Design and synthesis of FAJANU: A de novo C2 symmetric cyclopeptide family

Article abstract of DOI:10.1021/jm800047b

A novel cyclic peptide has been designed from several potent marine cytotoxic peptides, including IB-01212, luzopeptin, triostin, and thiocoraline. The FAJANU scaffold maintains C₂ symmetry, cyclic structure, and the construction of aromatic and aliphatic character at the N- and C-terminal extremes. A first six-member family was previously synthesized and evaluated biologically. Several analogues presented greater activity than IB-01212. Furthermore, on the basis of the most active candidate, we have performed a more exhaustive synthetic and structural analysis: (i) structure - activity relationship provided clues about the key elements in the framework, (ii) NMR assignment confirmed C₂ symmetry, and (iii) confocal images revealed its penetration and cellular localization.

Full text of DOI:10.1021/jm800047b

3194
J. Med. Chem. 2008, 51, 3194–3202
Design and Synthesis of FAJANU: a de Novo C2 Symmetric Cyclopeptide Family
Fayna Garcia-Martin,†,‡ Luis J. Cruz,*,†,| Ricard A. Rodriguez-Mias,†, Ernest Giralt,†,§ and Fernando Albericio*,†,‡,§
Institute for Research in Biomedicine, Barcelona Science Park, UniVersity of Barcelona, 08028 Barcelona, Spain, CIBER-BBN, Networking
Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, 08028 Barcelona, Spain, Department of Organic
Chemistry, UniVersity of Barcelona, 08028 Barcelona, Spain
ReceiVed January 17, 2008
A novel cyclic peptide has been designed from several potent marine cytotoxic peptides, including
IB-01212, luzopeptin, triostin, and thiocoraline. The FAJANU scaffold maintains C2 symmetry, cyclic
structure, and the construction of aromatic and aliphatic character at the N- and C-terminal extremes. A first
six-member family was previously synthesized and evaluated biologically. Several analogues presented greater
activity than IB-01212. Furthermore, on the basis of the most active candidate, we have performed a more
exhaustive synthetic and structural analysis: (i) structure-activity relationship provided clues about the key
elements in the framework, (ii) NMR assignment confirmed C2 symmetry, and (iii) confocal images revealed
its penetration and cellular localization.
Introduction
During recent years, several families of antitumoral cyclic
peptides with C2 symmetry have been isolated from marine
sources. All of these compounds show head-to-side chain
heterodetic (ester for Ser or thioester for Cys) bonds linking
the two backbones. Examples include the onchidin B,1,2
thiocoraline,3 triostin,4 IB-01212,5 sandramycin,6 and luzopeptin
A7 families. Furthermore, these peptides usually contain N-
methyl and D-amino acids in addition to other nonproteogenic
amino acids and, in some cases, aromatic heterocycles, a few
of which present a disulfide bridge, thereby converting them
into bicyclic peptides.8 Symmetric peptides with these charac-
teristics have also been isolated from terrestrial organisms such
as valinomycin, the related montanastatin, and korkormicin, all
Figure 1. Structure of IB-01212 (1) and its aza analogue (2).
of which are from the actinobacteria phylum.9
This collection of symmetric natural peptides presents a wide
range of biological activity. In some cases, symmetry has proved
to be suitable for interaction with highly symmetric complex
structures, as is the case of thiocoraline and triostin, which are
DNA intercalators. In other cases, symmetry has been considered
to be an instrument for the microorganisms themselves to
become more complex bioactive structures. Furthermore, in the
case of valinomycin, polar groups are oriented toward the center,
thereby allowing the delivery of selected ions through the cell
membrane.10 Syntheses of these peptides present an enormous
synthetic challenge, which can jeopardize the development of
drug discovery programs or even their industrial production.11–19
Here we explored a de novo C2-symmetric cyclopeptide family,
taking as the starting point the simplest cytotoxic IB-01212 (1)
and its more active analogue 2 (Figure 1),16,20 where both ester
bonds were replaced by amide ones.
Results and Discussion
Design, Synthesis, and Biological Screening of the New
FAJANU Peptide Family. Previous work by our group has
described the preparation of 1 analogues, where the substitution
of several amino acids using the same cyclic template, and the
presence of the amide bond instead of the ester bond to close
the cycle were examined.29 The result of that study was the
homodetic analogue 2, which was more stable than 1 and
showed a similar activity. In the present study, we designed a
new structure by increasing the macrocycle size and by adding
aromatic heterocycles with the purpose of improving antitumor
activity. The new structure shares the chemical feasibility of
IB-01212 analogues and the structural moieties relevant in
highly cytotoxic compounds such as triostin or thiocoraline.
NMe-Gly (Sar) a, a common amino acid in this kind of peptide,
* To whom correspondence should be addressed. Fax: 34934037126
(F.A.). E-mail: albericio@pcb.ub.us (F.A.).
†
Institute for Research in Biomedicine, Barcelona Science Park,
University of Barcelona.
‡
a Abbreviations: Abbreviations used for amino acids and the designations
of peptides follow the rules of the IUPAC-IUB Commission of Biochemical
Nomenclature in J. Biol. Chem. 1982, 247, 977-983. The following
additional abbreviations are used: aa, amino acid; Alloc, allyloxycarbonyl;
Boc, tert-butyloxycarbonyl; BPH, biphenyl-4-carboxylic acid; CHR, chromone
carboxylic acid; CTC, chlorotrityl chloride (Barlos) resin; Dab, diaminobu-
tiric acid; Dap, diaminopropyl carboxylic acid; DBU, 1,8-diazabicyclo-
[5.4.0]undec-7-ene; DIPEA, N,N-diisopropylethylamine; DIPCDI,
N′,N′′-diisopropylcarbodiimide; DKP, diketopiperazine; DMF, N,N-dim-
CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and
Nanomedicine.
§
Department of Organic Chemistry, University of Barcelona.
