Design and synthesis of novel DFG-Out RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors. 1. Exploration of [5,6]-fused bicyclic scaffolds

Article abstract of DOI:10.1021/jm300126x

To develop RAF/VEGFR2 inhibitors that bind to the inactive DFG-out conformation, we conducted structure-based drug design using the X-ray cocrystal structures of BRAF, starting from an imidazo[1,2-b]pyridazine derivative. We designed various [5,6]-fused bicyclic scaffolds (ring A, 1-6) possessing an anilide group that forms two hydrogen bond interactions with Cys532. Stabilizing the planarity of this anilide and the nitrogen atom on the six-membered ring of the scaffold was critical for enhancing BRAF inhibition. The selected [1,3]thiazolo[5,4-b]pyridine derivative 6d showed potent inhibitory activity in both BRAF and VEGFR2. Solid dispersion formulation of 6d (6d-SD) maximized its oral absorption in rats and showed significant suppression of ERK1/2 phosphorylation in an A375 melanoma xenograft model in rats by single administration. Tumor regression (T/C = -7.0%) in twice-daily repetitive studies at a dose of 50 mg/kg in rats confirmed that 6d is a promising RAF/VEGFR2 inhibitor showing potent anticancer activity.

Full text of DOI:10.1021/jm300126x

Article
pubs.acs.org/jmc
Design and Synthesis of Novel DFG-Out RAF/Vascular Endothelial
Growth Factor Receptor 2 (VEGFR2) Inhibitors. 1. Exploration of
[5,6]-Fused Bicyclic Scaffolds†
Masanori Okaniwa,*,‡ Masaaki Hirose,‡ Takashi Imada,‡ Tomohiro Ohashi,‡ Youko Hayashi,‡
Tohru Miyazaki,‡ Takeo Arita,‡ Masato Yabuki,‡ Kazuyo Kakoi,‡ Juran Kato,‡ Terufumi Takagi,‡
Tomohiro Kawamoto,‡ Shuhei Yao,‡ Akihiko Sumita,§ Shunichirou Tsutsumi,§ Tsuneaki Tottori,§
Hideyuki Oki,‡ Bi-Ching Sang,∥ Jason Yano,∥ Kathleen Aertgeerts,∥ Sei Yoshida,‡
and Tomoyasu Ishikawa*,‡
‡Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-Chome, Fujisawa,
Kanagawa 251-8555, Japan
§CMC Center, Takeda Pharmaceutical Company Limited, 17-85, Jusohonmachi 2-Chome, Yodogawa-ku, Osaka 532-8686, Japan
∥Structural Biology, Takeda California, Inc., 10410 Science Center Drive, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: To develop RAF/VEGFR2 inhibitors that bind to the inactive DFG-out conformation, we conducted structure-
based drug design using the X-ray cocrystal structures of BRAF, starting from an imidazo[1,2-b]pyridazine derivative. We designed
various [5,6]-fused bicyclic scaffolds (ring A, 1−6) possessing an anilide group that forms two hydrogen bond interactions with
Cys532. Stabilizing the planarity of this anilide and the nitrogen atom on the six-membered ring of the scaffold was critical for
enhancing BRAF inhibition. The selected [1,3]thiazolo[5,4-b]pyridine derivative 6d showed potent inhibitory activity in both BRAF
and VEGFR2. Solid dispersion formulation of 6d (6d-SD) maximized its oral absorption in rats and showed significant suppression
of ERK1/2 phosphorylation in an A375 melanoma xenograft model in rats by single administration. Tumor regression (T/C =
−7.0%) in twice-daily repetitive studies at a dose of 50 mg/kg in rats confirmed that 6d is a promising RAF/VEGFR2 inhibitor
showing potent anticancer activity.
mutation, particularly V600E, occurs in various carcinomas, in-
cluding malignant melanoma (∼60%), thyroid cancer (∼30%),
and colon cancer (∼15%).4,5 BRAF(V600E) kinase has approxi-
mately 13-fold more potent MEK phosphorylation activity than
does wild-type BRAF kinase, and the BRAF mutation is deeply
involved in the growth of these cancers.6 Hence, targeting the
Ras/RAF/MEK/ERK pathway may be a legitimate approach to
cancer treatment.7,8
INTRODUCTION
■
Signal transduction in the mitogen-activated protein kinase
(MAPK) or Ras/RAF/MEK/ERK pathway plays critical roles
in cellular activities, including proliferation, differentiation, and
survival. The pathway is controlled by extracellular signals
through membrane receptors such as receptor tyrosine kinases
(RTK)1 and is activated by oncogenic mutations in many types
of cancer.2 For example, there are many reports demonstrating
the correlation between RAS mutations and malignant tumors.3
According to these reports, the RAS gene is activated by muta-
tions at codon 12, 13, or 61 in various carcinomas, including
pancreatic cancer (∼90%), non-small-cell lung cancer (∼35%),
and liver cancer (∼30%), and so on. It is also known that BRAF
However, angiogenesis is also a critical process in solid tumor
progression because the tumors require significantly more oxygen,
Received: January 29, 2012
Published: February 29, 2012
© 2012 American Chemical Society
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Figure 1. Various BRAF inhibitors and their binding to the BRAF protein.
glucose, and other nutrients to sustain their rapid growth than do
normal tissues.9 Many cancer tissues secrete vascular endothelial
growth factor (VEGF) to promote angiogenesis from adjacent
blood vessels.10 The VEGF receptor 2 (VEGFR2) is expressed on
the surface of blood vessels, and it plays an important role in
tumor angiogenesis. VEGF/VEGFR2 inhibition has been demon-
strated as a cancer treatment method by using bevacizumab,11
a monoclonal antibody against VEGF, and several small mol-
ecule inhibitors of VEGFR2, such as sunitinib,12 axitinib,13 and
pazopanib.14
conformation of the ATP binding site. These type I inhibitors
of RAF are highly BRAF selective against other kinases, partic-
ularly VEGFR2.
Imidazo[1,2-b]pyridazine derivative 1a24 was identified as a
hit compound by kinase screening of our chemical library
(Figure 2A). Compound 1a showed significant inhibitory activi-
ties against BRAF and VEGFR2, with IC50 values of 43 nM and
3.1 nM, respectively. A molecular model was constructed using
the docking program GOLD, version 3.2,25a and the cocrystal
structure model of sorafenib19 with BRAF(V600E) was used to
examine the binding mode of imidazo[1,2-b]pyridazines.
Although the pyranyl group of 1a did not fit in this model
(vide infra), the simplified acetyl derivative 1b overlapped well
with sorafenib in the DFG-out conformation of BRAF(V600E)
(Figure 2B). An amide proton at the 2-position and a nitrogen
atom at the N-1 position of 2-aminoimidazo[1,2-b]pyridazine
were considered significant because they could interact with the
backbone CO and NH of Cys532 in the kinase hinge region
of the BRAF(V600E) protein. On the basis of this modeling,
novel DFG-out RAF inhibitors bearing [5,6]-fused bicyclic
rings (ring A, 1−6, Figure 3) were designed. An acyl group
(R1), which is smaller than pyran (1a), was considered suitable
because of space constraints in the binding site between the
indole side chain of Trp531 and Gly534. Additionally, the
benzamide moiety (ring C) linked to a central phenoxy group
(ring B) was also thought to be significant for binding with the
DFG-out conformation of BRAF. The amide NH between
rings B and C can interact with the carboxylate side chain of
Glu501, and the CO group of the amide interacts with the
backbone NH of Asp594 in the DFG motif. The benzamide
group (ring C) should occupy the hydrophobic back-pocket
region, where the phenyl group of Phe595 exists in the DFG-in
conformation (Figure 3).
Sorafenib15 (Figure 1) was initially developed as a C-RAF
(RAF-1) inhibitor, although its actual profile is a multikinase
inhibitor against VEGFR2, VEGFR3, and PDGFR-β kinases
involved in angiogenesis. Efficacy in the clinical studies was
thought to be primarily derived from inhibition of tumor
angiogenesis.16 Sorafenib was approved by the Food and Drug
Administration (FDA) for the treatment of hepatocellular
carcinoma and renal cell carcinoma with its efficacy likely due
to its antiangiogenesis activity.17 However, sorafenib showed in-
sufficient efficacy in metastatic melanoma phase 3 clinical trials,
most likely because of insufficient RAF inhibition in melanoma
tissues.18 One possible explanation may be that metastatic
melanoma is independent of angiogenesis. Another explanation
may be that the potency of sorafenib is insufficient for RAF
inhibition in melanoma tissues. Therefore, more potent dual
inhibitors against RAF and VEGFR2 may be beneficial for
patients suffering from various tumors, including metastatic
melanoma.
Over the past decade, efforts have been made to develop
drugs and optimize the effects of RAF kinase inhibitors by using
X-ray cocrystal structures of the BRAF protein with various
ligands. Sorafenib is the first reported RAF kinase inhibitor that
binds to the DFG-out “inactive” conformation of BRAF and
BRAF(V600E).19 Another RAF inhibitor, RAF265, has also
been reported as a RAF/VEGFR dual kinase inhibitor.20 These
two compounds are classified as type II inhibitors,21 which bind
to the DFG-out “inactive” conformation at the ATP binding
site and occupy the hydrophobic “back pocket” in kinases. In
contrast, vemurafenib (PLX4032)8b,22 and SB-59088523 are
classified as type I inhibitors, which bind to the DFG-in “active”
In this paper, the synthesis and structure−activity relation-
ships (SARs) of novel DFG-out RAF/VEGFR2 inhibitors
bearing various [5,6]-fused bicyclic scaffolds will be described.
CHEMISTRY
■
Imidazo[1,2-b]pyridazine26 derivatives 1a−n were prepared as
shown in Schemes 1 and 2. Compounds 1a−g with various acyl
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Figure 2. Design concept: (A) structures of hit compound 1a and its analogue 1b; (B) overlap modeling of 1b (yellow) and sorafenib (purple)
bound to BRAF(V600E) (PDB code 1UWJ).
Figure 3. Design of our DFG-out-type RAF inhibitors (left). Design of the [5,6]-fused bicyclic ring A (right).
groups (R1) were synthesized using the commercially available
ethyl 6-iodoimidazo[1,2-b]pyridazine-2-carboxylate 7 as the
starting material. SNAr displacement reaction of 7 with
3-aminophenol in the presence of potassium carbonate gave the
phenoxylated derivative 8 in 50% yield. Condensation of 8
with 3-(trifluoromethyl)benzoic acid using (1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide) hydrochloride (EDCI)
and 1-hydroxybenzotriazole (HOBt) as coupling reagents
afforded benzamide 9 in 86% yield. Hydrolysis of 9 with
8 N NaOH gave the corresponding carboxylic acid in 91%
yield. Subsequent Curtius rearrangement of the resulting
carboxylic acid using diphenylphosphorylazide (DPPA) and
tert-butanol gave the Boc-protected amine in 89% yield. The
Boc group was deprotected using 4 N HCl in EtOAc to provide
2-aminoimidazo[1,2-b]pyridazine derivative 10 in 67% yield.
Finally, the 2-amino group was acylated by the corresponding
carboxylic acids (a, c, and d) using EDCI and HOBt as the
coupling agents or the corresponding acid chlorides (b, e, f, and g)
to give the desired acylated 2-aminoimidazo[1,2-b]pyridazine
derivatives 1a−g in 42−81% yield (Scheme 1).
Cyclopropylcarbonyl-2-aminoimidazo[1,2-b]pyridazine de-
rivatives 1h−n were synthesized via a different route, for effic-
iently introducing various substituents (R3) (Scheme 2). The
reaction of commercially available 6-iodopyridazine-3-amine 11
with ethyl (chloroacetyl)carbamate in the presence of disodium
hydrogen phosphate gave ethyl 6-iodoimidazo[1,2-b]pyridazine-
2-carbamate 12 in 76% yield. Hydrolysis of ethyl carbamate with
aqueous barium hydroxide, followed by acylation of the resulting
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a
Scheme 1. Synthesis of N-Acylated 2-Aminoimidazo[1,2-b]pyridazines 1a−f
a
Reagents and conditions: (a) 3-aminophenol, K2CO3, DMF, 150 °C, 5 h (50%); (b) 3-(trifluoromethyl)benzoic acid, EDCI·HCl, HOBt, DMF,
t
room temp, 3 h (86%); (c) 8 N NaOH aq, MeOH, H2O, room temp, 4 h (91%); (d) DPPA, Et3N, BuOH, 100 °C, 14 h (89%); (e) 4 N HCl/
EtOAc, MeOH, room temp, 14 h (67%); (f) R1CO2H (a, c, and d), EDCI·HCl, HOBt, DMF, room temp (42−81%); (g) R1COCl (b, e, f, and g),
Et3N, THF, room temp (54−75%).
a
Scheme 2. Synthesis of Cyclopropyl Carbonylated 2-Aminoimidazo[1,2-b]pyridazines 1g−n
a
Reagents and conditions: (a) ethyl (chloroacetyl)carbamate, Na2HPO4, DMF, 110 °C, 4 h (76%); (b) Ba(OH)2·8H2O, H2O, NMP, 120 °C, 12 h
(71%); (c) cyclopropanecarbonyl chloride, DMA, room temp, 16 h (98%); (d) 3-aminophenol, K2CO3, DMF, 150 °C, 24 h (80%); (e) R3-
C6H4CO2H (h, l, and n), EDCI, HOBt, DMF, room temp (68−77%); (f) R3-C6H4COCl (i, j, and k), NMP, room temp (43−75%); (g) 1.4 M
methylmagnesium bromide in THF, toluene, room temp, 12 h (19%).
amine with cyclopropanecarbonyl chloride, was performed in
two steps to afford 2-cyclopropylcarbonylamino-6-iodoimidazo-
[1,2-b]pyridazine 13 in 70% yield. The reaction of 13 with
3-aminophenol in the presence of potassium carbonate afforded
the 6-phenoxylated derivative 14 in 80% yield. Amide-forming
reactions of 14 with the corresponding benzoic acids were
carried out in the presence of EDCI and HOBt to give 1h, 1l, and
1n in 68−77% yield or with the corresponding acid chlorides to
give 1i, 1j, and 1k in 43−75% yield. The tertiary alcohol derivative
1m was synthesized in 19% yield by the treatment of methyl ester
1l with methylmagnesium bromide.
Benzoic acids 15n−q were prepared by the method shown in
Scheme 3. The synthesis of 3-(1-cyano-1-methylethyl)benzoic
acid 15n began with cyanation of the corresponding com-
mercially available methyl-3-(bromomethyl)benzoate 16 with
potassium cyanide in the presence of 18-crown-6, and benzyl
cyanide 17 was obtained in 91% yield. Compound 17 was
dimethylated using iodomethane in the presence of sodium
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a
Scheme 3. Synthesis of Benzoic Acids 15n−q
a
Reagents and conditions: (a) potassium cyanide, 18-crown-6, CH3CN, room temp, 3 days (91%) or sodium cyanide, DMF, 80 °C, 1 h (79%); (b)
NaH, DMSO, room temp, then MeI, room temp (79−88%); (c) NaH, DMSO, room temp, then 1,2-dibromoethane, room temp (57−76%); (d)
LiOH·H2O, THF, MeOH, H2O, room temp (61−98%); (e) NBS, AIBN, CH3CN, 90 °C, 26 h (66%).
hydride, followed by hydrolysis with aqueous lithium hy-
droxide, to give the desired benzoic acid 15n in 77% yield in
two steps. Cyclopropyl derivative 15o was synthesized in two
steps from benzyl cyanide 17 by a similar method using 1,
2-dibromoethane; the obtained yield was 46% yield.
2-Chlorobenzoic acid (R3a = Cl) derivatives 15p and 15q
were synthesized using similar methods adopted for 15n and
15o. Bromination of the commercially available 3-methyl-
benzoate derivative 18 with N-bromosuccinimide (NBS) in the
presence of 2,2′-azobis(2-methylpropionitrile) (AIBN) gave the
benzyl bromide derivative 19 in 66% yield. Cyanation of 19
using sodium cyanide in N,N-dimethylformamide afforded
benzyl cyanide 20 in 79% yield. Subsequent alkylation of 20
with iodomethane or 1,2-dibromoethane in the presence of
sodium hydride afforded the corresponding alkylated deriva-
tives, which were hydrolyzed using aqueous lithium hydroxide
to give the desired benzoic acids 15p and 15q in 52−80% yield
in two steps.
4-difluoronitrobenzene 26 using 40% aqueous methylamine
solution. When N,N-dimethylformamide was used as a cosolvent
in this reaction, methylamine was allowed to react with the
2- and 4-fluorine groups of 26 to provide a complex mixture of
2-methylamino, 4-methylamino, and 2,4-bis(methylamino)
products. However, in the absence of N,N-dimethylformamide,
the desired 2-methylamino derivative 27b precipitated from the
reaction mixture. Thus, the N-methylated starting material 27b
could be selectively synthesized in 99% yield. Commercially
available 2-amino-4-fluoronitrobenzene 27a and the prepared
27b were allowed to react with tert-butyl (3-hydroxyphenyl)-
carbamate in the presence of potassium carbonate to afford
phenoxylated derivatives 28a,b in 69% and 100% yields, res-
pectively. Hydrogenation of the nitro group in 28a,b using
palladium/carbon gave the corresponding diamines in quantita-
tive yield. Subsequent treatment of the resulting diamines with
cyanogen bromide provided benzimidazol-2-amines in 69−100%
yield, which were acylated using cyclopropanecarbonyl chloride
to afford cyclopropyl carbonylated 2-aminobenzimidazoles 29a,b
in 98% and 65% yields, respectively. After the Boc groups of
29a,b were cleaved by TFA (88% yield), an amide coupling
reaction between the deprotected compounds and 15n was
carried out in the presence of EDCI and 4-(N,N-dimethylamino)-
pyridine (DMAP) to obtain the target benzimidazole derivatives
3a,b in 69% and 38% yields, respectively.
Imidazo[1,2-a]pyridine derivative27 2 was synthesized using
the method described in Scheme 4. The reaction of 5-bromo-
2-nitropyridine 21 with 3-nitrophenol in the presence of cesium
carbonate and subsequent hydrogenation using 10% palladium/
carbon provided aminophenoxylated aminopyridine 22 as the
dihydrochloride salt (yield of 54%, two steps) after treatment of
the product with 4 N HCl in EtOAc. Regioselective amide for-
mation between 22 and 3-(1-cyano-1-methylethyl)benzoyl
chloride, prepared in situ from 15n using oxalyl chloride, gave
benzamide 23 in 66% yield. Tosylation of the 2-amino group on
the pyridine ring, followed by alkylation with 2-iodoacetamide at
the nitrogen atom on the pyridine ring, provided the precursor
24 (yield, 63%) for the imidazo[1,2-a]pyridine scaffold in two
steps. The ring formation reaction of 24 proceeded in the pre-
sence of trifluoroacetic anhydride (TFAA) in dichloromethane to
give imidazo[1,2-a]pyridine-2-trifluoroacetamide 25 in 52% yield.
Cleavage of the trifluoroacetyl group with 1 N NaOH and sub-
sequent acylation of the resulting amino group using cyclopro-
panecarbonyl chloride gave the desired compound 2 (yield, 43%)
in two steps.
The synthesis of imidazo[4,5-b]pyridine derivatives29 4a,b is
shown in Scheme 6. Substituted benzoyl chloride, prepared by
the reaction of 15n with oxalyl chloride in situ, was allowed to
react with 3-aminophenol under Schotten−Baumann con-
ditions to provide the intermediate phenol 30 in 95% yield in
two steps. The regioselective reaction of 2,6-dichloro-3-nitro-
pyridine 31 with tert-butylamine proceeded at the 2-position to
give the tert-butylaminated compound 32a in 99% yield. Sub-
sequent coupling of 32a with 30 in the presence of potassium
carbonate afforded the coupled product 33a in 72% yield. The
nitro group of 33a was hydrogenated using palladium/carbon,
and the resulting aniline was treated with cyanogen bromide to
provide the imidazo[4,5-b]pyridine-2-amine derivative 34a in
82% yield in two steps. Subsequent acylation of the 2-amino
group of 34a with cyclopropanecarbonyl chloride, followed by
cleavage of the tert-butyl group with TFA, provided the desired
imidazo[4,5-b]pyridine derivative 4a in 79% yield in two steps.
Benzimidazole derivatives28 3a,b were synthesized as shown
in Scheme 5. For synthesis of the N-methylated compound
3b, introduction of a methyl group was regiocontrolled at
the first step by the regioselective SNAr displacement of 2,
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a
Scheme 4. Synthesis of Imidazo[1,2-a]pyridine 2
a
Reagents and conditions: (a) 3-nitrophenol, Cs2CO3, DMF, room temp, 12 h (54%); (b) H2, 10% Pd/C, MeOH, THF, EtOAc, room temp, 20 h,
then 4 N HCl/EtOAc (quant); (c) 3-(1-cyano-1-methylethyl)benzoyl chloride, DMA, room temp, 18 h (66%); (d) 4-methylbenzenesulfonyl
chloride, pyridine, 80 °C, 2 days (99%); (e) 2-iodoacetamide, DIEA, DMF, room temp, 48 h (63%); (f) TFAA, CH2Cl2, room temp, 16 h (52%);
(g) 1 N NaOH aq, EtOH, 45 °C, 12 h (quant); (h) cyclopropanecarbonyl chloride, DMA, room temp, 8 h (43%).
a
Scheme 5. Synthesis of 1H-Benzimidazole 3a,b
a
Reagents and conditions: (a) 40% methylamine solution, 0 °C, 1.5 h (99%); (b) tert-butyl (3-hydroxyphenyl)carbamate, K2CO3, DMF, 80−100 °C
(69−100%); (c) H2, 10% Pd/C, THF, MeOH, room temp (quant); (d) BrCN, THF, room temp (69−100%); (e) cyclopropanecarbonyl chloride,
DMAP, pyridine, room temp (65−98%); (f) TFA, reflux (88%); (g) 15n, EDCI·HCl, DMAP, pyridine (38−69%).
The corresponding N-methylated derivative 4b was prepared
using a method similar to that used to generate 4a. Regio-
selective amination of 2,6-dichloro-3-nitropyridine 31 with
methylamine provided the 2-methylaminated product 32b in
78% yield. The coupling reaction of 32b with 30 in the pre-
sence of potassium carbonate gave 33b in 88% yield. Reduction
of the nitro group of 33b and subsequent treatment with
cyanogen bromide provided the 1-methylimidazo[4,5-b]pyridine-
2-amine derivative 34b in 80% yield in two steps. Finally,
acylation of 34b with cyclopropanecarbonyl chloride provided
the desired N-methylated imidazo[4,5-b]pyridine derivative 4b
in 56% yield.
Condensation of 36 with 15n using EDCI in the presence of
DMAP in pyridine provided benzamide 37 in 99% yield. Re-
duction of the nitro group of 37 under hydrogenation conditions
using palladium/carbon gave aniline 38 in 98% yield. The
thiazole ring was constructed by the reaction of 38 with potas-
sium thiocyanate and bromine in acetic acid, and the obtained
yield was 87%. The obtained 2-amino-1,3-benzothiazole deriv-
ative was acylated using cyclopropanecarbonyl chloride to afford
the desired 1,3-benzothiazole derivative 5 in 88% yield.
