Luliberin analogues exhibiting a cytotoxic effect on tumor cells in vitro

Article abstract of DOI:10.1134/S1068162006050037

Luliberin analogues modified at the N-terminus were synthesized to search for drugs exerting a cytotoxic effect on cells of hormone-dependent tumors. A synthetic scheme effective in the preparation of analogues containing fatty acid residues was proposed. The cytotoxic effect of the peptides was studied on a number of cell lines of human tumors in vitro. The dependence of the antitumor effect on the length of peptide chain, amino acid sequence, and structure of the N-terminal group was demonstrated. Modification with palmitic acid was found to result in highly active compounds in the case of analogues containing more than ten aa, whereas modifications with lauric, caproic, or trimethylacetic acid led to compounds with significantly lower activities. Analogues of luliberin containing a palmitic acid residue and effectively inhibiting the growth of tumor cells in vitro were synthesized. Pleiades Publishing, Inc., 2006.

Full text of DOI:10.1134/S1068162006050037

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2006, Vol. 32, No. 5, pp. 413–419. © Pleiades Publishing, Inc., 2006.
Original Russian Text © S.V. Burov, T.V. Yablokova, M.Yu. Dorosh, Z.P. Shkarubskaya, M. Blank, N. Epstein, M. Fridkin, 2006, published in Bioorganicheskaya Khimiya, 2006,
Vol. 32, No. 5, pp. 459–466.
Luliberin Analogues Exhibiting a Cytotoxic Effect
on Tumor Cells In Vitro
a, 1
a
a
a
S. V. Burov , T. V. Yablokova , M. Yu. Dorosh , Z. P. Shkarubskaya ,
b
b
c
M. Blank , N. Epstein , and M. Fridkin
a
Institute of Macromolecular Compounds, Russian Academy of Sciences, Bol’shoi pr. 31, St. Petersburg, 119004 Russia
b
Chaim Sheba Medical Center, Tel-Hashomer, Israel
c
Weizmann Institute of Science, Rehovot, Israel
Received October 26, 2005; in final form, November 14, 2005
Abstract—Luliberin analogues modified at the N-terminus were synthesized to search for drugs exerting a
cytotoxic effect on cells of hormone-dependent tumors. A synthetic scheme effective in the preparation of ana-
logues containing fatty acid residues was proposed. The cytotoxic effect of the peptides was studied on a num-
ber of cell lines of human tumors in vitro. The dependence of the antitumor effect on the length of peptide chain,
amino acid sequence, and structure of the N-terminal group was demonstrated. Modification with palmitic acid
was found to result in highly active compounds in the case of analogues containing more than ten aa, whereas
modifications with lauric, caproic, or trimethylacetic acid led to compounds with significantly lower activities.
Analogues of luliberin containing a palmitic acid residue and effectively inhibiting the growth of tumor cells in
vitro were synthesized.
Key words: antitumor activity, luliberin, palmitic acid, synthetic peptides
DOI: 10.1134/S1068162006050037
INTRODUCTION
found on the cells of most adenocarcinomas including
pulmonary, intestinal, and ovarian adenocarcinomas
and adenocarcinomas of mammary gland, kidney, and
liver [6]. At the same time, they are present at signifi-
cantly lower levels or absent in normal tissues, except
for hypophysis. An increased expression of luliberin
receptors in the tumor cells along with peculiarities of
intracellular metabolism and signal transduction cre-
ates a basis for some selectivity of the action of thera-
peutics developed on the basis of luliberin analogues
Tumors of mammary gland, ovary, uterus, and pros-
tate gland are now the most abundant malignant dis-
2
eases. A characteristic feature of these tumors is their
appearance in hormone-depending organs and their
sensitivity to steroid hormones. The use of analogues of
luteinizing and follicle-stimulating releasing hormones
in the treatment of these tumors is a variant of therapy
of these diseases. Their long-term administration
results in a dramatic decrease in the concentration of
steroid hormones in blood and an inhibition of tumor
growth [1–3]. One of the drawbacks of this approach is
a gradual transformation of tumor with a loss of its sen-
sitivity to the hormonal treatment. At the same time, it
was demonstrated that cells of these tumors contained
[
7, 8]. In this case, the effect on a tumor may be com-
plex and may be associated with an inhibition of the
level of steroid hormones, induction of apoptosis, and,
possibly, with a stimulation of immune system.
