Design and synthesis of anti-cancer cyclopeptides containing triazole skeleton

Article abstract of DOI:10.1007/s00726-013-1663-1

We describe the design and synthesis of some hypothetical heptapeptides specifically to overcome the neoplastic activity of ras oncogene and their anti-cancer activities were studied. To improve the anti-cancer activity of the synthesized peptides, their

Full text of DOI:10.1007/s00726-013-1663-1

Amino Acids
DOI 10.1007/s00726-013-1663-1
ORIGINAL ARTICLE
Design and synthesis of anti-cancer cyclopeptides containing
triazole skeleton
Fatemeh Tahoori
Mahnaz Sadjadi
•
Saeed Balalaie
Parvin Boloori
•
Reza Sheikhnejad
•
•
Received: 4 November 2013 / Accepted: 29 December 2013
Ó Springer-Verlag Wien 2014
Abstract We describe the design and synthesis of some
hypothetical heptapeptides specifically to overcome the
neoplastic activity of ras oncogene and their anti-cancer
activities were studied. To improve the anti-cancer activity
of the synthesized peptides, their structure modifications
were done based on a sequential Ugi/Huisgen 1,3-Dipolar
cyclization reaction. The cyclopeptides which contained
triazole skeleton showed significant anti-cancer activity
against cancer cells with mutated ras oncogene such as
A549, PC3 and C26 cells. This study clearly shows the
importance of triazole skeleton in biological activity of the
peptides. It might be possible to overcome the difficulties
involved in making complex peptides by employing this
elegant chemistry.
been approved for the treatment of some cancers. The
efficacy of this cancer immunotherapy is, however, limited
by its large size and its nonspecific binding to the reticu-
loendothelial system that causes many undesirable side
effects (Aina et al. 2007). Furthermore, the drug research
and development has become very expensive and the
number of approved drugs has been declining in recent
years. Therefore, the demands for alternative approaches
are very high. This has contributed to the revival of pep-
tides as potential therapeutic drugs. A large number of
peptide-based drugs are now being marketed because new
synthetic strategies have been developed in recent years
(Vlieghe et al. 2010).
One classical strategy used in drug design is based on
the structure of receptor-binding pocket, called ‘‘rational
structure-based design’’ (Shoichet et al. 1993; Von Itzstein
et al. 1993). Most peptide drugs are designed this way.
Here we have used a novel strategy based on DNA–protein
binding criteria to design anti-cancer drugs. We focused
our interest on finding specific DNA–protein binding sites
along the promoter elements of ras oncogene. The precise
interactions between amino acid motifs of our designed
peptides and ras-specific regulatory sites within the CpG
islands might interfere with ras activity at transcriptional
level. The most active peptide is then selected based on its
in vitro anti-cancer activity to optimize its pharmaceutical
value by means of different chemical approaches. One such
approach would be the reduction of conformational space
by cyclization.
Keywords Ugi ligation ꢀ Ligation of peptides ꢀ Anti-
cancer activity ꢀ Cyclopeptides ꢀ Click reaction ꢀ Huisgen
1
,3-Dipolar reaction
Introduction
Several monoclonal antibodies such as Rituximab (anti-
CD20 antibody) and Herceptin (anti-HER-2 antibody) have
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-013-1663-1) contains supplementary
material, which is available to authorized users.
F. Tahoori ꢀ S. Balalaie (&)
Several hypothetical heptapeptides were designed based
on DNA–protein binding criteria known for regulation of
gene expression at transcriptional level. These peptides
designed to perhaps suppress ras oncogenic activities in
human cancer cells. The designed peptides 1–4 were tested
for their anti-cancer activities against A549, human lung
Peptide Chemistry Research Center, K. N. Toosi University
of Technology, P. O. Box 15875-4416, Tehran, Iran
e-mail: balalaie@yahoo.com
R. Sheikhnejad ꢀ M. Sadjadi ꢀ P. Boloori
Tofigh Daru Res. & Eng. Co., 61st. km 18 Karaj Highway,
3
7515-375 Tehran, Iran
123

F. Tahoori et al.
H
H
H
H
2
N-Ser- Ala-Pro-Pro-Pro-Arg-Lys -OH 1
N-Gly- Ala- Pro- Pro- Gly- Arg- Asp- OH 2
N-Arg- Pro- Pro- Gly- Ser- Pro- Ala- OH 3
N-Phe- Ala- Gly- Arg- Ser- Arg- Gly- OH 4
2010). Furthermore, a number of these compounds show a
broad spectrum of biological activities, displaying, for
example, antibacterial (Genin et al. 2000), herbicidal,
fungicidal (Wamhoff 1984), antiallergic (Buckle et al.
2
2
2
1986), or anti-HIV (Alvarez et al. 1994) properties.
Recently, 1,2,3-triazoles have also been used as catalysts
and ligands in transition metal-based catalyst systems
Scheme 1 Compositions of the designed heptapeptides 1–4
(Chan et al. 2004; Liu et al. 2005; Detz et al. 2006; Co-
lasson et al. 2007; Beyer et al. 2009; Hein et al. 2009; Duan
et al. 2009; Mager and Zeiler 2010). Since peptide 1
showed the best anti-cancer activity, we decided to modify
its structure using functionalized amino acids which could
form triazole scaffold.
cancer cells in vitro. The most active compound 1 is pro-
line-rich peptide (Ball et al. 2005) selected to further
improve its pharmaceutical potential with some specific
chemical modifications to create
Scheme 1).
Cyclic peptides are a unique class of compounds that
a cyclic peptide
(
Multicomponent reactions (MCR) have become
important tools in the preparation of structurally diverse
chemical libraries of drug-like polyfunctional compounds.
However, to ensure sufficient molecular diversity and
complexity of new chemical entities, there is a continuous
need for novel reactions with high efficiency and selec-
tivity in novel reaction media (D o¨ mling 2005, 2006;
Slobbe et al. 2012; Ruijter et al. 2011; D o¨ mling et al.
2012; Tietze et al. 2006; Tietze and Hauner 2000). We
intend to use the Ugi-4CR to construct products with
further functional groups which are prone to additional
ring closure reactions. This strategy allows us to prepare
in a very economic and ecologic way complex systems
(Bararjanian et al. 2010, 2011; Balalaie et al. 2011, 2012).
Recently, we showed that the Ugi-4CR is an efficient
approach for the synthesis of some novel GnRH analogs
with better anti-cancer activity (Arabanian et al. 2009;
Saleh-Abady et al. 2010). The 2D-NMR spectroscopic
data showed that this reaction affects the structure of
molecule on folding (Tahoori et al. 2010).
have made great contributions to the treatment of certain
diseases such as cancer. Penicillin, vancomycin, cyclo-
sporin, and echinocandins are well-known cyclic peptides.
Cilengitide is also a cyclic pentapeptide currently in clin-
ical trial for brain cancer, glioblastomas, and some other
cancers (Katsara et al. 2006; Mas-Moruno et al. 2010;
Boger 2001; Nicolaou et al. 1999; Rao et al. 1995; Chat-
terjee et al. 2005). Cyclic peptides, compared to linear
peptides, have been considered to have greater potential as
therapeutic agents. This may be due to their increased
chemical stability, receptor selectivity, as well as improved
pharmaco-dynamic properties. Considering these facts, we
decided to use one of the known cyclization methods to
prepare a unique cyclic heptapeptide. A reaction that would
seem ideal for conjugation of peptides and oligonucleo-
tides, due to the compatibility with many other functional
groups, is the copper(I) catalyzed 1,3-Dipolar cycloaddi-
tion between an azide and an alkyne, commonly referred to
as click chemistry (Rostovtsev et al. 2002; Kolb et al. 2001;
Moses and Moorhouse 2007; Wu and Fokin 2007; Kolb
and Sharpless 2003; Meldal and Tornoe 2008; Tornoe et al.
We report the design and synthesis of novel cyclopep-
tides through the Ugi ligation/click reaction to construct
cyclopeptides which have a triazole moiety and also lipo-
philic moieties (Scheme 2).
2
002). Moreover, triazole-modified peptidomimetics have
been shown as assembling protein-like oligomers and
nonpeptidic protein-mimetic foldamers (Angelo and Arora
2
005, 2007). Therefore, triazole-modified peptidomimetics
Results and discussion
have gained considerable attention for designing biological
effectors or foldamers (Horne et al. 2004; Kuijpers et al.
Reduction of conformational flexibility is important to
increase the affinity of a peptide for its natural receptor.
The first convenient approach to achieve this goal is head-
to-tail cyclization. To achieve this goal, chemical modifi-
cation of starting materials is important for many appli-
cations in biology and biotechnology. In order to
synthesize cyclopeptides, different strategies were studied.
Applying the well-known click chemistry is a known
approach to synthesize cyclopeptides with triazole moiety.
Functionalizing the scaffold with an alkyne moiety, and
also an azide group, to form triazole skeleton is an
approach to the synthesis of cyclopeptides.
2
004; Cantel et al. 2008). The synthesis of 1,2,3-triazoles
has grown in importance in medicinal (Chabre and Roy
008; Colombo and Peretto 2008; Hanselmann et al. 2010;
2
Moumne et al. 2010), material (Li et al. 2005; Rozkiewicz
et al. 2006; Wyszogrodzka and Haag 2008; Gadzikwa et al.
2
009; Golas and Matyjaszewski 2010; Bronisz 2005; Yue
et al. 2007; Fazio et al. 2008; Fletcher et al. 2008; Hua and
Flood 2010; Rawal et al. 2010), and biological researches
(
Hahn and Muir 2005; Heal et al. 2008; Ahsanullah et al.
009; Schneider 2010; Chemama et al. 2009; Nahrwold
et al. 2010; Michaels et al. 2010; Mamidyala and Finn
2
1
23

