Design and synthesis of novel DFG-out RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors: 2. Synthesis and characterization of a novel imide-type prodrug for improving oral absorption

Article abstract of DOI:10.1016/j.bmc.2012.06.015

As an alternative to the previously reported solid dispersion formulation for enhancing the oral absorption of thiazolo[5,4-b]pyridine 1, we investigated novel N-acyl imide prodrugs of 1 as RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors. Introducing N-acyl promoieties at the benzanilide position gave chemically stable imides. N-tert-Butoxycarbonyl (Boc) introduced imide 6 was a promising prodrug, which was converted to the active compound 1 after its oral administration in mice. Cocrystals of 6 with AcOH (6b) possessed good physicochemical properties with moderate thermodynamic solubility (19 μg/mL). This crystalline prodrug 6b was rapidly and enzymatically converted into 1 after its oral absorption in mice, rats, dogs, and monkeys. Prodrug 6b showed in vivo antitumor regressive efficacy (T/C = -6.4%) in an A375 melanoma xenograft model in rats. Hence, we selected 6b as a promising candidate and are performing further studies. Herein, we report the design, synthesis, and characterization of novel imide-type prodrugs.

Full text of DOI:10.1016/j.bmc.2012.06.015

Bioorganic & Medicinal Chemistry 20 (2012) 4680–4692
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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: 2. Synthesis and characterization
of a novel imide-type prodrug for improving oral absorption
Masanori Okaniwa a, , Takashi Imada a, Tomohiro Ohashi a, Tohru Miyazaki a, Takeo Arita a,
⇑
Masato Yabuki a, Akihiko Sumita b, Shunichirou Tsutsumi b, Keiko Higashikawa a, Terufumi Takagi a,
Tomohiro Kawamoto a, Yoshitaka Inui a, 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
a r t i c l e i n f o
a b s t r a c t
Article history:
As an alternative to the previously reported solid dispersion formulation for enhancing the oral absorp-
tion of thiazolo[5,4-b]pyridine 1, we investigated novel N-acyl imide prodrugs of 1 as RAF/vascular endo-
thelial growth factor receptor 2 (VEGFR2) inhibitors. Introducing N-acyl promoieties at the benzanilide
position gave chemically stable imides. N-tert-Butoxycarbonyl (Boc) introduced imide 6 was a promising
prodrug, which was converted to the active compound 1 after its oral administration in mice. Cocrystals
of 6 with AcOH (6b) possessed good physicochemical properties with moderate thermodynamic solubil-
Received 25 April 2012
Revised 4 June 2012
Accepted 5 June 2012
Available online 15 June 2012
Keywords:
RAF
VEGFR2
Imide-type prodrug
Oral absorption
ity (19 lg/mL). This crystalline prodrug 6b was rapidly and enzymatically converted into 1 after its oral
absorption in mice, rats, dogs, and monkeys. Prodrug 6b showed in vivo antitumor regressive efficacy
(T/C = À6.4%) in an A375 melanoma xenograft model in rats. Hence, we selected 6b as a promising can-
didate and are performing further studies. Herein, we report the design, synthesis, and characterization of
novel imide-type prodrugs.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Prodrug strategies other than the aforementioned formulation
technique have also been utilized for enhancing oral absorp-
In a previous report1, we described the synthesis and potent
activity 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 RAF/vascular endothelial growth factor
receptor 2 (VEGFR2) inhibitor. Compound 1 possesses potent
BRAF(V600E) inhibitory activity (IC50 = 7.0 nM) and VEGFR2 inhib-
itory activity (IC50 = 2.2 nM). Since 1 showed insufficient solubility
and an oral absorption profile, the solid dispersion (SD) formula-
tion technique2 was applied to maximize the oral bioavailability
of this compound. The SD formulation of this compound (1-SD)
demonstrated potent antitumor efficacy (T/C = À7.0%) in an A375
human melanoma xenograft model in rats based on the improved
solubility.
tion.3–5 According to these studies, an important point in prodrug
research is choosing the suitable promoiety that must be rapidly
cleaved by the enzymes in the body so that a bioactive compound
is released after oral administration. Additionally, in most cases,
solubility is a significant factor in achieving favorable oral absorp-
tion profiles.5 However, the choice of the promoiety is often lim-
ited and depends on the substructure of the active compounds.
For instance, when active compounds possess hydroxy or carbox-
ylic acid groups, the promoieties such as alkoxycarbonates6,7 or
phosphates8 have been used. When active compounds possess
amino groups or basic azoles, the promoieties such as phosphono
functionalities have been utilized.9–11 However, very few studies
have reported the introduction of acyl groups onto the amide
NH.12,13
Compound 1 possesses cyclopropanecarboxamide and benzani-
lide groups as amide substructures, and both these functional
groups are significant for its potent RAF/VEGFR2 inhibitory activ-
ity. In our optimization studies performed on 2-carboxamide
groups, cyclopropanecarboxamide 1 was selected because it was
stable against hydrolysis by human liver microsomes (Table 1).
However, acetamide 2 was comparatively less stable and under-
Abbreviations: 1H NMR, proton nuclear magnetic resonance; AUC, area under
the blood concentration/time curve; ERK, extracellular signal-regulated kinase; MC,
methylcellulose; MEK, mitogen-activated protein kinase; PK, pharmacokinetic;
VEGFR2, vascular endothelial growth factor receptor 2.
⇑
Corresponding authors. Tel.: +81 466 32 1158; fax: +81 466 29 4449 (M.O.); tel.:
+81 466 32 1155; fax: +81 466 29 4449 (T.I.).
E-mail addresses: masanori.okaniwa@takeda.com (M. Okaniwa), tomoyasu.ishik
awa@takeda.com (T. Ishikawa).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.015

M. Okaniwa et al. / Bioorg. Med. Chem. 20 (2012) 4680–4692
4681
Table 1
Profiles of [1,3]thiazolo[5,4-b]pyridine-2-acylamide 1, 2
F
N
S
O
Cl
HN
R1
N
O
N
H
CN
R1
Kinase IC50 (nM)
Cellular pMEKb IC50 (nM)
In vitro hydrolysisc
a
BRAF (V600E)
VEGFR2
Unchanged form Remaining (%)
Metabolite 3 formationd (%)
1
2
7.0 (6.0–8.3)
2.2 (2.0–2.4)
3.4 (3.1–3.7)
25
34
109.5
2.4
6.3 (5.8–6.9)
78.5
34.6
a
b
c
n = 2. 95% confidence index values.
Concentration producing 50% inhibition (IC50) against Raf substrate MEK phosphorylation in HT-29 (BRAFV600E mutant) cultured human colon cancer cell lines.
In vitro hydrolysis stability of each compound (30 M) was examined using human liver microsomes (1 mg protein/mL). Incubation was carried out for 60 min at 37 °C.
Metabolite 3 was identified as a 2-amino derivative (R1 = H).
l
d
Figure 1. Design of imide-type prodrugs.
went hydrolysis to give the deacetylated [1,3]thiazolo[5,4-b]pyri-
dine-2-amine 3 (R1 = H) as a major metabolite.1 Under these condi-
tions, the benzanilide moieties of 1 and 2 were very stable, and no
cleaved metabolite of benzanilide was observed.
2. Chemistry
Acetylation of 1 was initially examined, but nearly all the reac-
tion conditions afforded complex mixtures inseparable by conven-
tional silica gel column chromatography. Since 1 or the resulting
acetylated compounds appeared to be highly sensitive to the reac-
tion conditions used, we attempted the cyclopropanecarbonylation
of 1 using cyclopropanecarbonyl chloride, which is less reactive
than acetyl chloride. As expected, the cyclopropanecarbonylation
of 1 proceeded in the presence of DMAP in pyridine, and type B
imide product 4 was isolated in 63% yield in a regioselective manner
(Scheme 1). The chemical structure of 4 was determined by 1H NMR
and mass spectroscopic analysis. Further, 4 was obtained as chem-
ically stable crystals, as confirmed by powder X-ray diffractometry.
Next, we examined introduction of ethoxycarbonyl group and
Boc group into 1. Ethoxycarbonylation of 1 with diethyl dicarbon-
ate in pyridine afforded the ethoxycarbonylated compound 5 in
45% yield after conventional synthesis and purification by silica
gel column chromatography. The Boc group was introduced into
1 by following the similar procedure for ethoxycarbonylation to
give 6 in 63% yield. The chemical structures of 5 and 6 corre-
sponded to that of type B, and these compounds were also ob-
tained as chemically stable crystals in a regioselective manner.
To clarify the mechanism of regioselective monoacylation, we
On the basis of the results of in vitro hydrolysis studies by human
liver microsomes, wehypothesizedthat introductionofan enzymat-
ically unstable acyl moiety into 1 may produce a unique imide-type
prodrug of 1. Thus, we designed type A possessing an acyl group R2 in
the cyclopropanecarboxamide moiety and type B with an acyl group
R3 in the benzanilide moiety (Fig. 1). An initial synthetic investiga-
tion found that type A compounds were not suitable for develop-
ment because imide structure of type A was chemically too
instable to be isolated in general purification method (see Section
2). Therefore, our research focused on type B imide compounds. To
evaluate acyl groups for the imide-type prodrug B, we screened
the following: an enzymatically unstable acetyl group, a cyclopro-
panecarbonyl group and ethoxycarbonyl and tert-butoxycarbonyl
(Boc) groups, which afforded carbamate-type derivatives. For an
asymmetric bis-acyl imide prodrug B with two different acyl groups,
it is necessary to consider the regioselectivity of the enzymatic
cleavage reaction. The original benzanilide group of the bioactive
compound 1 must be more stable than the additional acyl groups.
The purpose of this study was the synthesis and evaluation of
novel imide-type prodrugs to improve the oral absorption of 1
and the evaluation of the physicochemical and biological profiles
of these compounds.
performed Boc-introducing reaction of
through the stepwise addition of di-tert-butyl dicarbonate (Boc2O)
1 in tetrahydrofuran

