Inhibition of Grb2 SH2 domain binding by non-phosphate-containing ligands. 2. 4-(2-Malonyl)phenylalanine as a potent phosphotyrosyl mimetic

Article abstract of DOI:10.1021/jm9904248

Nonhydrolyzable phosphotyrosyl (pTyr) mimetics serve as important components of many competitive Grb2 SH2 domain inhibitors. To date, the most potent of these inhibitors have relied on phosphonate-based structures to replace the 4-phosphoryl group of the

Full text of DOI:10.1021/jm9904248

J . Med. Chem. 2000, 43, 911-920
911
In h ibition of Gr b2 SH2 Dom a in Bin d in g by Non -P h osp h a te-Con ta in in g Liga n d s.
2. 4-(2-Ma lon yl)p h en yla la n in e a s a P oten t P h osp h otyr osyl Mim etic†
Yang Gao,‡,+ J uliet Luo,§,+ Zhu-J un Yao,‡ Ribo Guo,§ Hong Zou,§ J ames Kelley,‡ J ohannes H. Voigt,‡
Dajun Yang,§,¶ and Terrence R. Burke, J r.*,‡,¶
Laboratory of Medicinal Chemistry, Division of Basic Sciences, National Cancer Institute, National Institutes of Health,
Bethesda, Maryland 20892, and Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC
Received August 20, 1999
Nonhydrolyzable phosphotyrosyl (pTyr) mimetics serve as important components of many
competitive Grb2 SH2 domain inhibitors. To date, the most potent of these inhibitors have
relied on phosphonate-based structures to replace the 4-phosphoryl group of the parent pTyr
residue. Reported herein is the design and evaluation of a new pTyr mimetic, p-malonylphen-
ylalanine (Pmf), which does not contain phosphorus yet, in Grb2 SH2 domain binding systems,
approaches the potency of phosphonate-based pTyr mimetics. When incorporated into high
affinity Grb2 SH2 domain-directed platforms, Pmf is 15-20 times more potent than the closely
related previously reported pTyr mimetic, O-malonyltyrosine (OMT). Pmf-containing inhibitors
show inhibition constants as low as 8 nM in extracellular Grb2 binding assays and in whole
cell systems, effective blockade of both endogenous Grb2 binding to cognate erbB-2, and
downstream MAP kinase activation. Evidence is provided that use of an NR-oxalyl auxiliary
enhances effectiveness of Pmf and other inhibitors in both extracellular and intracellular
contexts. As one of the most potent Grb2 SH2 domain-directed pTyr mimetics yet disclosed,
Pmf may potentially have utility in the design of new chemotherapeutics for the treatment of
various proliferative diseases, including breast cancer.
Ch a r t 1
In tr od u ction
Protein-tyrosine kinases (PTKs) serve as cornerstones
for cellular signal transduction through creation of the
phosphotyrosyl (pTyr, 1) motif (Chart 1). By enzymatic
addition of phosphate to tyrosyl residues, information
is conveyed similar to “flipping a switch” or “converting
a binary 0 to 1.” Propagation of this information is
achieved by the actions of protein tyrosine binding (PTB)
and Src homology 2 (SH2) domains, whose binding
affinities for tyrosyl-containing ligands can be reversed
from low to high by introduction of the tyrosyl phos-
phate moiety.3 SH2 domain inhibitors may potentially
afford new approaches for the treatment of a variety of
diseases, including several cancers, where SH2 domains
frequently provide critical links between growth factor
receptor PTKs and downstream Ras-proteins that have
been implicated in oncogenic processes, including the
ErbB-2 (HER-2/neu) PTK found in a large proportion
of breast cancers.4 There is a significant overexpression
of mRNA for ErbB-2 in many breast cancers, with an
associated increase in the levels of its phosphorylated
gene product, p185erbB-2. Inhibitors of Grb2 SH2
domain binding could potentially uncouple p185erbB-2
from the Ras pathway and, in so doing, attenuate its
transforming effects.5,6 The possible therapeutic utility
of Grb2 SH2 domain inhibitors has made their develop-
ment an important area of research.1,7-22
Although pTyr residues play central roles in ligand
recognition and binding by SH2 domains, their hydro-
lytic lability to cellular phosphatases limits their utility
for inhibitor design. For this reason, pTyr mimetics have
become important tools for developing SH2 domain
antagonists, with a particular emphasis on phosphate
surrogates having been dictated by the essential role
of the phosphate group in pTyr binding affinity.23,24 For
the Grb2 SH2 domain, the phosphate moiety undergoes
key interactions with the Arg 67 and Arg 86 residues,
as well as with a number of Ser side chain hydroxyls
which line the pTyr binding pocket. Additional key
† For the previous paper in this series, see refs 1 and 2.
* To whom correspondence should be addressed. Mailing address:
Building 37, Room 5C06, National Institutes of Health, Bethesda, MD
20892. Tel: (301) 496-3597. Fax: (301) 402-2275. E-mail: tburke@
helix.nih.gov.
‡ National Cancer Institute.
§ Georgetown University Medical Center.
+ Authors contributed equally to the work.
¶
Senior authorship.
10.1021/jm9904248 CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/19/2000

912 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Gao et al.
Sch em e 1
Sch em e 2
interactions occur between the Arg 67 residue and the
carbonyl group of the pY-1 residue.25-28 In light of the
importance to overall affinity of interactions within the
pTyr binding pocket, various ways of maintaining these
without the use of phosphate groups have been exam-
ined.23,24 Until recently, the most potent of these ana-
logues have employed phosphonates of phenylalanine,
in which the phosphoryl ester oxygen has been replaced
either by a methylene (Pmp, 2)29 or by a substituted
methylene (F2Pmp, 3).30 Alternative compounds based
on non-phosphorus-containing phosphate mimetics have
generally been significantly less potent than phospho-
nates. Among such latter compounds are agents which
utilize carboxyl groups to introduce the anionic oxygen
functionality of the parent phosphate. Early carboxy-
based pTyr analogues include O-(2-malonyl)tyrosine
(OMT, 4),31 whose design was predicated on mimetics
of 4,5-dideoxyshikimate-3-phosphate (9) that employed
the malonyl group as a phosphate replacement in
compounds such as 10 and 11.32 Of note was the loss of
potency incurred in 11 by removal of the 2-malonyl ether
oxygen. When incorporated as a pTyr replacement in
high affinity Grb2 SH2 domain binding sequence, OMT
exhibited a greater than 100-fold reduction in potency
relative to the parent pTyr-containing peptide.8 More
recently the isomeric bis-carboxy analogue 5 as well as
monocarboxy-based pTyr mimetics 6 and 7 have also
demonstrated significantly reduced Grb2 SH2 domain
binding potency relative to the parent pTyr-containing
ligand.1 Because carboxy-based compounds may poten-
tially afford pharmacologically interesting alternatives
to phosphonate-based pTyr mimetics, developmental
efforts have continued. Accordingly, herein is reported
a new pTyr mimetic, p-(2-malonyl)phenylalanine (Pmf,
8), which exhibits Grb2 SH2 domain binding potency
approaching that of phosphonate-based mimetics.
eomers then performed. Reference peptides were pre-
pared using 14 and racemic D,L-leucine-amide resins.
The resulting free amino-containing dipeptide amides
showed good separation of diastereomers (diastereo-
meric retention time difference of 4 min). The procedure
was then repeated using enantiomerically pure L-
leucine-amide resin. Any observed diastereomeric con-
tamination evident in this latter peptide would arise
solely from 14. Less than 3% enantiomeric impurity was
observed, indicating at least 94% ee for title compound
14. Synthesis of naphthyl-containing tripeptides 25, 28,
and 29 was accomplished using Fmoc protocols similar
to a recent report.1 Coupling of free amine-containing
16 with appropriate protected amino acid analogues,
yielded intermediates 17a -19a , which were then N-
terminally deprotected and converted to either their
N-acetyl or N-(tert-butyl oxalyl) derivatives (Scheme 2).
