Structure-guided design of aminopyrimidine amides as potent, selective inhibitors of lymphocyte specific kinase: Synthesis, structure-activity relationships, and inhibition of in vivo T cell activation

Article abstract of DOI:10.1021/jm7010996

The lymphocyte-specific kinase (Lck), a member of the Src family of cytoplasmic tyrosine kinases, is expressed in T cells and natural killer (NK) cells. Genetic evidence, including knockout mice and human mutations, demonstrates that Lck kinase activity i

Full text of DOI:10.1021/jm7010996

J. Med. Chem. 2008, 51, 1681–1694
1681
Structure-Guided Design of Aminopyrimidine Amides as Potent, Selective Inhibitors of
Lymphocyte Specific Kinase: Synthesis, Structure–Activity Relationships, and Inhibition of in
Vivo T Cell Activation
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Erin F. DiMauro,* John Newcomb, Joseph J. Nunes, Jean E. Bemis, Christina Boucher, Lilly Chai, Stuart C. Chaffee,
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Holly L. Deak, Linda F. Epstein, Ted Faust, Paul Gallant, Anu Gore, Yan Gu, Brad Henkle, Faye Hsieh, Xin Huang,
Joseph L. Kim, Josie H. Lee, Matthew W. Martin, David C. McGowan, Daniela Metz, Deanna Mohn,
Kurt A. Morgenstern, Antonio Oliveira-dos-Santos, Vinod F. Patel, David Powers, Paul E. Rose, Stephen Schneider,
Susan A. Tomlinson, Yan-Yan Tudor, Susan M. Turci, Andrew A. Welcher, Huilin Zhao, Li Zhu, and Xiaotian Zhu
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Department of Medicinal Chemistry, Department of Molecular Structure, and Department of HTS and Molecular Pharmacology, Amgen Inc.,
One Kendall Square, Building 1000, Cambridge, Massachusetts 02139, and Department of HTS and Molecular Pharmacology, Department of
Inflammation, Department of Pharmaceutics, and Department of Pharmacokinetics and Drug Metabolism, Amgen, Inc., One Amgen Center
DriVe, Thousand Oaks, California 91320-1799
ReceiVed September 4, 2007
The lymphocyte-specific kinase (Lck), a member of the Src family of cytoplasmic tyrosine kinases, is
expressed in T cells and natural killer (NK) cells. Genetic evidence, including knockout mice and human
mutations, demonstrates that Lck kinase activity is critical for normal T cell development, activation, and
signaling. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated
autoimmune and inflammatory disease. With the aid of X-ray structure-based analysis, aminopyrimidine
amides 2 and 3 were designed from aminoquinazolines 1, which had previously been demonstrated to exhibit
potent inhibition of Lck and T cell proliferation. In this report, we describe the synthesis and structure–activity
relationships of a series of novel aminopyrimidine amides 3 possessing improved cellular potency and
selectivity profiles relative to their aminoquinazoline predecessors 1. Orally bioavailable compound 13b
inhibited the anti-CD3-induced production of interleukin-2 (IL-2) in mice in a dose-dependent manner (ED50
)
9.4 mg/kg).
Introduction
catalytic activity, have been observed in Lck knockout mice
and humans with Lck mutations. These findings suggest that a
small-molecule inhibitor of Lck activity could prove useful as
an immunosuppressive agent.
5
Signal transduction pathways mediated by the T cell receptor
a
1
(
TCR ) have been extensively researched. Novel immunosup-
pressive agents that target components of these pathways may
be useful as therapies for T-cell-mediated inflammatory or
autoimmune diseases such as rheumatoid arthritis, polyarthritis
scleroderma, inflammatory bowel disease, type I diabetes,
multiple sclerosis, ulcerative colitis, Crohn’s disease, Sjogren’s
disease, polymyositis, dermatomyositis, vasculitis, myasthenia
gravis, psoriasis, and lupus. Such therapies could also prove
advantageous in transplant organ or graft rejection.
The Src family of cytoplasmic protein tyrosine kinases
includes Src, Lck, Fyn, Lyn, Hck, Fgr, Blk, and Yes. Lck and
Fyn have been shown to have important roles in TCR-mediated
In recent years, we and others have disclosed several classes
6
of small-molecule Lck inhibitors. Some of these agents have
demonstrated oral bioavailability and inhibitory activities in
1
,6d–f,i–p
rodent models of T-cell-dependent immune responses.
2-Aminoquinazolines 1 (-L- ) -CONH-, -NHCO-, or
-NHCONH-) were identified via high-throughput screening
of our kinase-preferred compound collection as potent, ATP-
2
6
p
competitive inhibitors of Lck. Compounds with general
structure 1 also exhibited potent inhibitory activity against T-cell
proliferation and activation as measured by the human mixed
lymphocyte reaction (MLR) and the T cell receptor/anti-CD3-
induced production of interleukin-2 (IL-2). Extensive medicinal
chemistry efforts were invested toward understanding the SAR
of the 2-aminoquinazolines. These efforts led to the identification
of orally bioavailable compounds with inhibitory activity in the
anti-CD3-induced model of IL-2 production in mice and
moderate to good selectivity profiles. Analysis of several X-ray
cocrystal structures of aminoquinazolines 1 and Lck revealed
key H-bonding interactions in the ATP-binding site, which are
depicted in Figure 1. We sought to improve the selectivity of
our aminoquinazoline series by taking advantage of the sequence
differences among structurally related kinases in and around
the ATP-binding pocket (Table 1). Specifically, we targeted
structural modifications that would allow for an additional
H-bonding interaction with the threonine gatekeeper residue
3
4
signal transduction. TCR signals propagated by the stimulation
of Lck ultimately lead to gene regulation events triggering
cytokine release, proliferation, and survival of antigen specific
T cells, thereby amplifying immune responses. Defects in T
cell maturation and signaling, which affect expression and/or
*
To whom correspondence should be addressed. Phone: 617-444-5189.
Fax: 617-621-3907. E-mail: edimauro@amgen.com.
†
Department of Medicinal Chemistry.
Department of HTS and Molecular Pharmacology (Amgen at MA and
#
CA|).
Department of Molecular Structure.
Department of Pharmaceutics.
⊥
‡
Department of Inflammation.
Department of Pharmacokinetic and Drug Metabolism.
Abbreviations: Lck, lymphocyte-specific kinase; TCR, T cell receptor;
§
a
IL-2, interleukin 2; PTK, protein tyrosine kinase; MLR, human mixed
lymphocyte reaction; DFG, aspartic acid-phenylalanine-glycine; MAPK,
mitogen activated protein kinase; VEGF, vascular endothelial growth factor.
(
T316) in Lck. We hypothesized that such a new interaction
would increase selectivity over kinases with non-threonine
1
0.1021/jm7010996 CCC: $40.75
 2008 American Chemical Society
Published on Web 03/06/2008

1
682 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
DiMauro et al.
amides. Similarly, aminopyrimidine amide ureas (3, -L- )
-
NHCONH-, i.e., 23a,b) were prepared from 12 via reduction
of the nitro group and reaction of the derived aniline with
isocyanates. These synthetic routes proved to be quite general
such that a variety of reverse amides and ureas could be prepared
in moderate to good overall yields.
In some instances, unsubstituted 2-aminopyrimidine nitroben-
zamide 12a was converted to iodide 24 via a Sandmeyer reaction
1
0
(
Scheme 2). SNAr reactions of iodide 24 were accomplished
under thermal, basic conditions with alkylamines (i.e., 13e) or
under thermal, acidic conditions with anilines. The nitroben-
zamide products were then reduced to the corresponding anilines
and coupled to acids or acid chlorides to generate the final
compounds. Variations on the central aryl ring were typically
introduced early in the synthetic route beginning with ap-
propriately functionalized, commercially available benzoic acids
(i.e., 25, 26, 30). Final compounds were thus prepared following
standard procedures, as depicted in Scheme 2.
Figure 1. Design of aminopyrimidine amides 2 and 3 from amino-
quinazolines 1: -L- ) -CONH-, -NHCO-, or -NHCONH-.
Results and Discussion
Structure–Activity Relationships (SAR). To test our hy-
pothesis that the introduction of an H-bond donor moiety would
confer enhanced selectivity over kinases that possess a gate-
keeper residue other than threonine, we investigated a series of
direct 2-aminopyrimidine amide analogues of our nonselective
Table 1. Sequence Comparison at Gatekeeper Residue and Extended
Hydrophobic Pocket
a
Lck
Thr316
Met292
Leu295
Leu300
Ile355
Tyr360
Tie2
Jak3
P38R
KDR
a
Ile
Leu
Leu
Leu
Leu
Leu
Leu
Met
Ile
Ile
Ile
Val
Val
Leu
Leu
Ile
Phe
Cys
Ile
Met
Thr
Val
2-aminoquinazoline leads (Table 2). Replacement of the 2-ami-
Leu
Cys
noquinazoline with a 2-aminopyrimidine amide leads to a 10-
to 75-fold loss of potency on Lck, as evidenced by comparison
of the in vitro profiles for representative amides (34 vs 10a),
reverse amides (35 vs 13a), and ureas (36 vs 23a). The potency
loss is counteracted by the introduction of a 4-(4-methylpiper-
azino)anilino substituent at the 2-position in the pyrimidine
series (10b, 13b, 23b). We speculate that this gain in enzyme
potency results from additional stabilizing van der Waals
interactions between the aryl ring of the aniline substituent and
the two lipophilic residues (Leu251 of the glycine-rich loop and
Gatekeeper residue.
7
gatekeeper residues (namely, Tie-2, Jak3, and KDR). Support
for this hypothesis is provided by imatinib, which selectively
inhibits a subset of kinases (BCR-Abl, cKit, PDGFR) through
preferential binding to the inactive (DFG-out) conformation of
the protein and hydrogen bonding to the threonine gatekeeper
8
residue. Furthermore, a single point mutation at the Abl
gatekeeper residue (T315I) results in resistance to imatinib.
Toward this end, we designed aminopyrimidine amides with
9
Gly332) between which it is situated. Interestingly, reverse
9
amide 13b demonstrated greater selectivity than amide 10b and
greater cellular potency than urea 23b. Unfortunately, the
arylpiperazine 13b does not afford a substantial improvement
in selectivity over KDR relative to aminoquinazoline 35 (40-
fold vs 15-fold). In KDR, the valine gatekeeper residue is
relatively small and can presumably tolerate a proximal hydro-
philic amide moiety.
general structures 2 and 3. Herein, we describe synthesis, X-ray
structure, structure–activity relationships, and pharmacological
characterization of aminopyrimidine amides 3 and the identi-
fication of an orally bioavailable Lck inhibitor (13b) with dose-
dependent efficacy in a mouse model of T-cell receptor
stimulated IL-2 production.
X-ray crystallography was used to confirm our hypothesis
that a 2-aminopyrimidine amide binds to Lck in a manner simi-
lar to that observed for the 2-aminoquinazoline but also engages
the threonine gatekeeper in an H-bonding interaction (between
the amide NH and the oxygen of T316). The cocrystal structures
of Lck and aminopyrimidine amide 10b (Figure 2a) and Lck
and aminopyrimidine reverse amide 19 (Figure 2b) were solved
at 2.2 and 2.3 Å resolution, respectively. In both structures, the
inhibitor occupies the ATP-binding site and induces the protein
to assume a DFG-out conformation. The aminopyrimidine ring
makes two hydrogen bond contacts to the hinge region of the
enzyme at M319 (10b, 3.04 and 2.78 Å; 19, 2.99 and 2.89 Å).
As hypothesized, the pyrimidine amide moiety engages the
gatekeeper residue (T316) in an H-bond interaction (10b, 3.07
Å; 19, 3.11 Å), acting as an H-bond donor to the threonine
oxygen. The arylamide moiety makes hydrogen bond contacts
with the backbone NH of D382 (10b, 2.89 Å; 19, 2.97 Å) from
the DFG sequence and E288 (10b, 2.80 Å; 19, 2.99 Å)
from the C-helix. The aryl ring of the second amide thus
occupies the extended hydrophobic pocket, making van der
Chemistry
Aminopyrimidine amides (3, -L- ) -CONH-, i.e., 10a,b)
were prepared as depicted in Scheme 1. 2,5-Diaminopyrimidines
6
were prepared from 2-amino-5-nitropyrimidines 5, which were
1
either commercially available (5a, R ) H) or derived from
the SNAr reaction of 2-chloro-5-nitropyrimidine (4) and anilines
1
(
6
i.e., 6b, R ) 4-(4-methylpiperizino)aniline) or amines (i.e.,
c,d). Commercially available 3-iodo-4-methylbenzoyl chloride
11
(
7) was condensed with 3-(trifluoromethyl)aniline in the pres-
ence of triethylamine to provide iodoamide 8, which was then
subjected to palladium-catalyzed carbonylative esterification and
subsequent saponification to afford acid 9. Condensation of 9
with 2,5-diaminopyrimidines 6a,b provided the final compounds
1
-
0a,b. The synthesis of aminopyrimidine reverse amides (3,
L- ) -NHCO-, i.e., 13a-d, 14–22) was also accomplished
in a convergent manner (Scheme 1). Nitrobenzamides 12,
prepared by the reaction of 2,5-diaminopyrimidines 6 and acid
chlorides, were reduced to the corresponding anilines and
selectively coupled to acids or acid chlorides to generate reverse

