Template-constrained cyclic peptide analogues of somatostatin: Subtype-selective binding to somatostatin receptors and antiangiogenic activity

Article abstract of DOI:10.1016/S0968-0896(00)00135-8

β-Turns are a common secondary structure motif found in proteins that play a role in protein folding and stability and participate in molecular recognition interactions. Somatostatin, a peptide hormone possessing a variety of therapeutically-interesting b

Full text of DOI:10.1016/S0968-0896(00)00135-8

Bioorganic & Medicinal Chemistry 8 (2000) 2229±2241
Template-Constrained Cyclic Peptide Analogues of Somatostatin:
Subtype-Selective Binding to Somatostatin Receptors and
Antiangiogenic Activity
Daniel J. Suich,a,*,y Shaker A. Mousa,b Gurke Singh,c
George Liapakis,c Terry Reisinec and William F. DeGradoa,*
aDepartment of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104-6059, USA
bDuPont Pharmaceuticals Company, Wilmington, DE 19880, USA
cDepartment of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104, USA
Received 12 October 1999; accepted 4 May 2000
AbstractÐb-Turns are a common secondary structure motif found in proteins that play a role in protein folding and stability and
participate in molecular recognition interactions. Somatostatin, a peptide hormone possessing a variety of therapeutically-interest-
ing biological acitivities, contains a b-turn in its bioactive conformation. The b-turn and biological activities of somatostatin have
been succesfully mimicked in cyclic hexapeptide analogues. Two novel, structured, non-peptidic molecules were developed that are
capable of holding the bioactive tetrapeptide sequence of somatostatin analogues in a b-turn conformation, as measured by soma-
tostatin receptor (SSTR) binding. Template-constrained cyclic peptides in which the ends of the -Tyr-d-Trp-Lys-Val-tetrapeptide
were linked by sca€olds based on either an N,N0-dimethyl-N,N0-diphenylurea or a substituted biphenyl system (DJS631 and
DJS811, respectively), bound selectively to mouse SSTR2B and rat and human SSTR5 with anities as high as 1 nM. DJS811, at a
dose of 3 mg/kg/day, was shown in a mouse Matrigel model to inhibit angiogenesis to a level of 79%. The development of struc-
tured turn sca€olds allows b-turn sequences to be contained in the context of a compact structure, with less peptidic nature and
potentially greater bioavailability than cyclic hexapeptides. These systems can be used to study the determinants of b-turn forma-
tion, as well as to probe the importance of turn sequences occurring in molecular recognition interactions. The antiangiogenic
activity of DJS811 suggests that it may have antitumor activity as well. In addition, because SSTR2 is overexpressed on many types
of tumors, DJS631 and DJS811 may be useful in the development of agents for tumor imaging or the radiotherapy of cancer.
# 2000 Elsevier Science Ltd. All rights reserved.
Introduction
explore the requirements for turn formation, but also
directly as a therapeutic agent or as a probe of the bio-
logical processes mediated by its binding interactions.
Cyclic hexapeptides have been used for these purposes,
as they contain two fused b-turn structures, in which the
i residue of each turn corresponds to the i+3 residue of
the other. While the cyclic nature of these structures
gives them greater rigidity and hydrolytic stability than
their linear analogues, these compounds can still su€er,
due to their peptidic nature, from limited bioavailablity.
The b-turn is a common structural element in proteins
which not only contributes to protein folding and stability,
but also plays an important role in molecular recogni-
tion interactions.1 Molecular interactions in which b-
turns have been implicated as recognition elements
include the binding of peptide hormones to their
receptors,1 3 antibody-antigen recognition,4 7 post-trans-
lational modi®cation of proteins,1,8 and protein±protein
interactions.9 18 A means to contain the essential com-
ponents of the b-turn structure in the context of a small
molecule would be useful not only as a test bed to
One way to decrease the peptide content of a cyclic hexa-
peptide while further increasing its conformational rigid-
ity is to replace one of the b-turns with a synthetic
compound, which can either mimic the bioactive turn itself,
or serve as a structurally-constrained template or sca€old
to present the remaining peptide in a b-turn confor-
mation. A number of di€erent compounds have been
suggested as potential linkers for ®xing the conformation
*Corresponding authors. Tel.: +1-215-898-4590; fax: +1-215-573-7229;
e-mail: wdegrado@mail.med.upenn.edu
yCurrent address: Chiron Corporation, 4560 Horton St., Emeryville,
CA 94608, USA.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00135-8

2230
D. J. Suich et al. / Bioorg. Med. Chem. 8 (2000) 2229±2241
of cyclic peptides.19,20 Such linkers include derivatives
of 6-aminocaproic acids21 (vide infra), napthalene
derivatives,22,23 N-methyl-b-lactam derivatives,24 and
a-benzyl-o-aminomethylphenylacetic acid derivatives.25
b-Amino acids have also been used as templates for
incorporation into cyclic peptides.26,27 Kelly and co-
workers have similarly made use of dibenzofurans and
biaryl-based amino acids to encourage turn formation.19
A diphenylacetylene derivative has also been shown to
substitute well for a turn-forming dipeptide in b-hair-
pins.28 Nagai's bicyclic dipeptide turn mimetic BTD has
been incorporated into a number of di€erent bioactive
peptides and proteins,12,29,30 and benzodiazepine deri-
vatives have been incorporated into the cyclic peptide
gramicidin S with retention of activity.31 Finally, the
simple trans-dipeptide mimetic, m-aminomethylbenzoic
acid (mamb), has been independently developed by von
Itzstein32 and our own group33 to stabilize turn con-
formations in cyclic peptides. In the present work, we
have sought to extend this approach by developing struc-
turally-constrained sca€olds capable of presenting a tet-
rapeptide sequence such that the two central residues
adopt a turn conformation without any additional back-
bone constraints. A structure of this type would provide
a general system to study the parameters a€ecting b-
turn formation, as well as to generate bioactive mimet-
ics of b-turns with potentially greater bioavailability
and conformational rigidity than cyclic hexapeptides.
analogue agonists have been used therapeutically for the
control of acromegaly and certain endocrine tumors, and
as tumor imaging agents.36,38 While diabetes has been
treated with somatostatin agonists because of their abil-
ity to inhibit growth hormone release, antagonists of
SSTR5, capable of blocking the inhibition of insulin
release from the pancreas, would also be useful in the
treatment of type II diabetes.36,39,40 Somatostatin ana-
logues such as MK-678 (seglitide) and the cyclic octa-
peptides Sandostatin (octreotide, SMS 201-955) and
RC-160 (Table 1), all agonists, have been shown to inhibit
angiogenesis, the process by which new blood vessels are
formed.41,42 There has been substantial recent interest in
the inhibition of angiogenesis as a means of inhibiting
tumor growth and metastasis, as well as in the treatment
of diseases such as diabetic retinopathy and rheumatoid
arthritis.41 43 The interest in and therapeutic potential
of somatostatin analogues is underscored by e€orts to
generate libraries of somatostatin mimetics,44 and the
recent development of selective, non-peptide agonists of
somatostatin receptors.45 48
In this paper, we describe the design and synthesis of two
novel synthetic sca€olds capable of presenting a tetra-
peptide sequence in a b-turn conformation. Cyclic peptide
compounds in which these templates were used to dis-
play the -Tyr-d-Trp-Lys-Val- tetrapeptide turn sequence
from MK-678 bound to cloned somatostatin receptors
in vitro with high anity and selectivity. The more
potent of the analogues was further analyzed for its
ability to inhibit angiogenesis in a mouse model, and
exhibited strong antiangiogenic activity.
