New cyclic somatostatin analogues containing a pyrazinone ring: Importance of Tyr for antiproliferative activity

Article abstract of DOI:10.1016/j.bmcl.2008.10.001

Novel somatostatin analogues containing a pyrazinone ring, compounds 1 and 2, exhibited good antiproliferative activity on A431 tumor cells. To increase antitumor activity and binding affinity on somatostatin receptors (SSTRs), we substituted Tyr in the critical sequence, Tyr-d-Trp-Lys, with more hydrophobic aromatic residue. The substituted compounds dramatically lost antitumor activity, indicating that Tyr residue was an essential residue.

Full text of DOI:10.1016/j.bmcl.2008.10.001

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6199–6201
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New cyclic somatostatin analogues containing a pyrazinone ring:
Importance of Tyr for antiproliferative activity
Anna Miyazaki a, Yuko Tsuda a, Shoji Fukushima a, Toshio Yokoi a, Tibor Vántus b, Gyöngyi Bökönyi b,
Edit Szabó b, Anikó Horváth b, György Kéri b, Yoshio Okada a,
*
a Faculty of Pharmaceutical Sciences, Department of Medicinal Chemistry, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan
b Pathobiochemistry Research Group of Hungarian Academy of Science and Semmelweis University, H-1088 Budapest, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel somatostatin analogues containing a pyrazinone ring, compounds 1 and 2, exhibited good antipro-
Received 18 July 2008
Revised 19 September 2008
Accepted 1 October 2008
Available online 5 October 2008
liferative activity on A431 tumor cells. To increase antitumor activity and binding affinity on
somatostatin receptors (SSTRs), we substituted Tyr in the critical sequence, Tyr-D-Trp-Lys, with more
hydrophobic aromatic residue. The substituted compounds dramatically lost antitumor activity, indicat-
ing that Tyr residue was an essential residue.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Somatostatin analogue
Pyrazinone ring
Antiproliferative activity
The neuropeptide somatostatin (SRIF), a tetradecapeptide orig-
inally isolated from bovine hypothalamus and identified as a po-
tent inhibitor of GH secretion, exerts a wide variety of inhibitory
effects on the endocrine, exocrine, and neural function.1,2 It also
activates tyrosine phosphatases, thus indirectly inhibiting tyrosine
kinases which are involved in the regulation of cell proliferation,3
through a family of seven transmembrane G protein-coupled
receptors (SSTR1–SSTR5).4,5 The distribution of SSTRs ranges
widely throughout not only the organs in human body, but also
several tumor cells. The peptide-based analogues of somatostatin,
such as Octreotide6 and RC-160,7 which have high affinity to SSTR2
and SSTR5, have been developed for use in clinical therapies to
overcome the short therapeutic half-life of somatostatin, and used
in the diagnosis and treatment of gastrointestinal disorders,
including endocrine tumors.6,8 However, the use of these ana-
logues as antitumor agents has been limited because of their anti-
secretory effects and poor oral bioavailability. Numerous attempts
to discover the subtype-selective and orally stable analogues
internalized into the cytoplasm and nuclei, where it directly in-
duces apoptosis.13 The current study had two objectives. Firstly,
to use a pyrazinone ring for greater resistance to enzymatic degra-
dation, thereby enhancing the stability in the body and cellular
permeability through the gastrointestinal tract and blood–brain
barrier (BBB).14,15 Secondly, to enhance the antiproliferative activ-
ity by increasing the hydrophobicity of the molecule in order to in-
crease interaction between compounds and cell membranes and
SSTRs. We previously reported that somatostatin analogues con-
taining pyrazinone rings and active sequence (Tyr-D-Trp-Lys)
showed good antiproliferative activities on A431 tumor cells
(Fig. 1: 1 and 2). 16 In that paper, we also used aliphatic amino acids
instead of pyrazinone rings to compare the activities between the
two. While most cyclic somatostatin analogues have disulfide
bonds, these analogues have pyrazinone rings or aliphatic amino
acids for their ring formation. As the result, the analogues contain-
ing a pyrazinone ring exhibited more potent antiproliferative activ-
ity, although both conformations by CD spectra showed random
structures.16 Generally, almost somatostatin analogues form a b-
have proceeded; e.g., TT-232, [D-Phe-c(Cys-Tyr-D-Trp-Lys-Cys)-
Thr-NH2], was reported to have potent antiproliferative activity
without antisecretory action.9,10 The antiproliferative effect of
TT-232 is partially mediated by SSTR receptors (SSTR1 and 4).
