Synthesis and biological activity of cyclotetrapeptide analogues of the natural HDAC inhibitor FR235222

Article abstract of DOI:10.1016/j.bmc.2010.03.022

In the course of our ongoing efforts to discover new and more effective HDAC inhibitors useful for the development of promising anticancer candidates, we have recently undertaken a molecular modelling study on a small collection of FR235222 analogues, synthesized by us in the frame of a structure-activity relationship investigation, made in order to identify the key structural elements essential for the activity. Progress made in structure elucidation of HDAC active site, together with accurate docking calculations, provided new structural insights useful for a further refinement of the tetrapeptide scaffold which should assure an optimal interaction between the synthetic ligands and the biological target. Following the computer aided suggestions we synthesized six new cyclotetrapeptide analogues of the lead compound (3-8), bearing a carboxylic or an hydroxamic acid functionality as Zn binding moiety. Herein we describe their synthesis and their inhibition activity on different HDAC isoforms.

Full text of DOI:10.1016/j.bmc.2010.03.022

Bioorganic & Medicinal Chemistry 18 (2010) 3252–3260
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological activity of cyclotetrapeptide analogues of the natural
HDAC inhibitor FR235222
Stefania Terracciano a, Simone Di Micco a, Giuseppe Bifulco a, Paola Gallinari b, Raffaele Riccio a, Ines Bruno a,
*
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy
b Dipartimento di Oncologia, Istituto di Ricerche di Biologia Molecolare P. Angeletti, 00040 Pomezia, Roma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In the course of our ongoing efforts to discover new and more effective HDAC inhibitors useful for the
development of promising anticancer candidates, we have recently undertaken a molecular modelling
study on a small collection of FR235222 analogues, synthesized by us in the frame of a structure–activity
relationship investigation, made in order to identify the key structural elements essential for the activity.
Progress made in structure elucidation of HDAC active site, together with accurate docking calculations,
provided new structural insights useful for a further refinement of the tetrapeptide scaffold which should
assure an optimal interaction between the synthetic ligands and the biological target. Following the com-
puter aided suggestions we synthesized six new cyclotetrapeptide analogues of the lead compound (3–8),
bearing a carboxylic or an hydroxamic acid functionality as Zn binding moiety. Herein we describe their
synthesis and their inhibition activity on different HDAC isoforms.
Received 21 December 2009
Revised 8 March 2010
Accepted 11 March 2010
Available online 15 March 2010
Keywords:
Anticancer drugs
Cyclopeptides
HDAC
Olefin metathesis
Molecular docking
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
tors, HSP90 and several nuclear importers;4 for that reason,
clarifying the exact mechanism of HDAC inhibitors anticancer ef-
The posttranslational modification of histone core, known as
epigenetic mechanism, is fundamentally important to conforma-
tional changes of the chromatin and to the modulation of tran-
scriptional events.1 In particular the level of acetylation of
histone tails, which are controlled by two counteracting enzyme
families, HATs and HDACs, plays a crucial role in the regulation
of transcriptional machinery to switch the activity of genes in-
volved in cellular proliferation on and off.2 In the last years HDAC
inhibitors have emerged as precious tools in cancer chemopreven-
tion as they showed to induce growth arrest, differentiation, and/or
apoptotic cell death.3 Although many details of their mechanism of
action have been disclosed, several basic aspects are not fully
understood also because besides histones, the key substrates,
HDACs seem to regulate the functions of a growing list of non his-
tone proteins such as the oncosuppressor p53, transcription fac-
fects in such a complex network of cellular signalling is not a sim-
ple task. Another topic that still needs to be accomplished is to
identify the precise role played in tumorogenesis by the individual
HDAC isoforms. HDACs, in fact, can be divided into four groups:
classes I, II and IV are all Zn-dependent enzymes showing relevant
sequence similarity in their catalytic site which is represented by a
long and narrow channel with the Zn ion localized at the bottom;
in contrast, class III, also named sirtuins, show different structural
features, are NAD+-dependent deacetylase and are not considered
‘classical’ HDACs.3c,4,5 Concerning their localization in cellular
framework, class I and IV are basically nuclear proteins whereas
class II HDACs shuttle between the nucleus and the cytoplasm.3c,4,5
Despite the intense research in this area unfortunately very selec-
tive HDACi have not been yet identified so far as a consequence of
the limited structural information available, therefore the discov-
ery of small molecules able to selectively perturb th e individual
HDAC isoforms would be extremely useful to help clarify several
mechanistic details.
Abbreviations: Boc, tert-butyloxycarbonyl; ClTrt-Cl, 2-chlorotrityl chloride-resin;
DIEA, N,N-diisopropylethylamine; DCM, dichloromethane; DMF, N,N-dimethylform-
amide; Fmoc, 9-fluorenylmethyloxycarbonyl; Fmoc-
liglycine; Fmoc-Gly-OH, N- -Fmoc-glycine; Fmoc-
Fmoc- -Trp(Boc)-OH, N- -tryptophan; HATU, N-[(dimethylamino)-
-Fmoc-Nin-Boc-
L
-AllylGly-OH, N-
a-Fmoc-L-ally-
2. Discussion
a
D
-Pro-OH, N- -Fmoc-proline;
a
D
a
D
1H-1,2,3-triazolo-[4,5-b]pyridin-1-yl-methylene]-N-methyl-methanaminium hexa-
fluoroposphate N-oxide; HBTU, O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate; HOBt, N-hydroxybenzotriazole; NMM, N-methylmorpholine;
TFA, trifluoroacetic acid; TFE, 2,2,2-trifluoroethanol; TIS, triisopropylsilane.
* Corresponding author. Tel.: +39 089 969743; fax: +39 089 969702.
