Bicyclic tetrapeptides as potent HDAC inhibitors: Effect of aliphatic loop position and hydrophobicity on inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2014.06.031

Several histone deacetylase (HDAC) inhibiting bicyclic tetrapeptides have been designed and synthesized through intramolecular ring-closing metathesis (RCM) reaction and peptide cyclization. We designed bicyclic tetrapeptides based on CHAP31, trapoxin B a

Full text of DOI:10.1016/j.bmc.2014.06.031

Bioorganic & Medicinal Chemistry 22 (2014) 3862–3870
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bicyclic tetrapeptides as potent HDAC inhibitors: Effect of aliphatic
loop position and hydrophobicity on inhibitory activity
Md. Nurul Islam a,b, , Md. Shahidul Islam a,b, Md. Ashraful Hoque a,c, Tamaki Kato a, Norikazu Nishino a,
⇑
Akihiro Ito d, Minoru Yoshida d
a Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu 808-0196, Japan
b Department of Chemistry, Faculty of Science, University of Rajshahi, Rajshahi 6205, Bangladesh
c Department of Biochemistry and Molecular Biology, Faculty of Science, University of Rajshahi, Rajshahi 6205, Bangladesh
d Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Several histone deacetylase (HDAC) inhibiting bicyclic tetrapeptides have been designed and synthesized
through intramolecular ring-closing metathesis (RCM) reaction and peptide cyclization. We designed
bicyclic tetrapeptides based on CHAP31, trapoxin B and HC-toxin I. The HDAC inhibitory and p21 pro-
moter assay results showed that the aliphatic loop position as well as the hydrophobicity plays an impor-
tant role toward the activity of the bicyclic tetrapeptide HDAC inhibitors.
Received 16 April 2014
Revised 15 June 2014
Accepted 17 June 2014
Available online 26 June 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Histone deacetylase inhibitors
Bicyclic tetrapeptides
Ring-closing metathesis
Aliphatic loop position
1. Introduction
impact upon isoform selectivity.7 The cap groups of naturally
occurring cyclic peptides Trapoxins,8 Cyl-1 and Cyl-2,9 WF3161,10
Histone deacetylases (HDACs) are enzymes responsible for the
regulation of chromatin remodeling and gene transcription by
deacetylation of acetyl lysine residues in amino terminal tails of
histones.1–3 As overexpression of HDACs is associated with
carcinogenesis of cells, design and synthesis of novel HDAC inhib-
itors is a promising field on developing anticancer drugs. In fact,
there are a variety of HDAC inhibitors in clinical trials and two of
them, SAHA (Vorinostat) and FK228 (Romidepsin), are already in
the market. Both the drugs have demonstrated the therapeutic
benefit in cutaneous T-cell lymphoma (CTCL) and in some
malignancies.4,5 However, current HDAC inhibitors, including
SAHA and FK228, broadly inhibit the deacetylation activity of var-
ious HDAC enzymes including class I and II, which is associated
with reduced potency and toxic side effects.5,6 Therefore, it is desir-
able to release HDAC inhibitors that can selectively inhibit HDAC
isoform overexpressed in a cancer cell.
chlamydocin,11 and HC-toxins,12 may be helpful for selective HDAC
inhibitors design. Our extensive approaches using cyclic tetrapep-
tides CHAPs,13,14 SCOPs,15 and chlamydocin analogues with various
binding functional groups16 have shown that modification of the
cap group of HDAC inhibitors led to elevate significantly the
selectivity between HDAC isoforms.7 Macrocyclic HDAC inhibitors
possess the most complex cap groups which interact with HDAC
enzyme’s outer rim and have demonstrated excellent HDAC inhibi-
tion potency and isoform selectivity.17 The cyclic tetrapeptides
have the potential to interact with a large area of the enzyme sur-
face. The presence of hydrophobic groups in the tetrapeptide ring
is essential to the activities of cyclic tetrapeptide containing inhib-
itors.18 It is possible to customize the macrocyclic HDAC inhibitors
by creating a hydrophobic loop in cyclic tetrapeptide scaffold using
ring closing metathesis (RCM). The loop regions of the cyclic tetra-
peptides enhance the activity of the HDAC inhibitors by hydropho-
bic interaction with the enzyme surface. In a previous work, we
reported that incorporation of aliphatic loop to the cyclic tetrapep-
tide framework showed a potent activity and enhanced selectiv-
Interactions between the rim region of the enzyme and the cap
groups of inhibitors are of great importance to selective inhibitor
design. This region has less homology between isoforms compared
to the active site allowing the cap group to have a significant
ity.19 Furthermore, we have designed
a series of bicyclic
tetrapeptides to evaluate the importance of aliphatic loop length
in the cyclic tetrapeptide scaffold.20 The sequence and
configuration of amino acids in CHAP3121 were considered as the
basis for designing the inhibitors. Encouraged by the interesting
⇑
Corresponding author.
E-mail address: mnurulchem@gmail.com (M.N. Islam).
http://dx.doi.org/10.1016/j.bmc.2014.06.031
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

M. N. Islam et al. / Bioorg. Med. Chem. 22 (2014) 3862–3870
3863
inhibitory activities of two of those compounds (compounds 1 and
2, Fig. 1), in the present study, we have designed and synthesized
CHAP31 (LDLD configuration), trapoxin B (LLLD configuration)
and HC-toxin I (LDLD configuration) based bicyclic tetrapeptides
hydroxamic acid to demonstrate the effect of additional loop posi-
tion and hydrophobicity on the inhibitory activity of cyclic tetra-
peptide HDAC inhibitors. We also have designed and synthesized
two mono cyclic tetrapeptides to explore the effect of ring closing
on inhibitory activity of the inhibitors. We herein describe the
account on synthesis of bicyclic peptides using RCM reaction and
a brief description of interesting biological results.
groups of the adduct was 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 decarboxylation by refluxing
in toluene. After evaporation of toluene, the resulting oily
Boc-DL-Aen-OEt (14, 15) was purified by silica gel column chroma-
tography. Finally, Boc-DL-Aen-OEt was subjected to the action of
subtilisin Carlsberg from Bacillus licheniformis (Sigma) in a mixture
of dimethylformamide (DMF) and water (1:3, v/v). Boc-
9) was isolated as colorless oil. The recovered Boc-
(16, 17) was hydrolyzed by 1 M NaOH in EtOH to get Boc-
L
-Aen-OH (8,
-Aen-OEt
-Aen-OH
D
D
(10, 11) with excellent purity in quantitative yield.
Boc-L-Homoserine (18) and Boc-D-Homoserine (19) were
treated separately with NaH and allyl bromide in DMF to obtain
2. Results and discussion
2.1. Chemistry
Boc-
L
-2-amino-4-allyloxybutanoic acid (Boc-L-Aa4-OH) (12) and
Boc-
D
-2-amino-4-allyloxybutanoic acid (Boc-
D
-Aa4-OH) (13) in
quantitative yield (Scheme 2).
