Concise and enantioselective synthesis of Fmoc-Pmp(But) 2-OH and design of potent Pmp-containing Grb2-SH2 domain antagonists

Article abstract of DOI:10.1021/ol035078+

(Matrix presented) L-Phosphonomethylphenylalanine (L-Pmp) is an important phosphatase-resistant pTyr analogue. A most concise and stereoselective approach to the synthesis of the suitably protected Fmoc-Pmp(Bu ^t)₂-OH was developed in

Full text of DOI:10.1021/ol035078+

ORGANIC
LETTERS
2003
Vol. 5, No. 17
3095-3098
Concise and Enantioselective Synthesis
of Fmoc-Pmp(Bu )2-OH and Design of
t
Potent Pmp-Containing Grb2-SH2
Domain Antagonists
Peng Li,† Manchao Zhang,‡ Megan L. Peach,† Hongpeng Liu,‡ Dajun Yang,‡ and
,†
Peter P. Roller*
Laboratory of Medicinal Chemistry, National Cancer Institute, National Institutes of
Health, Frederick, Maryland 21702, and School of Medicine, UniVersity of Michigan,
Ann Arbor, Michigan 48109
proll@helix.nih.goV
Received June 13, 2003
ABSTRACT
L-Phosphonomethylphenylalanine (L-Pmp) is an important phosphatase-resistant pTyr analogue. A most concise and stereoselective approach
t
to the synthesis of the suitably protected Fmoc-Pmp(Bu )2-OH was developed in order to incorporate the functionally significant L-Pmp residue
into peptides and peptidomimetics efficiently using standard Fmoc protocol. With this key building block, we are able to efficiently synthesize
a series of potent Pmp-containing Grb2-SH2 domain antagonists, which can be used as chemotherapeutic leads for the treatment of erbB2-
overexpressed breast cancer.
The posttranscriptional O-phosphorylation on tyrosine resi-
dues of proteins plays important roles in cellular signal
transduction.1 Great efforts have been made to develop
phosphotyrosine (pTyr)-containing peptides and peptidomi-
metics to modulate aberrant cellular signaling for the
treatment of cancer and other diseases.2 However, the
chemical and enzymatic lability of pTyr restricts its ap-
plicability in the process of drug discovery. Therefore, a
number of pTyr mimetics have been developed with im-
proved hydrolytic stability.3 Among these mimetics, L-
phosphonomethylphenylalanine (L-Pmp),4 the structure of
which is shown in Figure 1, has been widely used for
designing biological and pharmacological probes, since Pmp
is not only phosphatase-resistant but also exhibits similar
biological properties to phosphotyrosine.5
† National Institutes of Health.
‡ Michigan University.
(1) (a) Pawson, T. Nature 1995, 373, 573-580. (b) Songyang, Z.;
Shoelson, S. E.; Chaudhuri, M.; Gish, G.; Pawson, T.; Haser, W. G.; King,
F.; Roberts, T.; Ratnofsky, S.; Lechleider, R. J. Cell 1993, 72, 767-778.
(c) Koch, C. A.; Anderson, D.; Moran, M. F.; Ellis, C.; Pawson, T. Science
1991, 252, 668-674.
(2) (a) Brugge, J. S. Science 1993, 260, 918-919. (b) Garc´ıa-Echeverr´ıa,
C. Curr. Med. Chem. 2001, 8, 1589-1604.
(3) Burke, T. R., Jr.; Yao, Z.-J.; Liu, D.-G.; Voigt, J.; Gao, Y.
Biopolymers 2001, 60, 32-44.
(4) Marseigne, I.; Roques, B. P. J. Org. Chem. 1988, 53, 3621-3624.
(5) Domchek, S. M.; Auger, K. R.; Chatterjee, S.; Burke, T. R., Jr.;
Shoelson, S. E. Biochemistry 1992, 31, 9865-9870.
10.1021/ol035078+ CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/19/2003