The synthesis of thiazolo[5,4-b]pyridine31 derivatives 6a−c
is shown in Scheme 8. Reaction of commercially available
2-chloro-5-nitropyridine 39 with Boc-protected aminophenol in
the presence of potassium carbonate provided a coupled pro-
duct, and subsequent reduction of the nitro group under standard
hydrogenation conditions provided the corresponding aniline 40
in 77% yield in two steps. Fused 1,3-thiazole ring construction
The synthesis of 1,3-benzothiazole derivative30 5 is shown
in Scheme 7. The reaction of 4-fluoronitrobenzene 35 with
3-aminophenol in the presence of potassium carbonate
gave the phenoxylated aniline derivative 36 in 95% yield.
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a
Scheme 6. Synthesis of Imidazo[4,5-b]pyridine 4
a
Reagents and conditions: (a) (COCl)2, cat. DMF, THF, room temp; (b) 3-aminophenol, NaHCO3, H2O, THF, room temp (96% in two steps);
(c) tert-butylamine, toluene, or methylamine, THF, room temp (78−99%); (d) 30, K2CO3, DMF, room temp, 18 h (72−88%); (e) H2, 10% Pd/C,
MeOH, THF, room temp, 12 h; (f) BrCN, THF, room temp, 18 h (80−82% in two steps); (g) cyclopropanecarbonyl chloride, DMAP, pyridine,
room temp (56−96%); (h) TFA, 80 °C, 2 h (82%).
a
Scheme 7. Synthesis of 1,3-Benzothiazole 5
a
Reagents and conditions: (a) 3-aminophenol, K2CO3, DMF, 80 °C, 8 h (95%); (b) 15n, EDCI·HCl, DMAP, pyridine, room temp, 4 h (99%); (c)
H2, 10% Pd/C, THF, MeOH, room temp, 14 h (98%); (d) KSCN, Br2, AcOH, room temp, 4 h (87%); (e) cyclopropanecarbonyl chloride, DMAP,
pyridine, room temp, 6 h (88%).
using potassium thiocyanate and bromine under conditions
similar to those used to generate 1,3-benzothiazole afforded the
2-aminothiazolo[5,4-b]pyridine derivative 41 in 88% yield. Acyla-
tion of the 2-amino group with cyclopropanecarbonyl chloride in
pyridine gave the precursor 42 for introducing benzamide moie-
ties, in 86% yield. Deprotection of the Boc group using TFA and
subsequent condensation using benzoic acid derivatives 15n−p
under the standard conditions provided the desired thiazolo[5,4-b]
pyridine derivatives 6a−c in 46−87% yield.
the corresponding acid chloride in situ using oxalyl chloride,
and the obtained acid chloride was allowed to react with 3-
amino-4-fluorophenol in the presence of aqueous sodium
bicarbonate to give an intermediate phenol 43 in 100% yield.
Compound 43 was allowed to react with 2-chloro-5-
nitropyridine 39 in the presence of potassium carbonate to
give a coupled product in 93% yield, and subsequent reduc-
tion of the nitro group using reduced iron and calcium chloride
in aqueous ethanol provided aniline 44 in 69% yield. Cycliza-
tion of 44 using potassium thiocyanate and bromine in acetic
acid gave thiazolo[5,4-b]pyridin-2-amine 45 in 69% yield.
Compound 6d was synthesized using a method similar to that
used for 6a−c in Scheme 9. Compound 15q was converted into
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a
Scheme 8. Synthesis of [1,3]Thiazolo[5,4-b]pyridine 6a−c
a
Reagents and conditions: (a) tert-butyl (3-hydroxyphenyl)carbamate, K2CO3, DMF, 70 °C, 2 h; (b) H2, 10% Pd/C, EtOH, THF, room temp, 7 h
(77% in two steps); (c) KSCN, Br2, AcOH, room temp, 1.5 h (88%); (d) cyclopropanecarbonyl chloride, pyridine, room temp, 1 h (86%); (e) TFA,
anisole, 0 °C, 1 h (79%); (f) 15n,o, SOCl2, DMAP, toluene, then pyridine (46−58%), or 15p, HATU, pyridine, room temp (87%).
a
Scheme 9. Synthesis of [1,3]Thiazolo[5,4-b]pyridine 6d
a
Reagents and conditions: (a) (COCl)2, cat. DMF, THF, room temp, 2.5 h, then 3-amino-4-fluorophenol, NaHCO3, THF, H2O, room temp, 1 h
(100%); (b) 39, K2CO3, DMF, room temp, 4 h (93%); (c) Fe(0), CaCl2, EtOH, H2O, 80 °C, 18 h (69%); (d) KSCN, Br2, AcOH, room temp, 6 h
(69%); (e) cyclopropanecarbonyl chloride, pyridine, THF, room temp, 3 h (96%).
Acylation of 45 with cyclopropanecarbonylchloride afforded
the desired compound 6d in 96% yield.
RESULTS AND DISCUSSION
■
To develop novel RAF/VEGFR2 inhibitors, we identified an
initial lead compound imidazo[1,2-b]pyridazine24 derivative 1a
from screening. Compound 1a showed good potency against
VEGFR2 and BRAF(V600E), with IC50 values of 3.1 and
43 nM, respectively. However, Western blotting assay for as-
sessing the phosphorylation level of the downstream MEK1/2
(pMEK) in HT-29 colon cancer cells revealed that its cellular
activity based on BRAF(V600E) inhibition was insufficient
(IC50 = 3800 nM). Thus, our initial medicinal chemistry efforts
were directed at exploring the alkylamide side chain (R1) and
hydrophobic “back pocket” benzamide moiety (R3) using the
imidazo[1,2-b]pyridazine scaffold 1 to enhance both BRAF-
(V600E) and cellular pMEK inhibitory activities.
Compound 6e, which was chlorinated at ring B, was pre-
pared by the synthetic route shown in Scheme 10. Reaction of
39 with 3-amino-4-chlorophenol in the presence of potassium
carbonate provided the phenoxylated compound 46 in 85%
yield. Protection of the anilino group of 46 by a trifluoroacetyl
group using trifluoroacetic anhydride, followed by reduction of
the nitro group using reduced iron in acetic acid, gave the tri-
fluoroacetylated compound 47 in 79% yield in two steps.
Cyclization of 47 using potassium thiocyanate and bromine in
acetic acid and subsequent acylation with cyclopropanecarbonyl
chloride provided the 2-acylated aminothiazolo[5,4-b]pyridine
derivative 48 in 42% yield in two steps. Deprotection of the tri-
fluoroacetyl group was achieved using sodium borohydride in
mixed solvent of methanol and ethanol to give the corres-
ponding aniline in 75% yield. Condensation with 15q under
standard conditions provided the desired thiazolo[5,4-b]-
pyridine derivative 6e in 64% yield.
Imidazo[1,2-b]pyridazines 1a−g, with various N-acyl groups
(R1) at the 2-position, were evaluated as shown in Table 1.
BRAF(V600E) modeling suggested that the R1 group exists in
the narrow space formed by the indole ring of Trp531 and
Gly534 and that the optimal size of the R1 group for fitting into
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a
Scheme 10. Synthesis of [1,3]Thiazolo[5,4-b]pyridine 6e
a
Reagents and conditions: (a) 3-amino-4-chlorophenol, K2CO3, DMF, room temp, 16 h (85%); (b) TFAA, THF, room temp, 1 h (87%); (c) Fe(0),
AcOH, 60 °C, 3 h (91%); (d) KSCN, Br2, AcOH, room temp, 16 h (72%); (e) cyclopropanecarbonyl chloride, pyridine, room temp, 1 h (59%);
(f) NaBH4, MeOH, EtOH, room temp, 1 h (75%); (g) 15q, (COCl)2, DMF, THF, then DMA, room temp, 2 h (64%).
the cyclopropyl derivative 1e showed potent cellular pMEK
activity with an IC50 of 310 nM. However, substitution of the
N-acyl group (R1) had negligible impact on the VEGFR2 in-
hibition, with compounds 1a−g showing nanomolecular-order
inhibitory activity against VEGFR2. Since the acetyl group of
1b was found to be sensitive to deacetylation32a by liver micro-
somes in an in vitro metabolism study (data not shown, but this
result was utilized in our next prodrug study32b), we chose the
cyclopropyl group (1e) as the representative R1 group.
Next, we investigated the effect of substituting R3 groups in
the “back pocket” of the benzene ring (ring C); the results are
summarized in Table 2. Our initial goal was to examine the sub-
stitution position of the trifluoromethyl group against BRAF-
(V600E). Alternating between the meta (1e), para (1h), and
ortho (1i) positions resulted in decreased BRAF inhibitory
activity, suggesting that meta substitution may be favorable for
inhibiting the BRAF protein.
Table 1. Structure−Activity Relationship of N-Acyl Groups
at 2-Amino Position (R1)
Replacement of the trifluoromethyl group with methoxy (1j)
and tert-butoxy (1k) groups helped in maintaining strong
BRAF(V600E) inhibitory activities comparable to that of 1e.
Additionally, 1k showed slightly increased cellular pMEK
inhibition, with an IC50 of 120 nM. In our model, wherein 1k
was bound to BRAF, the tert-butoxy group of 1k could occupy
the lipophilic space formed by Val504, Leu505, Ile513, Leu514,
and Thr508 in the back pocket to result in lipophilic van der
Waals interactions with the BRAF protein. On the basis of
BRAF inhibition studies, we assumed that bulky lipophilic
groups may stabilize the DFG-out binding conformation and
increase cellular activity. Therefore, to confirm our assumption,
we examined the introduction of bulky 1-hydroxy-1-methylethyl
(1m) and 1-cyano-1-methylethyl (1n) groups. Although 1m
showed reduced cellular pMEK inhibitory activity compared to
1k, the more lipophilic 1-cyano-1-methylethyl derivative 1n
demonstrated the most potent cellular activity among all the
derivatives in the imidazo[1,2-b]pyridazine series, with an IC50 of
55 nM. These results indicated that the lipophilic back pocket
moiety is a key pharmacophore of the DFG-out type BRAF
inhibitor for its potent pMEK inhibitory activity.
a
n = 2. Values in parentheses indicate the 95% confidence interval.
Concentration producing 50% inhibition (IC50) values against RAF
b
substrate MEK phosphorylation in HT-29 (BRAFV600E) cultured
human colon cancer cell lines. Not determined.
c
this narrow space would be smaller than that of a pyranyl
group. Therefore, we examined the SAR of R1 groups to deter-
mine their BRAF preference and their potency against cellular
pMEK activity. Replacement of the pyranyl group in 1a with a
methyl group in 1b resulted in significantly increased BRAF-
(V600E) inhibitory activity, with an IC50 of 4.1 nM. Com-
pounds bearing alkyl groups larger than those in 1b, such as
ethyl (1c) and isopropyl (1d) groups, exhibited decreased
BRAF(V600E) inhibitory activities, with IC50 values of 6.9 and
31 nM, respectively. Thus, 1b and 1c revealed more potent
cellular pMEK activity than did 1a. These results are consistent
with those of our modeling studies, suggesting that decreasing
the size of the N-acyl groups (R1) generally increased BRAF
and the cellular pMEK inhibitory activity. Because the R1 group
is located near a hinge in our BRAF model, bulky groups may
be unfavorable for binding the BRAF protein.
All the imidazo[1,2-b]pyridazine derivatives 1e−n showed
more potent activities against VEGFR2 than against BRAF-
(V600E). These results implied that VEGFR2 has a wider
range of molecular recognition in the back pocket than does
BRAF(V600E). Therefore, we moved to the exploration of
alternative [5,6]-fused bicyclic scaffolds to strengthen the
BRAF inhibitory activity.
Compounds 1e−g having a cycloalkyl group as R1 were
further evaluated. Similar to the alkyl side chain in 1b−d, de-
creasing the ring size from cyclopentyl (1g) to cyclopropyl (1e)
enhanced the BRAF(V600E) inhibitory activity. Additionally,
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Table 2. Structure−Activity Relationship of Back Pocket Region (R3)
a
b
n = 2. Values in parentheses indicate the 95% confidence interval. Concentration producing 50% inhibition (IC50) values against RAF substrate
c
MEK phosphorylation in HT-29 (BRAFV600E) cultured human colon cancer cell lines. Not determined.
To identify the appropriate scaffolds (ring A), novel [5,6]-
fused bicyclic derivatives 2−6, which had the same phenoxylated
back pocket moiety as does 1n, were designed and evaluated
(Table 3). Replacement of imidazo[1,2-b]pyridazine (1n) with
imidazo[1,2-a]pyridine (2) resulted in approximately 3-fold
reduction of the BRAF(V600E) and cellular pMEK inhibitory
activities. Similar tendencies were observed between benzimi-
dazole (3a) and imidazo[4,5-b]pyridine (4a) as well as between
1,3-benzothiazole (5) and [1,3]thiazolo[5,4-b]pyridine (6a).
These results suggested that the nitrogen atom adjacent to the
phenoxy group contributes to the enhanced BRAF(V600E)
inhibitory activities.
the steric hindrance between the N-3-methyl group and the 2-
cyclopropylcarboxamide moiety of 4b disrupted the planarity of
the C-2 carboxamide with ring A and might reduce BRAF in-
hibition. To confirm our hypothesis, we calculated the stable
dihedral angles of the amides in 3a,b and 4a,b using the MOE
program25 (see Experimental Section) to determine their mini-
mum potential energies (Figure 4). We found that the N-
methylated derivatives 3b and 4b preferred torsional conforma-
tions (energy, 3.2−4.2 kcal/mol) to the planar conformations of
3a and 4a. Accordingly, we focused on identifying scaffolds that
could stabilize the planarity of the C-2 carboxamide group with
ring A to enhance the BRAF inhibitory activity.
Additionally, alternating the fusion position of the imidazole
rings, such as 2 to 3a or 1n to 4a, maintained the BRAF(V600E)
inhibitory activity but reduced the cellular pMEK inhibitory
activity. Since the additional proton doner (NH) at the N-3 posi-
tion in 3a and 4a plausibly diminish those cellular permeability,
we examined the introduction of a methyl group at the N-3
position to mask the proton donor. However, the N-methylated
derivatives 3b and 4b showed dramatically decreased BRAF-
(V600E) inhibitory activity, with IC50 values of 960 and 200 nM,
respectively. Because the intermediate N-methylated imidazo-
[5,4-d]pyridine-2-amine 34b showed stronger BRAF(V600E)
inhibitory activity (IC50 = 35 nM) than did 4b, we assumed that
To stabilize this planarity, we designed fused 1,3-thiazole
scaffolds, 1,3-benzothiazole (5) and [1,3]thiazolo[5,4-b]pyridine
(6). On the basis of the intramolecular interactions between the
carbonyl group and sulfur by using the d-orbital of the sulfur
atom,33 we hypothesized that the planar conformations of the
C-2 carboxamide with their scaffolds may be stabilized more
effectively than those of the scaffolds 1−4. As expected, 5 and 6a
showed greater BRAF(V600E) inhibitory activity than did the
corresponding imidazole derivatives 3a and 4a. Particularly, the
[1,3]thiazolo[5,4-b]pyridine derivative 6a demonstrated the
most potent BRAF(V600E) and cellular pMEK activities, with
IC50 values of 5.6 and 6.2 nM, respectively. The inhibitory
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Table 3. Structure−Activity Relationship of Novel [5,6]-Fused Bicyclic Ring A
a
b
n = 2. Values in parentheses indicate the 95% confidence interval. Concentration producing 50% inhibition (IC50) values against RAF substrate
c
MEK phosphorylation in HT-29 (BRAFV600E) cultured human colon cancer cell lines. Not determined.
Figure 4. Stable dihedral angles of amides linked to ring A (3, 4) are calculated using ligand-based analysis to determine their minimal potential
energies. Compounds calculated are simplified analogues without the phenoxy moiety. Fused imidazoles without the methyl group (3a, 4a) are
categorized in the planar group. Fused imidazoles with a methyl group (3b, 4b) are categorized in the torsional group owing to steric hindrance
between 3-methyl and carbonyl. Dihedral angles (deg) and their internal energy gains (ΔE*) from their planar conformations are shown.
potency of 6a against BRAF(V600E) reached a level com-
[5,4-b]pyridine scaffold 6 as one of the most appropriate scaf-
parable to that against VEGFR2. Thus, we selected [1,3]thiazolo
folds for dual inhibition against BRAF(V600E) and VEGFR2.
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Because 6a showed sufficient in vitro potency to act as a
BRAF(V600E) and VEGFR2 dual kinase inhibitor, the pharma-
cokinetic (PK) profile of this compound was evaluated in mice
(Table 4). However, 6a showed poor drug exposure, with an
AUC0−8h of 0.393 μg h/mL, after oral administration at a dose
of 10 mg/kg in mice. To improve this poor oral absorption, we
initially substituted the dimethylcyanomethyl group (6a) with a
cyanocyclopropyl group (6b) so that the metabolized site was
blocked. The BRAF/VEGFR2 inhibitory activities remained
high, and this transformation was effective in enhancing oral ab-
sorption in mice while improving metabolic stability and slightly
increasing the solubility for 6b. Additionally, we attempted to
introduce an R3a group at the ortho position between benzamide
and the R3b group to increase the solubilities of 6a,b by twisting
the plane of benzamide (ring B) with ring C. As expected, 6c,
with a chlorine atom at the 2-position of benzamide (ring C),
showed improved solubility and oral absorption in mice while
maintaining the in vitro potency. Introduction of chlorine at R3a
proved to be effective for enhancing the poor solubility.
steric restriction around the 4-position, smaller R2 groups could
be more favorable to be introduced. The 6-chlorinated [1,3]-
thiazolo[5,4-b]pyridine derivative 6e showed reduced in vitro
potency against BRAF(V600E), but the 6-fluorinated [1,3]-
thiazolo[5,4-b]pyridine derivative 6d maintained sufficient in
vitro potency to act as a BRAF(V600E) and VEGFR2 dual
kinase inhibitor. Furthermore, 6d showed significantly im-
proved metabolic stability against human liver microsomes and
good PK profiles in mice. Therefore, we selected 6d as a prom-
ising candidate for further evaluation.
X-ray Cocrystal Structural Analysis of 6d with BRAF
and VEGFR2. We determined the X-ray cocrystal structures of
6d with BRAF and VEGFR2 proteins, respectively.34 The BRAF
cocrystal structure (PDB code 4DBN) revealed that 6d occupies
the ATP-binding site and stabilizes the inactive DFG-out con-
formation of BRAF by being accommodated to the back pocket
region (Figure 5A). As expected, the [1,3]thiazolo[5,4-b]-
pyridine-2-amine moiety is located in front of the hinge region
of BRAF and forms two significant hydrogen bonds between
the carbonyl of Cys532 and the NH of the 2-amide (2.7 Å)
and between the NH of Cys532 and the N-3 nitrogen (3.2 Å).
The cocrystal structure of BRAF supports our hypothesis
that the planar conformation of the 2-amide group with the
Finally, we optimized the substituents (R2) at the para or
6-position of the phenoxyl group (ring B) to enhance the meta-
bolic stability of 6c by blocking the metabolically labile site.
The results of our modeling studies showed that because of
Table 4. Structure−Activity Relationship of [1,3]Thiazolo[5,4-b]pyridine Derivatives 6a−e
a
b
n = 2. Values in parentheses indicate the 95% confidence interval. Concentration producing 50% inhibition (IC50) values against RAF substrate
c
MEK phosphorylation in HT-29 (BRAFV600E) cultured human colon cancer cell lines. 10 mmol/L solution of the compound in DMSO was
evaluated using the JP second fluid in the disintegration test (pH 6.8) containing bile acid. The sample solution was shaken at 37 °C for 24 h, and its
solubility was evaluated after filtration. Metabolism clearance of each compound was examined using liver microsomes and NADPH. Cassette
dosing of five compounds. Values shown are the mean of data from three mice. Compounds (10 mg/kg) were administered in 0.5% methylcellulose
in distilled water.
d
e
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Figure 5. Cocrystal structure of 6d with BRAF (PDB code 4DBN, 3.1 Å resolution) and VEGFR2 (PDB code 3VNT, 1.64 Å resolution): (A) DFG-
out conformation of BRAF (green cartoon) is stabilized by 6d; (B) back pocket region of BRAF (surface); (C) DFG-out conformation of VEGFR2
(magenta cartoon) is stabilized by 6d; (D) back pocket region of VEGFR2 (surface).
[1,3]thiazolo[5,4-b]pyridine scaffold may be stabilized through
sulfur−carbonyl interactions because the measured distance
(2.8 Å) between the sulfur and oxygen atoms is shorter than the
sum of the corresponding van der Waals radii of the oxygen and
sulfur atoms (3.32 Å).35 Interestingly, the indole side chain of
Trp531 is located near the 2-amide bond of 6d and possibly
forms π−π stacking interactions with the 2-amide. This type
of interaction is not observed in the cocrystal structure19 of
sorafenib, and it may be critical for enhancing the BRAF
inhibition of 6d. Structural differences of adenine sites between
4DBN (6d) and 1UWJ (sorafenib) is discussed in detail using
the figure that describes the overlapped structures in Supporting
Information. Additionally, the biphenyl amide moiety between
rings B and C of 6d forms two significant hydrogen bond inter-
actions with the backbone NH of Asp594 (3.0 Å) and the side
chain carboxylate of Glu501 in the C-helix (3.0 Å). These inter-
actions may be important for stabilizing the DFG-out inactive
conformation of BRAF. Furthermore, the 2-chloro-3-
(1-cyanocyclopropyl)benzene ring is located in a back pocket
that is twisted away from the carbonyl surface by 44.4° (Figure 5B).
The bulky cyanocyclopropyl group occupies the hydrophobic
back pocket formed by Val504, Thr508, Leu567, His574, and
Ile592 (indicated by the green area in Figure 5B); this hydro-
phobic interaction plays an important role enhancing the
BRAF(V600E) cellular activity.
Table 5. Kinase Selectivity of 6d
a
a
kinase
IC50 (nM)
kinase
PKA
IC50 (nM)
BRAF(wt)
C-RAF
FGFR3
PDGFRα
PDGFRβ
EGFR
Her2
12
>10000
>10000
6300
1.5
PKCθ
CHK1
CK1δ
22
12
>10000
>10000
2200
5.5
ERK1
>10000
>10000
>10000
>10000
>10000
420
CDK1
CDK2
Aurora B
p38α
2700
TIE2
240
c-Met
c-Kit
1100
JNK1
>10000
2900
Src
GSK3β
MEK1
MEKK1
IR
>10000
>10000
>10000
>10000
IKKβ
a
n = 2.
The cocrystal structure of VEGFR2 (PDB code 3VNT) also
revealed that 6d occupies the ATP binding site of VEGFR2 and
stabilizes the DFG-out conformation of VEGFR2 (Figure 5C).
Since similar hydrogen bond interactions (depicted in the red
dotted lines in Figure 5C), compared to those of BRAF, and
occupation of the 3-(1-cyanocyclopropyl)benzene moiety in
the back pocket region of VEGFR2 (Figure 5D) were observed,
6d was proved to be a DFG-out type RAF/VEGFR2 inhibitor.
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Figure 6. MAPK signal suppression effect by 2 h treatment of 6d (Western blotting) and its antiproliferative activity (GI50) in various cancer
cell lines possessing the BRAFV600E mutated gene.