Aminopyrimidine Amides as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1683
a
Scheme 1. Preparation of Aminopyrimidine Amides, Reverse Amides, and Ureas
a
(a) 4-(4-methylpiperazino)aniline, TFA, IPA, 80 °C (5b, 88%); (b) 3-morpholinopropan-1-amine, DIPEA, IPA, 70 °C (5c, 100%); (c) 1-methylpiperidin-
4
-amine, NaH, THF (5d, 100%); (d) 10% Pd/C, H2, CH3OH/EtOAc; (e) 3-(trifluoromethyl)aniline, NEt3, CH2Cl2 (84%); (f) CO(g), CH3OH, Pd(OAc)2,
dppp, NEt3, 70-100 °C (85%); (g) LiOH, THF/H2O (92%); (h) EDC-HCl, HATU, DIPEA, CH2Cl2 (10a, 65%; 10b, 91%); (i) (i) acid, SOCl2, 80 °C, (ii)
aniline, NEt3, CH2Cl2; (j) 1-isocyanato-3-(trifluoromethyl)benzene, benzene, 75 °C (23a, 20%; 23b, 46%).
Waals contacts to several lipophilic residues. This type of DFG-
out binding mode was previously observed for the parent
examined the effect on potency and selectivity of smaller tertiary
amine containing chains to the 2-position of the pyrimidine
(Table 3, 13c-e). Although these analogues proved quite
selective over KDR, Tie-2, and Jak3, their Lck kinase and
cellular potency was significantly less than 13b. As anticipated,
the H-bond donor to the hinge is important for potency; a
substantial loss in potency was also observed with the unsub-
stituted pyrimidine 37.
Selected SAR from variations on the arylamide is summarized
in Table 4. Moderate potency and good selectivity (vs KDR,
Tie-2, and Jak3) was observed with isoxazole 15, benzamide
16, and 2-methoxybenzamide 17. These three analogues pos-
sessed kinase selectivity profiles comparable to that of lead 13b
but were less potent. Compounds lacking the southern aryl ring,
such as acetamide 14, were also significantly (>50-fold) less potent
than 13b on Lck. Exceptional enzyme and cellular potency,
comparable to that of lead 13b, was consistently observed for meta-
2
-aminoquinazolines and is not observed for most other reported
1
2
Src-family kinase inhibitors such as PP1 and PP2. In view of
the marked similarity in binding mode between amides and
reverse amides with Lck, we are currently unable to offer a
rational explanation for the superior selectivity of 13b relative
to 10b. Similarities in the binding modes of aminopyrimidine
amides and quinazolines are demonstrated by the overlay of
two cocrystal structures with Lck (Figure 2c). As observed in
the cocrystal structures, substituents on the 2-amino group of
the pyrimidine can extend to the solvent front and may be
engaged in additional binding interactions with residues lining
1
3,6s,9
the hinge region and the glycine-rich loop.
In light of the gain in potency observed for 13b relative to
6
s
1
3a and the potential for additional H-bonding or van der
9
Waals interactions with residues lining the hinge region, we

1
684 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
DiMauro et al.
a
Scheme 2. Preparation of Aminopyrimidine Reverse Amides
a
(
a) isoamyl nitrite, CuI, CH2I2, THF, 70 °C (21%); (b) 2-morpholinoethanamine, DIPEA, IPA, 70 °C (100%); (c) 10% Pd/C, H2, CH3OH/EtOAc; (d)
3
-(trifluoromethyl)benzoyl chloride, NEt3, CH2Cl2 (63%); (e) (i) acid, SOCl2, 80 °C, (ii) aniline, NEt3, CH2Cl2; (f) EDC-HCl, HATU, DIPEA, DMF (42%);
(
g) BEt3, Pd(OAc)2, S-Phos, K3PO4, THF, 60 °C (20%).
or para-substituted derivatives, such as 18–22. Of these, 20 proved
most selective over KDR (>200-fold).
of IL-2 in 50% human whole-blood (WB IL-2) (Table 6).
Compounds 13b, 18-20, and 22 were very potent inhibitors of
IL-2 production in human whole blood. On the basis of the
general inhibitory activity in the p38R kinase assay (<10 nM)
for this series, three representative compounds, 13b, 18, and
19, were further counterscreened for inhibitory activity in the
lipopolysaccharide-induced production of TNFR in 50% human
whole blood (WB TNFR). The whole blood cellular selectivity
factor (WB IL-2 IC50/WB TNFR IC50) was substantial for all
three compounds (102-, 63-, and 685-fold, respectively). For
several compounds, the protein binding profiles were examined
in plasma from three species (Table 6).
Pharmacokinetic Profile. Selected PK data for discrete
intravenous (iv) and oral (po) dosing in male Sprague–Dawley
rats are shown in Table 7. Clearance ranged from low (22, 0.29
L/h/kg) to greater than hepatic blood flow (21, 6.64 L/h/kg),
and oral bioavailability was generally low (4–22%). Compound
13b exhibited a moderate rate of clearance, volume of distribu-
tion, oral exposure, and bioavailability. In light of the rat
pharmacokinetic profile and the exceptional in vitro potency,
An examination of the central aryl ring situated in the classical
hydrophobic pocket led to compounds with significantly im-
proved selectivity profiles (Table 5). On the basis of SAR from
the parent aminoquinazoline series, we surmised that a small
1
lipophilic substituent such as methyl or chloro at R was
important for cellular potency. We also discovered that the larger
ethyl substituent was tolerated with only a moderate (2- to
5
-fold) loss of Lck potency and afforded excellent selectivity
2
over p38R (i.e., 33). Small substituents at R , such as methyl
or chloro, were also better tolerated by Lck than by p38R (29
and 27). Unfortunately, none of these more selective analogues
demonstrated acceptable cellular potency. The narrow SAR is
consistent with expectations, given the lack of space in this
6
p,14
region of the binding site (see X-ray structures, Figure 2).
On account of their in vitro potencies and selectivity profiles,
selected aminopyrimidine amides were advanced into our
secondary cellular screening assay, which measures the com-
pound’s inhibitory effect on the anti-CD3 induced production

