Examination of phosphoryl-mimicking functionalities within a macrocyclic Grb2 SH2 domain-binding platform

Article abstract of DOI:10.1021/jm050059m

Reported herein are the design, synthesis, and Grb2 SH2 domain-binding affinities of several phosphoryl-mimicking groups displayed within the context of a conformationally constrained macrocyclic platform. With use of surface plasmon resonance techniques, single-digit nanomolar affinities were exhibited by phosphonic acid and malonyl-containing diacidic phosphoryl mimetics (for 4h and 4g, K_D = 1.47 and 3.62 nM, respectively). Analogues containing monoacidic phosphoryl mimetics provided affinities of K_D = 16-67 nM. Neutral phosphoryl-mimicking groups did not show appreciable binding.

Full text of DOI:10.1021/jm050059m

J. Med. Chem. 2005, 48, 3945-3948
3945
Examination of Phosphoryl-Mimicking
Functionalities within a Macrocyclic
Grb2 SH2 Domain-Binding Platform
Sang-Uk Kang,† Zhen-Dan Shi,† Karen M. Worthy,‡
Lakshman K. Bindu,‡
Pathirage G. Dharmawardana,§ Sarah J. Choyke,§
Donald P. Bottaro,§ Robert J. Fisher,‡ and
Terrence R. Burke, Jr.*,†
Figure 1. Structures of pTyr and pTyr mimeitcs.
Laboratory of Medicinal Chemistry, Center for Cancer
Research, National Cancer Institute, National Institutes of
Health, Frederick, Maryland 21702, Protein Chemistry
Laboratory, SAICsFrederick, Frederick, Maryland 21702,
and Urology Oncologic Branch, Center for Cancer Research,
National Cancer Institute, National Institutes of Health,
Bethesda, Maryland 20892
Received January 20, 2005
Abstract: Reported herein are the design, synthesis, and
Grb2 SH2 domain-binding affinities of several phosphoryl-
mimicking groups displayed within the context of a conform-
ationally constrained macrocyclic platform. With use of surface
plasmon resonance techniques, single-digit nanomolar affini-
ties were exhibited by phosphonic acid and malonyl-containing
diacidic phosphoryl mimetics (for 4h and 4g, KD ) 1.47 and
3.62 nM, respectively). Analogues containing monoacidic phos-
phoryl mimetics provided affinities of KD ) 16-67 nM. Neutral
phosphoryl-mimicking groups did not show appreciable bind-
ing.
Figure 2. Structures of macrocyclic Grb2 SH2 domain-
binding antagonists.
phosphoryl-mimicking charge for certain conformation-
ally constrained ligands.
SH2 domain-binding antagonists have been developed
by a number of groups as potential alternatives to
kinase inhibitors.1-4 Despite their central roles in SH2
domain recognition processes, phosphotyrosyl residues
(pTyr, 1, Figure 1) are contraindicated as components
of such inhibitors because of their hydrolytic lability and
the poor cellular bioavailability of the doubly ionized
phosphoryl moiety. Replacement of the phosphoryl ester
oxygen with either -CH2- or -CF2- has yielded
hydrolytically stable analogues such as Pmp (2) and
F2Pmp (3), respectively. However the anionic charge of
these phosphonate-based structures still presents prob-
lematic issues of bioavailability. Therefore, phosphoryl
mimetics continue to be sought that retain high binding
affinity, yet exhibit reduced net anionic charge.5,6
To date, monoacidic phosphoryl mimetics have tended
to exhibit less affinity than diacidic mimetics.7,8 This
can be attributed in part to the presence of two highly
conserved positively charged Arg residues within the
SH2 domain pTyr binding pocket.9 If one role served
by pTyr binding is to promote capture of conformation-
ally flexible peptides from solution and thereby allow
secondary ordering of the peptide along the protein
surface, the importance of interactions within the pTyr-
binding pocket may be diminished for ligands whose
solution conformations are preordered for binding. This
could be reflected in a reduced requirement for diacidic
Recently, a series of macrocyclic peptide mimetics
typified by 4h have been reported that bind to Grb2 SH2
domains with extremely rapid on-rates (Figure 2).10,11
The high kon values may indicate that solution confor-
mations are preordered for binding. Therefore, macro-
cycles such as 4 may provide qualitatively different
platforms for investigating phosphoryl mimicking func-
tionality than afforded by conventional nonconstrained
peptides. Since the 5-methylindolyl-containing 4 has
only been reported bearing a diacidic phosphonic acid-
based phosphoryl mimetic (4h), the current study was
undertaken to use this platform to examine additional
diacidic (4g), monoacidic (4c-f), and neutral (4a,b)
phosphoryl mimetics.
All analogues were prepared as shown in Scheme 1.
A characteristic feature of macrocycle construction was
the use of protected â-vinyl-containing pTyr mimetics
10,12 which was coupled to the 5-methylindolyl-contain-
ing dipeptide 1111 to yield metathesis precursors 12. The
synthesis of 10g has been reported previously.13 Ring
closure of 12 using second-generation Grubbs’ catalyst
[((PCy3)(Im(Mes)2)RudCHPh) (11))]14,15 provided the
protected macrocycles 14, which were converted to the
final products 4 by treatment with acid. In the case of
N-oxalyl-containing 4c, the nitro-containing precursor
14b was first reduced to the corresponding amine using
freshly prepared Al-Hg amalgam, then reacted directly
with tert-butyl oxalyl chloride/iPr2NEt to give the pro-
tected amide 15c, which was converted to 4c by treat-
ment with TFA (Scheme 2). The carboxymethyl ana-
* To whom correspondence should be addressed. Phone:
(301) 846-5906. Fax: (301) 846-6033. E-mail: tburke@helix.nih.gov.
† Laboratory of Medicinal Chemistry, National Cancer Institute.
‡ SAICsFrederick.
§ Urology Oncologic Branch, National Cancer Institute.
10.1021/jm050059m CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/13/2005