A simple means of determining the ability of a synthetic
sca€old to accomodate a tetrapeptide in the desired b-
turn conformation is to employ a bioactive peptide
sequence which requires such a conformation for high-
anity receptor binding. The ability of the constrained
peptide to bind to the receptor is then a direct measure
of how well the tetrapeptide adopts the required turn
conformation. For this purpose, we chose to mimic the
cyclic peptide hormone somatostatin (Table 1),34 for
which the bioactive conformation is known to be a b-
turn,35 and for which ®ve receptor subtypes have been
cloned and expressed.36 A potent cyclic hexapeptide
analogue of somatostatin, MK-678 (Table 1), contains
the sequence -Tyr-d-Trp-Lys-Val-, of which the central
-d-Trp-Lys- dipeptide forms the i+1 and i+2 residues
of a bII0-turn necessary for high anity receptor bind-
ing.37 Mimicry of somatostatin thus provides a simple
means of detecting the adoption of the desired turn
conformation. Additionally, the development of somato-
statin analogues is desirable due to the diverse, thera-
peutically-relevant biological activities of the natural
hormone. Somatostatin not only inhibits exocrine secre-
tions and the release of endocrine hormones such as
growth hormone, insulin, glucagon, and gastrin, but
also acts as a neurotransmitter and has direct anti-
proliferative e€ects on cells36 (vide infra). Somatostatin
Results and Discussion
Design of turn sca€olds
The ®rst role that a turn sca€old molecule must ful®ll is
that of geometry. Not only does the structure need to be
capable of joining the two ends of a tetrapeptide, it must
also bridge the gap in such a way as to allow the peptide
to adopt a turn conformation. A second requirement is
that the molecule itself contains signi®cant conforma-
tional constraint, such that it can hold the ends of the
tetrapeptide in the appropriate orientation without sig-
ni®cantly changing its own conformation. Two molecules
that could simultaneously ful®ll both of these require-
ments are shown in Figure 1. In Figure 1A is depicted an
N,N0-diphenylurea, which by virtue of its N,N0-dimethyl
substitution is expected to adopt a cisoid geometry
wherein the two aromatic rings lie adjacent and parallel
to each other, based on the X-ray structural analysis of
related compounds by Shudo and co-workers.49 The
plane of the rings is expected to be perpendicular to that
of the urea moiety (a). Given such a conformation, the
attached amide groups should then be adjacent to each
other as shown, at a distance capable of bridging the
ends of a tetrapeptide in a b-turn conformation.
Because the amides are directly attached to the aromatic
system, rotation about bonds b and c is expected to be
constrained to dihedral angles of Æ25ꢀ between the
plane of the amide and that of the aromatic ring. These
Table 1. Structures of somatostatin and analogues
Somatostatin-14
Ala-Gly-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-
Thr-Phe-Thr-Ser-Cys]-OH
MK-678
Sandostatin
RC-160
c[N-Me-Ala-Tyr-d-Trp-Lys-Val-Thr]
d-Phe-c[Cys-Phe-d-Trp-Lys-Thr-Cys]-Thr-ol
d-Phe-c[Cys-Tyr-d-Trp-Lys-Val-Cys]-Thr-NH2

D. J. Suich et al. / Bioorg. Med. Chem. 8 (2000) 2229±2241
2231
The ability of each of these sca€old structures to connect
the ends of a tetrapeptide adopting the bII0 turn con-
formation found in cyclic hexapeptide analogues of
somatostatin while still adopting a favorable conforma-
tion themselves was assessed and con®rmed by molecular
modeling. Structures were built in which each sca€old
was linked to the ends of the tetrapeptide -Ala-d-Ala-
Ala-Ala-. This tetrapeptide was used in lieu of the
actual peptide sequence of somatostatin analogues in
order to make the modeling less computationally inten-
sive, while still providing the constraint conferred by a
substituents in the peptide chain. When the ꢀ and angles
of the central -d-Ala-Ala- dipeptide of the structures were
restrained to those of a canonical bII0 turn (60ꢀ, 120ꢀ,
80ꢀ, 0ꢀ) and the structures were energy minimized,
reasonable conformations were obtained for the sca€old
moieties, in accordance with the considerations discussed
above (Figs 2 and 3). Analysis of these structures by
Figure 1. Parameters a€ecting conformational ¯exibility and con-
straint in designed b-turn sca€olds. Panel A depicts an N,N0-dimethyl-
N,N0-diphenylurea system in which constraint is conferred by the
cisoid geometry of the substituted urea a, restricted rotation about
bonds b and c, and hydrogen bond d. Panel B depicts a substituted
biphenyl system in which ¯exibility is provided by free rotation about
bonds e, f, and g, and constraint is imposed by restricted rotation
about bond h, steric clashes i and j between substituents ortho to the
interaromatic bond, and hydrogen bond k.
values for the dihedral angle were observed in a search
of relevant small-molecule crystal structures in the
Cambridge database, and can be explained by the bal-
ance of two opposing forces. Coplanarity is favored due
to overlap between the p systems of the amide and aro-
matic ring, but disfavored due to steric clashes between
the amide and hydrogens ortho to the point of attach-
ment. Finally, this structure should allow the ®rst and
last amide of an attached peptide to hydrogen bond to
each other as indicated by d. Such a hydrogen bond
corresponds to that between residues i and i+3 of a
cyclic hexapeptide, and its presence would provide further
conformational constraint on the system.
Figure 2. Energy-minimized model of template-constrained cyclic
peptide in which the N,N0-dimethyl-N,N0-diphenylurea sca€old links
the ends of the tetrapeptide -Ala-d-Ala-Ala-Ala-. The ꢀ and angles
of the i+1 (d-Ala) and i+2 residues of the peptide have been con-
strained to those of a canonical bII0-turn. The carbon atoms of each
sca€old are in yellow, and those of the peptides are in green; oxygens
atoms are red, nitrogens blue, and hydrogens cyan.
The biphenyl compound shown in Figure 1B was also
anticipated to ful®ll the requirements of appropriate
geometry and conformational constraint necessary for a
turn sca€old. The conformational space available to
this structure is limited by the restricted rotation about
the interaromatic single bond g due to a balance of
opposing e€ects. Overlap of the p systems of the rings
favors their coplanarity, whereas steric clashes between
substituents ortho to the interaromatic linkage such as
hydrogen atoms and the methylamino group (i and j)
disfavor coplanarity. For the same reasons discussed
above for the substituted urea system, the direct
attachment of the carboxyl group to the biphenyl
moiety (h) provides further constraint, with an expected
dihedral angle of Æ25ꢀ. Again, as for the substituted
urea system, the conformation of the biphenyl system
should allow hydrogen bonding to occur between
the ®rst and last amides of an attached peptide, pro-
viding additional constraint. At the same time, ¯ex-
ibility is present in this structure that should allow the
molecule to adopt a conformation capable of connecting
the ends of a tetrapeptide in a b-turn conformation.
Flexible connections are provided by the two rotatable
single bonds in the methylamino substiuent (e and f ), as
well as the soft potential asssociated with the inter-
aromatic single bond (g).
Figure 3. Energy-minimized model of template-constrained cyclic
peptide in which the substituted biphenyl sca€old links the ends of
the tetrapeptide -Ala-d-Ala-Ala-Ala-. The ꢀ and angles of the i+1
(d-Ala) and i+2 residues of the peptide have been constrained to
those of a canonical bII0-turn. The carbon atoms of each sca€old are
in yellow, and those of the peptides are in green; oxygens atoms are
red, nitrogens blue, and hydrogens cyan.

2232
D. J. Suich et al. / Bioorg. Med. Chem. 8 (2000) 2229±2241
dynamics and energy minimization without any restraints
on the peptide conformation revealed that while the sub-
stituted urea system is fairly rigid, the biphenyl system
confers signi®cant ¯exibility on the structure, an observa-
tion that is congruent with the presence of rotatable bonds
in the biphenyl system as noted in the discussion above.
Synthesis of cyclic peptide analogues of somatostatin
incorporating turn sca€olds
The cyclic peptide analogues of somatostatin incorpor-
ating the urea and biphenyl turn sca€olds discussed
above were synthesized as laid out in Schemes 1, 2, and 3.
Scheme 1. Synthesis of the N,N0-dimethyl-N,N0-diphenylurea sca€old derivatized with FmocVal.
Scheme 2. Synthesis of the substituted biphenyl sca€old derivatized with Fmoc-Val.
Scheme 3. Incorporation of Fmoc-Val-derivatized b-turn sca€olds into template-constrained cyclic peptide somatostatin analogues DJS631 and
DJS811.

