Use of 1,2,3-trisubstituted cyclopropanes as conformationally constrained peptide mimics in SH2 antagonists

Article abstract of DOI:10.1016/S0040-4039(00)01611-7

Novel conformationally constrained phosphotyrosine pseudopeptide derivatives of the tetrapeptide pY-E-E-I were prepared and evaluated as SH2 binding antagonists. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01611-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 9459–9464
Use of 1,2,3-trisubstituted cyclopropanes as
conformationally constrained peptide mimics in SH2
antagonists†
James P. Davidson and Stephen F. Martin*
Department of Chemistry and Biochemistry and Institute for Cellular and Molecular Biology,
The University of Texas, Austin, TX 78712, USA
Received 25 August 2000; accepted 18 September 2000
Abstract
Novel conformationally constrained phosphotyrosine pseudopeptide derivatives of the tetrapeptide
pY-E-E-I were prepared and evaluated as SH2 binding antagonists. © 2000 Elsevier Science Ltd. All
rights reserved.
Keywords: peptide mimics; cyclopropane; asymmetric synthesis.
Development of rigid replacements of peptide secondary structural elements is a common
strategy for designing non-peptidic, small molecules that will bind to enzyme active sites and
various biological receptors.1 Because such replacements may also be exploited to gain insights
regarding the biologically active conformation of flexible peptides, we invented the cyclo-
propane-derived isosteres II as a new class of mimics of the dipeptide array I. These novel
replacements, which may be abbreviated as –XaaC[COcpCO]Yaa–, were uniquely designed to
orient both the peptide backbone and the amino acid side chain by varying the stereochemistry
on the cyclopropane ring.2
* Corresponding author. E-mail: sfmartin@mail.utexas.edu
†
This article is dedicated to my friend Professor Harry H. Wasserman on the occasion of his 80th birthday and for
his many important contributions to organic chemistry and to the Tetrahedron journals.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01611-7

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To evaluate their efficacy in biological systems, we introduced peptide replacements related to
II into novel inhibitors of renin, HIV-1 protease, matrix metalloproteinases, Ras farnesyl-trans-
ferase as well as enkephalin analogues,3 and others have used such cyclopropane-derived mimics
in non-peptide fibrinogen receptor antagonists.4 These studies generally established the viability
of introducing trisubstituted cyclopropanes into biologically active analogues of peptides, but in
contrast to expectations, the potencies of the conformationally constrained ligands were
typically equal to, not superior to their flexible counterparts. Hence, the goal of preparing
tighter binding pseudopeptides through conformational constraint was not achieved, and the
fundamental question that arose at this juncture was: Why? Did the introduction of the
cyclopropane ring into the inhibitors not result in the expected entropic advantage, or did
unfavorable enthalpic factors override the entropic gains, thereby resulting in similar DGs of
binding? To address these issues, we decided to examine the binding affinities of 1–4, which are
conformationally constrained and flexible analogues of the phosphotyrosine-containing tetra-
peptide Ac-pTyr-Glu-Glu-Ile-OH (pY-E-E-I) (5), a well-known antagonist of SH2 domains.5
There had been numerous structural and thermodynamic studies of SH2 domains complexed
with various phosphotyrosine peptides,6 so it would be possible to correlate ligand structure with
the energetics of binding for this series of ligands. Using the available structural information,
preliminary modeling suggested that both 1 and 2 nicely mimicked all of the critical features of
a phosphotyrosine peptide ligand bound to the Src SH2 domain. We now report the details of
some of these studies.
The synthesis of the cyclopropane containing pseudopeptides 1 and 2 commenced with
conversion of 4-tert-butoxybromobenzene7 (6) into the corresponding iodide, which underwent
a palladium-catalyzed coupling with 2-propyne-1-ol to give an alkyne that was reduced to the
Z-alkene 7 in 81% overall yield (Scheme 1). The palladium-catalyzed coupling of the aryl
bromide 6 with 2-propyne-1-ol in the presence of (Ph3P)4Pd was very slow and proceeded in only
11% yield.8 The alcohol 7 was converted into the allylic diazoacetate 8 using a modified
Corey–Myers protocol in which acetonitrile was employed as the solvent.9 When 8 was heated
in the presence of Rh2[(5S)-MEPY]4,10 the cyclopropyl lactone 9 was obtained in 75% yield and
90% ee.
Ring opening of the lactone moiety of 9 via an aluminum mediated amidation with either
2,4-dimethoxybenzylmethylamine11 or dimethylamine according to the Weinreb procedure,12
followed by oxidation of the resulting amide alcohol with TPAP gave the all cis cyclopropyl
aldehydes 10 and 11 in 56 and 81% overall yields, respectively. Sequential epimerization alpha
to the aldehyde followed by Jones oxidation and global deprotection furnished the conforma-