3946 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Letters
Scheme 1
Scheme 2
ing under argon with hexamethyl disilazane under
argon. [Caution: vapors are flammable.] This was then
reacted with benzyl bromide, followed by a second
treatment with hexamethyl disilazane. Alkylation with
4-bromobenzyl bromide gave (4-bromobenzyl)benzylphos-
phinic acid, which was converted to 5e by esterification
with tert-butyl 2,2,2-trichloroacetimidate/BF3‚Et2.19,20
The corresponding tert-butyl (4-bromobenzyl)meth-
ylphosphinate 5f was prepared in a similar fashion by
sequential treatment of bis(trimethylsilyl)phosphonite
with 4-bromobenzyl bromide, then hexamethyl disila-
zane, then methyl iodide, and finally, tert-butyl 2,2,2-
trichloroacetimidate/BF3‚Et2.
logue 4d was obtained by lithium hydroxide mediated
decarboxylation of 4g.
Evaluation of binding affinity to recombinant Grb2
SH2 domain protein was accomplished using an enzyme
linked immunosorbent assay (ELISA) based competition
assay21,22 and a surface plasmon resonance (SPR) assay
that provided real-time kinetic data, including kon and
koff values (Table 1).23,24 On the basis of SPR data, the
highest affinities were exhibited by diacidic phosphoryl
mimetic-containing 4g and 4h,25,26 which showed en-
hanced binding on-rates (ka) that were more than 5-fold
greater than analogues having fewer acidic groups. The
highest affinity among the monoacidic phosphoryl mi-
metics was shown by the methylphosphinic acid 4f,
which had twice the affinity of the benzylphosphinic acid
4e or the carboxymethyl analogue 4d. The oxalylamido
analogue 4c showed the poorest affinity among the
monoacidic phosphoryl mimetics because of a dispro-
portionately rapid off-rate (kd). Compounds with un-
charged phosphoryl mimetics (4a and 4b) showed little
affinity (up to 1000 nM). ELISA-based IC50 values for
4d and 4g were in close agreement with SPR-derived
kD values. However, 4c,e,f gave lower IC50 values than
would be expected on the basis of SPR data, while 4h
gave a higher IC50 value. The reasons for these differ-
ences are not known. However, the ELISA IC50 values
are more dependent on experimental conditions than
Preparation of the key â-vinyl-containing pTyr mi-
metics 10 proceeded from the R,â-unsaturated oxazoli-
dinone-containing 716 through 8 by initial 1,4-addition
of vinylmagnesium bromide17 followed by conversion to
9 using stereoselective alkylation with R-bromo tert-
butyl acetate.12 With the exception of the nitro-contain-
ing 7b, construction of intermediates 7 was by Heck
reaction of 4-substituted phenylbromides (5) with known
(S)-N-acroyl-4-phenyl-2-oxazolidinone (6).17 The corre-
sponding Heck reaction of 4-bromonitrobenzene failed.
However the desired nitro-containing 7b can be ob-
tained by condensation of commercially available 4-nitro-
cinnamic acid with the Evans auxiliary (S)-4-phenyl-2-
oxazolidinone.
Starting aryl bromide 5a18 was obtained by reaction
of 4-bromophenol with tert-butyl 2,2,2-trichloroace-
timidate/BF3‚Et2O. For the synthesis of starting tert-
butyl phosphinate 5e, ammonium phosphinate was first
prepared by careful addition of 50% aqueous hypophos-
phorus acid to an equal-molar quantity of aqueous
ammonia at 0 °C. This was stirred for 1 h at room
temperature and then lyophilized to provide a hygro-
scopic white solid, which was dried over P2O5 under high
vacuum. The freshly prepared ammonium phosphinate
was converted to bis(trimethylsilyl)phosphonite by heat-

