Synthesis and biological evaluation of histone deacetylase inhibitors that are based on FR235222: A cyclic tetrapeptide scaffold

Article abstract of DOI:10.1016/j.bmcl.2008.03.047

We outline the synthesis of six novel derivatives that are based on a recently discovered HDAC inhibitor FR235222. Our work is the first report utilizing a novel binding element, guanidine, as metal coordinators in HDAC inhibitors. Further, we demonstrate that these compounds show cytotoxicity that parallels their ability to inhibit deacetylase activity, and that the most potent compounds maintain an l-Phe at position 1, and a d-Pro at position 4. Both inhibition of HDAC activity and cytotoxicity against the pancreatic cancer cell line BxPC3 are exhibited by these compounds, establishing that a guanidine unit can be utilized successfully to inhibit HDAC activity.

Full text of DOI:10.1016/j.bmcl.2008.03.047

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2549–2554
Synthesis and biological evaluation of histone
deacetylase inhibitors that are based on FR235222:
A cyclic tetrapeptide scaffold
Erinprit K. Singh,a Suchitra Ravula,a Chung-Mao Pan,a Po-Shen Pan,a
Robert C. Vasko,a Stephanie A. Lapera,a Sujith V. W. Weerasinghe,b
Mary Kay H. Pflumb and Shelli R. McAlpinea,*
aDepartment of Chemistry and Biochemistry, 5500 Campanile Drive, San Diego State University, San Diego, CA 92182, USA
bDepartment of Chemistry, 5101 Cass Avenue, Wayne State University, Detroit, MI 48202, USA
Received 6 February 2008; revised 14 March 2008; accepted 17 March 2008
Available online 20 March 2008
Abstract—We outline the synthesis of six novel derivatives that are based on a recently discovered HDAC inhibitor FR235222. Our
work is the first report utilizing a novel binding element, guanidine, as metal coordinators in HDAC inhibitors. Further, we dem-
onstrate that these compounds show cytotoxicity that parallels their ability to inhibit deacetylase activity, and that the most potent
compounds maintain an L-Phe at position 1, and a D-Pro at position 4. Both inhibition of HDAC activity and cytotoxicity against
the pancreatic cancer cell line BxPC3 are exhibited by these compounds, establishing that a guanidine unit can be utilized success-
fully to inhibit HDAC activity.
Ó 2008 Elsevier Ltd. All rights reserved.
Histone deacetylases (HDACs) are enzymes responsible
for the regulation of chromatin remodeling and gene
transcription by deacetylation of the amino-terminal
tails of histones. The inappropriate up-regulation of
HDACs is associated with carcinogenesis, and inhibitors
of HDACs have demonstrated efficacy against cancer
cell lines.1–3
rate to current chemotherapeutic treatments, there is an
immediate need for new drugs that provide additional
chemotherapeutic options to pancreatic cancer patients.
To date, HDACIs can be divided into five chemical fam-
ilies: hydroxamic acid derivatives, short chain fatty
acids, benzamides, electrophilic ketones, and cyclic tet-
rapeptides.1,11 These five families all inhibit the activity
of metal-dependent HDAC classes I and II. The phar-
macophore model for HDAC inhibition consists of
three elements: (1) a surface recognition unit binding
to the rim of the binding pocket, (2) a metal-binding do-
main, which chelates with the metal cation in the active
site, and (3) a linker that connects the surface recogni-
tion site to the metal-binding domain (Fig. 1). In most
natural products, one of three binding elements is typi-
cally found: alpha hydroxy ketones, epoxides, and N-
hydroxamic acids.11,12 There is a clear understanding
that HDACIs function by using the surface recognition
unit to bind to the HDAC protein surface near the metal
pocket, thus placing the metal-binding element inside
the binding pocket. However, for the FR235222 deriva-
tives a diversity of surface recognition and metal-bind-
ing elements and their effects on HDAC inhibition
have not been explored. Poor pharmacokinetic
There are already numerous HDAC inhibitors in clinical
trials.3,4 However, treating pancreatic cancers has been
unsuccessful with any drug currently on the market. Gi-
ven that HDACs are inappropriately up-regulated in
pancreatic cancers,5,6 HDAC inhibitors (HDACIs) have
tremendous potential for treating these drug-resistant
cancers. Pancreatic cancer is the fifth most deadly cancer
in U.S. Only 10% of patients are eligible for surgery,7
and less than 20% of pancreatic cancers respond to the
drug of choice (Gemzar) or other drugs on the mar-
ket.8,9 The 5-year survival rate for patients with pancre-
atic cancers is less than 5%.10 With such a low response
Keywords: HDAC; Guanidine; FR235222; Peptide; Pancreatic cancer.
*
Corresponding author. Tel.: +1 619 594 5580; fax: +1 619 594
4634; e-mail: mcalpine@sciences.sdsu.edu
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.047

