Design and synthesis of conformationally constrained Grb2 SH2 domain binding peptides employing α-methylphenylalanyl based phosphotyrosyl mimetics

Article abstract of DOI:10.1021/jm0492709

Previous work has shown that incorporation of either 1- aminocyclohexanecarboxylic acid (Ac₆c) or α-methyl-p- phosphonophenylalanine ((α-Me)Ppp) in the phosphotyrosyl (pTyr) C-proximal position (pY + 1 residue) of Grb2 SH2 domain binding peptid

Full text of DOI:10.1021/jm0492709

764
J. Med. Chem. 2005, 48, 764-772
Design and Synthesis of Conformationally Constrained Grb2 SH2 Domain
Binding Peptides Employing r-Methylphenylalanyl Based Phosphotyrosyl
Mimetics
Shinya Oishi,† Rajeshri G. Karki,† Sang-Uk Kang,† Xiangzhu Wang,† Karen M. Worthy,‡ Lakshman K. Bindu,‡
Marc C. Nicklaus,† Robert J. Fisher,‡ and Terrence R. Burke Jr.*,†
Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute, National Institutes of Health,
Frederick, Maryland 21702, and Protein Chemistry Laboratory, Science Applications International CorporationsFrederick,
Frederick, Maryland 21702
Received September 7, 2004
Previous work has shown that incorporation of either 1-aminocyclohexanecarboxylic acid (Ac6c)
or R-methyl-p-phosphonophenylalanine ((R-Me)Ppp) in the phosphotyrosyl (pTyr) C-proximal
position (pY + 1 residue) of Grb2 SH2 domain binding peptides confers high affinity. The
tetralin-based (S)-2-amino-6-phosphonotetralin-2-carboxylic acid (Atc(6-PO3H2)) simultaneously
presents structural features of both (R-Me)Ppp and Ac6c residues. The current study compares
the affinity of this tetralin hybrid Atc(6-PO3H2) versus Ac6c and (R-Me)Ppp residues when
incorporated into the pY + 1 position of a high-affinity Grb2 SH2 domain binding tripeptide
platform. The highest binding affinity (KD ) 14.8 nM) was exhibited by the (R-Me)Ppp-
containing parent, with the corresponding Ac6c-containing peptide being nearly 2-fold less potent
(KD ) 23.8 nM). The lower KD value was attributable primarily to a 50% increase in off-rate.
Replacement of the Ac6c residue with the tetralin-based hybrid resulted in a further 4-fold
decrease in binding affnity (KD ) 97.8 nM), which was the result of a further 6-fold increase
in off-rate, offset by an approximate 45% increase in on-rate. Therefore, by incorporation of
the key structural components found in (R-Me)Ppp into the Ac6c residue, the tetralin hybrid
does enhance binding on-rate. However, net binding affinity is decreased due to an associated
increase in binding off-rate. Alternatively, global conformational constraint of an (R-Me)Ppp-
containing peptide by â-macrocyclization did result in pronounced elevation of binding affinity,
which was achieved primarily through a decrease in the binding off-rate. Mathematical fitting
using a simple model that assumed a single binding site yielded an effective KD of 2.28 nM.
However this did not closely approximate the data obtained. Rather, use of a complex model
that assumed two binding sites resulted in a very close fit of data and provided KD values of
97 pM and 72 nM for the separate sites, respectively. Therefore, although local conformational
constraint in the pY + 1 residue proved to be deleterious, global conformational constraint
through â-macrocyclization achieved higher affinity. Similar â-macrocyclization may potentially
be extended to SH2 domain systems other than Grb2, where bend geometries are required.
The growth factor receptor bound protein 2 (Grb2) is
an SH2 domain containing noncatalytic module that
provides important connectivity in protein tyrosine
kinase (PTK)-dependent signaling associated with a
variety of cancers.1-6 Accordingly, significant effort has
been expended in developing Grb2 SH2 domain binding
antagonists as potential therapeutics.7-9 On the basis
of the preferred binding of Grb2 SH2 domains to
phosphotyrosyl (pTyr)-containing sites of the sequence
“pY-X-N”, tripeptide 1 (Figure 1) was identified by
Novartis scientists as a high-affinity ligand that is
characterized by a (pY + 1)-positioned10 1-aminocyclo-
hexanecarboxylic acid residue (Ac6c) (side chain shown
as 2, Figure 1).11 The Ac6c residue had been shown to
be optimal for the pY + 1 position due to its ability to
induce the â-bend conformation needed for binding and
to provide good van der Waals interactions with Phe
âD5 (F108) and Gln âD3 (Q106) residues.12 More
recently, a significant discovery related to the pY + 1
residue was reported by Garbay et al., who, using a
different peptide platform, showed that insertion of an
(R-methyl)pTyr residue (side chain shown as 3, Figure
1)13 or a pTyr mimetic such as R-methyl-p-phospho-
nophenylalanine ((R-Me)Ppp) (side chain shown as 4,
Figure 1)14 enhances the Grb2 SH2 domain binding
affinity of short peptide sequences through ionic inter-
actions of the negatively charged phosphate mimetic
with the positively charged guanidino group of the Arg
BG4 (R142) residue.15 To date, Ac6c and (R-Me)Ppp are
among the highest affinity pY + 1 residues identified
for Grb2 SH2 domain binding peptides. An unrelated
advance in Grb2 SH2 domain binding inhibitor design
is the induction of global conformational constraint
through macrocyclization originating at the pTyr mi-
metic â-position.16,17 Significant affinity enhancements
have been achieved in going from open chain analogues
such as 6 to macrocyclic variants such as 7.18-21
* Author to whom correspondence should be addressed. Address:
CCR, NCI; NIH; P.O. Box B, Bldg. 376 Boyles St.; Frederick, MD
21702-1201. Phone: (301) 846-5906. Fax: (301) 846-6033. E-mail:
tburke@helix.nih.gov.