Present address: Department of Tumor Immunology, NCMLS 278,
|
Radboud University Nijmegen Medical Centre, Geert Grooteplein 26/28,
6525 GA Nijmegen, The Netherlands.
Present address: Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston,
Massachusetts 02115
10.1021/jm800047b CCC: $40.75
2008 American Chemical Society
Published on Web 05/08/2008

A NoVel Cyclopeptide: FAJANU
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3195
Figure 2. General structure of the FAJANU peptide family and first
analogues synthesized.
was used to increase the size of the cycle, taking into account that
the NMe group does not change the hydrogen bond map. In a first
attempt, quinaldic acid, a heterocycle analogue present in other
cytotoxic compounds, was incorporated as end-capping in order
to mimic the aromatic ring of the Phe presented in 1.
The new class of compounds, called FAJANU peptides,
shows the following characteristics: (1) eight L-amino acid
peptide backbone, (2) symmetric cyclic structure, (3) two
antiparallel peptide chains linked by a homodetic amide bond,
(4) NMe amino acids in the cyclic peptide backbone, and (5)
two pharmacophore moieties linked to N-terminus chains. To
explore the potential of the new FAJANU structures, a set of
six peptides (Figure 2, see full structure in Figure 4) were
synthesized and their biological activity was evaluated.
Synthesis of the FAJANU Peptide Family. The most
convenient way to synthesize this kind of compound is by a
combination of solid-phase and solution methods.21–23 Three
strategies can be used for this purpose (Scheme 1):
(i) stepwise solid-phase synthesis, but with cyclization and
introduction of the heterocycle in solution, (ii) total sequential
stepwise solid-phase synthesis of the linear precursor and
then cyclization performed in solution, and (iii) [4 + 4]
convergent approach, taking advantage of the symmetry of
the molecule, followed by cyclization and introduction of
the heterocycle in solution.
Figure 3. RP-HPLC profile (H2O-ACN 5:5 to 0:10 in 15 min) and
MALDI-TOF (m/z calcd for C60H84N12O10, 1132.6; found 1133.7 [M
+ H]+, 1155.7 [M + Na]+, 1171.7 [M + K]+) of 7.
paramount importance24 and is more crucial when the
synthesis of peptides containing NMe amino acids is targeted.
In FAJANU (3–8), the choice of NMe-Gly (2) as the
C-component at the cyclization and solid-phase start point
avoids the risk of racemization during cyclization and favors
the parallel synthesis of all six analogues, because diversity
Xxx (1) is introduced at the third step.
Stepwise elongation was performed following methodolo-
gies previously developed by our group for the synthesis of
other cyclic peptides.25–27 For the preparation of NMe-
containing peptides, the Fmoc strategy as temporary amino-
protecting group is more convenient than the Boc strategy.
In this case, the formation of a Boc-oxazolonium ion can
occur by decomposing into an N-carboxyanhydride derivative,
which could react and thereby lead to polymerization.28
Removal of the Fmoc group from secondary amines is more
difficult than from primary ones, thus an extra DBU treatment
was carried out after standard treatment with piperidine.29,30
The ꢀ-amino function of the Dap was also protected with
the Fmoc group, reserving the Boc for the protection of the
R-amino function, which had been removed before the
heterocycle was introduced in solution at the end. At this
point, the above-mentioned side-reaction cannot occur.
We chose the CTC (chlorotrityl chloride, Barlos)31 resin
as solid support for several reasons, the main ones being:
(i) its lability in very mild acid conditions (1% TFA, HFIP,
TFE),32 because the use of higher TFA conditions during
cleavage could cause the loss of the N-Ac-NMe amino acid
terminus and cleavage of the NMesNMe amino acid bond;33
(ii) the absence of racemization during the incorporation of
the first residue into the resin, because this is done through
nucleophilic substitution and not through the activation of
the carboxylic group required for anchoring to hydroxyl-based
resins; (iii) this resin minimizes the formation of diketopi-
perazine (DKP).34,35 This is of relevance in this case because
of the presence of several consecutive NMe amino acids,
This first FAJANU family 3 to 8 (3–8) (Figure 2) was
synthesized using the stepwise linear method with cyclization
and heterocycle introduction in solution (Scheme 1i). In the
preparation of all cyclic peptides, the choice of cyclization
point (the carboxylic component in the cyclization step should
be the starting residue in the solid-phase synthesis) is of
ethylformamide; Fmoc, 9-fluorenylmethoxycarbonyl; DMSO, dimethyl
sulfoxide; GI50, 50% growth inhibition; h, hour; HATU, 1-[bis(dimethy-
lamino)methylene]-1H-1,2,3-triazolo-[4,5-b] pyridinium hexafluorophos-
phate 3-oxide; HFIP, hexafluoroisopropanol; HOAt, 1-hydroxy-7-azaben-
zotriazole; HOBt, 1-hydroxybenzotriazole; HQX, hydroxy quinoline carboxylic
acid; MALDI, matrix-assisted laser desorption ionization; MeCN, aceto-
nitrile; MeOH, methanol; MS, mass spectrometry; 1OH-NPH, 1-hydroxy-
2-naphthoic acid; 3OH-NPH, 3-hydroxy-2-naphthoic acid; NMe-aa, N-me-
thylated amino acid; Orn, ornithine; PIC, 2-picolinic acid; pip, piperidine;
PyAOP, (1H-7-azabenzotriazol-1-yloxy)tripyrrolidin-1-ylphosphonium hexaflu-
orophosphate; PyBOP, (1-Hydroxyhydroxy-1H-benzotriazolato-O)tri-1-pyr-
rolidinylphosphorus hexafluorophosphate; pip, piperidine; PYR, pyrrole-
2-carboxylic acid; QNL, quinaldic acid; QNX, 2-quinoxalinecarboxylic acid;
RP-HPLC, reverse phase high performance liquid chromatography; SAR,
structure–activity relationship; SPPS, solid-phase peptide synthesis; SRB,
sulforhodamine B; TIS, triisopropyl silane; TFA, trifluoroacetic acid; TFE,
trifluoroethanol; TOF, time-of-flight. Amino acid symbols denote L-
configuration. All reported solvent ratios are expressed as v/v, unless
otherwise stated.