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E. K. Singh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2549–2554
moiety.1,11 These guanidine and acetyl compounds are
1
derivatives of FR235222, which was isolated from the
fermentation broth of a fungus, Acremonium sp. No.
27082.21 Previous work by the Mori group reports that
FR235222 has potency against three types of lympho-
cytes (MLR, anti-CD3-blast, and TPA-blast) with IC50
values in the low nanomolar range thus demonstrating
promising anti-cancer properties.21 In this report we de-
scribe how changes to the surface recognition site at all
four positions (Fig. 1) impact the compound’s cytotox-
icity and HDAC activity. Literature precedence has
shown that a 4- to 5-atom chain is optimal in the linker
region of the molecule,2 therefore we have utilized both
a 4-atom chain and a 5-atom chain between the surface
recognition macrocyclic peptide and the carbonyl of the
acetyl or imine unit of the guanidine. Finally, we
explored how effective a guanidine unit would be as a
metal-binding unit compared to the acetyl moiety.
O
N
4
O
2
HN
HN HN
O
O
3
O
metal
binding
unit
surface
recognition
site
linker
FR235222 HDAC inhibitor
( )n
HO
R1
1
4
O
HN
NR
H3CO HN
O
O
O
3
H
2
N
( )n R5
BocHN
Fragment 1
n = 1 or 2
Fragment 2
We used a convergent solution phase route in order to
easily insert L- and D-amino acids in each position with-
in the derivatives. This route is also amenable to large-
scale synthesis for extensive biological studies. Scheme
1 depicts the two fragments involved in our synthetic
route, allowing us to form the linear tetrapeptide, which
is cyclized to generate the final product. The synthesis of
six novel HDACIs was completed using amino acids
shown in Scheme 1. Using 2(1-H-benzotriazole-1-yl)-
1,1,3-tetramethyl-uronium tetrafluoroborate (TBTU),
and diisopropylethylamine (DIPEA), acid-protected res-
idue 1(a–d), and N-Boc-protected residue 2 (Scheme 1)
were coupled to give the dipeptide MeO–1–2 (90–95%
yield). Deprotection of the acid on residue 1 using lith-
ium hydroxide gave the free acid 1–2 (85–95% yield)
as shown in Fragment 1. The synthesis of Fragment 2
(Fig. 1) was completed by coupling residue 3(a–b) to res-
idue 4(a–b) (Scheme 1) to give the dipeptide 3-4-Boc
(90–95% yield). The amine was deprotected on Frag-
ment 2 using TFA to give the free amine 3–4 (ꢀquanti-
tative yield). Fragments 1 and 2 were coupled using
multiple coupling agents yielding six examples of linear
tetrapeptides (yields ranged from 30% to 90% depending
on the substrate). Upon formation of the linear tetra-
peptide, an acid deprotection using lithium hydroxide
was performed (ꢀ85–95% yield), subsequently followed
by the deprotection of the amine on the linear tetrapep-
tide with TFA (ꢀquantitative yield). With a free acid
and free amine, cyclization of the linear tetrapeptide
was performed by dissolving it in 2:2:1 ratio of THF:CH
3CN:CH2Cl2 (0.007 M). Addition of DIPEA (6 equiv)
and three coupling agents (HATU, DEPBT, and TBTU
0.7 equiv ea) to the reaction gave a clear solution. Reac-
tions were usually complete in 2–4 h. A work-up with
methylene chloride and ammonium chloride, concentra-
tion in vacuo, purification via flash chromatography and
HPLC, provided the final products confirmed via LCMS
and H1NMR (yields ranged from ꢀ25% to 65% depend-
ing on the substrate). With the exception of compounds
5 and 6, a deprotection of amines on residues 3(a–b)
using hydrogenolysis were performed upon completion
of cyclization. This deprotection was accomplished by
dissolving the cyclized compound in EtOH (0.1 M)
and treating with hydrogen gas in the presence of 10%
NH
NH2
O
R = H or Me
R5
=
R
1 - R4 represent side chains
for amino acids at positions 1-4
Figure 1. Retrosynthetic approach for FR235222.
properties associated with several of the metal binding
domains led us to investigate the possibility of novel
structures utilizing guanidine and acetyl as metal-bind-
ing elements.1 Herein, we describe the synthesis of a
new family of cyclic peptides that are based on the
FR235222 natural product, a cyclic tetrapeptide struc-
turally related to Trapoxin, HC-toxin, Chlamydocin,
and apicidin.2,13,14 For the first time, it is explored
how a guanidine metal-binding element impacts the
HDAC inhibition and cytotoxicity.
There is extensive literature on derivatives containing the
three moieties found in natural product metal-binding
units. In addition to work exploring their potency, other
non-traditional metal binding units have been published
including, sulfur,15 N-formyl hydroxylamine,16 and
phosphorous-containing compounds.17 However, no
work has been published to date on guanidines as me-
tal-binding units in HDACIs. Guanidines represent a
very important class of compounds both biologically
and chemically. Their hydrophilic nature provides stabil-
ization of protein conformations via hydrogen bonding
and mediates solubility of natural products.18 With a
high pKa value of 12.5, arginine residues containing a
guanidinium side chain may not be considered optimal
metal-binding ligands. However, the highly acidic nature
of a metal cation found in the HDAC pocket can poten-
tially lower the pKa value of the guanidinium side chain,
allowing for coordination with the metal. In fact, several
recent reports document the stability of guanidine–metal
interactions,19,20 although additional studies are needed.
Despite their likely metal binding capabilities, guani-
dines have gone unexplored in the realm of HDACI as
potential metal-binding units.
Here we describe the design and synthesis of HDACI
incorporating the use of guanidines and compare them
to a more established metal-binding element, an acetyl

