2-{3-[4-(Alkylsulfinyl)phenyl]-1-benzofuran-5-yl}-5-methyl-1,3,4-oxadiazole derivatives as novel inhibitors of glycogen synthase kinase-3β with good brain permeability

6270 J. Med. Chem. 2009, 52, 6270–6286

DOI: 10.1021/jm900647e

2-{3-[4-(Alkylsulfinyl)phenyl]-1-benzofuran-5-yl}-5-methyl-1,3,4-oxadiazole Derivatives as Novel

Inhibitors of Glycogen Synthase Kinase-3β with Good Brain Permeability^†

Morihisa Saitoh,*^,‡Jun Kunitomo,^‡Eiji Kimura,^‡Hiroki Iwashita,^‡Yumiko Uno,^‡Tomohiro Onishi,^‡Noriko Uchiyama,^‡

Tomohiro Kawamoto,^‡Toshimasa Tanaka,^‡Clifford D. Mol,^§Douglas R. Dougan,^§Garret P. Textor,^§Gyorgy P. Snell,^§

Masayuki Takizawa,^‡Fumio Itoh,^‡and Masakuni Kori^‡

^‡Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd., 17-85 Jusohonmachi, 2-Chome, Yodogawa-ku, Osaka 532-8686,

Japan, and ^§Takeda San Diego, Inc., 10410 Science Center Drive, San Diego, California 92121

Received May 16, 2009

Glycogen synthase kinase 3β (GSK-3β) inhibition is expected to be a promising therapeutic approach

for treating Alzheimer’s disease. Previously we reported a series of 1,3,4-oxadiazole derivatives as potent

and highly selective GSK-3β inhibitors, however, the representative compounds 1a,b showed poor

pharmacokinetic profiles. Efforts were made to address this issue by reducing molecular weight and

lipophilicity, leading to the identification of oxadiazole derivatives containing a sulfinyl group, (S)-9b

and (S)-9c. These compounds exhibited not only highly selective and potent inhibitory activity against

GSK-3β but also showed good pharmacokinetic profiles including favorable BBB penetration. In

addition, (S)-9b and (S)-9c given orally to mice significantly inhibited cold water stress-induced

tau hyperphosphorylation in mouse brain.

Introduction

of microtubules, which then aggregate to form NFTs. Nor-

mally two to three residues on tau bear phosphate, however,

hyperphosphorylated tau protein in AD and other tauopa-

thies have close to nine phosphates.²Although phosphoryla-

tion of tau protein is regulated with balance in the activity

levels of kinases and phosphatases, hyperphosphorylation of

tau is thought to be induced mainly by the serine/threonine

kinase GSK-3β.³Overexpression of GSK-3β in cultured cells

and in transgenic mice results in hyperphosphorylation of

tau at several of the same sites seen in AD and inhibition

by lithium chloride attenuates phosphorylation in these

models.^4-11These studies suggest that GSK-3β is associated

with AD progression, and GSK-3β inhibitors are expected

to be promising therapeutic agents for AD. Many GSK-3β

inhibitors that are ATP competitive inhibitors have been

reported and their medicinal use has been reviewed in the

literature.^12,13Maleimide derivatives have been reported by

many groups.^14,15A variety of compounds with different

chemical structures have been reported, such as 2,5-diamino-

pyrimidine,¹⁶1-aza-9-fluorene,¹⁷and thiazolo[5,4-f]quinazo-

line derivatives.¹⁸In addition, natural product derived GSK-

3β inhibitors such as indirubins,¹⁹paullones,²⁰and manza-

mines²¹have been described. In this paper, we report the

design and synthesis of novel small molecular GSK-3β in-

hibitors having a 1,3,4-oxadiazole scaffold and the evaluation

of their in vivo efficacy against tau hyperphosphorylation in

mice. Previously, we reported a series of novel 1,3,4-oxadia-

zole derivatives as potent and highly selective GSK-3β in-

hibitors. Among them, 2-(1-benzofuran-5-yl)-1,3,4-oxadia-

zolederivative 1aand 2-(1H-benzimidazol-6-yl)-1,3,4-oxadia-

zole derivative 1b exhibited high potency with IC₅₀values of

3.5 and 2.3 nM, respectively (Figure 1).²²

Alzheimer’s disease (AD^a) is a dementia of middle- to old-

aged individuals and was discovered in 1906 by Alois Alzhei-

mer. AD has pathological features of neurofibrillary tangles

(NFT), senile plaque, and neuronal death. Neurofibrillary

degeneration appears to be required for the clinical expression

of the disease, and formation of NFT and neuronal death

correlate with the presence and or the degree of dementia in

AD.¹Thus, inhibition of NFT formation is a viable strategy

for the treatment of AD. NFT is composed of the abnormally

hyperphosphorylated microtubule-associated protein tau.

Hyperphosphorylation of tau protein causes destabilization

^†PDB codes for GSK-3β complexes 1b and (S)-9b are 3F88 and

3GB2, respectively.

*To whom corresponding should be addressed. Phone: þ81-6-6300-

6120. Fax: þ81-6-6300-6306. E-mail: Saitoh_Morihisa@takeda.co.jp.

^aAbbreviations: Ac, acetyl; AcOH, acetic acid; AD, Alzheimer’s

disease; AIBN, 2,2⁰-azobis(2-methylpropionitrile) BBB, blood-brain

barrier; n-BuOH, n-butanol; CDCl₃, deuterated chloroform; CDI, 1,1⁰-

carbonyldiimidazole; CWS; cold water stress; DBU, 1,8-diazabicyclo-

[5.4.0]undec-7-ene; DMA, N,N-dimethylacetamide; DMB, 2,4-dimeth-

oxybenzyl; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfox-

ide; DMSO-d₆, dimethyl sulfoxide-d₆; EDC, N-(3-dimethylaminopro-

pyl)-N⁰-ethylcarbodiimide hydrochloride; ERBB, v-erb-a erythroblastic

leukemia viral oncogene homologue; EtOAc, ethyl acetate; EtOH,

ethanol; FGFR, fibroblast growth factor receptor; GSK-3β, glycogen

synthase kinase 3β; HOBt, 1-hydroxybenzotriazole; HPLC, high-per-

formance liquid chromatography; IMAC, immobilized metal-chelate

affinity chromatography; m-CPBA, m-chloroperbenzoic acid; MeOH,

methanol; NBS, N-bromosuccinimide; NEt₃, triethylamine; NFT, neu-

rofibrillary tangles; NIS, N-iodosuccinimide; PDGFR, platelet-derived

growth factor receptor; PhCl, chlorobenzene; RIPA, radioimmunopre-

cipitation assay; rt, room temperature; TFA, trifluoroacetic acid;

Tf₂NPh, N-phenyl-bis(trifluoromethanesulfonimide); THF, tetrahy-

drofuran; TsCl, p-toluenesulfonyl chloride; p-TsOH, p-toluenesulfonic

acid monohydrate; VEGFR, vascular endothelial growth factor recep-

tor.

Although the X-ray cocrystal structure of compound 1b

with GSK-3β was not fully elucidated due to cleavage of the

r

pubs.acs.org/jmc

Published on Web 09/23/2009

2009 American Chemical Society

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6271

Figure 1. Design of compound (I) from compound 2.

the addition of hydrazine to 5. Reaction of the hydrazide 6 with

triethyl orthoacetate afforded oxadiazole 7. The 3-substituted

benzofurans 8a-d,f-j,l,n were prepared by reaction of 7 with

corresponding aryl boronic acid. Hydrolysis of 8d afforded

carboxylic acid 8e. Reduction of ketone 8j with NaBH₄gave

alcohol 8k. Reaction of phenol 8l with N-phenyl-bis(trifluoro-

methanesulfonimide) (PhNTf₂) and subsequent Pd-coupling

with dimethyl phosphite gave phosphate 8m. Oxidation of

methyl sulfides 8a,i and ethyl sulfide 8b with m-chloroperbenzoic

acid (m-CPBA) gave sulfoxides 9a-c and sulfone 9d (Scheme 1).

Coupling of 4-(methylthio)phenylboronic acid with 3-bro-

mobenzofuran intermediate 5 gave 10, which was treated with

hydrazine to afford hydrazide 11. Hydroxy-oxadiazole 12a

was obtained from hydrazide 11 by the reaction with 1,1⁰-

carbonyldiimidazole. Reaction of 11 with cyanogen bromide

gave amino-oxadiazole 12b. Sulfides 12a,b were oxidized to

sulfoxides 13a,b with oxone or m-CPBA. Sulfide 10 was

oxidized to sulfoxide 14, then reacted with hydrazine, carbon

disulfide, and triethylamine (NEt₃) followed by acidification

to yield 1,3,4-oxadiazole-2-thiol 15 (Scheme 2).

Figure 2. X-ray cocrystal structure of 1b in complex with GSK-3β.

The figure was prepared with PyMOL.²³

S-C bond in the X-ray beam to form the debenzylated

compound, some information could be gathered from the

partial structure solution. The structure (Figure 2) shows the

following major hydrogen bond interactions: the benzimida-

zole ring and the hinge region of GSK-3β, N3-nitrogen of the

oxadiazole ring and NH of Asp200, the N4-nitrogen of the

oxadiazole ring and a conserved water molecule, and the

4-methoxy group of the phenyl ring and Arg141.

Unfortunately, the bioavailability of compounds 1a and 1b

in rat was poor, probably due to poor solubility and instability

in rat liver microsomes. To improve the solubility and meta-

bolic stability of these derivatives, we focused on modification

of the highly lipophilic and metabolically labile S-benzyl

group, replacing it with a methyl group. The methyl oxadia-

zole derivative 2 was found to retain potent inhibitory activity

(IC₅₀=54 nM), while compound 2 still showed poor meta-

bolicstability andbioavailability. However, the lowmolecular

weight (MW 306.3) of compound 2 made us consider it

worthwhile to perform further optimization and SAR studies

on this series of compounds. Thus, we designed a series of

compounds based on this scaffold (I). On the basis of the X-

ray cocrystal structure of 1b and GSK-3β, we particularly

exploredother polarsubstituentsonthe phenyl ringinplace of

the methoxy group interacting with Arg141 in order to

improve metabolic stability and solubility and also modified

the hinge binding portion and the oxadiazole moiety.

Next, replacement of the benzofuran ring to other 6-5

fused heterocycles was performed. The syntheses are outlined

in Schemes 3-5. Ester 16²⁴and 4-(methylthio)aniline were

reacted in DMF to yield diphenyl amine 17. The nitro group

of 17 was reduced with sodium hydrosulfite to provide aniline

18, which was cyclized withformic acid toform benzimidazole

19. Reaction with hydrazine, followed by acetylation and

cyclization, afforded oxadiazole 22. Oxidation of 22 with

m-CPBA gave sulfoxide 23 (Scheme 3).

Esters 24^25-28were halogenated and coupled with

[4-(methylthio)phenyl]boronic acid to provide 26. Treatment

with hydrazide and cyclization gave oxadiazoles 28. Oxida-

tion of sulfides 28 afforded sulfoxides 29 (Scheme 4).

Condensation of carboxylic acid 30²⁹with acetohydrazide

and cyclization using p-toluenesulfonyl chloride (TsCl) pro-

vided oxadiazole 32. Coupling and oxidation of the sulfide

33 afforded sulfoxide 34 (Scheme 5).

The preparation of 1,2,4-triazole 36, 1,3,4-thiadiazole 39,

1,2,4-oxadiazoles 41 and 46, and pyridazine 51 were per-

formed according to Schemes 6-8. Oxadiazole 9b was reacted

with 2,4-dimethoxybenzylamine, followed by cleavage of

the 2,4-dimethoxybenzyl (DMB) group using trifluoroacetic

acid (TFA) to give triazole 36. Hydrazide 11 was condensed

with acetic acid (AcOH) and cyclized upon treatment with

Lawesson’s reagent to give thiadiazole 38, which was oxidized

to provide sulfoxide 39 (Scheme 6).

Chemistry

Commercially available 2,3-dihydro-1-benzofuran-5-car-

baldehyde 3 was directly converted to ester 4 by using iodine

and KOH. The resulting ester 4 was treated with N-bromo-

succinimide (NBS), followed by bromination with bromine

afforded 3-bromobenzofuran 5. Hydrazide 6 was prepared by

1,2,4-Oxadiazole 40 was synthesized from ester 10 and

acetamidoxime and subsequent oxidation yielded sulfoxide

41. Hydrolysis of 10 with NaOH followed by condensation

6272 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20

Saitoh et al.

Scheme 1^a

^aReagents and conditions: (a) I₂, KOH, MeOH, 0 °C, 86%; (b) (1) NBS, AIBN, PhCl, 80-85 °C, (2) Br₂, CH₂Cl₂, 0 °C∼rt, then KOH, MeOH-THF,

0 °C, 89%; (c) NH₂NH₂H₂O, MeOH, reflux, 95%; (d) CH₃C(OEt)₃, 120 °C, 97%; (e) Pd(PPh₃)₄, Na₂CO₃, DME-H₂O, ArB(OH)₂, reflux, 42-95%;

3

(f) 1 M NaOH, THF-MeOH, 60 °C, 83%; (g) NaBH₄, EtOH, 0 °C, 76%; (h) (1) PhNTf₂, NaH, THF, 92%, (2) HP(O)(OMe)₂, Pd(PPh₃)₄, i-Pr₂NEt,

toluene, 100 °C, 55%; (i) m-CPBA, CH₂Cl₂, 0 °C or rt, 26-84%.

Scheme 2^a

^aReagents and conditions: (a) 4-(methylthio)phenylboronic acid, Pd(PPh₃)₄, Na₂CO₃, DME-H₂O, reflux, 85%; (b) NH₂NH₂H₂O, MeOH, reflux,

3

95%; (c) CDI, Et₃N, DMF, 0 °C, 74% (12a); (d) BrCN, DMA, 79% (12b); (e) m-CPBA, CH₂Cl₂, 80% (13a); (f) oxone, THF-H₂O, 0 °C, 6% (13b);

(g) m-CPBA, CH₂Cl₂, 93%; (h) (1) NH₂NH₂H₂O, MeOH, reflux; (2) CS₂, NEt₃, EtOH, reflux, 47%.

3

with ammonium hydroxide afforded amide 43. Dehydra-

tion of 43 was accomplished with thionyl chloride to give

nitrile 44. Treatment of 44 with hydroxylamine hydrochlor-

ide and NaHCO₃gave the amidoxime intermediate, which

was acylated with acetic anhydride in dioxane to give

oxadiazole 45, followed by oxidation to yield sulfoxide 46

(Scheme 7).

Aldehyde 3 was converted to 3-bromobenzofuran 47, which

was condensed with methyl vinyl ketone to provide diketone

48. Reaction of diketone 48 with hydrazine afforded pyridazine

49, which was coupled with 4-(methylthio)phenylboronic

acid and subsequent oxidation gave sulfoxide 51 (Scheme 8).

Results and Discussion

The compounds synthesized were evaluated for GSK-3β

inhibitory activity in a non-RI kinase assay using Kinase-Glo

reagents and the results are shown in Tables 1-5. First,

we synthesized compounds with various substituents on the

3-phenyl ring (Table 1). Replacement of methoxy group of 2

with carboxylic acid or carboxamide (8e,f) showed similar

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6273

Scheme 3^a

^aReagents and conditions: (a) 4-(methylthio)aniline, DMSO, 110 °C, 93%; (b) Na₂S₂O₄, THF-EtOH, 0 °C∼rt, 100%; (c) HCO₂H, 100 °C, 74%;

(d) NH₂NH₂H₂O, EtOH, 86%; (e) AcCl, DMA, 0 °C, 100%; (f) TsCl, pyridine, 90 °C, 66%; (g) m-CPBA, CH₂Cl₂, 85%.