D. J. Suich et al. / Bioorg. Med. Chem. 8 (2000) 2229±2241
2233
In each case, the sca€old was synthesized in solution,
followed by coupling of Fmoc-l-valine (Fmoc=9-
¯uorenylmethyloxycarbonyl), corresponding to the last
residue of the tetrapeptide, using standard solution
phase methods. Next, these intermediates were extended
using solid-phase peptide synthesis methods to intro-
duce the remainder of the tetrapeptide sequence. After
cleavage of the linear, protected intermediates contain-
ing the sca€olds from the resin, cyclization and depro-
tection in solution a€orded the desired products.
tion of 10 mM, and the protecting groups were removed
from the side chains via catalytic reduction to a€ord the
desired template-constrained cyclic peptide somatostatin
analogues DJS631 and DJS811 as their TFA salts after
puri®cation by reverse-phase high pressure liquid chro-
matography (HPLC).
Binding of cloned somatostatin receptors (SSTRs) by
DJS631 and DJS811
As shown in Table 2, compounds DJS631 and DJS811
bound selectively to somatostatin receptors SSTR2B
(mouse, m) and SSTR5 (rat, r and human, h). In each
case, the compounds bound more tightly to SSTR2B
and with lower anity to SSTR5, and DJS811 bound to
the receptors more tightly than DJS631. The IC50s of
DJS631 and DJS811 for mSSTR2B, 7 and 1.0 nM,
respectively, compare favorably with that of somatostatin
for this receptor, 0.6 nM (Table 2).50 Both compounds
have a similar preference for binding mSSTR2B over
rSSTR5, with DJS631 binding 11-fold more potently to
mSSTR2B, and DJS811 with a 13-fold preference for
this receptor. DJS631 discriminates between rat and
human SSTR5 (IC50s of 80 and 220 nM, respectively) to
a greater extent than does DJS811 (IC50s of 13 and
15 nM, respectively). DJS811 is more similar to soma-
tostatin in this regard, which has nearly equal anity
for these two receptors (0.3 and 0.2 nM, respectively).36
The pattern of selectivity of DJS631 and DJS811 for
the cloned somatostatin receptors, in that they have
high anity for SSTR2 and SSTR5, but do not bind to
the others, is similar to that of analogues such as MK-678
and Sandostatin, except that these latter analogues do
have limited anity for SSTR3 (Table 2).36 Somatostatin,
by contrast, binds to all receptor subtypes with similar
anity, in the tenth-nanomolar to nanomolar range.36
For the urea sca€old, the t-butyl ester of 4-aminobenzoic
acid 1 was coupled to 4-nitroaniline with triphosgene to
a€ord the unsymmetrical urea intermediate 2. Methyla-
tion of 2 with CH3I, followed by catalytic reduction of the
nitro group a€orded the t-butyl ester-protected aniline
derivative 3. After coupling of Fmoc-l-Val-OH to 3 using
2 - (1H - benzotriazol - 1 - yl) - 1,1,3,3 - tetramethyluronium
hexa¯uorophosphate (HBTU), the t-butyl ester was
cleaved to a€ord the Fmoc-valine-derivatized urea scaf-
fold as the free acid (4) in 61% overall yield. The syn-
thesis of the Fmoc-valine-derivatized biphenyl sca€old
begins with the coupling of ethyl 3-iodobenzoate to the
commercially-available 2-formylbenzeneboronic acid
under Suzuki conditions to generate the biphenyl nucleus
5. Oxime formation using hydroxylamine hydrochloride,
followed by reduction with Zn0 a€orded the desired 2-
aminomethyl derivative 7 isolated as the hydrochloride
salt. The zwitterion resulting from saponi®cation of the
ethyl ester in 7 was coupled with Fmoc-l-Val-OH using
HBTU to yield the Fmoc-valine-derivatized biphenyl
sca€old as the free acid (8) in 30% overall yield.
Each of the Fmoc-valine-derivatized sca€old free acids
4 and 8 were coupled to Wang resin using diisopro-
pylcarbodiimide (DIPC) and 4-(dimethylamino)pyridine
(DMAP) in CH2Cl2. Using standard Fmoc peptide
synthesis methodology, Fmoc-l-Lys(Z)-OH (Z=benzyl-
oxycarbonyl), Fmoc-d-Trp-OH, and Fmoc-l-Tyr(2,6-
dichlorobenzyl)-OH were coupled in succession to the
resin-bound intermediates using HBTU. Cleavage of the
linear intermediates from the resin with a cocktail con-
taining tri¯uoroacetic acid (TFA) and cation scavengers
in CH2Cl2 a€orded side-chain-protected peptides 9 and 10
which incorporate the turn sca€olds at their C-termini.
These intermediates were cyclized in solution using 2-(7-
aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexa¯uorophosphate (HATU) at a peptide concentra-
The higher anity of DJS811 overall for the somatostatin
receptors compared with DJS631 could be attributed to
either of two factors. One is the greater hydrophobicity
of DJS811, a property inferred from its greater reten-
tion time by reverse-phase HPLC. A second is the
apparent greater ¯exibility of DJS811 compared with
DJS631 seen in the molecular modeling studies dis-
cussed previously. In the ®rst case, a more hydrophobic
ligand would be expected to bind with higher anity
to a lipophilic receptor than a more polar ligand, due to
the hydrophobic e€ect. A correlation between greater
Table 2. Anities of DJS631 and DJS811 for cloned somatostatin receptorsa
IC50 (nM)
Receptor
DJS631
DJS811
Somatostatin
MK-678
Sandostatin
hSSTR1
mSSTR2B
mSSTR3
hSSTR4
rSSTR5
hSSTR5
>1000 (2)
7 (3)
ꢁ1000 (2)
>1000 (1)
80 (3)
>1000 (2)
0.1
0.6
0.1
1.2
0.3
0.2
>1000
0.1
268
>1000
1.3
23
>1000
0.2
150
>1000
0.2
32
1.0 (3)
>1000 (2)
ND
13 (3)
220 (3)
15 (3)
aResults of radioligand displacement assay for DJS631 and DJS811 against cloned human (h), mouse (m), and rat (r) somatostatin receptors
(SSTRs). IC50 values calculated (in nM) represent the concentration of DJS631 or DJS811 capable of displacing half of the bound radioligand
[
125I]tyr11-somatostatin-14 from the receptor in vitro. The number of experiments is indicated in parentheses. Anities of somatostatin, MK-678,
and Sandostatin for these receptors measured by a similar method are shown for comparison.36,50

2234
D. J. Suich et al. / Bioorg. Med. Chem. 8 (2000) 2229±2241
hydrophobicity and higher potency for cyclic hexapeptide
somatostatin analogues has been observed.37 In the
second case, one would generally argue that ¯exibility in
a ligand would decrease its anity for a receptor, com-
pared with a ligand that is rigidly held in an optimal
binding conformation, due to the greater amount of
entropy loss upon binding for the more ¯exible molecule.
However, if the more rigid ligand is locked in a sub-
optimal binding conformation, a similar but more ¯exible
ligand may be able to o€set the negative e€ect of entropy
losses upon binding by having access to a conformation
optimal for receptor interaction, and as a result bind
more tightly than the rigid ligand. By either line of
reasoning, DJS811, being both more hydrophobic and
putatively more ¯exible than DJS631, would be expected
to bind with higher anity overall to the receptors,
which could explain the anities observed in Table 2.
The greater ¯exibility of DJS811 can also explain the
lower discrimination DJS811 displays in binding rat ver-
sus human SSTR5 than does DJS631. A ¯exible ligand
has a greater ability than a more rigid one to adjust to
subtle di€erences in the binding sites of various receptor
subtypes, and bind to them despite those di€erences.
ni®cant response to the treatment. Visualization of gel
slices stained by an immunohistochemical method sensi-
tive to the presence of platelet endothelial cell adhesion
molecule-1 (PECAM-1) protein found in blood vessels
con®rmed the inhibition of capillary growth in mice
treated with DJS811.
