Synthesis, solution structure, and bioactivity of six new simplified analogues of the natural cyclodepsipeptide jaspamide

Article abstract of DOI:10.1016/j.bmc.2005.05.042

Recently, we described the synthesis and the biological evaluation of three modified analogues of jaspamide (1), a natural cyclodepsipeptide possessing a potent antitumor activity as a consequence of its ability to interfere with actin cytoskeleton. To ob

Full text of DOI:10.1016/j.bmc.2005.05.042

Bioorganic & Medicinal Chemistry 13 (2005) 5225–5239
Synthesis, solution structure, and bioactivity of six new
simplified analogues of the natural cyclodepsipeptide jaspamide
Stefania Terracciano,a Ines Bruno,a Giuseppe Bifulco,a Elvira Avallone,b
Charles D. Smith,c Luigi Gomez-Palomaa and Raffaele Riccioa,*
a
`
Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Salerno, via Ponte Don Melillo, 84084 Fisciano (SA), Italy
b
`
Dipartimento di Chimica, Universita degli Studi di Salerno, via S. Allende, 84081 Baronissi (SA), Italy
cDepartment of Pharmacology, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA
Received 30 March 2005; revised 26 May 2005; accepted 26 May 2005
Available online 14 July 2005
Abstract—Recently, we described the synthesis and the biological evaluation of three modified analogues of jaspamide (1), a natural
cyclodepsipeptide possessing a potent antitumor activity as a consequence of its ability to interfere with actin cytoskeleton. To
obtain additional information on the potential pharmacophoric core of the target molecule, which is of fundamental importance
to discover new and more effective anticancer products, we decided to explore the biological effects of further structural modifica-
tions carried out on the parent molecule. The synthesis and the chemical characterization of six jaspamide analogues (2–7) are
reported and their conformational and biological properties are described.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
of antimitotic agents with improved pharmacological
profile. The natural compound showed ability to inter-
act with actin filaments and in particular to produce
structural and biophysical changes of cytoskeletal
dynamics, resulting in a potent antiproliferative activi-
ty.5–11 Despite the great interest reawakened by this
potentially therapeutic agent, much of the information
needed for a detailed understanding of the structural
requirements essential for the activity is still missing.
On the other hand, such information is absolutely neces-
sary to perform a rational design of new actin-targeted
inhibitors. With the aim of shedding more light on the
potential pharmacophoric core of this natural lead com-
pound, three modified analogues of jaspamide (8–10)
(Fig. 1) were recently synthesized and their biological
and conformational properties were described.12 Actual-
ly, our project was mainly inspired by the shared
conviction that fundamental to the bioactivity was the
Ala-N-Me-2BrTrp-b-Tyr tripeptidic portion,13 with the
polyketide chain acting as an appropriate linker, presum-
ably adding further conformational constrains to the
macrocyclic system, thus providing further assistance
to its intrinsic propensity to fold into a b-turn motif. In
the course of such study, we learned by fact that even
quite subtle structural differences in the arrangement of
the putative pharmacophoric region, easily translate into
loss of target selectivity of the intact natural product,
even though compounds 8–10 were nevertheless
Jaspamide (1), a cyclodepsipeptide isolated from marine
sponges of Jaspis genus,1–4 represents an attractive
molecular target for the development of a novel class
Abbreviations: Boc, tert-butyloxycarbonyl; ClTrt-Cl, 2-chlorotrityl-
chloride resin; DCC, N,N0-dicyclohexylcarbodiimide; DCM,
dichloromethane; DIEA, N,N-diisopropylethylamine; DMAP, 4-di-
methylaminopyridine; DMF, N,N-dimethylformamide; EBA, ethyl
bromoacetate; Fmoc, 9-fluorenylmethyloxycarbonyl; Fmoc-5-Ava-O-
H, N-a-Fmoc-5-aminopentanoic acid; Fmoc-8-Aoc-OH, N-a-Fmoc-8-
aminooctanoic acid; Fmoc-Ala-OH, N-a-Fmoc-L-alanine; Fmoc-D-
Trp(Boc)-OH, N-a-Fmoc-Nin-Boc-D-tryptophan; Fmoc-Val-OH, N-a-
Fmoc-L-valine; Fmoc-e-Ahx-OH, N-a-Fmoc-6-aminohexanoic acid;
Fmoc-b-Ala-OH, N-a-Fmoc-L-b-alanine; Fmoc-Phg-OH, N-a-Fmoc-
L-phenylglycine; Fmoc-Tyr(tBu)-OH, N-a-Fmoc-O-t-butyl-L-tyrosine;
HATU, N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-yl-me-
thylene]-N-methyl-methanaminium hexafluorophosphate N-oxide;
HBTU, O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate; Hepa, 3-(2-hydroxyethyl)-phenoxyacetic acid; HOBt,
N-hydroxybenzotriazole; NovaSynꢁ TGA resin, hydroxymethylphen-
oxyacetic acid linker; NMM, N-methylmorpholine; PyBop, benzo-
triazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate;
TFA, trifluoroacetic acid; TFE, 2,2,2-trifluoroethanol; TIS,
triisopropylsilane.
Keywords: Jaspamide; Cyclopeptides; Solid-phase synthesis; Actin
cytoskeleton; Cytotoxic.
*
Corresponding author. Tel.: +39 089 962818; fax: +39 089 962828;
e-mail: riccio@unisa.it
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.05.042

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S. Terracciano et al. / Bioorg. Med. Chem. 13 (2005) 5225–5239
Figure 1. Chemical structures of (a) the natural jaspamide (1) and its simplified analogues 2–7 and (b) the previously reported simplified analogues
8–10.
cytotoxic with IC50 values at micromolar levels.12 In the
design of these analogues, we intended to simplify those
structural features of the parent compound that were
considered most critical in terms of the challenge posed
by their chemical synthesis. On the basis of this criteri-
on, selected modifications, both on the polyketide and
on the tripeptide fragments, were implemented. Accord-
ingly, we first decided to replace the two unusual amino
acids D-N-methyl-2-bromotryptophan and L-b-tyrosine,
with D-tryptophan and L-valine or L-phenylglycine,
respectively (affording two series of analogues named
ÔcyclovalÕ and ÔcyclophgÕ). The rational behind such
two amino acidic substitutions was to take into due
account the diverse structural solutions that Nature
has used to fashion other effective agents capable of elic-
iting this kind of biological properties. Therefore, be-
sides jaspamide, dolyculide and chondramide
C
structures were also considered a useful source of inspi-
ration for this task. Instrumental for our design was a
recent report describing a computational approach
based on 3D alignments of microfilament-disrupting
natural agents, aimed at identifying a hypothetical array

S. Terracciano et al. / Bioorg. Med. Chem. 13 (2005) 5225–5239
5227
of molecular determinants that, if correctly oriented in
the space, should be capable to evoke target selectivity
and thus, ultimately, bioactivity.14 Hence, the valine iso-
propyl side chain of dolyculide and the phenyl ring of
the Phg residue of chondramide C can be both aligned
with the phenyl ring of the b-Tyr of jaspamide, suggest-
ing that these replacements are likely to be conservative
in the context of these chemical agents. Under these
premises, we have undertaken an additional exploration
on the chemistry of these jaspamide analogues, in the
hope of discovering new simplified actin-targeted cyto-
toxic products, or at least a series of molecules capable
to maintain as much as possible the potent antiprolifer-
ative effects displayed by this class of natural products.
Scheme 1. Synthetic scheme followed to obtain analogues 2–7.

