Discovery and optimization of a novel series of potent mutant B-Raf V600E selective kinase inhibitors

Article abstract of DOI:10.1021/jm301658d

B-Raf represents an attractive target for anticancer therapy and the development of small molecule B-Raf inhibitors has delivered new therapies for metastatic melanoma patients. We have discovered a novel class of small molecules that inhibit mutant B-Raf

Full text of DOI:10.1021/jm301658d

Article
pubs.acs.org/jmc
Discovery and Optimization of a Novel Series of Potent Mutant
B‑RafV600E Selective Kinase Inhibitors
Melissa M. Vasbinder,*,† Brian Aquila,† Martin Augustin,⊥ Huawei Chen,† Tony Cheung,† Donald Cook,†
Lisa Drew,† Benjamin P. Fauber,†,# Steve Glossop,∥ Michael Grondine,† Edward Hennessy,†
Jeffrey Johannes,† Stephen Lee,† Paul Lyne,† Mario Mortl,⊥ Charles Omer,† Sangeetha Palakurthi,†,∇
̈
Timothy Pontz,† Jon Read,§ Li Sha,†,○ Minhui Shen,† Stefan Steinbacher,⊥ Haixia Wang,† Allan Wu,‡
and Minwei Ye†
†Oncology iMED and ‡Discovery Sciences, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United
States
∥
§Discovery Sciences and Oncology iMED, AstraZeneca R&D Alderley Park, Cheshire, Macclesfield SK10 4TG, U.K.
⊥Proteros Biostructures GmbH, Bunsenstrasse 7a, D-82152 Planegg-Martinsried, Germany
S
* Supporting Information
ABSTRACT: B-Raf represents an attractive target for anticancer therapy and the development of small molecule B-Raf
inhibitors has delivered new therapies for metastatic melanoma patients. We have discovered a novel class of small molecules that
inhibit mutant B-RafV600E kinase activity both in vitro and in vivo. Investigations into the structure−activity relationships of the
series are presented along with efforts to improve upon the cellular potency, solubility, and pharmacokinetic profile. Compounds
selectively inhibited B-RafV600E in vitro and showed preferential antiproliferative activity in mutant B-RafV600E cell lines and
exhibited selectivity in a kinase panel against other kinases. Examples from this series inhibit growth of a B-RafV600E A375
xenograft in vivo at a well-tolerated dose. In addition, aminoquinazolines described herein were shown to display pERK elevation
in nonmutant B-Raf cell lines in vitro.
published contributing to this field, further supporting the
interest by many in the broader therapeutic potential for B-Raf
inhibitors.8−10
INTRODUCTION
■
The Raf family of intracellular serine/threonine protein kinases is
involved in the transmission of proliferative signals from cell
surface receptors to the cell nucleus through the mitogen-
activated protein kinase (MAPK) pathway.1 The RAS/RAF/
MEK/ERK pathway plays a key role in many cancers and is
activated in approximately 30% of all human tumors.2 B-Raf
somatic missense mutations have been reported in 66% of
malignant melanomas and in a lower occurrence in a wide range
of human cancers, with the most prevalent mutation V600E
found to be constitutively active in carcinoma cells.3 Selectively
targeting the mutant form of B-Raf with small molecule
inhibitors has been of great interest as a potential therapeutic
strategy. The 2011 FDA approval of vemurafenib (PLX4032/
RG7204, Plexxikon/Roche)4−7 for the treatment of metastatic
melanoma bearing the B-RafV600E mutation confirmed the
importance of B-Raf within the MAPK pathway and advanced
the treatment options for melanoma patients. Numerous other
clinical and preclinical small molecule inhibitors have been
We have reported previously an amidoheteroaryl class of
compounds to be potent inhibitors of mutant B-RafV600E with in
vivo activity.11 Compound 1 represents one such example that
potently inhibits B-RafV600E both in vitro (B-Raf V600E IC50 = 16
nM and A375 pERK IC50 = 40 nM, Figure 1) and in vivo. We also
identified additional compounds such as 2 (AZ628)12 to be
potent inhibitors of mutant B-RafV600E (B-Raf V600E IC50 = 34 nM
and A375 pERK IC50 = 15 nM, Figure 1), and 2 has been
reported by us and others for its ability to modulate the MAPK
pathway.13 Examples such as 1 and 2 are believed to bind to
mutant B-RafV600E in its DFG-out conformation.14 As part of our
screening efforts within AstraZeneca to identify inhibitors of B-
Raf, we screened a subset of our collection which was biased
Received: November 9, 2012
Published: February 11, 2013
© 2013 American Chemical Society
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Figure 1. AstraZeneca B-Raf inhibitors 1−3.
a
Scheme 1. Preparation of Aminoquinazolines 3 and 24−39
a
Reagents and conditions: (a) guanidine carbonate, DMA, 140 °C; (b) Pd(PPh3)4 (20 mol %), K2CO3, ArB(OH)2, DME/water, 90 °C; (c) 17−23,
Pd2(dba)3 (5 mol %), BINAP (10 mol %), Cs2CO3, dioxane, 100 °C.
toward small molecules known or expected to contain kinase
inhibitors. Aminoquinazoline 3 was identified as a singleton hit
from this effort and displayed reasonable biochemical (B-Raf
V600E IC50 = 77 nM) and cellular potency (A375 pERK IC50 = 190
nM), as shown in Figure 1. In contrast to previous inhibitors 1
and 2, the hit 3 was at least 5-fold less potent based on pERK
cellular potency. However, we did believe 3 represented an
excellent starting point for further optimization.
In this paper, we describe our efforts to further expand and
optimize the aminoquinazoline hit 3 into a DFG-in binding
inhibitor of mutant B-RafV600E. Examples from this inhibitor class
were optimized for cellular potency, physical properties, and rat
pharmacokinetics, resulting in examples with potent in vivo
activity in A375 xenograft models. We likewise found that the
aminoquinazoline compounds represented herein exhibited a
mutant B-RafV600E selective profile based on biochemical activity
and preferential cell panel antiproliferative activity over a range of
cell lines. The B-RafV600E selective nature of the compounds was
found to elevate pERK in nonmutant B-Raf cell lines in vitro, a
phenomenon which has been previously documented by others
in the literature as resulting from Raf dimerization events.15
Aminoquinazolines presented herein represent a new class of B-
RafV600E selective inhibitors that provide a complementary profile
to those we had previously reported (1 and 2), thus serving as
additional B-RafV600E selective tool compounds to further
interrogate the important role of mutant B-Raf within the
MAPK pathway.
Synthetic Chemistry. Aminoquinazolines were readily
accessed using the following synthetic route (Scheme 1).
Commercially available aldehyde 4 was converted to 6-
bromoaminoquinazoline 5 by treatment with guanidine
carbonate heating in DMA followed by Suzuki coupling with a
variety of boronic acids to afford amino derivatives 6−16.
Palladium-catalyzed coupling with the corresponding aryl
bromides generated the final aminoquinazoline analogs 3 and
24−39 (Tables 1 and 2).
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a
Scheme 2. Preparation of Aminoquinazolines 48−50 and 51−58
a
Reagents and conditions: (a) Pd2(dba)3 (10 mol %), BINAP (20 mol %), Cs2CO3, dioxane, 100 °C; (b) amine (1.1 equiv), MeOH, 3 Å MS, HOAc
(catalytic amount), NaBH3CN (1.6 equiv), rt, 12 h then 1 N NaOH; (c) 41−47, Xantphos (10 mol %), Cs2CO3 (3.0 equiv), dioxane, Pd(OAc)2,160
°C, microwave. For preparation of 51 and 53, see the Experimental Section. 1-[2-(4-Bromophenoxy)ethyl]pyrrolidine was commercially available.
a
Scheme 3. Preparation of Aminoquinazolines 67−71
a
Reagents and conditions: (a) method A or B [method A, N-(4-aminophenyl)-N-methylacetamide or N-(4-aminophenyl)acetamide (1.5 equiv),
MeCN, 125 °C, microwave, 30 min; method B, 59−61, propan-2-ol, 100 °C, 2 h]; (b) 4-pyridinylboronic acid (1.5 equiv), Cs2CO3 or K2CO3 (3.0
equiv), dioxane/water or DME/water (4/1), Pd(PPh3)4or PdCI2(dppf)-CH2CI2 (0.1 equiv), 100 °C.
As described in Scheme 2, benzyl amine analogs 48−50 (Table
2) were generated via the corresponding aminoquinazoline
aldehyde 40, followed by reductive amination with commercially
available amines. A variety of aryl bromides were utilized to
generate additional target analogs, including those containing
ethers (51, Table 2), reversed amides (52, Table 2), and α-
methyl benzyl amines and amides (53−58, Table 3). Aryl
bromides were either commercially available or readily
synthesized by standard conditions (see the Experimental
Section).
An alternative route to access similar aminoquinazoline
compounds containing either ethers (67, Table 2), reversed
amides (68−70, Table 2), or N-linked anilines (71, Table 3) was
accomplished, as reported in Scheme 3. Readily accessible 6-
bromo-2-chloroquinazoline16 was treated under either micro-
wave or thermal conditions (method A or B, respectively) with a
variety of anilines to afford bromides 62−66, which were then
subjected to palladium-catalyzed Suzuki coupling conditions
with 4-pyridinylboronic acid to afford 67−71.
Additional efforts to explore the effects of further diversifying
the α-methyl benzyl motif led to the generation of α-methyl
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a
Scheme 4. Preparation of Aminoquinazoline Analogues 75−84
a
Reagents and conditions: (a) (i) 72, Xantphos (10 mol %), Cs2CO3 (3.0 equiv), dioxane, Pd(OAc)2 (5 mol %), 100 °C, 4 h; (ii) 2 N HCl in ether.
(b) For compounds 75−76 and 78−80, (i) carboxylic acid, DIPEA, HATU, DMF, 60 °C; (ii) 4 M HCl in dioxane or 2 N HCl in ether, rt; for
compounds 81, 82, cyclohexane- or cyclopentane-1,3-dione (3 equiv), HOAc, DIPEA, MeOH, 60 °C, 12 h; for compound 83, piperidine-2,4-dione,
DMF, microwave, 150 °C; for preparation of 77, see the Experimental Section. (c) (i) NaBH4 (1.8 equiv), MeCN, benzyl carbonochloridate (1.2
equiv), 0 °C, dioxane; (ii) 4 M HCl in dioxane, rt. (d) (i) 73, HOAc, NMP, 105 °C, 6 h; (ii) Pearlman’s catalyst (15 mol %), MeOH, rt, H2, 1 atm.
amine intermediate 73, which was readily synthesized by
palladium-catalyzed coupling of 6 and 72 followed by BOC-
group deprotection. As described in Scheme 4, intermediate 73
served as a key building block for rapid synthesis of various α-
methyl amides and vinylogous amides such as 75−84 (Table 4).
The α-methyl amides (75−80) were prepared from coupling of
the corresponding carboxylic acids with amine 73 followed by
any required deprotection of BOC protecting groups to generate
the desired analogs. Compounds 81 and 82 were generated by
reacting 73 with cyclic 1,3-diketones such as cyclohexane-1,3-
dione or cyclopentane-1,3-dione with a catalytic amount of acid
or under microwave conditions in the presence of piperidine-2,4-
dione to yield 83. Vinylogous amide analog 84 was synthesized
by reduction of commercially available 3,5-dimethoxypyridine
followed by protection to afford intermediate 74. Treatment of
74 in the presence of amine 73 under acid-mediated conditions
followed by deprotection of the carbonylbenzyloxy protecting
group generated the target compound 84 (Scheme 4).
The chemistry we employed to probe substitution at the 7- and
8-positions of the aminoquinazoline is described in Scheme 5.
Readily accessible bromo chloride and amino intermediates such
as 88−90 were generated in accordance with known literature
procedures.17 Treatment of aniline 85 with 6-bromo-2-chloro-8-
methoxyquinazoline in the presence of refluxing propan-2-ol
followed by base generated 86, which after Suzuki coupling
afforded 87. Alternatively, the sequence of Suzuki coupling
(generating 91−93) followed by palladium-catalyzed coupling,
deprotection of the BOC group, and acetylation led to the
desired analogs (97−99). Further derivatization of 95 (R1 = Cl,
R = H) and 96 (R1 = H, R = Cl) by coupling with methyl boronic
acid followed by deprotection of the BOC group and acetylation
resulted in 100 and 101.
RESULTS AND DISCUSSION
■
Initial Hypothesis of Binding and Approach for
Compound Design and Optimization. We obtained a
cocrystal of compound 3 in kinase EphB4, which was used as a
surrogate protein due to the high binding site homology to B-
RafV600E. The structure confirmed 3 binding in a DFG-in manner,
making the canonical hydrogen-bond interactions with the hinge
region of the protein via the N3 of the quinazoline ring and the
aniline hydrogen.18 The binding mode oriented the 6-position 4-
pyridyl group in the selectivity pocket at the back of the ATP site
and the 4-substituent of the aniline ring directed into the solvent
channel of the ATP site (Figure 2A).
On the basis of the knowledge of the binding mode of 3 in
EphB4, the chemistry plan was focused on exploring the SAR of
this class of compounds. Our approach was 3-fold and examined
exploration of the A-, B-, and C-rings (Figure 2B). We prioritized
A- and C-ring exploration, as we believed these regions of the
molecule would influence most the cellular pERK potency and
physical properties without interfering with the B-ring of the
molecule, which was responsible for making the key interactions
to the hinge region of the protein.
SAR Development. We began our exploration with
modifications to the A-ring and found that a variety of steric
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a
Scheme 5. Preparation of Aminoquinazolines 87 and 97−101
a
Reagents and conditions: (a) 85, propan-2-ol, reflux, 3 h then DIPEA, reflux. (b) Pd(PPh3)4, Cs2CO3, 4-pyridinylboronic acid, dioxane/water (2/
1), 90 °C. (c) 72, Pd(OAc)2 (5 mol %), Xantphos (10 mol %), Cs2CO3, dioxane, 100 °C. (d) (i) HCl/THF or 4 N HCl/dioxane; (ii) NEt3, Ac2O,
rt. (e) (i) 95, 96, methyl boronic acid, K2CO3 (3.0 equiv), dioxane, Pd(OAc)2(4 mol %), PCy3-HBF4 (10 mol %), 100 °C, 12 h; (ii) HCl/THF; (iii)
NEt3, Ac2O, rt.
Figure 2. (A) Cocrystallization of 3 in EphB4, which was used as a surrogate to mutant B-Raf, illustrates the compound binding in a DFG-in manner in
the ATP site of the kinase domain of the protein. The structure has been deposited in the Protein Data Bank (reference code 4bb4). (B) Proposed plan
for aminoquinazoline exploration.
and electronic changes to the 4-pyridyl ring resulted in a
significant loss in cell potency (24−27, Table 1). Likewise,
replacing the 4-pyridyl with a phenyl group led to a much less
potent compound (28, A375 pERK IC50 = 3.80 μM). We
discovered that a 4-methyl-3-pyridyl group led to improvements
in both biochemical and cell potency (30, B-RafV600E IC50 = 0.023
μM, A375 pERK IC50 = 0.089 μM), achieving a 2-fold
improvement in cell potency vs the initial hit 3. However, a 3-
pyridyl or substituted 3-pyridyl ring was not tolerated (29, 31,
32). Further examples that explored the effects of various other
nitrogen-containing six-membered ring heterocycles also
resulted in a loss of cell potency, such as pyrimidine 33 (A375
pERK IC50 = 6.40 μM). Thus, while we identified tight SAR
around the 6-position of the aminoquinazoline in the initial A-
ring exploration, we were pleased to uncover the improvement in
cell potency (i.e, 30, Table 1).
We profiled the most potent analogs of this series (3 and 30)
early on to better understand the kinase selectivity profile. Both
compounds were screened against a Millipore panel of 83
kinases.19 We concluded the selectivity profile of 3 to be superior
to that of 30, as demonstrated by visualization of inhibition
profiles and S(10) selectivity scores of 0.01 and 0.11, respectively
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Table 1. A-Ring Exploration
a
b
Measured at Km ATP concentration. For experimental details, see the Supporting Information. All values are an average of at least two
independent dose−response curves. For experimental details, see the Supporting Information.
Figure 3. Millipore KinaseProfiler selectivity screening of a diverse kinase set (n = 83) at 1 μM of 3 and 30, with hits overlaid on the kinome phylogenetic
tree. Red circles indicate active hits ≤35% of control.
(Figure 3). The results revealed that the 4-pyridyl ring led to an
overall more selective profile, in particular against other tyrosine
and serine/threonine kinases. Therefore, based on the more
promiscuous nature of 30, we chose to continue our SAR
optimization keeping the 4-pyridyl A-ring constant.