† CCR, NCI, NIH.
‡ SAICsFrederick.
10.1021/jm0492709 CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/19/2005

Grb SH2 Binding Peptides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 765
Figure 1. Structures of analogues discussed.
The affinity-enhancing effects of Ac6c residues and
â-macrocyclization are in principle due to conforma-
tional restrictions leading to induction of â-bend geom-
etries, whereas the binding effects of pY + 1 (R-Me)pTyr
mimetics can be attributed predominately to ionic
interactions. Because these two modalities differ mecha-
nistically, the question arose as to what effects could
result from combining together features found individu-
ally in (R-Me)pTyr and â-bend-inducing motifs. As a
further consideration, it was realized that, although
several variants of (R-Me)pTyr that differ in the phos-
phoryl-mimicking 4-phenyl-substituted region have been
examined in the pY + 1 position,13,14 other modifications
of (R-Me)pTyr, particularly those that introduce aspects
of the Ac6c residue, have not been reported. Therefore,
studies were undertaken to address two issues. In the
first study, structural components of Ac6c and (R-Me)-
Ppp were combined into a single residue represented
by the tetralin-based (S)-2-amino-6-phosphonotetralin-
2-carboxylic acid (Atc(6-PO3H2, depicted by 5, Figure
1).22 Atc(6-PO3H2) may be seen both as an (R-Me)Ppp
residue that contains Ac6c as a structural component
(Figure 1) and as an (R-Me)Ppp variant that bears
conformational constraint of side chain ø1 and ø2 angles.22
For this study, open-chain peptides were prepared
having Ac6c, (R-Me)Ppp, and Atc(6-PO3H2) residues in
the pY + 1 position (peptides 6, 8, and 9, respectively).
A second study was undertaken in which the Ac6c
residue in macrocycle 7 was replaced with an (R-Me)-
Ppp residue to yield the corresponding globally con-
strained hybrid macrocycle 10. Target peptides 9 and
10 from the aforementioned studies explore the applica-
tion of local and global conformational constraint,
respectively, to Grb2 SH2 domain binding peptides
bearing pTyr mimetics in the pY + 1 position.
Synthesis
Coupling of asparagine 3-((1-naphthyl)propyl))amide
(11)23 with the sterically crowded N-Boc (R-Me)Phe(4-
PO3Et2)-OH (12)14,22 or with N-Boc (S)-2-amino-6-(di-
ethylphosphono)tetralin-2-carboxylic acid (Boc-Atc(6-
PO3Et2)-OH, 13)22 was accomplished using tetramethyl-
fluoroformamidinium hexafluorophosphate (TFFH)24 to
yield dipeptides 14 and 15, respectively (Scheme 1).
After trifluoroacetic acid (TFA)-mediated N-Boc depro-
tection of both 14 and 15, reaction with pTyr mimetic
1625 was conducted using TFFH activation to yield the
globally protected products 17 and 18, respectively.
Final products 8 and 9 were obtained following a two-
stage trimethylsilyliodide (TMSI) TFA deprotection
protocol. The Ac6c-containing peptide 6 has been re-
ported previously.25 Synthesis of (R-Me)Ppp-containing
macrocycle 10 (Scheme 2) was similar to previously
reported procedures.18,20
Results and Discussion
The objectives of the current work were to examine
the Grb2 SH2 domain binding effects incurred by: (1)
combining into a single analogue important structural
features of (R-Me)Ppp and Ac6c residues and (2) placing
an (R-Me)pTyr mimetic at the pY + 1 position of a
â-macrocyclized inhibitor. Objective 1 was accomplished
using the tetralin-based Atc(6-PO3H2) (5) as a substitute
for the pY + 1 Ac6c residue in the known high-affinity
(1-naphthyl)propylamide-containing pseudotripeptide 6.

766 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Oishi et al.
Scheme 1. Reagents and Conditions: (i) TFFH, i-Pr2EtN and either 12 or 13; (ii) (A) TFA, Anisole, (B) NaHCO3, (C)
13, TFFH, i-Pr2EtN; (iii) (A) TMSI, (B) TFA/H2O (95:5).
This latter peptide employs an R-carboxymethyl-con-
taining pTyr mimetic in the pY0 position.25 Synthesis
of the Atc(6-PO3H2)-containing title peptide 9 was
achieved using the globally protected tetralin analogue
13.22 The binding potency of 9 was compared with the
known Ac6c-containing peptide 625 and with the corre-
sponding (R-Me)Ppp-containing peptide 8. Evaluation
of Grb2 SH2 domain binding potency was accomplished
by plasmon resonance analysis using a Biacore instru-
ment, which measured the direct binding of synthetic
inhibitor to sensor-bound Grb2 SH2 domain protein.
Grb2 SH2 Domain Binding Affinities. Kinetic
binding data in the form of on- and off-rates and
associated KD values for 6, 8, and 9 are shown in Table
1. The highest binding affinity of these three peptides
was exhibited by the (R-Me)Ppp-containing 8 (KD ) 14.8
nM). The binding affinity of the Ac6c-containing 6 (KD
) 23.8 nM)26 was less than the affinity of 8, although
the difference between 6 and 8 was not as great as
would be expected based on the 10-fold potency en-
hancement incurred by replacing the pY + 1 Ac6c
residue with an (R-Me)Ppp residue to yield peptide 26
(Figure 2).14 It should be noted that peptides in the
current study are based on the 3-(1-naphthyl)propana-
mido parent 6, whereas analogues of the former study
were based on a different peptide (Figure 2). Relative
contributions to overall binding of the pY + 1 residue
could be different for these two peptides. Interestingly,
although the former study did report a prodrug-
protected form of Ac-pY-(RMe)pY-N-[3-(1-naphthyl)-

Grb SH2 Binding Peptides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 767
Scheme 2. Reagents and Conditions: (i) 12, TFFH, i-Pr2EtN (38% yield); (ii) (A) TFA, Anisole, (B) NaHCO3, (C) 21,
TFFH, i-Pr2EtN (8% yield); (iii) 23, CH2Cl2, Reflux (28% yield); (iv) (A) TMSI, (B) TFA/H2O (95:5) (29% yield).