Figure 7. VEGF signal suppression effect by 2 h treatment of 6d (Western blotting) in 293/KDR cell lines. The 293/KDR cells were untreated (−)
or treated (+) with VEGF-A (final concentration of 50 ng/mL) for 10 min.
a
Table 6. Mean Pharmacokinetic Parameters for 6d in Rats
dose (mg/kg)
route
CLtotal (mL h−1 kg−1
886 203
)
Vdss (mL/kg)
MRT (h)
AUC0−24h (μg h/mL)
Cmax, po (μg/mL)
F (%)
b
1
iv
1655 493
1.86 0.24
4.38 0.43
1.167 0.25
c
10
oral
8.23 1.04
1.29 0.07
70.5 8.9
a
b
c
Values shown are the mean SD of data from three rats. Delivered in polyethylene glycol/DMA (1/1). Solid dispersion (SD) powder, prepared
by spray dry method using hydroxypropylmethylcellulose (HP-55) (6d/HP-55 (1:4)); delivered in distilled water.
The stabilized planar conformation of the 2-amide group with
the [1,3]thiazolo[5,4-b]pyridine scaffold by the sulfur−carbonyl
interaction was also observed in the VEGFR2 cocrystal structure.
However, because of the replacement of the indole ring (Trp531)
in BRAF with the phenyl ring (Phe918) in VEGFR2, the VEGFR2
pocket appears to be spacious and more accommodating to the
various bicyclic fused rings with diversified and less planar amide
conformations compared to BRAF in Table 3.
Kinase Inhibitory Profiles of 6d. The inhibitory profiles
of 6d against 26 different kinases are summarized in Table 5.
Compound 6d exhibited potent inhibitory activity against not
only BRAF(V600E) (IC50 = 7.0 nM) but also wild-type BRAF
(12 nM) and C-RAF (1.5 nM). Additionally, other kinases
related to angiogenesis,36 such as FGFR3, PDGFRα, PDGFRβ,
were inhibited with IC50 values comparable to that of VEGFR2
inhibition. Several kinases, including Aurora B and Src, were
moderately inhibited with IC50 values ranging from 240 to
420 nM, and no significant inhibition was observed against the
remaining 19 kinases. Therefore, 6d was considered to have a char-
acter as a pan-RAF and angiogenesis-related kinases inhibitor.
In Vitro Pharmacology of 6d. To determine the potency of
6d as a BRAF(V600E) inhibitor, we investigated the in vitro
MAPK signal suppression effect of 6d in various cancer cells
harboring the BRAFV600E mutation (Figure 6). In several types of
colon, melanoma, and thyroid cancer cells, 6d suppressed both
phospho-MEK1/2 and phospho-ERK1/2 levels in a concentration-
dependent manner. Reflecting the inhibition of the down-
stream molecules in MAPK signal transduction, 6d demon-
strated antiproliferative activities among these cell lines, with
GI50 of 130−820 nM.
Next, the cellular VEGFR2 inhibitory activity of 6d was evaluated
in VEGFR2-overexpressing 293/KDR cells37 (Figure 7). Com-
pound 6d significantly inhibited the VEGFR2 phosphoryla-
tion induced by treatment with vascular endothelial growth
factor A (VEGF-A), with an IC50 of 0.53 nM. Furthermore, 6d
potently inhibited the VEGF-induced proliferation of HUVEC
at a GI50 of 19 nM. These results indicated that the cellular
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Figure 8. Mean (n = 2) phosphorylated ERK1/2 levels in F344 nude rats bearing A375 (BRAFV600E mutant) human melanoma xenograft tumors
after treatment with single dose of 6d-SD. Footnotes in the table portion indicate the following: (b) detected by Western blotting; (c) delivered in
distilled water; (d) solid dispersion formulation was delivered in distilled water; (e) dose of 6d is described.
VEGFR2 inhibitory activity (Table 5, Figure 7), this tumor
regression should include the efficacy based on the antiangio-
genesis potency and BRAF inhibitory activity.
CONCLUSION
■
We designed and developed RAF/VEGFR2 inhibitors bearing a
novel [1,3]thiazolo[5,4-b]pyridine scaffold 6 on the basis of
structure-based drug design using docking models of our lead
imidazo[1,2-b]pyridazines 1a,b with BRAF. Target compounds
were designed as DFG-out type binders for binding the inactive
conformation of BRAF. X-ray cocrystal structural analysis of the
representative compound 6d indicated that the two hydrogen
bonds in benzamide between rings B and C with Asp594 and
Glu501 play a key role in stabilizing the DFG-out conforma-
tion. Furthermore, the sulfur−carbonyl interactions were
important for the planar conformation of the 2-amide, together
with coordination of the π−π interaction with the indole ring of
Trp531, which was significant for enhancing BRAF inhibition.
The optimal compound 6d showed excellent in vitro potency as
a BRAF(V600E) and VEGFR2 dual kinase inhibitor.
Figure 9. Mean (n = 3) tumor volumes and body weights in F344 nude
rats bearing A375 human melanoma xenograft tumors dosed with 6d-SD
or vehicle. Footnotes in the table portion indicate the following: (b) com-
pound 6d-SD was administered orally twice daily for 14 days; (c) deliv-
ered in distilled water; (d) dolid dispersion formulation was delivered in
distilled water; (e) dose of 6d is described. P ≤ 0.05 vs control at day 14
(t-test).
In vitro pharmacologycal evaluation of 6d revealed potent
MAPK cascade suppression in various BRAFV600E mutant tumor
cell lines. Furthermore, 6d showed antiangiogenesis by
suppressing the VEGFR2 pathway in 293/KDR and VEGF-
stimulated HUVEC cells. Compound 6d-SD, prepared using a
spray-dried SD formulation, demonstrated a mechanism-based
in vivo PD effect as well as regressive antitumor efficacy in an
A375 human melanoma xenograft model in rats. These results
suggest that dual inhibition of BRAF(V600E) and VEGFR2
may provide strong antitumor efficacy and that 6d is a prom-
ising candidate for the treatment of various human cancers
harboring the BRAFV600E mutation.
antiangiogenesis activity of 6d resulted in enhanced in vivo
antitumor efficacy against various types of cancers.
Pharmacokinetic Profiles and in Vivo Studies of 6d in
Rats. To maximize the oral bioavailability of 6d, we applied a
spray-dried solid dispersion (SD) formulation38 to this com-
pound using hydroxypropyl methylcellulose phthalate (HPMCP,
HP-55). The obtained 6d-SD showed sufficient oral bioavailability
(F = 70.5%) at a dose of 10 mg/kg for 6d in rats (Table 6).
Reflecting the sufficient oral bioavailability, 6d-SD demon-
strated significantly decreased phosphorylation levels of ERK1/2
4 h after oral administration in an A375 (BRAFV600E mutant)
human melanoma xenograft model in rats (Figure 8).
Finally, we examined the antitumor efficacy of 6d-SD admin-
istered orally twice daily in an A375 melanoma xenograft model
in rats (Figure 9). Two weeks after administration at a dose of
10 mg/kg for 6d, we observed tumor regression with T/C
of −7.0% without severe toxicity. Since 6d also shows potent
EXPERIMENTAL SECTION
■
General Chemistry Information. The starting materials, re-
agents, and solvents for reactions were reagent-grade and were used as
purchased. Thin-layer chromatography (TLC) was carried out using
Merck Kieselgel 60, 63−200 mesh, F254 plates, or Fuji Silysia Chemical
Ltd., 100−200 mesh, NH plates. Chromatographic purification was
carried out using silica gel (Merck, 70−230 mesh) or basic silica gel
(Fuji Silysia Chemical Ltd., DM1020, 100−200 mesh). Melting points
were obtained using an OptiMelt melting point apparatus MPA100 and
used uncorrected. Proton nuclear magnetic resonance 1H NMR spectra
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were recorded using a Bruker AVANCE II (300 MHz) spectrometer
with tetramethylsilane (TMS) as an internal standard. The NMR data
are given as follows: chemical shift (δ) in ppm, multiplicity (where
applicable), coupling constants (J) in Hz (where applicable), and
integration (where applicable). Multiplicities are indicated by s (singlet),
d (doublet), t (triplet), q (quartet), dd (double doublets), dt, (double
triplet), ddd (double double doublet), br s (broad singlet), or
m (multiplet). MS spectra were collected with a Waters LC−MS
system (ZMD-1) and were used to confirm ≥95% purity of each
compound. The column used was an L-column 2 ODS (3.0 mm ×
50 mm i.d., CERI, Japan) with a temperature of 40 °C and a flow rate of
1.2 mL/min. Mobile phase A was 0.05% TFA in ultrapure water. Mobile
phase B was 0.05% TFA in acetonitrile which was increased linearly
from 5% to 90% over 2 min, 90% over the next 1.5 min, after which the
column was equilibrated to 5% for 0.5 min. Elemental analyses (Anal.)
and high-resolution mass spectrometry (HRMS) were carried out at
Takeda Analytical Laboratories, Ltd. Yields were not optimized.
N-{6-[3-({[3-(Trifluoromethyl)phenyl]carbonyl}amino)-
phenoxy]imidazo[1,2-b]pyridazin-2-yl}tetrahydro-2H-pyran-4-
carboxamide (1a). To a solution of N-{3-[(2-aminoimidazo[1,2-b]-
pyridazin-6-yl)oxy]phenyl}-3-(trifluoromethyl)benzamide 10 (150 mg,
0.36 mmol) in N,N-dimethylformamide (5 mL) were successively added
tetrahydro-2H-pyran-4-carboxylic acid (70.8 mg, 0.54 mmol), ED-
CI·HCl (104 mg, 0.54 mmol), HOBt (73.6 mg, 0.54 mmol), and tri-
ethylamine (76 μL, 0.54 mmol), and the reaction mixture was stirred at
room temperature for 14 h. The mixture was partitioned between ethyl
acetate (10 mL) and saturated aqueous NaHCO3 (5 mL). The organic
layer was dried over anhydrous Na2SO4, filtered, and evaporated. The
oily residue was purified with silica gel column chromatography (50−
100% ethyl acetate in n-hexane). Desired fractions were evaporated in
vacuo and the oily residue was crystallized with ethyl acetate/n-hexane
(1:4) to give 1a (155 mg, 81%) as a pale yellow amorphous solid.
1H NMR (DMSO-d6, 300 MHz): δ 1.52−1.81 (m, 4H), 2.56−2.83
(m, 1H), 3.25−3.40 (m, 2H), 3.80−3.97 (m, 2H), 7.03 (dd, J = 2.1, 7.7
Hz, 1H), 7.08 (d, J = 9.4 Hz, 1H), 7.46 (t, J = 8.1 Hz, 1H), 7.58−7.71
(m, 1H), 7.74 (t, J = 2.1 Hz, 1H), 7.75−7.86 (m, 1H), 7.98 (d, J =
8.1 Hz, 1H), 8.01−8.12 (m, 2H), 8.17−8.38 (m, 2H), 10.60 (s, 1H),
10.81 (s, 1H). HRMS (ESI) calcd for C26H22F3N5O4 [M + H]+ 526.1697.
Found: 526.1659.
N-(3-{[2-(Propionylamino)imidazo[1,2-b]pyridazin-6-yl]oxy}-
phenyl)-3-(trifluoromethyl)benzamide (1c). Compound 1c (116 mg)
was prepared in a similar manner to that described for 1a from 10
(150 mg, 0.363 mmol), using propionic acid (40 μL, 0.544 mmol),
EDCI·HCl (104 mg, 0.544 mmol), HOBt (73 mg, 0.544 mmol),
triethylamine (76 μL, 0.544 mmol), and N,N-dimethylformamide
(5 mL). Yield 68%, white crystals; mp 190−194 °C. 1H NMR
(DMSO-d6, 300 MHz): δ 1.07 (t, J = 7.5 Hz, 3H), 2.37 (q, J = 7.5 Hz,
2H), 7.01−7.11 (m, 2H), 7.46 (t, J = 8.2 Hz, 1H), 7.65−7.83 (m, 3H),
7.94−8.00 (m, 1H), 8.02 (s, 1H), 8.05 (d, J = 9.6 Hz, 1H), 8.22−8.30
(m, 2H), 10.59 (s, 1H), 10.75 (s, 1H). HRMS (ESI): calcd for
C23H18F3N5O3 [M + H]+ 470.1435. Found: 470.1457.
N-(3-{[2-(Isobutyrylamino)imidazo[1,2-b]pyridazin-6-yl]oxy}-
phenyl)-3-(trifluoromethyl)benzamide (1d). Compound 1d
(74 mg) was prepared in a similar manner to 1a from 10 (150 mg,
0.363 mmol), using 2-methylpropanoic acid (50 μL, 0.544 mmol),
EDCI·HCl (104 mg, 0.544 mmol), HOBt (73 mg, 0.544 mmol),
triethylamine (76 μL, 0.544 mmol), and N,N-dimethylformamide
(5 mL). Yield 42%, pale green crystals; mp 193 °C. 1H NMR (DMSO-
d6, 300 MHz): δ 1.08 (d, J = 7.0 Hz, 6H), 2.65−2.77 (m, 1H), 7.00−
7.11 (m, 2H), 7.46 (t, J = 8.1 Hz, 1H), 7.63−7.83 (m, 3H), 7.94−8.09
(m, 3H), 8.22−8.30 (m, 2H), 10.59 (s, 1H), 10.75 (s, 1H). MS (ESI)
m/z 484.15 (M + H)+. Anal. Calcd for C24H20F3N5O3: C, 59.63; H,
4.17; N, 14.49. Found: C, 59.53; H, 4.25; N, 14.55.
compound 1b (99 mg, 60%) as pale yellow crystals, mp 224−226 °C.
1H NMR (DMSO-d6, 300 MHz): δ 2.07 (s, 3H), 7.01−7.11 (m, 2H),
7.46 (t, J = 8.1 Hz, 1H), 7.63−7.83 (m, 3H), 7.95−8.01 (m, 2H), 8.06
(d, J = 9.6 Hz, 1H), 8.22−8.30 (m, 2H), 10.59 (s, 1H), 10.80 (s, 1H).
HRMS (ESI): calcd for C22H16F3N5O3 [M + H]+ 456.1278. Found:
456.1283.
N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]-
pyridazin-6-yl}oxy)phenyl]-3-(trifluoromethyl)benzamide (1e).
Compound 1e (132 mg) was prepared in a similar manner to 1b from
10, using cyclopropanecarbonyl chloride (45.5 mg, 0.44 mmol) and
pyridine (3 mL). Yield 75%, colorless crystals; mp 203−204 °C.
1H NMR (300 MHz, DMSO-d6): δ 0.61−1.04 (m, 4H), 1.67−2.04
(m, 1H), 7.03 (dd, J = 2.1, 7.6 Hz, 1H), 7.08 (d, J = 9.6 Hz, 1H), 7.46
(t, J = 8.1 Hz, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.73 (t, J = 2.1 Hz, 1H),
7.75−7.86 (m, 1H), 7.87−8.02 (m, 2H), 8.06 (d, J = 9.6 Hz, 1H),
8.19−8.41 (m, 2H), 10.59 (s, 1H), 11.09 (s, 1H). MS (ESI): m/z
482.2 (M + H)+. Anal. Calcd for C24H18F3N5O3: C, 59.88; H, 3.77; N,
14.55. Found: C, 59.91; H, 3.88; N, 14.37.
N-[3-({2-[(Cyclobutylcarbonyl)amino]imidazo[1,2-b]-
pyridazin-6-yl}oxy)phenyl]-3-(trifluoromethyl)benzamide (1f).
Compound 1f (98 mg) was prepared in a similar manner to 1b from
10 (150 mg, 0.362 mmol), using cyclobutanecarbonyl chloride (50 μL,
0.435 mmol), triethylamine (75 μL, 0.544 mmol), and tetrahydrofuran
(5 mL). Yield 54%, white amorphous solid. 1H NMR (CDCl3,
300 MHz): δ 1.83−2.11 (m, 2H), 2.15−2.46 (m, 4H), 3.14−3.28
(m, 1H), 6.88 (d, J = 9.4 Hz, 1H), 7.00−7.06 (m, 1H), 7.42−7.48
(m, 2H), 7.60−7.68 (m, 1H), 7.69−7.85 (m, 4H), 7.91−7.98 (m, 1H),
8.04−8.09 (m, 1H), 8.13 (s, 1H), 8.19 (s, 1H). HRMS (ESI): calcd for
C25H20F3N5O3 [M + H]+ 496.1591. Found: 496.1588.
N-[3-({2-[(Cyclopentylcarbonyl)amino]imidazo[1,2-b]-
pyridazin-6-yl}oxy)phenyl]-3-(trifluoromethyl)benzamide (1g).
Compound 1g (105 mg) was prepared in a manner similar to 1b from 10
(150 mg, 0.362 mmol), using cyclopentanecarbonyl chloride (53 μL, 0.435
mmol), triethylamine (75 μL, 0.544 mmol), and tetrahydrofuran (5 mL).
1
Yield 57%, white crystals; mp 215−217 °C. H NMR (DMSO-d6,
300 MHz): δ 1.47−1.90 (m, 8H), 2.81−2.95 (m, 1H), 7.00−7.10
(m, 2H), 7.46 (t, J = 8.2 Hz, 1H), 7.63−7.83 (m, 3H), 7.94−8.09
(m, 3H), 8.21−8.30 (m, 2H), 10.59 (s, 1H), 10.77 (s, 1H). HRMS
(ESI): calcd for C26H22F3N5O3 [M + H]+ 510.1748. Found: 510.1711.
N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]-
pyridazin-6-yl}oxy)phenyl]-4-(trifluoromethyl)benzamide (1h).
Compound 1h (110 mg) was prepared in a similar manner to 1a
from N-[6-(3-aminophenoxy)imidazo[1,2-b]pyridazin-2-yl]cyclo-
propanecarboxamide 14 (100 mg, 0.32 mmol), using, 4-(trifluoromethyl)-
benzoic acid 15h (63 mg, 0.33 mmol), EDCI·HCl (65 mg, 0.34 mmol),
HOBt (46 mg, 0.34 mmol), and N,N-dimethylformamide (5 mL).
1
Yield 68%, white crystals; mp 240−241 °C. H NMR (DMSO-d6,
300 MHz): δ 0.62−0.94 (m, 4H), 1.76−2.06 (m, 1H), 6.99−7.05
(m, 1H), 7.08 (d, J = 9.6 Hz, 1H), 7.45 (t, J = 8.1 Hz, 1H), 7.63−7.71
(m, 1H), 7.74 (t, J = 2.1 Hz, 1H), 7.92 (d, J = 8.2 Hz, 2H), 7.98
(s, 1H), 8.06 (d, J = 9.6 Hz, 1H), 8.13 (d, J = 8.2 Hz, 2H), 10.60
(s, 1H), 11.09 (s, 1H). MS (ESI): m/z 482.02 (M + H)+. Anal. Calcd
for C24H18F3N5O3: C, 59.88; H, 3.77; N, 14.55. Found: C, 59.92; H,
3.88; N, 14.43.
N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]-
pyridazin-6-yl}oxy)phenyl]-2-(trifluoromethyl)benzamide (1i).
To a solution of 2-(trifluoromethyl)benzoic acid 15i (110 mg,
0.581 mmol) in tetrahydrofuran (5 mL) was added oxalyl chloride
(84 μL, 0.969 mmol) followed by N,N-dimethylformamide (5 μL) at
4 °C. The reaction mixture was stirred at room temperature for 1.5 h
and evaporated in vacuo. The residue was diluted with NMP (5 mL),
and to the mixture was added 14 (150 mg, 0.484 mmol). The reaction
mixture was stirred at room temperature for 2 h. The mixture was
diluted with ethyl acetate (20 mL), washed with saturated NaHCO3
(20 mL) and brine (20 mL) successively, dried over anhydrous
MgSO4, filtered, and evaporated in vacuo. The residue was purified by
silica gel column chromatography (0−100% ethyl acetate in n-hexane)
to give compound 1i (176 mg, 75%) as a white amorphous solid.
1H NMR (DMSO-d6, 300 MHz): δ 0.77−0.84 (m, 4H), 1.86−
1.98 (m, 1H), 6.98−7.04 (m, 1H), 7.07 (d, J = 9.6 Hz, 1H), 7.43
N-{3-[(2-Acetamidoimidazo[1,2-b]pyridazin-6-yl)oxy]-
phenyl}-3-(trifluoromethyl)benzamide (1b). To a solution of 10
(150 mg, 0.362 mmol) in pyridine (3 mL) was added acetyl chloride
(31 μL, 0.435 mmol), and the reaction mixture was stirred at room
temperature for 5 h. The reaction mixture was concentrated in vacuo.
The resulting slurry was triturated with water (10 mL). The resulting
precipitate was collected and recrystallized from ethanol to give
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(t, J = 8.1 Hz, 1H), 7.51−7.57 (m, 1H), 7.64 (t, J = 2.1 Hz, 1H), 7.67−
7.76 (m, 2H), 7.76−7.88 (m, 2H), 7.98 (s, 1H), 8.05 (d, J = 9.6 Hz,
1H), 10.71 (s, 1H), 11.07 (s, 1H). HRMS (ESI): calcd for C24H18
F3N5O3 [M + H]+ 482.1435. Found: 482.1432.
Yield 77%, white crystals; mp 225−226 °C. H NMR (DMSO-d6,
300 MHz): δ 0.73−0.86 (m, 4H), 1.74 (s, 6H), 1.83−1.98 (m, 1H),
6.99−7.04 (m, 1H), 7.08 (d, J = 9.6 Hz, 1H), 7.45 (t, J = 8.1 Hz,
1H), 7.54−7.69 (m, 2H), 7.69−7.80 (m, 2H), 7.92 (dt, J = 7.8,
1.2 Hz, 1H), 7.98 (s, 1H), 8.00−8.10 (m, 2H), 10.45 (s, 1H), 11.09
(s, 1H). HRMS (ESI): calcd for C27H24N6O3 [M + H]+ 481.1983.
Found: 481.1969.
N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]-
pyridazin-6-yl}oxy)phenyl]-3-methoxybenzamide (1j).
Compound 1j (49 mg) was prepared in a similar manner to 1i from
14 (80 mg, 0.26 mmol) using 3-methoxybenzoic acid 15j (48 mg,
0.32 mmol), oxalyl chloride (32 μL, 0.37 mmol), N,N-dimethylforma-
mide (5 μL), tetrahydrofuran (2 mL), and NMP (2 mL). Yield 43%,
3-(1-Cyano-1-methylethyl)-N-[3-({2-[(cyclopropylcarbonyl)-
amino]imidazo[1,2-a]pyridin-6-yl}oxy)phenyl]benzamide (2).
To a solution of 3-(1-cyano-1-methylethyl)-N-[3-({2-[(trifluoroacetyl)-
amino]imidazo[1,2-a]pyridin-6-yl}oxy)phenyl]benzamide 25 (400 mg,
0.788 mmol) in ethanol (4.0 mL) was added 1 N NaOH (8.0 mL),
and the reaction mixture was stirred at 45 °C for 12 h. To the reaction
mixture was added water (100 mL), and the mixture was extracted
with ethyl acetate (200 mL). The organic layer was washed with brine
(100 mL) and dried over anhydrous Na2SO4. The insoluble material
was filtered off, and the filtrate was concentrated under reduced
pressure. The obtained residue was purified by basic silica gel column
chromatography (0−20% methanol in ethyl acetate), and the desired
fractions were combined and concentrated under reduced pressure to
give N-{3-[(2-aminoimidazo[1,2-a]pyridin-6-yl)oxy]phenyl}-3-
(1-cyano-1-methylethyl)benzamide (0.35 g, quantitative yield) as
1
white crystals; mp 186−187 °C. H NMR (DMSO-d6, 300 MHz):
δ 0.77−0.85 (m, 4H), 1.85−1.99 (m, 1H), 3.83 (s, 3H), 6.95−7.03
(m, 1H), 7.07 (d, J = 9.6 Hz, 1H), 7.12−7.21 (m, 1H), 7.37−7.49
(m, 3H), 7.48−7.56 (m, 1H), 7.61−7.70 (m, 1H), 7.73 (t, J = 2.1 Hz,
1H), 7.98 (s, 1H), 8.05 (d, J = 9.6 Hz, 1H), 10.34 (s, 1H), 11.09
(s, 1H). MS (ESI): m/z 444.02 (M + H)+. Anal. Calcd for
C24H21N5O4: C, 65.00; H, 4.77; N, 15.79. Found: C, 64.73; H, 4.87;
N, 15.69.
