Design and synthesis of novel DFG-out RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors: 3. Evaluation of 5-amino-linked thiazolo[5,4-d]pyrimidine and thiazolo[5,4-b]pyridine derivatives

Article abstract of DOI:10.1016/j.bmc.2012.07.032

Our aim was to discover RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors that possess strong activity and sufficient oral absorption, and thus, we selected a 5-amino-linked thiazolo[5,4-d]pyrimidine derivative as the lead compound because of its potential kinase inhibitory activities and its desired solubility. The novel tertiary 1-cyano-1-methylethoxy substituent was designed to occupy the hydrophobic region of 'back pocket' of BRAF on the basis of the X-ray co-crystal structure data of BRAF. In addition, we found that N-methylation of the amine linker could control the twisted molecular conformation leading to improved solubility. These approaches produced N-methyl thiazolo[5,4-b]pyridine-5-amine derivative 5. To maximize the in vivo efficacy, we attempted salt formation of 5. Our result indicated that the besylate monohydrate salt form (5c) showed significant improvement of both solubility and oral absorption. Owing to the improved physicochemical properties, compound 5c demonstrated regressive antitumor efficacy in a HT-29 xenograft model.

Full text of DOI:10.1016/j.bmc.2012.07.032

Bioorganic & Medicinal Chemistry 20 (2012) 5600–5615
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of novel DFG-out RAF/vascular endothelial growth
factor receptor 2 (VEGFR2) inhibitors: 3. Evaluation of 5-amino-linked
thiazolo[5,4-d]pyrimidine and thiazolo[5,4-b]pyridine derivatives
Masaaki Hirose a, , Masanori Okaniwa a, , Tohru Miyazaki a, Takashi Imada a, Tomohiro Ohashi a,
Yuta Tanaka a, Takeo Arita a, Masato Yabuki a, Tomohiro Kawamoto a, Shunichirou Tsutsumi b,
Akihiko Sumita b, Terufumi Takagi a, Bi-Ching Sang c, Jason Yano c, Kathleen Aertgeerts c, Sei Yoshida a,
⇑
⇑
Tomoyasu Ishikawa a,
⇑
a Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited: 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
b CMC Center, Takeda Pharmaceutical Company Limited: 17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan
c Structural Biology, Takeda California Inc., 10410 Science Center Drive, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Our aim was to discover RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors that pos-
sess strong activity and sufficient oral absorption, and thus, we selected a 5-amino-linked thiazolo[5,4-
d]pyrimidine derivative as the lead compound because of its potential kinase inhibitory activities and
its desired solubility. The novel tertiary 1-cyano-1-methylethoxy substituent was designed to occupy
the hydrophobic region of ‘back pocket’ of BRAF on the basis of the X-ray co-crystal structure data of
BRAF. In addition, we found that N-methylation of the amine linker could control the twisted molecular
conformation leading to improved solubility. These approaches produced N-methyl thiazolo[5,4-b]pyri-
dine-5-amine derivative 5. To maximize the in vivo efficacy, we attempted salt formation of 5. Our result
indicated that the besylate monohydrate salt form (5c) showed significant improvement of both solubil-
ity and oral absorption. Owing to the improved physicochemical properties, compound 5c demonstrated
regressive antitumor efficacy in a HT-29 xenograft model.
Received 13 June 2012
Revised 9 July 2012
Accepted 11 July 2012
Available online 23 July 2012
Keywords:
RAF
VEGFR2
Solubility
Oral absorption
Antitumor efficacy
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
and therefore they construct blood vessels by producing VEGF,
which stimulates adjacent VEGFR2. Thus, BRAF and VEGFR2 are
Members belonging to the RAF family are important molecules
involved in signal transduction of the mitogen-activated protein
kinase (MAPK) pathway associated with the cellular proliferation,
differentiation, and survival.1 Oncogenic mutations of BRAF are fre-
quently observed in many cancers and cause abnormal prolifera-
tion without any control by receptor tyrosine kinases (RTKs).2 In
addition, the signaling between the vascular endothelial growth
factor (VEGF) and VEGF receptor 2 (VEGFR2) plays a significant role
in the process of progression of solid tumors.3 Many solid tumors
require sufficient amount of nourishment for their progression
independently associated with exacerbation of solid tumors. Based
on the evidence, inhibitors of BRAF and VEGFR2 could be effective
therapeutic agents for cancer treatment.4
During drug discovery, the efficacy of a small molecule is gener-
ally determined not only by its binding affinity to target proteins
but also by its physicochemical properties. Despite the excellent
in vitro potency of the molecule, physicochemical properties such
as low solubility and low metabolic stability have a negative
impact on the pharmacokinetic (PK) profiles and the in vivo
efficacy of the molecule. Therefore, improvement of the physico-
chemical properties of the drug candidates by optimization of
highly potent lead compounds is a necessary process for drug
discovery.
Previously, we reported the design, synthesis, and character-
ization of 2-chloro-3-(1-cyanocyclopropyl)-N-[5-({2-[(cyclopro-
pylcarbonyl)amino][1,3]thiazolo[5,4-b]pyridin-5-yl}oxy)-2-fluoro-
phenyl]benzamide 1 as a highly potent RAF/VEGFR2 inhibitor
(IC50 values: BRAF, 7.0 nM and VEGFR2, 2.2 nM; Fig. 1).5 Although
this compound showed excellent in vitro cellular activity (pMEK
Abbreviations: 1H NMR, proton nuclear magnetic resonance; AUC, area under
the blood concentration/time curve; ERK, extracellular signal-regulated kinase;
HUVEC, human umbilical vein endothelial cells; MEK, mitogen-activated protein
kinase; PK, pharmacokinetic; VEGFR2, vascular endothelial growth factor receptor
2.
⇑
Corresponding authors. Tel.: +81 466 32 1029 (M.H.), +81 466 32 1158 (M.O.),
+81 466 32 1155 (T.I.).
E-mail addresses: masaaki.hirose@takeda.com (M. Hirose), masanori.okaniwa@
takeda.com (M. Okaniwa), tomoyasu.ishikawa@takeda.com (T. Ishikawa).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.032

M. Hirose et al. / Bioorg. Med. Chem. 20 (2012) 5600–5615
5601
Figure 1. Chemical structures of the previous RAF/VEGFR2 inhibitor 1 and lead compound 2a.
Figure 2. Stable conformations calculated for simplified methylated analog 2a0 (green) and non-methylated compound (gray). Simplified analog of 2a shows twisted
conformation between anilino benzene and [1,3]thiazolo[5,4-d]pyrimidine with increased dihedral angles of 46.5° and 6.2° compared to the almost planar conformation of
the non-methylated compound.
IC50 = 25 nM), the oral PK profiles were poor and in vivo efficacy
tion, shows twisted conformation between the anilino benzene
and [1,3]thiazolo[5,4-d]pyrimidine scaffold with increased dihe-
dral angles of 46.5° and 6.2° as a stable conformation compared
to the almost planar conformation of the corresponding non-meth-
ylated compound. Some studies reported that a twisted molecular
conformation contributes to augmenting the crystal packing en-
ergy, thus improving the dissolution rate as well as solubility.7–10
Thus, we hypothesized that twisted conformation of 5-amino-
linked moiety between the aniline benzene moiety and the bicyclic
scaffold may have an impact on improving solubility in aqueous
media. Furthermore, compound 2a showed potential inhibitory
activity against both BRAF and VEGFR2 (IC50 values: BRAF, 51 nM
and VEGFR2, 410 nM) as a lead compound. Therefore, we initiated
a lead optimization of 5-amino-linked chemotype (2a) using a
structure-guided drug design based on co-crystal structural infor-
mation of BRAF protein.
was weak because of its low thermodynamic solubility (3.6 lg/
mL) without assistance of formulation technology5 or prodrug
approach.6
Setting back to other potential chemotypes possessing suffi-
cient drug-like properties could be one of the solutions for drug
discovery. In particular, if X-ray co-crystal structure information
of the target protein is available, chemical modification starting
from other chemotypes could be strategic and productive. There-
fore, we explored an alternative chemotype to discover RAF/VEG-
FR2 inhibitors that possess strong activity as well as sufficient
solubility and oral PK profiles.
We found that 5-amino-linked [1,3]thiazolo[5,4-d]pyrimidine
derivative 2a possesses sufficient solubility (34 lg/mL) (Fig. 1).
We assumed that good solubility of 2a may be because of an in-
crease in the dihedral angle7 by introducing the methyl group at
the ortho position of the anilino benzene moiety (Fig. 2). Computa-
tional analysis for scanning stable conformations indicated that the
methylated analog 2a0, simplified compound of 2a for this calcula-
We reported that the in vitro cellular pMEK inhibitory activity
of our BRAF inhibitors was affected by the bulkiness of the substi-
tuent at the R3b position of ring C (Fig. 3).5 According to co-crystal
Figure 3. Design concept of DFG-out type RAF/VEGFR2 inhibitors.

5602
M. Hirose et al. / Bioorg. Med. Chem. 20 (2012) 5600–5615
Figure 4. Stable conformations calculated for simplified N-methyl analog (green) and NH analog (gray).
structure analysis of BRAF with 1, the 1-cyanocyclopropyl group as
R3b occupies the hydrophobic ‘back pocket’ region. We assumed
that this region could be important to stabilize the DFG-out
inactive conformation of BRAF. On the basis of this assumption,
we expected that the introduction of dimethyl groups at the adja-
cent position of terminal nitrile (2b,c) would enhance cellular
pMEK inhibition.
Next, we selected 2-fluoroanilide as ring B from compound 1
on the basis of our previous work,5 and examined 5-amino-linked
[1,3]thiazolo[5,4-b]pyridine scaffold (3–6) for the first time to
aspire for the enhancement of cellular inhibitory activity. We
calculated a twisting effect derived from N-methylation at the
R2 position (3–5). N-Methyl analog showed larger dihedral angles
between ring A (29.2°) and ring B (39.4°) than those of NH analog
(20.3° and 21.9°, respectively) (Fig. 4). N-Methyl derivatives 3–5
were expected to show twisted conformation between ring A
and B, similar to compound 2a–d. This twisting effect of N-meth-
ylation on solubility and PK profiles was verified using demethy-
lated compound 6. Finally, to further increase the thermodynamic
solubility of selected compound 5, salt formation with various
acids, such as hydrochloride (5a), sulfate (5b), and besylate (5c)
were evaluated. We have described these details in this
manuscript.
2. Chemistry
5-Amino-linked [1,3]thiazolo[5,4-d]pyrimidine11 derivatives
2a–d were synthesized by the route described in Scheme 1. Aniline
protection of 7 by tert-butoxycarbonyl (Boc) group using Boc2O
provided N-Boc derivative 8 in 89% yield. Hydrogenation of the
nitro group of 8 using 10% palladium on activated carbon (Pd/C)
provided aniline 9, which was reacted with commercially available
2-chloro-5-nitropyrimidin-4-yl thiocyanate 10 in the presence of
i-Pr2NEt in tetrahydrofuran (THF) to give 11 as a precursor mole-
cule for [1,3]thiazolo[5,4-d]pyrimidine. Subsequent reduction of
the nitro group of 11 using reduced iron (Fe(0)) in the presence
of CaCl2 resulted in ring formation reaction to afford [1,3]thiazol-
o[5,4-d]pyrimidine-2-amine derivative 12 in 35% yield in three
steps. Acylation of 12 using acetyl chloride in pyridine (98% yield)
followed by cleavage of N-Boc group using trifluoroacetic acid in
anisole provided aniline 13 in 83% yield. Amide coupling reaction
of 13 with various benzoic acids 17a–d in the presence of O-(7-aza-
benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophos-
phate (HATU) gave the target [1,3]thiazolo[5,4-d]pyrimidine
derivatives 2a–d in 46–65% yield.
The synthesis of 2-cyanoethylated and 2-cyano-2-methylpro-
pylated benzoic acid (17a, 17b) is shown in Scheme 2.
Scheme 1. Reagents and conditions: (a) Boc2O, THF, 70 °C, 15 h (89%); (b) H2, 10% Pd/C, EtOH, THF, room temp, 22 h; (c) 2-chloro-5-nitropyrimidin-4-yl thiocyanate 10, i-
Pr2NEt, THF, room temp, 30 min; (d) Fe(0), CaCl2, NMP, EtOH, water, 100 °C, 16 h (35% in three steps); (e) AcCl, pyridine, room temp, 1 h (98%); (f) TFA, anisole, 0 °C, 1 h (83%);
(g) benzoic acids, HATU, pyridine, room temp; (46–65%).

