Synthesis of somatostatin analogues containing C-terminal adamantane and their antiproliferative properties

Article abstract of DOI:10.1021/jm701599w

On the basis of the structure of somatostatin analogue TT-232 (1), which exhibited a highly potent antitumor activity, we synthesized small linear peptide derivatives and evaluated their antitumor and apoptotic activity. Of them, Boc-Tyr-D-Trp-1-adamantylamide (5) had the most potent cell antiproliferative activity in SW480 and A431 cell lines, which was supported in A431 cell lines by FACS analysis that demonstrated a major increase in DNA fragmentation in the subG1 fraction.

Full text of DOI:10.1021/jm701599w

J. Med. Chem. 2008, 51, 5121–5124
5121
Synthesis of Somatostatin Analogues Containing C-Terminal Adamantane and Their
Antiproliferative Properties
Anna Miyazaki,† Yuko Tsuda,† Shoji Fukushima,† Toshio Yokoi,† Tibor Va´ntus,‡ Gyo¨ngyi Bo¨ko¨nyi,‡ Edit Szabó,‡
Aniko´ Horva´th,‡ Gyo¨rgy Ke´ri,‡ and Yoshio Okada*,†
Faculty of Pharmaceutical Sciences, Department of Medicinal Chemistry, Kobe Gakuin UniVersity, Nishi-ku, Kobe, 651-2180, Japan, and
Pathobiochemistry Research Group of Hungarian Academy of Science and Semmelweis UniVersity, H-1088 Budapest, Hungary
ReceiVed December 19, 2007
On the basis of the structure of somatostatin analogue TT-232 (1), which exhibited a highly potent antitumor
activity, we synthesized small linear peptide derivatives and evaluated their antitumor and apoptotic activity.
Of them, Boc-Tyr-D-Trp-1-adamantylamide (5) had the most potent cell antiproliferative activity in SW480
and A431 cell lines, which was supported in A431 cell lines by FACS analysis that demonstrated a major
increase in DNA fragmentation in the subG1 fraction.
Introduction
The neuropeptide somatostatin (SRIF) exerts inhibitory effects
on the secretory processes in the endocrine and exocrine
systems. It also mediates tumor cell growth inhibition1-5 through
a family of seven transmembrane G-protein-coupled receptors
(SSTR1-SSTR5).6,7 SSTRs are distributed throughout human
body not only in normal cells but also in tumor cells. Native
somatostatin binds with high affinity to all SSTR subtypes,
whereas the commercially available somatostatin analogues bind
with varying degrees of activity;8 for example, cyclic and linear
octapeptide analogues bind to hSSTR 2, 3, and 5 subtypes with
high affinity but with reduced affinity to hSSTR1 and 4 subtypes.
Most of the somatostatin analogues developed for clinical use,
such as octreotide9-11 and RC-160,12 have high affinity to
SSTR2 and SSTR5 and act longer than somatostatin with use
in the diagnosis and treatment of gastrointestinal disorders,
including endocrine tumors.9,13-16 However, the use of these
analogues as antitumor agents has been limited because of their
antisecretory effects and poor oral bioavailability. Ke´ri et al.
reported that in vitro and in vivo, a selective somatostatin
structural analogue, TT-232 (1) [D-Phe-c(Cys-Tyr-D-Trp-Lys-
Cys)-Thr-NH2], had potent antiproliferative activity without
antisecretory action.17,18 Compound 1, which binds to SSTR1
and 4 receptors with low affinity but not to SSTR2, 3, and 5
receptors,19,20 exerts its effects through the tyrosine phospho-
rylation pathway, and further, 1 is internalized into the cytoplasm
and nuclei, where it directly induces apoptosis.20
Figure 1. Structures of somatostatin analogues 2-15.
hydrophobic moieties and N-terminal amino groups were
protected by tert-butyloxycarbonyl (t-Boc) groups to give
compounds 5-9. The resulting structures with D-Trp in the
middle of the peptide were expected to resist enzymatic
degradation. Being small and hydrophobic, these molecules
should exhibit potent cell permeability, and in this study, we
report the synthesis, physicochemical characterization, and the
biological activities of these new analogues.
