Potent reversible inhibitors of the protein tyrosine phosphatase CD45

Article abstract of DOI:10.1021/jm000447i

The cytosolic portion of CD45, a major transmembrane glycoprotein found on nucleated hematopoietic cells, contains protein tyrosine phosphatase activity and is critical for T-cell receptor-mediated T-cell activation. CD45 inhibitors could have utility in the treatment of autoimmune disorders and organ graft rejection. A number of 9,10-phenanthrenediones were identified that reversibly inhibited CD45-mediated p-nitrophenyl phosphate (pNPP) hydrolysis. Chemistry efforts around the 9,10-phenanthrenedione core led to the most potent inhibitors known to date. In a functional assay, the compounds were also potent inhibitors of T-cell receptor-ediated proliferation, with activities in the low micromolar range paralleling their enzyme inhibition. It was also discovered that the nature of modification to the phenanthrenedione pharmacophore could affect selectivity for CD45 over PTP1B (protein tyrosine phosphatase 1B) or vice versa.

Full text of DOI:10.1021/jm000447i

J . Med. Chem. 2001, 44, 1777-1793
1777
P oten t Rever sible In h ibitor s of th e P r otein Tyr osin e P h osp h a ta se CD45
Rebecca A. Urbanek,* Suzanne J . Suchard,† Gary B. Steelman, Katharine S. Knappenberger, Linda A. Sygowski,
Chris A. Veale, and Marc J . Chapdelaine
AstraZeneca Pharmaceuticals, 1800 Concord Pike, Wilmington, Delaware 19850
Received October 17, 2000
The cytosolic portion of CD45, a major transmembrane glycoprotein found on nucleated
hematopoietic cells, contains protein tyrosine phosphatase activity and is critical for T-cell
receptor-mediated T-cell activation. CD45 inhibitors could have utility in the treatment of
autoimmune disorders and organ graft rejection. A number of 9,10-phenanthrenediones were
identified that reversibly inhibited CD45-mediated p-nitrophenyl phosphate (pNPP) hydrolysis.
Chemistry efforts around the 9,10-phenanthrenedione core led to the most potent inhibitors
known to date. In a functional assay, the compounds were also potent inhibitors of T-cell
receptor-mediated proliferation, with activities in the low micromolar range paralleling their
enzyme inhibition. It was also discovered that the nature of modification to the phenanthrene-
dione pharmacophore could affect selectivity for CD45 over PTP1B (protein tyrosine phosphatase
1B) or vice versa.
In tr od u ction
diverse family of enzymes that, together with the
protein tyrosine kinases (PTKs), control the level of
intracellular tyrosine phosphorylation, thus regulating
many cellular functions.9 The PTKs are responsible for
phosphorylating tyrosine residues and were previously
believed to be the only critical players in controlling
levels of tyrosine phosphorylation. The PTPs are re-
sponsible for dephosphorylating these phosphotyrosine
residues, and in the past decade great advances have
been made in understanding the important role the
PTPs play in maintaining the balance of tyrosine
phosphorylation. Currently approximately 100 PTPs are
known, and it has been predicted that as many as 500
PTP genes exist in the human genome.10 The PTP
family is recognized by an invariant amino acid signa-
ture motif present at the active site, (H/V)C(X)5R(S/T),
where the cysteine and arginine residues are essential
for catalytic activity. Three-dimensional structural data
show there are conserved structural elements such as
the PTP motif and a conserved aspartic acid residue on
a surface loop, but there is little primary sequence
similarity in the PTP family.11
Inappropriate lymphocyte and monocyte/macrophage
activation are an underlying cause of organ transplant
rejection and autoimmune diseases such as multiple
sclerosis and rheumatoid arthritis. Unfortunately, cur-
rent therapies targeted at reducing immune-cell activa-
tion in these diseases also affect cells outside the
immune system and consequently carry the liability of
toxic side effects. For example, the immunosuppressants
cyclosporine and FK506, which inhibit IL-2 production
in T-cells by blocking the phosphatase activity of cal-
cineurin, can also cause renal toxicity, neurotoxicity,
and the increased risk of malignancy.1-3 Rapamycin
inhibits T-cell proliferation farther down the signaling
pathway by inhibiting the autocrine response of T-cells
to IL-2 by binding to and blocking a kinase essential
for cell cycle progression.4 In contrast to cyclosporine
and FK506, rapamycin’s antiproliferative effects are not
limited to cells of the immune system and can affect
growth factor-induced proliferation of fibroblasts, en-
dothelial cells, hepatocytes, and smooth muscle cells.5
Recent trials in renal transplantation reported an
increased incidence of hypertriglyceridemia, hypercho-
lesterolemia, thrombocytopenia, and leukopenia with
sirolimus (rapamycin) treatment.6 In addition, some
psoriasis patients developed capillary leak syndrome
following oral administration of sirolimus.7 Thus, even
though rapamycin represents a different class of im-
munosuppressive agent, it is not without significant
toxicity.7,8 Immunosuppressive drugs that more specif-
ically target the signaling pathways in the hematopoi-
etic cells responsible for initiating and maintaining
inflammation should have fewer and/or different side
effects than cyclosporine, FK506, or rapamycin.
Although there is little sequence similarity, the PTPs
do utilize the same mechanism of dephosphorylation,
which involves the formation of a covalent thiophos-
phate intermediate by the invariant cysteine residue in
the PTP signature motif. The invariant arginine residue
in the PTP motif plays a role in substrate binding and
transition state stabilization, and the invariant aspartic
acid residue on the surface loop may act as a general
acid in the first step and a general base in the second
step, Figure 1.11
CD45 is a family of transmembrane PTPs that are
expressed exclusively by hematopoietic cells. CD45 plays
a critical role in T-cell receptor (TCR)-mediated signal-
ing by regulating the phosphorylation and activity of
src-family protein tyrosine kinases and their sub-
strates.12 The cytoplasmic domain of CD45 possesses the
tyrosine phosphatase activity and is capable of dephos-
phorylating the negative regulatory site on p56lck and
The protein tyrosine phosphatases (PTPs) are a
* To whom correspondence should be addressed: Tel: 302-886-4818.
Fax: 302-886-5382. E-mail: rebecca.urbanek@astrazeneca.com.
† Present Address: Bristol-Myers Squibb Pharmaceutical Research
Institute, Immunology, Inflammation and Pulmonary Discovery, P.O.
Box 4000, Princeton, NJ 08543-4000.
10.1021/jm000447i CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/02/2001

1778 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11
Urbanek et al.
F igu r e 1. Dephosphorylation mechanism of the PTPs.
p59fyn src kinases.13 This allows the autophosphorylation
of the positive regulatory site on these molecules and
propagation of TCR-mediated signaling. CD45 can also
associate with cell surface receptors that lack a trans-
membrane domain and may work in concert with these
receptors to transduce an activation signal.14,15 Blockade
of CD45 by anti-CD45 antibodies inhibits T-cell activa-
tion in vitro,16 and T-cells from CD45 KO mice fail to
respond to antigen challenge.12
Recently, Lazarovits et al.17 reported that a mono-
clonal antibody against the RB isoform of CD45 inhibits
the alloreactivity of human CD4+ lymphocytes in vitro
and prevents renal and pancreatic islet rejection in
mice.18,19 Furthermore, lymphocytes from antibody-
treated animals showed reduced levels of steady state
mRNA transcripts for IFN-γ, TNFR, and ICAM-1. These
studies highlight the potential value of selective small-
molecule inhibitors of CD45 in the treatment of trans-
plant rejection and autoimmune diseases.
F igu r e 2. Hits in the high-throughput screen.
Development of potent small-molecule PTP inhibitors
is in its infancy, but it is an area of increased interest
due to a growing list of important physiological roles
played by many PTPs.11 To date, the majority of early
PTP inhibitors are compounds with a nonhydrolyzable
phosphotyrosine mimic, which may or may not be
incorporated into a peptide scaffold.20-34 The negatively
charged phosphoryl mimic would be expected to make
cell permeability difficult.35 The peptide portion of these
inhibitors would be expected to be metabolically labile
in vivo.
F igu r e 3. Structurally related inactive compounds.
active in the range of 400-2000 nM with parallel
functional activity inhibiting T-cell proliferation in the
range of 300-1600 nM. The antiproliferative effect was
shown not to be due to cytotoxicity.
The required presence of a 1,2-dione embedded in a
six-membered ring to maintain the desired inhibitory
activity became evident upon survey of similar readily
available compounds (Figure 3). A number of compounds
of type 7, where the central dione-containing ring of
9,10-phenanthrenedione was replaced with a lactone or
lactam, were tested but no CD45 inhibition was ob-
served at 10 µM. The mono-oxime of 9,10-phenan-
threnedione, 8, was also tested, and again no inhibition
of CD45 was observed. Benzil and several of its deriva-
tives, including compounds which contained electron-
withdrawing groups, and other 1,2-diones (compounds
of type 9 and 10) were void of CD45 inhibitory activity
at 10 µM. Simple phenanthrene and 1,2-dioxoacenaph-
thene 11 failed to inhibit pNPP hydrolysis by CD45.
The availability of crystal structures of protein ty-
rosine phosphatase 1B (PTP1B) has facilitated the
development of small-molecule PTP1B inhibitors.35-40
PTP1B has been shown to be a negative regulator of
the insulin receptor, thus its inhibition could be thera-
peutically useful in the treatment of diabetes and
obesity.41 In the present report, we describe our efforts
leading to potent and selective inhibitors of CD45.
Id en tifica tion of Lea d Ser ies a n d Scop e of Ea r ly
SAR
High-throughput screening of an archival collection
of compounds utilizing the cytosolic portion of CD45 and
p-nitrophenyl phosphate (pNPP) as the substrate af-
forded the compounds shown in Figure 2 that were