E. K. Singh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2549–2554
2551
O
OCH3
NHR
O
O
NHBoc
R
N
(a)
(c)
HO
R1
HO
NHBoc
+
R1
O
2
1
1-2 (Fragment 1)
O
R
N
R1
O
HO
NHBoc
( )n
R1
O
O
NR
N
(a)
1-2 (Fragment 1)
NHBoc
O
3
HN
O
( )n
MeO
( )n
( )n
OH
NH
R5
N
H
R5
O
HN
NH2
1. (a)
2. (c)
Linear Precursor
H3CO
O
HN
O
H
N
( )n
( )n
R5
BocN
HO
3-4 (Fragment 2)
4
O
R1
O
1
1. (d)
( )n
4
O
2. (b)
3. (e)
RN
N
HN
HN
O
3
H
metal
binding
unit
2
N
O
( )n
R5
surface
recognition
site
linker
Cyclized FR235222
Derivative
n = 1 or 2
NH
O
R = H or Me
R1 - R4 represent side chains
for amino acids at positions 1-4
R5 =
NH2
3
OMe
OMe
1
O
O
OMe
O
OMe
NH2
NH2
4
O
O
NH2
a
NH2
O
BocN
a
HO
b
d
BocN
b
HO
OMe
a
b
NH
NCBz
OMe
O
O
O
NH
CH3
NH
N
H
BzCHN
c
Scheme 1. Synthesis of FR235222 derivatives Reagents and conditions: (a) coupling agent (TBTU (1.2 equiv), and/or HATU (0.75 equiv)), DIPEA
(3 equiv), CH2Cl2 (0.1 M); (b) TFA (20%), anisole (2 equiv), CH2Cl2; (c) LiOH (4 equiv), MeOH; (d) LiOH (6 equiv), MeOH; (e) HATU (0.7 equiv),
DEPBT (0.7 equiv), TBTU (0.7 equiv), DIPEA (6 equiv), THF/CH3CN/DCM (2:2:1) 0.007 M.11
Pd/C. The reaction was stirred for 3–5 h under hydro-
gen, whereupon filtration with Celite yielded the final
product.22
N-methyl Phe (residue 1c) (compound 6), and the incor-
poration of a rigid binding element, tetrahydroisoquino-
line (residue 1d), (compound 4) (Fig. 2). Based on
previous precedents no changes were made to position
2 as changes to this position did not appear to effect po-
tency,2,11 and therefore a single ethyl moiety was utilized
at this position for all 6 compounds for ease of synthesis
(Fig. 2). It is well established that in position 4 it is
critical to maintain a D-amino acid in order to ensure
the appropriate binding of the macrocycle to the HDAC
binding site and for appropriate insertion of the linker
element.23 Further, studies have shown that a b-turn
element was important at this position, and indeed a
The amino acids (Scheme 1) were chosen based on work
done on other classes of tetrapeptide HDAC inhibitors.
It was demonstrated that altering the surface recogni-
tion unit at position 1 enhances potency relative to the
natural product depending on the amino acids in posi-
tions 2–4.2,23 Further, this position was known to toler-
ate both a D- and L-amino acid.2,23 Thus, our changes to
position 1 included: an L-Phe (residue 1a) (compounds 1
and 5), a D-Phe (residue 1b) (compounds 2 and 3), an