In this paper, we attempt to simplify the structure of 1 to
improve biological activity and cellular permeability and to
synthesize several new linear analogues (2-15, Figure 1). To
reduce the size of the active peptide and at the same time
improve its antiproliferative activity, we synthesized peptides
2-4, comprising only the essential biologically relevant amino
acid sequence, Tyr-D-Trp-Lys, and studied the resulting
structure-activity relationship. The studies on these compounds
revealed that placing hydrophobic moieties at the N- and
C-termini of the peptide was critical for the manifestation of
antiproliferative activity. Therefore, Lys was substituted with
Results and Discussion
Chemistry. Compounds 3 and 4 were prepared from Boc-
Tyr-D-Trp-Lys(Z)-OFma (2).21The OFm group of 2 was re-
moved by 20% piperidine/DMF to produce 3, which was converted
into 4 by HCl in dioxane. Each compound was purified by
semipreparative RP-HPLC. Compounds 5-14 were prepared as
follows: Boc-D-Trp-OH was coupled with the corresponding amine
by a mixed anhydride method. The Boc group of the resulting
compounds was removed by HCl in dioxane to produce their
a Abbreviations: OFm, 9-fluorenyl methyl ester; RP-HPLC, reversed-
phase high performance liquid chromatography; TFA, trifluoroacetic acid;
AcOEt, ethyl acetate; MeOH, methanol; AcOH, acetic acid; n-BuOH,
butanol; DMF, N,N-dimethylformamide; MTT, 3-(4,5-dimethylthiazol-2-
yl)-2,5-yldiphenyltetrazolium bromide; DIEA, N,N-diisopropylethylamine;
PyBop, benzotriazol-1-yloxy-tris-pyrrolidinophosphonium hexafluorophos-
phate.
* To whom correspondence should be addressed. Phone: +81-78-974-
1551. Fax: +81-78-974-5689. E-mail: okada@pharm.kobegakuin.ac.jp.
†
Kobe Gakuin University.
Pathobiochemistry Research Group of Hungarian Academy of Science
‡
and Semmelweis University.
10.1021/jm701599w CCC: $40.75
2008 American Chemical Society
Published on Web 08/05/2008

5122 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Brief Articles
Table 1. Antiproliferative Activities of Somatostatin Analogues 1-15 on
A431 Cells by MTT Test
Table 2. Antiproliferative Activities of Somatostatin Analogues 5, 6,
10, 11 on SW480 cells by MTT Test
concentration of compounda
concentration of compounda
compd
10 µM
25 µM
50 µM
compd
10 µM
25 µM
50 µM
1
50 ( 9.4
40 ( 4.6
2 ( 6.4
0 ( 4.6
0 ( 5.2
72 ( 1.4
26 ( 5.8
25 ( 6.1
3 ( 7.2
12 ( 3.7
19 ( 0.8
30 ( 5.4
7 ( 4.4
10 ( 7.2
0 ( 6.8
0 ( 9.5
60 ( 8.5
70 ( 4.9
7 ( 3.9
80 ( 7.4
80 ( 3.1
7 ( 1.9
1
13 ( 6.5
68 ( 1.0
7 ( 4.8
9 ( 4.4
0 ( 3.2
13 ( 2.2
47 ( 0.7
85 ( 2.6
53 ( 1.9
60 ( 1.3
15 ( 0.2
15 ( 1.6
88 ( 2.6
92 ( 1.0
95 ( 2.4
74 ( 3.9
57 ( 4.6
52 ( 5.5
cycloheximide
2
3
4
5
6
7
8
cycloheximide
5
6
10
11
0 ( 7.2
2 ( 6.2
0 ( 4.3
0 ( 6.7
90 ( 2.6
43 ( 5.7
21 ( 5.0
20 ( 6.0
18 ( 1.9
52 ( 5.8
76 ( 2.0
18 ( 7.6
5 ( 7.4
93 ( 1.7
95 ( 2.3
19 ( 3.9
31 ( 3.4
35 ( 5.7
91 ( 1.0
88 ( 9.0
40 ( 2.7
16 ( 1.0
22 ( 5.6
11 ( 6.8
a Antiproliferative activity of compounds is expressed as inhibition of
cellular proliferation (%) and listed as the mean ( SE.