M. Hirose et al. / Bioorg. Med. Chem. 20 (2012) 5600–5615
5603
Scheme 2. Reagents and conditions: (a) diethyl (cyanomethyl) phosphonate, K2CO3, THF, H2O, 60 °C, 1 h; (b) H2, 10% Pd/C, EtOH, THF, room temp, 2 h (88% in two steps); (c)
MeI, LHMDS, THF, ꢀ78 °C, 1 h (44%); (d) 2 N NaOH aq, MeOH, THF, 60 °C, 4 h (95–96%).
Horner–Wadsworth–Emmons reaction12 of the commercially
available formyl derivative 14 with diethyl (cyanomethyl) phos-
phonate in the presence of K2CO3 followed by hydrogenation of
double bond using 10% Pd/C provided the cyanoethylated benzo-
less than 50% yield under the same conditions as that of 20a.
The production of methoxycarbonylated compound 21c was de-
tected in 1H NMR as a byproduct. We assumed that methanolysis
of aliphatic nitrile may occur in competition with hydrolysis of
methyl benzoate. To reduce the formation of 21c, i-PrOH, a
bulkier alcohol than MeOH, was used as a solvent. i-PrOH signif-
icantly reduced side reaction, and these reaction conditions gave
21b as crystals in 77% yield after recrystallization from EtOH/H2O
(1:2).
ate 15 in 88% yield in two steps. Dimethylation of the
a-position
of the cyano group in 15 was accomplished with MeI and
lithium hexamethyldisilazide (LHMDS) under ꢀ78 °C conditions
to give 3-(2-cyano-2-methylpropyl)benzoate 16 in 44% yield.
Hydrolysis of 15, 16 with aqueous 2 N NaOH provided 17a,b in
95–96% yield.
The synthesis of N-methyl [1,3]thiazolo[5,4-b]pyridine deriva-
tives 3–5 is shown in Scheme 4. Boc introduction of 2-fluoro-5-
nitroaniline 22 with Boc2O in THF at 70 °C under reflux conditions
was found to be slow, and the reaction was not completed even
after 19 h. Because the nucleophilicity of aniline group was weak,
we performed the reaction under modified neat conditions at
80 °C. The reaction proceeded smoothly, but resulted in a mixture
of mono-Boc and di-Boc derivatives. Without purification, this
mixture was subjected to hydrogenation reaction of nitro groups
in the presence of 10% Pd/C to give the corresponding aniline deriv-
atives. Subsequent treatment of the mixed aniline derivatives with
K2CO3 resulted in conversion of di-Boc to mono-Boc to give com-
pound 23 in 63% yield in three steps as a single product. Treatment
of 23 with formic acid in the presence of acetic anhydride gave the
corresponding formamide followed by reduction with boran
dimethylsulfide complex to give monomethylated compound 24
in 73% yield in two steps. The reaction of 24 with commercially
available 2-chloro-5-nitropyridine in DMSO provided a coupled
product in 42% yield. Reduction of the nitro group under hydroge-
nation conditions afforded the corresponding aniline 25 in 87%
yield. Fused 1,3-thiazolo ring was constructed by reaction of
Phenoxy derivatives 21a,b were prepared from the corre-
sponding phenol derivatives 18a,b as described in Scheme 3. Cya-
nomethylation of the phenolic hydroxy group in 18a with
bromoacetonitrile in the presence of K2CO3 provided 19a in 86%
yield. Dimethylation of the
a-position of the cyano group in
19a was achieved under conditions similar to those of 16 in
Scheme 2. Compound 20a was obtained in 30% yield. Hydrolysis
of 20a with aqueous 2 N NaOH in a mixed solvent of MeOH/
THF (3:1) provided the target benzoic acid 21a in 51% yield. Chlo-
rinated derivative 21b was prepared under reaction conditions
similar to those used for the preparation for 21a. However, the
yields for each step were low. The optimized reaction conditions
in a dozen gram-scale preparation are also described in Scheme 3.
Starting from methyl 2-chloro-3-hydroxybenzoate 18b, alkylation
of the hydroxy group with chloroacetonitrile in the presence of
K2CO3 and NaI gave the cyanomethylated compound 19b in 92%
yield. In the next dimethylation, the base used was changed from
LHMDS to sodium hexamethyldisilazide (NaHMDS). The reaction
proceeded smoothly at between 0 and 20 °C to give 20b in 50%
yield. Subsequent hydrolysis of methyl ester in 20b gave 21b in
Scheme 3. Reagents and conditions: (a) bromoacetonitrile, K2CO3, acetone, reflux, 4 h (86%); (b) chloroacetonitrile, K2CO3, NaI, acetone, reflux, 13 h (92%); (c) MeI, LHMDS,
THF, ꢀ78 °C, 2 h, (30%); (d) MeI, NaHMDS, THF, room temp, 30 min (50%); (e) 2 N NaOH aq, MeOH, THF, room temp, 30 min (51%); (f) 2 N NaOH aq, i-PrOH, room temp, 2 h
(77%).

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M. Hirose et al. / Bioorg. Med. Chem. 20 (2012) 5600–5615
Scheme 4. Reagents and conditions: (a) Boc2O, 80 °C, 24 h; (b) H2 (1 atm), 10% Pd/C, EtOH, THF, room temp, 24 h; (c) K2CO3, MeOH, THF, 60 °C, 4 h (63% in three steps); (d)
Ac2O, HCO2H, THF, room temp, 16 h; (e) BH3ꢁSMe2, THF, room temp, 1.5 h (73% in two steps); (f) 2-chloro-5-nitropyridine, DMSO, 60 °C, 19 h (42%); (g) H2 (1 atm), 10% Pd/C,
EtOH, THF, room temp, 16 h (87%); (h) KSCN, Br2, AcOH, room temp, 1.5 h (48%); (i) AcCl, pyridine, room temp, 30 min; (j) TFA, anisole, 0 °C, 1 h (86% in two steps); (k) 21a,b,
HATU, pyridine, 90 °C, 48–59%; (l) cyclopropanecarbonyl chloride, pyridine, room temp, 1 h; (m) TFA, anisole, 0 °C, 1 h (75% in two steps).
aniline 25 with potassium thiocyanate and bromine in AcOH to
give the [1,3]thiazolo[5,4-b]pyridine derivative 26 in 48% yield.
Acetylation of the 2-amino group of 26 with acetyl chloride in
pyridine, followed by deprotection of the Boc group with trifluoro-
acetic acid (TFA) in the presence of anisole, gave the precursor
amine 27 for introducing benzamide moieties, in 86% yield in
two steps. Condensation of 27 with carboxylic acid 21a,b using
HATU in pyridine provided the desired [1,3]thiazolo[5,4-b]pyri-
dine-2-acetamide derivatives 3,4 in 48–59% yield. The 2-cyclopro-
80% yield in two steps. Condensation reaction of 28 with carboxylic
acid 21b provided 5 in 50% yield.
The synthesis of 6 was performed as shown in Scheme 5. Boc
introduction of aniline 22 was performed at 55 °C using 4 equiv of
Boc2O in the presence of Et3N to give the corresponding di-Boc com-
pound in 72% yield. The following reduction of the nitro group un-
der hydrogenation conditions gave 29 in 64% yield. The
commercially available [1,3]thiazolo[5,4-b]pyridine-2-amine 30
was acylated with cyclopropanecarbonyl chloride to give 31 in
30% yield. Next, Buchwald–Hartwig reaction13 of 31 with aniline
29 using Pd2(dba)3 and X-phos in the presence of tert-BuOK was
performed under microwave irradiation. During this reaction, one
Boc group of the di-Boc imide was cleaved to give the mono-Boc
panecarboxamide derivative
5 was synthesized in a similar
manner. Acylation of the 2-amino group in 26 with cyclopropane-
carbonyl chloride in pyridine, followed by deprotection of the Boc
group with TFA and anisole, afforded the aniline derivative 28 in
Scheme 5. Reagents and conditions: (a) Boc2O, Et3N, CH2Cl2, 55 °C, 12 h (72%); (b) 10% Pd/C, H2, MeOH, room temp, 12 h (64%); (c) cyclopropanecarbonyl chloride, THF, 0 °C,
2 h (30%); (d) Pd2(dba)3, X-phos, tert-BuOK, tert-BuOH, microwave, 90 °C, 35 min (63%); (e) 4 N HCl/EtOAc, 0 °C, 12 h (65%); (f) 21b, (COCl)2, cat. DMF, room temp, 1 h, then 33,
DMA, room temp, 2 h (66%).

M. Hirose et al. / Bioorg. Med. Chem. 20 (2012) 5600–5615
5605
product 32 in 63% yield. Deprotection of the N-Boc group (32) with
4 N HCl/EtOAc gave the corresponding aniline 33 in 65% yield. Fi-
nally, condensation reaction of aniline 33 with acid chloride pre-
pared by carboxylic acid 21b using oxalyl chloride in the presence
of catalytic DMF provided the desired derivative 6 in 66% yield.
[1,3]thiazolo[5,4-b]pyridine derivatives along with maintaining
the potent BRAF inhibition. Therefore, 2-fluorinated benzanilide
was selected instead of 4-methyl benzanilide (2a–d), and we
examined its adaptability to the 5-amino-linked chemotype. In
addition, N-methyl linker was designed, and we attempted to
achieve the twisted conformation between fused ring A and ring
B to increase solubility.
3. Results and discussion
The N-methyl [1,3]thiazolo[5,4-b]pyridine derivative 3 that we
designed showed better BRAF and VEGFR2 inhibitory activity as
well as more potent pMEK inhibitory activity with an IC50 value
of 30 nM than those of [1,3]thiazolo[5,4-d]pyrimidine 2c. However,
PK study indicated poor drug exposure of compound 3 with an AUC
The structure activity relationships (SAR) of 5-amino-linked
[1,3]thiazolo[5,4-d]pyrimidines 2a–d possessing various substitu-
ents on the ring C are described in Table 1. Cyanoethylated deriv-
ative 2a showed moderate BRAF inhibitory activity with IC50 value
of 51 nM, but weak cellular pMEK inhibitory activity (IC50
>500 nM). Our initial medicinal chemistry strategy to enhance cel-
lular activity was the introduction of dimethyl groups at the adja-
cent position of the terminal nitrile group in 2a to enhance
hydrophobic interaction at the back pocket region. (Fig. 3)
Dimethylation of the cyanoethyl group (2b) resulted in signifi-
cantly increased cellular pMEK inhibitory activity with IC50 value
of 150 nM, despite a slight reduction of BRAF inhibitory activity.
Next, to mask the metabolically labile benzylic methylene (2b)
by the oxygen atom, 3-(1-cyano-1-methylethoxy) derivative 2c
was evaluated. Compound 2c showed better metabolic stability
value of 0.311 lg h/mL after oral administration at a dose of
0–8h
10 mg/kg in mice. The introduction of a chlorine atom at the 2-po-
sition as an R3a group enhanced the oral absorption on the basis of
our empirical finding5, and thus, we attempted to apply our knowl-
edge to compound 4. Introduction of a chlorine atom at 2-position
dramatically improved the oral absorption of compound 4 (AUC0–
8h, 14.308 lg h/mL) without decreasing the other desirable phar-
macological effects. To achieve good PK profiles in humans, we ex-
plored the effects of replacement of acetamide (4, R1 = Me) with
cyclopropane carboxamide (5, R1 = cPr). As we expected, com-
pound 5 showed better metabolic stability (clearance rate: human
25
tion, compound 5 showed significant oral absorption with an
AUC0–8h value of 9.571 g h/mL in mice. Pharmacological profiles
lL/min/mg, mouse 32 lL/min/mg) than acetamide 4. In addi-
against human microsomes with the clearance rate of ꢀ13
lL/
min/mg, as we expected. Moreover, compound 2c exhibited fur-
ther enhancement of the cellular activity with IC50 value of 88
nM together with increase of BRAF inhibition comparable to those
of 2-chloro-3-(1-cyanocyclopropyl) derivative 2d.
l
of 5 (IC50 values: BRAF, 38 nM; VEGFR2, 7.5 nM; and cellular pMEK,
47 nM) were almost comparable to those of compound 4.
Finally, to verify the twisting effect of the N-methyl group on
the physicochemical profiles of 5, we evaluated the corresponding
NH derivative 6. Non-methylated derivative 6 showed significantly
With an aim of further enhancement of cellular pMEK inhibi-
tory activity, the finding of novel (1-cyano-1-methylethoxy)
substituent (2c) triggered us to apply this functionality to 5-
amino-linked [1,3]thiazolo[5,4-b]pyridine scaffold. The SAR of the
[1,3]thiazolo[5,4-b]pyridine-5-amine derivatives 3–6 possessing
the 3-(1-cyano-1-methylethoxy) group are shown in Table 2. The
previous SAR study5 indicated that introduction of the fluorine
atom at the 2-position of the benzanilide (ring B) was significant
to block the metabolic labile site of ring B in the 5-oxy-linked
decreased AUC value (0.008
lg h/mL) because of its low thermody-
namic solubility (0.91 g/mL). Interestingly, N-methylation not
l
only improved the oral PK profile of 6, but also showed an eightfold
increase in VEGFR2 inhibition (IC50 values: 5, 7.5 nM; 6, 64 nM).
Cellular VEGFR2 inhibitory activity was tested using HUVEC cells
stimulated by VEGF. Compound
5 showed potent growth
Table 1
Structure-activity relationships of 5-amino-linked [1,3]thiazolo[5,4-d]pyrimidines 2a–d possessing various ring C
Me
N
S
R3a
C
O
N
HN
O
B
R3b
Me
N
N
H
N
H
fused ring A
a
R3a
Kinase IC50 (nM)
R3b
BRAF (V600E)
VEGFR2
Compound
Cellular pMEKb IC50 (nM)
Human microsome stabilityc
(lL/min/mg)
CN
CN
2a
2b
2c
51
410
300
510
>500
150
88
35
100
73
56
Me Me
O
CN
-13
Me Me
Cl
2d
91
340
120
73
CN
a
b
c
n = 2.
Concentration producing 50% inhibition (IC50) values against RAF substrate MEK phosphorylation in HT-29 (BRAFV600E mutant) cultured human colon cancer cell lines.
Metabolism clearance of each compound was examined using human liver microsomes and NADPH.

