Utilization of a β-aminophosphotyrosyl mimetic in the design and synthesis of macrocyclic Grb2 SH2 domain-binding peptides

Article abstract of DOI:10.1021/jm030049q

Grb2 SH2 domains are protein-docking modules that exert important functions in both normal and pathogenic signal transduction processes. Development of synthetic Grb2 SH2 domain binding ligands is being pursued by several groups as potential new therapies

Full text of DOI:10.1021/jm030049q

J . Med. Chem. 2003, 46, 2621-2630
2621
Utiliza tion of a â-Am in op h osp h otyr osyl Mim etic in th e Design a n d Syn th esis of
Ma cr ocyclic Gr b2 SH2 Dom a in -Bin d in g P ep tid es
Kyeong Lee,† Manchao Zhang,‡ Hongpeng Liu,‡ Dajun Yang,‡ and Terrence R. Burke, J r.*,†
Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute, National Institutes of Health,
NCIsFrederick, P.O. Box B, Building 376 Boyles Street, Frederick, Maryland 21702, and Department of Internal Medicine,
University of Michigan Medical School, Ann Arbor, Michigan 48109
Received J anuary 29, 2003
Grb2 SH2 domains are protein-docking modules that exert important functions in both normal
and pathogenic signal transduction processes. Development of synthetic Grb2 SH2 domain
binding ligands is being pursued by several groups as potential new therapies for a variety of
diseases, including certain cancers. In these efforts, macrocyclization has been successfully
utilized to take advantage of preferential recognition by Grb2 SH2 domains of ligands in â-bend
conformations. Recent examples of this approach include olefin-metathesis-derived macrocycles
that employ ring closure at the â-position of key pTyr-mimicking residues. In the current study,
a novel phosphatase-stable â-amino-pTyr mimetic designated “Pmpâ” was utilized to prepare
variants of previously reported olefin-metathesis-derived macrocycles. An initial set of simplified
cyclic peptides lacking key naphthyl side chain functionality was first synthesized to determine
optimum ring size, with results indicating that a four-unit ring-closing segment was appropriate.
On the basis of these findings, macrolactamization was undertaken with a more highly
functionalized, naphthyl-containing γ-amino acid analogue. The resulting cyclic â-amino peptide
is the first of a new class of pTyr-mimetic-containing ligands that may have utility in the
development of antagonists of both Grb2 SH2 domains and other pTyr-dependent signaling
systems.
The growth factor receptor-bound protein 2 (Grb2) is
a noncatalytic adapter module comprising one SH2
domain and two SH3 domains and that serves functions
in a variety of signal transduction networks, including
the mitogenically important Ras pathway.1 Recent
promising results using kinase inhibitors as anticancer
agents2 highlight the potential therapeutic utility of
down-regulating signaling pathways, which include
Grb2-dependent processes involved with diseases such
as breast and kidney cancers.3 On the basis of the fact
that Grb2 SH2 domains preferentially associate with
peptide and protein ligands bearing “pTyr-Xxx-Asn”
sequences in â-bend conformations,4 an objective in the
development of Grb2-dependent signaling inhibitors has
been the creation of pTyr-mimetic-containing ligands
with preinduced “bend geometries”.5 For such purposes,
a pTyr + 1 R-aminocyclohexanecarboxylic acid (Ac6c)
residue has demonstrated particular utlility.6 One
example of an Ac6c-containing tripeptide mimetic is
shown by compound 1 (Figure 1).7 Recently, olefin-meta-
thesis-induced macrocyclizations have provided ring-
closed analogues such as 2 that contain additional con-
formational constraint.8 These macrocycles can exhibit
enhanced Grb2 SH2 domain binding affinity relative to
open-chain parents exemplified by 1.9 Encouraged by
these results, further work was undertaken to explore
methods of ring closure other than metathesis ap-
proaches.
traditional cyclic peptides10 or macrocycles where ring
closure is achieved through functionality originating
from amino acid R-positions.8 Maintenance of a similar
arrangement without reliance on olefin metathesis
chemistries could theoretically be achieved through
amide-based ring closure involving â-amino-containing
pTyr mimetics. We have recently reported the design
and synthesis of such a requisite â-amino analogue
(Pmpâ)11 as an isomeric variant of phosphonometh-
ylphenylalanine (Pmp),12 which is a widely used phos-
phatase-stable pTyr mimetic that can exhibit good SH2
domain binding affinity.13,14 In the first Grb2 SH2
domain directed application of Pmpâ, tripeptide mimetic
3 was synthesized wherein the aromatic functionality
that would normally occupy the C-terminus15 (refer to
structures 1 and 2) had been translocated to the
â-position of the Pmpâ residue.11 The poor binding
affinity of 3 (∼600 µM)16 indicated that the aryl-
containing â-amido functionality originating from Pmpâ
might be unsuitable in an open-chain configuration.
However, it remained to be seen whether ring closure
to structures such as 4 might result in reinstatement
of Grb2 SH2 domain binding affinity. Of particular note,
this genre of ring-closed compounds would represent
amido variants of metathesis-derived macrocycles dis-
cussed above. Accordingly, reported herein is the design
and synthesis of novel peptidomimetics that explore the
utility of such â-amido ring-closed macrocycles.
Macrocycles of type 2 are characterized by ring closure
at the pTyr mimetic â-position. This is distinct from
Ch em istr y
Prior to the synthesis of peptides of type 4, it was
noted that the geometries of â-amido-containing ring-
closing segments might differ from those of olefin-meta-
thesis-derived macrocycles such as 2. An initial study
* To whom correspondence should be addressed. Phone: (301) 846-
5906. Fax: (301) 846-6033. E-mail: tburke@helix.nih.gov.
† NCIsFrederick.
‡ University of Michigan Medical School.
10.1021/jm030049q CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/22/2003

2622 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Lee et al.
F igu r e 1. Examples of Grb2 SH2 domain binding compounds.
ring-open intermediates 17-20 could be derived from
a common precursor (16, Scheme 3). Synthesis of
17-20 necessitated preparation of Fmoc-protected
Pmpâ residue 13 in order to allow â-amino deprotection/
t
acylation while maintaining C-terminal Bu protection
(Scheme 3). Building block 13 was readily obtained from
common intermediate 11 by acid-catalyzed cleavage of
the N-(tert-butyl)sulfinyl group followed by saponifica-
tion and in situ Fmoc protection (82% yield, Scheme 2).
F igu r e 2. Two possible approaches toward ring closure using
a â-amino-pTyr mimetic.
Prior to preparing intermediates 18-20, a prelimi-
nary ring-closing study was undertaken using tetrapep-
tide 17, which can be obtained using commercially
available N-Fmoc-L-â-phenylalanine (Scheme 3). Tri-
peptide 15 was prepared by coupling H-Ac6c-Asn(OtBu)
(14) with N-Fmoc-L-â-phenylalanine in the presence of
diisopropylcarbodiimide (DIPCDI) and 1-hydroxyben-
zotriazole (HOBt). Fmoc deprotection of 15 followed by
HOBt active ester coupling with N-Boc-â-alanine pro-
vided Boc-protected tetrapeptide. Conversion of 15 to
17 was achieved by concomitant TFA-mediated removal
of N- and C-terminal protecting groups. In situ activa-
tion of 17 using pentafluorophenyl diphenylphosphinate
(FDPP) in the presence of N-methylmorpholine (NMM)23
proceeded smoothly to provide the desired macrocycle
21 in 80% yield. It should be noted that use of O-[7-
azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HATU) with N, N-diisopropyl-
ethylamine (DIPEA)24 gave a mixture of 21 and its
dehydration product.
was therefore undertaken to prepare a series of simpli-
fied cyclic â-amido-containing peptides (5-7) that lacked
the naphthyl functionality. The intent of this explor-
atory work was to determine the optimum ring size prior
to undertaking a synthetically more demanding fully
elaborated naphthyl-containing variant. Preliminary
efforts to effect ring closure were directed at condensa-
tion of the C-terminal carboxy-containing functionality
onto the Pmpâ â-amino group (Figure 2, ring closure
“A”). Illustrated in Scheme 1 is the attempted prepara-
tion of cyclic peptide 6 by this approach. Synthesis of
the required protected tripeptide sequence H-Pmpâ-
(OBn)2-Ac6c-Asn-â-Ala-OH (9) was achieved by Fmoc-
based solid-phase peptide synthesis on Wang resin,17,18
followed by acidic cleavage from the resin. The linear
precursor (9) for this route contained orthogonally
protected Pmpâ (12) that was prepared from methyl
ester 11, as previously described (Scheme 2).11,19 Desired
cyclization of the resulting carboxy-terminal peptide 9
did not occur in the presence of either N-[(dimethyl-
amino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene)-
N-methylmethanaminium hexafluorophosphate N-oxide
(HBTU)20,21 or diphenylphosphorylazide (DPPA)22 under
conditions of high dilution (5 × 10-4 M). Failure of
“route A” raised questions about the suitability of the
â-amino center for ring-closing amide bond formation.
Therefore, an alternative approach was envisioned
whereby acylation of the â-amino group with protected
amino-containing residues would proceed prior to ring
closure (“route B”, Figure 2). An advantage of this
protocol is the convergent manner in which penultimate
Following a similar strategy, phosphonate-protected
linear precursors 18-20 were obtained in a convergent
fashion from tripeptide 16, which was prepared by the
reaction of N-Fmoc-Pmpâ(OBn)2-OH (13) with H-Ac6c-
Asn(OtBu) (14) in the presence of 1-hydroxy-7-azaben-
zotriazole (HOAt) and ethyl(dimethylamino)propylcar-
bodiimide hydrochloride (EDC‚HCl).7,25 Ring closure of
18, 19, and 20 using FDPP under dilute conditions (1
× 10-4 M in DMF at room temperature) provided mac-
rocycles 22, 10, and 23, respectively, in 21-64% yield.
