Design and synthesis of 4-(α-hydroxymalonyl)phenylalanine as a new phosphotyrosyl mimetic and its use in growth factor receptor bound 2 Src-homology 2 (Grb2 SH2) domain-binding peptides

Article abstract of DOI:10.1021/jm050154v

A new phosphotyrosyl mimetic 4-(α-hydroxymalonyl)phenylalanine and its incorporation into a Grb2 SH2 domain-binding tripeptide are presented. In whole-cell studies using malonyl ethyl ester prodrug derivatives, it was observed that the 4-(α-hydroxymalonyl

Full text of DOI:10.1021/jm050154v

J. Med. Chem. 2005, 48, 5369-5372
5369
Brief Articles
Design and Synthesis of 4-(r-Hydroxymalonyl)phenylalanine as a New
Phosphotyrosyl Mimetic and Its Use in Growth Factor Receptor Bound 2
Src-Homology 2 (Grb2 SH2) Domain-Binding Peptides
Sang-Uk Kang,† Karen M. Worthy,‡ Lakshman K. Bindu,‡ Manchao Zhang,§ Dajun Yang,§ Robert J. Fisher,‡ and
Terrence R. Burke, Jr.*,†
Laboratory of Medicinal Chemistry, CCR, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702,
Protein Chemistry Laboratory, SAICsFrederick, Frederick, Maryland 21702, and University of Michigan Medical School,
Ann Arbor, Michigan 48109
Received February 17, 2005
A new phosphotyrosyl mimetic 4-(R-hydroxymalonyl)phenylalanine and its incorporation into
a Grb2 SH2 domain-binding tripeptide are presented. In whole-cell studies using malonyl ethyl
ester prodrug derivatives, it was observed that the 4-(R-hydroxymalonyl)phenylalanyl-
containing peptide exhibited greater efficacy than the nonhydroxylated 4-(malonyl)phenylalanyl-
containing congener in blocking the association of Grb2 with activated erbB-2 tyrosine kinase.
These results are consistent with de-esterification and at least partial intracellular decarboxy-
lation.
Introduction
Phosphotyrosyl (pTyr) mimetics are important com-
ponents of binding antagonists directed against the
growth factor receptor bound 2 (Grb2)1 SH2 domain.2
Compound 1 represents a pTyr-containing tripeptide
that was originally reported by Novartis Corp. as
exhibiting high-affinity Grb2 domain-binding affinity
(Figure 1).3 The related structure 2 was developed in
which the phosphoryl group has been replaced by a
malonyl moiety.4,5 However, a drawback of the malonyl-
based phosphoryl mimetic is its susceptibility to chemi-
cal decarboxylation leading to the less potent analogue
3.6 Indeed, under slightly acidic conditions appreciable
Figure 1. Structures of Grb2 SH2 domain-directed analogues
conversion of 2 to 3 is observed over the course of 24
h.7 On the basis of work in a p56lck SH2 domain-binding
system that showed conversion of a 4-(carboxymethyl)-
phenylalanyl pTyr mimetic to 4-(carboxy(R-hydroxym-
ethyl))phenylalanyl residue resulting in a doubling of
affinity,8 it was felt that the hydroxyl-containing 4 could
potentially exhibit higher affinity than 3 in a Grb2 SH2
domain-binding system. Furthermore, it was hoped that
the binding affinity of 4-(R-hydroxymalonyl)phenylala-
nyl-containing 5, which would yield 4 following decar-
boxylation, would be equivalent to or greater than
4-(malonyl)phenylalanyl-containing 2. The current study
was undertaken to prepare 5 and 4 and to compare their
affinities with 2 and 3. Additionally, ethyl esters 6 and
7 were prepared as potential prodrugs of 2 and 5 to
examine efficacy in a whole-cell system.
discussed in the text.
Synthesis
Of compounds 2-7 that are required for the current
study, 2 and 3 have been previously reported4-6 while
analogues 4-7 are new. Because 5 can be prepared from
7 by treatment with aqueous LiOH and because 4 can
be derived from 5 by decarboxylation in DMSO, ana-
logues 6 and 7 represent pivotal objectives to the overall
study. Key to the syntheses of 6 and 7 are the N-Fmoc-
protected pTyr mimetics 13a and 13b, respectively.
