Identification of potent non-peptide somatostatin antagonists with sst3 selectivity

Article abstract of DOI:10.1021/jm0108449

Using a solution-phase parallel synthesis strategy, a series of non-peptide somatostatin analogues were prepared, and their binding affinities to the five human somatostatin receptor subtypes (sst_1-5) were determined. Imidazolyl derivatives 2 w

Full text of DOI:10.1021/jm0108449

2990
J . Med. Chem. 2001, 44, 2990-3000
Id en tifica tion of P oten t Non -P ep tid e Som a tosta tin An ta gon ists w ith sst3
Selectivity
Lydie Poitout,* Pierre Roubert, Marie-Odile Contour-Galce´ra, Christophe Moinet, J acques Lannoy,
J acques Pommier, Pascale Plas, Dennis Bigg, and Christophe Thurieau*
Institut Henri Beaufour, 5 Avenue du Canada, F-91966 Les Ulis, France
Received February 8, 2001
Using a solution-phase parallel synthesis strategy, a series of non-peptide somatostatin
analogues were prepared, and their binding affinities to the five human somatostatin receptor
subtypes (sst1-5) were determined. Imidazolyl derivatives 2 were found to bind with moderate
affinity but with high selectivity to the sst3 receptor subtype. Further modifications of these
structures led to a more potent class of ligands, the tetrahydro-â-carboline derivatives 4. Among
these, compounds 4k (BN81644) and 4n (BN81674) bind selectively and with high affinity to
the sst3 receptor subtype (Ki ) 0.64 and 0.92 nM, respectively). Furthermore, 4k and 4n reverse
the inhibition of cyclic AMP accumulation induced by 1 nM somatostatin via sst3 receptors,
with IC50 ) 2.7 and 0.84 nM, respectively. The most potent compound 4n was shown to be a
competitive antagonist of human sst3 receptors by increasing the EC50 of SRIF-14-mediated
inhibition of cAMP accumulation with a KB of 2.8 nM (where KB is the concentration of
antagonist that shifts the agonist dose-response 2-fold). These new derivatives are, to our
knowledge, the first potent and highly selective non-peptide human sst3 antagonists known
and, as such, are useful tools for investigating the physiological role of sst3 receptors.
In tr od u ction
Somatostatin (somatotropin-release-inhibiting factor,
SRIF) is a peptide hormone originally isolated from the
hypothalamus as an inhibitor of growth hormone release
from the pituitary.1 Widely distributed in the central
nervous system, endocrine, and peripheral tissues, SRIF
comprises two physiological forms, a tetradecapeptide
(SRIF-14; Figure 1) and an N-terminal extended 28
F igu r e 1. Chemical structures of SRIF-14, lanreotide, and
amino acid peptide (SRIF-28). They exhibit multiple
biological effects2-4 which include the inhibition of the
pancreatic secretion of insulin and glucagon,5 and the
release of gastrin by the gut.6 In addition, SRIF pos-
sesses inhibitory effects on cell proliferation7 and is also
a neurotransmitter or neuromodulator, acting on cogni-
tive function,8 locomotor activity,9 and the release of
other neurotransmitters.10,11 The biological functions of
SRIF are mediated by a number of specific receptors.
To date, five SRIF receptor subtypes have been cloned
and characterized (sst1-5) with distinct distributions in
various tissues.12 They all belong to the G-protein-
coupled superfamily of receptors with seven hydrophobic
transmembrane domains. So far, only sst2 and sst5 have
been associated with specific physiological functions. sst2
receptors have a predominant role in mediating the
release of growth hormone (GH),13 while the inhibition
of insulin secretion from rat pancreatic islets is thought
to be mediated through sst5 receptors.14,15
octreotide.
(Figure 1) are now available for use in the treatment of
acromegaly, neuroendocrine tumors, and gastrointesti-
nal disorders. However, these analogues require parent-
eral administration. Recent efforts have therefore been
focused on the search for potent, selective, and orally
active non-peptide derivatives. On the basis of the
knowledge of the crucial role of the tripeptide sequence18
Phe7-Trp8-Lys9 in the binding at ssts, non-peptide
peptidomimetics, using a sugar or a benzodiazepinone
core to mimic the â-turn of SRIF and exhibiting micro-
molar affinities for SRIF receptors, have been re-
ported.19-22 More recently, a thiourea derivative, NNC
26-9100 (I; Figure 2) was described as a potent and
selective sst4 agonist,23 and a series of non-peptide
agonists, with moderate to high selectivity for each of
the five receptor subtypes, were also developed.24,25
Among them, the derivative L-054,522 (II; Figure 2) was
reported to bind selectively and with high affinity to the
sst2 receptor subtype.26,27
The limitations of therapeutic applications of SRIF-
14 due to its poor bioavailability and rapid proteolytic
degradation have led to the search for peptide analogues
with higher metabolic stability and improved selectivity
with respect to the different receptor subtypes. Long-
acting preparations of octreotide16 and lanreotide17
We initiated a synthetic program aimed at the iden-
tification of novel non-peptide SRIF agonists or antago-
nists for each subtype, since such compounds could be
useful not only as potential therapeutic agents but also
as tools for studying the physiological roles of individual
SRIF receptor subtypes.
* To whom correspondence should be addressed. Fax:
+33 1 69 07 38 02. Phone: +33 1 60 92 20 00. E-mail: lydie.poitout@
beaufour-ipsen.com and christophe.thurieau@beaufour-ipsen.com.
10.1021/jm0108449 CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/20/2001

Somatostatin Antagonists with sst3 Selectivity
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2991
side chains, we submitted imidazolyl compounds 3 to
Pictet-Spengler cyclization29 with various aldehydes to
afford the corresponding tetrahydro-â-carbolines 4. This
reaction was successfully carried out in parallel in
solution phase using resin scavengers to trap the excess
of the reagents.30 More than 400 compounds of structure
4 (as mixtures of C1-diastereomers) were readily syn-
thesized. Biological screening was carried out on the five
SRIF receptor subtypes and led to the discovery of a
set of compounds with high affinity and selectivity for
sst3 receptors.
To further enhance the potency and selectivity of
derivatives 4 for sst3 receptors, several structural
modifications were studied including the use of sym-
metric ketones instead of aldehydes in the Pictet-
Spengler reaction, to avoid formation of diastereomeric
mixtures. Replacement of the imidazole and indole rings
by thiazole and benzothiophene, respectively, was in-
vestigated as well as substitution on the indole moiety
and oxidation of the tetrahydro-â-carboline to its fully
aromatized counterpart.
We will describe herein the synthesis of a novel series
of tetrahydro-â-carbolines, their structure-activity re-
lationships (SARs), and their affinities for sst3 receptors.
The most potent compounds were then evaluated for
their agonist/antagonist properties.
F igu r e 2. Chemical structures of I (NNC 26-9100) and II (L-
054,522) and their inhibition constants (Ki) on somatostatin
receptor subtypes.
From our compound collection we chose different
templates suitable for parallel synthesis and displaying
various possibilities of attachments. Among them imi-
dazolyl derivatives 2 (Scheme 1) were selected. Using
solution-phase parallel synthesis, a library of more than
1500 members was synthesized, and some of these
imidazolyl derivatives were found to bind selectively to
the sst3 receptor subtype with moderate affinity.28 These
encouraging results validated the choice of imidazolyl
derivatives 2 as potential peptidomimetics as well as
the use of an indole moiety as a key side chain and
prompted us to develop chemistry around this structural
class to identify more potent compounds.
Ch em istr y
Imidazole derivatives 3a -f were prepared according
to Scheme 1.31,32 N-Boc-protected amino acids were
treated with cesium carbonate followed by condensation
with the appropriate R-bromoketone. Cyclization of the
resulting ketoesters of structure 1 using ammonium
acetate in refluxing xylene yielded the desired Boc-
protected scaffolds 2a -f, which upon acidic deprotection
afforded amines 3a -f. Pictet-Spengler cyclization with
various aldehydes was carried out in solution-phase
To introduce conformational rigidity into derivatives
2 and to provide a different spatial orientation to the
Sch em e 1a
a
Reagents: (a) Cs2CO3, EtOH/H2O; (b) R1COCH(Br)R2, DMF; (c) NH4OAc, xylene, reflux; (d) 1 N HCl/EtOAc; (e) R3CHO, TFA, CHCl3,
rt and then aminomethylpolystyrene resin; or R3COR4, acidic medium, reflux; (f) MnO2, CHCl3, reflux.

2992 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Poitout et al.
F igu r e 3. Set of compounds of structure 4 synthesized by solution-phase parallel synthesis.
Sch em e 2a
parallel synthesis. Condensation of amines 3a -f with
an excess of aldehyde in dichloromethane, in the pres-
ence of trifluoroacetic acid, followed by evaporation of
the mixture and subsequent trapping of the remaining
aldehyde by aminomethylpolystyrene resin, yielded the
desired tetrahydro-â-carbolines of structure 4 (R4 ) H).
The purity of the crude mixtures of diastereomers, as
determined by LC/MS, was in the range 70-97%.
