Evaluation of functional groups on amino acids in cyclic tetrapeptides in histone deacetylase inhibition

Article abstract of DOI:10.1007/s00726-011-0947-6

The naturally occurring cyclic tetrapeptide, chlamydocin, originally isolated from fungus Diheterospora chlamydosphoria, consists of α-aminoisobutyric acid, l-phenylalanine, d-proline and an unusual amino acid (S)-2-amino-8-((S)-oxiran-2-yl)-8-oxooctanoic acid (Aoe) and inhibits the histone deacetylases (HDACs), a class of regulatory enzymes. The epoxyketone moiety of Aoe is the key functional group for inhibition. The cyclic tetrapeptide scaffold is supposed to play important role for effective binding to the surface of enzymes. In place of the epoxyketone group, hydroxamic acid and sulfhydryl group have been applied to design inhibitor ligands to zinc atom in catalytic site of HDACs. In the research for more potent HDAC inhibitors, we replaced the epoxyketone moiety of Aoe with different functional groups and synthesized a series of chlamydocin analogs as HDAC inhibitors. Among the functional groups, methoxymethylketone moiety showed as potent inhibition as the hydroxamic acid. On the contrary, we confirmed that borate, trifruoromethylketone, and 2-aminoanilide are almost inactive in HDAC inhibition.

Full text of DOI:10.1007/s00726-011-0947-6

Amino Acids (2012) 42:2103–2110
DOI 10.1007/s00726-011-0947-6
ORIGINAL ARTICLE
Evaluation of functional groups on amino acids in cyclic
tetrapeptides in histone deacetylase inhibition
•
Md. Shahidul Islam Mohammed P. I. Bhuiyan Md. Nurul Islam
•
•
•
Tienabe Kipassa Nsiama Naoto Oishi Tamaki Kato
•
•
•
•
Norikazu Nishino Akihiro Ito Minoru Yoshida
Received: 22 February 2011 / Accepted: 18 May 2011 / Published online: 3 June 2011
Ó Springer-Verlag 2011
Abstract The naturally occurring cyclic tetrapeptide,
chlamydocin, originally isolated from fungus Diheteros-
pora chlamydosphoria, consists of a-aminoisobutyric acid,
L-phenylalanine, D-proline and an unusual amino acid (S)-
2-amino-8-((S)-oxiran-2-yl)-8-oxooctanoic acid (Aoe) and
inhibits the histone deacetylases (HDACs), a class of reg-
ulatory enzymes. The epoxyketone moiety of Aoe is the
key functional group for inhibition. The cyclic tetrapeptide
scaffold is supposed to play important role for effective
binding to the surface of enzymes. In place of the
epoxyketone group, hydroxamic acid and sulfhydryl group
have been applied to design inhibitor ligands to zinc atom
in catalytic site of HDACs. In the research for more potent
HDAC inhibitors, we replaced the epoxyketone moiety of
Aoe with different functional groups and synthesized a
series of chlamydocin analogs as HDAC inhibitors. Among
the functional groups, methoxymethylketone moiety
showed as potent inhibition as the hydroxamic acid. On the
contrary, we confirmed that borate, trifruoromethylketone,
and 2-aminoanilide are almost inactive in HDAC
inhibition.
Introduction
Chemical modifications of histones by dynamic acetylation
and deacetylation of e-amino groups of lysine residues by
histone acetyl transferases (HATs) and HDACs are
involved in chromatin remodeling and play a fundamental
role in epigenetics, the regulation of gene expression
(Yoshida et al. 2003; Grozinger and Schreiber 2002;
Kouzarides 1999; Hassig and Schreiber 1997). The level of
modification of histone acetylation and its effects have
received enormous interest; in parallel, growing evidences
support their importance for basic cellular function such as
DNA replication, transcription, differentiation, and apop-
tosis. The lysine residues are positively charged at physi-
ological pH. Acetylation of lysine residues of histone
neutralizes the positive charge and allows the DNA to
unwind from the Histone core. This unwound DNA is
transcriptionally active. On the other hand, deacetylation
restores the ionic interaction and causes DNA to return to
the transcriptionally repressed conformation. Deregulation
of HATs and HDACs has been implicated in the formation
and development of several human cancers by changing the
expression pattern of various genes. It is therefore proposed
that HDACs are a potential targets for the development of
small molecular anticancer agents.
