Highly potent cyclic disulfide antagonists of somatostatin

Article abstract of DOI:10.1021/jm9806289

The search for synthetic analogues of somatostatin (SRIF) which exhibit selective affinities for the five known receptor subtypes (sst_1-5) has generated a large number of potent agonist analogues. Many of these agonists display good subtype selectivities and affinities for the subtypes 2, 3, and 5, with very few selective for sst₁ or sst₄. Until the recent report by Bass and co-workers (Mol. Pharmacol. 1996, 50, 709-715; erratum Mol. Pharmacol. 1997, 51, 170), no true antagonists of somatostatin had been discovered, let alone any displaying differential receptor subtype selectivity. In this present study, we further explore the effect of this putative L,⁵D⁶ antagonist motif on somatostatin octapeptide analogues with a cyclic hexapeptide core. The most potent antagonist found to date is H- Cpa-cyclo[DCys-Tyr-DTrp-Lys-Thr-Cys]-Nal-NH₂, PRL-2970 (21), which has an IC₅₀ of 1.1 nM in a rat pituitary growth hormone in vitro antagonist assay versus SRIF (1 nM). This analogue bound to cloned human somatostatin subtype 2 receptors with a K(i) of 26 nM. The highest hsst₂ affinity analogue was H- Cpa-cyclo-[DCys-Pal-DTrp-Lys-Tle-Cys]-Nal-NH₂, PRL-2915 (15), with a K(i) of 12 nM (IC₅₀ = 1.8 nM). This analogue was also selective for hsst₂ over hsst₃ and hsst₅ by factors of 8 and 40, respectively, and had no agonist activity when tested alone at concentrations up to 10 μM. Regression analysis of the binding affinities versus the observed antagonist potencies revealed high correlations for hsst₂ (r = 0.65) and hsst₃ (r = 0.52) with a less significant correlation to hsst₅ (r = 0.40). This is quite different from the somatostatin agonist analogues which show a highly significant correlation to hsst₂ (r > 0.9). Receptor-selective somatostatin antagonists should provide valuable tools for characterizing the many important physiological functions of this neuropeptide.

Full text of DOI:10.1021/jm9806289

© Copyright 1999 by the American Chemical Society
Volume 42, Number 11
J une 3, 1999
Articles
High ly P oten t Cyclic Disu lfid e An ta gon ists of Som a tosta tin
Simon J . Hocart,* Rahul J ain,† William A. Murphy, J ohn E. Taylor,‡ and David H. Coy
Peptide Research Laboratories, Tulane University School of Medicine, New Orleans, Louisiana 70112, and
Biomeasure Inc., Milford, Massachusetts 01757
Received November 5, 1998
The search for synthetic analogues of somatostatin (SRIF) which exhibit selective affinities
for the five known receptor subtypes (sst1-5) has generated a large number of potent agonist
analogues. Many of these agonists display good subtype selectivities and affinities for the
subtypes 2, 3, and 5, with very few selective for sst1 or sst4. Until the recent report by Bass
and co-workers (Mol. Pharmacol. 1996, 50, 709-715; erratum Mol. Pharmacol. 1997, 51, 170),
no true antagonists of somatostatin had been discovered, let alone any displaying differential
receptor subtype selectivity. In this present study, we further explore the effect of this putative
6
L,5D antagonist motif on somatostatin octapeptide analogues with a cyclic hexapeptide core.
The most potent antagonist found to date is H-Cpa-cyclo[DCys-Tyr-DTrp-Lys-Thr-Cys]-Nal-
NH2, PRL-2970 (21), which has an IC50 of 1.1 nM in a rat pituitary growth hormone in vitro
antagonist assay versus SRIF (1 nM). This analogue bound to cloned human somatostatin
subtype 2 receptors with a Ki of 26 nM. The highest hsst2 affinity analogue was H-Cpa-cyclo-
[DCys-Pal-DTrp-Lys-Tle-Cys]-Nal-NH2, PRL-2915 (15), with a Ki of 12 nM (IC50 ) 1.8 nM).
This analogue was also selective for hsst2 over hsst3 and hsst5 by factors of 8 and 40, respectively,
and had no agonist activity when tested alone at concentrations up to 10 µM. Regression
analysis of the binding affinities versus the observed antagonist potencies revealed high
correlations for hsst2 (r ) 0.65) and hsst3 (r ) 0.52) with a less significant correlation to hsst5
(r ) 0.40). This is quite different from the somatostatin agonist analogues which show a highly
significant correlation to hsst2 (r > 0.9). Receptor-selective somatostatin antagonists should
provide valuable tools for characterizing the many important physiological functions of this
neuropeptide.
In tr od u ction
secretion of glucagon and insulin2 and the secretion of
gastrin from the gut.3 Additionally, SRIF acts as a
neurotransmitter or neuromodulator in the central
nervous system and peripheral tissues where it modu-
lates neuronal firing,4,5 release of other neurotransmit-
ters,6,7 motor activity,8 and cognitive processes.9 These
biological effects of SRIF, mostly inhibitory in nature,
are elicited through a series of G protein-coupled,
transmembrane receptors, of which five different recep-
tor subtypes have been characterized (sst1-5).10 These
five subtypes have similar affinities for the endogenous
The tetradecapeptide somatostatin (SRIF)1 is a potent
regulator of multiple biological functions. Although
SRIF was originally isolated from mammalian hypo-
thalamii and characterized as a potent physiological
inhibitor of growth hormone (GH) secretion from the
anterior pituitary, SRIF also inhibits the pancreatic
† Current address: Department of Medicinal Chemistry, National
Institute of Pharmaceutical Education and Research, Sector 67, S.A.S.
Nagar (Mohali), 160062 India.
‡ Biomeasure Inc.
10.1021/jm9806289 CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/06/1999

1864 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Hocart et al.
SRIF ligands but have differing distributions in various
tissues.11 The development of smaller, potent SRIF
agonists led to the discovery of differing affinities of the
various shortened ligands for the different receptor
subtypes.12,13 These selective agonists are currently
being used to probe the physiological role of each of the
receptors.14-16 Thus far, of the five receptor subtypes
for somatostatin, only sst2 and sst5 have been associated
with specific physiological functions. SST2 has a pre-
dominant role in mediating the release of GH as
evidenced by the positive correlation between the bind-
ing affinities of a large number of small agonist ana-
logues and their ability to inhibit GH secretion from
cultured rat anterior pituitaries.13 Inhibition of insulin
secretion from rat pancreatic islets is thought to be
mediated through sst5.17,18 However, elucidating the
physiological role of each of the receptor subtypes would
be greatly enhanced by the development of receptor
subtype-specific antagonists.11 Also, SRIF antagonists
might provide a novel route to increasing endogenous
levels of some hormones, notably growth hormone and
insulin.
in octapeptides containing a cyclic hexapeptide core and
demonstrate enhanced biological activity.
Resu lts a n d Discu ssion
Since the discovery of the tetradecapeptide hormone
somatostatin,1 much work has been expended to ascer-
tain the minimum structural requirements for biological
activity. This has resulted in a large number of small
peptide analogues, most of which are agonists.13,25-29
Although the native tetradecapeptide hormone binds to
each of the five receptor subtypes (sst1-5) with ap-
proximately equal affinity, the small peptide analogues
display markedly differing affinities for the five known
somatostatin receptors. Almost all of these analogues
contain the critical sequence DTrp8-Lys9, which lies in
the center of a â-bend and has been shown to be crucial
for receptor recognition. Most of these small analogues
are cyclic, and the majority of the structure-activity
relationships of these analogues can be explained in
terms of changes in the intracyclic bridging region. A
variety of cyclization schemes have been used to produce
analogues with differing pharmacological profiles in-
cluding: proline analogues and homologues,30 â and
higher homologous amino acids,26 modifications to cys-
tine disulfide bridging, including monosulfides31 and
trisulfides,32 and side-chain to side-chain bridging.33
Previously, we described a new series of linear soma-
tostatin analogues.13 These linear analogues have been
shown to maintain the critical â-bend conformation via
hydrophobic interactions between the terminal aromatic
residues, by low-temperature NMR spectroscopy.34
Some years ago, we reported the first partial antago-
nist of somatostatin: cyclo[Ahp-Phe-DTrp-Lys-Thr-
(Bzl)].19 This analogue was a weak antagonist at low
doses, stimulating growth in female rats,20 but was an
agonist at higher doses. However, the analogue does not
bind to cloned human sst receptors with any appreciable
affinity.21 However, a recent observation by Bass and
co-workers has appeared to realize the goal of pure
somatostatin antagonists.22,23 They noted that a weak
linear octapeptide agonist we reported recently contain-
ing 4-nitrophenylalanine (Npa), H-DPhe5-Npa6-Tyr7-
DTrp8-Lys9-Val10-Phe11-Thr12-NH2 (SRIF numbering),13
had a high affinity for sst2 but a weak effect on growth
hormone levels and thus was a good candidate from
which to derive an antagonist. From this compound they
designed disulfide-cyclized analogues employing Npa
and inverted chirality in positions 5 and 6 relative to
the agonists. These compounds were shown to antago-
nize the action of SRIF in a cAMP accumulation assay
and the somatostatin-stimulated growth of yeast cells
expressing the sst2 subtype. Of these antagonists, Ac-
Npa-cyclo[DCys-Tyr-DTrp-Lys-Thr-Cys]-DTyr-NH2 had
an affinity for sst2 comparable with that of the native
hormone.22,23 In our assay systems, the Bass analogue,
H -N p a -cyclo[D Cys -Tyr -D Tr p -Lys -Va l-Cys ]-Tyr -
NH2,22,23 gave an in vitro rat antagonist IC50 of 50 ( 18
nM and Ki’s for hsst2 and hsst5 of 21 ( 5 and 98 ( 24
nM, respectively.24 Recently, we reported on the general
Recently, an observation by Bass and co-workers22,23
led to the first competitive antagonists of somatostatin.