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S. Terracciano et al. / Bioorg. Med. Chem. 13 (2005) 5225–5239
more rigid linker (Chart 1). Thus, we envisioned the
replacement of the five carbons constituting the unsatu-
rated dimethyl-C3 segment with a suitable meta-disubsti-
tuted phenyl ring, which had to be elongated to form a
3-(2-hydroxyethyl)-phenoxyacetic acid to maintain in
our analogues the same number of atoms present in
jaspamide.
Chart 1. The replacement of the polyketide fragment of jaspamide
with the meta-substituted aromatic portion used for analogues 6 and 7.
2. Results and discussion
2.1. Synthesis
A convenient combination of solid- and solution-phase
synthetic techniques was used for the synthesis of the
six analogues 2–7. As concerning solid-phase chemistry,
the Fmoc/tBu protection scheme was adopted on a 2-
chlorotritylchloride resin solid support (analogues 2–4
and 6–7) and on NovaSynꢁTGA (analogue 5). Using
the 2-chlorotritylchloride resin, the first Fmoc-protected
amino acid was anchored to the linker by diisopropyl-
ethylamine (DIEA) treatment under anhydrous condi-
tions, followed by capping of the unreacted trityl
groups with methanol, according to the general proce-
dure a (see Section 3). On the other hand, when working
on the NovaSynꢁTGA resin, the attachment of the first
residue to the 4-hydroxymethylphenoxyacetic acid link-
er was achieved by a DMAP-catalyzed esterification
with the appropriate symmetrical anhydride, according
to the general procedure a0. The resulting loading degree
was determined by UV spectrophotometric analysis
using the general procedure a00. The resin was then sub-
mitted to several coupling-deprotection cycles to build
the protected linear peptides as precursors of the cyclic
analogues. All the Fmoc-protected amino acids were
activated by hydroxybenzotriazole/O-(benzotriazol-1-
To this end, we prepared by a combination of solution-
and solid-phase synthesis (Scheme 1) six more simplified
analogues (2–7) (Fig. 1) that from a structural viewpoint
can be divided in two groups: the first series of com-
pounds 2–4 share a common peptidic portion composed
by alanine, D-tryptophan, and phenylglycine while differ
for the variable-length open-chain linkers, in analogy
with the already reported compounds 8–10. In particu-
lar, in cyclopeptide 2 the b-alanine is joined together
with 5-aminopentanoic acid to the tripeptide, affording
a 19-membered macrocycle. In the analogue 3, a residue
of 6-aminohexanoic acid was employed as a spanned
chain to form a 16-membered cyclopeptide, and in the
analogue 4 an 8-aminooctanoic acid was used to connect
the N- and C-termini of our tripeptidic segment, thus
generating an 18-membered macrocycle. As concerning
the analogue 5, the polyketide-mimetic fragment com-
posed of b-alanine and 5-aminopentanoic acid is hooked
to a tripeptide moiety formed by alanine, D-tryptophan,
and a-tyrosine. The second series of analogues is based
on a more incisive modification at the polyketide level:
both compounds share, in fact, the same 3-(2-hydroxy-
ethyl)-phenoxyacetic acid spacer, synthesized in turn in
three steps from commercially available 3-(2-hydroxy-
ethyl)-phenol, to join the tripeptide Ala-D-Trp-Tyr (ana-
logue 6) and the Ala-D-Trp-Val peptidic moiety
(analogue 7), respectively. The choice of this peculiar
linker was made in an attempt of mimicking the geom-
etry of the central portion of the x-hydroxy-4-octenoic
acid polyketide found in jaspamide with a somewhat
yl)-N,N,N0,N0-tetramethyluronium
hexafluorophos-
phate (HOBt/HBTU) in the presence of N-meth-
ylmorpholine (NMM), as described in the general
procedure c; the progress of the amino acid coupling
was checked through the Kaiser and TNBS tests. 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 the general procedure b. After
each linear peptide was obtained, the Fmoc-protecting
group was removed from the N-terminal residue and
the protected peptide was cleaved from the 2-ClTrt resin
by using a 2:2:6 acetic acid/2,2,2-trifluoroethanol/dichlo-
romethane (AcOH/TFE/DCM) solvent mixture, accord-
ing to the procedure d (analogues 2–4 and 6–7); when
using the NovaSynꢁTGA resin (analogue 5), the cleav-
age was performed by treatment with TFA 95% (general
procedure d0) with concurrent side-chain deprotection.
The cyclization reactions of the linear precursors were
allowed to proceed in solution using O-(7-aza-
benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexa-
fluorophosphate (HATU) or PyBop and DIEA in
DCM/DMF, following general procedure e or e0. Final-
ly, after removing the protecting groups, purification on
semipreparative RP-HPLC yielded the pure cyclopep-
tides 2–7.

S. Terracciano et al. / Bioorg. Med. Chem. 13 (2005) 5225–5239
5229
2.2. Biological activity
In these experiments, A-10 cells were treated with either
jaspamide (1), as a positive control for microfilament
depolymerizing activity, or micromolar amounts of
compounds 2–7. Jaspamide (1) very potently caused
actin aggregation in these cells. This could be seen with
doses as low as 0.85 nM, and at 23 nM, there was
pronounced aggregation. Unfortunately, none of the
jaspamide analogues seemed to affect either microfila-
ments or microtubules. Indeed, while binucleated cells
were consistently and clearly present in samples treated
with jaspamide, no cellular aberration was observed
with compounds 2–7. This further biological investiga-
tion suggests that the structural simplifications involved
in these synthetic analogues gave rise to loss of target
selectivity of the parent compound, implying an
alternative mechanism of action for the observed cyto-
toxicity. In fact, in an attempt to gather more data on
such a different mode of action underlying the cytotoxic
activity displayed by analogue 7 (and very likely by ana-
logues 8–10 as well), we investigated the induction of
apoptosis exerted by this compound on Jurkat cells
(Fig. 2), as already reported for jaspamide itself.16,17
Biological evaluation of the synthetic compounds 2–7
against a minipanel of four cancer cell lines (T24, MCF-
7,15 NCI/ADR,15 and A-10) showed cell growth-inhibito-
ry activity with IC50 value ranging from 60 to 150 lg/mL
only for the analogue 7. Quite surprisingly, no significant
cytotoxicity toward any of the above cell lines was found
for the other compounds. From these preliminary results,
it is not easy to draw a clear-cut correlation between the
structural simplifications/modifications and the biologi-
cal activity. Nevertheless, a few observations can be
made. It seems that the substitution of b-Tyr with a-Tyr
or Phg produced a loss of activity which otherwise was re-
tained in the cycloval analogues (8–10) in which the b-Tyr
was replaced with Val. This consideration would be con-
firmed by the fact that the only active analogue (com-
pound 7) of the two series described herein contains a
valine in its tripeptidic portion; a comparison of the bio-
logical properties of 7 with those shown by the previously
described compound 10, another cycloval analogue dis-
playing the same macrocyclic size, suggests that the sub-
stitution of the 8-aminooctanoic acid with the less
flexible 3-(2-hydroxyethyl)-phenoxyacetic acid as polyke-
tide linker caused an improvement of the cytotoxicity.
Finally, with the aim of comparing the 3D features of
jaspamide with those of the simplified analogues 2–7,
their conformational properties in solution were thor-
oughly studied by restrained molecular mechanics
(MM) and molecular dynamics (MD) approaches using
NMR-derived constraints as input data to correctly
drive the calculations.
For the sake of completeness, we also assayed the six
new synthetic analogues on the cytoskeleton system.
Hence, their activity on microfilaments and microtu-
bules was examined in A-10 rat smooth-muscle cells.
Figure 2. Induction of Jurkat cell apoptosis by compound 7. Jurkat cells (1 · 106/mL) were incubated in 10% FCS-RPMI medium, in the presence of
100 lM of compound 7 and/or the indicated concentrations of recombinant TRAIL, for 24 h. Then, hypodiplody was analyzed by propidium iodide
incorporation in permeabilized cells and flow cytometry.18

