Spiro [1H-indene-1,4'-piperidine] derivatives as potent and selective non-peptide human somatostatin receptor subtype 2 (sst2) agonists [1]

Full text of DOI:10.1021/jm980194h

© Copyright 1998 by the American Chemical Society
Volume 41, Number 13
J une 18, 1998
Communications to the Editor
It has been recognized that the Trp8-Lys9 residue is
Sp ir o[1H-in d en e-1,4′-p ip er id in e]
Der iva tives As P oten t a n d Selective
Non -P ep tid e Hu m a n Som a tosta tin
Recep tor Su btyp e 2 (sst2) Agon ists
essential for bioactivity (GH release inhibition) based
on the studies of SRIF analogues.10 Smaller and highly
potent peptidyl SRIF analogues have been synthesized
and studied, and proved to be efficacious in GH release
inhibition in man. The best known of these is the cyclic
peptide, octreotide (H-D-Phe-<Cys-Phe-D-Trp-Lys-Thr-
Cys>Thr-ol, 1a , SMS 201-955, Sandostatin),11 which is
clinically used for the treatment of certain endocrine
tumors and acromegaly. A different class of cyclic
peptide, seglitide (c(N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe),
1b, MK-678),12 was also studied with insulin for the
treatment of insulin-dependent diabetes but not devel-
oped. Both peptides have poor oral bioavailability and
are administered by subcutaneous or intravenous injec-
tion. These agonists were discovered through the rat
pituitary GH release inhibition assay, which is now
known to be mediated through sst2. To extend their
usefulness, nonpeptide, orally active compounds that are
sst2 selective would be highly desirable. To date, only
two types of nonpeptide SRIF agonists have been
reported, one was based on carbohydrate cores as a
peptidomimetic scaffold,13 the other on a benzodiazepine
core.14,15 Both designs used templates to direct the Trp
and Lys side chains in approximate â-turn conforma-
tions to mimic their orientations in the cyclic peptides.
Although conceptually important, the agonists gener-
ated from these approaches were quite weak (IC50s in
the micromolar range in SRIF binding). In this paper
we describe our studies resulting in the development
of a class of small molecules (MW <550) that exhibited
potent SRIF agonist activities. Among them, a bis-
(aminomethyl)cyclohexane analogue, 2(R)-[(spiro[1H-
indene-1,4′-piperidin]-1′-ylcarbonyl)amino]-N-[3(S)-ami-
n om et h yl-1(R)-cycloh exylm et h yl]-3-(1H -in dol-3-
yl)propanamide (L-054,264, 2a), exhibited low nanomolar
binding to human sst2, functional potency, and high
selectivity (>1000-fold) toward the other receptor sub-
types. To the best of our knowledge, this is the first
potent and selective small molecule sst2 agonist re-
ported.
Lihu Yang,* Liangqin Guo, Alexander Pasternak,
Ralph Mosley, Susan Rohrer,† Elizabeth Birzin,†
Forrest Foor,† Kang Cheng,† J ames Schaeffer,† and
Arthur A. Patchett
Departments of Medicinal Chemistry and Biochemistry &
Physiology, Merck Research Laboratories,
Rahway, New J ersey 07065
Received March 30, 1998
Somatostatin (somatotropin release-inhibiting factor,
SRIF) is a widely distributed peptide occurring in two
biologically active forms, a tetradecapeptide SRIF14 (1)
1
and a 28-residue peptide SRIF28
.
SRIF has multiple
functions which includes modulating the secretion of
growth hormone (GH), insulin, glucagon, pancreatic
enzymes, and gastric acid, in addition to having potent
antiproliferative activity.2 In the central nervous sys-
tem, SRIF is a neurotransmitter, it increases cognitive
function, and it modulates locomotor activity.3 Because
of these properties, SRIF agonists may be used for the
treatment of acromegaly, diabetes, cancer, rheumatoid
arthritis, and Alzheimer’s disease among others. On the
other hand, the broad biological activities of SRIF
present the potential for side effects. Recently, five
SRIF receptor subtypes (sst1-5),4 all belonging to the
G-protein-linked receptor family, have been cloned and
characterized.5 Studies utilizing subtype selective SRIF
analogues6 in both in vivo and in vitro experiments
suggest that sst2 mediates the inhibition of growth
hormone release from pituitary somatotrophes, glucagon
release from the pancreas, and gastrin and acid secre-
tion,7 whereas sst5 selective analogues are believed to
inhibit insulin release.8 These results have especially
suggested the usefulness of sst2 selective analogues in
the treatment of acromegaly, retinopathy, and diabetes.9
† Department of Biochemistry & Physiology.
S0022-2623(98)00194-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/03/1998

