Interaction of aliphatic cap group in inhibition of histone deacetylases by cyclic tetrapeptides

Article abstract of DOI:10.1016/j.bmc.2007.09.021

Inhibitors of histone deacetylases (HDACs) are a promising class of anticancer agents that effect gene regulation. To know the interaction of aliphatic cap groups with HDACs, cyclic tetrapeptide and bicyclic peptide disulfide hybrids were synthesized without aromatic ring in their macrocyclic framework. Benzene ring of l-Phe in chlamydocin was replaced with several aliphatic amino acids and also a fused bicyclic tetrapeptide was synthesized by ring closing metathesis using Grubb's first generation catalyst. The inhibitory activities of the cyclic peptides against histone deacetylase enzymes were evaluated, which demonstrated most of them are interesting candidates as anticancer agents.

Full text of DOI:10.1016/j.bmc.2007.09.021

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 437–445
Interaction of aliphatic cap group in inhibition
of histone deacetylases by cyclic tetrapeptides
Norikazu Nishino,a,* Gururaj M. Shivashimpi,a Preeti B. Soni,a
Mohammed P. I. Bhuiyan,a Tamaki Kato,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 4 August 2007; revised 11 September 2007; accepted 12 September 2007
Available online 15 September 2007
Abstract—Inhibitors of histone deacetylases (HDACs) are a promising class of anticancer agents that effect gene regulation. To
know the interaction of aliphatic cap groups with HDACs, cyclic tetrapeptide and bicyclic peptide disulfide hybrids were synthesized
without aromatic ring in their macrocyclic framework. Benzene ring of L-Phe in chlamydocin was replaced with several aliphatic
amino acids and also a fused bicyclic tetrapeptide was synthesized by ring closing metathesis using Grubb’s first generation catalyst.
The inhibitory activities of the cyclic peptides against histone deacetylase enzymes were evaluated, which demonstrated most of them
are interesting candidates as anticancer agents.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
ment of HDACs is found to be linked to a number of
malignant diseases.6–9 Inhibition of HDAC enzyme activ-
ity proved to reverse and induce re-expression of differen-
tiation inducing genes. It is therefore proposed that
HDACs are potential targets for the development of
low molecular weight anticancer drugs.
Reversible acetylation and deacetylation of the e-amino
groups of lysine residues on core histone tails by histone
acetyl transferase (HAT) and histone deacetylase
(HDAC) enzymes play an important role in the epigenetic
regulation of gene expression by altering the chromatin
architecture and controlling the accessibility of DNA
and histones to transcriptional regulators.1–4 Acetylation
of lysine residues in N-terminal tails of core histones re-
duces the interaction with DNA. In addition, acetylated
histone tails are specifically recognized and bound by
bromodomain containing proteins such as components
of the basal transcription machinery or histone acetyl-
transferases.5 These activator complexes containing
HAT activity have been shown to induce activation of
transcription, while deacetylation is associated with a
condensed chromatin structure resulting in the repression
of gene transcription. Therefore, alteration of equilibrium
in histone acetylation leads to transcriptional deregula-
tion. This deviation in equilibrium of histone acetylation
status either due to HAT mutation or abnormal recruit-
Several structurally unrelated natural and synthetic
compounds have been reported so far as HDAC inhib-
itors. Among them trichostatin A (TSA),10 depsipeptide
FK228,11–13 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
FR-22549732 are examples of naturally occurring
HDAC inhibitors. Inhibitors like suberoylanilide
hydroxamic acid (SAHA),33 straight chain TSA and
SAHA analogs,34–36 scriptaid,37 and the benzamide
MS-27538,39 have also been designed and synthesized.
Chlamydocin is a naturally occurring cyclic tetrapeptide
containing an aromatic ring in its cyclic framework, and
inhibits histone deacetylases potently,40 whereas HC-
toxins19–23 are also cyclic tetrapeptides which do not
have aromatic ring in their cyclic frameworks are still
inhibiting the HDACs. Recently we have reported cyclic
tetrapeptide based HDAC inhibitors (CHAPs),41,42 and
SCOPs43 (Fig. 1).
Keywords: Histone deacetylases; Chlamydocin; Grubb’s alkene meta-
thesis; Bicyclic tetrapeptide; Inhibitors.
*
Corresponding author. E-mail: nishino@life.kyutech.ac.jp
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.09.021

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N. Nishino et al. / Bioorg. Med. Chem. 16 (2008) 437–445
Figure 1. Natural and synthetic inhibitors.
In the present study, to know the interaction of aliphatic
cap group with the surface of HDAC enzymes, we syn-
thesized cyclic tetrapeptide and bicyclic peptide disulfide
hybrids without aromatic ring in their capping group.