turn structure spanning residues D
-Trp and Lys.17 The pyrazinone
ring might allow the proper orientation of active sequence neces-
sary for activity. In fact, we reported the low-energy conforma-
11,12
It activates a phosphorylation cascade (the MAP kinase path-
tional searching paradigm of a 20,60-dimethyl-
L-tyrosine (Dmt)
way) and results in the induction of cyclin-dependent kinase inhib-
itors, causing cell cycle arrest. Furthermore, TT-232 might be
dimer ligand containing a pyrazinone ring.14 The pyrazinone ring
as a linker attempts to spatially align two Dmt pharmacophores
in order to closely face each other compared with the alkyl linker;
therefore, Dmt was well adapted to interact with the opioid recep-
tor.14 Furthermore, the advantage of a pyrazinone ring is that it is
* Corresponding author. Tel.: +81 78 974 1551; fax: +81 78 974 5689.
E-mail address: okada@pharm.kobegakuin.ac.jp (Y. Okada).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.001

6200
A. Miyazaki et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6199–6201
derivatives, 3-(40-aminobutyl)-6-(20-carboxyethyl)-5-methyl-2(1H)-
*
*
R
O
NH
pyrazinone (abbreviated as [Lys-Glu]) and 6-(40-aminobutyl)-3-(20-
carboxyethyl)-5-methyl-2(1H)-pyrazinone, (abbreviated as [Glu-
Lys]) were synthesized according to published procedures.14 Com-
pounds 3–6 were similarly prepared using the procedure reported
previously16 with one modification; namely, Dmt or Phe was used
instead of Tyr. Briefly, as shown in Scheme 1, the respective pyraz-
inone derivatives, Boc-[Lys-Glu]-OH or Boc-[Glu-Lys]-OH interme-
O
R1
R1
HN
CH
H2
C
R2
CH (CH2)4 NH2
NH
O
HN
CH
O
CH2
diate was coupled with H-Dmt/Phe-D-Trp-Lys(Z)-OFm by use of
HN
benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophos-
phate (PyBop) in N,N-dimethylformamide (DMF) containing N,N-
diisopropylethylamine (DIEA). After the removal of 9-fluorenyl
methyl ester (OFm) group by 20% piperidine/DMF and tert-butyl-
oxycarbonyl (Boc) group by HCl in dioxane, the resulting com-
pounds with free amino and carboxyl groups were purified by
semi-preparative reversed-phase (RP)-HPLC. The purified com-
pounds were cyclized using diphenylphosphoryl azide (DPPA) in
1 mM DMF containing triethylamine (TEA). After removal of the
benzyloxycarbonyl (Z) group by catalytic hydrogenation in 50%
acetic acid (AcOH) for 1 h, the crude products were purified by
semi-preparative RP-HPLC. The final compounds were analyzed
by MALDI-TOF mass spectrometry, 1H and 13C NMR, elemental
analysis.24
N
1: R1=H,
R2=OH
( )4
*
R=
3: R1=CH3,
5: R1=H,
R2=OH
R2=H
*
*
( )2
N
H
H
N
O
2: R1=H,
4: R1=CH3,
6: R1=H,
R2=OH
R2=OH
R2=H
O
( )4
*
R=
( )2
N
Figure 1. Structures of somatostatin analogues 1–6. Compounds 1, 3, and 5 are
[Lys-Glu]-pyrazinone cyclopeptides, compounds 2, 4, and 6 are [Glu-Lys]-pyrazi-
none cyclopeptides.
stable in body owing to cyclic structure and allows these com-
pounds to permeate the epithelial cell membranes in the gastroin-
testinal tracts and BBB.14,18,19 Thus, somatostatin analogues
containing pyrazinone ring should increase the bioavailability in
human body. As another approach, the cyclic analogues (3–6) were
prepared by substituting Tyr with Dmt or Phe in order to improve
the biological activity of 1 and 2. It is well known that Dmt is more
hydrophobic than Tyr and its replacement for Tyr in opioid pep-
tides and opioidomimetics dramatically enhanced opioid proper-
ties and biological activities in vitro and in vivo.15,20–22 We
postulated that these molecules should exhibit potent antiprolifer-
ative activity and bioavailability.