Aminopyrimidine Amides as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1685
a
Table 2. Aminoquinazolines vs Aminopyrimidine Amides
a
IC50 values are the mean of two or more separate determinations, in
duplicate.
1
3b appeared suitable for assessment via oral dosing in our
rodent model of T cell activation.
Selectivity Profile. Prior to in vivo testing, compound 13b was
screened for inhibitory activity against an extensive panel of kinases
15
(
Table 8). Excellent selectivity (>1000-fold) was observed over
a number of structurally diverse kinases. As expected, 13b was
not selective over other members of the Src family (Src, Fyn, Lyn),
p38R, Abl, or cFMS, all of which possess a threonine gatekeeper
residue. The Src-family kinases are broadly expressed in a variety
of tissues with high levels in hematopoietic cells. The lack of intra-
Src family selectivity remains a concern because of potential
adverse effects from multikinase inhibition. In particular, Lyn
knockout mice show defective BCR signaling and decreased
3
,16,17
peripheral B-cells.
The intra-Src family specificity
required for safe clinical application and/or therapeutic
treatment of autoimmune or inflammatory disease remains
an unknown at this time. Despite numerous reports of assorted
6
chemotypes with varying selectivity profiles, a small-
molecule inhibitor of Lck has yet to demonstrate successful
clinical application in autoimmune, inflammatory disease, or
transplant rejection. We believe that the overall kinase
selectivity profile offered by this new aminopyrimidine amide
class could pave the way to the identification of tool
compounds to answer such questions. Compound 13b was
also tested for its ability to inhibit IL-2 production in Jurkat
cells stimulated with phorbol 12-myritstyl 13-acetate (PMA)
in the presence of ionophore. In this assay, the IC50 was >20
µM, which provides further evidence that the inhibition of
kinases downstream of Lck is minimal.
Pharmacodynamic Profile. Compound 13b was further
evaluated as an inhibitor in the anti-CD3-induced IL-2 production
model in female BALB/c mice (Figure 3). Upon oral administration
at 10, 30, and 100 mg/kg, 13b exhibited statistically significant
dose-dependent inhibition of IL-2 production. In this study,
terminal plasma levels (at 2.5 h) of 13b were determined in
order to estimate the levels of exposure at each dose. At the
estimated ED50 (9.4 mg/kg) the mean terminal plasma
concentration (EC50) was ∼175 nM. This concentration
Figure 2. Cocrystal structures: (a) Lck and aminopyrimidine amide
1
0b; (b) Lck and aminopyrimidine reverse amide 19; (c) overlay of
aminopyrimidine amide 10b (green) with quinazoline 34 (blue) in Lck.
provides 25-fold coverage over the in vitro potency in the
50% human whole-blood IL-2 assay (IC50 ) 7 nM) at 2.5 h.
When adjusted for free fraction based on mouse protein
1
8
binding (Table 6), the EC50 (fu) for 13b is 4 nM, which is
consistent with the in vitro inhibitory activity in the absence
of whole blood (IL-2 IC50 ) 0.6 nM). 13b demonstrates
comparable in vivo efficacy to cyclosporine A (ED50 ) 10
6
i
mg/kg) in this model.
Conclusions
Aminopyrimidine amides 3, designed from aminoquinazolines
1, were demonstrated to afford improved potency and selectivity

1
686 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
DiMauro et al.
a
a
Table 3. SAR: Variations on the Pyrimidine Substituent
Table 5. SAR: Variations on the Central Aryl Ring
a
IC50 values are the mean of two or more separate determinations, in
duplicate.
a
IC50 values are the mean of two or more separate determinations, in
duplicate.
a
Table 4. SAR: Variations on the Arylamide
Table 6. Cellular Assay (WB IL-2) and Counterscreen (WB TNFR) in
a
5
0% Human Whole Blood and Protein Binding
IC50 (µM)
protein binding (%)
compd
WB IL-2
WB TNFR
human
mouse
rat
1
1
1
1
2
2
a
3a
3b
8
9
0
1.27
>10
0.718
1.01
1.37
0.007
0.016
0.002
0.013
0.043
96.7
96.4
98.1
95.8
96.8
97.8
98.2
98.4
98.1
98.8
99.4
97.7
98.6
98.9
98.3
2
IC50 values are the mean of two or more separate determinations, in
duplicate.
collection of novel aminopyrimidine amides, from which
compound 13b was identified as an orally active agent that
exhibited dose-dependent inhibitory activity (ED50 ) 9.4 mg/
kg) in the anti-CD3 induced production of IL-2 in mice.
Experimental Section
Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification. Quinazo-
lines 34–36 were prepared according to previously published
6
p
2 2 3
procedures. Dry organic solvents (CH Cl , CH CN, DMF, etc.)
were purchased from Aldrich packaged under nitrogen in Sure/
Seal bottles. Reactions were monitored using Agilent 1100 series
LCMS with UV detection at 254 nm and a low resonance
electrospray mode (ESI). Medium pressure liquid chromatography
(
MPLC) was performed on a CombiFlash Companion (Teledyne
Isco) with RediSep normal-phase silica gel (35 – 60 µm) columns
and UV detection at 254 nm. Preparative reversed-phase HPLC
was performed on a Gilson (215 liquid handler) YMC-Pack Pro
C18, 150 mm × 30 mm i.d. column, eluting with a binary solvent
2
system A and B using a gradient elution (A, H O with 0.1% TFA;
a
IC50 values are the mean of two or more separate determinations, in
3
B, CH CN with 0.1% TFA) with UV detection at 254 nm. Purity
was measured using Agilent 1100 series high-performance liquid
chromatography (HPLC) systems with UV detection at 254 nm
duplicate.
for the inhibition of Lck, T cell proliferation, and T cell
activation. Our hypothesis that the introduction of an amide
H-bond donor moiety would provide an additional site of
interaction between the ligand and the gatekeeper residue (T316)
of Lck was verified by X-ray crystallographic studies. This
interaction presumably contributes to the enhanced selectivity
