Discovery of sulfonylalkylamides: A new class of orally active factor Xa inhibitors

Available online at www.sciencedirect.com

Bioorganic & Medicinal Chemistry 16 (2008) 2243–2260

Discovery of sulfonylalkylamides: A new class of orally

active factor Xa inhibitors

Yasuhiro Imaeda,^*Toshio Miyawaki, Hiroki Sakamoto, Fumio Itoh, Noriko Konishi,

Katsuhiko Hiroe, Masaki Kawamura, Toshimasa Tanaka and Keiji Kubo

Pharmaceutical Research Division, Takeda Pharmaceutical Co., Ltd, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532-8686, Japan

Received 12 November 2007; revised 22 November 2007; accepted 27 November 2007

Available online 3 December 2007

Abstract—Factor Xa (FXa) is a trypsin-like serine protease involved in the coagulation cascade and has received great interest as a

potential target for the development of new antithrombotic agents. Most of amidine-type FXa inhibitors reported have been found

to show extremely poor oral bioavailability. Compound 1 is one of the ﬁrst reported non-amidine type FXa inhibitors. To discover

novel and orally active FXa inhibitors, we investigated ﬂexible linear linkers between the 6-chloronaphthalene ring and the 1-(pyri-

din-4-yl)piperidine moiety of 1 and found the orally active sulfonylalkylamide 2f with an FXa IC₅₀of 0.05 lM, comparable with

that of 1. Further modiﬁcation to reduce the CYP3A4 inhibitory activity of 2f resulted in the potent, selective, and orally active

2-methylpyridine analogue 2s (FXa IC₅₀of 0.061 lM), for which the liability of CYP3A4 inhibition was signiﬁcantly weakened

compared to 2f. Compound 2s also showed long lasting anticoagulant activity in cynomolgus monkeys.

1. Introduction

tion, with one molecule of FXa activating many mole-

cules of prothrombin to thrombin,⁴FXa inhibitors are

expected to be more eﬃcacious in interrupting the coag-

ulation cascade than direct thrombin inhibitors. More-

over, it was also shown recently in in vivo studies that

FXa inhibitors may have less bleeding risk than throm-

bin inhibitors.⁵Accordingly, FXa has emerged as an

attractive target enzyme for the development of new

antithrombotic agents.⁶

Abnormal blood clotting is responsible for a number of

thromboembolic diseases, including myocardial infarc-

tion, unstable angina, deep vein thrombosis, pulmonary

embolism, and ischemic stroke. While several anticoag-

ulants, such as warfarin (Coumadin), heparins, hirudin,

hirulog, and argatroban, are currently available for the

treatment and prevention of thrombus formation, some

of these lack oral bioavailability.¹Furthermore, the nor-

mal protocol for these therapies requires careful moni-

toring of clotting times to achieve eﬃcacy and

individual dose titration to minimize excessive bleeding.²

Therefore, there is a clear need to develop improved

anticoagulants which are orally active and have long

half-lives, but which do not cause unpredictable or seri-

ous bleeding complications.

A variety of low-molecular-weight FXa inhibitors have

been described. However, ﬁrst generation FXa inhibi-

tors^7,8(e.g., DX-9065a^8ashown in Fig. 1) incorporated

highly basic groups, such as amidine and/or guanidine

groups,⁸which imparted poor pharmacokinetic proper-

ties after oral administration.^7bTherefore, a range of

potent non-amidine (non-guanidine) type FXa inhibi-

tors have been extensively explored. Compound 1⁹is

one of the ﬁrst reported non-amidine type FXa inhibi-

tors (Fig. 1), and currently some non-amidine type

FXa inhibitors such as rivaroxaban¹⁰and apixaban¹¹

are being investigated in clinical trials.

Factor Xa (FXa), a trypsin-like serine protease convert-

ing prothrombin to thrombin, is located at the central

point linking the intrinsic and the extrinsic coagulation

pathways.³Since this process involves signal ampliﬁca-

A primary objective in our FXa inhibitor program is to

identify novel non-amidine type FXa inhibitors, bearing

new chemical frameworks, with selectivity and pharma-

cokinetic properties suﬃcient to demonstrate antithrom-

botic eﬃcacy in animals, especially in primates. To

Keywords: Factor Xa inhibitor; Anticoagulant; Sulfonylalkylamide;

CYP3A4.

*

Corresponding author. Tel.: +81 6 6300 6458; fax: +81 6 6300

6306; e-mail: Imaeda_Yasuhiro@takeda.co.jp

doi:10.1016/j.bmc.2007.11.073

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Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 2243–2260

Figure 1. Structure of DX-9065a and compound 1.

achieve this objective, we focused our attention on non-

amidine FXa inhibitor 1 as a starting point. Molecular

modeling studies on the binding mode of compound 1

with FXa have already been reported and showed the

following features: (1) the 6-chloronaphthalene moiety

takes the place of the amidinobenzene or the amidino-

naphthalene moieties of amidine type FXa inhibitors

as the S1 binding element in FXa,^7b,12(2) the basic 1-

(pyridin-4-yl)piperidine moiety is located in the S4 si-

te,^7b,12(3) the sulfonyl group is critical for the restricted

L-shaped conformation, which is essential for high aﬃn-

ity to FXa,^7b,11bcausing the aryl ring to turn 90ꢁ into the

S1 site and allowing the sulfonyl group to form a hydro-

gen bond with Ser195 or Gln192 in FXa,^7b(4) the piper-

azine ring adopts a chair conformation^7bplaying an

important role in presenting the S1 and S4 binding ele-

ments in favorable positions, and is not a mere spacer.¹²

In contrast, the molecular conformation of compound 1

appears to be highly rigid because the chemical structure

has four rings connected linearly by a direct bond or

one-atom spacer. The rigid conformation of 1 might

be expected to inﬂuence its pharmacokinetic and physi-

cochemical properties.¹³Therefore, we applied the fol-

lowing design strategy to generate new lead

compounds based on information gained from these

molecular modeling studies. We focused our synthetic

eﬀorts on the possibility of replacing the piperazine ring

of 1 with a ﬂexible linear linker and the sulfonamide

bond with the sulfonylmethylene bond, hoping to pro-

vide more desirable properties for an orally available

drug (Fig. 2). In this paper, we describe the synthesis,

structure–activity relationships, ex vivo anticoagulant

activities, selectivity, and pharmacokinetics of sul-

fonylalkylamides

inhibitors.¹⁴

2

as novel non-amidine FXa

2. Chemistry

The synthesis of sulfonylalkylamides 2a–d is depicted in

Scheme 1. Preparation of 4 was performed by diazotiza-

tion of 6-amino-2-naphthalenesulfonic acid (3) with 6 M

HCl and sodium nitrite followed by Sandmeyer reaction

to introduce a chlorine atom and sulfonylchlorination

with phosphorus pentachloride. Reduction of 4 with

lithium aluminum hydride (LAH) gave thiol 5. Alkyl-

ation of thiol 5 with N-Boc-bromoalkylamines 6a–c¹⁵

followed by oxidation with m-chloroperbenzoic acid

(mCPBA) aﬀorded alkylsulfones 7a–c. The resulting

compounds 7a–c were methylated with iodomethane to

yield corresponding N-methylcarbamates 8a–c. Depro-

tection of N-Boc-alkylamines 7a and 8a–c with triﬂuoro-

acetic acid (TFA) followed by condensation with

carboxylic acid 9¹⁶via the corresponding acyl chloride

aﬀorded the desired amides 2a–d.

Sulfonylalkylamides 2e–h were synthesized as shown in

Scheme 2. The required carboxylic acids 10a and 10b

were prepared from 5 by alkylation with ethyl 4-bromo-

butanoate or ethyl bromoacetate in the presence of so-

dium ethoxide followed by oxidation with mCPBA

and alkali hydrolysis. Michael addition of thiol 5 to

methyl acrylate, oxidation with mCPBA, and sequential

acid hydrolysis of the ester group aﬀorded 3-sulfonyl-

propionic acid 10c. The requisite amine 13 was synthe-

sized by reductive amination of ketone 12¹⁷with

methylamine. Amine 13 or 14 was condensed with

carboxylic acids 10a–c using 1-[3-(dimethylamino)pro-

pyl]-3-ethylcarbodiimide hydrochloride (WSC) and

1-hydroxybenzotriazole hydrate (HOBt) to aﬀord the

desired amides 2e–h.