In addition to assessing the kinase selectivity profile, we also
studied the biochemical and cell proliferation profile of 3.20 We
hypothesized on the basis of our findings from both biochemical
and cell proliferation data that aminoquinazoline 3 afforded a
different profile to previous inhibitors such as 2. Aminoquinazo-
line 3 was found to inhibit mutant B-RafV600E but was much less
potent against C-RafWT or inactive against B-RafWT under
saturated ATP conditions (Figure 4A), pointing to evidence for a
more mutant B-RafV600E selective profile. Compound 2 resulted
in a different biochemical profile affording a near equal inhibition
of B-RafV600E and C-Raf and a drop off in the B-RafWT potency.
We obtained similar results after comparing the cell proliferation
profile of compounds 2 and 3 against a panel of various cell lines.
Aminoquinazoline 3 resulted in preferential antiproliferative
activity in the presence of mutant B-RafV600E cell lines (Figure
4B). Compound 2, when treated under identical cell lines and
conditions, inhibited additional nonmutant B-Raf cell lines.
Thus, we identified early on the potential for the aminoquinazo-
line series to provide a different yet complementary profile to the
previous inhibitors such as 2 that we had discovered.
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Figure 4. (A) Biochemical inhibitory profile of 2 and 3. For a more detailed description of the biochemical differences of 2 and 3 with regard to substrate
ATP concentration, see the Supporting Information. (B) Antiproliferative activity of B-Raf inhibitors 2 and 3 against a panel of various cell lines. Cells
were incubated with DMSO or compounds 2 or 3 for 72 h and cell growth was determined by MTS assay, represented as log mean GI50 (average of three
replicates). Cell lines denoted with an asterisk (*) harbor the B-RafV600E mutation. For a complete listing of cell line type, further observations around the
proliferation profile, and additional MTS assay details, see the Supporting Information.
Table 2. C-Ring Modifications
Further exploration of the C-ring SAR involved modifications
toward the solvent channel, a region of the molecule that would
allow for optimization of the overall properties of the series. The
solubility for 3 was found to be 3 μM, so a key focus of our
optimization and SAR generation was around improving
solubility. Meta-substitution of the amido group was not
tolerated on the basis of the cellular potency; however, methyl
or fluoro substitution adjacent to the p-amido group was
acceptable, albeit with no improvement in solubility (Table 2,
34−36). We incorporated various basic amines, both acyclic and
cyclic, into the amide to serve as solubilizing groups. These
changes significantly improved the solubility; however, they
resulted in a slight decrease in potency (Table 2, analog 39, A375
pERK IC50 = 330 nM) or had no effect on potency (such as 37
and 38, with similar cellular potency to 3). In order to better
understand the functional group tolerance at the 2-position of
the quinazoline, we investigated other groups in addition to
amido-substituted phenyl. Diversifying beyond phenyl amides to
benzyl amine derivatives 48−50, while improving solubility, led
to reduced potency, in particular with example 50. Ethers such as
51 and 67 did not afford much benefit to potency, though
improved solubility could be achieved with the inclusion of basic
groups such as the pyrrolidine in 51. We also explored reversing
the amide with N-linked compounds such as 52 and 68−70
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(Table 2) or removing the amide altogether with N-linked analog
71 (Table 3). While not all of these changes led to favorable
improvements in cell potency, we were able to find some
examples where cell potency below 100 nM was achieved and
resulted in modest evidence for desirable solubility (52, A375
pERK IC50 = 45 nM, solubility 40 μM; 69, A375 pERK IC50 63
nM, solubility 7 μM; Table 2). It appeared from the SAR that N-
methyl amides were >6-fold more potent in the A375 pERK
cellular assay vs their desmethylated matched pairs (i.e., 52 vs 68
and 69 vs 70, Table 2). In contrast, N-methyl linked analogs such
as 71 were not tolerated on the basis of much weaker cell potency
(Table 3, A375 pERK IC50 = 855 nM). From our initial
exploration varying the functionality at the C-ring, we were able
to identify compounds with good solubility and improved
potency compared to initial hit 3. Next, we further explored
analogs 52 and 69 with the goal of maintaining the improve-
ments in potency while increasing the solubility.
Additional exploration of what else could be tolerated in the
solvent channel was instrumental to the optimization of this
series. We expanded on our prior knowledge that N-methyl
amides such as 52 and 69 improved potency while retaining
modest solubility. We were also concerned that analogs such as
52 and 69 could carry a potential mutagenic liability due to the
aromatic aniline functionality, and we sought to prioritize design
ideas to remove this functionality.21 We found that incorporating
an additional atom between the arene and the nitrogen was
tolerated, such as α-methyl amino derivative 53 (Table 3, A375
pERK IC50 = 49 nM). This compound had improved solubility
(Table 3, solubility 830 μM and human plasma protein binding
9.4% free) as compared to the hit 3 (solubility 3 μM and human
plasma protein binding 1.7% free).22 Further expanding this
theme to other α-methyl amides led to additional examples with
promising cell potency. Analogs 54−58 highlight the stereo-
chemical preference of the α-methyl group with respect to
cellular potency. We discovered retention of potency in the (R)-
enantiomer of each example (55 and 58, A375 pERK IC50 = 60
and 34 nM, respectively); however, these analogs suffered from
poor physical properties (solubility <10 μM and human protein
binding <2.3% free). On average, the (R)-enantiomers of both 54
and 57 were found to be 2−5 times more potent than the
corresponding (S)-enantiomers (i.e., 56 vs 55, A375 pERK IC50
= 160 vs 60 nM).23 We were optimistic about the
pharmacokinetic profiles of the α-methyl benzylic amino analogs
because examples 53, 55, and 58 had favorable rat liver
hepatocyte stabilities (Table 3, rat hepatocyte CLint 16, 11, and
6 μL/min/1E6, respectively). Likewise, human microsomal
stabilities for 53 and 58 were in a desirable range (Table 3).
Encouraged by these results, we expanded upon the (R)-α-
methyl amide motif.
We were successful in obtaining a cocrystal structure of 48 in
B-RafV600E, and our initial hypotheses from the cocrystallization
of 3 in EphB4 were confirmed (Figure 5). The structure revealed
Plans to improve upon the physical properties of analogs such
as 55 and 58 centered around expanding the diversity of the (R)-
α-methyl benzyl amides by incorporating solubilizing groups. We
explored basic groups off the amide functionality in an attempt to
improve solubility while the potency and in vitro PK profile that
we believed would be desirable were maintained. We found
amides containing cyclic amines to be most promising. A variety
of ring sizes were explored, and positioning of the nitrogen was
key for maintaining desirable cellular potency and solubility. For
example, 4-piperidino carboxamide 79 resulted in a significant
loss in cell potency (Table 4, A375 pERK IC50 = 688 nM),
whereas the 2-piperidino carboxamide 75 maintained desirable
cell potency (Table 4, A375 pERK IC50 = 45 nM). Compound 75
likewise had excellent solubility (>940 μM), human plasma
protein binding (5.1% free), and moderate in vitro rat clearance.
The 2-substituted piperidino carboxamide 75 was also found to
be more potent than the 3-piperidino analog 78 (Table 4, A375
pERK IC50 = 140 nM), whereas the 2-pyrrolidino carboxamide
80 was of similar cellular potency to 75 (Table 4, A375 pERK
IC50 = 74 nM).24 We also explored further elaboration of 75 with
various substituents to probe the importance of the free amine
and build upon the SAR. We believed attenuation of the basic
amine pKa could be a strategy to address pharmacokinetic
liabilities that may arise in vivo.25 Methyl substitution as in 76
was tolerated, yielding an identical potency to 75 with reduced
solubility (65 μM).26 Substitution to generate what we believed
would be a less basic moiety in hydroxyethyl analog 77 afforded a
potent inhibitor (A375 pERK IC50 = 25 nM) though with poor
solubility and higher predicted clearance as indicated by both rat
liver hepatocytes and human liver microsomal data (Table 4).
Figure 5. X-ray structure of 48 bound to the kinase domain of mutant B-
RafV600E. The structure has been deposited in the Protein Data Bank
(reference code 4H58), and detailed protein preparation, crystallization,
and freezing protocols are included in the Supporting Information.
binding to the kinase domain in a DFG-in conformation, making
the canonical hydrogen bond interaction to the N−H of C531 via
the N3 of the quinazoline ring and the backbone carbonyl of
E532 via the aniline hydrogen. The 4-substituent of the aniline
ring was directed into the solvent channel of the ATP site, and
the quinazoline and aniline rings were stacked against the indole
side chain of W530. The 6-position 4-pyridyl group was located
in the selectivity pocket at the back of the ATP site, above the
T528 gatekeeper residue. We speculated on the basis of the
structure that our observation of the 4-methyl-3-pyridyl analog
(30, Table 1), which resulted in significantly more potent activity
as compared to the desmethyl analog (29, Table 1), could be due
to anchoring of the methyl group over the T528 gatekeeper
residue to capitalize on a lipophilic interaction, thus locking into
place the 6-position 3-pyridyl ring into the selectivity pocket. We
also hypothesized that the preorganization gained by the
lipophilic interaction of the T528 methyl and methyl group of
the pyridyl ring in the selectivity pocket contributed to the
broader kinase activity of analogs such as 30, in particular by
other threonine-containing gatekeeper kinases. The X-ray crystal
structure of 48 thus helped support our plans to continue
exploring the C-ring substituents in the solvent channel. The X-
ray structure also suggested that additional exploration around
the B-ring at either the 7- or 8-position of the quinazoline would
be tolerated.
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Table 3. Additional C-Ring Modifications
a
b
Measured at Km ATP concentration. For experimental details, see the Supporting Information. All values are an average of at least two
c
independent dose−response curves. For experimental details, see the Supporting Information. Human PPB, rat hepatocyte, and human microsomal
stability details can be found in the Supporting Information.
Inspired by reports of vinylogous amide pharmacophores in a
series of pyrrolidine ether hNK1 antagonists,27 we found the
carboxamide could be replaced by a vinylogous amide and
derivatives such as 81 and 82 resulted in single digit nanomolar
cell potency (Table 4, 81−82 A375 pERK IC50 = 4−5 nM). Both
five- and six-membered rings were equally promising; however,
both resulted in poor solubility (Table 4, 4 μM). We
hypothesized the design and synthesis of close analogs such as
dihydropyridinones 83 and 84 incorporating nitrogen atoms into
the ring would improve the solubility with the expectation of
maintaining single digit nanomolar cell potency. We discovered
that analog 83 maintained potency however did little to improve
upon the solubility of prior compounds (Table 4). Analog 84 was
designed to combine features of the 2- and 3-piperidino
carboxamide profiles of 75 and 78 with the vinylogous amide
motif. Our hypothesis was that a combination of both features
would maintain a desirable potency and solubility profile.
However, 84 resulted in a less potent and still poorly soluble
analog (Table 4, A375 pERK IC50 = 63 nM, solubility 10 μM).
Our strategy to diversify beyond the (R)-α-methyl benzyl amides
to uncover analogs such as piperidino carboxamide 75 and
vinylogous amides 81 and 82 led to identification of promising
leads with in vitro PK profiles that we believed would translate
into an acceptable in vivo PK profile.
substitution at either the 7- or 8-position of the quinazoline
would be tolerated. These positions were oriented away from the
hinge binding region, whereas substitution of the aminoquinazo-
line at the 4- and 5-positions was expected to interfere with hinge
binding. We explored the electronic distribution of the
quinazoline ring as this might play a key role in modulating
PK. A limited number of groups representing electron-donating
and -withdrawing potential were investigated at the 7- and 8-
positions of the aminoquinazoline. We discovered 7-substitution
with chloro and methyl (Table 5, 98 and 100, A375 pERK IC50 =
430 and 573 nM, respectively) were detrimental to pERK cell
potency, resulting in a greater than 20-fold loss in potency as
compared to their 8-substituted matched pairs (Table 5, 99 and
101, respectively). Interestingly, both the 8- and 7-methoxy
substitutions were tolerated and resulted in potency similar to
that of 58 (Table 5, 87 and 97, A375 pERK IC50 = 10 and 25 nM,
respectively). These core substitutions did not lead to a desirable
solubility range, as most analogs had <10 μM solubility and high
human protein binding. Most examples exhibited acceptable rat
hepatocyte and human microsomal intrinsic clearance values.
Pharmacokinetic Properties of Leads. Aminoquinazo-
lines with desirable pERK cell potency (typically IC50 < 50 nM)
and in vitro rat hepatocyte and human microsomal stability
which should be predictive of modest clearance were progressed
into in vivo rat PK studies. We were encouraged to find that the
most potent analogs had low in vivo clearance and good
bioavailability (52 and 58, F = 60 and 100%, respectively, Table
Modifications to the core aminoquinazoline B-ring were also
examined to probe the SAR of this region. On the basis of our
knowledge of the aminoquinazoline binding mode, we believed
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Table 4. Amide Modifications
a
b
Measured at 5 mM ATP concentration. For experimental details, see the Supporting Information. All values are an average of at least two
c
independent dose−response curves. For experimental details, see the Supporting Information. Hu PPB, rat hepatocyte, and Hu microsomal stability
details can be found in the Supporting Information. 76 represents a mixture of diastereomers at the piperidinyl chiral center as the diasteromers
proved to be inseparable after various attempts at separation. Denotes not tested.
d
e
Table 5. B-Ring Core Modifications
IC50 (μM)
CLint
a
b
c
c
c
compd
R
R1
B-RafV600E
A375 pERK
solubility (μM) pH 7.4 Hu PPB Fu (%)
rat Hep (μL/min/1E6)
Hu Mic (μL/min/mg)
87
97
OMe
H
H
0.057
0.14
2.29
0.187
2.6
0.01
5
3.4
2.6
<1
<1
1.9
<1
2
4
3
5
2
8
OMe
Cl
0.025
0.43
<1
2
<4
8
98
H
99
Cl
H
0.02
<1
5
39
11
17
100
101
H
Me
H
0.573
0.025
Me
0.081
<1
5
a
b
Measured at 5 mM ATP concentration. For experimental details, see the Supporting Information. All values are an average of at least two
c
independent dose−response curves. For experimental details, see the Supporting Information. Hu PPB, rat hepatocyte, and human microsomal
stability details can be found in the Supporting Information.
6). However, a trend of short half-life and low volume of
distribution was also seen, a profile that might not achieve the
sustained exposure we would require to achieve target inhibition
in vivo. Analogs such as benzyl amine 53, which demonstrated
improved solubility and desirable potency, showed moderate−
high clearance in a manner similar to analog 84. Interestingly,
carboxamide 75 did lead to a desirable in vivo rat PK profile with
a longer half-life over a 24 h iv dosing period and larger volume of
distribution, though moderate clearance (Table 6). We believed
compounds 58 and 75 were excellent candidates for additional in
vivo evaluation.
Mutant B-RafV600E Selective Cell Profile Results in pERK
Elevation in Nonmutant B-Raf Cells. Additional experiments
to understand the effects of the mutant B-RafV600E selective
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a
Table 6. Rat PK of Lead Compounds
compd
CLobs (mL/min/kg)
Vdss (L/kg)
F (%)
t1/2 (h)
52
53
58
75
81
84
97
9
56
0.7
4.9
60
31
1
1.5
0.9
4.4
1.1
0.42
0.61
11
0.8
100
28
43
13
19
0.67
7.9
17
b
120
15
ND
b
0.67
ND
a
Han Wistar rat male; 10 mg/kg po (0.1% HPMC); 3 mg/kg iv (40%
b
DMA/40% TEG/20% saline). t1/2 represents the iv half-life. Denotes
not tested as a po dose, so F (%) was not determined.
profile are highlighted in Figure 6. An A549 cell line with B-RafWT
KRAS mutant background was evaluated for pERK response to
aminoquinazolines 3, 58, 69, 75, 81, and 87. Upon treatment of
compounds at 0.1-, 1-, 10-, and 100-fold the A375 pERK IC50, a
dose-dependent elevation of pERK was seen with no clear
margin to this effect, irrespective of the aminoquinazoline
chosen. This observation is in stark contrast to the effects seen
after a 75 min treatment with either 2 or Pfizer’s known selective
MEK1/2 inhibitor PD0325901 (102),28 both of which resulted
in a decreased pERK in a dose-dependent manner (Figure 6).
We wanted to understand how general this trend for elevated
pERK was in a variety of other B-RafWT and KRAS cellular
contexts, and thus selected lead aminoquinazoline 58 for
additional studies. Both cancer (melanoma, lung, and breast),
normal, and fibroblast cell lines were treated with 58 at 1-, 10-,
and 100-fold the A375 pERK IC50. Elevation of pERK was
observed in all cell lines after a 75 min treatment with 58. No
clear margin to the elevation of pERK in nonmutant B-Raf cell
lines, relative to decrease in pERK in A375 cell lines, was seen
over the range of doses investigated (Figure 7). In contrast to
aminoquinazoline compounds, 2 did not show pERK elevation in
any of the cell lines tested. MEK1/2 inhibitor 102 led to an
expected decrease, or near complete inhibition, of pERK in all
cell lines. Consistent with observations by others in this field,
there are differences in the ability of the various classes of B-
RafV600E inhibitors to lead to elevated pERK and MAPK pathway
activation in nonmutant B-Raf cells, an observation now well
documented to result from Raf dimerization.15,29
Figure 7. In vitro pERK evaluation in multiple nonmutant B-Raf cell
lines. Cells were collected following 75 min treatment with DMSO or
compound at the indicated doses (1-, 10-, and 100-fold the A375 pERK
IC50). Western blotting analysis was performed using antibodies against
the indicated proteins. pERK refers to phospho-ERK levels and tERK
represents total-ERK levels. 102 was used as a positive control in this
study.
