Nonpeptidic, monocharged, cell permeable ligands for the p56lck SH2 domain

Article abstract of DOI:10.1021/jm000446q

p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substra

Full text of DOI:10.1021/jm000446q

J . Med. Chem. 2001, 44, 2421-2431
2421
Non p ep tid ic, Mon och a r ged , Cell P er m ea ble Liga n d s for th e p 56lck SH2 Dom a in
J ohn R. Proudfoot,* Rajashehar Betageri, Mario Cardozo, Thomas A. Gilmore, Susan Glynn, Eugene R. Hickey,
Scott J akes, Alisa Kabcenell, Thomas M. Kirrane, Annette K. Tibolla, Susan Lukas, Usha R. Patel,
Rajiv Sharma, Mehran Yazdanian, and Neil Moss
Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, P.O. Box 368, Ridgefield, Connecticut 06877
Pierre L. Beaulieu, Dale R. Cameron, J ean-Marie Ferland, J ean Gauthier, J ames Gillard, Vida Gorys,
Martin Poirier, J ean Rancourt, Dominik Wernic, and Montse Llinas-Brunet
Boehringer Ingelheim (Canada) Ltd., Research and Development, 2100 Cunard Street, Laval, Quebec H7S 2G5, Canada
Received October 16, 2000
p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal
transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have
the potential to disrupt the interaction of p56lck with its substrates and derail the signaling
cascade that leads to the production of cytokines such as interleukin-2. Starting from the
quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently
disclosed (J . Med. Chem. 1999, 42, 722 and J . Med. Chem. 1999, 42, 1757) the design of the
modified dipeptide 3, which carries just two charges at physiological pH. Here we present the
elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good
binding affinity for the p56lck SH2 domain (Kd 1 µM) and good cell permeation, and this
combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early
event in T cell activation, namely calcium mobilization.
Sch em e 1
In tr od u ction
Src homology-2 (SH2) domains are protein modules
of about 100 amino acids that bind to phosphotyrosine-
containing peptide sequences. These domains function
as regulatory elements in cell signaling processes by
controlling the activity and localization of tyrosine
kinases and other proteins involved in signal transduc-
tion.1 They are attractive targets for drug discovery
efforts, especially in the areas of oncology and immunol-
ogy, and several reports have appeared recently on the
design of ligands for the SH2 domains of pp60c-src,2-4
Grb2,5,6 and p56lck.7
The lead structures for these efforts are usually
phosphotyrosine-containing peptides, and most of the
reported ligands are at least doubly charged at physi-
ological pH and often retain some peptidic character.
These two features are considered impediments to cell
permeation, and, although ligands with good binding
affinity to various SH2 domains are available, prodrug3c,8
and more exotic approaches9 have generally been needed
to deliver these ligands into cells.10 Recently, cellular
activity that does not rely on these approaches has been
reported for a monocharged ligand of the src SH2
domain.11
Our interest in interfering with the function of
p56lck,12 a tyrosine kinase that plays a critical role in
T cell activation, led us to design ligands for the SH2
domain of p56lck.13 Such ligands have the potential to
block the interaction of p56lck with its substrates such
as the T cell receptor and the tyrosine kinase ZAP70.
This could derail the signaling cascade that in CD4+
T
cells leads to the production of cytokines such as
interleukin-2 (IL-2), and, ultimately, to cell proliferation.
The ligands could therefore provide a novel means to
control the immune response.
* Address correspondence to J ohn R. Proudfoot at Department
of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals Inc.,
900 Ridgebury Road, PO Box 368, Ridgefield CT 06877. Phone:
(203) 798 5107. Fax: (203) 791 6072. E-mail: jproudfo@
rdg.boehringer-ingelheim.com.
The peptide 1, AcpYEEI,14 (Scheme 1) which binds
to the p56lck SH2 domain with high affinity (Kd ) 0.1
µM), served as the starting point for our efforts.15,16 Our
10.1021/jm000446q CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/22/2001

2422 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15
Proudfoot et al.
a
Ta ble 1
a
Number of determinations in brackets.
goal was the transformation of this molecule into a cell
permeable ligand with demonstrable efficacy in cellular
models of T cell activation. We viewed the five negative
charges at physiological pH, the metabolically unstable
phosphate group, and the peptidic nature of 1 as
undesirable characteristics. We previously described the
studies that led us to peptide 2 in which the two
C-terminal amino acids were replaced with a p-meth-
oxybenzyl group.7a Peptide 2, with two fewer carboxyl
groups, has affinity equal to 1 for the p56lck SH2
domain. Concurrent efforts to find a metabolically
stable, less charged, alternative for the phosphate group
gave us structure 3, which contains an acetic acid group
as a phosphate replacement.7b Replacement of the
doubly charged phosphate group with the singly charged
acetic acid group results in decreased binding affinity
of between 200- and 500-fold for the SH2 domain.7b,17
However, binding affinity is partly recovered, to the
extent of about 10-fold, with the introduction of lipo-
philic substituents, particularly naphthylacetyl groups
as shown, at the N terminus,7b a beneficial effect also
noted by others working this area.18 At this point we
had transformed the tetrapaptide 1 with five negative
charges at physiological pH into the dipeptide 3 with
two negative charges, albeit at the cost of a 50-fold
decrease in binding affinity. In addition, compound 3
is very poorly cell permeable (Table 1), and we expected
that substantial improvement in binding affinity and/
or permeability would be required before efficacy in
cellular assays could be demonstrated.
Our driving hypothesis was that a monocharged
ligand should provide the best balance between affinity
for the SH2 domain and physical properties that would
allow cell permeability and the demonstration of cellular
activity. Here we describe the continuation of our efforts,
and the elaboration of 3 to a cell permeable molecule,
with low micromolar binding affinity for the p56lck SH2
domain, and demonstrable cellular efficacy.
Design of a n Effective Ba ck bon e Mod ifica tion
The progress described in our previous two manu-
scripts, and outlined above, came from changes at the
N- and C-termini of the peptide 1 (Scheme 1). Our early
attempts to modify the P+1 region and dispense with
the carboxylate-containing P+1 side chain met with
mixed results. In the crystal structure of compound 1
bound to the p56lck SH2 domain, the P+1 carboxylic
acid containing side chain does not interact directly with

Nonpeptidic Ligands for the p56lck SH2 Domain
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15 2423
Sch em e 2
F igu r e 1. Two depictions of the bound conformation of 1.
favors a planar disposition of the pyridone and the
adjacent amide, which was predicted to be detrimental
for binding to the SH2 domain. For example, in the
structure of peptide 1 bound to the SH2 domain, the
P+1 N-H is almost orthogonal to the P+1 carbonyl
group (Figure 1) and partakes in an important hydrogen-
bonding interaction with the SH2 domain. Introduction
of a methyl substituent at the 4-position of the pyridone
ring, as in structure 5, ensures that the P+1 NH is
orthogonal to the plane of the ring and available for a
hydrogen-bonding interaction with the protein.
These conformational preferences for 4 and 5 were
confirmed by ab initio quantum mechanics calculations.
