Potent dipeptide inhibitors of the pp60(c-src) SH2 domain

Article abstract of DOI:10.1021/jm970853a

The design, synthesis, and evaluation of dipeptide analogues as ligands for the pp60(c-src) SH2 domain are described. The critical binding interactions between Ac-Tyr-Glu-N(n-C₅H₁₁)₂ (2) and the protein are established and

Full text of DOI:10.1021/jm970853a

1894
J . Med. Chem. 1998, 41, 1894-1908
P oten t Dip ep tid e In h ibitor s of th e p p 60c-sr c SH2 Dom a in
Gregory J . Pacofsky,*,† Karen Lackey,† Krystal J . Alligood,‡ J udd Berman,† Paul S. Charifson,§,1
Renae M. Crosby,‡ George F. Dorsey, J r.,† Paul L. Feldman,† Tona M. Gilmer,‡ Conrad W. Hummel,†,1
Steven R. J ordan,§,1 Christopher Mohr,§ Lisa M. Shewchuk,§ Daniel D. Sternbach,† and Marc Rodriguez†,1
Departments of Medicinal Chemistry, Cancer Biology, and Structural Chemistry, Glaxo Wellcome Inc., 5 Moore Drive,
Research Triangle Park, North Carolina 27709
Received December 22, 1997
The design, synthesis, and evaluation of dipeptide analogues as ligands for the pp60c-src SH2
domain are described. The critical binding interactions between Ac-Tyr-Glu-N(n-C5H11)2 (2)
and the protein are established and form the basis for our structure-based drug design efforts.
The effects of changes in both the C-terminal (11-27) and N-terminal (51-69) portions of the
dipeptide are explored. Analogues with reduced overall charge (92-95) are also investigated.
We demonstrate the feasibility of pairing structurally diverse subunits in a modest dipeptide
framework with the goal of increasing the druglike attributes without sacrificing binding
affinity.
Protein tyrosine kinases play a role in signal trans-
duction through phosphorylation of substrate proteins
as well as corresponding recognition and binding of
other phosphoproteins.2,3 The prototypical nonreceptor
tyrosine kinase pp60c-src contains a catalytic kinase
region, the SH1 (src homology 1) domain, which is
highly conserved among members of the src family, an
SH2 (src homology 2) domain4,5 that binds phospho-
tyrosine-containing proteins, and an SH3 (src homology
3) domain4,5 that recognizes proline-rich sequences.6
Overexpression or hyperactivation of pp60c-src has been
implicated in the development of human colon and
tion13 of a high-affinity (IC50 ≈ 1 µM) pentapeptide,
AcY*EEIE (1), bound to the src SH2 domain has
prompted the search for alternative peptide and/or
peptidomimetic ligands.24-27 These results reinforced
both the feasibility of a structure-based design approach
and our desire to identify lower-molecular-weight in-
hibitors using X-ray crystallographic studies to guide
target selection. A dipeptide, Ac-Tyr-Glu-N(n-C5H11 2
)
(2), emerged as our first success in this strategy and
served as the lead molecule for subsequent studies. This
result confirmed the viability of an approach to reduce
the size and complexity of SH2 ligands as part of a drug
discovery program.
breast carcinomas;2 thus, entities that modulate pp60c-src
-
regulated signal transduction pathways offer potential
value as antiproliferative agents.7-11
The src SH2 domain has been the subject of numerous
investigations. This domain is a region of approxi-
mately 100 amino acids that shares sequence similarity
with other members of the src family of proteins, as well
as other nonreceptor kinases (abl, lck, fyn, etc.),12 and
preferentially binds tyrosine-phosphorylated (Y*) pro-
teins. The SH2 domain may participate in the trans-
mission of signals through the formation of complexes
with specific phosphoproteins such as epidermal growth
factor receptor (EGFR),13 platelet-derived growth factor
receptor (PDGFR),14 and focal adhesion kinase (FAK).15
Alternatively, SH2 domains may act as adapters be-
tween phosphorylated receptors and other signaling
proteins16,17 or by regulating the activity of the kinase
domain.18 Thus, an entity that specifically disrupts or
inhibits protein-protein interactions involving the src
SH2 domain might interrupt signal transduction pro-
cesses perhaps making such inhibitors useful chemo-
therapeutic agents.
The emergence of information on the role of pp60c-src
in signal transduction and our ability to rapidly char-
acterize the interaction of ligands with the src SH2
domain at a molecular level prompted us to target SH2
inhibitors as novel cancer chemotherapeutics. The
results of our investigations including the successful
identification of dipeptide ligands are described herein.
The preferred binding sequence for the src SH2
domain has been determined,19 and structural studies
have defined the conformation of ligands complexed to
the src SH2 domain.20-23 The subsequent crystalliza-
Ch em istr y
† Department of Medicinal Chemistry.
‡ Department of Cancer Biology.
A synthetic route was developed to introduce diverse
C-terminal substitutions onto the dipeptide framework
§ Department of Structural Chemistry.
S0022-2623(97)00853-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/23/1998

Dipeptide Inhibitors of the pp60c-src SH2 Domain
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1895
Sch em e 1a
tial for epimerization at the carbon atom adjacent to
the reacting center,30 a number of methods were sur-
veyed to identify a procedure amenable to the synthesis
of these analogues. Ultimately, a protocol involving low-
temperature formation of the mixed anhydride (-78 °C,
4-methylmorpholine, isobutyl chloroformate, THF) fol-
lowed by the addition of the desired amine generated a
diverse set of amides with <5% of the epimeric adduct,
as determined by 1H NMR and reverse-phase HPLC
analyses.31 Removal of the phosphate-protecting groups
and concomitant unmasking of the glutamic acid deliv-
ered compounds 11-27.
The preparation of dipeptide analogues 51-69, con-
taining variations in the N-terminus of the dipeptide,
is depicted in Scheme 2. Conversion of amino acid 28
to the corresponding amide 29 was accomplished using
the mixed anhydride protocol.32 Removal of the nitrogen-
protecting group and subsequent coupling (DCC,
HOBT)33 with Z-Tyr-OH delivered 30. The phosphate
moiety was introduced as the diester [NaH, ClP(O)(Ot-
Bu)2]34 via alkylation of the phenolic oxygen to afford
dipeptide 31. Attachment of the N-terminal capping
group was achieved by removing the Z group by hydro-
genolysis, and the primary amine 32 was reacted with
electrophiles to produce 33-50. Finally, acid-catalyzed
deprotection of the phosphate produced the targets 51-
69; for 69 this treatment with acid also cleaved the BOC
group introduced during the synthesis of fragment 71.
a
Reagents: (a) HCl/dioxane; (b) BOC-Tyr-OH, BOP, HOBT,
Et3N; (c) AcOSuc; (d) [(BnO)2P(O)]2O; (e) HF-pyridine; (f) NMM,
ClCO2i-Bu, HNR1R2; (g) H2, Pd-C.
in the penultimate step (Scheme 1). Toward that end,
the key coupling intermediate 8 was synthesized. Re-
moval of the carbamate protecting group in 3 and
subsequent coupling of H-Glu(OBn)-OTMSE (4) with
BOC-Tyr-OH delivered dipeptide 5. Installation of the
N-terminal acetyl group yielded 6 which was phospho-
rylated with tetrabenzyl pyrophosphate.28 Subsequent
fluoride-catalyzed cleavage29 of the silyl group in 7 set
the stage for preparation of a series of C-terminal
analogues. Since such a strategy introduces the poten-
The synthesis of analogues 92-95, with variations in
the Y* + 1 position of the dipeptide framework, is shown
in Scheme 3. Conversion of the BOC-protected amino
acids 72-75 to the corresponding amides 76-79 was
accomplished using the mixed anhydride protocol.32
Sch em e 2a
a
Reagents: (a) NMM, ClCO2i-Bu, HN(n-C5H11)2; (b) H2, Pd-C; (c) Z-Tyr-OH, DCC, HOBT, Et3N; (d) NaH, ClP(O)(Ot-Bu)2; (e) R2CO2H,
(R2CO)2O, R2COCl, or R2NCO; (f) TFA; (g) SOCl2, DMF, pyridine; (h) PhCH2OH, Et3N, DMAP; (i) NaN3; (j) BOC2O, H2, Pd-C.

1896 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Pacofsky et al.
Sch em e 3a
a
Reagents: (a) NMM, ClCO2i-Bu, HN(n-C5H11)2; (b) HCl/dioxane; (c) BOC-Tyr-OH, DCC, HOBT, Et3N; (d) AcOH, DCC, HOBT, Et3N;
(e) NaH, ClP(O)(Ot-Bu)2; (f) HCl/Et2O.
Removal of the nitrogen-protecting group and subse-
quent coupling (DCC, HOBT)33 with BOC-Tyr-OH de-
livered the dipeptides 80-83; repetition of this sequence
using acetic acid in the final coupling procedure deliv-
ered 84-87. The phosphate moiety was introduced as
before followed by deprotection of the phosphate to
produce the targets 92-95.
framed by Ile217, Gly239, and Leu240; (3) interaction
between the Y* ring and Arg158 as well as hydrophobic
packing against the side-chain atoms of Lys206; and (4)
alignment of the phosphate with Ser180, Thr182, Arg158,
and Arg178.
Encouraged by the high-affinity binding obtained with
2, we prepared a series of dipeptide inhibitors varied
at the carboxyl terminus. A diverse set of amines were
chosen to help elucidate the gross binding requirements
of the lipophilic binding site. Compounds 9-14 (Table
1) were designed to determine the relative importance
of the n-pentyl chains. The unfunctionalized primary
amide 10 proved to bind weakly (ratio ) 200) when
compared with the pentapeptide 1. A dramatic im-
provement in binding affinity was observed with the
single C-terminal n-pentyl substituent present in 11.
A further, but comparatively smaller, increase in bind-
ing affinity was realized with the N-methyl, N-pentyl
substitution of 12 presumably due to an increase in
conformational rigidity35 of this tertiary amide. The
length of the alkyl chain was also varied, compounds
13 and 14, to discern the depth of this pocket. While
there were changes in binding affinity across the range
of alkyl groups studied, the optimum substitution of the
dipeptide was achieved using the di-n-pentyl amide as
in 2.
In the X-ray crystal structure of the dipeptide 2, one
of the n-pentyl chains faces the predominantly lipophilic
cavity, occupied by Ile in the case of 1, while the other
alkyl chain appears to be directed away from the protein
surface toward solvent. Compounds 15 and 16 were
designed to probe the use of differentially substituted
amides, that is, possessing one lipophilic fragment and
one more hydrophilic subunit, in an effort to accom-
modate both of the binding relationships mentioned
above simultaneously. This approach offered the po-
tential to generate a better overall solvation/desolvation
profile for the ligand during its association with the
protein. Unfortunately, no improvement in binding
versus 2 was observed with either 15 or 16.
Resu lts a n d Discu ssion
The X-ray crystallographic analysis of the pentapep-
tide 1 complexed to the src SH2 domain clearly estab-
lished the presence of two distinct binding regions on
the surface of the protein: (1) a pocket in which the
phosphotyrosine residue is bound and (2) a lipophilic
pocket in which the Ile residue at the Y* + 3 position is
bound. We reasoned that a dipeptide with appropriate
substitution at its carboxyl terminus might bind simi-
larly to the src SH2 domain if the phosphotyrosine-
containing subunit remained intact. Using an in vitro
binding assay,13 dipeptides were evaluated as inhibitors
of src SH3-SH2:phosphoprotein interactions using 1 as
the standard; for the purpose of comparison and to
normalize data from different experiments, results for
individual peptides are presented as a ratio, IC50(test)/
IC50(standard). A survey of hydrophobic amines pro-
duced Ac-Y*-E-N(n-C5H11)2 (2)23 which binds with high
affinity (ratio ≈ 8) to the src SH2 domain. The key
interactions between the lipophilic cleft of the src SH2
domain and the di-n-pentyl amide of 2 observed in the
crystal structure (Figure 1) marked the successful
replacement of the three C-terminal residues in 1 with
a fragment that would access the hydrophobic pocket,
occupied by Ile at the Y* + 3 position in 1, without
compromising interactions within the Y* binding pocket.
In fact, the X-ray structure mirrored that obtained with
the pentapeptide: (1) the dipeptide binding in an
extended conformation with a hydrogen bond between
its Tyr-NH and His204; (2) placement of a single
n-pentyl chain into the Y* + 3 hydrophobic pocket

