Discovery of a selective kinase inhibitor (TAK-632) targeting pan-RAF inhibition: Design, synthesis, and biological evaluation of C -7-substituted 1,3-benzothiazole derivatives

Article

pubs.acs.org/jmc

Discovery of a Selective Kinase Inhibitor (TAK-632) Targeting Pan-

RAF Inhibition: Design, Synthesis, and Biological Evaluation of C‑7-

Substituted 1,3-Benzothiazole Derivatives

Masanori Okaniwa,*^,†Masaaki Hirose,*^,†Takeo Arita,^†Masato Yabuki,^†Akito Nakamura,^†

Terufumi Takagi,^†Tomohiro Kawamoto,^†Noriko Uchiyama,^†Akihiko Sumita,^‡Shunichirou Tsutsumi,^‡

Tsuneaki Tottori,^‡Yoshitaka Inui,^†Bi-Ching Sang,^§Jason Yano,^§Kathleen Aertgeerts,^§,∥Sei Yoshida,^†

and Tomoyasu Ishikawa*^,†

^†Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1 Muraoka-Higashi 2-chome, Fujisawa, Kanagawa

251-8555, Japan

^‡CMC Center, Takeda Pharmaceutical Company Limited, 17-85 Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan

^§Structural Biology, Takeda California, Inc., 10410 Science Center Drive, San Diego, California 92121, United States

S

* Supporting Information

ABSTRACT: With the aim of discovering a selective kinase

inhibitor targeting pan-RAF kinase inhibition, we designed

novel 1,3-benzothiazole derivatives based on our thiazolo[5,4-

b]pyridine class RAF/VEGFR2 inhibitor 1 and developed a

regioselective cyclization methodology for the C-7-substituted

1,3-benzothiazole scaﬀold utilizing meta-substituted anilines.

Eventually, we selected 7-cyano derivative 8B (TAK-632) as a

development candidate and conﬁrmed its binding mode by

cocrystal structure with BRAF. Accommodation of the 7-cyano

group into the BRAF-selectivity pocket and the 3-(triﬂuoromethyl)phenyl acetamide moiety into the hydrophobic back pocket of

BRAF in the DFG-out conformation contributed to enhanced RAF potency and selectivity vs VEGFR2. Reﬂecting its potent

pan-RAF inhibition and slow oﬀ-rate proﬁle, 8B demonstrated signiﬁcant cellular activity against mutated BRAF or mutated

NRAS cancer cell lines. Furthermore, in both A375 (BRAF^V600E) and HMVII (NRAS^Q61K) xenograft models in rats, 8B

demonstrated regressive antitumor eﬃcacy by twice daily, 14-day repetitive administration without signiﬁcant body weight loss.

by our DFG-out type pan-RAF inhibitor, but not by DFG-in

type inhibitors, which signiﬁcantly induced the phosphorylation

of downstream MEK and ERK in CsFb cells. On the basis of

these results, our interest was directed toward DFG-out type

pan-RAF inhibitors to treat cancer patients including oncogenic

BRAF^V600Eas well as NRAS mutant in which BRAF (wt) and

CRAF are being involved in aberrant signal transduction.

Therefore, we initiated a drug discovery program aimed at

targeting DFG-out type pan-RAF inhibitors.

We have previously reported potent DFG-out type pan-RAF

inhibitors¹⁵⁻¹⁷and identiﬁed the thiazolo[5,4-b]pyridine

derivative 1 as an inhibitor of RAF and VEGFR2 kinases

(BRAF(V600E) IC₅₀, 5.6 nM; BRAF(wt), 12 nM; CRAF, 1.5

nM) and VEGFR2 (2.8 nM) (Figure 1).¹⁵Compound 1 also

showed potent cellular activity such as MAPK signal inhibition

in various cell lines possessing the BRAF^V600Emutation and

VEGFR2 phosphorylation inhibition in 293/KDR cells. In vivo

studies in a rat A375 xenograft model demonstrated regressive

antitumor eﬃcacy for compound 1 based on dual inhibition of

INTRODUCTION

■

The RAF family of protein kinases plays critical roles in cancer

progression.¹Recently, BRAF selective inhibitors such as

vemurafenib (PLX4032)²⁻⁴and dabrafenib (GSK2118436)⁵

have shown signiﬁcant clinical eﬃcacy in melanoma patients

bearing oncogenic BRAF^V600Emutation (Figure 1). However,

adverse events such as rapid development of squamous cell

carcinoma (SCC) and keratoacanthoma have been re-

ported.^3,5−8These observations were thought to be caused by

paradoxical activation of the MAPK pathway by BRAF selective

inhibitors in cells bearing wild-type BRAF (BRAF^wt).⁹⁻¹¹

Recent studies^10,12,13on this paradoxical activation reported

that RAF inhibitors instinctively transactivate RAF homodimers

(CRAF−CRAF) or heterodimers (CRAF−BRAF(wt)) and

activate RAS dependent MAPK signaling. Hence, it has been

reported that selective BRAF(V600E) inhibitors, like vemur-

afenib, have not shown potent antiproliferative activity against

cancer cell lines such as NRAS mutant melanoma in which RAS

dependent MAPK signaling is activated.¹⁴

To analyze this, our initial investigation (Supporting

Information, Table S1) revealed that the feedback activation

in ﬁbroblast CsFb cells (BRAF^wt) was signiﬁcantly suppressed

Received: May 26, 2013

A

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Figure 1. Chemical structures of RAF inhibitors including [1,3]thiazolo[5,4-b]pyridine derivative 1 and 1,3-benzothiazole derivative 2 as lead

compounds.

RAF and VEGFR2. However, a rat toxicological study of 1

showed pathological changes derived from angiogenesis

inhibition but not MAPK inhibition. These results imply that

compound 1 could be exerting antiangiogenesis activity on

vascular endothelial cells rather than MAPK inhibition on

cancer cells. On the basis of these results, we sought to increase

the pan-Raf kinase inhibitory potency relative to that for

VEGFR2 kinase.

sequently, we envisioned that the introduction of a suitable

substituent at the C-7 position of 1,3-benzothiazole 2¹⁵(Figure

1) could reduce VEGFR2 inhibitory activity by steric repulsion

between Phe1047 and the 7-substituent (Figures 3 and 4A). On

To better understand kinase selectivity, we utilized the X-ray

cocrystal structures of compound 1 bound to BRAF (PDB

code: 4DBN) and VEGFR2 (PDB code: 3VNT), which we

previously reported.¹⁸The two cocrystal structures were

superimposed and analyzed for diﬀerences. It was revealed

that there is a notable diﬀerence between BRAF and VEGFR2

in the conformations of the benzene ring of phenylalanine in

the DFG motif (Figure 2). The benzene ring of Phe595 in

BRAF (shown in green) is located below the thiazolo[5,4-

b]pyridine scaﬀold, whereas that of Phe1047 in VEGFR2

(shown in purple) is located in front of the N-7 position of

thiazolo[5,4-b]pyridine 1 with a distance of 3.2 Å. Con-

Figure 3. Structure of designed C-7-substituted 1,3-benzothiazole

derivatives and their predicted binding mode to the DFG-out

conformation of BRAF.

the basis of this hypothesis to increase BRAF selectivity over

VEGFR2, we planned to introduce an R¹substituent that would

be accommodated in this BRAF-selectivity pocket in front of

the C-7 position.

Additionally, we examined a new ring B−linker−ring C

moiety targeting the hydrophobic back pocket of BRAF

(Figures 3 and 4B). According to our crystallographic data,

the NHCO amide substructure connected to ring B could

apparently form two signiﬁcant hydrogen bonds with Glu501

and Asp594. Insertion of X (NH or CH₂) into the linker was an

important modiﬁcation because the additional amine (X = NH)

of ureides 4−6 could form an additional hydrogen bond with

the carboxylic acid of Glu501, and the methylene (X = CH₂) of

the acetamides 7−9 was considered ﬂexible enough that the

conformation of ring C could adjust to be accommodated into

the hydrophobic back pocket of BRAF.

Figure 2. Structural diﬀerences in the adenine sites of BRAF and

VEGFR2 bound to RAF/VEGFR2 inhibitor 1. The BRAF structure

(4DBN) is shown in green and the VEGFR2 stucture (3VNT) in

purple.

Our synthetic strategy for the preparation of the C-7-

substituted 1,3-benzothiazole scaﬀold is shown in Scheme 1.

B

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Figure 4. Design approaches employed for the ring A moiety and the ring B−linker−ring C moiety. (A) Ring A moiety: introduction of an R¹group

at the C-7 position for targeting a BRAF-selectivity pocket adjacent to Phe595. (B) Ring B−linker−ring C moiety targeting the hydrophobic back

pocket of BRAF: ureide derivatives 4−6 (linker: NHCONH) and acetamide derivatives 7−9 (linker: NHCOCH₂).

Scheme 1. Synthetic Strategy for the Preparation of C-7-Substituted 1,3-Benzothiazole Intermediates 11 by the Regioselective

Cyclization of Aniline 10

The C-7 unsubstituted (R¹= H) 1,3-benzothiazole derivative 2

was synthesized by Kaufmann’s method¹⁹from 4-phenoxylated

aniline intermediate 10 (R¹= H) using potassium thiocyanate

(KSCN) and bromine (Br₂).¹⁵For the construction of the 1,3-

benzothiazole scaﬀold, this methodology was frequently applied

to symmetrical anilines such as para-substituted anilines²⁰or

ortho-substituted anilines such as 2-methoxy-4-substituted

anilines.²¹In such cases, regioselectivity of the product formed

was either not an issue or was controlled by the presence of an

ortho substituent. In general, the electrophilic aromatic

substitution occurs in a regioselective manner, and the

regioselectivity is generally aﬀected by substituents on the

aromatic ring. Consequently, the regioselective cyclization of

meta-substituted aniline 10 with a variety of R¹substituents (R¹

≠ H) was of great interest in order to enable the eﬃcient

synthesis of desired C-7 isomer 11 (Scheme 1). However, to

the best of our knowledge, only a few publications describing

the preparation of C-7-substituted 1,3-benzothiazoles by

cyclization directly from meta-substituted anilines have been

reported (Supporting Information, Table S2).²²⁻²⁴Therefore,

we initiated synthetic studies on this scaﬀold.

In this article, we describe the design, synthesis, and

structure−activity relationships (SAR) of novel DFG-out type

selective kinase inhibitors targeting pan-RAF inhibition. In

addition, the characterization of our development candidate 8B

using surface plasmon resonance (SPR) spectroscopy and its

application to human cancer cells harboring either mutant

BRAF or mutant NRAS are also discussed.

Chemistry. The precursor anilines 10a−d for 1,3-

benzothiazole formation were prepared as shown in Scheme

2. S_NAr displacement of nitrobenzenes 14a−c with known

phenol derivative 13¹⁵in the presence of potassium carbonate

(K₂CO₃) in N,N-dimethylformamide (DMF) gave the

phenoxylated compounds 15a−c in 70−98% yield. Reduction

of the nitro group for 15a,c was carried out using reduced iron

(Fe(0)) in the presence of calcium dichloride (CaCl₂) in 90%

ethanol/water to avoid side reactions such as deﬂuorination

(10a) or nitrile reduction (10c) under hydrogenation

conditions using palladium on activated carbon (Pd/C). As a

result, the corresponding anilines 10a,c were obtained in 82−

99% yield. For compound 15b, hydrogenation of the nitro

group using Pd/C gave the corresponding aniline 10b in 92%

yield. In this reaction, 1-methylpyrrolidone (NMP) was used as

C

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a

Scheme 2

a

Reagents and conditions: (a) K₂CO₃, DMF, 25−80 °C, 12−18 h (70−98%); (b) Fe(0), CaCl₂, EtOH/H₂O (9:1), 80−100 °C (82−99%); (c) H₂,

10% Pd/C, NMP/MeOH/THF (2:4:1), room temp., 14 h (92%); (d) Cs₂CO₃, DMF, 80 °C, 16 h (82%).

Table 1

b

yield (%)

a

substrate

R¹

C-7 isomer 11

C-5 isomer 12

c

10a

10b

10c

10d

F

11a

11b

11c

11d

N.D.

90

12a

12b

12c

12d

57

CO₂Me

CN

5

c

81

N.D.

NO₂

73

N.D.

a

b

c

R⁴means the 3-(1-cyano-1-methylethyl)benzamide moiety. Isolated yield after conventional workup. Not optimized. Not detected.

a cosolvent to dissolve substrate 15b, which is insoluble in a

mixed solvent of tetrahydrofuran (THF) and methanol

(MeOH). The aniline derivative 10d was directly synthesized

from phenol 13 with 4-ﬂuoro-3-nitroaniline 14d (yield: 82%)

using cesium carbonate (Cs₂CO₃) in DMF.

Next, the regioselective 1,3-benzothiazole formation reaction

for anilines 10a−d was investigated. We applied Kaufmann’s

conditions¹⁹to this reaction; the results are shown in Table 1.

We ﬁrst determined the optimal amounts of KSCN and Br₂for

this reaction. The optimized stoichiometry was 4 and 1.5

equivalents relative to the anilines 10a−d, respectively. Lower

amounts of KSCN or bromine caused low yields. The reaction

of 10a (R¹= F) under these optimized conditions gave C-5

ﬂuorinated isomer 12a in 57% yield. In this experiment, the C-7

isomer 11a was not isolated after conventional workup.

Although the regioselectivity in 10a was undesirable for our

design concept, we continued our eﬀorts to achieve the desired

regioselectivity using other anilines 10b−d possessing various

R¹substituents. Interestingly, the ring formation reactions of

10b (R¹= CO₂Me), 10c (R¹= CN), and 10d (R¹= NO₂)

provided C-7 isomers 11b−d with our desired regioselectivity

in 73−90% isolated yields. Although the reaction of 10b (R¹=

CO₂Me) provided the C-5 isomer in 5% yield, the reaction of

10c (R¹= CN) and 10d (R¹= NO₂) did not provide C-5

isomers. The change of regioselectivity between F and other R¹

groups such as NO₂was also of great interest. This

regioselectivity, meta-ﬂuoroaniline provides a C-5 isomer²²

and meta-nitroaniline provides a C-7 isomer,²⁴is consistent

with other groups’ reports (Supporting Information, Table S2).

The ring formation reaction of thioureide derivatives using

D

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a

Scheme 3

a

Reagents and conditions: (a) cyclopropanecarbonyl chloride, pyridine, room temp., 2−4 h (68−79%); (b) LiOH·H₂O, THF, MeOH, H₂O, room

temp., 12 h (54%); (c) iso-butyl chloroformate, Et₃N, THF, 0°C, 0.5 h; then, NaBH₄, THF, MeOH, room temp., 2 h (55% in 2 steps).

bromine is also known as an alternative method to prepare the

1,3-benzothiazole ring. Some groups have reported that meta-

substituted thioureides can provide C-5 or C-7 benzothiazoles

in a regioselective manner (Supporting Information, Table

S3).²⁵⁻²⁸Interestingly, the tendency for regioselectivity

between both reactions is the same, and the direction of

cyclization is determined by an R¹substituent at the meta-

position (F, C-5, CO₂Me; NO₂, C-7). Regarding the cyano

group, our result is the ﬁrst example that 7-cyano-1,3-

benzothiazole can be prepared using the corresponding meta-

cyanoaniline in a regioselective manner.