The direct effect of luliberin analogues on tumor
on their surface the luliberin receptors that were pre- cells is low [9]. As a rule, they are used as vectors for
served even after the loss of sensitivity to steroid hor- the directed delivery of a cytotoxic agent to a target,
mones [4, 5]. Last years, the luliberin receptors were which permits a reduction of effective dose and undes-
ired side effects [10, 11]. The advantages of this
1
Corresponding author; phone/fax: (812) 323-3607;
approach are the action on not only the hormone-
e-mail: burov@hg.macro.ru.
dependent tumor cells but also on the hormone-inde-
2
Abbreviations: ATCC, American Type Cultures Collection;
pendent cells and the prevention of the tumor transition
Bzl(Cl ), 2,6-dichlorobenzyl; Cpa, 4-chlorophenylalanine; DGlu
2
(
AA), 4-(p-methoxybenzoil)-D-2-aminobutyric acid; hArg, into the state insensitive to the hormonal therapy. At the
homoarginine; Hcit, homocitrulline; GnRH, luliberin (gonadotro-
pin-releasing hormone); Hx, hexanoyl; Lau, lauryl; MTS, mesity-
lenesulfonyl; Nal, 3-(2-naphthyl)alanine; Nic, nicotinoyl; Pal, 3-
same time, some difficulties during the preparation of
hybrid preparations stimulate the search for the
luliberin analogues that themselves exhibit a cytotoxic
effect.
(
3-pyridyl)alanine; Pam, palmitoyl; Piv, pivaloyl; Z(Cl), 2-chlo-
robenzyloxycarbonyl.
4
13

4
14
BUROV et al.
Biologically active conformation of luliberin with primary increase in the level of steroid hormones
involves β-turn in the 5−8 site of the natural sequence [15, 16].
as the main structural element (Fig. 1) [12]. The intro-
The most widely used approach to preparation of
duction of D-amino acid residues in position 6, which therapeutic agents on the basis of luliberin analogues is
facilitate the β-turn formation, is widely used in the currently the multiple modifications with the use of
synthesis of highly effective luliberin analogues [13]. unnatural amino acid residues of various structures. A
An additional removal of His residue or a substitution number of highly active agonists (see below) are rec-
of D-amino acid residues for the His result in effective ommended abroad for practical use. No more than 3–
antagonists [14]. Both types of the analogues are used 5-amino acid residues of the natural sequence are
in clinical praxis. They exhibit higher or lesser effi- involved into the structure of these analogues. Such
ciency in dependence on the concrete tumor type. The strategy considerably increases the price of these prep-
disadvantage of luliberin agonists is the stimulation of arations and, in some cases, results in toxic or aller-
tumor growth at the early stage of therapy associated genic compounds [17, 18].
pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
10
Luliberin
1
2
3
4
5
6
7
8
9
Ac-DNal-DCpa-DPal-Ser-MeTyr-DAsn-Leu-Lys(iPr)-Pro-DAla-NH2
Ac-DNal-DCpa-DPal-Ser-Tyr-DHcit-Leu-Lys(iPr)-Pro-DAla-NH2
Ac-DNal-DCpa-DPal-Ser-Tyr-DCit-Leu-Arg-Pro-DAla-NH2
Abarelix
Antarelix
Cetrorelix
Ganirelix
Iturelix
Ac-DNal-DCpa-DPal-Ser-Tyr-DhArg(Et )-Leu-hArg(Et )-Pro-DAla-NH
2
2
2
Ac-DNal-DCpa-DPal-Ser-NicLys-DNicLys-Leu-Lys(iPr)-Pro-DAla-NH2
Ac-DNal-DCpa-DPal-Ser-Arg-DGlu(AA)-Leu-Arg-Pro-DAla-NH2
Nal-Glu
Structures of luliberin and several of its analogues used in medicine.
