Discovery of novel (4-piperidinyl)-piperazines as potent and orally active acetyl-CoA carboxylase 1/2 non-selective inhibitors: F-Boc and triF-Boc groups are acid-stable bioisosteres for the Boc group

Bioorganic & Medicinal Chemistry 19 (2011) 1580–1593

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Bioorganic & Medicinal Chemistry

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Discovery of novel (4-piperidinyl)-piperazines as potent and orally active

acetyl-CoA carboxylase 1/2 non-selective inhibitors: F-Boc and triF-Boc

groups are acid-stable bioisosteres for the Boc group

Tomomichi Chonan ^a,, Daisuke Wakasugi ^a, Daisuke Yamamoto ^b, Miyoko Yashiro ^c, Takahiro Oi ^a,

⇑

Hiroaki Tanaka ^a, Ayumi Ohoka-Sugita ^b, Fusayo Io ^b, Hiroko Koretsune ^b, Akira Hiratate ^a

^aMedicinal Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan

^bMolecular Function and Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan

^cResearch Computer System, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan

a r t i c l e i n f o

a b s t r a c t

Article history:

Novel (4-piperidinyl)-piperazine derivatives were synthesized and evaluated as ACC1/2 non-selective

inhibitors. Optimization of the substituents on the nitrogen of the piperidine ring led to the identiﬁcation

of the ﬂuorine substituted tert-butoxycarbonyl group. Advanced analog, 1,1,1-triﬂuoro-2-methylpropan-

2-yl 4-{4-[(2-amino-6-methyl-1-benzothiophen-3-yl)carbonyl]piperazin-1-yl}piperidine-1-carboxylate

(12c) showed potent inhibitory activities in enzyme-assay and cell-based assays. Compound 12c also

exhibited reduction of hepatic de novo fatty acid synthesis in rats after oral administration.

Received 17 December 2010

Revised 20 January 2011

Accepted 21 January 2011

Available online 27 January 2011

Keywords:

Acetyl-CoA carboxylase

Inhibitor

(4-Piperidinyl)-piperazine

tert-Butoxycarbonyl

F-Boc

Orally active

1. Introduction

ACC1/2 non-selective inhibitors is expected to reduce de novo fatty

acid synthesis and triglyceride (TG)-rich lipoprotein secretions in

Acetyl-CoA carboxylase (ACC) is a biotin-dependent homo olig-

omeric protein composed of a carboxyltransferase (CT), a biotin

carboxyl carrier protein and biotin carboxylase (BC) domains.

ACC is involved in the synthesis of malonyl-CoA from acetyl-CoA

in an ATP-dependent manner. Malonyl-CoA works not only as a

substrate for de novo fatty acid synthesis, but also as an allosteric

inhibitor of carnitine palmitoyl transferase (CPT-1), a key enzyme

that positively regulates mitochondrial b-oxidation. Therefore,

inhibition of ACC is expected to reduce de novo fatty acid synthesis

(FAS) and to enhance fatty acid b-oxidation (FAO) through disinhi-

bition of CPT-1, which might beneﬁt treatment of metabolic disor-

ders such as obesity and type 2 diabetes.¹

Two ACC isoforms, ACC1 and ACC2, have been cloned in rodents

and humans. ACC1 is predominantly expressed in lipogenic tissues

such as liver and adipose tissue, whereas ACC2 is predominantly

expressed in oxidative tissues such as liver, skeletal muscle and

heart.²Recent studies reported by Harada et al. indicate that hepa-

tic ACC2 could partially cover ACC1 function as a backup system.³

Consequently, reduction of malonyl-CoA levels in these tissues by

liver while increasing fatty acid b-oxidation in liver and skeletal

muscle. Therefore, an ACC1/2 non-selective inhibitor might pro-

vide a novel therapeutic approach for treating various metabolic

disorders.

Several classes of small molecule ACC1/2 non-selective inhibi-

tors have been reported to date (Fig. 1).⁴A piperidinylpiperidine

class of ACC1/2 non-selective inhibitors was disclosed by the Pﬁzer

group where compound 1 (CP-640186) was shown to signiﬁcantly

inhibit fatty acid synthesis in diabetic animal models.^1aA spiro-

chromanones class of ACC inhibitors 2, 3, and, 4 was disclosed

by Merck-Banyu,⁵Takeda,⁶and Pﬁzer,^7,8respectively. The Abbott

group disclosed the aryl-ether derivative 5 as an ACC2 selective

inhibitor.⁹

We have recently reported the structure–activity relationships

of a (4-piperidinyl)-piperazine class of ACC1/2 non-selective inhib-

itors, as represented by compound 6a (Fig. 2).¹⁰Compounds 6a and

6b showed potent inhibitory activity in the enzyme assay, whereas

their activity in the HepG2 cell-based assay was considerably

lower. Hence, we decided to further optimize compound 6 for

improved cell activity and in vivo activity. Our efforts were focused

on replacing the 2,6-diphenylpyridine and 1-acetyl-piperidine

moieties of compound 6a to improve the intrinsic potency and cell

⇑

Corresponding author. Tel.: +81 48 669 3108; fax: +81 48 652 7254.

E-mail address: tomomichi.chonan@po.rd.taisho.co.jp (T. Chonan).

doi:10.1016/j.bmc.2011.01.041

T. Chonan et al. / Bioorg. Med. Chem. 19 (2011) 1580–1593

1581

non-selective inhibitor 12c, which features, a novel ﬂuorine-

substituted tert-butoxycarbonyl (F-Boc) group. The F-Boc group

was found to be an acid-stable bioisostere for the Boc group.

2. Chemistry

The synthetic route for the target compounds is outlined in

Scheme 1. Commercially available 2-amino-benzo[b]thiophene-3-

carboxylic acid ethyl ester (21) was hydrolyzed to form 22. The

carboxylic acid 22 was coupled with tert-butoxy piperazine-1-

carboxylate in the presence of EDCI, followed by removal of the

tert-butoxycarbonyl (Boc) group to give 23. The piperazine group

of 23 was reductively alkylated with tert-butyl 4-oxopiperidine-

1-carboxylate followed by cleavage of the Boc group to furnish

24. The resulting diamine 24 was coupled with the desired acid

chloride or chloroformate to afford 25, which was treated with tri-

phosgene and ethylamine to give the target compounds 7a–j, 8a–f

and 9.

The synthetic route for the F-Boc intermediates is described in

Scheme 2. Carbamoylimidazole 28a was readily prepared from

ethyl ﬂuoroacetate (26) in two steps. Ethyl ﬂuoroacetate (26) was

reacted with excess methylmagnesium bromide to give 27a, which

was coupled with 1,1⁰-carbonyldiimidazole and treated with

methyl iodide to furnish carbamoylimidazolium salt 28a.¹¹Addi-

tion of 4-piperidone to the carbamoylimidazolium salt 28a yielded

carbamate 29a, which was reductively aminated with tert-butoxy

piperazine-1-carboxylate to give 30a. Removal of the Boc group of

30a gave the key intermediate 31a. The triﬂuoro-Boc intermediate

31b was prepared from commercially available 2-triﬂuoromethyl-

2-propanol (27b) in the same manner.

Figure 1. Structures of representative ACC inhibitors.

The preparation of the desired benzothiphene and thienopyri-

dine derivatives is illustrated in Scheme 3. The corresponding ben-

zothiophene carboxylic acids 22 and 32a–c or thienopyridine

carboxylic acids 33 and 34a–c¹²were coupled with the intermedi-

ates 31a and 31b to afford 35, 36, and 37. For compound 37,

the p-methoxy benzyl group was cleaved prior to the ﬁnal urea

formation. Treatment of 35, 36, and 38 with triphosgene and

ethylamine gave the target compounds 10a–b, 12b–c, 13–20.

3. Results and discussion

The derivatives were screened against partially puriﬁed human

liver ACC enzymes, and the subtype selectivity of the optimized

compounds was conﬁrmed using recombinant human ACC1 and

ACC2 enzymes. Among the tested compounds, selected potent

compounds were further evaluated for their ability to decrease

fatty acid synthesis (FAS) and increase fatty acid oxidation (FAO)

in HepG2 cells. FAS inhibition was assessed by measuring the de-

crease in [¹⁴C] acetate incorporation into cellular lipids,^1aand the

effects on FAO were evaluated by measuring the generation of

T₂O in the culture media.¹³

Activities resulting from modiﬁcation of the right hand moiety

of 6a are shown in Table 1. Replacement of the 1-acetylpiperidine

moiety with small alkyl groups such as methyl and ethyl groups re-

sulted in a reduction of potency. Of the amide derivatives 7b–h,

lipophilic bulky groups showed an increase in potency. The cyclo-

pentyl derivative 7h was 38-fold more potent than the methyl

derivative 7b. The cyclohexyl derivative 7i was equipotent to

compound 7a. The tert-butyl derivative 7j showed slightly reduced

potency compared to 7a. We were glad to ﬁnd that the carbamate

derivatives 8a–f had good inhibitory activity. Of them the tert-

butoxy derivative 8f was the most potent derivative. The corre-

sponding urea derivative 9 exhibited decreased potency relative

to 8f. The potent analog 8f, which has an IC₅₀value of 24 nM in

the enzyme assay, displayed potent inhibitory activity against fatty

Figure 2. Compound 6a, 6b and novel potent ACC1/2 inhibitor 7a.

permeability by reducing the molecular weight and molecular

complexity such as the number of rings. By the evaluation of a vari-

ety of heterocycles for the replacement of the 2,6-diphenylpyridine

moiety of compound 6a, the 2-ureido benzothiophene group as in

7a was found to be a suitable surrogate structure in terms of po-

tency and molecular weight.

Further modiﬁcation efforts were centered on compound 7a

and the identiﬁcation of smaller and more potent substituents than

the original 1-acetylpiperidin-4-yl group was attempted. Herein,

we report the identiﬁcation of potent and orally active ACC1/2

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T. Chonan et al. / Bioorg. Med. Chem. 19 (2011) 1580–1593

Scheme 1. Reagents and conditions: (a) NaOH, EtOH–THF, rt; (b) tert-butoxy piperazine-1-carboxylate, EDCI, HOBt, CHCl₃, rt; (c) 4 M HCl–AcOEt, AcOEt, rt; (d) tert-butyl 4-

piperidone-1-carboxylate, rt, CHCl₃, then NaBH(OAc)₃, rt; (e) 4 M HCl–AcOEt, AcOEt, rt; (f) (i) R¹COCl, Et₃N, CHCl₃, 0 °C; (g) (i) triphosgene, Et₃N, 70% ethylamine aq, CHCl₃,

0 °C; (ii) 4 M HCl–AcOEt, or 1 M maleic acid in AcOEt.

Scheme 2. Reagents and conditions: (a) MeMgBr, THF, ꢀ60 to 0 °C; (b) CDI, CHCl₃, rt; (c) MeI, CH₃CN, rt; (d) 4-piperidone triﬂuoroacetate, Et₃N, CH₃CN, rt; (e) tert-butoxy

piperazine-1-carboxylate, NaBH(OAc)₃, rt; (f) 4 M HCl–AcOEt, AcOEt, rt.

Scheme 3. Reagents and conditions: (a) 31a or 31b, EDCI, HOBt, Et₃N, DMF; (b) TFA, CHCl₃, rt; (c) (i) ethyl isocyanate, pyridine, 60 °C; (ii) 4 M HCl–AcOEt.

acid synthesis in HepG2 cells with an IC₅₀value of 79 nM. Although

compound 8f displayed attractive activity, 8f utilizes the tert-

butoxycarbonyl (Boc) group as a pharmacophore, which is readily

cleaved from an amino group under acidic conditions. In fact, only

23% of the parent was recovered after incubation of compound 8f

in simulated gastric media (pH 1.2) for 3 h (Table 2). Because of

the stability of the tert-butyl cation, the Boc group is labile to acidic

conditions. We thought that introduction of electron-withdrawing

groups to the tert-butyl group would destabilize the corresponding

cation structure, thus making the group stable under acidic condi-

tions. Hence, ﬂuorine-substituted derivatives 10a and 10b were

prepared and evaluated (Table 2.). The F-Boc and triF-Boc deriva-

tives 10a and 10b retained potency in both the enzyme and cell

assays. As anticipated, compounds 10a and 10b were stable after

incubation in the simulated gastric media. Monoﬂuoro substitution

was shown to have a signiﬁcant stabilizing effect.

The metabolic stabilities of compounds 10a and 10b were as-

sessed using a human microsomal incubation assay. Only 3% and

5% of the intact compounds were recovered for 10a and 10b,

respectively, indicating that compounds 10a and 10b are metabol-

ically unstable (Table 3). Similarly, the isopropyl carbamate deriv-

ative 8c showed very poor metabolic turnover (3%). The metabolite

identiﬁcation studies of 8c revealed that the main metabolic path-

way is the oxidation of the benzothiophene moiety.

Consequently, we evaluated the substituent effect on the

benzothiophene moiety of compounds 10a and 10b (Table 3).

The 5-methyl derivative 11 was equipotent to the parent 8c but

metabolically unstable, whereas the 6-methyl derivatives 12a–c

T. Chonan et al. / Bioorg. Med. Chem. 19 (2011) 1580–1593

1583

Table 1

Table 3

SAR of benzothiophene derivatives: variation of the amide, carbamate and urea

moieties

In vitro activities of the disubstituted 4-(4-piperidinyl)-piperazines 10a–b, 8c, 11–20

Compd

R²

R³

ACC IC₅₀(nM)

FAS IC₅₀(nM)

hMS^c(%)

a

b

R¹

ACC IC₅₀(nM)

FAS IC₅₀(nM)

a

b

Compd

10a

10b

8c

CH₂F

CF₃

H

CH₂F

CF₃

H

5-Me

6-Me

6-CF₃

6-F

17

31

28

32

60

32

58

98

62

38

27

12

117

41

45

61

81

208

254

628

73

3

5

3

6a

6b

7a

7b

7c

7d

7e

7f

76

33

74

1864

597

242

207

144

131

49

343

294

ND^c

309

146

450

ND^c

228

208

512

635

79

1-Acetylpiperidin-4-yl

Me

Et

n-Pr

n-Bu

iso-Bu

iso-Pr

c-Pentyl

c-Hex

tert-Bu

MeO

EtO

iso-PrO

c-PeO

c-HexO

tert-BuO

tert-BuNH

11

12a

12b

12c

13

14

15

16

17

18

19

77

84

87

7

17

51

29

13

ND^d

67

3

58

ND^d

67

7g

7h

7i

6-Cl

6-MeO

4-Aza

5-Aza

6-Aza

7-Aza

81

49

ND^d

152

67

7j

109

175

55

28

52

76

24

119

8a

8b

8c

8d

8e

8f

20

a

Inhibitory activity of compounds on the malonyl-CoA synthesis of human

ACC1/2. The IC₅₀value represents the mean from at least two independent

experiments.

b

Inhibitory activity of fatty acid synthesis in HepG2 cells.