The synthesis of sulfonylalkylamides 2i–n is shown in

Scheme 3. Alkylation of thiol 5 with 3-bromopropanol

followed by oxidation with mCPBA aﬀorded alcohol

15. Amines 16a–c were sulfonylated with 2,4-dini-

trobenzenesulfonyl chloride to sulfonamides 17a–c,

which were alkylated with alcohol 15 by the Mitsunobu

reaction¹⁸and then desulfonylation with propylamine to

give amines 18a–c.¹⁸Condensation of amines 18a–c with

carboxylic acid 9 via the corresponding acyl chloride

aﬀorded the desired amides 2i–k. Ester 2k was hydro-

lyzed to carboxylic acid 2l, which was condensed with

ammonia or morpholine to aﬀord amides 2m and 2n.

Sulfonylalkylamides 2o–v were synthesized as shown in

Scheme 4. Arylation of 1,4-dioxa-8-azaspiro[4.5]decane

Figure 2. Sulfonylalkylamides 2.

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 2243–2260

2245

Scheme 1. Synthesis of sulfonylakylamides 2a–d. Reagents (a) 6 M HCl, NaNO₂; (b) 6 M HCl, CuCl; (c) PCl₅, (d) LAH; (e) 5, Et₃N; (f) mCPBA; (g)

Mel, NaH; (h) TFA; (i) SOCl₂, then Et₃N.

Scheme 2. Synthesis of sulfonylakylamides 2e–h. Reagents (a) Br(CH₂)₃CO₂Et or BrCH₂CO₂Et, NaOH; (b) mCPBA; (c) NaOH, H₂O; (d) methyl

acrylate, Et₃N; (e) H₂O₂, AcOH; (f) H₂SO₄; (g) MeNH₂-HCl, NaBH₃CN, AcOH; (h) 10a–c, WSC, HOBt; (i) 10c, WSC, HOBt.

with 4-chloropyridines 19a–d^19–21in the presence of tri-

ethylamine in a sealed tube followed by hydrolysis of the

ketal groups with 4 M HCl produced piperidones 20a–d.

Reductive amination of 12 or 20a–d with methylamine,

glycine ethyl ester, ethyl 3-aminopropionate, or tert-bu-

tyl (2-aminoethyl)carbamate aﬀorded the requisite sec-

ondary amines 21a–g. Condensations of carboxylic

acid 10c with amines 21a–g were conducted under sev-

eral conditions. Amine 21a was condensed with carbox-

ylic acid 10c using HOBt and WSC to aﬀord amide 2o in

low yield. Carboxylic acid 10c was converted to acyl

chloride with thionyl chloride, which was coupled with

amine 21b to aﬀord amide 2p in low yield. The coupling

of carboxylic acid 10c and amines 21c–g using 4-(4,6-

dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chlo-

ride (DMTMM)²²as a dehydrating agent gave amides 2q

and 2s–v, respectively, in moderate to high yields. Re-

moval of the Boc group from 2q with TFA provided

amine 2r.

Sulfonylalkylamides 2w–aa were prepared as shown in

Scheme 5. Arylsulfonyl chlorides 22a–e^23–25were reduced

with sodium sulﬁte to give corresponding sodium aryl-

sulﬁnates 23a–e, which were then alkylated with bro-

mosuccinic acid and subsequently decarboxylated by

heat under basic conditions to aﬀord arylsulfonylprop-

ionic acids 24a–e. The coupling of carboxylic acids 24a–

e and amine 21d using DMTMM gave amides 2w–aa.

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Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 2243–2260

Scheme 3. Synthesis of sulfonylalkylamides 2i–n. Reagents (a) 3-bromopropanol, Et₃N; (b) mCPBA; (c) 2,4-dinitrobenzenesulfonyl chloride,

pyridine; (d) DEAD, PPh₃; (e) n-PrNH₂; (f) (1) 9, SOCl₂, (2) 18a–c, i-Pr₂NEt; (g) 2 M NaOH; (h) NH₄OH or morpholine, WSC, HOBt.

3. Results and discussion

from the linker, was found to exhibit comparable FXa

inhibitory activity to that of piperazine 1. Reduction

of the alkylene length in the linker of 2f resulted in de-

creased activity (2g). Piperazinamide 2h aﬀorded slightly

less potent FXa inhibitory activity than 2f. These results

suggest the importance of the distance between the 6-

chloronaphthalene ring and the 1-(pyridin-4-yl)piperi-

dine moiety for these activities. It is interesting that

the orientation of the amide bond aﬀects the optimal

length in the linker (2b and 2f).

The compounds thus synthesized were evaluated for

in vitro inhibitory potency against human FXa, ex-

pressed as IC₅₀values, and their activity in the prolon-

gation of human prothrombin time (PT), expressed as

the concentration of compound required to double the

clotting time (PT₂) in the PT assay. To estimate the oral

bioavailability of these compounds, the ex vivo PT pro-

longing activity was also determined 1 h after oral

administration to mice at a dose of 30 mg/kg and ex-

pressed as ratios of the PT of the compound-treated ani-

mals with that of control group.

To further improve the activities in this series, we inves-

tigated the eﬀect of substitution on the amide nitrogen

atoms in N-(3-sulfonylpropyl)amide 2b and 3-sulfonyl-

propanamide 2f. The results of in vitro FXa inhibition

and PT assays are shown in Table 2. The compounds

bearing alkyl groups larger than methyl (such as an ethyl

(2i) and an isopropyl (2j) group) exhibited slightly re-

duced FXa inhibitory potencies and diminished PT pro-

longing activities. Introduction of polar functionalities

such as ethoxycarbonylalkyl (2k, 2o, and 2p), carboxy-

methyl (2l), carbamoylmethyl (2m and 2n), or amino-

ethyl (2r) provided comparable FXa inhibitory activity

to 2b or 2f.

In vitro FXa inhibitory activities of the compounds hav-

ing varied linear linkers between the 6-chloronaphtha-

lene ring and the 1-(pyridin-4-yl)piperidine moiety are

listed in Table 1. The cleavage of the ethylene bond in

the piperazine ring of 1 and the replacement of the sul-

fonamide bond in 1 with a sulfonylmethylene bond led

to open chain N-(3-sulfonylpropyl)amide 2a, which

was found to be a less potent FXa inhibitor than 1.

Interestingly, N-methylation of the amide functionality

in 2a gave 2b with slightly increased FXa inhibitory

activity compared to 2a. Extension of the propylene

linkage of 2b by one atom, as in N-(3-sulfonylbu-

tyl)amide 2c, led to remarkably decreased activity,

whereas deletion of the methylene group in the linker

of 2b, as in 2d, aﬀorded decreased FXa inhibitory activ-

ity. Further investigation of the linker was performed by

inversion of the amide bond in 2b to lead to the 3-sul-

fonylbutanamide 2e with comparable FXa inhibitory

activity to that of 2b. Surprisingly, 3-sulfonylpropana-

mide 2f, in which the methylene group of 2e was deleted

The ex vivo PT prolonging activities in mice of a se-

lect set of the potent inhibitors were determined and

the results are shown in Table 2. The N-methyl deriv-

atives 2b and 2f signiﬁcantly prolonged mouse PT by

1.3- and 1.6-fold, respectively, whereas the compounds

with polar functionalities exhibited little to undetect-

able amounts of prolongation in the assay (2k, 2l,

2n–p, and 2r). Although each of the compounds had

a diﬀerent in vitro mouse PT prolonging activity, these

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 2243–2260

2247

Scheme 4. Synthesis of sulfonylakylamides 2o–v. Reagents: (a) Et₃N; (b) 4 M HCl; (c) R³NH₂, NaBH₃CN, AcOH; (d) MeNH₂-HCl, NaBH₃CN,

AcOH; (e) 10c, WSC, HOBt; (f) 10c, SOCl₂, (i-Pr)₂NEt; (g) 10c, DMTMM; (h) TFA.

Scheme 5. Synthesis of sulfonylakylamides 2w–aa. Reagents: (a) Na₂SO₃, NaHCO₃; (b) bromosuccinic acid, NaOH; (c) 21d, DMTMM.

results suggested that 2b and 2f would show good oral

bioavailability in mice. The N-methylamide 2f was

found to be the most potent human FXa inhibitor

with good oral bioavailability. Our strategy to dis-

cover novel and orally active FXa inhibitors by

increasing the ﬂexibility and converting the sulfon-

amide linkage to a sulfonyl group did not prove en-

tirely correct (for example 2f vs 2o).

In the course of further evaluation of 2f, it was found

to inhibit the human cytochrome P450 enzyme

CYP3A4 (81% inhibition at 1 lM) which is known

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Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 2243–2260

Table 1. In vitro activites of sulfonylalkylamides 2a–h

Compound

X

Human FXa,

a

IC₅₀(lM)

1

0.033

2a

2b

2c

2d

2e

2f

CH₂CH₂CH₂NHCO

CH₂CH₂CH₂NMeCO

CH₂CH₂CH₂CH₂NMeCO

CH₂CH₂NMeCO

0.47

0.095

0.84

0.14

0.050

0.72

CH₂CH₂CH₂CONMe

CH₂CH₂CONMe

CH₂CONMe

2g

2h

0.16

^aInhibitory activity against human FXa. IC₅₀values shown are the means of duplicate measurement.