3196 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Garcia-Martin et al.
Scheme 1. Three Methods Used for the Preparation of FAJANU Cyclopeptides: (i) Solid and Solution Strategy, (ii) Linear Strategy,
and (iii) 4 + 4 Convergent Strategy
Table 1. Cytotoxic Activity of the FAJANU Family against Several Tumor Cell Lines (GI50 0.1µM)43
activity (GI50) in vitro
compound
structure
breast44 MDA-MB-231 10-7
lung A549 10-7
colon HT29 10-7
1
2
3
4
5
6
7
8
12.3
7.1
29.0
28.6
41.6
6.4
6.5
40.8
{[QNL-Dap(&1,2)-(Me)Phe-(Me)Gly-(Me)Leu(&2,1)]2}
{[QNL-Dap((&1,2)-Pro-(Me)Gly-(Me)Leu(&2,1)]2}
med>{[QNL-Dap(&1,2)-(Me)Ala-(Me)Gly-(Me)Leu(&2,1)]2}
{[QNL-Dap(&1,2)-(Me)Val-(Me)Gly-(Me)Leu(&2,1)]2}
{[QNL-Dap(&1,2)-(Me)Leu-(Me)Gly-(Me)Leu(&2,1)]2}
{[QNL-Dap(&1,2)-(Me)Ile-(Me)Gly-(Me)Leu(&2,1)]2}
53.4
35.3
6.4
41.5
5.7
28.2
5.0
35.3
which are highly prone to form this stable cycle before the
incorporation of the third residue, thereby resulting in the
loss of the two first amino acids.36
Synthesis and Biological Evaluation of a Second
FAJANU-Based Library. On the basis of the most appropiate
candidate, peptide 7, we designed, synthesized, and evaluated
the biological activity of a new chemical library.
Another important issue in solid-phase synthesis is the choice
of coupling reagents. Thus, peptides containing NMe amino
acids, the amine of which has lower nucleophilicity, require
the concourse of the most efficient coupling reagents such as
those based on 7-aza-1-hydroxybenzotriazole (HOAt).37 Thus,
when an amino acid was incorporated into a secondary amine,
the HATU–HOAt–DIPEA strategy was preferred.37 These NMe
amino acids should be taking into account for the incorporation
of the third residue in order to minimize the formation of
DKPs.38 Thus, Fmoc removal from the second amino acid is
carried out in less time and the coupling of the third amino
acid is performed with DIPCDI-HOAt in the absence of bases,
which would otherwise increase the risk of DKP formation.39
Another critical step is the cyclization method in solution, the
PyAOP–HOAt–DIPEA40,41 at 1 mM concentration was chosen
as the most effective method to obtain the desired cyclic peptide
to avoid the guanidinylation that may occur when aminium salts,
such as HATU, are used.42 Finally, after Boc deprotection,
quinaldic acid was introduced using PyBOPHOAt–DIPEA.
Using this strategy, the overall yield for 7 after RP-HPLC
purification was 3.6%, with 99% purity as shown by RP-HPLC.
Figure 3 shows, as an example, the purity of one of the
analogues (7).
Exploring Synthetic Strategies. Prior to the generation of a
second library of FAJANU peptides based on 7, other synthetic
approaches were explored. To compare the results, 7 was used
as a model. In the second strategy (Scheme 1ii), where the
heterocycle was introduced on solid-phase, the Fmoc group was
used for the R-amine and the Alloc was chosen for the ꢀ-amine
side-chain. The overall yield for 7 was slightly lower (2.1%
yield, 92.1% purity) than that obtained when strategy 1i was
used. For the convergent approach (Scheme 1iii), the reagent
for the coupling of the fragments must be chosen carefully. The
phosphonium salt PyAOP offers the possibility to run long
coupling reactions without the formation of undesired byproduct
caused by the excesses presented. In this approach, the starting
point is changed in order to facilitate fragment coupling. Thus,
the synthesis began with the NMe-Leu (1) and the Dap was
introduced, well protected by NR-Boc and Nꢀ-Fmoc. Once the
four-amino-acid sequence was elongated in solid phase, 3/4 were
cleaved from the resin and coupled to the 1/4 remaining anchored
to the resin after removal of the Fmoc group.45 At the end of
the synthetic process, compound 7 was obtained with an overall
yield of 3.5% and 98% of purity.
Structure–Activity Relationship (SAR). Using the peptide
7 structure, we synthesized a second generation of cyclic
peptides using the strategies described above (characterization
is available in the Supporting Information). All the suitable
diversity points were examined taking into account symmetry
preservation. These analogues were divided into three groups
depending on the modification (Figure 4): (i) cycle length,
(ii) backbone amino acids, and (iii) aromatic moiety. Table 3
Biological Activity of the FAJANU Family. We evaluated
the cytotoxic activity of this first FAJANU family against
several tumor cell lines (Table 1). The closest analogue to 1
was active but did not show improved activity, while cyclic
proline (2) was noncytotoxic. Surprisingly, NMe-Val (6) was
not active, while the other hydrophobic residues were (5, 7,
and 8). Moreover, 7 [Xxx (1 and 5) ) NMe-Leu] was the
most active.

A NoVel Cyclopeptide: FAJANU
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3197
Figure 4. Structural scheme of the first and second generation of the FAJANU family.
summarizes the library and indicates the code, synthesis strategy,
and GI50 in three tumor cell lines in comparison with the peptide
of origin.
Analogues 9–11, which have a larger macrocycle than the
reference compound, differed in their activity. While 10 (2 Orn)
and 11 (2 Lys) were less active against all the cell lines, 9 (2

3198 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Garcia-Martin et al.
Figure 5. Live-cell confocal images. Peptide 7 internalization at 50 µM after 30 min incubation. (a) Cell membrane is marked in red (HeNe 543);
(b) identical field under UV (UV 350 nm), FAJANU 7 is shown in blue; (c) overlapping of both channels (control performed without peptide does
not show autofluorescence).