Design and synthesis of anti-cancer cyclopeptides
Scheme 2 Retrosynthetic approach for the synthesis of cyclopeptide 10 through sequential Ugi ligation/Huisgen 1,3-Dipolar reaction
The click reactions have the potential to be further
fortified when combined with multicomponent reactions.
The idea of using MCRs followed by a Huisgen [3+2]
copper-catalyzed reaction was investigated and a number
of pharmaceutically relevant heterocyclic compounds were
synthesized via classical multicomponent reactions com-
bined with click chemistry in separate steps such as
sequential Ugi/intermolecular alkyne–azide cycloaddition
prepared for click chemistry is the amino acid which
contains the azide group.
Since peptide 1 showed better anti-cancer activity
compared to other heptapeptides, compound 1 was selected
as a druggable molecule to be chemically modified. At first,
heptapeptide 1 was divided into a hexapeptide 7 which
contained an alkyne moiety and an amidated C-terminal
Lysine 6 which contains an azide moiety.
(
IAAC) (Ramachary and Barbas 2004; Akritopoulou-Zan-
The procedure for the synthesis of Fmoc-Lys(N )-
3
ze et al. 2004).
CONH 6 is shown in Scheme 3 in which the amine group
2
The sequence of Ugi isocyanide multicomponent reac-
tion, followed by post-condensation transformations, con-
stitutes an extremely powerful synthetic tool for the
preparation of structurally diverse complex molecules,
especially heterocyclic compounds (Orru and Ruijter 2010;
Aravind et al. 2011; De Graaff et al. 2012). Ultimately, this
one-pot sequential combination of multi-catalysis and
multicomponent approach should reduce the cost and waste
associated with pharmaceutical synthesis.
in the side chain was converted to azide moiety. To access
this molecule, Fmoc-Lys(Boc)-OH was selected as starting
material and the synthesis was done in four steps: (a) Am-
idation of terminal carboxylic acid was done using
ammonium chloride in the presence of TBTU as coupling
reagent and NMM as base to form compound 12. (b) The
Boc-protecting group was removed with trifluoroacetic
acid 13. (c) The conversion of side-chain amine group to
azide could be done using imidazoliumsulfonylazide 14 to
obtain compound 15. (d) Fmoc deprotection using 25 %
piperidine afforded H N-Lys(N )-CONH 6.
The wide variation in starting materials available for
IMCRs (isocyanide multicomponent reactions) opens up
versatile opportunities for the synthesis of compound
libraries. The significant potential of isocyanides for the
development of multicomponent reactions is a result of
their ability to take part in diverse bond formation pro-
cesses, their functional group tolerance, and the high levels
of chemo-, regio-, and stereoselectivity often observed.
According to our design, the copper(I)-catalyzed 1,3-
Dipolar cycloaddition between an azide and an alkyne was
used. The first moiety to be inserted in the Huisgen reaction
is an alkyne group. The alkyne moiety was added to the
side chain of serine and the other moiety which was
2
3
2
The imidazoliumsulfonylazide 14 was synthesized
according to the reported method (Johnasson and Pedersen
2012).
As shown in Scheme 4, Boc-Ser(O-Prop)-OH was pre-
pared through the reaction of Boc-Ser-OH and sodium
hydride followed by propargyl bromide reaction which led
to the desired Boc(O-Prop)-OH with 72 % yield.
The linear peptide analogue Boc-Ser(O-Prop)-Ala-Pro-
Pro–Pro-Arg(pbf)-OH was synthesized by standard solid-
phase peptide synthesis (SPPS) strategy on resin. It should
be noted that the initial experiments with Fmoc-Arg(Pbf)-
123