Fraction (%) remaining after 15-min incubation with human liver microsomes

9

775

c

a

Inhibitory activity of compounds on the malonyl-CoA synthesis of human

ACC1/2.

(1 mg protein/mL).

d

ND = No data.

b

Inhibitory activity of fatty acid synthesis in HepG2 cells.

ND = No data.

c

zyme and cell activities. In particular, compound 12c displayed no

discrepancy between these two assays.

The pharmacological proﬁles of 12c are summarized in Table 4

those of 6. Compound 12c similarly inhibited human and rat liver

cytosolic ACC activities. Regarding the isoform selectivity, com-

pound 12c favorably inhibited human recombinant ACC2 (rhACC2)

preferentially over human recombinant ACC1 (rhACC1).¹⁴In

HepG2 cell assays, compound 12c showed dose-dependent inhibi-

Table 2

In vitro activities of the disubstituted 4-(4-piperidinyl)-piperazines 8f, 10a and 10b

tion of fatty acid synthesis, with an IC₅₀of 0.06

lM, and increased

fatty acid oxidation, with an EC₅₀of 0.37 M. The pharmacokinetic

l

proﬁle of 12c was examined in Sprague–Dawley (SD) rats.

Compound 12c had excellent plasma exposure owing to good

bioavailability. Notably, the concentration of 12c in liver was quite

high (10300 ng/g tissue) compared to plasma (435 ng/mL) at 1 h

a

b

Compd

R²

ACC IC₅₀(nM)

FAS IC₅₀(nM)

Acid stability^c(%)

8f

10a

10b

CH₃

CH₂F

CF₃

24

17

31

79

61

81

23

98

100

Table 4

a

Inhibitory activity of compounds on the malonyl-CoA synthesis of human

Pharmacological proﬁles and plasma exposure of 6a and 12c

ACC1/2. The IC₅₀value represents the mean from at least two independent

experiments.

Pharmacological proﬁles

6a

12c

b

Inhibitory activity of fatty acid synthesis in HepG2 cells.

Enzyme assay^a

c

HPLC determination after 3 h incubation at 37 °C in aqueous solution (pH 1.2).

Human ACC1/2

Rat ACC1/2

Recombinant human ACC1

Recombinant human ACC2

76 nM

70 nM

101 nM

23 nM

58 nM

32 nM

192 nM

95 nM

exhibited a 2-fold decrease in potency relative to the compounds

8c, 10a, and 10b. However, the 6-methyl derivatives showed a sig-

niﬁcant improvement in metabolic stability. The 6-triﬂuoromethyl

derivative 13 and 6-ﬂuoro derivative 14 displayed decreased po-

tency and poor metabolic stability. The 6-chloro and 6-methoxy

derivatives 15 and 16 were equipotent to the parent 10b. Among

the thienopyridine derivatives 17–20, compound 17 was signiﬁ-

cantly potent, but its metabolic stability was poor. In the cell-based

FAS assay, the isopropoxy derivatives 8c, 11, and 12a exhibited an

8–10-fold decrease in inhibitory activity relative to the enzyme

activities. In contrast, the F-Boc and triF-Boc derivatives 10a, 10b,

12b, 12c, and 13–20 showed small discrepancies between the en-

Cell-based assay

HepG2 cell FAS (IC₅₀

HepG2 cell FAO (EC₅₀

)

0.34

0.58

l

M

0.06

0.37

l

M

)

Plasma exposure after po^b

C_max(ng/mL)

T_max(h)

107

4.00

1000

603

5.33

7480

AUC (ng/mL h)

a

Inhibitory activity of compounds on the malonyl-CoA synthesis of human

ACC1/2, rat ACC1/2, recombinant hACC1 and hACC2. The IC₅₀value represents the

mean from at least two independent experiments.

b

Plasma exposure after single oral administration of 6a and 12c at a dose of

10 mg/kg to male Sprague–Dawley rats.

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T. Chonan et al. / Bioorg. Med. Chem. 19 (2011) 1580–1593

after 10 mg/kg oral administration. Therefore, we selected the

compound 12c as a test compound for efﬁcacy studies in animal

models of hyperlipidemia.

triglyceride levels of fructose-drinking rats in a dose-dependent

manner with a minimum effective dose of 3–10 mg/kg (Fig. 3). In

contrast, Benzaﬁbrate, a PPAR-a agonist, reduced plasma triglycer-

Acute effects of compound 12c on the fatty acid synthesis were

examined in SD rats as described in Section 5.1. Consistent with its

potent intrinsic activity and high exposures in livers, compound

12c (10 mg/kg, po) potently suppressed de novo fatty acid synthe-

sis of livers (by 74.6%) at 1 h post administration.

Encouraged by the potent inhibitory effects found in the acute

model, we evaluated the chronic efﬁcacy of 12c in fructose-drinking

rats. Fructose feeding provides a dietary model of hypertriglycemia

because fructose stimulates hepatic de novo lipogenesis and VLDL

production in rats.^15,16Compound 12c reduced plasma and liver

ide levels but not liver triglyceride levels. These observations

suggest compound 12c, a potent non-selective ACC1/2 inhibitor,

effectively ameliorates hypertriglycemia and steatosis by dual

actions: blockage of de novo lipid synthesis and increase in fat

burning. At the same time, considerable skin irritation was

observed around the nose and all toes of the rats due to systemic

distribution. Considering that lipids are essential components of

skin barriers and several transgenic animals lacking lipid synthesis

enzymes (i.e., SCD-1,¹⁷DGAT-1¹⁸) develop skin damage, we could

not exclude the possibility that the skin irritations observed here

Figure 3. Effect of 12c and Bezaﬁbrate on the reduction of (A) plasma and (B) liver triglyceride levels in fructose-drinking rats. Plasma triglyceride levels were measured on

days 5 and 12. Liver triglyceride levels were measured on day 12. ^⁄Values signiﬁcantly different from Vehicle (p <0.05). ^⁄⁄Values signiﬁcantly different from Vehicle (p <0.01).

^⁄⁄⁄Values signiﬁcantly different from Vehicle (p <0.001). Data are means S.E.

T. Chonan et al. / Bioorg. Med. Chem. 19 (2011) 1580–1593

1585

were mechanism-based. For further understanding of the therapeu-

tic potential ACC inhibitors, an examination of the elaboration of

this study would be required.

addition of 2 M aqueous sodium hydroxide solution and extracted

with chloroform. The organic layer was dried over anhydrous mag-

nesium sulfate and concentrated in vacuo to afford 23 (3.87 g, 73%)

as a colorless amorphous. Compound 23 was used for the next step

without further puriﬁcation. ¹H NMR (200 MHz, CDCl₃): d 2.78–

3.02 (4H, m), 3.44–3.69 (4H, m), 5.22 (2H, br s), 7.05–7.16 (1H,

m), 7.23–7.43 (2H, m), 7.50–7.59 (1H, m).

4. Conclusion

In summary, a novel (4-piperidinyl)-piperazine class of com-

pounds as ACC1/2 non-selective inhibitors was designed, synthe-

sized and evaluated. Replacement of both substituents of the

(4-piperidinyl)-piperazine core of parent compound 6a gave novel

derivative 12c with a unique triF-Boc group that exhibited potent

ACC1/2 non-selective inhibitory activity and good oral activity.

Compound 12c potently reduced liver triglyceride levels and ame-

liorated hypertriglycemia of fructose-drinking rats. These results

suggest that ACC1/2 non-selective inhibitors might have potential

in the treatment of metabolic disorders such as obesity and type 2

diabetes.

5.2.3. (2-Amino-1-benzothiophen-3-yl)[4-(piperidin-4-yl)piper-

azin-1-yl]methanone (24)

A mixture of 23 (3.84 g, 14.6 mmol), tert-butyl 4-oxopiperidine-

1-carboxylate (2.93 g, 14.6 mmol) and sodium tri-acetoxy boro-

hidride (6.23 g, 29.4 mmol) in chloroform (40.0 mL) was stirred for

16 h at room temperature and quenched by addition of 2 M aqueous

sodium hydroxide solution in ice bath. The mixture was diluted

with water and extracted with chloroform twice. The combined

organics were washed with brine, dried over anhydrous magnesium

sulfate and concentrated in vacuo. The residue was puriﬁed by

NH silica gel column chromatography (50% ethyl acetate/n-

hexane) to afford tert-butyl 4-{4-[(2-amino-1-benzothiophen-3-

yl)carbonyl]piperazin-1-yl}piperidine-1-carboxylate (5.97 g, 91%)

as a colorless amorphous. ¹H NMR (600 MHz, CDCl₃): d 1.36–1.48

(11H, m), 1.75–1.81 (2H, m), 2.38–2.45 (1H, m), 2.49–2.55 (2H, m),

2.60 –2.75 (4H, m), 3.52–3.58 (2H, m), 3.60–3.67 (2H, m), 4.05–

4.24 (2H, m), 5.23 (2H, s), 7.09–7.13 (1H, m), 7.26–7.29 (1H, m),

7.37 (1H, d, J = 7.3 Hz), 7.54 (1H, d, J = 7.8 Hz).

To a solution of the carbamate (5.97 g, 13.4 mmol) in ethyl ace-

tate (30.0 mL) was added 4 M hydrogen chloride in ethyl acetate

(30.0 mL, 120 mmol). The reaction mixture was stirred for 2 days.

The resulting precipitate was obtained by ﬁltration and dissolved

in water the aqueous solution was basiﬁed by addition of 2 M

aqueous sodium hydroxide solution and extracted with chloro-

form. The organic layer was dried over anhydrous magnesium sul-

fate and concentrated in vacuo to afford 24 (4.24 g, 92%) as an off-

white amorphous. ¹H NMR (600 MHz, CDCl₃): d 1.36–1.46 (2H, m),

1.78–1.84 (2H, m), 2.35–2.42 (1H, m), 2.49–2.69 (6H, m), 3.12–3.18

(2H, m), 3.52–3.69 (4H, m), 5.22 (2H, s), 7.08–7.14 (1H, m), 7.25–

7.30 (1H, m), 7.37 (1H, d, J = 7.3 Hz), 7.54 (1H, d, J = 7.8 Hz); MS

(ESI): m/z 345 [M+H]⁺.

5. Experimental

5.1. Materials and methods

Unless otherwise noted, all solvents, chemicals, and reagents

were obtained commercially and used without puriﬁcation. Reac-

tions were monitored by TLC analysis using Merck Silica Gel 60

F₂₅₄thin-layer plates. Column chromatography was carried out

on a silica gel KANTO Reagents 60 N (spherical, neutral) or Fuji sily-

sia chemical Chromatorex NH. The ¹H NMR spectra were obtained

at 300 MHz on a Varian Instruments INOVA 300, 500 MHz on a

JEOL ECA500, or 600 MHz on a JEOL JNM-ECA600 with chemical

shift (d, ppm) reported relative to tetramethylsilane as an internal

standard. Electrospray Ionization (ESI) mass spectra were taken on

a micromass Platform-LC mass spectrometer or Shimadzu LCMS-

2010EV. Elemental analysis was performed using Perkin–Elmer

2400II or Yanaco MT-6 and are within 0.4% of theory unless other-

wise noted.

5.2. Chemistry

5.2.1. 2-Amino-1-benzothiophene-3-carboxylic acid (22)

To a solution of 2-amino-benzo[b]thiophene-3-carboxylic acid

ethyl ester (5.24 g, 23.6 mmol) in ethanol–tetrahydrofuran (1:1,

60.0 mL) was added 2 M aqueous sodium hydroxide solution

(60.0 mL) at room temperature, and the mixture was reﬂuxed for

8 h. After the removing solvents in vacuo, the residue was added

water and 2 M aqueous hydrochloric acid solution to adjusted pH

5.0. The resulting precipitate was obtained by ﬁltration, washed

with water and dried in vacuo to give 22 (4.34 g, 95%) as a pink

powder. ¹H NMR (600 MHz, DMSO-d₆): d 7.00–7.07 (1H, m),

7.18–7.25 (1H, m), 7.58 (1H, d, J = 7.3 Hz), 7.92 (2H, br s), 7.95–

8.02 (1H, m), 12.28 (1H, br s); MS (ESI): m/z 191 [MꢀH]⁺.

5.2.4. 1-(4-{4-[(2-Amino-1-benzothiophen-3-yl)carbonyl]-

piperazin-1-yl}piperidin-1-yl)propan-1-one (25c)

To a solution of 24 (150 mg, 0.44 mmol) in chloroform (3.0 mL)

were added triethylamine (0.06 mL, 0.44 mmol) and isobutyryl

chloride (0.05 ml, 0.44 mmol) at 0 °C and the reaction was stirred

for 1 h. The reaction mixture was added 1 M aqueous sodium

hydroxide solution (3.0 ml) and the organic layer was washed with

brine, dried over anhydrous magnesium sulfate. After concentra-

tion in vacuo, the residue was puriﬁed by NH silica gel column

chromatography (10% methanol/chloroform) to afford 25c

(123 mg, 71%) as a colorless powder. ¹H NMR (600 MHz, CDCl₃):

d 1.14 (3H, t, J = 7.4 Hz), 1.36–1.46 (2H, m), 1.81–1.89 (2H, m),

2.34 (2H, q, J = 7.4 Hz), 2.47–2.67 (6H, m), 2.97–3.04 (1H, m),

3.52–3.58 (2H, m), 3.60–3.67 (2H, m), 3.87–3.93 (1H, m), 4.63–

4.68 (1H, m), 5.24 (2H, s), 7.09–7.13 (1H, m), 7.25–7.30 (1H, m),

7.36 (1H, d, J = 8.3 Hz), 7.54 (1H, d, J = 7.8 Hz); MS (ESI): m/z 401

[M+H]⁺.

