Highly potent 4-amino-indolo[2,3-c]azepin-3-one-containing somatostatin mimetics with a range of sst receptor selectivities

Article abstract of DOI:10.1021/jm801205x

The synthesis, biological evaluation, and conformational analysis of 4-amino-indolo[2,3-c]azepin-3-one (Aia)- containing SRIF mimetics are reported. Different subtype selectivities are observed depending on the N- and C-terminal substituents of the D-Aia-

Full text of DOI:10.1021/jm801205x

J. Med. Chem. 2009, 52, 95–104
95
Highly Potent 4-Amino-indolo[2,3-c]azepin-3-one-Containing Somatostatin Mimetics with a
Range of sst Receptor Selectivities
Debby Feytens,† Magali De Vlaeminck,† Renzo Cescato,‡ Dirk Tourwe´,*,† and Jean Claude Reubi‡
Department of Organic Chemistry, Vrije UniVersiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium, DiVision of Cell Biology and
Experimental Cancer Research, Institute of Pathology, UniVersity of Berne, Berne, Switzerland
ReceiVed September 23, 2008
The synthesis, biological evaluation, and conformational analysis of 4-amino-indolo[2,3-c]azepin-3-one (Aia)-
containing SRIF mimetics are reported. Different subtype selectivities are observed depending on the N-
and C-terminal substituents of the D-Aia-Lys dipeptide mimetic. An sst5-selective analogue with subnanomolar
binding affinity was obtained that is the most potent agonist reported to date. A nonselective mimetic with
high potency was also identified. This study allows a better definition of the bioactive conformation of the
essential D-Trp side chain in the somatostatin pharmacophore.
Introduction
Somatotropin release-inhibiting factor (SRIFa) or somatostatin
is an endogenous cyclic tetradecapeptide. It was discovered by
Brazeau et al.1 in 1973 as a potent inhibitor of growth hormone
secretion. Somatostatin occurs in two forms, SRIF-14 (14 amino
acids) and SRIF-28 (28 amino acids), and is widely distributed
throughout the endocrine and central nervous systems and
peripheral tissues. SRIF exhibits several physiological functions
such as modulation of the release of growth hormone, insulin,
glucagon, and gastric acid.2-4 Moreover, it has been shown to
have potent antiproliferative effects and neurotransmitter activi-
ties.5
The effects of SRIF are mediated by five G-protein coupled
receptors, sst1-5, which have all been cloned and characterized.6
Because of the numerous biological functions of SRIF,
selective ligands can be important in the treatment of various
human diseases.
SAR studies on a large number of analogues revealed that
the Phe7-Trp8-Lys9 sequence (numbering of the residues follows
that of native SRIF) is critical for biological recognition.7,8
Several cyclic hexa- and octapeptide analogues containing a
D-Trp8-Lys9 sequence have been synthesized9-11 and developed
for clinical use, including octreotide (D-Phe-c[Cys-Phe-D-Trp-
Lys-Thr-Cys]-Thr-ol). Their poor oral availability and rapid
proteolysis are major drawbacks for their clinical use. Therefore,
extensive research toward nonpeptide analogues that are selec-
Figure 1
tive for each receptor subtype was carried out over the past
related analogue 3 suggested that subtype selectivity could
be modulated by modifications of the N- and C-terminal
substituents (Figure 1).
decade.12 The critical side chains of the Phe7-Trp8-Lys9 sequence
were displayed successfully on a variety of nonpeptide
scaffolds.13-15
To improve the affinity and selectivity, various new analogues
were designed by varying the N- and C-terminal substituents
in mimetics 2 and 3. The phenylacetyl or diphenylpropionyl
substituent was varied by removing a phenyl or a methylene
group, whereas the C-terminal benzyl amide was either elon-
gated or replaced by a (S)- or (R)-1-phenylethyl amide or a
1-(aminomethyl)naphtalene. Various N-substitutions in the D-
Trp-Lys or D-ꢀ-Me-Trp-Lys scaffolds developed by Merck have
been reported to result in a high sst2 affinity. These include a
4-phenylbenzoyl substituent17 and urea derivatives of a benz-
imidazolonepiperidine,18 4-spiroindanylpiperidine,18,19 and N-
substituted piperazines or isonipecotic acid,17,20 which mostly target
the Phe7 binding site. We have introduced these variations in
A series of analogues, based on the constrained D-Trp-scaffold
D-Aia 1, was developed in our group,16 and tests on the five
SRIF receptors revealed a new, highly potent sst4-5 selective
SRIF mimetic 2. The changes in affinity observed for the
* To whom correspondence should be addressed. Phone: 32-2-629-3295.
Fax: 32-2-629-3304. E-mail: datourwe@vub.ac.be.
†
Vrije Universiteit Brussels.
University of Berne.
‡
a Abbreviations: SRIF, somatostatin; Aia, 4-amino-indolo[2,3-c]azepin-
3-one; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; NMM, N-
methylmorpholine; TBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethylu-
ronium tetrafluoroborate; EBAW, EtOAc:n-BuOH:AcOH:H2O 1:1:1:1);
DMEM, Dulbecco’s modified Eagle’s medium; GDP, guanidine diphos-
phate; BSA, bovine serum albumin.
10.1021/jm801205x CCC: $40.75
2009 American Chemical Society
Published on Web 12/09/2008

96 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Feytens et al.