F. Tahoori et al.
2 3 2
Scheme 3 Synthesis of H N-Lys(N )-CONH 6
Scheme 4 Synthesis of Boc-
Ser(O-Prop)-OH 17
Scheme 5 Solid phase
hexapeptide synthesis of 7
OH coupled to the 2-chlorotrityl chloride resin (2-CTC
resin) and the peptides elongated more efficiently with high
purity. Coupling reactions were performed using protected
amino acids, activated with TBTU in the presence of
diisopropyl ethyl amine (DIPEA). Then, three S-proline
and one L-alanine was used in this sequence of the desired
peptide and finally Boc-Ser(O-Prop)-OH was added to the
peptide sequence to afford the protected hexapeptide 7.
Cleavage of the peptide from the surface of resin was done
using TFA (1 %) (Scheme 5).
decided to use Ugi ligation. Four-component reaction of
hexapeptide with carboxylic acid containing the propargyl
group, amidated functionalized lysine containing azide
moiety in the side chain as an amine group, 4-cyano-
benzaldehyde and cyclohexyl isocyanide in methanol led
to Ugi-ligated product 9.
All structures were confirmed based on spectroscopic
data and also high resolution mass spectrometry. Using
Ugi-4CR, a new stereocenter was created in the product
and the products formed as two diastereomers; the ratio of
diastereomers was 80:20. The diastereomers were sepa-
rated using column chromatography and compound 10 was
To twist and bring the two ends closer together for the
click reaction, based on our previous experience, we
1
23

Design and synthesis of anti-cancer cyclopeptides
2 3 2
Scheme 6 Synthesis of functionalized dipeptide H N-Arg(Pbf)-Lys(N )-CONH 19
separated as a major and pure stereoisomer. The anti-can-
cer activity of major and minor diastereomers was inves-
tigated and only the major diastereomer showed good
biological activity and will be discussed. The minor dia-
stereomer did not show good biological activity.
H N-Arg(Pbf)-Lys(N )-CONH 19 are shown in Scheme 6.
2 3 2
The amine group in the side chain of lysine was converted
to azide moiety using 14 and Fmoc-Lys(N )-OH 23 was
3
loaded on the surface of 2-chlorotrityl chloride resin in the
presence of DIPEA. Then Fmoc deprotection was done
using piperidine 25 % in DMF and Fmoc-Arg(Pbf)-OH
was added to the sequence using TBTU as coupling reagent
in the presence of DIPEA. The cleavage of the protected
dipeptide was done using 1 % TFA. The amidation of
C-terminal was done using ammonium chloride and TBTU
in the presence of NMM as base. Finally, Fmoc depro-
tection was carried out using 25 % piperidine and the
desired dipeptide 19 was formed.
1
,3-Dipolar cycloaddition reaction between an azide and
alkyne takes place in the presence of a Cu (I) catalyst under
mild conditions, resulting in the formation of a triazole link
connecting the two molecules. In peptide chemistry, the
increasing popularity of the click reaction is largely a result
of the unique properties of both azides and the resulting
triazoles. Interestingly, the triazole moiety formed by click
reaction has a unique similarity to an amide bond. The rel-
ative planarity, strong dipole moments, and hydrogen
bonding ability of triazole linkage make it as attractive as an
amide bond with added advantage that it is less prone to
hydrolytic cleavage. Triazole unit may impart rigidity,
lipophilicity, enhanced absorption and protease stability, and
act as an amide bond. Thus, the incorporation of the triazole
unit in the structure of peptide is an added advantage.
There are many reports for the cycloaddition of azides and
acetylenes which was done in the presence of CuI or
The pentapeptide 20 was synthesized based on the
standard SPPS method. The Ugi-4CR of pentapeptide 20 as
carboxylic acid, dipeptide 19 as amine, 4-cyano-benzal-
dehyde 5, cyclohexyl isocyanide 8 led to the formation of
compound 21 and finally using CuI.P(OEt) 18 (Langille
3
and Jamison 2006), cyclization was carried out and
cyclopeptide 22 was obtained (Scheme 7).
To access a cyclopeptide, at first the heptapeptide 27
was synthesized based on SPPS strategy, the sequence of
amino acids in this compound is the same as heptapeptide
1. There are only two changes: instead of Lys and Ser in
Cu(OAc) /sodium ascorbate. The reaction was checked
2
according to the reported methods based on CuI and
Cu(OAc) , but the yields were low (Johnasson and Pedersen
2
the sequence, Fmoc-Lys(N )-OH and Boc-Ser(O-Prop)-OH
3
2
012). Finally, CuI.P(OEt) was used as a reagent for the click
3
were used in C- and N-terminal, respectively. The 1,3-
reaction and cyclopeptide 10 was obtained in 75 % yield.
Based on the result of the experiment mentioned above
and based on the amino acid sequence in the structure of
peptide 1, another Ugi-4CR was designed. The two pen-
tapeptide 20 and dipeptide 19 segments were selected as
the carboxylic acid 20 and amine moiety 19, respectively.
The reaction sequences for the synthesis of dipeptide
Dipolar cycloaddition of peptide 27 was done according to
the standard method (CuI) as well as CuI. P(OEt) 18, but
3
in both cases the cyclization was not successful
(Scheme 8). It seems that the distance between alkyne and
azide moieties is very far. It shows that the Ugi-4CR could
affect the folding of molecule and cause efficient
cyclization.
123

F. Tahoori et al.
Scheme 7 Sequential Ugi ligation/Huisgen 1,3-Dipolar reaction to construct cyclopeptide 22
Scheme 8 Try for cyclization
of peptide 27
Peptide 27 had no biological activity. The click reaction
did not occur; perhaps due to the amino acid sequence of
this peptide or because the two ends failed to connect.
Compound 10 was highly active against lung cancer cells,
which induced apoptosis at a much lower dose than the
original heptapeptides. Yet, the same compound had no
anti-cancer activity before the click reaction (compound 9).
The chemical modifications before click reaction actually
affected the original heptapeptides 1 negatively. All pro-
ducts were less soluble in water; therefore, they were dis-
solved in a small volume of dimethyl sulfoxide (DMSO)
and then diluted with water for in vitro assays. Interest-
ingly, the products 21 and 22 had no anti-cancer activities;
this result shows that the selection of suitable segments for
Ugi-4CR has an important role in the biological activity of
products.
20-fold. The dose–response assay (Fig. 2) shows that
0.5 lg of compound 10 had the same or greater anti-
cancer activity than 10 lg of compound 1 (Fig. 1). The
biological activity of compound 10 was further examined
by its effect on different cell lines: A549 used was a well-
characterized human lung carcinoma cell line, known to
have mutated ras oncogene; C26, an aggressive colorectal
cancer cell line that contains constitutively activated ras
oncogene; PC3 is a cell line characteristic of prostatic
small cell carcinoma that also has mutated ras oncogene;
and noncancerous CHO, Chinese hamster ovary cell line
that does not have mutated ras oncogene. Cyclopeptide 10
showed great specificity against these cell lines. The
results (Fig. 3) show that 10 had significant anti-cancer
activity against all the three different cancer cell lines that
have mutated ras oncogene. Meanwhile, it had little or no
effect on noncancerous CHO cells and normal human
leukocytes.
The biological activities of products 1 (the unmodified
heptapeptide), 9, 10, 21, 22, and 27 were determined by
their effects on A549, human lung cancer cell line. The
results (Fig. 1) clearly indicate that cyclopeptide 10, the
final product in Scheme 9, shows significant anti-cancer
activity. The cyclization of our original heptapeptides by
this method has improved its biological activity up to
In conclusion, we have designed a hypothetical hep-
tapeptides specifically to overcome the neoplastic activity
of ras oncogene. This peptide showed the potential to be
druggable by inducing apoptosis in some specific cancer
cells known to have mutated ras oncogene. To improve
1
23

