Potent inhibition of Grb2 SH2 domain binding by non-phosphate- containing ligands

Article abstract of DOI:10.1021/jm980388x

Development of Grb2 Src homology 2 (SH2) domain binding inhibitors has important implications for treatment of a variety of diseases, including several cancers. In cellular studies, inhibitors of Grb2 SH2 domain binding have to date been large, highly cha

Full text of DOI:10.1021/jm980388x

J . Med. Chem. 1999, 42, 25-35
25
Articles
P oten t 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
Zhu-J un Yao,‡ C. Richter King,§ Tin Cao,§ J ames Kelley,‡ George W. A. Milne,‡ J ohannes H. Voigt,‡ 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, D.C.
Received J une 26, 1998
Development of Grb2 Src homology 2 (SH2) domain binding inhibitors has important impli-
cations for treatment of a variety of diseases, including several cancers. In cellular studies,
inhibitors of Grb2 SH2 domain binding have to date been large, highly charged peptides which
relied on special transport devices for cell membrane penetration. Work presented in the current
study examines a variety of pTyr mimetics in the context of a high-affinity Grb2 binding plat-
form. Among the analogues studied are new non-phosphorus-containing pTyr mimetics 23a
and 23b which, when incorporated into tripeptide structures 18f and 20f, are able to inhibit
Grb2 SH2 domain binding with affinities among the best yet reported for non-phosphorus-
containing SH2 domain inhibitors (IC50 values of 6.7 and 1.3 µM, respectively). The present
study has also demonstrated the usefulness of the NR-oxalyl group as an auxiliary which en-
hances the binding potency of both phosphorus- and non-phosphorus-containing pTyr mimetics.
When combined with the (phosphonomethyl)phenylalanine (Pmp) residue to give analogues
such as L-20d , potent inhibition of Grb2 SH2 domain binding can be achieved both in extra-
cellular assays using isolated Grb2 SH2 domain protein and in intracellular systems measuring
the association of endogenous Grb2 with its cognate p185erbB-2 ligand. These latter effects can
be achieved at micromolar to submicromolar concentrations without prodrug derivatization.
The oxalyl-containing pTyr mimetics presented in this study should be of general usefulness
for the development of other Grb2 SH2 domain antagonists, independent of the â-bend-
mimicking platform utilized for their display.
In tr od u ction
opment of SH2 domain inhibitors has therefore con-
cerned itself with three thematic areas: (1) interactions
within the pTyr binding pocket,4 (2) interactions with
recognition areas outside the pTyr pocket, and (3) bridg-
ing elements between structural features 1 and 2.5,6
Signal transduction is critical to normal cellular ho-
meostasis, with aberrations in some signaling pathways
having potentially adverse effects, including the promo-
tion of cancers and immune disorders. The phosphoty-
rosyl pharmacophore (pTyr, 1) serves a central role in
protein-tyrosine kinase (PTK)-mediated signal trans-
duction by providing key recognition features necessary
for assembly of multicomponent signaling complexes
through the actions of Src homology 2 (SH2), phospho-
tyrosine-binding (PTB), and newly discovered1 “STYX”
domains.2 For SH2 domains, recognition of pTyr-con-
taining ligands frequently depends on interaction of the
pTyr phosphate with two arginine residues in a well-
formed pocket. Additional, secondary binding interac-
tions are also provided by amino acids 2-3 residues
C-proximal to the pTyr residue, which introduce differ-
ential affinity toward SH2 domain subfamilies.3 Devel-
In the first area of investigation, pTyr mimetics have
been sought which are stable to phosphatases and which
offer the potential for cell membrane penetration. Phos-
phonate-based pTyr mimetics, such as (phosphonometh-
yl)phenylalanine (Pmp, 2)7 and (difluorophosphonometh-
yl)phenylalanine (F2Pmp, 3),8 which replace the tyrosyl
phosphate ester bond with a methylene unit, can retain
good SH2 domain binding affinity in extracellular prepa-
rations,9,10 and when administered into cells by micro-
injection11 or cell permeabilization techniques,12 they
have been shown to exhibit effects consistent with SH2
domain inhibition. However, while useful for pharmaco-
logical studies, such delivery techniques do not hold
promise for in vivo studies. A more traditional approach
using prodrug derivatization of the phosphonate moiety
has met with limited success when applied to F2Pmp-
containing peptides.13 These considerations have led to
the examination of non-phosphorus-containing pTyr
* To whom correspondence should be addressed at: Bldg 37, Rm
5C06, National Institutes of Health, Bethesda, MD 20892. Phone: (301)
496-3597. Fax: (301) 402-2275. E-mail: tburke@helix.nih.gov.
‡ National Institutes of Health.
§ Georgetown University Medical Center.
10.1021/jm980388x This article not subject to U.S. Copyright. Published 1999 by the American Chemical Society
Published on Web 12/05/1998

26 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 1
Yao et al.
mimetics,4,14,15 including O-malonyltyrosine (OMT, 4)16
and fluoro-O-malonyltyrosine (FOMT, 5),17 which may
offer alternative approaches to cell delivery. The present
study was undertaken to examine the application of
known pTyr mimetics such as F2Pmp and Pmp as well
as new pTyr mimetics, in the context of Grb2 (growth
factor receptor bound protein 2)18 SH2 domain binding
systems, with a particular emphasis on cell-based
studies without the need of prodrug derivatization.
Sch em e 2
Syn th esis
pTyr-containing â-bend mimetic 18b is within the
structural family of Grb2 SH2 domain antagonists
previously synthesized by solid-phase techniques.19,20
Despite repeated attempts, we were unable to achieve
the synthesis of 18b by solid-phase methods while
maintaining the N-terminal naphthylpropylamide. This
failure was most probably related to steric crowding
induced by the naphthylpropylamido group proximal to
the R-amino and carboxyl moieties of the â-bend-forming
1-aminocyclohexanecarboxylic acid residue (13). All
syntheses were therefore conducted by solution meth-
ods. Key naphthylpropylamine-containing dipeptide 14
was prepared by reacting amine 11 with the N-hydroxy-
succinimide active ester of N-Boc-L-asparagine to yield
the corresponding amide 12. The required naphthyl-
propylamine 11 has previously been reported;21 however
it was found expeditious to prepare it by the alternate
route shown in Scheme 1.22
Of note was tripeptide 17f, which required removal of
the 2-trimethylsilylethyl ester (tetrabutylammonium
fluoride) prior to TFA treatment.25 Synthesis of oxalyl-
containing analogues 20d and 20f was identical to that
described for analogues 18d and 18f, respectively,
Sch em e 3
Sch em e 1
After deprotection of 12a to amine 12b, coupling with
N-Boc-1-aminocyclohexanecarboxylic acid (13) provided
the protected dipeptide 14a (Scheme 2). Free dipeptide
amine 14b, obtained upon standard N-deprotection, was
coupled with Tyr (15a ), pTyr (15b), or pTyr mimetics
15c,23 15d ,24 15e,25 or 15f,25 all bearing suitable pro-
tecting groups.
except that N-acylation with tert-butyl oxylate was
performed rather than acetylation (Scheme 4).
Removal of amino protection and acetylation gave the
corresponding N-acetyl analogues 17a -f, which after
cleavage of remaining protecting groups and HPLC
purification, gave final tripeptides 18a -f (Scheme 3).