Table 2. Cytotoxic Activity of the FAJANU Family (GI50 0.1 µM) against Several Tumor Cell Lines
activity (GI50) in vitro
compound
structure
Breast MDA-MB-23144 107
Lung A549 107
Colon HT29 107
1
2
7
9
10
11
12
13
12.3
7.1
6.4
n.d.
58.0
n.d.
5.5
29.0
28.6
5.7
6.4
6.5
5.0
n.d.
24.4
n.d.
6.2
{[QNL-Dap(&1,2)-MeLeu-MeGly-MeLeu(&2,1)]2}
{[QNL-Dab(&1,2)-MeLeu-MeGly-MeLeu(&2,1)]2}
{[QNL-Orn(&1,2)-MeLeu-MeGly-MeLeu(&2,1)]2}
{[QNL-Lys(&1,2)-MeLeu-MeGly-MeLeu(&2,1)]2}
{[QNL-Dap(&1,2)-MeLeu-Gly-MeLeu(&2,1)]2}
{[QNL-Dap(&1)-MeLeu-MeGly-MeLeu(&2)]
n.d.
29.4
39.4
11.8
27.6
11.6
12.3
[QNL-Ser(&2)-MeLeu-MeGly-MeLeu(&1)]}
14
15
16
17
18
19
20
21
22
23
24
25
{[H-Dap(&1,2)-MeLeu-MeGly-MeLeu(&2,1)]2}
{[PIC-Dap(&1,2)-MeLeu-MeGly-MeLeu(&2,1)]2}
{[PYR-Dap(&1,2)-MeLeu-MeGly-MeLeu(&2,1)]2}
{[3OH-NPH-Dap(&1,2)-MeLeu-MeGly-MeLeu(&2,1)]2}
{[1OH-NPH-Dap(&1,2)-MeLeu-MeGly-MeLeu(&2,1)]2}
{[Fmoc-Dap(&1,2)-MeLeu-MeGly-MeLeu(&2,1)]2}
{[BPH-Dap(&1,2)-MeLeu-MeGly-MeLeu(&2,1)]2}
{[OH-COU-Dap(&1,2)-MeLeu-MeGly-MeLeu(&2,1)]2}
{[3-OH-Qui-Dap(&1,2)-MeLeu-MeGly-MeLeu(&2,1)]2}
{[CHR-Dap(&1,2)-MeLeu-MeGly-MeLeu(&2,1)]2}
{[HQX-Dap(&1,2)-MeLeu-MeGly-MeLeu(&2,1)]2}
{[QNX-Dap(&1,2)-MeLeu-MeGly-MeLeu(&2,1)]2}
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
23.1
n.d.
16.7
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
24
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
29.1
34.3
12.3
14.1
Dab) showed no activity. This general loss of cytotoxic activity
with increased macrocycle size could be explained by a major
flexibility freedom. These results indicate that appropriate molecule
size is required for biological activity. Consecutive change included
the incorporation of nonmethylated amino acids into the structure.
When NMe-Gly (2 and 6) was replaced by Gly (12), the structure
was less rigid, thereby resulting in maintained but lower cyto-
toxicity. Our results showed that NMe-Gly is crucial and both NMe-
Leu amino acids can be replaced but at the expense of one order
of activity. In addition to the above, we synthesized a depsipeptide.
Thus, an ester bond was added to the structure (13), which resulted
in a lower order of activity.
The quinaldic acid was replaced, which provided important
clues about the role of the heterocycle. As shown in analogue
14, heterocycle presence is crucial for antitumor activity. When
one-ring heterocyclic moiety (15 and 16) was introduced, no
cytotoxic activity was observed against any of the tumor cell
lines. Similar results were obtained when the heterocyclic was
replaced by a bicyclic moiety but with no heteroatom in the
rings (17, 18, and 20) or more distance between the amide bond
and the aromatic ring (19 and 21). Antitumor activity was
detected when the quinaldic acid was replaced by chromone-
2-acid (23), hydroxyquinaldic acid (24), and quinoxaline acid
(25), but not when a methyl-substituted heterocycle, such as
analogue 22, was introduced. Although further studies are
required, on the basis of these preliminary results, we can
conclude that a heteroatom-containing bicycle is the preferred
moiety for the ability to maintain cytotoxicity.
amino acids. Major conformation was assigned (Table 3) by
interpretation of the 1H and 13C 2D-NMR spectra: HSQC,
HMBC, TOCSY, and NOESY. Symmetry was rendered by a
duplicate numbering of peaks.
Internalization Study. Internalization study of 7 was shown
by confocal microscopy (Figure 5) with recording of optical
sections. Using a nonfixed A549 lung cell line, we studied
capacity of 50 µM of the cyclopeptide to penetrate the cell after
a 30 min incubation. The cell membrane was marked in red by
WGA (Figure 5a) and peptide 7 was detected in UV (Figure
5b, λmax 450 nm). Overlapping of both channels (Figure 5c)
showed a nonmembrane effect and the internalization of 7 in
the cytosol. On the basis of this result and on experiments that
demonstrated that our candidate 7 does not act as DNA
intercalator (please refer to Supporting Information), we propose
that, in contrast to other marine cyclic peptides,3 peptide 7 is
not a DNA bisintercalator.
Conclusions
Here we designed a novel hybrid cyclopeptide, FAJANU,
from potent cytotoxic cyclopeptides. Three strategies were
followed based on the solid-phase approach: the first was
elongation of the peptide sequence on solid-phase, where the
group was introduced in solution, the second was performed
entirely on solid-phase, and the third variant was based on a
convergent synthesis 4 + 4. In all cases, the desired peptide
was obtained; the solid and solution combination approach being
the most effective. The most active cyclopeptide, 7, was success-
fully characterized by HPLC–MS, MALDI–TOF, exact mass, and
NMR. The SAR analysis detected key features of cytotoxic activity,
which could be extrapolated for use in the design of more potent
NMR Study of Peptide 7. NMR spectra were recorded at
400 MHz in DMSO. Spectra showed several sets of peaks from
minor conformations as a result of cis–trans isomers of the NMe

A NoVel Cyclopeptide: FAJANU
Table 3. NMR Assignment of 7
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3199
0:10 of MeCN (0.05% TFA) in H2O (0.1% TFA) for 30 min, 20
mL/min, detection at 220 nm).
