Chlamydocin analogs bearing carbonyl group as possible ligand toward zinc atom in histone deacetylases

Article abstract of DOI:10.1016/j.bmc.2005.12.063

A series of chlamydocin analogs with various carbonyl functionalities were designed and synthesized as histone deacetylase (HDAC) inhibitors. Chlamydocin is a cyclic tetrapeptide containing an epoxyketone surrogate in the side chain which makes it irrever

Full text of DOI:10.1016/j.bmc.2005.12.063

Bioorganic & Medicinal Chemistry 14 (2006) 3438–3446
Chlamydocin analogs bearing carbonyl group as possible
ligand toward zinc atom in histone deacetylases
Mohammed P. I. Bhuiyan,a Tamaki Kato,a Tatsuo Okauchi,b Norikazu Nishino,a,*
Satoko Maeda,c Tomonori G. Nishinoc and Minoru Yoshidac
aGraduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu 808-0196, Japan
bFaculty of Engineering, Kyushu Institute of Technology, Kitakyushu 804-8550, Japan
cRIKEN, Saitama 351-0198, Japan
Received 12 December 2005; revised 28 December 2005; accepted 30 December 2005
Available online 24 January 2006
Abstract—A series of chlamydocin analogs with various carbonyl functionalities were designed and synthesized as histone deacetyl-
ase (HDAC) inhibitors. Chlamydocin is a cyclic tetrapeptide containing an epoxyketone surrogate in the side chain which makes it
irreversible inhibitor of HDACs, whereas apicidins are a class of cyclic tetrapeptides that contain an ethylketone moiety as zinc
ligand. We replaced the epoxyketone moiety of chlamydocin with several ketones and aldehyde to synthesize potent reversible
and selective HDAC inhibitors. The inhibitory activity of the cyclic tetrapeptides against histone deacetylase enzymes were evalu-
ated and the result showed most of them are potent inhibitors. Some of them have remarkable selectivity among the HDACs.
ꢀ 2006 Published by Elsevier Ltd.
1. Introduction
represent exciting opportunity for the development of
therapeutics for treatment of cancers.
Dynamic acetylation and deacetylation of the e-amino
groups of lysines by histone acetyl transferase (HAT)
and histone deacetylase (HDAC) enzymes play a funda-
mental role in the regulation of gene expression by alter-
ing the chromatin architecture and controlling the
accessibility of DNA to transcriptional regulators.1–4
Growing evidences support that the imbalance in his-
tone acetylation can lead to transcriptional deregulation
of genes that are involved in the control of cell cycle pro-
gression, differentiation, and/or apoptosis. Aberrant his-
tone acetylation caused by the disrupted HAT activity
or abnormal recruitment of HDACs has been related
to carcinogenesis.5–8 Inappropriate recruitment of
HDACs provides a molecular mechanism by which
genes necessary for proper differentiation or growth ar-
rest can be silenced leading to excessive proliferation.
Owing to the link between transcriptional repression
and the recruitment of HDACs, inhibitors of this enzy-
matic activity are expected to reverse repression and to
induce re-expression of differentiation-inducing genes.
Therefore design and synthesis of HDAC inhibitors
A wide variety of structurally unrelated natural and
synthetic compounds have so far been reported exhib-
iting HDAC inhibiting activity. Among them trichos-
tatin A (TSA),9 depsipeptide FK228,10–12 and the
cyclic tetrapeptide family including trapoxin (TPX),13
chlamydocin,14 TAN-1746,15 FR-235222,16 9,10-desep-
oxy-9-hydroxy-chlamydocin,17 HC toxins,18–22 Cyl-1,
Cyl-2,23–25 WF-3161,26 apicidin,27–30 FR-22549731 are
examples of naturally occurring HDAC inhibitors.
Inhibitors like suberoylanilide hydroxamic acid
(SAHA),32 straight chain TSA and SAHA ana-
logs,33–35 scriptaid,36 and the benzamide MS-27537,38
have also been designed and synthesized.
The epoxyketone functionality of 2-amino-8-oxo-9,10-
epoxydecanoic acids (Aoe) in trapoxin and other related
cyclic tetrapeptides is supposed to react irreversibly to
the functional nucleophile near the Zn atom of HDAC’s
active site.13 Among the Aoe-containing cyclic tetrapep-
tides, chlamydocin is found to be a highly potent histone
deacetylase (HDAC) inhibitor, inhibiting HDA activity
in vitro with an IC50 value of 1.3 nM.39 Though the
activity is mainly due to the epoxyketone moiety, there
are evidences which support that the cyclic tetrapeptide
framework also has a significant structural role.
*
Corresponding author. Tel./fax: +81 93 695 6061; e-mail:
nishino@life.kyutech.ac.jp
0968-0896/$ - see front matter ꢀ 2006 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2005.12.063

M. P. I. Bhuiyan et al. / Bioorg. Med. Chem. 14 (2006) 3438–3446
3439
The macrocycle provides specific hydrophobic interac-
tion with the rim of HDAC enzymes.40
chlamydocin framework equipped with ketones and alde-
hyde can show better inhibitory activity in terms of
reversibility and selectivity. For that, our strategy was
to replace the Aoe of chlamydocin with a terminal double
bond containing amino acid, 2-amino-8-nonenoic acid
(Ae9),44 and convert the olefin into aldehyde and various
ketones. The zinc binding functional groups, with the
proposed inhibitory mechanism, selected for this study
is shown in Figure 1.