They described the modification of a weak agonist with
high sst2 affinity to an antagonist by the inversion of
chirality at positions 5 and 6.22,23 In a previous study,21
5
6
we explored the effect of this putative L ,D antagonist
motif on various series of somatostatin agonist ana-
logues, both linear and cyclic. It was found that many
5
6
D ,L agonists could be converted into competitive an-
tagonists by applying this motif, the most potent of
which was H-Nal-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-
Nal-NH2 (compound 1; see Table 1). This antagonist had
poor selectivity for hsst2 with a binding affinity (Ki) of
81 nM and an IC50 of 15 nM against SRIF (1 nM) in a
rat in vitro antagonist bioassay (see Table 2). In
contrast, the most potent linear antagonist, H-Nal-
DCpa-Tyr-DTrp-Lys-Val-Phe-Thr-NH2, was 200-fold less
potent in the in vitro antagonist assay.21 To enhance
the potency and selectivity of somatostatin antagonists
and refine the structure-activity relationships, we
decided to investigate further our most potent cyclic
analogue, an octapeptide containing a cyclic hexapeptide
core (compound 1, see Table 1).
5
6
applicability of this putative L ,D antagonist motif on
somatostatin analogues of various topologies, both linear
and cyclic, but not containing Npa,21 the presence of
which was implied to be important for antagonism.22,23
It was found that Npa is not a structural requirement
for antagonism. It was also demonstrated that, indeed,
the above motif is capable of converting agonists with
different topologies into competitive antagonists with
varying affinities and potencies.21 The antagonists
derived from cyclic agonists were found to be consis-
tently more potent than those derived from linear
sequences.21 In this paper, we further explore the SRIF
antagonist paradigm by refining this structural motif
P osition 10 in Na l5,12 An a logu es. Previous modi-
fications to the critical â-bend region at position 7 led
to a marked increase in antagonist potency by the
replacement of Tyr by Pal.21 However, another critical
residue in the â-bend region is located at position 10.
In most somatostatin analogues published, this residue
is usually Val or the sterically similar, but hydrophilic,
Thr. Hence, we chose to investigate the effect of side-
chain steric bulk on the â-bend region by modifying
position 10. Replacement of Val with Gly (compound 2)

Cyclic Disulfide Antagonists of Somatostatin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1865
Ta ble 1. Analogue Amino Acid Sequences and Analytical Data
mass spectrum M - H+
HPLCc
d
e
no.
code
peptide sequence
calcda
obsdb
tR-1
tR-2
21
1
2
3
4
5
6
7
8
9
DC-38-48
PRL-2876
PRL-2874
PRL-2877
PRL-2879
PRL-2875
PRL-2889
PRL-2900
PRL-2891
PRL-2908
PRL-2882
PRL-2904
PRL-2903
PRL-2910
PRL-2915
PRL-3020
PRL-3052
PRL-3023
PRL-3024
PRL-2972
PRL-2970
PRL-2859
PRL-2872
PRL-2888
PRL-2858
PRL-2869
PRL-2857
PRL-2868
PRL-2894
PRL-2917
PRL-2918
PRL-2905
PRL-2907
PRL-2856
PRL-2862
PRL-3064
PRL-2855
PRL-2870
PRL-2902
PRL-2896
PRL-2878
PRL-2897
PRL-2898
PRL-2883
H-Nal-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-Nal-NH2
H-Nal-cyclo[DCys-Pal-DTrp-Lys-Gly-Cys]-Nal-NH2
H-Nal-cyclo[DCys-Pal-DTrp-Lys-Ala-Cys]-Nal-NH2
1137.4
1149.9
1193.1
1193.0
1195.2
1193.2
1232.6
1319.1
1181.3
1146.9
1159.1
1159.1
1162.7
1176.7
1164.8
1190.9
1164.8
1164.8
1163.8
1179.8
1114.3
1104.7
1116.8
1114.3
1104.2
1114.9
1103.7
1147.2
1161.1
1163.5
1236.0
1333.2
1259.0
1248.7
1257.9
1257.9
1261.8
1128.3
1133.0
1117.3
1204.1
1219.7
1149.4
1136.5
1150.5
1192.6
1192.6
1192.6
1192.6
1233.6
1319.4
1181.5
1146.4
1160.5
1160.5
1162.8
1176.9
1164.5
1189.6
1164.3
1165.7
1163.9
1180.5
1114.3
1103.3
1114.3
1114.3
1103.3
1114.3
1103.3
1147.1
1161.2
1161.2
1236.1
1330.1
1258.1
1247.1
1258.1
1258.1
1259.6
1128.3
1130.5
1117.4
1204.5
1219.5
1146.4
30.2
31.4
34.8
34.9
33.8
33.2
48.1
42.0
30.5
29.8
31.2
31.1
31.1
31.9
29.4
34.0
30.3
31.4
38.6
33.8
26.0
25.3
25.6
25.9
25.1
24.4
23.6
29.7
30.8
30.7
30.6
32.2
31.7
31.0
31.4
33.8
44.2
22.6
28.8
31.8
35.5
41.2
28.7
26.2
28.9
35.8
36.5
36.6
37.5
52.3
42.5
34.8
30.7
34.3
39.0
32.4
35.9
42.9
54.8
47.2
51.2
58.1
48.4
24.5
14.3
26.2
24.4
13.9
22.0
11.9
28.5
34.4
35.9
31.7
34.1
32.8
23.6
52.2
35.6
45.7
19.6
30.3
26.5
37.6
41.5
28.1
H-Nal-cyclo[DCys-Pal-DTrp-Lys-Leu-Cys]-Nal-NH2
H-Nal-cyclo[DCys-Pal-DTrp-Lys-Nle-Cys]-Nal-NH2
H-Nal-cyclo[DCys-Pal-DTrp-Lys-Ile-Cys]-Nal-NH2
H-Nal-cyclo[DCys-Pal-DTrp-Lys-Tle-Cys]-Nal-NH2
H-Nal-cyclo[DCys-Bta-DTrp-Lys-Val-Cys]-Nal-NH2
H-Nal-cyclo[DCys-3ITyr-DTrp-Lys-Val-Cys]-Nal-NH2
H-Trp-cyclo[DCys-Pal-DTrp-Lys-Tle-Cys]-Nal-NH2
H-Fpa-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-Nal-NH2
H-DFpa-cyclo[DCys-Pal-DTrp-Lys-Tle-Cys]-Nal-NH2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
35
H-Fpa-cyclo[DCys-Pal-DTrp-Lys-Tle-Cys]-Nal-NH2
H-Cpa-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-Nal-NH2
H-Cpa-cyclo[DCys-Pal-DTrp-Lys-Tle-Cys]-Nal-NH2
H-Cpa-cyclo[DCys-Pal-DTrp-Lys-Thr-Cys]-Nal-NH2
H-Cpa-cyclo[DCys-Pal-DTrp-Lys-Tba-Cys]-Nal-NH2
f
H-Cpa-cyclo[DCys-2Pal-DTrp-Lys-Thr-Cys]-Nal-NH2
f
H-Cpa-cyclo[DCys-D2Pal-DTrp-Lys-Thr-Cys]-Nal-NH2
H-Cpa-cyclo[DCys-Phe-DTrp-Lys-Thr-Cys]-Nal-NH2
H-Cpa-cyclo[DCys-Tyr-DTrp-Lys-Thr-Cys]-Nal-NH2
H-Fpa-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-Fpa-NH2
H-Fpa-cyclo[DCys-His-DTrp-Lys-Val-Cys]-Fpa-NH2
H-DFpa-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-DFpa-NH2
H-3Fpa-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-3Fpa-NH2
H-3Fpa-cyclo[DCys-His-DTrp-Lys-Val-Cys]-3Fpa-NH2
H-2Fpa-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-2Fpa-NH2
H-2Fpa-cyclo[DCys-His-DTrp-Lys-Val-Cys]-2Fpa-NH2
H-Cpa-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-Cpa-NH2
H-Cpa-cyclo[DCys-Pal-DTrp-Lys-Tle-Cys]-Cpa-NH2
H-DCpa-cyclo[DCys-Pal-DTrp-Lys-Tle-Cys]-DCpa-NH2
H-Bpa-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-Bpa-NH2
H-Iph-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-Iph-NH2
H-Pfp-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-Pfp-NH2
H-Pfp-cyclo[DCys-His-DTrp-Lys-Val-Cys]-Pfp-NH2
f
H-Pfp-cyclo[DCys-2Pal-DTrp-Lys-Val-Cys]-Pfp-NH2
f
H-Pfp-cyclo[DCys-D2Pal-DTrp-Lys-Val-Cys]-Pfp-NH2
H-Bip-cyclo[DCys-Tyr-DTrp-Lys-Ile-Cys]-Bip-NH2
H-Ypa-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-Ypa-NH2
H-Igl-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-Igl-NH2
H-Tic-cyclo[DCys-Tyr-DTrp-Lys-Val-Cys]-Tic-NH2
H-Nal-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-DDip-NH2
H-Nal-cyclo[DCys-Tyr-DTrp-Lys-Val-Cys]-DDip-NH2
H-Nal-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-Fpa-NH2
a
b
Theoretical molecular weight (M - H+, Da). Observed molecular weight (M - H+, Da). c Reversed-phase HPLC (C-18, 5 µm, 4.6 ×
d
250 mm, λ ) 215 nm) retention times (min). Each compound was found to have a purity of >97% in both HPLC systems. HPLC-1
elution system: A, 0.1% TFA; B, 0.1% TFA in 80% MeCN; 10% B to 70% B at 1% min- 1 and 1.5 mL min-1 e HPLC-2 elution system: C,
.
