Design, synthesis, and binding affinities of pyrrolinone-based somatostatin mimetics

Article abstract of DOI:10.1021/ol0476974

(Chemical Equation Presented) Tetrapyrrolinone somatostatin (SRIF) mimetics (cf. 1), based on a heterochiral (D,L-mixed) pyrrolinone scaffold, were designed, synthesized, and evaluated for biological activity. The iterative synthetic sequence, incorporati

Full text of DOI:10.1021/ol0476974

ORGANIC
LETTERS
2005
Vol. 7, No. 3
399-402
Design, Synthesis, and Binding
Affinities of Pyrrolinone-Based
Somatostatin Mimetics
Amos B. Smith, III,*,† Adam K. Charnley,† Eugen F. Mesaros,† Osamu Kikuchi,†
Wenyong Wang,† Andrew Benowitz,† Chi-Lien Chu,‡ Jin-Jye Feng,‡
Kuo-Hsin Chen,‡ Atsui Lin,‡ Fong-Chi Cheng,‡ Laurie Taylor,*,§ and
Ralph Hirschmann*,†
Department of Chemistry, UniVersity of PennsylVania,
Philadelphia, PennsylVania 19104, MDS Pharma SerVices-Taiwan Ltd.,
Pharmacology Laboratories, 158 Li-Teh Road, Peitou, Taipei, Taiwan 112, and
MDS Pharma SerVices, 22011 30th DriVe SE, Bothel, Washington 98021
smithab@sas.upenn.edu
Received November 9, 2004
ABSTRACT
Tetrapyrrolinone somatostatin (SRIF) mimetics (cf. 1), based on a heterochiral (D,L-mixed) pyrrolinone scaffold, were designed, synthesized,
and evaluated for biological activity. The iterative synthetic sequence, incorporating the requisite functionalized coded and noncoded amino
acid side chains, comprised a longest linear synthetic sequence of 23 steps. Binding affinities at two somatostatin receptor subtypes (hsst
4 and 5) reveal micromolar activity, demonstrating that the
of peptide -turns.
D,L-mixed pyrrolinone scaffold can be employed to generate functional mimetics
â
The design of novel â-turn mimetics holds great promise as
a tactic for drug discovery to overcome the pharmacokinetic
problems commonly associated with peptides. Research from
this laboratory has established the 3,5-linked (nitrogen
displaced) homochiral pyrrolinone scaffold (Figure 1) as a
competent â-strand/â-sheet peptidomimetic both in solution
and in the solid state.1 The biological relevance of the
pyrrolinone â-strand was subsequently demonstrated by the
design and synthesis of potent pyrrolinone-based inhibitors
of proteolytic enzymes, including HIV-1 protease,2 renin,3
and matrix metalloproteases.4 In addition, in collaboration
with Olson,5 we devised a high affinity peptide-pyrrolinone
hybrid ligand for the class II major histocompatability
† University of Pennsylvania.
‡ MDS Pharma Services-Taiwan Ltd.
§ MDS Pharma Services.
(1) (a) Smith, A. B., III; Keenan, T. P.; Holcomb, R. C.; Sprengeler, P.
A.; Guzman, M. C.; Wood, J. L.; Carroll, P. J.; Hirschmann, R. J. Am.
Chem. Soc. 1992, 114, 10672-10674. (b) Smith, A. B., III; Guzman, M.
C.; Sprengeler, P. A.; Keenan, T. P.; Holcomb, R. C.; Wood, J. L.; Carroll,
P. J.; Hirschmann, R. J. Am. Chem. Soc. 1994, 116, 9947-9962.
Figure 1. Backbone stereogenicity of a homochiral (DDD) and
heterochiral (LDL) polypyrrolinone chain.