The low macrolactamization of 18 (21%) may reflect
unfavorable ring strain in the resulting product (22).

â-Aminophosphotyrosyl Mimetic
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2623
Sch em e 1a
a
Reagents and conditions: (a) TFA/H2O/Et3SiH (95:2.5:2.5); (b) HBTU, HOBt, PIPEA, DMF (1 day), or DPPA, NMM, DMF (3 days).
Sch em e 2a
a
Reagents and conditions: (a) LiOH, MeOH, H2O; (b) (i) 4 N HCl/dioxane, MeOH, (ii) LiOH, MeOH, H2O, then CO2, Fmoc-OSu, dioxane.
Subsequent hydrogenolytic removal of phosphonate
benzyl protection gave target compounds 5, 6, and 7,
respectively. The linear des-â-amino peptide 27 was
prepared by deprotection of 26, which was obtained by
coupling H-Ac6C-Asn-NH2 24 and 4-phosphonomethyl-
caffeic acid analogue 258 under standard conditions
(Scheme 4).
Resu lts a n d Discu ssion
Although the stereoselective synthesis of â-amino
acids26-29 and their use for induction of turn-geom-
etries30-33 have found increasing importance in a variety
of contexts, the potential utility of these agents in the
development of SH2 domain directed ligands has yet to
be adequately explored. The recent convergence of two
findings (the high Grb2 SH2 domain binding potency
of macrocylic tetrapeptide mimetics of type 2 and the
availability of a new phosphatase-stable â-amino-pTyr
mimetic (Pmpâ, 12)) has allowed the design and syn-
thesis of 36 as the first member of a new class of cyclic
Grb2 SH2 domain binding ligand. Previous studies with
Grb2 SH2 domain-ligand binding ligands had shown
the importance of hydrophobic interactions in a region
proximal to the C-terminal pTyr + 2 Asn residue.34,35
This indicated that the design of â-macrocycles should
include suitably positioned hydrophobic functionality for
interaction with this hydrophobic patch.
In choosing a ring-closing segment that would serve
as an appropriate framework from which to append a
desired hydrophobic group, it was important to preserve
the bend geometry found in the parent “pTyr-Xxx-Asn”
sequence.4 Therefore, an initial set of analogues 5-7
were prepared to investigate effects of ring-closing
length on Grb2 SH2 domain binding potency. Analogue
27, which lacks functionalization at the pTyr mimetic
R-position, was chosen as a control for this study in
order to reduce synthetic complexity. This simplified
For the synthesis of the more fully elaborated mac-
rocycle 36 bearing naphthyl substitution, the novel
amino acid building block 33 was required (Scheme 5).
Allyl-containing propylamino derivative 30 was envi-
sioned as a chiral synthon that could provide 33 follow-
ing oxidation and γ-lactam formation. Amine 30 was
synthesized from 29, which had previously been pre-
pared from acylated Evans’ oxazolidinone 28 by protec-
tion in the presence of Boc anhydride and TEA.8
Oxidative cyclization of 30 to lactam 32 proceeded in a
two-step fashion by initial osmylation/NaIO4 oxidation
to lactol 31, followed by further oxidation using pyri-
dinium chlorochromate (PCC). Hydrolysis of lactam 32
using 1 N LiOH provided the desired Boc-protected
naphthymethyl-γ-amino acid (N-Boc-NM-GABA) 33 in
86% yield. Coupling of the requisite â-amino acid 33
with tripeptide 16 in the presence of HOAt yielded
open-chain 34 (Scheme 6) in 39% yield after treat-
ment with TFA. Macrocyclization of 34 using FDPP
provided benzyl-protected 35, which gave final product
36 in 26% yield from 34 following hydrogenolytic de-
benzylation.

2624 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Lee et al.
Sch em e 3a
a
Reagents and conditions: (a) (i) N-Fmoc-L-â-phe-OH or 13, HOBt, DIPCDI, DMF, (ii) piperidine, DMF; (b) (i) N-Boc-â-Ala-OH for 17
and 19, Boc-Gly-OH for 18, or Boc-4-Abu-OH for 20, HOBt, DIPCDI, DMF, (ii) TFA, CH2Cl2; (c) FDPP, NMM, DMF; (d) 10% Pd/C, H2,
40 psi.
Sch em e 4a
a
Reagents and conditions: (a) (i) 25, HOAt, EDC‚HCl, DMF, (ii) piperidine, DMF; (b) TFA/H2O/Et3Si (95:2.5:2.5).
pTyr mimetic has shown utility in earlier studies that
lead to the development of macrocycle 2.8 Evaluation
of Grb2 SH2 domain binding affinity was performed
using a previously reported enzyme linked immunosor-
bent assay (ELISA) based technique.36 As shown in
Table 1, peptides 5 and 6 containing two- and three-
unit ring-closing segments, respectively, were inactive.
Alternatively, ring closure with a four-unit segment
(peptide 7) provided affinity equivalent to that of the
parent 27. Since the bend-inducing Ac6c residue already
promotes a turn conformation in open-chain 27, it was
uncertain whether macrocyclization would significantly
enhance potency further through bend induction. In-
deed, the intent of this initial study was to ascertain
ring-closing length that would not disrupt proper bend
geometry, and within this context, peptide 7 appeared
to present an acceptable length. Having established a
satisfactory ring-closing length, introduction of naphthyl
functionality was achieved next by synthesis of macro-
cycle 36, which maintains the same ring-closing size as
presented in peptide 7. A resultant approximate 6-fold
enhancement in binding affinity (IC50 ) 7.9 µM for 36
compared to IC50 ) 48 µM for 7) was consistent with
previous studies showing the potential benefits of
hydrophobic interactions in the region occupied by the
naphthyl ring.
It was also apparent by the approximate 2 orders of
magnitude increase in potency in comparing 3 with 36
that appending the naphthyl group onto a ring-closing
segment offered significant binding interactions not
found in the ring-open parent analogue 3. However, it
seemed that macrocyclization via an amide spacer is less
advantageous than via the olefin spacer (IC50 ) 7.9 µM
for 36 versus IC50 ) 0.026 µM for 2a ). These results
are supported by molecular modeling studies. Figure 3
shows the binding of 2a (panel A) and 36 (panel B) to
the Grb2 SH2 domain as generated by molecular
mechanics calculations. These panels graphically high-

â-Aminophosphotyrosyl Mimetic
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2625
Sch em e 5a
a
Reagents and conditions: (a) (Boc)2O, TEA, MeOH; (b) (i) catalyst OsO4, NMO, THF/H2O (1:1), (ii) NaIO4, THF/H2O (10:1); (c) PCC,
molecular sieves, CH2Cl2; (d) LiOH, THF.
Sch em e 6a
a
Reagents and conditions: (a) (i) 33, HOAt, EDC‚HCl, DMF, (ii) TFA, CH2Cl2; (b) FDPP, NMM, DMF; (c) 10% Pd/C, MeOH,
H2 (40 psi).
Ta ble 1. Inhibition Data Obtained in Extracellular
ELISA-Based Grb2 SH2 Domain Binding Assays
light potential interactions of ligands with the hydro-
phobic region. Of note, interactions of 36 with the
hydrophobic patch (side chains of Leu â′D1, Lys âD6,
and Phe âE3) are less optimal than those of 2a . This is
exemplified by the naphthyl functionality of 36, which
is farther from the side chain of Phe âE3 compared with
2a . (Estimated distances are shown in panels A and B.)
Furthermore, the ring-closing olefin segment of 2a (pink
in panel B) can potentially afford additional favorable
interactions with the Lys âD6 side chain that are not
possible with the amide spacer found in 36. A visual
comparison of conformational differences, particularly
in the naphthyl portions of the ligands, that result from
a
compound
IC50 ( SD (µM)
2a
5
6
7
27
36
0.026 ( 0.002
.600
.600
47.9 ( 10.2
52.5 ( 5.3
7.9 ( 0.9
a
Values were obtained as described in the Experimental
Section.
alterations in ring-closing segments is shown in the
overlay of 2a and 36 (Figure 3C). The modest absolute

2626 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Lee et al.
F igu r e 3. Docking of 2a (A) and 36 (B) to the Grb2 SH2 domain as determined in the Experimental Section. Panel C shows the
overlay of energy-minimized conformations of 2a (green) and 36 (colored by atom). The ligands were extracted from energy-
minimized protein-ligand complexes.
based on an X-ray structure of the Grb2 SH2 domain com-
plexed with a hexapeptide inhibitor (PDB entry 1TZE). A
model of the binding site was constructed consisting of residues
between His 58 and Glu 152. Ligands were manually docked
into the active site based on the X-ray structure and confor-
mational minima obtained from random conformational analy-
sis. Minimizations were performed with MacroModel using
MMFF94 force field (Merck molecular force field)39 with a
continuum solvation model (a generalized Born surface area
approach40). The PRCG (Polak-Ribier conjugate gradient)
method was used with convergence criteria set to Gradient.
The ligand as well as protein residues within 5 Å were allowed
to move freely during energy minimization, while residues at
a distance between 5 and 10 Å were constrained by a parabolic
force constant of 50 kJ /Å and residues beyond 10 Å were
frozen.
Gen er a l Syn th etic P r oced u r es. Melting points were
determined on a Mel-Temp II melting apparatus and are
uncorrected. 1H NMR were obtained on a Varian 400 MHz
spectrometer, and chemical shifts (δ) are given relative to
tetramethylsilane. Fast atom bombardment mass spectra
(FABMS) were acquired with a VG Analytical 7070E mass
spectrometer under the control of a VG 2035 data system.
