Novel sst4-Selective Somatostatin (SRIF) Agonists. 3. Analogues Amenable to Radiolabeling

Article abstract of DOI:10.1021/jm030245x

After our discovery that H-c[Cys-Phe-Phe-DNal-Lys-Thr-Phe-Cys]-OH (ODN-8) had high affinity and marginal selectivity for human sst₃ (part 2 of this series: Erchegyi et al. J. Med. Chem., preceding paper in this issue) ¹¹ and that H-c

Full text of DOI:10.1021/jm030245x

J . Med. Chem. 2003, 46, 5597-5605
5597
Novel sst4-Selective Som a tosta tin (SRIF ) Agon ists. 3. An a logu es Am en a ble to
Ra d iola belin g
J udit Erchegyi,†,+ Beatrice Waser,‡,+ J ean-Claude Schaer,‡ Renzo Cescato,‡ J ean Franc¸ois Brazeau,†
J ean Rivier,*,† and J ean Claude Reubi‡
The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies,
10010 North Torrey Pines Road, La J olla, California 92037, and Division of Cell Biology and Experimental Cancer
Research, Institute of Pathology, University of Berne, CH-3012 Berne, Switzerland
Received May 20, 2003
After our discovery that H-c[Cys-Phe-Phe-DNal-Lys-Thr-Phe-Cys]-OH (ODN-8) had high affinity
and marginal selectivity for human sst3 (part 2 of this series: Erchegyi et al. J . Med. Chem.,
preceding paper in this issue)11 and that H-c[Cys-Phe-Phe-DTrp-Lys-Thr-Phe-Cys]-OH (ODT-
8, 3) had high affinity and marginal selectivity for human sst4, that H-c[Cys-Phe-Tyr-D-threo-
â-Me2Nal-Lys-Thr-Phe-Cys]-OH had high affinity for all sst’s except for sst1, and that H-c[Cys-
Phe-Tyr-L-threo-â-Me2Nal-Lys-Thr-Phe-Cys]-OH had high affinity for sst4 (IC50 ) 2.1 nM), with
more than 50-fold selectivity toward the other receptors (parts 1 and 2 of this series: Rivier et
al. and Erchegyi et al. J . Med. Chem., preceding papers in this issue), we found H-c[Cys-Phe-
Phe-Trp-Lys-Thr-Phe-Cys]-OH (OLT-8, 2), H-c[Cys-Phe-Phe-L-threo-â-MeTrp-Lys-Thr-Phe-Cys]-
OH (4) and H-c[Cys-Phe-Phe-D-threo-â-MeTrp-Lys-Thr-Phe-Cys]-OH (5) to have very high
affinity for sst4 (IC50 ) 0.7, 1.8, and 4.0 nM, respectively) and 5- to 10-fold selectivity versus
the other sst’s. From earlier work, we concluded that an L-amino acid at position 8 and a tyrosine
or 4-aminophenylalanine substitution at position 7 may lead to high sst4 selectivity. In fact,
[Tyr7]-2 (6) and [Tyr7]-3 (7) show ca. 5-fold selectivity for sst4, and [Aph7]-2 (8) and [Aph7]-3 (9)
have high sst4 affinity (IC50 ) 1.2 and 0.88 nM, respectively) and selectivity, suggesting that
indeed an L-residue at position 8 will direct selectivity toward sst4. Unexpectedly, [Ala7]-2 (10)
and [Ala7]-3 (11) have very high sst4 affinity (IC50 ) 0.84 and 0.98 nM, respectively) and
selectivity (>600- and 200-fold, respectively). The combination of Tyr2 and DTrp8 in analogues
14 and 22 did not affect the affinity of the analogues for sst4 (IC50 ) 1.2 and 1.1 nM, respectively)
but resulted in loss of selectivity, whereas the combination of Tyr2 and LTrp8 in H-Tyr-c[Cys-
Phe-Aph-Trp-Lys-Thr-Phe-Cys]-OH (13) and H-Tyr-c[Cys-Phe-Ala-Trp-Lys-Thr-Phe-Cys]-OH-
(19) retained high affinity (IC50 ) 1.9 and 1.98 nM, respectively) and sst4 selectivity (>50 and
>250, respectively). Interestingly, the same substitutions at positions 2 and 7, with L-threo-
â-MeTrp at position 8, yielded a much less selective analogue (20). Carbamoylation of the
N-terminus of most of these analogues resulted in slightly improved affinity, selectivity, or
both. Other amino acid substitutions in this series, such as those with Amp (25, 26), Orn (27),
or IAmp (29) at position 7, were also tolerated but with a 2- to 3-fold loss of affinity and
concomitant loss of selectivity. Analogous peptides with a tyrosine at position 11 (31-36) were
less selective than the corresponding peptides with a tyrosine at position 2. Several analogues
in this series compared favorably with the non-peptide L-803,087 (37) in terms of affinity and
selectivity. Analogues 8, 10, and 21 potently inhibited the forskolin-stimulated cAMP production
in sst4-transfected cells, therefore acting as full agonists. Cold monoiodination of 19 yielded
21, with retention of high sst4 selectivity and affinity (IC50 ) 3.5 nM). 125Iodinated 19 selectively
binds to sst4-transfected cells but not to sst1-3- or sst5-transfected cells. Binding in sst4-
transfected cells was completely displaced by SRIF-28 or the sst4-selective L-803,087.
In tr od u ction
and highly receptor-selective, (2) potent and highly
receptor-selective peptide tracers for binding assays and
scintigraphy studies, (3) potent and highly receptor-
selective peptide agonists, and (4) antagonists for in
vitro (mechanistic) and in vivo (functional/physiological)
studies. It is a particular challenge in the case of
somatostatin with five identified receptors.2-9 Progress
has been made recently toward achieving these goals:
we have described analogues that bind to sst1 receptors
that fulfill criteria 1-3,10 analogues that bind to sst3
that fulfill criteria 1, 2, and 4,11 and analogues that bind
to sst4 that fulfill criteria 1 and 3.1,12,13 Here we describe
our approach to the design of an analogue that binds to
As stated in the first paper in this series,1 the general
aim of our laboratories has been to study the tissue
distribution, mechanism of action, and physiological role
as well as pharmacological effects of the hypothalamic
releasing hormones in the endocrine, exocrine, and
nervous systems. To achieve these goals, we must
successfully design peptide analogues that are (1) potent
* To whom correspondence should be addressed. Phone: (858) 453-
4100. Fax: (858) 552-1546. E-mail: jrivier@salk.edu.
† The Salk Institute for Biological Studies.