afforded by these compounds relative to the parent amino-
quinazolines. Extensive SAR investigations resulted in a diverse
(
system A, Agilent Zorbax Eclipse XDB-C8 4.6 mm × 150 mm,
µm, 5-100% CH CN in H O with 0.1% TFA for 15 min at 1.5
mL/min; system B, Waters Xterra 4.6 mm × 150 mm, 3.5 µm,
-95% CH CN in H O with 0.1% TFA for 15 min at 1.0 mL/
5
3
2
5
3
2
1
min). 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. Chemical shifts are reported
6
in ppm from the solvent resonance (DMSO-d 2.49 ppm). Data

Aminopyrimidine Amides as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1687
a
Table 7. Mean PK Parameters Following Intravenous Dose (iv) or Oral Dose (po) in male Sprague–Dawley Rats
b
c
iv
po
compd
CL ((L/h)/kg)
Vss (L/kg)
t1/2 (h)
AUC0-∞ (ng· h/mL)
Cmax (ng/mL)
Tmax (h)
F (%)
1
1
1
2
2
2
3b
8
9
0
1
2
1.04
0.49
2.92
1.37
6.64
0.29
b
3.29
1.61
5.45
3.76
15.8
0.79
3.2
3.8
1.8
2.5
2.9
2.5
419
332
92
49
39
28
16
4.67
0.6
2.75
0.42
22
8
7
58
4
528
191
0.33
8
a
c
n ) 3 animals per study. Dosed iv at 0.5 mg/kg as a solution in DMSO. Dosed po at 2 mg/kg as a suspension in 1% Tween-80, 2% HPMC, 97%
water.
a
Table 8. Kinase Selectivity Profile of 13b
kinase
IC50 (µM)
Lck
Src
Fyn
Lyn
p38R
Abl
cFMS
KDR
Tie-2
JNK2
Syk
TrkA
IRK
Itk
CDK5
PAK2
RON
Jak3
0.0006
0.003
0.003
0.0009
0.005
0.002
0.040
0.024
9.95
4.29
>5
>5
>5
>25
>25
>25
>25
Figure 3. Effect of 13b on Anti-CD3 induced IL-2 production in
BALB/c mice. The 12 week old female BALB/c mice were pretreated
>25
Jak2
1.03
(
po) with 13b at 10, 30, or 100 mg/kg. After 1.0 h the mice were
PIM1
Aurora1
Aurora2
PKAR
PKBR
MSK1
P70S6K
ERK1
a
>30
>8.3
>25
challenged (iv) with anti-mouse CD3 monoclonal antibody (3 mg/
mouse). After 90 min of anti-CD3 challenge, blood was collected via
cardiac puncture. IL-2 was measured in serum using the BioSource
ELISA kit. Data points represent the mean ( SE; n ) 5 animals per
group: (/) p e 0.05 vs vehicle control by Mann–Whitney U-test. Mean
plasma concentrations for each dose are expressed in parentheses.
>85
>100
>100
>100
>100
IC50 values are the mean of two or more separate determinations, in
duplicate.
a red amorphous solid, which was used without further purification.
5
(
b (70 g, 224 mmol) was taken up in EtOH (500 mL), and Pd/C
10 g, 10% by weight wet) was added. The mixture was placed in
a stirred pressure vessel under 50 psi of H for 48 h. The catalyst
are reported as follows: chemical shift, multiplicity (s ) singlet, d
doublet, t ) triplet, q ) quartet, br ) broad, m ) multiplet),
2
)
was removed by suction filtration and washed with EtOH. The
organics were concentrated under reduced pressure and dried under
vacuum to afford the title compound (61.4 g, 96% yield) as a tan
amorphous solid, which was used without purification. H NMR
(400 MHz, DMSO-d ): δ 8.68 (s, 1H), 7.91 (s, 2H), 7.49–7.51 (m,
2H), 6.81–6.84 (m, 2H), 4.68 (br s, 2H), 3.0–3.02 (m, 4H),
coupling constants, and number of protons. Mass spectra were
obtained on a high resonance electrospray time-of-flight mass
spectrometer in positive ES ionization mode. Combustion analysis
was performed by Galbraith Laboratories, Inc., Knoxville, TN. For
final compounds that do not have elemental analysis data, the results
of HPLC in two diverse chromatographic conditions can be found
in the Supporting Information.
1
6
2.43–2.51 (m, 4H), 2.21 (s, 3H). MS (ESI, positive ion) m/z: 285
(MH ).
+
N2-(4-(4-Methylpiperazin-1-yl)phenyl)pyrimidine-2,5-di-
amine (6b). To a mixture of 4-(4-methylpiperazino)aniline (57.7
g, 302 mmol) and 4 (48.1 g, 302 mmol) in IPA (500 mL) was
added trifluoroacetic acid (46 mL, 603 mmol). The mixture was
heated to 80 °C for 4 h, then cooled to room temperature. The
solid was collected by suction filtration through a Buchner filtration
apparatus equipped with a micromembrane filter and washed with
N3-(2-Aminopyrimidin-5-yl)-4-methyl-N1-(3-(trifluorometh-
yl)phenyl)isophthalamide (10a). The title compound was prepared
from 6a (0.071 g, 0.371 mmol) and 9 (0.100 g, 0.309 mmol) using
the procedure described for the synthesis of 10b. Purification via
column chromatography on silica gel (gradient elution with 2-10%
CH
3
OH/CH
2
Cl
2
) afforded the title compound (0.083 g, 65%) as a
): δ 10.71 (s, 1H), 10.39
1
white solid. H NMR (400 MHz, DMSO-d
6
IPA (80 mL) and CH
afford the trifluroacetic acid salt of 5b as a pale-yellow amorphous
3
OH (100 mL), then dried under vacuum to
(s, 1H), 8.67 (s, 2H), 8.38 (s, 1H), 8.28 (d, J ) 1.9 Hz, 1H), 8.23
(d, J ) 8.5 Hz, 1H), 8.15 (dd, J ) 7.9, 1.9 Hz, 1H), 7.75 (t, J )
solid (114 g, 266 mmol, 88%). MS (ESI, negative ion) m/z: 315
7.9 Hz, 1H), 7.64 (d, J ) 8.0 Hz, 1H), 7.60 (d, J ) 8.0 Hz, 1H),
+
1
+
(
MH ). H NMR (400 MHz, DMSO-d
s, 1H), 9.18 (s, 2H), 7.63–7.61 (m, 2H), 7.02–7.04 (m, 2H),
.75–3.81 (m, 2H), 3.47–3.50 (m, 2H), 3.05–3.16 (m, 4H), 2.81
s, 3H). The TFA salt of 5b (96 g, 224 mmol) was suspended in
6
): δ 10.87 (br s, 1H), 10.73
6.70 (s, 2H), 2.61 (s, 3H). HRMS (C20
H
17
F
3
N
5
O
2
) : calcd,
(
3
(
416.132 89; found, 416.134 16. Anal. (C20
C, H, N.
H
16
F
3
N
5
O
2
3
·0.5CH OH)
4-Methyl-N3-(2-(4-(4-methylpiperazin-1-yl)phenylamino)py-
rimidin-5-yl)-N1-(3-(trifluoromethyl)phenyl)isophthalamide (10b).
A solution of 3-(trifluoromethyl)aniline (0.63 g, 3.9 mmol) and
water (400 mL), and 2 N NaOH was added until the aqueous layer
was pH 14. The solid was collected by suction filtration through a
Buchner filtration apparatus equipped with a micromembrane filter,
triethylamine (0.55 mL, 3.9 mmol) in CH
in an ice–water bath, and a solution of 7 (1.0 g, 3.6 mmol) in CH
(15 mL) was added slowly. The mixture was warmed to room
2
Cl
2
(10 mL) was cooled
washed with water, and dried under vacuum overnight to afford
Cl
2 2
+
5
b (70 g, 100% yield) [MS (ESI, positive ion) m/z: 315 (MH )] as