Potent Reversible Inhibitors of PTP CD45
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1779
appropriate peptide scaffold.32,33 When phosphonodif-
luoromethyl phenylalanine (F2Pmp) was substituted for
Pmp, a 1000-fold increase in potency was observed.34
Crystallography showed that this dramatic increase in
potency may be attributable to an unconventional
hydrogen bond between one of the fluorines and a
phenylalanine amido group in the active site of the
enzyme.43 Compounds 12 and 13 gave no inhibition of
CD45 at 10 µM.
F igu r e 4. Inactive R-difluoro compounds.
Our analysis of the combined sets of actives and
inactives led us to focus on compounds that possessed
a six-membered near-planar ring containing a 1,2-dione;
thus the chemistry focus would need to be incremental
changes to the core structure. We restricted our syn-
thetic efforts to compounds that would satisfy these
requirements.
P r op osed Mech a n ism of Action
As shown in Figure 5, it is hypothesized that the
active site cysteine of CD45 attacks a carbonyl of the
dione, thus rendering the enzyme catalytically inactive.
At first glance, this mechanism is analogous to the
inhibition of serine proteases by electrophilic carbo-
nyl-based inhibitors.44 The results indicate, however,
that more than just a single electrophilic carbonyl
group is required, as compounds 12 and 13 would be
expected to inhibit the enzyme under the simplest
variation of this scheme. There must be more to the
inhibition; one hypothesis is that when the hemi-
thioketal is flanked by a vicinal carbonyl group, as it
is in the proposed covalent inhibitor-enzyme adduct,
a thiophosphate ester transition state is mimicked.
This hypothesis gave us a starting point from which to
work.
F igu r e 5. Hypothesized inhibitory mechanism.
Sch em e 1. Synthesis of 4-Aryl-1,2-naphthalenedionesa
a
Reagents: Pd2dba3, P(o-tol)3, K2CO3, 20% H2O-THF, reflux.
Twelve different 4-amino-1,2-naphthalenediones were
evaluated and were shown to be inactive against CD45
at 10 µM. Furthermore, evaluation of a set of 1,4-
naphthalenediones did not lead to compounds of suf-
ficient activity to warrant further investigation.
Ch em istr y
The known R-difluoro compounds 12 and 1342 (Figure
4) were synthesized: first, to maintain the electron
deficiency of the remaining carbonyl; and second, to
determine if a parallel with work in the PTP1B inhibitor
design area could be drawn. Specifically, phosphonom-
ethyl phenylalanine (Pmp) is a nonhydrolyzable pTyr
mimic and inhibits PTP1B when incorporated into an
The 4-aryl-substituted naphthalenediones were pre-
pared according to Scheme 1. Suzuki couplings45 with
4-bromo-1,2-naphthalenedione 1446 and various aryl
boronic acids afforded 15.
The nitrated phenanthrenediones 16 were synthe-
sized via known methods as discussed in the Experi-
Sch em e 2. Synthesis of Nitrogen-Substituted 9,10-Phenanthrenedionesa
a
Reagents: (a) Raney Ni, 50 psi H2, THF; (b) R1COCl, Na2CO3, THF; (c) TsCl, DMAP, Et3N, CH2Cl2.

1780 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11
Sch em e 3. Synthesis of Glycine-Derived Compoundsa
Urbanek et al.
Although this reaction was successfully run using
various arylboronic acids, the utility of the products
generated was limited. Only when the arylboronic acid
was heterocyclic or ortho-substituted was the product
sufficiently soluble for further evaluation.
The synthesis of the peptides in Scheme 5 begins with
3, which was coupled with N-FMOC-L-asparagine tert-
butyl ester to give 25. The tert-butyl ester was hydro-
lyzed to the carboxylic acid, and this was attached to
2-chlorotrityl resin to give 26. Standard solid-phase
peptide chemistry was employed, followed by acetylation
of the terminal nitrogen and cleavage off of the resin
with TFA to afford 27.
a
Reagents: (a) EDC, DMAP, iPr2NEt, DMF; (b) TFA, CH2Cl2.
Sch em e 4. Suzuki Coupling To Afford
2-Aryl-9,10-phenanthrenedionesa
The solid-supported tetrapeptide fragments 28 seen
in Scheme 6 were assembled using standard solid-phase
peptide synthesis techniques. The fragments were
coupled with the 9,10-phenanthrenedione-carboxylic
acids 21 to give 29. The 6-chlorochlortrityl resin link
was then cleaved using TFA to afford the carboxy-linked
peptides 30.
a
Reagents: Pd2dba3, P(o-tol)3, K2CO3, 10% H2O-dioxane,
80 °C.
mental Section, and these were reduced to the amino
compounds 17 using Raney nickel hydrogenation. These
amino compounds were converted to the amides 18 and
sulfonamides 19 and 20 using standard chemical trans-
formations, Scheme 2.
The 9,10-phenanthrenedione-carboxylic acids 2147
were coupled with tert-butyl glycine hydrochloride to
give 22; in turn, a portion of the tert-butyl esters were
hydrolyzed to the carboxylic acids 23, Scheme 3.
Resu lts a n d Discu ssion
The in vitro inhibitory activity of our compounds
against the cytosolic portion of CD45 was evaluated
initially using pNPP as the substrate and observing
p-nitrophenol release.49 The rate of CD45 inhibition by
our compounds continued to change over the first 40
min of incubation as seen in Figure 6, suggesting a slow
on-rate and/or a complex binding interaction.
2-Bromo-9,10-phenanthrenedione 248 was used as a
building block to assemble the 2-aryl-9,10-phenan-
threnediones 24 via Suzuki couplings,45 Scheme 4.
Na p h th a len ed ion es. 1,2-Naphthalenedione, 6, was
identified as an inhibitor of CD45 in high-throughput
Sch em e 5. Synthesis of Peptidyl 9,10-Phenanthrenedionesa
a
Reagents: (a) FMOC-Asp-OtBu, N-methylmorpholine, THF; (b) TFA, CH2Cl2; (c) iPr2NEt, 2-chlorochlortrityl resin, CH2Cl2-DMF;
(d) piperidine, DMF; (e) FMOC-protected amino acid, HATU, iPr2NEt, DMF; (f) repeat steps d and e; (g) Ac2O, iPr2NEt, DMF; (h) TFA,
CH2Cl2.
Sch em e 6. Synthesis of Carboxy-Linked Tetrapeptide Inhibitorsa
a
Reagents: (a) HATU, iPr2NEt, DMF; (b) TFA.

Potent Reversible Inhibitors of PTP CD45
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1781
Ta ble 1. In Vitro Activity of the 1,2-Naphthalenedionesa
a
b
The reported values are the mean of all experiments, and the errors are standard errors of the mean. ND ) not determined.
cytotoxicity led us to expend chemistry efforts to further
this series.
The known compounds 34 and 35 were analyzed to
verify the surmised need for an electron-deficient car-
bonyl in our inhibitors.50 Surprisingly, these two com-
pounds are similarly potent to 6 in the enzyme inhibi-
tion assay, but inactive at inhibiting T-cell prolifera-
tion. Activity against the enzyme was surprising since
all 12 compounds tested with a 4-amino substituent on
the 1,2-naphthalenedione, including the three shown
here in the table (31-33), did not inhibit pNPP hy-
drolysis by CD45 at 10 µM. We envisaged similar
electronics between the 4-alkoxy- and 4-amino-1,2-naph-
F igu r e 6. Kinetic analysis of the hydrolysis of pNPP by CD45
alone (control) or in the presence of 1.5 µM compound 2. Note
the curvature in the reaction progress curve which indicates
the “slow-binding” nature of the inhibition of CD45 by the
compound.
thalenedione series and predicted, incorrectly, similar
enzymatic activities. 4-Bromo-1,2-naphthalenedione 1446
was active at inhibiting CD45 with an IC50 of 8.7 µM
but was only marginally active at inhibiting T-cell
proliferation.
The known indoles 36 and 3751 were active against
both the enzyme and T-cell proliferation. Both com-
pounds had favorable ratios of cytotoxicity to cell
screening. It inhibited CD45 and T-cell inhibition in
vitro in the single-digit micromolar range (Table 1).
Furthermore, it was not toxic to T-cells at 30 µM. The
4-fold margin of inhibition of T-cell proliferation to cell

1782 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11
Urbanek et al.
F igu r e 8. Kinetic analysis of the hydrolysis of pNPP by CD45
alone (control) or in the presence of 1.5 µM of compound 2.
(A) CD45 and compound were incubated for 90 min prior to
the addition of pNPP. (B) pNPP and compound were incubated
for 90 min prior to the addition of CD45. Note that preincu-
bation of CD45 with compound reduced the curvature in the
reaction progress curve consistent with the “slow-binding”
nature of the inhibition of CD45 by the compound.
F igu r e 7. (A) Lineweaver-Burk analysis of CD45 inhibition
by compound 5. The activity of CD45 was measured at steady
state as described in the Experimental Procedures section in
the presence of the following concentrations of compound: 9,
0 µM; 0, 0.25 µM; b, 0.5 µM; O, 0.75 µM; 2, 1.0 µM; 3, 1.5
µM. (B) Replot of the slope from the double-reciprocal plot
versus concentrations of compound.
proliferation (CC50/IC50 proliferation), with 20- and
8-fold margins, respectively. Again, this positive result
was unexpected considering the 3-position of indoles is
electron-rich. This effect could be explained by the added
aromaticity imparted by the indoles.
The 4-aryl-1,2-naphthalenediones 38-42, synthesized
to mimic the C ring of the 9,10-phenanthrenediones,
displayed levels of enzyme inhibition similar to those
of the other 1,2-naphthalenediones with no increase in
potency against CD45 relative to 6. On the other hand,
the 4-aryl-1,2-naphthalenediones were among the most
potent found at inhibiting T-cell proliferation. Even
though cell cytotoxicity was observed in the sub-30 µM
range, the ratios of cell cytotoxicity to inhibition of T-cell
proliferation for this group of compounds ranged from
as low as 18 to as high as 41.
The compounds 43-46 were also active against CD45
and displayed no cytotoxicity at 30 µM, and the subset
of compounds 43-45 inhibited T-cell proliferation in the
micromolar range. In general, based on our limited
exploration, the 1,2-naphthalenedione series displayed
a flat SAR against the isolated enzyme. The 1,2-
naphthalenedione series was functionally active and
displayed favorable cytoxicity characteristics; however,
chemical stability might be an issue for both synthetic
F igu r e 9. Reversible nature of CD45 inhibition by compound
4. CD45 was incubated with 5 mM pNPP in the presence of
compound, and the reaction was allowed to reach “steady state”
kinetics. At 60 min, 10-fold excess pNPP was added to the
reaction mixture to reverse inhibition.
intermediates and final products in this series. Coupled
with the fact that the 9,10-phenanthrenediones were
more potent at inhibiting CD45, the stability issues led
us to focus upon the 9,10-phenanthrenedione series.
9,10-P h en a n th r en ed ion es. Initially, a more de-
tailed kinetic analysis of the compounds in the 9,10-
phenanthrenedione series was undertaken. As shown
in Figure 7A, the effect of these compounds on the
CD45-catalyzed pNPP hydrolysis displayed the inter-
secting line pattern characteristic of a competitive in-
hibitor. However, the replot of the slope from the double-
reciprocal plot versus compound concentration was
hyperbolic, demonstrating a complex binding interaction
and suggesting that the compound was not only com-
peting at the substrate binding site (Figure 7B). This
is similar to a report by Puius et al.52 showing that pTyr
and bis-(para-phosphophenyl)methane substrates can