Scheme 1a
a Reagents and conditions: (a) Lys(Cbz)OMe, CH2Cl2, NMM, pH 6, MgSO4, NaBH3CN, 2 h; (b) EDC, pyridine, CH3CN:H2O 1:1, high dilution, overnight;
(c) MeOH, LiOH, 1.5 h; (d) PhCH2NH2, CH2Cl2, Net3, TBTU, pH 8, 1 h; (e) 1.0.5% H2O in TFA:CH3CN 2:1, 1 h, 2.N,N′-disuccinimidyl carbonate,
N,N′diisopropylethylamine, CH2Cl2, 30 min, followed by RNNH2 or 2. RNCOOH, CH2CL2, Net3, TBTU, pH 8, 1 h; (f) EtOH, HCl, 10% Pd/C, H2, 50 psi
or 36% HBr in AcOH, 1 h; (g) (1) 0.5% H2O in TFA:CH3CN 2:1, 1 h; (2) PhCH2COOH, CH2Cl2, NEt3, TBTU, pH 8, 1 h; (h) CH2Cl2, Net3, TBTU, RCNH2,
pH 8, 1 h.
peptidomimetic 2. The best analogues in respect of binding affinity
and selectivity were functionally tested for agonism and antagonism.
analogues, Boc-protection was removed first, followed by
coupling to phenyl acetic acid, yielding 11. After saponification
of the methyl ester in 11, various amines (Table 1) were coupled,
followed by Cbz-removal to 14a-d. For the synthesis of the
N-terminal variations set, a saponification of the methyl ester
in 6 was carried out, yielding 7, followed by a coupling reaction
with benzylamine, leading to 8. Further Boc-deprotection and
formation of the acyl or urea derivatives (Table 1), followed
by a final Cbz-deprotection, yielded analogues 10a-k (Table
1). Analogue 14e was obtained after Cbz-deprotection of 11
(Scheme 1). Compound 18 was synthesized starting from the
reductive amination of 4 with D-Lys(2-ClCbz)NHCH2Ph (Scheme
2). Subsequent cyclization, Boc-deprotection, and coupling to
phenylacetic acid yielded 17, which was eventually deprotected
to 18. Mimetic 21 was obtained according to Scheme 3. All
final products were purified with preparative HPLC resulting
in purities of 98% and higher.
Results
Two series of analogues were synthesized. In a first set, the
C-terminal benzyl amide was retained and the N-terminal acyl
group was changed (Scheme 1, 10a-k), whereas in the second
set, the N-terminal phenylacetyl group was kept constant while
the C-terminal part was varied (Scheme 1, 14a-e). Furthermore,
the influence of the configuration of the Lys residue in these
dipeptide mimetics was investigated (Scheme 2, 18).
Synthesis. Analogues 10a-k and 14a-d were synthesized
as shown in Scheme 1. Boc-2′-formyl-D-tryptophan 4 was
prepared by SeO2 oxidation of 2-(tert-butoxycarbonyl)-2,3,4,9-
tetrahydro-1H-beta-carboline-3-carboxylic acid (Boc-D-Tcc) as
described previously.21 Reductive amination with ε-Cbz-
protected lysine methyl ester and NaBH3CN, immediately
followed by cyclization using EDC/pyridine, yielded 6. In
contrast to our previous findings,16 the cyclization was complete
after overnight stirring. For the C-terminal variations set of
Determination of Somatostatin Receptor Affinity Profiles.
All compounds were tested for their ability to bind to cell
membrane pellet sections of cells expressing the five human

Aia-Containing Somatostatin Mimetics
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 97
Scheme 2a
a Reagents and conditions: (a) D-Lys(2-ClCbz)NHCH2Ph·TFA 22, CH2Cl2, NMM, pH 6, MgSO4, NaBH3CN, 2 h; (b) EDC, pyridine, CH3CN:H2O 1:1, high
dilution, overnight; (c) (1) 0.5% H2O in TFA:CH3CN 2:1, 1 h; (2) PhCH2COOH, CH2Cl2, NEt3, TBTU, pH 8, 1 h; (d) EtOH, HCl, 10% Pd/C, H2, 50 psi.
Table 1. Structure of N- and C-Terminal Substituents in 10 and 14
Scheme 3a
a Reagents and conditions: (a) CH2Cl2, NEt3, TBTU, PhCH2CH2NH2, pH 8, 1 h; (b) (1) 0.5% H2O in TFA:CH3CN 2:1, 1 h; (2) acetic anhydride, acetonitrile:
water 1:1, NEt3, pH 6, 2 h; (c) EtOH, HCl, 10% Pd/C, H2, 50 psi.
sst receptor subtypes in complete displacement experiments
using the universal somatostatin radioligand [125I]-[Leu8,D-
Trp22,Tyr25]-somatostatin-28. The resulting binding affinities are
shown in Table 2.

98 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Table 2. Binding Affinities of the SRIF Peptide Mimetics
Feytens et al.
a
IC50 (nM)
no.