Keywords Histone deacetylase inhibitor Á Chlamydocin Á
Epoxyketone Á Functional group design
The HDAC enzymes have been divided into four groups
based on function and DNA sequence similarity. The first
two groups involve a zinc atom at the active center to
employ metalloprotease-like mechanism for cleavage of
acetyl group. As natural inhibitors of HDACs, two different
types of metabolites have been reported. One group
includes trichostatin A (Yoshida et al. 1990), which bears
hydroxamic acid functional moiety at the end of the mol-
ecule. The second group, cyclic tetrapeptides, comprises
trapoxins (Kijima et al. 1993), chlamydocin (Closse and
Md. S. Islam Á M. P. I. Bhuiyan Á Md. N. Islam Á
T. K. Nsiama Á N. Oishi Á T. Kato Á N. Nishino (&)
Graduate School of Life Science and Systems Engineering,
Kyushu Institute of Technology, Wakamatsu,
Kitakyushu 808-0196, Japan
e-mail: nishino@life.kyutech.ac.jp
A. Ito Á M. Yoshida
RIKEN, Saitama 351-0198, Japan
123

2104
Md. S. Islam et al.
Fig. 1 Structures of
chlamydocin and related
inhibitors bearing different
functional groups
O
O
OH
O
H
O
HN
CH3
N
CF3
O
S
S
N
O
OH
O
O
O
N
H
HN
N
H
N
N
H
N
H
N
H
O
N
H
N
H
O
O
O
O
O
1
3
Chlamydocin
2
4
5
Hugenin 1974), Cyl-1 and Cyl-2 (Hirota et al. 1973a, b, c),
FR-235222 (Xie et al. 2005), TAN-1746 (Yoshimura et al.
1995), and apicidins (Darkin-Rattray et al. 1996; Meinke
and Liberator 2001; Han et al. 2000). These cyclic tetra-
peptides have typically an a,b-epoxyketone, ketone, or
hydroxyketone moiety, as a part of quite unusual amino
acid in the cyclic peptides, which mimic an acetylated
lysine residue and interact with the active-site zinc ion.
In the past decade, a considerable number of synthetic
analogs have been reported as cyclic-tetrapeptide HDAC
inhibitors. The design of these analogs have been done
either by changing the amino-acids sequence in the cyclic
tetrapeptide framework to achieve high affinity of the
inhibitors with the enzyme surface and/or by the intro-
duction of different type of functional groups as zinc
ligands to achieve a strong binding affinity to the enzyme’s
active site. The functional groups introduced so far are
mostly electron rich (having lone pair of electron or double
bond) and included hydroxamic acid (1, Furumai et al.
2001), retro-hydroxamic acid (2, Nishino et al. 2004),
hybrid disulfide (3, Nishino et al. 2003), methylketone
(4, Bhuiyan et al. 2006), and trifluoromethylketone (5, Jose
et al. 2004) as shown in Fig. 1. On the other hand, more
functional groups have been introduced to the non-peptide
HDAC inhibitors, such as 2-aminoanilide (Suzuki et al.
1999; Saito et al. 1999), thioacetate (Suzuki et al. 2004),
borate (Suzuki et al. 2009), phosphate (Kapustin et al.
2003), and so on.
In order to enhance the binding capacity to HDACs as
inhibitors, we attempted to derive novel artificial amino
acids and introduce them into the chlamydocin scaffold.
We designed and synthesized cyclic tetrapeptides (6–11)
containing amino acids with functional groups shown in
Fig. 2. o-Phenylenediamine was condensed to L-aminos-
uberoyl residue (6). The 2-amino-6-heptenoyl residue was
converted to the corresponding boric acid (7). L-Amino-7-
bromoheptanoyl residue was treated with sodium azide and
then reacted with acetylene for triazolyl product (8).
L-Lysine residue was condensed with acryloyl chloride and
chloroacetic acid to afford 9 and 10. The methoxymeth-
ylketone moiety in 11 was built from L-2-amino-8-none-
noyl residue.