On the other hand, apicidins are a class of cyclic tetra-
peptides that do not contain the classical electrophilic
epoxyketone moiety yet are potent inhibitors of
HDACs. SAR showed that the tryptophan residue in
the cyclic tetrapeptide and C-8 ketone in the side chain
are key constituents for HDACs’ inhibitory activity.28
Recently, we have reported a series of cyclic tetrapep-
tide-based HDAC inhibitors, CHAPs41,42 and SCOPs,43
by hybridizing the natural trapoxin, Cyl-1, Cyl-2,
WF3161, chlamydocin, and HC-toxins with trichostatin
and FK228, respectively.
2. Results and discussion
Our aim was to synthesize potent inhibitors of HDACs
with the chlamydocin-cyclic tetrapeptide scaffold.
Therefore, we intended to synthesize a chlamydocin ana-
log containing L-Ae9 using a solution-phase peptide syn-
thesis strategy as shown in Scheme 1. t-Butyl-protected
D-proline hydrochloride salt was coupled with Z-L-Phe
to give the protected dipeptide Z-L-Phe-D-Pro-OtBu in
quantitative yield. Z protection of the resulting dipep-
tide was then removed quantitatively by catalytic hydro-
genation using H2/Pd-C. The deprotected dipeptide was
In this paper, we proposed to synthesize conceptual
hybrids of apicidin and chlamydocin in which epoxyke-
tone moiety of chlamydocin is replaced by simple alde-
hyde and ketone groups as seen in apicidin. From the
SAR of naturally occurring cyclic tetrapeptide-based
inhibitors, it has been confirmed that a carbonyl group
at C-8 position is essential for the HDAC inhibitory
activity.14,29 Based on this fact, we expect that the
Figure 1. Proposed HDAC hydrolysis mechanism (a) and possible interaction of aldehyde and ketones (b–e) with Zn of HDAC. (a) Acetylated
lysine, (b) aldehyde, (c) methylketone, (d) bromomethylketone, and (e) hydroxymethylketone.
Scheme 1. Reagents: (a) Z-L-Phe, DCC, HOBt, Et3N; (b) H2/Pd-C; (c) Z-Aib, DCC, HOBt; (d) Boc-L-Ae9, DCC, HOBt; 4) (e) TFA; (f) HATU,
DIEA; (g) m-CPBA.

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M. P. I. Bhuiyan et al. / Bioorg. Med. Chem. 14 (2006) 3438–3446
again coupled with Z-Aib to yield the tripeptide Z-Aib-
L-Phe-D-Pro-OtBu in 87% yield. The N-terminal of the
tripeptide was deprotected and coupled with a Boc-L-
Aen9 to yield the linear tetrapeptide Boc-L-Ae9-Aib-L-
Phe-D-Pro-OtBu in excellent yields. After deprotection
of both the terminals by trifluoroacetic acid, the linear
tetrapeptide TFA salt was then cyclized under high
dilution conditions in dimethylformamide using
O-(7-azabenzotriazoyl-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) to give the cyclic tetra-
peptide cyclo(-L-Ae9-Aib-L-Phe-D-Pro-) in 87% yields
(Scheme 1). Except for the cyclization step the coupling
reagents used were DCC and HOBt throughout the
synthesis.
To know the influence of C-9 methyl group on HDAC
inhibitory activity, we synthesized a terminal aldehyde.
The synthesis was initiated by oxidizing the side-chain
double bond of 1 to the chlamydocin-diol analog (6)
using
microencapsulated
osmium
tetroxide
(MC-OsO4)45 and then oxidative cleavage of the diol 6
by sodium periodate led to yield the chlamydocin-alde-
hyde analog 7 (Scheme 3).
In order to explore the effect of a hydroxyl substituent
on the C-9 methyl group, the diol 6 was employed for
further modification to get a hydroxymethylketone
functionality. In addition, hydroxymethylketone is
the functional group in the chlamydocin analogs 9,10-
desepoxy-9-hydroxy-chlamydocin, TAN-1746, and
FR-235222.15–17 These compounds with a C-10 methyl
group could be compared with the synthesized
chlamydocin-hydroxymethylketone analog to evaluate
an effect of the terminal methyl group on the inhibitory
activity. First, the primary alcohol group was selectively
protected by t-butyldimethylsilyl chloride (TBDMSCl)
to get the corresponding 8. The protected cyclic tetra-
peptide was then oxidized to the ketone 9 using Dess–
Martin periodinane. TBDMS group of the ketone was
deprotected by the action of tetrabutylammonium fluo-
ride (TBAF) to get the desired hydroxymethylketone 10
(Scheme 4).
As the epoxides are fairly reactive groups and convenient
precursors, we planned to use the chlamydocin-epoxide
analog as an intermediate for ketone functionalization.
Accordingly, the side-chain double bond of 1 was oxi-
dized to the epoxide (2) using m-chloroperoxybenzoic
acid (Scheme 1). Initially, we checked the effect of halo-
gen substitution on the C-9 methyl group by synthesizing
a bromomethylketone from the epoxide 2. Regioselective
ring opening of the unsymmetrically substituted epoxide
was successfully carried out with lithium bromide
to yield the vicinal bromohydrin (3), which was subse-
quently employed for oxidation using Dess–Martin
periodinane reagent to obtain the chlamydocin-bro-
momethylketone analog (4) (Scheme 2). To synthesize
a C-8 methylketone, analogous to the apicidin’s
side-chain functionality, we reduced the terminal bro-
momethyl group of 4 by Zn-AcOH to derive the
chlamydocin-ketone analog (5) (Scheme 2).
