Novel 2-aminopyrimidine carbamates as potent and orally active inhibitors of Lck: Synthesis, SAR, and in vivo antiinflammatory activity

Article abstract of DOI:10.1021/jm060435i

The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and NK cells. Genetic evidence in both mice and humans demonstrates that Lck kinase activity is critical for signaling mediated by the T cell receptor (TCR), which leads to normal T cell development and activation. A small molecule inhibitor of Lck is expected to be useful in the treatment of T cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection. In this paper, we describe the synthesis, structure-activity relationships, and pharmacological characterization of 2-aminopyrimidine carbamates, a new class of compounds with potent and selective inhibition of Lck. The most promising compound of this series, 2,6-dimethylphenyl 2-((3,5-bis(methyloxy)-4-((3-(4-methyl-1-piperazinyl)propyl)- oxy)phenyl)amino)-4-pyrimidinyl(2,4-bis(methyloxy)phenyl)carbamate (43) exhibits good activity when evaluated in in vitro assays and in an in vivo model of T cell activation.

Full text of DOI:10.1021/jm060435i

J. Med. Chem. 2006, 49, 4981-4991
4981
Novel 2-Aminopyrimidine Carbamates as Potent and Orally Active Inhibitors of Lck:
Synthesis, SAR, and in Vivo Antiinflammatory Activity
,
†
,†
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†
Matthew W. Martin,* John Newcomb,* Joseph J. Nunes, David C. McGowan, David M. Armistead, Christina Boucher,
†
†
†
†
†
†
†
John L. Buchanan, William Buckner, Lilly Chai, Daniel Elbaum, Linda F. Epstein, Theodore Faust, Shaun Flynn,
Paul Gallant, Anu Gore, Yan Gu, Faye Hsieh, Xin Huang, Josie H. Lee, Daniela Metz, Scot Middleton, Deanna Mohn,
Kurt Morgenstern, Michael J. Morrison, Perry M. Novak, Antonio Oliveira-dos-Santos, David Powers, Paul Rose,
†
⊥
†
§
†
†
‡
‡
‡
†
†
†
†
+
†
†
†
+
†
+
†
‡
Stephen Schneider, Stephanie Sell, Yanyan Tudor, Susan M. Turci, Andrew A. Welcher, Ryan D. White, Debra Zack,
†
‡
†
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Huilin Zhao, Li Zhu, Xiaotian Zhu, Chiara Ghiron, Patricia Amouzegh, Monika Ermann, James Jenkins, David Johnston,
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Spencer Napier, and Eoin Power
Department of Chemistry Research and DiscoVery, Amgen Inc., One Kendall Square, Building 1000, Cambridge, Massachusetts 02139,
Department of Chemistry Research and DiscoVery, Department of Inflammation, Department of Pharmaceutics, and Department of
Pharmacokinetics & Drug Metabolism, Amgen Inc., One Amgen Center DriVe, Thousand Oaks, California 91320, and EVotec OAI,
1
51 Milton Park, Abingdon, Oxon OX14 4SD, UK
ReceiVed April 12, 2006
The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T
cells and NK cells. Genetic evidence in both mice and humans demonstrates that Lck kinase activity is
critical for signaling mediated by the T cell receptor (TCR), which leads to normal T cell development and
activation. A small molecule inhibitor of Lck is expected to be useful in the treatment of T cell-mediated
autoimmune and inflammatory disorders and/or organ transplant rejection. In this paper, we describe the
synthesis, structure-activity relationships, and pharmacological characterization of 2-aminopyrimidine
carbamates, a new class of compounds with potent and selective inhibition of Lck. The most promising
compound of this series, 2,6-dimethylphenyl 2-((3,5-bis(methyloxy)-4-((3-(4-methyl-1-piperazinyl)propyl)-
oxy)phenyl)amino)-4-pyrimidinyl(2,4-bis(methyloxy)phenyl)carbamate (43) exhibits good activity when
evaluated in in vitro assays and in an in vivo model of T cell activation.
Introduction
T cells serve pivotal roles in the adaptive immune response
by acting either as potentiators (CD4+ T helper cells) or
effectors (CD8+ cytolytic T effector cells) of immune reactions.
The immune specificity of these T cells is imparted by the T
a
1
cell receptor (TCR) for antigen, or CD3 complex. Signaling
pathways used by the TCR have been an intense area of research
in the discovery of novel immunosuppressive agents that might
serve as improved therapies for graft rejection and T cell-
mediated autoimmune diseases. One class of enzymes shown
to be important in TCR signal transduction is the protein kinases.
The Src-family of cytoplasmic tyrosine kinases is made up of
nine members: Src, Lck, Fyn, Lyn, Hck, Fgr, Blk, Yes, and
Figure 1. Structure of 2-aminopyrimidine carbamate 1.
A number of groups have previously reported the synthesis
1
3-25
and characterization of Lck kinase inhibitors.
Potent and
bioavailable Lck inhibitors have also been demonstrated to have
inhibitory activities in vivo in several models of T cell-dependent
2
2,26,27
immune responses.
Herein we describe the synthesis,
2
,3
structure-activity relationships, and pharmacological charac-
terization of 2-aminopyrimidine carbamates, a new class of
compounds with potent and selective inhibition of Lck.28 Lead
optimization led to inhibitors that demonstrated inhibition of T
cell activation in in vitro and in vivo biological assays.
Yrk. Of these Src-family kinases, Lck and Fyn have been
4
-6
shown to have important roles in TCR signal transduction.
TCR signals initiated by Lck ultimately lead to gene regulation
events triggering cytokine release, proliferation, and survival
of antigen specific T cells thereby amplifying specific immune
responses. Lck knock-out mice and patients with Lck mutations
effecting expression and/or catalytic activity show defects in T
cell maturation and signaling.7-12 These findings suggest that
a small molecule inhibitor of Lck kinase could be a useful
immunosuppressive for the treatment of graft rejection and/or
T cell-mediated autoimmune diseases.
Screening of our kinase-preferred compound collection
identified 2,6-dimethylphenyl-2-((4-(4-methyl-1-piperazinyl)-
phenyl)amino)-4-pyrimidinyl(phenyl)carbamate (compound 1,
Figure 1) as a potent inhibitor of Lck, with an IC50 of 14 nM in
an Lck kinase assay. Subsequently, we carried out structure-
activity relationship studies on 1 designed to identify substituents
for increased potency, selectivity, and optimal pharmacokinetic
properties. In the course of these studies, we utilized structural
biology to obtain enzyme:inhibitor cocrystal structures that
assisted in identifying the key components necessary for binding
to Lck.
*
To whom correspondence should be addressed: Tel: 617-444-5013.
Fax: 617-621-3908. E-mail: matmarti@amgen.com; Tel: 617-444-5055.
Fax: 617-621-3916. E-mail: jnewcomb@amgen.com.
†
⊥
Department of Chemistry Research and Discovery, Cambridge, MA.
Department of Pharmaceutics, Thousand Oaks, CA.
Department of Pharmacokinetics and Drug Metabolism, Thousand Oaks,
§
CA‡.
a
Department of Inflammation, Thousand Oaks, CA.
Department of Chemistry Research and Discovery, Thousand Oaks,
Abbreviations: Lck, lymphocyte-specific kinase; TCR, T cell receptor;
+
IL-2, interleukin 2; PTK, protein tyrosine kinase; CMGC, kinase group
containing CDK, MAPK, GSK3, and CLK families; AGC, kinase group
containing PKA, PKG, and PKC families.
CA|.
Evotec OAI.
1
0.1021/jm060435i CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/07/2006

4
982 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Martin et al.
Table 1. SAR: Variations of the Amine (IC50, µM)a
Scheme 1. Retrosynthetic Analysis of 2-Aminopyrimidine
Carbamates
Chemistry
Our strategy for the construction of the 2-aminopyrimidine
carbamates described in this paper is outlined in the retrosyn-
thetic analysis shown in Scheme 1. In this approach, carbamates
2
were prepared via the nucleophilic addition of anilines 3 to
2-chloropyrimidine carbamates 4 under acidic conditions. Car-
bamates 4, in turn, were accessed through the sequential addition
of amines 6 to 2,4-dichloropyrimidine (5) followed by acylation
with chloroformates 7. This concise route allowed for rapid
analoging, as each variation could be introduced by simply
employing the desired amine, chloroformate, and aniline.
The anilines required for the syntheses of the carbamates
shown in Tables 1 and 2 were either obtained from commercial
sources or prepared via one of the methods shown in Scheme
2
. The 4-alkoxy-substituted anilines 11 were synthesized from
the corresponding nitrophenols 8 via an alkylation with either
-bromo-2-chloroethane or 1-bromo-3-chloropropane. The re-
1
sulting alkyl chloride 9 then underwent nucleophilic displace-
ment with the desired amine (either dimethylamine, piperidine,
morpholine, or N-methylpiperazine) to afford alkoxynitro
derivative 10. Catalytic hydrogenation with palladium on carbon
afforded the desired alkoxyaniline 11. For the 4-(N-methylpip-
erazin-1-yl)aniline analogues (14a,b), 1-halo-4-nitrobenzene
derivatives 12 were heated with N-methylpiperazine to afford
the corresponding 1-methyl-4-(4-nitrophenyl)piperazines 13.
Reduction via catalytic hydrogenation with palladium on carbon
afforded the desired anilines 14.
a
IC50 values are the means of two or more separate determinations, in
duplicate.
For the synthesis of the 2-chloropyrimidine intermediates 16
and 18, one of the two methods outlined in Scheme 3 was used.
In the cases where the amine was an aniline, heating a solution
of 2,4-dichloropyrimidine (5), aniline 15, and diisopropylethy-
lamine in 2-propanol at 100 °C afforded the desired 4-anilino-
2
3 and 24 as shown in Scheme 5. 2-Chloropyrimidine-4-amines
(
(
16 or 18) were acylated with 2,6-dimethylphenyl chloroformate
20) to afford 2-chloropyrimidine carbamates (21 or 22) in good
yield. Heating compound 21 or 22 with anilines (11 or 14) in
the presence of trifluoroacetic acid in a solution of 2-propanol
promoted nucleophilic displacement of the chloride to afford
the desired 2-anilinopyrimidine carbamates (23 or 24) in good
yield.