‡ University of Berne.
+ Both contributed equally to this work.
10.1021/jm030245x CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/20/2003

5598 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Erchegyi et al.
sst4 and fulfills criteria 2 in addition to criteria 1 and
3, i.e., that is a potent sst4-selective agonist tracer.
and mass spectrometry. The measured masses obtained
using MALDI-MS were within 100 ppm of those calcu-
lated for the protonated molecule ions.
In the preceding papers,12,13 we described the chemi-
cal and biological characterization of a series of sst4-
selective agonists with H-c[Cys-Phe-Tyr-L-threo-â-Me2-
Nal-Lys-Thr-Phe-Cys]-OH, having >100-fold selectivity
for sst4 over the other four sst’s while retaining sst4
binding affinity (IC50 ) 2.7 nM) comparable to that of
SRIF-28 (IC50 ) 3.8 nM). Unfortunately, monoiodination
of this analogue resulted in significant loss of affinity
(IC50 ) 21 nM). Although we had achieved one of our
goals to generate a potent sst4-selective ligand (charac-
terized to be an agonist), we were unable to develop it
as a tracer for binding or localization studies.
The compounds were tested for their ability to bind
to 20-µm-thick cryostat sections of a membrane pellet
of cells transfected with the five human sst receptor
subtypes. For each of the tested compounds, complete
displacement experiments with the universal somato-
statin radioligand [Leu8,DTrp22,125ITyr25]SRIF-28 (30 000
cpm/100 µL), using increasing concentrations of the
unlabeled peptide ranging from 0.1 to 1000 nM, were
performed. The unlabeled SRIF-28 was run in parallel
using the same increasing concentrations as control.
IC50 values were calculated after quantification of the
data using a computer-assisted image-processing system
as described previously.18,19 Tissue standards that con-
tain known amounts of isotope, cross-calibrated to
tissue-equivalent ligand concentrations, were used for
quantitation.20 The advantages of the present method
using receptor autoradiography with sectioned cell
pellets compared to binding on cell homogenates are,
in addition to an economy on cells and a great flexibility,
the greater inter-assay reliability and reproducibility,
since sections of the same embedded pellet can be used
for successive experiments. As a minor disadvantage,
IC50 values are somewhat higher than those obtained
in the homogenate binding assay.18 The most potent and
selective analogues were then evaluated for their ago-
nist/antagonist properties, measuring inhibition of cyclic
adenosine monophosphate (cAMP) production. The
125monoiodinated form of the best sst4 analogues was
also used to label sst4-transfected cells.
Subsequent to the discovery that H-c[Cys-Phe-Tyr-
DNal-Lys-Thr-Phe-Cys]-OH (ODN-8) had high affinity
and marginal selectivity for human sst3,11 that ODT-8
(3) had high affinity and marginal selectivity for human
sst4,11 and the study presented in the accompanying
papers,1,12,13 we now find that OLT-8 (2) has very high
affinity for sst4 (IC50 ) 0.7 nM) and 5- to 10-fold
selectivity versus the other sst’s. This is in keeping with
a similar observation published recently in Lewis et al.’s
alanine scan of SRIF-NH2.14 In summary, although
circuitous, the rationale that led to the identification of
position 7 as important for sst4 selectivity came from
several independent observations. First, H-c[Cys-Phe-
LAgl(NâMe,benzoyl)-DTrp-Lys-Thr-Phe-Cys]-OH, with
IC50’s for sst1-5 ) >1000, 460, 447, 5.2, and 768 nM,
respectively, pointed to the importance of residue 7.1
Second, the relative lack of selectivity of H-c[DCys-Phe-
Tyr-D-threo-â-Me2Nal-Lys-Thr-Phe-Cys]-OH (IC50’s for
sst1-5 ) 545, 12, 14, 0.53, and 27 nM, respectively)
versus H-c[Cys-Phe-Tyr-L-threo-â-Me2Nal-Lys-Thr-Phe-
Cys]-OH (IC50’s for sst1-5 ) >10K, 339, 664, 3.5, and
668, respectively)12,13 suggested a critical role for an
L-configuration rather than D at position 8. Third, at
least in one series,1 substitution of LCys3 by DCys3 did
not bear significantly on either selectivity or activity
since the representative analogues H-c[Cys-Phe-Tyr-L-
threo-â-Me2Nal-Lys-Thr-Phe-Cys]-OH and H-c[DCys-
Phe-Tyr-L-threo-â-Me2Nal-Lys-Thr-Phe-Cys]-OH were
sst4-selective and were agonists in a cAMP accumulation
test.12,13 We concluded that an L-amino acid at position
8 and an optimized substitution at position 7 (such as
tyrosine or 4-aminophenylalanine, Aph) may lead to
increased sst4 selectivity and used this lead for further
SAR studies.
Considering the result of several observations de-
scribed in the preceding papers1,12,13 and outlined below,
we felt compelled to re-examine 20-year-old biological
results (based on the ability of somatostatin and ana-
logues to inhibit spontaneous secretion of growth hor-
mone by pituitary cells in culture)21 in light of our
present ability to test these analogues for their affinity
at each of the five identified somatostatin receptors.
First, we showed that H-c[Cys-Phe-Phe-DNal-Lys-Thr-
Phe-Cys]-OH (ODN-8) had high affinity and marginal
selectivity for human sst3,11 and that ODT-8 (3) had
high affinity and marginal selectivity for human sst4.
This suggested that a DTrp substitution might be more
favorable than a DNal at position 8 in the design of an
sst4-selective analogue, although we have already shown
that DNal-based analogues could be designed to be
highly selective for that receptor.12,13 We then concluded,
on the basis of the observation that H-c[Cys-Phe-Tyr-
D-threo-â-Me2Nal-Lys-Thr-Phe-Cys]-OH had high af-
finity for all sst’s except for sst1 and that H-c[Cys-Phe-
Tyr-L-threo-â-Me2Nal-Lys-Thr-Phe-Cys]-OH had high
affinity for sst4 (IC50 ) 2.1 nM), with more than 50-fold
selectivity toward the other receptors, that an L-config-
uration at position 8 may have a profound effect on
directing selectivity for sst4.12,13 This was confirmed
when we tested OLT-8 (2) with very high affinity for
sst4 (IC50 ) 0.7 nM) and 5- to 10-fold selectivity versus
the other sst’s. Interestingly, the binding affinity profile
of 4 and 5 is not significantly different than that of 2
and 3, respectively, suggesting that the additional
conformational constraint brought about by â-meth-
ylation at position 8 was ineffective in directing selectiv-
Resu lts a n d Discu ssion
All analogues shown in Table 1 were synthesized
either manually or automatically on a chloromethylated
resin using the Boc strategy and diisopropylcarbodiim-
ide (DIC) for amide bond formation and trifluoroacetic
acid (TFA) for Boc removal. The peptide resins were
treated with hydrogen fluoride (HF) in the presence of
scavengers to liberate the fully deblocked crude linear
peptides. Most amino acid derivatives used are com-
mercially available; the threo and erythro isomers of
â-methyltryptophan were prepared according to the
method of Snyder.15 Cyclization of the cysteines was
mediated by iodine in an acidic milieu. Purification was
carried out using multiple HPLC steps16 and character-
ized by HPLC,16 capillary zone electrophoresis (CZE),17

Novel sst4-Selective Somatostatin (SRIF) Agonists. 3
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5599
Ta ble 1. Physicochemical Properties and sst1-5 Binding Affinities (IC50, nM) of sst4-Selective Analogues and Control Peptides
MSc
purity (%)
IC50 (nM)d
sst3
M(mono) MH+(mono)
no.