1
688 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
DiMauro et al.
temperature and stirred for 20 h. The reaction mixture was
concentrated and purified via column chromatography on silica gel
6.88–6.90 (m, 2H), 3.05–3.07 (m, 4H), 2.54 (s, 3H), 2.44–2.47 (m,
4H), 2.22 (s, 3H). MS (ESI, positive ion) m/z: 448 (MH ).
+
(
gradient elution with 0-100% EtOAc/hexane) to afford 8 (1.219
N-(2-Amino-5-pyrimidinyl)-2-methyl-5-(((3-(trifluorometh-
yl)phenyl)carbonyl)amino)benzamide (13a). Reduction of 12a
according to the procedure described in the preparation of 23a
afforded 5-amino-N-(2-aminopyrimidin-5-yl)-2-methylbenzamide.
The title compound was prepared from 5-amino-N-(2-aminopyri-
midin-5-yl)-2-methylbenzamide (0.086 g, 0.353 mmol) and 3-(tri-
fluoromethyl)benzoyl chloride (0.052 mL, 0.353 mmol) using the
procedure described for the synthesis of 27. Purification via column
chromatography on silica gel (gradient elution with 0-100% 90:
10:1, CH Cl /CH OH/NH OH-CH Cl ) afforded the title com-
+
g, 84% yield) [MS (ESI, positive ion) m/z: 404 (MH )]. A 45 mL
stainless steel cylinder with glass liner was charged with 8 (1.22
g, 3.01 mmol) and CH
the mixture, and then bis(diphenylphosphino)propane (0.0683 g,
.165 mmol), triethylamine (1.00 mL, 7.17 mmol), and palla-
3
OH (15 mL). Argon was bubbled through
0
dium(II) acetate (0.0338 g, 0.150 mmol) were added. The cylinder
was capped with a pressure gauge, charged with CO gas (200 psi),
and heated to 70 °C for 20 h and then at 100 °C for 4 h. The reaction
mixture was cooled to room temperature and concentrated. The
residue was purified via column chromatography on silica gel
2
2
3
4
2
2
1
pound (0.060 g, 41% yield) as an off-white amorphous solid. H
(
2
gradient elution with 0-100% EtOAc/hexane) to afford methyl
NMR (400 MHz, DMSO-d ): δ 10.56 (s, 1H), 10.16 (s, 1H), 8.52
6
-methyl-5-((3-(trifluoromethyl)phenyl)carbamoyl)benzoate (0.858
(s, 2H), 8.33 (s, 1H), 8.28 (d, J ) 7.5 Hz, 1H), 7.99 (d, J ) 6.6
+
g, 85% yield) [MS (ESI, positive ion) m/z: 338 (MH )] as an orange
oil. Lithium hydroxide (0.154 g, 6 mmol) was added to a solution
of methyl 2-methyl-5-((3-(trifluoromethyl)phenyl)carbamoyl)benzoate
Hz, 1H), 7.99 (d, J ) 2.2 Hz, 1H), 7.85 - 7.77 (m, 2H), 7.32 (d, J
+
) 8.5 Hz, 1H), 6.55 (s, 2H), 2.37 (s, 3H). HRMS (C H F N O ) :
20
17
3
5
2
calcd, 416.132 89; found, 416.133 27.
(
0.858 g, 2.54 mmol) in THF (10 mL) and water (2 mL). The
2-Methyl-N-(2-((4-(4-methyl-1-piperazinyl)phenyl)amino)-5-
pyrimidinyl)-5-(((3-(trifluoromethyl)phenyl)carbonyl)amino)benz-
amide (13b). Reduction of 12a in a manner analogous to the method
described for the reduction of 12a in the preparation of 23a afforded
amino-2-methyl-N-(2-(4-(4-methylpiperazin-1-yl)phenylamino)py-
rimidin-5-yl)benzamide. The title compound was prepared from
5-amino-2-methyl-N-(2-(4-(4-methylpiperazin-1-yl)phenylamino)py-
rimidin-5-yl)benzamide (0.113 g, 0.271 mmol) and 3-(trifluorom-
ethyl)benzoyl chloride (0.044 mL, 0.298 mmol) using the procedure
described for the synthesis of 27. Trituration with CH Cl , filtration,
mixture was stirred at room temperature for 20 h. The layers were
separated, and the aqueous phase was acidified to pH 6 with 6 M
HCl (aq) and extracted with EtOAc. The combined organic phases
were dried over anhydrous Na
afford 9 (0.760 g, 92% yield) [MS (ESI, positive ion) m/z: 324
(
mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride (0.125 g, 0.650 mmol), 1-hydroxy-7-azabenzotriazole (0.0842
g, 0.619 mmol), diisopropylethylamine (0.323 mL, 1.86 mmol),
2 4
SO , filtered, and concentrated to
+
MH )] as a light-purple solid. A solution of 9 (0.200 g, 0.619
2
2
and 6b (0.176 g, 0.619 mmol) in CH
2
Cl
2
(4 mL) was stirred at
2 2
washing with CH Cl , and drying on high vacuum overnight
room temperature for 20 h. The reaction mixture was concentrated
and the residue was purified via column chromatography on silica
afforded the title compound (0.093 g, 58%) as a light-yellow
1
amorphous solid. H NMR (400 MHz, DMSO-d ): δ 10.58 (s, 1H),
6
gel (gradient elution with 0-100% 90:10:1, CH
NH OH-CH Cl ) to afford the title compound (0.332 g, 91% yield)
as a yellow solid. H NMR (400 MHz, DMSO-d
H), 10.46 (s, 1H), 9.38 (s, 1H), 8.74 (s, 2H), 8.26 (s, 1H), 8.19
2
Cl
2
/CH
3
OH/
10.35 (s, 1H), 9.37 (s, 1H), 8.73 (s, 2H), 8.33 (s, 1H), 8.29 (d, J )
7.9 Hz, 1H), 7.99 (d, J ) 6.9 Hz, 1H), 7.93 (d, J ) 2.1 Hz, 1H),
7.85 (dd, J ) 2.4, 8.2 Hz, 1H), 7.81 (t, J ) 7.8 Hz, 1H), 7.56 (d,
J ) 9.2 Hz, 2H), 7.33 (d, J ) 8.7 Hz, 1H), 6.89 (d, J ) 9.2 Hz,
2H), 3.06 (m, 4H), 2.47 (m, 4H), 2.39 (s, 3H), 2.23 (s, 3H). HRMS
4
2
2
1
6
): δ 10.61 (s,
1
(
d, J ) 1.4 Hz, 1H), 8.10 (d, J ) 8.1 Hz, 1H), 8.04 (dd, J ) 8.0,
+
1
7
9
2
5
.5 Hz, 1H), 7.62 (t, J ) 8.2 Hz, 1H), 7.56 (d, J ) 9.0 Hz, 2H),
(C H F N O ) : calcd, 590.248 58; found, 590.248 12.
3
1
31
3
7
2
.52 (d, J ) 8.1 Hz, 1H), 7.47 (d, J ) 7.6 Hz, 1H), 6.89 (d, J )
2-Methyl-N-(2-((3-(4-morpholinyl)propyl)amino)-5-pyrimidinyl)-
5-(((3-(trifluoromethyl)phenyl)carbonyl)amino)benzamide (13c). 4
(0.500 g, 3.13 mmol), 3-morpholinopropan-1-amine (1.35 g, 9.39
mmol), and N,N-diisopropylethylamine (3.4 mL, 4.7 mmol) were
taken up in IPA (20 mL) in a resealable Pyrex tube and heated at
70 °C for 20 h. After the mixture was cooled to room temperature,
the product precipitated out of solution and was collected by
filtration through a Buchner equipped with a micromembrane filter,
3
.0 Hz, 2H), 3.17 (d, J ) 3.7 Hz, 3H), 3.09 - 3.01 (m, 4H),
+
.48-2.42 (m, 4H), 2.22 (s, 3H). HRMS (C31
H
N
31
F
3
N
·CH
7
O
3
2
) : calcd,
90.248 58; found, 590.249 96. Anal. (C31
H
30
F
3
7
O
2
2
OH·H O)
C, H, N.
N-(2-Aminopyrimidin-5-yl)-2-methyl-5-nitrobenzamide (12a).
1 (0.66 g, 3.64 mmol) was heated to reflux in SOCl (6 mL) for
h. After the mixture was cooled to room temperature and
1
2
1
concentrated to dryness, the crude acid chloride was taken up in
CH Cl (25 mL). 6a (0.40 g, 3.64 mmol) was added to the solution,
washed with CH OH, and dried to afford 5c [MS (ESI, positive
+
2
2
ion) m/z: 268 (MH )] as a pale-yellow solid, which was used
and the mixture was allowed to stir at room temperature overnight.
The mixture became a thick white suspension. Triethylamine (0.700
mL, 4.73 mmol) was added. After 2 h, a thick white suspension
remained. The mixture was filtered through a Buchner apparatus
without further purification. Reduction of 5c in a manner analogous
to the method described for the reduction of 12a in the preparation
+
of 23a afforded 6c [MS (ESI, positive ion) m/z: 270 (M )] as a
thick rust-colored oil, which was used without purification. The
title compound was prepared from 6c (0.074 g, 0.20 mmol) and
3-(trifluoromethyl)benzoyl chloride (0.030 mL, 0.20 mmol) using
the procedure described for the synthesis of 27. Trituration with
CH Cl , filtration, washing with CH Cl , and drying under high
equipped with a micromembrane filter, washed with CH
2 2
Cl , and
dried on high vacuum to afford the title compound (962 mg, 97%)
+
[
MS (ESI, positive ion) m/z: 273 (M )] as an off-white solid, which
was used without further purification.
-Methyl-N-(2-(4-(4-methylpiperazin-1-yl)phenylamino)pyri-
midin-5-yl)-5-nitrobenzamide (12b). To a mixture of 11 (46.0 g,
81 mmol) in CH Cl (2 L) was added EDC (61.0 g, 318 mmol).
The mixture was allowed to stir for 30 min at room temperature.
b (60 g, 211 mmol) was added, and the mixture was allowed to
2
2
2
2
2
vacuum overnight afforded the title compound (0.062 g, 58%) as
1
6
a light-yellow amorphous solid. H NMR (400 MHz, DMSO-d ):
1
2
2
δ 10.56 (s, 1H), 10.15 (s, 1H), 8.54 (s, 2H), 8.32 (s, 1H), 8.28 (d,
J ) 7.2 Hz, 1H), 7.99 (d, J ) 8.2 Hz, 1H), 7.89 (s, 1H), 7.85-7.79
(m, 2H), 7.31 (d, J ) 8.6 Hz, 1H), 7.13 (m, 1H), 3.57 (m, 4H),
6
stir for 70 h. Water (2 L) was added. Then 2N NaOH was added
until the pH was 14. The organics were extracted into EtOAc (4
L). The aqueous layer was extracted with additional EtOAc, and
3.31-3.26 (m, 2H), 2.36 (s, 3H), 2.35-2.30 (m, 6H), 1.68 (m,
+
2H). HRMS (C27
H
30
F
3
N
6
O
3
) : calcd, 543.232 60; found, 543.235 18.
2-Methyl-N-(2-((1-methyl-4-piperidinyl)amino)-5-pyrimidinyl)-
5-(((3-(trifluoromethyl)phenyl)carbonyl)amino)benzamide (13d).
1-Methylpiperidin-4-amine (0.358 g, 3.13 mmol) and NaH (60%
dispersion in mineral oil) (0.126 g, 3.13 mmol) were taken up in
the combined organics were dried over anhydrous MgSO
concentrated under reduced pressure. The residue was taken up in
CH Cl . The solid formed was collected by suction filtration and
washed twice with CH Cl (250 mL). The solid was dried under
4
and
2
2
2
2
2
THF (16 mL) under an atmosphere of dry N . After the mixture
vacuum to obtain the title compound (47.6 g, 51%) as an orange
amorphous solid, which was used without further purification. H
was stirred at room temperature for 10 min, 4 (0.500 g, 3.13 mmol)
was added. The mixture was allowed to stir at room temperature
1
NMR (400 MHz, DMSO-d
6
): δ 10.53 (s, 1H), 9.40 (s, 1H), 8.71
for 1 h, then quenched with 1 N HCl, extracted with CH
concentrated to afford 5d [MS (ESI, positive ion) m/z: 239 (M )]
2 2
Cl , and
+
(
s, 2H), 8.39 (s, 1H), 8.26–8.29 (m, 1H), 7.55–7.65 (m, 3H),