Resu lts a n d Discu ssion
The present work examines non-phosphate-containing
pTyr mimetics in the context of Grb2 SH2 domain

Potent Inhibition of Grb2 SH2 Domain Binding
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 1 27
Sch em e 4
binding systems. The Grb2 SH2 domain affords an ideal
target for such a study because it provides critical links
between growth factor receptor PTKs and downstream
signaling events involving Ras proteins, which have
been directly implicated with oncogenic processes. Ex-
amples of this include members of the epidermal growth
factor receptor (EGFR) PTK family such as ErbB-2
(HER-2/neu), which are found in a large proportion of
breast cancers,26 the Met (hepatocyte growth factor/
scatter factor) PTK, which is overexpressed in many
human tumors,27 and the Bcr-Abl PTK, which is neces-
sary for Philadelphia chromosome-positive leukemia.28,29
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. In experimental cell systems such an over-
expression of p185erbB-2 leads to cell transformation,30,31
and signaling via Grb2 has also been shown to be
sufficient to transform such cells.32,33 Blockade of Grb2
SH2 domain binding could potentially provide a means
of uncoupling p185erbB-2 from the Ras pathway and, in
so doing, attenuate its transforming effects. Accordingly,
oblation of Grb2 function, either by elimination of the
entire Grb2 protein through antisense polynucleotides28
or by competitive blockade of Grb2 SH2 domain binding
interactions with pTyr-containing peptides,34,35 can
inhibit cell growth or EGF-induced Ras/mitogen-acti-
vated protein kinase signaling, respectively.
P la sm on Rea son a ce Bin d in g Stu d ies. To examine
pTyr mimetics in the context of Grb2 SH2 inhibitors,
the recently disclosed high-affinity pTyr-containing
tripeptide 18b19,20 was chosen as a model platform on
which to append phosphotyrosyl-mimicking analogues.
A series of new inhibitors 18c-f, 20d , and 20f incor-
porating pTyr mimetics 15c-f was prepared, and Grb2
SH2 domain binding affinities were determined by
surface plasmon reasonance (Figures 1 and 2).39,40 The
linear Shc(Y317) peptide D-D-P-S-pY-V-N-V-Q (24) was
employed as a reference and gave an IC50 of 1.3 µM in
this system. Consistent with previous reports,19,20 the
parent pTyr-containing 18b also exhibited very potent
binding affinity (IC50 ) 0.07 µM). The importance of the
“phosphate pharmacophore” to the overall binding of
18b was confirmed by the loss of all measurable binding
affinity upon removal of the phosphate group (compound
18a ).
A Novartis group has accordingly reported several
high-affinity pTyr-containing ligands, which, in addition
to having in common a pTyr residue that binds in the
pTyr pocket and an Asn residue in the pTyr + 2 position,
contain other features that enhance Grb2 binding
affinity.36 Among these features are elements which
induce â-bend conformations, as shown by cyclic peptide
21 (IC50 ) 0.37 µM)37 and â-bend-mimicking open-chain
tripeptides typified by 22 (IC50 ) 0.011 µM)19,20 which
utilize a 1-aminocyclohexanecarboxylic acid in the pTyr
+ 1 position to facilitate â-bend formation.38 This latter
type of structure provided an ideal starting point for
the current study, which is focused on interactions
within the pTyr binding pocket.

28 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 1
Yao et al.
show reduced inhibitory potency.10,14,42 The reasons for
the unexpectedly high potency of the Pmp-containing
inhibitor are not obvious. Also seen in Figure 2 is the
marked loss of potency for the D-Pmp-containing com-
pound D-18d 41 relative to its L-counterpart.
Carboxylate-based pTyr mimetics were examined
next. Non-phosphorus-containing pTyr mimetics such
as OMT (4) were originally designed to afford structural
alternatives to phosphorus-containing pTyr mimetics.16
In the current study, analogues 18e and 18f were
envisioned to function as variants of OMT in which one
malonyl carboxyl has been replaced with a hydrogen,
while the second carboxyl has been translocated to a
more distal location. Retention of two carboxyl groups
was deemed necessary in order to provide a dianionic
“phosphate pharmacophore” that could interact with two
critical arginines within the pTyr binding pocket. As
seen in Figure 1, appending the second carboxyl to a
flexible side chain resulted in a dramatic loss of binding
affinity (18e, IC50 . 100 µM). Anchoring the second
carboxyl directly onto the aryl ring resulted in signifi-
cantly better affinity (18f, IC50 ) 6.7 µM).
The reduced binding potency of 18f relative to phos-
phate-containing analogue 18b could potentially indi-
cate that interactions of the two carboxyls of 18f with
Arg67 (RA-helix) and Arg86 (âC-strand) within the pTyr
binding pocket do not faithfully approximate that
provided by the phosphorus-containing parent structure
18b. Note was therefore taken of the fact that the
tyrosyl R-nitrogen provides a site from which additional
critical interaction with Arg67 can be derived.36 To
examine whether introduction of anionic functionality
onto the tyrosyl R-nitrogen could allow beneficial inter-
action with the positively charged Arg67 residue, pTyr
mimetic 23a was modified by appending an NR-oxalyl
group to yield tricarboxylic analogue 23b. A conceptu-
ally similar approach has been used in the design of a
phosphorylated 2-(phenylmethyl)succinic acid pTyr mi-
metic.43 Figure 3C depicts the results of a molecular
dynamics simulation examining one possible manner in
which 23b could interact with the pTyr binding pocket.
Of particular note is the ability of the NR-oxalyl carboxyl
to bind to the RA-helix Arg67. Also evident are the ways
in which the aryl bis-carboxyls of 23b can provide key
recognition features displayed by the parent phosphate
group (Figure 3A). Introduction of mimetic 23b into the
general â-bend-mimicking platform was achieved in
straightforward fashion by N-terminal acylation using
tert-butyl oxylate prior to final TFA-mediated deprotec-
tion. Consistent with modeling expectations, increased
binding affinity was observed (20f, IC50 ) 1.3 µM). This
5-fold enhancement in affinity makes the non-phospho-
rus-containing 20f equivalent in potency to the reference
linear phosphopeptide 24, which was based on the
physiologically relevant Shc(Y317) sequence.
F igu r e 1. Effect of tyrosine modification on inhibition of Grb2
SH2 domain binding. Recombinant GST-Grb2 SH2 domain
was mixed with the compounds indicated, and the mixture was
allowed to flow across the surface-bound SHC phosphopeptide.
The amount of equilibrium binding (Ru(max)) was determined
and compared to binding when no inhibitor is present. As a
control, the SHC phosphopeptide sequence (D-D-P-S-pY-V-N-
V-Q) was used. Unmodified tyrosine (18a ) shows no inhibitory
activity. The most potent inhibitors contained phosphate (18b)
and phosphonate (18c).
F igu r e 2. Effect of stereochemistry and NR-oxalyl modifica-
tion on inhibition of Grb2 SH2 domain binding. Recombinant
GST-Grb2 SH2 domain was mixed with the compounds
indicated, and the mixture was allowed to flow across the
surface-bound SHC phosphopeptide. The amount of equilib-
rium binding (Ru(max)) was determined and compared to
binding when no inhibitor is present. As a control the phos-
phorylated 18b was used. The most potent compound was the
L-enantiomer of the NR-oxalyl-modified phosphonate (L-20d ).
Experiments were conducted with phosphonate-
containing analogues. The difluorophosphonate-based
analogue 18c (IC50 ) 0.08 µM) was found to be equally
potent to the pTyr-containing parent, which contrasts
with the previous demonstration of a 5-fold reduction
in potency in a Grb2 SH2 domain binding system for a
linear F2Pmp-containing peptide relative to its pTyr-
containing homologue.10 The nonfluorinated phospho-
nate-containing compound L-18d 41 showed inhibitory
potency equivalent to that of the F2Pmp-containing 18c,
which is also somewhat unexpected, since for other SH2
domain systems, Pmp-containing analogues frequently
The ability of NR-oxalyl functionality to enhance the
binding interaction of the carboxy pTyr mimetic sug-
gested that NR-oxalyl functionality could potentially
have benefitial effects on Pmp-containing inhibitors.