Dipeptide Inhibitors of the pp60c-src SH2 Domain
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1897
F igu r e 1. X-ray structure of 2 bound to the pp60c-src SH2 domain.
An alternative series of compounds were designed to
incorporate functionality in the amide that would fill
the Y* + 3 binding pocket and/or potentially form a
hydrogen-bonding interaction with amino acid residues
lining this pocket. To explore this idea of increasing
the size of the amide, variously substituted indoles were
prepared, and evaluation of this series revealed a
surprising tolerance for diversity in terms of binding
within the Y* + 3 pocket. For example, the indole 22
and both of the related, substituted indoles (23, 24) were
equipotent when linked through a two-carbon chain;
that is, no increase in binding affinity was observed with
the inclusion of a potential hydrogen bond partner
substituted on the indole ring. Interestingly, a 3-fold
improvement in binding affinity was observed with a
three-carbon tether (20, 21).
Significant improvements in binding affinity were
observed using modifications which optimized a hydro-
gen-bonding interaction between the constituents of the
C-terminal amide and a side-chain oxygen or backbone
carbonyl of residues lining the Y* + 3 binding pocket.
Initial studies showed that the simple carboxamide 18
bound weakly, when compared to the corresponding acid
19, suggesting a preference for ionizable functionality
deep within this pocket. More dramatic results were
observed when an alkyl chain terminating in a hydroxyl
group was utilized. For example, the hexanol 25 was
equipotent with 2; however, homologation with one and
two methylene units delivered heptanol 26 and octanol
27 with improved the binding affinity by 2- and 3-fold,
respectively, when compared with 2. While the X-ray
crystal analysis of 25 (Figure 2) confirmed a hydrogen-
bonding interaction between the alkanol and the hy-
droxyl group of Thr218, in practice this compound
displayed a propensity toward both internal lactoniza-
tion and dimerization with the Glu carboxylic acid.
While these compounds offered desirable solutions for
increasing binding potency from a structural perspec-
tive, their physical properties made them less interest-
ing for further investigations.
Our earlier work had established the binding proper-
ties of several dipeptides capped as the N-terminal
acetamide. Subsequent analysis of X-ray structures
reinforced several features about interactions between
this portion of these dipeptides and the protein in the
complexes: (1) the phosphotyrosine pocket and adjacent
space appeared sufficiently large to accommodate func-
tional groups other than acetyl to evaluate the relative
importance of steric and electronic variations on binding
affinity; (2) the array of hydrogen bonds in the Y* pocket
of the protein:ligand complex was highly conserved; (3)
the phosphotyrosine pocket was inadequately protected
from exposure to solvent.
Several N-terminal analogues of 2 were prepared in
an attempt to determine the impact of the above-
mentioned features on binding affinity (Table 2). In-
corporation of more sterically demanding aryl- and
heteroaryl-containing amides delivered dipeptides with
binding affinities comparable to that observed for 2.
These results suggested that binding relied on the
maintenance of a hydrogen-bonding interaction between
the acetamide carbonyl of the dipeptide and Arg158 of
the protein rather than the ability to fill the phospho-
tyrosine pocket. To attenuate this hydrogen bond and
determine the impact on binding affinity, a series of
electronically different N-terminal analogues, including
substituted benzamides and ureas, were synthesized
and evaluated as ligands for the src SH2 domain. The
affinities of these analogues on the whole were not
demonstrably different from the aforementioned amides;

1898 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Ta ble 1. C-Terminal Analogues of 2
Pacofsky et al.
a
Ratio ) IC50(test)/IC50(AcY*EEIE).
however, variations exist within each structural class.
The benzamide-based analogues 62-65 showed a mod-
est (3-5-fold) preference for electron-rich systems; the
anomalous result with 64 may be attributed to increased
solvation of the nitro group. In the urea series, a more
pronounced preference (6-13-fold) exists for the electron-
rich derivatives 66 and 67, as compared to analogue 68,
supporting the hypothesis that overall binding affinity
is influenced by the hydrogen bond strength of this
N-terminal carbonyl.
A network of hydrogen bonds anchors a phenyl
phosphate in the phosphotyrosine binding pocket of the
src SH2 domain which suggests the possibility for
increasing binding affinity by strengthening such in-
teractions between the ligand and the protein. In-
tramolecular association of the N-terminal components
of the ligand might further improve its binding by
capping the pocket and minimizing the need for solva-
tion of the Y* binding site. Compound 69 was designed
and prepared using this rationale as well as to explore
the potential for preorganization in this segment of the
ligand. This N-terminal aromatic system effectively
shields the pocket from solvent through a series of
hydrogen bonds; however, there is a decrease in binding
affinity for this compound. Closer examination of the
X-ray structure (Figure 3) shows that the distal oxygen
of the phosphate contacts the pendant amino group as
well as Thr182 and Ser180; the phosphate has rotated
from its normal position to accommodate this new array.
Also, the tyrosine-based phenyl ring of the peptide has
rotated to allow formation of an edge-to-face interaction
between the two aromatic rings of the ligand. Presum-
ably this combination of interactions has distorted the
binding trajectory of the phosphotyrosine resulting in
decreased affinity.
The work to date had relied exclusively on a Y* + 1
glutamate for binding to the src SH2 domain, and we
were aware of potential difficulties, such as poor cell
penetration properties, associated with such highly
charged compounds. To address these issues, we con-
sidered replacement of glutamic acid to reduce overall
charge while retaining, or improving, binding affinity.
Studies began with an analysis of the 2:SH2 complex
which showed the glutamic acid side chain packed

Dipeptide Inhibitors of the pp60c-src SH2 Domain
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1899
F igu r e 2. X-ray structure of 25 bound to the pp60c-src SH2 domain.
against the phenyl ring portion of Tyr205 and its
carboxyl terminus being canted toward an electrostatic
interaction with His204 to offset negative charge. This
proximity to Tyr205 and the reported ability of sulfur
to interact with aromatic systems36,37 prompted us to
install an amino acid with a sulfur-containing side
chain. Thus, a series of sulfur-based dipeptides (Table
3) were screened, with the methionine-containing dipep-
tide 92 having the highest affinity for the src SH2
domain. The X-ray analysis (Figure 4) of the complex
revealed improved hydrophobic stacking between the
methionine side chain and Tyr205 when compared to
the corresponding atoms of the glutamate in the parent
complex. Interestingly, the sulfur atom did not align
with the aromatic ring but rather was positioned to
interact with the phenolic hydroxyl group suggesting
that a shorter tether might orient the sulfur atom near
the π cloud. Unfortunately, a decrease in binding
affinity was realized in parallel studies with the S-
methylcysteine analogue 94 where the position of the
sulfur atom forced the terminal methyl group into the
π cloud. Noteworthy is the fact that a high-affinity
dipeptide with a neutral Y* + 1 residue emerged from
this study.
elucidate the structural requirements and preferences
of the protein. Confirmation of the ability to incorporate
diverse structural motifs into a modest dipeptide which
retains binding affinity remains the principal advantage
of this approach.
The inability of highly charged compounds of the type
described herein to penetrate cells would affect their
chemotherapeutic utility for intracellular targets. There-
fore, reduction of the overall charge of these dipeptides
has been an important focus of this program. The
strategy to use an uncharged residue adjacent to the
phosphotyrosine together with the information obtained
in the aforementioned studies has enabled the produc-
tion of potent inhibitors with reduced charge relative
to 2. Efforts to extend the scope of these results will be
the subject of further investigations.
Exp er im en ta l Section
Gen er a l Exp er im en ta l. Melting points were determined
on a Thomas-Hoover capillary melting point apparatus and
are uncorrected. 1H NMR data were obtained using a Varian
Unity Plus 300 or Unity Plus 400 spectrometer; chemical shifts
are reported in parts per million (ppm) and referenced to
solvent or internal TMS. Mass spectral data were obtained
using either a Perkin-Elmer Sciex API III or J EOL J MS mass
spectrometer. Elemental analyses were performed by Atlantic
Microlabs, Inc., Atlanta, GA. Unless noted otherwise, anhy-
drous solvents and reagents were purchased from commercial
suppliers and used as received. Flash chromatography was
carried out using EM Science silica gel 60, 230-400 mesh.
Isolated yields of purified materials are reported.
BOC-Glu (OBn )-OCH2CH2Si(CH3)3 (3). BOC-Glu(OBn)-
OH (5.00 g, 14.8 mmol) was dissolved in CH3CN (50 mL) at
room temperature. 2-(Trimethylsilyl)ethanol (4.20 mL, 29.3
mmol), 1,3-dicyclohexylcarbodiimide (DCC; 3.1 g, 15.0 mmol),
and pyridine (3.60 mL, 44.5 mmol) were added, and the
reaction solution was stirred for 18 h. A white precipitate
formed and was removed from the reaction by suction filtra-
tion. The filtrate was diluted with CH2Cl2 (150 mL) and
Con clu sion s
A strategy to identify dipeptide ligands for the pp60c-src
SH2 domain using structure-based design has yielded
compounds with binding affinities comparable to that
of pentapeptide 1. The dipeptide 2 utilized a simple
N,N-dialkyl amide to replace the amino acid residues
C-terminal to the phosphotyrosine in 1 and served as a
template for further modifications of the dipeptide
framework. Subsequent variations in both the C- and
N-terminal regions of 2 have produced analogues with
satisfactory binding affinities and have helped us

1900 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Ta ble 2. N-Terminal Analogues of 2
Pacofsky et al.
a
Ratio ) IC50(test)/IC50(AcY*EEIE).
Ta ble 3. Y* + 1 Analogues of 2
aqueous NaHCO3 (100 mL). The layers were separated, and
the organic phase was washed with 0.10 M aqueous HCl (1 ×
100 mL) and brine (1 × 100 mL), dried (Na2SO4), and
concentrated under reduced pressure. The residue was puri-
fied by flash chromatography, elution with 2:3 EtOAc-hexane,
to afford 5.30 g (81%) of 5 as a white foam.
Cm p d
R
Ra tioa
2
92
93
94
95
CH2CO2H
CH2SMe
CH2S(O2)Me
SMe
7.9
16.5
75.3
33.1
155.4
Ac-Tyr -Glu (OBn )-OCH2CH2Si(CH3)3 (6). A solution of 5
(1.55 g, 2.58 mmol) in HCl/dioxane (2.50 mL of a 4 M solution,
10.0 mmol) was stirred for 2 h. The volatiles were removed
in vacuo to afford a brown-yellow foam that was taken up in
CH2Cl2 (20 mL), treated with acetic acid N-hydroxysuccinimide
ester (AcOSuc) (570 mg, 3.61 mmol) followed by i-PrNEt2 (0.90
mL, 5.16 mmol), and stirred for 24 h. The reaction mixture
was diluted with EtOAc (100 mL) and washed with 100 mL
each of the following aqueous solutions: saturated NaHCO3,
1 M HCl, and brine. The organic phase was concentrated
under reduced pressure, and the residue was purified by flash
chromatography, elution with 75:25 EtOAc-hexane, to afford
1.20 g (86%) of 6 as an amorphous cream-colored solid.
Ac-Tyr [P (O)(OBn )2]-Glu (OBn )-OCH2CH2Si(CH3)3 (7).
A solution of 6 (610 mg, 1.12 mmol) in CH2Cl2 (8 mL) was
treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.35
mL, 2.34 mmol) and tetrabenzyl pyrophosphate (995 mg, 1.85
mmol). The reaction mixture was stirred for 3 h, diluted with
EtOAc (100 mL), and washed with 100 mL each of the
following aqueous solutions: saturated NaHCO3, 0.50 M HCl,
and brine. The organic phase was concentrated under reduced
pressure, and the residue was purified by flash chromatogra-
SCH2Ph
a
Ratio ) IC50(test)/IC50(AcY*EEIE).
washed (1 × 100 mL) with each of the following aqueous
solutions: 1 M aqueous HCl, saturated NaHCO3, and water.
The organic layer was dried (MgSO4) and concentrated under
reduced pressure to provide 6.00 g (93%) of 3 as a clear,
colorless, viscous oil.
HCl‚H-Glu (OBn )-OCH2CH2Si(CH3)3 (4). BOC-Glu(OBn)-
OCH2CH2Si(CH3)3 (6.00 g, 13.7 mmol) was treated with HCl/
dioxane (12.5 mL of a 4 M solution, 50.0 mmol) until 3 was no
longer detectable by TLC. The volatiles were removed in
vacuo, and the residue was solidified by trituration with Et2O
to provide 5.10 g (98%) of 4 as an amorphous, light-yellow solid.
BOC-Tyr -Glu (OBn )-OCH2CH2Si(CH3)3 (5). BOC-Tyr-OH
(3.39 g, 12.1 mmol) and 4 (4.05 g, 10.8 mmol) were combined
and dissolved in DMF (15 mL). To this solution was added
(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexaflu-
orophosphate (BOP) (4.83 g, 10.9 mmol) followed by i-PrNEt2
(4.55 mL, 26.1 mmol). The reaction mixture was stirred for 6
h and then diluted with EtOAc (100 mL) and saturated