For compound 4, calculations in a vacuum using Gauss-
ian HF/6-31G* basis set showed that the coplanar
conformation of the amide and pyridone ring is preferred
by about 5 kcal/mol over the orthogonal arrangement.
This conformation possesses the intramolecular hydro-
gen bond between the NH of amide and the carbonyl
group of the pyridone. In addition, the effect of solvent
was taken into account by computing the solvation free
energy for all rotamers. This thermodynamic quantity
was estimated using a continuum self-consistent reac-
tion field method (SCRF).21,22 Thus, when the effect of
solvent is taken into account, this planar conformation
remains the most stable by 3 kcal/mol. For compound
5, with the 4-methyl substituent, there is a calculated
energy minimum in a vacuum at about 65°, and the 90°
conformer is less favored by only about 1 kcal/mol.
Calculations indicate that for 5 the solvent effect
stabilizes the 90° conformer by about 1 kcal/mol relative
to vacuum, indicating that the binding conformer should
be favored in solution.
the protein.15 SAR studies showed that it could be
replaced by uncharged side chains without great cost
to binding affinity, if done in the context of tetrapep-
tides19 and pentapeptides.2a However, in the context of
molecules such as 2, we observed that incorporation of
an uncharged side chain (leucine being the best) led to
a 10-fold decrease in binding affinity.7a As we had
already lost binding affinity due to the phosphotyrosine
replacement, we felt that, in order to be successful, we
needed to identify a noncharged replacement for the
P+1 glutamic acid side chain that at least did not lose
binding affinity.
In the bound conformation of 1 we noted that the
CR-H of P+1 and the N-H of P+2 (indicated in Figure
1) are almost coplanar and point in the same direction.
Our earlier SAR studies had shown that methylation
is tolerated at the P+2 nitrogen atom in analogues of
1,19 and similar N-methylation is also tolerated in
compounds related to 2.7a The binding conformation of
1 depicted in Figure 1, and the tolerance of N-methy-
lation at P+2 prompted various cyclization strategies
directed toward rigidifying this region of the molecule.
Our most successful modification is illustrated in
structure 4, (Scheme 2). The P+1 side chain of 3 is
discarded, and the P+1 carbonyl group and the P+2
nitrogen atom are incorporated into a pyridone ring,20
attached to the P+1 CR. This modification was particu-
larly attractive since the charged side chain is elimi-
nated. Additionally, only one chiral center remains in
the proposed molecule. We hoped that the loss in
binding affinity due to the lack of a P+1 side chain
would be recovered by the rigidification of the molecule.
The conformational properties around the pyridone
moiety of 4 suggested that one additional structural
modification would be necessary. In structure 4, the P+1
NH can participate in a favorable electrostatic inter-
action with the carbonyl group of the pyridone ring. This
Syn th esis a n d Resu lts
The synthesis of the target ligands is outlined in
Scheme 3. The aminopyridinones IIIa and IIIb, precur-

2424 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15
Proudfoot et al.
Sch em e 3a
23 µM) than analogue 5, thus supporting the confor-
mational arguments used in the design of the pyridones.
Although we do not have crystal structures of our
ligands bound to the SH2 domain, we have generated a
model24 that illustrates what we believe to be the
important features involved in binding. In the model,
depicted in Figure 2, the p-methoxybenzyl group is
oriented toward the P+3 hydrophobic pocket. The
phenyl ring is perpendicular to the plane of Tyr181, in
position for a favorable edge-to-face aromatic-aromatic
interaction. The methoxy substituent is located deep in
the lipophilic pocket, with the methyl group in van der
Waals contact with Leu216, Ile193, and Ser194. The
pyridone ring lies on the surface of the protein, above
tyrosine 181, while the NH of the “out-of-plane” amide
group participates in a hydrogen bond with the back-
bone carbonyl of His180. The acetic acid group binds in
the phosphate recognition pocket, as previously de-
scribed.7b Figure 3 depicts an overlap of this model with
the crystal structure of the complex between the p56lck
SH2 domain and Ac-pTyr-Glu-Glu-Ile (ligand shown in
purple).
Although compound 5 represents a significant ad-
vance over compound 3, it still showed apparently poor
cell permeation properties when evaluated in a Caco-2
model25 of cell permeability (Table 1). Despite the
possibility that the apparent poor permeation of 5 could
in part be rationalized by an efflux mechanism, we
concentrated on identifying groups that might be ex-
pected to improve cell permeability (i.e., enhance de-
solvation). In general, the diffusion of molecules across
cell membranes is enabled by the shielding or elimina-
tion of hydrogen-bonding functionality.26,27 With regard
to structure 5, options for eliminating hydrogen-bonding
capacity were somewhat limited. Methylation of either
remaining amide nitrogen decreases binding affinity by
at least 10-fold,19 and we preferred avoiding a prodrug
approach with respect to the carboxylic acid. Since we
knew from our earlier SAR studies that small lipophilic
substituents are tolerated adjacent to the carboxylate
group,19 we targeted the R,R-dimethyl derivative 7,
expecting the methyl substituents to shield the polar
carboxylate functionality and enhance the cell perme-
ation properties relative to 5. Since both the 1- and
2-naphthylacetyl N-terminus substitution had been
effective in earlier series, analogue 8 was also synthe-
sized.
a
Conditions: (a) NaH, DMF, p-methoxybenzyl chloride; (b) H2,
10% Pd/C, ethanol; (c) EDC, CH2Cl2; (d) TFA, CH2Cl2; (e) naph-
thylacetic acid, EDC, CH2Cl2 or acetic anhydride, CH2Cl2; (f)
NaOH, THF, methanol, or LiOH, THF, H2O.
sors to the right-hand side of the targets, are derived
in a straightforward manner from 3-nitro-2-pyridone
and 4-methyl-3-nitro-2-pyridone, respectively, via N-
alkylation followed by reduction of the nitro group as
shown. The amino acid derivative IV7b,23 is coupled with
the amine IIIa or IIIb using standard conditions to give
the amide Va or Vb in good yield. Unblocking of the
N-terminus, followed by N-acylation and hydrolysis of
the methyl ester, all proceed under standard conditions
to give the compounds 4-6.
Compound 5 binds to the SH2 domain with a Kd of
1.3 µM (Table 1), i.e., it is 3-fold more potent than the
analogous compound 3 containing a glutamic acid
residue at P+1. This was an encouraging result. We had
finally dispensed with the charged side chain and even
improved binding affinity slightly in the process. Com-
pound 6, with an acetyl substituent at the N-terminus,
shows a decrease in binding affinity (Kd ) 7 µM)
consistent with that seen for N-acetyl vs N-naphthyl-
acetyl comparisons in earlier series. This result gave
us some confidence that these molecules were binding
in an analogous manner to our earlier structures. The
ligand lacking the methyl group at the 4-position of the
pyridone ring, compound 4, is considerably weaker (Kd)
The synthesis of compounds 7 and 8 is outlined in
Scheme 4. The dimethylacetic acid intermediate X was
synthesized in racemic form. It was derived in a
straightforward manner from a Heck reaction of N-Boc-
dehydroalanine benzyl ester28 with the iodophenylacetic
acid derivative VIIIc, followed by catalytic hydrogena-
tion to saturate the double bond and remove the benzyl
group. The only other difference of note from the
methods described in Scheme 3 is the use of the tert-
butyl ester as a protecting group for the carboxylic acid.
