Design, synthesis, and biological activities of potent and selective somatostatin analogues incorporating novel peptoid residues

Article abstract of DOI:10.1021/jm970393l

We report the synthesis, bioactivity, and structure-activity relationship studies of compounds related to the Merck cyclic hexapeptide c[Pro⁶-Phe⁷-D-Trp⁸-Lys⁹-Thr¹⁰-Phe¹¹], L-363,301 (the n

Full text of DOI:10.1021/jm970393l

J . Med. Chem. 1998, 41, 2679-2685
2679
Articles
Design , Syn th esis, a n d Biologica l Activities of P oten t a n d Selective
Som a tosta tin An a logu es In cor p or a tin g Novel P ep toid Resid u es
Thuy-Anh Tran,† Ralph-Heiko Mattern,† Michel Afargan,‡ Oved Amitay,‡ Ofer Ziv,‡ Barry A. Morgan,§
J ohn E. Taylor,§ Daniel Hoyer,| and Murray Goodman*,†
Department of Chemistry and Biochemistry, University of California at San Diego, La J olla, California 92093-0343,
Peptor, Ltd., Pharmacology & Pharmaceutical Research, Rehovot 76326, Israel, Biomeasure, Inc.,
Milford, Massachusetts 01757-3650, and Novartis Pharma AG, S-386/ 745, CH-4002 Basel, Switzerland
Received J une 13, 1997
We report the synthesis, bioactivity, and structure-activity relationship studies of compounds
related to the Merck cyclic hexapeptide c[Pro6-Phe7-D-Trp8-Lys9-Thr10-Phe11], L-363,301 (the
numbering in the sequence refers to the position of the residues in native somatostatin). The
Pro residue in this compound is replaced with arylalkyl peptoid residues. We present a novel
approach utilizing â-methyl chiral substitutions to constrain the peptoid side-chain conforma-
tion. Our studies led to molecules which show potent binding and increased selectivity to the
hsst2 receptor (weaker binding to the hsst3 and hsst5 receptors compared to L-363,301). In
vivo, these peptoid analogues selectively inhibit the release of growth hormone but have no
effect on the inhibition of insulin. The biological assays which include binding to five
recombinant human somatostatin receptors carried out in two independent laboratories and
in vivo inhibition of growth hormone and insulin provide insight into the relationship between
structure and biological activity of somatostatin analogues. Our results have important
implications for the study of other peptide hormones and neurotransmitters.
In tr od u ction
itself. In particular the studies of Veber and co-workers
resulted in the discovery of the cyclic hexapeptide
analogue c[Pro6-Phe7-D-Trp8-Lys9-Thr10-Phe11],7 L-363,-
301 (the numbering in the sequence refers to the
position of the residues in native somatostatin). This
molecule shows higher biological activity than native
somatostatin in inhibiting the release of growth hor-
mone, insulin, and glucagon. From NMR studies, Veber
proposed a type II′ â-turn about the tetrapeptide
sequence Phe7-D-Trp8-Lys9-Thr10 which is considered the
biological active portion, interacting with the receptor.
The Phe11-Pro6 dipeptide is contained within a type VI
â-turn which includes a cis peptide bond between the
Phe11 and Pro residues. This dipeptide unit, the so-
called bridging region, is important for maintaining the
proper orientation of the tetrapeptide portion and
contains a component of ligand-receptor interaction via
the phenyl ring.8 Hirschmann, Nicolaou, and their co-
workers designed peptidomimetics of somatostatin em-
ploying a â-D-glucose scaffold.9 They suggested that the
peptide backbone was not required for receptor binding
but served as a scaffold for supporting the side chains
of Phe7, Trp8, Lys9, and Phe11 in the required spatial
arrangement. The contribution of the dipeptide Phe11-
Pro6 to the binding affinities of somatostatin analogues
has been investigated thoroughly.10,11
Somatostatin, a tetradecapeptide hormone released
by the hypothalamus, plays important physiological
roles: it is a potent inhibitor of the release of several
hormones (i.e., glucagon, growth hormone, insulin,
gastrin) and regulates many other biological activities.1
It is also highly active in acromegalic patients, lowering
the plasma level of growth hormone, and therefore is of
potential therapeutic value in clinical treatment of
acromegaly and gastroenteropancreatic tumors.2 So-
matostatin induces its biological effects by interacting
with a family of structurally related receptors. Five
human somatostatin receptors have been cloned and
referred to as hsst1-5 receptors.3 The ligand SMS-201-
9954 (Octreotide, Sandostatin) and other clinically used
somatostatin analogues interact with the receptor sub-
types hsst2, -3, and -5 (we use the nomenclature
suggested by Hoyer et al.).5 The receptors hsst2 and -5
have been reported to mediate antiproliferative effects
of somatostatin on tumor cell growth and therefore may
mediate the clinical effects of Sandostatin in humans.6
The wide range of physiological roles of somatostatin
against a number of endocrine hormones and its very
short biological half-life have led to substantial efforts
to synthesize peptide analogues in search of molecules
exhibiting potent and selective biological activity and
longer duration of action as compared to the hormone
We have previously carried out extensive studies on
the structure-activity relationships of somatostatin
analogues related to L-363,301 in which the side-chain
torsions were constrained as a result of Câ-methyla-
tion.12,13 Results obtained from the conformational
† University of California at San Diego.
