Solid-phase assembly and in situ screening of protein tyrosine phosphatase inhibitors

Article abstract of DOI:10.1021/ol8006875

(Chemical Equation Presented) A highly efficient solid-phase strategy for assembly of small molecule inhibitors against protein tyrosine phosphatase 1B (PTP1B) is described. The method is highlighted by its simplicity and product purity. A 70-member combinatorial library of analogues of a known PTP1B inhibitor has been synthesized, which upon direct in situ screening revealed a potent inhibitor (K_i = 7.0 μM) against PTP1B.

Full text of DOI:10.1021/ol8006875

ORGANIC
LETTERS
2008
Vol. 10, No. 11
2295-2298
Solid-Phase Assembly and In Situ
Screening of Protein Tyrosine
Phosphatase Inhibitors
Rajavel Srinivasan, Lay Pheng Tan, Hao Wu, and Shao Q. Yao*
Departments of Chemistry and Biological Sciences, NUS MedChem Program of the
Office of Life Sciences, National UniVersity of Singapore, Singapore 117543
chmyaosq@nus.edu.sg
Received March 30, 2008
ABSTRACT
A highly efficient solid-phase strategy for assembly of small molecule inhibitors against protein tyrosine phosphatase 1B (PTP1B) is described.
The method is highlighted by its simplicity and product purity. A 70-member combinatorial library of analogues of a known PTP1B inhibitor
has been synthesized, which upon direct in situ screening revealed a potent inhibitor (Ki ) 7.0 µM) against PTP1B.
High-throughput enzymology occupies a pivotal role in
modern drug discovery programs.1 One of the main chal-
lenges in this field is the development of high-throughput
(HT) amenable chemical reactions that allow rapid synthesis
of diverse chemical libraries for the interrogation of different
classes of enzymes.2 One such reaction is the Cu(I)-catalyzed,
1,3-dipolar cycloaddition between an azide and an alkyne
fragment, also known as “click chemistry”, which was
pioneered by Sharpless and Meldal.3 Another class of
reactions possessing similar qualities is the amide bond-
forming reaction between an amine and a carboxylic acid
using suitable activating/coupling reagents. Numerous re-
search groups have recently used this reaction for solution-
phase, rapid assembly of small molecule inhibitors against
a variety of enzymes including HIV proteases,4a,b ꢀ-aryl
sulfotransferase,4c R-fucosidases,4d,e and SARS-3CL pro-
tease.4f The reaction is highly efficient, often generating the
desired products in nearly quantitative yields, thus allowing
in situ biological screening to be carried out directly in some
cases, even in the presence of excessive starting materials,
reagents, or byproduct (Figure 1a; Pathway A). The ef-
fectiveness of this method, however, had recently been
challenged, as it was discovered that unexpected byproduct
may give rise to false results.5 For example, Wong et al.
reported that an intermediate benzotriazole ester of the amide
(1) (a) Golebiowski, A.; Klopfenstein, S. R.; Portlock, D. E. Curr. Opin.
Chem. Biol. 2001, 5, 273–284. (b) Golebiowski, A.; Klopfenstein, S. R.;
Portlock, D. E. Curr. Opin. Chem. Biol. 2003, 7, 308–325.
(4) (a) Brik, A.; Lin, Y.-C.; Elder, J.; Wong, C.-H. Chem. Biol. 2002,
9, 891–896. (b) Lee, S.-G.; Chmielewski, J. Chem. Biol. 2006, 13, 421–
426. (c) Best, M. D.; Brik, A.; Chapman, E.; Lee, L. V.; Wong, C.-H.
ChemBioChem 2004, 5, 811–819. (d) Wu, C.-Y.; Chang, C.-F.; Chen, J. S.-
Y.; Lee, S.-T.; Wong, C.-H.; Lin, C.-H. Angew. Chem., Int. Ed. 2003, 42,
4661–4664. (e) Chang, C.-F.; Ho, C.-W.; Wu, C.-Y.; Chao, T.-A.; Wong,
C.-H.; Lin, C.-H. Chem. Biol. 2004, 11, 1301–1306. (f) Wu, C.-Y.; King,
K.-Y.; Kuo, C.-J.; Fang, J.-M.; Wu, Y.-T.; Ho, M.-Y.; Liao, C.-L.; Shie,
(2) (a) Hu, Y.; Uttamchandani, M.; Yao, S. Q. Comb. Chem. High-
Throughput Screen. 2006, 9, 203–212. (b) Sun, H.; Chattopadhaya, S.;
Wang, J.; Yao, S. Q. Anal. Bioanal. Chem. 2006, 386, 416–426.
(3) (a) Sharpless, K. B.; Manetsch, R. Exp. Opin. Drug DiscoVery 2006,
1, 525–538. (b) Kolb, H. C.; Sharpless, K. B. Drug DiscoVery Today 2003,
8, 1128–1137. (c) Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67,
3057–3062. (d) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless,
K. B. Angew. Chem., Int. Ed. 2002, 41, 2596–2599.
J.-J.; Liang, P.-H.; Wong, C.-H. Chem. Biol. 2006, 13, 261–268
.
10.1021/ol8006875 CCC: $40.75
Published on Web 05/06/2008
2008 American Chemical Society