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M. Okaniwa et al. / Bioorg. Med. Chem. 20 (2012) 4680–4692
Scheme 1. Synthesis of type B imide compounds 4–6. Reagents and conditions: (For 4) cyclopropanecarbonyl chloride (5.2 equiv), DMAP (5.0 equiv), pyridine, room temp,
1.5 h (63%); (For 5) diethyl dicarbonate (4.9 equiv), pyridine, room temp, 1 h (45%); (For 6) di-tert-butyl dicarbonate (3.0 equiv), pyridine, THF, room temp, 1 h (63%).
Figure 2. Proposed mechanism of regioselective mono-Boc introduction into 1 for preparing Boc prodrug 6: reactivity of two amides of 1 and chemical stability of two Boc
groups of 8.
in the presence of pyridine. Without pyridine, this reaction was ex-
tremely slow, indicating that pyridine plays a significant role in the
activation of amides by deprotonation and/or activation of Boc2O.
In optimal conditions in this reaction, more than 3 equiv of Boc2O
were required to convert 1 to 6 effectively. The results and our pro-
posed mechanism are summarized in Figures 2 and 3. The reaction
mixture was monitored by thin-layer chromatography (TLC) and
1H NMR analysis (DMSO-d6), in which the aminothiazole amide
and benzanilide protons were initially observed with different
chemical shifts of 12.7 and 10.58 ppm, respectively (Fig. 3A). The
Boc group was first introduced at the aminothiazole amide moiety
as type A. The formation of a mono-Boc compound 7 was detected
by 1H NMR 30 min after the addition of 1 equiv of Boc2O. The de-
crease in the height of the proton peak corresponding to the ami-
nothiazole amide (12.7 ppm) shown in Figure 3B suggested the
formation of 7 in the reaction mixture. A singlet peak correspond-
ing to the N-Boc group of aminothiazole imide was also detected at
1.66 ppm. Additionally, the formation of 7 was confirmed by the
appearance of an additional spot on the TLC plate (Rf: 0.6 in ethyl
acetate/n-hexane = 1:1) along with that of the starting material 1
(Rf: 0.2). These results suggested that the reactivity of aminothia-
zole amide should be higher than that of the benzanilide moiety.
Further monitoring of the reaction revealed a gradual decrease
in the intensity of the benzanilide peaks, as shown in Figure 3C,
indicating the formation of di-Boc compound 8. A peak due to the
N-Boc group of benzanilide also appeared at 1.14 ppm. In contrast,
the peak due to the aminothiazole amide (12.7 ppm) slowly recov-
ered after 160 min, and four distinguishable doublet peaks were ob-
served in the range 8.14–8.20 ppm. The peaks were considered to
be due to the protons at the 4-position of the thiazolo[5,4-b]pyri-
dine ring (1, 6–8). Since the produced aminothiazole imides 7 and
8 are reactive intermediates under the reaction conditions em-
ployed, we hypothesized that their Boc groups may be transferred
to other molecules to assist the ‘‘+Boc’’ reaction. Next, further addi-
tion of one equivalent of Boc2O accelerated the ‘+Boc’ reaction
(Fig. 3D). Three equivalents of Boc2O were necessary for the com-
plete conversion of 1 to 8, as shown inFigure 3E. In the work up pro-
cess using hydrochloric acid, selective Boc cleavage occurred at the
labile aminothiazole imide position to produce the desired mono-
Boc compound 6 (Rf: 0.3) in a regioselective manner (Fig. 3F). We
confirmed that this regioselective cleavage also occurred while
purifying a crude material by silica gel column chromatography.
Based on these results, it was found that the regioselectivity of this
mono-acylation was achieved by a cleavage reaction of the labile
aminothiazole imide in di-acylated compound.
[1,3]Thiazolo[5,4-b]pyridine acetamide derivative 2 and its
metabolite 9 (vide infra) were prepared using the route described
in Scheme 2. The reaction of commercially available 2-chloro-5-
nitropyridine 10 with 3-amino-4-fluorophenol in the presence of
potassium carbonate gave the phenoxylated compound 11 in 79%

M. Okaniwa et al. / Bioorg. Med. Chem. 20 (2012) 4680–4692
4683
d
(A)
(D)
d
Before
Boc2O (1 eq.)
addition
60 min after
second Boc2O
(1 eq.) addition
d
(ppm)
(ppm)
1.00 1.00
1
0.09 0.30
1
8.08 5.67
(B)
(E)
d
30 min after
first Boc2O
(1 eq.) addition
30 min after
d
d
third Boc2O
(1 eq.) addition
(ppm)
(ppm)
0.32 0.84
1
5.87
1.05
N.D. N.D.
1
9.03 9.01
(C)
(F)
d
d
120 min after
HCl addition
160 min after
first Boc2O
(1 eq.) addition
d
d
d
d
(ppm)
(ppm)
1.98
0.48 0.72
1
4.26
0.66 N.D.
1
3.05 9.18
Figure 3. Selected proton signals in 1H NMR analysis of reaction mixture (A–F). Representative chemical shifts (ppm) are described in black letters. Relative integral values of
proton signal areas are described in red letters; the sum of the integrals for the doublet due to C–H at the 4-position of thiazolo[5,4-b]pyridines (8.14–8.20 ppm) is set as 1 for
the standard.
Scheme 2. Synthesis of [1,3]thiazolo[5,4-b]pyridine 2 and 9. Reagents and conditions: (a) 3-amino-4-fluorophenol, K2CO3, DMF, room temp, 12 h (79%); (b) Boc2O, THF,
reflux, 12 h (90%); (c) H2, 10% Pd/C, MeOH, THF, room temp, 12 h (94%); (d) KSCN, Br2, AcOH, room temp, 12 h (90%); (e) acetyl chloride, pyridine, room temp, 12 h (52%); (f)
TFA, room temp, 30 min; (g) 2-chloro-3-(1-cyanocyclopropyl)benzoic acid, COCl2, cat. DMF, room temp; 30 min (58%); (h) cyclopropanecarbonyl chloride, DMAP, pyridine,
room temp, 12 h (59%).