These were prepared by nearly identical routes from
intermediates 9a and 9b, which were derived from
4-iodotoluene (8a) by reaction of diethyl malonate and
sodium hydride in the presence of CuBr (for 9a) or from
alkylation of toluene (8b) with diethyl ketomalonate and
SnCl4 (for 9b) (Scheme 1).9
Benzylic bromination of 9a to 10a was readily achieved
in 78% yield; however, similar bromination of 9b gave
an inseparable mixture of monobrominated (10b) and
dibrominated product 10c in a 10:3 ratio. Alkylation of
* To whom correspondence should be addressed. Phone: (301) 846-
5906. Fax: (301) 846-6033. E-mail: tburke@helix.nih.gov.
† National Cancer Institute.
‡ SAICsFrederick.
§ University of Michigan Medical School.
10.1021/jm050154v CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/12/2005

5370 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Brief Articles
Scheme 1a
a Reagents and conditions: (a) [for R ) I, X ) H, H] CuBr, NaH, HMPA, dioxane; (b) [for R ) H, X ) O] SnCl4; (c) NBS, AIBN, [for R
) H] CH2Cl2, reflux, [for R ) OH] CCl4; (d) LHMDS, HMPA, THF, -78 °C to room temperature; (e) H2, Pd-C, MeOH; (f) Fmoc-OSu,
NaHCO3, H2O, dioxane.
Scheme 2a
Table 1. SPR-Derived Steady-State Affinity Constants for
Binding to Grb2 SH2 Domain Protein
compd
Keq (µM)
1
2
3
4
5
0.240a,b
0.149a,c
6.11d,e
1.54
0.270
a Complex kinetics were observed. b ELISA IC50 value previ-
ously reported as 0.047 µM in ref 3. c ELISA IC50 value previously
reported as 0.07 µM in ref 4. d ELISA IC50 value previously
reported as 1 µM in ref 4. e SPR-derived IC50 value previously
reported as 0.6 µM in ref 6.
higher than what had previously been reported using
an ELISA assay (IC50 ) 0.047 µM).3 The binding affinity
of parent malonyl-containing 2 (Keq ) 0.149 µM) was
also higher than what had previously been reported by
ELISA techniques (IC50 ) 0.07 µM).4 The corresponding
decarboxylation product 3 exhibited less affinity in the
current assay (Keq ) 6.11 µM) than previously reported
using an ELISA assay (IC50 ) 1 µM)4 or using a Biacore
instrument, in which inhibitor in solution competed
with chip-bound pTyr-containing peptide for binding to
Grb2 SH2 domain protein in solution (IC50 ) 0.6 µM).6
Introduction of a hydroxyl group onto the malonyl
methylene of 2 resulted in a nearly 2-fold reduction in
binding affinity (5, Keq ) 0.270 µM). However the
corresponding decarboxylation product (4, Keq ) 1.54
µM) was approximately 4-fold more potent than the
congener 3, which lacked the hydroxyl group.
a Reagents and conditions: (a) TFA, SiEt3; (b) EtOH, H2SO4,
80 °C.
