Novel non-peptide ligands for the somatostatin sst3 receptor

Article abstract of DOI:10.1016/S0960-894X(01)00107-X

A series of imidazole derivatives has been prepared using high throughput parallel synthesis. Several compounds showed high affinity (K_i in 10^-6-10^-8M range) and selectivity at recombinant human somatostatin receptor subtype 3 (hsst₃).

Full text of DOI:10.1016/S0960-894X(01)00107-X

Bioorganic & Medicinal Chemistry Letters 11 (2001) 991–995
Novel Non-Peptide Ligands for the Somatostatin sst3 Receptor
Christophe Moinet,a,* Marie-Odile Contour-Galcera,a Lydie Poitout,a Barry Morgan,b
Tom Gordon,b Pierre Rouberta and Christophe Thurieaua,*
a
´
Institut Henri Beaufour, 5 Avenue du Canada, ZA de Courtaboeuf, F-91966 Les Ulis Cedex, France
bBiomeasure Inc., 27 Maple Street, Milford, MA 01757, USA
Received 29 May 2000; revised 5 February 2001; accepted 13 February 2001
Abstract—A series of imidazole derivatives has been prepared using high throughput parallel synthesis. Several compounds showed
high affinity (Ki in 10ꢀ6–10ꢀ8 M range) and selectivity at recombinant human somatostatin receptor subtype 3 (hsst3). # 2001
Elsevier Science Ltd. All rights reserved.
Somatostatin (SRIF) is a widely distributed peptide that
exhibits multiple biological functions such as inhibition
of the release of growth hormone, insulin, glucagon and
gastrin.1,2 SRIF also possesses antiproliferative and
neurotransmitter activities.3 Five somatostatin receptors
(sst1ꢀ5), all members of the G-protein coupled family,
have been cloned,4ꢀ6 however, the precise functional
activities of these receptors remain to be determined.7
Peptide analogues of SRIF, such as SMS 201-995
(Octreotide)8 and BIM 23014 (Lanreotide), have been
synthesized and developed for clinical use. Further stu-
dies on cyclic or linear peptide analogues9,10 are still
being reported but poor oral bioavailability, rapid pro-
teolysis and the relatively high cost of synthesis can be
considered as limitations for their clinical exploitation.
Consequently, considerable efforts have been reported
on the development of peptidomimetics11,12 of SRIF
which have the potential for oral bioavailability. Pio-
neering work on non-peptide structures designed to
mimic the peptide backbone of small cyclic peptides
with b-d-glucose,13 xylofuranose,14 mannitol,15 or ben-
zodiazepinone16 scaffolds have yielded compounds with
affinities for SRIF receptors in the micromolar range.
More recently, non-peptide structures with high affinity
and selectivity for the human somatostatin receptor
Imidazole dipeptide mimetics or ‘dipeptide azoles’ were
first described in the context of substance P antago-
nists,20 and more recently have been used to develop
endothelin antagonists,21 HIV protease inhibitors22 and
matrix metalloproteinase inhibitors.23 In the current
application, however, we were not interested in inserting
the dipeptide azole in a peptide sequence, but rather to
use it as an organic scaffold for rapid parallel synthesis
methods.
The derivatives 1 were prepared from commercially
available N-protected (Boc and Z) amino acids. Con-
densation of the cesium salts with a-bromoketones gave
access to ketoester intermediates that were then cyclized
with excess of ammonium acetate in refluxing xylene
with azeotropic removal of water. Removal of the ure-
thane N-protecting group (PG) was carried out either by
hydrogenolysis (palladium on charcoal in ethanol) for
the benzyloxycarbonyl group (Z) or by acidic treatment
(hydrochloric acid in ethyl acetate) for the tert-butylox-
ycarbonyl group (Boc) yielding compounds 2 (Scheme
1) which were isolated with yields ranged from 40 to
80% depending on the nature of R2 group. A variety of
aromatic, aliphatic and basic amino acids were used in
the synthesis of 2: those present in the SRIF b turn (Phe7,
Trp8, Lys9) were especially targeted.
17
subtypes have been identified, namely on sst2 and
sst4.18,19 In the present article, we report the design,
synthesis and biological evaluation of new potent
ligands for the sst3 receptor.
Among the enantiopure or racemic imidazole deriva-
tives 1 and 2 synthesized, indolyl derivatives exhibited
significant inhibition percentages (at 10ꢀ5 M) of radio-
ligand ([125I]Tyr11-SRIF) binding to the sst3 receptor
expressed in isolated membranes from CHO-K1 cells.24
A chiral preference for a d-Trp starting material was
discovered comparing the Ki values of 1a and 1c at the
*Corresponding author. Tel.: +33-160-922-000; fax: +33-169-073-
802; e-mail: christophe.thurieau@beaufour-ipsen.com; christophe.
moinet@beaufour-ipsen.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00107-X

