Synthesis of substituted imidazopyrazines as ligands for the human somatostatin receptor subtype 5

Article abstract of DOI:10.1016/S0960-894X(01)00051-8

A new preparation of trisubstituted imidazopyrazines and dihydroimidazopyrazines via parallel synthesis using aminoacids and bromoketones resulted in the discovery of non-peptidic sst₅ selective agonists.

Full text of DOI:10.1016/S0960-894X(01)00051-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 741±745
Synthesis of Substituted Imidazopyrazines as Ligands for the
Human Somatostatin Receptor Subtype 5
Marie-Odile Contour-Galcera,a,* Lydie Poitout,a Christophe Moinet,a Barry Morgan,b
Tom Gordon,b Pierre Rouberta and Christophe Thurieaua,*
a
Â
Institut Henri Beaufour, 5, Avenue du Canada, F-91966 Les Ulis Cedex, France
bBiomeasure Inc., 27 Maple Street, Milford, MA 01757, USA
Received 29 May 2000; revised 12 January 2001; accepted 19 January 2001
AbstractÐA new preparation of trisubstituted imidazopyrazines and dihydroimidazopyrazines via parallel synthesis using ami-
noacids and bromoketones resulted in the discovery of non-peptidic sst5 selective agonists. # 2001 Elsevier Science Ltd. All rights
reserved.
Somatostatin (SRIF) is a cyclic tetradecapeptide that
inhibits the release of various peptide hormones includ-
ing growth hormone (GH),1 insulin, glucagon2 and
gastrin.3 SRIF has been shown to inhibit cell prolifera-
tion4 and can also act as a neurotransmitter.5 The bio-
Fig. 1). It can be argued that the clinical use of this class
of therapeutics is limited by their lack of oral bioavail-
ability.14 Thus, the discovery of orally active com-
pounds which interact with somatostatin receptors
could be potentially bene®cial.
logical e€ects of SRIF are mediated through ®ve
6
distinct G protein-coupled receptor subtypes (sst1À5
)
Extensive investigations of structure±activity relation-
ships of peptide based SRIF analogues have shown that
the tetrapeptide sequence Phe7-d-Trp8-Lys9-Thr10 is
essential for biological activity.10,11 From the results of
NMR and crystallographic studies,12 several analogues
have been shown to adopt a b II0 turn around the Trp8-
Lys9 dipeptide, and non-peptide mimics involving the
use of a sugar13À15 or a benzodiazepinone16 core to
for which the respective physiological role remains to be
fully determined. The therapeutic applications of soma-
tostatin are limited by its low metabolic stability in vivo,
and this has led to the development of more stable pepti-
dic analogues such as the hexapeptide seglitide7 (MK 678,
I, Fig. 1) and the cyclic octapeptides octreotide8 (San-
dostatin1, II, Fig. 1) and lanreotide9 (Somatuline1, III,
Figure 1. Structures of MK 678 (I), octreotide (II), lanreotide (III), L-054,264 (IV), L-054,522 (V), and NNC 26-9100 (VI).
*Corresponding authors. Fax:+33-1-69-07-38-02; e-mail: m-odile.galcera.contour@beaufour- ipsen.com; christophe.thurieau@beaufour-ipsen.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00051-8

