Synthesis and characterization of a novel class of protein tyrosine phosphatase inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00019-6

Nonpeptidyl aryloxymethylphosphonates were prepared and evaluated as protein tyrosine phosphatase inhibitors. The results suggest that aryloxymethylphosphonates are effective nonhydrolyzable phosphotyrosine surrogates and provide further insight into the molecular mechanisms by which phosphate mimics inhibit phosphatase function. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00019-6

Bioorganic & Medicinal Chemistry Letters 10 (2000) 457±460
Synthesis and Characterization of a Novel Class of Protein
Tyrosine Phosphatase Inhibitors
Omar A. Ibrahimi, a,y Li Wu, b Kang Zhao a,{,* and Zhong-Yin Zhang b,*
aDepartment of Chemistry, New York University, 29 Washington Place, New York, 10003, USA
bDepartment of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
Received 23 November 1999; accepted 28 December 1999
AbstractÐNonpeptidyl aryloxymethylphosphonates were prepared and evaluated as protein tyrosine phosphatase inhibitors. The
results suggest that aryloxymethylphosphonates are e€ective nonhydrolyzable phosphotyrosine surrogates and provide further
insight into the molecular mechanisms by which phosphate mimics inhibit phosphatase function. # 2000 Elsevier Science Ltd. All
rights reserved.
Protein tyrosine phosphatases (PTPs), in conjunction
with protein tyrosine kinases (PTKs), regulate a myriad
of critical signaling events including proliferation, dif-
ferentiation, motility, development and metabolism.1
While PTKs have been extensively studied, the char-
acterization of the equally important PTPs has only
recently been undertaken.2 The precise roles of PTPs in
many of these signaling pathways are beginning to be
elucidated. For example, PTP1B has been implicated as
a negative regulator in the insulin-stimulated signaling
pathway.3,4 In addition, mice lacking PTP1B activity
exhibit increased sensitivity towards insulin and are
resistant to obesity.5 The design of speci®c nonpeptidyl
PTP1B inhibitors may provide a powerful tool in
understanding the in vivo role of this protein in signal
transduction. This inhibitor class may also prove thera-
peutically bene®cial in the treatment of Type II diabetes
mellitus.
phenylalanine (F2Pmp, 1b, X=CH2CH(NH2)CO2H).8±10
An interesting observation is that peptides bearing
F2Pmp are over 1000 times more potent PTP inhibitors
than the analogous peptides containing Pmp.9,10 This
has been attributed to a direct interaction between the
¯uorine atoms and PTP active site residues rather than
the lowering of the pKa value of the phosphonic acid.10
Indeed, speci®c interactions between one of the ¯uorine
atoms in di¯uoronaphthylmethyl phosphonic acid and
PTP1B have been observed.11
Biochemical studies indicate that the phenolic oxygen in
the substrate receives a proton from the general acid
(Asp181 in PTP1B) in the transition state.12 Moreover,
X-ray crystal structure of PTP1B/C215S in complex
with a pTyr-containing substrate reveals a hydrogen
bond between the phenolic oxygen and Asp181.13 It is
possible that the enhanced anity of F2Pmp over Pmp
arises from the ability of the ¯uorine atoms in F2Pmp to
interact with the PTP active site residues in a fashion
analogous to that involving the phenolic oxygen and
side chains in the active site of PTPs, which is lost in the
Pmp-containing molecules. As part of an ongoing e€ort
to identify e€ective and speci®c PTP inhibitors, we have
synthesized a class of compounds, aryloxymethyl-
phosphonates 3. We reasoned that the hydrogen bond-
ing interactions of PTP active site residues with the
phenolic oxygen in substrates 1c would be similarly
maintained in analogues 3. These phosphonate deriva-
tives allow us to test the rationale that the phenolic
oxygen plays a central role in enzyme±substrate inter-
actions and to explore other structural features that are
important for high anity binding to PTPs.