The synthesis of 1,3-benzothiazole derivatives 3b−f is shown

in Scheme 3. The acylation of 2-amino-1,3-benzothiazoles

11b−d with cyclopropanecarbonyl chloride in pyridine gave the

desired compounds 3b−d in 68−79% yield. Hydrolysis of the

methyl ester in 3b with lithium hydroxide monohydrate

(LiOH·H₂O) gave the corresponding carboxylic acid 3e in

54% yield. Reduction of the carboxyl group in 3e was achieved

via a mixed anhydride produced by the treatment of iso-butyl

chloroformate with 3e in the presence of triethylamine (Et₃N).

The subsequent reduction of the obtained anhydride with

sodium borohydride (NaBH₄) gave the desired hydroxymethyl

derivative 3f in 55% yield over 2 steps.

The synthesis of C-7 cyanated 1,3-benzothiazoles 4−9

possessing various ring B−linker−ring C moieties is shown in

Scheme 4. This route was planned for optimization of the back

pocket moiety. Therefore, we set the aniline derivatives 22A,B

as key intermediates for the synthesis of substituted phenyl

ureides 4−6 and phenyl acetamides 7−9 in the last step in the

route. The S_NAr displacement of 2-ﬂuoro-5-nitrobenzonitrile

14c with phenol 16A in the presence of K₂CO₃gave the

phenoxylated aniline 17A in 66% yield. Protection of the amine

in 17A by a triﬂuoroacetyl group was achieved in 67% yield

using triﬂuoroacetic anhydride (TFAA), followed by reduction

of the nitro group in 18A under palladium-mediated hydro-

genation conditions in a mixed solvent of NMP/MeOH to give

the desired compound 19A in 99% yield. The 1,3-benzothiazole

formation reaction of 19A using KSCN and bromine

successfully provided the desired 7-cyano-2-amino-1,3-benzo-

thiazole derivative 20A in 81% yield. The acylation of 20A with

cyclopropanecarbonyl chloride (82% yield) and subsequent

triﬂuoroacetyl cleavage from the resulting compound 21A with

LiOH·H₂O gave key intermediate 22A in 95% yield.

The corresponding ﬂuorinated key intermediate 22B (R²=

F) was prepared by a method similar to that used in the

preparation of 22A (R²= H). Reaction of 14c with

commercially available 16B in the presence of K₂CO₃followed

by protection of the amino group using TFAA gave the desired

compound 18B in 93% in 2 steps. Reduction of the nitro group

of 18B by hydrogenation in the presence of Pd/C was carefully

conducted, while monitoring the formation of deﬂuorinated

compound 19A. Ultimately, this reaction gave the desired

aniline 19B in 99% yield without the contamination with 19A.

The 1,3-benzothiazole cyclization reaction of 19B also

successfully provided the desired isomer 20B in 85% yield.

The acylation of 20B with cyclopropanecarbonyl chloride in

the presence of pyridine in THF gave 21B in comparatively

lower yield (55%) than that for the preparation of 21A. The

reason for the reduced yield in this reaction is thought to result

from cleavage of the triﬂuoroacetyl group in 20B. Introduction

of a ﬂuorine atom at the ortho-position of anilide 20B enhances

the acidity of the triﬂuoroacetamide moiety, which may activate

the nucleophilicity of the amide in its reaction with cyclo-

propanecarbonyl chloride to provide an asymmetrical imide at

the anilino moiety (ring B). The postulated asymmetrical imide

formed in situ should be easily cleaved under aqueous workup

conditions to give the corresponding bis-cyclopropanecarbony-

lated compound as a signiﬁcant byproduct. This contaminating

byproduct, fortunately, could be removed by conventional silica

gel column chromatography to give puriﬁed 21B. Since the

triﬂuoroacetamide of 21B was highly resistant against the

conventional alkaline hydrolysis conditions due to the presence

E

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a

Scheme 4

a

Reagents and conditions: (a) 16A,B, K₂CO₃, DMF, 25−60 °C, 2−4 h (66−100%); (b) TFAA, THF, room temp., 1−1.5 h (67−93%); (c) H₂, 10%

Pd/C, MeOH, room temp., 2−8 h (99%); (d) KSCN, Br₂, AcOH, room temp., 12 h (81−85%); (e) cyclopropanecarbonyl chloride, pyridine, THF

room temp., 6−16 h (55−82%); (f) LiOH·H₂O, THF/MeOH/H₂O (1:1:1), room temp., 18 h (95%); (g) NaBH₄, MeOH (2.4 v/v%), EtOH, room

temp., 0.5 h (85%); (h) phenyl isocyanate, DMF, room temp., 12 h (28−94%); (i) phenyl acetic acid, HATU, pyridine, 85 °C, 4 h (57−74%).

of the ortho-ﬂuorine group, hydride conditions using NaBH₄

were applied for cleavage of the triﬂuoroacetyl group. At ﬁrst,

deprotection using NaBH₄was found to be very slow in EtOH.

Optimal conditions were obtained by using 2−10 v/v% MeOH

as an additive to accelerate the reaction. Use of greater than 10

v/v% of MeOH lowered yields due to undesirable hydrolysis of

the cyclopropane carboxamide moiety at the 2-position.

Ultimately, cleavage of the triﬂuoroacetyl group from 21B

using NaBH₄in the presence of 2.4 v/v% MeOH in EtOH gave

deprotected aniline 22B in 85% yield.

Ureide formation from intermediate anilines 22A,B with the

corresponding isocyanate reagents was accomplished under

conventional conditions to give the desired ureide derivatives

4A, 5A,B, and 6A,B in 28−94% yield. Condensation of anilines

22A,B with the corresponding phenyl acetic acids using

hexaﬂuorophosphate, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-

tetramethyluronium (HATU), as a coupling reagent in the

presence of pyridine provided the desired acetamide derivatives

7A, 8A,B, and 9A in 57−74% yield.

RESULTS AND DISCUSSION

■

The 1,3-benzothiazoles 3b−f that possess various R¹

substituents at the C-7 position were evaluated, and the SAR

of 3b−f compared to C-7 unsubstituted 2 (R¹= H) is shown in

Table 2. Nitro derivative 3b (R¹= NO₂) retained potent BRAF

inhibition (IC₅₀= 25 nM), while demonstrating a 5-fold

reduction in VEGFR2 inhibitory activity (IC₅₀= 70 nM)

compared to that of compound 2. The ratio of VEGFR2 IC₅₀to

BRAF IC₅₀(V/B ratio) is a useful metric in discussing the

selectivity of our BRAF inhibitors. Introduction of a nitro group

F

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Table 2. Structure−Activity Relationship for C-7-Substituted 1,3-Benzothiazoles (R¹)

a

kinase IC₅₀(nmol/L)

b

R¹

BRAF (V600E)

VEGFR2

ratio VEGFR2/BRAF (V600E)

cellular pMEK IC₅₀(nmol/L)

2

H

25 (23−28)

25 (19−32)

13 (11−16)

12 (9.6−15)

290 (160−500)

24 (22−26)

14 (13−16)

70 (62−79)

76 (52−110)

33 (30−36)

15 (13−17)

0.56

2.8

240

29

3b

3c

3d

3e

3f

NO₂

CN

5.8

34

CO₂Me

CO₂H

CH₂OH

2.8

24

0.051

0.31

N.D.

170

7.4 (6.8−8.2)

a

b

n = 2. 95% conﬁdence intervals are shown in parentheses. Cellular phosphorylation level of the downstream MEK (pMEK) was evaluated in HT-

29 colon cancer cells (n = 1).

Table 3. Structure−Activity Relationship for Ureide Derivatives 4−6 Compared with that of Amide 3c

a

kinase IC₅₀(nmol/L)

b

c

ratio VEGFR2/BRAF

(V600E)

cellular pMEK IC₅₀

(nmol/L)

PO AUC mice

X

R²

R³

BRAF (V600E)

VEGFR2

(μg h/kg)

3c

bond

NH

H

F

m-C(CH₃)₂CN

o-CF₃

13 (11−16)

19 (16−23)

18 (5.6−58)

18 (12−26)

49 (17−140)

26 (13−51)

76 (52−110)

5.8

6.3

19

18

15

25

34

0.149

0.058

0.099

0.431

N.D.

4A

5A

6A

5B

6B

120 (82−170)

350 (310−410)

330 (250−430)

730 (650−810)

>500

75

m-CF₃

p-CF₃

110

22

m-CF₃

F

p-CF₃

660 (530−830)

b

81

0.158

a

n = 2. 95% conﬁdence intervals are shown in parentheses. Cellular phosphorylation level of the downstream MEK (pMEK) was evaluated in HT-

29 colon cancer cells (n = 1). Mean AUC_{0−8 h}values are shown (n = 2). Cassette dosing was of ﬁve compounds. Compounds (10 mg/kg) were

c

administered in 0.5% methyl cellulose in distilled water.

Table 4. Structure−Activity Relationship for Acetamide Derivatives 7−9

a

kinase IC₅₀(nmol/L)

b

c

ratio VEGFR2/BRAF

(V600E)

cellular pMEK IC₅₀

PO AUC mice

X

R²

R³

BRAF (V600E)

VEGFR2

(nmol/L)

(μg h/kg)

7A

8A

9A

8B

CH₂

H

F

o-CF₃

m-CF₃

p-CF₃

m-CF₃

14 (9.2−20)

1.3 (0.68−2.6)

9.5 (7.9−11)

2.4 (0.45−12)

100 (89−110)

150 (130−190)

190 (160−230)

7.1

120

20

>500

360

320

75

0.255

3.782

0.123

2.482

160 (140−180)

67

a

b

n = 2. 95% conﬁdence intervals are shown in parentheses. Cellular phosphorylation level of the downstream MEK (pMEK) was evaluated in HT-

29 colon cancer cells (n = 1). Mean AUC_{0−8 h}values are shown (n = 2). Cassette dosing was of ﬁve compounds. Compounds (10 mg/kg) were

c

administered in 0.5% methyl cellulose in distilled water.

at the C-7 position (3b) improved BRAF selectivity (V/B ratio

for NO₂, 2.8; for H, 0.56). Furthermore, the cellular inhibitory

activity of 3b toward MEK phosphorylation (IC₅₀= 29 nM)

was approximately 8-fold more potent than that for 2.

inhibitory activity of compounds 3c,d was equal to that of 3b

with an IC₅₀range of 24−34 nM. However, carboxylic acid

derivative 3e (R¹= CO₂H) and hydroxymethyl derivative 3f

(R¹= CH₂OH) showed diﬀerent SAR. Compound 3e showed

reduced BRAF inhibitory activity (IC₅₀= 290 nM), whereas

compound 3f retained BRAF inhibition with an IC₅₀value of

24 nM but with reduced cellular pMEK inhibition (IC₅₀= 170

nM). In addition, the V/B ratios of 3e and 3f dropped to 0.051

and 0.31, respectively. As a result, we found the C-7 position to

be signiﬁcant in modulating the V/B ratio, and nitro (3b),

Next, we evaluated the cyano derivative 3c (R¹= CN) and

the methoxycarbonyl derivative 3d (R¹= CO₂Me). Com-

pounds 3c and 3d showed a 2-fold increase in BRAF inhibitory

potency (3c, 13 nM; 3d, 12 nM) compared with that of the

nitro derivative 3b. The V/B ratio of 3c is about 2-fold that of

3d (V/B ratio for 3c, 5.8; for 3d, 2.8). The cellular pMEK

G

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cyano (3c), and methoxycarbonyl (3d) derivatives demon-

strated high V/B ratios as well as potent cellular pMEK

inhibition. The cyano derivative 3c had the highest V/B ratio

and was selected for further optimization.

In order to improve the V/B ratio further, we conducted

chemical modiﬁcation of the ring B−linker−ring C portion of

3c to explore appropriate linkers (X) targeted for selective RAF

inhibitors. First, we evaluated the ureide derivatives (X = NH,

R²= H) possessing a triﬂuoromethyl group with various

substitution positions such as o-CF₃(4A), m-CF₃(5A), and p-

CF₃(6A) (Table 3). Among them, the m-CF₃(5A) and p-CF₃

(6A) derivatives showed improved V/B ratios (V/B ratio for

m-, 19; for p-, 18) compared with that of the o-CF₃derivative

(V/B ratio for 4A: 6.3). Thus, we selected m- and p-CF₃groups

on ring C and attempted to introduce a ﬂuorine group at the

metabolically labile site of ring B on the basis of our previous

work.^15,17Compound 5B (R²= F, R³= m-CF₃) and 6B (R²=

F, R³= p-CF₃) showed comparable or slightly weaker BRAF

inhibition (5B IC₅₀, 49 nM; 6B, 26 nM) and V/B ratios similar

to those of the corresponding unsubstituted (R²= H)

derivatives 5A or 6A. Interestingly, the cellular pMEK

inhibitory potency of these ﬂuorinated compounds 5B and

6B increased, giving IC₅₀values of 22 and 81 nM, respectively.

However, in spite of attractive cellular activity with improved

BRAF selectivity, oral absorption for the ureide series was

found to be extremely low with AUC_{0−8 h}values of 0.058−

0.431 μg h/kg at a dose of 10 mg/kg in mice.

Figure 5. X-ray cocrystal structure of 8B bound to BRAF (2.93 Å

resolution).

reported.^15,18Furthermore, it was also shown that the

phenylacetamide linker of 8B forms signiﬁcant hydrogen

bonds between the carboxylate of Glu501 and the NH of the

acetamide (3.1 Å), and between the NH of Asp594 and the

carbonyl of the acetamide (2.8 Å). The methylene of the

phenylacetamide linker X in 8B seems to be important for

providing enough ﬂexibility to ﬁt into the back pocket of BRAF.

To clarify the importance of the phenylacetamide linker in

8B, we also obtained an X-ray cocrystal structure of ureide 5B

(PDB code: 4KSQ).³¹The only diﬀerence between 8B and 5B

is the linker X (8B, CH₂; 5B, NH) (Figure 6). This single

Next, we evaluated the acetamide derivatives (X = CH₂, R²=

H) having a CF₃group in three diﬀerent positions as shown in

Table 4. Meta-substitution of the CF₃group (8A) showed

notably enhanced BRAF inhibition, with potency at least 7-fold

greater than that for the ortho (7A)- and para-substituted (9A)

derivatives (IC₅₀values: m-, 1.3 nM; p-, 9.5 nM; o-, 14 nM).

Reﬂecting the remarkable increase in BRAF inhibitory potency

for 8A, we observed that the V/B ratio increased to 120. These

results encouraged us to synthesize ﬂuorine derivative 8B (R²=

F). As we expected, compound 8B showed a promising

enzymatic proﬁle for both BRAF inhibition (IC₅₀: 2.4 nM) and

BRAF selectivity (V/B ratio: 67). In addition, this ﬂuorinated

derivative also exhibited potent cellular pMEK activity with an

IC₅₀value of 75 nM. Furthermore, the pharmacokinetic (PK)

evaluation of m-CF₃derivatives 8A,B showed signiﬁcantly

improved oral absorption with respective AUC values of 3.782

and 2.482 μg h/kg in mice, compared with those of the o- and

p-CF₃derivatives. On the basis of promising BRAF potency,

selectivity, and good PK, compound 8B was selected for further

evaluation.