5.2.2. (2-Amino-1-benzothiophen-3-yl)(piperazin-1-yl)metha-

none (23)

A mixture of 22 (4.00 g, 20.7 mmol), 1-ethyl-3-(3-dimethylami-

nopropyl)carbodiimide hydrochloride (EDCI) (4.75 g, 24.8 mmol),

tert-butoxy piperazine-1-carboxylate (3.86 g, 20.7 mmol) and 1-

hydroxybenzotriazole monohydrate (HOBt) (3.36 g, 24.8 mmol) in

chloroform (60.0 mL) was stirred for 16 h. The reaction mixture

was washed with brine, the organic layer was dried over magne-

sium sulfate and concentration in vacuo. The residue was dissolved

in ethyl acetate (30.0 mL). To the mixture was added 4 M hydrogen

chloride in ethyl acetate (30.0 mL, 120 mmol) and the mixture was

stirred for 4 h. The resulting precipitate was obtained by ﬁltration

and dissolved in water. The aqueous solution was basiﬁed by

The following compounds 25a, 25d–p were prepared from the

corresponding starting materials in a similar manner to that de-

scribed for 25c.

5.2.5. 1-{4-[(4-{4-[(2-Amino-1-benzothiophen-3-yl)carbonyl]-

piperazin-1-yl}piperidin-1-yl)carbonyl]piperidin-1-yl}-

ethanone (25a)

Pale yellow amorphous (yield 51%); ¹H NMR (600 MHz, CDCl₃):

d 1.35–1.47 (2H, m), 1.60–1.96 (6H, m), 2.09 (3H, s), 2.47–2.76

1586

T. Chonan et al. / Bioorg. Med. Chem. 19 (2011) 1580–1593

(8H, m), 3.01–3.14 (2H, m), 3.51–3.69 (4H, m), 3.84–3.99 (2H, m),

4.54–4.67 (2H, m), 5.21–5.28 (2H, m), 7.08–7.14 (1H, m), 7.26–7.30

(1H, m), 7.36 (1H, d, J = 8.3 Hz), 7.55 (1H, d, J = 7.8 Hz); MS (ESI): m/

z 496 [MꢀH]^ꢀ.

5.2.12. 1-(4-{4-[(2-Amino-1-benzothiophen-3-yl)carbonyl]-

piperazin-1-yl}piperidin-1-yl)-2,2-dimethylpropan-1-one (25j)

Colorless amorphous (yield 64%); ¹H NMR (600 MHz, CDCl₃): d

1.27 (9H, s), 1.37–1.46 (2H, m), 1.83–1.89 (2H, m), 2.45–2.54

(3H, m), 2.61–2.67 (2H, m), 2.75–2.82 (2H, m), 3.52–3.68 (4H,

m), 4.44–4.50 (2H, m), 5.24 (2H, s), 7.09–7.13 (1H, m), 7.25–7.30

(1H, m), 7.37 (1H, d, J = 8.3 Hz), 7.54 (1H, d, J = 7.9 Hz); MS (ESI):

m/z 429 [M+H]⁺.

5.2.6. 1-(4-{4-[(2-Amino-1-benzothiophen-3-yl)carbonyl]-

piperazin-1-yl}piperidin-1-yl)butan-1-one (25d)

Colorless amorphous (yield 65%); ¹H NMR (600 MHz, CDCl₃): d

0.96 (3H, t, J = 7.3 Hz), 1.35–1.46 (2H, m), 1.61–1.69 (2H, m),

1.81–1.90 (2H, m), 2.28–2.32 (2H, m), 2.46–2.59 (4H, m), 2.60–

2.66 (2H, m), 2.97–3.04 (1H, m), 3.52–3.58 (2H, m), 3.59–3.68

(2H, m), 3.88–3.94 (1H, m), 4.63–4.69 (1H, m), 5.24 (2H, s), 7.09–

7.13 (1H, m), 7.25–7.30 (1H, m), 7.36 (1H, d, J = 7.8 Hz), 7.54 (1H,

d, J = 7.8 Hz); MS (ESI): m/z 415 [M+H]⁺.

5.2.13. Methyl 4-{4-[(2-amino-1-benzothiophen-3-yl)carbonyl]-

piperazin-1-yl}piperidine-1-carboxylate (25k)

Pale yellow amorphous (yield 59%); ¹H NMR (600 MHz, CDCl₃):

d 1.38–1.47 (2H, m), 1.76–1.83 (2H, m), 2.41–2.47 (1H, m),

2.48–2.55 (2H, m), 2.59–2.66 (2H, m), 2.72–2.82 (2H, m), 3.52–

3.58 (2H, m), 3.60–3.70 (5H, m), 4.05–4.31 (2H, m), 5.23 (2H, s),

7.09–7.12 (1H, m), 7.25–7.29 (1H, m), 7.36 (1H, d, J = 7.8 Hz),

7.54 (1H, d, J = 7.8 Hz).

5.2.7. 1-(4-{4-[(2-Amino-1-benzothiophen-3-yl)carbonyl]-

piperazin-1-yl}piperidin-1-yl)pentan-1-one (25e)

Colorless amorphous (yield 44%); ¹H NMR (600 MHz, CDCl₃): d

0.92 (3H, t, J = 7.57 Hz), 1.32–1.46 (4H, m), 1.56–1.63 (2H, m),

1.81–1.90 (2H, m), 2.29–2.35 (2H, m), 2.46–2.68 (6H, m), 2.97–

3.04 (1H, m), 3.51–3.68 (4H, m), 3.88–3.94 (1H, m), 4.63–4.70

(1H, m), 5.24 (2H, s), 7.09–7.13 (1H, m), 7.26–7.29 (1H, m), 7.36

(1H, d, J = 8.3 Hz), 7.55 (1H, d, J = 8.7 Hz); MS (ESI): m/z 429

[M+H]⁺.

5.2.14. Ethyl 4-{4-[(2-amino-1-benzothiophen-3-yl)carbonyl]-

piperazin-1-yl}piperidine-1-carboxylate (25l)

Colorless amorphous (yield 86%); ¹H NMR (600 MHz, CDCl₃): d

1.26 (3H, t, J = 7.1 Hz), 1.37–1.49 (2H, m), 1.75–1.85 (2H, m),

2.41–2.49 (1H, m), 2.49–2.56 (2H, m), 2.60–2.68 (2H, m), 2.71–

2.83 (2H, m), 3.51–3.70 (4H, m), 4.07–4.30 (4H, m), 5.19–5.27

(2H, m), 7.12 (1H, t, J = 6.9 Hz), 7.23–7.31 (1H, m), 7.37 (1H, d,

J = 7.8 Hz), 7.55 (1H, d, J = 7.8 Hz); MS (ESI): m/z 417 [M+H]⁺.

5.2.8. 1-(4-{4-[(2-Amino-1-benzothiophen-3-yl)carbonyl]-

piperazin-1-yl}piperidin-1-yl)-3-methylbutan-1-one (25f)

Colorless amorphous (yield 65%); ¹H NMR (600 MHz, CDCl₃): d

0.96 (6H, d, J = 6.9 Hz), 1.34–1.45 (2H, m), 1.82–1.90 (2H, m),

2.05–2.15 (1H, m), 2.17–2.25 (2H, m), 2.46–2.67 (6H, m), 2.98–

3.04 (1H, m), 3.52–3.68 (4H, m), 3.90–3.95 (1H, m), 4.66–4.70

(1H, m), 5.25 (2H, s), 7.09–7.13 (1H, m), 7.26–7.30 (1H, m), 7.36

(1H, d, J = 8.3 Hz), 7.54 (1H, d, J = 7.8 Hz); MS (ESI): m/z 429

[M+H]⁺.

5.2.15. Propan-2-yl 4-{4-[(2-amino-1-benzothiophen-3-yl)-

carbonyl]piperazin-1-yl}piperidine-1-carboxylate (25m)

Colorless amorphous (yield 56%); ¹H NMR (600 MHz, CDCl₃): d

1.24 (6H, d, J = 6.0 Hz), 1.37–1.48 (2H, m), 1.75–1.85 (2H, m),

2.40–2.48 (1H, m), 2.49–2.57 (2H, m), 2.61–2.68 (2H, m), 2.69–

2.81 (2H, m), 3.51–3.70 (4H, m), 4.07–4.31 (2H, m), 4.87–4.95

(1H, m), 5.19–5.27 (2H, m), 7.12 (1H, t, J = 7.6 Hz), 7.24–7.31 (1H,

m), 7.37 (1H, d, J = 8.3 Hz), 7.55 (1H, d, J = 7.8 Hz); MS (ESI): m/z

431 [M+H]⁺.

5.2.9. 1-(4-{4-[(2-Amino-1-benzothiophen-3-yl)carbonyl]-

piperazin-1-yl}piperidin-1-yl)-2-methylpropan-1-one (25g)

Colorless amorphous (yield 67%); ¹H NMR (600 MHz, CDCl₃): d

1.08–1.15 (6H, m), 1.35–1.47 (2H, m), 1.82–1.92 (2H, m), 2.46–

2.59 (4H, m), 2.60–2.67 (2H, m), 2.75–2.84 (1H, m), 2.98–3.06

(1H, m), 3.52–3.58 (2H, m), 3.60–3.68 (2H, m), 3.95–4.02 (1H,

m), 4.64–4.71 (1H, m), 5.24 (2H, s), 7.08–7.13 (1H, m), 7.24–7.30

(1H, m), 7.35–7.38 (1H, m), 7.53–7.56 (1H, m); MS (ESI): m/z 415

[M+H]⁺.

5.2.16. Cyclopentyl 4-{4-[(2-amino-1-benzothiophen-3-yl)-

carbonyl]piperazin-1-yl}piperidine-1-carboxylate (25n)

Pale yellow amorphous (yield 73%); ¹H NMR (600 MHz, CDCl₃):

d 1.35–1.46 (2H, m), 1.52–1.74 (6H, m), 1.74–1.89 (4H, m), 2.39–

2.47 (1H, m), 2.47–2.55 (2H, m), 2.59–2.67 (2H, m), 2.68–2.79

(2H, m), 3.50–3.58 (2H, m), 3.59–3.68 (2H, m), 3.96–4.34 (2H,

m), 5.06–5.11 (1H, m), 5.25 (2H, br s), 7.08–7.12 (1H, m), 7.25–

7.29 (1H, m), 7.36 (1H, d, J = 7.8 Hz), 7.54 (1H, d, J = 7.8 Hz); MS

(ESI): m/z 457 [M+H]⁺.

5.2.10. (2-Amino-1-benzothiophen-3-yl){4-[1-(cyclopentyl-

carbonyl)piperidin-4-yl]piperazin-1-yl}methanone (25h)

Colorless amorphous (yield 67%); ¹H NMR (600 MHz, CDCl₃): d

1.33–1.47 (2H, m), 1.51–1.65 (2H, m), 1.68–1.92 (8H, m), 2.45–

2.69 (6H, m), 2.83–2.94 (1H, m), 2.96–3.05 (1H, m), 3.48–3.71

(4H, m), 3.97–4.08 (1H, m), 4.63–4.71 (1H, m), 5.25 (2H, s), 7.07–

7.15 (1H, m), 7.24–7.30 (1H, m), 7.36 (1H, d, J = 7.3 Hz), 7.54 (1H,

d, J = 7.8 Hz); MS (ESI): m/z 441 [M+H]⁺.

5.2.17. Cyclohexyl 4-{4-[(2-amino-1-benzothiophen-3-yl)-

carbonyl]piperazin-1-yl}piperidine-1-carboxylate (25o)

Pale yellow amorphous (yield 80%); ¹H NMR (600 MHz, CDCl₃):

d 1.22–1.32 (1H, m), 1.33–1.47 (6H, m), 1.48–1.55 (1H, m), 1.65–

1.72 (2H, m), 1.75–1.87 (4H, m), 2.40–2.47 (1H, m), 2.48–2.56

(2H, m), 2.60–2.67 (2H, m), 2.69–2.81 (2H, m), 3.51–3.58 (2H,

m), 3.59–3.68 (2H, m), 4.11–4.27 (2H, m), 4.63–4.69 (1H, m),

5.23 (2H, br s), 7.09–7.13 (1H, m), 7.25–7.30 (1H, m), 7.36 (1H, d,

J = 8.3 Hz), 7.54 (1H, d, J = 7.8 Hz); MS (ESI): m/z 471 [M+H]⁺.

5.2.11. (2-Amino-1-benzothiophen-3-yl){4-[1-(cyclohexyl-

carbonyl)piperidin-4-yl]piperazin-1-yl}methanone (25i)

Colorless amorphous (yield 63%); ¹H NMR (600 MHz, CDCl₃):

d1.21–1.31 (3H, m), 1.33–1.58 (4H, m), 1.65–1.73 (3H, m), 1.76–

1.92 (4H, m), 2.43–2.57 (5H, m), 2.60–2.67 (2H, m), 2.96–3.04

(1H, m), 3.52–3.58 (2H, m), 3.60–3.68 (2H, m), 3.93–4.00 (1H,

m), 4.63–4.70 (1H, m), 5.23 (2H, s), 7.09–7.13 (1H, m), 7.25–7.30

(1H, m), 7.36 (1H, d, J = 7.8 Hz), 7.54 (1H, d, J = 7.8 Hz); MS (ESI);

m/z 455 [M+H]⁺.

5.2.18. 4-{4-[(2-Amino-1-benzothiophen-3-yl)- carbonyl]piper-

azin-1-yl}-N-tert-butylpiperidine-1-carboxamide (25p)

Colorless amorphous (yield 99%); ¹H NMR (600 MHz, CDCl₃): d

1.35 (9H, s), 1.40–1.52 (2H, m), 1.77–1.86 (2H, m), 2.38–2.46

(1H, m), 2.48–2.57 (2H, m), 2.60–2.69 (2H, m), 2.70–2.78 (2H,

m), 3.50–3.70 (4H, m), 3.89–3.97 (2H, m), 4.32 (1H, br s), 5.24

(2H, br s), 7.12 (1H, t, J = 7.6 Hz), 7.25–7.31 (1H, m), 7.37 (1H, d,

J = 7.3 Hz), 7.55 (1H, d, J = 7.8 Hz); MS (ESI): m/z 444 [M+H]⁺.