For this, we initially focused on the benzene ring of
L-Phe in chlamydocin to replace it by several aliphatic
amino acids. Recently, ring closing metathesis of
x-unsaturated amino acids to synthesize constrained
peptides catalyzed by ruthenium complexes has been re-
ported.44–48 On the basis of these reports, to increase the
size of aliphatic cap group we designed and synthesized
a fused bicyclic peptide. We synthesized a cyclic tetra-
peptide by successive introduction of Boc-L-2-amino-8-
nonenoate (L-Ae9) and Boc-D-2-amino-8-nonenoate
(D-Ae9) in the peptide macrocycle, and incorporated a
second aliphatic ring by the well-known ring closing al-
kene metathesis reaction between these two amino acids.
We herein describe the account on the synthesis of
chlamydocin analogues, bicyclic peptide by alkene
metathesis, and a brief description of interesting biolog-
ical results.
by L-2-amino-7-(2-pyridyl)-disulfidyl heptanoyl (L-Am7
(S2Py)), equipped with a thiol function protected as
disulfide hybrid.
Cyclic tetrapeptides were prepared according to general
Scheme 1 by the conventional solution phase method,
starting from the Z-imino acid tert-butyl ester. After
the removal of Z-protection by catalytic hydrogenation,
the free amine was extracted and used for condensation
with a Z-amino acid using DCC/HOBt. Boc-L-2-amino-
7-bromoheptanoic acid (Ab7)50 was incorporated to
prepare the linear tetrapeptide. After removal of both
the side protections by treating with trifluoroacetic acid,
cyclization reaction was carried out by the aid of HATU
in DMF (2 mM) with minimum amount of DIEA (2.5
equiv). The yield of cyclic tetrapeptides was 50–60%
after purification by silica gel chromatography. The cyc-
lic tetrapeptide containing Ab7 was reacted with potas-
sium thioacetate (Wako, Japan) to convert the bromide
to thioacetate ester. Subsequent treatment with methyl-
amine in the presence of 2,20-dipyridyl disulfide gave the
desired cyclic tetrapeptides.
2. Results and discussion
2.1. Chemistry
To explore the role of aromatic ring in the chlamydocin
framework, we replaced its L-Phe residue by incorporat-
ing several aliphatic hydrophobic amino acids like L-ala-
nine (L-Ala), L-leucine (L-Leu), L-isoleucine (L-Ile), and
L-norleucine (L-Nle) at the AA position in Scheme 1.
Following cyclic tetrapeptides were synthesized accord-
ing to the general method described above: cyclo(-L-Am7
(S2Py)-Aib-L-Ala-D-Pro-) (2), cyclo(-L-Am7(S2Py)-Aib-
L-Leu-D-Pro-) (3), cyclo(-L-Am7(S2Py)-Aib-L-Ile-D-
Pro-) (4), cyclo(-L-Am7(S2Py)-Aib-L-Nle-D-Pro-) (5).
Our aim was to synthesize potent inhibitors of HDACs.
Initially, we synthesized chlamydocin analogues by
replacing the phenyl alanine in the cyclic framework of
compound 149 (Fig. 2). The compound 1 has similar
cyclic framework as in chlamydocin, with the L-2-amino
-9,10-epoxy-8-oxodecanoic acid (L-Aoe) being replaced
Further, to increase the size of capping group and bury
the amide group between L-Phe and Aib in chlamydo-
cin by a hydrophobic jacket, we attempted to synthe-
size a fused bicyclic peptide inhibitor. In a recently
published paper,50 we demonstrated the synthesis and
use of a-amino acids with an easily replaceable bromo
group at the x-position in the side chain of a-carbon
atom. In light of this view, we synthesized Boc-L-Ae9
and Boc-D-Ae9 with a terminal double bond. Our syn-
Figure 2. Structure of cyclo(-L-Am7(S2Py)-Aib-L-Phe-D-Pro-) (1).49

N. Nishino et al. / Bioorg. Med. Chem. 16 (2008) 437–445
439
Scheme 1. Synthetic strategy of cyclic tetrapeptides 2–5.
thesis strategy starts from diethyl tertiarybutoxycar-
bonylaminomalonate with 7-bromo-1-heptene as illus-
trated in Scheme 2. 7-Bromo-1-heptene was coupled
with diethyl Boc-aminomalonate by using NaOEt in
ethanol. One of the ethyl ester groups of the adduct
is hydrolyzed by one equivalent of 1 M NaOH at low
temperature (0–5 ꢁC). The oily monoacid monoester
after work-up is then subjected to the decarboxylation
by refluxing in toluene. After evaporation of toluene,
the resulting oily Boc-DL-Ae9-OEt (6) was purified by
silica gel column chromatography. Finally Boc-DL-
Ae9-OEt was subjected to the action of subtilisin Carls-
berg from Bacillus licheniformis (Sigma) in a mixture of
DMF and water (1:3, v/v). Boc-L-2-Ae9-OH (7) was
isolated as colorless oil. The recovered Boc-D-Ae9-
OEt (8) was hydrolyzed by 1 M NaOH in EtOH to
get Boc-D-Ae9-OH (9) with excellent purity in quanti-
tative yield. Boc-protected a-amino acids containing al-
kyl side chains of different lengths with a terminal
double bond can be prepared by this method. The dou-
ble bond in the side chain is stable enough and variety
of functional group transformation is possible in the
double bond of these amino acids. Recently, we re-
ported the synthesis of chlamydocin analogues bearing
carbonyl group51 by incorporating Boc-L-Ae9-OH in
the cyclic scaffold and various functional group trans-
formations to give potent HDAC inhibitors.
as cis-, identified by calculating the coupling constant
of olefinic protons (Fig. 3).