3

Scheme 4^a

^aReagents and conditions: (a) NIS, CH₃CN, 80 °C, 73% (25b); (b) (1) Br₂, AcOH or CH₂Cl₂, (2) KOH, THF-EtOH, 0 °C, 85% (25a), 36% (25c), 30%

(25d); (c) 4-(methylthio)phenylboronic acid, Pd(PPh₃)₄, Na₂CO₃, DME, H₂O, reflux, 32-95%; (d) NH₂NH₂H₂O, MeOH or EtOH, reflux, 49-95%;

3

(e) CH₃C(OEt)₃, 120 °C, 93% (28a); (f) CH₃C(OEt)₃, DBU, n-BuOH, reflux, 67% (28c); (g) (1) AcCl, DMA, (2) TsCl, pyridine, 100 °C, 29% (28b), 58%

(28d); (h) m-CPBA, CH₂Cl₂, 41-86%.

activity to 2. However, the bioavailability and solubility of 8e

and 8f were improved. Although phenol 8l, ketone 8j, alcohol

8k, ester 8d, dimethyl phosphonate 8m, and 4-fluorophenyl

derivative 8c were less active than 2, 4-methylthio derivative

8a, methylsulfinyl derivative 9b, and methylsulfonyl deriva-

tive 8n exhibited equipotent or more potent activities with

IC₅₀values of 66, 35, and 42 nM, respectively. These results

point to the importance of electrostatic interaction with

Arg141 in determining inhibitory activity. Among these

compounds, methylsulfinyl derivative 9b exhibited good

solubility (38 μg/mL) and bioavailability (F=40%). Repla-

cing the methyl group of 9b or 8n with an ethyl group

resulted in similar inhibitory potency (9c,d), with 9c show-

ing good solubility (62 μg/mL) and bioavailability (F =

6274 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20

Saitoh et al.

Scheme 5^a

^aReagents and conditions: (a) AcNHNH₂, EDC, HOBt, DMF, 28%; (b) TsCl, pyridine, 90 °C, 42%; (c) 4-(methylthio)phenylboronic acid,

Pd(PPh₃)₄, Na₂CO₃, DME, H₂O, reflux, 52%; (d) m-CPBA, CH₂Cl₂, 67%.

Scheme 6^a

^aReagents and conditions: (a) 2,4-dimethoxybenzylamine, p-TsOH, xylene, 150 °C, 44%; (b) TFA, rt, 39%; (c) AcOH, EDC, HOBt, DMF, 75%;

(d) Lawesson’s reagent, toluene, 80 °C, 54%; (e) m-CPBA, CH₂Cl₂, 93%.

Scheme 7^a

^aReagents and conditions: (a) acetamidoxime, NaH, THF, 60 °C, 67%; (b) m-CPBA, CH₂Cl₂, 91%; (c) 1M NaOH aq, MeOH-THF, 96%; (d) (1) EDC,

HOBt, DMF; (2) aq NH₃, rt, 95%; (e) SOCl₂, DMF, 76%; (f) (1) NH₂OH HCl, NaHCO₃, MeOH, reflux; (2) Ac₂O, dioxane, 90 °C, 76%; (g) m-CPBA,

3

CH₂Cl₂, 87%.

85%). Conversion of the phenyl ring to pyridine showed

similar activity, improved solubility, and bioavailability

compared to 2. Shifting the methoxy or sulfinyl group at

the 4-postion to the 3-position resulted in decreased activity

(8h, 9a). It was observed that phenyl-substituted com-

pounds with lower log D values generally showed improved

solubility and metabolic stability. Because 4-methylsulfinyl

phenyl derivative 9b showed good solubility and bioavail-

ability, we modified other parts of 9b.

The effect of converting the benzofuran ring to other

heterocycles is shown in Table 2. Despite our expectations,

based on previous studies, that benzimidazole would be a

more suitable hinge binder, compound 23 showed a 5-fold

decrease in activity. It is possible that tighter binding of the

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6275

Scheme 8^a

^aReagents and conditions: (a) (1) NBS, AIBN, PhCl, 80 °C, 83%, (2) Br₂, CH₂Cl₂then KOH, EtOH, 0 °C, 44%; (b) methyl vinyl ketone, NEt₃,

3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide EtOH, 70 °C, 25%; (c) NH₂NH₂H₂O, EtOH, reflux, 23%; (d) [4-(methylthio)phenyl]boronic

3

acid, Na₂CO₃, Pd(PPh₃)₄, DME, H₂O, reflux, 68%; (e) m-CPBA, CH₂Cl₂, 87%.

Table 1. Effect of Substitutions on Phenyl Ring

Table 2. Effect of Hinge Binding Element

metabolic

stability

IC₅₀log

(nM)^aD^b(mL/min/mg)

human^c

Solubility^d

F^e

(%)

compd

R

(μg/mL)

2

Ph-4-OMe

54 3.39

50 1.18

69 1.67

180 2.47

100 2.74

179

0

0.14

54

nd^f

28

8e Ph-4-CO₂H

8f Ph-4-CONH₂

8l Ph-4-OH

121

170

157

85

32

1.1

0.13

26

nd^f

9.8

6.6

nd^f

44

8j Ph-4-COMe

8k Ph-4-C(OH)Me 160 2.68

8d Ph-4-CO₂Me 200 3.22

8m Ph-4-P(O)(OMe)₂280 2.28

248

0

0.07

31

8c Ph-4-F

110 3.32

66 3.86

35 1.75

42 1.69

35 2.13

38 2.04

77 1.87

74 3.41

210 1.85

155

75

1.0

0.06

38

nd^f

1.4

40

8a Ph-4-SMe

9b Ph-4-S(O)Me

8n Ph-4-S(O)₂Me

9c Ph-4-S(O)Et

9d Ph-4-S(O)₂Et

8g 4-Py

7

48

0.49

62

28

85

10

53

2.4

33

52

67

21

8h Ph-3-OMe

9a Ph-3-S(O)Me

191

75

0.61

>61

nd^f

10

^aIC₅₀values for GSK-3β shown are the mean of duplicate or

triplicate measurements. ^bMeasured at pH 7.4. ^cMetabolic stability in

human hepatic microsome ^dMeasured at pH 6.8. ^eBioavailability in a

rat cassette dosing at 0.1 mg/kg iv and 1 mg/kg po. ^fNot detected.

benzimidazole ring with the hinge amide could negatively

affect interactions between the oxadiazole nitrogen and the

conserved water or between the sulfoxide and Arg141. Ben-

zothiophene 29a and imidazo[1,2-a]pyridine derivative 29b

showed decrease in activity. Reduced electron density of the

heterocycles and altered direction of the two side chains might

affect the binding affinities as well. In the furopyridine series,

furo[3,2-b]pyridine 29c showed moderate activity, while furo-

[2,3-b]pyridine 29d and furo[2,3-c]pyridine 34 exhibited mark-

edly decreased activity.

^aIC₅₀values for GSK-3β shown are the mean of duplicate or

triplicate measurements.

4-thiadiazole 39 and 1,2,4-triazole 36 showed slightly de-

creased activity compared to 1,3,4-oxadiazole 9b. However,

pyridazine 51 showed markedly reduced potency, as did

isomers of 9b, 1,2,4-oxadiazoles 46, 41. These results suggest

that the adjacent nitrogen atom and ring size are closely fit to

the bindingpocket. Reduced potencyof compounds46and 41

might be due to reduced electron density of the nitrogen atom,

which resulted in weak hydrogen bond with the binding sites.

Substitution of the 1,3,4-oxadiazole ring and its impact on

inhibitory activity is displayed in Table 3. Interestingly, 1,3,

6276 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20

Table 3. Effect of Heterocyclic Rings

Saitoh et al.

Table 5. Inhibitory Activity of Enantiomers

IC₅₀

(nM)^a

compd

R

(R)-9b

(S)-9b

(R)-9c

(S)-9c

Me

Et

140

34

190

20

Et

^aIC₅₀values for GSK-3β shown are the mean of duplicate measure-

ments.

The effect of varying the methyl group on the oxadiazole

ring is shown in Table 4. Mercapto- and hydroxy-oxadiazoles

15, 13a showed reduced activity, while amino-oxadiazole 13b

showed 3-fold more potent activity compared to 9b. A dock-

ing study suggested that the amino group interacts with the

carboxyl group of Asp200. Unfortunately, amino-oxadiazole

13b had poor oral bioavailability, probably due to low

solubility and absorption (data not shown). We found that

in our newly designed scaffold (I) the introduction of appro-

priate polar substituents at the 4-position of the phenyl ring

improves inhibitory activity and biological properties, and the

introduction of a five-membered azole with an adjacent

nitrogen is suitable for interaction with the hydrogen bond

network composed of the surrounding amino acids (e.g.,

Lys85, Glu97, Asp200) and conserved water molecules. On

the basis of these results, sulfoxide derivatives 9b and 9c were

selected for further evaluation.

^aIC₅₀values for GSK-3β shown are the mean of duplicate measure-

ments.

Table 4. Effect of Substituents on the Oxadiazole

We measured the potencies for the enantiomers of 9b and

9c. Optical resolution of the racemates 9b and 9c was carried

out on a chiral column by high performance liquid chroma-

tography (HPLC). Absolute configurations were confirmed

by X-ray crystallographic analysis of (S)-9b and (S)-9c as

illustrated in Figure 3.

The S-isomers (S)-9b, (S)-9c were found to be eutomers as

shown in Table 5.

IC₅₀

(nM)^a

compd

R

Pharmacokinetic profiles in rats and brain concentrations

in mice of (S)-9b and (S)-9c were evaluated and the results are

shown in Table 6. (S)-9b and (S)-9c possessed good oral

absorption in rats with bioavailability (F %) of 72.8% and

65.5%, respectively. In addition, when (S)-9b and (S)-9c were

given orally at a dose of 3 mg/kg in mice, AUC_0-24hs were 734

9b

15

Me

SH

35

94

13a

13b

OH

NH₂

130

13

^aIC₅₀values for GSK-3β shown are the mean of duplicate or

triplicate measurements.

and 547 ng h/g, and K_pvalues (ratio of brain and plasma)

3

were 1.6 and 1.3, respectively (Table 6). Thus, these com-

pounds exhibited good pharmacokinetic profiles and favor-

able BBB permeability.

To determine kinase selectivity profiles, (S)-9b and (S)-9c

were tested for inhibitory activity against 10 serine/threonine

kinasesand12tyrosine kinases. Compounds(S)-9b and (S)-9c

showed no significant activity and IC₅₀values were greater

than 10 μM, indicating that these compounds are highly

selective GSK-3β inhibitors (Table 7).

The X-ray cocrystal structure of (S)-9b was obtained, and

analysis revealed that the N4 nitrogen atom of the oxadiazole

ring formed a hydrogen bond with the side chain of Lys85 of

the conserved Lys-Glu pair (Figure 4). The oxygen atom and

Figure 3. X-ray crystal structures of (S)-9b and (S)-9c.

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6277

Table 6. Pharmacokinetic Parameters in Rats and Brain or Plasma Concentrations in Mice

rats^a,b

mice^a,c

V_dss,iv

(mL/

kg)

CL_total,iv

(mL/min/

kg)

C_max,po

(ng/

mL)

AUC_0-24h,

brain

(ng h/

plasma

(ng h/

MRT_po

(h)

F^d

(%)

po

3

mL)

compd

(ng h/mL)

3

g)

(S)-9b

(S)-9c

1134

1650

27.4

28.4

396.9

289.6

1380.6

1229.1

2.19

3.03

72.8

65.5

734

547

457

409

^aData are expressed as the mean of three determinations. ^bDosed at 1 mg/kg iv and 3 mg/kg po in Crj:CD (SD) IGS rats. ^cDosed at 3 mg/kg po in

C57BL/6N mice. ^dBioavailability.

Table 7. Kinase Selectivity of (S)-9b, (S)-9c

IC₅₀(nM)

Ser/Thr kinase Tyr kinase

CDK1, CDK2, CDK5, CHK1, EGFR, ERBB2, Src, Lck, IR,

p38R, JNK1, MEKK1, IKKβ, TIE2, c-Kit, c-Met, VEGFR2,

compd

PKCθ, CK1δ

FGFR3, PDGFRR, PDGFRβ

(S)-9b

(S)-9c

>10000

Figure 5. Effect of (S)-9b and (S)-9c on cold water stress (CWS)

induced tau phosphorylation. (S)-9b and (S)-9c were orally admi-

nistered at a dose of 3 mg/kg 30 min before the CWS. Immunoblot

bands were quantified by densitometry. Data represent means (

SEM of 5 animals and are expressed as phospho-tau normalized to

total tau. Significance is defined as ### p e 0.001 (Student’s t test) in

comparison to control group and as *** p e 0.001 (Student’s t test)

in comparison to CWS-treated control group.

Arg141 led to identification of sulfoxide derivatives 9b and 9c.

Their eutomers (S)-9b and (S)-9c showed good pharmaco-

kinetic profiles including favorable BBB penetration and good

kinase selectivity. In addition, (S)-9b and (S)-9c administered

orally to mice significantly inhibited hyperphosphorylation of

tau protein in a CWS model. On the basis of these results,

compounds (S)-9b and (S)-9c were selected for further phar-

macological evaluation.

Figure 4. X-ray cocrystal structure of (S)-9b in complex with GSK-

3β. The figure was prepared with PyMOL.²³

the C2 hydrogen atom of the benzofuran ring interact with

main chain of Val135 and the hydrogen atom at the C7 also

interacts with carbonyl of Asp133 through a hydrogen bond.

The sulfoxide on the phenyl ring of (S)-9b was revealed to

interact with Arg141.

Experimental Section

Melting points were determined on a Buchi melting point

apparatus and were not corrected. Proton nuclear magnetic

resonance (¹H NMR) spectra were recorded on a Varian

Gemini-300 (300 MHz) or Bruker DPX300 (300 MHz) instru-

ments. Chemical shifts are reported as δ values (ppm) down-

field from internal tetramethylsilane of the indicated organic

solution. Peak multiplicities are expressed as follows. Abbre-

viations are used as follows: s, singlet; d, doublet; t, triplet;

q, quartet; dd, doublet of doublet; ddd, doublet of doublet

of doublets; dt, doublet of triplet; brs, broad singlet; m, multi-

plet. Coupling constants (J values) are given in hertz (Hz).