sst1
sst2
sst3
sst4
sst5
SS-28
2
1.9 ( 0.2 (12)
233 ( 74 (3)
253 ( 73 (3)
54 ( 12
27 ( 4 (3)
38 ( 3
2.0 ( 0.1 (12)
83 ( 2 (3)
292 ( 91 (3)<
10 ( 1
103 ( 42 (3)
22 ( 2
12 ( 2
242 ( 103 <
0.73 ( 0.23 (3)
2.1 ( 0.2 (3)
4.2 ( 0.2 (3)
327 ( 88 (3)
2.1 ( 0.7 (3)
183 ( 52 (4)
770 ( 177
1.7 ( 0.3 (12)
251 ( 16 (3)
>1000 (3)
153 ( 18
182 ( 44 (3)
162 ( 50
54 ( 15
>500
3.9 ( 1.0
2.0 ( 0.1 (12)
3.3 ( 0.2 (3)
7.0 ( 2.0 (3)
8.1 ( 0.8
1.3 ( 0.3 (3)
2.3 ( 0.1
2.6 ( 0.2 (12)
1.2 ( 0.1 (3)
5.0 ( 0.3
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
14a
14b
14c
14d
14e
18
3.4 ( 0.6
1.8 ( 0.1
3.7 ( 0.1
16 ( 0.3 (2)
8.8 ( 1.3
0.86 ( 0.30 (3)
1.4 ( 0.3 (3)
3.5 ( 0.9 (3)
8.0 ( 1.0 (3)
2.9 ( 0.3 (3)
0.60 ( 0.22 (4)
50 ( 12
3.2 ( 0.5 (3)
3.5 ( 1.1 (3)
13 ( 1
>1000
161 ( 75
14 ( 2
247 ( 42
76 ( 3 (3)
89 ( 7 (3)
372 ( 62 (3)
837 ( 47 (3)
379 ( 90 (3)
75 ( 15 (4)
630 ( 38
4.4 ( 1.0 (3)
6.9 ( 1.7 (3)
59 ( 23 (3)
2.1 ( 0.5
35 ( 6 (3)
2.4 ( 0.5 (4)
14 ( 1
1.0 ( 0.2 (3)
17 ( 2 (3)
2.4 ( 0.9
119 ( 16 (3)
260 ( 48 (3)
316 ( 75 (3)
9.1 ( 3.0
50 ( 5 (4)
403 ( 135
300 ( 22 (3)
105 ( 13 (3)
400 ( 97
183 ( 16 (3)
387 ( 94 (3)
962 ( 56
452 ( 105 (3)
215 ( 56 (3)
290 ( 45
>1000
105 ( 15 (3)
<
>1000
>1000 (3)
910 ( 162
515 ( 87 (3)
56 ( 7
7.0 ( 1.4 (3)
25 ( 1
0.56 ( 0.20 (3)
21
a The IC50 values were derived from competitive radioligand displacement assays using the nonselective [125]-[Leu8, D-Trp22, Tyr25]-somatostatin-28 as
radioligand. Mean values ( SEM; number in parenthesis represents number of experiments; all other values are n + 2.
Table 3. Lowest Energy Conformers of 10g
∆E pot
(kcal/mol)
ꢁ1 (D-Aia)
(deg)
ꢁ2 (D-Aia)
(deg)
ꢁ1 (Lys)
(deg)
conformation
conf1
conf2
conf3
conf4
conf5
0.00
0.71
0.95
1.03
1.05
-58
-55
-58
-55
176
-15
-19
-17
-21
6
-59
-60
-57
-57
-60
able to antagonize the 100 nM effect of SS-28. Thus 14a and
21 are full agonists for the sst5.
Molecular Modeling. The preferred conformations of the
nonselective analogue 10g and the sst5-selective analogue 21
were studied by molecular modeling using Macromodel 5.0.
The ꢁ2, ꢁ3, ꢁ4, and ꢁ5 of Lys were all fixed in the trans
conformation. After the conformational search using the low
mode (LMOD) method23 with the GB/SA solvation model of
Still et al.24 in water and subsequent minimization, the resulting
structures were clustered into families using an rmsd value of
0.2 Å. The lead structures of each family were considered and
those greater than 5 kcal/mol above the global minimum were
discarded. For both analogues, two values for the ꢁ1 of D-Aia
were found: trans and gauche (-) (g(-) for D-amino acids
corresponds to g(+) for L-amino acids).
A total of 8378 conformations were found for 10g (Table 3),
of which 503 lead structures remained after clustering. Only
structures with a potential energy of up to 5 kcal/mol above
the global minimum were considered (63 conformations). Of
those, 78% have ꢁ1 (D-Aia) ) gauche (-) and 22% have ꢁ1
(D-Aia) ) trans. For the Lys side chain, 49% has ꢁ1 (Lys) )
gauche (-) while in 35% a trans conformation is observed.
Only 16% have the ꢁ1 (Lys) ) gauche (+). As is shown in
Table 1, the lowest energy conformer of 10g has ꢁ1 (D-Aia) )
gauche (-) and ꢁ1 (Lys) ) gauche (-), the first structure having
a trans ꢁ1 (D-Aia) being 1.05 kcal/mol above the global
minimum.
Figure 2. Effect on [35S]GTPγS Binding. The stimulation of
[35S]GTPγS binding to sections of membrane pellets of CCL-sst5 cells
was performed as described in the Experimental Section. Sections of
membrane pellets of CCL-sst5 cells were treated either with 100 nM
SS-28, 1 nM or 1 µM 14a, 1 nM or 1 mM 21, or 100 nM SS-28 in the
presence of 10 mM 14a or 21. The bar-graphs represent the stimulated
specific [35S]GTPγS binding over basal level and were obtained in three
independent experiments in duplicates. The 1 nM conditions were
performed twice in duplicates.
To further characterize the analogues with the highest sst5
binding affinity, we functionally evaluated 14a and 21 for
agonism and antagonism in a [35S]GTPγS binding assay, using
CCL-sst5 cells, a cell line previously used by Hoyer and
colleagues in a similar assay.22 The specific binding of
[35S]GTPγS to membrane pellet sections of CCL-sst5 cells upon
stimulation with 14a or 21 alone or in the presence of SS-28
was determined by functional quantitative autoradiography. The
data presented in Figure 2 show that 14a and 21 stimulate
[35S]GTPγS binding in a dose dependent manner but are not
A total of 2200 conformations were found for 21, of which
423 lead structures remained after clustering. Conformations
greater than 5 kcal/mol above the global minimum were
discarded. The major part (84%) of the remaining structures
(55) has the D-Aia side chain in trans, while the Lys ꢁ1 is mainly
gauche (-) (69%) or trans (27%). As can be seen from Table

Aia-Containing Somatostatin Mimetics
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 99
Table 4. Lowest Energy Conformers of 21
for sst2 of all these analogues, except 10j, have increased
tremendously compared to 2, with subnanomolar sst2 affinity
for 10g. This indicates that the set of N-terminal substituents
that conferred high sst2 affinity in the D-Trp-Lys scaffold is
having a similar effect in the D-Aia-Lys scaffold and confirms
that an aromatic residue corresponding to Phe7 is important for
sst2 affinity. In general, a high potency at the sst5 is maintained
in all analogues. None of the analogues displayed high affinity
for sst1, but some showed low nanomolar affinity for sst3 (10g
and 10k) or for sst4 (10b, 10g, 10h, 10j). Again, although some
analogues in this N-terminal variations set showed high affinity
for a particular receptor subtype, affinity for another subtype
was always sufficiently high to prevent selectivity. As can be
seen in Table 2, 10g shows high potency for all receptors except
sst1. Only analogues 10i and 10k show a selectivity for sst5
over sst4 that is significantly higher than in 2, but in each of
those cases, selectivity of sst5 over sst2 or sst3 is lost.