Removal of carboxylic tert-butyl protection (TFA) yielded
the desired final compounds 25, 28, and 29. In a similar
fashion, 5-methylindolyl-containing compounds 30 and
31 were prepared starting from asparagine amide 2019
following conversion to dipeptide 21. All final products
were purified by HPLC (Scheme 3).
Syn th esis
All pTyr mimetics were utilized in the present study
as their NR-Fmoc derivatives, with orthogonal tert-butyl
protection of hydroxyl-bearing side chain functionality.
Except as indicated below, synthesis of these amino acid
analogues have already been reported,1,31,33-35 including
that of the title compound, NR-Fmoc 4-(bis((tert-butyl)-
oxycarbonyl)methyl)-L-phenylalanine.36 As shown in
Scheme 1, fluoro-Pmf analogue 15 was prepared by
electrophilic fluorination of iminolactone 1236 (NaH at
room temperature followed by N-fluorobenzenesulfon-
imide),37 followed by hydrogenolytic deprotection of the
resulting R-fluoro 13 to give free amino acid 14, which
was then treated with Fmoc-OSu/Na2CO3. To determine
the enantiomeric purity of final product 14, a leucine
amide dipeptide was prepared by solid-phase tech-
niques, with HPLC separation of any resulting diaster-
Resu lts a n d Discu ssion
In h ibition of Gr b2 SH2 Dom a in Bin d in g in Ex-
tr a cellu la r ELISA Assa ys. For Grb2 SH2 domain
inhibitors, the importance to overall affinity of interac-
tions within the pTyr binding pocket has resulted in
efforts to maintain high affinity without reliance on
hydrolytically labile phosphate functionality. Until re-
cently, the most potent Grb2 SH2 domain-directed pTyr
mimetics have relied on phosphonate-based structures
such as Pmp (2),1,7,19 with dicarboxy-based pTyr mimet-
ics such as O-malonyltyrosine (OMT, 4)8 and the

Inhibition of Grb2 SH2 Domain Binding
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 913
Ta ble 1. Inhibition of Grb2 SH2 Domain Bindinga
Sch em e 3
isomeric 538 exhibiting much less affinity. In the present
study, different arrangements of carboxyl groups were
appended onto phenylalanyl residues in order to main-
tain anionic interactions with SH2 domain Arg67 and
Arg86 residues, similar to those exhibited by the
OdP(O-)2 groups of either pTyr or Pmp. Using a
previously reported high affinity â-bend mimicking Grb2
SH2 domain binding platform,14 we have examined the
Grb2 SH2 domain binding potency of a series of pTyr
mimetics. Each analogue was prepared in both its NR-
acetyl and NR-oxalyl forms, predicated on our previous
observations that the N-oxalyl derivatives typically
exhibit greater binding potencies than their NR-acetyl
counterparts.1,39 In the present study, NR-oxalyl Pmp
analogue 24b (IC50 ) 50 nM; Table 1) served as a
reference, since Pmp is one of the most potent Grb2 SH2
domain-directed phosphorus-containing pTyr mimetics
reported to date. Using an ELISA-based extracellular
binding assay, the OMT residue (4) resulted in an
approximate 20-fold loss of potency (compound 25b ;
IC50 ) 1.1 µM) relative to the Pmp. Molecular dynamics
simulations comparing the Grb2 SH2 domain binding
of OMT and Pmp residues indicated that the OMT
malonyl group extends farther out from the aryl ring
than the phosphonate group which it mimics, resulting
in a degree of misalignment (Figure 1a).
In contrast to the over-extension of carboxyls observed
with OMT, a recent X-ray structure of the monocarboxy-
based pTyr mimetic, carboxymethylphenylalanine (Cmf,
7) bound to the p56lck SH2 domain, indicated good
alignment of the carboxyl and phosphoryl oxygens while
maintaining phenyl ring registry with the parent pTyr
residue.40 Indeed, molecular modeling dynamics simula-
tions of the Cmf residue bound within the Grb2 SH2
domain similarly showed good registry of carboxyl
oxygens as well as maintenance of phenyl ring registry
when compared to Pmp.39 Alternatively, in a fashion
similar to that seen with OMT, the carboxymethyl-
tyrosyl residue (Cmt, 6) showed misalignment due to
over-extension caused by the added carboxymethyl ether
a
Data were obtained using a Grb2 SH2 domain GST fusion
protein in an ELISA assay as described in the Experimental
Section. IC50 values were determined from serial dilutions of
inhibitor with each concentration done in triplicate. Numbers in
parentheses indicate the total number of ELISA experiments.
b
Plasmon resonance-based IC50 values have been reported previ-
ously (ref 1). c Single determinations were performed since results
are similar to previously reported plasmon resonance-based IC50
values (ref 16).
oxygen (Figure 1b). In agreement with this modeling
data, ELISA data showed that Cmt-containing analogue
26b (IC50 ) 19 µM) was approximately 20-fold less
potent than the corresponding Cmf-containing 27b
(IC50 ) 1 µM).39
Improvements in both predicted binding (Figure 1)
and actual binding (Table 1) of Cmf (7) relative to Cmt
(6) residues, induced by deletion of the bridging ether
oxygen, led us to examine a similar modification of the
OMT residue (4). This resulted in the new pTyr mimetic,
p-malonylphenylalanine Pmf (8), which upon subjection
to molecular modeling dynamics ligated to the Grb2 SH2
domain, compared favorably with a Pmp residue (Figure
1c). Subsequent ELISA binding analysis showed Pmf-
containing 28b (IC50 ) 70 nM) to be approximately
equipotent to Pmp (Table 1). These results indicated
that removing the ether oxygen from OMT to give Pmf
provided an approximate 15-fold increase in binding
potency, resulting in one of the most potent non-
phosphorus-containing pTyr mimetics yet reported for
Grb2 SH2 domain binding. This finding is somewhat

914 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Gao et al.
F igu r e 2. Effects of inhibitors on interaction of Grb2 with
tyrosine phosphorylated p185 erbB-2 in human breast cancer
cells MDA-MB-453 as described in the Experimental Section.
MDA-MB-453 cells were plated in 100 mm dishes cultured in
IME with 10% FBS medium overnight. Cells were treated with
inhibitors (25 µM) for 3 h, washed, and lysed and 500 µg of
protein immunoprecipitate was treated with Grb2 antibody.
Co-immunoprecipitated pTyr-containing p185 erbB-2 was
detected using pTyr antibody (Py99) and immunoblotting.
Western blotting with Grb2 MAb was done as a control.
isolated Grb2 SH2 domain constructs and short pTyr-
containing peptides. This type of data is a useful
indicator of protein-inhibitor interaction. However, in
physiological contexts, binding interactions occur be-
tween full Grb2 protein (which consists of an SH2
domain and two SH3 domains41) and phosphorylated
proteins, which include Shc and erbB-2 growth factor
receptor cytoplasmic domains. Therefore, effective in-
hibition by synthetic ligands in whole cell environments
differs from extracellular ELISA assays in two ways:
(1) In cellular systems ligands must cross cell mem-
branes prior to interacting with targets, and (2) both
the Grb2 SH2 domain host and cognate phosphorylated
erbB-2 proteins, with which the inhibitors must com-
pete, are much larger and more complex in the cellular
system than in the ELISA assay. To obtain a more
relevant measure of the inhibitory potency of synthetic
ligands, binding experiments were therefore conducted
in whole cells in which inhibition of binding of full
length Grb2 to native erbB-2 was measured following
treatment of cells with inhibitors. Shown in Figure 2
are results from these assays in which it is seen that
inhibition of cognate Grb2 SH2 domain binding was
observed with potencies varying roughly in the same
rank order as that observed in the ELISA assays. Of
note was the apparent ability of the NR-oxalyl group to
enhance cellular potency despite the added negative
charge which it imparts. Although the NR-oxalyl group
enhances binding potency in extracellular ELISA as-
says, it would have been expected in cellular assays that
the added charge could potentially hinder cell mem-
brane penetration, thereby offsetting binding enhance-
ment.