Following this procedure, more than 400 compounds
were prepared (Figure 3) and tested as mixtures of
diastereomers (ratios varied from 60:40 to 90:10 as
determined by LC/MS and/or 1H NMR). Compound 4f
was isolated, after recrystallization of the fumarate salt
of the mixture from methyl ethyl ketone, as the major
and pure diastereomer, which we assumed to be the
trans-1,3-tetrahydro-â-carboline.29 Pictet-Spengler cy-
clization of amines 3a -f with ketones required more
vigorous conditions and afforded the corresponding
tetrahydro-â-carbolines 4g-u . Oxidation of compounds
of general structure 4 (when R4 ) H) to 5 was achieved
using manganese dioxide in refluxing chloroform33
followed by filtration of the mixture on a Celite pad. This
convenient procedure allowed us to perform the reaction
in parallel and to prepare a set of 50 fully aromatized
carbolines of general structure 5.
a
Reagents: (g) (i) NH3/MeOH, (ii) NaHCO3, P2S5, (CH3OCH2)2;
(h) 2-bromoacetophenone, 90 °C; (j) 1 N HCl/EtOAc; (k) 5-nonanone,
1-butanol, reflux.
The thiazole derivative 9 (Scheme 2) was prepared
by conversion of Boc-D-Trp-OMe to the corresponding

Somatostatin Antagonists with sst3 Selectivity
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2993
Ta ble 1. Binding of Compounds 4a -u , 5a , 9, and 10 to Human sst3 Receptors
C3
config
compd
R2
R3
R4
R5
salt
Ki(sst3) (nM)a
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4p
4q
4r
4s
4t
R
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
2-NO2Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
200 ( 4.0
60 ( 8.1
R
4-MeOPh
4-NMe2Ph
4-pyridinyl
n-pentyl
R
33 ( 7.0
R
320 ( 76
11 ( 4.2
R
R
cyclohexyl
C4H4O4
2HCl
1.7 ( 0.11
84 ( 0.5
R
2-adamantyl
2-indanyl
R
13 ( 2.6
R
1-acetyl-4-piperidine
ethyl
71 ( 31
R
ethyl
11 ( 1.4
R
n-butyl
n-butyl
n-butyl
n-pentyl
n-hexyl
methyl
n-butyl
n-butyl
n-pentyl
n-butyl
n-butyl
n-butyl
n-butyl
n-pentyl
n-hexyl
cyclohexyl
n-butyl
n-butyl
n-pentyl
n-butyl
0.64 ( 0.12
0.79 ( 0.38
3.4 ( 1.0
0.92 ( 0.45
28 ( 6.0
R
C4H4O4
S
R
R
R
0.90 ( 0.41
17 ( 5.4
R/S
R/S
R/S
R
PhCH2O
Cl
CH3
H
3.4 ( 0.39
6.9 ( 1.2
5500 ( 3300
750 ( 83
3800 ( 2400
430 ( 190
4u
5a
9
HCl
10
a
Results are expressed as the mean ( SEM of 2-4 experiments performed in duplicate (compound concentrations ranged from 0.01
nM to 10 µM).
thioamide 6 using a saturated methanolic solution of
ammonia under pressure followed by treatment with
P2S5. Condensation of 6 with 2-bromoacetophenone and
subsequent cyclization to thiazole 7 afforded amine 8
after acidic deprotection. Pictet-Spengler cyclization
with 5-nonanone gave rise to the desired thiazolyl
tetrahydro-â-carboline 9. Compound 10 was prepared
analogously to derivative 4k , starting from Boc-D-
benzothienylalanine.
caused a 17-40-fold drop in affinity. There was a
preference for the R stereochemistry at the C3 center
since 4k was 6 times more potent than the S-isomer
4m . Substitution on the indole moiety did not increase
potency (4r , 4s, 4t), while replacing indole by ben-
zothiophene caused an important decrease in affinity
(10, Ki ) 430 nM), the latter result confirming the
importance of the indole ring as a mimic of Trp8 in SRIF.
Replacement of the basic imidazole moiety by a thiazole
(compound 9) resulted in a loss of activity (Ki ) 3800
nM), suggesting its key role in sst3 binding affinity.
Imidazole substitution seemed to be critical: biological
screening of libraries of tetrahydro-â-carbolines 4 with
various R1 groups (Figure 3) showed that compounds
with a phenyl substituent exhibited the highest affinity
whereas substitution on the phenyl ring resulted in a
dramatic drop in activity (percentage inhibition at 10
µM, <30%; data not shown). When R2 is a phenyl
instead of hydrogen, the compound 4u is about 8000
times less active than 4k .
Attempts to further increase the rigidity of the
tetrahydro-â-carboline template by synthesizing the
corresponding fully aromatized planar â-carbolines 5
resulted in a decrease in affinity, suggesting that
compounds 4 display an optimal binding conformation
and/or that the basic nitrogen is necessary for sst3
binding (compare 4f and 5a ).
Binding affinities of the most potent compounds 4f,
4k , 4m , and 4n toward sst1-5 receptors (Table 2)
revealed a 1000-10000-fold selectivity for sst3. The
P h a r m a cology
The affinity of the compounds for human SRIF
receptors was determined by measuring the inhibition
of [125I-Tyr11]SRIF-14 binding to membranes isolated
from CHO-K1 cells expressing each of the cloned human
SRIF receptor subtypes. The library screening was
performed in 96-well plate format. Percentage inhibi-
tions were first determined at 10 µM, and compounds
with greater than 70% inhibition at this concentration
were selected for inhibition constant (Ki) determination.
The biological activity of the derivatives 4 (Figure 3)
was confirmed by resynthesizing and retesting a se-
lected set of molecules with moderate to high affinity
for sst3 receptors (see Table 1, 4a -f).
Among this first set of compounds, 4e (Ki ) 11 nM)
and 4f (Ki ) 1.7 nM) were the most potent leads (Table
1). Removal of the C1 chiral center by using symmetrical
ketones (4k and 4n ) increased the activity (Ki ) 0.64
and 0.92 nM, respectively). Shortening the alkyl chain
of 4k (compound 4j) or extending it by two carbons (4p )

2994 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Poitout et al.
Ta ble 2. Binding Affinities (Ki, nM) of Selected
Tetrahydro-â-carbolines to Human sst1-5 Receptors
Ki (nM)
compd
4f
4k
4m
4n
4q
SRIF-14
sst1
sst2
sst3
sst4
sst5
1800
3700
>10000
>10000
4400
>10000
>10000
>10000
>10000
>10000
1.7 >10000
0.64
3.4
0.92 >10000
0.90 >10000
>10000
2200
3700
>10000
>10000
7100
9800
0.29
0.035 0.10
0.63
0.12
Ta ble 3. Inhibition of SRIF-14 (1 nM) Induced Reduction of
cAMP Accumulation Due to Forskolin (1 µM) in CHO-K1 Cells
Expressing the Human sst3 Receptor
compd
IC50 (nM)a
compd
IC50 (nM)a
4f
19 ( 9.7
2.7 ( 0.7
4s
4q
4t
43 ( 25
5.4 ( 4.2
450 ( 150
4k
4m
4n
6.8 ( 5.1
0.84 ( 0.27
a
Results are expressed as the mean ( SEM of three experi-
ments performed in triplicate (compound concentrations ranged
from 0.1 nM to 10 µM).
F igu r e 4. Inhibition by compound 4n of SRIF-14 decreased
cAMP accumulation induced by forskolin (1 µM) in CHO-K1
cells expressing the human sst3 receptor. The dose-response
reduction of cAMP levels induced by SRIF-14 was measured
with 1 nM (O), 10 nM (9), 100 nM (0), and 1000 nM (2)
compound 4n or without (b) compound 4n . Data represent the
mean ( standard error of three experiments performed in
duplicate. The inset shows the Schild regression analysis of
the data. DR is the ratio of the EC50 of SRIF-14 in the presence
of compound 4n over the EC50 of SRIF-14 alone. KB is the
concentration of compound 4n when log(DR - 1) ) 0.
functional activity of tetrahydro-â-carboline derivatives
4 with the highest binding activity was assessed by
measuring the modulation of intracellular adenosine
cyclic 3′-5′-monophosphate (cAMP) production stimu-
lated by forskolin on CHO-K1 cells expressing the
human sst3 receptor subtype.
While derivatives 4 were unable to reduce the cAMP
production elicited by forskolin as expected for agonists,
they blocked the inhibitory effect of 1 nM SRIF-14.
Compound 4n was the most potent antagonist with an
IC50 value of 0.84 nM (Table 3). In another set of
experiments, the dose-response decrease of cAMP
levels induced by SRIF-14 was measured in the presence
of increasing concentrations of 4n . Compound 4n in-
creased the EC50 of SRIF-14 in a dose-dependent man-
ner without affecting the maximal inhibition observed
(Figure 4). Schild regression analysis of the data led to
a KB value (concentration of compound 4n that shifts
SRIF-14 dose-response 2-fold) of 2.8 nM with a slope
factor of 0.9. These results demonstrate that compound
4n acts as a competitive antagonist at the human sst3
receptors. So far, very few cyclic peptide SRIF analogues
have been known to exhibit antagonist activity with
partial subtype selectivity.34-37 Meanwhile, during the
revision of this paper, Reubi et al. reported the discovery
of octapeptide derivatives as somatostatin sst3 receptor
antagonists.38 But, the limited number of sst3 selective
or partially selective ligands and the lack of common
structural features among them do not offer clear
assumptions for the factors determining agonist/antago-
nist activity at sst3 receptors.
probably due to its basicity compared to that of the
thiazole. In addition, the â-carboline moiety is necessary
for sst3 binding; its replacement by a benzothienopyri-
dine template proved to be detrimental to activity. On
the basis of these observations and the knowledge of
the crucial Phe7-Trp8-Lys9-Thr10 segment which com-
prises a â-turn, essential for SRIF binding, two hypoth-
eses may be proposed: (a) where the phenyl group and
â-carboline of structures 4 mimic the Phe7-Trp8 moiety
or (b) where the â-carboline and the imidazole, although
less basic than the terminal amine of lysine, mimic the
Trp8-Lys9 moiety of the tripeptide. However, as these
assumptions do not provide a satisfactory explanation
for their high selectivity and their antagonist behavior
at sst3 receptors, other hypotheses may be considered.