Element analyses were carried out by Takeda Analytical

Laboratories, and the results were within 0.4% of theoretical

values. LC/MS (ESI^þ) was performed on a ZQ-2000 apparatus

with acetonitrile/water mobile phase. Purities of test com-

pounds (15, 29d) were established by analytical HPLC using

the following conditions: column, Imtakt Cadenza CD-C18,

2.0 mm ꢀ 50 mm, S-3 μm; flow rate of 0.5 mL/min; solvent,

H₂O/0.1% TFA (A), MeCN/0.1% TFA (B); gradient condi-

tion: 10% B for 0.01 min, linear increase from 10% to 95% B

over 4 min, 100% B for 1.5 min. Analytical HPLC was

equipped with a LC-10ADvp pump and SPD-10A-vp (UV

detector) set at 254 and 220 nm. Preparative HPLC were

performed an automated Gilson HPLC system using a YMC

C-18 column (S-5 μM 50 mm ꢀ 20 mm i.d.) with 10-100%

gradient water-acetonitrile containing 0.1% TFA. Reaction

progress was determined by thin layer chromatography (TLC)

In vivo efficacy of (S)-9b and (S)-9c was examined using a

cold water stress model (CWS) in mice.³⁰We used the CWS

model since recent work has demonstrated the involvement of

GSK-3β in tau phosphorylation and the efficacy of lithium in

this model.^30-32CWS induced tau phosphorylation at several

GSK-3β directed sites such as pS199, pThr205, pThr231, and

pSer396. As the greatest increase in tau phosphorylation was

detected by the pThr205 antibody in our initial experiments,

we used this antibody for evaluating the efficacy of our

compounds. Compounds (S)-9b and (S)-9c significantly re-

duced tau phosphorylation by 35% and 38%, respectively

(Figure 5). We have observed that sulfoxide (S)-9b

was partially oxidized to sulfone 8n in plasma. However, oral

administration (30 mg/kg) of sulfone 8n did not significantly

reduce tau phosphorylation in the CWS model.

Conclusion

In this report, we described the synthesis and evaluation of

3-aryl benzofuran derivatives based on the previously reported

oxadiazole derivatives 1a and 1b. Removal of the S-benzyl

group and optimization of the methoxy group interacting with

6278 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20

Saitoh et al.

analysis on silica gel 60 F₂₅₄plate (Merck) or NH TLC plates

(Fuji Silysia Chemical Ltd.).

product was recrystallized from hexane/EtOAc to give 2 (0.21 g,

76%) as a colorless solid.

Methyl 2,3-dihydro-1-benzofuran-5-carboxylate (4). To a so-

lution of 3 (33.9 g, 229 mmol) and KOH (85%, 39.3 g, 596 mmol)

in MeOH (250 mL) at 0 °C was slowly added a solution of iodine

(75.6 g, 298 mmol) in MeOH (750 mL), and the mixture was

stirred overnight at room temperature. The reaction mixture

was diluted with EtOAc, and the organic layer was washed with

1 M sodium sulfite, water, and brine, dried over magnesium

sulfate, and concentrated in vacuo. The residue was purified by

basic silica gel column chromatography (hexane/EtOAc=5/1),

and the product was recrystallized from hexane/EtOAc to give

¹H NMR (CDCl₃) δ 2.64 (3H, s), 3.88 (3H, s), 7.02-7.08 (2H,

m), 7.56-7.61 (2H, m), 7.64 (1H, d, J=8.7 Hz), 7.80 (1H, s), 8.04

(1H, dd, J = 1.5, 8.7 Hz), 8.48 (1H, d, J = 1.5 Hz). Anal.

(C₁₈H₁₄N₂O₃) C, H, N.

The following compounds 8a-d,f-j,l,n were prepared in a

manner similar to that described for 2.

2-Methyl-5-{3-[4-(methylsulfanyl)phenyl]-1-benzofuran-5-yl}-

1,3,4-oxadiazole (8a). Yield 71%, colorless solid. ¹H NMR

(CDCl₃) δ 2.55 (3H, s), 2.64 (3H, s), 7.37-7.42 (2H, m),

7.57-7.61 (2H, m), 7.65 (1H, dd, J=0.8, 8.7 Hz), 7.85 (1H, s),

8.06 (1H, dd, J=1.7, 8.7 Hz), 8.48 (1H, dd, J=0.6, 1.7 Hz). Anal.

(C₁₈H₁₄N₂O₂S) C, H, N.

1

4 (35.0 g, 86%) as a colorless solid. H NMR (CDCl₃) δ 3.24

(2H, t, J=8.9 Hz), 3.87 (3H, s), 4.65 (2H, t, J=8.9 Hz), 6.79 (1H,

d, J=8.3 Hz), 7.85-7.89 (2H, m).

2-{3-[4-(Ethylsulfanyl)phenyl]-1-benzofuran-5-yl}-5-methyl-

1,3,4-oxadiazole (8b). Yield 72%, colorless solid. ¹H NMR

(CDCl₃) δ 1.38 (3H, t, J=7.3 Hz), 2.64 (3H, s), 3.02 (2H, q,

J=7.3 Hz), 7.46 (2H, d, J=8.5 Hz), 7.58 (2H, d, J=8.5 Hz), 7.65

(1H, dd, J=0.8, 8.7 Hz), 7.86 (1H, s), 8.05 (1H, dd, J=1.7,

8.7 Hz), 8.49 (1H, d, J=1.7 Hz). Anal. (C₁₉H₁₆N₂O₂S) C, H, N.

2-[3-(4-Fluorophenyl)-1-benzofuran-5-yl]-5-methyl-1,3,4-oxa-

diazole (8c). Yield 69%, colorless solid. ¹H NMR (CDCl₃) δ 2.64

(3H, s), 7.17-7.35 (2H, m), 7.59-7.67 (3H, m), 7.83 (1H, s), 8.06

(1H, dd, J=1.7, 8.7 Hz), 8.45 (1H, dd, J=0.6, 1.7 Hz). Anal.

(C₁₇H₁₁FN₂O₂) C, H, N.

Methyl 4-[5-(5-Methyl-1,3,4-oxadiazol-2-yl)-1-benzofuran-3-

yl]benzoate (8d). Yield 64%, colorless solid. ¹H NMR (CDCl₃)

δ 2.65 (3H, s), 3.97 (3H, s), 7.68 (1H, dd, J = 0.6, 8.7 Hz),

7.73-7.77 (2H, m), 7.95 (1H, s), 8.09 (1H, dd, J=1.7, 8.7 Hz),

8.16-8.20 (2H, m), 8.51 (1H, dd, J = 0.6, 1.7 Hz). Anal.

(C₁₉H₁₄N₂O₄) C, H, N.

4-[5-(5-Methyl-1,3,4-oxadiazol-2-yl)-1-benzofuran-3-yl]benzoic

acid (8e). To a solution of 8d (204 mg, 0.610 mmol) in THF

(6 mL) and MeOH (6 mL) was added 1 M NaOH (3 mL), and the

mixture was stirred at 60 °C for 24 h. After the reaction mixture

was cooled to room temperature, acidified with 1 M HCl, and

precipitated solid was collected. The crude solid was recrystal-

lized from EtOH to give 8e (163 mg, 83%) as a colorless solid.

¹H NMR (DMSO-d₆) δ 2.61 (3H, s), 7.89-7.94 (3H, m), 8.05

(1H, dd, J=1.7, 8.7 Hz), 8.10-8.14 (2H, m), 8.46 (1H, dd, J=

0.6, 1.7 Hz), 8.67 (1H, s), 13.08 (1H, brs). Anal. (C₁₈H₁₂N₂O₄)

C, H, N.

Methyl 3-Bromo-1-benzofuran-5-carboxylate (5). A mixture

of 4 (35.0 g, 196.4 mmol), NBS (38.9 g, 216.0 mmol), and AIBN

(0.645 g, 3.93 mmol) in PhCl (350 mL) was stirred at 85 °C for

2 h. The reaction mixture was cooled to room temperature,

diluted with EtOAc, and the organic layer was washed with

water, saturated aqueous sodium hydrogen carbonate, and

brine, dried over magnesium sulfate, and concentrated in vacuo.

The residue was purified by basic silica gel column chromatog-

raphy (hexane/EtOAc = 5/1) to give methyl 1-benzofuran-5-

carboxylate. To a mixture of methyl 1-benzofuran-5-carboxy-

late in CH₂Cl₂(150 mL) at 0 °C was slowly added bromine

(10.1 mL, 196.4 mmol), and the mixture was stirred at room

temperature for 1 h. The organic layer was washed with 1 M

sodium sulfite and brine, dried over magnesium sulfate, and

concentrated in vacuo to give methyl 2,3-dibromo-2,3-dihydro-

1-benzofuran-5-carboxylate. To a solution of methyl 2,3-dibro-

mo-2,3-dihydro-1-benzofuran-5-carboxylate in THF (250 mL)

at 0 °C was added a mixture of KOH (85%, 13.0 g, 196.4 mmol)

in MeOH (50 mL), and the mixture was stirred for 15 min. The

reaction mixture was diluted with EtOAc, and the organic layer

was washed with water and saturated aqueous sodium hydrogen

carbonate, dried over magnesium sulfate, and concentrated in

vacuo. The residue was purified by basic silica gel column

chromatography (THF), and the product was recrystallized

from hexane/EtOAc to give 5 (44.6 g, 89%) as a colorless solid.

¹H NMR (CDCl₃) δ 3.97 (3H, s), 7.53 (1H, dd, J=0.8, 8.7 Hz),

7.72 (1H, s), 8.09 (1H, dd, J=1.9, 8.7 Hz), 8.30 (1H, dd, J=0.8,

1.9 Hz).

4-[5-(5-Methyl-1,3,4-oxadiazol-2-yl)-1-benzofuran-3-yl]benzamide

1

(8f). Yield 65%, colorless solid. H NMR (DMSO-d₆) δ 2.61

3-Bromo-1-benzofuran-5-carbohydrazide (6). A solution of

5 (30.6 g, 120 mmol) and hydrazine hydrate (29.1 mL, 0.59 mol)

in MeOH (300 mL) was refluxed for 24 h. The reaction mixture

was cooled to 0 °C, and precipitated solid was filtered and

washed with cold MeOH to give 6 (29.2 g, 95%) as a colorless

solid. ¹H NMR (DMSO-d₆) δ 4.53 (2H, brs), 7.74 (1H, dd, J=

0.6, 8.7 Hz), 7.92 (1H, dd, J=1.7, 8.7 Hz), 8.07 (1H, dd, J=0.6,

1.7 Hz), 8.39 (1H, s), 9.92 (1H, brs).

2-(3-Bromo-1-benzofuran-5-yl)-5-methyl-1,3,4-oxadiazole (7).

A solution of 6 (25.5 g, 100 mmol) in triethyl orthoacetate

(150 mL) was stirred at 120 °C for 24 h. The reaction mixture

was cooled to room temperature, and precipitated solid was

filtered and washed with EtOAc. The solid was purified by basic

silica gel (THF), and the product was washed with hexane/THF

(1/1) to give 7 (27.0 g, 97%) as a colorless solid. ¹H NMR

(CDCl₃) δ 2.65 (3H, s), 7.62 (1H, dd, J=0.6, 8.9 Hz), 7.75 (1H,

s), 8.09 (1H, dd, J=1.7, 8.9 Hz), 8.24 (1H, dd, J=0.6, 1.7 Hz).

2-[3-(4-Methoxyphenyl)-1-benzofuran-5-yl]-5-methyl-1,3,4-

oxadiazole (2). A solution of 7 (0.25 g, 0.90 mmol), 4-methoxy-

phenylboronic acid (0.15 g, 1.00 mmol), sodium carbonate

(0.21 g, 2.00 mmol), and tetrakis(triphenylphosphine)palladium

(31.2 mg, 0.027 mmol) in DME (5 mL) and water (1 mL) under

nitrogen atmosphere was refluxed overnight. To the reaction

mixture was added water and extracted with EtOAc. The

combined organic layer was washed with brine, dried over

sodium sulfate, and concentrated in vacuo. The residue was

purified by silica gel column chromatography (EtOAc), and the

(3H, s), 7.46 (1H, brs), 7.84-7.88 (2H, m), 7.92 (1H, dd, J=0.6,

8.7 Hz), 8.03-8.08 (4H, m), 8.44 (1H, dd, J=0.6, 1.7 Hz), 8.63

(1H, s). Anal. (C₁₈H₁₃N₃O₃) C, H, N.

4-[5-(5-Methyl-1,3,4-oxadiazol-2-yl)-1-benzofuran-3-yl]pyridine

(8g). Yield 42%, colorless solid. ¹H NMR (CDCl₃) δ 2.66 (3H,

s), 7.60 (2H, dd, J=1.5, 4.5 Hz), 7.70 (1H, dd, J=0.6, 8.7 Hz),

8.03 (1H, s), 8.11 (1H, dd, J=1.7, 8.7 Hz), 8.54 (1H, dd, J=0.6,

1.7 Hz), 8.75 (2H, d, J=5.1 Hz). Anal. (C₁₆H₁₁N₃O₂) C, H, N.

2-[3-(3-Methoxyphenyl)-1-benzofuran-5-yl]-5-methyl-1,3,4-

oxadiazole (8h). Yield 77%, colorless solid. ¹H NMR (CDCl₃) δ

2.64 (3H, s), 3.90 (3H, s), 6.97 (1H, ddd, J=0.9, 2.6, 8.3 Hz), 7.18

(1H, dd, J=1.5, 2.6 Hz), 7.26 (1H, ddd, J=0.9, 1.5, 7.5 Hz), 7.44

(1H, dd, J=7.5, 8.3 Hz), 7.65 (1H, dd, J=0.6, 8.7 Hz), 7.86 (1H,

s), 8.07 (1H, dd, J=1.7, 8.7 Hz), 8.50 (1H, dd, J=0.6, 1.7 Hz).

Anal. (C₁₈H₁₄N₂O₃) C, H, N.

2-Methyl-5-{3-[3-(methylsulfanyl)phenyl]-1-benzofuran-5-yl}-

1,3,4-oxadiazole (8i). Yield 88%, colorless solid. ¹H NMR

(CDCl₃) δ 2.56 (3H, s), 2.64 (3H, s), 7.27-7.34 (1H, m),

7.42-7.46 (2H, m), 7.50-7.51 (1H, m), 7.66 (1H, dd, J=0.8,

8.7 Hz), 7.86 (1H, s), 8.07 (1H, dd, J=1.7, 8.7 Hz), 8.46 (1H, dd,

J=0.6, 1.7 Hz). Anal. (C₁₈H₁₄N₂O₂S) C, H, N.

1-{4-[5-(5-Methyl-1,3,4-oxadiazol-2-yl)-1-benzofuran-3-yl]-

phenyl}ethanone (8j). Yield 79%, colorless solid. ¹H NMR

(CDCl₃) δ 2.65 (3H, s), 2.67 (3H, s), 7.69 (1H, dd, J=0.6, 8.7

Hz), 7.75-7.80 (2H, m), 7.96 (1H, s), 8.06-8.14 (3H, m),

8.50-8.56 (1H, m). Anal. (C₁₉H₁₄N₂O₃) C, H, N.

Article

1-{4-[5-(5-Methyl-1,3,4-oxadiazol-2-yl)-1-benzofuran-3-yl]-

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6279

0.6, 8.7 Hz), 7.78-7.85 (4H, m), 7.94 (1H, s), 8.08 (1H, dd, J=

1.7, 8.7 Hz), 8.50 (1H, dd, J=0.6, 1.7 Hz). Anal. (C₁₈H₁₄N₂O₃S)

C, H, N.

phenyl}ethanol (8k). To a solution of 8j (0.30 g, 0.94 mmol) in

EtOH (3 mL) at 0 °C was added sodium tetrahydroborate (90%,

59 mg, 1.41 mmol) and the mixture was stirred overnight at

room temperature. The mixture was concentrated, diluted with

water, and extracted with EtOAc. The organic layer was washed

with brine, dried over magnesium sulfate, and concentrated in

vacuo. The residue was purified by basic silica gel column

chromatography (hexane/EtOAc=1/2 to 1/3), and the product

was recrystallized from hexane/EtOAc to give 8k (0.23 g, 76%) as

2-{3-[4-(Ethylsulfinyl)phenyl]-1-benzofuran-5-yl}-5-methyl-

1,3,4-oxadiazole (9c). Compound 9c was prepared in a manner

similar to that described for 9a in 82% yield as a colorless solid.