3-tert-Butoxy-N-[3-({2-[(cyclopropylcarbonyl)amino]-
imidazo[1,2-b]pyridazin-6-yl}oxy)phenyl]benzamide (1k).
Compound 1k (160 mg) was prepared in a similar manner to 1i from
14 (150 mg, 0.49 mmol), using 3-tert-butoxybenzoic acid 15k (110 mg,
0.58 mmol), oxalyl chloride (63 μL, 0.73 mmol), N,N-dimethylforma-
mide (5 μL), tetrahydrofuran (4 mL), and NMP (4 mL). Yield 57%,
1
colorless amorphous solid. H NMR (DMSO-d6, 300 MHz): δ 1.73
(s, 6H), 5.08 (s, 2H), 6.78 (dd, J = 2.1, 8.1 Hz, 1H), 6.87 (dd, J = 2.1,
9.6 Hz, 1H), 7.01 (s, 1H), 7.22 (d, J = 9.6 Hz, 1H), 7.34 (t, J = 8.2 Hz,
1H), 7.44 (s, 1H), 7.50−7.62 (m, 2H), 7.74 (d, J = 8.1 Hz, 1H), 7.88
(d, J = 7.5 Hz, 1H), 7.98 (s, 1H), 8.34 (d, J = 2.1 Hz, 1H), 10.34
(s, 1H). MS (ESI): m/z 412.05 (M + H)+.
1
white crystals; mp 206−207 °C. H NMR (DMSO-d6, 300 MHz): δ
0.74−0.87 (m, 4H), 1.33 (s, 9H), 1.85−1.98 (m, 1H), 6.90−7.03
(m, 1H), 7.07 (d, J = 9.6 Hz, 1H), 7.16−7.27 (m, 1H), 7.36−7.48 (m,
2H), 7.48−7.53 (m, 1H), 7.61−7.70 (m, 2H), 7.73 (t, J = 2.1 Hz, 1H),
7.98 (s, 1H), 8.05 (d, J = 9.6 Hz, 1H), 10.34 (s, 1H), 11.09 (s, 1H). MS
(ESI): 486.25 (M + H)+. Anal. Calcd for C27H27N5O4: C, 66.79; H, 5.61;
N, 14.42. Found: C, 66.68; H, 5.56; N,14.41.
To a solution of N-{3-[(2-aminoimidazo[1,2-a]pyridin-6-yl)oxy]-
phenyl}-3-(1-cyano-1-methylethyl)benzamide (200 mg, 0.486 mmol)
in N,N-dimethylacetamide (2.0 mL) was added cyclopropanecarbonyl
chloride (46 μL, 0.510 mmol), and the reaction mixture was stirred at
room temperature for 8 h. The reaction mixture was diluted with ethyl
acetate (100 mL), washed with 5% aqueous NaHCO3 (50 mL) and
brine (50 mL), and dried over anhydrous Na2SO4. The insoluble
material was filtered off, and the filtrate was concentrated under reduced
pressure. The residue was purified by basic silica gel column chroma-
tography (50−100% ethyl acetate in n-hexane). The desired fractions
were combined and concentrated under reduced pressure, and the
residue was triturated with ethyl acetate and i-Pr2O to give 2 (100 mg,
43%) as colorless crystals, mp 138−140 °C. 1H NMR (DMSO-d6,
300 MHz): δ 0.73−0.85 (m, 4H), 1.73 (s, 6H), 1.86−2.03 (m, 1H), 6.81
(dd, J = 2.4, 8.1 Hz, 1H), 7.10 (dd, J = 2.4, 9.6 Hz, 1H), 7.36 (t, J =
8.1 Hz, 1H), 7.44−7.52 (m, 2H), 7.57 (t, J = 7.8 Hz, 2H), 7.69−7.78
(m, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.98 (t, J = 1.5 Hz, 1H), 8.07 (s, 1H),
8.59 (d, J = 2.4 Hz, 1H), 10.35 (s, 1H), 10.98 (s, 1H). MS (ESI): m/z
480.2 (M + H)+. Anal. Calcd for C28H25N5O3: C, 70.13; H, 5.25; N,
14.60. Found: C, 70.26; H, 5.46; N, 14.56.
3-(1-Cyano-1-methylethyl)-N-[3-({2-[(cyclopropylcarbonyl)-
amino]-1H-benzimidazol-6-yl}oxy)phenyl]benzamide (3a). A
mixture of tert-butyl [3-({2-[(cyclopropylcarbonyl)amino]-1H-benzi-
midazol-6-yl}oxy)phenyl]carbamate 29a (1.58 g, 3.88 mmol) and tri-
fluoroacetic acid (50 mL) was refluxed at 80 °C for 1 h. The reaction
mixture was cooled at room temperature and evaporated in vacuo. The
residue was diluted with ethyl acetate (200 mL), washed with 0.1 N
HCl (100 mL) and saturated NaHCO3 (100 mL) successively, and
dried over anhydrous MgSO4. The solvent was evaporated in vacuo.
The residue was crystallized from methanol to give N-[6-(3-
aminophenoxy)-1H-benzimidazol-2-yl]cyclopropanecarboxamide
(1.06 g, 88%) as pale brown crystals, mp 213-214 °C. 1H NMR (DMSO-
d6, 300 MHz): δ 0.90−0.92 (m, 4H), 1.91−2.01 (m, 1H), 5.13 (br s,
2H), 6.07−6.10 (m, 2H), 6.22−6.26 (m, 1H), 6.79 (dd, J = 2.4, 8.7 Hz,
1H), 6.91−7.12 (m, 2H), 7.40 (br d, J = 8.4 Hz, 1H), 11.81 (br s, 1H),
11.99 (br s, 1H). HRMS (ESI): calcd for C17H16N4O2 [M + H]+
309.1346. Found: 309.1327.
Methyl 3-({[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]-
pyridazin-6-yl}oxy)phenyl]amino}carbonyl)benzoate (1l). Com-
pound 1l (270 mg) was prepared by a similar manner to 1a from 14
(250 mg, 0.81 mmol), using 3-(methoxycarbonyl)benzoic acid 15l
(150 mg, 0.83 mmol), EDCI·HCl (160 mg, 0.84 mmol), HOBt
(110 mg, 0.81 mmol), and N,N-dimethylformamide (8 mL). Yield
72%, white crystals; mp 216−217 °C. 1H NMR (DMSO-d6,
300 MHz): δ 0.74−0.85 (m, 4H), 1.84−1.98 (m, 1H), 3.91 (s, 3H),
6.97−7.05 (m, 1H), 7.08 (d, J = 9.6 Hz, 1H), 7.45 (t, J = 8.1 Hz, 1H),
7.63−7.79 (m, 3H), 7.98 (s, 1H), 8.06 (d, J = 9.6 Hz, 1H), 8.12−8.19
(m, 1H), 8.19−8.26 (m, 1H), 8.52 (t, J = 1.5 Hz, 1H), 10.59 (s, 1H),
11.09 (s, 1H). Anal. Calcd for C25H21N5O5: C, 63.69; H, 4.49; N,
14.85. Found: C, 63.48; H, 4.51; N, 14.76.
N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]-
pyridazin-6-yl}oxy)phenyl]-3-(1-hydroxy-1-methylethyl)-
benzamide (1m). To a solution of methyl 3-({[3-({2-
[(cyclopropylcarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)phenyl]-
amino}carbonyl)benzoate 1l (100 mg, 0.212 mmol) in tetrahydrofuran
(5 mL) was added dropwise a solution of 1.4 M methyl-
magnesium bromide in tetrahydrofuran/toluene (0.76 mL, 1.1 mmol)
under ice-cooling. After addition, the reaction mixture was stirred at room
temperature for 12 h. To the mixture was added water (10 mL), and the
mixture was treated with 1 N HCl (5 mL). The mixture was extracted
with ethyl acetate (30 mL), and the organic layer was washed with brine
(30 mL), dried over anhydrous Na2SO4, and filtered. The solvent was
evaporated under reduced pressure, and the residue was purified by silica
gel column chromatography (40−100% ethyl acetate in n-hexane) to give
1
compound 1m (19 mg, 19%) as a white amorphous solid. H NMR
(DMSO-d6, 300 MHz): δ 0.75−0.85 (m, 4H), 1.46 (s, 6H), 1.85−1.97
(m, 1H), 5.14 (s, 1H), 6.96−7.03 (m, 1H), 7.07 (d, J = 9.6 Hz, 1H),
7.37−7.50 (m, 2H), 7.67 (m, 2H), 7.73 (t, J = 2.1 Hz, 1H), 7.75−7.81
(m, 1H), 7.98 (s, 1H), 8.01 (t, J = 1.5 Hz, 1H), 8.05 (d, J = 9.6 Hz, 1H),
10.35 (s, 1H), 11.09 (s, 1H). HRMS (ESI): calcd for C26H25N5O4
[M + H]+ 472.1979. Found: 472.1941.
3-(1-Cyano-1-methylethyl)-N-[3-({2-[(cyclopropylcarbonyl)-
amino]imidazo[1,2-b]pyridazin-6-yl}oxy)phenyl]benzamide
(1n). Compound 1n (120 mg) was prepared by a similar manner to 1a
from 14 (100 mg, 0.32 mmol), using 3-(1-cyano-1-methylethyl)ben-
zoic acid 15n (62 mg, 0.33 mmol), EDCI·HCl (65 mg, 0.34 mmol),
HOBt (46 mg, 0.34 mmol), and N,N-dimethylformamide (5 mL).
Compound 3a (168 mg) was prepared in a similar manner to 1a
from N-[6-(3-aminophenoxy)-1H-benzimidazol-2-yl]cyclopropane-
carboxamide (156 mg, 0.506 mmol), using 3-(1-cyano-1-methylethyl)-
benzoic acid 15n (195 mg, 1.03 mmol), pyridine (5 mL), EDCI·HCl
(407 mg, 2.12 mmol), DMAP (58.2 mg, 0.476 mmol). Yield 69%,
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colorless crystals; mp 139−141 °C. H NMR (DMSO-d6, 300 MHz):
3-(1-Cyano-1-methylethyl)-N-[3-({2-[(cyclopropylcarbonyl)-
amino]-3-methyl-3H-imidazo[4,5-b]pyridin-5-yl}oxy)phenyl]-
benzamide (4b). To a solution of 34b (170 mg, 0.40 mmol) in
pyridine (2 mL) were added cyclopropanecarbonyl chloride (105 mg,
1.0 mmol) and DMAP (120 mg, 1.0 mmol) successively. The reaction
mixture was stirred at room temperature for 18 h. The mixture was
partitioned between ethyl acetate (50 mL) and water (50 mL). The
organic layer was dried over anhydrous MgSO4, filtered, and evap-
orated in vacuo. The residue was purified by silica gel column chroma-
tography (0−100% ethyl acetate in n-hexane) to give compound 4b
(110 mg, 56%) as colorless crystals, mp 132−134 °C. 1H NMR
(CDCl3, 300 MHz): δ 0.85−0.90 (m, 2H), 1.01−1.14 (m, 2H), 1.76
(s, 6H), 1.79−1.91 (m, 1H), 3.60 (s, 3H), 6.73 (d, J = 8.3 Hz, 1H),
6.96 (dt, J = 7.5, 1.9 Hz, 1H), 7.34−7.48 (m, 2H), 7.47−7.64 (m, 3H),
7.69 (ddd, J = 1.0, 2.0, 7.9 Hz, 1H), 7.77 (dt, J = 7.7, 1.2 Hz, 1H), 7.97
(t, J = 1.8 Hz, 2H), 12.13 (br s, 1H). HRMS (ESI): calcd for
C28H26N6O3 [M + H]+ 495.2139. Found: 495.2135.
δ 0.91−0.93 (m, 4H), 1.73 (s, 6H), 1.94−2.00 (m, 1H), 6.74 (dd, J =
1.8, 7.8 Hz, 1H), 8.85 (dd, J = 2.4, 8.4 Hz, 1H), 7.05−7.18 (m, 1H),
7.32 (t, J = 8.1 Hz, 1H), 7.41−7.59 (m, 4H), 7.72−7.75 (m, 1H), 7.88
(d, J = 7.8 Hz, 1H), 7.97−7.98 (m, 1H), 10.31 (br s, 1H), 11.84 (br s,
1H), 12.03 (br s, 1H). HRMS (ESI): calcd for C28H25N5O3 [M + H]+
480.2030. Found: 480.1995.
3-(1-Cyano-1-methylethyl)-N-[3-({2-[(cyclopropylcarbonyl)-
amino]-1-methyl-1H-benzimidazol-6-yl}oxy)phenyl]-
benzamide (3b). N-[6-(3-Aminophenoxy)-1-methyl-1H-benzimidazol-
2-yl]cyclopropanecarboxamide (1.02 g) was prepared in a similar
manner to intermediate of 3a, N-[6-(3-aminophenoxy)-1H-benzimi-
dazol-2-yl]cyclopropanecarboxamide from using tert-butyl [3-({2-
[(cyclopropylcarbonyl)amino]-1-methyl-1H-benzimidazol-6-yl}oxy)-
phenyl]carbamate 29b (1.52 g, 3.60 mmol), using trifluoroacetic acid
(50 mL). Yield 88%, pink amorphous solid. 1H NMR (CDCl3,
300 MHz): δ 0.93−0.98 (m, 2H), 1.11−1.13 (m, 2H), 1.94−1.99
(m, 1H), 3.68 (s, 3H), 6.34 (t, J = 2.1 Hz, 1H), 6.38−6.41 (m, 1H),
6.46−6.49 (m, 1H), 6.96 (d, J = 2.1 Hz, 1H), 7.03 (dd, J = 2.1, 8.4 Hz,
1H), 7.14 (t, J = 8.1 Hz, 1H), 7.28−7.37 (m, 1H). HRMS (ESI): calcd
for C18H18N4O2 [M + H]+ 323.1503. Found: 323.1487.
Compound 3b (97.3 mg) was prepared in a similar manner to 1a
from N-[6-(3-aminophenoxy)-1-methyl-1H-benzimidazol-2-yl]cyclo-
propanecarboxamide (166 mg, 0.514 mmol), using 3-(1-cyano-
1-methylethyl)benzoic acid 15n (316 mg, 1.67 mmol), pyridine
(5 mL), EDCI·HCl (456 mg, 2.38 mmol), and DMAP (38.9 mg, 0.318
mmol). Yield 38%, colorless crystals; mp 129−130 °C. 1H NMR
(DMSO-d6, 300 MHz): δ 0.82−0.92 (m, 4H), 1.73 (s, 6H), 1.90−2.05
(m, 1H), 3.54 (s, 3H), 6.76 (dd, J = 1.8, 7.8 Hz, 1H), 6.94 (d, J =
8.1 Hz, 1H), 7.31−7.37 (m, 2H), 7.41−7.60 (m, 4H), 7.72−7.75 (m,
1H), 7.90 (d, J = 7.8 Hz, 1H), 7.98−8.00 (m, 1H), 10.35 (br s, 1H),
10.89 (br s, 1H). HRMS (ESI): calcd for C29H27N5O3 [M + H]+
494.2187. Found: 494.2171.
3-(1-Cyano-1-methylethyl)-N-[3-({2-[(cyclopropylcarbonyl)-
amino]-3H-imidazo[4,5-b]pyridin-5-yl}oxy)phenyl]benzamide
(4a). A mixture of N-{3-[(2-amino-3-tert-butyl-3H-imidazo[4,5-b]-
pyridin-5-yl)oxy]phenyl}-3-(1-cyano-1-methylethyl)benzamide 34a
(0.23 g, 0.5 mmol), cyclopropanecarbonyl chloride (0.21 g, 2.0 mmol),
and DMAP (0.24 g, 2.0 mmol) in pyridine (2.0 mL) was stirred at room
temperature for 18 h. The reaction mixture was partitioned between
ethyl acetate (50 mL) and water (50 mL). The organic layer was dried
over anhydrous MgSO4 and filtered. The filtrate was concentrated in
vacuo, and the residue was purified by silica gel column chromato-
graphy (0−100% ethyl acetate in n-hexane) to give N-[3-({3-tert-butyl-
2-[(cyclopropylcarbonyl)amino]-3H-imidazo[4,5-b]pyridin-5-yl}oxy)-
phenyl]-3-(1-cyano-1-methylethyl)benzamide (0.15 g, 0.28 mmol, 56%)
as a colorless amorphous solid. The overreacted bis-(cyclopropylcarbonyl)
derivative was treated by 1 N NaOH and methanol (5:1) to give
additional N-[3-({3-tert-butyl-2-[(cyclopropylcarbonyl)amino]-3H-
imidazo[4,5-b]pyridin-5-yl}oxy)phenyl]-3-(1-cyano-1-methylethyl)-
3-(1-Cyano-1-methylethyl)-N-[3-({2-[(cyclopropylcarbonyl)-
amino]-1,3-benzothiazol-6-yl}oxy)phenyl]benzamide (5).
Potassium thiocyanate (8.09 g, 83.2 mmol) was added to acetic
acid (150 mL). To the solution was added a solution of N-[3-
(4-aminophenoxy)phenyl]-3-(1-cyano-1-methylethyl)benzamide 38
(7.34 g, 19.8 mmol) in acetic acid (150 mL). The mixture was stirred
at room temperature for 15 min. To the mixture was added a solution
of bromine (4.0 g, 25.0 mmol) in acetic acid (100 mL) at room tem-
perature over 30 min, and the reaction mixture was stirred at room
temperature for 4 h. The resulting yellow precipitate was removed by
filtration through a pad of Celite. The filtrate was concentrated under
reduced pressure. The residue was diluted with tetrahydrofuran
(100 mL) and ethyl acetate (200 mL), and the mixture was basified
with 2 N NaOH (200 mL). The organic layer was washed with
saturated NH4Cl (200 mL) and dried over anhydrous MgSO4.
The solvent was evaporated in vacuo to give N-{3-[(2-amino-1,
3-benzothiazol-6-yl)oxy]phenyl}-3-(1-cyano-1-methylethyl)-
1
benzamide (7.4 g, 87%) as a pale yellow amorphous solid. H NMR
(300 MHz, DMSO-d6): δ 1.73 (s, 6H), 6.73−6.76 (m, 1H), 6.96
(dd, J = 2.7, 8.7 Hz, 1H), 7.31−7.36 (m, 2H), 7.42−7.60 (m, 6H),
7.72−7.75 (m, 1H), 7.89 (d, J = 8.1 Hz, 1H), 7.98 (t, J = 1.8 Hz,
1H), 10.33 (br s, 1H). MS (ESI): m/z 429.05 (M + H)+.
To a solution of N-{3-[(2-amino-1,3-benzothiazol-6-yl)oxy]-
phenyl}-3-(1-cyano-1-methylethyl)benzamide (5.13 g, 12.0 mmol) in
pyridine (50 mL) were added cyclopropanecarbonyl chloride (2.5 mL,
27.6 mmol) and DMAP (89.3 mg, 0.731 mmol), successively, at 4 °C.
The reaction mixture was stirred at room temperature for 6 h. To the
mixture were added methanol (50 mL) and 2 N NaOH (10 mL)
successively, and the mixture was stirred at room temperature for 3 h.
The mixture was concentrated under reduced pressure, and the residue
was diluted with ethyl acetate (300 mL). The mixture was washed with
water (150 mL), 0.1 N HCl (100 mL), saturated NaHCO3 (100 mL),
and brine (100 mL), successively. The organic layer was dried over
anhydrous MgSO4. The solvent was evaporated in vacuo. The residue
was purified by silica gel column chromatography (5−50% ethyl
acetate in n-hexane). Desired fractions were combined and evaporated
in vacuo and the residue was crystallized from ethyl acetate/i-Pr2O to
give compound 5 (4.40 g, 88%) as colorless crystals, mp 120−121 °C.
1H NMR (DMSO-d6, 300 MHz): δ 0.94−0.97 (m, 4H), 1.73 (s, 6H),
1
benzamide (0.11 g, 40%) as a colorless amorphous solid. H NMR
(CDCl3, 300 MHz): δ 0.77−0.86 (m, 2H), 1.01−1.11 (m, 2H), 1.67−
1.73 (m, 1H), 1.76 (s, 15H), 6.74 (d, J = 8.5 Hz, 1H), 6.94 (dt, J = 6.8,
1.1 Hz, 1H), 7.31−7.55 (m, 4H), 7.59 (t, J = 2.1 Hz, 1H), 7.69 (d, J =
7.2 Hz, 1H), 7.78 (d, J = 7.9 Hz, 1H), 7.98 (t, J = 1.7 Hz, 1H), 8.11 (br
s, 1H), 12.86 (br s, 1H). MS (ESI): m/z 537 (M + H)+.
A solution of N-[3-({3-tert-butyl-2-[(cyclopropylcarbonyl)amino]-
3H-imidazo[4,5-b]pyridin-5-yl}oxy)phenyl]-3-(1-cyano-1-methylethyl)-
benzamide (0.23 g, 0.48 mmol) in trifluoroacetic acid (2 mL) was
stirred at 80 °C for 2 h. The mixture was cooled at room temperature
and evaporated in vacuo. The residue was partitioned between ethyl
acetate (50 mL) and 0.1 N NaOH (50 mL). The organic layer was dried
over anhydrous MgSO4 and filtered. The filtrate was concentrated in
vacuo. The residue was crystallized with acetone to give compound 4a
(188 mg, 82%) as colorless crystals, mp 180−181 °C. 1H NMR (CDCl3,
300 MHz): δ 0.41−1.19 (m, 4H), 1.74 (s, 6H), 2.53−2.60 (m, 1H),
6.77 (d, J = 8.3 Hz, 2H), 7.22−7.35 (m, 1H), 7.42−7.54 (m, 2H), 7.56−
7.79 (m, 3H), 7.91 (d, J = 7.9 Hz, 1H), 8.03 (t, J = 1.8 Hz, 1H), 9.60
(br s, 1H), 11.65 (br s, 1H), 13.23 (br s, 1H). HRMS (ESI): calcd for
C27H24N6O3 [M + H]+ 481.1983. Found: 481.1966.
1.93−2.01 (m, 1H), 6.80 (dd, J = 1.8, 8.1 Hz, 1H), 7.17 (dd, J = 2.4,
8.7 Hz, 1H), 7.37 (t, J = 8.1 Hz, 1H), 7.48 (t, J = 2.1 Hz, 1H), 7.55−
7.61 (m, 2H), 7.72−7.78 (m, 3H), 7.89 (d, J = 8.1 Hz, 1H), 7.98 (t, J =
1.8 Hz, 1H), 10.35 (br s, 1H), 12.63 (br s, 1H). HRMS (ESI): calcd
for C28H24N4O3S [M + H]+ 497.1642. Found: 497.1647.
