Synthetic and pharmacological studies on new simplified analogues of the potent actin-targeting Jaspamide

Article abstract of DOI:10.1016/j.bmc.2008.05.019

In the recent years, we focused our attention on the cyclodepsipeptide Jaspamide 1, an interesting marine metabolite, possessing a potent inhibitory activity against breast and prostate cancer, as a consequence of its ability to disrupt actin cytoskeleton dynamics. Although its biological profile has been well determined, many mechanistic details are still missing in terms of molecular target identification. For this reason, we decided to synthetically modify the natural metabolite, obtaining small arrays of unnatural variants useful to illuminate the structural requirements essential for the activity. Here, we report the synthesis of seven new Jaspamide analogues 2-8, containing, as the parent compound, a β-amino acid in the cyclopeptide backbone. Their biological profile is also described.

Full text of DOI:10.1016/j.bmc.2008.05.019

Bioorganic & Medicinal Chemistry 16 (2008) 6580–6588
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthetic and pharmacological studies on new simplified analogues
of the potent actin-targeting Jaspamide
a
a
b
Stefania Terracciano a, Ines Bruno a, , Elisabetta D’Amico , Giuseppe Bifulco , Angela Zampella ,
*
Valentina Sepe b, Charles D. Smith c, Raffaele Riccio a
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy
b Dipartimento di Chimica delle Sostanze Naturali, Università degli Studi di Napoli ‘‘Federico II”, Via D. Montesano 49, 80131 Napoli, Italy
c Hollings Cancer Center, Medical University of South Carolina, 86 Jonathan Lucas Street, Charleston, SC 29425, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In the recent years, we focused our attention on the cyclodepsipeptide Jaspamide 1, an interesting marine
metabolite, possessing a potent inhibitory activity against breast and prostate cancer, as a consequence of
its ability to disrupt actin cytoskeleton dynamics. Although its biological profile has been well deter-
mined, many mechanistic details are still missing in terms of molecular target identification. For this rea-
son, we decided to synthetically modify the natural metabolite, obtaining small arrays of unnatural
variants useful to illuminate the structural requirements essential for the activity. Here, we report the
synthesis of seven new Jaspamide analogues 2–8, containing, as the parent compound, a b-amino acid
in the cyclopeptide backbone. Their biological profile is also described.
Received 10 March 2008
Revised 6 May 2008
Accepted 7 May 2008
Available online 10 May 2008
Dedicated to the memory of Professor Luigi
Gomez-Paloma
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Cyclodepsipeptide
Cytoskeleton
Amino acids
Ring-closing metathesis
1. Introduction
the attractive class of cytoskeleton targeting agents, which, as it
has been well established, represent potential anticancer therapeu-
As a part of our investigations on bioactive natural metabolites,
which have shown to be precious tools in medicinal chemistry, re-
cently we focused our interest on the marine metabolite Jaspamide
1.1 From a structural point of view, Jaspamide is a representative
tics.2 In particular, Jaspamide showed to selectively interfere with
actin microfilaments and to produce potent antiproliferative ef-
fects.3 Although many efforts have been invested in this research
area, the mechanism of action of depsipeptide-based anti-actin
compounds, including chondramides,4 and doliculide,5 is quite un-
clear in terms of molecular aspects of their interaction with the
biological target, and, consequently, it is quite hard to explain
the similar effects of all these metabolites on actin, in absence of
a receptor mapping and further structural details. Previous investi-
gations made on Jaspamide indicated that, the two structural ele-
ments composing the molecule, the tripeptidic portion and the
polyketide fragment, play a cooperative role in eliciting the bioac-
tive shape of the compound.6 In fact, it seems that the tripeptide
moiety, in the macrocycle, is forced to adopt a preferential b-turn
conformation, indicating thus that the polyketide fragment is far
from accomplishing the mere function of spacer element, but it
likely plays a decisive role in generating geometric constrains
and, of consequence, inducing a selective conformation.
member of cyclodepsipeptide family that possesses an
L-Ala-D-N-
Me-2-BrTrp- -b-Tyr tripeptide fragment, which is linked to a x-
L
hydroxyacid of polyketide origin, containing four methyl groups,
located at 1–3 distance, as well as a trisubstituted double bond
(Fig. 1). Based on its biological behaviour, it can be included in
Abbreviations: Boc, tert-butyloxycarbonyl; ClTrt-Cl, 2-chlorotrityl chloride-
resin; DIEA, N,N-diisopropylethylamine; DCM, dichloromethane; DMF, N,N-dimeth-
ylformamide; Fmoc, 9-fluorenylmethyloxycarbonyl; Fmoc-8-Aoc-OH, N-a-Fmoc-8-
aminooctanoic acid; Fmoc-Ala-OH, N-a-Fmoc-
aminobutanoic acid; Fmoc-
-Trp(Boc)-OH, N-a-Fmoc-Nin-Boc-
b-Ala-OH, N-a-Fmoc-b- -alanine; Fmoc-b-Phe-OH, N-a-Fmoc-
Fmoc-b-Tyr-4-OMe-OH, Fmoc- -b-tyrosine; HATU, N-[(dimethylamino)-1H-1,2,3-
triazolo-[4,5-b]pyridin-1-yl-methylene]-N-methyl-methanaminium hexafluoro-
L
-alanine; Fmoc-Gaba, N-a-Fmoc-4-
-tryptophan; Fmoc-
-b-phenylalanine;
D
D
L
L
L
posphate N-oxide; HBTU, O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate; HOBt, N-hydroxybenzotriazole; NMM, N-methylmorpholine; TFA,
trifluoroacetic acid; TFE, 2,2,2-trifluoroethanol; 3-MePea, 3-methyl-4-pentenoic
acid; 4-Pea, 4-pentenoic acid; 4-Peo, 4-penten-1-ol.
* Corresponding author. Tel.: +39 089 969743; fax: +39 089 969702.
E-mail address: brunoin@unisa.it (I. Bruno).