Elemental analyses were obtained from Atlantic Microlab Inc.,
Norcross, GA. Amino acid analyses were carried out at Peptide
Technologies Inc., Gaithersburg, MD. Solvent was removed by
rotary evaporation under reduced pressure, and silica gel
column chromatography was performed using ICN SiliTech
32-63 D (60 Å). Anhydrous solvents were obtained com-
mercially and used without further drying. HPLC was con-
ducted using a Waters Corp. Prep LC4000 system having
photodiodide array detection. Binary solvent systems were as
indicated, where solvent system A was 0.1% aqueous TFA and
solvent system B was 0.1% TFA in acetonitrile, with a YMC
J ’sphere ODS-H80 column (4 µm particle size, 8 nm pore size),
20 mm diameter × 250 mm (flow rate of 10 mL/min), being
used for preparative work and a YMC J ’sphere ODS-H80
column (4 µm particle size, 8 nm pore size), 4.6 mm diameter
× 250 (flow rate of 1 mL/min), being used for analytical work.
affinity of analogue 36 is also consistent with its lack
of functionality at the pTyr mimetic R-position, since
functionality at this position is critical for optimum
binding interactions in the SH2 domain pTyr-binding
pocket.5 Introduction of such an R-functionality is the
objective of ongoing work.
The potential therapeutic value of the Grb2 SH2
domain binding ligands is dependent on their ability to
function in whole-cell systems. For analogue 2a , which
represents the olefin-containing homologue of â-amino
macrocycle 36, it had been shown previously that
despite high affinity in extracellular Grb2 SH2 domain
binding assays, markedly reduced potencies were ob-
served in whole-cell assays that examined intracellular
binding of Grb2 and growth inhibition.37 A more recent
study has shown that inclusion of carboxylic acid
functionality at the pTyr mimetic R-position (compound
2b) can significantly improve potencies in such whole-
cell assays. In the current study, it was therefore
expected that without the R-carboxlic acid functionality
compound 2a would exhibit poor inhibitory potencies
in cell-based assays.9 Indeed, side-by-side comparisons
of 2a with 36 showed that neither exhibited significant
inhibition of Grb2 SH2 domain binding to growth factor
activated erbB-2 in whole cells and that there was no
appreciable inhibition of growth of MDA-MB-453 breast
cancer cells.16
In conclusion, the current study presents the first
Grb2 SH2 domain binding ligands derived from car-
boxamido-based macrocyclization at the pTyr mimetic
â-position. This class of compound joins recently re-
ported olefin-metathesis-derived macrocycles such as 2
as new Grb2 SH2 domain binding motifs that may
provide leads for further development. Results of the
work presented herein may also find application in the
design of analogues directed at other signal transduction
targets that bind ligands in bend conformations, includ-
ing phosphotyrosine-binding (PTB) domains.38
N-F m oc-P m p â-OH (13). To a solution of 1111 (684.6 mg,
1.228 mmol) in MeOH (2.5 mL) was added 4 N HCl in dioxane
(1 mL) at 0 °C, and the resulting mixture was stirred at room
temperature (15 min). Volatiles were removed in vacuo, and
the resulting amine salt in dioxane/water (1:1, 6 mL) was
treated at room temperature with 1 N LiOH (6.1 mL, 6.1
mmol, 20 min). Then CO2 was introduced for several seconds
until the pH was adjusted to 8-8.5 (pH paper). Fmoc N-
hydroxysuccinimide ester (Fmoc-OSu) (496 mg, 1.47 mmol) in
dioxane was added, and the reaction mixture was stirred at
room temperature (2 h), diluted with cold 5% citric acid (10
mL), and subjected to an extractive workup (CH2Cl2) to provide
the crude product. Purification by silica gel column chroma-
tography (CH2Cl2/MeOH, 7:1) gave 13 as a white foam (665
Exp er im en ta l Section
Biologica l P r oced u r es. Inhibition of Grb2 SH2 domain
binding in extracellular assays using ELISA techniques was
performed as previously reported.36
Molecu la r Mod elin g. Molecular modeling studies were
carried out using MacroModel 8.0 (Schro¨dinger, L.L.C.) and
Sybyl 6.8 (Tripops, Inc.) on a Silicon Graphics Octane 2
workstation. Construction of protein-ligand complexes was

â-Aminophosphotyrosyl Mimetic
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2627
mg, 82% yield). 1H NMR (DMSO-d6): δ 7.41-7.17 (22H, m,
aromatic H), 4.91 (5H, m, OCH2Ph, CâH), 4.29-4.10 (3H, m,
aliphatic (Fmoc)), 3.72-3.47 (2H, m, CH2CO2), 3.29 (2H, d, J
) 21.6 Hz, CH2P(O)). FABMS (-Ve) m/z: 660 (M - H)-. Anal.
(C39H36NO7P‚0.1H2O) C, H, N.
mL), and the resulting mixture was stirred at room temper-
ature (2 h). Volatiles were removed and the resulting residue
was re-evaporated from toluene and then triturated with ether
and centrifuged to provide 17‚TFA in quantitative yield as a
white solid (100 mg). 1H NMR (D2O): δ 7.40-7.49 (5H, m, Ph),
5.26 (1H, t, J ) 7.6 Hz, CâH (Pmpâ)), 4.63 (1H, t, J ) 6 Hz,
CRH (Asn)), 3.25 (2H, t, J ) 6.8 Hz, CâH2 (â-Ala)), 2.90 (2H, d,
J ) 6.4 Hz, CH2C(O)N), 2.65-2.82 (4H, m, CH2 (Pmpâ), CRH2
(â-Ala)), 0.99-1.96 (10H, m, cyclohexyl). FABMS (+Ve) m/z:
476 (MH+).
H-Ac6c-Asn -(OtBu ) (14). To a mixture of H-Asn-(OtBu)‚HCl
(1.78 g, 7.92 mmol) and diisopropylethylamine (DIPEA) (1.38
mL, 7.92 mmol) in DMF (20 mL) was added a preformed HOBt
active ester solution, which was generated from the reaction
of N-Fmoc-1-aminocyclohexanecarboxylic acid (N-Fmoc-Ac6c-
OH)41 (2.92 g, 8.0 mmol), HOBt (1.13 g, 8.4 mmol), and DIPCDI
(1.38 mL, 8.8 mmol) in DMF (20 mL) (10 min). The mixture
was stirred overnight, then DMF was removed in vacuo, and
the residue was purified by silica gel flash column chroma-
tography (CH2Cl2/MeOH, 15:1) to give N-Fmoc-Ac6c-Asn-
Cyclo[â-Ala -â-P h e-Ac6c-Asn ] (21). To amine salt 17 (25
mg, 0.053 mmol) in DMF (53 mL) was added N-methylmor-
pholine (NMM) (0.06 mL, 0.53 mmol) and pentafluorophenyl
diphenylphosphinate (FDPP) (40.4 mg, 0.105 mmol), and the
resulting solution was stirred at room temperature for 24 h.
Solvent was removed and the resulting residue was purified
by silica gel flash chromatography (CH2Cl2/MeOH from 9:1 to
7:1) to give 6 as a white solid (19 mg, 80% yield). Mp >240 °C
1
(OtBu) (3.86 g, 91% yield). H NMR (DMSO-d6): δ 7.95-7.31
(11H, m, aromatic H, NH, NH2), 6.86 (1H, s, NH), 4.39 (1H,
m, CRH (Asn)), 4.22 (3H, aliphatic (Fmoc)), 1.43 (2H, m, CH2C-
(O)N), 1.17-1.99 (10H, m, cyclohexyl), 1.1 (9H, s, tBu). FABMS
(+Ve) m/z: 536 (MH+). To a solution of Fmoc-protected dipep-
tide (3.8 g, 7.09 mmol) in DMF (60 mL) was added piperi-
dine (3.5 mL), and the resulting solution was stirred at room
temperature (1 h). Solvent was removed, and the residue
was purified by silica gel flash chromatography (CH2Cl2/
MeOH from 100:1 to 7:1) to give 14 as a colorless oil (596
1
(dec). H NMR (CD3OD): δ 7.22-7.39 (5H, m, Ph), 5.49 (1H,
dd, J ) 3.2 and 12.4 Hz, CâH (Pmpâ)), 4.64 (1H, t, J ) 4.4 Hz,
CRH (Asn)), 4.05 (1H, m, CâH (â-Ala)), 2.98-301 (3H, m, CâH2
(â-Ala), CRH (â-Phe), CHHC(O)N), 2.85 (1H, dd, J ) 3.6 and
17.8 Hz, CRH (Pmpâ)), 2.54 (1H, dd, J ) 4 and 16.4 Hz, CHHC-
(O)N), 2.08-2.22 (2H, m, CâH2 (â-Ala)), 1.28-1.96 (10H, m,
cyclohexyl). FABMS (+Ve) m/z: 458 (MH+). Anal.(C39H36NO7P‚
0.5MeOH‚0.5H2O) C, H, N.
mg, 92% yield). 1H NMR (CD3OD): δ 4.57 (1H, dd, J
)
H-P m p â-Ac6c-Asn -(OtBu ) (16). To a solution of 14 (127 mg,
0.405 mmol), N-Fmoc-Pmpâ-OH (13) (268 mg, 0.405 mmol),
and HOAt (108 mg, 0.794 mmol) in DMF (10 mL) was added
a solution of 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide‚
HCl (EDC‚HCl) (113 mg, 0.591 mol) in DMF (10 mL) at 0 °C.