CHAP31 based bicyclic tetrapeptide 3 was synthesized accord-
To introduce alkyl loop to cyclic tetrapeptides, formation of
intramolecular covalent bond between alkyl side-chains of two
amino acids will be useful. Olefin metathesis have recently
appeared as a versatile and chemoselective means to create car-
bon–carbon bonds in a range of molecules including natural prod-
ucts, polymers, and biomolecules.22–26 Modern ruthenium olefin
metathesis catalysts tolerate many functional groups common in
biomolecules, including amides, alcohols, and carboxylic acids.27
ing to Scheme 3 by the conventional solution phase method.
H-D
-Pro-OtBu was condensed with 12 using DCC/HOBt. Boc protec-
tion was selectively removed by the reported procedure43 using
4 M HCl/dioxane, and free amino group was condensed with 13
by the same DCC/HOBt method to obtain linear tripeptides 21.
The linear tripeptide 22 with fused side ring were synthesized by
ring closing metathesis between D-Aa4 and L-Aa4 using Grubb’s
first generation Ru catalyst, in dichloromethane (DCM), followed
by catalytic hydrogenation. After selective deprotection of 22,
Boc protected amino suberic acid benzyl ester [Boc-L-Asu(OBzl)]
Several modified amino acids and peptides such as
a-helical
peptides,28–34 rigidified amino acids and peptides,35 antimicrobial
peptides36–38 and cyclic peptide MC4-ligand39 were synthesized
by the aid of intramolecular ring-closing metathesis (RCM) reac-
tion to alter the conformational and metabolic stability. Glucosy-
was incorporated to prepare the linear tetrapeptides 23. After
removal of both side protections by treating with trifluoroacetic
acid (TFA), cyclization reaction was carried out by the aid of N-
[(dimetylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-
N-methylmethanaminium hexafluorophosphate N-oxide (HATU)
in DMF under high dilution conditions with minimum amount of
diisopropylethyl amine (DIEA) (2.5 equiv) to obtain bicyclic tetra-
peptides 24. After benzyl (Bzl) deprotection by catalytic hydroge-
nation, the carboxyl group was condensed with hydroxylamine
benzyl ester, and finally Bzl protection was removed by catalytic
hydrogenation to obtain bicyclic tetrapeptide hydroxamic acids 3.
For the synthesis of trapoxin B based bicyclic tetrapeptide 4, the
ring closing metathesis was planned to be carried out after linear
tetrapeptide cyclization as shown in Scheme 4. Boc-tetrapeptide-
OtBu (25) synthesized by stepwise elongation from C-terminal
was deprotected by TFA and then cyclized by high dilution condi-
tion in DMF using HATU as described above. Cyclic tetrapaptide 26
obtained was subjected to RCM followed by catalytic hydrogena-
tion to yield a bicyclic tetrapeptide 27. Successive incorporation
lated porphyrins bearing
a cyclic pseudopentapeptide for
photodynamic therapy (PDT) of cancer, and inhibitors of Grb2
SH2 domain signaling, which was associated with breast cancer,
have also been synthesized.40,41 Recently, synthesis of apicidin
analogue has been reported.42 In a previous work, we designed
and synthesized bicyclic tetrapeptide S–S hybrid as HDAC inhibi-
tor.19 However in this work, we synthesized CHAP31, trapoxin B
and HC-toxin I based bicyclic tetrapeptide hydroxamic acids as
HDAC inhibitors (3–7, Fig. 2).
Our synthesis strategy started from the synthesis of building
block amino acid derivatives. Boc-
(Boc- -Ae7-OH) (8), Boc- -2-amino-7-octenoic acid (Boc-
OH) (9), Boc- -2-amino-6-heptenoic acid (Boc- -Ae7-OH) (10)
and Boc- -Ae8-OH) (11) were syn-
L
-2-amino-6-heptenoic acid
L
L
L
-Ae8-
D
D
D
-2-amino-7-octenoic acid (Boc-D
thesized by the reported procedure19 as illustrated in Scheme 1.
Bromoalkenes were separately coupled with diethyl Boc-
aminomalonate by using NaOEt in ethanol. One of the ethyl ester
O
O
O
NH HN
O
H
5
O
5
N
O
O
N
HN
N
HN
5
O
OH
O
O
O
NH HN
O
O
NH HN
O
O
NH
O
N
O
O
CHAP31
Trapoxin B
HC-toxin I
O
O
H
H
O
N
O
HN
5
N
N
HN
5
N
OH
OH
O
O
NH
O
HN
NH
O
O
HN
O
1
2
Figure 1. Natural and artificial cyclic peptide HDAC inhibitors.

3864
M. N. Islam et al. / Bioorg. Med. Chem. 22 (2014) 3862–3870
O
O
H
O
H
O
N
O
HN
HN
5
N
N
HN
5
N
OH
OH
H
N
O
NH
5
HN
O
O
OH
NH
O
NH
O
O
HN
O
O
NH
O
N
O
O
O
4
5
3
O
O
H
O
N
HN
5
N
H
N
O
OH
NH
5
HN
N
OH
O
NH
O
HN
O
O
NH
O
O
7
6
Figure 2. Designed HDAC inhibitors.
O
OEt
OEt
e
f
a-d
Boc
n-4
n-4
OEt
n-4
O
n-4
O
+
Boc
N
OH
OEt
OH
H
Boc
N
H
Boc
N
H
N
H
Boc
N
H
O
O
O
8, 9
: n = 7, 8
14, 15
16, 17
: n = 7, 8
: n = 7, 8
10 11
,
: n = 7, 8
Scheme 1. Synthesis of 2-amino-(nÀ1)-alkenoic acids (Aen): (a) Na, anhyd EtOH, reflux, 30 min.; (b) Br(CH2)nÀ4–CH@CH2, reflux, 6 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.