Molecular modeling dynamics simulations of NR-oxalyl
Pmp-containing 20d in the pTyr binding pocket showed
that while maintaining key binding interactions of the
pTyr-containing 18b, significant new interactions be-
tween the Arg67 residue and elements of the NR-oxalyl

Potent Inhibition of Grb2 SH2 Domain Binding
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 1 29
F igu r e 3. Interactions within the pTyr-binding pocket of ligated Grb2 SH2 domain, with particular note to interactions with
Arg67. (A) X-ray structure of bound KPFpYVNV peptide.47 Binding of the pTyr residue (truncated after the Phe residue) is shown
with water molecules omitted. (B) Binding of the NR-oxalyl L-Pmp residue used for compound 20d . (C) Binding of the NR-oxalyl
residue 23b used in compound 20f. Structures shown in panels B and C were derived as described in the Experimental Section.
group were evident (Figure 3A,B). These modeling
predictions were consistent with plasmon reasonance
binding which showed that oxalyl L-Pmp (L-20d )41 binds
approximately 2-fold higher affinity than L-18d (Figure
2). For the D-Pmp analogue (D-20d ),41 the enhancing
effect of the NR-oxalyl group is even more pronounced
(approximately 5-fold) relative to parent analogue D-18d
(Figure 2).
In h ibition of Gr b2 SH2 Dom a in Bin d in g in Cell-
F igu r e 4. Inhibition of Grb2 interaction with tyrosine phos-
Ba sed System s. Data in Figures 1 and 2 reflect
inhibition measured using isolated Grb2 SH2 domain
protein relative to a reference phopsho-SHC(Y317)
peptide. In physiological contexts however, Grb2 SH2
domains exist as part of larger Grb2 proteins which bind
to pTyr-containing ligands which are themselves protein
in nature. To examine the ability of synthetic analogues
phorylated p185erbB-2. Compounds showing activity in surface
plasmon resonance assays were tested for their ability to
inhibit interaction between intact Grb2 protein with a natural
protein target, p185erbB-2, in cell extracts. Cell lysates were
prepared, and Grb2 was immunoprecipitated in either the
presence or absence of inhibitor. Phosphotyrosinylated proteins
that coimmunoprecipitate were detected by immunoblotting
using specific anti-pTyr antibodies. Comparison of 18f with
20f shows the effect of NR-oxalyl modification on inhibitory
activity. The phosphate requirement for 18c is shown by the
nonphosphorylated 18a . All results were from one experiment
with identical amounts of cell lysate treated.
to inhibit the interaction of native Grb2 with p185erbB-2
,
two assays were used, one in which inhibitors were
introduced directly into cell lysates and one of which
required the compounds to cross membranes of intact
cells. In both cases, MDA-MB-453 cells were utilized.
These cells are derived from a human breast cancer
where there is amplification of the erbB-2 gene and
resultant overexpression of mRNA and protein. In these
cells there is a heavily phosphotyrosinylated protein
detectable by immuoblotting of cell lysates, which
inhibition is somewhat higher for 18c as compared to
20f. Removal of the oxalyl group (dicarboxy-based 18f)
resulted in a measurable loss of potency.
Compounds were also examined in a whole cell assay
in which inhibition of intracellular Grb2 binding was
measured following application of compounds to the
media of MDA-MB-453 cells. These conditions required
that compounds transit the cell membrane. As shown
in Figure 5, while the nonphosphorylated tyrosyl ana-
logue 18a showed no measurable inhibition even at 50
µM concentration, compounds 18c, 20f, 18d , and L-20d
corresponds to the overexpressed p185erbB-2 44
Interac-
.
tion of native Grb2 protein with ErbB2 was monitored
by immunoprecipitating Grb2 and detecting the amount
of p185erbB-2 which is coprecipitated using anti-phos-
photyrosine Western blotting.40 As shown in Figure 4,
when F2Pmp-containing 18c and tricarboxy-based 20f
are introduced into MDA-MB-453 cell lysates, there is
a clear dose-dependent reduction in the associated
p185erbB-2 bound to Grb2. Consistent with plasmon
reasonace binding data (Figures 1 and 2), the level of
were able to inhibit Grb2 interaction with p185erbB-2
.
In this assay, oxalyl L-Pmp-containing L-20d was clearly
the most potent analogue with densiometric scanning
of the blot indicating an IC50 value potentially as low

30 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 1
Yao et al.
Simulations Inc., San Diego, CA), using the cff91 force field,
which was modified to take into account the planarity of the
oxalylic amide group. The crystal structure of K-P-F-pY-V-N-
V-NH2 bound to Grb2 SH247 was used as the starting geom-
etry. The heptapeptide within the receptor was modified
appropriately to yield 20d and 20f in which the naphthyl group
was omitted. The resulting structure of 20d complexed to the
SH2 domain was then solvated with a 20-Å sphere of water
(673 molecules) centered around the spirocyclohexyl CR of 20d .
After 300 steps of energy minimization with the Polak-Ribiere
conjugated gradient algorithm (CG-PR), 50 ps of an NVT
molecular dynamics simulation (MD) at 298 K was performed.
The coordinates were saved every 1.0 ps. During minimization
and simulation, all atoms were held fixed except 20d , the
water molecules within a 15-Å sphere about the spirocyclo-
hexyl CR of 20d , and the side chains of the Grb2 SH2 protein
within 5 Å around the initial conformation of the heptapeptide.
The lowest-energy structure out of the 50 saved frames was
minimized with 3 steps of steepest decent (SD) and 115 steps
of CG-PG and is displayed in Figure 3B. For 20f, the structure
of the protein-peptide complex was subjected to a quenched
molecular dynamics simulation (QMD, NVT ensemble) at 800
K. The coordinates were saved every 200 fs and minimized by
300 steps of CG-PR. During the simulation, all atoms except
the side chain of the pTyr were held fixed. For the nonbonding
interactions, a cutoff of 1.0 Å, a spline width of 1.0 Å, and a
buffer width of 0.5 Å were used. The resulting 300 structures
were grouped into 19 clusters with an rmsd cutoff of 0.8 Å,
and the representative structures for each of the 19 clusters
were sorted by energy. The five lowest-energy structures
displayed a similar mode of binding. The next five structures
again showed a similar mode of binding, which was different
from the one of the first group. The lowest-energy structure
of each group was solvated with 687 and 670 water molecules,
respectively, and subjected to MD in the same way as 20d .
The total energy without solvent molecules for the lowest-
energy frames of both MD runs was compared after minimiza-
tion with 1 step of SD and 144 steps of CG-PR minimization
for the first group and 1 step of SD and 151 steps of CG-PR
minimization for the second group without applying con-
straints in both cases. For the structure from the first group,
a lower energy was obtained, and this complex is displayed in
Figure 3C. A constant dielectric constant of 1.00 and the cell
multipole method with fine accuracy were used for all non-
bonding interactions except from the QMD.
F igu r e 5. Interaction of Grb2 with p185erbB-2 can be potently
inhibited. Compounds showing activity in surface plasmon
resonance assays were tested for their ability to inhibit
interaction between intact Grb2 protein with a cellular target
by incubation of intact cells in culture. Cells were exposed to
inhibitors for 3 h, washed, and lysed, and Grb2 was immuno-
precipitated. pTyr-containing p185erbB-2 that co-immunopre-
cipitate were detected using specific pTyr antibodies and
immuoblotting. The most potent compound was the L-enanti-
omer of the phosphonate bearing NR-oxalyl modification. All
results shown were from a single experiment where plates
containing the same number of cells were treated.
as 0.5 µM. Comparison of the cellular data in Figure 5
with the cell-free IC50 values obtained by surface plas-
mon reasonance shows an approximate 50-fold reduc-
tion in potency incurred by cell membrane transit. While
evaluation of IC50 values is likely to vary with the time
of exposure to the compound, the cell line, and the exact
methodology used, these results suggest that oxalyl
L-Pmp-containing L-20d is a potent inhibitor of Grb2
when applied to intact cells.