On the basis of above observations, we synthesized a
bicyclic peptide disulfide hybrid. The cyclic tetrapeptide
cyclo(-L-Ab7-D-Ae9-L-Ae9-D-Pro-) (13) was synthesized
according to general method explained in Scheme 1. The
second ring was synthesized by ring closing metathesis
between D-Ae9 and L-Ae9 using Grubb’s first generation
Ru catalyst. Further, the alkene group in (14), which is a
mixture of cis and trans, was catalytically hydrogenated
to give aliphatic fused bicyclic peptide containing Ab7
(15). The bicyclic tetrapeptide 15 was reacted with
potassium thioacetate (Wako, Japan) to convert the
bromide into thioacetate ester (16). Further treatment
with methylamine in the presence of 2,20-dipyridyl disul-
fide gave the desired product (17) (Scheme 4). All the
synthesized compounds were characterized by 1H
NMR and high resolution FAB-MS. Purity of the com-
pounds was determined by HPLC analysis and all the
compounds showed purity above 97%.
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 cells with the aid
of 0.1 mM of dithioltritol (DTT), which is known to re-
duce SAS bond, but does not contribute to the enzyme
activity.13 In the presence of DTT, 2,20-dipyridyl disul-
fide did not effect the enzyme inhibition (unpublished
data). Detailed experimental procedure for the prepara-
tion and assay were performed according to the reported
methods.13,51 In addition, to know the inhibitory activ-
ity of these compounds in cell based condition, we car-
ried out p21 promoter assay according to the
literature. The results of HDAC inhibitory activity and
the p2151 promoter assay of the compounds are shown
in Table 1.
The interesting application of x-unsaturated amino
acids (Aens), synthesis of constrained peptides by ring
closing alkene metathesis (RCM) reaction, was done
as the methodology development for bicyclic peptides.
These cyclic peptides can also mimic the b-turns in the
naturally occurring bioactive proteins and peptides.
And also it is possible to get information about the rota-
tion of CAN (/) and CAC (w) r-bonds. We synthesized
a dipeptide Boc-D-Ae9-L-Ae9-OBzl (10) using the solu-
tion phase peptide synthesis protocol (Scheme 3). Cycli-
zation was done by Grubb’s first generation Ru catalyst
and the resulting cyclized product was a mixture of two
isomers. Two diastereomers cis (11) and trans (12) were
separated by HPLC and found in 1:2 ratio in 50% total
yield. The major one was obtained as trans- and minor
These compounds inhibit within nanomolar range. For
comparison, inhibitory activity of trichostatin A is also
shown. The compound 1 which showed good activity

440
N. Nishino et al. / Bioorg. Med. Chem. 16 (2008) 437–445
Scheme 2. Synthesis of Boc-L-Ae9-OH and Boc-D-Ae9-OEt. Reagents and conditions: (a) Na, anhyd EtOH, reflux, 30 min; (b) Br(CH2)5ACH@CH2,
reflux, 4 h; (c) NaOH aq, EtOH, 4 h; (d) toluene, reflux, 3 h; (e) subtilisin Carlsberg, H2O/DMF (1:3), 4 h; (f) 1 M NaOH aq, EtOH, 4 h.
Scheme 3. Synthesis of cyclic dipeptide. Reagents and conditions: (a) Bzl-Br, DMF, TEA, 6 h; (b) 2 M HCl/dioxane, 3 h; (c) Boc-D-Ae9-OH (9),
DMF, TEA, HBTU, HOBt, 4 h; (d) Grubb’s first generation Ru catalyst, 48 h, CH2Cl2.
is taken as standard for this study. In compounds 2–5,
when L-Phe is replaced by several aliphatic amino acids,
we expected decrease in activity as compared to 1. They
showed almost comparable in vitro activity. As the p21
promoter assay needs the cell permeability of the inhib-
itors, therefore compounds with more hydrophobicity
should exhibit greater activity toward the HDACs.
Compounds 2–5 show good activity in p21 assay but
they were 10-fold less active than 1. This explains the
fact that, although these compounds do not contain aro-
matic ring in their cap group, still there is favorable
Figure 3. Selected 1H NMR spectra of (a) cis and (b) trans
compounds.
interaction of the macrocyclic cap group with residues
at the rim of HDACs active site pocket.
Scheme 4. Synthesis of bicyclic peptide disulfide hybrid. Reagents: (a) Grubb’s first generation catalyst, DCM; (b) AcOH, Pd–C, H2; (c) AcSK,
DMF; (d) MeNH2/MeOH, 2,20-dipyridyl disulfide, DMF.