Results and discussion
Chemistry
Our aim was to evaluate the possible inhibitory function of
several chlamydocin analogs towards HDACs by introduc-
ing new and known functional groups in the chlamydocin
framework based on the inhibition mechanism. Therefore,
we proposed to synthesize chlamydocin analogs by replac-
ing the epoxyketone moiety of chlamydocin by different
functional groups. Synthesis of cyclic tetrapeptide was
carried out according to the general Scheme 1 by the
Fig. 2 Structures of novel
designed amino acid residues in
the cyclic tetrapeptides (6–11)
having different functional
groups. 6 2-aminoanilide,
7 boronic acid, 8 triazole,
9 acrylamide,
10 chloroacetamide,
11 methoxymethylketone
(Mmk)
123

Evaluation of functional groups on amino acids
2105
conventional solution phase method, starting from the Z-D-
Pro-OtBu. After the removal of Z-protection by catalytic
hydrogenation using H2/Pd–C, free amine was extracted and
coupled with Z-L-Phe-OH to give the protected dipeptide
Z-L-Phe-D-Pro-OtBu. The Z group of the resulting dipeptide
was deprotected by catalytic hydrogenation and coupled
with Z-Aib-OH to yield the tripeptide Z-Aib-L-Phe-D-Pro-
OtBu. The N-terminal of the tripeptide was deprotected and
coupled with (S)-7-bromo-2-(tert-butoxycarbonylamino)
heptanoic acid (Boc-L-7-bromo-Ahp-OH), (S)-2-(tert-but-
oxycarbonylamino)-6-heptenoic acid (Boc-L-Ae7-OH), Boc-
L-Lys(Z)-OH, (S)-2-(tert-butoxycarbonylamino)-8-nonenoic
acid (Boc-L-Ae9-OH) and (S)-8-(benzyloxy)-2-(tert-butoxy-
carbonylamino)-suberic acid (Boc-L-Asu(OBzl)-OH to yield
the relevant linear tetrapeptide. After deprotection of both
the terminals by trifluoroacetic acid (TFA), the linear tetrapep-
tide TFA salt was then cyclized under high dilution conditions in
dimethylformamide (DMF) using O-(7-azabenzotriazoyl-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)
or benzotriazole-1-yl-oxy-tris-(dimethylamino)-phospho-
nium hexafluorophosphate (BOP) to give the cyclic tetra-
peptide 12, 13, 15, 17, and 18, respectively. The yield of
cyclic tetrapeptides was 50–70% after purification by silica
gel chromatography. Terminal moieties of the synthesized
cyclic tetrapeptides were converted to several desired
groups depicted in Schemes 2, 3, 4, 5, and 6.
compound 8. The deprotection of Z group of compound 17
and subsequent treatment with acryloyl chloride and
2-chloroacetic acid yielded compounds 9 and 10, respec-
tively. The side chain double bond of 18 was oxidized to
epoxide 19 using m-CPBA. Regioselective ring opening of
the unsymmetrically substituted epoxide was carried with
sodium methoxide which was employed for oxidation with
Dess-Martin periodinane reagents to obtain compound 11.
All the final compounds were characterized by high reso-
lution FAB-MS. Purity of the compounds was determined
by HPLC analysis, and all the compounds showed purity
above 97%.
Enzyme inhibition and evaluation of biological activity
The synthesized compounds were assayed for HDAC
inhibitory activity using HDAC1, HDAC4, and HDAC6
enzymes prepared by 293T cells (Furumai et al. 2002). In
addition, to know the inhibitory activity of these com-
pounds in cell based condition, we carried out p21 pro-
moter assay according to the literature (Bhuiyan et al.
2006). Detailed experimental procedure for the preparation
and assay performed according to the reported methods.
The results of HDAC inhibitory activity and the p21 pro-
moter assay of chlamydocin (Furumai et al. 2001) and the
synthesized compounds are shown in Table 1. For com-
parison, inhibitory activity of chlamydocin is also shown.
Chlamydocin which showed good activity was taken as
standard for this study.
Catalytic hydrogenation of 12 gave the corresponding
acid which was coupled with o-phenylenediamine to obtain
compound 6. Regioselective addition of pinacolborane to
the alkene group of compound 13 was successfully carried
out, and the intermediate compound 14 was oxidized to
boronic acid using sodium periodate to obtain compound 7.