In our previous work, we synthesized a small collection of sim-
plified analogues, all containing the common dipeptide fragment
Ala-
a-Tyr,
D
-Trp, and, as third amino acid, replacing the unusual b-Tyr,
L
-Val and Phg.7 As concerning the polyketide-mimetic
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.019

S. Terracciano et al. / Bioorg. Med. Chem. 16 (2008) 6580–6588
6581
OH
O
12
9
HN
O
7
O
HN
Br
N 16
O
5
Me
19
3
NH
1
O
1
jaspamide
OCH3
O
O
O
HN
NH
NH
HN
O
NH
HN
NH
NH
O
O
HN
HN
O
O
HN
HN
HN
O
O
HN
O
NH
NH
O
NH
O
O
3
2
4
O
O
O
HN
NH
NH
HN
O
O
O
HN
O
O
HN
HN
HN
NH
NH
O
O
6
5
O
O
HN
O
HN
O
O
O
HN
HN
HN
O
HN
O
NH
NH
O
O
7
8
Figure 1. Structures of Jaspamide (1) and its new synthetic variants (2-8).
fragment, in some cases variable-length open chain linkers were
used to connect the N and C termini of the tripeptide portion; in
other cases an appropriately elongated meta-disubstituted phenyl
ring was used to generate 16, 18 and 19 membered macrocycles.
Unfortunately, even if all these synthetic compounds showed a
moderate cytotoxicity, they were found to completely loose the
target selectivity, resulting not active on actin cytoskeleton at all.
Such results indicated that the extreme simplification of the parent
compound was unsuccessful, by drastically affecting the biological
profile. Hence, starting from the assumption that the peptide core
can be considered as the pharmacophore, as first stage we worked
to design a new generation of Jaspamide analogues with the inten-
tion of reproducing more accurately this structural part of the mol-
ecule, considering, of course, some crucial simplifications which
can turn out useful on the synthetic plane. In more details we pro-
jected a small array of analogues, all containing as part of the pep-
tidic core a b-amino acid residue, which, in our opinion, could elicit
a b-turn conformational motif, that was supposed to be the bioac-
tive tridimensional arrangement7,8 As concerning the polyketide
moiety, we preferred to handle, in turn, the same available linear
linkers used for the previous compounds, where the macrocycle
closure has been realized through an amide bond formation (2–
5); furthermore, in analogues 6–8 we experienced the versatile
RCM reaction to generate the cyclopeptide backbone (Fig. 1).
2. Results and discussion
2.1. Design and preliminary conformational studies
In order to synthesize molecules containing a peptidic core con-
formationally similar to the Jaspamide, we projected and prelimi-
narily studied from a conformational point of view analogues 2

6582
S. Terracciano et al. / Bioorg. Med. Chem. 16 (2008) 6580–6588
and 3. In particular, an extensive conformational analysis was per-
formed in order to predict the geometrical features of 2 and 3 (see
Section 3.3). As it is clear from Figure 2, which reports a superpo-
sition of 2 and 3, respectively, with the X-ray structure of Jaspa-
mide, there is a very good alignment of the peptidic core of both
compounds with the natural product. Such result prompted us to
synthesize these two analogues and the others of the same family
in view of the expected biological activity which we hypothesized
to be related to the tripeptidic central core.
to Davies’ procedure.9 Thus, addition of lithium amide to commer-
cially available trans-methylcinnamate 9 afforded N-protected
methyl ester 10 in reasonable yields and excellent stereoselectivi-
ty. N-deprotection of both N-benzyl protection groups was readily
achieved by Pd(OH)2 promoted hydrogenolysis to give the corre-
sponding b-amino ester without compromising the stereoselectiv-
ity. Further ester hydrolysis (11) followed by Fmoc protection
furnished the corresponding N-protected b-amino acid 12, used
for the solid phase syntheses of compounds 2–3 and 7–8. For prep-
aration of N-Fmoc-b-Tyr-4OMe-OH 17, a slight different procedure
was applied (Scheme 1B). Benzylation of 4-methoxy cinnamic acid
13 furnished the benzyl ester 14 in quantitative yield. Conjugate
addition followed by global deprotection and N-Fmoc protection
afforded compound 17 that was used for preparation of cyclopep-
tide 4.
2.2. Synthesis of b-aminoacid synthons
The non-racemic chiral N-protected b-aminoacid 12 was pre-
pared by Michael conjugate addition of lithium (S)-N-benzyl-N-
(methylbenzyl)amide to conjugated ester 9 (Scheme 1A) according
Figure 2. Superposition of 2 (a) and 3 (b), respectively, with the X-ray structure of Jaspamide. In orange are represented the synthetic compounds.
A
NHFmoc
COOH
Ph
N
Ph
COOMe
NH2
COOMe
COOH
Ph
Ph
Ph
10
Ph
11
a
d
b,c
9
12
c) HCl 2M
d) FmocCl, Na2CO3, acetone:water 1:1
a) (
S
)-N-benzyl-N-(methylbenzyl)amide,
n
BuLi, THF
b) H2/Pd(OH)2, MeOH/AcOH/H20
B
Ph
N
Ph
COOBn
NH2
NHFmoc
COOH
COOR
COOH
b
c
d
MeO
MeO
MeO
MeO
16
15
17
13 R=H
a
14 R=Bn
c) H2/Pd(OH)2, MeOH/AcOH/H20
d) FmocCl, Na2CO3, acetone:water 1:1
a) BnBr,TEA, acetonitrile
b) ( )-N-benzyl-N-(methylbenzyl)amide, nBuLi, THF
S
Scheme 1. Preparation of b-aminoacids 12 and 17.

S. Terracciano et al. / Bioorg. Med. Chem. 16 (2008) 6580–6588
6583
2.3. Synthesis of cyclodepsipeptides
checked through the Kaiser and TNBS tests. After completion of
peptide assembly, the Fmoc protecting group was removed from
the N-terminal residue and the protected peptide was cleaved from
the resin using a 2:2:6 acetic acid/2,2,2-trifluoroethanol/dicholo-
romethane (AcOH/TFE/DCM) solvent mixture, according to proce-
dure d (81–97% overall yield with greater than 95% purity).
For the synthesis of analogues 2–5, the intramolecular cycliza-
tion was achieved in solution under highly dilute conditions using
A convenient combination of both solid-phase and solution ap-
proaches was used for the synthesis of the analogues 2–8 (Schemes
2 and 3). Solid-phase synthesis of the linear peptide sequences,
using the Fmoc/tBu protection scheme, was performed on a 2-chlo-
rotrityl chloride resin. Together with commercially available amino
acids residues, also two synthetically obtained b-Phe and b-Tyr
were used (see next section). Loading of the solid support was
achieved, under anhydrous conditions, with the first Fmoc-pro-
tected amino acid and an excess of diisopropylethlyamine (DIEA)
in DCM, followed by capping of the unreacted trityl groups with
methanol, according to general procedure a (see Section 3).