992
C. Moinet et al. / Bioorg. Med. Chem. Lett. 11 (2001) 991–995
Scheme 1. Reagents and conditions: (a) (i) Cs2CO3 (0.5 equiv), DMF/H2O or EtOH/H2O; (ii) BrCH2COR2 (1 equiv), DMF; (b) ammonium acetate
(20 equiv), xylene, reflux; (c) for PG=Z: Pd/C, EtOH, H2, and for PG=Boc: AcOEt, HCl 3 N.
sst3 receptor (210 and 3200 nM, respectively). The
selectivity of 1a was calculated to be superior to 47 over
the other somatostatin receptors. Then we have
observed that compound 1b (Ki=1400 nM) was about
7-fold less potent than 1a and that the removal of the
protecting group induced a decrease of potency at sst3
receptor (Ki of compound 2a superior to 5000 nM)
suggesting a great influence of substitution on primary
amine present in 2a for binding to sst3 receptor. In
addition, a parallel investigation on the nature of the R2
group (tert-Bu or substituted aromatic), starting from
Boc-d-Trp, allowed us to conclude that an unsub-
stituted phenyl moiety was a key element for affinity at
sst3 (data not shown).
succinimidylcarbonate,28,29 yielded bicyclic compounds
4 which were isolated and fully characterized. Com-
pounds 4, in excess, were submitted to nucleophilic
attack at room temperature with primary and secondary
amines, followed by quenching with aminomethylated
resin to yield additional ureas 3 with increased diversity.
Amides 5 were prepared by peptide coupling chemistry
using excess carboxylic acid (preactivated with carbo-
nyldiimidazole) followed by quenching with amino-
methylated resin and purification through a silica gel
pad. Condensation of imidazole derivatives 2 with
excess thioimidates30 at room temperature yielded ami-
dines 6 after quenching with aminomethylated resin. We
have observed that 2-propanol was the solvent of choice
for this conversion to proceed in good purity and yield,
compared to ethanol or dimethylformamide. Con-
densation of excess of imidazole derivatives 2 with
aldehydes yielded imines which were reduced in the
presence of Amberlite1 IRA-400 borohydride. Excess
primary amine 2 was removed by addition of carbox-
aldehyde resin giving access to pure secondary amines 7.
We were able to prepare rapidly greater than 1500
compounds using these solution-phase strategies with
UV purity of ca. 90%.31 According to the biological
results described above, the greatest affinities for the sst3
receptor were obtained from derivatives of 2-{(1R)-1-
amino-2-[indol-3-yl]ethyl}-4-phenyl-1H-imidazole (2a,
R8=H, Ki values are summarized in Table 1). No
compound in the series of secondary amines was more
potent than 1a. In the series of ureas, the corresponding
aliphatic urea 12 was 2-fold less potent than carbamate
1a whereas aromatic urea 16, our best compound found
in this series, exhibited a slightly better potency but a
lower selectivity (15-fold). Our best amide derivative 14
These initial encouraging results prompted us to
develop procedures for rapid parallel acylations of imi-
dazole derivatives 2 and particularly from 2a. These
chemistries were carried out in solution using resin sca-
venger methodologies (Scheme 2).25ꢀ27
Ureas 3 were synthesized by reaction with an excess of
isocyanate followed by quenching with an amino-
methylated resin. Alternatively, reaction with N,N0-di-
Scheme 2. Reagents and conditions: (a) R3NCO (1.2 equiv), dichloromethane, then aminomethylated resin, dichloromethane (R4=H) or (i) N,N0-
disuccinimidylcarbonate, acetonitrile; (ii) R3NHR4 (0.9 equiv), acetonitrile, then aminomethylated resin, tetrahydrofuran; (b) R5CO2H (1.1 equiv),
.
carbonyldiimidazole (1.1 equiv), tetrahydrofuran, then aminomethylated resin; (c) R6NHSMe HI (1.2 equiv), 2-propanol, then aminomethylated
resin, tetrahydrofuran; (d) R7CHO (0.85 equiv), methanol, borohydride resin (Amberlite1 IRA-400), then carboxaldehyde resin, dichloromethane.

