Novel, potent, and radio-iodinatable somatostatin receptor 1 (sst 1) selective analogues

Article abstract of DOI:10.1021/jm801314f

The proposed sst1 pharmacophore (J. Med. Chem. 2005, 48, 523-533) derived from the NMR structures of a family of mono- and dicyclic undecamers was used to design octa-, hepta-, and hexamers with high affinity and selectivity for the somatostatin sst₁

Full text of DOI:10.1021/jm801314f

J. Med. Chem. 2009, 52, 2733–2746
2733
Novel, Potent, and Radio-Iodinatable Somatostatin Receptor 1 (sst1) Selective Analogues
,†
,§
,‡
Judit Erchegyi, Renzo Cescato, Christy Rani R. Grace, Beatrice Waser,§ Ve´ronique Piccand,§ Daniel Hoyer,|
Roland Riek,‡ Jean E. Rivier,*,† and Jean Claude Reubi§
The Clayton Foundation Laboratories for Peptide Biology, Structural Biology Laboratory, The Salk Institute for Biological Studies,
10010 North Torrey Pines Road, La Jolla, California 92037, DiVision of Cell Biology and Experimental Cancer Research, Institute of
Pathology, UniVersity of Berne, Berne, CH 3010, Switzerland, Psychiatry/Neuroscience Research, WSJ-386/745, NoVartis Institutes for
Biomedical Research, Basel, CH 4002 Basel, Switzerland
ReceiVed October 17, 2008
The proposed sst1 pharmacophore (J. Med. Chem. 2005, 48, 523-533) derived from the NMR structures of
a family of mono- and dicyclic undecamers was used to design octa-, hepta-, and hexamers with high affinity
and selectivity for the somatostatin sst1 receptor. These compounds were tested for their in vitro binding
properties to all five somatostatin (SRIF) receptors using receptor autoradiography; those with high SRIF
receptor subtype 1 (sst1) affinity and selectivity were shown to be agonists when tested functionally in a
luciferase reporter gene assay. Des-AA1,4-6,10,12,13-[DTyr2,DAgl(NMe,2naphthoyl)8,IAmp9]-SRIF-Thr-NH2
(25) was radio-iodinated (125I-25) and specifically labeled sst1-expressing cells and tissues. 3D NMR structures
were calculated for des-AA1,4-6,10,12,13-[DPhe2,DTrp8,IAmp9]-SRIF-Thr-NH2 (16), des-AA1,2,4-6,10,12,13-[DAgl-
(NMe,2naphthoyl)8,IAmp9]-SRIF-Thr-NH2 (23), and des-AA1,2,4-6,10,12,13-[DAgl(NMe,2naphthoyl)8,IAmp9,Tyr11]-
SRIF-NH2 (27) in DMSO. Though the analogues have the sst1 pharmacophore residues at the previously
determined distances from each other, the positioning of the aromatic residues in 16, 23, and 27 is different
from that described earlier, suggesting an induced fit mechanism for sst1 binding of these novel, less
constrained sst1-selective family members.
Introduction
has antiproliferative activity and acts as a neurotransmitter or
neuromodulator in the brain.4–7a
Somatostatin (SRIFa) is a cyclic tetradecapeptide widely
distributed throughout the body with important regulatory effects
(mostly inhibitory) on a variety of endocrine and exocrine
functions. It was originally isolated from the hypothalamus and
characterized in 1973.2,3 Somatostatin is also found in the gut,
pancreas, in the nervous system, and in various exocrine and
endocrine glands. Somatostatin inhibits the release of growth
hormone from the anterior pituitary, insulin and glucagon from
the pancreas, and gastrin from the gastrointestinal tract. It also
SRIF interacts with five specific receptor subtypes (sst1-sst5)
that have been cloned and characterized, all belonging to the
G-protein-coupled receptor family.8–10 Cell lines recombinantly
expressing these cloned receptors are available to test SRIF
analogues for binding affinity, selectivity, and functional
effects.7,11–13 So far, only sst2 and sst5 receptors have been
clearly linked to specific physiological functions.14,15 For sst1
receptors, however, the cellular localization as well as the
distinct functions are still not fully understood, similarly to our
current lack of knowledge of sst3 or sst4 receptor function and
physiology.
* To whom correspondence should be addressed. Phone: (858) 453-4100.
Fax: (858) 552-1546. E-mail: jrivier@salk.edu.
†
The Clayton Foundation Laboratories for Peptide Biology.
Structural Biology Laboratory, The Salk Institute for Biological Studies.
Division of Cell Biology and Experimental Cancer Research, Institute
Sst1 receptors have been found in human cerebral cortex,16
human retina,17 neuroendocrine cells,14 endothelial cells of
human blood vessels (arteries and veins),18 and human
tumors.19–24 According to Lanneau25 and Olias,14 sst1 receptors
are involved in the intrahypothalamic regulation of growth
hormone (GH) secretion. The studies of Kreienkamp et al.
suggested an important role for sst1 in the control of basal GH
release in somatotrophs.26 Zatelli et al. have demonstrated that
sst1-selective activation inhibits hormone secretion and cell
viability in GH- and prolactin-secreting adenomas in vitro and
suggested that somatostatin analogues with affinity for sst1
receptors may be useful to control hormone hypersecretion and
reduce neoplastic growth of pituitary adenomas.27 Sst1 receptor
activation also modulates somatostatin release in basal ganglia.28
In hypothalamic, basal ganglia, and retinal functions, the sst1
receptor appears to act as an inhibitory autoreceptor located on
somatostatin neurons, whereas in the hippocampus, such a role
is still based on circumstantial evidence.
‡
§
of Pathology, University of Berne.
|
Psychiatry/Neuroscience Research, WSJ-386/745, Novartis Institutes
for Biomedical Research.
These authors contributed equally.
a Abbreviations: The abbreviations for the common amino acids are in
accordance with the recommendations of the IUPAC-IUB Joint Commission
on Biochemical Nomenclature (Eur. J. Biochem., 1984, 138, 9-37). The
symbols represent the L-isomer except when indicated otherwise. Additional
abbreviations: AA, amino acid; Agl, aminoglycine; ATP, adenosine 5′-
triphosphate; Boc, tert-butoxycarbonyl; Bzl, benzyl; Bzl(3Br), 3-bromoben-
zyl; cAMP, 3′,5′-cyclic adenosine monophosphate; Cbm, carbamoyl; CM,
chloromethyl; CZE, capillary zone electrophoresis; DIC, N,N′-diisopropy-
lcarbodiimide; DIPEA, diisopropylethylamine; DMEM, Dulbecco’s modified
Eagle’s medium; DMF, dimethylformamide; DQF-COSY, double quantum
filtered correlation spectroscopy; Fmoc, 9-fluorenylmethoxycarbonyl; HEPES,
4-(2-hydroxyethyl) piperazine-1-ethansulfonic acid; hhLys, homohomo-
lysine; HOBt, 1-hydroxybenzotriazole; IAmp, 4-(N-isopropyl)-aminometh-
ylphenylalanine; MBHA, 4-methylbenzhydrylamine; Mob, 4-methoxyben-
zyl; Nal, 3-(2-naphthyl)-alanine; NMP, N-methylpirrolidinone; NOESY,
nuclear Overhauser enhancement spectroscopy; PBS, phosphate buffered
saline; rmsd, root-mean-square deviation; ROESY, rotating frame nuclear
Overhauser enhancement spectroscopy; SRE, serum response element; SRIF,
somatostatin; SRIF-28, somatostatin-28; ssts, SRIF receptors; TEA, triethy-
lamine; TEAP, triethylammonium phosphate; TFA, trifluoroacetic acid;
TOCSY, total correlation spectroscopy; Z(2Br), 2-bromobenzyloxycarbonyl;
Z(2Cl), 2-chlorobenzyloxycarbonyl.
Sst1-selective analogues could possibly play a role in various
diseases. For instance, retinal disease therapeutics has been a
suggested indication for sst1.17,28–30 Recently, an sst1 antagonist
was shown to promote social interactions, reduce aggressive
10.1021/jm801314f CCC: $40.75
2009 American Chemical Society
Published on Web 04/07/2009

2734 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Erchegyi et al.
behavior, and stimulate learning.31,32 Matrone et al. demon-
strated the role of sst1-selective analogues in mediating the
inhibitory effect of SRIF on growth hormone secreting pituitary
tumors. They suggested that the sst1-selective analogues might
represent a further useful approach for the treatment of acrome-
galy in patients resistant or partially responsive to octreotide-
LAR or lanreotide treatment in vivo.33 It has also been reported
that in medullary thyroid carcinoma, calcitonin secretion and
gene expression can be reduced by treatment with sst1-selective
agonists by the reduction of cAMP response element binding
phosphorylation, suggesting that potent sst1-selective agonists
could have a therapeutic role in medullary thyroid carcinoma.34,35
Furthermore, because sst1-selective agonists were able to inhibit
endothelial activities, a potential therapeutic utility for admin-
istration of sst1-selective agonists in the proliferative diseases
involving angiogenesis has been suggested.18
Although numerous reports on the localization, physiological,
and therapeutic functions of sst1 receptors have been published,
it is still not clear which is the main sst1 receptor function and
the main sst1 related pathology. The design of more potent and
more (>100-fold) sst1-selective agonists and antagonists with
radio-labelable properties for in vitro binding assays and in vivo
scintigraphy studies as well as greater metabolic stability in
biological fluids than the native hormone could help to further
understand sst1-related biology and pathobiology.
We used an unresolved aminoglycine (Agl) derivative
Fmoc-D/LAgl(NMe,Boc)-OH46,47 as a template for the intro-
duction of a betidamino acid in the scaffolds (des-AA1,4-6,10,12,13
-
SRIF-Thr/Nal-NH2, des-AA1,2,4-6,10,12,13-SRIF-Thr/Nal-NH2, and
des-AA1,2,4-6,10,12,13-SRIF-NH2), which resulted in diastereomeric
mixtures that were separated by RP-HPLC48–50 and were fully
characterized. The absolute configuration of the Agl residue in
the diastereomers was deduced from enzymatic hydrolysis
studies with aminopeptidase M and proteinase K, respectively.
Comparison of the enzymatic cleavage patterns of the diaster-
eomers showed that the LAgl-containing analogue was hydro-
lyzed while the DAgl-containing analogue was not.50
The peptide resins were treated with anhydrous hydrogen
fluoride in the presence of scavengers (anisole and dimethyl-
sulfide) to liberate the fully deblocked crude peptides. Cycliza-
tion of the cysteines was mediated by iodine in an acidic milieu.
Purification was achieved using multiple RP-HPLC steps.49
Analytical RP-HPLC,49 capillary zone electrophoresis (CZE),51
and mass spectrometry were used to determine the purity and
identity of the analogues.
NMR Studies. The NMR samples of 16, 23, and 27 were
prepared by dissolving 2.5 mg of the peptide in 500 µL of
DMSO-d6. Assignment of the various proton resonances was
carried out using the standard procedure using DQF-COSY,
TOCSY, and NOESY experiments. Though 16 showed a single
set of resonances for all the protons, two sets of resonances
were observed for most of the protons for 23 and 27 in the
ratio 60:40, except for the HN and RH resonances of IAmp9.
This is due to the cis/trans isomerization of the amide bond
present in the side chain of the betidamino acid DAgl at position
8 of 23 and 27, as reported earlier.52 Almost complete
assignment of the chemical shifts of the various proton
resonances is carried out for 16, 23, and 27 and is shown in
parts A-C of Table 2. Amide resonances of Cys3 were not
observed for 23 and 27 in the NMR spectra due to fast exchange
with the solvent.
A reasonably large number of experimental NOEs is observed
for the three analogues in the NOESY spectrum measured with
a mixing time of 100 ms, leading to over 70 meaningful distance
restraints per analogue (Table 3, Figure 1). For all analogues,
structural restraints from NOEs were used as input for the
structure calculation with the program CYANA,53 followed
by restrained energy minimization using the program DIS-
COVER.54 The resulting bundle of 20 conformers per analogue
represents the 3D structure of each analogue. For each analogue,
the small residual violations in the distance constraints only for
the 20 refined conformers (Table 3) and the coincidence of
experimental NOEs and short interatomic distances (data not
shown) indicate that the input data represent a self-consistent
set and that the restraints are well satisfied in the calculated
conformers (Table 3). The deviations from ideal geometry are
minimal, and similar energy values were obtained for all 20
conformers of each analogue. The quality of the structures
determined is reflected by the small backbone rmsd values
relative to the mean coordinates of ∼0.5 Å (see Table 3 and
Figure 2).
Originally, we had identified the first generation of a
peptidic scaffold with selected amino acids (AA) deletions
(des-AA1,2,5-SRIF) that in combination with DTrp at position
8 and 4-(N-isopropyl)-aminomethylphenylalanine (IAmp) at
position 9 yielded des-AA1,2,5-[DTrp8,IAmp9]-SRIF (CH-
275)36 (2) (Table 1), a SRIF agonist that was 30-fold more
selective for sst1 versus sst2/4/5 and 10-fold versus sst3,
respectively.36 Our standard drug design approaches led to
additional very potent sst1-selective mono- and dicyclic
undecapeptides des-AA1,2,5-[DTrp8,IAmp9,Tyr11]-SRIF (3),37
des-AA1,2,5-[DTrp8,IAmp9,ITyr11]-SRIF (4),37 des-AA1,2,5
-
-
[DTrp8,IAmp9,ITyr11]-Cbm-SRIF
(5),1
des-AA1,2,5
cyclo(7-12)[Glu7,DTrp8,IAmp9,ITyr11,hhLys12]-SRIF (6),38
and des-AA1,2,5-[DAgl(NMe,2naphthoyl)8,IAmp9, Tyr11]-SRIF
(7)39 Table 1.1,37–40
In several drug design studies of different hypothalamic
releasing hormones (gonadotropin-releasing hormone (GnRH),41
corticotropin-releasing factor (CRF),42 (SRIF37,43,44), we had
demonstrated that substitutions of natural amino acids with
betidamino acids were compatible with biological activity.
Betidamino acids are monoacylated derivatives of R-aminogly-
cine. The synthesis of R-Fmoc,ꢀ-Boc-aminoglycine was orig-
inally described by Qasmi et al.,45 and the synthesis of the
methylated derivatives was published by our group.46 As we
demonstrated earlier, the substitution of DTrp8 by
DAgl(NMe,2naphthoyl)8 in the undecamer scaffold37 (3 versus
7, Table 1) had resulted in a 5-fold increase in binding affinity
at sst1.39
Here, we describe the rational design and optimization of a
novel class of peptidic somatostatin sst1-selective analogues
derived from the use of our published sst1-pharmacophore
model.1
Three-Dimensional Structure of H-DPhe2-c[Cys3-Phe7-DTrp8-
IAmp9-Phe11-Cys14]-Thr15-NH2 (16). Analogue 16 binds selec-
tively to sst1 with moderately high affinity (IC50 ∼ 30 nM). It
differs from octreotide by IAmp9, Phe11, and Thr15-NH2, and
the 3D NMR structure shows that the backbone has a ꢀ-turn of
type III’ around DTrp8 and IAmp9 (Figure 2A, Table 4). In all
of the calculated 20 conformers, there is a hydrogen bond
between the amide protons of Phe11 to the carbonyls of Phe7
Results and Discussion
Peptide Synthesis. All analogues shown in Table 1 were
synthesized either manually or automatically on a 4-methyl-
benzhydrylamine (MBHA) or chloromethylated (CM) resin
using the Boc-strategy and N,N′-diisopropylcarbodiimide (DIC)/
1-hydroxybenzotriazole (HOBt) for amide bond formation.