9
(Table 1). The growth inhibitory activities of 5, 6, 10, and 11
on SW480 human colon carcinoma cells were tested similarly
(Table 2). The peptides containing free NH2 groups (10 and
11) lost some of their antiproliferative activity; namely, they
exhibited less than 60% of the activity at the highest dose
compared to 5 and 6.
10
11
12
13
14
15
0 ( 6.5
8 ( 5.9
a Antiproliferative activity of compounds is expressed as inhibition of
Apoptosis. Cell proliferation assay data suggested that there
were at least four compounds (5, 6, 10, and 11) with very strong
effects in reducing the number of tumor cells when quantified
by the MTT assay. Another means to decrease the number of
cells is to induce apoptosis, which results in a drastic loss. To
test this hypothesis, we continued with our biological analysis
using flow cytometry (FACS), a method that is very efficient
in detecting and measuring DNA fragmentation, a major
hallmark of apoptosis. We detected an increase in the subG1
DNA fraction after the treatment with 5, which antiproliferation
studies revealed the greatest potency: 5 exhibited exceptionally
potent apoptosis effects (Figure 2), supporting our hypothesis
that 5 should have a potent antiproliferative effect through its
induction of apoptosis.
cellular proliferation (%) and listed as the mean ( SE.
corresponding amine hydrochlorides, which were coupled with
Boc-Tyr-OH by using PyBop as a coupling reagent to give
compounds 5-9. Boc groups were removed by HCl in dioxane
to produce compounds 10-14, which were purified by semi-
preparative RP-HPLC. In the case of the dipeptide (15), it was
prepared using Boc-Tyr-OH and H-D-Trp-OFm as starting
materials. All final compounds exhibited single peaks on
analytical HPLC with unique retention times (see Supporting
Information Table S3). All compounds were analyzed by
MALDI-TOF mass spectrometry, 1H and 13C NMR, elemental
analyses, and HPLC, in which all compounds (2-15) exhibited
greater than 98% purity (see Supporting Information).
Antiproliferative Activity. The growth inhibitory activity
of 2-15 on A431 human epidermal carcinoma cells was
measured by MTT test22 and summarized in Table 1.The linear
peptides (2-4) containing only the biologically important
sequence (-Tyr-D-Trp-Lys-), which is also present in molecule
1, were examined. Whereas 2 exhibited 10% antiproliferative
activity relative to 1, the inhibitory activity of 3 and 4 was
negligible. The HPLC analysis showed that 2 had a higher
degree of hydrophobicity than 3 and 4 (retention time: 46.8,
36.6, and 29.4 min, respectively). These data suggested that
protected N- and C-termini might contribute to manifestation
of the inhibitory activity. With these results in mind, we turned
our attention to shortening the peptide with hydrophobic
moieties at the N- and C-termini to produce 5-9. Nearly all
compounds exhibited antiproliferative activity in a dose-
dependent manner as shown in Table 1. Of these compounds,
5 and 6 proved to be the most potent; in fact, 5 exhibited 72%
inhibition at a dose of 10 µM, which was 1.5 times more potent
than 1. Peptides 5 and 6 have Boc protecting groups at the
N-termini and 1- or 2-adamantylamine at the C-termini, with
HPLC retention times of 43.0 and 42.0 min, respectively.
