Examination of acylated 4-aminopiperidine-4-carboxylic acid residues in the phosphotyrosyl+1 position of Grb2 SH2 domain-binding tripeptides

Article abstract of DOI:10.1021/jm0614073

A 4-aminopiperidine-4-carboxylic acid residue was placed in the pTyr+1 position of a Grb2 SH2 domain-binding peptide to form a general platform, which was then acylated with a variety of groups to yield a library of compounds designed to explore potential

Full text of DOI:10.1021/jm0614073

1978
J. Med. Chem. 2007, 50, 1978-1982
Examination of Acylated 4-Aminopiperidine-4-carboxylic Acid Residues in the
Phosphotyrosyl+1 Position of Grb2 SH2 Domain-Binding Tripeptides
Sang-Uk Kang,† Won Jun Choi,† Shinya Oishi,† Kyeong Lee,† Rajeshri G. Karki,† 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, Protein Chemistry Laboratory, Science Applications International Corporation-Frederick, Frederick, Maryland 21702
ReceiVed December 7, 2006
A 4-aminopiperidine-4-carboxylic acid residue was placed in the pTyr+1 position of a Grb2 SH2 domain-
binding peptide to form a general platform, which was then acylated with a variety of groups to yield a
library of compounds designed to explore potential binding interactions, with protein features lying below
the âD strand. The highest affinities were obtained using phenylethyl carbamate and phenylbutyrylamide
functionalities.
Introduction
Evaluation of Inhibitors in a Grb2 SH2 Domain-Binding
System. Kinetic binding data for peptides 8a-j were obtained
by surface plasmon resonance experiments that measured the
direct interaction of synthetic ligands with surface-bound Grb2
SH2 domain protein (Table 1).9 The pTyr+1 Ac6c-containing
peptide has a reported Grb2 SH2 domain-binding affinity of
24 nM.3 Methyl carbamate 8a represents the simplest analogue
Significant research has been devoted to enhancing binding
affinity, cellular potency, and metabolic stability of Grb2 SH2
domain binding antagonists.1 These efforts have included the
use of bend-inducing 1-aminocyclohexanecarboxylic acid (Ac6ca)
residues in the pTyr+1 position of “pTyr-Xxx-Asn”-contain-
ing peptides.2 However, little has been reported regarding
potential binding interactions that might result by extending
functionality outward from the Ac6c side chain ring.3 Residues
Val99, Phe101, Gln106, Phe108, Ser141, Asn143, and Gln144
of the Grb2 SH2 domain lie along and below the protein âD
strand (nomenclature based on structural homology among SH2
domains).4 These residues form a region proximal to the pTyr+1
position that could potentially interact with functionality derived
from suitably modified Ac6c residues. Starting from a previously
reported Ac6c-containing tripeptide inhibitor,5 replacement of
the Ac6c residue with 4-aminopiperidine-4-carboxylic acid
(Apc)6 and elaboration at the piperidinyl 1-position would yield
analogues of type 8 (Scheme 1) that could potentially interact
with this region of the protein.
examined in the current study and its binding affinity (KD
)
106 nM) serves as a reference for comparison with the remainder
of the series. Elaboration of the methyl carbamate to a
2-methoxyethyl carbamate (8b, KD ) 202 nM) resulted in a
loss of affinity, and introduction of an aromatic group resulted
in further loss of affinity when a one-methylene spacer was
employed (benzyl carbamate 8c, KD ) 320 nM). However,
adding an additional methylene group to 8c increased affinity
10-fold (phenylethyl carbamate 8d, KD ) 35.7 nM). Molecular
modeling simulations were performed on compounds 8a, 8c,
and 8d bound to the Grb2 SH2 domain protein with the Insight
II2000.0/Discover 97.0 modeling package (Molecular Simula-
tions, Inc., San Diego, CA) using the cff91 force field. When
the crystal structure of mAZ-pY-(RMe)pY-N-NH2 bound to
the Grb2 SH2 domain (1JYQ.pdb) was used, the peptide ligand
was modified using 3D-sketcher tools to yield inhibitors 8a,
8c, 8d, and 8j, and these were subjected to energy minimization
and molecular dynamics simulations (see the Supporting
Information). It was found that 8d exhibited the best extension
within the target region below the âD strand (Figure 1).