Radio-Iodinatable Sst1-SelectiVe Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2735

2736 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Table 2. Chemical Shifts (in ppm) of 16, 23, and 27 in DMSO-d6
Erchegyi et al.
residue
NH
RH
ꢀH
others
(A) Chemical Shifts (in ppm) of 16 in DMSO-d6
DPhe2
Cys3
8.00
9.28
8.55
8.66
8.88
7.72
8.65
8.11
4.19
5.39
4.71
4.23
4.24
4.90
5.20
4.25
3.26, 2.97
2.88, 2.76
2.87, 2.76
2.66, 2.50
3.09, 2.56
3.19, 3.00
2.84, 2.84
4.05
QD: 7.40; QE: 7.36
Phe7
QD: 6.99; QE: 7.03
DTrp8
IAmp9
Phe11
Cys14
Thr15
NH2
HD1: 6.86; HE3: 7.49; HE1: 10.73; HZ3: 7.04; HZ2: 7.36; HH2: 7.11
QD: 7.29; QE: 7.36; QT: 4.06; HH: 8.59; QK1: 1.15; QK2: 1.15; HI: 3.15
QD: 7.30; QE: 7.20
γCH3: 1.07; OH: 5.21
7.58,7.39
(B) Chemical Shifts (in ppm) of 23 in DMSO-d6
Cys3
major
minor
4.01
3.07, 2.89
Phe7
major
minor
8.87
5.03
4.75
3.01, 2.91
QD: 7.28; QE: 7.17
DAgl8
IAmp9
Phe11
Cys14
Thr15
NH2
major
minor
8.80
9.21
6.53
5.74
QG: 2.27; H1: 7.78; H3: 7.65; H4: 8.02; H5: 8.01; H6: 7.65; H7: 8.02; H8: 7.25
QG: 2.55; H1: 7.86; H3: 7.69; H4: 7.93; H5: 7.92; H6: 7.69; H7: 7.93; H8: 7.36
major
minor
8.24
4.27
2.92, 2.80
3.21, 2.93
3.07, 2.83
4.04
QD: 7.12; QE: 7.21; QT: 3.78; HH: 7.37; QK1: 1.23; QK2: 1.10; HI: 3.04
QE: 7.36; QT: 4.06; HI: 3.24
major
minor
8.39
8.12
4.57
4.48
QD: 7.29; QE: 7.21
major
minor
7.81
8.12
4.63
4.55
major
minor
7.67
7.60
4.10
γCH3: 1.03; OH: 4.90
7.17, 7.29
(C) Chemical Shifts (in ppm) of 27 in DMSO-d6
Cys3
major
minor
3.95
3.03, 2.82
Phe7
major
minor
8.75
5.02
4.75
3.01, 2.91
3.00, 2.87
QD: 7.28, QE: 7.41
DAgl8
IAmp9
Tyr11
Cys14
NH2
major
minor
8.86
9.23
6.53
5.71
QG: 2.34; H1: 7.81; H3: 7.62; H4: 8.00; H5: 7.89; H6: 7.64; H7: 8.00; H8: 7.28
QG: 2.62, H5: 8.01
major
minor
8.35
4.24
2.83, 2.83
3.08, 2.83
3.02, 2.80
QD: 7.10; QE: 7.21; QT: 3.81; HH: 7.34; QK1: 1.23; QK2: 1.11; HI: 3.07
HH: 7.21, QT: 4.07, HI: 3.23
major
minor
8.30
8.02
4.41
4.35
QD: 7.07; QE: 6.69
QD: 7.27
major
minor
7.62
4.41
major
7.27, 7.08
that stabilizes the ꢀ-turn. The side chains of DPhe2, Phe7, and
DTrp8 are in the gauche+ configuration, and the side chains of
IAmp9 and Phe11 are in the gauche- configuration (Table 4).
Table 3. Characterization of the NMR Structures of the Analogues
Studied by NMR
parameters
16
23
27
Three-Dimensional
Structure
of
H-c[Cys3-Phe7-
DAgl(NMe,2naphthoyl)8-IAmp9-Phe11-Cys14]-Thr15-NH2 (23).
Analogue 23 differs from 16 by the substitution of DTrp8 by
DAgl(NMe,2naphthoyl)8 and the deletion of DPhe2 at the
N-teminus (Table 1). With those modifications, 23 binds
selectively to sst1 with high affinity (IC50 ) 1 nM). The 3D
NMR structure of 23 shows that the angles around
DAgl(NMe,2naphthoyl)8 and IAmp9 do not represent any
standard ꢀ-turns (Figure 2B, Table 4). The side chains of Phe7,
DAgl(NMe,2naphthoyl)8, IAmp9, and Phe11 are in the gauche-
configuration (Table 4). In addition, the side chains of Phe7 and
DAgl(NMe,2naphthoyl)8 span a large conformational space
(Figure 2B, Table 4). In the major conformer, the side chain
methyl protons of DAgl(NMe,2naphthoyl)8 give rise to NOE/
ROEs to both the H1 and H8 protons of the naphthoyl group,
suggesting that DAgl(NMe,2naphthoyl)8 is in trans isomer in
the major conformer. On the basis of the large number of NOEs
restraints
NOE distances
angles (deg)
93
23
0.29
72
16
0.25
58
7
0.27
CYANA target function
rmsd (in Å)
backbone
overall
0.35 ( 0.15 0.75 ( 0.18 0.12 ( 0.06
1.12 ( 0.20 1.47 ( 0.31 1.92 ( 0.37
residual violations on distances
No g 0.1 (Å)
max (Å)
dihedral angles
No g 1.5, (deg)
max (deg)
0.50 ( 0.20 0.20 ( 0.06 0.40 ( 0.08
0.80 ( 0.15 0.06 ( 0.02 0.08 ( 0.01
0.0 ( 0.01 0.0 ( 0.0
0.1 ( 0.10 0.0 ( 0.0
0.0 ( 0.0
0.0 ( 0.0
CFF91 energies (kcal/mol)
total energy
van der Waals
218 ( 9
168 ( 8
50 ( 7
233 ( 8
181 ( 5
51 ( 4
202 ( 4
174 ( 6
28 ( 2
electrostatic

Radio-Iodinatable Sst1-SelectiVe Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2737
Figure 1. Survey of characteristic NOEs describing the secondary structure of analogues (A) 16, (B) 23, and (C) 27. Thin, medium, and thick bars
represent weak (4.5-6 Å), medium (3-4.5 Å), and strong (<3 Å) NOEs observed in the NOESY spectrum. One letter code represents the amino
acids in the sequence of the peptide, where f, g, w, and X represent DPhe, DAgl(NMe, 2naphthoyl), DTrp, and IAmp, respectively.
Figure 2. 3D NMR structures of analogues (A) H-DPhe-c[Cys-Phe-DTrp-IAmp-Phe-Cys]-Thr-NH2 (16), (B) H-c[Cys-Phe-DAgl(NMe,2naphthoyl)-
IAmp-Phe-Cys]-Thr-NH2 (23), and (C) H-c[Cys-Phe-DAgl(NMe,2naphthoyl)-IAmp-Tyr-Cys]-NH2 (27). For each analogue, 20 energy-minimized
conformers with the lowest target function are used to represent the 3D NMR structure. The bundle is obtained by overlapping the CR atoms of all
the residues. The backbone and the side chains are displayed, including the disulfide bridge. The amino acid side chains that are proposed to be
involved in sst1 binding are highlighted: light green, DTrp/DAgl at position 8; blue, IAmp at position 9; yellow, Phe at positions 7 and 11. In (D),
the side chains of sst1 binding analogue 5 (in dark green) are superimposed on the structure of 16 (in gray), 23 (in cyan), and 27 (in magenta). It
should be noted that the aromatic side chains at positions 7 and 11 of analogue 5 are in the other side of the peptide backbone compared to 16, 23,
and 27.
observed, the 3D NMR structure of the major conformer was
calculated and this is assumed to be the bioactive conformation.
conformer was calculated and this is assumed to be the bioactive
conformation. Moreover, the minor conformer was poorly
defined due to the few number of NOEs that could be identified
for this conformation.
Three-Dimensional
Structure
of
H-c[Cys3-Phe7-
DAgl(NMe,2naphthoyl)8-IAmp9-Tyr11-Cys14]-NH2 (27). The com-
position of 27 is different from that of 23, with Tyr11 replacing
Phe11 and the deletion of Thr15 at the C-terminus (Table 1), yet
it binds selectively to sst1 with high affinity (IC50 ) 6 nM).
The 3D NMR structure shows that the angles around
DAgl(NMe,2naphthoyl)8 and IAmp9 do not represent any
standard ꢀ-turns (Figure 2C, Table 4). The side chains of Phe7
and Tyr11 are in the gauche+ configuration, and the side chains
of DAgl(NMe,2naphthoyl)8 and IAmp9 are in the gauche-
Biological Testing. To determine their SRIF receptor-binding
properties, the compounds were tested for their ability to bind
to cryostat sections of a membrane pellet of cells expressing
the five human SRIF receptor subtypes (Table 1). For each of
the tested compounds, complete competition experiments were
carried out with the universal SRIF radioligand [Leu8,-
DTrp22,125ITyr25]SRIF-28(125I-[LTT]-SRIF-28).55 The results are
shown in Table 1. The most potent and selective analogues were
functionally evaluated for their agonist/antagonist properties
using a reporter gene assay that determines the biological activity
of the human sst1 receptor in CCL39-sst1-Luci cells, constitu-
tively expressing the human sst1 receptor as well as the luciferase
gene under the control of the serum response element (SRE).
The SRE is regulated by transcription factors and is activated
by many extracellular signals including ligands acting at
G-protein-coupled receptors.56,57 It has been shown that upon
configuration
(Table
4).
The
side
chains
of
DAgl(NMe,2naphthoyl)8 and IAmp9 span a large conformational
space (Figure 2C, Table 4). In the major conformer, the side
chain methyl protons of DAgl(NMe,2naphthoyl)8 give rise to
NOE/ROEs to both the H1 and H8 protons of the naphthoyl
group, suggesting that DAgl(NMe,2naphthoyl)8 is in trans
isomer in the major conformer. On the basis of the large number
of NOEs observed, the 3D NMR structure of the major