X-ray Cocrystal Structural Analysis of 8B with BRAF.

We determined the X-ray cocrystal structure of 8B bound to

BRAF kinase (Figure 5).²⁹The BRAF cocrystal structure (PDB

code: 4KSP) revealed that the optimal compound 8B is

accommodated by the DFG-out conformation of BRAF, as we

expected. Within the adenine site, the 1,3-benzothiazole-2-

amide moiety forms two hydrogen bonds between the carbonyl

of Cys532 and the NH of the 2-amide (3.0 Å), and between the

NH of Cys532 and the N-3 nitrogen (3.4 Å). Since the

measured distance between the carbonyl oxygen of the 2-amide

and the S-1 sulfur (2.9 Å) is shorter than the sum of the

corresponding van der Waals radii of the oxygen and sulfur

atoms (3.32 Å),³⁰the conformation of the 2-amide with respect

to the 1,3-benzothiazole scaﬀold is held in a planar

conformation by the sulfur−carbonyl interaction. These results

for 8B are consistent with those for 1 that we previously

Figure 6. X-ray cocrystal structures of BRAF bound to acetamide

derivative 8B and of BRAF bound to the corresponding ureide

derivative 5B. (A) Triﬂuoromethyl-substituted benzene of 8B in the

back pocket region. (B) Triﬂuoromethyl-substituted benzene of the

corresponding ureide 5B in the back pocket region. (C) Binding mode

of acetamide 8B with Glu501 and Asp594 (2.93 Å resolution). (D)

Binding mode of ureide 5B with Glu501 and Asp594 (3.30 Å

resolution).

change resulted in an approximately 20-fold increase in BRAF

inhibitory activity for 8B (IC₅₀: 2.4 nM) compared with that for

5B (IC₅₀: 49 nM). The cocrystal structural analysis revealed

that the m-CF₃-substituted benzene moieties of both 8B and

5B bind to the hydrophobic back pocket as we had envisioned

(Figure 6A,B). In the cocrystal structure of 8B, the phenyl-

acetamide linker anchors the benzene moiety into a twisted

orthogonal conformation with a dihedral angle of 88° at the

H

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CH₂position (Figure 6C). However, the ureide 5B shows a

decreased dihedral angle of 20° at the corresponding NH

position (Figure 6D). Since it is known that the acetamide

linker provides more ﬂexible conformations in a protein than

the ureide moiety does,³²the ﬂexibility of phenylacetamide

derivative 8B allows optimal hydrophobic interaction between

an 88° twisted m-CF₃-substituted benzene and a hydrophobic

back pocket. We assume that this conformational change could

account for the increase in RAF inhibition for 8B.

kinetics (low k_off) along with long residence time binding to

both BRAF and CRAF. In contrast, PLX4720,³³a precursor

analogue of vemurafenib (PLX4032), showed fast dissociation

kinetics and short residence time under the same assay

conditions (BRAF, k_off= 3.3 × 10⁻²s⁻¹; t_1/2= 21 s; CRAF,

k_{off (1)}= 2.2 × 10⁻³s⁻¹; t_{1/2 (1)}= 5.3 min; k_{off (2)}= 1.2 × 10⁻¹

s⁻¹; t_{1/2 (2)}= 5.8 s). These results suggested that 8B is a slow

oﬀ-rate, pan-RAF inhibitor.

Time Dependent Inhibition Properties of 8B against

BRAF and CRAF. Some reported kinase inhibitors have been

recognized as slow oﬀ-rate inhibitors with long residence times

bound to the target protein.^34,35These long residence times

have been used to explain the potent drug properties found for

these compounds.³⁶

Kinase Inhibitory Proﬁles of Compound 8B. The kinase

inhibitory proﬁle of compound 8B against 26 diﬀerent kinases

is summarized in Table 5. Compound 8B showed potent

Table 5. Kinase Selectivity of Compound 8B

We evaluated the biochemical activity of 8B against BRAF

and CRAF at two diﬀerent ATP concentrations of Km (low)

and 1.0 mM (high, corresponding to 200-times the BRAF K_m

and 2000-times the CRAF K_m) and two preincubation times (0

and 1 h) (Table 7). With 1 h of preincubation time, 8B

a

kinase

IC₅₀(nmol/L)

kinase

PKA

IC₅₀(nmol/L)

b

BRAF (wt)

C-RAF

FGFR3

PDGFRα

PDGFRβ

EGFR

Her2

8.3 (4.9−14)

>10000

1400

b

1.4 (0.75−2.6)

280

PKCθ

CHK1

CK1δ

610

>10000

790

120

ERK1

Table 7. Biochemical Activities of 8B against BRAF(V600E)

and CRAF with Diﬀering Preincubation Times (0 or 1 h)

and under Diﬀerent ATP Concentrations: K_m(Low) or 1000

μM (High)

>10000

740

CDK1

CDK2

Aurora B

p38α

580

TIE2

66

c-Met

>10000

3700

600

a

kinase IC₅₀(nmol/L)

c-Kit

JNK1

>10000

500

Src

GSK3β

MEK1

MEKK1

preincubation time:

ATP concentration:

BRAF(V600E):

0 h

1000 μM

1 h

1000 μM

IR

3700

b

K_m

IKKβ

>10000

15

58

62

2.2

1.5

3.3

5.0

a

b

n = 2. 95% conﬁdence intervals are in parentheses.

CRAF:

8.1

a

b

n = 2. K_mconcentration of ATP; BRAF(V600E), [ATP] = 5 μM.

CRAF: [ATP] = 0.5 μM.

single-digit nanomolar inhibitory activity against BRAF(wt) and

CRAF (BRAF(wt) IC₅₀, 8.3 nM; CRAF, 1.4 nM). In addition,

compound 8B inhibited Aurora B with an IC₅₀value of 66 nM.

Another 8 out of 26 kinases, such as PDGFRβ, FGFR3, GSK3β,

CDK2, P38α, PDGFRα, TIE2, and CDK1 were inhibited by 8B

with a range of IC₅₀values from 120−790 nM. CHK1, IKKβ,

and MEK1 were inhibited over an IC₅₀range of 1400−1700

nM. No signiﬁcant inhibition was observed against the other 12

kinases assayed. On the basis of these preclinical results,

compound 8B was found to be a selective kinase inhibitor

targeting the RAF family kinases.

Dissociation Kinetics of Compound 8B Using Surface

Plasmon Resonance Technology. To determine aﬃnities

and kinetic rate constants, compound 8B was injected over

immobilized BRAF(V600E) or CRAF, and surface plasmon

resonance (SPR) was used to analyze the interaction (Biacore

T100, http://www.biacore.com/). Compound 8B showed

similar equilibrium dissociation constants (K_D) against both

BRAF and CRAF by SPR (BRAF K_D, 1.6 × 10⁻⁹; CRAF, 0.52

× 10⁻⁹; Table 6). The dissociation rate constant (k_off) and the

residence time (t_1/2) of 8B were 1.9 × 10⁻⁵s⁻¹and 602 min for

BRAF, 9.0 × 10⁻⁵s⁻¹and 129 min for CRAF (Table 6). The

dissociation parameters indicate that 8B has slow dissociation

inhibited BRAF and CRAF in an ATP competitive manner (at

low ATP concentrations BRAF IC₅₀: 15 nM; CRAF: 8.1 nM).

The respective biochemical activity of 8B against BRAF and

CRAF reduced to IC₅₀values of 58 nM and 62 nM at high ATP

concentrations.

Next, we examined the activity of 8B with 1 h of

preincubation time for the assay. At low ATP concentration,

the inhibitory activity of 8B increased in a time-dependent

manner (BRAF IC₅₀, 2.2 nM; CRAF, 1.5 nM). At high ATP

concentration, 8B showed still potent inhibitory activity for

both BRAF and CRAF (IC₅₀: 3.3 nM and 5.0 nM, respectively).

These time-dependent inhibition properties with long residence

times suggested that 8B has the potential to demonstrate

eﬃcient inhibition of both BRAF and CRAF in cells.

In Vitro Pharmacology of Compound 8B. Reﬂecting the

potent BRAF (V600E) inhibitory activity in vitro, compound

8B inhibited phosphorylation of MEK (pMEK) in melanoma

A375 cells (BRAF^V600E) with an IC₅₀value of 12 nM (Table 8).

Downstream inhibition of ERK phosphorylation (pERK) was

also observed in A375 cells with an IC₅₀value of 16 nM. Next,

we also evaluated the cellular activity of 8B against human

melanoma HMVII cells (NRAS^Q61K/BRAF^G469V), which have

been reported to be insensitive to selective BRAF(V600E)

inhibitors such as vemurafenib.¹⁴In HMVII cells, signal

transduction of the MAPK cascade is thought to be activated

by mutated NRAS through the downstream BRAF(wt) and

CRAF. However, continuous inhibition of BRAF or CRAF by

the corresponding siRNA has been shown to induce down-

stream inhibition in cells with RAS mutation.¹²Interestingly,

Table 6. Dissociation Kinetics of 8B Measured by SPR

a

b

K_D

apparent k_off(sec⁻¹

)

dissociation half-life (min)

BRAF

1.6 × 10⁻⁹

5.2 × 10⁻¹⁰

1.9 × 10⁻⁵

9.0 × 10⁻⁵

602

129

CRAF:

a

b

K_D: equilibrium dissociation constant, K_off: dissociation rate

constant.

I

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Table 8. Cellular Activities of pan-RAF Inhibitor 8B

mean IC

⁵b⁰

(nmol/L)

a

mutation status

proliferation GI₅₀

c

cell line

BRAF

NRAS pMEK pERK

(nmol/L)

A375

V600E

G469V

12

49

16

50

66

HMVII

Q61K

200

a

Mutational status determined from the Sanger Institute database.

b

Numbers represent average IC₅₀values derived from a Western blot

c

assay after 2 h of 8B administration. (n = 2). Numbers represent

average GI₅₀derived from a ATPlite cell proliferation assay after 72 h

of 8B administration. (n = 3).

8B demonstrated strong inhibition of pMEK and pERK in

HMVII cells with IC₅₀values of 49 nM and 50 nM, respectively

(Table 8). Furthermore, antiproliferative activity of 8B was

potent in both A375 and HMVII cells with GI₅₀values of 66

nM and 200 nM, respectively. On the basis of these preclinical

results, compound 8B was identiﬁed as an antitumor drug

development candidate for patients with either BRAF^V600Eor

NRAS mutation, based on its apparent suppression of RAF

dimer feedback activation presumably caused by its slow oﬀ-

rate and pan-RAF inhibitory proﬁle.

Pharmacokinetic Proﬁle and in Vivo Studies of

Compound 8B in Rats. In a pharmacokinetic study,

compound 8B was administered using a solid dispersion

(SD) formulation based on our previous research¹⁵because 8B

showed low solubility (<0.02 μg/mL) in pH 6.8 phosphate

buﬀer.³⁷An SD formulation of 8B demonstrated dramatically

improved solubility (740 μg/mL) in pH 6.8 phosphate buﬀer

and exhibited signiﬁcant oral absorption (at a dose of 25 mg/

kg, AUC, 32.47 μg h/mL; F, 51.7%) in rats (Table 9). In a dog

PK study, 10 mg/kg administration of the SD formulation of

8B also showed superior oral bioavailability (F: 108%).

In vivo eﬃcacy of 8B was evaluated using an SD formulation

in a human melanoma A375 (BRAF^V600E) xenograft model in

F344 nude rats. Reﬂecting the potent in vitro pMEK inhibition,

oral single administration of 8B inhibited pERK in tumors at 8

h after its administration over a dose range of 1.9−24.1 mg/kg

(Figure 7). In particular, 9.7−24.1 mg/kg dosing with 8B

strongly inhibited pERK levels to 11% of the control. We

examined the antitumor eﬃcacy of 8B administered twice daily

for 14 days in an A375 xenograft model in rats (Figure 8).

Compound 8B exhibited dose-dependent antitumor eﬃcacy

without severe body weight reduction over a dose range of 3.9−

24.1 mg/kg. Signiﬁcant tumor regression was observed at 9.7

mg/kg and 24.1 mg/kg (T/C = −2.1% and −12.1%,

respectively).

Figure 7. Mean (n = 3; *P ≤ 0.025 compared with vehicle by the

Shirley−Williams’ test) phosphorylated ERK1/2 levels in the tumor in

a human melanoma A375 (BRAF^V600Emutant) xenograft model in

rats, at 8 h after oral administration of 8B. Data were detected by

Western blotting. The solid dispersion formulation (8B/hydrox-

ypropyl methylcellulose phthalate = 20:80) was delivered in distilled

water.

T/C values of 52%, 26%, and 0% at doses of 3.9 mg/kg, 9.7

mg/kg, and 24.1 mg/kg, respectively. This antitumor eﬃcacy

was dose-dependent and signiﬁcant (p < 0.025) compared with

that of the vehicle control at all three doses. In this model, the

HMVII tumor induced cancer cachexia and resulted in

approximately 6% body weight loss in the vehicle control

group over the 14-day administration period (body weight

change: −8.6 g for the vehicle and +16.5 g for nontumor

bearing). Interestingly, dose-dependent recovery of body

weight loss was observed. In particular, the group at 24.1

mg/kg showed no signs of body weight loss (body weight

change: +28.4 g). Recently, other DFG-out type (Type II)

inhibitors were reported to have the potential to induce RAF

dimerization and feedback activation in BRAF^wtcells, similar to

DFG-in type (Type I) inhibitors.³⁸Some inhibitors are not able

to inhibit activated MAPK signaling in BRAF^wtnormal cells at

their projected eﬃcacious doses and induce hyperplasia in

animal models.³⁹However, a study of our DFG-out type

inhibitor 8B using an A375 (BRAF^V600E) xenograft model

indicated that administration of 8B did not induce pERK

activation in normal skin tissues (BRAF^wt) at projected

eﬃcacious doses but slightly inhibited pERK at higher doses

of 25 and 50 mg/kg (Supporting Information, Figure S1).

Further characterization of how 8B inhibits the feedback

activation in BRAF^wtcells is of great interest. The results of

detailed biological studies of our compounds will be reported in

due course.

Next, antitumor eﬀects of 8B in a human melanoma HMVII

(NRAS^Q61K/BRAF^G469V) xenograft model were evaluated in rats

(Figure 9). In this study, 8B was orally administered as an SD

formulation and suppressed the growth of HMVII tumors with

CONCLUSIONS

We designed and synthesized selective kinase inhibitors

targeting pan-RAF kinase activity. On the basis of the X-ray

■

a

Table 9. Mean Pharmacokinetic Parameters for Compound 8B

dose (mg/kg)

animal

route

CL_total(mL/h/kg)

V_dss(mL/kg)

MRT (h)

AUC_0−24h(μg h/mL)

% F (%)

b

1

rat

iv

400 29

1872 71

4.69 0.25

6.27 0.37

2.23 0.27

7.12 0.76

2.512 0.18

32.47 4.68

1.429 0.13

15.48 2.00

c

25

rat

oral

iv

51.7 8.3

108 8.0

b

1

dog

704 68

1568 226

c

10

oral

a

b

c

Values shown are the mean SD of data (n = 3). Delivered in 1,3-butane-diol/DMA (1:1). Delivered as a solid dispersion formulation (8B/

hydroxypropyl methylcellulose phthalate = 20:80).