Compound 16 was obtained by treating compound 15 with
sodium azide. Treatment of 16 with acetylene yielded
In order to evaluate the inhibitory activity of triazole, we
introduced this new group in chlamydocin scaffold and
obtained compound 8. But this compound was inactive
which reveals that this group has no or very less binding
affinities for zinc. More recently, on the basis of the cata-
lytic mechanism, boronic acid containing HDAC inhibitors
bearing an a-amino acid moiety have been reported
(Suzuki et al. 2009). In order to compare the HDAC inhib-
itory activity, we introduced boronic acid in chlamydocin
scaffold and synthesized compound 7 which showed potent
activity against HDACs. As naturally occurring cyclic
tetrapeptides having epoxyketone moieties are irreversible
inhibitors, it has been proposed that epoxyketone moieties
undergo nucleophilic attack in the active sites. Guided by
this hypothesis, we synthesized compounds 9 containing
acrylamide group and compound 10 having chloroacet-
amide group. Both groups are susceptible to nucleophilic
attack, but these compounds displayed moderate activity.
This might be due to the less zinc-binding affinities for these
groups.
The cyclic tetrapeptide bearing methoxymethylketone
(11) showed the highest HDAC inhibition except the
hydroxamic acid (1), retrohydroxamate (2), and disulfide
(3) in the series of functional groups examined in the
Scheme 1 Synthesis of cyclic tetrapeptides
123

2106
Md. S. Islam et al.
Scheme 2 Reagents and
conditions: (a) MeOH, Pd–C,
H2, 12 h, 100%; (b) o-
phenylenediamine, BOP,
triethylamine, DMF, 12 h, 21%
Scheme 3 Reagents and conditions: (a) bis(1,5-cyclooctadiene)diiridium(I)dichloride ([Ir(cod)Cl]2), bis(diphenylphosphino)methane (DPPM),
pinacolborane, DCM, 48 h, 90%; (b) NH4OAc, NaIO4, Acetone, H2O, 20 h, 42%
Scheme 4 Reagents and conditions: (a) NaN3, DMF, 0°C, 4 h, 100%; (b) CuI, triethylamine, acetylene, DMF, 120 h, 55%
Scheme 5 Reagents and conditions: (a) AcOH, Pd–C, H2, 12 h, 72%; (b) acryloyl chloride, triethylamine, DCM, 12 h, 66%; (c) 2-chloroacetic
acid, DCC, DCM, 12 h, 67%
Scheme 6 Reagents and conditions: (a) m-chloroperoxybenzoic acid (m-CPBA), DCM, 15 h, 89%; (b) NaOMe/MeOH, 18 h, 68%; (c) Dess-
Martin periodinane (DMP), DCM, 8 h, 85%
123

Evaluation of functional groups on amino acids
2107
Table 1 HDAC inhibitory
No.
IC50 (lM)
EC1000 (lM)
activity and p21 promoter assay
data
HDAC1
HDAC4
HDAC6
Chlamydocin
0.00015
0.023
0.014
0.0040
1.1
–
1.100
0.065
0.39
–
1
0.034
0.033
0.0018
0.29
2.6
0.020
0.027
0.045
1.2
2
3
0.040
3.7
4
5
3.5
[100
[100
21
30.1
[25
3.6
6
24
33
7
2.1
0.15
52
8
65
[100
[100
[100
[100
86
9
15
19
[25
[25
0.043
10
11
13
18
0.038
0.028
chromatography was performed using silica gel 60
(230–400 mesh) eluting with solvents as indicated. All
compounds were routinely checked by thin-layer chroma-
tography (TLC) or high-performance liquid chromatogra-
phy (HPLC). TLC was performed on aluminum-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 A
50 mm, Merck). The mobile phases used were A: H2O with
0.1% TFA, B: CH3CN with 0.1% TFA using a solvent
gradient of A to B over 15 min with a flow rate of 2 mL/
min, with detection at 220 nm. FAB-mass spectra and
high-resolution mass spectra (HRMS) were measured on a
JEOL JMS-SX 102A instrument. In positive-mode MS anal-
ysis using 3-nitrobenzyl alcohol (NBA) as a matrix, boronic
acidisesterifiedwithNBA, andthemolecular weightdetected
corresponds to (M ? 2NBA-2H2O ? H)?. Amino acids
were coupled using standard solution phase chemistry with
1-hydroxybenzotriazole (HOBt), dicyclohexyl-carbodimide
(DCC), HATU or BOP.