C. Moinet et al. / Bioorg. Med. Chem. Lett. 11 (2001) 991–995
993
Table 1. Inhibition constants (Ki) of substituted imidazole derivatives on human sst3 receptor
Ki (nM)
b
Compound
R8
Purity (%)a
99
sst1
sst2
sst3
sst4
sst5
1a
>10,000
>10,000
210ꢁ40
1400ꢁ400
700ꢁ40
500ꢁ90
410ꢁ110
400ꢁ50
250ꢁ50
240ꢁ20
200ꢁ60
170ꢁ10
79ꢁ12
>10,000
>10,000
1870
8c
94
90
76
84
97
91
99
73
99d
86
91
—
1880
2840
5920
>10,000
6040
>10,000
5920
9c
1190
10c
11c
12
3430
6660
2120
2570
4220
3970
1130
>10,000
1520
>10,000
8680
>10,000
7090
>10,000
2360
13c
14
9100
>10,000
>10,000
>10,000
3470
>10,000
>10,000
>10,000
3840
>10,000
2250
15c
16
1550
2600
3650
17c
18c
SRIF-14
1730
691
2310
6180
36ꢁ5
5010
1620
—
0.36
0.049
0.14ꢁ0.02
0.59
0.14
aUV purity of crude products was determined by reverse phase HPLC (see ref 31).
bMeanꢁSEM. Number of experiences: n=2–3.
cIodohydrate salts.
dCompound purified on a small pad of silica gel because of insufficient initial purity (58%).

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C. Moinet et al. / Bioorg. Med. Chem. Lett. 11 (2001) 991–995
exhibited both potency and selectivity comparable to 1a
for sst3 receptor. In the amidine series, the phenylami-
dine derivative 13 is slightly less potent than 1a.
Removal of the aromatic moiety of 13 induced a nota-
ble decrease of potency (compound 9 is about 3-fold less
potent than 1a). Both the nature and the position of
substitution on the phenyl moiety of amidine 13
appeared to play crucial roles for biological activity.
Whether 2,4-difluoro substitution gave compound 15
with comparable potency than 1a, para-substitution
seemed to have more impact on potency. We have thus
identified an amidine derivative 18 with a p-methoxy
substitution which is about 6-fold more potent than 1a
whereas a bulker group (p-tBu) present in amidine deri-
vative 8 clearly decreased the affinity. p-CF3 (10) and p-
OCF3 (11) amidine derivatives were, respectively, about
14- and 11-fold less potent than 18. Then p-Cl sub-
stituted compound 17 was both 2-fold less potent and
less selective (about 8-fold) than 18.
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In a functional assay based on the accumulation of
adenosine cyclic 30-50-monophosphate (cAMP) in CHO-
K1 cells expressing human sst3 receptors,33 derivative 18
was not able to reduce the cAMP production elicited by
forskolin. It blocked the inhibitory effect of SRIF-14 on
forskolin-induced cAMP accumulation with a Kb value
(concentration that shifts SRIF-14 dose-response 2-
fold) of 420 nM, demonstrating its antagonist property.
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In conclusion, we have identified a novel series of selec-
tive ligands for the human somatostatin subtype 3
receptor with affinities in the nanomolar range. These
imidazole derivatives, obtained by rapid parallel synth-
esis from amino acids, may serve as new pharmacologi-
cal tools for future investigations on this biological target.
Further optimization of these leads to increase affinity
while retaining selectivity will be reported in due course.
20. Gordon, T. D.; Hansen, P. E.; Morgan, B. A.; Singh, J.;
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Acknowledgements
The authors gratefully acknowledge Drs. D. Giraud and
J. Camara for expert analytical characterization of
compounds.
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C. Moinet et al. / Bioorg. Med. Chem. Lett. 11 (2001) 991–995
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30. Reaction of thioamides with iodomethane in acetone at
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33. CHO-K1 confluent cells expressing the human sst3 recep-
tor were washed twice with RPMI 1640 containing 0.2% BSA
at 37 ꢂC and incubated for
5 min with 1 mM iso-
butylmethylxanthine at 37 ꢂC. Then, they were incubated for
20 min at 37 ꢂC with 1 mM forskolin and increasing con-
centrations of tested compound (agonist activity) or with 1
mM forskolin and increasing concentrations of SRIF-14 with
or without the tested compound (antagonist activity) at 0.1, 1
or 10 mM. Rapid aspiration and addition of 0.1 M HCl stop-
ped the reaction (Nickols, G. A. Eur. J. Pharmacol. 1985, 116,
137). Cyclic AMP levels were determined by radio-
immunoassay using Flash Plates (NEN Life Science Products).