Scheme 1. Concise Synthesis of Fmoc-L-Pmp(But)2-OHa
Figure 1. Structure of pTyr and pTyr mimetics.
During the past decade, several approaches have been
developed to synthesize racemic4,6 or enantiomerically pure
Pmp and its protected derivatives by using a chiral auxiliary7
or enzymatic desymmetrization8 or starting from a chiral
synthon.9 However, these synthetic methods are either too
tedious or not suitable for the synthesis of optically pure
Pmp and its derivatives in a large scale. Therefore, it is
significant to develop an efficient approach to the stereo-
selective synthesis of this key pTyr analogue for the
development of Pmp-containing pharmaceuticals and drug
candidates. Here, we would like to report a concise and
highly enantioselective approach to the synthesis of the
properly protected Pmp building block, Fmoc-L-Pmp(But)2-
OH 1 (Figure 1). This protection strategy can conveniently
and cleanly incorporate Pmp into a peptide using standard
Fmoc protocol.
t
a Reagents and conditions: (i) NaH, HPO3 Bu2, THF, reflux, 24
h, 94%; (ii) NBS, (PhCOO)2O, CCl4, reflux, 4 h, 67%; (iii) 2,
NaHMDS, THF-HMPA, -78 °C, 3 h, 78%; (iv) Pd black, H2,
2.5 bar, rt, overnight, 100%; (v) Fmoc-OSu, NaHCO3, CH3CN-
H2O, rt, overnight, 91%.
In our approach, diphenyloxazinone 210c was employed
as a chiral auxiliary to build the desired chirality of the
R-carbon of Pmp, by virtue of the high diastereoselectivity,
friendly chemical properties, and commercial availability of
the reagent 2.10 As shown in Scheme 1, the phosphonate
motif was constructed by refluxing 4-methylbenzylbromide
with sodium di-tert-butyl phosphite in THF and subsequent
bromination with N-bromosuccinimide.8a Subsequently, the
stereoselective enolate alkylation of the oxazinone 2 with
the obtained bromide 3 proceeded at -78 °C in the presence
of solvating agent HMPA. In this step, the enolate of 2 was
first generated with sodium hexamethyldisilylamide for 30-
40 min in THF-HMPA at -78 °C, followed by the addition
of compound 3 to afford the highly diastereoselective trans
alkylation product 4. Because of the intrinsic high reactivity
of the benzylic bromide 3, the alkylation reaction can proceed
smoothly and, generally, more than 98% de can be achieved
according to Williams’ work.10a After hydrogenolysis with
a catalytic amount of palladium black, di-(tert-butyl)-
phosphomethyl phenylalanine 5 was obtained, followed by
Fmoc protection to give Fmoc-L-Pmp(But)2-OH 1 in a 45%
overall yield via five steps. The enantiomeric purity of
compound 1 was determined by using HPLC to measure the
content of the synthesized diastereomers Fmoc-D/L-Pmp-Val-
NH2.9a,7a Compound 1 showed very good enantiomeric purity
(>95% ee). This is the most concise and stereoselective
approach to the synthesis of Pmp and its derivatives reported
to date, and the suitably protected Fmoc-L-Pmp(But)2-OH 1
is a very useful synthon for the synthesis of Pmp-containing
peptides and peptidomimetics using standard Fmoc protocol.
Using this key building block 1, we are able to synthesize
efficiently the Pmp-containing Grb2-SH2 domain antagonists
7-12, which were designed on the basis of the phage library-
derived cyclic peptide G1TE (Figure 2).11 Peptide 6 was
designed as a negative control. Compared to the Pmp-
containing peptide 7, compounds 8-11 were designed to
further constrain the conformation of these cyclic peptides.
To enhance cell permeability, peptide 12 was developed by
conjugating with a hydrophobic carrier peptide. Most im-
(6) (a) Burke, T. R., Jr.; Russ, P.; Lim, B. Synthesis 1991, 11, 1019-
1020. (b) Arslan, T.; Mamaev, S. V.; Mamaeva, N. V.; Hecht, S. M. J.
Am. Chem. Soc. 1997, 119, 10877-10887.
(7) (a) Liu, W.-Q.; Roques, B. P.; Garbay-Jauerguiberry, C. Tetrahe-
dron: Asymmetry 1995, 6, 647-650. (b) Cushman, M.; Lee, E.-S.
Tetrahedron Lett. 1992, 33, 1193-1196.
(8) (a) Baczko, K.; Liu, W.-O.; Roques, B. P.; Garbay-Jauerguiberry,
C. Tetrahedron 1996, 52, 2021-2030. (b) Garbay-Jauerguiberry, C.;
McCort-Tranchepain, I.; Barbe, B.; Ficheux, D.; Roques, B. P. Tetrahe-
dron: Asymmetry 1992, 3, 637-650. (c) Yokomatsu, T.; Minowa, T.;
Murano, T.; Shibuya, S. Tetrahedron 1998, 54, 9341-9356.
(9) (a) Yao, Z.-J.; Gao, Y.; Burke, T. R., Jr. Tetrahedron: Asymmetry
1999, 10, 3727-3734. (b) Dow, D. L.; Bechle, B. M. Synlett 1994, 293-
294.
(10) (a) Williams, R. M.; Im, M.-N. J. Am. Chem. Soc. 1991, 113, 9276-
9286 and literature cited therein. (b) Bender, D. M.; Williams, R. M. J.
Org. Chem. 1997, 62, 6690-6691 and literature cited therein. (c) Benzyl
(2R,3S)-(-)-6-oxo-2,3-diphenyl-4-morpholine carboxylate 2 is commercially
available from Aldrich Chemical Co.: catalog No. 33,187-2.
(11) (a) Oligino, L.; Lung, F.-D. T.; Sastry, L.; Bigelow, J.; Cao, T.;
Curran, M.; Burke, T. R.; Wang, S.-M.; Krag, D.; Roller, P. P.; King, C.
R. J. Biol. Chem. 1997, 272, 29046-29052. (b) Long, Y.-Q.; Voigt, J. H.;
Lung, F.-D. T.; King, C. R.; Roller, P. P. Bioorg. Med. Chem. Lett. 1999,
9, 2267-2272. (c) Lung, F.-D. T.; Long, Y.-Q.; King, C. R.; Varady, J.;
Wu, X.-W.; Wang, S.; Roller, P. P. J. Pept. Res. 2001, 57, 447-454.
3096
Org. Lett., Vol. 5, No. 17, 2003