RESULTS AND DISCUSSION
carcinoma of prostate in vivo and its in vitro ineffi-
ciency, which indicated the absence of its direct
cytotoxic effect [19]. At the same time, peptide (II)
modified with a palmitoyl residue was effective in
The goal of this study is the preparation of luliberin
analogues with cytotoxic effect on the cells of human
adenocarcinoma. We introduced minimum number of
D-amino acids with a plain structure (D-Phe, D-Lys) growth inhibition of the cells of ovary carcinoma in
into the natural sequence, which differs our approach vitro and prostate carcinoma in vivo [20]. Therefore,
from that used usually.
we pay a substantial attention to the choice of struc-
The basis of this study was our previous result: a ture of the N-terminal group in new luliberin ana-
high antitumor effect of peptide (I) on the model of logues.
H-Pro-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
Pam-Pro-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
Piv-Pro-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
(I)
(II)
(III)
Lau-Pro-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
H-Pro-DNal-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
(IV)
(V)
Pam-Pro-DNal-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
H-Pro-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-NHEt
(VI)
(VII)
(VIII)
(IX)
(X)
(XI)
(XII)
(XIII)
(XIV)
(XV)
Pam-Pro-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-NHEt
H-Pro-Gly-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
H-Gly-Pro-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
Pam-Pro-Gly-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
Lau-Pro-Gly-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
Hx-Pro-Gly-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
Pam-Gly-Pro-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
Pam-Pro-Gly-Pro-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
Pam-DPro-Gly-Pro-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2 (XVI)
Pam-Lys-Pro-Gly-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
Pam-Lys-Gly-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
(XVII)
(XVIII)
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006

LULIBERIN ANALOGUES EXHIBITING A CYTOTOXIC EFFECT
415
Pam-DLys-Pro-Gly-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2 (XIX)
Pam-DLys-Gly-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
Pam-DPro-Gly-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
Pam-Pro-Pro-Pro-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
(XX)
(XXI)
(XXII)
Pam-DPro-Pro-Pro-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2 (XXIII)
Pam-Pro-Gly-DLeu-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
Pam-Pro-Gly-DPhe-Ala-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
Pam-Pro-Ala-DPhe-Pro-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2
(XXIV)
(XXV)
(XXVI)
Amino acid sequences of the synthesized luliberin analogues
We prepared a series of analogues (III)–(XXVI). erature data about a high antagonistic activity of the
The peptides were synthesized by the solid phase luliberin analogues containing aromatic D-amino acid
method on an NPS-4000 semiautomatic synthesizer residues in position 2 [23]. One can see from Table 2
using the Boc/Bzl-strategy. Analogues (III)–(VI) and that the elongation of peptide chain up to 11 aa residues
(
IX)–(XXVI) were obtained using 4-methylbenzhy- [analogues (XI) and (XIV)] or its truncation [analogue
drylaminopolymer; compounds (VII) and (VIII), on (VIII)] give rise to highly active substances.