N. Nishino et al. / Bioorg. Med. Chem. 16 (2008) 437–445
Table 1. HDAC inhibitory activity and p21 promoter assay data
441
resolution 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 mea-
sured 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 resonances were confirmed, when possible, by
correlated spectroscopy. Amino acids were coupled
using standard solution-phase chemistry with dic-
yclohexyl-carbodiimide (DCC), 2-(1H-benzatriazol-1-
Compound
IC50 (nM)
p21 promoter assay
HDAC1 HDAC4 HDAC6 EC1000 (nM)
TSA52
190
39
94
58
38
38
35
200
18
52
33
23
24
20
280
404
430
230
310
280
4900
190
45
149
2
750
630
260
270
160
3
4
5
17
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU), or O-(7-azabenzotriazoyl-1-yl)-1,1,3,3-tetram-
To increase the size of macrocyclic cap group, we syn-
thesized compound 17. This compound showed good
activity in both cell free and cell based conditions in
nanomolar range. This is probably because of the pres-
ence of a big flexible aliphatic fused ring which interacts
extensively with the rim residues of HDACs. Also cell
permeability is enhanced in this case, which is reflected
in its p21 activity data. Further, compound 17 was
poorly inhibited by HDAC6 compared to HDAC1
and HDAC4 (HDAC6/HDAC1 = 140) and its selectiv-
ity was better as compared to 1 (HDAC6/HDAC1
= 10.3). Thus, 17 shows better selectivity than the other
compounds listed in Table 1.
ethyluronium hexafluorophosphate (HATU).
4.1.1. Synthesis of cyclo(-L-Am7(S2Py)-Aib-L-Ala-D-
Pro-) (2). To a cooled solution of H-D-Pro-OtBu
(256 mg, 1.50 mmol), Z-L-Ala-OH (336 mg, 1.50 mmol),
and HOBtÆH2O (230 mg, 1.50 mmol) in dimethylform-
amide (DMF) (3 mL) was added DCC (375 mg,
1.80 mmol). The mixture was stirred at room tempera-
ture for 8 h. After completion of the reaction, DMF
was evaporated and the residue was dissolved 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 concen-
trated to remain an oily substance, which was purified
by silica gel chromatography using a mixture of chloro-
form and methanol (99:1) to yield Z-L-Ala-D-Pro-OtBu
(471 mg, 80%) as colorless oil. To the protected dipep-
tide (471 mg, 1.25 mmol) dissolved in acetic acid
(5 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
2 M Na2CO3 solution and dried over anhydrous
Na2CO3. Evaporation of ethyl acetate gave H-L-Ala-D-
Pro-OtBu (300 mg, 99%). To a cooled solution of H-L-
Ala-D-Pro-OtBu (300 mg, 1.22 mmol) and Z-Aib-OH
(300 mg, 1.22 mmol) in DMF (4 mL) were successively
added DCC (302 mg, 1.50 mmol), HOBtÆH2O (190 mg,
1.22 mmol). The product Z-Aib-L-Ala-D-Pro-OtBu
(460 mg, 80%) was obtained in the same manner as de-
scribed earlier as white foam. The tripeptide Z-Aib-L-
Ala-D-Pro-OtBu (460 g, 1.00 mmol) was subjected to
catalytic hydrogenation with Pd–C (50 mg) in acetic acid
(5 mL). The free amine was taken into ethyl acetate
(25 mL) by the aid of 2 M Na2CO3 solution (10 mL).
After drying over anhydrous Na2CO3, ethyl acetate
solution was evaporated to obtain H-Aib-L-Ala-D-Pro-
OtBu (327 mg, 99%). The N-terminal free tripeptide H-
Aib-L-Ala-D-Pro-OtBu (327 mg, 1.00 mmol) was cou-
pled with Boc-L-Ab7-OH (330 mg, 2.30 mmol) accord-
ing 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-Aib-L-Ala-D-
Pro-OtBu (473 mg, 80%) as a white foam. Boc-L-Ab7-
Aib-L-Ala-D-Pro-OtBu (474 mg, 0.75 mmol) was dis-
solved in TFA (3 mL) and left standing on ice for 3 h.
After evaporation of TFA, the residue was solidified
3. Conclusion
In order to develop HDAC inhibitors with aliphatic cap-
ping group, we replaced aromatic ring of phenylalanine
in the chlamydocin framework by several aliphatic ami-
no acids. We also successfully developed a method to
synthesize bicyclic tetrapeptide by ring closing metathe-
sis using Grubb’s first generation catalyst. Our synthetic
strategy allowed the convenient preparation of several
analogues of chlamydocin. The cell free and cell based
assay studies showed that most of the synthesized com-
pounds were potently inhibited HDACs and compound
17 has some selectivity among HDAC1 and HDAC6.