Potent Reversible Inhibitors of PTP CD45
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1783
Ta ble 2. Inhibitory Trend in a Series of Amino-9,10-phenanthrenedionesa
pNPP
lck
prolif.
compd
R1
R2
R3
R4
IC50, µM
IC50, µM
IC50, µM
CC50, µM
47
3
48
49
50
51
NH2
H
H
H
NH2
H
H
H
H
H
NH2
H
H
H
H
H
NH2
H
>30
>30
7.8 ( 0.8
0.2 ( 0.6
0.2 ( 0.1
0.2 ( 0.1
0.5 ( 0.2
1.0 ( 0.3
9.6 ( 0.6
5.0 ( 1.0
17.0 ( 3.5
9.0 ( 2.0
9.0 ( 2.3
18.0 ( 2.5
0.4 ( 0.1
3.7 ( 1.5
0.8 ( 0.2
0.4 ( 0.1
0.4 ( 0.1
2.3 ( 0.9
10.5 ( 2.2
2.9 ( 1.5
4.7 ( 1.2
3.2 ( 0.7
H
NHC(O)(CH2)2CO2Me
H
H
NHC(O)(CH2)2CO2Me
H
a
The reported values are the mean of all experiments, and the errors are standard errors of the mean.
interact with more than one site on PTP1B. In addition
to occupying the canonical pTyr-binding site (the active
site), these substrate molecules also interact with a low-
affinity noncatalytic aryl phosphate-binding site adja-
cent to the active site.
Preincubating CD45 with compound for 90 min before
the addition of substrate resulted in a linear rate of
pNPP hydrolysis as seen in Figure 8A. In contrast,
preincubating compound with substrate for 90 min had
no effect on the kinetics of substrate hydrolysis (Fig-
ure 8B). These findings further illustrate that these
compounds are slow-binding inhibitors of CD45. It
remains unresolved whether the compounds are under-
going hydration, there is a change in the conformation
of the enzyme, and/or the compounds bind to multiple
sites on the enzyme during the first 40 min of incuba-
tion.
To rule out the possibility that the phenanthrenedi-
ones were simply inactivating CD45 by direct or indirect
oxidation of the catalytic cysteine residue, we conducted
experiments to determine if the compounds were re-
versible inhibitors. The inhibition of CD45 by our
compounds was completely reversed by addition of
excess substrate (Figure 9). It should be emphasized
that the renewed turnover of pNPP occurs readily even
in the absence of additional reductant. This is in striking
contrast to pervanadate, a commonly used inhibitor of
PTPs, which is a potent oxidizing agent that causes
irreversible inhibition of PTP1B.53
One of the first trends observed with the 9,10-
phenanthrenedione series was the positional depen-
dence of the amino-group effect, Table 2. 1-Amino-9,10-
phenanthrenedione 47, with an electron-donating amino
group ortho-positioned to one of the carbonyls, is inac-
tive against the isolated enzyme. The 2- and 4-amino-
9,10-phenanthrenediones, 3 and 49, where the meta-
amino groups cannot be in resonance with the nearby
carbonyl, are active with pNPP IC50s in the submicro-
molar range. The conjugated para-position 3-amino
group in 48 causes a loss of roughly an order of
magnitude in potency relative to the two meta-posi-
tioned amino-substituted congeners 3 and 49. Interest-
ingly, when the 1- and 3-amino groups of 47 and 48 are
converted into the electron-withdrawing succinamyl
methyl esters 50 and 51, the enzyme activity is restored.
No crisp parallel trend exists in the functional activity
measurement opposite T-cell proliferation inhibition by
these compounds. With the exception of 47, all com-
pounds in Table 2 are functionally active with submi-
cromolar IC50s. The 8 µM antiproliferative activity of
47, in the absence of enzymatic activity, is explicable
and attributable to the cytotoxicity of the compound.
The other five compounds of Table 2 have safety
margins between 18 and 85 with respect to the ratio of
cytotoxicity to functional activity.
Many of the compounds were also evaluated in
endpoint assays using a synthetic phosphotyrosyl pep-
tide corresponding to phosphorylated lck505 [ATEGQ-
p YQPQP] as the substrate.54,55 This peptide corresponds
to the negative regulatory pTyr site of p56lck which is
dephosphorylated by CD45 in vitro.12 The same rank
order of inhibitory capacity was observed when the lck
10-mer was used as substrate rather than pNPP, Table
2. Several lessons were learned from this small subset
of compounds: (a) electron-donating groups in direct
conjugation with the proximal carbonyl decrease or
completely abolish inhibitory activity, which follows the
hypothesized mechanism of inhibition; (b) the lck 10-
mer data follow the same trend as pNPP data but are
about 10-fold less potent; (c) inhibition of T-cell prolif-
eration and cytotoxicity can be separated.
Results with a more diverse set of 9,10-phenan-
threnediones are shown in Table 3. The introduction of
a nitro group onto the phenanthrenedione core, namely
compounds 4, 5, 52, and 53, gives compounds with
enzymatic inhibitory capability that are similar to the
parent 9,10-phenanthrenedione 1. It was encouraging
that the cytotoxicity of these compounds was lower than
that of the parent structure. The 2,5-dinitro 54, inter-
estingly, is devoid of CD45 inhibitory activity, antipro-
liferative activity, and cytotoxicity.
Several carboxylic acid-substituted 9,10-phenanthrene-
diones were synthesized to potentially interact with the
invariant Arg residue located at the active site. The
carboxylic acids 55 and 56 were active against the
enzyme but functionally inactive, presumably due to cell
permeability issues. The glycine-derived carboxylic acids
57 and 58 maintained enzymatic activity, inhibited
T-cell proliferation, and displayed no T-cell cytotoxicity
at 30 µM. The tert-butyl ester 59 displayed a higher level
of T-cell proliferation inhibition than its acid counter-
part 57, consistent with a neutral species’ more facile
passive diffusion across a membrane compared to its
anionic counterpart.
Next a number of amides that reversed the position
of the NH and CdO relative to what it had been in

1784 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11
Ta ble 3. In Vitro Activity of the Phenanthrenedionesa
Urbanek et al.

Potent Reversible Inhibitors of PTP CD45
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1785
Ta ble 3 (Continued)
a
b
The reported values are the mean of all experiments, and the errors are standard errors of the mean. POA ) position of attachment.
c ND ) not determined.
compounds 57-60 and the two sulfonamides 19 and 20
were synthesized from 2- or 3-amino-9,10-phenan-
threnedione. Among the amides made and tested were
amide-ester variants, with a varying number of meth-
ylene spacer groups between the amide and the ester
functionality (compounds 61-64). In general, all these
variants maintained the submicromolar enzyme inhibi-
tion of the of the glycine acids regardless of the number
of methylene spacers introduced. Unfortunately, modest
increases in inhibition of T-cell proliferation were offset
by a parallel increase in cytotoxicity (below 30 µM).
Of the simple alkyl amides made, the pivalamide 65
is the most potent compound at inhibiting pNPP hy-
drolysis and T-cell proliferation, with IC50 values of 200
and 100 nM, respectively. Compound 65 also main-
tained a 35-fold margin in inhibition of proliferation over
cytotoxicity. It should be noted that the alkyl amides
and benzoyl amides (compounds 69-72) inhibit CD45