importantly, the inhibitor was cell-permeable and showed
good cellular activity in PTP1B-expressing COS-7 cells.7b
Inspired by this work, we recently developed our own version
of a cell-permeable bidentate PTP1B inhibitor using “click”
chemistry (Figure 1b, center), which shows comparable
inhibitory activity (IC50 ) 4.7 µM against PTP1B). In the
current work, we have successfully developed our second-
generation, cell-permeable PTP1B inhibitors by using a solid-
phase amide-forming reaction to rapidly link the core and
the peripheral groups together. Unlike traditional solution-
phase approaches,4 our method delivers pure bidentate
inhibitors at the end of synthesis and therefore is suitable
for direct in situ screening. By screening these inhibitors
against PTP1B, we have uncovered a candidate molecule
which possesses comparable inhibition against PTP1B
(Figure 1b, right; Ki ) 7.0 µM).
In our strategy, we used the commercially available
4-formyl-3-methoxyphenoxy (FMP) resin to capture various
amine fragments via reductive amination, followed by
attaching the isoxazole warhead (Scheme 1). Key advantages
of our method include the following: (i) it is a traceless
approach allowing the use of exact same sets of starting
material as in solution-phase synthesis; (ii) it is solid-phase,
enabling a large library to be constructed efficiently; (iii) it
is robust, giving high-quality products which in most cases
are spectroscopically pure enough to be used directly for
biological screening. The synthesis of the acid-containing
warheads, A and B, started from the commercially available
4-hydroxyacetophenone 1, which underwent benzylation to
give 2 (83% yield). Subsequently, condensation between 2
and dimethyloxalate in the presence of NaOMe, followed
by cyclization of the resulting product, gave the isoxazole
carboxylic methylester, 3, in modest yield (42% in two
steps).8 Conversion of 3 to 5 was carried out first by base
hydrolysis, followed by t-Bu ester formation (85% in two
steps). Next, the benzyl ether on 5 was deprotected by H2
hydrogenolysis (in Pd/C) giving 6, followed by O-alkylation
with two different linkers to afford 7a and 7b (93 and 63%
yield, respectively). Subsequent deprotection of the benzyl
esters gave the two acid-containing warheads, A and B,
respectively. To start the assembly on solid-phase, two sets
of 35 aromatic amines were treated with FMP resin in the
presence of Na(OAc)3BH/2% glacial acetic acid in DCE to
give the corresponding secondary aromatic amines 10.
Reductive amination proceeded smoothly with a variety of
aromatic amines bearing different substituents including
-OH, -OR, -SR, -F, -Cl, -OCF3, -CO2R, and -R.
Benzyl, napthyl, and anthracenyl amines too underwent
reductive amination smoothly. Subsequent amide bond-
forming reaction between the resin-bound secondary aromatic
amines and the acid warheads (A and B) was found to be
highly challenging and required extensive optimizations. A
variety of coupling reagents including HATU, PyBop,
HBTU, EDC, and DIC were attempted, but none gave the
desired products in satisfactory yield and purity. Fortunately,
by in situ conversion of A and B into their corresponding
acid chlorides, 8a and 8b, with 1 equiv of oxalyl chloride
(with DIEA in DCM), we were able to successfully couple
Figure 1. (a) Two strategies using amide bond-forming reaction.
(b) Various cell-permeable PTP1B inhibitors. The bidentate inhibi-
tors contain a core (in red) and a peripheral group (in blue).
bond-forming reaction was found to be responsible for the
inhibitory activity toward SARS-3CL proteases.4f To avoid
such potential complications, we aim to develop solid-phase,
amide bond-forming reactions using the same sets of starting
materials (Figure 1a; Pathway B). Herein, we report a
traceless solid-phase approach for rapid assembly of protein
tyrosine phosphatase (PTP) inhibitors using amide bond-
forming reaction (Figure 1a; Pathway B).
Protein tyrosine phosphatases (PTPs) are a main class of
signaling enzymes.6a PTP1B is the prototype of all PTPs
and has been identified as a key player in major human
diseases such as diabetes, obesity, and cancer.6b–d The elegant
work of Zhang et al. that indicates PTP1B possesses a unique
secondary binding site next to its primary phosphotyrosine
binding pocket6b has enabled the development of potent and
specific bidentate PTP1B inhibitors.7,8 One of the most potent
and cell-permeable PTP1B inhibitors identified to date, as
shown in Figure 1b (left), was developed by scientists from
Abbott Laboratories.7b This inhibitor contains a core iso-
xazole group which serves as a cell-permeable bioisostere
of phosphotyrosine and a hydrophobic aromatic group that
binds to the secondary site in PTP1B. It was reasonably
potent (Ki ) 5.7 µM) and selective toward PTP1B over its
closest analogue, TCPTP (>30-fold in selectivity). More
(5) Brik, A.; Wu, C.-Y.; Wong, C.-H. Org. Biomol. Chem. 2006, 4,
1446–1457.
(6) (a) Hunter, T. Cell 2000, 100, 113–127. (b) Zhang, Z.-Y. Curr. Opin.
Chem. Biol. 2001, 5, 416–423. (c) Johnson, T. O.; Ermolieff, J.; Jirousek,
M. R. Nat. ReV. Drug DiscoVery 2002, 1, 696–709. (d) Bialy, L.; Waldmann,
H. Angew. Chem., Int. Ed. 2005, 44, 3814–3839.
(7) (a) Guo, X.-L.; Shen, K.; Wang, F.; Lawrence, D. S.; Zhang, Z.-Y.
J. Biol. Chem. 2002, 277, 41014–41022. (b) Liu, G.; et al. J. Med. Chem.
2003, 46, 4232–4235. (c) Liu, G.; et al. J. Med. Chem. 2003, 46, 3437–
3440
(8) Srinivasan, R.; Uttamchandani, M.; Yao, S. Q. Org. Lett. 2006, 8,
713–716
.
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Org. Lett., Vol. 10, No. 11, 2008