4. Experimental
4.1. General
Unless otherwise noted, all solvents and reagents were
reagent grade and used without purification. Flash chro-
matography was performed using silica gel 60 (230–
400 mesh) eluting with solvents as indicated. All
compounds were routinely checked by thin layer chro-
matography (TLC) and/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 gradi-
ent of A–B over 15 min with a flow rate of 2 mL/min,
with detection at 220 nm. FAB-mass spectra and high

442
N. Nishino et al. / Bioorg. Med. Chem. 16 (2008) 437–445
by trituration with ether to yield TFAÆH-L-Ab7-Aib-L-
Ala-D-Pro-OH (460 mg, 99%). To a volume of DMF
(100 mL) TFAÆH-L-Ab7-Aib-L-Ala-D-Pro-OH (460 mg,
0.75 mmol), HATU (342 mg, 0.90 mmol), and DIEA
(0.24 mL, 1.80 mmol) were added in five aliquots with
30 min time interval, while the solution was stirred vig-
orously. After the final addition the reaction mixture
was allowed to stir for an additional hour. Completion
of the cyclization reaction was monitored by HPLC
and then DMF was evaporated under reduced pressure.
The crude cyclic tetrapeptide was dissolved in ethyl ace-
tate and was successively washed with 10% citric acid,
4% sodium bicarbonate, and brine. Finally the ethyl ace-
tate solution was dried over anhydrous MgSO4 and fil-
tered. After evaporation of ethyl acetate, the residue
was purified by silica gel chromatography using a mix-
ture of chloroform and methanol (99:1, v/v) to yield cy-
clo(-L-Ab7-Aib-L-Ala-D-Pro-) (200 mg, 60%) as a white
foam after drying in vacuo. The cyclic tetrapeptide cy-
clo(-L-Ab7-Aib-L-Ala-D-Pro-) (200 mg, 0.45 mmol) was
dissolved in DMF (2 mL), then potassium thioacetate
(80 mg, 0.70 mmol) was added and stirred for 4–5 h.
The solvent was evaporated and the residue was dis-
solved in ethyl acetate and washed with 10% citric acid
and brine. The ethyl acetate solution was dried over
anhydrous MgSO4 and evaporated to thioester cyclo
(-L-Am7(acetyl)-Aib-L-Ala-D-Pro-) (197 mg, 89%). To
a solution of cyclo(-L-Am7(acetyl)-Aib-L-Ala-D-Pro-)
(197 mg, 0.40 mmol) in DMF (2 mL), under argon
atmosphere, 2,20-dipyridyldisulfide (200 mg, 0.90 mmol)
and 40% solution of CH3NH2/MeOH (0.22 mL,
2.00 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 chloroform and methanol (99:1, v/v) to yield cyclo(-L-
Am7(S2Py)-Aib-L-Ala-D-Pro-) as white foam (50 mg,
30%), HPLC, tR 5.98 min. HR-FAB-MS [M+H]+
584.2336 for C29H38O4N5S2 (calcd 584.2287). 1H
NMR (500 MHz, CDCl3): dH 1.27 (m, 2H), 1.34 (s, 3H),
1.36 (d, J = 7 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).
(t, 1H), 4.71 (dd, J = 2, 2.5 Hz, 1H), 5.98 (s, 1H), 7.08
(m, 1H), 7.21 (d, J = 7.2 Hz, 1H), 7.31 (d, J = 8 Hz,
1H), 7.72 (m, 1H), 8.46 (dd, J = 4 Hz, 1H).
4.1.3. Synthesis of cyclo(-L-Am7(S2Py)-Aib-L-Ile-D-Pro-)
(4). These compounds were synthesized using DL-
2MePhe instead of Aib according the procedure
described for 3, to yield cyclo(-L-Am7(S2Py)-Aib-L-Ile-
D-Pro-) (40 mg, 22%) as a white foam. HPLC, tR
7.88 min. HR-FAB-MS [M+H]+ 564.2659 for
1
C27H42O4N5S2 (calcd 564.2678). H NMR (500 MHz,
CDCl3): dH 0.86 (d, J = 7 Hz, 3H), 0.92 (t, 3H) 1.16
(m, 1H), 1.29 (m, 1H), 1.35 (s, 3H), 1.41 (m, 2H),
1.61–1.69 (m, 7H), 1.78 (s, 1H), 1.84 (m, 1H), 1.93 (m,
1H), 2.02 (m, 1H), 2.26 (m, 1H), 2.38 (m, 1H), 2.77 (t,
2H), 3.51 (m, 1H), 3.89 (m, 1H), 4.17 (ddd, J = 10.2,
10.2, 7.6 Hz, 1H), 4.55 (t, 1H), 4.74 (dd, J = 2,
2 Hz),1H), 5.97 (s, 1H), 7.08 (m, 1H), 7.19 (d,
J = 10.2 Hz, 1H), 7.31 (d, J = 10.5 Hz, 1H), 7.72 (d,
J = 8 Hz, 1H), 8.46 (dd, J = 5 Hz, 1H).