1786 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11
Ta ble 4. In Vitro Activity of Heterocyclic Analoguesa
Urbanek et al.
compounds 88-105, where the peptide chain is attached
to 2-carboxy-9,10-phenanthrenedione: T-cell prolifera-
tion was inhibited by varying degrees. These compounds
also show no cytotoxicity at 30 µM. In contrast, com-
pounds 106-121, those linked to the 3-position of the
dione core (the most “lck-like”), have essentially lost
functional activity.
Selectivity Stu d ies. The large number of PTPs in
the human body make it important to demonstrate
selectivity for CD45 over other phosphatases. To exam-
ine this issue, the lck 10-mer was used as the substrate
and a subset of phenanthrenediones were compared for
activity against CD45 and PTP1B, a tyrosine phospha-
tase that is ca. 40% sequence homologous with CD45.
The lck peptide was derived from the COOH terminus
of p56lck (ATEGQp YQPQP), a potential in vivo substrate
of CD45.58 This specific phosphopeptide has been used
by others to characterize CD45 and other PTPases for
specificity and selectivity.55 In kinetic assays, the Km
of CD45 for pNPP, the substrate used in the initial
studies, is 4.8 mM, with a catalytic efficiency, measured
as kcat/Km, of 1.1 × 104 s-1 M.-1 With the lck peptide as
substrate, the Km value changed to 130 µM and kcat/Km
rises to 76 × 104 s-1 M,-1 a vast improvement over
pNPP.55 However, the rank order potency of the com-
pounds tested did not change with lck peptide substrate,
giving us confidence that these inhibitors would block
the activity of CD45 toward its physiological substrate.
a
The reported values are the mean of all experiments, and the
errors are standard errors of the mean.
with similar IC50s. However, the alkyl amides were
slightly more potent inhibitors of T-cell proliferation
relative to the benzoyl amides.
A small subset of 2-aryl-9,10-phenanthrenediones
73-78 was also investigated. These possess IC50s
against the isolated enzyme and against T-cell prolif-
eration in the 1 µM range, with variable cytotoxicity.
To improve the physical properties of the 9,10-phe-
nanthrenediones, a few heterocyclic variants were made,
Table 4. The furanyl-, thiophenyl-,56 and pyridyl-57 com-
pounds 79-82 produced sustained CD45 inhibition, but
they lost potency relative to 1. They all maintained
functional activity (inhibition of T-cell proliferation)
while exhibiting variable cytotoxicity.
The Km of CD45 for the lck 10-mer was also deter-
mined using the end-point assay. In these assays, the
ratio of lck to CD45 is about 3-fold higher than that used
in the kinetic assays. The Km value of CD45 for lck in
this assay was 43 µM. The Km values for PTP1B and
FAP (Fas-associated phosphatase) for lck were similar
to that obtained for CD45, with Km values of 46 and 38
µM, respectively.
The first seven entries in Table 7 show that diversely
substituted phenanthrenediones are inhibitors of CD45
with IC50s in the single-digit micromolar range, while
not inhibiting PTP1B. The 2-linked benzoyl amides 68
and 70, however, inhibit both enzymes essentially
equipotently. Compound 71, which is merely a positional
isomer of 70, regains its CD45 activity and loses the
PTP1B activity. The 2-aryl-9,10-phenanthrenediones 73,
74, and 77 show a reversal of selectivity, as these are
now selective for PTP1B over CD45. The data in Table
7 demonstrate that, by making a simple structural
modification (location or absence of an aryl group off of
the 2-position), modulation of this series of inhibitors’
selectivity for CD45 versus PTP1B is attainable. These
findings follow what is known in the PTP1B inhibitor
design area; Burke et al. showed that PTP1B has
multiple binding interactions possible for polyaromatic
systems.39 It has also been shown more recently that
the active site of PTP1B can accommodate substituted
and extended aromatic systems, and these aromatic
systems also interact beyond the catalytic site, which
could lead to higher potency and phosphatase specific-
ity.35 This “plasticity” of PTP1B to allow binding of large
hydrophobic groups seems to be attributable to the
conformation of a single invariant amino acid residue
at the active site.59 Most of the non-peptide small-
molecule inhibitors of PTP1B and other PTPs have an
aromatic moiety as the core structure.
P ep tid yl In h ibitor s. As discussed previously, early
PTP inhibitors focused on the incorporation of a non-
hydrolyzable phosphotyrosine mimic into an appropriate
peptide scaffold. In an attempt to increase the binding
potency, various peptides were appended to the 9,10-
phenanthrenedione core. The dione core was incorpo-
rated into peptides that correspond to the sequence Ala-
Thr-Glu-Gly-Gln-pTyr -Gln-Pro-Gln-Pro surrounding the
critical tyrosine residue in the src kinase p56lck, a
physiological substrate of CD45. Table 5 shows com-
pounds where the peptide chain was built to the
N-terminus of the lck peptide. These compounds inhib-
ited CD45 in the single-digit micromolar range but were
only marginally active or not active in inhibiting T-cell
proliferation, presumably because of poor cellular per-
meability. Table 6 shows compounds where the peptide
chain was appended to 2- or 3-carboxy-9,10-phenan-
threnedione and progressed to the C-terminus of the lck
peptide. More diversity was built into this subset of
peptides, particularly those linked to the 2-carboxy, to
take advantage of other potential recognition elements
in the enzyme. These compounds, like the compounds
in Table 5, were able to maintain, but not increase,
potency against CD45. One interpretation of these
results is that the amino acid residues do not make
favorable contacts with the enzyme. On the other hand,
no serious repulsions exist since enzyme inhibition is
maintained. One interesting trend is noteworthy in

Potent Reversible Inhibitors of PTP CD45
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1787
Ta ble 5. In Vitro Activity of 9,10-Phenanthrenedione-Incorporated Peptidyl Inhibitorsa
compd
AA1..AAn-Ac
pNPP IC50, µM
prolif. IC50, µM
CC50, µM
83
84
85
86
87
Gln
Gln-Gly-Glu
Gln-Gly-Glu-Thr-Ala
Gln-Gly-Glu-Thr-Ala-Thr
Gln-Gly-Glu-Thr-Ala-Thr-Phe
1.1 ( 0.2
1.5 ( 0.3
2.0 ( 0.3
1.8 ( 0.3
1.4 ( 0.2
>30
NDb
ND
ND
ND
ND
>30
21.4 ( 2.5
23.0 ( 7.0
>30
a
b
The reported values are the mean of all experiments, and the errors are standard errors of the mean. ND ) not determined.
Ta ble 6. In Vitro Activity of Additional Peptidyl Inhibitorsa
pNPP
prolif.
pNPP
prolif.
IC50
µM
,
IC50
µM
,
CC50
,
IC50
µM
,
IC50
µM
,
CC50,
µM
compd POAb AA1-AA2-AA3-AA4-AA5
µM compd POAb AA1-AA2-AA3-AA4-AA5
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
Gly-Gly-Pro-Glu-Gly 0.6 ( 0.3 19.3 ( 7.9 >30 105
Glu-Gly-Pro-Glu-Gly 1.4 ( 0.0 8.8 ( 0.5 >30 106
Arg-Gly-Pro-Glu-Gly 0.9 ( 0.0 3.3 ( 0.2 >30 107
Gly-Glu-Pro-Glu-Gly 1.9 ( 0.3 9.0 ( 2.9 >30 108
Glu-Glu-Pro-Glu-Gly 1.1 ( 0.0 14.2 ( 5.7 >30 109
Arg-Glu-Pro-Glu-Gly 1.4 ( 0.2 5.0 ( 1.6 >30 110
Gly-Arg-Pro-Glu-Gly 1.0 ( 0.6 5.5 ( 3.3 >30 111
Glu-Arg-Pro-Glu-Gly 0.7 ( 0.1 3.0 ( 1.5 >30 112
Arg-Arg-Pro-Glu-Gly 0.6 ( 0.2 11.0 ( 5.1 >30 113
Gly-Gly-Pro-Arg-Gly 0.9 ( 0.3 16.0 ( 3.8 >30 114
Glu-Gly-Pro-Arg-Gly 0.7 ( 0.1 23.0 ( 1.5 >30 115
Arg-Gly-Pro-Arg-Gly 1.1 ( 0.2 14.0 ( 1.5 >30 116
Gly-Glu-Pro-Arg-Gly 0.8 ( 0.2 20.0 ( 2.5 >30 117
Glu-Glu-Pro-Arg-Gly 1.0 ( 0.3 16.0 ( 6.7 >30 118
Arg-Glu-Pro-Arg-Glu 0.9 ( 0.2 20.0 ( 5.5 >30 119
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Arg-Arg-Pro-Arg-Gly 1.3 ( 0.3 21.0 ( 8.7 >30
Glu-Gln-Pro-Gln-Pro 0.6 ( 0.1 >30
Gly-Gln-Pro-Gln-Pro 1.2 ( 0.1 >30
Gln-Gln-Pro-Gln-Pro 0.7 ( 0.4 >30
Gly-Glu-Pro-Gln-Pro 0.7 ( 0.0 >30
>30
>30
>30
>30
Gln-Glu-Pro-Gln-Pro 1.1 ( 0.3
5.0 ( 2.4 >30
Gly-Gln-Gly-Gln-Pro 1.1 ( 0.1 20.0 ( 1.8 >30
Gln-Gln-Gly-Gln-Pro 0.9 ( 0.4 14.5 ( 7.2 >30
Glu-Gln-Gly-Gln-Pro 0.6 ( 0.1
7.0 ( 2.5 >30
Gly-Glu-Gly-Gln-Pro 0.7 ( 0.1 10.5 ( 3.2 >30
Gln-Glu-Gly-Gln-Pro 1.1 ( 0.2 16.0 ( 10.3 >30
Gln-Gln-Pro-Glu-Gly 0.9 ( 0.1 >30
Gly-Gln-Gly-Glu-Pro 0.9 ( 0.01 >30
Gly-Gln-Pro-Gln-Gly 1.0 ( 0.1 >30
Gln-Gln-Pro-Gln-Gly 1.1 ( 0.1 >30
Gly-Glu-Pro-Gln-Gly 0.7 ( 0.0 >30
Gly-Gln-Pro-Glu-Gly 0.7 ( 0.1 >30
NDc
>30
>30
>30
>30
>30
Gly-Arg-Pro-Arg-Gly 1.4 ( 0.0 >30
>30 120
Glu-Arg-Pro-Arg-Gly 0.8 ( 0.2 19.0 ( 5.1 >30 121
a
b
The reported values are the mean of all experiments, and the errors are standard errors of the mean. POA ) position of attachment.
c ND ) not determined.
Ta ble 7. PTP1B Selectivitya
Ta ble 8. Further Selectivity Dataa
CD45 lck
IC50, µM
PTP1B lck
IC50, µM
CD45 CD45 FAP FAP cathep. cathep. cathep.
compd
pNPP
compd IC50
lck
IC50
pNPP
IC50
lck
IC50
B
IC50
L
IC50
S
IC50
b
b
b,c
b,d
b,e
1
59
3
3.1 ( 1.2
4.9 ( 1.5
2.3 ( 0.9
3.8 ( 0.3
5.2 ( 2.6
4.5 ( 2.8
3.1 ( 0.7
11.0 ( 0.9
11.0 ( 5.2
5.8 ( 2.0
>30
>30
>30
3
57
64
63
50
70
65
82
0.4
0.8
0.3
0.5
0.4
0.6
0.2
1.4
2.3
1.9
2.9
3.7
4.7
11
4.7
15
12
9.9
4.4
0.6
7
>30
>30
>30
>30
>30
20
>10
>10
>10
>10
>10
>10
>10
>10
0.6
0.9
5.4
6.0
>10
>10
>10
ND
9
>30
65
67
79
82
68
70
71
73
74
77
>30
>30
>10
NDf
0.4
>30
>30
18.0 ( 2.5
18.0 ( 0.5
>30
3.8
3.1
>30
>30
0.5
0.6
0.4
0.4
14
a
b
All data are given in µM. The value is the mean of experi-
ments run in triplicate. c CBZ-L-lysine p-nitrophenyl ester used
19.0 ( 1.0
18.0 ( 1.6
16.0 ( 1.8
>30
>30
d
as substrate. Z-Phe-Arg-NH-Mec used as substrate. e Z-Val-Val-
Arg-NH-Mec used as substrate. f ND ) not determined.
a
The reported values are the mean of all experiments, and the
errors are standard errors of the mean.
of compounds was tested against proteases that have
active site cysteine residues and also against another
tyrosine phosphatase, FAP, as shown in Table 8.
Although some compounds do show activity against
cathepsin L and S, this is not seen for all compounds,
The selectivity seen between CD45 and PTP1B was
very encouraging, but to further address the concern
that the phenanthrenediones would be nonspecific and
react with cysteine residues in general, a diverse subset