The reaction mixture was warmed to room temperature and
stirred for 24 h. After removal of volatiles, the residue was
dissolved in DMF and treated with piperidine (0.2 mL) for 2
h. The reaction mixture was concentrated to dryness and
purified by silica gel flash chromatography (CH2Cl2/MeOH
from 30:1 to 7:1) to give 16 as a white solid (246 mg, 83% yield).
Mp 202-204 °C. 1H NMR (CD3OD): δ 7.25-7.37 (14H, m,
aromatic H), 4.95 (m, 4H, CH2Ph), 4.55 (1H, m, CâH (Pmpâ)),
4.31 (1H, dd, J ) 5.6 and 8.6 Hz, CRH (Asn)), 3.28 (2H, d, J )
23.6 Hz, CH2P(O)), 2.59-2.83 (4H, m, CRH2 (Pmpâ), CH2C(O)-
5.2 and 6.2 Hz, CRH (Asn)), 2.80 (1H, dd, J ) 6.0 and 16 Hz,
CH2C(O)N), 2.69 (1H, dd, J ) 5.2 and 16 Hz, CH2C(O)N),
1.34-1.90 (10H, m, cyclohexyl), 1.46 (9H, s, tBu). FABMS
(+Ve) m/z: 314 (MH+).
H-â-P h e-Ac6c-Asn -(OtBu ) (15). To a solution of 14 (143
mg, 0.448 mmol) in DMF (0.5 mL) was added a preformed
active ester solution that was generated by the reaction of
N-Fmoc-L-â-Phe-OH42 (175.5 mg, 0.453 mmol), HOBt (64 mg,
0.475 mmol), and DIPCDI (0.08 mL, 0.498 mol) in DMF (1.5
mL) (10 min), and the resulting mixture was stirred overnight.
DMF was removed in vacuo and the residue was purified by
silica gel flash chromatography (hexanes/EtOAc/1 M NH3/
MeOH, 10:5:1) to give N-Fmoc-â-Phe-Ac6c-Asn-(OtBu) (239 mg,
78% yield) as a white solid. Mp 167-169 °C. 1H NMR (DMSO-
d6): δ 7.22-7.89 (17H, m, aromatic H, NH, NH2), 6.88 (1H, s,
NH), 4.94 (1H, m, CâH (Pmpâ)), 4.33 (1H, m, CRH (Asn)), 4.18-
4.34 (3H, m, aliphatic (Fmoc)), 2.54-2.68 (2H, m, CH2 (Pmpâ)),
2.44 (d, 2H, J ) 5.6 Hz, CH2C(O)N), 1.28-1.92 (10H, m,
t
NH), 2.19-1.16 (19H, m, cyclohexyl, Bu). FABMS (+Ve) m/z:
735 (MH+). Anal. (C39H51N4O8P‚1MeOH‚0.5H2O) C, H, N.
H-Gly-P m p â-Ac6c-Asn -OH‚TF A (18). To a solution of 16
(152 mg, 0.207 mmol) in DMF (1 mL) was added a preformed
active ester solution generated by the reaction of N-Boc-Gly-
OH (40 mg, 0.228 mmol), HOBt (34 mg, 0.251 mmol), and
DIPCDI (0.04 mL, 0.262 mol) in DMF (2 mL) (10 min), and
the resulting mixture was stirred overnight. DMF was re-
moved in vacuo and the residue was purified by silica gel flash
chromatography (CH2Cl2/MeOH from 30:1 to 20:1) to give
N-Boc-Gly-Pmpâ-Ac6c-Asn-(OtBu) as a foam (99 mg, 85% yield).
1H NMR (DMSO-d6): δ 8.27 (1H, d, J ) 8.4 Hz, NH), 7.72
(1H, s, NH), 7.67 (1H, d, J ) 8 Hz, NH), 7.17-7.38 (15H, m,
aromatic H, NH), 6.90 (2H, m, NH2), 5.18 (1H, m, CâH (Pmpâ)),
4.95 (4H, m, CH2Ph), 4.33 (1H, m, CRH (Asn)), 3.53 (2H, d, J
) 6 Hz, CH2 (Gly)), 3.30 (2H, d, J ) 21.6, CH2P(O)), 2.52-
2.69 (2H, m, CH2C(O)), 2.43 (2H, dd, J ) 2 and 5.4 Hz, CRH2
(Pmpâ)), 1.91 (2H, m, cyclohexyl), 1.52 (2H, m, cyclohexyl), 1.37
t
cyclohexyl), 1.34 (9H, s, Bu). FABMS (+Ve) m/z: 683 (MH+).
To a solution of Fmoc-protected tripeptide (235 mg, 0.344
mmol) in CH3CN/DMF (2:1, 6 mL) was added piperidine
(0.14 mL), and resulting solution was stirred at room temper-
ature for 2 h. Solvent was removed, and the residue was
purified by silica gel flash chromatography (CH2Cl2/MeOH
from 15:1 to 7:1) to give 15 as a colorless oil (127 mg, 80%
1
yield). H NMR (CD3OD): δ 7.25-7.39 (5H, m, Ph), 4.48 (1H,
t, J ) 5.2 Hz, CâH (Pmpâ)), 4.34 (1H, dd, J ) 4.8 and 9 Hz,
CRH (Asn)), 2.62-2.75 (4H, m, 2H, m, CH2 (Pmpâ), CH2C(O)N),
1.23-2.10 (10H, m, cyclohexyl), 1.42 (9H, s, tBu). FABMS
(+Ve) m/z: 461 (MH+).
H-â-Ala -â-P h e-Ac6c-Asn -OH‚TF A (17). To a solution of 15
(94 mg, 0.204 mmol) in DMF (0.5 mL) was added the
preformed active ester solution that was generated by the
reaction of N-Boc-â-Ala-OH38 (38.9 mg, 0.206 mmol), HOBt (29
mg, 0.216 mmol), and DIPCDI (0.035 mL, 0.226 mol) in DMF
(1 mL) (10 min), and the resulting mixture was stirred at room
temperature (overnight). DMF was removed in vacuo and the
residue was purified by silica gel flash chromatography
(hexanes/EtOAc/1 M NH3/MeOH, 4:2:1) to give N-Boc-â-Ala-
â-Phe-Ac6c-Asn-(OtBu) as a foam (127 mg, 98% yield). 1H NMR
(CD3OD): δ 7.22-7.38 (5H, m, Ph), 5.33 (1H, m, CâH (â-Phe)),
4.49 (1H, m, CRH (Asn)), 2.72-2.79 (4H, m, CRH2 (â-Ala), CRH2
(â-Phe)), 2.75 (1H, dd, J ) 5.2 and 15.8 Hz, CHHC(O)N), 2.64
(1H, dd, J ) 4.8 and 16 Hz, CHHC(O)N), 2.41 (2H, m, CRH2
(â-Ala)), 2.03 (2H, m, CâH2 (â-Ala)), 1.23-2.04 (10H, m,
t
t
(9H, s, Bu), 1.36 (9H, s, Bu), 1.27-1.04 (5H, m, cyclohexyl).
FABMS (+Ve) m/z: 735 (MH+). To a solution of Boc-protected
tetrapeptide (101 mg, 0.122 mmol) in CH2Cl2 (2 mL) was added
TFA (4 mL), and the resulting mixture was stirred at room
temperature for 2 h. Volatiles were removed and the resulting
residue was coevaporated with toluene, treated with ether, and
centrifuged to provide 18‚TFA as a white solid (84 mg, 93%
yield). 1H NMR (DMSO-d6): δ 8.79 (1H, d, J ) 8 Hz, NH),
7.87 (1H, s, NH), 7.63 (1H, d, J ) 8 Hz, NH), 7.15-7.37 (16H,
m, aromatic H, NH2), 6.86 (2H, m, NH2), 5.22 (1H, m, CâH
(Pmpâ)), 4.95-4.98 (4H, m, CH2Ph), 4.85 (1H, d, J ) 7.2, CRH
(Asn)), 4.37 (2H, m, CH2 (Gly)), 2.74 (1H, dd, J ) 7.2 and 14.6
Hz, CH2C(O)N), 2.58 (1H, dd, J ) 7.2 and 14.6 Hz, CH2C(O)N),
2.46-2.54 (2H, m, CRH2 (Pmpâ)), 1.83-2.20 (3H, m, cyclohexyl),
0.95-1.55 (m, 7H, cyclohexyl). FABMS (+Ve) m/z: 736 (MH+).
t
t
cyclohexyl), 1.45 (9H, s, Bu), 1.42 (9H, s, Bu). FABMS (+Ve)
m/z: 631 (MH+). To a solution of Boc-protected tetrapeptide
(119 mg, 0.188 mmol) in CH2Cl2 (2 mL) was added TFA (2.5

2628 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Lee et al.