Fig. 3 Possible interaction of methoxymethylketone with Zn in
HDAC
present study. The inhibition mechanism is estimated as
shown in Fig. 3. The interaction of the ketone as acetate
form is enhanced by methoxy group. The selectivity of
cyclic tetrapeptide inhibitors for HDAC 1 and 4 over
HDAC 6 could be explained by fact that HDAC 6 has a
˚
relatively wide rim (17.5 A) compared to HDAC 1 (12.5
˚
A) (Butler et al. 2010). The tetrapeptide scaffold is well
fit to the rim region of HDAC 1 and 4 which is less in the
case of HDAC 6. Thus, for HDAC inhibition, we dis-
covered novel functional group methoxymethylketone,
which could be easily synthesized in three steps from
2-amino-8-nonenoic acid-containing cyclic tetrapeptide
precursor.
Synthesis of linear tripeptide H-Aib-L-Phe-D-Pro-OtBu
To a cooled solution of H-D-Pro-OtBu (3.10 g, 18.1 mmol)
and Z-L-Phe-OH (5.41 g, 18.1 mmol) in DMF (36.0 mL),
HOBt.H2O (2.77 g, 18.1 mmol) and DCC (4.48 g,
21.7 mmol) were added, and the mixture was stirred at
room temperature for 12 h. After completion of the reac-
tion, DMF was evaporated, and the residue was dissolved
in ethyl acetate and washed with 10% citric acid, 4%
sodium bicarbonate and brine, respectively, and then dried
over anhydrous MgSO4. After evaporation of the ethyl
acetate, the residue was purified by silica gel chromatog-
raphy using a mixture of chloroform and methanol (99:1)
Experimental
General
Unless otherwise noted, all solvents and reagents were
of reagent grade and used without purification. Flash
123

2108
Md. S. Islam et al.
to yield Z-L-Phe-D-Pro-OtBu (7.52 g, 92%, HPLC, rt
6.55 min) as a colorless oil. The protected dipeptide
(7.52 g, 16.6 mmol) was dissolved in acetic acid (85 mL).
Pd–C (0.85 g) was added, and the mixture was stirred
under hydrogen atmosphere for 12 h. The reaction was
monitored by TLC and HPLC. After completion of the
reaction, Pd–C was filtered off, and the acetic acid was
evaporated. The residue was dissolved in ethyl acetate, and
the organic phase was washed with saturated sodium car-
bonate solution and dried over anhydrous Na2CO3. Evap-
oration of ethyl acetate gave H-L-Phe-D-Pro-OtBu (5.53 g,
83%, HPLC, rt 5.43 min) which was coupled with Z-Aib-
OH (3.25 g, 13.7 mmol) following the same procedure
described above to get Z-Aib-L-Phe-D-Pro-OtBu (7.01 g,
95%, HPLC, rt 8.03 min) as a white foam. The tripeptide
Z-Aib-L-Phe-D-Pro-OtBu (1.06 g, 2.0 mmol) was subjected
to hydrogenation in a similar manner as described for H-L-
Phe-D-Pro-OtBu to obtain H-Aib-L-Phe-D-Pro-OtBu
(0.880 g, 100%, HPLC, rt 4.56 min).
Cyclo(-L-Asu(2-aminoanilinyl)-Aib-L-Phe-D-Pro-)(6) Com-
pound 12 (5.35 g, 9.0 mmol) was dissolved in methanol
(45 mL) and Pd–C (0.45 g) was added. The solution was
stirred under hydrogen overnight. After filtration of Pd–C,
methanol was evaporated to yield cyclo(-L-Asu-Aib-L-
Phe-D-Pro-) (4.5 g, 100%). HPLC, rt 4.68 min. To a
cooled solution of cyclo(-L-Asu-Aib-L-Phe-D-Pro-) (0.20 g,
0.40 mmol) and o-phenylenediamine (0.21 g, 2.0 mmol) in
DMF (2 mL), triethylamine (0.084 mL, 0.6 mmol) and BOP
(0.265 g, 0.6 mmol) were added, and the mixture was stirred
at room temperature for 12 h. After completion of the reac-
tion, the compound was purified according to the procedure
reported for 12 to yield compound 6 as solid crystal (0.05 g,
21%). HPLC, rt 6.03 min, HRMS (FAB) calcd for
C32H43N6O5 [(M ? H)?] 591.3295, found 591.3278.