OH
O
OH
O
a
a
OH
OH
OH
OH
Boc
N
H
Boc
N
H
Boc
N
H
Boc
N
H
O
O
O
O
18
12
19
13
Scheme 2. Synthesis of Boc-
L
-2-amino-4-allyloxybutanoic acid (Boc-
L
-Aa4-OH) and Boc-
D-2-amino-4-allyloxybutanoic acid (Boc-D-Aa4-OH): (a) 60% NaH, allyl bromide,
DMF, 12 h.
of hydroxamic acid benzyl ester and catalytic hydrogenation gave
The length of aliphatic loop in compounds 4, 5 and in reference
the desired compound 4.
compound 1 is the same (10 methylenes), but they are different in
amino acid configuration and the aliphatic loop position. Com-
pounds 4 and 5 are similar in activity against HDAC1 and HDAC4,
but not as potent and selective as compound 1. However, a sharp
difference in cell based activity was observed among compounds
1, 4 and 5. As compared with compound 1, the decrease in activity
of 4 and 5 are seventy-fold and eight-fold, respectively. In our pre-
vious work, we reported that the cell based activity increases with
the increase in hydrophobicity of the inhibitors having the same
loop position.20 This fact is supported by the activity of compound
3. Compound 3, which has loop size similar to that of compound 1
but two oxygen atoms in the loop, showed considerably low activ-
ity against HDACs l, 4 and 6. Compound 3 also has lowest p21 pro-
moter-inducing activity. The ether bonds present in the loop of
compound 3 has different conformation and hydrophobicity to that
of compound 1 having simple saturated alkyl chain (–CH2–). The
reduced activity is due to the change in structure of the cap group
and hydrophobicity. Interestingly, the magnitude of hydrophobic-
ities expressed by HPLC retention times are the order of 5 > 1 > 4
although the order of cell based activity is 1 > 5 ꢀ 4. Compounds
1, 4 and 5 have different loop position and peptide backbone
conformation. These differences strongly affect the p21
HC-toxin
I
based bicyclic tetrapeptide
5
was synthesized
-Pro-OtBu (29)
through fragment condensation of H-Asu(OBzl)-
D
and Boc-protected linear dipeptide with fused ring (31) by DCC/
HOBt, and following cyclization and incorporation of hydroxamic
acid gave the desired compound 5 (Scheme 5).
Monocyclic tetrapeptides 6 and 7 (Fig. 2) were synthesized in a
similar manner to their corresponding bicyclic tetrapeptides
excluding RCM. All the compounds 3–7 were characterized by 1H
NMR and high resolution FAB-MS.
2.2. Enzyme inhibition and biological activity
Compounds 3–7 were assayed for HDAC inhibitory activity
using HDAC1, HDAC4 and HDAC6 prepared from 293T cells. To
examine the inhibitory activity in cell based condition, we carried
out p21 promoter assay. Preparation and assays of HDACs and p21
promoter were performed according to the reported methods.16
The detail experimental procedures are described in the Section 4
of this paper. CHAP31 was used as the reference compound.21 Most
of the compounds showed activity in nanomolar range. The results
are summarized in Table 1.

M. N. Islam et al. / Bioorg. Med. Chem. 22 (2014) 3862–3870
3865
O
O
O
a
O
4
b-d
+
OH
H
N
N
O
O
N
HN
O
Boc
N
H
Boc
N
H
Boc
N
H
O
O
O
O
O
O
21
20
12
O
4
O
O
O
O
O
e, f
b, c, g
H
N
N
O
O
N
Boc
Boc
N
H
N
H
N
H
N
H
O
O
O
O
5
O
O
22
O
OBzl
23
O
O
H
N
5
5
O
O
N
HN
OBzl
N
HN
OH
h, i
f, j, k
O
O
NH HN
NH HN
O
O
O
O
O
O
O
O
4
4
24
3
Scheme 3. Synthesis of CHAP31 based bicyclic tetrapeptide 3. Reagents and conditions: (a) DCC, HOBt, DMF, 12 h, 80%; (b) 4 M HCl/dioxane, 35 min, 73%; (c) saturated
Na2CO3; (d) Boc- -Aa4-OH, DCC, HOBt, DMF, 12 h, 89%; (e) Grubbs’ first generation catalyst, DCM, 48 h, 52%; (f) AcOH, Pd–C, H2, 12 h, 75%; (g) Boc- -Asu(OBzl)-OH, DCC, HOBt,
D
L
DMF, 12 h, 79%; (h) TFA, 3 h, 92%; (i) HATU, DIEA, DMF, 4 h, 65%; (j) HClÁH2NOBzl, DCC, HOBt, TEA, DMF, 91%; (k) Pd–BaSO4, AcOH, H2, 72%.
O
OBzl
O
5
O
N
HN
OBzl
O
4
4
5
H
N
N
O
O
Boc
a, b
NH HN
O
N
H
N
H
4
O
O
O
O
4
5
25
26
O
O
H
N
O
O
N
N
HN
5
HN
HN
OH
OH
O
O
e, f
c, d
NH
O
NH
O
HN
O
O
4
27
Scheme 4. Synthesis of trapoxin B based bicyclic tetrapeptide 4. Reagents and conditions: (a) TFA, 3 h, 97%; (b) HATU, DIEA, DMF, 4 h, 41%; (c) Grubbs first generation
catalyst, DCM, 48 h; (d) AcOH, Pd–C, H2, 12 h; (e) HClÁH2NOBzl, DCC, HOBt, TEA, DMF, 24 h, 68%; (f) Pd–BaSO4, AcOH, H2, 24 h, 67%.
promoter-inducing activity. Although many drug molecules are
transported across biological membranes via passive diffusion at
a rate related to their hydrophobicity, carrier-mediated and active
uptake of drugs is far more common than is usually assumed. In
this case, drugs having better interaction with carrier proteins
show better cell permeability.44 The p21 assay results of com-
pounds 1, 4 and 5 may be explained on the basis of carrier medi-
ated transport of drug into the cell. It can be assumed that, due
to structural difference, compound 1 has highest interaction with
carrier followed by compound 5 and 4. These results indicate that
not only the hydrophobicity but also the loop position and peptide
backbone conformation is important for inhibitory activity of the
bicyclic peptides.
compounds for CHAP31, trapoxin and HC-toxin I based bicyclic
peptides (1, 4 and 5), respectively. For CHAP31 and HC-toxin based
inhibitors, activity in both cell free and cell based conditions
improved upon ring closing. However, for trapoxin based inhibitors
4 and 6 opposite trend was observed. Small structural changes to
the cap group can have a profound influence on the activity of an
HDAC inhibitor.18 Ring closing causes the structural change in
the cyclic tetrapeptide cap group and hence affects the activity of
the inhibitors. The structural change is favorable for interaction
between inhibitor cap group and enzyme surface for the former
case and the reverse for the latter. HDAC inhibitors with macrocy-
clic cap groups have facility to bind with multiple grooves in the
rim region of HDAC enzymes.18 Peptides with open and close ali-
phatic side chains have the opportunity to bind efficiently in the
rim region of HDAC enzymes as both have macrocyclic cap groups.