1788 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11
Urbanek et al.
5,6-Dioxo-5,6-d ih yd r o-n a p h th a len e-1-ca r boxylic a cid
m eth yl ester (43): orange solid; H NMR (300 MHz, CDCl3)
and none of the compounds tested were active against
cathepsin B. The active site cysteine residue of cysteine
proteases such as the cathepsins B, L, and S is readily
oxidized, so these findings further support that the
phenanthrenediones are not acting as oxidizing agents
in the enzyme inhibition assays.60 Likewise, with the
exception of compound 70, these compounds are far less
potent against FAP when using either pNPP or the lck
10-mer as the substrate. These data, combined with the
findings on PTP1B, indicate these compounds are not
simple “thiol traps” and that it is possible to synthesize
inhibitors with selectivity among tyrosine phosphatases.
In summary, a series of 9,10-phenanthrenediones has
been developed that inhibit the protein tyrosine phos-
phatase CD45 and T-cell proliferation in the 1 µM range.
These inhibitors were found to be reversible, competitive
inhibitors of CD45-induced pNPP hydrolysis. Selectivity
for CD45 over PTP1B is attainable by a simple struc-
tural modification, and selectivity for CD45 over FAP
and three evaluated cysteine proteases is also achiev-
able. The compounds presented in this paper are the
most potent small-molecule inhibitors of CD45 known
to date.
1
δ 8.62 (1H, d, J ) 11 Hz), 8.29 (1H, d, J ) 7.8 Hz), 8.20 (1H,
d, J ) 7.8 Hz), 7.58 (1H, t, J ) 7.8 Hz), 6.56 (1H, d, J ) 11
Hz), 3.99 (3H, s); HPLC 3.69 min. HRMS theor. [M + H]:
217.0501 amu; obs. [M + H]: 217.0518 amu; deviation of 7.9
ppm.
5,6-Dioxo-5,6-d ih yd r o-n a p h th a len e-2-ca r boxylic a cid
m eth yl ester (44): orange solid; 1H NMR (300 MHz, CDCl3)
δ 8.17 (2H, m), 8.05 (1H, s), 7.52 (1H, d, J ) 10.2 Hz), 6.53
(1H, d, J ) 10.2 Hz), 3.99 (3H, s); HPLC 3.61 min.
6-Ben zoyl-[1,2]n a p h th oqu in on e (45): orange solid; 1H
NMR (300 MHz, CDCl3) δ 8.23 (1H, d, J ) 7.8 Hz), 7.86 (1H,
dd, J ) 1.5, 7.8 Hz), 7.83-7.79 (3H, m), 7.67 (1H, m), 7.57-
7.51 (3H, m), 6.54 (1H, d, J ) 10.2 Hz); HPLC 5.93 min. HRMS
theor. [M + H]: 263.0708 amu; obs. [M + H]: 263.072 amu;
deviation of 4.5 ppm.
7-Hyd r oxy-[1,2]n a p h th oqu in on e (46). This compound
was prepared by the method of Teuber and Goetz:62 red solid;
1H NMR (300 MHz, DMSO-d6) δ 10.50 (1H, broad s), 7.56 (1H,
d, J ) 9.8 Hz), 7.40 (1H, d, J ) 8.1 Hz, 7.31 (1H, d, J ) 2.7
Hz), 7.05 (1H, dd, J ) 8.1, 2.7 Hz), 6.16 (1H, d, J ) 9.9 Hz).
Anal. Calcd. for C10H6O3‚0.1H2O: C, 68.26; H, 3.55. Found:
C, 67.96, 67.89; H, 3.64, 3.65.
Compounds 4, 5, 53, and 54 were synthesized as described
by Schmidt,63 taking into account the results disclosed by Ray
and Francis.64
2-Nit r o-p h en a n t h r en e-9,10-d ion e (4): orange solid; 1H
NMR (300 MHz, DMSO-d6) δ 8.80 (1H, d, J ) 2.8 Hz), 8.69
(1H, d, J ) 8.5 Hz), 8.60 (1H, dd, J ) 2.4, 8.7 Hz), 8.51 (1H,
d, J ) 8.3 Hz), 8.22 (1H, dd, J ) 1.4, 7.7 Hz), 7.93 (1H, ddd,
J ) 1.6, 8.1, 8.1 Hz), 7.73 (1H, dd, J ) 7.5, 7.5 Hz). Anal.
(C14H7NO4) C, H, N.
4-Nit r o-p h en a n t h r en e-9,10-d ion e (5): orange solid; 1H
NMR (300 MHz, DMSO-d6) δ 8.27 (1H, dd, J ) 1.5, 7.9 Hz),
8.17 (1H, dd, J ) 1.3, 7.9 Hz), 8.10 (1H, dd, J ) 1.5, 7.8 Hz),
7.79-7.71 (2H, m), 7.63 (1H, ddd, J ) 0.9, 7.7, 7.7 Hz), 7.46
(1H, d, J ) 8.0 Hz). Anal. (C14H7NO4‚0.25H2O) C, H, N.
2,7-Din itr o-p h en a n th r en e-9,10-d ion e (53): yellow solid;
1H NMR (300 MHz, tfashake-DMSO-d6) δ 8.77 (2H, d, J ) 2.7
Hz), 8.76 (2H, d, J ) 9.1 Hz), 8.62 (2H, dd, J ) 2.7, 9.1 Hz).
Anal. (C14H6N2O6) C, H, N.
Exp er im en ta l Section
Ch em istr y. 1H NMR spectra were obtained at 300 MHz
using a Bruker DPX 300 spectrometer and were referenced to
TMS. Analytical HPLC was done on an HP 1100 HPLC, with
a C18 Dynamax column (5 cm × 4.6 mm, 3 µM particle size,
100 Å pore size), and a flow rate of 0.5 mL/min, 20-60% CH3-
CN in H2O over 7.5 min, holding at 60% CH3CN for 2.5 min,
while monitoring at 254 and 210 nm. Mass spectral data were
obtained on a Micromass QTOF mass spectrometer. Silica gel
chromatography was performed with ICN silica 32-63, 60 Å.
Thin-layer chromatography was done on silica gel 60 F-254
(0.25 mm thickness) plates, and visualization was accom-
plished with UV light. Elemental analyses (C, H, N) were
performed on an Exeter Analytical CE-440 elemental analyzer,
and all compounds are within (0.4% of theory unless other-
wise indicated. Unless otherwise noted, all materials were
obtained commercially and used without further purification.
Compounds 1, 6, 31, 32, and 33 were purchased from
commercial sources and used as received.
4-(2-Ch lor o-p h en yl)-[1,2]n a p h th oqu in on e (42). To a
solution of 14 (350 mg, 1.48 mmol) in THF (20 mL) and H2O
(5 mL) was added 2-chlorophenylboronic acid (231 mg, 1.48
mmol), followed by tri-o-tolylphosphine (45 mg, 148 µmol) and
K2CO3 (614 mg, 4.44 mmol). The mixture was deoxygenated
with bubbling N2 for about 10 min, at which time the N2 line
was removed and tris(dibenzylideneacetone)dipalladium(0) (68
mg, 74 µmol) was added. The resultant mixture was heated
to reflux for 2 h under N2, at which point no starting bromide
was detectable by TLC (hexanes:ethyl acetate, 1:1, v/v). The
mixture was cooled to room temperature, and the THF
evaporated under reduced pressure. The dried material was
dissolved in ethyl acetate, washed sequentially with saturated
aqueous ammonium chloride, H2O, and brine, dried over Na2-
SO4, filtered, and evaporated under reduced pressure. The
isolated material was chromatographed on silica gel (hexanes-
ethyl acetate, 4:1, v/v) and dried to yield 42 as an orange
solid: 1H NMR (300 MHz, CDCl3) δ 8.21 (1H, dd, J ) 6.9, 2.1
Hz), 7.56-7.52 (3H, m), 7.48-7.44 (2H, m), 7.44 (1H, m), 6.91
(1H, dd, J ) 2, 6 Hz), 6.39 (1H, s); HPLC 7.45 min. HRMS
theor. [M + H]: 269.0369 amu; obs. [M + H]: 269.0366 amu;
deviation of -1.4 ppm.
2,5-Din itr o-p h en a n th r en e-9,10-d ion e (54): yellow solid;
1H NMR (300 MHz, DMSO-d6) δ 8.67 (1H, d, J ) 2.5 Hz), 8.54
(1H, dd, J ) 2.5, 9.1 Hz), 8.36 (1H, dd, J ) 1.5, 8.1 Hz), 8.26
(1H, dd, J ) 1.3, 7.8 Hz), 7.87 (1H, dd, J ) 7.8, 7.8 Hz), 7.73
(1H, d, J ) 8.8 Hz).
3-Nitr o-p h en a n th r en e-9,10-d ion e (52). This compound
was prepared as described by Braithwaite and Holt:65 orange
solid; 1H NMR (300 MHz, TFA-d1-DMSO-d6) δ 9.02 (1H, d, J
) 1.7 Hz), 8.50 (1H, d, J ) 8.1 Hz), 8.29 (2H, m), 8.13 (1H, dd,
J ) 1.1, 7.6 Hz), 7.85 (1H, ddd, J ) 1.3, 7.7, 7.7 Hz), 7.64 (1H,
dd, J ) 7.4, 7.4 Hz). Anal. (C14H7NO4‚0.1H2O) C, H, N.
2,7-Dia m in o-p h en a n th r en e-9,10-d ion e: Compound 53
(3.0 g, 10 mmol) was suspended in THF (200 mL), and Raney
nickel (ca. 1 g) was added. The reaction was hydrogenated on
a Parr shaker under 50 psi H2 for 2.5 h, at which time the
mixture was filtered through a plug of diatomaceous earth,
and the filtrate was evaporated under rotary evaporation. The
product was chromatographed on silica gel (15% to 100%
EtOAc in CH2Cl2 as eluant) and dried to afford 2,7-diamino-
phenanthrene-9,10-dione (1.1 g, 4.6 mmol, 46%) as a black
solid. 1H NMR (300 MHz, TFA-d1-DMSO-d6) δ 8.77 (2H, d, J
) 2.7 Hz), 8.76 (2H, d, J ) 9.1 Hz), 8.62 (2H, dd, J ) 2.7, 9.1
Hz). Anal. Calcd for C14H6N2O6: C, 56.39; H, 2.02; N, 9.39.
Found: C, 56.20, 56.44; H, 2.17, 2.14; N, 8.89, 8.95.
The compounds 3, 47, 48, and 49 were synthesized from the
appropriate nitro compound according to the general procedure
given for 2,7-diamino-phenanthrene-9,10-dione.
2-Am in o-p h en a n th r en e-9,10-d ion e (3): black solid; 1H
NMR (300 MHz, DMSO-d6) δ 8.00 (1H, d, J ) 7.8 Hz), 7.92-
7.87 (2H, m), 7.65 (1H, ddd, J ) 1.7, 8.0, 8.0 Hz), 7.31 (1H,
Compounds 38-41 were synthesized by the general proce-
dure given for compound 42. Please see the supporting
material for characterization data.
Compounds 43-45 were prepared using the procedures as
described by Barton et al.61