F igu r e 1. Comparison of the Grb2 SH2 domain ligand
binding modes based on molecular modeling as described in
the Experimental Section. Protein backbone atoms are super-
imposed. Heavy atoms of both ligands and one set of interact-
ing side chains are shown in each panel. Dashed lines in ligand
color, or white for ligands colored by atom type, show hydrogen
bonds: (a) 24b and 25b (red); (b) 26b and 27b (magenta); (c)
24b and 28b (yellow).
counter to earlier uses of malonyl-based phosphate
mimetics in 4,5-dideoxyshikimate-3-phosphate ana-
logues, where removal of the ether oxygen from com-
pound 10 significantly reduced potency (compound 11).32
To potentially compensate for the loss of hydrogen
bonding functionality incurred by loss of the ether
oxygen in Pmf, fluorine was introduced at the malonyl
R-methylene in a modification similar to the previously
reported synthesis of fluoromalonyltyrosine (FOMT).37
This resulted in a reduction in potency (compound 29b;
IC50 ) 170 nM), as was observed when carboxydifluo-
romethyl phenylalanine was prepared by addition of
fluorines to Cmf.34,39
In tr a cellu la r In h ibition of MAP Kin a se Activa -
tion . MAP kinase activation is a downstream event
associated with erbB-2/Grb2 signaling through the Ras
pathway, occurring distal to the Grb2-dependent activa-
tion of membrane-bound Ras. Therefore, measurement
of inhibition of MAP kinase activation by exogenously
administered Grb2 SH2 domain antagonists indicates
the cumulative effectiveness of these agents in discon-
necting the Ras pathway from erbB-2 signaling. In the
present study, inhibition of MAP kinase was measured
by MAP kinase specific antibody in MDA-MB-453 cells
treated with growth factor heregulin (HRG). Experi-
Wh ole Cell Assa ys: In tr a cellu la r In h ibition of
Gr b2 SH2 Dom a in Bin d in g. ELISA data present in
Table 1 and discussed above are from extracellular
assays which measure inhibition of binding between

Inhibition of Grb2 SH2 Domain Binding
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 915
Ta ble 2. Inhibition of Grb2 SH2 Domain Bindinga
F igu r e 3. Effects of Grb2 inhibitors on activation of MAP
kinase in human breast cancer cells MDA-MB-453 as described
in the Experimental Section.
ments were repeated at least two times for all com-
pounds, with representative results from active ana-
logues shown in Figure 3. Inhibition of MAP kinase
activation was observed in response to extracellular
administration of Grb2 antagonists in a fashion ap-
proximating that seen both for extracellular ELISA
binding (Table 1) and intracellular binding of Grb2 to
p185erbB-2 (Figure 2). Of note was the general ability
of NR-oxalyl derivatives to elicit significantly greater
inhibition than their NR-acetyl counterparts, with Pmf-
containing 28b reducing MAP kinase activation to near
basal levels when administered extracellularly at a
concentration of 25 µM.
a
Data were obtained using a Grb2 SH2 domain GST fusion
protein in an ELISA assay as described in the Experimental
Section. IC50 values were determined from serial dilutions of
inhibitor with each concentration done in triplicate. Numbers in
parentheses indicate the total number of ELISA experiments.
hibitor potencies on the order of those observed using
the phosphorus-containing pTyr mimetic, phosphono-
methyl phenylalanine (Pmp). Both Pmp- and Pmf-
containing inhibitors show potent inhibition in extra-
cellular Grb2 binding assays, as well as in cell-based
assays which measure endogenous Grb2 binding to
erbB-2, MAP kinase activation. Evidence is also pro-
vided that use of an N-oxalyl auxiliary enhances ef-
fectiveness of inhibitors in both extracellular and in-
tracellular contexts. Because cell membrane penetration
of charged SH2 domain inhibitors has traditionally been
a limiting factor in whole cell or animal assays, the
favorable results in cell-based assays of Pmf-containing
compounds such as 28b may render these analogues of
particular value. Such agents may have utility as
potential chemotherapeutics for the treatment of various
proliferative diseases, including breast cancer.
5-Meth ylin d olyl An a logu es. The focus of our efforts
on the development of pTyr mimetics which can bind
with high affinity in the Grb2 SH2 domain pTyr binding
pocket has utilized the naphthyl-containing â-bend
mimicking dipeptide construct 16 as a display platform
previously disclosed by Furet et al. to exhibit high Grb2
SH2 domain affinity when N-terminally coupled to NR-
acetyl pTyr.14 More recently it has been shown that
replacement of the naphthyl portion of 16 by a 5-meth-
ylindolyl moiety provides a significant enhancement in
Grb2 SH2 domain binding potency.19 Using the 5-meth-
ylindolyl construct, the title pTyr mimetic Pmf was
therefore examined as its NR-acetyl and NR-oxalyl
construct (compounds 31a and 31b, respectively) and
compared to the corresponding Pmp analogues 30a and
30b. As shown in Table 2, in extracellular ELISA
binding assays NR-acetyl 30a (IC50 ) 4 nM; previously
Exp er im en ta l Section
reported as IC50 ) 0.4 nM19) and NR-oxalyl 30b (IC50
)
Molecu la r Mod elin g. All simulations were performed with
the Insight II 97.0/Discover 3.0 modeling package,42 using the
cff91 force field, which was modified to take into account the
planarity of the oxalylamido group. A constant dielectric
constant of 1.00 and the cell multipole method with fine
accuracy was used for all nonbonding interactions except from
the QMD simulations. The crystal structure of K-P-F-pY-V-
N-V-NH2 bound to the Grb2 SH2 domain25 was used as the
starting geometry. The structure of the heptapeptide was
modified to yield 24b within the receptor. The naphthyl ring
was positioned into the hydrophobic pocket comprised by Lys
109, Leu 111, and Phe 119. All receptor and ligand atoms but
the hydroxy hydrogens of Ser 88, 90, and 96, the oxalyl group,
and the modified pTyr residue were held fixed during 14
quenched molecular dynamics simulations (QMD, NVT en-
semble, 60 ps, different random seed for each simulation) at
800 K. The coordinates were saved every 200 fs and minimized
by 300 steps of the Polak-Ribiere conjugated gradient algo-
rithm (CG-PR). During the MD simulation the contribution
of the vdw interactions was scaled to 2% and that of the
Coulombic interactions to 20% in order to sample the complete
conformational space. For each of the 14 runs the conformation
2 nM) were substantially more potent than their cor-
responding naphthyl-containing homologues 24a and
24b (Table 1). Pmf-containing analogues 31a and 31b
(IC50 ) 12 nM and 8 nM, respectively) also showed
potencies in the same range, although slightly reduced.
In whole cell assays following extracellular administra-
tion of inhibitor, NR-oxalyl 31b was the most potent of
the indolyl analogues in both inhibiting Grb2 association
with phosphorylated ebrB-2 growth factor receptor
(Figure 2) and in downstream MAP kinase activation
(Figure 3).
Con clu sion s
Reported herein is the design and evaluation of a new
pTyr mimetic, p-malonyl phenylalanine (Pmf), which in
a Grb2 SH2 domain binding system is 15-20 times
more potent than the previously reported O-malonyl
tyrosine (OMT). Incorporation of Pmf into high affinity
Grb2 SH2 domain-directed platforms can provide in-

916 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Gao et al.
with the lowest energy was stored. Comparison of the resulting
14 structures showed convergence. The converged structure
served as the starting geometry for 300-step CG-PR minimiza-
tion followed by seven 50 ps MD (NVT-ensemble) simulations
at 298 K with seven different random seed numbers, where
the complex was solvated by a sphere of water with a radius
of 20 Å, centered around the nitrogen of Lys 109. During
minimizations and simulations, all atoms were held fixed
except the ligand, the water molecules in a radius of 16 Å
around the nitrogen of Lys 109, and the side chains of the Grb2
SH2 domain within 6 Å around the initial conformation of the
heptapeptide of the X-ray structure. The coordinates were
saved every 1 ps and minimized by 300 steps of CG-PR. The
frame with lowest energy of the 350 obtained solvated struc-
tures was determined and is depicted in Figure 1a. The same
QMD procedure and solvated MD simulations were repeated
for 25b, 26b, 27b, and 28b (parts a, b, and c of Figure 1,
respectively).
specific antibody, e.g., phospho-p44/42 MAP kinase antibody
(New England BioLabs) and visualized with ECL (Amersham,
Arlington Heights, IL). To evaluate the equal loading of the
proteins, blots were subsequently stripped and reprobed with
a monoclonal antibody recognizing the total Grb2 protein or
Hsp-70 protein.