E-mail address: brunoin@unisa.it (I. Bruno).
Recently we had the occasion to explore the chemical and the
pharmacological properties of FR235222,6 a natural product iso-
lated from the fermentation broth of a fungus, Acremonium sp.
and possessing a potent HDAC inhibition activity. This metabolite
belongs to cyclopeptide class of HDAC inhibitors which includes,
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.022

S. Terracciano et al. / Bioorg. Med. Chem. 18 (2010) 3252–3260
3253
among others, apicidins,7 FK228,8 H-C toxin,9 azumamides,10 trap-
oxins11 and chlamydocin.12 The other chemical classes of HDACi13
are carboxylates, benzamides, electrophilic ketones and hydroxa-
mic acids like vorinostat (also known as SAHA)14 which has been
recently approved for treatment of cutaneous T-cell lymphoma.
Encouraged by the interesting biological properties of FR235222,
we decided first to undertake its total synthesis15 and afterwards
we moved our attention to explore some structure–activity rela-
tionships developing a first-generation of modified analogues
whose biological behaviour was fully evaluated.16 In the course
of this biological screening we succeeded to identify a new hit,
compound 2, which showed an inhibition profile better than the
parent molecule (Fig. 1). At the same time we invested many ef-
forts in refining, at quantum mechanical level, the protein model
represented by HDLP enzyme,17 the bacterial homologue of human
HDAC, in order to obtain more predictive affinity properties of the
screened compounds. We then utilized this optimized model to
dock the structures of the synthesized compounds in the binding
pocket of the enzyme, and the collected data provided suggestions
for new structural refinements of the tetrapeptide scaffold which
should enhance affinity properties for the target.18
Because AUTODOCK 3.0.5 software22 does not allow rotations of
the cyclic portion during the calculations, a preliminary conforma-
tional search was performed on structures 4, 6 and 8 through
Monte Carlo Multiple Minimum (MCMM) method,23 to get the glo-
bal minimum conformations of the putative ligands and to use
these as starting points in the docking studies. In particular, the
cis- and trans-isomers, around the peptide bond between proline
and tryptophan, have been considered in the conformational
search. Compounds 3 and 7 present the same tetrapeptide core
of compound 2, whose experimental structure has been previously
determined.18 A comparison of 1H and 2D-NOESY spectra revealed
an expected superimposition of the resonances, thus the structures
of 3 and 7 were built from the experimental representative confor-
mations of cis- and trans-isomers found for 2.18 The cis- and trans-
isomers of 3, 4 and 6–8 were analysed to evaluate if one or both
isomers could be interacting with the biological target. In this sec-
tion we will describe the detailed docking results for both isomers
of compound 6, as they gave the best calculated binding affinity
(4.83 ꢀ 10ꢁ12 Mꢁ1 for cis and 8.65 ꢀ 10ꢁ12 Mꢁ1 for trans-isomer).
The analysis of the theoretical results revealed that the cis- and
trans-isomers of 6 showed an overlapped docked conformations
in the binding site of HDLP (Fig. 3), establishing the same interac-
tions with the macromolecular counterparts.
The side chain of tryptophan involved in the cis–trans isomer-
ism was accommodated in a deep pocket delimited by the Q192,
Y186, A197, F200, and K267 residues (Fig. 4), enveloping the indole
ring and strongly contributing to the complex stability as found for
2.18
Moreover, the NH group of the indole formed a hydrogen bond
with the oxygen in the side chain of Q192, also showed by the
docked structure of 2.18 The remainder aromatic group interacted
with an opposite hydrophobic cavity of protein surface, formed
by N20, H21, P22 and Y91 (Fig. 4) and it was hydrogen bonded with
the side chains of H21 and Y91. The carboxylate moiety interacted
with the catalytic portion of the enzyme coordinating the zinc ion
For example, the introduction of more than a bulky aromatic
amino acid residue as tryptophan to the peptide backbone has
shown to increase the ligand-enzyme complex stability, in addi-
tion, when these bulky residues were alternate in the sequence,
an optimal interaction of the binders with the counterpart hydro-
phobic protein surface18 was assured. Taking into account the com-
puter aided suggestions we synthesized six new compounds 3–8
(Fig. 2), in which we also introduced more simplified Zn binding
domains, in order to reduce the time of the synthetic procedures.
In more details compounds 3–6 present, instead of the rare Ahoda
(2S,9R-2-amino-9-hydroxy-8-oxodecanoic acid) residue joined to
the cyclopeptide scaffold, a carboxylic acid functionalized spacer,
identical to that present in the potent HDAC inhibitor Azumamide
E
10 in contrast compounds 7 and 8, possess the hydroxamic acid19
functionality (Fig. 2) as Zn chelating element.
in bidentate manner (Fig. 3) and established a hydrogen bond with
H
e
2 of H131, whereas the carbon chain formed van der Waals con-
tacts with the 11 Å channel conducting to the zinc ion. The proline
side chains pointed outside the protein, without giving any further
interactions. Compound 4 is constituted by the same residues of 6,
except for the replacement of a tryptophan by a phenylalanine.
Even though, the docking poses for cis- and trans-isomers of 4
and 6 showed the same spatial orientations in the binding site of
HDLP (Fig. S2), the presence of a phenylalanine gave less extended
van der Waals interactions with a consequent lower binding affin-
ity for the protein. Compound 8 shared the same tetrapeptide core
of 6, indeed the common structural moiety followed closely the
docked conformations of 6 cis- and trans-isomers in the binding
site (Fig. S5), differing only for the coordination to the prosthetic
group of the enzyme and for the carbon linker spatial orientation.