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M. Okaniwa et al. / Bioorg. Med. Chem. 20 (2012) 4680–4692
Table 2
Physicochemical properties and mouse PK profiles of imide-type prodrugs 4–6
F
N
O
O
Cl
HN
S
N
O
N
CN
O
R3
4–6
R3
Crystallinitya (%)
Melting pointb (°C)
Solubilityc
3.2
(lg/mL)
Mouse PK AUC0–8 h
Active compound 1
(lg h/mL)
d
Prodrug intact
e
4
69
190
220
0.511
n.d.
5
6
EtO
tert-BuO
74
44
0.79
49
0.097
4.183
–
f
134 (dec.)
0.062
a
b
c
Determined using powder X-ray diffractometry.
Determined using differential scanning calorimetry.
The Japanese Pharmacopoeia 2nd fluid for disintegration test (pH 6.8) (JP2) containing 20 mmol/L of bile acid.
d
Cassette dosing of five compounds. Values shown are mean SD of data from three mice. Compounds (10 mg/kg) were administered in 0.5% methylcellulose in distilled
water.
e
Not detected.
Decomposition.
f
Table 3
Physicochemical properties of active compound 1 and N-Boc prodrug crystals 6, 6a–b
Solvate
Solid form
Crystallinitya (%)
Melting pointb (°C)
Solubilityc(
lg/mL)
Initial dissolution rated g/mm2/min)
(l
1
6
6a
6b
Solvent free
Crystal
Semicrystal
Crystal
55
44
76
77
213
3.6
49
4.2
19
0.043
n.d.
0.072
0.101
Monohydrate
Ethanol (1/1)e
Acetic acid (1/1)e
134 (dec.)f
138 (dec.)f
141 (dec.)f
Crystal
a
b
c
d
e
f
Determined using powder X-ray diffractometry.
Determined using differential scanning calorimetry.
The Japanese Pharmacopoeia 2nd fluid for disintegration test (pH 6.8) (JP2)16 containing 20 mmol/L of bile acid.
The initial dissolution rates were measured using rotating disk method.
Theoretical number of cosolvent was determined by single crystal analysis and described in parenthesis (host/solvate).
Decomposition.
yield. Boc protection of the anilino group of 11 (90% yield) and sub-
sequent reduction of the nitro group under conventional hydroge-
nation conditions afforded the aminopyridine derivative 12 in 94%
yield. Cyclization of 12 with potassium thiocyanate and bromine
afforded [1,3]thiazolo[5,4-b]pyridine-2-amine derivative 13 in
90% yield. Acylation of the 2-amino group of 13 with acetyl chlo-
ride in pyridine gave 14 in 52% yield. Cleavage of the N-Boc group
of 14 using trifluoroacetic acid and subsequent acylation of the
resulting amino group with 2-chloro-3-(1-cyanocyclopropyl)ben-
zoic acid1 under standard conditions provided the desired acetam-
ide 2 in 58% yield in 2 steps.
detected in the blood. Although the solubility (3.2 lg/mL) and oral
absorption level of 4 were insufficient, these results motivated the
evaluation of other imide-type compounds 5 and 6. The Boc
derivative 6 showed better solubility (49
lg/mL) than did the
ethoxycarbonyl derivative 5 (0.79 g/mL). From our previous
l
investigation, we asserted that modification of the benzanilide
substructure of 1 had a significant impact on its solubility.1 Intro-
Metabolite 9 was synthesized in 59% yield by acylation of 13
with cyclopropanecarbonyl chloride in the presence of DMAP in
pyridine.
3. Results and discussion
3.1. Physicochemical properties and mouse PK profiles of
imide-type prodrugs
Physicochemical properties and mouse PK profiles of synthe-
sized imide compounds 4–6 are summarized in Table 2. After oral
administration of 10 mg/kg of cyclopropanecarbonylated imide 4
in mice, the bioactive compound 1 was observed to have an AUC
Figure 4. Initial dissolution ratea of active compound 1 and prodrug 6a–b. aJP216
value of 0.511 lg h/mL. In this study, intact compound 4 was not
containing 200 mmol/mL of bile acid was used as a solvent.

M. Okaniwa et al. / Bioorg. Med. Chem. 20 (2012) 4680–4692
4685
duction of a chlorine atom into the ortho-position of the benzoyl
moiety enhanced the solubility by twisting the planar conforma-
tion of the benzanilide. In this prodrug research, the introduction
of an N-Boc group into benzanilide moiety was reflected in a de-
creased melting point for 6 (mp = 134 °C) as compared to that of
the active compound 1 (mp = 213 °C), which implied a decrease
in crystal packing energy. We assumed that the bulky lipophilic
tert-butyl group interferes with intermolecular hydrogen bonding
crystals of 6 were considered to be semicrystalline. Hence, we var-
ied the crystallization solvents in order to obtain stable crystals.
However, despite numerous crystallization trials, no stable unsol-
vated crystals were obtained. Some stable solvated cocrystals were
obtained when using EtOH, AcOH, acetone, DMSO, and i-PrOH. For
the pharmaceutical use, residual solvents must be controlled for
the safety of drugs. Thus, we selected cocrystals with EtOH (6a)
and AcOH (6b) for further physicochemical evaluation based on
the guidelines for residual solvents by the International Conference
on Harmonization (ICH).15
or p–p stacking interactions, thereby decreasing the crystal pack-
ing energy and increasing the solubility.14
Because of the enhanced solubility, the exposure of 1 was dra-
matically improved (AUC 4.183 lg h/mL) in a pharmacokinetic
(PK) study after oral administration (dose: 10 mg/kg) of 6 in mice.
In this study, the uncleaved, intact 6 was observed to have a low
We summarized the physicochemical properties of the prodrug
cocrystals of 6a and 6b and compared these properties with those
of the solvent-free monohydrate 6 and bioactive compound 1, as
shown in Table 3. Both 6a and 6b showed sufficient crystallinities
AUC value (0.062
moiety of 6 is rapidly cleaved after oral absorption and that 6 func-
l
g h/mL). These results suggest that the Boc pro-
of 76% and 77%, respectively. However, the solubility of 6b (19
mL) was significantly higher than that of 6a (4.2 g/mL).
lg/
l
tions effectively as a prodrug of 1.
Next, we compared the dissolution rates of active compound 1
and prodrugs 6a,b using the Japanese Pharmacopoeia 2nd fluid for
disintegration test (pH 6.8) (JP2)16 containing 200 mmol/mL of bile
acid, as shown in Figure 4. As expected, initial dissolution rates of
For further evaluation, we conducted a polymorph study of pro-
drug 6, which was initially crystallized as a monohydrate from 90%
MeOH in H2O. The crystallinity of 6 remained low (44%), and the
Figure 5. Binding mode of cocrystals determined using single-crystal X-ray structure analysis. Presumed hydrogen bonds (HB) are shown as dotted lines. Numeric data express
the hydrogen bond distance (Å) between the donor (D) and acceptor (A) atoms with estimated standard deviations in parentheses. (A) Ethanol cocrystals 6a (CCDC 862986). The
ethanol molecule forms two hydrogen bonds (HB1,2) with two adjacent host molecules. (B) AcOH cocrystals 6b (CCDC 863435). The AcOH molecule forms two hydrogen bonds
(HB3,4) with a single host molecule.

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M. Okaniwa et al. / Bioorg. Med. Chem. 20 (2012) 4680–4692
6a (0.072
better than that of 1 (0.043
l
g/mm2/min) and 6b (0.101
l
g/mm2/min) were slightly
that the AcOH molecule in 6b should exist in the un-ionized molec-
ular form. This indicates that 6b is not a salt but an AcOH solvate.
l
g/mm2/min). We suspected that the
enhanced dissolution rate of 6b would be reflected by the im-
proved oral absorption of 1 after oral administration of 6b.
3.3. In vivo biological profiles of imide-type prodrug 6b
3.2. Single-crystal X-ray structural analysis of 6a and 6b
The selected prodrug 6b was evaluated by in vivo testing in a
human melanoma A375 xenograft model in rats. The prodrug 6b
showed a dose-dependent inhibitory activity against phosphoryla-
tion of downstream ERK1/2 (pERK1/2) 4 h after treatment at doses
of 12.9, 32.3, and 129 mg/kg, as shown in Figure 7A, which are
equivalent to doses of 10, 30, and 100 mg/kg of 1. On the basis of
these results, we performed anti-tumor efficacy studies with
twice-daily oral administration in rats (Fig. 7B). Reflecting the
pERK1/2 inhibitory effects, 6b exhibited potent anti-tumor efficacy
at a dose of more than 32.3 mg/kg (30 mg/kg as 1) in a dose-
Although we confirmed the enhanced solubility and initial dis-
solution rate of 6b, we could not clarify how the physicochemical
properties of 6a (with EtOH) and 6b (with AcOH) were different.
Thus, we conducted single-crystal X-ray structural analysis of the
cocrystals of 6a and 6b to compare the structural differences be-
tween these two compounds, as shown in Figure 5.17
The cocrystal structure of 6a (Fig. 5A) shows that the EtOH mol-
ecule functions as a hub of intermolecular interaction for 6. The hy-
droxy group of EtOH is within the hydrogen bonding distance
between two adjacent host molecules of 6 and coordinates two
hydrogen bonds, HB1 and HB2, with 6. The HB1 and HB2 distances
are 2.845 and 2.844 Å, respectively. Interestingly, these hydrogen
bond networks connect each crystal lattice.
dependent manner. Interestingly, at
a dose of 129 mg/kg
(100 mg/kg as 1), 6b demonstrated tumor regression efficacy (T/
C: À6.4%) without severe body weight loss.
3.4. Pharmacokinetic profiles of prodrug 6b in various animals
However, the cocrystal structure of 6b (Fig. 5B) shows that the
AcOH molecule is located near the aminothiazole moiety and
forms bidentate hydrogen bonds (HB3 and HB4) with a single host
molecule 6. The HB3 and HB4 distances are 2.809 and 2.712 Å,
respectively. The hydrogen bond network in 6b does not extend
beyond the crystal lattice, as opposed to that in 6a. The solubility
and dissolution rate of 6b are higher than those of 6a because of
the lower crystal lattice energy of the former. Thus, we assume that
the hydrogen bond networks of the synthon connect to each other
in 6a with increased crystal lattice energy, thus negatively impact-
ing the solubility of 6a. However, the crystal motif of 6b may be
advantageous for dissolution because there are no hydrogen bonds
between the synthons.
To further evaluate 6b as a prodrug of 1, PK profiles were inves-
tigated in various animals (Table 4). In these studies, the active
compound 1 was the main product in all the animals. Nearly all
of 6b was converted into 1, but a minor amount of metabolite 9
was also observed. For example, at a dose of 64.6 mg/kg in nude
rats,
1.80
9
l
was detected and found to have an AUC value of
g h/mL. In this experiment, the exposure of active compound
g h/mL. In view of the weak BRAF inhibitory
1 was AUC0–24 h 4.28
l
activity (IC50 = 2000 nM) of metabolite 9, we reasoned that the
antitumor efficacy based on pERK inhibition shown in Figure 7
should be mainly derived from the treatment of active compound
1 released from prodrug 6b.
In the cocrystal structure of 6b, the distances between hydrogen
and the donor oxygen (D–H), and hydrogen and the acceptor azole
(H. . .A) were measured using discrete Fourier transform analysis,
as shown in Figure 6. Although an observed D–H distance of
1.151 Å was slightly longer than a typical O–H bond length
(1.015 Å) in carboxylic acids,18 the hydrogen atom was observed
near the donor oxygen atom as compared to a distance of
1.563 Å from the acceptor nitrogen atom. These results suggest
Furthermore, it was found that 6b functions more effectively as
a prodrug of 1 in dogs and monkeys than in rats. The efficacious
AUC values for 1 were 25.33 and 9.90 lg h/mL in dogs and mon-
keys, respectively. Interestingly, the production levels of the unde-
sired metabolite 9 were low in these non-rodent animals. The 40
and 330-times higher ratio of AUC values of 1 against that of 9
were achieved in dogs and monkeys, but in rats the ratio was only
2.4 times. These results revealed that the cleavage ratio of the
HB4
Hydrogen Bond Distance (Å)
D…A D–H H…A
HB4 OH (AcOH) :N (Thiazolo) 2.712 (5) 1.151 (4) 1.563 (4)
Hydrogen Bond Angle (°)
D–H…A
Donor (D)
Acceptor (A)
175.9 (3)
Figure 6. Single crystal structure of 6b with AcOH, showing hydrogen peaks (light green) observed after discrete Fourier transformation.

