Discovery of aminoquinazolines as potent, orally bioavailable inhibitors of Lck: Synthesis, SAR, and in vivo anti-inflammatory activity

Article abstract of DOI:10.1021/jm0605482

The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and natural killer (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. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. Screening of our kinase-preferred collection identified aminoquinazoline 1 as a potent, nonselective inhibitor of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminoquinazolines possessing in vitro mechanism-based potency. Optimized, orally bioavailable compounds 32 and 47 exhibit anti-inflammatory activity (ED₅₀ of 22 and 11 mg/kg, respectively) in the anti-CD3-induced production of interleukin-2 (IL-2) in mice.

Full text of DOI:10.1021/jm0605482

J. Med. Chem. 2006, 49, 5671-5686
5671
Discovery of Aminoquinazolines as Potent, Orally Bioavailable Inhibitors of Lck: Synthesis,
SAR, and in Vivo Anti-Inflammatory Activity
,
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Erin F. DiMauro,* John Newcomb,* Joseph J. Nunes, Jean E. Bemis, Christina Boucher, John L. Buchanan,
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William H. Buckner, Victor J. Cee, Lilly Chai, Holly L. Deak, Linda F. Epstein, Ted Faust, Paul Gallant,
Stephanie D. Geuns-Meyer, Anu Gore, Yan Gu, Brad Henkle, Brian L. Hodous, Faye Hsieh, Xin Huang, Joseph L. Kim,
Josie H. Lee, Matthew W. Martin, Craig E. Masse, 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, Ryan D. White, Huilin Zhao, Li Zhu, and
Xiaotian Zhu|
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§
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†
†
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‡
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Department of Medicinal Chemistry, Department of HTS and Molecular Pharmacology and Department of Molecular Structure,
One Kendall Square, Building 1000, Cambridge, Massachusetts 02139, and 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 May 10, 2006
The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T
cells and natural killer (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. Selective inhibition of Lck is expected to offer a new therapy for the treatment
of T-cell-mediated autoimmune and inflammatory disease. Screening of our kinase-preferred collection
identified aminoquinazoline 1 as a potent, nonselective inhibitor of Lck and T cell proliferation. In this
report, we describe the synthesis and structure-activity relationships of a series of novel aminoquinazolines
possessing in vitro mechanism-based potency. Optimized, orally bioavailable compounds 32 and 47 exhibit
anti-inflammatory activity (ED50 of 22 and 11 mg/kg, respectively) in the anti-CD3-induced production of
interleukin-2 (IL-2) in mice.
Introduction
Different populations of T cells serve pivotal roles in the
adaptive immune response by acting as potentiators (CD4+ T
helper cells) or effectors (CD8+ cytolytic T effector cells) of
immune reactions. The immune specificity of a particular T cell
a
is imparted by the specificity of the T cell receptor (TCR ) for
1
antigen, or CD3 complex. Signal transduction pathways used
by the TCR have been extensively researched. Novel immu-
nosuppressive agents that target components of these pathways
might serve as improved therapies for graft rejection and/or
T-cell-mediated autoimmune disease.
Figure 1. Activity of 2-aminoquinazoline lead 1.
Protein kinases have been shown to play important roles in
TCR signal transduction. The Src family of cytoplamic tyrosine
Genetically modified mice lacking Lck expression and patients
with Lck mutations affecting expression and/or catalytic activity
show defects in T cell maturation and signaling. These findings
kinases is made up of eight members: Src, Lck, Fyn, Lyn, Hck,
2
Fgr, Blk, and Yes. Lck and Fyn have been shown to have
4
3
important roles in TCR signal transduction. TCR signals
suggest that a small-molecule inhibitor of Lck kinase activity
could prove to be useful in the treatment of graft rejection
and/or T-cell-mediated autoimmune diseases.
potentiated by Lck ultimately lead to gene regulation events
triggering cytokine release, proliferation, and survival of antigen
specific T cells, thereby amplifying specific immune responses.
The synthesis and characterization of several classes of small
5
molecule Lck inhibitors has been described. In addition, potent
*
To whom correspondence should be addressed. For E.F.D.: phone,
6
17-444-5189; fax, 617-621-3907; e-mail, edimauro@amgen.com. For
and bioavailable Lck antagonists have been shown to have
inhibitory activities in vivo in models of T-cell-dependent
J.N.: phone, 617-444-5055; fax, 617-621-3916; e-mail, jnewcomb@
amgen.com.
5j,6
†
immune responses. Herein, we describe the synthesis, struc-
ture-activity relationships, and pharmacological characterization
of aminoquinazolines with potent and selective inhibition of Lck.
This activity translates into inhibition of in vitro and in vivo
assays of T cell activation.
Department of Medicinal Chemistry, Amgen Inc., MA.
Department of HTS and Molecular Pharmacology.
Department of Molecular Structure.
Department of Pharmaceutics, Amgen Inc., CA.
∇
|
⊥
‡
Department of Inflammation, Amgen Inc., CA
§
Department of Pharmacokinetics and Drug Metabolism, Amgen Inc.,
CAa.
As a result of our screening efforts in a homogeneous time-
resolved fluorescent (HTRF) kinase assay, we identified ami-
noquinazoline 1 as a potent inhibitor of Lck (Figure 1).
Compound 1 also exhibited potent inhibitory activity in two
Abbreviations: Lck, lymphocyte-specific kinase; TCR, T cell receptor;
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.
1
0.1021/jm0605482 CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/22/2006

5
672 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
DiMauro et al.
Scheme 2. Preparation of 2-Amino-6-bromoquinazolinesa
Scheme 1. Retrosynthetic Analysis of 2,6-Quinazolines
a
(a) Guanidine carbonate, DIEA, NMP, 155 °C; (b) MeNH2(g), p-TSA,
820 psi, 160 °C; (c) isoamyl nitrite, CuI, CH2I2, THF, reflux; (d) aniline,
TFA, IPA, 80 °C; (e) 2-morpholinoethanamine, DIEA, IPA, 70 °C.
Scheme 3. Preparation of 2-Amino-6-arylquinazolinesa
cellular assays: the human mixed lymphocyte reaction (MLR)
and the T cell receptor/anti-CD3-induced production of inter-
leukin-2 (IL-2). This lead compound suffered from poor
selectivity, low solubility (26 µg/mL in fasted state simulated
intestinal fluid at pH 6.8 (SIF)), and marginal bioavailability in
rats (2%). We therefore initiated the development of chemistry
toward this class of compounds to investigate the SAR and to
improve the physicochemical and pharmacokinetic (PK) proper-
ties. Toward this end, we have identified orally bioavailable
Lck inhibitors (32 and 47) with good efficacy in a mouse model
of inflammation.
Chemistry
Retrosynthetic analysis of the 2,6-disubstituted quinazoline
target compounds 2 is depicted in Scheme 1. We envisioned a
convergent synthetic route with final step Suzuki coupling of
functionalized quinazoline bromides 3 and boronic esters 4 that
would allow for the preparation of a diverse set of compounds.
2
2
-Substituted quinazoline bromides 3 could arise from 5-bromo-
-fluorobenzaldehyde (5). Boronic ester benzamides 4 could
originate from the coupling of iodobenzoic acids 6 and anilines.
In the forward sense, this modular synthetic route was typically
employed.
a
(
a) Bis(pinacolato)diboron, 10% Pd(dppf)Cl2, KOAc, DMF, 70 °C; (b)
SOCl , 80 °C; (c) 3-(trifluoromethyl)benzenamine, NEt , CH Cl , 60 °C;
2
3
2
2
The synthesis of 6-bromoquinazolin-2-amine (3a) was ac-
complished using modifications to the established method of
(d) 6-bromo-N-methylquinazolin-2-amine (3b), 10% Pd(dppf)Cl2, K2CO3,
CH3CN/H2O, 60 °C; (e) 3-(trifluoromethyl)benzoyl chloride; (f) 1-isocy-
anato-3-(trifluoromethyl)benzene; (g) O,O-dipyridin-2-yl carbonothioate,
CH2Cl2; (h) 4-(trifluoromethyl)benzene-1,2-diamine, PS-DCC, THF, 70 °C.
7
Hynes and Campbell. Acid-promoted condensation with meth-
ylamine gas in a high-pressure reactor afforded 6-bromo-N-meth-
8
ylquinazolin-2-amine (3b). A Sandmeyer reaction was used to
such that a variety of quinazoline bromides 3 and boronic esters
4 could be coupled in moderate to good yields. Iodide 10 was
converted to the boronic ester 11 using the Miyaura protocol.9
Suzuki coupling to 3b afforded 6-(5-amino-2-methylphenyl)-
N-methylquinazolin-2-amine (12). This versatile intermediate
was used in the preparation of reversed amides 13, ureas 14,
and aminobenzimidazoles 15 as shown in Scheme 3.
prepare 6-bromo-2-iodoquinazoline (7) from 3a. An SNAr reac-
tion of iodide 7 with anilines was accomplished under thermal,
acidic conditions to afford 2-anilino-6-bromoquinazolines such
as 3c and with amines under thermal, basic conditions to afford
2
-amino-6-bromoquinazolines such as 3d (Scheme 2).
A variety of 2-amino-6-arylquinazolines were synthesized
according to the methods outlined in Scheme 3. Synthesis of
-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo-
For rapid SAR at the aniline amide or for variation on the
central ring, an alternative synthetic route, proceeding through
acid intermediates 18, was occasionally employed (Scheme 4).
The halides 16 were either commercially available or prepared
according to established methods.
4
ic acid (8) was accomplished using modifications to the
9
established method of Miyaura et al. Preparation of boronic
ester amides, such as 4a, was accomplished by initial heating
of 8 in thionyl chloride, followed by reaction of the crude acid
chloride with the aniline in the presence of triethylamine. Suzuki
coupling of boronate 4a and 3b afforded 2-amino-6-aryl-quinazo-
line (9). These Suzuki conditions proved to be quite general
Most of the anilines used in the preparation of boronic esters
4 were commercially available. Others were prepared either by
1
0
established methods or by short reaction sequences, some of
which are illustrated in Scheme 5.

Aminoquinazolines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5673
Scheme 4. Alternative Route to Quinazolinesa
Figure 2. Cocrystal structure of Lck and 1.
a
(a) 10% Pd(dppf)Cl2, K2CO3, CH3CN/H2O, 60 °C; (b) SOCl2, 80 °C;
Table 1. Sequence Comparison in Extended Hydrophobic Pocket
(c) 3-(trifluoromethyl)benzenamine, NEt3, CH2Cl2, 60 °C.
Lck
Thr316a Met292 Leu295 Leu300 Ile355 Tyr360
Scheme 5. Preparation of Anilinesa
Zap70
Jak3
p38R
KDR
Met
Met
Thr
Val
Met
Leu
Leu
Leu
Leu
Leu
Met
Ile
Ile
Ile
Val
Val
Lue
Leu
Ile
Phe
Cys
Ile
Leu
Cys
a
Gatekeeper residue.
the extended hydrophobic pocket among structurally related
kinases (Table 1). Thus, all compounds were counterscreened
for inhibitory activity against a panel of kinases that included
the proteins Zap70, Jak3, and p38R, all of which are involved
in immune cell signaling pathways. Zap70 is a nonreceptor
tyrosine kinase of the Syk family that functions immediately
downstream of Lck in the T cell signaling pathway. Like Lck,
its expression is restricted to T cells and NK cells.13 All
compounds presented herein were >25 µM vs Zap70. Further
downstream is Janus kinase 3 (Jak3), which mediates signal
transduction via the γ chain of lymphokine surface receptors.14
Jak3 is under investigation as the target of the small-molecule
immunosuppressive agent 3-[4-methyl-3-[N-methyl-N-(7H-pyr-
rolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropioni-
1
5
trile (CP-690550). Mitogen-activated protein kinase (MAPK)
p38R is a key signaling protein in the stress-activated signal
transduction cascade that results in the production of pro-
inflammatory cytokines such as TNFR and IL-1â.16 A number
of small-molecule p38 inhibitors have advanced into clinical
trials in the past few years. We also counterscreened against
the structurally related VEGF receptor-2 (KDR), a receptor
tyrosine kinase that is responsible for regulating the growth and
differentiation of the vascular system and its components.17
Selectivity over KDR is important with respect to establishing
a practical therapeutic window.
1
6
a
1
2
(
a)1-Methylimidazolidin-2-one, N ,N -dimethylethane-1,2-diamine, CuI,
K2CO3, toluene, 110 °C; (b) 2-morpholinoethanamine, benzene, 40 °C; (c)
0% Pd/C, H2, CH3OH, EtOAc; (d) chloroacetyl chloride, NEt3, CH2Cl2,
1
reflux; (e) diethylamine, K2CO3, THF, 50 °C; (f) 3-chloropivaloyl chloride,
NEt3, CH2Cl2, 35 °C; (g) K2CO3, acetone, 50 °C.
Results and Discussion
Select SAR from variations on the aryl ring of the amide is
summarized in Table 2. During the course of our investigations,
we observed a general trend toward improved metabolic
Structure-Activity Relationships (SAR). The cocrystal
structure of Lck and 1 was solved at 2.0 Å resolution (Figure
). The inhibitor occupies the ATP binding site and forces the
1
8
2
stability, solubility, and oral bioavailability with 2-NHMe-
1
1
protein to assume an extended “DFG-out” conformation. The
quinazoline ring makes hydrogen bond contacts to the linker
region of the enzyme at Met319. The amide moiety makes
hydrogen bond contacts with the backbone NH of Asp382 from
the DFG sequence and Glu288 from the C-helix. The aryl ring
of the amide thus sits deep within the extended hydrophobic
pocket, making several van der Waals contacts to lipophilic
residues. This DFG-out binding mode is not observed for most
quinazolines compared to 2-NH -quinazolines (i.e., 9 vs 1).
2
Furthermore, 2-NHMe-quinazolines generally possessed in vitro
profiles similar to those of their corresponding 2-NH -quinazo-
2
lines. Therefore, most synthetic efforts were focused on
structural modifications to 9. A study of aryl ring substitution
revealed a slight improvement in selectivity with 2,3-disubsti-
tuted amides such as 27 and 28. According to X-ray analysis
of compounds similar to 27 and 28, a substituent on C2 forces
the aryl ring to twist out of plane with respect to the amide
(dihedral angle was ∼20°). Substitution at the amide 4-position
(29 and 30) had little effect on potency and did not improve
1
2
reported Src-family kinase inhibitors such as PP1 and PP2.
We initially sought to improve the selectivity of our lead com-
pound by taking advantage of the sequence differences within

5
674 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
DiMauro et al.
Table 2. SAR: Variations on the Amide Aryl
Figure 3. Cocrystal structure of Lck and 36.
selectivity. 2,3,4-Trisubstituted amides such as 31 were typically
less potent. The 2,5-disubstitution pattern (32-37) was generally
well tolerated with a few exceptions (i.e., 33). X-ray analysis
of compounds in this series bound to Lck, revealed that the
CF3 substituent at C5 was buried in a highly lipophilic pocket,
allowing the substituent at C2 to project toward solvent.
Therefore, we prepared several compounds with tertiary-amine-
containing groups at this position (i.e., 32, 35, and 36) in an
effort to improve solubility. The incorporation of a urea or amide
functionality at C2 afforded compounds with improved selectiv-
ity over KDR and Jak3 (34-37) relative to 9.
X-Ray analysis of compound 36 bound to Lck (Figure 3)
brought to light a plausible reason for the observed selectivity
over KDR (566-fold). The substituent on C2 forces the aryl
ring to twist significantly out of plane with respect to the amide
in order to prevent clashing of the 2-substituent with Glu288
of the C-helix. When these compounds bind to KDR, two
potential van der Waals clashes may occur: (1) between the
aryl ring and a Leu methyl group (equivalent of Met292 in Lck)
and (2) between the CF3 and a Val methyl group (equivalent of
Leu300 in Lck). Both of these apparent clashes result from the
aniline ring rotation upon binding to Lck.
A brief examination of modifications to the central aryl ring
revealed narrow SAR in this portion of the scaffold (Table 3).
A small lipophilic substituent such as methyl or chloro para to
2
the amide (R ) is important for cell potency. Small substituents
3
at R , such as fluoro, were well tolerated but generally did not
improve cellular potency or physicochemical properties. The
narrow SAR was consistent with expectations, given the lack
of space in this region of the binding site (see X-ray structures,
1
1,19
Figures 2 and 3).
We next examined a number of variations to the amide func-
tionality such as reversed amides, ureas, and aminobenzimida-
zoles exemplified by 13-15 (Table 4). Analogues from these
series generally offered no improvement in potency or selectivity
over the lead series (i.e., 9). Truncated analogues such as 45,
lacking the H-bond donor/acceptor moiety and aryl ring to
occupy the extended hydrophobic pocket, were less potent.
The effect of substitution on the linker-binding portion of
the quinazoline is summarized in Table 5. X-Ray crystal-
lographic studies revealed that the quinazolines bind Lck in such
an orientation that a substituent off the 2-amino position would
2
0
presumably be exposed to solvent (Figure 3). As expected,
substantial variation at this position was tolerated with little
effect on potency. Compounds with a lipophilic substituent such
as methyl, phenyl, or cyclopropyl on the amine were highly
potent (9, 54, 55). The introduction of a tertiary-amine-