5% MeCN in TEAP (0.1 M, pH 3); D, 20% C in MeCN, 20% D to 80% D at 1% min-1 and 1.5 mL min-1
chirality.
.
f Tentative assignment of 2Pal
was accompanied by a large (100-fold) reduction in in
vitro potency to 1.5 µM, though with only a halving of
affinity for hsst2 (see Table 2). Ala10 (compound 3)
caused a smaller loss in antagonist activity than Gly
(IC50 ) 425 nM), with a 2.5-fold reduction in affinity
for hsst2, and markedly less affinity for hsst5 (3, Ki )
1860 nM). The substitution of Leu in position 10 (4) was
accompanied by a decrease in antagonist activity (IC50
) 48 nM) and a marked reduction in affinity for hsst2
relative to compound 1. Nle10 (5) resulted in a similar
IC50 to that of the Leu10 analogue but restored the
affinity for hsst2 and hsst3. Replacement of Val10 with
Ile (6) gave an antagonist with potency and affinity for
hsst3 similar to that of the parent compound (1) al-
though it abolished affinity for hsst2. The unusual amino
acid tert-leucine (Tle10) caused the antagonist potency
to double relative to Val10 (7, IC50 ) 8.4 nM), and the
affinity for hsst2 and hsst4 was increased. In an exten-
sion of the previous studies involving aromatic modifi-
cations to position 7,21 the replacement of Pal in
analogue 1 with the sulfur analogue of tryptophan (Bta)
was accompanied by marked reductions in IC50 and Ki
for hsst2, hsst3, and hsst5 (compound 8). The insertion
of a nonradioactive version of the moiety frequently
generated in radiolabeling peptides, 3-I-Tyr7 (9), in place
of Pal7 in compound 1 retained the antagonist activity,
but the binding affinities for types 2 and 3 were
approximately halved.
Ar om a tic5,Na l12 An a logu es. In the agonist series
of analogues with similar topology, the presence of large
hydrophobic aromatic amino acids in position 5 is
important for enhanced biological activity.13 Addition-
ally, many analogues also contain a hydrophobic aro-
matic amino acid at position 12. In a previous paper,21
we reported that although both the agonists and an-
tagonists share a propensity for terminal hydrophobic
amino acids, the preferred chirality differs. In general,
agonists of this topological class prefer a D amino acid
in position 5, whereas the antagonists favor L amino
acids in the same location. To study the effect of steric

1866 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Hocart et al.
Ta ble 2. Binding Affinities (Ki) for the Cloned Human sst1-5 Receptors and Rat Antagonist IC50 Data versus Substitution at
Positions 5, 7, 10, and 12 in H-W5-cyclo[DCys-X7-DTrp-Lys-Y10-Cys]-Z12-NH2
antagonist
IC50 ( SEM (n)b
(nM)
substitution
Kia ( SEM (nM)
no.
5
7
10
12
hsst1
hsst2
hsst3
hsst4
>1000
1570
>1000
728
672
1040
145
hsst5
1
2
3
4
5
6
7
8
9
Nal
Nal
Nal
Nal
Nal
Nal
Nal
Nal
Nal
Trp
Fpa
DFpa
Fpa
Cpa
Cpa
Cpa
Cpa
Cpa
Cpa
Cpa
Cpa
Fpa
Fpa
DFpa
3Fpa
3Fpa
2Fpa
2Fpa
Cpa
Cpa
DCpa
Bpa
Iph
Pal
Val
Gly
Ala
Leu Nal
Nle
Ile
Tle
Val
Val
Tle
Val
Tle
Tle
Val
Tle
Thr
Nal
Nal
Nal
348 ( 158
413 ( 73
81 ( 12
163 ( 11
203 ( 30
543 ( 11
76 ( 16
171 ( 46
192 ( 24
379 ( 30
243 ( 54
126 ( 13
152 ( 71
371 ( 125
524 ( 175
15 ( 3.6 (5)
382 ( 214 1510 ( 425 (2)
Pal
Pal
Pal
Pal
Pal
Pal
Bta
3ITyr
Pal
Pal
Pal
Pal
Pal
Pal
Pal
Pal
2Pale
1200 ( 196
415 ( 76
1860
968 ( 166
814
847 ( 94
631 ( 98
1400
468 ( 117
2250
287
260
535 ( 116
240
425 ( 25 (2)
48 ( 21 (5)
38 ( 11 (3)
12 ( 5.8 (3)
8.4 ( 0.9 (4)
403 ( 144 (3)
20 (1)
Nal
Nal
Nal
Nal
Nal
Nal
Nal
Nal
Nal
Nal
Nal
Nal
846 ( 639
172 ( 54
>1000
>1000
35 ( 5.5
418 ( 112
194 ( 0.5
84 ( 6.2
35 ( 8.1
132 ( 18
26 ( 3.1
25 ( 9.7
12 ( 1.3
12 ( 2.2
166 ( 66
14 ( 1.1
233 ( 2.0
51 ( 18
>1000
>1000
>1000
484
709 ( 328 >1000
482 ( 142 >1000
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
210 ( 42
>1000
32 ( 3.0 (2)
6.5 ( 1.8 (4)
20 ( 7.5 (2)
2.5 ( 0.6 (11)c
2.6 ( 0.9 (4)
1.8 ( 0.5 (5)c
2.6 ( 0.7 (3)
125 ( 15 (2)
2.7 ( 1.0 (3)
135 ( 41 (2)
1.4 ( 0.2 (7)
1.1 ( 0.2 (7)
87 ( 36 (5)
105 ( 5 (2)
74 ( 19 (3)
77 ( 28 (3)
137 ( 22 (3)
114 ( 17 (3)
393 ( 92 (3)
3.0 ( 0.8 (6)
5.9 ( 1.6 (3)
71 ( 13 (3)
2.7 ( 0.4 (4)
4.0 ( 1.0 (6)
292 ( 135 (5)
271 ( 185 >1000
555 ( 16 888
>1000
>1000
>1000
>1000
1400
238
1030
1690
443 ( 202
>1000
435 ( 322
1950 ( 1190
>1000
231 ( 102 >1000
85 ( 1.2
100 ( 57
38 ( 2.4
175
53 ( 1.3
108 ( 60
290 ( 56
93 ( 4.8
120 ( 2.5
269 ( 87
1590
895
520
>1000
140 ( 4.6
110 ( 34
70 ( 12
204 ( 49
87 ( 50
48 ( 11
369
173
694
748
734
Tba Nal
Thr
ndd
Nal
Nal
Nal
Nal
Fpa
Fpa
DFpa
3Fpa
3Fpa
2Fpa
2Fpa
Cpa
Cpa
DCpa
Bpa
Iph
Pfp
Pfp
Pfp
Pfp
Bip
Ypa
Igl
>1000
D2Pale Thr
nd
Phe
Tyr
Pal
His
Pal
Pal
His
Pal
His
Pal
Pal
Pal
Pal
Pal
Pal
His
2Pald
Thr
Thr
Val
Val
Val
Val
Val
Val
Val
Val
Tle
Tle
Val
Val
Val
Val
Val
>1000
>1000
>1000
847
26 ( 6.8
20 ( 2.7
31 ( 9.5
106 ( 9.6
117 ( 48
103 ( 25
241 ( 11
176 ( 88
32 ( 4.3
19 ( 2.6
56 ( 6.0
16 ( 4.8
32 ( 3.7
633 ( 148
637 ( 71
838 ( 665 >1000
495 ( 174 >1000
397 ( 193 >1000
82 ( 13
60 ( 16
186 ( 45
37 ( 9.5
385 ( 72
>1000
1580
382 ( 181 >1000
972 ( 411
139 ( 50
140
153
66
>1000
>1000
>1000
51 ( 42
58 ( 13
301
1640
2880
3310
436
1190 ( 321
57 ( 19
374 ( 114
575 ( 155
525 ( 199
69 ( 12
18 ( 4.3
111 ( 20
124 ( 48
170 ( 25
93 ( 32
48 ( 25
960
Pfp
Pfp
Pfp
Pfp
Bip
Ypa
Igl
176
>1000
926 ( 228 2410 ( 880 (3)f
568 ( 422
34 ( 11
>1000
692 ( 78
nd
nd
918 ( 342
1380 ( 383
689
765 ( 82 (2)
D2Pald Val
nag
Tyr
Pal
Pal
Tyr
Pal
Tyr
Pal
Ile
>1000
>1000
736 ( 265
1030 ( 26
2230 ( 1230 1350 ( 176 >1000
2110 ( 1150 (3)
51 ( 25 (3)
81 ( 60 (2)
nah
91 ( 29 (3)
65 ( 23 (3)
150 ( 50 (2)
Val
Val
Val
Val
Val
Val
99 ( 6.4
252 ( 31
345 ( 73
37 ( 4.7
16 ( 0.6
69 ( 6.2
103 ( 77
568 ( 108
>1000
682 ( 318
304
469 ( 421
Tic
Tic
802 ( 120 >1000
1600 ( 599 >1000
1560 ( 557 >1000
>1000
Nal
Nal
Nal
DDip
DDip
Fpa
>1000
725
461
376
>1000
1060
166 ( 41
>1000
a
b
Expressed as the mean ( SEM; single values indicate the results of one binding experiment. Rat in vitro antagonist IC50 (nM)
versus SRIF (1.0 nM), expressed as the mean ( SEM of n separate dose-response curves. c No agonist activity when tested alone at
concentrations up to 10 µM. Not determined. e Tentative assignment of 2Pal chirality. f Not a full antagonist at doses up to 10 µM.
d
g
h
Not an antagonist at doses up to 10 µM. Not an antagonist at doses up to 100 µM; found to be an agonist with IC50 ) 403 nM.
bulk at the N-terminal exocyclic position, we made a
series of Nal12 analogues containing a variety of aro-
matic amino acids in position 5.