Peptides 7, 8, and 9 have 1-naphthylamine, tert-butylamine, and
aminomethylcoumarine at their C termini, with their retention
times of 39.4, 37.2, and 37.1 min, respectively. Their antipro-
liferative activity was less than 30% (Table 1) on A431 cells
compared to that of 5 and 6. Removing Boc protecting groups
from the N-termini of peptides (5-9) resulted in compounds
(10-14) in which the peptides containing 1- and 2-adamanty-
lamine (10 and 11) exhibited the highest activity on A431 cells,
similar to that observed with 1. Removal of the hydrophobic
moieties from N- and C-termini produced H-Tyr-D-Trp-OH (15),
which exhibited only 10% of the activity compared to 5 and 6
Microscopic Study. On the basis of the cell proliferation and
FACS analysis data, four of the most active compounds (5, 6,
10, and 11) were selected to visualize the morphological changes
in tumor cells after the indicated treatment. Not surprisingly,
microscopic visualization showed once again that 5 was the
peptide that indeed changed cell morphology, thereby indicating
directly revealing that apoptosis had been induced (see Sup-
porting Information Figure S1).
Conclusions
In this study, the new linear somatostatin analogues were
prepared containing the biologically essential amino acid
sequence (-Tyr-D-Trp-Lys-) present in the tumor-selective
heptapeptide 1. Compounds 5-14, substitutions of the C-
terminal Lys of the Tyr-D-Trp-Lys sequence with various
hydrophobic and bulky residues (adamantyl-, naphthyl-, tert-
butyl-, 4-methylcoumarinyl-) with or without Boc group at the
N-termini, indicated that the compounds containing adamanty-
lamine (5, 6, 10, and 11) showed the most potent antiprolif-
erative activity on A431 cells, even greater than 1. In addition,
5 with a Boc group was the most active analogue in the
induction of apoptosis. These results indicate that the potent
activity in the tested cell lines might be attributed to the Tyr-
D-Trp sequence coupled with a hydrophobic and bulky residue
at the N-terminus and at the C-terminus, which thus increased
hydrophobicity in the whole molecule. Our data further suggest
that not only hydrophobicity but also aliphatic properties and
rigidity are important at the C-terminus. On the basis of these
structure-activity studies, we were able to develop and then
select a highly promising apoptosis inducing dipeptide amide.
Further studies are underway exploring the detailed mechanism

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 5123
Boc-Tyr-D-Trp-Lys(Z)-OH (3). The title compound was pre-
pared from Boc-Tyr-D-Trp-Lys(Z)-OFm (2) (1.2 g, 1.31 mmol) and
20% piperidine/DMF (40 mL), and the crude product was recrystal-
lized from ether. Yield 377 mg (29.0%), mp 108-112 °C, [R]D25
+9.76°(c 1.0, MeOH), Rf ) 0.47 (CHCl3/MeOH/H2O ) 8:3:1).
HCl·H-Tyr-D-Trp-Lys(Z)-OH (4). The title compound was
prepared from Boc-Tyr-D-Trp-Lys(Z)-OH (3) (795 mg, 1.09 mmol)
and 4 M HCl/dioxane (1.4 mL, 5.45 mmol). The crude product
was purified with semipreparative RP-HPLC and lyophilized from
1 M HCl. Yield 146 mg (20.1%), amorphous, [R]2D5 -83.5°(c 1.0,
H2O), Rf ) 0.38 (n-BuOH/AcOH/H2O ) 4:1:5).
General Procedure for Synthesis of Boc-Tyr-D-Trp-X [X:
1-Adamantylamide (5), 2-Adamantylamide (6), 1-Naphthyl-
amide (7), tert-Butylamide (8), and 4-Methylcoumarin-7-
ylamide (9)]. Boc-Tyr-OH (576 mg, 2.05 mmol) and H-D-Trp-X
[prepared from Boc-D-Trp-X (1.86 mmol) and 4 M HCl/dioxane
(2.3 mL, 9.32 mmol)] in DMF (50 mL) were coupled using PyBop
reagent (1.1 g, 2.05 mmol) and DIEA (1.9 mL, 11.2 mmol) at room
temperature overnight. After removal of the solvent, the residue
was extracted with AcOEt, which was washed with 10% citric acid,
5% NaHCO3, and water and dried over Na2SO4. After removal of
Na2SO4 by filtration, the solvent of the filtrate was evaporated.