2
-chloropyrimidine 16. Alternatively, for alkylamines such as
benzylamines, more mild conditions could be used. In these
cases, addition of a benzylamine, such as 17, to a 0 °C solution
of the 2,4-dichlopyrimidine (5) in 2-propanol and warming the
solution to room temperature afforded the desired product 18
in good yield.
Results and Discussion
The desired 2,6-dimethylphenyl chloroformate (20) was
readily prepared as shown in Scheme 4. A 0 °C solution of
Structure-Activity Relationships (SAR). All compounds
were screened for inhibitory activity against Lck in a homoge-
neous time-resolved fluorescent (HTRF) kinase assay. For the
purposes of determining selectivity, the compounds were also
tested against related kinases Src, p38R, and KDR. Selected
compounds were tested for cellular activity by measuring the
2
,6-dimethylphenol (19) in dichloromethane was sequentially
treated with a solution of triphosgene in dichloromethane and
then pyridine. The mixture was slowly warmed to room
temperature, affording 2,6-dimethylphenyl chloroformate (20)
in quantitative yield.
The three previously described building blocks could be
combined to form the desired 2-aminopyrimidine carbamates
a
inhibition of T cell receptor-induced IL-2 production in human
T cells.

Potent and Orally ActiVe Inhibitors of Lck
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4983
Table 2. SAR: Variations of the Aniline Side Chain (IC50, µM)a
a
IC50 values are the means of two or more separate determinations, in duplicate. b IL-2 Sec.:Anti-CD3/CD28-induced T cell IL-2 secretion assay. c IC50
value is for human mixed lymphocyte reaction; compound 34 was not tested in the T cell receptor-induced IL-2 secretion assay.
2
-Aminopyrimidine carbamate 1 had an IC50 of 14 nM in an
improvement (or loss) in potency and selectivity relative to the
parent phenyl group. Interestingly, 2,4-disubstituted phenyl
groups (28) showed a similar potency increase on Lck as
2-substituted variants. However, the disubstitution led to greater
than 300-fold selectivity over p38R and KDR. Bulkier 2,4
derivatives, such as 27, enhanced selectivity to greater than
1000-fold versus these two kinases. We also explored substitut-
ing the phenyl group with a benzyl substituent. As exemplified
by compound 29, 2,5-disubstituted benzyl groups were equi-
potent with the 2,4-disubstituted phenyl derivatives 27 and 28,
Lck kinase assay and experienced a 300-fold loss in potency
when tested in a T cell receptor-induced IL-2 cellular assay (IC50
)
3.9 µM). Our initial work focused on exploring variants of
the phenyl group in carbamate 1 and studying the effect this
would have on potency and selectivity.29 As shown in Table 1,
phenyl groups with a 2-substituent, such as 25, provided a 10-
fold potency increase over the unsubstituted phenyl. 4-Substi-
tuted phenyl groups, such as in 26, and larger aromatic groups,
such as naphthyl (30) or biphenyl (31), showed no significant

4
984 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Martin et al.
Scheme 2. Synthesis of Aniline Side Chainsa
a
(a) K2CO3, MeCN, reflux; (b) K2CO3, MeCN, 80 °C; (c) H2, 10% Pd/C, EtOH, rt, 16 h; (d) 100 °C.
Scheme 3. Synthesis of 2-Chloro-pyrimidine-4-aminesa
Scheme 5. Synthesis of 2-Aminopyrimidine Carbamatesa
a
(a) (i-Pr)2NEt, THF, rt; (b) CF3CO2H, i-PrOH, 100 °C, 16 h.
position was well tolerated. The main area of differentiation
between these compounds was in their selectivity profile and
cellular activity. The introduction of electron-donating or
electron-withdrawing groups on the aniline (compounds 33, 34,
and 35, respectively) afforded compounds with similar selectiv-
ity to 28, but with a 10-fold increase in cellular activity
a
(
a) (i-Pr)2NEt, rt f 100 °C, 24 h; (b) i-PrOH, 0 °C f rt, 15 h.
Scheme 4. Synthesis of Chloroformatesa
(presumably due to improved solubility and/or cell permeability).
Replacement of the N-methylpiperazine with 2-(dimethylamino)-
ethoxy or 2-(dimethylamino)propoxy groups (36 and 37) also
improved cell potency. In these cases p38R selectivity was
improved, while selectivity against KDR remained unchanged
(
200-fold for both 36 and 37). Replacing the dimethylamino
a
(
a) Triphosgene, pyridine, CH2Cl2, 0 °C f rt, 100%.
group on the alkoxy side chain with a cyclic amine, such as
piperidine (compound 41), had little impact on potency or
selectivity.
We also investigated the impact of combining substituted
anilines with variations on the alkoxy side chain. For 3-fluoro-
substituted anilines (38, 44, and 45), KDR selectivity was
improved slightly, but with a small loss in cell potency, relative
to 36, 37, and 41. 3,5-Dimethoxy derivative 39 showed good
cellular potency (0.29 µM) and selectivity over KDR and p38R
though there was some loss in selectivity. For this reason, we
chose to conduct further SAR studies employing anilines with
the 2,4-disubstitution.
In an effort to further improve the enzyme activity and
optimize the cellular potency of 2,4-dimethoxyaniline-derived
carbamate 28, we varied the aniline group in the 2-position of
the pyrimidine ring. As shown in Table 2, all variations led to
potent Lck inhibitors, indicating that chemical diversity at this

Potent and Orally ActiVe Inhibitors of Lck
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4985
Figure 2. X-ray structure of 2-aminopyrimidine carbamate 43 bound to Lck.
Table 3. Selectivity Profile of Pyrimidine Carbamate 43
(
200-fold for both). Replacing the N-methylpiperazine with a
kinase
Lck
IC50 (µM)a
morpholine (compound 40) saw a slight decrease in cell potency,
but a 10-fold increase in selectivity over p38R (relative to
compound 39). Increasing the alkoxy side chain to a propoxy
linker (compounds 42 and 43) had the most significant effect.
0.0006
0.10
>40
2.4
0.11
>25
0.24
1.2
Btk
CDK2
c-Met
FGF-1R
IGF-1R
JAK2
JAK3
JNK3
KDR
p38R
PKCθ
Src
3
-Methoxy derivative 42 maintained selectivity over KDR and
p38R (200-fold and 2000-fold, respectively) with a slight
improvement in cell potency (0.10 µM). 3,5-Dimethoxy deriva-
tive 43 also maintained selectivity over KDR and p38R (200-
fold and 800-fold, respectively), but with a significant increase
in cell potency (0.061 µM). Because it afforded such a marked
increase in cell potency, we chose to further explore the
properties of pyrimidine carbamate 43.
>5
0.14
0.53
>5
0.001
0.20
0.20
0.37
Syk
Tie-2
Zap-70
Structural Confirmation. In an effort to confirm the binding
elements responsible for potent inhibition of the target protein
in this series of compounds, we obtained a cocrystal structure
of Lck with carbamate 43, one of the most promising pyrimidine
carbamates in terms of potency and selectivity. Consistent with
findings from other researchers, three key H-bond interactions
a
IC50 values are the means of two or more separate determinations, in
duplicate.
Table 4. Cellular Profile of Pyrimidine Carbamate 43
cell assaya
IC50 (µM)b
between the inhibitor 43 and the linker region of the protein
are observed (Figure 2).1
6,19,25
The core pyrimidine in carbamate
IL-2 Sec.
T Cell Prolif.
huWB IL-2
huMLR
TCR ú-chain
PMA/iono
JKT
0.061
0.49
3.0
0.21
0.78
1.8
4
3 binds to the linker region through two hydrogen bonds:
Methionine 319 donates a backbone NH to the N3 pyrimidine
acceptor and the carbonyl from Glu 317 accepts the CH in the
4
3
-position.30 A third interaction is made between the linker Met
19 carbonyl and the NH substituent at position 2 of the
1.7
pyrimidine. The aniline side chain extends out of the enzyme,
with the N-methylpiperazine moiety fully exposed to the solvent.
The aryl ether group is rotated 114° from the plane due to the
presence of the 2,6-substituents, which appropriately orientates
the aromatic ring to enter the deep hydrophobic pocket. This is
consistent with previous findings that have shown the 2,6-
a IL-2 Sec.:Anti-CD3/CD28-induced T cell IL-2 secretion assay; T Cell
Prolif.: Anti-CD3/CD28-induced T cell proliferation assay; huWB IL-2:
Anti-CD3/CD28-induced T cell IL-2 secretion assay using human whole
blood; huMLR: human mixed lymphocyte reaction; TCR ú-chain: T cell
receptor ú-chain phosphorylation assay; PMA/iono: Anti-CD3/CD28-
induced T cell IL-2 secretion assay stimulated with phorbol myristic acid
and calcium ionophore; JKT: Jurkat proliferation/survival assay. See
Experimental Section for a description of each assay. b IC50 values are the
means of two or more separate determinations, in duplicate.
1
5,17
substitution to be a key element responsible for potency.
Interestingly, the 2-methoxy group on the aniline fills the small
pocket adjacent to the ribose-binding pocket. This novel
interaction could explain the improved potency of the 2,4-
dimethoxy aniline derivatives relative to compounds lacking a
(
>100-fold). As expected, due to the close homology of Lck
to other members of the Src family, no selectivity was shown
against Src.
2-substituent on the aniline ring.