M. Okaniwa et al. / Bioorg. Med. Chem. 20 (2012) 4680–4692
4687
Figure 7. In vivo studies of 6b in A375 human melanoma xenograft models in F344 nude rats. (A) Mean phosphorylated ERK1/2 levels in tumor tissues were detected (n = 2)
4 h after treatment with a single dose. (B) Mean tumor volumes were determined (n = 4); P 60.025 versus control at day 14 (ShirleyÀWilliams test).
imide-type prodrug 6b is species-dependent, but the cleavage
reaction primarily produced the desired bioactive compound 1 in
these selected animals.
To investigate the differences among various species and pre-
dict human PK profiles, we examined the in vitro metabolism
using hepatic microsomes, as shown in Table 5. In rodents,
the in vitro formation of 9 was comparable to that of 1 in rats
(ratio of formation 1/9 = 0.76), but the latter was dominantly
formed in mice (ratio of formation 1/9 = 19). Prodrug 6b was
converted into 1 in dogs (ratio = 3.3) and monkeys (ratio = 3.0).
Notably, in humans, prodrug 6b selectively produced 1 with a
ratio of 152.
A comparison of the ratio of in vitro formation and in vivo AUC
is shown in Figure 8. Since the AUC ratios were generally higher
Table 4
Selected oral pharmacokinetic profiles of 6b in various animals
F
N
S
O
Cl
HN
O
N
O
N
H
CN
i
1
F
ii
N
N
S
Active compound
O
O
Cl
HN
O
BRAF(V600E) IC50 = 7.0 nM
N
O
CN
ii
i
O
F
N
HN
S
N
O
NH
H3C
CH3
CH3
O
6b
O
O
(AcOH cocrystal)
9
H3C
CH3
CH3
Metabolite
BRAF(V600E) IC50 = 2000 nM
Species
Dose (mg/kg)
AUC0–24 h
(l
g h/mL)a
Ratio AUC0À24 h 1/9
Prodrug 6
Active compound 1
Metabolite 9
Mice
32.3b
12.9b
64.6b
32.3b
32.3b
0.37 0.06
0.02 0.00
0.08 0.02
0.53 0.12
0.53 0.23
4.90 1.56
1.39 0.30
4.28 1.03
25.33 4.11
9.90 3.52
0.50 0.17
0.57 0.06
1.80 0.24
0.63 0.23
0.03 0.06
9.8
2.4
2.4
40
Rats (Nude)
Rats (Nude)
Dogs
Monkeys
330
a
Values shown are mean SD of data from three animals.
Delivered in 0.5% methylcellulose in distilled water.
b

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M. Okaniwa et al. / Bioorg. Med. Chem. 20 (2012) 4680–4692
Table 5
Difference in the formation of active compound 1 and metabolite 9 from prodrug 6b by hepatic microsomes between different species
Species
In vitro formationa
Metabolite 9 formation (%)
Active compound 1 formation (%)
Ratio 1/9
Mice
Rats
Dogs
Monkeys
Humans
52.3
15.3
2.3
1.8
15.2
2.8
20.2
0.7
0.6
0.1
19
0.76
3.3
3.0
152
a
Hepatic microsomes (4 mg protein/mL) and 6b (10
l
mol/L) were used. Reactions were carried out at 37 °C for 2 h.
1000
100
10
1
Ratio of in vitro formation levels
Ratio of AUC values
0.1
Mice
Rats
Dogs Monkeys Human
Figure 8. Comparison of ratio between in vitro formation levels by microsome and AUC values in various animals.
than those in a hepatic microsomal study performed on rats, dogs,
and monkeys, PK in humans for 6b may be a promising as the pro-
drug of the active compound 1.
was carried out using Merck Kieselgel 60, 63–200 mesh, F254 plates
or Fuji Silysia Chemical Ltd, 100–200 mesh, NH plates. Chromato-
graphic 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 nu-
clear magnetic resonance (1H NMR) spectra were obtained using a
Bruker AVANCE II (300 MHz) spectrometer with tetramethylsilane
(TMS) as the internal standard. The data are given as follows:
chemical shift (d) in ppm, multiplicity (where applicable), coupling
constants (J) in Hz (where applicable), and integration (where appli-
cable). Multiplicities are indicated by s (singlet), d (doublet), t (trip-
let), q (quartet), dd (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 P95% purity of each compound.
The column used was an L-column 2 ODS (3.0 Â 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 spectroscopy (HRMS) were carried
out at Takeda Analytical Laboratories, Ltd. Yields were not
optimized.
4. Conclusion
Toenhance the oral absorption of active compound 1, we designed
and investigated novel N-acyl imide-type prodrugs. We found that an
N-acyl promoiety introduced at the benzanilide position provided
chemically stable type B imides. Particularly, introduction of N-Boc
into the imide 6 resulted in significant improvement of the solubility
and oral absorption in mice. Furthermore, cocrystals of 6b with AcOH
hadgood physicochemical propertieswith moderatethermodynamic
solubility(19 lg/mL)andshowed potentinvivo antitumor regressive
efficacy (T/C = À6.4%) ina humanmelanoma A375 xenograftmodel in
rats based on the potent BRAF(V600E) inhibitory activity of 1
(IC50 = 7.0 nM). As discussed in our previous reports, the antitumor
efficacy of the prodrug of 1 was thought to include the additional effi-
cacy derived from the potent VEGFR2 inhibitory activity of 1
(IC50 = 2.2 nM). Pharmacokinetics studies on 6b in various animals
showed that this compound functions effectively as a prodrug of 1
in dogs and monkeys than in rats.
Thus, we conclude that the Boc-introduced imide compound
practically functions as a prodrug for improving thermodynamic
solubility and oral absorption, and prodrug 6b is a promising can-
didate for a RAF/VEGFR2 inhibitor.
5.2. N-(5-{[2-(Acetylamino)[1,3]thiazolo[5,4-b]pyridin-5-
yl]oxy}-2-fluorophenyl)-2-chloro-3-(1-cyanocyclopropyl)
benzamide (2)
5. Experimental section
5.1. General chemistry information
A solution of tert-butyl (5-{[2-(acetylamino)[1,3]thiazolo[5,4-
b]pyridin-5-yl]oxy}-2-fluorophenyl)carbamate (14, 0.65 g, 1.54
mmol) in trifluoroacetic acid (5 mL) was stirred at room tempera-
ture for 30 min. The reaction mixture was concentrated under
The starting materials, reagents, and solvents were reagent-
grade and used as purchased. Thin-layer chromatography (TLC)