An e€ective strategy towards the design of PTP inhibi-
tors is the replacement of the labile phosphotyrosine
(pTyr) with a nonhydrolyzable analogue. The most
commonly used phosphorus-based pTyr analogues
are phosphonomethyl phenylalanine (Pmp, 1a, X=
CH2CH(NH2)CO2H)6,7 and phosphonodi¯uoromethyl
*Corresponding authors. Tel.: +1-718-430-4288; fax: +1-718-430-
8922; e-mail: zyzhang@aecom.yu.edu; Tel.: +1-858-812-1534; fax:
+1-858-812-1584; e-mail: kang.zhao@nifg.novartis.com
yPresent address: NYU School of Medicine, 550 1st Avenue, New
York 10016, USA.
{Present address: Novartis Institute of Functional Genomics, 3115
Merry®eld Row, San Diego, CA 92121, USA.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00019-6

458
O. A. Ibrahimi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 457±460
Synthesis
Biochemical Characterization
Compounds of type 3 were prepared via facile two-pot
syntheses from readily available phenol derivatives
(Scheme 1). Compounds of type 2 were treated with the
corresponding equivalent of NaH (60% in oil; 1.1 equiv
for 2a, 2d±f; 2.2 equiv for 2b±c) in DMSO for 30 min to
give dark red solutions. Diethyl p-tolylsulfonyloxy-
methyl phosphonate (1.1 equiv for 2a±b, 2d±f; 2.2 equiv
for 2c), prepared according to the literature procedure,14
was then added and the mixture was stirred overnight.15
Alternatively, diethyl p-chlorophenylsulfonyloxymethyl
phosphonate can be used to give the desired products
with slightly increased yields.14 Cleavage of the phos-
phonate diesters followed a modi®ed procedure.16 After
addition of excess trimethylsilyl bromide (TMSBr) to
the diester intermediates, the mixture was stirred for 1 h.
TMSBr was then evaporated under reduced pressure
and followed by the stepwise addition of EtOAc and
deionized water to the reaction mixture. The two layers
were subsequently mixed followed by removal of the
organic layer. The EtOAc wash was repeated two addi-
tional times. Final products 3 were then concentrated by
the removal of water under reduced pressure. No fur-
ther puri®cation was needed.17 Isolated yields for all
four steps ranged from 71 to 87%.
The e€ect of the aryloxymethylphosphonates on the
PTP1B and the dual speci®city phosphatase VHR
catalyzed p-nitrophenyl phosphate hydrolysis reaction
was examined at 30 ꢀC and pH 7.0.18 The aryl-
oxymethylphosphonates were not hydrolyzed by the
PTPs and inhibited the PTP reaction reversibly and
competitively with respect to the substrate (data not
shown). The Ki values are summarized in Table 1. It is
interesting to note that the Ki value for 3a is similar
to that of di¯uorobenzyl phosphonate (1b, X=H).19
In addition, the Ki value of 3a is also similar to the
Km of phenyl phosphate.20 Thus insertion of a methyl-
ene group between the phenolic oxygen and the phos-
phonate group does not disrupt the hydrogen bonding
interactions between the phenolic oxygen and the
enzyme active site. This also suggests that the additional
spacer of the methlylene group in 3a has no adverse
e€ects on the ability of the phosphate group and the
aromatic moiety to interact with the enzyme. Thus,
aryloxymethylphosphonates, like F2Pmp, are e€ective
nonhydrolyzable pTyr mimics that when attached to an
appropriate peptide template should yield high anity
PTP inhibitors.
Scheme 1. Synthesis of aryloxymethylphosphonates.

O. A. Ibrahimi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 457±460
459
Table 1. Inhibition constants of PTP1B and VHR with aryloxy-
methylphosphonates
thereby enhancing both inhibitor anity and speci®city.
Because of their relative ease of preparation without loss
of inhibitor potency, the aryloxymethylphosphonates
provide an excellent alternative to di¯uoromethyl-
enephosphonates as nonhydrozable pTyr surrogates.
Moreover, the ability of aryloxymethylphosphonates to
serve as nonhydrolyzable pTyr mimetics should make
them very useful in studying the role of SH2 domains, in
addition to PTPs, in cell signaling. Currently, we are
exploring methods to incorporate this moiety into
appropriate peptide templates.