Table 1. In Vitro Grb2 SH2 Domain Binding Affinitya
(R-Me)Ppp structural components into the Ac6c residue
ka (M-1 s-1
)
kd (s-1
)
KD (nM)
did result in an increase in the rate of binding, this was
accompanied by an unfavorable elevation in binding off-
rate.
no.
6
8
9
4.18 × 106 ( 2.88 × 105 9.91 × 10-2 ( 2.94 × 10-3 23.8 ( 1.8
4.50 × 106 ( 1.96 × 104 6.65 × 10-2 ( 1.61 × 10-4 14.8 ( 0.08
6.09 × 106 ( 2.41 × 105 5.94 × 10-1 ( 2.26 × 10-3 97.8 ( 1.8
The second objective of the current study was to
examine the effects of introducing global conformational
constraint into open-chain 8 through conversion to the
â-macrocycle 10. An analysis of six independent experi-
ments using a simple binding model provided a KD value
of 2.28 nM. The major contribution to affinity enhance-
ment was provided by a more than 10-fold decrease in
dissociation rate relative to open-chain 8. However, it
was noted that mathematical fitting using a simple
model that assumed a single binding site did not closely
approximate the data obtained, whereas use of a
complex model that assumed two binding sites resulted
in a very close fit of data. Figure 3 shows a single data
set that has been fit according to simple (red) and
complex (blue) models. It can be seen that the overall
KD value of 4.3 nM obtained from the simple model
actually represents the combination of affinities derived
from binding to two sites with KD values of 97 pM and
10 2.03 × 106 ( 4.27 × 103 4.63 × 10-3 ( 8.81 × 10-6 2.28 ( 0.01
a Determined by plasmon resonance and evaluated by global
analysis of at least four independent data sets as described in the
Experimental Section.
propanamide], the binding affinity of the unprotected
peptide was not provided.14
Examination of relative association and dissociation
rates (ka and kd, respectively, Table 1) showed that the
lower affinity of 6 was due primarily to a 50% increase
in its rate of dissociation relative to 8. Alternatively,
combining features of the Ac6c residue and the (R-Me)-
Ppp residue into the Atc(6-PO3H2) residue in analogue
9 resulted in a net loss of inhibitory potency (KD ) 97.8
nM) despite a 35-45% enhancement in rate of associa-
tion. The loss of binding affinity of 9 was attributed to
a 6- to 9-fold increase in dissociation rate compared to
6 and 8, respectively. Therefore, although combining key

768 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Oishi et al.
be referred to as an “equitorial” orientation (“conforma-
tion 1”, Figure 4B). The second study placed the plane
of the cyclohexyl ring perpendicular to that of the pY0
phenyl ring in what will be referred to as an “axial”
orientation (“conformation 2”, Figure 4C). The interac-
tions of the inhibitors with the Grb2 SH2 domain were
measured using tools available within Insight II and are
shown in Table 3. All compounds were found to make
key hydrogen bonding (H-bonding) interactions with
residues Arg67, Arg86, Ser90, His107, Lys109, and
Leu120, with the carboxamide moiety of the pY + 2 Asn
residue making two H-bonding interactions in a biden-
tate manner with the backbone carbonyl and NH of
Lys109 as well as with the backbone carbonyl of Leu120.
In addition, the backbone amide of the pY + 1 residue
was H-bonded to the carbonyl of His107. The importance
of these interactions for high-affinity binding is indi-
cated by their presence in all reported crystal structures
of ligated Grb2 SH2 domains. For compound 8, the
acidic phosphonate hydroxyls of the pY0 residue make
five H-bonding interactions: one each with the side
chain hydroxyl groups of Ser88 and Ser90, one each with
the guanidino group of Arg67 and Arg86, and one with
the backbone amide of Ser90. The R-CH2COOH group
of 8 interacts with the side chain of Arg67 through two
H-bonds. The pY + 1 phosphonate group of 8 makes two
H-bonding interactions: one with the backbone amide
of Arg142 and another with the side chain hydroxyl
group of Ser141. In contrast, the macrocycle 10 has
enhanced electrostatic interactions due to stronger
interactions with the side chain hydroxyl of Ser90, the
side chain guanidino group of Arg67, and the backbone
amide and side chains of Arg142 and Asn143. The
binding conformation of 10 is also stabilized by an
intramolecular H-bond. Compound 9 has greater elec-
trostatic interactions in conformation 1 than those seen
in conformation 2, giving higher electrostatic interac-
tions than those obtained with compound 8. Binding of
9 in a conformation 1 orientation could potentially result
in higher binding affinity than that exhibited by 8.
However, because conformation 1 is approximately 10
kcal/mol higher in energy than conformation 2, it would
most likely not be the major conformer in solution. In
addition to exhibiting weaker electrostatic interactions
as compared to those of 8, conformation 2 of 9 also has
a lower van der Waals interaction energy. Consistent
with experimentally observed binding data (Table 1),
these results indicate that binding of 9 in a conformation
2 orientation would result in lower binding affinity than
that for conformation 1 or that for compound 8.