M. Okaniwa et al. / Bioorg. Med. Chem. 20 (2012) 4680–4692
4689
reduced pressure, and the residue was diluted with ethyl acetate
5.4. Ethyl {[2-chloro-3-(1-cyanocyclopropyl)phenyl]carbonyl}
(100 mL), washed with 0.1 N NaOH (100 mL), and dried over anhy-
drous MgSO4. The insoluble material was filtered off, and the filtrate
was concentrated under reduced pressure. The obtained residue
was triturated with diethyl ether to give N-[5-(3-amino-4-fluoro-
phenoxy)[1,3]thiazolo[5,4-b]pyridin-2-yl]acetamide (0.49 g, quan-
titative yield) as white amorphous solid. 1H NMR (DMSO-d6,
300 MHz): d 2.27 (s, 3H), 4.03 (br s, 2H), 6.40–6.46 (m, 1H), 6.61
(dd, J = 3.0, 7.5 Hz, 1H), 6.89 (d, J = 8.7 Hz, 1H), 6.97 (dd, J = 8.7,
10.8 Hz, 1H), 7.92 (d, J = 8.7 Hz, 1H), 11.66 (br s, 1H). HRMS (ESI):
calcd for C14H11FN4O2S [M+H]+ 319.0660. Found: 319.0641.
[5-({2-[(cyclopropylcarbonyl)amino][1,3]thiazolo[5,4-b]
pyridin-5-yl}oxy)-2-fluorophenyl]carbamate (5)
To a mixture of compound 1 (151 mg, 0.278 mmol) in pyridine
(1.5 mL) was added diethyl dicarbonate (197 lL, 1.36 mmol) at
room temperature. The reaction mixture was stirred at room tem-
perature for 1 h. To the mixture was added water (5 mL), and the
mixture was extracted with ethyl acetate (3 Â 5 mL). The organic
layer was washed with water (5 mL) and brine (2 mL), succes-
sively, and dried over anhydrous MgSO4. The insoluble was filtered
off, and the filtrate was concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (mesh
To a solution of 2-chloro-3-(1-cyanocyclopropyl)benzoic acid1
(0.33 g, 1.5 mmol) in oxalyl chloride (1.5 mL) was added DMF
(100
l
L), and the mixture was stirred at room temperature for
60 lm, 100 g, eluent 5–80% ethyl acetate in n-hexane). Desired
30 min, and concentrated to dryness under reduced pressure. This
material was dissolved in a mixture of N,N-dimethylacetamide/tet-
rahydrofuran (1:1, 3 mL), and the solution was added dropwise to a
solution of N-[5-(3-amino-4-fluorophenoxy)[1,3]thiazolo[5,4-
b]pyridin-2-yl]acetamide (0.33 g, 1.00 mmol) in N,N-dimethylacet-
amide (3 mL) under ice-cooling. The reaction mixture was stirred
at room temperature for 3 h, and poured into water (100 mL),
and the mixture was extracted with ethyl acetate (2 Â 100 mL).
The combined organic layers were dried over anhydrous Na2SO4.
The insoluble material was filtered off, and the filtrate was concen-
trated under reduced pressure. The obtained residue was purified
by silica gel column chromatography (0–100% ethyl acetate/n-hex-
ane), and recrystallized from methanol to give 2 (280 mg, 54%) as
colorless crystalline solid; mp 213–215 °C. 1H NMR (DMSO-d6,
300 MHz): d 1.39 (dd, J = 5.1, 7.5 Hz, 2H), 1.82 (dd, J = 5.4, 7.5 Hz,
2H), 2.30 (s, 3H), 6.94–6.99 (m, 1H), 7.03 (d, J = 8.7 Hz, 1H), 7.19
(dd, J = 9.0, 10.5 Hz, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.50 (dd, J = 1.8,
7.8 Hz, 1H), 7.68 (dd, J = 1.8, 7.8 Hz, 1H), 7.97 (d, J = 8.7 Hz, 1H),
8.06 (d, J = 3.0 Hz, 1H), 8.39 (dd, J = 2.7, 6.6 Hz, 1H), 9.18 (br s,
1H). HRMS (ESI): calcd for C25H17ClFN5O3S [M+H]+ 522.0797.
Found: 522.0803.
fractions were combined and evaporated under reduced pressure.
Crystallization of the residue was carried out from ethyl acetate,
and the obtained crystals were collected by filtration, dried under
vacuum to give 5 as white crystalline solid (77.6 mg, 45%); mp
220 °C. 1H NMR (DMSO-d6, 300 MHz): d 0.90 (t, J = 7.1 Hz, 3H),
0.93–1.00 (m, 4H), 1.39–1.49 (m, 2H), 1.75–1.85 (m, 2H), 1.93–
2.06 (m, 1H), 4.03 (q, J = 7.1 Hz, 2H), 7.16 (d, J = 8.7 Hz, 1H), 7.32–
7.40 (m, 1H), 7.43–7.51 (m, 2H), 7.51–7.58 (m, 1H), 7.59–7.70 (m,
2H), 8.19 (d, J = 8.7 Hz, 1H), 12.70 (br s, 1H). Powder X-ray diffrac-
tion (Cu-K
11.76, 12.62, 13.32, 14.14, 14.54, 14.92. Anal. Calcd for
30H23ClFN5O4S: C, 58.11; H, 3.74; N, 11.29. Found: C, 58.10; H,
a radiation, diffraction angle: 2h (°)): 6.88, 9.68, 10.52,
C
3.86; N, 11.08.
5.5. tert-Butyl {[2-chloro-3-(1-cyanocyclopropyl)phenyl]
carbonyl}[5-({2-[(cyclopropylcarbonyl)amino][1,3]thiazolo
[5,4-b]pyridin-5-yl}oxy)-2-fluorophenyl]carbamate
monohydrate (6)
To a solution of 1 (3.0 g, 5.47 mmol) in pyridine (55 mL) was
added dropwise di-tert-butyl dicarbonate (3.58 g, 16.4 mmol) in
THF (17 mL) at room temperature. The mixture was stirred at room
temperature for 1 h. It was then diluted with water (120 mL) and
ethyl acetate (240 mL). The organic layer was washed with brine
(120 mL) and dried over anhydrous Na2SO4. The insoluble was
filtered off, and the filtrate was concentrated under reduced
pressure. The residue was purified by silica gel column chromatog-
5.3. 2-Chloro-3-(1-cyanocyclopropyl)-N-(cyclopropylcarbonyl)-
N-[5-({2-[(cyclopropylcarbonyl)amino][1,3]thiazolo[5,4-b]
pyridin-5-yl}oxy)-2-fluorophenyl]benzamide (4)
To a mixture of compound 11 (3.5 g, 6.39 mmol) and DMAP
(3.87 g, 31.7 mmol) in pyridine (35 mL) was added cyclopropane-
carbonyl chloride (3.0 mL, 33.1 mmol) at 10 °C. The reaction mix-
ture was stirred at room temperature for 1.5 h. The mixture was
poured into cooled water (70 mL), and the mixture was extracted
with ethyl acetate (3 Â 80 mL). The organic layer was washed
with water (50 mL) and brine (30 mL), successively, and dried
over anhydrous MgSO4. The insoluble was filtered off, and the fil-
trate was concentrated under reduced pressure. The residue was
raphy (mesh 60 lm, 100 g, eluent 5–80% ethyl acetate in n-hexane).
Desired fractions were combined and evaporated in vacuo to give
crude 6 as white amorphous powder (2.25 g, 63%). Crystallization
of the crude 6 (1.0 g) was carried out from methanol/water (9:1,
10 mL), and the obtained crystals were collected by filtration, dried
at 75 °C under vacuum to give 6 (0.95 g, 95%) as monohydrate white
crystalline solid; mp 134 °C (dec). 1H NMR (DMSO-d6, 300 MHz): d
0.84–1.02 (m, 4H), 1.14 (s, 9H), 1.32–1.55 (m, 2H), 1.68–1.88 (m,
2H), 1.90–2.08 (m, 1H), 7.16 (d, J = 8.7 Hz, 1H), 7.27–7.40 (m, 1H),
7.42–7.55 (m, 3H), 7.60–7.76 (m, 2H), 8.20 (d, J = 8.7 Hz, 1H),
purified by silica gel column chromatography (mesh 60 lm,
100 g, eluent 5–80% ethyl acetate in n-hexane). Desired fractions
were combined and evaporated under reduced pressure. Crystal-
lization of the residue was carried out from ethyl acetate, and the
obtained crystals were collected by filtration, dried under vac-
uum to give 4 as white crystalline solid (2.47 g, 63%); mp
12.70 (s, 1H). Powder X-ray diffraction (Cu-Ka radiation, diffraction
angle: 2h (°)): 2.86, 5.84, 9.90, 10.96, 11.2, 11.64, 12.78, 13.26, 14.9,
15.4, 15.8, 16.56, 17.28, 17.8, 18.46, 18.82, 19.1, 19.98, 20.38, 21.66,
22.02, 22.64, 22.9, 23.12, 23.5, 23.8, 24.78. Anal. Calcd for
190 °C. 1H NMR (DMSO-d6, 300 MHz):
d
0.84–1.03 (8H, m),
C
32H27ClFN5O5SÁH2O: C, 57.70; H, 4.39; N, 10.51. Found: C, 57.90;
1.33–1.44 (m, 2H), 1.72–1.80 (m, 2H), 1.81–1.93 (m, 1H), 1.93–
2.06 (m, 1H), 7.13 (d, J = 8.7 Hz, 1H), 7.29–7.64 (m, 6H), 8.19
(d, J = 8.7 Hz, 1H), 12.71 (br s, 1H). Powder X-ray diffraction
H, 4.39; N, 10.37.
5.6. tert-Butyl {[2-chloro-3-(1-cyanocyclopropyl)phenyl]
carbonyl}[5-({2-[(cyclopropylcarbonyl)amino][1,3]thiazolo
[5,4-b]pyridin-5-yl}oxy)-2-fluorophenyl]carbamate ethanol
(1/1) solvate (6a)
(Cu-Ka radiation, diffraction angle: 2h (°)): 2.52, 2.76, 4.22,
4.86, 5.24, 7.46, 8.46, 9.76, 10.18, 10.54, 11.36, 11.62, 12.38,
13.04, 13.52, 13.84, 14.14, 14.78, 15.28, 15.56, 15.88, 16.32,
16.6, 16.8, 17.34, 17.8, 18.68, 19.12, 19.54, 20.02, 21.02, 21.56,
21.98, 22.7, 22.94, 23.4, 23.8, 24.5, 24.86. Anal. Calcd for
Crystallization of 6 (50 mg) was carried out from ethanol/water
(9:1, 0.44 mL), and the obtained crystals were collected by filtra-
tion, dried at 75 °C under vacuum to give 6a (45 mg, 84%) as white
C31H23ClFN5O4S: C, 60.44; H, 3.76; N, 11.37. Found: C, 60.26; H,
3.84; N, 11.25.