Syn th esis. Gen er al Syn th etic Meth ods. Elemental analy-
ses were obtained from Atlantic Microlab Inc., Norcross, GA,
and fast atom bombardment mass spectra (FABMS) were
acquired with a VG Analytical 7070E mass spectrometer under
the control of a VG 2035 data system. Where indicated,
FABMS matrixes used were glycerol (Gly) or nitrobenzoic acid
(NBA). 1H NMR data were obtained on Bruker AC250 (250
MHz) and are reported in ppm relative to TMS and referenced
to the solvent in which they were run. Solvent was removed
by rotary evaporation under reduced pressure, and silica gel
chromatography was performed using Merck silica gel 60 with
a particle size of 40-63 µm. Anhydrous solvents were obtained
commercially and used without further drying. HPLCs were
conducted using a Waters Prep LC4000 system having pho-
todiode array detection and binary solvent systems as indi-
cated where A ) 0.1% aqueous TFA and B ) 0.1% TFA in
acetonitrile and either Vydac C18 (10 µm) Peptide & Protein
or Advantage C18 (5 µ) columns (preparative size, 20 mm
diameter × 250 mm long with a flow rate of 10 mL/min;
semipreparative size, 10 mm diameter × 250 mm long, with
a flow rate of 2 mL/min).
Biologica l. Cells a n d Cell Cu ltu r e. Cell lines were
obtained from the American Type Culture Collection (Rock-
ville, MD) and Lombardi Cancer Center, Georgetown Univer-
sity Medical Center. Cells were routinely maintained in
improved minimal essential medium (IMEM, Biofluids, Rock-
ville, MD) with 10% fetal bovine serum. Cultures were
maintained in a humidified incubator at 37 °C and 5% CO2.
A. In h ibition of Gr b2 SH2 Dom a in Bin d in g Usin g
ELISA Tech n iqu es. A biotinated phosphopeptide encompass-
ing a Grb2 SH2 domain binding sequence derived from SHC
protein, was bound at 20 ng/mL to 96-well plates overnight.
Nonspecific interactions were inhibited by 5% bovine serum
albumin containing TBS. Samples of recombinant purified
Grb2 SH2-GST fusion protein were preincubated with serial
dilutions of inhibitors, then added into each well. After
extensive washing with 0.1% bovine serum albumin in TBS,
bound Grb2 SH2 domain was detected using anti-GST anti-
bodies and goat anti-mouse antibody conjugated to alkaline
phosphatase. Quantitation of bound alkaline phosphatase was
achieved by a colorimetric reaction employing p-nitrophenyl
phosphate as substrate.
Ben zyl (3S,5S,6R)-3-[4-(Bis((ter t-bu tyl)oxyca r bon yl)-
flu or om et h yl)p h en ylm et h yl]-(-)-6-oxo-5,6-d ip h en yl-4-
m or p h olin e-ca r boxyla te (13). To the suspension of 60%
NaH in oil (150 mg, 3.75 mmol) in anhydrous THF (30 mL)
was added a solution of benzyl (3S,5S,6R)-3-[4-(bis((tert-butyl)-
oxycarbonyl)methyl)phenylmethyl]-(-)-6-oxo-5,6-diphenyl-4-
morpholine-carboxylate36 (2.075 g, 3.0 mmol) in anhydrous
THF (30 mL) at 0 °C, and then the resulting mixture was
stirred at room temperature (1 h). The solution was cooled to
0 °C, and benzenesulfonamino fluoride (950 mg, 3.0 mmol) in
anhydrous THF (20 mL) was added dropwise. The reaction
mixture was stirred (0 °C, 2.5 h), then quenched with saturated
aqueous ammonium chloride (50 mL), subjected to an extrac-
tive workup (EtOAc), dried (Na2SO4), and taken to dryness.
Purification by silica gel flash chromatography (EtOAc-
hexanes, from 1:10 to 1:6) provided 13 as a white solid (1.96
B. In h ibition of Gr b2 SH2 Dom a in Bin d in g in Wh ole
Cells. ErbB2 overexpressing breast cancer cells, MDA-MB-
453, were treated with inhibitors (25 µM) for 3 h in serum-
free IMEM medium (Gibco). Cells were washed twice with PBS
to remove inhibitor, then cell lysates were prepared using 1%
Triton X-100 in PBS containing 0.2 mM NaVO4. Grb2 and
associated Grb2-binding proteins were immunoprecipitated
from each lysate (500 µg) with anti-Grb2 antibodies and
collected using protein A Sepharose. Immunoprecipitated
proteins were separated by SDS-PAGE on 8-16% gradient
gels (Novex), and pTyr-containing proteins were detected by
Western blotting using anti-phosphotyrosine antibodies (Up-
state Biochemicals Inc.). Previous experiments have shown
that a major tyrosine phosphorylated protein in these cells is
the p185 erbB-2, which is overexpressed as a consequence of
gene amplification. Western blotting with Grb2 MAb was done
as a control.
1
g, 92%): H NMR (CDCl3; two conformers were observed in a
ratio of 4:9 at 23 °C) major conformer δ 7.60 (2H, d, J ) 8.1
Hz), 7.41∼7.00 (12 H, m), 6.81 (1H, d, J ) 7.3), 6.56 (2H, m),
6.48 (2H, d, J ) 7.3 Hz), 5.35 (1H, dd, J ) 2.4, 6.10 Hz,
-OOCCH-N), 5.08 (1H, d, J ) 12.2 Hz, OCH2Ph), 4.99 (1H, d,
J ) 12.7 Hz, OCH2Ph), 4.80 (1H, d, J ) 2.93 Hz, -PhCHOOC),
4.15 (1H, d, J ) 2.93 Hz, -PhCHN-), 3.57 (1H, dd, J ) 6.4,
13.7 Hz, -CH2-CHNCOO), 3.42 (1H, dd, J ) 2.4, 13.4 Hz, -CH2-
CHNCOO), 1.52 (9H, s), 1.41 (9H, s), minor confomer δ 7.54
(2H, d, J ) 8.1 Hz), 7.41∼7.00 (13 H, m, overlapping),
6.56∼6.64 (4H, m, overlapping), 5.32 (1H, d, J ) 12.2 Hz,
OCH2Ph), 5.26 (1H, dd, J ) 2.9, 6.4 Hz, -OOCCH-N), 5.11 (1H,
d, J ) 12.0 Hz, OCH2Ph), 4.97 (1H, overlapping, -PhCHOOC),
4.36 (1H, d, J ) 2.7 Hz, -PhCHN-), 3.57 (1H, dd, J ) 6.8, 13.7
Hz, -CH2-CHNCOO), 3.37 (1H, dd, J ) 2.9, 13.7 Hz, -CH2-
CHNCOO), 1.52 (9H, s, overlapping), 1.43 (9H, s); FABMS
C. In h ibition of MAP K Activa tion in Wh ole Cells. The
state of threonine and tyrosine phosphorylation of cellular
MAP kinase (MAPK) was determined using a polyclonal
antibody specifically recognizing the phosphorylated threonine
and tyrosine residues of MAPK. Briefly, MDA-MB-453 cells
were plated in 12-well plates and cultured in serum-free
medium overnight. Cells were treated with 25 µM of various
compounds for 4 h and then followed by addition of 10 nM of
Heregulin stimulation. Cells were then washed twice with ice-
cold PBS and lysed in 1 mL of lysis buffer (50 mM Tris-HCl,
pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 5 mM
EDTA, 5 mM EGTA, 1 mM PMSF, 50 µg/mL approtinin, 50
µg/mL leupeptin, and 2 mM sodium orthovanadate). A protein
concentration was determined by BCA method (Pierce, Rock-
ford, IL). Protein (50 µg) was subjected to 8-20% SDS-PAGE
gel (Novex, San Diego, CA) and transferred to nitrocellulose
membrane. Activation of MAP kinase was detected with a
(+VE, NBA) m/z 598.8 [MH+ - 2 C4H8], 554.8 [MH+
-
-
2C4H8 - CO2], 510.7 [MH+ - 2C4H8 - 2CO2]. Anal. (C42H44
FNO8) C, H, N.