J

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Figure 8. Antitumor activity of a solid dispersion of 8B (p.o., b.i.d.) in

human melanoma A375 (BRAF^V600Emutant) bearing F344 nude rats

(A) Mean (n = 4; *P ≤ 0.025 compared with the vehicle at day 14 by a

one-tailed Shirley Williams’ test) tumor volumes. (B) Mean (n = 4; *P

≤ 0.025 compared with the vehicle at day 14 by a one-tailed Shirley

Williams’ test) body weight change. The solid dispersion formulation

(8B/hydroxypropyl methylcellulose phthalate =20:80) was delievered

in distilled water.

Figure 9. Antitumor activity of a solid dispersion of 8B in human

melanoma HMVII (NRAS^Q61K/BRAF^G469Vmutant) bearing F344 nude

rats (A) Mean (n = 4; *P ≤ 0.025 compared with the vehicle at day 14

by a one-tailed Shirley Williams’ test) tumor volumes. (B) Mean (n =

4; *P ≤ 0.025 compared with the vehicle at day 14 by a one-tailed

Shirley Williams’ test) body weight change. The solid dispersion

formulation (8B/hydroxypropyl methylcellulose phthalate = 20:80)

was delievered in distilled water.

cocrystal structures of compound 1 with both BRAF (PDB

code: 4DBN) and VEGFR2 (PDB code: 3VNT), C-7

substitution of lead compound 2 proved to be a fruitful

approach to enhancing RAF inhibitory potency relative to

VEGFR2. These novel compounds were eﬃciently prepared by

the regioselective C-7-substituted 1,3-benzothiazole ring

formation reaction of meta-substituted anilines with KSCN

and bromine. Various substituents, such as NO₂, CN, and

CO₂Me, provided good regioselectivity. Among these scaﬀolds,

the 7-cyano derivatives showed enhanced BRAF selectivity over

VEGFR2 as well as potent cellular pMEK inhibitory activity.

Subsequent optimization of the ring B−linker−ring C

domain provided the m-CF₃-substituted phenylacetamide

derivative 8B as a potent pan-RAF inhibitor with favorable in

vitro activity (BRAF(V600E) IC₅₀, 2.4 nM; BRAF(wt), 8.3 nM;

CRAF, 1.4 nM; pMEK (A375) IC₅₀, 12 nM; pMEK (HMVII),

49 nM; V/B ratio, 67). In addition, compound 8B was shown

preclinically to be a selective kinase inhibitor targeting pan-RAF

kinase activity by testing against a panel of kinases. X-ray

cocrystal structure analysis revealed that 8B occupies the ATP

site of BRAF in the DFG-out conformation. Furthermore, the

ﬂexible acetamide linker provides a more twisted conformation

for ring C that can interact more eﬀectively with the

hydrophobic back pocket than is observed for the ureide

derivative (dihedral angles of 88° for the acetamide linker and

20° for the ureide linker).

An SPR study revealed that compound 8B is a slow oﬀ-rate

inhibitor of both BRAF and CRAF. Reﬂecting the slow oﬀ-rate

property, compound 8B demonstrated potent inhibitory

activity even at high ATP concentration (BRAF(V600E) IC₅₀,

3.3 nM; CRAF, 5.0 nM) with 1 h of preincubation in a

biochemical assay.

K

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hexane), and the obtained solution was concentrated under reduced

pressure. The residue was recrystallized from EtOAc/n-hexane to give

3b (706 mg, 68%) as pale-yellow crystals; mp 143−145 °C. ¹H NMR

(DMSO-d₆, 300 MHz): δ 0.98 (d, J = 4.2 Hz, 4H), 1.73 (s, 6H), 1.96−

2.08 (m, 1H), 3.81 (s, 3H), 6.72−6.79 (m, 1H), 7.25 (d, J = 8.7 Hz,

1H), 7.31−7.42 (m, 2H), 7.49−7.63 (m, 2H), 7.69−7.77 (m, 1H),

7.88 (dt, J = 7.7, 1.3 Hz, 1H), 7.97 (t, J = 1.7 Hz, 1H), 8.01 (d, J = 8.7

Hz, 1H), 10.31 (s, 1H), 12.69 (br s, 1H). MS (ESI+) m/z 555.25 (M

+ H)⁺. Anal. Calcd for C₃₀H₂₆N₄O₅S·0.4H₂O: C, 64.13; H, 4.81; N,

9.97. Found: C, 64.17; H, 4.81; N, 9.84.

In vitro pharmacological studies revealed that compound 8B

possesses signiﬁcant cellular activity against BRAF^V600Eand

NRAS mutated cancer cell lines. In vivo pharmacological

evaluation in a melanoma A375 (BRAF^V600E) xenograft model

in rats revealed potent inhibition of the MAPK cascade in

tumor cells after a single oral administration of 8B at doses of

4−25 mg/kg. Furthermore, twice daily, 14 days of repetitive

administration of 8B demonstrated regressive antitumor

eﬃcacy at doses of 4−25 mg/kg without body weight changes.

Moreover, compound 8B induced regression in HMVII

(NRAS^Q61K) tumors and restored body weight loss derived

from HMVII tumor-induced cachexia.

These results suggested that slow oﬀ-rate pan-RAF inhibitors

could represent a second generation of RAF inhibitors to be

tested in the clinic for the treatment of human cancer harboring

either BRAF^V600Eor NRAS mutation. Along with another pan-

RAF inhibitor MLN2480 under phase I clinical study by

Takeda,⁴⁰we selected compound 8B (TAK-632) as an

alternative development candidate.

N-[3-({7-Cyano-2-[(cyclopropylcarbonyl)amino]-1,3-benzothia-

zol-6-yl}oxy)phenyl]-3-(1-cyano-1-methylethyl)benzamide (3c). To

a solution of N-{3-[(2-amino-7-cyano-1,3-benzothiazol-6-yl)oxy]-

phenyl}-3-(1-cyano-1-methylethyl)benzamide 11c (150 mg, 0.33

mmol) in pyridine (2 mL) was added cyclopropanecarbonyl chloride

(59 μL, 0.66 mmol), and the mixture was stirred at room temperature

for 2 h. The reaction mixture was concentrated under reduced

pressure. The residue was suspended in EtOAc (50 mL), washed with

5% aqueous NaHCO₃solution (50 mL) and brine (50 mL),

successively, and dried over anhydrous Na₂SO₄. Insoluble material

was ﬁltered oﬀ, and the ﬁltrate was concentrated under reduced

pressure. The obtained residue was puriﬁed by silica gel column

chromatography (30−100% EtOAc in n-hexane), and the obtained

solution was concentrated under reduced pressure. The residue was

recrystallized from EtOAc to give 3c (119 mg, 69%) as colorless

EXPERIMENTAL SECTION

■

General Chemistry Information. The starting materials,

reagents, and solvents for reactions were of reagent grade and were

used as purchased. Thin-layer chromatography (TLC) was carried out

using Merck Kieselgel 60, 63−200 mesh, F254 plates, or Fuji Silysia

Chemical Ltd., 100−200 mesh, NH plates. Chromatographic

puriﬁcation was carried out using silica gel (Merck, 70−230 mesh)

or basic silica gel (Fuji Silysia Chemical Ltd., DM1020, 100−200

mesh). Melting points were obtained using an OptiMelt melting point

apparatus MPA100 and are 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 (δ) in

ppm, multiplicity (where applicable), coupling constants (J) in Hz

(where applicable), and integration (where applicable). Multiplicities

are indicated by s (singlet), d (doublet), t (triplet), q (quartet), dd

(doublet of doublets), dt, (doublet of triplets), ddd (doublet of

doublet of doublets), br s (broad singlet), or m (multiplet). MS

spectra were collected with a Waters LC-MS system (ZMD-1) and

were used to conﬁrm ≥95% purity of each compound. The column

used was an L-column 2 ODS (3.0 × 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

and 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.

1

crystals; mp 273−275 °C. H NMR (DMSO-d₆, 300 MHz): δ 0.90−

1.09 (m, 4H), 1.74 (s, 6H), 1.96−2.10 (m, 1H), 6.93 (dd, J = 2.1, 7.7

Hz, 1H), 7.20 (d, J = 8.9 Hz, 1H), 7.45 (t, J = 8.1 Hz, 1H), 7.54−7.68

(m, 3H), 7.70−7.81 (m, 1H), 7.91 (d, J = 7.9 Hz, 1H), 8.00 (t, J = 1.7

Hz, 1H), 8.05 (d, J = 8.9 Hz, 1H), 10.43 (s, 1H), 13.01 (br s, 1H). MS

(ESI) m/z 522.25 (M + H)⁺. Anal. Calcd for C₂₉H₂₃N₅O₃S: C, 66.78;

H, 4.44; N, 13.43. Found: C, 66.57; H, 4.46; N, 13.40.

The following compounds (3d) were prepared from the

corresponding 2-amino-1,3-benzothiazole derivatives (11d) with

cyclopropanecarbonyl chloride by a method similar to that described

for 3c.

3-(1-Cyano-1-methylethyl)-N-[3-({2-[(cyclopropylcarbonyl)-

amino]-7-nitro-1,3-benzothiazol-6-yl}oxy)phenyl]benzamide (3d).

Yield 79%, yellow crystals (recrystallized from EtOAc/n-hexane), mp

223−225 °C. ¹H NMR (DMSO-d₆, 300 MHz): δ 0.95−1.05 (m, 4H),

1.73 (s, 6H), 1.98−2.09 (m, 1H), 6.81−6.92 (m, 1H), 7.33−7.47 (m,

2H), 7.49−7.65 (m, 3H), 7.71−7.78 (m, 1H), 7.90 (dt, J = 7.8, 1.2 Hz,

1H), 7.99 (t, J = 1.7 Hz, 1H), 8.15 (d, J = 8.7 Hz, 1H), 10.38 (s, 1H),

12.90 (br s, 1H). MS (ESI) m/z 542.25 (M + H)⁺. Anal. Calcd for

C₂₈H₂₃N₅O₅S: C, 62.10; H, 4.28; N, 12.93. Found: C, 61.83; H, 4.36;

N, 12.70.

6-[3-({[3-(1-Cyano-1-methylethyl)phenyl]carbonyl}amino)-

phenoxy]-2-[(cyclopropylcarbonyl)amino]-1,3-benzothiazole-7-car-

boxylic Acid (3e). To a solution of 3b (570 mg, 1.02 mmol) in a mixed

solvent of THF/MeOH/H₂O (3:1:1, 10 mL) was added LiOH·H₂O

(150 mg, 3.66 mmol), and the mixture was stirred at room

temperature for 12 h. The reaction mixture was neutralized with 1

N HCl, diluted with EtOAc/THF (1:1, 200 mL), and washed with

water (100 mL). The organic layer was concentrated under reduced

pressure. The obtained residue was puriﬁed by silica gel column

chromatography (0−10% MeOH in EtOAc), and the obtained

solution was concentrated under reduced pressure to give 3e (300

Methyl 6-[3-({[3-(1-Cyano-1-methylethyl)phenyl]carbonyl}-

amino)phenoxy]-2-[(cyclopropylcarbonyl)amino]-1,3-benzothia-

zole-7-carboxylate (3b). To a solution of methyl 2-amino-6-[3-({[3-

(1-cyano-1-methylethyl)phenyl]carbonyl}amino)phenoxy]-1,3-benzo-

thiazole-7-carboxylate 11b (0.92 g, 1.88 mmol) in pyridine (5 mL) was

added cyclopropanecarbonyl chloride (371 μL, 4.1 mmol), and the

mixture was stirred at room temperature for 2 h. The reaction mixture

was concentrated under reduced pressure. The obtained residue was

diluted with EtOAc (100 mL), washed with water (100 mL) and brine

(100 mL), successively, and dried over anhydrous Na₂SO₄. Insoluble

material was ﬁltered oﬀ, and the ﬁltrate was concentrated under

reduced pressure. The obtained residue was suspended in MeOH (10

mL), and Na₂CO₃(250 mg) was added to the mixture. The mixture

was stirred at room temperature for 4 h. The reaction mixture was

diluted with EtOAc (100 mL), washed with water (100 mL) and brine

(100 mL), successively, and dried over anhydrous Na₂SO₄. Insoluble

material was ﬁltered oﬀ, and the ﬁltrate was concentrated under

reduced pressure. The obtained residue was successively puriﬁed by

basic silica gel column chromatography (40−100% EtOAc in n-

hexane) and silica gel column chromatography (40−60% EtOAc in n-

1

mg, 54%) as a colorless amorphous solid. H NMR (DMSO-d₆, 300

MHz): δ 0.90−1.01 (m, 4H), 1.72 (s, 6H), 1.94−2.09 (m, 1H), 6.70

(dd, J = 2.0, 8.0 Hz, 1H), 7.21 (d, J = 8.7 Hz, 1H), 7.27−7.36 (m, 2H),

7.47−7.61 (m, 2H), 7.68−7.77 (m, 1H), 7.87 (d, J = 7.7 Hz, 1H),

7.92−7.99 (m, 2H), 10.30 (s, 1H), 12.61 (s, 1H), 13.55 (br s, 1H). MS

(ESI) m/z 541.25 (M + H)⁺. Anal. Calcd for C₂₉H₂₄N₄O₅S·0.8H₂O:

C, 62.76; H, 4.65; N, 10.09. Found: C, 62.75; H, 4.58; N, 9.93.

3-(1-Cyano-1-methylethyl)-N-[3-({2-[(cyclopropylcarbonyl)-

amino]-7-(hydroxymethyl)-1,3-benzothiazol-6-yl}oxy)phenyl]-

benzamide (3f). To a solution of 3e (200 mg, 0.369 mmol) in THF (8

mL) were added triethylamine (101 μL, 0.738 mmol) and iso-butyl

chloroformate (96 μL, 0.738 mmol) at 4 °C, and the mixture was

stirred at 4 °C for 30 min. Insoluble material was ﬁltered oﬀ, and the

L

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ﬁltrate was concentrated under reduced pressure. The obtained residue

was dissolved in THF (2 mL), and to the mixture were added NaBH₄

(42 mg, 1.10 mmol) and MeOH (2 mL), and the mixture was stirred

at room temperature for 2 h. The reaction mixture was concentrated

under reduced pressure. The obtained residue was diluted with EtOAc

(20 mL), washed with 1 N HCl (5 mL), 5% aqueous NaHCO₃

solution (10 mL) and brine (5 mL), successively, and dried over

anhydrous Na₂SO₄. Insoluble material was ﬁltered oﬀ, and the ﬁltrate

was concentrated under reduced pressure. The obtained residue was

puriﬁed by silica gel column chromatography (20−60% EtOAc in n-

hexane), and the obtained solution was concentrated under reduced

pressure to give 3f (108 mg, 55%) as pale yellow crystals; mp 152−153

Hz, 1H), 9.01 (s, 1H), 9.13 (s, 1H), 13.00 (s, 1H). HRMS (ESI) calcd

for C₂₆H₁₈F₃N₅O₃S 538.1155 [M + H]⁺. Found: 538.1107.