Table 2. In vitro and ex vivo activites of sulfonylalkylamides 2b, 2f, 2i–p, and 2r

Compound

A/B

R

In vitro

Ex vivo

Mouse PT, ratio^c

a

b

Human FXa, IC₅₀(lM)

Human PT, PT₂(lM)

Mouse, PT₂(lM)

2b

2i

A

Me

Et

0.095

0.12

0.25

1.7

3.2

8.4

1.9

1.8

2.1

17

1.3

NT^d

11

NT^d

1.0

2j

i-Pr

2k

2l

CH₂CO₂Et

CH₂CO₂H

CH₂CO₂NH₂

0.064

0.054

0.12

14

NT^d

1.0

NT^d

2m

2n

A

0.085

1.6

26

1.0

2f

2o

B

Me

0.050

0.060

0.037

0.095

0.13

1.0

1.1

8.0

10

1.6

1.1

CH₂CO₂Et

CH₂CH₂CO₂Et

CH₂CH₂NH₂

2p

2r

DX-9065a

0.85

0.63

0.69

14

1.0

1.1^e(1.4)^f

8.6

6.8

^aSee corresponding footnote in Table 1.

^bPT (prothrombin time) is deﬁned as the concentration of compound required to double the time to clot formation in the PT assay. PT₂values

shown are means of duplicate measurement.

^cEx vivo mouse PT prolonging activity was determined 1 h after oral administration of the PT of the compound at a dose of 30 mg/kg and expressed

as ratios of the compound-treated mice to that of control group (n = 3–5).

^dNT means ‘not tested’.

^e30 mg/kg po (n = 5).

^f100 mg/kg po (n = 5).

to be a primary factor responsible for the metabolism

of most drugs.²⁶Drugs that show CYP inhibition

inﬂuence the pharmacokinetics of other co-adminis-

tered drugs and can cause undesired eﬀects in

patients.

It might be reasoned that the inhibitory potency toward

CYP3A4 is attributed to coordination of the pyridine

ring to the heme.²⁷We thought that the introduction

of a substituent at the 2- or 6-position of the pyridine

ring might prevent it from coordinating and reduce the

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 2243–2260

Table 3. In vitro and ex vivo activites of sulfonylalkylamides 2f and 2s–v

2249

Compound

R¹

R²

In vitro

CYP3A4,

Ex vivo

Human FXa,

a

IC₅₀(lM)

Human PT,

b

PT₂(lM)

Mouse PT,

b

PT₂(lM)

Mouse PT,

ratio^d

% inhibition at 1 lM^c

2f

2s

2t

2u

2v

H

Me

H

0.05

0.061

0.11

1.0

1.6

1.5

3.6

1.6

81

40

21

49

40

8.0

4.8

6.4

NT^e

4.1

1.6

1.1

NT^e

1.3

H

CH₂OH

NH₂

Me

H

Me

0.079

0.063

^a,bSee corresponding footnotes in Tables 1 and 2.

^cInhibitory activity against human CYP3A4.

^d,eSee corresponding footnotes in Table 2.

CYP3A4 inhibitory potency.²⁸Some 2- and 6-substi-

tuted pyridine analogues such as 2-methyl (2s), 2-

hydroxymethyl (2t), 2-amino (2u), and 2,6-dimethyl

(2v) pyridines were examined and the results are shown

in Table 3. The inhibitory potencies of these compounds

against CYP3A4 were successfully lowered with little ef-

fect on FXa inhibitory potencies and the human PT pro-

longing activities. Among these compounds, 2s was the

most potent and orally active inhibitor with reduced

CYP3A4 inhibition.

Table 4. In vitro activities of sulfonylalkylamides 2w–2aa

Compound

Ar

Human FXa,

a

IC₅₀(lM)

2s

0.061

>5.0

To understand the inﬂuence of the 6-chloronaphtha-

lene moiety on the FXa inhibitory activity in this ser-

ies, we investigated the eﬀect of 6-chloronaphthalene

replacements in compound 2s (Table 4). 7- and 5-

chloronaphthalene analogues 2w and 2x displayed sig-

niﬁcant drops in activity. Replacement of the chlorine

atom with the isosteric methyl group resulted in a

slight loss in activity (2y vs. 2s). Surprisingly, 5-

and 6-chlorobenzothiophene analogues 2z and 2aa

showed remarkably decreased activity, while some po-

tent FXa inhibitors bearing the 5- and 6-chlorobenzo-

thiophene moieties have been reported.^25,29The

diﬀerence in the binding mode between this series

and the other FXa inhibitors in the S1 region might

be responsible for this result. These ﬁndings conﬁrm

the importance of the 6-chloronaphthalene moiety

for the FXa inhibitory activity in our sulfonylalkyla-

mide series.

2w

2x

>5.0

2y

0.72

>5.0

2z

2aa

^aSee corresponding footnotes in Table 1.

Since many trypsin-like proteases have essential physio-

logical functions,³⁰an important consideration for the

development of FXa inhibitors is the ability of the mol-

ecule to bind selectively. In several enzyme inhibition as-

says, compound 2s displayed >200-fold selectivity for

FXa versus several coagulation (thrombin, kallikrein)

and ﬁbrinolytic enzymes (plasmin, tissue plasminogen

activator (t-PA)) (Table 5). Furthermore, 2s showed

no signiﬁcant binding to trypsin.

The pharmacokinetic proﬁle of compound 2s was evalu-

ated in cynomolgus monkeys. As illustrated in Table 6,

2s displayed good oral bioavailability (66.7%) after oral

administration at a dose of 1 mg/kg in fasted monkeys.

Recently, several FXa inhibitors were reported to have

good oral bioavailability in monkeys.^17,31

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Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 2243–2260

Table 5. Selectivity proﬁle for compound 2s

Serine protease

FXa

Thrombin

7.5

Kallikrein

3.0

Trypsin

>200

Plasmin

>200

t-PA

67

K_i(lM)

0.015

Data are expressed as the means of three determinations.

Table 6. In vivo cynomolgus monkey pharmacokinetics of compound 2s (n = 3, fasted)^a

iv dose

1 mg/kg

po dose

1 mg/kg

C_5min(lg/mL)

0.565 0.283

C_max(lg/mL)

0.134 0.032

AUC (lg h/mL)

1.579 0.799

T_max(h)

MRT (h)

V_d(ss)(L/kg)

CL_total(L h/kg)

0.734 0.303

B.A. (%)

4.54 0.32

3.285 1.270

MRT (h)

AUC (lg h/mL)

1.030 0.449

2.00 1.73

6.60 0.97

66.7 4.2

^aValues are expressed as means SD of three determinations.

Table 7. Ex vivo PT prolonging activities of compound 2s in cynomolgus monkeys at 3 mg/kg, po (n = 3, fasted)

Dose

PT (% of basal value)^a

0.5 h

1 h

2 h

4 h

8 h

3 mg/kg

142.0 41.22

133.6 30.48

146.9 18.71

144.6 5.20

117.7 0.64

^aValues are expressed as means SEM of three determinations.

The ex vivo PT prolonging activity of 2s was evaluated

in cynomolgus monkeys, since the in vitro concentration

response of PT in cynomolgus monkey plasma was

found to be similar to that in human plasma (the PT₂

in cynomolgus monkey plasma was 1.1 lM). As shown

in Table 7, 2s displayed potent and long lasting PT pro-

longing activity after oral administration at a dose of

3 mg/kg.

hydrogen bond could not be observed between 2s and

Gly216 and/or Gly219.

4. Conclusion

To discover orally active FXa inhibitors in primates,

increasing the conformational ﬂexibility of the linker be-

tween the 6-chloronaphthalene ring and the 1-(pyridin-

4-yl)piperidine moiety of 1 led to orally active sul-

fonylalkylamides 2b and 2f. Further modiﬁcation to

diminish the interaction with CYP3A4 resulted in 2-

methylpyridine analogue 2s, which is a potent, selective,

and orally bioavailable FXa inhibitor with reduced

CYP3A4 inhibitory activity, which demonstrated a long

duration of anticoagulant activity in cynomolgus mon-

keys. Various other optimization strategies using this

sulfonylalkylamide series will be reported in due course.