Incorporation of constrained amino acids in a SRIF
mimetic has been shown to provide more potent com-
pounds with altered activity and selectivity profiles
toward sst1-5.
39 Thus, the conformational rigidity of the
tetrahydro-â-carboline scaffold may play an important
role in selective binding at sst3. In addition, specific
lipophilic and electrostatic interactions between the
constrained derivatives 4 and crucial residues of the sst3
receptor subtype, located in the extracellular and/or
transmembrane domains, may be considered. A se-
quence alignment of the five human SRIF receptor
subtypes revealed that several amino acids are con-
served or conservatively exchanged in sst1, sst2, sst4, and
sst5 but not in sst3, particularly in the extracellular loop
III and in transmembrane domain VII (e.g., Glu286-
Glu287, Phe290-Phe291). Site-directed mutagenesis
may be helpful in determining whether the latter
residues are essential for tetrahydro-â-carboline 4 bind-
ing. This method was successfully used by Kaupmann
et al.,40 where advantage was taken between the binding
Con clu sion
Our approach consisted of the synthesis of an imida-
zolyl scaffold displaying various possibilities of attach-
ment and which was amenable to parallel synthesis.
The choice of these imidazolyl derivatives allowed us
to introduce, via a Pictet-Spengler cyclization, a con-
strained indole moiety. Despite the knowledge of the
crucial role of the indole group for somatostatin receptor
binding, the high sst3 selectivity of these new tetra-
hydro-â-carboline derivatives 4 remains puzzling. SAR
analysis suggests a key role of the imidazole moiety

Somatostatin Antagonists with sst3 Selectivity
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2995
ter t-Bu tyl (1S)-2-(1H-In d ol-3-yl)-1-(4-p h en yl-1H-im id a -
zol-2-yl)eth ylca r ba m a te (2b). Using the same procedure as
described for 2a , Boc-L-Trp-OH (50 g, 164 mmol) gave an oil.
Crystallization in dichloromethane afforded 53 g (80%) of 2b
as a white solid: mp 208-210 °C; 1H NMR (DMSO-d6) δ 1.32
(s, 9H), 3.15 (dd, 1H, 3J ) 7 Hz, 2J ) 14 Hz), 3.35 (m, 1H),
4.88 (m, 1H), 6.94-7.77 (m, 12H), 10.77 (s, 1H), 11.87 (s, 1H).
ter t-Bu tyl 2-[5-(Ben zyloxy)-1H-in d ol-3-yl]-1-(4-p h en yl-
1H-im id a zol-2-yl)eth ylca r ba m a te (2c). Using the same
procedure as described for 2a , Boc-5-BzO-Trp-OH (7 g, 17
mmol) gave an oil. Purification by flash chromatography using
AcOEt/heptane (1:1) as the eluent afforded 3.8 g (44%) of 2c
as an off-white foam: 1H NMR (DMSO-d6) δ 1.32 (s, 9H), 3.11
selectivity of octreotide (Figure 2) for sst2 over sst1
receptors, thus identifying two crucial residues located
in the transmembrane domains VI and VII.
In conclusion, the tetrahydro-â-carboline derivatives
BN 81644 (4k ; Ki ) 0.64 nM, IC50 ) 2.7 nM) and BN
81674 (4n ; Ki ) 0.92 nM, IC50 ) 0.84 nM, KB ) 2.8 nM)
are believed to be the first non-peptide sst3 antagonists
known so far. These new highly potent and selective sst3
antagonists are useful tools for investigating the physi-
ological role of the sst3 receptors. Few biological data
are so far available: expression studies have revealed
a high level of sst3 receptor mRNA in the cerebellum,41,42
gastrointestinal tract,43 rat adrenal PC12 cells,44 mouse
insulinoma MIN6 cells,45 and human J urkat T cells.46
Moreover, a subtype-selective induction of apoptosis by
sst3 receptors has been reported,47 suggesting a possible
effect of sst3 antagonists as anti-apoptotic agents.
Studies are in progress to evaluate the therapeutic
potential of this new class of non-peptidic somatostatin
receptor ligands.
3
2
(dd, 1H, J ) 7.5 Hz, J ) 14 Hz), 3.30 (m, 1H), 4.85 (m, 1H),
4.99 (m, 2H), 6.75-7.78 (m, 16H), 10.61 (s, 1H), 11.80 (s, 1H).
ter t-Bu tyl 2-(5-Meth yl-1H-in d ol-3-yl)-1-(4-p h en yl-1H-
im id a zol-2-yl)eth ylca r ba m a te (2d ). Using the same proce-
dure as described for 2a , Boc-5-Me-Trp-OH (6.6 g, 20 mmol)
gave 2.2 g (27%) of 2d as an off-white foam: 1H NMR (DMSO-
3
2
d6) δ 1.39 (s, 9H), 2.39 (s, 3H), 3.19 (dd, 1H, J ) 6.5 Hz, J )
14 Hz), 3.30 (m, 1H), 4.92 (m, 1H), 6.90-7.83 (m, 11H), 10.64
(s, 1H), 11.84 (s, 1H).
ter t-Bu tyl 2-(5-Ch lor o-1H-in d ol-3-yl)-1-(4-p h en yl-1H-
im id a zol-2-yl)eth ylca r ba m a te (2e). A mixture of 5-Cl-Trp-
OH (10 g, 42 mmol) and di-tert-butyl dicarbonate (10.05 g, 46
mmol) in dioxane (120 mL) was refluxed for 2 h and then
cooled to room temperature. The solvent was evaporated and
the resulting oil crystallized in diisopropyl ether to afford 12
g (84%) of Boc-5-Cl-Trp-OH.
Using the same procedure as described for 2a , Boc-5-Cl-Trp-
OH (12 g, 35 mmol) gave an oil which was crystallized in
diisopropyl ether/heptane to afford 10 g (65%) of 2e as an off-
white foam: 1H NMR (DMSO-d6) δ 1.36 (s, 9H), 3.17 (dd, 1H,
3J ) 6.5 Hz, 2J ) 14 Hz), 3.30 (m, 1H), 4.90 (m, 1H), 7.05-
7.83 (m, 11H), 11.01 (s, 1H), 11.85 (s, 1H).
ter t-Bu t yl (1R)-1-(4,5-Dip h en yl-1H -im id a zol-2-yl)-2-
(1H-in d ol-3-yl)eth ylca r ba m a te (2f). Using the same pro-
cedure as described for 2a , Boc-D-Trp-OH (9.1 g, 30 mmol) and
2-bromo-2-phenylacetophenone (8.26 g, 30 mmol) gave an oil.
Purification by flash chromatography using dichloromethane/
methanol (97:3) as the eluent followed by crystallization of the
resulting oil in diisopropyl ether afforded 7.2 g (50%) of 2f as
an off-white powder: mp 143-145 °C; 1H NMR (CDCl3) δ 1.43
(s, 9H), 3.45 (dd, 1H, 3J ) 6 Hz, 2J ) 14 Hz), 3.65 (m, 1H),
5.07 (m, 1H), 5.47 (s, 1H), 7.01-7.62 (m, 15H), 8.09 (s, 1H),
9.48 (s, 1H).
Exp er im en ta l Section
Ch em istr y. Anhydrous dichloromethane and N,N-dimeth-
ylformamide (DMF) were purchased in septum-sealed bottles,
and all other solvents were of reagent grade and used as
received from various commercial sources. Aminomethylpoly-
styrene resin was purchased from Novabiochem. Analytical
thin-layer chromatography was performed on silica gel 60 F254
coated glass plates, visualized under UV light. Melting points
were determined using a Bu¨chi 535 apparatus and are
uncorrected. 1H and 13C NMR spectra were recorded on a
Bru¨cker ARX 400 spectrometer. Chemical shifts are expressed
in parts per million (ppm) relative to tetramethylsilane, and
spin multiplicities are given as s (singlet), d (doublet), dd
(double of doublets), t (triplet), or m (multiplet). Optical
rotation was measured at 20 °C with a J asco DIP-370 pola-
rimeter. LC/MS was recorded on a Micromass Plateform II.
Elemental analyses were performed on a Fison EA 1108
apparatus. The percentage of water included in the elemental
analyses was determined by a coulometric Karl Fisher titration
using a Metrohm 737 KF coulometer.