Table 1
Antiproliferative activity on A431 and SW480 cells by MTT assay.
Compound
A431a
25
SW480a
25
10
lM
l
M
50
lM
10
l
M
l
M
50 lM
TT-232
Cyclohexiinide
50 9.4
40 4.6
19 3.0
28 9.0
60 8.5
70 4.9
36 5.0
42 3.0
80 7.4
80 3.1
56 35
58 36
13 6.5
68 1.0
24 5.7
35 4.6
47 0.7
85 2.6
23 1.5
31 8.9
16 1.0
11 3.2
88 2.6
92 1.0
1
2
3
4
5
6
7
3.7
35 4.7
14 2.0
0
0
1.9
4.3
0
0
1.2
2.5
0
0
4.5
2.5
8
2
0
2
3.7
5.0
5.5
3.8
The unnatural amino acid, Dmt was synthesized in our labora-
tory according to the procedure of Dygos et al.23 Enantiomeric pur-
ity (>98%) was ascertained both by HPLC using a chiral column
7
0
4
7.9
5.6
3.6
31 2.3
6.8
28 6.7
4.9
25 6.4
2.3
7
4
3.3
4.5
0
0
0
[CROWNPAC CR(+)] and by enzymatic reaction with L- and D-amino
acid oxidases, followed by amino acid analysis. Two pyrazinone
a
Antiproliferative activity of compounds are expressed as inhibition of cellular
proliferation (%) and listed as the mean SE.
9-fluorenylmethanol,
.
EDC HCl, DMAP,
DMF
i) HCl/dioxane
ii) TEA, DMF
i) HCl/dioxane
Boc-
-D-Trp-Lys(Z)-OFm
Boc-Lys(Z)-OH
Boc-Lys(Z)-OFm
Boc-D-Trp-Lys(Z)-OFm
X
ii) Boc-Tyr-OH,
PyBop, DIEA, DMF
TEA, IBCF,
THF, -15ºC
X=Tyr, Dmt, Phe
Boc-D-Trp-OH
O
O
C
H
N
H
N
i) 20% piperidine/DMF
ii) HCl/dioxane
PyBop, DIEA, DMF
Boc
*
R
*
C
OH
Boc
*
R
*
X
D-Trp-Lys(Z)-OFm
HCl/dioxane
Boc-
-D-Trp-Lys(Z)-OFm
X
R
*
*
O
NH
N
1: R1=H,
3: R1=CH3,
5: R1=H,
R2=OH
R1
R1
( )4
O
*
HN
HC
R=
i) TEA, DPPA, DMF
ii) H2/Pd-Black, AcOH/H2
R2=OH
R2=H
H2
C
*
( )2
N
R2
CH (CH2)4 NH2
NH
O
(
O
H
H
2: R1=H,
4: R1=CH3,
6: R1=H,
R2=OH
R2=OH
R2=H
O
O
N
HN
)
4
*
CH
CH2
R=
O
*
(
)
2
N
HN
Scheme 1. The synthetic route to cyclic compounds 1–6.

A. Miyazaki et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6199–6201
6201
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Their antiproliferative activities on A431 cells (human epithelial
tumor cells) and SW480 cells (human colon carcinoma cells) on
which the SSTRs are expressed were measured by 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Table
1).25 Each analogue was tested at concentrations of 10, 25, and
50 lM. TT-232 and cycloheximide, used as external standards as
a well known and efficient apoptosis inducer and cell proliferation
inhibitor were used as positive controls.