(Figure 8C) and displayed excellent tolerability with no
significant body weight loss.30
CONCLUSION
■
The aminoquinazoline DFG-in class of compounds were shown
to be potent and selective B-RafV600E inhibitors that evolved from
a singleton hit identified from a kinase subset screening effort.
Aminoquinazolines were optimized for B-RafV600E cellular
potency, physical properties, and in vivo rat pharmacokinetics.
We have demonstrated that a lead compound from this series, 58,
exhibits in vivo tumor growth inhibition in an A375 xenograft
model. A key finding from this class of compounds was the B-
RafV600E selective profile, which, similar to previous reports by
others in the field, resulted in the elevation of pERK, thus
highlighting the potential for multiple novel chemotypes to
generate such a profile. Compounds such as the aminoquinazo-
line series described herein, in addition to the complementary
profile afforded by analogs such as 2, provide useful tools to
further interrogate the important role of B-Raf within the MAPK
pathway.31
In Vivo Activity of Lead Aminoquinazolines. Lead
compound 58 was progressed for additional in vivo profiling. A
dose-dependent decrease in pERK was observed in an A375
mouse xenograft PD study with 58 starting at the 10 mg/kg dose
as observed by Western blot and quantitation of the percent
reduction in pERK (Figure 8A,B). In addition, a dose-dependent
increase in drug concentration in the plasma was observed (see
the Supporting Information). Compound 58 was progressed
further into an in vivo efficacy study in an A375 mutant B-
RafV600E xenograft model dosed 25 mg/kg orally twice daily for
10.5 days. 58 demonstrated significant tumor growth inhibition
EXPERIMENTAL SECTION
■
Chemistry. 1H NMR spectra were recorded on either Bruker 300 or
400 MHz NMR spectrometers using deuterated DMSO (DMSO-d6)
unless otherwise stated. Temperatures are given in degrees Celsius
(°C); operations were carried out at room temperature or ambient
temperature, that is, in the range 18−25 °C. Chemical shifts are
expressed in parts per million (ppm, δ units). Coupling constants are
Figure 6. Evaluation of pERK in A549 cell lines. Cells were collected following a 75 min treatment with DMSO or compound at the indicated doses
(0.1-, 1-, 10-, and 100-fold the A375 pERK IC50). pERK refers to phospho-ERK levels and tERK represents total-ERK levels. Western blotting analyses
were performed using antibodies against the indicated proteins. 102 was used as a positive control in this study.
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Figure 8. (A) A375 xenograft dose−response mouse PD of compound 58 at 2 h post po single dose. Tumors and plasma were harvested 2 h after dosing.
Western blotting was performed on tumor samples to determine pERK inhibition. An equal amount of protein was loaded into each well and total ERK
(tERK) was used for further normalization. (B) Percent reduction in pERK from the A375 xenograft dose−response mouse PD. For additional details
including plasma concentration information, see the Supporting Information. (C) A375 mouse xenograft tumor growth inhibition study with 58 (n = 5)
dosed at 25 mg/kg po BID for 10.5 days. The vehicle group (n = 8) received 0.5% HPMC. Tumor volume and body weights were measured twice
weekly. For additional details, see the Supporting Information.
given in units of hertz (Hz). Splitting patterns describe apparent
multiplicities and are designated as s (singlet), d (doublet), dd
(doublet−doublet), t (triplet), q (quartet), m (multiplet), and br s
(broad singlet). Mass spectroscopy analyses were performed with an
Agilent 1100 equipped with Waters columns (Atlantis T3, 2.1 × 50 mm,
3 μm or Atlantis dC18, 2.1 × 50 mm, 5 μm) eluted with a gradient
mixture of H2O−acetonitrile with formic acid or ammonium acetate.
The purity determination of all reported compounds was performed
with an Agilent 1100 equipped with Waters columns (Atlantis T3, 2.1 ×
50 mm, 3 μm; or Atlantis dC18, 2.1 × 50 mm, 5 μm) eluted for >10 min
with a gradient mixture of H2O−acetonitrile with formic or trifluoro-
acetic acid at wavelengths of 220, 254, and 280 nm. All final compounds
analyzed were >95% pure unless otherwise indicated. Reverse-phase
chromatography was performed with Gilson systems using a YMC-
AQC18 reverse-phase HPLC column with dimension 20 mm/100 and
50 mm/250 in water/MeCN with 0.1% TFA, 0.1% ammonium acetate,
or 0.1% formic acid as mobile phase. Most of the reactions described
were monitored by thin-layer chromatography on 0.25 mm E. Merck
silica gel plates (60F-254), visualized with UV light or LC−MS. Flash
column chromatography was performed on an ISCO system MPLC
Combi-flash systems (4700 Superior Street, Lincoln, NE) unless
otherwise mentioned using silica gel cartridges.
N-(2-Methoxyethyl)-4-[(6-pyridin-4-ylquinazolin-2-yl)-
amino]benzamide (3). 6-Pyridin-4-ylquinazolin-2-amine (6, 50 mg,
0.225 mmol), 4-bromo-N-(2-methoxyethyl)benzamide (17, 58 mg,
0.225 mmol), Cs2CO3 (220 mg, 0.675 mmol, 3.0 equiv), and BINAP
(14 mg, 0.023 mmol, 10 mol %) in dioxane (2 mL) were treated with
Pd2(dba)3 (11 mg, 0.012 mmol, 5 mol %). The reaction mixture was
heated to 100 °C for 12 h. The reaction was then quenched with 10%
NaOH (aq) and extracted with EtOAc. The organics were dried with
NaCl (satd) and then Na2SO4 (solid). The solvent was removed under
reduced pressure and the resulting solid was purified by a Gilson HPLC
(0.1% TFA in CH3CN and water) to afford 60 mg (52%) of the title
compound. 1H NMR: 10.38 (s, 1H), 9.45 (s, 1H), 8.86 (d, 2H), 8.61 (d,
1H), 8.38 (m, 2H), 8.22 (d, 2H), 8.07 (d, 2H), 7.87 (m, 3H), 3.44 (m,
4H), 3.27 (s, 3H). LC−MS: m/z 400.
H2O (5:1, 4 mL) were treated with Pd(Ph3P)4 (206 mg, 0.179 mmol, 20
mol %). The reaction was stirred at 90 °C for 12 h. The reaction was
quenched with 10% NaOH and extracted with EtOAc. The combined
organics were dried with NaCl (satd) and then Na2SO4 (s). The solvents
were removed under reduced pressure. The crude product was purified
by column chromatography utilizing an ISCO system (EtOAc−MeOH)
to yield 100 mg (51%) of the title compound. LC−MS: m/z 223.
Following a similar procedure to that of 6, 7−16 were synthesized.
6-(2-Methylpyridin-4-yl)quinazolin-2-amine (7). The title
compound was synthesized from 6-bromoquinazolin-2-amine (5) and
(2-methylpyridin-4-yl)boronic acid. LC−MS: m/z 237.
6-(3-Methoxypyridin-4-yl)quinazolin-2-amine (8). The title
compound was synthesized from 6-bromoquinazolin-2-amine (5) and
(3-methoxypyridin-4-yl)boronic acid. LC−MS: m/z 253.
6-(3-Methylpyridin-4-yl)quinazolin-2-amine (9). The title
compound was synthesized from 6-bromoquinazolin-2-amine (5) and
(3-methylpyridin-4-yl)boronic acid. LC−MS: m/z 237.
6-(3-Chloropyridin-4-yl)quinazolin-2-amine (10). The title
compound was synthesized from 6-bromoquinazolin-2-amine (5) and
(3-chloropyridin-4-yl)boronic acid. 1H NMR: 7.71−7.65 (m, 4H),
7.58−7.45 (m, 5H).
6-Phenylquinazolin-2-amine (11). The title compound was
synthesized from 6-bromoquinazolin-2-amine (5) and phenyl boronic
acid. LC−MS: m/z 222.
6-Pyridin-3-ylquinazolin-2-amine (12). The title compound was
synthesized from 6-bromoquinazolin-2-amine (5) and pyridin-3-
ylboronic acid. LC−MS: m/z 223.
6-(4-Methylpyridin-3-yl)quinazolin-2-amine (13). The title
compound was synthesized from 6-bromoquinazolin-2-amine (5) and
(4-methylpyridin-3-yl)boronic acid. LC−MS: m/z 237.
6-(6-Methoxypyridin-3-yl)quinazolin-2-amine (14). The title
compound was synthesized from 6-bromoquinazolin-2-amine (5) and
(6-methoxypyridin-3-yl)boronic acid. LC−MS: m/z 253.
6-(6-Fluoropyridin-3-yl)quinazolin-2-amine (15). The title
compound was synthesized from 6-bromoquinazolin-2-amine (5) and
(6-fluoropyridin-3-yl)boronic acid. LC−MS: m/z 256.
6-(Pyrimidin-5-yl)quinazolin-2-amine (16). The title compound
was synthesized from 6-bromoquinazolin-2-amine (5) and pyrimidin-5-
ylboronic acid. LC−MS: m/z 224.
4-Bromo-N-(2-methoxyethyl)benzamide (17). 2-Methoxyethyl-
amine (10 mL) at 0 °C was treated with 4-bromobenzoyl chloride (2.0 g,
9.1 mmol). After 15 min, 10% HCl was added to the reaction mixture.
The resulting white solid (2.00 g, 85%) was collected by vacuum
6-Bromoquinazolin-2-amine (5). 2-Fluoro-5-bromobenzalde-
hyde (4, 1.0 g, 4.9 mmol) and guanidine carbonate (1.3 g, 7.4 mmol,
1.5 equiv) were dissolved in DMA and heated to 140 °C for 5 h. The
reaction was treated with H2O and the resulting precipitate was
collected by vacuum filtration. LC−MS: m/z 225.
6-Pyridin-4-ylquinazolin-2-amine (6). 6-Bromoquinazolin-2-
amine (5, 200 mg, 0.89 mmol), 4-pyridinylboronic acid (165 mg, 1.34
mmol, 1.5 equiv), and K2CO3 (370 mg, 2.68 mmol, 3.0 equiv) in DME/
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filtration. 1H NMR: 8.59 (t, 1H), 7.78 (d, 2H), 7.66 (d, 2H), 3.42 (m,
4H), 3.25 (s, 3H).
7.87−7.80 (m, 5H), 7.53 (t, 2H), 7.42 (t, 1H), 3.44 (m, 4H), 3.28 (s,
3H). LC−MS: m/z 399.
N-(2-Methoxyethyl)-4-[(6-pyridin-3-ylquinazolin-2-yl)-
amino]benzamide (29). The title compound was synthesized from 4-
bromo-N-(2-methoxyethyl)benzamide (17) and 6-pyridin-3-ylquinazo-
lin-2-amine (12) following a similar procedure to that of 3. 1H NMR:
10.29 (s, 1H), 9.42 (s, 1H), 9.03 (s, 1H), 8.61 (m, 1H), 8.40 (m, 1H),
8.36 (m, 1H), 8.23 (m, 2H), 8.08 (d, 2H), 7.84 (m, 3H), 7.54 (m, 1H),
3.44 (m, 4H), 3.27 (s, 3H). LC−MS: m/z 400.
3-Bromo-N-(2-methoxyethyl)benzamide (18). 2-Methoxyethyl-
amine (0.435 mL, 5.0 mmol), 3-bromobenzoic acid (1.00 g, 5.0 mmol),
and DIPEA (1.31 mL, 7.5 mmol) were dissolved in DMF (10 mL)
followed by the addition of HATU (2.85 g, 7.5 mmol). The reaction
mixture was stirred for 12 h at rt whereupon the mixture was extracted
with saturated NH4Cl solution and washed with EtOAc three times. The
organic layers were dried over magnesium sulfate, filtered, and
concentrated in vacuo to afford the crude mixture that after purification
using an ISCO system (EtOAc−MeOH) provided the title compound.
LC−MS: m/z 259.
4-Bromo-N-(2-methoxyethyl)-2-methylbenzamide (19). The
title compound was synthesized from 4-bromo-2-methylbenzoic acid
and 2-methoxyethylamine using a method analogous to that of 18. LC−
MS: m/z 273.
4-Bromo-2-fluoro-N-(2-methoxyethyl)benzamide (20). The
title compound was synthesized from 4-bromo-2-fluorobenzoic acid
and 2-methoxyethylamine using a method analogous to that of 18. LC−
MS: m/z 277.
4-Bromo-N-[2-(dimethylamino)ethyl]benzamide (21). The
title compound was synthesized from 4-bromobenzoyl chloride and
N,N-dimethylethane-1,2-diamine using a method analogous to that of
17. LC−MS: m/z 272.
4-Bromo-N-[2-(1-methylpyrrolidin-2-yl)ethyl]benzamide
(22). The title compoumd was synthesized from 4-bromobenzoyl
chloride and [2-(1-methylpyrrolidin-2-yl)ethyl]amine using a method
analogous to that of 17. LC−MS: m/z 312.
4-Bromo-N-(2-pyrrolidin-1-ylethyl)benzamide (23). The title
compound was synthesized from 4-bromobenzoic acid and (2-
pyrrolidin-1-ylethyl)amine using a method analogous to that of 18.
LC−MS: m/z 298.
N-(2-Methoxyethyl)-4-{[6-(2-methylpyridin-4-yl)quinazolin-
2-yl]amino}benzamide (24). The title compound was synthesized
from 4-bromo-N-(2-methoxyethyl)benzamide (17) and 6-(2-methyl-
pyridin-4-yl)quinazolin-2-amine (7) following a similar procedure to
that of 3. 1H NMR: 10.30 (s, 1H), 9.42 (s, 1H), 8.54 (d, 1H), 8.44 (s,
1H), 8.38 (m, 1H), 8.27 (d, 1H), 8.08 (m, 2H), 7.84 (m, 2H), 7.72 (s,
1H), 7.62 (m, 2H), 3.44 (m, 4H), 3.27 (s, 3H), 2.56 (s, 3H). LC−MS:
m/z 414. Purity: 94%.
N-(2-Methoxyethyl)-4-{[6-(3-methoxypyridin-4-yl)-
quinazolin-2-yl]amino}benzamide (25). The title compound was
synthesized from 4-bromo-N-(2-methoxyethyl)benzamide (17) and 6-
(3-methoxypyridin-4-yl)quinazolin-2-amine (8) following a similar
procedure to that of 3. 1H NMR: 10.27 (s, 1H), 9.41 (s, 1H), 8.52 (s,
1H), 8.38 (m, 1H), 8.33 (d, 1H), 8.19 (m, 1H), 8.07 (d, 3H), 7.85 (d,
2H), 7.78 (d, 1H), 7.49 (d, 1H), 3.94 (s, 3H), 3.44 (m, 4H), 3.27 (s,
3H). LC−MS: m/z 430.
N-(2-Methoxyethyl)-4-{[6-(3-methylpyridin-4-yl)quinazolin-
2-yl]amino}benzamide (26). The title compound was synthesized
from 4-bromo-N-(2-methoxyethyl)benzamide (17) and 6-(3-methyl-
pyridin-4-yl)quinazolin-2-amine (9) following a similar procedure to
that of 3. 1H NMR: 10.28 (s, 1H), 9.41 (s, 1H), 8.55 (s, 1H), 8.50 (d,
1H), 8.42−8.35 (m, 1H), 8.10−8.02 (m, 3H), 7.93−7.78 (m, 4H), 7.36
(d, 1H), 3.47−3.41 (m, 4H), 3.27 (s, 3H), 2.32 (s, 3H). LC−MS: m/z
414.
4-{[6-(3-Chloropyridin-4-yl)quinazolin-2-yl]amino}-N-(2-
methoxyethyl)benzamide (27). The title compound was synthe-
sized from 4-bromo-N-(2-methoxyethyl)benzamide (17) and 6-(3-
chloropyridin-4-yl)quinazolin-2-amine (10) following a similar proce-
dure to that of 3. 1H NMR: 10.34 (s, 1H), 9.47 (s, 1H), 8.80 (s, 1H),
8.65 (d, 1H), 8.46−8.35 (m, 1H), 8.22−8.15 (m, 1H), 8.09 (d, 2H), 8.01
(dd, 1H), 7.91−7.81 (m, 3H), 7.64 (d, 1H), 3.50−3.40 (m, 4H), 3.28 (s,
3H). LC−MS: m/z 434. Purity: 90%.