2738 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Erchegyi et al.
Table 4. Torsion Angles φ, Ψ, and ꢁ1 (in deg) of the Bundle of 20 Energy Minimized Conformers
analogue
angle
DPhe2
Cys3
Phe7
DTrp8
IAmp9
Phe11
Cys14
Thr15
16
φ
-174 ( 5
46 ( 2
-125 ( 11
-108 ( 12
145 ( 5
1 ( 32
60 ( 7
31 ( 4
27 ( 31
63 ( 1
14 ( 2
-109 ( 1
-68 ( 11
-68 ( 24
-168 ( 19
52 ( 34
100 ( 18
-100 ( 37
-54 ( 75
134 ( 47
102 ( 76
Ψ
ꢁ1
-147 ( 1
9 ( 30
analogue
angle
DPhe2
Cys3
Phe7
DAgl8
IAmp9
Phe11
Cys14
Thr15
23
φ
-84 ( 62
170 ( 10
-41 ( 50
-13 ( 62
-74 ( 13
-59 ( 93
-117 ( 33
-158 ( 11
-150( 6
48 ( 7
26 ( 2
-155 ( 4
-96 ( 26
-172 ( 2
-102 ( 12
146 ( 64
-98 ( 81
-137 ( 40
Ψ
ꢁ1
179 ( 60
99 ( 61
analogue
angle
DPhe2
Cys3
Phe7
DAgl8
IAmp9
Tyr11
Cys14
Thr15
27
φ
-156 ( 8
157 ( 1
27 ( 1
95 ( 4
-54 ( 1
-112 ( 102
166 ( 3
42 ( 1
-18 ( 80
-110 ( 8
-35 ( 3
141 ( 9
-57 ( 6
Ψ
ꢁ1
132 ( 9
34 ( 13
-85 ( 0
-109 ( 0
cophore has two aromatic side chains, at position 6 or 7 and
11, in addition to the DTrp8 and IAmp9 pair. The sst1 selectivity
is achieved mainly through the amino acid IAmp at position 9,
which replaces Lys, present in most of the somatostatin
analogues. The side chain of IAmp is longer than the side chain
of Lys, it has an aromatic group (amino-methylphenylalanine)
extended by a relatively bulky aliphatic group (isopropyl).
Hence, we conclude that IAmp cannot be accommodated in a
smaller binding pocket that will fit a smaller side chain as that
of Lys9 critical for sst2-5 binding. We propose that only in the
sst1 receptor structure is the binding pocket large enough to
accommodate the side chain of IAmp (comparing the residues
in the models of the transmembrane regions of the somatostatin
receptors based on the crystal structure of rhodopsin) and
therefore analogues with IAmp at position 9 show selectivity
for sst1.
Figure 3. SRIF analogue 25 is a potent sst1 agonist when tested in
the luciferase reporter gene assay. The assay was performed as described
in Experimental Procedures. CCL39-sst1-Luci cells were treated with
increasing concentrations (0.1 nmol/L, 1 nmol/L, 10 nmol/L, 100 nmol/
L, and 1 µmol/mL) of SRIF (b), 33 (9), or 25 ([). The stimulation of
the luciferase reporter gene activity by the compounds is expressed as
% stimulation of the 1 µmol/mL SRIF effect. Shown are the
dose-response curves of the compounds. The sst3-antagonist 35 (2),
used as negative control at 1 µmol/mL, shows no effect in the luciferase
reporter gene assay.
We wanted to identify smaller peptidic molecules than the
undecamers, which would retain selectivity as well as good
affinity at sst1 receptors and possibly would act as antagonists
having greater metabolic stability than the previously published
analogues.36–39,59 As we published earlier, the hypothesis that
IAmp9 by itself might establish sst1 selectivity in the otherwise
potent pan-somatostatin octapeptide des-AA1,2,4,5,12,13-[DTrp8]-
SRIF (ODT-8) (8)50 failed (Table 1). Substitution of Lys9 in 8
by IAmp9 resulted in des-AA1,2,4,5,12,13-[DTrp8, IAmp9]-SRIF (9),
which showed no binding affinity to any SRIF receptor,37 most
probably due to steric effects perturbing the alignment of the
DTrp8-IAmp9 side chains with respect to the other aromatic side
chains.
agonist binding, SRIF receptors mediate an increase of luciferase
expression via SRE in this reporter gene assay.32,58 Figure 3
shows that stimulating CCL39-sst1-Luci cells with somatostatin
analogues activates the luciferase gene in a concentration-
dependent manner. SRIF, 23, and 25 exhibit EC50 values of 0.93,
3.9, and 0.37 nM, respectively, while the sst1- agonist des-AA1,5-
[Tyr2,DTrp8,IAmp9]-SRIF (CH-288) (33)37 exhibits an EC50
Because the backbone conformations of the sst1-selective
analogues have a hairpin-like structure similar to that of the
sst2/3/5-selective octreotide-based analogues, we decided to
introduce IAmp at position 9 in the octreotide scaffold with
the additional substitutions (an aromatic side chain at positions
6 or 7 and at position 11) indispensable to fulfill the sst1
pharmacophore’s requirements (see below).
value
of
33
nM.
Des-AA1,2,4,5,12,13[DCys3,Tyr7,-
DAgl8(NMe,2naphthoyl)]-Cbm-SRIF(Sst3-ODN-8) (35),43 a short-
sized somatostatin analogue with no affinity to sst1 receptor used
as a negative control, was unable to stimulate luciferase gene
expression in CCL39-sst1-Luci cells. The results of the reporter
gene assay are summarized in Table 1.
With the aim of searching for sst1-selective antagonists, we
first substituted Lys9 with IAmp9 in the published sst2-antagonist
des-AA1,4-6,11-13-[Cpa2,DCys3,Tyr7,DTrp8]-SRIF-2Nal-NH2
(12),60,61 which resulted in (des-AA1,4-6,11-13-[Cpa2,DCys3,-
Tyr7,DTrp8,IAmp9]-SRIF-2Nal-NH2 (13) with no binding af-
finity to sst1/4/5 and low binding affinity to sst2/3 (ca. 350 nM).
On the basis of the sst1 pharmacophore, it was expected that
the introduction of IAmp alone would not be sufficient for sst1
binding (Table 1) because 13 does not have two of the aromatic
residues at positions 6 or 7 and 11 necessary for sst1 binding.
Although there is an aromatic residue at position 7 in octreotide-
Structure-Activity Relationship Studies. The proposed sst1
receptor pharmacophore1 derived from the NMR structures of
the family of the undecamers (mono and dicyclic)37,38 compared
with the previously proposed sst2, sst2,5, and sst4 pharmacophores
are shown in Figure 4.
With a well-defined pharmacophore, we were now in a
position to design improved analogues following a rational
approach. In this regard, we designed putative sst1-selective
octa-, hepta-, and hexamers derived from the known undecam-
ers.1 Figure 4A shows that the sst1-selective agonist-pharma-

Radio-Iodinatable Sst1-SelectiVe Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2739
Figure 4. Schematic drawings of agonist pharmacophores for receptor-selective analogues binding to the somatostatin receptors: (A) sst1, (B) sst2,
(C) sst2/5, and (D) sst4. The amino acid side chains, which are part of the pharmacophores, and the range of distances between the corresponding
Cγ atoms of the side chains are shown.
based analogues, they do not have Phe at position 11. Hence,
we replaced Thr at position 10 by Phe at 11 (as in SRIF), leaving
four residues and the two Cys residues in the cycle. This
analogue (16) exhibited similar affinity to sst1 (IC50 ) ∼30 nM)
as 8, but it is more selective because it shows low affinity to
the other four receptors (see Table 1). Therefore analogue 16
was selected as lead for further derivatization.
Our SAR studies focused on positions 2, 3, 8, 9, 11, and 15
of the analogue 16 (SRIF numbering). Affinities to the five SRIF
receptors were determined in receptor binding assay and are
summarized in Table 1.
analogues, which contained 11 residues, 27, containing only 6
residues, is a much smaller and appealing molecule.
Although des-AA1,4-6,12,13-[Tyr2,DTrp8,IAmp9]-SRIF-Thr-NH2
(14), 15, and 17-20 (Table 1) showed IC50 for sst1 in the 500
nM range, their binding affinities are significantly lower
compared to that of SRIF-28 (IC50 for sst1 ) 2.9 nM), 8 (IC50
for sst1 ) 27 nM), and 5 (IC50 for sst1 ) 2.5 nM).1 The lower
affinities of these analogues could be explained based on the
knowledge of the sst1 pharmacophore and be due to one or more
of the following reasons: (1) the distances between the side
chains of the residues involved in binding may be slightly
different from the distances required by the sst1 pharmacophore
(the distances reported for efficient receptor-ligand interactions
are as follows: between DTrp8 and IAmp9 is 7-8 Å, between
DTrp8 and Phe6/7 is 6-7.5 Å, between DTrp8 and Phe11 is
9.5-12 Å, between IAmp9 and Phe6/7 is 9-11 Å, between
IAmp9 and Phe11 is 8-10 Å, and between Phe6/7 and Phe11 is
6-7.5 Å, (Figure 4A)1) that the side chains possibly occupy
the binding pocket partially, (2) the conformational rigidity in
the side chain of DTrp at position 8 is in such a way that it
cannot occupy completely the available binding pocket, (3)
although the analogues have two aromatic residues, the aromatic
side chains could be on the front side of the peptide backbone
(when displayed from N to the C-terminus), resulting in µM
binding affinity.
SAR at position 2 where DPhe, Cpa, Tyr, and DTyr were
substituted show that these residues can be eliminated with only
small differences in binding affinity to sst1 (des-AA1,2,4-6,10,12,13
-
[DTrp8,IAmp9]-SRIF-Thr-NH2 (15) versus 16, des-AA1,2,4-6,10,12,13
-
[DTrp8,IAmp9,ITyr11]-SRIF-Thr-NH2 (17) versus des-AA1,4-6,10,12,13
-
[DPhe2,DTrp8,IAmp9,ITyr11]-SRIF-Thr-NH2 (18), des-AA1,2,4-6,10,12,13
-
[DCys3,DTrp8,IAmp9,ITyr11]-SRIF-2Nal-NH2 (19) versus des-
AA1,4-6,10,12,13-[Cpa2,DCys3,DTrp8,IAmp9,ITyr11]-SRIF-2Nal-
NH2 (20), des-AA1,2,4-6,10,12,13-[LAgl(NMe,2naphthoyl)8,IAmp9]-
SRIF-Thr-NH2
(24)
versus
des-AA1,4-6,10,12,13
-
[DTyr2,LAgl(NMe,2naphthoyl)8,IAmp9]-SRIF-Thr-NH2 (26), and
des-AA1,2,4-6,10,12,13-[DCys3,DAgl(NMe,2naphthoyl)8,IAmp9]-SRIF-
2Nal-NH2
(29)
versus
des-AA1 , 4 - 6 , 1 0 , 1 2 , 1 3
-
[Tyr2,DCys3,DAgl(NMe,2naphthoyl)8,
IAmp9]-SRIF-2Nal-NH2 (31)). We suggest that the individual
nature of these amino acids (despite the fact that their side chains
are not involved in binding) is responsible for the differences in
affinity. SAR at position 3 suggests that the substitution of Cys
with DCys has little effect on sst1 binding affinity and function.
SAR at position 8 shows the unique nature of Agl8-containing
analogues that are all highly selective for sst1 over the other four
receptor subtypes (23, 25, 27, 29). Interestingly, for this scaffold,
a D-configuration is significantly more favorable than the L-
conformation. SAR at position 9 shows that the introduction of
IAmp results in more active and selective analogues (des-
AA1,4-6,10,12,13-[Cpa2,DCys3,DAgl(NMe,2naphthoyl)8]-SRIF-2Nal-
NH2 (21) versus 31). SAR at position 11 shows that the substitution
of Phe as in 15 or 16 with I-Tyr results in some loss in sst1 binding
affinity (17 and 18). SAR at position 15 shows that this residue
can also be eliminated from some structures without loss of binding
affinity and selectivity as in 27. This deletion along with other most
favorable substitutions yielded the hexapeptide H-c[Cys3-Phe7-
DAgl(NMe,2naphthoyl)8-IAmp9-Tyr11-Cys14]-NH2 (27). This pep-
tide selectively binds to sst1 with an average IC50 ) 6.0 nM, (IC50
>1000 nM at sst2, 317 nM at sst3, 481 nM at sst4, and >1000 nM
at sst5, respectively). Compared to our previously published
In the octreotide pharmacophore, the side chains of DTrp8
and Lys9 are in close proximity (4-6 Å),62,63 (Figure 4C),
whereas in the sst1-pharmacophore, the side chains of DTrp8
and IAmp9 are farther apart (7-8 Å) (Figure 4A). Because the
side chain of aminoglycine (Agl) has been shown to span a
larger volume than the DTrp side chain, we introduced acylated
Agl at position 8.52 This substitution in the shortened analogues
(21, 23, 25, 27, 29, and 31) improved the binding affinity and
selectivity for sst1 as well. Some of these analogues also exhibit
binding to sst4. The introduction of Phe11 (21, 23, 25, 29, and
31) results in an aromatic group close to Lys9/IAmp9 as well as
DTrp/DAgl8, which satisfies the sst4 pharmacophore partially
and allows binding (Figure 4D, Table 5).
Following up on the observation by Bass et al.64,65 and Hocart
et al.61 that one could turn an octreotide-based sst2 agonist
scaffold characterized by a DAaa2-LCys3 to an antagonist
scaffold characterized by a LAaa2-DCys3, we tested 31 for
agonism/antagonism. In our functional luciferase reporter gene
assay, 31 along with 23, 25, 27, and 29, were agonists at human
sst1 (Table 1). The best analogue in this series is 25, with an
EC50 value of 0.37 nM in the luciferase reporter gene assay,
with a binding IC50 of 0.19 nM, and 5000-fold selectivity versus

