A two stage click-based library of protein tyrosine phosphatase inhibitors

Article abstract of DOI:10.1016/j.bmc.2006.09.036

Protein tyrosine phosphatases (PTPs) are important regulators of signal transduction pathways. Potent and selective PTP inhibitors are useful for probing these pathways and also may serve as drugs for the treatment of a variety of diseases including type

Full text of DOI:10.1016/j.bmc.2006.09.036

Bioorganic & Medicinal Chemistry 15 (2007) 458–473
A two stage click-based library of protein tyrosine
phosphatase inhibitors
Jian Xie and Christopher T. Seto*
Department of Chemistry, Brown University, 324 Brook St. Box H, Providence, RI 02912, USA
Received 31 August 2006; revised 18 September 2006; accepted 20 September 2006
Available online 12 October 2006
Abstract—Protein tyrosine phosphatases (PTPs) are important regulators of signal transduction pathways. Potent and selective PTP
inhibitors are useful for probing these pathways and also may serve as drugs for the treatment of a variety of diseases including type
2 diabetes and infection by the bacterium Yersinia pestis. In this report Cu(I)-catalyzed ‘click’ cycloaddition reactions between azides
and alkynes were employed to generate two sequential libraries of PTP inhibitors. In the first round library methyl 4-azidobenzoyl-
formate was reacted with 56 mono- and diynes. After hydrolysis of the methyl esters, the resulting a-ketocarboxylic acids were
assayed in crude form against the Yersinia PTP and PTP1B. Four compounds were selected for further evaluation, and one com-
pound was chosen as the lead for generation of the second round library. This lead compound was modified by conversion of an
alcohol into an azide group, and the resulting azide was reacted with the same 56 mono- and diynes that were used in the first gen-
eration library. After screening the crude inhibitors against the Yersinia PTP and PTP1B, four compounds were selected and eval-
uated in pure form against the Yersinia PTP, PTP1B, TCPTP, LAR, and CD45. The best bis(a-ketocarboxylic acid) inhibitor 34 had
an IC50 value of 550 nM against the Yersinia PTP and an IC50 value of 710 nM against TCPTP. The most potent inhibitor contain-
ing a single a-ketocarboxylic acid group 32 had IC50 values of 2.1, 5.7, and 2.6 lM against the Yersinia PTP, PTP1B, and TCPTP,
respectively.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
a variety of diseases including type II diabetes, cancer,
dysfunctions of the immune system, and infection by
pathogenic bacteria. For example, PTP1B, the first iden-
tified PTP,4 is a negative regulator of insulin-induced
glucose metabolism.5 This PTP dephosphorylates the
insulin receptor and thus shuts down the insulin signal-
ing cascade. CD45 and TCPTP are two phosphatases
that are involved in cytokine signaling and they play
important roles in the immune response.6–9 Finally, a
variety of pathogenic bacteria including Yersinia pestis
and Salmonella typhimurium utilize highly active PTPs
as part of their virulence mechanisms. The Y. pestis bac-
terium injects its phosphatase into host cells using a type
III secretion system, where it targets several focal adhe-
sion proteins.
1.1. PTP inhibitors
Protein tyrosine kinases (PTKs) and protein tyrosine
phosphatases (PTPs) control intracellular signal trans-
duction pathways by regulating the phosphorylation
state of tyrosine residues in proteins.1 While the struc-
tures and biological functions of PTKs have been stud-
ied in detail, until recently PTPs have received less
scrutiny. PTPs are characterized by a conserved active
site sequence (H/V)C(X)5R(S/T) called the PTP signa-
ture motif.2 Based on this unique sequence, a recent sur-
vey of the human genome revealed the existence of 107
PTP genes and 81 active protein tyrosine phosphatases.3
Since PTPs regulate signal transduction, defective or
inappropriate PTP activities play an important role in
PTPs are attractive targets for drug development since
4% of the ‘druggable genome’ is thought to be phospha-
tases.10 Potent and selective PTP inhibitors should be
useful for probing signal transduction pathways and
also as drugs for the treatment of PTP-related diseases.
As a result, there is increasing effort to develop PTP
inhibitors, especially since a PTP1B knockout mouse
validated this enzyme as a target for the treatment of
Keywords: Library; Protein tyrosine phosphatase; Inhibitor; Click
Chemistry.
*
Corresponding author. Tel.: +1 401 863 3587; fax: +1 401 863
9368; e-mail: Christopher_Seto@brown.edu
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.036

J. Xie, C. T. Seto / Bioorg. Med. Chem. 15 (2007) 458–473
459
type II diabetes and perhaps obesity.11,12 A number of
O
non-hydrolyzable phosphate mimics have been devel-
oped as PTP inhibitors including aryl a-ketocarboxylic
acids,13–17 2-(oxalylamino)benzoic acids,18 difluorom-
ethylenesulfonates,19,20 squaric acids,21 difluoromethyl
enephosphonates,22 and O-malonyltyrosine.23 However,
since all PTPs share the same signature motif in the cat-
alytic domain, it is usually necessary to extend inhibitors
outside of the catalytic site to gain selectivity and im-
prove potency. Incorporating pTyr mimics into peptide
templates is one method to improve the activity of
inhibitors.15,19,22,24
O
N
OH
N
N
R
1
Figure 1. General structure of the inhibitors.
PTP1B contains a second non-catalytic pTyr binding
site that is formed mainly by Arg24, Arg254, Gly259,
Gln262, and Met258.25 These residues, except for
Arg254 and Gln262, are not as conserved in many PTPs
compared to the residues that make up the active site.
This secondary site can be exploited to design inhibitors
that simultaneously target both the active site and the
secondary binding site. Such inhibitors often show
greatly improved potency and selectivity.26,27 Other
peripheral residues, such as Arg47, Asp48, Lys41,
Phe52, and Ala27, also can be targeted by extended
inhibitors.14,16,17,28–31 Our research group has recently
developed two libraries of extended bidentate PTP
inhibitors that are derived from a-ketocarboxylic
acids.16,17 These libraries, which were designed to target
the active site and also peripheral residues, led to the
identification of several potent and selective inhibitors.
4-azidobenzoylformate was reacted with 56 mono- and
diynes in the presence of Cu(I). Hydrolysis of the methyl
esters gave a-ketoacid inhibitors, which were screened in
crude form against the Yersinia PTP and PTP1B. We
selected a lead inhibitor and modified its structure to
incorporate an azide. This compound served as the
starting point for the second generation library, which
was prepared by reacting it with the 56 mono- and diy-
nes. This two-stage approach yielded several inhibitors
with IC50 values in the low micromolar range against
the Yersinia PTP and PTP1B.
2. Chemistry
The first generation library required a molecule such as
compound 4 (Scheme 1) that incorporated an azide
group to participate in the click reaction, and an a-keto-
carboxylic acid that functions as a phosphate mimic and
is designed to bind in the active site of PTPs. Compound
2 was prepared from 40-acetamidoacetophenone using
the procedure of Domagala and Haskell.43 Reaction of
the aromatic amine with sodium nitrite and trifluoroace-
tic acid (TFA) gave the corresponding aryl diazonium
salt, which was further converted to aryl azide 3 with
sodium azide.44 Azide 3 does not react with alkynes such
as propiolic acid under a variety of Cu(I)-catalyzed reac-
tion conditions.32,45–48 This low reactivity may be
caused by complexation of Cu(I) with the a-ketoacid
(Fig. 2). Similar complexes have been documented in
the literature.49–54 To avoid this problem we esterified
the a-ketoacid to give compound 4.
1.2. Combinatorial libraries using click chemistry
The Cu(I)-catalyzed [3+2] azide–alkyne cycloaddition
reaction, which Sharpless has developed into the most
successful example of click chemistry, offers an expedi-
ent method to connect two components together with
high yield and purity.32–35 This reaction has become a
powerful method for generating combinatorial libraries,
and it has found increasing applications in bioconjuga-
tion,36–38 lead discovery, and lead optimization.39–41
Wong used this reaction to prepare a library of 85 hu-
man a-1,3-fucosyltransferase inhibitors, and they identi-
fied a nanomolar inhibitor that displays excellent
selectivity.39 Sharpless and coworkers used the active
site of acetylcholine esterase to bind 49 azide/alkyne
pairs and to catalyze the cycloaddition reaction in the
absence of Cu(I).40 Interestingly, only one pair was
aligned correctly in the active site to allow the cycload-
dition reaction to occur. For this pair, the enzyme served
as a reaction vessel that catalyzed the formation of an
extremely potent inhibitor with a dissociation constant
of 77 fM.
The first generation library was synthesized as outlined
in Scheme 2. Azide 4 was reacted in a 1:1 ratio with
50 alkynes and in a 2:1 ratio with six diynes (Fig. 3) to
O
O
O
O
O
O
Yao recently synthesized and screened a click-based li-
brary of PTP inhibitors that yielded a compound with
an IC50 value of 4.7 lM against PTP1B.42 Our current
studies are designed to investigate binding interactions
between inhibitors and residues outside of the PTP cat-
alytic site. To accomplish this goal, we have constructed
a library of a-ketoacid-based inhibitors using a two-
stage approach. Figure 1 shows the general structure
of the inhibitors. In the first generation library methyl
OH
OH
OMe
c, d
a, b
NH
N
2
N
3
3
3
2
4
Scheme 1. Reagents and conditions: (a) NaNO2, TFA, 0 ꢁC; (b) NaN3,
Et2O; (c) SOCl2, C6H6, reflux; (d) MeOH.