N-(2-Methoxyethyl)-4-(6-phenylquinazolin-2-ylamino)-
benzamide (28). The title compound was synthesized from 4-bromo-
N-(2-methoxyethyl)benzamide (17) and 6-phenylquinazolin-2-amine
(11) following a similar procedure to that of 3. 1H NMR: 10.23 (s, 1H),
9.42 (s, 1H), 8.37 (m, 1H), 8.28 (m, 1H), 8.21 (m, 1H), 8.08 (d, 2H),
N-(2-Methoxyethyl)-4-{[6-(4-methylpyridin-3-yl)quinazolin-
2-yl]amino}benzamide (30). The title compound was synthesized
from 4-bromo-N-(2-methoxyethyl)benzamide (17) and 6-(4-methyl-
pyridin-3-yl)quinazolin-2-amine (13) following a similar procedure to
that of 3. 1H NMR: 10.26 (s, 1H), 9.40 (s, 1H), 8.47 (m, 2H), 8.38 (m,
1H), 8.07 (m, 2H), 8.00 (s, 1H), 7.84 (m, 4H), 7.37 (d, 1H), 3.44 (m,
4H), 3.27 (s, 3H), 2.32 (s, 3H). LC−MS: m/z 414.
N-(2-Methoxyethyl)-4-{[6-(6-methoxypyridin-3-yl)-
quinazolin-2-yl]amino}benzamide (31). The title compound was
synthesized from 4-bromo-N-(2-methoxyethyl)benzamide (17) and 6-
(6-methoxypyridin-3-yl)quinazolin-2-amine (14) following a similar
procedure to that of 3. 1H NMR: 10.23 (s, 1H), 9.38 (s, 1H), 8.61 (m,
1H), 8.37 (m, 1H), 8.25 (m, 1H), 8.16 (m, 2H), 8.08 (m, 2H), 7.83 (m,
3H), 6.98 (d, 1H), 3.91 (s, 3H), 3.45 (m, 4H), 3.27 (s, 3H). LC−MS:
m/z 430.
4-{[6-(6-Fluoropyridin-3-yl)quinazolin-2-yl]amino}-N-(2-
methoxyethyl)benzamide (32). The title compound was synthe-
sized from 4-bromo-N-(2-methoxyethyl)benzamide (17) and 6-(6-
fluoropyridin-3-yl)quinazolin-2-amine (15) following a similar proce-
dure to that of 3. 1H NMR: 10.27 (s, 1H), 9.40 (s, 1H), 8.69 (bs, 1H),
8.38 (m, 3H), 8.21 (m, 1H), 8.06 (d, 2H), 7.84 (m, 3H), 7.35 (m, 1H),
3.45 (m, 4H), 3.27 (s, 3H). LC−MS: m/z 418.
N-(2-Methoxyethyl)-4-(6-(pyrimidin-5-yl)quinazolin-2-
ylamino)benzamide (33). The title compound was synthesized from
4-bromo-N-(2-methoxyethyl)benzamide (17) and 6-(pyrimidin-5-yl)-
quinazolin-2-amine (16) following a similar procedure to that of 3. 1H
NMR: 10.33 (s, 1H), 9.42 (s, 1H), 9.29 (s, 2H), 9.23 (s, 1H), 8.47 (m,
1H), 8.33 (m, 1H), 8.07 (m, 2H), 7.87 (m, 3H), 3.46 (m, 4H), 3.28 (s,
3H). LC−MS: m/z 401.
N-(2-Methoxyethyl)-3-[(6-pyridin-4-ylquinazolin-2-yl)-
amino]benzamide (34). The title compound was synthesized from 6-
pyridin-4-ylquinazolin-2-amine (6) and 3-bromo-N-(2-methoxyethyl)-
1
benzamide (18) following a similar procedure to that of 3. H NMR:
10.17 (s, 1H), 9.41 (s, 1H), 8.68 (d, 2H), 8.45 (m, 3H), 8.29 (d, 1H),
8.16 (d, 1H), 7.84 (d, 2H), 7.78 (d, 1H), 7.44 (m, 2H), 3.58 (m, 4H),
3.29 (s, 3H). LC−MS: m/z 400.
N-(2-Methoxyethyl)-2-methyl-4-[(6-pyridin-4-ylquinazolin-
2-yl)amino]benzamide (35). The title compound was synthesized
from 4-bromo-N-(2-methoxyethyl)-2-methylbenzamide (19) and 6-
pyridin-4-ylquinazolin-2-amine (6) following a similar procedure to that
of 3. 1H NMR: 10.12 (s, 1H), 9.41 (s, 1H), 8.68 (d, 2H), 8.44 (m, 1H),
8.28 (d, 1H), 8.16 (m, 1H), 7.92 (d, 1H), 7.83 (m, 4H), 7.33 (d, 1H),
3.45 (m, 4H), 3.28 (s, 3H), 2.38 (s, 3H). LC−MS: m/z 414.
2-Fluoro-N-(2-methoxyethyl)-4-[(6-pyridin-4-ylquinazolin-2-
yl)amino]benzamide (36). The title compound was synthesized from
4-bromo-2-fluoro-N-(2-methoxyethyl)benzamide (20) and 6-pyridin-4-
ylquinazolin-2-amine (6) following a similar procedure to that of 3. 1H
NMR: 10.52 (s, 1H), 9.47 (s, 1H), 8.69 (d, 2H), 8.48 (s, 1H), 8.33 (d,
1H), 8.16 (d, 1H), 8.02 (m, 1H), 7.86 (m, 3H), 7.71 (m, 2H), 3.44 (m,
4H), 3.27 (s, 3H). LC−MS: m/z 418.
N-[2-(Dimethylamino)ethyl]-4-[(6-pyridin-4-ylquinazolin-2-
yl)amino]benzamide (37). The title compound was synthesized from
4-bromo-N-[2-(dimethylamino)ethyl]benzamide (21) and 6-pyridin-4-
ylquinazolin-2-amine (6) following a similar procedure to that of 3. 1H
NMR: 10.31 (s, 1H), 9.43 (s, 1H), 8.68 (d, 2H), 8.46 (m, 1H), 8.28 (m,
2H), 8.07 (m, 2H), 7.84 (m, 5H), 3.36 (m, 2H), 2.39 (m, 2H), 2.17 (s,
6H). LC−MS: m/z 413.
N-[2-(1-Methylpyrrolidin-2-yl)ethyl]-4-[(6-pyridin-4-ylquina-
zolin-2-yl)amino]benzamide (38). The title compound was
synthesized from 4-bromo-N-[2-(1-methylpyrrolidin-2-yl)ethyl]-
benzamide (22) and 6-pyridin-4-ylquinazolin-2-amine (6) following a
similar procedure to that of 3. 1H NMR: 10.30 (s, 1H), 9.43 (s, 1H), 8.69
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(d, 2H), 8.46 (s, 1H), 8.30 (m, 2H), 8.07 (d, 2H), 7.85 (m, 5H), 3.27 (m,
2H), 2.92 (m, 1H), 2.20 (s, 3H), 2.00 (m, 4H), 1.62 (m, 2H), 1.44 (m,
2H). LC−MS: m/z 453.
4-[(6-Pyridin-4-ylquinazolin-2-yl)amino]-N-(2-pyrrolidin-1-
ylethyl)benzamide (39). The title compound was synthesized from 4-
bromo-N-(2-pyrrolidin-1-ylethyl)benzamide (23) and 6-pyridin-4-
ylquinazolin-2-amine (6) following a similar procedure to that of 3.
1H NMR: 10.31 (s, 1H), 9.43 (s, 1H), 8.69 (d, 2H), 8.46 (s, 1H), 8.28
Compounds 49 and 50 were prepared following a similar procedure
to that of 48.
N-(4-{[(2-Methoxyethyl)(methyl)amino]methyl}phenyl)-6-
pyridin-4-ylquinazolin-2-amine (49). The title compound was
synthesized from 4-[(6-pyridin-4-ylquinazolin-2-yl)amino]-
benzaldehyde (40) and (2-methoxyethyl)methylamine. 1H NMR:
10.01 (s, 1H), 9.37 (s, 1H), 8.67 (d, 2H), 8.41 (d, 1H), 8.25 (m, 1H),
8.18 (s, 1H), 7.92 (d, 2H), 7.83 (d, 2H), 7.75 (d, 1H), 7.24 (d, 2H), 3.66
(s, 2H), 3.47 (m, 2H), 3.20 (s, 3H), 2.66 (m, 2H), 2.27 (s, 3H). LC−
MS: m/z 422 (M + Na).
(m, 2H), 8.07 (d, 2H), 7.84 (m, 5H), 3.35 (m, 2H), 2.41−2.53 (m, 4H),
1.67 (m, 6H). LC−MS: m/z 439.
N,N-Dimethyl-N′-{4-[(6-pyridin-4-ylquinazolin-2-yl)amino]-
benzyl}ethane-1,2-diamine (50). The title compound was synthe-
sized from 4-[(6-pyridin-4-ylquinazolin-2-yl)amino]benzaldehyde (40)
and N,N-dimethylethane-1,2-diamine. 1H NMR: 10.00 (s, 1H), 9.38 (s,
1H), 8.69 (d, 2H), 8.43 (d, 1H), 8.26 (m, 1H), 7.92 (d, 2H), 7.84 (d,
2H), 7.76 (d, 1H), 7.28 (d, 2H), 3.67 (s, 2H), 2.54 (m, 2H), 2.33 (m,
2H), 2.12 (s, 6H). LC−MS: m/z 397 (M − H).
6-Pyridin-4-yl-N-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-
quinazolin-2-amine (51). The title compound was synthesized from
6-pyridin-4-ylquinazolin-2-amine (6) and 1-[2-(4-bromophenoxy)-
ethyl]pyrrolidine (commercially available) using a method analogous
to the preparation of 3. 1H NMR: 9.86 (s, 1H), 9.33 (s, 1H), 8.67 (d,
2H), 8.39 (s, 1H), 8.21 (m, 1H), 7.83 (m, 4H), 7.70 (d, 1H), 6.95 (d,
2H), 4.04 (m, 2H), 2.79 (m, 2H), 2.53 (m, 4H), 1.68 (m, 4H). LC−MS:
m/z 412. Purity: 90%.
4-[(6-Pyridin-4-ylquinazolin-2-yl)amino]benzaldehyde (40).
6-Pyridin-4-ylquinazolin-2-amine (6, 2 g, 9.0 mmol), 4-bromobenzal-
dehyde (1.83 g, 9.9 mmol), Cs2CO3 (8.8 g, 27 mmol, 3.0 equiv), and
BINAP (1.12 g, 1.8 mmol, 0.2 equiv) in dioxane (60 mL) were treated
with Pd2(dba)3 (825 mg, 0.9 mmol). The reaction mixture was heated to
100 °C for 3 h. The reaction was cooled and filtered. The crude mixture
was purified on an ISCO system (EtOAc/Et3N) to yield 1.5 g (51%) of
the title compound. 1H NMR: 10.61 (s, 1H), 9.87 (s, 1H), 9.48 (s, 1H),
8.69 (m, 2H), 8.49 (m, 1H), 8.33 (m, 1H), 8.25 (d, 2H), 7.87 (m, 5H).
LC−MS: m/z 327.
N-(4-Bromophenyl)-3-methoxy-N-methylpropanamide (41).
The title compound was synthesized from 3-methoxypropanoic acid and
(4-bromophenyl)methylamine using a method analogous to that of 18.
LC−MS: m/z 273.
[1-(4-Bromophenyl)ethyl](2-methoxyethyl)amine (42). To a
solution of 1-(4-bromophenyl)ethanone (500 mg, 2.51 mmol) and (2-
methoxyethyl)amine (188 mg, 2.51 mmol) in toluene (13 mL) were
added diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (907
mg, 3.51 mmol), thiourea (19 mg, 0.25 mmol), and 5 Å molecular sieves
(∼5 g). The reaction was heated at 50 °C under nitrogen for
approximately 40 h. The reaction mixture was filtered, solvent was
evaporated under reduced pressure, and the residue was purified by an
ISCO system (EtOAc/hexane, TLC with I2) to give 120 mg of a
colorless oil (19% yield). 1H NMR (CDCl3): 7.39 (d, 2H), 7.17 (d, 2H),
3.69 (m, 1H), 3.41 (m, 2H), 3.30 (s, 3H), 2.59 (m, 2H), 1.29 (d, 3H).
N-(1-(4-Bromophenyl)ethyl)-3-methoxypropanamide (43).
The title compound was synthesized from 1-(4-bromophenyl)-
ethylamine and 3-methoxypropanoyl chloride using a method
analogous to that of 17. LC−MS: m/z 287.
N-[(1R)-1-(4-Bromophenyl)ethyl]-3-methoxypropanamide
(44). The title compound was synthesized from [(1R)-1-(4-
bromophenyl)ethyl]amine and 3-methoxypropanoic acid using a
method analogous to that of 18. LC−MS: m/z 287.
N-[(1S)-1-(4-Bromophenyl)ethyl]-3-methoxypropanamide
(45). The title compound was synthesized from [(1S)-1-(4-
bromophenyl)ethyl]amine and 3-methoxypropanoic acid using a
method analogous to that of 18. LC−MS: m/z 287.
N-(1-(4-Bromophenyl)ethyl)acetamide (46). The title com-
pound was synthesized from 1-(4-bromophenyl)ethanamine and acetyl
chloride using a method analogous to that of 17. LC−MS: m/z 243.
(R)-N-(1-(4-Bromophenyl)ethyl)acetamide (47). The title com-
pound was synthesized from (R)-1-(4-bromophenyl)ethanamine and
acetyl chloride using a method analogous to that of 17. LC−MS: m/z
243.
N-(4-{[(2-Methoxyethyl)amino]methyl}phenyl)-6-pyridin-4-
ylquinazolin-2-amine (48). 4-[(6-Pyridin-4-ylquinazolin-2-yl)-
amino]benzaldehyde (40, 70 mg, 0.21 mmol) and (2-methoxyethyl)-
amine (18 mg, 0.24 mmol) in 5 mL of MeOH (with 3 Å molecular
sieves) were stirred at room temperature, whereupon a few drops of
acetic acid were added. NaBH3CN (22 mg, 1.6 equiv) was then added
and the reaction mixture was stirred at room temperature overnight
followed by quenching with NaOH (1 N, aq, ∼5 mL). The reaction
mixture was extracted with EtOAc, and the water layers were then
extracted with EtOAc three times. The combined organic layers were
washed with water and brine, evaporated, and purified by Gilson HPLC
(0.1% 10 mM ammonium acetate in CH3CN and water) to give 25 mg
(95%) of the title compound. 1H NMR: 9.99 (s, 1H), 9.36 (s, 1H), 8.67
(d, 2H), 8.41 (d, 1H), 8.25 (m, 1H), 7.91 (d, 2H), 7.83 (d, 2H), 7.75 (d,
1H), 7.27 (d, 2H), 3.66 (s, 2H), 3.39 (m, 2H), 3.23 (s, 3H), 2.63 (m,
2H). LC−MS: m/z 384 (M−H).
3-Methoxy-N-methyl-N-(4-(6-(pyridin-4-yl)quinazolin-2-
ylamino)phenyl)propanamide (52). The title compound was
synthesized from N-(4-bromophenyl)-3-methoxy-N-methylpropana-
mide (41) and 6-pyridin-4-ylquinazolin-2-amine (6) using a method
analogous to the preparation of 3, except with Xantphos and
palladium(II) acetate and by heating in a microwave at 160 °C for 1
h. 1H NMR: 10.2 (s, 1H), 9.41 (s, 1H), 8.68 (d, 2H), 8.44 (s, 1H), 8.28
(d, 1H), 8.06 (d, 2H), 7.83 (m, 3H), 7.28 (d, 2H), 3.48 (t, 2H), 3.14 (m,
6H), 2.27 (t, 2H). LC−MS: m/z 414.
N-(4-{1-[(2-Methoxyethyl)amino]ethyl}phenyl)-6-pyridin-4-
ylquinazolin-2-amine (53). The title compound was synthesized
from [1-(4-bromophenyl)ethyl](2-methoxyethyl)amine (42) and 6-
pyridin-4-ylquinazolin-2-amine (6) using a method analogous to the
preparation of 3. 1H NMR: 9.97 (s, 1H), 9.36 (s, 1H), 8.67 (d, 2H), 8.41
(d, 1H), 8.25 (m, 2H), 7.90 (d, 2H), 7.83 (d, 2H), 7.74 (d, 1H), 7.27 (d,
2H), 3.68 (m, 1H), 3.34 (m, 2H), 3.21 (s, 3H), 2.43 (m, 2H), 1.25 (d,
3H). LC−MS: m/z 422 (M + Na).