2740 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Erchegyi et al.
Table 5. Distances between Cγ Atoms (in Å) of Selected Residues for the Analogues Studied by NMR and the sst1/sst4 Pharmacophoresa
F7- X8
F7- IAmp9
F7- F11
X8- IAmp9
X8- F11
IAmp9-F11
Sst1pharmacophore
Sst4pharmacophore
16
23
27
6.0-7.5
9.0-11.0
6.0-7.5
7.0-8.0
4.5-6.5
5.3-6.7
6.9-8.1
4.5-7.1
9.5-12.0
5.5-9.5
8.2-8.8
9.6-11.2
9.0-9.8
8.0-10.0
4.5-6.5
8.0-8.6
5.8-6.6
6.4-8.3
5.9-7.0
5.9-7.8
5.2-6.8
9.4-9.8
7.3-10.6
8.5-10.0
5.1-6.8
6.6-11.0
6.4-7.7
a X refers to either DTrp or DAgl(NMe, 2naphthoyl) residues.
performed on adjacent sections of sst1-expressing human prostate
cancer. The relatively weak 125I-25-labeling is difficult to explain
because the binding affinity of the cold iodinated compound
des-AA1,4-6,10,12,13-[DnatITyr2,DAgl(NMe,2naphthoyl)8,IAmp9]-
SRIF-Thr-NH2 (34) is in the low nanomolar range (IC50 = 1.0
nM for 34). sst2-expressing tumors, used as negative controls,
were completely negative for 125I-25 binding (Table 6) despite
an extremely high density of sst2 receptors in these tumors. This
further indicates the high sst1 specificity of the tracer.
The sst1 pharmacophore reported previously by our group
showed that the side chains of four different residues are
important for the binding of these analogues.1 They are the
indole ring of DTrp at position 8, IAmp side chain at position
9, and two aromatic side chains at positions 6 or 7 and 11. The
distances between the side chains of these residues should be
close to the values given in Table 5.1 In addition, the sst1
pharmacophore was different from the other SRIF receptor
pharmacophores63,66,67 in the positioning of the aromatic side
chains. In all of the other SRIF receptor pharmacophores, the
aromatic side chains are present at the front side of the peptide
backbone when the structure of the peptide is oriented from N-
to C-terminus. In contrast, in the sst1 pharmacophore, both of
the aromatic side chains are present at the back side of the
peptide backbone.1 Therefore, any peptide that has the sst1
pharmacophore that involves the four residues at the proposed
distance should bind to sst1.
To verify the identity of the sst1 pharmacophore, the 3D
structures of three analogues, 16, 23, and 27, that bind selectively
to sst1 with nM affinity were studied. All these analogues have
IAmp at position 9, which is crucial for selectivity to sst1. As
shown in Figure 2, the other residues important for sst1 binding,
namely DTrp/DAgl,8 Phe7 and Phe/Tyr11, are present in all of
the three analogues. The distances between the corresponding
Cγ atoms of the side chains of the four residues are given in
Table 5. Comparing the distances with the sst1 pharmacophore
suggests that all of the three analogues have the sst1 pharma-
cophore, except for a small discrepancy, but a detailed com-
parison of the structures show that the positioning of the side
chains of the aromatic groups at position 7 and 11 is not exactly
identical to that described for the sst1 pharmacophore.1 In the
structures of the three analogues studied here, the aromatic side
chains are present in the front side of the peptide backbone
(Figure 2). Because these peptides have high binding affinity
for sst1, the binding of these peptides to sst1 could be due to
the inherent flexibility observed in these peptides. The presence
of DAgl at position 8 introduces a large flexibility to the peptide
backbone and in turn to the side chains of 23 and 27, compared
to 16, which has DTrp at position 8. This is also reflected in
the number of NOEs observed for 23 and 27, which is less
compared to the number of NOEs observed for 16 (Table 3).
In addition, the chemical shift values observed for the aromatic
protons of Phe7 and Phe11 are very similar for 23 and 27 (parts
B and C of Table 2), indicating a random coil or less structured
elements compared to 16 (part A of Table 2). Comparing the
chemical shift dispersion of the aromatic region of similar sst2-
selective octreotide-type SRIF analogues63 with these sst1-
Figure 5. 125I-25 specifically labels the sst1 expressing cell line.
Autoradiograms showing total binding of 125I-25 in sst1 (A), sst2 (C),
sst3 (E), sst4 (G), and sst5 (I) receptor expressing cells. Only sst1 cells
are labeled. Autoradiograms showing nonspecific binding of 125I-25 in
sst1 (B), sst2 (D), sst3 (F), sst4 (H), and sst5 (J) receptor expressing
cells (in the presence of an excess of unlabeled 25).
sst2/5, 500-fold selectivity versus sst3, and 100-fold selectivity
versus sst4, respectively. SRIF and the sst1-agonist 33 exhibit
EC50 values of 0.93 and 33 nM, respectively. Figure 3 shows
that SRIF, 25 and 33 are agonists because they effectively
stimulate luciferase expression in the reporter gene assay. It is
noteworthy to mention that 25 is more potent than SRIF (EC50:
0.37 nM versus EC50: 0.93 nM), whereas the standard sst1
agonist 33 is approximately 100-fold less potent than either of
them. We have used 35,3,4 a short-sized somatostatin analogue
with no affinity to sst1 receptors, as a negative control, in this
test; it was found to be inactive at a concentration of 1 µM.
Analogue 25 was radio-iodinated with 125I and found to bind
to sections of membrane pellet of sst1-expressing transfected
cells with high specificity, as shown in Figure 5. Moreover, we
evaluated the ability of the 125I-25 to bind to sst1-expressing
human cancers. Table 6 shows that this radioligand was able to
label virtually all tested sst1-expressing tumors, including a
selection of well characterized sst1-expressing prostate cancers,
mesenchymal cancers, bronchial carcinoids, and gastroentero-
pancreatic tumors. Figure 6 and Table 6 show that compared
to the strong 125I-[LTT]-SRIF-28 labeling, found to be fully
displaceable by 33 in these tissues and therefore indicative of
sst1 expression, the 125I-25 labeling was noticeably weaker when

Radio-Iodinatable Sst1-SelectiVe Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2741
Table 6. 125I-25 Specifically Labels sst1-Expressing Human Cancersa
125I-[LTT]-SRIF-28 binding displaceable by 33
125I-25 binding displaceable by 33
tumor type
no. of cases
incidence
density
incidence
density
A: sst1-expressing tumors
prostate cancer
mesenchymal tumors
bronchial carcinoids
gastroenteropancreatic tumors
B: sst2-expressing tumors
8
7
6
4
8
8/8
7/7
6/6
+++
++/+++
++
++
-
7/8
6/7
6/6
4/4
0/8
+
++
+ /++
+/++
-
4/4
0/8b
a +++, ++, + means high, moderate, or low receptor density, respectively. b 125I-[LTT]-SRIF-28-binding in these tumors is fully displaceable by octreotide.
Figure 7. Amide and aromatic region of the 1D proton NMR spectra
of analogues des-AA1,4-6,11-13-[DPhe2,DTrp8]-SRIF-Thr-NH2 (octreotide
amide) (11), 16, 23, and 27. The chemical shift dispersion of the
aromatic region of analogues 11 and 16 are comparable, while those
Figure 6. 125I-25 labels an sst1 receptor expressing human prostate
of 23 and 27 suggest that these peptides are less structured in DMSO
cancer. (A) Hematoxylin eosin stained tumor section (bar: 1 mm). (B,C)
compared to analogues 11 and 16.
Autoradiograms showing total binding of 125I-[LTT]-SRIF-28 with
strong tumor labeling (B) that is displaceable by the sst1-selective 33
This was the case because it was based on the structures of
(C), indicating sst1 expression. (D,E,F) Autoradiograms showing total
several high affinity ligands of differing primary sequences.1
The most potent of these sst1-selective analogues (25) in respect
of affinity, selectivity, and functionality could be radio-iodinated.
While this tracer is highly specific for labeling sst1-expressing
tumors with very high binding affinity, the sensitivity of the
assay using this radioligand remains below expectations. This
is somewhat disappointing, but it should be kept in mind that
agonist radioligands are not devoid of caveats. We have shown
previously that although 125I-[LTT]-SRIF-28, [125I]Tyr10-CST14,
or [125I]Tyr3-octreotide are excellent radioligands showing high
affinity and high specific binding in studies using membranes
of recombinantly expressed SRIF receptors in CCL-39 or other
cells,68,69 it is clear that [125I]Tyr10-CST14 is by far inferior as a
radioligand compared to 125I-[LTT]-SRIF-28 or [125I]Tyr3-
octreotide in receptor autoradiography when using slices of brain
or peripheral tissues known to express SRIF receptors,70
illustrating the fact that all agonists are not equal.
binding of 125I-25 with clear tumor labeling (D) that is displaceable by
an excess of unlabeled 25 (E) or 33 (F).
selective analogues suggests that the latter are less structured
in DMSO (Figure 7). On the basis of the conformational
flexibility of 23 and 27 (parts B and C of Table 2, it is suggested
that the aromatic side chains could move to the backside of the
peptide backbone for sst1 binding. The higher binding affinity
of 23 and 27 compared to that of 16 could be due to the larger
flexibility of the peptide backbone of the former, resulting in a
better fit of 23 and 27 into the sst1 binding pocket compared to
16, which shows a single conformation with much less flexibility
for its peptide backbone (parts A-D of Figure 2, Table 5).
Conclusions
To conclude, using a pharmacophore derived from the
structures of undecamers,1 we were able to design somatostatin
heptamers and hexamers with improved or similar affinity and
sst1 selectivity compared to the previously published undecam-
ers. Although the positioning of the aromatic side chains in these
shortened analogues is not exactly identical to that found for
the sst1 pharmacophore proposed earlier, backbone flexibility
is suggested as a mechanism for induced fit necessary for sst1
binding.
Experimental Procedures
Chemistry: Starting Materials. The Boc-Cys(Mob)-CM resin
with a capacity of 0.3-0.5 mequiv/g was obtained according to
published procedures.71 All NR-tert-butoxycarbonyl (BOC) pro-
tected amino acids with side chain protection were purchased from
Bachem Inc. (Torrance, CA), Chem-Impex International (Wood
Dale, IL), Novabiochem (San Diego, CA), or Reanal (Budapest,
Hungary). The side chain protecting groups were as follows:
Cys(Mob), Lys[Z(2Cl)] Ser(Bzl), Thr(Bzl), Tyr[Z(2Br)], and m-I-
Tyr[Bzl(3Br)]. Boc-IAmp(Z),72 Fmoc-D/LAgl(ΝMe,Boc),46 and
These results demonstrate that rational and efficient SAR in
peptides is possible. In this case, it was critical that the structure
of the original pharmacophore be well-defined and accurate.