Given the receptor subtype selectivity of DJS811, there
are at least two possible explanations as to its mode of
action in inhibiting new blood vessel formation. One
possibility is the inhibition of growth hormone release,
as has been suggested for the observed antiangiogenic
e€ect of Sandostatin and RC-160 in the chicken chor-
ioallantoic membrane model,42 as well as the inhibition
of retinal neovascularization by MK-678.41 High anity
agonist binding to SSTR2 has been shown to mediate the
inhibition of growth hormone release.36,52 Given its
high anity for SSTR2, DJS811 may also inhibit
angiogenesis by this mechanism. Another mechanism by
which the antiangiogenic activity may be mediated is the
stimulation of tyrosine phosphatase activity induced by
binding of agonist analogues of somatostatin to
SSTR253,54 or possibly SSTR5.55,56 Stimulation of such
tyrosine phosphatase activity by the binding of DJS811 to
SSTR2 or SSTR5 could counteract the e€ects of endo-
thelial cell transmembrane receptor tyrosine kinases
which mediate the angiogenic process.57,58
Antiangiogenic activity of DJS811
Using a mouse model in which capillary growth is
induced in an implanted gel matrix called Matrigel,51
the ability of DJS811 to inhibit angiogenesis was tested.
A positive control, in which mice (eight animals) were
subcutaneously injected with Matrigel containing basic
®broblast growth factor (bFGF) gave a value of
2.9Æ0.4 g/dL hemoglobin content after a period of 7
days (Table 3). For similarly-treated mice (10 animals)
that in addition received an infusion of 3 mg/kg/day of
DJS811, hemoglobin content of the Matrigel after the
same time period was only 0.68Æ0.41 g/dL, which
represents a 79Æ18% inhibition of angiogenesis. The
negative control group, which received Matrigel
implants, but no bFGF and no DJS811 (10 animals)
gave a hemoglobin content of 0.08Æ.01 g/dL. Impor-
tantly, every mouse treated with DJS811 showed a sig-
Conclusions
We have synthesized two novel structured sca€olds
designed to present a tetrapeptide sequence in a turn
conformation without additional backbone constraints.
Sca€olds based on both N,N0-dimethyl-N,N0-diphenyl-
urea and substituted biphenyl systems were designed
using molecular modeling techniques, taking into con-
sideration both conformational constraint and ¯exibility
within these systems. The designed sca€old molecules
were synthesized readily in good overall yield, and were
then incorporated into cyclic peptides containing the
bioactive tetrapeptide sequence from the somatostatin
analogue MK-678. Both of these template-constrained
cyclic peptides DJS631 and DJS811 bound selectively
to cloned somatostatin receptors mSSTR2 and rat and
human SSTR5 with high anity, indicating that the
tetrapeptide sequence was indeed adopting a b-turn
conformation about the central -d-Trp-Lys- dipeptide
as intended. The exact conformation of the tetrapeptide
sequence and the sca€olds in these molecules is cur-
rently under further investigation by NMR. This work
parallels that of van Binst and co-workers, who have
demonstrated SSTR2-selective binding of both diaster-
eomers of a template-constrained cyclic peptide contain-
ing a similar tetrapeptide sequence and racemic a-benzyl-
o-aminomethylphenylacetic acid as a template.25,59 The
development of structured turn sca€olds allows turn
sequences to be contained in the context of a compact
structure. These molecules might display greater stability
and bioavailability than their cyclic hexapeptide counter-
parts, and can be used to probe the importance of turn
sequences occurring in molecular recognition interac-
tions. Additionally, such systems can be used to study
Table 3. Antiangiogenic activity of DJS811 in the mouse Matrigel
modela
Positive control
Treated
3.3
2.8
2.6
3.7
3.0
3.0
2.5
2.4
0.81
0.16
0.04
0.38
0.56
0.63
1.05
0.78
1.27
1.16
MeanÆSD
2.9Æ0.4
0.68Æ0.41
Inhibition level: 79Æ18%
aValues for hemoglobin content, reported in g/dL, of bFGF-treated
Matrigel implanted in mice for 7 days. Each listed value is from an
individual animal. The treated group received 3 mg/kg/day of DJS811,
while the positive control group did not. A 79% level of inhibition of
angiogenesis was calculated from these data as described in the text.

D. J. Suich et al. / Bioorg. Med. Chem. 8 (2000) 2229±2241
2235
the determinants of turn formation in proteins and
peptides by varying the tetrapeptide that is incorpor-
ated. For example, the importance of the relative spatial
orientation of the ends of the peptide, enforced by the
sca€old, could be compared to the role of local interac-
tions such as chirality and side chain±side chain interac-
tions in determining the turn conformation.21,60,61 We
have also shown DJS811 to be a potent inhibitor of
angiogenesis in a mouse Matrigel model, suggesting that
this compound may have antitumor activity as well; this
possibility is currently under investigation. Finally,
because somatostatin receptors, particularly SSTR2, are
overexpressed on many types of tumor cells, both DJS631
and DJS811 may be useful in the development of agents
for tumor imaging or radiotherapy of cancer.62,63
(d, 2H, J=9.0 Hz), 1.62 (s, 9H). HRMS: calcd for
C11H14NO4 (M+H)+, 224.0923; found, 224.0922.
tert-Butyl 4-aminobenzoate (1). tert-Butyl 4-nitrobenzo-
ate (22.32 g, 100 mmol) was dissolved in tetrahydrofuran
(THF) (120 mL) and H2O (40 mL), and Pd/C (10 wt%
Pd, 1.064 g, 1 mol% Pd) was added. The reaction vessel
was purged with Ar, then H2, and pressurized with H2 to
53 psi. The reaction mixture was allowed to shake at room
temperature for 19h. The reaction vessel was purged with
Ar, the reaction mixture was ®ltered through Celite, and
the ®lter bed was washed with CH3CN. The ®ltrate was
concentrated in vacuo, and to the resulting solid residue
was added saturated NaHCO3 and EtOAc. The pH of
the mixture was adjusted to 10 with aqueous NaOH, the
layers were shaken together and separated, and the aque-
ous layer was extracted three times more with EtOAc.
The organic layers were combined, dried with MgSO4,
and the solvent was evaporated to a€ord 1 (15.41 g,
Experimental
1
Molecular modeling
80%) as a light-orange crystalline solid. H NMR (ace-
tone-d6, 300 MHz) d 7.68 (d, 2H, J=8.7 Hz), 6.64 (d,
2H, J=8.7 Hz), 5.31 (br. s, 2H), 1.52 (s, 9H). HRMS:
calcd for C11H15NO2 (M)+, 193.1103; found, 193.1098.
The software packages Insight II 95.0/Discover 2.9.7
(Biosym/MSI) and Sybyl 6.03 (Tripos Associcates) were
utilized in visualizing and energy minimizing structures.
Sybyl was utilized for the dynamics and energy minimiza-
tion studies, using the tripos force ®eld, a temperature of
800 K, a dielectric constant of 15, and a 50 ps run time
with snapshots taken every 50 fs for the dynamics runs.
The 500 lowest energy structures were energy minimized
and analyzed.
1-(40-Nitrophenyl)-3-(400-tert-butylcarboxyphenyl)urea (2).
To 375 mL anhydrous THF under Ar was added 4-
nitroaniline (12.24g, 88.60 mmol) with stirring. After dis-
solution of the solid, the reaction mixture was cooled to
0 ꢀC. Triphosgene (8.76 g, 29.5 mmol) was added, then
diisopropylethylamine (DIEA) (30.87mL, 177.2 mmol)
was added in three portions over 5 min. The reaction
mixture was allowed to stir for 5 min at 0 ꢀC, then at
room temperature for 45 min. The reaction mixture was
again cooled to 0 ꢀC. 1 (15.41 g, 79.74 mmol) was dis-
solved in 125 mL of anhydrous THF and added to the
reaction mixture. The cooling bath was removed, and the
reaction mixture was allowed to stir for an additional
42 h. The solvent was evaporated, and the residue was
dissolved in CH2Cl2. The solution was extracted three
times with 1 N NaHSO4, at which point a solid began
precipitating from the organic layer. The organic layer
suspension was washed once with brine, and enough
THF was added to the organic layer to bring the solid
back into solution. The organic layer was dried with
MgSO4 and the solvent was evaporated. The residue
was triturated with Et2O, the mixture was ®ltered, and
the solid was washed several times with Et2O. The resi-
dual solvent was removed from the solid under vacuum
Synthesis of DJS631 and DJS811
General. The purity of peptide derivatives was assessed
and the progress of reactions involving peptide derivatives
was monitored using analytical reverse-phase HPLC on a
C18 column, with mobile phases consisting of water and
CH3CN containing 0.1% TFA. Puri®ed synthetic inter-
mediates were characterized by 1H NMR and high-reso-
lution mass spectrometry (HRMS), and were judged to
be pure by either thin-layer chromatography (silica gel)
or reverse-phase HPLC, as appropriate.
tert-Butyl 4-nitrobenzoate. To a slurry of 4-nitrobenzoic
acid (16.71 g, 100 mmol) in 200 mL pyridine was added
tosyl chloride (38.13 g, 200 mmol) with stirring. Once
the solid was completely dissolved, the solution was
chilled to 0 ꢀC, and tert-butanol (7.41 g, 100 mmol)
was added. After stirring at 0 ꢀC for 2 h, the reaction
mixture was allowed to warm to room temperature.