The synthesized ketones, aldehyde, and intermediate
epoxide were assayed for HDAC inhibitory activity
using HDAC1, HDAC4, and HDAC6 prepared from
293T cells.12 Detailed experimental procedure for the en-
zyme preparation and assay methods are given in the
Scheme 2. Reagents: (a) LiBr, AcOH; (b) Dess–Martin periodinane; (c) Zn, AcOH.
Scheme 3. Reagents: (a) MC-OsO4, N-methylmorpholine-N-oxide; (b) NaIO4.

M. P. I. Bhuiyan et al. / Bioorg. Med. Chem. 14 (2006) 3438–3446
3441
Scheme 4. Reagents: (a) TBDMSCl, DIEA, DMAP; (b) Dess–Martin periodinane; (c) TBAF.
experimental section. As a control experiment, the
chlamydocin analogs with double bond and diol were
also examined for HDAC inhibitory activity. The results
of the HDAC inhibitory activity and the p21 promoter
assay of the compounds are shown in Table 1.
hydroxy-chlamydocin, TAN-1746, and FR-23522215–17
as possible metabolic products from the epoxyketone
moiety also showed potent HDAC inhibitory activity.
The hydroxymethylketone 10 was specific toward
HDAC4 compared with HDAC1 and 6. It is about four
times more active toward HDAC4 than HDAC1 and 15
times more active than HDAC6. The compound was
most selective among the inhibitors listed in Table 1.
The chlamydocin-epoxide analog 2 was active in micro-
molar concentrations, whereas dehydro-chlamydocin in
which the C-8 carbonyl group of chlamydocin is re-
placed by hydroxyl group was inactive.14 This result
indicates that a C-8 carbonyl as well as a C-8,9 epoxide
can inhibit HDACs.
The chlamydocin analogs bearing carbonyl groups were
examined for hyperacetylation of H3 and H4 histone
proteins as a result of HDAC inhibition. We observed
a concentration-dependent inhibition profile in the Wes-
tern blot analysis of H3 and H4 acetylation. The efficien-
cies were in order of compounds 10 ꢁ 4 > 5.
The chlamydocin-aldehyde (7), which lacks the C-9
methyl group, shows moderate HDAC inhibitory activ-
ity. A higher activity of chlamydocin-bromomethylke-
tone (4) than that of the corresponding methylketone
(5) reveals that the presence of a ꢀI group with high
polarizability (soft basicity) and nucleophilicity on C-9
methylene increases the inhibitory activity.
3. Conclusion
In order to develop novel ketone-based HDAC inhibi-
tors, design and synthesis of a series of chlamydocin
analogs equipped with various carbonyl functionalities
have been performed successfully by using unique amino
acid L-2-amino-8-nonenoic acid. Our synthetic strategy
allowed the convenient preparation of the simple func-
tional group for potent HDAC inhibition. These inhib-
itors have displayed potent HDAC inhibitory activity
under cell free and cell-based conditions. Some of the
synthesized inhibitors have selectivity among the
HDAC1, HDAC4, and HDAC6. Considering the activ-
ity and selectivity together, the hydroxymethylketone
group as a zinc ligand is most promising among the
compounds synthesized.
The chlamydocin-hydroxymethylketone analog 10 in
which a hydrogen of the C-9 methyl group of 5 was re-
placed by a hydroxyl group was about fivefold more ac-
tive than the parent methylketone (5) (Table 1). Whereas
the corresponding diol 6 in which the C-8 position had a
hydroxyl group instead of the carbonyl was inactive.
This observation further supports the fact that a C-8 car-
bonyl group is essential and an atom carrying a lone pair
of electrons, b to the C-8 carbonyl group, has positive ef-
fects on the activity. It should be noted that the hydrox-
ymethylketone moiety found in 9,10-desepoxy-9-
Table 1. HDAC inhibitory activity and p21 promoter activity data for
the chlamydoc in analogs
4. Experimental
4.1. General
Compound
IC50 (lM)
EC1000 (lM)
HDAC1
0.0190
>100
4.69
HDAC4
HDAC6
0.0280
TSA46
0.0200
>100
4.05
0.190
>250
22.7
Unless otherwise noted, all solvents and reagents were of
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 chromatography
(TLC) or high-performance liquid chromatography
(HPLC). TLC was performed on aluminum-backed
silica gel plates (Merck DC-Alufolien Kieselgel 60
1
2
>100
>100
4
0.0619
1.08
0.0595
0.290
0.621
3.70
>100
14.1
0.887
0.344
1.20
5
6
>100
>100
NT
7
10
4.33
0.223
1.29
0.0604
20.9
0.234
NT, not tested.

