBzl)-L-A1in-L-Phe-D-Pro-) (77mg and
0.116mmol) was deprotected by catalytic hydrogenation
using Pd–C (50mg) to yield cyclo(-L-Asu-L-A1in-L-Phe-
D-Pro-) (63mg, 94%). It was then coupled with hydrox-
ylamine hydrochloride as reported earlier to yield
cyclo(-L-Asu(NHOH)-L-A1in-L-Phe-D-Pro-) (11) (48mg,
82%). HPLC, rt 16.0min, 1H NMR (500MHz,
DMSO-d6): d 10.32 (s, 1H), 8.65 (s, 1H), 8.16 (d, 1H),
7.65 (br, 1H), 7.09–7.29 (m, 9H), 6.67 (d, 1H), 4.94
(m, 1H), 4.58–4.84 (m, 1H), 4.22–4.31 (m, 1H), 3.42
(m, 1H), 3.35 (m, 1H), 3.20(m, 1H), 3.16 (m, 1H),
2.79–2.95 (m, 2H), 1.15–2.50(m, 16H); HR FAB-MS
[M+H]+ 590.2957 for C32H40O6N5 (calcd 590.2979).
1
HPLC, rt 17.4min, H NMR (500MHz, DMSO-d6) d:
Conformer 1, 7.85 (d, 1H), 7.72 (d, 1H), 7.16–7.31 (m,
5H), 6.95 (d, 1H), 4.96 (m, 1H), 4.73 (d, 1H), 4.33 (m,
1H), 3.40–3.52 (m, 2H), 3.03–3.25 (m, 2H), 2.80–2.94
(m, 2H), 2.18–2.46 (m, 2H), 1.26–1.98 (m, 22H); Con-
former 2, 8.60–8.65 (m, 1H), 7.16–7.31 (m, 5H), 6.82
(d, 1H), 4.85 (br, 1H), 4.50(m, 1H), 4.33 (m, 1H),
3.40–3.52 (m, 2H), 3.03–3.25 (m, 2H), 2.80–2.94 (m,
2H), 2.18–2.46 (m, 2H), 1.26–1.98 (m, 22H); HR-
FABMS [M+H]+ 570.3264 for C30H44O6N5 (calcd
570.3292).
4.1.5. Synthesis of cyclo(-L-Asu(NHOH)-Acc8-L-Phe-D-
Pro-) (7). This compound was synthesized according to
the procedure reported for 3 using Acc8 instead of Aib.
1
HPLC, rt 18.4min, H NMR (500MHz, DMSO-d6) d:
Conformer 1, 7.67–7.92 (br, 1H), 7.13–7.28 (m, 6H),
6.84–6.93 (br, 1H), 4.92 (br, 1H), 4.81 (br, 1H), 4.24
(br, 1H), 3.34–3.49 (br, 2H), 2.98–3.12 (br, 2H), 2.69–
2.85 (br, 2H), 2.21–2.41 (br, 4H), 1.21–1.90(br, 22H);
Conformer 2, 7.67–7.92 (br, 1H), 7.13–7.28 (m, 6H),
6.84–6.93 (br, 1H), 4.68 (br, 1H), 4.45 (br, 1H), 4.24
(br, 1H), 3.34–3.49 (br, 2H), 2.98–3.12 (br, 2H), 2.69–
2.85 (br, 2H), 2.21–2.41 (br, 4H), 1.21–1.90(br, 22H);
HR-FABMS [M+H]+ 584.3455 for C31H46O6N5 (calcd
584.3448).
cyclo(-L-Asu(OBzl)-D-A1in-L-Phe-D-Pro-)
(225mg,
0.339mmol) was deprotected and then coupled with
hydroxylamine hydrochloride as reported earlier to yield
cyclo(-L-Asu(NHOH)-D-A1in-L-Phe-D-Pro) (12) (179
mg, 0.312mmol). 129mg (70%) HPLC, rt 17.0min,
1H NMR (500MHz, DMSO-d6): d 10.32 (s, 1H),
8.95 (d, 1H), 8.83 (br, 1H), 8.64 (br, 1H), 7.16–7.29
4.1.6. Synthesis of cyclo(-L-Asu(NHOH)-A2in-L-Phe-D-
Pro-) (8). This compound was synthesized according to
the procedure reported for 3 using A2in instead of Aib.