460
J. Xie, C. T. Seto / Bioorg. Med. Chem. 15 (2007) 458–473
I
give triazoles 5. Cu(I) was generated in situ using a com-
bination of CuSO4 and sodium ascorbate in the presence
of the ligand tris(benzyltriazolylmethyl)amine
(TBTA).32 Alkynes A1–A54 were obtained from com-
mercial sources, while alkynes A55 and A56 were pre-
pared by coupling of aminoalcohols 8 and 9 with
propiolic acid (Scheme 3).55 Aminoalcohol 8 was ob-
tained by reduction of racemic amino acid 7.56 After
the cycloaddition reactions were complete, the methyl
L
Cu
n-2
O
O
O
N
3
Figure 2. Possible complex between a-ketoacid 3 and Cu(I).
O
O
O
O
N
O
OMe
OH
O
OMe
a
b
N
N
N
N
3
N
N
R
R
4
5
6
Scheme 2. Reagents: (a) alkyne, CuSO4Æ5H2O, sodium ascorbate, EtOH, t-BuOH, H2O, TBTA; (b) 1 N NaOH, then 1 N HCl.
OR
O
n
OH
O
A8 R=H
A10
A1 n=3
A2 n=5
A6
A7
A9 R=CH
A3
3
A4
A5
O
O
H N
2
O
O
O
O
NH
HN
2
N
HN
A14
OH
A16
OH
O
A11
A12
A13
A15
A17
A18
RO
n
H N
2
A19 n=0
A20 n=1
A21 n=2
N
O
H N
2
A25 R=H
A26 R=CH
A22
A23
A27
A24
A32
A28
3
F C
3
N
F C
3
F
CF
Br
3
Br
A34
A35
O
A33
A30
A31
A29
OH
OH
CF
O
S
O
O
3
N
4
N
A40
O
A41
A37
A38
A36
A39
X
Ph
N
N
N
S
N
OH
O
O
Ph
A48
A42
A47
A46
A45
A43 X=H
A44 X=Cl
NH
OH
A56
O
n
O
O
A52 n=3
A53 n=4
A54 n=5
OH
A51
N
N
A49
A50
H
H
A55
Figure 3. Alkynes used to synthesize the library.

J. Xie, C. T. Seto / Bioorg. Med. Chem. 15 (2007) 458–473
461
a
b
O
OH
OH
OH
H N
2
N
H
H N
2
O
7
8
A55
b
NH
OH
NH
OH
O
H N
2
N
H
9
A56
Scheme 3. Reagents and condition: (a) LiAlH4, THF, reflux; (b) propiolic acid, DCC, CH2Cl2.
ester groups were saponified followed by neutralization
of the reaction mixtures to give the crude inhibitors 6.
The reactions were then diluted with DMSO to give a
10 mM stock solution of inhibitor based on the concen-
trations of starting materials used in the cycloaddition
reactions, and with the assumption that the reactions
proceeded to completion.
inhibitors were assayed against the two PTPs to obtain
IC50 values.
The enzyme assays demonstrated that, from the first
generation library, compound 13 emerged as the
most potent mono-a-ketoacid inhibitor. As a result,
we chose this compound as the initial lead for devel-
opment of the second generation library. For this li-
brary, we needed to prepare compound 22 (Scheme
4), which incorporates all of the structural elements
of inhibitor 13, but also has an additional azide
group attached to the 40-position of the biphenyl
unit. The synthesis began with compound 18, which
was prepared using the procedure of Makarov and
co-workers.57 Sonogashira coupling converted the
aryl iodide into the TMS-protected alkyne 19, which
was deprotected to give alkyne 20.58 The Cu(I)-cata-
lyzed cycloaddition reaction between azide 4 and al-
kyne 20 proceeded smoothly to give triazole 21.
However, sodium nitrite oxidation of the amino
group in 21, followed by substitution of the resulting
diazonium salt with sodium azide, gave a product,
possibly compound 22, which was insoluble in all
standard organic solvents including DMSO. Since
this compound was insoluble, it could not be used
as a viable building block for the second generation
library.
The crude inhibitors were screened at 100 lM against
the Yersinia PTP and PTP1B. Four compounds (11,
13, 15, and 17, corresponding to alkynes A16, A46,
A50, and A56) (Fig. 4) were selected for resynthesis on
a larger scale and complete characterization. The pure
O
O
O
OR
O
OR
12 R = CH
13 R = H
3
10 R = CH
11 R = H
3
N
N
N
N
N
N
OH
O
The inhibition studies indicated that compound 17
(Fig. 4) had similar activity against the Yersinia PTP
as compound 13, although it was less active against
PTP1B. This observation prompted us to choose 17 as
an alternate lead compound for synthesis of the second
generation library. We were unable to convert com-
pound 16 directly to azide 26 (Scheme 5) using a variety
of reaction conditions.59–67 Instead, we opted to incor-
porate the azide early on in the synthesis. Boc-tryptoph-
anol 23 was converted to the corresponding azide 24
using a Mitsunobu reaction.68 After removal of the
Boc group, amine 25 was coupled with compound 10
using N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
(EDC) and 4-dimethylaminopyridine (DMAP) to give
compound 26.
O
O
14 R = CH
15 R = H
O
N
3
O
N
OR
OR
16 R = CH
17 R = H
3
O
RO
O
N
H
N
N
N
N
N
N
NH
N
O
OH
Figure 4. Four inhibitors selected for further evaluation, and their
methyl ester precursors.

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J. Xie, C. T. Seto / Bioorg. Med. Chem. 15 (2007) 458–473
TMS
I
a
b
H N
2
H N
2
H N
2
18
19
20
O
O
OMe
OMe
O
O
O
O
OMe
c
d, e
N
N
N
insoluble
N
N
N
N
3
4
NH
N
3
2
21
22
Scheme 4. Reagents and conditions: (a) ethynyltrimethylsilane, Pd(PPh3)2Cl2, CuI, TEA; (b) 10% NaOH, MeOH; (c) 20, CuSO4Æ5H2O, sodium
ascorbate, EtOH, t-BuOH, H2O, TBTA; (d) NaNO2, TFA, 0 ꢁC; (e) NaN3, diethyl ether.
a
NH
NH
OH
b
NH
N
N
3
3
BocHN
BocHN
H N
2
23
24
25
O
O
O
O
OMe
OMe
c
H
N
N
N
N
N
N
N
OH
NH
10
26
O
O
N
3
Scheme 5. Reagents: (a) PPh3, diisopropyl azodicarboxylate (DIAD), ZnN6, toluene; (b) TFA, CH2Cl2; (c) 25, EDC, DMAP, CH2Cl2.
For the second generation library, azide 26 was reacted
with the 56 mono- and diynes shown in Figure 3 using
conditions that were similar to those used for the first
generation library (Scheme 6). After saponification of
the methyl esters, the crude inhibitors 28 were screened
against the Yersinia PTP and PTP1B. Compounds 30,
32, 34, and 36 (Fig. 5), corresponding to alkynes A39,
A48, A49, and A50, were resynthesized on a larger scale,
fully characterized, and assayed in pure form against the
Yersinia PTP, PTP1B, TCPTP, CD45, and LAR.
3. PTP inhibition studies
The crude inhibitors from the first generation library
were screened at 100 lM against the Yersinia PTP and
PTP1B (Fig. 6). Assay mixtures contained p-nitrophen-
ylphosphate (p-NPP) as the substrate, 50 mM 3,3-dim-
ethylglutarate buffer at pH 7.0, 1 mM EDTA, 50 mM
NaCl, and 10% DMSO. Compound 3 was included as
a control and it showed 26% and 0% inhibition of the
Yersinia PTP and PTP1B, respectively. We performed

J. Xie, C. T. Seto / Bioorg. Med. Chem. 15 (2007) 458–473
463
O
O
O
O
O
O
N
OH
OMe
OMe
a
b
N
N
N
N
N
N
N
NH
NH
NH
N
NH
NH
NH
26
27
28
O
O
O
N
N
N
3
N
N
R
R
N
N
Scheme 6. Reagents: (a) alkyne, CuSO4Æ5H2O, sodium ascorbate, acetone, t-BuOH, H2O, TBTA; (b) 1 N NaOH, then 1 N HCl.
O
O
O
N
O
N
OR
OR
29 R = CH
30 R = H
31 R = CH
32 R = H
3
3
H
N
H
N
N
N
N
N
NH
NH
O
O
N
N
N
N
O
S
O
N
N
OMe
O
O
RO
OR
33 R = CH
34 R = H
O
O
3
NH
HN
N
N
N
H
N
H
N
N
N
N
O
O
O
N
N
N
N
N
N
O
O
O
N
RO
OR
35 R = CH
36 R = H
3
HN
NH
N
N
N
N
H
N
H
N
N
O
O
N
N
N
N
N
N
Figure 5. Four inhibitors selected for further evaluation, and their methyl ester precursors.