PTP1B
Ki (mM)
VHR
Ki (mM)
Compound
3a
3b
3c
3d
3e
3f
3.3‹0.2
1.1‹0.1
0.047‹0.005
1.3‹0.2
1.3‹0.1
26‹5
2.6‹0.4
1.3‹0.2
4.8‹0.5
5.7‹0.5
0.57‹0.08
0.41‹0.09
It is also possible that potent and selective non-peptidyl
low molecular weight inhibitors of PTP1B can be
obtained when a properly functionalized phosphate
surrogate is attached to an appropriate aromatic fra-
mework, which e€ectively occupies the pTyr pocket and
interacts with the immediate surroundings beyond the
catalytic site, thereby enhancing both inhibitor anity
and speci®city. As shown in Table 1, addition of a pro-
panoic acid (3b) or an aromatic ring(s) to 3a (3d±f)
increases the anity for both PTP1B and VHR.
Moreover, it appears that both PTP1B and VHR do
not discriminate between 3d and 3e, consistent with an
early study with di¯uronaphthalenylmethyl phosphonic
acids.21 These results indicate that there is an inherent
plasticity in PTP active sites to accommodate com-
pounds signi®cantly larger than pTyr. Remarkably,
compound 3c bearing two aryloxymethylphosphonates
exhibited a Ki value that was 70-fold more potent than
that of the mono derivative (3a). Compound 3c was also
nearly 30-fold more selective toward PTP1B than VHR.
The high anity of 3c for PTP1B may be the result of
additional binding interactions between the second ary-
loxymethylphosphonate and the second non-catalytic
aryl phosphate binding site in PTP1B.22 Alternatively,
the second aryloxymethylphosphonate in 3c may also
form salt bridges with Arg47 in PTP1B as the acidic
amino acids at P-1 and P-2 do.13 The fact that the Ki
for 3c is comparable to those of bis-di¯urobenzyl
phosphonate methane23 and other bis-aryldi¯uoro-
phosphonates24 is consistent with the conclusion that
aryloxymethylphosphonates are e€ective nonhydrolyz-
able phosphotyrosine surrogates.
Acknowledgements
We thank Mr. Nduka M. Amankular for his excellent
assistance in the synthesis of compounds 3a±3f. We also
thank Ms. Saba Al-Zabin for her assistance in the pre-
paration of this manuscript. This work was supported in
part by a Research Award from the American Diabetes
Association (Z.-Y. Z.). O.I. is a recipient of the MSTP
fellowship administered by the NIH. K. Z. acknowledges
the donors of the Petroleum Research Fund, adminis-
tered by the American Chemical Society, and NSF
Faculty Early Career Development Program for their
®nancial support.
References and Notes
1. Hunter, T. Cell. 1995, 80, 225.
2. Zhang, Z. Y. CRC Crit. Rev. Biochem. Mol. Biol. 1998, 33, 1.
3. Ahmad, F.; Li, P. M.; Meyerovitch, J.; Goldstein, B. J. J.
Biol. Chem. 1995, 270, 20503.
4. Kenner, K. A.; Anyanwu, E.; Olefsky, J. M.; Kusari, J. J.
Biol. Chem. 1996, 271, 19810.
5. Elchelby, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.;
Collins, S.; Lee Loy, A.; Normandin, D.; Cheng, A.; Himms-
Hagen, J.; Chan, C.-C.; Ramachandran, C.; Gresser, M. J.;
Tremblay, M. L.; Kennedy, B. P. Science 1999, 283, 1544.
6. Zhang, Z.-Y.; Maclean, D.; McNamara, D. J.; Dobrusin, E.
M.; Sawyer, T. K.; Dixon, J. E. Biochemistry 1994, 33, 2285.
7. Chatterjee, S.; Goldstein, B. J.; Csermely, P.; Shoelson, S. E.
In Peptides (Proceedings of the Twelfth American Peptide
Symposium); Leiden, Netherlands, 1992, pp 553.
8. Burke, Jr., T. R.; Smyth, M.; Nomizu, M.; Otaka, A.;
Roller, P. P. J. Org. Chem. 1993, 58, 1336.
9. Burke, Jr., T. R.; Kole, H. K.; Roller, P. P. Biochem. Bio-
phys. Res. Commun. 1994, 204, 129.