5606
M. Hirose et al. / Bioorg. Med. Chem. 20 (2012) 5600–5615
Table 2
Profiles of 5-amino-linked [1,3]thiazolo[5,4-b]pyridines 3–6
F
N
S
R3a
C
O
HN
O
B
O
CN
R1
N
X
N
H
Me Me
fused ring A
R1
X
R3a
Kinase IC50 (nM)
BRAF (V600E) VEGFR2
Cellular pMEKb IC50 (nM)
Microsome stabilityc
(
l
L/min/mg)
Mouse PKd
a
Human
Mouse
AUC PO (lg h/mL)
3
4
5
6
Me
Me
cPr
cPr
N-Me
N-Me
N-Me
NH
H
23
45
38
15
10
14
7.5
64
30
22
47
87
22
45
25
55
0.311
14.308
9.571
0.008
Cl
Cl
Cl
25
32
N.D.e
N.D.e
a
b
c
n = 2.
Concentration producing 50% inhibition (IC50) values against RAF substrate MEK phosphorylation in HT-29 (BRAFV600E mutant) cultured human colon cancer cell lines.
Metabolism clearance of each compound was examined using liver microsomes and NADPH.
Mice cassette dosing of five compounds.
Not determined.
d
e
the main chain atoms of Cys532 and the nitrogen atoms of carbox-
Table 3
amide and fused-thiazolo ring, and presents planar conformation
between the core scaffold (ring A) and carboxamide by carbonyl–
sulfur interaction. The benzanilide moiety (ring B and ring C)
anchors to DFG-out conformation of BRAF by forming 2 hydrogen
bonds with the carboxylate of Glu501 from helix C and the back-
bone NH from Asp594 as a DFG motif. The novel 2-chloro-3-(1-
cyano-1-methylethoxy)benzene moiety (ring C) occupies the
hydrophobic back pocket lined by residues Val504, Leu505,
Thr508, Ile513, Leu514, Leu567, His574, Ile592, and Gly593. These
results are consistent with those of our initial design based on
modeling.
Kinase selectivity of compound 5
a
a
Kinase
IC50 (nM)
Kinase
IC50 (nM)
BRAF(wt)
C-RAF
69
23
PKA
PKCh
>10,000
>10,000
>10,000
>10,000
>10,000
3700
FGFR3
86
CHK1
CK1d
ERK1
CDK1
CDK2
Aurora B
PDGFR
a
43
PDGFRb
EGFR
Her2
TIE2
c-Met
c-Kit
Src
91
>10,000
1200
840
>10,000
430
>10,000
4000
p38a
45
JNK1
>10,000
>10,000
1000
130
GSK3b
MEK1
MEKK1
IR
IKKb
>10,000
8100
3.3. Salt formation study of compound 5
>10,000
a
n = 2.
We investigated the ability of salt formation of compound 5
with optimal acids to enhance oral absorption along with improv-
ing solubility. Although 5-oxy-linked [1,3]thiazolo[5,4-b]pyridine
derivative 1 did not form the salt, 5-amino-linked [1,3]thiazol-
o[5,4-b]pyridine derivative 5 formed salts with various acids.
Among the salts, hydrochloride (5a), sulfate (5b), and besylate
monohydrate (5c) were suitable forms for their characterization.
Powder X-ray diffractometry (XRD) experiments revealed that
these 3 salts formed crystalline state with crystallinity values of
42–81% (Table 4). Melting point of each compound was deter-
mined by differential scanning calorimetry (DSC). Melting points
of 5-amino-linked derivative 5 (165 °C) and its salts 5a–c (128–
179 °C) were much lower than that of 5-oxy-linked derivative 1
(213 °C). The 5-amino-linked derivative 5 and its salts 5a–c
showed higher thermodynamic solubility15 than the 5-oxy-linked
derivative 1 consistent with the above results. In particular, the
inhibition against HUVEC cells with IC50 value of 1.2 nM. Thus, we
selected compound 5, characterized as a VEGFR2-dominant RAF/
VEGFR2 inhibitor, for further development.
3.1. Kinase inhibitory profile of compound 5
The inhibitory profiles of compound 5 against 26 different ki-
nases are summarized in Table 3. The inhibitory activity of com-
pound 5 against RAF family kinases, including BRAF(V600E), was
comparable to that against the angiogenesis-related kinases such
as FGFR3, PDGFR
In addition, compound 5 inhibited p38
kinases, including Src, c-Kit, and Tie2 were moderately inhibited by
compound 5; range of IC50 values, 130–840 nM. Further, no signif-
icant inhibition was observed against the remaining 17 kinases.
Thus, compound 5 was considered a pan-RAF and angiogenesis-
related kinases inhibitor characterized by dominant VEGFR2 inhi-
bition (IC50 = 7.5 nM).
a
, and PDGFRb; range of IC50 values, 23–91 nM.
a; IC50 value, 45 nM. Several
thermodynamic solubility (31–37
derivative 5 was enhanced by salt formation (5a–c) compared to
that of the free base 5 (18 g/mL). The AUC0–8h value of besylate
monohydrate 5c (23.246 g h/mL) was the highest among the
lg/mL) of the 5-amino-linked
l
l
AUC0–8h values of the other salts and therefore we selected 5c as
a promising candidate for further evaluation.
3.2. X-ray co-crystal structural analysis of compound 5 with
BRAF
3.4. Pharmacokinetic profiles and in vivo studies of 5c in rats
The mode of binding of compound 5 to BRAF was determined by
X-ray co-crystal structural analysis (Fig. 5).14 The results of the
analysis revealed that compound 5 occupies the ATP-binding site
and stabilizes the inactive ‘DFG-out’ conformation of BRAF. Thiaz-
olo[5,4-b]pyridine-2-cyclopropanecarboxamide moiety anchors to
the kinase hinge region by forming 2 hydrogen bonds between
Oral administration of 5c at a dose of 25 mg/kg showed good
oral bioavailability (%F = 38.4%) with an AUC value of 5.77 lg h/
mL in rats. Selected PK profiles of 5c in rats are shown in Table 5.
We evaluated in vivo activity of compound 5c in a HT-29 hu-
man colorectal cancer xenograft model in nude rats after oral

M. Hirose et al. / Bioorg. Med. Chem. 20 (2012) 5600–5615
5607
Figure 5. Corystal structure of compound 5 with BRAF (PDB code: 4FC0, 2.95 Å resolution). (A) DFG-out conformation of BRAF is stabilized by 5. (B) Back pocket region of
BRAF (surface).
Table 4
Selected physicochemical properties and pharmacokinetic profile of 5 and its salts 5a–c, compared with 1
Compound
Salt
Crystallinitya (%)
Melting pointb (°C)
Thermodynamic solubilityc
(l
g/mL)
Mouse PKd
AUC PO (lg h/mL)
5
Free base
Hydrochloride
Sulfate
38
56
42
81
55
165
164
179
128
213
18
31
35
37
3.6
9.571
5a
5b
5c
1
19.846
15.917
23.246
1.902
BesylateꢁH2O
Free base
a
b
c
Determined by powder X-ray diffractometry.
Determined by differential scanning calorimetry.
The Japanese Pharmacopoeia 2nd fluid for disintegration test (pH 6.8) (JP2)15 containing 20 mmol/L of bile acid.
d
Cassette dosing of five compounds. Values shown are mean of data from three mice. Compounds (10 mg/kg) were administered in 0.5% methylcellulose in distilled water.
Table 5
Meana pharmacokinetic parameters for 5c in rats
Dose (mg/kg)
Route
CLtotal (mL/h/kg)
1750 433
Vdss (mL/kg)
MRT (h)
AUC0–24h
(
lg h/mL)
Cmax PO (
lg/mL)
%F (%)
1b
iv
Oral
5517 1581
3.13 0.23
5.26 0.55
0.60 0.17
5.77 2.97
25c
0.66 0.24
38.4 19.9
a
b
c
Values shown are mean SD of data from 3 rats.
Delivered in polyethylene glycol:DMA (1/1).
Delivered in 0.5% methylcellulose in distilled water.
(B)
(A)
800
600
400
200
0
Phosphorylated ERK1/2 level
vehicle 5c, 25 mg/kg
: vehicle
: 5c, (25 mg/kg, qd)
**
T/C = 70 %
10
15
20
25
30
Days after inoculation
Figure 6. In vivo studies of 5c (25 mg/kg, qd) in a BRAF(V600E) bearing HT-29 human colorectal cancer xenograft models in F344 nude rats. (A) The levels of phosphorylated
ERK1/2 were reduced in tumor tissues (n = 2) in 4 h after treatment with single dose of 25 mg/kg of 5c. (B) Antitumor efficacy of 5c was determined (n = 3) at a dose of 25 mg/
kg/day for consecutive 14 days. Data are plotted as mean tumor volume (in mm3) standard error of the mean, ⁄⁄P 60.01 versus control group at day 14 (t-test).
administration of 5c at a dose of 25 mg/kg in a conventional 0.5%
methylcellulose suspension. Reflecting the good oral PK profile
based on improved solubility, compound 5c significantly sup-
pressed the phosphorylation of ERK on the basis of BRAF inhibition
in tumor tissues (Fig. 6A). Repetitive administration of 5c at the
same dose (25 mg/kg, qd) for 2 weeks showed tumor regression
with T/C value of ꢀ70% without lethal toxicity (Fig. 6B). Because
compound 5 showed a greater inhibition of VEGFR2 than that of