compd
HPLCa CZEb
calcd
obsd
sst1
sst2
sst4
sst5
1
somatostatin-28, SRIF-28
98
97
98 3146.5
98 1078.44
98 1078.44
98 1092.46
90 1092.46
96 1094.44
98 1094.44
97 1093.45
98 1093.45
>98 1002.41
94 1002.41
99 1241.50
>98 1256.51
98 1256.51
92 1136.46
>98 1136.46
92 1299.52
97 1425.43
99 1165.47
98 1179.49
99 1291.38
97 1165.47
97 1179.49
96 1208.49
98 1270.53
91 1313.55
98 1251.52
99 1313.55
96 1355.58
>98 1355.58
98 1094.44
98 1094.44
99 1137.44
98 1109.45
90 1152.45
98 1152.45
3147.7
3.2 ( 0.2
(36)
5.3 ( 0.7
(3)
27 ( 3.4
(4)
21
(17; 25)
49
(40; 58)
27 ( 6.8
(3)
117 ( 37
(3)
213 ( 19
(4)
450 ( 135
(3)
>1000
(3)
>1000
(3)
13
(14; 11)
270 ( 39
(4)
517 ( 159
(3)
650 ( 115
(3)
840 ( 101
(4)
347 ( 120
(3)
>1000
(2)
>1000
(4)
88
(88; 87)
>1000
(2)
330 ( 112
(3)
>1000
(400; 2792)
>1000
(3)
>1000
(2)
>1000 ( 50
(3)
99
(60; 137)
>1000
(3)
323 ( 63
(3)
397 ( 165
(3)
6.5 ( 1.8
(3)
10.3 ( 0.9
(3)
55
(43; 67)
327 ( 37
(3)
757 ( 94
(3)
>1000
(3)
2.3 ( 0.1
(37)
130 ( 65
(3)
41 ( 8.7
(6)
27
(27, 26)
40
(24; 54)
54 ( 16
(3)
25.7 ( 6
(3)
347 ( 61
(3)
71 ( 2.1
(3)
807 ( 146
(3)
183 ( 18
(3)
179
(150; 207)
260 ( 20
(3)
56 ( 5.8
(3)
>1000
(3)
120 ( 15
(3)
550 ( 276
(3)
>1000
(2)
3.5 ( 0.2
(36)
13 ( 0.7
(3)
13 ( 3.2
(4)
330
(210; 449)
53
(44; 61)
22 ( 1.7
(3)
35.7 ( 8
(3)
>1000
(3)
271 ( 120
(3)
750 ( 278
(3)
897 ( 103
(3)
57
(66; 47)
135 ( 25
(4)
263 ( 97
(3)
780 ( 62
(3)
650 ( 161
(3)
210 ( 36
(3)
325
(500; 150)
624 ( 213
(4)
2.5 ( 0.1
(35)
0.7 ( 0.3
(3)
1.8 ( 0.7
(4)
1.8
2.4 ( 0.2
(34)
14 ( 4.1
(3)
46 ( 27
(3)
221
2
3
4
5
6
7
8
9
H-c[Cys-Phe-Phe-Trp-Lys-Thr-Phe-
Cys]-OH (OLT-8)
H-c[Cys-Phe-Phe-DTrp-Lys-Thr-Phe-
Cys]-OH (ODT-8)
H-c[Cys-Phe-Phe-L-threo-â-MeTrp-
Lys-Thr-Phe-Cys]-OH
H-c[Cys-Phe-Phe-D-threo-â-MeTrp-
Lys-Thr-Phe-Cys]-OH
H-c[Cys-Phe-Tyr-Trp-Lys-Thr-Phe-
Cys]-OH
H-c[Cys-Phe-Tyr-DTrp-Lys-Thr-Phe-
Cys]-OH
H-c[Cys-Phe-Aph-Trp-Lys-Thr-Phe-
Cys]-OH
1079.4
1079.2
1093.4
1093.5
1095.4
1095.0
1094.3
1094.3
1003.4
1003.5
1242.4
1257.5
1257.6
1137.4
1137.2
1300.6
1426.4
1166.3
1180.5
1292.3
1166.4
1180.5
1209.5
1271.6
1314.5
1252.5
1314.5
1356.6
1356.6
1095.4
1095.3
1138.8
1110.36
1153.44
1153.4
>94
98
(2.7; 0.9)
(190; 251)
90
4
42
(5.2; 2.9)
1.3 ( 0.1
(3)
1.8 ( 0.7
(3)
1.2 ( 0.1
(4)
0.88 ( 0.3 30 ( 7.4
(3) (3)
0.84 ( 0.2 633 ( 186
(3) (3)
0.98 ( 0.1 199 ( 56
(4)
(28; 52)
63 ( 7
(3)
20.3 ( 2.9
(3)
1000 ( 91
(4)
97
>98
98
H-c[Cys-Phe-Aph-DTrp-Lys-Thr-Phe- >98
Cys]-OH
10 H-c[Cys-Phe-Ala-Trp-Lys-Thr-Phe-
Cys]-OH
11 H-c[Cys-Phe-Ala-DTrp-Lys-Thr-Phe-
Cys]-OH
12 H-Tyr-c[Cys-Phe-Phe-Trp-Lys-Thr-
Phe-Cys]-OH
13 H-Tyr-c[Cys-Phe-Aph-Trp-Lys-Thr-
Phe-Cys]-OH
>98
93
99
98
99
91
>98
92
97
99
96
99
98
99
84
97
91
96
93
>98
98
99
99
96
99
86
90
(4)
1.6
19
(1; 2.1)
1.9 ( 0.3
(4)
1.2 ( 0.3
(3)
1.5 ( 0.07 >1000
(3)
1.1 ( 0.2
(3)
1.2 ( 0.3
(3)
8.8 ( 1.3
(4)
2.0 ( 0.5
(4)
0.9
(1.2; 0.6)
3.5 ( 0.8
(4)
1.1 ( 0.2
(3)
5.6
(5.5; 5.6)
2.4 ( 0.5
(3)
2.4 ( 0.2
(3)
3.2 ( 0.6
(3)
(18; 20)
663 ( 157
(4)
34 ( 8.6
(3)
14 H-Tyr-c[Cys-Phe-Aph-DTrp-Lys-Thr-
Phe-Cys]-OH
15 H2N-CO-c[Cys-Phe-Aph-Trp-Lys-
Thr-Phe-Cys]-OH
(3)
103 ( 15