peptide 12a using standard Fmoc protocol on PAL resin,
the Pmp residue can be cleanly incorporated using the
coupling reagent HATU with the addition of HOAt to
accelerate the reaction. To reduce the amount of deletion
peptide, a double coupling strategy was adopted considering
the steric hindrance of 1-amino-1-cyclohexanecarboxylic acid
(Ach). After the completion of peptide elongation, the
N-terminal was capped with 10 equiv of chloroacetic
anhydride to give compound 12b. No base was added in the
process of the chloroacetylation to avoid any chance of
racemization. After the peptide was cleaved from the PAL
resin by treatment with TFA containing 2.5% each (v/v) of
triethylsilane (TES) and deionized water, the linear chloro-
acetylated peptide underwent intramolecular thioetherification
in dilute buffer solution to afford the carrier-conjugated
26mer cyclic peptidomimetic 12 (Scheme 2).
Figure 2. Structure of Pmp-containing G1TE analogues 7-11.
portantly, the carrier-conjugated cyclic peptide 12 can be
successfully synthesized from compound 1. Previously, we
tried to synthesize peptide 12 using the commercially
available Fmoc-L-Pmp-OH, but no product was obtained due
to various side reactions caused by the free P-OH groups.
As shown in Scheme 2, after the construction of the 23mer
The synthetic strategy of compound 12 can also be
employed for the synthesis of G1TE analogues, 6-9.
Sulfoxides 10 and 11 were obtained by treating the thioether
9 with 5% H2O2 aqueous solution overnight. The (R)-
configured sulfoxide 10 can be easily separated as the major
product from its diastereoisomer 11 using HPLC, in a ratio
of 1.4:1. Compared to the (S)-configured sulfoxide 11, the
(R)-configured sulfoxide 10 is more thermodynamically
stable and thus given as the major product under this mild
oxidization condition. The absolute configuration of the two
sulfoxide isomers was determined by CD spectra according
to the guidelines described in the literature and confirmed
by extensive molecular modeling.12
Scheme 2. Illustrative Synthesis of Compound 12a
Extracellular ELISA-based binding assays indicate that
incorporating Pmp into the third position of G1TE analogues
tremendously improves the potency of the resulting peptides.
For instance, the Pmp3-containing peptide 7 is 20 000-fold
more potent than the corresponding Tyr3-containing analogue
6, as shown in Table 1. Replacing the Met8 with the more
Table 1. Inhibitory Activities of Pmp-Containing G1TE
Analogues Evaluated by ELISA Assaysa
amino acid compositions
compdsb AA1 AA3 AA4 AA8
AA10
IC50 (nM)c
G1TE
6
Glu Tyr
Ala Tyr
Glu Met
Ach Met
Cys
> 105 d
Cys
Cys
2.65 × 105
((1.20 × 105)
7
8
9
10
11
12e
Ala Pmp Ach Met
Ala Pmp Ach 2-Nal Cys
Ala Pmp Ach NPG Cys
Ala Pmp Ach NP Gf Cys(OR ) 8.08 ((0.76)
Ala Pmp Ach NP Gf Cys(OS ) 18.5 ((3.6)
Ala Pmp Ach NP Gf Cys
13.3 ((0.08)
2.38 ((0.90)
13.4 ((2.0)
125 ((35)
a In extracellular ELISA binding assays, a biotin-labeled SHC-phospho-
peptide competed with our G1TE analogues. b Structures of these G1TE
analogues are shown in Figure 2. c Values are means of three experiments;
standard deviation is given in parentheses. d Biacore assay indicates that
G1TE exhibits inhibitory activity with an IC50 ) 20 µM.11b e Sequence of
the carrier13 and the structure of the carrier-conjugated peptide 12 are shown
in Scheme 2. f NPG stands for L-neopentylglycine.
a Reagents and conditions: (i) SPPS, Fmoc chemistry, ABI
synthesizer; (ii) 1, HATU, HOAt, DIEA, DMF, rt, 1 h, double
coupling; (iii) SPPS, Fmoc chemistry, ABI synthesizer; (iv)
(ClCH2CO)2O, rt, overnight; (v) TFA:TES:H2O ) 40:1:1, rt, 2 h;
(vi) NH4OAc, NH4OH, pH ) 8.2, CH3CN-H2O, rt, 8 h.
Org. Lett., Vol. 5, No. 17, 2003
3097