4.1.4. Synthesis of cyclo(-L-Am7(S2Py)-Aib-L-Nle-D-
Pro-) (5). This compound was synthesized according to
the procedure described to yield cyclo(-L-Am7(S2Py)-
Aib-L-Nle-D-Pro-) (80 mg 64%). HPLC, tR 6.92 min.
HR-FAB-MS [M+H]+ 584.2336 for C29H38O4N5S2
(calcd 584.2287). 1H NMR (500 MHz, CDCl3): dH
0.90 (t, 3H), 1.29–1.13 (m, 6H), 1.34 (s, 3H), 1.40 (m,
3H), 1.60–1.69 (m, 8H), 1.76 (s, 3H), 1.84 (m, 1H),
1.90 (m, 1H), 2.50 (m, 1H), 2.38 (m, 1H), 2.77 (t, 2H),
3.52 (m, 1H), 3.92 (m, 1H), 4.17 (ddd, J = 10.0, 10.1,
8 Hz, 1H), 4.72 (dd, J = 2, 2.5 Hz, 1H), 4.84 (dd,
J = 8.5, 8 Hz, 1H), 5.97 (s, 1H), 7.09 (m, 2H), 7.30 (d,
J = 10 Hz, 1H), 7.65 (m, 1H), 7.72 (d, J = 8 Hz, 1H),
8.46 (dd, J = 5 Hz, 1H).
4.1.5. Synthesis of Boc-L-2-amino-8-nonenoic acid (7).
Metallic sodium (4.60 g, 200 mmol) was dissolved in
absolute ethanol (200 mL) and diethyl tert-butoxycar-
bonylaminomalonate (55.06 g, 200 mmol) was added
to the solution. The reaction mixture was refluxed for
30 min for the complete removal of the labile hydrogen
and then 7-bromo-1-heptene (35.4 g, 200 mmol) was
added and refluxed for 4 h. The reaction mixture was
cooled on an ice bath and hydrolysis was carried out
by the treatment of 1 M aq NaOH (200 mL, 200 mmol).
After selective hydrolysis for 3 h at 0 ꢁC, ethanol was
evaporated and the unreacted 7-bromo-1-heptene was
removed by extraction with diethyl ether under basic
condition. Then the aqueous solution was extracted with
ethyl acetate at pH 3–4, by adding solid citric acid.
Finally ethyl acetate layer was washed with brine and
dried over anhydrous MgSO4. Ethyl acetate was evapo-
rated to obtain a colorless oil of monoester monoacid
(59.0 g, 87%). HPLC, tR 10.12 min. It was then dissolved
in toluene (175 mL) and refluxed for 3 h. The oily ester
after evaporation of toluene is then subjected to silica-
gel column chromatographic purification to get Boc-
DL-Ae9-OEt (6) as colorless oil (43.5 g, 83%). The result-
ing oily mass was suspended into a mixture of water
(450 mL) and DMF (150 mL) (1:3, v/v) solvent system
at 37 ꢁC using a mechanical stirrer and pH was adjusted
at about 7–8 by adding 1 M aq ammonia solution. Then
4.1.2. Synthesis of cyclo(-L-Am7(S2Py)-Aib-L-Leu-D-
Pro-) (3). These compounds were synthesized using L-
leucine instead of L-Phe according the method described
earlier, to yield cyclo(-L-Am7(S2Py)-Aib-L-Leu-D-Pro-)
(154 mg, 39%) HPLC, tR 8.10 min. HR-FAB-MS
[M+H]+ 564.2665 for C27H42O4N5S2 (calcd 564.2678).
1H NMR (500 MHz, CDCl3): dH 0.92 (d, J = 7 Hz,
3H), 1.01 (d, J = 7.2 Hz, 3H) 1.15 (m, 1H), 1.27 (m,
1H), 1.38 (s, 3H), 1.41 (m, 3H), 1.59–1.67 (m, 6H),
1.78 (s, 1H), 1.86 (m, 1H), 1.91 (m, 1H), 2.02 (m, 1H),
2.28 (m, 1H), 2.41 (m, 1H), 2.77 (t, 2H), 3.53 (m, 1H),
3.91 (m, 1H), 4.18 (ddd, J = 10, 10.1, 7.7 Hz, 1H), 4.53

N. Nishino et al. / Bioorg. Med. Chem. 16 (2008) 437–445
443
subtilisin Carlsberg from B. licheniformis (60 mg, 1 mg
enzyme per mmol of substrate) was added and pH was
maintained at 7–8 by continuous addition of 1 M aq
ammonia solution. The reaction was completed within
4 h. Water and DMF were evaporated and Boc-D-
Ae9-OEt (8) (22.2 g, 51%) was extracted with diethyl
ether under basic condition. Then the aqueous solution
was extracted with ethyl acetate as described above to
tR 13.22 min. HR-FAB-MS [M+H]+ 487.3214 for
C28H43O5N2 (calcd 487.3172). 1H NMR (500 MHz,
CDCl3): dH 1.17, 1.25, 1.33 and 1.64 (m, 12H) 1.42 (s,
9H), 1.78 and 1.62 (m, 2H), 1.87 and 1.65 (m, 2H),
1.94 and 2.02 (m, 4H), 4.15 (m, 1H), 4.78 (m, 1H),
5.17 (ABq, J = 12.5 Hz, 2H), 5.24 (d, J = 7.5 Hz, 1H),
5.34 (m, 2H), 6.49 (d, J = 7.5 Hz, 1H), 7.35 (m, 5H)
and trans isomer (12) (30.5 mg, 32%). HPLC, tR
15.02 min. HR-FAB-MS [M+H]+ 487.3151 for
C28H43O5N2 (calcd 487.3172). 1H NMR (500 MHz,
CDCl3): dH 1.07, 1.23 and 1.49 (m, 12H) 1.43 (s, 9H),
1.79 (m, 2H), 1.95 (m, 2H), 1.93 and 2.03 (m, 4H),
4.23 (m, 1H), 4.67 (m, 1H), 5.17 (ABq, J = 12.2 Hz,
2H), 5.23 (d, J = 8 Hz, 1H), 5.32 (m, 2H), 6.51 (d,
J = 7 Hz, 1H), 7.35 (m, 5H).