¹H NMR (CDCl₃) δ1.27 (3H, t, J = 7.3 Hz), 2.65 (3H, s),

2.78-3.06 (2H, m), 7.68 (1H, d, J=8.7 Hz), 7.73-7.78 (2H, m),

7.79-7.85 (2H, m), 7.94 (1H, s), 8.08 (1H, dd, J=1.7, 8.7 Hz),

8.51 (1H, d, J=1.1 Hz). Anal. (C₁₉H₁₆N₂O₃S) C, H, N.

2-{3-[4-(Ethylsulfonyl)phenyl]-1-benzofuran-5-yl}-5-methyl-

1,3,4-oxadiazole (9d). To a solution of 8b (0.30 g, 0.89 mmol) in

CH₂Cl₂(5 mL) at 0 °C was added 69-75% m-CPBA (0.63 g,

2.67 mmol), and the mixture was stirred for 4 days at room

temperature. The reaction mixture was concentrated and purified

bybasic silica gel column chromatography(hexane/EtOAc=1/1),

and the product was recrystallized from hexane/EtOAc to give

9d (86 mg, 26%) as a colorless solid. ¹H NMR (CDCl₃) δ 1.35

(3H, t, J=7.4 Hz), 2.65 (3H, s), 3.19 (2H, q, J=7.3 Hz), 7.70 (1H,

dd, J=0.6, 8.7 Hz), 7.87 (2H, d, J=8.5 Hz), 7.98 (1H, s), 8.05

(2H, d, J=8.5 Hz), 8.10 (1H, dd, J=1.7, 8.7 Hz), 8.49-8.51 (1H,

m). Anal. (C₁₉H₁₆N₂O₄S) C, H, N.

Methyl 3-[4-(Methylsulfanyl)phenyl]-1-benzofuran-5-carboxy-

late (10). Compound 10 was prepared in a manner similar to

that described for 2 in 85% yield as a colorless solid. ¹H NMR

(CDCl₃) δ 2.55 (3H, s), 3.95 (3H, s), 7.37-7.41 (2H, m),

7.55-7.60 (3H, m), 7.83 (1H, s), 8.08 (1H, dd, J=1.7, 8.7 Hz),

8.53 (1H, dd, J=0.6, 1.7 Hz). Anal. (C₁₇H₁₄O₃S) C, H, N.

3-[4-(Methylsulfanyl)phenyl]-1-benzofuran-5-carbohydrazide

(11). A solution of 10 (7.46 g, 25.0 mmol) and hydrazine hydrate

(3.06 mL, 125 mmol) in MeOH (100 mL) was refluxed for 3 days.

After cooling, the precipitate was collected by filtration and

recrystallized from MeOH to give 11 (7.11 g, 95%) as a colorless

solid. ¹H NMR (DMSO-d₆) δ 2.54 (3H, s), 4.52 (2H, brs),

7.40-7.44 (2H, m), 7.71-7.78 (3H, m), 7.89 (1H, dd, J=1.7,

8.7 Hz), 8.38 (1H, dd, J=0.4, 1.7 Hz), 8.45 (1H, s), 9.91 (1H, brs).

Anal. (C₁₆H₁₄N₂O₂S) C, H, N.

1

a colorless solid. H NMR (CDCl₃) δ 1.58 (3H, d, J=0.8 Hz),

1.86-1.94 (1H, m), 2.57-2.68 (3H, m), 4.94-5.05 (1H, m),

7.53 (2H, d, J=7.9 Hz), 7.61-7.69 (3H, m), 7.86 (1H, s), 8.05

(1H, dd, J = 1.5, 8.7 Hz), 8.49 (1H, d, J = 1.5 Hz). Anal.

(C₁₉H₁₆N₂O₃) C, H, N.

4-[5-(5-Methyl-1,3,4-oxadiazol-2-yl)-1-benzofuran-3-yl]phenol

1

(8l). Yield 95%, colorless solid. H NMR (DMSO-d₆) δ 2.60

(3H, s), 6.95 (2H, d, J=8.3 Hz), 7.56 (2H, d, J=8.3 Hz), 7.86

(1H, d, J=8.7 Hz), 8.00 (1H, dd, J=1.7, 8.7 Hz), 8.36 (2H, s).

Anal. (C₁₇H₁₂N₂O₃0.2H₂O) C, H, N.

3

Dimethyl {4-[5-(5-Methyl-1,3,4-oxadiazol-2-yl)-1-benzofur-

an-3-yl]phenyl}phosphonate (8m). To a solution of 8l (380 mg,

1.30 mmol) and Tf₂NPh (511 mg, 1.43 mmol) in THF (15 mL)

was slowly added a 60% suspension of sodium hydride in

mineral oil (57.2 mg, 1.43 mmol), and the mixture was stirred

for 30 min at room temperature. The reaction mixture was

poured into water and extracted with EtOAc. The organic layer

was washed with brine, dried over magnesium sulfate, and

concentrated in vacuo. The residue was purified by basic silica

gel column chromatography (hexane/THF = 1/2), and the

product was recrystallized from hexane/THF to give 4-[5-(5-

methyl-1,3,4-oxadiazol-2-yl)-1-benzofuran-3-yl]phenyl trifluoro-

methanesulfonate (507 mg, 92%) as a colorless solid.

A mixture of 4-[5-(5-methyl-1,3,4-oxadiazol-2-yl)-1-benzo-

furan-3-yl]phenyl trifluoromethanesulfonate (391 mg, 0.90 mmol),

dimethyl phosphate (0.165 mL, 1.80 mmol), tetrakis(triphenyl-

phosphine)palladium (104 mg, 0.09 mmol), and N-ethyldiiso-

propylamine (0.314 mL, 1.80 mmol) in toluene (5 mL) under

argon atmosphere was stirred for 2 h at 100 °C. After cooling to

room temperature, the mixture was diluted with water and

extracted with EtOAc. The organic layer was washed with

1 M HCl and brine, dried over magnesium sulfate, and concen-

trated in vacuo. The residue was purified by basic silica gel

column chromatography (EtOAc), and the product was recrys-

tallized from hexane/EtOAc to give 8m (190 mg, 55%) as a

colorless solid. ¹H NMR (CDCl₃) δ 2.65 (3H, s), 3.82 (6H, d, J=

11.1 Hz), 7.68 (1H, dd, J = 0.6, 8.7 Hz), 7.76-7.81 (2H, m),

7.91-7.99 (3H, m), 8.09 (1H, dd, J=1.7, 8.7 Hz), 8.50 (1H, dd,

J=0.6, 1.7 Hz). Anal. (C₁₉H₁₇N₂O₅P) C, H, N.

5-{3-[4-(Methylsulfanyl)phenyl]-1-benzofuran-5-yl}-1,3,4-oxa-

diazol-2-ol (12a). To a solution of 11 (0.25 g, 0.83 mmol) and

NEt₃(0.17 mL, 1.25 mmol) in DMF (5 mL) at 0 °C was added

CDI (0.27 g, 1.68 mmol), and the mixture was stirred at 0 °C for

1 h. The reaction mixture was diluted with water and extracted

with EtOAc/THF (1/1) solution. The organic layer was washed

with brine, dried over magnesium sulfate, and concentrated in

vacuo. The residue was recrystallized from hexane/EtOAc to

give 12a (0.20 g, 74%) as a colorless solid. ¹H NMR (DMSO-d₆)

δ 2.54 (3H, s), 7.44 (2H, d, J=8.6 Hz), 7.70 (2H, d, J=8.6 Hz),

7.85 (2H, d, J=1.1 Hz), 8.20 (1H, t, J=1.1 Hz), 8.50 (1H, s),

12.59 (1H, brs). Anal. (C₁₇H₁₂N₂O₃S) C, H, N.

2-Methyl-5-{3-[4-(methylsulfonyl)phenyl]-1-benzofuran-5-yl}-

1,3,4-oxadiazole (8n). Yield 85%, colorless solid. ¹H NMR

(CDCl₃) δ 2.65 (3H, s), 3.13 (3H, s), 7.70 (1H, dd, J=0.6, 8.7 Hz),

7.85-7.89 (2H, m), 7.98 (1H, s), 8.08-8.12 (3H, m), 8.50 (1H,

dd, J=0.6, 1.7 Hz). Anal. (C₁₈H₁₄N₂O₄S) C, H, N.

2-Methyl-5-{3-[3-(methylsulfinyl)phenyl]-1-benzofuran-5-yl}-

1,3,4-oxadiazole (9a). To a solution of 8i (774 mg, 2.40 mmol)

in CH₂Cl₂(15 mL) was added 69-75% m-CPBA (888 mg,

3.60 mmol), and the mixture was stirred for 5 min at room

temperature. The reaction mixture was concentrated in vacuo,

and the residue was purified by basic silica gel column chroma-

tography (EtOAc) and the product was recrystallized from

CH₂Cl₂/MeOH to give 9a (503 mg, 59%) as a colorless solid.

¹H NMR (CDCl₃) δ 2.65 (3H, s), 3.14 (3H, s), 7.70 (1H, dd, J=

0.6, 8.7 Hz), 7.73-7.78 (1H, m), 7.97-8.01 (3H, m), 8.10 (1H,

dd, J=1.7, 8.7 Hz), 8.18-8.19 (1H, m), 8.46 (1H, dd, J=0.6,

1.7 Hz). Anal. (C₁₈H₁₄N₂O₃S) C, H, N.

5-{3-[4-(Methylsulfanyl)phenyl]-1-benzofuran-5-yl}-1,3,4-oxa-

diazol-2-amine (12b). To a solution of 11 (1.60 g, 5.36 mmol) in

DMA (25 mL) was added cyanogen bromide (0.62 g, 5.90 mmol),

and the mixture was stirred for 6 h at room temperature. Then to

the mixturewas added cyanogenbromide (0.17 g, 1.61 mmol) and

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

mixture was diluted with water and precipitated solid was filtered.

The solid was dissolved in EtOAc/THF (1/1) solution, then

washed with brine, dried over magnesium sulfate, and concen-

trated in vacuo. The residue was purified by basic silica gel

column chromatography (THF), and the product was recrystal-

lized from hexane/THF to give 12b (1.37 g, 79%) as a colorless

solid. ¹H NMR (DMSO-d₆) δ 2.54 (3H, s), 7.23 (2H, brs), 7.44

(2H, d, J=8.3 Hz), 7.69 (2H, d, J=8.3 Hz), 7.80-7.90 (2H, m),

8.21 (1H, brs), 8.47 (1H, s). Anal. (C₁₇H₁₃N₃O₂S) C, H, N.

5-{3-[4-(Methylsulfinyl)phenyl]-1-benzofuran-5-yl}-1,3,4-oxa-

diazol-2-ol (13a). Compound 13a was prepared in a manner

similar to that described for 9a in 80% yield as a colorless solid.

¹H NMR (CDCl₃) δ 2.81 (3H, s), 7.80-7.95 (4H, m), 7.96 (2H,

d, J=8.4 Hz), 8.24 (1H, s), 8.62 (1H, s), 12.59 (1H, s). Anal.

(C₁₇H₁₂N₂O₄S) C, H, N.

2-Methyl-5-{3-[4-(methylsulfinyl)phenyl]-1-benzofuran-5-yl}-

1,3,4-oxadiazole (9b). Compound 9b was prepared in a manner

similar to that described for 9a in 84% yield as a colorless solid.

¹H NMR (CDCl₃) δ 2.65 (3H, s), 2.81 (3H, s), 7.69 (1H, dd, J=

6280 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20

Saitoh et al.

5-{3-[4-(Methylsulfinyl)phenyl]-1-benzofuran-5-yl}-1,3,4-oxa-

diazol-2-amine (13b). To a solution of 12b (0.25 g, 0.77 mmol) in

THF (10 mL) at 0 °C was added oxone (0.24 g, 0.77 mmol) in

water (3 mL), and the mixture was stirred at 0 °C for 30 min and

at room temperature for 1 h. The reaction mixture was diluted

with water, and precipitated solid was filtered and purified by

reverse phase preparative HPLC to give 13b (15 mg, 6%) as a

colorless solid. ¹H NMR (DMSO-d₆) δ 2.81 (3 H, s), 7.24 (2H, s),

7.84-7.92 (4H, m), 7.93-7.98 (2H, m), 8.25 (1H, s), 8.59 (1H, s).

Anal. (C₁₇H₁₃N₃O₃S) C, H, N.

Methyl 3-[4-(Methylsulfinyl)phenyl]-1-benzofuran-5-carboxy-

late (14). The compound 14 was prepared in a manner similar to

that described for 9a in 93% yield as a colorless solid. ¹H NMR

(CDCl₃) δ 2.80 (3H, s), 3.96 (3H, s), 7.61 (1H, dd, J=0.6, 8.9 Hz),

7.77-7.84 (4H, m), 7.92 (1H, s), 8.12 (1H, dd, J=1.7, 8.9 Hz),

8.54 (1H, dd, J=0.6, 1.7 Hz). Anal. (C₁₇H₁₄O₄S) C, H, N.

5-{3-[4-(Methylsulfinyl)phenyl]-1-benzofuran-5-yl}-1,3,4-oxa-

diazole-2-thiol (15). A solution of 14 (2.23 g, 7.10 mmol) and

hydrazine hydrate (1.76 mmol, 36.3 mmol) in MeOH (25 mL)

was refluxed for 4 days. The solvent was removed, and the

residue was dissolved in EtOH (30 mL). To the solution were

added carbon disulfide (1.28 mL, 21.3 mmol) and NEt₃(1.19 mL,

8.52 mmol), and the mixture was refluxed for 4 h. The reaction

mixture was cooled to room temperature, diluted with water and

1 M HCl, and extracted with EtOAc. The organic layer was

washed with brine, dried over sodium sulfate, and concentrated

in vacuo. The crude solid was recrystallized from THF to give 15

(1.19 g, 47%) as a pale-yellow solid. ¹H NMR (DMSO-d₆) δ 2.82

(3H, s), 7.85-7.89 (2H, m), 7.92 (1H, dd, J = 0.8, 8.9 Hz),

7.94-8.00 (3H, m), 8.33 (1H, dd, J=0.8, 1.5 Hz), 8.65 (1H, s),

14.75 (1H, brs). HPLC purity: 96%

Methyl 3-{[4-(Methylsulfanyl)phenyl]amino}-4-nitrobenzoate

(17). To a solution of 16 (10.64 g, 53.4 mmol) in DMSO (100 mL)

was added 4-(methylthio)aniline (14.87 g, 106.8 mmol), and the

mixture was stirred at 110 °C for 6 h. The reaction mixture was

cooled to room temperature and diluted with EtOAc. The organic

layer was washed with water and brine, dried over magnesium

sulfate, and concentrated in vacuo. The crude solid was recrystal-

lized from hexane/EtOAc to give 17 (15.88 g, 93%) as a brown solid.