Design and synthesis of anti-cancer cyclopeptides
The effect of peptides on A549, lung cancer cell line
Experimental section
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
General
Commercially available chemicals were used as received
unless otherwise stated. Flash column chromatography
was carried out using silica Gel 60 (particle size
0.04–0.06 mm/230–400 mesh). The abbreviations are
given in separate place. The mass spectra were recorded
by EI-mass (70 eV), mass (ESI-triple quadrupole), mass
(ESI-ion trap), HRMS (ESI-FT-ICR), HRMS (MALDI-
FT-ICR). The purification of peptides was done using
preparative HPLC (column C18, 7 lm). NMR spectra
were recorded at 500, 300 MHz in CDCl , DMSO-d and
1
9
10
21
22
27
Peptides (10 µg/well)
Fig. 1 The anti-cancer activities of synthesized peptides against
human lung carcinoma cells, A549
3
6
D O.
2
the anti-cancer activity of this peptide, we chemically
modified it by cyclization reaction. The reaction could be
categorized as a sequential Ugi/Huisgen 1,3-Dipolar
cyclization reaction. We have now constructed a cyclo-
peptide that contains a triazole motif. The existence of a
triple bond opens an avenue to a diversity of subsequent
compounds accessible by different reactions. The anti-
cancer activity of all products was examined in vitro and
only cyclopeptide 10 with triazole skeleton showed sig-
nificant anti-cancer activity against cancer cells with
mutated ras oncogene such as A549, PC3 and C26 cells.
Cyclopeptide 10 had little or no activity on Chinese
hamster cells, CHO that does not express ras oncogene.
This study clearly shows the importance of triazole
skeleton in biological activity of the peptides. It might
be possible to overcome the difficulties involved in
making complex peptides by employing this elegant
chemistry.
Synthetic procedures
General procedure for the synthesis of heptapeptides-
COOH (1–4)
Synthesis was carried out using 2-chlorotrityl chloride resin
(
1.0 mmol/g) following the standard Fmoc strategy. Fmoc-
Lys(Boc)-OH (4.687 g, 10 mmol) was attached to the
-CTC resin (5.000 g) with DIPEA (6.85 mL, 40 mmol) in
2
anhydrous DCM:DMF (50 mL, 1:1) at room temperature
for 2 h. After filtration, the remaining trityl chloride groups
were capped by a solution of DCM/MeOH/DIPEA (17:2:1,
120 mL) for 30 min. Then, it was filtered and washed
thoroughly with DCM (1 9 20 mL), DMF (4 9 20 mL)
and MeOH (5 9 20 mL). The loading capacity was
determined by weight after drying the resin under vacuum
and was 1.0. The resin-bound Fmoc-amino acid was
Scheme 9 Sequential Ugi ligation/Huisgen 1,3-Dipolar reaction to construct cyclopeptide 10
123

F. Tahoori et al.
The effect of 10 on A549, lung cancer cell line
The same procedure was used for the synthesis of peptide
2–4.
1
00
This procedure was used for the synthesis of hexapep-
tide 7, only Fmoc-Ser(O-prop)-OH was used instead of
t
Fmoc-Ser( Bu)-OH.
80
60
40
20
0
HRMS (ESI) heptapeptides
?
m/z [M?H] Calcd for C H N O 752.44189, Found
1
3
3 58 11 9
?
7
52.44183. [M?Na]
Calcd for C H N NaO
33 57 11 9
0
0.5 µg
1.5 µg
5 µg
7
74.42409, Found 774.42400.
?
Peptide Concentration
2
Found 669.33266.
m/z [M?H] Calcd for C H N O 669.33284,
27 45 10 10
Fig. 2 The dose-dependent anti-cancer activity of cyclopeptide 10
against human lung carcinoma cells, A549
?
3
Found 681.36879.
m/z [M?H] Calcd for C H N O 681.36893,
29 49 10 9
?
4
Found 750.40269.
m/z [M?H] Calcd for C H N O 750.40298,
31 52 13 9
The effect of 10 on different cell lines
1
00
90
80
70
60
50
40
30
20
10
0
HRMS (ESI-FT-ICR)
?
HRMS (ESI-FT-ICR) 7 m/z: [M?H]
C H N O S 1,014.49667, Found
M?Na] Calcd for C H N NaO S 1,036.47904, Found
Calcd for
1,014.49665,
4
8 72 9 13
?
[
4
8
?
71
9
13
1,036.47899, [M?K]
Calcd for C H KN O S
48 71 9 13
1,052.45305, Found 1,052.45299.
A549
Pc3
C26
CHO
LK
Cell Lines
Fmoc-Lys(Boc)-CONH 12
2
Fig. 3 The specificity of cyclopeptide 10 against different cell lines,
A549, PC3, C26, CHO, and LK
A solution of Fmoc-Lys(Boc)-OH 11 (4.396 g, 9.4 mmol)
and ammonium chloride (1.069 g, 20 mmol) in N-methyl-
2-pyrrolidinone (3 mL) was magnetically stirred and then
TBTU (4.815 g, 15 mmol) in N-methyl morpholine
(5.5 mL, 50 mmol) was added to the mixture. The mixture
was stirred for 12 h at room temperature.
washed with DMF (3 9 20 mL) and treated with 25 %
piperidine in DMF (65 mL) for 30 min and the resin was
washed with DMF (3 9 20 mL). Then a solution of Fmoc-
Arg(Pbf)-OH (4.866 g, 7.5 mmol), TBTU (2.407 g,
7
.5 mmol), and DIPEA (3.0 mL, 17.5 mmol) in 30 mL
A yellow solution was formed. The reaction progress
was monitored using thin layer chromatography (1:2:10,
DMF was added to the resin-bound free amine and shaken
for 1 h at room temperature. After completion of coupling,
resin was washed with DMF (4 9 20 mL). The coupling
was repeated as in the same way as for other amino acids of
their sequences. In all cases for the presence or absence of
free primary amino groups, Kaiser Test was used. Fmoc
determination was done using UV spectroscopy method.
After completion of couplings, resin was washed with
DMF (4 9 20 mL). The produced heptapeptide was
cleaved from resin by treatment of TFA (1 %) in DCM
H O:MeOH:ethyl acetate). The reaction was completed
2
after 12 h. Then, by slow addition of 70 mL H O, a yellow
2
discretion was formed. The deposition was filtered and
dried. Mass of discretion was 4.830 g (9.4 mmol) with
94 % yield (Arabanian et al. 2010).
1
m.p. 158–161 °C; HNMR (300 MHz, DMSO-d )
6
t
d = 1.26–1.28 (m, 2H, CH ), 1.35 (s, 9H, Bu), 1.56–1.59
2
(m, 2H, CH ), 1.89 (quin, 2H, CH ), 2.85–2.90 (m, 2H,
2
2
CH NH), 3.28 (t, 1H, J = 7.0 Hz, CH fluorene),
2
(
(
275 mL) and neutralization with pyridine (4 %) in MeOH
85 mL). The solvent was removed under reduced pressure
4.20–4.29 (m, 3H, CHa and CH O), 6.76 (brs, 1H, NH–
CO–CH fluorenyl), 6.96 (brs, 1H, NH-Boc), 7.31 (t, 1H,
2
2
and precipitated in water. The precipitate was filtered and
dried. Final deprotection was done using TFA (95 %) and
reagent K (TFA/TES/Water 95:2.5:2.5). The excess TFA/
DCM was removed under reduced pressure. The desired
peptide was precipitated in diisopropyl ether. The purifi-
cation was done using preparative HPLC (Column C18).
J = 7.3 Hz, H–Ar), 7.32–7.37 (m, 2H, H–Ar), 7.40 (t, 1H,
J = 7.0 Hz, H–Ar), 7.71 (d, 1H, J = 6.3 Hz, H–Ar), 7.82
(d, 1H, J = 7.5 Hz, H–Ar), 7.87 (d, 2H, J = 7.3 Hz, H–
1
3
Ar) ppm; C NMR (75 MHz, DMSO-d ) d = 17.2, 22.9
6
(CH ), 28.3 (C(CH ) ), 29.2 (CH ), 31.5 (CH NH),46.7
2
3 3
2
2
a
(CH fluorene), 54.4 (CH –CONH ), 65.6 (CH O–), 77.3
2
2
1
23