T. Chonan et al. / Bioorg. Med. Chem. 19 (2011) 1580–1593

1587

5.2.19. 1-Ethyl-3-(3-{[4-(1-propanoylpiperidin-4-yl)piperazin-

5.2.23. 1-Ethyl-3-(3-{[4-(1-pentanoylpiperidin-4-yl)piperazin-

1-yl]carbonyl}-1-benzothiophen-2-yl)urea (7c)

1-yl]carbonyl}-1-benzothiophen-2-yl)urea (7e)

To a solution of 25c (120 mg, 0.29 mmol) in chloroform (5.0 mL)

were added triethylamine (0.08 mL, 0.58 mmol) and triphosgene

(28 mg, 0.096 mmol) at 0 °C. The reaction mixture was stirred for

5 min and added 70% aqueous ethylamine solution (0.07 mL,

0.87 mmol) at room temperature. After stirring for 10 min, the

solvent was removed in vacuo and the residue was puriﬁed by

NH silica gel column chromatography (ethyl acetate) to afford 7c

(66 mg, 47%) as a colorless powder. ¹H NMR (600 MHz, CDCl₃): d

1.07–1.17 (6H, m), 1.36–1.46 (2H, m), 1.81–1.89 (2H, m), 2.34

(2H, q, J = 7.5 Hz), 2.46–2.60 (4H, m), 2.61–2.69 (2H, m), 2.97–

3.04 (1H, m), 3.24–3.30 (2H, m), 3.51–3.73 (4H, m), 3.87–3.94

(1H, m), 4.63–4.70 (1H, m), 5.02–5.08 (1H, m), 7.19–7.23 (1H,

m), 7.31–7.35 (1H, m), 7.43 (1H, d, J = 7.8 Hz), 7.72 (1H, d,

J = 7.8 Hz), 9.42 (1H, br s).; MS (ESI): m/z 472 [M+H]⁺. Anal. Calcd

for C₂₄H₃₃N₅O₃Sꢁ0.2H₂O: C, 60.66; H, 7.08; N, 14.74. Found: C,

60.94; H, 6.95; N, 14.44.

Colorless powder (yield 54%); ¹H NMR (600 MHz, CDCl₃): d 0.93

(3H, t, J = 7.3 Hz), 1.03 (3H, t, J = 7.1 Hz), 1.32–1.47 (4H, m), 1.56–

1.66 (2H, m), 1.80–1.90 (2H, m), 2.30–2.36 (2H, m), 2.47–2.70

(6H, m), 2.97–3.06 (1H, m), 3.18–3.26 (2H, m), 3.54–3.72 (4H,

m), 3.88–3.95 (1H, m), 4.64–4.70 (1H, m), 5.20–5.26 (1H, m),

7.17–7.23 (1H, m), 7.30–7.36 (1H, m), 7.42 (1H, d, J = 8.3 Hz),

7.72 (1H, d, J = 7.8 Hz), 9.41 (1H, br s); MS (ESI): m/z 500 [M+H]⁺.

Anal. Calcd for C₂₆H₃₇N₅O₃S: C, 62.50; H, 7.46; N, 14.02. Found C,

62.58; H, 7.53; N, 13.96.

5.2.24. 1-Ethyl-3-[3-({4-[1-(3-methylbutanoyl)piperidin-4-

yl]piperazin-1-yl}carbonyl)-1-benzothiophen-2-yl]urea (7f)

Colorless powder (yield 47%); ¹H NMR (600 MHz, CDCl₃): d 0.96

(6H, d, J = 6.4 Hz), 1.02 (3H t, J = 7.1 Hz), 1.34–1.47 (2H, m), 1.81–

1.89 (2H, m), 2.06–2.15 (1H, m), 2.18–2.24 (2H, m), 2.46–2.69

(6H, m), 2.97–3.05 (1H, m), 3.17–3.25 (2H, m), 3.51–3.71 (4H,

m), 3.89–3.96 (1H, m), 4.64–4.72 (1H, m), 5.23–5.29 (1H, m),

7.17–7.24 (1H, m), 7.30–7.35 (1H, m), 7.42 (1H, d, J = 7.8 Hz),

7.72 (1H, d, J = 7.8 Hz), 9.40 (1H, br s); MS (ESI): m/z 500 [M+H]⁺.

Anal. Calcd for C₂₆H₃₇N₅O₃S: C, 62.50; H, 7.46; N, 14.02. Found:

C, 62.50; H, 7.61; N, 13.84.

The following compounds 7a, 7b, 7d–j, 8a–f, and 9 were

prepared from the corresponding starting materials in a similar

manner to that described for 7c. Compounds 7a, 7h, 8d and 8f were

obtained as maleate.

5.2.20. 1-{3-[(4-{1-[(1-Acetylpiperidin-4-yl)carbonyl]piperidin-

4-yl}piperazin-1-yl)carbonyl]-1-benzothiophen-2-yl}-3-ethyl-

urea maleate (7a)

5.2.25. 1-Ethyl-3-[3-({4-[1-(2-methylpropanoyl)piperidin-4-

yl]piperazin-1-yl}carbonyl)-1-benzothiophen-2-yl]urea (7g)

Colorless powder (yield 67%); ¹H NMR (600 MHz, CDCl₃): d

1.06–1.16 (9H, m), 1.34–1.47 (2H, m), 1.81–1.92 (2H, m), 2.46–

2.59 (4H, m), 2.62–2.71 (2H, m), 2.76–2.84 (1H, m), 2.98–3.07

(1H, m), 3.24–3.31 (2H, m), 3.50–3.73 (4H, m), 3.94–4.03 (1H,

m), 4.64–4.72 (1H, m), 5.03–5.09 (1H, m), 7.19–7.23 (1H, m),

7.31–7.36 (1H, m), 7.44 (1H, d, J = 7.8 Hz), 7.72 (1H, d, J = 7.8 Hz),

9.41 (1H, br s); MS (ESI): m/z 486 [M+H]⁺. Anal. Calcd for

The corresponding free amine was obtained as a pale yellow

amorphous (yield 82%); ¹H NMR (600 MHz, CDCl₃): d 0.99–1.07

(3H, m), 1.34–1.47 (2H, m), 1.58–1.96 (6H, m), 2.10 (3H, s), 2.46–

2.77 (8H, m), 3.02–3.15 (2H, m), 3.17–3.26 (2H, m), 3.53–3.67

(4H, m), 3.85–3.99 (2H, m), 4.54–4.68 (2H, m), 5.27 (1H, br s),

7.17–7.24 (1H, m), 7.30–7.36 (1H, m), 7.42 (1H, d, J = 8.3 Hz),

7.72 (1H, d, J = 8.3 Hz), 9.40 (1H, br s); MS (ESI): m/z 569 [M+H]⁺.

To a solution of the free amine (549 mg, 0.97 mmol) in ethylacetate

(4.0 mL) and ethanol (1.0 ml)was added 0.1 M ethylacetate maleic

acid solution (9.7 mL, 0.97 mmol) at room temperature. After the

stirring overnight, the resultant precipitate was collected by ﬁltra-

tion (562 mg) as a colorless powder. Anal. Calcd for C₂₉H₄₀N₆O₄Sꢁ

1.0C₄H₄O₄ꢁ3.0H₂O: C, 53.65; H, 6.82; N, 11.37. Found: C, 53.76;

H, 6.45; N, 10.98.

C

₂₅H₃₅N₅O₃Sꢁ0.2H₂O: C, 61.37; H, 7.29; N, 14.31. Found: C, 61.66;

H, 7.19; N, 14.04.

5.2.26. 1-[3-({4-[1-(Cyclopentylcarbonyl)piperidin-4-yl]pipera-

zin-1-yl}carbonyl)-1-benzothiophen-2-yl]-3-ethylurea maleate

(7h)

The corresponding free amine was obtained as a colorless pow-

der (yield 84%); ¹H NMR (600 MHz, CDCl₃): d 1.07 (3H, t, J = 7.1 Hz),

1.35–1.47 (2H, m), 1.52–1.63 (2H, m), 1.69–1.92 (8H, m), 2.48–2.60

(4H, m), 2.62–2.71 (2H, m), 2.90 (1H, q, J = 8.0 Hz), 3.02 (1H, t,

J = 11.9 Hz), 3.22–3.29 (2H, m), 3.56–3.74 (4H, m), 4.03 (1H, d,

J = 13.8 Hz), 4.69 (1H, d, J = 11.9 Hz), 5.13 (1H, br s), 7.22 (1H, t,

J = 7.6 Hz), 7.34 (1H, t, J = 7.6 Hz), 7.44 (1H, d, J = 8.3 Hz), 7.73

(1H, d, J = 8.3 Hz), 9.43 (1H, br s); MS (ESI): m/z 512 [M+H]⁺. The

maleate was prepared from the free amine in a similar manner

5.2.21. 1-(3-{[4-(1-Acetylpiperidin-4-yl)piperazin-1-yl]carbonyl}-

1-benzothiophen-2-yl)-3-ethylurea (7b)

Colorless powder (yield 38%); ¹H NMR (600 MHz, CDCl₃): d

0.93–1.00 (3H, m), 1.35–1.49 (2H, m), 1.79–1.89 (2H, m), 2.09 (s,

3 H), 2.47–2.68 (6H, m), 3.02–3.09 (1H, m), 3.14–3.21 (2H, m),

3.54–3.67 (4H, m), 3.82–3.89 (1H, m), 4.61–4.67 (1H, m), 5.43

(1H, br s), 7.19–7.24 (1H, m), 7.30–7.35 (1H, m), 7.41 (1H, d,

J = 7.8 Hz), 7.72 (1H, d, J = 8.3 Hz), 9.39 (1H, br s); MS (ESI): m/z

458 [M+H]⁺. Anal. Calcd for C₂₃H₃₁N₅O₃Sꢁ0.2H₂O: C, 59.90; H,

6.86; N, 15.19. Found: C, 59.91; H, 6.86; N, 15.03.

to that described for 7a. Anal. Calcd for

C₄H₄O₄ꢁ2.0H₂O: C, 56.09; H, 6.83; N, 10.55. Found: C, 56.09; H,

C

₂₇H₃₇N₅O₃Sꢁ1.0-

6.76; N, 10.52.

5.2.22. 1-(3-{[4-(1-Butanoylpiperidin-4-yl)piperazin-1-yl]-

carbonyl}-1-benzothiophen-2-yl)-3-ethylurea (7d)

5.2.27. 1-[3-({4-[1-(Cyclohexylcarbonyl)piperidin-4-yl]piper-

azin-1-yl}carbonyl)-1-benzothiophen-2-yl]-3-ethylurea (7i)

Colorless powder (yield 76%); ¹H NMR (600 MHz, CDCl₃): d

1.03–1.10 (2H, m), 1.21–1.32 (4H, m), 1.33–1.61 (5H, m), 1.62–

1.73 (3H, m), 1.76–1.91 (4H, m), 2.41–2.57 (4H, m), 2.61–2.70

(2H, m), 2.97–3.04 (1H, m), 3.21–3.28 (2H, m), 3.51–3.73 (4H,

m), 3.94–4.00 (1H, m), 4.64–4.71 (1H, m), 5.08–5.15 (1H, m),

7.19–7.23 (1H, m), 7.31–7.35 (1H, m), 7.43 (1H, d, J = 7.8 Hz),

7.72 (1H, d, J = 7.8 Hz), 9.41 (1H, br s); MS (ESI): m/z 526 [M+H]⁺.

Anal. Calcd for C₂₈H₃₉N₅O₃Sꢁ0.7H₂O: C, 62.47; H, 7.56; N, 13.01.

Found: C, 62.68; H, 7.52; N, 12.52.

Colorless powder (yield 14%); ¹H NMR (600 MHz, CDCl₃): d

0.93–0.99 (3H, m), 1.04–1.15 (3H, m), 1.34–1.46 (2H, m),

1.60–1.69 (2H, m), 1.78–1.91 (2H, m), 2.24–2.33 (2H, m),

2.42–2.59 (4H, m), 2.61–2.69 (2H, m), 2.98–3.04 (1H, m),

3.24–3.30 (2H, m), 3.52–3.78 (4H, m), 3.88–3.94 (1H, m),

4.64–4.69 (1H, m), 5.04–5.09 (1H, m), 7.19–7.23 (1H, m),

7.31–7.35 (1H, m), 7.43 (1H, d, J = 7.8 Hz), 7.72 (1H, d, J = 7.8 Hz),

9.41(1H, br s); MS (ESI): m/z 486 [M+H]⁺. Anal. Calcd for

C

₂₅H₃₅N₅O₃Sꢁ0.4H₂O: C, 60.93; H, 7.32; N, 14.21. Found: C, 60.95;

H, 7.34; N, 14.33.

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T. Chonan et al. / Bioorg. Med. Chem. 19 (2011) 1580–1593

5.2.28. 1-[3-({4-[1-(2,2-Dimethylpropanoyl)piperidin-4-yl]piper-

azin-1-yl}carbonyl)-1-benzothiophen-2-yl]-3-ethylurea (7j)

Pale yellow amorphous (yield 84%); ¹H NMR (600 MHz, CDCl₃):

d 1.07 (3H, t, J = 7.1 Hz), 1.28 (9H, s), 1.36–1.46 (2H, m), 1.83–1.89

(2H, m), 2.47–2.56 (3H, m), 2.63–2.70 (2H, m), 2.74–2.83 (2H, m),

3.21–3.29 (2H, m), 3.55–3.73 (4H, m), 4.44–4.51 (2H, m), 5.13 (1H,

br s), 7.18–7.25 (1H, m), 7.30–7.37 (1H, m), 7.43 (1H, d, J = 8.3 Hz),

7.72 (1H, d, J = 7.8 Hz), 9.42 (1H, br s); MS (ESI): m/z 500 [M+H]⁺.

Anal. Calcd for C₂₆H₃₇N₅O₃Sꢁ0.3H₂O: C, 61.83; H, 7.50; N, 13.87.

Found: C, 61.86; H, 7.43; N, 13.86.