this mixture with Williams lactone 1110 allowed ready
separation of the desired 12b in 30% overall yield from
9b. Similar alkylation of 10a gave the lactone adduct
12a in extremely low yield (14%). Since the reaction
proceeds in higher yield when the malonyl moiety bears
t
bis-tBu ester protection,5 the known Bu-protected 14
was prepared and converted to desired diethyl ester 12a
(Scheme 2). Finally, hydrogentation of lactones 12a and
12b gave the corresponding free amino acids, which
were converted to the protected pTyr mimetics 13a and
13b, respectively, by treatment with Fmoc-OSu. Syn-
thesis of final products 6 and 7 was by known meth-
ods.11
Grb2 SH2 Domain-Binding Affinities in
Extracellular Assays
Inhibition by Ester Prodrugs 6 and 7 of Grb2
Binding to p185erbB-2 in MDA-MB-453 Breast
Cancer Cells in Culture
By use of previously reported procedures,4 produgs 6
and 7 were examined in cultured MDA-MB-453 breast
cancer cells overnight. It was found that nonhydroxy-
lated 6 is less potent than the hydroxyl-containing 7 in
blocking the intracellular association of Grb2 with the
p185erbB-2 tyrosine kinase (Figure 2). These results are
the reverse of what would be expected on the basis of
Determination of Grb2 SH2 domain-binding affinities
by surface plasmon resonance (SPR) was accomplished
using Biacore instruments as previously described.11
Recombinant Grb2 SH2 domain protein was coupled to
sensor chips, and steady-state equilibrium constants
were determined for binding of ligands in solution to
the chip-bound protein (Table 1). Reference compound
1 provided a Keq of 0.240 nM, which was somewhat

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5371
stirring continued at room temperature (2 h). The mixture was
poured into ice-cold dilute aqueous HCl, extracted with CHCl3,
and dried (MgSO4). Residue was purified by silica gel flash
1
chromatography to give 9b as a colorless oil (99% yield). H
NMR (CDCl3) δ 7.53 (2 H, d, J ) 8.2 Hz), 7.18 (2 H, d, J ) 8.6
Hz), 4.30 (4 H, m), 2.35 (3 H, s), 1.29 (6 H, t, J ) 7.22 Hz). 13
C
NMR (CDCl3) δ 170.2, 138.6, 133.2, 128.9, 126.7, 80.1, 63.1,
21.3, 14.1. FAB-MS (+VE) m/z 267 (MH+). Anal. (C14H18O5)
C, H, O.
Diethyl 2-[4-(Bromomethyl)phenyl]propane-1,3-dioate
(10a). To a stirred solution of 9a (3.0 g, 12.0 mmol) in CH2Cl2
(100 mL) were added N-bromosuccinimide (NBS) (2.2 g, 12.2
mmol) and AIBN (197 mg, 1.2 mmol), and the mixture was
stirred at 85 °C (1 h). Additional AIBN (100 mg, 0.6 mmol)
was added, and stirring continued at 85 °C (1 h). The mixture
was cooled to room temperature, volatiles were removed by
rotary evaporation, and the residue was purified by silica gel
flash to give 10a as a colorless oil (3 g, 78% yield based on a
5% yield of recovered 9a). 1H NMR (CDCl3) δ 7.39 (4 H, s),
4.61 (1 H, s), 4.48 (2 H, s), 4.15-4.28 (4 H, m), 2.34 (3 H, s),
1.26 (6 H, t, J ) 7.0 Hz). 13C NMR (CDCl3) δ 185.7, 168.1,
137.9, 133.2, 129.9, 129.5, 62.1, 57.8, 33.1, 14.2. FAB-MS
(+VE) m/z 329 (MH+). Anal. (C14H17BrO4‚0.1H2O) C, H.
Diethyl 2-[4-({(3S,5S,6R)-2-Oxo-5,6-diphenyl-4-[benzyl-
oxycarbonyl]morpholin-3-yl}methyl)phenyl]propane-1,3-
dioate (12a). To a stirred solution of Williams lactone 11 (3.6
g, 9.36 mmol) in anhydrous THF (200 mL) with HMPA (10
mL) at -78 °C was added 1 M LHMDS in hexanes (9.36 mL,
9.36 mmol), and the mixture was stirred at -78 °C (30 min).
To this was transferred via cannula a precooled solution of
10a (2.8 g, 8.5 mmol) in THF (50 mL). Then cooling was
removed, and the mixture was stirred at room temperature
(1 h). The mixture was poured into cold aqueous NH4Cl
solution and extracted (EtOAc), the combined extracts were
dried (MgSO4), and solvent was removed by rotary evapora-
tion. Purification by silica gel flash chromatography gave 12a
as a colorless oil (750 mg, 14% yield) (see below for spectral
data).
Figure 2. SDS gels showing the inhibition of binding of
p185erbB-2 to Grb2 (p25) by analogues 5 and 2 in whole cells.
