Peptidic α-ketocarboxylic acids and sulfonamides as inhibitors of protein tyrosine phosphatases

Article abstract of DOI:10.1021/jo034113n

One common approach tbr designing protein tyrosine phosphatase (PTPase) inhibitors is to incorporate a nonhydrolyzable phosphotyrosine (pTyr) mimic into a peptide substrate for PTPases. This report describes the synthesis of three such nonhydrolyzable pTy

Full text of DOI:10.1021/jo034113n

epidermal growth factor receptor Ac-DADEpYL-NH2
(EGFR988-993).9 A number of investigators have shown
that the Ac-DADE-X-L-NH2 peptide, where X denotes a
non-hydrolyzable pTyr analogue, is a useful template for
designing PTPase inhibitors.10 One of the most effective
pTyr mimics that has been inserted into this peptide is
difluoromethylphosphonophenylalanine (F2Pmp).11 The
difluoromethylenephosphonic acid (DFMP) group has
also been used as the basis for a number of other
nonpeptidic PTPase inhibitors.12 Despite the potency of
the DFMP group, the efficacy of phosphonates is ham-
pered by their inability to penetrate into cells. This low
permeability is likely to be due to their high charge at
physiological pH.13
To improve bioavailability, efforts have been directed
toward the development of less-charged, non-phosphorus-
containing inhibitors. These efforts have resulted in pTyr
mimics that include L-O-(2-malonyl)tyrosine (OMT),14 its
fluorinated counterpart 4′-O-[2-(2-fluoromalonyl)]-L-ty-
rosine (FOMT),15 and 2-(oxalylamino)benzoic acids.16
Recently, we reported that R-ketoacids such as phenyl-
glyoxylic acid (1) can serve as a new class of pTyr
mimic.17,18 While several pTyr mimics that contain a
single carboxylic acid group have been found to be
moderately good ligands of src homology (SH2) domains,
few have shown good affinity for PTPases.19
During our investigations of new phosphate analogues
that can be used for PTPase inhibition, we also became
interested in sulfonamides. Sulfonamides are a common
functional group in medicinal chemistry, and they have
served in a variety of applications such as carbonic an-
hydrase inhibitors20 and bacteriostatic agents.21 Com-
pounds such as benzylsulfonamide 2 may possess a si-
milar geometry to that of aryl phosphates and aryl-
methylenephosphonates. As a result, investigation of
sulfonamides may to lead to noncharged PTPase inhibi-
tors that have improved bioavailability. Here, we report
the synthesis of pTyr analogues that incorporate an
R-ketoacid or sulfonamide group that are then used in
the synthesis of Ac-DADE-X-L-NH2-based inhibitors
Peptidic r-Ketocarboxylic Acids and
Sulfonamides as Inhibitors of Protein
Tyrosine Phosphatases
Yen Ting Chen,† Jian Xie,† and Christopher T. Seto*
Department of Chemistry, Brown University, 324 Brook St.
Box H, Providence, Rhode Island 02912
christopher_seto@brown.edu
Received January 28, 2003
Abstract: One common approach for designing protein
tyrosine phosphatase (PTPase) inhibitors is to incorporate
a nonhydrolyzable phosphotyrosine (pTyr) mimic into a
peptide substrate for PTPases. This report describes the
synthesis of three such nonhydrolyzable pTyr mimics that
contain R-ketoacid, R-hydroxyacid, and methylenesulfona-
mide functional groups in place of the phosphate. These pTyr
mimics were incorporated into the peptide sequence Ac-Asp-
Ala-Asp-Glu-X-Leu-NH2, where X is the pTyr mimic, and
analyzed for activity against the Yersinia PTPase and
PTP1B.
There is considerable interest in the development of
inhibitors for protein tyrosine phosphatases (PTPases)
because PTPases are associated with a variety of human
diseases.1,2 For example, overexpression of the human
PTPases PTP1B, LAR, and PTPR leads to abnormal
insulin resistance that is often associated with type II
diabetes.3 As a consequence, several studies have shown
that inhibitors of PTP1B are potential therapeutic agents
for the treatment of both diabetes and obesity.4,5 In a
second example, the Yersinia pestis bacterium, the
causative agent of the bubonic plague, employs the
PTPase YopH that is essential for its virulence.6 Thus,
inhibitors of YopH are potential antibacterial agents that
can be targeted for this organism.
The active site of PTPases selectively binds phospho-
tyrosine (pTyr),7 but the amino acids that flank a pTyr
residue are also crucial for recognition of substrates by
PTPases.8,9 One peptide that is a good substrate for a
variety of PTPases is the sequence derived from the
(10) Burke, T. R., Jr.; Yao, Z.-J.; Liu, D.-G.; Voigt, J.; Gao, Y.
Biopolymers 2001, 60, 32.