20, respectively.35 The introduction of Cpa5 in place of
Nal5 (14) resulted in a potent analogue with an IC50 of
2.6 nM with increased affinity for hsst2, hsst3, and hsst5
relative to the parent analogue 1. As in the case of the
Nal12 analogues (1 and 7), the incorporation of Tle10 with
Cpa5 (15) led to an improvement in hsst2 affinity, but
the IC50 was not improved significantly. Cpa5,Thr10 (16)
was equipotent with Cpa5,Val10 (14) in the antagonist
assay, although its hsst2 binding affinity was improved
by a factor of 2. Substitution of the bulky tert-butyl-
alanine in position 10 (17) led to a marked reduction in
antagonist activity and affinity for hsst2, although the
affinities for hsst1 and hsst5 were improved relative to
Val10 (14).
The substitution of Trp for Nal5 with Tle10 (10) caused
a 4-fold loss of antagonist activity and affinity for hsst5
with a smaller reduction in affinity for hsst2 relative to
parent compound 7. The placement of Fpa5 (11) in
compound 1 instead of Nal5 caused a halving in IC50 to
6.5 nM, and the affinity for hsst2 was increased to 35
nM. Inversion of the chirality of Fpa5 (12) was ac-
companied by a 3-fold loss of antagonistic activity as
expected when compared with the L analogue 12 (IC50
) 20 nM). The additional replacement of Val10 by Tle10,
together with Fpa5 (13), was accompanied by a decrease
in IC50 to 2.5 nM and a slight increase in hsst2 affinity
relative to the parent analogue 7. This compound (13)
was the first antagonist to show some selectivity for
hsst2, with hsst2/hsst3 and hsst2/hsst5 Ki ratios of 8 and
3-Pyridylalanine in position 7 was shown in the
previous paper to be superior to Tyr7 in terms of the
antagonistic potency.21 Interestingly, during the syn-
thesis of an o-Pal analogue Cpa5,2Pal7,Thr10 (18), the
incorporation of Boc-2Pal was accompanied by an

Cyclic Disulfide Antagonists of Somatostatin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1867
intense ninhydrin-like blue coloration during the coup-
ling. The coloration washed out following the completion
of the reaction, and the synthesis completed as usual.
HPLC chromatography of the cleaved, crude peptide
showed the presence of two closely eluting peaks with
identical peak areas and molecular weights, suggesting
racemization had occurred during the coupling of 2Pal.
However, these diastereoisomers were separated readily
during the standard chromatographic purification step.
The optical configuration of each diastereoisomer was
tentatively inferred from a comparison of the HPLC
elution behaviors with two model pairs of somatostatin
analogues, synthesized separately as diastereoisomers
of known optical configuration,36 and of the observed
biological activities. In each model case, the L configu-
ration diastereoisomer eluted before the D form from a
TFA/C-18 HPLC system. The faster eluting analogue,
tentatively identified as the L2Pal7 diastereoisomer (18),
was equipotent with Pal7 (16), and the affinity for hsst2
was not significantly different. The slower eluting
analogue, tentatively identified as the D2Pal7 diastereo-
isomer (19), demonstrated a major loss of antagonism
(IC50 ) 135 nM) with reductions in the affinities for all
the receptor subtypes. Replacement of Pal in position 7
with Phe (20) in the Cpa5,Thr10 parent analogue (16)
was accompanied by a halving of the IC50 to 1.4 nM and
a 4-fold drop in affinity for hsst2 to 51 nM. The
substitution of Tyr7 (21) produced a highly active
antagonist with an IC50 of 1.1 nM and increased affinity
for hsst5, though with one-half the affinity for hsst2 as
Pal7 (16).
the affinity for hsst3. Bromophenylalanine substituted
in both terminal positions (32) gave an analogue with
a similar IC50, but with increased affinity for hsst2 and
hsst5 relative to the Cpa analogue (29). Iodophenylala-
nine (33) also gave a similar IC50 to the Cpa analogue
(29). However, this analogue had the highest affinities
for hsst3 and hsst5 of the halogenated phenylalanine
analogues.
Incorporation of the hydrophobic amino acid pen-
tafluorophenylalanine (Pfp) in positions 5 and 12 (34)
produced a poor antagonist (IC50 ) 292 nM) compared
with the Nal parent compound (1). The analogue
displayed weak affinity for receptor subtypes 3 and 4,
with little affinity for the other subtypes. Replacement
of Pal7 by His (35) further reduced the antagonism such
that the analogue could not fully block the action of
SRIF at the doses tested (up to 10 µM). However,
affinity for hsst1 was markedly improved. Incorporation
of Boc-2Pal in this Pfp5,12 analogue was again ac-
companied by racemization, leading to a pair of dia-
stereoisomers, which were readily separated by HPLC.
Chirality was tentatively assigned as before. The faster
eluting analogue, tentatively assigned as L2Pal7 (36),
gave a weak antagonist analogue which bound weakly
to hsst3 but did not bind to hsst2. The slower eluting
analogue, tentatively assigned as D2Pal7 (37), produced
a compound which, as expected, was not an antagonist
at doses up to 10 µM. The bulky biphenylalanine (Bip)
in both terminal positions with Ile10 (38) produced a very
weak antagonist (IC50 ) 2.1 µM) with little affinity for
any of the receptor subtypes. Ypa5,12 (39) gave a modest
antagonist with an IC50 of 51 nM. Substitution of Igl in
both positions (40) produced a moderate antagonist with
an IC50 of 81 nM but with weak affinity for all receptor
types. Unexpectedly, the constrained Trp analogue,
Tic5,12,Tyr7 (41), was a feeble agonist (IC50 ) 403 nM)
with weak affinity for hsst2. This compound exhibited
no antagonism at the highest dose tested (100 µM).
Ar om a tic5,12 An a logu es. To probe the effect of steric
bulk at both exocyclic positions, we made a series of
analogues, doubly substituted with various aromatic
amino acids of differing sizes. The substitution of
fluorophenylalanine (Fpa) in both exocyclic positions 5
and 12 with Pal7 (22) produced a moderately active
antagonist with an IC50 of 87 nM. Replacement of Pal
by His (23) had little effect on the antagonism and
affinity for hsst2, while markedly reducing the affinity
for hsst1. Inversion of the chirality of both Fpa moieties
together with Pal7 (24), also had little effect on antago-
nism but noticeably reduced affinities for hsst2, hsst3,
and hsst5. Incorporation of the m-Fpa isomer, 3Fpa, in
the terminal positions 5 and 12 (25) also produced an
analogue with a similar antagonist activity to Fpa5,12
(22), but inferior hsst2, hsst3, and hsst5 affinities.
However, the affinity for hsst1 was much improved to
82 nM. The additional incorporation of His7 with
In the previous paper, we investigated the effect of
the branched amino acid diphenylalanine in position 5.21
Substitution of DDip at position 12 with Nal5 gave an
antagonist with an IC50 of 91 nM and an affinity for
hsst2 of 37 nM (42), slightly more active than the His7
analogue reported in the previous paper.21 The affinity
for sst2 doubled, but the antagonism was unchanged on
the additional substitution of Tyr7 (43). The incorpora-
tion of Fpa12 with Nal5 (44) reduced the antagonism by
20-fold from the Fpa5,Nal12 analogue (11) demonstrating
a distinct preference for a larger, unbranched hydro-
phobic moiety at the C-terminus.
3Fpa5,12 (26) again reduced antagonist activity (IC50
)
137 nM) and increased hsst1 affinity. In the case of the
o-Fpa isomer (2Fpa) in positions 5 and 12 (27), the
antagonism was decreased relative to the para analogue
(22). The additional substitution of His7 (28) increased
the affinity for hsst1, although the antagonist activity
was reduced as expected (IC50 ) 393 nM).