13C δ (ppm)
1H δ (ppm)
J (Hz)
5.6
1H NMR and 13C NMR spectroscopy was performed on a Varian
Mercury 400 apparatus.
NMe-Leu (1 and 5) CRH
55.2
38.6
38.7
22.6
22.6
30.4
172.5
5.07 (t)
CꢀH2
CγH
CδH3
Cδ′H3
N-CH3
CO
1.35–1.65 (m)
1.89–1.91 (m)
0.79 (2d)
0.81 (2d)
3.06 (s)
Chemical Synthesis. General Methods. Manual solid-phase
peptide elongation and other solid-phase manipulations were carried
out in polypropylene syringes, each fitted with a polyethylene
porous disk. Solvents and soluble reagents were removed by suction.
Fmoc removal was carried out with piperidine-DMF (1:5) (1 × 1
min, 2 × 10 min), and for removal of the Fmoc group in a
secondary amine, the piperidine protocol used was piperidine-DMF
(1:5) (1 min, 2 × 15 min) and DBU-piperidine-DMF-toluene
(5:5:70:20) (2 × 5 min). Previous washings of the resin were
performed with CH2Cl2 (5 × 1 min), DMF (5 × 1 min), and CH2Cl2
(1 × 15 min). Washings between deprotection, coupling, and
subsequent deprotection steps were carried out with DMF (5 ×
0.5 min), CH2Cl2 (5 × 0.5 min), and DMF (5 × 0.5 min) using 10
mL of solvent/g of resin each time. After the first amino acid
coupling, the remaining chloride functions were methoxylated by
treatment with MeOH (1 mg of resin:1µL of MeOH) for 30 min.
The couplings of the amino acids were verified by several tests.
The ninhydrin test was used for primary amines and the De Clercq46
or Chloranil47 tests for secondary amines, the former being more
sensitive.
5.2
5.2
NMe-Gly (2 and 6) CRH2
49.6
37.0
171.6
4.59/4.62 (2s)
3.07 (s)
N-CH3
CO
NMe-Leu (3 and 7) CRH
51.3
37.3
38.7
23.5
23.5
30.8
169.5
5.11 (t)
5.6
CꢀH2
CγH
CδH3
Cδ′H3
N-CH3
CO
1.35–1.65 (m)
1.89–1.91 (m)
0.86 (2d)
0.91 (2d)
2.72 (s)
5.2
5.2
Dap (4 and 8)
CRH
CꢀH2
NH
49.5
49.5
5.68 (t)
3.75/3.79 (d)
9.02 (s)
5.6
13.6
CO
169.2
Cleavage was performed by TFA-CH2Cl2 (1:99) (5 × 1 min).
Filtrate was collected on H2O (1 mL/100 mg resin) in 50 mL
centrifuge tubes, and the solvents were partially removed under
pressure. The peptides were precipitated by adding cold tert-
butylmethyl ether and centrifuged (5 min × 4000 rpm); the solution
was then decanted and the solid was triturated with cold tert-
butylmethyl ether, which was again decanted. This process was
repeated twice. Next, the peptide was solved in H2O-MeCN (1:1)
and the solution was lyophilized.
Solution reactions were performed in round-bottomed flasks
followed by convenient workup. Organic solvent extracts were dried
over anhydrous MgSO4, followed by solvent removal under reduced
pressure at temperatures below 40 °C.
QNL
C2
163.2
119.0
138.5
131.0
128.8
128.7
131.2
129.8
146.5
C3H
C4H
C4a
C5H
C6H
C7H
C8H
C8a
8.57–8.62(m)
8.15–8.17 (m)
7.71–7.74 (m)
8.09–8.12 (m)
7.87–7.91 (m)
8.16 (d)
6.8
analogues. Structural determination by NMR revealed that 7
presented C2 symmetry. In addition, internalization of the peptide
was shown by a confocal technique.
Strategy 1: Synthesis of Compound 7 Backbone. Heterocycle
Addition in Solution (Scheme 1 Supporting Information). The first
strategy started with the incorporation onto the CTC resin (1.0 mmol/
g) of the Fmoc-NMeGly-OH (1 equiv) directly with DIPEA (1.6
equiv) in CH2Cl2. Next, 5 min later, more DIPEA (3.2 equiv) was
added. The mixture was stirred for 1 h. The remaining chloride
functions were methoxylated as described in the General Methods
section. After washings and Fmoc removal, the Fmoc-NMeLeu-
OH (3 equiv) was coupled using HATU–HOAt–DIPEA (3:3:9) in
DMF. After 90 min of coupling, the Chloranil test was negative.