Aminoquinazolines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5675
Table 5. SAR: Variations on the Quinazoline
Table 3. SAR: Variations on the Central Aryl Ring
Table 4. SAR: Variations on the H-Bonding Moiety
A few of the most potent and/or selective compounds were
advanced into our secondary cellular screening assay, which
measures the compound’s inhibitory effect on the anti-CD3
containing solubilizing group at this position led to a marked
improvement in solubility (10- to 15-fold increase). The H-bond
donor to the linker is important for cellular potency as
demonstrated by a comparison of the data for 49 and 50.
Although compounds 27 and 48, containing 2-Me, 3-CF3
amides, offered improved selectivity relative to their monosub-
stituted analogues 9 and 47, their cellular activity was signifi-
cantly lower. We were delighted to observe improved selectivity
over KDR with some compounds from this series (51-53). We
are presently unable to offer a structural explanation for this
apparent selectivity, but it may be due to differences in
electropotential surfaces in this region of the kinases (Figure
4
). Thus, a positively charged tertiary amine salt is expected to
interact favorably with polar surfaces (red ) oxygen) and
unfavorably with highly lipophilic surfaces (gray ) carbon).
Figure 4. Electrostatic potential surfaces of Lck (a) and KDR (b).

5
676 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
DiMauro et al.
Table 6. Cellular Assay (WB IL-2) and Countersceen (WB TNFR) in
Human Whole Blood and Protein Binding
human
protein
mouse
protein
rat
protein
WB IL-2 WB TNFR
cmpd
(µM)
(µM)
binding (%) binding (%) binding (%)
1
9
0.216
0.250
0.113
2.31
1.16
0.272
8.4
>10.0
>2.5
>2.5
s
>2.5
>2.5
s
98.7
96.2
s
99.4
97.0
s
97.6
97.8
s
3
3
4
4
5
5
2
7
6
7
2
3
s
s
s
99.5
95.0
s
97.9
97.1
s
95.8
95.3
s
7.3
s
s
s
s
Table 7. Mean PK Parameters Following Intravenous Dose (iv) or Oral
Dose (po) in Male Sprague-Dawley Ratsa
iv
po
CL
V
ss
t
1/2
AUC0-∞
C
max
t
max
F
cmpd (mL h- kg-1
1
)
(mL kg-1) (h) (ng h mL-1) (ng mL-1) (h) (%)
1
9
2
6
7
1105b
1641c
1546
3582
7626
3005
5041
2.7
2.3
6.5
2.0
1.8
91e
19
75
121
67
4
4
6
4
3
2
24
55
11
38
e
819
1369
d
f
3
4
4
837
1337c
2881c
305e
668e
128
a
n ) 3 animals per study. b Dosed iv at 2 mg/kg as a solution in DMSO.
c
d
Dosed iv at 1 mg/kg as a solution in DMSO. Dosed iv at 5 mg/kg as a
e
solution in DMSO. Dosed po at 5 mg/kg as a suspension in 1% Tween-
8
0, 2% HPMC, 97% water. f Dosed po at 2 mg/kg as a suspension in 1%
Tween-80, 2% HPMC, 97% water.
Table 8. Kinase Selectivity Profiles of 32 and 47
IC50 (µM)
IC50 (µM)
kinase
32
47
kinase
32
>25
>5
0.868
0.017
0.009
>5
>25
>25
>25
>25
47
Lck
Src
Syk
Tyk2
Itk
0.0005
0.017
1.11
0.0002
0.002
0.292
1.83
JNK2
JNK3
Jak3
KDR
p38R
Aurora1
Aurora2
PKBR
PKAâ
Zap-70
0.173
0.145
0.072
0.001
0.003
2.73
>5
3.33
>25
>25
>25
>10
>25
0.545
>25
Figure 5. Effect of 32 and 47 on anti-CD3 induced IL-2 production
in BALB/c mice. The 12 week old female BALB/c mice were pre-
treated (po) with 32 or 47 at 10, 30, or 100 mg/kg. After 1.0 h the
mice were challenged (iv) with antimouse CD3 monoclonal antibody
(3 mg/mouse). After 90 min of anti-CD3 challenge, blood was collected
via cardiac puncture. IL-2 levels were 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.
Btk
>5
CDK5
MSK1
Pak2
JNK1
>25
>25
>25
>25
>25
>25
0.389
induced production of IL-2 in 50% human whole blood (WB
IL-2). In this assay, compounds 9, 32, and 47 were submicro-
molar inhibitors of IL-2 production (Table 6). Other promising
compounds such as 37, 52, and 53 were less potent inhibitors
of IL-2 production in human whole blood. On the basis of their
inhibitory activity in the p38R kinase assay (<10 nM), potent
compounds 9, 32, and 47 were further counterscreened for
inhibitory activity in the lipopolysaccharide-induced produc-
tion of tumor necrosis factor R in human whole-blood (WB
TNFR). Gratifyingly, potency was marginal in this assay
was observed over a number of structurally diverse kinases
including Syk, Tyk2, Itk, Btk, CDK5, Msk1, Pak2, Jnk1,
Aurora1, Aurora2, PKBR, PKAâ, and Zap70. As expected, these
compounds are not selective over other members of the Src
family, including Src kinase.
Pharmacodynamic Profiles. On the basis of their PK and
selectivity profiles, compounds 32 and 47 were further evaluated
to ascertain in vivo activity upon oral administration at 10, 30,
and 100 mg/kg. Both compounds exhibited statistically signifi-
cant dose-dependent inhibition of anti-CD3-induced IL-2 pro-
duction in female BALB/c mice (Figure 5). Terminal plasma
levels (at 2.5 h) of compounds 32 and 47 were also determined
in these experiments to estimate the levels of exposure at each
dose. At the estimated ED50 values for 32 (22 mg/kg) and 47
(11 mg/kg) the mean terminal plasma concentrations (EC )
(>2.5 µM).
Pharmacokinetic Profiles. On account of their in vitro
potencies, several aminoquinazolines were selected for phar-
macokinetic (PK) profiling. PK data for discrete intravenous
(iv) and oral (po) dosing in male Sprague-Dawley rats are shown
in Table 7. Compound 32 exhibited a low rate of clearance and
a high volume of distribution, while both parameters were
relatively high for compound 47. However, both 32 and 47
exhibited improved oral exposures and bioavailabilities relative
to structurally related compounds from this series (i.e., 1, 9,
and 46).
Selectivity Profile. Prior to in vivo testing, compounds 32
and 47 were screened for inhibitory activity against an extensive
panel of kinases (Table 8).21 Excellent selectivity (>1000-fold)
5
0
were ∼1400 and 630 nM, respectively. These levels are in an
adequate range relative to the in vitro potency of both
compounds in the whole-blood IL-2 assay (IC ) 113 and 272
50
nM, respectively). When adjusted for free fraction based on
mouse protein binding (Table 6), the EC50 (fu) for 47 is ∼18
nM, which is consistent with the in vitro inhibitory activity in
the absence of whole blood (IL-2 IC50 ) 21 nM).

Aminoquinazolines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5677
Conclusions
completion, the heat source was removed and the mixture was
allowed to cool to below 100 °C, then quenched by the addition of
ice (2 kg) and water (4 L). The resulting bronze solid was stirred
for an additional 30 min, then was isolated by vacuum filtration,
washing with water (1 L) and then denatured EtOH (1 L). The
solids were then transferred to a 5 L flask and stirred in denatured
EtOH (2 L) for 2 h before refiltering. Subsequent washes in EtOH
(0.5 L), in a 1:1 mixture of toluene/EtOH (0.5 L), and then in
toluene (0.5 L) and drying afforded the title compound (168 g, 48%)
as a pale-yellow solid. Analytical data were identical to that
previously reported.7
As a result of our screening efforts, aminoquinazoline 1 was
identified as a potent inhibitor of Lck and T cell proliferation.
X-ray crystallographic studies revealed an unusual DFG-out
binding mode, which presumably contributes to the marked
potency. Extensive SAR investigations resulted in a diverse
collection of novel aminoquinazolines and revealed the structural
features that contribute to potency, selectivity, and acceptable
pharmacokinetic properties for this series. Optimized, orally
bioavailable compounds 32 and 47 exhibit anti-inflammatory
activity (ED50 ) 22 and 11 mg/kg, respectively) in the anti-
CD3 induced production of IL-2 in mice. These findings
demonstrate the potential of this highly tractable chemical series
and have paved the way for the advancement of more selective
compounds, which will be reported in due course.
6
-Bromo-N-methylquinazolin-2-amine (3b). A 1 L high-pres-
sure reactor was charged with aminoquinazoline 3a (20.0 g,
9.3 mmol), p-toluenesulfonic acid monohydrate (33.9 g, 179
8
mmol), and methylamine (∼150 g). The reactor was slowly heated
to 150 °C (820 psi, internal pressure). After 24 h, the reactor was
cooled to room temperature, and excess methylamine was slowly
vented. The crude residue was taken up in CH
washed with saturated aqueous NaHCO and brine. The organic
fraction was concentrated in vacuo and purified by silica gel
chromatography (1-3% CH OH in CH Cl ) to afford the title
compound (14.6 g, 68%) as a yellow solid. H NMR (400 MHz,
DMSO-d ): δ 9.07 (s, 1H), 8.05 (d, J ) 2.3 Hz, 1H), 7.77 (dd, J
2 2
Cl (1.0 L) and
Experimental Section
3
Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification. Dry
organic solvents (CH Cl , CH CN, DMF, etc.) were purchased from
2 2 3
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
3
2
2
1
6
) 9.1, 2.3 Hz, 1H), 7.52 (s, 1H), 7.43 (d, J ) 8.8 Hz, 1H), 2.89 (d,
9 8 3
J ) 4.8 Hz, 3H). MS, m/z (C H BrN ): calcd, 237.0; found, 238.0
(MH).
6-Bromo-N-phenylquinazolin-2-amine (3c). In a resealable
Pyrex tube, 6-bromo-2-iodoquinazoline (7) (1.4 g, 4.1 mmol) and
aniline (0.45 mL, 4.9 mmol) were taken up in IPA. 2,2,2-
Trifluoroacetic acid (0.63 mL, 8.2 mmol) was added, and the
tube was sealed. The suspension was stirred at 70 °C overnight.
Then triethylamine (∼1 mL) was added to neutralize the mix-
ture, and the mixture was concentrated. The residue was puri-
(
215 liquid handler), YMC-Pack Pro C18, 150 mm × 30 mm i.d.
column, eluting with a binary solvent system A and B using a
gradient elution (system A consisting of H O with 0.1% TFA;
system B consisting of CH CN with 0.1% TFA) with UV detection
2
3
at 254 nm. Purity was measured using Agilent 1100 series high-
performance liquid chromatography (HPLC) systems with UV
detection at 254 nm (system A consisting of Agilent Zorbax Eclipse
fied by silica gel chromatography (3-10% CH
3
OH in CH
afford the title compound (1.1 g, 88%). H NMR (400 MHz,
DMSO-d
2 2
Cl ) to
1
6
): δ 10.02 (s, 1H), 9.29 (s, 1H), 8.19 (d, J ) 2.5 Hz,
XDB-C8 4.6 mm × 150 mm, 5 µm, 5-100% CH
3
CN in H
2
O with
1H), 7.97 (d, J ) 8.6 Hz, 2H), 7.91 (dd, J ) 8.8, 2.3 Hz, 1H), 7.62
0.1% TFA for 15 min at 1.5 mL/min; system B consisting of Waters
(d, J ) 8.8 Hz, 1H), 7.34 (t, J ) 7.8 Hz, 2H), 7.01 (t, J ) 7.2 Hz,
Xterra 4.6 mm × 150 mm, 3.5 µm, 5-95% CH
3
CN in H
2
O with
1H). MS, m/z (C14
+ 2).
3
H10BrN ): calcd, 300.1; found, 300, 302 (M, M
0.1% TFA for 15 min at 1.0 mL/min). Melting points were obtained
1
using an OptiMelt and are uncorrected. 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 in ppm from the solvent
6-Bromo-N-(2-morpholinoethyl)quinazolin-2-amine (3d). In
a resealable Pyrex tube, iodide 7 (0.130 g, 0.388 mmol) was
dissolved in IPA (3 mL), and DIEA (0.102 mL, 0.582 mmol) and
2-morpholinoethanamine (0.260 mL, 1.94 mmol) were added. The
tube was sealed, and the mixture was heated at 80 °C for 2 h. After
cooling to room temperature, the mixture was concentrated to a
yellow residue and purified by silica gel chromatography (0-20%
resonance (DMSO-d
chemical shift, multiplicity (s ) singlet, d ) doublet, t ) triplet, q
quartet, br ) broad, m ) multiplet), coupling constants, and
6
, 2.49 ppm). Data are reported as follows:
)
number of protons. Mass spectra were obtained on a high-resonance
electrospray time-of-flight mass spectrometer in positive ES ioniza-
tion mode. Combustion analysis was performed by Galbraith
Laboratories, Inc., Knoxville, TN.
CH
3 2 2
OH in CH Cl ) to afford the title compound (0.125 g, 95%) as
1
a yellow amorphous solid. H NMR (400 MHz, DMSO-d
6
): δ
9.08 (s, 1H), 8.05 (d, J ) 2.3 Hz, 1H), 7.78 (dd, J ) 8.8, 2.3 Hz,
1H), 7.40 (d, J ) 8.6 Hz, 2H), 3.54-3.61 (m, 4H), 3.49 (q, J )
6.3 Hz, 2H), 2.51-2.58 (m, 2H), 2.38-2.47 (m, 4H). MS, m/z
3
-(2-Aminoquinazolin-6-yl)-4-methyl-N-(3-(trifluoromethyl)-
phenyl)benzamide (1). The title compound was prepared from acid
8a and 3-(trifluoromethyl)benzenamine using a method analogous
4
(C14H17BrN O): calcd, 337; found, 337, 339 (M, M + 2).
1
6-Bromo-N-(3-morpholinopropyl)quinazolin-2-amine (3e). The
title compound was prepared from iodoquinazoline 7 and 3-mor-
pholinopropan-1-amine using a method analogous to the preparation
to the preparation of compound 46, giving an off-white amorphous
solid. Yield: 0.526 g, 83%. HPLC purity: 99% (system A). H
1
NMR (400 MHz, DMSO-d
6
): δ 10.51 (s, 1H), 9.18 (s, 1H), 8.24
of compound 3d, giving a pale-yellow amorphous solid. Yield: 0.14
1
(
(
8
s, 1H), 8.08 (d, J ) 8.5 Hz, 1H), 7.97 (d, J ) 1.6 Hz, 1H), 7.94
g, 89%. H NMR (400 MHz, DMSO-d
6
): δ 8.97 (s, 1H), 7.94 (d,
dd, J ) 8.0, 1.8 Hz, 1H), 7.87 (d, J ) 1.9 Hz, 1H), 7.78 (dd, J )
J ) 2.3 Hz, 1H), 7.66 (dd, J ) 9.1, 2.3 Hz, 1H), 7.52 (s, 1H), 7.28
.6, 2.0 Hz, 1H), 7.60 (t, J ) 8.0 Hz, 1H), 7.52 (d, J ) 8.3 Hz,
(d, J ) 9.1 Hz, 1H), 3.45-3.50 (m, 4H), 3.29 (q, J ) 6.3 Hz, 2H),
2
H), 7.45 (d, J ) 7.5 Hz, 1H), 6.93 (s, 2H), 2.37 (s, 3H). HRMS
2.23-2.29 (m, 6H), 1.59-1.69 (m, 2H). MS, m/z (C15
4
H19BrN O):
+
(
(
C
23
23
H
H
18
F
F
3
N
N
4
O) : calcd, 423.14272; found, 423.14320. Anal.
O) C, H, N.
calcd, 351; found, 351, 353 (M, M + 2).
C
17
3
4
6-Bromo-N-methyl-N-(3-morpholinopropyl)quinazolin-2-
amine (3f). NaH (0.026 g, 0.640 mmol, 60% dispersion in mineral
oil) was added to a solution quinazoline 3e (0.023 g, 0.640 mmol)
in THF (3.5 mL) and DMF (0.3 mL). After the mixture was stirred
at room temperature for 10 min, MeI (0.04 mL, 0.640 mmol) was
added at once. The mixture was allowed to stir at room temperature
for 3 h and then quenched with water. The mixture was then
6-Bromoquinazolin-2-amine (3a). A stirred mixture of guani-
dine carbonate (281 g, 1.56 mol), NEt(i-Pr) (540 mL, 3.12 mol),
2
and NMP (2 L) was heated to 150-160 °C with a heating mantle.
A solution of aldehyde 5 (250 g, 1.20 mol) in NMP (100 mL) was
added dropwise via addition funnel over 1 h while maintaining
reflux. Upon complete addition, the mixture was maintained at
1
50-160 °C for an additional 1-2 h until consumption of the
extracted several times with CH
were washed with brine, dried over Na
2
Cl
2
. The combined organic extracts
SO , filtered, and concen-
aldehyde was complete as determined by LC analysis. Upon
2
4