Con clu sion s
Development of Grb2 SH2 domain binding inhibitors
has important implications for treatment of a variety
of cancers. To date, Grb2 SH2 domain blockers used in
cellular contexts have been large, highly charged pep-
tides which relied on special transport devices for cell
membrane penetration.34,35,45 The work presented in the
current study has focused on pTyr mimetics and their
potential application to cell-based studies. New non-
phosphorus-containing pTyr mimetics 23a and 23b have
been introduced, which, when incorporated into high-
affinity â-bend-mimicking platforms 18f and 20f, are
able to exhibit Grb2 SH2 domain with low micromolar
affinity. Compounds 18f and 20f are relatively compact,
highly lipophilic molecules which represent improve-
ments over previously reported non-pTyr-containing
competitive antagonists to Grb2 SH2 domains that have
utilized much larger peptide structures.40,45,46 The present
study has also demonstrated the usefulness of the NR-
oxalyl group as an auxiliary which enhances the binding
potency of both phosphorus and non-phosphorus-
containing pTyr mimetics. When combined with the
Pmp residue to give analogues such as L-20d , potent
inhibition of Grb2 SH2 domain binding can be achieved
in extracellular assays. More importantly, when admin-
istered to cells without prodrug derivatization, intra-
cellular inhibition of Grb2 SH2 domain binding is
achieved at micromolar to submicromolar concentra-
tions. The oxalyl-containing pTyr mimetics presented
in this study should be of general usefulness for the
development of other Grb2 SH2 domain antagonists,
independent of the particular â-bend-mimicking plat-
form utilized for their display.
Biologica l. A. In h ibition of SH2 Dom a in Bin d in g
Usin g Su r fa ce P la sm on Reson a n ce (SP R). The methods
used were designed to measure a solution IC50 for peptide
inhibition of the Grb2 SH2 domain. The approach minimizes
the “left shift” encountered in SPR experiments that is
observed when binding equilibrium constants are determined
from association and dissociation rates at the SPR surface. In
this study the SPR serves simply as a detector of free Grb2
SH2 in solution. The IC50 values are determined by mixing
peptide with recombinant Grb2 SH2 domains and then mea-
suring the amount of binding at equilibrium to the immobilized
SHC phosphopeptide. The methods were essentially as previ-
ously described for the quantitative comparison of binding
constants for the binding of other SH2 domains with phos-
phopeptides in solution.39 In our experiments the immobilized
phase was generated using SHC phosphopeptide, biotin-
DDPSpYVNVQ (Quality Controlled Biochemicals, QCB), >90%
purity by C18 HPLC and of the appropriate molecular weight
(1453) by mass spectrometry. This peptide was attached to SA5
chips at 2 nM. Binding of GST-Grb2 was conducted at 200 nM
in HBS buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 0.01%
Triton X-100) at flow rate of 5 µL/min for 10 min. Total Ru
change for GST-Grb2 SH2 binding to SHC phosphopeptide in
the absence of inhibitors was 150-250 Ru. Compounds tested
as inhibitors at the indicated concentrations were premixed
with the GST-Grb2 SH2 prior to introduction onto the immo-
bilized chip monitored. The SHC phosphopeptide, DDPSpYVN-
VQ, was obtained from QCB. Equilibrium binding Ru was
determined at 20 s following the last GST-Grb2 SH2 flowing
Exp er im en ta l Section
Molecu la r Mod elin g. All simulations were performed with
the Insight II 97.0/Discover 3.0 modeling package (Molecular

Potent Inhibition of Grb2 SH2 Domain Binding
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 1 31
across the chip. Two experiments were conducted using
separate sensor chips and peptide dilutions.
h). Solid was removed by filtration and washed with ether;
then the combined ether solution was dried (Na2SO4) and
taken to dryness, yielding 9 as an oil51 (9.36 g, 100%): 1H NMR
(CDCl3) δ 8.08 (1H, dd, J ) 2.4, 7.4 Hz), 7.88 (1H, m), 7.74
(1H, d, J ) 7.6 Hz), 7.34-7.57 (4H, m), 3.76 (2H, t, J ) 6.4
Hz), 3.20 (2H, t, J ) 7.5 Hz), 2.05 (2H, m), 1.75 (1H, brs).
B. In h ibition of Gr b2 SH2 Dom a in Bin d in g in Cell
Extr a cts. Cell lysates were prepared from serum-treated
erbB-2-overexpressing breast (MDA-MB-453) cancer cells us-
ing 1% Triton X-100 in PBS containing 0.2 mM NaVO4.
Lysates were incubated with the indicated concentration of
inhibitory compounds for 30 min. Grb2 and associated Grb2-
binding proteins were immunoprecipitated from each lysate
(5 mg) with anti-Grb2 antibodies and collected using protein
A Sepharose using methods previously described.48 Immuno-
precipitated proteins were separated by SDS-PAGE on 8-16%
gradient gels (Novagen). pTyr-containing proteins were de-
tected by Western blotting using anti-phosphotyrosine anti-
bodies (Upstate Biochemicals Inc.). Previous experiments have
shown that a major tyrosine phosphorylated protein in these
cells is the p185erbB-2, which is overexpressed as a consequence
of gene amplification.44
C. In h ibition of Gr b2 SH2 Dom a in Bin d in g in Wh ole
Cells. MDA-MB-453 cells were treated with the indicated
compounds of for 3 h in serum-free media, IMEM (Gibco). Cells
were washed twice with PBS to remove the inhibitory com-
pound. To determine the amount of phosphotyrosine-bound
p185erbB-2, cells were then treated as described described
above; cells were lysed, Grb2 was immunoprecipitated, and
phosphotyrosine-containing proteins were detected using im-
munoblotting and anti-phosphotyrosine antibody.
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 a Bruker AC250
spectrometer (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. Anhy-
drous solvents were obtained commercially and used without
further drying. Preparative HPLC were conducted using a
Waters Prep LC4000 system having photodiode array detec-
tion.
Eth yl 3-(Na p h th -1-yl)p r op -2-en oa te (7). To a suspension
of NaH (60% in mineral oil, 2.4 g, 60 mmol) in anhydrous ether
(200 mL) was slowly added a solution of triethyl phosphonoac-
etate (12.33 g, 55 mmol) in anhydrous ether (25 mL) over 30
min at 0 °C, and the mixture was stirred at 0 °C (1 h). A
solution of 1-naphthaldehyde (6) (90-95%; 8.44 g, ∼54 mmol)
in ether (25 mL) was added, and the reaction was allowed to
warm to room temperature and then stirred (8 h). After an
extractive aqueous workup, the resulting organic layers were
dried (Na2SO4) and solvent removed to provide 7 as a colorless
oil49 (12.47 g, quantitative based on aldehyde): 1H NMR
(CDCl3) δ 8.62 (1H, d, J ) 15.9 Hz), 8.29 (1H, d, J ) 8.1 Hz),
7.97 (2H, m), 7.85 (1H, d, J ) 7.1 Hz), 7.54-7.70 (3H, m), 6.62
(1H, d, J ) 15.9 Hz), 4.41 (1H, q, J ) 7.1 Hz), 1.47 (3H, t, J )
7.1 Hz).
3-(Na p h th -1-yl)p r op yl 1-Azid e (10). To a solution of 9 (9.0
g, 48.4 mmol) in anhydrous CH2Cl2 (100 mL) were added MsCl
(4.5 mL, 58.1 mmol) and triethylamine (10.1 mL, 72.6 mmol)
under argon at 0 °C. The mixture was stirred first at 0 °C (2
h) and then at room temperature (30 min) and then subjected
to an aqueous extractive workup. The resulting organic layer
was dried (Na2SO4) and taken to dryness to provide crude 10
(12.55 g, 98%). This was taken up in dry DMF (100 mL), stirred
with sodium azide (3.78 g, 58.1 mmol) at 90 °C (3 h), then
cooled to room temperature, and poured into ice-water (200
mL). The mixture was extracted with dichloromethane (3 ×
50 mL), washed with brine (100 mL), dried (Na2SO4), and
taken to dryness. The residue was purified by silica gel
chromatography using a mixture of hexanes and ethyl acetate
(20:1, v/v) to afford 10 as a clear oil (8.38 g, 82% based on the
alcohol 9): 1H NMR (CDCl3) δ 8.04 (1H, dd, J ) 1.5, 7.8 Hz),
7.88 (1H, dd, J ) 2.2, 7.4 Hz), 7.76 (1H, d, J ) 8.0 Hz), 7.33-
7.58 (4H, m), 3.39 (2H, t, J ) 6.7 Hz), 3.19 (2H, t, J ) 7.4 Hz),
2.06 (2H, m).
