Small molecule somatostatin receptor subtype-2 antagonists

Article abstract of DOI:10.1016/S0960-894X(01)00568-6

The first potent small molecule sst2 antagonists are reported. Altering known sst2 agonist molecules yielded compounds with high sst2 binding affinity and full antagonist activity. Compound 7a, for example, displaced somatostatin binding to the sst2 recep

Full text of DOI:10.1016/S0960-894X(01)00568-6

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2731–2734
Small Molecule Somatostatin Receptor Subtype-2 Antagonists
Bruce A. Hay,* Bridget M. Cole, Frank DiCapua, Glen W. Kirk, Marianne C. Murray,
Rona A. Nardone, Dennis J. Pelletier, Anthony P. Ricketts,
Alan S. Robertson and Todd W. Siegel
Global Research and Development, Groton Laboratories, Pfizer Inc, Eastern Pt. Road, Groton, CT 06340-5146, USA
Received 1 June 2001; accepted 3 August 2001
Abstract—The first potent small molecule sst2 antagonists are reported. Altering known sst2 agonist molecules yielded compounds
with high sst2 binding affinity and full antagonist activity. Compound 7a, for example, displaced somatostatin binding to the sst2
receptor with an IC50=2.9 nM and antagonized somatostatin action with an IC50=29 nM. # 2001 Elsevier Science Ltd. All rights
reserved.
The somatostatin peptide is known to have multiple
functions in the endocrine system. Some of the many
physiological functions of somatostatin include the
inhibition of growth hormone, glucagon, and insulin
secretion, and the regulation of gastrin secretion from
the gastrointestinal tract.1 There are five main receptor
subtypes that have been cloned from human tissue.2 Of
these, the sst2 receptor subtype has attracted some of
the most interest because of the role it plays in growth
hormone regulation.
Based on experience with our series, we introduced
small changes in the scaffolding of the L-054,522 series
of sst2 agonists in an attempt to discover structurally
related antagonists.
Compounds were made by standard amide and urea
coupling methods as shown in Scheme 1. d-Trp-Lys(-
BOC)-O-t-Bu was synthesized by an EDC mediated
coupling of Z-d-Trp and HLys(BOC)-O-t-Bu followed
by hydrogenolysis of the Z group. The BOC protected
derivatives of ureas 7a–d were formed by reaction of the
d-Trp-Lys(BOC)-O-t-Bu substrate with N,N0-disucci-
midyl carbonate followed by the requisite mono-
substituted piperazine. The BOC protected derivative of
amide 7e was formed by an EDC mediated coupling of
d-Trp-Lys(BOC)-O-t-Bu with the N-substituted iso-
nipecotic acid. The BOC groups were removed by reac-
tion with 5% TFA in DCM at room temperature for 10
min to yield the finished products 7a–e.
We were interested in developing a small molecule sst2
antagonist in the belief that this would upregulate
growth hormone levels in livestock. Starting from our
initial high-throughput screening lead 1, we were able to
develop compounds such as 2 with good receptor bind-
ing affinities (IC50=85 nM). It was intriguing to find
that we could make both functional agonists and
antagonists in this series, and that function would often
flip between agonist and antagonist with small changes
in the molecule (Fig. 1).
The sst2 receptor binding assay was conducted as pre-
viously described6 with minor modification. The assay
utilized Neuro2A cells transiently expressing the full
length porcine sst2 amino acid sequence.7 Membranes
were incubated with [125I]-somatostatin 14 (15 nCi) and
test compounds for 1 h at 37 ꢀC, before vacuum filtra-
tion through glass fiber filters and quantitation of
bound radioactivity by liquid scintillation counting.
Cyclic AMP (cAMP) content of pituitary cells was used
to differentiate somatostatin agonists from antagonists
as previously described,8 with minor modifications.
GH4C1 cells at 1–2ꢁ106/mL were incubated for 20 min
at 37 ꢀC with test compound dose-titrations in the pre-
sence of 100 nM vasoactive intestinal peptide (VIP) and
A series of small molecule sst2 agonists has recently
been disclosed by Merck scientists.3 These molecules,
exemplified by L-054,522 (3), are very potent and selec-
tive agonists that inhibit growth hormone release in
vitro. However, no antagonists were reported in this
series. Indeed, small molecule sst2 antagonists have
been elusive in the literature.4 The only examples of sst2
antagonists are peptide derivatives of somatostatin,5
exemplified by 4 and 5.
*Corresponding author. Tel.:+1-860-441-4403; fax:+1-860-441-6952;
e-mail: bruce_a_hay@groton.pfizer.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00568-6