Replacement of the carbamoyl oxygen of 8d with a methylene
yielded the corresponding phenylbutyrylamide 8e (KD ) 27 nM).
Addition of one methylene to 8e gave the phenylvaleramide
8f, which suffered a significant loss of binding affinity (KD )
2180 nM). This indicated that a chain extension of 3 units
beyond the carbonyl was preferable to 4 units. Accordingly,
the indole-containing analogue 8g was prepared to present a
3-unit extension between the phenyl ring and the amide carbonyl
(the indolyl nitrogen is considered as part of the extending
chain). Likewise, analogue 8h was prepared as a urea variant
of carbamate 8d. The affinities of 8g (KD ) 458 nM) and 8h
(KD ) 272 nM) were less than 8d, as was the affinity of
sulfonamide 8i (KD ) 2140 nM).
To explore these potential interactions, preparation of a small
library of Apc-containing final products (8) was accomplished
using the known intermediates 1,7 2,8 and 4,5 as shown in
Scheme 1. Of note, coupling 3 with the protected pTyr mimetic
4 required HOAt (1-hydroxy-7-azabenzotriazole) active ester
methodology due to steric hindrance.5 Hydrogenolytic cleavage
of the Apc 1-amino-Cbz group in 5 gave the free piperidino-
containing analogue 6, which upon acylation, yielded the series
of analogues 7. The products 7a-d and 7j were prepared using
chloroformates, while HOBT active ester coupling was em-
ployed for compound 7e, and acid chlorides were used for 7f,
7g, and 7i. The synthesis of urea 7h employed an isocyanate.
Treatment of the tert-butyl-protected products 7 with TFA gave
the final products 8a-j, which were purified by HPLC.
* To whom correspondence should be addressed. Laboratory of Medicinal
Chemistry, Center for Cancer Research, National Cancer Institute, National
Institutes of Health, NCI-Frederick, P.O. Box B, Bldg. 376 Boyles St.,
Frederick, Maryland 21702-1201. Tel.: (301) 846-5906. Fax: (301) 846-
6033. E-mail: tburke@helix.nih.gov.
† Center for Cancer Research.
The analogue 8j contains a 4-carboxybenzyl carbamate
moiety. Previous work has shown that pTyr or pTyr mimetics
placed at the pTyr+1 position can promote high affinity binding
by interacting with the Grb2 SH2 domain Arg142 residue.3,10
‡ Science Applications International Corporation.
a Abbreviations: pTyr, phosphotyrosyl; Ac6c, aminocyclohexanecar-
boxylic acid; Apc, 4-aminopiperidine-4-carboxylic acid; HOAt, 1-hydroxy-
7-azabenzotriazole.
10.1021/jm0614073 CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/20/2007

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1979
Scheme 1. Synthesis of Analogues Used in the Current Study
Table 1. Grb2 SH2 Domain-Binding Affinities of Synthetic Inhibitors Determined by Surface Plasmon Resonance
a Placement of R groups is as shown in Scheme 1. b Data was obtained by surface plasmon resonance experiments as described in the Supporting Information.9
Although the structure of the 4-carboxybenzyl carbamoyl moiety
represents a departure from the more traditional pTyr mimetics
used in these prior studies, modeling studies showed that
interaction of the 4-carboxybenzyl group with the Arg142
residue was possible (Figure 1D). However, the poor affinity
of 8j (KD ) 871 nM) indicated that the desired interaction of
the 4-carboxybenzyl group with the Arg142 residue was not
achieved.
Conformationally-Constrained Amide Surrogates. The
phenylbutyrylamide side chain of the most potent analogue 8e
could potentially bind in two distinct conformations due to
rotational restriction about the amide C(dO)sN bond. This type
of rotamer would not be expected for the carbamoyl linkage in
the homologous structure 8d. As one approach toward examin-
ing whether rotamers about the phenylbutyrylamide bond at the
Apc 4-position might contribute differently to the affinity

1980 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Brief Articles
Figure 1. Structures of analogues 8a, 8c, 8d, and 8j (in gold) docked into the Grb2 SH2 domain. The protein is shown in ribbon depiction, with
important side chain groups shown in ball and stick rendering.