Design and synthesis of anti-cancer cyclopeptides
(
–OC(CH ) ), 120.1, 125.3, 127.0, 127.6, 140.7, 143.8,
3
stirring for 10 min at 0 °C, the solution was added to the
EtOAc solution over 20 min. After stirring for 10 min, the
resulting suspension was filtered and the precipitate washed
with EtOAc (4 9 10 mL) and dried under suction for
30 min affording the title compound as a white solid
(2.250 g, 70 %) with spectral characteristics in accordance
3
1
43.9 (C Ar), 155.5 (fluorenyl-CH -CONH-), 155.9
2
t
(
–OCO Bu), 173.9 (CONH ) ppm; IRmmax(neat) 3,313,
2
-
,055 (NH), 1,687 (C=O), 1,511 cm ; MS (70 eV): m/z
1
3
?
?
?
(
%):467 (10) [M ], 394 (74) [M -C H O], 366 (67) [M -
4 9
C H NO ].
5
9
2
with literature data (Goddard-Borger and Stick 2007).
1
Fmoc-Lys-CONH 13
2
H NMR(300 MHz, D O) d = 7.46 (dd, 1H, J = 2.1 Hz,
2
4
5
J = 1.2 Hz, CH ), 7.88 (t, 1H, J = 2.1 Hz, CH ), 9.15 (t,
2
13
Fmoc-Lys(Boc)-CONH 12 (2.935 g, 8.0 mmol) was dis-
2
1H, J = 1.2 Hz, CH ) ppm; C NMR (75 MHz, D O)
2
4
5
2
solved in 50 % (v/v) TFA in CH Cl (100 mL). Then tri-
2
d = 119.8 (C ), 121.8 (C ), 137.0 (C ) ppm; IRm
(KBr)
max
2
-
1
ethylsilane (1.4 mL, 9.0 mmol) was added to the mixture
as scavenger. The reaction mixture was stirred for 3 h at
room temperature. Then, the solvent was removed under
the reduced pressure, the pH of the mixture was adjusted
via addition of NaOH (1 N). The precipitate was filtered
and washed with water. 2.490 g of product was achieved.
3,111 (NH), 2,167 (N ), 1,428 (SO ) cm
3
.
2
Fmoc-Lys(N )-CONH 15
3
2
A
suspension of Fmoc-Lys-CONH2 13 (2.910 g,
7.9 mmol) with potassium carbonate (2.722 g, 19.7 mmol)
and copper sulfate pentahydrate (0.017 g, 0.07 mmol) in
methanol (50 mL) was prepared in a proper round bottle.
The imidazole-1-sulfonyl azide hydrochloride (2.000 g,
9.5 mmol) was added to the suspension slowly. The above
mixture was stirred for 20 h at room temperature. After
completion of reaction, solvent was removed under vac-
The yield at this step was 93 % (Diaz-Mochon et al. 2005).
1
HNMR (300 MHz, DMSO-d ) d = 1.32–1.34 (m, 2H,
6
CH ), 1.43–1.50 (m, 2H, CH ), 1.89 (quin, 2H, CH ),
2
2
2
2
.67–2.87 (m, 2H, CH –NH ), 3.29 (t, 1H, J = 7.0 Hz, CH
2 2
fluorene), 3.50–3.81 (brs, 2H, NH ), 4.20–4.27 (m, 3H,
2
–
CH O, CHa) 6.26 (s, 1H, CONH), 6.95 (m, 2H, CONH,
2
fluorenyl CH CONH), 7.31 (t, 1H, J = 7.3 Hz, H–Ar),
2
uum, then 120 mL H O was added to reaction mixture and
2
7
.32–7.37 (m, 2H, H–Ar), 7.40 (t, 1H, J = 7.0 Hz, H–Ar),
.71 (d, 1H, J = 6.2 Hz, H–Ar), 7.82 (d, 1H, J = 7.5 Hz,
pH was diminished to 2 by consumption of concentrated
HCl. The achieved acidic solution was extracted by eth-
ylacetate (3 9 20 mL) and finally the whole organic phase
was washed by brine. Organic phase was dried by anhy-
7
1
3
H–Ar), 7.87 (d, 2H, J = 7.4 Hz, H–Ar) ppm;
C
NMR(75 MHz, DMSO-d ) d = 17.2, 22.1, 28.9 (CH ),
6
2
a
3
1
1
0.1 (CH –NH ), 33.8 (CH fluorene), 53.9 (CH –CONH ),
2
drous MgSO and the solvent was distilled. Yellow oil
4
2
2
09.7, 111.2, 115.2, 119.1, 119.9, 121.3, 123.3, 127.3,
28.9, 137.4, 139.4, 142.5 (C Ar), 173.9 (fluorenyl-CH2-
(2.000 g, 65 %) was formed.
1
H NMR(300 MHz, DMSO-d ) d = 1.29–1.37 (m, 2H,
6
CONH-), 176.7 (CONH ) ppm; IRm
(neat) 3,055, 2,984
CH ), 1.42–1.59 (m, 2H, CH ), 1.59–1.71 (m, 2H, CH ),
2
2
max
1
2
2
-
NH), 2,854 (CH), 1,681 (CO) cm ; MS (70 eV): m/z (%):
(
3.32 (t, 2H, J = 6.9 Hz, CH N ), 3.68–3.73 (m, 1H, CH
2 3
?
66 (6) [M ], 351 (16) [M -NH ], 337 (12) [M -
?
?
a
3
fluorene), 3.85–3.92 (m, 1H, CH –CONH ), 4.20–4.25 (m,
2
2
(
H C=NH )].
2
2H, -fluorenyl-CH -O), 6.99 (brs, 1H, NH-COOCH flu-
2
2
2
orenyl), 7.28–7.35 (m, 3H, H–Ar), 7.36–7.50 (m, 3H, H–Ar
and CONH ), 7.59 (brs, 1H, CONH ), 7.72 (d, 1H,
Imidazole-1-sulfonyl azide hydrochloride 14
2
2
J = 7.2 Hz, H–Ar), 7.78 (d, 2H, J = 7.4 Hz, H–Ar) ppm;
1
3
A round bottle containing suspension of sodium azide
C NMR (75 MHz, DMSO-d ) d = 22.5, 28.3, 31.7
6
a
(
1.001 g, 15.4 mmol) and acetonitrile (20 mL) was placed
(CH ), 47.1 (CH fluorene), 51.1 (CH N ), 56.8 (CH –
2 2 3
in ice bath. When the reaction mixture was cooled, sulfuryl
chloride (1.25 mL, 15.4 mmol) was dropped slowly. Then,
ice bath was removed. And reaction mixture was stirred for
CONH ), 67.2 (CH –O–), 120.1, 125.1, 127.1, 127.8,
2 2
141.3, 143.6, 143.8 (C–Ar), 156.3 (COOCH fluorenyl),
2
177.0 (CONH ) ppm; IRm
(neat) 3,380, 3,351, 2,943
max
2
-
(NH), 2,105 (N ), 1,775, 1,681 (C=O) cm ; MS(70 eV):
1
2
0 h at room temperature. The reaction mixture was cooled
to 0 °C (ice bath) again and imidazole (2.000 g,
9.3 mmol) was added over 10 min. The reaction mixture
3
?
m/z (%):393 (70) [M ].
2
was stirred for 4 h at room temperature. Then it was diluted
H N-Lys(N )-CONH 6
2
2
3
with EtOAc (20 mL) and washed with H O (2 9 20 mL).
2
The organic phase was washed by saturated NaHCO3
Fmoc-Lys(N )-CONH 15 (2.001 g, 5.1 mmol) was dis-
3 2
solution (2 9 20 mL). The organic phase was separated
solved in ethylacetate (8 mL) and then piperidine (2.0 mL,
20 mmol) was added. The reaction mixture was stirred at
room temperature. After 3 h, the reaction mixture was
extracted by water (3 9 10 mL). The aqueous phases were
and dried with anhydrous MgSO and cooled to 0 °C (ice
4
bath). Acetyl chloride (2.098 mL, 29.4 mmol) was added
dropwise to ice cold EtOH (5 mL) over 10–15 min. After
123