5608
M. Hirose et al. / Bioorg. Med. Chem. 20 (2012) 5600–5615
BRAF, we supposed that this strong antitumor efficacy would be
achieved by the strong inhibition against both BRAF and VEGFR2.
5.2. N-(3-{[2-(Acetylamino)[1,3]thiazolo[5,4-d]pyrimidin-5-yl]
amino}-4-methylphenyl)-3-(2-cyanoethyl) benzamide (2a)
To a mixture of 3-(2-cyanoethyl)benzoic acid 17a (74 mg,
0.420 mmol) and HATU (160 mg, 0.420 mmol) in pyridine (4 mL)
was added 13 (120 mg, 0.38 mmol) at room temperature. The mix-
ture was stirred at room temperature for 1 h. It was then parti-
tioned between EtOAc (20 mL) and aqueous NaHCO3 solution
(15 mL). The aqueous layer was extracted with EtOAc (5 mL). The
combined organic layer was washed with brine (5 mL), dried over
anhydrous Na2SO4 and concentrated in vacuo. The resulting resi-
due was purified with basic silica gel column chromatography
(12 g, 70–100% EtOAc in n-hexane) to give 2a (117 mg, 65%) as pale
yellow amorphous solid: 1H NMR (300 MHz, DMSO-d6) d 2.19 (3H,
s), 2.19 (3H, s), 2.83–2.92 (2H, m), 2.94–3.04 (2H, m), 7.20 (1H, d,
J = 8.7 Hz), 7.44–7.56 (3H, m), 7.79–7.88 (2H, m), 7.92 (1H, d,
J=2.1 Hz), 8.75 (1H, s), 9.00 (1H, br s), 10.17 (1H, br s), 12.35 (1H,
br s). HRMS (ESI): Calcd for C24H21N7O2S [M+H]+ 472.1550. Found:
472.1521.
4. Conclusion
We performed structure-guided drug design to determine DFG-
out type RAF/VEGFR2 inhibitors that possess potent activity to-
gether with sufficient oral PK profiles. The binding mode analysis
of N-methyl [1,3]thiazolo[5,4-b]pyridine derivative 5 validated by
BRAF X-ray co-crystal structure revealed that the novel 2-chloro-
3-(1-cyano-1-methylethoxy)benzene moiety (ring C) occupied
the hydrophobic back pocket of BRAF. In addition, we found that
N-methylation of the aniline moiety for twisting the conformation
between fused ring A and ring B contributed not only to improving
the solubility but also to enhancing the VEGFR2 inhibitory activity
of 5. On the basis of this effect, we characterized compound 5 as a
VEGFR2-dominant RAF/VEGFR2 inhibitor. Salt formation of free
base 5 with besylate monohydrate (5c) was identified by single
X-ray crystallographic analysis. Compound 5c showed better ther-
modynamic solubility and oral PK profile in mice than that of its
free base 5 and other salts 5a,b.
Pharmacokinetic evaluation of 5c in rats showed significant oral
absorption at a dose of 25 mg/kg. In a HT-29 human colorectal can-
cer xenograft model in nude rats, single administration of com-
pound 5c showed significant suppression of the phosphorylated
ERK levels at a dose of 25 mg/kg. In the similar xenografted model,
5c demonstrated tumor regression with a T/C value of ꢀ70% by
repetitive oral administration at a dose of 25 mg/kg, qd without
lethal toxicity. We supposed that this potent antitumor efficacy
of 5c was achieved by the strong inhibitory activity against both
BRAF and VEGFR2 on the basis of in vitro pharmacological profiles.
Thus, compound 5c was selected as a promising candidate of RAF/
VEGFR2 inhibitor.
5.3. N-(3-{[2-(Acetylamino)[1,3]thiazolo[5,4-d]pyrimidin-5-yl]
amino}-4-methylphenyl)-3-(2-cyano-2-methyl
propyl)benzamide (2b)
Compound 2b (108 mg) was prepared from 13 (120 mg,
0.382 mmol), 3-(2-cyano-2-methylpropyl)benzoic acid 17b
(86 mg, 0.420 mmol) and HATU (160 mg, 0.420 mmol) by the
method similar to that described for 2a. Yield: 56%; colorless crys-
tals; recrystallized from MeOH; mp 289–290 °C. 1H NMR
(300 MHz, DMSO-d6) d 1.33 (6H, s), 2.19 (3H, s), 2.19 (3H, s), 2.94
(2H, s), 7.20 (1H, d, J = 8.5 Hz), 7.46–7.57 (3H, m), 7.83 (1H, s),
7.85–7.91 (1H, m), 7.92 (1H, d, J = 2.3 Hz), 8.74 (1H, s), 8.99 (1H,
br s), 10.19 (1H, br s), 12.35 (1H, br s). HRMS (ESI): Calcd for
C
26H25N7O2S [M+H]+ 500.1863. Found: 500.1836.
5. Experimental section
5.4. N-(3-{[2-(Acetylamino)[1,3]thiazolo[5,4-d]pyrimidin-5-yl]
amino}-4-methylphenyl)-3-(1-cyano-1-
5.1. General chemistry information
methylethoxy)benzamide (2c)
The starting materials, reagents, and solvents for reactions were
reagent-grade and were used as purchased. Thin-layer chromatog-
raphy (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 sil-
ica gel (Merck, 70–230 mesh) or basic silica gel (Fuji Silysia Chem-
ical 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
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 (d) 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 sin-
glet), or m (multiplet). MS spectra were collected with a Waters
LC–MS system (ZMD-1) and were used to confirm P95% purity
of each compound. The column used was an L-column 2 ODS
(3.0 ꢂ 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 spec-
troscopy (HRMS) were carried out at Takeda Analytical Laborato-
ries, Ltd Yields were not optimized.
Compound 2c (61 mg) was prepared from 13 (83 mg,
0.265 mmol), 3-(1-cyano-1-methylethoxy)benzoic acid 21a
(85 mg, 0.345 mmol) and HATU (120 mg, 0.318 mmol) by the
method similar to that described for 2a. Yield: 46%; colorless crys-
tals; recrystallized from THF/n-hexane; mp 276–277 °C. 1H NMR
(300 MHz, DMSO-d6) d 1.74 (6H, s), 2.19 (3H, s), 2.19 (3H, s), 7.20
(1H, d, J = 8.4 Hz), 7.40 (1H, ddd, J = 8.0, 2.3, 1.1 Hz), 7.51 (1H, dd,
J = 8.4, 2.1 Hz), 7.56 (1H, t, J = 8.0 Hz), 7.70 (1H, t, J = 2.3 Hz), 7.79
(1H, dt, J = 8.0, 1.1 Hz), 7.92 (1H, d, J = 2.1 Hz), 8.74 (1H, s), 9.00
(1H, br s), 10.24 (1H, br s), 12.35 (1H, br s). HRMS (ESI): Calcd for
C
25H23N7O3S [M+H]+ 502.1656. Found: 502.1623.
5.5. N-(3-{[2-(Acetylamino)[1,3]thiazolo[5,4-d]pyrimidin-5-yl]
amino}-4-methylphenyl)-2-chloro-3-(1-
cyanocyclopropyl)benzamide (2d)
Compound 2d (121 mg) was prepared from 13 (120 mg,
0.382 mmol),
2-chloro-3-(1-cyanocyclopropyl)benzoic
acid
(110 mg, 0.500 mmol) and HATU (189 mg, 0.500 mmol) by the
method similar to that described for 2a. Yield: 61%; off-white crys-
tals; recrystallized from EtOAc; mp 264–266 °C. 1H NMR
(300 MHz, DMSO-d6) d 1.40–1.51 (2H, m), 1.74–1.86 (2H. m),
2.18 (3H, s), 2.19 (3H, s), 7.20 (1H, d, J = 8.3 Hz), 7.41–7.53 (2H,
m), 7.59 (1H, dd, J = 7.8, 1.8 Hz), 7.64 (1H, dd, J = 7.6, 1.8 Hz), 7.90
(1H, d, J = 2.1 Hz), 8.73 (1H, s), 8.98 (1H, br s), 10.50 (1H, br s),
12.35 (1H, br s). HRMS (ESI): Calcd for C25H20ClN7O2S [M+H]+
518.1160. Found: 518.1125.

M. Hirose et al. / Bioorg. Med. Chem. 20 (2012) 5600–5615
5609
5.6. N-{5-[[2-(Acetylamino)[1,3]thiazolo[5,4-b]pyridin-5-yl]
(methyl)amino]-2-fluorophenyl}-3-(1-cyano-1-
methylethoxy)benzamide (3)
7.54–7.62 (1H, m), 7.79 (1H, d, J = 9.0 Hz), 7.85 (1H, dd, J = 6.9,
2.7 Hz), 10.51 (1H, s), 12.48 (1H, s). Anal. Calcd for
C28H25Cl2FN6O3S: C, 54.64; H, 4.09; N, 13.65; Cl, 11.52. Found: C,
54.66; H, 3.98; N, 13.63; Cl, 11.32. Powder X-ray diffraction
(Cu-Ka radiation, diffraction angle: 2h(°)): 4.08, 14.06, 16.22,
18.22, 18.60, 21.06, 23.08, 23.54, 23.92, 24.06, 25.34.
Compound
3
(61 mg) was prepared from 27 (80 mg,
0.241 mmol), 21a (99 mg, 0.482 mmol) and HATU (183 mg,
0.482 mmol) by the method similar to that described for 2a. Yield:
48%; off-white crystals; recrystallized from THF/n-hexane; mp
203–204 °C. 1H NMR (300 MHz, DMSO-d6) d 1.74 (6H, s), 2.17
(3H, s), 3.43 (3H, s), 6.63 (1H, d, J = 8.9 Hz), 7.24 (1H, ddd, J = 8.9,
4.5, 2.7 Hz), 7.39 (1H, dd, J = 10.2, 8.9 Hz), 7.43 (1H, ddd, J = 8.0,
2.5, 0.9 Hz), 7.57 (1H, t, J = 8.0 Hz), 7.60 (1H, dd, J = 6.9, 2.7 Hz),
7.73 (1H, t, J = 1.9 Hz), 7.75–7.85 (2H, m), 10.26 (1H, br s), 12.16
(1H, br s). Anal. Calcd for C26H23FN6O3S: C, 60.22; H, 4.47; N,
16.21. Found: C, 60.18; H, 4.53; N, 16.16. HRMS (ESI): Calcd for
5.10. 2-Chloro-3-(1-cyano-1-methylethoxy)-N-{5-[{2-[(cyclopropyl-
carbonyl)amino][1,3]thiazolo[5,4-b]pyridin-5-yl}(methyl)amino]-
2-fluorophenyl}benzamide sulfate (5b)
To a mixture of sulfuric acid (26.5 lL, 0.518 mmol) in n-heptane
(6 mL) was added a solution of 5 (150 mg, 0.259 mmol) in EtOAc
(6 mL). The mixture was diluted with EtOAc (12 mL) and stirred
at 50 °C for 30 min. It was then evaporated under reduced pres-
sure. The residue was recrystallized from THF/n-heptane (1:1,
20 mL). The precipitate was collected by filtration to give 5b
(140 mg, 80%) as pale yellow crystals. 1H NMR (300 MHz, DMSO-
d6) d 0.74–1.06 (4H, m), 1.79 (6H, s), 1.92–2.09 (1H, m), 3.43 (3H,
s), 6.64 (1H, d, J = 8.9 Hz), 7.16–7.24 (1H, m), 7.33–7.65 (4H, m),
7.79 (1H, d, J = 8.9 Hz), 7.85 (1H, dd, J = 6.9, 2.5 Hz), 10.51 (1H, s),
12.47 (1H, s). Anal. Calcd for C28H24ClFN6O3S+1.0H2SO4: C, 49.67;
H, 3.87; N, 12.41. Found: C, 49.27; H, 3.91; N, 12.18. Powder X-
ray diffraction (Cu-Ka radiation, diffraction angle: 2h(°)): 3.72,
11.38, 13.64, 18.14, 18.80, 19.04, 23.00.
C
26H23FN6O3S [M+H]+ 519.1609. Found: 519.1594.
5.7. N-{5-[[2-(Acetylamino)[1,3]thiazolo[5,4-b]pyridin-5-yl]
(methyl)amino]-2-fluorophenyl}-2-chloro-3-(1-cyano-1-
methylethoxy)benzamide (4)
Compound
4
(78 mg) was prepared from 27 (80 mg,
0.241 mmol), 21b (133 mg, 0.554 mmol) and HATU (211 mg,
0.554 mmol) by the method similar to that described for 2a. Yield:
59%; off-white crystals; recrystallized from EtOH; mp 181–182 °C.
1H NMR (300 MHz, DMSO-d6) d 1.79 (6H, s), 2.17 (3H, s), 3.43 (3H,
s), 6.64 (1H, d, J = 9.1 Hz), 7.21 (1H, ddd, J = 8.7, 4.0, 2.7 Hz), 7.34–
7.42 (2H, m), 7.50 (1H, t, J = 8.0 Hz), 7.58 (1H, dd, J = 8.0, 1.6 Hz),
7.79 (1H, d, J = 9.1 Hz), 7.85 (1H, dd, J = 6.9, 2.7 Hz), 10.50 (1H, br
s), 12.17 (1H, br s). Anal. Calcd for C26H22ClFN6O3S+0.5H2O: C,
55.56; H, 4.12; N, 14.95. Found: C, 55.76; H, 4.04; N, 14.91.
5.11. 2-Chloro-3-(1-cyano-1-methylethoxy)-N-{5-[{2-[(cyclopropyl-
carbonyl)amino][1,3]thiazolo[5,4-b]pyridin-5-yl}(methyl)amino]-
2-fluorophenyl}benzamide besylate hydrate (5c)
To a solution of 5 (4.17 g, 7.20 mmol) in EtOAc (55 mL) was
added a solution of benzenesulfonic acid (90%, 1.52 g, 8.64 mmol)
in EtOAc (5 mL) at 55 °C. It was then cooled and evaporated under
reduced pressure. The residue was crystallized from THF/n-hep-
tane (1:1, 100 mL) to give crude besylate salt (5.02 g). The resulting
crude salt was recrystallized from a mixed solvent of acetone
(115 mL) and n-heptane (100 mL), and the resulting precipitate
was collected by filtration to give 5c (4.59 g, 84%) as colorless crys-
tals. 1H NMR (300 MHz, DMSO-d6) d 0.73–1.08 (4H, m), 1.79 (6H, s),
1.90–2.08 (1H, m), 3.43 (3H, s), 6.64 (1H, d, J = 9.0 Hz), 7.13–7.25
(1H, m), 7.25–7.45 (5H, m), 7.50 (1H, t, J = 7.8 Hz), 7.54–7.64 (3H,
m), 7.79 (1H, d, J = 9.0 Hz), 7.85 (1H, dd, J = 7.0, 2.6 Hz), 10.50
(1H, s), 12.47 (1H, s). Anal. Calcd for C34H30ClF N6O6S2+1.0H2O: C,
54.07; H, 4.27; N, 11.13. Found: C, 54.03; H, 4.31; N, 11.08. Powder
X-ray diffraction (Cu-Ka radiation, diffraction angle: 2h(°)): 4.08,
8.28, 12.50, 16.54, 21.00, 23.36, 24.62.
5.8. 2-Chloro-3-(1-cyano-1-methylethoxy)-N-{5-[{2-[(cyclopropyl-
carbonyl)amino][1,3]thiazolo[5,4-b]pyridin-5-yl}(methyl)amino]-
2-fluorophenyl}benzamide (5)
Compound
5 (123 mg) was prepared from 28 (150 mg,
0.420 mmol), 2-chloro-3-(1-cyano-1-methylethoxy)benzoic acid
21b (201 mg, 0.840 mmol) and HATU (319 mg, 0.840 mmol) by
the method similar to that described for 2a. Yield: 50%; colorless
crystals; recrystallized from EtOAc; mp 178–179 °C. 1H NMR
(300 MHz, DMSO-d6) d 0.89–0.99 (4H, m), 1.92–2.03 (1H, m),
3.43 (3H, s), 6.64 (1H, d, J = 8.9 Hz), 7.20 (1H, ddd, J = 8.9, 4.2,
2.8 Hz), 7.34–7.42 (2H, m), 7.50 (1H, t, J = 8.0 Hz), 7.58 (1H, dd,
J = 8.0, 1.5 Hz), 7.79 (1H, d, J = 8.9 Hz), 7.85 (1H, dd, J = 7.0,
2.8 Hz), 10.50 (1H, br s), 12.46 (1H, br s). Anal. Calcd for
C
28H24ClFN6O3S: C, 58.08; H, 4.06; N, 14.51. Found: C, 57.81; H,
4.06; N, 14.45. HRMS (ESI): Calcd for C28H24ClFN6O3S [M + H]+
579.1376. Found: 579.1337. Powder X-ray diffraction (Cu-Ka radi-
ation, diffraction angle: 2h(°)): 2.78, 8.46, 10.90, 11.34, 12.64,
13.66, 14.22, 22.94.
5.12. 2-Chloro-3-(1-cyano-1-methylethoxy)-N-[5-({2-[(cyclopropyl-
carbonyl)amino][1,3]thiazolo[5,4-b]pyridin-5-yl}amino)-2-fluoro-
phenyl]benzamide (6)
To a solution of 21b (278 mg, 1.16 mmol) in THF (2.5 mL) were
5.9. 2-Chloro-3-(1-cyano-1-methylethoxy)-N-{5-[{2-
[(cyclopropylcarbonyl)amino][1,3]thiazolo[5,4-b]pyridin-5-
yl}(methyl)amino]-2-fluorophenyl}benzamide hydrochloride
(5a)
added DMF (25 lL) and oxalyl chloride (125 lL, 1.46 mmol), and
the mixture was stirred at room temperature for 1 h. The reaction
mixture was concentrated under reduced pressure to give
2-chloro-3-(1-cyano-1-methylethoxy)benzoyl chloride as pale yel-
low oil. To a solution of 2-chloro-3-(1-cyano-1-methylethoxy)ben-
zoyl chloride in DMA (4.0 mL) was added 33 (200 mg, 0.582 mmol),
and the mixture was stirred at room temperature for 2 h. To the
reaction mixture was added saturated aqueous NaHCO3 (20 mL),
and the mixture was extracted with EtOAc (50 mL). The organic
layer was washed with water (20 mL) and brine (20 mL), and dried
over anhydrous Na2SO4, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
(20–100% EtOAc in n-hexane) and crystallized from n-hexane/
EtOAc (1:1) to give 6 (216 mg, 66%) as pale purple crystals: mp
To a solution of 5 (100 mg, 0.173 mmol) in a mixed solvent of
EtOAc (8 mL) and EtOH (20 mL) was added 4 N HCl/EtOAc (88 lL,
0.352 mmol) at 50 °C. The mixture was stirred at room tempera-
ture for 30 min. It was then evaporated under reduced pressure.
The residue was triturated with EtOH for 2 h, and the precipitate
was collected by filtration to give 5a (78.8 mg, 74%) as colorless
crystals. 1H NMR (300 MHz, DMSO-d6) d 0.77–1.05 (4H, m), 1.79
(6H, s), 1.89–2.07 (1H, m), 3.43 (3H, s), 6.64 (1H, d, J = 9.0 Hz),
7.12–7.30 (1H, m), 7.32–7.44 (2H, m), 7.50 (1H, t, J = 7.8 Hz),