Figure 2. Structure of reference peptide 25.
Figure 3. Plasmon resonance data derived from the binding
of 10 to Grb2 SH2 domain protein showing mathematical
fitting using either simple (red) or complex (blue) models.
Table 2. Kinetic Analysis of Binding Data Shown in Figure 3
Using Simple or Complex Binding Modelsa
no.
ka (M-1 s-1
)
kd (s-1
)
KD (nM)
10
10A
10B
1.5 × 106
6.5 × 106
2.3 × 105
6.5 × 10-3
6.3 × 10-4
1.8 × 10-2
4.3
0.097
72
a Determined by plasmon resonance. Analysis is of a single data
set using either a simple binding model (for 10) or a complex
binding model (for 10A and 10B). Data used for these analyses
are shown in Figure 3.
72 nM (Table 2). The mechanistic basis for this complex
binding behavior is unclear, although it may indicate
that in addition to the expected binding orientation
shown in Figure 4D peptide 10 may be interacting with
other sites or in nonstandard modes. Because this type
of complex binding behavior was not observed with the
flexible, open-chain peptides 6-9, such secondary bind-
ing may be dependent on the macrocyclic nature of 10.
Molecular Modeling. The interactions of peptides
6, 8, 9, and 10 with the Grb2 SH2 domain protein were
examined using molecular modeling techniques. The
results of these calculations are shown graphically in
Figure 4. Modeling of 9 was conducted in two separate
studies. In one study, the plane of the cyclohexyl ring
was parallel to that of the pY0 phenyl ring in what will
For Grb2 SH2 domain bound ligands containing an
Ac6c residue at the pY + 1 position, what we have
referred to as an axial cyclohexyl ring conformation has
been reported to predominate.27 The higher binding
affinity of the originally reported analogue 26 that
contains a pY + 1 (R-Me)Ppp residue (Figure 2)14
potentially may be attributable to a combination of
favorable electrostatic and hydrophobic interactions. As
can be seen from Table 3, the presence of the (1-
naphthyl)propylamide moiety in compounds 6, 8, 9, and
10 contributes substantially to more favorable van der
Waals energies as compared to those of 25. However,
25 exhibits more favorable electrostatic interactions
than these former analogues. Enhancing electrostatic

Grb SH2 Binding Peptides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 769
Figure 4. Hypothetical Grb2 SH2 domain binding modes of 8 (A), 9 with the pY + 1 cyclohexylphenyl ring in equatorial (B) and
axial (C) orientations, and 10 (D). Hydrogen bonds are represented as dotted lines in black. For reasons of clarity, only key
residues and atoms are shown.
Table 3. Calculated Energy Parameters Associated with Figure 4
compound 8 (A)a
compound 9 (B)a
compound 9 (C)a
compound 6
(kcal/mol)
compound 10 (D)a
compound 25
(kcal/mol)
parameter
(kcal/mol)
(kcal/mol)
(kcal/mol)
(kcal/mol)
van der Walls energy
coulomb energy
-49.517
-50.942
-45.905
-83.406
-40.188
-19.479
-39.897
-53.996
-40.617
-93.296
-38.355
-111.332
a The letter in parentheses refers to the panel in Figure 4 associated with the indicated parameters.
interactions in 8 and 9 could potentially result in higher
binding affinity.
above and confirmed that the final positions of ligands
within the binding site are the most stable conforma-
tions.
It should be noted that additional extensive modeling
studies were performed, including conformational
searches of the ligands using the Monte Carlo multiple
minimum (MCMM) approach in Macromodel 8.0, flex-
ible docking using GLIDE, and energy evaluations using
ab initio methods in Gaussian 03. Although the results
of these studies were not presented, they validated the
results from the molecular dynamics simulations shown
Conclusions
Structure-activity studies have previously identified
the Ac6c and (R-Me)Ppp residues as being among
optimal pY + 1 residues for high Grb2 SH2 domain
binding affinity. It was therefore of interest to investi-
gate the binding effects of combining the structural
features of these two residues into a single tetralin-

770 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Oishi et al.
cyclohexyl ring perpendicular to that of the pY0 phenyl ring)
orientations (conformation 1 and conformation 2, respectively).
The resulting protein-ligand structures were solvated with a
30 Å sphere of H2O (3160 molecules) and subjected to 1000
steps of energy minimization with the steepest descent method
followed by 500 steps of the Polak-Ribiere conjugate-gradient
algorithm (CG-PR). The minimized complex was subjected to
50 ps of an NVT molecular dynamics (MD) simulation at 298
K. The coordinates were saved every 100 fs and subsequently
minimized by 500 steps of CG-PR. During minimization and
simulation, all atoms were held fixed except for those within
a 10 Å sphere around the inhibitors, including H2O molecules.
For each inhibitor, the frame with the lowest energy among
the 500 minimized frames is depicted in Figure 4. For purposes
of comparison, peptide 25 was also subjected to minimization
and MD simulation as described above. The intermolecular
interaction energies between the bound inhibitor and the
protein in the lowest energy frame were evaluated using tools
available within Insight II30 and are shown in Table 3.
General Synthetic. Reactions were carried out under
argon. Anhydrous solvents were purchased from Aldrich
Chemical Corporation and used without further drying. Com-
bustion analyses were obtained from Atlantic Microlab, Inc.,
Norcross, GA. 1H NMR spectra were obtained using a Varian
400 MHz spectrometer and are reported in ppm relative to
TMS and referenced to the solvent in which they were run.
Fast atom bombardment mass spectra (FABMS) were acquired
with a VG analytical 7070E mass spectrometer. HPLC separa-
tions were conducted using a Waters Prep LC4000 system with
photodiode array detection and a J-sphere ODS-H80 column
(20 mm × 250 mm) with a solvent system consisting of 0.1%
aqueous TFA (v/v, solvent A)/0.1% TFA in MeCN (v/v, solvent
B).