3-(1-Cyano-1-methylethyl)-N-[3-({2-[(cyclopropylcarbonyl)-
amino][1,3]thiazolo[5,4-b]pyridin-5-yl}oxy)phenyl]benzamide
(6a). Compound 6a (111 mg) was prepared in a similar manner to 1i
from N-[5-(3-aminophenoxy)[1,3]thiazolo[5,4-b]pyridin-2-yl]cyclo-
propanecarboxamide 42 (126 mg, 0.387 mmol), using 3-(1-cyano-1-
methylethyl)benzoic acid 15n (147 mg, 0.778 mmol), thionyl chloride
(100 μL, 1.37 mmol), toluene (5 mL), DMAP (10 mg, 0.0819 mmol),
1
and pyridine (3 mL). Yield 58%, colorless crystals; mp 234 °C. H
NMR (DMSO-d6, 300 MHz): δ 0.94−0.97 (m, 4H), 1.74 (s, 6H),
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1.95−2.04 (m, 1H), 6.93 (ddd, J = 0.9, 2.4, 8.1 Hz, 1H), 7.15 (d, J =
8.1 Hz, 1H), 7.43 (t, J = 8.4 Hz, 1H), 7.57−7.65 (m, 3H), 7.75 (ddd,
J = 0.9, 2.1, 7.8 Hz, 1H), 7.92 (dt, J = 7.2, 1.5 Hz, 1H), 8.02 (t, J =
1.8 Hz, 1H), 8.18 (d, J = 8.7 Hz, 1H), 10.41 (br s, 1H), 12.68 (br s,
1H). MS (ESI): m/z 498.15 (M + H)+. HRMS (ESI): calcd for
C27H23N5O3S [M + H]+ 498.1594. Found: 498.1580.
column chromatography (eluent, ethyl acetate) and triturated with
i-Pr2O to give N-[5-(3-amino-4-chlorophenoxy)[1,3]thiazolo[5,4-b]-
pyridin-2-yl]cyclopropanecarboxamide (2.00 g, 75%) as white crystals,
mp 237−238 °C. 1H NMR (DMSO-d6, 300 MHz): δ 0.88−1.10
(m, 4H), 1.90−2.10 (m, 1H), 5.49 (br s, 2H), 6.32 (dd, J = 2.7, 8.6 Hz,
1H), 6.54 (d, J = 2.7 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H), 7.21 (d, J =
8.6 Hz, 1H), 8.15 (d, J = 8.6 Hz, 1H), 12.69 (br s, 1H). MS (ESI):
m/z 361.0 (M + H)+.
3-(1-Cyanocyclopropyl)-N-[3-({2-[(cyclopropylcarbonyl)-
amino][1,3]thiazolo[5,4-b]pyridin-5-yl}oxy)phenyl]benzamide
(6b). Compound 6b (82 mg) was prepared in a similar manner to 1i
from N-[5-(3-aminophenoxy)[1,3]thiazolo[5,4-b]pyridin-2-yl]cyclo-
propanecarboxamide 42 (118 mg, 0.362 mmol), using 3-(1-cyano-
1-methylethyl)benzoic acid 15o (151 mg, 0.808 mmol), thionyl
chloride (200 μL, 2.74 mmol), toluene (5 mL), DMAP (10 mg, 0.0819
mmol), and pyridine (2 mL). Yield 46%, colorless crystals; mp 208−
215 °C. 1H NMR (DMSO-d6, 300 MHz): δ 0.94−0.97 (m, 4H), 1.61
(dd, J = 5.4, 8.4 Hz, 2H), 1.81 (dd, J = 5.4, 8.4 Hz, 2H), 1.97−2.01 (m,
1H), 6.91−6.95 (m, 1H), 7.15 (d, J = 8.7 Hz, 1H), 7.42 (t, J = 8.4 Hz,
1H), 7.52−7.59 (m, 2H), 7.62−7.64 (m, 2H), 7.81 (br s, 1H), 7.86
(dt, J = 6.0, 2.4 Hz, 1H), 8.18 (d, J = 8.7 Hz, 1H), 10.40 (br s, 1H),
12.70 (br s, 1H). MS (ESI): m/z 495.46 (M + H)+. HRMS (ESI):
calcd for C27H21N5O3S [M + H]+ 496.1438. Found: 496.1406.
2-Chloro-3-(1-cyanocyclopropyl)-N-[3-({2-[(cyclopropylcar-
bonyl)amino][1,3]thiazolo[5,4-b]pyridin-5-yl}oxy)phenyl]-
benzamide (6c). Compound 6c (169 mg) was prepared in a similar
manner to 1a from N-[5-(3-aminophenoxy)[1,3]thiazolo[5,4-b]-
pyridin-2-yl]cyclopropanecarboxamide 42 (120 mg, 3.68 μmol),
using 2-chloro-3-(1-cyclopropyl)benzoic acid 15p (90 mg, 405 μmol),
HATU (154 mg, 405 μmol), and pyridine (3 mL). Yield 87%, colorless
Compound 6e (151 mg) was prepared in a similar manner to 1i
from N-[5-(3-amino-4-chlorophenoxy)[1,3]thiazolo[5,4-b]pyridin-2-
yl]cyclopropanecarboxamide (150 mg, 0.416 mmol), using 2-chloro-
3-(1-cyanocyclopropyl)benzoic acid 15q (184 mg, 0.830 mmol),
oxalyl chloride (88 μL, 1.04 mmol), N,N-dimethylformamide (15 μL),
tetrahydrofuran (4.6 mL), and N,N-dimethylacetamide (4.6 mL).
1
Yield 64%, white crystals; mp 138−140 °C. H NMR (DMSO-d6,
300 MHz): δ 0.90−1.00 (m, 4H), 1.40−1.50 (m, 2H), 1.75−1.82 (m,
2H), 1.95−2.05 (m, 1H), 7.15 (dd, J = 3.0, 9.0 Hz, 1H), 7.21 (d,
J = 8.7 Hz, 1H), 7.40−7.55 (m, 1H), 7.55−7.70 (m, 4H), 8.20 (d, J =
8.7 Hz, 1H), 10.38 (br s, 1H), 12.71 (br s, 1H). HRMS (ESI): calcd
for C27H19Cl2N5O3S [M + H]+ 564.0658. Found: 564.0660.
Ethyl 6-(3-Aminophenoxy)imidazo[1,2-b]pyridazine-2-car-
boxylate (8). A mixture of ethyl 6-iodoimidazo[1,2-b]pyridazine-
2-carboxylate 7 (13.6 g, 42.8 mmol), 3-aminophenol (7.02 g, 64.3 mmol),
potassium carbonate (10.2 g, 64.3 mmol), and N,N-dimethylformamide
(100 mL) was stirred at 150 °C for 5 h. After the mixture was cooled at
room temperature, ethyl acetate (300 mL) and water (300 mL) were
added to the reaction mixture, and the resulting insoluble material was
filtered through a pad of Celite. The aqueous layer was extracted with
ethyl acetate (5 × 200 mL). The combined organic layer was washed
with saturated aqueous NaHCO3 (300 mL) and dried over anhydrous
MgSO4. The solvent was evaporated under reduced pressure and
the residue was purified by basic silica gel column chromatography
(0−50% ethyl acetate/n-hexane) to give 8 (6.41 g, 50%) as colorless
1
crystals; mp 226−227 °C. H NMR (DMSO-d6, 300 MHz): δ 0.88−
0.99 (m, 4H), 1.38−1.49 (m, 2H), 1.78−1.84 (m, 2H), 1.93−2.04 (m,
1H), 6.93 (ddd, J = 0.9, 2.3, 8.1 Hz, 1H), 7.15 (d, J = 8.7 Hz, 1H), 7.41
(t, J = 8.1 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.51−7.56 (m, 1H), 7.58−
7.63 (m, 2H), 7.66 (dd, J = 1.7, 7.6 Hz, 1H), 8.17 (d, J = 8.7 Hz, 1H),
10.70 (br s, 1H), 12.70 (br s, 1H). HRMS (ESI): calcd for
C27H20ClN5O3S [M + H]+ 530.1048. Found: 530.1033.
1
crystals, mp 138−139 °C. H NMR (DMSO-d6, 300 MHz): δ 1.30
(t, J = 7.2 Hz, 3H), 4.29 (q, J = 7.2 Hz, 2H), 5.33 (br s, 2H), 6.32−
6.50 (m, 3H), 7.07 (t, J = 8.0 Hz, 1H), 7.18 (d, J = 9.6 Hz, 1H), 8.20
(d, J = 9.6 Hz, 1H), 8.61 (s, 1H). MS (ESI): m/z 299.00 (M + H)+.
Anal. Calcd for C15H14N4O3: C, 60.40; H, 4.73; N, 18.78. Found: C,
60.28; H, 4.65; N, 18.63.
2-Chloro-3-(1-cyanocyclopropyl)-N-[5-({2-[(cyclopropylcarbonyl)
amino][1,3]thiazolo[5,4-b]pyridin-5-yl}oxy)-2-fluorophenyl]-
benzamide (6d). To a mixture of N-{5-[(2-amino[1,3]thiazolo
[5,4-b]pyridin-5-yl)oxy]-2-fluorophenyl}-2-chloro-3-(1-cyanocyclopropyl)-
benzamide 45 (9.0 g, 18.8 mmol) and pyridine (2.3 mL, 28.1 mmol)
in tetrahydrofuran (90 mL) was added dropwise cyclopropanecarbonyl
chloride (1.89 mL, 20.8 mmol) at 4 °C. The reaction mixture was
stirred at room temperature for 3 h. An additional amount of cyclo-
propanecarbonyl chloride (0.063 mL, 0.69 mmol) was added to the
mixture. The reaction mixture was stirred at room temperature for an
additional 12 h. To the mixture were added water (100 mL) and
saturated NaHCO3 (100 mL), and the mixture was stirred at room
temperature for 30 min. The resulting precipitate was collected by
filtration, washed with water (200 mL), and dried under vacuum to
give compound 6d (9.85 g, 96%) as colorless crystals, mp 213 °C.
1H NMR (DMSO-d6, 300 MHz): δ 0.77−1.08 (m, 4H), 1.35−
1.53 (m, 2H), 1.73−1.88 (m, 2H), 1.92−2.07 (m, 1H), 7.07 (dt,
J = 8.7, 3.6 Hz, 1H), 7.17 (d, J = 8.7 Hz, 1H), 7.38 (dd, J = 9.1,
10.2 Hz, 1H), 7.43−7.50 (m, 1H), 7.55−7.69 (m, 2H), 7.78 (dd,
J = 3.0, 6.4 Hz, 1H), 8.18 (d, J = 8.7 Hz, 1H), 10.57 (s, 1H), 12.70
(s, 1H). MS (ESI): m/z 548.0 (M + H)+. Anal. Calcd for
C27H19ClFN5O3S: C, 59.18; H, 3.49; N, 12.78. Found: C, 59.01; H,
3.53; N, 12.65.
2-Chloro-N-[2-chloro-5-({2-[(cyclopropylcarbonyl)amino]-
[1,3]thiazolo[5,4-b]pyridin-5-yl}oxy)phenyl]-3-(1-cyanocyclo-
propyl)benzamide (6e). Under ice-cooling, to a suspension of
sodium borohydride (5.63 g, 149 mmol) in methanol (66 mL) was added
in small portions N-(5-{4-chloro-3-[(trifluoroacetyl)amino]phenoxy}-
[1,3]thiazolo[5,4-b]pyridin-2-yl)cyclopropanecarboxamide 48 (3.4 g,
7.44 mmol), and the reaction mixture was stirred at room tem-
perature for 1 h. The reaction mixture was diluted with ethyl acetate
(150 mL) and partitioned with water (200 mL). The organic layer was
concentrated under reduced pressure. The residue was diluted with
ethyl acetate (200 mL), washed with brine (100 mL), and the organic
layer was dried over anhydrous MgSO4, filtered, and concentrated
under reduced pressure. The residue was purified by basic silica gel
Ethyl 6-(3-{[3-(Trifluoromethyl)benzoyl]amino}phenoxy)-
imidazo[1,2-b]pyridazine-2-carboxylate (9). A mixture of 8
(4.63 g, 15.5 mmol), 3-(trifluoromethyl)benzoic acid (4.42 g,
23.2 mmol), HOBt (3.13 g, 23.2 mmol), EDCI·HCl (4.44 g,
23.2 mmol) in N,N-dimethylformamide (90 mL) was stirred at
room temperature for 3 h. Saturated aqueous ammonium chloride
(90 mL) was added to the reaction mixture, and the mixture was
extracted with ethyl acetate (300 mL). The separated organic layer was
dried over anhydrous MgSO4 and filtered through a pad of basic silica
gel using ethyl acetate as eluting solvent. The filtrate was concentrated
under reduced pressure. The resulting slurry was triturated with ethyl
acetate/i-Pr2O (1:1) and the resulting precipitate was collected by
filtration to give 9 (6.31 g, 86%) as colorless crystals, mp 218−219 °C.
1H NMR (DMSO-d6, 300 MHz): δ 1.29 (t, J = 7.1 Hz, 3H), 4.29 (q,
J = 7.1 Hz, 2H), 7.10 (dd, J = 2.0, 8.2 Hz, 1H), 7.30 (d, J = 9.8 Hz,
1H), 7.49 (t, J = 8.2 Hz, 1H), 7.66−7.73 (m, 1H), 7.76−7.84 (m, 2H),
7.95−8.01 (m, 1H), 8.23−8.31 (m, 3H), 8.62 (s, 1H), 10.63 (s, 1H).
MS (ESI): m/z 471.10 (M + H)+. Anal. Calcd for C23H17F3N4O4: C,
58.73; H, 3.64; N, 11.91. Found: C, 58.63; H, 3.78; N, 11.85.
N-{3-[(2-Aminoimidazo[1,2-b]pyridazin-6-yl)oxy]phenyl}-3-
(trifluoromethyl)benzamide (10). A mixture of 9 (5.81 g, 12.3
mmol) and 8 N NaOH (20 mL) in methanol (100 mL) was stirred at
room temperature for 4 h. The mixture was acidified with 5 N HCl
to pH 5 and extracted with 25% tetrahydrofuran in ethyl acetate
(3 × 300 mL). The combined organic layer was dried over anhydrous
MgSO4. The insoluble was filtered off, and the filtrate was evaporated
under reduced pressure to give 6-(3-{[3-(trifluoromethyl)benzoyl]-
amino}phenoxy)imidazo[1,2-b]pyridazine-2-carboxylic acid (4.95 g,
91%) as a colorless solid, mp 229−231 °C. 1H NMR (DMSO-d6, 300
MHz): δ 7.10 (dd, J = 1.8, 8.2 Hz, 1H), 7.27 (d, J = 9.8 Hz, 1H), 7.49
(t, J = 8.2 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.75−7.84 (m, 2H), 7.98
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(d, J = 7.7 Hz, 1H), 8.21−8.32 (m, 3H), 8.52 (s, 1H), 10.62 (s, 1H),
12.87 (br s, 1H). MS (ESI): m/z 443.05 (M + H)+. Anal. Calcd for
C21H13F3N4O4: C, 57.02; H, 2.96; N, 12.67. Found: C, 56.98; H, 3.02;
N, 12.57.
300 MHz): δ 5.59 (s, 2H), 7.23 (d, J = 8.9 Hz, 1H), 7.34−7.42
(m, 2H). MS (ESI): m/z 260.95 (M + H)+.
To a solution (10 mL) of 6-iodoimidazo[1,2-b]pyridazine-2-amine
(10 g, 38.4 mmol) in N,N-dimethylacetamide (120 mL) was added
cyclopropanecarbonyl chloride (4.20 mL, 46.1 mmol) at 4 °C. The
reaction mixture was stirred at room temperature for 16 h. Water (240 mL)
was added to the reaction mixture at 4 °C. The resulting precipitate
was collected by filtration and washed with water (200 mL) to give
compound 13 (12.3 g, 98%) as pale yellow crystals, mp 248−250 °C.
1H NMR (DMSO-d6, 300 MHz): δ 0.79−0.87 (m, 4H), 1.89−2.00 (m,
1H), 7.49 (d, J = 9.2 Hz, 1H), 7.73 (d, J = 9.2 Hz, 1H), 8.23 (s, 1H), 11.19
(s, 1H). MS (ESI): m/z 328.95 (M + H)+. Anal. Calcd for C10H9IN4O: C,
36.61; H, 2.76; N, 17.08. Found: C, 36.89; H, 2.84; N, 17.20.
N-[6-(3-Aminophenoxy)imidazo[1,2-b]pyridazine-2-yl]-
cyclopropanecarboxamide (14). A mixture of 13 (12.3 g, 37.4
mmol), 3-aminophenol (8.18 g, 74.9 mmol), and potassium carbonate
(11.9 g, 74.9 mmol) in N,N-dimethylformamide (120 mL) was stirred
at 150 °C for 24 h. The mixture was cooled to room temperature and
diluted with ethyl acetate (150 mL) and water (150 mL). The mixture
was filtered through a pad of Celite. The filtrate was partitioned
between ethyl acetate (150 mL) and saturated aqueous NaHCO3
(150 mL). The aqueous layer was extracted with ethyl acetate (4 ×
100 mL). The organic layers were combined and dried over anhydrous
MgSO4, filtered, and evaporated in vacuo. The residue was purified by
silica gel column chromatography (50−100% ethyl acetate in n-
hexane) to give compound 14 (9.24 g, 80%) as pale brown crystals,
mp 237−239 °C. 1H NMR (DMSO-d6, 300 MHz): δ 0.74−0.86
(m, 4H), 1.87−1.98 (m, 1H), 5.29 (s, 2H), 6.30 (dd, J = 1.7, 8.0 Hz,
1H), 6.35 (t, J = 1.7 Hz, 1H), 6.43 (dd, J = 1.7, 8.0 Hz, 1H), 6.95 (d,
J = 9.8 Hz, 1H), 7.05 (t, J = 8.0 Hz, 1H), 7.92−8.04 (m, 2H), 11.06
(s, 1H). MS (ESI): m/z 328.94 (M + H)+. Anal. Calcd for
C16H15N5O2·0.1H2O: C, 61.77; H, 4.92; N, 22.51. Found: C, 61.68;
H, 4.93; N, 22.50.
To a suspension of 6-(3-{[3-(trifluoromethyl)benzoyl]amino}-
phenoxy)imidazo[1,2-b]pyridazine-2-carboxylic acid (3.00 g,
6.78 mmol) in tetrahydrofuran (200 mL) and tert-butanol (200 mL)
was added triethylamine (1.89 mL, 13.5 mmol) at room temperature,
and the mixture was stirred at room temperature for 10 min. To the
mixture was added diphenylphosphorylazide (2.17 mL, 10.1 mmol).
The reaction mixture was stirred at room temperature for 10 min and
then stirred at 100 °C for 14 h. After the mixture was cooled at room
temperature, ethyl acetate (400 mL) was added to the mixture. The
mixture was washed with saturated aqueous NaHCO3 (200 mL), and
the separated organic layer was washed with brine (200 mL) and dried
over anhydrous MgSO4. The solvent was evaporated under reduced
pressure and the residue was purified by silica gel column
chromatography (0−95% ethyl acetate in n-hexane) to give tert-butyl
[6-(3-{[3-(trifluoromethyl)benzoyl]amino}phenoxy)imidazo[1,2-b]-
pyridazin-2-yl]carbamate (3.10 g, 89%) as colorless crystals, mp 123−
1
125 °C. H NMR (DMSO-d6, 300 MHz): δ 1.46 (s, 9H), 6.99−7.07
(m, 2H), 7.45 (t, J = 8.1 Hz, 1H), 7.64−7.83 (m, 4H), 7.94−8.05
(m, 2H), 8.22−8.30 (m, 2H), 10.05 (br s, 1H), 10.60 (br s, 1H). MS
(ESI): m/z 514.15 (M + H)+. Anal. Calcd for C25H22F3N5O4·0.8H2O:
C, 56.88; H, 4.51; N, 13.27. Found: C, 56.65; H, 4.55; N, 13.15.
A mixture of tert-butyl [6-(3-{[3-(trifluoromethyl)benzoyl]-
amino}phenoxy)imidazo[1,2-b]pyridazin-2-yl]carbamate (400 mg,
0.779 mmol), 4 N HCl in ethyl acetate (10 mL), and methanol
(10 mL) was stirred at room temperature for 14 h. The solvent was
neutralized with 8 N NaOH (5 mL). The resulting mixture was
extracted with ethyl acetate (50 mL). The organic layer was washed
with water (50 mL) and brine (50 mL), successively, and dried over
anhydrous MgSO4. The solvent was evaporated under reduced pres-
sure, and the residue was purified by silica gel column chromatography
(50−100% ethyl acetate in n-hexane) to give 10 (215 mg, 67%) as pale
3-(1-Cyano-1-methylethyl)benzoic Acid (15n). To a solution of
methyl 3-(cyanomethyl)benzoate 17 (7.0 g, 40 mmol) in dimethylsulf-
oxide (80 mL) was slowly added sodium hydride (60% in oil, 4.8 g,
120 mmol) under ice-cooling. The reaction mixture was stirred at room
temperature for 20 min. To the mixture was added iodomethane
(7.5 mL, 120 mmol), and the mixture was stirred at room temperature
for 16 h. Under ice-cooling, the reaction mixture was diluted with water
(80 mL). The mixture was extracted with ethyl acetate (200 mL), and
the organic layer was washed with water (100 mL) and brine (100 mL),
dried over anhydrous Na2SO4, and filtered. The solvent was evapo-
rated under reduced pressure, and the residue was purified by silica gel
column chromatography (5−50% ethyl acetate in n-hexane) to give
methyl 3-(1-cyano-1-methylethyl)benzoate (6.4 g, 79%) as a colorless
oil. 1H NMR (DMSO-d6, 300 MHz): δ 1.72 (s, 6H), 3.89 (s, 3H), 7.61
(t, J = 7.8 Hz, 1H), 7.84 (ddd, J = 1.2, 2.1, 7.8 Hz, 1H), 7.95 (dt, J = 7.8,
1.2 Hz, 1H), 8.08 (t, J = 1.5 Hz, 1H). MS (ESI): m/z 204.06 (M + H)+.
To a solution of methyl 3-(1-cyano-1-methylethyl)benzoate (2.8 g,
14 mmol) in tetrahydrofuran (30 mL) were added lithium hydroxide
monohydrate (0.98 g, 24 mmol), methanol (10 mL), and water
(10 mL), and the mixture was stirred at room temperature for 18 h. The
solvent was evaporated under reduced pressure, and the residue was
diluted with water (15 mL). The mixture was adjusted to pH 3 by
adding 1 N HCl slowly. The resulting white precipitate was collected by
filtration and washed with water (100 mL) to give the compound 15n
1
green crystals, mp 158−160 °C. H NMR (DMSO-d6, 300 MHz):
δ 5.29−5.35 (m, 2H), 6.80 (d, J = 9.4 Hz, 1H), 6.93−7.00 (m, 1H),
7.16 (s, 1H), 7.43 (t, J = 8.2 Hz, 1H), 7.62−7.68 (m, 2H), 7.70−
7.82 (m, 2H), 7.97 (d, J = 7.9 Hz, 1H), 8.21−8.29 (m, 2H), 10.55
(s, 1H). MS (ESI): m/z 414.16 (M + H)+. Anal. Calcd for
C20H14F3N5O2·0.3H2O: C, 57.36; H, 3.51; N, 16.72. Found: C, 57.36;
H, 3.49; N, 16.62.