Initially, selective removal of the N-Boc group presented
some difficulty, but we eventually found that HCl in
dioxane was effective in removing the Boc group while
leaving the ester intact.
The effects of dimethyl substitution adjacent to the
carboxylate group and the P-1 amide are illustrated in
Table 1. The comparison of dimethyl derivative 7RS (Kd

Nonpeptidic Ligands for the p56lck SH2 Domain
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15 2425
F igu r e 2. Some interactions of compound 6 with the p56lck SH2 domain.
F igu r e 3. Overlap of compound 6 and AcpYEEI bound to the p56lck SH2 domain.
) 1.2 µM) with 5 (Kd ) 1.3 µM) shows that, while the
additional methyl groups adjacent to the carboxyl group
have little effect on binding affinity to the SH2 domain,29
they improve cell permeation (22 × 10-6 vs 11 × 10-6
cm/s when measured in the BfA direction, Table 1).
The 2-naphthylacetyl analogue 8RS shows a 3-fold
enhancement in binding affinity (Kd ) 0.3 µM) with
permeability properties equivalent to 7RS. Note that
both compounds are efflux pump substrates. Additional
dimethyl substitution adjacent to the P-1 amide, il-
lustrated with compound 9RS, results in a small
decrease in binding affinity versus 8RS (9RS Kd ) 1.2
µM, 8RS Kd ) 0.3 µM) and improved cell permeation
over 8RS in the AfB direction (6 × 10-6 vs 0.19 × 10-6
cm/s). This indication of further improved permeability
along with good affinity prompted the resolution of 9RS
by chiral HPLC into enantiomers 9R and 9S. The S
enantiomer, 9S, was identified as the active enanti-
omer,30 and permeation data in the BfA Caco-2 experi-
ment confirmed the expected good cell permeability
properties.
In h ibition of Ca In flu x
Now that we had demonstrated good binding affinity
along with acceptable cell permeability in compound 9S,
we wanted to show as conclusively as possible that our
compounds would inhibit T cell activation by the
intended mechanism, i.e., binding to the lck SH2
domain. It is not uncommon for compounds to cause
unpredicted effects when tested in cell based assays, so
we felt it would be prudent to test the much less active
enantiomer 9R as a control along with 9S. This would
increase our confidence that we were inhibiting by the
desired mechanism.
Engagement of the T cell receptor (TCR) leads to an
increase in cytosolic calcium levels resulting from the
release of calcium from intracellular stores and subse-
quent influx of extracellular calcium. An antagonist of

2426 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15
Proudfoot et al.
Sch em e 4a
signaling dependent, there is strong evidence that
compound 9S inhibits the early events of T cell signal
transduction via the intended mechanism.
Activation of the receptor mediated signal transduc-
tion pathway can lead to the induction of multiple genes
and ultimately to cellular proliferation. The effects of
compounds 9R and 9S on the synthesis and secretion
of one of these gene products, IL-2, was also examined.
Human CD4+ T lymphocytes, purified from isolated
peripheral blood mononuclear cells, were stimulated
with antibodies to CD3 and CD28 in the presence or
absence of compound. Following an overnight incuba-
tion, the cell supernatants were removed, and secreted
IL-2 was quantified by ELISA. Compound 9S inhibited
IL-2 secretion in a dose dependent fashion with an IC50
of 9.4 µM (Table 2). However, the enantiomer 9R, which
binds much less effectively to the lck SH2 domain, also
blocked cytokine secretion to a similar extent. Compa-
rable results were obtained for the two compounds when
inhibition of cell proliferation was monitored. The
similar profile for the two enantiomers in this longer
duration assay suggests that nonspecific effects may be
occurring in addition to the early effects on T cell
activation.
Con clu sion
Recently, several research groups have tackled the
problem of designing cell permeable ligands for various
SH2 domains. Given the nature of the natural ligands,
i.e., multiply charged phospho-peptides, this has proven
to be a difficult task. We have shown that it is possible
to design ligands for the p56lck SH2 domain that have
binding affinity in the low micromolar range and that
possess good cell permeability. Our success derives from
a decision to pursue inhibitors that contain no more
than one negative charge at physiological pH and from
commitment to lessen the peptidic character of the lead
molecules. The net result of our efforts is the transfor-
mation of a metabolically unstable, quintuply charged
tetrapeptide 1 into an essentially nonpeptidic, mono-
charged species, 9S. This molecule displays good binding
affinity (Kd ) 1 µM) and good cell permeation properties.
This combination of binding affinity and cell perme-
ability allowed us to demonstrate clear-cut inhibitory
effects on a very early event in T cell activation, namely
calcium mobilization.
a
Conditions: (a) I2, NaIO3, concentrated H2SO4, AcOH, 55 °C;
(b) oxalyl chloride, DMF, CH2Cl2, KOtBu; (c) Pd(OAc)2, NaHCO3,
(Bu)4NCl, DMF, benzyl (2-tbutoxycarbonylamino)acrylate; (d) Pd/
C, ethanol; (e) EDC, CH2Cl2, IIIb; (f) HCl/dioxane; (g) EDC,
appropriate carboxylic acid, CH2Cl2; (h) TFA.
Ta ble 2
inhibition of Ca+2
influx in J urkat
T cells
inhibition of IL-2
production from
CD4+ T cells
(EC50, µM)
inhibition of
proliferation of
CD4+ T cells
(EC50, µM)
compd
(EC50, µM)
3
5
>100
>100
7RS 51.0 ( 16.1 (n ) 4)
8RS 30.5 ( 2.9 (n ) 4)
9S
9R
10.1 ( 0.8 (n ) 4)
9.4 ( 4.2 (n ) 9) 8.7 ( 3.6 (n ) 4)
29% inhibition @ 50 µM 11.3 ( 1.0 (n ) 3) 16.6 ( 4.8 (n ) 3)
57% inhibition @ 50 µM
the lck SH2 domain should abrogate the release of
calcium from intracellular stores and prevent the influx
of extracellular calcium. The ability of the two enanti-
omers 9S and 9R to inhibit the function of the lck SH2
domain in intact cells was assessed in J urkat cells (a
human T cell leukemic line), loaded with the calcium-
sensitive indicator dye, Fluo-3. Changes in fluorescence
following antibody mediated TCR cross-linking were
monitored over time. Compound 9S inhibited receptor-
mediated increase in intracellular calcium concentration
with an EC50 of 10 µM (Table 2). Application of the
inactive enantiomer 9R had a substantially weaker
Exp er im en ta l Deta ils
General experimental details have been previously de-
scribed.7a,7b
1-(4-Meth oxyben zyl)-4-m eth yl-3-n itr o-2-p yr id on e (IIb).