Cyclo(-L-Ae7-Aib-L-Phe-D-Pro-) (13) This compound
was synthesized according to the procedure reported for
compound 12 using Boc-L-Ae7-OH instead of Boc-L-
Asu(OBzl)-OH. The product is white foam (0.768 g, 82%).
HPLC, rt 7.91 min, LC–MS calcd for C25H34N4NaO4
[(M ? Na)?] 477.3, found 477.5.
Synthesis of cyclic tetrapeptides
Cyclo(-L-7-(pinacolboranyl)-Ahp-Aib-L-Phe-D-Pro-) (14)
A
Cyclo(-L-Asu(OBzl)-Aib-L-Phe-D-Pro-) (12) The N-ter-
minal free tripeptide H-Aib-L-Phe-D-Pro-OtBu (8.1 g,
20 mmol) was coupled with Boc-L-Asu(OBzl)-OH (7.56 g,
20 mmol) according to the method described above, and
the fully protected crude linear tetrapeptide was purified by
silica gel chromatography using a mixture of chloroform
and methanol (99:1) to yield Boc-L-Asu(OBzl)-Aib-L-Phe-
D-Pro-OtBu (9.7 g, 12.7 mmol, 64%). Boc-L-Asu(OBzl)-
Aib-L-Phe-D-Pro-OtBu (9.6 g, 12.5 mmol) was dissolved in
TFA (50 mL) at 0°C and kept for 3 h. After evaporation of
TFA, the residue was solidified by precipitating with ether
to yield H-L-Asu(OBzl)-Aib-L-Phe-D-Pro-OH as TFA salt
(9.0 g, 12.5 mmol, 100%). To a volume of DMF
(1,200 mL) solvent TFA salt (9.0 g, 12.5 mmol), HATU
(7.13 g, 18.7 mmol) and N,N-diisopropylethylamine
(DIEA) (7.61 mL, 43.7 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 cyclization reaction was monitored by HPLC, and then
DMF was evaporated under vacuo. The crude cyclic tet-
rapeptide was dissolved in ethyl acetate, and the solution
was washed successively by 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 chloroform
and methanol (99:1) to yield compound 12 (5.35 g,
9.0 mmol, 72%). HPLC, rt 7.98 min.
solution of [Ir(cod)Cl]2 (0.034 g, 0.05 mmol) and DPPM
(0.038 g, 0.10 mmol) in anhydrous and degassed dichloro-
methane (DCM) (5.0 mL) was flashed with Ar for 5 min
and stirred for 10 min at room temperature. After that,
to the solution, cyclo(-L-Ae7-Aib-L-Phe-D-Pro-) (0.227 g,
0.50 mmol) and pinacolborane (113 lL, 0.75 mmol) were
added, and the mixture was flashed with Ar for 5 min, then
stirred at room temperature for 2 days. The reaction mixture
was concentrated and purified according to the procedure
reported for 12 to give compound 14 (0.260 g, 90%) as a
white solid. HPLC, rt 8.94 min, LC–MS calcd for
C31H48BN4O6 [(M ? H)?] 583.4, found 583.5.
Cyclo(-L-7-(boronyl)-Ahp-Aib-L-Phe-D-Pro-) (7) To
a
solution of cyclo(-L-7-(pinacolboranyl)-Ahp-Aib-L-Phe-D-
Pro-) (0.200 g, 0.34 mmol) in acetone/H2O (4 mL/2 mL),
NaIO4 (0.215 g, 1.0 mmol) and NH4OAc (0.770 g,
1.0 mmol) were added, and the suspension was stirred at
room temperature for 20 h. Then acetone was evaporated,
and the reaction mixture was poured into water and
extracted with ethyl acetate. 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 (98:2) to yield compound 7 (0.070 g,
42%) as a white solid. HPLC, rt 6.10 min, LC–MS calcd
for C25H37BN4NaO6 [(M ? Na)?] 523.3, found 523.5.
HRMS (FAB) calcd for C39H48O10N6B [(M ? 2NBA
2H2O ? H)? 771.3525, found 771.3587.