2176 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
Communications to the Editor
Sch em e 1. General Synthesis of SRIF Agonist 2
Directed screening was used to discover the original
lead from which the compounds of this report were
developed. Extensive studies with the cyclic peptides
have established that the key pharmacophoric elements
of SRIF include Phe7-Trp8-Lys9 and Phe11.16 The atomic
coordinates of the side chain orientations in L-363,377
(c(Pro-Tyr-D-Trp-Lys-Thr-Phe), 1c), which is a cyclic
peptide related to 1b, provided the initial probe for
similarity searches. The peptide backbone of cyclic
SRIF analogues serves as a scaffold for the side chains
and is not itself important for activity.17 Three-
dimensional similarity searches18 of the Merck flexi-
base19 afforded 2b, which was found to be a modest
inhibitor of SRIF binding to murine sst2 (Ki ) 200 nM)
and human sst2 (Ki ) 615 nM). Details of the searching
strategy will be reported elsewhere.
a
Reagents: (a) Boc2O/DCM/MeOH; (b) DSC/DCM-THF, DIEA,
and then spiro(1H-indene-1,4′-piperidine) and DIEA; (c) LiOH/
THF/MeOH/H2O; (d) EDC-HOBT/DCM; (e) HCl/EtOAc.
Sch em e 2. Synthesis of 4aa
Ch em istr y. The synthetic methods used in the
preparation of these SRIF agonists are shown in Scheme
1. The mono-Boc-protected diamine 4 can be readily
prepared by reacting di-tert-butyl dicarbonate (Boc2O)
and the diamine 3 in methanol. Treatment of D-
tryptophan methyl ester with 1 equiv of N,N′-disuccin-
imidyl carbonate (DSC) and diisopropylethylamine
(DIEA) in THF and dichloromethane (DCM) followed
by addition of spiro(indene-1,4′-piperidine)20 and DIEA
gave the desired urea 5. Saponification of the ester
afforded the carboxylic acid 6. Coupling of the acid 6
with mono-Boc-protected diamines 4 using 1-hydroxy-
benzotriazole (HOBT) and 1-(3-(dimethylamino)propyl)-
3-ethylcarbodiimide hydrochloride (EDC) in DCM yielded
the Boc-protected precursor 7, which, upon acidic depro-
tection, afforded compound 2. Careful separation of the
reaction intermediate 7, where the diamine is 1,3-bis-
(aminomethyl)cyclohexane, by collecting the fast-moving
fractions gave a single compound 2a after Boc depro-
tection.
[3(R)-Aminomethyl-1(S)-cyclohexylmethyl]carbamic
acid tert-butyl ester (4a ) was prepared according to
Scheme 2. 3-Cyanobenzoic acid (8) was reduced to
3-(aminomethyl)benzoic acid followed by Boc protection
and hydrogenation of the benzene ring to give the
predominantly (>10:1) cis-N-Boc-3-(aminomethyl)cyclo-
hexanecarboxylic acid 9. Chiral resolution with (S)-R-
methylbenzylamine in ethyl acetate gave the optically
pure 1R,3S compound 10. Its stereochemistry was
established through an X-ray crystal structure of the
chiral salt 10.21 The carboxylic acid was converted to
amine 4a through a four-step procedure involving
borane reduction, mesylation, azide displacement, and
a
Reagents: (f) Raney Ni/NH3-EtOH, 1000 psi; Boc2O, NaOH/
H2O-dioxane; (g) 50 psi, H2, PtO2, AcOH; (h) (S)-R-methylbenzy-
lamine, EtOAc, 60-25 °C; (i) BH3/THF; (j) MsCl, DMAP, DIEA,
DCM; (k) NaN3, DMF-H2O, 60 °C; (l) H2, 10% Pd/C, EtOH.
palladium-catalyzed hydrogenation. Incorporation of
amine 4a resulted in 2a establishing its absolute
stereochemistry.
Resu lts a n d Discu ssion . The compounds reported
here were first tested for their specific binding to the
five cloned human somatostatin receptors (sst1-5), ex-
pressed in CHO-K1 cell lines. Binding affinities for
sst1-4 were determined by the displacement of [125I]-
Tyr11-SRIF14 from the corresponding receptors. Simi-
larly, radiolabeled SRIF28 was used for sst5 assay since
it has a significantly greater affinity at this receptor
subtype (8-fold).5e The functional activity was then
investigated, first by the inhibition of forskolin-stimu-
lated cAMP accumulation in stably transfected mouse
L20ZH cells22 and then by measuring the compound’s
ability to inhibit the release of growth hormone in vitro
(rat pituitary cell assay). The latter assay is especially
relevant to the sst2 receptor, and it had been used for
the earlier SRIF analogue work before the receptors
were cloned.12 Without exception, all of the compounds
studied in this paper were full agonists (Table 1).There
is a marked preference for the D stereochemistry in the
current compounds since the D-Trp analogue (2c) is
almost 30-fold more potent than the L isomer 2d ; such
a difference was not observed with cyclic peptide SRIF