3-(Na p h th -1-yl)p r op yla m in e (11). To a solution of 10
(1.19 g, 5.63 mmol) in anhydrous ether (20 mL) was added a
solution of LiAlH4 in THF (1 M, 6 mL, 6 mmol) under argon
at 0 °C. The reaction mixture was stirred at room temperature
(1 h), quenched at 0 °C by addition of 1 N aqueous NaOH (0.86
mL), then stirred (1 h; 0 °C), and filtered through a pad of
Celite. The filtrate was dried (Na2SO4) and taken to dryness
to provide 11 as a clear oil (1.04 g, 100%):21,22 1H NMR (DMSO)
δ 8.09 (1H, dd, J ) 2.0, 7.3 Hz), 7.90 (1H, dd, J ) 2.4, 7.0 Hz),
7.75 (1H, d, J ) 7.8 Hz), 7.34-7.57 (4H, m), 3.06 (2H, t, J )
7.8 Hz), 2.62 (2H, t, J ) 6.9 Hz), 1.72 (2H, tt, J ) 6.9, 8.0 Hz),
1.50 (2H, brs).
3-(Na p h th -1-yl)p r op a n a m id o-N-(ter t-bu toxyca r bon yl)-
L-a sp a r a gin e (12a ). Freshly prepared amine 11 (525 mg, 2.84
mmol) and N-Boc-L-Asn N-hydroxysuccinimide ester (935 mg,
2.84 mmol) in anhydrous dimethoxyethane (10 mL) were
stirred at room temperature. The reaction mixture solidified
after 10 min and was allowed to stand (1 h) and then treated
with cold H2O (30 mL). The resulting white solid was collected
by filtration, washed with H2O (5 × 10 mL), and dried in vacuo
to provide 12a in a quantitative yield: 1H NMR (DMSO) δ
8.12 (1H, d, J ) 7.3 Hz), 7.94 (2H, m), 7.80 (1H, d, J ) 7.3
Hz), 7.43-7.62 (4H, m), 7.32 (1H, s), 6.93 (2H, m), 4.26 (1H,
dt, J ) 7.1, 6.9 Hz), 3.20 (2H, dt, J ) 6.3, 6.1 Hz), 3.07 (2H, t,
J ) 7.5 Hz), 2.45 (2H, m), 1.83 (2H, tt, J ) 7.3, 7.1 Hz), 1.41
(9H, s); FABMS (+VE, Gly) m/z 400 (MH+).
Com p ou n d 14a . A solution of 12a (398 mg, 1 mmol) in 10%
(v/v) TFA in dichloromethane (11 mL) was stirred at room
temperature (overnight); then solvent was removed and resi-
due dried under vacuum to give crude amine 12b as its TFA
salt. An active ester solution prepared by stirring N-Boc-1-
amino-1-cyclohexanecarboxylic acid (13) (243 mg, 1 mmol),
HOBT‚H2O (153 mg, 1 mmol), and dicyclohexylcarbodiimide
(DCC) (240 mg, 1.15 mmol) in anhydrous DMF (3 mL) at room
temperature (30 min) was added to a solution of 12a ‚TFA and
diisopropylethylamine (DIPEA) (0.348 mL, 2 mmol) in DMF
(2 mL), and the reaction mixture was stirred at room temper-
ature (overnight). Solvent was removed under high vacuum,
and the residue was chromatographed over silica gel using first
EtOAc:CHCl3 (1:10) and then MeOH:CHCl3 (1:10) to provide
14a as a white foam (353 mg, 67% based on 12a ): 1H NMR
(DMSO) δ 8.08 (2H, m), 7.90 (1H, m), 7.75 (1H, m), 7.62 (1H,
m), 7.50 (2H, m), 7.39 (3H, m), 7.27 (1H, s), 6.92 (1H, s), 4.38
(1H, m), 3.45 (1H, m), 3.24 (1H, m), 3.03 (2H, t, J ) 6.7 Hz),
2.73 (1H, m), 2.44 (1H, dd, J ) 5.1, 16.1 Hz), 1.69-2.05 (6H,
m), 1.40-1.65 (6H, m), 1.34 (9H, s); FABMS (+VE, Gly) m/z
525 (MH+).
Eth yl 3-(Na p h th -1-yl)p r op a n oa te (8). A solution of 7 (12
g, 53.1 mmol) in 95% EtOH (180 mL) was hydrogenated over
10% Pd/C (600 mg) at room tempeature using an H2-filled
balloon (overnight). The reaction mixture was filtered and
taken to dryness to give a 8 as a clear oil50 (11.9 g, 98%): 1H
NMR (CDCl3) δ 8.05 (1H, d, J ) 7.8 Hz), 7.86 (1H, dd, J )
2.7, 7.6 Hz), 7.75 (1H, d, J ) 7.6 Hz), 7.35-7.58 (4H, m), 4.17
(2H, q, J ) 7.1 Hz), 3.44 (2H, t, J ) 8.1 Hz), 2.77 (2H, t, J )
8.3 Hz), 1.26 (3H, t, J ) 7.1 Hz).
3-(Na p h th -1-yl)p r op a n -1-ol (9). To a suspension of LiAlH4
(1.91 g, 50.4 mmol) in anhydrous ether (50 mL) was added
dropwise a solution of 8 (11.5 g, 50.4 mmol) in ether (50 mL)
over 30 min at 0 °C, under argon and then the mixture was
stirred at room temperature (overnight). The reaction mixture
was cooled to 0 °C and quenched by slow (30 min) addition of
H2O (7.2 mL, 0.4 mol); then the mixture was stirred at 0 °C (1
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
16a a n d 16d -f. Treatment of 14a with TFA:triethylsilane

32 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 1
Yao et al.
(100: 2) at room temperature (1 h) and removal of TFA
provided 14b‚TFA, which was partitioned between aqueous
NaHCO3 and EtOAC to provide free amine 14b [FABMS
(+VE, Gly) m/z 425 [M + H]+; HRMS calcd for C24H33N4O3
(MH+) m/z 425.2552, found 425.2557]. To a solution of 14b
(106 mg, 0.25 mmol) in anhydrous DMF (1 mL) was added an
active ester solution formed by reacting appropriately pro-
tected pTyr mimetic 15a , 15d ,24 15e,25 or 15f25 (0.3 mmol),
HOBT‚H2O (41 mg, 0.3 mmol), and DIPCDI (47 µL, 0.3 mmol)
in anhydrous DMF (0.5 mL) at room temperature (10 min).
The combined reaction mixture was then stirred at room
temperature overnight. Solvent was removed under high
vacuum, and residue was purified by silica gel chromatography
(EtOAc in CHCl3) to provide products as white foams. Com-
pound 16a : 90% yield; FABMS (+VE, NBA) m/z 866 (MH+).
Compound 16d : 69% yield (an unresolved mixture of diaster-
eomers, epimeric at the Pmp R-carbon); FABMS (+VE, NBA)
m/z 982.8 (MH+). Compound 16e: 79% yield; FABMS (+VE,
NBA) m/z 1054.6 (MH+). Compound 16f: 79% yield; FABMS
(+VE, NBA) m/z 1068.3 (MH+).
Gen er a l P r oced u r e for Con ver sion of Com p ou n d s 16a
a n d 16d -f to Th eir N-Acetyl Der iva tives 17a a n d 17d -
f, Resp ectively. To solutions of 16a and 16d -f (0.1-0.2
mmol) in anhydrous MeCN (2.0 mL) was added piperidine (4
equiv), and the solutions were stirred at room temperature (1
h). Solvent and excess piperidine were removed under high
vacuum; then residues were taken up in anhydrous MeCN and
treated with N-acetylimidazole (4 equiv) at room temperature.