‡ Peptor, Ltd.
§ Biomeasure, Inc.
| Novartis Pharma AG.
S0022-2623(97)00393-2 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/18/1998

2680 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Tran et al.
Ch a r t 1. Structures of L-363,301, Sandostatin, NPhe, (R)-âMeNphe, and (S)-âMeNphe Analogues
analysis of these analogues revealed the features of a
side-chain topology of analogues that bind well to
somatostatin receptors.
addition, a model developed in our group has shown that
the aromatic side chain of the D-Phe residue of San-
dostatin is in spatial proximity of the bridging region.15
We envisioned that the incorporation of a peptoid
residue containing an aromatic side chain into position
6 can occupy some of the conformational space available
to Phe11 in L-363,301 and/or the D-Phe in Sandostatin.
We made further attempts to constrain the peptoid
side chain by incorporation of novel chiral â-methylated
peptoid residues. Two side-chain-methylated peptoid
residues, (R)- and (S)-N(RMe)benzylglycines [(R)- and
(S)-âMeNphe], were designed and incorporated into
position 6 to give the two analogues c[(R)-âMeNphe6-
Phe7-D-Trp8-Lys9-Thr10-Phe11] [(R)-âMeNph e compound]
and c[(S-âMeNphe6-Phe7-D-Trp8-Lys9-Thr10-Phe11] [(S)-
âMeNp h e compound]. The conformational analysis of
our peptoid analogues using 1H NMR and computer
simulations is described in the accompanying paper.17
Design Ra tion a le
In our current studies we investigate the role of the
bridging region by incorporating novel peptoid residues.
Peptoids represent a new class of imino acid residues
that are not found in nature and have been shown to
possess significant proteolytic stability.14
Our studies of somatostatin analogues are based on
the Merck compound c[Pro6-Phe7-D-Trp8-Lys9-Thr10
-
Phe11], L-363,301. An arylalkyl peptoid residue, N-
benzylglycine (“Nphe”), is incorporated into position 6
of L-363,301 resulting in c[Nphe6-Phe7-D-Trp8-Lys9-
Thr10-Phe11] (Np h e com p ou n d ) (Chart 1). The ratio-
nale of this design is based on several considerations.
Peptoid structures can induce cis amide bonds as does
Pro6 in L-363,301. The functional groups in the peptoid
resi-
Syn th esis
due can be readily altered, and therefore structural ri-
gidity, complexity, and diversity of the designed ana-
logues can be increased easily. NMR studies on L-363,-
301 have established that in solution the phenyl ring
of Phe11 is positioned close to the proline ring.10 In
The synthesis of the peptoid residues was achieved
by alkylation of benzyl-, (R)-R-methylbenzyl-, or (S)-R-
methylbenzylamines with ethyl bromoacetate.18 The
routes of synthesis of the peptoid analogues are shown
in Scheme 1. The syntheses of these three analogues

Somatostatin Analogues with Novel Peptoid Residues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2681
Sch em e 1. Synthesis of c[(R)-âMeNphe-Phe-D-Trp-Lys-Thr-Phe]
Ta ble 1. In Vitro Inhibition of Radioligand Binding to Human Recombinant Receptors: Ki (nM) ( SEMa,b
compound
hsst1
hsst2
hsst3
hsst4
hsst5
hsst5/hsst2
hsst3/hsst2
L-363,301
Phe-Nphe
Phe-(R)-âMeNphe
Phe-(S)-âMeNphe
>1000
>1000
>1000
>1000
5.10 ( 0.76
6.98 ( 0.83
2.33 ( 0.41
29.5 ( 2.49
129 ( 51
253 ( 57
425 ( 100
797 ( 125
>1000
25.0 ( 6.8
100.7 ( 45.5
33.5 ( 12.5
87.0 ( 22.6
4.9
14.4
14.4
2.9
25.3
36.2
182.4
27.0
>1000
>1000
987 ( 13
a
b
Data obtained from J ohn Taylor. Binding assays were carried out with cell membranes from CHO-K1 cells.
were designed around the reaction between the tripep-
tide Cbz-D-Trp-Lys(Boc)-Thr(tBu)-OH19 and the other
tripeptide Boc-Phe-Nxaa-Phe-OBzl (Nxaa ) Nphe, (R)-
or (S)-âMeNphe) fragments incorporating the bridging
region. The Boc-Phe-Nxaa-Phe-OBzl tripeptides were
synthesized in solution in a stepwise manner with
benzyl and ethyl ester protection for the C-terminals
and Boc protection for the R-amino groups. Ethyl[(N,N-
dimethylamino)propyl]carbodiimide (EDC) was used as
coupling agent in the presence of hydroxybenzotriazole
(HOBt) to prepare the protected tripeptides. For the
more hindered coupling of Boc-Phe-OH with (R)- or (S)-
âMeNphe-OEt, the highly efficient coupling reagent
PyBroP was used instead of EDC/HOBt.