The docked 3D arrangement of 5 is very similar to that calculated
for 4, 6 and 8 with the glycine instead of the proline (Fig. S3). In this
3. Docking studies
The X-ray structure of HDLP17 was used as a macromolecule
model in docking calculations, applying refined enzyme calculation
parameters, as described in our previous study on cyclopeptidic
HDAC inhibitors,20 related to the electrostatic and van der Waals
terms of binding energy. We also applied these protein parameters,
previously optimized,20 in a recent analysis on other HDAC bioac-
tive tetrapeptide ligands18,20 and on cyclopeptide mimetics,21
reaching a good qualitative accordance between the results of the-
oretical KD and biological assays.
In the molecular modelling calculations the carboxylic function
of 3–6 was considered as dissociated at physiological pH and, con-
sequently, deprotonated in the simulations.
2nd generation of FR235222 analogues
Natural compound
1st generation of FR235222 analogues
L-Phe
R2
NH HN
NH HN
O
O
N
O
N
(2R, 4S)-4-MePro
O
R1
O
NH
O
NH
O
O
O
O
NH HN
O
O
O
R3
NH HN
L-Iva
HN
O
R
(2S, 9R)-Ahoda
OH
O
OH
FR235222
3-8
2
Lead compound
Figure 1. FR235222, 2 and second-generation analogues.

3254
S. Terracciano et al. / Bioorg. Med. Chem. 18 (2010) 3252–3260
2nd Generation of FR235222 Analogues
O
O
O
O
N
N
H
N
H
N
H
NH
O
O
O
NH
N
N
O
NH
HN
O
HN
HN
O
O
O
O
O
H
H
N
H
N
N
O
OH
OH
OH
NH HN
O
O
O
O
O
OH
HN
HN
HN
HN
6
4
5
3
O
O
N
N
H
NH
O
N
O
NH
HN
O
O
H
N
NHOH
O
NH HN
O
O
O
NHOH
HN
HN
8
7
O
O
OH
NH HN
NH HN
O
O
O
Azumamide E
Figure 2. Chemical structures of analogues 3–8 and of Azumamide E.
Figure 3. Superimposition of cis- and trans-isomers of 6 in the zinc-binding site. The green mesh represents the hydrophobic pocket of the protein. The Zn2+ is represented by
a CPK sphere in red brick. The cis- and trans-isomers are depicted by sticks (respectively, orange and yellow) and balls (by atom type: C, gray; polar H, sky blue; N, blue; O,
red). The figure highlights the similar interactions with the 11 Å deep hydrophobic channel of the esenoic carbon chain, and the close 3D arrangements of the proline and
tryptophans residues on the receptor surface hydrophobic pockets.
case, also, the aromatic side chains were well accommodated into
opposite hydrophobic hollows.
Compounds 3 and 7 structurally differed from 2 only for the lin-
ker and the metal binder. Thus, as expected, the calculated bioac-
tive conformations were very similar to that found for their
progenitor 2.18 In particular, for the cis-conformations of 3 and 7
the tryptophan side chain is accommodated in a deep hydrophobic
cavity constituted by Q192, Y186, A197, F200, and K267, whereas

S. Terracciano et al. / Bioorg. Med. Chem. 18 (2010) 3252–3260
3255
3–8 (Scheme 1). Solid-phase synthesis of the linear peptide se-
quences, using standard N-Fmoc/tBu SPPS strategy, was performed
on a 2-chlorotrityl chloride resin.24 The solid support was loaded,
under anhydrous conditions, with the first Fmoc-protected amino
acid in presence of an excess of diisopropylethlyamine (DIEA) in
DCM, followed capping of the unreacted trityl groups with metha-
nol, according to general procedure a (see Section 7) (Scheme 1a).
The obtained loading degree was determined by UV spectrophoto-
metric analysis, using the general procedure a’. The resin was then
submitted to coupling-deprotection cycles to build the linear pro-
tected peptides. For this purpose all the Fmoc-protected amino acids
were activated by HOBt and HBTU in presence of N-methylmorpho-
line (NMM) or DIEA, as described in the general procedure c, and the
progress of the amino acid coupling was checked through the Kaiser
test. Fmoc deprotection, before each coupling step, was achieved by
treatment of the resin-anchored peptide with a 20% solution of
piperidine in N,N-dimethylformamide (DMF), according to general
procedure b. To avoid the undesired diketopiperazine formation at
the dipeptide level, as consequence of the presence of proline
residue, during Fmoc removal in piperidine, we performed a fast
Fmoc deprotection cycle at the level of the second coupling reaction
(see Section 7). The next step consisted of Fmoc protecting group re-
moval from the N-terminal residue, then the protected peptide was
cleaved from the resin 2-ClTrt resin by using a 2:2:6 acetic acid/
2,2,2-trifluoroethanol/dicholoromethane (AcOH/TFE/DCM) solvent
mixture, according to procedure d. The linear tetrapeptides (3a–
8a), obtained in high yields (88–95%), were cyclized, in DCM/DMF
under highly dilute conditions with HATU as coupling reagent and
DIEA, following general procedure e, to give the desired cyclopep-
tides (3b–8b) (Scheme 1a).
Removal of Nin-Boc protecting group was achieved using 95%
aqueous trifluoroacetic acid (TFA), according to procedure f.
Finally the advanced precursors 3c–8c were submitted to olefin
cross-metathesis reactions either with 4-pentenoic acid 9, to give
the final compounds 3–6, or with its hydroxamate derivative 10
(see Section 7) to give 7d–8d (Scheme 1b). Metathesis reactions
were carried out using microwave irradiation, in order to speed
up the reaction rate.25 As experienced in our previous studies, we
used Grubbs’ second-generation catalyst, which was found to be
more stable to air and more reactive in our reaction conditions.26
The optimal conditions found were: 18% mol of Grubbs’ second-
generation catalyst, DCM as the solvent and microwave heating
at 300 W, 60 °C, for two 30 min periods (general procedure g).