The resulting loading degree was determined by UV spectro-
photometric analysis, using the general procedure a0 (0.17–
0.53 mmol gꢀ1). The Fmoc protecting group, before each coupling
step, was removed by treatment with a 20% solution of piperidine
in N,N-dimethylformamide (DMF), according to general procedure
b. The resin was then submitted to coupling-deprotection steps in
order to obtain the desired linear sequences as precursors of the
cyclic analogues. All the coupling steps were performed using clas-
sical SPPS conditions with hydroxybenzotriazole/O-(benzotriazol-
1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate (HOBt/
HBTU) in presence of N-methylmorpholine (NMM), as described
in the general procedure c; the amino acid coupling degree was
O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
hexa-
fluorophosphate (HATU) and DIEA in DCM/DMF, following general
procedure e (Scheme 2).
Peptide cyclic structures were confirmed, on crude products,
through analytical reversed-phase HPLC (RP-HPLC) and ESI-MS
mass spectrometry analysis. Finally, after removing the protecting
groups, purification on semi-preparative RP-HPLC on a C18 col-
umn, (UV–vis detection), yielded the pure cyclopeptides 2–5 (the
cyclopeptides were obtained in 22–30% yield calculated after two
HPLC purification steps). Their structures were fully elucidated
by ESI-MS and NMR spectroscopic analysis.
The synthesis of the advanced intermediates 6b–8b (Scheme 2)
followed the SPPS protocol up to 6a–8a. After cleavage from the
resin, according the general procedure d (greater than 82% yield),
ester bond formation at the C terminus of the linear peptide with
4-penten-1-ol, was performed in solution, with HATU, as coupling
reagent, in basic conditions. Finally, the intramolecular cyclization
FmocAA1
Fmoc-AA1-COOH
a
d
b, c cycles
Cl
Cl
PS
=
HOOC-AA1-AA2-AA3 AAn-NH2
AA1-AA2-AA3 AAn-NH2
AA1-AA2-AA3 AAn
ClTrt-Cl-resin
81-97%
= side-chain
AA1-AA2-AA3 AAn
f
e
protecting group
2-5
a) DIEA, DCM
c) HOBt-HBTU-NMM, DMF
d) AcOH/TFE/DCM
e) HATU, DIEA, DCM
f) TFA/H2O/TIS
22-30% (2 HPLC purification steps)
b) 20% piperidine/DMF
2= NH2-Gaba-β Ala-Ala-D-Trp- β-Phe-COOH
3= NH2-8-Aoc-Ala-D-Trp-β-Phe-COOH
4= NH2-Gaba-β-Ala-Ala-D-Trp-β-Tyr-4-OMe-COOH
5= NH2-Gaba-β-Ala-Ala-D-Trp-β-Ala-COOH
Scheme 2. General synthetic approach followed for analogues 2-5.
O
R
Fmoc-AA1-COOH
HOOC-AA1-AA2-AA3-NH
8a R= CH3
6a-7a R= H
O
O
O
O
R
R
O
O
-AA1-AA2-AA3-NH
-AA1-AA2-AA3-NH
g
h,f
Ring-closing
8b R= CH3
6b-7b R= H
6-7 R= H
8 R= CH3
metathesis
h) Grubbs catalyst 2nd generation,
DCM, MW
20-33% yield after 2 HPLC
purification steps
g) HATU, DIEA, DCM
6= NH2-4-Peo-4-Pea-Ala-D-Trp-β-Ala-COOH
7= NH2-4-Peo-4-Pea-Ala-D-Trp-β-Phe-COOH
8= NH2-4-Peo-3-MePea-Ala-D-Trp-β-Phe-COOH
f) TFA/H2O/TIS
Scheme 3. General synthetic approach followed for analogues 6-8.

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S. Terracciano et al. / Bioorg. Med. Chem. 16 (2008) 6580–6588
was performed through Ring Closing Methatesis (RCM) reaction,
which represents a suitable approach for the construction of vari-
able-sized macrocyclic ring systems, especially in the synthesis of
natural products.10 As this procedure has high functional-groups
tolerance, it has emerged as an efficient approach for obtaining
modified cyclopeptides.
on a C18 column and the structures of cyclodepsipeptides 6–8
(20–33% yield after 2 purification HPLC steps) were confirmed by
MS and NMR analysis.
2.4. Biological activity
In our previous studies on cyclopeptidomimetics synthesis we
experienced the efficiency of second-generation Grubbs’ catalyst,
which was found to be more stable and more reactive in our reac-
tion conditions (Fig. 3).
The antiproliferative activity of the compounds 2–8 was evalu-
ated on kidney A498 adenocarcinoma cells. A moderate cytotoxic-
ity (IC50 = 25 lM) was observed only for compounds 7 and 8. The
synthetic compounds 2–8 were also tested for their activity on
the microfilaments. A498 cells were treated with either cytochala-
sin B as positive control for microfilament depolymerising activity,
or 100 lmol amounts of compounds 2-8. Whilst binucleated cells
were consistently and clearly present in samples treated with cyto-
chalasin B, no cellular aberration was observed with compounds
2–8, even if a modest change on the actin morphology was ob-
served for compound 8 (Fig. 4).
These results appeared quite unexpected, considering the good
alignment found between two of our synthetic compounds and
Jaspamide in our preliminary conformational studies. Presumably
the extreme simplification of the polyketide moiety, proposed in
order to allow more synthetic accessibility of our analogues, re-
sulted to be detrimental for the biological activity. This hypothesis
is based on the consideration that the structural variations made
on the peptide portion, such as the substitution of the two rare
amino acids (b-Tyr and N-methyl-2-brTrp) with commercially
available or, at least more synthetically accessible residues, were
suggested by previous investigations made on other natural ac-
tin-targeting agents5b which show, for instance, the absence of
bromine atom on Trp residue. Our conclusion was that, even if
the three peptide portions seem to play a crucial role in eliciting
bioactivity, as it has emerged by several investigations, the impor-
tance of the polypropionate unit cannot be underestimated in
endowing the molecule with the proper lipophilicity and in assur-
ing the right orientation of the tripeptide backbone in the space,
also in accordance with the parallel findings by Maier’s group.12
In any case, despite the disappointing biological response of our
synthetic compounds, we definitely gained further information
On the basis of the reported data regarding the successful use of
microwave in RCM,11 we used MW irradiation instead of conven-
tional heating, in order to speed up the reaction, as well as to in-
crease reaction yield. Hence for the synthesis of analogues 6–8,
we used the following optimized procedure: refluxing DCM as sol-
vent, 10 mol% of second generation Grubbs’ catalyst, microwave
heating at 300 W for two 40 min periods. A second addition of
fresh 10 mol% catalyst was performed after the first forty minutes.