10.1021/ol0476974 CCC: $30.25
© 2005 American Chemical Society
Published on Web 01/08/2005

complex (MHC) protein HLA-DR1, which Wiley and co-
workers6 subsequently demonstrated, via X-ray analysis, to
bind with remarkable similarity to the native peptide. More
recently, we disclosed that heterochiral (D,L-mixed)7 tetra-
pyrrolinones adopt â-turn-like conformations in solution.8
This result suggests that the pyrrolinone scaffold is capable
of mimicking both the â-strand and â-turn conformations
of peptides simply by modification of the backbone stereo-
genicity.
A stringent test for a peptidomimetic would be to devise
an active ligand for a biologically important receptor. It is,
however, important to recognize that observation of affinity
with a receptor known to recognize a particular conformation
(i.e., â-turn) does not necessarily establish the active
conformation but, when taken together with physical data,
provides circumstantial evidence about the bioactive con-
formation of the ligand. With this caveat in mind, we chose
to test the biological relevance of the pyrrolinone â-turn via
the design and synthesis of pyrrolinone-based somatostatin
(SRIF-14) mimetics, both because of our long standing
interest in nonpeptide somatostatin mimetics9 and because
the â-turn of SRIF-14 has been shown to be necessary and
sufficient for both receptor binding and signal transduction.10
Somatostatin (Somatotropin Release Inhibiting Factor,
SRIF-14) is an endogenous, cyclic tetradecapeptide hormone
with numerous biological activities, including the regulation
of both endocrine secretion (i.e., growth hormone, insulin,
glucagon, and secretin) and exocrine secretion (i.e., gastric
acid).11 In addition, SRIF acts both as a neurotransmitter in
cell signaling and as an inhibitor of cell proliferation. To
date five human somatostatin receptor subtypes (hsst 1-5)
belonging to the G-protein coupled receptor (GPCR) family
have been identified.12 The short biological half-life (<3
min)13 of SRIF-14 has led to intense efforts to design stable,
orally bioavailable peptide and nonpeptide mimetics of
somatostatin.14 The former, but not the latter, has been
achieved. Numerous studies, principally by the Merck
group,10 revealed that the biologically active pharmacophore
of SRIF comprises a type I â-turn projecting the essential
Phe7, Trp8, and Lys9 side chains with appropriate trajectories
to interact with the receptor. Peptidal peptidomimetics were
subsequently pioneered by Spatola.15 Research initiated in
1987 at the University of Pennsylvania has gone a long way
to put designed nonpeptidal peptidomimetics of neuropeptide
hormones solidly on the map.9 From the beginning, we
appreciated that there is an important difference in the
molecular nature of the interaction of peptides with enzymes
versus those with receptors such as the G-protein coupled
receptors (GPCRs). The former involve the interaction of
the side chains and the peptide backbone of both the enzymes
and their peptidal substrates. For binding and signal trans-
duction of G-protein coupled receptors with their peptidal
ligands, only side-chain interactions are thought to be
required. Having initially envisioned the polypyrrolinones
for use as protease inhibitors, the scaffold was designed to
interact with the backbone of the proteins, in addition to
providing side-chain interactions.
Nonpeptide peptidomimetics incorporating tryptophan and
lysine mimicking side chains attached to a variety of turn
scaffolds have produced a wide spectrum of biologically
active SRIF mimetics (including scaffolds based on â-D-
glucose,9 benzodiazepines,16 spirolactams,17 tripeptide het-
erocycles,18 and â-peptides19).
We envisioned that a D,L-mixed tetrapyrrolinone would
provide the requisite turn scaffold8 upon which to design
SRIF mimetics as ligands for their G-protein coupled
receptors. Incorporation of side chains onto the â-turn mimic
defined by the Phe7, Trp8, Lys9, Thr10 sequence suggested
a pool of potential SRIF mimetics (Figure 2). Monte Carlo
conformational searches20 predicted that the desired turn
structure would be the low energy conformation of hetero-
chiral oligopyrrolinones. For example, Figure 3 displays a
(2) (a) Smith, A. B., III; Hirschmann, R.; Pasternak, A.; Akaishi, R.;
Guzman, M. C.; Jones, D. R.; Keenan, T. P.; Sprengeler, P. A.; Darke, P.