The reaction mixture was degassed with argon after the first
30 minutes to give off any dissolved ethene. The structures of the
desired compounds 3–6 and 7d–8d were confirmed by analytical
RP-HPLC and mass spectrometry. Compounds 7d–8d were sub-
jected first to Trt groups deprotection by TFA (1% in DCM) and then
to catalytic hydrogenation, yielding expected hydroxamate 7–8,
according to general procedure h and i.
Figure 4. Three-dimensional models of the interactions formed by cis- and trans-
isomers of (respectively a, and b) with HDLP binding site. The protein is
6
represented by molecular surface and ribbon. The ligands are depicted by sticks
(yellow) and balls (coloured by atom type: C, gray; polar H, white; N, dark-blue; O,
red). The figure highlights essential interactions: the side chains of tryptophans are
located in hydrophobic pockets. The esenoic carbon chain establishes interactions
with the 11 Å hydrophobic channel, with the zinc ion contained at the bottom and
with amino acids of catalytic site.
the five-membered ring of proline was in contact with the side
chains of Y91, E92 and G140 residues. The predicted bioactive con-
formations of the trans-isomers of 3 and 7, was rotated by 180° re-
spect to the docked cis conformations (Figs. S1 and S4) inverting
the positions of tryptophan and proline into the surface cavities
of the macromolecule.
Compared to compound 3 and 7, structure 6 interacted with both
aromatic side chains of tryptophan within the protein, increasing
the affinity for the biological target. The same considerations could
be made for 4, 5 and 8. Thus, the modification of the primary se-
quence of these new tetrapeptides seems to favour the interactions
with the surface hydrophobic cavities of the enzyme. Moreover, the
presence of bulky groups was important for the affinity to the mac-
romolecule, because they increase the van der Waals contacts with
the macromolecular counterparts, and the indole ring in all cases
gave hydrogen bonds with the protein amino acids. Based on the ob-
tained structure–activity relationships, we moved to the synthesis
and biological evaluations of compounds 3–8.
The crude products were purified by semi-preparative RP-HPLC
on a C18 column and the structures of 3–8 were confirmed by MS
and NMR analysis.
5. Biological activity
To evaluate the potency and the selectivity of these compounds
as HDAC inhibitors their activity on distinct HDAC isotypes was
compared with that of the parent compound 2 (Table 1). The test
system included class IIa-specific assays relying on the use of
HDAC4, 5, and 7 catalytic domains purified to homogeneity from
bacterial cells,27,28 and on a class IIa-specific in vitro substrate.27,29
This assay format makes it possible to measure the intrinsic cata-
lytic activity of class IIa HDACs in the absence of contaminant
endogenous class I members.
4. Chemistry
A convenient combination of both solid and solution phase syn-
thetic approaches were used for the synthesis of the six analogues

3256
S. Terracciano et al. / Bioorg. Med. Chem. 18 (2010) 3252–3260
1) SPPS of the linear tetrapeptides
Cycles of
Loading with
C-terminal amino acid
Nα deprotection
Cleavage
and coupling
a
b,c,b
d
Fmoc-AA1-OH
Fmoc-AA1
HOOC-AA1-AA2-AA3-AA4-NH2
AA1-AA2-AA3-AA4
[3a-8a]
Cl
PS
a) DIPEA (4equiv), DCM, 2h; b) 20% piperidine/DMF (5/10 min);
=
Cl
c) HOBt-HBTU (3equiv)-NMM/DIPEA (4equiv), DMF, 2h; d) AcOH/TFE/DCM (2:2:6).
= side chain protecting group
ClTrt-Cl-resin
2) Cyclizazion under highly dilute conditions
R2
O
R1
O
NH HN
O
e, f
HOOC-AA1-AA2-AA3-AA4-NH2
[3c-8c]
R3
NH HN
O
e) HATU (2equiv), DIPEA (2.5equiv), DCM/DMF (7.8 x 10-5M), 2h.
f) TFA/H20/TIS (95:4:1)
Scheme 1a. General synthetic approach for analogues 3c–8c.
microwave–assisted Cross Methatesis (CM) reaction
R2
O
R2
O
O
R1
O
NH HN
NH HN
O
R1
O
R3
O
OH
NH HN
g
R3
OH
O
O
NH HN
9
O
+
[3-6]
O
O
NHOTrt
[3c-8c]
R2
R2
O
10
O
R1
O
NH HN
NH HN
NH HN
O
R1
O
R3
O
O
R3
NH HN
h, i
g)18% mol Grubbs catalyst 2nd generation, DCM,
300W, 30 min, 2 cycles; h) TFA 1% in DCM; i) H2, Pd/C
NHOTrt
NHOH
O
O
[7d-8d]
Scheme 1b. Synthetic strategy for compounds 3–8.
[7-8]
Our reference compound 2 displayed submicromolar inhibitory
activity on class I HDACs 1, 2, and 3. Its potency was similar to that
measured on deacetylation reactions catalyzed by HDACs present
in the nuclear extract (NE) from HeLa cells.
In contrast with molecular docking predictions, compounds 4
and 5 showed no measurable inhibitory activity to a concentration
class I HDACs. Class IIb HDACs 6 and 10 have been found to be resis-
tant to cyclic peptide inhibitors such as trapoxin and CHAPS,30,31 and
to be selectively inhibited by non peptidic compounds.32,33 Class IIa
HDACs 4, 5, 7, 9 have been less extensively characterized in terms of
inhibition profile. However recently described class IIa-specific
inhibitors34,35 do not include peptidic compounds.
up to 50 lM on reactions catalyzed by the HeLa nuclear HDACs,
6. Conclusion
while compound 6 showed only a weak activity at the same condi-
tions. In contrast, 3 displayed a moderate, broad-spectrum HDAC
inhibitor activity.