After an additional 2 h of stirring, another portion of
tert-butanol (7.41 g, 100 mmol) was added. The reaction
mixture was allowed to stir for an additional 14 h. The
solvent was evaporated, and the residue was partitioned
between EtOAc and saturated NaHCO3. The organic
layer was washed three times with saturated NaHCO3,
and the pooled aqueous layers were washed once with
EtOAc. The organic layers were pooled and washed
once with brine, four times with 1 N NaHSO4, once
more with brine, and ®nally dried with MgSO4. The
solvent was evaporated to give the title compound
(22.32 g, 100%) as a light-yellow solid. 1H NMR
(CDCl3, 300 MHz) d 8.26 (d, 2H, J= 9.0 Hz), 8.15
1
to yield 2 (28.50 g, 100%) as a light-yellow solid. H
NMR (acetone-d6, 300 MHz) d 9.54 (s, 1H), 9.27 (s, 1H),
8.18 (d, 2H, J= 9.2 Hz), 7.88 (d, 2H, J=8.8 Hz), 7.80 (d,
2H, J=9.2 Hz), 7.65 (d, 2H, J=8.8 Hz), 1.56 (s, 9H).
HRMS: calcd for C18H19N3O5 (M)+, 357.1325; found,
357.1335.
1-(40-Nitrophenyl)-3-(400-tert-butylcarboxyphenyl)-1,3-di-
methylurea (2a). To 100 mL of anhydrous dimethylfor-
mamide (DMF) under Ar was added 2 (9.10g, 25.5 mmol)
with stirring. After dissolution of 2, the reaction mixture
was cooled to 0 ꢀC in a bath, and NaH (60 wt%, 2.80 g,
70.0 mmol) was added in a single portion. After a fur-
ther 5 min of stirring, CH3I (17.4 mL, 280 mmol) was
added, and the reaction mixture was allowed to stir at

2236
D. J. Suich et al. / Bioorg. Med. Chem. 8 (2000) 2229±2241
0 ꢀC for 15 min. The cooling bath was removed, and the
reaction mixture was allowed to stir for 3 h at room
temperature. The reaction was quenched by the addition
of 1 N NaHSO4 to a pH of 5. The solvent was evapor-
ated in vacuo, and the residue was puri®ed by silica gel
¯ash column chromatography (eluted with 35% EtOAc in
hexanes). The solvent was evaporated to yield 2a (8.24 g,
84%) as a yellow solid. 1H NMR (acetone-d6, 300 MHz) d
8.00 (d, 2H, J=9.2 Hz), 7.74 (d, 2H, J=8.8 Hz), 7.24 (d,
2H, J=9.2 Hz), 7.16 (d, 2H, J=8.7Hz), 3.32 (s, 3H), 3.25
(s, 3H), 1.52 (s, 9H). HRMS: calcd for C20H24N3O5
(M+H)+, 386.1716; found, 386.1717.
Hz), 4.07 (dd, 1H, J=7.0 Hz, 8.9 Hz), 3.18 (s, 3H), 3.12
(s, 3H), 2.14 (m, 1H), 1.51 (s, 9H), 0.97 (d, 6H, J=
6.8 Hz). HRMS: calcd for C40H44N4O6Na (M+Na)+,
699.3158; found, 699.3153.
1-(9000-Fluorenylmethyloxycarbonyl-L-valyl-(40-aminophe-
nyl))-3-(400-carboxyphenyl)-1,3-dimethylurea (4). Com-
pound 3a (3.19 g, 4.71 mmol) was dissolved in CH2Cl2
(20 mL) with stirring, and anisole (2 mL) was added.
The reaction mixture was cooled on an ice bath to 0 ꢀC,
and TFA (20 mL) was added. The reaction was allowed
to stir for 9 h at 0 ꢀC, and then for 1 h at room tem-
perature. The solvent was evaporated in vacuo, and the
residue was coevaporated several times with CH2Cl2.
The residue was partitioned between CH2Cl2 and 1 N
NaHSO4, and the organic layer was extracted two more
times with 1 N NaHSO4. The aqueous layers were
combined and extracted three times with CH2Cl2. The
organic layers were pooled, dried with MgSO4, and the
solvent was removed in vacuo to a€ord 4 (2.85g, 97%) as
an o€-white foam. 1H NMR (acetone-d6, 300 MHz) d 9.26
(s, 1H), 7.85 (d, 2H, J=7.5 Hz), 7.77 (d, 2H, J=8.7 Hz),
7.71 (dd, 2H, J=6.8Hz, 6.8 Hz), 7.46 (d, 2H, J=8.8 Hz),
7.39 (dd, 2H, J=7.4Hz, 7.4 Hz), 7.28 (d, 2H, J=7.6 Hz),
7.05 (d, 2H, J=8.8Hz), 6.95 (d, 2H, J=8.9Hz), 6.67 (d,
1H, J=9.0Hz), 4.32 (m, 2H), 4.23 (dd, 1H, J=7.0Hz,
14.1Hz), 4.07 (dd, 1H, J=6.9 Hz, 9.0 Hz), 3.19 (s, 3H),
3.14 (s, 3H), 2.12 (m, 1H), 0.96 (d, 6H, J=6.8 Hz).
HRMS: calcd for C36H36N4O6Na (M+Na)+, 643.2533;
found, 643.2547.
1-(40 -Aminophenyl)-3-(400 -tert-butylcarboxyphenyl)-1,3-
dimethylurea (3). Compound 2a (8.21 g, 21.3 mmol) was
dissolved in 150 mL of THF and 50mL of H2O, and Pd/C
(10 wt%, 227 mg, 1 mol% Pd) was added. The reaction
vessel was purged with Ar, then H2, and pressurized with
H2 to 55 psi. The reaction mixture was shaken at room
temperature for 18 h, then the reaction vessel was purged
with Ar, and the reaction mixture was ®ltered through
Celite. The ®lter bed was washed with H2O and THF.
The ®ltrate was concentrated in vacuo, and the residue
was partitioned between EtOAc and 1 N NaHSO4. The
organic layer was extracted nine more times with 1 N
NaHSO4, and 10 times with 1 N HCl. The aqueous
layers were pooled and the pH was adjusted to 10.5 with
the addition of aqueous NaOH. The aqueous solution
was extracted four times with EtOAc, adjusting the pH
up to 10.5 with aqueous NaOH after the ®rst extraction.
The organic layers were pooled, dried with MgSO4, and
the solvent was evaporated in vacuo to a€ord 3 (7.32 g,
97%) as a brown foam. 1H NMR (acetone-d6, 300 MHz)
d 7.72 (d, 2H, J=8.9 Hz), 7.00 (d, 2H, J=8.9 Hz), 6.69
(d, 2H, J=8.8 Hz), 6.43 (d, 2H, J=8.8 Hz), 4.54 (br. s,
2H), 3.12 (s, 3H), 3.05 (s, 3H), 1.54 (s, 9H). HRMS: calcd
for C20H25N3O3 (M)+, 355.1890; found, 355.1830.
Coupling of 4 to Wang resin to generate resin-bound in-
termediate 9. Wang resin (0.85 mmol/g substitution
level, 1.18 g, 1 mmol), 4 (931 mg, 1.5 mmol), and DMAP
(12.2 mg, 0.1 mmol) were dissolved/suspended in 12 mL
CH2Cl2 with stirring using anhydrous conditions under
Ar. To the reaction mixture was added DIPC (266 mL,
1.7 mmol), and the reaction mixture was allowed to stir
under Ar for 12 h. The reaction mixture was ®ltered and
the resin washed with CH2Cl2 and MeOH, alternating
between the two. The resin was dried under vacuum, then
a small portion was deprotected with 20% piperidine/
DMF and used to determine the level of derivatization
using the picric acid assay. The A358 of the ®ltrate indi-
cated a derivatization level of 52%. The total weight of
derivatized resin 9 was 1.65 g.