Nr-Fm oc-4-(bis((ter t-bu tyl)oxyca r bon yl)flu or om eth yl)-
L-p h en yla la n in e (15). A solution of 13 (1.76 g, 2.49 mmol)
in THF-EtOH (1:1, 20 mL), with 2 drops of AcOH added to
promote reaction, was hydrogenated over 150 mg of Pd black
(45 psi H2, room temperature, 5 h). Catalyst was removed by
filtration, and combined organics were concentrated to give
crude free amino acid 14 as a white sticky solid. A mixture of
crude 14, Fmoc-OSu (881 mg, 2.49 mmol), and Na2CO3 (791
mg, 74.5 mmol) in 35 mL of dioxane-water (1:1) was stirred
at room temperature (overnight), then cooled to 0 °C, acidified
with 0.2 M HCl (120 mL), and subjected to an extractive

Inhibition of Grb2 SH2 Domain Binding
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 917
workup (EtOAc). Removal of solvent and purification by silica
gel chromatography (CHCl3-EtOAc-MeOH) to provided prod-
uct 15 as a white solid (1.28 g, 85% from 13): 1H NMR (DMSO)
δ 12.70 (1H, br s), 7.87 (2H, d, J ) 7.3 Hz), 7.79 (1H, d, J )
8.6 Hz), 7.65 (2H, t, J ) 7.3 Hz), 7.44∼7.23 (8H, m), 4.22∼4.10
(4H, m), 3.12 (1H, dd, J ) 3.7, 13.4 Hz), 2.90 (1H, dd, J )
10.7, 13.7 Hz), 1.42 (9H, s), 1.42 (9H, s); FABMS (+VE, NBA)
m/z 618 [M - H]. HR-FABMS calcd for C35H37FNO8 (M - H)
m/z, 618.2503; found, 618.2484.
anhydrous acetonitrile or DMF (3 mL) and treated overnight
with N-acetylimidazole (55 mg, 0.5 mmol). Solvent was
removed, and residues were purified by silica gel chromatog-
raphy (CHCl3-EtOAc-MeOH) to provide products as white
foams.
Com p ou n d 17c (99%): 1H NMR (CDCl3) δ 8.28∼8.02 (2H,
m), 7.90∼7.45 (3H, m), 7.90∼7.45 (3H, m), 7.64∼7.34 (4H, m),
7.10 (2H, d, J ) 8.6 Hz), 6.89 (2H, d, J ) 8.6 Hz), 6.57 (1H,
brd, J ) 6.8 Hz), 6.49 (1H, brs), 5.76 (1H, brs), 5.02 (1H, s),
4.84∼4.72 (1H, m), 4.68∼4.58 (1H, m), 3.48∼3.36 (2H, m),
3.20∼2.84 (5H, m), 2.65 (1H, dd, J ) 8, 15 Hz), 2.15∼1.15 (12H,
m), 1.58 (18H, s); FABMS (+VE, NBA) m/z 844 [MH+].
Com p ou n d 18c (94% yield): 1H NMR (CDCl3) δ 8.03 (2H,
m), 7.81 (1H, m), 7.63 (3H, m), 7.46∼7.05 (9H, m), 6.65 (2H,
m), 4.67 (2H, m), 4.40 (1H, s), 3.34 (2H, m), 3.12∼2.89 (5 H,
m), 2.55 (1H, dd, J ) 4.60, 15.1 Hz), 1.81 (3H, s), 2.04∼1.15
(12H, m), 1.46 (18H, s); FABMS (+VE, NBA) m/z 828 [MH+],
772 [MH+ - C4H8].
Deter m in a tion of En a n tiom er ic P u r ity of 15. Enantio-
meric purity of 15 was determined using previously reported
procedures.34 L-Leu-Rink amide resin (12.5 mg, 5 µmol based
on 0.4 mmol/g; Bachem Corp., Torrance, CA) was washed well
with several 1 mL portions of N-methyl-2-pyrrolidinone (NMP),
and Fmoc amino protection was removed by treatment with
20% piperidine in NMP (0.5 mL, 1 min; then 0.5 mL, 20 min).
The deblocked resin was washed well with NMP (10 × 1 mL),
then coupled overnight with a solution of active ester formed
by reacting 15 (12.5 µmol), 1-hydroxybenzotriazole (HOBt), and
1,3-diisopropyl-carbodiimide (DIPCDI) in NMP (1.0 mL, 10
min). The resin was washed with NMP (10 × 1 mL), and the
N-terminal Fmoc protection was removed by treatment with
20% piperidine in NMP (0.5 mL, 5 min). The deblocked resin
was washed with NMP (10 × 1 mL) and dichloromethane
(10 × 2 mL), then dipeptide was cleaved using a mixture of
TFA (1.85 mL), H2O (100 mL), and triethylsilane (50 µL) (1
h), and the resulting dipeptide was analyzed using a semi-
preparative Vydac column with linear gradient from 5% B to
30% B over 25 min). An identical procedure repeated using
d,L-Leu-Rink amide resin, provided a reference mixture of
diastereomeric D,L-leucine dipeptides, which gave HPLC re-
tention times for diastereomeric peaks at 16.61 min and 20.64
min. On this basis, diastereomeric contamination in the
L-leucine dipeptide accounted for area less than 3% of that
observed for the major diastereomer, indicating that greater
than 97% of a single enantiomer was present in 15.
Com p ou n d 19c (94% yield): 1H NMR (CDCl3) δ 8.05 (2H,
m), 7.80 (1H, m), 7.66 (2H, m), 7.55∼7.05 (9H, m), 6.62 (1H,
brs), 6.37 (1H, d, J ) 6.10 Hz), 5.83 (1H, s), 4.69 (2H, m), 3.34
(2H, m), 3.15∼2.85 (5H, m), 2.55 (1H, dd, J ) 4.15, 15.14 Hz),
2.10∼0.83 (12H, m), 1.81 (3H, s), 1.50 (18H, s); FABMS (+VE,
NBA) m/z 846 [MH+].
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
17d -19d . To solutions of tripeptides 17a -19a (0.05 mmol)
in anhydrous acetonitrile (2 mL) was added piperidine (40 µL,
0.4 mmol), and the solutions were stirred at room temperature
(3 h). Solvent and excess piperidine were removed under high
vacuum, crude free amines 17b-19b were dissolved in anhy-
drous DMF (2 mL), and diisopropyl ethylamine (22 µL, 0.125
mmol) was added followed by tert-butyl oxalyl chloride (16 µL,
0.125 mmol). The reaction mixtures were stirred at room
temperature (overnight), and then the mixtures were concen-
trated and purified by silica gel chromatography (CHCl3-
EtOAc-MeOH) to provide products as white foams.
Com p ou n d 17d (94%): 1H NMR (CDCl3) δ 8.18∼8.09 (1H,
m), 8.04 (1H, d, J ) 8.1 Hz), 7.95∼7.90 (1H, m), 7.85∼7.66
(2H, m), 7.60∼7.48 (2H, m), 7.44 (2H, d, J ) 4.7 Hz), 7.23 (2H,
d, J ) 8.5 Hz), 6.95 (2H, d, J ) 8.5 Hz), 6.49 (1H, brs), 6.17
(1H, brs), 5.59 (1H, brs), 5.02 (1H, s), 4.84∼4.72 (1H, m),
4.68∼4.58 (1H, m), 3.48∼3.36 (2H, m), 3.25∼2.96 (5H, m), 2.58
(1H, dd, J ) 5.1, 15.0 Hz), 2.05∼0.97 (12H, m), 2.15∼1.15 (18H,
s), 1.56 (9H, s); FABMS (+VE, NBA) m/z 930.6 [MH+].