∆E pot
(kcal/mol)
ꢁ1 (D-Aia)
(deg)
ꢁ2 (d-Aia)
ꢁ1 (Lys)
conformation
(deg)
(deg)
conf1
conf2
conf3
conf4
conf5
0.00
0.29
0.94
1.17
1.58
175
176
175
175
176
6
5
6
6
5
-59
-60
-62
-60
-60
4, the lowest energy conformer of 21 has ꢁ1 (D-Aia) ) 175°
and ꢁ1 (Lys) ) -59°. The first conformer with a gauche (-)
conformation for the D-Aia side chain is 3.0 kcal/mol above
the global minimum.
Discussion
Starting from the structure of the previously reported highly
potent SRIF mimetic 2, new analogues containing the in-
dolo[2,3-c]azepin-3-one ring system as a constraint for Trp were
designed and synthesized. As can be seen from Table 2, N-
and C-terminal variation of the substituents on the D-Aia-Lys
scaffold results in analogues having a low nanomolar potency
but a range of sst receptor subtype selectivities.
Some C-terminal variations were also examined (14a-e). In
analogue 14a, the alkyl chain of the benzyl amide is elongated
with one carbon. Compared to 2, this results in a decrease of
sst2 affinity while binding to all the other receptors is enhanced.
The affinity for sst5 is in the subnanomolar range, which is the
highest of all analogues in this set. To explore the orientation
of the aromatic moiety at the C-terminus, analogues 14b and
14c were designed. Whereas in 14b a general loss of affinity
for all receptors is observed, analogue 14c becomes highly
potent at sst4, with however retention of substantial potency at
sst5.
The importance of the aromatic moiety at the N-terminus in
2 was examined by removing it (analogue 10a). As shown in
Table 2, compared to 2, the affinity of 10a for sst1 is retained
while it has decreased for all the other receptors. Affinities for
sst4 and sst5 are still in the low nanomolar range, showing that
the aromatic residue at the N-terminus is not necessary for
binding to sst4 and sst5. The length of the carbon chain was
then investigated by extending it (10b) and shortening it (10c).
In both cases, high affinity for sst4 and sst5 is retained while
binding to sst1-3 is increased compared to 2. Apparently, the
sst1 receptor best accommodates the shorter benzoyl substituent
in 12c because it shows the highest increase in affinity (233
nM in 2 to 27 nM in 10c). However, also the extension of the
chain in 10b results in a strong increase in sst1 affinity (54 nM),
which is in this case accompanied by an increase in sst2 affinity
(83 nM in 2 to 10 nM in 10b). The introduction of a 4-chloro
substituent in the benzoyl group, to give analogue 10d, which
was reported to increase mainly sst3 affinity in a series of
tryptophan-based mimetics,25 significantly changed the affinity
for sst2 when compared to 10c. In contrast, the introduction of
a 4-phenyl substituent, leading to the biphenyl-4-carboxamide
10e, has a more pronounced influence: the affinity for the sst1,4,5
is substantially reduced, while that for sst2,3 is markedly
increased. This is consistent with the previously reported sst2
selectivity of a biphenyl-4-carboxamide-substituted mimetic.17
A shortened version of 3, analogue 10f, was prepared.
Compared to 3, binding affinities toward sst1,2,3 decreased.
Mainly affinity for sst4 increased while that for sst5 was retained,
resulting in a loss of sst5 versus sst4 selectivity. Compared to 2,
this analogue was less potent for all receptors.
As discussed above, none of the analogues resulting from
variations of the phenylacetyl substituent in 2 showed an
improved selectivity profile. The removal of the phenyl sub-
stituent to give acetyl-substituted analogue 10a maintains the
low nanomolar affinity for sst4 and sst5, but the benzoyl
substituent in 10c induces the highest potency at these receptors,
but also at sst1.
A set of N-terminal acyl and urea substituents, leading to
analogues 10g, 10h, 10i, 10j, and 10k, was selected from potent
sst2-selective antagonists17 and agonists18-20,26,27 developed
previously. These 4-substituted piperidine or piperazine sub-
stituents carry an aromatic ring that is intended to target the
Phe7 binding site. Accordingly, as is shown in Table 2, affinities
The design of 14d was based on a naphthyl containing sst5-
selective mimetic synthesized by Souers et al.28 Compound 14d
shows a 2-fold decreased affinity for sst5, but an improved sst5
versus sst4 selectivity compared to 2. Surprisingly, the cyclic
thioether ꢀ-turn mimetic described by Souers et al. had a
preferred D-configuration for the Lys residue.28 In our case, the
D-Lys epimer of 2, analogue 18, showed a general large decrease
of affinity, especially for sst1 and sst2.
Because several of the reported somatostatin mimetics contain
a Lys methyl ester,26,27,29 methyl ester 14e was investigated.