Boc-(R-Me)Phe(4-PO3Et2)-Asn-(CH2)3-(1-naphthyl) (14).
To a stirred solution of (S)-asparagine 3-(1-naphthyl)propyl-
amide (11)23 (200 mg, 0.668 mmol) in dry DMF (2 mL) were
added Boc-(R-Me)Phe(4-PO3Et2)-OH (12)14,22 (305 mg, 0.734
mmol), tetramethylfluoroformamidinium hexafluorophosphate
(TFFH)24 (194 mg, 0.734 mmol), and i-Pr2NEt (0.254 mL, 1.46
mmol) at 0 °C, and stirring was continued for 24 h at room
temperature. The mixture was extracted with EtOAc, and the
extract was washed successively with saturated citric acid
solution, brine, saturated NaHCO3 solution, and brine and
dried over Na2SO4. Concentration followed by flash chroma-
tography over silica gel using CH2Cl2/MeOH (100:0 to 10:1)
provided 14 as colorless semisolid (287 mg, 61% yield). 1H
NMR (CDCl3): δ 1.33 (m, 6 H), 1.37 (s, 9 H), 1.48 (s, 2 H),
1.98 (m, 2 H), 2.23 (dd, J ) 15.6 and 5.1 Hz, 1 H), 2.98 (d, J
) 13.6 Hz, 1 H), 3.02 (dd, J ) 15.6 and 3.9 Hz, 1 H), 3.09 (t,
J ) 7.5 Hz, 2 H), 3.28 (m, 1 H), 3.30 (d, J ) 13.1 Hz, 1 H),
3.38 (m, 1 H), 4.12 (m, 4 H), 4.62 (m, 1 H), 4.86 (s, 1 H), 5.32
(br, 1 H), 6.05 (br, 1 H), 7.24 (dd, J ) 8.2 and 3.9 Hz, 2 H),
7.30-7.56 (m, 5 H), 7.68 (d, J ) 8.5 Hz, 1 H), 7.74 (dd, J )
13.1 and 8.2 Hz, 2 H), 7.81 (m, 2 H), 8.02 (d, J ) 7.3 Hz, 1 H).
FABMS m/z: 697 (MH+). Anal. Calcd for C36H49N4O8P‚
0.8H2O: C, 60.80; H, 7.17; N, 7.88. Found: C, 60.92; H, 7.12;
N, 7.92.
based analogue as shown by 5, Figure 1. Indeed,
replacement of the pY + 1 Ac6c residue with this hybrid
resulted in a 35-45% enhancement in binding on-rate.
However, overall binding affinity was reduced due to a
concomitant 6- to 9-fold increase in off-rates. Molecular
modeling calculations indicated that electrostatic inter-
actions may be major contributors to these binding
effects. It can be concluded that local conformational
constraint of the pY + 1 (R-Me)Ppp residue of peptide
8 failed to enhance overall binding affinity. Alterna-
tively, global conformational constraint of the (R-Me)-
Ppp-containing peptide 8 by â-macrocyclization to 10
did result in pronounced elevation of binding affinity,
with the effect being achieved primarily through a
decrease in the binding off-rate. Of note, mathematical
fitting using a simple model that assumed a single
binding site for 10 yielded an effective KD of 2.28 nM.
However, this did not closely approximate the data
obtained. Rather, use of a complex model that assumed
two binding sites resulted in a very close fit of data and
provided KD values of 97 pM and 72 nM for the separate
sites, respectively. The value of global conformational
constraint through â-macrocyclization may potentially
be extended to SH2 domain systems other than Grb2
where bend geometries are required.
Experimental Section
Plasmon Resonance Analysis of Grb2 SH2 Domain
Binding Affinity. Binding experiments were performed on
a BIACORE S51 instrument (Biacore, Inc., Piscataway, NJ).
All biotinylated Grb2 SH2 domain protein (b-Grb2) was
expressed and purified (Protein Expression Laboratory and
The Protein Chemistry Laboratory, SAICsFrederick). The
b-Grb2 was immobilized onto the carboxymethyl-5′-dextran
surface (CM5 sensor chip, Biacore, Inc.) by amine coupling.
The lyophilized b-Grb2 was reconstituted in 50% DMSO in
H2O to make a stock solution of 1 mg/mL and stored at -80°
C. A 1:12.5 dilution of b-Grb2 was used for immobilization and
prepared by dilution in acetate buffer pH 5.0, with 5% DMSO.
Phosphate-buffered saline (PBS, 1×, pH 7.4) was used as the
running buffer.
An immobilization wizard was used to facilitate immobiliza-
tion targeting. For b-Grb2, 2500-5000 resonance units (RU)
of protein were captured on the CM5 sensor chip. Small
molecule ligands were serially diluted in running buffer to
concentrations of 1.25-1500 nM, as described in each sensor-
gram, and injected at 25 °C at a flow rate of 30 µL/min for 1
min, and dissociation was monitored for an additional 3 min.