L.; Emini, E. A.; Holloway, M. K.; Schleif, W. A. J. Med. Chem. 1994,
37, 215-218. (b) Smith, A. B., III; Hirschmann, R.; Pasternak, A.; Yao,
W.; Sprengeler, P. A.; Holloway, M. K.; Kuo, L. C.; Chen, Z.; Darke, P.
L.; Schleif, W. A. J. Med. Chem. 1997, 40, 2440-2444. (c) Smith, A. B.,
III; Hirschmann, R.; Pasternak, A.; Guzman, M. C.; Yokoyama, A.;
Sprengeler, P. A.; Darke, P. L.; Emini, E. A.; Schleif, W. A. J. Am. Chem.
Soc. 1995, 117, 11113-11123.
(3) Smith, A. B., III; Akaishi, R.; Jones, D. R.; Keenan, T. P.; Guzman,
M. C.; Holcomb, R. C.; Sprengeler, P. A.; Wood, J. L.; Hirschmann, R.;
Holloway, M. K. Biopolymers 1995, 37, 29-53.
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R.-Q.; Hirschmann, R. F. Org. Lett. 2000, 2, 3809-3812.
(12) (a) Yamada, Y.; Post, S.; Wang, K.; Tager, H.; Bell, G.; Seino, S.
Proc. Natl. Acad. Sci. U.S.A.. 1992, 89, 251-255. (b) Yasuda, K.; Rens-
Domiano, S.; Breder, C.; Law, S.; Saper, C.; Reisine, T.; Bell, G. J. Biol.
Chem. 1992, 267, 20422-20428. (c) O’Carroll, A.-M.; Lolait, S.; Konig,
M.; Mahan, L. Mol. Pharmacol. 1992, 42, 939-946. (d) Bruno, J.; Xu, Y.;
Song, J.; Berelowitz, M. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 11151-
11155. (e) Rohrer, L.; Faulf, F.; Bruns, C.; Buttner, R.; Schule, R. Proc.
Natl. Acad. Sci. U.S.A. 1993, 90, 4196-4200.
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Guzman, M. C.; Hirschmann, R.; Schweiger, E. J.; Bolin, D. R.; Nagy, Z.;
Campbell, R. M.; Cox, D. C.; Olson, G. L. J. Am. Chem. Soc. 1999, 121,
9286-9298.
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P. A.; Smith, A. B., III; Hirschmann, R. F.; Wiley: D. C. J. Am. Chem.
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(7) While D,L descriptors are not directly applicable to the pyrrolinone
units, their use simplifies comparison with peptidal structures.
(8) Smith, A. B., III; Wang, W.; Sprengeler, P. A.; Hirschmann, R. J.
Am. Chem. Soc. 2000, 122, 11037-11038.
(9) (a) Hirschmann, R.; Nicolaou, K. C.; Pietranico, S.; Leahy, E. M.;
Salvino, J.; Arison, B.; Cichy, M. A.; Spoors, G. P.; Shakespeare, W. C.;
Sprengeler, P. A.; Hamley, P.; Smith, A. B., III; Reisine, T.; Raynor, K.;
Maechler, L.; Donaldson, C.; Vale, W.; Freidinger, R. M.; Cascieri, M. R.;
Strader, C. D. J. Am. Chem. Soc. 1993, 115, 12550-12568 and references
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Nat. Acad. Sci. U.S.A. 1978, 2636-2640.
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400
Org. Lett., Vol. 7, No. 3, 2005

polypyrrolinone synthesis suggested the use of the dimethyl
acetal based pyrrolinone synthetic protocol, developed in our
laboratory.8 The requisite building blocks are illustrated in
Figure 2. Not unexpectedly, however, and despite our best
efforts, complications with introduction of the indole side
chain led us to focus on several aromatic surrogates for the
indole ring. Naphthyl- and phenyl-based side chains have
been employed successfully as SRIF indole replacements
without significantly decreasing biological activity9,22 and,
as such, held greater promise for ready incorporation into
the polypyrrolinone-based scaffold (vide infra).