The fully protected linear hexapeptides were obtained
from fragment condensation reactions between Cbz-D-
Trp-Lys(Boc)-Thr(tBu)-OH and H-Phe-NXaa-Phe-OBzl
using EDC/HOBt. Upon reductive removal of the ben-
zyloxycarbonyl and benzyl ester protections, the depro-
tected linear hexapeptides were allowed to cyclize at 1
mM concentration using DPPA/K2HPO4. Simultane-
ous acidolysis of tert-butyl ether and Nꢀ-Boc protections
from the protected cyclic compounds was carried out
with TFA in the presence of scavengers to give the
desired cyclic hexapeptides, which were purified by
reversed-phased HPLC. Detailed synthesis and char-
acterization of all new compounds are given in the
Supporting Information.
Biologica l Resu lts a n d Discu ssion
The somatostatin analogues were tested in vitro for
their specific binding to the five human somatostatin
receptors expressed in CHO cell lines (Table 1, hsst1-5
receptors; Table 2, hsst2-4 receptors) hSF9 cells (Table
2, hsst1) or CCL-39 cell lines (Table 2, hsst5 receptor)
and in vivo for their ability to inhibit the release of
growth hormone and insulin in rats. The in vitro tests
were carried out in two independent laboratories, J .T.
(Table 1) and D.H. (Table 2). The binding potencies of
the peptoid analogues and the ratios of the binding
potencies to the hsst5 and hsst2 as well as the ratios of
the binding potencies to the hsst3 and hsst2 are given
in Tables 1 and 2. Both sets of binding constants show
that the Nphe and the (R)-âMeNphe compounds bind
more effectively than the (S)-âMeNphe to the hsst2
receptor. The binding constants for the hsst2 receptor
obtained by J .T. suggest that the (S)-âMeNphe binds 1
order of magnitude weaker than the (R)-âMeNphe and
4 times weaker than the Nphe compound, while the data
obtained by D.H. show that the Nphe and the (R)-
âMeNphe compounds are equipotent and 3-fold more
active in the hsst2 receptor compared to the (S)-

2682 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Tran et al.
Ta ble 2. In Vitro Inhibition of Radioligand Binding to Human Recombinant Receptors: Kd (nM) ( SEMa,b
compound
hsst1
hsst2
hsst3
hsst4
hsst5
hsst5/hsst2
hsst3/hsst2
L-363,301
Phe-Nphe
Phe-(R)-âMeNphe
Phe-(S)-âMeNphe
4570 ( 106
7080 ( 333
3550 ( 431
13183 ( 1254
0.76 ( 0.05
2.95 ( 0.71
2.04 ( 0.75
6.17 ( 1.34
525 ( 37
282 ( 27
407 ( 50
339 ( 68
8913 ( 420
4680 ( 210
4168 ( 195
2630 ( 460
7.6 ( 1.5
69 ( 3
288 ( 7
115 ( 3
10
690
23.4
141.2
18.6
158.6
199.5
54.9
a
b
Data obtained from Daniel Hoyer. Binding assays were carried out with cell membranes from CHO cells for hsst2-4 receptors,
hSF9 cells for the hsst1 receptor, and CCL-39 cells for the hsst5 receptor.
F igu r e 1. Effect of somatostatin analogues on GH release in
rats: 1, control, n ) 20; 2, Sandostatin, 100 µg/kg, n ) 24; 3,
RC-160 (H-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Trp-NH2), 100
µg/kg, n ) 10; 4, L-363,301, 100 µg/kg, n ) 10; 5, Nphe
analogue, 100 µg/kg, n ) 15; 6, (R)-âMeNphe analogue, 100
µg/kg, n ) 15; 7, (S)-âMeNphe analogue, 100 µg/kg, n ) 10.
F igu r e 2. Effect of somatostatin analogues on insulin release
in rats: 1, control, n ) 15; 2, Sandostatin, 100 µg/kg, n ) 10;
3, RC-160 (H-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Trp-NH2),
100 µg/kg, n ) 10; 4, L-363,301, 100 µg/kg, n ) 10; 5, Nphe
analogue, 100 µg/kg, n ) 10; 6, (R)-âMeNphe analogue, 100
µg/kg, n ) 10; 7, (S)-âMeNphe analogue, 100 µg/kg, n ) 10.
âMeNphe. The results obtained for the hsst5 receptor
indicate considerable variations in binding constants.