5
678 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
DiMauro et al.
trated to afford the crude title compound as an orange solid, which
was used without further purification. MS, m/z (C16H21BrN O):
4
calcd, 365; found, 365 (M).
4-Methyl-N-(4-(1-methylpiperidin-4-yloxy)phenyl)-3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (4d). The title
compound was prepared from acid 8 and 4-(1-methylpiperidin-4-
yloxy)benzenamine using a method analogous to the preparation
of compound 4f and was used without further purification. MS,
6
-Bromo-N-(2-(piperidin-1-yl)ethyl)quinazolin-2-amine (3g).
The title compound was prepared from iodide 7 and 2-(piperidin-
-yl)ethanamine using a method analogous to the preparation of
compound 3d. The crude material was used without further
purification. MS, m/z (C15 ): calcd, 335; found, 335, 337
M, M + 2).
-Bromo-N-(2-(pyrrolidin-1-yl)ethyl)quinazolin-2-amine (3h).
The title compound was prepared from iodide 7 and 2-(pyrrolidin-
-yl)ethanamine using a method analogous to the preparation of
compound 3d and was used without further purification. MS, m/z
): calcd, 321; found, 321 (M).
-Bromo-N-(1-methylpiperidin-4-yl)quinazolin-2-amine (3i).
1
2 4
m/z (C26H35BN O ): calcd, 450.4; found, 451.2 (MH).
N-(4-Chloro-3-(trifluoromethyl)phenyl)-4-methyl-3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (4e). The title
compound was prepared from acid 8 and 4-chloro-3-(trifluoro-
methyl)benzenamine using a method analogous to the preparation
of compound 4f and was used without further purification. MS,
H19BrN
4
(
6
m/z (C21
N-(4-Chloro-2-methyl-3-(trifluoromethyl)phenyl)-4-methyl-3-
4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (4f). Acid
(0.300 g, 1.14 mmol) and SOCl (2.34 mL, 32.0 mmol) were
3 3
H22BClF NO ): calcd, 439.7; found, 440 (MH).
1
(
14 4
(C H17BrN
6
8
2
combined in a resealable Pyrex tube. The tube was sealed and
heated to 60 °C for 1 h. Upon completion (as judged by quenching
an aliquot with methanol and analysis by LCMS), the reaction
mixture was diluted with toluene and the solvent was removed in
The title compound was prepared from iodide 7 and 1-methylpi-
peridin-4-amine using a method analogous to the preparation of
compound 3d, giving a pale-yellow amorphous solid. Yield: 0.072
1
g, 50%. H NMR (400 MHz, DMSO-d
6
): δ 9.08 (s, 1H), 8.03 (d,
2 2
vacuo. The crude acid chloride was dissolved in CH Cl (5.7 mL)
J ) 2.3 Hz, 1H), 7.76 (dd, J ) 9.1, 2.3 Hz, 1H), 7.50 (d, J ) 7.8
in a 25 mL round-bottom flask, and 4-chloro-2-methyl-3-(trifluoro-
Hz, 1H), 7.39 (d, J ) 8.6 Hz, 1H), 3.72-3.85 (m, 1H), 2.76 (d, J
methyl)benzenamine1 (0.286 g, 1.37 mmol) and triethylamine
0c
)
11.6 Hz, 2H), 2.17 (s, 3H), 1.98 (t, J ) 10.9 Hz, 2H), 1.87 (d,
(
∼0.1 mL) were added. The mixture was stirred under nitrogen at-
4
J ) 11.4 Hz, 2H), 1.50-1.62 (m, 2H). MS, m/z (C14H17BrN ):
mosphere at room temperature overnight. The solvent was removed
in vacuo, and the residue was partitioned between ethyl acetate
calcd, 321.0; found, 321 (M).
6-Bromo-N-cyclopropylquinazolin-2-amine (3j). The title com-
3
and saturated aqueous NaHCO . The layers were separated, and
pound was prepared from iodide 7 and cyclopropylamine using a
method analogous to the preparation of compound 3d, giving a
the aqueous layer was extracted with EtOAc. The combined organic
extracts were washed with brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo to afford the title compound,
which was used in the subsequent reaction without further purifica-
1
yellow amorphous solid. Yield: 0.454 g, 77%. H NMR (400 MHz,
DMSO-d
6
): δ 9.09 (s, 1H), 8.07 (d, J ) 2.0 Hz, 1H), 7.74-7.82
m, 2H), 7.45 (d, J ) 8.8 Hz, 1H), 2.80-2.89 (m, 1H), 0.68-0.75
m, 2H), 0.50-0.56 (m, 2H). MS, m/z (C11 ): calcd, 264.0;
(
(
3 3
tion. MS, m/z (C22H24BClF NO ): calcd, 453.7; found, 454.1 (MH).
H10BrN
3
4-Methyl-N-(2-(1-methylpiperidin-4-yloxy)-5-(trifluorometh-
found, 264 (M).
-Methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-
3-(trifluoromethyl)phenyl)benzamide (4a). The title compound
yl)phenyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benza-
mide (4g). The title compound was prepared from acid 8 and 2-(1-
methylpiperidin-4-yloxy)-5-(trifluoromethyl)benzenamine using a
method analogous to the preparation of compound 4f and was used
4
(
was prepared from acid 8 and 3-(trifluoromethyl)benzenamine using
a method analogous to the preparation of compound 4f and was
used without purification. MS, m/z (C21H23BF NO ): calcd, 405.2;
3 3
without further purification. MS, m/z (C27
18.4; found, 519.2 (MH).
-Methyl-N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)-3-
4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (4h). The
title compound was prepared from acid 8 and 2-(piperidin-1-yl)-
-(trifluoromethyl)benzenamine using a method analogous to the
preparation of compound 4f and was used without further purifica-
tion. MS, m/z (C26 ): calcd, 489.4; found, 489.5 (MH).
-Methyl-N-(2-(3-methyl-2-oxoimidazolidin-1-yl)-5-(trifluo-
3 2 4
H34BF N O ): calcd,
5
found, 406.2 (MH).
4
4-Methyl-N-(2-methyl-3-(trifluoromethyl)phenyl)-3-(4,4,5,5-
(
tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (4b). The title
compound was prepared from acid 8 and 2-methyl-3-(trifluoro-
methyl)benzenamine using a method analogous to the preparation
5
of compound 4f and was purified by column chromatography (100%
3 2 3
H33BF N O
1
CH
MHz, DMSO-d
dd, J ) 7.8, 1.8 Hz, 1H), 7.62 (t, J ) 8.1 Hz, 2H), 7.45 (t, J ) 8.1
2
Cl
2
). Mp 155.0-159.0 °C. Yield: 1.53 g, 60%. H NMR (400
4
6
): δ 10.16 (s, 1H), 8.25 (d, J ) 1.8 Hz, 1H), 7.98
romethyl)phenyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzamide (4i). The title compound was prepared from acid 8
and aniline 21 using a method analogous to the preparation of
compound 4f and was used without further purification. MS, m/z
(C H BF N O ): calcd, 503.2; found, 504.2 (MH).
(
Hz, 1H), 7.36 (d, J ) 8.1 Hz, 1H), 2.55 (s, 3H), 2.30 (s, 3H), 1.34
(s, 12H).
N-(2,3-Dihydro-1H-inden-4-yl)-4-methyl-3-(4,4,5,5-tetramethyl-
25
29
3
3
4
1,3,2-dioxaborolan-2-yl)benzamide (4c). N-(2,3-Dihydro-1H-in-
1-(2-(4-Methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl-
)benzamido)-4-(trifluoromethyl)phenyl)-3-(2-morpholinoethyl)-
urea (4j). The title compound was prepared from acid 8 and ani-
line 23, using a method analogous to the preparation of com-
pound 4f, and was used without further purification. MS, m/z
(C H BF N O ): calcd, 576.3; found, 577.3 (MH).
den-4-yl)-3-iodo-4-methylbenzamide was prepared from 3-iodo-4-
methylbenzoic acid and 2,3-dihydro-1H-inden-4-amine using a
method analogous to the preparation of N-(3-iodo-4-methylphenyl)-
3-(trifluoromethyl)benzamide (step 1 of the procedure for the
preparation of 13). It was isolated by trituration with CH Cl and
2
2
28
36
3
4
5
filtration through a B u¨ chner micromembrane apparatus to afford
the crude product as a white solid, which was used without further
purification. MS, m/z (C17H16INO): calcd, 377.2; found, 378 (MH).
N-(2,3-Dihydro-1H-inden-4-yl)-3-iodo-4-methylbenzamide (0.53
g, 1.4 mmol), bis(pinacolato)diboron (0.46 g, 1.8 mmol), KOAc
N-(2-(2-(Diethylamino)acetamido)-5-(trifluoromethyl)phenyl)-
4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benza-
mide (4k). The title compound was prepared from acid 8 and ani-
line 25, using a method analogous to the preparation of com-
pound 4f, and was used without further purification. MS, m/z
(
0.48 g, 4.9 mmol), Pd(dppf)Cl
DMF (4.3 mL) were combined in a sealed tube and heated to 80
C for 8 h. Upon completion (as judged by LCMS), the reaction
mixture was cooled to room temperature, and the solvent was
evaporated in vacuo. The residue was taken up in CH Cl , washed
with water, dried over Na SO , filtered, and concentrated in vacuo
to provide a thick, brown residue. This residue was passed through
a plug of silica (100% CH Cl ) to afford the title compound as a
white amorphous solid, which was used without further purification.
MS, m/z (C23 ): calcd, 377.2; found, 378 (MH).
2
‚CH
2
Cl
2
(0.10 g, 0.14 mmol), and
3 3 4
(C27H35BF N O ): calcd, 533.3; found, 534.2 (MH).
N-(2-(3,3-Dimethyl-2-oxoazetidin-1-yl)-5-(trifluoromethyl)-
phenyl)-4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl-
)benzamide (4l). The title compound was prepared from acid 8
and aniline 26, using a method analogous to the preparation of
compound 4f, and was used without further purification. MS, m/z
°
2
2
2
4
3 2 4
(C26H30BF N O ): calcd, 502.2; found, 503.1 (MH).
2
2
2-Fluoro-4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)-N-(3-(trifluoromethyl)phenyl)benzamide (4m). 5-Bromo-
2-fluoro-4-methyl-N-(3-(trifluoromethyl)phenyl)benzamide was pre-
H28BNO
3