Substitution of chlorophenylalanine in both exocyclic
positions (29) produced a 5-fold increase in antagonist
activity (IC50 ) 3 nM) relative to the Nal5,12 parent
compound (1). Replacement of Val10 by Tle10 (30) halved
the antagonism and doubled the affinity for hsst2. The
inversion of the chirality of the Cpa5,12 moieties, together
with Tle10 (31), reduced the antagonism by more than
10-fold relative to the L form (30) and markedly reduced
Cor r ela tion of Bin d in g Affin ities w ith Agon ist
a n d An ta gon ist Activity. Some years ago, we reported
a highly significant correlation (r ) 0.93) between the
binding affinity for cloned mouse sst2 receptors and the
growth hormone release-inhibiting potency of the so-
matostatin agonists in a rat in vitro pituitary cell
assay.13 These agonists have since been reassayed
against both the cloned rat and cloned human sst1-5
receptors, and the correlation results versus the rat in
vitro growth hormone release-inhibiting potencies are
given in Table 3.37 All ill-defined Ki values (e.g. >1000)
were dropped from the correlation analyses. With the
cloned human receptor affinity data, a strong correlation
was found between the rat in vitro growth hormone

1868 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Hocart et al.
Ta ble 3. Correlations between Binding Affinities (Ki) for
Cloned Rat and Human Somatostatin Receptors sst1-5 and Rat
Agonist IC50 Potencies for Agonists and between Cloned
Human Somatostatin Receptors sst1-5 and Rat Antagonist IC50
Potencies for Antagonists
substituted analogues. The chloro, bromo, and iodo
analogues (29, 32, and 33, respectively) gave similar
IC50 values and were 20-fold more potent than the fluoro
analogue (22). Interestingly, the hsst2 binding affinities
for the para-halogenated compounds were quite similar.
o-, m-, and p-fluoro substitution (27, 25, and 22,
respectively) had little effect on the antagonist IC50, but
the para compound (22) had a much greater affinity for
hsst2. Pentafluoro substitution was accompanied by a
marked reduction in hsst2 affinity and antagonism. The
â-branched analogue diphenylalanine (Dip) was the
least active analogue of the group with little binding
affinity for any receptor subtype.
correlation coefficient (r)
receptor
species
sst1
sst2
sst3
sst4
sst5
Agonistsa
0.94
0.91
rat
human
ndc
0.02
0.89
0.14
nd
0.11
0.57
0.35
Antagonistsb
0.65 0.52
human
0.02
0.07
0.40
a
34 agonists taken from ref 13 with recent rat and human
b
receptor subtype binding data from ref 49. Pooled antagonist
A rank ordering of the most potent analogues in the
rat in vitro antagonist assay (IC50 < 5 nM, see Table 5)
is dominated by peptides containing a large hydrophobic
amino acid, most often Nal, at the C-terminus, with a
halogenated phenylalanine analogue at the N-terminus.
The most active compounds (IC50 < 2 nM) contain Nal12
exclusively, with the smaller Cpa at position 5. This is
in contrast to agonist octapeptide analogues, which
frequently contain smaller, or hydrophilic, amino acids
at the C-terminus.13 Although compounds 20 and 21
were found to be the most potent in the rat antagonist
bioassay, they are inferior to compounds 15 and 13 in
terms of their receptor subtype selectivities. These latter
analogues (15 and 13) display selectivities for hsst2 over
hsst3 by a factor of 8 and for hsst2 over hsst5 by factors
of 40 and 20, respectively. The substitutions at positions
7 and 10 are responsible for the differences in selectivity
between the most selective compound (15) and the most
potent antagonists (20 and 21). Analogues 13 and 15
have also been found to be devoid of any agonist activity
when tested alone at concentrations up to 10 µM.
Subtype-selective agonists have now been described for
all receptor subtypes except type 4.38,39 These are the
first potent antagonists to display partial subtype
selectivity. We are continuing to investigate the struc-
ture-activity relationships of the somatostatin antago-
nists to further refine the subtype selectivities and
antagonistic potencies to help explain these receptor
correlation observations. As more selective analogues
are discovered and additional functional subtype-selec-
tive assays developed, the role of each of the receptor
subtypes may be further probed and potential pharma-
ceutical agents developed.
binding data from this paper (41 unique antagonists) and the prior
paper (26 antagonists) in ref 21 (Hocart et al. J . Med. Chem. 1998,
41, 1146-1154). c Not determined.
release-inhibiting potency of the agonists and hsst2 (r
) 0.91) with a less significant correlation to hsst5 (r )
0.35). No significant correlations were found with hsst1,
hsst3, or hsst4 (r’s < 0.16). For the agonist cloned rat
receptor binding affinity data, again the highest cor-
relation was obtained with subtype 2 (r ) 0.94), but
significant correlations were also found with rsst3 and
rsst5 (r ) 0.89 and 0.57, respectively).
In the case of somatostatin antagonists, these current
analogues (41 compounds) and the antagonist analogues
from our prior publication21 (26 antagonists) show a
different pattern of correlations between the rat in vitro
antagonist potencies and the binding affinities for the
cloned human receptor subtypes. These data are sum-
marized in Table 3. The antagonists show a reduced
preference for hsst2 and major correlations for hsst3 and
hsst5 relative to the agonists. This correlation pattern
with the cloned human receptors is more like that found
with rat receptors in the agonist series.
The study of SRIF agonists during the last 2 decades
has produced a large number of agonist analogues of
various ring sizes and some linear compounds.13,25-28
More recently, SRIF agonists and antagonists have both
been developed using cloned human receptor binding
affinities to guide the synthesis of more active and
selective compounds.13,21 This has culminated in the
synthesis of receptor subtype-selective agonist ana-
logues.12,13 The few potent antagonists thus far discov-
ered have not yet produced truly subtype-specific ana-
logues, although as can be seen from this work, hsst2
partially selective analogues are beginning to be des-
cribed. Thus it is not so surprising to find the lack of a
dominant correlation between the observed rat in vitro
antagonist IC50 values and the measured Ki affinities
for any one of the cloned human receptor subtypes. It
is hoped that routine functional assays for all five SRIF
subtypes will be developed and thus provide an invalu-
able aid in the future refinement of more specific
analogues.
Exp er im en ta l Section
Abbr evia tion s. The nomenclature for the somatostatin
receptor subtypes is in accordance with the recommendations
of IUPHAR,40 in which sst4 refers to the receptor originally
cloned by Bruno et al.41 and sst5 refers to the receptor cloned
by O′Carroll et al.17,42 Abbreviations of the common amino
acids are in accordance with the recommendations of IUPAC-
IUB.43 Additional abbreviations: Bip, biphenylalanine; Bpa,
4-bromophenylalanine; Bta, benzothienylalanine; Cpa, 4-chlo-
rophenylalanine; Dip, 3,3-diphenylalanine; Fpa, 4-fluorophe-
nylalanine; 3Fpa, 3-fluorophenylalanine; 2Fpa, 2-fluorophe-
nylalanine; Igl, 2-indanylglycine; Iph, 4-iodophenylalanine; 3-I-
Tyr, 3-iodotyrosine; Nal, 3-(2-naphthyl)alanine; Npa, 4-nitro-
phenylalanine; 2Pal, 2-pyridylalanine; Pal, 3-pyridylalanine;
Pfp, pentafluorophenylalanine; Tba, tert-butylalanine; Tic,
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; Tle, tert-leu-
cine; Ypa, 4-cyanophenylalanine.
Con clu sion
The substitution of the same hydrophobic aromatic
amino acid at both exocyclic positions shows a prefer-
ence for the heavier, halogen-substituted phenylala-
nines (analogues ranked by increasing IC50 are shown
in Table 4). Among the p-halogen-substituted phenyl-
alanines, the fluoro compound (22) was much less active
in the antagonist assay than the heavier halogen-
Ma ter ia ls. 4-Methylbenzhydrylamine hydrochloride resin
(0.41 or 0.29 mequiv g-1) was obtained from Advanced
ChemTech Inc., Louisville, KY. NR-tert-Butyloxycarbonyl (Boc)-

Cyclic Disulfide Antagonists of Somatostatin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1869
Ta ble 4. Effect of the Double Substitution of an Amino Acid at Positions 5 and 12 in H-X5-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-X12-NH2
antagonist
Ki ( SEM (nM)
IC50 ( SEM (n)b
no.a
X
hsst1
hsst2
hsst3
hsst4
3310
301
436
hsst5
65
139 ( 50
48 ( 25
524 ( 175
304
748
469 ( 421
369
(nM)
32
29
33
1
39
25
40
22
27
34
36c
Bpa
Cpa
Iph
Nal
Ypa
3Fpa
Igl
Fpa
2Fpa
Pfp
374 ( 114
16 ( 4.8
32 ( 4.3
32 ( 3.6
81 ( 12
99 ( 6.4
117 ( 48
252 ( 31
20 ( 2.7
241 ( 11
633 ( 148
1410 ( 406
57 ( 19
50 ( 42
18 ( 4.3
2.7 ( 0.4 (4)
3.0 ( 0.8 (6)
4.0 ( 1.0 (6)
15 ( 3.6 (5)
51 ( 25 (3)
77 ( 28 (3)
81 ( 60 (2)
87 ( 36 (5)
114 ( 17 (3)
292 ( 135 (5)
500 ( 120 (2)
>1000
575 ( 155
348 ( 158
>1000
171 ( 45
103 ( 77
495 ( 174
568 ( 108
120 ( 2.5
385 ( 72
111 ( 20
1300 ( 294
>1000
>1000
>1000
82 ( 13
736 ( 265
435 ( 322
186 ( 45
525 ( 199
682 ( 318
>1000
>1000
176
1580
960
2030
Dip
>1000
>1000
a
Compounds are ranked by mean IC50 values. b Antagonist IC50 (nM) versus SRIF (1.0 nM). c Compound 36 (RJ -01-76) from the previous
antagonist paper: Hocart et al. J . Med. Chem. 1998, 41, 1146-1154 (ref 21).