To demonstrate the effect of ring closing on inhibitory activity,
compounds 2, 6 and 7 were used as open chain reference

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M. N. Islam et al. / Bioorg. Med. Chem. 22 (2014) 3862–3870
OBzl
OBzl
5
a, b
O
O
5
N
O
N
OBzl
O
Boc
H2N
N
H
7
O
O
O
O
O
5
O
N
e
O
28
29
Boc
N
N
H
N
H
H
O
O
i
7
O
c, d
O
4
4
H
32
OH
OBzl
Boc
N
N
H
N
Boc
N
H
H
O
O
O
f, g
d, h
5
30
31
Scheme 5. Synthesis of HC-toxin I based bicyclic tetrapeptide 5. Reagents and conditions: (a) 4 M HCl/dioxane, 30 min; (b) saturated Na2CO3, 68% (c) Grubbs first generation
catalyst, DCM, 48 h; (d) AcOH, Pd–C, H2, 12 h; (e) DCC, HOBt, DMF, 12 h, 72%; (f) TFA, 3 h, 95%; (g) HATU, DIEA, DMF, 4 h, 51%; (h) HClÁH2NOBzl, DCC, HOBt, TEA, DMF, 81%; (i)
Pd–BaSO4, AcOH, H2, 24 h, 66%.
Table 1
HDAC inhibitory activity and p21 promoter activity data for bicyclic tetrapeptide hydroxamic acids and reference compounds
Compounds
Loop size
HPLCa retention time (min)
IC50 (nM)
HDAC4
HDAC6/HDAC1b
p21 promoter assay, EC1000 (nM)
HDAC1
HDAC6
CHAP3121
—
—
0.4
9.1
25
31
25
25
37
43
3
13
32.5
45.1
13.6
7.7
9.2
19.6
3.7
22
7.2
13
2200
510
58
1
2
3
4
5
6
7
–(CH2)10
—
–[(CH2)2-O-(CH2)4-O-(CH2)2]–
–(CH2)10
–(CH2)10
—
—
–
6.03
7.51
4.31
5.80
7.06
6.55
7.67
5.5
12
41
26
31
15
43
410
340
240
230
490
140
570
-
-
80
80
13.3
a
HPLC analysis was performed on a Hitachi instrument equipped with a Chromolith performance RP-18e column (4.6 Â 100 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–B over 15 min with a flow rate of 2 mL/min, with detection at 220 nm.
b
Selectivity for HDAC1 over HDAC6.
As a result, there is no drastic change in inhibition activity of the
inhibitors with open and closed aliphatic side chain. However,
upon ring closing the aliphatic side chains become rigidified and
more selective to some enzymes. This assumption is supported
by the activity data of HDAC inhibitors for which significant
increased in specificity for HDAC1 over HDAC6 was observed upon
ring closing.
visualized by UV light or charring. Analytical HPLC was performed
on a Hitachi instrument equipped with a chromolith performance
RP-18e column (4.6 Â 100 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–B over 15 min with a flow rate
of 2 mL/min, with detection at 220 nm. HR FAB-mass spectra were
measured on a JEOL JMS-SX 102A instrument. NMR spectra were
recorded on a JEOL JNM A500 MHz spectrometer in DMSO-d6
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. Ring closing metathesis (RCM) was per-
form by the aid of benzylidene-bis(tricyclohexylphosphine)dichlo-
roruthenium (Grubbs first generation catalyst). Coupling reactions
were performed by standard solution-phase chemistry using
dicyclohexylcarbodiimide (DCC), N-[(dimetylamino)-1H-1,2,3-
triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium
hexafluorophosphate N-oxide (HATU).
3. Conclusion
We synthesized bicyclic tetrapeptides, by the aid of olefin
metathesis, and monocyclic tetrapeptides hydroxamic acid to
develop HDAC inhibitors with good selectivity and potency. We
showed that although hydrophobicity is important, loop position
as well as amino acid sequence in the cyclic tetrapeptide scaffold
is also crucial for inhibitory activity. Bicyclic tetrapeptides possess
complex cap groups and thus capable of making numerous interac-
tions with the enzyme surface. It is possible to customize the mac-
rocyclic HDAC inhibitors by ring closing metathesis to improve
potency and selectivity.
4.1.1. Synthesis of Boc-L-2-amino-6-heptenoic acid (Boc-L-Ae7-
OH) (8)
4. Experimental
4.1. General
Metallic sodium (4.0 g, 165 mmol) was dissolved in absolute
ethanol (165 mL) and diethyl tert butoxycarbonylaminomalonate
(45.42 g, 165 mmol) was added to the solution. The reaction mix-
ture was refluxed for 30 min for the complete removal of the labile
hydrogen and then 5-bromo-1-pentene (25 g, 165 mmol) was
added and refluxed for 6 h. The reaction mixture was cooled on
an ice bath and hydrolysis was carried out by the treatment of
1 M aq NaOH (165 mL, 165 mmol). After selective hydrolysis for
4 h at 0 °C, ethanol was evaporated and the unreacted 5-bromo-
1-pentene was removed by extraction with diethyl ether under
basic condition. Then the aqueous solution was extracted with
Unless otherwise noted, all solvents and reagents were reagent
grade and used without purification. Flash chromatography was
performed using silica gel 60 (230–400 mesh) eluting with sol-
vents as indicated. All compounds were routinely checked by thin
layer chromatography (TLC) and/or high performance liquid
chromatography (HPLC). TLC was performed on aluminum-backed
silica gel plates (Merck DC-Alufolien Kieselgel 60 F254) with spots

M. N. Islam et al. / Bioorg. Med. Chem. 22 (2014) 3862–3870
3867
ethyl acetate at pH 3–4, by adding solid citric acid. Finally, ethyl
4.1.7. Synthesis of bicyclic tetrapeptide hydroxamic acid with
acetate layer was washed with brine and dried over anhydrous
MgSO4. Ethyl acetate was evaporated to obtain a colorless oil of
monoester monoacid (42.5 g, 82%). HPLC, retention time
7.55 min. It was then dissolved in toluene (180 mL) and refluxed
for 3 h. The oily ester after evaporation of toluene is then subjected
oxygen in the loop (3)
To a cooled solution of H-D L-
-Pro-OtBu (0.65 g, 3.8 mmol), Boc-
Aa4-OH (1.08 g, 4.18 mmol) and HOBtÁH2O (0.581 g, 3.8 mmol) in
DMF (8 mL), DCC (0.94 g, 4.56 mmol) was added. The mixture
was stirred for 12 h at room temperature. 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 concentrated to remain an oily sub-
stance, which was purified by silica gel chromatography using a
to silica gel column chromatographic purification to get Boc-DL
-
Ae7-OEt (14) as colorless oil (30.8 g, 84%). The resulting oily mass
was suspended into a mixture of water (342 mL) and DMF
(114 mL) (1:3, v/v) solvent system at 38 °C using a mechanical stir-
rer and pH was adjusted at about 7–8 by adding 1 M aq ammonia
solution. Then subtilisin Carlsberg from B. licheniformis (57 mg)
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-Ae7-OEt (16)
(15.6 g, 50.5%) was extracted with diethyl ether under basic condi-
tion. Then the aqueous solution was extracted with ethyl acetate as
mixture of chloroform and methanol (99:1) to yield Boc-L-Aa4-D-
Pro-OtBu (20) (1.25 g, 80%) as oil. The protected dipeptide
(1.24 g, 3.0 mmol) was dissolved in 4 M HCl/dioxane (10 mL) and
the mixture was kept at room temperature for 35 min. The reaction
was monitored by TLC. After completion of the reaction, HCl/diox-
ane was evaporated. The residue was dissolved in EtOAc and
washed with saturated Na2CO3, dried over anhydrous Na2CO3
described above to get Boc-
L-Ae7-OH (8) as colorless oil (13.6 g,
49%). HPLC, retention time 7.05 min.