To a stirred suspension of Ib (2.0 g, 13 mmol) in DMF (60 mL)
was added NaH (60% suspension in oil, 0.52 g). After 1 h,
4-methoxybenzyl chloride (1.8 mL) was added, and the mixture
was stirred overnight at room temperature. Ethyl acetate was
added, and the organic phase was washed with water, dried,
filtered, and evaporated. Chromatography of the residue over
silica gel (ethyl acetate/hexane) gave IIb (2.0 g, 7.3 mmol,
56%): 1H NMR δ CDCl3 7.30-7.26 (3H), 6.89 (2H, d, J ) 9),
6.05 (1H, d, J ) 7), 5.08 (2H, s), 3.80 (3H, s), 2.24 (3H, s);
MS(CI) 275 (MH+). Anal. (C14H14N2O4) C, H, N.
effect on the activation induced calcium influx (EC50
>
50 µM), supporting action of 9S via antagonism of the
lck SH2 domain. In addition, 9S had no effect on calcium
influx induced by a T cell receptor independent mech-
anism, that of treatment with the calcium ATPase
inhibitor thapsigargin31 (data not shown). Since the
results from the calcium assay and from phosphoryla-
tion experiments (to be published in a separate ac-
count)32 showed efficacy that was enantiomer and TCR
3-Am in o-1-(4-m e t h oxyb e n zyl)-4-m e t h yl-2-p yr id on e
(IIIb). A mixture of IIb (1.5 g, 5.5 mmol) and 10% Pd/C (0.15
g) in ethanol (150 mL) was hydrogenated at 40 psi in a Parr
apparatus for 5.5 h. The catalyst was removed by filtration,

Nonpeptidic Ligands for the p56lck SH2 Domain
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15 2427
and the solvent was evaporated to give IIIb (1.3 g, 5.3 mmol,
96%): 1H NMR δ CDCl3 7.24 (2H, d, J ) 9), 6.85 (2H, d, J )
9), 6.69 (1H, d, J ) 7), 5.97 (1H, d, J ) 7), 5.08 (2H, s), 4.04
(2H, br s), 3.78 (3H, s), 2.04 (3H, s); MS(CI) 245 (MH+). Anal.
(C14H16N2O2) C, H, N.
3-Am in o-1-(4-m eth oxyben zyl)-2-pyr idon e (IIIa) was syn-
thesized in an analogous manner: 1H NMR δ CDCl3 7.26 (2H,
d, J ) 9), 6.86 (2H, d, J ) 9), 6.72 (1H, dd, J ) 2, 7), 6.50 (1H,
dd, J ) 2, 7), 6.04 (1H, apparent t, J ) 7), 5.10 (2H, s), 4.22
(2H, br s), 3.78 (3H, s,); MS(CI) 231 (MH+). Anal. (C13H14N2O2)
C, H, N.
2,2-Dim eth yl-2-n a p h th yla cetic Acid . 2-Na p h th yla cetic
Acid Ben zyl Ester . To a stirred solution of 2-naphthylacetic
acid (1 g, 5.4 mmol) in acetonitrile (10 mL) were added DBU
(0.97 mL, 6.4 mmol) and benzyl bromide (0.77 mL, 6.4 mmol).
After 3 h, the mixture was concentrated, and the residue was
taken up in ethyl acetate. The organic phase was washed with
10% citric acid, 10% NaHCO3, and brine, dried (MgSO4), and
concentrated to give 2-naphthylacetic acid benzyl ester as a
white solid (1.5 g, 100%).
2,2-Dim eth yl-(2-n a p h th yl)a cetic Acid Ben zyl Ester .
2-Naphthylacetic acid benzyl ester (0.750 g, 2.72 mmol) in THF
(4 mL) was slowly added to a cold (0 °C) solution of sodium
bis(trimethylsilyl)amide (1 M in THF, 4.1 mL) and iodo-
methane (0.25 mL). After 30 min at room temperature,
additional sodium bis(trimethylsilyl)amide (4.1 mL) and io-
domethane (0.25 mL) were added. After 1 h, ethyl acetate was
added, and the mixture was washed with 10% citric acid, 10%
NaHCO3, and brine. The solvent was evaporated, and flash
column chromatography of the residue (10% ethyl acetate/
hexane) gave 2,2-dimethyl-(2-naphthyl)acetic acid, bezyl ester
as a colorless oil (0.46 g, 1.5 mmol, 55%).
was taken on directly to the next step: 1H NMR δ CDCl3 9.33
(1H, br s), 7.28-7.25 (6H, m), 7.09 (1H, d, J ) 7), 6.88 (2H, d,
J ) 9), 6.75 (2H, d, J ) 8), 6.10 (1H, d, J ) 7), 5.07 (2H, m),
3.81 (3H, s), 3.81 (1H, m), 3.72 (3H, s), 3.64 (2H, s), 3.35 (1H,
m), 2.77 (1H, m), 2.16 (3H, s); MS(EI) 463 (M+•). Anal.
(C26H29N3O5) C, H, N.
3-[2′-(S)-(1′′′-Na p h th yla cetyl)a m in o-3′-(4′′-m eth oxyca r -
b on ylm et h yl)b en zen e]p r op a n oyla m in o-1-(4-m et h oxy-
ben zyl)-4-m eth yl-2-p yr id on e (VIIb). To a stirred solution
of 1-naphthylacetic acid (0.041 g, 0.22 mmol) in methylene
chloride (2 mL) cooled on ice was added EDC (0.046 g, 0. 24
mmol). After 20 min, VIb (0.10 g, 0.22 mmol) in methylene
chloride (1 mL) was added, followed by Hunig’s base (0.03 g).
The mixture was stirred on ice for 1 h and allowed to warm to
room temperature overnight. The mixture was diluted with
ethyl acetate, washed with water, dried, filtered, and evapo-
rated. Chromatography of the residue over silica gel (methyl-
ene chloride/methanol, 19/1) gave VIIb (0.12 g, 0.19 mmol,
1
86%): mp 154-155 °C; H NMR δ CDCl3 7.93-7.78 (4H, m),
7.51-7.34 (4H, m), 7.24 (2H, d, J ) 9), 7.06 (1H, d, J ) 7),
6.93 (2H, d, J ) 8), 6.89 (2H, d, J ) 9), 6.75 (2H, d, J ) 8),
6.04 (1H, d, J ) 7), 5.96 (1H, d, J ) 8), 5.05 (2H, s), 4.85 (1H,
m), 4.04 (2H, m), 3.82 (3H, m), 3.71 (3H, s), 3.53 (2H, s), 2.99-
2.80 (2H, m), 2.00 (3H, s); MS(CI) 632 (MH+). Anal. (C38H37N3O6‚
0.5H2O) C, H, N.
3-[2′-(S)-(1′′′-Na p h th yla cetyl)a m in o-3′-(4′′-ca r boxym e-
t h yl)b en zen e]p r op a n oyla m in o-1-(4-m et h oxyb en zyl)-4-
m eth yl-2-p yr id on e (5). To a solution of VIIb (0.07 g, 0.11
mmol) in methanol/THF (1/1, 5 mL) was added aqueous NaOH
(1 M, 0.15 mL). The mixture was stirred at room temperature
overnight and acidified with 1 M aqueous HCl. The solvents
were evaporated, and the residue was taken up in ethyl
acetate/water. The organic phase was dried, filtered, and
evaporated to give 5 (0.065 g, 0.11 mmol, 100%): mp 224-
2,2-Dim eth yl-(2-n a p h th yl)a cetic Acid . A mixture of 2,2-
dimethyl-(2-naphthyl)acetic acid, benzyl ester (0.46 g, 1.5
mmol) and 10% Pd/C (0.040 g) in ethanol (5 mL) was
hydrogenated at 1 atm for 16 h. The catalyst was removed by
filtration, and the solvent was evaporated to give 2,2-dimethyl-
(2-naphthyl)acetic acid (0.33 g, 1.5 mmol, 100%).