1-(9000-Fluorenylmethyloxycarbonyl-L-valyl-(40-aminophe-
nyl))-3-(400 -tert-butylcarboxyphenyl)-1,3-dimethylurea
(3a). Fmoc-l-valine (3.39 g, 10.0 mmol) and HBTU
(3.79 g, 10.0 mmol) were partially dissolved in DMF
(10 mL) with stirring. DIEA (1.74 mL, 10.0 mmol) was
added, and the solution was allowed to stir for an addi-
tional 20 min. The solution was transferred to a ¯ask
containing 3 (1.78 g, 5.0 mmol), DMF (5 mL) was added,
and the reaction mixture was allowed to stir at room
temperature for 22 h. The solvent was evaporated in
vacuo, and the residue was partitioned between EtOAc
and 1 N NaHSO4. The organic layer was extracted two
more times with 1 N NaHSO4, once with brine, three
times with saturated NaHCO3, and once with brine.
The organic layer was dried with MgSO4 and the sol-
vent was evaporated in vacuo. The residue was puri®ed
by silica gel ¯ash column chromatography, eluting with
1% MeOH in CH2Cl2. The solvent was removed in
vacuo to yield 3a (3.28 g, 97%) as an o€-white foam. 1H
NMR (acetone-d6, 300 MHz) d 9.26 (s, 1H), 7.85 (d, 2H,
J=7.5 Hz), 7.70 (d, 2H, J=8.8 Hz), 7.70 (d, 2H,
J=ꢁ8 Hz), 7.47 (d, 2H, J=8.9 Hz), 7.39 (dd, 2H, J=
7.4 Hz, 7.4 Hz), 7.29 (dd, 2H, J=7.3 Hz, 7.3 Hz), 7.02
(d, 2H, J=8.8 Hz), 6.95 (d, 2H, J=8.8 Hz), 6.67 (d, 1H,
J=8.9 Hz), 4.32 (m, 2H), 4.23 (dd, 1H, J=7.2 Hz, 14.3
Linear peptide derivative 11. Derivatized Wang resin 9
(1.65 g, equivalent to 1 mmol of original Wang resin)
was washed with DMF in a fritted synthesis vessel and
then deprotected with 20% piperidine/DMF for 25min
with gentle mixing. The solution was removed via suction
and the resin was washed with DMF and CH2Cl2,
alternating between the two and ending with DMF. A
Kaiser ninhydrin test performed on a resin sample was
positive. Fmoc-l-Lys(Z)-OH (1.01g, 2.00mmol) and
HBTU (759mg, 2 mmol) were partially dissolved in DMF
(3 mL) with stirring, and DIEA (523 mL, 3 mmol) was
added. After stirring for 15min, the solution was added to
the resin. Additional DMF (1.0 mL) was used to rinse
the reaction vessel and was then added to the resin to
make a free-¯owing slurry. The slurry was mixed gently
for 2 h, then the solution was removed via suction. The

D. J. Suich et al. / Bioorg. Med. Chem. 8 (2000) 2229±2241
2237
resin was washed with DMF, CH2Cl2, and ®nally DMF.
A Kaiser test performed on a resin sample was negative.
The above procedure was repeated to couple Fmoc-d-
Trp-OH (853 mg, 2.00 mmol) and then Fmoc-l-Tyr
(2,6-dichlorobenzyl)-OH (1.12 g, 2.00 mmol). In each
case the derivatized resin gave a negative Kaiser test.
The resin was deprotected with 20% piperidine/DMF,
the solution was removed via suction, and the resin was
washed with DMF, CH2Cl2, DMF, CH2Cl2, and air
dried. The resin was suspended in triisopropylsilane:
anisole:TFA:CH2Cl2 (6:4:25:15) (25 mL), and was stirred
for 2 h. The resin was ®ltered and washed with TFA and
CH2Cl2, and the ®ltrate was concentrated in vacuo. The
residue was co-evaporated several times with CH2Cl2,
and then triturated with pentane:Et2O (2:1). The residue
was dissolved in THF, concentrated in vacuo, and the
residue was coevaporated several times with CH2Cl2.
The residue was puri®ed by preparative reverse-phase
HPLC on a C18 column, using mobile phases of water
and CH3CN containing 0.1% TFA. The eluate was
lyophilized, the residue was coevaporated from THF,
and the residual solvent was removed in vacuo to a€ord
the TFA salt of 11 (294 mg, 44%) as a tan solid. HRMS:
calcd for C62H68N9O10Cl2 (M+H)+, 1168.4466; found,
1168.4492.
Ethyl 3-iodobenzoate. 3-Iodobenzoic acid (10.0 g, 40.3
mmol) and DMAP (1.48 g, 12.1 mmol) were dissolved in
EtOH (130 mL) with stirring, and the solution was
cooled to 0 ꢀC on an ice bath. Dicyclohexylcarbodiimide
(DCC) (9.98 g, 48.4 mmol) was added, and the reaction
was allowed to stir for 16 h, gradually warming to room
temperature. The reaction mixture was ®ltered, the solid
washed with EtOAc, and the ®ltrate was concentrated in
vacuo. The residue was puri®ed by silica gel ¯ash column
chromatography, eluting with 3% EtOAc in hexanes.
The solvent was removed in vacuo to a€ord the title
compound (10.69 g, 96%) as a yellow oil. 1H NMR
(CDCl3, 300 MHz) d 8.38 (s, 1H), 8.01 (d, 1H,
J=7.8 Hz), 7.88 (d, 1H, J=7.8 Hz), 7.18 (dd, 1H,
J=7.9 Hz, 7.9 Hz), 4.38 (q, 2H, J=7.1 Hz), 1.40 (t, 3H,
J=7.1 Hz). HRMS: calcd for C9H9O2I (M)+, 275.9647;
found, 275.9643.
3-Ethylcarboxy-20-formylbiphenyl (5). Ethyl 3-iodo-
benzoate (5.06 g, 18.3 mmol) was dissolved in toluene
(33 mL) with stirring. Aqueous 2 M Na2CO3 (16.7 mL)
was added, the reaction mixture was purged with Ar, and
Pd(PPh3)4 (578 mg, 0.5 mmol, 3 mol% Pd) was added,
repurging the reaction mixture with Ar. 2-Formylben-
zeneboronic acid (2.50 g, 16.7 mmol) was dissolved in
EtOH (8.3 mL) and added to the reaction mixture, and
the reaction mixture was purged with Ar again. The
reaction mixture was heated to re¯ux and allowed to stir
for 14 h. The solvent was evaporated from the reaction
mixture in vacuo, and the residue was puri®ed by silica gel
¯ash column chromatography, eluting with 5% EtOAc in
hexanes. The solvent was removed in vacuo to a€ord 5
Cyclization of 11 to 11a. Protected linear peptide deriva-
tive 11 (134mg, 104 mmol) and HATU (43.7 mg, 114
mmol) were dissolved in anhydrous DMF (10.4 mL) in an
oven-dried ¯ask, and the solid was dissolved with stirring.
DIEA (63.7 mL, 365 mmol) was added, and the reaction
vessel was purged with Ar. The reaction mixture was
allowed to stir for 20 h, the solvent was removed in
vacuo, and the residue was partitioned between EtOAc
and saturated NaHCO3. The EtOAc layer was extracted
two times more with saturated NaHCO3, once with
brine, three times with 1 N NaHSO4, and once with
brine. The organic layer was dried with MgSO4, the
solvent was removed in vacuo, and the residue was
coevaporated twice with CH2Cl2 to a€ord the protected
cyclic product 11a (105 mg, 88%) as a yellow-brown
solid. HRMS: calcd for C62H65N9O9Cl2Na (M+Na)+,
1172.4180; found, 1172.4196.