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S. Terracciano et al. / Bioorg. Med. Chem. 13 (2005) 5225–5239
2.3. Conformational analysis of analogues 2–7
the most intense and significative ROESY cross-peak
volumes into internuclear distances. The so-obtained
distances were used as experimental restraints for
the successive molecular mechanics and dynamics
calculations.
2.3.1. NMR spectroscopy and molecular mechanics and
dynamics calculations. A detailed NMR analysis, based
on a set of homonuclear and heteronuclear correlation
2D experiments (see Section 3), was carried out on ana-
logues 2–7 to obtain the sequence-specific assignment of
A preliminary round of free molecular mechanics and
dynamics calculations was run at 500 K to properly mon-
itor the conformational space. Subsequently, MM and
MD calculations were driven at 300 K by using the
restraints collected during the NMR analysis, providing
1
the H and 13C resonances of the six cyclopeptides (see
Tables 1 and 2). Successively, a careful analysis of the
,
ROESY data, based on a distance calibration (rꢀ6
two-spin approximation), was performed, to convert
Table 1. 1H NMR data for analogues 2–7 (600 MHz, DMSO-d6)
2
3
4
5
6
7
Phg
Phg
Phg
Tyr
Tyr
Val
a
b
c
d
e
NH
5.30
a
b
c
d
e
NH
5.40
a
b
c
d
e
NH
5.23
a
b
c
d
e
NH
4.17
2.57, 2.94
a
b
c
d
e
NH
4.18
2.74, 2.90
a
b
3.88
1.92
0.77
0.80
8.12
7.52a
7.36a
7.32
7.34a
7.38a
7.32
7.41a
7.35a
7.30
c1-CH3
c2-CH3
NH
7.01a
6.60a
8.52
6.96
6.63
8.19
8.29
8.29
8.11
Trp
Trp
Trp
Trp
Trp
Trp
a
4.43
a
4.28
a
4.36
a
4.26
a
4.34
a
4.57
b
1-NH
2.90, 3.72
10.84
7.14
b
1-NH
2.94, 3.23
10.83
7.12
b
1-NH
3.02, 3.19
10.86
7.30
b
1-NH
2.68a
10.74
7.05
b
1-NH
2.77, 3.01
10.80
7.05
b
1-NH
2.87, 3.08
10.81
7.11
2
2
2
2
2
2
3
3
3
3
3
3
4
4
4
4
4
4
5
7.61
6.97
7.05
7.32
5
7.53
7.00
7.05
7.32
5
7.61
6.99
7.15
7.33
5
7.49
6.99
7.02
7.28
5
7.55
6.99
7.06
7.31
5
7.58
6.96
7.03
7.29
6
6
6
6
6
6
7
7
7
7
7
7
8
8
8
8
8
8
9
NH
9
NH
9
NH
9
NH
9
NH
9
NH
8.29
8.64
8.32
8.09
8.12
8.14
Ala
Ala
Ala
Ala
Ala
Ala
a
b
NH
4.17
0.97
8.16
a
b
NH
4.16
1.03
8.28
a
b
NH
4.30
0.92
8.03
a
b
NH
4.37
0.97
7.91
a
b
NH
4.29
1.06
8.26
a
b
NH
4.34
1.04
8.13
b-Ala
Ahx
Aoc
b-Ala
Hepab
Hepab
a
b
NH
2.28, 2.34
3.22, 3.42
7.31
a
b
c
d
e
NH
2.06, 2.20
1.30, 1.63
1.36, 1.44
1.41a
a
b
c
d
e
f
NH
2.00, 2.20
1.10a
a
b
NH
2.03, 2.43
3.04, 3.25
7.71
a
4.47, 4.58
6.74
a
4.47, 4.58
6.74
10
20
30
40
50
60
100
200
10
20
30
40
50
60
100
200
1.19a
1.40, 1.60
1.30a
5-Ava
a
b
c
d
NH
2.85, 3.42
7.38
5-Ava
a
b
c
d
NH
6.84
7.16
6.80
7.15
1.93, 2.15
1.21, 1.42
1.30a
2.82, 3.17
7.50
1.91, 2.03
1.43, 1.49
1.34a
6.77
2.85a
4.17, 4.31
6.75
2.82, 2.87
4.09, 4.40
2.99, 3.08
8.05
2.95, 3.06
7.42
a 2H.
b

S. Terracciano et al. / Bioorg. Med. Chem. 13 (2005) 5225–5239
Table 2. 13H NMR data for analogues 2–7 (600 MHz, DMSO-d6)
5231
2
3
4
5
6
7
Phg
Phg
Phg
Tyr
Tyr
Val
a
b
c
d
e
58.1
138.8
127.8
128.7
128.1
a
b
c
d
e
58.2
139.2
128.3
128.6
129.2
a
b
c
d
e
58.0
138.6
127.6
128.4
127.3
a
b
c
d
e
55.3
36.7
a
b
c
d
e
55.1
36.0
a
b
59.3
29.8
129.2
130.6
115.3
c1-CH3
c2-CH3
19.20
19.17
130.9
115.6
Trp
Trp
Trp
Trp
Trp
Trp
a
b
2
3
4
5
6
7
8
9
54.4
28.0
a
b
2
3
4
5
6
7
8
9
55.0
26.7
a
b
2
3
4
5
6
7
8
9
54.8
27.2
a
b
2
3
4
5
6
7
8
9
54.9
27.6
a
b
2
3
4
5
6
7
8
9
54.24
27.69
121.54
a
b
2
3
4
5
6
7
8
9
53.8
29.2
124.5
124.2
124.5
124.7
124.0
119.0
118.8
121.9
110.9
119.0
118.9
121.2
111.0
118.3
118.5
123.8
110.6
119.0
118.9
121.4
111.9
118.90
118.73
121.41
111.92
120.4
120.0
121.46
111.7
Ala
Ala
Ala
Ala
Ala
Ala
a
b
49.4
17.1
a
b
50.0
17.5
a
b
48.36
17.84
a
b
47.81
19.12
a
b
48.50
18.24
a
b
49.5
20.0
b-Ala
Ahx
Aoc
b-Ala
Hepaa
Hepaa
a
b
35.8
35.5
a
b
c
d
e
35.0
24.4
27.5
27.6
38.4
a
b
c
d
e
35.5
25.2
28.2
28.6
38.4
37.5
a
b
35.7
35.9
a
67.80
158.5
116.1
141.0
121.8
129.8
114.1
34.6
a
69.1
158.5
115.1
140.6
121.8
129.7
114.0
34.6
10
20
30
40
500
600
1
10
20
30
40
500
600
1
5-Ava
5-Ava
a
b
c
d
36.2
23.5
29.4
38.2
a
b
c
d
35.8
23.7
28.7
39.3
f
200
65.41
200
65.3
a
the final ensembles of structures representing analogues
2–7.
analogues 8–10 were inspected. Actually, the torsion
angle values found for those compounds were not in
agreement with a b-turn of type II, resulting in values
quite different from the corresponding angles observed
for jaspamide, suggesting, considering that analogues
8–10 do not affect the cytoskeleton, a crucial role of this
b-turn type II region for specifically targeting actin
microfilaments.
2.3.2. Conformational analysis on analogues 2–7. In a
previous study, we carried out a comparative structural
analysis of analogues 8–10 with respect to jaspamide to
gain insight into its possible molecular determinants
underlying the induced actin hyperpolymerization, and
hence to determine what may be the key elements of
the parent compound ultimately responsible for its
potent antiproliferative activity. To this end, we first
compared the three above-mentioned derivatives to the
X-ray structure of jaspamide. Subsequently, since jasp-
amide was reported to contain in its D-Trp-b-Tyr region
a b-turn of type II13 considered critical to its bioactivity,
the corresponding regions of the NMR structures of
In analogy with the above approach, we have also com-
pared the NMR solution structures of compounds 2–7
with the X-ray structure of jaspamide (Figs. 3–8). The
results indicate that among all of these simplified deriv-
atives, only compounds 5 and 7 reveal a good superim-
position of the backbone in the segment Ala-D-Trp, even
if a quite poor correlation is observed for the region in

5232
S. Terracciano et al. / Bioorg. Med. Chem. 13 (2005) 5225–5239
Figure 3. Superposition of the NMR solution structure of analogue 2
(gray) and the X-ray structure of jaspamide (black).
Figure 6. Superposition of the NMR solution structure of analogue 5
(gray) and the X-ray structure of jaspamide (black).
Figure 7. Superposition of the NMR solution structure of analogue 6
(gray) and the X-ray structure of jaspamide (black).
Figure 4. Superposition of the NMR solution structure of analogue 3
(gray) and the X-ray structure of jaspamide (black).
Figure 5. Superposition of the NMR solution structure of analogue 4
(gray) and the X-ray structure of jaspamide (black).
Figure 8. Superposition of the NMR solution structure of analogue 7
(gray) and the X-ray structure of jaspamide (black).