In conclusion a small array of carboxy and hydroxamate deriv-
atives of the known potent HDAC inhibitors FR235222 has been
developed. In compounds 3–8 we modified both the composition
of tetrapeptidic core and the functional linker, in order to expand
the chemical diversity and to simplify the synthetic procedure.
Unfortunately, even though molecular modelling studies showed
that the peptidic scaffold efficiently fit with the protein counter-
Compounds 7 and 8 showed a more restricted spectrum of activ-
ity than 3, displaying a moderate propensity towards HDACs 4, 5,
and 6, and a weak to undetectable activity on class I HDAC1, 2, 3.
Compound 7 was also selective over HDAC8, therefore showing class
II-specificity. Most of the well- known HDAC inhibitors either do not
discriminate between class I and class II members, or are specific for

S. Terracciano et al. / Bioorg. Med. Chem. 18 (2010) 3252–3260
3257
Table 1
Biological activities on individual HDAC isoforms
Compound
HDAC subtype
HeLa NE
1
2
3
4
5
6
7
8
e
IC50
(lM)
TSA
2 (Lead)
0.02 (0.009)
0.133 (0.0057) 0.0675 (0.0021) 0.179 (0.030)
0.0247 (0.0004)
22 (1)
50% inh.a (4.7)
NDb
9.4 (0.65)
33.6 (5.5)
NDb
NAc
NAc
23 (0.88)
3
4
5
6
7
8
23.6 (2.1)
10.2 (2.65)
11.4 (4.53)
37% inh.d (2.83) NAc
NAc
NAc
30% inh.a (6.2)
NDb
42% inh.d (3.81) 30% inh.d (10.61) 14.7% inh.d (1.77) 9.6 (0.14)
73 (1) 10.65 (2.33)
34% inh.a (8.5) 23.5% inh.a (3.5)
31 (4.24) 2 (0.17)
NAc
NAc
NDb
31 (4.74) 24.7 (5.77) 48.5% inh.a (10.61) 27.6 (3.25)
a
b
c
% inhibition at 100
Not determined.
lM.
Not active up to 50
l
M.
M.
Values are means of 2–3 experiments. Standard deviations are in parenthesis.
d
e
% inhibition at 50
l
part, the biological data are not very satisfactory, as the tested
molecules showed only a moderate HDAC inhibition activity.
Probably, the modification of the functional domain could be
responsible of the activity decrease. However, we observed that
variations in the amino acid composition of the peptidic core seem
to affect the isoform profile, in fact compounds 7 and 8 displayed a
major specificity towards class II HDAC enzymes. Even though the
role played by each HDAC isotype in carcinogenesis still remains
unclear, it is worthy to note that this kind of specificity has not pre-
viously reported in cyclopeptidic compounds.
All microwave irradiation experiments were carried out in a
dedicated CEM-Discover Focused Microwave Synthesis apparatus,
operating with continuous irradiation power from 0 to 300 W uti-
lizing the standard absorbance level of 300 W maximum power.
The Discover™ system offers controllable ramp time, hold time
(reaction time) and uniform stirring. The temperature was moni-
tored using the CEM-Discover built-in-vertically-focused IR tem-
perature sensor.
All NMR spectra (1H, HMBC, HSQC, TOCSY, COSY, ROESY) were
recorded on a Bruker Avance DRX600, at T = 298 K. Compounds
3–8 were dissolved in 0.5 mL DMSO-d6 (99.95%, Carlo Erba, 99.95
atom% D) (1H NMR d = 2.50 ppm, 13C NMR d = 39.5 ppm). NMR data
were processed on a Silicon Graphics Indigo 2 workstation using
UXNMR software. Chemical shifts are expressed as d (ppm) (see
Supplementary data).
7. Experimental section
7.1. General information
All chemicals were purchased from commercial sources and
were used as received without further purification. Unless speci-
fied, solvents were reagent grade; they were purchased from Al-
drich, Fluka, Carlo Erba. CH2Cl2 and DMF used for solid-phase
reactions were synthesis grade (dried over activated molecular
sieves (4 Å)). H2O and CH3CN were HPLC grade. 2-Chlorotrityl chlo-
ride resin (100–200 mesh), 1% DVB, (ClTrt-Cl, loading level:
8. Computational details
All ligands were built and their geometries optimized through
MacroModel 8.5 software36 package and using the OPLS-AA force
field.37 For 4, 6 and 8 both cis and trans-isomers, around the pep-
tide bond formed by amine and carboxylic groups, respectively,
of proline and tryptophan residues, were considered. Monte Carlo
Multiple Minimum (MCMM) method of the MacroModel package36
was used in order to allow a full exploration of the conformational
space of 4, 6 and 8 cis and trans-isomers, with a 50 kJ molꢁ1 upper
energy limit and 10,000 steps. The so obtained geometries were
optimized using the Polak–Ribier conjugate gradient algorithm
(PRCG, 9 ꢀ 107 steps, maximum derivative less than 0.001 kcal/
mol). A GB/SA (generalized Born/surface area)38 solvent treatment
was used, mimicking the presence of H2O, in the geometry optimi-
zation and in the conformational search steps. The protein
(1C3R.pdb)17a for the docking calculations was previously pre-
pared20 using MacroModel software: all hydrogen were added,
bond order and missing atoms were checked by visual inspection,
and the charges of side chains were assigned considering their pKa.