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Scheme 1. (a) Mg, THF, D; then I2, 0°C. (b) Pd(PPh3)4, CuI, pyrrolidine, 2-propyne-1-ol, 0°C“rt. (c) H2, Lindlar
catalyst, quinoline. (d) TsNNHCOCl, DMA, CH3CN, −20°C; then Et3N. (e) Rh2[(S)-MEPY]4, CH2Cl2, D. (f) AlMe3,
2,4-(MeO)2C6H3CH2NH2MeCl. (g) AlMe3, Me2NH2Cl. (h) TPAP, NMO. (i) Et3N, MeOH. (j) Jones oxidation. (k)
CF3CO2H. (l) TBS–Cl, NMM; 1H-tetrazole, i-Pr2NP(OBn)2 then t-BuOOH. (m) DCC, HOBT, CH2Cl2,
HꢀE(OBn)ꢀE(OBn)ꢀI(OBn). (n) H2, Pd
tionally constrained tyrosine acids 12 (65% yield) and 13 (73% yield). The phenolic groups of 12
and 13 were phosphorylated,13 and subsequent coupling of the cyclopropanecarboxylic acids
thus obtained with the benzyl protected tripeptide H2N-Glu(OBn)-Glu(OBn)-Ile-CO2Bn in the
presence of DCC and HOBT followed by exhaustive debenzylation by hydrogenolysis furnished
the targeted pseudopeptides 1 and 2 in 47 and 58% overall yields.
The syntheses of the flexible analogues of 5 began with the conversion of 6 into 14 via a
one-pot Heck/hydrogenation procedure (Scheme 2). The acid 14 was converted to the chiral
imide 15 in 82% yield following a procedure by Mathre.14 Reaction of the sodium enolate of 15
with either a-bromo-N,N-dimethylacetamide15 or a-bromo-N-(2,4-dimethoxybenzyl)-N-
methylacetamide16 gave the alkylated products with high diastereoselectivity (>95%); removal of
the chiral auxiliary with LiOBn provided 16 and 17 in 63 and 66% overall yields, respectively.
Global deprotection by sequential reaction with CF3CO2H and catalytic hydrogenolysis gave the
flexible tyrosine acid replacements 18 and 19 in nearly quantitative yield (98–99%). Transforma-
tion of 18 and 19 into the flexible pseudopeptides 3 and 4 was achieved by sequential
phosphorylation, peptide coupling and deprotection in 44–53% overall yields according the
procedure developed for the syntheses of 1 and 2.
Once compounds 1–5 were in hand, their binding affinities for the p56lck SH2 domain were
determined in a competitive binding assay using the Pharmacia Biacore instrument according to
a standard experimental protocol.17 The measured affinities are summarized in Table 1.
The conformationally constrained analogues 1 and 2 are both slightly more potent than their
flexible counterparts 3 and 4, and the binding affinities of 1 and the parent tetrapeptide 5 are
comparable. These results suggest that 1–5 bind in similar geometries to the SH2 domain, so it
seems likely that the cyclopropane ring in 1 and 2 correctly mimic the bound orientation of the
phosphotyrosine moiety. The biggest difference in binding potency is a factor of about 2.7

9462
Scheme 2. (a) PdCl2, CH2ꢁCHCO2H, H2O, Na2CO3, D, 24 h; then H2, Pd/C, 8 h. (b) Me3CCOCl, Et3N, 0°C then
(S)-(−)-4-benzyl-2-oxazolidinone, LiCl, rt. (c) NaHMDS, −78°C then a-bromo acetamide. (d) LiOBn, −78°C“rt. (e)
CF3CO2H, rt. (f) H2, Pd/C, EtOH. (g) TBS–Cl, NMM; 1H-tetrazole, i-Pr2NP(OBn)2 then t-BuOOH. (h) DCC,
HOBT, CH2Cl2, HꢀE(OBn)ꢀE(OBn)ꢀI(OBn). (i) H2, Pd
Table 1
Competitive binding assays with p56lck SH2 domain
Entry
Dissociation constant (Kd nM)
Relative error (nM)
1
2
3
4
5
119
255
318
389
119
18
25
30
31
24
favoring the cyclopropane derivative 1 over its flexible counterpart 3. This corresponds to an
approximate energy difference of 0.6 kcal/mol, somewhat less than would be expected purely on
the basis of the number of restricted rotors in 1.18 Owing to the limitations of a competitive
binding assay, it is impossible to determine if any of this energy is a result of an entropic effect
due to the pre-organization induced by the cyclopropane. On the other hand, the technique of
isothermal titration calorimetry provides an efficient means of obtaining the entire thermody-
namic profile of the binding event. However, because there is a significant conformational
change that occurs in the p56lck SH2 domain upon complexation with phosphotyrosine
ligands,16 interpreting the results of any calorimetric studies with this protein would be
problematic. Hence, the thermodynamics associated with the formation of complexes of 1 and
3 with the Src SH2 domain, which does not to undergo any significant conformational changes
upon ligand binding,6b will be pursued instead. The results of these investigations will be
reported in due course.

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Acknowledgements
We thank the National Institutes of Health, The Robert A. Welch Foundation, the Texas
Advanced Research Program, Pfizer, Inc. and Merck Research Laboratories for their generous
support of our research. We are also thankful to Neil Moss, Scott Jakes, and Susan Lucas of
Boehringer Ingelheim Pharmaceuticals, Inc. for performing the bioassays of 1–5.
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