In order to remove the evolving ethylene during the RCM, a gentle
stream of an inert gas was employed.
To find the best cyclization conditions we performed several tri-
als and finally we selected the very high 0.3 mM dilution. The
structures of the desired compounds were confirmed by mass
spectrometry and analytical RP-HPLC. After RCM, all cyclodepsi-
peptides were deprotected, using TFA in the presence of scaveng-
ers. Crude products were purified by semi-preparative RP-HPLC
N
N
Cl
Ru
PCy3
Cl
Grubbs second-generation
Figure 3. Grubbs’ catalysts structure.
Figure 4. Microfilaments immunofluorescence assays on A-498 kidney adenocarcinoma cells of Jaspamide analogues 2-8.

S. Terracciano et al. / Bioorg. Med. Chem. 16 (2008) 6580–6588
6585
useful to optimize the rational design of a new generation of ana-
logues, as these data represent the only guidelines in absence of a
detailed receptor mapping and well-defined structural determi-
nants which could clarify at molecular level the nature of interac-
tion of Jaspamide with its biological target.
operating with continuous irradiation power from 0 to 300 W uti-
lizing the standard absorbance level of 300 W maximum power.
The reaction mixture was carried out in 100 mL round-bottomed
flask containing a magnetic stirring and fitted with a reflux
condenser.
The DiscoverTM system also offers controllable ramp time, hold
time (reaction time) and uniform stirring. The temperature was
monitored using the CEM-Discover built-in-vertically-focused IR
temperature sensor. After the irradiation period, the reaction ves-
sel was cooled rapidly (60–120 s) to ambient temperature by air
jet cooling.
3. Experimental
3.1. General
Unless specified otherwise, all starting materials, reagents, and
solvents were commercially available and used without further
purification. DCM and DMF used for solid-phase reactions were
synthesis grade (dried over activated 4 Å molecular sieves). Water
and CH3CN were HPLC grade. 2-Chlorotrityl chloride resin (100–
200 mesh), 1% DVB, (ClTrt-Cl, loading level: 1.04 and 1.4 mmol/
g), HOBt, and HBTU were purchased from Novabiochem. Fmoc-
amino acids were obtained from Novabiochem, Neosystem and
Fluka. HATU was purchased from Fluka. Solid-phase peptide syn-
theses, using the Fmoc-t-Bu strategy, were carried out on a poly-
propylene ISOLUTE SPE column on a VAC MASTER system, a
manual parallel synthesis device purchased from Stepbio. All solid
phase reactions were run under a nitrogen atmosphere with dry
solvents. For estimation of Fmoc amino acids on the resin, absor-
bance at 301 nm was read employing a Shimadzu UV 2101 PC.
Analytical and semipreparative reverse phase HPLC with UV detec-
tor system operating at 220 and 250 nm was performed on a Jupi-
ter C-18 column (250 ꢁ 4.60 mm, 5l, 300 Å; 250 ꢁ 10.00 mm, 10l,
300 Å, respectively). The binary solvent system (A/B) was as fol-
lows: 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B).
All ring-closing metathesis (RCM) reactions were carried out
under an argon atmosphere with dry, degassed solvents under
anhydrous conditions. Grubbs catalyst 2nd generation was pur-
chased from Aldrich.
3.4. Representative procedure for lithium amide addition
n-BuLi (2 equiv) was added dropwise to a stirred solution of (S)-
N-benzyl-N-(methylbenzyl)amide (2 equiv) in anhydrous THF at
0 °C and stirred for 15 min under argon. The solution was cooled
at ꢀ78 °C and a solution of a,b-unsaturated ester in THF was added
via cannula and stirred at ꢀ78 °C for 12 h before the addition of
saturated aqueous ammonium chloride. The resulting mixture
was portioned between brine and ether and the combined organic
extracts dried (MgSO4), filtered and concentrated in vacuo before
purification on silica gel column chromatography.
3.4.1. Compound 10
Purification with hexane/ethyl acetate 996:4 (68% yield, 95%
de). 1H NMR (400 MHz, CDCl3): d 1.33 (3H, d, J = 7.0 Hz), 2.70
(1H, dd, J = 9.0 e 15.2 Hz), 2.83 (1H, dd, J = 9.0 e 15.2 Hz), 3.56
(3H, s), 3.84 (2H, q, J = 14.8 Hz) 4.15 (1H, q, J = 7.07 Hz), 4.59 (1H,
dd, J = 6.0 e 9.4 Hz), 7.27–7.56 (15H, ovl); 13C NMR (100 MHz,
CDCl3): d 15.7, 37.3, 50.6, 51.3, 56.6, 59.1, 126.5–128.2, 141.2,
141.6, 144.0, 172.0. ESI-MS: m/z 374.2 [M+H]+.
3.4.2. Compound 15
All the NMR spectra (1H, HMBC, HSQC, TOCSY, COSY, ROESY)
were recorded on a Bruker Avance DRX600 spectrometer at
T = 298 K. The compounds 2–8 were dissolved in 0.5 mL of
99.95% d6-DMSO (Carlo Erba, 99.95 Atom % D) (1H, d = 2.50 ppm;
13C d = 39.5 ppm). The NMR data were processed on a Silicon Gra-
phic Indigo 2 workstation using UXNMR software.
Electrospray mass spectrometry (ES-MS) was performed on a
LCQ DECA ThermoQuest (San Josè, CA, USA) mass spectrometer.
High resolution mass spectra were acquired on a Q-Tof ULTIMA
(Waters, Manchester UK) calibrated with [Glu]-Fibrinopeptide B
fragments using standard experimental conditions.
Purification with hexane/ethyl acetate 99:1 (75% yield, 98% de).
1H NMR (400 MHz, CDCl3): d 1.39 (3H, d, J = 6.4 Hz), 2.64 (1H, dd,
J = 9.0 e 15.0 Hz), 2.71 (1H, dd, J = 5.4 e 15.0 Hz), 3.71 (1H, m),
3.81 (3H, s), 3.84 (1H, q, J = 7.0 Hz), 4.14 (1H, q, 7.2 Hz), 4.93 (2H,
m), 6.87 (2H, d, J = 8.0 Hz), 7.09–7.46 (16H, ovl); 13C NMR
(100 MHz, CDCl3): d 15.7, 37.3, 50.6, 51.3, 56.6, 59.1, 126.5–
128.2, 141.2, 141.6, 144.0, 145.9, 159.3, 172.0.