Rep r esen ta tive P r oced u r e for th e Syn th esis of Im i-
d a zole Der iva tives of Str u ctu r e 2: P r ep a r a tion of ter t-
Bu t yl (1R)-2-(1H -In d ol-3-yl)-1-(4-p h en yl-1H -im id a zol-2-
yl)eth ylca r ba m a te (2a ). A solution of Boc-D-Trp-OH (50 g,
164 mmol) and cesium carbonate (27 g, 83 mmol) in EtOH (350
mL) was shaken for about 30 min at room temperature, and
then evaporated under reduced pressure. To the resulting salt
in DMF (600 mL) was added 2-bromoacetophenone (32.6 g,
164 mmol). The mixture was stirred for about 1 h at room
temperature under argon and then concentrated under re-
duced pressure. Ethyl acetate (400 mL) was added, the mixture
filtered, and the CsBr washed with ethyl acetate. The filtrate
was then concentrated under reduced pressure. A solution of
the resulting oil and ammonium acetate (246 g, 320 mmol) in
xylene (2 L) was refluxed for 45 min. Excess NH4OAc and H2O
were removed using a Dean-Stark trap. The mixture was then
cooled to room temperature, diluted with ethyl acetate (1 L),
and washed twice with water, 10% NaHCO3 aqueous solution
until basic pH, and brine. The organic layer was then dried
(Na2SO4), and concentrated under reduced pressure until ca.
400 mL of solvent remained. The organic layer was allowed
to stand at room temperature, upon which crystals appeared
that were collected by filtration and washed with diethyl ether
to afford 57.5 g (89%) of 2a as a white powder: mp 208-210
ter t-Bu tyl (1R)-2-(1-Ben zoth iop h en -3-yl)-1-(4-p h en yl-
1H-im id a zol-2-yl)eth ylca r ba m a te (2g). Using the same
procedure as described for 2a , Boc-D-3-benzothienylalanine (5
g, 15 mmol) gave an oil. Crystallization in isopentane afforded
4 g (64%) of 2g as a white powder: mp 116-120 °C; 1H NMR
3
(DMSO-d6) δ 1.30 (s, 9H), 3.30 (m, 1H), 3.59 (dd, 1H, J ) 6
2
Hz, J ) 12 Hz), 5.03 (m, 1H), 7.19-7.96 (m, 12H), 11.93 (s,
1H).
Rep r esen ta tive P r oced u r e for th e Syn th esis of Am in o
Im id a zole Der iva tives 3. P r ep a r a tion of (1R)-2-(1H-
In d ol-3-yl)-1-(4-p h en yl-1H -im id a zol-2-yl)-1-et h a n a m in e
(3a ). Into a suspension of 2a (57 g, 142 mmol) in ethyl acetate
(500 mL) at 0 °C was bubbled dry HCl until TLC (eluent ethyl
acetate, 100%) showed complete disappearance of starting
material. The resulting mixture was then evaporated under
reduced pressure. Diethyl ether was added to the resulting
solid, and the mixture was filtered. The dihydrochloride was
washed with dichloromethane followed by diethyl ether to
afford 57 g (99%) of a white foam.
3a was used in the next step either as its dihydrochloride
salt or as its free base.
1
°C; H NMR (DMSO-d6) δ 1.32 (s, 9H), 3.15 (dd, 1H, 3J ) 7
The free base 3a was obtained after treatment of the salt
with 10% NaHCO3 aqueous solution and the usual workup:
1H NMR (CD3OD) δ 3.36 (dd, 1H, 3J ) 7 Hz, 2J ) 14.3 Hz),
3.45 (dd, 1H, 3J ) 7 Hz, 2J ) 14.3 Hz), 4.51 (t, 1H, 3J ) 7 Hz),
6.95-7.68 (m, 11H); [R]D ) -114° (c 1.2, DMSO).
(1S)-2-(1H-In d ol-3-yl)-1-(4-p h en yl-1H-im id a zol-2-yl)-1-
eth a n a m in e Dih yd r och lor id e (3b). Using the same proce-
Hz, 2J ) 14 Hz), 3.35 (m, 1H), 4.88 (m, 1H), 6.94-7.77 (m,
12H), 10.77 (s, 1H), 11.87 (s, 1H); 13C NMR (DMSO-d6) δ 28.34,
30.22, 49.99, 78.04, 110.55, 111.37, 112.47, 118.30, 118.49,
120.90, 123.54, 124.01, 124.36, 125.93, 127.64, 128.48, 128.93,
135.21, 136.13, 139.53, 149.21, 155.19; [R]D ) +6.0° (c 1.3,
DMSO). Anal. (C24H26N4O2‚0.75H2O) C, H, N.

2996 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Poitout et al.
dure as described for 3a , 2b (53 g, 132 mmol) gave 49 g (98%)
of dihydrochloride 3b as a hygroscopic foam: 1H NMR (DMSO-
d6) δ 3.65 (dd, 1H, J ) 6 Hz, J ) 14 Hz), 3.80 (dd, 1H, J )
10 Hz, 2J ) 14. Hz), 4.98 (m, 1H), 6.95-8.03 (m, 11H), 9.31 (s,
3H), 11.02 (s, 1H).
afforded 805 mg (83%) of the title product as a foam: diaster-
eomeric ratio 88:12; 1H NMR (DMSO-d6) δ 3.10 (m, 2H, CH2),
3.73 (s, 3H, OCH3), 4.16 (m, 1H, H3), 5.24; 5.23 (2s, 1H, H1),
6.89-7.74 (m, 14H), 10.64; 10.69 (2s, 1H), 11.91; 12.13 (2s,
1H). Anal. (C27H24N4O‚0.5H2O) C, H, N.
3
2
3
2-[5-(Ben zyloxy)-1H-in d ol-3-yl]-1-(4-p h en yl-1H-im id a -
zol-2-yl)eth yla m in e Dih yd r och lor id e (3c). Using the same
procedure as described for 3a , 2c (3.8 g, 7.5 mmol) gave 2.8 g
(78%) of dihydrochloride 3c as an off-white powder: mp 180-
(3R)-1-(4-Dim et h yla m in op h en yl)-3-(4-p h en yl-1H -im i-
d a zol-2-yl)-2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e (4c). Free
base 3a (800 mg, 2.64 mmol), 4-(dimethylamino)benzaldehyde
(788 mg, 5.28 mmol), and TFA (2.0 mL, 26.4 mmol) gave an
oil. Basic workup followed by flash chromatography on silica
gel using AcOEt as the eluent afforded 998 mg (87%) of the
1
2
3
182 °C; H NMR (DMSO-d6) δ 3.62 (dd, 1H, J ) 14 Hz, J )
2
3
5.5 Hz), 3.74 (dd, 1H, J ) 14 Hz, J ) 10 Hz), 4.99 (m, 3H),
1
6.74-8.05 (m, 15H), 9.31 (m, 3H), 10.89 (s, 1H).
title product as a foam: diastereomeric ratio 58:42; H NMR
(DMSO-d6) δ 2.85, 2.88 (2s, 6H, N(CH3)2), 3.06 (m, 2H, CH2),
4.25 (m, 1H, H3), 5.23 (s, 1H, H1), 6.72-7.78 (m, 14H), 10.25
(s, 1H), 12.01 (2s, 1H). Anal. (C28H27N5‚0.5H2O) C, H, N.
2-(5-Meth yl-1H-in d ol-3-yl)-1-(4-p h en yl-1H-im id a zol-2-
yl)et h yla m in e Dih yd r och lor id e (3d ). Using the same
procedure as described for 3a , 2d (1.6 g, 3.8 mmol) gave 1.12
g (75%) of dihydrochloride 3d as a hygroscopic foam: 1H NMR
(DMSO-d6) δ 2.25 (s, 3H), 3.62 (dd, 1H, J ) 14 Hz, J ) 5.5
Hz), 3.74 (dd, 1H, 2J ) 14 Hz, 3J ) 10 Hz), 4.94 (m, 1H), 6.84-
8.01 (m, 10H), 9.31 (m, 3H), 10.88 (s, 1H).
2-(5-Ch lor o-1H-in d ol-3-yl)-1-(4-p h en yl-1H-im id a zol-2-
yl)eth yla m in e Dih yd r och lor id e (3e). Using the same pro-
cedure as described for 2a , 2e (10 g, 23 mmol) gave 6.5 g (69%)
of dihydrochloride 3e as a white solid: mp 198-200 °C; 1H
NMR (DMSO-d6) δ 3.59 (dd, 1H, 2J ) 14 Hz, 3J ) 5.5 Hz),
3.74 (dd, 1H, 2J ) 14 Hz, 3J ) 10 Hz), 4.94 (m, 1H), 6.84-8.01
(m, 10H), 9.20 (m, 3H), 11.21 (s, 1H).
(3R )-3-(4-P h e n yl-1H -im id a zol-2-yl)-1-(4-p yr id in yl)-
2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e (4d ). A solution of free
amine 3a (1,5 g, 5 mmol), pyridine-4-carbaldehyde (954 µL,
10 mmol), and TFA (3.85 mL, 50 mmol) in dichloromethane
(25 mL) was stirred for 2 h at room temperature and
evaporated under reduced pressure. The resulting residue was
diluted in EtOAc (80 mL), washed successively with 10%
NaHCO3 aqueous solution and brine, dried (Na2SO4), and
evaporated to yield an oil. Flash chromatography on silica gel
using dichloromethane/ethanol (90:10) as the eluent afforded
250 mg (13%) of 4d as an off-white powder: mp 204-206 °C;
2
3
1
diastereomeric ratio 85:15; H NMR (CD3OD) δ 3.20 (m, 2H,
(1R)-1-(4,5-Dip h en yl-1H -im id a zol-2-yl)-2-(1H -in d ol-3-
yl)-1-et h a n a m in e Dih yd r och lor id e (3f). Using the same
procedure as described for 3a , 2f (3.6 g, 7.5 mmol) gave 2.4 g
(71%) of dihydrochloride 3f as an off-white solid: mp 260-
3
2
CH2), 4.25, 4.48 (2 dd, 1H, J ) 4.9 Hz, J ) 10 Hz, H3), 5.38,
5.43 (2s, 1H, H1), 7.01-7.71 (m, 12H), 8.49-8.55 (m, 2H). Anal.