4690
M. Okaniwa et al. / Bioorg. Med. Chem. 20 (2012) 4680–4692
crystalline solid; mp 138 °C (dec). 1H NMR (DMSO-d6, 300 MHz): d
0.82–1.00 (m, 4H), 1.06 (t, J = 7.0 Hz, 3H), 1.14 (s, 9H), 1.35–1.58
(m, 2H), 1.71–1.85 (m, 2H), 1.94–2.07 (m, 1H), 3.38–3.53 (m,
2H), 4.34 (t, J = 5.1 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 7.26–7.41 (m,
1H), 7.41–7.57 (m, 3H), 7.60–7.74 (m, 2H), 8.19 (d, J = 8.7 Hz,
trated under reduced pressure. The obtained residue was purified
by silica gel column chromatography (50–100% ethyl acetate/n-
hexane), and crystallized from diethyl ether. The obtained crystals
were collected by filtration, dried under vacuum to give 11 (7.90 g,
79%) as yellow powder. 1H NMR (DMSO-d6, 300 MHz): d 3.88 (br s,
2H), 6.43–6.48 (m, 1H), 6.57 (dd, J = 2.7, 9.0 Hz, 1H), 6.99 (dd,
J = 0.6, 9.0 Hz, 1H), 7.04 (dd, J = 8.7, 10.5 Hz, 1H), 8.46 (dd, J = 3.0,
9.0 Hz, 1H), 9.06 (dd, J = 0.3, 2.7 Hz, 1H). HRMS (ESI): calcd for
1H), 12.70 (br s, 1H). Powder X-ray diffraction (Cu-K
a radiation,
diffraction angle: 2h (°)): 5.98, 9.76, 10, 11.64, 12.04, 12.98,
13.32, 14.46, 14.78, 15.16, 15.9, 16.56, 16.98, 17.76, 18.16, 18.58,
18.94, 19.32, 19.62, 20.08, 20.42, 20.98, 21.2, 21.46, 22.22, 22.56,
23.28, 23.44, 23.9, 24.32, 24.78. Anal. Calcd for C32H27ClFN5O5SÁ1.0
EtOHÁ0.5H2O: C, 58.07; H, 4.87; N, 9.96. Found: C, 57.90; H, 4.73; N,
10.06.
C
11H8FN3O3 [M+H]+ 250.0622. Found: 250.0607.
5.10. tert-Butyl {5-[(5-aminopyridin-2-yl)oxy]-2-fluorophenyl}
carbamate (12)
5.7. tert-Butyl {[2-chloro-3-(1-cyanocyclopropyl)phenyl]
carbonyl}[5-({2-[(cyclopropylcarbonyl)amino][1,3]thiazolo
[5,4-b]pyridin-5-yl}oxy)-2-fluorophenyl]carbamate acetic acid
(1/1) solvate (6b)
A solution of 11 (7.43 g, 30 mmol) and di-tert-butyl dicarbonate
(10.9 g, 50 mmol) in tetrahydrofuran (50 mL) was refluxed for 12 h.
After cooling at room temperature, the reaction mixture was
poured into water (200 mL), and the mixture was extracted with
ethyl acetate (2 Â 100 mL). The combined organic layers were
dried over anhydrous Na2SO4. The insoluble material was filtered
off, and the filtrate was concentrated under reduced pressure.
The obtained residue was triturated with diethyl ether to give
tert-butyl {2-fluoro-5-[(5-nitropyridin-2-yl)oxy]phenyl}carbamate
(9.41 g, 90%) as white amorphous solid. 1H NMR (DMSO-d6,
300 MHz): d 1.51 (s, 9H), 6.73–6.79 (m, 1H), 6.81 (br s, 1H), 7.03
Crystallization of 6 (200 mg) was carried out from acetic acid/
water (4:1, 1.0 mL), and the obtained crystals were collected by fil-
tration, dried at 60 °C under vacuum to give 6b (199 mg, 91%) as
white crystalline solid; mp 141 °C (dec). 1H NMR (DMSO-d6,
300 MHz): d 0.85–1.05 (m, 4H), 1.14 (s, 9H), 1.38–1.53 (m, 2H),
1.74–1.85 (m, 2H), 1.90 (s, 3H), 1.94–2.07 (m, 1H), 7.17 (d,
J = 8.7 Hz, 1H), 7.28–7.40 (m, 1H), 7.42–7.57 (m, 3H), 7.61–7.73
(m, 2H), 8.20 (d, J = 8.7 Hz, 1H), 11.95–12.93 (br s, 2H). Powder X-
(d, J = 9.0 Hz, 1H), 7.13 (dd,
J = 9.0, 10.5 Hz, 1H), 8.02 (d,
J = 4.5 Hz, 1H), 8.47 (dd, J = 3.0, 9.0 Hz, 1H), 9.04 (d, J = 2.7 Hz, 1H).
A suspension of tert-butyl {2-fluoro-5-[(5-nitropyridin-2-yl)
oxy]phenyl}carbamate (3.49 g, 10 mmol) and 10% palladium-
carbon (1.0 g) in methanol/tetrahydrofuran (1:1, 40 mL) was vigor-
ously stirred at room temperature under hydrogen atmosphere for
12 h. The insoluble material was filtered off, and the filtrate was con-
centrated under reduced pressure. The residue was poured into
water (200 mL), and the mixture was extracted with ethyl acetate
(2 Â 100 mL). The combined organic layers were dried over anhy-
drous Na2SO4. The insoluble material was filtered off, and the filtrate
was concentrated under reduced pressure, and the residue was trit-
urated with diethyl ether to give 12 (3.00 g, 94%) as white amor-
phous solid. 1H NMR (DMSO-d6, 300 MHz): d 1.50 (s, 9H), 3.49 (br
s, 2H), 6.65–6.70 (m, 1H), 6.71 (br s, 1H), 6.75 (d, J = 8.7 Hz, 1H),
7.02 (dd, J = 9.0, 10.5 Hz, 1H), 7.07 (dd, J = 3.0, 8.4 Hz, 1H), 7.68 (d,
J = 3.0 Hz, 1H), 7.88 (d, J = 2.7 Hz, 1H). MS (ESI): m/z 320 (M+H)+.
ray diffraction (Cu-Ka radiation, diffraction angle: 2h (°)): 6.16,
7.72, 8.4, 8.78, 9.1, 10.84, 11.16, 11.64, 12.44, 12.64, 12.92, 13.38,
13.98, 14.26, 14.58, 15.4, 16.1, 16.42, 17.06, 17.74, 17.98, 18.48,
18.8, 19.72, 20.04, 20.34, 20.72, 21.08, 21.58, 21.92, 22.54, 22.94,
23.62, 24.04, 24.28, 24.82. Anal. Calcd for
1.0AcOH: C, 57.67; H, 4.41; N, 9.89. Found: C, 57.58; H, 4.47; N, 9.85.
C
32H27ClFN5O5SÁ
5.8. tert-Butyl (5-{[2-(cyclopropylcarbonyl)amino[1,3]
thiazolo[5,4-b]pyridin-5-yl]oxy}-2-fluorophenyl)carbamate (9)
To a solution of tert-butyl {5-[(2-amino[1,3]thiazolo[5,4-b]pyri-
din-5-yl)oxy]-2-fluorophenyl}carbamate 13 (1.13 g, 3.0 mmol) and
DMAP (1.22 g, 10 mmol) in pyridine (10 mL) was added
dropwise cyclopropanecarbonyl chloride (1.05 g, 10 mmol) under
ice-cooling, and the reaction mixture was stirred at room temper-
ature for 12 h. To the reaction mixture was added water (200 mL),
and the mixture was extracted with ethyl acetate (2 Â 100 mL).
The combined organic layers were dried over anhydrous MgSO4.
The insoluble material was filtered off, and the filtrate was concen-
trated under reduced pressure. The obtained residue was triturated
with diethyl ether to give 9 (0.78 g, 59%) as pale yellow amorphous
solid. Crystallization of the solid was carried out from ethyl ace-
tate/n-heptane (1:1), and the obtained crystals were collected by
filtration, dried under vacuum to give colorless crystalline solid;
mp 268–270 °C. 1H NMR (DMSO-d6, 300 MHz): d 1.02–1.10 (m,
2H), 1.21–1.28 (m, 2H), 1.50 (s, 9H), 1.58–1.67 (m, 1H), 6.76–
6.81 (m, 2H), 6.95 (d, J = 8.7 Hz, 1H), 7.08 (dd, J = 9.0, 10.8 Hz,
1H), 7.96 (d, J = 3.6 Hz, 1H), 7.97 (br s, 1H), 10.12 (br s, 1H). MS
(ESI): m/z 445 (M+H)+. Anal. Calcd for C21H21FN4O4S: C, 56.75; H,
4.76; N, 12.61. Found: C, 56.51; H, 4.83; N, 12.47.
5.11. tert-Butyl {5-[(2-amino[1,3]thiazolo[5,4-b]pyridin-5-yl)
oxy]-2-fluorophenyl}carbamate (13)
To a solution of 12 (3.00 g, 9.4 mmol) and potassium thiocyanate
(3.93 g, 40 mmol) in acetic acid (40 mL) was added dropwise bro-
mine (2.40 g, 15 mmol) under ice-cooling, and the mixture was stir-
red at room temperature for 12 h. The yellow insoluble material was
filtered off, and the filtrate was concentrated under reduced pres-
sure. To the residue was added saturated NaHCO3 (200 mL), and
the mixture was extracted with ethyl acetate (2 Â 100 mL). The
combined organic layers were dried over anhydrous Na2SO4. The
insoluble material was filtered off, and the filtrate was concentrated
under reduced pressure. The obtained residue was triturated with
diethyl ether to give 13 (3.20 g, 90%) as pale yellow amorphous solid.
1H NMR (DMSO-d6, 300 MHz): d 1.51 (s, 9H), 5.49 (br s, 2H), 6.71–
6.76 (m, 2H), 6.85 (d, J = 8.4 Hz, 1H), 7.06 (dd, J = 9.0, 10.5 Hz, 1H),
7.73 (d, J = 8.7 Hz, 1H), 8.30 (br s, 1H). HRMS (ESI): calcd for
5.9. 2-Fluoro-5-[(5-nitropyridin-2-yl)oxy]aniline (11)
A mixture of 2-chloro-5-nitropyridine (10, 6.34 g, 40 mmol), 3-
amino-4-fluorophenol (5.08 g, 40 mmol) and potassium carbonate
(5.52 g, 40 mmol) in DMF (20 mL) was stirred at room temperature
for 12 h. The reaction mixture was poured into water (200 mL), and
the mixture was extracted with ethyl acetate (2 Â 100 mL). The
combined organic layers were dried over anhydrous Na2SO4. The
insoluble material was filtered off, and the filtrate was concen-
C
17H17FN4O3S [M+H]+ 377.1078. Found: 377.1070.
5.12. tert-Butyl (5-{[2-(acetylamino)[1,3]thiazolo[5,4-b]pyridin-
5-yl]oxy}-2-fluorophenyl)carbamate (14)
To a solution of 13 (1.13 g, 3.0 mmol) and DMAP (1.22 g,
10 mmol) in pyridine (10 mL) was added dropwise acetyl chloride