and EtOAc was evaporated to get H-
oil (0.68 g, 73%). To a cooled solution of H-
(0.68 g, 2.18 mmol), Boc- -Aa4-OH (0.621 g, 2.40 mmol) and
L
-Aa4-
D
-Pro-OtBu as heavy
-Aa4-
-Pro-OtBu
L
D
4.1.2. Synthesis of Boc-
L
-2-amino-7-octenoic acid (Boc-
L
-Ae8-
D
OH) (9)
HOBtÁH2O (0.334 g, 2.81 mmol) in DMF (5 mL), DCC (0.542 g,
Compound 9 was synthesized by the same procedure as
described for compound 8 using 6-bromo-1-hexene instead of 5-
bromo-1-pentene. HPLC, retention time 8.04 min.
2.62 mmol) was added and stirred for 12 h at room temperature.
The product Boc-D-Aa4-L-Aa4-D
-Pro-OtBu (21) was obtained in
the same manner as described earlier as heavy oil (0.825 g, 89%),
HPLC, retention time 7.02 min. To a solution of linear tripeptide
4.1.3. Synthesis of Boc-
D
-2-amino-6-heptenoic acid (Boc-
D-Ae7-
Boc-D-Aa4-L-Aa4-D
-Pro-OtBu (21) (0.815 g, 1.47 mmol) in anhy-
OH) (10)
drous and degassed DCM (185 mL), a solution of Grubbs first gen-
eration ruthenium catalyst (0.241 g, 0.29 mmol) in anhydrous and
degassed DCM (35 mL) was added. The reaction mixture was stir-
red at room temperature for 48 h. After the completion of reaction,
DCM was evaporated and the residue was purified by silica gel
chromatography using a mixture of chloroform and methanol
(99:1) to yield linear tripeptide with fused cycle as foam (0.40 g,
52%) which on catalytic hydrogenation in presence of Pd–C
(0.04 g) in AcOH (10 mL) yield compound 22 (0.37 g, 75%), HPLC,
retention time 9.02 min. The protected tripeptide 22 (0.365 g,
0.69 mmol) was dissolved in 4 M HCl/dioxane (4 mL) and the mix-
ture was kept at room temperature for 35 min. The reaction was
monitored by TLC. After completion of the reaction, HCl/dioxane
was evaporated. The residue was dissolved in EtOAc and washed
with saturated Na2CO3, dried over anhydrous Na2CO3 and EtOAc
was evaporated to get tripeptide free amine (0.180 g, 61%). To a
The ester Boc-
luted using 5 mg of subtilisin Carlsberg from B. licheniformis using
the procedure described in Section 4.1.1. The pure -ester (14.4 g,
53 mmol) was dissolved in ethanol (60 mL) and hydrolyzed by
1 M NaOH (60 mL, 60 mmol) for 4 h at 0 °C. After completion of
the reaction, ethanol was evaporated and the aqueous solution of
Boc-D-Ae7-OH was acidified to pH 3 by solid citric acid. Then the
aqueous solution was extracted with ethyl acetate. Finally ethyl
acetate layer was washed with brine, dried over anhydrous MgSO4,
D-Ae7-OEt (16) (15.6 g, 557.5 mmol) was re-reso-
D
and evaporated to obtain Boc-
(12.75 g, 99%). HPLC, retention time 7.00 min.
D-Ae7-OH (10) as colorless oil
4.1.4. Synthesis of Boc-
OH) (11)
Compound 11 was synthesized by the same procedure as
described for compound 10. HPLC retention time 8.08 min.
D-2-amino-7-octenoic acid (Boc-D-Ae8-
cooled solution of the free amine (0.180 g, 0.42 mmol), Boc-L-
Asu(OBzl)-OH (0.190 g, 0.50 mmol) and HOBtÁH2O (0.064 g,
0.42 mmol) in DMF (2 mL), DCC (0.104 g, 0.50 mmol) was added
and stirred for 12 h at room temperature. The product linear tetra-
peptide (23) (0.260 g, 79%) was obtained in a similar manner as
described earlier as foam. HPLC, retention time 8.68 min. The pro-
tected tetrapeptide (23) (0.255 g, 0.32 mmol) was dissolved in TFA
(3 mL) at 0 °C and kept for 3 h. After evaporation of TFA, the residue
was solidified using ether and petroleum ether to yield TFA salt of
the linear tetrapeptide (0.215 g, 92%). To DMF solvent 290 mL), the
TFA salt (0.215 g, 0.29 mmol), HATU (0.167 g, 0.44 mmol), and
DIEA (0.125 mL, 0.73 mmol) were added in separate five portions
in every 30 min with stirring, for the cyclization reaction. After
completion of the reaction, DMF was evaporated under vacuo,
the residue was dissolved in ethyl acetate and washed with citric
acid (10%) solution, sodium bicarbonate (4%) solution, and brine,
respectively. It was then dried over anhydrous MgSO4 and filtered.
After evaporation of ethyl acetate, the residue was purified by silica
gel chromatography using a mixture of chloroform and methanol
(99:1) to yield the bicyclic tetrapeptide (24) (0.115 g, 65%). HPLC,
retention time 6.18 min. Compound 24 (0.105 g, 0.17 mmol) was
dissolved in acetic acid (5 mL) and Pd–C (0.03 g) was added. The
mixture was stirred under H2 for 12 h. After filtration of Pd–C,
4.1.5. Synthesis of Boc-
-Aa4-OH) (12)
To a cooled solution of Boc-
(2.5 g, 11.4 mmol) in DMF (30 mL), 60% NaH (1.0 g, 25.1 mmol)
was added and stirred until the evolution of H2 gas was stopped.
Allyl bromide (1.06 mL, 12.5 mmol) was added and the reaction
mixture was stirred for 12 h at room temperature. After
evaporation of DMF, 4% NaHCO3 (50 mL) was added and the
mixture was washed with diethyl ether to remove unreacted allyl
bromide. The aqueous layer was acidified with solid citric acid to
L-2-amino-4-allyloxybutanoic acid (Boc-
L
L-Homoserine (Boc-L-Hse-OH) (18)
pH 3, and Boc-
ethyl acetate layer was washed with brine and dried over MgSO4.