The data obtained by J .T. show that the Nphe and the
(S)-âMeNphe have reduced potencies at the hsst5 re-
ceptor compared to L-363,301, while the (R)-âMeNphe
compound is insignificantly less potent in the hsst5
compared to L-363,301. The binding constants mea-
sured by D.H., on the other hand, suggest a reduced
potency at the hsst5 receptor for the (R)-âMeNphe
compared to the Nphe and (S)-âMeNphe. Since the two
laboratories use different cell lines for the hsst5 recep-
tor and different radioligands as well as slightly differ-
ent protocols (see the Experimental Section), it is
difficult to compare the two results in quantitative
terms.
In qualitative terms the results obtained from both
laboratories are consistent at the hsst3 receptor. All
three peptoid analogues show reduced potency at the
hsst3 receptor compared to L-363,301. Both sets of
binding data indicate that the peptoid analogues, espe-
cially the Nphe and (R)-âMeNphe, are hsst2-selective
somatostatin analogues.
The changes in binding suggest critical topochemical
requirements for the peptoid side chains. Spatial prox-
imities between the three aromatic side chains of Phe11,
Nxaa,6 and Phe7 appear to influence the selectivity and
binding potency of these compounds. It is reasonable
to assume that aromatic stacking in positions 11, 6, and
7 of the cyclic hexapeptides plays an important role in
stabilizing the active conformations of these peptides.
Indeed, Veber proposed a hydrophobic stacking of Phe11-
Pro6-Phe7 side chains based on marked upfield shifts
in the proton NMR spectrum.8
on the release of insulin and growth hormone were
examined in vivo in rats. Sandostatin, RC-160 (H-D-
Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Trp-NH2), and L-363,-
301 served as reference compounds. Figure 1 shows
that the peptoid analogues Nphe, (R)-âMeNphe, and (S)-
âMeNphe are as active as Sandostatin, RC-160, and
L-363,301 in the inhibition of growth hormone. These
results are in good agreement with the hypothesis that
hsst2 receptor is responsible for the inhibition of the
GH release.5,20
Figure 2 demonstrates that analogues Nphe, (R)-
âMeNphe, and (S)-âMeNphe have no effect on the inhi-
bition of insulin compared to Sandostatin, RC-160, and
L-363,301 under these experimental conditions. The
statistical evaluation and results of tests at different
concentrations are given in Figures 1S-3S in the Sup-
porting Information. The incorporation of the arylalkyl
peptoid residues results in a loss of inhibition of insulin
release while these analogues remain active in the
inhibition of GH release. This observation could suggest
that the phenyl ring of the peptoid residue at position
6 may interact directly with the receptor responsible for
the GH release while it prevents interaction with the
receptor associated with insulin release. Detailed un-
derstanding of these changes could prove valuable for
the design of functionally selective analogues.
Con clu sion s
Modifications in the bridging region resulted in so-
matostatin analogues that are selective in the in vitro
binding to the human somatostatin receptor subtype
hsst2. Peptoid residues were designed which effectively
replace the proline of L-363,301 and have led to the
The effects of the peptoid analogues of somatostatin

Somatostatin Analogues with Novel Peptoid Residues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2683
receptor subtypes, in ice-cold 50 mM Tris-HCl and centrifuging
twice at 39000g (10 min), with an intermediate resuspension
in fresh buffer. The final pellets were resuspended in 10 mM
Tris-HCl for assay. For the hsst1,3,4,5 assays, aliquots of the
membrane preparations were incubated (30 min/37 °C) with
0.05 nM [125I-Tyr11]SRIF-14 in 50 mM HEPES (pH 7.4)
containing BSA (10 mG/mL), MgCl2 (5 mM), Trasylol (200 kIU/
mL), bacitracin (0.02 mg/mL), and phenylmethanesulfonyl
fluoride (0.02 mg/mL). The final assay volume was 0.3 mL.
For the hsst2 assay, [125I]MK-678 (0.05 nM) was employed as
the radioligand and the incubation time was 90 min/25 °C.
The incubations were terminated by rapid filtration through
GF/C filters (presoaked in 0.3% poly(ethylenimine)) using a
Brandel filtration manifold. Each tube and filter were then
washed three times with 5-mL aliquots of ice-cold buffer.
Specific binding was defined as the total radioligand bound
minus that bound in the presence of 1000 nM SRIF-14
(hsst1,3,4,5) or 1000 nM MK-678 (hsst2).
2. D.H. (Ta ble 2). Cell culture and expression of human
SRIF receptors in CHO or CCL39 cells were as reported
previously.23 Insect SF9 cells expressing hsst1 receptors were
obtained as pellets. Cells were washed twice with 10 mmol
of HEPES (pH 7.6) and harvested and membranes prepared
as described.24 Membranes were resuspended into 10 mmol/L
HEPES (pH 7.6) containing 5 mmol/L MgCl2, 10 mg/mL
bacitracin, and 0.5% (w/v) bovine serum albumin (BSA).