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M. Hirose et al. / Bioorg. Med. Chem. 20 (2012) 5600–5615
226–228 °C. 1H NMR (DMSO-d6) d 0.86–1.00 (4H, m), 1.80 (6H, s),
1.91–2.04 (1H, m), 6.92 (1H, d, J = 8.9 Hz), 7.20 (1H, t, J = 10.2,
9.3 Hz), 7.39 (1H, dd, J = 7.0, 1.5 Hz), 7.46–7.63 (2H, m), 7.63–
7.73 (1H, m), 7.90 (1H, d, J = 8.9 Hz), 8.16 (1H, dd, J = 7.0, 2.7 Hz),
9.40 (1H, s), 10.35 (1H, s), 12.47 (1H, br s). Anal. Calcd for
temperature for 1 h. It was then partitioned between EtOAc
(50 mL) and aqueous NaHCO3 solution (50 mL). The aqueous layer
was extracted with EtOAc (10 mL). The combined organic layer
was washed with brine (10 mL), and passed through a pad of basic
silica gel (10 g). The filtrate was concentrated and recrystallized
from EtOAc to give tert-butyl (3-{[2-(acetylamino)[1,3]thiazol-
C
27H22ClFN6O3S: C, 57.39, H, 3.92, N, 14.87. Found: C, 57.34; H,
3.99; N, 14.69. HRMS (ESI): Calcd for C27H22ClFN6O3S [M+H]+
565.1219. Found: 565.1198.
o[5,4-d]pyrimidin-5-yl]amino}-4-methylphenyl)
carbamate
(1.31 g, 98%) as pale purple solid; mp 218–219 °C.1H NMR
(300 MHz, DMSO-d6) d 1.46 (9H, s), 2.12 (3H, s), 2.18 (3H, s), 7.07
(1H, d, J = 8.4 Hz), 7.18 (1H, dd, J = 8.4, 2.1 Hz), 7.60 (1H, d,
J = 2.1 Hz), 8.71 (1H, s), 8.88 (1H, br s), 9.23 (1H, br s), 12.33 (1H,
br s).
5.13. tert-Butyl (4-methyl-3-nitrophenyl)carbamate (8)
To a solution of 4-methyl-3-nitroaniline 7 (15.0 g, 98.6 mmol)
in THF (40 mL) was added dropwise Boc2O (25.8 g, 118 mmol) in
THF (20 mL) over 30 min at 70 °C. The reaction mixture was stirred
at 70 °C for 15 h and concentrated under reduced pressure. The
resulting residue was recrystallized with EtOAc/n-hexane to give
compound 8 (22.3 g, 89%) as pale yellow crystals; mp 86–87 °C.
1H NMR (300 MHz, DMSO-d6) d 1.48 (9H, s), 2.43 (3H, s), 7.38
(1H, d, J = 8.6 Hz), 7.58 (1H, dd, J = 8.6, 2.3 Hz), 8.22 (1H, d,
J = 2.3 Hz), 9.76 (1H, br s). Anal. Calcd for C12H16N2O4: C, 57.13;
H, 6.39; N, 11.10. Found: C, 57.31; H, 6.46; N, 11.22.
A mixture of tert-butyl (3-{[2-(acetylamino)[1,3]thiazolo[5,4-
d]pyrimidin-5-yl]amino}-4-methylphenyl) carbamate (1.27 g) and
anisole (2 mL) in TFA (20 mL) was stirred at 0 °C for 1 h. It was then
concentrated, and the residue was partitioned a mixed solvent of
EtOAc/THF (1:1, 50 mL) and aqueous NaHCO3 solution (50 mL).
The aqueous layer was extracted with a mixed solvent of EtOAc/
THF (1:1, 2 ꢂ 10 mL). The combined organic layer was washed
with brine (5 mL), dried over anhydrous Na2SO4 and concentrated
in vacuo to give 13 (795 mg, 83%) as purple solid; mp 257–258 °C.
1H NMR (300 MHz, DMSO-d6) d 2.03 (3H, s), 2.18 (3H, s), 4.83 (2H,
br s), 6.31 (1H, dd, J = 8.2, 2.4 Hz), 6.76 (1H, d, J = 2.4 Hz), 6.84 (1H,
d, J = 8.2 Hz), 8.67 (1H, br s), 8.70 (1H, s), 12.31 (1H, br s).
5.14. tert-Butyl {3-[(2-amino[1,3]thiazolo[5,4-d]pyrimidin-5-
yl)amino]-4-methylphenyl}carbamate (12)
To a solution of 8 (10.1 g, 39.8 mmol) in EtOH (60 mL) and THF
(20 mL) was added 10% Pd/C (2.12 g). The mixture was stirred at
room temperature for 22 h under H2 atmosphere (3 atm). The mix-
ture was diluted with EtOAc (80 mL) and passed through a pad of
Celite. The filtrate was concentrated in vacuo to give tert-butyl (3-
amino-4-methylphenyl) carbamate 9 as colorless solid (8.82 g).
This material was used for the next reaction without further puri-
fication. 1H NMR (300 MHz, DMSO-d6) d 1.44 (9H, s), 1.95 (3H, s),
4.72 (2H, br s), 6.48 (1H, d, J = 7.9 Hz), 6.73 (1H, d, J = 7.9 Hz),
6.82 (1H, s), 8.90 (1H, br s).
To a solution of 9 in THF (200 mL) were added i-Pr2NEt
(10.4 mL, 59.8 mmol) and 2-chloro-5-nitropyrimidin-4-yl thiocya-
nate 10 (9.49 g, 43.8 mmol) at 10 °C. The mixture was stirred at
room temperature for 30 min. The mixture was partitioned be-
tween EtOAc (150 mL) and aqueous NaHCO3 solution (200 mL).
The aqueous layer was extracted with EtOAc (2 ꢂ 30 mL). The com-
bined organic layer was washed with brine (20 mL), dried over
anhydrous Na2SO4 and concentrated in vacuo to give tert-butyl
{4-methyl-3-[(5-nitro-4-thiocyanatopyrimidin-2-
5.16. Methyl 3-(2-cyanoethyl)benzoate (15)
To
a suspension of methyl 2-formylbenzoate 14 (2.00 g,
12.2 mmol) and K2CO3 (2.02 g, 14.6 mmol) in THF (20 mL)/water
(0.4 mL) was added diethyl (cyanomethyl)phosphonate (2.29 mL,
14.6 mmol). The reaction mixture was stirred at 60 °C for 1 h,
and then partitioned between EtOAc (50 mL) and water (50 mL).
The separated aqueous layer was extracted with EtOAc (20 mL).
The combined organic layers were washed with brine (10 mL),
dried over Na2SO4 and concentrated under reduced pressure to
give methyl 3-(2-cyanoethenyl)benzoate as colorless solid (ca.
5:1 E/Z mixture). This material was used for the next reaction with-
out further purification.
A mixture of methyl 3-(2-cyanoethenyl)benzoate and 10% Pd/C
(648 mg, 0.609 mmol, 648 mg) in EtOH/THF (3:1, 60 mL) was stir-
red at room temperature under H2 atmosphere (1 atm) for 2 h. The
mixture was passed through Celite pad and the filtrate was con-
centrated under reduced pressure. The residue was purified by sil-
ica gel chromatography (30 g, 10–25% EtOAc in n-hexane) to give
compound 15 as colorless oil (2.03 g, 88% in two steps). 1H NMR
(300 MHz, DMSO-d6) d 2.85 (2H, dt, J = 6.9, 1.2 Hz), 2.93–3.00
(2H, m), 3.86 (3H, s), 7.49 (1H, t, J = 7.6 Hz), 7.60 (1H, dt, J = 7.6,
1.5 Hz), 7.86 (1H, dt, J = 7.6, 1.5 Hz), 7.91 (1H, t, J = 1.5 Hz).
yl)amino]phenyl}carbamate 11 as gray solid.
To a mixture of 11 and reduced iron (6.68 g, 120 mmol) in EtOH
(120 mL) and NMP (80 mL) was added a solution of calcium
chloride (13.3 g, 120 mmol) in water (20 mL). The mixture was
stirred at 100 °C for 16 h. It was the cooled, and passed through a
pad of Celite. The insoluble was washed with EtOAc (200 mL).
The filtrate and washings were combined and evaporated. The res-
idue was partitioned between EtOAc (200 mL) and water (350 mL).
The aqueous layer was extracted with EtOAc (2 ꢂ 80 mL). The com-
bined organic layer was washed with brine (50 mL) and passed
through a pad of silica gel (50 g). The filtrate was concentrated to
give 12 (5.24 g, 35% in three steps) as gray solid; mp 212–213 °C.
1H NMR (300 MHz, DMSO-d6) d 1.46 (9H, s), 2.11 (3H, s), 7.04
(1H, d, J = 7.8 Hz), 7.14 (1H, d, J = 7.8 Hz), 7.43–7.77 (3H, m), 8.26
(1H, s), 8.48 (1H, br s), 9.20 (1H, br s).
5.17. Methyl 3-(2-cyano-2-methylpropyl)benzoate (16)
To a solution of methyl 3-(2-cyanoethyl)benzoate 15 (510 mg,
2.70 mmol) and iodomethane (0.671 mL, 10.8 mmol) in THF
(15 mL) was added dropwise a solution of LHMDS (1.1 M in THF,
7.35 mL, 8.09 mmol) in THF (10 mL) at –78 °C over 30 min. The
mixture was stirred at –78 °C for 1 h, and then poured into a mix-
ture of EtOAc (50 mL) and aqueous NH4Cl (50 mL). The mixture
was partitioned and the separated organic layer was washed with
brine (10 mL), dried over anhydrous Na2SO4 and concentrated un-
der reduced pressure. The residue was purified by silica gel chro-
matography (12 g, 5–15% EtOAc in n-hexane) to give 16 as
yellow oil (260 mg, 44%). 1H NMR (300 MHz, DMSO-d6) d 1.30
(6H, s), 2.94 (2H, s), 3.86 (3H, s), 7.52 (1H, t, J = 7.8 Hz), 7.58 (1H,
dt, J = 7.8, 1.6 Hz), 7.87–7.93 (2H, m).
5.15. N-{5-[(5-Amino-2-methylphenyl)amino][1,3]thiazolo[5,4-
d]pyrimidin-2-yl}acetamide (13)
To a solution of 12 (1.20 g, 3.22 mmol) in pyridine (20 mL) was
added acetyl chloride at 0 °C. The mixture was stirred at room