N-{7-Cyano-6-[4-ﬂuoro-3-({[4-(triﬂuoromethyl)phenyl]-

carbamoyl}amino)phenoxy]-1,3-benzothiazol-2-yl}-

cyclopropanecarboxamide (6B). Yield 51%, colorless crystals

1

(recrystallized from acetone/n-hexane), mp 176−177 °C. H NMR

(DMSO-d₆, 300 MHz): δ 0.84−1.22 (m, 4H), 1.86−2.07 (m, 1H),

6.68−6.92 (m, 1H), 7.13 (d, J = 8.9 Hz, 1H), 7.36 (dd, J = 9.1, 11.0

Hz, 1H), 7.55−7.73 (m, 4H), 7.91−8.13 (m, 2H), 8.87 (d, J = 2.5 Hz,

1H), 9.51 (s, 1H), 12.99 (s, 1H). MS (ESI) m/z 556.1 (M + H)⁺.

Anal. Calcd for C₂₆H₁₇F₄N₅O₃S·1.0H₂O: C, 54.45; H, 3.34; N, 12.21.

Found: C, 54.69; H, 3.23; N, 12.09.

1

N-{7-Cyano-6-[3-({[2-(triﬂuoromethyl)phenyl]acetyl}amino)-

phenoxy]-1,3-benzothiazol-2-yl}cyclopropanecarboxamide (7A).

To a solution of 2-(2-(triﬂuoromethyl)phenyl)acetic acid (138 mg,

0.68 mmol) in DMF (2 mL) was added 22A (120 mg, 0.34 mmol) at

room temperature. The mixture was stirred at room temperature for

12 h and was diluted with EtOAc (10 mL). The mixture was washed

with aqueous 5% NaHCO₃solution (5 mL) and brine (5 mL),

successively, dried over anhydrous Na₂SO₄, ﬁltered, and evaporated.

The residue was puriﬁed by silica gel column chromatography (30−

100% EtOAc in n-hexane). Desired fractions were combined and

evaporated in vacuo. The crude material was crystallized from EtOAc

°C. H NMR (DMSO-d₆, 300 MHz) δ 0.90−1.00 (m, 4H), 1.73 (s,

6H), 1.93−2.07 (m, 1H), 4.74 (d, J = 5.1 Hz, 2H), 5.65 (t, J = 5.3 Hz,

1H), 6.66−6.77 (m, 1H), 7.09 (d, J = 8.5 Hz, 1H), 7.33 (t, J = 8.2 Hz,

1H), 7.37 (t, J = 2.2 Hz, 1H), 7.51 (dd, J = 0.9, 8.3 Hz, 1H), 7.57 (t, J

= 7.8 Hz, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.69−7.79 (m, 1H), 7.83−

7.92 (m, 1H), 7.97 (t, J = 1.8 Hz, 1H), 10.33 (s, 1H), 12.51 (br s, 1H).

MS (ESI) m/z 527.20 (M

+

H)⁺. Anal. Calcd for

C₂₉H₂₆N₄O₄S·0.25H₂O: C, 65.58; H, 5.03; N, 10.55. Found: C,

65.65; H, 5.16; N, 10.42.

N-{7-Cyano-6-[3-({[2-(triﬂuoromethyl)phenyl]carbamoyl}amino)-

phenoxy]-1,3-benzothiazol-2-yl}cyclopropanecarboxamide (4A).

To a solution of 1-isocyanato-2-(triﬂuoromethyl)benzene (63 μL,

0.44 mmol) in DMF (2 mL) was added N-[6-(3-aminophenoxy)-7-

cyano-1,3-benzothiazol-2-yl]cyclopropanecarboxamide 22A (120 mg,

0.34 mmol) at room temperature. The mixture was stirred at room

temperature for 12 h and was diluted with EtOAc (10 mL). The

mixture was washed with 5% aqueous NaHCO₃(5 mL) and brine (5

mL), successively, dried over anhydrous Na₂SO₄, ﬁltered, and

evaporated. The resulting material was puriﬁed by basic silica gel

column chromatography (60−100% EtOAc in n-hexane). Desired

fractions were combined and evaporated in vacuo. The oily residue

was crystallized from EtOAc (4 mL). The crystalline solid was

collected by ﬁltration using 50% EtOAc in n-hexane to give 4A (107

mg, 58%) as colorless crystals; mp 181−183 °C. ¹H NMR (DMSO-d₆,

300 MHz): δ 0.89−1.07 (m, 4H), 1.93−2.13 (m, 1H), 6.64−6.90 (m,

1H), 7.10−7.23 (m, 2H), 7.24−7.33 (m, 1H), 7.32−7.43 (m, 2H),

7.62 (t, J = 7.6 Hz, 1H), 7.67 (d, J = 7.9 Hz, 1H), 7.88 (d, J = 8.1 Hz,

1H), 8.03 (d, J = 9.1 Hz, 1H), 8.10 (s, 1H), 9.53 (s, 1H), 13.00 (s,

1H). MS (ESI) m/z 538.2 (M + H)⁺. Anal. Calcd for

C₂₆H₁₈F₃N₅O₃S·0.5H₂O: C, 57.14; H, 3.50; N, 12.81, Found: C,

57.43; H, 3.79; N, 12.42.

1

to give 7A (135 mg, 74%) as colorless crystals; mp 205−206 °C. H

NMR (DMSO-d₆, 300 MHz): δ 0.82−1.12 (m, 4H), 1.94−2.14 (m,

1H), 3.90 (s, 2H), 6.85 (td, J = 4.5, 2.5 Hz, 1H), 7.16 (d, J = 9.1 Hz,

1H), 7.28−7.57 (m, 5H), 7.57−7.78 (m, 2H), 8.02 (d, J = 8.9 Hz,

1H), 10.35 (s, 1H), 12.98 (br s, 1H). MS (ESI) m/z 537.2 (M + H)⁺.

Anal. Calcd for C₂₇H₁₉F₃N₄O₃S: C, 60.44; H, 3.57; N, 10.44. Found:

C, 60.28; H, 3.63; N, 10.42.

The following compounds (8A,B and 9A) were prepared from the

corresponding aniline derivatives (22A,B) with the corresponding

phenylacetic acids by a method similar to that described for 7A.

N-{7-Cyano-6-[3-({[3-(triﬂuoromethyl)phenyl]acetyl}amino)-

phenoxy]-1,3-benzothiazol-2-yl}cyclopropanecarboxamide (8A).

Yield 57%, colorless crystals (recrystallized from EtOAc/n-heptane),

1

mp 183−184 °C. H NMR (DMSO-d₆, 300 MHz): δ 0.91−1.11 (m,

4H), 1.93−2.11 (m, 1H), 3.77 (s, 2H), 6.79−6.91 (m, 1H), 7.15 (d, J

= 9.0 Hz, 1H), 7.31−7.43 (m, 2H), 7.43−7.48 (m, 1H), 7.50−7.65 (m,

3H), 7.67 (s, 1H), 8.02 (d, J = 9.0 Hz, 1H), 10.38 (s, 1H), 12.99 (s,

1H). MS (ESI) m/z 537.0 (M + H)⁺. Anal. Calcd for C₂₇H₁₉F₃N₄O₃S:

C, 60.44; H, 3.57; N, 10.44. Found: C, 60.28; H, 3.59; N, 10.42.

N-{7-Cyano-6-[4-ﬂuoro-3-({[3-(triﬂuoromethyl)phenyl]acetyl}-

amino)phenoxy]-1,3-benzothiazol-2-yl}cyclopropanecarboxamide

(8B). Yield 60%, colorless crystals (recrystallized from EtOAc/n-

heptane), mp 209−210 °C. ¹H NMR (DMSO-d₆, 300 MHz): δ 0.89−

1.05 (m, 4H), 1.97−2.13 (m, 1H), 3.88 (s, 2H), 6.97 (dt, J = 8.7, 3.6

Hz, 1H), 7.08 (d, J = 9.0 Hz, 1H), 7.37 (dd, J = 9.1, 10.6 Hz, 1H),

7.49−7.64 (m, 3H), 7.68 (s, 1H), 7.83 (dd, J = 3.0, 6.4 Hz, 1H), 7.99

(d, J = 9.0 Hz, 1H), 10.21 (s, 1H), 12.97 (s, 1H). MS (ESI) m/z

555.50 (M + H)⁺. Anal. Calcd for C₂₇H₁₈F₄N₄O₃S: C, 58.48; H, 3.27;

N, 10.10. Found: C, 58.40; H, 3.18; N, 9.99.

The following compounds (5A,B and 6A,B) were prepared from

the corresponding aniline derivatives (22A,B) with the corresponding

isocyanate reagents by a method similar to that described for 4A.

N-{7-Cyano-6-[3-({[3-(triﬂuoromethyl)phenyl]carbamoyl}amino)-

phenoxy]-1,3-benzothiazol-2-yl}cyclopropanecarboxamide (5A).

Yield 28%, colorless crystals (recrystallized from EtOAc/n-hexane),

1

mp 269−270 °C. H NMR (DMSO-d₆, 300 MHz): δ 0.83−1.12 (m,

4H), 1.91−2.16 (m, 1H), 6.77 (dd, J = 2.0, 8.0 Hz, 1H), 7.15 (d, J =

9.0 Hz, 1H), 7.20−7.44 (m, 4H), 7.44−7.66 (m, 2H), 7.96 (s, 1H),

8.03 (d, J = 9.0 Hz, 1H), 9.01 (s, 1H), 9.08 (s, 1H), 12.99 (s, 1H).

HRMS (ESI) calcd for C₂₆H₁₈F₃N₅O₃S: 538.1155 [M + H]⁺. Found:

538.1117.

N-{7-Cyano-6-[3-({[4-(triﬂuoromethyl)phenyl]acetyl}amino)-

phenoxy]-1,3-benzothiazol-2-yl}cyclopropanecarboxamide (9A).

Yield 69%, colorless crystals (recrystallized from EtOAc), mp 233−

235 °C. ¹H NMR (DMSO-d₆, 300 MHz): δ 0.90−1.08 (m, 4H),

1.86−2.19 (m, 1H), 3.76 (s, 2H), 6.70−6.99 (m, 1H), 7.15 (d, J = 9.1

Hz, 1H), 7.30−7.41 (m, 2H), 7.45 (d, J = 1.7 Hz, 1H), 7.53 (d, J = 8.0

Hz, 2H), 7.68 (d, J = 8.0 Hz, 2H), 8.03 (d, J = 9.1 Hz, 1H), 10.39 (s,

1H), 13.00 (s, 1H). MS (ESI) m/z 537.2 (M + H)⁺. Anal. Calcd for

C₂₇H₁₉F₃N₄O₃S: C, 60.44; H, 3.57; N, 10.44, Found: C, 60.35; H,

3.81; N, 10.20.

N-[3-(4-Amino-2-ﬂuorophenoxy)phenyl]-3-(2-cyanopropan-2-yl)-

benzamide (10a). To a solution of 3-(2-cyanopropan-2-yl)-N-[3-(2-

ﬂuoro-4-nitrophenoxy)phenyl]benzamide 15a (2.03 g, 4.83 mmol) in

90% EtOH in water were added Fe(0) (2.97 g, 53.1 mmol) and CaCl₂

(95%, 1.41 g, 12.1 mmol). The mixture was reﬂuxed for 12 h, and was

then cooled and ﬁltered through a pad of Celite. The ﬁltrate was

concentrated under reduced pressure. The residue was partitioned

between EtOAc (300 mL) and 5% aqueous NaHCO₃solution (300

mL). The organic layer was washed with water (2 × 200 mL) and

N-{7-Cyano-6-[4-ﬂuoro-3-({[3-(triﬂuoromethyl)phenyl]-

carbamoyl}amino)phenoxy]-1,3-benzothiazol-2-yl}-

cyclopropanecarboxamide (5B). Yield 85%, colorless crystals

(recrystallized from EtOAc/n-hexane), mp 252−254 °C. ¹H NMR

(DMSO-d₆, 300 MHz): δ 0.98 (d, J = 4.9 Hz, 4H), 2.00 (br s, 1H),

6.69−6.94 (m, 1H), 7.09 (d, J = 8.9 Hz, 1H), 7.22−7.43 (m, 2H), 7.52

(d, J = 5.1 Hz, 2H), 7.85−8.10 (m, 3H), 8.85 (s, 1H), 9.46 (s, 1H),

12.97 (br s, 1H). HRMS (ESI) calcd for C₂₆H₁₇F₄N₅O₃S: 556.1061

[M + H]⁺. Found: 556.1049.

N-{7-Cyano-6-[3-({[4-(triﬂuoromethyl)phenyl]carbamoyl}amino)-

phenoxy]-1,3-benzothiazol-2-yl}cyclopropanecarboxamide (6A).

Yield 94%, colorless crystals (recrystallized from EtOAc), mp 217−

218 °C. ¹H NMR (DMSO-d₆, 300 MHz): δ 0.93−1.08 (m, 4H),

1.99−2.07 (m, 1H), 6.74−6.85 (m, 1H), 7.17 (d, J = 9.0 Hz, 1H),

7.20−7.28 (m, 1H), 7.32−7.45 (m, 2H), 7.62 (s, 4H), 8.05 (d, J = 9.0

M

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brine (200 mL), successively, dried over anhydrous Na₂SO₄, ﬁltered,

and evaporated. The residue was puriﬁed by silica gel column

chromatography (30−100% EtOAc in n-hexane). Desired fractions

were combined and evaporated to give 10a (1.94 g, 99%) as yellow

and the mixture was stirred at room temperature for 3 h. The resulting

yellow insoluble material was ﬁltered oﬀ and washed with AcOH. The

ﬁltrate and washings were combined, and the mixture was

concentrated under reduced pressure. The obtained residue was

suspended in EtOAc (200 mL), washed successively with saturated

aqueous NaHCO₃solution (100 mL) and brine (2 × 100 mL), and

dried over anhydrous Na₂SO₄. Insoluble material was ﬁltered oﬀ, and

the ﬁltrate was concentrated under reduced pressure. The residue was

puriﬁed by basic silica gel column chromatography (40−100% EtOAc

in n-hexane), and the obtained solution was concentrated under

reduced pressure to give 11b (1.15 g, 90%) as a colorless solid; mp

115−116 °C. ¹H NMR (DMSO-d₆, 300 MHz) δ 1.73 (s, 6H), 3.74 (s,

3H), 6.65−6.73 (m, 1H), 7.06 (d, J = 8.7 Hz, 1H), 7.25−7.38 (m,

2H), 7.44−7.63 (m, 5H), 7.67−7.77 (m, 1H), 7.82−7.91 (m, 1H),

7.97 (t, J = 1.7 Hz, 1H), 10.29 (s, 1H). HRMS (ESI) calcd for

C₂₆H₂₂N₄O₄S: 487.1435 [M + H]⁺. Found: 487.1406.