464
J. Xie, C. T. Seto / Bioorg. Med. Chem. 15 (2007) 458–473
Figure 6. Assays of the first round library members at 100 lM against the Yersinia PTP (open bars) and PTP1B (solid bars). Designators on the
x-axis refer to the library members generated from the alkynes shown in Figure 3.
a series of other controls for the enzyme assays that
demonstrated that none of starting materials, reagents
or solvents used during preparation of the library were
significant inhibitors of the PTPs. One potential compli-
cation in the assay of the crude inhibitors is that a-keto-
acid 3, which is a modest PTP inhibitor, could be formed
during the library synthesis by saponification of com-
pound 4 if compound 4 was not completely consumed
by reaction with the alkyne. However, TLC analyses
of the cycloaddition reactions showed complete con-
sumption of azide 4. Thus, the concentration of a-keto-
acid 3, if it was present in the crude assay solutions, was
low.
A46–A48 and A56 showed good activity against the
Yersinia PTP, and unlike most of the other compounds,
A46 was also active against PTP1B. Comparison of A55,
which does not inhibit either enzyme, with A56 suggests
that the indole N–H group in A56 may be an important
contributor to binding of this compound.
Bidentate PTP inhibitors that incorporate more than
one non-hydrolyzable phosphotyrosine mimic often
show increased potency when compared to their mono-
dentate analogs. This observation prompted us to in-
clude the diynes A49–A54 in the library. Bidentate
inhibitors derived from flexible diynes such as A51–
A54 were not very active. They likely encounter too
large a conformational entropic penalty for binding,
which offsets any advantage they gain from having
two a-ketoacid groups. By contrast, the diynes A49
and A50 that incorporate rigid aromatic spacers have
a lower entropic penalty to binding and thus have good
activity against both PTPs.
Several groups of inhibitors showed activity in the range
of 40% inhibition or better. For example, small aliphatic
amines such as A11 and A12, and aromatic amines
including A23 and A24 had reasonable activity. The
inhibitor derived from A25, which is the phenol analog
of aniline A24, was also active. The aromatic sulfoxide
A39 and the 2-alkoxybenzothiazoles A43 and A44 inhib-
it the Yersinia PTP, but like the other compounds from
A1 to A45, they are not significant inhibitors of PTP1B.
Inhibitors with large aromatic surface areas such as
Based upon the screening data we selected inhibitors de-
rived from alkynes A46, A49, A50, and A56, which dis-
played greater than 60% inhibition of one or both PTPs,

J. Xie, C. T. Seto / Bioorg. Med. Chem. 15 (2007) 458–473
465
for resynthesis and evaluation in purified form. Howev-
er, the inhibitor generated from A49 was insoluble in
aqueous DMSO, and thus assay data are not available
for this compound. Compound 3 and compound 11 (de-
rived from A16) were included in the assays as controls.
crude inhibitors (Table 1). As expected, compounds 3
and 11 are modest inhibitors of the Yersinia PTP with
IC50 values of 220 and 160 lM, respectively. In contrast,
compounds 13 and 15 are good inhibitors of both PTPs.
As predicted from the screening studies, compound 17
has good activity against the Yersinia enzyme, but is a
modest inhibitor of PTP1B.
The IC50 values for the purified inhibitors are fully con-
sistent with the data obtained from screening of the
We initially selected inhibitor 13 as the basis for the sec-
ond generation library. However as noted above, the
solubility characteristics of the requisite azide-contain-
ing analog of 13, compound 22, made it an unsuitable
building block for the library. Inhibitor 17 provided
an alternate lead compound that ultimately proved suc-
cessful. We did not base the second generation library
on the bidentate inhibitor 15 because we wanted to ob-
tain inhibitors with a low charge state at physiological
pH to improve their chances of cell permeability. Com-
pounds such as 15 with two a-ketoacid groups will have
a double negative charge at neutral pH and are less
likely to diffuse through the cell membrane than related
compounds that bear only a single negative charge.
Table 1. Inhibition of phosphatases by compounds 3, 11, 13, 15, and
17a
Inhibitor (alkyne)b
IC50 (lM)
Yersinia PTP
PTP1B
3
220 20
160 20
11 (A16)
13 (A46)
15 (A50)
17 (A56)
45
25
58
7
3
4
84
77
5
4
500 15
a Assays were performed in 50 mM 3,3-dimethylglutarate buffer at pH
7.0, 1 mM EDTA, 50 mM NaCl, and 10% DMSO.
b Number in brackets refers to the alkyne used to generate the
inhibitor.
Figure 7. Assays of the second round library members at 10 lM against the Yersinia PTP (open bars) and PTP1B (solid bars). Designators on the x-
axis refer to the library members generated from the alkynes shown in Figure 3.

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J. Xie, C. T. Seto / Bioorg. Med. Chem. 15 (2007) 458–473
The second generation library, which was based on
inhibitor 17, was screened against the Yersinia PTP
and PTP1B at a concentration of 10 lM (Fig. 7). Com-
pound 17 was used as a control. The hits from this sec-
ond library with activities in the range of 40% inhibition
or better are significantly more potent than inhibitors
identified from the first round of screening since the sec-
ond library was assayed at a 10-fold lower concentration
compared to the first. It is interesting to note that many
of the alkynes that yielded active inhibitors in the first
generation library also gave active inhibitors in the sec-
ond library. This result is somewhat surprising since the
functional groups associated with each alkyne occupy
different regions of the active site in the two libraries.
Examples include inhibitors derived from aniline A24,
aromatic sulfoxide A39, benzothiazoles A43 and A44,
biphenyl A46, and naphthalene A48. One plausible
explanation for this observation is that these particular
compounds incorporate structural features that are well
suited to form strong hydrogen bonding or hydrophobic
interactions with residues near the active site of the
PTPs.
PTP1B and TCPTP because the active sites of these
two enzymes have high homology.69 The residues that
constitute the secondary binding site in PTP1B, such
as Arg24, have no counterparts in LAR and CD45.
Moreover, LAR has no residue corresponding to
Arg47 in PTP1B, which plays an important role for
inhibitor binding in a number of studies. Thus, it is rea-
sonable that inhibitors 30 and 32 have the lowest activ-
ities against LAR.
The most active inhibitors that we identified in this
study are the bis-a-ketoacids 34 and 36. In particular
compound 34, with a central 1,4-disubstituted benzene
ring, is a potent PTP inhibitor. It has an IC50 of
550 nM against the Yersinia PTP and an IC50 of
710 nM against TCPTP. It is approximately 2-fold more
active against the Yersinia PTP, PTP1B, and TCPTP
when compared with inhibitor 36 that incorporates a
1,3-disubstituted benzene ring at its center. Compounds
34 and 36 have similar activities against CD45, and like
the mono-a-ketoacid inhibitors, neither inhibits LAR.
We performed a Lineweaver–Burk analysis of com-
The only monoalkyne that yielded a hit in the second li-
brary, but not the first, is o-trifluoromethylphenylacety-
lene A34. Similar to the first library, the rigid diynes A49
and A50 yielded active bidentate inhibitors. However,
unlike the first library, three of the flexible diynes
(A52–A54) also gave inhibitors with significant activity.
These compounds, which incorporate two a-ketoacids,
may be able to bind simultaneously to the catalytic site
and to the secondary pTyr binding site or to other near-
by basic residues. This binding mode, with two sets of
salt-bridging interactions between enzyme and inhibitor,
would lead to improved activity.
We selected compounds 30, 32, 34, and 36 (Fig. 5), cor-
responding to alkynes A39, A48, A49, and A50, respec-
tively, for resynthesis and further evaluation. In crude
form, these compounds all gave greater than 60% inhibi-
tion of the Yersinia PTP at 10 lM concentration. After
resynthesis and characterization, we measured IC50 val-
ues for these inhibitors against five PTPs; the Yersinia
PTP, PTP1B, TCPTP, CD45, and LAR (Table 2).
The inhibition profiles of the two mono-a-ketoacids (30
and 32) are remarkably similar. Both compounds give
IC50 values in the range of 2–3 lM against the Yersinia
PTP and TCPTP, 6–8 lM against PTP1B, 15–17 lM
against CD45, and no inhibition of LAR at up to
100 lM. Inhibitors often have similar potencies against
Figure 8. Inhibition of TCPTP by compound 34. The activity of
TCPTP was measured at pH 7.0 as described in Section 5 in the
presence of the following concentrations of compound 34: (Ç) 0 lM;
(j) 1 lM; (·) 1.5 lM; (m) 2 lM. Substrate concentrations used in the
assays were 1.0, 2.5, 5.0, and 7.5 mM.
Table 2. Inhibition of phosphatases by compounds 30, 32, 34, and 36a
Inhibitor (alkyne)b
IC50 (lM)
Yersinia PTP
PTP1B
TCPTP
CD45
15.8
LAR
30 (A39)
32 (A48)
34 (A49)
36 (A50)
2.1 0.8
2.1 0.8
7.4 0.2
5.7 0.5
2.1 0.2
4.1 0.4
2.6 0.3
2.6 0.3
2
0% at 100 lM
0% at 100 lM
0% at 100 lM
0% at 100 lM
16.8 1.3
8.4 1.7
8.5 1.2
0.55 0.02
1.3 0.1
0.71 0.03
1.8 0.1
a Assays were performed in 50 mM 3,3-dimethylglutarate buffer at pH 7.0, 1 mM EDTA, 50 mM NaCl, and 10% DMSO.
b Number in brackets refers to the alkyne used to generate the inhibitor.