(3)
317 ( 92
(3)
990
(480; 1500)
692 ( 166
(4)
393
(350; 435)
>1000
(2)
51 ( 20
(3)
629
(410; 847)
>1000
(3)
342
(260; 424)
247 ( 27
(3)
458
(550; 365)
50 ( 5
(3)
397 ( 152
(3)
84 ( 23
(3)
16 H2N-CO-c[Cys-Phe-Aph-DTrp-Lys-
Thr-Phe-Cys]-OH
17 H2N-CO-Tyr-c[Cys-Phe-Aph-Trp-
Lys-Thr-Phe-Cys]-OH
18 H2N-CO-(m-I)-Tyr-c[Cys-Phe-Aph-
Trp-Lys-Thr-Phe-Cys]-OH
19 H-Tyr-c[Cys-Phe-Ala-Trp-Lys-Thr-
Phe-Cys]-OH
20 H-Tyr-c[Cys-Phe-Phe-L-threo-â-Me-
Trp-Lys-Thr-Phe-Cys]-OH
21 H-(m-I)-Tyr-c[Cys-Phe-Ala-Trp-Lys-
Thr-Phe-Cys]-OH
22 H-Tyr-c[Cys-Phe-Ala-DTrp-Lys-Thr-
Phe-Cys]-OH
23 H-Tyr-c[Cys-Phe-Ala-D-threo-â-Me-
Trp-Lys-Thr-Phe-Cys]-OH
24 H2N-CO-Tyr-c[Cys-Phe-Ala-Trp-
Lys-Thr-Phe-Cys]-OH
25 H-Tyr-c[Cys-Phe-Amp-Trp-Lys-Thr-
Phe-Cys]-OH
26 H2N-CO-Tyr-c[Cys-Phe-Amp-Trp-
Lys-Thr-Phe-Cys]-OH
27 H2N-CO-Tyr-c[Cys-Phe-Orn-Trp-
Lys-Thr-Phe-Cys]-OH
28 H2N-CO-Tyr-c[Cys-Phe-Amp-DTrp-
Lys-Thr-Phe-Cys]-OH
29 H2N-CO-Tyr-c[Cys-Phe-IAmp-Trp-
Lys-Thr-Phe-Cys]-OH
30 H2N-CO-Tyr-c[Cys-Phe-IAmp-DTrp-
Lys-Thr-Phe-Cys]-OH
31 H-c[Cys-Phe-Phe-Trp-Lys-Thr-Tyr-
Cys]-OH
32 H-c[Cys-Phe-Phe-DTrp-Lys-Thr-Tyr-
Cys]-OH
33 H2N-CO-c[Cys-Phe-Phe-DTrp-Lys-
Thr-Tyr-Cys]-OH
34 H-c[Cys-Phe-Aph-Trp-Lys-Thr-Tyr-
Cys]-OH
35 H2N-CO-c[Cys-Phe-Aph-Trp-Lys-
Thr-Tyr-Cys]-OH
622 ( 85
(4)
48
(18; 77)
>1000
(2)
57 ( 1.7
(3)
46
116
(75;156)
1025
(550; 1500)
347 ( 120
(3)
381
(34; 57)
700 ( 92
(3)
(110; 652)
980 ( 73
(3)
259
326
(250; 267)
118 ( 42
(3)
(319-332)
197 ( 48
(3)
>1000
(420; 3722)
>1000
(3)
110 ( 17
(4)
>1000
(3)
10 ( 1.7
(3)
8.2 ( 4.4
(3)
16
(9.5; 22)
247 ( 86
(3)
587 ( 150
(3)
561
2.1
(250; 872)
29 ( 22
(3)
130 ( 32
(3)
12 ( 1.5
(3)
43 ( 13
(3)
15.5 ( 6
(3)
52
(35; 68)
170 ( 25
(3)
>1000
(3)
227 ( 108
(3)
(2.2; 2.0)
2.6 ( 0.2
(3)
2.3 ( 0.5
(4)
2.3 ( 0.4
(3)
1.3 ( 0.2
(3)
0.51 ( 0.1 4.8 ( 0.33
(3)
24 ( 6.5
(3)
(3)
1.0
7.6
(1.1; 0.9)
1.1 ( 0.2
(3)
3 ( 0.4
(3)
4.3 ( 2.4
(3)
(9.5; 5.7)
240 ( 45
(3)
557 ( 70
(3)
110 ( 12
(3)
36 H2N-CO-c[Cys-Phe-Aph-DTrp-Lys-
Thr-Tyr-Cys]-OH
623 ( 340
(3)
>10K
(4)
37 L-803,087 (Merck)26
>1000
(4)
>10K
(4)
4.2 ( 1.9
(3)
>10K
(4)
a
Percent purity determined by HPLC using 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 on a Vydac C18 column (0.21 × 15 cm, 5 µm particle size, 300 Å pore size). Detection at 214 nm.
b
Capillary zone electrophoresis (CZE) was done using a Beckman P/ACE system 2050 controlled by an IBM Personal System/2 model
50Z and using a ChromJ et integrator; field strength of 15 kV at 30 °C. Mobile phase: 100 mM sodium phosphate (85:15, H2O:CH3CN),
pH 2.50, on a Supelco P175 capillary (363 µm o.d. × 75 µm i.d. × 50 cm length). Detection at 214 nm. c The calculated m/z of the monoisotope
d
compared with the observed [M + H]+ monoisotopic mass. The IC50 values (nM) were derived from competitive radioligand displacement
assays reflect the affinities of the analogues for the cloned somatostatin receptors using the nonselective 125I-[Leu8,DTrp22,Tyr25]SRIF-28,
as the radioligand. Mean value ( SEM when N g 3 (shown in parentheses). Otherwise, mean with single values in parentheses.