The following compounds (11c,d and 12a) were prepared from the

corresponding aniline derivatives (10a,c,d) using KSCN, Br₂, and

AcOH by a method similar to that described for 11b.

N-{3-[(2-Amino-7-cyano-1,3-benzothiazol-6-yl)oxy]phenyl}-3-(1-

cyano-1-methylethyl)benzamide (11c). Yield 81%, yellow solid, mp

179−182 °C. ¹H NMR (DMSO-d₆, 300 MHz): δ 1.74 (s, 6H), 6.81−

6.91 (m, 1H), 7.04 (d, J = 8.7 Hz, 1H), 7.41 (t, J = 8.1 Hz, 1H), 7.49−

7.67 (m, 4H), 7.69−7.80 (m, 1H), 7.84−7.95 (m, 3H), 8.00 (t, J = 1.7

Hz, 1H), 10.39 (s, 1H). HRMS (ESI) calcd for C₂₅H₁₉N₅O₂S:

454.1332 [M + H]⁺. Found: 454.1306.

1

crystals; mp 77−78 °C. H NMR (DMSO-d₆, 300 MHz): δ 1.74 (s,

6H), 5.34 (s, 2H), 6.35−6.44 (m, 1H), 6.49 (dd, J = 2.4, 13.2 Hz, 1H),

6.61−6.73 (m, 1H), 6.80−7.03 (m, 1H), 7.28 (t, J = 8.1 Hz, 1H), 7.36

(t, J = 2.1 Hz, 1H), 7.41−7.50 (m, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.67−

7.79 (m, 1H), 7.83−7.94 (m, 1H), 7.99 (t, J = 1.8 Hz, 1H), 10.31 (s,

1H). MS (ESI) m/z 390.05 (M + H)⁺.

Methyl 5-amino-2-[3-({[3-(1-cyano-1-methylethyl)phenyl]-

carbonyl}amino)phenoxy]benzoate (10b). To a solution of methyl

2-[3-({[3-(1-cyano-1-methylethyl)phenyl]carbonyl}amino)phenoxy]-

5-nitrobenzoate 15b (4.00 g, 8.70 mmol) in NMP/MeOH/THF

(2:4:1) (70 mL) was added 10% Pd/C (400 mg), and the mixture was

stirred at room temperature for 14 h under a hydrogen atmosphere (1

atm). Insoluble material was ﬁltered oﬀ, and the ﬁltrate was

concentrated under reduced pressure. The obtained residue was

diluted with EtOAc (200 mL), washed successively with water (2 ×

100 mL) and brine (2 × 100 mL), and dried over anhydrous Na₂SO₄.

Insoluble material was ﬁltered oﬀ, and the ﬁltrate was concentrated

under reduced pressure. The obtained residue was puriﬁed by basic

silica gel column chromatography (eluate: EtOAc), and the obtained

solution was concentrated under reduced pressure to give 10b (3.42 g,

92%) as a pale yellow oil. ¹H NMR (DMSO-d₆, 300 MHz): δ 1.74 (s,

6H), 2.69 (s, 3H), 5.33 (s, 2H), 6.44−6.63 (m, 1H), 6.71−6.96 (m,

2H), 7.07 (d, J = 2.6 Hz, 1H), 7.19−7.32 (m, 2H), 7.39−7.44 (m,

1H), 7.57 (t, J = 7.7 Hz, 1H), 7.68−7.80 (m, 1H), 7.83−7.94 (m, 1H),

7.98 (t, J = 1.7 Hz, 1H), 10.28 (s, 1H). MS (ESI) m/z 430.20 (M +

H)⁺.

The following compounds (10c) were prepared from the

corresponding nitro derivatives (15c) using Fe(0) and CaCl₂by a

method similar to that described for 10a.

N-[3-(4-Amino-2-cyanophenoxy)phenyl]-3-(1-cyano-1-

methylethyl)benzamide (10c). Yield 82%, yellow oil. ¹H NMR

(DMSO-d₆, 300 MHz): δ 1.74 (s, 6H), 5.48−5.66 (br s, 2H), 6.65−

6.80 (m, 1H), 6.86−7.05 (m, 3H), 7.34 (t, J = 8.1 Hz, 1H), 7.42 (t, J =

2.1 Hz, 1H), 7.48−7.55 (m, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.69−7.81

(m, 1H), 7.84−7.94 (m, 1H), 8.00 (t, J = 1.7 Hz, 1H), 10.35 (s, 1H).

MS (ESI) m/z 397.15 (M + H)⁺.

N-{3-[(2-Amino-7-nitro-1,3-benzothiazol-6-yl)oxy]phenyl}-3-(1-

cyano-1-methylethyl)benzamide (11d). Yield 73%, yellow solid; mp

202−204 °C. ¹H NMR (DMSO-d₆, 300 MHz): δ 1.73 (s, 6H), 6.71−

6.88 (m, 1H), 7.21 (d, J = 8.7 Hz, 1H), 7.36 (t, J = 8.2 Hz, 1H), 7.44

(t, J = 2.1 Hz, 1H), 7.51−7.63 (m, 2H), 7.67−7.78 (m, 2H), 7.83−

7.93 (m, 3H), 7.98 (t, J = 1.7 Hz, 1H), 10.34 (s, 1H). MS (ESI) m/z

474.20 (M + H)⁺. Anal. Calcd for C₂₄H₁₉N₅O₄S·0.2H₂O: C, 60.42; H,

4.10; N, 14.68. Found: C, 60.48; H, 4.14; N, 14.49.

N-{3-[(2-Amino-5-ﬂuoro-1,3-benzothiazol-6-yl)oxy]phenyl}-3-(2-

cyanopropan-2-yl)benzamide (12a). Yield 57%, yellow crystals, mp

1

112−113 °C. H NMR (DMSO-d₆, 300 MHz): δ 1.73 (s, 6H), 6.74

(dd, J = 2.0, 7.8 Hz, 1H), 7.25−7.37 (m, 2H), 7.40 (t, J = 2.0 Hz, 1H),

7.49−7.55 (m, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.61−7.69 (m, 3H),

7.70−7.78 (m, 1H), 7.85−7.92 (m, 1H), 7.98 (t, J = 1.6 Hz, 1H),

10.32 (s, 1H). HRMS (ESI) calcd for C₂₄H₁₉FN₄O₂S: 447.1286 [M +

H]⁺. Found: 447.1250.

N-[3-(4-Amino-2-nitrophenoxy)phenyl]-3-(1-cyano-1-

methylethyl)benzamide (10d). To a mixture of 3-(1-cyano-1-

methylethyl)-N-(3-hydroxyphenyl)benzamide 13 (20 g, 71.3 mmol)

and 4-ﬂuoro-3-nitroaniline 14d (10.9 g, 69.9 mmol) in DMF (150

mL) was added Cs₂CO₃(33.8 g, 104 mmol), and the mixture was

stirred at 80 °C for 16 h. The reaction mixture was cooled to room

temperature, and insoluble material was ﬁltered oﬀ and washed with

EtOAc. The ﬁltrate and washings were combined, and the mixture was

concentrated under reduced pressure. The obtained residue was

diluted with EtOAc (300 mL), washed successively with water (300

mL) and brine (2 × 150 mL), and dried over anhydrous Na₂SO₄.

Insoluble material was ﬁltered oﬀ, and the ﬁltrate was concentrated

under reduced pressure. The obtained residue was puriﬁed by basic

silica gel column chromatography (eluate: EtOAc), and the obtained

solution was concentrated under reduced pressure. The residue was

crystallized from EtOAc/n-hexane to give 10d (23.8 g, 82%) as red-

3-(1-Cyano-1-methylethyl)-N-[3-(2-ﬂuoro-4-nitrophenoxy)-

phenyl]benzamide (15a). To a mixture of 13 (2.0 g, 7.13 mmol) and

3,4-diﬂuoronitrobenzene 14a (1.19 g, 7.49 mmol) in DMF (15 mL)

was added K₂CO₃(1.48 g, 10.7 mmol) at room temperature. The

mixture was stirred at 80 °C for 18 h, and the mixture was partitioned

between EtOAc (200 mL) and water (200 mL). The organic layer was

washed with water (200 mL) and brine (200 mL), successively, and

dried over anhydrous Na₂SO₄. Insoluble material was ﬁltered oﬀ, and

the ﬁltrate was concentrated under reduced pressure. The obtained

residue was puriﬁed by silica gel column chromatography (EtOAc/n-

hexane), and the obtained solution was concentrated under reduced

pressure to give 15a (2.09 g, 70%) as a yellow oil. ¹H NMR (DMSO-

d₆, 300 MHz): δ 1.60−1.65 (m, 2H), 1.80−1.84 (m, 2H), 6.52 (dt, J =

6.6, 2.4 Hz, 1H), 7.09−7.14 (m, 2H), 7.32−7.33 (m, 1H), 7.54−7.56

(m, 2H), 7.80 (s, 1H), 7.84−7.88 (m, 1H), 9.43 (s, 1H), 10.16 (br s,

1H). MS (ESI) m/z 420.10 (M + H)⁺.

1

orange crystals; mp 137−138 °C. H NMR (DMSO-d₆, 300 MHz): δ

1.74 (s, 6H), 5.71 (s, 2H), 6.61−6.74 (m, 1H), 6.93 (dd, J = 2.7, 8.7

Hz, 1H), 7.06 (d, J = 8.7 Hz, 1H), 7.20 (d, J = 2.7 Hz, 1H), 7.31 (t, J =

8.1 Hz, 1H), 7.39 (t, J = 2.1 Hz, 1H), 7.45−7.52 (m, 1H), 7.58 (t, J =

7.8 Hz, 1H), 7.68−7.79 (m, 1H), 7.90 (dt, J = 1.5, 7.8 Hz, 1H), 7.99

(t, J = 1.8 Hz, 1H), 10.33 (s, 1H). MS (ESI) m/z 417.15 (M + H)⁺.

Methyl 2-amino-6-[3-({[3-(1-cyano-1-methylethyl)phenyl]-

carbonyl}amino)phenoxy]-1,3-benzothiazole-7-carboxylate (11b).

Potassium thiocyanate (1.02 g, 10.5 mmol) was suspended in AcOH

(10 mL), and the mixture was stirred at room temperature for 10 min.

A solution of 10b (1.13 g, 2.62 mmol) in AcOH (10 mL) was added

to the obtained solution, and the mixture was further stirred at room

temperature for 10 min. A solution of bromine (460 mg, 2.88 mmol)

in AcOH (5 mL) was slowly added dropwise to the obtained solution,

The following compounds (15b,c and 17A,B) were prepared from

the 4-ﬂuoronitrobenzene derivatives (14b,c) and the corresponding

phenols (13 and 16A,B) using K₂CO₃and DMF by a method similar

to that described for 15a.

Methyl 2-[3-({[3-(1-Cyano-1-methylethyl)phenyl]carbonyl}-

amino)phenoxy]-5-nitrobenzoate (15b). Yield 98%, colorless

1

crystals, mp 144−145 °C. H NMR (DMSO-d₆, 300 MHz): δ 1.75

(s, 6H), 3.88 (s, 3H), 6.90−6.99 (m, 1H), 7.14 (d, J = 9.3 Hz, 1H),

7.43−7.53 (m, 1H), 7.55−7.64 (m, 1H), 7.64−7.71 (m, 2H), 7.72−

7.81 (m, 1H), 7.88−7.95 (m, 1H), 8.02 (t, J = 1.7 Hz, 1H), 8.41 (dd, J

= 3.0, 9.3 Hz, 1H), 8.64 (d, J = 3.0 Hz, 1H), 10.49 (s, 1H). MS (ESI)

m/z 460 (M + H)⁺.

N

dx.doi.org/10.1021/jm400778d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

3-(1-Cyano-1-methylethyl)-N-[3-(2-cyano-4-nitrophenoxy)-

phenyl]benzamide (15c). Yield 94%, yellow oil. ¹H NMR (DMSO-d₆,

300 MHz): δ 1.75 (s, 6H), 7.01−7.19 (m, 2H), 7.55 (t, J = 8.1 Hz,

1H), 7.61 (t, J = 7.8 Hz, 1H), 7.68−7.80 (m, 2H), 7.81 (t, J = 2.1 Hz,

1H), 7.89−7.99 (m, 1H), 8.03 (t, J = 1.7 Hz, 1H), 8.48 (dd, J = 2.8, 9.4

Hz, 1H), 8.88 (d, J = 2.8 Hz, 1H), 10.56 (1H, s). MS (ESI) m/z 427

(M + H)⁺.

7.59 (m, 1H), 7.63 (d, J = 8.9 Hz, 1H), 7.92 (s, 2H), 11.30 (s, 1H).

MS (ESI) m/z 379 (M + H)⁺. Anal. Calcd for C₁₆H₉F₃N₄O₂S·0.5H₂O:

C, 49.61; H, 2.60; N, 14.46. Found: C, 49.65; H, 2.86; N, 14.30.

N-{5-[(2-Amino-7-cyano-1,3-benzothiazol-6-yl)oxy]-2-ﬂuoro-

phenyl}-2,2,2-triﬂuoroacetamide (20B). Yield 85%, pale yellow

1

crystals, mp 251−253 °C. H NMR (DMSO-d₆, 300 MHz): δ 7.00

(d, J = 8.9 Hz, 1H), 7.10−7.19 (m, 1H), 7.26 (dd, J = 3.1, 6.1 Hz, 1H),

7.42 (t, J = 9.5 Hz, 1H), 7.62 (d, J = 8.9 Hz, 1H), 7.91 (s, 2H), 11.34

(s, 1H). MS (ESI) m/z 397 (M + H)⁺. Anal. Calcd for C₁₆H₈F₄N₄O₂S:

C, 48.49; H, 2.03; N, 14.14. Found: C, 48.26; H, 2.26; N, 13.78.

The following compounds (21A,B) were prepared from the

corresponding 2-amino-1,3-benzothiazole derivatives (20A,B) with

cyclopropanecarbonyl chloride by a method similar to that described

for 3c.

2-(3-Aminophenoxy)-5-nitrobenzonitrile (17A). Yield 66%, yellow

1

crystals (recrystallized from EtOAc/n-hexane), mp 131−133 °C. H

NMR (DMSO-d₆, 300 MHz): δ 5.48 (s, 2H), 6.31−6.37 (m, 1H), 6.38

(t, J = 2.2 Hz, 1H), 6.51−6.58 (m, 1H), 7.03 (d, J = 9.4 Hz, 1H),

7.11−7.20 (m, 1H), 8.45 (dd, J = 2.8, 9.4 Hz, 1H), 8.82 (d, J = 2.8 Hz,

1H). MS (ESI) m/z 255.95 (M + H)⁺. Anal. Calcd for C₁₃H₉N₃O₃:C,

61.18; H, 3.55; N, 16.46. Found: C, 61.12; H, 3.46; N, 16.63.