1
226 °C (ethyl acetate/methanol); H NMR δ DMSO 9.48 (1H,
s), 8.51 (1H, d, J ) 8), 7.88-7.13 (14H, m), 7.51-7.34 (4H,
m), 6.89 (2H, d, J ) 9), 6.15 (1H, d, J ) 7), 5.03 (2H, m), 4.75
(1H, m), 3.89 (2H, m), 3.73 (3H, s), 3.53 (2H, s), 3.21 (1H, m),
2.87 (1H, m), 1.88 (3H, s). Anal. (C37H35N3O6‚H2O) C, H, N.
3-[2′-(S)-tBu toxyca r bon yla m in o-3′-(4′′-m eth oxyca r bon -
ylm eth yl)ben zen e]pr opan oylam in o-1-(4-m eth oxyben zyl)-
4-m eth yl-2-p yr id on e (Vb). A mixture of IV7b,23 (1.0 g, 3
mmol) and EDC (0.63 g, 3.3 mmol) in methylene chloride (5
mL) was stirred at 0 °C for 20 min. 3-Amino-1-(4-methoxy-
benzyl)-4-methyl-2-pyridone (IIIb) (0.80 g, 3.3 mmol) was
added, and after 1 h, the mixture was allowed to warm to room
temperature and was stirred at room temperature overnight.
The mixture was diluted with ethyl acetate, washed with
water, dried, filtered, and evaporated. Chromatography of the
residue over silica gel (ethyl acetate/hexane 1/4) gave Vb (0.45
g, 0.80 mmol, 27%) as a noncrystalline solid: 1H NMR δ CDCl3
7.95 (1H, br s), 7.26-7.21 (6H, m), 7.06 (1H, d, J ) 7), 6.86
(2H, d, J ) 8), 6.06 (1H, d, J ) 7), 5.10 (1H, br), 5.07 (1H, d,
J ) 15), 5.00 (1H, d, J ) 15), 4.59 (1H, m), 3.79 (3H, s), 3.68
(3H, s), 3.58 (2H, s), 3.27 (1H, m), 3.08 (1H, m), 2.08 (3H, s),
1.38 (9H,s); MS(EI) 563 (M+•). Anal. (C31H37N3O7) C, H, N.
3-[2′-(S)-tBu toxyca r bon yla m in o-3′-(4′′-m eth oxyca r bon -
y lm e t h y l)b e n z e n e ]p r o p a n o y la m i n o -1-(4-m e t h o x y -
ben zyl)-2-p yr id on e (Va ) was obtained as a noncrystalline
solid in an analogous manner using IIIa as a coupling
partner: 1H NMR δ CDCl3 8.93 (1H, br s), 8.34 (1H, dd, J )
2, 7), 7.22-7.12 (6H, m), 7.00 (1H, dd, J ) 2, 7), 6.86 (2H, d,
J ) 9), 6.20 (1H, apparent t, J ) 7), 5.08-5.02 (3H), 4.54 (1H,
br), 3.77 (3H, s), 3.67 (3H, s), 3.57 (2H, s), 3.20 (1H, dd, J ) 6,
9), 3.05 (1H, m), 1.38 (9H, s). Anal. (C30H35N3O7) C, H, N.
3-[2′-(S)-Am in o-3′-(4′′-m eth oxycar bon ylm eth yl)ben zen e]-
p r op a n oyla m in o-1-(4-m et h oxyb en zyl)-4-m et h yl-2-p yr i-
d on e (VIb). To a solution of Vb (0.40 g, 0.71 mmol) in
methylene chloride (3 mL) was added TFA, and the mixture
was stirred at room temperature overnight. The solvents were
evaporated, and the residue was taken up in methylene
chloride and aqueous sodium bicarbonate. The organic phase
was dried (Na2SO4), filtered, and evaporated. Chromatography
of the residue over silica gel (methylene chloride/methanol)
gave VIb (0.40 g, 0.86 mmol, >100%) as a sticky solid which
3-[2′-(S)-Acetyla m in o-3′-(4′′-ca r boxym eth yl)ben zen e]-
p r op a n oyla m in o-1-(4-m et h oxyb en zyl)-4-m et h yl-2-p yr i-
d on e (6). To a solution of VIb (0.10 g, 0.22 mmol) in methylene
chloride (2 mL), cooled on ice, was added a few drops of Hunig’s
base followed by acetic anhydride (0.045 g). The mixture was
allowed to warm to room temperature overnight. The solvent
was evaporated, and the residue was triturated with ether and
collected by filtration to give VIIc: mp 158-159 °C; 1H NMR
δ CDCl3 7.82 (1H, s), 7.28-7.22 (6H, m), 7.08 (1H, d, J ) 7),
6.89 (2H, d, J ) 8), 6.22 (1H, d, J ) 8), 6.09 (1H, d, J ) 7),
5.08 (1H, d, J ) 14), 5.03 (1H, d, J ) 4), 4.95 (1H, m), 3.81
(3H, m), 3.70 (3H, s), 3.61 (2H, s), 3.25-3.18 (2H, m), 2.07 (3H,
s), 2.02 (3H, s); MS(CI) 506 (MH+). Anal. (C28H31N3O6‚0.5H2O)
C, H, N.
To a solution of VIIc (0.065 g, 0.13 mmol), prepared above,
in methanol (3 mL) was added aqueous NaOH (1 M, 0.14 mL).
The mixture was stirred at room temperature overnight and
acidified with 1 M aqueous HCl. The solvent was evaporated,
and the residue was partitioned between ethyl acetate and
water. The organic phase was separated, dried, filtered, and
evaporated to give 6 (0.06 g, 0.012 mmol, 55% over two steps
from VIb): mp 190-192 °C (ethyl acetate/methanol); 1H NMR
δ DMSO 9.40 (1H, s), 8.14 (1H, d, J ) 8), 7.63 (1H, d, J ) 7),
7.27 (4H, m), 7.15 (2H, d, J ) 8), 6.89 (2H, d, J ) 9), 6.14 (1H,
d, J ) 7), 5.12 (2H, m), 4.74 (1H, m), 3.72 (3H, s), 3.51 (2H, s),
3.08 (1H, m), 2.78 (1H, m), 1.88 (3H, s), 1.76 (3H, s); MS(EI)
491 (M+•). Anal. (C27H29N3O6‚0.25H2O) C, H, N.