H-â-Ala -P m p â-Ac6c-Asn -OH‚TF A (19). To a solution of 16
(149 mg, 0.203 mmol) in DMF (1 mL) was added a preformed
active ester solution formed by the reaction of N-Boc-â-Ala-
OH (38.8 mg, 0.205 mmol), HOBt (30.5 mg, 0.225 mmol), and
DIPCDI (0.035 mL, 0.225 mol) in DMF (1.5 mL) (10 min), and
the resulting mixture was stirred overnight. DMF was re-
moved in vacuo and the resulting residue was purified by silica
gel flash chromatography (CH2Cl2/MeOH from 18:1 to 12:1)
to give N-Boc-â-Ala-Pmpâ-Ac6c-Asn-(OtBu) as a foam (181
mg, 98% yield). 1H NMR (CD3OD): δ 7.21-7.35 (14H, m,
aromatic H), 5.32 (1H, t, J ) 6.8 Hz, CâH (Pmpâ)), 4.89-4.95
(4H, m, CH2Ph), 4.48 (1H, t, J ) 4.8 Hz, CRH (Asn)), 3.25 (2H,
d, J ) 23.9 Hz, CH2P(O)), 2.60-2.85 (6H, m), 2.38 (2H, m,
CH2C(O)N), 1.93-2.06 (2H, m, cyclohexyl), 1.18-1.73 (26H,
m, tBu, cyclohexyl). FABMS (+Ve) m/z: 906 (MH+). To a
solution of Boc-protected tetrapeptide (112 mg, 0.123 mmol)
in CH2Cl2 (2 mL) was added TFA (3 mL), and the resulting
mixture was stirred at room temperature for 2 h. Volatiles
were removed, and the resulting residue was coevaporated
from toluene, treated with ether, and centrifuged to give
19‚TFA as a white solid (91 mg, 99% yield). 1H NMR
(DMSO-d6): δ 8.79 (1H, d, J ) 8 Hz, NH), 7.87 (1H, s, NH),
7.63 (1H, d, J ) 8 Hz, NH), 7.15-7.37 (16H, m, aromatic H,
NH), 6.86 (2H, m, NH2), 5.22 (1H, m, CâH (Pmpâ)), 4.95-4.98
(4H, m, CH2Ph), 4.84 (1H, m, CRH (Asn)), 4.37 (2H, m, CâH2
(â-Ala)), 2.68-2.98 (8H, m, CH2P(O), CH2C(O)N, CRH2 (â-Ala),
CRH2 (Pmpâ)), 1.83-2.20 (3H, m, cyclohexyl), 0.95-1.55 (7H,
m, cyclohexyl). FABMS (+Ve) m/z: 750 (MH+).
cyclohexyl). FABMS (+Ve) m/z: 718 (MH+). HR-FABMS calcd
for C37H44N5O8PCs: 850.1982 (MCs). Found: 850.1980.
Cyclo[â-Ala -P m p â(OBn )2-Ac6c-Asn ] (10). Following a pro-
cedure similar to that reported above for the synthesis of
21, reaction of amine salt 19 (39 mg, 0.052 mmol), NMM
(0.058 mL, 0.52 mmol), and FDPP (40 mg, 0.10 mmol) in
DMF (52 mL) provided benzyl-protected cyclic peptide 10 (24
mg, 63% yield). 1H NMR (CD3OD): δ 7.22-7.34 (14H, m,
aromatic H), 5.23 (1H, m, CâH (Pmpâ)), 4.94 (4H, m, CH2Ph),
4.76 (1H, dd, J ) 4.8 and 7.4 Hz, CRH (Asn)), 3.64-3.54
(2H, m, CâH (â-Ala)), 3.28 (2H, d, J ) 21.6 Hz, CH2P(O)), 2.86
(3H, m, CHHC(O)N, CRH2 (Pmpâ)), 2.77 (1H, dd, CHHC(O)N),
2.35 (1H, m, CRH (â-Ala)), 2.23 (1H, m, CRH (â-Ala)), 1.86-
2.03 (3H, m, cyclohexyl), 1.31-1.66 (7H, m, cyclohexyl).
FABMS (+Ve) m/z: 732 (MH+). Anal. (C38H46N5O8P‚0.6H2O‚
1CH3OH) C, H, N.
Cyclo[Abu -P m p â(OBn )2-Ac6c-Asn ] (23). Following a pro-
cedure similar to that reported above for the synthesis of 21,
reaction of amine salt 20 (98 mg, 0.128 mmol), NMM (0.14
mL, 1.28 mmol), and FDPP (98.4 mg, 0.256 mmol) in DMF
(128 mL) provided benzyl-protected cyclic peptide 23 (40
mg, 42% yield). 1H NMR (CD3OD): δ 7.19-7.32 (14H, m,
aromatic H), 5.29 (1H, d, J ) 10.8 Hz, CâH (Pmpâ)), 4.90 (4H,
m, CH2Ph), 4.60 (1H, dd, J ) 4.4 and 9.2 Hz, CRH (Asn)), 3.48
(1H, m, CγH (Abu)), 3.04 (1H, m, CγH (Abu)), 2.69-2.87 (3H,
m, CH2C(O)N, CRH2 (Pmpâ)), 2.57 (1H, dd, J ) 12.8 and 14
Hz, CH2C(O)N), 2.27 (1H, m, CRH (Abu), 2.14 (1H, m, CRH
(Abu)), 1.86-1.98 (4H, m, CRH2 (Abu), cyclohexyl), 1.67-1.84
(2H, m, cyclohexyl), 1.58 (4H, m, cyclohexyl), 1.41 (1H, m,
cyclohexyl), 1.29 (1H, m, cyclohexyl). FABMS (+Ve) m/z: 746
(MH+). Anal. (C39H48N5O8P‚2H2O) C, H, N.
H-Abu -P m p â-Ac6c-Asn -OH‚TF A (20). To a solution of 16
(127 mg, 0.173 mmol) in DMF (1 mL) was added a preformed
active ester solution formed by the reaction of N-Boc-4-
aminobutyric acid (N-Boc-Abu-OH) (35.6 mg, 0.175 mmol),
HOBt (26 mg, 0.19 mmol), and DIPCDI (0.33 mL, 0.21 mol)
in DMF (1.5 mL) (10 min), and the resulting mixture was
stirred at room temperature overnight. DMF was removed in
vacuo, and the residue was purified by silica gel flash chro-
matography (CH2Cl2/MeOH from 20:1 to 15:1) to give N-Boc-
Abu-Pmpâ-Ac6c-Asn-(OtBu) as a white solid (128 mg, 84%
Cyclo[Gly-P m p â-Ac6c-Asn ] (5). A solution of benzyl-
protected cyclic peptide 22 (32.8 mg, 0.045 mmol) in MeOH
(10 mL) was hydrogenated over 10% Pd/C (30 mg) at 40 psi of
H2 in a Parr shaker (overnight). The mixture was filtered
through Celite, the pad was washed with aqueous MeOH, and
the combined filtrate was concentrated to a white solid, which
was purified on reverse-phase C18 silica gel (H2O/MeOH, 9:1)
and lyophilized to give 5 as a white solid (12.4 mg, 51% yield).
Mp 262-265 °C. Purity >99% (as determined by reverse-phase
HPLC, gradient elution from 0% to 90% of solvent B over 30
1
yield). Mp 199-200 °C. H NMR (DMSO-d6): δ 8.25 (1H, d, J
) 8.4 Hz, NH), 7.72 (1H, s, NH), 7.68 (1H, d, J ) 8 Hz, NH),
7.20-7.36 (15H, m, aromatic H, NH), 6.78 (1H, t, J ) 5.2 Hz,
NH), 5.20 (1H, dd, J ) 8.4 and 15 Hz, CâH (Pmpâ)), 4.96 (4H,
m, 2 CH2Ph), 4.34 (1H, m, CRH (Asn)), 3.31 (2H, d, J ) 21.6
Hz, CH2P(O)), 2.87 (2H, dd, J ) 6.4 and 12.8 Hz, CγH2 (Abu)),
2.66 (2H, dd, J ) 6.4 and 13.8 Hz, CH2C(O)N), 2.43 (2H, d, J
) 5.6 Hz, CRH2 (Abu)), 2.06 (2H, t, J ) 8 Hz, CRH2 (Pmpâ)),
1.99 (1H, m, CâH (Abu)), 1.89 (1H, m, CâH (Abu)), 1.05-1.59
1
min, tR ) 13.44 min). H NMR (D2O): δ 7.32 (4H, m, phenyl),
5.15 (1H, t, J 5.6, CâH (Pmpâ)), 4.88 (1H, t, J ) 8 Hz, CRH
(Asn)), 3.95 (1H, d, J ) 15.6 Hz, CRH (Gly)), 3.84 (1H, d,
J ) 15.6 Hz, CRH (Gly)), 2.94-3.05 (3H, m, CH2P(O) and
CHC(O)N), 2.81-2.90 (2H, m, CRH2 (Pmpâ)), 2.73 (1H, dd, J
) 7.6 and 15.4 Hz, CHC(O)N), 2.12 (1H, m, cyclohexyl), 1.90
(3H, m, cyclohexyl), 1.57 (4H, m, cyclohexyl), 1.47 (1H, m,
cyclohexyl), 1.35 (1H, m, cyclohexyl). FABMS (-Ve) m/z: 536
(M - H). HR-FABMS calcd for C23H31N5O8P: 536.1944 (M -
H). Found: 536.1910.
t
(28H, m, Bu, cyclohexyl). FABMS (+Ve) m/z: 920 (MH+). To
a solution of Boc-protected tetrapeptide (119 mg, 0.129 mmol)
in CH2Cl2 (2 mL) was added TFA (4 mL), and the resulting
mixture was stirred at room temperature (2.5 h). Volatiles
were removed, and the resulting residue was coevaporated
with toluene. Then the residue was treated with ether and
centrifuged to give 20‚TFA as a white solid (98 mg, 99% yield).
1H NMR (DMSO-d6): δ 8.39 (1H, d, J ) 8.4 Hz, NH), 7.18-
7.38 (14H, m, aromatic H), 6.89 (1H, s, NH), 5.21 (1H, m, CâH
(Pmpâ)), 4.95 (4H, m, CH2Ph), 4.88 (1H, d, J ) 7.2 Hz, CRH
(Asn)), 4.39 (2H, m, CγH2 (Abu)), 2.66-2.97 (4H, m, CRH2
(Pmpâ), CH2C(O)N), 2.42-2.51 (2H, m, CRH2 (Abu)), 2.00 (H,
m, CâH2 (Abu)), 1.32-1.88 (10H, m, cyclohexyl). FABMS
(+Ve) m/z: 764 (MH+).