Aminopyrimidine Amides as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1689
)+: calcd, 460.245 55; found, 460.244 07. Anal.
as a tan amorphous solid, which was used without further
HRMS (C25
30 7 2
H N O
purification. Reduction of 5d in a manner analogous to the method
(C25
H
29
N
7
O
2
2
·3H O) C, H, N.
described for the reduction of 12a in the preparation of 23a afforded
5-Methyl-N-(4-methyl-3-((2-(4-(4-methylpiperazin-1-yl)phe-
nylamino)pyrimidin-5-yl)carbamoyl)phenyl)isoxazole-4-carbox-
amide (15). The title compound was prepared from 5-amino-2-
methyl-N-(2-(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-5-
yl)benzamide (0.100 g, 0.24 mmol) and 5-methylisoxazole-4-
carbonyl chloride (0.038 g, 0.26 mmol) using the procedure
described for the synthesis of 27. Trituration with CH
washing with CH Cl , and drying under high vacuum overnight
afforded the title compound (0.112 g, 86% yield) as an off-white
+
6
d [MS (ESI, positive ion) m/z: 285 (MH )] as a tan solid, which
was without further purification. The title compound was prepared
from 6d (0.063 g, 0.185 mmol) and 3-(trifluoromethyl)benzoyl
chloride (0.027 mL, 0.185 mmol) using the procedure described
for the synthesis of 27. Trituration with CH
with CH Cl , and drying under high vacuum overnight afforded
the title compound as an off-white amorphous solid (0.065 g, 68%).
2 2
Cl , filtration, washing
2 2
Cl , filtration,
2
2
2
2
1
6
H NMR (400 MHz, DMSO-d ): δ 10.58 (s, 1H), 10.22 (s, 1H),
1
8
8
7
2
.59 (s, 2H), 8.32 (s, 1H), 8.29 (d, J ) 8.8 Hz, 1H), 7.99 (d, J )
.2 Hz, 1H), 7.93 (d, J ) 2.3 Hz, 1H), 7.83-7.79 (m, 2H),
.33-7.30 (m, 2H), 3.88 (br s, 1H), 3.40 (br s, 2H), 3.03 (br s,
6
amorphous solid. H NMR (400 MHz, DMSO-d ): δ 10.35 (s, 1H),
10.16 (s, 1H), 9.45 (s, 1H), 9.10 (s, 1H), 8.74 (s, 2H), 7.88 (d, J )
2.4 Hz, 1H), 7.71 (dd, J ) 2.0, 8.5 Hz, 1H), 7.62 (d, J ) 9.2 Hz,
H), 2.73 (br s, 3H), 2.36 (s, 3H), 2.06 (br s, 2H), 1.69 (br s, 2H).
2H), 7.31 (d, J ) 8.2 Hz, 1H), 7.96 (d, J ) 8.5 Hz, 2H), 2.70 (s,
+
+
HRMS (C26
H
28
F
3
N
6
O
2
) : calcd, 513.222 04; found, 513.223 82.
3H), 2.37 (s, 3H). HRMS (C28
27.251 78.
-Methyl-N-(2-((4-(4-methyl-1-piperazinyl)phenyl)amino)-5-
H
31
N
8
O
3
) : calcd, 527.251 36; found,
5
2
-Methyl-N-(2-((2-(4-morpholinyl)ethyl)amino)-5-pyrimidinyl)-
-(((3-(trifluoromethyl)phenyl)carbonyl)amino)benzamide (13e).
2a (0.200 g, 0.732 mmol), CuI (0.140 g, 0.732 mmol), and CH
2
5
1
pyrimidinyl)-5-((phenylcarbonyl)amino)benzamide (16). The title
compound was prepared from 5-amino-2-methyl-N-(2-(4-(4-meth-
ylpiperazin-1-yl)phenylamino)pyrimidin-5-yl)benzamide (0.080 g,
2 2
I
(
0.30 mL, 3.73 mmol) were taken up in THF (3.7 mL) in a 16 mm
100 mm resealable Pyrex tube. The tube was sealed, and the
×
0
.19 mmol) and benzoyl chloride (0.022 mL, 0.19 mmol) using
the procedure described for the synthesis of 27. Trituration with
CH Cl , filtration, washing with CH Cl , and drying under high
mixture was heated at 70 °C for 2 h. After cooling to room
temperature, the crude reaction mixture was taken up in 1:1
EtOAc/1 N HCl, and the layers were separated. The organics were
2
2
2
2
vacuum overnight afforded the title compound (0.053 g, 53% yield)
as a light-yellow amorphous solid. H NMR (400 MHz, DMSO-
washed with saturated aqueous NH
anhydrous Na SO , filtered, and concentrated. The residue was
purified via column chromatography on silica gel (elution with
00% EtOAc) to afford 24 (0.060 g, 21% yield) [MS (ESI, positive
4
Cl and then dried over
1
2
4
d
6
): δ 10.37 (s, 1H), 10.34 (s, 1H), 9.37 (s, 1H), 8.73 (s, 2H), 7.98
m, 3H), 7.63-7.53 (m, 6H), 7.31 (d, J ) 8.3 Hz, 1H), 6.89 (d, J
) 9.2 Hz, 2H), 3.05 (m, 4H), 2.45 (m, 4H), 2.37 (s, 3H), 2.22 (s,
(
1
+
ion) m/z: 385 (MH )] as a light-yellow solid. 24 (0.060 g, 0.156
mmol), 2-morpholinoethanamine (0.200 g, 1.5 mmol), and N,N-
diisopropylethylamine (0.041 mL, 0.234 mmol) were taken up in
IPA (2.5 mL) in a 16 mm × 100 mm resealable Pyrex tube and
heated at 70 °C for 4 h. The mixture was concentrated and purified
by preparative thin layer chromatography on silica gel (elution with
+
3
H). HRMS (C30
Anal. (C30
-Methyl-5-(((2-(methyloxy)phenyl)carbonyl)amino)-N-(2-((4-
4-methyl-1-piperazinyl)phenyl)amino)-5-pyrimidinyl)benza-
H
32
N
7
O
2
) : calcd, 522.261 20; found, 522.260 61.
H
31
N
7
O
2
·1.5CH OH·0.5 CH CN) C, H, N.
3
3
2
(
mide (17). The title compound was prepared from 5-amino-2-
methyl-N-(2-(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-5-
yl)benzamide (0.100 g, 0.24 mmol) and 2-methoxybenzoyl chloride
(0.037 mL, 0.24 mmol) using the procedure described for the
synthesis of 27. Purification via column chromatography on silica
2
.5% CH
3 2 2
OH/CH Cl ) to afford 2-methyl-N-(2-(2-morpholinoet-
hylamino)pyrimidin-5-yl)-5-nitrobenzamide (0.060 g, 100%) [MS
+
(
ESI, positive ion) m/z: 387 (M )] as yellow amorphous solid.
Reduction of 2-methyl-N-(2-(2-morpholinoethylamino)pyrimidin-
-yl)-5-nitrobenzamide in a manner analogous to the method
described for the reduction of 12a in the preparation of 23a afforded
-amino-2-methyl-N-(2-(2-morpholinoethylamino)pyrimidin-5-yl)-
gel (gradient elution with 0-20% CH
3 2 2
OH/CH Cl ) afforded the
5
title compound (0.092 g, 70% yield) as a yellow amorphous solid.
1
6
H NMR (400 MHz, DMSO-d ): δ 10.34 (s, 1H), 10.22 (s, 1H),
5
9
7
1
.36 (s, 1H), 8.73 (s, 2H), 7.92 (s, 1H), 7.75 (d, J ) 8.3 Hz, 1H),
.64 (d, J ) 7.6 Hz, 1H), 7.57-7.50 (m, 3H), 7.28 (d, J ) 8.3 Hz,
H), 7.20 (d, J ) 8.3 Hz, 1H), 7.08 (t, J ) 7.5 Hz, 1H), 6.89 (d,
+
benzamide [MS (ESI, positive ion) m/z: 357 (M )] as a light-yellow
foam, which was used without further purification. The title
compound was prepared from 5-amino-2-methyl-N-(2-(2-morpholi-
noethylamino)pyrimidin-5-yl)benzamide (0.060 g, 0.168 mmol) and
J ) 8.8 Hz, 2H), 3.91 (s, 3H), 3.05 (m, 4H), 2.46 (m, 4H), 2.36 (s,
+
3
5
H), 2.22 (s, 3H). HRMS (C31
52.271 44. Anal. (C31
-(((3-Fluoro-5-(trifluoromethyl)phenyl)carbonyl)amino)-2-
H
34
N
7
O
3
) : calcd, 552.271 76; found,
3
-(trifluoromethyl)benzoyl chloride (0.025 mL, 0.168 mmol) using
the procedure described for the synthesis of 27. Preparative thin
layer chromatography on silica gel (elution with 5% CH OH/
CH Cl ) afforded the title compound (0.056 g, 63%) as a light-
yellow amorphous solid. H NMR (400 MHz, DMSO-d
s, 1H), 10.17 (s, 1H), 8.55 (s, 2H), 8.33 (s, 1H), 8.29 (d, J ) 8.1
34 7 3
H N O ) H, N. C: calcd, 67.5; found, 66.7.
5
3
methyl-N-(2-((4-(4-methyl-1-piperazinyl)phenyl)amino)-5-pyri-
midinyl)benzamide (18). The title compound was prepared from
2
2
1
6
): δ 10.58
5
-amino-2-methyl-N-(2-(4-(4-methylpiperazin-1-yl)phenylamino)py-
rimidin-5-yl)benzamide (0.100 g, 0.24 mmol) and 3-fluoro-5-
trifluoromethyl)benzoyl chloride (0.040 mL, 0.26 mmol) using the
procedure described for the synthesis of 27. Trituration with CH Cl
filtration, washing with CH Cl , and drying under high vacuum
overnight afforded the title compound (0.065 g, 44% yield) as a
(
Hz, 1H), 7.99 (d, J ) 7.8 Hz, 1H), 7.90 (d, J ) 2.0 Hz, 1H),
(
7
1
.85-7.79 (m, 2H), 7.32 (d, J ) 8.3 Hz, 1H), 6.93 (t, J ) 5.8 Hz,
2
2
,
H), 3.58 (m, 4H), 3.42-3.37 (m, 2H), 2.49-2.45 (m, 2H), 2.41
+
2
2
(m, 4H), 2.37 (s, 3H). HRMS (C26
H F N O
28 3 6 3
) : calcd, 529.216 95;
found, 529.220 00. Anal. (C26
H F N O
27 3 6 3
3
·2CH OH) H, C. N: calcd,
1
light-yellow amorphous solid. H NMR (400 MHz, DMSO-d
0.72 (s, 1H), 10.35 (s, 1H), 9.37 (s, 1H), 8.72 (s, 2H), 8.07 (m,
6
): δ
1
3.37; found, 14.28.
1
5-(Acetylamino)-2-methyl-N-(2-((4-(4-methyl-1-piperazinyl)phe-
1H), 8.01 (m, 1H), 7.85 (s, 1H), 7.74 (dd, J ) 1.8, 8.6 Hz, 1H),
7.64 (t, J ) 8.9 Hz, 1H), 7.56 (d, J ) 8.9 Hz, 2H), 7.33 (d, J ) 8.5
Hz, 1H), 6.89 (d, J ) 9.1 Hz, 2H), 3.05 (m, 4H), 2.46 (m, 4H),
nyl)amino)-5-pyrimidinyl)benzamide (14). The title compound
was prepared from 5-amino-2-methyl-N-(2-(4-(4-methylpiperazin-
+
1
-yl)phenylamino)pyrimidin-5-yl)benzamide (0.100 g, 0.24 mmol)
and acetyl chloride (0.019 mL, 0.26 mmol) using the procedure
described for the synthesis of 27. Trituration with CH Cl , filtration,
washing with CH Cl , and drying under high vacuum overnight
afforded the title compound (0.108 g, 96%) as an off-white
2.38 (s, 3H), 2.22 (s, 3H). HRMS (C H F N O ) : calcd,
3
1
30
4
7
2
608.239 16; found, 608.238 31. Anal. (C H F N O ) C, N. H:
calcd, 4.81; found, 5.23.
3
1
29
4
7
2
2
2
2
2
2-Methyl-N-(4-methyl-3-(((2-((4-(4-methyl-1-piperazinyl)phe-
nyl)amino)-5-pyrimidinyl)amino)carbonyl)phenyl)-3-(trifluo-
romethyl)benzamide (19). The title compound was prepared from
5-amino-2-methyl-N-(2-(4-(4-methylpiperazin-1-yl)phenylamino)py-
rimidin-5-yl)benzamide (0.080 g, 0.191 mmol) and 2-methyl-3-
(trifluoromethyl)benzoyl chloride (0.047 g, 0.210 mmol) using the
1
amorphous solid. H NMR (400 MHz, DMSO-d
1
7
6
): δ 10.32 (s, 1H),
0.07 (s, 1H), 9.45 (s, 1H), 8.73 (s, 2H), 7.75 (d, J ) 1.6 Hz, 1H),
.62 (d, J ) 8.9 Hz, 2H), 7.56 (dd, J ) 1.8, 8.1 Hz, 1H), 7.23 (d,
J ) 8.2 Hz, 1H), 6.96 (d, J ) 8.8 Hz, 2H), 3.85-3.39 (br m, 4H),
.25-2.91 (br m, 4H), 2.81 (br s, 3H), 2.33 (s, 3H), 2.05 (s, 3H).
3
2 2
procedure described for the synthesis of 27. Trituration with CH Cl ,