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M.-O. Contour-Galcera et al./ Bioorg.Med.Chem.Lett.11 (2001) 741±745
742
mimic the b-turn have been described as ligands for
somatostatin receptors. Recently, tryptophan deriva-
tives (L-054,264 (IV), L-054,522 (V), Fig. 1) were
reported to be potent and selective sst2 agonists with
inhibition constants (Ki) of 1.6 and 0.01 nM, respec-
tively.17,18 2-Pyridylthioureas have also been identi®ed
as potent sst4 agonists. Among them, NNC 26-9100 (VI,
Fig. 1) exhibited a Ki value of 6 nM at the sst4 receptor
and shows good selectivity with respect to other SRIF
receptor subtypes.19
a€orded the imidazole derivative (4), after work up and
puri®cation by ¯ash chromatography. Following this
procedure, more than 15 imidazole derivatives (4) were
prepared. N-alkylation of the imidazole ring was then
carried out using a solution-phase parallel strategy. The
nucleophilic character of the free nitrogen atom of imi-
dazole was enhanced by polymer supported N-methyl
morpholine,25 which permitted substitution with a-bro-
moketone (5). The resulting N-alkylated compound (6)
was isolated after addition of aminomethyl polystyrene
resin to the reaction mixture to trap any excess of bro-
moketone. The acidic cleavage of the tert-butoxy car-
bonyl group allowed spontaneous cyclization to yield
the dihydroimidazopyrazine (7). When R1 was a 3-
methyl-indole group, the imidazopyrazine (7) could be
puri®ed and characterized. In all other cases, the inter-
mediate (7) was not stable and was oxidized to the cor-
responding fully aromatized imidazopyrazine (8) either
by air oxidation in methanolic solution or by addition
of an oxidation agent such as manganese dioxide or
chromic acid supported on resin (Magtrieve1).26 The
dihydro intermediate (7) could be reduced to the tetra-
hydroimidazopyrazine by treatment with borohydride
supported on resin in MeOH.27 The secondary amino
group was acylated in chloroform in the presence of
morpholinomethylpolystyrene resin to a€ord N-sub-
stituted tetrahydroimidazopyrazines (9).
Knowing that dipeptides can be mimicked by ®ve-
membered heterocycles such as thiazoles, oxazoles or
imidazoles20 and also that the imidazole ring is an
excellent amide isostere,21 we focused our synthetic
e€orts on the development of an imidazole chemistry
leading to a series of imidazopyrazine derivatives. One
of our goals was to access this sca€old through parallel
synthesis in order to produce rapidly a large number of
structurally diverse compounds. As an example, we wish
to report the synthesis of trisubstituted imidazopyr-
azines and their binding pro®les at somatostatin recep-
tors, and particularly the sst5 subtype.
Chemistry
The synthesis of imidazo[1,2-a]pyrazine has been the
subject of a recent review.22 Several approaches,
including the condensation of an a-halocarbonyl com-
pound with a 2-aminopyrazine derivative,23 and the
reaction of an a-aminoalcohol with a chloropyrazine,
followed by oxidation of the alcohol to a ketone and
dehydrative ring closure24 have been described.
The structural diversity of imidazopyrazines obtained
by this strategy was inhibited by the limited molecular
diversity of commercially available bromoketones,
which are used in two occasions in our strategy. There-
fore, e€orts were focused on the synthesis of original
and diverse bromoketones. Two di€erent methods were
applied allowing the rapid preparation of 15 additional
bromoketones. In the ®rst case, a carboxylic acid was
activated as an acyl chloride or a mixed anhydride28 and
then transformed into a diazoketone using trimethyl-
silyldiazomethane. Bromination was then carried out
using HBr in dichloromethane. In the second case, a
methylketone was converted to the corresponding
We developed an original strategy (Scheme 1) where the
imidazole core is ®rst created from an N-protected a-
aminoacid,20 and alkylated with an a-bromoketone.
Subsequent cleavage of the N-protecting group cyclizes
to the desired bicyclic species. As described in Scheme 1,
the N-protected a-aminoacid (1) was transformed into
its cesium salt and condensed with an appropriate
a-bromoketone (2). Cyclization of the resulting keto-
ester (3) using ammonium acetate in re¯uxing xylene
29
bromoketone using CuBr2 or poly(vinylpyridinium
hydrobromide perbromide) resin30 as brominating
agent. We were particularly attracted to the preparation
of methyl-4-bromo-2,2-dimethyl-3-oxobutanoate (10)
Scheme 1. (a) (i) Cs2CO3 (0.5 equiv), EtOH/H2O (1:1); (ii) bromoketone (2) (1 equiv), DMF; (b) NH4OAc (20 equiv), xylene, 150 ꢀC, 1 h, Dean±
Stark; (c) polymer supported N-methyl-morpholine (2 equiv), DMF; (d) 10% TFA in DCM; (e) DMSO or MeOH, 24 h, 20 ꢀC or Magtrieve1
,
MeOH, 3 h, 40 ꢀC; (f) (i) polymer supported borohydride (4 equiv), MeOH, pH 5; (ii) R4COCl (1.3 equiv), CHCl3, polymer supported N-methyl-
morpholine (2 equiv).

Â
M.-O. Contour-Galcera et al./ Bioorg.Med.Chem.Lett.11 (2001) 741±745
743
(Scheme 2) and conversion to the imidazole inter-
Results and Discussion
mediate (11). The acid derivative (12) could be easily
coupled to various primary or secondary amines, giving
4-substituted imidazoles (13). This procedure dramatically
increased the diversity at the R2 position.
We have prepared libraries of dihydroimidazopyrazines
(7) and imidazopyrazines (8) incorporating side chains
of the somatostatin b-turn. More than 300 individual
Scheme 2. (a) CuBr2 (2.5 equiv), AcOEt, 80 ꢀC, 5 h; (b) (i) Cs2CO3 (0.5 equiv), EtOH/H2O (1:1); (ii) bromoketone (9) (1 equiv), DMF; (iii) NH4OAc
ꢀ
ꢀ
0
.
(20 equiv), xylene, 150 C, 1 h, Dean±Stark; (c) LiOH H2O (6.6 equiv), THF, 3 h, 80 C; (d) R2 NH2 (1.1 equiv), CDI (1.2 equiv), THF, 1 h.
Table 1. Inhibition constant (Ki) values of imidazopyrazines on human SRIF receptors
Compound
R1
R2
R3
Ki (nM)
a
a
a
a
b
sst1
sst2
sst3
sst4
sst5
7a
>10,000
>10,000
>10,000
>10,000
>1000
8a
8b
8c
8d
8e
8f
>5000
>5000
4000
>5000
>5000
3200
>5000
>5000
10,000
2500
>5000
>5000
>5000
>5000
>10,000
>10,000
7200
2300Æ740
430Æ69
3400
7900
4600
8600
1200
1200Æ300
540Æ20
>10,000
9700
>10,000
9500
8900
>10,000
>10,000
>10,000
7200
8g
8h
8i
3700
360Æ21
3300
6000
3100
790Æ130
600Æ53
2900
2100
1700
aData represent single values from one binding experiment.
bData represent the meanÆSEM of 2±3 experiments.