J. Xie, C. T. Seto / Bioorg. Med. Chem. 15 (2007) 458–473
467
pound 34 against TCPTP, which shows that the inhibi-
tor is either a reversible competitive or perhaps mixed
inhibitor of the phosphatase (Fig. 8). Fitting of the data
to kinetic equations for competitive, non-competitive
and uncompetitive inhibition gives a best fit to the com-
petitive scenario.
the appropriate safety precautions including a fume
hood and blast shield.
5.2. Compound 3
To a solution of 4.20 g (25.5 mmol) of compound 2 in
50 mL of TFA at 0 ꢁC was added 3.50 g (50.7 mmol)
of sodium nitrite in one portion. After the mixture was
stirred for 2 h, 8.27 g (127 mmol) of sodium azide was
added slowly over 20 min followed by the addition of
60 mL of Et2O. The resulting mixture was stirred in
the dark for an additional 2 h and the temperature
was allowed to rise to room temperature. After the sol-
vent was evaporated, the residue was dissolved in
100 mL of 1 N HCl and extracted with EtOAc (3·
100 mL). The combined organic phases were washed
with brine (100 mL) and dried over MgSO4. The solvent
was then removed under reduced pressure. The residue
was purified by chromatography using 5% acetic acid
in methylene chloride as the eluent. Compound 3 was
obtained as yellow solid (4.10 g, 84%). 1H NMR
(300 MHz, acetone-d6) d 6.35 (br s, 1H), 7.29 (d,
J = 6.8 Hz, 2H), 8.10 (d, J = 6.8 Hz, 2H); 13C NMR
(75 MHz, acetone-d6) d 119.9, 129.6, 132.2, 147.1,
165.2, 186.4; HRMS-ESI (MꢀH+) calcd for
C8H4N3O3 190.0253. Found: 190.0250.
4. Conclusions
In this report we have described the synthesis and
screening of a two stage library of a-ketoacid-based
PTP inhibitors. In the first stage, the Cu(I)-catalyzed
cycloaddition reaction was used to append 56 different
alkynes and diynes to an azide-containing pharmaco-
phore. The best mono-ketoacid inhibitor (13) identified
during the first round of screening showed a 5-fold
improvement in activity against the Yersinia PTP when
compared to the parent compound (3). The best biden-
tate inhibitor from the first round (15) was nine times
more potent than compound 3. In the second stage of li-
brary synthesis, we selected a mono-ketoacid lead com-
pound from the first stage and modified its structure to
incorporate an azide group. We performed a second
round of click reactions to couple this azide with the
56 alkynes and diynes. Screening of the second stage li-
brary identified a mono-ketoacid (32) and a bis-ketoacid
(34) that are 100 and 400 times more potent than the
parent compound 3, respectively.
5.3. Compound 4
A mixture of 3.0 g (15.7 mmol) of compound 3 and
2.3 mL (31.4 mmol) of thionyl chloride in 100 mL of
anhydrous benzene was heated at reflux for 2 h. The sol-
vent was removed under reduced pressure. The material
was dissolved in 50 mL of methanol and the solution
was stirred for 10 min. After the solvent was removed
under reduced pressure, the residue was purified by flash
chromatography (1:1 methylene chloride/hexane). Com-
This two stage library represents an efficient strategy
to advance from a poorly active lead compound (3,
IC50 = 220 lM) to a nanomolar inhibitor of two
important PTPs. It has the added advantage of requir-
ing a relatively small number of building blocks, in
this case 56 alkynes and diynes, which can be used
for both stages of library synthesis. This strategy
should be useful for extending inhibitors so that they
take advantage of binding interactions with regions of
the enzyme that are outside of the immediate region
of the active site.
1
pound 4 was obtained as yellow solid (2.8 g, 87%). H
NMR (300 MHz, CDCl3) d 3.95 (s, 3H), 7.09 (d,
J = 8.6 Hz, 2H), 8.01 (d, J = 8.7 Hz, 2H); 13C NMR
(75 MHz, CDCl3) d 52.9, 119.3, 129.1, 132.2, 146.9,
163.7, 184.1; HRMS-ESI (M+Na+) calcd for
C9H7N3NaO3 228.0385. Found: 228.0375.
5. Experimental
5.4. Compound 8
5.1. General methods
To a mixture of 0.60 g (2.8 mmol) of racemic 3-(1-naph-
thyl)alanine in 40 mL of THF was added 0.30 g
(7.9 mmol) of lithium aluminum hydride. The mixture
was heated at reflux for 12 h. After the solution was
cooled to room temperature, the solvent was evaporated
under reduced pressure. The residue was suspended in
100 mL of EtOAc and washed with water (100 mL)
and brine (100 mL). The organic phase was separated
and dried over MgSO4. Filtration followed by evapora-
tion afforded compound 8 as white solid (0.50 g, 89%).
The product was used in the next reaction without fur-
1H NMR and 13C NMR spectra were acquired on Bru-
ker Avance-300 or Avance-400 instruments. Chemical
shifts are reported relative to TMS (d = 0.00 ppm) for
1H NMR and CDCl3 (d = 77.0 ppm) for 13C NMR.
Mass spectra were recorded on either a Shimadzu
LCMS-QP8000 or an Applied Biosystems QSTAR elec-
trospray mass spectrometer. Dry solvents were obtained
from a Solvent Dispensing System. All reagents were
used as received. Compounds 2 and 18 were prepared
using procedures from the literature.43,57 Several com-
pounds reported herein (14, 21, 31, 33, and 35) are insol-
uble in all solvents that are commonly used to prepare
samples for mass spectrometric analysis. As a result,
mass spectral data have not been provided for these five
compounds. Caution: All reactions involving metal
azides or hydrazoic acid should be performed using
1
ther purification. H NMR (300 MHz, CDCl3) d 2.07
(br s, 3H), 2.93 (dd, J = 15.0, 9.9 Hz, 1 H), 3.29–3.37
(m, 2H), 3.51 (dd, J = 7.8, 6.9 Hz, 1H), 3.72 (dd,
J = 10.5, 3.6 Hz, 1H), 7.34–7.56 (m, 4H), 7.78 (d,
J = 8.1 Hz, 1H), 7.84–7.91 (m, 1H), 8.04–8.09 (m, 1H);
13C NMR (75 MHz, CDCl3) d 38.0, 53.3, 66.5, 123.8,