Boc-Tyr-D-Trp-1-adamantylamide (5). The crude product was
purified by open column silica gel chromatography (AcOEt/hexane
) 2:1) and precipitated with petroleum ether. Yield 1.14 g (82.6%),
mp 140-143 °C, [R]2D5 +14.7°(c 1.0, MeOH), Rf ) 0.64 (AcOEt/
hexane ) 1:1).
Boc-Tyr-D-Trp-2-adamantylamide (6). The crude product was
purified by open column silica gel chromatography (AcOEt/hexane
) 2:1) and precipitated with petroleum ether. Yield 1.19 g (86.2%),
mp 138-141 °C, [R]2D5 +11.0°(c 1.0, MeOH), Rf ) 0.51 (AcOEt/
hexane ) 1:1).
Boc-Tyr-D-Trp-1-naphthylamide (7). The crude product was
recrystallized from MeOH. Yield 825 mg (59.8%), mp 237-239
°C, [R]2D5 +18.0°(c 1.0, MeOH), Rf ) 0.64 (CHCl3/MeOH/AcOH
) 90:8:2).
Boc-Tyr-D-Trp-tert-butylamide (8). The crude product was
purified by open column silica gel chromatography (AcOEt/hexane
) 2:1) and precipitated with petroleum ether. Yield 854 mg
(87.6%), mp 113-115 °C, [R]2D5 +15.0°(c 1.0, MeOH), Rf ) 0.57
(AcOEt/hexane ) 1:1).
Boc-Tyr-D-Trp-4-methylcoumarin-7-ylamide (9). The crude
material was purified by open column silica gel chromatography
(3-5% MeOH in CHCl3) and precipitated with petroleum ether.
Yield 286 mg (27.8%), mp 232-235 °C, [R]2D5 +99.6°(c 1.0,
MeOH), Rf ) 0.43 (AcOEt/hexane ) 1:1).
General Procedure for Synthesis of HCl·H-Tyr-D-Trp-X
[X: 1-Adamantylamide (10), 2-Adamantylamide (11), 1-Naph-
thylamide (12), tert-Butylamide (13), and 4-Methylcoumarin-7-
ylamide (14)]. Boc-Tyr-D-Trp-X (0.166 mmol) was treated with 4
M HCl/dioxane (0.4 mL, 1.67 mmol) at room temperature for 1 h.
Dry ether was added to a solution to form a precipitate, which was
collected by filtration and washed with dry ether. The crude product
was purified by semipreparative RP-HPLC and lyophilized from 1
M HCl.
HCl·H-Tyr-D-Trp-1-adamantylamide (10). Yield 150 mg
(83.8%), amorphous, [R]D25 +24.6°(c 1.0, MeOH), Rf ) 0.81 (n-
BuOH/AcOH/pyridine/H2O ) 4:1:1:2).
HCl·H-Tyr-D-Trp-2-adamantylamide (11). Yield 78.5 mg
(87.9%), amorphous, [R]D25 +28.3°(c 1.0, MeOH), Rf ) 0.82 (n-
BuOH/AcOH/pyridine/H2O ) 4:1:1:2).
HCl · H-Tyr-D-Trp-1-naphthylamide (12). Yield 73.5 mg
(82.2%), amorphous, [R]D25 +28.2°(c 1.0, MeOH), Rf ) 0.72 (n-
BuOH/AcOH/pyridine/H2O ) 4:1:1:2).
HCl·H-Tyr-D-Trp-tert-butylamide (13). Yield 71.7 mg (81.8%),
amorphous, [R]2D5 +35.5°(c 1.0, MeOH), Rf ) 0.75 (n-BuOH/
AcOH/pyridine/H2O ) 4:1:1:2).