After all amine had reacted (from 3 to 24 h) solvent was
removed, and residues were purified by silica gel chromatog-
raphy (EtOAc in CHCl3) to provide pure products as resins in
quantitative yield, except where noted. Compound 17a: FABMS
(+VE, Gly) m/z 686 (MH+); HRMS calcd for C39H52N5O6 m/z
686.3918 [M + H]+, found 686.3882. Compound L-17d : faster,
29% yield.41 Compound D-17d : slower, 18% yield.41 Compound
17e: FABMS (+VE, NBA) m/z 874.6 (MH+). Compound 17f:
FABMS (+VE, NBA) m/z 885.5 (MH+).
protected tripeptide as a white foam (197 mg, 92%): 1HNMR
(DMSO, 250 MHz): δ 8.20 (1H, s), 8.00-8.15 (2H, m), 7.90
(1H, m), 7.73 (1H, m), 7.36-7.50 (10H, m), 7.21 (1H, d, J )
7.3 Hz), 6.91 (1H, s), 4.36 (2H, m), 4.10 (4H, m), 3.38-3.50
(2H, m), 3.17 (2H, m), 3.04 (2H, t, J ) 7.8 Hz), 2.66 (2H, m),
1.35-2.10 (12H, m), 1.30 (9H, s), 1.21 (3H, t, J ) 7.1 Hz), 1.20
(3H, t, J ) 7.1 Hz).
A portion of this material (130 mg, 0.152 mmol) in dichlo-
romethane (1 mL) with TFA (1 mL) was stirred at room
temperature (1 h); then solvent was removed. The resulting
foam was dissloved in DMF (1 mL) containing triethylamine
(46 µL, 0.334 mmol) and 1-acetylimidazole (25 mg, 0.230
mmol), and the mixture was stirred at room temperature (2
h) and then concentrated. Chromatographic purification of the
residue on silica gel (CHCl3:MeOH, 15:1) provided 17c as a
white foam (122 mg, 100%): 1H NMR (DMSO) δ 8.25 (1H, m),
7.87-8.13 (4H, m), 7.74 (1H, t, J ) 4.9 Hz), 7.36-7.53 (10H,
m), 6.91 (1H, s), 4.70 (1H, m), 4.36 (1H, m), 6.91 (1H, s), 4.70
(1H, m), 4.36 (1H, m), 4.09 (4H, m), 3.41-3.52 (2H, m), 3.16
(2H, m), 3.04 (2H, t, J ) 8.0 Hz), 2.56-2.63 (2H, m), 1.74 (3H,
s), 1.20 (6H, t, J ) 7.1 Hz), 1.15-2.01 (12H, m).
F in a l P r od u ct 18a . A solution of 17a (0.21 mmol) in TFA
(1.9 mL) with H2O (0.1 mL) and triethylsilane (50 µL) was
stirred at room temperature (1 h), then taken to dryness, and
purified by HPLC using a Prep NovaPak HR C18 6-µm radial
compression cartridge (200 mm × 40 mm diameter) with a
flow rate of 50 mL/min and a linear gradient from 10% to 50%
B over 20 min (solvent A, 0.1% aqueous TFA; solvent B, 0.1%
TFA in MeCN). Product 18a was obtained as a white solid
(118 mg, 88% yield): 1H NMR (DMSO) δ 8.12-8.23 (3H, m),
7.93-8.00 (2H, m), 7.80 (1H, dd, J ) 4.3, 4.7 Hz), 7.54 (3H,
m), 7.43 (3H, m), 7.00-7.50 (1H, br), 7.07 (2H, d, J ) 7.5 Hz),
6.94 (1H, s), 6.67 (2H, d, J ) 7.5 Hz), 4.58 (1H, m), 4.40 (1H,
m), 2.94-3.29 (5H, m), 2.55-2.79 (3H, m), 2.05-1.10 (12H,
m), 1.81 (3H, s); FABMS (+VE, Gly) m/z 630 (MH+); HR-
FABMS calcd for C35H44N5O6 (MH+) m/z 630.33292, found
630.3339.
Com p ou n d 18b. A solution of L-17b (48.7 mg, 0.059 mmol),
thioanisole (0.100 mL), TFA (0.5 mL), and dichloromethane
(0.5 mL) was stirred at room temperature (overnight), then
solvent was evaporated, and the residue was dried under
vacuum. The crude product was dissolved in MeCN:water (1:
1, 2 mL), filtered, and purified by HPLC, using a Vydac C18
column (250 mm × 20 mm diameter) with a flow rate of 20
mL/min and a linear gradient from 10% to 100% B over 40
min (solvent A, 0.1% aqueous TFA; solvent B, 0.1% TFA in
MeCN). Product 18b was obtained as a white solid (26 mg,
62%): 1H NMR (DMSO) δ 8.22 (2H, m), 8.07 (1H, m), 7.91
(2H, m), 7.74 (1H, m), 7.36-7.53 (6H, m), 7.17 (2H, d, J ) 8.3
Hz), 7.03 (2H, d, J ) 8.3 Hz), 6.90 (1H, s), 4.64 (1H, m), 4.35
(1H, m), 3.17 (3H, m), 2.72 (1H, m), 2.59 (1H, m), 1.76 (3H, s),
1.13-2.07 (12H, m) ppm; FABMS (-VE, Gly) m/z 708 (M -
H); HR-FABMS calcd for C35H43N5O9P (M - H) m/z 708.2798,
found 708.2803.
Com p ou n d 18c. A mixture of 17c (70 mg, 0.088 mmol),
TFA (1.2 mL), thioanisole (140 µL, 1.2 mmol), TMSBr (155
µL, 1.2 mmol), and m-cresol (92 µL, 0.876 mmol) was stirred
at room temperature (24 h); then the mixture was concentrated
under argon and treated with cold ether:hexane (1:1; 7 mL)
to provide a precipitate. Supernatant was removed following
centrifugation, and solid was washed three times by resus-
pension and centrifugation. The resulting solid was purified
by HPLC as indicated for the purification of 18a using a linear
gradient from 5% to 50% B over 25 min to provide 18c as a
white solid (43 mg, 66%yield): 1H NMR (DMSO) δ 8.29 (1H,
d, J ) 7.4 Hz), 8.25 (1H, s), 8.09 (1H, t, J ) 6.1 Hz), 7.99 (1H,
d, J ) 8.1 Hz), 7.89 (1H, dt, J ) 1.9, 5.4 Hz), 7.74 (1H, t, J )
4.6 Hz), 7.33-7.54 (10H, m), 6.91 (1H, s), 4.65 (1H, m), 4.33
(1H, q, J ) 6.6 Hz), 3.17 (2H, m), 3.04 (3H, m), 2.81 (1H, dd,
J ) 13.8, 10.7 Hz), 2.65 (1H, dd, J ) 6.3, 15.1 Hz), 2.56 (1H,
dd, J ) 4.9, 15.1 Hz), 1.77 (3H, s), 1.10-2.05 (12H, m); FABMS
(-VE, Gly) m/z 742 (M - H).