(11) Burke, T. R., Jr.; Kole, H. K.; Roller, P. P. Biochem. Biophys.
Res. Commun. 1994, 204, 129.
† These authors contributed equally to this work.
* Corresponding author.
(1) Tonks, N. K.; Neel, B. G. Curr. Opin. Cell Biol. 2001, 5, 416.
(2) Burke, T. R., Jr.; Zhang, Z.-Y. Biopolymers 1998, 47, 225.
(3) Goldstein, B. J.; Li, P.-M.; Ding, W.; Ahmad, F.; Zhang, W.-R.
Vitam. Horm. 1998, 54, 67.
(4) Elchebly, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.; Collins,
S.; Loy, A. L.; 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.
(12) Ripka, W. C. Annu. Rep. Med. Chem. 2000, 35, 231.
(13) Kole, H. K.; Smyth, M. S.; Russ, D. L.; Burke, T. R., Jr. Biochem.
J. 1995, 311, 1025.
(14) Kole, H. K.; Akamatsu, M.; Ye, B.; Yan, X.; Barford, D.; Roller,
P. P.; Burke, T. R., Jr. Biochem. Biophys. Res. Commun. 1995, 209,
817.
(15) 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.
(16) Andersen, H. S.; Iversen, L. F.; Jeppesen, C. B.; Branner, S.;
Norris, K.; Rasmussen, H. B.; Moller, K. B.; Moller, N. P. H. J. Biol.
Chem. 2000, 275, 7101.
(17) Chen, Y. T.; Onaran, M. B.; Doss, C. J.; Seto, C. T. Bioorg. Med.
Chem. Lett. 2001, 11, 1935.
(5) Klaman, L. D.; Boss, O.; Peroni, O. D.; Kim, J. K.; Martino, J.
L.; Zabolotny, J. M.; Moghal, N.; Lubkin, M.; Kim, Y.-B.; Sharpe, A.
H.; Stricker-Krongrad, A.; Shulman, G. I.; Neel, B. G.; Kahn, B. B.
Mol. Cell Biol. 2000, 20, 5479.
(6) Bliska, J. B.; Guan, K. L.; Dixon, J. E.; Falkow, S. Proc. Natl.
Acad. Sci. U.S.A. 1991, 88, 1187.
(18) Chen, Y. T.; Seto, C. T. J. Med. Chem. 2002, 45, 3946.
(19) Gao, Y.; Wu, L.; Luo, J. H.; Guo, R.; Yang, D.; Zhang, Z.-Y.;
Burke, T. R., Jr. Bioorg. Med. Chem. Lett. 2000, 10, 923.
(20) Ilies, M.; Supuran, C. T.; Scozzafava, A.; Casini, A.; Mincione,
F.; Menabuoni, L.; Caproiu, M. T.; Maganu, M.; Banciu, M. D. Bioorg.
Med. Chem. 2000, 8, 2145.
(7) Jia, Z.; Barford, D.; Flint, A. J.; Tonks, N. K. Science 1995, 268,
1754.
(8) Vetter, S. W.; Keng, Y.-F.; Lawrence, D. S.; Zhang, Z.-Y. J. Biol.
Chem. 2000, 275, 2265.
(9) Zhang, Z. T.; Maclean, D.; McNamara, D. J.; Sawyer, T. K.;
Dixon, J. E. Biochemistry 1994, 33, 2285.
(21) Bock, L.; Miller, G. H.; Schaper, K.-J.; Seydel, J. K. J. Med.
Chem. 1974, 17, 23.
10.1021/jo034113n CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/22/2003
J. Org. Chem. 2003, 68, 4123-4125
4123

SCHEME 1a
a Reagent: (a) NBS, benzoyl peroxide, CCl4, reflux, 61%; (b) compound 7, LiHMDS, THF, HMPA, -78 °C to room temperature, 45%;
(c) H2 (1 atm), Pd(OH)2/C, THF, MeOH, 91%; (d) Fmoc-OSu, NaHCO3, 1:1 H2O:dioxane, 79%; (e) MnO2, CH2Cl2, 45%.
SCHEME 2a
a Reagents: (a) Na2SO3, acetone, H2O, 59%; (b) PCl5, CH2Cl2;
(c) NH4OH, CH2Cl2, 53% (two steps); (d) 6 N HCl; propylene oxide,
88%; (e) Fmoc-OSu, NaHCO3, 1:1 H2O:dioxane, 85%.