3442
M. P. I. Bhuiyan et al. / Bioorg. Med. Chem. 14 (2006) 3438–3446
F254) with spots visualized by UV light or charring. Ana-
lytical HPLC was performed on a Hitachi instrument
equipped with a chromolith performance RP-18e col-
umn (4.6 Æ 50 mm, Merck). The mobile phases used were
A: H2O with 10% CH3CN and 0.1% TFA, B: CH3CN
with 0.1% TFA using a solvent gradient of A to B over
15 min with a flow rate of 2 mL/min, with detection at
220 nm. FAB-mass spectra and high-resolution mass
spectra (HRMS) were measured on a JEOL JMS-SX
102A instrument. NMR spectra were recorded on a
JEOL JNM A500 MHz spectrometer. Unless otherwise
stated, all NMR spectra were measured in CDCl3 solu-
coupled with Boc-L-Ae9-OH (5.43 g, 20.0 mmol) accord-
ing to the method described earlier and the fully protect-
ed crude linear tetrapeptide was purified by silica gel
chromatography using a mixture of chloroform and
methanol (99:1) to yield Boc-L-Ae9-Aib-L-Phe-D-Pro-
OtBu (12.3 g, 94%) as a white foam. Boc-L-Ae9-Aib-L-
Phe-D-Pro-OtBu (12.3 g, 18.8 mmol) was dissolved in
TFA (50 mL) at 0 ꢁC and kept for 3 h. After evaporation
of TFA, the residue was solidified by titration with ether
to yield H-L-H-L-Ae9-Aib-L-Phe-D-Pro-OH as TFA salt
(11.6 g, 100%). HPLC, rt 5.96 min. HR-FAB MS
[M+H]+ 501.3050 for C27H41O5N4 (calcd 501.3077). To
a volume of DMF (800 mL) H-L-Ae9-Aib-L-Phe-D-
Pro-OH Æ TFA (8.00 mmol, 4.92 g), HATU (3.64 g,
9.60 mmol), and DIEA (4.45 mL, 25.6 mmol) were add-
ed 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 moni-
tored by HPLC and then DMF was evaporated under re-
duced pressure. The crude cyclic tetrapeptide 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 cyclo(-L-Ae9-Aib-L-Phe-D-Pro-)
(1) (3.36 g, 87%) as a white foam after drying in vacuo.
1
tions with reference to TMS. All H shifts are given in
parts per million (s, singlet; d, doublet; t, triplet; m, mul-
tiplet). Assignments of proton resonances were con-
firmed, when possible, by selective homonuclear
decoupling experiments or by correlated spectroscopy.
Amino acids were coupled using standard solution-
phase chemistry with dicyclohexyl-carbodimide (DCC)
or O-(7-azabenzotriazoyl-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate (HATU).
4.1.1. Synthesis of cyclo(-L-Ae9-Aib-L-Phe-D-Pro-) (1).
To a cooled solution of HCl Æ H-D-Pro-OtBu (12.5 g,
60.0 mmol) and Z-L-Phe-OH (18.0 g, 60.0 mmol) in
dimethylformamide (DMF) (120 mL), HOBt Æ H2O
(9.19 g, 60.0 mmol), DCC (14.9 g, 72.0 mmol) and tri-
ethylamine (8.52 mL, 60.0 mmol) were added and the
mixture was stirred at room temperature for 8 h. After
completion of the reaction, 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 chromatography using a mixture
of chloroform and methanol (99:1) to yield Z-L-Phe-D-
Pro-OtBu (27.2 g, 100%, HPLC, rt 9.66 min) as a color-
less oil. The protected dipeptide (27.2 g, 60.0 mmol) was
dissolved in acetic acid (120 mL). Pd-C (3.50 g) was add-
ed and the mixture was stirred under hydrogen atmo-
sphere 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 organ-
ic phase was washed with saturated sodium bicarbonate
solution and dried over anhydrous MgSO4. Evaporation
of ethyl acetate gave H-L-Phe-D-Pro-OtBu (16.4 g, 86%,
HPLC, rt 5.60 min.), which was coupled with Z-Aib-OH
(13.1 g, 55.0 mmol) following the same procedure de-
scribed above and purified by silica gel chromatography
using a mixture of chloroform and methanol (99:1) to
obtain Z-Aib-L-Phe-D-Pro-OtBu (24.1 g, 87%, HPLC,
rt 8.95 min) as a white foam. The tripeptide Z-Aib-L-
Phe-D-Pro-OtBu (24.1 g, 44.8 mmol) was dissolved in
acetic acid (90 mL) and Pd-C (2.50 g) was added. The
mixture was stirred under hydrogen for 12 h. 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, washed with 4% sodium bicarbonate,
and dried over anhydrous MgSO4. Evaporation of ethyl
acetate gave H-Aib-L-Phe-D-Pro-OtBu (18.1 g, 100%,
HPLC, rt 5.68 min). The N-terminal free tripeptide
H-Aib-L-Phe-D-Pro-OtBu (8.07 g, 20.0 mmol) was
HPLC, rt 9.34 min, HR-FAB MS [M+H]+ 483.2971
25
for C27H39O4N4 (calcd 483.2971), ½aꢂ ꢀ73.6 (c 0.1,
D
MeOH), 1H NMR (500 MHz, CDCl3): dH 1.28 (m,
2H), 1.32 (m, 2H), 1.34 (s, 3H), 1.38 (m, 2H), 1.63
(m, 1H), 1.73 (m, 1H), 1.76 (m, 1H), 1.77 (s, 3H), 1.80
(m, 1H), 2.03 (m, 2H), 2.18 (m, 1H), 2.32 (m, 1H), 2.95
(dd, J = 13.3, 5.7 Hz, 1H), 3.23 (m, 1H), 3.26 (dd,
J = 13.5, 10.0 Hz, 1H), 3.86 (m, 1H), 4.18 (m, 1H), 4.66
(m, 1H), 4.97 (m, 2H), 5.16 (ddd, J = 10.1, 10.1,
5.9 Hz, 1H), 5.79 (ddt, J = 17.2, 10.2, 6.5, 1H), 5.91 (s,
1H), 7.08 (d, J = 10.5 Hz, 1H), 7.21 (m, 3H), 7.27 (m,
2H), 7.52 (d, J = 10.5 Hz, 1H).
