A novel phosphotyrosine mimetic 4′-carboxymethyloxy-3′-phosphonophenylalanine (Cpp): Exploitation in the design of nonpeptide inhibitors of pp60Src SH2 domain

Article abstract of DOI:10.1016/S0960-894X(01)00446-2

The novel phosphotyrosine (pTyr) mimetic 4′-carboxymethyloxy-3′-phosphonophenylalanine (Cpp) has been designed and incorporated into a series of nonpeptide inhibitors of the SH2 domain of pp60^c-Src (Src) tyrosine kinase. A 2.2 A X-ray crystal structure of 1a bound to a mutant form of Lck SH2 domain provides insight regarding the structure-activity relationships and supports the design concept of this new pTyr mimetic.

Full text of DOI:10.1016/S0960-894X(01)00446-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2319–2323
A Novel Phosphotyrosine Mimetic 40-Carboxymethyloxy-
30-phosphonophenylalanine (Cpp): Exploitation in the Design of
Nonpeptide Inhibitors ofpp60 Src SH2 Domainy
Noriyuki Kawahata, Michael G. Yang, George P. Luke, William C. Shakespeare,
Raji Sundaramoorthi, Yihan Wang, Daniel Johnson, Taylor Merry, Shelia Violette,
Wei Guan, Catherine Bartlett, Jeremy Smith, Marcos Hatada, Xiaode Lu,
David C. Dalgarno, Charles J. Eyermann, Regine S. Bohacek and Tomi K. Sawyer*
ARIAD Pharmaceuticals, Inc., Cambridge, MA 02139-4234, USA
Received 30 November 2000; accepted 14 June 2001
Abstract—The novel phosphotyrosine (pTyr) mimetic 40-carboxymethyloxy-30-phosphonophenylalanine (Cpp) has been designed
and incorporated into a series of nonpeptide inhibitors of the SH2 domain of pp60c-Src (Src) tyrosine kinase. A 2.2 A X-ray crystal
structure of 1a bound to a mutant form of Lck SH2 domain provides insight regarding the structure–activity relationships and
supports the design concept of this new pTyr mimetic. # 2001 Elsevier Science Ltd. All rights reserved.
The ubiquitous nature of protein kinases and their sig-
nificant roles in signal transduction have prompted the
examination of these proteins as therapeutic targets. In
particular, inhibitors of pp60c-Src (Src) tyrosine kinase
have been identified as potential therapeutics for
osteoporosis1ꢀ3 and cancer.4ꢀ6 The kinase possesses a
noncatalytic domain referred to as the Src homology
domain-2 (SH2) that is characterized by a sequence-
specific phosphotyrosine (pTyr) binding region. The
SH2 domain is known to be essential for propagating
intracellular signals through pTyr recognition leading to
an assemblage of multiprotein complexes.7 Src SH2
inhibitors have been developed to block such signal
transduction pathways.8ꢀ10
effective nonpeptidic template to replace the Glu-Glu-
Ile substructure of the above phosphopeptide. Never-
theless, the pharmacological application of pTyr con-
taining analogues is limited by the metabolic liabilities
of the phosphate group. Extensive efforts have resulted
in a large number of nonhydrolyzable pTyr ana-
logues.11ꢀ17 Our recent efforts to obtain novel non-
hydrolyzable pTyr analogues have shown that the
incorporation of a phosphonate in the 30-position of
pTyr yielded high affinity ligands for Src SH2.18 In an
effort to further explore novel 30,40-difunctionalized
pTyr mimics, the design and synthesis of 40-carboxy-
methyloxy-30-phosphonophenylalanine (Cpp) modified
series of nonpeptide inhibitors of the Src SH2 domain
was undertaken.
The X-ray crystal structure of the pTyr containing pen-
tapeptide Ac-pTyr-Glu-Glu-Ile-Glu complexed with Src
SH2 has been solved.8 Such data have shown a distinct
pTyr binding region, hydrophobic pockets and potential
electrostatic and hydrogen bonding contacts and have
provided a starting point for the design of nonpeptidic
ligands for SH2 domains. Recently, Lunney et al.9 and
Sawyer10 have described a modified benzamide as an
The novel pTyr mimetic was assembled by coupling a
functionalized aryl iodide to an iodo-zinc alanine deri-
vative (Scheme 1). To prepare the aryl iodide, diethyl(2-
hydroxyphenyl)phosphonate (2) was obtained by treat-
ing diethyl(2-iodophenyl)phosphate with butyllithium.19
Regioselective iodination was successfully accomplished
using chloramine-T and sodium iodide.20 Protection of
the phenol as a benzyl ether gave 3, which was then used
for the coupling to Boc-protected iodo-zinc alanine
derivative.21 The benzyl group of 4 was removed by
hydrogenation and saponification gave the Boc-protected
*Corresponding author. Fax: +1-617-494-8144; e-mail: tomi.sawyer
@ariad.com
yPresented as a poster at the 16th American Peptide Symposium.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00446-2