Marseigne and Roques,24 was allowed to react with so-
dium sulfite to give the corresponding sodium sulfonate
13. Sulfonate 13 was treated with phosphorus pentachlo-
(compounds 3 and 4). The activity of these peptide-based
inhibitors is evaluated against two PTPases, the Yersinia
ride to give the sulfonyl chloride, which was then con-
PTPase and PTP1B.
verted to sulfonamide 14 by reaction with ammonium
Compound 11 (Scheme 1) is a protected form of the
hydroxide. Compound 14 was heated at reflux in 6 N HCl
R-ketoacid-containing pTyr analogue that is suitable for
to effect the hydrolysis of the ester and N-acetyl groups,
use in Fmoc-based solid-phase peptide synthesis. We
and to decarboxylate the resulting dicarboxylic acid.
began the synthesis of compound 11 by bromination of
R-ketoester 5 with N-bromosuccinimide and benzoyl
Workup of the reaction with propylene oxide in ethanol
gave free amino acid 15. Finally, the amine was protected
peroxide to give benzyl bromide 6. Compound 5 was
with an Fmoc group to give compound 16 as a racemic
prepared according to the procedure of Nimitz and
mixture.
Mosher.22 To synthesize the amino acid in stereochemi-
Hexapeptides 3 and 4 were synthesized on Rink amide
cally pure form, we employed the diphenyloxazinone
chiral auxiliary 7 that has been developed by Williams
and Im.23 Treatment of compound 7 with LiHMDS gave
the corresponding glycine-based enolate. Alkylation of
this enolate with benzyl bromide 6 afforded oxazinone 8
with the desired stereochemistry. Subsequent hydrogen-
ation of 8 with palladium hydroxide on carbon removed
the chiral auxiliary to generate free amino acid 9. During
this reaction, the ketone was also reduced to give the
R-hydroxy ester as a mixture of two diastereomers. After
the amine was protected with an Fmoc group, the ketone
was regenerated by oxidation with MnO2 to give com-
pound 11.
resin25 with use of manual solid-phase methods with
Fmoc chemistry. Resin cleavage and side-chain depro-
tections were performed with a solution of 95% trifluo-
roacetic acid, 2.5% H2O, and 2.5% triisopropylsilane.
During the cleavage and deprotection of peptide 3, we
observed that the ketoacid moiety was reduced to the
corresponding R-hydroxyacid to give peptide 17 as a 1:1
mixture of two diastereomers. This type of ionic reduction
of ketones to alcohols mediated by trifluoroacetic acid and
triisopropylsilane has been studied by a number of inves-
tigators.26 When the cleavage and deprotection was
performed in the absence of triisopropylsilane, the reac-
tion yielded the desired ketoacid-containing peptide 3 in
diastereomerically pure form. Since we prepared the
sulfonamide-based amino acid 16 as a racemic mixture,
The synthesis of the racemic sulfonamide-containing
amino acid 16 is shown in Scheme 2. Compound 12,
which was prepared according to the procedure of
(24) Marseigne, I.; Roques, B. P. J. Org. Chem. 1988, 53, 3621.
(25) Rink, H. Tetrahedron Lett. 1987, 28, 3787.
(26) Mayr, H.; Dogan, B. Tetrahedron Lett. 1997, 38, 1013 and
references therein.
(22) Nimitz, J. S.; Mosher, M. S. J. Org. Chem. 1981, 46, 211.
(23) Williams, R. M.; Im, M.-N. J. Am. Chem. Soc. 1991, 113, 9276.
4124 J. Org. Chem., Vol. 68, No. 10, 2003

TABLE 1. Inhibition of Phosphatases by Peptide
Inhibitors 3, 4, and 17
have better activity than other Ac-DADE-X-L-NH2-
basedinhibitors that contain pTyr analogues with a single
carboxylic acid.19 Of the pTyr analogues that are monoan-
ionic, only the difluoromethylenesulfonates that have
been developed by Taylor and co-workers show higher
activity.28
IC50 (µM)
compd
Yersinia PTPase
PTP1B
2
370 ( 60
150 ( 15
370 ( 50
2100 ( 400
180 ( 20
NDc
3
270 ( 30
>2500
NDc
4aa
4ba
17b
Burke and co-workers have suggested a rationale for
why some carboxylic acid-based peptide inhibitors may
have low affinity for phosphatases.19 While the peptide
chain provides favorable binding interactions with resi-
dues outside of the catalytic site of PTPases, it limits the
geometrical freedom and depth of insertion of the aryl
phosphate analogue into the active site. The optimal
positioning of aryl R-ketoacids in the active site may be
somewhat different than that of aryl phosphates. There-
fore, by restricting the binding geometry of the R-ketoacid
segment in peptide 3 into the same geometry as is
observed for aryl phosphates, the R-ketoacid moiety may
not be able to make optimal contacts with the active site
residues. This results in relatively weak binding com-
pared to compounds such as difluoromethylenephospho-
nates that closely mimic the geometry of the natural aryl
phosphate substrates. In conclusion, we have synthesized
three new nonhydrolyzable pTyr mimics, and investi-
gated their activity within the context of peptide-based
inhibitors. These compounds are not as active as many
dianionic pTyr analogues, but they are more active than
most of the monoanionic analogues that have been
reported to date.