4.1.2. Synthesis of chlamydocin-epoxide analog (2). To a
chilled solution of the cyclic tetrapeptide 1 (4.73 g,
9.80 mmol) in anhydrous dichloromethane (200 mL) in
a solution of purified m-chloroperoxybenzoic acidꢀ (m-
CPBA) (3.38 g, 19.6 mmol) in 100 mL of anhydrous
dichloromethane was added in aliquots for 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 solution was then washed with 4% sodium
bicarbonate and brine followed by drying over anhy-
drous magnesium sulfate and filtration. After evapora-
tion of dichloromethane, the residue was purified by
silica gel chromatography using a mixture of chloroform
and methanol (99:1) to yield the white foam 2 (4.33 g,
89%). HPLC, rt 7.13 min. HR-FAB MS [M+H]+
ꢀ
Commercially available m-CPBA (75–85%) was washed thoroughly
with phosphate buffer of pH 7.5 and the residue dried under reduced
pressure.

M. P. I. Bhuiyan et al. / Bioorg. Med. Chem. 14 (2006) 3438–3446
3443
25
D
499.2891 for C27H39O5N4 (calcd 499.2920), ½aꢂ ꢀ66.9
(500 MHz, CDCl3): dH 1.31 (m, 2H), 1.32 (m, 3H),
1.34 (s, 3H), 1.62 (m, 2H), 1.75 (m, 1H), 1.77 (s, 3H),
1.79 (m, 1H), 1.80 (m, 1H), 2.17 (m, 1H), 2.32 (m,
1H), 2.65 (t, J = 7.5 Hz, 2H), 2.95 (dd, J = 13.5,
6.0 Hz, 1H), 3.21 (m, 1H), 3.26 (dd, J = 13.5, 10.0 Hz,
1H), 3.86 (m, 1H), 3.87 (s, 2H), 4.18 (m, 1H), 4.66 (m,
1H), 5.16 (ddd, J = 10.2, 10.2, 5.5 Hz, 1H), 5.93 (s,
1H), 7.10 (d, J = 10.5 Hz, 1H), 7.22 (m, 3H), 7.27 (m,
2H), 7.50 (d, J = 10.5 Hz, 1H).
1
(c 0.1, MeOH), H NMR (500 MHz, CDCl3): dH 1.28
(m, 2H), 1.32 (m, 2H), 1.34 (s, 3H), 1.38 (m, 1H), 1.46
(m, 1H), 1.52 (m, 2H) 1.64 (m, 1H), 1.74 (m, 1H), 1.76
(m, 1H), 1.77 (s, 3H), 1.80 (m, 1H), 2.18 (m, 1H), 2.32
(m, 1H), 2.46 (m, 1H), 2.74 (m, 1H), 2.89 (m, 1H),
2.95 (dd, J = 13.5, 6.0 Hz, 1H), 3.23 (m, 1H), 3.26 (dd,
J = 13.5, 10.0 Hz, 1H), 3.86 (m, 1H), 4.19 (m, 1H),
4.66 (m, 1H), 5.16 (ddd, J = 10.2, 10.2, 5.8 Hz, 1H),
5.94 (s, 1H), 7.10 (d, J = 10.0 Hz, 1H), 7.21 (m, 3H),
7.27 (m, 2H), 7.51 (d, J = 10.5 Hz, 1H).
4.1.5. Synthesis of chlamydocin-methylketone analog (5).
To a stirred mixture of activated zinc powder (1.00 g) in
glacial acetic acid (3.0 mL), a solution of the bromom-
ethylketone 4 (112 mg, 0.194 mmol) in 1.0 mL of glacial
acetic acid was added dropwise. After 1 h, diethyl ether
was added to dilute the reaction mixture and zinc pow-
der was filtered off. The organic phase was washed with
4% sodium bicarbonate and brine, respectively, and
finally dried over anhydrous MgSO4 and filtered. Ether
was evaporated to get the crude methylketone, which
was purified by silica gel chromatography using a mix-
ture of chloroform and methanol (99:1) to yield the cor-
responding pure 5 (91.0 mg, 93%) as a white foam.
4.1.3. Synthesis of chlamydocin-bromohydrin analog (3).
To a solution of the epoxide 2 (2.49 g, 5.00 mmol) in
anhydrous tetrahydrofuran (50 mL), glacial acetic
(99.99+%) and anhydrous lithium bromide (695 mg,
8.00 mmol) were added. The reaction solution was stir-
red for 5 h at room temperature. After completion, the
reaction was quenched by adding water. Ethyl acetate
was added to the reaction mixture and the organic layer
was washed with 4% sodium bicarbonate followed by
brine. The organic phase was dried over anhydrous
MgSO4, filtered, and evaporated. The crude bromohy-
drin 3 was then purified by silica gel chromatography
using a mixture of chloroform and methanol (99:2) to
yield the corresponding pure 3 (2.69 g, 93%) as a white
foam. HPLC, rt 6.68 min. HR-FAB MS [M+H]+
HPLC, rt 7.24 min. HR-FAB MS [M+H]+ 499.2897
25
for C27H39O5N4 (calcd 499.2920), ½aꢂ ꢀ68.1 (c 0.1,
D
MeOH), 1H NMR (500 MHz, CDCl3): dH 1.31 (m,
3H), 1.32 (m, 2H), 1.34 (s, 3H), 1.57 (m, 1H), 1.66
(m, 1H), 1.74 (m, 1H), 1.77 (s, 3H), 1.79 (m, 2H), 2.18
(m, 1H), 2.13 (s, 3H), 2.32 (m, 1H), 2.41 (t, J = 7.5 Hz,
2H), 2.95 (dd, J = 13.5, 6.0 Hz, 1H), 3.21 (m, 1H),
3.26 (dd, J = 13.5, 10.0 Hz, 1H), 3.86 (m, 1H), 4.18
(m, 1H), 4.66 (m, 1H), 5.16 (ddd, J = 10.2, 10.2,
5.5 Hz, 1H), 5.90 (s, 1H), 7.09 (d, J = 10.5 Hz, 1H),
7.21 (m, 3H), 7.27 (m, 2H), 7.50 (d, J = 10.5 Hz, 1H).