468
J. Xie, C. T. Seto / Bioorg. Med. Chem. 15 (2007) 458–473
125.4, 125.7, 126.0, 127.3, 127.4, 128.9, 132.1, 134.0,
134.9; HRMS-ESI (M+H+) calcd for C13H16NO
202.1232. Found: 202.1238.
in 5 mL of ethanol and 5 mL of tert-butyl alcohol
was added 10 mg (0.0189 mmol) of TBTA followed
by the addition of 53 mg of CuSO4Æ5H2O
(0.212 mmol, dissolved in 2.5 mL of water) and
209 mg of sodium ascorbate (1.05 mmol, dissolved in
2.5 mL of water). The resulting mixture was allowed
to stir for 24 h. Water (10 mL) was then added to
the mixture and the precipitate was collected by filtra-
tion and washed with water (3· 50 mL). After the sol-
id was dried under vacuum, compound 10 was
obtained as a grey powder (500 mg, 87%). 1H NMR
(300 MHz, DMSO-d6) d 3.96 (s, 3H), 8.22 (m, 4H),
9.58 (s, 1H), 13.42 (br s, 1H); 13C NMR (75 MHz,
DMSO-d6) d 53.6, 121.0, 128.0, 132.3, 132.4, 140.9,
141.5, 161.8, 163.6, 185.2; HRMS-ESI (M+H+) calcd
for C12H10N3O5 276.0620. Found: 276.0619.
5.5. Compound A55
To a solution of 35 mg (0.498 mmol) of propiolic acid in
20 mL of methylene chloride was added 123 mg
(0.598 mmol) of dicyclohexylcarbodiimide (DCC). The
solution was stirred at 0 ꢁC for 10 min. Compound 8
(100 mg, 0.498 mmol) was then added and the resulting
mixture was allowed to stir for another 3.5 h. After the
mixture was filtered, the solution was concentrated to
dryness. The residue was purified by flash chromatogra-
phy (10:1 methylene chloride/methanol) to give com-
pound A55 as a yellow powder (56 mg, 44%). 1H
NMR (300 MHz, CDCl3) d 2.76 (s, 1H), 2.90–3.71 (m,
5H), 4.39 (m, 1H), 6.85 (d, J = 7.8 Hz, 1H), 7.33–7.79
(m, 5H), 7.85 (d, J = 7.8 Hz, 1H), 8.27 (d, J = 8.4 Hz,
1H); 13C NMR (75 MHz, CDCl3) d 33.9, 52.5, 62.4,
73.9, 123.9, 125.4, 125.8, 126.4, 127.6, 128.8, 132.1,
133.6, 133.9, 152.6; HRMS-ESI (M+Na+) calcd for
C16H15NNaO2 276.1000. Found: 276.0992.
5.9. Representative procedure for the synthesis of
compounds 11, 13, 15, and 17: compound 11
To a solution of 50 mg of compound 10 (0.18 mmol)
in 0.5 mL of DMSO was added 0.5 mL of 1 N NaOH.
The solution was allowed to stir for 15 min. After
0.5 mL of 1 N HCl was added to quench the reaction,
the mixture was poured into 20 mL of ice water. The
solid was collected by filtration and washed with
water (2· 20 mL). After the solid was dried under vac-
uum, compound 11 was obtained as grey powder
(36 mg, 76%). 1H NMR (300 MHz, DMSO-d6) d
8.18 (d, J = 9.0 Hz, 2H), 8.26 (d, J = 9.0 Hz, 2H),
9.57 (s, 1H); 13C NMR (75 MHz, DMSO-d6) d
121.6, 128.4, 132.3, 132.9, 141.2, 141.9, 162.2, 166.3,
188.1; HRMS-ESI (MꢀH+) calcd for C11H6N3O5
260.0309. Found: 260.0300.
5.6. Compound A56
Compound A56 was prepared from 1.0 g (5.3 mmol) of
L-tryptophanol 9 by procedures analogous to the prepa-
ration of compound A55 (yellow powder, 0.73 g, 58%,
eluent: 20:1 methylene chloride/methanol). 1H NMR
(300 MHz, acetone-d6) d 3.02–3.20 (m, 2H), 3.49 (s,
1H), 3.72–3.74 (m, 2H), 4.35–4.47 (m, 2H), 7.05–7.24
(m, 3H), 7.44 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 7.8 Hz,
1H), 7.90 (d, J = 8.1 Hz, 1H), 10.08 (s, 1H); 13C NMR
(75 MHz, acetone-d6) d 26.4, 52.9, 62.9, 74.0, 78.1,
111.4, 111.5, 118.7, 118.8, 121.4, 123.4, 127.9, 136.8,
152.4; HRMS-ESI (M+Na+) calcd for C14H14N2NaO2
265.0953. Found: 265.0961.
5.10. Compound 12
Compound 12 was obtained from 52 mg (0.25 mmol)
of compound 4, 45 mg (0.25 mmol) of A46, and
4 mg (0.0076 mmol) of TBTA by procedures analo-
gous to the preparation of compound 10 (grey pow-
der, 90 mg, 93%). 1H NMR (300 MHz, DMSO-d6) d
3.99 (s, 3H), 7.41 (d, J = 6.0 Hz, 1H), 7.45–7.56 (m,
2H), 7.76 (d, J = 6.0 Hz, 2H), 7.85 (d, J = 6.0 Hz,
2H), 8.07 (d, J = 6.0 Hz, 2H), 8.26 (br s, 4H), 9.57
(s, 1H); 13C NMR (75 MHz, DMSO-d6) d 54.1,
120.8, 126.8, 127.5, 128.2, 128.6, 129.8, 129.9,
132.4, 133.0, 140.3, 141.0, 141.8, 148.3, 164.2,
185.7.
5.7. Synthesis of the first round library
To a 1 dram vial containing a solution of 5 mg
(0.024 mmol) (or 10 mg, 0.049 mmol when using diynes)
of compound 4, 0.024 mmol of alkyne or diyne, 0.4 mg
(0.76 lmol) of TBTA in 0.15 ml of ethanol and
0.15 mL of tert-butyl alcohol was added 0.3 mg Cu-
SO4Æ5H2O (0.0012 mmol, dissolved in 75 lL of water)
followed by the addition of 1 mg of sodium ascorbate
(0.0049 mmol, dissolved in 75 lL of water). The mixture
was stirred for 24 h at room temperature. Once the reac-
tions were determined to be complete by TLC analysis,
50 lL of 1 N NaOH was added. The solution was stirred
for 15 min and then 50 lL of 1 N HCl was added to neu-
tralize the reaction mixture. Based on the assumption
that the yield of the reaction was 100%, DMSO
(1.89 mL) was added to the vial to make a 10 mM stock
solution of the inhibitor.
5.11. Compound 13
Compound 13 was obtained from 50 mg (0.13 mmol) of
compound 12 by procedures analogous to the prepara-
tion of compound 11 (yellow powder, 45 mg, 93%). H
1
NMR (300 MHz, DMSO-d6) d 7.04 (t, J = 6.0 Hz,
1H), 7.48–7.53 (m, 2H), 7.76 (d, J = 6.0 Hz, 2H), 7.84
(d, J = 9.0 Hz, 2H), 8.06 (d, J = 9.0 Hz, 2H), 8.20 (br
s, 4H), 9.54 (s, 1H); 13C NMR (75 MHz, DMSO-d6) d
120.6, 120.7, 120.8, 126.8, 127.5, 128.7, 128.6, 129.9,
132.0, 132.2, 140.3, 140.9, 141.1, 148.2, 167.3; HRMS-
ESI (MꢀH+) calcd for C22H14N3O3 368.1035. Found:
368.1030.
5.8. Representative procedure for the synthesis of
compounds 10, 12, 14, and 16: compound 10
To a solution of 430 mg (2.10 mmol) of compound 4
and 147 mg (2.10 mmol) of propiolic acid dissolved