Dipeptide Inhibitors of the pp60c-src SH2 Domain
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1901
F igu r e 3. X-ray structure of 69 bound to the pp60c-src SH2 domain.
phy, eluting with 3:2 EtOAc-hexane, to afford 790 mg (88%)
of 7 as a viscous oil.
Ac-Tyr [P (O)(OH)2]-Glu -N(n -C6H13)2 (13): 1H NMR (300
MHz, CD3OD) δ 0.92 (m, 6H), 1.32 (m, 12H), 1.50 (m, 2H),
1.63 (m, 2H), 1.79 (m, 1H), 1.90 (s, 3H), 1.97 (m, 1H), 2.40 (m,
2H), 2.84 (m, 1H), 3.12 (m, 2H), 3.22 (m, 1H), 3.48 (m, 2H),
4.59 (m, 1), 4.82 (m, 1H), 7.10 (d, 2H, J ) 8.3 Hz), 7.22 (d, 2H,
J ) 8.3 Hz); MS (ESI) m/z 598 (M - H). Anal. (C28H46N3O9-
P‚1.5H2O) C, H, N.
Ac-Tyr [P (O)(OH)2]-Glu -N[(n -C5H11)(CH2CH2OH)] (15):
1H NMR (300 MHz, CD3OD) δ 0.98 (m, 3H), 1.39 (m, 4H), 1.6
(m, 1H), 1.70 (m, 1H), 1.95 (m, 4H), 2.06 (m, 1H), 2.41 (m,
1H), 2.54 (m, 1H), 2.87 (m, 1H), 3.14 (m, 1H), 3.38 (m, 3H),
3.61 (m, 4H), 4.62 (m, 1H), 7.17 (d, 2H), 7.24 (d, 2H); MS (FAB)
m/z 544 (M - H). Anal. (C23H36N3O10P) C, H, N.
Ac-Tyr [P (O)(OH)2]-Glu -N[(n -C5H11)(CH2CH2CO2H)] (16):
1H NMR (300 MHz, CD3OD) δ 0.95 (m, 3H), 1.31 (m, 6H), 1.60
(m, 2H), 1.81 (m, 1H), 1.89 (s, 3H), 1.91 (m, 1H), 2.38 (m, 2H),
2.47 (m, 1H), 2.70 (m, 1H), 2.82 (m, 1H), 3.04 (m, 1H), 3.37
(m, 2H), 3.42 (m, 1H), 3.57 (m, 1H), 3.65 (m, 1H), 4.55 (m,
1H), 4.85 (1H), 7.12 (d, 2H, J ) 8.4 Hz), 7.16 (d, 2H, J ) 8.4
Hz); MS (ESI) m/z 574 (MH+). Anal. (C24H36N3O11P‚1.5H2O)
C, H, N.
Ac-Tyr [P (O)(OBn )2]-Glu (OBn )-OH (8). To a solution of
7 (400 mg, 0.50 mmol) in CH2Cl2 (5 mL), in a plastic reaction
vessel, at 0 °C was added HF‚pyridine (0.40 mL), and the
reaction mixture was stirred for 2 h while being followed
closely by TLC; significant dephosphorylation occurred with
longer reaction times. The reaction mixture was diluted with
CH2Cl2 (250 mL) and H2O (200 mL), and the layers were
separated. The aqueous layer was extracted with CH2Cl2 (1
× 150 mL), and the combined organic layers were treated with
KF (10 g) followed by Na2SO4 and then filtered. The filtrate
was concentrated under reduced pressure, and the residue was
purified by column chromatography, elution with 95:4:1 CH2-
Cl2-MeOH-AcOH, to provide 150 mg (44%) of 8 as a white
foam.
Compounds 11-27 were synthesized using the following
method described for 14.
Ac-Tyr [P (O)(OH)2]-Glu -N(n -C7H15
) (14). To a solution
2
of 8 (46 mg, 0.07 mmol) in THF (1 mL) at -70 °C were added
4-methylmorpholine (21 µL, 0.19 mmol) and isobutyl chloro-
formate (9 µL, 0.069 mmol). This solution was stirred for 5
min prior to the addition of HN(n-C7H15)2 (53 µL, 0.20 mmol).
The reaction mixture was warmed to -50 °C and stirred for
2.5 h. At the low temperature, the reaction mixture was
diluted with EtOAc (25 mL) and saturated aqueous NaHCO3
(25 mL). The biphasic solution was warmed to room temper-
ature, and the layers were separated. The organic layer was
washed with H2O (1 × 30 mL) and brine (1 × 30 mL), dried
(Na2SO4), and concentrated under reduced pressure. The
residue was purified by column chromatography, elution with
4:1 EtOAc-hexane, to provide a clear viscous oil. This
material was dissolved in EtOH (1.5 mL), treated with 10%
Pd-C, and stirred under H2 (1 atm) for 6.5 h. The reaction
mixture was purged with N2 and filtered through a pad of
Celite, and the filtrate was concentrated under reduced
pressure to provide 23 mg (57%) of 14 as an amorphous white
solid: MS (ESI) m/z 628 (M + H). Anal. (C30H50N3O9P‚1.5H2O)
C, H, N.
Ac-Tyr [P (O)(OH)2]-Glu -N[(n -C5H11)(CH2CH2CO2Et)] (17):
1H NMR (300 MHz, CD3OD) δ 0.95 (m, 3H), 1.32 (m, 9H), 1.66
(m, 2H), 1.97 (s, 3H), 2.40 (m, 2H), 2.57 (m, 1H), 2.71 (m, 1H),
2.84 (m, 1H), 3.10 (m, 2H), 3.39 (m, 1H), 3.60 (m, 2H), 4.13
(m, 2H), 4.59 (m, 1H), 5.86 (m, 1H), 7.13 (d, 2H, J ) 8.4 Hz),
7.22 (d, 2H, J ) 8.4 Hz); MS (FAB) m/z 602 (MH+), 624 (M +
Na). Anal. (C26H40N3O11P‚1.0H2O) C, H, N.
Ac-Tyr [P (O)(OH)2]-Glu -NH[(CH2)6CONH2] (18): 1H NMR
(300 MHz, CD3OD) δ 1.35 (m, 4H), 1.48 (m, 2H), 1.89 (m, 1H),
1.92 (s, 3H), 2.04 (m, 1H), 2.20 (m, 2H), 2.34 (m, 2H), 2.84 (m,
1H), 3.15 (m, 3H), 4.28 (m, 1H), 4.53 (m, 1H), 7.11 (d, 2H),
7.23 (d, 2H); MS (FAB) m/z 559 (MH+); HRFAB-MS calcd for
C
23H36N4O10P (MH+) 559.216 12, found 559.216 12 (MH+).
Anal. (C23H35N4O10P‚1.5H2O‚1i-PrOH) C, H, N.
Ac-Tyr [P (O)(OH)2]-Glu -NH[(CH 2)6CO2H] (19): 1H NMR
(300 MHz, CD3OD) δ 1.31 (m, 6H), 1.48 (m, 2H), 1.59 (m, 2H),
1.84 (m, 1H), 1.89 (s, 3H), 2.05 (m, 1H), 2.33 (m, 2H), 2.38 (m,
2H), 2.85 (dd, 1H), 3.16 (m, 3H), 4.30 (m, 1H), 4.58 (m, 1H),

1902 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Pacofsky et al.
F igu r e 4. X-ray structure of 92 bound to the pp60c-src SH2 domain.
7.14 (d, 2H), 7.24 (d, 2H), 7.80 (t, 1H, exchangeable), 8.19 (d,
1H, exchangeable); MS (ESI) m/z 572 (M - H). Anal.
(C24H36N3O11P‚1H2O‚0.5i-PrOH) C, H, N.
Ac-Tyr [P (O)(OH)2]-Glu -N[(CH3)[3-(3-p r op yl-2-ca r boxa -
m id oin d ole)]] (20): 1H NMR (300 MHz, DMSO-d6) δ 1.78
(m, 1H), 1.80 (s, 3H), 1.95 (m, 3H), 2.25 (m, 2H), 2.69 (m, 2H),
2.80 (m, 1H), 3.00 (m, 4H), 3.51 (m, 2H), 4.51 (m, 1H), 4.80
(m, 1H), 6.99 (br s, 2H), 7.04 (d, 2H), 7.14 (d, 2H), 7.22 (m,
2H), 7.59 (m, 4H), 8.22 (d, 1H). MS (FAB) m/z 646 (MH+).
Anal. (C29H36N5O10P‚2H2O) C, H, N.
Ac-Tyr [P (O)(OH )2]-Glu -N[(CH 3)[3-(3-p r op ylin d ole)]]
(21): 1H NMR (300 MHz, DMSO-d6) δ 1.78 (m, 1H), 1.80 (s,
3H), 1.92 (m, 2H), 2.23 (m, 2H), 2.70 (m, 2H), 2.96 (m, 4H),
3.42 (m, 2H), 4.50 (m, 1H), 4.80 (m, 1H), 6.95 (m, 1H), 7.04
(m, 4H), 7.14 (d, 2H), 7.32 (d, 1H), 7.50 (d, 1H), 7.60 (m, 2H),
10.38 (br s, 1H); MS (FAB) m/z 603 (MH+). Anal. (C28H35N4-
O9P‚1H2O‚0.5i-PrOH) C, H, N.
Ac-Tyr [P (O)(OH)2]-Glu -N[(CH3)[2-(3-eth ylin dole)]] (22):
1H NMR (300 MHz, DMSO-d6) δ 1.77 (m, 2H), 1.81 (s, 3H),
2.21 (m, 2H), 2.79 (m, 2H), 2.97 (m, 5H), 3.61 (m, 2H), 4.51
(m, 1H), 4.77 (m, 1H), 7.06 (m, 7H), 7.34 (m, 1H), 7.58 (m,
2H), 10.45 (br s, 1H); MS (FAB) m/z 589 (MH+). Anal.
(C27H33N4O9P‚1H2O‚0.5i-PrOH) C, H, N.
Ac-Tyr [P (O)(OH )2]-Glu -NH [2-(3-e t h yl-5-h yd r oxyin -
d ole)] (23): 1H NMR (300 MHz, CD3OD) δ 1.87 (m, 1H), 1.91
(s, 3H), 2.20 (m, 1H), 2.3 (m, 2H), 2.85 (m, 3H), 3.06 (m, 1H),
3.48 (m, 2H), 4.30 (m, 1H), 4.56 (m, 1H), 6.63 (d, 1H), 6.93 (s,
1H), 7.02 (s, 1H), 7.12 (m, 3H), 7.20 (d, 2H), 7.80 (t, 1H,
exchangeable), 8.19 (d, 1H, exchangeable), 10.40 (br s, 1H);
MS (FAB) m/z 591 (MH+). Anal. (C26H31N4O10P‚0.5H2O‚1i-
PrOH) C, H, N.
Ac-Tyr [P (O)(OH )2]-Glu -NH [2-(3-e t h yl-5-m e t h oxyin -
d ole)] (24): 1H NMR (300 MHz, CD3OD) δ 1.86 (m, 1H), 1.90
(s, 3H), 2.30 (m, 2H), 2.81 (m, 1H), 2.90 (m, 2H), 3.04 (dd, 1H),
3.48 (m, 2H), 3.82 (s, 3H), 4.29 (m, 1H), 4.53 (m, 1H), 6.72 (d,
1H), 7.06 (s, 2H), 7.12 (d, 2H), 7.19 (m, 3H), 7.81 (t, 1H,
exchangeable), 8.17 (d, 1H, exchangeable), 10.35 (br s, 1H);
MS (ESI) m/z 603 (M - H). Anal. (C27H33N4O10P‚2.25H2O)
C, H, N.
Ac-Tyr [P (O)(OH)2]-Glu -N(CH3)[(CH2)6OH] (25): 1H NMR
(300 MHz, DMSO-d6) δ 1.30 (m, 4H), 1.46 (m, 4H), 1.73 (m,
1H), 1.80 (s, 3H), 1.91 (m, 1H), 2.22 (m, 2H), 2.79 (dd, 1H),
2.91 (m, 3H), 3.00 (dd, 1H), 3.31 (m, 2H), 3.42 (m, 2H), 4.48
(m, 1H), 4.78(m, 1H), 7.03 (d, 2H), 7.17 (d, 2H), 7.59 (d, 2H,
exchangeable); MS (FAB) m/z 546 (MH+). Anal. (C26H31N4-
O
10P‚0.5i-PrOH) C, H, N.
Ac-Tyr [P (O)(OH)2]-Glu -N(CH3)[(CH2)7OH] (26): 1H NMR
(300 MHz, DMSO-d6) δ 1.29 (m, 6H), 1.53 (m, 4H), 1.78 (m,
1H), 1.80 (s, 3H), 1.98 (m, 1H), 2.30 (m, 2H), 2.92 (m, 5H),
3.39 (m, 4H), 4.52 (m, 1H), 4.80 (m, 1H), 7.08 (d, 2H), 7.19 (d,
2H), 7.62 (br s, 2H, exchangeable); MS (FAB) m/z 560 (MH+).
Anal. (C24H38N3O10P‚0.5H2O) C, H, N.
Ac-Tyr [P (O)(OH)2]-Glu -N(CH3)[(CH2)8OH] (27): 1H NMR
(300 MHz, DMSO-d6) δ 1.34 (m, 8H), 1.48 (m, 4H), 1.77 (m,
1H), 1.80 (s, 3H), 1.90 (m, 1H), 2.23 (m, 2H), 2.80 (m, 1H),
2.93 (m, 4H), 3.37 (m, 4H), 4.50 (m, 1H), 4.78 (m, 1H), 7.03 (d,
2H), 7.14 (d, 2H), 7.60 (br d, 2H, exchangeable); MS (ESI) m/z
574 (MH+). Anal. (C25H40N3O10P‚1H2O) C, H, N.
Z-Glu (Ot-Bu )-N(n -C5H11
) (29). Isobutyl chloroformate
2
(4.25 mL, 32.8 mmol) was added to a solution of 28 (10.1 g,
29.8 mmol) and 4-methylmorpholine (9.82 mL, 89.5 mmol) in
THF (150 mL) at -20 °C. The mixture was stirred for 5 min,
treated with HN(n-C5H11)2 (6.03 mL, 29.8 mmol), and allowed
to warm to room temperature over 14 h. The mixture was
diluted with saturated aqueous NaHCO3 (ca. 50 mL) and
concentrated under reduced pressure. The residue was ex-
tracted with EtOAc (2 × 150 mL). The combined organic
extracts were washed with saturated aqueous NaHCO3 (1 ×
50 mL), H2O (1 × 50 mL), and saturated aqueous NaCl (1 ×
50 mL), dried (Na2SO4), and concentrated under reduced
pressure. The residue was purified by flash chromatography,
elution with hexanes-EtOAc (4:1), to provide 11.3 g (80%) of
29 as an oil.
Z-Tyr -Glu (Ot-Bu )-N(n -C5H11)2 (30). A mixture of 29 (11.3
g, 23.7 mmol) and 10% Pd-C (200 mg) in MeOH (120 mL)
was stirred under H2 (1 atm) for 4 h. The reaction mixture
was purged with N2 and filtered through a pad of Celite with
additional MeOH (ca. 100 mL), and the filtrate was concen-