1
690 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
DiMauro et al.
6
as a yellow solid. H NMR (400 MHz, DMSO-d ): δ 10.11 (s,
1
filtration, washing with CH
overnight afforded the title compound (0.068 g, 59%) as an off-
white amorphous solid. H NMR (400 MHz, DMSO-d
2
Cl
2
, and drying under high vacuum
1H), 9.09 (s, 1H), 8.89 (s, 1H), 8.52 (s, 2H), 8.03 (s, 1H), 7.62 (d,
J ) 1.9 Hz, 1H), 7.58 (d, J ) 7.8 Hz, 1H), 7.52 (t, J ) 7.8 Hz,
1H), 7.45 (dd, J ) 8.3, 2.1 Hz, 1H), 7.32 (d, J ) 7.3 Hz, 1H), 7.22
1
6
): δ 10.62
(
s, 1H), 10.34 (s, 1H), 9.36 (s, 1H), 8.71 (s, 2H), 7.90 (d, J ) 1.9
Hz, 1H), 7.83 (d, J ) 7.7 Hz, 1H), 7.71 (t, J ) 7.3 Hz, 2H),
7
(d, J ) 8.2 Hz, 1H), 6.54 (s, 2H), 2.32 (s, 3H). HRMS
+
.56-7.52 (m, 3H), 7.31 (d, J ) 8.5 Hz, 1H), 6.88 (d, J ) 8.8 Hz,
(C20
H
18
F
3
N
6
O
2
) : calcd, 431.143 78; found, 431.143 01.
2
H), 3.05 (m, 4H), 2.46 (m, 7H), 2.36 (s, 3H), 2.22 (s, 3H). HRMS
2-Methyl-N-(2-((4-(4-methyl-1-piperazinyl)phenyl)amino)-5-
pyrimidinyl)-5-((((3-(trifluoromethyl)phenyl)amino)car-
bonyl)amino)benzamide (23b). In a 2 L stainless steel pressure
vessel with overhead stirring was charged a mixture of 12b (25 g,
55.9 mmol), EtOH (300 mL), EtOAc (200 mL), and Pd/C (15 g,
+
(
C
32
H
33
F
3
N
7
O
2
) : calcd, 604.264 68; found, 604.264 68.
2,2-Difluoro-N-(4-methyl-3-(((2-((4-(4-methyl-1-piperazinyl)phe-
nyl)amino)-5-pyrimidinyl)amino)carbonyl)phenyl)-1,3-benzo-
dioxole-5-carboxamide (20). The title compound was prepared
from 5-amino-2-methyl-N-(2-(4-(4-methylpiperazin-1-yl)phenylami-
no)pyrimidin-5-yl)benzamide (0.080 g, 0.191 mmol) and 2,2-
difluorobenzo[d][1,3]dioxole-5-carbonyl chloride (0.046 g, 0.210
mmol) using the procedure described for the synthesis of 27.
Purification via column chromatography on silica gel (gradient
10% by weight wet). The reactor was charged with 65 psi of H
2
for 72 h. The catalyst was removed by suction filtration and washed
with EtOH. The organics were concentrated under reduced pressure
and dried under vacuum to obtain 5-amino-2-methyl-N-(2-(4-(4-
methylpiperazin-1-yl)phenylamino)pyrimidin-5-yl)benzamide [MS
+
elution with 0-100% 90:10:1, CH
afforded the title compound (0.069 g, 60% yield) as a light-yellow
2
Cl
2
/CH
3
OH/NH
4
OH-CH
2
Cl
2
)
(ESI, positive ion) m/z: 418 (MH )], which was used without
purification. The title compound was prepared from 5-amino-2-
methyl-N-(2-(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-5-
yl)benzamide (0.071 g, 0.170 mmol) and 1-isocyanato-3-(trifluo-
romethyl)benzene (0.026 mL, 0.187 mmol) using the procedure
described for the synthesis of 23a. Purification via column
chromatography on silica gel (gradient elution with 0-100% 90:
1
amorphous solid. H NMR (400 MHz, DMSO-d
0.34 (s, 1H), 9.37 (s, 1H), 8.72 (s, 2H), 7.99 (s, 1H), 7.92 - 7.89
m, 2H), 7.79 (dd, J ) 1.6, 8.3 Hz, 1H), 7.60 (d, J ) 8.3 Hz, 1H),
.56 (d, J ) 8.8 Hz, 2H), 731 (d, J ) 8.3 Hz, 1H), 6.89 (d, J ) 8.8
Hz, 2H), 3.08 (m, 4H), 2.55 (m, 4H), 2.37 (s, 3H), 2.29 (s, 3H).
6
): δ 10.40 (s, 1H),
1
(
7
+
HRMS (C31
H
30
F
2
N
7
O
4
) : calcd, 602.232 19; found, 602.232 34.
10:1, CH
pound as a light-yellow solid (0.047 g, 46%). H NMR (400 MHz,
DMSO-d ): δ 10.29 (s, 1H), 9.35 (s, 1H), 9.08 (s, 1H), 8.89 (s,
2
Cl
2
/CH
3
OH/NH
4
OH-CH
2 2
Cl ) afforded the title com-
1
5
-(((4-(1,1-Dimethylethyl)phenyl)carbonyl)amino)-2-methyl-
N-(2-((4-(4-methyl-1-piperazinyl)phenyl)amino)-5-pyrimidinyl)-
benzamide (21). The title compound was prepared from 5-amino-
2
6
1H), 8.71 (s, 2H), 8.03 (s, 1H), 7.64 (s, 1H), 7.58-7.44 (m, 5H),
7.31 (d, J ) 7.6 Hz, 1H), 7.23 (d, J ) 8.3 Hz, 1H), 6.89 (d, J )
-methyl-N-(2-(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-
-yl)benzamide (0.080 g, 0.191 mmol) and 4-tert-butylbenzoyl
5
8.7 Hz, 2H), 3.05 (br s, 4H), 2.46 (br s, 4H), 2.33 (s, 3H), 2.22 (s,
+
chloride (0.042 mL, 0.210 mmol) using the procedure described
for the synthesis of 27. Trituration with CH Cl , filtration, washing
with CH Cl , and drying under high vacuum overnight afforded
the title compound (0.058 g, 53%) as a white amorphous solid. H
NMR (400 MHz, DMSO-d ): δ 10.33 (s, 1H), 10.28 (s, 1H), 9.36
s, 1H), 8.72 (s, 2H), 7.95 (d, J ) 1.8 Hz, 1H), 7.91 (d, J ) 8.5
3H). HRMS (C31
H
32
F
3
N
8
O
2
) : calcd, 605.259 48; found, 605.260 56.
2
2
2,6-Dichloro-N-(2-((4-(4-methyl-1-piperazinyl)phenyl)amino)-
5-pyrimidinyl)benzamide (27). 2,6-Dichlorobenzoyl chloride (0.061
mL, 0.43 mmol) was added to a suspension of N2-(4-(4-methylpip-
erazin-1-yl)phenyl)pyrimidine-2,5-diamine (0.11 g, 0.39 mmol) and
CH Cl (2 mL). The solution was stirred at room temperature for
2
2
1
6
(
2
2
Hz, 2H), 7.81 (dd, J ) 2.0, 8.6 Hz, 1H), 7.56 (d, J ) 8.5 Hz, 4H),
7
2
10 min, and then triethylamine (0.070 mL, 0.50 mmol) was added.
The suspension was stirred at room temperature overnight. The
reaction mixture was filtered through a Buchner apparatus equipped
with a micromembrane filter, washed with CH Cl , and dried under
.29 (d, J ) 8.5 Hz, 1H), 6.89 (d, J ) 9.0 Hz, 2H), 3.05 (m, 4H),
.47 (m, 4H), 2.36 (s, 3H), 2.23 (s, 3H), 1.32 (s, 9H). HRMS
+
(
C
34
H
40
N
7
O
2
) : calcd, 578.323 80; found, 578.324 08.
2
2
5
-(((3,5-Bis(methyloxy)phenyl)carbonyl)amino)-2-methyl-
vacuum overnight to afford the title compound (0.141 g, 79%) as
1
N-(2-((4-(4-methyl-1-piperazinyl)phenyl)amino)-5-pyrimidinyl)-
benzamide (22). The title compound was prepared from 5-amino-
-methyl-N-(2-(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-
6
a pale-yellow solid. H NMR (400 MHz, DMSO-d ): δ 10.78 (s,
1H), 9.44 (s, 1H), 8.68 (s, 2H), 7.93-7.51 (m, 5H), 6.89 (d, J )
2
5
8.8 Hz, 2H), 3.06 (m, 4H), 2.45 (m, 4H), 2.22 (s, 3H). HRMS
+
-yl)benzamide (0.080 g, 0.191 mmol) and 3,5-dimethoxybenzoyl
(C22
(C22
H
H
23
C
C
l2
N
N
6
O) : calcd, 457.130 49; found, 457.130 83. Anal.
O) C, H, N.
chloride (0.042 g, 0.210 mmol) using the procedure described for
the synthesis of 27. Trituration with CH Cl , filtration, washing
with CH Cl , and drying under high vacuum overnight afforded
the title compound (0.030 g, 27%) as an off-white amorphous solid.
22
l2
6
2
2
2,6-Dimethyl-N-(2-((4-(4-methyl-1-piperazinyl)phenyl)ami-
no)-5-pyrimidinyl)-3-(((3-(trifluoromethyl)phenyl)carbonyl)amino)ben-
zamide (29). 28 was prepared from 26 (0.300 g, 1.5 mmol) and 6b
(0.48 g, 1.7 mmol) using the procedure described for the synthesis
of 12a. Purification via column chromatography on silica gel
(gradient elution with 3-10% CH OH/CH Cl ) afforded 28 (0.73
2
2
1
6
H NMR (400 MHz, DMSO-d ): δ 10.32 (s, 1H), 10.28 (s, 1H),
9
2
1
2
3
5
.36 (s, 1H), 8.72 (s, 2H), 7.92 (d, J ) 1.9 Hz, 1H), 7.82 (dd, J )
.0, 8.5 Hz, 1H), 7.55 (d, J ) 8.8 Hz, 2H), 7.30 (d, J ) 8.1 Hz,
H), 7.12 (d, J ) 2.2 Hz, 2H), 6.88 (d, J ) 9.2, 2H), 6.72 (t, J )
3
2
2
+
g, 90%) [MS (ESI, positive ion) m/z: 462 (MH )] as a yellow solid.
Reduction of 28 in a manner analogous to the method described
for the reduction of 12a in the preparation of 23a afforded crude
3-aniline, which was purified via preparative reverse-phase HPLC
.0 Hz, 1H), 3.82 (s, 6H), 3.05 (m, 4H), 2.46 (m, 4H), 2.37 (s,
H), 2.22 (s, 3H). HRMS (C32
82.282 62.
+
H
36
N
7
O
4
) : calcd, 582.282 33; found,
N-(2-Amino-5-pyrimidinyl)-2-methyl-5-((((3-(trifluorometh-
(eluting with 10–90% (0.1% trifluoroacetic acid in CH
over 15 min). Clean fractions were combined and partitioned
between CH Cl and saturated aqueous NaHCO . The aqueous
phase was separated and extracted with CH Cl . The combined
organic phases were washed with brine, dried over anhydrous
Na SO , filtered, and concentrated to afford 3-amino-2,6-dimethyl-
3
CN)-water
yl)phenyl)amino)carbonyl)amino)benzamide (23a). 12a (0.300
g, 1.1 mmol) and 10% Pd/C (∼0.150 g) were combined in EtOAc
2
2
3
(
24 mL) and CH
3
OH (6 mL). The mixture was purged with H
2
,
2
2
then allowed to stir under 1 atm of H
filtered through a pad of Celite, and washing with CH
CH Cl and drying afforded 5-amino-N-(2-aminopyrimidin-5-yl)-
-methylbenzamide [MS (ESI, positive ion) m/z: 244 (MH )] as a
pale-yellow amorphous solid, which was used without purification.
2
for 24 h. The mixture was
3
OH and
2
4
2
2
N-(2-(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-5-yl)ben-
+
+
2
zamide (0.550 g, 80%) [MS (ESI, positive ion) m/z: 432.1 (MH )]
as a light-yellow solid. The title compound was prepared from
3-amino-2,6-dimethyl-N-(2-(4-(4-methylpiperazin-1-yl)phenylami-
no)pyrimidin-5-yl)benzamide (80 mg, 0.2 mmol) and 3-(trifluo-
romethyl)benzoyl chloride (0.04 mL, 0.2 mmol) using the procedure
described for the synthesis of 27 and purified via preparative
reverse-phase HPLC (eluting with 10–90% (0.1% trifluoroacetic
5
0
3
-Amino-N-(2-aminopyrimidin-5-yl)-2-methylbenzamide (0.108 g,
.444 mmol) was taken up in benzene (8 mL), and 1-isocyanato-
-(trifluoromethyl)benzene (0.062 mL, 0.444 mmol) was added. The
mixture was allowed to stir at room temperature overnight. The
reaction mixture was concentrated and the residue was purified via
column chromatography on silica gel (gradient elution with 0-5%
acid in CH
combined and partitioned between CH
3
CN)-water over 15 min). Clean fractions were
3 2 2
CH OH/CH Cl ) to afford the title compound (0.040 g, 20% yield)
2
Cl and saturated aqueous
2