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B. A. Hay et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2731–2734
Figure 1. sst2 agonists and antagonists.
Scheme 1. (a) HLys(BOC)-O-t-Bu.HCl, DMAP, EDC, HOBt, DCM; (b) H2, Pd/C, MeOH; (c) N,N0-disuccimidyl carbonate, DIEA, THF; (d)
benzenesulfonylpiperazine, DIEA, THF; (e) 4-benzenesulfonylisonipecotic acid, DMAP, EDC, HOBt, DCM; (f) 5% TFA, DCM, 10 min.
10 nM somatostatin 14. Following incubation, cAMP
content was determined using Adenylyl Cyclase Activa-
tion FlashPlate1 plates (NEN) in comparison with cAMP
standards. VIP increased cAMP content of the GH4C1
cells, and somatostatin caused a partial inhibition. Soma-
tostatin antagonists increased cAMP content in compar-
ison to control wells containing VIP and somatostatin
alone. Somatostatin agonists decreased cAMP content.
pos Associates. A three-dimensional structure was
determined for each of the compounds, and conformers
were generated by systematically rotating every rota-
table bond in 5 ꢀ increments. The conformers were then
clustered into families, and the lowest energy member of
each family was taken to be representative of that clus-
ter. Conformations were not determined for the long
alkylamine chains corresponding to the lysine residue;
the flexible nature of this chain allows it to be placed in
an equivalent orientation in each of the compounds.
Moreover, we chose to model it in its extended form
All calculations were performed using standard proto-
cols available through the SYBYL forcefield from Tri-