The structures of 3 and 7 presenting an overlapping 1H proton and
2D-NOESY spectra with compound 2 reported in our previous
studies of FR235222 analogues.18 Thus, structures of 3 and 7 were
built from the previously determined experimental structures of
cis- and trans-isomers of 2, leaving unaltered the found 3D arrange-
ments of the common portions.
1.4 mmolgꢁ1), Fmoc-
OH, Fmoc-Gly-OH, Fmoc-
D
-Pro-OH, Fmoc-L-Phe-OH, Fmoc-L-Trp-(Boc)-
L
-AllylGly-OH, HOBt, and HBTU were pur-
chased from Novabiochem and Neosystem. HATU, was purchased
from Fluka.
Solid-phase peptide syntheses, using the Fmoc-tBu strategy,
were carried out on a polypropylene ISOLUTE SPE column on a
VAC MASTERsystem, a manual parallel synthesis device purchased
from Stepbio. All solid phase reactions were run under a nitrogen
atmosphere with dry solvents. All cross-metathesis (CM) reactions
were carried out under an argon atmosphere with dry, degassed
solvents under anhydrous conditions. Grubbs catalyst second-gen-
eration was purchased from Aldrich.
For estimation of Fmoc amino acids on the resin, absorbance at
k = 301 nm was recorded on a Shimadzu UV 2101 PC.
Electrospray mass spectrometry (ES-MS) was performed on a
LCQ DECA ThermoQuest (San Josè, California, USA) mass spectrom-
eter. High resolution mass spectra (HRMS) were acquired on a Q-
Tof ULTIMA (Waters, Manchester UK) calibrated with [Glu]-Fibri-
nopeptide B fragments using standard experimental conditions.
Analytical and semipreparative reversed-phase HPLC was
AUTODOCK 3.0.522 was used for all docking calculations. HDLP
(histone deacetylase-like protein) is a metalloprotein, so a non-
bonded model for metallic center according to the nonbonded Zn
parameters of Stote and Karplus39 (zinc radius = 1.10 Å, well
depth = 0.25 kcal/mol) was used. In order to have an accurate
performed on a Jupiter C18 column (250 ꢀ 4.60 mm, 5
l
, 300 Å,
flow rate = 1 mLminꢁ1; 250 ꢀ 10.00 mm, 10
l, 300 Å, flow rate =
5 mL minꢁ1, respectively). The binary solvent system (A/B) was
as follows: 0.1% TFA in water (A) and 0.1% TFA in CH3CN (B). The
absorbance was detected at 220–240 nm.

3258
S. Terracciano et al. / Bioorg. Med. Chem. 18 (2010) 3252–3260
weight of the electrostatics, the previously derived partial charge
of Zn = 1.175 and of the amino acids involved in the catalytic cen-
ter (A169, H170, D168, D258), were used.20 In particular the above
charges were calculated at DFT theory level using the chelpg
method.40
tion of hexane (15 times volume) to remove acetic acid as an azeo-
trope, the filtrate was concentrated and lyophilized (83–95%
overall yield).
(e) Cyclization: The cyclization step was performed in solution
at a concentration of 7.8 ꢀ 10ꢁ5 M with HATU (2.0 equiv) and DIEA
(2.5 equiv) in DCM/DMF.44 The solution was stirred at 4 °C for 1 h
and then allowed to warm to room temperature for 1 h. The cycli-
zation reaction was monitored via HPLC and ES-MS spectra. After
2 h the solvent was removed under reduced pressure.
Compound 3b: A portion of the linear peptide (71.8 mg,
For all the docking calculations a grid box size of 66 ꢀ 64 ꢀ 64
points spaced by 0.375 Å, centred between Zn2+ and H170 and cov-
ering the catalytic centre surface of HDLP was used. For all the
docked structures, all bonds were treated as active torsional bonds
except the amide bonds. In order to achieve a representative con-
formational space during the docking calculations, eleven calcula-
tions consisting of 256 runs were performed, obtaining 2816
structures (256 ꢀ 6). The Lamarckian genetic algorithm was used
for dockings. An initial population of 600 randomly placed individ-
uals, a maximum number of 5 ꢀ 106 energy evaluations, and a
maximum number of 6 ꢀ 106 generations were taken into account.
A mutation rate of 0.02 and a crossover rate of 0.8 were used, and
the local search frequency was set up at 0.26.
0.11 mmol) was dissolved in DCM/DMF (1410 mL/800
HATU (83.6 mg, 0.22 mmol, 2.0 equiv) and DIEA (47.0
l
L) with
L,
l
0.27 mmol, 2.5 equiv).
RP HPLC: Jupiter C-18 column (250 ꢀ 4.60 mm, 5
l, 300 Å),
from 5% B to 100% B over 30 min, flow rate of 1 mL/min,
k = 220 nm. tR 22.7 min; ES-MS: m/z 628.4 [M+H]+, 650.4 [M+Na]+.
Compound 4b: A portion of the linear peptide (61.4 mg,
0.09 mmol) was dissolved in DCM/DMF (1153 mL/730
lL) with
Results differing by less than 2 Å in positional root-mean-
square deviation (rmsd) were clustered together and represented
by the result with the most favourable free energy of binding.
All the 3D models were depicted using the Phyton software:41
molecular surfaces are rendered using Maximal Speed Molecular
Surface (MSMS)42 and, in Figure 3, the hydrophobic pocket of the
protein is represented as an isocontour.43
HATU (68.4 mg, 0.18 mmol, 2.0 equiv) and DIEA (38.3
0.22 mmol, 2.5 equiv).
l
L,
RP HPLC: Jupiter C-18 column (250 ꢀ 4.60 mm, 5
l, 300 Å),
from 5% B to 100% B over 30 min, flow rate of 1 mL/min,
k = 240 nm. tR 26.6 min; ES-MS: m/z 628.3 [M+H]+.