This resulted in a decrease of all binding affinities except toward
sst4, which results in an analogue that is quite selective except
versus sst5. The high binding affinities of phenylethylamide 14a
for sst4,5 and the lower affinities for sst1-3 of the N-acetylated
10a led to the design of the N-acetyl-D-Aia-Lys-phenylethyla-
mide analogue 21. This mimetic indeed shows an excellent
potency for sst5 in the subnanomolar range, combined with a
10-fold selectivity of sst5 over sst4 and higher selectivities over
the other receptors. 14a and 21 have also been tested functionally
in a [35S]GTPγS binding assay where they both behaved as full
agonists. Analogue 21 confirms that the presence of an aromatic
moiety at the N-terminus is not necessary for binding to sst4
and sst5. An iminosugar based sst4-selective mimetic with
affinity in the micromolar range was very recently reported by
Chagnault et al.30 Adding a benzyl substituent resulted in a
substantial increase in sst5 affinity, indicating that this aromatic
residue makes an important binding interaction with sst5 but
not with sst4. In our case, removing the aromatic moiety at the
N-terminus does not have any influence on sst4 or sst5 binding
affinities. This shows that hypotheses concerning SAR studies
may be mimetic dependent, as was also observed above for the
Lys configuration in the Souers mimetics.27
Only a few potent and sst5-selective SRIF peptidic analogues
have been reported: two cyclic peptides, PTR 3046, published
by Gilon et al.31 (IC50 sst5 ) 67 nM), and a dicarba-containing
analogue 23 recently reported by d’Addona et al.32 (IC50 sst5 )

100 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Feytens et al.
Figure 4. Octreotide 28.
as agonists. Recently, the first class of sst5-selective antagonists
was designed at Hoffmann-La Roche by a chemogenomics
approach.34,35 The most potent analogue 27 (Ki ) 13 nM) is
shown in Figure 3. These comparisons confirm that 21 is one
of the most potent sst5-selective nonpeptidic mimetics reported
to date exhibiting agonistic properties. Its selectivity profile is
different from that of the previously reported sst5-selective
ligands.
Conformational Analysis. Many previously reported subtype
selective cyclic peptide analogues contain a type II′ ꢀ-turn
around the D-Trp8-Lys9 residues and many somatostatin mi-
metics were designed on a ꢀ-turn mimetic template. A com-
prehensive conformational study on 4-amino-1,2,4,5-tetrahydro-
2-benzazepin-3-ones showed that these structures do not adopt
turn conformations but rather prefer an extended conformation.36
An NMR study of 2 and 3 indeed indicated that these
compounds do not adopt ꢀ-turns,16 nevertheless, the upfield
position of the Lys CγH2, which is ascribed to a close proximity
of the Trp and Lys side chains in the peptidic analogues and
which correlates with high receptor affinity,37 was observed.
This upfield chemical shift of the Lys γ protons is also observed
in the newly synthesized compounds 10a-k, 14a-e, and 21
while it is absent in D-Lys containing 18. This is reflected in
the binding profiles, 18 is the analogue with the lowest binding
affinities for all the receptors, with complete loss of binding to
sst1 and sst2.
To better understand the high binding affinities of our
analogues, despite the absence of a ꢀ-turn, compounds 10g
(nonselective analogue) and 21 (sst5-selective) were subjected
to a conformational analysis. The results were compared to data
obtained for sst2,3,5-selective octreotide 2838 (Figure 4) and the
sst5-selective dicarba-analogue 23 recently published by d’Addona
et al.32
For analogue 10g, the conformational search resulted in a
lowest energy conformer with ꢁ1 (D-Aia) ) -58° and ꢁ1 (Lys)
) -59° (Table 3). Four more conformers were found within
1.1 kcal/mol above this global minimum, three of which have
ꢁ1 (D-Aia) in gauche (-) and one with ꢁ1 (D-Aia) in trans. A
gauche (-) value for the lysine side chain was found in all
four. Because of coinciding signals of the azepinone and lysine
alpha protons, NMR data in MeOD could not identify the ꢁ1
value of D-Aia. The observed upfield shift of the lysine γ protons
however indicates a close proximity of these hydrogens with
the indole ring, which is only possible when the D-Aia adopts
a trans conformation. According to the molecular modeling,
the first conformer with ꢁ1 of D-Aia in trans has an energy of
1.05 kcal/mol above the global minimum, the majority (78%)
having a gauche (-) conformation. This discrepancy may be
due to a solvent effect but also indicates that 10g can easily
switch from a trans to a gauche (-) D-Aia conformation.
NMR data of 21 in MeOD clearly show that the solution
conformation of this analogue has the ꢁ1 (D-Aia) in trans. This
Figure 3. Previously reported sst5-selective agonists and antagonist.
12 nM). These peptides show an excellent sst5 selectivity (at
least 24-fold), but their affinity toward sst5 is at least 18 times
less than in analogue 21.
An sst5-selective nonpeptidic mimetic 24 (Figure 3) was
reported previously by Merck.26 This analogue shows a sub-
nanomolar affinity for sst5 (IC50 ) 0.4 nM) and the selectivity
of sst5 over sst1 is 8-fold. The naphthyl containing thioether 25
(Figure 3), which was already mentioned,28 has an affinity of
87 nM for sst5 combined with a high selectivity of sst5 over the
other receptors. A benzoxazepine somatostatin mimetic 26
(Figure 3) was reported to have excellent sst5 affinity (IC50
)
0.3 nM) and a selectivity of 200 and more over the other receptor
subtypes.15,33 These three mimetics have all been shown to act

Aia-Containing Somatostatin Mimetics
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 101
Figure 5. Superposition of 21 (yellow) with 23 (pink).
is in good agreement with the molecular modeling results (Table
4), where the five lowest energy conformers all have the ꢁ1 of
the D-Aia residue in trans. All these structures have the lysine
side chain in gauche (-).
The most remarkable difference between 10g and 21 is the
different ability to adopt the g(-) conformation of the D-Aia
residue. This increased flexibility of 10g compared to 21 may
allow it to adapt more easily to the different receptor subtypes.
For octreotide 28, a conformational equilibrium is assumed
based on NMR studies38 as well as X-ray crystallography.39
One conformer shows an antiparallel ꢀ-sheet structure while in
the other two, the C-terminal residues form a 310 helix-like fold.
In both conformations, the D-Trp side chain adopts a trans
conformation while the Lys ꢁ1 is gauche (-) or trans. NMR
studies on dicarba-analogues32 such as 23 show a similar
equilibrium and indicate that this equilibrium is shifted to the
helical conformations in sst5-selective analogues.