F. Tahoori et al.
washed by fresh ethylacetate (1 9 10 mL) again. Aqueous
triethylphosphite (0.9 mL, 5.3 mmol) and toluene
(5.5 mL) in 10 min. The round bottle was sealed to
protect from light; after 1 h stirring at room temperature,
the reaction mixture was passed through Celite and was
concentrated in vacuum. Further purification was done
using recrystallization in toluene and hexane; the white
crystal (1.100 g, 57 %) was formed (Langille and Jami-
son 2006).
phase was dried. Yellow viscous oil (0.850 g, 98 %) was
obtained.
1
H NMR (300 MHz, DMSO-d ) d = 1.32–1.45 (m, 2H,
6
CH ), 1.47–1.60 (m, 4H, 2CH ), 2.92 (m, 3H, CH-NH and
2
2
2
a
CH CONH ), 3.29 (t, 2H, J = 6.7 Hz, CH N ), 6.80 (brs,
2
2 3
1
3
2
H, CONH ) ppm;
2
C NMR (75 MHz, DMSO-d )
6
a
d = 22.5, 23.9, 28.3 (CH ), 43.9 (CH N ), 53.6 (CH ),
2
2 3
1
1
78.2 (C=O) ppm; IRmmax (neat) 3,485, 3,469, 3,420 (NH),
,098 (N ), 1,697 (C=O) cm ; MS (70 eV): m/z (%): 170
H NMR (300 MHz, CDCl ) d = 4.11 (quin, 2H, CH ),
3
2
-
1
2
1.29 (t, 3H, J = 7.0 Hz, CH ) ppm. [Compare to P (OEt) :
3 3
3
?
5) [M -H], 127 (14) [M -CONH ], 85 (90) [M -
?
?
1
(
H NMR (500 MHz, CDCl ) 3.88 (6H, q, J = 7.0), 1.28
3
2
?
CH N O], 56 (94) [C H NO ].
(9H, t, J = 7.0)].
2
4
2 4
Boc-Ser(O-Prop)-OH17
General procedure for the synthesis of peptide 9
via Ugi-4CR
N-tert-butoxycarbonyl-L-serine 16 (2.000 g, 10 mmol)
was dissolved in DMF (5 mL) and the solution was
placed in ice bath. Sodium hydride [0.880 g, 22 mmol,
A
solution of Fmoc-Lys(N )-CONH
3
6
(0.260 g,
2
1.5 mmol) and 4-cyano-benzaldehyde (0.200 g, 1.5 mmol)
in MeOH (5 mL) was added for the formation of imine.
After 1 h, Boc-Ser(O-Prop)-Ala-Pro-Pro–Pro-Arg(Pbf)-
COOH 7 (1.520 g, 1.5 mmol) was added, this reaction
followed with addition of cyclohexyl isocyanide (0.2 mL,
1.5 mmol). The mixture was stirred for 48 h. After com-
pletion of the reaction, solvent was evaporated in vacuum.
The crude oil was purified by flash column chromatogra-
phy. The product 9 was obtained as a white solid (1.730 g,
6
0 % (w/w) dispersion in mineral oil] was added slowly
and the reaction mixture stirred for 1 h at 0 °C. Then,
propargyl bromide (1.3 mL, 11 mmol) was added drop-
wise to the mixture; yellow solution was formed. The
solution was placed in ice bath for 1 h. Then, ice bath
was removed and the solution was stirred at room tem-
perature for 4 h. The progress of reaction was followed
by thin layer chromatography (30:20:1, petro-
leum ether:ethylacetate:acetic acid). Water (15 mL) was
added to the mixture which was then washed with
diethylether (3 9 10 mL). The aqueous phase was acid-
ified to pH 3 by adding 10 % HCl. The solution was
extracted from acidic solution with ethylacetate
82 %).
?
HRMS (ESI-FT-ICR) m/z: [M?Na]
Calcd for
C H N NaO S, 1,430.72662, Found 1,430.72659.
6
9
98 16
14
General procedure for the synthesis of cyclopeptide 10
through Huisgen 1,3-Dipolar cycloaddition reaction
(
3 9 25 mL). The organic phase was dried using mag-
nesium sulfate and the solvent was removed under vac-
uum. Orange viscous oil was achieved and purified by
column chromatography (30:20:1, petroleum ether:ethyl-
acetate:acetic acid). The solvent was evaporated afford-
ing a pale yellow, viscous oil (1.820 g, 75 %), with
spectral characteristics in accordance with literature data
Freshly prepared 9 (1.500 g, 1.07 mmol) was dissolved in
CH Cl (1,000 mL). N,N-Diisopropylethylamine (0.55 mL,
2
2
3.41 mmol) and copper(I) iodide triethylphosphite 18
(0.370 g, 1.61 mmol) were added to the reaction mixture.
The reaction mixture was stirred and protected from light for
5 days at room temperature. The solvent was evaporated.
The residue was redissolved in CH Cl and purified by flash
(
Brink et al. 2006; Jacobsen et al. 2011).
1
t
H NMR (500 MHz, CDCl ) d = 1.44 (s, 9H, Bu), 2.45
3
2
2
(
t, 1H, J = 2.6 Hz, CCH, rotamer 1), 2.49 (t, 1H,
column chromatography (14:1:1 CH Cl :acetone:MeOH)
2 2
J = 2.4 Hz, CCH, rotamer 2) 3.78 (dd,1H, J = 9.4 Hz,
J = 3.6 Hz, CH2 serine), 3.98 (dd,1H, J = 9.5 Hz,
affording the title compound as a yellow solid (1.130 g,
?
75 %). HRMS (ESI-FT-ICR) m/z: [M?Na] Calcd for
J = 3.3 Hz, CH2 serine), 4.14 (d, 2H, J = 2.3 Hz, O–
a
C H N NaO S 1,430.57978; Found 1,430.57972.
69
98 16
14
CH CCH, rotamer 1), 4.49–4.51(m, 1H, CH ), 4.75 (d, 2H,
2
J = 2.4 Hz, O–CH CCH, rotamer 2), 5.38 (d, 1H,
2
Fmoc-Lys-OH 23
J = 8.6 Hz, NH) ppm; IRmmax (neat) 3,440 (COOH), 3,294
-
1
(
NH), 1,715, 1,692 (C=O) cm
.
A 50 % (v/v) solution of TFA in CH Cl (16 mL) was
2
2
added to Fmoc-Lys(Boc)-OH 11 (4.680 g, 10 mmol) and
then triethylsilane (1.6 mL, 10 mmol) as a scavenger was
added to reaction mixture. The mixture was stirred for 2 h
at room temperature. The solvent and bulk of excess TFA
were removed under vacuum. The solution of sodium
Copper(I) iodide triethyl phosphate 18
Copper iodide (1.000 g, 5.3 mmol) prepared by Nishiz-
awa method was added to a round bottle containing
1
23