5600 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Erchegyi et al.
ity. Prior studies by Huang et al.,22 using â-MeTrp in
SRIF hexapeptide scaffold cyclo[Pro6-Phe7-DTrp8-Lys9-
Thr10-Phe11], showed that the corresponding analogue
with the (2R,3S)-â-MeTrp8 (D-threo) substitution exhib-
ited a “folded” conformation and significant changes in
the receptor binding affinity by restricting the confor-
mational freedom around the side chain. An IC50 < 1
nM for receptor binding (inhibition of 125I-MK 678 on
AtT-20 cells23) was reported. In that assay, the (2R,3R)-
â-MeTrp8- and the (2S,3S)-â-MeTrp8-containing ana-
OH (20) showed significant loss of selectivity (20-fold
with respect to sst2) yet retained high affinity for sst4
(IC50 ) 0.9 nM).
We had shown earlier that carbamoylation of the
N-terminus of most SRIF analogues resulted in slightly
improved affinity, selectivity, or both and confirmed
these results with the synthesis of 15-17 and 24. As
expected, the three analogues with an LTrp8 (15, 17, and
24) are highly potent and sst4-selective, whereas the
DTrp8-containing analogue 16 is less selective than 15.
logues showed extremely low binding affinity (IC50
>
Because of our need for radioiodinatable analogues,
we synthesized the cold iodinated analogues of our most
promising tracer precursors. In this case, cold mono-
iodination of 17 and 19 yielded 18 and 21, with slightly
decreased affinity for sst4 and little effect to the binding
affinity toward the other four receptors. We radiolabeled
19 with 125I, purified the monoiodinated from the
diiodinated analogues using HPLC, and tested its
binding to sst1-5 cells. As seen in Figure 1, 125I-19 was
able to strongly label sst4 cells, whereas it did not bind
to sst1-3 or sst5 cells. Binding was detected both in sst4-
transfected CCL39 and HEK293 cells. In both cell types,
binding of 125I-19 was found to be specific for soma-
tostatin receptors of the sst4 subtype, since the radio-
ligand could be completely displaced not only by 10 but
also by SRIF-28 and by the sst4-selective non-peptide
L-803,087 (Figure 2).
Because the Tyr2 substitution did not negatively
impact either affinity or selectivity, we investigated the
effect of substitutions other than Tyr, Aph, or Ala at
position 7 in the parent [Tyr2]OLT-8 (12). Interestingly,
substitutions by basic amino acids such as 4-amino-
methylphenylalanine (Amp) (25, 26), ornithine (27), or
isopropyl-4-aminomethylphenylalanine (IAmp) (29) were
well tolerated, but with a 2- to 3-fold loss of affinity and
concomitant loss of selectivity. As in other pairs of
analogues (8-9, 10-11, 13-14, and 15-16), substitution
of LTrp8 with DTrp8 in 29-30 resulted in increased
affinity for sst2 and sst5, to the detriment of selectivity.
Tyrosine substitutions in SRIF and shorter analogues
have traditionally been inserted at the N-terminus of
the analogues (i.e., position 2 with deletion of residue
1) and at positions 7 and 11, where these were well
tolerated, and not as a substitution of Phe6, which was
less favorable.24 We therefore substituted Phe11 by Tyr11
in both OLT-8 and ODT-8 to yield 31 and 32, respec-
tively. In fact, this substitution was well tolerated in
both analogues with the same tendency for sst4 selectiv-
ity as had been shown for OLT-8 (2) and ODT-8 (3)
themselves. Carbamoylation of 32 to yield 33, however,
led to some loss of affinity at sst1 and sst2. In this series,
substitution of Phe7 by Aph7 led to dramatic changes
in receptor selectivity, since 34 has retained the high
affinity of its parent 31 with significant loss of affinity
at sst1-3 and sst5. Carbamoylation of 34 led to 35 with
increased sst4 selectivity and only limited loss of affinity.
1000 nM), while the (2S,3R)-â-MeTrp8-containing ana-
logue (L-threo) had an intermediate affinity (IC50 > 10
nM). Unfortunately, these results were published before
the discovery of the sst’s and selectivity could not be
assessed. Upon these observations, we synthesized
analogues 4, 5, 20, and 23 containing only the threo
isomers of â-MeTrp.
Substitution of Phe7 in 2 and 3 by Tyr yielded 6 and
7, which showed similar binding affinity profiles, namely
that the introduction of DTrp8 in this octapeptide
scaffold tends to increase potency at all receptors,
whereas the L-isomer tends to favor sst4 selectivity. We
concluded from these results that an L-amino acid at
position 8 and an optimized substitution at position 7
(such as tyrosine, 6) may lead to high sst4 selectivity.
In fact, H-c[Cys-Phe-Aph-Trp-Lys-Thr-Phe-Cys]-OH (8)
and H-c[Cys-Phe-Aph-DTrp-Lys-Thr-Phe-Cys]-OH (9)
have high sst4 affinity (IC50 ) 1.2 and 0.88 nM,
respectively) and selectivity (>150-fold and 40-fold,
respectively), suggesting that, indeed, an L-residue at
position 8 will direct selectivity toward sst4 and that
concomitant substitutions at position 7 could improve
selectivity. Unpredictably, H-c[Cys-Phe-Ala-Trp-Lys-
Thr-Phe-Cys]-OH (10) and H-c[Cys-Phe-Ala-DTrp-Lys-
Thr-Phe-Cys]-OH (11) have very high sst4 affinity
(IC50 ) 0.84 and 0.98 nM, respectively) and selectivity
(>600- and 200-fold, respectively), although, as men-
tioned earlier, there were recently published indications
that [Ala7]SRIF-NH2 was indeed sst4-selective, with
moderate affinity.14 Clearly, with such a simple ana-
logue, we had achieved one of our stated goals of
generating a potent, highly sst4-selective ligand. Whether
this analogue could be derivatized to yield an iodinat-
able tracer became our next priority. Because it is
generally admitted that extensions at the N-terminus
of SRIF and analogues are compatible with high po-
tency, we made a series of tyrosinated analogues. The
parent 12 also showed significant selectivity for sst4 and
high affinity for that receptor (IC50 ) 1.6 nM). Addition-
ally, we found that the combination of Tyr2 and DTrp8,
such as in H-Tyr-c[Cys-Phe-Aph-DTrp-Lys-Thr-Phe-
Cys]-OH (14) and H-Tyr-c[Cys-Phe-Ala-DTrp-Lys-Thr-
Phe-Cys]-OH (22) or H-Tyr-c[Cys-Phe-Ala-D-threo-â-
MeTrp-Lys-Thr-Phe-Cys]-OH (23), did not significantly
affect the affinity of the analogue for sst4 (IC50 ) 1.2,
1.1, and 5.6 nM, respectively) but resulted in loss of
selectivity, whereas the combination of Tyr2 and LTrp8,
such as in H-Tyr-c[Cys-Phe-Aph-Trp-Lys-Thr-Phe-Cys]-
OH (13) and H-Tyr-c[Cys-Phe-Ala-Trp-Lys-Thr-Phe-
Cys]-OH (19), retained high affinity (IC50 ) 1.9 and 1.98
nM, respectively) and sst4 selectivity (>50 and >250,
respectively). Unexpected was the observation that
H-Tyr-c[Cys-Phe-Ala-L-threo-â-MeTrp-Lys-Thr-Phe-Cys]-
It has not escaped our attention that analogues with
>100- and 25-fold sst4 selectivity and high affinity, such
as 16 or 36, respectively, with DTrp8 and a blocked
N-terminus could be more stable in a biological milieu
than the highly sst4-selective and potent 19, for ex-
ample.