Scheme 1. Traceless Solid-Phase Synthesis of Bidendate PTP1B Inhibitors
them, giving the resin-bound inhibitors 11 with excellent
purity (>95% in most cases). Finally, cleavage of the
products from the resin using an optimized TFA cleavage
cocktail (TFA/DCM/water ) 10:9:1) gave a total of 70
bidentate PTP1B inhibitors, A1-A35 and B1-B35. Most
crude products were characterized by LC-MS/NMR and used
for direct in situ screening for PTP1B inhibition.
Next, the inhibitory activity of the bidentate inhibitors was
determined using a standard fluorescence microplate assay
as previously reported.8 First, an inhibitor fingerprint of the
70-member library against PTP1B was obtained, from which
six potent hits (A21, A26, A34, A35, B7, and B25) were
identified (Figure 2). Detailed inhibition studies were then
carried out to obtain the IC50/Ki of these compounds against
PTP1B and TCPTP, and results are summarized in Table 1.
The best inhibitor against PTP1B was found to be A26, with
IC50 and Ki values of 10.3 and 7.0 µM, respectively.
Significantly, it also showed a 10-fold selectivity over TCPTP.
It is interesting to note that A26, as well as the other good
inhibitors of PTP1B, as shown in Figure 2, contains a bulky
aromatic group. This coincides reasonably with previously
known cell-permeable inhibitors of PTP1B developed using
other strategies, such as fragment-based or click chemistry
approaches (Figure 1b),7,8 thus further validating our solid-
phase amide-forming methodology as a feasible strategy for
future discovery of other enzyme inhibitors.
In conclusion, we have developed a solid-phase amide
bond-forming approach for rapid assembly of bidentate
Table 1. Inhibition of the Six Selected Inhibitors
IC50 (Ki) in µM
enzyme
PTP1B
A21
A26
A34
A35
B7
B25
19.6
(9.5)
174.5
10.3
(7.0)
105.4
23.0
(10.3)
124.4
35.9
60.5
28.7
Figure 2. Six candidate hits identified against PTP1B.
TCPTP
103.5
205.1
489.1
Org. Lett., Vol. 10, No. 11, 2008
2297

PTP1B inhibitors. Products generated were of consistently
high quality and required no further purification. We identi-
fied a compound whose inhibition against PTP1B was
comparable to that of other known PTP1B inhibitors.7,8 We
are currently extending the strategy to the synthesis of other
enzyme inhibitors.
Science, Technology, and Research (A*Star) of Singapore.
The authors thank Prof. Harry Charbonneau (Purdue Uni-
versity) for the TCPTP construct.
Supporting Information Available: Experimental details
and characterization of compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. Funding support was provided by
National University of Singapore (NUS) and the Agency for
OL8006875
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Org. Lett., Vol. 10, No. 11, 2008