Dipeptide Inhibitors of the pp60c-src SH2 Domain
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1903
(4-Cl)P h CO-Tyr [P (O)(Ot -Bu )2]-Glu (Ot -Bu )-N(n -C5-
trated under reduced pressure to provide 8.15 g (90% yield) of
H-Glu(Ot-Bu)-N(n-C5H11)2 as an oil. A solution of H-Glu(Ot-
H11)2 (43): prepared in this fashion from 32 and 4-chloroben-
Bu)-N(n-C5H11
)
2
(8.10 g, 23.7 mmol) in CH2Cl2 (20 mL) was
zoic acid; 470 mg (90%) as a white foam.
added to a solution of Z-Tyr-OH (7.46 g, 23.7 mmol), DCC (5.37
g, 26.1 mmol), and HOBT (3.52 g, 26.1 mmol) in CH2Cl2 (80
mL) and DMF (20 mL) at 0 °C. Et3N (4.00 mL, 28.4 mmol)
was added, and the mixture was allowed to warm to room
temperature over 16 h. The reaction mixture was filtered
through a pad of Celite using CH2Cl2 (ca. 100 mL), and the
filtrate was concentrated under reduced pressure. The residue
was diluted with H2O and extracted with EtOAc (2 × 150 mL).
The combined organic extracts were washed with H2O (3 ×
50 mL) and saturated aqueous NaCl (1 × 50 mL), dried (Na2-
SO4), and concentrated under reduced pressure. The residue
was purified by flash chromatography, elution with hexanes-
EtOAc (2:1), to provide 11.8 g (78%) of 30 as a foam.
(4-CF 3)P h CO-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-N(n -C5-
H11
) (44): prepared in this fashion from 32 and 4-(trifluo-
2
romethyl)benzoic acid; 476 mg (88%) as a foam.
(4-NO2)P h CO-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-N(n -C5-
11)2 (45): prepared in this fashion from 32 and 4-nitrobenzoic
acid; 319 mg (69%) as a white foam.
(4-MeO)P h CO-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-N(n -C5-
11)2 (46): prepared in this fashion from 32 and 4-methoxy-
benzoic acid; 301 mg (66%) as a white foam.
(4-BOCNH CH 2)P h CH 2CO-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-
Bu )-N(n -C5H11)2 (50): prepared in this fashion from 32 and
71; 301 mg (60%) as a yellow foam.
H
H
P h CH2OCH2CO-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-N(n -C5-
Z-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-N(n -C5H11)2 (31). A so-
lution of 30 (1.00 g, 1.56 mmol) in THF (8 mL) was added to
a suspension of NaH (78 mg of 60% oil dispersion, 1.96 mmol)
in THF (6 mL) at 0 °C, and the mixture was stirred for 30
min. A solution of di-tert-butyl phosphorochloridate (429 mg,
1.88 mmol) in THF (2 mL) was added, and the mixture was
allowed to warm to room temperature over 4 h. The mixture
was treated with saturated aqueous NaHCO3 (ca. 20 mL) and
extracted with EtOAc (2 × 50 mL). The combined organic
extracts were washed with H2O (2 × 25 mL) and saturated
aqueous NaCl (1 × 25 mL), dried (Na2SO4), and concentrated
under reduced pressure. The residue was purified by flash
chromatography, elution with hexanes-EtOAc (3:2), to provide
730 mg (56%) of 31 as an oil.
H-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-N(n -C5H11)2 (32). A mix-
ture of 31 (1.13 g, 1.36 mmol) and 10% Pd-C (100 mg) in
MeOH (14 mL) was stirred under H2 (1 atm) for 3 h. The
reaction mixture was purged with N2 and filtered through a
pad of Celite with additional MeOH (ca. 20 mL), and the
filtrate was concentrated under reduced pressure to provide
935 mg (98%) of 32 as an oil.
P h CO-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-N(n -C5H11)2 (33).
A solution of 32 (436 mg, 0.63 mmol) and benzoic acid (77 mg,
0.63 mmol) in CH2Cl2 (2.75 mL) and DMF (1.25 mL) was
treated with DCC (142 mg, 0.69 mmol) and HOBT (93 mg,
0.69 mmol) at 0 °C. Et3N (0.11 mL, 0.79 mmol) was added,
and the mixture was allowed to warm to room temperature
overnight. The reaction mixture was filtered through a pad
of Celite using CH2Cl2 (ca. 10 mL), and the filtrate was
concentrated under reduced pressure. The residue was diluted
with H2O and extracted with EtOAc (2 × 25 mL). The
combined organic extracts were washed with saturated aque-
ous NaHCO3 (1 × 10 mL), H2O (2 × 10 mL), and saturated
aqueous NaCl (1 × 10 mL), dried (Na2SO4), and concentrated
under reduced pressure. The residue was purified by flash
chromatography, elution with hexanes-EtOAc (2:1), to provide
441 mg (88%) of 33 as a foam.
H
11)2 (40). Benzyloxyacetyl chloride (0.13 mL, 0.79 mmol) was
added to a suspension of 32 (530 mg, 0.76 mmol) and NaH
(36 mg of 60% oil dispersion, 0.90 mmol), in THF (15 mL) at
0 °C. The mixture was stirred for 3 h at 0 °C, treated with
saturated aqueous NaHCO3 (ca. 5 mL), concentrated under
reduced pressure, and extracted with EtOAc (2 × 25 mL). The
combined organic extracts were washed with saturated aque-
ous NaHCO3 (1 × 5 mL), H2O (1 × 5 mL), and saturated
aqueous NaCl (1 × 5 mL), dried (Na2SO4), and concentrated
under reduced pressure. The residue was purified by flash
chromatography, elution with hexanes-EtOAc (gradient, 3:2-
1:5), to provide 336 mg (53%) of 40 as a white foam.
CF 3CO-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-N(n -C5H11)2 (42).
Trifluoroacetic anhydride (0.11 mL, 0.79 mmol) was added to
a mixture of 32 (530 mg, 0.76 mmol) and NaH (36 mg of 60%
oil dispersion, 0.90 mmol) in THF (15 mL) at 0 °C. The
mixture was stirred for 3 h at 0 °C, treated with saturated
aqueous NaHCO3 (ca. 5 mL), concentrated under reduced
pressure, and extracted with EtOAc (2 × 25 mL). The
combined organic extracts were washed with saturated aque-
ous NaHCO3 (1 × 5 mL), H2O (1 × 5 mL), and saturated
aqueous NaCl (1 × 5 mL), dried (Na2SO4), and concentrated
under reduced pressure. The residue was purified by flash
chromatography, elution with hexanes-EtOAc (gradient, 3:2-
1:5), to provide 261 mg (44%) of 42 as a white foam.
(4-NO2)P h NH CO-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-N(n -
C5H11
) (49). A catalytic amount of DMAP was added to a
2
solution of 32 (590 mg, 0.85 mmol) and 4-nitrophenyl isocy-
anate (154 mg, 0.94 mmol) in CH2Cl2 (3 mL) at 0 °C, and the
mixture was allowed to warm to room temperature overnight.
The reaction mixture was concentrated under reduced pres-
sure, and the residue was purified by flash chromatography,
elution with hexanes-EtOAc (gradient, 3:2-1:5), to provide
491 mg (67%) of 49 as a foam.
(4-MeO)P h NH CO-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-N(n -
C5H11)2 (48): prepared in this fashion from 32 and 4-methoxy-
phenyl isocyanate; 266 mg (34%) as a foam.
P h CH 2CO-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-N(n -C5H 11
)
2
(4-NH )P h NH CO-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-N(n -
C5H11
)
(247). A mixture of 49 (229 mg, 0.27 mmol) and 10%
(34): prepared in this fashion from 32 and phenylacetic acid;
320 mg (60%) as a white foam.
P h CH 2CH 2CO-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-N(n -C5-
2
Pd-C (44 mg) in MeOH (2.6 mL) was stirred under H2 (1 atm)
for 4 h. The reaction mixture was purged with N2 and filtered
through a pad of Celite with additional MeOH (ca. 100 mL),
and the filtrate was concentrated under reduced pressure. The
oily residue was purified by flash chromatography, elution with
MeOH-CH2Cl2 (gradient, 1:19-1:4), to provide 204 mg (93%)
of 47 as a white foam.
H11
) (35): prepared in this fashion from 32 and 3-phenyl-
2
propionic acid; 272 mg (57%) as a white foam.
P h S C H 2C O -T y r [P (O )(O t -B u )2]-G lu (O t -B u )-N (n -C 5-
H
11)2 (36): prepared in this fashion from 32 and (phenylthio)-
acetic acid; 290 mg (63%) as a white foam.
(4-OH)P h CH CH2CO-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-N(n -
(37): p2repared in this fashion from 32 and 3-(4-
Z-Tyr [P (O)(OH)2]-Glu -N(n -C5H11
) (51). TFA (0.40 mL,
2
C5H11
)
2
5.19 mmol) was added to a solution of 31 (200 mg, 0.24 mmol)
in CH2Cl2 (2 mL). The mixture was stirred for 16 h and
concentrated under reduced pressure to give a foam. This
foam was washed with hexane (3 × 5 mL) and Et2O (2 × 5
mL), and excess solvent was removed in vacuo to provide 138
mg (86%) of 51 as a white foam: 1H NMR (400 MHz, DMSO-
d6) δ 8.20 (d, J ) 8.4 Hz, 1H), 7.47 (d, J ) 8.6 Hz, 1H), 7.27
(comp, 7H), 7.05 (d, J ) 8.0 Hz, 2H), 4.95 (comp, 2H), 4.73
(ddd, J ) 8.8, 8.8, 4.5 Hz, 1H), 4.26 (m, 1H), 3.37 (comp, 3H),
3.27 (m, 1H), 3.19 (dt, J ) 13.2, 7.0 Hz, 1H), 2.92 (dd, J )
13.8, 3.3 Hz, 1H), 2.68 (m, 1H), 2.28 (dd, J ) 7.4, 7.0 Hz, 2H),
hydroxyphenyl)propionic acid; 375 mg (52%) as a white foam.
(3-P yr id yl)CH CH2CO-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-
N(n -C5H11
)
(38):2prepared in this fashion from 32 and 3-(3-
2
pyridyl)propionic acid; 685 mg (66%).
P h CH CH2CH2CO-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-N(n -
C5H11)2 (239): prepared in this fashion from 32 and 4-phenyl-
butyric acid; 316 mg (65%) as a colorless oil.
P h CH2SCH2CO-Tyr [P (O)(Ot-Bu )2]-Glu (Ot-Bu )-N(n -C5-
H
11)2 (41): prepared in this fashion from 32 and (benzylthio)-
acetic acid; 307 mg (62%) as a colorless oil.