2742 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Erchegyi et al.
Fmoc-DAgl(Boc)73 were synthesized in our laboratory. All reagents
and solvents were reagent grade or better and used without further
purification.
1). The diastereomers of the Agl containing peptides could be
separated by preparative RP-HPLC.
Determination of the Stereochemistry of Agl in the
Peptide Synthesis. Peptides were synthesized by the solid-phase
approach with Boc chemistry either manually or on a CS-Bio
peptide synthesizer model CS536. Boc-Cys(Mob)-CM resin with
a capacity of 0.3-0.5 mequiv/g or 4-methylbenzhydrylamine
(MBHA) resin with a capacity of 0.4 mequiv/g was used,
respectively. Couplings of the protected amino acids were mediated
by diisopropylcarbodiimide (DIC) and (HOBt) in DMF for 1 h and
monitored by the qualitative ninhydrin test.74 A 3 equiv excess of
the protected amino acids based on the original substitution of the
resin was used in most cases. Boc removal was achieved with
trifluoroacetic acid (60% in CH2Cl2, 1-2% ethanedithiol or
m-cresol) for 20 min. An isopropyl alcohol (1% m-cresol) wash
followed TFA treatment, and then successive washes with triethy-
lamine solution (10% in CH2Cl2), methanol, triethylamine solution,
methanol, and CH2Cl2 completed the neutralization sequence. D/L-
Agl(NMe,2naphthoyl) residue was formed on the resin. In short:
after Fmoc-D/LAgl(ΝMe,Boc)-OH was coupled, the Boc protecting
group was removed with TFA, 3 equiv 2naphthoyl chloride, and 3
equiv DIPEA were used to acylate the free secondary amino group
of the side chain. Removal of the ΝR-Fmoc protecting group with
20% piperidine in DMF in two successive 5 and 15 min treatments
was followed by the standard elongation protocol until the comple-
tion of the peptide.
Peptides. Because the L and D enantiomers of Fmoc-D/
LAgl(ΝMe,Boc)46 used for the synthesis of peptides (21, des-
AA1,4-6,10,12,13-[Cpa2,DCys3,LAgl(NMe,2naphthoyl)8]-SRIF-2Nal-
NH2 (22), 23-25, 27, des-AA1,2,4-6,10,12,13-[LAgl(NMe,2naphthoyl)8,
IAmp9,Tyr11]-SRIF-NH2 (28), 29, des-AA1,2,4-6,10,12,13-[DCys3,LAgl-
(NMe,2naphthoyl)8,IAmp9]-SRIF-2Nal-NH2 (30), 31, and des-
AA1,4-6,10,12,13-[Tyr2,DCys3,LAgl(NMe,2naphthoyl)8,IAmp9]-SRIF-
2Nal-NH2 (32)) were not resolved initially, two diastereomers were
generated, isolated, characterized, and tested. Separation of the L-
from the DAgl-containing peptides was achieved using RP-HPLC.
The absolute configuration of the Agl was deduced from enzymatic
hydrolysis studies with trypsin, aminopeptidase M, and proteinase
K (Roche Diagnostics Corp., Indianapolis, IN), respectively, as we
published earlier.50 All enzymatic hydrolyses were carried out
according to the protocols suggested by the manufacturers.
Trypsin, which is highly specific toward positively charged side
chains with lysine and arginine, was used to determine the
stereochemistry of 21 and 22. Trypsin (Roche, 5 µg) in 0.05% TFA
(20 µL) was added to peptide (0.02 µmol) dissolved in 0.046 M
Tris buffer (200 µL, pH ) 8.1) containing 0.01 M CaCl2. The
hydrolysis was followed by RP-HPLC for 6 days. Trypsin was able
to open the ring structure of 21 and 22, but the rate of reactions
was very much different. We suggest that 22 contains the L-isomer
of the Agl derivative and 21 contains the D-isomer of the Agl
derivative because 55% of 22 versus only 28% of 21 were
hydrolyzed in 6 days.
All of the peptides were cleaved from the resin support with
simultaneous side chain deprotection by anhydrous HF containing
the scavengers anisole (10% v/v) and methyl sulfide (5% v/v) for
60 min at 0 °C. The diethyl ether precipitated crude peptides were
cyclized in 75% acetic acid (200 mL) by addition of iodine (10%
solution in methanol) until the appearance of a stable orange color.
Forty minutes later, ascorbic acid was added to quench the excess
iodine.
Peptides with L-amino acids at their N-terminus and IAmp at
position 9 instead of Lys (23 versus 24 and 27 versus 28) were
digested with aminopeptidase M, which is a metalloprotease, and
can hydrolyze peptides at a free R-amino group of L-amino acids
(except X-Pro bonds and the amino groups of Asp, Gln, or ꢀAla)
to determine the absolute configuration of Agl. The treatment of
24 and 28 with aminopeptidase M at room temperature for 48 h
resulted in many very hydrophilic products followed by RP-HPLC,
indicating that these peptides have been completely hydrolyzed,
providing evidence that these analogues contained the L-enantiomer
of Agl in their sequence. Compounds 23 and 27 were hydrolyzed
only into two products, suggesting that these two peptides contained
the D-enantiomer of Agl in their sequence, resulting in more
resistance to the enzymatic hydrolysis. To confirm this conclusion,
these analogues were also digested with proteinase K, which is a
serine protease that exhibits very broad cleavage specificity. The
predominant site of cleavage is the peptide bond adjacent to the
carboxyl group of hydrophobic aliphatic and aromatic amino acids
with blocked R amino groups. Peptides were dissolved (20 µg) at
a concentration of 0.3 mg/mL in 10 mM Tris buffer pH 7.8.
Proteinase K (5 µL, 6 U) was added and reacted for 16 h at 37 °C.
An aliquot (5 µL) was quenched with 45 µL 0.1 N HCl prior to
analysis by HPLC for determination of degradation products. The
aliquots were screened using microbore RP-HPLC under buffer
system A; 0.1%TFA/H2O, buffer system B, 70% CH3CN in A at a
flow rate of 0.05 mL/min under gradient conditions 10-80% B
over 30 min. Fragments of 23 identified as H-Phe-Agl(NMe,-
2naphthoyl)-IAmp-Phe-Cys-Thr-NH2andH-Phe-Agl(NMe,2naphthoyl)-
IAmp-Phe-OH were confirmed by MALDI-MS. These observations
verified that 23 contained the DAgl. The mass of fragments of 24
were found to be <500 Da, indicating the presence of LAgl.
Analogues 25, 26, 29, 30, and 31 and 32 contain a D-residue at
position 2 (DTyr) or 3 (DCys), respectively, therefore enzymatic
hydrolysis by aminopeptidase M could not be expected for any of
these compounds. Instead, these analogues were digested with
proteinase K as described above.
Purification and Characterization of the Analogues. The
crude, lyophilized peptides were purified by preparative RP-HPLC49
on a 5 cm × 30 cm cartridge, packed in the laboratory with
reversed-phase Vydac C18 silica (15-20 µM particle size, 300 Å)
using a Waters Prep LC 4000 preparative chromatograph system
with a Waters 486 tunable absorbance UV detector and Huston
Instruments Omni Scribe chart recorder. The peptides eluted with
a flow rate of 100 mL/min using a linear gradient of 1% B per 3
min increase from the baseline % B. Eluent A ) 0.25 N TEAP pH
2.25, eluent B ) 60% CH3CN, 40% A. As a final step, all peptides
were rechromatographed in a 0.1% TFA solution and CH3CN on
the same cartridge at 100 mL/min (gradient of 1% CH3CN/min).
The collected fractions were screened by analytical RP-HPLC on
a system using two Waters 501 HPLC pumps, a Schimadzu SPD-
6A UV detector, a Rheodyne model 7125 injector, a Huston
Instruments Omni Scribe chart recorder, and a Vydac C18 column
(0.46 cm × 25 cm, 5 µm particle size, 300 Å pore size). The
fractions containing the product were pooled and subjected to
lyophilization. The purity of the final peptide was determined by
analytical RP-HPLC performed with a linear gradient using 0.1 M
TEAP pH 2.5 as eluent A and 60% CH3CN/40% A as eluent B on
a Hewlett-Packard series II 1090 liquid chromatograph connected
to a Vydac C18 column (0.21 cm × 15 cm, 5 µm particle size, 300
Å pore size). The capillary zone electrophoresis (CZE) analysis of
the peptides was performed on a Beckman P/ACE System 2050;
field strength of 15 kV at 30 °C on an Agilent µSil bare fused-
silica capillary (75 µm id × 40 cm length).51 Mass spectra (MALDI-
TOF-MS) were measured on an ABI-Perseptive DE-STR instru-
ment. The instrument employs a nitrogen laser (337 nm) at a
repetition rate of 20 Hz. The applied accelerating voltage was 20
kV. Spectra were recorded in delayed extraction mode (300 ns
delay). All spectra were recorded in the positive reflector mode.
Spectra were sums of 100 laser shots. Matrix R-cyano-4-hydroxy-
cinnamic acid was prepared as saturated solutions in 0.3% TFA in
50% CH3CN. The observed monoisotopic (M + H)+ values of each
peptide corresponded with the calculated (M + H)+ values (Table
The digestion of 25 with proteinase K for 4 days resulted in
four products, which were identified as H-cyclo[Cys-Phe-
Agl(NMe,2naphthoyl)-IAmp-Phe-Cys]-Thr-NH2, H-Phe-Agl(NMe,-
2naphthoyl)-IAmp-Phe-Cys-Thr-NH2, H-Phe-Agl(NMe,2naphthoyl)-
IAmp-Phe-OH, and the starting material H-DTyr-cyclo[Cys-Phe-
Agl(NMe,2naphthoyl)-IAmp-Phe-Cys]-Thr-NH2, confirmed by MAL-