Aminopyrimidine Amides as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1691
NaHCO
CH Cl . The combined organic phases were washed with brine,
dried over anhydrous Na SO , filtered, and concentrated to afford
the title compound (0.023 g, 20%) as a light-yellow solid. H NMR
400 MHz, DMSO-d ) δ ppm 10.55 (s, 1H), 10.29 (s, 1H), 9.44 (s,
H), 8.78 (s, 2H), 8.37 (m, 2H), 8.05 (d, J ) 8.8 Hz, 1H), 7.86 (t,
3
. The aqueous phase was separated and extracted with
layer chromatography TLC (eluting three times with 95:5:0.5,
2
2
CH
2 2 3 4 2 2
Cl /CH OH/NH OH-CH Cl ) to afford the title compound
2
4
(0.010 g, 0.016 mmol, 20%) as an off-white solid. MS (ESI, positive
1
+
1
6
ion) m/z: 616 (MH ). H NMR (400 MHz, DMSO-d ): δ 10.42 (s,
(
1
6
1H), 10.37 (s, 1H), 9.36 (s, 1H), 8.71 (s, 2H), 7.99 (s, 1H),
7.87-7.94 (m, 2H), 7.82 (d, J ) 9.3 Hz, 1H), 7.60 (d, J ) 8.3 Hz,
1H), 7.55 (d, J ) 8.2 Hz, 2H), 7.35 (d, J ) 8.0 Hz, 1H), 6.88 (d,
J ) 8.5 Hz, 2H), 3.06 (s, 4H), 2.67-2.78 (m, 2H), 2.42-2.50 (m,
J ) 7.8 Hz, 1H), 7.62 (d, J ) 8.8 Hz, 2H), 7.37 (d, J ) 8.2 Hz,
1
4
H), 7.24 (d, J ) 8.1 Hz, 1H), 6.94 (d, J ) 9.0 Hz, 2H), 3.12 (m,
H), 2.53 (m, 4H), 2.38 (s, 3H), 2.29 (s, 3H), 2.26 (s, 3H). HRMS
4H), 2.24 (s, 3H), 1.17 (t, J ) 7.4 Hz, 3H). HRMS (C32H -
32
+
+
(
C
32
H
33
F
3
N
7
O
2
) : calcd, 604.264 23; found, 604.265 65.
F
2
N
7
O
4
) : calcd, 616.247 84; found, 616.248 29.
N-(4-Chloro-3-(((2-((4-(4-methyl-1-piperazinyl)phenyl)amino)-
-pyrimidinyl)amino)carbonyl)phenyl)-2,2-difluoro-1,3-benzo-
dioxole-5-carboxamide (32). 30 (0.150 g, 0.874 mmol) was added
to a solution of 2,2-difluoro-1,3-benzodioxole-5-carbonyl chloride
2-Methyl-N-(5-pyrimidinyl)-5-(((3-(trifluoromethyl)phenyl)-
5
carbonyl)amino)benzamide (37). 5-Amino-2-methyl-5-nitro-N-
(pyrimidin-5-yl)benzamide was prepared from 26 and pyrimidin-
5-amine according to the procedure used for the synthesis of 12b.
Reduction of 5-amino-2-methyl-5-nitro-N-(pyrimidin-5-yl)benza-
mide in a manner analogous to the method described for the
reduction of 12a in the preparation of 23a afforded 5-amino-2-
methyl-N-(pyrimidin-5-yl)benzamide [MS (ESI, positive ion) m/z:
(
2 2
0.202 g, 0.918 mmol) in CH Cl (9.0 mL). The mixture was stirred
at room temperature for 2.5 h to afford a white suspension, and
triethylamine (0.16 mL, 1.14 mmol) was added. The resulting
yellow solution was stirred at room temperature for 16 h. The
reaction mixture was concentrated to afford an off-white solid. This
+
228 (M )], which was used without further purification. The title
solid was partitioned between CH
was separated and extracted with CH
phases were washed with brine, dried over anhydrous Na
filtered, and concentrated to afford 31 (0.193 g, 62%) as an off-
2
Cl
2
and water. The aqueous phase
Cl . The combined organic
SO
2
2
2
4
,
1
white solid. H NMR (400 MHz, DMSO-d
6
): δ 10.43 (s, 1H), 8.01
(
d, J ) 1.4 Hz, 1H), 7.97 (s, 1H), 7.90 (dd, J ) 8.3, 1.4 Hz, 1H),
.77-7.84 (m, 1H), 7.58 (d, J ) 8.5 Hz, 1H), 7.36 (d, J ) 8.5 Hz,
7
1
+
H). MS (ESI, positive ion) m/z: 355.9 (MH ). A resealable tube
was charged with 6b (0.100 g, 0.35 mmol), 31 (0.16 g, 0.46 mmol),
2H), 7.86 (s, 1H), 7.81 (s, 1H), 7.37 (s, 1H), 2.40 (s, 3H). HRMS
+
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.10
(C20
H
16
F
3
N
4
O
2
) : calcd, 401.121 99; found, 401.123 99.
g, 0.53 mmol), 1-hydroxy-7-azabenzotriazole (0.048 g, 0.35 mmol),
and DMF (2.5 mL). N,N-Diisopropylethylamine (0.214 mL, 1.2
mmol) was added, and the system was flushed with argon. The
tube was sealed and the mixture stirred at room temperature for
Biological Materials and Methods. Lck Kinase Assay. The
Lck HTRF kinase assay involves 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 ATP was 0.5 µM (Km(app)
) 0.6 ( 0.1 µM), and the final 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 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% Tween-20. Prior to reading,
steptavidin allophycocyanin (SA-APC) was added at a final
concentration in the assay of 0.0004 mg/mL, along with europilated
antiphosphotyrosine Ab (Eu-anti-PY) at a final concentration 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. Reported IC50 values are the mean of two or more separate
determinations run in duplicate.
1
7 h. The reaction mixture was concentrated to afford an orange-
brown oil. This oil was partitioned between CH Cl and water, and
saturated aqueous NaHCO was added. The aqueous layer was
separated and extracted with CH Cl . The combined organic phases
were washed with brine, dried over anhydrous Na SO , filtered,
2
2
3
2
2
2
4
and concentrated to afford an orange-brown oil. This material was
purified via column chromatography on silica gel (gradient elution
with 0-100% 90:10:1, CH
an orange solid. This material was dissolved in 1:1 (DMSO/CH
and further purified via preparative reverse-phase HPLC (eluting
with 10-90% (0.1% trifluoroacetic acid in CH CN-water over 15
min). Clean fractions were combined and partitioned between
EtOAc and saturated aqueous NaHCO . The aqueous phase was
separated and extracted with EtOAc. The combined organic phases
were washed with brine, dried over anhydrous Na SO , filtered,
and concentrated to afford the title compound (0.092 g, 42%) as a
2
Cl
2
/CH
3
OH/NH
4
OH-CH
2
Cl
2
) to afford
3
OH)
3
3
2
4
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
+
1
yellow solid. MS (ESI, positive ion) m/z: 622 (MH ). H NMR
400 MHz, DMSO-d ): δ 10.56 (s, 2H), 9.40 (s, 1H), 8.70 (s, 2H),
.02 (d, J ) 12.8 Hz, 2H), 7.90 (d, J ) 8.6 Hz, 2H), 7.45-7.72
m, 4H), 6.88 (d, J ) 8.5 Hz, 2H), 3.05 (s, 4H), 2.41-2.48 (m,
(
6
8
m
the kinase and measured K values for the substrates.
(
4
Human Mixed Lymphocyte Reaction (MLR). The purpose of
this assay is to test the potency of T cell activation inhibitors in an
in vitro model of allogeneic T cell stimulation. Human peripheral
+
H), 2.22 (s, 3H). HRMS (C30
2 7 4
H27ClF N O ) : calcd, 622.177 56;
found, 622.176 69.
5
N-(4-Ethyl-3-(((2-((4-(4-methyl-1-piperazinyl)phenyl)amino)-
-pyrimidinyl)amino)carbonyl)phenyl)-2,2-difluoro-1,3-benzo-
blood lymphocytes (hPBL, 2 × 10 /well) were incubated with
5
mitomycin C treated B lymphoblastoid cells (JY cell line (ATCC,
5
dioxole-5-carboxamide (33). A resealable tube was charged with
2 (0.050 g, 0.080 mmol), palladium(II) acetate (0.0018 g, 0.0080
Rockville, MD), 1 × 10 /well) as allogeneic stimulators in the
3
presence or absence of dilutions of potential inhibitor compound
in 96-well round-bottom tissue culture plates. These cultures were
mmol), S-Phos (0.0066 g, 0.016 mmol), potassium phosphate (0.068
g, 0.32 mmol), and THF (1.0 mL). A solution of triethylborane (1
M in THF, 0.12 mL, 0.12 mmol) was added, and the system was
flushed with argon. The tube was sealed and the mixture stirred at
incubated at 37 °C in 5% CO for 6 days total. The proliferative
2
3
response of the hPBL was measured by H-thymidine incorporation
overnight between days 5 and 6 after initiation of culture. Cells
3
6
0 °C for 18 h. The THF was removed in vacuo, and DMF (2.0
were harvested onto glass fiber filters, and H-thymidine incorpora-
mL) was added. Palladium(II) acetate (0.0018 g, 0.0080 mmol),
S-Phos (0.0066 g, 0.016 mmol), potassium phosphate (0.068 g, 0.32
mmol), and triethylborane (0.12 mL, 0.12 mmol) were added, the
system was evacuated and purged with argon, and the tube was
sealed. The mixture stirred at 100 °C for 6 h. The reaction mixture
was filtered through a pad of Celite and concentrated to afford a
yellow-green solid. This material was purified via preparative thin
tion into DNA was analyzed by liquid scintillation counter.
Anti-CD3/CD28-Induced T Cell IL-2 Secretion and Proli-
feration Assay (IL-2). 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

1
692 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
DiMauro et al.
anti-CD3 and an anti-CD28 antibody in 96-well tissue culture plates
mouse IL-2, and additional references. 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
5
% CO , then secreted IL-2 in the supernatants was quantified by
2
cytokine ELISA (Pierce/Endogen, St. Louis, MO). The cells
remaining in the wells were then pulsed with H-thymidine
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(
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Supporting Information Available: Elemental analysis and
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Aminopyrimidine Amides as Inhibitors
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