the Merrifield resin. The hydrophobic N-terminal group
was attached by the carbodiimide method using free
Note that only few luliberin analogues containing
more than 10 amino acid residues are described in liter-
palmitic, lauric, and trimethylacetic acids or the corre-
ature [24]. First of all, this is associated with an impor-
sponding derivatives of N-terminal amino acid resi-
tant role of the N-terminal amino acid in the exhibition
dues. Peptides (III)–(VI) and (IX)–(XXVI) were
of biological activity of this releasing-hormone. For
example, on the basis of conformational calculations,
cleaved from polymer (with the simultaneous deprotec-
tion) by treating with 1 M solution of trifluoromethane-
Momany [25] proposed that certain spatial orientation
sulfonic acid in trifluoroacetic acid in the presence of
of the pyroglutamic acid ring should be realized for the
thioanisole and ethanedithiol. Analogues (VII) and
exhibition of agonistic or antagonistic activity. At the
(VIII) were cleaved from the polymeric support by the
same time, there are data about the possibility of intro-
duction of N-terminal Pro, whose structure is similar to
that of pyroglutamic acid, or unnatural amino acids in
the analogue sequences (in the case of luliberin antag-
onists) [26, 27].
treatment with anhydrous ethylamine [21] at 0°C with
the subsequent removal of protecting groups under the
above-described conditions. After deprotection, the
peptides were desalted by gel filtration on a Sephadex
G-15 column eluted with 50% acetic acid. They were
further purified by preparative HPLC. All the prepared
compounds were characterized by analytical HPLC and
mass spectrometry (Table 1).
The proper choice of N-terminal group was impor-
tant for the further studies, especially on the basis of the
The cytotoxic activity of the analogues synthesized
was studied on various lines of human adenocarcinoma
cells (ATCC), including adenocarcinomas of mammary
gland, prostate, ovaries, intestine, and liver, which con-
tained the luliberin receptors [6]. The F-89 normal
fibroblasts were used as control cells. The tumor cells
were incubated in the presence of different amounts of
the studied peptides for 72 h. The efficiencies of the
analogues were evaluated according to staining of the
viable cells (Hemacolor assay [22]). The results
obtained for the Colo-205 cell line, proved to be the
most sensitive to the peptides (Fig. 1), are given in
Table 2.
We chose the starting structure for the modification
on the basis of comparison of the antitumor activities of
peptides (V), (VII), (IX), and (X) with those of ana-
logues (VI), (VIII), (XI), (XIV), (XXIV). We analyzed
the influence on the efficacy of action of the peptides of
both the length of peptide chain and the role of the
amino acid residue in position 2 of the natural
sequence. Substitution of D-leucine for D-phenylala-
nine [peptide (XXIV)] results in a significant decrease
in the activity, which is in a good agreement with the lit-
Viable cells, %
1
00
8
6
4
2
0
0
0
0
MDA MB-231
HCT-116
Colo-205
0
3.125 × 10–6 1.25 × 10–5
5 × 10–5
1 × 10–4
Concentration of the peptide, å
7.8125 × 10–6 6.25 × 10–6 2.5 × 10–5
Fig. 1. Sensitivity of various lines of tumor cells to the
action of analogue (XI).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006

4
16
BUROV et al.
Table 1. Yields and physicochemical characteristics of
luliberin analogues
We synthesized peptides (III), (IV), (XII), and
XIII) to study the effect of the N-terminal group struc-
(
ture on the antitumor effect of analogues in vitro. A
comparison of the efficiency of these compounds and
analogues (I), (II), (V), (VI), (VII), (VIII), and (IX)–
(XI) confirms the importance of the palmityl residue
for the cytotoxic effect of the analogues. Shortening of
the carbon chain of the N-terminal group [peptides
Molecular mass
CH CN, Yield, mg
3
%
Peptide
*
(%)
determined
by MS**
calculated
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
III)
28
46
23
68
22
55
17
19
54
40
29
56
46
48
40
39
38
41
48
47
48
48
48
49
15 (24)
23 (34)
23 (38)
26 (36)
20 (27)
13 (15)
16 (26)
15 (25)
32 (44)
24 (34)
33 (49)
26 (36)
29 (38)
35 (45)
23 (28)
31 (42)
34 (43)
24 (32)
36 (48)
23 (29)
21 (27)
23 (32)
21 (33)
31 (42)
1243.71
1341.82
1209.67
1447.90
1130.66
1368.89
1216.67
1216.67
1454.90
1398.84
1314.74
1454.90
1551.95
1551.95
1583.00
1485.94
1583.00
1485.94
1454.90
1591.99
1591.99
1420.92
1428.89
1468.92
1243.70
1341.98
–***
IV)
(IV), (XII), and (XIII)] or the use of branched trimeth-
V)
ylacetic acid [peptide (III)] result in a dramatic
decrease in the effect. Moreover, none of the examined
analogues, except the palmitoyl derivatives, exhibit any
cytotoxic effect. Hence, we used palmitoyl group for
the further modification of N-terminal amino group.