2552
E. K. Singh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2549–2554
O
O
O
N
O
NH
N
O
NH
N
O
NH
NH HN
O
NH HN
O
O
H
N
4
NH HN
O
O
H
N
NH2
NH
3
NH2
NH
H
N
O
1
2
NH2
NH
Compound 1
Compound 3
Compound 2
O
O
O
N
N
O
O
NH
N
N
O
N
NH HN
O
O
H
NH HN
O
3
O
N
NH2
NH
NH HN
O
O
O
5
O
1
N
CH3
2
4
H
N
H
CH3
Compound 4
Compound 5
O
Compound 6
N
O
NH
NH
HN
O
O
O
N
OCH3
Apicidin
Figure 2. FR235222 derivatives (compounds 1–6) and Apicidin.
D-proline (residue 4a) or a D-piperdinyl carboxylate res-
idue (residue 4B) was considered optimal.2,23 Thus, we
included a D-proline (compounds 1, 2, 4, and 6), and a
D-piperdinyl carboxylate moiety (compound 3) as part
of our exploratory work on this new class of derivatives.
potent cyclic tetrapeptide HDACI (IC50 value of
13 2 nM) was used as a positive control (0.3 0.3%
deacetylase activity) and DMSO was used as a negative
control (set to 100% deacetylase activity) (Fig. 3). Com-
pound 5 was the most potent compound, and inhibited
deacetylase activity to 38 2% at 200 lM concentration
relative to the DMSO control. This compound con-
We also made modifications to the linker region at posi-
tion 3 (Fig. 2). Previous work had shown that a 4- to 5-
atom chain was optimal in the linker region of the
molecule in order to place the electronegative binding
element at the appropriate position in the HDAC pock-
et.2 Thus, we chose two different residues for substitu-
tion at this position, where one contained a 4-atom
chain (residue 3a) leading to the guanidine unit (com-
pounds 1–4), and the other utilized a 5-atom chain lead-
ing to the protected lysine binding element (residue 3b)
(compounds 5 and 6). Finally, we utilized two different
binding elements: guanidine (3a) (compounds 1–4) and
N-acetyl (3b) (compounds 5 and 6). Note: the nitrogen
of the guanidine and the nitrogen of the side chain lysine
are considered part of the linker unit, whereas the ami-
dine of the guanidine and the carbonyl of the acetyl are
considered part of the metal-binding element.
FR235222 Derivatives - Deacetylase Activity
100%
80%
60%
40%
20%
0%
A
1
2
3
4
5
6
0.3% 97% 97% 98% 83% 38% 86%
HDAC 200uM
Figure 3. HDAC inhibition assay of FR235222 derivatives. Each
column represents the average of three independent trials at 200 lM of
apicidin or compounds 1–6, with standard error of 0.3%, 4%, 5%,
4%, 1%, 2%, and 6%, respectively. A, Apicidin. All data were
normalized to the 4% DMSO control (100%).
The six compounds were assayed at 200 lM concentra-
tion against endogenous HDACs from HeLa cell ly-
sates, as previously described.24 Apicidin (Fig. 2) a