2320
N. Kawahata et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2319–2323
30-diethylphosphonotyrosine derivative (5). The stereo-
chemical integrity of 5 was confirmed by coupling to
optically pure (R)- and (S)-1-phenethylamine using 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride (EDC) and 1-hydroxy-7-azabenzotriazole (HOAt).
A comparison of the diastereomers by NMR indicated
that this novel pTyr mimetic is obtained in high
enantiomeric purity (>97%). The carboxylic acid 5 was
then coupled to the benzamide templates9,10,22 using
EDC and HOAt. Exchange of the Boc group for an
acetyl, O-alkylation and deprotection using iodo-
trimethylsilane (TMSI) gave the desired final products
1a–c after purification by reversed-phase HPLC (RP-
HPLC) and lyophilization. A monoethylphosphonate
analogue 1h was obtained by quenching the TMSI-
mediated deprotection prior to full deprotection of the
phosphonate.
A des-acetylamino series was synthesized to explore the
effect of removing the acetylamino moiety thereby
eliminating a chiral center (Scheme 2). Additionally,
modifications to the carboxymethyl group were examined
within this series. Methyl-3-(4-hydroxyphenyl)propionate
was phosphorylated and an ortho-transfer of phosphate
was carried out in a manner similar to that described
above except without lithium halogen exchange to give
Scheme 1. Synthesis and incorporation of the Cpp residue: (a) Et2O3PCl, K2CO3, MeCN, 95%; (b) BuLi, THF, ꢀ78 ꢁC, 93%; (c) chloramine-T,
NaI, DMF, 73%; (d) BnBr, Cs2CO3, MeCN, 91%; (e) (PhCN)2PdCl2 (o-tol)3P, DMA, THF, 63%; (f) H2, Pd/C; (g) LiOH; (h) EDC, HOAt, NMM,
75%; (i) TFA, DCM then Ac2O, NMM, 83%; (j) BrCH2COOt-Bu, Cs2CO3, MeCN, 89%; (k) TMSI, MeCN.
Scheme 2. Synthesis of the des-acetylamino series: (a) Et2O3PCl, K2CO3, MeCN, 96%; (b) (i) LDA, THF, ꢀ78 ꢁC, 65%; (ii) LiOH, MeOH, THF,
90%; (c) EDC, HOAt, NMM, 82%; (d) BrCH2CO2t-Bu, Cs2CO3, 89%; (e) TMSI, MeCN; (f) Tf2NPh, TEA, 88%; (g) BrCH2CN, Cs2CO3, 95%;
(h) NaN3, NH4Cl.

N. Kawahata et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2319–2323
2321
Table 1. SH2 binding affinities (IC50, mM) of 1a–k in the fluorescence polarization23 assay
Compound
R1
R2
R3
R4
R5
SH2 binding (IC50
Yes
)
Src
Zap70
1a
1b
1c
1d
1e
1f
1g
1h
1ia
1j
Ac-HN–
Ac-HN–
Ac-HN–
H–
H–
H–
HO2C–CH2–
HO2C–CH2–
HO2C–CH2–
HO2C–CH2–
F3CO2S–
tetrazole–H2C–
H–
HO2C–CH2–
HO2C–CH2–
HO2C–CH2–
H2O3P–
H2O3P–
H2O3P–
H2O3P–
H2O3P–
H2O3P–
H2O3P–
H2O3P–
EtHO3P–
H2O3P–
H–
H–
H3C–
H–
H3C–
H3C–
H3C–
H3C–
H–
H3C–
H3C–
H–
H3C–
H3C–
H3C–
H3C–
H3C–
—
3.6
3.2
378
35.4
>500
>500
>500
7.3
>500
370
110
94.5
>500
303
>500
>500
>500
156
>500
>500
2.6
>500
>500
>500
>500
>500
>500
>500
>500
>500
—
H–
Ac-HN–
Ac-HN–
Ac-HN–
Ac-HN–
—
H–
H–
H3C–
H3C–
1k
H–
2.7
441
Binding affinity assessed by IC50 (mM) using the fluorescent analogue of Ac-pTyr-Glu-Glu-Ile-Glu as described in Lynch et al.23
aControl compound (Ac-Cpp-benzylamide).
8.19 Coupling to the gem-dimethyl benzamide template
followed by O-derivatization and deprotection provided
the des-acetylamino analogues 1d–g after RP-HPLC
purification and lyophilization.
and inhibit rabbit osteoclast-mediated resorption of
dentine. Compound 1a was observed to weakly inhibit
the resorption of dentine slices by rabbit osteoclasts
(Table 2). An increase in the inhibitory properties was
observed for 1h and it is believed that this is due to
enhanced cellular permeability of the compound.
The binding affinities of 1a–k to the Src SH2 domain
(Table 1) were determined using a fluorescence polar-
ization (FP) binding assay.23 The binding affinity of 1a
is comparable to the corresponding pTyr-containing
analogue 1k. Noteworthy, 1a is noticeably more selec-
tive than 1k for the Src SH2 versus the Yes SH2. Addi-
tionally, the dramatic increase in binding affinity of 1a
relative to 1j highlights the importance of the 30-phos-
phonate. Similar binding affinity and Src SH2 selectivity
were observed for 1b, which possesses the geminal
dimethyl benzamide template. The importance of the
methyl groups of the benzamide template is highlighted
by the diminished binding affinity of 1c relative to 1a
and 1b. It is also interesting to note that the monoethyl
phosphonate analogue 1h displays a decrease in binding
affinity of only 2-fold relative to 1a. The 10-fold loss
in binding affinity of 1d indicates the importance of
the acetylamino group. Replacement of the carboxy-
methyloxy group with a triflate, tetrazolemethylether, or
hydrogen (1e–g, respectively) abolishes activity indicat-
ing the importance of the carboxylic acid functionality.
Compound 1a was co-crystallized with a mutant Lck
SH2 (S164C substituted Lck119-225) that possesses
matched binding pocket residues with that of Src SH2
(Fig. 1).25 The 40-carboxymethylether moiety of the Cpp
is observed to occupy the phosphate binding pocket.
Relative to 1k, the phenyl ring of the Cpp residue of 1a
is shifted out of the aromatic binding pocket. This may
result from the increase in the number of atoms between
the aromatic ring and the carboxylic acid relative to the
phosphate of pTyr. This positioning of the aromatic
ring allows for the extension of the Lys bD6 side chain
(Fig. 2a and b) and places the side-chain e-amino group
The rabbit osteoclast pitting assay24 was performed to
assess the ability of the compound to penetrate the cells
Table 2. Cellular antiresportive activity in the rabbit osteoclast pitting
assay24
Compound
% Inhibitiona
1a
1h
1k
+
+++
++
aWhere ‘+’=0–35% inhibition at 100 mM, ‘++’=36–70% inhibition
at 100 mM and ‘+++’=71–100% inhibition at 100 mM.
Figure 1. X-ray crystallographic structure of 1a bound to a mutant of
Lck (119-225) SH2 (Lck119-225 S164C) (2.2 A).25