1904 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Pacofsky et al.
1.85 (m, 1H), 1.68 (m, 1H), 1.56 (m, 2H), 1.45 (m, 2H), 1.39-
1.15 (comp, 8H), 0.89 (t, J ) 7.1 Hz, 3H), 0.85 (t, J ) 7.2 Hz,
3H); MS (ESI) m/z 662 (M - H, 100); HRFAB-MS calcd for
(3-P yr id yl)CH 2CH 2CO-Tyr [P (O)(OH )2]-Glu -N(n -C5-
H
11)2 (57): prepared in this fashion from 38; 55 mg (33%) as
1
a foam; H NMR (400 MHz, DMSO-d6) δ 8.48 (d, J ) 5.5 Hz,
1H), 8.23 (s, 1H), 8.22 (d, J ) 8.5 Hz, 1H), 8.08 (d, J ) 8.5 Hz,
1H), 7.89 (d, J ) 8.0 Hz, 1H), 7.55 (dd, J ) 8.0, 5.5 Hz, 1H),
7.11 (d, J ) 8.5 Hz, 2H), 6.99 (d, J ) 8.2 Hz, 2H), 4.68 (ddd,
J ) 8.8, 8.8, 4.5 Hz, 1H), 4.51 (ddd, J ) 9.4, 9.0, 3.8 Hz, 1H),
3.34 (m, 2H), 3.25 (m, 1H), 3.09 (m, 1H), 2.88 (m, 1H), 2.79
(m, 2H), 2.59 (dd, J ) 13.8, 10.7 Hz, 1H), 2.42 (m, 2H), 2.23
(m, 2H), 1.83 (m, 1H), 1.66 (m, 1H), 1.54 (m, 2H), 1.43 (m,
2H), 1.34-1.15 (comp, 8H), 0.86 (t, J ) 7.0 Hz, 3H), 0.83 (t, J
) 7.1 Hz, 3H); MS (FAB) m/z 663 (M + H, 100); HRFAB-MS
calcd for C32H47N4O9P (MH+) 663.3159, found 663.3157 (MH+).
Anal. (C32H47N4O9P‚0.5TFA) C, H, N.
C
32H46N3O10P (MH+) 664.2999, found 664.3005 (MH+). Anal.
(C32H46N3O10P‚0.5H2O) C, H, N.
P h CO-Tyr [P (O)(OH)2]-Glu -N(n -C5H11)2 (52): prepared in
this fashion from 33; 255 mg (75%) as a white foam; 1H NMR
(400 MHz, DMSO-d6) δ 8.50 (d, J ) 8.3 Hz, 1H), 8.26 (d, J )
8.5 Hz, 1H), 7.76 (d, J ) 7.3 Hz, 1H), 7.50 (dd, J ) 7.3, 7.3 Hz,
1H), 7.43 (dd, J ) 7.7, 7.3 Hz, 2H), 7.30 (d, J ) 8.6 Hz, 2H),
7.03 (d, J ) 8.2 Hz, 2H), 4.73 (ddd, J ) 13.1, 8.8, 4.4 Hz, 1H),
4.67 (dd, J ) 8.3, 3.5 Hz, 1H), 3.34 (m, 2H), 3.26 (m, 1H), 3.09
(m, 1H), 3.02 (dd, J ) 13.1, 3.4 Hz, 1H), 2.92 (m, 1H), 2.28
(dd, J ) 7.2, 7.0 Hz, 2H), 1.84 (m, 1H), 1.69 (m, 1H), 1.55 (m,
2H), 1.42 (m, 2H), 1.34-1.15 (comp, 8H), 0.85 (t, J ) 7.0 Hz,
3H), 0.83 (t, J ) 7.2 Hz, 3H); MS (FAB) m/z 634 (M + H, 68),
105 (100); HRFAB-MS calcd for C31H44N3O9P (MH+) 634.2893,
found 634.2894 (MH+). Anal. (C31H44N3O9P‚0.25H2O) C, H,
N.
P h CH2CH2CH2CO-Tyr [P (O)(OH)2]-Glu -N(n -C5H11)2 (58):
1
prepared in this fashion from 39; 97 mg (39%) as a foam; H
NMR (400 MHz, DMSO-d6) δ 8.10 (d, J ) 8.3 Hz, 1H), 7.98 (d,
J ) 8.4 Hz, 1H), 7.24 (dd, J ) 7.5, 7.4 Hz, 2H), 7.18 (d, J )
8.4 Hz, 2H), 7.15 (d, J ) 7.5 Hz, 1H), 7.12 (d, J ) 7.3 Hz, 2H),
7.01 (d, J ) 8.4 Hz, 2H), 4.76 (ddd, J ) 8.8, 8.8, 4.5 Hz, 1H),
4.51 (ddd, J ) 9.2, 9.2, 3.8 Hz, 1H), 3.37 (m, 2H), 3.24 (m,
1H), 3.07 (m, 1H), 2.92 (dd, J ) 13.7, 3.8 Hz, 1H), 2.67 (dd, J
) 13.7, 10.4 Hz, 1H), 2.44 (t, J ) 7.5 Hz, 2H), 2.24 (t, J ) 6.3
Hz, 2H), 2.03 (m, 2H), 1.82 (m, 1H), 1.67 (t, J ) 7.5 Hz, 2H),
1.62 (m, 1H), 1.53 (m, 2H), 1.41 (m, 2H), 1.34-1.15 (comp, 8H),
0.86 (t, J ) 7.1 Hz, 3H), 0.83 (t, J ) 7.2 Hz, 3H); MS (FAB)
m/z 676 (M + H, 45), 158 (100); HRFAB-MS calcd for
P h CH 2CO-Tyr [P (O)(OH )2]-Glu -N(n -C5H 11
)
(53): pre-
2
pared in this fashion from 34; 218 mg (91%) as a white foam;
1H NMR (400 MHz, DMSO-d6) δ 8.21 (d, J ) 8.4 Hz, 1H), 8.20
(d, J ) 8.3 Hz, 1H), 7.21 (dd, J ) 7.2, 7.0 Hz, 2H), 7.16 (d, J
) 7.2 Hz, 1H), 7.13 (d, J ) 8.3 Hz, 2H), 7.07 (d, J ) 7.0 Hz,
2H), 6.98 (d, J ) 8.2 Hz, 2H), 4.71 (ddd, J ) 9.0, 8.8, 4.2 Hz,
1H), 4.55 (ddd, J ) 9.0, 8.8, 3.8 Hz, 1H), 3.36 (comp, 4H), 3.24
(m, 1H), 3.12 (dt, J ) 13.3, 7.0, 6.8 Hz, 1H), 2.92 (dd, J ) 13.8,
4.2 Hz, 1H), 2.69 (dd, J ) 13.8, 8.8 Hz, 1H), 2.23 (dd, J ) 7.7,
7.6 Hz, 2H), 1.80 (m, 1H), 1.73-1.59 (comp, 6H), 1.29-1.14
(comp, 8H), 0.88 (t, J ) 7.1 Hz, 3H), 0.84 (t, J ) 7.1 Hz, 3H);
MS (FAB) m/z 648 (M + H, 40), 158 (100); HRFAB-MS calcd
for C32H46N3O9P (MH+) 648.3050, found 642.3047 (MH+).
Anal. (C32H46N3O9P) C, H, N.
C
34H50N3O9P (MH+) 676.3363, found 676.3360 (MH+). Anal.
(C34H50N3O9P‚0.5H2O) C, H, N.
P h CH 2OCH 2CO-Tyr [P (O)(OH)2]-Glu -N(n -C5H11
) (59):
2
prepared in this fashion from 40; 74 mg (28%) as a white foam;
1H NMR (400 MHz, DMSO-d6) δ 8.31 (d, J ) 8.4 Hz, 1H), 7.72
(d, J ) 8.3 Hz, 1H), 7.33 (comp, 5H), 7.14 (d, J ) 8.6 Hz, 2H),
7.00 (d, J ) 8.2 Hz, 2H), 4.71 (ddd, J ) 8.8, 8.8, 4.3 Hz, 1H),
4.60 (ddd, J ) 8.6, 8.6, 4.3 Hz, 1H), 4.44 (s, 2H), 3.83 (ABq,
J AB ) 15.0 Hz, ∆νAB ) 15.0 Hz, 2H), 3.36 (m, 2H), 3.25 (m,
1H), 3.10 (m, 1H), 2.97 (dd, J ) 13.7, 4.0 Hz, 1H), 2.83 (dd, J
) 13.8, 9.0 Hz, 1H), 2.25 (m, 2H), 1.85 (m, 1H), 1.69 (m, 1H),
1.54 (m, 2H), 1.45 (m, 2H), 1.34-1.11 (comp, 8H), 0.85 (t, J )
7.0 Hz, 3H), 0.84 (t, J ) 7.1 Hz, 3H); MS (FAB) m/z 678 (M
+ H, 62), 158 (100); HRFAB-MS calcd for C33H48N3O10P (MH+)
678.3156, found 678.3160 (MH+). Anal. (C33H48N3O10P‚0.5TFA)
C, H, N.
P h CH2CH2CO-Tyr [P (O)(OH)2]-Glu -N(n -C5H11)2 (54): pre-
pared in this fashion from 35; 84 mg (41%) as a foam; 1H NMR
(400 MHz, DMSO-d6) δ 8.17 (d, J ) 8.3 Hz, 1H), 8.02 (d, J )
8.4 Hz, 1H), 7.22 (dd, J ) 7.4, 7.3 Hz, 2H), 7.13 (comp, 5H),
7.00 (d, J ) 8.4 Hz, 2H), 4.69 (ddd, J ) 8.8, 8.8, 4.5 Hz, 1H),
4.52 (ddd, J ) 9.0, 9.0, 4.0 Hz, 1H), 3.34 (m, 2H), 3.25 (m,
1H), 3.09 (m, 1H), 2.89 (dd, J ) 13.8, 4.0 Hz, 1H), 2.69 (t, J )
7.7 Hz, 2H), 2.64 (m, 1H), 2.33 (t, J ) 7.7 Hz, 2H), 2.24 (dd, J
) 7.5, 7.5 Hz, 2H), 1.84 (m, 1H), 1.66 (m, 1H), 1.54 (m, 2H),
1.43 (m, 2H), 1.34-1.15 (comp, 8H), 0.85 (t, J ) 7.1 Hz, 3H),
0.83 (t, J ) 7.2 Hz, 3H); MS (FAB) m/z 662 (M + H, 25), 158
(100); HRFAB-MS calcd for C33H48N3O9P (MH+) 662.3206,
found 662.3194 (MH+). Anal. (C33H48N3O9P‚0.5H2O) C, H, N.
P h CH2SCH2CO-Tyr [P (O)(OH)2]-Glu -N(n -C5H11
)
(60):
2
prepared in this fashion from 41; 107 mg (44%) as a foam; 1H
NMR (400 MHz, DMSO-d6) δ 8.25 (d, J ) 8.4 Hz, 1H), 8.13 (d,
J ) 8.2 Hz, 1H), 7.28 (dd, J ) 7.4, 7.3 Hz, 2H), 7.22 (comp,
3H), 7.18 (d, J ) 8.4 Hz, 2H), 7.01 (d, J ) 8.3 Hz, 2H), 4.71
(ddd, J ) 8.8, 8.8, 4.4 Hz, 1H), 4.56 (ddd, J ) 8.8, 8.8, 4.0 Hz,
1H), 3.64 (ABq, J AB ) 13.0 Hz, ∆νAB ) 13.3 Hz, 2H), 3.35 (m,
2H), 3.26 (m, 1H), 3.08 (m, 1H), 3.00 (s, 2H), 2.95 (dd, J )
13.9, 4.0 Hz, 1H), 2.73 (dd, J ) 13.9, 9.5 Hz, 1H), 2.26 (m,
2H), 1.84 (m, 1H), 1.68 (m, 1H), 1.55 (m, 2H), 1.44 (m, 2H),
1.34-1.15 (comp, 8H), 0.85 (t, J ) 7.2 Hz, 3H), 0.83 (t, J ) 7.3
Hz, 3H); MS (FAB) m/z 694 (M + H, 22), 158 (100); HRFAB-
MS calcd for C33H48N3O9PS (MH+) 694.2927, found 694.2921
(MH+). Anal. (C33H48N3O9PS‚0.5H2O) C, H, N.
P h SCH2CO-Tyr [P (O)(OH)2]-Glu -N(n -C5H11
) (55): pre-
2
pared in this fashion from 36; 90 mg (94%) as a white foam;
1H NMR (400 MHz, DMSO-d6) δ 8.29 (d, J ) 8.3 Hz, 1H), 8.26
(d, J ) 8.2 Hz, 1H), 7.24 (comp, 4H), 7.14 (m, 1H), 7.09 (d, J
) 8.3 Hz, 2H), 6.98 (d, J ) 8.4 Hz, 2H), 4.68 (dd, J ) 13.2, 8.9
Hz, 1H), 4.55 (ddd, J ) 8.3, 7.3, 4.0 Hz, 1H), 3.64 (s, 2H), 3.34
(m, 2H), 3.23 (m, 1H), 3.10 (m, 1H), 2.89 (dd, J ) 14.0, 4.0 Hz,
1H), 2.70 (dd, J ) 14.0, 9.2 Hz, 1H), 2.23 (m, 2H), 1.83 (m,
1H), 1.66 (m, 1H), 1.55 (m, 2H), 1.44 (m, 2H), 1.34-1.11 (comp,
8H), 0.84 (t, J ) 7.0 Hz, 3H), 0.86 (t, J ) 7.2 Hz, 3H); MS
(FAB) m/z 680 (M + H, 55), 158 (100); HRFAB-MS calcd for
CF 3CO-Tyr [P (O)(OH)2]-Glu -N(n -C5H11
) (61): prepared
2
32H46N3O9PS (MH+) 680.2771, found 680.2778 (MH+). Anal.
in this fashion from 42; 95 mg (46%) as a foam; 1H NMR (400
MHz, DMSO-d6) δ 9.57 (d, J ) 8.5 Hz, 1H), 8.49 (d, J ) 8.3
Hz, 1H), 7.24 (d, J ) 8.5 Hz, 2H), 7.04 (d, J ) 8.4 Hz, 2H),
4.71 (ddd, J ) 8.8, 8.8, 4.6 Hz, 1H), 4.58 (dd, J ) 8.0, 3.5 Hz,
1H), 3.35 (m, 2H), 3.28 (m, 1H), 3.09 (m, 1H), 2.99 (dd, J )
13.8, 3.5 Hz, 1H), 2.83 (dd, J ) 13.7, 5.7 Hz, 1H), 2.26 (m,
2H), 1.84 (m, 1H), 1.69 (m, 1H), 1.56 (m, 2H), 1.43 (m, 2H),
1.34-1.11 (comp, 8H), 0.87 (t, J ) 7.0 Hz, 3H), 0.83 (t, J ) 7.1
Hz, 3H); MS (FAB) m/z 626 (M + H, 65), 158 (100); HRFAB-
MS calcd for C26H39F3N3O9P (MH+) 626.2454, found 626.2460
(MH+). Anal. (C26H39F3N3O9P‚0.5H2O) C, H, N.
C
(C32H46N3O9PS‚0.5H2O) C, H, N.
(4-OH )P h CH 2CH 2CO-Tyr [P (O)(OH )2]-Glu -N(n -C5H 11
)
2
(56): prepared in this fashion from 37; 75 mg (93%) as a white
foam; 1H NMR (400 MHz, DMSO-d6) δ 9.10 (br s, 1H), 8.15 (d,
J ) 8.4 Hz, 1H), 7.98 (d, J ) 8.3 Hz, 1H), 7.13 (d, J ) 8.3 Hz,
2H), 7.01 (d, J ) 8.2 Hz, 2H), 6.90 (d, J ) 8.4 Hz, 2H), 6.61 (d,
J ) 8.3 Hz, 2H), 4.69 (ddd, J ) 8.8, 8.8, 4.4 Hz, 1H), 4.51 (ddd,
J ) 8.9, 8.9, 4.1 Hz, 1H), 3.34 (m, 2H), 3.23 (m, 1H), 3.08 (m,
1H), 2.89 (dd, J ) 13.9, 4.1 Hz, 1H), 2.65 (dd, J ) 13.9, 9.7
Hz, 1H), 2.56 (m, 2H), 2.27 (comp, 4H), 1.84 (m, 1H), 1.66 (m,
1H), 1.53 (m, 2H), 1.43 (m, 2H), 1.34-1.15 (comp, 8H), 0.86
(t, J ) 7.0 Hz, 3H), 0.83 (t, J ) 7.1 Hz, 3H); MS (FAB) m/z
(4-Cl)P h CO-Tyr [P (O)(OH)2]-Glu -N(n -C5H11
) (62): pre-
2
pared in this fashion from 43; 131 mg (49%) as a foam; 1H
NMR (400 MHz, DMSO-d6) δ 8.58 (d, J ) 8.3 Hz, 1H), 8.27 (d,
J ) 8.3 Hz, 1H), 7.76 (d, J ) 8.5 Hz, 2H), 7.49 (d, J ) 8.5 Hz,
2H), 7.27 (d, J ) 8.5 Hz, 2H), 7.00 (d, J ) 8.4 Hz, 2H), 4.67
678 (M + H, 27), 158 (100); HRFAB-MS calcd for C33H48N3O10
P
-
(MH+) 678.3156, found 678.3154 (MH+). Anal. (C33H48N3O10
P‚0.5H2O) C, H, N.