3.5. Representative procedure for N-deprotection and
hydrolysis
The N-protected b-amino esters arising from conjugate addition
were dissolved in MeOH/acetic acid/H20 (10:1:0.25, 2 mL/
0.1 mmol) and the solution poured in the pressure bottle of a Parr
hydrogenation apparatus. Pd (OH)2 Degussa E101 (20 equiv) was
added and the mixture shaken under 6 atm of H2 for 12 h. The bot-
tle was degassed, the catalyst was filtered and washed several
times with MeOH. The solvent was evaporated and the crude prod-
uct was immediately subjected to acidic hydrolysis without purifi-
cation. b-Amino ester was dissolved in 2 M HCl (1 mL/0.2 mmol)
and heated at 140 °C for 16 h. The solvent was removed in vacuo
and the resulting material was chromatographed on silica gel elut-
ing with DCM/MeOH 99:1.
3.2. Computational details
In order to allow a full exploration of the conformational space
of the molecules 2 and 3, MM/MD calculations at different temper-
atures (300 K, 500 K, 700 K/10 ns) were performed using the
AMBER force field (MacroModel software package) All the so-ob-
tained structures (in number of 100) were minimized using the Po-
lak-Ribier Conjugate Gradient algorithm (PRCG, 1000 steps,
maximum derivative less than 0.05 kcal/mol). This led to the selec-
tion of the lowest energy minimum conformer for the molecules; a
parallel analysis was performed using the MonteCarlo Multiple
Minimum (MCMM) method (50 K steps) of the MacroModel pack-
age,13 leading to the same results obtained by MM/MD
calculations.
3.5.1. Compound 11
25
White amorphous solid; ½dꢂD þ 6:8ðc0:9; H2OÞ; ESI-MS: m/z
166.1 [M+H]+.
3.3. Microwave irradiation experiments
3.5.2. Compound 16
25
White amorphous solid; ½dꢂD ꢀ 0:6ðc0:1; H2OÞ; ESI-MS: m/z
All microwave irradiation experiments were carried out in a
dedicated CEM-Discover Focused Microwave Synthesis apparatus,
196.1 [M+H]+.

6586
S. Terracciano et al. / Bioorg. Med. Chem. 16 (2008) 6580–6588
3.6. Representative procedure for N-Fmoc protection
(3–4 equiv) and NMM (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, 3ꢁ 1.5 min), and DCM (3 mL, 3ꢁ 1.5 min).
Na2CO3 (3 equiv) was added to a solution of free b-amino acids
in acetone/H2O 1:1 (2 ml/0.2 mmol) and stirred for 30 min before
FmocCl was added (1.1 equiv). After stirring overnight the mixture
was extracted with EtOAc for 3 times. The combined organic layers
were dried (MgSO4), filtered and concentrated in vacuo. Silica gel
chromatography (hexane/ethyl acetate 9:1) furnished N-protected
b-amino acids in good yields.
3.7.5. Cleavage
The dried peptide resin was treated for 2 h, under stirring, with
the following cleavage mixture: AcOH/TFE/DCM (2:2:6;
10 lL ꢁ 1 mg of resin). Then the resin was filtered off and washed
with neat cleavage mixture (3 mL, 3ꢁ 1.5 min). After addition of
hexane (15 times volume) to remove acetic acid as an azeotrope,
the filtrate was concentrated and lyophilized (81–97% overall
yield).
3.6.1. Compound 12
Yield (75%). 1H NMR (400 MHz, CDCl3): d 2.90 (2H, m), 3.63 (3H,
s), 4.13 (1H, m), 4.21 (1H, t, J = 6.6), 4.41 (2H, d, 6.6 Hz), 7.29–7.33
(7H, m), 7.39 (2H, t, 6.4 Hz), 7.58 (2H, d, 6.4 Hz), 7.76 (2H, d,
6.4 Hz); 13C NMR (100 MHz, CDCl3): d 40.6, 47.5, 52.1, 67.0,
120.2, 125.3, 126.4, 127.3, 127.9, 129.0, 141.5, 144.0, 155.9,
172.0. ESI-MS: m/z 388.1 [M+H]+.
3.7.6. Cyclization
The cyclization step was performed in solution at a concentra-
tion of 7.7 ꢁ 10ꢀ5 M with HATU (2.0 equiv) and DIEA (2.5 equiv)
in DCM. The solution was stirred at 4 °C for 1 h and then allowed
to warm to room temperature for 1 h. The solvent was removed
under reduced pressure.
3.6.2. Compound 17
Yield (69%). 1H NMR (400 MHz, CDCl3): d 2.81 (1H, dd, J = 5.9 e
15.2 Hz) 2.91 (1H, dd, J = 5.2 e 15.2 Hz), 3.80 (3H, s), 4.09 (1H, m),
4.21 (1H, t, J = 6.3 Hz), 4.39 (2H, d, 6.3 Hz), 6.87 (2H, d, J = 7.0 Hz),
7.23 (2H, d, 7.0 Hz), 7.30 (2H, t, 6.2 Hz), 7.40 (2H, t, 67.3 Hz), 7.58
(2H, d, 6.2 Hz), 7.76 (2H, d, 7.3 Hz); 13C NMR (100 MHz, CDCl3): d
30.9, 41.0, 47.5, 51.4, 55.5, 61.0, 67.0, 114.3, 120.2, 125.3, 127.3,
127.6, 128.0, 141.5, 155.8, 172.0. ESI-MS: m/z 418.2 [M+H]+.
3.7.7. Final deprotection
Final deprotection was carried out with TFA/H2O/TIS
(95:2.5:2.5; 100lL ꢁ 1 mg of resin) for 1 h, under stirring. The
crude product was purified by semipreparative RP HPLC (22–30%
yield calculated after two HPLC purification steps) and character-
ized by ES-MS and NMR spectra.
3.7. General procedures for the synthesis of compounds 2–8
3.7.8. Ester bond formation
The coupling reaction was performed by using 4-penten-1-ol
(1.6 equiv), HATU (3.0 equiv) and DIEA (3.8 equiv) in DCM at a con-
centration of 0.2 M. The solution was stirred at 4 °C for 1 h and
then allowed to warm to room temperature overnight. The solvent
was removed under reduced pressure. The crude product was ana-
lyzed by analytical RP HPLC and ESI-MS.