In comparison with the compounds containing Tyr (1 and 2),
the replacement by Dmt (3 and 4) or Phe (5 and 6) resulted in a se-
vere or total loss of antiproliferative activity on A431 cells.
Although Dmt-containing opioid peptides and opioidomimetics
showed enhanced opioid receptor affinity and biological activity,
the decreased antiproliferative activity in the current study indi-
cates that the structures of the binding site of these receptors are
quite different from those of opioid receptors in spite of comprising
the same G protein-coupled receptor family. The hydrophobicity of
compounds were measured by RP-HPLC and the result was Phe (5
and 6) > Dmt (3 and 4) > Tyr (1 and 2) (data not shown). The more
hydrophobic compounds had almost no activity, though the li-
gand–receptor interaction might be activated by hydrophobicity.
On the other hand, it may also be conjectured that Tyr interacts
with SSTRs through H bonding through the OH functionality. How-
ever, the binding site of SSTRs was not accommodated by the
added bulkiness of Dmt, unlike opioid receptors. On SW480 cells
the activities of all compounds were not much different, but the
compounds containing Tyr exhibited more potent activity as ex-
pected. The difference of potency between both cells might be
due to the number of SSTRs expressed on respective tumor cells
and the SSTRs subtype. These results suggest that the Tyr residue
in structure of the cyclic peptides 1 and 2 is crucial for antitumor
activity.
13. Rohrer, S. P.; Birzin, E. T.; Mosley, R.; Berk, S. C.; Hutchins, S.; Shen, D. M.;
Xiong, Y.; Hayes, E.; Parmar, R.; Foor, F.; Mitra, S.; Degrado, S.; Shu, M.; Klopp,
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Chem. 2004, 47, 2599.
15. Jinsmaa, Y.; Okada, Y.; Tsuda, Y.; Shiotani, K.; Sasaki, Y.; Ambo, A.; Bryant, D. S.;
Lazarus, H. L. J. Pharmacol. Exp. Ther. 2004, 309, 432.
16. Miyazaki, A.; Yokoi, T.; Tachibana, Y.; Enomoto, R.; Lee, E.; Bokonyi, G.; Keri, G.;
Tsuda, Y.; Okada, Y. Tetrahedron Lett. 2004, 45, 6323.
17. Freidinger, M. R.; Perlow, S. D.; Randall, C. W.; Saperstein, R.; Arison, H. B.;
Veder, F. D. Int. J. Pept. Protein Res. 1984, 23, 142.
18. Shiotani, K.; Li, T.; Miyazaki, A.; Tsuda, Y.; Bryant, D. S.; Ambo, A.; Sasaki, Y.;
Lazarus, H. L.; Okada, Y. Bioorg. Med. Chem. Lett. 2006, 16, 5793.
19. Igarashi, K.; Murabayashi, Y.; Hotta, K.; Kitamura, Y.; Kasuya, F.; Shiotani, K.; Li,
T.; Miyazaki, A.; Tsuda, Y.; Okada, Y.; Fukushima, S. J. Chromatogr. B 2004, 806,
53.
20. Salvadori, S.; Attila, M.; Balboni, G.; Bianchi, C.; Bryant, S. D.; Crescenzi, O.;
Guessiini, R.; Picone, D.; Tancredi, T.; Temussi, P. A.; Lazarus, L. H. Mol. Med.
1995, 1, 678.
21. Lazarus, L. H.; Bryant, S. D.; Cooper, P. S.; Guerrini, R.; Balboni, G.; Salvadori, S.
Drug Discovery Today 1998, 3, 284.
22. Bryant, S. D.; Salvadori, S.; Cooper, P. S.; Lazarus, L. H. Trends Pharmacol. Sci.
1999, 19, 42.
23. Dygos, J. H.; Yonan, E. E.; Scaros, M. G.; Goodmonson, O. J.; Getman, D. P.;
Periana, R. A.; Beck, G. R. Synthesis 1992, 741.