3-[2′-(S)-Am in o-3′-(4′′-m eth oxycar bon ylm eth yl)ben zen e]-
p r op a n oyla m in o-1-(4-m et h oxyb en zyl)-2-p yr id on e H y-
d r och lor id e (VIa ‚HCl). A solution of Va (0.62 g, 1.1 mmol)
in 4 N HCl/dioxane (7.5 mL) was left at room temperature for
16 h. The solvent was evaporated. Ethyl acetate was added,
and the solvent was evaporated to give the hydrochloride salt
of VIa as a solid (0.52 g, 1.1 mmol): 1H NMR δ DMSO 10.13
(1H, s), 8.37 (3H, br s), 8.18 (1H, dd, J ) 2, 7), 7.64 (1H, dd, J

2428 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15
Proudfoot et al.
) 2, 7), 7.32 (2H, d, J ) 9), 7.27-7.19 (4H, m), 6.91 (2H, d, J
) 9), 6.32 (1H, apparent t, J ) 7), 5.10 (m, 2H), 4.57 (m, 1H),
3.73 (3H, s), 3.64 (2H, s), 3.60 (3H, s), 3.18-3.02 (2H, m); MS-
(CI) 450. Anal. (C25H27N3O5‚HCl‚H2O) C, H, N.
(18.1 g, 52.1 mmol, 81%): mp 60-62 °C; 1H NMR δ CDCl3
7.63 (2H, d, J ) 8), 7.09 (2H, d, J ) 8), 1.58 (6H, s), 1.37 (9H,
s). Anal. (C14H19O2I) C, H.
2-tBu toxyca r bon yla m in o-3-[4′-(1′′-tbu toxyca r bon yl-1′′-
m eth yl)eth yl]ben zen ep r op en oic Acid , Ben zyl Ester (IX).
A mixture of benzyl (2-tbutoxycarbonylamino)acrylate (8.00 g,
27.9 mmol), VIIIc (7.03 g, 20.3 mmol), sodium bicarbonate
(4.03 g), tetrabutylammonium chloride hydrate (6.47 g), and
palladium acetate (0.41 g) in dimethylformamide (100 mL) was
degassed and covered with argon three times. The mixture was
heated under argon at 80 °C for 5 h, cooled, diluted with ethyl
acetate/hexane, and washed with water. The organic phase
was dried, filtered, and evaporated. Chromatography of the
residue over silica gel (ethyl acetate/hexane 98/2 to 9/1) gave
IX as an oil that solidified upon trituration with hexane (7.24
3-[2′-(S)-(1′′′-Na p h th yla cetyl)a m in o-3′-(4′′-m eth oxyca r -
b on ylm et h yl)b en zen e]p r op a n oyla m in o-1-(4-m et h oxy-
ben zyl)-2-p yr id on e (VIIa ). To a solution of 1-naphthylacetic
acid (0.21 g, 0.11 mmol) in methylene chloride (2 mL) cooled
on ice was added EDC (0.22 g, 0.11 mmol). After 15 min, VIa ‚
HCl (0.40 g, 0.82 mmol) in methylene chloride (10 mL) was
added. After 3 h at room temperature, the mixture was
fractionated directly over silica gel (methylene chloride to
methylene chloride/ethyl acetate 3/1) to give VIIa (0.31 g, 0.50
mmol, 61%): mp 151-153 °C (ethyl acetate/hexane); 1H NMR
δ CDCl3 8.85 (1H, s), 8.25 (1H, dd, J ) 2, 7), 7.92-7.23 (9H,
m), 6.99 (1H, dd, J ) 2, 7), 6.90 (2H, d, J ) 9), 6.85 (2H, d, J
) 9), 6.54 (2H, d, J ) 8), 6.22 (1H, d, J ) 8), 6.18 (1H, apparent
t, J ) 7), 5.80 (1H, d, J ) 8) 5.11 (2H, s), 4.79 (1H, m), 4.09
(1H, d, J ) 16), 4.00 (1H, d, J ) 16), 3.80 (3H, m), 3.69 (3H,
s), 3.49 (2H, s), 2.88 (2H, d, J ) 6); Anal. (C38H37N3O6) C, H,
N.
3-[2′-(S)-(1′′′-Na p h th yla cetyl)a m in o-3′-(4′′-ca r boxym e-
t h yl)b en zen e]p r op a n oyla m in o-1-(4-m et h oxyb en zyl)-2-
p yr id on e (4). To a stirred solution of VIIa (0.15 g, 0.24 mmol)
in THF (3 mL) was added a solution of lithium hydroxide
hydrate (0.013 g) in water (0.3 mL). After 3.5 h at room
temperature, ethyl acetate and aqueous sodium bicarbonate
were added. The aqueous phase was separated, acidified with
1 N KHSO4, and extracted with ethyl acetate. The organic
phase was dried, filtered, and evaporated. Trituration with
ether gave 4 (0.076 g, 0.12 mmol, 50%) as a solid that was
collected by filtration: mp 187-190 °C; 1H NMR δ DMSO 9.53
(1H, s), 8.75 (1H, d, J ) 8), 8.23 (1H, dd, J ) 2, 7), 7.89-7.85
(2H, m), 7.77 (1H, d, J ) 8), 7.57 (1H, dd, J ) 2, 7), 7.45 (1H,
t, J ) 7), 7.38-7.13 (9H, m), 6.91 (2H, d, J ) 9), 6.31 (1H,
apparent t, J ) 7), 5.12 (2H, s), 4.77 (1H, m), 3.91 (2H, s),
3.74 (3H, s), 3.34 (2H, s), 3.12 (1H, m), 2.88 (1H, m). Anal.
(C36H33N3O6‚0.5H2O) C, H, N.
1
g, 14.6 mmol, 72%): mp 81-83 °C; H NMR δ CDCl3 7.51-
7.28 (9H, m), 6.13 (1H, br), 5.28 (1H, s), 1.51 (6H, s), 1.36 (9H,
s); MS(EI) 495 (M +•). Anal. (C29H37NO6) C, H, N.
2-(R,S)-t Bu t oxyca r b on yla m in o-3-[4′-(1′′-t b u t oxyca r b -
on yl-1′′-m eth yl)eth yl]ben zen ep r op a n oic Acid (X). A mix-
ture of IX (7.39 g, 14.9 mmol) and 10% Pd/C (0.20 g) in ethanol
(75 mL) was hydrogenated at 45 psi in a Parr apparatus for
26 h. The catalyst was removed by filtration, and the solvent
was evaporated. The residue crystallized from hexane giving
X (4.67 g, 11.5 mmol, 77%): mp 105-107 °C; 1H NMR δ CDCl3
7.28 (2H, d, J ) 8), 7.13 (2H, d, J ) 8), 4.92 (1H, d, J ) 8),
4.59 (1H, m), 3.19-3.05 (2H, m), 1.51 (6H, s), 1.41 (9H, s), 1.37
(9H, s); MS(CI) 425 (M + NH4+). Anal. (C22H33NO6) C, H, N.