2-(3-Amino-4-ﬂuorophenoxy)-5-nitrobenzonitrile (17B). Yield

100%, beige crystals, mp 140−141 °C. ¹H NMR (DMSO-d₆, 300

MHz): δ 5.55 (s, 2H), 6.33−6.46 (m, 1H), 6.60 (dd, J = 3.0, 7.6 Hz,

1H), 7.02 (d, J = 9.4 Hz, 1H), 7.13 (dd, J = 8.7, 11.1 Hz, 1H), 8.44

(dd, J = 2.7, 9.4 Hz, 1H), 8.83 (d, J = 2.7 Hz, 1H). MS (ESI) m/z

273.95 (M + H)⁺. Anal. Calcd for C₁₃H₈FN₃O₃: C, 57.15; H, 2.95; N,

15.38. Found: C, 57.22; H, 2.96; N, 15.34.

N-(7-Cyano-6-{3-[(triﬂuoroacetyl)amino]phenoxy}-1,3-benzo-

thiazol-2-yl)cyclopropanecarboxamide (21A). Yield 82%, colorless

crystals (recrystallized from EtOAc), mp 243−244 °C. ¹H NMR

(DMSO-d₆, 300 MHz): δ 0.80−1.13 (m, 4H), 1.92−2.11 (m, 1H),

6.93−7.15 (m, 1H), 7.22 (d, J = 8.9 Hz, 1H), 7.35−7.73 (m, 3H), 8.06

(d, J = 9.0 Hz, 1H), 11.0−12.1 (br s, 1H), 12.2−13.4 (br s, 1H). MS

(ESI) m/z 446.95 (M + H)⁺.

N-(7-Cyano-6-{4-ﬂuoro-3-[(triﬂuoroacetyl)amino]phenoxy}-1,3-

benzothiazol-2-yl)cyclopropanecarboxamide (21B). Yield 55%,

colorless crystals (recrystallized from EtOAc/n-hexane), mp 242−

243 °C. ¹H NMR (DMSO-d₆, 300 MHz): δ 0.95−1.05 (m, 4H),

1.97−2.09 (m, 1H), 7.16 (d, J = 9.0 Hz, 1H), 7.19−7.28 (m, 1H), 7.36

(dd, J = 3.0, 6.2 Hz, 1H), 7.46 (t, J = 9.5 Hz, 1H), 8.05 (d, J = 9.0 Hz,

1H), 11.36 (s, 1H), 12.99 (s, 1H). MS (ESI) m/z 465.1 (M + H)⁺.

Anal. Calcd for C₂₀H₁₂F₄N₄O₃S: C, 51.73; H, 2.60; N, 12.06. Found:

C, 52.11; H, 2.85; N, 12.08.

N-[3-(2-Cyano-4-nitrophenoxy)phenyl]-2,2,2-triﬂuoroacetamide

(18A). To a solution of 17A (18 g, 70.5 mmol) in THF (150 mL) was

added triﬂuoroacetic anhydride (11.8 mL, 84.9 mmol) at 0 °C, and the

mixture was stirred at room temperature for 1.5 h. The reaction

mixture was diluted with EtOAc (500 mL), washed successively with

water (150 mL), 5% aqueous NaHCO₃solution (500 mL), and brine

(500 mL), and dried over anhydrous Na₂SO₄. Insoluble material was

ﬁltered oﬀ, and the ﬁltrate was concentrated under reduced pressure.

The obtained residue was puriﬁed by silica gel column chromatog-

raphy (10−40% EtOAc in n-hexane), and the obtained solution was

concentrated under reduced pressure to give 18A (16.6 g, 67%) as

N-[6-(3-Aminophenoxy)-7-cyano-1,3-benzothiazol-2-yl]-

cyclopropanecarboxamide (22A). Compound 21A (1.06 g, 2.37

mmol) was dissolved in a mixed solvent of THF/MeOH/H₂O (1:1:1)

(75 mL), LiOH·H₂O (1.05 g, 25.7 mmol) was added, and the mixture

was stirred at room temperature for 18 h. The reaction mixture was

neutralized with 1 N HCl and concentrated under reduced pressure.

The obtained precipitates were repeatedly washed with water to give

1

beige crystals; mp 190−191 °C. H NMR (DMSO-d₆, 300 MHz) δ

7.09 (d, J = 9.3 Hz, 1H), 7.17−7.22 (m, 1H), 7.54−7.63 (m, 1H),

7.63−7.72 (m, 2H), 8.42−8.49 (m, 1H), 8.89 (d, J = 2.6 Hz, 1H),

11.46 (br s, 1H). MS (ESI) m/z 351 (M + H)⁺. Anal. Calcd for

C₁₅H₈F₃N₃O₄: C, 51.29; H, 2.30; N, 11.96. Found: C, 51.46; H, 2.33;

N, 12.04.

1

22A (0.79 g, 95%) as colorless crystals; mp 233−234 °C. H NMR

(DMSO-d₆, 300 MHz): δ 0.90−1.14 (m, 4H), 1.96−2.11 (m, 1H),

5.33 (s, 2H), 6.18−6.30 (m, 2H), 6.37−6.49 (m, 1H), 6.98−7.07 (m,

1H), 7.10 (d, J = 9.1 Hz, 1H), 8.00 (d, J = 9.1 Hz, 1H), 12.96 (br s,

1H). MS (ESI) m/z 351.05 (M + H)⁺. Anal. Calcd for

C₁₈H₁₄N₄O₂S·0.5H₂O: C, 60.15; H, 4.21; N, 15.59. Found: C,

60.31; H, 4.19; N, 15.57.

The following compound (18B) was prepared from the aniline

derivative (17B) using triﬂuoroacetic anhydride by a method similar to

that described for 18A.

N-[5-(2-Cyano-4-nitrophenoxy)-2-ﬂuorophenyl]-2,2,2-triﬂuoroa-

1

cetamide (18B). Yield 93%, colorless crystals, mp 168−169 °C. H

NMR (DMSO-d₆, 300 MHz): δ 7.06 (d, J = 9.4 Hz, 1H), 7.35−7.45

(m, 1H), 7.51−7.63 (m, 2H), 8.47 (dd, J = 2.8, 9.4 Hz, 1H), 8.88 (d, J

= 2.8 Hz, 1H), 11.51 (s, 1H). Anal. Calcd for C₁₅H₇F₄N₃O₄: C, 48.79;

H, 1.91; N, 11.38. Found: C, 48.85; H, 2.02; N, 11.30.

N-[6-(3-Amino-4-ﬂuorophenoxy)-7-cyano-1,3-benzothiazol-2-yl]-

cyclopropanecarboxamide (22B). To a solution of NaBH₄(1.46 g,

38.6 mmol) in EtOH (20 mL) was added MeOH (0.5 mL). To this

suspension was added 21B (900 mg, 1.93 mmol) at 0 °C. The reaction

mixture was stirred at 0 °C for 0.5 h then at room temperature for 0.5

h. The mixture was diluted with EtOAc (20 mL), and the resulting

mixture was concentrated in vacuo. The resulting residue was diluted

with EtOAc (200 mL), washed successively with 5% aqueous

NaHCO₃solution (100 mL) and brine (2 × 100 mL), and dried

over anhydrous Na₂SO₄. Insoluble material was ﬁltered oﬀ, and the

ﬁltrate was concentrated under reduced pressure. The obtained residue

was puriﬁed by basic silica gel column chromatography (eluent:

EtOAc), and the obtained solution was concentrated under reduced

pressure. The residue was recrystallized from EtOAc/n-hexane to give

The following compounds (19A,B) was prepared from the nitro

derivatives (18A,B) using H₂and 10% Pd/C by a method similar to

that described for 10b.

N-[3-(4-Amino-2-cyanophenoxy)phenyl]-2,2,2-triﬂuoroaceta-

1

mide (19A). Yield 99%, pale yellow oil. H NMR (DMSO-d₆, 300

MHz): δ 5.55 (s, 2H), 6.81 (d, J = 8.1 Hz, 1H), 6.88−6.94 (m, 2H),

6.96−7.03 (m, 1H), 7.22 (t, J = 2.1 Hz, 1H), 7.32−7.42 (m, 1H),

7.41−7.50 (m, 1H), 11.28 (br s, 1H). MS (ESI) m/z 321.95 (M +

H)⁺. Anal. Calcd for C₁₅H₁₀F₃N₃O₂: C, 56.08; H, 3.14; N, 13.08.

Found: C, 56.28; H, 3.17; N, 13.13.

N-[5-(4-Amino-2-cyanophenoxy)-2-ﬂuorophenyl]-2,2,2-triﬂuor-

oacetamide (19B). Yield 99%, gray crystals, mp 136−137 °C. ¹H

NMR (DMSO-d₆, 300 MHz): δ 5.53 (s, 2H), 6.84−7.00 (m, 4H), 7.09

(dd, J = 3.2, 6.2 Hz, 1H), 7.33 (t, J = 9.5 Hz, 1H), 11.20 (br s, 1H).

MS (ESI) m/z 340.00 (M + H)⁺. Anal. Calcd for C₁₅H₉F₄N₃O₂: C,

53.11; H, 2.67; N, 12.39. Found: C, 52.92; H, 2.80; N, 12.13.

The following compounds (20A,B) were prepared from the

corresponding aniline derivatives (19A,B) using KSCN, Br₂, and

AcOH by a method similar to that described for 11b.

1

22B (611 mg, 85%) as colorless crystals; mp 196−197 °C. H NMR

(DMSO-d₆, 300 MHz): δ 0.89−1.07 (m, 4H), 1.95−2.08 (m, 1H),

5.40 (s, 2H), 6.16−6.35 (m, 1H), 6.49 (dd, J = 3.0, 7.6 Hz, 1H), 6.96−

7.11 (m, 2H), 8.00 (d, J = 8.9 Hz, 1H), 12.94 (br s, 1H). MS (ESI) m/

z 369.1 (M + H)⁺. Anal. Calcd for C₁₈H₁₃FN₄O₂S: C, 58.69; H, 3.56;

N, 15.21. Found: C, 58.63; H, 3.58; N, 15.00.

ASSOCIATED CONTENT

* Supporting Information

■

S

N-{3-[(2-Amino-7-cyano-1,3-benzothiazol-6-yl)oxy]phenyl}-2,2,2-

triﬂuoroacetamide (20A). Yield 81%, yellow crystals, mp 263−264

°C. ¹H NMR (DMSO-d₆, 300 MHz): δ 6.89−7.00 (m, 1H), 7.06 (d, J

= 8.9 Hz, 1H), 7.35 (t, J = 2.1 Hz, 1H), 7.44 (t, J = 8.1 Hz, 1H), 7.51−

Information related to the feedback activation in ﬁbroblast

CsFb cells, reported regioselective/nonselective C-7 substituted

1,3-benzothiazole ring formation reaction of meta-substituted

O

dx.doi.org/10.1021/jm400778d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Lee, R. J.; Grippo, J.; Puzanov, I.; Kim, K. B.; Ribas, A.; McArthur, G.

A.; Sosman, J. A.; Chapman, P. B.; Flaherty, K. T.; Xu, X.; Nathanson,

K. L.; Nolop, K. Clinical efficacy of a RAF inhibitor needs broad target

blockade in BRAF-mutant melanoma. Nature 2010, 467, 596−599.

(3) Chapman, P. B.; Hauschild, A.; Robert, C.; Haanen, J. B.;

Ascierto, P.; Larkin, J.; Dummer, R.; Garbe, C.; Testori, A.; Maio, M.;

Hogg, D.; Lorigan, P.; Lebbe, C.; Jouary, T.; Schadendorf, D.; Ribas,

A.; O’Day, S. J.; Sosman, J. A.; Kirkwood, J. M.; Eggermont, A. M. M.;

Dreno, B.; Nolop, K.; Li, J.; Nelson, B.; Hou, J.; Lee, R. J.; Flaherty, K.

T.; McArthur, G. A. For the BRIM-3 Study Group. Improved survival

with Vemurafenib in melanoma with BRAF V600E mutation. N. Engl.

J. Med. 2011, 364, 2507−2516.

anilines and regioselective cyclization of meta-substituted

thioureides, and assay protocols used in kinase enzyme assays,

cellular assays, SPR assay, in vivo studies, solubility study,

pharmacokinetic studies, and structural biology studies. This

material is available free of charge via the Internet at http://

pubs.acs.org.

Accession Codes

PDB accession codes are 4KSP for BRAF with 8B and 4KSQ

for BRAF with 5B.

AUTHOR INFORMATION

Corresponding Authors

*(M.O.) Phone: +81-466-32-1158. Fax: +81-466-29-4448. E-

mail: masanori.okaniwa@takeda.com.

■

(4) Ribas, A.; Flaherty, K. T. BRAF targeted therapy changes the

treatment paradigm in melanoma. Nat. Rev. Clin. Oncol. 2011, 8, 426−

433.

(5) Hauschild, A.; Grob, J. J.; Demidov, L. V.; Jouary, T.; Gutzmer,

R.; Millward, M.; Rutkowski, P.; Blank, C. U.; Miller, W. H.;

Kaempgen, E., Jr.; Martín-Algarra, S.; Karaszewska, B.; Mauch, C.;

Chiarion-Sileni, V.; Martin, A. M.; Swann, S.; Haney, P.; Mirakhur, B.;

Guckert, M. E.; Goodman, V.; Chapman, P. B. Dabrafenib in BRAF-

mutated metastatic melanoma: a multicentre, open-label, phase 3

randomised controlled trial. Lancet 2012, 380, 358−365.

*(M.H.) Phone: +81-466-32-1029. Fax: +81-466-29-4448. E-

mail: masaaki.hirose@takeda.com.

*(T.I.) Phone: +81-466-32-1155. Fax: +81-466-29-4449. E-

mail: tomoyasu.ishikawa@takeda.com.

Present Address

^∥K.A.: Dart Neuroscience, 10420 Wateridge Circle, San Diego,

CA 92121.

(6) Flaherty, K. T.; Puzanov, I.; Kim, K. B.; Ribas, A.; McArthur, G.

A.; Sosman, J. A.; O’Dwyer, P. J.; Lee, R. J.; Grippo, J. F.; Nolop, K.;

Chapman, P. B. Inhibition of mutated, activated BRAF in metastatic

melanoma. N. Engl. J. Med. 2010, 363, 809−819.

Notes

The authors declare no competing ﬁnancial interest.

(7) Anforth, R. M.; Blumetti, T. C. M. P.; Kefford, R. F.; Sharma, R.;

Scolyer, R. A.; Kossard, S.; Long, G. V.; Fernandez-Penas, P.

̃

ACKNOWLEDGMENTS

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Cutaneous manifestations of dabrafenib (GSK2118436): a selective

inhibitor of mutant BRAF in patients with metastatic melanoma. Br. J.

Dermatol. 2012, 167, 1153−1160.

(8) Falchook, G. S.; Long, G. V.; Kurzrock, R.; Kim, K. B.; Arkenau,

T. H.; Brown, M. P.; Hamid, O.; Infante, J. R.; Millward, M.; Pavlick,

A. C.; O’Day, S. J.; Blackman, S. C.; Curtis, C. M.; Lebowitz, P.; Ma,

B.; Ouellet, D.; Kefford, R. F. Dabrafenib in patients with melanoma,

untreated brain metastases, and other solid tumours: a phase 1 dose-

escalation trial. Lancet 2012, 379 (9829), 1893−1901.