Figure 2. FACS analysis data of 5. Compound 5 induces apoptotic
cell death in A431 epidermal carcinoma cells. Cells were treated with
5 (30 µM) for 48 h or left untreated and then quantified for their DNA
content after propidium iodide (PI) staining. Values shown in boxes
are the percent of cells with less DNA content than that of G1 phase
cells (sub-G1). The experiment was independently repeated three times.
In FACS analysis, FL2-H represents the fluorescence channel 2 and
counts represent the counted cells during the analysis. M1 shows the
apoptotic fraction of investigated cells (in %), and 5 induced strong
apoptosis (∼62%) even above the background (spontaneous apoptosis)
of 8.4%.
of action and potential applications of our simplified 1 analogues
with Tyr-D-Trp as an important biological core template.
Experimental Section
Boc-Tyr-D-Trp-Lys(Z)-OFm (2). Boc-Tyr-OH (1.70 mmol) and
H-D-Trp-Lys(Z)-OFm [prepared from Boc-D-Trp-Lys(Z)-OFm (1.2
g, 1.55 mmol) and 4 M HCl/dioxane (1.9 mL, 7.75 mmol)] were
coupled with PyBop (1.70 mmol) in DMF (100 mL) containing
DIEA (1.1 mL, 6.20 mmol). The mixture was stirred at 0 °C for
10 min and then room temperature overnight. After removal of the
solvent, the residue was extracted with AcOEt, which was washed
with 10% citric acid, 5% NaHCO3 solution, and water and dried
over Na2SO4. After removal of Na2SO4 by filtration, the solvent of
the filtrate was evaporated and the crude product was purified by
open column silica gel chromatography. The compound was
crystallized from a suitable solvent and filtered. The crude product
was purified by open column silica gel chromatography (AcOEt/
hexane ) 2:1). The compound was precipitated with hexane. Yield
745 mg (56.1%), mp 97-103 °C, [R]2D5 -10.7°(c 1.0, MeOH), Rf
) 0.64 (CHCl3/MeOH/AcOH ) 90:8:2).
HCl·H-Tyr-D-Trp-4-methylcoumarin-7-ylamide (14). Yield
84.0 mg (93.3%), amorphous, [R]2D5 -24.6°(c 1.0, MeOH), Rf )
0.74 (n-BuOH/AcOH/pyridine/H2O ) 4:1:1:2).

5124 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Brief Articles
(4) Colas, B.; Cambilleu, C.; Buscail, L.; Zeggari, M.; Esteve, J. P.; Lautre,
V.; Thomas, F.; Vaysse, N.; Susini, C. Stimulation of a membrane
tyrosine phosphatase activity by somatostatin analogues in rat pan-
creatic acinar cells. Eur. J. Biochem. 1992, 207, 1017–1024.
(5) Pan, M. G.; Florio, T.; Stock, P. J. G-Protein activation of a hormone-
stimulated phosphatase in human tumor cells. Science 1992, 256,
1215–1217.
(6) Breader, C. D.; Yamada, Y. Y.; Yasuda, K.; Seino, S.; Saper, C. B.;
Bell, G. I. Differential expression of somatostatin receptor subtypes
in brain. J. Neurosci. 1992, 12, 3920–3934.
HCl·H-Tyr-D-Trp-OH (15). Boc-Tyr-D-Trp-OFm (211 mg,
0.232 mmol) was treated with 20% piperidine/DMF (10 mL)
followed by treatment with 4 M HCl/dioxane (0.6 mL, 2.32 mmol)
to give the title compound, which was crystallized from ether. The
crude product was purified by semipreparative RP-HPLC and
lyophilized from 1 M HCl. Yield 65.8 mg (70.2%), amorphous,
[R]2D5 +26.3°(c 1.0, MeOH), Rf ) 0.46 (n-BuOH/AcOH/pyridine/
H2O ) 4:1:1:2).