E. K. Singh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2549–2554
2553
tained an acetyl-binding unit, and like the natural prod-
uct, it possessed an L-Phe at position 1 and a proline res-
idue at position 4. Since compound 5 is an acetyl-lysine
mimic, the ability for compound 5 to act as an HDAC
mimic was assessed; this information can be found as
a part of the supplementary materials. Compounds 4
and 6 also displayed some potency at 200 lM concentra-
tion, with 83 1% and 86 6% activity, respectively,
relative to the DMSO control. Compounds 5 and 6 only
differ in the presence of an N-methyl at the position 1,
suggesting that this single methyl group substitution
influences potency by altering the conformation of the
macrocyclic surface recognition unit.11 Because com-
pound 4 contains a guanidine binding element, the data
suggest that guanidine is an appropriate choice when
designing new HDACI. It should be noted that com-
pounds 2 and 3 contain a D-Phe residue at position 1
and do not show appreciable deacetylase activity at
200 lM concentration, suggesting that the stereochemis-
try is important at position 1. In addition, compound 4
with a tetrahydroisoquinoline moiety at 1 demonstrated
greater potency than compound 1, which incorporated
an L-Phe, suggesting that more rigid side chain is impor-
tant for potency. In total, the data highlight the impor-
tant role of position 1 in governing inhibitor potency,
aiding future design efforts.
This compound contains an L-Phe at position 1, and a
D-Pro at position 4. Finally, we show that a guanidine
unit can be utilized successfully to inhibit HDAC
activity. Future derivatives will incorporate 5-atom
linkers with guanidines as metal binding elements as
well as metal binding elements with electron-withdraw-
ing groups. Their effectiveness will be described in the
near future.
Acknowledgment
We thank San Diego State University for financial sup-
port. Partial support was provided to R.C.V. by the Ho-
well Foundation.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2008.03.047.
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The deacetylase activity correlated with the cytotoxicity
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In summary, we outlined the synthesis of 6 novel
derivatives that are based on a recently discovered
HDAC inhibitor, FR235222. We show the use of a
novel binding element, guanidine, where our work is
the first to report these units being utilized as metal
coordinators in HDAC inhibitors. Further, we demon-
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80%
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20%
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A
1
2
3
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98%
27%
17%
20%
0%
38%
19%
BxPC3 5uM
Figure 4. Cytotoxicity assays of FR235222 derivatives. Data
points = average four wells from three assays (5 lM). Error = 5%,
A, Apicidin, control, DMSO.

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