M. Okaniwa et al. / Bioorg. Med. Chem. 20 (2012) 4680–4692
4691
(0.79 g, 10 mmol) under ice-cooling, and the reaction mixture was
stirred at room temperature for 12 h. To the reaction mixture was
added water (200 mL), and the mixture was extracted with ethyl
acetate (2 Â 100 mL). The combined organic layers were dried over
anhydrous MgSO4. The insoluble material was filtered off, and the
filtrate was concentrated under reduced pressure. The obtained
residue was purified by silica gel column chromatography
(0–100% ethyl acetate/n-hexane), and triturated with diethyl ether
to give 14 (0.65 g, 52%) as white amorphous solid. 1H NMR (DMSO-
d6, 300 MHz): d 1.52 (s, 9H), 2.24 (s, 3H), 6.76–6.80 (m, 1H), 6.81–
6.89 (m, 1H), 6.97 (dd, J = 4.5, 8.7 Hz, 1H), 7.09 (dd, J = 9.0, 10.5 Hz,
1H), 7.93 (d, J = 8.7 Hz, 1H), 7.98 (br s, 1H), 10.45 (br s, 1H). MS
(ESI): m/z 419 (M+H)+.
radiation generated at 50 mA and 40 kV. Sample was placed on sil-
icone plate at room temperature. Data was collected from 2° to 35°
(2h) at a step size of 0.02° and scanning speed of 6°/min. The crys-
tallinity of each sample was calculated by Hermans method.19 The
procedure is described as follows: (1) calculate all peak areas from
the XRD pattern of the sample; (2) subtract background area from
the area of (1) by the blank analysis; (3) subtract the halo pattern
derived from amorphous in the sample from the area of (2); (4) cal-
culate the crystallinity of the sample, [Area of 3]/[Area of 2] Â 100.
5.16. Thermal analysis
Differential scanning calorimetry (DSC) was performed using a
DSC EXSTAR 6200 system (Seiko Instruments, Chiba, Japan). A
DSC thermogram was obtained in a closed aluminum pan using a
sample weight of ca 3 mg and a heating rate of 5 °C/min under a
nitrogen flow at 50 mL/min.
5.13. Mechanism analysis of Boc introducing reaction of 1
To a mixture of compound 1 (300 mg, 0.547 mmol) and pyri-
dine (0.88 mL, 10.9 mmol) in tetrahydrofuran (2.12 mL) was added
di-tert-butyl dicarbonate (119 mg, 0.545 mmol) at room tempera-
ture. The reaction mixture was stirred at room temperature. Dur-
5.17. Thermodynamic solubility
ing the reaction, test samples (50
were taken out at 1, 30, 60, 160 min. Then, additional amount of
di-tert-butyl dicarbonate (119 mg, 0.545 mmol) was added at room
temperature. During the further reaction, test samples (50 lL) of
l
L) of the reaction mixture
About 0.5 mg of sample was added to 0.5 mL of a dissolving sol-
vents, which was 20 mmol/L bile acid in the JP 2nd fluid for disinte-
gration solution (pH 6.8)16, and the sample solution were shaken at
37 °C for 18 h. The solution was filtered through a membrane filter
the reaction mixture were taken out at additional 30 and 60 min.
The final di-tert-butyl dicarbonate (119 mg, 0.545 mmol) was
added to the reaction mixture. The mixture was stirred at room
(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, Mil-
ford, MA, USA) and UV detector (W2487, Waters) operated at
temperature for additional 30 min, and 50 lL of the reaction mix-
ture was sampled at 30 min. To the reaction mixture was added
1 N HCl (15 mL) and 1,2-dimethoxyethane (15 mL) at room tem-
perature. The mixture was stirred at room temperature, and sam-
pled at 2 h after 1 N HCl addition. The reaction mixture was
warming up to 35 °C and stirred at 35 °C for 2 h, then sampled.
230 nm. The packaged column was Shiseido MG-III ODS (3 lm,
4.6 Â 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) acetonitrile. 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.
The volume of each samples was 50 lL. The sampled mixture
was partitioned between ethyl acetate (1.0 mL) and water
(0.2 mL). Ethyl acetate layer was sampled (0.7 mL), and evaporated
under reduced pressure. The resulting oily residue was analyzed by
TLC (ethyl acetate/n-hexane = 1:1) and 1H NMR spectroscopy.
5.18. Intrinsic dissolution rate
The initial dissolution rates were measured using rotating disk
method.20 About 20 mg of drug substance was compressed at the
force of 20 kN/cm2 using a single tablet punch press to obtain a
7 mm diameter disk. The solvent for the dissolution test was
250 mL of 200 mmol/L bile acid in the JP 2nd fluid for disintegra-
tion solution (pH 6.8).16 The disk was rotated at 200 rpm at
37 °C. At each time interval a 0.50 mL portion of an aliquot of the
solution was withdrawn from the flask and diluted with acetoni-
trile to provide twice diluted solution. The compound concentra-
tion was analyzed by HPLC.
5.14. In vitro hydrolysis test
In vitro hydrolysis stability of compound 6b was tested using
hepatic microsomes obtained from rats, dogs, monkeys and hu-
mans (Table 5). The incubation mixtures were prepared under
ice-cold conditions by adding 20
tein/mL), 50 L of potassium phosphate buffer (50 mmol/L, pH 7.4)
and 29 L of ultrapure water in an eppendorf tube. The reactions
were initiated by adding 1 L of compound solution (10 mol/L)
to the incubation mixtures. Incubations were conducted at 37 °C
for 2 h and terminated by adding 100 L of the ice-cold acetoni-
lL of the microsomes (4 mg pro-
l
l
l
l
5.19. X-Ray crystallographic analysis.17
l
trile. The zero-time incubations which served as the controls were
terminated by adding the ice-cold acetonitrile before adding com-
pound. The samples were mixed by vortex mixing vigorously and
centrifuging at 3000Âg 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.
In vitro hydrolysis stability of compound 1 and 2 was tested
using incubation mixture (1 mg protein/mL) and compound solu-
tion (30 lmol/L) (Table 1). Incubations were conducted at 37 °C
A single crystal (0.14Â0.08Â0.04 mm) of ethanol cocrystal 6a
was obtained by recrystallization from ethanol. A single crystal
(0.24 Â 0.15 Â 0.08 mm) of acetic acid cocrystal 6b was obtained
by recrystallization from acetic acid. All measurements were made
on a Rigaku R-AXIS RAPID diffractometer using graphite mono-
chromated Cu-K
a radiation. The structures were solved by direct
methods using the program SIR92.21 The structures were then re-
fined by the full-matrix least-squares refinement (SHELXL9722) with
anisotropic temperature factors for the non-hydrogen atoms and
isotropic temperature factors for the hydrogen atoms. Crystal data
for 6a; C32H27ClFN5O5SÁC2H5OH; M = 694.18; triclinic, space group
P-1 (#2), a = 9.1155(2), b = 12.7039(2), c = 15.2671(3) Å,
for 60 min.
5.15. Powder X-ray diffractometry (XRD)
a
= 96.0441(7), b = 107.4964(7),
Å3, Z = 2, Dc = 1.382 g/cm3, R1 = 0.051, wR2 = 0.172 for 3606 ob-
served reflections with I.2 (I). Crystal data for 6b;
32H27ClFN5O5SÁC2H4O2; M = 708.16; triclinic, space group P-1
c = 93.7673(7)°, V = 1667.94(6)
Powder X-ray diffraction patterns were collected using a RINT
2100 Ultima+ (Rigaku, Tokyo, Japan) with Cu K
r
a
(k = 1.5418 Å)
C