Com p ou n d 17b. A solution of 14a (168 mg, 0.320 mmol)
in 20% (v/v) TFA in dichloromethane (3.6 mL) was stirred at
room temperature (overnight); then solvent was removed and
residue was dried to give crude 14b‚TFA as a syrup. An active
ester solution formed by reacting N-Ac-L-Tyr((tert-BuO)2PO)-
OH (146 mg, 0.352 mmol), HOBT‚H2O (54 mg, 0.352 mmol),
and DCC (73 mg, 0.352 mmol) in DMF (1 mL) (20 min) was
added to a solution of 14b‚TFA and DIPEA (0.113 mL, 0.64
mmol) in DMF (2 mL), and the reaction mixture was stirred
at room temperature (7 h). Solvent was removed under high
vacuum and the residue was chromatographed on silica gel,
elueting with MeOH:CHCl3 (1:40, 1:20, then 1:10) to provide
L-17b41 as a white foam (60.4 mg). Also obtained was the
diastereomer D-17b41 (29 mg) resulting from partial racem-
ization at the tyrosyl R-carbon. Additionally, an unseparated
mixture of L-17b and D-17b (79.2 mg) was collected, providing
a 64% combined yield of tyrosyl-coupled product. L-17b: 1H
NMR (DMSO) δ 8.22 (2H, m), 8.10 (1H, m), 7.97 (1H, d, J )
8.1 Hz), 7.89 (1H, m), 7.74 (1H, m), 7.49 (3H, m), 7.37 (3H,
m), 7.21 (2H, d, J ) 8.5 Hz), 7.03 (2H, d, J ) 8.3 Hz), 6.90
(1H, s), 4.60 (1H, m), 4.35 (1H, m), 3.16 (2H, m), 3.03 (4H, m),
2.73 (1H, t, J ) 11.4 Hz), 2.59 (1H, dd, J ) 6.6, 11.9 Hz), 1.75
(3H, s), 1.43 (18H, s), 1.17-2.05 (12H, brm). D-17b: 1HNMR
(DMSO) δ 8.49 (1H, s), 8.45 (1H, d, J ) 5.4 Hz), 8.09 (1H, m),
7.90 (1H, m), 7.76 (1H, t, J ) 4.6 Hz), 7.65 (1H, d, J ) 8.1
Hz), 7.27-7.52 (8H, m), 7.06 (2H, d, J ) 8.3 Hz), 6.87 (1H, s),
4.57 (1H, m), 4.42 (1H, dt, J ) 5.1, 9.3 Hz), 3.28 (1H, m), 3.05
(3H, m), 2.87 (2H, m), 2.59 (2H, m), 1.77 (3H, s), 1.43 (18H, s),
1.15-2.00 (12H, brm); FABMS (+VE, NBA) m/z 822 (MH+).
Com p ou n d 17c. To a solution of amine 14b (106 mg, 0.25
mmol) in DMF (1 mL) was added an activated ester solution
formed by reacting L-N-Boc-F2Pmp(OEt)2-OH (15c)23 (135 mg,
0.30 mmol), HOBt‚H2O (46 mg, 0.30 mmol), and DIPCDI (47
µL, 0.30 mmol) in DMF (1.0 mL) (20 min), and the combined
solution was stirred at room temperature (overnight). DMF
was removed under high vacuum and residue was chromato-
graphed on silica gel (CHCl3:MeOH, 25:1) to provide the Boc-
F in a l P r od u ct L-18d . Treatment of L-17d (0.035 mmol) as
described above for the preparation of 18a and HPLC purifica-

Potent Inhibition of Grb2 SH2 Domain Binding
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 1 33
tion of crude product as described therein provided L-18d as
a white solid (17 mg, 69% yield):41 1H NMR (DMSO) δ 8.27
(2H, m), 8.12 (1H, m), 8.01 (1H, d, J ) 8.1 Hz), 7.94 (1H, m),
7.80 (1H, dd, J ) 4.3, 4.7 Hz), 7.55 (3H, m), 7.43 (3H, m), 7.18
(4H, m), 6.96 (1H, s), 4.67 (1H, m), 4.41 (1H, m), 3.23 (2H, m),
3.08 (3H, m), 2.94 (2H, d, J ) 21.4 Hz), 2.55-2.85 (3H, m,
partially covered by DMSO peaks), 1.11-2.25 (12H, m), 1.80
(3H, s); FABMS (-VE, Gly) m/z 706 (M - H); HR-FABMS
calcd for C36H45N5O8P (M - H) m/z 706.3006, found 706.2960.
F in a l P r od u ct D-18d . Treatment of D-17d (0.035 mmol)
as described above and HPLC purification of crude product
as described therein provided D-18d as a white solid (14 mg,
84% yield):41 1H NMR (DMSO) δ 8.57 (1H, s), 8.47 (1H, d, J )
5.1 Hz), 8.15 (1H, m), 7.95 (1H, m), 7.81 (1H, dd, J ) 4.2, 4.7
Hz), 7.66 (1H, d, J ) 8.1 Hz), 7.56 (2H, m), 7.45 (3H, m), 7.34
(1H, s), 7.24 (4H, m), 6.95 (1H, s), 4.62 (1H, m), 4.47 (1H, m),
3.34 (2H, m), 2.82-3.15 (3H, m), 2.95 (2H, d, J ) 21.4 Hz),
2.50-2.75 (3H, m), 1.84 (3H, s), 0.75-2.18 (12H, m); FABMS
(-VE, Gly) m/z 706 (M - H); HR-FABMS calcd for C36H45N5O8P
(M - H) m/z 706.3006, found 706.3057.
F in a l P r od u ct 18e. Treatment of 17e (0.20 mmol) as
described above for the synthesis of 18a and HPCL purification
of crude product as described therein provided pure 18e as a
white solid (114 mg, 75% yield): 1H NMR (DMSO) δ 8.23 (2H,
m), 8.12 (1H, m), 7.93-8.00 (2H, m), 7.79 (1H, t, J ) 4.3 Hz),
7.53 (3H, m), 7.42 (3H, m), 6.93 (2H, s), 6.78 (2H, s), 4.71 (2H,
s), 4.68 (2H, s), 4.58 (1H, m), 4.38 (1H, m), 3.20 (2H, m), 3.08
(2H, t, J ) 6.4 Hz), 2.95 (1H, dd, J ) 13.7, 2.4 Hz), 2.73 (1H,
dd, J ) 13. 6, 10.3 Hz), 2.63 (2H, t, J ) 5.4 Hz), 1.82 (3H, s),
1.10-2.05 (12H, m); FABMS (-VE, Gly) m/z 760 (M - H); HR-
FABMS calcd for C39H46N5O11 (M - H) m/z 760.3194, found
760.3155.
F in a l P r od u ct 18f. A solution of 17f (0.075 mmol) and
tetrabutylammonium fluoride (TBAF) (1.0 M in THF, 300 µL)
in anhydrous DMF (2 mL) was stirred at room temperature
(overnight) and then taken to dryness under high vacuum.
Residue was dissolved in a mixture of TFA (1.9 mL), H2O (0.1
mL), and triethylsilane (50 µL), stirred at room temperature
(1 h), then taken to dryness, and purified by HPLC using a
Prep NovaPak HR C18 6-µm radial compression cartridge (200
mm × 40 mm diameter) as described above for 18a and 18e
to provide 18f as its mono-TBAF salt (36 mg) and di-TBAF
salt (17 mg). A portion (16 mg) of mono-TBAF salt was
repurified by HPLC to provide 18f as its free diacid (11 mg):
1H NMR (DMSO) δ 8.34 (1H, s), 8.24 (1H, d, J ) 6.8 Hz), 8.11
(1H, m), 8.02 (1H, d, J ) 8.1 Hz), 7.95 (1H, m), 7.78 (1H, m),
7.70 (1H, s), 7.54 (3H, m), 7.34-7.43 (4H, m), 6.95 (1H + 1H,
d + s, J ) 8.5 Hz), 4.79 (2H, s), 4.64 (1H, m), 4.40 (1H, m),
3.40 (2H, m, partially covered by water peak), 3.22 (1H, m),
3.00-3.10 (3H, m), 2.50-2.80 (2H, m, partially covered by
DMSO), 1.79 (3H, s), 2.05-1.1 (12H, m); FABMS (-VE, Gly)
m/z 730 (M - H); HR-FABMS calcd for C38H44N5O10 (M - H)
m/z 730.3088, found 730.2997.
Hz), 2.72 (1H, dd, J ) 4.6 13.7 Hz), 2.67 (2H, m, partially
covered by DMSO), 1.35 (18H, s), 1.05-2.04 (12H, m).