Ta ble 5. Potent Analogues with Rat Antagonist IC50 < 5 nM.
antagonist
substitution
Kib ( SEM (nM)
IC50 ( SEM (n)c
no.a
5
7
10
12
hsst1
>1000
443 ( 202
>1000
>1000
>1000
1400
hsst2
hsst3
hsst4
hsst5
(nM)
21
20
15
13
14
16
32
18
29
33
Cpa
Cpa
Cpa
Fpa
Cpa
Cpa
Bpa
Cpa
Cpa
Iph
Tyr
Phe
Pal
Pal
Pal
Pal
Pal
2Pald
Pal
Pal
Thr
Thr
Tle
Tle
Val
Thr
Val
Thr
Val
Val
Nal
Nal
Nal
Nal
Nal
Nal
Bpa
Nal
Cpa
Iph
26 ( 6.8
51 ( 18
12 ( 1.3
26 ( 3.1
25 ( 9.7
12 ( 2.2
16 ( 4.8
14 ( 1.1
32 ( 4.3
32 ( 3.7
93 ( 4.8
290 ( 56
100 ( 57
231 ( 102
85 ( 1.2
38 ( 2.4
57 ( 19
>1000
>1000
895
>1000
1590
48 ( 11
87 ( 50
520
535 ( 116
240
139 ( 4.6
66
70 ( 12
139 ( 50
48 ( 25
1.1 ( 0.2 (7)
1.4 ( 0.2 (7)
1.8 ( 0.5 (5)
2.5 ( 0.6 (11)
2.6 ( 0.9 (4)
2.6 ( 0.7 (3)
2.6 ( 0.4 (4)
2.7 ( 1.0 (3)
3.0 ( 0.8 (6)
4.0 ( 1.0 (6)
>1000
3310
374 ( 114
1030
53 ( 1.3
51 ( 42
>1000
>1000
301
575 ( 155
18 ( 4.3
436
a
b
All compounds are ranked by mean IC50 values. Expressed as the mean ( SEM; single values indicate the results of one binding
experiment. c Rat in vitro antagonist IC50 (nM) versus SRIF (1.0 nM), expressed as the mean ( SEM of n separate dose-response curves.
Tentative assignment of 2Pal chirality.
d
protected amino acids were purchased from Bachem Inc.,
Torrance, CA, Advanced ChemTech Inc., and Synthetech Inc.,
Albany, OR. The reactive side chains of the amino acids were
masked with one of the following groups: Cys, 4-methylben-
zyloxycarbonyl; His, 3-Bom; Lys, 2-chlorobenzyloxycarbonyl;
Ser and Thr, O-benzyl; Tyr, O-2,6-dichlorobenzyl. All reagents
and solvents were ACS grade or better and used without
further purification.
subjected to lyophilization. Each peptide was obtained as a
fluffy white powder of constant weight by lyophilization from
aqueous acetic acid. The purity of the final peptides was
assessed at 215 nm by analytical RP-HPLC in two systems.
Analytical RP-HPLCs were recorded using a Vydac C-18
support (4.6 × 250 mm, 5 µm, 300 Å pore size, Liquid
Separations Group). The two linear gradient systems were
used at a flow rate of 1.5 mL min-1: HPLC-1, A, 0.1% TFA;
B, 0.1% TFA in 80% MeCN; 10% B to 70% B at 1% min-1; and
HPLC-2, C, 5% MeCN in TEAP (0.1 M, pH 3); D, 20% C in
MeCN, 20% D to 80% D at 1% min-1. Column eluent was
monitored at 215 nm. The retention time and purity of each
peptide were assessed by the Rainin Dynamax HPLC Method
Manager. Each peptide was found to have a purity of g97%.
The HPLC retention time results are given in Table 1.
Am in o Acid An a lysis. The peptides were hydrolyzed in
vacuo (110 °C, 20 h) in 4 M methanesulfonic acid containing
0.2% 3-(2-aminoethyl)indole (Pierce). Amino acid analyses
were performed on the hydrolysates following derivatization
with o-phthaldialdehyde reagent (Sigma Chemical Co.) using
an automatic HPLC system (Rainin Instrument Co.) fitted
with a 100 × 4.6 mm, 3 µm C18 axial compression column
with integral guard column (Microsorb AAAnalysis, Type O;
Rainin Instrument Co.) The derivatized primary amino acids
were eluted using a binary gradient of buffer A, 0.10 M sodium
acetate containing 4.5% v/v methanol and 0.5% v/v tetrahy-
drofuran at pH 7.2, and buffer B, methanol. The gradient
sequence: 0% A at 0 min, 35% A at 16.5 min, 90% A at 30
min, and 90% A at 33 min was used with a flow rate of 1.0
mL min-1 at ambient temperature. Eluent was monitored at
340 nm and integrated by the Dynamax HPLC Method
Manager (Rainin). Standard retention times were as follows:
Asp, 6.6 min; Arg, 19.9 min; Trp, 25.4 min; and Lys, 29.5 min.
Each peptide produced the expected analytical results for the
primary amino acids. Cysteine was not quantified. (Results
not shown.)
P ep tid e Syn th esis. The somatostatin antagonists were
assembled on 4-methylbenzhydrylamine-functionalized, 1%
cross-linked polystyrene resin (0.29 or 0.41 mequiv g-1), in
0.25- or 0.5-mmol scale on a CS Bio Co. (San Carlos, CA; Model
No. CS 136) or an Advanced ChemTech (model 200) synthe-
sizer, using the following protocol: deblocking, 40% TFA (5
min, 19 min); DCM wash cycle (three washes); neutralization,
10% DIEA (1 min, 5 min); DMF wash cycle; DCM wash cycle
(two washes); double coupling; first with 1,3-diisopropylcar-
bodiimide esters (3 equiv), 30 min in DCM; DCM wash (three
washes); second coupling with preformed TBTU esters (3
equiv), 90 min in DMF, with a catalytic amount of DIEA; DMF
wash (one wash); DCM wash (three washes). Coupling reac-
tions were monitored qualitatively with the ninhydrin test.
P ep tid e Clea va ge. The peptides were cleaved from the
resin support with simultaneous side-chain deprotection by
acidolysis using anhydrous hydrogen fluoride containing the
scavengers anisole (∼30% v/v) and dithiothreitol (∼0.6% w/v)
for 45 min at 0 °C. The peptides were cyclized in 90% acetic
acid (∼500 mL) with a slight excess of I2 (15 min). Excess I2
was then removed by the addition of ascorbic acid.
P u r ifica tion . The crude peptides were purified by prepara-
tive RP-HPLC on C-18 bonded silica gel using axial compres-
sion columns (Dynamax-300 Å, 5 or 8 µm, 21.4 × 250 mm). A
linear gradient elution system at a flow rate of 20 mL min-1
was employed: A, 0.1% TFA; B, 0.1% TFA in 80% MeCN; 20%
B to 50% B at 1% min-1. The separations were monitored by
analytical RP-HPLC at 215 nm and TLC on silica gel plates
(Merck F60) with ninhydrin visualization. The fractions
containing the product were pooled, concentrated in vacuo, and
Ma ss Sp ectr om etr y. The peptides were analyzed by
matrix-assisted laser desorption/ionization time-of-flight mass

1870 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Hocart et al.
spectrometry using a LaserMat 2000 mass spectrometer
(Thermal Bioanalysis, San J ose, CA) using R-cyano-4-hydroxy-
cinnamic acid as the matrix with Substance P (1348.7 Da) as
an internal standard. In each case, the spectra consisted of a
major M - H+ ion peak for the internal standard, the expected
analyte M - H+ peak, and a few peaks associated with the
matrix (<500 Da). The results are given in Table 1.
intermediate resuspension in fresh buffer. The final pellets
were resuspended in Tris-HCl (10 mM) for assay. Aliquots of
the membranes were incubated (30 min at 37 °C) with 0.05
nM [125I]Tyr11-SRIF (types 1, 3, 4, 5) or [125I]MK-678 (type 2)
in 50 nM HEPES (pH 7.4) containing BSA (10 mg mL-1),
MgCl2 (5 mM), Trasylol (200 kIU mL-1), bacitracin (0.02 mg
mL-1), and phenylmethanesulfonyl fluoride (0.02 mg mL-1).
The final assay volume was 0.3 mL, and incubations were
terminated by rapid filtration through GF/C filters presoaked
in 0.3% poly(ethylenimine) using a Brandel rapid filtration
module. Each tube and filter were then washed with aliquots
of cold buffer (3 × 5 mL). Specific binding was defined as the
total radioligand bound minus that bound in the presence of
1.0 µM SRIF. The following total radioligand binding and
nonspecific binding (nsb) values were typically obtained with
these assay systems: hsst1, 7000 cpm total versus 3500 cpm
nsb; hsst2, 9000 cpm total versus 1000 cpm nsb; hsst3, 8000
cpm total versus 1000 cpm nsb; hsst4, 6000 cpm total versus
3500 cpm nsb; and hsst5, 7500 cpm total versus 3500 cpm nsb.
The binding affinities expressed as Ki values ( SEM (nM) for
each of the five receptor subtypes are given in Table 2.