Potent Reversible Inhibitors of PTP CD45
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1789
dd, J ) 7.0, 7.0 Hz), 7.19 (1H, d, J ) 2.8 Hz), 6.91 (1H, dd, J
) 2.5, 8.6 Hz), 5.87 (2H, s). Anal. (C14H9NO2‚0.33H2O) C, H,
N.
1-Am in o-p h en a n th r en e-9,10-d ion e (47): purple solid; 1H
NMR (300 MHz, TFA-d1-DMSO-d6) δ 8.25 (1H, d, J ) 8.2 Hz),
8.05 (1H, dd, J ) 1.2, 7.8 Hz), 7.77 (1H, ddd, J ) 1.5, 7.2, 8.6
Hz), 6.97 (1H, dd, J ) 2.1, 7.4 Hz); HPLC 5.13 min. HRMS
theor. [M + H]: 224.0712 amu; obs. [M + H]: 224.0718 amu;
deviation of 2.9 ppm.
s), 9.29 (1H, dd, J ) 5.7, 5.7 Hz), 8.71 (1H, s), 8.39 (1H, d, J
) 7.9 Hz), 8.13 (1H, d, J ) 8.3 Hz), 8.07 (1H, dd, J ) 1.2, 7.5
Hz), 7.98 (1H, dd, J ) 1.6, 8.2 Hz), 7.85 (1H, ddd, J ) 1.3, 8.7,
8.7 Hz), 7.59 (1H, dd, J ) 7.5, 7.5 Hz), 4.03 (2H, d, J ) 5.6
Hz). Anal. (C17H11NO5‚1.0H2O) C, H, N.
Compound 57 was prepared analogously to compound 58.
[(9,10-Dioxo-9,10-d ih yd r o-p h en a n th r en e-2-ca r bon yl)-
1
a m in o]-a cetic a cid (57): orange solid; H NMR (300 MHz,
DMSO-d6) δ 12.69 (1H, s), 9.22 (1H, dd, J ) 6.0, 6.0 Hz), 8.54
(1H, d, J ) 1.9 Hz), 8.46 (1H, d, J ) 8.2 Hz), 8.39 (1H, d, J )
8.0 Hz), 8.23 (1H, dd, J ) 1.9, 8.4 Hz), 8.08 (1H, dd, J ) 1.1,
7.5 Hz), 7.82 (1H, ddd, J ) 1.5, 8.0, 8.0 Hz), 7.59 (1H, dd, J )
8.0, 8.0 Hz), 3.97 (2H, d, J ) 5.8 Hz). Anal. (C17H11NO5) C, H,
N.
N-(9,10-Dioxo-9,10-dih ydr o-ph en an th r en -2-yl)-2-flu or o-
ben za m id e (70). To a solution of 3 (50 mg, 240 µmol) in THF
(10 mL) was added an excess of Na2CO3 (1 g), followed by
2-fluorobenzoyl chloride (46 µL, 384 µmol). The mixture was
shaken overnight and then filtered, and the solvent evapo-
rated. The resulting material was purified on a silica gel
column, using CH2Cl2-10% EtOAC/CH2Cl2 as the eluant to
yield the pure amide 70 as a red solid: 1H NMR (300 MHz,
DMSO-d6) δ 10.80 (1H, s), 8.45 (1H, d, J ) 2.4 Hz), 8.30 (1H,
d, J ) 9 Hz), 8.23 (1H, d, J ) 8 Hz), 8.09 (1H, dd, J ) 2.3, 8.9
Hz), 8.02 (1H, dd, J ) 1.3, 7.7 Hz), 7.80-7.70 (2H, m), 7.62
(1H, m), 7.50 (1H, dd, J ) 7.3, 7.3 Hz), 7.38 (2H, m); HPLC
6.97 min. HRMS theor. [M + H]: 346.0879 amu; obs. [M +
H]: 346.0882 amu; deviation of 0.7 ppm.
3-Am in o-p h en a n th r en e-9,10-d ion e (48): brown solid; 1H
NMR (300 MHz, DMSO-d6) δ 7.99 (2H, m), 7.83 (1H, d, J )
8.4 Hz), 7.78 (1H, d, J ) 7.2 Hz), 7.53 (1H, dd, J ) 7.2, 7.2
Hz), 7.30 (1H, s), 6.89 (2H, s), 6.64 (1H, dd, J ) 1.5, 8.6 Hz).
Anal. (C14H9O2N‚0.10H2O) C, H, N.
1
4-Am in o-p h en a n th r en e-9,10-d ion e (49): black solid; H
NMR (300 MHz, DMSO-d6) δ 8.56 (1H, d, J ) 8.0 Hz), 7.94
(1H, dd, J ) 1.4, 7.8 Hz), 7.73 (1H, ddd, J ) 1.6, 7.8, 7.8 Hz),
7.41 (1H, ddd, J ) 1.0, 7.9, 7.9 Hz), 7.36 (1H, dd, J ) 3.2, 5.9
Hz), 7.20 (2H, m), 5.84 (2H, s). Anal. (C14H9NO2) C, H, N.
2-Br om o-p h en a n th r en e-9,10-d ion e (2). This compound
1
was prepared as described by Bhatt:48 orange solid; H NMR
(300 MHz, DMSO-d6) δ 8.30 (1H, d, J ) 7.9 Hz), 8.27 (1H, d,
J ) 8.6 Hz), 8.07 (1H, d, J ) 2.4 Hz), 8.04 (1H, d, J ) 1.0, 7.6
Hz), 7.96 (1H, dd, J ) 2.7, 8.9 Hz), 7.79 (1H, ddd, J ) 1.7, 8.2,
8.2 Hz), 7.56 (1H, dd, J ) 7.6, 7.6 Hz). Anal. (C14H7O2) C, H.
Compounds 55 and 56 were prepared as described by
Langenbeck et al.47
9,10-Dioxo-9,10-d ih yd r o-p h en a n t h r en e-2-ca r b oxylic
a cid (55): yellow solid; 1H NMR (300 MHz, DMSO-d6) δ 13.45
(1H, s), 8.51 (1H, d, J ) 1.9 Hz), 8.46 (1H, d, J ) 8.2 Hz), 8.37
(1H, d, J ) 8.0 Hz), 8.24 (1H, dd, J ) 2.0, 8.2 Hz), 8.08 (1H,
dd, J ) 0.75, 7.6 Hz), 7.83 (1H, dd, J ) 7.2, 7.2 Hz), 7.61 (1H,
dd, J ) 7.5, 7.5 Hz). Anal. (C15H8O4‚0.4 H2O) C, H.
Compounds 50, 51, 61-69, 71, and 72 were prepared
analogously to the procedure used for the preparation of
compound 70. Please see the Supporting Information for
characterization data.
N-(9,10-Dioxo-9,10-dih ydr o-ph en an th r en e-2-yl)-4-m eth -
yl-N-[(4-m eth ylph en yl)su lfon yl]-ben zen esu lfon am ide (20).
To a solution of 3 (200 mg, 900 µmol) in CH2Cl2 (10 mL) under
N2 was added Et3N (630 µL, 4.48 mmol), p-toluenesulfonyl
chloride (TsCl, 340 mg, 1.79 mmol), and a catalytic amount of
N,N-(dimethylamino)pyridine. The resultant solution was
stirred overnight, after which time it was diluted with ethyl
acetate (25 mL) and washed sequentially with saturated
aqueous NH4Cl, water, and brine and then dried over Na2-
SO4. Filtration followed by evaporation under reduced pressure
yielded a product which was purified by silica gel chromatog-
raphy using CH2Cl2 as the eluant; the first material eluted
from the column was the ditosylate 20 (39 mg, 73 µmol, 8%)
which was dried to a yellow solid: 1H NMR (300 MHz, DMSO-
d6) δ 8.39 (1H, d, J ) 8.7 Hz), 8.31 (1H, d, J ) 8.0 Hz), 8.06
(1H, dd, J ) 1.2, 7.7 Hz), 7.81 (1H, dd, J ) 7.5, 7.5 Hz), 7.73
(4H, d, J ) 8.3 Hz), 7.60 (1H, dd, J ) 7.5, 7.5 Hz), 7.54 (1H,
m), 7.52 (4H, d, J ) 8.3 Hz), 7.36 (1H, dd, J ) 2.4, 8.5 Hz),
2.50 (6H, s); HPLC 9.85 min. HRMS theor. [M + H]: 532.0889
amu; obs. [M + H]: 532.087 amu; deviation of -3.5 ppm.
N-(9,10-Dioxo-9,10-dih ydr o-ph en an th r en e-2-yl)-4-m eth -
yl-ben zen esu lfon a m id e (19). A second material which elut-
ed from the column was the monotosylate 19 (53 mg, 140
mmol, 16%) which was dried to an orange solid: 1H NMR (300
MHz, DMSO-d6) δ 10.77 (1H, s), 8.17 (1H, d, J ) 9 Hz), 8.12
(1H, d, J ) 8.1 Hz), 7.96 (1H, dd, J ) 1.5, 7.8 Hz), 7.78-7.29
(4H, m), 7.48 (1H, s), 7.46 (1H, t, J ) 8.4 Hz), 7.38 (2H, d, J
) 8.1 Hz), 2.32 (3H, s); HPLC 7.14 min. HRMS theor. [M +
H]: 378.08 amu; obs. [M + H]: 378.0798 amu; deviation of
-0.5 ppm.
9,10-Dioxo-9,10-d ih yd r o-p h en a n t h r en e-3-ca r b oxylic
a cid (56): orange solid; 1H NMR (300 MHz, DMSO-d6) δ 13.66
(1H, br s), 8.71 (1H, s), 8.35 (1H, d, J ) 8.0 Hz), 8.12 (1H, d,
J ) 8.0 Hz), 8.08-8.02 (2H, m), 7.81 (1H, dd, J ) 7.5, 7.5 Hz),
7.58 (1H, dd, J ) 7.5, 7.5 Hz). Anal. (C15H8O4‚0.2H2O) C, H.
[(9,10-Dioxo-9,10-d ih yd r o-p h en a n th r en e-3-ca r bon yl)-
a m in o]-a cetic Acid ter t-Bu tyl Ester (60). To a solution of
56 (1.1 g, 4.5 mmol) in anhydrous DMF (10 mL) under N2 was
added tert-butyl glycine hydrochloride (760 mg, 4.6 mmol),
EDC (1.1 g, 6.0 mmol), DMAP (60 mg, 500 µmol), and
diisopropylethylamine (2.1 mL, 12 mmol), and the resultant
solution was stirred for 16 h. The reaction was diluted with
EtOAc and washed with 1 M HCl, water, saturated aqueous
NaHCO3, and brine. The organic layer was dried over MgSO4,
filtered, concentrated, and chromatographed on silica gel (3:1
EtOAc:CH2Cl2, v/v) to afford a yellow solid (500 mg). This solid
was subsequently recrystallized from refluxing EtOAc to afford
pure 60 (310 mg, 31%): yellow solid; 1H NMR (300 MHz,
DMSO-d6) δ 9.29 (1H, dd, J ) 6.1, 6.1 Hz), 8.71 (1H, s), 8.38
(1H, d, J ) 8.0 Hz), 8.13 (1H, d, J ) 8.3 Hz), 8.07 (1H, dd, J
) 1.5, 7.9 Hz), 7.97 (1H, dd, J ) 1.5, 8.3 Hz), 7.85 (1H, ddd, J
) 1.5, 7.8, 7.8 Hz), 7.58 (1H, ddd, J ) 7.2, 7.2 Hz), 4.00 (2H,
d, J ) 6.1 Hz), 1.45 (9H, s). Anal. (C21H19NO5‚0.2H2O) C, H,
N.
Compound 59 was prepared analogously to compound 60.
[(9,10-Dioxo-9,10-d ih yd r o-p h en a n th r en e-2-ca r bon yl)-
a m in o]-a cetic a cid ter t-bu tyl ester (59): yellow solid; 1H
NMR (300 MHz, DMSO-d6) δ 9.23 (1H, dd, J ) 5.7, 5.7 Hz),
8.54 (1H, d, J ) 1.9 Hz), 8.46 (1H, d, J ) 8.7 Hz), 8.39 (1H, d,
J ) 7.9 Hz), 8.22 (1H, dd, J ) 2.3, 8.7 Hz), 8.07 (1H, dd, J )
1.6, 7.7 Hz), 7.82 (1H, ddd, J ) 1.5, 7.9, 7.9 Hz), 7.59 (1H, dd,
J ) 7.4, 7.4 Hz), 3.94 (2H, d, J ) 6.3 Hz), 1.44 (9H, s). Anal.
(C21H19NO5‚0.1H2O) C, H, N.
[(9,10-Dioxo-9,10-d ih yd r o-p h en a n th r en e-3-ca r bon yl)-
a m in o]-a cetic Acid (58). To a solution of 60 (200 mg, 550
µmol) in CH2Cl2 (10 mL) under N2 was added TFA (10 mL).
This mixture was stirred for 2 h and concentrated under
reduced pressure. The residue was dissolved in CH2Cl2 and
evaporated once again to rid residual TFA. The material was
recrystallized from refluxing EtOAc to afford 58 (70 mg,
47%): orange solid; 1H NMR (300 MHz, DMSO-d6) δ 12.7 (1H,
2-Ben zofu r a n -2-yl-p h en a n th r en e-9,10-d ion e (73). To a
mixture of 2 (150 mg, 520 µmol) in dioxane (5 mL) and H2O
(0.5 mL) were added benzo[b]furan-2-boronic acid (169 mg, 1.4
mmol), tris(dibenzylideneacetone)-dipalladium(0) (48 mg, 52
µmol), tri-o-tolylphosphine (32 mg, 1.4 mmol), and K2CO3 (220
mg, 1.57 mmol). The resultant mixture was heated at 80 °C
for 18 h, and the solid material was removed by filtration. The
soluble material was purified using a Rainin preparative
HPLC on a Rainin phenyl column (25 cm × 21.4 mm, 8 µM
particle size, 60 Å), 40 mL/min, with 50%-80% dioxane/H2O
for 40 min, followed by 80%-50% for 5 min. If the purity at
this stage was not sufficient, the material was then chromato-