5.2.33. Cyclohexyl 4-[4-({2-[(ethylcarbamoyl)amino]-1-benzo-

thiophen-3-yl}carbonyl)piperazin-1-yl]piperidine-1-carboxy-

late (8e)

Colorless amorphous (yield 80%); ¹H NMR (600 MHz, CDCl₃): d

0.99–1.07 (3H, m), 1.22–1.32 (2H, m), 1.33–1.47 (5H, m), 1.48–

1.55 (1H, m), 1.64–1.73 (2H, m), 1.74–1.88 (4H, m), 2.40–2.59

(3H, m), 2.60–2.69 (2H, m), 2.69–2.82 (2H, m), 3.18–3.26 (2H,

m), 3.48–3.76 (4H, m), 4.10–4.30 (2H, m), 4.63–4.70 (1H, m),

5.14–5.21 (1H, m), 7.18–7.23 (1H, m), 7.30–7.35 (1H, m), 7.43

(1H, d, J = 7.8 Hz), 7.72 (1H, d, J = 7.8 Hz), 9.41 (1H, br s); MS

(ESI): m/z 542 [M+H]⁺. Anal. Calcd for C₂₈H₃₉N₅O₄Sꢁ0.5H₂O: C,

61.07; H, 7.32; N, 12.72. Found: C, 60.80; H, 7.30; N, 12.51.

5.2.29. Methyl 4-[4-({2-[(ethylcarbamoyl)amino]-1-benzothio-

phen-3-yl}carbonyl)piperazin-1-yl]piperidine-1-carboxylate

(8a)

5.2.34. tert-Butyl 4-[4-({2-[(ethylcarbamoyl)amino]-1-benzo-

thiophen-3-yl}carbonyl)piperazin-1-yl]piperidine-1-carboxy-

late (8f)

Colorless powder (yield 37%); ¹H NMR (600 MHz, CDCl₃): d

1.07–1.13 (3H, m), 1.37–1.47 (2H, m), 1.75–1.83 (2H, m), 2.42–

2.56 (3H, m), 2.62–2.69 (2H, m), 2.72–2.83 (2H, m), 3.24–3.31

(2H, m), 3.52–3.71 (7H, m), 4.06–4.31 (2H, m), 5.01–5.06 (1H,

m), 7.19–7.23 (1H, m), 7.31–7.35 (1H, m), 7.42–7.45 (1H, m),

7.71–7.73 (1H, m), 9.41 (1H, br s); MS (ESI): m/z 474 [M+H]⁺. Anal.

Calcd for C₂₃H₃₁N₅O₄Sꢁ0.2H₂O: C, 57.89; H, 6.63; N, 14.68. Found:

C, 57.93; H, 6.54; N, 14.38.

The corresponding free amine was obtained as a colorless pow-

der (yield 51%); ¹H NMR (600 MHz, CDCl₃): d 1.01 (3H, t, J = 7.1 Hz),

1.37–1.45 (2H, m), 1.46 (9H, s), 1.76–1.81 (2H, m), 2.41–2.47 (1H,

m), 2.50–2.59 (2H, m), 2.63–2.76 (4H, m), 3.17–3.25 (2H, m), 3.57–

3.66 (4H, m), 4.10–4.29 (2H, m), 5.35 (1H, br s), 7.21 (1H, t,

J = 7.1 Hz), 7.33 (1H, t, J = 7.3 Hz), 7.43 (1H, d, J = 7.8 Hz), 7.73

(1H, d, J = 7.8 Hz), 9.43 (1H, br s); MS (ESI): m/z 516 [M+H]⁺. The

maleate was prepared from the free amine in a similar manner

5.2.30. Ethyl 4-[4-({2-[(ethylcarbamoyl)amino]-1-benzothio-

phen-3-yl}carbonyl)piperazin-1-yl]piperidine-1-carboxylate

(8b)

to that described for 7a. Anal. Calcd for

C

₂₆H₃₇N₅O₄Sꢁ1.0-

C₄H₄O₄ꢁ1.7H₂O: C, 54.40; H, 6.76; N, 10.57. Found: C, 54.42; H,

6.73; N, 10.72.

Colorless powder (yield 17%); ¹H NMR (600 MHz, CDCl₃): d

0.97–1.06 (3H, m), 1.26 (3H, t, J = 7.1 Hz), 1.37–1.48 (2H, m), 1.60

(2H, s), 1.74–1.85 (2H, m), 2.42–2.58 (3H, m), 2.61–2.70 (2H, m),

2.72–2.84 (2H, m), 3.17–3.25 (2H, m), 3.52–3.73 (2H, m), 4.07–

4.31 (4H, m), 5.20–5.27 (1H, m), 7.22 (1H, t, J = 8.0 Hz), 7.34 (1H,

t, J = 7.6 Hz), 7.43 (1H, d, J = 8.3 Hz), 7.73 (1H, d, J = 7.8 Hz), 9.41

(1H, br s); MS (ESI): m/z 488 [M+H]⁺. Anal. Calcd for C₂₄H₃₃N₅-

O₄Sꢁ0.8H₂O: C, 57.42; H, 6.95; N, 13.95. Found: C, 57.63; H, 6.87;

N, 13.69.

5.2.35. N-tert-Butyl-4-[4-({2-[(ethylcarbamoyl)amino]-1-benzo-

thiophen-3-yl}carbonyl)piperazin-1-yl]piperidine-1-carbox-

amide (9)

Colorless amorphous (yield 81%); ¹H NMR (600 MHz, CDCl₃): d

0.93–1.01 (3H, m), 1.34 (9H, s), 1.40–1.50 (2H, m), 1.55–1.67 (2H,

m), 1.74–1.84 (2H, m), 2.39–2.46 (1H, m), 2.47–2.59 (2H, m),

2.60–2.68 (2H, m), 2.69–2.77 (2H, m), 3.12–3.23 (2H, m), 3.53–

3.75 (2H, m), 3.88–3.96 (2H, m), 4.33 (1H, br s), 5.30–5.38 (1H,

m), 7.20 (1H, t, J = 7.6 Hz), 7.33 (1H, t, J = 7.6 Hz), 7.42 (1H, d,

J = 7.8 Hz), 7.72 (1H, d, J = 7.8 Hz), 9.41 (1H, br s); MS (ESI): m/z

515 [M+H]⁺: Anal. Calcd for C₂₆H₃₈N₆O₃Sꢁ0.2H₂O: C, 60.25; H,

7.47; N, 16.04. Found; C, 60.15; H, 7.44; N, 16.04.

5.2.31. Propan-2-yl 4-[4-({2-[(ethylcarbamoyl)amino]-1-benzo-

thiophen-3-yl}carbonyl)piperazin-1-yl]piperidine-1-carboxy-

late (8c)

Colorless powder (yield 69%); ¹H NMR (600 MHz, CDCl₃): d

0.99–1.06 (3H, m), 1.24 (6H, d, J = 6.0 Hz), 1.37–1.47 (2H, m),

1.55–1.64 (2H, m), 1.75–1.84 (2H, m), 2.42–2.49 (1H, m), 2.49–

2.58 (2H, m), 2.61–2.69 (2H, m), 2.70–2.81 (2H, m), 3.18–3.25

(2H, m), 3.54–3.72 (2H, m), 4.09–4.30 (2H, m), 4.88–4.95 (1H,

m), 5.20–5.26 (1H, m), 7.22 (1H, t, J = 7.6 Hz), 7.34 (1H, t,

J = 6.9 Hz), 7.43 (1H, d, J = 7.8 Hz), 7.73 (1H, d, J = 7.8 Hz), 9.42

(1H, br s); MS (ESI): m/z 502 [M+H]⁺. Anal. Calcd for

5.2.36. 1-Fluoro-2-methylpropan-2-ol (27a)

To a solution of ethyl ﬂuoroacetate (21.2 g, 200 mmol) in tetra-

hydrofuran (200 mL) was added dropwise 3.0 M diethylether

methyl magnesium bromide solution (128 ml, 414 mmol) at

ꢀ60 °C and the reaction was stirred for 1 h under argon atmo-

sphere. The mixture was allowed to warm to 0 °C and stirred for

4 h. To this was added water (100 mL), concd hydrochloride

(36.0 mL) and sodium chloride (20.0 g), then extracted with Ether

(200 mL). The organic layer was dried over sodium sulfate and dis-

tilled under normal pressure at 71–102 °C to afford a solution of

27a in tetrahydrofuran (123 mmol, 62%). ¹H NMR (200 MHz,

CDCl₃): d 1.25 (3H, s), 1.26 (3H, s), 4.20 (2H, d, J = 47.5 Hz).

C

₂₅H₃₅N₅O₄Sꢁ0.8H₂O: C, 58.19; H, 7.15; N, 13.57. Found: C, 58.45;

H, 7.05; N, 13.32.

5.2.32. Cyclopentyl 4-[4-({2-[(ethylcarbamoyl)amino]-1-benzo-

thiophen-3-yl}carbonyl)piperazin-1-yl]piperidine-1-carboxy-

late (8d)

Pale yellow amorpous (yield 54%); ¹H NMR (600 MHz, CDCl₃): d

0.98–1.08 (3H, m), 1.22–1.32 (2H, m), 1.34–1.46 (2H, m), 1.64–1.74

(4H, m), 1.74–1.89 (4H, m), 2.37–2.58 (3H, m), 2.59–2.69 (2H, m),

2.69–2.80 (2H, m), 3.18–3.27 (2H, m), 3.40–3.78 (4H, m), 3.98–4.33

(2H, m), 5.06–5.11 (1H, m), 5.11–5.18 (1H, m), 7.18–7.23 (1H, m),

7.31–7.35 (1H, m), 7.43 (1H, d, J = 7.8 Hz), 7.72 (1H, d, J = 7.8 Hz),

9.41 (1H, br s); MS (ESI): m/z 528 [M+H]⁺; The maleate was pre-

pared from the free amine in a similar manner to that described

for 7a. Anal. Calcd for C₂₇H₃₇N₅O₄Sꢁ1.0H₂O: C, 59.43; H, 7.20; N,

12.83. Found: C, 59.70; H, 7.05; N, 12.75.

5.2.37. 1-{[(1-Fluoro-2-methylpropan-2-yl)oxy]carbonyl}-3-

methyl-1H-imidazol-3-ium iodide (28a)

A mixture of 27a (10.1 g, 110 mmol) and 1,1⁰-carbonyldiimidaz-

ole (CDI) (35.7 g, 220 mmol) in chloroform (220 mL) was stirred at

room temperature for 13 h under nitrogen atmosphere. The reac-

tion mixture was poured into water (200 mL) and the organic layer

was washed with brine (100 mL). The aqueous layer was extracted

with chloroform (100 mL). The combined organics were dried over

anhydrous magnesium sulfate and concentrated in vacuo to afford

1-ﬂuoro-2-methylpropan-2-yl-1H-imidazole-1-carboxylate (21.7

T. Chonan et al. / Bioorg. Med. Chem. 19 (2011) 1580–1593

1589

g, 99.9 mmol) as a colorless oil. ¹H NMR (200 MHz, CDCl₃) d 1.64

(3H, s), 1.65 (3H, s), 4.56 (2H, d, J = 47.5 Hz), 7.06 (1H, s), 7.38

(1H, s), 8.09 (1H, s).

m), 2.66–2.88 (2H, m), 3.43 (4H, br s), 3.98–4.23 (2H, m); MS (ESI):

m/z 424 [M+H]⁺.

A mixture of 1-ﬂuoro-2-methylpropan-2-yl-1H-imidazole-1-

carboxylate (21.7 g, 99.9 mmol) and iodomethane (29.0 mL,

466 mmol) in acetonitrile (132 mL) was stirred at room tempera-

ture for 32 h. The solvent and excess iodomethane were removed

in vacuo to give 28a (30.1 g, 91.7 mmol) as a brown amorphous.

¹H NMR (200 MHz, CDCl₃) d 1.75 (3H, s), 1.76 (3H, s), 4.29

(3H, s), 4.68 (2H, d, J = 47.0 Hz), 7.46 (1H, m), 7.71 (1H, m), 10.66

(1H, s).

5.2.43. 1-Fluoro-2-methylpropan-2-yl 4-(piperazin-1-

yl)piperidine-1-carboxylate dihydrochloride (31a)

To a solution of 30a (18.0 g, 46.5 mmol) in ethyl acetate

(210 mL) was added 4 M hydrogen chloride in ethyl acetate

(70 mL, 279 mmol) and the mixture was stirred for 2 h. The solvent

was removed in vacuo and the residue was added Ether (200 mL).

The resulting precipitate was obtained by ﬁltration and dried in va-

cuo to afford 31a (14.2 g, 85%) as an colorless powder. ¹H NMR

(600 MHz, DMSO-d₆): d 1.40 (3H, s), 1.40 (3H, s), 1.56 (2H, br s),

1.99–2.18 (2H, m), 2.64–2.88 (2H, m), 3.20–3.75 (10H, m), 3.96–

4.16 (2H, m) 4.49 (2H, d, J = 47.7 Hz); MS (ESI): m/z 288 [M+H]⁺.

Anal. Calcd for C₁₄H₂₆FN₃O₂ꢁ2.0HClꢁ0.2H₂O: C, 46.21; H, 7.87; N,

11.55. Found: C, 46.14; H, 7.84; N, 11.52.

5.2.38. 3-Methyl-1-{[(1,1,1-triﬂuoro-2-methylpropan-2-yl)oxy]-

carbonyl}-1H-imidazol-3-ium iodide (28b)

Compound 28b was prepared from commercially available

1,1,1-triﬂuoro-2-methylpropan-2-ol using the procedure described

for 28a as a colorless powder. ¹H NMR (600 MHz, CDCl₃): d 1.92

(6H, s), 4.31 (3H, s), 7.65–7.68 (2H, m), 10.22 (1H, s).

5.2.44. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-(piperazin-1-

yl)piperidine-1-carboxylate (31b)

To a solution of 30b (11.5 g, 26.3 mmol) in methanol (160 mL)

was added 4 M hydrogen chloride in ethyl acetate (80 mL,

320 mmol) and the mixture was stirred for 5 h. The solvent was re-

moved in vacuo and the residue was dissolved in water the aque-

ous solution was basiﬁed by addition of 2 M aqueous sodium

hydroxide solution and extracted with chloroform. The organic

layer was dried over anhydrous magnesium sulfate and concen-

trated in vacuo to afford 31a as a colorless powder (7.73 g,

91%). ¹H NMR (600 MHz, CDCl₃): d 1.36–1.51 (2H, m), 1.67 (6H,

m), 1.76–1.91 (2H, m), 2.33 –2.41 (1H, m), 2.54 (4H, br s), 2.66–

2.86 (2H, m), 2.91 (4H, t, J = 4.81 Hz), 3.97–4.24 (2H, m); MS

(ESI): m/z 324 [M+H]⁺.