M. Hirose et al. / Bioorg. Med. Chem. 20 (2012) 5600–5615
5611
5.18. 3-(2-Cyanoethyl)benzoic acid (17a)
at 0 °C over 1 h. The reaction mixture was stirred at room temper-
ature for 30 min, and then poured into a mixture of EtOAc (150 mL)
and aqueous NH4Cl (150 mL). The separated aqueous layer was ex-
tracted with EtOAc (50 mL). The combined organic layers were
washed with brine (30 mL), dried over MgSO4 and concentrated
under reduced pressure. The residue was dissolved in EtOAc
(100 mL) and passed through a pad of silica gel (100 g, EtOAc:
250 mL). The filtrate was concentrated under reduced pressure
and the residue was purified by basic silica gel column chromatog-
raphy (400 g, 0–15% EtOAc in n-hexane) to give 20b as pale yellow
oil (8.55 g, 50%). 1H NMR (300 MHz, DMSO-d6) d 1.70 (6H, s), 3.80
(3H, s), 7.44 (1H, t, J = 7.9 Hz), 7.51 (1H, dd, J = 7.9, 1.8 Hz), 7.59
(1H, dd, J = 7.9, 1.8 Hz).
To a solution of 15 (169 mg, 0.893 mmol) in MeOH/THF (3:1,
4 mL) was added 2 N NaOH (0.893 mL, 1.79 mmol). The reaction
mixture was stirred at 60 °C for 4 h, and then acidified with 6 N
hydrochloric acid (0.5 mL). The mixture was partitioned between
EtOAc (20 mL) and 1 N hydrochloric acid (15 mL). The separated
aqueous layer was extracted with EtOAc (5 mL). The combined or-
ganic layers were washed with brine (5 mL), dried over anhydrous
Na2SO4 and concentrated under reduced pressure to give com-
pound 17a (151 mg, 96%) as colorless crystals; mp 153–154 °C.
1H NMR (300 MHz, DMSO-d6) d 2.80–2.89 (2H, m), 2.91–3.00
(2H, m), 7.46 (1H, t, J = 7.6 Hz), 7.56 (1H, dt, J = 7.6, 1.5 Hz), 7.83
(1H, dt, J = 7.6, 1.5 Hz), 7.89 (1H, t, J = 1.5 Hz), 12.94 (1H, br s).
5.24. 3-(1-Cyano-1-methylethoxy)benzoic acid (21a)
5.19. 3-(2-Cyano-2-methylpropyl)benzoic acid (17b)
Compound 21a (1.01 g) was prepared from 20a (2.07 g,
9.44 mmol) and 2 N NaOH (9.44 mL, 18.9 mmol) by the method
similar to that described for 17a. Yield, 51%, colorless crystals;
crystallized from EtOAc/n-hexane; mp 93–94 °C. 1H NMR
(300 MHz, DMSO-d6) d 1.71 (6H, s), 7.42 (1H, ddd, J = 8.0, 2.4,
1.2 Hz), 7.53 (1H, t, J = 8.0 Hz), 7.69–7.73 (1H, m), 7.77 (1H, dt,
J = 8.0, 1.2 Hz), 13.10 (1H, br s).
Compound 17b (230 mg) was prepared from 16 (260 mg,
1.20 mmol) and 2 N NaOH (1.20 mL, 2.39 mmol) by the method
similar to that described for 17a. Yield: 95%; colorless crystals;
crystallized from EtOAc; mp 144–145 °C. 1H NMR (300 MHz,
DMSO-d6) d 1.31 (6H, s), 2.93 (2H, s), 7.43–7.58 (2H, m), 7.81–
7.92 (2H, m), 12.98 (1H, br s).
5.20. Methyl 3-(cyanomethoxy)benzoate (19a)
5.25. 2-Chloro-3-(1-cyano-1-methylethoxy)benzoic acid (21b)
To a suspension of methyl 2-hydroxybenzoate 18a (5.00 g,
32.9 mmol) and K2CO3 (6.81 g, 49.3 mmol) in acetone (60 mL)
was added bromoacetonitrile (2.63 mL, 39.4 mmol). The reaction
mixture was stirred at 60 °C for 4 h, and then partitioned between
EtOAc (100 mL) and aqueous NaHCO3 (100 mL). The separated
aqueous layer was extracted with EtOAc (30 mL). The combined or-
ganic layers were washed with brine (10 mL), dried over anhy-
drous MgSO4 and concentrated under reduced pressure. The
residue was purified by basic silica gel chromatography (100 g,
10–20% EtOAc in n-hexane) to give compound 19a as colorless so-
lid (5.43 g, 86%). 1H NMR (300 MHz, DMSO-d6) d 3.87 (3H, s), 5.27
(2H, s), 7.37 (1H, ddd, J = 8.1, 2.6, 1.3 Hz), 7.54 (1H, t, J = 8.1 Hz),
7.59 (1H, dd, J = 2.6, 1.3 Hz), 7.68 (1H, dt, J = 8.1, 1.3 Hz).
To a solution of 20b (19.0 g, 74.7 mmol) in i-PrOH (250 mL) was
added 2 N aqueous NaOH (44.8 mL, 89.7 mmol). The reaction mix-
ture was stirred at room temperature for 2 h, and then concen-
trated under reduced pressure. The residue was dissolved in
EtOAc/water (5:1, 360 mL) and acidified with 1 N hydrochloric acid
(90 mL). The separated aqueous layer was extracted with EtOAc
(50 mL). The combined organic layers were washed with brine
(30 mL), dried over anhydrous MgSO4 and concentrated under re-
duced pressure. The residue was crystallized with EtOH/water
(35 mL/60 mL) to give 21b (13.9 g, 77%) as colorless crystals; mp
131–133 °C. 1H NMR (300 MHz, DMSO-d6) d 1.77 (6H, s), 7.47
(1H, t, J = 7.9 Hz), 7.55 (1H, dd, J = 7.9, 1.9 Hz), 7.61 (1H, dd,
J = 7.9, 1.9 Hz), 13.55 (1H, br s).
5.21. Methyl 2-chloro-3-(cyanomethoxy)benzoate (19b)
5.26. tert-Butyl (5-amino-2-fluorophenyl)carbamate (23)
Compound 19b (57.8 g) was prepared from methyl 2-chloro-3-
hydroxybenzoate 18b (52.0 g, 279 mmol), K2CO3 (57.9 g,
419 mmol), sodium iodide (62.8 g, 419 mmol), and chloroacetonit-
rile (19.5 mL, 307 mmol) by the method similar to that described
for 19a. Yield: 92%; colorless amorphous solid. 1H NMR
(300 MHz, DMSO-d6) d 3.87 (3H, s), 5.34 (2H, s), 7.43–7.52 (3H, m).
A mixture of 2-fluoro-5-nitroaniline 22 (28.7 g, 184 mmol) and
Boc2O (100 g, 460 mmol) was stirred at 80 °C for 24 h. After cool-
ing, the reaction mixture was directly purified by basic silica gel
column chromatography (500 g, 0–10% EtOAc in n-hexane) to give
a mixture of tert-butyl (2-fluoro-5-nitrophenyl)carbamate and di-
tert-butyl (2-fluoro-5-nitrophenyl)imidodicarbonate as yellow oil.
This material was used for the next reaction without further
purification.
5.22. Methyl 3-(1-cyano-1-methylethoxy)benzoate (20a)
To a solution of the crude mixture in EtOH/THF (10:1, 660 mL)
was added 10% Pd/C (12.0 g, 11.2 mmol). The reaction mixture
was stirred at room temperature under H2 atmosphere (1 atm)
for 24 h, then passed through a pad of Celite and concentrated un-
der reduced pressure to give a mixture of 23 and di-tert-butyl (5-
amino-2-fluorophenyl)imidodicarbonate.
To a solution of the crude mixture in MeOH/THF (4:1, 250 mL)
was added K2CO3 (25.4 g, 184 mmol). The reaction mixture was
stirred at 60 °C for 4 h, then passed through a pad of Celite. The fil-
trate was concentrated under reduced pressure and the residue
was partitioned between EtOAc (300 mL) and water (200 mL).
The separated aqueous layer was extracted with EtOAc (100 mL).
The combined organic layers were washed with brine (100 mL),
dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The residue was crystallized with EtOAc/n-hexane to give
Compound 20a (2.07 g) was prepared from 19a (6.00 g,
31.4 mmol), iodomethane (15.6 mL, 251 mmol) and LHMDS
(1.1 M in THF, 62.8 mL, 69.0 mmol) by the method similar to that
described for 17. Yield: 30%; yellow oil. 1H NMR (300 MHz,
DMSO-d6) d 1.72 (6H, s), 3.87 (3H, s), 7.47 (1H, ddd, J = 8.0, 2.4,
1.2 Hz), 7.58 (1H, t, J = 8.0 Hz), 7.71–7.74 (1H, m), 7.80 (1H, dt,
J = 8.0, 1.2 Hz).
5.23. Methyl 2-chloro-3-(1-cyano-1-methylethoxy)benzoate
(20b)
To a solution of 19b (15.2 g, 67.4 mmol) and iodomethane
(12.6 mL, 202 mmol) in THF (200 mL) was added dropwise a solu-
tion of NaHMDS (1.9 M in THF, 78.0 mL, 148 mmol) in THF (50 mL)