Communications to the Editor
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13 2177
Ta ble 1. sst2 Receptor Binding Affinities of 2
Ta ble 2. Binding Affinities (Ki, nM) of SRIF Agonists at sst1-5
Receptors
entry
Trp
-NHXNH2
sst2 Ki (nM)a
1c
2b
2c
2d
2e
2f
2g
2h
2i
0.5
615
300
10000
815
46
225
53
1108
D/L
D
L
D
D
D
D
D
-NH(CH2)4NH2
-NH(CH2)4NH2
-NH(CH2)4NH2
-NH(CH2)3NH2
-NH(CH2)5NH2
-NH(CH2)6NH2
-Lys-NH2
The unconstrained amino side chain of these com-
pounds is an obvious site for improvement,24 and
fortunately, such studies proved to be very fruitful. We
sought to decrease flexibility by introducing ring restric-
tions and thereby identify an optimal binding conforma-
tion. Extensive studies with different cyclohexane and
piperidine-based diamines confirmed that a five-carbon
spacer between the two nitrogens is essential for high
potency.25 Also important was the observation that the
side chain preferred a fully extended form. Among
them, commercially available 1,3-bis(aminomethyl)-
cyclohexane showed the best potency enhancement. The
diamine is a mixture of cis and trans isomers with cis
being predominant. As can be seen from Table 2, the
isomeric mixture 2j is 10-fold more potent than the
linear compound 2f. More importantly, compound 2j
exhibited higher receptor selectivity toward sst2 than
2f. The single isomer 2a showed marked improvement
in sst2 affinity (Ki ) 1.6 nM) and selectivity (>1000-
fold) in the receptor binding assay over the other four
receptors. Compound 2a showed full agonism on sst2
in the inhibition of forskolin-stimulated accumulation
of cAMP with an IC50 of 2 nM, and it also inhibited the
release of growth hormone in the rat pituitary assay
with IC50 of 6 nM. The other isomer 2k was less active
than 2a . A comparison of our small molecule SRIF
agonists with SRIF14 is shown in Table 2.
-Lys-OH
a
All compounds were examined over a range of concentrations
from 0.01 nM to 10000 nM using full log dilutions and seven-point
titrations to determine IC50 values. All experiments were per-
formed in triplicate with replicate values varying less than 10%.
Compounds were tested in at least two completely separate
experiments. Reported values represent averages from pooled
experiments.
and its agonists. In fact, (D-Trp8)-SRIF14 has the same
potency as its native form, and the peptidyl SRIF
analogues were mostly indiscriminate toward the ster-
eochemistry of Trp8.23 Shortening the amino side chain
of 2c caused a 2-3-fold drop in affinity (2e), while
extending it by one carbon resulted in more than 6-fold
increase in binding (2f). Further lengthening the chain
caused a drop in affinity (2g). The optimal linear amine
is 1,5-diaminopentane with five carbons separating the
amino groups, as is the case for lysine, suggesting
correspondence at the D-Trp8 and Lys9 residues of the
cyclic SRIF agonists. It is worth noting that the
incorporation of a lysine amide (2h ) did not further
improve potency and incorporation of a lysine free acid
(2i) renders the compound quite inactive. Possibly
intramolecular hydrogen bonding causes the lysine
amide to have a slightly different conformation than the
Trp8-Lys9 amide has when presented in a cyclic peptide.
The excellent potency and selectivity for sst2 which
was achieved with 2a may be due to a combination of
F igu r e 1. Stereoview of the superposition of 2a and the cyclic peptide 1c.

2178 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 13
Communications to the Editor
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factors. Potency presumably derives from three critical
binding elements of SRIF, the Trp8-Lys9 mimetic and a
lipophilic spiroindene moiety capable of binding in the
receptor pockets occupied by Phe6 and/or Phe11 of the
native ligand. Improved selectivity as well as potency
arises from the use of a ring to enforce conformational
rigidity onto the amino side chain to reduce the flex-
ibility of the lysine surrogate, making it fit particularly
well to sst2. Our work demonstrates that one can
approach the agonist potency of the large peptide SRIF
with a small molecule like 2a , and improved selectivity
is an added bonus. Figure 1 depicts the superposition
of 2a onto the peptide probe originally used to identify
the lead compound. The conformation of 2a was derived
by energy minimization using MMFF94(s).26 As shown
in the figure, the D-Trp bis(aminomethyl)cyclohexane
amide portion of the 2a maps to the Trp8-Lys9 of 1c.
The spiroindene reaches the space occupied by either
the Tyr7 or the Pro. These modeling studies provided
us a convenient way to visualize and compare our small
molecule ligands with the cyclic peptides in three
dimensions and generated stimulating ideas in the
design of even more potent and selective compounds.
In summary, 2a is a structurally novel, potent and
selective sst2 receptor agonist. The further exploitation
of the SAR and optimization of this class of SRIF
agonists will be reported subsequently.
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