5.2.39. 1-Fluoro-2-methylpropan-2-yl 4-oxopiperidine-1-

carboxylate (29a)

Compound 28a (30.1 g, 91.7 mmol) was dissolved in chloroform

(164 mL) and added to a solution of 4-piperidone triﬂuoroacetate

(24.7 g, 116 mmol) and triethylamine (32.3 mL, 232 mmol) in chlo-

roform (300 mL) at 0 °C. The reaction mixture was allowed to

warm to room temperature for 2 h and poured into water

(200 mL). The organic layer was washed with water (100 mL)

and brine (100 mL), dried over anhydrous magnesium sulfate and

concentrated in vacuo. The residue was puriﬁed by silica gel

column chromatography (10% ethyl acetate/n-hexane) to afford

29a (14.5 g, 73%) as a colorless powder. ¹H NMR (200 MHz, CDCl₃):

d

1.50 (3H, s), 1.51 (3H, s), 2.45 (4H, t, J = 6.2 Hz), 3.73

(4H, t, J = 6.4 Hz), 4.50 (2H, d, J = 47.4 Hz); MS (ESI): m/z 218

5.2.45. 1-Fluoro-2-methylpropan-2-yl 4-{4-[(2-amino-1-

benzothiophen-3-yl)carbonyl]piperazin-1-yl}piperidine-1-

carboxylate (35a)

[M+H]⁺.

A mixture of 22 (120 mg, 0.60 mmol), EDCI (138 mg, 0.72

mmol), 31a (216 mg, 0.60 mmol) and HOBt (97 mg, 0.72 mmol)

in N,N-dimethylformamide (6.0 mL) was stirred for 20 h. The reac-

tion mixture was diluted with ethyl acetate and washed with

brine, the organic layer was dried over magnesium sulfate and con-

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

chromatography (5% methanol/chloroform) to afford 35a (228 mg,

82%) as a pale yellow amorphous. ¹H NMR (600 MHz, CDCl₃): d

1.38–1.48 (8H, m), 1.72–1.86 (2H, m), 2.38–2.83 (7H, m), 3.49–

3.71 (4H, m), 4.03–4.25 (2H, m), 4.47 (2H, d, J = 47.7 Hz), 5.18–

5.27 (2H, m), 7.11 (1H, t, J = 7.1 Hz), 7.25–7.30 (1H, m), 7.36 (1H,

d, J = 7.8 Hz), 7.54 (1H, d, J = 7.8 Hz); MS (ESI): m/z 463 [M+H]⁺.

The following compounds 35b, 36a–h, 37a–d were prepared

from the corresponding starting materials in a similar manner to

that described for 35a.

5.2.40. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-oxopiperidine-1-

carboxylate (29b)

Compound 29b was prepared from 28b using the procedure de-

scribed for 29a as a colorless powder. ¹H NMR (600 MHz, CDCl₃): d

1.73 (6H, s), 2.49 (4H, br s), 3.75 (4H, br s); MS (ESI): m/z 254

[M+H]⁺.

5.2.41. tert-Butyl 4-(1-{[(1-ﬂuoro-2-methylpropan-2-yl)oxy]-

carbonyl}piperidin-4-yl)piperazine-1-carboxylate (30a)

A mixture of 29a (14.0 g, 64.4 mmol) and 1-(tert-butoxycar-

bonyl)piperidone (12.1 g, 65.0 mmol) in CHCl₃(322 mL) was

stirred at room temperature for 1.5 h. To this was added sodium

tri-acetoxyborohidride (27.6 g, 130 mmol) and the reaction

mixture was stirred for 5 h. The reaction mixture was added 3 M

aqueous sodium hydroxide solution (70 mL) to basify. The organic

layer was washed with brine (100 mL), dried over anhydrous

magnesium sulfate and concentrated in vacuo. The residue was

puriﬁed by silica gel column chromatography (5% methanol/chlo-

roform) to afford 30a (19.1 g, 49.6 mmol) as a colorless powder.

¹H NMR (200 MHz, CDCl₃) d 1.30–1.53 (16H, m), 1.70–1.86 (3H,

m), 2.31–2.55 (5H, m), 2.61–2.86 (2H, m), 3.36–3.48 (4H, m),

4.04–4.23 (2H, m), 4.47 (2H, d, J = 47.7 Hz); MS (ESI): m/z 388

[M+H]⁺.

5.2.46. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-{4-[(2-amino-1-

benzothiophen-3-yl)carbonyl]piperazin-1-yl}piperidine-1-

carboxylate (35b)

Colorless amorphous (87%); ¹H NMR (600 MHz, CDCl₃): d 1.37–

1.49 (2H, m), 1.68 (6H, s), 1.76–1.87 (2H, m), 2.39–2.48 (1H, m),

2.48–2.57 (2H, m), 2.58–2.68 (2H, m), 2.68–2.87 (2H, m), 3.50–

3.70 (4H, m), 3.98–4.23 (2H, m), 5.24 (2H, s), 7.09–7.13 (1H, m),

7.25–7.30 (1H, m), 7.36 (1H, d, J = 7.8 Hz), 7.54 (1H, d, J = 7.8 Hz);

MS (ESI): m/z 499 [M+H]⁺.

5.2.42. tert-Butyl 4-(1-{[(1,1,1-triﬂuoro-2-methylpropan-2-

yl)oxy]carbonyl}piperidin-4-yl)piperazine-1-carboxylate (30b)

Compound 30b was prepared from 29b using the procedure de-

scribed for 30a as a colorless powder. ¹H NMR (200 MHz, CDCl₃) d

1.37–1.50 (11H, m), 1.69 (6H, s), 1.76–1.87 (2H, m), 2.37–2.56 (5H,

5.2.47. Propan-2-yl 4-{4-[(2-amino-5-methyl-1-benzothiophen-

3-yl)carbonyl]piperazin-1-yl}piperidine-1-carboxylate (36a)

Pale yellow amorphous (51%); ¹H NMR (600 MHz, CDCl₃): d 1.23

(6H, d, J = 6.0 Hz), 1.38–1.47 (2H, m), 1.77–1.82 (2H, m), 2.39–2.54

1590

T. Chonan et al. / Bioorg. Med. Chem. 19 (2011) 1580–1593

(6H, m), 2.61–2.78 (4H, m), 3.51–3.70 (4H, m), 4.11–4.27 (2H, m),

4.87–4.94 (1H, m), 5.14–5.19 (2H, m), 6.94 (d, J = 7.79 Hz, 1H), 7.18

(1H, s), 7.42 (1H, d, J = 7.8 Hz); MS (ESI): m/z 445 [M+H]⁺.

m), 2.59–2.67 (2H, m), 2.69–2.85 (2H, m), 3.50–3.58 (2H, m),

3.59–3.67 (2H, m), 3.83 (3H, s), 4.00–4.20 (2H, m), 5.03–5.10 (2H,

m), 6.90 (1H, dd, J = 8.9, 2.5 Hz), 7.09 (1H, d, J = 2.3 Hz), 7.24–7.28

(1H, m); MS (ESI): m/z 529 [M+H]⁺.

5.2.48. Propan-2-yl 4-{4-[(2-amino-6-methyl-1-benzothiophen-

3-yl)carbonyl]piperazin-1-yl}piperidine-1-carboxylate (36b)

Colorless amorphous (51%); ¹H NMR (600 MHz, CDCl₃): d 1.23

(6H, d, J = 6.0 Hz), 1.37–1.45 (2H, m), 1.76–1.82 (2H, m), 2.39

(3H, s), 2.40–2.54 (3H, m), 2.60–2.78 (4H, m), 3.51–3.66 (4H, m),

4.12–4.25 (2H, m), 4.87–4.93 (1H, m), 5.12–5.17 (2H, m), 7.10

(1H, d, J = 9.7 Hz), 7.24–7.27 (1H, m), 7.35 (1H, s); MS (ESI): m/z

445 [M+H]⁺.

5.2.55. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-[4-({6-ﬂuoro-2-

[(4-methoxybenzyl)amino]-1-benzothiophen-3-yl}carbonyl)-

piperazin-1-yl]piperidine-1-carboxylate (37a)

Pale yellow amorphous (80%); ¹H NMR (600 MHz, CDCl₃): d

1.35–1.50 (2H, m), 1.68 (6H, s), 1.74–1.87 (2H, m), 2.38–2.53

(3H, m), 2.56–2.66 (2H, m), 2.67–2.86 (2H, m), 3.45–3.62 (4H,

m), 3.80 (3H, s), 4.00–4.21 (2H, m), 4.38 (2H, d, J = 6.0 Hz), 6.86–

6.91 (3H, m), 6.98–7.04 (1H, m), 7.22–7.32 (4H, m); MS (ESI):

m/z 637 [M+H]⁺.

5.2.49. 1-Fluoro-2-methylpropan-2-yl 4-{4-[(2-amino-6-methyl-

1-benzothiophen-3-yl)carbonyl]piperazin-1-yl}piperidine-1-

carboxylate (36c)

Colorless amorphous (73%); ¹H NMR (600 MHz, CDCl₃): d 1.37–

1.50 (8H, m), 1.75– 1.83 (2H, m), 2.38–2.54 (6H, m), 2.59–2.82 (4H,

m), 3.51–3.67 (4H, m), 4.04–4.22 (2H, m), 4.47 (2H, d, J = 47.2 Hz),

5.12–5.17 (2H, m), 7.08–7.11 (1H, m), 7.24–7.27 (1H, m), 7.35 (1H,

s); MS (ESI): m/z 477 [M+H]⁺.

5.2.56. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-[4-({2-[(4-meth-

oxybenzyl)amino]thieno[3,2-b]pyridin-3-yl}carbonyl)piper-

azin-1-yl]piperidine-1-carboxylate (37b)

Yellow amorphous (94%); ¹H NMR (600 MHz, CDCl₃): d 1.39–

1.51 (2H, m), 1.69 (6H, s), 1.77–1.91 (2H, m), 2.40–2.49 (1H, m),

2.58–2.91 (6H, m), 3.56–3.74 (4H, m), 3.81 (3H, s), 3.99–4.24

(2H, m), 4.44 (2H, d, J = 5.5 Hz), 6.87–6.95 (3H, m), 7.32 (2H, d,

J = 8.7 Hz), 7.61–7.68 (1H, m), 7.75–7.80 (1H, m), 8.44–8.47 (1H,

m); MS (ESI): m/z 620 [M+H]⁺.

5.2.50. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-{4-[(2-amino-6-

methyl-1-benzothiophen-3-yl)carbonyl]piperazin-1-yl}piper-

idine-1-carboxylate (36d)

Colorless amorphous (64%); ¹H NMR (600 MHz, CDCl₃): d 1.43

(2H, br s), 1.68 (6H, s), 1.76–1.85 (2H, m), 2.39 (3H, s), 2.40–2.46

(1H, m), 2.48–2.54 (2H, m), 2.58–2.66 (2H, m), 2.68–2.85 (2H,

m), 3.50–3.57 (2H, m), 3.59–3.68 (2H, m), 4.00–4.21 (2H, m),

5.13–5.18 (2H, s), 7.10 (1H, d, J = 7.3 Hz), 7.23–7.27 (1H, m), 7.35

(1H, s); MS (ESI): m/z 513 [M+H]⁺.

5.2.57. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-[4-({2-[(4-meth-

oxybenzyl)amino]thieno[3,2-c]pyridin-3-yl}carbonyl)piper-

azin-1-yl]piperidine-1-carboxylate (37c)

Pale yellow amorphous (82%); ¹H NMR (600 MHz, CDCl₃): d

1.36–1.50 (2H, m), 1.69 (6H, s), 1.76–1.88 (2H, m), 2.39–2.48

(1H, m), 2.50–2.87 (6H, m), 3.57 (4H, br s), 3.81 (3H, s), 4.00–

4.24 (2H, m), 4.43 (2H, d, J = 6.0 Hz), 6.90 (2H, d, J = 8.7 Hz),

7.28–7.34 (3H, m), 7.49 (1H, d, J = 5.5 Hz), 8.22 (1H, d, J = 5.0 Hz),

8.66 (1H, s); MS (ESI): m/z 620 [M+H]⁺.

5.2.51. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-(4-{[2-amino-6-

(triﬂuoromethyl)-1-benzothiophen-3-yl]carbonyl}piperazin-1-

yl)piperidine-1-carboxylate (36e)

Pale yellow amorphous (76%); ¹H NMR (600 MHz, CDCl₃): d

1.37–1.50 (2H, m), 1.68 (6H, s), 1.75–1.88 (2H, m), 2.40–2.48

(1H, m), 2.49–2.58 (2H, m), 2.60–2.88 (4H, m), 3.47–3.71 (4H,

m), 3.99–4.22 (2H, m), 5.48 (2H, br s), 7.42 (1H, d, J = 8.7 Hz),

7.51 (1H, d, J = 7.3 Hz), 7.80 (1H, s); MS (ESI): m/z 567 [M+H]⁺.

5.2.58. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-[4-({2-[(4-meth-

oxybenzyl)amino]thieno[2,3-b]pyridin-3-yl}carbonyl)piper-

azin-1-yl]piperidine-1-carboxylate (37d)

Pale yellow amorphous (63%); ¹H NMR (600 MHz, CDCl₃): d

1.36–1.50 (2H, m), 1.69 (6H, s), 1.76–1.87 (2H, m), 2.39–2.55

(3H, m), 2.58–2.67 (2H, m), 2.69–2.88 (2H, m), 3.42–3.64 (4H,

m), 3.81 (3H, s), 4.00–4.23 (2H, m), 4.44 (2H, d, J = 6.0 Hz),

6.89 (2H, d, J = 8.71 Hz),, 7.16–7.21 (1H, m), 7.28–7.35 (3H, m),

7.53–7.58 (1H, m), 8.17–8.22 (1H, m); MS (ESI): m/z 620

[M+H]⁺.