Binding affinities alone may not reflect subtle differences in
binding kinetics, because similar KD values may arise from
markedly different rates of association and dissociation. Interac-
tion kinetic maps that plot association rates (ka) versus dis-
sociation rates (kd), with an overlay of KD isovalues, can allow
visual discrimination of classes of compounds based on similar
binding behaviors.12 Plotting 8a-j in this fashion indicated three
groups of compounds (Figure 3): Group A compounds (8d and
8e) display slow dissociate rates and relatively higher association
rates. Although conversion of the carbamate functionality of
8d into the amide group of 8e introduced significant changes
in rotational flexibility and hydrogen-bonding potential, little
difference was observed in the actual binding affinities of 8d
and 8e. Instead, the structural change resulted in a marked
decrease in the rate of dissociation for 8e. This may mean that
8e is a more desirable inhibitor than 8d, because low dissociation
Figure 2. Use of pTyr+1 2-amino-2-carboxytetralin-based residues
rates have been hypothesized to enhance the efficacy of drug-
target interactions.12 The Group B compound (8a) exhibits a
higher association rate, but this is also accompanied by a more
rapid dissociation rate. Group C compounds (8b, 8c, and 8f-j)
exhibit slower association rates as well as moderate to rapid
dissociation rates.
as conformationally constrained mimetics of amide rotamers arising
from the phenylbutyrylamide of 8e.
of compound 8e, the tetralin-based analogues “mimetic A” and
“mimetic B” were designed as conformationally constrained
amide replacements (Figure 2).11 However, the low affinity of
both mimetic A and mimetic B (KD values > 10 µM) indicated
that the tetralin platforms lacked critical functionality needed
to function as conformationally constrained amide bond sur-
rogates.
Conclusions
When a 4-piperidinyl variant of the known pTyr+1 residue
Ac6c was used, the current study investigated the effects of
extending functionality outward from the Ac6c residue to take

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1981
amine 6. Aliquots of the stock solution were reacted with appropri-
ate acylating agents in the presence of diisopropylethylamine to
yield protected intermediates 7a-j. Yields and spectral data are
provided as Supporting Information. For intermediates 7a-d and
7j, acylations were performed using chloroformates, while HOBT
active ester coupling was employed for intermediate 7e, and acid
chlorides were used for 7f, 7g, and 7i. The synthesis of urea 7h
employed the appropriate isocyanate. Treatment of the tert-butyl-
protected products 7 with a mixture of TFA/H2O/ethanedithiol (3:
0.1:0.1) gave the globally deprotected final products 8a-j. Physical
data and combined yields from 6 following HPLC purification are
provided below.
Final Product 8a: 64% yield; 1H NMR (DMSO-d6) δ 8.62 (1H,
s), 8.11 (1H, m), 7.99 (1H, d, J ) 8.2 Hz), 7.92 (1H, m), 7.77 (1H,
t, J ) 4.7 Hz), 7.53-7.49 (2H, m), 7.46-7.39 (4H, m), 7.13-7.11
(2H, m), 6.96-6.93 (3H, m), 4.35 (1H, m), 3.68 (2H, m), 3.59
(3H, s), 3.25-3.02 (7H, m), 2.89 (2H, d, J ) 21.3 Hz), 2.71 (1H,
dd, J ) 6.4 and 14.6 Hz), 2.53-2.42 (2H, m), 2.33 (1H, m), 2.01-
1.66 (8H, m). FAB-MS (-VE) m/z 766 (M - H). HRMALDI-MS
(+VE) calcd for C37H46N5O11NaP [M+Na], 790.2824; found,
790.2787.
Figure 3. Interaction kinetic maps for Grb2 SH2 domain-binding
inhibitors 8a-e plotting association rate (ka) versus dissociation rate
(kd) with an overlay of KD isovalues. Data was organized into three
groups, A, B, and C, depending on a combination of these factors.
Final Product 8b: 52% yield; 1H NMR (DMSO-d6) δ 8.63 (1H,
s), 8.11 (1H, d, J ) 7.6 Hz), 8.00 (1H, d, J ) 8.2 Hz), 7.92 (1H,
m), 7.78 (1H, t, J ) 4.7 Hz), 7.54-7.41 (6H, m), 7.12 (2H, m),
6.95 (3H, m), 4.36 (1H, m), 4.11 (2H, m), 3.69 (2H, m), 3.51 (2H,
t, J ) 4.5 Hz), 3.26 (3H, s), 3.26-3.00 (7H, m), 2.89 (2H, d, J )
21.5 Hz), 2.71 (1H, dd, J ) 6.4 and 15.8 Hz), 2.54-2.43 (2H, m),
2.33 (1H, m), 2.01-1.70 (8H, m). FAB-MS (-VE) m/z 810 (M -
H). HRMALDI-MS (+VE) calcd for C39H50N5O12NaP [M + Na],
834.3121; found, 834.3086.