Aminoquinazolines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5679
pared from 5-bromo-2-fluoro-4-methylbenzoic acid22 and 3-(trifluoro-
methyl)benzenamine using a method analogous to the preparation
of N-(3-iodo-4-methylphenyl)-3-(trifluoromethyl)benzamide (step
with water and then three times with saturated NaCl, dried over
Na
brown residue. This material was purified by silica gel chroma-
tography (2-10% CH OH in CH Cl ) to afford the title compound
(1.3 g, 42%) as an off-white amorphous solid. H NMR (400 MHz,
DMSO-d ): δ 6.92 (d, J ) 2.6 Hz, 1H), 6.81 (d, J ) 8.1 Hz, 1H),
6.54 (dd, J ) 8.0, 2.6 Hz, 1H), 4.80 (br s, 2H), 2.27 (s, 3H), 1.27
(s, 12 H). MS, m/z (C13 ): calcd, 233.1; found, 234.1 (MH).
2 4
SO , filtered, and concentrated in vacuo to provide a thick,
1
of the procedure for the preparation of 13). It was purified by
3
2
2
1
silica gel chromatography (2-30% EtOAc in hexanes). Yield:
1
0
8
8
.330 g, 51%. H NMR (400 MHz, DMSO-d
6
): δ 10.75 (s, 1H),
6
.18 (s, 1H), 7.94 (s, 1H), 7.92 (d, J ) 6.7 Hz, 1H), 7.62 (t, J )
.0 Hz, 1H), 7.50-7.46 (m, 2H), 2.42 (s, 3H). MS (ES, negative
H20BNO
2
ion), m/z (C15
H10BrF
4
NO): calcd, 376.2; found, 375 (M - 1).
6-(5-Amino-2-methylphenyl)-N-methylquinazolin-2-amine (12).
Bromide 3b (0.14 g, 0.61 mmol), boronate 11 (0.17 g, 0.73 mmol),
2 M Na CO (0.9 mL, 1.8 mmol), Pd(PPh ) (0.070 g, 0.060 mmol),
2 3 3 4
toluene (5.0 mL), and EtOH (1.0 mL) were combined in a sealed
tube and heated to 80 °C for 4.5 h. Upon completion (as judged
by LCMS), the reaction mixture was cooled to room temperature
and filtered to remove a gray solid. The filtrate was taken up in
The title compound was prepared from 5-bromo-2-fluoro-4-
methyl-N-(3-(trifluoromethyl)phenyl)benzamide, using a method
analogous to the preparation of compound 4c, and was used without
4 3
further purification. MS, m/z (C21H22BF NO ): calcd, 423.2l; found,
4
24 (MH).
-Bromo-2-iodoquinazoline (7). Aminoquinazoline 3a (5.00 g,
2.3 mmol), CuI (4.30 g, 22.3 mmol), and CH (9.0 mL, 114
6
2
2
I
2
CH
and concentrated in vacuo to provide a thick, brown residue. This
material was passed through a plug of silica gel (10% CH OH in
CH Cl ) to afford the title compound as a tan amorphous solid,
2 2 2 4
Cl and washed with water, then dried over Na SO , filtered,
mmol) were dissolved in THF (100 mL), and isoamyl nitrite (85%)
9.0 mL, 68 mmol) was added. After purging with N , the mixture
(
2
3
was heated to reflux for 2.5 h. After cooling to room temperature,
the crude reaction mixture was partitioned between EtOAc (500
mL) and 1 N HCl (500 mL). After separation, the aqueous portion
was extracted three times with EtOAc and the combined organic
2
2
16 4
which was used without further purification. MS, m/z (C16H N ):
calcd, 264.3; found, 265 (MH).
N-(4-Methyl-3-(2-(methylamino)quinazolin-6-yl)phenyl)-3-
(trifluoromethyl)benzamide (13). A solution of 3-iodo-4-methyl-
aniline (0.20 g, 0.86 mmol), 3-(trifluoromethyl)benzoyl chloride
extracts were washed twice with saturated aqueous NH
over Na SO , filtered, and concentrated to a purple oil. The oil
was passed through a plug of silica (100% CH Cl ) and concentrated
to afford the title compound (2.60 g, 35%) as an off-white
4
Cl, dried
2
4
2
2
(0.19 g, 0.13 mL, 0.90 mmol), and CH
room temperature for 30 min to afford an off-white suspension.
NEt (0.11 g, 0.16 mL, 1.1 mmol) was added, and the resulting so-
2 2
Cl (8.0 mL) was stirred at
1
amorphous solid. H NMR (400 MHz, DMSO-d
.48 (d, J ) 2.3 Hz, 1H), 8.18 (dd, J ) 9.0, 2.15 Hz, 1H), 7.91 (d,
J ) 8.8 Hz, 1H). MS, m/z (C BrIN ): calcd, 334.9; found, 335
MH).
-Methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ben-
zoic Acid (8). Iodide 6a (5.00 g, 19.1 mmol), bis(pinacolato)diboron
7.26 g, 28.6 mmol), KOAc (9.36 g, 95.4 mmol), Pd(dppf)Cl
CH Cl (1.30 g, 1.78 mmol), and DMF (100 mL) were combined
6
): δ 9.34 (s, 1H),
3
8
lution was stirred at room temperature for 16 h. The reaction mixture
was concentrated, and the resulting solid was partitioned between
H
8 4
2
(
CH
2
Cl
2
and water. The organic phase was separated and washed
SO , filtered, and concentrated
4
with brine, dried over anhydrous Na
2
4
to afford N-(3-iodo-4-methylphenyl)-3-(trifluoromethyl)benz-
1
(
2
‚
amide (0.31 g, 89%) as an off-white solid. H NMR (400 MHz,
6
DMSO-d ): δ 10.47 (s, 1H), 8.34-8.33 (m, 2H), 8.26 (d, J ) 7.8
2
2
in a sealed tube and heated to 80 °C for 6 h. Upon completion (as
judged by LCMS), the reaction mixture was cooled to room
temperature, and the solvent was evaporated in vacuo. The residue
was dissolved in EtOAc (150 mL) and 2 N HCl (150 mL). The
organic phase was separated, and the aqueous phase was extracted
with EtOAc (2 × 150 mL). The combined organic extracts were
washed with brine, dried over magnesium sulfate, mixed with
activated charcoal, and filtered through a pad of silica gel. The
solvent was removed in vacuo to provide a brown oil. The oil was
dissolved in EtOAc and 2 N NaOH. The organic layer was separated
and extracted with 2 N NaOH (2 × 100 mL). The water layer was
washed with EtOAc until the extracts were clear (6×). The aqueous
layer was acidified to pH 3 with 6 N HCl, and a precipitate formed.
Hz, 1H), 7.98 (d, J ) 8.3 Hz, 1H), 7.80 (t, J ) 7.6 Hz, 1H), 7.73
(dd, J ) 8.3, 1.8 Hz, 1H), 7.33 (d, J ) 8.3 Hz, 1H), 2.36 (s, 3H).
A resealable tube was charged with boronate 17b (0.080 g, 0.26
mmol), N-(3-iodo-4-methylphenyl)-3-(trifluoromethyl)benzamide
(0.11 g, 0.28 mmol), K
and water (0.5 mL). Pd(dppf)Cl
was added, and the system was purged with argon. The tube was
sealed, and the mixture was stirred at room temperature for 18 h.
2
CO
3
(0.06 g, 0.42 mmol), DMF (2.5 mL),
‚CH Cl (0.020 g, 0.030 mmol)
2
2
2
The reaction mixture was partitioned between CH
The aqueous phase was separated and extracted with CH
combined organic phases were washed with brine, dried over
anhydrous Na SO , filtered, and concentrated to afford a brown
oil. This oil was purified via preparative thin layer silica gel chro-
matography (eluting with 95:5:0.5 CH Cl /CH OH/NH OH) to
afford the title compound (0.07 g, 59%) as a tan amorphous solid.
2
Cl
2
and water.
2
Cl . The
2
2
4
The precipitate was filtered and dried to yield the title compound
2
2
3
4
1
(
2.7 g, 54%) as an off-white amorphous solid. H NMR (400 MHz,
1
DMSO-d
6
): δ 12.78 (br s, 1H), 8.23 (d, J ) 1.8 Hz, 1H), 7.89
6
HPLC purity: 96% (system A). H NMR (400 MHz, DMSO-d ):
(dd, J ) 7.9, 1.8 Hz, 1H), 7.30 (d, J ) 8.1 Hz, 1H), 2.52 (s, 3H),
δ 10.53 (s, 1H), 9.21 (s, 1H), 8.36-8.42 (m, 1H), 8.34 (d, J ) 7.6
4
1.31 (s, 12H). MS, m/z (C14H20BO ): calcd, 263.1; found, 263.1
Hz, 1H), 8.03 (d, J ) 8.0 Hz, 1H), 7.76-7.88 (m, 6H), 7.62 (d, J
(MH).
) 8.3 Hz, 1H), 7.39 (d, J ) 9.1 Hz, 1H), 2.98 (d, J ) 4.7 Hz, 3H),
+
4
-Methyl-3-(2-(methylamino)quinazolin-6-yl)-N-(3-(trifluo-
2.33 (s, 3H). HRMS (C24
H
20
F N
3 4
O) : calcd, 437.158 37; found,
romethyl)phenyl)benzamide (9). The title compound was prepared
from boronate 4a and bromide 3b using a method analogous to
the preparation of compound 33. Yield: 0.044 g, 36%. HPLC
437.158 66. Anal. (C24
H
19
F
3
N
4
O‚CH
3
2
OH‚H O) C, H, N.
1-(4-Methyl-3-(2-(methylamino)quinazolin-6-yl)phenyl)-3-(3-
(trifluoromethyl)phenyl)urea (14). 1-Isocyanato-3-(trifluoro-
methyl)benzene (0.0170 mL, 0.119 mmol) was added to a suspen-
sion of aniline 12 (0.030 g, 0.113 mmol) in benzene (2 mL). The
mixture was allowed to stir at room temperature overnight and then
filtered through a micromembrane B u¨ chner apparatus, washing with
benzene to afford the title compound (0.028 g, 56%) as an off-
1
purity: 100% (system A); 100% (system B). H NMR (400 MHz,
DMSO-d
6
): δ 10.50 (s, 1H), 9.13 (s, 1H), 8.21 (s, 1H), 8.06 (d, J
)
8.4 Hz, 1H), 7.94 (s, 1H), 7.90 (d, J ) 8.0 Hz, 1H), 7.89 (s,
1
4
4
H), 7.84 (d, J ) 2.0 Hz, 1H), 7.77-7.42 (m, 5H), 2.90 (d, J )
+
.8 Hz, 3H), 2.34 (s, 3H). HRMS (C24
37.158 37; found, 437.158 80.
20 3 4
H F N O) : calcd,
1
white solid. HPLC purity: 99% (system A). H NMR (400 MHz,
4
-Methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ben-
6
DMSO-d ): δ 9.15 (s, 1H), 9.04 (s, 1H), 8.79 (s, 1H), 8.02 (s,
zenamine (11). Iodide 10 (3.0 g, 13 mmol), bis(pinacolato)diboron
3.6 g, 14 mmol), KOAc (4.4 g, 45 mmol), Pd(dppf)Cl
0.94 g, 1.3 mmol), and DMSO (35 mL) were combined in a sealed
1H), 7.77 (d, J ) 4.0 Hz, 1H), 7.70 (dd, J ) 8.0, 4.0 Hz, 1H),
(
(
2 2
‚CH Cl
2
7.58-7.49 (m, 4H), 7.41-7.22 (m, 4H), 2.92 (s, J ) 4.0 Hz, 3H),
+
2.22 (s, 3H). HRMS (C24
H
21
F N
3 5
O) : calcd, 452.169 27; found,
tube and heated to 80 °C for 10 h. Upon completion (as judged by
LCMS), the reaction mixture was cooled to room temperature, and
the solvent was evaporated in vacuo. The residue was taken up in
EtOAc and filtered to remove a gray solid. The filtrate was washed
452.169 83.
N-Methyl-6-(2-methyl-5-(5-(trifluoromethyl)-1H-benzo[d]imi-
dazol-2-ylamino)phenyl)quinazolin-2-amine (15). 6-(5-Amino-
2-methylphenyl)-N-methylquinazolin-2-amine (0.50 g, 1.9 mmol)

5
680 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
DiMauro et al.
was taken up in CH Cl
2
2
(∼12 mL). To the solution was added O,O-
1-(2-Amino-4-(trifluoromethyl)phenyl)-3-methylimidazolidin-
2-one (21). A resealable Pyrex tube was charged with bromide 20
(1.1 g, 4.5 mmol), 1-methylimidazolidin-2-one (0.59 g, 5.8 mmol),
dipyridin-2-yl carbonothioate (0.44 g, 1.9 mmol). The light-brown
solution was stirred at room temperature for 3 h. The crude reaction
mixture was filtered through a B u¨ chner apparatus with a micro-
2 3
K CO (1.23 g, 9.0 mmol), and CuI (0.85 g, 0.45 mmol). To the
1
2
membrane filter, and the filtrate was washed with CH
to afford 6-(5-isothiocyanato-2-methylphenyl)-N-methylquinazolin-
-amine as a pale-yellow powder (crop 1). MS, m/z (C17 S):
calcd, 306.4; found, 307 (MH). The mother liquors were washed
with water, then dried over Na SO , filtered, and concentrated to
Cl
2 2
and dried
mixture was added toluene (3.0 mL) and N ,N -dimethylethane-
1,2-diamine (96 µL, 0.90 mmol). The tube was sealed and heated
at 110 °C for 24 h. After cooling to room temperature, the mixture
was diluted with EtOAc and washed with water and brine. The
2
14 4
H N
2
4
organic fraction was dried with Na
and purified by silica gel chromatography (5-10% CH
2 2
CH Cl
2
SO
4
, concentrated in vacuo,
OH in
) to afford the title compound (0.70 g, 60%). H NMR (400
MHz, DMSO-d ) δ 7.15 (d, J ) 8.1 Hz, 1H), 7.04 (s, 1H), 6.83 (d,
afford 6-(5-isothiocyanato-2-methylphenyl)-N-methylquinazolin-2-
amine as a yellow-tan solid (crop 2). Crops 1 and 2 were used
without further purification.
3
1
6
In a 16 mm × 120 mm resealable Pyrex tube 6-(5-isothiocyanato-
J ) 8.1 Hz, 1H), 5.46 (s, 2H), 3.63-3.53 (m, 2H), 3.48-3.41 (m,
2H), 2.75 (s, 3H). MS, m/z (C11H F N O): calcd, 259.1; found,
12 3 3
2
-methylphenyl)-N-methylquinazolin-2-amine (0.080 g, 0.26 mmol),
PS-DCC (0.49 g, 0.78 mmol), and 4-(trifluoromethyl)-1,2-phen-
ylenediamine (0.070 g, 0.39 mmol) were taken up in THF (6 mL).
The tube was sealed, and the mixture was stirred at 70 °C overnight.
After cooling, the crude reaction mixture was filtered through a
260.2 (MH).
1-(2-Amino-4-(trifluoromethyl)phenyl)-3-(2-morpholino-
ethyl)urea (23). To a solution of isocyanate 22 (0.735 g, 3.17
mmol) in benzene (10 mL) at room temperature was added
2-morpholinoethanamine (0.433 g, 3.33 mmol). The resulting
yellow mixture was heated at 40 °C for 16 h. After the mixture
was cooled to room temperature, the yellow precipitate was removed
by filtration and rinsed with hexanes. The crude 1-(2-morpholino-
ethyl)-3-(2-nitro-4-(trifluoromethyl)phenyl)urea was advanced with-
out further purification. MS, m/z (C14H F N O ): calcd, 362.1;
17 3 4 4
found, 363.1 (MH).
To a solution of crude 1-(2-morpholinoethyl)-3-(2-nitro-4-(trifluoro-
methyl)phenyl)urea (0.970 g, 2.68 mmol) in CH OH (30 mL) and
medium glass frit, and PS-DCC was washed with CH
Concentration and purification by silica gel chromatography
2 2
Cl .
(
3-80% 90:10:1 CH
2 2 2 2
Cl in CH Cl ) afforded the title compound
(0.070 g, 58%) as a pale-yellow solid. HPLC purity: 99% (system
1
A). H NMR (400 MHz, DMSO-d
s, 0.5H) [NH-tautomers], 9.73 (s, 0.5 H) and 9.68 (s, 0.5 H) [NH-
tautomers], 9.16 (s, 1H), 7.81 (s, 1H), 7.71 (m, 3H), 7.57 (m, 2H),
.42 (m, 2H), 7.30 (m, 2H), 2.93 (d, J ) 4.2 Hz, 3H), 2.23 (s, 3H).
6
): δ 11.33 (s, 0.5H) and 11.20
(
7
+
HRMS (C24
H F N
20 3 6
) : calcd, 449.169 61; found, 449.169 39. Anal.
O) H, N. C: calcd, 61.80; found, 62.34.
-(4,4,5,5-Tetramethyl-1,3,2-dioxaboralan-2-yl)quinazolin-2-
amine (17a). A pressure flask was charged with Pd (dba) (0.082
g, 0.089 mmol), (o-biphenyl)PCy (0.094 g, 0.270 mmol), and
3
(C
24
H
19
F N ‚H
3 6 2
EtOAc (10 mL) was added 10% Pd/C (0.400 g). The mixture was
6
exposed to an atmosphere of H
was filtered, concentrated, and purified by silica gel chromatography
(5-10% CH OH in CH Cl ) to afford the title compound (0.650
g, 73%). H NMR (400 MHz, DMSO-d ) δ 7.93 (s, 1 H), 7.68 (d,
2
(balloon). After 24 h, the mixture
2
3
2
3
2
2
1
dioxane (10 mL). The flask was purged with argon, and the mixture
was stirred for 10 min at ambient temperature. To this stirred
solution was added 6-bromoquinazolin-2-amine (3a) (1.0 g, 4.5
mmol), bis(pinacolato)diboron (1.4 g, 5.4 mmol), KOAc (0.70 g,
6
J ) 8.3 Hz, 1H), 6.99 (s, 1H), 6.83 (d, J ) 8.7 Hz, 1H), 6.30 (t,
J ) 5.2 Hz, 1H), 5.11 (s, 2H), 3.62-3.55 (m, 4H), 3.18-3.25 (m,
2H), 2.42-2.35 (m, 6H). MS, m/z (C14
found, 333.1 (MH).
19 3 4 2
H F N O ): calcd, 332.2;
7.1 mmol), and dioxane (10 mL). The mixture was again purged
with argon, and the flask was sealed and heated to 80 °C for a
period of 15 h. The reaction mixture was then cooled to ambient
temperature and filtered through Celite, and the cake was washed
N-(2-Amino-4-(trifluoromethyl)phenyl)-2-(diethylamino)-
acetamide (25). To aniline 24 (7.00 g, 34.0 mmol) in CH Cl (300
mL) at 0 °C was added chloroacetyl chloride (3.80 mL, 47.6 mmol)
followed by NEt (9.50 mL, 68.0 mmol). The solution was heated
2
2
2 2
with CH Cl . The filtrate was concentrated in vacuo, and the crude
3
solid was triturated with hexanes to afford the title compound as
an orange solid, which was used without further purification. H
at reflux for 48 h. After the mixture was cooled to room temperature,
water was added, and the organic fraction was washed with brine,
1
NMR (400 MHz, DMSO): δ 9.17 (s, 1H), 8.17 (s, 1H), 7.85 (d, J
dried with Na
was purified by silica gel chromatography (70-100% hexanes/
CH Cl ) to afford 2-chloro-N-(2-nitro-4-(trifluoromethyl)phenyl)-
acetamide (9.10 g, 95%). H NMR (400 MHz, DMSO-d
(s, 1H), 8.11-8.17 (m, 1H), 8.03-8.10 (m, 1H), 4.44 (s, 2H). MS,
m/z (C ClF ): calcd, 282.0; found, 283.1 (MH).
2 4
SO , filtered, and concentrated in vacuo. The residue
)
8.4 Hz, 1H), 7.36 (d, J ) 8.4 Hz, 1H), 7.06 (br s, 2H), 1.31 (s,
3 2
12H). MS, m/z (C14H18BN O ): calcd, 271.1; found, 272.1 (MH).
2
2
1
N-Methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
quinazolin-2-amine (17b). The title compound was prepared from
bromide 3b using a method analogous to the preparation of
6
) δ 8.36
9
H
6
3 2 3
N O
compound 17a, giving an orange amorphous solid. Yield: 1.15 g,
To a solution of 2-chloro-N-(2-nitro-4-(trifluoromethyl)phenyl)-
acetamide (0.750 g, 2.65 mmol) in THF (15 mL) was added
K CO (0.730 g, 5.30 mmol) and diethylamine (0.30 mL, 2.90
2 3
mmol). After heating at 50 °C for 16 h, the mixture was partitioned
1
9
1
6%. H NMR (400 MHz, DMSO-d
H), 7.70 (d, J ) 8.59 Hz, 1H), 7.39 (s, 1H), 7.28 (s, 1H), 2.74 (d,
6
): δ 8.98 (s, 1H), 8.01 (s,
J ) 4.7 Hz, 3H), 1.16 (s, 12H).
-(2-Amino-6-quinazolinyl)-4-methylbenzoic Acid (18a). A
flask was charged with boronate 17a (0.50 g, 1.8 mmol), 3-iodo-
-methylbenzoic acid (0.39 g, 1.5 mmol), Pd(dppf)Cl ‚CH Cl (0.12
g, 0.14 mmol), and K CO (0.32 g, 2.4 mmol). The flask was then
3
between EtOAc and water. The organic fraction was washed with
brine, dried with Na
was taken up in CH OH (10 mL), and a slurry of 10% Pd/C (0.200
g) in EtOAc (2 mL) was added. After exposure to an atmosphere
of H (balloon) for 24 h, the mixture was filtered and concentrated
to afford the crude title compound, which was advanced without
2 4
SO , and concentrated in vacuo. The residue
4
2
2
2
3
2
3
charged with DMF (14 mL) and water (4 mL), and the solution
was purged with argon for a period of 10 min. The solution was
stirred at ambient temperature for a period of 15 h, then filtered
through a B u¨ chner funnel, and washed with EtOAc. The filtrate
2
18 3 3
further purification. MS, m/z (C13H F N O): calcd, 289.1; found,
290.2 (MH).
was concentrated in vacuo and then redissolved in EtOAc/H
1:1; 30 mL). The aqueous layer was separated and washed with
2
O
1-(2-Amino-4-(trifluoromethyl)phenyl)-3,3-dimethylazetidin-
2-one (26). To a solution of aniline 24 (3.00 g, 14.9 mmol) in
(
EtOAc and then acidified to pH ∼3 with 1 N HCl. The fine tan
CH
2
Cl
2
(75 mL) was added 3-chloropivaloyl chloride (2.52 g,
(4.15 mL, 29.8 mmol). The solu-
precipitate obtained was centrifuged in 250 mL ultracentrifuge vials
19.4 mmol) followed by NEt
3
(
at 3000 rpm for 15 min) and then washed with 2 × 25 mL of dis-
tion was heated at 35 °C for 24 h before cooling to room
temperature and adding water (100 mL). The organic layer was
separated, washed with brine, and dried with Na SO . After
2 4
concentration in vacuo, the crude 3-chloro-2,2-dimethyl-N-(2-nitro-
tilled water. After the aqueous washes were decanted, the resulting
light-tan solid was lyophilized for a period of 15 h to yield the
title compound as a light-tan solid (0.28 g, 70%). H NMR (400
1
MHz, DMSO): δ 9.18 (s, 1H), 7.82 (m, 3H), 7.73 (dd, J ) 1.6,
4-(trifluoromethyl)phenyl)propanamide was advanced without fur-
ther purification. MS, m/z (C12H12ClF N O ): calcd, 324.1; found,
3 2 3
325.1 (MH).
2
.0 Hz, 1H), 7.47 (dd, J ) 8.8, 8.0 Hz, 2H), 7.12 (br s, 2H), 2.32
(
s, 3H). MS, m/z (C16
H
13
3 2
N O ): calcd, 279.1; found, 280.1 (MH).