J. Xie, C. T. Seto / Bioorg. Med. Chem. 15 (2007) 458–473
469
5.12. Compound 14
CuI in 20 mL of triethylamine was added 0.44 g
(4.5 mmol) of ethynyltrimethylsilane. The mixture was
stirred at room temperature for 1.5 h and then poured
into 100 mL of saturated NH4Cl. After extraction with
EtOAc (2· 100 mL), the combined organic phases were
washed with water (100 mL), brine (100 mL) and dried
over MgSO4. The solvent was then removed under re-
duced pressure. The residue was purified by flash chro-
matography (1:1 EtOAc/hexanes) to give compound 19
Compound 14 was obtained from 33 mg (0.16 mmol) of
compound 4, 10 mg (0.079 mmol) of A50, and 3 mg
(0.0057 mmol) of TBTA by procedures analogous to
the preparation of compound 10 (grey powder, 41 mg,
1
95%). H NMR (300 MHz, DMSO-d6) d 3.98 (s, 6H),
7.68 (t, J = 7.5 Hz, 1H), 7.98 (d, J = 9.0 Hz, 2H), 8.27
(s, 8H), 8.63 (s, 1H), 9.63 (s, 2H); 13C NMR (75 MHz,
DMSO-d6) d 54.0, 120.8, 121.1, 123.0, 126.4, 130.8,
131.5, 132.4, 132.9, 141.8, 148.3, 164.2, 185.7.
1
as a yellow powder (0.92 g, 85%). H NMR (300 MHz,
CDCl3)
d 0.30 (s, 9H), 3.77 (s, 2H), 6.76 (d,
J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.51 (s, 4H);
13C NMR (75 MHz, CDCl3) d 0.1, 94.3, 105.4, 115.4,
120.8, 126.0, 127.9, 130.5, 132.4, 141.2, 146.3; HRMS-
ESI (M+) calcd for C17H19NSi 265.1287. Found:
265.1281.
5.13. Compound 15
Compound 15 was obtained from 41 mg (0.064 mmol)
of compound 14 by procedures analogous to the prepa-
ration of compound 11 (grey powder, 30 mg, 78%). H
1
NMR (300 MHz, DMSO-d6) d 7.67 (t, J = 7.5 Hz,
1H), 7.99 (d, J = 6.0 Hz, 2H), 8.18–8.28 (m, 8H), 8.64
(s, 1H), 9.62 (s, 2H); 13C NMR (75 MHz, DMSO-d6)
d 120.9, 121.1, 123.1, 126.4, 130.8, 131.6, 132.4, 141.4,
148.2, 167.3; HRMS-ESI (MꢀH+) calcd for
C26H15N6O6 507.1053. Found: 507.1042.
5.17. Compound 20
To a solution of 0.80 g (3.0 mmol) of compound 19 dis-
solved in 30 mL of methanol was added 7 mL of 10%
aqueous NaOH. The solution was allowed to stir for
2 h. The organic solvent was then removed under re-
duced pressure and the resulting slurry was extracted
with 100 mL of CH2Cl2. The organic phase was washed
with water (100 mL), brine (100 mL) and dried over
MgSO4. The solvent was then evaporated to give
0.58 g (99%) of compound 20 as yellow solid. 1H
NMR (300 MHz, CDCl3) d 3.19 (s, 1H), 3.79 (s, 2H),
6.77 (d, J = 8.7 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.57
(dd, J = 13.1, 8.5 Hz, 4H); 13C NMR (75 MHz, CDCl3)
d 77.6, 84.0, 115.4, 119.8, 126.2, 128.0, 130.3, 132.6,
141.6, 146.4; HRMS-ESI (M+) calcd for C14H11N
193.0891. Found: 193.0886.
5.14. Compound 16
Compound 16 was obtained from 190 mg (0.927 mmol)
of compound 4, 224 mg (0.926 mmol) of A56, and 15 mg
(0.0285 mmol) of TBTA by procedures analogous to the
preparation of compound 10 (yellow powder, 398 mg,
96%). 1H NMR (300 MHz, acetone-d6) d 3.21 (d,
J = 6.9 Hz, 2H), 3.77–3.80 (m, 2H), 4.02 (s, 3H), 4.23
(s, 1H), 4.50–4.51 (m, 1H), 7.02 (t, J = 7.2 Hz, 1H),
7.10 (t, J = 6.9 Hz, 1H), 7.27 (s, 1H), 7.38 (d,
J = 8.1 Hz, 1H), 7.80 (d, J = 7.5 Hz, 2H), 8.24 (d,
J = 8.7 Hz, 2H), 8.28 (d, J = 9.0 Hz, 2H), 9.12 (s, 1H),
10.05 (s, 1H); 13C NMR (75 MHz, acetone-d6) d 27.1,
52.4, 53.0, 63.2, 111.7, 112.0, 119.0, 119.2, 121.0,
121.6, 123.7, 124.8, 128.4, 132.2, 132.8, 137.2, 141.5,
144.9, 159.6, 164.1, 185.3; HRMS-ESI (M+Na+) calcd
for C23H21N5NaO5 470.1440. Found: 470.1454.
5.18. Compound 21
Compound 21 was obtained from 0.62 g (3.0 mmol) of
compound 4, 0.58 g (3.0 mmol) of compound 20,
48 mg (0.091 mmol) of TBTA, 75 mg (0.30 mmol) of
CuSO4Æ5H2O and 240 mg (1.21 mmol) of sodium ascor-
bate by procedures analogous to the preparation of
compound 10 (yellow solid, 1.13 g, 94%). 1H NMR
5.15. Compound 17
(300 MHz, DMSO-d6)
d 3.98 (s, 3H), 6.66 (d,
Compound 17 was obtained from 50 mg (0.112 mmol)
of compound 16 by procedures analogous to the prepa-
ration of compound 11 (yellow powder, 40 mg, 83%). 1H
NMR (300 MHz, DMSO-d6) d 2.55 (s, 1H), 2.94–3.05
(m, 2H), 3.48–3.61 (m, 2H), 4.27–4.32 (m, 1H), 6.95 (t,
J = 7.5 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.16 (d,
J = 2.1 Hz, 1H), 7.32 (d, J = 9.0 Hz, 1H), 7.67 (d,
J = 6.0 Hz, 1H), 8.18 (d, J = 9.0 Hz, 2H), 8.24 (d,
J = 9.0 Hz, 2H), 8.34 (d, J = 9.0 Hz, 1H), 9.44 (s, 1H),
10.79 (s, 1H); 13C NMR (75 MHz, DMSO-d6) d 27.3,
52.6, 63.3, 112.2, 119.0, 119.4, 121.5, 121.7, 124.0,
125.9, 128.4, 132.4, 132.8, 137.0, 141.3, 145.0, 159.7,
166.3, 188.2; HRMS-ESI (MꢀH+) calcd for
C22H18N5O5 432.1308. Found: 432.1318.
J = 5.7 Hz, 2H), 7.46 (d, J = 6.9 Hz, 2H), 7.70 (d,
J = 7.2 Hz, 2H), 7.96 (d, J = 6.9 Hz, 2H), 8.24 (s, 4H),
9.48 (s, 1H); 13C NMR (75 MHz, DMSO-d6) d 54.0,
115.2, 120.2, 120.7, 126.6, 126.7, 127.9, 132.3, 132.9,
141.5, 141.8, 148.6, 164.2, 185.7.
5.19. Compound 24
To a solution of 1.08 g (3.74 mmol) of Boc-tryptopha-
nol and 1.96 g (7.48 mmol) of PPh3 in 40 mL of tolu-
ene was added 0.86 g (2.80 mmol) of ZnN6Æ2Py. DIAD
(1.51 g, 7.48 mmol) was then added dropwise into the
slurry. The reaction mixture was stirred for 1 h and
then the solid was removed by filtration. After the tol-
uene was removed under reduced pressure, the residue
was purified by flash chromatography (2.5:1 hexanes/
EtOAc) to give compound 24 as white solid (1.11 g,
94%). 1H NMR (300 MHz, CDCl3) d 1.54 (s, 9H),
2.95–3.02 (m, 2H), 3.29–3.44 (m, 2H), 4.15 (br s,
5.16. Compound 19
To a slurry of 1.2 g (4.1 mmol) of compound 18, 85 mg
(0.12 mmol) of PdCl2(PPh3)2, and 40 mg (0.21 mmol) of