Com p ou n d 18d (50% yield): 1H NMR (CDCl3) δ 8.02 (2H,
m), 7.83∼7.21 (13H, m), 6.69 (1H, s), 6.38 (1H, s), 4.67 (2H,
m), 4.40 (1H, s), 3.43∼2.88 (7 H, m), 2.56 (1H, dd, J ) 4.9,
14.9 Hz), 2.05∼0.94 (12H, m), 1.46 (18H, s), 1.46 (9H, s);
FABMS (+VE, NBA) m/z 914 [MH+].
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
17a -19a . To the solution of 161 (46 mg, 0.1 mmol) in
anhydrous DMF (2 mL) was added an active ester solution
formed by reacting NR-Fmoc/O-(tert-butyl)-protected pTyr mi-
metics OMT,31 Pmf,36 or 15 (0.1 mmol), HOBt‚H2O (14 mg, 0.1
mmol), and DIPCDI (16 µL, 0.1 mmol) in anhydrous DMF (2
mL) at room temperature (10 min). The resulting reaction
mixtures were stirred at room temperature (overnight), then
solvent was removed under high vacuum, and residues were
purified by silica gel chromatography (CHCl3:EtOAc:MeOH)
to provide products as white foams.
Com p ou n d 17a (68% yield): 1H NMR (CDCl3) δ 8.14∼8.02
(2H, m), 7.92∼7.64 (6H, m), 7.58∼7.30 (9H, m), 7.08 (2H, d,
J ) 8.1 Hz), 6.91 (2H, d, J ) 8.1 Hz), 6.88 (1H, brs), 6.43 (1H,
brs), 5.59 (1H, brs), 5.43 (1H, d, J ) 6.8 Hz), 5.01 (1H, s),
4.83∼4.70 (1H, m), 4.25∼4.16 (2H, m), 3.48∼3.32 2H, m),
3.18∼2.85 (5H, m), 2.64 (1H, dd, J ) 8.1,15.4 Hz), 2.15∼1.15
(12H, m), 1.58 (18H, s); FABMS (+VE, NBA) m/z 1024.9
[MH+].
Com p ou n d 18a (100% yield): 1H NMR (CDCl3) δ 8.01 (2H,
m), 7.81∼7.71 (6H, m), 7.50∼6.95 (13H, m), 6.58 (1H, s), 5.56
(3H, m), 4.69 (1H, m), 4.55∼4.40 (1H, m), 4.40 (1H, s), 4.31
(2H, d, J ) 6.84 Hz), 4.09 (1H, m), 3.34 (1H, m), 3.12∼2.88 (5
H, m), 2.63 (1H, dd, J ) 4.4, 15.1 Hz), 2.05∼1.11 (12H, m),
1.46 (18H, s); FABMS (+VE, NBA) m/z 1008 [MH+].
Com p ou n d 19a (97% yield): 1H NMR (CDCl3) δ 8.30∼8.14
(1H, s, br), 8.07∼8.00 (2H, m), 7.83∼7.64 (5H, m), 7.55∼7.10
(14H, m), 6.85∼6.76 (1H, s, br), 5.70∼5.60 (1H, s, br), 5.40 (1H,
m), 4.68 (1H, m), 4.55∼4.30 (3H, m), 4.14 (1H, t, J ) 6.6 Hz),
3.38 (2H, m), 3.15∼2.62 (6H, m), 2.10∼1.00 (12H, m), 1.50
(18H, s); FABMS (+VE, NBA) m/z 1026 [MH+].
Com p ou n d 19d (90% yield): 1H NMR (CDCl3) δ 8.06 (2H,
m), 7.85∼7.24 (12H, m), 6.80 (1H, s), 6.57 (1H, s), 5.66 (1H,
s), 4.75∼4.60 (2H, m), 3.34 (2H, m), 3.18∼2.95 (5H, m), 2.53
(1H, dd, J ) 4.6, 14.9 Hz), 2.05∼0.97 (12H, m), 1.50 (18H, s),
1.47 (9H, s); FABMS (+VE, NBA) m/z 932.8 [MH+].
Gen er a l P r oced u r e for th e Con ver sion of P r otected
Der iva tives 17c-19c a n d 17d -19d to F in a l P r od u cts 25a ,
28a , 29a a n d 25b, 28b, 29b, Resp ectively. Protected ana-
logues 17c-19c and 17d -19d (0.05∼0.1 mmol) were treated
(1 h) with solutions of TFA (1.90 mL):H2O (0.10 mL):trieth-
ylsilane (0.05 mL), taken to dryness, and purified by HPLC to
yield products as white solids.
Com p ou n d 25a . Purification by preparative HPLC (Ad-
vantage column; linear gradient from 10% B to 90% B over 20
min; retention time, 15.6 min) yielded product as a white
powder (62% yield): 1H NMR (DMSO-d6) δ 8.28∼8.21 (2H,
brs), 8.17∼8.10 (1H, m), 8.04∼7.92 (2H, m), 7.79 (1H, t, J )
4.2 Hz), 7.58∼7.50 (2H, m), 7.46∼7.38 (2H, m), 7.20 (2H, d,
J ) 8.6 Hz), 6.93 (1H, brs), 6.87 (2H, d, J ) 8.6 Hz), 5.34 (1H,
s), 4.96∼4.58 (1H, m), 4.45∼4.35 (1H, m), 3.28∼2.98 (5H, m),
2.85∼2.60 (2H, m), 2.05∼1.10 (12H, m), 1.80 (3H, s); semi-
preparative HPLC retention time: 21.6 min (Vydac column;
linear gradient from 10% B to 90% B over 20 min); FABMS
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
17c-19c. To solutions of tripeptides 17a -19a (0.05 mmol) in
anhydrous acetonitrile (2 mL) was added piperidine (40 µL,
0.4 mmol), and the solutions were stirred at room temperature
(3 h). Solvent and excess piperidine were removed under high
vacuum, and crude free amines 17b-19b were dissolved in
(-VE, Gly), m/z 730 [M - H]. HR-FABMS calcd for C37H44
FN5O8 [M - H - CO2] m/z, 686.3190; found, 686.3182.
-

918 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Gao et al.
Com p ou n d 28a . Purification by preparative HPLC (Ad-
vantage column; linear gradient from 5% B to 50% B over 15
min; retention time, 19.2 min) yielded product as a white
powder (73% yield): 1H NMR (DMSO-d6) δ 8.25 (2H, m), 8.08
(1H, m), 7.99 (1H, m), 7.90 (1H, m), 7.74 (1H, m), 7.51∼7.18
(10H, m), 6.90 (1H, s), 4.65 (1H, m), 4.60 (1H, s), 4.36 (1H, m),
3.35∼2.98 (5H, m), 2.82∼2.56 (3H, m), 1.78 (3H, s), 2.08∼1.12
(12H, m); semipreparative HPLC retention time: 25.19 min
(Vydac column; linear gradient from 0% B to 50% B over 15
min, then from 50% B to 60% B over 15 min); FABMS (-VE,
Gly) m/z 714 [M - H], 670 [M - H - CO2], 626 [M - H -
2CO2]. HR-FABMS calcd for C38H45N5O9 [M - H] m/z, 714.3139;
found, 714.3199.
graphic purification of the concentrated residue (CHCl3-
EtOAC, 10:1 to CHCl3-MeOH, 20:1) provided 21a as a light-
yellow solid (4.89 g, 96%): 1H NMR (DMSO-d6) δ 8.08 (1H, d,
J ) 7.3 Hz), 7.89 (2H, d, J ) 7.6 Hz), 7.78 (1H, s), 7.65 (2H,
dd, J ) 8.0, 9.6 Hz), 7.50∼7.20 (9H, m), 6.92 (1H, s), 6.85 (1H,
d, J ) 8.3 Hz), 6.21 (1H, d, J ) 2.7 Hz), 4.38 (1H, m), 4.30∼4.15
(3H, m), 4.05 (2H, m), 2.96 (2H, m), 2.64 (1H, dd, J ) 15.1,
6.6 Hz), 2.54∼2.46 (1H, dd, partially covered by DMSO peaks),
2.33 (3H, s), 1.98∼1.20 (12H, brm); FABMS (+VE, NBA) m/z
650 [MH+].