579.2156 for C27H40O5N479Br (calcd 579.2182) and
25
D
581.2098 for C27H40O5N481Br (calcd 581.2162), ½aꢂ
ꢀ56.8 (c 0.1, MeOH), 1H NMR (500 MHz, CDCl3):
dH 1.30 (m, 2H), 1.31 (m, 2H), 1.34 (s, 3H), 1.35 (m,
1H), 1.46 (m, 1H), 1.54 (m, 2H), 1.63 (m, 1H), 1.74
(m, 1H), 1.77 (s, 3H), 1.79 (m, 1H), 1.80 (m, 1H), 2.09
(s, 1H), 2.17 (m, 1H), 2.32 (m, 1H), 2.95 (dd, J = 13.5,
6.0 Hz, 1H), 3.21 (m, 1H), 3.26 (dd, J = 13.5, 10.0 Hz,
1H), 3.38 (m, 1H), 3.53 (m, 1H), 3.77 (m, 1H), 3.85
(m, 1H), 4.20 (m, 1H), 4.67 (m, 1H), 5.16 (ddd,
J = 10.2, 10.2, 5.5 Hz, 1H), 6.07 (d, J = 8.5 Hz, 1H),
7.14 (d, J = 10.5 Hz, 1H), 7.22 (m, 3H), 7.27 (m, 2H),
7.52 (d, J = 10.5 Hz, 1H).
4.1.6. Synthesis of chlamydocin-diol analog (6). The cyc-
lic tetrapeptide cyclo(-L-Ae9-Aib-L-Phe-D-Pro-) (1)
(1.79 g, 3.70 mmol) was dissolved in a mixture of ace-
tone, acetonitrile, and water (3:1:1, 30 mL ) to which
4-methylmorpholine-N-oxide
(NMO)
(900 mg,
7.70 mmol) and microencapsulated osmium tetroxide
(MC-OsO4, Wako) (760 mg, ꢃ0.300 mmol, 10 mol%)
were added. The reaction mixture was stirred for 18 h
and HPLC monitoring showed complete conversion of
the double bond into vicinal diol. The catalyst was re-
moved by filtration and washed thoroughly with metha-
nol. The methanol washings were combined with the
filtrate and the mixture was evaporated. The crude diol
was purified by silica gel chromatography using a mix-
ture of chloroform and methanol (95:5) to yield the cor-
responding pure diol 6 (1.94 g, 100%) as a white foam.
4.1.4. Synthesis of chlamydocin-bromomethylketone ana-
log (4). To a solution of the bromohydrin 3 (209 mg,
0.360 mmol) in anhydrous dichloromethane (4.0 mL)
Dess–Martin periodinane (458 mg, 1.08 mmol) was add-
ed. The reaction mixture was stirred at 25 ꢁC for 3 h.
TLC and HPLC showed complete conversion of the
starting bromohydrin. The reaction solution was then
diluted by adding diethyl ether (4.0 mL) followed by
careful addition of a saturated solution of sodium bicar-
bonate containing 804 mg of sodium thiosulfate penta-
hydrate (Na2S2O3 Æ 5H2O). After stirring for 10 min.
the suspension became a clear solution. The organic
layer was separated from the aqueous layer and was
washed with brine, and dried over anhydrous MgSO4.
The filtered organic phase was evaporated to get a crude
bromomethylketone which was then purified by silica
gel chromatography using a mixture of chloroform
and methanol (99:1) to yield the corresponding white
foamy ketone 4 (185 mg, 89%). HPLC, rt 7.33 min.
HR-FAB MS [M+H]+ 577.2051 for C27H38O5N479Br
(calcd 577.2026) and 579.1997 for C27H38O5N481Br
HPLC, rt 5.37 min. HR-FAB MS [M+H]+ 517.3034
25
for C27H41O6N4 (calcd 517.3026), ½aꢂ ꢀ65.2 (c 0.1,
D
MeOH), 1H NMR (500 MHz, CDCl3): dH 1.31 (m,
2H), 1.32 (m, 2H), 1.34 (s, 3H), 1.35 (m, 1H), 1.41 (m,
1H), 1.44 (m, 2H), 1.63 (m, 1H), 1.74 (m, 1H), 1.77 (s,
3H), 1.79 (m, 1H), 1.81 (m, 1H), 2.17 (m, 1H), 2.32
(m, 1H), 2.95 (dd, J = 13.5, 6.0 Hz, 1H), 3.21 (m, 1H),
3.26 (dd, J = 13.5, 10.0 Hz, 1H), 3.44 (m, 1H), 3.65
(m, 1H), 3.70 (m, 1H), 3.85 (m, 1H), 4.19 (m, 1H),
4.67 (m, 1H), 5.16 (ddd, J = 10.2, 10.2, 5.5 Hz, 1H),
6.14 (d, J = 5 Hz, 1H), 7.15 (d, J = 10.0 Hz, 1H), 7.22
(m, 3H), 7.27 (m, 2H), 7.51 (d, J = 10.0 Hz, 1H).