S. Terracciano et al. / Bioorg. Med. Chem. 13 (2005) 5225–5239
5233
Table 3. Peptide backbone dihedral angles observed for analogues 2–
10 in the segment corresponding to the L-Ala-D-N-Me-2BrTrp-b-Tyr
region of jaspamide in comparison with the ones measured for the
X-ray structure of jaspamide (1x) and for the NMR conformational
ensemble 1i–1vi in the corresponding tripeptidic segment
and the polyketide portions in eliciting bioactivity. If
from one side it is likely true that the tripeptidic segment
of the molecule may well contain the main functional
determinants for exerting the bioactivity, it is also true
that the role of the polyketide portion cannot be under-
estimated to the mere function of a linker that has only
to join the two N- and C-termini of the tripeptide. In
other words, the importance of the polyketide portion
may have well been mistaken or misunderstood.
Compound
/
wi
/
i+1
wi+1
i
1x
1i
1ii
1iii
1iv
1v
1vi
8
ꢀ156.5
ꢀ162.0
ꢀ160.2
ꢀ156.6
ꢀ162.3
ꢀ154.6
ꢀ136.6
ꢀ83.0
+160.0
+166.7
+91.7
+109.4
+87.6
+90.4
+88.5
+84.2
+84.8
+90.3
+92.6
+86.8
+78.1
+88.9
+110.4
+59.3
+97.9
+154.8
+122.0
ꢀ26.7
ꢀ60.6
ꢀ70.6
+85.7
ꢀ65.6
+162.6
+161.1
+86.3
ꢀ71.3
ꢀ61.2
3. Experimental section
ꢀ92.0
+140.9
+105.2
+101.2
+142.3
+130.9
+70.8
ꢀ141.1
ꢀ117.5
ꢀ135.2
ꢀ122.8
ꢀ69.2
3.1. General experimental procedures
9
ꢀ89.9
10
2
ꢀ98.6
All the NMR spectra (1H, HMBC, HSQC, TOCSY,
COSY, and ROESY with tmix = 0.4 s) were recorded
on a Bruker Avance DRX600, on a Bruker Avance
300 MHz, or on a Bruker DRX 400 spectrometers at
T = 298 K. The compounds (2–7) were dissolved in
0.5 mL of 99.95% DMSO-d6 (Carlo Erba, 99.95 Atom
% D) (1H, d = 2.50 ppm; 13C, d = 39.5 ppm). The
NMR data were processed on a Silicon Graphic Indigo
2 workstation using UXNMR software.
ꢀ89.5
3
ꢀ81.4
4
ꢀ100.1
ꢀ146.40
ꢀ73.5
+47.0
5
+137.8
ꢀ31.0
+101.6
ꢀ138.7
ꢀ105.0
ꢀ130.2
6
7
+69.2
which the b-Tyr is replaced by a a-Tyr or a L-Val,
respectively.
Electrospray mass spectrometry (ES-MS) was per-
In Table 3, the / and w dihedral angles of analogues 2–7
describing the backbone torsions of the L-Ala-D-Trp-L-
Val region are reported and compared to the corre-
sponding angles measured for jaspamide in its X-ray
structure and in its NMR-derived conformational
ensemble proposed by Inman and Crews.19 Angles
`
formed on a LCQ DECA TermoQuest (San Jose, Cali-
fornia, USA) mass spectrometer.
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 was performed on a Jupiter C-18 column
/
i+1
and wi+1, characteristic of the D-Trp-b-Tyr region
˚
of jaspamide, are indeed reminiscent of a b-turn of type
II, being around +90ꢂ and 0ꢂ, respectively. In analogues
2–7, the angles /i+1 are found in a large range, from
+59.3 of 4 to 154.8 of 6, and only compounds 2, 3,
and 5 present an angle of about 90ꢂ; the little superim-
posibility in the conformations of these analogues is also
confirmed by the values of angles wi+1, which range
from +47.0ꢂ of analogue 4 to ꢀ138.7ꢂ of analogue 5.
These values are not in agreement with a b-turn of type
II and, in addition, they are quite different from the cor-
responding angle observed for jaspamide.
(250 · 4.60 mm, 5 lm, 300 A; 250 · 10.00 mm, 10 lm,
300 A, respectively).
˚
All starting materials, reagents, and solvents were com-
mercially available and used without further purifica-
tion. Unless specified, solvents were of reagent grade;
they were purchased from Aldrich, Fluka, and Carlo
Erba. DCM and DMF used for solid-phase reactions
˚
were synthesis grade (dried over activated 4 A molecular
sieves). H2O and CH3CN were of HPLC grade. 2-Chlo-
rotritylchloride resin (100–200 mesh), 1% DVB, (ClTrt-
Cl, loading level: 1.04 and 1.4 mmol/g), NovaSynꢁ TGA
resin (loading level: 0.24 mmol/g), Fmoc-Ala-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-
Val-OH, DCC, DMAP, HOBt, HBTU, and PyBop were
purchased from Novabiochem. Fmoc-b-Ala-OH, Fmoc-
5-Ava-OH, Fmoc-e-Ahx-OH, Fmoc-8-Aoc-OH, and
Fmoc-Phg-OH were obtained from Neosystem. HATU
was purchased from Fluka. Solid-phase peptide synthe-
ses, using the Fmoc-t-Bu strategy, were carried out on a
polypropylene ISOLUTE SPE column on a VAC MAS-
TER system, a manual parallel synthesis device pur-
chased from Stepbio.
Overall, such comparative analysis accounts for the
rather poor bioactivity profile shown by these simplified
analogues, emphasizing how small must be the region of
chemical space, both in terms of geometrical/conforma-
tional features and allowed functional groups, in which
macrocycles of this type are still endowed with the same
bioactivity displayed by the parent compound. This con-
sideration would in turn discourage further attempts of
producing additional simplified molecules of this kind.
However, to be fair and to put under the right perspec-
tive such unsuccessful (at least from a pharmaceutical
viewpoint) attempts, one has also to take into account
that no 3D model at the atomic level or any other chem-
ical information is yet available on the forces intervening
in the interaction between jaspamide and its biological
target. A final consideration that emerges from the
whole of our model studies and structural comparisons
has to do with the relative importance of the peptide
3.2. Computational details
A conformational search was performed for all the com-
pounds by: (1) a step of free molecular dynamics at high
temperature (500 K, 5.0 ps); (2) a round of restrained
molecular dynamics and minimization at high tempera-

5234
S. Terracciano et al. / Bioorg. Med. Chem. 13 (2005) 5225–5239
ture (500 K, 5.0 ps), the restraints were obtained using a
distance calibration (rꢀ6, two-spin approximation) to
convert the most intense and significant ROESY cross-
peak volumes into internuclear distances; and (3) a final
step of restrained molecular dynamics and minimization
at 300 K (5.0 ps).
(b) Fmoc deprotection. 20% piperidine in DMF (3 mL,
1 · 1.5 min), 20% piperidine in DMF (3 mL,
1 · 10 min); washings in DMF 2 · 3 mL, DCM
2 · 3 mL, and DMF 2 · 3 mL (1.5 min each).
(c) Peptide coupling conditions. The coupling reaction
was promoted by a HOBt/HBTU in DMF coupling pro-
tocol: Fmoc-amino acid (3–4 equiv), HOBt (3–4 equiv),
HBTU (3–4 equiv), and NMM (4–5 equiv) were stirred
under N2 in 2.5 mL of DMF for 2 h. After each cou-
pling, washings were carried out with DMF (3 mL,
3 · 1.5 min) and DCM (3 mL, 3 · 1.5 min).
During the simulations, the consistent-valence force
field (CVFF)20 was applied. The effect of the solvent
(DMSO) was incorporated in the calculations by consid-
ering it as a continuous dielectric medium, characterized
by a dielectric constant of 47. Minimizations and molec-
ular dynamics simulations were performed in the Dis-
cover module of Insight II.21
(d) Cleavage (ClTrt-Cl). 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 res-
in). Then, the resin was filtered off and washed with neat
cleavage mixture (3 mL, 3 · 1.5 min). After addition of
hexane (15 times volume) to remove acetic acid as an azeo-
trope, the filtrate was concentrated and lyophilized.
3.3. General procedures for the synthesis of compounds 2–7
(a) Loading of the resin (ClTrt-Cl). The ClTrt-Cl resin
was placed in a 25 mL polypropylene ISOSOLUTE syr-
inge on a VAC MASTER system, swollen in 3 mL of
DMF for 1 h, and then washed with 2 · 3 mL of DCM.
(d0) Cleavage (NovaSynꢁ TGA). The dried peptide resin
was treated for 1 h, under stirring, with the following
cleavage mixture: TFA/H2O (95:5; 10 lL · 1 mg of resin).
Then, the resin was filtered off andwashed with neat cleav-
age mixture (3 mL, 3 · 1.5 min). The filtrate volume was
reduced under a N2 stream and then added with a 20-fold
excess of cold diethyl ether. A white precipitate appeared
soon after ether addition and ether peptide mixture was
kept at 0 ꢂC for 2 h. The precipitate was collected by filtra-
tion through a 0.45 lm PTFE membrane filter (PALL
Corporation/Gelman Laboratory) installed on a glass
47 mm filter holder with a vacuum aspirator; the precipi-
tated peptide was washed with cold ether. The filter was
transferred to a glass vessel, the solid was removed by
HPLC-grade water (some drops of glacial acetic acid)
and ultrasounds, and the suspension was lyophilized.
A solution of Fmoc-AA-OH (1 equiv) and DIEA (4
equiv) in 2.5 mL of dry DCM was added and the mix-
ture was stirred for 2 h with a N2 stream. The mixture
was then removed, and the resin was washed with 3·
DCM/MeOH/DIEA (17:2:1), and sequentially with the
following washing/treatments: DCM 3 · 3 mL, DMF
2 · 3 mL, and DCM 2 · 3 mL (1.5 min each).
(a0) Loading of the resin (NovaSynꢁ TGA). The resin was
placed into a 25 mL polypropylene ISOSOLUTE syringe
on a VAC MASTER system, swollen in 5 mL of DMF for
1 h. The symmetric anhydride of the first Fmoc-AA-OH
(10 equiv) was prepared by dissolving it in dry DCM
(12 mL) and DMF (few drops); DCC (5 equiv) was added
at 0 ꢂC and the mixture was stirred for 25 min. The reac-
tion mixture was filtered and the filtrate was evaporated
under reduced pressure to give the desired product. It
was dissolved in DMF and added to the swelled polymeric
support; DMAP (1 equiv) was added and the resulting
mixture was stirred for 4 h. The support was washed with
DMF 3 · 3 mL, DCM 3 · 3 mL, and Et2O (1.5 min each)
and then dried in vacuo over KOH.
(e) Cyclization. The cyclization step was performed in
solution at a concentration of 7.7 · 10ꢀ4 M with HATU
(2.0 equiv) and DIEA (2.5 equiv) in DCM. The solution
was stirred at 4 ꢂC for 1 h and then allowed to warm to
room temperature overnight. The solvent was removed
under reduced pressure.
(a00) Estimation of the level of first residue attachment.
The loading of the resin was determined by UV quanti-
fication of the Fmoc-piperidine adduct.
(e0) Cyclization. The cyclization step was performed in
solution at a concentration of 7.7 · 10ꢀ3 M with PyBop
(3.0 equiv) and DIEA (6.0 equiv) in DMF. The solution
was stirred at 4 ꢂC for 1 h and then allowed to warm to
room temperature overnight. The solvent was removed
under reduced pressure.
The assay was performed on duplicate samples: 0.4 mL
of piperidine and 0.4 mL of DCM were added to two
dried samples Fmoc amino acid-resin in two volumetric
flasks of 25 mL. The reaction was allowed to proceed for
30 min at rt and then 1.6 mL of MeOH was added and
the solutions were diluted to 25 mL volume with DCM.
A reference solution was prepared in a 25 mL volumet-
ric flask using 0.4 mL of piperidine, 1.6 mL of MeOH
and DCM to volume. The solutions were shaked and
the absorbance of the samples versus the reference solu-
tion was measured at 301 nm. The substitution level (ex-
pressed in millimoles of amino acid per gram of resin)
(f) Side-chain deprotection. Final deprotection was car-
ried 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 and character-
ized by ES-MS and NMR spectra.
3.4. Synthesis of analogues 2–4, 6, and 7
3.4.1. Anchoring of N-Fmoc-AA-OH to the resin [2a–4a,
6a, 7a]. This step was accomplished by using the general
procedure a described above. In the following are
was calculated from the equation: mmol/g = (A301
7800) · (25 mL/g of resin).
/