Radio-Iodinatable Sst1-SelectiVe Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2743
Table 7. Separation of the D- from the L-Agl-Containing Peptides by
Structure Determination. The chemical shift assignment of the
major/minor conformer was obtained by the standard procedure
using DQF-COSY and TOCSY spectra for intraresidual assignment
and the NOESY spectrum was used for the sequential assignment.85
The collection of structural restraints is based on the NOEs and
RP-HPLCa
compd
HPLC-column
gradient
retention time (min)
21
22
23
24
25
26
27
28
29
30
31
32
Vydac C18
Vydac C18
Vydac C18
Vydac C18
Gemini C18
Gemini C18
Vydac C18
Vydac C18
Gemini C18
Gemini C18
Vydac C18
Vydac C18
20-80%B in 40 min
20-80%B in 40 min
30- 60%B in 30 min
30-60%B in 30 min
20-60%B in 40 min
20-60%B in 40 min
30-70%B in 30 min
30-70%B in 30 min
40-70%B in 30 min
40-70%B in 30 min
30-70%B in 40 min
30-70%B in 40 min
32.33
35.26
17.93
23.39
30.56
34.15
11.75
14.91
20.28
25.10
27.68
32.75
3
vicinal JNHR couplings. Dihedral angle constraints were obtained
3
1
from the JNHR couplings, which were measured from the 1D H
NMR spectra and from the intraresidual and sequential NOEs along
with the macro GRIDSEARCH in the program CYANA.53 The
calibration of NOE intensities versus 1H-1H distance restraints and
appropriate pseudoatom corrections to the nonstereo specifically
assigned methylene, methyl, and ring protons were performed using
the program CYANA. On an average, approximately 70 NOE
constraints and 15 angle constraints were utilized while calculating
the conformers (Table 3). A total of 100 conformers were initially
generated by CYANA, and a bundle containing 20 CYANA
conformers with the lowest target function values were utilized for
further restrained energy minimization using the program DIS-
COVER with steepest decent algorithm.54 The resulting energy
minimized bundle of 20 conformers was used as a basis for
discussing the solution conformation of the different SRIF ana-
logues. The structures were analyzed using the program MOL-
MOL.86
a HPLC buffer system: A ) TEAP (pH 2.5) and B ) 60% CH3CN/40%
A with a gradient slope of 1% B/min at flow rate of 0.2 mL/min.
DI-MS. After the digestion of 26 with proteinase K for 4 days, no
starting material was detected by HPLC, one of the fragments was
identified as H-DTyr-Cys-Phe-Agl(NMe,2naphthoyl)-IAmp-Phe-
Cys-OH and all of the other detected fragments had a mass of less
than 500, suggesting that this peptide contains the L-isomer of Agl.
The digestion of 29 with proteinase K for 7 days resulted in
three products, which were identified as H-cyclo[DCys-Phe-
Biology: Reagents. All reagents were of the best grade available
and were purchased from common suppliers. Lactalbumin hydroly-
sate was from HyClone, ATP from Sigma-Aldrich, D-luciferin from
Roche Diagnostics, and coenzyme A from Calbiochem.
Cell Lines. Chinese hamster lung fibroblasts (CCL39) stably
expressing the human sst1 and the luciferase reporter gene under
the control of the serum response element (CCL39-sst1-Luci) were
from D. Hoyer (Novartis, Basel, Switzerland) and grown in DMEM
with GlutaMAX-I/Ham’s F-12 Nut. Mix. with GlutaMAX-I (1:1)
supplemented with 10% fetal bovine serum, 100U/mL penicillin,
100 µg/mL streptomycin, and 400 µg/mL geneticin (G418-sulfate)
at 37 °C and 5% CO2. All culture reagents were from Gibco BRL,
Life Technologies.
Receptor Autoradiography. Cell membrane pellets were pre-
pared as previously described and stored at -80 °C.87 Receptor
autoradiography was performed on 20 µm thick cryostat (Microm
HM 500, Walldorf, Germany) sections of the membrane pellets,
mounted on microscope slides, and then stored at -20 °C. For each
of the tested compounds, complete competition experiments with
the universal SRIF radioligand [Leu8, D-Trp22, 125I-Tyr25]-SRIF-28
Agl(NMe,2-naphthoyl)-IAmp-Phe-Cys]-OH,
H-DCys-Phe-
Agl(NMe,2naphthoyl)-IAmp-Phe-OH, and the starting material
H-cyclo[DCys-Phe-Agl(NMe,2naphthoyl)-IAmp-Phe-Cys]-Nal-
NH2, confirmed by MALDI-MS. After the digestion of 30 with
proteinase K for 47 h, a small amount of the starting material was
detected by HPLC and the fragments were identified as H-Agl(NMe,-
2naphthoyl)-IAmp-Phe-OH and H-IAmp-Phe-Cys-Nal-NH2 con-
firmed by MALDI-MS, suggesting that this peptide contains the
L-isomer of Agl.
The digestion of 31 with proteinase K for 47 h resulted in three
fragments, which were identified as H-cyclo[DCys-Phe-
Agl(NMe,2naphthoyl)-IAmp-Phe-Cys]-Nal-NH2, H-DCys-(Cys-
Nal-NH2)-Phe-Agl(NMe,2naphthoyl)-IAmp-Phe-OH, and H-DCys-
Phe-Agl(NMe,2naphthoyl)-IAmp-Phe-OH and the starting material
confirmed by MALDI-MS. After the digestion of 32 with proteinase
K for 47 h, a small amount of the starting material was detected
by HPLC and no other fragments could be identified, suggesting
that this peptide contains the L-isomer of Agl.
Overall, aminoglycine-containing enantiomers were relatively
easy to separate using HPLC under gradient conditions (Table 7).
(
125I-[LTT]-SRIF-28) (2000 Ci/mmol; ANAWA, Wangen, Swit-
1
zerland) using 15000 cpm/100 µL and increasing concentrations
of the unlabeled peptide ranging from 0.1 to 1000 nM were
performed. As control, unlabeled SRIF-28 was run in parallel using
the same increasing concentrations. The sections were incubated
with 125I-[LTT]-SRIF-28 for 2 h at room temperature in 170 mmol/L
Tris-HCl buffer (pH 8.2) containing 1% BSA, 40 mg/L bacitracin,
and 10 mmol/L MgCl2 to inhibit endogenous proteases. The
incubated sections were washed twice for 5 min in cold 170 mmol/L
Tris-HCl (pH 8.2) containing 0.25% BSA. After a brief dip in 170
mmol/L Tris-HCl (pH 8.2), the sections were dried quickly and
exposed for 1 week to Kodak BioMax MR film. IC50 values were
calculated after quantification of the data using a computer-assisted
image processing system as described previously.88 Tissue standards
(Autoradiographic [125I] and/or [14C] microscales, GE Healthcare,
Little Chalfont, UK) that contain known amounts of isotope, cross-
calibrated to tissue-equivalent ligand concentrations, were used for
quantification.88–91
NMR Experiments. The H NMR spectra were recorded on a
Bruker 700 MHz spectrometer operating at proton frequency of
700 MHz. Chemical shifts were measured using DMSO (δ ) 2.49
ppm) as an internal standard. The 2D spectra were acquired at 298
K. Resonance assignments of the various proton resonances have
been carried out using total correlation spectroscopy (TOCSY),75,76
double-quantum filtered spectroscopy (DQF-COSY),77 and nuclear
Overhauser enhancement spectroscopy (NOESY).78–80 The TOCSY
experiments employed the MLEV-17 spin-locking sequence sug-
gested by Davis and Bax,75 applied for a mixing time of 50 or 70
ms. The NOESY experiments were carried out with a mixing time
of 100 or 150 ms. The TOCSY and NOESY spectra were acquired
using 800 complex data points in the ω1 dimension and 1024
complex data points in the ω2 dimension and were subsequently
zero-filled to 1024 × 2048 before Fourier transformation. The DQF-
COSY spectra were acquired with 1024 × 4096 data points and
were zero-filled to 2048 × 4096 before Fourier transformation. The
TOCSY, DQF-COSY, and NOESY spectra were acquired with 16,
16, and 24 scans, respectively, with a relaxation delay of 1 s. The
signal from the residual water of the solvent was suppressed using
presaturation during the relaxation delay and during the mixing time.
The TOCSY and NOESY data were multiplied by a 75° shifted
sine-function in both dimensions. To differentiate exchange peaks
from the NOEs, rotating frame NOESY (ROESY)81,82 spectra were
measured at 150, 250, and 400 ms. All spectra were processed using
the software PROSA.83 The spectra were analyzed using the
software X-EASY.84
The analogue 25 was 125I-monoradiolabeled (2000 Ci/mmol,
ANAWA, Switzerland) and used as radioligand in binding studies
on cell membrane pellet sections of cells stably expressing sst1
receptors and on tissue sections, following the same protocol as
described above for 125I-[LTT]-SRIF-28.
Luciferase Assay. The luciferase reporter gene assay was
performed as described previously by Hoyer and colleagues32,58
with minor modifications. CCL39-sst1-Luci cells were seeded at
25000 cells per well in 96-well plates. After 24 h, the cells were
washed once with PBS and then serum-deprived for 24 h in assay