123

Evaluation of functional groups on amino acids
2109
Cyclo(-L-7-bromo-Ahp-Aib-L-Phe-D-Pro-) (15) This com-
pound was synthesized according to the procedure reported
for compound 12 using Boc-L-7-bromo-Ahp-OH instead of
Boc-L-Asu(OBzl)-OH. The product is white foam (0.42 g,
60%). HPLC, rt 6.19 min, LC–MS calcd for
C25H36BrN4O4 [(M ? H)?] 535.2, found 535.3.
in DCM (1 mL), DCC (0.052 g, 0.25 mmol) was added, and
the mixture was stirred gently. After 2 h, cyclo(-L-Lys-Aib-L-
Phe-D-Pro-) (0.080 g, 0.17 mmol) was added, and the mixture
was stirred for 12 h at room temperature. After completion of
the reaction, the compound was purified as described for
compound12 to yield compound 10 (0.063 g, 67%). HPLC, rt
6.07 min, HRMS (FAB) calcd for C26H37ClN5O5 [(M ? H)?]
534.2483, found 534.2532.
Cyclo(-L-7-azide-Ahp-Aib-L-Phe-D-Pro-) (16) To a coo-
led solution of cyclo(-L-7-bromo-Ahp-Aib-L-Phe-D-Pro-)
(0.107 g, 0.20 mmol) in DMF (1 mL), NaN3 (0.016 g,
0.25 mmol) was added, and the mixture was stirred gently
for 4 h at room temperature. After completion of the
reaction, DMF was evaporated, and the residue was dis-
solved in ethyl acetate and washed with brine and then
dried over anhydrous MgSO4. After evaporation of the
ethyl acetate, the residue was precipitated with ether to
yield compound 16 (0.10 g, 100%). HPLC, rt 6.09 min,
HRMS (FAB) calcd for C25H36N7O4 [(M ? H)?]
498.2829, found 498.2822.
Cyclo(-L-Ae9-Aib-L-Phe-D-Pro-) (18) This compound
was synthesized according to the procedure reported for
compound 12 using Boc-L-Ae9-OH instead of Boc-L-
Asu(OBzl)-OH. The product is white foam (3.36 g, 87%).
HPLC, rt 9.34 min., HRMS (FAB) calcd for C27H39O4N4
[(M ? H)?] 483.2971, found 483.2971.
Cyclo(-L-Ae9(epo)-Aib-L-Phe-D-Pro-) (19) To a chilled
solution of compound 18 (4.72 g, 9.77 mmol) in anhydrous
DCM (200 mL) a solution of purified m-chloroperoxy-
benzoic acid (3.37 g, 19.54 mmol) in 100 mL of anhy-
drous DCM was added in aliquots over 30 min with
stirring. The reaction mixture was allowed to stir for 18 h
at room temperature. TLC and HPLC showed almost
complete conversion of the olefin into epoxide. The solu-
tion was then washed with 4% sodium bicarbonate and
brine followed by drying over anhydrous MgSO4 and fil-
tration. The compound was purified to yield cyclo(-L-
Ae9(epo)-Aib-L-Phe-D-Pro-) (4.33 g, 89%). HPLC, rt
7.13 min. HRMS (FAB) calcd for C27H39O5N4
[(M ? H)?] 499.2920, found 499.2891.
Cyclo(-L-7-(1H-1,2,3-triazol-1-yl)-Ahp-Aib-L-Phe-D-Pro-)
(8) To a solution of cyclo(-L-7-azide-Ahp-Aib-L-Phe-D-
Pro-) (0.10 g, 0.20 mmol) in DMF (2 mL), CuI (0.8 mg,
4 lmol) and triethylamine (0.22 mmol, 31 lL) were
added. Acetylene gas was passed through the mixture
for 120 h. DMF was evaporated, and the residue was
dissolved in ethyl acetate and washed with brine and
then dried over anhydrous MgSO4. After evaporation of
the ethyl acetate, the residue was purified by HPLC
(30-55% CH3CN, 50 mm column) to yield compound
8 (0.029 g, 55%). HPLC, rt 5.67 min, HRMS (FAB)
calcd for C27H38N7O4 [(M ? H)?] 524.2985, found
524.3020.
Cyclo(-L-Ae9(Mmk)-Aib-L-Phe-D-Pro-) (11) The epoxide
(499 mg, 1.0 mmol) was dissolved in 0.5 M sodium
methoxide in methanol (2 mL). The reaction mixture was
stirred at 28°C for 16 h and then quenched by adding acetic
acid. Ethyl acetate was added to the reaction solution, and
the compound was purified as described for compound 12
to yield the corresponding ether (361 mg, 68%). HPLC, rt
6.27 min, HRMS (FAB) calcd for C28H43O6N4
[(M ? H)?] 531.3183, found 531.3204. To a solution of
vicinal alkoxy ether (318 mg, 0.60 mmol) in anhydrous
DCM (12 mL) DMP (1.53 g, 3.6 mmol) was added. The
reaction mixture was stirred at 25°C for 3 h. TLC and
HPLC showed complete conversion of the starting alcohol.