Aminoquinazolines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5681
A mixture of 3-chloro-2,2-dimethyl-N-(2-nitro-4-(trifluorometh-
provided the title compound (0.078 g, 57%). HPLC purity: 98%
1
yl)phenyl)propanamide (3.89 g, 12.0 mmol) and K
9.0 mmol) in acetone (100 mL) was heated to 50 °C. After 72 h,
the mixture was filtered and concentrated in vacuo. The residue
was taken up in CH OH (25 mL), and a slurry of 10% Pd/C (0.200
g) in EtOAc (5 mL) was added. After exposing the mixture to an
atmosphere of H (balloon) for 24 h, the mixture was filtered and
2
CO
3
(4.00 g,
(system A). H NMR (400 MHz, DMSO-d
6
): δ 10.27 (s, 1H), 9.16
2
(s, 1H), 7.96 (s, 1H), 7.93 (dd, J ) 8.2, 1.7 Hz, 1 H), 7.86 (d, J )
1.8 Hz, 1H), 7.76 (dd, J ) 8.7, 2.0 Hz, 1H), 7.58 (m, 3H), 7.52 (d,
3
J ) 8.0 Hz, 1H), 7.42 (br s, 1H), 2.93 (d, J ) 4.7 Hz, 3H), 2.37 (s,
+
3H), 2.35 (q, J ) 3.3 Hz, 3H). HRMS (C25
H21ClF
3
N
4
O) : calcd,
2
485.135 05; found, 485.134 99. Anal. (C25
H20ClF
3
N
4
O) C, H, N.
concentrated in vacuo. The residue was purified by silica gel
4-Methyl-3-(2-(methylamino)quinazolin-6-yl)-N-(2-(1-meth-
ylpiperidin-4-yloxy)-5-(trifluoromethyl)phenyl)benzamide (32).
The title compound was prepared from boronate 4g and bromide
3b, using a method analogous to the preparation of compound 33.
chromatography (10-20% hexanes/EtOAc) to afford the title
1
compound (1.75 g, 35% over three steps). H NMR (400 MHz,
DMSO-d
.0 Hz, 1H), 5.93 (s, 2H), 3.58 (s, 2H), 1.31 (s, 6H). MS, m/z
O): calcd, 258.1; found, 259.1 (MH).
-Methyl-N-(2-methyl-3-(trifluoromethyl)phenyl)-3-(2-
methylamino)quinazolin-6-yl)benzamide (27). The title com-
6
) δ 7.09 (d, J ) 8.2 Hz, 1H), 7.05 (s, 1H), 6.86 (d, J )
1
7
Yield: 0.435 g, 63%. HPLC purity: 99% (system A). H NMR
(C H
12 13
F N
3 2
(400 MHz, DMSO-d ): δ 9.60 (s, 1H), 9.14 (s, 1H), 8.27 (d, J )
6
4
1.8 Hz, 1H), 7.86-7.91 (m, 3H), 7.78 (dd, J ) 8.8, 1.8 Hz, 1H),
(
7.58 (d, J ) 7.8 Hz, 1H), 7.48-7.55 (m, 2H), 7.44 (s, 1H), 7.33
(d, J ) 8.6 Hz, 1H), 4.66 (s, 1H), 2.93 (d, J ) 4.8 Hz, 3H), 2.40-
pound was prepared from boronate 4b and bromide 3b, using a
method analogous to the preparation of compound 33. Yield: 0.087
2.48 (m, 2H), 2.38 (s, 3H), 2.15-2.26 (m, 2H), 1.99 (s, 3H), 1.85-
1
+
g, 49%. HPLC purity: 99% (system A). H NMR (400 MHz,
1.94 (m, 2H), 1.71-1.80 (m, 2H). HRMS (C30
H
31
F
3
N
5
O
2
) : calcd,
DMSO-d
8.0 Hz, 1H), 7.86 (d, J ) 1.8 Hz, 1H), 7.78 (dd, J ) 8.6, 1.9
Hz, 1H), 7.62 (m, 3H), 7.51 (d, J ) 8.0 Hz, 1H), 7.45 (m, 2H),
6
): δ 10.17 (s, 1H), 9.16 (s, 1H), 7.97 (s, 1H), 7.93 (d, J
550.24244; found: 550.24249. Anal. (C30
H. N: calcd, 12.04; found, 11.48.
H
30
F
3
N
5
O
2
3
‚CH OH) C,
)
4-Methyl-3-(2-(methylamino)quinazolin-6-yl)-N-(2-(piperidin-
1-yl)-5-(trifluoromethyl)phenyl)benzamide (33). To a resealable
glass tube was added boronate 4h (0.30 g, 0.61 mmol), bromide
3b (0.097 g, 0.41 mmol), and K CO (0.34 g, 2.5 mmol), followed
2
.93 (d, J ) 4.8 Hz, 3H), 2.37 (s, 3H), 2.32 (s, 3H). HRMS
+
(C
25
H
H
22
F
F
3
N
N
4
O) : calcd, 451.174 02; found, 451.174 33. Anal.
O) H, N. C: calcd, 66.66; found, 66.16.
(C
25
21
3
4
2
3
N-(2,3-Dihydro-1H-inden-4-yl)-4-methyl-3-(2-(methylamino)-
quinazolin-6-yl)benzamide (28). The title compound was prepared
from boronate 4c and bromide 3b, using a method analogous to
by CH
3
CN (3 mL) and H
2
O (1 mL). Pd(dppf)Cl
2
2 2
‚CH Cl (0.030
g, 0.041 mmol) was then added, and the tube was sealed. The
mixture was heated to 60 °C for 3 h. After cooling to room
the preparation of compound 33. Yield: 0.094 g, 55%. HPLC pur-
temperature, the mixture was partitioned between CH
and the aqueous phase was extracted with CH Cl . The combined
organic phases were dried over MgSO , and the solvent was
evaporated. The crude product was purified on the Gilson MPLC
system, using CH CN/H O/0.1% TFA as the solvent system. The
product fractions were collected and basified with saturated aqueous
NaHCO . The product was extracted with CH Cl , and the or-
ganic layers were dried over MgSO , filtered, and concentrated in
2 2
Cl and brine,
1
ity: 99% (system A). H NMR (400 MHz, DMSO-d
6
): δ 9.94 (s,
2
2
1
H), 9.15 (s, 1H), 7.92-7.86 (m, 3H), 7.77 (d, J ) 8.6, 1H), 7.57
d, J ) 8.9 Hz, 1H), 7.48 (d, J ) 8.1 Hz, 1H), 7.42 (br s, 1 H),
.20 (d, J ) 8.0 Hz, 1H), 7.13 (t, J ) 7.4 Hz, 1H), 7.09 (d, J ) 8.0
Hz, 1H), 2.92-2.88 (m, 5H), 2.81 (t, J - 7.4 Hz, 2H), 2.36 (s,
4
(
7
3
2
+
3
H), 2.00-1.90 (m, 2H). HRMS (C26
found, 409.202 17. Anal. (C26 O‚0.5H
-Methyl-3-(2-(methylamino)quinazolin-6-yl)-N-(4-(1-meth-
H
25
N
4
O) : calcd, 409.202 29;
2
3
2
2
H
24
N
4
O) C, H, N.
4
4
vacuo to afford the title compound (0.09 g, 27%) as an amorphous
1
ylpiperidin-4-yloxy)phenyl)benzamide (29). The title compound
was prepared from boronate 4d and bromide 3b, using a method
yellow solid. HPLC purity: 97% (system A). H NMR (400 MHz,
DMSO-d ): δ 9.72 (s, 1H), 9.15 (s, 1H), 8.44 (s, 1H), 7.90 (d, J )
6
analogous to the preparation of compound 33. Yield: 0.697 g, 81%.
8.7 Hz, 1H), 7.87 (s, 2H), 7.81 (dd, J ) 8.7, 2.0 Hz, 1H), 7.58 (t,
J ) 7.9 Hz, 2H), 7.49 (dd, J ) 8.5, 2.0 Hz, 1H), 7.40 (d, J ) 8.3
1
HPLC purity: 100% (system A). H NMR (400 MHz, DMSO-d
6
):
δ 10.1 (s, 1H), 9.16 (s, 1H), 7.93-7.85 (m, 3H), 7.78 (d, J ) 8.8
Hz, 1H), 7.65 (d, J ) 8.8 Hz, 2H), 7.58 (d, J ) 9.8 Hz, 1H), 7.48
Hz, 1H), 2.94-2.87 (m, 8H), 2.40 (s, 3H), 1.66 (m, 4H), 1.50 (m,
+
2H). HRMS (C29
H
29
F
3
N
5
O) : calcd, 520.2318; found, 520.231 71.
(
(
3
d, J ) 8.1 Hz, 1H), 7.41 (s, 1H), 6.93 (d, J ) 8.6 Hz, 2H), 4.31
s, 1H), 2.93 (d, J ) 4.8 Hz, 3H), 2.66-2.57 (m, 3H), 2.36 (s,
Anal. (C29
H F
28 3
5 3
N O‚0.5CH OH) C, H, N.
4-Methyl-N-(2-(3-methyl-2-oxoimidazolidin-1-yl)-5-(tri-
fluoromethyl)phenyl)-3-(2-(methylamino)quinazolin-6-yl)-
benzamide (34). The title compound was prepared from boronate
4i and bromide 3b, using a method analogous to the preparation of
H), 2.22-2.15 (m, 4H), 1.88-1.96 (m, 2H), 1.57-1.67 (m, 2H).
+
HRMS (C29
H N O
32 5 2
) : calcd, 482.255 05; found, 482.256 53. Anal.
OH) H, N. C: calcd, 68.23; found, 68.82.
N-(4-Chloro-3-(trifluoromethyl)phenyl)-4-methyl-3-(2-
methylamino)quinazolin-6-yl)benzamide (30). The title com-
(
C
H
29 31
N O
5
2
‚2CH
3
compound 33. Yield: 0.135 g, 81%. HPLC purity: 100% (system
1
(
A). H NMR (300 MHz, DMSO-d
6
): δ 10.53 (s, 1H), 9.14 (s, 1H),
pound was prepared from boronate 4e and bromide 3b, using a
method analogous to the preparation of compound 33. Yield: 0.061
g, 33%. HPLC purity: 99% (system A). 1HNMR (400 MHz,
8.24 (s, 1H), 7.89-7.43 (m, 9H), 3.90 (t, J ) 7.7 Hz, 2H), 3.49 (t,
J ) 7.7 Hz, 2H), 2.92 (d, J ) 4.4 Hz, 3H), 2.71 (s, 3H), 2.37 (s,
+
3H). HRMS (C28
H
26
F
3
N
6
O
2
) : calcd, 535.206 39; found, 535.206 16.
DMSO-d
6
): δ 10.6 (s, 1H), 9.16 (s, 1H), 8.36 (d, J ) 2.2 Hz,
Anal. (C28
H
25
F
3
N
6
O
2
2
‚H O) H, N. C: calcd, 60.86; found, 61.34.
1
9
8
7
H), 8.15 (dd, J ) 8.5, 2.0 Hz, 1H), 7.96 (s, 1H), 7.93 (d, J )
1-(2-(4-Methyl-3-(2-(methylamino)quinazolin-6-yl)benzamido)-
4-(trifluoromethyl)phenyl)-3-(2-morpholinoethyl)urea (35). The
title compound was prepared from boronate 4j and bromide 3b,
using a method analogous to the preparation of compound 33.
Yield: 0.055 g, 30%. HPLC purity: 100% (system A).
NMR (300 MHz, DMSO-d
.4 Hz, 1H), 7.86 (s, 1H), 7.78 (d, J ) 8.0 Hz, 1H), 7.72 (d, J )
.8 Hz, 1H), 7.58 (d, J ) 8.5 Hz, 1H), 7.53 (d, J ) 7.8 Hz, 1H),
.42 (br s, 1H), 2.93 (d, J ) 4.7 Hz, 3H), 2.37 (s, 3H). HRMS
+
1
(C
24
H
19ClF
18ClF
3
N
N
4
O) : calcd, 471.119 40; found, 471.118 95. Anal.
H
(C
24
H
3
4
O‚H
2
O) C, N. H: calcd, 4.12; found, 3.69.
6
): δ 10.20 (s, 1H), 9.12 (s, 1H), 8.32
N-(4-Chloro-2-methyl-3-(trifluoromethyl)phenyl)-4-methyl-3-
(s, 1H), 8.15 (d, J ) 5.5 Hz, 1H), 8.00-7.91 (m, 2H), 7.90-7.65
(
(
(
2-(methylamino)quinazolin-6-yl)benzamide (31). Boronate 4f
0.128 g, 0.283 mmol), 6-bromo-N-methylquinazolin-2-amine
0.074 g, 0.31 mmol), Pd(dppf)Cl
(m, 3H), 7.61-7.42 (m, 4H), 6.91 (br s, 1H), 3.49 (m, 4H), 3.15
(m, 2H), 2.87 (d, J ) 4.5 Hz, 3H), 2.38-2.20 (m, 9H). HRMS
+
2
‚CH Cl
2
(0.021 g, 0.028 mmol),
(C31
(C31
H
H
33
F
F
3
N
N
7
O
O
3
) : calcd, 608.259 15; found, 608.258 79. Anal.
2
Na
2
CO (0.43 mL, 2.0 M solution in water), and dioxane (1.9 mL)
3
32
3
7
3 2
‚H O) C, H, N.
were combined in a resealable tube. The tube was sealed and heated
to 80 °C overnight. Upon completion (as judged by LCMS), the
reaction mixture was diluted with ethyl acetate and water. The
aqueous layer was separated and extracted twice with ethyl acetate.
The combined organic extracts were washed with brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. Silica gel
N-(2-(2-(Diethylamino)acetamido)-5-(trifluoromethyl)phenyl)-
4-methyl-3-(2-(methylamino)quinazolin-6-yl)benzamide (36). The
title compound was prepared from boronate 4k and bromide 3b,
using a method analogous to the preparation of compound 33.
1
Yield: 0.060 g, 25%. HPLC purity: 100% (system A). H NMR
(300 MHz, DMSO-d
8.24 (s, 1H), 8.02-7.82 (m, 2H), 7.77-7.61 (m, 3H), 7.51-7.35
6
): δ 9.84 (s, 1H), 9.69 (s, 1H), 8.99 (s, 1H),
2 2 3 4 2 2
chromatography (90:10:1 CH Cl /CH OH/NH OH in CH Cl )