(C25H21N5‚0.5H2O) C, H, N.
1
2
3
262 °C; H NMR (DMSO-d6) δ 3.64 (dd, 1H, J ) 14 Hz, J )
6 Hz), 3.74 (dd, 1H, 2J ) 14 Hz, 3J ) 9 Hz), 4.88 (m, 1H), 6.93-
7.47 (m, 15H), 9.25 (s, 3H), 11.07 (s, 1H).
(3R)-1-(n -P en tyl)-3-(4-p h en yl-1H-im id a zol-2-yl)-2,3,4,9-
tetr a h yd r o-1H-â-ca r bolin e (4e). Free base 3a (500 mg, 1.65
mmol), hexanal (395 µL, 3.30 mmol), and TFA (1.27 mL, 33.2
mmol) gave an oil. Basic workup followed by flash chroma-
tography on silica gel using AcOEt/heptane (90:10) as the
eluent afforded 320 mg (50%) of the title product as an off-
(1R)-2-(1-Ben zoth ioph en -3-yl)-1-(4-ph en yl-1H-im idazol-
2-yl)-1-eth a n a m in e Dih yd r och lor id e (3g). Using the same
procedure as described for 3a , 2g (4,0 g, 9.5 mmol) gave 3.0 g
(80%) of dihydrochloride 3g as an off-white powder: mp 190-
1
white foam: diastereomeric ratio 65:35; H NMR (DMSO-d6)
1
2
3
192 °C; H NMR (DMSO-d6) δ 3.80 (dd, 1H, J ) 14 Hz, J )
δ 0.85 (s, 3H), 1.33-1.46 (m, 6H), 1.63 (m, 1H), 2.04 (m, 1H),
2.95 (m, 2H, CH2), 4.10 (m, 1H, H3), 4.19 (m, 1H, H1), 6.93-
7.80 (m, 10H), 10.73 (s, 1H), 11.97 (s, 1H). Anal. (C25H28N4‚
0.25H2O) C, H, N.
2
3
5.5 Hz), 3.93 (dd, 1H, J ) 14 Hz, J ) 10 Hz), 5.05 (m, 1H),
7.35-8.05 (m, 11H), 9.40 (m, 3H).
Gen er a l P r oced u r e for Tetr a h yd r o-â-ca r bolin e 4 Li-
br a r y Syn th esis. In a vial, to a solution of an amine of
structure 3 (0.05 mmol) in chloroform (0.8 mL) were succes-
sively added an aldehyde (0.10 mmol) and TFA (0.5 mmol).
The mixture was stirred for 5 h at room temperature on an
orbital shaker and then concentrated under reduced pressure.
The resulting residue was diluted in THF (3 mL), and
aminomethylpolystyrene resin (loading 2.75 mmol/g, 0.15
mmol) was added. The resulting suspension was shaken
overnight on an orbital shaker at room temperature and then
filtered on cartridges with frits. The filtrate was concentrated
under reduced pressure, and the resulting residue was filtered
on a silica gel pad (with ethyl acetate as the eluent) to afford
the tetrahydro-â-carboline of structure 4 as a mixture of
diastereomers. An aliquot was analyzed by LC/MS.
(3R)-1-Cycloh exyl-3-(4-ph en yl-1H-im idazol-2-yl)-2,3,4,9-
tetr a h yd r o-1H-â-ca r bolin e F u m a r a te (4f). A solution of
free amine 3a (3.6 g, 12 mmol), cyclohexanecarboxaldehyde
(2.68 g, 24 mmol), and TFA (9.2 mL, 120 mmol) in dichlo-
romethane (90 mL) was stirred for 2 h at room temperature
and evaporated under reduced pressure. The resulting residue
was diluted in dichloromethane (100 mL), washed successively
with 10% NaHCO3 aqueous solution and brine, dried (Na2SO4),
and evaporated under reduced pressure to give an oil. Flash
chromatography on silica gel using AcOEt/heptane (70:30) as
the eluent afforded 2.15 g (45%) of the title product as its free
base (diastereomeric ratio 85:15). To the free base (1.18 g, 3
mmol) in methyl ethyl ketone (15 mL) was added fumaric acid
(345 mg, 3 mmol). The mixture was warmed to 50 °C to obtain
a solution. The white crystals obtained when the solution was
allowed to stand overnight at room temperature were collected
by filtration, washed with methyl ethyl ketone and diethyl
ether, and dried to afford 1.2 g (80%) of white crystals of 4f as
its fumarate salt: mp 200-202 °C; one single diastereomer;
1H NMR (DMSO-d6) δ 1.18-1.79 (m, 10H), 2.10 (m, 1H), 2.92
(t, 1H, 2J ) 3J ) 11 Hz, H4), 3.01 (dd, 1H, 2J ) 4 Hz, H4_),
4.12 (dd, 1H, 3J ) 11 Hz, 3J ) 4 Hz, H3), 4.22 (s, 1H, H1), 6.62
(s, 2H), 6.94-7.78 (m, 10H), 10.71 (s, 1H); 13C NMR (DMSO-
d6) δ 26.36, 26.83, 27.50, 29.68, 40.94, 52.96, 58.52, 108.09,
111.32, 115.17, 117.62, 118.63, 120.78, 124.55, 126.34, 127.11,
128.76, 134.21, 134.47, 135.28, 136.50, 137.85, 149.92, 166.63.
Anal. (C26H28N4, C4H4O4‚0.25H2O) C, H, N.
The following compounds were prepared using this proce-
dure. However, for confirmation of chemical structure and
biological activity, they were resynthesized on a larger scale
and purified by flash chromatography on silica gel.
(3R)-1-(2-Nit r op h en yl)-3-(4-p h en yl-1H-im id a zol-2-yl)-
2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e (4a ). Free base 3a (900
mg, 2.97 mmol), 2-nitrobenzaldehyde (896 mg, 5.95 mmol), and
TFA (2.25 mL, 29.7 mmol) gave an oil. Basic workup followed
by flash chromatography on silica gel using AcOEt/heptane
(60:40) as the eluent afforded 1.0 g (78%) of the title product
as a yellow foam: diastereomeric ratio 57:43; 1H NMR (DMSO-
d6) δ 3.10 (m, 2H, CH2), 4.09, 4.34 (2m, 1H, H3), 5.85, 5.91
(2m, 1H, H1), 6.98-8.03 (m, 14H), 10.41; 10.76 (2s, 1H), 11.82;
12.05 (2s, 1H). Anal. (C26H21N5O2‚1H2O) C, H, N.
(3R)-1-(4-Meth oxyp h en yl)-3-(4-p h en yl-1H-im id a zol-2-
yl)-2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e (4b). Free base 3a
(700 mg, 2.3 mmol), 4-anisaldehyde (562 µL, 4.62 mmol), and
TFA (1.7 mL, 23 mmol) gave an oil. Basic workup followed by
flash chromatography on silica gel using AcOEt as the eluent
Rep r esen ta tive P r oced u r e for P ictet-Sp en gler Cy-
cliza tion w ith Keton es. Meth od A: P r ep a r a tion of (3R)-
1,1-Dibu tyl-3-(4-p h en yl-1H-im id a zol-2-yl)-2,3,4,9-tetr a h y-
dr o-1H-â-car bolin e as Its Fr ee Base (4k) an d Its Fu m ar ate
Sa lt (4l). A mixture of dihydrochloride 3a (10 g, 33 mmol),
1-butanol (150 mL), and 5-nonanone (28.4 g, 165 mmol) was

Somatostatin Antagonists with sst3 Selectivity
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2997
1
refluxed for 4 h, and 10 mL of 1-butanol was then removed
using a Dean-Stark apparatus. After being refluxed for a
further 2 h, the mixture was heated at 100 °C overnight and
then cooled to room temperature. The solvent was evaporated
and the resulting residue partitioned between ethyl acetate
(100 mL) and 10% NaHCO3 solution (50 mL). After decanta-
tion, the organic layer was washed successively with 10%
NaHCO3 aqueous solution (50 mL) and water and then dried
(MgSO4). Evaporation of the solvent afforded a brown residue
which was purified by flash chromatography on silica gel using
dichloromethane/AcOEt (90:10) as the eluent. The pure frac-
tions were collected and concentrated under reduced pressure
to give, after being washed with diisopropyl ether, 3.6 g (26%)
of a white powder as the free base 4k (26%): mp 160-162 °C.
Anal. (C28H34N4‚1.75H2O) C, H, N.
solid: mp 198-200 °C; H NMR (DMSO-d6) δ 2.93-3.07 (m,
3H), 3.24, 3.36 (AB, 2H, J ) 16 Hz), 3.73 (d, 1H, J ) 16 Hz),
4.27 (m, 1H, H3), 6.95-7.73 (m, 14H), 10.91 (s, 1H), 11.83 (s,
1H); [R]D ) +43.0° (c 1.4, DMSO). Anal. (C28H24N4‚0.25H2O)
C, H, N: calcd 13.31, found 12.50.