5.2.52. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-{4-[(2-amino-

thieno[2,3-c]pyridin-3-yl)carbonyl]piperazin-1-yl}piperidine-

1-carboxylate (36f)

Pale yellow amorphous (69%); ¹H NMR (600 MHz, CDCl₃): d

1.38–1.49 (2H, m), 1.68 (6H, s), 1.77–1.86 (2H, m), 2.42–2.48

(1H, m), 2.48–2.57 (2H, m), 2.60–2.69 (2H, m), 2.69–2.87 (2H,

m), 3.47–3.67 (4H, m), 4.00–4.23 (2H, m), 5.75–5.80 (2H, m),

7.20 (1H, d, J = 5.5 Hz), 8.38 (1H, d, J = 6.0 Hz), 8.71 (1H, s); MS

(ESI): m/z 500 [M+H]⁺.

5.2.59. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-{4-[(2-amino-6-

ﬂuoro-1-benzothiophen-3-yl)carbonyl]piperazin-1-yl}piper-

idine-1-carboxylate (38a)

To a solution of 37a (590 mg, 0.927 mmol) in chloroform

(3.00 mL) was added triﬂuoroacetic acid (3.00 mL). The reaction

mixture was stirred at room temperature for 1 h, and poured

3 M sodium hydroxide aqueous solution. The organic layer was

washed with brine and dried over anhydrous magnesium sulfate,

concentrated. The residue was puriﬁed by NH silica gel column

chromatography (20% ethyl acetate/n-hexane) to afford 38a as a

pale yellow amorphous (40%). ¹H NMR (600 MHz, CDCl₃): d

1.36–1.51 (2H, m), 1.67 (6H, s), 1.76–1.88 (2H, m), 2.38–2.57

(3H, m), 2.58–2.87 (4H, m), 3.47–3.68 (4H, m), 3.98–4.24 (2H,

m), 5.15 (2H, br s), 6.99–7.06 (1H, m), 7.23–7.33 (2H, m); MS

(ESI): m/z 517 [M+H]⁺.

5.2.53. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-{4-[(2-amino-6-

chloro-1-benzothiophen-3-yl)carbonyl]piperazin-1-yl}piper-

idine-1-carboxylate (36g)

Colorless amorphous (87%); ¹H NMR (600 MHz, CDCl₃):

d 1.37–1.49 (2H, m), 1.68 (6H, s), 1.76–1.87 (2H, m), 2.40–2.56

(3H, m), 2.58–2.67 (2H, m), 2.68–2.87 (2H, m), 3.45–3.69 (4H,

m), 3.98–4.23 (2H, m), 5.27 (2H, br s), 7.22–7.29 (2H, m), 7.52

(1H, d, J = 1.8 Hz); MS (ESI): m/z 533 [M+H]⁺.

5.2.54. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-{4-[(2-amino-6-

methoxy-1-benzothiophen-3-yl)carbonyl]piperazin-1-yl}piper-

idine-1-carboxylate (36h)

The following compounds 38b–d were prepared from the corre-

sponding starting materials in a similar manner to that described

for 38a.

Colorless amorphous (87%). ¹H NMR (600 MHz, CDCl₃): d 1.43

(2H, br s), 1.64–1.85 (8H, m), 2.42–2.48 (1H, m), 2.49–2.57 (2H,

T. Chonan et al. / Bioorg. Med. Chem. 19 (2011) 1580–1593

1591

5.2.60. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-{4-[(2-aminothi-

eno[3,2-b]pyridin-3-yl)carbonyl]piperazin-1-yl}piperidine-1-

carboxylate (38b)

5.2.65. Propan-2-yl 4-[4-({2-[(ethylcarbamoyl)amino]-5-methyl-

1-benzothiophen-3-yl}carbonyl)piperazin-1-yl]piperidine-1-

carboxylate maleate (11)

Pale yellow amorphous (92%); ¹H NMR (600 MHz, CDCl₃): d

1.37–1.52 (2H, m), 1.69 (6H, s), 1.76–1.91 (2H, m), 2.40–2.50

(1H, m), 2.52–2.90 (6H, m), 3.32–3.94 (4H, m), 3.99–4.25 (2H,

m), 5.73 (2H, br s), 6.98 (1H, dd, J = 8.0, 4.8 Hz), 7.80 (1H, d,

J = 7.8 Hz), 8.42–8.50 (1H, m); MS (ESI): m/z 500 [M+H]⁺.

Colorless powder (69%); ¹H NMR (600 MHz, CDCl₃): d 1.06 (3H,

t, J = 7.1 Hz), 1.24 (6H, d, J = 6.4 Hz), 1.37–1.46 (2H, m), 1.76–1.82

(2H, m), 2.42–2.55 (6H, m), 2.64–2.79 (4H, m), 3.20–3.28 (2H,

m), 3.54–3.74 (4H, m), 4.13–4.26 (2H, m), 4.87–4.94 (1H, m),

5.16 (1H, s), 7.02–7.06 (1H, m), 7.22 (1H, s), 7.58–7.61 (1H, m),

9.36 (1H, s); MS (ESI): 516 [M+H]⁺. Anal. Calcd for C₂₆H₃₇N₅O₄Sꢁ-

C₄H₄O₄ꢁ1.5H₂O: C, 54.70; H, 6.73; N, 10.63. Found: C, 54.52; H,

6.25; N, 10.32.

5.2.61. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-{4-[(2-aminothi-

eno[3,2-c]pyridin-3-yl)carbonyl]piperazin-1-yl}piperidine-1-

carboxylate (38c)

Colorless amorphous (95%); ¹H NMR (600 MHz, CDCl₃): d 1.38–

1.50 (2H, m), 1.68 (6H, s), 1.76–1.88 (2H, m), 2.41–2.49 (1H, m),

2.52–2.88 (6H, m), 3.61 (4H, br s), 3.99–4.24 (2H, m), 5.51 (2H,

br s), 7.50 (1H, d, J = 6.0 Hz), 8.28 (1H, d, J = 5.0 Hz), 8.66 (1H, s);

MS (ESI): m/z 500 [M+H]⁺.

5.2.66. Propan-2-yl 4-[4-({2-[(ethylcarbamoyl)amino]-6-methyl-

1-benzothiophen-3-yl}carbonyl)piperazin-1-yl]piperidine-1-

carboxylate (12a)

Colorless amorphous (37%); ¹H NMR (600 MHz, CDCl₃): d 1.08

(3H, t, J = 7.1 Hz), 1.23 (6H, d, J = 6.4 Hz), 1.36–1.46 (2H, m),

1.76–1.82 (2H, m), 2.40–2.55 (7H, m), 2.62–2.78 (4H, m), 3.21–

3.30 (2H, m), 3.53–3.70 (3H, m), 4.14–4.26 (2H, m), 4.87–4.94

(1H, m), 5.13 (1H, s), 7.15 (1H, d, J = 8.3 Hz), 7.32 (1H, d,

J = 8.3 Hz), 7.52 (1H, s), 9.36 (1H, s); MS (ESI): 516 [M+H]⁺. Anal.

Calcd for C₂₆H₃₇N₅O₄Sꢁ0.9H₂O: C, 58.71; H, 7.35; N, 13.17. Found

C, 58.81; H, 7.39; N, 13.02.

5.2.62. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-{4-[(2-aminothi-

eno[2,3-b]pyridin-3-yl)carbonyl]piperazin-1-yl}piperidine-1-

carboxylate (38d)

Pale yellow amorphous (92%); ¹H NMR (600 MHz, CDCl₃): d 1.38–

1.51 (2H, m), 1.68 (6H, s), 1.76–1.88 (2H, m), 2.41–2.57 (3H, m),

2.59–2.88 (4H, m), 3.46–3.55 (2H, m), 3.60–3.71 (2H, m), 4.01–

4.23 (2H, m), 5.53 (2H, br s), 7.17–7.24 (1H, m), 7.58 (1H, d,

J = 6.4 Hz), 8.25 (1H, d, J = 6.4 Hz); MS (ESI): m/z 500 [M+H]⁺.

5.2.67. 1-Fluoro-2-methylpropan-2-yl 4-[4-({2-[(ethylcarbam-

oyl)amino]-6-methyl-1-benzothiophen-3-yl}carbonyl)piper-

azin-1-yl]piperidine-1-carboxylate hydrochloride (12b)

5.2.63. 1-Fluoro-2-methylpropan-2-yl 4-[4-({2-[(ethylcarba-

moyl)amino]-1-benzothiophen-3-yl}carbonyl)piperazin-1-yl]-

piperidine-1-carboxylate hydrochloride (10a)

Pale red powder (77%); ¹H NMR (600 MHz, CDCl₃): d 1.06 (3H, t,

J = 7.1 Hz), 1.38–1.50 (8H, m), 1.74–1.82 (2H, m), 2.39–2.55 (6H,

m), 2.61–2.84 (4H, m), 3.20–3.29 (2H, m), 3.53–3.71 (4H, m),

4.05–4.24 (2H, m), 4.47 (2H, d, J = 47.2 Hz), 5.17 (1H, s), 7.15 (1H,

d, J = 8.3 Hz), 7.31 (1H, d, J = 8.3 Hz), 7.52 (1H, s), 9.35 (1H, s); MS

(ESI): 548 [M+H]⁺. Anal. Calcd for C₂₇H₃₈FN₅O₄Sꢁ1.0HClꢁ1.0H₂O: C,

53.85; H, 6.86; N, 11.63. Found: C, 53.69; H, 6.71; N, 11.40.

To a solution of 35a (228 mg, 0.493 mmol) in pyridine (6.00 mL)

was added ethyl isocyanate (0.195 mL, 2.47 mmol). The reaction

mixture was heated at 60 °C for 17 h and concentrated. To this

was added chloroform and washed with water (5 times). The or-

ganic layer was washed with brine and dried over anhydrous mag-

nesium sulfate, then concentrated. The residue was puriﬁed by NH

silica gel column chromatography (ethyl acetate) to afford the free

amine of 7a as a colorless amorphous (80%). ¹H NMR (600 MHz,

CDCl₃): d 1.06 (3H, t, J = 7.1 Hz), 1.37–1.49 (8H, m), 1.74–1.83

(2H, m), 2.40–2.57 (3H, m), 2.59–2.83 (4H, m), 3.20–3.27 (2H,

m), 3.51–3.71 (4H, m), 4.05–4.23 (2H, m), 4.47 (2H, d, J =

47.2 Hz), 5.14 (1H, s), 7.21 (1H, t, J = 7.6 Hz), 7.33 (1H, t, J =

7.6 Hz), 7.43 (1H, d, J = 8.3 Hz), 7.72 (1H, d, J = 8.0 Hz), 9.41 (1H,

s); MS (ESI): m/z 534 [M+H]⁺.

5.2.68. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-[4-({2-[(ethyl-

carbamoyl)amino]-6-methyl-1-benzothiophen-3-yl}carbonyl)-

piperazin-1-yl]piperidine-1-carboxylate (12c)

Colorless powder (76%); ¹H NMR (600 MHz, CDCl₃): d 1.00 (3H,

t, J = 6.9 Hz), 1.36–1.48 (2H, m), 1.68 (6H, s), 1.75–1.85 (2H, m),

2.39–2.47 (4H, m), 2.48–2.57 (2H, m), 2.59–2.67 (2H, m), 2.68–

2.86 (2H, m), 3.15–3.23 (2H, m), 3.51–3.72 (4H, m), 3.99–4.22

(2H, m), 5.29 (1H, t, J = 5.3 Hz), 7.15 (1H, d, J = 8.3 Hz), 7.28–7.32

(1H, m), 7.52 (1H, s), 9.36 (1H, s); MS (ESI): 584 [M+H]⁺. Anal. Calcd

for C₂₇H₃₆F₃N₅O₄Sꢁ1.0HClꢁ1.0H₂O: C, 50.82; H, 6.16; N, 10.97.

Found: C, 50.93; H, 5.93; N, 10.76.

To a solution of the free amine (210 mg, 0.394 mmol) in ethyl

acetate (10.0 mL) was added 4.0 M ethylacetate hydro chloride

solution (0.108 mL, 0.433 mmol) at room temperature. After the

stirring for 2 h, the resultant precipitate was collected by ﬁltration

(184 mg) to afford 10a as a colorless powder. Anal. Calcd for

5.2.69. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-(4-{[2-amino-6-

(triﬂuoromethyl)-1-benzothiophen-3-yl]carbonyl}piperazin-1-

yl}piperidine-1-carboxylatehydrochloride (13)

C

₂₆H₃₆FN₄O₄Sꢁ1.0HClꢁ1.0H₂O: C, 53.10; H, 6.68; N, 11.91. Found:

C, 52.94; H, 6.39; N, 11.70.

Pale yellow powder (70%); ¹H NMR (600 MHz, CDCl₃): d ppm

0.99–1.10 (3H, m), 1.37–1.50 (2H, m), 1.68 (6H, s), 1.75–1.89

(2H, m), 2.40–2.60 (3H, m), 2.61–2.89 (4H, m), 3.20–3.29 (2H,

m), 3.44–3.80 (4H, m), 3.98–4.25 (2H, m), 5.23 (1H, s), 7.50 (1H,

d, J = 8.3 Hz), 7.56 (1H, d, J = 8.3 Hz), 7.99 (1H, br s), 9.53 (1H, s);

MS (ESI): 638 [M+H]⁺. Anal. Calcd for C₂₇H₃₃F₆N₅O₄Sꢁ

1.0HClꢁ0.5H₂O: C, 47.47; H, 5.16; N, 10.25. Found: C, 47.40; H,

4.94; N, 10.13.

The following compounds 10b, 11, 12a–c, 13–20 were prepared

from the corresponding starting materials in a similar manner to

that described for 10a.