Design and synthesis of anti-cancer cyclopeptides
hydroxide (1 M) was added to the residue; the product was
precipitated at pH 6. The precipitate was filtered and
washed with water and dried affording a white powder
attached to the 2-CTC resin (1.000 g) with DIPEA
(1.37 mL, 8 mmol) in anhydrous DCM:DMF (10 mL, 1:1)
at room temperature for 2 h. After filtration, the resin was
capped by a solution of DCM/MeOH/DIPEA (17:2:1,
24 mL) for 30 min. Then, it was filtered and washed
thoroughly with DCM (1 9 7 mL), DMF (4 9 7 mL). The
resin-bound Fmoc-amino acid was treated with 25 %
piperidine in DMF (15 mL) for 30 min and the resin was
washed with DMF (3 9 7 mL). Then a solution of Fmoc-
Arg(Pbf)-OH (0.971 g, 1.5 mmol), TBTU (0.480 g,
1.5 mmol), and DIPEA (0.6 mL, 3.5 mmol) in 7 mL DMF
was added to the resin-bound free amine and shaken for 1 h
at room temperature. After completion of coupling, resin
was washed with DMF (4 9 7 mL) and DCM (3 9 7 mL).
The produced dipeptide was cleaved from resin by treat-
ment of TFA (1 %) in DCM (55 mL) and neutralization
with pyridine (4 %) in MeOH (17 mL). The solvent was
removed under reduced pressure and precipitated in water
and dried affording a powder (1.202 g).
(
3.500 g, 95 %).
1
mp: 247–249 °C;
H
NMR (300 MHz, CDCl3)
d = 1.41–1.83 (m, 4H, 2CH ), 1.94 (quin, 2H, CH ),
2
2
3.01–3.10 (m, 2H, CH NH ), 3.36 (t, 1H, J = 7.1 Hz, CH
2 2
fluorene),4.18 (t, 1H, J = 6.5 Hz, CHCOOH), 4.35–4.40 (m,
H, –OCH ), 4.76 (brs, 2H, NH ), 5.94 (d, 1H, J = 7.6 Hz,
2
2
2
NHCOOCH fluorenyl), 7.28 (t, 2H, J = 7.3 Hz, H–Ar), 7.37
2
(
t, 2H, J = 7.3 Hz, H–Ar), 7.73 (d, 2H, J = 7.4 Hz, H–Ar),
.57 (d, 2H, J = 6.9 Hz, H–Ar) ppm; IRmmax (KBr): 3,330
NH, COOH), 3,052 (CH aromatic), 2,940 (CH aliphatic),
7
(
-1
1
,689 (C=O), 1,596 (C=C) cm .
Fmoc-Lys(N )-OH 24
3
A suspension of Fmoc-Lys-OH 23 (2.947 g, 8 mmol) with
potassium carbonate (2.730 g, 19.7 mmol) and copper
sulfate pentahydrate (0.017 g, 0.07 mmol) in methanol
?
Mass (ESI-triple quadrupole) m/z: [M?H] Found for
(
50 mL) was prepared in a proper round bottle. The
imidazole-1-sulfonyl azide hydrochloride (2.000 g,
.5 mmol) was added to the suspension slowly. The mix-
C H N O S 803.10000.
40 51 8 8
9
Amidation of C-terminal of dipeptide 26
ture was stirred for 20 h at room temperature. After com-
pletion of reaction, the solvent was removed under
The dipeptide 25 (1.200 g, 1.5 mmol) and N-methyl mor-
pholine (0.8 mL, 7.5 mmol) were added to a solution of
vacuum. Then 120 mL H O was added to reaction mixture
2
and the pH was diminished to 2 by consumption of con-
centrated HCl. The achieved acidic solution was extracted
by ethylacetate (3 9 20 mL) and finally the whole organic
phase was washed by brine. The organic phase was dried
by magnesium sulfate and the solvent was distilled. Yellow
oil (2.360 g, 75 %) was formed (Sabido 2009).
TBTU (0.740 g, 2.3 mmol) and NH Cl (0.160 g,
4
3.0 mmol) in NMP (3 mL). The mixture was stirred
overnight. The dipeptide was precipitated in water and the
C-terminal amidated dipeptide 26 was dried affording a
yellow powder (1.040 g, 87 %).
1
mp: 224–228 °C; H NMR (300 MHz, DMSO-d6)
General procedure for Fmoc deprotection of amidated
d = 1.27–1.73 (m, 6H, 3CH ), 3.26 (t, 2H, J = 6.5 Hz,
2
dipeptide 19
CH N ), 4.22 (t, 1H,J = 7.0 Hz, CH fluorene), 4.43 (d, 2H,
2
3
a
J = 6.8 Hz, –OCH ), 4.50–4.54 (m, 1H, CH COOH), 5.58
2
Purified amidated dipeptide 26 (0.96 g, 1.2 mmol) was
added to ethylacetate (2 mL), and then piperidine (0.5 mL,
(
d, 1H, J = 8.2 Hz, fluorenylCH OOCNH), 7.32 (t, 2H,
2
J = 7.3 Hz, H–Ar), 7.41 (t, 2H, J = 7.3 Hz, H–Ar), 7.55
d, 1H, J = 7.3 Hz, H–Ar), 7.60 (d, 1H, J = 7.3 Hz, H–
5
mmol) was added. The reaction mixture was stirred at
(
room temperature. After 3 h, reaction mixture was
extracted by water (3 9 10 mL). The aqueous phases were
washed by fresh ethylacetate (1 9 10 mL) again. Aqueous
phase was dried using natrium sulfate. Yellow viscous oil
1
Ar), 7.76 (d, 2H, J = 7.3 Hz, H–Ar) ppm; C NMR
3
(
75 MHz, DMSO-d ) d = 22.9, 27.8, 30.3 (CH ), 50.5 (CH
6
2
a
fluorene), 54.9 (CH N ), 55.5 (CH COOH), 61.1(OCH ),
2
2
3
1
1
20.0, 120.1 121.2, 123.9, 125.3, 127.1, 127.6, 129.5,
35.3, 140.7, 143.8, 143.9 (C–Ar), 156.2 (CONH), 171.8
(
0.62 g, 90 %) was obtained.
Mass (ESI-triple quadrupole) m/z: [M?H] Found for
C H N O S 580.10000.
?
(
COOH) ppm; IRmmax (KBr): 3,455 (NH, COOH), 3,152
CH aromatic), 2,089 (N ), 1,743 (C=O carboxylic acid),
2
5 42 9 5
(
3
-
1
1
,670 (CONH) cm
.
General procedure for the synthesis of pentapeptides-
COOH 20
General procedure for preparation of dipeptide 25
The synthesis of pentapeptide 25 was done using the
standard Fmoc SPPS strategy. At first, Fmoc-Pro-OH was
loaded on the surface of resin. The two times Fmoc-Pro-
OH, Fmoc-Ala-OH, and Boc-Ser(O-Prop)-OH were loaded
The synthesis of dipeptide was carried out using 2-chlo-
rotrityl chloride resin (1.0 mmol/g) following the standard
Fmoc strategy. Fmoc-Lys(N )-OH (0.790 g, 2 mmol) was
3
123