The crude product (0.15 mmol) was then dissolved in DMF
(1 mL) containing DIEA (44 µL, 0.25 mmol) and tert-butylox-
alyl chloride (0.23 mmol) (prepared by reaction of oxalyl
chloride with tert-butyl alcohol). After 10 min the reaction
mixture was concentrated and purified by silica gel chroma-
tography (CHCl3:MeOH, from 30:1 to 20:1) to provide L-Pmp-
containing diastereomer L-19d (59.4 mg, 44%) (assignment of
L-Pmp configuration was based on analogy to the N-acetyl
compound L-17d ), followed by the D-Pmp-containing diaste-
reomer D-19d (52.3 mg, 39%). For L-19d : 1H NMR (DMSO) δ
8.69 (1H, d, J ) 8.1 Hz), 8.32 (1H, s), 8.08 (1H, m), 7.97 (1H,
d, J ) 7.8 Hz), 7.89 (1H, m), 7.74 (1H, t, J ) 4.8 Hz), 7.49
(3H, m), 7.38 (3H, m), 7.16 (4H, m), 6.92 (1H, s), 4.63 (1H, m),
4.38 (1H, m), 2.85-3.25 (8H, m), 2.67 (1H, dd, J ) 6.8, 15.9
Hz), 2.55 (1H, dd, J ) 5.4, 15.9 Hz, partially covered by DMSO
peaks), 1.06-2.05 (12H, m), 1.41 (9H, s), 1.31 (18H, s); FABMS
(+VE, Gly) m/z 928 (M + Na), 906 (MH+).
Com p ou n d 19f. A solution of 16f (88 mg, 0.082 mmol) in
anhydrous MeCN (2 mL) with piperidine (27 µL, 0.33 mmol)
was stirred at room temperature (1 h), then taken to dryness,
and placed under high vacuum. Residue was dissolved in
anydrous DMF (0.5 mL) and treated overnight with an
activated ester solution formed by stirring (10 min) oxalic acid
mono-tert-butyl ester (24 mg, 0.16 mmol), HOBT‚H2O (22 mg,
0.16 mmol), and DIPCDI (26 µL, 0.16 mmol) in anhydrous
DMF (0.5 mL). Solvent was removed under high vacuum and
residue purified by silica gel chromatography (MeOH in
EtOAc) to provide 19f as a syrup (45 mg, 56% yield): 1H NMR
(CDCl3) δ 8.13 (2H, m), 7.98 (1H, m), 7.80-7.69 (2H, m), 7.60-
7.27 (4H, m), 6.80 (2H, m), 6.42 (1H, bs), 5.78 (1H, bs), 4.80-
4.66 (3H, m), 4.62 (2H, s), 4.50-4.40 (3H, m), 3.50-3.35 (2H,
m), 3.25-3.00 (6H, m), 2.15-1.15 (14H, m), 1.56 (18H, s), 0.14
(9H, s); FABMS (+VE, NBA) m/z 975.2 (MH+).
F in a l P r od u ct 20d . A solution of L-19d (45 mg, 0.0497
mmol) in dichloromethane (0.5 mL) with TFA (0.5 mL) and
triethylsilane (20 µL) was stirred at room temperature (2 h);
then the mixture was concentrated and dried under vacuum.
Residue was purified by HPLC using an Advantage C18 5-µm
column (20 mm diameter × 250 mm) with a binary solvent
mixture of A (0.1% aqueous TFA) and B (0.1% TFA in MeCN)
and a linear gradient over 25 min from 5% to 60% B. Final
product L-20d was obtained as a white solid (31 mg, 84%): 1H
NMR (DMSO) δ 8.77 (1H, d, J ) 8.1 Hz), 8.32 (1H, s), 8.08
(1H, m), 7.97 (1H, d, J ) 8.1 Hz), 7.91 (1H, m), 7.75 (1H, m),
7.50 (3H, m), 7.39 (3H, m), 7.14 (4H, m), 6.91 (1H, s), 4.68
(1H, m), 4.37 (1H, m), 3.16 (3H, m), 3.03 (3H, m), 2.89 (2H, d,
J ) 21.2 Hz), 2.68 (1H, dd, J ) 6.3, 14.6 Hz), 2.53 (1H, dd, J
) 4.9, 14.6 Hz), 1.15-2.05 (12H, m) (-COOH and P(O)(OH)2
did not show); FABMS (-VE, Gly) m/z 736 [(M - H)-], 692
[(M - H - CO2)-], 664 [(M - H - CO2 - CO)-], 322, 212, 79
[(PO3)-]; HR-FABMS calcd for C36H44N5O10P (M - H) m/z
736.2748, found 736.2817.
Com p ou n d 19d . A mixture of diastereomers epimeric at
the Pmp R-carbon (16d ) (150 mg, 0.15 mmol) was treated with
piperidine (48 µL, 0.45 mmol) in acetonitrile (2 mL) at room
temperature (2 h), then solvent was removed, and residue was
dried under high vacuum. At this stage a portion of crude
product was purified by silica gel chromatography (CHCl3:
MeOH, 20:1 to 15:1) to afford two diastereomeric free amines,
the L-Pmp-containing compound followed by the D-Pmp-
containing coumpound.41 For the L-Pmp-containing isomer: 1H
NMR (DMSO) δ 8.18 (1H, s), 8.08 (2H, m), 7.90 (1H, m), 7.75
(1H, m), 7.64 (1H, m), 7.50 (2H, m), 7.41 (3H, m), 7.01-7.12
(4H, m), 6.92 (1H, s), 4.36 (1H, m), 3.51 (1H, m), 2.90-3.27
(5H, m, partially covered by water peak), 2.94 (2H, d, J ) 21.3
Hz), 2.50-2.75 (3H, m, partially covered by DMSO), 1.35 (18H,
s), 1.10-2.00 (12H, m); FABMS (+VE, Gly) m/z 800 (M + Na),
778 (MH+), 722 (MH+ - C4H8). For the D-Pmp-containing
isomer: 1H NMR (DMSO) δ 8.32 (1H, s), 8.09 (2H, m), 7.89
(1H, m), 7.74 (1H, m), 7.64 (1H, m), 7.44-7.55 (3H, m), 7.40
(1H, s), 7.38 (1H, d, J ) 1.7 Hz), 7.06-7.15 (4H, m), 6.97 (1H,
s), 4.35 (1H, m), 3.52 (1H, m), 3.18 (2H, m), 3.05 (2H, t, J )
7.6 Hz), 2.95 (2H, d, J ) 21.5 Hz), 2.85 (1H, dd, J ) 1.2, 13.7
D-20d was prepared as above from D-19d in 69% yield: 1H
NMR (DMSO) δ 8.95 (1H, d, J ) 7.1 Hz), 8.45 (1H, s), 8.08
(1H, m), 7.89 (1H, m, 7.77 (1H, dd, J ) 9.3, 2.4 Hz), 7.65 (1H,
d, J ) 7.6 Hz), 7.50 (2H, m), 7.38 (4H, m), 7.16 (4H, m), 6.91
(1H, s), 4.66 (1H, m), 4.40 (1H, m), 3.16 (2H, m), 3.02 (4H, m),
2.90 (2H, d, J ) 21.5 Hz), 2.56 (2H, m), 0.94-2.08 (12H, m)
(-COOH and P(O)(OH)2 did not show); FABMS (+VE, Gly)
m/z 738 (MH+).
F in a l P r od u ct 20f. A solution of 19f (37 mg, 0.038 mmol)
in MeCN (1.0 mL) with 1.0 M TBAF in THF (0.12 mmol) was
stirred at room temperature (2 days), then solvent was
removed, and the residue was dried under vacuum. The
resulting material was dissolved in a mixture of TFA (950 µL),
H2O (50 µL), and triethylsilane (25 µL) and stirred at room
temperature (3 h), then taken to near dryness. Treatment with
cold ether gave crude product as a white solid, which was dried
under an argon stream and purified twice by HPLC as
described above for the purification of 18f, providing 20f as a
white solid (17 mg, 58%): 1H NMR (DMSO) δ 8.79 (1H, d, J )
8.1 Hz), 8.4 (1H, s), 8.06 (1H, m), 7.98 (1H, d, J ) 7.5 Hz),

34 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 1
Yao et al.
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