24. Physicochemical data of compounds 3–6. c{[Lys-Glu]-Dmt-
D
-Trp-Lys}ꢀHCl (3):
Yield 41.6 mg (78.0%), amorphous, [
a
]
D
25 +121° (c 1.0, H2O), Rf 0.71 (n-butanol/
AcOH/pyridine/H2O = 4:1:1:2), TOF-MS m/z: Calcd [M+H]+. 741.9. Found:
741.9. Anal. Calcd for C40H53ClN8O6ꢀ4H2O: C, 56.6; H, 7.24; N, 13.2. Found. C,
56.6; H, 6.86; N, 13.5. 1H NMR (500 MHz, dimethyl sulfoxide-d6 (DMSO-d6)) d
10.7 (1H, s), 9.08 (1H, s), 8.23 (1H, d, J = 8.3 Hz), 7.97 (1H, d, J = 8.1 Hz), 7.88
(1H, d, J = 8.6 Hz), 7.73 (2H, br), 7.69 (1H, t, J = 7.1 Hz), 7.54 (1H, d, J = 7.9 Hz),
7.32 (1H, d, J = 8.1 Hz), 7.06 (1H, t, J = 8.0 Hz), 7.00–6.98 (2H, m), 6.36 (2H, s),
4.64 (1H, q-like, J = 7.9 Hz), 4.53 (1H, q-like, J = 7.7 Hz), 4.00 (1H, td, J = 14 and
8.4 Hz), 3.17 (1H, dq, J = 15 and 7.2 Hz), 2.85–2.78 (3H, m), 2.75–2.58 (6H, m),
2.58–2.37 (3H, m), 2.25–2.20 (1H, m), 2.18 (6H, s), 2.12 (3H, s), 1.55–1.45 (2H,
In conclusion, the newly designed and synthesized somato-
statin analogues contained the cyclic conformation due to a pyraz-
inone ring and the biologically essential amino acid sequence in
TT-232. Their antiproliferative activities were tested on two differ-
ent tumor cell lines, which initially indicated that Tyr, rather than
the more hydrophobic residues Dmt or Phe, was an important ele-
ment. We conclude that the exhibition of antiproliferative activity
through SSTRs needs not only hydrophobicity, but also a Tyr resi-
m), 1.45–1.27 (4H, m), 1.25–1.15 (2H, m), 0.95–0.85 (2H, br).
25
c{[Glu-Lys]-Dmt-
D-Trp-Lys}ꢀHCl (4): Yield 26.5 mg (61.5%), amorphous, [a]
D
+85.8° (c 1.0, H2O), Rf 0.61 (n-butanol/AcOH/pyridine/H2O = 4:1:1:2), TOF-MS
m/z: Calcd [M+H]+. 741.9. Found: 741.7. Anal. Calcd for C40H53ClN8O6 3.5H2O:
C, 57.2; H, 7.19; N, 13.3. Found: C, 57.0; H, 6.89; N, 13.3. 1H NMR (500 MHz,
DMSO-d6) d 10.7 (1H, s), 8.23 (1H, d, J = 7.5 Hz), 8.01 (1H, d, J = 8.6 Hz), 7.85
(1H, d, J = 6.9 Hz), 7.74 (2H, br), 7.67 (1H, br), 7.55 (1H, d, J = 7.9 Hz), 7.31 (1H,
d, J = 8.1 Hz), 7.05 (1H, t, J = 7.6 Hz), 6.97 (1H, t, J = 7.5 Hz), 6.94 (1H, d,
J = 2.0 Hz), 6.36 (2H, s), 4.78 (1H, br), 4.70 (1H, q-like, J = 7.7 Hz), 4.02 (1H, q-
like, J = 7.0 Hz), 3.64–3.55 (1H, m), 2.96–2.86 (1H, m), 2.85–2.72 (3H, m), 2.70–
2.61 (3H, m), 2.60–2.48 (3H, m), 2.54 (6H, s), 2.24–2.12 (2H, m), 1.89 (3H, s),
due. The three amino acid residues, Tyr-
D-Trp-Lys is surely useful
for the design of somatostatin analogues with antitumor activity,
and we intend to develop this sequence as core template.