1
(3.21 g, 76%) as a light yellow oil. H NMR (CDCl3,
300 MHz) d 9.96 (s, 1H), 8.16±8.04 (m, 3H), 7.68 (dd, 1H,
J=7.5Hz, 7.5 Hz), 7.59±7.52 (m, 3H), 7.46 (d, 1H,
J=7.5 Hz), 4.41 (q, 2H, J=7.1 Hz), 1.41 (t, 3H, J=7.1 Hz).
HRMS: calcd for C16H15O3 (M+H)+, 255.1021; found,
255.1011.
3-Ethylcarboxy-20-hydroxyiminobiphenyl (6). Compound
5 (3.18 g, 12.5 mmol) was dissolved in THF (30 mL)
with stirring, and pyridine (1.52 mL, 18.8 mmol) was
.
added. HONH2 HCl (1.13 g, 16.3 mmol) was added,
followed by EtOH (40 mL), and the reaction mixture
was allowed to stir for 14 h. The reaction mixture was
concentrated in vacuo, and the residue was partitioned
between 1 N NaHSO4 and EtOAc. The aqueous layer was
extracted three times more with EtOAc, the EtOAc layers
were combined, dried with MgSO4, and the solvent evap-
orated in vacuo. The residue was puri®ed by silica gel
¯ash column chromatography, eluting with 15% EtOAc
in hexanes. The eluate was concentrated in vacuo
Template-constrained cyclic peptide DJS631. Protected
cyclic peptide 11a (40 mg, 34.7mmol) was dissolved in
MeOH (12.5 mL), H2O (1.5 mL), 88% HCOOH (1.65
mL) with stirring, and the solution was sparged with Ar.
To the solution was added 10 wt% Pd/C (55.5 mg,
52.2 mmol Pd, 150 mol% Pd), and the reaction mixture
was again sparged with Ar. The reaction mixture was
allowed to stir under Ar for 30 h, and the mixture was
®ltered through Celite. The ®lter bed was washed with
H2O and MeOH, and the ®ltrate was concentrated in
vacuo. The residue was coevaporated with H2O/MeOH,
several times with MeOH, and then puri®ed by pre-
parative reverse-phase HPLC on a C18 column with
mobile phases consisting of water and CH3CN containing
0.1% TFA. The eluate was lyophilized, the residue was
taken up into H2O, ®ltered, and lyophilized again to
a€ord the TFA salt of DJS631 (15.7 mg, 46%) as an o€-
white solid. HRMS: calcd for C47H55N9O7Na (M+
Na)+, 880.4122; found, 880.4147.
1
to a€ord 6 (3.07 g, 91%) as a white solid. H NMR
(acetone-d6, 300 MHz) d 10.39 (s, 1H), 8.07 (d, 1H,
J=7.2 Hz), 8.00±7.93 (m, 3H), 7.66±7.58 (m, 2H), 7.52±
7.36 (m, 3H), 4.37 (q, 2H, J=7.1 Hz), 1.36 (t, 3H,
J=7.1 Hz). HRMS: calcd for C16H16NO3 (M+H)+,
270.1130; found, 270.1137.
3-Ethylcarboxy-20-aminomethylbiphenyl, hydrochloride salt
(7). Compound 6 (2.98 g, 11.1 mmol) was dissolved with
stirring in 1:1 EtOH:HOAc (66 mL) and the solution was
cooled to 0 ꢀC on an ice bath. Zn dust (10.85 g,

2238
D. J. Suich et al. / Bioorg. Med. Chem. 8 (2000) 2229±2241
166.0 mmol) was added to the reaction mixture in small
portions over the course of 30 min. The ice bath was
removed, and the mixture was allowed to stir for 28 h.
The reaction mixture was ®ltered, and the solid was
washed with EtOH. The ®ltrate was concentrated in
vacuo, and the residue was dissolved in EtOAc and
saturated Na2CO3. The pH of the aqueous layer was
adjusted to ꢁ12 with 1 N NaOH, and the layers were
shaken together and separated. The aqueous layer was
extracted three times more with EtOAc, and the organic
layers were combined, dried with MgSO4, and con-
centrated in vacuo. The residue was dissolved in EtOAc,
and anhydrous HCl gas was bubbled through the solu-
tion with stirring for 10 min, during which time a solid
precipitated. The mixture was cooled to 0 ꢀC on an ice
bath, and then ®ltered. The solid was washed with ice-
cold EtOAc, and residual solvent was removed from
it under vacuum to a€ord 7 (2.23 g, 69%) as a white
powder. 1H NMR (dimethylsulfoxide-d6, 300 MHz) d
8.48 (bs, 3H), 8.01 (d, 1H, J=7.4 Hz), 7.89 (s, 1H), 7.76±
7.61 (m, 3H), 7.54±7.44 (m, 2H), 7.32 (d, 1H,
J=7.2 Hz), 4.33 (q, 2H, J=7.1 Hz), 3.90 (d, 2H,
J=5.0 Hz), 1.32 (t, 3H, J=7.1 Hz). HRMS: calcd for
C16H18NO2 (M+H)+, 256.1338; found, 256.1350.
Coupling of 8 to Wang resin to generate resin-bound
intermediate 10. The derivatization reaction was carried
out in essentially the same fashion as that for the coupling
of 4 to Wang resin to generate 9. Wang resin (1.00g,
850 mmol) was derivatized with compound 8 (700mg,
1.28mmol) using DIPC (399 mL, 2.55mmol) and DMAP
(20.8 mg, 170 mmol) in CH2Cl2 (12 mL) for 48 h. During
®ltration, the resin was washed with DMF and MeOH
rather than CH2Cl2 and MeOH. The derivatization level
of the resin 10, measured by the picric acid assay, was
36%, and the total weight of the resin was 1.16 g.
Linear peptide derivative 12. Linear peptide intermediate
12 was derived from resin-bound intermediate 10 in
essentially the same fashion as peptide intermediate 11 was
derived from resin-bound intermediate 9. To resin-bound
intermediate 10 (1.16 g, equivalent to 850 mmol original
Wang resin) were coupled in succession Fmoc-l-Lys(Z)-
OH (641 mg, 1.28 mmol), Fmoc-d-Trp-OH (544 mg,
1.28 mmol), and Fmoc-l-Tyr(2,6-dichlorobenzyl)-OH
(717 mg, 1.28 mmol), using HBTU (484 mg, 1.28 mmol)
and DIEA (370 mL, 2.12 mmol) in DMF (3.5 mL), and
coupling times of 2±2.5 h. The peptide was deprotected,
cleaved from resin, and puri®ed by preparative reverse-
phase HPLC as described for the synthesis of 11. The
eluate was lyophilized, the residue was coevaporated
from THF, and the residual solvent was removed in
vacuo to a€ord the TFA salt of 12 (124 mg, 33%) as a tan
solid. HRMS: calcd for C60H63N7O9Cl2Na (M+Na)+,
1118.3962; found, 1118.3913.
3-Carboxy-2-(900 -¯uorenylmethyloxycarbonyl-L -valyl-
aminomethyl)biphenyl (8). Compound
7
(817 mg,
2.80 mmol) was dissolved in THF (20 mL) with stirring.
10 N NaOH (280 mL) was added, followed by 1 N
NaOH (20 mL), and the mixture was heated to re¯ux.
The reaction mixture was stirred at re¯ux for 8 h. The
reaction mixture was allowed to cool, and concentrated
HCl (aq) was added to the mixture with stirring until
the solution had a pH of 1. The organic solvent was
evaporated in vacuo, and the remaining aqueous solu-
tion was lyophilized to give solid 7a. Fmoc-l-Val-OH
(1.90 g, 5.60 mmol) and HBTU (2.12 g, 5.60 mmol) were
partially dissolved in DMF (6 mL) with stirring, and
DIEA (1.46 mL, 8.40 mmol) was added. The solution
was allowed to stir for 15 min, and was then added to
the lyophilized residue 7a, which was dissolved with stir-
ring. Additional DMF (3 mL) was added, and the reac-
tion mixture was allowed to stir for 24 h. The solvent was
removed in vacuo, and the residue was partitioned
between CH2Cl2 and 3.7% HCl (aq). The CH2Cl2 layer
and the solid suspended in it were separated from the
aqueous layer, and the aqueous layer was extracted
three times more with CH2Cl2. The organic layers were
pooled and the solvent evaporated in vacuo. The residue
was puri®ed by silica gel ¯ash column chromatography,
eluting with 1% MeOH/1% HOAc in CH2Cl2. The eluate
was concentrated in vacuo, coevaporating four times
with THF/toluene, three times with CH2Cl2, and twice
with EtOAc. The residue was triturated with EtOAc,
and the solid was ®ltered and washed with EtOAc.