S. Terracciano et al. / Bioorg. Med. Chem. 13 (2005) 5225–5239
5235
reported the amounts, loading levels, and other experi-
mental details employed.
3.5.2.1. [2b]. First coupling: Fmoc-5-Ava-OH
(154.0 mg, 0.45 mmol), HOBt (69.4 mg, 0.45 mmol),
HBTU (172.0 mg, 0.45 mmol), and NMM (66.8 lL,
0.60 mmol). After incorporation of Fmoc-5-Ava-OH,
the Fmoc-protecting group was removed according to
the general procedure b.
To the ClTrt-Cl resin (427.9 mg, 1.04 mmol/g loading le-
vel, 2a; 462.7 mg, 1.04 mmol/g loading level, 3a; 442.6 mg,
1.04 mmol/g loading level, 4a; 319.0 mg, 1.40 mmol/g
loading level, 6a; and 383.0 mg, 1.40 mmol/g loading
level, 7a) were added the corresponding C-terminal N-a-
Fmoc-AA-OH (N-a-Fmoc-b-Ala-OH: 53.2 mg, 0.17 mmol,
2a; Fmoc-e-Ahx-OH: 65.3 mg, 0.18 mmol, 3a; Fmoc-8-
Aoc-OH: 67.1 mg, 0.17 mmol, 4a; Fmoc-Tyr(tBu)-OH:
59.7 mg, 0.13 mmol, 6a; and Fmoc-Val-OH: 52.5 mg,
0.15 mmol, 7a) in DCM (2.5 mL) and DIEA (118.4 lL,
0.68 mmol, 2a; 125.4 lL, 0.72 mmol, 3a; 122.6 lL,
0.70 mmol, 4a; 90.5 lL, 0.52 mmol, 6a; and 106.6 lL,
0.61 mmol, 7a), in order to obtain a lower substitution
level. The reaction was quenched with DCM/MeOH/
DIEA (17:2:1), the resin was then filtered, washed, and
dried under vacuum over KOH for 24 h according to
the procedure a. The substitution level of Fmoc-AA-O-
ClTrt resins, determined spectrophotometrically by
Fmoc cleavage, following the protocol a00 outlined in gen-
eral procedures, was: 0.38 mmol/g (2a), 0.33 mmol/g (3a),
0.31 mmol/g (4a), 0.32 mmol/g (6a), and 0.33 mmol/g (7a).
Second coupling: Fmoc-Phg-OH (169.4 mg, 0.45 mmol),
HOBt (69.4 mg, 0.45 mmol), HBTU (172.0 mg,
0.45 mmol), and NMM (66.8 lL, 0.60 mmol), followed
by N-a-deprotection.
Third coupling: Fmoc-D-Trp(Boc)-OH (238.8 mg,
0.45 mmol), HOBt (69.4 mg, 0.45 mmol), HBTU
(172.0 mg, 0.45 mmol), and NMM (66.8 lL, 0.60 mmol),
followed by N-a-deprotection.
Fourth coupling: Fmoc-Ala-OH (141.2 mg, 0.45 mmol),
HOBt (69.4 mg, 0.45 mmol), HBTU (172.0 mg,
0.45 mmol), and NMM (66.8 lL, 0.60 mmol), followed
by N-a-deprotection.
3.5.2.2. [3b]. First coupling: Fmoc-Phg-OH (209.0 mg,
0.56 mmol), HOBt (86.0 mg, 0.56 mmol), HBTU
(212.4 mg, 0.56 mmol), and NMM (77.0 lL, 0.70 mmol),
followed by N-a-deprotection accomplished with the
general procedure b.
3.5. Synthesis of analogue 5
3.5.1. Anchoring of N-Fmoc-AA-OH to the resin [5a].
This step was accomplished by using the general proce-
dure a0 described above. In the following are reported
the amounts, loading levels, and other experimental
details employed.
Second coupling: Fmoc-D-Trp(Boc)-OH (294.8 mg,
0.56 mmol), HOBt (86.0 mg, 0.56 mmol), HBTU
(212.4 mg, 0.56 mmol), and NMM (77.0 lL, 0.70 mmol),
followed by N-a-deprotection.
Third coupling: Fmoc-Ala-OH (174.3 mg, 0.56 mmol),
HOBt (86.0 mg, 0.56 mmol), HBTU (212.4 mg,
0.56 mmol), and NMM (77.0 lL, 0.70 mmol), followed
by N-a-deprotection.
To the N-a-Fmoc-b-Ala-OH (532.3 mg, 1.71 mmol) in
DCM (12 mL) and DMF (500 lL) was added at 0 ꢂC
DCC (176.4 mg, 0.85 mmol) in DCM (1 mL) and the mix-
ture was stirred for 25 min. The reaction mixture was
filtered and the filtrate was evaporated under reduced
pressure. The symmetric anhydride was dissolved in
DMF (4 mL) and added to the swelled NovaSynꢁ TGA
resin (714.0 mg, 0.24 mmol/g loading level); DMAP
(21.3 mg, 0.171 mmol) was added and the resulting mix-
ture was stirred for 4 h according to the procedure a0.
The substitution level of Fmoc-b-Ala-O-NovaSynꢁ
TGA resin, determined spectrophotometrically by Fmoc
cleavage, following the protocol a00 outlined in general
procedures, was 0.20 mmol/g (5a).
3.5.2.3. [4b]. First coupling: Fmoc-Phg-OH (179.2 mg,
0.48 mmol), HOBt (73.5 mg, 0.48 mmol), HBTU
(182.1 mg, 0.48 mmol), and NMM (66.0 lL, 0.60 mmol),
followed by N-a-deprotection according to the general
procedure b.
Second coupling: Fmoc-D-Trp(Boc)-OH (253.0 mg,
0.48 mmol), HOBt (73.5 mg, 0.48 mmol), HBTU
(182.1 mg, 0.48 mmol), and NMM (66.0 lL, 0.60 mmol),
followed by N-a-deprotection.
3.5.2. Synthesis of linear peptide on the solid support [2b–
7b]. The assembly of the linear peptides was performed
manually, in the C ! N direction, adopting the Fmoc
protection scheme and using HOBt/HBTU as activation
reagents. After swelling Fmoc-AA-O-resin in DMF
(3 mL, 1 h), the Fmoc-protecting group was removed by
treatment with 20% piperidine in DMF according to the
general procedure b.
Third coupling: Fmoc-Ala-OH (149.5 mg, 0.48 mmol),
HOBt (73.5 mg, 0.48 mmol), HBTU (182.1 mg,
0.48 mmol), and NMM (66.0 lL, 0.60 mmol), followed
by N-a-deprotection.
3.5.2.4. [5b]. First coupling: Fmoc-5-Ava-OH
(175.1 mg, 0.51 mmol), HOBt (78.5 mg, 0.51 mmol),
HBTU (194.6 mg, 0.51 mmol), and NMM (75.2 lL,
0.68 mmol), followed by N-a-deprotection according to
the general procedure b.
After the N-a-Fmoc deprotection, each amino acid was
introduced by a single coupling step, using a 3- to 4-fold
excess of the Fmoc amino acid and of the activation re-
agents, following the coupling protocol c. The Kaiser or
TNBS test was used to assess coupling efficiency.
Second coupling: Fmoc-Tyr(t-Bu)-OH (235.7 mg,
0.51 mmol), HOBt (78.5 mg, 0.51 mmol), HBTU