3.7.1. Loading of the resin
The ClTrt-Cl resin was placed in a 25 mL polypropylene ISOSO-
LUTE syringe on a VAC MASTER system, swollen in 3 mL of DMF for
1 h, and then washed with 2ꢁ 3 mL of DCM.
A solution of Fmoc-AA-OH (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 3ꢁ DCM/MeOH/DIEA (17:2:1), and sequentially
with the following washing/treatments: DCM 3ꢁ 3 mL, DMF 2ꢁ
3 mL, DCM 2ꢁ 3 mL (1.5 min each).
3.7.9. RCM under microwave irradiation
Optimized microwave ring closing metathesis.
To a stirred solution (0.3 mM) of the linear precursor in dry
DCM was purged argon for 10 min followed by the addition of
10% mol of Grubbs catalyst 2nd. The mixture was refluxed at
60 °C under an argon atmosphere and irradiated at a maximum
power (300 W) for 40 min. The reaction mixture was then de-
gassed with N2 to dry off any dissolved ethene, an additional 10%
mol catalyst was added and the solution was stirred and subjected
to MW irradiation (300 W) for a further 40 min. Finally, the solu-
tion was concentrated in vacuo and the crude cyclodepsipeptide
was purified by RP-HPLC.
The crude products were purified by semipreparative reverse
phase HPLC (on a Jupiter C-18 column: 250 ꢁ 10.00 mm, 10l,
300 Å, flow rate = 4 mL/min) using the following gradient condi-
tions (20–33% yield after 2 purification HPLC steps) and character-
ized by ES-MS and NMR spectra.
3.7.2. Estimation of the level of first residue attachment
The loading of the resin was determined by UV quantification of
the Fmoc-piperidine adduct.
The assay was performed on duplicate samples: 0.4 mL of piper-
idine and 0.4 mL of DCM were added to two dried samples Fmoc
amino acid-resin (ꢃ3.0 mg) in two volumetric flasks of 25 mL.
The reaction mixture was allowed to proceed for 30 min at rt
and then 1.6 mL of MeOH was added and the solutions were di-
luted to 25 mL volume with DCM. A reference solution was pre-
pared in a 25 mL volumetric flask 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 gꢀ1 = (A301/7800) ꢁ (25 mL gꢀ1 of resin). The obtained load-
3.7.10. Cyclo [b-Phe-D-Trp-Ala-b-Ala-Gaba] (2)
RP-HPLC tR = 13.35 min. from 15% B to 85% B over 35 min;
3.5 mg (27% yield) as a white solid; ES-MS, calcd for C30H36N6O5
560.6; found m/z = 561.1 [M+H]+, 583. 3 [M+Na]+, 599. 1 [M+K]+.
HRMS calcd for C30H37N6O5 [M+H]+ 561.2820; found 561.2560.
1H and 13C NMR data (600 MHz, DMSO-d6): b-Phe dH: a 5.24, b
2.53 (2H), Ar 7.05–7.26, NH 8.46; Trp dH: a 4.58, b 2.86, 3.05, NHin
10.82, 2 7.12, 5 7.65, 6 6.97, 7 7.05, 8 7.32, NH 8.12; Ala dH: a 4.33, b
0.97, NH 8.00; b-Ala dH: a 2.22, 2.39, b 3.27, 3.35, NH 7.37; Gaba dH:
a 2.80 (2H), b 1.49 (2H), c 3.15 (2H); b-Phe dC: a 49.83, b 42.16, Ar
126.00–128.40; Trp dC, a 52.67, b 27.27, 2 122.92, 5 118.02, 6
117.75, 7 120.43, 8 110.70; Ala dC: a 47.18, b 17.00; b-Ala dC: a
34.50, b 34.76; Gaba dC: a 37.18, b 24.78, c 37.08.
ing degree was 0.17–0.53 mmol gꢀ1
.
3.7.3. Fmoc deprotection
About 20% piperidine in DMF (3 mL, 1ꢁ 1.5 min), 20% piperidine
in DMF (3 mL, 1ꢁ 10 min); washings in DMF 2ꢁ 3 mL, DCM 2ꢁ
3 mL, DMF 2ꢁ 3 mL, (1.5 min each).
3.7.4. Peptide coupling conditions
The coupling reaction was promoted by a HOBt/HBTU in DMF
coupling protocol: Fmoc-amino acid, 4-pentenoic acid and 3-
methyl-4-pentenoic acid (3–4 equiv), HOBt (3–4 equiv), HBTU

S. Terracciano et al. / Bioorg. Med. Chem. 16 (2008) 6580–6588
6587
3.7.11. Cyclo[b-Phe-D-Trp-Ala-8-Aoc] (3)
The RP-HPLC analysis showed a main peak at tR = 19.70 min.
from 5% B to 100% B over 45 min; 6.9 mg (23% yield) as a slightly
O
8
yellow solid; ES-MS, calcd for
C31H39N5O4 545.6; found m/
7
6
z = 546.2 [M+H]+, 568.4 [M+Na]+, 584.2 [M+K]+.
HRMS calcd for C31H40N5O4 [M+H]+ 546.3075; found 546.2985.
The minor peak eluting at tR 20.51 min was shown to contain a
diastereoisomeric compound, ES-MS, m/z = 546.2 [M+H]+.
1H and 13C NMR data (600 MHz, DMSO-d6): b-Phe dH: a 5.25, b
2.41 (2H), Ar 7.04–7.28, NH 8.71; Trp dH: a 4.63, b 2.80, 3.00, NHin
10.75, 2 6.93, 5 7.57, 6 6.96, 7 7.04, 8 7.30, NH 8.06; Ala dH: a 4.44, b
0.89, NH 7.86; 8-Aoc dH: a 1.93, 2.13, b 1.13 (2H), c 1.15, 1.25, d
1.56 (2H), e 1.35 (2H), f 2.98 (2H), NH 7.76; b-Phe dC: a 51.00, b
44.13, Ar 126.69–129.15; Trp dC: a 53.51, b 28.99, 2 124.44, 5
119.20, 6 118.80, 7 121.62, 8111.85; Ala dC: a 47.62, b 19.20;
Aoc dC: a 35.71, b 25.39, c 29.00, d 25.80, e 28.70, f 39.21.