Experiments were conducted as described in ref 4. The p185
bands were obtained by immunopreciptating with Grb2 anti-
bodies and immunoblotting for p185erbB-2. The intensity of the
p25 bands, which were obtained by immunopreciptating with
Grb2 antibodies and immunoblotting for Grb2, indicates
uniform expression of Grb2 at each concentration of inhibitor.
the Grb2 SH2 domain-binding potencies of the free
malonyl-containing species 2 and 5, respectively. One
possible explanation for the observed results is that at
least a component of the active intracellular species is
the monocarboxy analogues 3 and 4, which could arise
through de-esterification and intracellular decarboxy-
lation.
Conclusion
The current study presents a new R-hydroxymalonyl-
containing phosphotyrosyl mimetic and its incorporation
into a Grb2 SH2 domain-binding peptide. It was ratio-
nalized that introduction of a hydroxyl group into the
malonyl methylene could potentially enhance the bind-
ing affinity of the decarboxylation product. Indeed, for
the decarboxylation products, a 4-fold enhancement of
binding affinity was observed for R-hydroxyl-containing
4 relative to its nonhydroxylated counterpart 3. It was
also hoped that the parent R-hydroxymalonyl-containing
species (5) would exhibit binding affinity equal to or
greater than its nonhydroxylated counterpart (2). How-
ever, it was found that 2 displayed an approximate
2-fold higher affinity than 5. Nonetheless, in whole-cell
studies using ethyl ester prodrug derivatives of 2 and
5, it was observed that the hydroylated species exhibited
higher efficacy than the nonhydroxylated species in
blocking the association of Grb2 with activated erbB-2
tyrosine kinase.
Synthesis of 12a from 14. To a stirred solution of 14 (346
mg, 0.5 mmol) in CH2Cl2 (8 mL) at 0 °C was added SiEt3 (1
mL) and TFA (2 mL). Then the mixture was stirred at room
temperature (2 h). Solvent was removed by rotary evaporation,
and residue was placed under high vacuum. Then the residue
was dissolved in EtOH (5 mL), concentrated H2SO4 (2 drops)
was added, and the mixture was refluxed (2 h). The mixture
was cooled to 0 °C, acid was neutralized by addition of
saturated aqueous NaHCO3, and the mixture was extacted
with EtOAc. The combined organic phase was purified by silica
gel flash chromatography to give 12a (76 mg, 24% yield) as a
colorless oil along with decarboxylated material (130 mg, 46%
1
yield). H NMR (CDCl3) (two conformers were observed in a
ratio of 1:0.5): major conformer, δ 7.41 (2 H, d, J ) 8.0 Hz),
7.24 (2 H, d, J ) 8.0 Hz), 7.04-7.21 (10 H, m, overlapping),
6.46-6.81 (5 H, m, overlapping), 5.34 (1H, dd, J ) 2.7 and 6.1
Hz, -NCHCOO-), 4.96-5.11 (2 H, m, -OCH2Ph, overlap-
ping), 4.80 (1 H, d, J ) 3.1 Hz, -CHPhOOC-), 4.60 (1 H, s,
CH(CO2Et)2), 4.10-4.28 (5 H, m, -NCHPh-, (OCH2CH3) ×
2, overlapping), 3.75(1 H, dd, J ) 6.1 and 13.7 Hz, one of -CH2-
CH(N)COO-), 3.39 (1 H, dd, J ) 2.9 and 13.7 Hz, the other of
-CH2CH(N)COO-, overlapping), 1.26 (3 H, m, OCH2CH3,
overlapping), 1.15-1.19 (3 H, m, OCH2CH3, overlapping);
minor conformer, δ 7.34 (2H, d, J ) 8.0 Hz), 7.04-7.21 (12 H,
m, overlapping), 6.46-6.81 (5 H, m, overlapping), 5.24 (1 H,
dd, J ) 3.1 and 6.4 Hz, -NCHCOO-), 4.96-5.11 (3 H, m,
-OCH2Ph, CHPhOOC-, overlapping), 4.58 (1 H, s CH(CO2-
Et)2), 4.36 (1 H, d, J ) 3.1 Hz, NCHPh-,), 4.10-4.30 (4 H, m,
-(OCH2CH3) × 2, overlapping), 3.54 (1 H, dd, J ) 7.0 and 13.9
Hz, one of -CH2CH(N)COO-), 3.34 (1 H, dd, J ) 3.3 and 13.9
Hz, CH2CH(N)COO-), 1.26 (3 H, m, OCH2CH3, overlapping),
1.15-1.19 (3 H, m, OCH2CH3, overlapping). FAB-MS (+VE)
m/z 636 (MH+). Anal. (C38H37NO8‚0.2H2O) C, H, N.