To predict the binding mode of these inhibitors, a

molecular modeling study of 2s was carried out using

the program GOLD and the X-ray structure of FXa

complexed with the inhibitor RPR128515, reported by

Sanoﬁ-Aventis³²(Fig. 3). In the model, compound 2s

adopted nicely the L-shape conformation needed for

FXa binding. There is no interaction between the

strongly basic pyridine ring and the carboxyl group of

Asp189 at the bottom of the S1 pocket, while the 6-chlo-

ronaphthyl group is situated in the hydrophobic S1 re-

gion and a close contact is observed between the

chlorine atom and the benzene ring of Tyr228.^10,12,33

Compound 2s represents a parallel example in FXa inhi-

bition wherein a basic group, thought to be essential in

forming the salt bridge with Asp189, can be eliminated

and replaced by a neutral group capable of contributing

enough favorable interaction to produce good aﬃnity to

the enzyme. The pyridine ring is located in the hydro-

phobic S4 site and makes hydrophobic contacts with

the aromatic rings of Tyr99, Phe174, and Trp215. The

sulfone oxygen is involved in a hydrogen bond with

the Gln192 main chain nitrogen. The linker in 2s seems

to control the conformation to ﬁt the FXa binding sites

without direct interaction with FXa. Recently, other

researchers have reported that the important interaction

for high aﬃnity to FXa is not an ionic interaction with

Asp189, but hydrogen bonding interactions with the

carbonyl groups in the amide linkages of Gly216 and/

or Gly219.^12,32,33c,34It is interesting to note that a

5. Experimental

5.1. Chemistry

Melting points were determined with a Yanagimoto

melting point apparatus and are uncorrected. ¹H

NMR spectra were recorded on a Varian Gemini-

200 spectrometer. Chemical shifts are given in d values

(ppm) using tetramethylsilane as the internal standard.

Reactions were followed by TLC on Silica gel 60 F

254 precoated TLC plates (E. Merck) or NH TLC

plates (Fuji Silysia Chemical Ltd.). Chromatographic

separations were carried out on silica gel 60 (0.063–

0.200 or 0.040–0.063 mm, E. Merck) or basic silica

gel (Chromatorex^ꢂNH, 100–200 mesh, Fuji Silysia

Chemical Ltd.) using the indicated eluents. Com-

pounds 3, 6a, 6c, 14, 16a–c, 19a and solvents were

commercially available and used as received. Yields

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 2243–2260

2251

at room temperature for 10 min and then reﬂuxed for

3 h. The reaction mixture was cooled to 0 ꢁC and cau-

tiously quenched with EtOAc (50 mL) and 5.5 M HCl

(400 mL). The organic layer was separated and dried

over anhydrous Na₂SO₄. The solvent was evaporated

in vacuo and the residue was puriﬁed by silica gel chro-

matography (EtOAc) to give 5 (38.5 g, 86%) as a beige

1

powder. H NMR (CDCl₃) d: 3.60 (1H, s), 7.34–7.48

(2H, m), 7.61 (1H, s), 7.66 (1H, s), 7.68–7.81 (2H, m).

5.1.3. tert-Butyl{3-[(6-chloronaphthalen-2-yl)sulfonyl]pro-

pyl}carbamate (7a). To a solution of 5 (0.73 g, 3.75

mmol) and Et₃N (1.05 mL, 7.50 mmol) in DMF

(20 mL) was added 6a (0.85 g, 3.57 mmol). The resulting

mixture was stirred at 100 ꢁC for 5 h under argon atmo-

sphere and then cooled to room temperature. The reac-

tion mixture was poured into water (50 mL) and stirred

at room temperature overnight. The pale yellow precip-

itate was collected by ﬁltration. The precipitate was par-

titioned between brine and EtOAc, and ﬁltered through

a Celite pad. The organic layer was separated and dried

over anhydrous MgSO₄. The solvent was evaporated

and the residue was suspended in EtOAc (50 mL).

mCPBA (70%, 2.19 g, 8.90 mmol) was added to the

solution and the resulting mixture was stirred at room

temperature overnight. The reaction mixture was

washed with saturated aqueous NaHCO₃solution and

brine, and dried over anhydrous Na₂SO₄. The solvent

was evaporated in vacuo and the residue was crystallized

from Et₂O to give 7a (0.80 g, 58%) as colorless crystals.

¹H NMR (CDCl₃) d: 1.40 (9H, s), 1.85–2.05 (2H, m),

3.15–3.35 (4H, m), 4.68 (1H, br s), 7.59 (1H, dd,

J = 2.2 and 8.4 Hz), 7.85–8.00 (4H, m), 8.46 (1H, s).

Figure 3. Binding model of compound 2s in FXa.

are unoptimized. Chemical intermediates were charac-

terized by H NMR.

1

5.1.1. 6-Chloro-2-naphthalenesulfonyl chloride (4). To a

suspension of 3 (44.6 g, 200 mmol) in water (200 mL)

was added Na₂CO₃(11.1 g, 10.4 mmol) portionwise un-

der reﬂux to give a clear solution. The solution was

cooled in an ice-bath to form a ﬁne precipitate. Concen-

trated HCl (43 mL) was slowly added followed by drop-

wise addition of

a solution of NaNO₂(16.6 g,

240 mmol) in water (100 mL) at 65 ꢁC over 30 min.

The reaction mixture was stirred at 0–5 ꢁC for a further

1 h and the pale orange precipitate was collected by ﬁl-

tration. The diazonium salt was added to an ice-cooled

solution of CuCl (23.8 g, 240 mmol) in 28% HCl

(100 mL). The resulting mixture was stirred at room

temperature for 1 h and then at 60 ꢁC for 30 min. After

the reaction mixture was cooled to 0 ꢁC, it was adjusted

to pH 1–2 with 50% aqueous KOH. The precipitate was

collected by ﬁltration and the solid was suspended in hot

water (200 mL). The mixture was basiﬁed with 50%

aqueous KOH. The insoluble material was collected by

ﬁltration and dried to give a solid (16.8 g). The ﬁltrate

was concentrated in vacuo to give a solid (19.7 g).

The following compounds 7b and 7c were prepared in a

manner similar to that described for 7a.

5.1.4. tert-Butyl{4-[(6-chloronaphthalen-2-yl)sulfonyl]butyl}-

carbamate (7b). ¹H NMR (CDCl₃) d: 1.39 (9H, s), 1.48–

1.90 (4H, m), 3.00–3.28 (4H, m), 4.54 (1H, br s), 7.59

(1H, dd, J = 1.8 and 8.8 Hz), 7.82–8.00 (4H, m), 8.46

(1H, s).

5.1.5. tert-Butyl{2-[(6-chloronaphthalen-2-yl)sulfonyl]ethyl}-

carbamate (7c). H NMR (CDCl₃) d: 1.37 (9H, s), 3.39

(2H, t, J = 5.8 Hz), 3.57 (2H, t, J = 5.8 Hz), 5.19 (1H,

br s), 7.60 (1H, dd, J = 1.9 and 8.9 Hz), 7.88–7.97 (4H,

m), 8.47 (1H, d, J = 1.2 Hz).

1

The second solid (19.7 g) was heated to 100–110 ꢁC for

1.5 h with PCl₅(40.3 g). The reaction mixture was

cooled to room temperature and ice (19.7 g) was added

portionwise. The precipitate was collected by ﬁltration

and puriﬁed by silica gel chromatography (CHCl₃/hex-

ane = 1/3) to give 4 (3.07 g, 6%) as a light-brown

powder.

5.1.6. tert-Butyl{3-[(6-chloronaphthalen-2-yl)sulfonyl]pro-

pyl}methylcarbamate (8a). To an ice-cooled solution of

7a (4.91 g, 12.8 mmol) in THF (50 mL) was added

NaH (60% in oil; 0.56 g, 14.0 mmol). After the mixture

was stirred at room temperature for 2 h, iodomethane

(4.00 mL, 64.2 mmol) was added. The resulting mixture

was stirred at room temperature for 36 h and then con-

centrated in vacuo. The residue was diluted with water.

The precipitate was collected by ﬁltration, dried, and

recrystallized from EtOAc–hexane to give 8a (4.79 g,

The ﬁrst solid (16.8 g) was heated to 100–110 ꢁC for 4 h

with PCl₅(33.8 g). The reaction mixture was cooled to

room temperature and ice was added portionwise. The

precipitate was collected by ﬁltration and puriﬁed by sil-

ica gel chromatography (CHCl₃/hexane = 1/3) to give 4

(9.05 g, 17%). ¹H NMR (CDCl₃) d: 7.66 (1H, dd,

J = 2.1 and 8.7 Hz), 7.97–8.07 (4H, m), 8.59 (1H, s).

1

94%) as colorless crystals. H NMR (CDCl₃) d: 1.39

5.1.2. 6-Chloro-2-naphthalenethiol (5). To a solution of 4

(60.0 g, 230 mmol) in THF (700 mL) was added LiAlH₄

(17.4 g, 460 mmol) and the resulting mixture was stirred

(9H, s), 1.90–2.04 (2H, m), 2.80 (3H, s), 3.12–3.20

(2H, m), 3.31 (2H, t, J = 6.7 Hz), 7.60 (1H, dd, J = 1.8

and 9.0 Hz), 7.86–7.97 (4H, m), 8.46 (1H, s).