The reaction mixture was then diluted by adding diethyl
ether (12 mL) followed by careful addition of a saturated
solution of sodium bicarbonate containing 2.68 g of
sodium thiosulfate pentahydrate (Na2S2O3Á5H2O). After
stirring for 10 min, the suspension became a clear solution.
The organic layer was separated from the aqueous layer
and purified as described for compound 12 to yield the
corresponding ketone (270 mg, 85%). HPLC, rt 6.16 min.
HRMS (FAB) calcd for C28H41O6N4 [(M ? H)?]
529.3026, found 529.3026.
Cyclo(-L-Lys(Z)-Aib-L-Phe-D-Pro-) (17) This compound
was synthesized according to the procedure reported for
compound 12 using Boc-L-Lys(Z)-OH instead of Boc-L-
Asu(OBzl)-OH. The product is white foam (1.20 g, 71%).
Cyclo(-L-Lys(2-propenoyl)-Aib-L-Phe-D-Pro-) (9) Cyclo
(-L-Lys(Z)-Aib-L-Phe-D-Pro-) (0.30 g, 0.5 mmol) was
subjected to hydrogenation as described for H-L-Phe-D-Pro-
OtBu to yield cyclo(-L-Lys-Aib-L-Phe-D-Pro-) (0.16 g,
72%). HPLC, rt 4.85 min. To a cooled solution of cyclo(-L-
Lys-Aib-L-Phe-D-Pro-) (0.080 g, 0.17 mmol) in DCM
(1 mL), triethylamine (35 lL, 0.25 mmol) and acryloyl
chloride (20 lL, 0.25 mmol) were added and stirred for
12 h at room temperature. After completion of the reaction,
DCM was evaporated, and the residue was purified as
described for compound 12 to yield compound 9 (0.059 g,
66%). HPLC, rt 5.96 min, HRMS (FAB) calcd for
C27H38N5O5 [(M ? H)?] 512.2873, found 512.2780.
Cyclo(-L-Lys(2-chloroacetyl)-Aib-L-Phe-D-Pro (10) To a
cooled solution of 2-chloroacetic acid (0.048 g, 0.50 mmol)
123

2110
Md. S. Islam et al.
Hirota A, Suzuki A, Suzuki H, Tamura S (1973b) Isolation and
biological activity of cyl-2, a metabolite of Cylindrocladium
scoparium. Agric Biol Chem 37:643–647
Hirota A, Suzuki A, Tamura S (1973c) Characterization of four amino
acids constituting cyl-2, a metabolite from Cylindrocladium
scoparium. Agric Biol Chem 37:1185–1189
Jose B, Oniki Y, Kato T et al (2004) Novel histone deacetylase
inhibitors: cyclic tetrapeptide with trifluoromethyl and penta-
fluoroethyl ketones. Bioorg Med Chem Lett 14:5343–5346. doi:
10.1016/j.bmcl.2004.08.016
Conclusion
In order to develop HDAC inhibitors with various func-
tional groups, we replaced the epoxyketone moiety of
chlamydocin scaffold with several known and new func-
tional groups and evaluated these functional groups on the
scaffold as HDAC inhibitory activity. The cell free and
cell-based assay study showed that one of the functional
groups potently inhibited HDACs in inhibition mechanism.
Most of the functional groups have selectivity towards
HDAC1 and HDAC4.
´
Kapustin GV, Fejer G, Jennifer L, Gronlund JL et al (2003)
Phosphorus-based SAHA analogues as histone deacetylase
inhibitors. Org Lett 5:3053–3056. doi:10.1021/ol035056n
Kijima M, Yoshida M, Suguta K et al (1993) Trapoxin, an antitumor
cyclic tetrapeptide, is an irreversible inhibitor of mammalian
histone deacetylase. J Biol Chem 268:22249–22435
Conflict of interest The authors declare that they have no conflict
of interest.
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Meinke PT, Liberator P (2001) Histone deacetylase: a target for
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