Surface regeneration was not used. Samples of differing
concentrations of small molecule ligands were injected in
increasing concentration, with every injection being performed
in duplicate within each experiment. To subtract background
noise from each data set, all samples were also run over an
unmodified reference surface, and injections of running buffer
were performed throughout every experiment (“double refer-
encing”). Up to six data sets were fit to a simple 1:1 interaction
model or to a surface heterogeneity model (“complex model”)
for compound 10, using the global data analysis program
CLAMP.28 The mean of the ratio and associated error were
calculated according to known procedures.29
Boc-Atc(6-PO3Et2)-Asn-(CH2)3-(1-naphthyl) (15). Cou-
pling of 11 (200 mg, 0.668 mmol) with Boc-(S)-2-amino-6-
(diethylphosphono)tetralin-2-carboxylic acid (Boc-Atc(6-PO3-
Et2)-OH) 1322 in a manner similar to that described above for
the preparation of 14, provided 15 as a colorless semisolid (154
1
mg, 32% yield). H NMR (CDCl3): δ 1.34 (m, 15 H), 2.00 (m,
2 H), 2.31 (m, 1 H), 2.48 (dd, J ) 15.3 and 4.8 Hz, 1 H), 2.58
(m, 1 H), 2.72 (m, 1 H), 2.85 (d, J ) 17.0 Hz, 1 H), 2.92 (m, 1
H), 3.13 (m, 3 H), 3.25 (m, 1 H), 3.32 (d, J ) 16.8 Hz, 1 H),
3.44 (m, 1 H), 4.12 (m, 4 H), 4.78 (m, 1 H), 4.92 (s, 1 H), 5.33
(br, 1 H), 6.10 (br, 1 H), 7.14 (dd, J ) 7.8 and 4.1 Hz, 1 H),
7.32-7.74 (m, 8 H), 7.83 (d, J ) 7.8 Hz, 1 H), 8.05 (d, J ) 8.0
Hz, 1 H), 8.31 (d, J ) 8.2 Hz, 1 H). FABMS m/z: 709 (MH+).
Anal. Calcd for C37H49N4O8P‚0.5H2O: C, 61.91; H, 7.02; N,
7.81. Found: C, 61.92; H, 7.02; N, 7.81.
Molecular Modeling. Simulations were performed with
the Insight II 2000.0/Discover 97.0 modeling package (Molec-
ular Simulations, Inc., San Diego, CA), using the cff91 force
field.30,31 The crystal structure of peptide 25 bound to Grb2
SH2 domain protein (PDB code 1JYQ15) was used as the
starting geometry. The structure of 25 was first modified to
yield inhibitors 8, 9, and 10. In the case of 9, the conformation
of the pY + 1 cyclohexyl ring was modeled separately in
equitorial (the plane of the cyclohexyl ring being parallel to
that of the pY0 phenyl ring) and axial (the plane of the
[(2R)-2-(tert-Butyloxycarbonylmethyl)-3-(4-di-tert-
butylphosphonomethyl)phenylpropionyl]-(r-Me)Phe-

Grb SH2 Binding Peptides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 771
(4-PO3Et2)-Asn-(CH2)3-(1-naphthyl) (17). Protected peptide
14 (316 mg, 0.453 mmol) was treated with TFA/anisole (10:1,
5.5 mL) for 2 h at room temperature. The mixture was
concentrated and neutralized with saturated NaHCO3 solution.
The mixture was extracted with EtOAc, and the extract was
washed with saturated NaHCO3 solution and brine and dried
over Na2SO4. Concentration under reduced pressure gave the
corresponding amine. To a stirred solution of the amine in dry
DMF (1.4 mL) were added protected pTyr mimetic 1625 (307
mg, 0.498 mmol), TFFH (131 mg, 0.498 mmol), and i-Pr2NEt
(0.173 mL, 0.997 mmol) at 0 °C, and stirring was continued
for 2 days at room temperature. The mixture was extracted
with EtOAc, and the extract was washed successively with 5%
citric acid solution, brine, saturated NaHCO3 solution, and
brine, and dried over Na2SO4. Concentration followed by flash
chromatography over silica gel using CH2Cl2/MeOH (100:0 to
10:1) provided 17 as a colorless semisolid (260 mg, 54% yield).
1H NMR (400 MHz, CDCl3): δ 1.31 (m, 6 H), 1.36 (s, 9 H),
1.40 (s, 9 H), 1.41 (s, 9 H), 1.44 (s, 3 H), 2.04 (m, 2 H), 2.21
(dd, J ) 17.7 and 3.3 Hz, 1 H), 2.47 (m, 3 H), 2.77 (m, 1 H),
2.84-3.04 (m, 5 H), 3.14 (m, 2 H), 3.24 (d, J ) 13.6 Hz, 1 H),
3.37 (m, 2 H), 3.73 (m, 4 H), 4.55 (m, 1 H), 5.33 (br, 1 H), 6.29
(br, 1 H), 6.48 (s, 1 H), 6.98 (d, J ) 7.9 Hz, 2 H), 7.17 (m, 4 H),
7.32-7.58 (m, 6 H), 7.69 (m, 3 H), 7.82 (d, J ) 7.6 Hz, 1 H),
8.07 (d, J ) 8.1 Hz, 1 H). FABMS m/z: 1049 (MH+). Anal.
Calcd for C55H78N4O12P2‚H2O: C, 61.90; H, 7.56; N, 5.25.
Found: C, 61.85; H, 7.55; N, 5.32.