Clearly, we have not synthesized and tested all
possible combinations of substitutions at positions 2, 7,

Novel sst4-Selective Somatostatin (SRIF) Agonists. 3
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5601
F igu r e 2. sst4 specificity of the binding of 125I-19 in sections
of sst4-expressing CCL39 cell pellets. (A) Total binding of 125I-
19. (B) Binding in the presence of 10 (10-6 M). (C) Binding in
the presence of SRIF-28 (1) (10-6 M). (D) Binding in the
presence of L-803,087 (37) (10-6 M). Full displacement of 125I-
19 is seen with all three compounds.
concentration of 100 nM, with an EC50 ) 0.49 nM; it
was used as control. Our best sst4 analogue, 10, was
tested in this system and shown to have full agonistic
properties similar to those of SRIF-28, with an EC50
)
1.12 nM. The sst3-selective sst3-ODN-811 was used as
negative control in these experiments (Figure 3A).
Analogues 8 and 21 are also agonists in this system,
with EC50 values ) 0.29 and 1.13 nM, respectively
(Figure 3B). Analogue 8 was tested in this system
because of its high affinity and selectivity and the fact
that it differed significantly at position 7 from 10 (basic
aromatic side chain versus the neutral aliphatic nature
of the alanine side chain) and could therefore have been
an antagonist. We tested 21 because it was going to be
used as a tracer after radiolabeling and we wanted to
confirm, indirectly, the agonistic activity of its parent
10. Comparable results on cAMP production were
obtained in sst4-transfected CCL39 cells with these
compounds (data not shown).
The present study describes a family of potent pep-
tidic analogues of somatostatin with agonistic properties
and high selectivity for the sst4-receptor subtype. With
its high sst4 affinity, comparable to that of the natural
SRIF-28, the best compound, 10, is significantly better
than any other peptidic sst4-selective analogue and
compares favorably with the recently discovered non-
peptide agonist L-803,087 reported by Rohrer et al.26
This series of novel analogues, having a 4-aminophen-
ylalanine or even better, an alanine at position 7,
combined with the native tryptophan residue at position
8 of the OLT-8 octapeptide scaffold, demonstrates that
extraordinary sst4 receptor selectivity can be achieved
with rather simple substitutions. These findings how-
ever, leave open the challenge of identifying sst4-
selective antagonists.
F igu r e 1. Binding of 125I-19 to sections containing cell pellets
from sst1-expressing CHO-K1 cells, sst2-expressing CCL39
cells, sst3-expressing CCL39 cells, sst4-expressing CCL39 cells,
and sst5-expressing CHO-K1 cells. All sections were incubated
with 30 000 cpm/100 µL of ligand. Strong specific labeling with
the ligand is seen only in sst4 cells (G). (A, C, E, G, I)
Autoradiograms showing total binding of 125I-19 to sections of
cell pellets expressing sst1 (A), sst2 (C), sst3 (E), sst4 (G), and
sst5 (I). (B, D, F, H, K) Autoradiograms showing nonspecific
binding of 125I-19 (in the presence of 10 (10-6 M).
8, and 11 of OLT-8 and ODT-8 with and without
carbamoylation of the N-terminus. However, several
analogues in this series compared favorably with the
non-peptide L-803,087 (37) in terms of affinity and
selectivity. sst4-selective analogues such as 8-11, 16,
17, and 34 have more than twice the binding affinity of
SRIF-28. Many others are equipotent to SRIF at sst4.
Finally, others may exhibit long duration of action in
vivo once a clear function for sst4 is assigned and an in
vivo test is validated (see below).
To distinguish agonists from antagonists, the effect
of the best sst4-selective analogue on forskolin-stimu-
lated cAMP production in sst4-expressing HEK293
cells25 was evaluated. Figure 3 illustrates the data. The
agonist SRIF-28 potently inhibited forskolin-stimulated
cAMP accumulation by more than 91% at a peptide
The present study also permitted us to obtain a
radioligand closely related to the structures of our best
sst4 compounds. This radioligand 125I-19 binds only to
sst4 cells and not to cells expressing the other four sst’s,
as shown by the complete displacement of the radio-
ligand by SRIF-28 or by the sst4-selective L-803,087.
Such sst4-selective agonist radioligands may be useful

5602 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Erchegyi et al.
chelators with isotopes without significant modification
of their binding affinity, to be used as clinical tools;35,36
(c) a peptide will poorly cross the blood-brain barrier.
If it is to be used for systemic application as a drug or
radioligand in clinical indications involving peripheral
targets, it will be a great advantage if it does not
penetrate into the brain, an organ known to contain
sst4.27
To conclude, this study indicates that simple substi-
tutions in the somatostatin molecule will yield potent
sst4-selective agonists that may be developed as tools
for preclinical and clinical studies. An important sst4-
selective analogue (19) that can be easily radiolabeled
has been identified. The fact that derivatives of the
above retain high selectivity and affinity after carbam-
oylation of the N-terminus (24 and 26, for example)
suggests that substitution of these analogues with
chelating agents at the N-terminus may open the door
to sst4-selective radiotherapeutics (work in progress).