(9) Hall-Jackson, C. A.; Eyers, P. A.; Cohen, P.; Goedert, M.; Boyle,

F. T.; Hewitt, N.; Plant, H.; Hedge, P. Paradoxical activation of Raf by

a novel Raf inhibitor. Chem. Biol. 1999, 6, 559−568.

(10) Poulikakos, P. I.; Zhang, C.; Bollag, G.; Shokat, K. M.; Rosen, N.

RAF inhibitors transactivate RAF dimers and ERK signalling in cells

with wild-type BRAF. Nature 2010, 464, 427−430.

(11) Su, F.; Viros, A.; Milagre, C.; Trunzer, K.; Bollag, G.; Spleiss, O.;

Reis-Filho, J. S.; Kong, X.; Koya, R. C.; Flaherty, K. T.; Chapman, P.

B.; Kim, M. J.; Hayward, R.; Martin, M.; Yang, H.; Wang, Q.; Hilton,

H.; Hang, J. S.; Noe, J.; Lambros, M.; Geyer, F.; Dhomen, N.;

Niculescu-Duvaz, I.; Zambon, A.; Niculescu-Duvaz, D.; Preece, N.;

Robert, L.; Otte, N. J.; Mok, S.; Kee, D.; Ma, Y.; Zhang, C.; Habets, G.;

Burton, E. A.; Wong, B.; Nguyen, H.; Kockx, M.; Andries, L.; Lestini,

B.; Nolop, K. B.; Lee, R. J.; Joe, A. K.; Troy, J. L.; Gonzalez, R.;

Hutson, T. E.; Puzanov, I.; Chmielowski, B.; Springer, C. J.; McArthur,

G. A.; Sosman, J. A.; Lo, R. S.; Ribas, A.; Marais, R. RAS mutations in

cutaneous squamous-cell carcinomas in patients treated with BRAF

inhibitors. N. Engl. J. Med. 2012, 366 (3), 207−215.

(12) Hatzivassiliou, G.; Song, K.; Yen, I.; Brandhuber, B. J.;

Anderson, D. J.; Alvarado, R.; Ludlam, M. J.; Stokoe, D.; Gloor, S.

L.; Vigers, G.; Morales, T.; Aliagas, I.; Liu, B.; Sideris, S.; Hoeflich, K.

P.; Jaiswal, B. S.; Seshagiri, S.; Koeppen, H.; Belvin, M.; Friedman, L.

S.; Malek, S. RAF inhibitors prime wild-type RAF to activate the

MAPK pathway and enhance growth. Nature 2010, 464 (7287), 431−

435.

We thank Tohru Miyazaki, and Takashi Imada for their support

in the synthesis reactions. We express our gratitude to Yuka

Hasegawa, Akiko Nakayama, Kazuyo Kakoi, and Katherine

Galvin for their helpful support in biological evaluation. We

thank Koji Mori, Shuuhei Yao, Hiroshi Miki, Taeko Yoshida,

and Hidehisa Iwata for their assistance in evaluating the kinase

inhibitory activities. We thank Yuuki Koudono for his assistance

in preparing the SD formulation. We thank Douglas R. Cary for

his helpful support in proofreading.

ABBREVIATIONS USED

■

¹H NMR, proton nuclear magnetic resonance; AUC, area under

the blood concentration−time curve; CL_total, clearance; DMA,

N,N-dimethylacetamide; ERK, extracellular signal-regulated

kinase; ESI, electrospray ionization; HPLC, high-performance

liquid chromatography; HRMS, high-resolution mass spectros-

copy; K_D, equilibrium dissociation constants; k_off, dissociation

rate constant; MAPK, mitogen-activated protein kinase; MEK,

mitogen-activated protein kinase; MRT, mean residence time;

MS, mass spectroscopy; NMP, 1-methylpyrrolidone; PD,

pharmacodynamic; PDB, protein data bank; PK, pharmacoki-

netic; SAR, structure−activity relationships; SCC, squamous

cell carcinoma; SD, solid dispersion; SD, standard deviation;

SPR, surface plasmon resonance; TFAA, triﬂuoroacetic

anhydride; TLC, thin-layer chromatography; VDss, steady

state volume of distribution; VEGF, vascular endothelial growth

factor; VEGFR2, vascular endothelial growth factor receptor 2;

wt, wild-type

REFERENCES

■

(13) Heidorn, S. J.; Milagre, C.; Whittaker, S.; Nourry, A.; Niculescu-

Duvas, I.; Dhomen, N.; Hussain, J.; Reis-Filho, J. S.; Springer, C. J.;

Pritchard, C.; Marais, R. Kinase-dead BRAF and oncogenic RAS

cooperate to drive tumor progression through CRAF. Cell 2010, 140

(2), 209−221.

(1) Maurer, G.; Tarkowski, B.; Baccarini, M. Raf kinases in cancer-

roles and therapeutic opportunities. Oncogene 2011, 30, 3477−3488.

(2) Bollag, G.; Hirth, P.; Tsai, J.; Zhang, J.; Ibrahim, P. N.; Cho, H.;

Spevak, W.; Zhang, C.; Zhang, Y.; Habets, G.; Burton, E. A.; Wong, B.;

Tsang, G.; West, B. L.; Powell, B.; Shellooe, R.; Marimuthu, A.;

Nguyen, H.; Zhang, K. Y. J.; Artis, D. R.; Schlessinger, J.; Su, F.;

Higgins, B.; Iyer, R.; D’Andrea, K.; Koehler, A.; Stumm, M.; Lin, P. S.;

(14) Yang, H.; Higgins, B.; Kolinsky, K.; Packman, K.; Go, Z.; Iyer,

R.; Kolis, S.; Zhao, S.; Lee, R.; Grippo, J. F.; Schostack, K.; Simcox, M.

P

dx.doi.org/10.1021/jm400778d | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

E.; Heimbrook, D.; Bollag, G.; Su, F. RG7204 (PLX4032), a selective

BRAFV600E inhibitor, displays potent antitumor activity in preclinical

melanoma models. Cancer Res. 2010, 70, 5518−5527.

(15) Okaniwa, M.; Hirose, M.; Imada, T.; Ohashi, T.; Hayashi, Y.;

Miyazaki, T.; Arita, T.; Yabuki, M.; Kakoi, K.; Kato, J.; Takagi, T.;

Kawamoto, T.; Yao, S.; Sumita, A.; Tsutsumi, S.; Tottori, T.; Oki, H.;

Sang, B. -C.; Yano, J.; Aertgeerts, K.; Yoshida, S.; Ishikawa, T. Design

and synthesis of novel DFG-out RAF/vascular endothelial growth

factor receptor 2 (VEGFR2) inhibitors. 1. Exploration of [5,6]-fused

bicyclic scaffolds. J. Med. Chem. 2012, 55, 3452−3478.

(16) Okaniwa, M.; Imada, T.; Ohashi, T.; Miyazaki, T.; Arita, T.;

Yabuki, M.; Sumita, A.; Tsutsumi, S.; Higashikawa, K.; Takagi, T.;

Kawamoto, T.; Inui, Y.; Yoshida, S.; Ishikawa, T. 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. Bioorg. Med. Chem.

2012, 20, 4680−4692.

(17) Hirose, M.; Okaniwa, M.; Miyazaki, T.; Imada, T.; Ohashi, T.;

Tanaka, Y.; Arita, T.; Yabuki, M.; Kawamoto, T.; Tsutsumi, S.; Sumita,

A.; Takagi, T.; Sang, B. C.; Yano, J.; Aertgeerts, K.; Yoshida, S.;

Ishikawa, T. 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. Bioorg. Med. Chem. 2012, 20, 5600−5615.

(18) For BRAF, DOI: 10.2210/pdb4dbn/pdb. For VEGFR2,

DOI:10.2210/pdb3vnt/pdb.

(19) Kaufmann, H. P.; Weber, E. Arzneimittelsynthetische studien

IV. Synthese schwefelhaltiger verbindungen. Arch. Pharm. 1929, 192−

211.

(20) Stuckwisch, C. G. Derivatives of 2-amino-6-methoxybenzothia-

zole. J. Am. Chem. Soc. 1949, 71, 3417.

(21) Ismail, I. A.; Sharp, D. E.; Chedekel, M. R. Synthesis of

benzothiazoles. α-Amino-(4-hydroxy-6-benzothiazolyl)propionic acid.

J. Org. Chem. 1980, 45, 2243−2246.

(22) Charris, J.; Monasterios, M.; Dominguez, J.; Infante, W.; De

Castro, N. Synthesis of some 5-nitro-2-furfurylidene derivatives and

their antibacterial and antifungal activities. Heterocycl. Commun. 2002,

8 (3), 275−280.

(23) Tomasi, A.; Morgan, B. P.; McDonald, A.; Roof, D.; Maxon, M.

Compounds, Compositions, and Methods. WIPO Patent Application

WO 2004/098494, Nov 18, 2004.

data. A medicinal chemistry focused analysis. J. Chem. Inf. Model. 2008,

48 (1), 1−24.

(33) Tsai, J.; Lee, J. T.; Wang, W.; Zhang, J.; Cho, H.; Mamo, S.;

Bremer, R.; Gillette, S.; Kong, J.; Haass, N. K.; Sproesser, K.; Li, L.;

Smalley, K. S.; Fong, D.; Zhu, Y. L.; Marimuthu, A.; Nguyen, H.; Lam,

B.; Liu, J.; Cheung, I.; Rice, J.; Suzuki, Y.; Luu, C.; Settachatgul, C.;

Shellooe, R.; Cantwell, J.; Kim, S. H.; Schlessinger, J.; Zhang, K. Y.;

West, B. L.; Powell, B.; Habets, G.; Zhang, C.; Ibrahim, P. N.; Hirth,

P.; Artis, D. R.; Herlyn, M.; Bollag, G. Discovery of a selective inhibitor

of oncogenic B-Raf kinase with potent antimelanoma activity. Proc.

Natl. Acad. Sci. U.S.A. 2008, 105, 3041−3046.

(34) Wood, E. R.; Truesdale, A. T.; McDonald, O. B.; Yuan, D.;

Hassell, A.; Dickerson, S. H.; Ellis, B.; Pennisi, C.; Horne, E.; Lackey,

K.; Alligood, K. J.; Rusnak, D. W.; Gilmer, T. M.; Shewchuk, L.

Aunique structure for epidermal growth factor receptor bound to

GW572016 (Lapatinib): relationships among protein conformation,

inhibitor off-rate, and receptor activity in tumor cells. Cancer Res. 2004,

64, 6652−6659.

(35) Iwata, H.; Imamura, S.; Hori, A.; Hixon, M. S.; Kimura, H.;

Miki, H. Biochemical characterization of TAK-593, a novel VEGFR/

PDGFR inhibitor with a two-step slow binding mechanism.

Biochemistry 2011, 50, 738−751.

(36) Copeland, R. A.; Pompliano, D. L.; Meek, T. D. Drug-target

residence time and its implications for lead optimization. Nat. Rev.

Drug Discovery 2006, 5 (9), 730−739.

(37) The Ministry of Health, Labour and Welfare. The Ministry of

Health, Labour and Welfare Ministerial Notiﬁcation No. 285. General

Tests/Reagents, Test Solutions. The Japanese Pharmacopoeia, English

version, Ministry of Health, Labour and Welfare: Tokyo, Japan, 2006;

Vol. 15, p. 239.

(38) Lavoie, H.; Thevakumaran, N.; Gavory, G.; Li, J. J.; Padeganeh,

A.; Guiral, S.; Duchaine, J.; Mao, D. Y. L.; Bouvier, M.; Sicheri, F.;

Therrien, M. Inhibitors that stabilize a closed RAF kinase domain

conformation induce dimerization. Nat. Chem. Biol. 2013, 9 (7), 428−

436.

(39) Carnahan, J.; Beltran, P. J.; Babij, C.; Le, Q.; Rose, M. J.;

Vonderfecht, S.; Kim, J. L.; Smith, A. L.; Nagapudi, K.; Broome, M. A.;

Fernando, M.; Kha, H.; Belmontes, B.; Radinsky, R.; Kendall, R.;

Burgess, T. L. Selective and potent Raf inhibitors paradoxically

stimulate normal cell proliferation and tumor growth. Mol. Cancer

Ther. 2010, 9, 2399−2410.

(40) Galvin, K. MLN2480: A Novel pan-RAF Inhibitor for Treatment

of BRAF Mutant and Wild-Type Tumors, 103rd Annual Meeting of

American Association for Cancer Research (AACR), Chicago, IL, Mar

31−Apr 4, 2012; Abstract.

(24) Malik, J. K.; Manvi, F. V.; Nanjwade, B. K.; Singh, S. Synthesis

and screening of some new 2-amino substituted-benzothiazole

derivatives for antifungal activity. Drug Invention Today 2009, 1 (1),

32−34.

(25) Schnur, R. C.; Fliri, A. F. J.; Kajiji, S.; Pollack, V. A. N-(5-

fluorobenzothiazol-2-yl)-2-guanidinothiazole-4-carboxamide. A novel,

systemically active antitumor agent effective against 3LL Lewis Lung

carcinoma. J. Med. Chem. 1991, 34, 914−918.

(26) Wang, M.; Gao, M.; Mock, B. H.; Miller, K. D.; Sledge, G. W.;

Hutchins, G. D.; Zheng, Q. H. Synthesis of carbon-11 labeled

fluorinated 2-arylbenzothiazoles as novel potential PET cancer imaging

agents. Bioorg. Med. Chem. 2006, 14 (24), 8599−8607.

(27) Wang, M.; Jiang, S.; Hou X. [1,2,3] Piazthiole-7-formhydrazide

Derivative as I-Type Plant Disease-Resisting Revulsive. SIPO Patent

Application CN 1389114, Jan 08, 2003.

(28) Gyorkos, A. C.; Corrette, C. P.; Cho, S. Y.; Turner, T. M.; Pratt,

S. A.; Aso, K.; Kori, M.; Gyoten, M. Preparation of Nitrogen-

Containing Fused Heterocyclic Compounds Like Benzimidazoles and

Benzothiazoles as CRF Receptor Antagonists. WIPO Patent

Application WO 2005/044793, May 19, 2005.

(29) The coordinates and structure factors have been deposited with

the Protein Data Bank with accession code 4KSP for BRAF with 8B.

(30) Bondi, A. van der Waals volumes and radii. J. Phys. Chem. 1964,

68, 441−451.

(31) The coordinates and structure factors have been deposited with

the Protein Data Bank with accession code 4KSQ for BRAF with 5B.

(32) Brameld, K. A.; Kuhn, B.; Reuter, D. C.; Stahl, M. Small

molecule conformational preferences derived from crystal structure

Q

dx.doi.org/10.1021/jm400778d | J. Med. Chem. XXXX, XXX, XXX−XXX

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Discovery of a selective kinase inhibitor (TAK-632) targeting pan-RAF inhibition: Design, synthesis, and biological evaluation of C -7-substituted 1,3-benzothiazole derivatives

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