For crude membrane preparations, cells were harvested by
washing with 10 mM HEPES, pH 7.5, scrapping off the culture
plates with 4 mL of the same buffer, and centrifugation at 4
°C for 5 min at 2500g. The cell pellet was either stored at
-80 °C or directly used. The cells were resuspended in binding
assay buffer (10 mM HEPES, pH 7.5, 0.5% (w/v) BSA) by
homogenization with the polytron at 50 Hz for 20 s.
preparation of somatostatin analogues with modified
biological activities with respect to binding to isolated
receptors and inhibition of GH and insulin release. A
structure-activity relationship study was carried out
in order to optimize the side-chain orientation of the
analogues, and the (R)-âMeNphe analogue was found
to be the most potent compound in this series with
regard to the binding to the hsst2 receptor. Indeed, the
position 6 in L-363,301 peptide could be the most
interesting position in terms of alteration of binding
affinity. These studies clearly demonstrate a novel
approach using side-chain-methylated peptoids to elu-
cidate bioactive conformations of somatostatin ana-
logues.
The finding that injections of our analogues into rats
did not influence insulin secretion indicates that under
these given experimental conditions the peptoid-con-
taining compounds might selectively inhibit the GH
compared with Sandostatin, RC-160, and L-363,301
which are very potent in the inhibition of both GH and
insulin.7 The peptoid analogues inhibit the secretion
of growth hormone in doses comparable to that of RC-
160, L363,301, and Sandostatin. While RC-160, L-363,-
301, and Sandostatin inhibit the release of insulin at
these doses (100 µg/kg), the peptoid analogues have no
effect on insulin secretion. This group of new target
molecules should be very useful for delineating the roles
played by the somatostatin receptors in mediating the
variety of important physiological processes under the
inhibitory control of somatostatin. The fact that rela-
tively simple modifications of biologically active peptides
led to potent and selective analogues opens new exciting
opportunities in drug design. These results are fully
consistent with the results of our receptor-based ap-
proach to drug design.21 These analogues will be an
important guide in efforts to design therapeutically
useful somatostatin analogues.
Ra d ioliga n d Bin d in g Assa y. Following radioligands were
used for human SRIF receptor binding:
[
125I]LTT-SRIF28
(hsst1 SF9, hsst3 CHO, hsst5 CCL-39), [125I]CGP 23996 (hsst4,
CHO), and [125I]Tyr3-octreotide (hsst2, CHO).
In competition experiments, 150 µL of the cell or membrane
homogenate was incubated with 50 µL of [125I]LTT-SRIF28
,
[
125I]SRIF14, [125I]CGP 23996, or [125I]Tyr3-octreotide (2175 Ci/
mmol, 25-35 pM, final concentration each), respectively, in
binding assay buffer containing MgCl2 (5 mM), the protease
inhibitor bacitracin (5µg/mL), and either 50 µL of binding assay
buffer (total binding) or 50 µL of various peptide concentra-
tions. Nonspecific binding was determined in the presence of
50 µg of SRIF14 (1 µM). After 1 h at room temperature, the
incubation was terminated by vacuum filtration through glass
fiber filters presoaked in 0.3% (w/v) poly(ethylenimine). The
filters were rinsed twice with 5 mL of ice-cold 10 mM Tris/
HCl buffer, pH 7.4, and dried. Bound radioactivity was
measured in a γ-counter (80% counting efficiency).
Da ta An a lysis. Competition and saturation curves from
experiments performed in triplicate determination were ana-
lyzed as described previously.25 Data were analyzed by
nonlinear regression curve fitting with the computer program
SCTFIT.26 The data are reported as pKD values (-log mol/L).
Protein concentrations were determined by the method of
Bradford27 with bovine serum albumin as a standard.
An im a l Stu d ies: Gr ow th Hor m on e a n d In su lin Tests.
An im a ls a n d Tr ea tm en t. In vivo studies were perfomed
with Wistar male rats (Harlan, Israel), weighing 180-220 g.
Animals were maintained as outlined in the Guide for the Care
and Use of Laboratory Animals (NIH Publication no. 85-23,
1985). The animals were allowed free access to food and water
until 18 h before the experiment, when all the food (but not
the water) was withdrawn. All animals were housed in cages
with wide mesh wire bottom to prevent coprophagia (feeding
on excrement).
Exp er im en ta l Section
Abbreviations used are those recomended by the IUPAC-
IUB Commission: Bzl, benzyl; Boc, (tert-butyloxy)carbonyl;
tBu, tert-butyl; Cbz, benzyloxycarbonyl; DPPA, diphenyl phos-
phorazidate; EDC, 1-ethyl-3-[3′-(dimethylamino)propyl]carbo-
diimide; GH, growth hormone; HOBt, 1-hydroxybenzotriazole;
HPLC, high-pressure liquid chromatography; PyBroP, bromo-
tris(pyrrolidino)phosphonium hexafluorophosphate; hsst re-
ceptor, human somatostatin receptor; TFA, trifluoroacetic acid.