3-Methoxy-N-(1-{4-[(6-pyridin-4-ylquinazolin-2-yl)amino]-
phenyl}ethyl)propanamide (54). The title compound was synthe-
sized from N-(1-(4-bromophenyl)ethyl)-3-methoxypropanamide (43)
and 6-pyridin-4-ylquinazolin-2-amine (6) using a method analogous to
the preparation of 3, using Xantphos and palladium(II) acetate and by
heating in a microwave at 160 °C for 1 h. 1H NMR: 9.98 (s, 1H), 9.36 (s,
1H), 8.68 (d, 2H), 8.42 (s, 1H), 8.25 (d, 2H), 7.89 (d, 2H), 7.84 (d, 2H),
7.76 (d, 1H), 7.27 (d, 2H), 4.9 (m, 1H), 3.52 (t, 2H), 3.21 (s, 3H), 2.35
(t, 2H), 1.34 (s, 3H). LC−MS: m/z 428. Purity: 93%.
The following compounds 55−58 were prepared in a similar fashion
as 54.
3-Methoxy-N-((1R)-1-{4-[(6-pyridin-4-ylquinazolin-2-yl)-
amino]phenyl}ethyl)propanamide (55). The title compound was
synthesized from N-[(1R)-1-(4-bromophenyl)ethyl]-3-methoxypropa-
namide (44) and 6-pyridin-4-ylquinazolin-2-amine (6). 1H NMR: 9.98
(s, 1H), 9.36 (s, 1H), 8.67 (d, 2H), 8.41 (s, 1H), 8.24 (d, 2H), 7.89 (d,
2H), 7.83 (d, 2H), 7.75 (d, 1H), 7.27 (d, 2H), 4.90 (m, 1H), 3.52 (t,
2H), 3.21 (s, 3H), 2.35 (t, 2H), 1.34 (d, 3H). LC−MS: m/z 428.
3-Methoxy-N-((1S)-1-{4-[(6-pyridin-4-ylquinazolin-2-yl)-
amino]phenyl}ethyl)propanamide (56). The title compound was
synthesized from N-[(1S)-1-(4-bromophenyl)ethyl]-3-methoxypropa-
namide (45) and 6-pyridin-4-ylquinazolin-2-amine (6). 1H NMR: 9.98
(s, 1H), 9.36 (s, 1H), 8.67 (d, 2H), 8.41 (s, 1H), 8.25 (d, 2H), 7.89 (d,
2H), 7.83 (d, 2H), 7.74 (d, 1H), 7.27 (d, 2H), 4.9 (m, 1H), 3.52 (t, 2H),
3.21 (s, 3H), 2.35 (t, 2H), 1.3 (d, 3H). LC−MS: m/z 428.
N-(1-(4-(6-(Pyridin-4-yl)quinazolin-2-ylamino)phenyl)ethyl)-
acetamide (57). The title compound was synthesized from N-(1-(4-
bromophenyl)ethyl)acetamide (46) and 6-pyridin-4-ylquinazolin-2-
1
amine (6). H NMR: 9.98 (s, 1H), 9.36 (s, 1H), 8.67 (d, 2H), 8.41
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(s, 1H), 8.25 (m, 2H), 7.90 (d, 2H), 7.83 (d, 2H), 7.75 (d, 1H), 7.27 (d,
2H), 4.88 (m, 1H), 1.83 (s, 3H), 1.33 (d, 3H). LC−MS: m/z 384.
Purity: 90%.
(m, 3H), 7.75−7.64 (m, 2H), 6.94 (d, 2H), 4.10−4.03 (m, 2H), 3.65 (t,
2H), 3.31 (s, 3H). LC−MS: m/z 373.
3-Methoxy-N-(4-(6-(pyridin-4-yl)quinazolin-2-ylamino)-
phenyl)propanamide (68). The title compound was synthesized
from N-(4-(6-bromoquinazolin-2-ylamino)phenyl)-3-methoxypropa-
namide (63) and 4-pyridinylboronic acid using a method similar to
that of 6, except with PdCl2(dppf)·CH2Cl2 as catalyst. Purification was
performed by Gilson HPLC (0.1% ammonium acetate in CH3CN and
(R)-N-(1-(4-(6-(Pyridin-4-yl)quinazolin-2-ylamino)phenyl)-
ethyl)acetamide (58). The title compound was synthesized from (R)-
N-(1-(4-bromophenyl)ethyl)acetamide (47) and 6-pyridin-4-ylquina-
zolin-2-amine (6). 1H NMR: 9.98 (s, 1H), 9.36 (s, 1H), 8.67 (d, 2H),
8.41 (s, 1H), 8.25 (m, 2H), 7.90 (d, 2H), 7.83 (d, 2H), 7.76 (d, 1H), 7.27
(d, 2H), 4.88 (m, 1H), 1.83 (s, 3H), 1.33 (d, 3H). LC−MS: m/z 384.
[4-(2-Methoxyethoxy)phenyl]amine (59). 4-Aminophenol (2.2
g, 19.8 mmol) and potassium carbonate (5.5 g, 39.6 mmol) were
dissolved in DMF. To the reaction mixture was added 1-chloro-2-
methoxyethane (2 mL, 21.8 mmol) and the mixture was stirred
overnight at 80 °C. The resulting solids were filtered, and the filtrate was
washed with brine, dried over Na2SO4, concentrated under reduced
pressure, and purified by an ISCO system (50−100% EtOAc in hexanes)
to afford 538 mg of the title compound (16% yield). LC−MS: m/z 168.
N-(4-Aminophenyl)-3-methoxypropanamide (60). The title
compound was synthesized from deprotection of tert-butyl {4-[3-
methoxypropanoyl)amino]phenyl}carbamate intermediate, which was
synthesized in analogy to 18 from tert-butyl (4-aminophenyl)carbamate
and 3-methoxypropanoic acid and was carried forward crude into the
BOC group deprotection using standard acid mediated conditions. LC−
MS: m/z 403.
N-(2-Methoxyethyl)-N-methylbenzene-1,4-diamine (61). To a
solution of N-(2-methoxyethyl)-N-methyl-4-nitroaniline (prepared
from 1-bromo-4-nitrobenzene and 2-methoxyethylamine using a
method analogous to the preparation of 3, 700 mg, 3.3 mmol) in
ethanol (8 mL) was added SnCl2·H2O (1.8 g, 8.3 mmol), and the
reaction was stirred overnight at 70 °C. To the reaction mixture was then
added 4.0 M NaOH. The mixture was extracted (2×) with EtOAc, and
the combined organic extracts were dried over Na2SO4 and
concentrated under reduced pressure to afford 505 mg of a green oil,
which was taken on crude immediately to the next reaction.
6-Bromo-N-[4-(2-methoxyethoxy)phenyl]quinazolin-2-
amine (62). To [4-(2-methoxyethoxy)phenyl]amine (59, 100 mg,
0.598 mmol) in propan-2-ol (3 mL) was added 6-bromo-2-
chloroquinazoline (prepared in analogy to ref 16; 131 mg, 0.538
mmol). The reaction mixture was stirred for 2 h at 100 °C and then
allowed to cool to room temperature. The title compound was formed as
a precipitate from the solution yielding 123 mg (56% yield). LC−MS:
m/z 388.
1
water) to yield a yellow solid (16 mg, 0.04 mmol, 30.6% yield). H
NMR: 10.17 (s, 1H), 9.98 (s, 1H), 9.39 (s, 1H), 8.99 (d, 2H), 8.72 (s,
1H), 8.50 (d, 2H), 8.43 (d, 1H), 7.90 (d, 2H), 7.81 (d, 1H), 7.62−7.55
(m, 2H), 3.61 (t, 2H), 3.24 (s, 3H), 2.54 (t, 2H). LC−MS: m/z 400.
N-Methyl-N-{4-[(6-pyridin-4-ylquinazolin-2-yl)amino]-
phenyl}acetamide (69). The title compound was synthesized from N-
{4-[(6-bromoquinazolin-2-yl)amino]phenyl}-N-methylacetamide (64)
and 4-pyridinylboronic acid using a similar method to that of 68, except
with cesium carbonate as base, dioxane/water (4:1) as solvent, and with
reaction temperature of 80 °C. Purification was performed by Gilson
1
HPLC (0.1% ammonium acetate in CH3CN and water). H NMR:
10.20 (s, 1H), 9.41 (s, 1H), 8.68 (d, 2H), 8.45 (s, 1H), 8.28 (m, 1H),
8.06 (d, 2H), 7.82 (m, 3H), 7.29 (d, 2H), 3.14 (s, 3H), 1.78 (s, 3H).
LC−MS: m/z 370.
N-{4-[(6-Pyridin-4-ylquinazolin-2-yl)amino]phenyl}-
acetamide (70). The title compound was synthesized from N-{4-[(6-
bromoquinazolin-2-yl)amino]phenyl}acetamide (65) and 4-pyridinyl-
boronic acid using a similar method to that of 69. 1H NMR: 9.96 (s, 1H),
9.87 (s, 1H), 9.35 (s, 1H), 8.68 (m, 2H), 8.40 (s, 1H), 8.25 (m, 1H), 7.86
(m, 5H), 7.53 (m, 2H), 2.02 (s, 3H). LC−MS: m/z 356.
N-(2-Methoxyethyl)-N-methyl-N′-(6-pyridin-4-ylquinazolin-
2-yl)benzene-1,4-diamine (71). The title compound was synthesized
from N′-(6-bromoquinazolin-2-yl)-N-(2-methoxyethyl)-N-methylben-
zene-1,4-diamine (66) and 4-pyridinylboronic acid using a similar
method to that of 69. 1H NMR: 9.86 (s, 1H), 9.30 (s, 1H), 8.78 (d, 2H),
8.49 (s, 1H), 8.28 (d, 1H), 8.08 (d, 2H), 7.80 (s, 2H), 7.69 (d, 1H), 6.83
(s, 2H), 3.48 (bs, 7H), 2.94 (s, 3H). LC−MS: m/z 386.
(R)-tert-Butyl 1-(4-Bromophenyl)ethylcarbamate (72). (R)-1-
(4-Bromophenyl)ethanamine (5 g, 24.99 mmol), BOC anhydride (6.00
g, 27.94 mmol), and THF (50 mL) were stirred at room temperature for
12 h. The resulting reaction mixture was concentrated under reduced
pressure to afford a white solid which was used directly in the next step.
1H NMR: 7.50 (m, 2H), 7.24 (m, 2H), 4.48−4.66 (m, 1H), 1.35 (s, 9
H), 1.27 (d, J = 7.16 Hz, 3H). LC−MS: m/z 200 (M + H − BOC).
(R)-N-(4-(1-Aminoethyl)phenyl)-6-(pyridin-4-yl)quinazolin-2-
amine Hydrochloride (73). The title compound was synthesized from
(R)-tert-butyl 1-(4-bromophenyl)ethylcarbamate (72) and 6-pyridin-4-
ylquinazolin-2-amine (6) using a method analogous to the preparation
of 3, with Xantphos and palladium(II) acetate and by heating at 100 °C
for 4 h. Purification on an ISCO system (100% hexanes to 100% ethyl
acetate) afforded (R)-tert-butyl 1-(4-(6-(pyridin-4-yl)quinazolin-2-
ylamino)phenyl)ethylcarbamate, which was carried forward and treated
with 2 N HCl in ether solution followed by concentration under reduced
pressure to afford the title compound. 1H NMR: 10.20−10.43 (m, 1H),
9.44 (s, 1H), 8.97 (d, J = 5.27 Hz, 2H), 8.65−8.76 (m, 1H), 8.25−8.49
(m, 6H), 8.03 (m, 2H), 7.85 (d, J = 8.67 Hz, 1H), 7.49 (m, 2H), 4.39 (m,
1H), 1.52 (d, J = 6.59 Hz, 3H). LC−MS: m/z 342.
Benzyl 3-Hydroxy-5-oxo-5,6-dihydropyridine-1(2H)-carbox-
ylate (74). To a 50 mL round-bottomed flask was added 3,5-
dimethoxypyridine (0.983 g, 7.06 mmol) and NaBH4 (0.481 g, 12.72
mmol) in acetonitrile (20.54 mL) to give a colorless suspension. Benzyl
carbonochloridate (1.256 mL, 8.48 mmol) was added, dropwise, at 0 °C
over 20 min to produce a milky mixture. The reaction mixture was
stirred for an additional 30 min at 0 °C (whereby LC−MS indicated the
consumption of starting material) and then poured into 1 M aqueous
NaOH (20 mL). The reaction was extracted with EtOAc (3 × 20 mL),
the organic fractions were combined and rinsed with brine (20 mL),
dried over Na2SO4, filtered, and concentrated under reduced pressure to
yield an oily mixture. Exposure of the mixture to MeOH produced small
crystals, which were isolated via vacuum filtration. The resulting filtrate
was concentrated under reduced pressure, exposed to MeOH (5 mL),
cooled at 0 °C for 30 min, and the resulting solid was isolated via
N-(4-(6-Bromoquinazolin-2-ylamino)phenyl)-3-methoxypro-
panamide (63). The title compound was synthesized from 6-bromo-2-
chloroquinazoline (prepared in analogy to ref 16) and N-(4-amino-
phenyl)-3-methoxypropanamide (60) using a method analogous to 62.
LC−MS: m/z 403.
N-{4-[(6-Bromoquinazolin-2-yl)amino]phenyl}-N-methylace-
tamide (64). 6-Bromo-2-chloroquinazoline (prepared in analogy to ref
16; 100 mg, 0.412 mmol, 1.0 equiv), N-(4-aminophenyl)-N-
methylacetamide (101 mg, 0.617 mmol, 1.5 equiv), and acetonitrile
(5.0 mL) were added to a microwave vial which was heated in a
microwave at 125 °C for 30 min. The reaction was then concentrated to
afford a crude solid that was purified by an ISCO system (100% hexanes
to 100% EtOAc) to obtain a yellow solid. LC−MS: m/z 372.
N-{4-[(6-Bromoquinazolin-2-yl)amino]phenyl}acetamide
(65). The title compound was synthesized from 6-bromo-2-
chloroquinazoline (prepared in analogy to ref 16) and N-(4-
aminophenyl)acetamide using a method similar to that of 64. LC−
MS: m/z 372.
N′-(6-Bromoquinazolin-2-yl)-N-(2-methoxyethyl)-N-methyl-
benzene-1,4-diamine (66). The title compound was synthesized
from 6-bromo-2-chloroquinazoline (prepared in analogy to ref 16) and
N-(2-methoxyethyl)- N-methylbenzene-1,4-diamine (61) prepared by a
method similar to that of 62. LC−MS: m/z 389.
N-[4-(2-Methoxyethoxy)phenyl]-6-pyridin-4-ylquinazolin-2-
amine (67). The title compound was synthesized from 4-pyridinylbor-
onic acid and 6-bromo-N-[4-(2-methoxyethoxy)phenyl]quinazolin-2-
amine (62) using a procedure analogous to that of 6. 1H NMR: 9.88 (s,
1H), 9.32 (s, 1H), 8.66 (s, 2H), 8.39 (s, 1H), 8.22 (d, 1H), 7.90−7.80
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vacuum-filtration. The filtrate was concentrated via reduced pressure to
yield the dimethyl enol ether intermediate, which was subsequently
dissolved in 1,4-dioxane (20 mL). Then 4 M aqueous HCl was added
(10 mL) and reaction mixture was stirred at 23 °C for 3 h. The reaction
was then extracted with EtOAc (3 × 20 mL), and the organic extracts
were combined, rinsed with brine (30 mL), dried over Na2SO4, filtered,
and concentrated under reduced pressure to yield the title compound
(75% purity by LC−MS), which was taken forward as the crude material
to the next step. LC−MS: m/z 248.
(m, 1H), 8.46 (m, 2H), 7.91 (m, 5H), 7.28 (m, 2H), 4.88 (m, 1H), 3.67
(m, 2H), 2.94 (m, 2H), 1.95 (m, 1H), 1.73 (m, 2H), 1.58 (m, 2H), 1.37
(m, 3H). LC−MS: m/z 453.
(R)-N-(1-(4-(6-(Pyridin-4-yl)quinazolin-2-ylamino)phenyl)-
ethyl)piperidine-4-carboxamide Hydrochloride (79). The title
compound was synthesized from 1-(tert-butoxycarbonyl)piperidine-4-
carboxylic acid and (R)-N-(4-(1-aminoethyl)phenyl)-6-(pyridin-4-yl)-
quinazolin-2-amine hydrochloride (73) using a method similar to that of
75. 1H NMR: 10.21 (s, 1H), 9.41 (s, 1H), 9.24 (d, J = 9.80 Hz, 1H), 9.00
(d, J = 6.97 Hz, 2H), 8.69−8.86 (m, 2H), 8.52 (d, J = 6.97 Hz, 2H), 8.45
(dt, J = 8.76, 2.03 Hz, 2H), 7.89 (m, 2H), 7.80 (d, J = 9.04 Hz, 1H), 7.29
(m, 2H), 4.89 (m, 1H), 3.59−3.75 (m, 1H), 3.39−3.54 (m, 1H), 3.17−
3.31 (m, 2H), 2.73−2.94 (m, 2H), 1.63−1.94 (m, 3H), 1.36 (d, J = 6.97
Hz, 3 H). LC−MS: m/z 453.