Antiproliferative Activity. Two cell lines are well-characterized,
fast-growing, and represent excellent model systems for antipro-
liferative studies. As a routine and quantitative test method, the
MTT assay was applied for the quantification of the differences in
the number of cells after treatment with the compounds in addition
to cycloheximide and 1 that served as positive controls, screening
the 14 compounds derived from 1. Each analogue was tested at
10, 25, and 50 µM. Cycloheximide was used as an external standard
as a well-known and efficient apoptosis inducer and cell prolifera-
tion inhibitor. Antiproliferative activitiy was examined using the
MTT.
MTT Analysis. MTT assay is based on the mitochondrial
dehydrogenase enzyme that cleaves the tetrazolium rings of the
pale-yellow MTT in viable cells and that form a dark-blue formazan
crystals which is largely impermeable to membranes resulting in
its accumulation in healthy cells. Solubilization of the cells by the
addition of DMSO results in the liberation of the crystals which
are solubilized. The amount of surviving cells is directly propor-
tional to the level of the formazan product created. The color can
be quantified using a colorimetric assay using an ELISA reader at
540 nm based on the reduction of the soluble yellow MTT
tetrazolium salt to a blue insoluble formazan product using the
mitochondrial succinic dehydrogenase in live cells.
(7) Bruno, J. F.; Xu, Y.; Song, J.; Berelowitz, M. Tissue distribution of
somatostatin receptor subtype messenger ribonucleic acid in the rat.
Endocrinology 1993, 133, 2561–2567.
(8) Lamberts, S. W. Somatostatin and somatostatin analogues: diagnostic
and therapeutic uses. Curr. Opin. Oncol. 2002, 14, 53–57.
(9) Bauer, W.; Briner, U.; Doepfner, W.; Haller, R.; Huguenin, R.;
Marbach, P.; Petcher, T. SMS 201-995: a very potent and selective
octapeptide analogue of somatostatin with prolonged action. Life Sci.
1982, 31, 1133–1140.
(10) Setyono-Han, B.; Henkelman, M. S.; Foekens, J. A.; Kllin, G. M.
Direct inhibitory effects of somatostatin (analogues) on the growth of
human breast cancer cells. Cancer Res. 1987, 47, 1566–1570.
(11) Schally, A. V.; Redding, T. W. Somatostatin analogs as adjuncts to
agonist of luteinizing hormone-releasing hormone in the treatment of
experimental prostate cancer. Proc. Natl. Acad. Sci. U.S.A. 1987, 84,
7275–7279.
(12) Cai, R. Z.; Szoke, B.; Lu, R.; Fu, D.; Redding, T. W.; Schally, A. V.
Synthesis and biological activity of highly potent octapeptide analogs
of somatostatin. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 1896–1900.
(13) Kaupmann, K.; Bruns, C.; Raulf, F.; Weber, H. P.; Mattes, H.; Lubbert,
H. Two amino acids, located in transmembrane domains VI and VII,
determine the selectivity of the peptide agonist SMS 201-995 for the
SSTR2 somatostatin receptor. EMBO J. 1995, 14, 727–735.
(14) Srkalovic, G.; Cai, R. Z.; Schally, A. V. Evaluation of receptors for
somatostatin in various tumors using different analogs. J. Clin.
Endocrinol. Metab. 1990, 70, 661–669.
(15) Weckbecker, G.; Raulf, F.; Stolz, B.; Bruns, C. Somatostatin analogs
for diagnosis and treatment of cancer. Pharmacol. Ther. 1993, 60,
245–264.
(16) Hofland, L. J.; Visser-Wisselaar, H. A.; Lamberts, S. W. Somatostatin
analogs: clinical application in relation to human somatostatin receptor
subtypes. Biochem. Pharmacol. 1995, 50, 287–297.