[
125I]CGP 23996 (c[Lys-Asu-Phe-Phe-Trp-Lys-Thr-(125I-Tyr)-
Thr-Ser]), [125I]Tyr3-octreotide (D-Phe-c[Cys-(125I-Tyr)-D-Trp-
Lys-Thr-Cys]-Thr-OH), [125I]Leu8, D-Trp22, and Tyr25(LTT)-
SRIF28 (Ser-Ala-Asn-Ser-Asn-Pro-Ala-Leu-Ala-Pro-Arg-Glu-
Arg-Lys-Ala-Gly-c[Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-(125I-
Tyr)-Thr-Ser-Cys]-OH) were purchased from ANAWA AG
(Wangen, Switzerland).
Recep tor Bin d in g Assa ys. 1. J .T. (Ta ble 1). Sta ble
Exp r ession of h sst Recep tor Su btyp es. The complete
coding sequences of genomic fragments of the hsst1-4 receptor
genes and a cDNA clone for the hsst5 were subcloned into
mammalian expression vector pCMV. Clonal cell lines stably
expressing the hsst1-5 receptors were obtained by transfection
into CHO-K1 cells (ATCC) using the calcium phosphate
coprecipitation method.22 The plasmid pRSV-neo (ATCC) was
included as a selectable marker. Clonal cell lines were selected
in RPMI 1640 media containing 0.5 mg/mL G418 (Gibco), ring-
cloned, and expanded into culture.
Experiments were carried out under Nembutal anesthesia
(60 mg/kg ip), to stimulate GH release (at time 0). All drugs
were dissolved in saline (0.9% NaCl) and administrated sc (10
min following anesthesia), at a final dose of 100 µg/kg. The
control group received 1 mL/kg saline (0.9% NaCl). At 25 min
femoral cannulation was performed, and 0.5 g/kg L-Arg (dis-
Ra d ioliga n d Bin d in g Assa ys. Membranes for in vitro
receptor binding assays were obtained by homogenizing (Poly-
tron setting 6, 15 s) the CHO-K1 cells, expressing the hsst

2684 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Tran et al.
types SSTR1 and SSTR2. Proc. Natl. Acad. Sci. U.S.A. 1994,
91, 2315-2319. (b) Buscail, L.; Esteve, J .-P.; Saint-Laurent, N.;
Bertrand, V.; Reisine, T.; O’Carroll, A.-M.; Bell, G. I.; Schally,
A. V.; Vaysse, N.; Susini, C. Inhibition of cell proliferation by
the somatostatin analogue RC-160 is mediated by somatostatin
receptor subtypes SSTR2 and SSTR5 through different mech-
anisms. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 1580-1584.
(7) Veber, D. F.; Freidinger, R. M.; Perlow, D. S.; Palaveda, W. J .,
J r.; Holly, F. W.; Strachan, R. G.; Nutt, R. F.; Arison, B. J .;
Homnick, C.; Randall, W. C.; Glitzer, M. S.; Saperstein, R.;
Hirschmann, R. A potent cyclic hexapeptide analogue of soma-
tostatin. Nature 1981, 292, 55-58.
(8) Veber, D. F. Design of a Highly Active Cyclic Hexapeptide Ana-
logue of Somatostatin. In Peptides, Synthesis, Structure and
Function; Proceedings of the Seventh American Peptides Sym-
posium; Rich, D. H., Gross, V. J ., Eds.; Pierce Chemical Co.:
Rockford, IL, 1983; pp 685-694.
(9) Hirschmann, R.; Nicolaou, K. C.; Pietranico, S.; Salvino, J .;
Leahy, E. M.; Sprengeler, P. A.; Furst, G.; Strader, C. D.;
Cascieri, M. A.; Candelore, M. R.; Donalson, C.; Vale, W.;
Maechler, L. Nonpeptidal peptidomimetics with a â-D-glucose
scaffolding - A partial somatostatin agonist bearing a close
structural relationship to a potent, selective substance-P an-
tagonist. J . Am. Chem. Soc. 1992, 114, 9217-9218.
(10) Veber, D. F. Design and discovery in the development of peptide
analogues. In Peptides: Chemistry and Biology; Proceedings of
the Twelfth American Peptides Symposium; Smith, J . A., Rivier,
J . E., Eds.; ESCOM: Leiden, 1992; pp 3-14.
solved in saline) was administrated intravenous in order to
stimulate insulin release. The “none” group did not receive
L-Arg stimulation. Blood was collected 5 min following L-Arg
stimulation from the inferior vena cava for GH and insulin
measurements. Plasma heparin (15 U/mL) was obtained from
blood samples and stored at -20 °C until assay.
Hor m on e Assa ys. Rat growth hormone (rGH) 125I levels
were determined by appropiate Amersham’s assay system. The
standard in this kit has been calibrated against a reference
standard preparation (NIH-RP2) obtained from the National
Institute of Diabetes and Digestive and Kidney Diseases
(NIDDK). Rat insulin (rInsulin) 125I levels were measured by
appropiate Linco’s assay system.
All samples were measured by duplicate. All samples were
diluted 1:1 with assay buffer. Plasma concentrations were
calculated from standard curve after consideration of the
dilution factor.