10. Chen, L.; Wu, L.; Otaka, A.; Smyth, M. S.; Roller, P. P.;
Burke, T. R.; den Hertog, J.; Zhang, Z.-Y. Biochem. Biophys.
Res. Commun. 1995, 216, 976.
11. Burke, Jr., T. R.; Ye, B.; Yan, X.; Wang, S.; Jia, Z.; Chen,
L.; Zhang, Z.-Y.; Barford, D. Biochemistry 1996, 35, 15989.
12. Hengge, A. C.; Sowa, G.; Wu, L.; Zhang, Z.-Y. Biochem-
istry 1995, 34, 13982.
13. Jia, Z.; Barford, D.; Flint, A. J.; Tonks, N. K. Science
1995, 268, 1754.
14. Cornforth, S. J.; Wilson, J. R. H. J. Chem. Soc. Perkin.
Trans. 1 1994, 1897.
15. Merlo, V.; Roberts, S. M.; Storer, R.; Bethell, R. C. J.
Chem. Soc. Perkin. Trans. 1 1994, 1477.
16. Landry, D. W.; Zhao, K.; Yang, G. X. Q.; Glickman, M.;
Georgiadis, T. M. Science 1993, 259, 1899.
17. All intermediates and ®nal compounds had satisfactory
1H, 13C NMR spectra.
Conclusion
In summary, we have described the synthesis and char-
acterization of a novel class of PTP inhibitors, aryloxy-
methylphosphonates, which act as nonhydrolyzable
pTyr surrogates. The ability of the aryloxymethlypho-
sphonates to inhibit PTPs as well as the di¯uoro-
methylenephenyl phosphonates implies that the active
site of PTPs may interact with the ether oxygen in aryl-
oxymethylphosphonates in a manner analogous to that
involving the phenolic oxygen of substrates. We have
also demonstrated that potent and selective small mole-
cule PTP inhibitors can be obtained with properly
functionalized aryloxymethylphosphonates. These mole-
cules e€ectively occupy the pTyr pocket and interact with
the immediate surroundings beyond the catalytic site,

460
O. A. Ibrahimi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 457±460
18. The PTP activity was assayed at 30 ꢀC in a reaction mix-
ture (0.2 mL) containing appropriate concentrations of pNPP
as substrate. The bu€er used was pH 7.0, 50 mM 3,3-di-
methylglutarate, 1 mM EDTA. The ionic strength of the
solution was kept at 0.15 M using NaCl. The reaction was
initiated by addition of enzyme and quenched after 2±3 min by
addition of 1 mL of 1 N NaOH. The nonenzymatic hydrolysis
of the substrate was corrected by measuring the control with-
out the addition of enzyme. The amount of product p-nitro-
phenol was determined from the absorbance at 405 nm using a
pattern were evaluated using a direct curve-®tting program
KINETASYST (IntelliKinetics, State College, PA).
19. Yao, Z.-J.; Ye, B.; Wu, X.-W.; Wang, S.; Wu, L.; Zhang,
Z.-Y.; Burke, T. R. Bioorg. Med. Chem. 1998, 6, 1799.
20. Zhang, Z.-Y. J. Biol. Chem. 1995, 270, 11199.
21. Kole, K. H.; Smyth, M. S.; Russ, P. L.; Burke, T. R. Bio-
chem. J. 1995, 311, 1025.
22. Puius, Y. A.; Zhao, Y.; Sullivan, M.; Lawerence, D. S.;
Almo, S. C.; Zhang, Z.-Y. Proc. Natl. Acad. Sci. USA 1997,
94, 13420.
23. Taylor, S. D.; Kotoris, C. C.; Dinaut, A. N.; Want, Q.;
Ramachandran, C.; Huang, Z. Bioorg. Med. Chem. 1998, 6,
1457.
24. Taing, M.; Keng, Y.-F.; Shen, K.; Wu, L.; Lawerence, D.
S.; Zhang, Z.-Y. Biochemistry 1999, 38, 3793.
1
molar extinction coecient of 18,000 M cm 1. Inhibition
constants for the PTP inhibitors was determined in the fol-
lowing manner. The initial rate at eight di€erent pNPP con-
centrations (0.2±5 Km) was measured at three di€erent ®xed
inhibitor concentrations. The inhibition constant and inhibition