(R)-N-((R)-1-(4-(6-(Pyridin-4-yl)quinazolin-2-ylamino)-
phenyl)ethyl)piperidine-2-carboxamide Hydrochloride (75). In
a 10 mL vial, (R)-N-(4-(1-aminoethyl)phenyl)-6-(pyridin-4-yl)-
quinazolin-2-amine hydrochloride (73, 5.567 g, 12.35 mmol), (R)-1-
(tert-butoxycarbonyl)piperidine-2-carboxylic acid hydrochloride (3.94
g, 14.82 mmol), DIPEA (9.71 mL, 55.57 mmol), and HATU (9.39 g,
24.70 mmol) were combined in DMF (61.7 mL) to give a yellow
solution. The reaction was stirred for 18 h at 60 °C, poured into 10%
aqueous K2HPO4 (60 mL), and extracted with EtOAc (3 × 40 mL). The
organic extracts were combined and rinsed with saturated NH4Cl (3 ×
30 mL) and brine (40 mL), dried over Na2SO4, filtered, and
concentrated under reduced pressure to yield a yellow foam. HCl (4
M) in dioxane (31.2 mL, 125 mmol) and MeOH (6.24 mL) were added
to afford a yellow solution. The reaction was stirred for 4 h at 23 °C and
then evaporated to dryness, followed by addition of toluene,
concentration under reduced pressure, and then drying under high
(R)-N-((R)-1-(4-(6-(Pyridin-4-yl)quinazolin-2-ylamino)-
phenyl)ethyl)pyrrolidine-2-carboxamide Hydrochloride (80).
The title compound was synthesized from BOC-D-proline and (R)-N-
(4-(1-aminoethyl)phenyl)-6-(pyridin-4-yl)quinazolin-2-amine hydro-
1
chloride (73) using a method similar to that of 75. H NMR: 10.21
(br s, 1H), 10.07 (s, 1H), 9.34−9.42 (m, 1H), 9.10−9.23 (m, 1H), 8.80
(d, J = 5.27 Hz, 2H), 8.41−8.58 (m, 2H), 8.32 (dd, J = 9.04, 2.07 Hz,
1H), 8.05−8.13 (m, 2H), 7.95 (s, 1H), 7.77 (d, J = 8.85 Hz, 1H), 7.31 (d,
J = 8.67 Hz, 2H), 4.95 (quin, 1H), 4.15−4.32 (m, 1H), 3.63−3.76 (m,
1H), 3.41−3.54 (m, 1H), 2.30−2.43 (m, 1H), 1.69−1.96 (m, 3H), 1.43
(d, J = 6.97 Hz, 3 H). LC−MS: m/z 439.
1
(R)-3-(1-(4-(6-(Pyridin-4-yl)quinazolin-2-ylamino)phenyl)-
ethylamino)cyclohex-2-enone (81). To (R)-N-(4-(1-aminoethyl)-
phenyl)-6-(pyridin-4-yl)quinazolin-2-amine hydrochloride (73, 0.298 g,
0.66 mmol) and cyclohexane-1,3-dione (0.222 g, 1.98 mmol), acetic acid
(0.038 mL, 0.66 mmol), and DIPEA (0.358 mL, 2.05 mmol) was added
MeOH (3.31 mL) to give a yellow solution. The reaction was stirred for
12 h at 60 °C. The reaction was quenched with 1 M aqueous NaOH (30
mL). The resulting thick oil was triturated with a mixture of MTBE (10
mL) and EtOAc (6 mL). The resultant yellow solid was isolated via
vacuum filtration, rinsed with MTBE (2 × 5 mL), and dried under high-
vacuum to yield the title compound. 1H NMR: 10.01 (s, 1H), 9.31−9.40
(m, 1H), 8.63−8.75 (m, 2H), 8.42 (d, J = 2.07 Hz, 1H), 8.26 (dd, J =
8.85, 2.26 Hz, 1H), 7.92 (m, 2H), 7.81−7.87 (m, 2H), 7.77 (d, J = 8.85
Hz, 1H), 7.40 (d, J = 6.78 Hz, 1H), 7.27 (m, 2H), 4.67 (s, 1H), 4.42
(quin, 1H), 2.39 (m, 2H), 2.01 (m, 2H), 1.73−1.86 (m, 2H), 1.41 (d, J =
6.78 Hz, 3H). LC−MS: m/z 436.
vacuum to yield the title compound. H NMR: 9.99 (s, 1H), 9.37 (s,
1H), 8.68 (d, J = 5.09 Hz, 2H), 8.42 (s, 1H), 8.26 (d, J = 7.35 Hz, 1 H),
7.67−8.01 (m, 6H), 7.28 (d, J = 8.29 Hz, 2H), 4.90 (m, 1H), 3.06 (d, J =
8.85 Hz, 1H), 2.92 (d, J = 12.43 Hz, 1H), 2.10−2.24 (m, 1H), 1.73 (d, J
= 9.42 Hz, 2H), 1.17−1.53 (m, 7H). LC−MS: m/z 453.
1-Methyl-N-((R)-1-(4-(6-(pyridin-4-yl)quinazolin-2-ylamino)-
phenyl)ethyl)piperidine-2-carboxamide Hydrochloride (76).
The title compound was synthesized from 1-methylpiperidine-2-
carboxylic acid and (R)-N-(4-(1-aminoethyl)phenyl)-6-(pyridin-4-yl)-
quinazolin-2-amine hydrochloride (73) using a method analogous to
the preparation of 75. 1H NMR (MeOH-d4), reported as a mixture of
diastereomers: 9.23 (s, 1H), 8.49−8.64 (m, 2H), 8.24 (m, 1H), 8.15 (m,
1H), 7.67−7.91 (m, 5 H), 7.22−7.40 (m, 2H), 4.96−5.10 (m, 1H),
3.13−3.18 (m, 2H), 2.74−3.03 (m, 1H), 2.58−2.72 (m, 1H), 2.46−2.54
(m, 3H), 1.97−2.07 (m, 1H), 1.80 (m, 2H), 1.57−1.74 (m, 2H), 1.41−
1.55 (m, 3H). LC−MS: m/z 467.
(R)-3-(1-(4-(6-(Pyridin-4-yl)quinazolin-2-ylamino)phenyl)-
ethylamino)cyclopent-2-enone (82). The title compound was
synthesized from (R)-N-(4-(1-aminoethyl)phenyl)-6-(pyridin-4-yl)-
quinazolin-2-amine hydrochloride (73) and cyclopentane-1,3-dione
using a method analogous to the preparation of 81. 1H NMR: 10.02 (s,
1H), 9.38 (s, 1H), 8.65−8.72 (m, 2H), 8.42 (d, J = 2.07 Hz, 1H), 8.26
(dd, J = 8.76, 2.17 Hz, 1H), 8.01 (d, J = 6.97 Hz, 1H), 7.93 (m, 2H),
7.81−7.86 (m, 2H), 7.77 (d, J = 8.85 Hz, 1H), 7.30 (m, 2H), 4.67 (s,
1H), 4.41 (quin, 1H), 2.52−2.58 (m, 2H), 2.04−2.15 (m, 2H), 1.44 (d, J
= 6.78 Hz, 3H). LC−MS: m/z 422.
(R)-1-(2-Hydroxyethyl)-N-((R)-1-(4-(6-(pyridin-4-yl)-
quinazolin-2 ylamino)phenyl)ethyl)piperidine-2-carboxamide
(77). In a 10 mL vial, (R)-N-((R)-1-(4-(6-(pyridin-4-yl)quinazolin-2-
ylamino)phenyl)ethyl)piperidine-2-carboxamide hydrochloride (75,
0.463 g, 0.82 mmol), 2-(tert-butyldimethylsilyloxy)acetaldehyde
(0.158 g, 0.91 mmol), acetic acid (0.047 mL, 0.82 mmol), and DIPEA
(0.460 mL, 2.64 mmol) were combined in MeOH (4.12 mL) to afford a
yellow solution. Sodium cyanoborohydride (0.078 g, 1.24 mmol) was
added and the reaction was stirred for 4 h at 23 °C. LC−MS indicated
that the TBDMS group was cleaved under the reaction conditions, and
all of the starting material was consumed. The reaction was poured into
1 M aq NaOH (30 mL) and extracted with EtOAc (3 × 20 mL). The
organic extracts were combined and rinsed with brine (20 mL), dried
over Na2SO4, filtered, and concentrated under reduced pressure to yield
a pale yellow solid which was purified via an ISCO system (10% MeOH/
DCM) to yield the title compound. 1H NMR (DMSO-d6 with a drop of
MeOH added for dissolution): 9.32 (s, 1H), 8.58−8.70 (m, 2H), 8.34
(br s, 1H), 8.20 (dd, J = 8.85, 2.07 Hz, 1H), 7.89 (m, J = 8.48 Hz, 2H),
7.71−7.83 (m, 3H), 7.30 (m, J = 8.48 Hz, 2H), 4.88 (q, J = 6.91 Hz, 1H),
3.37−3.64 (m, 2H), 2.69−2.82 (m, 1H), 2.56−2.65 (m, 1H), 2.13−2.31
(m, 1H), 1.96−2.13 (m, 1H), 1.59−1.81 (m, 2H), 1.42−1.59 (m, 3H),
1.37 (d, J = 6.97 Hz, 3H), 1.12−1.33 (m, 2H). LC−MS: m/z 497.
(S)-N-((R)-1-(4-(6-(Pyridin-4-yl)quinazolin-2-ylamino)-
phenyl)ethyl)piperidine-3-carboxamide Hydrochloride (78).
The title compound was synthesized from (S)-1-(tert-butoxycarbonyl)-
piperidine-3-carboxylic acid and (R)-N-(4-(1-aminoethyl)phenyl)-6-
(pyridin-4-yl)quinazolin-2-amine hydrochloride (73) using a method
similar to that of 75 except using 2 N HCl in ether for BOC group
deprotection. 1H NMR: 10.17 (s, 1H), 9.40 (m, 2H), 8.98 (m, 2H), 8.70
(R)-4-(1-(4-(6-(Pyridin-4-yl)quinazolin-2-ylamino)phenyl)-
ethylamino)-5,6-dihydropyridin-2(1H)-one (83). The title com-
pound was synthesized from (R)-N-(4-(1-aminoethyl)phenyl)-6-
(pyridin-4-yl)quinazolin-2-amine (73-free base) and piperidine-2,4-
dione using a method analogous to the preparation of 81, except using
DMF as solvent and heating in a microwave at 150 °C for 30 min.
Compound was purified by an ISCO system (0−40% methanol/ethyl
1
acetate gradient). H NMR (MeOH-d4): 7.48 (s, 1H), 6.89−6.95 (m,
2H), 6.46 (d, J = 2.02 Hz, 1H), 6.40 (dd, J = 8.84, 2.27 Hz, 1H), 6.19 (d, J
= 8.59 Hz, 2H), 6.06−6.11 (m, 2H), 6.02 (d, J = 8.84 Hz, 1H), 5.65 (d, J
= 8.59 Hz, 2H), 2.88 (s, 1H), 2.82 (q, J = 6.65 Hz, 1H), 1.72−1.75 (m,
2H), 0.89 (t, J = 6.95 Hz, 2H), −0.13 (d, J = 6.82 Hz, 3H). LC−MS: m/z
437. Purity: 90%.
(R)-5-(1-(4-(6-(Pyridin-4-yl)quinazolin-2-ylamino)phenyl)-
ethylamino)-1,2-dihydropyridin-3(6H)-one (84). (i) (R)-N-(4-(1-
Aminoethyl)phenyl)-6-(pyridin-4-yl)quinazolin-2-amine (73-free base,
1.016 g, 2.98 mmol), benzyl 3-hydroxy-5-oxo-5,6-dihydropyridine-
1(2H)-carboxylate (74, 0.883 g, 3.57 mmol), and acetic acid (0.170 mL,
2.98 mmol) were combined in NMP (6.97 mL) to give a yellow solution.
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The reaction was heated at 105 °C for 6 h, cooled to ambient
temperature, and poured into saturated aq NH4Cl (40 mL). The
mixture was extracted with EtOAc (3 × 30 mL), and the organic
fractions were combined, rinsed with 1 M aqueous NaOH (30 mL) and
brine (30 mL), dried over Na2SO4, filtered, and concentrated under
reduced pressure to yield a dark orange oil which was purified via
column chromatography (SiO2, 10% MeOH/DCM) to yield (R)-
benzyl-5-oxo-3-(1-(4-(6-(pyridin-4-yl)quinazolin-2-ylamino)phenyl)-
ethylamino)-5,6-dihydropyridine-1(2H)-carboxylate which was carried
forward crude into the next step. LC−MS: m/z 570. (ii) In a 20 mL
round-bottomed flask, (R)-benzyl 5-oxo-3-(1-(4-(6-(pyridin-4-yl)-
quinazolin-2-ylamino)phenyl)ethylamino)-5,6-dihydropyridine-
1(2H)-carboxylate (0.380 g, 0.53 mmol) and Pearlman’s catalyst (0.056
g, 0.08 mmol) were combined in MeOH (2.66 mL) to give a yellow
suspension. The reaction was stirred for 18 h at 23 °C under a hydrogen
balloon (1 atm). The reaction was filtered, and the filtrate was
concentrated over Celite and subjected to column chromatography
(SiO2, 18% MeOH/DCM) to yield the title compound as a yellow solid.
1H NMR (MeOH-d4): 8.94−9.12 (m, 1H), 8.40−8.59 (m, 2H), 8.08
6-Bromo-7-methoxyquinazolin-2-amine (88). 2-Amino-5-
bromo-4-methoxybenzaldehyde (prepared as described in ref 17; 180
mg, 0.78 mmol) was dissolved in decalin (5 mL). Guanidine carbonate
(0.4 g, 2.2 mmol) was added, and the mixture was heated at 210 °C for 3
h. The mixture was allowed to cool and was purified by silica gel
chromatography to give the product as a yellow solid (110 mg, 55%). 1H
NMR: 8.90 (s, 1H), 8.05 (s, 1H) 3.92 (s, 3H), 6.87 (s, 1H). LC−MS: m/
z 254.
6-Bromo-7-chloroquinazolin-2-amine (89). To 2-amino-5-
bromo-4-chlorobenzaldehyde (prepared as described in ref 17; 6.44 g,
27.5 mmol) and guanidine carbonate (7.45 g, 41.35 mmol) was added
DMA (55 mL), and the resulting mixture was placed in a 140 °C oil bath.
After 4 h, the reaction was allowed to cool and was then diluted with
H2O (200 mL), precipitating an orange-colored amorphous solid that
was isolated by suction-filtration. The filter cake was washed with H2O
and dried in air to give a yellow solid (6.54 g) which was suspended in
refluxing MeOH (100 mL) for 16 h. The resulting mixture was allowed
to cool and was suction-filtered, washed with MeOH, and dried in air to
give the product as a yellow solid (4.73 g, 67%). 1H NMR: 9.11 (s, 1H),
8.27 (s, 1H), 7.63 (s, 1H), 7.20 (s, 2H). LC−MS: m/z 259.
6-Bromo-8-chloroquinazolin-2-amine (90). The title compound
was synthesized from 2-amino-5-bromo-3-chlorobenzaldehyde (pre-
pared as described in ref 17) using a method analogous to the
preparation of 89, to afford a yellow solid (7.38 g, 84% yield). 1H NMR:
9.13 (s, 1H), 7.99−8.11 (m, 2H), 7.38 (br. s., 2H); m/z 259.
7-Methoxy-6-(pyridin-4-yl)quinazolin-2-amine (91). The title
compound was synthesized from 6-bromo-7-methoxyquinazolin-2-
amine (88) and 4-pyridinylboronic acid using a method analogous to
the preparation of 87, but with Cs2CO3 and dioxane/water (2/1) as
solvent (94% yield). LC−MS: m/z 253.
7-Chloro-6-(pyridin-4-yl)quinazolin-2-amine (92). The title
compound was synthesized from 6-bromo-7-chloroquinazolin-2-amine
(89) using a method analogous to the preparation of 6, except with
(PPh3)2PdCl2 (4.0 mol %) as catalyst and dioxane/water (2/1) as
solvent (3.42 g, 77%). 1H NMR: 9.18 (s, 1H), 8.64−8.75 (m, 2H), 7.94
(s, 1H), 7.60 (s, 1H), 7.48−7.58 (m, 2H), 7.21 (s, 2H). LC−MS: m/z
257.
8-Chloro-6-(pyridin-4-yl)quinazolin-2-amine (93). The title
compound was synthesized from 6-bromo-8-chloroquinazolin-2-amine
(90) following the procedure described for the preparation of 92, to
afford the title compound as a yellow solid (3.41 g, 99%) that was
contaminated with a minor amount of the bis-4-pyridyl product derived
from coupling at both the bromo and chloro positions, which was used
without further purification. LC−MS: m/z 257.
(m, 2H), 7.48−7.86 (m, 5H), 7.26 (m, 2H), 5.06 (s, 1H), 4.30−4.59 (m,
1H), 3.42−3.62 (m, 2H), 3.14−3.25 (m, 2H), 1.50 (m, 3H). LC−MS:
m/z 437.