1790 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11
Urbanek et al.
graphed on a silica gel column using CH2Cl2 as the eluant:
purple solid; H NMR (300 MHz, CDCl3) δ 8.64 (1H, d, J )
mL) was added for 7 min and drained. The resin was washed
with DMF (10 × 10 mL). A portion of this material (ca. 15%)
was removed and treated with a solution of Ac2O (1 mL) and
diisopropylethylamine (2 mL) in DMF (5 mL) for 2 h. The
solution was drained, washed with DMF (10 × 5 mL), CH2Cl2
(5 × 5 mL), and Et2O (5 × 5 mL), and dried under a N2 stream.
The resin was treated with a solution of TFA containing 2%
H2O and 2% thioanisole (5 mL) for 2 h. The solids were
removed, and the solution was concentrated to a red oil.
Addition of Et2O (10 mL) induced precipitation of a red solid,
which was collected by vacuum filtration. The material was
purified by preparative HPLC on a C18 Dynamax column (21.4
mm × 25 cm, 60 Å) using a gradient of 10% to 26% acetonitrile
in H2O with 0.1% TFA. Fractions containing pure product were
combined and lyophilized to give the product 84 (40 mg) as a
red solid: HPLC 2.69 min. HRMS theor. [M + H]: 695.2313
amu; obs. [M + H] 695.2307 amu; deviation of -0.9 ppm.
1
1.8 Hz), 8.21 (2H, ddd, J ) 1.2, 7.8, 7.8 Hz), 8.10 (1H, d, J )
8.7 Hz), 8.05 (1H, d, J ) 7.8 Hz), 7.75 (1H, dd, J ) 7.8, 7.8
Hz), 7.64 (1H, d, J ) 7.5 Hz), 7.57 (1H, d, J ) 8.1 Hz), 7.50
(1H, dd, J ) 7.8, 7.8 Hz), 7.35 (1H, ddd, J ) 1.2, 6.6, 6.6 Hz),
7.31-7.28 (2H, m); HPLC 10.97 min. HRMS theor. [M + H]:
325.0865 amu; obs. [M + H]: 325.0886 amu; deviation of 6.6
ppm.
Compounds 74-78 were prepared using the general proce-
dure reported for compound 73. Please see the Supporting
Information for characterization details.
Com p ou n d 84. FMOCAsp(OH)OtBu (3.3 g, 8 mmol) and
N-methylmorpholine (900 µL, 8 mmol) were dissolved in dry
THF (20 mL) and chilled to -10 °C. Isobutylchloroformate (910
µL, 7 mmol) was added dropwise, maintaining the reaction
temperature below -7 °C. To this mixture was added a
suspension of 3 (1.12 g, 5 mmol) in dry THF (40 mL), and the
reaction was allowed to warm to ambient temperature. The
disappearance of 3 was monitored by TLC (Rf ) 0.42, 25:75
EtOAc:CH2Cl2, v/v on a silica gel plate), and reaction was
complete after 7 d. The solvent was removed by rotary
evaporation, and the residue was partitioned between EtOAc
and saturated aqueous NH4Cl. The organic phase was washed
with saturated aqueous NaHCO3 and brine and then dried
over anhydrous MgSO4. This mixture was filtered, and the
filtrate was concentrated to a red-brown solid and chromato-
graphed on silica gel with 15:85, EtOAC:CH2Cl2, v/v. This gave
FMOCAsp-2-aminophenanthrene-9,10-dione (1.92 g, 3.1 mmol,
63%) as a red solid. This intermediate was dissolved in CH2-
Cl2 (100 mL), and TFA (30 mL) was added. After 1 h the
volatiles were removed by rotary evaporation. Residual TFA
was removed by dissolving the material in CH2Cl2 and
concentration on the rotary evaporator twice. This was dis-
solved in dry CH2Cl2 (100 mL), and diisopropylethylamine (5
mL) and dry DMF (10 mL) were added under N2. 2-Chloro-
chlortrityl resin (4.5 g of 1.2 mmol/g, 5.5 mmol) was added to
the mixture and mixed by shaking. After 3 h, MeOH (10 mL)
was added, and the shaking was continued for an additional
10 min. The resin was collected by vacuum filtration and
washed with CH2Cl2 (5 × 10 mL), DMF (2 × 10 mL), CH2Cl2
(2 × 10 mL), and MeOH (3 × 10 mL). The resin was dried for
64 h in vacuo. An incorporation of 2.3 mmol of FMOCAsp(3-
aminophenanthrene-9,10-dione) was calculated based on the
increase of mass to 5.8 g. The resin was swelled in DMF (25
mL) for 1 h and then washed with DMF (5 × 25 mL). The
resin was treated with 20% piperidine in DMF (10 mL) for 3
min, drained, then was treated with more 20% piperidine in
DMF (10 mL) for 7 min and drained. The resin was washed
with DMF (10 × 10 mL). A solution of FMOCGln(trt)OH (6.72
g, 11 mmol), HATU (3.8 g, 10 mmol), and diisopropylethyl-
amine (3.5 mL) in DMF (15 mL) was added to the resin and
mixed with gentle nitrogen bubbling for 2 h. The solution was
drained, and the resin was washed with DMF (10 × 10 mL).
The resin was treated with 10 mL of 20% piperidine in DMF
(10 mL) for 3 min and drained, and then another portion of
20% piperidine in DMF (10 mL) was added for 7 min and
drained. The resin was washed with DMF (10 × 10 mL). A
portion of the resin (ca. 10%) was removed for other reactions,
and the remaining resin was treated with a solution of
FMOCGlyOH (1.93 g, 11 mmol), HATU (3.8 g, 10 mol), and
diisopropylethylamine (3.5 mL) in DMF (15 mL) for 1 h. The
reaction was drained and washed with DMF (10 × 10 mL).
The resin was treated with 20% piperidine in DMF (10 mL)
for 3 min, drained, then treated with additional 20% piperidine
in DMF (10 mL) for 7 min, and drained. The resin was washed
with DMF (10 × 10 mL). A portion of the resin (about 10%)
was removed for other reactions, and the remaining resin was
treated with a solution of FMOCGlu(tBu)OH (4.68 g, 11 mmol),
HATU (3.8 g. 10 mmol), and diisopropylethylamine (3.5 mL)
in DMF (15 mL) for 16 h. The solution was drained, and the
resin was washed with DMF (10 × 10 mL). The resin was
treated with 20% piperidine in DMF (10 mL) for 3 min and
drained, and then an additional 20% piperidine in DMF (10
Compounds 83 and 85-87 were prepared in an analogous
manner to compound 84. Please see the Supporting Informa-
tion for characterization data.
Com p ou n d 106. 6-Chlorochlortrityl resin (6.19 g, 7 mmol)
was swelled in dry CH2Cl2 (30 mL), and
a solution of
FMOCprolineOH (1.18 g, 3.5 mmol) and diisopropylethylamine
(1.8 mL, 10.5 mmol) in dry CH2Cl2 (10 mL) was added. The
reaction was gently mixed under a nitrogen atmosphere for
30 min, and then MeOH (3 mL) was added to cap the
unreacted sites. After 15 min, the resin was transferred to a
fritted glass funnel and washed with DMF (2 × 50 mL), CH2-
Cl2 (2 × 50 mL), MeOH (2 × 50 mL), and Et2O (2 × 50 mL).
The resin was dried in vacuo at 50 °C to constant weight. A
portion of this material (1.5 g, 0.25 mmol) was swelled in DMF
(5 mL) for 15 min and then washed with DMF (3 × 5 mL).
The resin was treated with 20% piperidine in DMF (5 mL) for
3 min, drained, and then treated with another 20% piperidine
in DMF (5 mL) for 7 min and drained. The resin was washed
with DMF (10 × 5 mL). A solution of FMOCglutamine(Trt)-
OH (610 mg, 1 mmol), HATU (380 mg, 1 mmol), and diiso-
propylethylamine (350 µL) in DMF (5 mL) was added to the
resin and mixed with a gentle nitrogen bubbling for 2 h. The
solution was drained, and the resin was washed with DMF
(10 × 5 mL). The resin was treated with 20% piperidine in
DMF (5 mL) for 3 min and drained, and then another portion
of 20% piperidine in DMF (5 mL) was added, mixed for 7 min,
and then drained. The resin was washed with DMF (10 × 5
mL). A solution of FMOCprolineOH (340 mg, 1 mmol), HATU
(380 mg, 1 mmol), and diisopropylethylamine (350 µL) in DMF
(5 mL) was added to the resin and mixed with a gentle nitrogen
bubbling for 16 h. The solution was drained, and the resin was
washed with DMF (10 × 5 mL). The resin was treated with
20% piperidine in DMF (5 mL) for 3 min, drained, treated with
another portion of 20% piperidine in DMF (5 mL) for 7 min,
and drained. The resin was washed with DMF (10 × 5 mL). A
solution of FMOCglutamineOH (370 mg, 1 mmol), HATU (380
mg, 1 mmol), and diisopropylethylamine (350 µL) in DMF (5
mL) was added to the resin and mixed with a gentle nitrogen
bubbling for 2.5 h. The solution was drained and the resin
washed with DMF (10 × 5 mL). The resin was treated with
20% piperidine in DMF (5 mL) for 3 min, drained, then treated
with another 20% piperidine in DMF (5 mL) for 7 min, and
drained. The resin was washed with DMF (10 × 5 mL). A
solution of FMOCglutamateOH(OtBu) (830 mg, 1 mmol),
HATU (380 mg, 1 mmol), and diisopropylethylamine (350 µL)
in DMF (5 mL) was added to the resin and mixed with a gentle
nitrogen bubbling for 2 h. The solution was drained, and the
resin was washed with DMF (10 × 5 mL). The resin was
treated with 20% piperidine in DMF (5 mL) for 3 min, drained,
then treated with another 20% piperidine in DMF (5 mL) for
7 min, and drained. The resin was washed with DMF (10 × 5
mL). A solution of 56 (250 mg, 1 mmol), HATU (380 mg, 1
mmol), and diisopropylethylamine (350 µL) in DMF (5 mL)
was added to the resin and mixed with a gentle nitrogen
bubbling for 16 h. The solution was drained, and the resin was
washed with DMF (10 × 5 mL), CH2Cl2 (5 × 5 mL), and Et2O
(5 × 5 mL) and then dried under a stream of N2. The resin