Analogues such as 16 and 36 may have the additional
advantage of being stable in a biological milieu. Finally,
potent and sst4-selective analogues selected from the
three series described in this and the two preceding
papers have unique conformations determined by NMR
in dimethyl sulfoxide (a solvent that may mimic the
receptor’s environment), thus suggesting general struc-
tural parameters responsible for sst4 selectivity.37
Exp er im en ta l Section
Abbr evia tion s. The abbreviations for the common amino
acids are in accordance with the recommendations of the
IUPAC-IUB J oint Commission on Biochemical Nomenclature
(Eur. J . Biochem. 1984, 138, 9-37). The symbols represent
the L-isomer except when indicated otherwise. Additional
abbreviations: Agl, aminoglycine; Amp, 4-aminomethylphen-
ylalanine; Aph, 4-aminophenylalanine; â-MeTrp, â-methyl-
tryptophan; Boc, tert-butoxycarbonyl; Bzl, benzyl; Z(2Br),
2-bromobenzyloxycarbonyl; Z(2Cl), 2-chlorobenzyloxycarbon-
yl; Cbm, carbamoyl (H2N-CO-); CZE, capillary zone electro-
phoresis; DIC, N,N′-diisopropylcarbodiimide; DIPEA, diiso-
propylethylamine; Fmoc, 9-fluorenylmethoxycarbonyl; HOBt,
1-hydroxybenzotriazole; IAmp, isopropyl-4-aminomethylphen-
ylalanine; (m-I)-Tyr, monoiodinated tyrosine; Mob, 4-meth-
oxybenzyl; Nal, 3-(2-naphthyl)-alanine; â-Me2Nal, â-methyl-
3-(2-naphthyl)alanine; NMP, N-methylpirrolidinone; OBzl,
benzyl ester; SRIF, somatostatin-14; sst’s, somatostatin recep-
tors; TEA, triethylamine; TEAP, triethylammonium phos-
phate; TFA, trifluoroacetic acid.
Sta r tin g Ma ter ia ls. Boc-Cys(Mob)-CM resin, with a capac-
ity of 0.3-0.4 mequiv/g, was prepared by the method reported
by Horiki et al.38 All Boc-NR-protected amino acids with side-
chain protection {Cys(Mob), Lys[Z(2Cl)], Orn(Z), Thr(Bzl), Tyr-
[Z(2Br)], and (m-I)-Tyr(3-BrBzl)} were commercially available
(Bachem Inc., Torrance, CA; Chem Impex, Wood Dale, IL;
Reanal, Budapest, Hungary), except for Boc-Aph(Fmoc), Boc-
Amp(Z), and Boc-IAmp(Z),39 which were synthesized in our
laboratory. Additionally, the threo and erythro isomers of
â-methyltryptophan were prepared according to the method
reported by Snyder.15 In short, indole (50 mmol) in glacial
acetic acid (30 mL) was reacted with isopropylamine (56 mmol)
and a solution of acetaldehyde (53 mmol) in benzene (10 mL)
at 0 °C for 3 days to form 3-(isopropylaminoethylidene)indole
(13.5 mmol, 27.1% yield). The 3-(isopropylaminoethylidene)-
indole (11 mmol) was reacted with dibenzyl acetamidomalo-
nate (11 mmol) and sodium methoxide (18.5 mmol) in toluene
(15 mL) at 85 °C for 12 h, resulting in dibenzyl(3-indolyleth-
ylidene)acetamidomalonate (9 mmol, 90% yield), which was
converted to ammonium (3-indolylethylidene)acetamidomalo-
nate upon hydrogenolysis with 10% Pd/C catalyst (0.15 g) in
F igu r e 3. Effect of various concentrations of the sst4-selective
agonist 10 and the sst3-selective antagonist sst3-ODN-8 (A),
as well as the sst4-selective agonists 8 and 21 (B), in compari-
son with SRIF-28 as control, on forskolin-stimulated cAMP
accumulation in HEK293 cells expressing sst4. In (A) concen-
tration-response curves were obtained with increasing con-
centrations of SRIF-28 (b) and 10 (9). sst3-ODN-8 (2), as
negative control, has no effect. In (B) the effects of 8 (2) and
21 (9) are shown in comparison to SRIF-28 (b). Data are
expressed as % forskolin-stimulated cAMP accumulation. Each
plot represents the mean of two independent experiments.
Analogues 10, 8, and 21 behave as agonists.
in localizing sst4 in human tissues and deciphering the
functions mediated by sst4. Presently, the sst4 system
is poorly understood, specifically in humans. Available
information on sst4 includes the observation that sst4
is present in the rat brain, where somatostatin may play
a neurotransmitter role,27 and that sst4 is also found in
the rat lungs and in human placenta.28,29
It will also be worth evaluating the role of sst4 in
cancer. It is known that the sst4 mRNA can be detected
in some human tumors.30-33 However, the incidence of
the sst4 protein appears to be extremely low34 and its
importance not well established in human tumors.
Therefore, a confirmation of the presence or absence of
the receptor protein, using sensitive receptor binding
assays with selective radioligands such as 125I-19, would
be welcome. Those studies are underway.
The peptidic nature of our sst4 agonists results in a
number of advantageous properties: (a) their structures
can be modified easily and rationally to obtain more
potent or more stable compounds; (b) the relatively large
molecules allow the addition of further isotopes, or even

Novel sst4-Selective Somatostatin (SRIF) Agonists. 3
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5603
95% ethanol (25 mL) and addition of ammonium hydroxide
(10 mL). The yield of this product was 96%. Decarboxylation
and the formation of the unresolved 2-acetamido-3-(3-indolyl)-
butyric acid (acetyl-â-methyltryptophan) was achieved by re-
fluxing the ammonium (3-indolylethylidene)acetamidomalo-
nate (6.4 mmol) in 6 mL of a pyridine:water (1:1) solvent
mixture for 5 h. After dilution of the reaction mixture with
water and acidification with 10% sulfuric acid to pH 3, the
threo isomers were separated by fractional crystallization (45%
yield) from the erythro isomers, which were obtained by ethyl
acetate extractions of the mother liquor (41% yield). Hydrolysis
of the separated acetyl threo and erythro isomers by refluxing
with 4 N sulfuric acid for 5 h and neutralizing with saturated
solution of Ba(OH)2 resulted in the formation of the threo-
(2S,3R and 2R,3S) and the erythro-(2S,3S and 2R,3R) isomers
of 2-amino-3-(3-indolyl)butyric acid (â-methyltryptophan,
â-MeTrp), with a yield of 64.5% and 54%, respectively. After
preparative HPLC purification,16 both diastereomers showed
>95% purity, determined by CZE; the threo isomers migrated
faster than the erythro isomers.16,40 The threo isomers could
be distinguished from the erythro isomers on the basis of the
proton coupling constants between CR-H and Câ-H, deter-
mined from 1H NMR spectra. Diastereomers with larger
coupling constant (J H â ) 7.16 Hz) were assigned the erythro-
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 of iodine.