470
J. Xie, C. T. Seto / Bioorg. Med. Chem. 15 (2007) 458–473
1H), 4.94–5.05 (m, 1H), 7.00 (d, J = 1.2 Hz, 1H), 7.15–
7.28 (m, 2H), 7.39 (d, J = 7.8 Hz, 1H), 7.69 (d,
J = 7.8 Hz, 1H), 8.70 (s, 1H); 13C NMR (75 MHz,
CDCl3) d 28.5, 50.8, 53.3, 70.2, 79.9, 110.8, 111.5,
118.8, 119.6, 122.1, 123.2, 127.6, 136.4, 155.6;
HRMS-ESI (M+Na+) calcd for C16H21N5NaO2
338.1593. Found: 338.1585.
were determined to be complete by TLC analysis,
100 lL of 1 N NaOH was added. The solution was
stirred for 15 min and then 100 lL of 1 N HCl was
added to neutralize the reaction mixture. Based on
the assumption that the yield of the reaction was
100%, DMSO (1.418 mL) was added to the vial to
make a 5 mM stock solution of the inhibitor.
5.20. Compound 25
5.23. Representative procedure for the synthesis of
compounds 29, 31, 33, and 35: compound 29
Compound 24 (1.1 g, 3.5 mmol) was stirred with 50 mL
of 50% TFA in methylene chloride for 30 min. The sol-
vent was then removed and the residue was purified by
flash chromatography (20:1 methylene chloride/metha-
nol) to give compound 25 as a brown oil (0.45 g,
To a solution of 25 mg (0.053 mmol) of compound 26
and 9.5 mg (0.053 mmol) of A39 dissolved in 1 mL of
acetone and 1 mL of tert-butyl alcohol was added
1 mg (0.0019 mmol) of TBTA followed by the addi-
tion of 1.3 mg of CuSO4Æ5H2O (0.053 mmol, dissolved
in 0.5 mL of water) and 5.3 mg of sodium ascorbate
(0.027 mmol, dissolved in 0.5 mL of water). The
resulting mixture was allowed to stir for 24 h. Water
(10 mL) was then added to the mixture and the pre-
cipitate was collected by filtration and washed with
water (2· 20 mL). After the solid was dried under
vacuum, compound 29 was obtained as grey powder
(30 mg, 87%). 1H NMR (300 MHz, acetonitrile-d3) d
2.31 (s, 3H), 3.17–3.20 (m, 2H), 4.00 (s, 3H), 4.62–
4.81 (m, 3H), 7.06 (t, J = 6.9 Hz, 1H), 7.14 (t,
J = 6.9 Hz, 1H), 7.20 (d, J = 2.4 Hz, 1H), 7.32 (d,
J = 8.4 Hz, 2H), 7.40 (d, J = 8.1 Hz, 1H), 7.64 (t,
J = 7.8 Hz, 2H), 7.78 (d, J = 8.4 Hz, 2H), 8.02 (d,
J = 9.0 Hz, 2H), 8.24 (d, J = 9.0 Hz, 2H), 8.39 (s,
1H), 8.65 (s, 1H), 9.18 (s, 1H); 13C NMR (75 MHz,
acetonitrile-d3) d 20.6, 27.4, 50.0, 52.9, 53.6, 110.1,
111.4, 118.4, 119.1, 120.7, 121.6, 123.7, 124.4, 127.4,
127.5, 128.0, 129.9, 131.8, 132.7, 136.5, 137.7, 140.8,
143.4, 145.3, 148.2, 159.2, 163.4, 184.9; HRMS-ESI
(M+Na+) calcd for C32H28N8NaO6S 675.1750.
Found: 675.1760.
1
60%). H NMR (400 MHz, CDCl3) d 1.79 (br s, 2H),
2.80 (dd, J = 14.0, 7.6 Hz, 1H), 2.98 (dd, J = 14.4,
5.2 Hz, 1H), 3.26–3.34 (m, 2H), 3.47 (dd, J = 11.2,
3.6 Hz, 1H), 7.07 (d, J = 1.6 Hz, 1H), 7.17 (t,
J = 7.2 Hz, 1H), 7.24 (t, J = 7.2 Hz, 1H), 7.40 (d,
J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 8.37 (br s,
1H); 13C NMR (75 MHz, CDCl3) d 30.9, 52.3, 57.6,
111.3, 111.9, 118.8, 119.6, 122.2, 122.8, 127.5, 136.4;
HRMS-ESI (M+H+) calcd for C11H14N5 216.1249.
Found: 216.1257.
5.21. Compound 26
To a solution of 0.38 g (1.4 mmol) of compound 10,
0.27 g (1.4 mmol) of EDC, and 0.17 g (1.4 mmol) of
DMAP dissolved in 50 mL of methylene chloride
was added 0.30 g (1.4 mmol) of compound 25. The
reaction mixture was stirred overnight at room tem-
perature. The solvent was then removed and the resi-
due was dissolved in 100 mL of Et2O. The solution
was washed with 100 mL of 1 N HCl and 100 mL of
brine. The organic layer was dried over MgSO4 and
concentrated to dryness. The residue was purified by
flash chromatography (100:2 methylene chloride/acetic
acid) to afford compound 26 as grey powder (0.41 g,
62%). 1H NMR (300 MHz, acetone-d6) d 2.90–3.25
(m, 2H), 3.65–3.72 (m, 2H), 4.02 (s, 3H), 4.60–4.71
(m, 1H), 7.01–7.13 (m, 2H), 7.30 (d, J = 2.1 Hz, 1H),
7.39 (d, J = 8.1 Hz, 1H), 7.74 (d, J = 7.8 Hz, 1H),
7.96–7.99 (d, J = 8.4 Hz, 1H), 8.22–8.27 (m, 4H),
9.10 (s, 1H), 10.09 (br s, 1H); 13C NMR (75 MHz,
CDCl3) d 27.8, 49.8, 52.5, 53.5, 110.8, 111.4, 118.5,
118.8, 120.7, 121.4, 123.4, 124.5, 127.8, 131.7, 132.5,
136.8, 141.1, 144.3, 159.2, 163.7, 184.9; HRMS-ESI
(M+Na+) calcd for C23H20N8NaO4 495.1505. Found:
495.1502.
5.24. Representative procedure for the synthesis of
compounds 30, 32, 34, and 36: compound 30
To a solution of 30 mg of compound 29 (0.046 mmol)
dissolved in 0.5 mL of DMSO was added 0.5 mL of
1 N NaOH. The solution was allowed to stir for
15 min. After 0.5 mL of 1 N HCl was added to
quench the reaction, the mixture was poured into
20 mL of ice water. The solid was collected by filtra-
tion and washed with water (2· 20 mL). After the
solid was dried under vacuum, compound 30 was
obtained as grey powder (25 mg, 85%). 1H NMR
(300 MHz, DMSO-d6) d 2.24 (s, 3H), 3.01–3.19 (m,
2H), 4.65–4.88 (m, 3H), 6.96 (t, J = 6.9 Hz, 1H),
7.06 (t, J = 6.9 Hz, 1H), 7.21 (d, J = 1.8 Hz, 1H),
7.31–7.34 (m, 3H), 7.61 (d, J = 7.8 Hz, 1H), 7.70
(d, J = 8.4 Hz, 2H), 8.12 (d, J = 8.7 Hz, 2H), 8.18
(d, J = 8.7 Hz, 2H), 8.89 (d, J = 8.7 Hz, 1H), 8.92
(s, 1H), 9.29 (s, 1H), 10.89 (s, 1H); 13C NMR
(75 MHz, DMSO-d6) d 21.8, 28.5, 50.6, 54.1, 110.8,
112.3, 119.2, 121.3, 121.8, 124.4, 126.0, 128.0,
128.1, 128.9, 129.6, 130.8, 132.0, 133.9, 137.0,
138.2, 140.6, 144.4, 145.5, 148.0, 159.8, 167.7,
5.22. Synthesis of the second round library
To a 1 dram vial containing a solution of 5 mg
(0.011 mmol) (or 10 mg, 0.022 mmol, when using diy-
nes) of compound 26, 0.011 mmol of alkyne or diyne,
0.5 mg (0.94 nmol) of TBTA in 0.2 ml of acetone and
0.1 mL of tert-butyl alcohol was added 0.3 mg Cu-
SO4Æ5H2O (0.0012 mmol, dissolved in 100 lL of
water) and 1 mg of sodium ascorbate (0.0049 mmol,
dissolved in 100 lL of water). The mixture was stir-
red for 24 h at room temperature. Once the reactions
191.0;
C31H26N8NaO6S 661.1594. Found: 661.1610.
HRMS-ESI
(M+Na+)
calcd
for