¹H NMR (CDCl₃) δ 2.53 (3H, s), 3.89 (3H, s), 7.21 (2H, d, J=

8.7 Hz), 7.31-7.34 (3H, m), 7.85 (1H, d, J=1.5 Hz), 8.25 (1H, d, J=

8.7 Hz), 9.42 (1H, s).

Methyl 4-Amino-3-{[4-(methylsulfanyl)phenyl]amino}benzoate

(18). To a solution of sodium hydrosulfite (55.7 g, 320 mmol) in

water (125 mL) at 0 °C was added dropwise a solution of 17

(6.37 g, 20.0 mmol) in THF (100 mL) and EtOH (50 mL), and the

mixture was stirred at room temperature for 1 h. The reaction

mixture was basified by saturated aqueous sodium hydrogen

carbonate, concentrated, and the water layer was extracted with

EtOAc. The organic layer was washed with saturated aqueous

sodium hydrogen carbonate and brine, dried over sodium sulfate,

and concentrated in vacuo. The residue was purified by basic

silica gel column chromatography (hexane/EtOAc=5/1 to 0/1) to

give 18 (5.77 g, 100%) as a brown oil. ¹H NMR (CDCl₃) δ 2.43

(3H, s), 3.83 (3H, s), 4.22 (2H, s), 5.15 (1H, s), 6.66 (2H, d, J=8.7

Hz), 6.76 (1H, d, J=8.4 Hz), 7.22 (2H, d, J=8.7 Hz), 7.73-7.79

(2H, m).

hydrate (9.70 mL, 200 mmol) in EtOH (70 mL) was refluxed

overnight. The reaction mixture was cooled to room tempera-

ture and precipitated solid was filtered and washed with EtOH

to give 20 (5.14 g, 86%) as a colorless solid. ¹H NMR (DMSO-

d₆) δ 2.58 (3H, s), 4.47 (2H, s), 7.53 (2H, d, J=8.7 Hz), 7.67 (2H,

d, J=8.7 Hz), 7.80 (2H, s), 8.06 (1H, s), 8.65 (1H, s), 9.83 (1H, s).

N⁰-Acetyl-1-[4-(methylsulfanyl)phenyl]-1H-benzimidazole-6-car-

bohydrazide (21). To a solution of 20 (1.0 g, 3.35 mmol) in DMA

(5 mL) at 0 °C was added acetyl chloride (0.26 mL, 3.69 mmol),

and the mixture was stirred at room temperature for 1 h. To the

mixture was added EtOAc and precipitated solid was collected

and washed with EtOAc to give 21 (1.14 g, 100%) as a colorless

solid. ¹H NMR (DMSO-d₆) δ 1.92 (3H, s), 2.58 (3H, s), 7.53 (2H,

d, J=8.7 Hz), 7.69 (2H, d, J=8.7 Hz), 7.87 (2H, s), 8.12 (1H, s),

8.82 (1H, s), 9.89 (1H, s), 10.38 (1H, s).

6-(5-Methyl-1,3,4-oxadiazol-2-yl)-1-[4-(methylsulfanyl)phe-

nyl]-1H-benzimidazole (22). To a solution of 21 (761 mg,

2.24 mmol) in pyridine (10 mL) was added TsCl (1.56 g, 8.00 mmol),

and the mixture was stirred overnight at 90 °C. The reaction

mixture was cooled to room temperature, and the mixture was

concentrated and diluted with chloroform. The organic layer

was washed with saturated aqueous sodium hydrogen carbo-

nate and brine, dried over sodium sulfate, and concentrated in

vacuo. The residue was purified by basic silica gel (hexane/

EtOAc=4/1 to 1/1), and the product was recrystallized from

hexane/EtOAc to give 22 (428 mg, 66%) as a colorless solid. ¹H

NMR (CDCl₃) δ 2.58 (3H, s), 2.63 (3H, s), 7.43-7.49 (4H, m),

7.95-8.02 (2H, m), 8.19 (2H, s). Anal. (C₁₇H₁₄N₄OS 0.3H₂O)

3

C, H, N.

6-(5-Methyl-1,3,4-oxadiazol-2-yl)-1-[4-(methylsulfinyl)phenyl]-

1H-benzimidazole (23). The compound 23 was prepared in a

manner similar to that described for 9a in 85% as a colorless

solid. ¹H NMR (CDCl₃) δ 2.64 (3H, s), 2.85 (3H, s), 7.74 (2H, d,

J = 8.7 Hz), 7.94 (2H, d, J = 8.7 Hz), 7.98-8.05 (2H, m),

8.26-8.28 (2H, m). Anal. (C₁₇H₁₄N₄O₂S 0.5H₂O) C, H, N.

3

Methyl 3-Bromo-1-benzothiophene-5-carboxylate (25a). Bro-

mine (2.06 mL, 40.2 mmol) was added dropwise to a stirred

solution of 24a (5.15 g, 26.8 mmol) in AcOH (50 mL), and the

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

mixture was diluted with EtOAc, and the organic layer was

washed with 1 M sodium sulfite and saturated aqueous sodium

hydrogen carbonate, dried over magnesium sulfate, and con-

centrated in vacuo. The residue was purified by silica gel column

chromatography (hexane/EtOAc=5/1), and the product was

recrystallized from hexane/EtOAc to give 25a (6.20 g, 85%) as a

1

colorless solid. H NMR (CDCl₃) δ 3.99 (3H, s), 7.52 (1H, s),

7.90 (1H, dd, J=0.6, 8.5 Hz), 8.08 (1H, ddd, J=0.4, 1.5, 8.5 Hz),

8.53 (1H, dd, J=0.6, 1.5 Hz). Anal. (C₁₀H₇BrO₂S) C, H, N.

Methyl 3-Iodoimidazo[1,2-a]pyridine-6-carboxylate (25b). To

a solution of 24b (8.81 g, 50.0 mmol) in acetonitrile (500 mL) at

0 °C was added NIS (12.4 g, 55.0 mmol), and the mixture was

stirred at 80 °C for 5 h. The mixture was cooled to room

temperature, and precipitated solid was filtered to give 25b

(11.0 g, 73%) as a pale-yellow solid. ¹H NMR (CDCl₃) δ 4.01

(3H, s), 7.69 (1H, d, J=10.0 Hz), 7.80 (1H, s), 7.85 (1H, dd,

J=2.5, 10.0 Hz), 8.91 (1H, s).

Ethyl 3-Bromofuro[3,2-b]pyridine-5-carboxylate (25c). Bro-

mine (7.82 mL, 157 mmol) was added dropwise to a solution

of 24c (0.30 g, 1.57 mmol) in CH₂Cl₂(5 mL), and the mixture

was stirred at room temperature for 3.5 h. The mixture was

concentrated, diluted with THF, cooled to 0 °C, and treated

dropwise with 1 M KOH in EtOH solution. After stirring at

room temperature for 5 min, the mixture was poured into water

and extracted with EtOAc. The organic layer was washed with

brine, dried over sodium sulfate, and concentrated in vacuo. The

residue was purified by silica gel column chromatography

(hexane/EtOAc =17/3 to 1/1) to give 25c (0.15 g, 36%) as a

colorless solid. ¹H NMR (CDCl₃) δ 1.48 (3H, t, J=7.2 Hz), 4.51

(2H, q, J=7.2 Hz), 7.89 (1H, d, J=8.7 Hz), 8.00 (1H, s), 8.22

(1H, d, J=8.7 Hz).

Methyl

1-[4-(Methylsulfanyl)phenyl]-1H-benzimidazole-6-

carboxylate (19). A solution of 18 (5.78 g, 20.0 mmol) in formic

acid (50 mL) was stirred overnight at 100 °C. After the mixture

was cooled to room temperature, EtOAc was added and the

organic layer was washed with saturated aqueous sodium

hydrogen carbonate and brine, dried over magnesium sulfate,

and concentrated in vacuo. The crude solid was recrystallized

from hexane/EtOAc to give 19 (4.44 g, 74%) as a colorless solid.

¹H NMR (CDCl₃) δ 2.58 (3H, s), 3.94 (3H, s), 7.42-7.48 (4H,

m), 7.88 (1H, d, J=8.4 Hz), 8.05 (1H, dd, J=1.2, 7.2 Hz), 8.20

(2H, d, J=5.2 Hz).

1-[4-(Methylsulfanyl)phenyl]-1H-benzimidazole-6-carbohydra-

zide (20). A solution of 19 (5.96 g, 20 mmol) and hydrazine

Article

Ethyl 3-Bromofuro[2,3-b]pyridine-5-carboxylate (25d). The

compound 25d was prepared in a manner similar to that

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6281

AcOEt. The organic layer was washed with saturated aqueous

sodium hydrogen carbonate and brine, dried over magnesium

sulfate, and concentrated in vacuo. The residue was purified by

basic silica gel column chromatography (hexane/EtOAc=4/1 to

1/1), and the product was recrystallized from hexane/chloro-

form to give 28b (160 mg, 40%) as a yellow solid.

1

described for 25c in 30% yield as a colorless solid. H NMR

(CDCl₃) δ 1.45 (3H, t, J=7.2 Hz), 4.47 (2H, q, J=7.2 Hz), 7.83

(1H, s), 8.56 (1H, d, J=2.1 Hz), 9.06 (1H, d, J=2.1 Hz). Anal.

(C₁₀H₈BrNO₃) C, H, N.

¹H NMR (CDCl₃) δ 2.57 (3H, s), 2.63 (3H, s), 7.42-7.52

(4H, m), 7.75-7.83 (3H, m), 8.95 (1H,s). Anal. (C₁₇H₁₄N₄OS)

C, H, N.

The following compounds 26a-d were prepared in a similar

manner to that described for 2.

Methyl 3-[4-(Methylsulfanyl)phenyl]-1-benzothiophene-5-car-

1

boxylate (26a). Yield 76%, colorless solid. H NMR (CDCl₃)

5-(5-Methyl-1,3,4-oxadiazol-2-yl)-3-[4-(methylsulfanyl)phe-

nyl]furo[3,2-b]pyridine (28c). To asolution of 27c (0.20 g, 0.68 mmol

and triethyl orthoacetate (0.27 mL, 1.49 mmol) was added

n-BuOH (5 mL), and the mixture was stirred refluxed for 1.5 h.

To the mixture was added DBU (0.10 mL, 0.68 mmol), and the

mixture was refluxed overnight. The reaction mixture was cooled

to room temperature and precipitated solid was recrystallized from

δ 2.56 (3H, s), 3.94 (3H, s), 7.38-7.42 (2H, m), 7.44 (1H, s),

7.49-7.53 (2H, m), 7.95 (1H, dd, J = 0.6, 8.5 Hz), 8.05 (1H,

dd, J = 1.5, 8.5 Hz), 8.56 (1H, dd, J = 0.6, 1.5 Hz). Anal.

(C₁₇H₁₄O₂S₂) C, H, N.

Methyl 3-[4-(Methylsulfanyl)phenyl]imidazo[1,2-a]pyridine-6-

carboxylate (26b). Yield 42%, colorless solid. ¹H NMR (CDCl₃)

δ 2.57 (3H, s), 3.94 (3H, s), 7.41-7.50 (4H, m), 7.65-7.69 (1H,

m), 7.74-7.77 (2H, m), 9.02 (1H, t, J=1.2 Hz).

1

hexane/EtOAc to give 28c (0.15 g, 67%) as a colorless solid. H

NMR (CDCl₃) δ 2.55 (3H, s), 2.71 (3H, s), 7.40 (2H, d, J=8.5 Hz),

7.95 (1H, d, J=8.7 Hz), 8.06 (2H, d, J=8.5 Hz), 8.21 (1H, s), 8.28

(1H, d, J=8.7 Hz). Anal. (C₁₇H₁₃N₃O₂S) C, H, N.

Ethyl 3-[4-(Methylsulfanyl)phenyl]furo[3,2-b]pyridine-5-car-

1

boxylate (26c). Yield 32%, colorless solid. H NMR (CDCl₃)

δ 1.48 (3H, t, J=7.2 Hz), 2.54 (3H, s), 4.51 (2H, q, J=7.2 Hz),

7.38 (2H, d, J=8.7 Hz), 7.88 (1H, d, J=8.7 Hz), 8.09 (2H, d, J=

8.7 Hz), 8.18 (1H, d, J = 8.7 Hz), 8.21 (1H, s). Anal.

(C₁₇H₁₅NO₃S) C, H, N.

5-(5-Methyl-1,3,4-oxadiazol-2-yl)-3-[4-(methylsulfanyl)phe-

nyl]furo[2,3-b]pyridine (28d). The compound 28d was prepared

in a manner similar to that described for 7 in 58% as a colorless

solid.

¹H NMR (DMSO-d₆) δ 2.55 (3H, s), 2.63 (3H, s), 7.45 (2H, d,

J=8.5 Hz), 7.77 (2H, d, J=8.5 Hz), 8.71 (1H, s), 8.82 (1H, d, J=

2.1 Hz), 8.98 (1H, d, J=2.1 Hz).

Ethyl 3-[4-(Methylsulfanyl)phenyl]furo[2,3-b]pyridine-5-car-

boxylate (26d). Yield 95%, colorless solid. H NMR (CDCl₃)

1

δ 1.44 (3H, t, J=7.2 Hz), 2.55 (3H, s), 4.46 (2H, q, J=7.2 Hz),

7.39 (2H, d, J=8.7 Hz), 7.58 (2H, d, J=8.7 Hz), 7.94 (1H, s), 8.79

(1H, d, J=1.9 Hz), 9.07 (1H, d, J=2.3 Hz).

The following compounds 29a-d were prepared in a similar

manner to that described for 9a.

The following compounds 27a-d were prepared in a similar

manner to that described for 11.

3-[4-(Methylsulfanyl)phenyl]-1-benzothiophene-5-carbohydra-

2-Methyl-5-{3-[4-(methylsulfinyl)phenyl]-1-benzothiophen-5-yl}-

1,3,4-oxadiazole (29a). Yield 86%, colorless solid. ¹H NMR

(CDCl₃) δ 2.64 (3H, s), 2.84 (3H, s), 7.58 (1H, s), 7.74-7.78 (2H,

m), 7.81-7.85 (2H, m), 8.05 (1H, dd, J=0.8, 8.5 Hz), 8.08 (1H,

dd, J = 1.5, 8.5 Hz), 8.50 (1H, dd, J = 0.8, 1.5 Hz). Anal.

(C₁₈H₁₄N₂O₂S₂) C, H, N.

1

zide (27a). Yield 95%, colorless solid. H NMR (DMSO-d₆)

δ 2.56 (3H, s), 4.51 (2H, brs), 7.41-7.46 (2H, m), 7.59-7.63 (2H,

m), 7.85 (1H, dd, J=1.5, 8.5 Hz), 7.89 (1H, s), 8.14 (1H, d, J=

8.5 Hz), 8.31 (1H, d, J = 1.5 Hz), 9.91 (1H, brs). Anal.

(C₁₆H₁₄N₂OS₂) C, H, N.

6-(5-Methyl-1,3,4-oxadiazol-2-yl)-3-[4-(methylsulfinyl)phe-

nyl]imidazo[1,2-a]pyridine (29b). Yield 82%, colorless solid. ¹H

NMR (CDCl₃) δ 2.65 (3H, s), 2.84 (3H, s), 7.75-7.81 (2H, m),

7.83-7.89 (5H, m), 9.02 (1H, t, J=1.5 Hz). Anal. (C₁₇H₁₄N₄-

3-[4-(Methylsulfanyl)phenyl]imidazo[1,2-a]pyridine-6-carbo-

hydrazide (27b). Yield 76%, colorless solid. ¹H NMR (DMSO-

d₆) δ 2.56 (3H, s), 4.52 (2H, s), 7.45-7.48 (2H, d, J=8.7 Hz),

7.64-7.72 (4H, m), 7.81 (1H, s), 8.90 (1H, s), 9.56 (1H, s).