Experimental Section
Diethyl 2-(4-Methylphenyl)propane-1,3-dioate (9a). To
a stirred suspension of NaH (2.2 g, 55 mmol) in 200 mL of
dioxane at 0 °C was slowly added diethyl malonate (7.6 mL,
50 mmol). The mixture was stirred at room temperature (30
min). Then CuBr (8.6 g, 60 mmol) and 4-iodotoluene (16.4 g,
75 mmol) were added, and the mixture was heated to 95-98
°C (1 h). Then HMPA (5 mL) was added and stirring continued
(9 h). Inorganics were removed by filtration, volatiles were
evaporated under reduced pressure, and residue was purified
by silica gel flash chromatography to provide 9a as a colorless
oil (4.38 g, 35% yield). 1H NMR (CDCl3) δ 7.28 (2 H, d, J ) 8.2
Hz), 7.16 (2 H, d, J ) 8.0 Hz), 4.57 (1 H, s), 4.15-4.27 (4 H,
m), 2.34 (3 H, s), 1.26 (6 H, t, J ) 7.2 Hz). 13C NMR (CDCl3)
δ 185.7, 168.5, 138.2, 130.1, 129.5, 129.3, 61.9, 57.8, 21.3, 14.2.
FAB-MS (+VE) m/z 251 (MH+). Anal. (C14H18O4) C, H, O.
Diethyl 2-Hydroxy-2-(4-methylphenyl)propane-1,3-
dioate (9b). To a solution of diethyl ketomalonate (15.4 mL,
100 mmol) in toluene (53.3 mL, 500 mmol) at 0 °C was added
SnCl4 (14 mL, 120 mmol) over 20 min, and the mixture was
stirred at 0 °C (10 min). Then the ice bath was removed, and
Diethyl 2-[4-({(3S,5S,6R)-2-Oxo-5,6-diphenyl-4-[benzyl-
oxycarbonyl]morpholin-3-yl}methyl)phenyl]-2-hydroxy-
propane-1,3-dioate (12b). A mixture of 9b (2.66 g, 10 mmol),

5372 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Brief Articles
NBS (1.87 g, 10.5 mmol), and AIBN (164 mg, 1 mmol) in CCl4
(50 mL) was stirred at reflux (1 h). Then the mixture was
cooled to room temperature and evaporated to dryness and
the residue was purified by silica gel flash chromatography to
yield 10a contaminated with dibrominated byproduct. To a
stirred solution of Williams lactone 11 (5 g, 13 mmol) in
anhydrous THF (120 mL) with HMPA (15 mL) at -78 °C was
added 1 M LHMDS in THF (15 mL, 15 mmol). The mixture
was stirred at -78 °C and then transferred via cannula to a
stirred solution of crude 10a in THF (40 mL) at -78 °C.