Cellular Activity. To further understand the cellular proper-
ties of pyrimidine carbamate 43, it was subjected to a series of
functional cell assays. As shown in Table 4, carbamate 43
exhibited good potency in the T cell receptor-induced IL-2
secretion assay (IL-2 Sec.) and also inhibited subsequent T cell
proliferation (T Cell Prolif.) in the same human T cells. When
the IL-2 secretion assay was performed using human whole
blood (huWB IL-2), the potency shifted significantly. This is
likely due to the relatively high protein binding displayed by
this compound (98.7% in humans; 98.4% in rats). Pyrimidine
carbamate 43 also inhibited a human mixed lymphocyte reaction
Selectivity Profile. To evaluate carbamate 43 in an in vivo
setting, it was critical to understand the selectivity profile of
this molecule. To that end, carbamate 43 was assayed against
an extended panel of tyrosine and serine/threonine kinases
3
1
a
(
Table 3). Within the protein tyrosine kinase (PTK) group,
43 was selective (>100-fold) over representatives from the Syk,
Tie, JakA, VEGFR, FGFR, Tec, InsR, and Met families. Similar
selectivity was seen over kinases outside of the PTK group
a
a
(
CMGC and AGC ). Namely, kinases from the MAPK, CDK,
and PKC families all showed excellent levels of selectivity

4
986 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Martin et al.
Table 5. Pharmacokinetic Parameters for Compound 43 Following iv
Dose in Sprague-Dawley Rats
consistent with the IC50 of compound 43 in the in vitro human
whole-blood IL-2 secretion assay (∼3 µM; Table 4).
a
b
dose (mg/kg) AUC0f∞ (ng h/mL) CL (L/h/Kg) Vss (L/kg) t1/2 (h)
Conclusions
1
568
1.83
11.3
4.6
a
n ) 3 animals per study. b Dosed as a solution in DMSO.
The novel 2-aminopyrimidine carbamates have been shown
to be potent and selective inhibitors of Lck. Our investigation
of the SAR of this series of compounds combined with structural
analysis has identified the key components necessary for kinase
inhibition. Optimization efforts led to 2-aminopyrimidine car-
bamate 43, which showed inhibition of IL-2 production in in
vitro assays and an in vivo model of T cell activation. Future
studies will aim to improve the cellular potency and pharma-
cokinetic properties with the hope of increasing the in vivo
efficacy.
Table 6. Pharmacokinetic Parameters for Compound 43 Following Oral
Dose in Sprague-Dawley Rats
a
doseb
Cmax
tmax
AUC0f∞
bioavailability,
(mg/kg)
(ng/mL)
(h)
(ng h/mL)
F (%)
5
22
4.0
175
6
a
n ) 2 animals per study. b Dosed as an aqueous suspension in 1%
Tween 80 in 2% HPMC.
Experimental Section
Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification. 4-(3-
Piperidin-1-ylpropoxy)phenylamine (11j) was prepared as previ-
ously reported.32 3,5-Dimethoxy-4-(4-methylpiperazin-1-yl)aniline
was obtained from Evotec OAI, Oxon, UK. Anhydrous solvents
were obtained from Aldrich and used directly. All reactions
involving air- or moisture-sensitive reagents were performed under
a nitrogen or argon atmosphere. All final compounds were purified
to >95% purity as determined by high-performance liquid chro-
matography (HPLC). Purity was measured using Agilent 1100
Series high performance liquid chromatography (HPLC) with UV
detection at 254 nm (15 min; 1.5 mL/min flow rate; 0 to 100%
Figure 3. Inhibition of anti-CD3-induced IL-2 production in mice.
Mice were dosed p.o. 1 h prior to challenge with compound 43 (eight
per group) as described in the Experimental Section. Mice were then
challenged i.v. with anti-mouse CD3ꢀ monoclonal antibody. Ninety
minutes after challenge, IL-2 levels in serum were determined by
ELISA. Data points represent the mean IL-2 levels/group ( the standard
error. P values were determined vs vehicle control by Mann-Whitney
U-test. * indicates p e 0.05.
0
.1% TFA in CH
3
2
CN/100-0% 0.1% TFA in H O). Silica gel
columns were performed using either glass columns packed with
silica gel (200-400 mesh, Aldrich Chemical) or prepacked silica
1
gel cartridges (Biotage). H NMR spectra were recorded on a Bruker
AV-400 (400 MHz) spectrometer at ambient temperature or on a
Varian 400 MHz or on a Varian 300 MHz spectrometer. All
observed protons are reported as parts per million (ppm) downfield
from tetramethylsilane (TMS) or other internal reference in the
appropriate solvent indicated. Data are reported as follows: chemi-
cal shift, multiplicity (s ) singlet, d ) doublet, t ) triplet, q )
quartet, br ) broad, m ) multiplet), coupling constants, and number
of protons. Low-resolution mass spectral (MS) data were determined
on an Agilent 1100 Series LCMS with UV detection at 254 nm
and a low resonance electrospray mode (ESI). High-resolution mass
spectra were obtained on a high resonance electrospray time-of-
flight mass spectrometer. Combustion analysis was performed by
Atlantic Microlab, Inc., Norcross, GA, or Galbraith Laboratories,
Knoxville, TN, and were within 0.4% of calculated unless otherwise
noted.
(
huMLR) in the same potency range as the other in vitro cell
assays utilizing purified human cells. This compound also
displayed inhibition of a mechanism-based biochemical cell
assay probing Lck-dependent TCR ú-chain phosphorylation
(TCR ú-chain). As expected, the compound showed much
reduced potency when IL-2 was induced in a receptor-
independent fashion by stimulating with phorbol ester and
calcium ionophore (PMA/iono). In addition, the compound
displayed potency in the µM range when tested in a general
proliferation assay using the human T cell line, Jurkat (JKT).
With this promising in vitro data, we set out to examine the in
vivo properties of pyrimidine carbamate 43.
Pharmacokinetic Profile. The pharmacokinetic properties
of carbamate 43 were assessed in the rat following both
intravenous (i.v.) and oral (p.o.) dosing (Tables 5 and 6,
respectively). The compound displayed a moderate clearance
N,N-Dimethyl-3-(4-nitrophenoxy)propylamine (10a). Potas-
sium carbonate (24.9 g, 180 mmol) was added to a solution of
4
3
-nitrophenol (10 g, 72 mmol) in acetonitrile (100 mL). 1-Bromo-
-chloropropane (113.2 g, 720 mmol) was added, and the mixture
(
1.83 L/h/kg) and high volume of distribution (Vss ) 11.3 L/kg)
was stirred under reflux overnight. The reaction was cooled to room
temperature, the solids were filtered off, and the solvent was
evaporated under reduced pressure to afford crude 3-(4-nitrophe-
noxy)propyl chloride (9a) (13 g) which was used without purifica-
tion.
A sealed tube was charged with 3-(4-nitrophenoxy)propyl
chloride (9a) (2.00 g, 9.27 mmol), potassium carbonate (7.69 g,
46.4 mmol), and acetonitrile (15 mL). Dimethylamine hydrochloride
after i.v. dosing. After p.o. dosing, the tmax of compound 43
was found to be 4.0 h with a relatively low bioavailability (6%).
In Vivo Studies of 2-Aminopyrimidine Carbamate 43.
Despite the low bioavailability, the compound was tested in a
short-term T-cell receptor dependent mouse model. A single
dose of pyrimidine carbamate 43 was administered at 100, 30,
and 10 mg/kg orally to mice and evaluated for its ability to
inhibit anti-CD3-mediated IL-2 secretion in serum (Figure 3).
Terminal plasma levels of compound 43 were determined to
derive a pharmacokinetic-pharmacodynamic relationship. The
compound showed a dose-dependent inhibition of IL-2 produc-
tion with an ED50 estimated at 80 mg/kg. Based on the measured
plasma levels from the three dose groups, the estimated
concentration of carbamate 43 at the ED50 was calculated to be
(3.78 g, 46.4 mmol) was added, and the reaction was mixture stirred
overnight at 80 °C. The mixture was cooled to room temperature,
and water and dichloromethane were added. The aqueous layer was
separated and extracted with dichloromethane. The combined
organics were dried over anhydrous sodium sulfate, filtered, and
concentrated to afford N,N-dimethyl-3-(4-nitrophenoxy)propylamine
1
(
3
10a) (1.18 g, 57%). H NMR (400 MHz, CDCl ) δ ppm 1.95 (t,
J ) 7 Hz, 2 H), 2.20 (s, 6 H), 2.35-2.45 (m, 2 H), 4.05 (t, J ) 7
Hz, 2 H), 6.90 (d, J ) 8 Hz, 2 H), 8.10 (d, J ) 8 Hz, 2 H).
1
.5 µM. The estimated compound levels at the ED50 are

Potent and Orally ActiVe Inhibitors of Lck
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4987
4
-(3-(Dimethylamino)propoxy)aniline (11a). A suspension of
palladium on carbon (10%, 0.400 g) in a solution of N,N-dimethyl-
-(4-nitrophenoxy)propylamine (10a) (4.4 g, 19.6 mmol) in ethanol
50 mL) was stirred under a hydrogen (g) atmosphere for 16 h.
δ ppm 1.30-1.40 (m, 2 H), 1.40-1.50 (m, 4 H), 1.70-1.80 (m, 2
H), 2.25-2.40 (m, 6 H), 3.90 (t, J ) 7 Hz, 2 H), 6.25-6.30 (m, 1
H), 6.35-6.40 (m, 1 H), 6.75-6.85 (m, 1 H).