25
1
(calcd 579.2005), ½aꢂ ꢀ57.4 (c 0.1, MeOH), H NMR
D

3444
M. P. I. Bhuiyan et al. / Bioorg. Med. Chem. 14 (2006) 3438–3446
4.1.7. Synthesis of chlamydocin-aldehyde analog (7). To a
stirred solution of the diol 6 (32.0 mg, 0.0620 mmol) in a
mixture of tetrahydrofuran (1.0 mL) and water
(0.5 mL), sodium periodate (NaIO4) (40.0 mg,
0.186 mmol) was added. Stirring was continued for
1 h, while HPLC showed the almost complete conver-
sion of the starting diol into product. The reaction solu-
tion was dissolved into ethyl acetate, washed with water
and brine, dried over anhydrous MgSO4, filtered, and
evaporated. The crude product was purified by silica
gel chromatography using a mixture of chloroform
and methanol (98:2) to yield the corresponding pure
aldehyde 7 (27.0 mg, 90%) as a white foam. HPLC, rt
sodium bicarbonate (50 mL) containing 14.4 g of sodium
thiosulfate pentahydrate (Na2S2O3Æ5H2O). After stirring
for 10 min. the suspension became a clear solution. The
organic layer was separated from the aqueous layer
and was washed with brine, and dried over anhydrous
MgSO4. The filtered organic phase was evaporated to
get a crude TBDMS protected hydroxymethylketone,
which was then purified by silica gel chromatography
using a mixture of chloroform and methanol (99:1) to
yield the purified ketone 9 (1.77 g, 86%) as a white foam.
HPLC, rt 11.06 min. HR-FAB MS [M+H]+ 629.3763 for
25
C33H53O6N4Si (calcd 629.3734), ½aꢂ ꢀ54.4 (c 0.1,
D
MeOH), 1H NMR (500 MHz, CDCl3): dH 0.09 (m,
6H), 0.92 (m, 6H), 1.31 (m, 2H), 1.32 (m, 2H), 1.34 (s,
3H), 1.58 (m, 2H), 1.64 (m, 1H), 1.74 (m, 1H), 1.77 (s,
3H), 1.79 (m, 1H), 1.81 (m, 1H), 2.17 (m, 1H), 2.32 (m,
1H), 2.48 (t, J = 7.0 Hz, 2H), 2.95 (dd, J = 13.5, 6.0 Hz,
1H), 3.21 (m, 1H), 3.26 (dd, J = 13.5, 10.0 Hz, 1H),
3.85 (m, 1H), 4.15 (s, 2H), 4.18 (m, 1H), 4.66 (m, 1H),
5.16 (ddd, J = 10.2, 10.2, 5.5 Hz, 1H), 5.94 (s, 1H), 7.09
(d, J = 10.5 Hz, 1H), 7.22 (m, 3H), 7.27 (m, 2H), 7.51
(d, J = 10.0 Hz, 1H).
5.66 min. HR-FAB MS [M+H]+ 485.2747 for
25
C26H37O5N4 (calcd 485.2764), ½aꢂ
ꢀ71.6 (c 0.1,
D
MeOH), 1H NMR (500 MHz, CDCl3): dH 1.29 (m,
2H), 1.32 (m, 2H), 1.34 (s, 3H), 1.36 (m, 1H), 1.63 (m,
1H), 1.65 (m, 1H), 1.74 (m, 1H), 1.77 (s, 3H), 1.79 (m,
2H), 2.18 (m, 1H), 2.32 (m, 1H), 2.43 (m, 2H), 2.95
(dd, J = 13.5, 6.0 Hz, 1H), 3.21 (m, 1H), 3.26 (dd,
J = 13.5, 10.0 Hz, 1H), 3.86 (m, 1H), 4.18 (m, 1H),
4.66 (m, 1H), 5.16 (ddd, J = 10.2, 10.2, 5.5 Hz, 1H),
5.92 (s, 1H), 7.11 (d, J = 10.5 Hz, 1H), 7.22 (m, 3H),
7.27 (m, 2H), 7.49 (d, J = 10.5 Hz, 1H), 9.76 (m, 1H).
4.1.10. Synthesis of chlamydocin-hydroxymethylketone
analog (10). To a cooled stirred solution of the TBDMS
protected ketone 9 (1.27 g, 2.02 mmol) in tetrahydrofu-
ran (THF) (50 mL), tetrabutylammonium fluoride
(TBAF) (3.10 mL, 3.10 mmol) was added and stirring
was continued for 25 min. The reaction was quenched
by adding a solution of ammonium chloride (1.27 g in
25 mL water) with continuous stirring followed by addi-
tion of 50 mL of water and stirring. The aqueous layer
was washed thoroughly with dichloromethane and the
combined dichloromethane layer was washed with brine
followed by drying over anhydrous MgSO4, filtration,
and evaporation. The crude residue was purified by sil-
ica gel chromatography using a mixture of chloroform
and methanol (98:2) to yield the corresponding pure
hydroxymethylketone 10 (1.03 g, 99%) as a white foam
after drying in vacuo. HPLC, rt 5.41 min. HR-FAB
4.1.8. Synthesis of TBDMS protected chlamydocin-diol
analog (8). To a stirred solution of the diol 6 (1.94 g,
3.73 mmol) in anhydrous dichloromethane (30 mL)
diisopropylethylamine (DIEA) (5.60 mL, 32.2 mmol),
4-dimethylaminopyridine (DMAP)
0.460 mmol), and t-butyldimethylsilyl
(56.0 mg,
chloride
(TBDMSCl) (793 mg, 5.22 mmol) were added. Stirring
was continued for 8 h, while HPLC showed the almost
complete conversion of the starting diol into product.