VI)
1447.70
–***
VII)
VIII)
IX)
1369.09
–***
Considerable difference in the activities of peptides
XIV) and (XI) points out to the importance of the
X)
–***
(
XI)
1454.70
1398.70
1314.50
1454.80
1551.46
1551.70
1582.80
1485.80
1582.80
1485.50
1454.80
1591.84
1591.52
1420.66
1428.84
1468.70
amino acid sequence in the N-terminal part of the mol-
ecule. Next, we investigated the effect of the nature and
number of amino acid residues located between the
palmitoyl group and position 1 in the releasing-hor-
mone structure on the efficiency of cytotoxic effect of
analogues (XV)–(XXIII) in vitro. We found that the
incorporation of Pro-Gly and D-Pro-Gly sequences
[peptides (XV) and (XVI)] into the sequence of ana-
logue (II) decreases the activity. A similar effect is
observed in the case of attachment of Lys residue to the
N-terminal Pro in peptide (XI). At the same time, trun-
cation of the amino acid sequence and/or application of
D-Lys [analogues (XVIII)–(XX)] result in the highly
effective compounds. Note that the presence of addi-
tional ε-amino group in the side chain can be used for
the synthesis of conjugates with the antitumor agents
with no influence on the active site of the releasing hor-
mone.
XII)
XIII)
XIV)
XV)
XVI)
XVII)
XVIII)
XIX)
XX)
XXI)
XXII)
XXIII)
XXIV)
XXV)
XXVI)
Significant influence of point modifications on the
cytotoxic effect of the analogues along with the preser-
vation of the physicochemical properties of peptide
*
Concentration of acetonitrile corresponding to the product
elution in the course of the gradient HPLC.
+
*
* Calculated for [M + H] .
[
substitution ofAla for Pro and Gly residues in peptides
*
** Peptides served as precursors for the obtaining of ana-
logues (VI), (VIII), (XI), and (XIV) and were used
without the MS characterization.
(
XXV) and (XXVI), respectively] indicate the specific-
ity of analogues and the possible participation of the
luliberin receptors in this process. The absence of cyto-
toxic effect on the normal human fibroblasts (they are
data on the antitumor activity of peptides (I) and used as control cells and do not contain the correspond-
(
II) [20].
ing receptors) in the analogues at the doses lethal for
the tumor cells additionally confirms this proposal. The
typical dependence of the number of viable Colo-205
cells on the effective dose is given in Fig. 2 for peptides
The modification of peptide antigens by the residue
of palmitic acid is widely used for the enhancement of
immune response. However, there are many reports (XX) and (XXV).
about the apoptosis-inducing effect of palmitic acid and
its selective cytotocxic action on tumor cells [28–30].