Dipeptide Inhibitors of the pp60c-src SH2 Domain
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1905
(m, 2H), 3.32 (m, 2H), 3.24 (m, 1H), 3.07 (m, 1H), 2.99 (dd, J
) 13.7, 3.3 Hz, 1H), 2.87 (dd, J ) 13.7, 11.3 Hz, 1H), 2.25 (dd,
J ) 7.3, 7.0 Hz, 2H), 1.84 (m, 1H), 1.67 (m, 1H), 1.51 (m, 2H),
1.40 (m, 2H), 1.34-1.11 (comp, 8H), 0.83 (t, J ) 7.0 Hz, 3H),
0.80 (t, J ) 7.2 Hz, 3H); MS (FAB) m/z 668 (M + H, 30), 158
(100); HRFAB-MS calcd for C31H43ClN3O9P (MH+) 668.2504,
found 668.2498 (MH+). Anal. (C31H43ClN3O9P‚0.5H2O) C, H,
N.
(4-NO2)P h NHCO-Tyr [P (O)(OH)2]-Glu -N(n -C5H11)2 (68):
prepared in this fashion from 49; 137 mg (65%) as a foam; 1H
NMR (400 MHz, DMSO-d6) δ 9.46 (s, 1H), 8.45 (d, J ) 8.6 Hz,
1H), 8.11 (d, J ) 9.2 Hz, 2H), 7.56 (d, J ) 9.3 Hz, 2H), 7.11 (d,
J ) 8.4 Hz, 2H), 7.01 (J ) 8.2 Hz, 2H), 6.54 (d, J ) 8.0 Hz,
1H), 4.73 (ddd, J ) 8.9, 8.9, 4.3 Hz, 1H), 4.55 (dd, J ) 12.2,
7.6 Hz, 1H), 3.35 (m, 2H), 3.26 (M, 1H), 3.13 (m, 1H), 2.98
(dd, J ) 13.8, 4.3 Hz, 1H), 2.80 (dd, J ) 13.8, 7.6 Hz, 1H),
2.28 (m, 2H), 1.85 (m, 1H), 1.69 (m, 1H), 1.55 (m, 2H), 1.45
(m, 2H), 1.34-1.15 (comp, 8H), 0.85 (J ) 7.0 Hz, 3H), 0.83 (t,
J ) 7.1 Hz, 3H); MS (FAB) m/z 694 (M + H, 37), 158 (100);
HRFAB-MS calcd for C31H44N5O11P (MH+) 694.2853, found
679.2850 (MH+). Anal. (C31H44N5O11P‚0.5H2O) C, H, N.
(4-H 2N C H 2)P h C H 2C O -T y r [P (O )(O H )2]-G lu -N (n -C 5-
H11)2 (69): prepared in this fashion from 50; 157 mg (73%) as
a white foam; 1H NMR (400 MHz, DMSO-d6) δ 8.50 (br s, 2H),
8.31 (d, J ) 8.5 Hz, 1H), 8.24 (d, J ) 8.4 Hz, 1H), 7.28 (d, J )
8.1 Hz, 2H), 7.09 (d, J ) 8.4 Hz, 2H), 6.96 (d, J ) 8.4 Hz, 2H),
6.94 (d, J ) 8.0 Hz, 2H), 4.71 (dd, J ) 13.2, 8.8 Hz, 1H), 4.48
(ddd, J ) 10.0, 10.0, 3.2 Hz, 1H), 3.87 (br s, 2H), 3.50 (d, J )
13.2 Hz, 1H), 3.36 (m, 2H), 3.30 (m, 1H), 3.17 (d, J ) 13.2 Hz,
1H), 3.10 (m, 1H), 2.92 (d, J ) 11.2 Hz, 1H), 2.63 (m, 1H),
2.25 (m, 2H), 1.83 (m, 1H), 1.66 (m, 1H), 1.55 (m, 2H), 1.44
(m, 2H), 1.36-1.15 (comp, 8H), 0.87 (t, J ) 6.9 Hz, 3H), 0.83
(t, J ) 7.2 Hz, 3H); MS (FAB) m/z 677 (M + H, 100); HRFAB-
MS calcd for C33H50N4O9P (MH+) 677.3315, found 677.3323
(MH+). Anal. (C33H49N4O9P‚0.6TFA) C, H, N.
4-[[(ter t-Bu t oxyca r b on yl)a m in o]m et h yl]p h en yla cet -
ic Acid (71). Thionyl chloride (2.25 mL, 30.8 mmol) was
added to a suspension of 4-(bromomethyl)phenylacetic acid (70)
in PhCH3 (200 mL), DMF (10 mL), and pyridine (2.70 mL, 33.4
mmol) at 0 °C. The reaction mixture was stirred for 5 min,
then warmed to room temperature, stirred for an additional
75 min, and concentrated under reduced pressure. The residue
was dissolved in CH2Cl2 (200 mL), cooled to 0 °C, treated with
benzyl alcohol (3.20 mL, 30.9 mmol), Et3N (3.10 mL, 22.1
mmol), and a catalytic amount of DMAP, and stirred at
ambient temperature overnight. The reaction mixture was
washed with half-saturated aqueous NaHCO3 (1 × 50 mL),
H2O (1 × 50 mL), 2 N aqueous NaOH (1 × 50 mL), and
saturated aqueous NaCl (1 × 50 mL), dried (Na2SO4), and
concentrated under reduced pressure. The residue was passed
through a plug of silica gel (230-400 mesh), elution with
hexanes-EtOAc (gradient, 5-10%), to provide 1.51 g (22%)
of the benzyl ester as a brown amorphous solid that was
dissolved in DMF (50 mL) and treated with sodium azide (605
mg, 9.30 mmol). The reaction mixture was stirred at room
temperature overnight, diluted with H2O (ca. 50 mL), and
extracted with EtOAc (2 × 175 mL). The combined organic
extracts were washed with H2O (4 × 100 mL), half-saturated
aqueous NaHCO3 (1 × 100 mL), and saturated aqueous NaCl
(1 × 150 mL), dried (Na2SO4), and concentrated under reduced
pressure to give 1.28 g (98%) of the azide as a yellow oil. The
azide (653 mg, 2.32 mmol) was dissolved in MeOH (25 mL),
treated with BOC2O (767 mg, 3.51 mmol) and 10% Pd-C (36
mg), and then stirred under H2 (1 atm) overnight. The
reaction mixture was flushed with N2 and filtered through a
pad of Celite with MeOH (ca. 25 mL), and the filtrate was
concentrated under reduced pressure. The residue was dis-
solved in EtOAc (25 mL), treated with 10% aqueous KHSO4,
and stirred vigorously for 5 min. The layers were separated,
and the aqueous phase was extracted with EtOAc (2 × 10 mL).
The combined organic extracts were washed with H2O (1 ×
10 mL) and saturated aqueous NaCl (1 × 10 mL), dried (Na2-
SO4), and concentrated under reduced pressure to give an oil
that was triturated with Et2O and heptane to provide 141 mg
(23%) of 71 as an amorphous white solid.
(4-CF 3)P h CO-Tyr [P (O)(OH)2]-Glu -N(n -C5H11)2 (63): pre-
pared in this fashion from 44; 48 mg (21%) as a foam; 1H NMR
(400 MHz, DMSO-d6) δ 8.77 (d, J ) 8.4 Hz, 1H), 8.33 (d, J )
8.4 Hz, 1H), 7.95 (d, J ) 8.2 Hz, 2H), 7.82 (d, J ) 8.4 Hz, 2H),
7.30 (d, J ) 8.6 Hz, 2H), 7.03 (d, J ) 8.4 Hz, 2H), 4.73 (comp,
2H), 3.34 (m, 2H), 3.28 (m, 1H), 3.10 (m, 1H), 3.03 (dd, J )
13.9, 3.7 Hz, 1H), 2.91 (m, 1H), 2.28 (dd, J ) 7.2, 7.0 Hz, 2H),
1.86 (m, 1H), 1.70 (m, 1H), 1.55 (m, 2H), 1.42 (m, 2H), 1.34-
1.15 (comp, 8H), 0.85 (t, J ) 7.0 Hz, 3H), 0.82 (t, J ) 7.1 Hz,
3H); MS (FAB) m/z 702 (M + H, 45), 173 (100); HRFAB-MS
calcd for C32H43F3N3O9P (MH+) 702.2767, found 702.2759
(MH+). Anal. (C32H43F3N3O9P‚0.75H2O) C, H, N.
(4-NO2)P h CO-Tyr [P (O)(OH)2]-Glu -N(n -C5H11)2 (64): pre-
pared in this fashion from 45; 111 mg (93%) as a white foam;
1H NMR (400 MHz, DMSO-d6) δ 8.89 (d, J ) 8.4 Hz, 1H), 8.37
(d, J ) 8.4 Hz, 1H), 8.28 (d, J ) 8.7 Hz, 2H), 7.99 (d, J ) 8.8
Hz, 2H), 7.30 (d, J ) 8.5 Hz, 2H), 7.03 (d, J ) 8.3 Hz, 2H),
4.73 (comp, 2H), 3.33 (m, 2H), 3.27 (m, 1H), 3.10 (m, 1H), 3.04
(m, 1H), 2.90 (dd, J ) 12.5, 12.5 Hz, 1H), 2.28 (dd, J ) 7.1,
7.0 Hz, 2H), 1.86 (m, 1H), 1.70 (m, 1H), 1.55 (m, 2H), 1.42 (m,
2H), 1.34-1.11 (comp, 8H), 0.85 (t, J ) 6.9 Hz, 3H), 0.82 (t, J
) 7.1 Hz, 3H); MS (FAB) m/z 679 (M + H, 35), 158 (100);
HRFAB-MS calcd for C31H44N4O11P (MH+) 679.2744, found
679.2751 (MH+). Anal. (C31H44N4O11P‚0.25H2O) C, H, N.
(4-MeO)P h CO-Tyr [P (O)(OH)2]-Glu -N(n -C5H11)2 (65): pre-
pared in this fashion from 46; 126 mg (53%) as a white foam;
1H NMR (400 MHz, DMSO-d6) δ 8.35 (d, J ) 8.4 Hz, 1H), 8.22
(d, J ) 8.4 Hz, 1H), 7.76 (d, J ) 8.8 Hz, 2H), 7.29 (d, J ) 8.4
Hz, 2H), 7.02 (d, J ) 8.4 Hz, 2H), 6.96 (d, J ) 8.7 Hz, 2H),
4.72 (ddd, J ) 8.8, 7.0, 4.6 Hz, 1H), 4.65 (ddd, J ) 9.6, 9.6, 3.8
Hz, 1H), 3.78 (s, 3H), 3.35 (m, 2H), 3.25 (m, 1H), 3.07 (m, 1H),
3.01 (dd, J ) 13.8, 3.8 Hz, 1H), 2.91 (dd, J ) 12.3, 11.4 Hz,
1H), 2.27 (dd, J ) 7.0, 7.0 Hz, 2H), 1.84 (m, 1H), 1.69 (m, 1H),
1.55 (m, 2H), 1.42 (m, 2H), 1.34-1.11 (comp, 8H), 0.85 (t, J )
7.0 Hz, 3H), 0.82 (t, J ) 7.1 Hz, 3H); MS (FAB) m/z 664 (M +
H, 30), 135 (100); HRFAB-MS calcd for C32H46N3O10P (MH+)
664.2999, found 664.3014 (MH+). Anal. (C32H46N3O10P‚0.5TFA)
C, H, N.
(4-NH2)P h NHCO-Tyr [P (O)(OH)2]-Glu -N(n -C5H11)2 (66):
1
prepared in this fashion from 47; 78 mg (49%) as a foam; H
NMR (400 MHz, DMSO-d6) δ 8.66 (s, 1H), 8.33 (d, J ) 8.4 Hz,
1H), 7.26 (d, J ) 8.7 Hz, 2H), 7.09 (d, J ) 8.4 Hz, 2H), 7.00 (d,
J ) 8.4 Hz, 2H), 6.92 (d, J ) 8.8 Hz, 2H), 6.22 (d, J ) 8.0 Hz,
1H), 4.72 (ddd, J ) 8.9, 8.9, 4.3 Hz, 1H), 4.49 (dd, J ) 12.4,
7.8 Hz, 1H), 3.36 (m, 2H), 3.26 (m, 1H), 3.12 (m, 1H), 2.94
(dd, J ) 14.0, 4.3 Hz, 1H), 2.76 (dd, J ) 14.0, 7.8 Hz, 1H),
2.25 (m, 2H), 1.85 (m, 1H), 1.68 (m, 1H), 1.53 (m, 2H), 1.45
(m, 2H), 1.34-1.11 (comp, 8H), 0.85 (t, J ) 6.8 Hz, 3H), 0.84
(t, J ) 7.0 Hz, 3H); MS (ESI) m/z 662 (M - H, 100); HRFAB-
MS calcd for C31H46N5O9P (MH+) 664.3111, found 664.3107
(MH+). Anal. (C31H46N5O9P‚0.67TFA) C, H, N.
(4-MeO)P h NHCO-Tyr [P (O)(OH)2]-Glu -N(n -C5H11)2 (67):
prepared in this fashion from 48; 107 mg (55%) as a foam; 1H
NMR (400 MHz, DMSO-d6) δ 8.47 (s, 1H), 8.32 (d, J ) 8.3 Hz,
1H), 7.22 (d, J ) 8.9 Hz, 2H), 7.11 (d, J ) 8.4 Hz, 2H), 7.01 (d,
J ) 8.4 Hz, 2H), 6.78 (d, J ) 9.0 Hz, 2H), 6.12 (d, J ) 8.0 Hz,
1H), 4.72 (ddd, J ) 8.8, 8.8, 4.4 Hz, 1H), 4.50 (dd, J ) 13.6,
7.8 Hz, 1H), 3.67 (s, 3H), 3.34 (m, 2H), 3.26 (m, 1H), 3.13 (m,
1H), 2.94 (dd, J ) 12.6, 4.4 Hz, 1H), 2.75 (dd, J ) 13.6, 8.0
Hz, 1H), 2.25 (m, 2H), 1.82 (m, 1H), 1.68 (m, 1H), 1.53 (m,
2H), 1.45 (comp, 2H), 1.34-1.18 (comp, 8H), 0.86 (t, J ) 6.8
Hz, 3H), 0.84 (t, J ) 7.0 Hz, 3H); MS (FAB) m/z 679 (M + H,
43), 158 (100); HRFAB-MS calcd for C32H47N4O10P (MH+)
679.3108, found 664.3102 (MH+). Anal. (C32H47N4O10P‚
0.25H2O) C, H, N.
BOC-Cys(Me)-OH (74). BOC2O (4.36 g, 20.0 mmol) was
added portionwise to a solution of H-Cys(Me)-OH (2.70 g, 20.0
mmol) and NaOH (1.20 g, 30.0 mmol) in H2O (20 mL) and 1,4-
dioxane (20 mL). The reaction mixture was stirred for 3 h
and acidified (pH 4) with 10% aqueous KHSO4, and the
aqueous phase was extracted with EtOAc (2 × 100 mL). The
combined organic extracts were washed with saturated aque-