5
682 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
DiMauro et al.
(
2
5
m, 3H), 5.36 (br s, 1H), 3.25-3.09 (m, 5H), 2.70-2.55 (m, 4H),
1% NEt
3
in EtOAc) provided the title compound (0.030 g, 68%)
+
.38 (s, 3H), 1.11-0.96 (m, 6H). HRMS (C30
H
32
F
3
N
6
O
2
) : calcd,
as a yellow solid. HPLC purity: 99.3% (system A); 99.7% (sys-
1
65.253 34; found, 565.253 32. Anal. (C30
H F
31 3
6 2
N O ) H, N. C:
6
tem B). H NMR (400 MHz, DMSO-d ): δ 10.67 (s, 1H), 9.21 (s,
calcd, 63.82; found, 63.01.
1H), 8.33 (s, 1H), 8.27 (s, 1H), 8.23 (s, 1H), 8.15-8.09 (m, 2H),
N-(2-(3,3-Dimethyl-2-oxoazetidin-1-yl)-5-(trifluoromethyl)-
phenyl)-4-methyl-3-(2-(methylamino)quinazolin-6-yl)benz-
amide (37). The title compound was prepared from boronate 4l
and bromide 3b using a method analogous to the preparation of
compound 33. Yield: 0.09 g, 56%. HPLC purity: 100% (system
A). H NMR (400 MHz, DMSO-d
8.01-7.95 (m, 2H), 7.70-7.47 (m, 4H), 6.97 (s, 2H). HRMS
+
(C22
H
16
F
3
N
4
O) : calcd, 409.127 07; found, 409.127 013.
5-(2-Aminoquinazolin-6-yl)-2-fluoro-N-(3-(trifluoromethyl)-
phenyl)benzamide (40). 2-Fluoro-5-iodo-N-(3-(trifluoromethyl)-
phenyl)benzamide was prepared from 2-fluoro-5-iodobenzoic acid,
using a method analogous to the preparation of 3-iodo-N-(3-
1
6
) δ 10.63 (s, 1H), 9.14 (s, 1H),
1
8
.13 (s, 1H), 7.95-7.84 (m, 3H), 7.82-7.76 (m, 1H), 7.75-7.71
(trifluoromethyl)phenyl)benzamide. Yield: 2.4 g, 52%. H NMR
(
(
(
(
m, 1H), 7.65-7.50 (m, 3H), 7.44 (br s, 1H), 3.66 (s, 2H), 2.92
3
(400 MHz, CDCl ): δ 8.50 (dd, J ) 7.3, 2.3 Hz, 1H), 8.45 (s,
d, J ) 4.6 Hz, 3H), 2.38 (s, 3 H), 1.29 (s, 6H). HRMS
1H), 7.96 (s, 1H), 7.87-7.83 (m, 2H), 7.52 (dd, J ) 7.7, 7.7 Hz,
1H), 7.45 (d, J ) 7.8 Hz, 1H), 7.00 (dd, J ) 11.8, 8.5 Hz, 1H).
+
C
C
29
H
H
27
F
F
3
N
N
5
O
O
2
) : calcd, 534.211 14; found, 534.211 34. Anal.
29
26
3
5
2
‚CH
3
OH) C, N. H: calcd, 5.56; found, 5.06.
8 4
MS, m/z (C14H F INO): calcd, 409.12; found, 409.9 (MH).
3
-(2-(Methylamino)quinazolin-6-yl)-N-(3-(trifluoromethyl)-
phenyl)benzamide (38). The title compound was prepared from
-((3-(trifluoromethyl)phenyl)carbamoyl)phenylboronic acid and
A mixture of 2-fluoro-5-iodo-N-(3-(trifluoromethyl)phenyl)benz-
amide (0.095 g, 0.23 mmol), boronate 17a (0.066 g, 0.23 mmol),
Pd(PPh ) (0.013 g, 0.010 mmol), Na CO (2.0 M solution in water,
3 4 2 3
3
bromide 3b using a method analogous to the preparation of
compound 39. Purification by silica gel chromatography (gradient,
0.35 mL, 0.69 mmol), 0.5 mL of EtOH, and 3 mL of toluene was
heated in a sealed tube at 80 °C for 16 h. The mixture was cooled
to ambient temperature and was added to EtOAc and water. The
aqueous layer was extracted once with EtOAc. The combined
5
-9% acetone/CH
2 2
Cl ) provided the title compound as a light-
yellow amorphous solid. Yield: 0.024 g, 48%. HPLC purity: 100%
1
(system A); 99.7% (system B). H NMR (400 MHz, DMSO-d
6
):
organic layers were washed once with brine, dried with MgSO
filtered, and concentrated. Purification by silica gel chromatography
(7/7/7/1 CH Cl /hexanes/t-BuOMe/CH OH/NH OH) provided the
title compound (0.057 g, 58%) as an orange solid. HPLC purity:
4
,
δ 10.67 (s, 1H), 9.19 (s, 1H), 8.35 (s, 1H), 8.28 (s, 1H), 8.24 (s,
1
2
4
H), 8.17-8.09 (m, 2H), 8.02-7.95 (m, 2H), 7.70-7.45 (m, 5H),
2
2
3
4
+
.92 (d, J ) 4.4 Hz, 3H). HRMS (C23
18 3 4
H F N O) : calcd,
1
23.142 72; found, 423.142 96.
3
95% (system A). H NMR (300 MHz, CDCl ): δ 9.12 (s, 1H),
3
-(2-Aminoquinazolin-6-yl)-N-(3-(trifluoromethyl)phenyl)-
benzamide (39). To a stirring suspension of 3-iodobenzoic acid
10.0 g, 40.3 mmol) in CH Cl (300 mL) at 0 °C was added 3
8.65 (d, J ) 16.5 Hz, 1H), 8.50 (dd, J ) 7.4, 2.5 Hz, 1H), 8.05-
7.95 (m, 3H), 7.90-7.81 (m, 2H), 7.69 (d, J ) 8.8 Hz, 1H), 7.54
(
2
2
(dd, J ) 7.9 Hz, 1H), 7.46 (d, J ) 8.0 Hz, 1H), 7.35 (dd, J ) 11.9,
+
drops of DMF followed by oxalyl chloride (3.52 mL, 40.3 mmol).
The mixture was allowed to warm to room temperature over several
hours and was allowed to stir overnight. The solvent was removed
in vacuo, and the residue was taken up in THF (200 mL).
8.6 Hz, 1H), 5.27 (s, 2H). HRMS (C22
H
15
F
4
N
4
O) : calcd,
14 4 4
427.117 65; found, 427.117 62. Anal. (C22H F N O) C, H, N.
3-(2-Aminoquinazolin-6-yl)-4-chloro-N-(3-(trifluoromethyl)-
phenyl)benzamide (41). 3-Bromo-4-chloro-N-(3-(trifluoromethyl)-
phenyl)benzamide was prepared from 3-bromo-4-chlorobenzoic acid
and 3-(trifluoromethyl)benzoyl chloride using a method analogous
to the preparation of N-(3-iodo-4-methylphenyl)-3-(trifluoromethyl)-
3
-(Trifluoromethyl)benzenamine (5.09 mL, 40.7 mmol) was added,
resulting in a slight exotherm that was controlled by a water bath.
After 5 h, the mixture was concentrated in vacuo and the residue
purified by silica gel chromatography (10% EtOAc/hexanes) to give
-iodo-N-(3-(trifluoromethyl)phenyl)benzamide (5.20 g, 33%) as
a white solid. H NMR (400 MHz, CDCl
benzamide (step 1 of the preparation of 13). Yield: 0.085 g, 14%.
1
3
3 4
H NMR (400 MHz, CH OH-d ): δ 8.30 (d, J ) 4.0 Hz, 1H),
1
3
): δ 8.21 (s, 1H), 7.93-
8.16 (d, J ) 4.0 Hz, 1H), 7.93 (d, J ) 8.0 Hz, 2H), 7.70 (d, J )
8.0, 1H), 7.56 (t, J ) 8.0 Hz, 1H), 7.45 (m, 1H).
7
1
3
.83 (m, 5H), 7.51 (dd, J ) 8.0, 8.0 Hz, 1H), 7.44 (d, J ) 7.9 Hz,
H), 7.26 (dd, J ) 7.8, 7.8 Hz, 1H). MS, m/z (C14
8 3
H F INO): calcd,
A heterogeneous mixture of 3-bromo-4-chloro-N-(3-(trifluoro-
methyl)phenyl)benzamide (0.085 g, 0.22 mmol), boronate 17a
90.1; found, 391.9 (MH).
To a solution of 3-iodo-N-(3-(trifluoromethyl)phenyl)benzamide
0.43 g, 1.1 mmol) in THF (30 mL) under argon at 0 °C was added
(0.090 g, 0.34 mmol), 2.2 M K
glycol dimethyl ether (1.0 mL), and Pd(dppf)Cl
g, 0.011 mmol) was heated to 80 °C for 15 h. The cooled reaction
mixture was diluted with EtOAc and washed with brine, dried with
2
CO
3
(0.30 mL, 0.67 mmol), ethylene
Cl (0.0090
(
2
‚CH
2
2
MeMgCl (3.0 M solution in THF, 1.9 mL, 5.6 mmol) dropwise.
The resulting light-yellow solution was stirred for 45 min and cooled
to -78 °C, and t-BuLi (1.7 M solution in pentane, 3.3 mL, 5.6
mmol) was added dropwise. The solution was allowed to stir for 5
min, and trimethoxyborane (1.2 mL, 10 mmol) was added dropwise.
The solution was allowed to stir for 90 min and was then sealed
and stored at 0 °C overnight. The reaction was quenched by addition
Na
2
SO
4
, and concentrated to dryness. The solid residue was purified
Cl :CH OH:NH OH) to afford
by preparative TLC (90:10:1, CH
2
2
3
4
the title compound (0.012 g, 12%) as a light-yellow solid. HPLC
purity: 98.3% (system A); 99% (system B). H NMR (400 MHz,
1
3 4
CH OH-d ): δ 9.12 (s, 1H), 8.13 (s, 1H), 8.00 (d, J ) 2.3 Hz,
of saturated aqueous Na
2
SO
3
(5 mL) and 10% aqueous NaHSO
SO was added until
4
1H), 7.94-7.89 (m, 3H), 7.87 (dd, J ) 8.6, 2.0 Hz, 1H), 7.67 (d,
(25 mL). Additional saturated aqueous Na
2
3
J ) 8.597 Hz, 1H), 7.55 (d, J ) 9.0 Hz, 1H), 7.51 (t, J ) 8.0 Hz,
+
the yellow color disappeared. The aqueous material was extracted
four times with EtOAc. The combined organic extracts were dried
1H), 7.39 (d, J ) 7.8 Hz, 1H). HRMS (C22
H15ClF
3
N
4
O) : calcd,
443.088 10; found, 443.087 84.
with Na
2
SO
4
, filtered, and concentrated. Purification by silica gel
OH in CH Cl ) provided
2-Fluoro-4-methyl-5-(2-(methylamino)quinazolin-6-yl)-N-(3-
(trifluoromethyl)phenyl)benzamide (42). The title compound was
prepared from boronate 4m and bromide 3b, using a method
chromatography (gradient, 2.5-10% CH
the title compound (0.27 g, 82%) as a yellow foam. The material
was homogeneous by HPLC but exhibited a complex H NMR
spectrum, presumably due to the presence of anhydrides. MS, m/z
3
2
2
1
analogous to the preparation of compound 33. Yield: 0.064 g, 84%.
1
HPLC purity: 99% (system A). H NMR (400 MHz, DMSO-d
6
):
(
C
14
H
10BF
3
NO
A mixture of 3-((3-(trifluoromethyl)phenyl)carbamoyl)phen-
ylboronic acid (0.033 g, 0.110 mmol), bromide 3a (0.024 g, 0.11
mmol), Pd(PPh (0.004 g, 0.003 mmol), Na CO (2.0 M solution
in water, 0.110 mL, 0.210 mmol), EtOH (0.100 mL), and toluene
0.700 mL) was heated in a sealed tube at 90 °C for 2 h. The mixture
was cooled to room temperature and was added to EtOAc and 2.0
M aqueous Na CO . The organic layer was washed once with brine,
dried with Na SO , filtered, and concentrated. Purification by
silica gel chromatography (gradient, 40-100% EtOAc/hexanes, then
3
): calcd, 308.0; found, 310.1 (MH).
δ 10.70 (s, 1H), 9.14 (s, 1H), 8.20 (s, 1H), 7.96 (d, J ) 7.8 Hz,
1H), 7.84 (d, J ) 2.1 Hz, 1H), 7.75 (dd, J ) 8.7, 2.1 Hz, 1H),
7.65-7.52 (m, 3H), 7.46 (m, 1H), 7.44 (br s, 1H), 7.39 (m, 1H),
+
)
4
2
3
2.92 (d, J ) 4.8 Hz, 3H), 2.36 (s, 3H). HRMS (C24
H
19
F
4
N
4
O) :
3
calcd, 455.148 95; found, 455.149 26. Anal. (C24
CH OH) C, H, N.
18 4 4
H F N O‚
(
3
5-(2-Aminoquinazolin-6-yl)-2-fluoro-4-methyl-N-(3-(trifluo-
romethyl)phenyl)benzamide (43). The title compound was pre-
pared from boronate 4m and bromide 3a, using a method analogous
to the preparation of compound 33. Yield: 0.083 g, 79%. HPLC
2
3
2
4