Figure 6. Superposition of 10g (brown) with 28 (blue).
sst5 affinity.41 Therefore, the observed effect in the D-Aia-Lys
analogues reported here, yielding a potent sst5 agonist, may be
mainly the result of the conformational constraint imposed by
the D-Aia residue.
Conclusion
On the basis of the structure of the potent sst4,5-selective SRIF
mimetic 2, a series of new analogues was synthesized and their
binding affinities for the five somatostatin receptors were
determined. Depending on the N- or C-terminal substituents of
the D-Aia-Lys dipeptide mimetic, potent analogues with a range
of different subtype selectivities were obtained. Because some
of the analogues show a low nanomolar affinity for each one
of the receptor subtypes, except for sst1, it can be concluded
that the D-Aia-Lys scaffold represents a pharmacophore that is
universally recognized by sst2-5. The most interesting analogue
is 21, which shows a subnanomolar affinity for sst5 and at least
a 10-fold selectivity over the other receptors and behaves as a
full agonist. sst5-selective agonists may find important applica-
tions because the sst5 receptor is believed to mediate the
inhibition of growth hormone and adrenocorticotropin secre-
tion.42 It also inhibits insulin and amylase secretion in the
pancreas. Some tumors like GH-expressing pituitary adenoma,
kidney cancer, and thyroid cancer overexpress sst5.43,44 sst5 has
also been reported to play a role in learning and memory.45
A potent nonselective mimetic 10g was identified. Analogues
that bind equally well to all five subtypes are very interesting
as drugs for long-term therapy in tumors that express several
different somatostatin receptor subtypes. They can also be
effective in nononcological indications characterized by distur-
bances of the physiological somatostatin system. When labeled
with radioactive elements, they can identify tissues expressing
all five subtypes.46 Molecular modeling studies were carried
out on 21 and 10g, showing that these analogues have a different
ability to adopt a trans or gauche (-) D-Aia side chain
For octreotide 28,38 the proposed conformations of the Lys
side chain are gauche (-) or trans as is the case for our
analogues 10g and 21. The conformations found for 23 also
have the lysine side chain in gauche (-).32 The similarity
between the sst5-selective analogues 21 and 23 is reflected in
the good backbone overlaps resulting from superimposing the
lowest energy conformer of 21 (conf 1 in Table 4) with a
representative NMR-derived conformation of 23 as is shown
in Figure 5.
Superpositions of 10g (Conf 5 in Table 3) with octreotide
28 also show a good overlap except for the N-terminal part
(Figure 6).
Both analogues 10g and 21 have similar values for the ꢁ1 of
the D-Trp and Lys residues. The cyclic nature of the Aia scaffold
also results in a well defined ꢁ2 value, which is different from
the one proposed in 23 and octreotide but is consistent with the
observed high field shift of the Lys γ methylene protons in the
NMR spectra. It can be mentioned that the D-Aia-Lys scaffold
not only constrains the D-Trp side chain but also involves an
alkylation of the Lys NR. NR-methylation of Lys9 was shown
not to cause dramatic effects on the receptor affinity and potency
in octapeptide somatostatin agonists.40 In contrast in an oc-
tapeptide somatostatin antagonist, it substantionally increased

102 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Feytens et al.
General Procedure for the Boc-Deprotection. Boc-protected
amine (1.8 mmol) was dissolved in a TFA:water mixture (95:5, 13
mL) and acetonitrile was added (6 mL). The reaction was stirred
for 1 h and the mixture was evaporated. Crude TFA-salts were
used in the next reactions.
General Procedure for the Saponification. Methyl ester (4.05
mmol) was dissolved in MeOH (96 mL) and aqueous LiOH (1M,
4 equiv, 16.2 mmol, 16.2 mL) was added. The reaction was
followed with TLC (silica, EtOAc) and after 1.5 h it was complete.
MeOH was evaporated and water (100 mL) and EtOAc (50 mL)
were added. After extraction, the aqueous layer was acidified to
pH 4 and extracted with EtOAc (3 × 50 mL). The organic layers
were combined and washed with brine (3 × 100 mL). The organic
phase was dried over MgSO4, and after filtration, the mixture was
evaporated.
General Procedure for the Formation of Amide Bonds. Acid
(54 mmol) was dissolved in dry CH2Cl2 (250 mL). NEt3 (3 equiv,
162 mmol, 22.5 mL) and TBTU (1.1 equiv, 59.4 mmol, 19.07 g)
were added and the mixture was stirred at room temperature for
10 min. Amine TFA-salt (1.1 equiv, 59.4 mmol) was added and
the pH was kept at 8 by the addition of NEt3. The reaction was
stirred for 1 h. The solution was extracted with HCl (1M, 3 × 80
mL), NaHCO3 (saturated, 3 × 80 mL) and brine (3 × 80 mL). The
organic layer was dried over MgSO4, and after filtration, the residue
was evaporated. No further purification was necessary.
General Procedure for the Formation of Urea Bonds. A
suspension of amine TFA-salt (55.4 mmol), N,N′-disuccinimidyl
carbonate (1.1 equiv, 60.9 mmol, 15.6 g) and N,N-diisopropylethy-
lamine (2 equiv, 110.8 mmol, 18.3 mL) in CH2Cl2 (500 mL) was
prepared. The solution was stirred at room temperature for 30 min,
and a clear solution was formed. Piperidine or piperazine derivative
(1 equiv, 55.4 mmol) was added and the pH was kept at 8 by the
addition of DIEA. Stirring was continued overnight. The solution
was extracted with HCl (1M, 3 × 15 mL), NaHCO3 (saturated, 3
× 15 mL) and brine (3 × 15 mL). The organic layer was dried
over MgSO4, and after filtration, the solution was evaporated. No
further purification was necessary.