5236
S. Terracciano et al. / Bioorg. Med. Chem. 13 (2005) 5225–5239
(194.6 mg, 0.51 mmol), and NMM (75.2 lL, 0.68 mmol),
followed by N-a-deprotection.
3.5.3.1. [2c]. The HPLC analysis showed a main peak
at tR 18.0 min; ES-MS: m/z 679.1 [M+H]+, 701.4
[M+Na]+. The minor peak eluting at tR 17.0 min was
shown to contain a mixture of 2c along with a diastereo-
isomeric compound.
Third coupling: Fmoc-D-Trp(Boc)-OH (270.1 mg, 0.51
mmol), HOBt (78.5 mg, 0.51 mmol), HBTU (194.6 mg,
0.51 mmol), and NMM (75.2 lL, 0.68 mmol), followed
by N-a-deprotection.
3.5.3.2. [3c]. The HPLC analysis showed a main peak
at tR 20.1 min; ES-MS: m/z 622.2 [M+H]+. The minor
peak eluting at tR 18.6 min was shown to contain a mix-
ture of 3c along with a diastereoisomeric compound.
Fourth coupling: Fmoc-Ala-OH (159.7 mg, 0.51 mmol),
HOBt (78.5 mg, 0.51 mmol), HBTU (194.6 mg,
0.51 mmol), and NMM (75.2 lL, 0.68 mmol), followed
by N-a-deprotection.
3.5.3.3. [4c]. The HPLC analysis showed a main peak
at tR 20.7 min; ES-MS: m/z 650.1 [M+H]+, 672.1
[M+Na]+. The minor peak eluting at tR 19.8 min was
shown to contain a mixture of 4c along with a diastereo-
isomeric compound.
3.5.2.5. [6b]. First coupling: Fmoc-D-Trp(Boc)-OH
(195.0 mg, 0.37 mmol), HOBt (56.6 mg, 0.37 mmol),
HBTU (140.3 mg, 0.37 mmol), and NMM (50.7 lL,
0.46 mmol), followed by N-a-deprotection according to
the general procedure b.
3.5.3.4. [6c]. The HPLC analysis showed a main peak
at tR 25.21 min; ES-MS: m/z 773.0 [M+H]+, 795.2
[M+Na]+, 811.3 [M+K]+.
Second coupling: Fmoc-Ala-OH (115.2 mg, 0.37 mmol),
HOBt (56.6 mg, 0.37 mmol), HBTU (140.3 mg,
0.36 mmol), and NMM (50.7 lL, 0.45 mmol), followed
by N-a-deprotection.
3.5.3.5. [7c]. The HPLC analysis showed a main peak
at tR 22.39 min; ES-MS: m/z 653.2 [M+H]+, 675.3
[M+Na]+, 691.3 [M+K]+.
Third coupling: Hepa (80.0 mg, 0.41 mmol), HOBt
(58.7 mg, 0.38 mmol), HBTU (137.8 mg, 0.36 mmol),
and NMM (50.7 lL, 0.46 mmol).
3.5.4. Cleavage from the resin and side-chains depro-
tection [5c]. After chain assembly, the partially pro-
tected peptide was deprotected from Boc and
cleaved from the resin according to the general pro-
cedure d0.
3.5.2.6. [7b]. First coupling: Fmoc-D-Trp(Boc)-OH
(252.8 mg, 0.48 mmol), HOBt (73.5 mg, 0.48 mmol),
HBTU (182.1 mg, 0.48 mmol), and NMM (66.0 lL,
0.60 mmol), followed by N-a-deprotection according
to the general procedure b.
The crude product (89.1 mg) was identified as 5c, NH2-
Ala-Tyr-5-Ava-b-Ala-COOH, on the basis of ES-MS
(m/z 609.2 [M+H]+, 631.1 [M+Na]+, and 647.0
1
Second coupling: Fmoc-Ala-OH (149.4 mg, 0.48 mmol),
HOBt (73.5 mg, 0.48 mmol), HBTU (182.1 mg,
0.48 mmol), and NMM (66.0 lL, 0.60 mmol), followed
by N-a-deprotection.
[M+K]+) and H NMR experiments.
3.5.5. Synthesis of the cyclopeptides [2–7d], and final
deprotection to obtain 2–7. The cyclization step was
accomplished, for each peptide, by following the proce-
dure e (2–4d) or e0 (6 and 7d) described above.
Third coupling: Hepa (115.0 mg, 0.58 mmol), HOBt
(73.5 mg, 0.48 mmol), HBTU (182.1 mg, 0.48 mmol),
and NMM (66.0 lL, 0.60 mmol).
3.5.5.1. [2d]. The crude linear peptide (117.0 mg,
0.17 mmol) was dissolved in DCM (220.0 mL) with
HATU (128.0 mg, 0.34 mmol, 1.98 equiv) and DIEA
(74.0 lL, 0.43 mmol, 2.5 equiv). The solution was stirred
for 1 h on an ice bath and then the mixture was allowed
to warm up at room temperature overnight. The cycliza-
tion reaction was monitored via HPLC and ES-MS
spectra. After 20.0 h, the solvent was removed under
reduced pressure.
3.5.3. Cleavage of the protected peptide from the resin [2–
4c, 6–7c]. After chain assembly, the partially protected
peptide was cleaved from the resins according to the
general procedure d.
The dried peptide resin was treated for 2 h with the
cleavage mixture AcOH/TFE/DCM (2:2:6) under stir-
ring, followed by an additional treatment with fresh
reagent (3 mL, 3 · 1.5 min). Then, the resin was removed
by filtration and the filtrate was condensed, lyophilized,
and characterized by analytical RP-HPLC and ES-MS.
Side-chain deprotection was carried out with TFA/H2O/
TIS 95:2.5:2.5 (100 lL · 1 mg of resin) for 1 h under
stirring. The cleavage mixture was evaporated and
lyophilized, yielding 254.0 mg of crude cyclopeptide. A
portion of the crude cyclopeptide (160.0 mg) was puri-
The crude product (117.0 mg 2c; 100.0 mg 3c; 98.0 mg
4c; 100.0 mg 6c; and 115.0 mg 7c) was analyzed by
RP-HPLC Jupiter C-18 column (250 · 4.60 mm, 5 lm,
fied by semipreparative RP-HPLC on a Jupiter C-18
˚
column (250 · 10.00 mm, 10 lm, 300 A), using
a
˚
300 A) using the following gradient: from 5% B to
55 min gradient from 10:90 to 45:55 of CH3CN/H2O
(each containing 0.1% TFA) at flow rate of 4 mL/min
and detecting at 250 nm. The HPLC purification yielded
the analogue 2, c-[Ala-Trp-Phg-5-Ava-b-Ala], as a
yellow oil (17.1 mg; tR = 33.10 min; ES-MS, m/z
100% B over 30 min at flow rate of 1 mL/min. The bina-
ry solvent system (A/B) was as follows: 0.1%
TFA in water (A) and 0.1% TFA in acetonitrile (B).
The absorbance was detected at 240 nm.