An t a gon ism of t h e in Vit r o SR IF In h ib it ion of GH
Relea se. Anterior pituitaries from adult male rats were
collected and dispersed by a previously described trypsin/
DNase method.44 The dispersed cells were diluted with sterile-
filtered Dulbecco’s modified Eagle medium (MEM; Gibco
Laboratories, Grand Island, NY), which was supplemented
with 2.5% fetal calf serum (Gibco), 3% horse serum (Gibco),
10% fresh rat serum (stored on ice for no longer than 1 h) from
the pituitary donors, 1% MEM nonessential amino acids
(Gibco), gentamycin (10 ng mL-1; Sigma), and nystatin (10,000
U mL-1; Gibco). The cells were randomly plated at a density
of approximately 200 000 cells/well (Costar cluster 24; Roch-
ester Scientific Co., Rochester, NY). The plated cells were
maintained in the above Dulbecco’s medium in a humidified
atmosphere of 95% air/5% CO2 at 37 °C for 4-5 days. In
preparation for a hormone challenge, the cells were washed
with medium 199 (Gibco, 3 × 1 mL). Each dose of analogue (6
doses/plate) was tested in the presence of SRIF (1 nM) in
triplicate wells in a total volume of 1 mL of medium 199
containing 1% BSA (fraction V; Sigma Chemical Co.). All wells
contained GHRH(1-29)NH2 (1 nM). A GHRH(1-29)NH2 (1
nM)-stimulated control group and an SRIF (1 nM) with
GHRH(1-29)NH2 (1 nM)-inhibited control group were in-
cluded on each cell culture plate. After the plates were
incubated in an air/carbon dioxide atmosphere (95/5%, 3 h at
37 °C), the medium was removed and stored at -20 °C until
assayed for hormone content. Growth hormone in media was
measured by a standard double-antibody RIA using compo-
nents generously supplied by the NHPP, NIDDK, NICHHD,
and USDA. Antagonist IC50’s versus SRIF (1 nM) were
calculated using Sigmaplot (J andel Scientific, San Rafael, CA).
Values are expressed as the mean IC50 (nM) ( SEM from (n)
separate dose-response curves and are given in Table 2.
Ack n ow led gm en t. We would like to thank Dr.
Barry Morgan of Biomeasure Inc. for his useful discus-
sions and comments, Ms. Etchie Yauger for her excellent
support of this work in performing the purification of
many peptides reported herein and amino acid analysis
of all the peptides, Dr. Ning-Yi J iang and Mr. J oseph
Fuselier for the synthesis and purification of some of
the peptides, Ms. Vienna Mackey for her assistance with
the in vitro antagonist biological assays, and Ms. Karyn
Van Buren for her expeditious administrative support.
Refer en ces
(1) Brazeau, P.; Vale, W.; Burgus, R.; Ling, N.; Butcher, M.; Rivier,
J .; Guillemin, R. Hypothalamic Polypeptide that Inhibits the
Secretion of Immunoreactive Pituitary Growth Hormone. Science
1973, 179, 77-79.
(2) Mandarino, L.; Stenner, D.; Blanchard, W.; Nissen, S.; Gerich,
J .; Ling, N.; Brazeau, P.; Bohlen, P.; Esch, F.; Guillemin, R.
Selective Effects of Somatostatin -14, -25, and -28 on in vivo
Insulin and Glucagon Secretion. Nature 1981, 291, 76-77.
(3) Bloom, S.; Mortimer, C.; Thorner, M.; Besser, G.; Hall, R.;
Gomez-Pan, A.; Roy, U.; Russel, R.; Coy, D.; Kastin, A.; Schally,
A. Inhibition of Gastrin and Gastric Acid Secretion by Growth
Hormone Release-Inhibiting Hormone. Lancet 1974, 2, 1106-
1109.
F u n ction a l Exp r ession of th e Clon ed Hu m a n Som a -
tosta tin Recep tor s. The genomic clones containing the
17,45-48
human somatostatin receptors (hsst1-5
)
were kindly
provided by Dr. Graeme I. Bell (University of Chicago). The
hsst1, hsst2, hsst3, hsst4, and hsst5 cDNAs were isolated as a
1.5-kb PstI-XmnI fragment, 1.7-kb BamHI-HindIII fragment,
2.0-kb NcoI-HindIII fragment, 1.4-kb NheI-NdeI fragment, and
1.2-kb HindIII-XbaI fragment, respectively, each containing
the entire coding region of the full-length receptors. These
fragments were independently subcloned into the correspond-
ing restriction endonuclease sites in the mammalian expres-
sion vector pCMV5, downstream from the human cytomega-
lovirus (CMV) promoter, to produce the expression plasmids
pCMV5/hsst1, pCMV5/hsst2, pCMV5/hsst3, pCMV5/hsst4, and
pCMV5/hsst5. For transfection into CHO-K1 cells, a plasmid,
pRSV-neo (American Type Culture Collection, Rockville, MD),
carrying the neomycin mammalian cell selectable marker was
added.
(4) Renaud, L.; Martin, J .; Brazeau, P. Depressant Action of TRH,
LHRH and SRIF on Activity of Central Neurons. Nature 1975,
255, 233-235.
(5) Dodd, J .; Kelly, J . Is Somatostatin an Excitatory Trans-
mitter in the Hippocampus? Nature 1978, 273, 674-677.
(6) Chesselet, M. F.; Reisine, T. Somatostatin Regulates Dopamine
Release in Rat Striatal Slices and Cat Caudate Nuclei. J .
Neurosci. 1983, 3, 232-236.
(7) Gothert, M. Somatostatin Selectively Inhibits Noradrenaline
Release from Hypothalamic Neurons. Nature 1980, 288, 86-
88.
(8) Havlicek, V.; Rezek, M.; Freisen, H. Somatostatin and Thyrotro-
pin Releasing Hormone Central Effect on Sleep and Motor
Systems. Pharmacol. Biochem. Behav. 1976, 4, 455-459.
(9) Schettini, G. Brain Somatostatin: Receptor-Coupled Transduc-
ing Mechanisms and role in Cognitive Functions. Pharmacol.
Res. 1991, 23, 203-215.
Recep tor Exp r ession a n d Tr a n sfection . Transfections
were performed by the calcium phosphate method. CHO-K1
cells were maintained in R-MEM (Gibco) supplemented with
10% fetal calf serum and transfected with each of the expres-
sion plasmids using calcium phosphate precipitation. Clones
that had inherited the expression plasmid were selected in
R-MEM supplemented with 500 µg mL-1 Geneticin (G418;
Gibco). Independent CHO-K1 clones were picked by glass-ring
cloning and expanded in culture in the selective media.
Membranes were prepared from the isolated clones, and hsst
(10) Reisine, T.; Bell, G. I. Molecular Properties of Somatostatin
Receptors. Neuroscience 1995, 67, 777-790.
(11) Reisine, T.; Bell, G. I. Molecular Biology of Somatostatin
Receptors. Endocrine Rev. 1995, 16, 427-442.
(12) Coy, D. H.; Taylor, J . E. Receptor-Specific Somatostatin Ana-
logues: Correlations with Biological Activity. Metabolism 1996,
34 (Suppl. 1), 21-23.
(13) Raynor, K.; Murphy, W. A.; Coy, D. H.; Taylor, J . E.; Moreau,
J .-P.; Yasuda, K.; Bell, G. I.; Reisine, T. Cloned Somatostatin
Receptors: Identification of Subtype-Selective Peptides and
Demonstration of High Affinity Binding of Linear Peptides. Mol.
Pharmacol. 1993, 43, 838-844.
expression was initially assessed for binding with [125I]Tyr11
SRIF and [125I]MK-678 (for sst2).
-
Ra d ioliga n d Bin d in g Assa ys. Cell membranes of the five
cell types were obtained from homogenates (Polytron setting
6, 15 s) of the corresponding CHO-K1 cells in ice-cold Tris-
HCl (50 mM) and centrifuged (39000g, 10 min × 2), with an
(14) Rossowski, W. J .; Coy, D. H. Specific Inhibition of Rat Pancreatic
Insulin and Glucagon Release by Receptor-Selective Somatosta-
tin Analogues. Biochem. Biophys. Res. Commun. 1994, 205, 341-
346.

Cyclic Disulfide Antagonists of Somatostatin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 1871
(15) Rossowski, W. J .; Gu, Z. F.; Akarca, U. S.; J ensen, R. T.; Coy,
D. H. Characterization of Somatostatin Receptor Subtypes
Controlling Rat Gastric Acid and Pancreatic Amylase Release.
Peptides 1994, 15, 1421-1424.
(16) Murthy, K. S.; Coy, D. H.; Makhlouf, G. M. Somatostatin
Receptor-Mediated Signaling in Smooth Muscle - Activation of
Phospholipase C-â3 by Gâgamma and Inhibition of Adenyl
Cyclase by GRilGRo. J . Biol. Chem. 1996, 271, 23458-23463.
(17) O’Carroll, A.-M.; Raynor, K.; Lolait, S. J .; Reisine, T. Charac-
terization of Cloned Human Somatostatin Receptor SSTR5. Mol.
Pharmacol. 1994, 46, 291-298.
(18) Rossowski, W.; Coy, D. H. Potent Inhibitory effects of a Type
Four Receptor Selective Somatostatin Analogue on Rat Insulin
Release. Biochem. Biophys. Res. Commun. 1993, 197, 366-371.
(19) Fries, J . L.; Murphy, W. A.; Sueiras-Diaz, J .; Coy, D. H.
Somatostatin Antagonist Analogue Increases GH, Insulin and
Glucagon Release in the Rat. Peptides 1982, 3, 811-814.
(20) Spencer, G. S. G.; Hallett, K. G. Somatostatin Antagonist
Analogue Stimulates Growth in Rats. Life Sci. 1985, 37, 27-
30.