Aminoquinazolines
purity: 99% (system A). H NMR (DMSO-d
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5683
1
6
, 400 MHz): δ 10.69
δ 10.52 (s, 1H), 9.17 (s, 1H), 8.24 (s, 1H), 8.08 (d, J ) 8.8 Hz,
1H), 7.97 (s, 1H), 7.94 (dd, J ) 7.7, 1.9 Hz, 1H), 7.87 (d, J ) 1.8
Hz, 1H), 7.78 (dd, J ) 8.7, 1.9 Hz, 1H), 7.63-7.50 (m, 3H), 7.46
(d, J ) 8.3 Hz, 1H), 7.32-7.26 (m, 1H), 3.62-3.57 (m, 4H), 3.54
(
(
(
s, 1H), 9.16 (s, 1H), 8.20 (s, 1H), 7.96 (d, J ) 8.1 Hz, 1H), 7.84
d, J ) 1.9 Hz, 1H), 7.75 (dd, J ) 8.7, 2.0 Hz, 1H), 7.64-7.59
m, 2H), 7.52-7.48 (m, 2H), 7.39 (d, J ) 11.4 Hz, 1H), 6.93 (s,
+
2
H), 2.36 (s, 3H). HRMS (C23
H
17
F N
4 4
O) : calcd, 441.133 30;
(q, J ) 6.6 Hz, 2H), 2.56 (t, J ) 6.6 Hz, 2H), 2.46 (s, 4H), 2.37
+
found, 441.133 59. Anal. (C23
H
16
4 4
F N O) C, H. N: calcd, 12.72;
(s, 3H). HRMS (C29
H
29
F N O
3 5 2
) : calcd, 536.226 79; found,
found: 13.42.
536.226 28. Anal. (C29
found, 12.19.
H
28
F
3
N
5
2 2
O ‚0.5H O) C, H. N: calcd, 12.86;
3-(2-Aminoquinazolin-6-yl)-2-methyl-N-(3-(trifluoromethyl)-
phenyl)benzamide (44). 3-Iodo-2-methyl-N-(3-(trifluoromethyl)-
phenyl)benzamide was prepared from 3-iodo-2-methylbenzoic acid
and 3-(trifluoromethyl)benzenamine, using a method analogous to
the preparation of 3-iodo-N-(3-(trifluoromethyl)phenyl)benzamide.
4-Methyl-N-(2-methyl-3-(trifluoromethyl)phenyl)-3-(2-(2-mor-
pholinoethylamino)quinazolin-6-yl)benzamide (48). The title
compound was prepared from boronate 4b and bromide 3d, using
a method analogous to the preparation of compound 33. Yield:
1
1
Yield: 0.073 g, 30%. HPLC purity: 95% (system A). H NMR
0.092 g, 97%. HPLC purity: 99% (system A). H NMR (400 MHz,
(
(
(
1
4
400 MHz, CDCl
3
): δ 7.95 (d, J ) 8.0 Hz, 1H), 7.91 (s, 1H), 7.82
6
DMSO-d ): δ 10.16 (s, 1H), 9.17 (s, 1H), 7.97 (d, J ) 1.8 Hz,
d, J ) 8.0 Hz, 1H), 7.63 (s, 1H), 7.49 (t, J ) 8.0 Hz, 1H), 7.43
1H), 7.94 (dd, J ) 8.0, 2.0 Hz, 1H), 7.86 (d, J ) 1.9 Hz, 1H), 7.78
(dd, J ) 8.0, 1.9 Hz, 1H), 7.65-7.60 (m, 2H), 7.58-7.50 (m, 2H),
7.48-7.42 (m, 1H), 7.27 (br m, 1H), 3.59 (m, 4H), 3.53 (m, 2H),
d, J ) 8.0 Hz, 1H), 7.40 (d, J ) 8.0 Hz, 1H), 6.95 (t, J ) 8.0 Hz,
11 3
H), 2.55 (s, 3H). MS, m/z (C15H F INO): calcd, 405.15; found,
06.0 (MH).
2.56 (m, 2H), 2.46 (m, 4H), 2.37 (s, 3H), 2.32 (s, 3H). HRMS
+
The title compound was prepared from boronate 17a and 3-iodo-
-methyl-N-(3-(trifluoromethyl)phenyl)benzamide, using a method
(C30
(C30
H
H
31
F
F
3
N
N
5
O
O
2
) : calcd, 550.242 44; found, 550.242 71. Anal.
2
2
30
3
5
3
‚CH OH) C, H, N.
analogous to the preparation of compound 40. Purification by silica
gel chromatography (3% CH OH in CH Cl ) provided the title
compound as an off-white amorphous solid. Yield: 0.073 g, 30%.
HPLC purity: 95% (system A). H NMR (400 MHz, DMSO-d ):
6
δ 10.77 (s, 1H), 9.18 (s, 1H), 8.28 (s, 1H), 7.96 (d, J ) 8.0 Hz,
4-Methyl-N-(2-methyl-3-(trifluoromethyl)phenyl)-3-(2-(3-mor-
pholinopropylamino)quinazolin-6-yl)benzamide (49). The title
compound was prepared from boronate 4b and bromide 3e, using
a method analogous to the preparation of compound 33. Yield:
0.089 g, 82%. HPLC purity: 99% (system A). H NMR (400 MHz,
6
DMSO-d ): δ 10.17 (s, 1H), 9.16 (s, 1H), 7.96 (s, 1H), 7.93 (dd,
3
2
2
1
1
1
7
3
H), 7.77 (d, J ) 4.0 Hz, 1H), 7.69 (dd, J ) 12.0, 4.0 Hz, 1H),
.60 (t, J ) 8.0 Hz, 1H), 7.42-7.52 (m, 5H), 6.93 (s, 2H), 2.28 (s,
J ) 8.1, 1.8 Hz, 1H), 7.85 (d, J ) 1.8 Hz, 1H), 7.77 (dd, J ) 8.6,
2.0 Hz, 1H), 7.63 (dd, J ) 7.7, 3.7 Hz, 2H), 7.56-7.49 (m, 3H),
7.45 (t, J ) 7.8 Hz, 1H), 3.62-3.57 (m, 4H), 3.43 (q, J ) 6.6 Hz,
+
H). HRMS (C23
H
18
F
3
N
4
O) : calcd, 423.142 72; found, 423.143 18.
Anal. (C23
-o-Tolylquinazolin-2-amine (45). A mixture of bromide 3a
3.02 g, 13.5 mmol), o-tolylboronic acid (2.2 g, 16 mmol),
Na CO (6.0 g, 57 mmol), and Pd(PPh Cl (0.055 g, 0.80 mmol)
in 7:3:2 DME/H O/EtOH (120 mL) was heated at 80 °C. After 3
h, the reaction mixture was cooled to room temperature and
partitioned between EtOAc and H O. The aqueous layer was
extracted with EtOAc, and the combined organic layers were
washed with brine and dried over Na SO . Purification by silica
17 3 4
H F N O) C, H, N.
6
2H), 2.42-2.35 (m, 9H), 2.32 (s, 3H), 1.84-1.70 (m, 2H). HRMS
+
(
(C31
(C31
H
H
33
F
F
3
N
N
5
O
O
2
) : calcd, 564.258 09; found, 564.257 58. Anal.
2
3
)
2
2
32
3
5
2
‚H O) C, H, N.
2
3
2
4-Methyl-3-(2-(methyl(3-morpholinopropyl)amino)quinazo-
lin-6-yl)-N-(2-methyl-3-(trifluoromethyl)phenyl)benzamide (50).
The title compound was prepared from boronate 4b and bromide
3f, using a method analogous to the preparation of compound 33.
2
2
4
Yield: 0.063 g, 45%. HPLC purity: 100% (system A); 100%
1
gel chromatography (10-100% EtOAc in hexanes) afforded the
(system B). H NMR (400 MHz, DMSO-d
6
): δ 10.20 (s, 1H), 9.29
title compound (2.0 g, 62%) as a yellow amorphous solid. HPLC
(d, J ) 18.1 Hz, 1H), 7.99-7.80 (m, 4H), 7.67-7.45 (m, 5H),
3.95 (m, 2H), 3.87 (m, 1H), 3.80 (m, 1H), 3.61 (m, 2H), 3.56 (m,
1 H), 3.46 (m, 2H), 3.30 (br s, 1H), 3.26 (br s, 1H), 3.17 (m, 1H),
1
purity: 94% (system A). H NMR (400 MHz, DMSO-d
(
7
6
): δ 9.15
s, 1H), 7.76 (d, J ) 1.9 Hz, 1 H), 7.69 (dd, J ) 8.7, 1.9 Hz, 1H),
.47 (d, J ) 8.7 Hz, 1H), 7.35-7.27 (m, 4H), 6.88 (s, 2H), 2.28
2.57 (s, 3H), 2.40 (s, 3H), 2.34 (s, 3H), 2.13 (m, 1H), 0.97 (m,
+
+
(s, 3H). HRMS (C15H N
14 3
) : calcd, 236.118 22; found, 236.118 47.
1H). HRMS (C32
H
35
F
3
N
5
O
2
) : calcd, 578.27374; found: 578.27136.
Anal. (C15
H
13
N
3
‚0.33H
2
O) C, H, N.
4-Methyl-N-(2-methyl-3-(trifluoromethyl)phenyl)-3-(2-(2-(pi-
peridin-1-yl)ethylamino)quinazolin-6-yl)benzamide (51). The title
compound was prepared from boronate 4b and bromide 3g, using
a method analogous to the preparation of compound 33. Yield:
3-(2-Aminoquinazolin-6-yl)-4-methyl-N-(2-methyl-3-(trifluo-
romethyl)phenyl)benzamide (46). To a flame-dried dispos-
able sealed tube was added acid 18a (0.15 g, 0.54 mmol) fol-
lowed by 3 mL of SOCl
1
2
. The mixture was purged with N
2
, and
0.094 g, 67%. HPLC purity: 99% (system A). H NMR (400 MHz,
the tube was sealed and heated at 80 °C for 1 h, then allowed to
cool to room temperature. The mixture was then transferred to a
6
DMSO-d ): δ 10.16 (s, 1H), 9.15 (s, 1H), 7.93-7.75 (m, 4H),
7.60-7.43 (m, 5H), 7.22 (br s, 1H), 3.50 (m, 2H), 2.40-2.37 (m,
5
0 mL pear-shaped flask with anhydrous CH
Cl
2 2
and concentrated.
6H), 2.35 (s, 3H), 2.29 (s, 3H), 1.49 (m, 4H), 1.38 (m, 2H). HPLC
+
At this point, 6 mL of anhydrous toluene was added and the mixture
was concentrated (repeat). The resulting crude acid chloride was
purity: 99%. HRMS (C31
H F
33 3
N
5
O) : calcd, 548.263 17; found,
548.263 28. Anal. (C31
found, 5.85.
H
32
F N
3 5
3
O‚CH OH) C, N. H: calcd, 6.26;
then taken up in 4 mL of CH
was added followed by 2-methyl-3-(trifluoromethyl)benzenamine
0.12 g, 0.67 mmol) and an additional 1 mL of CH Cl . The mixture
was stirred at room temperature for 2.5 h, concentrated, and purified
OH/CH Cl
2 2 3
Cl , and NEt (0.23 mL, 1.6 mmol)
4-Methyl-N-(2-methyl-3-(trifluoromethyl)phenyl)-3-(2-(2-(pyr-
rolidin-1-yl)ethylamino)quinazolin-6-yl)benzamide (52). The title
compound was prepared from boronate 4b and bromide 3h, using
a method analogous to the preparation of compound 33. Yield:
(
2
2
using MPLC (ISCO, 0-3% CH
3
2
2
over 30 min, 3-5%
1
over 15 min), providing the title compound (0.15 g, 65%) as an
0.061 g, 57%. HPLC purity: 99% (system A). H NMR (400 MHz,
1
off-white solid. HPLC purity: 100% (system A). H NMR (300
6
DMSO-d ): δ 10.17 (s, 1H), 9.17 (s, 1H), 7.96 (s, 1H), 7.93 (dd,
MHz, DMSO-d
H), 7.86 (d, J ) 1.9 Hz, 1H), 7.77 (dd, J ) 8.6, 2.1 Hz, 1H), 7.62
dd, J ) 7.8, 2.6 Hz, 2H), 7.52 (s, 1H), 7.50 (s, 1H), 7.44 (t, J )
.8 Hz, 1H), 6.94 (s, 2H), 2.36 (s, 3H), 2.31 (s, 3H). HRMS
6
): δ 10.16 (s, 1H), 9.17 (s, 1H), 7.97-7.91 (m,
J ) 8.1, 1.5 Hz, 1H), 7.85 (d, J ) 1.8 Hz, 1H), 7.76 (dd, J ) 8.6,
2.0 Hz, 1H), 7.63 (dd, J ) 8.0, 4.2 Hz, 2H), 7.56-7.49 (m, 2H),
7.45 (t, J ) 7.8 Hz, 1H), 7.38 (d, J ) 8.1 Hz, 1H), 3.91-3.80 (m,
1H), 2.78 (d, J ) 10.9 Hz, 2H), 2.37 (s, 3H), 2.32 (s, 3H), 2.19 (s,
2
(
7
+
(C
24
H
H
20
F
F
3
N
N
4
O) : calcd, 437.158 37; found, 437.159 07. Anal.
O‚H O) C, H, N.
3H), 2.07-1.97 (m, 2H), 1.95-1.87 (m, 2H), 1.65-1.52 (m, 2H).
+
(C
24
19
3
4
2
HRMS (C30
Anal. (C30
H
31
F
3
N
5
5
O) : calcd, 534.247 52; found, 534.247 29.
4
-Methyl-3-(2-(2-morpholinoethylamino)quinazolin-6-yl)-N-
H
30
F
3
N
2
O‚H O) H, N. C: calcd, 65.32; found, 65.93.
(3-(trifluoromethyl)phenyl)benzamide (47). The title compound
4-Methyl-N-(2-methyl-3-(trifluoromethyl)phenyl)-3-(2-(1-
methylpiperidin-4-ylamino)quinazolin-6-yl)benzamide (53). The
title compound was prepared from boronate 4b and bromide 3i,
using a method analogous to the preparation of compound 33.
was prepared from boronate 4a and bromide 3d, using a method
analogous to the preparation of compound 33. Yield: 0.050 g, 17%.
1
HPLC purity: 100% (system A). H NMR (400 MHz, DMSO-d
6
):