General Procedure for the Acetylation. Amine TFA-salt (0.89
mmol) was dissolved in acetonitrile:water 1:1(10 mL) and the pH
was adjusted to 6 by the addition of NEt3. Acetic acid anhydride
(5 equiv, 4.46 mmol, 0.42 mL) was added in three portions while
the pH was kept at 6. The reaction was stirred for 2 h at room
temperature. The solution was evaporated and after the addition of
EtOAc (25 mL), it was extracted with HCl (1M, 3 × 15 mL),
NaHCO3 (saturated, 3 × 15 mL), and brine (3 × 15 mL). The
organic layer was dried over MgSO4, and after filtration, the solution
was evaporated. No further purification was necessary.
General Procedure for the Deprotection of Cbz and 2-Cl Cbz.
Cbz-protected compound (0.64 mmol) was dissolved in EtOH (50
mL). HCl (1equiv, 0.64 mmol, 0.064 mL) and 10% Pd/C (20 wt
%) were added and the mixture was hydrogenated overnight in a
Parr-apparatus at 20-50 psi. The catalyst was filtered off and the
crude product was purified by preparative HPLC.
Procedure for the Cbz-deprotection of 9d. Dry HBr in AcOH
(36%, 1.5 g) was added to Cbz-protected compound 9d (0.41 mmol,
0.292 g). A CaCl2-tube was put on the flask and the mixture was
allowed to stand at room temperature for 1 h. Dry ether was added
to precipitate the amine hydrobromide. The supernatant liquid was
decanted and the solid triturated with ether, filtered, and washed
with ether.
conformation. The analogues reported here are the first examples
of peptidomimetics in which the side chain conformation of
the essential D-Trp residue has been constrained to allow only
a limited range of ꢁ1 and ꢁ2 values. Many models for the
bioactive conformation required for selective recognition by the
somatostatin receptor subtypes have been proposed.32,47-50
The results reported here should allow a better definition of the
D-Trp orientation in these models and exclude the gauche (+)
ꢁ1 orientation.
Experimental Section
General. Lys(ε-Cbz)OMe and Boc-D-Lys(ε-2-ClCbz)OH were
purchased from Novabiochem (La¨ugelfinger, Switzerland). D-Lys(ε-
2-ClCbz)NHCH2Ph was synthesized using the standard procedures
for amide formation and Boc-deprotection.51 Boc-2′-formyl tryp-
tophan 4 was prepared as described previously.21 Synthesis and
characterization of compound 8 were published previously.16 Thin
layer chromatography (TLC) was performed on plastic sheet
precoated with silica gel 60F254 (Merck, Darmstadt, Germany) using
specified solvent systems. Mass spectrometry (MS) was recorded
on a VG Quattro II spectrometer using electrospray (ESP) ionization
(positive or negative ion mode). Data collection was done with
Masslynx software. Analytical RP-HPLC was performed using an
Agilent 1100 Series system (Waldbronn, Germany) with a Supelco
Discovery BIO Wide Pore (Bellefonte, PA, USA) RP C-18 column
(25 cm × 4.6 mm, 5 µm) using UV detection at 215 nm. The mobile
phase (system 1: water/acetonitrile, system 2: water/methanol)
contained 0.1% TFA. The standard gradient consisted of a 20 min
run from 3 to 97% acetonitrile (system 1) or methanol (system 2)
at a flow rate of 1 mL/min. Preparative HPLC was performed on
a Gilson apparatus and controlled with the software package
Unipoint. The reverse phase C18-column (Discovery BIO Wide
Pore 25 cm × 21.2 mm, 10 µm) was used under the same conditions
as the analytical RP-HPLC, but with a flow rate of 20 mL min-1
.
Nuclear magnetic resonance (NMR): 1H NMR and 13C NMR spectra
were recorded at 250 and 63 MHz, respectively, on a Bruker Avance
250 spectrometer or at 500 and 125 MHz on a Bruker Avance II
500. Calibration was done with TMS (tetramethylsilane) or residual
solvent signals as an internal standard. The solvent used is
mentioned in all cases and the abbreviations used are: s (singlet),
d (doublet), dd (double doublet), t (triplet), q (quadruplet), br s
(broad singlet), m (multiplet), Lys (lysine protons), azep (azepinone
protons), arom (aromatic protons). Optical rotations were measured
on a Perkin-Elmer 241 polarimeter. Infrared spectral data were
obtained using an Avatar 370 FT-IR.
General Procedure for the Reductive Amination. Boc-2′-
formyl Trp 4 (1.0 g, 3.0 mmol) was suspended in dichloromethane
(45 mL) and Lys(Cbz)OMe ·HCl (1.05 equiv, 3.2 mmol, 1.04 g)
or (D)Lys(2-ClCbz)NHBn ·TFA (1.05 equiv, 3.2 mmol, 1.75 g) were
added. The pH was adjusted to 6 by the addition of N-methyl
morpholine. MgSO4 (20 wt %, 0.20 g) and NaBH3CN (2.5 equiv,
7.5 mmol, 0.47 g) were added and the reaction was stirred at room
temperature for 1.5 to 3 h.
The reaction mixture was evaporated and used without further
purification in the cyclization reaction.
General Procedure for the Cyclization. Synthesis of 6 and
16. Crude amine 5 or 15 (9.0 mmol) was dissolved acetonitrile:
water 1:1 (600 mL) and the solution was cooled to 0 °C. Pyridine
(2.5 equiv, 22.5 mmol, 1.8 mL) and EDC ·HCl (1.5 equiv, 13.5
mmol, 2.58 g) were added and the mixture was stirred at 0 °C for
1 h. Stirring was continued at room temperature overnight (5) or
during 4 days (15, 3 more equiv of pyridine and EDC ·HCl were
added).
Acetonitrile was evaporated and 150 mL of EtOAc were added.