1906 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Pacofsky et al.
ous NaCl (1 × 50 mL), dried (Na2SO4), and concentrated under
reduced pressure to give 4.55 g (97%) of 74 as an oil that
solidified on standing.
at 0 °C in THF (0.7 mL), and the mixture was stirred for 30
min. A solution of di-tert-butyl phosphorochloridate (75 mg,
0.33 mmol) in THF (0.5 mL) was added, and the mixture was
allowed to warm to room temperature over 16 h, then treated
with saturated aqueous NaHCO3 (ca. 5 mL), and poured into
EtOAc (ca. 50 mL). The layers were separated, and the organic
phase was washed with saturated aqueous NaHCO3 (1 × 10
mL) and saturated aqueous NaCl (1 × 10 mL), dried (Na2-
SO4), and concentrated under reduced pressure. The residue
was purified by flash chromatography, elution with 50:1
EtOAc-MeOH, to provide 85 mg (57%) of 88 as a foam.
Ac-Tyr [P (O)(Ot-Bu )2]-Met(O2)-N(n -C5H11)2 (89): prepared
in this fashion from 85; 81 mg (54%) as a colorless oil.
BOC-Met-N(n -C5H11
) (76). Isobutyl chloroformate (0.46
2
mL, 3.53 mmol) was added to a solution of 72 (800 mg, 3.20
mmol) and 4-methylmorpholine (1.10 mL, 9.60 mmol) in THF
(32 mL) at -20 °C. The mixture was stirred for 5 min, treated
with HN(n-C5H11)2 (0.65 mL, 3.20 mmol), and allowed to warm
to room temperature over 3.5 h. The mixture was added to
EtOAc (ca. 100 mL), washed with saturated aqueous NaHCO3
(1 × 50 mL), H2O (1 × 50 mL), and saturated aqueous NaCl
(1 × 50 mL), dried (Na2SO4), and concentrated under reduced
pressure. The residue was purified by flash chromatography,
elution with hexanes-EtOAc (5:1), to provide 1.17 g (94%) of
76 as an oil.
Ac-Tyr [P (O)(Ot-Bu )2]-Cys(Me)-N(n -C5H 11
)
(90): pre-
2
pared in this fashion from 86; 236 mg (59%) as a white foam.
BOC-Met(O2)-N(n -C5H11
) (77): prepared in this fashion
2
Ac-Tyr [P (O)(Ot-Bu )2]-Cys(Bn )-N(n -C5H 11 (91): pre-
)
2
from 73; 960 mg (96%) as a solid.
pared in this fashion from 87; 270 mg (66%) as an oil.
BOC-Cys(Me)-N(n -C5H11 (78): prepared in this fashion
)
2
Ac-Tyr [P (O)(OH)2]-Met-N(n -C5H11)2 (92). HCl (0.51 mL
of a 1 M solution in Et2O, 0.51 mmol) was added to a solution
of 88 (70 mg, 0.10 mmol) in CH2Cl2 (1.0 mL). The mixture
was stirred for 3 h and concentrated under reduced pressure.
Excess solvent was removed in vacuo to provide 58 mg (98%)
of 92 as a foam: 1H NMR (300 MHz, CDCl3) δ 7.75 (br s, 1H),
7.06 (comp, 5H), 5.05 (m, 1H), 4.75 (m, 1H), 3.51 (m, 1H), 3.36
(m, 2H), 3.10 (m, 2H), 2.85 (m, 1H), 2.48 (m, 2H), 2.07 (s, 3H),
1.92 (m, 2H), 1.88 (s, 3H), 1.63 (m, 2H), 1.52 (m, 2H), 1.43-
1.18 (comp, 8H), 0.92 (t, J ) 6.6 Hz, 3H), 0.87 (t, J ) 6.9 Hz,
3H); MS (FAB) m/z 574 (M + H, 43), 158 (100); HRFAB-MS
from 74; 1.35 g (70%) as a colorless oil.
BOC-Cys(Bn )-N(n -C5H11 (79): prepared in this fashion
)
2
from 75; 1.67 g (95%) as a colorless oil.
BOC-Tyr -Met-N(n -C5H11 (80). HCl (3.0 mL of a 4 M
)
2
solution in dioxane, 11.9 mmol) was added to a solution of 76
(1.15 g, 2.96 mmol) in CH2Cl2 (6 mL). The mixture was stirred
for 14 h and concentrated under reduced pressure, and the
residue was washed with Et2O (3 × 5 mL). Excess solvent
was removed in vacuo to provide 950 mg (98%) of the
hydrochloride as a foam. A solution of HCl-H-Met-N(n-C5H11
)
2
calcd for
C
26H44N3O7PS (MH+) 574.2716, found 574.2713
(1.02 g, 3.14 mmol) in CH2Cl2 (10 mL) was added to a solution
of BOC-Tyr-OH (883 mg, 3.14 mmol), DCC (712 mg, 3.46
mmol), and HOBT (467 mg, 3.46 mmol) in CH2Cl2 (15 mL)
and DMF (7 mL) at 0 °C. Et3N (0.53 mL, 3.77 mmol) was
added, and the mixture was allowed to warm to room tem-
perature over 16 h. The reaction mixture was filtered through
a pad of Celite using EtOAc (ca. 75 mL), and the filtrate was
concentrated under reduced pressure. The residue was diluted
with EtOAc (ca. 150 mL), washed with H2O (3 × 25 mL) and
saturated aqueous NaCl (1 × 25 mL), dried (Na2SO4), and
concentrated under reduced pressure. The residue was puri-
fied by flash chromatography, elution with hexanes-EtOAc
(2:1), to provide 1.40 g (81%) of 80 as a foam.
BOC-Tyr -Met(O2)-N(n -C5H11)2 (81): prepared in this fash-
ion from 77; 935 mg (75%) as a white foam.
BOC-Tyr -Cys(Me)-N(n -C5H11)2 (82): prepared in this fash-
ion from 78; 1.56 g (90%) as a foam.
BOC-Tyr -Cys(Bn )-N(n -C5H11)2 (83): prepared in this fash-
ion from 79; 1.72 g (80%) as a white foam.
(MH+). Anal. (C26H44N3O7PS‚0.5H2O) C, H, N.
Ac-Tyr [P (O)(OH)2]-Met(O2)-N(n -C5H11)2 (93): prepared in
this fashion from 89; 51 mg (91%) as an off-white foam; 1H
NMR (300 MHz, CDCl3) δ 8.03 (br s, 1H), 7.43 (br s, 1H), 7.04
(comp, 4H), 5.05 (m, 1H), 4.73 (m, 1H), 3.48 (m, 1H), 3.33 (m,
2H), 3.10 (comp, 4H), 2.91 (s, 3H), 2.82 (m, 1H), 2.19 (m, 2H),
1.82 (s, 3H), 1.63 (m, 2H), 1.49 (m, 2H), 1.41-1.13 (comp, 8H),
0.90 (t, J ) 6.8 Hz, 3H), 0.86 (t, J ) 7.0 Hz, 3H); MS (FAB)
m/z 606 (M + H, 85), 136 (100); HRFAB-MS calcd for
C
26H44N3O9PS (MH+) 606.2614, found 606.2615 (MH+). Anal.
(C26H44N3O9PS‚0.5H2O) C, H, N.
Ac-Tyr [P (O)(OH)2]-Cys(Me)-N(n -C5H11)2 (94): prepared
in this fashion from 90; 176 mg (96%) as a white foam; 1H
NMR (400 MHz, CDCl3) δ 7.87 (br s, 1H), 7.30 (br s, 1H), 7.08
(comp, 4H), 5.04 (dd, J ) 14.9, 7.7 Hz, 1H), 4.76 (dd, J ) 7.7,
6.6 Hz, 1H), 3.49 (m, 1H), 3.38 (ddd, J ) 14.9, 14.9, 7.7 Hz,
2H), 3.14 (m, 1H), 3.03 (dd, J ) 13.8, 5.5 Hz, 1H), 2.87 (comp,
2H), 2.72 (dd, J ) 13.8, 5.5 Hz, 1H), 2.11 (s, 3H), 1.89 (s, 3H),
1.65 (comp, 2H), 1.53 (comp, 2H), 1.41-1.20 (comp, 8H), 0.92
(t, J ) 7.0 Hz, 3H), 0.87 (t, J ) 7.0 Hz, 3H); MS (FAB) m/z
560 (M + H, 55), 158 (100); HRFAB-MS calcd for C25H42N3O7-
PS (MH+) 560.2559, found 560.2549 (MH+). Anal. (C25H42N3O7-
PS) C, H, N.
Ac-Tyr -Met-N(n -C5H11
) (84). HCl (2.52 mL of a 4 M
2
solution in dioxane, 10.0 mmol) was added to a solution of 80
(1.39 g, 2.52 mmol) in CH2Cl2 (5 mL). The mixture was stirred
for 3 h and concentrated under reduced pressure to provide
1.19 g (97%) of the hydrochloride as a foam. A solution of HCl-
H-Tyr-Met-N(n-C5H11
) in CH2Cl2 (2 mL) was added to a
2
Ac-Tyr [P (O)(OH)2]-Cys(Bn )-N(n -C5H11
) (95): prepared
2
in this fashion from 91; 220 mg (98%) as a white foam; 1H
NMR (400 MHz, CDCl3) δ 7.66 (br s, 1H), 7.27 (comp, 5H),
7.05 (comp, 4H), 6.84 (br s, 1H), 5.00 (ddd, J ) 14.2, 8.0 Hz,
1H), 4.78 (dd, J ) 13.8, 8.0 Hz, 1H), 3.71 (s, 2H), 3.52 (ddd, J
) 13.8, 8.0, 7.5 Hz, 1H), 3.23 (m, 2H), 3.09 (m, 1H), 2.99 (dd,
J ) 13.8, 5.5 Hz, 1H), 2.88 (dd, J ) 14.2, 5.5 Hz, 1H), 2.79
(dd, J ) 13.6, 8.0 Hz, 1H), 2.63 (dd, J ) 13.6, 6.0 Hz, 1H),
1.89 (s, 3H), 1.61-1.46 (comp, 4H), 1.39-1.19 (comp, 8H), 0.92
(t, J ) 7.2 Hz, 3H), 0.85 (t, J ) 7.0 Hz, 3H); MS (FAB) m/z
636 (M + H, 78), 158 (100); HRFAB-MS calcd for C31H46N3O7-
PS (MH+) 636.2872, found 636.2858 (MH+). Anal. (C31H46N3O7-
PS) C, H, N.
solution of AcOH (31 mg, 0.51 mmol), DCC (116 mg, 0.56
mmol), and HOBT (76 mg, 0.56 mmol) in CH2Cl2 (2 mL) and
DMF (1 mL) at 0 °C. Et3N (0.11 mL, 0.79 mmol) was added,
and the reaction mixture was allowed to warm to room
temperature over 18 h. The mixture was filtered through a
pad of Celite using EtOAc (ca. 50 mL), and the filtrate was
concentrated under reduced pressure. The residue was diluted
with EtOAc (ca. 75 mL), washed with H2O (3 × 20 mL) and
saturated aqueous NaCl (1 × 20 mL), dried (Na2SO4), and
concentrated under reduced pressure. The residue was puri-
fied by flash chromatography, elution with EtOAc, to provide
208 mg (82%) of 84 as a foam.
Ac-Tyr -Met(O2)-N(n -C5H11)2 (85): prepared in this fashion
from 81; 210 mg (46%) as a white foam.
Ac-Tyr -Cys(Me)-N(n -C5H11)2 (86): prepared in this fashion
from 82; 520 mg (88%) as a white foam.
Ac-Tyr -Cys(Bn )-N(n -C5H11)2 (87): prepared in this fashion
from 83; 535 mg (88%) as a white foam.
Ac-Tyr [P (O)(Ot-Bu )2]-Met-N(n -C5H11)2 (88). A solution
of 84 (108 mg, 0.22 mmol) in THF (1 mL) was added to a
suspension of NaH (313 mg of 60% oil dispersion, 0.77 mmol)
SH2 ELISA. The evaluation of peptides using ELISA,
including the expression and purification of recombinant
proteins, was conducted as described previously.13 Briefly,
autophosphorylated epidermal growth factor receptor tyrosine
kinase (EGFR-TK) in buffer, in the presence of 1 mM Na3-
VO4, was diluted to 0.66 µM, and a 50-µL aliquot was placed
in each well of 96-well Nunc Maxisorp plates (USA/Scientific
Plastics, Ocala, FL). After incubation, the plates were washed,
and the wells were filled with blocking buffer and incubated