2322
N. Kawahata et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2319–2323
Figure 2. Overlapped representations of 1a and 1k bound to a mutant of Lck (119-225) SH2 (Lck119-225 S164C) (X-ray crystallography, 2.2 A):
(a) (left) displays the ‘side’ view and (b) (right) the ‘top’ view relative to the plane of the Cpp aromatic ring.
within hydrogen bonding distance to the 30-phospho-
nak, D.; Sternbach, D.; Mehrotra, M.; Peel, M.; Shampine, L.;
Davis, R.; Robbins, J.; Patel, I. R.; Kassel, D.; Burkhart, W.;
nate of the Cpp residue. This may account for the sub-
Moyer, M.; Bradshaw, T.; Berman, J. J. Biol. Chem. 1994,
stantial increase in binding affinity of 1a over 1j and
269, 31711.
supports the design concept of this new pTyr mimetic.
9. Lunney, E. A.; Para, K. S.; Rubin, J. R.; Humblet, C.;
The relative positions of the benzamide template remain
similar between 1a and 1k.
Fergus, J. H.; Marks, J. S.; Sawyer, T. K. J. Am. Chem. Soc.
1997, 119, 12471.
10. Sawyer, T. K. Biopolymers 1998, 47, 243.
In conclusion, the novel pTyr mimetic Cpp has been
designed and synthetically incorporated into nonpeptide
inhibitors of the Src SH2 domain. X-ray structural
studies suggest that both the 40-carboxymethylether and
the 30-phosphonate groups of the Cpp residue provide
effective intermolecular contacts with the Src SH2 pTyr
binding pocket. Specifically, the 30-phosphonate of the
Cpp residue dramatically improves binding affinity (cf.
1a vs 1j). The binding affinity and cellular activity of 1h
highlight the effectiveness of the monoethyl ester analo-
gue of the Cpp residue in this lead series. These results
provide the basis for further studies to exploit the con-
cept of multifunctional group modification of pTyr in
the design of SH2 inhibitors.
11. Burke, T. R., Jr.; Smyth, M. S.; Otaka, A.; Nomizu, M.;
Roller, P. P.; Wolf, G.; Case, R.; Shoelson, S. E. Biochemistry
1994, 33, 6490.
12. Burke, T. R., Jr.; Ye, B.; Akamatsu, M.; Ford, H., Jr.;
Yan, X.; Kole, H. K.; Wolf, G.; Shoelson, S. E.; Roller, P. P.
J. Med. Chem. 1996, 39, 1021.
13. Stankovic, C. J.; Surendran, N.; Lunney, E.; Plummer,
M. S.; Para, K.; Shahipour, A.; Fergus, J. H.; Marks, J. S.;
Herrera, R.; Hubbell, S. E.; Humblet, C.; Saltiel, A. R.; Stew-
art, B. H.; Sawyer, T. K. Bioorg. Med. Chem. Lett. 1997, 7,
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14. Yao, Z.-J.; King, C. R.; Cao, T.; Kelley, J.; Milne, G.;
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