Sta tistica l Meth od s. Plasma hormone concentrations
were expressed as means and standard error of the means
(mean ( SEM). One-way ANOVA followed by Fisher’s con-
trast was used to compare between groups. A P value of <0.05
was considered statistically significant. Each group consisted
of n ) 10-12 subjects; animals were divided randomly between
groups.
(11) Freidinger, R. M.; Perlow, D. S.; Randall, W. C.; Saperstein, R.;
Arison, B. H.; Veber, D. F. Conformational modifications of cyclic
hexapeptide somatostatin analogues. Int. J . Pept. Protein Res.
1984, 23, 142-150.
(12) Yamazaki, T.; Ro, S.; Goodman, M.; Chung, N. N.; Schiller, P.
W. A Topochemical Appraoch To Explain Morphiceptin Bioac-
tivity. J . Med. Chem. 1993, 36 (6), 708-719.
(13) (a) Huang, Z.; He, Y.-B.; Raynor, K.; Tallent, M.; Reisine, T.;
Goodman, M. Main chain and side chain chiral methylated
somatostatin analogues: Syntheses and conformational analy-
ses. J . Am. Chem. Soc. 1992, 114, 9390-9401. (b) He, Y.-B.;
Huang, Z.; Raynor, K.; Reisine, T.; Goodman, M. Conformations
of somatostatin related cyclic hexapeptides incorporating specific
R-methylated and â-methylated residues. J . Am. Chem. Soc.
1993, 115, 8066-8072.
Ack n ow led gm en t. We wish to thank the National
Institute of Health (DK 15410) for their support of this
research. R.M. gratefully acknowledges a DFG postdoc-
toral fellowship. We are indebted to Dr. David Coy,
Tulane University School of Medicine, New Orleans, for
his invaluable suggestions.
Su p p or tin g In for m a tion Ava ila ble: Complete experi-
mental descriptions for synthesis of Nphe, (R)-âMeNphe, and
(S)-âMeNphe analogues and supplementary Scheme 1 for the
preparation of Nphe compound. (18 pages). Ordering informa-
tion can be found on any current masthead page.
(14) Miller, S. M.; Simon, R. J .; Ng, S.; Zuckerman, R. N.; Kerr, J .
M.; Moos, W. H. Comparison of the proteolytic susceptibilities
of homologous L-amino acid, D-amino acid, and N-substituted
glycine peptide and peptoid oligomers. Drug Dev. Res. 1995, 35,
20-32.
(15) Melacini, G.; Zhu, Q.; Goodman, M. Multiconformational NMR
analysis of Sandostatin (octreotide): Equilibrium between beta-
sheet and partially helical structures. Biochemistry 1997, 36,
1233-1241.
(16) Hruby, V. J .; Toth, G.; Gehrig, C. A.; Kao, L. F.; Knapp, R.; Lui,
G. K.; Yamamura, H. I.; Krammer, T. H.; Davis, P.; Burks, T.
F. Topographically designed analogues of (D-Pen,D-Pen5) en-
kephalin. J . Med. Chem. 1991, 34, 1823-1830.
(17) Mattern, R.-H.; Tran, T.-A.; Goodman, M. Conformational
Analyses of Somatostatin-Related Cyclic Hexapeptides Contain-
ing Peptoid Residues. J . Med. Chem. 1998, 41, 2686-2692.
(18) (a) Mouna, A. M.; Nguyen, C.; Rage, I.; Xie, J . Preparation of
Boc N-alkyl glycines for peptoid synthesis. Synth. Commun.
1994, 24, 2429-2435. (b) Simon, R. J .; Kania, R. S.; Zuckermann,
R. N.; Huebner, V. D.; J ewell, D. A.; Banville, S.; Ng, S.; Wang,
L.; Rosenberg, S.; Marlowe, C. K.; Spellmeyer, D. C.; Tan, R.;
Frankel, A. D.; Santi, D. V.; Cohen, F. E.; Bartlett, P. A. Peptoids
- A modular approach to drug discovery. Proc. Natl. Acad. Sci.
U.S.A. 1992, 89, 9367-9371.
(19) Spencer, J . R.; Delaet, N. G. J .; Toy-Palmer, A.; Antonenko, V.
V.; Goodman, M. Unusual peptide bond cleavage reactions
during acidolytic deprotection reactions. J . Org. Chem. 1993, 58,
1635-1638.
(20) (a) Reisine, T.; Bell, G. Molecular biology of somatostatin
receptors. Endocrine Rev. 1995, 16, 427-442. (b) Rossowski, W.;
Coy, D. Potent inhibitory effects of a type four receptor-selective
somatostatin analogue on rat insulin release. Biochem. Biophys.
Res. Commun. 1993, 197, 366-371. (c) Rossowski, W.; Coy, D.
Specific inhibition of rat pancreatic insulin or glucagon release
by receptor-selective somatostatin analogues. Biochem. Biophys.
Res. Commun. 1994, 205, 341-346.