(R)-N-(1-(4-Aminophenyl)ethyl)acetamide (85). (i) (R)-1-(4-
Bromophenyl)ethanamine (25 g, 0.125 mol) was treated with acetic
anhydride (100 mL). The reaction mixture was heated to 50 °C for 4 h.
The organics were removed under reduced pressure to afford (R)-N-(1-
(4-bromophenyl)ethyl)acetamide (30 g, 99% yield), which was used
directly in the next step. (ii) A solution of (R)-N-(1-(4-bromophenyl)-
ethyl)acetamide (30 g, 120 mmol), diphenylmethanimine (27.2 g, 150
mmol, 1.3 equiv), Pd2(dba)3-CH2Cl2 (6.5 g, 6.3 mmol, 0.53 equiv), 2-
(dicyclohexylphosphino)-2′,4′,6′-triisopropyl-1,1′-biphenyl (X-Phos,
6.1 g, 8.2 mmol, 0.068 equiv), and Cs2CO3 (122 g, 375 mmol, 3
equiv) in 1,4-dioxane (1.5 L) was heated to 100 °C for 12 h. The
reaction mixture was cooled to 25 °C and poured into water, followed by
extraction with EtOAc. The organics were dried over Na2SO4 (s) and
removed under reduced pressure. The resulting product was purified by
silica gel chromatography (petroleum ether/ethyl acetate, 1:1) to afford
(R)-N-(1-(4-(Diphenylmethyleneamino)phenyl)ethyl)acetamide (33.6
g, 78% yield). LC−MS: m/z 343. (iii) (R)-N-(1-(4-
(diphenylmethyleneamino)phenyl)ethyl)acetamide (33.5 g, 97.8
mmol) in Et2O (300 mL) was treated with 1 N HCl (300 mL). The
reaction mixture was stirred at 25 °C for 12 h. The reaction mixture was
concentrated under reduced pressure and the resulting residue was
triturated with Et2O. The resulting solid was dissolved in a mixture of
10% w/v aq K2HPO4 and EtOAc. The organics were washed with NaCl
(satd) and then dried with Na2SO4 (s). The organics were removed
under reduced pressure to afford the title compound (17.2 g, 98% yield).
LC−MS: m/z 179.
(R)-N-(1-(4-(6-Bromo-8-methoxyquinazolin-2-ylamino)-
phenyl)ethyl)acetamide (86). 6-Bromo-2-chloro-8-methoxyquina-
zoline (prepared as described in ref 17; 100 mg, 0.366 mmol) and (R)-
N-(1-(4-aminophenyl)ethyl)acetamide (85, 98 mg, 0.549 mmol, 1.5
equiv) were dissolved in 2-propanol (20 mL), and the mixture was
heated to reflux for 3 h. DIPEA (0.140 mL, 0.805 mmol, 2.2 equiv) was
then added and the mixture was heated at reflux for 12 h. The crude
mixture was then concentrated under reduced pressure. The resulting
suspension was washed with methanol, and the solid was collected by
filtration and then dried under high vacuum to afford the title compound
(110 mg, 73% yield), which was carried forward crude into the next step.
(R)-N-(1-(4-(8-Methoxy-6-(pyridin-4-yl)quinazolin-2-
ylamino)phenyl)ethyl)acetamide (87). The title compound was
synthesized from (R)-N-(1-(4-(6-bromo-8-methoxyquinazolin-2-
ylamino)phenyl)ethyl)acetamide (86) and 4-pyridinylboronic acid
using a method analogous to the preparation of 6, except using
Cs2CO3 as base and 1,4-dioxane/water (2:1) as solvent. The crude
product was purified by column chromatography (DCM/MeOH) to
afford 80 mg (80%) of the title compound. 1H NMR: 10.01 (s, 1H), 9.32
(s, 1H), 8.68 (s, 2H), 8.23 (d, 1H), 7.97 (m, 5H), 7.64 (s, 1H), 7.23 (m,
2H), 4.90 (m, 1H), 4.08 (s, 3H), 1.83 (s, 3H), 1.33 (d, 3H). LC−MS:
m/z 414.
(R)-tert-Butyl 1-(4-(7-Methoxy-6-(pyridin-4-yl)quinazolin-2
ylamino)phenyl)ethylcarbamate (94). The title compound was
synthesized from (R)-tert-butyl 1-(4-bromophenyl)ethylcarbamate
(72) and 7-methoxy-6-(pyridin-4-yl)quinazolin-2-amine (91) using a
method analogous to the preparation of 3, with Xantphos and
palladium(II) acetate followed by purification on an ISCO system
(CH2Cl2:MeOH = 30:1) to afford the title compound (0.41 g, 73%). 1H
NMR: 10.07 (s, 1H), 9.14 (s, 1H), 8.60−8.62 (m, 2H), 7.93 (s, 1H),
7.85−7.88 (m, 2H), 7.56−7.58 (m, 2H), 7.22−7.25 (m, 2H), 7.15 (s,
1H), 4.52−4.61 (m, 1H), 3.96 (s, 3H), 1.35 (s, 9H), 1.29 (d, J = 7.1 Hz,
3H).
(R)-tert-Butyl 1-(4-(7-Chloro-6-(pyridin-4-yl)quinazolin-2-
ylamino)phenyl)ethylcarbamate (95). The title compound was
synthesized from 7-chloro-6-(pyridin-4-yl)quinazolin-2-amine (92)
using a method analogous to the preparation of 3, with Xantphos and
heating at 90 °C followed by purification on an ISCO system
(CH2Cl2:MeOH = 30:1) to afford the product (2.73 g, 49%). 1H
NMR: 10.07 (s, 1 H), 9.35 (s, 1H), 8.67−8.76 (m, 2H), 8.05 (s, 1H),
7.82−7.94 (m, 3H), 7.52−7.61 (m, 2H), 7.31−7.38 (m, 1H), 7.23−7.30
(m, 2H), 4.54−4.65 (m, 1H), 1.38 (s, 9H), 1.31 (d, J = 7.1 Hz, 3H).
LC−MS: m/z 476.
(R)-tert-Butyl 1-(4-(8-Chloro-6-(pyridin-4-yl)quinazolin-2-
ylamino)phenyl)ethylcarbamate (96). The title compound was
synthesized from 8-chloro-6-(pyridin-4-yl)quinazolin-2-amine (93)
1
using a method analogous to the preparation of 95 (2.92 g, 51%). H
NMR: 10.29 (s, 1H), 9.41 (s, 1H), 8.66−8.72 (m, 2H), 8.46−8.49 (m,
2012
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Article
1H), 8.41−8.45 (m, 1H), 8.02−8.12 (m, 2H), 7.84−7.90 (m, 2H),
7.25−7.40 (m, 3H), 4.55−4.67 (m, 1H), 1.37 (s, 9H), 1.32 (d, J = 7.07
Hz, 3H). LC−MS: m/z 476.
7.86 (m, 2H), 7.24−7.33 (m, 2H), 4.85−4.95 (m, 1H), 2.69 (s, 3H),
1.84 (s, 3H), 1.35 (d, J = 7.1 Hz, 3H). LC−MS: m/z 398.
(R)-N-(1-(4-(7-Methoxy-6-(pyridin-4-yl)quinazolin-2-
ylamino)phenyl)ethyl)acetamide (97). (i) (R)-tert-Butyl 1-(4-(7-
methoxy-6-(pyridin-4-yl)quinazolin-2-ylamino)phenyl)ethylcarbamate
(94, 0.40 g, 0.8 mmol) was added to HCl/THF (50 mL) and the mixture
was allowed to stir at room temperature overnight. The mixture was
concentrated, and the solid residue was washed with hexanes to afford
(R)-N-(4-(1-aminoethyl)phenyl)-7-methoxy-6-(pyridin-4-yl)-
quinazolin-2-amine dihydrochloride (0.34 g, 98%, m/z 373), which was
carried forward into the next step. (ii) (R)-N-(4-(1-Aminoethyl)-
phenyl)-7-methoxy-6-(pyridin-4-yl)quinazolin-2-amine dihydrochlor-
ide (102 mg, 0.23 mmol) was added to EtOAc (5 mL) and triethylamine
(300 μL, 2.2 mmol) followed by acetic anhydride (100 μL, 1.1 mmol).
The resulting mixture was allowed to stir at room temperature. After 1 h,
MeOH (1 mL) was added to the heterogeneous mixture to improve
overall solubility. After stirring at room temperature overnight, the
mixture was diluted with H2O (10 mL). The biphasic, heterogeneous
mixture was suction-filtered, and the filter cake was washed with EtOAc
and H2O to afford the title compound (40 mg, 42%). 1H NMR: 9.82 (s,
1H), 9.16 (s, 1H), 8.61−8.68 (m, 2H), 8.21−8.27 (m, 1H), 7.95 (s, 1H),
7.87−7.93 (m, 2H), 7.56−7.62 (m, 2H), 7.23−7.30 (m, 2H), 7.16 (s,
1H), 4.84−4.93 (m, 1H), 3.97 (s, 3H), 1.84 (s, 3H), 1.35 (d, J = 6.8 Hz,
3H). LC−MS: m/z 414.
(R)-N-(1-(4-(7-Chloro-6-(pyridin-4-yl)quinazolin-2-ylamino)-
phenyl)ethyl)acetamide (98). The title compound was synthesized
from (R)-tert-butyl 1-(4-(7-chloro-6-(pyridin-4-yl)quinazolin-2-
ylamino)phenyl)ethylcarbamate (95) using a method similar to that
for the preparation of 97, except with 4 N HCl/dioxane for BOC group
deprotection, followed by treatment with acetic anhydride as described
for the preparation of 97. 1H NMR: 10.09 (s, 1H), 9.35 (s, 1H), 8.68−
8.74 (m, 2H), 8.21−8.28 (m, 1 H), 8.06 (s, 1H), 7.85−7.94 (m, 3H),
7.55−7.58 (m, 2H), 7.25−7.30 (m, 2H), 4.84−4.94 (m, 1 H), 1.84 (s,
3H), 1.34 (d, J = 7.1 Hz, 3H). LC−MS: m/z 418.
ASSOCIATED CONTENT
■
S
* Supporting Information
Tables SI1−SI4, Figure SI1, protein expression, purification,
crystallization, and structure determination for 3 and 48,
biological and DMPK assay details, pharmacodynamic protocol,
and A375 mouse xenograft assay protocol details. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
PDB ID for 3−EphB4 complex, 4bb4; PDB ID for the 48−B-Raf
complex, 4H58.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 1-781-839-4886. Fax: 1-781-839-4230. E-mail: melissa.
vasbinder@astrazeneca.com.
Present Addresses
#Discovery Chemistry, Genentech, Inc., One DNA Way, South
San Francisco, CA 94080.
∇Dana-Farber Cancer Institute, 450 Brookline Avenue, Room
348, Harvard Institutes of Medicine, Boston, MA 02215.
○Infection iMED, AstraZeneca R&D Boston, 35 Gatehouse
Drive, Waltham, MA 02451.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to thank Mukta Bagul, Robert Godin,
Geraldine Hamilton, Erika Keeton, and Mali Wald for their early
contributions to this work and to Claudio Chuaqui, Corinne
Reimer, and David Scott for useful discussions and assistance
during the preparation of the manuscript.
(R)-N-(1-(4-(8-Chloro-6-(pyridin-4-yl)quinazolin-2-ylamino)-
phenyl)ethyl)acetamide (99). The title compound was synthesized
from (R)-tert-butyl 1-(4-(8-chloro-6-(pyridin-4-yl)quinazolin-2-
ylamino)phenyl)ethylcarbamate (96) using a method similar to that
for the preparation of 98. 1H NMR: 10.29 (s, 1 H), 9.41 (s, 1 H), 8.65−
8.73 (m, 2H), 8.45−8.49 (m, 1H), 8.41−8.44 (m, 1H), 8.20−8.26 (m,
1H), 8.03−8.13 (m, 2H), 7.84−7.90 (m, 2H), 7.27−7.33 (m, 2H),
4.85−4.95 (m, 1H), 1.85 (s, 3H), 1.35 (d, J = 6.8 Hz, 3H). LC−MS: m/z
418.
ABBREVIATIONS USED
■
BINAP, 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl; CLint, in
vitro intrinsic clearance; CLobs, measured in vivo clearance; DFG,
sequence of the three amino acids aspartic acid-phenylalanine-
glycine; DIPEA, diisopropylethylamine; DME, ethylene glycol
dimethyl ether; EtOAc, ethyl acetate; Fu, fraction unbound;
HATU, (Dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo-
[4,5-b]pyridin-3-yloxy)methaniminium hexafluorophosphate;
Hep, hepatocytes; HOAc, acetic acid; HPMC, hydroxypropyl
methylcellulose; Hu, human; LC−MS, liquid chromatography−
mass spectrometry; Mic, microsomes; MS, molecular sieves;
MPLC, medium-pressure liquid chromatography; mut, mutant;
Pd2(dba)3, tris(dibenzylideneacetone)dipalladium; Pd(OAc)2,
palladium(II) acetate; PdCl2(dppf)-CH2Cl2, 1,1′-bis-
(diphenylphosphino)ferrocene-palladium(II)dichloride di-
chloromethane complex; PCy3·HBF4, tricyclopentyl phosphine
tetrafluoroborate; satd, saturated; TBDMS, tert-butyldimethylsi-
loxy; TEG, triethylene glycol; TGI, tumor growth inhibition;
(R)-N-(1-(4-(7-Methyl-6-(pyridin-4-yl)quinazolin-2-ylamino)-
phenyl)ethyl)acetamide (100). (i) A test tube equipped with a stir
bar was charged with (R)-tert-butyl 1-(4-(7-chloro-6-(pyridin-4-yl)-
quinazolin-2-ylamino)phenyl)ethylcarbamate (95, 238 mg, 0.50 mmol),
methylboronic acid (63 mg, 1.05 mmol), Pd(OAc)2 (4.4 mg, 3.9 mol
%), PCy3·HBF4(19.3 mg, 10.5 mol %), and potassium carbonate (281
mg, 2.03 mmol). The tube was evacuated and back-filled with N2, and
then dioxane (1.5 mL) and water (0.5 mL) were added. The mixture was
allowed to stir at room temperature for a few minutes and was then
placed in a 100 °C oil bath. After heating for 12 h, the reaction was
allowed to cool, and the mixture was partitioned between EtOAc and
water. The aqueous layer was extracted with EtOAc, and the combined
organics were concentrated under reduced pressure. The resulting solid
residue was used without further purification in the next step. LC−MS:
m/z 456. (ii and iii) BOC-deprotection and acylation were
1
accomplished using a method analogous to the preparation of 97. H
NMR: 9.87 (s, 1H), 9.27 (s, 1H), 8.65−8.71 (m, 2H), 8.21−8.27 (m,
1H), 7.87−7.93 (m, 2H), 7.81 (s, 1H), 7.62 (s, 1H), 7.46−7.53 (m, 2H),
7.23−7.29 (m, 2H), 4.83−4.93 (m, 1H), 2.41 (s, 3H), 1.84 (s, 3H), 1.34
(d, J = 7.1 Hz, 3H). LC−MS: m/z 398.
V
dss , volume of distribution; Xantphos, 4,5-bis-
(diphenylphosphino)-9,9-dimethylxanthene.
(R)-N-(1-(4-(8-Methyl-6-(pyridin-4-yl)quinazolin-2-ylamino)-
phenyl)ethyl)acetamide (101). The title compound was synthesized
from (R)-tert-butyl 1-(4-(8-chloro-6-(pyridin-4-yl)quinazolin-2-
ylamino)phenyl)ethylcarbamate (96) using a method analogous to
the preparation of 100. 1H NMR: 10.01 (s, 1H), 9.34 (s, 1H), 8.64−8.71
(m, 2H), 8.19−8.29 (m, 2H), 8.17 (s, 1H), 7.96−8.04 (m, 2H), 7.79−
REFERENCES
■
(1) Peyssonnaux, C.; Eychene, A. The Raf/MEK/ERK pathway: New
concepts of activation. Biol. Cell 2001, 93, 53−62.
(2) Wellbrook, C.; Karasarides, M.; Marais, R. The RAF proteins take
centre stage. Nat. Rev. Mol. Cell Biol. 2004, 5, 875−885.
2013
dx.doi.org/10.1021/jm301658d | J. Med. Chem. 2013, 56, 1996−2015

Journal of Medicinal Chemistry
Article
(3) Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg,
S.; Teague, J.; Woffendin, H.; Garnett, M. J.; Bottomley, W.; Davis, N.;
Dicks, E.; Ewing, R.; Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.;
Kosmidou, V.; Menzies, A.; Mould, C.; Parker, A.; Stevens, C.; Watt, S.;
Hooper, S.; Wilson, R.; Jayatilake, H.; Gusterson, B. A.; Cooper, C.;
Shipley, J.; Hargrave, D.; Pritchard-Jones, K.; Maitland, N.; Chenevix-
Trench, G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.; Cossu, A.;
Flanagan, A.; Nicholson, A.; Ho, J. W. C.; Leung, S. Y.; Yuen, S. T.;
Weber, B. L.; Seigler, H. F.; Darrow, T. L.; Paterson, H.; Marais, R.;
Marshall, C. J.; Wooster, R.; Stratton, M. R.; Futreal, P. A. Mutations of
the BRAF gene in human cancer. Nature 2002, 417, 949−954.