4692
M. Okaniwa et al. / Bioorg. Med. Chem. 20 (2012) 4680–4692
(#2), a = 9.158(4), b = 13.669(6), c = 13.891(6) Å,
a
= 91.75(3),
administered orally to non-fasted mice at a dose of 10 mg/kg. For
single dosing PK study of 6b, the compound was suspended in
0.5% methylcellulose solution for oral administration. Blood sam-
ples were taken from the femoral vein at the designated time
points after dosing, and centrifuged to obtain the plasma fractions.
The plasma samples were de-proteinized with acetonitrile contain-
ing an internal standard. After centrifugation, the supernatant was
diluted with a mixture of 0.01 mol/L ammonium formate solution
and acetonitrile (9:1, v/v) and centrifuged again. The compound
concentrations in the supernatant were measured by LC/MS/MS.
b = 92.17(3),
c
= 96.83(3)°, V = 1724(4) Å3, Dc = 1.364 g/cm3, Z = 2,
R1 = 0.076, wR2 = 0.168 for 3032 observed reflections with I.2r (I).
5.20. In vivo xenograft model
All experiments were approved by the Takeda Animal Care and
Use Committee (Approved No.: TEACUC-00004191, TEACUC-2719).
Human melanoma cell line A375 (purchased from American Type
Culture Collection (ATCC)) was proliferated in Dulbecco’s modified
Eagle medium (DMEM) (Invitrogen) supplemented with 10% heat-
inactivated fetal bovine serum. The cells were cultured in tissue
culture dishes in a humidified incubator at 37 °C in an atmosphere
of 5% CO2. Compound 6b was suspended in vehicle solution includ-
ing 0.5% methylcellulose (Shin-Etsu Chemical) solution. Six-week
old female athymic nude rats (F344/N Jcl-rnu/rnu female (CLEA
Japan, Inc.)) were received subcutaneous injections with 3 Â 106
Acknowledgments
The authors would like to thank Yukiko Watanabe for her assis-
tance in PK evaluation. The authors would like to thank Yoichi Nag-
ano for his assistance in crystallographic data deposition to the
Cambridge Crystallographic Data Centre.
A375 cells in 100 lL of Hanks’ balanced salt solution (Invitrogen).
When tumors reached a volume of 150–250 mm3, mice were orally
administrated with vehicle or compound 6b (12.9, 32.3, 64.6 and
129 mg/kg). At 4 h after administration, the tumor was collected
and homogenized in the lysis buffer (1% NP-40, 0.5 % sodium
deoxycholate, 1 % SDS, 97.5 % Dulbecco’s phosphate-buffered sal-
ines (DPBS) (GIBCO) with Protease Inhibitor Cocktail Set 3 (Calbio-
chem) and Phosphatase Inhibitor Cocktail 2 (Sigma)). The protein
concentration in the tumor lysate was determined by bicinchoni-
nic acid (BCA) protein assay kit (Thermo), and adjusted to
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.06.015.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
References and notes
1.25
l
g/
l
L. An equal volume of 2Â SDS sample buffer (BioRad)
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residual solvents. Q3C(R4). February 2009.
16. The Ministry of Health, Labour and Welfare Ministerial Notification No. 285,
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17. CCDC 862986 (6a) and CCDC 863435 (6b) contain the supplementary
crystallographic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
18. Allen, F. H.; Kennard, O.; Watson, D. G. J. Chem. Soc., Perkin Trans. II 1987, S1.
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was added to the above-mentioned protein solution and incubated
at 95 °C for 5 min. The cell lysates were applied to SDS–PAGE, and
the protein was transferred onto Sequi-Blot™ polyvinylidene
difluoride TPVDF) membrane with the iBlot™ Dry Blotting System
(Invitrogen). The cells membrane were blocked with the Starting
Block Blocking Buffer (PIERCE), and reacted overnight with anti-
phosphorylated ERK1/2 (Ser217/221) (Cell signaling) diluted
1000-fold with Can Get SignalÒ Immunoreaction Enhancer Solu-
tion I (TOYOBO). The membrane was washed with TBS containing
0.05% Tween 20 (Wako Pure Chemical Industries, Ltd.), and reacted
at room temperature for 1 h with horseradish peroxidase (HRP) la-
beled rabbit IgG polyclonal antibody (GE Healthcare) diluted 2000-
fold with Can Get SignalÒ Immunoreaction Enhancer Solution II
(TOYOBO). The membrane was washed in the same manner as
above, chemical luminescence of a phosphorylated MEK1/2 protein
labeled with the antibody, which was caused by ECL-plus Detec-
tion Reagent (GE Healthcare), was detected by Luminescent Image
Analyzer LAS-1000 (FUJIFILM Corporation). Taking the lumines-
cence of the control group free of the test compound as 100%,
the concentration (IC50 value) of the compound necessary for
inhibiting the residual luminescence to 50% of the control group
was calculated using PCP software (SAS Institute). For tumor
growth experiments, rats were randomized into 5 groups (n = 4)
and orally administrated with vehicle or compound 6b (12.9,
32.3, 64.6 and 129 mg/kg) twice-daily for two weeks. Tumor vol-
umes were calculated as volume = L Â l2 Â 1/2, where L was taken
to be the longest diameter across the tumor and l the correspond-
ing perpendicular. Treatment over control (T/C, %), an index of anti-
tumor 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 tu-
mor volume assessment.
22. Sheldrick, G. M. SHELXL97. Program for the refinement of crystal structures.
University of Göttingen, Germany, 1997.
5.21. Pharmacokinetic studies
For oral cassette dosing PK study, test compounds were dis-
solved in a mixture of DMSO and 1,3-butylene glycol and were