3-[2′-(R,S)-t Bu t oxyca r b on yla m in o-3′-[4′′-(1′′′-t b u t oxy-
ca r b on yl-1′′′-m et h yl)et h yl]b en zen e]p r op a n oyla m in o-1-
(4-m eth oxyben zyl)-4-m eth yl-2-p yr id on e (XI). To a stirred
solution of X (1.56 g, 3.83 mmol) in methylene chloride (5 mL)
at 0 °C was added EDC (0.80 g, 4.2 mmol). After 10 min IIIb
(1.16 g, 4.75 mmol) was added, and the mixture was stirred
at room temperature for 4 h. Additional EDC (0.21 g) was
added, and the mixture was stirred for 2 days. The mixture
was fractionated directly over silica gel (CH2Cl2 to 10% EtOAc/
CH2Cl2) to give XI (1.63 g, 2.57 mmol, 67%): mp 134-136 °C
1
4-Iodo-(1′-tbu toxycar bon yl-1′-m eth yl)eth ylben zen e (VI-
IIc). To a stirred solution of ethyl phenylacetate (32.8 g, 200
mmol) in THF (1 L) under argon was added sodium bistri-
methylsilylamide (2 M in THF, 100 mL). After 45 min, methyl
iodide (13 mL) was added over 15 min. After 30 min, additional
sodium bistrimethylsilylamide (2 M in THF, 100 mL) was
added, followed, after 30 min, by methyl iodide (14 mL). After
30 min, the mixture was diluted with hexane and washed with
water. The organic phase was dried, filtered, and evaporated
to give (1′-ethoxycarbonyl-1′-methyl)ethylbenzene (VIIIa ) (28.0
g, 146 mmol, 73%) which was taken on directly to the next
step. A mixture of VIIIa (28.0 g, 146 mmol), iodine (24.7 g),
sodium iodate (7.1 g), and concentrated sulfuric acid (4 mL)
in acetic acid (200 mL) was stirred and heated at 55 °C for 90
h. The solvent was evaporated, and the residue was partitioned
between water and hexane. The organic phase was washed
with aqueous sodium thiosulfate, dried, filtered, and evapo-
rated. The residue was taken up in ethanol (200 mL) and water
(100 mL), and potassium hydroxide (20 g) was added. The
mixture was heated under reflux for 5 h and cooled to room
temperature. The mixture was washed with hexane. The
aqueous phase was acidified with concentrated hydrochloric
acid and extracted with hexane. The organic phase was dried,
filtered, and evaporated to give 4-iodo-(1′-carboxy-1′-methyl)-
ethylbenzene (VIIIb) as a solid (19.6 g, 67 mmol, 46%): mp
(Et2O); H NMR δ CDCl3 7.96 (1H, br s), 7.28-7.16 (6H, m),
7.06 (1H, d, J ) 7), 6.86 (2H, d, J ) 9), 6.06 (1H, d, J ) 7),
5.08 (1H, br), 5.07 (1H, d, J ) 14), 5.00 (1H, d, J ) 14), 4.59
(1H, m), 3.79 (3H, s), 3.28 (1H, m), 3.05 (1H, m), 2.07 (3H, s),
1.49 (6H, s), 1.38 (9H, s), 1.35 (9H, s). Anal. (C36H47N3O7) C,
H, N.
3-[2′-(R,S)-Na p h th yla cetyla m in o-3′-[4′′-(1′′′-ca r boxy-1′′′-
m et h yl)et h yl]b en zen e]p r op a n oyla m in o-1-(4-m et h oxy-
ben zyl)-4-m eth yl-2-p yr id on e (8RS). To a solution of XI
(0.32 g) in dioxane (0.5 mL) cooled on ice was added HCl in
dioxane (4 M, 1 mL). The mixture was allowed to warm to
room temperature over 45 min, then diluted with ethyl acetate,
washed with aqueous sodium bicarbonate, dried (Na2SO4),
filtered, and evaporated. The XII (0.30 g) obtained was carried
on directly to the next step. To a stirred solution of XII (0.30
g) in CH2Cl2 (3 mL) at room temperature were added 2-naph-
thylacetic acid (0.095 g) and EDC (0.098 g). After 1 h, the
mixture was diluted with ethyl acetate, washed with water,
dried, filtered, and evaporated, and the residue was taken up
in TFA. After 2 h, the TFA was removed by evaporation, and
the residue, dissolved in ethyl acetate, was washed with water,
dried, filtered, and evaporated. Chromatography of the residue
over silica gel (CH2Cl2/EtOH/AcOH 97/2.5/0.5) gave 8RS (0.14
g,) as an oil that solidified on trituration with ether: mp 208-
210 °C (Et2O/ CH2Cl2); 1H NMR δ CDCl3 8.81 (1H, br s), 7.12-
7.73 (12H, m), 6.89 (4H, m), 6.27 (1H, d, J ) 7), 6.14 (1H, d,
J ) 7), 5.07 (2H, s), 4.82 (1H, m), 3.80 (3H, s), 3.45 (2H, s),
3.11 (1H, m), 2.78 (1H, m), 2.00 (3H, s), 1.58 (3H, s), 1.56 (3H,
s). Anal. (C39H39N3O6) C, H, N.
1
106-110 °C (hexane); H NMR δ CDCl3 7.66 (2H, d, J ) 8),
7.15 (2H, d, J ) 8), 1.57 (6H, s). Anal. (C10H11O2I) C, H.
To a solution of VIIIb (18.7 g, 64.3 mmol) in methylene
chloride (150 mL) was added dimethylformamide (0.5 mL)
followed by oxalyl chloride (10 mL), dropwise. After 1 h, the
solvent was evaporated, and the residue was taken up in THF
7RS was prepared in an analogous manner: mp 149-151
°C (Et2O/ CH2Cl2); 1H NMR δ CDCl3 8.80 (1H, br s), 7.08-
7.81 (12H, m), 6.87 (2H, d, J ) 9), 6.72 (2H, d, J ) 8), 6.14
(2H, m), 5.07 (2H, s), 4.79 (1H, m), 3.80 (3H, s), 3.80 (1H, d, J
) 16), 3.71 (1H, d, J ) 16), 3.01 (1H, m), 2.68 (1H, m), 1.97
(3H, s), 1.59 (3H, s), 1.57 (3H, s). Anal. (C39H39N3O6‚0.25H2O)
C, H, N.
t
(100 mL) and cooled on ice. Potassium butoxide (1 M in THF,
75 mL) was added over 20 min. The mixture was diluted with
hexane, washed with water, dried, filtered, and evaporated.
The residue was triturated with methanol/water to give 4-iodo-
(1′-tbutoxycarbonyl-1′-methyl)ethylbenzene (VIIIc) as a solid

Nonpeptidic Ligands for the p56lck SH2 Domain
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15 2429
3-[2′-(R,S)-(Dim eth yl)-n a p h th yla cetyla m in o-3′-[4′′-(1′′′-
ca r boxy-1′′′-m eth yl)eth yl]ben zen e]p r op a n oyla m in o-1-(4-
m eth oxyben zyl)-4-m eth yl-2-p yr id on e (9RS) was prepared
in an analogous manner using 2,2-dimethylnaphthylacetic acid
cross-linked the T cell receptor. Changes in cytoplasmic
calcium concentration were monitored by measurement of
fluorescence over time on a luminescence spectrometer (SLM
Amico Bowman Series 2). The EC50 values were calculated
based on the percent of control fluorescence at six doses of
compound.