[(2R)-2-(tert-Butyloxycarbonylmethyl)-3-(4-di-tert-bu-
tylphosphonomethyl)phenylpropionyl]-Atc(6-PO3Et2)-
Asn-(CH2)3-(1-naphthyl) (18). Coupling of 15 (120 mg, 0.169
mmol) with 16 by a procedure similar to that described above
for the preparation of the protected peptide 17 from 14,
provided 18 as a colorless semisolid (64 mg, 35% yield). 1H
NMR (CDCl3): δ 1.31 (m, 6 H), 1.33 (s, 9 H), 1.38 (s, 9 H),
1.39 (s, 9 H). 1.94-2.11 (m, 2 H), 2.14 (dd, J ) 17.5 and 3.4
Hz, 1 H), 2.25 (m, 1 H), 2.46 (m, 3 H), 2.64 (dd, J ) 15.1 and
5.1 Hz, 1 H), 2.74 (m, 1 H), 2.78-2.91 (m, 4 H), 2.92-3.04 (m,
3 H), 3.13 (m, 2 H), 3.38 (m, 3 H), 4.09 (m, 4 H), 4.71 (m, 1 H),
5.32 (br, 1 H), 6.19 (br, 1 H), 6.27 (s, 1 H), 6.89 (d, J ) 8.0 Hz,
2 H), 7.11 (m, 3 H), 7.38 (m, 2 H), 7.42-7.60 (m, 5 H), 7.70
(dd, J ) 6.8 and 2.9 Hz, 1 H), 7.79 (d, J ) 8.2 Hz, 1 H), 7.84
(d, J ) 8.0 Hz, 1 H), 8.09 (d, J ) 7.3 Hz, 1 H). FABMS m/z:
1061 (MH+). Anal. Calcd for C56H78N4O12P2‚H2O: C, 62.32; H,
7.47; N, 5.19. Found: C, 62.00; H, 7.44; N, 5.55.
[(2R)-3-(4-Phosphonomethyl)phenyl-2-(carboxymeth-
yl)propionyl]-(r-Me)Phe(4-PO3H2)-Asn-(CH2)3-(1-naph-
thyl) (8). To a stirred solution of protected peptide 17 (30 mg,
0.0285 mmol) in MeCN (1 mL) were added thioanisole (0.100
mL) and trimethylsilyliodide (TMSI) (0.711 mL) at 0 °C. The
mixture was stirred for 30 min at 0 °C and an additional 1 h
at room temperature. After concentration, the residue was
dissolved in 95% TFA solution (10 mL), and stirring was
continued for 2 h at room temperature. The mixture was
concentrated and extracted with H2O, and the extract was
washed with Et2O. The aqueous solution was purified by
preparative HPLC using a linear gradient 30% to 35% B over
20 min to provide 8 as a colorless powder (13 mg, 55% yield).
1H NMR (DMSO-d6): δ 1.17 (s, 3 H), 1.87 (m, 2 H), 2.01 (dd,
J ) 16.9 and 4.8 Hz, 1 H), 2.41 (dd, J ) 15.2 and 10.7 Hz, 1
H), 2.47-2.73 (m, 4 H), 2.86-3.31 (m, 9 H), 4.28 (m, 1 H), 6.89
(s, 1 H), 7.03 (m, 4 H), 7.16 (m, 2 H), 7.34 (s, 1 H), 7.38-7.45
(m, 6 H), 7.58 (t, J ) 5.3 Hz, 1 H), 7.76 (dd, J ) 6.8 and 2.1
Hz, 1 H), 7.85 (d, J ) 7.8 Hz, 1 H), 7.90 (m, 1 H), 8.13 (d, J )
7.8 Hz, 1 H), 8.48 (s, 1 H). FABMS m/z: 823 [(M - H)-].
J ) 8.3 Hz, 1 H), 8.15 (d, J ) 8.0 Hz, 1 H), 8.71 (s, 1 H).
FABMS m/z: 835 [(M - H)-].
(2S)-3-(4-Bisethoxyphosphono)phenyl-2-(tert-butoxy-
carbonyl)amino-2-methyl-N-((1S)-1-{N-[(2S)-2-(naphthyl-
methyl)pent-4-enyl]carbamoyl}-2-carbamoylethyl)pro-
pylamide (20). Coupling of (2S)-2-amino-N1-[(2S)-2-(1-naph-
thalenylmethyl)-4-pentenyl]butanediamide (19)17 (300 mg,
0.883 mmol) with Boc-(R-Me)Phe(4-PO3Et2)-OH (7) 12 in a
manner similar to that described above for the preparation of
14 provided 20 as a colorless semisolid (258 mg, 38% yield).
1H NMR (400 MHz, CDCl3): δ 1.29 (s, 9 H), 1.33 (m, 6 H),
1.45 (s, 3 H), 2.04-2.20 (m, 3 H), 2.23 (dd, J ) 1.5 and 5.3 Hz,
1 H), 2.95-3.10 (m, 5 H), 3.29 (d, J ) 13.4 Hz, 1 H), 3.44 (m,
1 H), 4.13 (m, 4 H), 4.62 (m, 1 H), 4.82 (s, 1 H), 5.02-5.13 (m,
2 H), 5.34 (br, 1 H), 5.83 (m, 1 H), 6.02 (br, 1 H), 7.23 (m, 2
H), 7.29-7.53 (m, 5 H), 7.66-7.84 (m, 5 H), 8.00 (d, J ) 8.5
Hz, 1 H). FABMS m/z: 737 (MH+).
N-{(1S)-2-(4-Bisethoxyphosphono)phenyl-1-methyl-1-
[N-((1S)-1-{N-[(2S)-2-(naphthylmethyl)pent-4-enyl]-
carbamoyl}-2-carbamoylethyl)carbamoyl]ethyl}-(2S,3R)-
2-(tert-butoxycarbonylmethyl)-3-{4-[bis(tert-butoxy)-
phosphonomethyl]phenyl}pente-4-enamide (22). Cou-
pling of (2S,3S)-3-(4-{[bis(tert-butoxy)phosphono]methyl}-
phenyl)-2-{[(tert-butyl)oxycarbonyl]methyl}pent-4-enoic acid
(21)18 (120 mg, 0.169 mmol) with 20 by a procedure similar to
that described above for the preparation of the protected
peptide 17 from 14 provided 22 as a colorless semisolid (30
mg, 8% yield). 1H NMR (400 MHz, CDCl3): δ 1.19 (s, 3 H),
1.32 (t, J ) 7.0 Hz, 6 H), 1.40 (s, 9 H), 1.41 (s, 9 H), 1.42 (s, 9
H), 2.02-2.24 (m, 3 H), 2.39 (dd, J ) 18.0 and 9.7 Hz, 1 H),
2.54 (m. 3 H), 2.82-3.19 (m, 8 H), 3.24 (m, 1 H), 3.41 (m, 1
H), 4.11 (m, 4 H), 4.64 (m, 1 H), 4.93 (m, 2 H), 5.10 (m, 2 H),
5.64 (m, 1 H), 5.87 (m, 1 H), 6.43 (s, 1 H), 6.64 (d, J ) 7.8 Hz,
1 H), 6.80-7.20 (m, 7 H), 7.32-7.56 (m, 5 H), 7.67 (m, 3 H),
7.83 (d, J ) 7.8 Hz, 1 H), 8.06 (d, J ) 8.3 Hz, 1 H). FABMS
m/z: 1115 (MH+).