3
(
1-(2-Fluoro-4-nitrophenyl)-4-methylpiperazine (13a). A sealed
The catalyst was filtered off and the solvent removed under reduced
pressure to afford 4-(3-(dimethylamino)propoxy)aniline (11a) (3.8
g, 100%) as a brown oil. H NMR (400 MHz, DMSO-d
tube was charged with N-methylpiperazine (30 mL, 27.1 g, 270
mmol) and cooled in ice/water. 3,4-Difluoronitrobenzene (2.0 g,
1
6
) δ ppm
12.8 mmol) was added, and the mixture was heated at 100 °C
1
3
8
.95 (t, 2H, J ) 6.5 Hz, 2 H), 2.25 (s, 6 H), 2.35-2.45 (m, 2 H),
overnight. The reaction mixture was concentrated to remove excess
N-methylpiperazine, and the residue dissolved in 1 M hydrochloric
acid (30 mL). The solution was washed with dichloromethane (2
.95 (t, J ) 6.5 Hz, 2 H), 4.70 (s, 2 H), 6.90 (d, J ) 8 Hz, 2 H),
.10 (d, J ) 8 Hz, 2 H), 6.65 (d, J ) 8 Hz, 2 H), 6.75 (d, J ) 8
Hz, 2 H).
×
20 mL). The aqueous phase was separated, basified with 5 M
4-(2-(Dimethylamino)ethoxy)aniline (11b). 4-(2-(Dimethylami-
sodium hydroxide (10 mL), and then extracted with dichlo-
romethane (2 × 20 mL). The combined organic phases were dried
over anhydrous sodium sulfate, filtered, and concentrated to afford
no)ethoxy)aniline (11b) was synthesized from 4-nitrophenol, dim-
ethylamine hydrochloride, and 1-bromo-2-chloroethane according
to the procedure outlined for the synthesis of 4-(3-(dimethylamino)-
propoxy)aniline (11a). H NMR (400 MHz, CDCl
s, 6 H), 2.65 (t, J ) 7 Hz, 2 H), 3.9 (t, J ) 7 Hz, 2 H), 6.5-7 (m,
1
-(2-fluoro-4-nitrophenyl)-4-methylpiperazine (13a) (1.5 g, 49%)
1
3
) δ ppm 2.25
1
as a low melting solid. H NMR (CDCl
m, 4H), 3.33 (m, 4H), 6.92 (m, 1H), 7.91 (m, 1H), 8.00 (m, 1H).
-(2-Fluoro-4-aminophenyl)-4-methylpiperazine (14a). 1-(2-
3
) δ ppm 2.39 (s, 3H), 2.63
(
2
(
H), 6.65-6.75 (m, 2H).
-Fluoro-4-(2-(4-methylpiperazin-1-yl)ethoxy)aniline (11c).
-Fluoro-4-(2-(4-methylpiperazin-1-yl)ethoxy)aniline (11c) was
1
3
Fluoro-4-aminophenyl)-4-methylpiperazine (14a) was synthesized
from 1-(2-fluoro-4-nitrophenyl)-4-methylpiperazine (13a) via hy-
drogenation over 10% palladium on carbon as detailed for the
3
synthesized from 2-fluoro-4-nitrophenol, 4-methylpiperazine, and
-bromo-2-chloroethane according to the procedure outlined for the
1
1
synthesis of 4-(3-(dimethylamino)propoxy)aniline (11a). H NMR
1
synthesis of 4-(3-(dimethylamino)propoxy)aniline (11a). H NMR
400 MHz, CDCl
(
(
CDCl
m, 2 H), 6.33 (m, 2 H), 6.75 (m, 1 H).
-Methoxy-4-(4-methylpiperazin-1-yl)aniline (14b). 3-Meth-
oxy-4-(4-methylpiperazin-1-yl)aniline (14b) was synthesized from
-chloro-5-nitroanisole according to the two-step procedure outlined
for the synthesis of 1-(2-fluoro-4-aminophenyl)-4-methylpiperazine
3
) δ ppm 2.29 (s, 3 H), 2.53 (m, 4 H), 2.94 (m, 4 H), 3.48
(
3
) δ ppm 2.20 (s, 3 H), 2.30-2.40 (m, 4 H), 2.40-
.65 (m, 4 H), 2.75 (t, J ) 7 Hz, 2 H), 4 (t, J ) 7 Hz, 2 H),
.25-6.3 (m, 1 H), 6.3-6.35 (m, 1 H), 6.75-6.85 (m, 1 H).
,5-Dimethoxy-4-(2-(4-methylpiperazin-1-yl)ethoxy)aniline
2
6
3
3
2
(
(
11d). 3,5-Dimethoxy-4-(2-(4-methylpiperazin-1-yl)ethoxy)aniline
11d) was synthesized from 2,6-dimethoxy-4-nitrophenol, 4-me-
thylpiperazine, and 1-bromo-2-chloroethane according to the pro-
cedure outlined for the synthesis of 4-(3-(dimethylamino)propoxy)-
1
(14a). H NMR (CDCl
3
) δ ppm 2.40 (s, 3 H), 2.50-2.70 (m, 4 H),
.90-3.10 (m, 4 H), 3.80 (s, 3 H), 6.20-6.40 (m, 2 H), 6.80-
.90 (m, 1 H). MS (ESI, pos. ion) m/z: 222 (M + 1).
,6-Dimethylphenyl Chloroformate (20). A solution of 2,6-
2
6
1
aniline (11a). H NMR (400 MHz, CDCl
3
) δ ppm 2.25 (s, 3 H),
2
2
.40-2.70 (m, 8 H), 2.75 (t, J ) 7 Hz, 2 H), 3.70 (s, 6 H), 3.90 (t,
J ) 7 Hz, 2 H), 5.90 (s, 2 H).
,5-Dimethoxy-4-(2-morpholinoethoxy)aniline (11e). 3,5-
dimethylphenol (1.2 g, 10 mmol) in dichloromethane (20 mL) was
cooled to 0 °C, and a solution of triphosgene (1.07 g, 3.6 mmol) in
3
15 mL of dichloromethane was added with stirring. Pyridine (0.80
Dimethoxy-4-(2-morpholinoethoxy)aniline (11e) was synthesized
from 2,6-dimethoxy-4-nitrophenol, morpholine, and 1-bromo-2-
chloroethane according to the procedure outlined for the synthesis
mL, 10 mmol) was added dropwise, and the reaction was allowed
to warm to room-temperature overnight. The mixture was diluted
with ethyl acetate and partitioned between ethyl acetate and 1 N
HCl (aq). The organic phase was separated and washed with 1 N
HCl (aq) and brine, dried over anhydrous sodium sulfate, filtered,
and concentrated to afford 2,6-dimethylphenyl chloroformate (20)
1
of 4-(3-(dimethylamino)propoxy)aniline (11a). H NMR (400 MHz,
CDCl
3
) δ ppm 2.50-2.55 (m, 4 H), 2.70 (t, J ) 7 Hz, 2 H), 3.60-
.70 (m, 4 H), 3.70 (s, 6 H), 4.95 (t, J ) 7 Hz, 2 H), 5.8 (s, 2 H).
-Methoxy-4-(3-(4-methylpiperazin-1-yl)propoxy)aniline (11f).
-Methoxy-4-(3-(4-methylpiperazin-1-yl)propoxy)aniline (11f) was
3
3
as a yellow oil (1.84 g, 100%). The product was used without further
3
1
purification. H NMR (CDCl
3
) δ ppm 2.2 (s, 6 H), 7.15 (m, 3 H).
synthesized from 2-methoxy-4-nitrophenol, 4-methylpiperazine, and
-bromo-3-chloropropane according to the procedure outlined for
1
2,6-Dimethylphenyl 2-Chloropyrimidin-4-yl-(2,4-dimethoxy-
phenyl)carbamate (22a). A solution of 2,4-dichloropyrimidine (3.0
g, 20 mmol), 2,4-dimethoxyaniline (3.1 g, 20 mmol), and N,N-
diisopropylethylamine (7.8 g, 10 mL, 60 mmol) in 2-propanol (20
mL) was stirred at room temperature for 48 h. The resulting
suspension was partitioned between saturated aqueous potassium
carbonate solution and ethyl acetate. The organic phase was
separated, dried over anhydrous sodium sulfate, filtered, and
concentrated to afford crude 2-chloro-N-(2,4-dimethoxyphenyl)-
pyrimidin-4-amine (16a) which was used without purification. MS
(ESI, pos. ion) m/z: 266 (M + 1).
1
the synthesis of 4-(3-(dimethylamino)propoxy)aniline (11a). H
NMR (400 MHz, CDCl ) δ ppm 1.90-2.10 (m, 2 H), 2.35 (s, 3
3
H), 2.40-2.60 (m, 10 H), 3.80 (s, 3 H), 4.00 (t, J ) 7 Hz, 2 H),
6
8
.20 (dd, J ) 2, 8 Hz, 1 H), 6.30 (d, J ) 2 Hz, 1 H), 6.80 (d, J )
Hz, 1 H).
3
,5-Dimethoxy-4-(3-(4-methylpiperazin-1-yl)propoxy)anil-
ine (11g). 3,5-Dimethoxy-4-(3-(4-methylpiperazin-1-yl)propoxy)-
aniline (11g) was synthesized from 2,6-dimethoxy-4-nitrophenol,
-methylpiperazine, and 1-bromo-3-chloropropane according to the
4
procedure outlined for the synthesis of 4-(3-(dimethylamino)-
1
propoxy)aniline (11a). H NMR (400 MHz, CDCl
3
) δ ppm 1.80-
.90 (m, 2 H), 2.20 (s, 3 H), 2.30-2.60 (m, 10 H), 3.70 (s, 6 H),
.85 (t, J ) 7 Hz, 2 H), 5.90 (s, 2 H).
-Fluoro-4-(3-(4-methylpiperazin-1-yl)propoxy)aniline (11h).
-Fluoro-4-(3-(4-methylpiperazin-1-yl)propoxy)aniline (11h) was
2-Chloro-N-(2,4-dimethoxyphenyl)pyrimidin-4-amine (16a) (5.3
g, 20 mmol) from above was added to a solution of 2,6-
dimethylphenyl chloroformate (3.7 g, 20 mmol) in tetrahydrofuran
(75 mL). N,N-Diisopropylethylamine (7.8 g, 10.0 mL, 60 mmol)
was added and the mixture stirred at room temperature for 48 h.