Therefore, we decided to use the N-terminal palmitoyl
group for increasing the antitumor activity of luliberin
analogues. In addition, such modification could facili-
tate the peptide penetration through the blood-brain
Thus, luliberin analogues with a high cytotoxic
activity towards various cell lines of human adenocar-
cinoma were obtained. Biological trials demonstrated
that the use of palmitic acid in the design of analogues
with increased efficiency is promising. Further studies
of both the antitumor activity of the synthesized ana-
barrier. This property is particularly important for the logues in vivo and the possibility of their use as carriers
therapy of brain tumors.
for cytotoxic agents are of interest.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006

LULIBERIN ANALOGUES EXHIBITING A CYTOTOXIC EFFECT
417
EXPERIMENTAL
Table 2. Cytotoxic effect of luliberin analogues on the Co-
lo-205 cells of human intestinal carcinoma
Reagents for peptide synthesis and amino acid
derivatives from Sigma, Fisher Scientific, and Bachem
United States); Reanal (Hungary); Saxon Bichemicals
GMBH (Germany); 4-methylbenzhydrylaminopoly-
Peptide
GnRH
mer (Sigma, United States); and Merrifield’s polymer (III)
Dose, 10–4
M
Cytotoxic effect*
(
>1
–
–
>1
(
Novabiochem, Switzerland) were used in this study.
(
(
(
IV)
>1
–
Dimethylformamide was distilled before use and stored
over 4 Å molecular sieve. Melting points were deter-
mined on a Boetius hot plate (Germany). Homogeneity
V)
>1
–
VI)
0.5
>1
+
of the prepared derivatives of the N-terminal amino acid (VII)
–
residues was determined by TLC on precoated Merck F
54 plates (Germany) in the following chromato-
graphic systems: (A) 1 : 3 1% ammonia–sec-butanol,
B) 9 : 2 chloroform–methanol, and (C) 90 : 7 : 3 chlo-
(
(
(
VIII)
0.5
>1
++++
–
2
IX)
X)
>1
–
(
roform–methanol–acetic acid. The substances were (XI)
0.2
>1
++++
–
detected on TLC plates in UV light at 254 nm and by
the treatment with sulfuric acid and subsequent heat-
ing.
The solid phase peptide synthesis was achieved on
an NPS-4000 semiautomatic synthesizer (Neosystem
Laboratories, France).
(XII)
(XIII)
(XIV)
(XV)
>1
–
1
+++
++
++
++
+++
++++
++++
++
++
++
++
++++
++
0.5
0.5
0.5
0.5
0.35
0.5
0.5
0.5
0.5
0.5
0.35
0.5
(XVI)
Peptides were desalted by gel filtration on Sephadex (XVII)
G-15 (Pharmacia, Sweden). HPLC was carried out on a
System Gold chromatograph (Beckman, United States)
equipped with a Zorbax 300SB-C18 column ((4.6 ×
(XVIII)
(XIX)
(XX)
1
50 mm, 5 µm) for the analytical chromatography and
Discovery C18 column (10 × 250 mm, 5 µm) for the (XXI)
preparative chromatography. The analytical and prepar-
(XXII)
(XXIII)
(XXIV)
ative columns were eluted with a gradient of acetoni-
trile in 0.1% H PO at a flow rate of 1 ml/min and a gra-
3
4
dient of acetonitrile in 0.1% TFA at a flow rate of
5
ml/min, respectively (the acetonitrile percents corre- (XXV)
sponding to the elution of the products are given in
Table 1). The products were detected at 230 nm. Ana-
logues modified with palmitic, lauric, and trimethylace-
tic acids were preliminarily purified by the method of
solid phase extraction on a LiChroprep RP-18 column
(XXVI)
*
Numbers of + signs are proportional to a decrease in the number
of viable tumor cells determined according to a change in optical
absorption of eluate at 650 nm.
(
15 × 250 mm, Merck, Germany). Amino acid analysis
was carried out on a Microtechna T339M analyzer
Czech Republic) after the peptide hydrolysis in 6 N
with the content of amino groups of 1 mmol/g. The side
functions of Ser, Tyr,Arg, and Lys were blocked by Bzl,
(
HCl at 110°ë for 24 h. The mass spectra were recorded
on a MX-5303 time-off-flight mass spectrometer at
electrospray ionization (FINEPKhF, Russian Academy
of Sciences) and reflect mode.