HPLC, rt 17.8min, H NMR (500MHz, DMSO-d6) d:
Conformer 1, 8.65 (br, 1H), 8.29 (br, 1H), 7.80(d,
1

N. Nishino et al. / Bioorg. Med. Chem. 12 (2004) 5777–5784
5783
(m, 10H), 4.99 (d, 1H), 4.48 (m, 1H), 4.35 (m, 1H), 3.48
(br, 1H), 2.73–3.32 (m, 4H), 1.28–2.29 (m, 17H); HR
FAB-MS [M+H]+ 590.2978 for C32H40O6N5 (calcd
590.2979).
the procedure reported for 3 using A2in instead of Aib.
HPLC, rt 17.9min, H NMR (500MHz, DMSO-d6): d
8.00 (br, 1H), 7.78 (d, 1H), 7.11–7.20 (m, 4H), 6.23 (d,
1H), 5.15 (m, 1H), 4.56 (m, 1H), 4.24 (m, 1H), 3.80–
3.88 (m, 2H), 3.08 (m, 1H), 2.88 (m, 1H), 3.09 (m,
1H), 2.72–2.88 (m, 2H), 0.78–1.93 (m, 24H); HR FAB-
MS [M+H]+ 570.3309 for C30H44O6N5 (calcd 570.3292).
1
4.1.10. Synthesis of cyclo(-L-Asu(NHOH)-L-Pro-L-Phe-
D-Pro-) (13). This compound was synthesized according
to the procedure reported for 3 using L-Pro instead of
1
Aib. HPLC, rt 15.4min, H NMR (500MHz, DMSO-
4.1.16. Synthesis of cyclo(-D-Asu(NHOH)-L-Pro-D-Phe-
Aib-) (19). This compound was synthesized according to
the procedure reported for 3. HPLC, rt 15.2min, H
d6): d 8.61 (br, 1H), 8.15 (br, 1H), 7.13–7.29 (m, 5H),
3.80–4.75 (m, 6H), 2.61–3.05 (m, 4H), 1.14–1.94 (m,
18H); CD (methanol) k ([h]M) 222 (25,000), 211
(17,000) nm; HR FAB-MS 528.2853 [M+H]+ for
C27H38O6N5 (calcd 528.2822).
1
NMR (500MHz, DMSO-d6) d: Conformer 1, 7.95–
8.07 (m, 1H), 7.56–7.58 (m, 1H), 7.12–7.32 (m, 6H),
4.58–4.74 (m, 3H), 3.41–3.57 (m, 2H), 2.73–3.08 (m,
4H), 1.16–2.23 (m, 18H); Conformer 2, 8.66–8.68 (m,
1H), 7.95–8.07 (m, 1H), 7.12–7.32 (m, 5H), 6.74 (br,
1H), 4.58–4.74 (m, 1H), 4.48 (m, 1H), 4.16 (m, 1H),
3.41–3.57 (m, 2H), 2.73–3.08 (m, 4H), 1.16–2.23 (m,
18H); CD (methanol) k ([h]M) 242 (59,000), 228
(À23,000), 221 (500), 213 (À13,000), 197 (À36,000)
nm; HR FAB-MS [M+H]+ 516.2850for C 26H38O6N5
(calcd 516.2822).