The resulting suspension was diluted with ethyl acetate, and the
mixture was partitioned between ethyl acetate and saturated aqueous
sodium carbonate solution. The organic phase was separated and
washed with brine, dried over anhydrous sodium sulfate, filtered,
and concentrated to afford an orange oil. This oil was purified via
column chromatography on silica gel (eluting with dichloromethane)
to afford 2,6-dimethylphenyl 2-chloropyrimidin-4-yl-(2,4-dimethox-
1
3
3
3
synthesized from 2-fluoro-4-nitrophenol, 4-methylpiperazine, and
-bromo-3-chloropropane according to the procedure outlined for
1
1
the synthesis of 4-(3-(dimethylamino)propoxy)aniline (11a). H
NMR (400 MHz, CDCl ) δ ppm 1.80-1.90 (m, 2 H), 2.2 (s, 3 H),
.30-2.55 (m, 10 H), 3.90 (t, J ) 7 Hz, 2 H), 6.30 (m, 1 H), 6.40
m, 1 H), 6.70 (m, 1 H).
-Fluoro-4-(3-(piperidin-1-yl)propoxy)aniline (11i). 3-Fluoro-
3
2
(
3
1
4
-(3-(piperidin-1-yl)propoxy)aniline (11i) was synthesized from
yphenyl)carbamate (22a) (3.4 g, 41%) as an orange solid. H NMR
2
-fluoro-4-nitrophenol, piperidine, and 1-bromo-3-chloropropane
6
(400 MHz, DMSO-d ) δ ppm 2.08 (s, 6 H), 3.79 (s, 3 H), 3.83 (s,
3 H), 6.64 (dd, J ) 8.65, 2.59 Hz, 1 H), 6.75 (d, J ) 2.53 Hz, 1
H), 7.04-7.12 (m, 3 H), 7.37 (d, J ) 8.72 Hz, 1 H), 8.08 (d, J )
according to the procedure outlined for the synthesis of 4-(3-
1
3
(dimethylamino)propoxy)aniline (11a). H NMR (400 MHz, CDCl )

4
988 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Martin et al.
5
4
.81 Hz, 1 H), 8.66 (d, J ) 5.94 Hz, 1 H). MS (ESI, pos. ion) m/z:
14 (M + 1).
2.45 (t, 4 H), 2.99 (t, J ) 4.00 Hz, 4 H), 3.78 (s, 3 H), 6.56 (d, J
) 8.59 Hz, 2 H), 6.98 (d, J ) 8.84 Hz, 2 H), 7.04-7.11 (m, 3 H),
7.11-7.16 (m, 1 H), 7.29 (d, J ) 8.084 Hz, 1 H), 7.42-7.48 (m,
2 H), 7.54 (t, J ) 7.83 Hz, 1 H), 8.35 (d, J ) 5.56 Hz, 1 H), 9.26
2,6-Dimethylphenyl 2,5-Dimethoxybenzyl(2-chloropyrimidin-
4
-yl)carbamate (22b). A solution of 2,4-dichloropyrimidine (3.16
+
g, 21.2 mmol) in 2-propanol (40 mL) was cooled to 0 °C, and N,N-
diisopropylethylamine (2.74 g, 3.70 mL, 21.2 mmol) was added
dropwise via syringe. 2,5-Dimethoxybenzylamine (3.2 mL, 21.2
mmol) was added dropwise via syringe, and the ice bath was
removed. The mixture was stirred at room temperature for 2.5 days
and was then concentrated. The residue was purified via column
chromatography on silica gel (eluting with 1:1 hexanes-ethyl
acetate) to afford N-(2,5-dimethoxybenzyl)-2-chloropyrimidin-4-
amine (18a) (3.99 g, 67%) as an off-white solid. MS (ESI, pos.
ion) m/z: 280 (M + 1).
(s, 1 H). HPLC Purity: 96%. HRMS ([M + H] ) calcd: 539.27652
found: 539.27577.
2,6-Dimethylphenyl 4-(Methyloxy)phenyl(2-((4-(4-methyl-1-
1
piperazinyl)phenyl)amino)-4-pyrimidinyl)carbamate (26). H
NMR (400 MHz, DMSO-d ) δ ppm 2.11 (s, 6 H), 2.23 (s, 3 H),
6
2.44-2.48 (m, 4 H), 2.95-3.02 (m, 4 H), 3.86 (s, 3 H), 6.53 (d, J
) 8.97 Hz, 2 H), 6.99 (d, J ) 7.45 Hz, 2 H), 7.06-7.15 (m, 5 H,)
7.40-7.47 (m, 3 H), 8.35 (d, J ) 5.68 Hz, 1 H), 9.30 (s, 1 H).
+
HRMS (M + H]
) calcd: 539.27652 found: 539.27594. Anal.
2
(C H N O ‚0.9 H O): C, H, N.
31
34
6
3
2-Chloro-N-(2,5-dimethoxybenzyl)pyrimidin-4-amine (18a) (0.300
2,6-Dimethylphenyl 4-Fluoro-2-((1-methylethyl)oxy)phenyl-
g, 1.07 mmol) from above was added to a solution of 2,6-
dimethylphenyl chloroformate (0.198 g, 1.07 mmol) in tetrahydro-
furan (2.0 mL). N,N-Diisopropylethylamine (0.138 g, 0.19 mL, 1.07
mmol) was added and the mixture stirred at room temperature for
(
2-((4-(4-methyl-1-piperazinyl)phenyl)amino)-4-pyrimidinyl)car-
1
bamate (27). H NMR (400 MHz, DMSO-d
(
4
Hz, 1 H), 6.64 (d, J ) 8.84 Hz, 2 H), 7.00 (dd, J ) 8.34, 2.65 Hz,
1
)
8
6
) δ ppm 0.80-1.31
m, 6 H), 2.18 (s, 6 H), 2.27-2.31 (s, 3 H), 2.52 (t, J ) 5.00 Hz,
H), 3.057 (t, J ) 4.80 Hz, 4 H), 4.71 (ddd, J ) 12.19, 6.00, 5.81
17 h. The resulting suspension was diluted with ethyl acetate and
the mixture was partitioned between ethyl acetate and saturated
aqueous sodium carbonate solution. The organic phase was
separated and washed with brine, dried over anhydrous sodium
sulfate, filtered, and concentrated to afford an orange oil. This oil
was purified via column chromatography on silica gel (gradient
elution with 0-25% ethyl acetate-hexane) to afford 2,6-dimeth-
H), 7.06 (d, J ) 8.84 Hz, 2 H), 7.11-7.19 (m, 3 H), 7.26 (dd, J
11.24, 2.65 Hz, 1 H), 7.53 (d, J ) 5.81 Hz, 1 H), 7.58 (dd, J )
.34, 6.57 Hz, 1 H), 8.41 (d, J ) 5.81 Hz, 1 H), 9.30 (s, 1 H).
+
HRMS (M + H] ) calcd: 585.29839 found: 585.29849. Anal.
): C, H, N.
,6-Dimethylphenyl-2,4-bis(methyloxy)phenyl(2-((4-(4-meth-
yl-1-piperazinyl)phenyl)amino)-4-pyrimidinyl)carbamate (28).
33 6 3
(C H37FN O
2
ylphenyl 2,5-dimethoxybenzyl-(2-chloropyrimidin-4-yl)carbamate
1
(22b) (0.351 g, 77%) as a white solid. H NMR (400 MHz, DMSO-
1
H NMR (400 MHz, DMSO-d
6
) δ ppm 2.09 (s, 6 H), 2.23 (s, 3
d
6
6
) δ ppm 1.98 (s, 6 H), 3.63 (s, 3 H), 3.69 (s, 3 H), 5.36 (s, 2 H),
H), 2.43-2.48 (m, 4 H), 2.96-3.02 (m, 4 H), 3.76 (s, 3 H), 3.87
(
.66 (d, J ) 3.03 Hz, 1 H), 6.83 (dd, J ) 8.78, 3.09 Hz, 1 H), 6.95
s, 3 H), 6.55 (d, J ) 8.97 Hz, 2 H), 6.66-6.71 (m, 1 H), 6.82 (d,
(
8
d, J ) 8.84 Hz, 1 H), 7.09 (s, 3 H), 8.09 (d, J ) 5.94 Hz, 1 H),
J ) 2.53 Hz, 1 H), 7.00-7.12 (m, 5 H), 7.33 (d, J ) 8.59 Hz, 1
H), 7.46 (d, J ) 5.81 Hz, 1 H), 8.33 (d, J ) 5.68 Hz, 1 H), 9.28
.68 (d, J ) 5.94 Hz, 1 H). MS (ESI, pos. ion) m/z: 428 (M + 1).
General Procedure for the Synthesis of 2-Aminopyrimidine
(s, 1 H). HRMS ([M+Na]+) calcd: 591.26902 found: 591.2679.
Carbamates. Synthesis of 2,6-Dimethylphenyl-2,4-bis(methyl-
oxy)phenyl(2-((4-((2-(dimethylamino)ethyl)-oxy)phenyl)amino)-
Anal. (C32
,6-Dimethylphenyl (2,5-Bis(methyloxy)phenyl)methyl(2-((4-
4-methyl-1-piperazinyl)phenyl)amino)-4-pyrimidinyl)carbam-
H N
36 6
O
4
2
‚0.2 H O): C, H, N.