(17) Keri, G.; Erchegyi, J.; Horvath, A.; Mezo, I.; Idei, M.; Vantus, T.;
Balogh, A.; Vadasz, Z.; Bokonyi, G.; Seprodi, J.; Teplan, I.; Csuka,
O.; Tejeda, M.; Gaai, D.; Szegedi, Z.; Szende, B.; Roze, C.; Kalthoff,
H.; Ullrich, A. A tumor-selective somatostatin analog (TT-232) with
strong in vitro and in vivo antitumor activity. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 12513–12518.
Apoptosis (Flow Cytometric Analysis). A431 cells were
incubated at 37 °C for 24 h in DMEM supplemented with 10%
FCS using 24-well plates, seeding 50 000 cells per well. After
treatment, the cells were washed, fixed with 70% ethanol, and stored
at -20 °C or used for propidium iodide (PI) staining. The stained
cells were subjected to flow cytometry (FACS Calibur, BD
Biosciences) to detect and quantify apoptosis. Cells with their DNA
content less than that of G1 phase cells (sub-G1) were assumed to
be apoptotic. The analysis was performed with CellQuest software.
(18) Keri, G.; Mezo, I.; Vadasz, Z.; Horvath, A.; Idei, M.; Vantus, T.;
Balogh, A.; Bokonyi, G.; Bajor, T.; Teplan, I.; Tamas, J.; Mak, M.;
Horvath, J.; Csuka, O. Structure-activity relationship studies of novel
somatostatin analogs with antitumor activity. Pept. Res. 1993, 6, 281–
288.
(19) Stetak, A.; Lankenau, A.; Vantus, T.; Csermely, P.; Ullrich, A.; Keri,
G. The antitumor somatostatin analogue TT-232 induces cell cycle
arrest through PKCδ and c-Src. Biochem. Biophys. Res. Commun.
2001, 285, 483–488.
(20) Vantus, T.; Keri, G.; Krivickiene, Z.; Valius, M.; Stetak, A.; Keppens,
S.; Csermely, P.; Bauer, I. P.; Bokonyi, G.; Declercq, W.; Vandena-
beele, P.; Merlevede, W.; Vandenheede, R. J. The somatostatin
analogue TT-232 induces apoptosis in A431 cells sustained activation
of stress-activated kinases and inhibition of signaling to extracellular
signal-regulated kinases. Cell. Signalling 2001, 13, 717–725.
(21) Miyazaki, A.; Yokoi, T.; Tachibana, Y.; Enomoto, R.; Lee, E.;
Bokonyi, G.; Keri, G.; Tsuda, Y.; Okada, Y. Design and synthesis of
novel type somatostatin analogs with antiproliferative activities on
A431 tumor cells. Tetrahedron Lett. 2004, 45, 6323–6327.
(22) Mosmann, T. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J. Immunol.
Methods 1983, 65, 55–63.
Acknowledgment. This work was supported in part by
KAKENHI (Grant 17790026), by a Grant-in-Aid (C) from Kobe
Gakuin University, and by NEXT “Academic Frontier” Project
(2006) and in part by Hungarian Grants JAP-6/02, OTKA
49478, 60197, Cell Kom Ret-06/2006. T.V. is a Bolyai
Postdoctoral Fellow of the Hungarian Academy of Sciences.
1
Supporting Information Available: Synthetic procedures, H
and 13C NMR, elemental analysis, and HPLC data for all the new
compounds, and biological assay procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) Brazeau, P.; Vale, W.; Burgus, R.; Ling, N.; Butcher, M.; Rivier, J.;
Guillemin, R. Hypothalamic polypeptide that inhibits the secretion of
immnoreactive pituitary growth hormone. Science 1973, 179, 77–79.
(2) Reichilin, S.; Somatostatin, N. N. Engl. J. Med. 1983, 309, 1556–
1563.
(3) Patel, Y. C.; Srikant, C. B. Somatostatin mediation of adenohypo-
physical secretion. Annu. ReV. Physiol. 1986, 48, 551–567.
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