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 3947
Table 1. Grb2 SH2 Domain-Binding Results
a Obtained by SPR experiments as described in ref 23, where “n” refers to the number of surfaces fit to a global analysis of a simple
Langmuir binding isotherm and where the errors in ka and kd reflect the error of the global fit to these paramenters. KD and associated
SE values were calculated as described in ref 24, with the SE values also being the errors of the global analysis. b Obtained by
ELISA-based experiments as described in ref 22, wherein IC50 values ( the standard error of the mean (SE) were obtained as the mean
of “n” independent experiments performed with nine doses per curve with each dose done in quadruplicate. c Value as reported in ref 25.
the SPR-derived KD values, and the SPR data should
be used as the more reliable indicator of binding affinity.
domain-binding antagonist yet reported having a mono-
acidic phosphoryl mimetic.
In the interpretation of these results, care should be
taken in equating the number of acidic groups contained
in a phosphoryl mimetic with formal charge, since
complete ionization may not occur at a given pH. For
example, it is known that because of elevated pKa2
values, bis-acidic phosphonic acids are only partially
ionized at neutral pH, in contrast to phosphoryl groups,
which are di-ionized at pH 7.28 Differences in ionization
states between phosphoryl groups and phosphonic acids
have been used to at least partially explain reduction
in SH2 domain-binding affinity often observed when the
pTyr phosphate group is replaced with a phosphonic
acid moiety.29 Additionally, factors other than anionic
charge, such as hydrogen-bonding acceptor/donor prop-
erties, may contribute to variations in affinity exhibited
by different phosphoryl mimetics.
Significant effort has been directed toward reducing
phosphoryl-mimicking charge on SH2 domain binding
antagonists. For example, on the basis of a phosphate-
containing p56lck SH2 domain-binding dipeptide mi-
metic (KD ) 40 nM), a group from Boehringer Ingelheim
Pharmaceuticals obtained low micromolar KD values
using several monoacidic phosphoryl mimetics, includ-
ing carboxymethyl and oxalylamido-based analogues.8
In our own hands, open-chain analogues containing
monocarboxy-based pTyr mimetics have been shown to
exhibit Grb2 SH2 domain IC50 values in the low micro-
molar to submiromolar range.7 Novartis Corporation
has reported low micromolar to submicromolar affinity
constants for monoacidic phosphinic acid based pTyr
mimetics that include benzyl and methyl phosphinates
upon which analogues 4e and 4f of the current study
were predicated.27 In all of these prior reports, affinities
of inhibitors bearing monoacidic phosphoryl mimetics
were in the submicromolar range at best. Accordingly,
to our knowledge, 4f is the most potent Grb2 SH2
It is of note that although large losses of affinity are
observed for 4a and 4b, which lack any phosphoryl-
mimicking acidic groups, only modest losses of affinity
occurred for monoacidic phosphoryl mimetics (i.e., a

3948 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12
Letters
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10-fold reduction for 4f compared to 4h). This may be
attributed to the differential roles in pTyr recognition
played by the ArgâB5 residue, which is situated in the
rear of the binding pocket compared to the more
anteriorly located ArgRA2 residue. Thermodynamic
studies have shown that while the ArgâB5 residue is
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peptides, the ArgRA2 residue is less important.30 Data
presented in Table 1 highlight the need for high-affinity
binding of at least one anionic interaction with the
ArgâB5 residue. It is of note that cyclic Grb2 SH2
domain-binding peptides have been reported that are
devoid of phosphate-mimicking functionality at the Y0
position. However, these peptides contain acidic residues
at the Y-2 amino acid position that are hypothesized to
bond with the ArgâB5 residue, thereby highlighting the
importance of this critical interaction.31
In conclusion, by examination of a variety of phos-
phoryl mimicking functionalities within the context of
a conformationally constrained Grb2 SH2 domain-
binding platform, ligands have been identified that
contain monoacidic phosphoryl mimetics that exhibit
low nanomolar affinities. The current study may facili-
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Supporting Information Available: Detailed experi-
mental procedures for the preparation of final products 4a-g
and results from elemental analysis. This material is available
free of charge via the Internet at http://pubs.acs.org.
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JM050059M