Potent Reversible Inhibitors of PTP CD45
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 11 1791
was treated with TFA containing 2% thioanisole and 2% water
(5 mL) for 3 h. The solids were filtered off, and the filtrate
was evaporated to a red oil. Addition of Et2O (10 mL) induced
precipitation, and this solid was collected by vacuum filtration
to give the product (300 mg) as a pink solid. The material was
purified by preparative HPLC on a C18 Dynamax column (21.4
mm × 25 cm, 60 Å) using a gradient of 10% to 26% acetonitrile
in water with 0.1% TFA. Fractions containing pure product
were combined and lyophilized to give 106 (100 mg) as a yellow
solid: HPLC 2.55 min. Anal. (C40H45N7O13‚1.0H2O‚0.5CF3-
CO2H) C, H, N.
plate containing RPMI with compound and incubated over-
night at 37 °C in an incubator containing 5% CO2. The dilution
scheme and culture media were the same as those used in the
T-cell proliferation assay. After the incubation period, cells
were washed with Dulbecco’s phosphate- buffered saline (D-
PBS) and incubated with 1 µM Calcein-AM for 30-45 min in
D-PBS as described in the technical sheet provided with the
LIVE/DEAD Viability/Cytotoxicity Kit from Molecular Probes.
Percent viability was assessed on a fluorescent plate reader
(excitation filter 485/20 nm; emission filter 530/25 nm) where
the 100% control value is the fluorescence intensity observed
in the absence of test compound. The results from this assay
are expressed as CC50, i.e., the concentration of compound that
is cytotoxic for 50% of the cells. The CC50 was calculated using
GraphPad Prism software (GraphPad Software, San Diego,
CA). Methanol was used as a positive control (100% cytotox-
icity) in these assays. Cyclosporine was not cytotoxic at 100
nM, the highest concentration tested in our assays.
Compounds 88-105 and 107-121 were synthesized accord-
ing to the general procedure given for compound 106. Please
see the Supporting Information for characterization data.
Biology. pNP P Assa y for P h osp h a ta se Activity. CD45
and PTP1B enzymes were purchased from BIOMOL (Ply-
mouth Meeting, PA). FAP enzyme was obtained from BioSig-
nal (Montreal, Canada). Phosphatase activity was assayed in
96-well plates in a buffer containing 20 mM imidazole, pH 7.0,
50 mM NaCl, 2.5 mM EDTA, 5 mM DTT, and 10 µg/mL BSA
using pNPP as a substrate. In some experiments, 10 mM
glutathione was substituted for DTT. Compounds were tested
in a range from 30 to 0.01 µM, with a final concentration of 1
or 5% DMSO depending upon the solubility of the compound.
PTPase activity was measured by following the increase in
absorbance at 405 nm on a SpectraMax Plus spectrophoto-
metric plate reader (Molecular Devices, Sunnyvale, CA).
Lck P ep tid e Assa y for P h osp h a ta se Activity. The lck
COOH terminal peptide TEGQpYQPQP was obtained from
Cambridge Research Biochemicals (Wilmington, DE). Phos-
phatase activity was assayed in 96-well plates in a buffer
containing 25 mM HEPES, pH 7.2, 5 mM DTT, and 10 µg/mL
BSA using the lck peptide as a substrate. Compounds were
tested in a range from 30 to 0.01 µM at a final concentration
of 5% DMSO. Phosphatase enzyme was incubated with
substrate, in the presence or absence of compound, at room
temperature for 1.5 h. At the end of the incubation period,
BIOMOL “Green Reagent” (BIOMOL), Plymouth Meeting, PA)
was added to each well, the plates were incubated for 30 min
at room temperature, and the absorbance was read at 620 nm.
To estimate the Km values for the different phosphatase
enzymes, the lck 10-mer was varied over a concentration range
of 25-600 µM. Km values were calculated with GraphPad
Prism software (GraphPad Software, San Diego, CA).
Ack n ow led gm en t. We thank Paul Warwick for
development of the synthesis of the lck 10-mer, Lisa
Schaffter for the mass spectral analyses, Bruce Gomes
for discussions of the kinetics of these compounds, and
David Aharony, Smita Ghanekar, George Booth, and
Nicola Clarkson for the cathepsin selectivity assays.
Su p p or tin g In for m a tion Ava ila ble: Characterization
data for compounds 38-41, 50, 51, 61-69, 71, 72, 74-78, 83,
85-105, and 107-121. This material is available free of
charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Mihatsch, M.; Kyo, M.; Morozumi, K.; Yamaguchi, Y.; Nickeleit,
V.; Ryffel, B. The side-effects of cyclosporine-A and tacrolimus.
Clin. Nephrol. 1998, 49, 356-363.
(2) Hojo, M.; Morimoto, M.; Maluccio, T.; Asano, T.; Morimoto, K.;
Lagman, M.; Shimbo, T.; Suthanthiran, M. Cyclosporine induces
cancer progression by a cell-autonomous mechanism. Nature
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