get Boc-L-Ae9-OH (7) as colorless oil (19.2 g, 49%).
22:4
D
HPLC, tR 8.38 min. ½aꢀ
3.0 (c = 1, MeOH). HRMS
[M+H]+ 272.1893 for C14H26NO4 (calcd 272.1862) H
NMR (500 MHz, CDCl3): dH 1.29–1.39 (m, c, d and e,
6H), 1.49 (s, 9H), 1.67 (m, 1H), 1.86 (m, 1H), 2.06 (m,
2H), 4.29 (m, 1H), 4.92–5.01 (m, 3H), 5.79 (ddt,
J = 17.2, 10.2, 6.5 Hz, 1H).
1
4.1.6. Synthesis of Boc-D-2-amino-8-nonenoic acid (9).
The ester Boc-D-Ae9-OEt (8) (22.2 g, 74.1 mmol) was
re-resoluted using 5 mg of subtilisin Carlsberg from
B. licheniformis using the procedure described in exper-
imental Section 4.1.5. The pure D-ester (20.0 g,
66.8 mmol) was dissolved in ethanol (70 mL) and hydro-
lyzed by 1 M NaOH (70 mL, 70 mmol) for 4 h at 0 ꢁC.
After completion of the reaction, ethanol was evapo-
rated and the aqueous solution of Boc-D-Ae9-OH (9)
was acidified to pH 3 by solid citric acid. Then the aque-
ous solution was extracted with ethyl acetate. Finally
ethyl acetate layer was washed with brine, dried over
anhydrous MgSO4, and evaporated to obtain Boc-D-
4.1.8. Synthesis of bicyclic disulfide hybrid (17). The pro-
tected dipeptide Boc-L-Ae9-D-Pro-OtBu (4.00 g,
9.43 mmol) synthesized according to the procedure as
described earlier was dissolved in 4 N HCl/dioxane
(40 mL) and the reaction solution was kept at room tem-
perature for 30 min. After evaporation of HCl/dioxane,
the residue was dissolved in ethyl acetate and washed
with 10% sodium carbonate to get the free amine H-L-
Ae9-D-Pro-OtBu (1.88 g, 61%). HPLC, tR 5.68 min.
MALDI-TOF-MS [M+Na]+ 325.7 for C18H33O3N2
(calcd 325.2). The free amine H-L-Ae9-D-Pro-OtBu
(1.88 g, 5.80 mmol) was coupled with Boc-D-Ae9-OH
(1.63 g, 6.00 mmol) following the same procedure men-
tioned earlier to obtain Boc-D-Ae9-L-Ae9-D-Pro-OtBu
(3.26 g, 97%). HPLC, tR 11.60 min. The tripeptide
Boc-D-Ae9-L-Ae9-D-Pro-OtBu (3.26 g, 5.60 mmol) was
treated with 4 N HCl/dioxane (20 mL) as described
above. The resulting free amine H-D-Ae9-L-Ae9-D-Pro-
OtBu was extracted into ethyl acetate and obtained as
a colorless oil (1.91 g, 72%, HPLC, tR 7.91 min). The
N-terminal deprotected tripeptide H-D-Ae9-L-Ae9-D-
Pro-OtBu (1.24 g, 2.59 mmol) and Boc-L-Ab7-OH
(1.26 g, 3.90 mmol) were condensed using the DCC/
HOBt protocol to afford Boc-L-Ab7-D-Ae9-L-Ae9-D-
Pro-OtBu (1.30 g, 63%), after work-up and flash chro-
matography. HPLC, tR 12.55 min. MALDI-TOF-MS
[M+Na]+ 805.4 for C39H67O7N479Br Na (calcd 805.4).