Cyclo[â-Ala -P m p â-Ac6c-Asn ] (6). Hydrogenolytic deben-
zylation of protected cyclic peptide 10 as described above for
the synthesis of 5 provided final product 6 as a white solid
(10.2 mg, 59% yield). Mp >275 °C (dec). Purity 95% (as
determined by reverse-phase HPLC, gradient elution from 0%
to 90% of solvent B over 20 min, tR ) 5.31 min). 1H NMR
(D2O): δ 7.35 (4H, m, phenyl), 5.29 (1H, dd, J ) 3.6 and 12.2
Hz, CâH (Pmpâ)), 4.82 (1H, m, CRH (Asn)), 3.64 (1H, m, CâH
(â-Ala)), 3.52 (1H, m, CâH (â-Ala)), 3.22 (2H, d, J ) 21.6 Hz,
CH2P(O)), 2.94 (2H, m, CRH2 (Pmpâ)), 2.74 (2H, m, CH2C(O)N),
2.43 (2H, t, J ) 5.6 Hz, CRH (â-Ala)), 2.02 (1H, m, cyclohexyl),
1.88 (3H, m, cyclohexyl), 1.29-1.56 (6H, m, cyclohexyl).
FABMS (-Ve) m/z: 550 (M - H). HR-FABMS calcd for
Cyclo[Gly-P m p â(OBn )2-Ac6c-Asn ] (22). Following a pro-
cedure similar to that reported above for the synthesis of
21, reaction of amine salt 18 (87.5 mg, 0.119 mmol), NMM
(0.13 mL, 1.19 mmol), and FDPP (91.4 mg, 0.238 mmol) in
DMF (119 mL) provided benzyl-protected cyclic peptide 22
(18 mg, 21% yield). H NMR (CD3OD): δ 7.15-7.32 (14H,
m, aromatic H), 5.15 (1H, t, J ) 5.6 Hz, CâH (Pmpâ)), 4.89
(4H, m, CH2Ph), 4.73 (1H, m, CRH (Asn)), 3.62 (2H, m, CH2
(Gly)), 3.22 (2H, d, J ) 21.6 Hz, CH2P(O)), 2.72-2.87 (3H, m,
CH2C(O)N, CâH2 (Pmpâ)), 2.56 (1H, dd, J ) 6.4 and 15 Hz,
CH2C(O)N), 2.05 (1H, m, cyclohexyl), 1.81-1.89 (2H, m,
cyclohexyl), 1.22-1.53 (6H, m, cyclohexyl), 0.86 (1H, m,
C
24H33N5O8P: 550.2067 (M - H). Found: 550.2056.
Cyclo[Abu -P m p â-Ac6c-Asn ] (7). Hydrogenolytic debenzy-
lation of protected cyclic peptide 23 (38.3 mg, 0.051 mmol) as
described above for the synthesis of 5 provided final product
7 as a white solid (15.6 mg, 54% yield). Mp >282 °C (dec).
Purity >99% (as determined by reverse-phase HPLC, gradient
elution from 0% to 90% of solvent B over 30 min, tR ) 11.35
1
min). H NMR (D2O): δ 7.17 (4H, s, phenyl), 5.14 (1H, dd, J
) 2.4 and 12.4 Hz, CâH (Pmpâ)), 4.45 (1H, dd, J ) 4.4 and
10.4 Hz, CRH (Asn)), 3.36 (1H, m, CγH (Abu)), 2.94 (1H, m,

â-Aminophosphotyrosyl Mimetic
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2629
CγH (Abu)), 2.82-2.87 (3H, m, CH2P(O), CHC(O)N), 2.75 (1H,
dd, J ) 2.4 and 14.6 Hz, CRH (Pmpâ)), 2.60 (1H, dd, J ) 10.4
and 15.6 Hz, CHC(O)N), 2.52 (1H, dd, J ) 12.8 and 14.4 Hz,
CRH (Pmpâ)), 2.09-2.22 (2H, m, CRH (Abu)), 1.31-1.82 (12H,
m, CâH (Abu, cyclohexyl). FABMS (-Ve) m/z: 564 (M - H),
79. HR-FABMS calcd for C25H35N5O8P: 564.2249 (M - H).
Found: 564.2223.
diol intermediate), which was used without further purifica-
tion. [No detectable aldehyde proton was apparent in the 1H
NMR spectrum.] To a mixture of 31 (245 mg, 0.75 mmol) and
molecular sieves (4 Å, 300 mg) in CH2Cl2 at room temperature
was added pyridinium chlorochromate (PCC, 323 mg), and the
mixture was stirred at room temperature (overnight). To this
was added Celite and ether. The resulting mixture was filtered
through a silica gel pad, and the pad was washed with ether
(4 × 100 mL). The combined filtrate was concentrated and
purified by silica gel flash chromatography (hexanes/EtOAc
from 4:1 to 3:1) to yield lactam 32 as a yellow oil (184 mg,
Desa m in o-P m p (OtBu )-Ac6c-Asn -NH2 (26). To a solution
of 24 (113 mg, 0.443 mmol) and desamino-Pmp(OtBu)-OH (25)7
(162 mg, 0.456 mmol) in DMF (4 mL) was added HOAt (0.5-
0.7 M in DMF, 1.2 mL) followed by a solution of EDC‚HCl (110
mg, 0.576 mol) in DMF (4 mL) at 0 °C. Then the mixture was
warmed to room temperature and stirred at room temperature
for 24 h. The reaction mixture was evaporated to dryness and
purified by silica gel flash chromatography (CH2Cl2/MeOH
from 20:1 to 9:1) to provide 26 as a white solid (224 mg, 85%
1
76% yield). H NMR (CDCl3): δ 7.28-7.97 (7H, m, aromatic-
H), 3.78 (1H, dd, J ) 7.6 and 11.5 Hz, 5-H), 3.54 (1H, dd, J )
6.4 and 11 Hz, 5-H), 3.20 (2H, d, J ) 7.6 Hz, 4′-H), 2.79 (1H,
m, 4-H), 2.61 (1H, dd, J ) 8 and 17.4 Hz, 3-H), 2.39 (1H, dd,
t
J ) 7.6 and 17.4 Hz, 3-H), 1.51 (9H, s, Bu). FABMS (+Ve)
m/z: 326 (MH+). Anal. (C20H23NO3) C, H, N.
1
yield). Mp 117-119 °C. H NMR (CDCl3): δ 8.15 (1H, d, J )
8 Hz, NH (Asn)), 7.43 (1H, s, NH (Ac2C), 7.27 (2H, m, Cγ(O)-
NH2 (Asn)), 7.05-7.11 (4H, m, phenyl), 6.46 (2H, br d, J )
19.6 Hz, CONH2 (Asn)), 4.64 (1H, m, CRH (Asn)), 2.94 (2H, d,
J ) 2.94 Hz, CH2P(O)), 2.84 (3H, m, CâH2C(O)N (Asn), CâH2
(desamino-Pmp)), 2.63 (1H, m, CâH2C(O)N (Asn), 2.56 (2H,
m, CRH2 (Pmp)), 1.13-2.04 (19H, m, cyclohexyl, tBu). FABMS
(+Ve) m/z: 595 (MH+). HR-FABMS calcd for C29H47N4O7P:
595.3261 (MH+). Found: 595.3231.
(3R)-3-(ter t-Bu toxyca r bon yla m in om eth yl)-4-n a p h th a -
len -1-ylbu tyr ic Acid (N-Boc-NM-GABA) (33). To a solution
of 32 (153 mg, 0.474 mmol) in THF (2.5 mL) was added 1 M
LiOH (1.9 mL, 1.9 mmol), and the resulting solution was
stirred at room temperature (2 h). THF was evaporated, and
the residue was washed with ether (2 × 10 mL). Then the
aqueous layer was acidified with 3 N HCl and extracted with
ether (3 × 10 mL). The combined organic layer was dried
(Na2SO4), filtered, and evaporated to provide 33 as a white
foam (140 mg, 86% yield). 1H NMR (CDCl3): δ 7.31-8.07 (7H,
m, aromatic-H), 3.24 (2H, br s, 3′-H), 3.54 (2H, m, 4-H), 2.55
Desa m in o-P m p -Ac6c-Asn -NH2 (27). Treatment of tBu-
protected phosphonate 26 (48 mg, 0.081 mmol) with a solution
of TFA (3.7 mL) and triethylsilane (TES) (0.05 mL) in H2O
(0.1 mL) at room temperature (1 h) with subsequent removal
of solvent and purification by reverse-phase HPLC (gradient
elution from 0% to 95% of solvent B over 35 min, tR ) 21.49
min) provided 27 as a white solid (25 mg, 65% yield). Mp 142-
144 °C. Purity 96% (as determined by reverse-phase HPLC,
gradient elution from 0% to 90% of solvent B over 30 min, tR
) 29.29 min). 1H NMR (D2O): δ 7.27 (4H, m, phenyl), 4.63
(1H, dd, J ) 5.2 and 9.4 Hz, CRH (Asn)), 3.16 (2H, d, J ) 21.2
Hz, CH2P(O)), 2.94 (2H, t, J ) 6.8 Hz, CâH (desamino-Pmp)),
2.86 (1H, dd, J ) 4.8 and 7.8 Hz, CHC(O)), 2.70 (3H, m, CRH2
(desamino-Pmp, CHC(O)N), 2.11-1.87 (10H, m, cyclohexyl).
FABMS (-Ve) m/z: 481 (M - H). HR-FABMS calcd for
t
(1H, m, 3-H), 2.44 (2H, br s, 2-H), 1.43 (9H, s, Bu). FABMS
(+Ve) m/z: 344 (MH+). FABMS (-Ve) m/z: 342 (M - H). Anal.