Dipeptide Inhibitors of the pp60c-src SH2 Domain
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1907
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Cytoplasmic Signaling Proteins. Science 1991, 252, 668-674.
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T. M. Involvement of pp60c-src with Two Major Signaling
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pp60c-src Protein Kinase is an Early Event in Colonic Carcino-
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Increase in Activity and Level of pp60c-src in Progressive Stages
of Human Colorectal Cancer. J . Clin. Invest. 1993, 91, 53-60.
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Hennipman, A.; Michels, A. A.; Staal, G. E. J . Characterization
of Protein Tyrosine Kinases from Human Breast: Involvement
of the c-src Oncogene Product. Cancer Res. 52, 4773-4778.
(12) Pawson, T.; Gish, G. D. SH2 and SH3 Domains: From Structure
to Function. Cell 1992, 71, 359-362.
(13) Gilmer, T.; Rodriguez, M.; J ordan, S.; Crosby, R.; Alligood, K.;
Green, M.; Kimery, M.; Wagner, C.; Kinder, D.; Charifson, P.;
Hassell, A. M.; Willard, D.; Luther, M.; Rusnak, D.; Sternbach,
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J .; Patel, I. R.; Kassel, D.; Burkhart, W.; Moyer, M.; Bradshaw,
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S. A.; Claesson-Welsh, L.; Heldin, C.-H. Identification of Two
J uxtamembrane Auto-phosphorylation Sites in PDGF â-Recep-
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overnight. Finally, the plates were washed, wrapped in
aluminum foil, and stored at -20 °C for up to 8 weeks.
Binding of the glutathione S-transferase (GST) src SH3-
SH2 fusion protein to autophosphorylated EGFR was quan-
titated by enzyme-linked immunosorbent assay (ELISA). The
fusion protein was diluted in blocking buffer containing 1 mM
Na3VO4 and 5 mM dithiothreitol (DTT), and 50 µL of each
dilution was added in triplicate to wells in EGFR-TK-coated
plates. After incubation, the plates were washed, and the
presence of bound src fusion protein was detected by affinity-
purified anti-src antibody. After incubation, the plates were
washed, and anti-src antibody was detected by alkaline
phosphatase-linked goat anti-mouse IgG, Fc-specific (J ackson
ImmunoResearch Laboratories, Inc., West Grove, PA); the
plates were incubated and washed as before. Following a final
wash with alkaline phosphatase (AP) buffer, 50 µL of 1 mg/
mL p-nitrophenyl phosphate in AP buffer was placed in each
well and incubated. The plates were read on a Molecular
Devices (Menlo Park, CA) UVmax plate reader at 405 nm.
For the competitive ELISA, aqueous peptide stock solutions,
pH 7.0, were serially diluted in buffer containing 1 mM Na3-
VO4 and 5 mM DTT and incubated with the GST src SH3-
SH2 fusion protein. Each peptide solution (50 µL) was added
to wells of EGFR-TK-coated plates in triplicate before process-
ing as described above. The activity is expressed as a
percentage of the activity shown by the control: y ) 100 ×
(ODobs - ODblank)/(ODmax - ODblank). Data available for
nonlinear curve fitting were evaluated to determine the IC50
values. If a nonlinear fit could not be obtained, the IC50 values
were obtained by linear interpolation.
Using this competition assay and 1 as the standard, peptides
were tested as inhibitors of src SH3-SH2:phosphoprotein
interactions. The IC50 of 1 was 0.67 ( 0.04 µM. For the
purpose of comparison and to normalize data from different
experiments, results for individual peptides are presented as
a ratio: IC50(test)/IC50(standard).
Cr ysta llogr a p h y. Crystallographic data of peptides bound
to the human pp60c-src SH2 domain were obtained as reported
previously.23 Briefly, human pp60c-src SH2:dipeptide crystals
were grown by the hanging drop vapor diffusion method where
protein was mixed with an equimolar amount of peptide. Data
were collected on a Rigaku RAXIS IIC area detector mounted
on a Siemens rotating anode X-ray generator. Using the
structure of the SH2:1 complex13 as a starting model, ad-
ditional structures were solved. Structures were manually
rebuilt using FRODO,39 refined with X-PLOR,40 and evaluated
using PROCHECK.41
(20) Waksman, G.; Kominos, D.; Robertson, S. C.; Pant, N.; Balti-
more, D.; Birge, R. B.; Cowburn, D.; Hanafusa, H.; Mayer, B.
J .; Overduin, M.; Resh, M. D.; Rios, C. B.; Silverman, L.;
Kuriyan, J . Crystal Structure of the Phosphotyrosine Recogni-
tion Domain SH2 of v-src Complexed with Tryosine-Phospho-
rylated Peptides. Nature 1992, 358, 646-653.
Ack n ow led gm en t. We would like to thank Rod
Davis and Larry Shampine for providing low- and high-
resolution mass spectral data. We thank Dr. Mike Cory
for preparing figures in the manuscript.
(21) Kuriyan, J .; Cowburn, D. Structures of SH2 and SH3 Domains.
Curr. Opin. Struct. Biol. 1993, 3, 828-837.
(22) Waksman, G.; Shoelson, S. E.; Pant, N.; Cowburn, D.; Kuriyan,
J . Binding of a High Affinity Phosphotyrosyl Peptide to the Src
SH2 Domain: Crystal Structures of the Complexed and Peptide-
free Forms. Cell 1993, 358, 779-790.
(23) Charifson, P. S.; Shewchuk, L. M.; Rocque, W.; Hummel, C. W.;
J ordan, S. R.; Mohr, C.; Pacofsky, G. J .; Peel, M. R.; Rodriguez,
M.; Sternbach, D. D.; Consler, T. G. Peptide Ligands of pp60c-src
SH2 Domains: A Thermodynamic and Structural Study. Bio-
chemistry 1997, 36, 6283-6293.
Su p p or tin g In for m a tion Ava ila ble: Atomic coordinates
are available from the Protein Data Bank,38 Brookhaven
National Laboratory for (compound, PDB file code): 1, 1SHD;
2, 1A1B. Coordinates for compounds 25, 69, and 92 will be
submitted. Spectral data for compounds 3-8, 28-50, 71, 74,
and 76-91 (12 pages). Ordering information is given on any
current masthead page.
(24) Plummer, M. S.; Lunney, E. A.; Para, K. S.; Vara Prasad, J . V.
N.; Shahripour, A.; Singh, J .; Stankovic, C. J .; Humblet, C.;
Fergus, J . H.; Marks, J . S.; Sawyer, T. K. Hydrophobic D-Amino
Acids in the Design of Peptide Ligands for the pp60src SH2
Domain Drug Des. Discovery 1996, 13, 75-81.
(25) Plummer, M. S.; Lunney, E. A.; Para, K. S.; Shahripour, A.;
Stankovic, C. J .; Humblet, C.; Fergus, J . H.; Marks, J . S.;
Herrera, R.; Hubbell, S.; Saltiel, A.; Sawyer, T. K. Design of
Peptidomimetic Ligands for the pp60src SH2 Domain. Bioorg.
Med. Chem. 1997, 5, 41-47.
(26) Shahripour, A.; Para, K. S.; Plummer, M. S.; Lunney, E. A.;
Holland, D. R.; Rubin, J . R.; Humblet, C.; Fergus, J . H.; Marks,
J . S.; Saltiel, A.; Sawyer, T. K. Structure-Based Design of Novel,
Peptide Ligands Targeting the pp60src SH2 Domain. Bioorg. Med.
Chem. Lett. 1997, 7, 1107-1112.
Refer en ces
(1) Current addresses: P.S.C., Vertex Pharmaceuticals, 130 Waverly
St., Cambridge, MA 02139-4242. C.W.H., Amgen Boulder, Inc.,
Mail Drop AB/4A, 3200 Walnut St., Boulder, CO 80301. S.R.J .,
Amgen, Mail Stop 14-2B, 1840 Dehavilland Dr., Thousand Oaks,
CA 91320. M.R., NEOSYSTEM, 7 Rue de Boulogne, 67100
Strasbourg, France.
(2) Schlessinger, J .; Ullrich, A. Growth Factor Signaling by Receptor
Tyrosine Kinases. Neuron 1992, 9, 383-391.
(3) Schlessinger, J .; Ullrich, A. Signal Transduction by Receptors
with Tyrosine Kinase Activity. Cell 1990, 61, 203-212.
(4) Margolis, B. Proteins with SH2 Domains: Transducers in the
Tyrosine Kinase Signaling Pathway. Cell Growth Differ. 1992,
3, 73-80.

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