5.2.64. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-[4-({2-[(ethylcar-

bamoyl)amino]-1-benzothiophen-3-yl}carbonyl)piperazin-1-yl]-

piperidine-1-carboxylate hydrochloride hydrochloride (10b)

Colorless powder (84%); ¹H NMR (600 MHz, CDCl₃): d 1.10 (3H,

t, J = 7.3 Hz), 1.38–1.48 (2H, m), 1.56 (6H, s), 1.76–1.86 (2H, m),

2.40–2.56 (3H, m), 2.60–2.87 (4H, m), 3.24–3.31 (2H, m), 3.49–

3.74 (4H, m), 3.98–4.22 (2H, m), 5.04 (1H, t, J = 5.3 Hz), 7.21 (1H,

t, J = 7.6 Hz), 7.33 (1H, t, J = 7.6 Hz), 7.44 (1H, d, J = 7.8 Hz), 7.72

(1H, d, J = 7.8 Hz), 9.41 (1H, s); MS (ESI): 570 [M+H]⁺. Anal. Calcd

for C₂₆H₃₄F₃N₅O₄Sꢁ1.0HClꢁ0.75H₂O: C, 50.40; H, 5.94; N, 11.30.

Found: C, 50.62; H, 5.71; N, 11.25.

5.2.70. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-[4-({2-[(ethylcar-

bamoyl)amino]-6-ﬂuoro-1-benzothiophen-3-yl}carbonyl)-

piperazin-1-yl]piperidine-1-carboxylate (14)

Pale yellow powder (74%); ¹H NMR (600 MHz, CDCl₃):

d

1.03–1.09 (3H, m), 1.36–1.50 (2H, m), 1.68 (6H, s), 1.74–1.87 (2H,

m), 2.40–2.58 (3H, m), 2.59–2.89 (4H, m), 3.19–3.28 (2H, m),

1592

T. Chonan et al. / Bioorg. Med. Chem. 19 (2011) 1580–1593

3.44–3.76 (4H, m), 3.98–4.23 (2H, m), 5.11 (1H, br s), 7.04–7.12

(1H, m), 7.33–7.39 (1H, m), 7.40–7.44 (1H, m), 9.35 (1H, s); MS

(ESI): 588 [M+H]⁺. Anal. Calcd for C₂₆H₃₃F₄N₅O₄Sꢁ1.0HClꢁ1.0H₂O:

C, 48.63; H, 5.65; N, 10.91. Found: C, 48.84; H, 5.35; N, 10.84.

5.2.76. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-[4-({2-[(ethylcar-

bamoyl)amino]thieno[2,3-b]pyridin-3-yl}carbonyl)piperazin-

1-yl]piperidine-1-carboxylate hydrochloride (20)

Pale yellow powder (62%); ¹H NMR (600 MHz, CDCl₃): d 1.17 (t,

J = 7.1 Hz, 3H), 1.37–1.50 (2H, m), 1.68 (6H, s), 1.75–1.88 (2H, m),

2.40–2.56 (3H, m), 2.62–2.88 (4H, m), 3.34 (2H, dd, J = 7.3,

5.5 Hz), 3.47–3.56 (2H, m), 3.60–3.75 (2H, m), 4.00–4.23 (2H, m),

5.15–5.23 (1H, m), 7.24–7.29 (1H, m), 7.64–7.71 (1H, m),

8.39–8.46 (1H, m), 9.63 (1H, s); MS (ESI): 571 [M+H]⁺. Anal. Calcd

for C₂₅H₃₃F₃N₆O₄Sꢁ2.0HClꢁ2.0H₂O: C, 44.18; H, 5.78; N, 12.37.

Found: C, 44.45; H, 5.57; N, 12.33.

5.2.71. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-[4-({6-chloro-2-

[(ethylcarbamoyl)amino]-1-benzothiophen-3-yl}carbonyl)-

piperazin-1-yl]piperidine-1-carboxylate hydrochloride (15)

Pale yellow powder (87%); ¹H NMR (600 MHz, CDCl₃): d 1.04–

1.11 (3H, m), 1.36–1.50 (2H, m), 1.68 (6H, s), 1.75–1.86 (2H, m),

2.41–2.57 (3H, m), 2.60–2.87 (4H, m), 3.22–3.30 (2H, m), 3.47–

3.75 (4H, m), 3.98–4.23 (2H, m), 5.10 (1H, br s), 7.28–7.31 (1H,

m), 7.32–7.36 (1H, m), 7.70 (1H, d, J = 8.3 Hz), 9.40 (1H, s); MS

(ESI): 604 [M+H]⁺. Anal. Calcd for C₂₆H₃₃ClF₃N₅O₄Sꢁ1.0HClꢁ

1.0H₂O: C, 47.42; H, 5.51; N, 10.63. Found: C, 47.77; H, 5.33; N,

10.65.

5.2.77. HPLC analysis

For the chemical stability test, the residual amount was ana-

lyzed using reverse-phase HPLC with a CAPCELL PAK C18 UG120

(5

l

m particle size, / 4.6 ꢂ 150 mm; SHISEIDO), then eluted at

1.0 mL/min with acetonitrile–H₂O buffer solution and monitored

using UV absorbance at 240 nm.

5.2.72. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-[4-({2-[(ethyl-

carbamoyl)amino]-6-methoxy-1-benzothiophen-3-yl}carbo-

nyl)piperazin-1-yl]piperidine-1-carboxylate hydrocloride (16)

¹H NMR (600 MHz, CDCl₃): d 1.09 (3H, t, J = 7.3 Hz), 1.37–1.48

(2H, m), 1.68 (6H, s), 1.75–1.85 (2H, m), 2.40–2.57 (3H, m), 2.59–

2.68 (2H, m), 2.68–2.86 (2H, m), 3.23–3.30 (2H, m), 3.51–3.71

(4H, m), 3.86 (3H, s), 4.00–4.21 (2H, m), 5.04 (1H, t, J = 5.5 Hz),

6.96 (1H, dd, J = 8.7, 2.3 Hz), 7.22 (1H, d, J = 2.29 Hz), 7.33 (1H, d,

J = 8.7 Hz), 9.29 (1H, s); MS (ESI): 600 [M+H]⁺. Anal. Calcd for

5.3. Biology

5.3.1. Preparation of human liver ACC

Human liver cytosol was obtained from KAC Co. The cytosol was

saturated with ammonium sulfate (ﬁnal 30%). The resulting precip-

itate was dissolved in 50 mM MOPS buffer (pH 7.5, 250 mM

sucrose, 2.0 mM EDTA, 2.0 mM DTT, 5% glycerol), and then loaded

onto a RESOURCE Q anion-exchange column (GE Healthcare) that

was pre-equilibrated with 50 mM MOPS buffer. The ACC fractions

were eluted with 50 mM MOPS buffer containing 250 mM NaCl

and pooled as partially puriﬁed human ACCs.

C

₂₇H₃₆F₃N₅O₅Sꢁ1.0HClꢁ0.5H₂O: C, 50.27; H, 5.94; N, 10.86. Found:

C, 50.04; H, 5.90; N, 10.53.

5.2.73. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-[4-({2-[(ethyl-

carbamoyl)amino]thieno[3,2-b]pyridin-3-yl}carbonyl)piper-

azin-1-yl]piperidine-1-carboxylate hydrochloride (17)

Pale brown powder (78%); ¹H NMR (600 MHz, CDCl₃): d 0.89–

1.03 (3H, m), 1.40–1.52 (2H, m), 1.69 (6H, s), 1.78–1.93

(2H, m), 2.43–2.90 (7H, m), 3.13–3.26 (2H, m), 3.42–3.58 (2H,

m), 3.80–3.93 (2H, m), 4.00–4.26 (2H, m), 5.48 (1H, s), 7.07–7.12

(1H, m), 7.96–8.01 (1H, m), 8.52–8.57 (1H, m), 9.87 (1H, s); MS

(ESI): 571 [M+H]⁺. Anal. Calcd for C₂₅H₃₃F₃N₆O₄Sꢁ2.0HClꢁ

2.0H₂O: C, 44.18; H, 5.78; N, 12.37. Found: C, 44.49; H, 5.53; N,

12.36.

5.3.2. ACCs enzyme assay

ACCs were preincubated with a test compound or vehicle

(DMSO) in the assay buffer (60.6 mM Tris acetate, pH 7.5,

1.32 lM b-mercaptoethanol, 5 mM magnesium acetate, 8 mM

magnesium sulfate, 10 mM sodium citrate and 1 mg/mL BSA) at

37 °C for 30 min. The enzyme reaction was started by adding the

substrate mixture; 2.12 mM ATP, NaHCO₃(18.2, 12 or 0.35 mM

for liver ACC, rhACC1 or rhACC2, respectively) and [¹⁴C] acetyl-

CoA (25, 35 or 10 lM for liver ACC, rhACC1 or rhACC2, respec-

tively). The enzyme reaction was terminated by adding 10 mM

carnitin and 1.88 U/mL carnitine acetyltransferase (SIGMA). Under

these conditions, the residual [¹⁴C] acetyl-CoA was converted to

5.2.74. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-[4-({2-[(ethyl-

carbamoyl)amino]thieno[3,2-c]pyridin-3-yl}carbonyl)pipera-

zin-1-yl]piperidine-1-carboxylate hydrochloride (18)

Pale yellow powder (84%); ¹H NMR (600 MHz, CDCl₃): d 1.03–

1.13 (3H, m), 1.36–1.51 (2H, m), 1.69 (6H, s), 1.76–1.86 (2H, m),

2.42–2.50 (1H, m), 2.51–2.90 (6H, m), 3.27 (2H, dd, J = 7.3,

5.5 Hz), 3.52–3.72 (4H, m), 4.00–4.24 (2H, m), 5.30 (1H, s), 7.67

(1H, d, J = 4.6 Hz), 8.36 (1H, t, J = 5.5 Hz), 8.77 (1H, s), 9.56 (1H,

s); MS (ESI): 571 [M+H]⁺. Anal. Calcd for C₂₅H₃₃F₃N₆O₄Sꢁ2.0HClꢁ

2.0H₂O: C, 44.18; H, 5.78; N, 12.37. Found: C, 44.27; H, 5.71; N,

12.27.

[

¹⁴C] acetyl-carnitine. The reaction mixture (40

lL) was then

mixed with 90

lL of 50% (w/v) cation-exchage resin in a 96-well

ﬁltration plate. In this procedure, [¹⁴C] acetyl-carnitine, a positively

charged substrate, was absorbed to the resin. Radioactivity of the

eluate containing a negatively charged [¹⁴C] malonyl-CoA was

measured as ACCs activities.

5.3.3. Fatty acid synthesis in HepG2 cells

HepG2 cells were seeded into a 24-well plate at 3 ꢂ 10⁵cells/

well in DMEM containing 10% FBS and maintained for 48 h. Cells

were starved in serum-free DMEM containing 1.5% BSA for 1 h with

5.2.75. 1,1,1-Triﬂuoro-2-methylpropan-2-yl 4-[4-({2-[(ethylcar-

bamoyl)amino]thieno[2,3-c]pyridin-3-yl}carbonyl)piperazin-1-

yl]piperidine-1-carboxylate hydrochloride (19)

or without a test compound. 4 l

Ci of [³H] acetate was added to

each well. After 2-h incubation, the medium was removed and

the cells were washed with PBS two times. The cell were saponiﬁed

by addition of 0.5 mL of 0.5 M KOH in H₂O and 0.5 mL of 5 M KOH

in MeOH, followed by incubation for 2 h at 70 °C. Following sapon-

iﬁcation, the reaction mixture was washed three times with hex-

ane, and the organic phase was discarded. The aqueous phase

was acidiﬁed to pH <2 by HCl, and then fatty acids were extracted

with hexane. The radioactivity of the fatty acids was measured

using a liquid scintillation counter.

Colorless powder (86%); ¹H NMR (600 MHz, CDCl₃): d 1.00–

1.08 (3H, m), 1.38–1.48 (2H, m), 1.68 (6H, s), 1.76–1.85 (2H, m),

2.42–2.59 (3H, m), 2.61–2.87 (4H, m), 3.22–3.29 (2H, m), 3.49–

3.69 (4H, m), 4.00–4.22 (2H, m), 5.51 (1H, s), 7.29 (1H, t,

J = 5.5 Hz), 8.46 (1H, d, J = 5.5 Hz), 8.93 (1H, s), 9.70 (1H, s); MS

(ESI): 571 [M+H]⁺. Anal. Calcd for C₂₅H₃₃F₃N₆O₄Sꢁ2.0HClꢁ

4.0H₂O: C, 41.96; H, 6.06; N, 11.74. Found: C, 42.12; H, 5.74; N,

11.75.

T. Chonan et al. / Bioorg. Med. Chem. 19 (2011) 1580–1593

1593

5.3.4. Fatty acid oxidation in HepG2 cells

tional Animal Care and Use Committee of Taisho Pharmaceutical

Co., Ltd and were in accordance with the Guidelines for Propre

Conduct of Animal Experiments (Science Council of Japan, 2006).

HepG2 cells were seeded into a 24-well plate at 1.5 ꢂ 10⁵cells/

well in DMEM containing 10% FBS and maintained for 48 h. The

medium was removed and replaced with 20 mM HEPES (pH 7.4)

buffer containing 140 mM NaCl, 5.0 mM KCl, 2.5 mM MgCl₂,

1 mM CaCl₂and 0.2% BSA with or without a test compound.

Acknowledgments

After 1 h, 1.5

l

Ci of [³H] palmitic acid was added to each well. After

The authors thank Yoshiki Fukasawa for collecting DMPK data.

The authors also thank Drs. Nagaaki Sato and Shigeru Tokita for

editing this manuscript.

a 1-h incubation, the medium was transferred to a tube and then

10% TCA was added. After centrifugation, the supernatants were

removed and mixed with 1% (w/v) charcoal in a 96-well ﬁltration

plate. The radioactivity of [³H] H₂O in the eluate was measured

using a MicroBeta (Perkin Elmer).

Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.bmc.2011.01.041.

5.3.5. Expression and puriﬁcation of rhACC1 and rhACC2

N-terminally c-Myc tagged recombinant human ACC1 (1–2346

a.a.) and ACC2 (149–2458 a.a. deleted N-terminal mitochondrial

anchoring domain [1–148 a.a.]) were expressed in baculovirus/

Sf9 systems. The tagged ACCs were puriﬁed using a c-Myc tagged

protein mild puriﬁcation kit (MBL).

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Article Doi

Discovery of novel (4-piperidinyl)-piperazines as potent and orally active acetyl-CoA carboxylase 1/2 non-selective inhibitors: F-Boc and triF-Boc groups are acid-stable bioisosteres for the Boc group

DOI: 10.1016/j.bmc.2011.01.041

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