Ethyl (6-Iodoimidazo[1,2-b]pyridazin-2-yl)carbamate (12).
To a solution of 6-iodopyridazine-3-amine 11 (20.0 g, 90.5 mmol)
in N,N-dimethylacetamide (160 mL) were added ethyl (chloroacetyl)-
carbamate (25.5 g, 153 mmol) and disodium hydrogenphosphate
(32.1 g, 226 mmol), and the reaction mixture was stirred at 110 °C for
4 h. After the mixture was cooled at room temperature, water
(700 mL) was added to the mixture, and the precipitated crystals were
filtered and washed with water (200 mL), acetonitrile (100 mL), and
n-hexane (200 mL), successively, to give compound 12 (22.8 g, 76%)
as black crystals, mp 215−216 °C. 1H NMR (DMSO-d6, 300 MHz): δ
1.26 (t, J = 7.1 Hz, 3H), 4.17 (q, J = 7.1 Hz, 2H), 7.48 (d, J = 9.2 Hz,
1H), 7.71 (d, J = 9.2 Hz, 1H), 8.07 (s, 1H), 10.51 (s, 1H). MS (ESI):
m/z 332.87 (M + H)+.
N-(6-Iodoimidazo[1,2-b]pyridazin-2-yl)cyclopropanecarbo-
xamide (13). To a solution of barium hydroxide octahydrate (28.5 g,
90.3 mmol) in water (400 mL) was added a solution of ethyl
(6-iodoimidazo[1,2-b]pyridazin-2-yl)carbamate 12 (20.0 g, 60.2 mmol)
in NMP (200 mL). The reaction mixture was stirred at 120 °C for 12 h.
After cooling to 80 °C, the mixture was diluted with water (200 mL),
tetrahydrofuran (200 mL), and ethyl acetate (400 mL). The mixture
was stirred at room temperature for 20 min. The insoluble was filtered
off, and the filtrate was washed with water (200 mL) and brine
(200 mL), successively, and dried over anhydrous MgSO4. The solvent
was evaporated under reduced pressure. The residual slurry was
triturated with water (200 mL), and the resulting crystals were collected
by filtration to give 6-iodoimidazo[1,2-b]pyridazine-2-amine (11.0 g,
71%) as orange crystalline needles, mp 173−175 °C. 1H NMR (DMSO-d6,
1
(2.5 g, 98%) as white crystals, mp 166−168 °C. H NMR (DMSO-d6,
300 MHz): δ 1.72 (s, 6H), 7.57 (t, J = 7.8 Hz, 1H), 7.78 (dt, J = 7.8, 1.5
Hz, 1H), 7.92 (dt, J = 7.8, 1.5 Hz, 1H), 8.08 (t, J = 1.5 Hz, 1H), 13.19
(s, 1H). Anal. Calcd for C11H11NO2: C, 69.83; H, 5.86; N, 7.40. Found:
C, 69.64; H, 5.95; N, 7.34.
3-(1-Cyanocyclopropyl)benzoic Acid (15o). Methyl 3-(1-
cyanocyclopropyl)benzoate (1.30 g) was prepared in a similar manner
to the intermediate of 15n, methyl 3-(1-cyano-1-methylethyl)benzoate
from methyl 3-(cyanomethyl)benzoate 17 (1.5 g, 8.56 mmol), using
sodium hydride (60% in oil, 1.03 g, 25.7 mmol), 1,2-dibromoethane
(2.41 g, 12.84 mmol), and dimethylsulfoxide (30 mL). Yield 76%,
1
colorless oil. H NMR (CDCl3, 300 MHz): δ 1.38−1.56 (m, 2H),
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1.74−1.82 (m, 2H), 3.93 (s, 3H), 7.40−7.49 (m, 1H), 7.55−7.62 (m,
1H), 7.88 (t, J = 1.5 Hz, 1H), 7.96 (dt, J = 7.8, 1.5 Hz, 1H).
Compound 15o (0.73 g) was prepared in a similar manner to 15n
from methyl 3-(1-cyanocyclopropyl)benzoate (1.28 g, 6.36 mmol), using
lithium hydroxide monohydrate (0.443 g, 10.8 mmol), tetrahydrofuran
(12 mL), methanol (4 mL), and water (6 mL). Yield 61%, colorless
n-hexane) to give compound 19 (3.42 g, 66%) as a colorless oil.
1H NMR (CDCl3, 300 MHz): δ 3.94 (s, 3H), 4.64 (s, 2H), 7.31 (t, J =
7.7 Hz, 1H), 7.58 (dd, J = 1.7, 7.7 Hz, 1H), 7.71 (dd, J = 1.7, 7.7 Hz,
1H). MS (ESI): m/z 264.90 (M + H)+.
Methyl 2-Chloro-3-(cyanomethyl)benzoate (20). To a solution
of 19 (748 mg, 2.84 mmol) in N,N-dimethylformamide (7 mL) was
added sodium cyanate (412 mg, 8.41 mmol), and the reaction mixture
was stirred at 80 °C for 1 h under nitrogen atmosphere. The reaction
mixture was diluted with a mixed solvent of ethyl acetate and n-hexane
(1:1, 200 mL). The mixture was washed with water (200 mL) and
brine (200 mL), successively, dried over anhydrous MgSO4, and
filtered. The filtrate was evaporated under reduced pressure, and the
residue was purified by silica gel column chromatography (2−20%
ethyl acetate in n-hexane) and recrystallized from ethyl acetate and
n-hexane to give compound 20 (470 mg, 79%) as white crystals, mp
68−69 °C. 1H NMR (CDCl3, 300 MHz): δ 3.91 (s, 2H), 3.95 (s, 3H),
7.39 (t, J = 7.8 Hz, 1H), 7.66−7.72 (m, 1H), 7.76−7.81 (m, 1H). Anal.
Calcd for C10H8ClNO2: C, 57.30; H, 3.85; N, 6.68. Found: C, 57.20;
H, 3.80; N, 6.74.
1
crystals; mp 192−193 °C. H NMR (CDCl3, 300 MHz): δ 1.43−1.53
(m, 2H), 1.73−1.84 (m, 2H), 4.26 (br s, 1H), 7.49 (t, J = 7.8 Hz, 1H),
7.66 (dt, J = 7.8, 1.5 Hz, 1H), 7.94 (t, J = 1.5 Hz, 1H), 8.04 (dt, J = 7.8,
1.5 Hz, 1H). Anal. Calcd for C11H9NO2·0.3H2O: C, 68.60; H, 5.02; N,
7.27. Found: C, 68.64; H, 4.99; N, 7.34.
2-Chloro-3-(1-cyano-1-methylethyl)benzoic Acid (15p).
Methyl 2-chloro-3-(1-cyanocyclopropyl)benzoate (1.99 g) was
prepared in a similar manner to the intermediate of 15n, methyl
3-(1-cyano-1-methylethyl)benzoate from methyl 2-chloro-3-
(cyanomethyl)benzoate 20 (2.00 g, 9.54 mmol), using sodium hydride
(60% in mineral oil, 1.14 g, 28.6 mmol), iodomethane (1.78 mL,
28.6 mmol), and dimethylsulfoxide (20 mL). Yield 88%, colorless oil.
1H NMR (CDCl3, 300 MHz): δ 1.90 (s, 6H), 3.95 (s, 3H), 7.33−7.40
(m, 1H), 7.57−7.64 (m, 2H). MS (ESI): m/z 238.00 (M + H)+.
Compound 15p (1.43 g) was prepared in a similar manner to 15n
from methyl 2-chloro-3-(1-cyanocyclopropyl)benzoate (1.67 g,
7.02 mmol), using lithium hydroxide monohydrate (501 mg, 11.9
mmol), tetrahydrofuran (24 mL), methanol (8 mL), and water
(8 mL). Yield 91%, white crystals; mp 124−125 °C. 1H NMR (CDCl3,
300 MHz): δ 1.92 (s, 6H), 7.41 (t, J = 7.8 Hz, 1H), 7.67 (dd, J = 1.6,
7.8 Hz, 1H), 7.85 (dd, J = 1.6, 7.8 Hz, 1H). Anal. Calcd for
C11H10ClNO2·0.1H2O: C, 58.60; H, 4.56; N, 6.21. Found: C, 58.56;
H, 4.49; N, 6.14.
2-Chloro-3-(1-cyanocyclopropyl)benzoic Acid (15q). Methyl
2-chloro-3-(1-cyanocyclopropyl)benzoate (25.7 g) was prepared in
a similar manner to the intermediate of 15n, methyl 3-(1-cyano-1-
methylethyl)benzoate from methyl 2-chloro-3-(cyanomethyl)benzo-
ate 20 (40.0 g, 191 mmol), using sodium hydride (60% in mineral oil,
22.9 g, 573 mmol), 1,2-dibromoethane (34 mL, 395 mmol), and dime-
thylsulfoxide (400 mL). Yield 57%, colorless crystals; mp 63−64 °C. 1H
NMR (CDCl3, 300 MHz): δ 1.33−1.39 (m, 2H), 1.76−1.83 (m, 2H),
3.95 (s, 3H), 7.32 (t, J = 7.7 Hz, 1H), 7.50 (dd, J = 1.7, 7.7 Hz, 1H),
7.74 (dd, J = 1.7, 7.7 Hz, 1H). MS (ESI): m/z 236 (M + H)+. Anal.
Calcd for C12H10ClNO2: C, 61.16; H, 4.28; N, 5.94. Found: C, 60.97;
H, 4.32; N, 5.93.
Compound 15q (22.1 g) was prepared in a similar manner to 15n
from methyl 2-chloro-3-(1-cyanocyclopropyl)benzoate (25.7 g, 109
mmol), using lithium hydroxide monohydrate (6.69 g, 159 mmol),
tetrahydrofuran (210 mL), methanol (70 mL), and water (70 mL).
Yield 91%, white crystals; mp 155−156 °C. 1H NMR (CDCl3,
300 MHz): δ 1.32−1.42 (m, 2H), 1.79−1.87 (m, 2H), 4.47 (br s, 1H),
7.37 (t, J = 7.7 Hz, 1H), 7.56 (dd, J = 1.7, 7.7 Hz, 1H), 7.95 (dd, J =
1.7, 7.7 Hz, 1H). Anal. Calcd for C11H8ClNO2: C, 59.61; H, 3.64; N,
6.32. Found: C, 59.57; H, 3.66; N, 6.23.
Methyl 3-(Cyanomethyl)benzoate (17). To a solution of methyl
3-(bromomethyl)benzoate 16 (10.0 g, 44 mmol) in acetonitrile
(100 mL) were added potassium cyanate (5.7 g, 87 mmol) and 18-
crown-6 (1.0 g), and the reaction mixture was stirred at room tem-
perature for 3 days. The reaction mixture was filtered, and the solvent
was evaporated under reduced pressure. The residue was purified by
silica gel column chromatography (5−30% ethyl acetate in n-hexane).
The combined desired fractions were concentrated under reduced
pressure to give compound 17 (7.0 g, 91%) as a colorless oil. 1H NMR
(DMSO-d6, 300 MHz): δ 3.88 (s, 3H), 4.17 (s, 2H), 7.57 (t, J =
7.6 Hz, 1H), 7.61−7.69 (m, 1H), 7.88−7.95 (m, 1H), 7.97 (br s, 1H).
MS (ESI): m/z 176.03 (M + H)+.
5-(3-Aminophenoxy)pyridine-2-amine Dihydrochloride (22).
To a mixture of 5-bromo-2-nitropyridine 21 (20.5 g, 101 mmol)
and cesium carbonate (50 g, 153 mmol) in N,N-dimethylformamide
(200 mL) was added dropwise a solution of 3-nitrophenol (15.5 g, 111
mmol) in N,N-dimethylformamide (100 mL) over 1 h, and the reac-
tion mixture was stirred at room temperature for 12 h. The reaction
mixture was concentrated under reduced pressure, and the residue
was diluted with water (300 mL) and extracted with ethyl acetate
(600 mL). The organic layer was washed with 5% aqueous NaHCO3
(300 mL) and brine (300 mL) and dried over anhydrous Na2SO4. The
insoluble material was filtered off, and the filtrate was concentrated
under reduced pressure. The residue was purified by basic silica gel
column chromatography (10−40% ethyl acetate in n-hexane), and the
desired fractions were concentrated under reduced pressure to give
2-nitro-5-(3-nitrophenoxy)pyridine (14.28 g, 54%) as colorless crystals,
1
mp 113−114 °C. H NMR (DMSO-d6, 300 MHz): δ 7.69−7.87 (m,
3H), 8.10 (t, J = 2.1 Hz, 1H), 8.17 (dt, J = 7.7, 1.8 Hz, 1H), 8.38 (d,
J = 9.0 Hz, 1H), 8.53 (d, J = 2.7 Hz, 1H). MS (ESI): m/z 262.07
(M + H)+. Anal. Calcd for C11H7N3O5: C, 50.58; H, 2.70; N, 16.09.
Found: C, 50.51; H, 2.69; N, 16.06.
To a solution of 2-nitro-5-(3-nitrophenoxy)pyridine (14.0 g, 53.6
mmol) in methanol/tetrahydrofuran/ethyl acetate (5:1:1, 1.4 L) was
added 10% palladium/carbon (1.4 g), and the reaction mixture was
stirred at room temperature under hydrogen atmosphere (1.0 atm) for
20 h. The insoluble material was filtered off, and the filtrate was
concentrated in vacuo. The residue was diluted with ethyl acetate (300
mL), and to the mixture was added dropwise slowly 4 N HCl in ethyl
acetate (30 mL). The resulting colorless precipitate was collected by
filtration, washed with i-Pr2O (100 mL) and n-hexane (100 mL), and
dried under vacuum to give the compound 22 (15.2 g, quantitative
yield) as colorless solid. 1H NMR (DMSO-d6, 300 MHz): δ 6.69−6.83
(m, 2H), 6.85−6.95 (m, 1H), 7.09 (d, J = 9.6 Hz, 1H), 7.33 (t, J = 8.0
Hz, 1H), 7.86 (dd, J = 2.7, 9.6 Hz, 1H), 7.98 (d, J = 2.7 Hz, 1H), 8.15
(br s, 3H), 10.02 (br s, 3H).
N-{3-[(6-Aminopyridin-3-yl)oxy]phenyl}-3-(1-cyano-1-
methylethyl)benzamide (23). Compound 23 (3.44 g) was prepared
in a similar manner to 1i from 5-(3-aminophenoxy)pyridine-2-amine
dihydrochloride 22 (3.5 g, 12.7 mmol), using 3-(1-cyano-1-
methylethyl)benzoic acid 15n (2.66 g, 14.0 mmol), oxalyl chloride
(1.63 mL, 19.1 mmol), N,N-dimethylformamide (20 μL), tetrahy-
drofuran (28 mL), and N,N-dimethylacetamide (50 mL). Yield 66%,
1
colorless crystals; mp 143−144 °C. H NMR (DMSO-d6, 300 MHz):
δ 1.74 (s, 6H), 5.91 (s, 2H), 6.51 (d, J = 8.9 Hz, 1H), 6.66−6.77 (m,
1H), 7.23 (dd, J = 2.7, 8.9 Hz, 1H), 7.30 (t, J = 8.1 Hz, 1H), 7.38 (t,
J = 2.1 Hz, 1H), 7.43−7.52 (m, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.68−
7.82 (m, 2H), 7.84−7.94 (m, 1H), 7.99 (t, J = 1.8 Hz, 1H), 10.33
(s, 1H). MS (ESI): m/z 373.10 (M + H)+. Anal. Calcd for C22H20N4O2: C,
70.95; H, 5.41; N, 15.04. Found: C, 70.93; H, 5.46; N, 15.01.
Methyl 3-(Bromomethyl)-2-chlorobenzoate (19). To a solu-
tion of methyl 2-chloro-3-methylbenzoate 18 (3.60 g, 19.4 mmol) in
acetonitrile (60 mL) were added 1-bromopyrrolidine-2,5-dione (11.5 g,
64.3 mmol) and AIBN (960 mg, 5.84 mmol), and the reaction mixture
was stirred at 90 °C for 26 h. The reaction mixture was concentrated
under reduced pressure, and the insoluble material was filtered off. The
filtrate was concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (0−5% ethyl acetate in
N-{3-[(1-(2-Amino-2-oxoethyl)-6-{[(4-methylphenyl)-
sulfonyl]imino}-1,6-dihydropyridin-3-yl)oxy]phenyl}-3-(1-
cyano-1-methylethyl)benzamide (24). To a solution of 23 (2.5 g,
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6.71 mmol) in pyridine (60 mL) was added 4-methylbenzenesulfonyl
chloride (1.34 g, 7.05 mmol) under ice-cooling, and the reaction
mixture was stirred at 80 °C for 2 days. After the mixture was cooled at
room temperature, water (200 mL) was added to the mixture. The
mixture was extracted with ethyl acetate (300 mL). The organic layer
was washed with brine (300 mL) and dried over anhydrous Na2SO4.
The insoluble material was filtered off, and the filtrate was
concentrated under reduced pressure to give 3-(1-cyano-1-methyl-
ethyl)-N-{3-[(6-{[(4-methylphenyl)sulfonyl]amino}pyridin-3-yl)oxy]-
phenyl}benzamide (3.48 g, 99%) as colorless crystals, mp 156−
157 °C. 1H NMR (DMSO-d6, 300 MHz): δ 1.74 (s, 6H), 2.34 (s, 3H),
6.75 (dd, J = 2.4, 8.1 Hz, 1H), 7.15 (d, J = 9.0 Hz, 1H), 7.27−7.45 (m,
4H), 7.46−7.68 (m, 3H), 7.71−7.83 (m, 3H), 7.89 (d, J = 7.8 Hz,
1H), 7.99 (s, 1H), 8.02 (d, J = 3.0 Hz, 1H), 10.37 (s, 1H), 11.07 (br s,
1H). MS (ESI): m/z 527.2 (M + H)+. Anal. Calcd for C29H26N4O4S: C,
66.14; H, 4.98; N, 10.64. Found: C, 66.17; H, 5.01; N, 10.45.
tert-Butyl [3-(3-Amino-4-nitrophenoxy)phenyl]carbamate
(28a). To a solution of tert-butyl (3-hydroxyphenyl)carbamate
(6.89 g, 32.9 mmol) and 5-fluoro-2-nitroaniline 27a (5.09 g, 32.6 mmol)
in N,N-dimethylformamide (100 mL) was added potassium carbonate
(11.2 g, 80.9 mmol), and the reaction mixture was stirred at 100 °C for
14 h. The mixture was cooled at room temperature and diluted with
ethyl acetate/n-hexane (1:1, 250 mL). The mixture was washed with
water (2 × 150 mL) and brine (100 mL), successively, and dried over
anhydrous MgSO4. The solvent was evaporated in vacuo, and the
residue was purified with silica gel column chromatography (2−30%
ethyl acetate in n-hexane) to give compound 28a (7.7 g, 69%) as yellow
crystals, mp 130−131 °C. 1H NMR (CDCl3, 300 MHz): δ 1.54 (s, 9H),
6.13 (br s, 2H), 6.19 (d, J = 2.7 Hz, 1H), 6.35 (dd, J = 2.4, 6.3 Hz, 1H),
6.57 (br s, 1H), 6.74−6.78 (m, 1H), 7.11−7.16 (m, 1H), 7.28−7.35
(m, 2H), 8.12 (d, J = 9.6 Hz, 1H).
tert-Butyl {3-[3-(Methylamino)-4-nitrophenoxy]phenyl}-
carbamate (28b). Compound 28b (21.8 g) was prepared in a
similar manner to 28a from 27b (8.58 g, 50.4 mmol), using tert-butyl
(3-hydroxyphenyl)carbamate (11.0 g, 52.3 mmol), N,N-dimethylfor-
mamide (200 mL), and potassium carbonate (28.6 g, 207 mmol). Yield,
quantitative; orange amorphous solid. 1H NMR (CDCl3, 300 MHz): δ
1.51 (s, 9H), 2.92 (d, J = 5.1 Hz, 3H), 6.22 (dd, J = 2.7, 9.6 Hz, 1H),
6.29 (d, J = 2.4 Hz, 1H), 6.64 (br s, 1H), 6.75 (ddd, J = 0.9, 2.4, 8.1 Hz,
1H), 7.12 (d, J = 8.4 Hz, 1H), 7.27−7.33 (m, 2H), 8.14 (d, J = 9.6 Hz,
1H), 8.19 (br d, J = 4.8 Hz, 1H).
To a solution of 3-(1-cyano-1-methylethyl)-N-{3-[(6-{[(4-
methylphenyl)sulfonyl]amino}pyridin-3-yl)oxy]phenyl}benzamide
(3.2 g, 6.08 mmol) in N,N-dimethylformamide (20 mL) was added
N-ethyl-N-isopropylpropan-2-amine (1.11 mL, 6.38 mmol), and the
reaction mixture was stirred at room temperature for 15 min.
2-Iodoacetamide (1.18 g, 6.38 mmol) was added to the reaction mixture,
and the mixture was stirred at room temperature for 48 h. The
reaction mixture was concentrated under reduced pressure, and to the
residue was added 5% aqueous NaHCO3 (150 mL). The mixture was
extracted with ethyl acetate (300 mL). The organic layer was washed
with brine (150 mL) and dried over anhydrous Na2SO4. The insoluble
material was filtered off, and the filtrate was concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (50−100% ethyl acetate in n-hexane) and triturated
with a mixed solvent of ethyl acetate, i-Pr2O, and n-hexane (1:1:1,
20 mL) to give compound 24 (2.23 g, 63%) as colorless crystals, mp 128−
tert-Butyl [3-({2-[(Cyclopropylcarbonyl)amino]-1H-benzimi-
dazol-6-yl}oxy)phenyl]carbamate (29a). A mixture of 28a
(5.51 g, 15.6 mmol), 10% palladium/carbon (875 mg), tetrahydrofuran
(20 mL), and methanol (100 mL) was stirred at room temperature
under hydrogen atmosphere for 18 h. The insoluble was filtered off and
the filtrate was evaporated in vacuo to give tert-butyl [3-(3,4-diamino-
phenoxy)phenyl]carbamate (5.17 g, quant) as a purple amorphous solid.
1H NMR (CDCl3, 300 MHz): δ 1.49 (s, 9H), 3.24 (br s, 2H), 3.49
1
130 °C. H NMR (DMSO-d6, 300 MHz): δ 1.74 (s, 6H), 2.34 (s, 3H),
(br s, 2H), 6.37−6.43 (m, 3H), 6.61 (ddd, J = 0.9, 2.4, 8.1 Hz, 1H), 6.66
(d, J = 8.1 Hz, 1H), 6.90 (t, J = 2.1 Hz, 1H), 7.07−7.11 (m, 1H), 7.17
(t, J = 8.1 Hz, 1H).