P u r ifica tion of P ep tid es. The crude, lyophilized peptides
were purified by preparative RP-HPLC16 on a 5-cm × 30-cm
cartridge, packed in the laboratory with reversed-phase 300
Å Vydac C18 silica (15-20 µm particle size) using a Waters
Associates Prep LC/System 500A system. 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). All
peptides were subjected to a second purification step, carried
out with eluents A ) 0.1% TFA in water and B ) 60% CH3-
CN/40% A on the same cartridge using a linear gradient of
1% B per min increase from the baseline % B. Analytical HPLC
screening of the purification was performed on a Vydac C18
column (0.46 × 25 cm, 5 µm particle size, 300 Å pore size)
connected to a Rheodyne injector, two Beckman 100A pumps,
a model 420 system controller programmer, a Kratos 750 UV
detector, and a Houston Instruments D-5000 strip chart
recorder. The fractions containing the product were pooled and
subjected to lyophilization.
Ch a r a cter iza tion of SRIF An a logu es. (See Table 1
footnote.) The purity of the final peptides 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 chro-
matograph connected to a Vydac C18 column (0.21 × 15 cm, 5
µm particle size, 300 Å pore size), controller model 362, and a
Think J et printer. CZE analysis was performed as described
earlier.17 Each peptide was found to have a purity of >90% by
HPLC and CZE. Mass spectra (MALDI-MS) were measured
on an ABI-Perseptive DE-STR instrument. 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-hydroxycinnamic
acid was prepared as saturated solutions in 0.3% trifluoro-
acetic acid and 50% acetonitrile. The observed monoisotopic
(M + H)+ values of each peptide corresponded with the
calculated (M + H)+ values.
-H
R
(2S,3S and 2R,3R)-â-MeTrp configuration, and diastereomers
with smaller coupling constant (J H ) 4.41 Hz) the threo-
-H
R
â
(2S,3R and 2R,3S)-â-MeTrp configuration.41,42 These isomers
were converted to their NR-tert-butoxycarbonyl derivatives. For
peptide synthesis, the unresolved NR-tert-butoxycarbonyl threo
isomers were used to yield the epimeric peptide mixtures
separated by RP-HPLC. The absolute configuration at the
R-carbon of the threo-â-MeTrp in compounds 4, 5, 20, and 23
(Table 1) was determined by enzymatic hydrolysis with
trypsin, carboxypeptidase B, and carboxypeptidase A in three
consecutive steps. Trypsin was able to quantitatively open the
ring structure of compounds 4 and 20 within 2 h, whereas it
took 2 days for partial opening of 5 and 23 after the lysine
residue to yield the linear free acids, which eluted earlier on
RP-HPLC and showed an 18-mass-unit increase. We further
hydrolyzed the linear derivatives of 4 and 20 with carboxy-
peptidase B, which cleaved quantitatively the C-terminus
lysine within 2 h, as shown by MS and a shift in RP-HPLC
retention time toward the more hydrophobic region. Finally,
these products were completely digested with carboxypeptidase
A in 20 min, suggesting that all amino acids in the remaining
sequence were of the L-configuration. One of the free amino
acids (â-MeTrp) could be identified as one of the hydrolysis
product by RP-HPLC. This procedure is described in more
detail in the preceding papers.12,13
R ecep t or Au t or a d iogr a p h y. CHO-K1, CCL39, and
HEK293 cells stably expressing the cloned five human sst’s
were grown as described previously.18,25 All cell culture re-
agents were supplied by Gibco BRL, Life Technologies (Grand
Island, NY). The receptor autoradiographical experiments
were performed as reported earlier.18
All reagents and solvents were ACS grade and were used
without further purification.
Ad en yla te Cycla se Activity. Modulation of forskolin-
stimulated adenylate cyclase activity was determined using a
radioimmunoassay measuring intracellular cAMP levels by
competition binding. sst4-expressing HEK293 and CCL39
cells11,25,44 were subcultured in poly-D-lysine-coated 96-well
culture plates at 2 × 104 cells/well and grown for 24 h. Culture
medium was removed from the wells, and 100 µL of fresh
medium containing 0.5 mM 3-isobutyl-I-methylxanthine (IBMX)
was added to each well. Cells were incubated for 30 min at 37
°C. Medium was then removed and replaced with fresh
medium containing 0.5 mM IBMX, with or without 10 µM
forskolin and various concentrations of peptides. Cells were
incubated for 30 min at 37 °C. After removal of medium, cells
were lysed, and cAMP accumulation was determined using a
commercially available cAMP scintillation proximity assay
(SPA) system according to the instructions from the manu-
facturer (RPA 538, Amersham Biosciences, Little Chalfont,
UK). cAMP data were expressed as percentage of stimulation
over the nonstimulated level. Values of EC50 (the agonist
concentration causing 50% of its maximal effect) were derived
from concentration-response curves.
P ep tid e Syn th esis. Peptides were synthesized by the solid-
phase approach, either manually or on a CS-Bio peptide
synthesizer, model CS536.43 Peptide couplings were mediated
for 1 h by DIC in CH2Cl2 or NMP and monitored by the
qualitative ninhydrin test. A 3-equiv excess of amino acid
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% m-cresol) for 20 min. An isopropyl alcohol
(1% m-cresol) wash followed TFA treatment, and then suc-
cessive washes with triethylamine solution (10% in CH2Cl2),
methanol, triethylamine solution, methanol, and CH2Cl2 com-
pleted the neutralization sequence. The ureido group (Cbm)
at the N-terminus of 15-18, 24, 26-30, 33, 35, 36 was
introduced on the resin. The N-terminal Boc group of the fully
assembled peptide was deprotected with TFA in the usual
manner,10,18 and after neutralization, the carbamoylation
proceeded with NaOCN (100 mg, 0.65 mmol) in NMP (4 mL)
and glacial acetic acid (3 mL per gram of initial resin). The
mixture was agitated at room temperature for 30 min, and
ninhydrin test indicated a complete reaction. The completed
peptide was then cleaved from the resin by HF containing the
scavengers anisole (10% v/v) and methyl sulfide (5% v/v) for
Ack n ow led gm en t. This work was supported in part
by NIH Grants DK-50124 and DK-59953. We thank

5604 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
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