1
in the coupling reaction: mp 157-161 °C (Et2O/CH2Cl2); H
NMR δ CDCl3 8.66 (1H, br s), 7.01-7.79 (12H, m), 6.87 (2H,
d, J ) 8), 6.69 (2H, d, J ) 8), 6.14 (1H, d, J ) 7), 5.95 (1H, d,
J ) 7), 5.07 (1H, d, J ) 14), 5.00 (1H, d, J ) 14), 4.82 (1H, m),
3.79 (3H, s), 3.04 (1H, m), 2.67 (1H, m), 2.05 (3H, s), 1.56 (3H,
s), 1.53 (6H, s), 1.41 (3H, s). Anal. (C41H43N3O6‚0.5H2O), C, H,
N.
Activa tion of Hu m a n CD4+ T Cells. Purified human
CD4+ T lymphocytes were suspended in RPMI 1640 supple-
mented with 1% fetal bovine serum, nonessential amino acids,
1 mM sodium pyruvate, 0.29 mg/mL L-glutamine, penicillin,
and streptomycin, and 5 × 104 cells were added per well to a
96-well flat bottom plate. Compounds were diluted from DMSO
stock solutions into medium and added to the cells, and the
mixtures were preincubated for 1 h at 37 °C. The cells were
activated by the addition of 0.12 ng/well mouse anti-human
CD3, 100 ng/well rat anti-human CD28 (Clone YTH913.12),
and 2 × 105 goat anti-mouse IgG coated magnetic beads/well
(Dynal) in a final volume of 210 µL/well. The cells were
incubated at 37 °C in a humidified atmosphere of 5% CO2
overnight. A total of 10 µL/well of conditioned medium was
removed, and the amount of interleukin-2 secreted by the T
cells was quantitated utilizing an ELISA kit (R&D Systems)
according to the manufacturers instructions. To measure
proliferation, the cells were pulse labeled 48 h following
activation with 0.5 µCi/well [3H]-thymidine (Amersham) and
incubated a further 18 h at 37 °C. The cells were harvested
on a 96-well plate harvester, and the incorporated radiolabeled
thymidine determined using a â-plate counter.
3-[2′-(S)-(Dim et h yl)-n a p h t h yla cet yla m in o-3′-[4′′-(1′′′-
ca r boxy-1′′′-m eth yl)eth yl]ben zen e]p r op a n oyla m in o-1-(4-
m eth oxyben zyl)-4-m eth yl-2-p yr id on e (9S): mp 115-117
°C; MS 674 (MH+). Anal. (C41H43N3O6‚2H2O) C, H, N. ee 99.6%
by chiral HPLC.
3-[2′-(R)-(Dim et h yl)-n a p h t h yla cet yla m in o-3′-[4′′-(1′′′-
ca r boxy-1′′′-m eth yl)eth yl]ben zen e]p r op a n oyla m in o-1-(4-
m eth oxyben zyl)-4-m eth yl-2-p yr id on e (9R): mp 117-119
°C; MS 674 (MH+). Anal. (C41H43N3O6. 2H2O) C, H, N. ee 98.7%
by chiral HPLC.
Ca co-2 P er m ea bility Assa y. Caco-2 cells, originating from
a human colorectal carcinoma, were obtained from American
Tissue Culture Collection, Rockville, MD. The cells were grown
at 37 °C in an atmosphere of 5% CO2 in Dulbecco’s Modified
Eagle Medium (DMEM) containing 10% fetal calf serum, 1%
nonessential amino acids, penicillin (100 Units/ml), and strep-
tomycin (100 µg/mL). All culture media and reagents were
from Gibco BRL Products, Gaithersburg, MD.
EC50 values for either IL-2 production or proliferation were
calculated from data fit to a sigmoidal curve using nonlinear
regression techniques.
Caco-2 cells were seeded at a density of 80 000 cells/cm2 in
six-well plates on polycarbonate filters (Costar, Transwell cell
culture inserts, diameter 24.5 mm, pore size 3.0 µm) coated
with rat tail collagen type I. The cells were allowed to grow
and differentiate for up to 25 days. Cells of passage numbers
23 to 50 were used throughout.
Molecu la r Mod elin g. The crystal structure of the p56lck
SH2 domain complexed with Ac-(p-carboxymethyl)Phe-Glu-
Glu-Ile17 was used to provide the starting geometry. The initial
position of the Ac-phenylacetic portion of the ligand was the
same as in the crystal structure of the complex with the
tetrapeptide. The pyridone and p-methoxybenzyl groups were
modeled with manual docking procedures. As previously,7a,7b
the p-methoxybenzyl group was oriented toward the P+3
pocket. This initial model was optimized by performing a
molecular dynamics (MD) simulation (1 nanosecond (ns)) using
CharmM.33 The electrostatic Coulombic potential was com-
puted with a distance dependent dielectric using CharmM
charges for the protein, and MNDO ESP charges using
MOPAC34 for the ligand. The MD was carried out in explicit
solvent, using a TIP3P35 solvent cap of 25 Å around the
inhibitor. In this MD protocol, all the atoms within 13 Å of
the inhibitor were allowed to move freely. The rest of the
protein was constrained using a potential harmonic constraint
of 5 kcal/Å2, and the remaining solvent molecules were
maintained within the sphere limit by applying the CharmM
DROP restraint option. Several structures, as well as the
average structure, were collected along the 1 ns trajectory for
further analysis.
Prior to permeability experiments, the culture medium was
replaced with the transport medium, Hank’s Balanced Salt
Solution (HBSS), pH 7.4, and equilibrated for 30 min at 37
°C. All wells in six-well clusters received 2.5 mL of HBSS. Drug
solutions were prepared in HBSS at a final concentration of
0.01 to 0.1 mM. All permeability experiments were performed
in an incubator at 37 °C and an atmosphere of 5% CO2. To
initiate permeability experiments, the apical side of the
monolayers received 1.5 mL of drug solutions. The amount of
solute permeated was determined by either moving the inserts
to new wells containing fresh medium or taking a sample from
the basolateral side and replacing it with fresh medium at
discrete time intervals. Transport rates were then determined
by plotting the cumulative amount permeated as a function
of time. Samples were analyzed by HPLC (Hewlett-Packard,
HP 1090). The apparent permeability coefficient, PCaco-2, was
then determined according to eq 1
PCaco-2 ) J /ACo
(1)
Ack n ow led gm en t. We thank Dr. Denice Spero, Dr.
Xiao-J un Wang, and Michel Emmanuel for assistance
in the scale-up of various compounds. We also thank
Kelli Chessic and Katherine Briggs for assistance in
running the Caco-2 model.
where J was the rate of appearance of the drug in the receiver
chamber, Co was the initial concentration of the solute in the
donor chamber, and A was the surface area of the filter. Mass
balance was calculated for each experiment. In all experi-
ments, the mass balance was more than 90%. Mannitol
permeability was used to assess the integrity of Caco-2 cell
monolayers. Permeability coefficients were determined at least
in triplicates, and the mean and standard deviations are
reported.
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Nonpeptidic Ligands for the p56lck SH2 Domain
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15 2431
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a
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