Sequential deprotection, and cyclization yielded cyclo
(-L-Ab7-D-Ae9-L-Ae9-D-Pro-) 13 (380 mg, 62%). HPLC,
tR 11.43 min. HR-FAB-MS [M+H]+ 609.3038 for
C30H50O4N479Br (calcd 609.3015) and 611.3006 for
C30H50N4O481Br (calcd 611.2995). The cyclic tetrapep-
tide 13 (235 mg, 0.38 mmol) was treated with Grubb’s
first generation catalyst (64 mg, 0.08 mmol) in anhy-
drous dichloromethane (50 mL) according to the similar
procedure as described earlier to yield an isomeric mix-
ture of fused bicyclic tetrapeptides 14 (100 mg, 45%).
HPLC, tR 10.10 min. HR-FAB-MS [M+H]+ 581.2714
for C28H46O4N479Br (calcd 581.2702). To the bicyclic
peptide 14 dissolved in acetic acid (10 mL), Pd–C
(100 mg) was added and the mixture was stirred under
hydrogen atmosphere for 10 h. The reaction was moni-
tored by TLC and HPLC. After completion of the reac-
tion, Pd–C was filtered off and the acetic acid was
evaporated to give 15 (90.6 mg, 98%). HPLC, tR
9.35 min. HR-FAB-MS [M+H]+ 583.2861 for
C28H48O4N479Br (calcd 583.2859). Further, compound
Ae9-OH (9) as colorless oil (17.9 g, 99%). HPLC, tR
8.33 min. ½aꢀ
22:1
D
2.8 (c = 1, MeOH).
4.1.7. Synthesis of cyclic dipeptide by alkene ring closing
metathesis (11) and (12). To a cooled solution of HClÆH-
L-Ae9-OBzl (900 mg, 3.00 mmol), Boc-D-Ae9-OH
(820 mg, 3.00 mmol) in DMF (10 mL), HBTU (1.71 g,
4.50 mmol), HOBtÆH2O (459 mg, 3.00 mmol), and tri-
ethyl amine (1.30 mL, 9.00 mmol) were added succes-
sively. The mixture was stirred at room temperature
for 4 h. After completion of the reaction, DMF was
evaporated and the residue was dissolved in ethyl ace-
tate and washed with 10% citric acid, 4% sodium bicar-
bonate, and brine, respectively. The ethyl acetate
solution was dried over anhydrous MgSO4 and concen-
trated to remain an oily substance, which was purified
by silica gel chromatography using a mixture of chloro-
form and methanol (99:1) to yield Boc-D-Ae9-L-Ae9-
OBzl (10) (1.40 g, 89%) as colorless foam. HPLC, tR
12.68 min. HR-FAB-MS [M+H]+ 515.3453 for
C30H47O5N2 (calcd 515.3485). To a solution of linear
dipeptide Boc-D-Ae9-L-Ae9-OBzl (107 mg, 0.20 mmol)
in anhydrous and degassed dichloromethane (25 mL),
a solution of Grubb’s first generation ruthenium catalyst
(33 mg, 0.04 mmol) in anhydrous and degassed dichlo-
romethane (5 mL) was added. The reaction mixture
was stirred at room temperature for 48 h. After the com-
pletion of reaction, dichloromethane was evaporated.
Finally diastereomers were separated by HPLC [col-
umn: YMC pack, ODS-A (250 · 10 mm), condition:
Gradient condition from solvent A to B 75–80% over
25 min with 220 nm detection, Sol. A; 10% acetonitrile
with 0.1% TFA and Sol. B; 100% acetonitrile with
0.1% TFA] to yield cis isomer (11) (17 mg, 18%). HPLC,

444
N. Nishino et al. / Bioorg. Med. Chem. 16 (2008) 437–445
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15 was transformed to its thioester 16 (41 mg, 81%).
HPLC, tR 9.00 min. HR-FAB-MS [M+H]+ 579.3578
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1
646.3478 for C33H52O4N5S2 (calcd 646.3461) H NMR
(500 MHz, CDCl3): dH 1.14–1.42 (m, 29H), 1.61 (m,
1H), 1.69 (m, 2H), 1.81 (m, 3H), 1.90–1.99 (m, 2H),
2.13 (m, 1H), 2.28 (m, 1H), 2.39 (m, 1H), 2.77 (t, 2H),
3.52 (ddd, J = 11.5, 11.5, 7.7 Hz, 1H), 4.08 (m, 1H),
4.24 (ddd, J = 10.5, 10.25, 7.25 Hz, 1H), 4.84 (m, 1H),
6.12 (d, J = 9.5 Hz, 1H), 6.22 (d, J = 10 Hz, 1H), 7.08
(m, 1H), 7.11 (d, J = 10 Hz, 1H), 7.65 (m, 1H), 7.72
(d, J = 7 Hz, 1H), 8.46 (dd, J = 1, 1 Hz, 1H).
Acknowledgment
This study was supported by the Program for Promo-
tion of Fundamental Studies in Health Sciences of the
National Institute of Biomedical Innovation (NIBIO).
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