5612
M. Hirose et al. / Bioorg. Med. Chem. 20 (2012) 5600–5615
23 (21 g) as beige crystals. The filtrate was concentrated under re-
duced pressure, and the residue was purified by basic silica gel col-
umn chromatography (200 g, 5–20% EtOAc in n-hexane) to give 23
(5.3 g) as yellow crystals; mp 94–95 °C. Combined yield: 63% in
three steps. 1H NMR (300 MHz, DMSO-d6) d 1.44 (9H, s), 4.90
(2H, br s), 6.23 (1H, ddd, J = 8.8, 4.0, 2.8 Hz), 6.80 (1H, dd,
J = 10.9, 8.8 Hz), 6.84 (1H, dd, J = 6.9, 2.8 Hz), 8.57 (1H, br s). Anal.
Calcd for C11H15 FN2O2: C, 58.40; H, 6.68; N, 12.38. Found: C,
58.32; H, 6.73; N, 12.44.
5.29. tert-Butyl {5-[(2-amino[1,3]thiazolo[5,4-b]pyridin-5-
yl)(methyl)amino]-2-fluorophenyl}carbamate (26)
To a solution of potassium thiocyanate (1.26 g, 13.0 mmol) in
AcOH (30 mL) was added 25 (1.08 g, 3.25 mmol). To the resulting
mixture was added dropwise a solution of bromine (545 mg,
3.41 mmol) in AcOH (5 mL) at 10 °C over 10 min, and the reaction
mixture was stirred at room temperature for 1 h. Additional
amount of bromine (260 mg, 1.62 mmol) was added and the mix-
ture was stirred for 30 min, and then passed through a pad of Celite
and concentrated under reduced pressure. The residue was parti-
tioned between EtOAc/THF (1:1, 100 mL) and aqueous NaHCO3
(50 mL). The separated aqueous layer was extracted with EtOAc
(20 mL). The combined organic layers were washed with brine
(10 mL), dried over anhydrous Na2SO4 and concentrated under re-
duced pressure. The residue was purified by basic silica gel column
chromatography (30 g, 60–80% EtOAc in n-hexane) to give 26
(613 mg, 48%) as red amorphous solid. 1H NMR (300 MHz,
DMSO-d6) d 1.44 (9H, s), 3.31 (3H, s), 6.44 (1H, d, J = 8.8 Hz), 6.97
(1H, ddd, J = 8.7, 4.2, 2.6 Hz), 7.21 (1H, dd, J = 10.6, 8.7 Hz), 7.30
(2H, br s), 7.40 (1H, d, J = 8.8 Hz), 7.52 (1H, dd, J = 7.1, 2.6 Hz),
9.04 (1H, br s).
5.27. tert-Butyl [2-fluoro-5-(methylamino)phenyl]carbamate
(24)
A mixture of Ac2O (5.01 mL, 53.0 mmol) and formic acid
(8.34 mL, 221 mmol) was stirred at room temperature for 30 min.
To a solution of 23 (10.0 g, 44.2 mmol) in THF (60 mL) was added
the abovementioned reaction mixture at 10 °C. The resulting mix-
ture was stirred at room temperature for 16 h. The resulting mix-
ture was partitioned between EtOAc (100 mL) and aqueous
saturated NaHCO3 (100 mL). The separated aqueous layer was ex-
tracted with EtOAc (30 mL). The combined organic layer was
washed with brine (10 mL), and then passed through a pad of silica
gel (100 g) using 50% EtOAc in n-hexane. The filtrate was concen-
trated in vacuo to dryness. The resulting residue was dissolved into
THF (100 mL). To the mixture was added borane–dimethyl sulfide
complex (11.7 mL, 111 mmol) at room temperature. The mixture
was stirred at room temperature for 1.5 h. Then, MeOH (20 mL)
and AcOH (10 mL) were added. The mixture was stirred at room
temperature for 30 min. The resulting mixture was concentrated
in vacuo, and the residue was partitioned between EtOAc
(100 mL) and aqueous NaHCO3 (100 mL). The separated aqueous
layer was extracted with EtOAc (30 mL). The combined organic
layer was washed with brine (20 mL), dried over anhydrous
Na2SO4 and concentrated in vacuo. The residue was purified with
silica gel column chromatography (200 g, 0–10% EtOAc in n-hex-
ane) to give 24 (7.80 g, 73%) as yellow oil. 1H NMR (300 MHz,
DMSO-d6) d 1.45 (9H, s), 2.61 (3H, d, J = 5.1 Hz), 5.52 (1H, q,
J = 5.1 Hz), 6.20 (1H, ddd, J = 8.8, 3.7, 2.8 Hz), 6.80 (1H, dd, J = 6.8,
2.8 Hz), 6.89 (1H, dd, J = 10.7, 8.8 Hz), 8.62 (1H, br s).
5.30. N-{5-[(3-Amino-4-fluorophenyl)(methyl)amino]-
[1,3]thiazolo[5,4-b]pyridin-2-yl}acetamide (27)
Compound 27 (546 mg) was prepared from 26 (740 mg,
1.90 mmol), acetyl chloride (0.339 mL, 4.75 mmol), pyridine
(20 mL) and TFA/anisole (20:1, 21 mL) by the method similar to
that described for 13. Yield: 86%; pale purple crystals; recrystal-
lized from EtOAc; mp 262–266 °C. 1H NMR (300 MHz, DMSO-d6)
d 2.17 (3H, s), 3.34 (3H, s), 5.28 (2H, br s), 6.40–6.47 (1H, m),
6.50 (1H, d, J = 9.1 Hz), 6.67 (1H, dd, J = 8.4, 2.6 Hz), 7.05 (1H, dd,
J = 11.3, 8.4 Hz), 7.72 (1H, d, J = 9.1 Hz), 12.12 (1H, br s). Anal. Calcd
for C15H14FN5OS: C, 54.37; H, 4.26; N, 21.13. Found: C, 54.47; H,
4.31; N, 20.98.
5.31. N-{5-[(3-Amino-4-fluorophenyl)(methyl)amino]-
[1,3]thiazolo[5,4-b]pyridin-2-yl}cyclopropane carboxamide
(28)
5.28. tert-Butyl {5-[(5-aminopyridin-2-yl)(methyl)amino]-2-
fluorophenyl}carbamate (25)
Compound 28 (55 mg) was prepared from 26 (80 mg,
0.21 mmol), cyclopropanecarbonyl chloride (24
lL, 0.27 mmol),
To a solution of 24 (2.13 g, 8.87 mmol) in DMSO (5.9 mL) was
added 2-chloro-5-nitropyridine (1.69 g, 10.6 mmol). The reaction
mixture was stirred at 60 °C for 19 h, and then partitioned between
EtOAc (50 mL) and aqueous NaHCO3 (50 mL). The separated aque-
ous layer was extracted with EtOAc (20 mL). The combined organic
layers were washed with brine (10 mL), dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The residue
was purified by basic silica gel column chromatography (100 g,
5–15% EtOAc in n-hexane) to give tert-butyl {2-fluoro-5-
pyridine (1.0 mL) and TFA/anisole (10:1, 2.2 mL) by the method
similar to that described for 13. Yield: 75%; colorless crystals;
recrystallized from EtOAc; mp 240–242 °C. 1H NMR (300 MHz,
DMSO-d6) d 0.90–0.97 (4H, m), 1.90–2.05 (1H, m), 3.33 (3H, s),
5.27 (2H, br s), 6.40-6.47 (1H, m), 6.51 (1H, d, J = 9.0 Hz), 6.67
(1H, dd, J = 8.2, 2.5 Hz), 7.05 (1H, dd, J = 11.3, 8.6 Hz), 7.71 (1H, d,
J = 9.0 Hz), 12.41 (1H, br s). Anal. Calcd for C17H16FN5OS: C,
57.13; H, 4.51; N, 19.59. Found: C, 57.01; H, 4.45; N, 19.37.
[methyl(5-nitropyridin-2-yl)amino]phenyl}carbamate
(1.35 g,
5.32. Di-tert-butyl (5-amino-2-fluorophenyl)imidodicarbonate
(29)
42%) as yellow amorphous solid. 1H NMR (300 MHz, DMSO-d6) d
1.45 (9H, s), 3.48 (3H, s), 6.43 (1H, d, J = 9.5 Hz), 7.12 (1H, ddd,
J = 8.8, 4.2, 2.6 Hz), 7.36 (1H, dd, J = 10.7, 8.8 Hz), 7.69 (1H, dd,
J = 7.1, 2.6 Hz), 8.17 (1H, dd, J = 9.5, 2.7 Hz), 9.04 (1H, d,
J = 2.7 Hz), 9.22 (1H, br s).
To a solution of 2-fluoro-5-nitroaniline 22 (15.6 g, 100 mmol) in
dichloromethane (200 mL) were added di-tert-butyl bicarbonate
(87.2 g, 400 mmol) and Et3N (20.4 g, 200 mmol), and the mixture
was stirred at 55 °C for 12 h. The reaction mixture was concen-
trated under reduced pressure, and the residue was purified by sil-
ica gel column chromatography (10% ethyl acetate in petroleum
ether) to give di-tert-butyl (2-fluoro-5-nitrophenyl)imidodicarbon-
ate (19.3 g, 72%) as yellow powder. 1H NMR (CDCl3) d 1.44 (18H, s),
8.12–8.16 (1H, m), 8.21–8.25 (2H, m).
Compound 25 (1.08 g) was prepared from tert-butyl {2-fluoro-
5-[methyl(5-nitropyridin-2-yl)amino]phenyl}carbamate (1.35 g,
3.73 mmol) and 10% Pd/C (198 mg) by the method similar to that
described for 10. Yield: 87%; purple amorphous solid. 1H NMR
(300 MHz, DMSO-d6) d 1.43 (9H, s), 3.21 (3H, s), 4.81 (2H, br s),
6.58 (1H, dd, J = 8.7, 0.6 Hz), 6.73 (1H, ddd, J = 8.9, 4.1, 2.8 Hz),
6.88 (1H, dd, J = 8.7, 2.8 Hz), 7.08 (1H, dd, J = 10.6, 8.9 Hz), 7.28
(1H, dd, J = 7.1, 2.8 Hz), 7.66 (1H, dd, J = 2.8, 0.6 Hz), 8.87 (1H, br s).
To a solution of di-tert-butyl (2-fluoro-5-nitrophenyl)imidodi-
carbonate (256 mg, 0.718 mmol) in MeOH (10 mL) was added

M. Hirose et al. / Bioorg. Med. Chem. 20 (2012) 5600–5615
5613
10% Pd/C (50 mg), and the mixture was stirred under H2 atmo-
sphere at room temperature for 12 h. Insoluble was filtered off
by through a pad of Celite, and the filtrate was concentrated under
reduced pressure to give 29 (150 mg, 64%) as colorless powder: 1H
NMR (CDCl3) d 1.42 (18H, s), 3.55 (2H, br s), 6.46–6.54 (1H, m),
6.55–6.59 (1H, m), 6.85–6.92 (1H, m).
was expressed in Sf9 insect cells (Invitrogen, Life Technologies,
Inc.) and purified by immobilized metal-chelate affinity chroma-
tography (IMAC). Protein bound to the IMAC column was eluted
using a buffer containing 250 mM imidazole. The tag was removed
by cleavage with rTEV protease in 40 mM imidazole, followed by a
reverse IMAC purification. Flow through fractions containing the
target protein were pooled and the protein was concentrated to a
final concentration of 2.6 mg/mL in a delivery buffer of 25 mM bis-
trispropane pH7.0, 250 mM NaCl, 40 mM Imidazole, 1 mM TCEP,
25% Glycerol. 1 mM of 5 was added to the protein to grow macro
seed crystals or diffraction quality crystals, respectively. Small
macro seed crystals were washed with well solution and trans-
5.33. N-(5-Chloro[1,3]thiazolo[5,4-b]pyridin-2-yl)
cyclopropanecarboxamide (31)
To
1.50 mmol) in dichloromethane (5 mL) were added oxalyl chloride
(190 mg, 1.50 mmol) and DMF (40 L), and the mixture was stirred
a
solution of cyclopropanecarboxylic acid (129 mg,
l
ferred by hand to a fresh drop containing 1 lL well solution + 1 lL
at room temperature for 1 h. The reaction mixture was added to a
solution (10 mL) of commercially available 30 (185 mg,
1.00 mmol) in THF at 0 °C, and the mixture was stirred for 2 h.
To the reaction mixture was added water (10 mL), and the mixture
was extracted with EtOAc (3 ꢂ 30 mL). The organic layer was con-
centrated under reduced pressure to give 31 (76 mg, 30 %) as col-
orless powder; 1H NMR (CDCl3) d 1.02–1.06 (2H, m), 1.23–1.30
(2H, m), 1.51–1.69 (1H, m), 7.38 (1H, d, J = 8.7 Hz), 7.92 (1H, d,
J = 8.7 Hz), 9.93 (1H, br s).
B-RAF protein in delivery buffer + 1 mM compound 5. The crystal-
lization condition for the macro seed crystals was 14 % PEG 8000,
0.8 M Lithium Cl, 0.06 M Tris_base and 0.04 M Tris Cl, while the
crystallization conditions to support growth of the macro seeds
was 7.8 % PEG 8000, 0.8 M Lithium Cl, 0.07 M Tris_base and
0.03 M Tris Cl. Crystals were harvested and frozen by flash freezing
in liquid nitrogen using 25% ethylene glycol in mother liquor. The
crystals diffracted to 2.95 Å with clear omit electron density for the
compound. X-ray diffraction data were collected at the Advanced
Photon Source (APS) beam line ID-23B, and processed using the
program HKL2000.16 The structure was determined by molecular
replacement using MOLREP,17 utilizing the published coordinates
of B-RAF with accession code 1UWH.18 Subsequent structure
refinement and model building were performed utilizing REFMAC
and XtalView.19,20 Compound 5 was bound into the active site
and clearly visible in the electron density maps. The figure was
generated using PyMol Molecular Graphics System, Version 1.3
(Schrödinger, LLC).
5.34. tert-Butyl [5-({2-[(cyclopropylcarbonyl)amino][1,3]-
thiazolo[5,4-b]pyridin-5-yl}amino)-2-fluorophenyl]carbamate
(32)
To a mixture of 31 (76 mg, 0.30 mmol), 29 (98 mg, 0.300 mmol),
tris(dibenzylideneacetone)dipalladium (57 mg, 0.06 mmol), dicy-
clohexyl(20,40,60-triisopropylbiphenyl-2-yl)phosphine
(X-phos)
(15 mg, 0.03 mmol) and potassium tert-butoxide (145 mg,
1.40 mmol) was added tert-butanol (20 mL) under nitrogen atmo-
sphere, and the mixture was stirred under a microwave irradiation
at 90 °C for 35 min. To the reaction mixture was added water
(5 mL), and the mixture was extracted with ethyl acetate
(3ꢂ50 mL). The organic layer was dried over anhydrous sodium
sulfate, and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (50% EtOAc in
petroleum ether) to give 32 (84 mg, 63%) as brown powder. 1H
NMR (CDCl3) d 0.93–1.00 (2H, m), 1.25–1.28 (2H, m), 1.50 (9H, s),
1.54–1.69 (1H, m), 6.80–6.85 (3H, m), 6.97–7.05 (1H, m), 7.16–
7.22 (1H, m), 7.77 (1H, d, J = 8.7 Hz), 8.02 (1H, dd, J = 7.2, 2.4 Hz),
11.56 (1H, br s).
5.37. Determination of BRAF(V600E) kinase inhibitory activity
Recombinant BRAF(V600E) protein was expressed as N-termi-
nal FLAG-tagged protein using a baculovirus expression system.
Recombinant GSTP1-MEK1 (K96R) protein was prepared using
FreeStyle 293 expression system (Invitrogen Life Technologies,
Carlsbad, CA). Test compounds (2.5
oxide (DMSO) were added to 37.5
l
L) dissolved in dimethyl sulf-
l
L of a reaction solution (25 mM
HEPES (pH 7.5), 10 mM magnesium acetate, 1 mM dithiothreitol)
containing 25 ng of BRAF(V600E) enzyme and 250 ng of recombi-
nant protein GSTP1-MEK1 (K96R) prepared using FreeStyle 293
expression system (Invitrogen), and the mixture was incubated
at room temperature for 10 min. Ten microliters of ATP solution
5.35. N-{5-[(3-Amino-4-fluorophenyl)amino][1,3]thiazolo[5,4-
b]pyridin-2-yl}cyclopropanecarboxamide (33)
(2.5
ture, and the mixture was reacted at room temperature for
20 min. The reaction was quenched by adding 50 L of ice-cooled
lM ATP, 0.1 lCi [c-
32P]ATP) was added to the obtained mix-
To a solution of 4 N hydrogen chloride in ethyl acetate (50 mL)
was added 32 (3.40 g, 7.67 mmol), and the mixture was stirred at
0 °C for 12 h. The reaction mixture was neutralized with saturated
aqueous NaHCO3, and extracted with EtOAc (3 ꢂ 50 mL). The or-
ganic layer was concentrated under reduced pressure to give 33
(1.70 g, 65%) as purple powder. 1H NMR (DMSO-d6) d 0.92–0.97
(4H, m), 1.96–2.00 (1H, m), 5.08 (2H, br s), 6.73–6.78 (1H, m),
6.85–6.92 (2H, m), 7.16 (1H, dd, J = 8.4, 2.4 Hz), 7.84 (1H, d,
J = 9.0 Hz), 8.97 (1H, s), 12.41 (1H, s).
l
20% trichloroacetic acid (Wako Pure Chemical Industries, Ltd) to
the reaction solution. The reaction solution was allowed to stand
at 4 °C for 30 min, and the acid-precipitable fraction was trans-
ferred to GF/C filter plate (Millipore Corporation) using cell har-
vester (PerkinElmer). The plates were dried at 45 °C for 60 min,
and 40 lL of MicroScinti 0 (PerkinElmer) was added thereto. The
radioactivity was measured using TopCount (PerkinElmer). The ki-
nase inhibitory rate (%) of the test compounds was calculated by
the following formula:
5.36. Expression, purification, crystallization and structure
determination
Inhibitory rate ð%Þ ¼ ð1 ꢀ ðcount of test compound ꢀ blankÞ
ꢃ ðcontrol ꢀ blankÞÞ ꢂ 100
The kinase domain of human BRAF (residues 445–726) and hu-
man p50Cdc37 were cloned into the dual expression vector pFast-
Bac Dual (Invitrogen, Life Technologies, Inc.). The encoded BRAF
contained an N-terminal purification tag followed by a recombi-
nant Tobacco Etch Virus (rTEV) protease cleavage site. The protein
The count of the solution reacted without addition of the com-
pound was used as a ‘control’, and the count of the solution with-
out the compound and enzyme was used as a ‘blank’. The
concentration of inhibitor producing 50% inhibition of the kinase