Cooling was removed, and the mixture was stirred at room
temperature (16 h) and then poured into cold aqueous NH4Cl
solution. The mixture was extracted (EtOAc), and the com-
bined organic phase was dried (MgSO4) and reduced in volume
to give starting Williams lactone 11 as a solid, which was
removed by filtration. The filtrate was taken to dryness and
purified by silica gel flash chromatography to give 12b as a
white solid (30% yield over two steps). Mp 53 °C. 1H NMR
(CDCl3) (two conformers were observed in a ratio of 1:0.5):
major conformer, δ 7.69 (2 H, d, J ) 8.4 Hz), 7.36-7.45 (2 H,
m, overlapping), 7.04-7.28 (10 H, m, overlapping), 6.46-6.81
(5 H, m, overlapping), 5.35 (1 H, m, -NCHCOO-), 5.03 (2 H,
m, -OCH2Ph, overlapping), 4.79 (1 H, d, J ) 3.1 Hz,
-CHPhOOC-), 4.07 (1H, d, J ) 2.9 Hz, -NCHPh-), 3.93-
4.40 (4 H, m, (OCH2CH3) × 2, overlapping), 3.77 (1 H, dd, J )
6.1 and 13.7 Hz, one of -CH2CH(N)COO-), 3.35-3.44 (1 H,
m, -CH2CH(N)COO-, overlapping), 1.22-1.32 (3 H, m,
OCH2CH3, overlapping), 1.06-1.12 (3 H, m, OCH2CH3, over-
lapping); minor conformer, δ 7.62 (2 H, d, J ) 8.4 Hz), 7.36-
7.45 (2 H, m, overlapping), 7.04-7.28 (10 H, m, overlapping),
6.46-6.81 (5 H, m, overlapping), 5.27 (1 H, m, -NCHCOO-),
5.03 (2 H, m, -OCH2Ph, overlapping), 4.95 (1 H, d, J ) 2.9
Hz, -CHPhOOC-), 3.93-4.40 (5 H, m, -NCHPh-, (OCH2-
CH3) × 2, overlapping), 3.57 (1 H, dd, J ) 6.4 and 13.7 Hz,
one of -CH2CH(N)COO-), 3.35-3.44 (1 H, m, -CH2CH(N)-
COO-, overlapping), 1.22-1.32 (3 H, m, OCH2CH3, overlap-
ping), 1.06-1.12 (3 H, m, OCH2CH3, overlapping). FAB-MS
(+VE) m/z 652 (MH+).
to provide the intermediate free amino acid. To this was added
dioxane (10 mL) and H2O (10 mL) along with NaHCO3 (541
mg, 6.44 mmol) and Fmoc-Osu (725 mg, 2.15 mmol), and the
mixture was stirred at room temperature (20 h). The mixture
was diluted with CHCl3, and ice was added followed by dilute
HCl to attain pH 3. The mixture was extracted well with
CHCl3, and the combined organic phase was dried (MgSO4),
concentrated, and purified by silica gel flash chromatography
to afford 13b as a white solid (95% yield from 12b). Mp 80-
82 °C. 1H NMR (DMSO-d6) δ 7.88 (2 H, d, J ) 7.4 Hz), 7.66 (2
H, t, J ) 7.2 Hz), 7.28-7.42 (5 H, m), 7.17 (2 H, d, J ) 8.4
Hz), 7.07 (1 H, s), 4.26(1 H, m), 4.14 (6 H, m), 3.94 (1 H, m),
3.08 (1 H, dd, J ) 3.9, 13.3 Hz), 2.91 (1 H, dd, J ) 7.4, 14.2
Hz), 1.15 (6 H, m). FAB-MS (-VE) m/z 560 (M - H).
References
(1) Lowenstein, E. J.; Daly, R. J.; Batzer, A. G.; Li, W.; Margolis,
B.; Lammers, R.; Ullrich, A.; Skolnik, E. Y.; Barsagi, D.;
Schlessinger, J. The SH2 and SH3 domain-containing protein
Grb2 links receptor tyrosine kinases to Ras signaling. Cell 1992,
70, 431-442.
(2) Fretz, H.; Furet, P.; Garcia-Echeverria, C.; Rahuel, J.; Schoepfer,
J. Structure-based design of compounds inhibiting Grb2-SH2
mediated protein-protein interactions in signal transduction
pathways. Curr. Pharm. Des. 2000, 6, 1777-1796.
(3) 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.
(4) Gao, Y.; Luo, J.; Yao, Z.-J.; Guo, R.; Zou, H.; Kelley, J.; Voigt, J.