2
4-pyrimidinyl)carbamate (36). A resealable tube was charged with
(
2,6-dimethylphenyl 2-chloropyrimidin-4-yl(2,4-dimethoxyphenyl)-
1
ate (29). H NMR (400 MHz, DMSO-d
.23 (s, 3 H), 2.43-2.48 (m, 4 H), 2.98-3.05 (m, 4 H), 3.61 (s, 3
H), 3.77 (s, 3 H), 5.36 (s, 2 H), 6.62 (d, J ) 2.91 Hz, 1 H), 6.68
6
) δ ppm 2.02 (s, 6 H),
carbamate (22a) (8.50 g, 20.5 mmol), 4-(2-(dimethylamino)ethoxy)-
aniline (11b) (4.07 g, 22.6 mmol), trifluoroacetic acid (7.01 g, 4.74
mL, 61.5 mmol), and 2-propanol (160 mL). The system was flushed
with argon, the tube was sealed, and the mixture was heated at
2
(
d, J ) 8.21 Hz, 2 H), 6.83 (dd, J ) 8.84, 3.03 Hz, 1 H), 6.99 (d,
J ) 8.84 Hz, 1 H), 7.05-7.12 (m, 3 H), 7.30 (d, J ) 7.07 Hz, 2
1
00 °C for 24 h. The reaction mixture was cooled to room
H), 7.38 (d, J ) 5.68 Hz, 1 H), 8.36 (d, J ) 5.68 Hz, 1 H), 9.34
temperature and concentrated. The residue was diluted with ethyl
acetate and partitioned between ethyl acetate and saturated aqueous
potassium carbonate solution. The organic layer was separated,
washed with brine, dried over anhydrous sodium sulfate, and
concentrated. The residue was purified via column chromatography
on silica gel (eluting with 90:10:1 dichloromethane/methanol/
ammonium hydroxide) to afford 2,6-dimethylphenyl 2,4-bis(m-
ethyloxy)phenyl(2-((4-((2-(dimethyl-amino)ethyl)oxy)phenyl)-amino)-
+
(
s, 1 H). HRMS (M + H] ) calcd: 583.30273 found: 583.30187.
Anal. Calcd for C33
C, 67.59; H, 6.77; N, 14.19.
,6-Dimethylphenyl 2-((4-(4-Methyl-1-piperazinyl)phenyl)-
38 6 4
H N O : C, 68.02; H, 6.57; N, 14.42. Found:
2
1
amino)-4-pyrimidinyl(2-naphthalenyl)carbamate (30). H NMR
400 MHz, DMSO-d
(
2
6
6
) δ ppm 2.15 (s, 6 H), 2.22 (s, 3 H), 2.36-
.45 (m, 4 H), 2.65-2.75 (m, 4 H), 5.71-5.84 (m, 2 H), 6.63-
.72 (m, 2 H), 7.02-7.12 (m, 3 H), 7.53-7.57 (m, 1 H), 7.58-
4
-pyrimidinyl)carbamate (36) (4.52 g, 40%) as an off-white solid.
1
7.68 (m, 3 H), 8.02-8.07 (m, 1 H), 8.08-8.17 (m, 3 H), 8.39 (d,
6
H NMR (400 MHz, DMSO-d ) δ ppm 2.09 (s, 6 H), 2.22 (s, 6
+
J ) 5.68 Hz, 1 H), 9.27 (s, 1 H). HRMS (M + H] ) calcd:
H,) 2.60 (t, J ) 5.81 Hz, 2 H), 3.76 (s, 3 H), 3.87 (s, 3 H), 3.94 (t,
J ) 5.94 Hz, 2 H), 6.53 (s, 1 H), 6.56 (s, 1 H), 6.69 (dd, J ) 8.59,
2
7
5
34 6 2
59.28160 found: 559.28133. Anal. Calcd for C34H N O : C,
73.10; H, 6.13; N, 15.04. Found: C, 72.09; H, 6.42; N, 14.21.
.53 Hz, 1 H,) 6.83 (d, J ) 2.53 Hz, 1 H), 7.02-7.14 (m, 5 H),
.34 (d, J ) 8.59 Hz, 1 H), 7.47 (d, J ) 5.81 Hz, 1 H), 8.34 (d, J
2,6-Dimethylphenyl-1,1′-biphenyl-4-yl(2-((4-(4-methyl-1-pip-
1
)
+
5.81 Hz, 1 H), 9.35 (s, 1 H). MS (ESI, pos. ion) m/z: 558 (M
erazinyl)phenyl)amino)-4-pyrimidinyl)carbamate (31). H NMR
+
1). HRMS ([M + H] ) calcd: 558.27110 found: 558.27066.
6
(400 MHz, DMSO-d ) δ ppm 2.12-2.19 (m, 9 H), 2.21-2.27 (m,
Anal. (C31
H N
35 5
O
5
): C, H, N.
4 H), 2.59-2.65 (m, 4 H), 6.28-6.34 (m, 2 H), 6.85-6.92 (m, 2
H), 7.05-7.14 (m, 3 H), 7.40-7.46 (m, 1 H), 7.49-7.57 (m, 3 H),
7.59-7.65 (m, 2 H), 7.85-7.91 (m, 2 H), 7.93-7.99 (m, 2 H),
The following compounds were synthesized according to the
general procedure detailed above:
+
2
,6-Dimethylphenyl 2-((4-(4-Methyl-1-piperazinyl)phenyl)-
8.38 (d, J ) 5.81 Hz, 1 H), 9.33 (s, 1 H). HRMS (M + H] )
1
36 36 6 2
calcd: 585.29725 found: 585.29747. Anal. (C H N O ): C, H,
amino)-4-pyrimidinyl(phenyl)carbamate (1). H NMR (400 MHz,
CDCl ) δ ppm 2.18 (s, 6 H), 2.41 (s, 3 H), 2.72 (2, 4 H), 3.13 (s,
H), 6.63 (d, J ) 8.72, 2 H), 6.89 (br s, 3 H), 7.03 (s, 3 H), 7.34
N.
3
4
2,6-Dimethylphenyl 1,1′-Biphenyl-3-yl(2-((4-(4-methyl-1-pip-
1
(
d, J ) 8.45, 2 H), 7.50-7.58 (m, 3 H), 7.63 (d, J ) 5.78 Hz, 1
erazinyl)phenyl)amino)-4-pyrimidinyl)carbamate (32). H NMR
H), 8.51 (d, J ) 5.78 Hz, 1 H). HPLC Purity: 97%. HRMS (M +
H] ) calcd: 509.26595 found: 509.26542.
(400 MHz, DMSO-d ) δ ppm 2.15 (s, 6 H), 2.20 (s, 3 H), 2.35-
6
+
2.41 (m, 4 H), 2.80-2.86 (m, 4 H), 6.37-6.45 (m, 2 H), 6.93-
2
,6-Dimethylphenyl 2-(Methyloxy)phenyl(2-((4-(4-methyl-1-
7.01 (m, 2 H), 7.04-7.13 (m, 3 H), 7.35-7.42 (m, 1 H), 7.43-
7.52 (m, 4 H), 7.63-7.69 (m, 1 H), 7.75 (s, 1 H), 7.77 (s, 1 H),
7.87-7.95 (m, 2 H), 8.39 (d, J ) 5.68 Hz, 1 H), 9.31 (s, 1 H).
1
piperazinyl)phenyl)amino)-4-pyrimidinyl)carbamate (25).
NMR (400 MHz, DMSO-d ) δ ppm 2.10 (s, 6 H), 2.23 (s, 3 H),
H
6

Potent and Orally ActiVe Inhibitors of Lck
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4989
+
HRMS (M + H] ) calcd: 585.29725 found: 585.29669. Anal.
1.46-1.54 (m, 4 H), 1.78-1.87 (m, 2 H), 2.09 (s, 6 H), 2.28-
2.41 (m, 6 H), 3.75 (s, 3 H), 3.85-3.91 (m, 5 H), 6.51 (s, 1 H),
6.54 (s, 1 H), 6.69 (dd, J ) 8.59, 2.53 Hz, 1 H), 6.82 (d, J ) 2.53
Hz, 1 H), 7.04-7.12 (m, 5 H), 7.34 (d, J ) 8.59 Hz, 1 H), 7.47 (d,
J ) 5.81 Hz, 1 H), 8.34 (d, J ) 5.81 Hz, 1 H), 9.34 (s, 1 H).
(C
36
H
36
N
6
O
2
2
‚0.9 H O): C, H, N.
,6-Dimethylphenyl 2,4-Bis(methyloxy)phenyl(2-((3-(methyl-
2
oxy)-4-(4-methyl-1-piperazinyl)phenyl)amino)-4-pyrimidinyl)-
1
carbamate (33). H NMR (400 MHz, DMSO-d
H), 2.21 (s, 3 H), 2.38-2.47 (m, 4 H), 2.80-2.91 (m, 4 H), 3.66
s, 3 H), 3.75 (s, 3 H), 3.88 (s, 3 H), 6.31-6.39 (m, 1 H), 6.68 (dd,
J ) 8.72, 2.53 Hz, 1 H), 6.71-6.78 (m, 1 H), 6.79-6.85 (m, 2 H),
.03-7.12 (m, 3 H), 7.33 (d, J ) 8.59 Hz, 1 H), 7.45 (d, J ) 5.68
Hz, 1 H), 8.34 (d, J ) 5.81 Hz, 1 H), 9.23 (s, 1 H). HPLC purity:
6
) δ ppm 2.09 (s,
+
6
HRMS (M + H] ) calcd: 612.31805 found: 612.31809. Anal.
(
41 5 5
(C35H N O ): C, H, N.
2,6-Dimethylphenyl 2,4-Bis(methyloxy)phenyl(2-((3-(methyl-
7
oxy)-4-((3-(4-methyl-1-piperazinyl)propyl)oxy)phenyl)amino)-
1
4-pyrimidinyl)carbamate (42). H NMR (400 MHz, DMSO-d
6
)
+
1
00%. HRMS (M + H] ) calcd: 599.29764 found: 599.29696.