1′-Acetyle-(3R)-(4-p h en yl-1H-im id a zol-2-yl)-2,3,4,9-tet-
r a h yd r osp ir o[â-ca r b olin e-1,4′-p ip er id in e] (4i). Using
method B, free base 3a (1.5 g, 5 mmol), N-acetylpiperidone
(1.23 mL, 10 mmol), and TFA (3.9 mL, 50 mmol) gave a brown
residue which was purified, after basic workup, by flash
chromatography on silica gel using dichloromethane/methanol
(95:5) as the eluent. The pure fractions were collected and
concentrated under reduced pressure to give, after crystal-
lization in dichloromethane/diethyl ether, 0.3 g (14%) of the
1
title compound as a gray powder: mp 248-250 °C; H NMR
(DMSO-d6) δ 1.63-1.93 (m, 3H), 2.06 (s, 3H, CH3), 2.20-2.40
(m, 2H), 2.88-3.25 (m, 3H), 3.62-3.69 (m, 2H), 4.19 (m, 1H),
4.29 (m, 1H), 6.95-7.82 (m, 10H), 10.82 (s, 1H), 11.89 (s, 1H);
[R]D ) +44.4° (c 1.2, DMSO). Anal. (C26H27N5O‚0.5 H2O) C,
H, N.
The free base 4k (1.3 g, 3 mmol) was dissolved in acetone
(5 mL). Fumaric acid (448 mg, 3 mmol) was added, and the
mixture was warmed to 50 °C to obtain a solution. The solution
was allowed to stand overnight, upon which white crystals
appeared. Diethyl ether (20 mL) was added, and the suspen-
sion was filtered to afford 1.05 g of the fumarate salt 4l as a
(3R)-1,1-Dieth yl-3-(4-p h en yl-1H-im id a zol-2-yl)-2,3,4,9-
tetr a h yd r o-1H-â-ca r bolin e (4j). Using method B, free base
3a (1.5 g, 5 mmol), 3-pentanone (1 mL, 10 mmol), and TFA
(3.9 mL, 50 mmol) gave a brown residue which was purified,
after basic workup, by flash chromatography on silica gel using
AcOEt/heptane (70:30) as the eluent. The pure fractions were
collected and concentrated under reduced pressure to give,
after crystallization in dichloromethane and being washed with
isopentane, 0.5 g (27%) of the title compound as a white
powder: mp 218-220 °C; 1H NMR (CD3OD) δ 0.79 (t, 3H),
1.02 (t, 3H), 1.76-2.05 (m, 4H), 2.94 (dd, 1H, 2J ) 14 Hz, 3J )
11 Hz, H4), 3.10 (dd, 1H, 2J ) 14 Hz, 3J ) 4 Hz, H4′), 4.47 (dd,
1H, 3J ) 11 Hz, 3J ) 4 Hz, H3), 6.92-7.71 (m, 10H). Anal.
(C24H26N4) C, H, N.
(3R)-1,1-Dip en tyl-3-(4-p h en yl-1H-im id a zol-2-yl)-2,3,4,9-
tetr a h yd r o-1H-â-ca r bolin e (4n ). Using method B, free base
3a (1.5 g, 5 mmol), 6-undecanone (2 mL, 10 mmol), and TFA
(3.9 mL, 50 mmol) gave a brown residue which was purified,
after basic workup, by flash chromatography on silica gel using
AcOEt/heptane (70:30) as the eluent. The pure fractions were
collected and concentrated under reduced pressure to give,
after crystallization in isopentane, 0.4 g (17%) of the title
compound as a white powder: mp 170-172 °C; 1H NMR
(DMSO-d6) δ 0.80 (t, 3H), 0.86 (t, 3H), 1.12-1.51 (m, 12H),
1.62-1.88 (m, 4H), 2.09 (s, 1H, NH), 2.78 (dd, 1H, 2J ) 14 Hz,
3J ) 11 Hz, H4), 3.00 (dd, 1H, 3J ) 3 Hz, 2J ) 14 Hz, H4′), 4.26
(m, 1H, H3), 6.92-7.79 (m, 10H), 10.67 (s, 1H), 11.95 (s, 1H);
[R]D ) +37.1° (c 1.1, DMSO). Anal. (C30H38N4‚0.25H2O) C, H,
N.
1
white solid: mp 168-170 °C; H NMR (DMSO-d6) δ 0.80 (t,
3H), 0.86 (t, 3H), 1.22-1.44 (m, 8H), 1.70-1.90 (m, 4H), 2.88
(dd, 1H, 2J ) 14 Hz, 3J ) 11 Hz, H4), 3.04 (dd, 1H, 2J ) 14 Hz,
3J ) 3.3 Hz, H4_), 4.37 (m, 1H, H3), 6.61 (s, 2H), 6.93-7.78 (m,
10H), 10.74 (s, 1H); 13C NMR (DMSO-d6) δ 14.28, 22.95, 23.19,
25.52, 26.32, 27.81, 38.49, 48.90, 58.01, 106.94, 111.24, 117.87,
118.54, 120.84, 124.59, 126.38, 126.70, 128.76, 134.11, 134.50,
136.27, 138.70, 149.71, 166.69; [R]D ) +6.8° (c 1.3, DMSO).
Anal. (C28H34N4, C4H4O4‚0.75H2O) C, H, N.
Meth od B: P r ep a r a tion of (3S)-1,1-Dibu tyl-3-(4-p h en -
yl-1H -im id a zol-2-yl)-2,3,4,9-t et r a h yd r o-1H -â-ca r b olin e
(4m ). A solution of free base 3b (3 g, 10 mmol), 5-nonanone
(3.5 mL, 20 mmol), and TFA (7.7 mL, 100 mmol) in dichloro-
ethane (90 mL) was refluxed for 8 h. The mixture was then
cooled to room temperature and evaporated under reduced
pressure. The resulting residue was partitioned between
dichloromethane (100 mL) and 10% NaHCO3 aqueous solution
(50 mL). After decantation, the organic layer was washed
successively with 10% NaHCO3 aqueous solution (50 mL) and
water and dried (MgSO4). Evaporation of the solvent afforded
a brown residue which was purified by flash chromatography
on silica gel using AcOEt/heptane (70:30) as the eluent. The
pure fractions were collected and concentrated under reduced
pressure to give, after being washed with isopentane, 1.0 g
(23%) of a white powder as the free base 4m : mp 136-138
°C; 1H NMR (DMSO-d6) δ 0.80 (t, 3H), 0.86 (t, 3H), 1.22-1.42
(m, 8H), 1.62-1.90 (m, 4H), 2.08 (s, 1H), 2.78 (dd, 1H, 2J ) 14
Hz, 3J ) 11 Hz, H4), 3.04 (dd, 1H, 2J ) 14 Hz, 3J ) 6 Hz, H4_),
4.37 (m, 1H, H3), 6.93-7.78 (m, 10H), 10.67 (s, 1H), 11.95 (s,
1H); [R]D ) -20.8° (c 1.9, MeOH). Anal. (C28H34N4‚0.25H2O)
C, H, N.
(3R)-1,1-Dih exyl-3-(4-p h en yl-1H-im id a zol-2-yl)-2,3,4,9-
tetr a h yd r o-1H-â-ca r bolin e (4p ). Using method A, dihydro-
chloride 3a (1.12 g, 3 mmol) and dihexyl ketone (2.69 g, 15
mmol) gave a brown residue which was purified, after basic
workup, by flash chromatography on silica gel using dichlo-
romethane/AcOEt (90:10) as the eluent. The pure fractions
were collected and concentrated under reduced pressure to
give, after crystallization in diethyl ether, 0.2 g (14%) of 4p
as a white powder: mp 166-167 °C; 1H NMR (DMSO-d6) δ
0.80 (t, 3H), 0.83 (t, 3H), 1.10-1.49 (m, 16H), 1.61-1.84 (m,
(3R)-3-(4-P h en yl-1H -im id a zol-2-yl)sp ir o[â-ca r b olin e-
1,2′-a d a m a n tyl] Dih yd r och lor id e (4g). Using method A,
dihydrochloride 3a (1.12 g, 3 mmol) and 2-adamantanone (2.25
g, 15 mmol) gave a brown residue which was purified, after
basic workup, by flash chromatography on silica gel using
AcOEt as the eluent. The pure fractions were collected and
concentrated under reduced pressure. The resulting oil was
dissolved in diethyl ether, and 1 N HCl in ethyl acetate was
added. The dihydrochloride was collected by filtration and
washed with diethyl ether to give 0.2 g (15%) of 4g as a white
2
3
4H), 2.05 (s, 1H, NH), 2.77 (dd, 1H, J ) 14 Hz, J ) 11 Hz,
H4), 2.99 (dd, 1H, 2J ) 14 Hz, 3J ) 4 Hz, H4′), 4.23 (m, 1H,
H3), 6.93-7.78 (m, 10H), 10.65 (s, 1H), 11.93 (s, 1H); [R]D
+26.8° (c 1.6, DMSO). Anal. (C32H42N4) C, H, N.
)
1
solid: mp 158-160 °C; H NMR (DMSO-d6) δ 1.61-2.56 (m,
13H), 2.80 (m, 1H), 3.37-3.63 (m, 2H), 3.63 (m, 1H), 7.03-
8.18 (m, 10H), 10.79 (s, 1H); [R]D ) +19.5° (c 1.4, DMSO). Anal.