advantage of potential interactions with an unexplored region
of the Grb2 protein lying beneath the âD strand. Carbamoyl,
amido, urea, and sulfonamido linkages to the 4-piperidinyl
nitrogen were prepared. Tethering phenyl rings at varying
distances from the piperidinyl nitrogen indicated that the best
affinities were provided by a three-unit chain, with a phenyl-
butyryl amide group provided the highest affinity. Because SH2
domains share a high degree of structural homology, the current
approach may be useful in preparing binding inhibitors directed
at other SH2 domains.
Final Product 8c: 76% yield; 1H NMR (DMSO-d6) δ 8.64 (1H,
s), 8.11 (1H, m), 8.00 (1H, d, J ) 8.2 Hz), 7.91 (1H, m), 7.77 (1H,
t, J ) 4.9 Hz), 7.54-7.30 (11H, m), 7.12 (2H, m), 6.94 (3H, m),
5.08 (2H, s), 4.35 (1H, m), 3.73 (2H, m), 3.24-2.91 (7H, m), 2.89
(2H, d, J ) 21.3 Hz), 2.70 (1H, dd, J ) 6.8 and 15.8 Hz), 2.53-
2.43 (2H, m), 2.33 (1H, m), 2.01-1.70 (8H, m). FAB-MS (-VE)
m/z 842 (M - H). HRMALDI-MS (+VE) calcd for C43H20N5O11-
NaP [M + Na], 866.3137; found, 866.3101.
Experimental Section
Final Product 8d: 65% yield; 1H NMR (DMSO-d6) δ 8.61 (1H,
s), 8.11 (1H, m), 7.99 (1H, d, J ) 8.0 Hz), 7.92 (1H, m), 7.77 (1H,
t, J ) 4.5 Hz), 7.53-7.48 (2H, m), 7.46-7.39 (4H, m), 7.31-7.19
(5H, m), 7.11 (2H, m), 6.95 (3H, m), 4.35 (1H, m), 4.17 (2H, m),
3.66 (2H, m), 3.25-3.00 (7H, m), 2.91-2.86 (4H, m), 2.71 (1H,
dd, J ) 6.3 and 15.6 Hz), 2.54-2.42 (2H, m), 2.33 (1H, m), 1.99
(1H, m), 1.92-1.78 (6H, m), 1.67 (1H, m). FAB-MS (-VE) m/z
856 (M - H). HRMALDI-MS (+VE) calcd for C44H52N5O11NaP
[M + Na], 880.3293; found, 880.3261.
Synthesis of Dipeptide 3. Reacting 17 and commercially
available NR-Fmoc-(4-N-Cbz-piperidinyl)carboxylic acid8 according
to protocols similar to those reported for the synthesis of the
corresponding Ac6c-containing congener, the dipeptide 3 was
obtained in 89% yield as a white solid (mp 169-171 °C). 1H NMR
(DMSO-d6) δ 8.43 (1H, s), 8.06 (1H, d, J ) 8.4 Hz), 7.90 (1H,
m), 7.83 (1H, t, J ) 5.5 Hz), 7.75 (1H, d, J ) 7.8 Hz), 7.56-7.47
(2H, m), 7.43-7.29 (8H, m), 6.87 (1H, s), 5.07 (2H, s), 4.46 (1H,
m), 3.75 (2H, m), 3.21-3.10 (4H, m), 3.02 (2H, t, J ) 7.6 Hz),
2.57-2.42 (2H, m), 1.91-1.73 (4H, m), 1.33 (2H, t, J ) 14.3 Hz).
FAB-MS (+VE) m/z 560 (MH+).