2252

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 2243–2260

The following compounds 8b and 8c were prepared in a

manner similar to that described for 8a.

5.1.12. N-{2-[(6-Chloronaphthalen-2-yl)sulfonyl]ethyl}-N-

methyl-1-(pyridin-4-yl)piperidine-4-carboxamide (2d).

1

Yield 70%, pale yellow amorphous powder. H NMR

(CDCl₃) d: 1.71–1.82 (4H, m), 2.58–2.73 (1H, m),

2.80–2.94 (2H, m), 3.22 (3H, s), 3.47 (2H, t,

J = 6.5 Hz), 3.79 (2H, t, J = 6.5 Hz), 3.88 (2H, m),

6.64 (2H, d, J = 6.5 Hz), 7.59 (1H, dd, J = 2.0 and

8.8 Hz), 7.90–7.97 (4H, m), 8.25 (2H, d, J = 6.5 Hz),

8.46 (1H, s). Anal. Calcd for C₂₄H₂₆N₃O₃SClÆ0.7H₂O:

C, 59.48; H, 5.70; N, 8.67. Found: C, 59.47; H, 5.68;

N, 8.53.

5.1.7. tert-Butyl{4-[(6-chloronaphthalen-2-yl)sulfonyl]butyl}-

methylcarbamate (8b). ¹H NMR (CDCl₃) d: 1.39 (9H, s),

1.45–1.85 (4H, m), 2.78 (3H, s), 3.18 (4H, t, J = 6.6 Hz),

7.59 (1H, dd, J = 1.8 and 8.8 Hz), 7.85–8.00 (4H, m),

8.46 (1H, s).

5.1.8. tert-Butyl{2-[(6-chloronaphthalen-2-yl)sulfonyl]ethyl}-

methylcarbamate (8c). ¹H NMR (CDCl₃) d: 1.35 (9H, s),

2.85 (3H, s), 3.38–3.46 (2H, m), 3.59 (2H, br s), 7.58

(1H, d, J = 8.7 Hz), 7.87–7.95 (4H, m), 8.46 (1H, s).

5.1.13. 4-[(6-Chloronaphthalen-2-yl)sulfonyl]butanoic acid

(10a). To a solution of 5 (0.97 g, 4.98 mmol) and sodium

ethoxide (0.48 g, 7.05 mmol) in EtOH (20 mL) was

added ethyl 4-bromobutyrate (1.07 g, 5.49 mmol). The

resulting mixture was stirred at 60 ꢁC for 1 h and then

concentrated in vacuo. The residue was partitioned be-

tween water and EtOAc. The organic layer was sepa-

rated, washed with water, dried over anhydrous

MgSO₄, and concentrated in vacuo. The residual solid

was dissolved in EtOAc (20 mL) and a solution of

mCPBA (2.38 g, 13.8 mmol) in EtOAc (20 mL) was

added dropwise. The resulting mixture was stirred at

room temperature for 1.5 h. The reaction mixture was

washed with saturated aqueous NaHCO₃solution and

brine, dried over anhydrous MgSO₄, and concentrated

in vacuo. The residual oil was dissolved in 1 M NaOH

(10 mL) and MeOH (40 mL). The resulting mixture

was stirred at room temperature for 1.5 h and then con-

centrated in vacuo. The residue was diluted with water

and acidiﬁed with 1 M HCl. The precipitate was col-

lected by ﬁltration to give 10a (1.21 g, 76%) as a color-

less needle. ¹H NMR (CDCl₃) d: 1.97–2.15 (2H, m),

2.55 (2H, t, J = 7.0 Hz), 3.27 (2H, t, J = 7.6 Hz), 7.60

(1H, dd, J = 8.7 and 1.7 Hz), 7.87–7.98 (4H, m), 8.48

(1H, s).

5.1.9. N-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]propyl}-

N-methyl-1-(pyridin-4-yl)piperidine-4-carboxamide (2b).

A mixture of 8a (0.33 g, 0.83 mmol) in toluene (2 mL)

and TFA (2 mL) was stirred at room temperature for

30 min. The solvent was evaporated in vacuo and the

residue was diluted in CH₂Cl₂(20 mL). A mixture of

9¹⁶(0.35 g, 1.70 mmol) and thionyl chloride (2 mL)

was reﬂuxed for 30 min and then concentrated in vacuo.

The residue and triethylamine (4 mL) were added to the

solution and the resulting mixture was stirred at room

temperature for 15 h. The reaction mixture was concen-

trated in vacuo and the residue was partitioned between

EtOAc and water. The organic layer was separated,

washed with water, aqueous NaHCO₃, and brine, dried

over anhydrous Na₂SO₄, and concentrated in vacuo.

The residue was puriﬁed by basic silica gel chromatogra-

phy (EtOAc to 20/1 EtOAc/MeOH) to give 2b (60 mg,

14%) as a colorless amorphous powder. ¹H NMR

(CDCl₃) d: 1.70–2.20 (5H, m), 2.60–3.00 (4H, m), 3.09

(3H, s), 3.09–3.22 (2H, m), 3.40–3.65 (2H, m), 3.80–

4.00 (2H, m), 6.65 (2H, d, J = 6.6 Hz), 7.55–7.68 (1H,

m), 7.82–8.00 (4H, m), 8.24 (2H, d, J = 6.6 Hz), 8.46

(1H, s). Anal. Calcd for C₂₅H₂₈ClN₃O₃SÆ0.1H₂O: C,

61.55; H, 5.83; N, 8.61. Found: C, 61.29; H, 5.85; N,

8.54.

5.1.14. [(6-Chloronaphthalen-2-yl)sulfonyl]acetic acid

(10b). Compound 10b was prepared in a manner similar

1

to that described for 10a. H NMR (CDCl₃+ DMSO-

The following compounds 2a, 2c, and 2d were prepared

in a manner similar to that described for 2b.

d₆) d: 4.20 (2H, s), 7.59 (1H, dd, J = 2.2 and 8.8 Hz),

7.90–8.05 (4H, m), 8.54 (1H, s).

5.1.10. N-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]propyl}-

1-(pyridin-4-yl)piperidine-4-carboxamide (2a). Yield

85%, colorless powder. Mp 202–205 ꢁC. ¹H NMR

(CDCl₃) d: 1.28–1.78 (5H, m), 2.15–2.40 (1H, m),

2.68–2.90 (2H, m), 2.98–3.18 (2H, m), 3.20–3.48 (3H,

m), 3.78–3.98 (2H, m), 6.76 (2H, d, J = 6.6 Hz), 7.70–

8.00 (3H, m), 8.05–8.35 (5H, m), 8.62 (1H, s). Anal.

Calcd for C₂₄H₂₆ClN₃O₃SÆ0.4H₂O: C, 60.15; H, 5.64;

N, 8.77. Found: C, 60.11; H, 5.43; N, 8.57.

5.1.15. Methyl 3-[(6-chloronaphthalen-2-yl)thio]propano-

ate (11). A mixture of 5 (6.30 g, 32.4 mmol), methyl

acrylate (2.90 g, 33.7 mmol), and Et₃N (0.90 mL,

6.46 mmol) in EtOAc (75 mL) was stirred at room tem-

perature for 2.5 h. The solvent was evaporated in vacuo

and the residue was washed with hexane to give 11

1

(8.88 g, 98%) as a colorless solid. H NMR (CDCl₃) d:

2.68 (2H, t, J = 7.4 Hz), 3.27 (2H, t, J = 7.4 Hz), 3.68

(3H, s), 7.39–7.48 (2H, m), 7.65–7.71 (2H, m), 7.74–

7.77 (2H, m).

5.1.11. N-{4-[(6-Chloronaphthalen-2-yl)sulfonyl]butyl}-

N-methyl-1-(pyridin-4-yl)piperidine-4-carboxamide (2c).

Yield 88%, colorless amorphous powder. ¹H NMR

(CDCl₃) d: 1.55–1.90 (8H, m), 2.54–3.00 (3H, m), 3.02

(3H, s), 3.25 (2H, t, J = 7.5 Hz), 3.36 (2H, t,

J = 6.4 Hz), 3.74–3.98 (2H, m), 6.65 (2H, d,

J = 6.6 Hz), 7.58 (1H, dd, J = 2.2 and 8.8 Hz), 7.82–

8.00 (4H, m), 8.25 (2H, d, J = 6.5 Hz), 8.45 (1H, s).

Anal. Calcd for C₂₆H₃₀ClN₃O₃S: C, 62.54; H, 6.05; N,

8.40. Found: C, 62.27; H, 6.05; N, 8.49.

5.1.16.