Bzl(Cl ), Mts, and Z(Cl) protecting groups, respec-
2
tively. Boc-group served as a temporary protection for
α
the N -function.
The peptide chain was elongated on a NPS-4000
semiautomatic peptide synthesizer (Neosystem Labo-
ratories, France) according to the following protocol
The antitumor activity was studied on the following
lines of human adenocarcinoma cells (ATCC, United
States), including the MCF7 and MDA MB-231 cells of
adenocarcinoma of mammary gland, the DU-145,
PC-3, 22RV-1, and LNCaP cells of adenocarcinoma of
prostate, the OVCAR3 ovarian adenocarcinoma cells,
the SW-48, HT-29, and Colo-205 intestinal adenocarci-
noma cells, and the Hep G2 cells of liver adenocarci-
noma. The viable tumor cells were counted according
to the change in optical absorption at 650 nm (ELISA
reader EL 800 BioTek Instruments, United States).
for every cycle: (1) CH Cl (1 min); (2) 65%
2
2
TFA/CH Cl (1 and 15 min); (3) CH Cl (2 × 0 min); (4)
2
2
2
2
CH Cl (2 × 1 min); (5) DMF (1 min); (6) 5%
2
2
TEA/DMF (1 and 2 min); (7) DMF (3 × 1 min); (8) cou-
pling (2 h); (9) DMF (2 × 1 min); and (10) CH Cl
2
2
(1 min).
Fourfold excesses of the active esters of the corre-
sponding Boc-amino acids with 1-hydroxybenzotriaz-
Peptides (III)–(VI) and (IX)–(XXVI) were syn- ole were used for the couplings. The esters were
thesized on the 4-methylbenzhydrylaminopolymer obtained in situ from the corresponding Boc-amino
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006

4
18
BUROV et al.
Viable cells, %
ture using tenfold excess of the corresponding free acid.
After two h, the coupling was repeated. In the course of
the peptide (III) obtaining, the residue of trimethylace-
tic acid was attached according to the standard protocol
with the use of equivalent amount of
1
00
8
6
4
2
0
0
0
0
1
-hydroxy-7-aza-benzotriazole to increase the acyla-
tion efficiency.
The peptides were cleaved from the polymer with
the simultaneous deprotection by the treatment with
1 M solution of trifluoromethanesulfonic acid in TFA
(11 ml/g of resin) in the presence of 2 : 1 thioanisole–
ethanedithiol mixture (1.5 ml/g of resin) for 2 h. The
reaction mixture was diluted with cool ether, and the
precipitate was filtered on a Schott filter. The peptide
was separated from the polymer by its dissolution in
TFA and repeatedly precipitated with ether. The precip-
itate was filtered, washed with ether, and dried in a vac-
uum. The deprotected peptides were desalted on a col-
umn (15 × 500 mm) with Sephadex G-15 in 50% acetic
acid and, further, purified by rpHPLC.
(
(
XX)
XXV)
0
1.125 × 10–6 1.25 × 10–5 5 × 10–5
.56 × 10–6 6.25 × 10–6 2.5 × 10–5 1 × 10–4
Concentration of the peptide, å
1
Fig. 2. Dependence of the percent of the Colo-205 viable
cells on the concentration of analogues.
Peptides (VII) and (VIII) were synthesized on a
Merrifield polymer (the content of chloromethyl groups
was 1.2 mmol/g) loaded with Boc-Pro-OH
acids (1 equiv), diisopropylcarbodiimide (1 equiv) and
(
0.83 mmol/g) [31] using the protocol of solid phase
1
-hydroxybenzotriazole (1 equiv) at 0°C for 30-min
synthesis described above. The peptides were cleaved
from the polymer by the treatment with anhydrous eth-
ylamine at 0°ë for 4 h [21]. Ethylamine was removed
in a vacuum, and the residues were dried in a vacuum.
The products were dissolved in acetic acid and the
polymer was filtered off. The filtrates were evaporated,
under stirring.