5
3
4
1
2
O
4-Pea-4-Peo
3.7.15. Cyclo[b-Phe-D-Trp-Ala-4-Pea-4-Peo] (7)
RP-HPLC tR = 26.50 min. from 5% B to 100% B over 60 min; 1.9 mg
(20% yield) as a slightly brown oil; ES-MS, calcd for C31H36N4O5
544.6; found m/z = 545.2 [M+H]+.
3.7.12. Cyclo [b-Tyr-4-OMe-D-Trp-Ala-b-Ala-Gaba] (4)
RP-HPLC tR = 12.00 min. from 5% B to 100% B over 55 min;
HRMS calcd for C31H37N4O5 [M+H]+ 454.2758; found 545.2527.
The E isomer was obtained with 83% selectivity as determined
by HPLC.
2.9 mg (22% yield) as a slightly yellow solid; ES-MS, calcd for
C
31H38N6O6 590.6; found m/z = 591.4 [M+H]+, 613.2 [M+Na]+.
HRMS calcd for C31H39N6O6 [M+H]+ 591.2926; found 591.2829.
1H and 13C NMR data (600 MHz, DMSO-d6): b-Tyr-4-OMe dH: a
RP-HPLC of the Z isomer tR = 27.56 min. from 5% B to 100% B
over 60 min.
5.19, b 2.40 (2H), Ar 6.77–7.00, 4-OMe 3.72, NH 8.65; Trp dH: a
4.57, b 2.85, 3.03, NHin 10.77, 2 6.95, 5 7.64, 6 6.97, 7 7.02, 8
7.30, NH 8.07;Ala dH: a 4.32, b 0.97, NH 7.97; b-Ala dH: a 2.38
(2H), b 3.12, 3.37, NH 7.50; Gaba dH: a 1.90 (2H), b 1.20 (2H), c
1H and 13C NMR data (600 MHz, DMSO-d6): b-Phe dH: d 5.28, b
2.69, 2.76, Ar 7.24–7.70, NH 8.89; Trp dH: d 4.61, b 2.95 (2H), NHin
10.74, 2 7.00, 5 7.62, 6 6.95, 7 7.03, 8 7.31, NH 8.09; Ala dH: d 4.44, b
0.83, NH 7.77; 4-Pea-4-Peo dH:2 2.01, 2.30, 3 2.04, 2.24, 4-CH 5.30
(overlapping signals), 5-CH 5.30 (overlapping signals), 6 1.87, 7
1.50, 8 3.90, 4.05; b-Phe dC: d 49.84, b 42.50, Ar 126.66–128.01;
Trp dC: d 52.72, b 28.00, 2 123.54, 5 118.70, 6 117.92, 7 120.84, 8
112.90; Ala dC: d 47.37, b 19.36; 4-Pea-4-Peo dC: 2 33.68, 3
27.00, 4-CH 128.47, 5-CH 128.47, 6 27.97, 7 27.48, 8 63.40.
2.93, 3.08, NH 7.78; b-Tyr-4-OMe dC:
a 49.50, b 43.27, Ar
113.20–130.00, 4-OMe 54.85; Trp dC: a 52.72, b 27.75, 2 123.26,
5 118.32, 6 118.06, 7 120.60, 8 110.92; Ala dC: a 47.46, b 17.00;
b-Ala dC: a 34.73, b 35.21; Gaba dC: a 33.00, b 28.25, c 37.53.
3.7.13. Cyclo [b-Ala-D-Trp-Ala-b-Ala-Gaba] (5)
RP-HPLC tR = 13.84 min from 5% B to 100% B over 50 min;
3.0 mg (30% yield) as a slightly yellow solid; ES-MS, calcd for
24H32N6O5 484.5; found m/z = 485.2 [M+H]+, 507.3 [M+Na]+,
O
8
C
523.2 [M+K]+.
7
HRMS calcd for C24H33N6O5 [M+H]+ 485.2507; found 485.2587.
1H and 13C NMR data (600 MHz, DMSO-d6): b-Ala dH: a 1.96
(2H), b 3.37 (2H), NH 7.99; Trp dH: a 4.42, b 2.90, 3.14, NHin
10.78, 2 7.13, 5 7.60, 6 6.95, 7 7.03, 8 7.29, NH 8.11; Ala dH: a
4.29, b 0.90, NH 7.89; b-Ala dH: a 2.16, 2.34, b 3.20, 3.35, NH
7.59; Gaba dH: a 1.96 (2H), b 1.55 (2H), c 3.00 (2H), NH 7.66; b-
Ala dC: a 32.60, b 35.33; Trp dC: a 53.07, b 27.10, 2 123.50, 5
118.27, 6 117.94, 7 120.55, 8 110.91; Ala dC: a 47.33, b17.41; b-
Ala dC: a 34.98, b 35.02; Gaba dC: a 32.60, b 25.16, c 37.25.
6
5
3
4
1
2
O
4-Pea-4-Peo
3.7.14. Cyclo [b-Ala-D-Trp-Ala-4-Pea-4-Peo] (6)
RP-HPLC tR = 31.05 min. from 10% B to 60% B over 65 min;
1.7 mg (23% yield) as a white solid; ES-MS, calcd for C25H32N4O5
468.5; found m/z = 469.3 [M+H]+, 491.2 [M+Na]+.
3.7.16. Cyclo[b-Phe-D-Trp-Ala-3-MePea-4-Peo] (8)
RP-HPLC tR = 38.56 min. from 5% B to 20% B over 5 min and then
from 20% B to 60% B over 50 min; slightly brown oil; ES-MS, calcd for
HRMS calcd for C25H33N4O5 [M+H]+ 469.2445; found 469.2338.
The E isomer was obtained with 90% selectivity as determined
by HPLC.
C
32H38N4O5 558.6; found m/z = 559.2 [M+H]+, 581.3 [M+Na]+.
HRMS calcd for C32H39N4O5 [M+H]+ 559.2915; found 559.2603.
The E isomer was obtained with 90% selectivity as determined
RP-HPLC of the Z isomer tR = 32.15 min. from 10% B to 60% B
over 65 min.
by HPLC.
RP-HPLC of the Z isomer tR = 39.80 min. from 5% B to 20% B over
5 min and then from 20% B to 60% B over 50 min.