3-[(6-Chloronaphthalen-2-yl)sulfonyl]propanoic

acid (10c). A mixture of 11 (8.88 g, 31.6 mmol) and

30% H₂O₂(6 mL) in AcOH (60 mL) was reﬂuxed for

30 min. To the reaction mixture was added 98%

H₂SO₄(6 mL) and reﬂuxing was continued for a further

1.5 h. The reaction mixture was diluted with water, and

the precipitate was collected by ﬁltration and dried. The

crude product was puriﬁed by silica gel chromatography

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 2243–2260

2253

(EtOAc). The product was recrystallized from IPE-hex-

ane to give 10c (8.20 g, 87%) as colorless crystals. H

NMR (CDCl₃) d: 2.82 (2H, t, J = 7.5 Hz), 3.25 (2H, t,

J = 7.5 Hz), 7.60 (1H, dd, J = 2.0 and 9.0 Hz), 7.91–

7.97 (4H, m), 8.47 (1H, s).

m), 6.58–6.72 (2H, m), 7.55–7.65 (1H, m), 7.80–8.00

(4H, m), 8.20–8.34 (2H, m), 8.49 (1H, s). Anal. Calcd

for C₂₃H₂₄ClN₃O₃S: C, 60.32; H, 5.28; N, 9.18. Found:

C, 60.17; H, 5.25; N, 9.19.

1

5.1.21. 1-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]propanoyl}-

4-(pyridin-4-yl)piperazine (2h). Yield 48%, colorless

powder. Mp 216–217 ꢁC. H NMR (CDCl₃) d: 2.85–

3.00 (2H, m), 3.27 (2H, t, J = 5.4 Hz), 3.38 (2H, t,

J = 5.4 Hz), 3.50–3.75 (6H, m), 6.64 (2H, d,

J = 6.6 Hz), 7.58 (1H, dd, J = 2.2 and 8.8 Hz), 7.88–

8.00 (4H, m), 8.32 (2H, d, J = 6.6 Hz), 8.48 (1H, s).

Anal. Calcd for C₂₂H₂₂ClN₃O₃SÆ0.1H₂O: C, 59.28; H,

5.02; N, 9.43. Found: C, 59.16; H, 5.00; N, 9.37.

5.1.17. N-Methyl-1-(pyridin-4-yl)piperidin-4-amine (13).

To a solution of 12¹⁷(0.88 g, 4.99 mmol), acetic acid

(0.30 g, 5.00 mmol), and methylamine hydrochloride

(0.37 g, 5.48 mmol) in MeOH (10 mL) was added por-

tionwise NaBH₃CN (0.34 g, 5.41 mmol). The reaction

mixture was stirred at room temperature for 17 h and

then concentrated in vacuo. The residue was partitioned

between aqueous K₂CO₃solution and CH₂Cl₂. The or-

ganic layer was separated, dried over anhydrous

Na₂SO₄, and the solvent was evaporated in vacuo to

give 13 (0.96 g, quant.) as a brown oil. ¹H NMR

(CDCl₃) d: 1.22–1.50 (2H, m), 1.90–2.10 (2H, m), 2.47

(3H, s), 2.51–2.70 (1H, m), 2.84–3.02 (2H, m), 3.75–

3.92 (2H, m), 6.66 (2H, d, J = 6.6 Hz), 8.25 (2H, d,

J = 6.6 Hz).

1

5.1.22. 3-[(6-Chloronaphthalen-2-yl)sulfonyl]propan-1-ol

(15). Compound 15 was prepared in a manner similar

1

to that described for 7a. H NMR (CDCl₃) d: 1.94–

2.10 (2H, m), 3.25–3.38 (2H, m), 3.76 (2H, t,

J = 6.0 Hz), 7.59 (1H, dd, J = 2.2 and 8.8 Hz), 7.80–

8.00 (4H, m), 8.49 (1H, s).

5.1.18. 4-[(6-Chloronaphthalen-2-yl)sulfonyl]-N-methyl-

N-[1-(pyridin-4-yl)piperidin-4-yl]butanamide (2e). To a

solution of 10a (0.16 g, 0.51 mmol) and HOBt (0.08 g,

0.52 mmol) in DMF (5 mL) was added WSC (0.15 g,

0.79 mmol) and the mixture was stirred at room temper-

ature for 1 h. 15 (0.12 g, 0.42 mmol) was added and the

resulting mixture was stirred at room temperature for

14 h. The reaction mixture was concentrated in vacuo,

and the residue was basiﬁed with 1 M NaOH and ex-

tracted with EtOAc. The extract was washed with brine,

dried over anhydrous Na₂SO₄, and concentrated in va-

cuo. The residue was crystallized from Et₂O to give 2e

5.1.23. N-Isopropyl-2,4-dinitrobenzenesulfonamide (17b).

To a solution of 16b (0.36 g, 6.09 mmol) and pyridine

(0.53 mL, 6.55 mmol) in CH₂Cl₂(15 mL) was added

2,4-dinitrobenzenesulfonyl chloride (1.07 g, 4.01 mmol)

and the resulting mixture was stirred at room tempera-

ture for 30 min. The reaction mixture was acidiﬁed to

pH 2 with 1 M HCl, concentrated in vacuo, and parti-

tioned between water and EtOAc. The organic layer

was separated, washed with water and brine, dried over

anhydrous Na₂SO₄, and concentrated in vacuo. The res-

idue was puriﬁed by silica gel chromatography (hexane/

EtOAc = 1/1) and the product was crystallized from

hexane–EtOAc to give 17b (0.46 g, 40%) as colorless

1

(0.22 g, 90%) as colorless crystals. Mp 185–186 ꢁC. H

1

NMR (CDCl₃) d: 1.55–1.90 (4H, m), 2.00–2.25 (2H,

m), 2.45–2.74 (2H, m), 2.79 (3H, s), 2.82–3.02 (2H, m),

3.34 (2H, t, J = 7.2 Hz), 3.88–4.05 (2H, m), 4.52–4.80

(1H, m), 6.65 (2H, d, J = 6.6 Hz), 7.59 (1H, dd, J = 1.8

and 8.8 Hz), 7.80–8.00 (4H, m), 8.26 (2H, d,

J = 6.6 Hz), 8.47 (1H, s). Anal. Calcd for

C₂₅H₂₈ClN₃O₃SÆ0.1H₂O: C, 61.55; H, 5.83; N, 8.61.

Found: C, 61.44; H, 5.70, N, 8.76.

needles. H NMR (CDCl₃) d: 1.19 (6H, d, J = 6.6 Hz),

3.62–3.82 (1H, m), 5.18 (1H, d, J = 7.2 Hz), 8.40 (1H,

d, J = 8.8 Hz), 8.56 (1H, dd, J = 2.2 and 8.8 Hz), 8.68

(1H, d, J = 2.2 Hz).

The following compounds 17a and 17c were prepared in

a manner similar to that described for 17b.

1

5.1.24. N-Ethyl-2,4-dinitrobenzenesulfonamide (17a). H

The following compounds 2f–h were prepared in a man-

ner similar to that described for 2e.

NMR (CDCl₃) d: 1.25 (3H, t, J = 7.0 Hz), 3.20 (2H, q,

J = 7.0 Hz), 8.40 (1H, d, J = 8.8 Hz), 8.56 (1H, dd,

J = 2.2 and 8.8 Hz), 8.68 (1H, d, J = 2.2 Hz).

5.1.19. 3-[(6-Chloronaphthalen-2-yl)sulfonyl]-N-methyl-

N-[1-(pyridin-4-yl)piperidin-4-yl]propanamide (2f). Yield

60%, colorless powder. Mp 175–176 ꢁC. ¹H NMR

(CDCl₃) d: 1.50–1.95 (4H, m), 2.70–3.08 (4H, m), 2.83

(3H, s), 3.58 (2H, t, J = 7.9 Hz), 3.80–4.10 (2H, m),

4.46–4.72 (1H, m), 6.65 (2H, d, J = 6.6 Hz), 7.60 (1H,

dd, J = 1.8 and 8.8 Hz), 7.80–8.00 (4H, m), 8.26 (2H,

d, J = 6.6 Hz), 8.49 (1H, s). Anal. Calcd for

C₂₄H₂₆ClN₃O₃SÆ0.2H₂OÆ0.1Et₂O: C, 60.67; H, 5.75; N,

8.70. Found: C, 60.69; H, 5.72; N, 8.88.

5.1.25. Ethyl N-[(2,4-dinitrophenyl)sulfonyl]glycinate

(17c). ¹H NMR (CDCl₃) d: 1.19 (3H, t, J = 7.2 Hz),

4.07 (2H, q, J = 7.2 Hz), 4.08 (2H, d, J = 5.8 Hz), 6.14

(1H, t, J = 5.8 Hz), 8.31 (1H, d, J = 8.8 Hz), 8.55 (1H,

dd, J = 2.2 and 8.8 Hz), 8.76 (1H, d, J = 2.2 Hz).