Final Product 8e: 65% yield; 1H NMR (DMSO-d6) δ 8.65 (1H,
d, J ) 13.3 Hz), 8.11 (1H, m), 8.00 (1H, t, J ) 8.8 Hz), 7.92 (1H,
m), 7.78 (1H, t, J ) 4.9 Hz), 7.55-7.39 (6H, m), 7.28 (2H, m),
7.19-7.11 (5H, m), 6.98-6.92 (3H, m), 4.35 (1H, m), 4.05 (1H,
m), 3.56 (1H, m), 3.27-2.68 (11H, m), 2.60-2.24 (4H, m), 2.40-
2.22 (3H, m), 2.05-1.61 (9H, m). HRMALDI-MS (+VE) calcd
for C45H54N5O10NaP [M + Na], 878.3501; found, 878.3513.
Final Product 8f: 60% yield; 1H NMR (DMSO-d6) δ 8.64 (1H,
d, J ) 8.2 Hz), 8.11 (1H, d, J ) 8.0 Hz), 8.00 (1H, t, J ) 7.0 Hz),
7.91 (1H, m), 7.77 (1H, t, J ) 4.9 Hz), 7.55-7.39 (6H, m), 7.27
(2H, m), 7.16 (5H, m), 6.96 (3H, m), 4.35 (1H, m), 4.03 (1H, m),
3.61 (1H, m), 3.27-2.97 (7H, m), 2.89 (2H, d, J ) 21.5 Hz), 2.82-
2.68 (2H, m), 2.60-2.29 (8H, m), 2.04-1.75 (6H, m), 1.65-1.46
(4H, m). FAB-MS (-VE) m/z 868 (M - H). HRMALDI-MS
(+VE) calcd for C46H56N5O10NaP [M + Na], 892.3657; found,
892.3697.
Synthesis of Protected Tripeptide Mimetic 5. Similar to a
previously reported procedure,5 to a solution of dipetide 3 (784
mg, 1.40 mmol) in DMF (2 mL) was added an active ester solution
formed by reacting 45 (988 mg, 2.10 mmol), HOAt (0.5 M in DMF,
4.20 mL, 2.10 mmol), and EDCI‚HCl (425 mg, 3.36 mmol) in DMF
(3 mL; 15 min at room temperature), and the mixture was stirred
at 40 °C (21 h). Solvent was removed by vacuum distillation, and
the remaining residue was purified by silica gel column chroma-
tography to provide 5 as a white solid (835 mg, 59% yield), mp
1
153-155 °C. H NMR (CDCl3) δ 8.06 (1H, d, J ) 8.0 Hz), 7.88
(1H, d, J ) 7.8 Hz), 7.82 (1H, m), 7.68 (1H, dd, J ) 3.1 and 6.4
Hz), 7.49 -7.40 (3H, m), 7.37-7.29 (6H, m), 7.19-7.09 (3H, m),
6.99 (2H, d, J ) 7.8 Hz), 6.92 (1H, s), 6.46 (1H, s), 5.49 (1H, s),
5.10 (2H, s), 4.60 (1H, s), 3.92 (2H, m), 3.35 (2H, m), 3.12 (2H,
m), 3.04-2.88 (7H, m), 2.66 (1H, m), 2.49 (2H, m), 2.25 (1H, m),
2.13-1.80 (6H, m), 1.41 (9H, s), 1.39 (9H, s), 1.34 (9H, s). FAB-
MS (+VE) m/z 1012 (MH+).
Final Product 8g: 57% yield; 1H NMR (DMSO-d6) δ 8.64 (1H,
s), 8.10 (1H, d, J ) 8.2 Hz), 7.97 (1H, d, J ) 7.4 Hz), 7.90 (1H,
m), 7.77 (1H, m), 7.54-7.29 (9H, m), 7.11 (2H, m), 6.94 (4H, m),
6.28 (1H, d, J ) 2.5 Hz), 4.35 (3H, m), 4.05 (1H, m), 3.53 (1H,
m), 3.24-2.97 (7H, m), 2.92-2.68 (5H, m), 2.53-2.43 (2H, m),
2.36 (3H, s), 2.36-2.29 (1H, m), 2.00-1.64 (8H, m). FAB-MS
(-VE) m/z 893 (M - H). HRMALDI-MS (+VE) calcd for
C47H55N6O10NaP [M + Na], 917.3610; found, 917.3636.