Removal of Fmoc-protecting group and washings were carried out
as described above for secondary amines. The Boc-Dap(Fmoc)-
OH (3 equiv) was coupled with DIPCDI (3 equiv) and HOAt (3
equiv). After overnight coupling, the Fmoc-protecting group in the
lateral chain was removed. The other amino remained protected
by the Boc group until the end of the backbone formation. The
fourth amino acid, Fmoc-NMeLeu-OH (3 equiv) was coupled with
PyBOP–HOAt–DIPEA (3:3:9) in DMF. After 2 h of coupling, more
PyBOP (3 equiv) was added and the reaction continued for 60 min
more. Following Fmoc removal as described above for secondary
amines, the second Fmoc-NMeGly-OH (3 equiv) was coupled with
HATU–HOAt–DIPEA (3:3:9) in DMF for 90 min. The Fmoc was
removed and the Fmoc-NMeLeu-OH (3 equiv) was coupled using
HATU (3 equiv), HOAt (3 equiv), and DIPEA (9 equiv) in DMF
for 90 min. To continue the backbone formation, the second Boc-
Dap(Fmoc)-OH (3 equiv) was incorporated using PyBOP–HOAt–
DIPEA (3:3:9) in DMF for 2 h after Fmoc deprotection. Subse-
quently, the last amino acid was added: Fmoc-NMeLeu-OH (3
equiv) was coupled with PyBOP (3 equiv) and HOAt (3 equiv)
with DIPEA (9 equiv) as base in DMF. Again, after 2 h of coupling,
more PyBOP (3 equiv) was added, and the reaction was left to
stand for a further 60 min. After Fmoc removal, the peptide was
cleaved from the resin as described in the General Methods section,
thereby obtaining a solid-white peptide in a 70% yield, which was
Experimental Section
General. Protected L-N-methyl amino acids were obtained from
Luxembourg Industries (Tel Aviv, Israel), Neosystem (Strasbourg,
France), Calbiochem-Novabiochem AG (Laüfelfingen, Switzerland),
Bachem AG (Bubendorf, Switzerland), and Iris BioTech GmbH
(Germany). PyBOP was supplied by Calbiochem-Novabiochem
AG. CTC resin was obtained from Rohm & Haas (PA). DIPCDI
was obtained from Fluka Chemika (Buchs, Switzerland), TBTU
and HOBt from Albatross Chem. Inc. (Montreal, Canada), HOAt
and PyAOP were supplied by Applied Biosystems (Foster City,
CA), and solvents for peptide synthesis, RP-HPLC, and HPLC-
MS were obtained from Scharlau (Barcelona, Spain). Trifluoroacetic
acid was supplied by KaliChemie (Bad Wimpfen, Germany). Other
chemicals used in this study were obtained from Aldrich (Milwau-
kee, WI) and were of the highest purity available. HPLC was
performed on a Waters Alliance 2695 chromatographic system
(Waters, MA) with a PDA 995 detector and a reverse-phase
Symmetry C18 column (4.6 mm × 150 mm, 5 µm). Linear
gradients of 0.045% TFA in H2O and 0.036% in MeCN were run
at a flow rate of 1.0 mL/min, and the gradient went from from 5:5
to 0:10 MeCN in H2O unless indicated otherwise. HPLC-MS was
performed on a Waters Alliance 2796 with UV/vis detector 2487
and an ESI-MS Micromass ZQ chromatographic system (Waters),
using a reverse-phase Symmetry 300 C18 column (3.9 mm × 150
mm, 5 µm). Then 0.1% formic acid in H2O and 0.07% formic acid
in MeCN were used as eluents. Mass spectra were recorded on a
MALDI Voyager DE RP time-of-flight (TOF) spectrometer (PE
Biosystems, Foster City, CA) using AcH as matrix. Exact mass
spectra were recorded by the mass spectrometry unit at the
University of Santiago de Compostela.
Purification was performed in a semipreparative RP-HPLC
(reverse-phase Symmetry C18 column, linear gradient from 3:7 to

3200 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Garcia-Martin et al.
characterized by RP-HPLC (tR 10.48 min, 90%) and HPLC-MS
(m/z calcd for C50H92N10O13, 1041.3; found 1040.6 [M + H]+).
Furthermore, the peptide was cycled using the PyAOP protocol (3
equiv), HOAt (3 equiv), and DIPEA (9 equiv) at a 1 mM
concentration in CH2Cl2-DMF (9:1). Total coupling was followed
by HPLC-MS (tR 15.45 min, m/z calcd for C50H90N10O12, 1023.3;
found 1024.9 [M + H]+). Next, the Boc group was removed by
TFA-H2O-TIS (95:2.5:2.5), (1 mL of cocktail:50 mg of peptide)
for 1 h and characterized by RP-HPLC (tR 11.22 min, 76%) and
HPLC-MS (m/z calcd for C40H74N10O8, 824.0; found 824.7 [M +
H]+, 413.2 [M+2H]2+). After lyophilization by H2O-MeCN
(1:1), the quinaldic acid (3 equiv) was introduced using PyBOP (3
equiv), HOAt (3 equiv), and DIPEA (9 equiv) for 3 h in DMF-
CH2Cl2 (1:9). The total incorporation was followed by HPLC-MS.
Once the peptide was obtained, solvents were evaporated and the
cyclic peptide was dissolved in MeCN-H2O (1:1) and purified by
semipreparative RP-HPLC (tR 12.8 min) to give the title product
(3.6% yield, 99%). MALDI-TOF-MS (m/z calcd for C60H84N12O10,
1132.6; found 1132.9 [M + H]+, 1155.8 [M + Na]+, 1171.8 [M
+ K]+) and exact mass spectra ESI-TOF (m/z calcd for
C60H84N12O10, 1132.6; found 1133.65, 1134.65, 1135.66, 1136.66,
1137.67) confirmed the desired peptide.
Strategy 2: Linear Synthesis of 7 (Scheme 2 Supporting In-
formation). A solution of Fmoc-NMeLeu-OH (1 equiv) and DIPEA
(1.5 equiv) in CH2Cl2 was added to the CTC resin (1.0 mmol/g),
and after 5 min, more DIPEA (3 equiv) was added. The mixture
was stirred for 1 h. The Fmoc-protecting group was removed, and
the Fmoc-NMeGly-OH (2 equiv) was coupled using HATU (2
equiv), HOAt (2 equiv), and DIPEA (6 equiv) in DMF. After 1 h
of coupling, the De Clercq test was negative. Removal of the Fmoc-
protecting group and washings were carried out as described in
the General Methods section. The Fmoc-NMeLeu-OH (4 equiv)
was coupled with DIPCDI (2 equiv) and a minimum amount of
DIPEA (DIPCDI-DIPEA/19:1) in CH2Cl2 using the asymmetric
anhydride method. After overnight coupling, the De Clercq test
was negative and Fmoc was removed. The fourth amino acid, Fmoc-
Dap(Alloc)-OH (4 equiv), was coupled with HATU (2.5 equiv),
HOAt (2.5 equiv), and DIPEA (5 equiv) in DMF for 1 h. Once the
Fmoc group was removed, the heterocycle (quinaldic acid, 2 equiv)
was coupled in the sequence with PyBOP–HOAt–DIPEA (2:2:6)
in DMF overnight. Next, the Alloc group of the Dap was removed
using a reduction procedure, which consisted of reduction by PdPh3
(0.1 equiv) and PhSiMe3 (10 equiv) in CH2Cl2 (3 × 15 min) under
Ar atmosphere;48 washings with CH2Cl2 followed each treatment.