After evaporation of ethyl acetate, Boc- -Hse(Allyl)-OH (12)
L-Hse(Allyl)-OH was extracted in ethyl acetate. The
L
(2.5 g, 81%) was obtained as heavy oil. HPLC retention time
5.57 min.
4.1.6. Synthesis of Boc-
-Aa4-OH) (13)
Compound 13 was synthesized by the same procedure as
described for compound 12 using Boc- -Hse-OH (19) instead of
Boc- -Hse-OH (18). HPLC retention time 5.56 min.
D-2-amino-4-allyloxybutanoic acid (Boc-
D
D
L

3868
M. N. Islam et al. / Bioorg. Med. Chem. 22 (2014) 3862–3870
acetic acid was evaporated to yield bicyclic tetrapeptide carboxylic
acid (0.088 g, 100%). HPLC, retention time 4.84 min. The bicyclic
tetrapeptide carboxylic acid (0.078 g, 0.15 mmol) was dissolved
in DMF (2 mL) at 0 °C, and O-Benzylhydroxylamine hydrochloride
(0.021 g, 0.30 mmol), HOBtÁH2O (0.046 g, 0.30 mmol), triethyl-
amine (0.043 mL, 0.30 mmol) and DCC (0.074 g, 0.36 mmol) were
added. The mixture was stirred for 24 h. The product (0.08 g,
91%) obtained was dissolved in acetic acid (5 mL), and Pd–BaSO4
(0.10 g) was added. The mixture was stirred under H2 for 24 h.
After filtration of Pd–BaSO4, acetic acid was evaporated and crys-
tallized with ether to yield compound 3 (0.106 g, 72%). HPLC,
retention time 4.31 min. 1H NMR (500 MHz, DMSO-d6): dH 1.08–
1.73 (m, 12H), 1.89–1.98 (m, 5H), 2.18–2.24 (m, 2H), 3.20–3.69
(m, 12H), 3.97 (ddd, J = 9.5, 9.5, 3.5 Hz, 1H), 4.20 (m, 2H), 4.43
(m, 1H), 4.70 (d, J = 7.5 Hz, 1H), 4.82 (m, 1H), 6.88 (d, J = 9.5 Hz,
1H), 7.15 (d, J = 9.5 Hz, 1H), 8.50 (d, J = 8.5 Hz, 1H), 8.63 (s, 1H),
10.31 (s, 1H). HR FAB-MS [M+H]+ 540.3043 for C25H42N5O8 (calcd
540.3033).
acid (10%) solution, sodium bicarbonate (4%) solution, and brine,
respectively. It was then dried over anhydrous MgSO4 and filtered.
After evaporation of ethyl acetate, the residue was purified by silica
gel chromatography using a mixture of chloroform and methanol
(99:1) to yield the cyclic tetrapeptide 26 (0.290 g, 41%). HPLC,
retention time 6.12 min. To a solution of 26 (0.270 g, 0.424 mmol)
in anhydrous and degassed DCM (55 mL), a solution of Grubbs first
generation ruthenium catalyst (0.073 g, 0.09 mmol) in anhydrous
and degassed DCM (5 mL) was added. The reaction mixture was
stirred at room temperature for 48 h. After the completion of reac-
tion, DCM was evaporated and the residue was purified by silica
gel chromatography using a mixture of chloroform and methanol
(99:1) to yield bicyclic tetrapeptide as foam which on catalytic
hydrogenation in presence of Pd–C (0.050 g) in AcOH (5 mL) yield
bicyclic tetrapeptide carboxylic acid (27) (0.090 g, 40%). HPLC,
retention time 7.47 min. Compound 27 (0.090 g, 0.17 mmol)
was dissolved in DMF (1 mL) at 0 °C, and O-Benzylhydroxylamine
hydrochloride
(0.042 g,
0.26 mmol),
HOBtÁH2O
(0.027 g,
0.17 mmol), triethylamine (0.038 mL, 0.26 mmol) and DCC
(0.054 g, 0.26 mmol) were added. The mixture was stirred for
24 h. The product bicyclic tetrapeptide hydroxamic acid benzyl
ester (0.073 g, 68%) was obtained in a similar manner as described
earlier. The bicyclic tetrapeptide hydroxamic acid benzyl ester
(0.073 g, 0.12 mmol) was dissolved in acetic acid (5 mL), and Pd–
BaSO4 (0.100 g) was added. The mixture was stirred under H2 for
24 h. After filtration of Pd–BaSO4, acetic acid was evaporated and
crystallized with ether to yield compound 4 (0.042 g, 67%). HPLC,
retention time 5.80 min. 1H NMR (500 MHz, DMSO-d6, 40 °C): dH
1.07–1.13 (m, 2H),1.16–1.39 (m, 16H), 1.43–1.54 (m, 4H), 1.64–
1.78 (m, 4H), 1.81 (s, 2H), 1.91–1.98 (m, 3H), 3.29–3.41 (m, 4H),
3.78 (m, 1H), 4.14 (ddd, J = 11, 11, 3.6 Hz, 1H), 4.35 (dd, J = 11.6,
7.9 Hz, 1H), 5.06 (d, J = 7.9 Hz, 1H), 8.33 (d, J = 7.6 Hz, 1H), 8.44
(d, J = 11 Hz, 1H), 8.90 (s, 1H). HR FAB-MS [M+Na]+ 558.3187 for
4.1.8. Synthesis of trapoxin based bicyclic tetrapeptide
hydroxamic acid (4)
To a cooled solution of H-D L-
-Pro-OtBu (0.960 g, 5.6 mmol), Boc-
Ae8-OH (1.58 g, 6.16 mmol) and HOBtÁH2O (0.857 g, 5.6 mmol) in
DMF (12 mL), DCC (1.38 g, 6.72 mmol) was added. The mixture
was stirred for 12 h at room temperature. 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 concentrated to remain an oily sub-
stance, which was purified by silica gel chromatography using a
mixture of chloroform and methanol (99:1) to yield Boc-L-Ae8-D-
Pro-OtBu (2.16 g, 93%) as oil. The protected dipeptide (2.15 g,
5.24 mmol) was dissolved in 4 M HCl/dioxane (25 mL) and the
mixture was kept at room temperature for 35 min. The reaction
was monitored by TLC. After completion of the reaction HCl/diox-
ane was evaporated. The residue was dissolved in EtOAc and
washed with saturated Na2CO3, dried over anhydrous Na2CO3
C27H45N5O6Na (calcd 558.3268).
4.1.9. Synthesis of HC-toxin-I based bicyclic tetrapeptide
hydroxamic acid (5)
and EtOAc was evaporated to get H-
oil (1.44 g, 88%). To a cooled solution of H-
(1.44 g, 4.64 mmol), Boc- -Ae8-OH (1.31 g, 5.10 mmol) and
L
-Ae8-
D
-Pro-OtBu as heavy
-Ae8-
-Pro-OtBu
4.1.9.1. Synthesis of H-
L
-Asu(OBzl)-
D
-Pro-OtBu (29).