,6-Dimethylphenyl 2-((3,5-bis(methyloxy)-4-(4-methyl-1-pip-
erazinyl)phenyl)amino)-4-pyrimidinyl(2,4-bis(methyloxy)phenyl)-
carbamate (34). 1H NMR (400 MHz, DMSO-d ) δ ppm 2.08 (s,
H), 2.20 (s, 3 H), 2.36 (s, 4 H), 2.95 (s, 4 H), 3.62 (s, 6 H), 3.78
s, 3 H), 3.81 (s, 3 H), 6.61 (dd, J ) 8.72, 2.40 Hz, 1 H), 6.72 (d,
δ ppm 1.78-1.88 (m, 2 H), 2.093 (s, 6 H), 2.24 (s, 3 H), 2.36-
2.47 (m, 6 H), 2.50 (m, 4 H), 3.65 (s, 3 H), 3.76 (s, 3 H), 3.83-
3.91 (m, 5 H), 6.40 (s, 1 H), 6.68 (dd, J ) 8.59, 2.53 Hz, 1 H),
6.76 (m, 1 H), 6.80 (d, J ) 2.53 Hz, 1 H), 6.84-6.92 (m, 1 H),
7.01-7.14 (m, 3 H), 7.34 (d, J ) 8.59 Hz, 1 H), 7.43 (d, J ) 5.81
Hz, 1 H), 8.35 (d, J ) 5.81 Hz, 1 H), 9.22 (s, 1 H). HPLC Purity:
2
6
6
(
+
J ) 2.40 Hz, 1 H), 6.89 (s, 2 H), 7.03-7.10 (m, 3 H), 7.15 (d, J
100%. HRMS (M + H] ) calcd: 657.33951 found: 657.33974.
)
1
5.81 Hz, 1 H), 7.33 (d, J ) 8.59 Hz, 1 H), 8.36 (d, J ) 5.68 Hz,
2,6-Dimethylphenyl 2,4-Bis(methyloxy)phenyl(2-((3-fluoro-4-
((3-(4-methyl-1-piperazinyl)propyl)oxy)phenyl)amino)-4-pyrim-
+
H), 9.14 (s, 1 H). HPLC purity: 100%. HRMS (M + H] )
1
calcd: 629.3009 found: 629.30863.
idinyl)carbamate (44). H NMR (400 MHz, DMSO-d ) δ ppm
6
2
,6-Dimethylphenyl 2,4-Bis(methyloxy)phenyl(2-((3-fluoro-4-
4-methyl-1-piperazinyl)phenyl)amino)-4-pyrimidinyl)carbam-
ate (35). H NMR (400 MHz, DMSO-d
1.79-1.88 (m, 2 H), 2.09 (s, 6 H), 2.15 (s, 3 H), 2.23-2.44 (m, 10
H), 3.76 (s, 3 H), 3.85 (s, 3 H), 3.92-3.98 (m, 2 H), 6.63-6.69
(m, 1 H), 6.70-6.77 (m, 1 H), 6.79 (d, J ) 2.53 Hz, 1 H), 6.87-
6.93 (m, 1 H), 7.04-7.14 (m, 4 H), 7.34 (d, J ) 8.46 Hz, 1 H),
7.51 (d, J ) 5.81 Hz, 1 H), 8.38 (d, J ) 5.81 Hz, 1 H), 9.51 (s, 1
(
1
6
) δ ppm 2.09 (s, 6 H),
2
.22-2.29 (m, 3 H), 2.86-2.93 (m, 4 H), 3.76 (s, 3 H), 3.85 (s, 3
H), 6.59-6.69 (m, 2 H), 6.80 (d, J ) 2.53 Hz, 1 H), 6.90 (d, J )
+
8
7
.34 Hz, 1 H), 6.99-7.12 (m, 4 H), 7.34 (d, J ) 8.59 Hz, 1 H),
H). HRMS (M + H] ) calcd: 645.31952 found: 645.31855. Anal.
.51 (d, J ) 5.81 Hz, 1 H), 8.38 (d, J ) 5.68 Hz, 1 H), 9.51 (s, 1
(C35
6 5 2
H41FN O ‚0.5 H O): C, H, N.
+
H). HRMS (M + H] ) calcd: 587.27766 found: 587.27708. Anal.
2,6-Dimethylphenyl 2,4-Bis(methyloxy)phenyl(2-((3-fluoro-4-
((3-(1-piperidinyl)propyl)oxy)phenyl)amino)-4-pyrimidinyl)car-
(C H35FN O
32 6 4
): C, H, N.
,6-Dimethylphenyl 2,4-Bis(methyloxy)phenyl(2-((4-((3-(dimeth-
ylamino)propyl)oxy)phenyl)amino)-4-pyrimidinyl)carbamate (37).
2
6
bamate (45). 1H NMR (400 MHz, DMSO-d ) δ ppm 1.34-1.42
(m, 2 H), 1.46-1.53 (m, 4 H), 1.79-1.87 (m, 2 H), 2.09 (s, 6 H),
2.28-2.40 (m, 6 H), 3.76 (s, 3 H), 3.85 (s, 3 H), 3.91-3.99 (m, 2
H), 6.66 (dd, J ) 8.72, 2.53 Hz, 1 H), 6.73 (t, J ) 9.35 Hz, 1 H),
6.79 (d, J ) 2.53 Hz, 1 H), 6.87-6.94 (m, 1 H), 7.02-7.14 (m, 4
H), 7.34 (d, J ) 8.59 Hz, 1 H), 7.51 (d, J ) 5.81 Hz, 1 H), 8.38
1
H NMR (400 MHz, DMSO-d
.13 (m, 6 H), 2.17 (s, 6 H), 2.37 (t, J ) 7.07 Hz, 2 H), 3.77 (s, 3
H), 3.84-3.94 (m, 5 H), 6.54 (d, J ) 8.84 Hz, 2 H), 6.70 (dd, J )
.59, 2.53 Hz, 1 H), 6.83 (d, J ) 2.53 Hz, 1 H), 7.02-7.17 (m, 5
6
) δ ppm 1.75-1.89 (m, 2 H), 2.05-
2
8
+
H), 7.34 (d, J ) 8.59 Hz, 1 H), 7.48 (d, J ) 5.81 Hz, 1 H), 8.35
(d, J ) 5.68 Hz, 1 H), 9.51 (s, 1 H). HRMS (M + H] ) calcd:
(
(
d, J ) 5.81 Hz, 1 H), 9.33 (s, 1 H). HPLC Purity: 96%. HRMS
630.30862 found: 630.30831. Anal. (C35
H, N.
5 5 2
H40FN O ‚0.25 H O): C,
+
M + H] ) calcd: 572.28675 found: 572.28597.
2
,6-Dimethylphenyl-2,4-bis(methyloxy)phenyl(2-((3-fluoro-4-
2,6-Dimethylphenyl 2-((3,5-Bis(methyloxy)-4-((3-(4-methyl-
1-piperazinyl)propyl)-oxy)phenyl)amino)-4-pyrimidinyl(2,4-bis-
(methyloxy)phenyl)carbamate (43). A resealable tube was charged
with 2,6-dimethylphenyl 2-chloropyrimidin-4-yl(2,4-dimethoxyphe-
nyl)carbamate (22a) (0.27 g, 0.55 mmol), 3,5-dimethoxy-4-(3-(4-
methylpiperazin-1-yl)propoxy)aniline (11g) (0.19 g, 0.65 mmol),
trifluoroacetic acid (0.29 g, 0.20 mL, 1.2 mmol), and 2-propanol
(3.0 mL). The system was flushed with argon, and the tube was
sealed. The mixture was heated at 100 °C for 18 h. The reaction
mixture was cooled to room temperature and concentrated to afford
a brown oil. The oil was diluted with ethyl acetate and partitioned
between ethyl acetate and saturated aqueous potassium carbonate.
The organic layer was separated, washed with brine, dried over
anhydrous sodium sulfate, and concentrated to afford 2,6-dimeth-
ylphenyl 2-((3,5-bis(methyloxy)-4-((3-(4-methyl-1-piperazinyl)-pro-
pyl)oxy)phenyl)amino)-4-pyrimidinyl(2,4-bis(methyloxy)phenyl)-
(
(2-(4-methyl-1-piperazinyl)ethyl)oxy)phenyl)amino)-4-pyrim-
idinyl)carbamate (38). H NMR (400 MHz, DMSO-d
1
6
) δ ppm
2
.09 (s, 6 H), 2.16 (s, 3 H), 2.24-2.39 (m, 4 H), 2.66 (t, J ) 5.81
Hz, 3 H), 3.77 (s, 3 H), 3.84 (s, 3 H), 4.02 (t, J ) 5.81 Hz, 2 H),
6
1
.62-6.69 (m, 1 H), 6.72-6.81 (m, 2 H), 6.91 (d, J ) 10.11 Hz,
H), 7.02-7.16 (m, 4 H), 7.34 (d, J ) 8.59 Hz, 1 H), 7.50 (d, J
)
(
5.68 Hz, 1 H), 8.38 (d, J ) 5.68 Hz, 1 H), 9.52 (s, 1 H). HRMS
+
39
M + H] ) calcd: 631.30387 found: 631.30582. Anal. (C34H -
FN O
6 5
‚0.5 H
,6-Dimethylphenyl 2-((3,5-Bis(methyloxy)-4-((2-(4-methyl-
-piperazinyl)ethyl)oxy)phenyl)amino)-4-pyrimidinyl(2,4-bis(m-
2
O): C, H, N.