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
22a -23a . Synthesis of tripeptides 22a and 23a were ac-
complished by coupling free amine 21b (obtained by reacting
21a with piperidine in DMF followed by removal of solvent
under high vacuum) with appropriately protected amino acids
in fashions similar to that described above for the synthesis
of 17a and 17b, respectively.
Com p ou n d 29a . Purification by preparative HPLC (Ad-
vantage column; linear gradient from 10% B to 90% B over 20
min; retention time, 15.4 min) yielded product as a white
powder (86% yield): 1H NMR (DMSO-d6) δ 8.30∼8.20 (2H, m),
8.08 (1H, m), 7.99 (1H, d, J ) 8.1 Hz), 7.88 (1H, m), 7.74 (1H,
t, J ) 4.9 Hz), 7.55∼7.22 (10H, m), 6.90 (1H, s), 4.65 (1H, m),
4.35 (1H, m), 3.30∼2.55 (8H, m), 1.77 (3H, s), 2.00∼1.00 (12H,
m); semipreparative HPLC retention time: 22.78 min (Vydac
column; linear gradient from 0% B to 90% B over 25 min);
FABMS (-VE, Gly) m/z 732.9 [M - H], 688.9 [M - H - CO2],
644.9 [M - H - 2CO2]. HR-FABMS calcd for C37H43FN5O7
[M - H - CO2] m/z, 688.3147; found, 688.3121.
Com p ou n d 22a (52% yield): 1H NMR (CDCl3) δ 8.37 (1H,
s), 8.32 (1H, s), 8.10∼7.90 (4H, m), 7.80∼7.65 (2H, m),
7.60∼6.90 (14H, m), 6.36∼6.30 (1H, m), 4.57∼4.35 (2H, m),
4.23∼4.10 (4H, m), 3.38 (1H, s), 3.35 (1H, s), 3.16∼2.60 (4H,
m), 2.39 (3H, s), 2.10∼0.84 (12H, m), 1.38 (18H, s); FABMS
(+VE, NBA) m/z 1003.7 (MH+).
Com pou n d 23a (quantitative): 1H NMR (CDCl3) δ 8.06∼6.94
(21H, m), 6.56 (1H, s), 6.32 (1H, d, J ) 2.7 Hz), 4.62 (1H, m),
4.46 (1H, m), 4.40 (1H, s), 4.34 (2H, d, J ) 6.8 Hz), 4.12 (3H,
m), 3.30∼2.80 (5H, m), 2.64 (1H, dd, J ) 3.4, 14.65 Hz), 2.40
(3H, s), 2.10∼0.84 (12H, m), 1.45 (18H, s); FABMS (+VE, NBA)
m/z 1011.6 [MH+], 955.6 [MH+ - C4H8].
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
22c a n d 23c. Synthesis of acetylated tripeptides 22c and 23c
were accomplished by piperidine treatment of NR-Fmoc pro-
tected 22a and 23a followed by acetylation with N-acetylim-
idazole and chromatographic purification similar to that
described above for the conversion of 17a and 18a to 17c and
18c, respectively.
Com p ou n d 22c (quantitative): 1H NMR (DMSO-d6) δ 8.30
(1H, s), 8.26 (1H, d, J ) 7.3 Hz), 8.04∼7.96 (1H, m), 7.72 (1H,
brs), 7.55∼6.90 (10H, m), 6.33 (1H, d, J ) 1.6 Hz), 4.75∼4.62
(1H, m), 4.43∼4.32 (1H, m), 4.18 (2H, t, J ) 6.8 Hz), 3.15∼2.60
(6H, m), 2.40 (3H, s), 2.06∼1.15 (12H, m), 1.79 (3H, s), 1.40
(18H, s); FABMS (+VE, NBA) m/z 823 [MH+].
Com p ou n d 25b. Purification by preparative HPLC (Ad-
vantage column; linear gradient from 10% B to 90% B over 20
min; retention time, 15.6 min) yielded product as a white
powder (71% yield): 1H NMR (DMSO-d6) δ 8.82 (1H, d, J )
7.7 Hz), 8.35 (1H, brs), 8.18∼8.10 (1H, m), 8.04 (1H, d, J )
8.1 Hz), 7.97∼7.92 (1H, m), 7.79 (1H, t, J ) 4.7 Hz), 7.60∼7.52
(2H, m), 7.46∼7.40 (2H, m), 7.22 (2H, d, J ) 8.1 Hz), 6.96 (1H,
brs), 6.86 (2H, d, J ) 8.1 Hz), 5.35 (1H, s), 4.74∼4.70 (1H, m),
4.48∼4.35 (1H, m), 3.23∼2.65 (7H, m), 2.10∼1.10 (12H, m);
semipreparative HPLC retention time: 15.1 min (Vydac
column; linear gradient from 10% B to 90% B over 20 min);
FABMS (-VE, Gly) m/z 760 [M - H]. HR-FABMS calcd for
C
37H42FN5O10 [M - H - CO2] m/z, 716.2932; found, 716.2879.
Com p ou n d 28b. Purification by preparative HPLC (Ad-
vantage column; linear gradient from 5% B to 50% B over 15
min; retention time, 18.5 min) yielded product as a white
powder (52% yield): 1H NMR (DMSO-d6) δ 8.82 (1H, s), 8.08
(1H, m), 8.00 (1H, d, J ) 7.3 Hz), 7.89 (1H, m), 7.74 (1H, m),
7.53∼7.20 (10H, m), 6.92 (1H, s), 4.73 (1H, m), 4.60 (1H, s),
4.36 (1H, m), 3.25∼2.93 (6H, m), 2.69 (1H, dd, J ) 6.8, 15.4
Hz), 2.52 (1H, m), 2.08∼1.12 (12H, m); semipreparative HPLC
retention time: 24.35 min (Vydac column; linear gradient from
0% B to 50% B over 15 min, then from 50% B to 60% B over
15 min); FABMS (-VE, Gly) m/z 744 [M - H], 700 [M - H -
CO2]. HR-FABMS calcd for C37H42N5O9 [M - H - CO2] m/z,
700.2983; found, 700.2966.
Com p ou n d 23c (quantitative): 1H NMR (CDCl3) δ 8.08
(1H, d, J ) 7.8 Hz), 7.66 (2H, m), 7.36∼6.94 (10 H, m), 6.32
(1H, d, J ) 2.9 Hz), 6.03 (1H, s), 4.70∼4.55 (2H, m), 4.41 (1H,
s), 4.11 (2H, t, J ) 6.6 Hz), 3.25∼3.05 (3H, m), 3.00∼2.85 (2H,
m), 2.65 (1H, dd, J ) 4.4 Hz), 2.40 (3H, s), 2.10∼1.05 (12H,
m), 1.86 (3H, s), 1.46 (18H, s); FABMS (+VE, NBA) m/z 831
[MH+], 775 [MH+ - C4H8].
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
22d a n d 23d . Synthesis of tripeptides 22d and 23d were
accomplished by piperidine treatment of NR-Fmoc protected
22a and 23a followed by acylation with tert-butyl oxalyl
chloride and chromatographic purification similar to that
described above for the conversion of 17a and 18a to 17d and
18d , respectively.
Com p ou n d 22d (quantitative): 1H NMR (DMSO-d6) δ 8.78
(1H, d, J ) 4.7 Hz), 8.44∼8.31 (2H, m), 8.07∼7.96 (2H, m),
7.66∼7.16 (7H, m), 6.95 (2H, d, J ) 9.0 Hz), 6.33 (1H, d, J )
3.8 Hz), 4.80∼4.66 (1H, m), 4.46∼4.34 (1H, m), 4.18 (1H, t,
J ) 6.4 Hz), 3.25∼2.60 (6H, m), 2.40 (3H, s), 2.10∼1.05 (12H,
m), 1.48 (9H, s), 1.37 (18H, s); FABMS (+VE, NBA) m/z 909
[MH+].