NDc
a 4a and 4b are two different diastereomers of peptide 4 that
differ in stereochemistry of the sulfonamide-containing amino acid.
b Mixture of two diastereomers that differ in the stereochemistry
of the hydroxyl group of the R-hydroxyacid moiety. c Not deter-
mined.
peptide 4 was obtained as a mixture of two diastereo-
mers. These diastereomers were separated by reverse-
phase HPLC and assayed separately.
Assays were performed with p-nitrophenyl phosphate
(p-NPP) as the substrate at 25 °C in pH 5.5 acetate buffer
(100 mM) that contained 100 mM NaCl and 10% DMSO
to increase the solubility of the inhibitors. As shown in
Table 1, benzylsulfonamide (2) has an IC50 value against
the Yersinia PTPase of 370 µM. We then investigated the
potential of the arylmethylenesulfonamide within the
context of a peptide-based inhibitor. However, one dias-
tereomer of this peptide (4a) has an IC50 value against
the Yersinia PTPase of 370 µM. Thus, we did not observe
any improvement in activity over the simple unsubsti-
tuted benzylsulfonamide inhibitor 2. Compound 4a is a
millimolar inhibitor of PTP1B, while its diastereomer 4b
is a millimolar inhibitor against the Yersinia enzyme.
In previous work, we have found that aryl R-ketocar-
boxylic acids comprise a new class of inhibitors for protein
tyrosine phosphatases.17,18 For example, phenylglyoxylic
acid 1 is a 2.7 mM inhibitor of the Yersinia PTPase.17
This value can be improved upon significantly by adding
electron-donating substituents to the phenyl ring, or by
exchanging the phenyl ring for more electron-rich aro-
matic groups. In light of these studies, we synthesized
peptide 3 that incorporates an aryl R-ketoacid as a mimic
of phosphotyrosine. During the synthesis of peptide 3,
we found that cleavage of the peptide from the solid
support with a combination of trifluoroacetic acid and
triisopropylsilane produced the corresponding R-hydroxy-
carboxylic acid-containing peptide 17. This failed reaction
was somewhat fortuitous since it provided us with an
additional compound for evaluation. Our earlier studies
demonstrated that R-hydroxyacids retain some activity
against PTPases.17
The R-hydroxyacid-containing peptide 17 is a reason-
able inhibitor of the Yersinia PTPase with an IC50 value
of 180 µM, while the R-ketoacid-containing peptide 3 has
similar activity with an IC50 value of 150 µM (Table 1).
Thus, peptide 3 represents a 15-fold improvement over
the simple phenylglyoxylic acid (1) starting point. These
compounds are not as potent as other peptide-based
inhibitors that incorporate doubly charged phosphoty-
rosine mimics such as fluoro-O-malonyl tyrosine and
difluorophosphonomethyl phenylalanine.27 However, they
Experimental Section
PTPase Assays. The phosphatase activities of the Yersinia
PTPase and PTP1B (purchased from Calbiochem) were assayed
with use of p-NPP as the substrate and the reaction progress
was monitored by UV spectroscopy. Initial rates were deter-
mined by monitoring the hydrolysis of p-NPP at 420 nm, from
10 to 120 s after mixing. Assay solutions contained 1 mM EDTA,
100 mM NaCl, 100 mM acetate at pH 5.5, and 10% DMSO.
Substrate concentrations were 2.5 and 1.2 mM for the Yersinia
PTPase and PTP1B, respectively. IC50 values were calculated
by using a Dixon analysis. Data analysis was performed with
the commercial graphing package Grafit (Erithacus Software
Ltd.). The Km values under these conditions were found to be
2.5 mM for the Yersinia PTPase and 0.62 mM for PTP1B. The
Km value for PTP1B in the absence of DMSO and in acetate
buffer at pH 5.5 is reported to be 0.75 mM.29
Acknowledgment. This research was supported by
the NIH NIGMS (Grant GM57327).
Supporting Information Available: Experimental pro-
cedures and characterization for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JO034113N
(28) Leung, C.; Grzyb, J.; Lee, J.; Meyer, N.; Hum, G.; Jia, C.; Liu,
S.; Taylor, S. D. Bioorg. Med. Chem. 2002, 10, 2309.
(29) Iversen, L. F.; Andersen, H. S.; Branner, S.; Mortensen, S. B.;
Peters, G. H.; Norris, K.; Olsen, O. H.; Jeppesen, C. B.; Lundt, B. F.;
Ripka, W.; Moller, K. B.; Moller, N. P. H. J. Biol. Chem. 2000, 275,
10300.
(27) Groves, M. R.; Yao, Z.-J.; Roller, P. P.; Burke, T. R., Jr.; Barford,
D. Biochemistry 1998, 37, 17773.
J. Org. Chem, Vol. 68, No. 10, 2003 4125