General Procedures for the Synthesis of Final Products 8a-
8j. Protected tripeptide mimetic 5 in MeOH was hydrogenated over
10% Pd‚C (50% by weight of 5) using a balloon filled with H2 gas
(overnight). The reaction mixture was filtered and taken to dryness
then dissolved in CH2Cl2 to provide a 0.1 M stock solution of free

1982 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Brief Articles
Final Product 8h: 67% yield; 1H NMR (DMSO-d6) δ 8.58 (1H,
s), 8.12 (1H, d, J ) 7.8 Hz), 7.97-7.91 (2H, m), 7.78 (1H, m),
7.55-7.47 (3H, m), 7.44-7.39 (3H, m), 7.30-7.27 (2H, m), 7.18
(3H, m), 7.12 (2H, m), 6.97-6.92 (3H, m), 6.60 (1H, br s), 4.35
(1H, m), 3.64 (2H, m), 3.25-2.90 (8H, m), 2.91 (2H, d, J ) 21.1
Hz), 2.79-2.69 (4H, m), 2.55-2.45 (2H, m), 2.35 (1H, dd, J )
10.0 and 13.7 Hz), 2.03-1.63 (8H, m). FAB-MS (-VE) m/z 855
(M - H). HRMALDI-MS (+VE) calcd for C44H53N6O10NaP [M
+ Na], 879.3453; found, 879.3416.
(b) Furet, P.; Gay, B.; Caravatti, G.; Garcia-Echeverria, C.; Rahuel,
J.; Schoepfer, J.; Fretz, H. Structure-based design and synthesis of
high affinity tripeptide ligands of the Grb2-SH2 domain. J. Med.
Chem. 1998, 41, 3442-3449.
(3) Oishi, S.; Karki, R. G.; Kang, S.-U.; Wang, X.; Worthy, K. M.; Bindu,
L. K.; Nicklaus, M. C.; Fisher, R. J.; Burke, T. R., Jr. Design and
synthesis of conformationally constrained Grb2 SH2 domain binding
peptides employing R-methylphenylalanyl based phosphotyrosyl
mimetics. J. Med. Chem. 2005, 48, 764-772.
(4) (a) Eck, M. J.; Shoelson, S. E.; Harrison, S. C. Recognition of a
high-affinity phosphotyrosyl peptide by the Src homology-2 domain
of P56(lck). Nature 1993, 362, 87-91. (b) Waksman, G.; Shoelson,
S. E.; Pant, N.; Cowburn, D.; Kuriyan, J. Binding of a high affinity
phosphotyrosyl peptide the the Src SH2 domain: Crystal structures
of the complexed and peptide-free forms. Cell 1993, 72, 779-790.
(5) Wei, C.-Q.; Li, B.; Guo, R.; Yang, D.; Burke, T. R., Jr. Development
of a phosphatase-stable phosphotyrosyl mimetic suitably protected
for the synthesis of high affinity Grb2 SH2 domain-binding ligands.
Bioorg. Med. Chem. Lett. 2002, 12, 2781-2784.
(6) Wysong, C. L.; Yokum, T. S.; McLaughlin, M. L.; Hammer, R. P.
4-Aminopiperidine-4-carboxylic acid: A cyclic R,R-disubstituted
amino acid for preparation of water soluble highly helical peptides.
J. Org. Chem. 1996, 61, 7650-7651.
(7) Yao, Z. J.; King, C. R.; Cao, T.; Kelley, J.; Milne, G. W. A.; Voigt,
J. H.; Burke, T. R. Potent inhibition of Grb2 SH2 domain binding
by non-phosphate-containing ligands. J. Med. Chem. 1999, 42, 25-
35.
Final Product 8i: 32% yield; 1H NMR (DMSO-d6) δ 8.61 (1H,
s), 8.11 (1H, d, J ) 7.7 Hz), 8.02 (1H, d, J ) 8.1 Hz), 7.91 (1H,
m), 7.77 (1H, t, J ) 5.0 Hz), 7.55-7.20 (11H, m), 7.10 (2H, m),
6.92 (3H, m), 4.36 (1H, m), 3.43 (2H, m), 3.30 (2H, m), 3.19 (2H,
m), 3.07 (5H, m), 2.94 (2H, m), 2.88 (2H, d, J ) 21.5 Hz), 2.71
(1H, dd, J ) 6.6 and 15.8 Hz), 2.54-2.42 (2H, m), 2.31 (1H, m),
2.01-1.79 (8H, m). FAB-MS (-VE) m/z 876 (M - H). HRM-
ALDI-MS (+VE) calcd for C43H52N5O11NaP [M + Na], 900.3014;
found, 900.3039.