(C20H25NO4‚0.2H2O) C, H, N.
Tetr a p ep tid e‚TF A (34). To a solution of 16 (260 mg, 0.230
mmol), 33 (122 mg, 0.354 mmol), and HOAt (0.5-0.7 M in
DMF, 1.4 mL) in DMF (8 mL) was added a solution of EDC‚
HCl (99 mg, 0.517 mmol) in DMF (2 mL) at 0 °C. The reaction
mixture was warmed to room temperature and then stirred
for 22 h. After removal of volatiles, the residue was purified
by silica gel flash chromatography (CH2Cl2/MeOH from 40:1
to 20:1) to provide intermediate Boc-protected tetrapeptide as
a white solid (96 mg, 39% yield), which was used without
further purification. Mp 247-249 °C. 1H NMR (CDCl3):
δ
C
21H31N4O7P: 481.1873 (M - H). Found: 481.1852.
7.11-7.79 (21H, m, aromatic H), 5.38 (1H, br s, CâH (Pmpâ)),
4.82-4.88 (4H, m, CH2Ph), 4.59 (1H, m, CRH (Asn)), 3.04-
3.17 (4H, m, CH2P(O), CγH2 (NM-GABA)), 2.90 (1H, m, CâH
(NM-GABA)), 2.66-2.80 (5H, m, CH2C(O)N, CRH2 (Pmpâ),
CHH-naphthyl), 2.56-2.36 (3H, m, CHH-naphthyl, CRH2
(NM-GABA)), 1.92 (2H, m, cyclohexyl), 1.62-1.71 (2H, m,
cyclohexyl), 1.18-1.48 (24H, m, cyclohexyl, tBu). FABMS
(+Ve) m/z: 1060 (MH+). To a solution of Boc-protected tet-
rapeptide (89 mg, 0.084 mmol) in CH2Cl2 (2 mL) was added
TFA (3 mL), and the resulting mixture was stirred at room
temperature (2 h). Volatiles were removed, the resulting
residue was coevaporated with toluene, and then the residue
was treated with ether and centrifuged to provide 34‚TFA as
a white solid (80 mg, quantitative). 1H NMR (DMSO-d6): δ
8.57 (1H, d, J ) 8.4 Hz, NH), 7.15-8.11 (24H, m, aromatic H,
NH × 4), 6.90 (2H, d, NH), 5.27 (1H, m, CâH (Pmpâ)), 4.91
(4H, dd, J ) 2.8 and 4.8 Hz, CH2Ph), 4.84 (1H, d, J ) 7.2 Hz,
CRH (Asn)), 4.39 (2H, m, CγH2 (NM-GABA)), 3.02 (3H, m, CâH
(NM-GABA), CH2C(O)N), 2.88 (2H, m, CRH2 (Pmpâ)), 2.72 (2H,
m, CH2-naphthyl), 2.21 (2H, m, CRH2 (NM-GABA)), 1.32-1.52
(10H, m). FABMS (+Ve) m/z: 904 (MH+).
(2S)-2-(Na p h t h a len -1-ylm et h ylp en t -4-en yl)ca r b a m ic
Acid ter t-Bu tyl Ester (30). To a solution of 298 (1.3 g, 5.8
mmol) and triethylamine (2.4 mL, 17.3 mmol) in MeOH (12
mL) was added Boc anhydride (2 mL, 8.67 mmmol) at 0 °C.
Then the mixture was warmed to room temperature and
stirred at room temperature overnight. Volatiles were re-
moved, and the residue was dissolved in EtOAc and washed
with H2O. The organic layer was dried (Na2SO4), filtered,
concentrated to dryness, and then purified by silica gel flash
chromatography (petroleum ether/EtOAc from 30:1 to 15:1) to
provide 30 as a colorless oil (1.76 g, 94% yield). 1H NMR
(CDCl3): δ 7.32-7.95 (7H, m, aromatic-H), 5.84 (1H, m, 4-H),
5.09-5.13 (2H, m, 5-H), 3.23 (1H, m, 1-H), 3.14-2.99 (3H, m,
t
1-H, 2′-H), 2.18-2.06 (3H, m, 2-H, 3-H), 1.47 (9H, s, Bu).
FABMS (+Ve) m/z: 326 (MH+). Anal. (C21H27NO2) C, H, N.
(3S)-N-Boc-4-(n a p h th ylm eth yl)p yr r olid in e-2-on e (32).
To a solution of 30 (440 mg, 1.35 mmol) in 50% aqueous THF
(20 mL) was added 4-methymorpholine N-oxide (NMO, 190
mg, 1.62 mmol)) and OsO4 (4% in H2O, 0.5 mL) at 0 °C, and
the resulting mixture was stirred vigorously at room temper-
ature for 6 h. Additional NMO (190 mg) was added, and
stirring was continued overnight. The mixture was poured into
1 M Na2S2O3 and extracted with EtOAc. The organic layer was
concentrated and purified on silica gel flash chromatography
(hexanes/EtOAc from 4:1 to 1:2) to give diol intermediate (367
mg, 76% crude yield) and the recovered starting material 30
(81 mg, 18% yield). To a solution of the diol intermediate (350
mg, 0.97 mmol) in THF (20 mL) was added a suspension of
NaIO4 (287.5 mg, 1.62 mmol) in H2O (2 mL) at 0 °C, and the
mixture was stirred for 3.5 h, poured into ice/water, and
extracted with EtOAc. The organic layer was concentrated and
purified on silica gel flash chromatography (hexanes/EtOAc,
4:1) to give lactol 31 as a colorless oil (246 mg, 77% yield from
Ben zyl-P r otected Cyclop ep tid e (35). Following a proce-
dure similar to that reported above for the synthesis of 21,
reaction of amine salt 34 (76 mg, 0.084 mmol), NMM (0.092
mL, 0.84 mmol), and FDPP (64 mg, 0.168 mmol) in DMF (82
mL) provided benzyl-protected cyclic peptide 35 (32 mg, 48%
1
yield). H NMR (CD3OD): δ 7.16-8.40 (21H, m, aromatic H),
5.31 (1H, d, J ) 12 Hz, CâH (Pmpâ)), 4.88 (4H, m, CH2Ph),
4.65 (1H, m, CRH (Asn)), 3.78 (1H, dd, J ) 12.8 and 6.8 Hz,
CγH (NM-GABA)), 3.34-3.37 (1H, m, CâH (NM-GABA)), 3.22
(2H, d, J ) 21.6 Hz, CH2P(O)), 3.03 (1H, m, CγH (NM-GABA),
2.49-2.91 (6H, m, CH2C(O)N, CH2-naphthyl, CRH (Pmpâ)),
1.99-2.15 (3H, m, CRH (NM-GABA), cyclohexyl), 1.29-1.85

2630 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Lee et al.
(9H, m, cyclohexyl). FABMS (+Ve) m/z: 886 (MH+). HR-
FABMS calcd for C50H56N5O8PCs: 1018.2921 (MCs). Found:
1018.2962.
(16) Unpublished data.
(17) Chang, J . K.; Shimizu, M.; Wang, S.-S. Fully automated solid
phase synthesis of protected peptide hydrazides on recycling
hydroxymethyl resin. J . Org. Chem. 1976, 41, 3255-3258.
(18) Fmoc-â-Ala Wang resin (100-200 mesh) loaded at 0.83 mmol/g
was obtained from Novabiochem, San Diego, CA.
(19) Lee, K.; Zhang, M.; Yang, D.; Burke, T. R., J r. Design and
synthesis of a novel phosphatase-stable â-amino acid pTyr
mimetic and its utilization in the preparation of novel Grb2 SH2
domain ligands. Presented at the 224th National Meeting of the
American Chemical Society, Boston, MA, August 2002.
(20) Knorr, R.; Trzeciak, A.; Bannwarth, W. New coupling reagents
in peptide chemistry. Tetrahedron Lett. 1989, 30, 1927-1930.
(21) Bernatowicz, M. S.; Daniels, S. B.; Koster, H. A comparison of
acid labile linkage agents for the synthsis of peptide C-terminal
amides. Tetrahedron Lett. 1989, 30, 4645-4648.
(22) Nomizu, M.; Otaka, A.; Burke, T., R., J r.; Roller, P. P. Synthesis
of phosphonomethyl-phenylalanine and phosphotyrosine con-
taining cyclic peptides as inhibitors of protein tyrosine kinase/
SH2 interactions. Tetrahedron 1994, 50, 2691-2728.
(23) Samy, R.; Kim, H. Y.; Brady, M.; Toogood, P. L. Total synthesis
of motuporin and 5-[L-Ala]-motuporin. J . Org. Chem. 1999, 64,
826-831.
Cyclop ep tid e (36). Hydrogenolytic debenzylation of pro-
tected cyclic peptide 35 as described above for the synthesis
of 5 provided final product 36 as a white solid (16 mg, 62%
yield). Mp >255 °C (dec). Purity >99% (as determined by
reverse-phase HPLC, gradient elution from 0% to 90% of
1
solvent B over 35 min, tR ) 10.01 min). H NMR (DMSO-d6):
δ 8.37 (1H, d, J ) 8 Hz, NH), 8.33 (1H, s, NH), 8.07 (1H, d, J
) 8.8 Hz, NH), 7.91 (1H, d, J ) 7.6 Hz, NH), 7.78 (1H, dd, J
) 2.4 and 7 Hz, NH), 7.10-7.56 (11H, m, aromatic-H), 6.85
(1H, s, NH), 6.52 (1H, s, NH), 5.13 (1H, m, CâH (Pmpâ)), 4.24
(1H, m, CRH (Asn)), 3.08-3.18 (3H, m, CγH2 (NM-GABA), CâH
(NM-GABA)), 2.89 (2H, d, J ) 20.8 Hz, CH2P(O)), 2.75 (6H,
m, CH2C(O)N, CâH2 (Pmpâ), CH2-naphthyl), 1.92-2.10 (2H,
m, CRH2 (NM-GABA)), 1.16-1.82 (10H, m, cyclohexyl). FABMS
(-Ve) m/z: 704 (M - H). HR-FABMS calcd for C36H44N5O8-
PCs: 838.1982 (MCs). Found: 838.2020.