To a
L
D
cooled solution of H- L-
D
-Pro-OtBu (0.555 g, 3.25 mmol), Boc-
L
Asu(OBzl)-OH (0.907 g, 3.25 mmol) and HOBtÁH2O (0.497 g,
3.25 mmol) in DMF (7 mL), DCC (0.803 g, 3.9 mmol) was added.
The mixture was stirred for 12 h at room temperature. 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 concentrated to
remain an oily substance, which was purified by silica gel chroma-
tography using a mixture of chloroform and methanol (99:1) to
HOBtÁH2O (0.710 g, 4.64 mmol) in DMF (10 mL), DCC (1.15 g,
5.57 mmol) was added and stirred for 12 h at room temperature.
The product Boc-D-Ae8-L-Ae8-D
-Pro-OtBu was obtained in a similar
manner as described earlier as heavy oil (2.14 g, 82%). HPLC, reten-
tion time 8.74 min. The protected tripeptide (1.00 g, 1.82 mmol)
was dissolved in 4 M HCl/dioxane (10 mL) and the mixture was
kept at room temperature for 30 min. The reaction was monitored
by TLC. After completion of the reaction HCl/dioxane was evapo-
rated. The residue was dissolved in EtOAc and washed with satu-
rated Na2CO3, dried over anhydrous Na2CO3 and EtOAc was
evaporated to get tripeptide free amine (0.780 g, 95%). To a cooled
yield Boc-L-Asu(OBzl)-D
-Pro-OtBu (28) (1.40 g, 80%) as oil. The pro-
tected dipeptide (28) (1.40 g, 2.60 mmol) was dissolved in 4 M HCl/
dioxane (13 mL) and the mixture was kept at room temperature for
30 min. The reaction was monitored by TLC. After completion of
the reaction HCl/dioxane was evaporated. The residue was
dissolved in EtOAc and washed with saturated Na2CO3, dried over
anhydrous Na2CO3 and EtOAc was evaporated to get
solution of the free amine (0.780 g, 1.74 mmol), Boc-L-Asu(OBzl)-
OH (0.660 g, 1.74 mmol) and HOBtÁH2O (0.266 g, 1.74 mmol) in
DMF (4 mL), DCC (0.430 g, 2.09 mmol) was added and stirred for
12 h at room temperature. The product linear tetrapeptide (25)
(0.930 g, 65%) was obtained in a similar manner as described ear-
lier as foam. HPLC, retention time 9.21 min. The protected tetra-
peptide 25 (0.920 g, 1.14 mmol) was dissolved in TFA (4 mL) at
0 °C and kept for 3 h. After evaporation of TFA, the residue was
solidified using ether and petroleum ether to yield TFA salt of the
linear tetrapeptide (0.835 g, 97%). To DMF solvent (550 mL), the
TFA salt (0.835 g, 1.11 mmol), HATU (0.633 g, 1.66 mmol), and
DIEA (0.48 mL, 2.78 mmol) were added in separate five portions
in every 30 min with stirring for the cyclization reaction. After
completion of the reaction, DMF was evaporated under vacuo,
the residue was dissolved in ethyl acetate and washed with citric
H-L-Asu(OBzl)-D
-Pro-OtBu (29) as heavy oil (0.840 g, 68%).
4.1.9.2. Synthesis of linear dipeptide with fused ring
(31). To -Ae8-OBzl (1.26 g,
cooled solution of HClÁH-
4.44 mmol), Boc- -Ae8-OH (1.14 g, 4.44 mmol), triethylamine
a
D
L
(0.639 mL, 4.44 mmol) and HOBtÁH2O (0.679 g, 4.44 mmol) in
DMF (10 mL), DCC (1.10 g, 5.33 mmol) was added. The mixture
was stirred for 12 h at room temperature. 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

M. N. Islam et al. / Bioorg. Med. Chem. 22 (2014) 3862–3870
3869
over anhydrous MgSO4 and concentrated to remain an oily sub-
stance, which was purified by silica gel chromatography using a
1H), 4.60 (d, J = 7 Hz, 1H), 7.2 (d, J = 4 Hz, 1H), 7.62 (d, J = 8.5 Hz,
1H), 8.12 (d, J = 8.8 Hz, 1H); FAB-MS [M+H]+ 538.3635 for
mixture of chloroform and methanol (99:1) to yield Boc-
L
-Ae8-
C27H48N5O6 (calcd 538.3605).
D
-Ae8-OBzl (30) (1.60 g, 80%) as crystalline solid. To a solution of
linear dipeptide (0.973 g, 2 mmol) in anhydrous and degassed
DCM (250 mL), a solution of Grubbs first generation ruthenium
catalyst (0.330 g, 0.4 mmol) in anhydrous and degassed DCM
(50 mL) was added. The reaction mixture was stirred at room tem-
perature for 48 h. After the completion of reaction, DCM was evap-
orated and the residue was purified by silica gel chromatography
using chloroform to get fused cyclic dipeptide which on catalytic
hydrogenation in presence of Pd–C (0.150 g) in AcOH (7 mL)
yielded compound (31) (0.465 g, 63%).
4.1.11. Synthesis of reference compound 7
This compound was synthesized in
described for compound 5 using Boc- -Ae7-OBzl and Boc-
OH instead of Boc- -Ae8-OBzl and Boc- -Ae8-OH, respectively,
a
similar manner as
-Ae7-
D
L
D
L
and by excluding RCM step. HPLC, retention time 7.67 min. 1H
NMR (500 MHz, DMSO-d6, 40 °C) dH 0.84 (m, 6H), 1.19–1.32 (m,
16H), 1.47 (m, 5H), 1.55–1.75 (m, 5H), 1.92 (t, J = 7.3, 2H), 2.20
(m, 1H), 2.50 (m, 2H), 3.53 (dd, J = 9.5, 8.5 Hz, 1H), 3.74 (ddd,
J = 9.5, 9.3, 4 Hz, 1H), 4.24 (m, 2H), 4.63 (m, 1H), 4.71 (d,
J = 7.3 Hz, 1H), 6.99 (d, J = 9.8 Hz, 1H), 7.19 (d, J = 9.5 Hz, 1H),
8.37 (d, J = 8.8, 1H); FAB-MS [M+H]+ 538.3545 for C27H48N5O6
(calcd 538.3605).
4.1.9.3. Synthesis of bicyclic tetrapeptide hydroxamic acid
(5).