B. A. Hay et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2731–2734
2733
because of the known activity of a more rigid form3 of
3. Each of the representative conformers was minimized
and placed into an ensemble. This ensemble of con-
formations was then compared to the NMR structure of
Sandostatin1 (octreotide acetate),9 and a pharmaco-
phore was determined by finding common conforma-
tions that were consistent with the solution structure.
It is widely accepted that the Phe7-Trp8-Lys9 residues of
somatostatin are critical for bioactivity. These same
residues or suitable mimics are found in essentially all of
the peptide analogues of somatostatin, including the
0
0
commercial product Sandostatin1 (as Phe3 -Trp4 -
0
DLys5 ) and in all of the smaller sst2 peptidomimetic
agonists reported to date. Compounds 2 and 3, for
example, each have a terminal aryl group, a Trp or
modified Trp residue, and a Lys residue that correspond
to the somatostatin Phe7, Trp8, and Lys9 residues,
respectively. Our strategy was to meld certain features
of structures 2 and 3 in order to retain the potency of 3
and the antagonist activity of 2. Starting with the ago-
nist 6, a simple des-methyl analogue of L-054,522 (3),
we synthesized a series of derivatives where the benz-
imidazolonepiperidine group of 6 was replaced with an
N-substituted piperazine or an N-substituted iso-
nipecotic acid, yielding structures 7a–e10 (Table 1). As
we hoped, structures 7a–e retained most of the binding
affinity of 6, but were all full antagonists (Table 2, Fig.
2). All compounds had binding affinities in the low
nanomolar range, with functional potency ranging from
12 to 7700 nM.11
Table 1. Structural data for compound 7a–e
Entry
A
B
R1
Molecular modeling indicated that antagonists 7a–e
would have the terminal aryl group (Phe7 equivalent) in
a position closer to the antagonist 2 than the agonist 6,
as shown in Figure 3. The orientation of the terminal
aryl group in antagonist 2 is expected to be different
from agonist 6 because of the different stereochemistry
at the Trp center. While antagonists 7a–e have the same
Trp stereochemistry as agonist 6, it appears that the
sulfonamide sp2 nitrogen in 7a, 7b, and 7e (or amide sp2
nitrogen in 7c and 7d) can alter the geometry of 7a–e
enough to place the terminal aryl group in the hypo-
thesized antagonist binding pocket.
7a
7b
7c
7d
7e
N
N
N
N
CH
SO2
SO2
CO
CO
SO2
H
CH3
H
CH3
H
Table 2. Sst2 receptor binding and functional data for compounds 1–
2, 5–6 and 7a–e. Binding data are from duplicate 12-point titrations,
each with triplicate wells per concentration. Functional data are from
individual experiments, or are means from 2–4 replicate experiments
Entry
Sst2 Binding
IC50 (nM)
Functional activity
Functional
IC50 (nM)
Small structural changes have also been responsible for
a functional change in a peptidic series of somatostatin
analogues. Hocart et al.5 found that changing the chir-
ality of DCys6 to l in cyclic peptide 4 switched function
from antagonist to agonist. Their molecular modeling
studies indicated that this would dramatically change
the orientation of the exocyclic Nal residue with no
apparent change in the relative positions of the Tyr7,
DTrp8, and Lys9 residues, contrary to what we observed
1
2
5
6
7a
7b
7c
7d
7e
>2000
85
Antagonist
Antagonist
Antagonist
Agonist
Antagonist
Antagonist
Antagonist
Antagonist
Antagonist
6500
1200
270
6.5
4.1
0.26
2.9
9.2
6.9
4.4
3.2
29
7700
1600
67
120
Figure 2. Agonism (6) and antagonism (5 and 7b) of somatostatin
action in GH4C1 cells. Data are meanꢂstandard error from three to
four experiments.
Figure 3. Overlap of three structures from the sst2 pharmacophore.
Agonist 6 is shown in turquoise and antagonists 2 and 7a are shown in
orange and yellow, respectively.

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B. A. Hay et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2731–2734
with 6 versus 7a–e. It may be that 7a–e and 4 are hitting
different antagonist binding pockets, or perhaps the
inversion at Cys6 induces subtle changes in the con-
formation of 4 in the Tyr7, DTrp8 and Lys9 region.
Parmar, R. M.; Foor, F.; Mitra, S. W.; Degrado, S. J.; Shu,
M.; Klopp, J. M.; Cai, S.-J.; Blake, A.; Chan, W. W. S.; Pas-
ternak, A.; Yang, L.; Patchett, A. A.; Smith, R. G.; Chapman,
K. T.; Schaeffer, J. M. Science 1998, 282, 737.
4. Liu, J.; Underwood, D. J.; Cascieri, M. A.; Rohrer, S. P.;
Cantin, L.-D.; Chicchi, G.; Smith, A. B.; Hirschmann, R. J.
Med. Chem. 2000, 43, 3827.
5. (a) Hocart, S. J.; Jain, R.; Murphy, W. A.; Taylor, J. E.;
Morgan, B.; Coy, D. H. J. Med. Chem. 1998, 41, 1146. (b)
Morgan, B.; Murphy, W.; Coy, D. H. WO 9824807, Chem.
Abstr. 1998, 1998, 129 6803. (c) Hocart, S. J.; Jain, R.; Mur-
phy, W. A.; Taylor, J. E.; Coy, D. H. J. Med. Chem. 1999, 42,
1863. (d) Bass, R. T.; Buckwalter, B. L.; Patel, B. P.; Pausch,
M. H.; Price, L. A.; Strnad, J.; Hadcock, J. R. Mol. Pharma-
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In summary, we have discovered the first small molecule
sst2 antagonists. An analysis of the in vivo effects of
these molecules is the next step in defining their prac-
tical utility.
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for certain compounds a particularly high degree of receptor
occupancy is required for effective modulation of signal
transduction.