(4) Bollag, G.; Hirth, P.; Tsai, J.; Zhang, J.; Ibrahim, P. N.; Cho, H.;
Spevak, W.; Zhang, C.; Zhang, Y.; Habets, G.; Burton, E. A.; Wong, B.;
Tsang, G.; West, B. L.; Powell, B.; Shellooe, R.; Marimuthu, A.; Nguyen,
H.; Zhang, K. Y. J.; Artis, D. R.; Schlessinger, J.; Su, F.; Higgins, B.; Iyer,
R.; D’Andrea, K.; Koehler, A.; Stumm, M.; Lin, P. S.; Lee, R. J.; Grippo,
J.; Puzanov, I.; Kim, K. B.; Ribas, A.; McArthur, G. A.; Sosman, J. A.;
Chapman, P. B.; Flaherty, K. T.; Xu, X.; Nathanson, K. L.; Nolop, K.
Clinical efficacy of a RAF inhibitor needs broad target blockade in
BRAF-mutant melanoma. Nature 2010, 467, 596−599.
(5) Chapman, P. B.; Hauschild, A.; Robert, C.; Haanen, J. B.; Ascierto,
P.; Larkin, J.; Dummer, R.; Garbe, C.; Testori, A.; Maio, M.; Hogg, D.;
Lorigan, P.; Lebbe, C.; Jouary, T.; Schadendorf, D.; Ribas, A.; O’Day, S.
J.; Sosman, J. A.; Kirkwood, J. M.; Eggermont, A. M.; Dreno, B.; Nolop,
K.; Li, J.; Nelson, B.; Hou, J.; Lee, R. J.; Flaherty, K. T.; McArthur, G. A.
Improved survival with vemurafenib in melanoma with BRAF V600E
mutation. N. Engl. J. Med. 2010, 364, 2507−2516.
(6) Flaherty, K. T.; Puzanov, I.; Kim, K. B.; Ribas, A.; McArthur, G. A.;
Sosman, J. A.; O’Dwyer, P. J.; Lee, R. J.; Grippo, J. F.; Nolop, K.;
Chapman, P. B. Inhibition of mutated, activated BRAF in metastatic
melanoma. N. Engl. J. Med. 2010, 363, 809−819.
N.; Rudolph, J. Potent and selective pyrazolo[1,5-a]pyrimidine based
inhibitors of B-RafV600E kinase with favorable physicochemical and
pharmacokinetic properties. Bioorg. Med. Chem. Lett. 2012, 22, 1165−
1168. (c) Mathieu, S.; Gradl, S. N.; Ren, L.; Wen, Z.; Aliagas, I.;
Gunzner-Toste, J.; Lee, W.; Pulk, R.; Zhao, G.; Alicke, B.; Boggs, J. W.;
Buckmelter, A. J.; Choo, E. F.; Dinkel, V.; Gloor, S. L.; Gould, S. E.;
Hansen, J. D.; Hastings, G.; Hatzivassiliou, G.; Laird, E. R.; Moreno, D.;
Ran, Y.; Voegtli, W. C.; Wenglowsky, S.; Grina, J.; Rudolph, J. Potent
and selective aminopyrimidine-based B-Raf inhibitors with favorable
physicochemical and pharmacokinetic properties. J. Med. Chem. 2012,
55, 2869−2881. (d) Packard, G. K.; Papa, P.; Riggs, J. R.; Erdman, P.;
Tehrani, L.; Robinson, D.; Harris, R.; Shevlin, G.; Perrin-Ninkovic, S.;
Hilgraf, R.; McCarrick, M. A.; Tran, T.; Fleming, Y.; Bai, A.; Richardson,
S.; Katz, J.; Tang, Y.; Leisten, J.; Moghaddam, M.; Cathers, B.; Zhu, D.;
Sakata, S. Discovery and optimization of thieno[2,3-d]pyrimidines as B-
Raf inhibitors. Bioorg. Med. Chem. Lett. 2012, 22, 747−752.
(11) Lyne, P. D.; Aquila, B.; Cook, D. J.; Dakin, L. A.; Ezhuthachan, J.;
Ioannidis, S.; Pontz, T.; Su, M.; Ye, Q.; Zheng, X.; Block, M. H.; Cowen,
S.; Deegan, T. L.; Lee, J. W.; Scott, D. A.; Custeau, D.; Drew, L.;
Poondru, S.; Shen, M.; Wu, A. Identification of amidoheteroaryls as
potent inhibitors of mutant (V600E) B-Raf kinase with in vivo activity.
Bioorg. Med. Chem. Lett. 2009, 19, 1026−1029.
(12) Shen, M.; Wu, A.; Aquila, B.; Lyne, P.; Drew, L. Linking molecular
characteristics to the pharmacological response of a panel of cancer cell
lines to the BRAF inhibitor, AZ628. Los Angeles, CA. AACR 98th
Meeting (2007).
(13) Montagut, C.; Sharma, S. V.; Shioda, S. T.; McDermott, U.;
Ulman, M.; Ulkus, L. E.; Dias-Santagata, D.; Stubbs, H.; Lee, D. Y.;
Singh, A.; Drew, L.; Haber, D. A.; Settleman, J. Elevated CRAF as a
potential mechanism of acquired resistance to BRAF inhibition in
melanoma. Cancer Res. 2008, 68, 4853−4861.
(7) Flaherty, K.; Puzanov, I.; Sosman, J.; Kim, K.; Ribas, A.; McArthur,
G.; Lee, R. J.; Grippo, J. F.; Nolop, K.; Chapman, P. Phase I study of
PLX4032: Proof of concept for V600E BRAF mutation as a therapeutic
target in human cancer. J. Clin. Oncol. 2009, 27 (15S (May 20
Supplement)), 9000.
(14) For references comparing DFG-in and DFG-out binding, see:
(a) Nagar, B.; Bornmann, W. G.; Pellicena, P.; Schindler, T.; Veach, D.
R.; Miller, W. T.; Clarkson, B.; Kuriyan, J. Crystal structures of the kinase
domain of c-Abl in complex with the small molecule inhibitors
PD173955 and imatinib (STI-571). Cancer Res. 2002, 62, 4236−4243.
(b) Tokarski, J. S.; Newitt, J. A.; Chang, C. Y. J.; Cheng, J. D.; Wittekind,
M.; Kiefer, S. E.; Kish, K.; Lee, F. Y. F.; Borzillerri, R.; Lombardo, L. J.;
Xie, D.; Zhang, Y.; Klei, H. E. The structure of dasatinib (BMS-354825)
bound to activated ABL kinase domain elucidates its inhibitory activity
against imatinib-resistant ABL mutants. Cancer Res. 2006, 66, 5790−
5797.
(15) For recent studies highlighting MAPK pathway activation in cells
containing wild type B-Raf through Raf dimerization, see: (a) Heidorn,
S. J.; Milagre, C.; Whittaker, S.; Nourry, A.; Niculescu-Duvas, I.;
Dhomen, N.; Hussain, J.; Reis-Filho, J. S.; Springer, C. J.; Pritchard, C.;
Marais, R. Kinase-dead BRAF and oncogenic RAS cooperate to drive
tumor progression through CRAF. Cell 2010, 140, 209−221.
(b) Poulikakos, P. I.; Zhang, C.; Bollag, G.; Shokat, K. M.; Rosen, N.
RAF inhibitors transactivate RAF dimers and ERK signaling in cells with
wild-type BRAF. Nature 2010, 464, 427−430. (c) Hatzivassiliou, G.;
Song, K.; Yen, I.; Brandhuber, B. J.; Anderson, D. J.; Alvarado, R.;
Ludlam, M. J. C.; Stokoe, D.; Gloor, S. L.; Vigers, G.; Morales, T.;
Aliagas, I.; Liu, B.; Sideris, S.; Hoeflich, K. P.; Jaiswal, B. S.; Seshagiri, S.;
Koeppen, H.; Belvin, M.; Friedman, L. S.; Malek, S. RAF inhibitors
prime wild-type RAF to activate the MAPK pathway and enhance
growth. Nature 2010, 464, 431−436. (d) Carnahan, J.; Beltran, P. J.;
Babij, C.; Le, Q.; Rose, M. J.; Vonderfecht, S.; Kim, J. L.; Smith, A. L.;
Nagapudi, K.; Broome, M. A.; Fernando, M.; Kha, H.; Belmontes, B.;
Radinsky, R.; Kendall, R.; Burgess, T. L. Selective and potent Raf
inhibitors paradoxically stimulate normal cell proliferation and tumor
growth. Mol. Cancer Ther. 2010, 9, 2399−2410.
(8) For description of dabrafenib (GSK2118436, GlaxoSmithKline), a
selective B-Raf inhibitor in advanced clinical studies, see: (a) Rheault, T.
R.; Stellwagen, J. C.; Adjabeng, G. M.; Hornberger, K. R.; Petrov, K. G.;
Waterson, A. G.; Dickerson, S. H.; Mook, R. A.; Laquerre, S. G.; King, A.
J.; Rossanese, O. W.; Arnone, M. R.; Smitherman, K. N.; Kane-Carson,
L. S.; Han, C.; Moorthy, G. S.; Moss, K. G; Uehling, D. E. Discovery of
dabrafenib: a selective inhibitor of Raf kinases with antitumor activity
against B-Raf-driven tumors. ACS Med. Chem. Lett. 2013,
DOI: 10.1021/ml4000063. (b) Kefford, R.; Arkenau, H. T.; Brown,
M. P.; Millward, M.; Infante, J. R.; Long, G. V.; Ouellet, D.; Curtis, M.;
Lebowitz, P. F.; Falchook, G. S. Phase I/II study of GSK2118436, a
selective inhibitor of oncogenic mutant BRAF kinase, in patients with
metastatic melanoma and other solid tumors. J. Clin. Oncol. 2010, 28 (15
Suppl (May 20 Supplement)), 8503.
(9) Recent reviews of B-Raf inhibitors and B-Raf targeted therapy:
(a) Zambon, A.; Niculescu-Duvaz, I.; Niculescu-Duvaz, D.; Marais, R.;
Springer, C. J. Small molecule inhibitors of BRAF in clinical trials. Bioorg.
Med. Chem. Lett. 2012, 22, 789−792. (b) Kim, D. H.; Sim, T. Novel
small molecule Raf kinase inhibitors for targeted cancer therapeutics.
Arch. Pharm. Res. 2012, 35, 605−615. (c) Nimmagadda, N.; Tawbi, H.
RAF kinase inhibitors for the treatment of melanoma. Drugs Future
2011, 36, 63−68. (d) Ribas, A.; Flaherty, K. T. BRAF targeted therapy
changes the treatment paradigm in melanoma. Nat. Rev. Clin. Oncol.
2011, 8, 426−433.
(10) For a selection of representative recent examples of B-Raf
inhibitors, see the following and references therein: (a) Ren, L.;
Ahrendt, K. A.; Grina, J.; Laird, E. R.; Buckmelter, A. J.; Hansen, J. D.;
Newhouse, B.; Moreno, D.; Wenglowsky, S.; Dinkel, V.; Gloor, S. L.;
Hastings, G.; Rana, S.; Rasor, K.; Risom, T.; Sturgis, H. L.; Voegtli, W.
C.; Mathieu, S. The discovery of potent and selective pyridopyrimidin-7-
one based inhibitors of B-RafV600E kinase. Bioorg. Med. Chem. Lett. 2012,
22, 3387−3391. (b) Ren, L.; Laird, E. R.; Buckmelter, A. J.; Dinkel, V.;
Gloor, S. L.; Grina, J.; Newhouse, B.; Rasor, K.; Hastings, G.; Gradl, S.
(16) Jones, J. D. Quinazoline derivatives useful as intermediates. Patent
WO1992015569, 1992.
(17) Jain, R.; Lin, X.; Ng, S. C.; Pfister, K. B.; Ramurthy, S.; Rico, A.;
Subramanian, S.; Wang, X. M. 2-Arylaminoquinazolines for treating
proliferative diseases. Patent WO2009153313A1, 2009.
(18) An EphB4 co-crystallization of 3 was readily obtained and used as
a surrogate to B-RafV600E due to its identical gatekeeper and similar
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binding site and selectivity pocket. We used this surrogate to guide initial
hypotheses around the binding mode. Detailed protein preparation,
crystallization, and freezing protocols are included in the Supporting
Information of the following report: Bardelle, C.; Coleman, T.; Cross,
D.; Davenport, S.; Kettle, J. G.; Ko, E. J.; Leach, A. G.; Mortlock, A.;
Read, J.; Roberts, N. J.; Robins, P.; Williams, E. J. Inhibitors of the
tyrosine kinase EphB4. Part 2: Structure-based discovery and
optimization of 3,5-bis substituted anilinopyrimidines. Bioorg. Med.
Chem. Lett. 2008, 18, 5717−5721.
(19) The KinaseProfiler panel testing was performed at Millipore, with
data overlaid against the kinome phylogenetic tree using Millipore’s
DART visualization tool.
(20) For additional information around the biochemical profiles of 2
and 3, cell line background, and tumor type, see the Supporting
Information.
(21) For a recent paper describing the concern around aromatic
anilines and potential mutagenic concerns, see the following and
references therein: Birch, A. M.; Groombridge, S.; Law, R.; Leach, A. G.;
Mee, C. D.; Schramm, C. Rationally designing safer anilines: The
challenging case of 4-aminobiphenyls. J. Med. Chem. 2012, 55, 3923−
3933.
(22) Chiral resolution of the enantiomers of 53 was performed and
there was a 2-fold difference in cellular potency between the two
enantiomers. Absolute confirmation of (R)- and (S)-enantiomers was
not determined.
(23) The (S)-enantiomer of 57 (not shown) had an IC50 = 190 nM in
the A375 pERK cell assay.
(24) We chose to switch to screening at 5 mM ATP concentration in
the B-RafV600E enzyme assay as we continued our lead optimization of
the aminoquinazoline series and discontinued screening at Km ATP
concentrations (as reported by enzyme potencies shown in Tables
1−3). We believed the higher ATP concentration was more
representative of cellular ATP levels and provided a more robust
enzyme-cell correlation. For additional details around the advantages of
screening kinase inhibitors at various ATP concentrations, see the
following paper and references therein: Knight, Z. A.; Shokat, K. M.
Features of selective kinase inhibitors. Chem. Biol. 2005, 12, 621−637.
(25) Morgenthaler, M.; Schweizer, E.; Hoffmann-Roder, A.; Benini, F.;
̈
Martin, R. E.; Jaeschke, G.; Wagner, B.; Foischer, H.; Bendels, S.;
Zimmerli, D.; Schneider, J.; Diederich, F.; Kansy, M.; Muller, K.
̈
Predicting and tuning physicochemical properties in lead optimization:
Amine basicities. Chem. Med. Chem. 2007, 2, 1100−1115.
(26) Work was done to identify the stereochemical preference at the
carboxamide stereogenic center and in each instance the more potent
diastereomer is represented in Table 4. We were unable to separate the
diastereomers of compound 76 and data shown is for the mixture of
diastereomers.
(27) Lin, P.; Chang, L.; DeVita, R. J.; Young, J. R.; Eid, R.; Tong, X.;
Zheng, S.; Ball, R. G.; Tsou, N. N.; Chicchi, G. G.; Kurtz, M. M.; Tsao,
K-L. C.; Wheeldon, A.; Carlson, E. J.; Eng, W.; Burns, H. D.;
Hargreavesd, R. J.; Mills, S. G. The discovery of potent, selective, and
orally bioavailable hNK1 antagonists derived from pyrrolidine. Bioorg.
Med. Chem. Lett. 2007, 17, 5191−5198.
(28) For additional information on 102, see the following and
references therein: Haura, E. B.; Ricart, A. D.; Larson, T. G.; Stella, P. J.;
Bazhenova, L.; Miller, V. A.; Cohen, R. B.; Eisenberg, P. D.; Selaru, P.;
Wilner, K. D.; Gadgeel, S. M. A phase II study of PD-0325901, an oral
MEK inhibitor, in previously treated patients with advanced non−small
cell lung cancer. Clin. Cancer Res. 2010, 16, 2450−2457.
(29) Wang, X.; Kim, J. Conformation-specific effects on Raf kinase
inhibitors. J. Med. Chem. 2012, 55, 7332−7341.
(30) The kinase selectivity profile of 58 was similar to that of 3 with
S(10) selectivity score of 0.01. For a full listing of the kinase panel data,
see the Supporting Information.
(31) Additional details around compound 2 will be reported in a future
manuscript.
2015
dx.doi.org/10.1021/jm301658d | J. Med. Chem. 2013, 56, 1996−2015