3-[4-(Methylsulfanyl)phenyl]furo[3,2-b]pyridine-5-carbohy-

drazide (27c). Yield 82%, colorless solid. ¹H NMR (DMSO-d₆)

δ 2.54 (3H, s), 4.64 (2H, d, J=4.3 Hz), 7.38 (2H, d, J=8.5 Hz),

8.07 (1H, d, J=8.7 Hz), 8.23 (1H, d, J=8.7 Hz), 8.32 (2H, d,

J=8.5 Hz), 8.97 (1H, s), 9.93 (1H, brs). Anal. (C₁₅H₁₃N₃O₂S)

C, H, N.

3-[4-(Methylsulfanyl)phenyl]furo[2,3-b]pyridine-5-carbohy-

drazide (27d). Yield 49%, colorless solid. ¹H NMR (DMSO-d₆)

δ 2.54 (3H, s), 4.60 (2H, brs), 7.43 (2H, d, J=8.5 Hz), 7.78 (2H, d,

J=8.5 Hz), 8.63 (1H, s), 8.76-8.84 (2H, m), 10.06 (1H, brs).

2-Methyl-5-{3-[4-(methylsulfanyl)phenyl]-1-benzothiophen-5-

yl}-1,3,4-oxadiazole (28a). The compound 28a was prepared in a

manner similar to that described for 7 in 93% as a colorless solid.

¹H NMR (CDCl₃) δ 2.57 (3H, s), 2.63 (3H, s), 7.39-7.43 (2H,

m), 7.47 (1H, s), 7.50-7.54 (2H, m), 8.02 (1H, dd, J=0.8, 8.5

Hz), 8.07 (1H, dd, J=1.5, 8.5 Hz), 8.49 (1H, dd, J=0.8, 1.5 Hz).

Anal. (C₁₈H₁₄N₂OS₂) C, H, N.

6-(5-Methyl-1,3,4-oxadiazol-2-yl)-3-[4-(methylsulfinyl)phe-

nyl]imidazo[1,2-a]pyridine (28b). To a solution of 27b (505 mg,

1.69 mmol) in DMA (4 mL) was added acetyl chloride (0.132 mL,

1.86 mmol), and the mixture was stirred at room temperature for

1.5 h. The reaction mixture was diluted with EtOAc and

precipitated white solid was collected to give N⁰-acetyl-

3-[4-(methylsulfanyl)phenyl]imidazo[1,2-a]pyridine-6-carbohy-

drazide (425 mg, 74%). A mixture of N⁰-acetyl-3-[4-(methyl-

sulfanyl)phenyl]imidazo[1,2-a]pyridine-6-carbohydrazide (425 mg,

1.25 mmol) and TsCl (714 mg, 3.74 mmol) in pyridine (5 mL)

was stirred at 100 °C for 1 day under argon atmosphere. After

cooled to room temperature, the mixture was diluted with

O₂S H₂O) C, H, N.

3

5-(5-Methyl-1,3,4-oxadiazol-2-yl)-3-[4-(methylsulfinyl)phe-

nyl]furo[3,2-b]pyridine (29c). Yield 41%, colorless solid. ¹H

NMR (CDCl₃) δ 2.71 (3H, s), 2.78 (3H, s), 7.81 (2H, d, J =

8.7 Hz), 8.00 (1H, d, J = 8.7 Hz), 8.27-8.35 (4H, m). Anal.

(C₁₇H₁₃N₃O₃S) C, H, N.

5-(5-Methyl-1,3,4-oxadiazol-2-yl)-3-[4-(methylsulfinyl)phe-

nyl]furo[2,3-b]pyridine (29d). Yield 41%, colorless solid. ¹H

NMR (DMSO-d₆) δ 2.64 (3H, s), 2.81 (3H, s), 7.82-7.91 (2H,

m), 7.98-8.08 (2H, m), 8.83 (1H, s), 8.89 (1H, d, J=2.1 Hz), 9.01

(1H, d, J=1.9 Hz). HPLC purity: 96%

N⁰-Acetyl-3-bromofuro[2,3-c]pyridine-5-carbohydrazide (31).

To a solution of 30 (1.58 g, 6.53 mmol) and acetohydrazine

(611 mg, 7.83 mmol) in DMF (10 mL) were added EDC (1.50 g,

7.83 mmol) and HOBt (1.06 g, 7.83 mmol), and the mixture was

stirred at room temperature for 2 days. The reaction mixture

was added into water, and precipitated solid was collected to

give 31 (0.55 g, 28%) as a colorless solid. ¹H NMR (CDCl₃) δ

1.93 (3H, s), 8.20 (1H, d, J=0.9 Hz), 8.70 (1H, s), 9.12 (1H, d, J=

0.9 Hz), 10.06 (1H, s), 10.46 (1H, s).

3-Bromo-5-(5-methyl-1,3,4-oxadiazol-2-yl)furo[2,3-c]pyridine

(32). The compound 32 was prepared in a manner similar to that

described for 22 in 42% as a colorless solid. ¹H NMR (CDCl₃)

δ 2.69 (3H, s), 7.90 (1H, s), 8.51 (1H, d, J=0.9 Hz), 9.00 (1H, d,

J=0.9 Hz).

5-(5-Methyl-1,3,4-oxadiazol-2-yl)-3-[4-(methylsulfanyl)phe-

nyl]furo[2,3-c]pyridine (33). The compound 33 was prepared in a

manner similar to that described for 2 in 52% as a colorless solid.

¹H NMR (DMSO-d₆) δ 2.55 (3H, s), 2.63 (3H, s), 7.46 (2H, d, J=

8.3 Hz), 7.76 (2H, d, J=8.3 Hz), 8.61 (1H, s), 8.80 (1H, s), 9.21

(1H, s).

6282 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20

Saitoh et al.

5-(5-Methyl-1,3,4-oxadiazol-2-yl)-3-[4-(methylsulfinyl)phe-

nyl]furo[2,3-c]pyridine (34). The compound 34 was prepared in a

manner similar to that described for 9a in 67% as a colorless

solid. ¹H NMR (DMSO-d₆) δ 2.64 (3H, s), 2.82 (3H, s), 7.88 (2H,

d, J=8.4 Hz), 8.03 (2H, d, J=8.4 Hz), 8.66 (1H, d, J=0.9 Hz),

8.92 (1H, s), 9.24 (1H, d, J = 0.9 Hz). Anal. (C₁₇H₁₃N₃O₃S)

C, H, N.

4-(2,4-Dimethoxybenzyl)-3-methyl-5-{3-[4-(methylsulfinyl-

phenyl]-1-benzofuran-5-yl}-4H-1,2,4-triazole (35). To a solu-

tion of 9b (0.80 g, 2.36 mmol), 2,4-dimethoxybenzylamine

(5.34 mL, 35.5 mmol), and p-TsOH (46 mg, 0.24 mmol) in

xylene (10 mL) was stirred overnight at 150 °C. The reaction

mixture was diluted with water and extracted with EtOAc.

The organic layer was washed with brine, dried over sodium

sulfate, and concentrated in vacuo. The residue was purified

by basic silica gel (hexane/EtOAc = 1/1 to 0/1), and the

product was recrystallized from hexane/EtOAc to give 35

(0.51 g, 44%) as a colorless solid. ¹H NMR (DMSO-d₆) δ 2.34

(3 H, s), 2.80 (3 H, s), 3.65 (3 H, s), 3.75 (3 H, s), 5.08 (2 H, s),

6.46 (1 H, dd, J=1.9 8.3 Hz), 6.51 (1 H, d, J=8.3 Hz), 6.60

(1 H, d, J=1.9 Hz), 7.62 (1 H, dd, J=1.5, 8.3 Hz), 7.66-7.75

(4 H, m), 7.84 (1 H, d, J=8.3 Hz), 7.87 (1 H, d, J=1.5 Hz), 8.59

(1 H, s).

3-Methyl-5-{3-[4-(methylsulfinyl)phenyl]-1-benzofuran-5-yl}-

4H-1,2,4-triazole (36). A mixture of 35 (0.405 g, 0.83 mmol) in

TFA (10 mL) was stirred overnight at room temperature. The

mixture was concentrated, and saturated aqueous sodium hy-

drogen carbonate was added and extracted with EtOAc. The

organic layer was washed with brine, dried over magnesium

sulfate, and concentrated in vacuo. The residue was purified by

basic silica gel column chromatography (EtOAc/MeOH=1/0 to

97/3), and the product was recrystallized from acetone to give 36

(0.11 g, 39%) as a colorless solid. ¹H NMR (DMSO-d₆) δ 2.40

(3 H, s), 2.82 (3 H, s), 7.78 (1 H, d, J=8.7 Hz), 7.87 (2 H, d, J=

8.4 Hz), 7.97 (2 H, d, J=8.4 Hz), 8.06 (1 H, dd, J=1.7, 8.7 Hz),

8.48-8.50 (1 H, m), 8.54 (1 H, s), 13.77 (1 H, brs). Anal.

(C₁₈H₁₅N₃O₂S) C, H, N.

N⁰-Acetyl-3-[4-(methylsulfanyl)phenyl]-1-benzofuran-5-carbo-

hydrazide (37). To a solution of 11 (1.0 g, 3.35 mmol) and AcOH

(0.29 mL, 5.03 mmol) in DMF (10 mL) were added EDC (0.86 g,

5.03 mmol) and HOBt (0.68 g, 5.03 mmol), and the reaction

mixture was stirred at room temperature for 15 h. The mixture

was diluted with water and extracted with EtOAc. The organic

layer was washed with water, dried over magnesium sulfate, and

concentrated in vacuo. The residue was crystallized from hex-

ane/Et₂O to give 37 (0.85 g, 75%) as a colorless solid. ¹H NMR

(DMSO-d₆) δ 1.92 (3H, s), 2.53 (3H, s), 7.40 (2H, d, J=8.4 Hz),

7.70-7.80 (3H, m), 7.90 (1H, d, J=8.7 Hz), 8.41 (1H, s), 8.45

(1H, s), 9.88 (1H, brs), 10.41 (1H, brs).

for 30 min. Then to the mixture was added 10 (0.70 g, 2.35 mmol),

and the mixture was stirred at 60 °C for 2 h. The reaction mixture

was diluted with water and extracted with EtOAc. The organic

layer was washed with water and dried over magnesium sulfate

and concentrated in vacuo. The residue was purified by silica gel

column chromatography (hexane/EtOAc=3/1 to 2/1), and the

product was recrystallizedfrom EtOAc to give 40 (0.51 g, 67%) as

a colorless solid. ¹H NMR (CDCl₃) δ 2.43 (3H, s), 3.32 (3H, s),

7.45 (2H, d, J=8.4 Hz), 7.72 (2H, d, J=8.4 Hz), 7.92 (1H, d, J=

8.7 Hz), 8.10-8.20 (1H, m), 8.55-8.60 (2H, m). Anal.

(C₁₈H₁₄N₂O₂S) C, H, N.

3-Methyl-5-{3-[4-(methylsulfinyl)phenyl]-1-benzofuran-5-yl}-

1,2,4-oxadiazole (41). The compound 41 was prepared in a

manner similar to that described for 9a in 91% as a colorless

solid. ¹H NMR (DMSO-d₆) δ 2.44 (3H, s), 2.82 (3H, s), 7.87 (2H,

d, J=8.4 Hz), 7.95 (2H, d, J=9.0 Hz), 7.99 (1H, d, J=8.4 Hz),

8.14 (1H, d, J = 8.4 Hz), 8.58 (1H, s), 8.67 (1H, s). Anal.

(C₁₈H₁₄N₂O₃S) C, H, N.

3-[4-(Methylsulfanyl)phenyl]-1-benzofuran-5-carboxylic Acid

(42). To a solution of 10 (3.3 g, 10.2 mmol) in MeOH (20 mL)

and THF (10 mL) was added 1 M NaOH aqueous solution (20.5 mL,

20.5 mmol), and the mixture was stirred at room temperature for

17 h. Then 1 M HCl aqueous solution (22 mL) was added and

concentrated in vacuo. The residue was diluted with water and

precipitated solid was filtered to give 42 (2.8 g, 96%) as a white

1

solid. H NMR (DMSO-d₆) δ 2.54 (3H, s), 7.43 (2H, d, J =

8.1 Hz), 7.68 (2H, d, J=8.1 Hz), 7.76 (1H, d, J=9.0 Hz), 7.99

(1H, d, J=9.0 Hz), 8.44 (1H, s), 8.48 (1H, s), 12.99 (1H, brs).

Anal. (C₁₆H₁₂O₃S) C, H, N.

3-[4-(Methylsulfanyl)phenyl]-1-benzofuran-5-carboxamide (43).

The compound 43 was prepared in a manner similar to that

described for 31 in 95% as a colorless solid. ¹H NMR (DMSO-

d₆) δ 2.54 (3H, s), 7.37 (1H, brs), 7.41 (2H, d, J = 8.7 Hz),

7.70-7.80 (3H, m), 7.92 (1H, d, J=8.7 Hz), 8.13 (1H, brs), 8.41

(1H, s), 8.44 (1H, s). Anal. (C₁₆H₁₃NO₂S) C, H, N.

3-[4-(Methylsulfanyl)phenyl]-1-benzofuran-5-carbonitrile (44). To

a solution of 43 (2.35 g, 8.29 mmol) in DMF (10 mL) was added

dropwise thionyl chloride (1.21 mL, 16.6 mmol), and the

mixture was stirred at room temperature for 5 h. The react-

ion mixture was quenched by the addition of saturated aqueous

sodium hydrogen carbonate and extracted with EtOAc.

The organic layer was washed with water, dried over magnes-

ium sulfate, and concentrated in vacuo. This residue was

purified by silica gel column chromatography (toluene), and

the product was recrystallized from cold hexane/Et₂O to give

44 (1.67 g, 76%) as a colorless solid. ¹H NMR (DMSO-d₆) δ 2.53

(3H, s), 7.38 (2H, d, J=8.1 Hz), 7.73 (2H, d, J=8.1 Hz), 7.80

-8.00 (2H, m), 8.45 (1H, s), 8.58 (1H, s). Anal. (C₁₆H₁₁NOS) C,

H, N.

5-Methyl-3-{3-[4-(methylsulfanyl)phenyl]-1-benzofuran-5-yl}-

1,2,4-oxadiazole (45). A solution of 44 (1.32 g, 4.97 mmol),

hydroxylamine hydrochloride (1.04 g, 14.9 mmol), and sodium

hydrogen carbonate (1.25 g, 14.9 mmol) in MeOH (20 mL) was

refluxed for 6 h and concentrated. The residue was added to

water and precipitated solid was filtered and washed with water.