1H and 13C NMR data (600 MHz, DMSO-d6): b-Ala dH: a 2.43,
2.53, b 3.16, 3.48, NH 8.03, Trp dH: a 4.48, b 2.87, 3.08, NHin, 2
7.09, 5 7.59, 6 6.95, 7 7.03, 8 7.29, NH 8.06; Ala dH: a 4.33, b
0.90, NH 7.79; 4-Pea-4-Peo dH: 2 2.05, 2.23, 3 2.06, 2.25, 4-CH
5.31 (overlapping signals), 5-CH 5.31 (overlapping signals), 6
1.88, 7 1.51, 8 3.88, 3.98; b-Ala dC: a 34.22, b 35.50; Trp dC: a
53.23, b 28.29, 2 123.99, 5 118.88, 6 118.42, 7 121.20, 8 111.43;
4-Pea-4-Peo dC: 2 34.94, 3 27.92, 4-CH 129.55, 5-CH 129.55, 6
28.43, 7 27.79, 8 63.39.
1H and 13C NMR data (600 MHz, DMSO-d6): b-Phe dH: d 5.30, b
2.68, 2.78, Ar 7.22–7.34, NH 8.90; Trp dH: d 4.63, b 2.77, 2.95, NHin
10.75., 2 7.02,5 7.66, 6 6.97, 7 7.05, 8 7.30, NH 8.14; Ala dH: d 4.47, b
0.80, NH 7.75; 3-MePea-4-Peo dH:2 1.78, 2.11,3 2.46, 3-Me 0.94,4-
CH 5.23,5-CH 5.31,6 1.87 (2H), 7 1.52 (2H), 8 3.93, 4.09; b-Phe dC: d
49.80, b 42.27, Ar 126.37–128.89; Trp dC: d 53.00, b 28.68, 2
121.10, 5 118.96, 6 118.36, 7 121.24, 8 111.41; Ala dC: d 47.07,

6588
S. Terracciano et al. / Bioorg. Med. Chem. 16 (2008) 6580–6588
b 47.07; 3-MePea-4-Peo dC: 2 43.82,3 33.96, 3-Me 21.82,4-CH
134.70,5-CH 128.06,6 28.66, 7 28.20, 8 64.20.
References and notes
1. (a) Zabriskie, T. M.; Klocke, J. A.; Ireland, C. M.; Marcus, A. H.; Molinski, T. F.;
Faulkner, D. J.; Xu, C.; Clardy, J. C. J. Am. Chem. Soc. 1986, 108, 3123–3124; (b)
Crews, P.; Manes, L. V.; Boehler, M. Tetrahedron Lett. 1986, 27, 2797–2800; (c)
Braekman, J. C.; Daloze, D.; Moussiaux, B.; Riccio, R. J. Nat. Prod. 1987, 50, 994–
995.
2. (a) Fenteany, G.; Zhu, S. Curr. Top. Med. Chem. 2003, 3, 593–616; (b) Giganti, A.;
Friederich, E. Prog. Cell Cycle Res. 2003, 5, 511–525; (c) Allingham, J. S.;
Klenchin, V. A.; Rayment, I. Cell. Mol. Life Sci. 2006, 63, 2119–2134.
3. (a) Senderowicz, A. M.; Kaur, G.; Sainz, E.; Laing, C.; Inman, W. D.; Rodriguez, J.;
Crews, P.; Malspeis, L.; Grever, M. R.; Sausville, E. A.; Duncan, K. L. J. Natl. Cancer
Inst. 1995, 87, 46–51; (b) Bubb, M. R.; Senderowicz, A. M.; Dunkan, K. L.; Korn,
E. D. J. Biol. Chem 1994, 269, 14869–14871; (c) Spector, I.; Breat, F.; Shochet, N.;
Bubb, M. Microsc. Res. Tech. 1999, 47, 18–37; (d) Bubb, M. R.; Spector, I.; Beyer,
B.; Fosen, K. A. J. Biol. Chem. 2000, 275, 5163–5170; (e) Visegràdy, B.; Lorinczy,
D.; Hild, G.; Somogyi, B.; Nyitrai, M. FEBS Lett. 2004, 565, 163–166; (f)
Visegràdy, B.; Lorinczy, D.; Hild, G.; Somogyi, B.; Nyitrai, M. FEBS Lett. 2005,
579, 6–10.
O
8
7
6
5
Me
1
4
3
2
O
3-MePea-4-Peo
4. (a) Kunze, B.; Jansen, R.; Sasse, F.; Hofle, G.; Reichenbach, H. J. Antibiot. 1995,
1262; (b) Holzinger, A.; Lutz-Meindl, U. Cell. Motil. Cytoskeleton 2001, 48, 87–
95.
3.8. Biological tests
5. (a) Ishiwata, H.; Nemoto, T.; Ojija, M.; Yamada, K. J.Org. Chem. 1994, 59, 4710–
4711; (b) Bai, R.; Covell, D. G.; Chunfeng, L.; Gosh, A. K.; Hamel, E. J. Biol. Chem.
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6687–6700; (b) Hu, T-S.; Tannert, R.; Arndt, H. D.; Waldmann, H. Chem.
Commun. 2007, 3942–3944.
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R. J. Nat. Prod. 2004, 67, 1325–1331; (b) Terracciano, S.; Bruno, I.; Bifulco, G.;
Avallone, E.; Smith, C. D.; Gomez-Paloma, L.; Riccio, R. Bioorg. Med. Chem. 2005,
13, 5225–5239.
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1991, 38, 324–334.
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Org. Lett. 1999, 1, 953–956; (c) Fustner, A. Angew. Chem. Int. Ed. 2000, 39, 3012–
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references reported therein.
3.8.1. Cytotoxicity assay
A498 kidney adenocarcinoma cells were maintained in a R-
MEM medium containing 10% fetal bovine serum and 50 lg/mL
gentamycin at 37 °C in an atmosphere of 5% CO2 and 95% air. To
determine the cytotoxicities of the test compounds, cells were pla-
ted into 96-well tissue culture plates at approximately 15% conflu-
ency and were allowed to attach and recover for 24 h. The cells
were then treated in triplicate with varying concentrations of the
test compound for 48 h, and cell survival was assayed using the
sulforhodamine B (SRB) binding assay.14 The percentage of cell sur-
vival was calculated as the percentage of SRB binding as compared
with control cultures.
3.8.2. Immunofluorescence assays
A-498 cells were grown to near-confluency on glass coverslips
and treated with the indicated compounds for 16 h. Microtubules
and microfilaments were stained with monoclonal anti-b-tubulin
and monoclonal anti-actin antibodies, respectively, and visualized
with fluorescein-conjugated anti-mouse IgG as previously
described.15
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Acknowledgment
Financial support by the University of Salerno and MIUR (Rome)
is gratefully acknowledged.