2
1
1
ethyloxy)phenyl)carbamate (39). H NMR (400 MHz, DMSO-
d
6
) δ ppm 2.07-2.12 (m, 6 H), 2.15 (s, 3 H), 2.19-2.42 (m, 8 H),
2
.57 (t, J ) 5.94 Hz, 2 H), 3.64 (s, 6 H), 3.79 (s, 3 H), 3.82 (s, 3
H), 3.85 (t, J ) 6.06 Hz, 2 H), 6.62 (dd, J ) 8.59, 2.53 Hz, 1 H),
1
6
.73 (d, J ) 2.53 Hz, 1 H), 6.94 (s, 2 H), 7.03-7.12 (m, 3 H),
carbamate (43) (0.189 g, 42%) as an off-white solid. H NMR (400
7
)
.14 (d, J ) 5.81 Hz, 1 H), 7.34 (d, J ) 8.59 Hz, 1 H), 8.37 (d, J
6
MHz, DMSO-d ) δ ppm 1.67-1.76 (m, 2 H), 2.10 (s, 6 H), 2.15
+
5.56 Hz, 1 H), 9.15 (s, 1 H). HRMS (M + H] ) calcd: 673.33442
(s, 3 H), 2.24-2.38 (m, 6 H), 2.38-2.45 (m, 3 H), 3.64 (s, 6 H),
3.74-3.88 (m, 8 H), 6.62 (dd, J ) 8.65, 2.59 Hz, 1 H), 6.73 (d, J
) 2.53 Hz, 1 H), 6.94 (s, 2 H), 7.02-7.17 (m, 5 H), 7.34 (d, J )
8.59 Hz, 1 H), 8.37 (d, J ) 5.68 Hz, 1 H), 9.14 (s, 1 H). MS (ESI,
found: 673.33418. Anal. (C36
,6-Dimethylphenyl 2-((3,5-Bis(methyloxy)-4-((2-(4-morpholi-
nyl)ethyl)oxy)phenyl)amino)-4-pyrimidinyl(2,4-bis(methyloxy)-
H
44
N
6
7
O ‚0.7 H
2
O): C, H, N.
2
1
+
phenyl)carbamate (40). H NMR (400 MHz, DMSO-d
.08 (s, 6 H), 2.40-2.48 (m, 4 H), 2.54-2.63 (m, 2 H), 3.51-
.60 (m, 4 H), 3.63 (s, 6 H), 3.78 (s, 3 H), 3.81 (s, 3 H), 3.83-
.90 (m, 2 H), 6.57-6.65 (m, 1 H), 6.72 (d, J ) 2.53 Hz, 1 H),
.94 (s, 2 H), 7.02-7.16 (m, 4 H), 7.34 (d, J ) 8.59 Hz, 1 H),
.36 (d, J ) 5.68 Hz, 1 H), 9.16 (s, 1 H). HRMS (M + H] )
calcd: 660.30279 found: 660.30315. Anal. (C35
O): C, H, N.
,6-Dimethylphenyl-2,4-bis(methyloxy)phenyl(2-((4-((3-(1-pi-
peridinyl)propyl)oxy)phenyl)amino)-4-pyrimidinyl)carbamate
6
) δ ppm
pos. ion) m/z: 687 (M + 1). HRMS (M + H] ) calcd: 687.35007
2
3
3
6
8
found: 687.34959. Anal. (C37
H N
46 6
7 2
O ‚0.3 H O): C, H, N.
Lck Kinase Assay. The Lck HTRF kinase assay involved ATP-
dependent phosphorylation of a biotinylated substrate peptide of
gastrin in the presence or absence of inhibitor compound. The final
concentration of gastrin was 1.2 µM. The final concentration of
+
H
41
N
5
O
8
‚0.7
ATP was 0.5 µM (K
m
app ) 0.6 ( 0.1 µM), and the final
H
2
concentration of Lck (a GST-kinase domain fusion (AA 225-509))
was 250 pM. Buffer conditions were as follows: 50 mM HEPES
pH ) 7.5, 50 mM NaCl, 20 mM MgCl, 5 mM MnCl, 2 mM DTT,
0.05% BSA. The assay was quenched and stopped with 160 µL of
2
1
(
6
41). H NMR (400 MHz, DMSO-d ) δ ppm 1.34-1.42 (m, 2 H),

4
990 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Martin et al.
detection reagent. Detection reagents were as follows: Buffer made
of 50 mM Tris, pH ) 7.5, 100 mM NaCl, 3 mM EDTA, 0.05%
BSA, 0.1% Tween20. Prior to reading, Streptavidin allophycocyanin
at 3000 rpm in the centrifuge for 5 min to remove nuclei before 25
µL of supernatant was transferred to new tubes for the Igen ú-chain
phosphorylation detection assay.
(
SA-APC) was added at a final concentration in the assay of 0.0004
A master mix was made containing (vol. per sample tube): 21
µL of Igen dilution buffer (PBS, 1% BSA, 0.1% tween-20; Igen/
BioVeris; Gaithersburg, MD), 4 µL of the capture antibody, anti-
human CD247 mAb, clone G3 (BD Coulter, San Diego, CA)
coupled with Dynabeads M-280, 200 µL of the detection antibody,
ruthenylated p-CD3-ú (415.9A; Santa Cruz Biotech., Santa Cruz,
CA) at 1 to 16000 dilution in Igen dilution buffer. 25 µL per sample
lysate was transferred from the lysate plate to a tube including the
negative and positive controls. Sample tubes were shaken at room
temperature overnight. Tubes then were placed in the carousel of
Igen tube analyzer and run on the analyzer. Signal-to-noise ratio
was generally around 15-20.
The procedure of coupling the capture antibody, anti-human
CD247 mAb, clone G3 to Dynabeads M-280 was based on the
protocol provided by Dynal Biotech when the beads were pur-
chased. The procedure of p-CD3-ú ruthenylation is based on the
protocol provided by Bio Veris when BV-TAG NHS-Ester was
purchased.
Pharmacokinetic Studies. Sprague-Dawley rats were admin-
istered a solution of compound in DMSO at the indicated doses
i.v. For oral dosing, a suspension in 2% hydroxypropyl methyl-
cellulose with 1% Tween-80 was administered. Samples were taken
at the indicated times and analyzed for parent compound by LC-
MS method.
Anti-CD3-Induced IL-2 Production in Mice. 12 week old (20
g) BALB/c mice were dosed 1 h prior to challenge with compound
p.o. (8 per group) at the indicated doses in 2% hydroxypropyl
methylcellulose with 1% Tween-80. Mice were then challenged
i.v. with anti-mouse CD3ꢀ monoclonal antibody (145.2C11, BD
PharMingen, San Diego, CA; 3 µg/mouse) diluted in PBS. Ninety
minutes after anti-CD3 challenge, blood was collected via cardiac
puncture. IL-2 levels were measured in serum by ELISA (Bio-
Source, Camarillo, CA). Data points represent the mean IL-2 levels/
group ( the standard error. P values were determined vs vehicle
control by Mann-Whitney U-test.
mg/mL, along with europilated anti-phosphotyrosine Ab (Eu-anti-
PY) at a final conc of 0.025 nM. The assay plate was read in a
Discovery fluorescence plate reader with excitation at 320 nm and
emission at 615 and 655 nm.
Assays for other kinases were done in a similar way as described
above, varying the concentrations of enzyme, peptide substrate, and
ATP added to the reaction, depending on the specific activity of
m
the kinase and measured K s for the substrates.
Human Mixed Lymphocyte Reaction (huMLR). The purpose
of this assay was to test the potency of T cell activation inhibitors
in an in vitro model of allogeneic T cell stimulation. Human
5
peripheral blood lymphocytes (hPBL; 2 × 10 /well) were incubated
with mitomycin C-treated B lymphoblastoid cells (JY cell line
5
(
ATCC, Rockville, MD); 1 × 10 /well) as allogeneic stimulators
in the presence or absence of dilutions of potential inhibitor
compound in 96-well round-bottom tissue culture plates. These
cultures were incubated at 37 °C in 5% CO for 6 days total. The
2
3
proliferative response of the hPBL was measured by H-thymidine
incorporation overnight between days 5 and 6 after initiation of
culture. Cells were harvested onto glass fiber filters and 3H-
thymidine incorporation into DNA was analyzed by liquid scintil-
lation counter.
Jurkat Proliferation/Survival Assay. The purpose of this assay
was to test the general antiproliferative/cytotoxic effect of com-
pounds on the Jurkat human T cell line (ATCC, Rockville, MD).
5
Jurkat cells (1 × 10 /well) were plated in 96-well flat-bottom tissue
culture plates with or without compound dilutions and cultured for
7
2
2 h at 37 °C in 5% CO . Viable cell number was determined during
the last 4 h of culture by adding 10 µL/well WST-8 dye (Alexis;
San Diego, CA). WST-8 dye conversion relied on active mito-
chondrial electron transport for reduction of the tetrazolium dye.
The dye conversion was read by OD at 450-600 nM.
Anti-CD3/CD28-Induced T Cell IL-2 Secretion and Prolif-
eration Assay. The purpose of this assay was to test the potency
of T cell receptor (TCR; CD3) and CD28 signaling pathway
inhibitors in human T cells. T cells were purified from human
peripheral blood lymphocytes (hPBL) and preincubated with or
without compound prior to stimulation with a combination of an
anti-CD3 and an anti-CD28 antibody in 96-well tissue culture plates
(
5
by cytokine ELISA (Pierce/Endogen, St. Louis, MO). The cells
remaining in the wells were then pulsed with H-thymidine
overnight to assess the T cell proliferative response. Cells were
harvested onto glass fiber filters and H-thymidine incorporation
Supporting Information Available: Analysis data for the
compounds synthesized and data collection and refinement statistics
for the X-ray structure of compound 43 bound to Lck. This material
is available free of charge via the Internet at http://pubs.acs.org.
5
1 × 10 T cells/well). Cells were cultured for ∼20 h at 37 °C in
% CO
2
and then secreted IL-2 in the supernatants was quantified
References
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1) Kane, L. P.; Lin, J.; Weiss, A. Signal transduction by the TCR for
3
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3
Potential targets for drug discovery. Annu. ReV. Immunol. 1997, 15,
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71-404.
into DNA was analyzed by liquid scintillation counter. For
comparison purposes, phorbol myristic acid (PMA) and calcium
ionophore were used in combination to induce IL-2 secretion from
purified T cells. Potential inhibitor compounds were tested for
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(
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2
430-2437.
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6
TCR ú-Chain Phosphorylation Assay. Jurkat cells (1.25 × 10 /
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RPMI + 10% FCS and then preincubated with 50 µL compound
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1
concentration of 1.2 µg/mL for 30 min. 20 µL of GAM-IgG was
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(
(
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