4.83 (s, 2H), 6.76 (dd, J = 2.4, 7.8 Hz, 1H), 7.28 (d, J = 8.1 Hz, 2H),
7.32−7.46 (m, 3H), 7.48 (t, J = 2.1 Hz, 1H), 7.59 (t, J = 7.8 Hz, 2H),
7.68 (d, J = 8.1 Hz, 2H), 7.71−7.82 (m, 3H), 7.86−7.94
(m, 1H), 8.01 (t, J = 1.8 Hz, 1H), 8.13 (d, J = 2.4 Hz, 1H), 10.41
(s, 1H). MS (ESI): m/z 584.15 (M + H)+. Anal. Calcd for C31H29N5O5S:
C, 63.79; H, 5.01; N, 12.00. Found: C, 63.64; H, 5.24; N, 11.79.
N-{3-[(2-Aminoimidazo[1,2-a]pyridin-6-yl)oxy]phenyl}-3-(1-
cyano-1-methylethyl)benzamide (25). To a solution of 24 (1.00 g,
1.72 mmol) in dichloromethane (8 mL) was added trifluoroacetic acid
anhydride (6 mL), and the reaction mixture was stirred at room tem-
perature for 16 h. The reaction mixture was concentrated under
reduced pressure, and to the mixture was added 5% aqueous NaHCO3
(150 mL), and the mixture was extracted with ethyl acetate (150 mL).
The organic layer was washed with brine (150 mL) and dried over
anhydrous Na2SO4. The insoluble material was filtered off, and the
filtrate was concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (30−60% ethyl acetate
in n-hexane) and triturated with i-Pr2O and n-hexane to give
compound 25 (450 mg, 52%) as colorless crystals, mp 130−132 °C.
1H NMR (DMSO-d6, 300 MHz): δ 1.73 (s, 6H), 6.84 (dd, J = 2.4, 7.5
To a solution of tert-butyl [3-(3,4-diaminophenoxy)phenyl]-
carbamate (3.00 g, 9.51 mmol) in tetrahydrofuran (150 mL) was
added cyanogen bromide (2.93 g, 27.7 mmol). The reaction mixture
was stirred at room temperature for 3 days. The reaction mixture was
diluted with ethyl acetate (300 mL). The mixture was washed with
saturated NaHCO3 (2 × 100 mL) and brine (100 mL), successively,
and dried over anhydrous MgSO4. The solvent was evaporated in
vacuo to give tert-butyl {3-[(2-amino-1H-benzimidazol-6-yl)oxy]-
phenyl}carbamate (4.37 g, quant) as brown crystals, mp 117−
119 °C. 1H NMR (CDCl3, 300 MHz): δ 1.44 (s, 9H), 4.16 (br s, 2H),
6.59−6.69 (m, 2H), 6.77−6.90 (m, 2H), 6.96−6.98 (m, 2H), 7.05
(d, J = 8.4 Hz, 1H), 7.14 (t, J = 8.1 Hz, 1H). MS (ESI): m/z 341.05
(M + H)+.
To a solution of tert-butyl {3-[(2-amino-1H-benzimidazol-6-yl)oxy]-
phenyl}carbamate (1.30 g, 3.82 mmol) in pyridine (50 mL) were added
cyclopropanecarbonyl chloride (1 mL, 11.0 mmol) and DMAP
(21.3 mg, 0.174 mmol). The reaction mixture was stirred at room tem-
perature for 16 h. To the reaction mixture were added methanol
(30 mL) and 8 N NaOH (3 mL), successively, and the mixture was
stirred at room temperature for 2.5 h. The mixture was evaporated in
vacuo, and the residue was dissolved in methanol (5 mL) and ethyl
acetate (50 mL). The mixture was washed with 0.1 N HCl (50 mL) and
saturated NaHCO3 (50 mL), successively, and dried over anhydrous
MgSO4. The solvent was evaporated in vacuo. The residue was
crystallized from ethyl acetate and i-Pr2O to give the compound 29a
(1.53 g, 98%) as a colorless crystalline solid, mp 144−145 °C. 1H NMR
(DMSO-d6, 300 MHz): δ 0.90−0.92 (m, 4H), 1.42 (s, 9H), 1.92−1.99
(m, 1H), 6.51−6.55 (m, 1H), 6.80 (dd, J = 2.1, 8.4 Hz, 1H), 7.02−7.21
(m, 3H), 7.36−7.41 (m, 2H), 9.36 (br s, 1H), 11.83 (br s, 1H), 12.01
(br s, 1H). MS (ESI): m/z 409.15 (M + H)+.
Hz, 1H), 7.22 (dd, J = 2.4, 9.6 Hz, 1H), 7.38 (t, J = 8.1 Hz, 1H), 7.51
(t, J = 2.4 Hz, 1H), 7.54−7.68 (m, 3H), 7.70−7.79 (m, 1H), 7.89 (d,
J = 8.1 Hz, 1H), 7.99 (t, J = 1.8 Hz, 1H), 8.27 (s, 1H), 8.66 (d, J =
2.4 Hz, 1H), 10.36 (s, 1H), 12.48 (s, 1H). MS (ESI): m/z 508.10
(M + H)+.
5-Fluoro-N-methyl-2-nitroaniline (27b). Methylamine solution
(40%, 38.4 g) was added dropwise to 2,4-difluoro-1-nitrobenzene 26
(25.0 g, 157 mmol) at 0 °C over 15 min. After completion of the addi-
tion, the reaction mixture was stirred at 0 °C for 1.5 h. To the mixture
was added water (500 mL) and the resulting crystals were collected by
filtration to give compound 27b (26.4 g, 99%) as a yellow crystalline
1
solid, mp 105−106 °C. H NMR (DMSO-d6, 300 MHz): δ 2.94 (d,
J = 4.8 Hz, 3H), 6.53 (ddd, J = 2.7, 7.8, 10.2 Hz, 1H), 6.78 (dd, J = 2.7,
12.3 Hz, 1H), 8.17 (dd, J = 6.3, 9.6 Hz, 1H), 8.32 (br s, 1H). Anal.
Calcd for C7H7FN2O2: C, 49.41; H, 4.15; N, 16.46. Found: C, 49.15;
H, 4.20; N, 16.45.
tert-Butyl [3-({2-[(Cyclopropylcarbonyl)amino]-1-methyl-1H-
benzimidazol-6-yl}oxy)phenyl]carbamate (29b). tert-Butyl {3-[4-
amino-3-(methylamino)phenoxy]phenyl}carbamate (10.1 g) was pre-
pared in a similar manner to the intermediate of 29a, tert-butyl [3-(3,
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4-diaminophenoxy)phenyl]carbamate from 28b (10.9 g, 30.4 mmol),
using 10% palladium/carbon (1.8 g), tetrahydrofuran (150 mL), ethanol
(100 mL), and hydrogen (3 atm). 1H NMR (CDCl3, 300 MHz): δ 1.49
(s, 9H), 2.81 (s, 4H), 2.88 (s, 1H), 2.95 (s, 1H), 6.31 (dd, J = 2.7,
8.1 Hz, 1H), 6.37 (d, J = 2.4 Hz, 1H), 6.47 (br s, 1H), 6.60−6.67
(m, 2H), 6.89 (t, J = 2.1 Hz, 1H), 7.11−7.20 (m, 2H). MS (ESI): m/z
330.05 (M + H)+.
tert-Butyl {3-[(2-amino-1-methyl-1H-benzimidazol-6-yl)oxy]-
phenyl}carbamate (7.46 g) was prepared in a similar manner to the
intermediate of 29a, tert-butyl {3-[(2-amino-1H-benzimidazol-6-yl)-
oxy]phenyl}carbamate from tert-butyl {3-[4-amino-3-(methylamino)-
phenoxy]phenyl}carbamate (10.1 g, 30.4 mmol), using tetrahydrofur-
an (200 mL) and cyanogen bromide (4.33 g, 40.9 mmol). Yield, 69%
in two steps; black amorphous solid. 1H NMR (DMSO-d6, 300 MHz):
δ 1.43 (s, 9H), 3.33 (br s, 2H), 3.46 (s, 3H), 6.47−6.50 (m, 2H), 6.65
(dd, J = 2.4, 8.4 Hz, 1H), 6.92 (d, J = 2.4 Hz, 1H), 7.10−7.19 (m, 3H),
9.34 (br s, 1H). MS (ESI): m/z 355.05 (M + H)+.
Compound 29b (1.89 g) was prepared in a similar manner to 29a
from tert-butyl {3-[(2-amino-1-methyl-1H-benzimidazol-6-yl)oxy]-
phenyl}carbamate (2.45 g, 6.91 mmol), using pyridine (100 mL),
cyclopropanecarbonyl chloride (3.0 mL, 33.1 mmol), and DMAP
(258 mg, 2.11 mmol). Yield 65%, pale brown amorphous solid. 1H NMR
(CDCl3, 300 MHz): δ 0.85−0.90 (m, 2H), 1.06−1.11 (m, 2H), 1.50
(s, 9H), 1.80−1.90 (m, 1H), 3.60 (m, 3H), 6.56 (br s, 1H), 6.64−6.68
(m, 1H), 6.90−6.94 (m, 2H), 7.08−7.11 (m, 2H), 7.20−7.33 (m, 2H),
8.63−8.65 (m, 1H). MS (ESI): m/z 423.15 (M + H)+.
3-(1-Cyano-1-methylethyl)-N-(3-hydroxyphenyl)benzamide
(30). Compound 30 (13.0 g) was prepared in a similar manner to 1i
from 3-(1-cyano-1-methylethyl)benzoic acid 15n (10.0 g, 52.8 mmol),
using tetrahydrofuran (200 mL), N,N-dimethylformamide (80 μL),
oxalyl chloride (6.28 mL, 72.0 mmol), 3-aminophenol (5.24 g,
48.0 mmol), NaHCO3 (6.05 g, 72.0 mmol), and water (60 mL). Yield
96%, white crystals; mp 172 °C. 1H NMR (CDCl3, 300 MHz): δ 1.75
(s, 6H), 6.49−6.55 (m, 1H), 7.08−7.18 (m, 2H), 7.30−7.34 (m, 1H),
7.59 (t, J = 7.8 Hz, 1H), 7.72−7.77 (m, 1H), 7.88−7.93 (m, 1H), 8.01
(t, J = 1.7 Hz, 1H), 9.43 (s, 1H), 10.18 (s, 1H). MS (ESI): m/z 281
(M + H)+.
(200 mL) and water (200 mL). The organic layer was dried over
anhydrous MgSO4 and filtered. The filtrate was concentrated in vacuo
and the residue was purified by silica gel column chromatography
(0−100% ethyl acetate in n-hexane) to give compound 33a (1.70 g,
1
3.59 mmol, 72%) as a yellow solid. H NMR (CDCl3, 300 MHz):
δ 1.19 (s, 9H), 1.78 (s, 6H), 6.26 (d, J = 9.0 Hz, 1H), 6.77−7.04
(m, 1H), 7.34−7.50 (m, 2H), 7.50−7.59 (m, 1H), 7.65 (t, J = 2.1 Hz,
1H), 7.70 (ddd, J = 1.0, 2.0, 7.9 Hz, 1H), 7.77 (dt, J = 7.7, 1.4 Hz, 1H),
7.90 (s, 1H), 7.98 (t, J = 1.7 Hz, 1H), 8.43 (d, J = 9.0 Hz, 1H), 8.63
(s, 1H). MS (ESI): m/z 474 (M + H)+.
3-(1-Cyano-1-methylethyl)-N-(3-{[6-(methylamino)-5-nitro-
pyridin-2-yl]oxy}phenyl)benzamide (33b). Compound 33b
(3.96 g) was prepared in a similar manner to 33a from 32b (1.88 g,
10 mmol), using 30 (2.80 g, 10 mmol), potassium carbonate (1.38 g,
10 mmol), and N,N-dimethylformamide (80 mL). Yield 88%, yellow
1
amorphous solid. H NMR (CDCl3, 300 MHz): δ 1.78 (s, 6H), 2.92
(d, J = 4.9 Hz, 3H), 6.21 (d, J = 9.0 Hz, 1H), 6.90−7.10 (m, 1H),
7.38−7.47 (m, 2H), 7.48−7.60 (m, 1H), 7.66−7.75 (m, 2H), 7.79
(dt, J = 7.7, 1.3 Hz, 1H), 7.93 (s, 1H), 7.99 (t, J = 1.7 Hz, 1H), 8.32−
8.54 (m, 2H). MS (ESI): m/z 432.15 (M + H)+.
N-{3-[(2-Amino-3-tert-butyl-3H-imidazo[4,5-b]pyridin-5-yl)-
oxy]phenyl}-3-(1-cyano-1-methylethyl)benzamide (34a).
A mixture of N-(3-{[6-(tert-butylamino)-5-nitropyridin-2-yl]oxy}-
phenyl)-3-(1-cyano-1-methylethyl)benzamide 33a (1.42 g,
3.0 mmol) and 10% palladium/carbon (0.50 g) in methanol
(10 mL) and tetrahydrofuran (5 mL) was stirred at room temperature
under hydrogen atmosphere for 2 h. The catalyst was filtered through
a pad of Celite. The filtrate was concentrated in vacuo, and the residue
was partitioned between ethyl acetate (200 mL) and water (200 mL).
The organic layer was dried over anhydrous MgSO4 and filtered. The
filtrate was concentrated in vacuo to give N-(3-{[5-amino-6-(tert-
butylamino)pyridin-2-yl]oxy}phenyl)-3-(1-cyano-1-methylethyl)-
1
benzamide (1.33 g, quant) as a purple amorphous solid. H NMR
(CDCl3, 300 MHz): δ 1.29 (s, 9H), 1.77 (s, 6H), 2.85 (br s, 2H),
4.30 (br s, 1H), 6.07 (d, J = 7.9 Hz, 1H), 6.89 (d, J = 7.9 Hz, 2H),
7.28−7.40 (m, 2H), 7.40−7.57 (m, 2H), 7.65−7.81 (m, 3H), 7.95
(t, J = 1.8 Hz, 1H). MS (ESI): m/z 444.30 (M + H)+.
The obtained solid (1.33 g) was dissolved in tetrahydrofuran (10 mL).
To the solution was added cyanogen bromide (0.74 g, 7.00 mmol), and
the reaction mixture was stirred at room temperature for 18 h. The
reaction was quenched by the addition of saturated NaHCO3 (50 mL).
The mixture was partitioned between ethyl acetate (200 mL) and water
(200 mL). The organic layer was dried over anhydrous MgSO4 and
filtered. The filtrate was concentrated in vacuo. The residue was purified
by silica gel column chromatography (0−100% ethyl acetate in n-hexane)
to give compound 34a (1.15 g, 2.45 mmol, 82% in two steps) as a pale
N-tert-Butyl-6-chloro-3-nitropyridin-2-amine (32a). To a
solution of 2,6-dichloro-3-nitropyridine 31 (25.2 g, 131 mmol) in
toluene (150 mL) was added dropwise a solution of tert-butylamine
(9.63 g) in toluene (100 mL) at 0 °C over 30 min. The reaction mix-
ture was stirred at room temperature for 6 h. To the reaction mixture
was added an additional amount of tert-butylamine (10.0 g), and the
mixture was stirred at room temperature for an additional 8 h. To the
reaction mixture was added water (300 mL). The organic layer was
washed with saturated ammonium chloride (200 mL) and brine
(200 mL), successively, dried over anhydrous MgSO4, filtered, and
evaporated in vacuo to give compound 32a (29.6 g, 99%) as an orange
1
black amorphous solid. H NMR (CDCl3, 300 MHz): δ 1.76 (s, 15H),
4.78 (br s, 2H), 6.69 (d, J = 8.3 Hz, 1H), 6.92 (ddd, J = 1.0, 2.2, 8.1 Hz,
1H), 7.35 (t, J = 8.1 Hz, 1H), 7.40−7.54 (m, 3H), 7.56 (d, J = 8.3 Hz,
1H), 7.70 (ddd, J = 1.1, 2.1, 7.9 Hz, 1H), 7.76 (dt, J = 7.6, 1.4 Hz, 1H),
7.91 (s, 1H), 7.96 (t, J = 1.7 Hz, 1H). MS (ESI): m/z 469 (M + H)+.
N-{3-[(2-Amino-3-methyl-3H-imidazo[4,5-b]pyridin-5-yl)-
oxy]phenyl}-3-(1-cyano-1-methylethyl)benzamide (34b).
N-(3-{[5-Amino-6-(methylamino)pyridin-2-yl]oxy}phenyl)-3-(1-cyano-
1-methylethyl)benzamide (3.54 g) was prepared in a similar manner to
the intermediate of 34a, N-(3-{[5-amino-6-(tert-butylamino)pyridin-
2-yl]oxy}phenyl)-3-(1-cyano-1-methylethyl)benzamide from compound
33b (3.96 g, 8.83 mmol), using 10% palladium/carbon (1.0 g), tetra-
hydrofuran (5 mL), and methanol (10 mL) to give a purple amorphous
solid. 1H NMR (CDCl3, 300 MHz): δ 1.73 (s, 6H), 2.63−3.27 (m, 5H),
4.42 (br s, 1H), 5.95 (d, J = 7.9 Hz, 1H), 6.80−6.95 (m, 2H), 7.27−
7.33 (m, 1H), 7.36 (t, J = 2.1 Hz, 1H), 7.40−7.51 (m, 2H), 7.62−7.71
(m, 1H), 7.75 (dd, J = 7.7, 1.7 Hz, 1H), 7.95 (t, J = 1.8 Hz, 1H), 8.12
(s, 1H). MS (ESI): m/z 402.15 (M + H)+.
1
crystalline solid, mp 87 °C. H NMR (DMSO-d6, 300 MHz): δ 1.50
(s, 9H), 6.81 (d, J = 8.5 Hz, 1H), 8.28 (s, 1H), 8.44 (d, J = 8.7 Hz,
1H). Anal. Calcd for C9H12ClN3O2: C, 47.07; H, 5.27; N, 18.30.
Found: C, 46.97; H, 5.22; N, 18.26.
6-Chloro-N-methyl-3-nitropyridin-2-amine (32b). To a solu-
tion of 2,6-dichloro-3-nitropyridine 31 (3.86 g, 20 mmol) was added
dropwise 2 M methylamine solution in tetrahydrofuran (15 mL,
30 mmol) at 0 °C. The reaction mixture was stirred at room tem-
perature for 1 h. The mixture was partitioned between ethyl acetate
(300 mL) and water (300 mL). The organic layer was dried over
anhydrous MgSO4, filtered, and evaporated in vacuo. The residue was
purified by silica gel column chromatography (0−100% ethyl acetate
in n-hexane) to give compound 32b (2.94 g, 78%) as a yellow solid.
1H NMR (CDCl3, 300 MHz): δ 3.18 (d, J = 4.9 Hz, 3H), 6.62 (d, J =
8.5 Hz, 1H), 8.25−8.40 (m, 2H). MS (ESI): m/z 187.95 (M + H)+.
N-(3-{[6-(tert-Butylamino)-5-nitropyridin-2-yl]oxy}phenyl)-3-
(1-cyano-1-methylethyl)benzamide (33a). A mixture of 2-tert-
butylamino-3-nitro-6-chloropyridine 32a (1.15 g, 5.0 mmol),
3-(1-cyano-1-methylethyl)-N-(3-hydroxyphenyl)benzamide 30
(1.40 g, 5.0 mmol), and potassium carbonate (0.69 g, 5.0 mmol) in
N,N-dimethylformamide (5 mL) was stirred at room temperature for
18 h. The reaction mixture was partitioned between ethyl acetate
Compound 34b (0.34 g) was prepared in a similar manner to 34a
from N-(3-{[5-amino-6-(methylamino)pyridin-2-yl]oxy}phenyl)-3-(1-
cyano-1-methylethyl)benzamide (0.40 g), using cyanogen bromide
(0.53 g, 5.0 mmol) and tetrahydrofuran (10 mL). Yield, 80% in
1
two steps; pale brown solid. H NMR (CDCl3, 300 MHz): δ 1.73
(s, 6H), 3.53 (s, 3H), 4.82 (s, 2H), 6.64 (d, J = 8.3 Hz, 1H), 6.90
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(dd, J = 2.1, 7.9 Hz, 1H), 7.29−7.38 (m, 1H), 7.38−7.44 (m, 1H),
7.48 (t, J = 7.2 Hz, 2H), 7.56 (d, J = 8.3 Hz, 1H), 7.67 (dd, J = 1.0,
7.8 Hz, 1H), 7.72−7.80 (m, 1H), 7.95 (t, J = 1.7 Hz, 1H), 8.21 (br s, 1H).
MS (ESI): m/z 427.20 (M + H)+.
to 5 from tert-butyl {3-[(2-amino[1,3]thiazolo[5,4-b]pyridin-5-yl)-
oxy]phenyl}carbamate 41 (1.00 g, 2.79 mmol), using cyclopropane-
carbonyl chloride (327 μL, 3.63 mmol) and pyridine (30 mL). Yield
86%, pale yellow crystals; mp 199 °C. 1H NMR (DMSO-d6,
300 MHz): δ 0.86−1.03 (m, 4H), 1.45 (s, 9H), 1.92−2.05 (m, 1H),
6.68−6.79 (m, 1H), 7.08 (d, J = 8.7 Hz, 1H), 7.20−7.38 (m, 3H), 8.15
(d, J = 8.7 Hz, 1H), 9.48 (br s, 1H), 12.66 (br s, 1H).
3-(4-Nitrophenoxy)aniline (36). Compound 36 (21.8 g) was pre-
pared in a similar manner to 8 from 1-fluoro-4-nitrobenzene 35 (14.1 g,
100 mmol), using 3-aminophenol (11.2 g, 102 mmol), potassium
carbonate (26.5 g, 191 mmol), and N,N-dimethylformamide (150 mL).
A solution of tert-butyl [3-({2-[(cyclopropylcarbonyl)amino][1,3]-
thiazolo[5,4-b]pyridin-5-yl}oxy)phenyl]carbamate (900 mg, 2.11
mmol) and anisole (1 mL) in trifluoroacetic acid (10 mL) was stirred
at 0 °C for 1 h. The reaction mixture was concentrated under reduced
pressure, and to the residue was added saturated aqueous NaHCO3
(50 mL), and the mixture was extracted with tetrahydrofuran/ethyl
acetate (1:1, 50 mL and then 15 mL). The combined organic layers
were washed with brine (5 mL) and dried over anhydrous Na2SO4.
The insoluble material was filtered off, and the filtrate was con-
centrated under reduced pressure. The residue was recrystallized from
tetrahydrofuran/ethyl acetate to give compound 42 (542 mg, 79%)
as pale yellow crystals, mp 261−263 °C. 1H NMR (DMSO-d6,
300 MHz): δ 0.87−1.03 (m, 4H), 1.92−2.05 (m, 1H), 5.25 (br s, 2H),
6.23 (ddd, J = 0.8, 2.4, 7.9 Hz, 1H), 6.28 (t, J = 2.4 Hz, 1H), 6.40 (ddd,
J = 0.8, 2.4, 7.9 Hz, 1H), 6.97−7.08 (m, 2H), 8.11 (d, J = 8.7 Hz, 1H),
12.67 (br s, 1H). MS (ESI): m/z 327.1 (M + H)+. Anal. Calcd for
C16H14N4O2S·0.1H2O: C, 58.56; H, 4.36; N, 17.07. Found: C, 58.55;
H, 4.40; N, 17.03.