5614
M. Hirose et al. / Bioorg. Med. Chem. 20 (2012) 5600–5615
activities (IC50 values) and 95% confidence intervals (95% CI) for
BRAF(V600E) were analyzed using GraphPad Prism version 5.01,
GraphPad Software (USA).
tumor lysate was determined by bicinchoninic acid (BCA) protein
assay kit (Thermo), and adjusted to 1.25 g/ L. An equal volume
of SDS sample buffer (BioRad) was added to the above-mentioned
protein solution and incubated at 95 °C for 5 min. Western blotting
was carried out in a manner similar to ‘intracellular MEK/ERK phos-
phorylation inhibitory activity’.
l
l
5.38. Measurement of inhibitory activities against VEGFR2
VEGFR2 kinase activity was determined by use of an anti-phos-
photyrosine antibody with quantitation performed through the
AlphaScreenÒ system (PerkinElmer, USA). Enzyme reactions were
performed in 50 mM Tris–HCl pH 7.5, 5 mM MnCl2, 5 mM MgCl2,
0.01% Tween-20 and 2 mM DTT, containing 10 lM ATP, 0.1 lg/
mL biotinylated poly-GluTyr (4:1) and 0.1 nM of VEGFR2 (Milli-
5.41. Antitumor efficacy study
Treatments started after 1–3 weeks from inoculation when the
estimated tumor mass was 300–500 mm3. Compound 5c was sus-
pended in 0.5% methyl cellulose solution in distilled water, and the
resulting suspension was orally administered to nude rats. All ani-
mals were treated with vehicle (diluted water) or 5c suspension
once a day for 14 consecutive days by oral gavages. Tumor volumes
were calculated as volume = L ꢂ l2 ꢂ 1/2, where L was taken to be
the longest diameter across the tumor and l was taken to be the
corresponding perpendicular. Treatment over control (T/C, %), an
index of antitumor efficacy, was calculated by comparison of the
mean change in tumor volume over the treatment period for the
control and treated groups. Body weight was also measured on
the day of tumor volume assessment.
pore, UK).
Prior to catalytic initiation with ATP, compound and enzyme
were incubated for 5 min at room temperature. The reactions were
quenched by the addition of 25
lL of 100 mM EDTA, 10
lg/mL
AlphaScreen streptavidine donor beads and 10
lg/mL acceptor
beads in 62.5 mM HEPES pH 7.4, 250 mM NaCl, and 0.1% BSA.
Plates were incubated in the dark overnight and then read by EnVi-
sion 2102 Multilabel Reader (PerkinElmer). Wells containing the
substrate and the enzyme without compound were used as total
reaction control. Wells containing biotinylated poly-GluTyr (4:1)
and enzyme without ATP were used as basal control. The concen-
tration of inhibitor producing 50% inhibition of the kinase activities
(IC50 values) and 95% confidence intervals (95% CI) for VEGFR2
were analyzed using GraphPad Prism version 5.01, GraphPad Soft-
ware (USA). Sigmoidal dose–response (variable slope) curves were
fitted using non-linear regression analysis, with the top and bot-
tom of the curve constrained at 100 and 0, respectively.
5.42. Microsome stability
In vitro oxidative metabolic studies of compounds were carried
out using hepatic microsomes obtained from humans. Briefly, the
incubation mixtures were prepared under ice-cold conditions by
adding 50
38 L of ultrapure water, 1
tein/mL), and 1 L of the compound solution (1
eppendorf tube at the final concentrations indicated. The reactions
were initiated by adding 10 L of the NADPH-generating system
l
L of potassium phosphate buffer (50 mmol/L, pH 7.4),
L of the microsomes (0.2 mg pro-
mol/L) in an
l
l
5.39. Cell proliferation assay
l
l
HT29 cells were seeded into a 96-well plate at 3000 cells/well
and were incubated overnight at 37 °C in a 5% CO2 incubator. Var-
ious concentrations of the test compounds were added, and the
cells were cultured for a further 3 days. Cell viability was assessed
by relative cellular ATP levels using CellTiter-Glo luminescent cell
viability assay (Promega) according to the manufacture’s instruc-
tion. The IC50 values and 95% confidence intervals (95% CI) were
calculated from a dose–response curve generated by least-squares
linear regression of the response.
l
(final concentrations of 5 mmol/L MgCl2, 5 mmol/L glucose 6-phos-
phate, 0.5 mmol/L b-NADP+, and 1.5 units/mL glucose-6-phosphate
dehydrogenase) to the incubation mixtures. For the samples
without NADPH, ultrapure water was substituted for the NADPH-
generating system. Incubations were conducted at 37 °C for
20 min in a final volume of 100
100 L of ice-cold acetonitrile. For each species, zero-time incuba-
tions which served as the controls were terminated by adding
100 L of ice-cold acetonitrile before adding 1 L of the compound
lL and terminated by adding
l
For HUVECs, cells (3 ꢂ 103 cells/well) were seeded into 96-well
plates and cultured overnight. Compounds were added the plates
with VEGF (final concentration: 60 ng/mL) and the cells were cul-
tured for 5 days. Cellular proliferation was assessed by adding Cell
l
l
solution. The samples were mixed by vortex mixing vigorously and
centrifuging at 3000ꢂg for 10 min at 4 °C. The supernatant frac-
tions were subjected to high performance liquid chromatography
(HPLC) with an UV detector. All incubations were made in
duplicate.
Counting Kit-8 reagent (10 lL/well; DOJINDO Laboratories, Kuma-
moto, Japan), incubation for several hours, and measurement of the
absorbance at 450 nm with a microplate reader (BioRad Labs, Her-
cules, CA). The concentration causing 50% inhibition (IC50) was cal-
culated from the dose–response curve generated by least-squares
linear regression analysis of the response using SAS software and
the NLIN procedure (version 5.0; SAS Institute Japan, Inc., Tokyo,
Japan).
5.43. Thermodynamic solubility
About 0.5 mg of sample was added to 0.5 mL of a dissolving
solvents, which was 20 mmol/L bile acid in the JP 2nd fluid for
disintegration solution (pH 6.8),15 and the sample solution were
shaken at 37 °C for 18 h. The solution was filtered through a mem-
5.40. In vivo pharmacodynamic (PD) study
brane filter (0.45 lm). The filtrate was diluted with acetonitrile
and analyzed by high-performance liquid chromatography (HPLC).
The sample solutions were analyzed with an HPLC system (W2695,
Waters, Milford, MA, USA) and UV detector (W2487, Waters) oper-
ated at 230 nm. The packaged column was Shiseido MG-III ODS
At 10 days after transplantation, compound 5c was suspended in
0.5% methyl cellulose solution in distilled water, and the resulting
suspension was orally administered to nude rats having an en-
grafted tumor with a tumor volume of 300–600 mm3. After 4 h after
administration of 5c, the tumor was collected under ether anesthe-
sia and the tumor was homogenized in radio-immunoprecipitation
assay (RIPA) buffer (1% NP-40, 0.5% sodium deoxycholate, 1% SDS,
97.5% Dulbecco’s phosphate buffered saline (DPBS) (GIBCO) with
Protease Inhibitor Cocktail Set 3 (Calbiochem) and Phosphatase
Inhibitor Cocktail 2 (Sigma)). The protein concentration in the
(3
lm, 4.6 mm ꢂ 75 mm, Shiseido, Tokyo, Japan) operated at
40 °C at a flow rate of 1.0 mL/min. The two mobile phases used
were (A) 50 mmol/mL ammonium acetate buffer and (B) acetoni-
trile. The elution program started at 100:0 = A:B, ramped linearly
to 0:100 = A:B at 5 min, held there until 7 min, and returned to
100:0 = A:B at 8 min.

M. Hirose et al. / Bioorg. Med. Chem. 20 (2012) 5600–5615
5615
5.44. Pharmacokinetic studies
(DOE) Office of Science by Argonne National Laboratory, supported
by the U.S. DOE under Contract No. DE-AC02-06CH11357. Finally,
the authors express their gratitude to Yukiko Watanabe for her
assistance in PK evaluation.
For oral cassette dosing PK study, test compounds were dis-
solved in a mixture of DMSO and 1,3-butylene glycol and were
administered orally to non-fasted mice at a dose of 10 mg/kg. For
single dosing PK study of 5c, the compound was dissolved in a mix-
ture of dimethylacetamide and 1,3-butanediol (1:1, v/v) and was
administered intravenously to non-fasted rats at a dose of 1 mg/
kg. Compound 5c was suspended in 0.5% methylcellulose solution
for oral administration and was administered orally to non-fasted
rats at a dose of 25 mg/kg.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.07.032.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
Blood samples were taken from the femoral vein at the desig-
nated time points after dosing, and centrifuged to obtain the plas-
ma fractions. The plasma samples were de-proteinized with
acetonitrile containing an internal standard. After centrifugation,
the supernatant was diluted with a mixture of 0.01 mol/L ammo-
nium formate solution and acetonitrile (9:1, v/v) and centrifuged
again. The compound concentrations in the supernatant were mea-
sured by LC/MS/MS.
References and notes
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6. Okaniwa, M.; Imada, T.; Ohashi, T.; Miyazaki, T.; Arita, T.; Yabuki, M.; Sumita,
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Local energy minimum conformations of compounds in Figures
2 and 4 were calculated with molecular mechanic analysis in MOE
(version 2006.0804 and 2009.1002, respectively). During the min-
imization procedure, the following conditions were adopted. The
MMFF94s force field21 was set for the molecular mechanic calcula-
tion. The dielectric constant was set to 4 ꢂ r, where r is the inter-
atomic distance. Atomic charges of the compounds were assigned
by using the AM1-BCC method.
11. Naito, T.; Inoue, S. Chem. Pharm. Bull. 1958, 6, 338.
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Acknowledgements
14. The coordinates and structure factors have been deposited with the Protein
Data Bank with accession code 4FC0.
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Japanese pharmacopoeia English version, 2006, 15, p 239.
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The authors thank Shuhei Yao, Noriko Uchiyama, Hiroshi Miki,
Taeko Yoshida, and Hidehisa Iwata for their assistance in evaluat-
ing the kinase inhibitory activities. The authors also thank Kazuyo
Kakoi, Juran Kato and Kenichi Iwai for their assistance in evaluating
the cellular pMEK inhibitory activities, and Akira Hori and Yuichi
Kakoi for their assistance in evaluating the cellular VEGFR2 inhib-
itory activities. The authors are grateful to Garret Textor, Matt
Kroeger, and Gyorgy Snell from Takeda California for their assis-
tance in determining the crystal structure of BRAF with 5. The
authors acknowledge the Advanced Photon Source, an Office of Sci-
ence User Facility operated for the U.S. Department of Energy
19. Collaborative Computational Project, Number 4. Acta Crystallogr.
Crystallogr. 1994, 50, 760.
D Biol.
20. McRee, D. E. J. Struct. Biol. 1999, 125, 156.
21. Halgren, T. A. J. Comput. Chem. 1999, 20, 720.