Toward this end, tetrapyrrolinones (1-3) were envisioned
to arise from four R,R-disubstituted amino ester precursors
(A-D), exploiting an iterative C to N extension of the
pyrrolinone chain. The requisite building blocks were readily
prepared via the enantioretentive alkylation tactic developed
by Karady23a and Seebach.23b Toward this end, amino esters
derived from phenylalanine and valine were prepared via
procedures developed early in our pyrrolinone synthetic
program;8 the amino esters possessing noncoded amino acid
side chains (R- and â-naphthyl), (-)-CR and (-)-Câ, and
the lysine mimic (-)-B were prepared exploiting our
“universal oxazolidinones synthetic protocol.”24 Experimental
details for construction of A-D are available in the Sup-
porting Information.
Figure 2. Pool of prospective pyrrolinone-based SRIF mimetics
and the requisite building blocks.
With the necessary amino esters in hand, we began
iterative construction of the tetrapyrrolinones (Scheme 1).
Condensation of (+)-A with hydrocinnamaldehyde employ-
ing azeotropic removal of water afforded the corresponding
imine, which upon treatment with KHMDS undergoes
metallo-enamine promoted cyclization to generate mono-
pyrrolinone (-)-5, effectively capping the C-terminal pyr-
rolinone with a benzyl group. Hydrolysis of the dimethyl
acetal then affords the corresponding monopyrrolinone
aldehyde, which is condensed with amino ester (-)-B and
in turn subjected to the same KHMDS protocol to provide
bispyrrolinone (+)-6, a common intermediate for prospective
â-turn mimetics 1-3.
stereoview of the low energy conformation of prospective
pyrrolinone SRIF mimetic 2, overlayed with the solution
structure of cyclic hexapeptide L-363,301, a potent SRIF
mimetic.21 Particularly pleasing is the excellent overlap of
the tryptophan and lysine mimicking side chains that occupy
the critical i + 1 and i + 2 positions of SRIF-14.
The third iterative pyrrolinone ring construction, the point
of synthetic diversion, permits incorporation of three aromatic
tryptophan-mimicking side chains into the growing poly-
pyrrolinone rings. Yields here were modest. Not withstanding
the modest yields, the fourth pyrrolinone synthetic iteration
introduced a benzyl side chain in each case, employing amino
ester (-)-D for trispyrrolinones (-)-7 and (-)-8 and the
reduced congener (+)-10 for trispyrrolinone (-)-9. Staudinger
reduction or hydrogenation of the azide to generate the lysine
side chain mimic provided tetrapyrrolinone SRIF mimetics
1-3. The syntheses of 1-3 were achieved via a longest
(22) (a) Roher, S. P.; Birzin, E. T.; Mosley, R. T.; Berk, S. C.; Hutchins,
S. M.; Shen, D.-M.; Xiong, Y.; Hayes, E. C.; 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.; Pasternak, A.; Yang, L.; Patchett, A. A.; Smith, R. G.;
Chapman, K. T.; Schaeffer, J. M. Science 1998, 282, 737-740. (b) Reubi,
J. C.; Shaer, J.-C.; Wenger, S.; Hoeger, C.; Erchegyi, J.; Waser, B.; Rivier,
J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13973-13978. (c) Michel, A.;
Scarso, A.; Loffet, A. Peptides 1984: Proceedings of the 18th European
Peptide Symposium; Ragnarsson, U., Ed.; Almquvist and Wiksell Intl.:
Stockholm, Sweden, 1984; pp 585-589.
Figure 3. Stereoview: overlay of L-363,301 (black) with 2 (gray).
The initial target selected for synthesis was tetrapyrroli-
none 4, incorporating an indole side chain. Experience with
(21) (a) Nutt, R. F.; Colton, C. D.; Saperstein, R.; Veber, D. F. In
Somatostatin; Reichlin, S., Ed.; Plenum: New York, 1987; p 83. (b) Veber,
D. F. In Peptides-Chemistry and Biology: Proceedings of the 12th American
Peptide Symposium; Smith, J. A., Rivier, J. E., Eds.; ESCOM: Leiden,
1992; p 3.