hydrophobic 2-naphthylalanine (2-Nal) gives the 2-Nal8-
containing peptide 8, with an IC50 ) 2.38 nM, which is 5.6-
fold more potent than the corresponding analogue 7. The
improved binding affinity of compound 8 is attributed to the
more favorable intramolecular van der Waals interactions
of the hydrophobic naphthyl side chain of 2-Nal8 with the
side chains of Leu2 and Cys,10 as indicated by molecular
modeling. Such interactions stabilize the favored conforma-
tion of the peptide and allow other residues such as Pmp3,
Ach,4 Asn5, Val6, and Tyr9 in the peptide to bind to Grb2-
SH2 protein optimally. To further constrain the backbone
conformation of these Pmp-containing G1TE analogues,
compounds 10 and 11 were designed and synthesized by
oxidizing the flexible thioether linkage of peptide 9 into
relatively rigid sulfoxides. Interestingly, only the (R)-
configured sulfoxide 10 showed higher inhibitory potency
in comparison with the thioether precursor 9, while the other
diastereoisomer 11 is even less active than thioether 9 due
to an unfavorable conformational change caused by repulsion
between the oxygen of the (S)-configured sulfoxide and the
carbonyl oxygen of the N-acetyl group according to molec-
ular modeling and CD spectra.12e
Figure 3. Inhibition of the association of Grb2 with p185erbB2 in
MDA-MB453 cancer cells by treatment with compound 12.
Figure 3. Compound 12 also exhibits a moderate antimito-
genic effect in breast cancer cell cultures. The growth of
MDA-MB-453 cancer cells was inhibited by 40% after
treatment with a 5 µM concentration of inhibitor 12.
In conclusion, we have developed a concise and stereo-
selective approach to the synthesis of the suitably protected
L-Pmp building block Fmoc-L-Pmp(tBu)2-OH, which is very
useful in the synthesis of Pmp-containing peptides and
peptidomimetics. Using this key synthon, we are able to
efficiently synthesize a series of potent Pmp-containing Grb2-
SH2 domain antagonists, which are typically at least 104-
fold more potent than the lead G1TE.
Whole cell assays show that the carrier-conjugated cyclic
peptidomimetic 12 can penetrate cell membranes and ef-
fectively inhibit the association of Grb2 protein with the
p185erbB2 complex in erbB2-overexpressing MDA-MB-453
cancer cells at low micromolar concentrations, as shown in
Supporting Information Available: Experimental details
for the preparation of compound 1 and G1TE analogues
6-11 and characterization data thereof, detailed information
about CD spectral analysis of compounds 9-11, and
molecular modeling. This material is available free of charge
via the Internet at http://pubs.acs.org.
(12) (a) Ottenheijm, H. C. J.; Liskamp, R. M. J.; Helquist, P.; Lauher, J.
W.; Shekhani, M. S. J. Am. Chem. Soc. 1981, 103, 1720-1723. (b) Kubec,
R.; Musah, R. A. Phytochemistry 2001, 58, 981-985. (c) Van den Broek,
L. A. G. M.; Breuer, M. L.; Liskamp, R. M. J.; Ottenheijm, H. C. J. J.
Org. Chem. 1987, 52, 1511-1517. (d) Liskamp, R. M. J.; Zeegers, H. J.
M.; Ottenheijm, H. C. J. J. Org. Chem. 1981, 46, 5408-5413. (e) Detailed
information about CD spectra analysis and molecular modeling is described
in Supporting Information.
(13) Lindgren, M.; Hallbrink, M.; Prochiantz, A.; Langel, U. Trends
Pharmacol. Sci. 2000, 21, 99-103.
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