Residual solvent was evaporated from the solid in vacuo
Cyclization of 12 to 12a. The cyclization of 12 to 12a
was carried out in the same fashion as the cyclization of
11 to 11a. Peptide derivative 12 (122 mg, 101 mmol) was
treated with HATU (42.1 mg, 111 mmol) and DIEA
(61.4 mL, 353 mmol) in anhydrous DMF (10.1 mL) for
14 h. The reaction mixture was worked up as described
for the cyclization of 11, to a€ord 12a (88 mg, 81%) as a tan
solid. HRMS: calcd for C60H61N7O8Cl2Na (M+Na)+,
1100.3848; found, 1100.3865.
Template-constrained cyclic peptide DJS811. The de-
protection of 12a to a€ord DJS811 was carried out in
the same fashion as the deprotection of 11a to a€ord
DJS631. Protected cyclic peptide 12a (85 mg, 79 mmol)
was treated for 18 h with 88% HCOOH (1 mL) and
10 wt% Pd/C (126 mg, 118 mmol Pd, 150 mol% Pd) in
MeOH (10 mL) and H2O (1 mL) under positive pressure
of Ar. The reaction mixture was worked up and puri®ed
by preparative reverse-phase HPLC as described for the
deprotection of 11a to a€ord the TFA salt of DJS811
(35.4 mg, 50%) as a white solid. HRMS: calcd for
C45H51N7O6Na (M+Na)+, 808.3798; found, 808.3807.
Radioligand binding
1
to a€ord 8 (1.00 g, 65%) as an o€-white solid foam. H
Receptor binding assays on cloned somatostatin recep-
tors were performed using human SSTR1, mouse
SSTR2B, mouse SSTR3, human SSTR4, and both rat
and human SSTR5 expressed in mammalian cells. Cells
were harvested, membranes were prepared, binding of
DJS631 and DJS811 was assayed, and IC50s were cal-
culated essentially as described.64 (3-[125I]iodotyrosyl11)
NMR (dimethylsulfoxide-d6, 300 MHz) d 8.44 (t, 1H,
J=5.1Hz), 7.94±7.86 (m, 4H), 7.75±7.70 (m, 2H), 7.54±
7.18 (m, 11H), 4.31±4.18 (m, 5H), 3.86±3.80 (m, 1H),
1.96±1.89 (m, 1H), 0.82 (dd, 6H, J=6.3 Hz). HRMS:
calcd for C34H32N2O5Na (M+Na)+, 571.2209; found,
571.2221.

D. J. Suich et al. / Bioorg. Med. Chem. 8 (2000) 2229±2241
2239
Somatostatin-14(tyr11), obtained from Amersham, was
used as the receptor labeling agent.
Drabkin reagent solution (Hgb kit, Sigma #525A). The
absorbance at 540 nm of the resulting colored mixture
was read on a spectrophotometer (the absorbance
intensity at this wavelength is proportional to total
hemoglobin content). Hemoglobin concentration was
calculated from a ®ve-point standard curve. Hemoglo-
bin data is presented as grams of hemoglobin/dL and as
percent inhibition of angiogenesis: % inhibition=100[1
(treated negative control)/(positive control negative
control)].
Matrigel angiogenesis animal model
Matrigel is an extract from murine tumor containing
laminin, collagen IV, heparin sulfate, and proteoglycans.
When the extract is supplemented with growth factors
and injected subcutaneously into mice, an intense angio-
genic response can be observed. This model has been
used to study the stimulation of angiogenesis by a variety
of factors.51,65,66
Immunohistochemistry and visualization
Matrigel with attached peritoneal lining was placed in
cold M 1 embedding matrix (Lipshaw) and snap frozen
in 2-methylbutane chilled on dry ice. Thin sections, 10mm
thick, were placed on (poly-l-lysine)-coated slides (Poly-
science), air dried and stored in a dessicated container at
80 ꢀC. Immediately before use, the slides were air
dried at room temperature, placed in MeOH:3% H2O2
(4:1) for 20 min, and then rinsed in 0.05 M Tris±HCl
(pH 7.4) (Tris=tris(hydroxymethyl)aminomethane)
bu€er. Tissue was digested with 0.05% Pronase (Cal-
biochem) and 0.11% ethylenediaminetetraacetic acid
(EDTA) in PBS for 20 min at 37 ꢀC, then rinsed twice in
Tris bu€er. Next, tissue samples were ®xed in 4 ꢀC ace-
tone for 8 min and rinsed three times in Tris bu€er. Rat
anti-mouse monoclonal CD31 (PECAM PharMingen
#01951D) or as a control IgG2a, k isotype standard
(PharMingen #11021D) was applied at 5 mg/mL, and
the slides were incubated overnight at room tempera-
ture. Slides were rinsed twice in Tris bu€er, and biotin-
labeled goat anti-rat IgG (H+L, 5 mg/mL, Southern
Biotechnology #3050-08) was applied. The slides were
incubated at room temperature for 40 min, and were
then rinsed three times in Tris bu€er. Streptavidin-
alkaline phospatase conjugate (10±20 units/mL, Boeh-
ringer-Mannheim #1089161) was applied for 40min at
room temperature, and the slides were rinsed three times
in Tris bu€er. HistoMark Red (Kirkegaard & Perry
Laboratories #55-69-00) was used to visualize the alka-
line-phosphatase-labeled antigens. The endothelial cell
content of the Matrigel was observed using the Optomax
image analysis system at 20Â magni®cation. Fields were
selected in a consistent pattern using the peritoneal lin-
ing for orientation.
Animals
Animal studies were conducted in a facility fully accre-
dited by the American Association for the Accreditation
of Laboratory Animal Care. Female C57BI/6 mice, 6±8
weeks old (Charles River Laboratory) were housed 10 per
cage in a room controlled to 20Æ2 ꢀC and 50Æ10% rela-
tive humidity, with a 12h light/dark cycle. The animals
were fed a standard pelleted mouse chow (Agway), and
water was available ad libitum.
Mini-pump implantation
Mini-pumps were purchased from Alza (#2002). The
pumps were ®lled 3 h prior to implantation and incubated
in ꢁ5 mL of phosphate-bu€ered saline (PBS) at 37 ꢀC to
allow the ¯ow to start. Animals were anesthetized with
25 mg/kg ketamine and 5 mg/kg Rompun mix, and a
small 2 cm incision was made on the dorsal skin near the
scapula of the mouse. Using forceps, a 1 inch tunnel was
made subcutaneously, into which the pump was inserted;
the wound was closed with a clip.
Matrigel preparation and use
Matrigel (Collaborative Biomedical) was allowed to
thaw overnight on ice at 4 ꢀC, and aliquots of the
thawed gel were placed into cold polypropylene tubes.
For both the positive control and treated animals,
bFGF (Gibco) was added to the gel (500 ng/mL); no
bFGF was added to the negative control gel aliquots.
Protein tyrosine kinase inhibitors were dissolved in
dimethylsulfoxide and added directly to the gel aliquots.
The tubes were capped and rotated end over end at 4 ꢀC
for at least 3 h. Gel aliquots (0.5 mL) were injected sub-
cutaneously into the ventral midline of the mice; the
liquid gel hardened once inside the animal. After 7 days,
the mice were sacri®ced, and the gel with the attached
peritoneal lining was removed by dissecting away the
skin from the gel. The gel samples were then assayed for
angiogenesis, using a hemoglobin and an immunohisto-
chemical assay.
Acknowledgements
This work was supported by grant CHE-34646 to William
F. DeGrado. D. J. S. was supported in part by the DuPont
Merck Pharmaceutical Co. postdoctoral fellows program.
References and Notes
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and crushed with a pestle. They were allowed to sit
overnight at 4 ꢀC and were then spun in a microfuge.
Aliquots of the lysate (200 mL) were added to 1.3 mL of
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