A mixture of the solid and acetic anhydride (0.94 mL, 9.95

mmol) in dioxane (20 mL) was refluxed at 90 °C for 15 h. The

mixture was added into saturated aqueous sodium hydrogen

carbonate and extracted with EtOAc. The organic layer was

washed with water and brine, dried over magnesium sulfate, and

concentrated in vacuo. The residue was purified by silica gel

column chromatography (toluene), and the product was recrys-

tallized from acetone/Et₂O to give 45 (1.22 g, 76%) as a colorless

solid. ¹H NMR (DMSO-d₆) δ 2.54 (3H, s), 3.32 (3H, s), 7.44 (2H,

d, J=8.4 Hz), 7.69 (2H, d, J=8.4 Hz), 7.85 (1H, d, J=8.7 Hz),

8.03 (1H, d, J = 8.7 Hz), 8.44 (1H, s), 8.48 (1H, s). Anal.

(C₁₈H₁₄N₂O₂S) C, H, N.

2-Methyl-5-{3-[4-(methylsulfanyl)phenyl]-1-benzofuran-5-yl}-

1,3,4-thiadiazole (38). The mixture of compound 37 (0.76 g,

2.23 mmol), Lawesson’s reagent (1.81 g, 4.46 mmol), and

toluene (100 mL) was stirred at 80 °C for 1 h and purified by

column chromatography (basic silica gel, EtOAc). The crude

product was crystallized from EtOAc to give 38 (0.41 g, 54%) as

a colorless solid. ¹H NMR (DMSO-d₆) δ 2.54 (3H, s), 2.78 (3H,

s), 7.43 (2H, d, J=8.1 Hz), 7.72 (2H, d, J=8.1 Hz), 7.84 (1H, d, J

=8.4 Hz), 7.95 (1H, dd, J=2.1, 9.0 Hz), 8.37 (1H, s), 8.49 (1H, s).

Anal. (C₁₈H₁₄N₂OS₂) C, H, N.

2-Methyl-5-{3-[4-(methylsulfinyl)phenyl]-1-benzofuran-5-yl}-

1,3,4-thiadiazole (39). Compound 39 was prepared in a manner

similar to that described for 9a in 93% as a colorless solid. ¹H

NMR (DMSO-d₆) δ 2.79 (3H, s), 2.81 (3H, s), 7.80-7.90 (3H,

m), 7.95-8.05 (3H, m), 8.42 (1H, d, J=1.8 Hz), 8.61 (1H, s).

Anal. (C₁₈H₁₄N₂O₂S₂) C, H, N.

3-Methyl-5-{3-[4-(methylsulfanyl)phenyl]-1-benzofuran-5-yl}-

1,2,4-oxadiazole (40). To a solution of acetoamidoxime (0.19 g,

2.58 mmol) in THF (15 mL) was added a 60% suspension of

sodium hydride in mineral oil (103 mg, 2.58 mmol). The reaction

mixture was stirred at room temperature for 15 min and at 60 °C

5-Methyl-3-{3-[4-(methylsulfinyl)phenyl]-1-benzofuran-5-yl}-

1,2,4-oxadiazole (46). Compound 46 was prepared in a manner

similar to that described for 9a in 87% as a colorless solid. ¹H

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6283

NMR (DMSO-d₆) δ 2.69 (3H, s), 2.82 (3H, s), 7.80-7.95 (3H,

m), 7.95 (2H, d, J=8.4 Hz), 8.06 (1H, d, J=8.7 Hz), 8.48 (1H, s),

8.60 (1H, s). Anal. (C₁₈H₁₄N₂O₃S) C, H, N.

3-Bromo-1-benzofuran-5-carbaldehyde (47). To a solution of 3

(1.0 g, 6.75 mmol) in PhCl (20 mL) were added NBS (1.44 g,

8.1 mmol) and AIBN (22 mg, 0.13 mmol), and the mixture was

stirred at 80 °C for 1 h. The mixture was cooled, washed with

saturated aqueous sodium bicarbonate, dried over magnesium

sulfate, and concentrated in vacuo to give 1-benzofuran-5-

carbaldehyde (0.82 g, 83%) as a pale-orange oil.

-89.9 (c 0.49, MeOH); t_R=25.4 min (CHIRALPAK AS 4.6

mmIDꢀ250 mmL, EtOH, 0.4 mL/min); 99.9% ee.

Optical Resolution of 9c. Compound 9c (530 mg, 1.50 mmol)

was separated by preparative HPLC (CHIRALCEL OJ

(0.50 cm ꢀ 50 cm), hexane/EtOH 1:1, 80 mL/min, UV254 nm)

to afford (R)-9c (249 mg, 47% yield) and (S)-9c (245 mg, 46%

yield). (R)-9c: [R]²⁵_Dþ127.2; t_R=19.8 min (CHIRALCEL OJ

4.6 mmIDꢀ250 mmL, hexane/EtOH 1:1, 0.5 mL/min); 99.9%

ee. (S)-9c: [R]²⁵-130.4; t_R=30.5 min (CHIRALCEL OJ 4.6

D

mmIDꢀ250 mmL, hexane/EtOH 1:1, 0.5 mL/min); 99.9% ee.

X-ray Crystallographic Data. Data obtained for (S)-9b and

(S)-9c are included in Supporting Information. X-ray crystal-

lographic data for compounds (S)-9b and (S)-9c have been

deposited with the Cambridge Crystallographic Data Center

as CCDC 711975 and CCDC 711976, respectively. The crystal-

lographic data can be obtained free of charge by writing to

CCDC, 12 Union Road, CAMBRIDGE, CB2, IEZ, UK (fax

(þ44) 1223 336033, e-mail: deposit@ccdc.cam.ac.uk).

To a solution of 1-benzofuran-5-carbaldehyde (0.8 g, 5.47 mmol)

in CH₂Cl₂(5 mL) was added dropwise bromine (0.36 mL, 7.12

mmol), and the mixture was stirred at room temperature for 1 h.

The reaction mixture was concentrated in vacuo and diluted

with EtOH (5 mL). Potassium hydroxide (0.68 g, 12.0 mmol)

was added, and the mixture was stirred at 60 °C for 30 min and

then diluted with water and extracted with EtOAc. The organic

layer was washed with water, dried over magnesium sulfate, and

concentrated in vacuo. The residue was purified by silica

gel column chromatography (hexane/acetone = 20:1), and

the product was recrystallized from cold hexane to give the 47

(0.19 g, 15%) as a colorless solid. ¹H NMR (CDCl₃) δ 7.62 (1H,

d, J=8.7 Hz), 7.76 (1H, s), 7.94 (1H, dd, J=1.8, 8.7 Hz), 8.10

(1H, d, J = 1.8 Hz), 10.10 (1H, s). Anal. (C₉H₅BrO₂)

C, H, N.

1-(3-Bromo-1-benzofuran-5-yl)pentane-1,4-dione (48). A solu-

tion of 47 (225 mg, 0.10 mmol), methyl vinyl ketone (0.10 mL,

0.12 mmol), 3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium

bromide (63 mg, 0.025 mmol), NEt₃(0.21 mL, 0.15 mmol),

and EtOH (5 mL) was stirred at 70 °C for 1 h. The mixture was

diluted with water and extracted with EtOAc. The organic layer

was washed with water, dried over magnesium sulfate, and

concentrated in vacuo. This residue was purified by silica gel

column chromatography (hexane/acetone=3/1) to give the 48

(73 mg, 25%) as a colorless oil. ¹H NMR (CDCl₃) δ 2.28 (3H, s),

2.93 (2H, t, J=6.3 Hz), 3.37 (2H, t, J=6.3 Hz), 7.54 (1H, d, J=

8.7 Hz), 7.72 (1H, s), 8.04 (1H, dd, J=1.5, 8.4 Hz), 8.21 (1H, s).

3-(3-Bromo-1-benzofuran-5-yl)-6-methylpyridazine (49). To a

solution of 48 (2.24 g, 7.60 mmol) in EtOH (20 mL) was added

hydrazine hydrate (0.40 mL, 8.35 mmol), and the mixture was

refluxed for 5 h. The reaction mixture was added to water and

extracted with EtOAc. The organic layer was washed with water

dried over magnesium sulfate and concentrated in vacuo. This

residue was purified by silica gel column chromatography

(hexane/acetone=3/1 to 1/1), and the product was recrystallized

from hexane/Et₂O to give 49 (0.51 g, 23%) as a colorless solid.

¹H NMR (300 MHz, DMSO-d₆) δ 2.78 (3H, s), 7.40 (1H, d, J=

8.7 Hz), 7.61 (1H, d, J=8.7Hz), 7.71(1H, s), 7.83(1H, d, J=8.7 Hz),

8.14 (1H, d, J = 8.7 Hz), 8.21 (1H, s). Anal. (C₁₃H₉BrN₂O)

C, H, N.

Expression, Purification, Crystallization, and Structure Deter-

mination. The gene for human GSK-3β (residues 48 to 433) was

cloned into a pSXB100 baculovirus expression vector with an

n-terminal 6-Histidine tag containing an rTEV protease clea-

vage site and expressed and purified essentially as described

previously.²²Briefly, the expressed protein was purified by

immobilized metal-chelate affinity chromatography (IMAC),

the eluted protein treated with rTEV protease to remove the tag,

and subjected to a second round of IMAC purification followed

by dialysis and further purification by cation exchange chroma-

tography to obtain the singly phosphorylated protein species.

Inhibitor in DMSO solution was added slowly, with stirring, to

the dilute protein (∼1 mg/mL) to a final concentration of 1 mM,

and the enzyme inhibitor complex further concentrated to

10-15 mg/mL for crystallization experiments. Cocrystals of

the complex are grown at 4 °C by mixing 50 nL of enzyme:

inhibitor solution with 50 nL of precipitant solution containing

20% PEG 3350 and 0.15 M NaCl and are flash-frozen by direct

immersion in liquid nitrogen using ethylene glycol as a cryopro-

tectant. X-ray diffraction data were collected at the Advanced

Light Source in Berkeley, CA, on Beamline 5.0.3 at a wavelength

of 1.0 A. Diffraction data for the cocrystal belong to the

orthorhombic space group C222₁with unit cell dimensions

85.3 A ꢀ 104.1 A ꢀ 110.0 A and extend to 2.4 A resolution

with an R_mergeof 0.066. The structures were solved by molecular

replacement with AMoRe³³using the previously reported

structure of GSK-3β (PDB entry 3F88) and refined with RE-

FMAC within the CCP4 suite of programs.³⁴The final refined

crystallographic statistics for the structure are, R=22.3 (R_free

,

28.9), with root-mean-square deviations (rmsd) in the bond

lengths of 0.012 A, and in the bond lengths of 1.44°. The

structure has been deposited with the RCSB structure database

with accession codes 3GB2.

3-Methyl-6-{3-[4-(methylsulfanyl)phenyl]-1-benzofuran-5-

yl}pyridazine (50). The compound 50 was prepared in a manner

similar to that described for 2 in 68% as a colorless solid. H

GSK-3β and CDK5 Kinase Assay. The human GSK-3β

expressed as N-terminal FLAG-tagged proteins using baculo-

virus expression system (Takeda Pharmaceutical Company

Ltd., Osaka, Japan). The human p35/cyclin dependent kinase

5 (CDK5) was purchased from Millipore Corp. (Bedford, MA),

which was expressed as N-terminal GST fusion protein using

baculovirus expression system. The kinase assay was performed

in a reaction mixture that contained 25 mM HEPES, pH 7.5,

10 mM magnesium acetate, 1 mM dithiothreitol, and 0.01%

BSA and serially diluted test compounds. The assay was con-

ducted in a 96-well plate assay format. The end amount of

enzyme was 20 ng/well for CDK5 or 40 ng/well for GSK-3β. The

end amount of substrate was 1 ng/well of CDK5 substrate

peptide for CDK5 (Calbiochem, La Jolla, CA) for CDK5 or

100 ng/well of GSK-3β substrate peptide (Millipore Corp.) for

GSK-3β.

1

NMR (DMSO-d₆) δ 2.54 (3H, s), 2.67 (3H, s), 7.43 (2H, d, J=

8.7 Hz), 7.66 (1H, d, J=8.7 Hz), 7.76 (2H, d, J=8.7 Hz), 7.82

(1H, d, J=8.7 Hz), 8.17 (1H, d, J=8.7 Hz), 8.26 (1H, d, J=

8.7 Hz), 8.45 (1H, s), 8.58 (1H, s).

3-Methyl-6-{3-[4-(methylsulfinyl)phenyl]-1-benzofuran-5-

yl}pyridazine (51). Compound 51 was prepared in a manner

similar to that described for 9a in 87% as a colorless solid. ¹H

NMR (DMSO-d₆) δ 2.67 (3H, s), 2.81 (3H, s), 7.67 (1H, d, J=

9.0 Hz), 7.84 (2H, d, J=8.4 Hz), 7.86 (1H, d, J=8.4 Hz), 8.03

(2H, d, J=8.1 Hz), 8.20 (1H, d, J=9.0 Hz), 8.29 (1H, d, J=

8.4 Hz), 8.58 (1H, s), 8.62 (1H, s). Anal. (C₂₀H₁₆N₂O₂S) C, H, N.

Optical Resolution of 9b. Compound 9b (1.0 g, 2.96 mmol)

was separated by preparative HPLC (CHIRALPAK AS (0.50

cm ꢀ 50 cm), EtOH, 45 mL/min, UV254 nm) to afford (R)-9b

(0.49 g, 49% yield) and (S)-9b (0.49 g, 49% yield). (R)-9b: [R]²⁵

All the kinase reactions were started by addition of the ATP-

solution (final 500 nM) and were incubated for 90 min at 37 °C

for GSK-3β or for 45 min at room temperature for CDK5. The

D

þ86.9 (c 0.55, MeOH); t_R=17.9 min (CHIRALPAK AS 4.6

mmIDꢀ250 mmL, EtOH, 0.4 mL/min); 99.9% ee. (S)-9b: [R]²⁵

D

6284 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20

Saitoh et al.

reactions were terminated by the Kinase-Glo reagent contained

EDTA (50 μL/well, Promega Corp., Madison, WI). Ten min-

utes after addition of the Kinase-Glo reagent, the luminescence

was measured on a Wallac ARVO 1420 (PerkinElmer, Shelton,

CT). The reaction window was calculated from the difference of

the average signals obtained from the control (5% DMSO) and

the background wells. The inhibition with compounds was

expressed by the inhibitor concentration that produced 50%

inhibition (IC₅₀) of the activity without compound. The IC₅₀

values were obtained by linear regression analysis with a

GraphPad Prism (version 3.02 for Windows, GraphPad soft-

ware, Inc. San Diego, CA). The best-fit lines were obtained by

analyzing the logistic fitting equation.

using the baculovirus expression system. The cytoplasmic do-

mains of v-erb-a erythroblastic leukemia viral oncogene homo-

logue 2 (ERBB2) and epidermal growth factor receptor (EGFR)

were expressed as N-terminal peptide (DYKDDDD)-tagged

protein using baculovirus expression system. These expressed

kinase proteins were purified by using anti-FLAG M2 affinity

gel (Sigma-Aldrich, St. Louis, MO). Fibroblast growth factor

receptor 3 (FGFR3), platelet-derived growth factor receptor

alpha (PDGFRR), PDGFRβ, TIE2, c-Met, c-Kit, Src, insulin

receptor (IR), and lymphocyte-specific protein tyrosine kinase

(Lck) were purchased from Millipore Corp. Assays for 10

tyrosine kinases except ERBB2 and EGFR using antiphospho-

tyrosine antibody were performed in 384-well plates using the

Alphascreen system (PerkinElmer) at room temperature.