Acknowledgments
This work was supported in part by KAKENHI (17790026), by a
Grant-in-Aid (C) from Kobe Gakuin University, by NEXT ‘Academic
Frontier’ Project (2006), and in part by Hungarian Grants JAP-6/02,
OTKA 49478, 60197, Cell Kom Ret-06/2006, Tibor Vántus is a Bolyai
Postdoctoral Fellow of the Hungarian Academy of Sciences.
1.56–1.47 (1H, m), 1.46–1.32 (6H, m), 1.28–1.18 (1H, m), 1.08–0.94 (2H, m).
25
c{[Lys-Glu]-Phe-
D
-Trp-Lys}ꢀHCl (5): Yield 43.5 mg (61.7%), amorphous, [a]
D
+77.8° (c 1.0, H2O), Rf 0.65 (n-butanol/AcOH/pyridine/H2O = 4:1:1:2), TOF-MS
m/z: Calcd [M+H]+. 697.8. Found: 697.8. Anal. Calcd for C38H49ClN8O5 4H2O: C,
56.7; H, 7.13; N, 13.9. Found: C, 56.5; H, 7.03; N, 14.1. 1H NMR (500 MHz,
DMSO-d6) d 10.8 (1H, s), 8.48 (1H, d, J = 8.1 Hz), 8.24 (1H, d, J = 8.2 Hz), 7.83
(1H, d, J = 8.3 Hz), 7.75 (2H, br), 7.69 (2H, br), 7.33 (1H, d, J = 8.1 Hz), 7.17 (1H,
d, J = 1.6 Hz), 7.12–7.05 (4H, m), 7.01 (1H, t, J = 7.5Hz), 6.93 (2H, d-like,
J = 6.7 Hz), 4.63–4.54 (2H, m), 4.12 (1H, dt, J = 8.5 and 5.7 Hz), 3.17 (1H, m),
3.02 (1H, dd, J = 14 and 5.4 Hz), 2.93 (1H, m), 2.86–2.75 (2H, m), 2.74–2.65 (3H,
m), 2.57–2.37 (5H, m), 2.24 (1H, s), 2.10 (3H, s), 1.60–1.41 (5H, m), 1.40–1.30
References and notes
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(2H, m), 1.25 (1H, m), 1.07 (2H, m).
25
c{[Glu-Lys]-Phe-
D
-Trp-Lys}ꢀHCl (6): Yield 45.5 mg (57.3%), amorphous, [a]
D
+41.9° (c 1.0, H2O), Rf 0.48 (n-butanol/AcOH/pyridine/H2O = 4:1:1:2), TOF-MS
m/z: Calcd [M+H]+. 697.8. Found: 697.5. Anal. Calcd for C38H49ClN8O5ꢀ3H2O: C,
58.0; H, 7.04; N, 14.2. Found: C, 57.9; H, 6.78; N, 14.5. 1H NMR (500 MHz,
DMSO-d6) d 10.7 (1H, s), 8.23 (1H, d, J = 7.5 Hz), 8.01 (1H, d, J = 8.6 Hz), 7.85
(1H, d, J = 6.9 Hz), 7.74 (2H, br), 7.67 (1H, br), 7.55 (1H, d, J = 7.9 Hz), 7.31 (1H,
d, J = 8.1 Hz), 7.05 (1H, t, J = 7.6 Hz), 6.97 (1H, t, J = 7.5 Hz), 6.94 (1H, d,
J = 2.0 Hz), 6.36 (2H, s), 4.78 (1H, br), 4.70 (1H, q-like, J = 7.7 Hz), 4.02 (1H, q-
like, J = 7.0 Hz), 3.64–3.55 (1H, m), 2.96–2.86 (1H, m), 2.85–2.72 (3H, m), 2.70–
2.61 (3H, m), 2.60–2.48 (3H, m), 2.54 (6H, s), 2.24–2.12 (2H, m), 1.89 (3H, s),
1.56–1.47 (1H, m), 1.46–1.32 (6H, m), 1.28–1.18 (1H, m), 1.08–0.94 (2H, m).
25. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
8. Kaupmann, K.; Bruns, C.; Raulf, F.; Weber, H. P.; Mattes, H.; Lubbert, H. EMBO J.
1995, 14, 727.