Peptides (IV), (XI), (XII), (XV), (XVI), and
(
XXI)–(XXVI) were synthesized with the use of Pam-
Pro-OH, Pam-D-Pro-OH, and Lau-Pro-OH derivatives
prepared by the procedures described below.
Pam-ONSu. A solution of DCC (6.39 g, 0.031 mol) and the residues were dried in a vacuum. The deprotec-
in a minimum volume of THF was added to a solution tion, desalting, and purification of the peptides were
of palmitic acid (7.95 g, 0.031 mol) and HONSu carried out by the procedure described above for ana-
(
4.28 g, 0.037 mol) in a minimum volume of THF at logues (III)–(VI) and (IX)–(XXVI). Amino acid com-
0
°C under stirring. The reaction mixture was kept for positions and molecular masses calculated from the
h at 0°C and overnight at room temperature. N,N'- mass spectrometry data (Table 1) corresponded to the
1
Dicyclohexylurea was filtered off, and the filtrate was theoretical values for all the synthesized peptides.
evaporated. The residue was dissolved in ethyl acetate,
Cytotoxic effect of the synthesized analogues was
washed with 5% solution of Na SO (three times) and
2
4
determined on various cell lines of human adenocarci-
noma in the test of staining viable cells (Hemacolor
assay [22]). Normal F-89 fibroblasts were used as con-
water, and dried with Na SO . The solvent was evapo-
2
4
rated, and the product was crystallized from hexane;
yield 5.91 g (54%); mp 82–84°C; R 0.92 (B).
f
4
trol cells. The examined cells (10 cells per well) were
Pam-Pro-OH. Triethylamine (2.7 ml, 19.2 mmol)
placed into wells of a 96-well polystyrene plate (Cell-
Star, Greiner Bio-One) in a Dulbecco’s Modified Eagle
Medium (DMEM, 200 µl) and incubated at 37°C for
was added to a solution of Pam-ONSu (3.4 g,
9
.6 mmol) and proline (1.1 g, 9.6 mmol) in a minimum
volume of DMF at 0°C. The reaction mixture was
1
6 h in a CO -incubator. On the next day, the cultural
2
stirred at room temperature for 24 h, neutralized with
medium was removed, and the solution of examined
peptide in DMEM (200 µl per well) was added to the
wells. The plates were incubated at 37°C for 72 h. The
cells were fixed with methanol and colored by a Hema-
color reagent (Merck, Germany). The reagent was
eluted with 0.5% SDS in PBS, and the optical absorp-
tion was determined at 650 nm. The number of viable
cells was calculated for every concentration of the
1
N H SO , and evaporated. The residue was dissolved
2 4
in water and acidified to pH 4. The precipitated solid
was filtered and washed with water; yield 4.2 g (92%);
mp 53°C; R 0.39 (A), 0.74 (B), 0.57 (C).
f
Pam-D-Pro-OH and Lau-Pro-OH were prepared by
the similar procedure and coupled according to the pro-
tocol of the solid phase synthesis described above.
Preparation of peptides (VI), (XIII), (XIV), and examined peptide (%) by the equation: (ODEXP/OD ) ×
K
(
XVII)–(XX). N-Terminal group was introduced by 100, where ODEXP and OD are optical absorption in
K
carbodiimide method in a 1 : 10 DMSO–CH Cl mix- the presence of the peptide in the tested concentration
2
2
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006

LULIBERIN ANALOGUES EXHIBITING A CYTOTOXIC EFFECT
419
and in the absence of the peptide. Every test was carried 16. Harrison, G.S., Wierman, M.E., Nett, T.M., and
Glode, L.M., Endocrine-Related Cancer, 2004, vol. 11,
pp. 725–748.
out no less than three times.
1
7. Chester, A.E., Fairchild, D.G., and Depass, L.R., Fun-
dam. Appl. Toxicol., 1991, vol. 17, pp. 505–518.
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