F. Tahoori et al.
on the surface of resin. The peptide was removed from the
surface of 2-CTC resin using 1 % TFA based on the known
procedure.
surface. The peptide was removed from the surface of resin
using TFA (1 %) based on the known procedure.
?
HRMS (MALDI-FT-ICR) m/z: [M?H] Calcd for
?
HRMS (ESI-FT-ICR) m/z = C H N O [M?H]
2
C H N O S
54 82 13 14
1168.58514,
Found
1,168.58489;
9
44
6
9
?
[M?Na] Calcd for C H N NaO S 1,190.57059,
Found 606.31390, Calc. 606.31399, C H N NaO
2
9
43
6
9
6
54 81 13
?
14
?
M?Na] Found 628.29535, Calc. 628.29536, C H KN
[
Found 1,190.57048; [M?K] Calcd for C H KN O S
54 81 13 14
2
9
44
?
O [M?K] Found 644.26965, Calc. 644.26971.
1,206.52828, Found 1,206.52901.
9
General procedure for the synthesis of peptide 21
via Ugi-4CR
General in vitro experiments
Cancer cells were all seeded at 5,000 cells/well in a
6-well plate and the culture was maintained in RPMI 1640
A solution of H N-Arg(Pbf)-Lys(N )-NH 19 (0.500 g,
3
2
2
9
0
0
.86 mmol)
and
4-cyano-benzaldehyde
(0.120 g,
supplemented with 10 % fetal bovine serum, 1 % L-gluta-
mine, 100 units/mL penicillin, and 100 lg/mL streptomy-
cin overnight. The media was replaced with fresh media
containing up to 50 lg of peptides and incubated for 48 h
.86 mmol) in MeOH (3 mL) was added for the formation
of imine. After 1 h, Boc-Ser(O-Prop)-Ala-Pro-Pro–Pro-
COOH 20 (0.520 g, 0.86 mmol) was added, this reaction
was followed with addition of cyclohexyl isocyanide
in a humidified atmosphere of 95 % air and 5 % CO at
2
(
0.11 mL, 0.86 mmol). The mixture was stirred for 48 h.
3
7 °C until the control cultures were confluent. The media
Further purification was done using flash column chroma-
tography. The desired product was achieved as yellow oil
was then removed and the plate was washed two times with
phosphate-buffered saline (PBS). Serum-free media
(
1.030 g, 85 %).
(100 lL) containing 0.5 mg/mL MTT dye was added into
?
Mass (ESI-ion trap) m/z: [M?Na]
Found for
each well and incubated at 37 °C for 2 h. The media with
dye was removed, washed with PBS and the reactive dye
was solved by addition of 100 lL dimethylsulfoxide
C H N NaO S 1430.33.
6
9
98 16
14
General procedure for the synthesis of cyclopeptide 22
via Huisgen 1,3-Dipolar cycloaddition reaction
(DMSO). The absorbance was read using an automatic
multiwell spectrophotometer. The experiment was always
performed in triplicates.
Freshly prepared 21 (1.100 g, 0.8 mmol) was dissolved in
CH Cl (900 mL). Then, N,N-diisopropylethylamine
2
2
Acknowledgments S. B. gratefully acknowledges Iran National
Science Foundation (INSF). We are also thanking Iranian ministry of
health and medicinal education and also presidential office deputy of
science and technology for financial support.
(
(
0.4 mL, 2.4 mmol) and copper(I) iodide triethylphosphite
0.272 g, 1.2 mmol) were added to reaction mixture. The
reaction mixture was stirred and protected from light for
days at room temperature. The solvent was evaporated.
The residue was redissolved in CH Cl and purified by
5
Conflict of interest Meanwhile, I certify that there is no conflict of
interest with any financial organization regarding the material dis-
cussed in the manuscript.
2
2
flash column chromatography (14:1:1 CH Cl :ace-
2
2
tone:MeOH) affording the title compound as a yellow solid
(
0.820 g, 73 %).
Mass (ESI-ion trap) m/z: [M?H] Found for C H
9 98
?
6
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6 14
?
HRMS (ESI-FT-ICR) m/z: [M-(C H N )] Calcd for
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10
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(
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