(24) Downing, S. V.; Aguilar, E.; Meyers, A. I. Total synthesis of
bistratamide D. J . Org. Chem. 1999, 64, 826-831.
(25) Angell, Y. M.; Garc´ıa-Echeverr´ıa, C.; Rich, D. H. Comparative
studies of the coupling of N-methylated, sterically hindered
amino acids during solid-phase peptide synthesis. Tetrahedron
Lett. 1994, 35, 5981-5984.
Ack n ow led gm en t. Appreciation is expressed to Dr.
J ames Kelley of the LMC for mass spectral analysis and
to the Komen Foundation for Breast Cancer for financial
support (for D.Y.).
(26) Cole, D. C. Recent stereoselective synthetic approaches to beta-
amino acids. Tetrahedron 1994, 50, 9517-9582.
Refer en ces
(27) Cardillo, G.; Tomasini, C. Asymmetric synthesis of beta-amino
acids and alpha-substituted beta-amino acids. Chem. Soc. Rev.
1996, 25, 117-128.
(28) J uaristi, E.; Lopez-Ruiz, H. Recent advances in the enantiose-
lective synthesis of beta-amino acids. Curr. Med. Chem. 1999,
6, 983-1004.
(29) Abdel-Magid, A. F.; Cohen, J . H.; Maryanoff, C. A. Chemical
process synthesis of beta-amino acids and esters. Curr. Med.
Chem. 1999, 6, 955-970.
(1) Tari, A. M.; Lopez-Berestein, G. GRB2: A pivotal protein in
signal transduction. Semin. Oncol. 2001, 28, 142-147.
(2) Traxler, P.; Bold, G.; Buchdunger, E.; Caravatti, G.; Furet, P.;
Manley, P.; O’Reilly, T.; Wood, J .; Zimmermann, J . Tyrosine
kinase inhibitors: From rational design to clinical trials. Med.
Res. Rev. 2001, 21, 499-512.
(3) Garbay, C.; Liu, W. Q.; Vidal, M.; Rogues, B. P. Inhibitors of
RAS signal transduction as antitumor agents. Biochem. Phar-
macol. 2000, 60, 1165-1169.
(4) Rahuel, J .; Gay, B.; Erdmann, D.; Strauss, A.; GarciaEcheverria,
C.; Furet, P.; Caravatti, G.; Fretz, H.; Schoepfer, J .; Grutter, M.
G. Structural basis for specificity of GRB2-SH2 revealed by a
novel ligand binding mode. Nat. Struct. Biol. 1996, 3, 586-589.
(5) Fretz, H.; Furet, P.; Garcia-Echeverria, C.; Rahuel, J .; Schoepfer,
J . Structure-based design of compounds inhibiting Grb2-SH2
mediated protein-protein interactions in signal transduction
pathways. Curr. Pharm. Des. 2000, 6, 1777-1796.
(6) Garcia-Echeverria, C.; Gay, B.; Rahuel, J .; Furet, P. Mapping
the X + 1 binding site of the Grb2-SH2 domain with alpha,-
alpha-disubstituted cyclic alpha-amino acids. Bioorg. Med.
Chem. Lett. 1999, 9, 2915-2920.
(7) Wei, C.-Q.; Li, B.; Guo, R.; Yang, D.; Burke, T. R., J r. Develop-
ment of a phosphatase-stable phosphotyrosyl mimetic suitably
protected for the synthesis of high affinity Grb2 SH2 domain-
binding ligands. Bioorg. Med. Chem. Lett. 2002, 12, 2781-2784.
(8) Gao, Y.; Wei, C.-Q.; Burke, T. R., J r. Olefin Metathesis in the
Design and Synthesis of a Globally Constrained Grb2 SH2
Domain Inhibitor. Org. Lett. 2001, 3, 1617-1620.
(9) Wei, C.-Q.; Gao, Y.; Lee, K.; Guo, R.; Li, B.; Zhang, M.; Yang,
D.; Burke, T. R., J r. Macrocyclization in the design of Grb2 SH2
domain-binding ligands exhibiting high potency in whole cell
systems. J . Med. Chem. 2003, 46, 244-254.
(10) Ettmayer, P.; France, D.; Gounarides, J .; J arosinski, M.; Martin,
M. S.; Rondeau, J . M.; Sabio, M.; Topiol, S.; Weidmann, B.;
Zurini, M.; Bair, K. W. Structural and conformational require-
ments for high-affinity binding to the SH2 domain of Grb2. J .
Med. Chem. 1999, 42, 971-980.
(11) Lee, K.; Zhang, M.; Yang, D.; Burke, T. R., J r. Design and
synthesis of a beta-amino phosphotyrosyl mimetic suitably
protected for peptide synthesis. Bioorg. Med. Chem. Lett. 2002,
12, 3399-3401.
(12) Marseigne, I.; Roques, B. P. Synthesis of new amino acids
mimicking sulfated and phosphorylated tyrosine residues. J .
Org. Chem. 1988, 53, 3621-3624.
(30) DeGrado, W. F.; Schneider, J . P.; Hamuro, Y. The twists and
turns of beta-peptides. J . Pept. Res. 1999, 54, 206-217.
(31) Mohle, K.; Gunther, R.; Thormann, M.; Sewald, N.; Hofmann,
H. J . Basic conformers in beta-peptides. Biopolymers 1999, 50,
167-184.
(32) Schumann, F.; Mueller, A.; Koksch, M.; Mueller, G.; Sewald, N.
Are â-amino acids γ-turn mimetics? Exploring a new design
principle for bioactive cyclopeptides. J . Am. Chem. Soc. 2000,
122, 12009-12010.
(33) Gademann, K.; Seebach, D. Synthesis of cyclo-beta-tripeptides
and their biological in vitro evaluation as antiproliferatives
against growth of human cancer cell lines. Helv. Chim. Acta
2001, 84, 2924-2937.
(34) Fretz, H.; Furet, P.; Schoepfer, J .; Garcia-Echeverria, C.; Gay,
B.; Rahuel, J .; Caravatti, G. Targeting a hydrophobic patch on
the surface of the Grb2-SH2 domain leads to high-affinity
phosphotyrosine-containing peptide ligands. Presented at the
15th American Peptide Symposium, Nashville, TN, J une 14-
19, 1997; Abstract P422.
(35) Schoepfer, J .; Gay, B.; End, N.; Muller, E.; Scheffel, G.; Caravatti,
G.; Furet, P. Convergent synthesis of potent peptide inhib-
itors of the Grb2-SH2 domain by palladium catalyzed coupling
of a terminal alkyne. Bioorg. Med. Chem. Lett. 2001, 11, 1201-
1203.
(36) Gao, Y.; Luo, J .; Yao, Z.-J .; Guo, R.; Zou, H.; Kelley, J .; Voigt, J .
H.; Yang, D.; Burke, T. R., J r. Inhibition of Grb2 SH2 domain
binding by non-phosphate containing ligands. 2. 4-(2-Malonyl)-
phenylalanine as a potent phosphotyrosyl mimetic. J . Med.
Chem. 2000, 43, 911-920.
(37) Gao, Y.; Voigt, J .; Wu, J . X.; Yang, D.; Burke, T. R., J r.
Macrocyclization in the design of a conformationally constrained
Grb2 SH2 domain inhibitor. Bioorg. Med. Chem. Lett. 2001, 11,
1889-1892.
(38) Shoelson, S. E. SH2 and PTB domain interactions in tyrosine
kinase signal transduction. Curr. Opin. Chem. Biol. 1997, 1,
227-234.
(13) Burke, T. R., J r.; Smyth, M. S.; Otaka, A.; Nomizu, M.; Roller,
P. P.; Wolf, G.; Case, R.; Shoelson, S. E. Nonhydrolyzable
phosphotyrosyl mimetics for the preparation of phosphatase-
resistant SH2 domain inhibitors. Biochemistry 1994, 33, 6490-
6494.
(39) Halgren, T. A. Merck molecular force field. I. Basis, form, scope,
parameterization, and performance of MMFF94. J . Comput.
Chem. 1996, 17, 490-519.
(14) Yao, Z. J .; King, C. R.; Cao, T.; Kelley, J .; Milne, G. W. A.; Voigt,
J . H.; Burke, T. R. Potent inhibition of Grb2 SH2 domain binding
by non-phosphate-containing ligands. J . Med. Chem. 1999, 42,
25-35.
(15) Furet, P.; Gay, B.; Caravatti, G.; GarciaEcheverria, C.; Rahuel,
J .; Schoepfer, J .; Fretz, H. Structure-based design and synthesis
of high affinity tripeptide ligands of the Grb2-SH2 domain. J .
Med. Chem. 1998, 41, 3442-3449.
(40) Qiu, D.; Shenkin, P. S.; Hollinger, F. P.; Still, W. C. The GB/SA
continuum model for solvation. A fast analytical method for the
calculation of approximate Born radii. J . Phys. Chem. 1997, 101,
3005-3014.
(41) Purchased from Advanced Chemtech, Louisville, KY.
(42) Purchased from Peptech Corp., Cambridge, MA.
J M030049Q