2744 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Erchegyi et al.
receptor targeting of tumors. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
16436–16441.
(14) Olias, G.; Viollet, C.; Kusserow, H.; Epelbaum, J.; Meyerhof, W.
Regulation and function of somatostatin receptors. J. Neurochem. 2004,
89, 1057–1091.
medium (DMEM with GlutaMax-I/Ham’s F12 Nut. Mix. with
GlutaMax-I (1:1) containing 5 g/L lactalbumin hydrolysate and 20
mM HEPES) at 37 °C and 5% CO2. The cells were then treated in
triplicate at 37 °C and 5% CO2 with assay medium alone (basal)
or with assay medium containing the compounds to be tested at
concentrations between 0.1 nM and 1 µM for 5 h. The cells were
then washed with PBS and lysed in lysis buffer (100 mM KPi-
buffer containing 0.2% Triton X-100 and 1 mM DL-dithiothreitol).
Luciferase activity was measured using a luminometer by injecting
first the ATP reagent (10 mM ATP, 35 mM glycyl-glycine, and 20
mM MgCl2, pH 7.8) and then the luciferin reagent (0.47 mM
D-luciferin, 0.27 mM coenzyme A, 35 mM glycyl-glycine, and 20
mM MgCl2, pH 7.8). Measuring parameters: delay time, 5 s;
measurement time, 10 s. The 1 µM SRIF-14 effect minus the basal
effect was set as 100%. Stimulation of luciferase reporter gene
activity by the different compounds is expressed as % stimulation
of the 1 µM SRIF-14 effect. As positive control for the luciferase
reporter gene activity, 10% fetal bovine serum in assay medium
was used.
(15) Weckbecker, G.; Lewis, I.; Albert, R.; Schmid, H. A.; Hoyer, D.;
Bruns, C. Opportunities in somatostatin research: biological, chemical,
and therapeutic aspects. Nat. ReV. Drug DiscoVery 2003, 2, 999–1017.
(16) Piwko, C.; Thoss, V. S.; Schupbach, E.; Kummer, J.; Langenegger,
D.; Probst, A.; Hoyer, D. Pharmacological characterisation of human
cerebral cortex somatostatin SRIF1 and SRIF2 receptors. Naunyn-
Schmiedeberg’s Arch. Pharmacol. 1997, 355, 161–167.
(17) Thermos, K. Functional mapping of somatostatin receptors in the retina:
a review. Vision Res. 2003, 43, 1805–1815.
(18) Bocci, G.; Culler, M. D.; Fioravanti, A.; Orlandi, P.; Fasciani, A.;
Colucci, R.; Taylor, J. E.; Sadat, D.; Danesi, R.; Del Tacca, M. In
vitro antiangiogenic activity of selective somatostatin subtype-1
receptor agonists. Eur. J. Clin. InVest. 2007, 37, 700–708.
(19) Reubi, J. C.; Schaer, J. C.; Waser, B.; Hoeger, C.; Rivier, J. A selective
analog for the somatostatin sst1-receptor subtype expressed by human
tumors. Eur. J. Pharmacol. 1998, 345, 103–110.
(20) Reubi, J. C.; Waser, B.; Laissue, J. A.; Gebbers, J. O. Somatostatin
and vasoactive intestinal peptide receptors in human mesenchymal
tumors: in vitro identification. Cancer Res. 1996, 56, 1922–1931.
(21) Reubi, J. C.; Waser, B.; Schaer, J.-C.; Laissue, J. A. Somatostatin
receptor sst1-sst5 expression in normal and neoplastic human tissues
using receptor autoradiography with subtype-selective ligands. Eur.
J. Nucl. Med. 2001, 28, 836–846.
(22) Reubi, J. C.; Schaer, J. C.; Waser, B.; Mengod, G. Expression and
localization of somatostatin receptor SSTR1, SSTR2, and SSTR3
mRNAs in primary human tumors using in situ hybridization. Cancer
Res. 1994, 54, 3455–3459.
(23) Reubi, J. C.; Waser, B.; Schaer, J. C.; Markwalder, R. Somatostatin
receptors in human prostate and prostate cancer. J. Clin. Endocrinol.
Metab. 1995, 80, 2806–2814.
(24) Miller, G. M.; Alexander, J. M.; Bikkal, H. A.; Katznelson, L.; Zervas,
N. T.; Klibanski, A. Somatostatin receptor subtype gene expression
in pituitary adenomas. J. Clin. Endocrinol. Metab. 1995, 80, 1386–
1392.
(25) Lanneau, C.; Bluet-Pajot, M. T.; Zizzari, P.; Csaba, Z.; Dournaud, P.;
Helboe, L.; Hoyer, D.; Pellegrini, E.; Tannenbaum, G. S.; Epelbaum,
J.; Gardette, R. Involvement of the sst1 somatostatin receptor subtype
in the intrahypothalamic neuronal network regulating growth hormone
secretion: an in vitro and in vivo antisense study. Endocrinology 2000,
141, 967–979.
(26) Kreienkamp, H.-J.; Akgu¨n, E.; Baumeister, H.; Meyerhof, W.; Richter,
D. Somatostatin receptor subtype 1 modulates basal inhibition of
growth hormone release in somatotrophs. FEBS Lett. 1999, 462, 464–
466.
(27) Zatelli, M. C.; Piccin, D.; Tagliati, F.; Ambrosio, M. R.; Margutti,
A.; Padovani, R.; Scanarini, M.; Culler, M. D.; degli Uberti, E. C.
Somatostatin receptor subtype 1 selective activation in human growth
hormone (GH)- and prolactin (PRL)-secreting pituitary adenomas:
effects on cell viability, GH, and PRL secretion. J. Clin. Endocrinol.
Metab. 2003, 88, 2797–2802.
(28) Vasilaki, A.; Papasava, D.; Hoyer, D.; Thermos, K. The somatostatin
receptor (sst(1)) modulates the release of somatostatin in the nucleus
accumbens of the rat. Neuropharmacology 2004, 47, 612–618.
(29) Dal Monte, M. D.; Petrucci, C.; Vasilaki, A.; Cervia, D.; Grouselle,
D.; Epelbaum, J.; Kreienkamp, H. J.; Richter, D.; Hoyer, D.; Bagnoli,
P. Genetic deletion of somatostatin receptor 1 alters somatostatinergic
transmission in the mouse retina. Neuropharmacology 2003, 45, 1080–
1092.
(30) Thermos, K.; Bagnoli, P.; Epelbaum, J.; Hoyer, D. The somatostatin
sst1 receptor: an autoreceptor for somatostatin in brain and retina.
Pharmacol. Ther. 2006, 110, 455–464.
(31) Hoyer, D.; Dixon, K.; Gentsch, C.; Vassout, A.; Enz, A.; Jaton, A.;
Nunn, C.; Schoeffter, P.; Neumann, P.; Troxler, T.; Pfaeffli, P. NVP-
SRA880, a somatostatin sst1 receptor antagonist promotes social
interactions, reduces aggressive behaviour and stimulates learning.
Pharmacologist 2002, 44, A254.
(32) Hoyer, D.; Nunn, C.; Hannon, J.; Schoeffter, P.; Feuerbach, D.;
Schuepbach, E.; Langenegger, D.; Bouhelal, R.; Hurth, K.; Neumann,
P.; Troxler, T.; Pfaeffli, P. SRA880, in vitro characterization of the
first non-peptide somatostatin sst(1) receptor antagonist. Neurosci. Lett.
2004, 361, 132–5.
(33) Matrone, C.; Pivonello, R.; Colao, A.; Cappabianca, P.; Cavallo, L. M.;
Del Basso De Caro, M. L.; Taylor, J. E.; Culler, M. D.; Lombardi,
G.; Di Renzo, G. F.; Annunziato, L. Expression and function of
somatostatin receptor subtype 1 in human growth hormone secreting
pituitary tumors deriving from patients partially responsive or resistant
to long-term treatment with somatostatin analogs. Neuroendocrinology
2004, 79, 142–148.
Acknowledgment. The project described was supported in
part by award no. 5R01DK59953-05 from the National Institute
of Diabetes and Digestive and Kidney Diseases. The content is
solely the responsibility of the authors and does not necessarily
represent the official views of the National Institute of Diabetes
and Digestive and Kidney Diseases or the National Institutes
of Health. We are indebted to R. Kaiser and C. Miller for
technical assistance in the synthesis and characterization of the
peptides and Dr. W. Fisher, and William Low for mass
spectrometric analyses. J.R. is the Dr. Frederik Paulsen Chair
in Neurosciences Professor.
References
(1) Grace, C. R. R.; Durrer, L.; Koerber, S. C.; Erchegyi, J.; Reubi, J. C.;
Rivier, J. E.; Riek, R. Somatostatin receptor 1 selective analogues: 4.
Three-dimensional consensus structure by NMR. J. Med. Chem. 2005,
48, 523–533.
(2) Brazeau, P.; Vale, W. W.; Burgus, R.; Ling, N.; Butcher, M.; Rivier,
J. E.; Guillemin, R. Hypothalamic polypeptide that inhibits the
secretion of immunoreactive pituitary growth hormone. Science 1973,
179, 77–79.
(3) Guillemin, R.; Gerich, J. E. Somatostatin: physiological and clinical
significance. Ann. ReV. Med. 1976, 27, 379–388.
(4) Tannenbaum, G. S.; Epelbaum, J. Somatostatin. In Handbook of
PhysiologysThe Endocrine System; Kostyo, J. L., Goodman, H. M. ,
Eds.; Oxford University Press: New York, 1999; Vol. V: Hormonal
Control of Growth, pp 221-265.
(5) Janecka, A.; Zubrzycka, M.; Janecki, T. Review: Somatostatin Analogs.
J. Pept. Res. 2001, 58, 91–107.
(6) Schally, A. V.; Comaru-Schally, A. M.; Nagy, A.; Kovacs, M.;
Szepeshazi, K.; Plonowski, A.; Varga, J. L.; Halmos, G. Hypothalamic
hormones and cancer. Front. Neuroendocrinol. 2001, 22, 248–291.
(7) Hannon, J. P.; Nunn, C.; Stolz, B.; Bruns, C.; Weckbecker, G.; Lewis,
I.; Troxler, T.; Hurth, K.; Hoyer, D. Drug design at peptide receptors:
somatostatin receptor ligands. J. Mol. Neurosci. 2002, 18, 15–27.
(8) Hoyer, D.; Epelbaum, J.; Feniuk, W.; Humphrey, P. P. A.; Meyerhof,
W.; O’Caroll, A. M.; Patl, Y.; Reisine, T.; Reubi, J. C.; Schindler,
M.; Schonbrunn, A.; Taylor, J. E.; Vezzani, A. Somatostatin receptors.
In The IUPHAR Compendium of Receptor Characterization and
Classification, 2nd ed.; Girdlestrom, D. , Ed.; IUPHAR Media:
London, 2000; pp354-364.
(9) Csaba, Z.; Dournaud, P. Cellular biology of somatostatin receptors.
Neuropeptides 2001, 35, 1–23.
(10) Patel, Y. C. Somatostatin and its receptor family. Front. Neuroendo-
crinol. 1999, 20, 157–198.
(11) Siehler, S.; Hoyer, D. Characterisation of human recombinant soma-
tostatin receptors. 4. Modulation of phospholipase C activity. Naunyn-
Schmiedeberg’s Arch. Pharmacol. 1999, 360, 522–532.
(12) Cescato, R.; Schulz, S.; Waser, B.; Eltschinger, V.; Rivier, J. E.;
Wester, H.-J.; Culler, M.; Ginj, M.; Liu, Q.; Schonbrunn, A.; Reubi,
J. C. Internalization of sst2, sst3, and sst5 receptors: effects of
somatostatin agonist and antagonists. J. Nucl. Med. 2006, 47, 502–
511.
(13) Ginj, M.; Zhang, H.; Waser, B.; Cescato, R.; Wild, D.; Erchegyi, J.;
Rivier, J.; Ma¨cke, H. R.; Reubi, J. C. Radiolabeled somatostatin
receptor antagonists are preferable to agonists for in vivo peptide