5.1.26. 3-[(6-Chloronaphthalen-2-yl)sulfonyl]-N-isopro-

pylpropan-1-amine (18b). To a solution of 17b (0.29 g,

1.00 mmol), 15 (0.28 g, 1.00 mmol), and triphenylphos-

phine (0.32 g, 1.22 mmol) in THF (5 mL) was added

diethyl azodicarboxylate (DEAD; 40% toluene solution;

0.30 mL, 1.22 mmol) and the resulting mixture was stir-

red at room temperature for 5 h. The reaction mixture

was concentrated in vacuo and the residue was acidiﬁed

to pH 2 with 1 M HCl and extracted with EtOAc. The

5.1.20. 2-[(6-Chloronaphthalen-2-yl)sulfonyl]-N-methyl-

N-[1-(pyridin-4-yl)piperidin-4-yl]acetamide (2g). Yield

45%, colorless powder. Mp 189–190 ꢁC. ¹H NMR

(CDCl₃) d: 1.23–2.00 (8H, m), 2.70–3.17 (4H, m),

3.50–3.65 (2H, m), 3.70–4.10 (4H, m), 4.13–4.18 (2H,

2254

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 2243–2260

organic extract was washed with water and brine, dried

over anhydrous Na₂SO₄, and concentrated in vacuo.

The residue obtained was dissolved in EtOAc (50 mL)

and mCPBA (70%, 2.95 g, 12.0 mmol) was added to

the solution and stirred at room temperature for 1 h.

The reaction mixture was washed with aqueous NaH-

CO₃solution and brine, dried over anhydrous Na₂SO₄,

and concentrated in vacuo. The residue was puriﬁed by

silica gel chromatography (hexane/EtOAc = 2/1) and the

product was crystallized from EtOAc–Et₂O. The prod-

uct was dissolved in CH₂Cl₂(10 mL) and isopropyl-

amine (1 mL) was added. The resulting mixture was

stirred at room temperature for 30 min. The reaction

mixture was concentrated in vacuo and the residue

was partitioned between water and EtOAc. The organic

layer was separated, washed with aqueous NaHCO₃

solution and brine, dried over anhydrous Na₂SO₄, and

concentrated in vacuo. The residue was puriﬁed by basic

silica gel chromatography (EtOAc to 30/1 EtOAc/

m), 4.00–4.20 (1H, m), 6.60–6.70 (2H, m), 7.56 (1H,

dd, J = 1.8 and 8.8 Hz), 7.80–8.00 (4H, m), 8.20–8.30

(2H, m), 8.47 (1H, s). Anal. Calcd for C₂₇H₃₂ClN₃O_3-

SÆ0.5H₂O: C, 62.00; H, 6.36; N, 8.03. Found: C, 62.29;

H, 6.34; N, 8.20.

5.1.31. Ethyl N-{3-[(6-chloronaphthalen-2-yl)sulfonyl]pro-

pyl}-N-{[1-(pyridin-4-yl)piperidin-4-yl]carbonyl}glycinate

(2k). Compound 2k was prepared in a manner similar to

that described for 2b in 61% yield as a colorless powder.

Mp 169–171 ꢁC. ¹H NMR (CDCl₃) d: 1.23 (1.5H, t,

J = 7.2 Hz), 1.29 (1.5H, t, J = 7.2 Hz), 1.65–2.15 (6H,

m), 2.35–2.60 (0.5H, m), 2.70–3.02 (2.5H, m), 3.15–

3.32 (2H, m), 3.54 (1H, t, J = 6.6 Hz), 3.67 (1H, t,

J = 7.4 Hz), 3.80–3.96 (2H, m), 3.99 (1H, s), 4.10 (1H,

s), 4.12 (1H, q, J = 7.2 Hz), 4.21 (1H, q, J = 7.2 Hz),

6.64 (2H, d, J = 4.8 Hz), 7.55–7.68 (1H, m), 7.82–8.00

(4H, m), 8.25 (2H, d, J = 5.8 Hz), 8.45 (0.5H, s), 8.47

(0.5H, s). Anal. Calcd for C₂₈H₃₂ClN₃O₅S: C, 60.26;

H, 5.78; N, 7.53. Found: C, 60.28; H, 6.05; N, 7.63.

1

MeOH) to give 18b (0.16 g, 55%) as a colorless oil. H

NMR (CDCl₃) d: 1.08 (6H, d, J = 6.2 Hz), 1.92–2.12

(2H, m), 2.75–3.00 (1H, m), 2.80 (2H, t, J = 7.0 Hz),

3.25–3.38 (2H, m), 7.57 (1H, dd, J = 2.0 and 8.8 Hz),

7.90–8.00 (4H, m), 8.48 (1H, s).

5.1.32. N-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]propyl}-

N-{[1-(pyridin-4-yl)piperidin-4-yl]carbonyl}glycine (2l).

A solution of 2k (0.44 g, 0.78 mmol) in 2 M NaOH

(0.80 mL, 1.60 mmol) and MeOH (50 mL) was stirred

at room temperature for 2 h. The reaction mixture was

concentrated in vacuo and the residue was puriﬁed by

CHP-20 chromatography (water to 1/9 MeOH/water)

to give 2l (0.40 g, 92%) as a colorless amorphous pow-

The following compounds 18a and 18c were prepared in

a manner similar to that described for 18b.

5.1.27. 3-[(6-Chloronaphthalen-2-yl)sulfonyl]-N-ethylpro-

1

pan-1-amine (18a). H NMR (CDCl₃) d: 1.04 (3H, t,

der. H NMR (CD₃OD) d: 1.50–2.10 (6H, m), 2.75–

J = 7.1 Hz), 1.80–2.00 (2H, m), 2.57 (2H, q,

J = 7.1 Hz), 2.69 (2H, t, J = 7.0 Hz), 3.20–3.35 (2H,

m), 7.58 (1H, dd, J = 1.8 and 8.8 Hz), 7.55–8.00 (4H,

m), 8.46 (1H, s).

3.75 (7H, m), 3.91 (2H, s), 4.10–4.30 (2H, m), 7.09

(2H, d, J = 7.8 Hz), 7.65 (1H, dd, J = 1.8 and 8.8 Hz),

7.90–8.20 (6H, m), 8.54 (1H, s). Anal. Calcd for

C₂₆H₂₈ClN₃O₅SÆH₂O: C, 56.98; H, 5.52; N, 7.67.

Found: C, 57.21; H, 5.29; N, 7.84.

5.1.28.

Ethyl

N-{3-[(6-chloronaphthalen-2-yl)sulfo-

nyl]propyl}glycinate (18c). ¹H NMR (CDCl₃) d: 1.25

(3H, t, J = 7.0 Hz), 1.80–2.00 (2H, m), 2.71 (2H, t,

J = 6.6 Hz), 3.20–3.35 (2H, m), 3.31 (2H, s), 4.15 (2H,

q, J = 7.0 Hz), 7.59 (1H, dd, J = 2.0 and 8.8 Hz), 7.90–

8.00 (4H, m), 8.47 (1H, s).

5.1.33. N-{3-[(2-Amino-2-oxoethyl)sulfonyl]propyl}-N-(6-

chloronaphthalen-2-yl)-1-(pyridin-4-yl)piperidine-4-carbox-

amide (2m). To a solution of 2l (0.09 g, 0.16 mmol) and

HOBt (0.03 g, 0.16 mmol) in DMF (3 mL) was added

WSC (0.05 g, 0.25 mmol) and the mixture was stirred

at room temperature for 1 h. Ammonia solution (25%,

0.1 mL) was added and the resulting mixture was stirred

at room temperature for 18 h. The reaction mixture was

concentrated in vacuo and the residue was partitioned

between water and EtOAc. The organic layer was sepa-

rated, washed with brine, dried over anhydrous Na₂SO₄,

and concentrated in vacuo. The residue was crystallized

from DMF–EtOAc to give 2m (0.05 g, 57%) as colorless

5.1.29. N-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]propyl}-

N-ethyl-1-(pyridin-4-yl)piperidine-4-carboxamide

(2i).