(C29H30N4‚2HCl‚0.75H2O) C, H, N.
(3R)-1-Cycloh exyl-1-m et h yl-3-(4-p h en yl-1H-im id a zol-
2-yl)-2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e (4q). Using method
B, free base 3a (1.5 g, 5 mmol), cyclohexyl methyl ketone (1.4
mL, 10 mmol), and TFA (3.9 mL, 50 mmol) gave a brown
residue which was purified, after basic workup, by flash
chromatography on silica gel using AcOEt/heptane (70:30) as
the eluent. The pure fractions were collected and concentrated
under reduced pressure to give, after crystallization in diethyl
ether, 0.2 g (10%) of the title compound as a white powder:
mp 154-156 °C; 1H NMR (DMSO-d6) δ 0.92-1.32 (m, 6H), 1.48
(s, 3H, CH3), 1.60 (m, 2H), 1.79 (m, 2H), 1.95 (m, 2H), 2.77
2′,3′-Dih yd r o-(3R)-(4-p h en yl-1H-im id a zol-2-yl)sp ir o[â-
ca r bolin e-1,2′-1H-in d en e] (4h ). Using method A, dihydro-
chloride 3a (1.12 g, 3 mmol) and 2-indanone (1.98 g, 15 mmol)
gave a brown residue which was purified, after basic workup,
by flash chromatography on silica gel using AcOEt/heptane
(80:20) as the eluent. The pure fractions were collected and
concentrated under reduced pressure to give, after crystal-
lization in diisopropyl ether, 0.3 g (24%) of 4h as a white

2998 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Poitout et al.
(dd, 1H, 2J ) 14 Hz, 3J ) 11 Hz, H4), 2.97 (dd, 1H, 2J ) 14 Hz,
3J ) 4 Hz, H4′), 4.19 (m, 1H, H3), 6.88-7.79 (m, 10H), 10.65
(s, 1H). Anal. (C27H30N4‚0.25H2O) C, H, N.
H) (0.05 mmol) and MnO2 (5 equiv, w/w) in chloroform was
refluxed for 5 h. The mixture was cooled to room temperature
and filtered on a Celite pad. The filtrate was evaporated under
reduced pressure to afford the corresponding fully aromatized
â-carboline of structure 5.
Ben zyl 1,1-Dibu tyl-3-(4-ph en yl-1H-im idazol-2-yl)-2,3,4,9-
tetr a h yd r o-1H-â-ca r bolin -6-yl Eth er (4r ). Using method A,
dihydrochloride 3c (2.8 g, 5.8 mmol) and 5-nonanone (5 mL,
29 mmol) gave a brown residue which was purified, after basic
workup, by flash chromatography on silica gel using AcOEt/
heptane (1:1) as the eluent. The pure fractions were collected
and concentrated under reduced pressure to give, after crystal-
lization in diisopropyl ether, 230 mg (7%) of 4r as a white
powder: mp 138-140 °C; 1H NMR (CDCl3) δ 0.84 (t, 3H), 0.88
(t, 3H), 1.25-1.62 (m, 8H), 1.75-1.95 (m, 5H), 2.95 (dd, 1H,
1-Cycloh exyl-3-(4-p h en yl-1H -im id a zol-2-yl)-9H -â-ca r -
bolin e (5a ). A suspension of free base 1-cyclohexyl-3-(4-
phenyl-1H-imidazol-2-yl)-2,3,4,9-tetrahydro-1H-â-carboline 4f
(300 mg, 0.75 mmol) and MnO2 (1.8 g) in chloroform (15 mL)
was refluxed for 5 h. The mixture was cooled to room
temperature and filtered on a Celite pad. The filtrate was
evaporated under reduced pressure to afford a solid. Flash
chromatography on silica gel using AcOEt/heptane (1:1) gave,
after crystallization in diisopropyl ether, 205 mg (69%) of the
title compound as a white powder: mp 168-170 °C; 1H NMR
(DMSO-d6) δ 1.32-1.60 (m, 3H), 1.81-2.04 (m, 7H), 3.32 (s,
1H), 7.19-8.83 (m, 11H), 11.61 (s, 1H), 12.21 (s, 1H); 13C NMR
(DMSO-d6) δ 25.90, 26.43, 31.20, 41.07, 108.50, 112.13, 114.08,
119.51, 121.60, 122.04, 124.54, 126.14, 128.17, 128.39, 133.07,
135.16, 138.19, 141.02, 147.61, 149.41. Anal. (C26H24N4‚
0.25H2O) C, H, N.
3
2
3
2J ) 15 Hz, J ) 11 Hz, H4), 3.45 (dd, 1H, J ) 15 Hz, J ) 4
3
3
Hz H4′), 4.48 (dd, 1H, J ) 4 Hz, J ) 11 Hz, H3), 5.12 (s, 2H),
6.91-7.73 (m, 16H). Anal. (C35H40N4O) C, H, N.
1,1-Dib u t yl-6-ch lor o-3-(4-p h e n yl-1H -im id a zol-2-yl)-
2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e (4s). Using method A,
dihydrochloride 3e (1.2 g, 2.6 mmol) and 5-nonanone (2.23 mL,
13 mmol) gave a brown residue which was purified, after basic
workup, by flash chromatography on silica gel using dichlo-
romethane/AcOEt (90:10) as the eluent. The pure fractions
were collected and concentrated under reduced pressure to
give, after crystallization in diisopropyl ether, 500 mg (42%)
ter t-Bu tyl (1R)-2-Am in o-1-(1H-in dol-3-ylm eth yl)-2-th io-
oxoeth ylca r ba m a te (6). To a reactor under 200 psi of
pressure were added methyl (2R)-2-[(tert-butoxycarbonyl)-
amino]-3-(1H-indol-3-yl)propanoate (6.2 g, 22 mmol) and metha-
nol (120 mL) saturated with NH3. The solution was stirred at
85 °C for 24 h and then cooled to room temperature. The
solvent was evaporated and the product precipitated by the
addition of diisopropyl ether. Filtration gave 5.4 g (81%) of a
white powder (mp 142-143 °C). To a solution of the amide (5
g, 16 mmol) in 85 mL of 1,2-dimethoxyethane were added
NaHCO3 (5.2 g, 62 mmol) and then P2S5 (7.3 g, 32 mmol) over
a period of 45 min. The mixture was stirred overnight and the
solvent evaporated. The resulting residue was suspended in
ethyl acetate, washed successively with water and 10%
NaHCO3 aqueous solution, and dried (MgSO4). The solvent was
evaporated and the crude product precipitated by addition of
isopentane/diisopropyl ether (1:1). Filtration gave 4.3 g (84%)
of the title product as an off-white foam: 1H NMR (DMSO-d6)
1
of 4s as a white powder: mp 152-154 °C; H NMR (DMSO-
d6) δ 0.79 (t, 3H), 0.87 (t, 3H), 1.18-1.42 (m, 8H), 1.62-1.92
(m, 4H), 2.10 (s, 1H), 2.78 (dd, 1H, 2J ) 15 Hz, 3J ) 11 Hz,
H4), 2.99 (dd, 1H, 2J ) 15 Hz, 3J ) 4 Hz, H4′), 4.25 (m, 1H,
H3), 7.0-7.79 (m, 9H), 10.91 (s, 1H), 11.95 (s, 1H). Anal.
(C28H33ClN4) C, H, N.
6-Met h yl-1,1-d ip en t yl-3-(4-p h en yl-1H -im id a zol-2-yl)-
2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e (4t). Using method A,
dihydrochloride 3d (0.9 g, 2.3 mmol) and 6-undecanone (2.35
mL, 11.5 mmol) gave a brown residue which was purified, after
basic workup, by flash chromatography on silica gel using
AcOEt/heptane (1:1) as the eluent. The pure fractions were
collected and concentrated under reduced pressure to give,
after crystallization in diisopropyl ether, 0.35 g (35%) of 4t as
1
an off-white powder: mp 202-204 °C; H NMR (DMSO-d6) δ
2
3
δ 1.29 (s, 9H), 2.93 (dd, 1H, J ) 14.5 Hz, J ) 11 Hz), 3.14
(dd, 1H, 2J ) 14.5 Hz, 3J ) 4 Hz), 4.47 (m, 1H), 6.72-7.64 (m,
6H), 9.17 (s, 1H), 9.62 (s, 1H), 10.81 (s, 1H).
0.79 (t, 3H), 0.84 (t, 3H), 1.16-1.48 (m, 12H), 1.63-1.85 (m,
2
3
4H), 2.35 (s, 3H), 2.79 (dd, 1H, J ) 15 Hz, J ) 11 Hz, H4),
2
3
2.97 (dd, 1H, J ) 15 Hz, J ) 4 Hz H4′), 3.31 (s, 1H), 4.27 (m,
1H, H3), 6.82-7.77 (m, 9H), 10.54 (s, 1H). Anal. (C31H40N4‚
0.25H2O) C, H, N.
ter t-Bu tyl (1R)-2-(1H-In d ol-3-yl)-1-(4-p h en yl-1,3-th ia -
zol-2-yl)eth ylca r ba m a te (7). A mixture of 6 (2.24 g, 7 mmol)
and 2-bromoacetophenone (1.4 g, 7 mmol) was heated until
complete melting (90 °C). The temperature was kept at 90 °C
for 10 min and then the mixture allowed to cool to room
temperature. Ethyl acetate (50 mL) and water (25 mL) were
added. After decantation, the organic layer was washed with
10% NaHCO3 aqueous solution and water and dried (MgSO4).