2-[(1S,5S,9R,13S)-3,12,15-Triaza-13-(4-Bisethoxy-
phosphono)phenylmethyl-9-[4-bis(tert-butoxy)phospho-
no-methyl]phenyl-13-methyl-5-naphthylmethyl-2,11,14-
trioxocyclopentadec-7-enyl]acetamide (24). To a solution
of 22 (25 mg, 0.022 mmol) in CH2Cl2 (5.5 mL) was added
ruthenium catalyst 23 (9.5 mg, 0.011 mmol) in CH2Cl2 (1.8
mL) under argon. The reaction was stirred at 45 °C for 24 h.
The crude reaction mixture was evaporated in vacuo, and the
residue was purified by silica gel chromatography using
CH2Cl2/MeOH (10:1) to provide 24 as a colorless powder (7
1
mg, 28% yield). H NMR (400 MHz, CDCl3): δ 1.18-1.46 (m,
33 H), 1.59 (s, 3 H), 1.78-1.91 (m, 2 H), 2.14 (m, 2 H), 2.30
(m, 1 H), 2.63 (dd, J ) 13.2 and 10.0 Hz, 1 H), 2.68-3.13 (m,
8 H), 3.34 (dd, J ) 13.7 and 4.6 Hz, 1 H), 3.75 (dd, J ) 13.0
and 5.6 Hz, 1 H), 3.86 (m, 1 H), 4.11 (m, 4 H), 4.55 (m, 1 H),
4.98 (br, 1 H), 5.36 (ddd, J ) 14.8, 10.2 and 2.8 Hz, 1 H), 5.74
(dd, J ) 14.8 and 11.8 Hz, 1 H), 5.84 (br, 1 H), 6.32 (s, 1 H),
7.05-7.52 (m, 12 H), 7.64-7.82 (m, 4 H), 8.16 (d, J ) 8.6 Hz,
1 H). FABMS m/z: 1087 (MH+).
2-[(1S,5S,9R,13S)-3,12,15-Triaza-13-(4-phosphono)-
phenylmethyl-9-(4-phosphonomethyl)phenyl-13-methyl-
5-naphthylmethyl-2,11,14-trioxocyclopentadec-7-enyl]-
acetamide (10). Treatment of 24 (6.5 mg, 0.0301 mmol) as
described above for the preparation of 8 provided 10 as
colorless powder (1.5 mg, 29% yield). 1H NMR (400 MHz,
DMSO-d6): δ 1.90-2.22 (m, 8 H), 2.39 (m, 1 H), 2.60 (m, 1 H),
2.64-2.88 (m, 6 H), 3.08 (d, J ) 12.8 Hz, 1 H), 3.14-3.26 (m,
2 H), 3.57 (m, 1 H), 4.01 (m, 1 H), 4.26 (m, 1 H), 5.46 (m, 1 H),
5.81 (m, 1 H), 7.02 (s, 1 H), 7.16-7.29 (m, 6 H), 7.37-7.59 (m,
8 H), 7.76 (m, 1 H), 7.90 (d, J ) 8.1 Hz, 1 H), 8.17 (d, J ) 8.6
Hz, 1 H), 8.29 (d, J ) 8.2 Hz, 1 H), 8.89 (s, 1 H). LRMS (FAB)
m/z: 861 [(M-H)-].
[(2R)-3-(4-Phosphonomethyl)phenyl-2-(carboxymeth-
yl)propionyl]-Atc(6-PO3H2)-Asn-(CH2)3-(1-naphthyl) (9).
Treatment of 18 (32 mg, 0.0301 mmol) as described above for
the preparation of 8 provided 9 as colorless powder (11 mg,
Acknowledgment. Appreciation is expressed to
Drs. James Kelley and Christopher Lai of the LMC for
mass spectral analysis and to Dr. Megan Peach for
helpful molecular modeling discussions. Support was
provided by the Japan Society for the Promotion of
1
43% yield). H NMR (DMSO-d6): δ 1.78-2.06 (m, 4 H), 2.18
(m, 1 H), 2.32-2.54 (m, 3 H), 2.66 (m, 1 H), 2.75-3.30 (m, 12
H), 4.42 (m, 1 H), 6.82 (d, J ) 7.8 Hz, 2 H), 6.95 (s, 1 H), 7.02
(m, 2 H), 7.15 (dd, J ) 7.8 and 3.6 Hz, 1 H), 7.35-7.57 (m, 8
H), 7.78 (dd, J ) 5.8 and 3.6 Hz, 1 H), 7.92 (m, 1 H), 8.08 (d,

772 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Oishi et al.
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(20) Shi, Z.-D.; Wei, C.-Q.; Lee, K.; Liu, H.; Zhang, M.; Araki, T.;
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JM0492709