2-Chloro-3-(1-cyanocyclopropyl)-N-(2-fluoro-5-hydroxyphenyl)-
benzamide (43). Compound 43 (23.4 g) was prepared in a similar
manner to 1i from 2-chloro-3-(1-cyanocyclopropyl)benzoic acid 15q
(16.0 g, 72.2 mmol), using oxalyl chloride (7.2 mL, 84.0 mmol), N,
N-dimethylformamide (0.1 mL), tetrahydrofuran (150 mL), 3-amino-
4-fluorophenol (9.00 g, 70.8 mmol), NaHCO3 (13.9 g, 166 mmol),
water (100 mL), and tetrahydrofuran (50 mL). Yield 100%, brown
crystals; mp 188−189 °C. 1H NMR (DMSO-d6, 300 MHz): δ
1.40−1.49 (m, 2H), 1.76−1.85 (m, 2H), 6.52−6.63 (m, 1H), 7.07
(dd, J = 9.0, 10.5 Hz, 1H), 7.39 (dd, J = 2.9, 6.5 Hz, 1H), 7.47
(d, J = 7.6 Hz, 1H), 7.53−7.60 (m, 1H), 7.64 (dd, J = 1.7, 7.6 Hz, 1H),
9.48 (s, 1H), 10.30 (s, 1H). MS (ESI): m/z 331.1 (M + H)+. Anal.
Calcd for C17H12ClFN2O2: C, 61.73; H, 3.66; N, 8.47. Found: C,
61.68; H, 3.93; N, 8.15.
N-{5-[(5-Aminopyridine-2-yl)oxy]-2-fluorophenyl}-2-chloro-
3-(1-cyanocyclopropyl)benzamide (44). A mixture of 43 (23.0 g,
69.6 mmol), 2-chloro-5-nitropyridine 39 (12.2 g, 77.1 mmol), and
potassium carbonate (11.5 g, 83.1 mmol) in N,N-dimethylformamide
(70 mL) was stirred at room temperature for 4 h. The mixture was
diluted with N,N-dimethylformamide (130 mL). Water (250 mL) was
added to the mixture. The mixture was stirred at room temperature for
1 h. The resulting precipitate was collected by filtration and washed
with water (250 mL) to give 2-chloro-3-(1-cyanocycropropyl)-N-{2-
fluoro-5-[(5-nitropyridin-2-yl)oxy]phenyl}benzamide (29.3 g, 93%) as
gray crystals, mp 201−202 °C. 1H NMR (DMSO-d6, 300 MHz):
δ 1.38−1.51 (m, 2H), 1.74−1.86 (m, 2H), 7.08−7.19 (m, 1H), 7.32
(d, J = 9.1 Hz, 1H), 7.42 (dd, J = 9.0, 10.3 Hz, 1H), 7.47 (t, J = 7.6 Hz,
1H), 7.56−7.62 (m, 1H), 7.65 (dd, J = 1.7, 7.6 Hz, 1H), 7.85 (dd, J =
2.8, 6.4 Hz, 1H), 8.65 (dd, J = 2.6, 9.1 Hz, 1H), 9.06 (d, J = 2.6 Hz,
1H), 10.62 (s, 1H). MS (ESI): m/z 453.1 (M + H)+. Anal. Calcd for
C22H14ClFN4O4: C, 58.35; H, 3.12; N, 12.37. Found: C, 58.34; H,
3.32; N, 12.57.
1
Yield 95%, yellow crystalline solid; mp 79 °C. H NMR (DMSO-d6,
300 MHz): δ 5.39 (br s, 2H), 6.26 (ddd, J = 0.9, 2.4, 7.8 Hz, 1H), 6.31
(t, J = 2.1 Hz, 1H), 6.48 (ddd, J = 0.9, 2.1, 8.1 Hz, 1H), 7.07−7.14
(m, 3H), 8.24 (dt, J = 7.2, 3.6 Hz, 2H). MS (ESI): m/z 271.95
(M + H)+. Anal. Calcd for C12H10N2O3: C, 62.60; H, 4.38; N, 12.17.
Found: C, 62.63; H, 4.39; N, 12.19.
3-(1-Cyano-1-methylethyl)-N-[3-(4-nitrophenoxy)phenyl]-
benzamide (37). Compound 37 (8.10 g) was prepared in a similar
manner to 1i from 36 (4.69 g, 20.4 mmol), using 15h (3.98 g,
21.0 mmol), EDCI·HCl (4.69 g, 24.5 mmol), DMAP (151 mg, 1.24
mmol), and pyridine (100 mL). Yield 99%, yellow amorphous solid.
1H NMR (DMSO-d6, 300 MHz): δ 1.75 (s, 6H), 6.97 (dd, J = 1.8, 8.4
Hz, 1H), 7.20 (dt, J = 10.5, 3.3 Hz, 2H), 7.49 (t, J = 7.8 Hz, 1H), 7.60
(t, J = 7.2 Hz, 1H), 7.67−7.71 (m, 2H), 7.76 (d, J = 8.7 Hz, 1H), 7.93
(d, J = 7.8 Hz, 1H), 8.02 (t, J = 1.5 Hz, 1H), 8.29 (dt, J = 10.5, 3.3 Hz,
2H), 10.49 (br s, 1H). MS (ESI): m/z 402.05 (M + H)+.
N-[3-(4-Aminophenoxy)phenyl]-3-(1-cyano-1-methylethyl)-
benzamide (38). To a solution of 37 (8.10 g, 20.2 mmol) in tetra-
hydrofuran (50 mL) and methanol (50 mL) was added 10% palladium/
carbon (555 mg), and the reaction mixture was stirred at room tem-
perature under hydrogen atmosphere (2.5 atm) for 14 h. The insoluble
was filtered off and the filtrate was concentrated in vacuo to give com-
1
pound 38 (7.34 g, 98%) as a pale gray amorphous solid. H NMR
(DMSO-d6, 300 MHz): δ 1.74 (s, 6H), 5.00 (br s, 2H), 6.58−6.67 (m,
3H), 6.77−6.81 (m, 2H), 7.27 (t, J = 8.1 Hz, 1H), 7.37 (t, J = 2.1 Hz,
1H), 7.43−7.46 (m, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.74 (ddd, J = 0.9,
2.1, 7.8 Hz, 1H), 7.90 (dt, J = 8.1, 1.2 Hz, 1H), 7.99 (t, J = 1.5 Hz, 1H),
10.30 (br s, 1H). MS (ESI): m/z 372.10 (M + H)+.
tert-Butyl {3-[(5-Aminopyridin-2-yl)oxy]phenyl}carbamate
(40). To a suspension of tert-butyl (3-hydroxyphenyl)carbamate
(3.02 g, 14.4 mmol) and potassium carbonate (2.99 g, 21.7 mmol) in
N,N-dimethylformamide (35 mL) was added 2-chloro-5-nitropyridine
39 (2.52 g, 15.9 mmol), and the reaction mixture was stirred at 70 °C
for 2 h. To the reaction mixture was added water (100 mL), and the
mixture was extracted with ethyl acetate (100 mL, 50 mL). The com-
bined extracts were washed with brine (20 mL) and dried over an-
hydrous MgSO4. The insoluble material was filtered off, and the filtrate
was concentrated under reduced pressure to give tert-butyl {3-[(5-
nitropyridin-2-yl)oxy]phenyl}carbamate as a yellow solid. To a solution
of tert-butyl {3-[(5-nitropyridin-2-yl)oxy]phenyl}carbamate in ethanol/
tetrahydrofuran (4:1, 100 mL) was added 10% palladium/carbon
(1.54 g), and the reaction mixture was stirred at room temperature for
7 h under hydrogen atmosphere (1.0 atm). The insoluble material was
filtered off, and the filtrate was concentrated under reduced pressure.
The residue was recrystallized from methanol to give compound 40
(3.35 g, 77% in two steps from 39) as brown crystalline solid, mp 166−
1
167 °C. H NMR (DMSO-d6, 300 MHz): δ 1.45 (s, 9H), 5.11 (br s,
2H), 6.52 (dd, J = 1.5, 7.4 Hz, 1H), 6.74 (d, J = 8.7 Hz, 1H), 7.07 (dd,
J = 2.2, 8.7 Hz, 1H), 7.10−7.23 (m, 3H), 7.55 (d, J = 2.2 Hz, 1H), 9.36
(br s, 1H).
tert-Butyl {3-[(2-Amino[1,3]thiazolo[5,4-b]pyridin-5-yl)oxy]-
phenyl}carbamate (41). Compound 41 (3.51 g) was prepared in
a similar manner to 5 from 40 (3.33 g, 11.1 mmol), using potassium
thiocyanate (4.30 g, 44.2 mmol), AcOH (60 mL), and bromine
(1.85 g, 11.6 mmol). Yield 88%, pale yellow crystals; mp 181−182 °C.
1H NMR (DMSO-d6, 300 MHz): δ 1.45 (s, 9H), 6.62−6.70 (m, 1H),
6.88 (d, J = 8.9 Hz, 1H), 7.18−7.29 (m, 3H), 7.62 (br s, 2H), 7.71
(d, J = 8.9 Hz, 1H), 9.43 (br s, 1H). MS (ESI): m/z 359.1 (M + H)+.
N-[5-(3-Aminophenoxy)[1,3]thiazolo[5,4-b]pyridin-2-yl]-
cyclopropanecarboxamide (42). The intermediate tert-butyl
[3-({2-[(cyclopropylcarbonyl)amino][1,3]thiazolo[5,4-b]pyridin-
5-yl}oxy)phenyl]carbamate (1.02 g) was prepared in a similar manner
A mixture of 2-chloro-3-(1-cyanocycropropyl)-N-{2-fluoro-5-[(5-
nitropyridin-2-yl)oxy]phenyl}benzamide (6.60 g, 14.6 mmol), reduced
iron (1.68 g, 30.0 mmol), calcium chloride (3.33 g, 30.0 mmol), water
(80 mL), and ethanol (20 mL) was stirred at 80 °C for 18 h. The
reaction mixture was cooled at room temperature. To the mixture
were added water (250 mL), 1 N NaOH (250 mL), ethyl acetate
(300 mL), and Celite (33 g), successively, and the mixture was stirred
for 15 min. The mixture was filtered through a pad of Celite. The
insoluble was washed with ethyl acetate (100 mL). The filtrate and
washings were combined, and the separated organic layer was dried
over anhydrous MgSO4. The solvent was evaporated in vacuo. The
resulting slurry was triturated with diethyl ether to give compound 44
1
(4.23 g, 69%) as pale yellow crystals, mp 186−187 °C. H NMR
3475
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Journal of Medicinal Chemistry
Article
(CDCl3, 300 MHz): δ 1.35−1.42 (m, 2H), 1.80−1.85 (m, 2H), 3.45−
3.57 (br s, 2H), 6.82 (d, J = 8.4 Hz, 1H), 6.82−6.88 (m, 1H), 7.02
(d, J = 8.4 Hz, 1H), 7.12 (dd, J = 7.5, 8.4 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H),
7.50 (dd, J = 1.5, 7.5 Hz, 1H), 7.66 (dd, J = 1.8, 7.8 Hz, 1H), 7.70 (d, J =
3.0 Hz, 1H), 7.97−8.03 (br s, 1H), 8.28 (dd, J = 3.0, 6.6 Hz, 1H). MS
(ESI): m/z 423.1 (M + H)+. Anal. Calcd for C22H16ClFN4O2·0.25H2O: C,
61.83; H, 3.89; N, 13.11. Found: C, 61.94; H, 3.84; N, 12.83.
collected and dried over anhydrous MgSO4, filtered, and concentrated
under reduced pressure. The residual oil was dissolved in toluene, and
the solution was purified by silica gel column chromatography (20−80%
ethyl acetate in n-hexane) to give compound 47 (10.9 g, 91%) as a
brown oil. 1H NMR (CDCl3, 300 MHz): δ 3.57 (br s, 2H), 6.83 (d, J =
8.7 Hz, 1H), 6.93 (dd, J = 2.8, 8.7 Hz, 1H), 7.11 (dd, J = 2.8, 8.7 Hz, 1H),
7.39 (d, J = 8.7 Hz, 1H), 7.69 (d, J = 3.0 Hz, 1H), 8.11 (d, J = 3.0 Hz,
1H), 8.41 (br s, 1H). MS (ESI): m/z 332.0 (M + H)+.
N-(5-{4-Chloro-3-[(trifluoroacetyl)amino]phenoxy}[1,3]-
thiazolo[5,4-b]pyridin-2-yl)cyclopropanecarboxamide (48).
To a mixture of 47 (12 g, 36.2 mmol) and potassium thiocyanate
(14.1 g, 145 mmol) in acetic acid (145 mL) was added dropwise
bromine (8.67 g, 54.3 mmol) under ice-cooling. The reaction mixture
was stirred at room temperature for 16 h. The reaction mixture was
filtered through Celite, and the filtrate was concentrated under
reduced pressure. The residual oil was dissolved in ethyl acetate
(100 mL), and to the mixture was slowly added saturated NaHCO3
(150 mL), and the mixture was partitioned. The organic layer was dried
over anhydrous MgSO4, filtered, and concentrated under reduced
pressure. The residual solid was collected by filtration using i-Pr2O
(100 mL) and dried under vacuum to give N-{5-[(2-amino[1,3]-
thiazolo[5,4-b]pyridin-5-yl)oxy]-2-chlorophenyl}-2,2,2-trifluoroaceta-
mide (10.1 g, 72%) as pale yellow crystals, mp 164−165 °C. 1H NMR
(DMSO-d6, 300 MHz): δ 6.98 (d, J = 8.5 Hz, 1H), 7.16 (dd, J = 2.8,
8.8 Hz, 1H), 7.27 (d, J = 2.8 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.66
(br s, 1H), 7.75 (d, J = 8.5 Hz, 1H), 11.30 (br s, 1H). MS (ESI): m/z
389.0 (M + H)+. Anal. Calcd for C14H8ClF3N4O2S: C, 43.25; H, 2.07;
N, 14.41. Found: C, 43.01; H, 2.17; N, 14.28.
To a solution of N-{5-[(2-amino[1,3]thiazolo[5,4-b]pyridin-5-yl)oxy]-
2-chlorophenyl}-2,2,2-trifluoroacetamide (5.0 g, 12.86 mmol) in pyridine
(25 mL) was added dropwise cyclopropanecarbonyl chloride (1.28 mL,
14.2 mmol) under ice-cooling, and the reaction mixture was stirred at
room temperature for 1 h. The reaction mixture was treated with
saturated NaHCO3 (100 mL) and diluted with ethyl acetate (100 mL),
and the organic layer was collected. The organic layer was dried over
anhydrous MgSO4, filtered, and concentrated under reduced pressure.
The residual solid was crystallized from ethyl acetate (30 mL),
collected by filtration, and dried under vacuum to give compound 48
(3.46 g, 59%) as white crystals, mp 235−236 °C. 1H NMR (DMSO-d6,
300 MHz): δ 0.86−1.07 (m, 4H), 1.90−2.10 (m, 1H), 7.20 (d, J = 8.7
Hz, 1H), 7.26 (dd, J = 2.7, 8.9 Hz, 1H), 7.38 (d, J = 2.7 Hz, 1H), 7.66
(d, J = 8.9 Hz, 1H), 8.20 (d, J = 8.7 Hz, 1H), 11.34 (br s, 1H), 12.72
(br s, 1H). MS (ESI): m/z 457.0 (M + H)+.
N-{5-[(2-Amino[1,3]thiazolo[5,4-b]pyridin-5-yl)oxy]-2-fluoro-
phenyl}-2-chloro-3-(1-cyanocyclopropyl)benzamide (45). The
mixture of potassium thiocyanate (3.89 g, 40 mmol) in acetic acid
(50 mL) was stirred at room temperature for 10 min. To the mixture
was added 44 (4.23 g, 10 mmol), and the mixture was stirred at room
temperature for an additional 5 min. To the mixture was added a
solution of bromine (2.40 g, 15 mmol) in acetic acid (50 mL) at
15 °C. The resulting mixture was stirred at room temperature for 6 h.
The resulting yellow precipitate was removed by filtration through a
pad of Celite and washed with acetic acid (50 mL). The filtrate and
washings were combined and concentrated under reduced pressure.
The residue was diluted with 0.1 N NaOH (100 mL), and the mixture
was extracted with ethyl acetate (100 mL). The organic layer was dried
over anhydrous MgSO4. The solvent was evaporated in vacuo. The
residue was purified with silica gel column chromatography (0−100%
ethyl acetate in n-hexane). Desired fractions were combined and evap-
orated in vacuo. And the resulting slurry was triturated with diethyl
ether to give the compound 45 (3.32 g, 69%) as pale yellow crystals,
mp 215−216 °C. 1H NMR (300 MHz, DMSO-d6): δ 1.41−1.49
(m, 2H), 1.75−1.85 (m, 2H), 6.95 (d, J = 8.5 Hz, 1H), 6.97−7.03
(m, 1H), 7.33 (dd, J = 9.3, 10.2 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.56−
7.67 (m, 4H), 7.69 (dd, J = 2.9, 6.5 Hz, 1H), 7.73 (d, J = 8.5 Hz, 1H),
10.53 (s, 1H). HRMS (ESI): calcd for C23H15ClFN5O2S [M + H]+
480.0692. Found: 480.0692.
2-Chloro-5-[(5-nitropyridin-2-yl)oxy]aniline (46). To a solu-
tion of 2-chloro-5-nitropyridine 39 (4.76 g, 30 mmol) and 3-amino-4-
chlorophenol (4.31 g, 30 mmol) in N,N-dimethylformamide (15 mL)
was added potassium carbonate (4.15 g, 30 mmol), and the reaction
mixture was stirred at room temperature for 16 h. To the reaction
mixture was added ethyl acetate (80 mL), and the mixture was washed
successively with water (50 mL) and saturated brine (50 mL). The
organic layer was dried over anhydrous MgSO4 and concentrated
under reduced pressure. The residual solid was recrystallized from
ethyl acetate/n-hexane (1:1) (30 mL), and the crystals were collected
by filtration, washed with i-Pr2O (20 mL), and dried under vacuum to
give compound 46 (6.74 g, 85%) as brown crystals, mp 123−124 °C.
1H NMR (CDCl3, 300 MHz): δ 4.19 (br s, 2H), 6.48 (dd, J = 2.7, 8.6
Hz, 1H), 6.58 (d, J = 2.7 Hz, 1H), 7.01 (d, J = 9.0 Hz, 1H), 7.29 (d,
J = 8.6 Hz, 1H), 8.47 (dd, J = 2.7, 9.0 Hz, 1H), 9.05 (d, J = 2.7 Hz,
1H). MS (ESI): m/z 266.0 (M + H)+. Anal. Calcd for C11H8ClN3O3:
C, 49.73; H, 3.04; N, 15.82. Found: C, 49.73; H, 3.13; N, 15.64.
N-{5-[(5-Aminopyridin-2-yl)oxy]-2-chlorophenyl}-2,2,2-tri-
fluoroacetamide (47). A solution of 46 (6.5 g, 24.5 mmol) in tetra-
hydrofuran (50 mL) and trifluoroacetic anhydride (3.73 mL, 26.9 mmol)
was stirred at room temperature for 1 h. The reaction mixture was con-
centrated under reduced pressure, and the residue was dissolved in ethyl
acetate (80 mL). The solution was washed with saturated NaHCO3
(50 mL). The organic layer was dried over anhydrous MgSO4, filtered,
and concentrated under reduced pressure. The residual solid was collect-
ed by filtration using i-Pr2O (30 mL) and dried under vacuum to give
N-{2-chloro-5-[(5-nitropyridin-2-yl)oxy]phenyl}-2,2,2-trifluoroacetamide
ASSOCIATED CONTENT
■
S
* Supporting Information
Information methods used in kinase enzyme assays, cellular
assays, in vivo studies, computational studies, structural biology
studies, solubility study, microsomal study, and pharmacokinetic
studies. This material is available free of charge via the Internet at
http://pubs.acs.org.
Accession Codes
†PDB accession codes are 4DBN for the BRAF cocrystal
structure and 3VNT for the VEGFR2 cocrystal structure.
1
(7.73 g, 87%) as white crystals, mp 117−118 °C. H NMR (CDCl3,
300 MHz): δ 6.95−7.19 (m, 2H), 7.52 (dd, J = 1.5, 9.0 Hz, 1H), 8.20−
8.30 (m, 1H), 8.40−8.60 (m, 2H), 9.00−9.10 (m, 1H). MS (ESI): m/z
362.0 (M + H)+. Anal. Calcd for C13H7ClF3N3O4: C, 43.17; H, 1.95;
N, 11.62. Found: C, 43.17; H, 2.06; N, 11.54. To a solution of N-
{2-chloro-5-[(5-nitropyridin-2-yl)oxy]phenyl}-2,2,2-trifluoroacetamide
(13 g, 35.9 mmol) in acetic acid (200 mL) was added reduced iron (10 g,
179 mmol). The mixture was stirred at 60 °C for 3 h. After cooling at
room temperature, the reaction mixture was concentrated under reduced
pressure. The concentrate was diluted with ethyl acetate (150 mL), and
to the mixture was slowly added saturated NaHCO3 (200 mL). The
mixture was filtered through Celite. The organic layer of the filtrate was
AUTHOR INFORMATION
■
Corresponding Author
*For M.O.: phone, +81-466-32-1158; fax, +81-466-29-4449;
e-mail, masanori.okaniwa@takeda.com. For T.I.: phone, +81-
466-32-1155; fax, +81-466-29-4449; e-mail, tomoyasu.ishikawa@
takeda.com.
Notes
The authors declare no competing financial interest.
3476
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Journal of Medicinal Chemistry
Article
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A. H.; Hinkle, K. W.; Hunter, R. N. III; Johnson, J. H.; Knick, V. B.;
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ACKNOWLEDGMENTS
■
The authors thank Naoki Tomita, Osamu Kurasawa, and Yuta
Tanaka for their support in the synthesis reactions. The authors
also thank Kouji Mori, Noriko Uchiyama, Hiroshi Miki, Taeko
Yoshida, and Hidehisa Iwata for their assistance in evaluating the
kinase inhibitory activities, and Akira Hori and Yuichi Kakoi for
their assistance in evaluating the cellular VEGFR2 inhibitory
activities. The authors are grateful to Garret Textor, Matt Kroeger,
and Gyorgy Snell from Takeda San Diego for their assistance in
determining the crystal structure of BRAF with 6d. The authors
acknowledge the Advanced Photon Source, an Office of Science
User Facility operated for the U.S. Department of Energy (DOE)
Office of Science by Argonne National Laboratory, supported by
the U.S. DOE under Contract DE-AC02-06CH11357. Finally, the
authors express their gratitude to Yukiko Watanabe for her
assistance in PK evaluation.
ABBREVIATIONS USED
■
1H NMR, proton nuclear magnetic resonance; AUC, area under
the blood concentration time curve; CLtotal, clearance; ERK,
extracellular signal-regulated kinase; FDA, Food and Drug
Administration; FGFR, fibroblast growth factor receptor; HPLC,
high-performance liquid chromatography; HUVEC, human
umbilical vein endothelial cell; MAPK, mitogen-activated protein
kinase; MEK, mitogen-activated protein kinase; MS, mass
spectrometry; PD, pharmacodynamic; PDB, Protein Data Bank;
PDGFR, platelet-derived growth factor receptor; PK, pharmaco-
kinetic; RTK, receptor tyrosine kinase; SAR, structure−activity
relationship; SD, solid dispersion; SD, standard deviation; VDss,
steady state volume of distribution; VEGF, vascular endothelial
growth factor; VEGFR2, vascular endothelial growth factor
receptor 2
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