Com p ou n d 23d (94% yield): 1H NMR (CDCl3) δ 7.96 (1H,
d, J ) 7.8 Hz), 7.68 (1H, d, J ) 7.1 Hz), 7.50 (1H, m), 7.38∼7.24
(8H, m), 7.18 (1H, d, J ) 2.9 Hz), 7.00 (1H, d, J ) 8.3 Hz),
6.36 (1H, d, J ) 2.9 Hz), 6.03 (1H, s, br), 4.66 (2H, m), 4.41
(1H, s), 4.17 (2H, t, J ) 6.8 Hz), 3.26 (2H, m), 3.12 (2H, d,
J ) 7.3 Hz), 3.08∼3.00 (1H, dd, J ) 4.6, 15.1 Hz), 2.58∼2.50
(1H, dd, J ) 4.9, 15.14 Hz), 2.43 (3H, s), 2.10∼1.00 (12H, m),
1.51 (9H, s), 1.47 (18H, s); FABMS (+VE, NBA) m/z 917 [MH+],
861 [MH+ - C4H8].
Com p ou n d 29b. Purification by preparative HPLC (Ad-
vantage column; linear gradient from 10% B to 90% B over 20
min; retention time, 14.9 min) yielded product as a white
powder (63% yield): 1H NMR (DMSO-d6) δ 8.85 (1H, d, J )
7.8 Hz), 8.32 (1H, s), 8.08 (1H, m), 7.99 (1H, d, J ) 8.1 Hz),
7.88 (1H, m), 7.83 (1H, t, J ) 4.9 Hz), 7.55∼7.22 (10H, m),
6.91 (1H, s), 4.37 (1H, m), 4.30 (1H, m), 3.25∼2.91 (7H, m),
2.68 (1H, dd, J ) 6.4, 15.4 Hz), 2.06∼0.93 (12H, m); semi-
preparative HPLC retention time: 22.05 min (Vydac column;
linear gradient from 0% B to 90% B over 25 min); FABMS
(-VE, Gly) m/z 762 [M - H], 718 [M - H - CO2]. HR-FABMS
calcd for C37H41FN5O9 [M - H - CO2] m/z, 718.2888; found,
718.2849.
Syn th esis of Dip ep tid e 21a . A mixture of NR-Fmoc
1-aminocyclohexanylcarboxylic acid (2.85 g, 7.80 mmol), HOBt‚
H2O (1.19 g, 5.80 mmol) and DIPCDI (1.22 mL, 7.80 mmol) in
DMF (40 mL) was stirred at room temperature (1 h), then
freshly prepared 3-(N-(5-methylindolyl))propanamido-L-aspar-
agine (20)19 (7.80 mmol) in DMF (10 mL) was added, and the
reaction mixture was stirred at room temperature (overnight).
The mixture was poured into ice-water (200 mL) and sub-
jected to an extractive workup (EtOAc). Silica gel chromato-
Gen er a l P r oced u r e for th e Con ver sion of P r otected
Der iva tives 22c, 23c a n d 22d , 23d to F in a l P r od u cts 30a ,

Inhibition of Grb2 SH2 Domain Binding
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 919
(6) Williams, E. J .; Dunican, D. J .; Green, P. J .; Howell, F. V.;
Derossi, D.; Walsh, F. S.; Doherty, P. Selective inhibition of
growth factor-stimulated mitogenesis by a cell-permeable Grb2-
binding peptide. J . Biol. Chem. 1997, 272, 22349-22354.
(7) 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.
31a a n d 30b, 31b, Resp ectively. Protected analogues 22c,
23c and 22d , 23d (∼0.1 mmol) were treated (1 h) with
solutions of TFA (1.90 mL):H2O (0.10 mL):1,2-ethanedithiol
(0.05 mL), taken to near dryness, and triturated with anhy-
drous ether to yield solids which were suspended in ether,
collected, and dried to provide crude 30a , 31a and 30b, 31b,
respectively, as white or slightly pink solids. Final products
were obtained by HPLC purification.
(8) Ye, B.; Akamatsu, M.; Shoelson, S. E.; Wolf, G.; Giorgetti-Peraldi,
S.; Yan, X. J .; Roller, P. P.; Burke, T. R. L-O-(2-malonyl)-
tyrosine: A new phosphotyrosyl mimetic for the preparation of
Src homology 2 domain inhibitory peptides. J . Med. Chem. 1995,
38, 4270-4275.
Com p ou n d 30a . Purification by preparative HPLC (Ad-
vantage column; linear gradient from 30% B to 90% B over 20
min; retention time, 11.5 and 12.1 min) yielded product as a
white powder (27% yield): 1H NMR (D2O) δ 8.08 (1H, brs),
7.56∼7.38 (3H, m), 7.33∼7.05 (6H, m), 5.75∼5.60 (1H, m),
4.70∼4.55 (1H, m), 4.53∼4.40 (1H, m), 4.33∼4.20 (2H, m),
3.90∼2.70 (6H, m), 2.55 (1.5H, s), 2.45 (1.5H, s), 2.20∼1.0 (15H,
m); semipreparative HPLC retention time: 8.4 and 10.4 min
(Vydac column; linear gradient from 40% B to 90% B over 20
min) [Note: compound exists as two interconverting forms on
HPLC]; FABMS (-VE, Gly) m/z 709.2 (M - H). HR-FABMS
calcd for C35H46N6O8P [M - H] m/z, 709.3115; found, 709.3091.
Com p ou n d 30b. Purification by preparative HPLC (Ad-
vantage column; linear gradient from 10% B to 90% B over 20
min; retention time, 15.3 and 15.7 min) yielded product as a
white powder (67% yield): 1H NMR (D2O) δ 8.28∼8.20 (1H,
m), 7.58∼7.05 (9H, m), 5.75∼5.60 (1H, m), 4.70∼4.45 (1H, m),
4.30∼4.14 (1H, m), 3.90∼2.70 (6H, m), 2.55∼2.40 (3H, m),
2.25∼1.12 (12H, m); semipreparative HPLC retention time:
19.0 and 21.0 min (Vydac column; linear gradient from 10%
B to 90% B over 20 min) [Note: compound exists as two
interconverting forms on HPLC]; FABMS (-VE, Gly) m/z 739.2
(M - H). HR-FABMS calcd for C35H44N6O10P [M - H] m/z,
739.2857; found, 739.2881.
Com p ou n d 31a . Purification by preparative HPLC (Vydac
column; linear gradient from 10% B to 90% B over 20 min;
retention time, 16.0 min) yielded product as a light pink
powder (54% yield): 1H NMR (DMSO) δ 8.30∼8.20 (2H, m),
7.97 (1H, d, J ) 7.6 Hz), 7.45∼7.15 (9H, m), 6.95∼6.75 (2H,
m), 6.28 (1H, d, J ) 2.9 Hz), 4.70∼4.60 (1H, m), 4.60 (1H, s),
4.30 (1H, m), 4.13 (2H, t, J ) 6.6 Hz), 3.10∼2.57 (6H, m), 2.35
(3H, s), 2.00∼1.00 (12H, m), 1.79 (3H, s); semipreparative
HPLC retention time: 26.20 min (Vydac column; linear
gradient from 10% B to 90% B over 30 min); FABMS (-VE,
Gly) m/z 717 [M - H], 673 [M - H - CO2]. HR-FABMS calcd
for C37H46N6O9 [M - H] m/z, 717.3248; found, 717.3221.
Com p ou n d 31b. Purification by preparative HPLC (Vydac
column; linear gradient from 10% B to 90% B over 20 min;
retention time, 15.4 min) yielded product as a light pink
powder (47% yield): 1H NMR (DMSO) δ 8.90∼8.75 (1H, m),
8.29 (1H, m), 8.00∼7.85 (1H, m), 7.50∼7.07 (9H, m), 7.00∼6.69
(3H, m), 4.71 (1H, m), 4.49 (1H, s), 4.32 (1H, m), 4.15∼4.04
(2H, m), 3.25∼2 0.55 (6H, m), 2.35∼2.17 (3H, several single
peaks), 2.00∼1.11 (12H, m); semipreparative HPLC retention
time: 24.67 min (Vydac column; linear gradient from 10% B
to 90% B over 30 min); FABMS (-VE, Gly) m/z 747 [M - H],
703 [M - H - CO2]. HR-FABMS calcd for C36H43N6O9 [M -
H - CO2] m/z, 703.3092; found, 703.3092.
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