Compound 5b: A portion of the linear peptide (92.5 mg,
0.15 mmol) was dissolved in DCM/DMF (1923 mL/1.0 mL) with
HATU (114.1 mg, 0.30 mmol, 2.0 equiv) and DIEA (65.3
0.37 mmol, 2.5 equiv).
lL,
8.1. General procedures for the synthesis of compound s 3–8
RP HPLC: Jupiter C-18 column (250 ꢀ 4.60 mm, 5
l, 300 Å),
(a) Loading of the resin: The ClTrt-Cl resin was placed in a 25 mL
polypropylene ISOSOLUTE syringe on a VAC MASTER system, swol-
len in 3 mL of DMF for 1 h, and then washed with 2 ꢀ 3 mL of DCM.
A solution of Fmoc-AA-OH (1.1 equiv) and DIEA (4 equiv) in
2.5 mL of dry DCM was added and the mixture was agitated for
2 h with a N2 stream. The mixture was then removed, and the resin
was washed with 3ꢀ DCM/MeOH/DIEA (17:2:1), and sequentially
with the following washing/treatments: DCM 3 ꢀ 3 mL, DMF
2 ꢀ 3 mL, DCM 2 ꢀ 3 mL (1.5 min each).
from 5% B to 100% B over 30 min, flow rate of 1 mL/min,
k = 220 nm. tR 22.6 min; ES-MS: m/z 588.0 [M+H]+, 610.2 [M+Na]+.
Compound 6b:
0.09 mmol) was dissolved in DCM/DMF (1152 mL/700
HATU (68.4 mg, 0.18 mmol, 2.0 equiv) and DIEA (38.3
A
portion of the linear peptide (70.4 mg,
L) with
L,
l
l
0.22 mmol, 2.5 equiv).
RP HPLC: Jupiter C-18 column (250 ꢀ 4.60 mm, 5
l, 300 Å),
from 5% B to 100% B over 30 min, flow rate of 1 mL/min,
k = 220 nm. tR 27.2 min; ES-MS: m/z 767.2 [M+H]+, 789.0
[M+Na]+.
(a0) Estimation of the level of first residue attachment: Resin load-
ing was determined by UV quantification of the Fmoc-piperidine
adduct.
Compound 7b: A portion of the linear peptide (62.8 mg,
0.09 mmol) was dissolved in DCM/DMF (1153 mL/700
HATU (68.4 mg, 0.18 mmol, 2.0 equiv) and DIEA (38.3
0.22 mmol, 2.5 equiv).
l
L) with
L,
The assay was performed ona duplicate samples:0.4 mL of piper-
idine and 0.4 mL of DCM were added to two dried samples Fmoc
amino acid-resin (ꢂ3.0 mg) in two volumetric flasks of 25 mL. The
reaction was allowed to proceed for 30 min at rt and than 1.6 mL
of MeOH were added and the solutions were diluted to 25 mL
volume with DCM. A reference solution was prepared in a 25 mL vol-
umetricflask using 0.4 mL of piperidine, 1.6 mL of MeOH and DCM to
volume. The solutions were shaken and the absorbance of the
samples versus the reference solution was measured at 301 nm.
The substitution level (expressed in mmol of amino acid/g of resin)
was calculated from the equation: mmol gꢁ1 = (A301/7800) ꢀ
(25 mL gꢁ1 of resin). The obtained loading degree was: 0.28–
l
RP HPLC: Jupiter C-18 column (250 ꢀ 4.60 mm, 5
l, 300 Å),
from 5% B to 100% B over 30 min, flow rate of 1 mL/min,
k = 220 nm. tR 22.6 min; ES-MS: m/z 628.4 [M+H]+, 650.4 [M+Na]+.
Compound 8b:
0.08 mmol) was dissolved in DCM/DMF (1025 mL/500
HATU (60.4 mg, 0.16 mmol, 2.0 equiv) and DIEA (34.8
A
portion of the linear peptide (63.0 mg,
L) with
L,
l
l
0.20 mmol, 2.5 equiv).
RP HPLC: Jupiter C-18 column (250 ꢀ 4.60 mm, 5
l, 300 Å),
from 5% B to 100% B over 30 min, flow rate of 1 mL/min,
k = 220 nm. tR 27.1 min; ES-MS: m/z 767.2 [M+H]+, 789.4 [M+Na]+.
(f) Boc deprotection: Side-chain deprotection was carried out by
treatment with TFA/H2O/TIS (95:4:1, 10 ml ꢀ 1 mg) for 20 min
while stirring.
0.40 mmol gꢁ1
.
(b) Fmoc deprotection: 20% piperidine in DMF (3 mL,
1 ꢀ 1.5 min, 1 ꢀ 5 min or 1 ꢀ 10 min); washings in DMF
2 ꢀ 3 mL, DCM 2 ꢀ 3 mL, DMF 2 ꢀ 3 mL, (1.5 min each).
The crude product was purified by semipreparative RP HPLC on
(c) Peptide coupling conditions: The coupling reaction was pro-
moted by a HOBt/HBTU in DMF coupling protocol: Fmoc-amino
acid, (3–4 equiv), HOBt (3–4 equiv), HBTU (3–4 equiv) and NMM
(4–5 equiv) or DIEA (4–5 equiv) were agitated under N2 in 2.5 mL
of DMF for 2 h. After each coupling, washings were carried out with
DMF (3 mL, 3 ꢀ 1.5 min), and DCM (3 mL, 3 ꢀ 1.5 min).
a Jupiter C-18 column (250 ꢀ 10.00 mm, 10
l, 300 Å).