S. Terracciano et al. / Bioorg. Med. Chem. 13 (2005) 5225–5239
5237
561.1 [M+H]+, 583.3 [M+Na]+, and 599.2 [M+K]+;
C30H36N6O5).
analogue 5; c-[Ala-Trp-Tyr-5-Ava-b-Ala] (10.7 mg;
tR = 16.09 min; ESI-MS, m/z 591.3 [M+H]+, 613.4
[M+Na]+; and 629.4 [M+K]+; C31H38N6O6).
3.5.5.2. [3d]. The crude linear peptide (100.0 mg,
0.16 mmol) was dissolved in DCM (205.0 mL) with
HATU (122.0 mg, 0.32 mmol, 1.98 equiv) and DIEA
(70.5 lL, 0.40 mmol, 2.5 equiv). The solution was
stirred for 1 h on an ice bath and then the mixture
was allowed to warm up at room temperature over-
night. The cyclization reaction was monitored via
HPLC and ES-MS spectra. After 20 h, the solvent
was removed under reduced pressure. Side-chain depro-
tection was accomplished by using the general proce-
dure f. The reaction mixture was evaporated and
lyophilized, yielding 217.6 mg of crude cyclopeptide.
3.5.5.5. [6d]. The crude linear peptide (100.0 mg,
0.13 mmol) was dissolved in DMF (17.3 mL) with PyBop
(210.7 mg, 0.40 mmol, 3.0 equiv) and DIEA (141.0 lL,
0.81 mmol, 6.0 equiv). The solution was stirred for 1 h
on an ice bath and then the mixture was allowed to warm
up at room temperature overnight. The cyclization reac-
tion was monitored via HPLC and ESI-MS spectra. After
24 h, the solvent was evaporated under reduced pressure.
Side-chain deprotection was carried out by using the gen-
eral procedure f. The reaction mixture was evaporated
and lyophilized; a portion of this crude cyclodepsipeptide
(130.0 mg) was then purified by semipreparative RP-
HPLC on a Jupiter C-18 column (250 · 10.00 mm,
A
was purified by semipreparative RP-HPLC on a Jupiter
portion of this crude cyclopeptide (100.0 mg)
˚
˚
C-18 column (250 · 10.00 mm, 10 lm, 300 A), using a
10 lm, 300 A), using a 85 min gradient from 10:90 to
57 min gradient from 10:90 to 45:55 of CH3CN/H2O
(each containing 0.1% TFA) at flow rate of 4 mL/min
and detecting at 250 nm. The purification yielded, as
a pale yellow oil, the analogue 3, c-[Ala-Trp-Phg-e-
Ahx] (9.0 mg; tR = 38.11 min; ESI-MS, m/z 504.2
[M+H]+, 526.3 [M+Na]+; C28H33N5O4).
70:30 of CH3CN/H2O (each containing 0.1% TFA) at
flow rate of 4 mL/min and detecting at 260 nm. The
HPLC purification yielded, as yellow oil, the analogue
6; c-[Hepa-Ala-Trp-Tyr] (7.2 mg; tR = 41.19 min; ESI-
MS, m/z 599.3 [M+H]+, 621.4 [M+Na]+; C33H34N4O7).
3.5.5.6. [7d]. The crude linear peptide (115.0 mg,
0.18 mmol) was dissolved in DMF (24.2 mL) with
PyBop (280.9 mg, 0.54 mmol, 3.0 equiv) and DIEA
(188.0 lL, 1.08 mmol, 6.0 equiv). The solution was
stirred for 1 h on an ice bath and then the mixture was
allowed to warm up at room temperature overnight.
The cyclization reaction was monitored via HPLC and
ESI-MS spectra. After 24 h, the solvent was evaporated
under reduced pressure. Side-chain deprotection was
carried out by using the general procedure f. The reac-
tion mixture was evaporated and lyophilized; a portion
of this crude cyclodepsipeptide (240.0 mg) was then
purified by semipreparative RP-HPLC on a Jupiter
3.5.5.3. [4d]. The crude linear peptide (98.0 mg,
0.15 mmol) was dissolved in DCM (199.0 mL) with
HATU (116.7 mg, 0.30 mmol, 1.98 equiv) and DIEA
(67.5 lL, 0.39 mmol, 2.5 equiv). The solution was stirred
for 1 h on an ice bath and then the mixture was allowed
to warm up at room temperature overnight. The cycliza-
tion reaction was monitored via HPLC and ESI-MS
spectra. After 20 h, the solvent was evaporated under re-
duced pressure. Side-chain deprotection was carried out
by using the general procedure f. The reaction mixture
was evaporated and lyophilized, yielding 214.4 of crude
cyclopeptide. A portion of this crude cyclopeptide
(130.0 mg) was then purified by semipreparative RP-
HPLC on a Jupiter C-18 column (250 · 10.00 mm,
˚
C-18 column (250 · 10.00 mm, 10 lm, 300 A), using a
60 min gradient from 10:90 to 70:30 of CH3CN/H2O
(each containing 0.1% TFA) at flow rate of 4 mL/min
and detecting at 260 nm. The HPLC purification yield-
ed, as yellow oil, the analogue 7; c-[Hepa-Ala-Trp-Val]
(7.0 mg; tR = 38.22 min; ESI-MS, m/z 535.3 [M+H]+,
557.4 [M+Na]+; and 573.4 [M+K]+; C29H34N4O6).
˚
10 lm, 300 A), using a 73 min gradient from 10:90 to
55:45 of CH3CN/H2O (each containing 0.1% TFA)
at flow rate of 4 mL/min and detecting at 250 nm.
The HPLC purification yielded, as a pale yellow oil,
the analogue 4; c-[Ala-Trp-Phg-8-Aoc] (10.3 mg; tR =
45.48 min; ESI-MS, m/z 532.3 [M+H]+, 554.4
[M+Na]+; C30H37N5O4).
3.5.6. Synthesis of 3-(2-hydroxyethyl)-phenoxyacetic acid
ethyl ester (11). To a stirred solution of commercially
3.5.5.4. [5]. The crude linear peptide (87.5 mg,
0.14 mmol) was dissolved in a DCM/DMF mixture
(199.0 mL/50.0 mL) with HATU (105.4 mg, 0.28 mmol,
1.98 equiv) and DIEA (61.0 lL, 0.35 mmol, 2.5 equiv).
The solution was stirred for 1 h on an ice bath and then
the mixture was allowed to warm up at room tempera-
ture overnight. The cyclization reaction was monitored
via HPLC and ESI-MS spectra. After 20 h, the reaction
mixture was evaporated and lyophilized, yielding 191.0
of crude cyclopeptide. A portion of this crude cyclopep-
tide (150.0 mg) was then purified by semipreparative
RP-HPLC on a Jupiter C-18 column (250 · 10.00 mm,
available
3-hydroxyphenethyl
alcohol
(0.640 g,
4.63 mmol) in 25 mL of acetone, potassium carbonate
(3.20 g, 23.1 mmol) and, after 10 min, ethylbromoace-
tate (0.62 mL, 0.56 mmol) were added. The resulting
mixture was refluxed overnight. Chloroform (20 mL)
was added and potassium carbonate was filtered off
and repeatedly washed several times with chloroform.
The filtrate was concentrated in vacuo to afford 11
(1.01 g, 97%) as a pale yellow oil which was used in
the next step without further purification.
Rf = 0.75 (30% petroleum ether in ethyl acetate).
˚
10 lm, 300 A), using a 50 min gradient from 15:85 to
65:35 of CH3CN/H2O (each containing 0.1% TFA)
at flow rate of 4 mL/min and detecting at 250 nm.
The HPLC purification yielded, as a white solid, the
1H NMR (CDCl3, 400 MHz) d: 1.29 (3H, t, J = 7.1 Hz,
–CH2CH3); 2.83 (2H, t, J = 6.5 Hz, Ar–CH2CH2–OH);
3.83 (2H, t, J = 6.5 Ar–CH2CH2–OH); 4.26 (2H, q,