Enzyme reactions were performed in 50 mM Tris-HCl, pH

7.5, 5 mM MnCl₂, 5 mM MgCl₂, 0.01% Tween-20, 2 mM

dithiothreitol, and 0.1 μg/mL biotinylated poly-GluTyr (4:1)

containing optimized concentration of enzyme, ATP as des-

cribed below. Prior to the kinase reaction, compound and

enzyme were incubated for 5 min at room temperature. The

reactions were initiated by the addition of ATP. After the

reaction period as described below at room temperature, the

reactions were stopped by the addition of 25 μL of 100 mM

EDTA, 10 μg/mL Alphascreen streptavidine donor beads, and

10 μg/mL acceptor beads described below in 62.5 mM HEPES,

pH 7.4, 250 mM NaCl, and 0.1% BSA. The plates were

incubated in the dark for more than 12 h and then read by

EnVision 2102 Multilabel Reader (PerkinElmer). The well

containing substrate and enzyme without compound was used

as total reaction control. The reaction conditions for these 10

kinases were optimized for each kinase: VEGFR2 (19 ng/mL of

enzyme, 10 μM ATP, 10 min reaction, PY-100 conjugated

acceptor beads (PY-100)); FGFR3 (20 ng/mL of enzyme,

20 μM ATP, 10 min reaction, PY-100); PDGFRR (50 ng/mL

of enzyme, 10 μM ATP, 30 min reaction, PT66 conjugated

acceptor beads (PT66)); PDGFRβ (50 ng/mL of enzyme, 20 μM

ATP, 60 min reaction, PT66); TIE2 (20 ng/mL of enzyme, 2 μM

ATP, 10 min reaction, PT66); c-Met (1 ng/mL of enzyme, 2 μM

ATP, 10 min reaction, PT66); c-Kit (10 ng/mL of enzyme, 20 μM

ATP, 20 min reaction, PT66); Src (0.33 ng/mL of enzyme, 2 μM

ATP, 10 min reaction, PY-100); IR (100 ng/mL of enzyme,

10 μM ATP, 60 min reaction, PT66); Lck (100 ng/mL of enzyme,

2 μM ATP, 30 min reaction, PY-100). Assays for ERBB2 and

EGFR kinase using radio labeled [γ-³²P] ATP (GE Healthcare)

were performed in 96-well plates. ERBB2 and EGFR kinase

reactions were performed in 50 mM Tris-HCl, pH 7.5, 5 mM

MnCl₂, 0.01% Tween-20, and 2 mM dithiothreitol containing

0.9 μCi of [γ-³²P] ATP per reaction, 50 μM ATP, 5 μg/mL poly-

Glu-Tyr (4:1), 0.1% DMSO, and 0.25 μg/mL of ERBB2 or

EGFR cytoplasmic domains in a total volume of 50 μL. Prior to

the kinase reaction, compound and enzyme were incubated for

5 min at room temperature. The kinase reactions were initiated

by the addition of ATP. After the kinase reaction for 10 min

(ERBB2) and 5 min (EGFR) at room temperature, the reactions

were terminated by the addition of 10% trichloroacetic acid

(final concentration). The [γ-³²P]-phosphorylated proteins were

filtrated in Harvest plate (Millipore Corp.) with a Cell harvester

(PerkinElmer) and washed free of [γ-³²P] ATP with 3% phos-

phoric acid. The plate was dried, followed by the addition of

25 μL of MicroScint0 (PerkinElmer). The radioactivity was

counted by a Topcount scintillation counter (PerkinElmer).

In Vivo Evaluation in Cold Water Stress Model. Compounds

were suspended in 0.5% methylcellulose and were administered

orally in a volume of 3 mL/kg of body weight 30 min before the

CWS. Mice (N=5) were immersed up to the neck in cold water

(1-2 °C) for 4 min and then were returned to individual cages.

Mice were sacrificed by decapitation 10 min after CWS, the

brains were immediately removed, and tissues were dissected.

Antibodies. The following antibodies were used for immuno-

blotting at appropriate concentrations as recommended by the

Serine/Threonine Kinase Profiling by IC₅₀Measurement. As-

says for 10 serine/threonine kinases using radio labeled [γ-³³P]

ATP (GE Healthcare, Piscataway, NJ) were performed in 96-

well plates. Mitogen-activated protein kinase p38 alpha (p38R),

protein kinase C theta (PKCθ) and Jun N-terminal kinase 1

(JNK1) were expressed as N-terminal FLAG tagged protein

using baculovirus expression system. I kappa B kinase β (IKKβ)

and MEK kinase 1 (MEKK1) were expressed as C-terminal

FLAG tagged protein using baculovirus expression system.

Casein kinase 1 delta (CK1δ) was expressed as N-terminal

GST fusion protein using Escherichia coli expression system.

Checkpoint kinase 1 (CHK1) was expressed as N-terminal GST

fusion protein using baculovirus expression system. CDK1/

CycB and CDK2/CycA were expressed as C-terminal 6xHis-

tagged CDK1 or CDK2, and N-terminal GST-tagged cyclin B

or cyclin A protein using the baculovirus expression system.

The reaction conditions were optimized for each kinase: p38R

(100 ng/well of enzyme, 1 μg/well of myelin basic protein (MBP)

(Wako Pure Chemical Industries, Osaka, Japan), 0.1 μCi/well of

[γ-³³P] ATP, 60 min reaction at 30 °C); MEKK1 (25 ng/well of

enzyme, 1 μg/well of MBP, 0.1 μCi/well of [γ-³³P] ATP, 60 min

reaction at 30 °C); PKCθ (25 ng/well of enzyme, 2 μg/well of

MBP, 0.1 μCi/well of [γ-³³P] ATP, 60 min reaction at 30 °C);

JNK1 (10 ng/well of enzyme, 1 μg/well of c-Jun, 0.1 μCi/well of

[γ-³³P] ATP, 60 min reaction at 30 °C); IKKβ (20 ng/well of

enzyme, 1 μg/well of IκBR, 0.1 μCi/well of [γ-³³P] ATP, reaction

at room temperature); CDK1/CycB (4.2 ng/well of enzyme,

1 μg/well of Histone H1 (Calbiochem), 0.2 μCi/well of [γ-³³P]

ATP, 20 min reaction at room temperature); CDK2/CycA

(1.8 mUnits/well of enzyme, 1 μg/well of Histone H1, 0.2 μCi/

well of [γ-³³P] ATP, 20 min reaction at room temperature);

CK1δ (120 ng/well of enzyme, 2.4 μM of CK1tide (Millipore

Corp.), 0.2 μCi/well of [γ-³³P] ATP, 20 min reaction at room

temperature); CHK1 (30 ng/well of enzyme, 25 μM of CHKtide

(Millipore Corp.), 0.2 μCi/well of [γ-³³P] ATP, 10 min reaction

at room temperature). Except for the PKCθ, enzyme reactions

were performed in 25 mM HEPES, pH 7.5, 10 mM magnesium

acetate, 1 mM dithiothreitol, and 500 nM ATP containing

optimized concentration of enzyme, substrate, and radiolabeled

ATP as described above in a total volume of 50 μL. For the

PKCθ, enzyme reactions were performed in 25 mM HEPES, pH

7.5, 10 mM magnesium acetate, 1 mM dithiothreitol, lipid

activator (Millipore Corp.).

Prior to the kinase reaction, compound and enzyme were

incubated for 5 min at reaction temperature as described above.

The kinase reactions were initiated by the addition of ATP. After

the reaction period as described above, the reactions were termi-

nated by the addition of 10% trichloroacetic acid (final con-

centration). The [γ-³³P]-phosphorylated proteins were filtrated in

Harvest Plate (Millipore Corp.) with a Cell Harvester (Perkin-

Elmer) and then free of [γ-³³P] ATP was washed out with 3%

phosphoric acid. The plates were dried, followed by the addition of

40 μL of MicroScint0 (PerkinElmer). The radioactivity was

counted by a TopCount scintillation counter (PerkinElmer).

Tyrosine Kinase Profiling by IC₅₀Measurement. The cyto-

plasmic domain of vascular endothelial growth factor receptor 2

(VEGFR2) was expressed as N-terminal FLAG-tagged proteins

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6285

manufacturers. Rabbit polyclonal antibodies recognizing phos-

pho-Thr205 tau was purchased from Biosource International

(Camarillo, CA). A rabbit polyclonal antibody recognizing total

tau, tau Ab-3, was purchased from Lab Vision Corporation

(Fremont, CA).

Animals. Male C57BL/6Njcl mice, 8-12 weeks of age, (Clea

Japan, Tokyo, Japan) were used for the CWS model. All animals

used in this study were housed in groups and allowed free access

to food and water and were cared for in accordance with the

Principles and Guidelines on Animal Experimentation of the

Pharmaceutical Research Division of Takeda Pharmaceutical

Company Ltd.

Preparation of Hippocampal Protein Extract. Hippocampi

were immediately homogenized in ice cold radioimmunopreci-

pitation assay (RIPA) buffer (50 mM Tris-HCl (pH 7.6), 5 mM

ethylenediamine tetra acetic acid (EDTA), 1 mM ethylene glycol

tetraacetic acid (EGTA), 30 mM NaF, 5 mM sodium diphos-

phate, 2 μM pepstatin A, 100 mM NaCl, 1% NP-40, 0.25%

sodium deoxycholate, 1 μM microcystin LR, 40 μM leupeptin,

100 μM 4-(2-aminoethyl)benzenesulfonyl fluoride (ABSF),

2 mM sodium orthovanadate, 1 μM MG115), and centrifuged

at 20000g for 10 min at 4 °C. The supernatants were transferred

to fresh tubes. The protein concentrations were determined by

BCA protein assay (PIERCE, Rockford, IL).

SDS-PAGE and Western Blotting. Samples containing 3-10 μg

of protein were separated on a 10% SDS-polyacrylamide gel and

transferred to a polyvinylidene fluoride (PVDF) membrane. The

membrane was washed once with 0.1% Tween-20 containing Tris-

buffered saline (TBS-T) for 15 min before being treated with Block-

Ace (DS Pharma Biomedical, Tokyo, Japan) for 45 min and then

probed with primary antibody in TBS-T containing 3% bovine

serum albumin (BSA). The membrane was washed 3 times with

TBS-T and then incubated with the antirabbit IgG horseradish

peroxidase-linked species-specific F(ab⁰)2 fragment (GE Health-

care, Piscataway, NJ) in TBS-T for 1 h. After washing 3 times with

TBS-T, the blot was developed with ImmunoStar Reagents (Wako

Pure Chemical Industries). The images obtained with a CCD

camera (LAS-1000plus Luminescent Image Analyzer; Fuji Film,

Tokyo, Japan) were quantified by densitometry (Image Gauge

version 3.46; Fuji Film).

Brain and Plasma Concentration in Mice. Compounds (S)-9b

and (S)-9c were administered to nonfasted Jcl: C57BL/6N mice

(male, 8 weeks old, n=3) orally (3 mg/kg, 0.5% methylcellulose

suspension). At 15 and 30 min and 1, 2, 4, 8, 24 h after oral

administration, blood and brain samples were collected. The

blood samples were centrifuged to obtain the plasma fraction.

The brain samples were homogenized in saline to obtain the

brain homogenate. The plasma and brain homogenate samples

were deproteinized with acetonitrile containing an internal

standard. After centrifugation, the supernatant obtained was

diluted with 0.01 mol/L ammonium acetate and centrifuged

again. The compound concentration in the supernatant was

measured by LC/MS/MS with an API4000 triple quadrupole

mass spectrometer (MDS Sciex). The mass spectrometer was

equipped with a turbo ionspray source and operated in positive

ion mode. The HPLC conditions were as follows: column,

Chiralcel OJ-RH (2.1 mm ꢀ 150 mm); mobile phase, (A) 0.01 mol/

L ammonium acetate/(B) acetonitrile=6/4; gradient condition,

0-7 min: 40% B; 7-7.2 min: 40-85% B; 7.2-13.5 min: 85% B;

13.5-14 min: 85-40% B; 14-30 min: 40% B; flow rate, 0.2 mL/

min; column temperature, 40 °C.

Bioavailability Analysis. Test compounds were administered

as a cassette dosing to nonfasted rats. After oral and intravenous

administration, blood samples were collected. The blood sam-

ples were centrifuged to obtain the plasma fraction. The plasma

samples were deproteinized with acetonitrile containing an

internal standard. After centrifugation, the supernatant was

diluted with 0.01 mol/L ammonium acetate and centrifuged

again. The compound concentrations in the supernatant were

measured by LC/MS/MS.

Solubility Determination. Small volumes of the compound

DMSO solutions were added to the aqueous buffer (pH 6.8).

After incubation, precipitates were separated from by filtration

through a filter plate. The filtrates were analyzed for compound

in solution by HPLC analysis.

Acknowledgment. We thank Drs. Shant Salakian and Bi-

Ching Sang for molecular biology and protein expression,

Masashi Yamaguchi and Hisashi Fujita for pharmacokinetic

studies, Dr. Sachiko Itono for docking model studies, and

Keiko Higashikawa for X-ray crystal structure of (S)-9b and

(S)-9c.

Statistical Analyses. Statistical analysis was performed by

statistical analysis software (SAS preclinical package, version

5.0; SAS Institute, Inc., Cary, NC). Values were expressed as

mean ( SEM and statistically analyzed using an unpaired

Student’s t test. In the CWS model, the inhibitory rate of each

stressed group was calculated by defining the mean value of the

normal group as 0% and that of the CWS-treated control group

as 100%.

Supporting Information Available: Elemental analysis data.

This material is available free of charge via the Internet at http://

pubs.acs.org.

Pharmacokinetic Studies. Compounds (S)-9b and (S)-9c were

administered to nonfasted Crl:CD(SD)IGS rats (male, 8 weeks

old, n=3) intravenously (1 mg/kg, DMA/1, 3-butandiol) and

orally (3 mg/kg, 0.5% methylcellulose suspension). At 5, 10, 15,

and 30 min and 1, 2, 4, 8, 24 h after intravenous administration

or at 15 and 30 min and 1, 2, 4, 8, 24 h after oral administration,

blood samples were collected from tail vein. The blood samples

were centrifuged to obtain the plasma fraction. The plasma

samples were deproteinized with acetonitrile containing

an internal standard. After centrifugation, the supernatant

obtained was diluted with 0.01 mol/L ammonium acetate and

centrifuged again. The compound concentration in the super-

natant was measured by LC/MS/MS with an API4000 triple

quadrupole mass spectrometer (MDS Sciex). The mass spectro-

meter was equipped with a turbo ionspray source and operated

in positive ion mode. The HPLC conditions were as follows:

column, Chiralcel OJ-RH (2.1 mm ꢀ 150 mm); mobile phase,

(A) 0.01 mol/L ammonium acetate/(B) acetonitrile=6/4; gra-

dient condition, 0-7 min: 40%B; 7-7.2 min: 40-85% B;

7.2-13.5 min: 85% B; 13.5-14 min: 85-40% B; 14-30 min:

40% B; flow rate, 0.2 mL/min; column temperature, 40 °C.

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Article Doi

2-{3-[4-(Alkylsulfinyl)phenyl]-1-benzofuran-5-yl}-5-methyl-1,3,4-oxadiazole derivatives as novel inhibitors of glycogen synthase kinase-3β with good brain permeability

DOI: 10.1021/jm900647e

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Article abstract of DOI:10.1021/jm900647e

Full text of DOI:10.1021/jm900647e

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