H.; Yang, D.; Burke, T. R., Jr. Inhibition of Grb2 SH2 domain
binding by non-phosphate containing ligands. 2. 4-(2-Malonyl)-
phenylalanine as a potent phosphotyrosyl mimetic. J. Med.
Chem. 2000, 43, 911-920.
(5) Gao, Y.; Burke, T. R., Jr. Stereoselective preparation of L-4-(2′-
malonyl)phenylalanine suitably protected for Fmoc-based syn-
thesis of potent signal transduction inhibitory ligands. Synlett
2000, 134-136.
(6) Burke, T. R., Jr.; Luo, J.; Yao, Z.-J.; Gao, Y.; Milne, G. W. A.;
Guo, R.; Voigt, J. H.; King, C. R.; Yang, D. Monocarboxylic
phosphotyrosyl mimetics in the design of Grb2 SH2 domain
inhibitors. Bioorg. Med. Chem. Lett. 1999, 9, 347-352.
(7) Unreported results.
(8) Beaulieu, P. L.; Cameron, D. R.; Ferland, J. M.; Gauthier, J.;
Ghiro, E.; Gillard, J.; Gorys, V.; Poirier, M.; Rancourt, J.; Wernic,
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Ingraham, R.; Jakes, S.; Kabcenell, A.; Kirrane, T.; Lukas, S.;
Patel, U.; Proudfoot, J.; Sharma, R.; Tong, L.; Moss, N. Ligands
for the tyrosine kinase p56lck SH2 domain: Discovery of potent
dipeptide derivatives with monocharged, nonhydrolyzable phos-
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(9) Ghosh, S.; Pardo, S. N.; Salomon, R. G. Ester enolates from
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(2S)-3-{4-[Bis(ethoxycarbonyl)methyl]phenyl}-2-[(flu-
oren-9-ylmethoxy)carbonylamino]propanoic Acid (13a).
A solution of 12a (636 mg, 1.0 mmol) in MeOH (10 mL) was
hydrogenated over 10% Pd-C (200 mg) using a hydrogen-filled
balloon (17 h). Catalyst was removed by filtration through
Celite 545, and the filtrate was evaporated to provide the
intermediate free amino acid. To this was added dioxane (8
mL) and H2O (8 mL) along with NaHCO3 (252 mg, 3.00 mmol)
and Fmoc-Osu (725 mg, 2.15 mmol), and the mixture was
stirred at room temperature (1.5 h). The mixture was diluted
with CHCl3, and ice was added. Then dilute HCl was added
to attain pH 3. The mixture was extracted well with CHCl3,
and the combined organic phase was dried (MgSO4), concen-
trated, and purified by silica gel flash chromatography to afford
(10) Williams, R. M.; Im, M. N. Asymmetric synthesis of monosub-
stituted and R,R-disubstituted R-amino acids via diastereose-
lective glycine enolate alkylations. J. Am. Chem. Soc. 1991, 113,
9276-86.
1
13a as a solid (284 mg, 52% from 12a). Mp 137-138 °C. H
NMR (DMSO-d6) δ 7.88 (2 H, d, J ) 7.4 Hz), 7.71 (1 H, d, J )
8.4 Hz), 7.66 (2H, t, J ) 8.2 Hz), 7.38-7.43 (2 H, m), 7.25-
7.33 (6 H, m), 4.87 (1 H, s), 4.02-4.25 (8 H, m), 3.08 (1 H, dd,
J ) 4.4, 14.0 Hz), 2.88 (1 H, dd, J ) 10.8, 14.0 Hz), 1.11-1.17
(6 H, m). FAB-MS (+VE) m/z 546 (MH+). Anal. (C38H37NO8)
C, H, N.
(2S)-3-{4-[Bis(ethoxycarbonyl)hydroxymethyl]phenyl}-
2-[(fluoren-9-ylmethoxy)carbonylamino]propanoic Acid
(13b). A suspension of 12b (1.4 g, 2.15 mmol) in MeOH (20
mL) was hydrogenated over 10% Pd-C (280 mg) using a
hydrogen-filled balloon (19 h). Catalyst was removed by
filtration through Celite 545, and the filtrate was evaporated
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