J. Xie, C. T. Seto / Bioorg. Med. Chem. 15 (2007) 458–473
471
5.25. Compound 31
7.65 (d, J = 7.6 Hz, 2H), 7.85 (s, 4H), 8.19 (s, 8H),
8.57 (s, 2H), 8.94 (d, J = 8.4 Hz, 2H), 9.34 (s, 2H),
10.88 (s, 2H); 13C NMR (75 MHz, DMSO-d6) d 29.0,
51.0, 53.4, 111.0, 112.3, 119.2, 121.5, 121.8, 122.9,
124.4, 126.2, 126.4, 128.2, 131.0, 132.3, 137.0, 140.0,
141.1, 144.6, 146.6, 159.8; HRMS-ESI (MꢀH+) calcd
for C54H41N16O8 1041.3293. Found: 1041.3265.
Compound 31 was obtained from 25 mg (0.053 mmol)
of compound 26, 9.6 mg (0.053 mmol) of A48, and
1 mg (0.0076 mmol) of TBTA by procedures analogous
to the preparation of compound 29 (grey powder,
1
31 mg, 90%). H NMR (400 MHz, DMSO-d6) d 3.11–
3.17 (m, 2H), 3.87 (s, 3H), 3.95 (s, 3H), 4.73–4.77 (m,
3H), 6.99 (t, J = 7.6 Hz, 1H), 7.07 (t, J = 7.4 Hz, 1H),
7.16 (dd, J = 8.8, 2.0 Hz, 1H), 7.25 (d, J = 2.0 Hz, 1H),
7.31 (d, J = 2.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.65
(d, J = 7.6 Hz, 1H), 7.82–7.89 (m, 3H), 8.19 (s, 4H),
8.25 (s, 1H), 8.60 (s, 1H), 8.95 (d, J = 8.8 Hz, 1H),
9.36 (s, 1H), 10.89 (br s, 1H ); 13C NMR (75 MHz,
CDCl3) d 28.6, 51.0, 54.0, 56.0, 59.2, 119.2, 120.0,
121.4, 121.9, 122.8, 124.2, 124.4, 124.9, 126.2, 126.8,
128.2 (two overlapping peaks), 129.3, 130.3, 130.8,
132.7, 132.8, 134.6, 137.0, 141.3, 144.6, 147.1, 157.3,
158.2, 159.9, 164.0, 185.6.
5.29. Compound 35
Compound 35 was obtained from 30 mg (0.064 mmol)
of compound 26, 4 mg (0.032 mmol) of A50 and 2 mg
(0.0076 mmol) of TBTA by procedures analogous to
the preparation of compound 29 (grey powder, 33 mg,
1
97%). H NMR (400 MHz, DMSO-d6) d 3.15–3.19 (m,
4H), 3.95 (s, 6H), 4.68–4.83 (m, 6H), 6.98 (t,
J = 7.4 Hz, 2H), 7.06 (t, J = 7.2 Hz, 2H), 7.24 (s, 2H),
7.33 (d, J = 8 Hz, 2H), 7.44 (t, J = 7.6 Hz, 1H), 7.64
(d, J= 7.6 Hz, 2H), 7.70 (d, J = 7.6 Hz, 2H), 8.18 (s,
8H), 8.28 (s, 1H), 8.61 (s, 2H), 8.91 (d, J = 8.4 Hz,
2H), 9.34 (s, 2H), 10.85 (s, 2H); 13C NMR (75 MHz,
DMSO-d6) d 28.5, 51.0, 53.5, 54.0, 111.0, 112.3, 119.2,
121.4, 121.8, 122.5, 123.1, 124.4, 125.3, 126.2, 128.2,
130.3, 132.2, 132.7, 137.0, 141.4, 144.6, 146.7, 159.8,
164.0, 185.6.
5.26. Compound 32
Compound 32 was obtained from 31 mg (0.047 mmol)
of compound 31 by procedures analogous to the prepa-
ration of compound 30 (grey powder, 27 mg, 89%). H
1
NMR (400 MHz, DMSO-d6) d 3.09–3.17 (m, 2H), 3.87
(s, 3H), 4.73–4.85 (m, 3H), 7.02 (t, J = 7.4 Hz, 1H),
7.08 (t, J = 7.4 Hz, 1H), 7.16 (dd, J = 8.8, 2.0 Hz, 1H),
7.26 (d, J = 2.0 Hz, 1H), 7.28 (d, J = 2.0 Hz, 1H), 7.34
(d, J = 7.6 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.82–7.90
(m, 4H), 8.08–8.16 (m, 3H), 8.26 (s, 1H), 8.61 (s, 1H),
8.98 (d, J = 8.8 Hz, 1H), 9.35 (s, 1H), 10.89 (s, 1H);
13C NMR (75 MHz, CDCl3) d 28.6, 51.0, 53.4, 56.0,
106.8, 111.1, 112.3, 119.2, 120.0, 121.5, 121.8, 122.7,
124.2, 124.4, 124.9, 126.2, 126.9, 128.2 (two overlapping
peaks), 129.3, 130.3, 131.8, 134.6, 137.0, 140.9, 144.6,
147.1, 158.2, 159.9; HRMS-ESI (MꢀH+) calcd for
C35H27N8O5 639.2104. Found: 639.2118.
5.30. Compound 36
Compound 36 was obtained from 33 mg (0.031 mmol)
of compound 35 by procedures analogous to the prepa-
ration of compound 30 (grey powder, 26 mg, 81%). H
1
NMR (300 MHz, DMSO-d6) d 3.01–3.21 (m, 4H),
4.68–4.84 (m, 6H), 7.00 (t, J = 7.4 Hz, 2H), 7.06 (t,
J = 7.5 Hz, 2H), 7.23 (s, 2H), 7.33 (d, J = 8.1 Hz, 2H),
7.43 (t, J = 7.6 Hz, 1H), 7.64 (d, J= 7.5 Hz, 2H), 7.70
(d, J = 7.8 Hz, 2H), 8.16 (s, 8H), 8.27 (s, 1H), 8.60 (s,
2H), 8.90 (d, J = 8.4 Hz, 2H), 9.28 (s, 2H), 10.85 (s,
2H); 13C NMR (75 MHz, DMSO-d6) d 28.6, 50.9,
53.5, 111.0, 112.3, 119.2, 121.2, 121.6, 121.8, 122.5,
123.0, 124.4, 125.3, 126.2, 128.2, 130.3, 131.8, 132.2,
137.0, 141.0, 144.5, 146.6, 159.8; HRMS-ESI (MꢀH+)
calcd for C54H41N16O8 1041.3293. Found: 1041.3260.
5.27. Compound 33
Compound 33 was obtained from 30 mg (0.064 mmol)
of compound 26, 4 mg (0.032 mmol) of A49 and 2 mg
(0.0076 mmol) of TBTA by procedures analogous to
the preparation of compound 29 (grey powder, 34 mg,
100%). 1H NMR (400 MHz, DMSO-d6) d 3.05–3.16
(m, 4H), 3.95 (s, 6H), 4.68–4.82 (m, 6H), 6.98 (t,
J = 7.2 Hz, 2H), 7.07 (t, J = 7.2 Hz, 2H), 7.24 (s, 2H),
7.33 (d, J = 8.0 Hz, 2H), 7.64 (d, J = 7.6 Hz, 2H), 7.84
(s, 4H), 8.19 (s, 8H), 8.56 (s, 2H), 8.92 (d, J = 8.4 Hz,
2H), 9.36 (s, 2H), 10.86 (s, 2H); 13C NMR (75 MHz,
DMSO-d6) d 28.5, 51.0, 53.4, 54.0, 111.0, 112.3, 119.2,
121.4, 121.8, 122.9, 124.4, 126.3, 126.4, 128.2, 131.0,
132.7, 137.0, 141.4, 144.6, 146.6, 159.8, 164.0, 185.6.
5.31. PTP assays
The phosphatase activities of the Yersinia PTP, PTP1B,
LAR, TCPTP, and CD45 were assayed using p-nitro-
phenyl phosphate (p-NPP) as the substrate at room tem-
perature and the reaction progress was monitored by
UV spectroscopy. Initial rates were determined by mon-
itoring the hydrolysis of p-NPP at 405 nm, from 10 to
130 s after mixing. Assay solutions contained 50 mM
3,3-dimethylglutarate at pH 7.0, 1 mM EDTA, 50 mM
NaCl, and 10% DMSO. For percent inhibition assays,
the substrate concentrations were kept at 2.9 and
2.0 mM for Yersinia PTP and PTP1B, respectively.
For IC50 assays, the substrate concentrations were kept
at Km. The Km values in this buffer were determined to
be 2.9, 2.0, 2.3, 2.1, and 7.0 mM, for the Yersinia PTP,
PTP1B, LAR, TCPTP, and CD45, respectively. IC50
values were calculated using a Dixon analysis. Data
analysis was performed using the commercial graphing
package Grafit (Erithacus Software, Ltd.). This pro-
5.28. Compound 34
Compound 34 was obtained from 34 mg (0.032 mmol)
of compound 33 by procedures analogous to the prepa-
ration of compound 30 (grey powder, 28 mg, 85%). H
1
NMR (400 MHz, DMSO-d6) d 3.06–3.17 (m, 4H),
4.68–4.83 (m, 6H), 6.99 (t, J = 7.6 Hz, 2H), 7.07 (t,
J = 7.6 Hz, 2H), 7.25 (s, 2H), 7.34 (d, J = 8.0 Hz, 2H),

472
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