Final Product 8j: 55% yield; 1H NMR (DMSO-d6) δ 8.65 (1H,
s), 8.11 (1H, m), 8.01 (1H, d, J ) 8.4 Hz), 7.96-7.90 (3H, m),
7.77 (1H, t, J ) 5.1 Hz), 7.57-7.40 (8H, m), 7.12 (2H, m), 6.97-
6.93 (3H, m), 5.16 (2H, s), 4.36 (1H, m), 3.75 (2H, m), 3.25-3.01
(7H, m), 2.89 (2H, d, J ) 21.3 Hz), 2.71 (1H, dd, J ) 6.3 and 15.8
Hz), 2.54-2.43 (2H, m), 2.33 (1H, m), 2.02-1.72 (8H, m). FAB-
MS (-VE) m/z 886 (M - H). HRMALDI-MS (+VE) calcd for
C44H50N5O13NaP [M + Na], 910.3035; found, 910.3006.
(8) Available from Pharmacore Inc., High Point, NC.
(9) Oishi, S.; Karki, R. G.; Shi, Z.-D.; Worthy, K. M.; Bindu, L.; Chertov,
O.; Esposito, D.; Frank, P.; Gillette, W. K.; Maderia, M. A.; Hartley,
J.; Nicklaus, M. C.; Barchi, J. J., Jr.; Fisher, R. J.; Burke, T. R., Jr.
Evaluation of macrocyclic Grb2 SH2 domain-binding peptide mi-
metics prepared by ring-closing metathesis of C-terminal allylglycines
with an N-terminal â-vinyl-substituted phosphotyrosyl mimetic.
Bioorg. Med. Chem. 2005, 13, 2431-2438.
(10) (a) Nioche, P.; Liu, W.-Q.; Broutin, I.; Charbonnier, F.; Latreille,
M.-T.; Vidal, M.; Roques, B.; Garbay, C.; Ducruix, A. Crystal
structures of the SH2 domain of Grb2: Highlight on the binding of
a new high-affinity inhibitor. J. Mol. Biol. 2002, 315, 1167-1177.
(b) Liu, W. Q.; Vidal, M.; Gresh, N.; Rogues, B. P.; Garbay, C.
Small peptides containing phosphotyrosine and adjacent R-Me-
phosphotyrosine or its mimetics as highly potent inhibitors of Grb2
SH2 domain. J. Med. Chem. 1999, 42, 3737-3741. (c) Liu, W.-Q.;
Vidal, M.; Olszowy, C.; Million, E.; Lenoir, C.; Dhotel, H.; Garbay,
C. Structure-activity relationships of small phosphopeptides, inhibi-
tors of Grb2 SH2 domain, and their prodrugs. J. Med. Chem. 2004,
47, 1223-1233.
Acknowledgment. The authors wish to thank Drs. James
A. Kelley and Christopher Lai of the Laboratory of Medicinal
Chemistry, NCI, for FAB-MS data. This research was supported
in part by the Intramural Research Program of the NIH, Center
for Cancer Research, NCI-Frederick.
Supporting Information Available: Spectroscopic data for
compounds 7a-j, synthetic experimental procedures for the acyl-
ating species used to synthesize compounds 7j, and synthetic
procedures for mimetics A and B, as well as details of the docking
protocol used to generate Figure 1 and procedures for Biacore
binding analysis. This material is available free of charge via the
Internet at http://pubs.acs.org.
(11) Synthetic procedures for the preparation of mimetics A and B are
provided in the Supporting Information.
(12) Markgren, P. O.; Schaal, W.; Hamalainen, M.; Karlen, A.; Hallgerg,
A.; Samuelsson, B.; Danielson, U. H. Relationships between structure
and interaction kinetics for HIV-1 protease inhibitors. J. Med. Chem.
2002, 45, 5430-5439.
References
(1) Burke, T. R., Jr. Development of Grb2 SH2 domain signaling
antagonists: A potential new class of antiproliferative agents. Int. J.
Pept. Res. Ther.. 2006, 12, 33-48.
(2) (a) Garcia-Echeverria, C.; Gay, B.; Rahuel, J.; Furet, P. Mapping
the X+1 binding site of the Grb2-SH2 domain with R,R-disubstituted
cyclic R-amino acids. Bioorg. Med. Chem. Lett. 1999, 9, 2915-2920.
JM0614073