5238
S. Terracciano et al. / Bioorg. Med. Chem. 13 (2005) 5225–5239
Scheme 2. Synthetic scheme followed to obtain 3-(2-hydroxyethyl)-phenoxyacetic acid (12).
J = 7.1 Hz, –CH2CH3); 4.61 (2H, s, ArOCH2COOEt);
6.76 (1H, br d, J = 8.0 Hz, H-6); 6.80 (1H, br s, H-2);
6.86 (1H, br d, J = 8.0 Hz, H-4); 7.23 (1H, t like,
J = 8.0 Hz, H-5).
MEM (A-10 cells) medium containing 10% fetal bovine
serum and 50 lg/mL gentamicin at 37 ꢂC in an atmo-
sphere of 5% CO2 and 95% air. To determine the cyto-
toxicities of the test compounds, cells were plated into
96-well tissue culture plates at approximately 15% con-
fluency, and were allowed to attach and recover for
24 h. The cells were then treated in triplicate with vary-
ing concentrations of the test compound for 48 h, and
cell survival was assayed using the sulforhodamine B
(SRB) binding assay.22 The percentage of cell survival
was calculated as the percentage of SRB binding as com-
pared with control cultures.
13C NMR (CDCl3, 400 MHz) d: 14.1, 39.1, 61.3, 63.4,
65.3, 112.3, 115.6, 122.4, 129.6, 140.3, 158.0, 168.9.
3.5.7. Synthesis of 3-(2-hydroxyethyl)-phenoxyacetic acid
(12). To a stirred solution of 3-(2-hydroxyethyl)-phe-
noxyacetic acid ethyl ester (11) (1.01 g, 4.51 mmol) in
methanol (20 mL), a solution of potassium hydroxide
(0.280 g, 5.00 mmol) in water (10 mL) was added, and
the resulting mixture was stirred for 3 h. The reaction
mixture was diluted with water (10 mL) and was neu-
tralized with HCl (1 N). Methanol was evaporated in
vacuo, and the aqueous layer was extracted with diethyl
ether (3 · 20 mL). The combined organic layers were
washed with brine, dried over MgSO4, filtered, and
evaporated in vacuo to afford 3-(2-hydroxyethyl)-phe-
noxyacetic acid (12) (0.630 g, 0.321 mmol, 70%) as a
pale yellow oil (see Scheme 2).
3.6.3. Immunofluorescence assays. A-10 cells were grown
to near-confluency on glass coverslips in 24-well plates,
and then treated with the test compounds for 24 h.
Microtubules were incubated with monoclonal anti-b-
tubulin, followed by fluorescein-conjugated anti-mouse
IgG as previously described.23,24 In similar experiments,
microfilaments were incubated with monoclonal anti-b-
actin, followed by fluorescein-conjugated anti-mouse
IgG. Cytoskeletal structures were then visualized by
epi-fluorescence and digital images were recorded of
representative fields.
Rf = 0.11 (0.1% acetic acid in diethyl ether).
1H NMR (CDCl3, 400 MHz) d: 2.85 (2H, t, J = 6.4 Hz,
Ar–CH2CH2–OH); 3.86 (2H, t, J = 6.5 Hz, Ar–
CH2CH2–OH); 4.68 (2H, s, ArOCH2COOH); 6.79
(1H, br d, J = 8.0 Hz, H-6); 6.82 (1H, br s, H-2); 6.90
3.6.4. Analysis of hypodiploid (apoptotic) nuclei. The hu-
man lymphoblastoid cell line Jurkat was obtained from
American Type Culture Collection (ATCC, LGC Pro-
mochem, Middlesex, UK) and cultured in RPMI medi-
um supplemented with 10% heat-inactivated fetal calf
serum (FCS). Recombinant TRAIL was obtained from
Alexis Biochemicals, San Diego, CA. Apoptosis was
analyzed by propidium iodide incorporation in perme-
abilized cells and flow cytometry as described.18 Briefly,
cells (5 · 105) were washed in PBS and resuspended in
500 lL of a solution containing 0.1% sodium citrate,
0.1% Triton X-100, and 50 lg/mL propidium iodide
(Sigma Chemical Co., St. Louis, MO). Following incu-
bation at 4 ꢂC for 30 min in the dark, cell nuclei were
analyzed with a Becton–Dickinson FACScan flow
cytometer. Cellular debris was excluded from analysis
by raising the forward scatter threshold, and the DNA
content of the nuclei was registered on a logarithmic
scale. The percentage of the elements in the hypodiploid
region was calculated.
(1H, br d, J = 8.0 Hz, H-4); 7.26 (1H,
J = 8.0 Hz, H-5).
t like,
13C NMR (CDCl3, 400 MHz) d: 39.0, 63.4, 64.8, 112.5,
115.5, 122.7, 129.7, 140.4, 157.0, 172.2.
3.6. Biological tests
3.6.1. Materials. MCF-7 breast carcinoma cells and
NCI/ADR cells, an MDR line that overexpresses P-gly-
coprotein, were obtained from the Division of Cancer
Treatment of the National Cancer Institute, USA. T24
human bladder carcinoma (HTB-4) and A-10 rat aortic
smooth-muscle (CRL-1476) cells were from the Ameri-
can Type Culture Collection. Sulforhodamine B, anti-
bodies against b-tubulin (T-4026), and antibodies
against b-actin (A-2228) were obtained from Sigma
Chemical Company (St. Louis, MO). RPMI-1640, a-
MEM, and fetal bovine serum were from Gibco-BRL
(Grand Island, NY).
Acknowledgments
The University of Salerno and the Ministry of Instruc-
tion, University and Research (MIUR, Rome), are
gratefully acknowledged for financial support of this
3.6.2. Cytotoxicity assay. Cells were maintained in
RPMI-1640 (MCF-7, NCI/ADR, and T24 cells) or a-

S. Terracciano et al. / Bioorg. Med. Chem. 13 (2005) 5225–5239
5239
Fenteany, G.; Zhu, S. Curr. Top. Med. Chem. 2003, 3,
593–616.
project through the funds ex-60% and PRIN 2001, 2003
programs, respectively. The use of instrumental facilities
of the Competence Center for Diagnostics and Molecu-
lar Pharmaceutics, sponsored by Regione Campania
POR funds, is gratefully acknowledged. Finally, we
thank Prof. Caterina Turco and Ms. Rita Di Giacomo
(University of Salerno) for performing apoptosis assays
on Jurkat cells.
11. (a) McGrath, J. L.; Tardy, Y.; Dewey, C. F., Jr.; Meister,
J. J.; Hartwig, J. H. Biophys. J. 1998, 75, 2070–2078; (b)
Cramer, L. P. Curr. Biol. 1999, 9, 1095–1105.
12. Terracciano, S.; Bruno, I.; Bifulco, G.; Copper, J. E.;
Smith, C. D.; Gomez-Paloma, L.; Riccio, R. J. Nat. Prod.
2004, 67, 1325–1331.
13. Kahn, M.; Nakanishi, H.; Su, T.; Lee, J. H.; Johnson, M.
Int. J. Pept. Protein Res. 1991, 38, 324–334.
14. Bai, R.; Covell, D. G.; Liu, C.; Ghosh, A. K.; Hamel, E.
J. Biol. Chem. 2002, 277, 32165–32171.
References and notes
15. Fairchild, C. R.; Ivy, S. P.; Kao-Shan, C. S.; Whang-Peng,
J.; Rosen, N.; Israel, M. A.; Melera, P. W.; Cowan, K. H.;
Golsith, M. E. Cancer Res. 1987, 47, 5141–5148.
16. Posey, C. S.; Bierer, B. E. J. Biol. Chem. 1999, 274, 4259–
4265.
17. Odaka, C.; Sanders, M. L.; Crews, P. Clin. Diagn. Lab.
Immun. 2000, 7, 947–952.
18. Nicoletti, I.; Migliorati, G.; Pagliacci, M. C.; Grignani,
F.; Riccardi, C. A. J. Immunol. Methods 1991, 139, 271–
279.
1. 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.
2. Crews, P.; Manes, L. V.; Boehler, M. Tetrahedron Lett.
1986, 27, 2797–2800.
3. Braekman, J. C.; Daloze, D.; Moussiaux, B.; Riccio, R.
J. Nat. Prod. 1987, 50, 994–995.
4. Zampella, A.; Giannini, C.; Debitus, C.; Roussakis, C.;
DÕAuria, M. V. J. Nat. Prod. 1999, 62, 332–334.
5. Stingl, J.; Andersen, R. J.; Emerman, J. T. Cancer
Chemother. Pharmacol. 1992, 30, 401–406.
19. Inman, W.; Crews, P. J. Am. Chem. Soc. 1989, 11, 2822–
2829.
20. Dauber-Osguthorpe, P.; Roberts, V. A.; Osguthorpe, D.
J.; Wolff, J.; Genest, M.; Hagler, A. T. Proteins: Struct.
Funct. Genet. 1998, 4, 31–47.
21. Accelerys Inc. (US, UK) 9685 Scranton Rd., San Diego,
CA 92121-3752.
22. Skehan, P.; Storeng, P.; Scudiero, D.; Monks, A.;
McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.;
Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107–1112.
23. Smith, C. D.; Zhang, X.; Mooberry, S. L.; Patterson, G.
M.; Moore, R. E. Cancer Res. 1994, 54, 3779–3784.
24. Zhang, X.; Minale, L.; Zampella, A.; Smith, C. D. Cancer
Res. 1997, 57, 3751–3758.
6. 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.
7. Takeuchi, T.; Ara, G.; Sausville, E. A.; Teicher, B. Cancer
Chemother. Pharmacol. 1998, 42, 491–496.
8. Bubb, M. R.; Senderowicz, A. M.; Dunkan, K. L.; Korn,
E. D. J. Biol. Chem. 1994, 269, 14869–14871.
9. Bubb, M. R.; Spector, I.; Beyer, B.; Fosen, K. A. J. Biol.
Chem. 2000, 275, 5163–5170.
10. (a) Spector, I.; Breat, F.; Shochet, N.; Bubb, M.
Microsc. Res. Tech. 1999, 47, 18–37; (b) Jordan, M. A.;
Wilson, L. Curr. Opin. Cell Biol. 1998, 10, 123–130; (c)