The layers were separated and the organic phase was extracted with
HCl (1M, 3 × 100 mL), NaHCO3 (saturated, 3 × 100 mL) and
brine (3 × 100 mL). The organic layer was dried over MgSO4,
and the crude mixture was purified by column chromatography
(silica, EtOAc:CH2Cl2 gradient). Yield (6) ) 37%. Yield (16) )
40%.
Determination of Somatostatin Receptor Binding Affinity
Profiles. Cell membrane pellets were prepared and receptor
autoradiography was performed on 20 µm thick pellet sections
(mounted on microscope slides), as described in detail previously.22,52
For each of the tested compounds, complete displacement experi-
ments were performed with the universal somatostatin radioligand
[
125I]-[Leu8,D-Trp22,Tyr25]-somatostatin-28 (2000 Ci/mmol; Anawa,
Wangen, Switzerland) using 15000 cpm/100 µL and increasing
concentrations of the unlabeled compounds ranging from 0.1 to
1,000 nmol/L. Somatostatin-28 was run in parallel as control using
the same increasing concentrations. IC50 values were calculated after

Aia-Containing Somatostatin Mimetics
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 103
(5) Gillies, G. Somatostatin: The neuroendocrine story. Trends Pharmacol.
Sci. 1997, 18, 87–95.
(6) Hoyer, D.; Bell, G. I.; Epelbaum, J.; Fenuik, W.; Humphrey, P. P. A.;
O’Carroll, A.-M.; Patel, Y. C.; Schonbrunn, A.; Taylor, J. E.; Reisine,
T. Classification and nomenclature of somatostatin receptors. Trends
Pharmacol. Sci. 1995, 16, 86–88.
quantification of the data using a computer-assisted image process-
ing system.22,52 Tissue standards containing known amounts of
isotopes, cross-calibrated to tissue-equivalent ligand concentrations,
were used for quantification.52
[35S]GTPγS Binding Assay. Chinese hamster lung fibroblasts
(CCL39) stably expressing the human sst5 (CCL-sst5) were kindly
provided by D. Hoyer (Novartis Pharma, Basel, Switzerland) and
were grown in DMEM with GlutaMAX-I/Ham’s F-12 Nut. Mix.
with GlutaMAX-I (1:1) supplemented with 10% (v/v) fetal bovine
serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 400 µg/
mL Geneticin (G418-sulfate), and cultured at 37 °C and 5% CO2.
All culture reagents were from Gibco BRL, Life Technologies
(Grand Island, NY). Cell membrane pellets of CCL-sst5 were
prepared as previously described and stored at -80 °C.53
[35S]GTPγS binding assay was performed on 20 µm thick cryostat
(Microm HM 500, Walldorf, Germany) sections of the membrane
pellets, mounted on microscope slides. The sections were first
preincubated for 10 min at room temperature in assay buffer (100
mM Tris-HCl buffer (pH 8.2), 1% BSA, 40 mg/L bacitracin 10
mM MgCl2) and then for 30 min in assay buffer containing 50 µM
GDP. Subsequently the sections were incubated for 2 h at room
temperature in assay buffer containing 50 µM GDP, 40 pM (100000
cpm/ml) [35S]GTPγS (1250 Ci/mmol; PerkinElmer, Waltham, MA),
and the absence (basal level) or presence (stimulated) of the
compounds to be tested at the concentrations indicated. At the end
of the incubation, the sections were washed on ice with assay buffer.
After a brief dip in assay buffer without BSA and bacitracin to
remove excess salts, the sections were dried and exposed to Kodak
BioMax MR films. The relative optical densities of the pellets were
determined using the MCID Basic 7.0 program (Imaging Research
Inc.). Values were expressed as percentage over basal level.
Molecular Modeling. The calculations were carried out using
Macromodel 5.054 with Maestro 8.0 as a graphic interface.
Analogues 10g and 21 were built using the following constraints:
ꢁ2 (Lys) ) ꢁ3 (Lys) ) ꢁ4 (Lys) ) ꢁ5 (Lys) ) 180°. The MM3*
force field55 was used for energy minimization in combination with
the GB/SA solvation model of Still et al.,24 using MacroModel’s
default parameters for an aqueous medium. Conformational searches
were carried out using the pure low mode search.23 Structures were
generated and minimized by means of the Polak-Ribie`re conjugate
gradient method as implemented in MacroModel, using a gradient
convergence criterion of 0.1 kJ/mol Å. The resulting conformations
were again minimized to an energy convergence of 0.01 kJ/mol Å.
Duplicate structures and those greater than 50 kJ/mol above the
global minimum were discarded. The remaining structures were
clustered into families using Xcluster 1.7. A rmsd value of 0.2 Å
was used.
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(16) Feytens, D.; Cescato, R.; Reubi, J. C.; Tourwe, D. New sst(4/5)-
selective somatostatin peptidomimetics based on a constrained tryp-
tophan scaffold. J. Med. Chem. 2007, 50, 3397–3401.
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Acknowledgment. D. Feytens is a research assistant of the
Fund for Scientic Research Flanders (FWO-Vlaanderen). We
thank Dr. M. Kofod-Hansen from Novo Nordisk for a gift of
1,2-dihydro-1-methanesulfonyl spiro[3H-indole-3,4-piperidine].
Supported by FWO-grant no. G.0008.08. We thank Prof. A.
Carotenuto for providing us the coordinates of 23.
(21) Pulka, K.; Feytens, D.; Van den Eynde, I.; De Wachter, R.; Kosson,
P.; Misicka, A.; Lipkowski, A.; Chung, N. N.; Schiller, P. W.; Tourwe,
D. Synthesis of 4-amino-3-oxo-tetrahydroazepino[3,4-b]indoles: new
conformationally constrained Trp analogs. Tetrahedron 2007, 63,
1459–1466.
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ics. J. Am. Chem. Soc. 1990, 112, 6127–6129.
Supporting Information Available: Characterizations of com-
pounds 6-22. HPLC-purity data of compounds 6-22. HPLC-
chromatogram traces of compounds 10a-k, 14a-e, 18, and 21.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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