The reaction solution was washed with water and brine,
dried over anhydrous MgSO4, filtered and evaporated to
get the crude primary hydroxyl-protected diol. The res-
idue was purified by silica gel chromatography using a
mixture of chloroform and methanol (98:2) to yield
the corresponding pure monoprotected diol 8 (2.15 g,
91%) as a white foam. HPLC, rt 10.74 min. HR-FAB
MS [M+H]+ 631.3895 for C33H55O6N4Si (calcd
MS [M+H]+ 515.2835 for C27H39O6N4 (calcd
25
D
515.2870), ½aꢂ ꢀ65.1 (c 0.1, MeOH), 1H NMR
1
631.3891), H NMR (500 MHz, CDCl3): dH 0.08 (m,
(500 MHz, CDCl3): dH 1.31 (m, 2H), 1.32 (m, 3H),
1.34 (s, 3H), 1.64 (m, 1H), 1.65 (br, 1H), 1.74 (m, 1H),
1.77 (s, 3H), 1.79 (m, 1H), 1.81 (m, 1H), 1.82 (m, 1H),
2.17 (m, 1H), 2.32 (m, 1H), 2.40 (t, J = 7.5 Hz, 2H),
2.95 (dd, J = 13.5, 6.0 Hz, 1H), 3.21 (m, 1H), 3.27 (dd,
J = 13.5, 10.0 Hz, 1H), 3.86 (m, 1H), 4.18 (m, 1H),
4.23 (s, 2H), 4.67 (m, 1H), 5.16 (ddd, J = 10.2, 10.2,
5.5 Hz, 1H), 6.03 (s, 1H), 7.14 (d, J = 10.0 Hz, 1H),
7.21 (m, 3H), 7.27 (m, 2H), 7.50 (d, J = 10.0 Hz, 1H).
6H), 0.91 (m, 6H), 1.31 (m, 2H), 1.32 (m, 2H) 1.34 (s,
3H), 1.36 (m, 2H), 1.45 (m, 2H), 1.64 (m, 1H), 1.74
(m, 1H), 1.77 (s, 3H), 1.79 (m, 1H), 1.81 (m, 1H), 1.84
(br, 1H), 2.17 (m, 1H), 2.32 (m, 1H), 2.95 (dd,
J = 13.5, 6.0 Hz, 1H), 3.21 (m, 1H), 3.26 (dd, J = 13.5,
10.0 Hz, 1H), 3.38 (m, 1H), 3.61 (m, 2H), 3.85 (m,
1H), 4.18 (m, 1H), 4.66 (m, 1H), 5.16 (ddd, J = 10.2,
10.2, 5.5 Hz, 1H), 5.93 (d, J = 6.0 Hz, 1H), 7.08 (d,
J = 10.5 Hz, 1H), 7.22 (m, 3H), 7.27 (m, 2H), 7.52 (d,
J = 10.5 Hz, 1H).
4.2. Preparation of HDACs and assay for enzyme activity
4.1.9. Synthesis of TBDMS protected chlamydocin-
hydroxymethylketone analog (9). To a solution of the
protected diol 8 (2.07 g, 3.29 mmol) in anhydrous dichlo-
romethane (65 mL), Dess–Martin periodinane (8.15 g,
19.3 mmol) was added. The reaction mixture was stirred
at 25 ꢁC for 3 h. TLC and HPLC showed complete con-
version of the starting compound. The reaction solution
was then diluted by adding diethyl ether (65 mL)
followed by careful addition of a saturated solution of
293T cells (1–2 · 106) were grown in a 100-mm dish for
24 h and transiently transfected with 10 lg each of the
vector pcDNA3-HDAC1 for human HDAC1,
pcDNA3-HDAC4 for human HDAC4, or pcDNA3-
mHDA2/HDAC6 for mouse HDAC6, using the Lipo-
fectAMINE2000 reagent (Invitrogen). After successive
cultivation in DMEM for 24 h, the cells were washed
with PBS and lysed by sonication in lysis buffer contain-
ing 50 mM Tris–HCl (pH 7.5), 120 mM NaCl, 5 mM

M. P. I. Bhuiyan et al. / Bioorg. Med. Chem. 14 (2006) 3438–3446
3445
EDTA, and 0.5% NP40. The soluble fraction collected
References and notes
by microcentrifugation was precleared by incubation
with protein A/G plus agarose beads (Santa Cruz Bio-
technologies, Inc.). After the cleared supernatant had
been incubated for 1 h at 4 ꢁC with 4 lg of an anti-
FLAG M2 antibody (Sigma–Aldrich Inc.) for HDAC1,
HDAC4, and HDAC6, the agarose beads were washed
three times with lysis buffer and once with histone deace-
tylase buffer consisting of 20 mM Tris–HCl (pH 8.0),
150 mM NaCl, and 10% glycerol. The bound proteins
were released from the immune complex by incubation
for 1 h at 4 ꢁC with 40 lg of the FLAG peptide (Sig-
ma-Aldrich Inc.) in histone deacetylase buffer
(200 lL). The supernatant was collected by centrifuga-
tion. For the enzyme assay, 10 lL of the enzyme frac-
tion was added to 1 lL of fluorescent substrate (2 mM
Ac-KGLGK(Ac)-MCA) and 9 lL of histone deacetyl-
ase buffer, and the mixture was incubated at 37 ꢁC for
30 min. The reaction was stopped by the addition of
30 lL of trypsin (20 mg/ml) and incubated at 37 ꢁC for
15 min. The released aminomethyl coumarin (AMC)
was measured using a fluorescence plate reader. The
50% inhibitory concentrations (IC50) were determined
as means with SD calculated from at least three indepen-
dent dose response curves.
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