5
684 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
DiMauro et al.
1
Yield: 0.090 g, 85%. HPLC purity: 99% (system A). H NMR
peripheral blood lymphocytes 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 (1 ×
(
400 MHz, DMSO-d
6
): δ 10.29 (s, 1H), 9.29 (s, 1H), 8.08-8.04
(
1
m, 2H), 7.97 (d, J ) 1 Hz, 1H), 7.90-7.88 (dd, J ) 8.0, 1.0 Hz,
5
H), 7.77-7.73 (m, 2H), 7.68-7.50 (m, 4H), 3.97 (m, 1H), 2.90
10 T cells/well). Cells were cultured for ∼20 h at 37 °C in 5%
(
(
(
(
m, 2H), 2.49 (s, 3H), 2.44 (s, 3H), 2.31 (s, 3H), 2.17-2.11
2
CO , and then secreted IL-2 in the supernatants was quantified by
m, 2H), 2.05-2.00 (m, 2H), 1.76-1.67 (m, 2H). HRMS
cytokine ELISA (Pierce/Endogen, St. Louis, MO). The cells
+
3
C
C
30
H
H
31
F
F
3
N
N
5
O) : calcd, 534.247 52; found, 534.247 48. Anal.
remaining in the wells were then pulsed with [ H]thymidine
30
30
3
5
O‚H
2
O) C, H. N: calcd, 12.70; found, 12.17.
-Methyl-N-(2-methyl-3-(trifluoromethyl)phenyl)-3-(2-
overnight to assess the T cell proliferative response. Cells were
3
4
harvested onto glass fiber filters, and [ H]thymidine incorporation
(
phenylamino)quinazolin-6-yl)benzamide (54). The title com-
into DNA was analyzed by a liquid scintillation counter. For
comparison purposes, phorbol myristic acid (PMA) and calcium
ionophore can be used in combination to induce IL-2 secretion from
purified T cells. Potential inhibitor compounds can be tested for
inhibition of this response as described above for anti-CD3 and
-CD28 antibodies.
Human whole-blood anti-CD3/CD28-induced IL-2 secretion
assays were run in a similar fashion as described above using whole
blood from normal volunteers diluted 50% in tissue culture medium
prior to stimulation.
pound was prepared from boronate 4b and bromide 3c, using a
method analogous to the preparation of compound 33. Yield: 0.020
g, 12%. HPLC purity: 99% (system A). H NMR (400 MHz,
1
6
DMSO-d ): δ 10.19 (s, 1H), 9.96 (s, 1H), 9.38 (s, 1H), 8.01 (m,
4
1
1
(
5
H), 7.96 (dd, J ) 8.2, 2.0 Hz, 1H), 7.91 (dd, J ) 8.6, 2.0 Hz,
H), 7.77 (d, J ) 8.6 Hz, 1H), 7.63 (m, 2H), 7.54 (d, J ) 8.1 Hz,
H), 7.45 (m, 1H), 7.35 (m, 2H), 7.01 (m, 1H), 2.40 (s, 3H), 2.33
+
s, 3H). HRMS (C30
13.190 08. Anal. (C30
0.45.
H
H
24
F
F
3
N
N
4
O) : calcd, 513.189 67; found,
O) C, H. N: calcd, 10.93; found,
23
3
4
1
Human Whole-Blood LPS-Induced TNFr. Compounds were
preincubated with heparinized human whole blood diluted in RPMI
1640 with L-glutamine (GIBCO) supplemented with 10% v/v human
serum AB (Gemini BioSciences) and 1% pen/strep at a final whole-
blood dilution of 50% in 96-well flat-bottom plates (Falcon) for 1
h at 37 °C. LPS (List Biological Laboratories, 0.1 µg/mL final)
was subsequently added. Plates were then incubated overnight (18
h) at 37 °C. Secreted cytokines were measured by ELISA.
Pharmacokinetic Studies. Male Sprague-Dawley rats were
administered the compound intravenously as a solution in DMSO
or orally as a suspension in 2% hydroxypropylmethylcellulose with
1% Tween-80, at the indicated doses. Samples were taken at various
times after dosing and analyzed for parent compound by LCMS.
Anti-CD3 Induced IL-2 Production in Mice. The 12 week old
(20 g) BALB/c mice were dosed, per os, 1 h prior to challenge
with compound (8 per group) at the indicated doses in 2%
hydroxypropylmethylcellulose with 1% Tween-80. Mice were then
challenged intravenously with antimouse CD3 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 (BioSource, Camarillo, CA). Data points represent the
mean IL-2 levels/group ( the standard error. P values were
determined vs vehicle control by the Mann-Whitney U-test.
SIF Solubility Assay. The solubility media was fasted state
simulated intestinal fluid (SIF), pH 6.8, containing 5 mM sodium
taurochol, 1.5 mM lecithin, 2.9 mM KH PO , and 0.22 M KCl.
3
-(2-(Cyclopropylamino)quinazolin-6-yl)-4-methyl-N-(2-meth-
yl-3-(trifluoromethyl)phenyl)benzamide (55). The title compound
was prepared from boronate 4b and bromide 3j, using a method
analogous to the preparation of compound 33. Yield: 0.086 g, 91%.
1
HPLC purity: 99% (system A). H NMR (400 MHz, DMSO-d
6
):
δ 10.17 (s, 1H), 9.18 (s, 1H), 7.97 (s, 1H), 7.94 (dd, J ) 7.9, 1.6
Hz, 1H), 7.88 (d, J ) 1.8 Hz, 1H), 7.79 (dd, J ) 8.7, 2.0 Hz, 1H),
7
1
2
.67 (d, J ) 3.8 Hz, 1H), 7.64-7.60 (m, 3H), 7.51 (d, J ) 8.0 Hz,
H), 7.45 (t, J ) 7.8 Hz, 1H), 2.92-2.86 (m, 1H), 2.37 (s, 3H),
.32 (s, 3H), 0.77-0.72 (m, 2H), 0.57-0.54 (m, 2H). HRMS
+
(
(
C
C
27
H
H
24
F
F
3
N
N
4
O) : calcd, 477.18967; found, 477.19025. Anal.
27
23
3
4
3
O‚0.5CH OH) C, H, N.
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 µM ( 0.1), 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,
streptavidin allophycocyanin (SA-APC) was added at a final
concentration in the assay of 0.0004 mg/mL, along with europilated
anti-phosphotyrosine Ab (Eu-anti-PY) at a final concentration of
.025 nM. The assay plate was read in a Discovery fluorescence
plate reader with excitation at 320 nm and emission at 615 and
55 nm.
2
4
The Symyx solubility system (Santa Clara, CA) consisted of a liquid
handling robot and an Agilent 1100 HPLC. An experiment template
was created for solubility measurements of 24 compounds on a
96-well plate. Samples were prepared assuming a screening criterion
of 200 µg/mL solubility. A stock solution was first prepared for
each of the 24 compounds on a 24-well plate. A mixed solution of
0
6
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
50/50 CH OH/DME (v/v) was used as the stock solvent. The weight
3
the kinase and measured K
m
values for the substrates.
of the solid sample (i.e., 1 mg, to the nearest 0.01 mg) was directly
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
imported, and the appropriate volume of the stock solvent was
calculated and dispensed into the 4 mL vial for each compound by
the liquid handing robot to obtain a stock concentration of 500 µg/
mL. From each of the 24 compound stock solutions, volumes of
200, 250, 250, and 250 µL were robotically transferred to an array
of four 1 mL glass vials on a 96-well plate: one vial for the
calibration standard and the other three vials for solubility samples.
After evaporation of the stock solvent using a centrifugal evaporator,
500 µL of the solubility media was added to the corresponding
sample vials on the 96-well plate for each compound to give a
calculated concentration of 250 µg/mL. A stir bar was added to
each vial using a 96-well stir bar dispenser, and the vials were sealed
with a 96-well cap mat. The samples were equilibrated by stirring
at 100 rpm for 24 h and subsequently left standing unstirred for an
additional 24-48 h at room temperature. After equilibration, 500
5
blood lymphocytes (hPBL, 2 × 10 /well) were incubated with
mitomycin C-treated B lymphoblastoid cells (JY cell line (ATCC,
5
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
2
incubated at 37 °C in 5% CO for 6 days total. The proliferative
3
response of the hPBL was measured by [ H]thymidine incorporation
overnight between days 5 and 6 after initiation of culture. Cells
3
were harvested onto glass fiber filters, and [ H]thymidine incor-
poration into DNA was analyzed by liquid scintillation counter.
Anti-CD3/CD28-Induced T Cell IL-2 Secretion and Prolif-
eration 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
µL of 50/50 CH
3
OH/DME was added to the corresponding standard
vials on the same plate for each compound to give a calibration

Aminoquinazolines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5685
concentration of 200 µg/mL. Immediately before solubility mea-
(4) (a) Molina, T. J.; Kishihara, K.; Siderovskid, D. P.; van Ewijk, W.;
Narendran, A.; Timms, E.; Wakeham, A.; Paige, C. J.; Hartmann,
K.-U.; Veillette, A.; Davidson, D.; Mak, T. W. Profound block in
surement by HPLC, the sealed 96-well plate was centrifuged at
1650 rpm for 10 min using a plate centrifuge to allow separation
lck
thymocyte development in mice lacking p56 . Nature 1992, 357,
of the compound supernatant from undissolved solid. The solubility
samples and the calibration standards in 1 mL vials on a 96-well
plate were injected directly onto the HPLC column after centrifug-
ing. A fast gradient method was developed for HPLC throughput
starting with 100% water containing 0.04% trifluoroacetic acid
161-164. (b) Straus, D. B.; Weiss, A. Genetic evidence for the
involvement of the lck tyrosine kinase in signal transduction through
the T cell antigen receptor. Cell 1992, 70, 585-593. (c) Levin, S.
D.; Anderson, S. J.; Forbush, K. A.; Perlmutter, R. M. A Dominant-
negative transgene defines a role for p56lck in thymopoiesis. EMBO
J. 1993, 12, 1671-1680. (d) Goldman, F. D.; Ballas, Z. K.; Schutte,
B. C.; Kemp, J.; Hollenback, C.; Noraz, N.; Taylor, N. Defective
expression of p56lck in an infant with severe combined immunode-
ficiency. J. Clin. InVest. 1998, 102, 421-429. (e) Hubert, P.;
Bergeron, F.; Ferreira, V.; Seligmann, M.; Oksenhendler, E.; Debre,
(TFA) and holding for 0.1 min, then ramping to 100% acetonitrile
containing 0.04% TFA in 0.1 min and holding for 1.0 min. A
Phenomenex Synergi Hydro-RP C-18 column (10 mm × 2.0 mm,
2
µm) was chosen for retention and resolution of both polar and
lck
nonpolar compounds. The injection volume was 2 µL, with a
detection wavelength of 220 nm. For each compound, its solubility
was quantified through individual standard peak area calibration
at a concentration equal to the solubility screening criterion (i.e.,
P.; Autran, B. Defective p56 activity in T cells from an adult patient
+
with idiopathic CD4 lymphocytopenia. Int. Immunol. 2000, 12,
4
49-457. (f) Seddon, B.; Zamoyska, R. TCR signals mediated by
Src family kinases are essential for the survival of naive T cells. J.
Immunol. 2002, 169, 2997-3005.
200 µg/mL).
(
5) (a) Hanke, J. H.; Gardner, J. P.; Dow, R. L.; Changelian, P. S.;
Brissette, W. H.; Weringer, E. J.; Pollok, B. A.; Connelly, P. A.
Discovery of a novel, potent, and Src family selective tyrosine kinase
inhibitor. J. Biol. Chem. 1996, 271, 695-701. (b) Trevillyan, J. M.;
Chiou, X. G.; Ballaron, S. J.; Tang, Q. M.; Buko, A.; Sheets, M. P.;
Smith, M. L.; Putnam, B.; Wiedeman, P.; Tu, N.; Madar, D.; Smith,
H. T.; Gubbins, E. J.; Warrior, U. P.; Chen, Y.-W.; Mollison, K.
W.; Faltynek, C. R.; Djuric, S. W. Inhibition of p56lck tyrosine kinase
by isothiazoles. Arch. Biochem. Biophys. 1999, 364, 19-29. (c)
Arnold, L. D.; Calderwood, D. J.; Dixon, R. W.; Johnston, D. N.;
Kamens, J. S.; Munschauer, R.; Rafferty, P.; Ratnofsky, S. E. Pyrrolo-
Protein Binding Assay. Plasma filtrate was used for the
calibration curve and QC samples. To generate plasma filtrate, an
aliquot of plasma was filtered through Millipore Centriplus (a filter)
by spinning at 3000g (4980 rpm) at 4 °C for 24 h with a Beckman
Avanti J-251 ultracentrifuge. An aliquot of 10 µL of test compound
(1 mg/mL in DMSO) was added to 1990 µL of human, rat, and
mouse plasma to give final concentration of 5 mg/mL (12.9 µM).
Separate QC samples were prepared by adding 10 µL of the test
compound spiked plasma sample (5 µg/mL) to 490 µL of plasma
filtrate, in duplicate. The plasma samples including the QC sam-
ples were then incubated at 37 °C for 15 min. Following incuba-
tion, 800 µL was transferred to the centrifuge tube (in duplicate)
and spun at 120 000 rpm for 3 h at 37 °C with a TLA-120.2 rotor
[
2,3-d]pyrimidines containing an extended 5-substituent as potent and
selective inhibitors of Lck I. Bioorg. Med. Chem. Lett. 2000, 10,
167-2170. (d) Snow, R. J.; Cardozo, M. G.; Morwick, T. M.;
2
Busacca, C. A.; Dong, Y.; Eckner, R. J.; Jacober, S.; Jakes, S.;
Kapadia, S.; Lukas, S.; Panzenbeck, M.; Peet, G. W.; Peterson, J.
D.; Prokopowicz, A. S., III; Sellati, R.; Tolbert, R. M.; Tschantz,
M. A.; Moss, N. Discovery of 2-phenylamino-imidazo[4,5-h]iso-
quinolin-9-ones: a new class of inhibitors of Lck kinase. J. Med.
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(
Optima TLX ultracentrifuge, Beckman Coulter). The top por-
tion below the lipid layer of the centrifuged plasma sample was
aspirated (100 µL in triplicate) for LCMS analysis. The QC sam-
ples (100 µL in duplicate) were also aspirated for LCMS analy-
sis. The concentrations of the samples (n ) 6) and QCs (n ) 2)
were determined from a linear regression of peak area ratios (ana-
lyte peak area/IS peak area) versus the theoretical concentra-
tions of the calibration standards. The percent of binding was
calculated as
%
protein binding )
concentration of the centrifuged plasma sample
× 100
mean concentration of the QC
1
-
D. R.; Schieven, G. L.; Barrish, J. C.; Wityak, J. Discovery of
lck
2
-amino-heteroaryl-benzothiazole-6-anilides as potent p56 inhibi-
tors. Bioorg. Med. Chem. Lett. 2003, 13, 2587-2590. (h) Goldberg,
D. R.; Butz, T.; Cardozo, M. C.; Echner, R. J.; Hammach, A.; Huang,
J.; Jakes, S.; Kapadia, S.; Kashem, M.; Lukas, S.; Morwick, T. M.;
Panzenbeck, M.; Patel, U.; Pav, S.; Peet, G. W.; Peterson, J. D.;
Prokopowics, A. S.; Snow, R. J.; Sellati, R.; Takahashi, H.; Tan, J.;
Tschantz, M. A.; Wang, X.-J.; Wang, Y.; Wolak, J.; Moss, N.
Optimization of 2-phenylaminoimidazo[4,5-h]isoquinolin-9-ones: orally
active inhibitors of lck kinase. J. Med. Chem. 2003, 46, 1337-1349.
(i) Chen, P.; Norris, D.; Das, J.; Spergel, S. H.; Wityak, J.; Leith,
L.; Zhao, R.; Chen, B.-C.; Pitt, S.; Pang, S.; Shen, D. R.; Zhang, R.;
De Fex, H. F.; Doweyko, A. M.; McIntyre, K. W.; Shuster, D. J.;
Behnia, K.; Schieven, G. L.; Barrish, J. C. Discovery of novel
Acknowledgment. We thank our colleagues Jay Larrow and
Perry Novak for providing substantial quantities of 3a, 3b, 7,
, and 11; Stuart Chaffee, Brad Herberich, and Rob Rzasa for
the synthesis of 40, 44, and 45, respectively; and Helming Tan
and Maggie Wacker for the SIF solubility assay.
8
Supporting Information Available: Elemental analysis and
HPLC data, X-ray methods and data, exposures for 32 and 47, and
additional references. This material is available free of charge via
the Internet at http://pubs.acs.org.
2
-(aminoheteroaryl)-thiazole-5-carboxamides as potent and orally
active Src-family kinase p56 inhibitors. Bioorg. Med. Chem. Lett.
004, 14, 6061-6066. (j) Chen, P.; Doweyko, A. M.; Norris, D.;
Lck
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