(32) Chen, L.; Skinner, S. R.; Gordon, T. D.; Taylor, J . E.; Barany,
G.; Morgan, B. A. Isolation, Characterization and Synthesis of
a Trisulfide Related to the Somatostatin Analogue Lanreotide.
In Peptides: Frontiers of Peptide Science, Proceedings of the
Fifteenth American Peptide Symposium; Tam, J . P., Kaumaya,
P. T. P., Eds.; Kluwer: Dordrecht, The Netherlands, 1999; pp
275-276.
(33) Thurieau, C.; J aniak, P.; Krantic, S.; Guyard, C.; Pillon, A.;
Kucharczyk, N.; Vilaine, J .-P.; Fauche`re, J .-L. A New Soma-
tostatin Analogue with Optimized Ring Size Inhibits Neointimal
Formation Induced by Balloon Injury in Rats Without Altering
Growth Hormone Release. Eur. J . Med. Chem. 1995, 30, 115-
122.
(34) Van Binst, G.; Tourwe´, D. Backbone Modifications in Soma-
tostatin Analogues: Relation Between Conformation and Activ-
ity. Pept. Res. 1992, 5, 8-13.
(35) Rossowski, W. J .; Cheng, B.-L.; J iang, N.-Y.; Coy, D. H.
Examination of Somatostatin Involvement in the Inhibitory
Action of GIP, GLP-1, Amylin and Adrenomedullin on Gastric
Acid Release using a new SRIF Antagonist Analogue. Br. J .
Pharmacol. 1998, 125, 1081-1087.
(21) Hocart, S. J .; J ain, 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.
(22) Bass, R. T.; Buckwalter, B. L.; Patel, B. P.; Pausch, M. H.; Price,
L. A.; Strnad, J .; Hadcock, J . R. Identification and Characteriza-
tion of Novel Somatostatin Antagonists. Mol. Pharmacol. 1996,
50, 709-715.
(36) The model diastereoisomeric peptides were H-Nal-D&LCys-His-
DTrp-Lys-Val-Cys-Nal-NH2 and H-DNal-Cys-His-DTrp-Lys-Val-
Cys-D&LNal-NH2 which had retention times in the TFA HPLC
system of LCys, 30.3; DCys, 30.9; LNal, 36.6; DNal, 37.8 min,
respectively.
(23) Bass, R. T.; Buckwalter, B. L.; Patel, B. P.; Pausch, M. H.; Price,
L. A.; Strnad, J .; Hadcock, J . R. Identification and Characteriza-
tion of Novel Somatostatin Antagonists, Erratum. Mol. Phar-
macol. 1997, 51, 170.
(24) Coy, D. H.; Murphy, W. A.; Rossowski, W. J .; Hocart, S. J .; J ain,
R.; Fuselier, J .; Taylor, J . E. Somatostatin Receptor Antagonists
(37) Binding data not shown.
(38) Reisine, T.; Bell, G. I. Molecular Properties of Somatostatin
Receptors Neuroscience 1995, 67, 777-790.
(39) Liapakis, G.; Hoeger, C.; Rivier, J .; Reisine, T. Development of
a
Selective Agonist at the Somatostatin Receptor Subtype
SSTR1. J . Pharmacol. Exp. Ther. 1996, 276, 1089-1094.
(40) Hoyer, D.; Bell, G. I.; Berelewitz, M.; Epelbaum, J .; Feniuk, W.;
Humphrey, P. P. A.; O’Carroll, A.-M.; Patel, Y. C.; Schonbrunn,
A.; Taylor, J . E.; Reisine, T. Classification and Nomenclature of
Somatostatin Receptors. Trends Pharmacol. Sci. 1995, 16, 86-
88.
(41) Bruno, J .-F.; Xu, Y.; Song, J .; Berelowitz, M. Molecular Cloning
and Functional Expression of a Novel Brain Specific SRIF
Receptor. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 11151-11155.
(42) O’Carroll, A.-M.; Lolait, S. J .; Konig, M.; Mahan, L. C. Molecular
Cloning and Expression of a Pituitary SRIF Receptor with
Preferential Affinity for SRIF-28. Mol. Pharmacol. 1992, 42,
939-946.
(43) IUPAC-IUB Commission of Biochemical Nomenclature (CBN)
Symbols for Amino-Acid Derivatives and Peptides, Recom-
mendations 1971. Eur. J . Biochem. 1972, 27, 201-207.
(44) Murphy, W. A.; Taylor, J .; Moreau, J .-P.; Coy, D. H. Novel
Heptapeptide Somatostatin Analogue Displays Antitumor Activ-
ity Independent of Effects on Growth Hormone Secretion. Pept.
Res. 1989, 2, 128-132.
(45) Yamada, Y.; Post, S. R.; Wang, K.; Tager, H. S.; Bell, G. I.; Seino,
S. Cloning and Functional Characterization of a Family of
Human and Mouse Somatostatin Receptors Expressed in Brain,
Gastrointestinal Tract and Kidney. Proc. Natl. Acad. Sci. U.S.A.
1992, 89, 251-255.
based an
a mixed Neuromedin B Antagonist/Somatostatin
Agonist. In Peptides: Frontiers of Peptide Science, Proceedings
of the Fifteenth American Peptide Symposium; Tam, J . P.,
Kaumaya, P. T. P., Eds.; Kluwer: Dordrecht, The Netherlands,
1999; pp 526-529.
(25) Brady, S. F.; Nutt, R. F.; Holly F. W.; Paleveda, W. J .; Strachan,
R. G.; Bergstrand, S. J .; Veber, D. F.; Saperstein, R. Synthesis
and Biological Activity of Somatostatin Analogues of Reduced
Ring Size. In Peptides: Synthesis, Structure, Function, Proceed-
ings of the Seventh American Peptide Symposium; Rich, D. H.,
Gross, E., Eds.; Pierce Chemical Co.: New York, 1981; pp 653-
656.
(26) Veber, D. F. Design of a Highly Active Cyclic Hexapeptide
Analogue of Somatostatin. In Peptides: Synthesis, Structure,
Function. Proceedings of the Seventh American Peptide Sympo-
sium; Rich, D. H., Gross, E., Eds.; Pierce Chemical Co.: New
York, 1981; pp 685-694.
(27) Veber, D. F.; Saperstein, R.; Nutt, R. F.; Freidinger, R. M.; Brady,
S. F.; Curley, P.; Perlow, D. S.; Paleveda, W. J .; Colton, C. D.;
Zacchei, A. G.; Tocco, D. J .; Hoff, D. R.; Vandlen, R. L.; Gerich,
J . E.; Hall, L.; Mandarino, L.; Cordes, E. H.; Anderson, P. S.;
Hirschmann, R. A Super Active Cyclic Hexapeptide Analogue
of Somatostatin. Life Sci. 1984, 34, 1371-137.
(28) Veber, D. F. Design and Discovery in the Development of Peptide
Analogues. In Peptides: Chemistry and Biology. Proceedings of
the Twelfth American Peptide Symposium; Smith, J . A., Rivier,
J . E., Eds.; ESCOM: Leiden, 1992; pp 3-14.
(29) Murphy, W. A.; Heiman, M. L.; Lance, V.; Mezo, I.; Coy, D. H.
Octapeptide Analogues of Somatostatin Exhibiting Greatly
Enhanced in vivo and in vitro Inhibition of Growth Hormone
Secretion in the Rat. Biochem. Biophys. Res. Commun. 1985,
132, 922-928.
(30) Freidinger, R. M.; Schwenk Perlow, D.; Randall, W. C.; Saper-
stein, R.; Arison, B. H.; Veber, D. F. Conformational Modifica-
tions of Cyclic Hexapeptide Somatostatin Analogues. Int. J . Pept.
Protein Res. 1984, 23, 142-150.
(31) O¨ sapay, G.; Prokai, L.; Ho-Seung, K.; Medzihradszky, K. F.; Coy,
D. H.; Liapakis, G.; Reisine, T.; Melacini, G.; Zhu, Q.; Wang, S.
H.-H.; Mattern, R.-H.; Goodman, M. Lanthionine-Somatostatin
Analogues: Synthesis, Characterization, Biological Activity, and
Enzymatic Stability Studies. J . Med. Chem. 1997, 40, 2241-
2251.
(46) Yasuda, K.; Rens-Domiano, S.; Breder, C. D.; Law, S. F.; Sapel,
C. B.; Reisine, T.; Bell, G. I. Cloning of a Novel Somatostatin
Receptor, SSTR-3, Coupled to Adenyl Cyclase. J . Biol. Chem.
1992, 267, 20422-20428.
(47) Yamada, Y.; Reisine, T.; Law, S. F.; Ihara, Y.; Kubota, A.;
Kagimoto, S.; Seino, M.; Seino, Y.; Bell, G. I.; Seino, S. Soma-
tostatin Receptors, an Expanding Gene Family: Cloning and
Functional Characterization of Human SSTR3, a Protein Coupled
to Adenylate Cyclase. Mol. Pharmacol. 1992, 42, 2136-2142.
(48) Rohrer, L.; Raulf, F.; Bruns, C.; Buettner, R.; Hofstaedter, F.;
Schule, R. Cloning and Characterization of a Fourth Human
Somatostatin Receptor. Proc. Natl. Acad. Sci. U.S.A. 1993, 90,
4196-4200.
(49) Taylor, J . E. Personal communication.
J M9806289