Compound 3c: from 5% B to 100% B over 40 min, flow rate of
5 mL/min, k = 240 nm. tR 20.0 min; ES-MS: m/z 528.3 [M+H]+,
550.4 [M+Na]+.
Compound 4c: from 5% B to 100% B over 30 min, flow rate of
5 mL/min, k = 220 nm. tR 21.1 min; ES-MS: m/z 528.1 [M+H]+.
Compound 5c: from 5% B to 100% B over 50 min, flow rate of
5 mL/min, k = 220 nm. tR 21.4 min; ES-MS: m/z 488.2 [M+H]+.
Compound 6c: from 5% B to 100% B over 50 min, flow rate of
5 mL/min, k = 240 nm. tR 21.8 min; ES-MS: m/z 567.3 [M+H]+.
(d) Cleavage: The dried peptide resin was treated for 2 h, under
stirring, with the following cleavage mixture: AcOH/TFE/DCM
(2:2:6; 10
l
L ꢀ 1 mg of resin). Then the resin was filtered off and
washed with neat cleavage mixture (3 mL, 3 ꢀ 1.5 min). After addi-

S. Terracciano et al. / Bioorg. Med. Chem. 18 (2010) 3252–3260
3259
Table 2
Calculated and observed molecular weights (by MS and HPLC analysis) and HPLC tR data for compounds 3–8
Compound
Formula
ES-MS [M+H]+
HRMS
HPLC
Calcd
Found
Calcd for [M+H]+
Found
tR (min)
3
4
5
6
7
8
C33H37N5O6
C33H37N5O6
C30H33N5O6
C35H38N6O6
C33H40N6O6
C35H41N7O6
599.6
599.6
559.6
638.7
616.7
655.7
600.2
600.1
560.2
639.3
617.2
656.4
600.2822
600.2822
560.2509
639.2931
617.3088
656.3197
600.2902
600.2902
560.2404
639.2824
617.2998
656.2999
33.57a
35.31b
33.50c
32.89d
26.20e
30.90f
The binary solvent system (A/B) was as follows: 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B).
a
5–65% of solvent system (A/B) for 70 min.
5–60% of solvent system (A/B) for 70 min.
5–55% of solvent system (A/B) for 60 min.
5–70% of solvent system (A/B) for 75 min.
5–100% of solvent system (A/B) for 30 min.
5–100% of solvent system (A/B) for 95 min.
b
c
d
e
f
Compound 7c: from 5% B to 100% B over 40 min, flow rate of
5 mL/min, k = 240 nm. tR 19.8 min; ES-MS: m/z 528.3 [M+H]+,
550.4 [M+Na]+.
Compound 8c: from 5% B to 100% B over 50 min, flow rate of
5 mL/min, k = 240 nm. tR 21.8 min; ES-MS: m/z 567.3 [M+H]+,
589.0 [M+Na]+.
mains were expressed and purified from Escherichia coli as de-
scribed.27,28 Full-length HDAC8 was purified from E. coli soluble
extracts as described.46 IC50 values were determined using the
HDAC fluorescent activity assay (BIOMOL Research Laboratories,
Plymouth Meeting, PA). Assays were performed in 96-well plates
according to the manufacturer’s instructions by using either the
commercial Fluor de Lys substrates KI-104 (HDAC1, 2, 3, 6) and
KI-178 (HDAC8), or the trifluoro acetyl lysine substrate (HDAC4,
5, 7).27 HDACi activities were evaluated by pre-incubating com-
pounds and recombinant enzymes in assay buffer at rt for 15 min,
before substrate addition. Mean values of at least two independent
assays are reported. Standard deviation values were <20%.
(g) Optimized microwave cross-metathesis reactions: The olefins
(1.1–1.2 equiv) were stirred in dry DCM (0.19 M) in a microwave
vial. Grubb’s second-generation catalyst (18 mol %) was added,
and the mixture stirred and subjected to microwave radiation,
300 W, 80 °C, for 30 min. The reaction mixture was then degassed
with argon for 2 min and mixture stirred and subjected to MW
radiation for a further 30 min at the same conditions. After the irra-
diation period, the reaction vessel was cooled rapidly (60–120 s) to
ambient temperature by air jet cooling. The mixture was dried and
then purified by HPLC chromatography.
(h) Trt deproptection: Removal of trytil group was performed in
solution with TFA/TIS/DCM (1:1:98) for 30 min under stirring.
(i) Catalytic hydrogenation: To the cyclopeptides dissolved in
MeOH, 10% Pd–C was added and the mixture was stirred under
hydrogen atmosphere overnight. The reaction was monitored by
TLC, HPLC and ESMS. After completion of the reaction, the catalyst
was filtered off and MeOH was evaporated.
Acknowledgements
Financial support by the University of Salerno, MIUR (Rome)
and PRIN-06 are gratefully acknowledged. We also thank to C. Pao-
lini e M.C. Nardi for technical support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.03.022.
The crude products were purified by semipreparative reverse
phase HPLC (on a Jupiter C-18 column: 250 ꢀ 10.00 mm, 10
l,
References and notes
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8.2. Synthesis of compound 10
To a stirred solution of 4-pentenoic acid 9 (150 mg, 1.50 mmol,
153.3
hydrochloride (413 mg, 1.50 mmol), HOBt (229,65 mg, 1.50 mmol),
DIC (232,26 L, 1.50 mmol) and DIEA (1.50 mmol, 261.18 L), at
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18 column: 250 ꢀ 10.00 mm, 10
l, 300 Å, flow rate = 5 mL/min)
using the following gradient: 15–100% of solvent system (A/B)
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cell extracts as previously described.27 HDAC4, 5, 7 catalytic do-

3260
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