4.1.11. Synthesis of cyclo(-L-Asu(NHOH)-Aib-L-Phe-D-
Pip-) (14). This compound was synthesized as reported
for compound 3 using DL-Pip instead of D-Pro. After
cyclization reaction, the cyclic peptide containing D-
Pip was separated using column chromatography. The
benzyl protection of Asu was removed by catalytic
hydrogenation as earlier and coupled with hydroxyl
amine to yield cyclo(-L-Asu(NHOH)-Aib-L-Phe-D-Pip-)
125mg (74%), 1H NMR (500MHz, DMSO-d6): d
10.33 (s, 1H), 8.65 (br, 1H), 7.90 (d, 1H), 7.15–7.38
(m, 6H), 6.20(d, 1H), 5.12 (m, 2H), 4.19 (m, 1H), 3.65
(m, 1H), 3.03 (m, 1H), 2.90 (m, 2H), 2.72 (m, 2H),
1.91–1.95 (m, 4H), 1.26–1.56 (m, 16H); HR FAB-MS
[M+H]+ 530.2960 for C27H40O6N5 (calcd 530.2979).
4.1.17. Synthesis of cyclo(-L-Asu(NHOH)-L-Pro-D-Phe-
Aib-) (20). This compound was synthesized according to
the procedure reported for 3. HPLC, rt 13.1min, H
1
NMR (500MHz, DMSO-d6): d 8.60(br, 1H), 7.16–
7.40 (br, 7H), 6.49 (br, 1H), 3.90–4.50 (br, 3H), 2.60–
3.20(br, 8H), 1.1–02.20(br, 16H); HR FAB-MS
[M+H]+ 516.2827 for C26H38O6N5 (calcd 516.2822).
4.1.12. Synthesis of cyclo(-L-Asu(NHOH)-Acc5-L-Phe-D-
Pip-) (15). This compound was synthesized according to
the procedure reported for 3 using Acc5 instead of Aib.
4.1.18. Synthesis of cyclo(-L-Asu(NHOH)-Aib-L-Phe-L-
Pro-) (21). This compound was synthesized according to
the procedure reported for 3. HPLC, rt 13.2min, H
1
1
HPLC, rt 17.4min, H NMR (500MHz, DMSO-d6): d
7.82 (d, 1H), 7.66 (br, 1H), 7.17–7.24 (m, 5H), 6.18 (d,
1H), 5.12 (m, 2H), 4.22 (m, 1H), 3.70(m, 1H), 2.88–
3.05 (m, 4H), 2.60 (m, 1H), 2.33 (m, 2H), 1.27–1.94
(m, 20H); HR FAB-MS [M+H]+ 556.3179 for
C29H42O6N5 (calcd 556.3135).
NMR (500MHz, DMSO-d6): d 8.60(br, 1H), 7.9–0
8.20(br, 2H), 7.13–7.26 (br, 6H), 4.60–4.90(br, 2H),
4.22 (br, 1H), 3.40 (br, 2H), 2.60–3.20 (br, 6H), 1.05–
1.98 (br, 16H); CD (methanol) k ([h]M) 198 (À76,000)
nm; HR FAB-MS [M+H]+ 516.2831 for C26H38O6N5
(calcd 516.2822).
4.1.13. Synthesis of cyclo(-L-Asu(NHOH)-Acc8-L-Phe-D-
Pip-) (16). This compound was synthesized according to
the procedure reported for 3 using Acc8 instead of Aib.
4.2. MHC class-I molecule up-regulation assay
1
HPLC, rt 20.0min, H NMR (500MHz, DMSO-d6): d
The activity of cyclic tetrapeptides to induce the expres-
sion of MHC class-I molecules was determined by the
method reported previously.25 Briefly, 24h after 5000
B16/BL6 cells had been introduced into each well of a
96-well microplate (200lL), test samples were added.
After an additional 72h of incubation, the cell-surface
expression of the MHC class-I molecules was measured
by a cell ELISA method. The degree of MHC class-I up-
regulating activity of the compounds was compared
with respect to their concentrations for 2-fold up-regula-
tion (C·2) of the MHC class-I molecules.