Evaporation of the solvent afforded a residue which was
purified by flash chromatography on silica gel using dichlo-
romethane/AcOEt (95:5) as the eluent. The pure fractions were
collected and evaporated to give 1.1 g (38%) of the title
compound as a foam: 1H NMR (DMSO-d6) δ 1.33 (s, 9H), 3.20
(dd, 1H, 2J ) 14.5 Hz, 3J ) 10 Hz), 3.48 (dd, 1H, 2J ) 14.5 Hz,
3J ) 4.6 Hz), 5.07 (m, 1H), 6.97-7.97 (m, 12H), 10.82 (s, 1H).
(3R )-1,1-Dib u t yl-3-(4,5-d ip h e n yl-1H -im id a zol-2-yl)-
2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e (4u ). Using method A,
dihydrochloride 3f (1.30 g, 2.9 mmol) and 5-nonanone (2.5 mL,
14.5 mmol) gave a brown residue which was purified, after
basic workup, by flash chromatography on silica gel using
dichloromethane/AcOEt (95:5) as the eluent. The pure fractions
were collected and concentrated under reduced pressure to
give, after crystallization in diisopropyl ether, 0.6 g (38%) of
1
4u as a white solid: mp 150-152 °C; H NMR (DMSO-d6) δ
0.80 (t, 3H), 0.87 (t, 3H), 1.19-1.48 (m, 8H), 1.68-1.90 (m,
2
3
4H), 2.16 (s, 1H), 2.85 (dd, 1H, J ) 15 Hz, J ) 11 Hz, H4),
3.01 (dd, 1H, 2J ) 15 Hz, 3J ) 4 Hz, H4′), 4.25 (m, 1H, H3),
6.94-7.51 (m, 14H), 10.70 (s, 1H), 12.20 (s, 1H); [R]D ) +3.4°
(c 1.5, DMSO). Anal. (C34H38N4‚0.25H2O) C, H, N.
(1R)-2-(1H -In d ol-3-yl)-1-(4-p h en yl-1,3-t h ia zol-2-yl)-1-
eth a n a m in e Hyd r och lor id e (8). To a solution of 7 (1.2 g,
2.85 mmol) in ethyl acetate (10 mL) was added 20 mL of a 1
N HCl solution in ethyl acetate. The mixture was stirred for
2 h at 20 °C and then heated to 50 °C for a further 2 h. The
crystals which formed on cooling were collected by filtration
and washed with diethyl ether to give an 89% yield of the title
product as an orange powder: mp 170-172 °C; 1H NMR
(DMSO-d6) δ 3.38 (dd, 1H, 2J ) 14.5 Hz, 3J ) 10 Hz), 3.56 (dd,
(3R)-1,1-Dibu tyl-3-(4-p h en yl-1H-im id a zol-2-yl)-1,2,3,4-
t et r a h yd r o[1]b en zot h ien o[2,3-c]p yr id in e (10). Using
method A, dihydrochloride 3g (1 g, 2.5 mmol) and 5-nonanone
(2.23 mL, 13 mmol) gave a brown residue which was purified,
after basic workup, by flash chromatography on silica gel using
AcOEt/heptane (1:1) as the eluent. The pure fractions were
collected and concentrated under reduced pressure to give,
after crystallization in diisopropyl ether, 0.1 g (9%) of 10 as a
2
3
3
3
1
1H, J ) 14.5 Hz, J ) 5 Hz), 5.03 (dd, 1H, J ) 10 Hz, J ) 5
Hz), 6.95-8.07 (m, 11H), 8.95 (s, 3H), 11.04 (s, 1H). Anal.
(C19H17N3S‚HCl‚0.75H2O) C, H, N, S: calcd 8.68, found 7.98.
white solid: mp 198-200 °C; H NMR (DMSO-d6) δ 0.80 (t,
3H), 0.89 (t, 3H), 1.19-1.48 (m, 8H), 1.62-1.90 (m, 4H), 2.88
(dd, 1H, 2J ) 15 Hz, 3J ) 11 Hz, H4), 3.16 (dd, 1H, 2J ) 15 Hz,
3J ) 4 Hz, H4′), 4.36 (m, 1H, H3), 6.94-7.92 (m, 10H), 12.03
(s, 1H); [R]D ) +36.5° (c 1.5, DMSO). Anal. (C28H33N3S‚
0.75H2O) C, H, S, N: calcd 9.19, found 8.68.
(3R)-1,1-Dib u t yl-3-(4-p h en yl-1,3-t h ia zol-2-yl)-2,3,4,9-
t et r a h yd r o-1H -â-ca r b olin e H yd r och lor id e (9). Using
method A, hydrochloride 8 (210 mg, 0.59 mmol) and 5-nonanone
(0.52 mL, 3 mmol) gave a brown residue which was purified,
after basic workup, by flash chromatography on silica gel using
Gen er a l P r oced u r e for â-Ca r bolin e Libr a r y Syn th esis.
A suspension of tetrahydro-â-carboline of structure 4 (R4 )

Somatostatin Antagonists with sst3 Selectivity
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2999
dichloromethane/AcOEt (97:3) as the eluent. The pure fractions
were collected and concentrated under reduced pressure. The
resulting oil was dissolved in diethyl ether, and 1 N HCl in
ethyl acetate was added. The hydrochloride was collected by
filtration and washed with diethyl ether to give 85 mg (30%)
of the title product as an orange powder: mp 134-136 °C; 1H
NMR (DMSO-d6) δ 0.90 (m, 6H), 1.28-1.51 (m, 8H), 2.10-
2.41 (m, 4H), 3.38 (m, 2H), 5.55 (m, 1H, H3), 7.03-8.30 (m,
10H), 9.74 (s, 1H), 9.99 (s, 1H), 11.26 (s, 1H). Anal. (C28H33N3S‚
HCl‚0.25H2O) C, H, N, S.
Ack n ow led gm en t. We thank J ose Camara, Denis
Giraud, Gilles Mario, and Karine Suchet for analytical
support.
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HindIII, 1.4-kb NheI-NdeI, and 1.2-kb HindIII-XbaI for sst1,
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Hu m a n Som a tosta tin Recep tor Bin d in g Assa y. CHO-
K1 cells stably expressing each distinct SRIF receptor subtype
were collected with 0.5 mM EDTA, and centrifuged at 500g
for 5 min at 4 °C. The pellet was resuspended in 50 mM Tris,
pH 7.4, and centrifuged twice at 500g for 5 min at 4 °C. The
cells were lysed by sonication and centrifuged at 39000g for
10 min at 4 °C. The pellet was resuspended in the same buffer
and centrifuged at 50000g for 10 min at 4 °C, and membranes
in the resulting pellet were stored at -80 °C. An aliquot was
taken for the assessment of protein content by Bradford’s
method.52 Competitive inhibition experiments of [125I-Tyr11]-
SRIF-14 (NEN Life Science Products) binding were run in
polypropylene 96-well plates. Cell membranes (10-20 µg of
protein/well) were incubated in 50 mM HEPES (pH 7.4), 0.2%
bovine serum albumin (BSA), 5 mM MgCl2, 200 KIU/mL
Trasylol, 0.02 mg/mL bacitracin, and 0.02 mg/mL phenyl-
methanesulfonyl fluoride with 0.05-0.1 nM [125I-Tyr11]SRIF-
14 for 50-90 min at 37 °C depending on the receptor subtype.
Bound [125I-Tyr11]SRIF-14 was separated from free [125I-Tyr11]-
SRIF-14 by filtration through 96-well GF/C glass fiber filter
plates (Unifilter, Packard) presoaked with 0.1% (w/v) poly-
ethylenimine, using a Packard Filtermate harvester. Filters
were washed three times with 50 mM HEPES at 0-4 °C and
assayed for radioactivity using a Packard Topcount scintilla-
tion counter. Specific binding was obtained by subtracting
nonspecific binding (determined in the presence of 0.1 µM
SRIF-14) from total binding. Binding data were analyzed by
computer-assisted nonlinear regression analysis (data analysis
toolbox, MDL Information Systems).
F u n ction a l Assa y. CHO-K1 cells expressing the human
sst3 receptor were used at confluence in 96-well culture plates.
The cells were washed twice with 100 µL/well of RPMI 1640
containing 0.2% BSA at 37 °C and incubated for 5 min with 1
µM isobutylmethylxanthine at 37 °C. Then, the plates were
incubated for 20 min at 37 °C with 1 µM forskolin and
increasing concentrations of the tested compound (measure-
ment of agonist activity), or with 1 µM forskolin, 1 nM SRIF,
and increasing concentrations of the tested compound (mea-
surement of antagonist activity). Rapid aspiration and addition
of 100 µL/well of 0.1 M HCl stopped the reaction.53 The potency
of the most potent antagonist was also assessed by performing
dose-response measurements of SRIF-14 activity (concentra-
tion ranged from 1 pm to 1 µM) in the presence of fixed
concentrations of the tested compound, leading to the deter-
mination of the KB value (affinity of the antagonist expressed
as the concentration that shifts the agonist dose-response
2-fold). Cyclic AMP levels were determined by radioimmu-
noassay using Flash Plates (NEN Life Science Products).
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