Once the Alloc had been removed, the other four amino acids were
introduced into the sequence as follows: Fmoc-NMeLeu-OH (2
equiv) was added using PyBOP–HOAt–DIPEA (2:2:6) as coupling
reagents, once again in DMF for 3 h. After Fmoc removal, Fmoc-
NMeGly-OH was coupled as previously described. Once the Fmoc
group had been removed, the Fmoc-NMeLeu-OH (2 equiv) was
introduced using HATU–HOAt–DIPEA (2:2:6) as coupling reagents
in DMF for 1 h. The Fmoc was removed, and the last amino acid
was incorporated: Fmoc-Dap(Alloc)-OH (2 equiv) with HATU (2
equiv), HOAt (2 equiv), and DIPEA (6 equiv) in DMF during 2 h.
Quinaldic acid incorporation and Alloc removal was done as
described above. Cleavage from the CTC resin was performed as
described in the General Methods section. Finally, the peptide was
cyclized using PyAOP (2 equiv), HOAt (2 equiv), and DIPEA (6
equiv) in CH2Cl2–DMF (9:1) to give the desired peptide in a 2.1%
yield of the title compound with a purity of 91% as analyzed by
HPLC (tR 12.4 min). MALDI-TOF-MS, m/z calcd 1132.6 for
C60H84N12O10; found 1132.7 [M + H]+, 1156.1 [M + Na]+, 1172.1
[M + K]+.
removed using the protocol described in the General Methods
section. Next, the Fmoc-NMeGly-OH (2 equiv) was coupled using
HATU (2 equiv), HOAt (2 equiv), and DIPEA (6 equiv) in DMF.
After 1 h of coupling, the De Clercq test was negative. Removal
of the Fmoc-protecting group and washings were carried out as
described above. The Fmoc-NMeLeu-OH (3 equiv) was coupled
with DIPCDI (3 equiv) and HOAt (3 equiv) in CH2Cl2, thereby
avoiding DKP formation. After leaving the reaction to rest
overnight, the De Clercq test was negative and Fmoc was removed.
The fourth amino acid, the Boc-Dap(Fmoc)-OH (1.9 equiv), was
coupled with HATU (1.9 equiv), HOBt (1.9 equiv), and DIPEA as
base for 1 h in DMF. A second coupling was not necessary
following the De Clercq test.
At this point the resin was divided into two parts, called
fragments A and B:Fragment A. Three-quarters of the resin was
cleaved using TFA-CH2Cl2 (1:99, 5 × 0.5 min). The solution was
evaporated and precipitated as described in the General Methods
section. After lyophilization with H2O-MeCN (1:1), the fragment
was characterized by RP-HPLC (tR 8.63 min, 91%) and MALDI-
TOF (m/z calcd for C40H58N6O8, 750.4; found 750.7 [M + H]+,
774.7 [M + Na]+, 790.7 [M + K]+), which verified the desired
fragment.Fragment B. In the remaining quarter of the resin, the
Fmoc group was removed.Convergent Coupling of Fragment A and
B. Fragment A (3 equiv) was coupled to the remaining quarter of
the resin using PyAOP (3 equiv), HOAt (3 equiv), and DIPEA (9
equiv) in DMF overnight. The ninhydrin test confirmed the total
coupling of Fragment A onto B. Next, the Fmoc group was removed
in solid-phase. The peptide was cleaved from the resin as described
above for fragment A and again lyophilized.Cyclization. The crude
linear peptide was dissolved in CH2Cl2-DMF (9:1, 1 mM); next,
PyAOP (3 equiv), HOAt (3 equiv), and DIPEA (9 equiv) were
added. The mixture was allowed to stir until cyclization was
completed, which was followed by RP-HPLC. The solvent was
removed by evaporation under reduced pressure. The cyclic peptide
was dissolved in H2O-MeCN (1:1), the fragment was characterized
by HPLC-MS (tR 12.27 min, m/z calcd for C50H90N10O12, 1022.7;
found 1022 [M +H ]+), which verified the desired cyclic
peptide.Boc Removal. Boc was removed by TFA-H2O-TIS
(95:2.5:2.5, 1 mL of cocktail for each 50 mg of peptide) for 1 h.
Peptide precipitation was performed as described in the General
Methods section.Quinaldic Acid Coupling. The heterocycle was
added in solution as follows: quinaldic acid (4 equiv) was coupled
by PyBOP (4 equiv), HOAt (4 equiv), and DIPEA (12 equiv) until
the reaction was completed, which was followed by RP-HPLC. The
cyclic peptide was dissolved in H2O–MeCN (1:1) and purified by
semipreparative RP-HPLC (tR 12.6 min, same conditions as
described in the General Methods section) to give the title product
(1.2% yield, 99%). MALDI-TOF-MS (m/z calcd for C60H84N12O10,
1132.6; found 1133.8 [M + H]+, 1155.9 [M + Na]+, 1171.9 [M
+ K]+) verified the desired cyclopeptide.
Cell Growth Inhibition Assay. A colorimetric assay using
sulforhodamine B (SRB) was adapted to perform a quantitative
measurement of cell growth and viability, following a previously
described method.49 The cells were seeded in 96-well microtiter
plates at 5 × 103 cells/well in aliquots of 195 µL of RPMI medium
and were left to grow in a drug-free medium for 18 h to allow
attachment to the plate surface. Afterward, samples were added in
aliquots of 5 µL (dissolved in DMSO–H2O, 3:7). After 72 h of
exposure, the antitumor effect was measured by the SRB methodol-
ogy. Cells were fixed by adding 50 µL of cold 50% (wt/vol)
trichloroacetic acid (TCA) and were incubated for 60 min at 4 °C.