Radio-Iodinatable Sst1-SelectiVe Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2745
(34) Zatelli, M. C.; Tagliati, F.; Piccin, D.; Taylor, J. E.; Culler, M. D.;
Bondanelli, M.; degli Uberti, E. C. Somatostatin receptor subtype
1-selective activation reduces cell growth and calcitonin secretion in
a human medullary thyroid carcinoma cell line. Biochem. Biophys.
Res. Commun. 2002, 297, 828–834.
(35) Zatelli, M. C.; Piccin, D.; Tagliati, F.; Bottoni, A.; Luchin, A.; Vignali,
C.; Margutti, A.; Bondanelli, M.; Pansini, G. C.; Rosa Pelizzo, M.;
Culler, M. D.; Degli Uberti, E. C. Selective activation of somatostatin
receptor subtypes differentially modulates secretion and viability in
human medullary thyroid carcinoma primary cultures: potential clinical
perspectives. J. Clin. Endocrinol. Metab. 2006, 91, 2218-2224.
(36) Liapakis, G.; Hoeger, C.; Rivier, J.; Reisine, T. Development of a
selective agonist at the somatostatin receptor subtype SSTR1. J. Phar-
macol. Exp. Ther. 1996, 276, 1089–1094.
(55) Cescato, R.; Erchegyi, J.; Waser, B.; Piccand, V.; Maecke, H. R.;
Rivier, J. E.; Reubi, J. C. Design and in vitro characterization of highly
sst2-selective somatostatin antagonists suitable for radiotargeting.
J. Med. Chem. 2008, 51, 4030–4037.
(56) Hill, C. S.; Treisman, R. Differential activation of c-fos promoter
elements by serum, lysophosphatidic acid, G proteins and polypeptide
growth factors. EMBO J. 1995, 14, 5037–5047.
(57) Treisman, R. Journey to the surface of the cell: Fos regulation and
the SRE. EMBO J. 1995, 14, 4905–4913.
(58) Nunn, C.; Cervia, D.; Langenegger, D.; Tenaillon, L.; Bouhelal, R.;
Hoyer, D. Comparison of functional profiles at human recombinant
somatostatin sst2 receptor: simultaneous determination of intracellular
Ca2+ and luciferase expression in CHO-K1 cells. Br. J. Pharmacol.
2004, 142, 150–160.
(59) Reubi, J. C.; Schaer, J.-C.; Waser, B.; Hoeger, C.; Rivier, J. A selective
analog for the somatostatin sst1-receptor subtype expressed by human
tumors. Eur. J. Pharmacol. 1998, 345, 103–110.
(60) Hocart, S. J.; Jain, R.; Murphy, W. A.; Taylor, J. E.; Coy, D. H. Highly
potent cyclic disulfide antagonists of somatostatin. J. Med. Chem. 1999,
42, 1863–1871.
(61) Hocart, S. J.; Jain, R.; Murphy, W. A.; Taylor, J. E.; Morgan, B.;
Coy, D. H. Potent antagonists of somatostatin: Synthesis and biology.
J. Med. Chem. 1998, 41, 1146–1154.
(37) Rivier, J. E.; Hoeger, C.; Erchegyi, J.; Gulyas, J.; DeBoard, R.; Craig,
A. G.; Koerber, S. C.; Wenger, S.; Waser, B.; Schaer, J.-C.; Reubi,
J. C. Potent somatostatin undecapeptide agonists selective for soma-
tostatin receptor 1 (sst1). J. Med. Chem. 2001, 44, 2238–2246.
(38) Rivier, J. E.; Kirby, D. A.; Erchegyi, J.; Waser, B.; Eltschinger, V.;
Cescato, R.; Reubi, J. C. Somatostatin receptor 1 selective analogues:
3. Dicyclic peptides. J. Med. Chem. 2005, 48, 515–522.
(39) Erchegyi, J.; Hoeger, C. A.; Low, W.; Hoyer, D.; Waser, B.;
Eltschinger, V.; Schaer, J.-C.; Cescato, R.; Reubi, J. C.; Rivier, J. E.
Somatostatin receptor 1 selective analogues: 2. N-Methylated scan.
J. Med. Chem. 2005, 48, 507–514.
(40) Erchegyi, J.; Hoeger, C.; Wenger, S.; Waser, B.; Schaer, J.-C.; Reubi,
J. C.; Rivier, J. E. N-Methyl scan of a sst1-selective somatostatin
(SRIF) analog. In PeptidessThe WaVe of the Future: 2nd International
Peptide Symposium/17th American Peptide Symposium, San Diego,
CA, June 10-14, 2001; Lebl, M. , Houghten, R. A. , Eds.; Springer:
Norwell, MA, 2001; pp719-720.
(41) Jiang, G.; Miller, C.; Koerber, S. C.; Porter, J.; Craig, A. G.;
Bhattacharjee, S.; Kraft, P.; Burris, T. P.; Campen, C. A.; Rivier, C. L.;
Rivier, J. E. Betidamino acid scan of the GnRH antagonist acyline.
J. Med. Chem. 1997, 40, 3739–3748.
(42) Cervini, L.; Theobald, P.; Corrigan, A.; Craig, A. G.; Rivier, C.; Vale,
W.; Rivier, J. Corticotropin releasing factor (CRF) agonists with
reduced amide bonds and Ser7 substitutions. J. Med. Chem. 1999,
42, 761–768.
(43) Reubi, J. C.; Schaer, J.-C.; Wenger, S.; Hoeger, C.; Erchegyi, J.; Waser,
B.; Rivier, J. SST3-selective potent peptidic somatostatin receptor
antagonists. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13973–13978.
(44) Erchegyi, J.; Wenger, S.; Waser, B.; Eltschinger, V.; Cescato, R.;
Reubi, J. C.; Koerber, S. C.; Grace, R. C. R.; Riek, R.; Rivier, J. E.
Use of betidamino acids in drug design. In Understanding Biology
Using Peptides, The 19th American Peptide Symposium, San Diego,
CA, June 18-23, 2005; Blondelle, S. E. , Ed.; Springer: Norwell, MA,
2005; pp517-518.
(62) Melacini, G.; Zhu, Q.; Osapay, G.; Goodman, M. A refined model
for the somatostatin pharmacophore: conformational analysis of
lanthionine-sandostatin analogs. J. Med. Chem. 1997, 40, 2252–2258.
(63) Grace, C. R. R.; Erchegyi, J.; Koerber, S. C.; Reubi, J. C.; Rivier, J.;
Riek, R. Novel sst2-selective somatostatin agonists. Three-dimensional
consensus structure by NMR. J. Med. Chem. 2006, 49, 4487–4496.
(64) Bass, R. T.; Buckwalter, B. L.; Patel, B. P.; Pausch, M. H.; Price,
L. A.; Strnad, J.; Hadcock, J. R. Identification and characterization of
novel somatostatin antagonists. Mol. Pharmacol. 1996, 50, 709–715.
(65) Bass, R. T.; Buckwalter, B. L.; Patel, B. P.; Pausch, M. H.; Price,
L. A.; Strnad, J.; Hadcock, J. R. Identification and characterization of
novel somatostatin antagonists. Mol. Pharmacol. 1997, 51, 170
Erratum.
(66) Grace, C. R. R.; Erchegyi, J.; Koerber, S. C.; Reubi, J. C.; Rivier, J.;
Riek, R. Novel sst4-selective somatostatin (SRIF) agonists. Part IV:
Three-dimensional consensus structure by NMR. J. Med. Chem. 2003,
46, 5606–5618.
(67) Grace, C. R. R.; Erchegyi, J.; Reubi, J. C.; Rivier, J. E.; Riek, R. ;
Three-dimensional consensus of structure of sst2-selective somatostatin
(SRIF) antagonists by NMR. Biopolymers 2008, 89 1077-1087.
(68) Siehler, S.; Seuwen, K.; Hoyer, D. [125I]Tyr10-cortistatin14 labels all
five somatostatin receptors. Naunyn-Schmiedeberg’s Arch. Pharmacol.
1998, 357, 483–489.
(69) Siehler, S.; Seuwen, K.; Hoyer, D. Characterisation of human
recombinant somatostatin receptors. 1. Radioligand binding studies.
Naunyn-Schmiedeberg’s Arch. Pharmacol. 1999, 360, 488–499.
(70) Fehlmann, D.; Langenegger, D.; Schuepbach, E.; Siehler, S.; Feuer-
bach, D.; Hoyer, D. Distribution and characterisation of somatostatin
receptor mRNA and binding sites in the brain and periphery. J. Physiol.
(Paris) 2000, 94, 265–281.
(71) Horiki, K.; Igano, K.; Inouye, K. Amino acids and peptides. Part 6.
Synthesis of the Merrifield resin esters of N-protected amino acids
with the aid of hydrogen bonding. Chem. Lett. 1978, 2, 165–168.
(72) Rivier, J. E.; Jiang, G.; Porter, J.; Hoeger, C.; Craig, A. G.; Corrigan,
A. Z.; Vale, W. W.; Rivier, C. L. GnRH antagonists: novel members
of the azaline B family. J. Med. Chem. 1995, 38, 2649–2662.
(73) Sypniewski, M.; Penke, B.; Simon, L.; Rivier, J. (R)-tert-Butoxycar-
bonylamino-fluorenylmethoxycarbonyl-glycine from (S)-Benzyloxy-
carbonyl-serine or from papain resolution of the corresponding amide
or methyl ester. J. Org. Chem. 2000, 65, 6595–6600.
(45) Qasmi, D.; Rene´, L.; Badet, B. An R-aminoglycine derivative suitable
for solid phase peptide synthesis using Fmoc strategy. Tetrahedron
Lett. 1993, 34, 3861–3862.
(46) Jiang, G.-C.; Simon, L.; Rivier, J. E. Orthogonally protected N-methyl-
substituted R-aminoglycines. Protein Pept. Lett. 1996, 3, 219–224.
(47) Rivier, J. E.; Jiang, G.-C.; Koerber, S. C.; Porter, J.; Craig, A. G.;
Hoeger, C. Betidamino acids: versatile and constrained scaffolds for
drug discovery. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2031–2036.
(48) Hoeger, C. A.; Galyean, R. F.; Boublik, J.; McClintock, R. A.; Rivier,
J. E. Preparative reversed phase high performance liquid chromatog-
raphy. II. Effects of buffer pH on the purification of synthetic peptides.
Biochromatography 1987, 2, 134–142.
(49) Miller, C.; Rivier, J. Peptide chemistry: Development of high-
performance liquid chromatography and capillary zone electrophoresis.
Biopolymers Pept. Sci. 1996, 40, 265–317.
(50) Rivier, J.; Erchegyi, J.; Hoeger, C.; Miller, C.; Low, W.; Wenger, S.;
Waser, B.; Schaer, J.-C.; Reubi, J. C. Novel sst4-selective somatostatin
(SRIF) agonists. Part I: Lead identification using a betide scan. J. Med.
Chem. 2003, 46, 5579–5586.
(51) Miller, C.; Rivier, J. Analysis of synthetic peptides by capillary zone
electrophoresis in organic/aqueous buffers. J. Pept. Res. 1998, 51, 444–
451.
(52) Gairi, M.; Saiz, P.; Madurga, S.; Roig, X.; Erchegyi, J.; Koerber, S. C.;
Reubi, J. C.; Rivier, J. E.; Giralt, E. Conformational analysis of a
potent SSTR3-selective somatostatin analogue by NMR in water
solution. J. Peptide Sci. 2006, 12, 82–91.
(53) Gu¨ntert, P.; Mumenthaler, C.; Wu¨thrich, K. Torsion angle dynamics
for NMR structure calculation with the new program DYANA. J. Mol.
Biol. 1997, 273, 283–298.
(54) Hagler, A. T. , Theoretical simulation of conformation, energetics and
dynamics of peptides. In The Peptides: Analysis, Synthesis, Biology;
Udenfriend, S., Meienhofer, J., Hruby, V. J. , Eds.; Academic Press:
Orlando, FL, 1985; Vol. 7, pp213-299.
(74) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Color test
for detection of free terminal amino groups in the solid-phase synthesis
of peptides. Anal. Biochem. 1970, 34, 595–598.
(75) Davis, D. G.; Bax, A. Assignment of complex 1H NMR spectra via
two-dimensional homonuclear Hartmann-Hahn spectroscopy. J. Am.
Chem. Soc. 1985, 107, 2820–2821.
(76) Braunschweiler, L.; Ernst, R. R. Coherence transfer by isotropic
mixing: application to proton correlation spectroscopy. J. Magn. Reson.
1983, 53, 521–528.
(77) Rance, M.; Sorensen, O. W.; Bodenhausen, B.; Wagner, G.; Ernst,
R. R.; Wu¨thrich, K. Improved spectral resolution in COSY 1H NMR
spectra of proteins via double quantum filtering. Biochem. Biophys.
Res. Commun. 1983, 117, 479–485.
(78) Kumar, A.; Wagner, G.; Ernst, R. R.; Wu¨thrich, K. Buildup rates of
the nuclear Overhauser effect measured by two-dimensional proton
magnetic resonance spectroscopy: implications for studies of protein
conformation. J. Am. Chem. Soc. 1981, 103, 3654–3658.
(79) Macura, S.; Ernst, R. R. Elucidation of cross-relaxation in liquids by
two-dimensional NMR spectroscopy. Mol. Phys. 1980, 41, 95–117.

2746 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Erchegyi et al.
(80) Macura, S.; Huang, Y.; Suter, D.; Ernst, R. R. Two-dimensional
chemical exchange and cross-relaxation spectroscopy of coupled
nuclear spins. J. Magn. Reson. 1981, 43, 259–281.
(81) Bax, A.; Davis, D. G. MLEV-17-based two-dimensional homonuclear
magnetization transfer spectroscopy. J. Magn. Reson. 1985, 65, 355–
360.
(82) Bothner-By, A. A.; Stephens, R. L.; Lee, J.; Warren, C. D.; Jeanloz,
R. W. Structure determination of a tetrasaccharide: transient nuclear
Overhauser effects in the rotating frame. J. Am. Chem. Soc. 1984,
106, 811–813.
(83) Gu¨ntert, P.; Dotsch, V.; Wider, G.; Wu¨thrich, K. Processing of multi-
dimensional NMR data with the new software PROSA. J. Biolmol.
NMR 1992, 2, 619–629.
(87) Reubi, J. C.; Schaer, J. C.; Waser, B.; Wenger, S.; Heppeler, A.;
Schmitt, J. S.; Ma¨cke, H. R. Affinity profiles for human somatostatin
receptor sst1-sst5 of somatostatin radiotracers selected for scinti-
graphic and radiotherapeutic use. Eur. J. Nucl. Med. 2000, 27, 273–
282.
(88) Reubi, J. C.; Kvols, L. K.; Waser, B.; Nagorney, D. M.; Heitz, P. U.;
Charboneau, J. W.; Reading, C. C.; Moertel, C. Detection of
somatostatin receptors in surgical and percutaneous needle biopsy
samples of carcinoids and islet cell carcinomas. Cancer Res. 1990,
50, 5969–5977.
(89) Reubi, J. C. In vitro identification of vasoactive intestinal peptide
receptors in human tumors: implications for tumor imaging. J. Nucl.
Med. 1995, 36, 1846–1853.
(90) Baskin, D. G.; Wimpy, T. H. Calibration of [14C]plastic standards for
quantitative autoradiography of [125I]labeled ligands with Amersham
Hyperfilm beta-max. Neurosci. Lett. 1989, 104, 171–177.
(91) Miller, J. A.; Zahniser, N. R. The use of 14C-labeled tissue paste
standards for the calibration of 125I-labeled ligands in quantitative
autoradiography. Neurosci. Lett. 1987, 81, 345–350.
(84) Eccles, C.; Gu¨ntert, P.; Billeter, M.; Wu¨thrich, K. Efficient analysis
of protein 2D NMR spectra using the software package EASY.
J. Biomol. NMR 1991, 1, 111–130.
(85) Wu¨thrich; K. In NMR of Proteins and Nucleic Acids; J. Wiley & Sons:
New York, 1986.
(86) Koradi, R.; Billeter, M. MOLMOL: a program for display and analysis
of macromolecular structures. PDB Newsletter 1998, 84, 5–7.
JM801314F