10.34 (br, 1H), 8.66 (br, 1H), 9.84 (d, 1H), 7.12–7.28
(m, 6H), 6.25 (d, 1H), 5.10–5.16 (m, 2H), 4.22 (m,
1H), 3.67 (m, 1H), 3.04 (m, 1H), 2.86 (m, 1H), 2.72
(m, 1H), 1.20–1.95 (m, 30H); HR FAB-MS [M+H]+
598.3627 for C32H48O6N5 (calcd 598.3605).
4.1.14. Synthesis of cyclo(-L-Asu(NHOH)-A2in-L-Phe-D-
Pip-) (17). This compound was synthesized according to
the procedure reported for 3 using A2in instead of Aib.
HPLC, rt 19.0min, 1H NMR (500MHz, DMSO-d6): d
10.29 (s, 1H), 8.61 (s, 1H), 8.01 (d, 1H), 7.89 (br, 1H),
7.08–7.22 (m, 9H), 6.22 (m, 1H), 5.11 (m, 2H), 4.22
(m, 1H), 3.72–3.76 (br, 2H), 3.38 (m, 1H), 3.04–3.11
(m, 3H), 2.88–2.92 (m, 1H), 2.71–2.73 (m, 1H), 1.86–
1.92 (m, 6H), 1.19–1.56 (m, 10H); HR FAB-MS
[M+H]+ 604.3126 for C33H42O6N5 (calcd 604.31351).
4.3. Circular dichroism
CD spectra were recorded on a JASCO J-820spectro-
polarimeter (Tokyo, Japan) using a quartz cell of
1mm light path length at room temperature. Peptide
solution (0.1mM) was dissolved in methanol and CD
spectra were recorded in terms of molar ellipticity,
[h]M (degcm2 dmolÀ1).
4.1.15. Synthesis of cyclo(-L-Asu(NHOH)-A2in-L-Ile-D-
Pip-) (18). This compound was synthesized according to

5784
N. Nishino et al. / Bioorg. Med. Chem. 12 (2004) 5777–5784
11. Umehara, K.; Nahahara, K.; Kiyota, S.; Iwami, M.;
Okamoto, M.; Tanaka, H.; Kohsaka, M.; Aoki, H.;
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12. Darkin-Rattray, S. J.; Gurnett, A. M.; Myers, R. W.;
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4.4. NMR spectroscopy and structure calculation
NMR spectra were recorded on a JEOL spectrometer
operating at 500MHz in DMSO-d6 or CDCl3 using
TMS as internal standard. Spectra were recorded at var-
iable temperatures such as, 298, 303, 313, 323, and
333K, respectively. Assignments of proton resonances
were confirmed, when possible, by COSY, HOHAHA,
and NOESY experiments. Mixing times for NOESY
experiments were 200 and 400ms. Number of scans var-
ied between 32 and 64, and the number of points in t2
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M.; Goto, T.; Okuhara, M. J. Antibiot. 1994, 47, 301–310.
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Nishigaki, F.; Kawamura, I.; Shimomura, K. J. Antibiot.
1994, 47, 315–323.
16. Furumai, R.; Matsuyama, A.; Kobashi, N.; Lee, K.-H.;
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1
dimension was 512. H resonances were assigned using
3
a
standard procedures. JNH–C coupling constants for
nonoverlapping signals were determined from 1D spec-
tra with high digital resolution.
H
4.5. Molecular modeling studies
All calculations were performed on a Silicon Graphics
computer. The distance geometry program was used to
generate structures consistent with the distance con-
straints derived from the NOEs. Temperature coefficient
of NH protons indicating hydrogen bonds and / angles
calculated from JNH–Ha were used to filter out structures
that did not meet the experimental data. An error of
30ꢁ was tolerated for the / angles calculated from
JNH–Ha at this stage of refinement. Energy minimization
and molecular dynamics calculation were carried out
using the CHARMm program of Insight II using
CHARMm forcefield.
17. Gore, S. D.; Carducci, M. A. Exp. Opin. Invest. Drugs
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