To a cooled solution of H-L-Asu(OBzl)-D
-Pro-OtBu (29)
(0.540 g, 1.25 mmol), compound 31 (0.446 g, 1.20 mmol) and
HOBtÁH2O (0.184 g, 1.20 mmol) in DMF (3 mL), DCC (0.296 g,
1.44 mmol) was added. The mixture was stirred for 12 h at room
temperature. After completion of the reaction, the product linear
tetrapeptide (32) (0.670 g, 72%) was obtained in a similar manner
as described earlier as foam. HPLC, retention time 9.21 min. The
protected tetrapeptide 32 (0.660 g, 0.84 mmol) was dissolved in
TFA (3 mL) at 0 °C and kept for 3 h. After evaporation of TFA, the
residue was solidified using ether and petroleum ether to yield
TFA salt of the linear tetrapeptide (0.580 g, 95%). To DMF solvent
(800 mL), the TFA salt (0.580 g, 0.80 mmol), HATU (0.456 g,
1.20 mmol), and DIEA (0.384 mL, 2 mmol) were added in separate
five portions in every 30 min with stirring, for the cyclization reac-
tion. After completion of the reaction, DMF was evaporated under
vacuo and the product bicyclic tetrapeptide benzyl ester (0.250 g,
51%) was obtained in a similar manner as described earlier as foam.
HPLC, retention time 9.21 min. The bicyclic tetrapeptide benzyl
ester (0.230 g, 0.376 mmol) was dissolved in acetic acid (5 mL)
and Pd–C (0.050 g) was added. The mixture was stirred under H2
for 12 h. After filtration of Pd–C, acetic acid was evaporated to yield
bicyclic tetrapeptide carboxylic acid (0.195 g, 95%). The bicyclic
tetrapeptide carboxylic acid (0.165 g, 0.317 mmol) was dissolved
in DMF (2 mL) at 0 °C, and O-Benzylhydroxylamine hydrochloride
(0.76 g, 0.476 mmol), HOBtÁH2O (0.049 g, 0.317 mmol), triethyl-
amine (0.069 mL, 0.476 mmol) and DCC (0.098 g, 0.476 mmol)
were added. The mixture was stirred for 24 h. After completion
of the reaction, DMF was evaporated under vacuum and the prod-
uct bicyclic tetrapeptide hydroxamic acid benzyl ester (0.160 g,
81%) was obtained in a similar manner as described earlier as foam.
The bicyclic tetrapeptide hydroxamic acid benzyl ester (0.160 g,
0.256 mmol) was dissolved in acetic acid (5 mL), and Pd–BaSO4
(0.100 g) was added. The mixture was stirred under H2 for 24 h.
After filtration of Pd–BaSO4, acetic acid was evaporated and crys-
tallized with ether to yield bicyclic tetrapeptide hydroxamic acid
(5) (0.090 g, 66%). HPLC, retention time 7.06 min. 1H NMR
(500 MHz, DMSO-d6, 40 °C): dH 1.05–1.79 (m, 24H), 1.83–2.08 (m,
10H), 3.37–3.45 (m, 1H), 3.55 (m, 1H), 3.62 (ddd, J = 9.5, 9.5,
3.8 Hz, 1H), 4.34–4.43 (m, 2H), 4.64 (m, 1H), 4.72 (d, J = 7.3 Hz,
1H), 6.85 (d, J = 10.1 Hz, 1H), 7.16 (d, J = 9.8 Hz, 1H), 8.75 (d,
J = 8.6, 1H),8.63 (s, 1H), 10.27 (s, 1H). HR FAB-MS [M+H]+
536.3445 for C27H48N5O6 (calcd 536.3448).
4.2. Preparation of HDACs and assay for enzyme activity
293T cells (1–2 Â 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
LipofectAMINE2000 reagent (Invitrogen). After successive cultiva-
tion in DMEM for 24 h, the cells were washed with PBS and lysed
by sonication in lysis buffer containing 50 mM Tris–HCl (pH 7.5),
120 mM NaCl, 5 mM EDTA, and 0.5% NP40. The soluble fraction col-
lected by microcentrifugation was precleared by incubation with
protein A/G plus agarose beads (Santa Cruz Biotechnologies, 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 deacetylase buffer
consisting of 20 mM Tris–HCl (pH 8.0), 150 mM NaCl, and 10% glyc-
erol. The bound proteins were released from the immune complex
by incubation for 1 h at 4 °C with 40
Aldrich Inc.) in histone deacetylase buffer (200
was collected by centrifugation. For the enzyme assay, 10
enzyme fraction was added to 1 L of fluorescent substrate (2 mM
Ac-KGLGK(Ac)-MCA) and 9 L of histone deacetylase buffer, and
the mixture was incubated at 37 °C for 30 min. The reaction was
stopped by the addition of 30 L of trypsin (20 mg/ml) and incu-
l
g of the FLAG peptide (Sigma–
L). The supernatant
L of the
l
l
l
l
l
bated 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 independent dose response curves.
4.3. The p21 promoter assay
The human wild-type p21 promoter luciferase fusion plasmid,
WWP-Luc, was a kind gift from Dr. B. Vogelstein. A luciferase
reporter plasmid (pGW-FL) was constructed by cloning the 2.4 kb
genomic fragment containing the transcription start site into Hin-
dIII and SmaI sites of the pGL3-Basic plasmid (Promega Co., Madi-
son, WI). Mv1Lu (mink lung epithelial cell line) cells were
transfected with the pGW-FL and a phagemid expressing neomy-
cin/kanamycin resistance gene (pBKCMV, Stratagene, La Jolla, CA)
with the Lipofectamine reagent (Life Technology, Rockville, MD,
4.1.10. Synthesis of reference compound 6
USA). After the transfected cells had been selected by 400 lg/mL
This compound was synthesized in
described for compound using Boc-
Boc-
a
similar manner as
Geneticin (G418, Life Technology), colonies formed were isolated.
One of the clones was selected and named MFLL-9. MFLL-9
expressed a low level of luciferase, whose activity was enhanced
by TSA in a dose-dependent manner. MFLL-9 cells (1 Â 105) cul-
tured in a 96-well multi-well plate for 6 h were incubated for
18 h in the medium containing various concentrations of drugs.
The luciferase activity of each cell lysate was measured with a
4
L-Ae7-OH instead of
L
-Ae8-OH and by excluding RCM step. HPLC, retention time
6.55 min. 1H NMR (500 MHz, DMSO-d6) dH 0.82–0.88 (m,
6H),1.16–1.33 (m, 16H), 1.43–1.69 (m, 9H), 1.71–1.84 (m, 2H),
1.89–1.97 (m, 2H), 2.01–2.18 (m, 2H), 3.74 (dd, J = 13.2, 7.5 Hz,
1H), 3.88 (m, 1H), 4.08 (m, 1H), 4.29 (m, 1H), 4.38(d, J = 8.2 Hz,

3870
M. N. Islam et al. / Bioorg. Med. Chem. 22 (2014) 3862–3870
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