Refer en ces
(1) (a) Brazeau, P.; Vale, W.; Burgus, R.; Ling, N.; Bucher, M.;
Rivier, J .; Guillemin, R. Hypothalamic polypeptide that inhibits
the secretion of the immunoreactive growth hormone. Science
1973, 179, 77-79. (b) Koerker, D. J .; Harker, L. A.; Goodner, C.
J . Effects of somatostatin on hemostasis in baboons. N. Engl. J .
Med. 1975, 96, 749-754. (c) Gerich, J . E.; Lovinger, R.; Grodsky,
G. M. Glucagon and Insulin Release from the Perfused Rat
Pancreas in Response to Arginine, Isoproterenol and Theophyl-
line: Evidence for a Preferential Effect on Glucagon Secretion.
Endocrinology 1975, 96, 749-754.
(2) (a) Hall, R.; Schally, A. V.; Evered, D.; Kastin, A. J .; Mortimer,
C. H.; Tunbrigde, W. M. G.; Besser, G. M.; Coy, D. H.; Goldie,
D. J .; McNeilly, A. S.; Phenekos, C.; Weightmen, D. Action of
growth-hormone- release inhibitory in healthy men and acrome-
galy. Lancet 1973, 2, 581. (b) Lamberts, S. W. J .; Krenning, E.
P.; Reubi, J . C. The role of somatostatin and its analogues in
diagnosis and treatment of tumors. Endocrine Rev. 1991, 12,
450-482.
(3) Bell, G. I.; Reisine, T. Molecular Biology of Somatostatin
Receptors. Trends Neurosci. 1993, 16, 34-38.
(4) Bauer, W.; Briner, U.; Dopfner, W.; Haller, R.; Huguenin, R.;
Marbach, P.; Petcher, T. J .; Pless, J . SMS-201-995: A very potent
and selective octapeptide analogue of somatostatin with pro-
longed action. Life Sci. 1982, 31, 1133-1140.
(5) (a) Hoyer, D.; Bell, G. I.; Berelowitz, M.; Epelbaum, J .; Feniuk,
W.; Humphrey, P. P. A.; O′Carroll, A.-M.; Patel, Y. C.; Schon-
brunn, A.; Taylor, J . E.; Reisine, T. Classification and nomen-
clature of somatostatin receptors TiPS 1995 16, 86-88. (b)
Raynor, K.; Murphy, W.; Coy, D.; Taylor, J .; 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. (c) Raynor, K.; O′Carroll, A.-M.; Kong, H.; Yasuda,
K.; Mahan, L.; Bell, G. I.; Reisine, T. Characterization of cloned
somatostatin receptors SSTR4 and SSTR5. Mol. Pharmacol.
1993, 44, 385-392.
(6) (a) Buscail, L.; Delesque, N.; Esteve, J .-P.; Saint-Laurent, N.;
Prats, H.; Clerc, P.; Robberecht, D.; Bell, G. I.; Liebow, C.;
Schally, A. V.; Vaysse, N.; Susini, C. Stimulation of tyrosine
phosphatase and inhibition of cell proliferation by somatostatin
analogues - Mediation by human somatostatin receptor sub-
(21) J iang, X.; Goodman, M. Modeling Somatostatin Receptor-ligand
Complexes. Proceedings of the 15th American Peptide Sympo-
sium; in press.
(22) Ausabel, F. M.; Brent, R.; Kingston, R. E.; Moore, D. D.; Seidman,
J . G.; Smith, J . A.; Struhl, K. Current Protocols in Molecular
Biology; J ohn Wiley &Sons: New York, 1987.

Somatostatin Analogues with Novel Peptoid Residues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2685
(23) Kaupmann, K.; Bruns, C.; Hoyer, D.; Seuwen, K.; Lu¨bbert, H.
Distribution and second messenger coupling of four somatostatin
receptor subtypes expressed in brain. FEBS Lett. 1993, 331, 53-
59.
(24) Hoyer, D.; Neijt, H. C. Identification of serotonin 5-HT3 recogni-
tion sites in membranes of N1E-115 neuroblastoma cells by
radioligand binding. Mol. Pharmacol. 1988, 33, 303-309.
(25) Hoyer, D.; Engel, G.; Kalkman, H. O. Molecular pharmacology
of 5-HT1 and 5-HT2 recognition sites in rat and pig brain
membranes: radioligand binding studies with [3H]5-HT, [3H]8-
OH-DPAT, (-)[125I]iodocyanopindolol, [3H]mesulergine and [3H]-
ketanserin. Eur. J . Pharmacol. 1985, 118, 13-23.
(26) De Lean. SCTFIT: A Computer Program for Simultaneous
Analysis of Saturation and Competition Curves; Howard Hughes
Medical Institute, Duke University Medical Center: Durham,
NC, 1979.
(27) Bradford, M. M. A rapid and sensitive method for the quanti-
tation of microgram quantities of protein utilizing the prin-
ciple of protein-dye binding. Anal. Biochem. 1976, 72, 248-
254.
J M970393L