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M.-O. Contour-Galcera et al./ Bioorg.Med.Chem.Lett.11 (2001) 741±745
744
compounds were rapidly prepared by parallel synthesis
methods. The purity of the compounds was determined
by LC/MS.31 UV purity of the compounds presented
here is above 80%. Competitive inhibition of [125I-
Tyr11]SRIF-14 (NEN Life Science Products) binding to
membranes isolated from CHO-K1 cells stably expres-
sing each human SRIF receptor subtype, was measured
in 96-well plates by an adaptation of the method of
Shimon and al.32 Compounds were tested at 10 mM.
Inhibition constants (Ki) were determined for compounds
eliciting more than 70% inhibition at 10 mM.
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In conclusion, a series of diverse imidazopyrazines and
dihydroimidazopyrazines has been synthesized, and
their binding anity to each of the human somatostatin
receptor subtypes have been determined. Higher a-
nities were obtained with a basic group in the R1 posi-
tion, and we hypothesize that this might be mimicking
the side chain of the Lys9 residue of the b-turn. These
novel preferential non-peptidic sst5 ligands were shown
to be agonists. Sst5 receptors have a predominant role in
inhibiting the release of insulin from pancreatic islets35
and GH from pituitary cells.32 It was suggested that sst5
agonists may be e€ective in the treatment of GH and/or
prolactin secreting adenomas.36,37 Our results validate
the hypothesis that small heterocyclic sca€olds can pro-
vide SRIF receptor ligands with agonist characteristics.
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We wish to thank the Analytical Department for
performing the LC/MS analyses.

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M.-O. Contour-Galcera et al./ Bioorg.Med.Chem.Lett.11 (2001) 741±745
745
25. Morpholinomethyl-polystyrene HL (200±400 mesh), 2%
DVB, supplier: Novabiochem.
26. Chromic acid, polymer-supported, supplier: Aldrich Che-
mical Company, Inc.
(3H, m), 3.51±3.62 (1H, m), 3.25 (2H, t, J=7.44), 3.05±3.10
(4H, m), 2.86 (2H, t, J=7.45), 1.93±2.20 (2H, m), 1.65±1.70
(6H, m), 1.55 (6H, s), 1.22±1.26 (2H, m). MS (ESI) m/z 490.2
(M+H)+.
27. Borohydride, polymer-supported (on Amberlite1 IRA-
400), supplier: Aldrich Chemical Company, Inc.
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31. Mass spectra were acquired on a single quadrupole elec-
trospray mass spectrometer (Micromass, Platform model) and
HPLC retention times were acquired on an HPLC system (HP
1100) equipped with a photodiode array UV detector.
32. Shimon, I.; Taylor, J. E.; Dong, J. Z.; Bitonte, R. A.; Kim,
S.; Morgan, B.; Coy, D. H.; Culler, M. D. J.Clin.Invest. 1997,
99, 789.
34. CHO-K1 con¯uent cells expressing the human sst5 recep-
tor were washed twice with RPMI 1640 containing 0.2% BSA
at 37 ꢀC and incubated for
5 min with 1 mM iso-
butylmethylxanthine at 37 ꢀC. Then, they were incubated for
20 min at 37 ꢀC with 1 mM forskolin and increasing con-
centrations of sst5 agonist. Rapid aspiration and addition of
0.1 M HCl stopped the reaction (Nickols, G. A. Eur.J.Phar-
macol. 1985, 116, 137). Cyclic AMP levels were determined by
radioimmunoassay using Flash Plates (NEN Life Science
Products).
35. Rossowski, W. J.; Coy, D. H. Biochem.Biophys.Res.
Commun. 1993, 197, 366.
36. Shimon, I.; Yan, X.; Taylor, J. E.; Weiss, M. H.; Culler,
M. D.; Melmed, S. J.Clin.Invest. 1997, 100, 2386.
37. Jaquet, P.; Oua®k, L.; Saveanu, A.; Gunz, G.; Fina, F.;
Dufour, H.; Culler, M. D.; Moreau, J. P.; Enjalbert, A. J.
Clin.Endocrinol.Metab. 1999, 84, 3268.
33. Selected data for compound 8g: IR (KBr) n 2936, 1678,
1
1208, 1137, 800, 722 cmÀ1. H NMR (400 MHz, DMSO-d6, d
(TMS), J (Hz)): 8.90 (1H, s), 8.7 (1H, d, J=7.2), 8.11 (1H, s),
8.09 (1H, d, J=6.8), 8.01 (1H, s), 7.67±7.83 (2H, m), 7.41±7.50