Plates were washed with deionized H2O and dried; 100 µL of SRB
solution (0.4 wt %/vol in 1% acetic acid) was added to each
microtiter well and incubated for 10 min at room temperature.
Unbound SRB was removed by washing with 1% acetic acid. Plates
were air-dried, and the bound stain was solubilized with Tris buffer.
Optical densities were read on an automated spectrophotometer plate
reader at a single wavelength of 490 nm. Data analyses were
automatically generated by LIMS implementation. Using control
OD values (C), test OD values (T), and time zero OD values (T0),
Strategy 3: Convergent Synthesis of 7 (Scheme 3 Sup-
porting Information). The third strategy parts from the same resin,
which is divided at a later stage. The first four amino acids were
coupled as follows: a solution of Fmoc-NMe-Leu-OH (1 equiv)
and DIPEA (1 equiv) in CH2Cl2 was added to the CTC resin (1.0
mmol/g), and 5 min later, more DIPEA (2 equiv) was added. The
mixture was stirred for 1 h. The remaining chloride functions were
methoxylated. After washings, the Fmoc-protecting group was

A NoVel Cyclopeptide: FAJANU
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3201
(14) Chakavrarty, P. K.; Olsen, R. K.; Synthesis of triostin, A. Tetrahedron
Lett. 1978, 19, 1613–1616.
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antibiotics. II. Synthesis of triostin A. Bull. Chem. Soc. Jpn. 1984,
57, 2203–2210.
(16) Cruz, J. L.; Cuevas, C.; Cañedo, L. M.; Giralt, E.; Albericio, F. Total
solid-phase synthesis of marine cyclodepsipeptide IB-01212. J. Org.
Chem. 2006, 71, 3339–3344.
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preliminary DNA binding properties. J. Am. Chem. Soc. 1993, 115,
11624–11625.
the drug concentration that caused 50% growth inhibition (GI50
value) was calculated from the equation, 100 × [(T - T0)/C -
T0)] ) 50.
Confocal Microscopy. Lung tumor cell line A 549 was used as
model for the confocal assay. A 549 was settled onto glass-bottom
microwell dishes (35 mm Petri dish, 14 mm microwell, 1.5 cover
glass). Cells were seeded at a concentration of 104 cells/dish. After
24 h, cells were incubated for 30 min with 7 and WGA (TRITC)
C was added just before recording the image.
Confocal studies were performed on a LEICA UV microscope
with a 63× objective lens. The peptide was excited at 350 nm UV
laser and its emission was recorded at 450 nm. When no peptide
was in the sample under identical conditions, no fluorescence was
detected.
(18) Ciufolini, M. A.; Valognes, D.; Xi, N. Total synthesis of Luzopeptin
E2. Angew. Chem., Int. Ed. 2000, 39, 2493–2495.
(19) Dunlap, W. C.; Battershill, C. N.; Liptrot, C. H.; Cobb, R. E.; Bourne,
D. G.; Jaspars, M.; Long, P. F.; Newman, D. J. Biomedicinals from
the phytosymbionts of marine invertebrates: A molecular approach.
Methods 2007, 42, 358–376.
(20) Cruz, L. J.; Francesch, A.; Cuevas, C.; Albericio, F. Synthesis and
structure-activity relationship of cytotoxic marine cyclodepsipeptide
IB-01212 analogues. Chem. Med. Chem. 2007, 2, 1076–1084.
(21) Garcia-Echevarria, C.; Albericio, F.; Giralt, E.; Pons, M. Design,
synthesis, and complexing properties of (1Cys-1′Cys,4Cys-4′Cys)-
dithiobis(Ac-L-lCys-L-Pro-D-Val-L-4Cys-NH2). The first example of
a new family of ion-binding peptides. J. Am. Chem. Soc. 1993, 115,
11663–11670.
(22) Caba, J. M.; Rodriguez, I. M.; Manzanares, I.; Giralt, E.; Albericio,
F. Solid-phase total synthesis of trunkamide A(1). J. Org. Chem. 2001,
66, 7568–7574.
(23) López-Macià, A.; Jiménez, J. C.; Royo, M.; Giralt, E.; Albericio, F.
Synthesis and structure determination of Kahalalide F (1,2). J. Am.
Chem. Soc. 2001, 123, 11398–11401.
(24) Davies, J. S. The cyclization of peptides and depsipeptides. J. Pept.
Sci. 2003, 9, 471–501.
(25) Bayo, N.; Jimenez, J. C.; Rivas, L.; Nicolas, E.; Albericio, F. Solid-
phase synthesis of the cyclic lipononadepsipeptide [N-Mst(Ser1),
D-Ser4, L-Thr6, L-Asp8, L-Thr9] syringotoxin. Chem.sEur. J. 2003,
9, 1096–1103.
Acknowledgment. We thank Dr. Núria Bayó-Puxan for
fruitful discussions that originated part of this work. They also
thank Elisabet Prats-Alfonso for her assistance with electro-
phoresis assays and Dr. Andres Francesc and Pharma Mar S.A
(Madrid) for carrying out the biological screening. This study
was partially supported by CICYT (CTQ2006-03794/BQU), the
Instituto de Salud Carlos III (CB06-01-0074), the Generalitat
de Catalunya (2005SGR 00662), the Institute for Research in
Biomedicine, and the Barcelona Science Park.
Supporting Information Available: Characterization of the
FAJANU family, gel electrophoresis analysis and synthetic strategy
schemes. This material is available free of charge via the Internet
at http://pubs.acs.org
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(45) Alternatively, in the 4 + 4 strategy, if the heterocycle was
incorporated in solid-phase before the coupling of the protected
segment, NR-Alloc, Nꢀ-Fmoc was required and FAJANU 7 was
JM800047B