(23) (a) Karady, S.; Amato, J. S.; Weinstock, L. M. Tetrahedron Lett.
1984, 25, 4337-4340. (b) Seebach, D.; Fadel, A. HelV. Chim. Acta 1985,
68, 1243-1250.
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Hirschmann, R. Tetrahedron Lett. 1997, 38, 3809-3812.
Org. Lett., Vol. 7, No. 3, 2005
401

Scheme 1. Preparation of SRIF Mimetics 1-3
linear sequence of 23 steps [12 steps from (+)-A] in an
based SRIF mimetics suggests that micromolar activity
provides sufficient and relevant validation for the potential
of nonpeptidal scaffolds. In addition, as observed with our
â-D-glucose turn mimics, optimization of mimetics with
micromolar activity can lead to nanomolar ligands.9 There-
fore, the observation that SRIF mimetics 1-3 displace [125I]-
SRIF14 binding to somatostatin receptors in vitro at low
micromolar concentrations supports the potential utility of
the pyrrolinone scaffold as a â-turn peptidomimetic. Equally
important, these results establish polypyrrolinones as a
privileged scaffold capable of generating functional mimetics
of multiple peptidal secondary structures. Efforts to develop
more potent â-turn peptidomimetics based on the 3,5-linked
pyrrolinone scaffold continue in our laboratory.
overall yield of ca. 1.0%.
Binding affinities of the three tetrapyrrolinone SRIF
mimetics were determined via a radioligand binding assay
with the [125I]-SRIF14 ligand at two somatostatin receptors25
(hsst 426 and 527) (Table 1). All three designed ligands
Table 1. Binding Affinities of Pyrrolinone-Based SRIF
Mimetics
ligand
IC50 hsst 4
IC50 hsst 5
(-)-1
(+)-2
(+)-3
SRIF-14
2.14 µM
4.04 µM
2.05 µM
0.111 nM
2.44 µM
1.27 µM
38% at 10 µM
0.362 nM
Acknowledgment. Financial support was provided by the
NIH (National Institute of Allergy and Infectious Diseases)
through grant no. AI-42010. Additional funding was provided
by Merck Research Laboratories and the University of
Pennsylvania.
possessed low micromolar affinity for hsst 4, with (-)-1 and
(+)-3 exhibiting an IC50 of approximately 2 µM and (+)-2
possessing an IC50 of 4 µM. At the hsst 5 receptor, only the
naphthyl-containing compounds displaced binding of [125I]-
SRIF14. Despite the modest binding affinities of these
compounds relative to SRIF, our experience with the glucose-
Supporting Information Available: Schemes for the
preparation of the amino ester building blocks and experi-
mental details for all synthetic transformations. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL0476974
(25) Methods employed in this study have been adapted from the
scientific literature to maximize reliability and reproducibility. Reference
standards were run as an integral part of each assay to ensure the validity
of the results obtained. IC50 values were determined by a nonlinear, least
squares regression analysis using Data Analysis Toolbox (MDL Information
Systems, San Leandro, CA).
(26) (a) Liapakis, G.; Fitzpatrick, D.; Hoeger, C.; Rivier, J.; Vandlen,
R.; Reisine, T. J. Biol. Chem. 1996, 271, 20331-20339. (b) Patel, Y. C.;
Srikant, C. B. Endocrinology 1994, 135, 2814-2817.
(27) (a) Greenwood, M. T.; Hukovic, N.; Kumar, U.; Panetta, R.; Hjorth,
S. A.; Srikant, C. B.; Patel, Y. C. J. Pharmacol. Exp. Ther. 1997, 52, 807-
814. (b) Patel, Y. C.; Srikant, C. B. Endocrinology 1994, 135, 2814-2817.
402
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