Compound 2i was prepared in a manner similar to that

described for 2b in 92% yield as a colorless amorphous

powder. ¹H NMR (CDCl₃) d: 1.07 (0.75H, t,

J = 7.2 Hz), 1.22 (2.25H, t, J = 7.2 Hz), 1.60–2.20 (6H,

m), 2.55–2.78 (1H, m), 2.78–3.00 (2H, m), 3.10–3.26

(2H, m), 3.26–3.60 (4H, m), 3.78–4.00 (2H, m), 6.64

(2H, d, J = 6.6 Hz), 7.55–7.70 (1H, m), 7.80–8.10 (4H,

m), 8.24 (2H, d, J = 6.6 Hz), 8.46 (1H, s). Anal. Calcd

for C₂₆H₃₀ClN₃O₃SÆH₂O: C, 60.28; H, 6.23; N, 8.11.

Found: C, 60.41; H, 6.16; N, 8.18.

1

crystals. Mp 222–225 ꢁC. H NMR (DMSO-d₆+ D₂O)

d: 1.35–2.00 (6H, m), 2.50–3.00 (3H, m), 3.20–4.00

(6H, m), 3.78 (1H, s), 4.00 (1H, s), 6.70 (1H, d,

J = 6.0 Hz), 6.76 (1H, d, J = 6.0 Hz), 7.69 (1H, dd,

J = 2.0 and 8.8 Hz), 7.88–8.02 (1H, m), 8.05–8.40 (5H,

m), 8.57 (0.5H, s), 8.60 (0.5H, s). Anal. Calcd for

C₂₆H₂₉ClN₄O₄SÆ0.5H₂O: C, 58.04; H, 5.62; N, 10.41.

Found: C, 58.32; H, 5.59; N, 10.23.

5.1.30. N-{3-[(6-Chloronaphthalen-2-yl)sulfonyl]propyl}-

N-isopropyl-1-(pyridin-4-yl)piperidine-4-carboxamide (2j).

Compound 2j was prepared in a manner similar to that

described for 2b in 54% yield as a colorless amorphous

powder. ¹H NMR (CDCl₃) d: 1.12 (1.2H, d,

J = 6.6 Hz), 1.25 (4.8 H, d, J = 6.6 Hz), 1.60–2.20 (6H,

m), 2.60–2.80 (1H, m), 2.80–3.00 (2H, m), 3.21 (2H, t,

J = 7.5 Hz), 3.35 (2H, t, J = 7.7 Hz), 3.80–4.00 (2H,

5.1.34. N-(6-Chloronaphthalen-2-yl)-N-{3-[(2-morpholin-

4-yl-2-oxoethyl)sulfonyl]propyl}-1-(pyridin-4-yl)piperidine-

4-carboxamide (2n). Compound 2n was prepared in a

manner similar to that described for 2m in 79% yield

as a colorless powder. Mp 240–241 ꢁC. ¹H NMR

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 2243–2260

2255

(DMSO-d₆+ DCl) d: 1.35–2.00 (6H, m), 2.55–4.30

(19H, m), 7.13 (2H, d, J = 6.4 Hz), 7.72 (1H, dd,

J = 2.2 and 8.8 Hz), 7.95 (1H, dt, J = 1.8 and 9.6 Hz),

8.10–8.36 (5H, m), 8.61 (1H, s). Anal. Calcd for

C₃₀H₃₅ClN₄O₅S: C, 60.14; H, 5.89; N, 9.35. Found: C,

59.86; H, 5.88; N, 9.13.

5.1.41.

yl]amino}ethyl)carbamate (21c). Compound 21c was pre-

tert-Butyl

(2-{[1-(pyridin-4-yl)piperidin-4-

1

pared in a manner similar to that described for 13. H

NMR (CDCl₃) d: 1.25–1.50 (2H, m), 1.45 (9H, s),

1.90–2.05 (2H, m), 2.65–3.05 (5H, m), 3.15–3.30 (2H,

m), 3.75–3.92 (2H, m), 4.91 (1H, br s), 6.60–6.72 (2H,

m), 8.20–8.28 (2H, m).

5.1.35. 1-(2-Methylpyridin-4-yl)piperidin-4-one (20a). A

mixture of 19a (15.1 g, 118 mmol), 1,4-dioxa-8-azaspi-

ro[4.5]decane (14.0 g, 97.8 mmol), and Et₃N (30.0 g,

296 mmol) in EtOH (150 mL) was heated at 150 ꢁC for

13 h in a sealed tube. The reaction mixture was cooled

to room temperature and concentrated in vacuo. The res-

idue was basiﬁed with 4.5 M NaOH and K₂CO₃, and ex-

tracted with EtOAc (3 · 100 mL). The combined organic

layer was washed with brine, dried over anhydrous

Na₂SO₄, and concentrated in vacuo. The residue was

washed with hexane and the residual solid was dissolved

in acetone (30 mL). To this was added 4 M HCl

(52.8 mL, 211 mmol) at 50 ꢁC and the resulting mixture

was stirred at 50 ꢁC for 5 h. The reaction mixture was

concentrated in vacuo. The residue was diluted with

1 M NaOH, basiﬁed to pH 11 with K₂CO₃, and ex-

tracted with CH₂Cl₂(3 · 100 mL). The extract was dried

over anhydrous Na₂SO₄and concentrated in vacuo to

5.1.42. N-Methyl-1-(2-methylpyridin-4-yl)piperidin-4-

amine (21d). Compound 21d was prepared in a manner

similar to that described for 13. H NMR (CDCl₃) d:

1.30–1.48 (2H, m), 1.92–2.02 (2H, m), 2.44 (3H, s),

2.47 (3H, s), 2.54–2.70 (1H, m), 2.84–2.98 (2H, m),

6.49–6.54 (2H, m), 8.14 (1H, d, J = 6.0 Hz).

1

5.1.43.

{4-[4-(Methylamino)piperidin-1-yl]pyridin-2-

yl}methanol (21e). Compound 21e was prepared in a

manner similar to that described for 13. ¹H NMR

(CDCl₃) d: 1.36 (2H, m), 1.98 (2H, m), 2.47 (3H, s),

2.61 (1H, m), 2.94 (2H, m), 3.86 (2H, m), 4.63 (2H, s),

6.60 (2H, m), 8.19 (1H, d, J = 6.0 Hz).

5.1.44. 4-[4-(Methylamino)piperidin-1-yl]pyridin-2-amine

(21f). Compound 21f was prepared in a manner similar

to that described for 13. ¹H NMR (CDCl₃) d: 1.34 (2H,

m), 1.95 (2H, m), 2.46 (3H, s), 2.58 (1H, m), 2.87 (2H,

m), 3.76 (2H, m), 4.19 (2H, br s), 4.63 (2H,s), 5.87

(1H, d, J = 2.4 Hz), 6.20 (1H, dd, J = 2.4 and 6.2 Hz),

7.80 (1H, d, J = 6.2 Hz).

1

give 20a (9.45 g, 51%) as a yellow oily solid. H NMR

(CDCl₃) d: 2.49 (3H, s), 2.56 (4H, t, J = 6.3 Hz), 3.74

(4H, t, J = 6.3 Hz), 6.54–6.61 (2H, m), 8.23 (1H, d,

J = 5.8 Hz).

The following compounds 20b–d were prepared in a

manner similar to that described for 20a.

5.1.45. 1-(2,6-Dimethylpyridin-4-yl)-N-methylpiperidin-4-

amine (21g). Compound 21g was prepared in a manner

similar to that described for 13. H NMR (CDCl₃) d:

1.40 (2H, m), 2.00 (2H, m), 2.47 (9H, s), 2.69 (1H, m),

2.98–3.12 (4H, m), 3.88 (1H, m), 6.43 (2H, s).

1

5.1.36. 1-[2-(Hydroxymethyl)pyridin-4-yl]piperidin-4-one

(20b). ¹H NMR (CDCl₃) d: 2.57 (4H, t, J = 6.1 Hz), 3.76

(4H, t, J = 6.3 Hz), 4.68 (2H, s), 6.62–6.67 (2H, m), 8.27

(1H, d, J = 5.4 Hz).

5.1.46. Ethyl N-{3-[(6-chloronaphthalen-2-yl)sulfonyl]pro-

panoyl}-N-[1-(pyridin-4-yl)piperidin-4-yl]glycinate (2o).

Compound 2o was prepared in a manner similar to that

described for 2e in 9% yield as a colorless amorphous

1

5.1.37. 1-(2-Aminopyridin-4-yl)piperidin-4-one (20c). H

NMR (CDCl₃) d: 2.53 (4H, t, J = 6.0 Hz), 3.68 (4H, t,

J = 6.0 Hz), 5.91 (1H, d, J = 2.2 Hz), 6.23 (1H, dd,

J = 2.2 and 6.2 Hz), 7.87 (1H, d, J = 6.2 Hz).

1

powder. H NMR (CDCl₃) d: 3.91 (3H, s), 7.79 (1H,

d, J = 8.8 Hz), 8.13–8.17 (3H, m), 8.34 (2H, d,

J = 8.4 Hz), 8.50 (1H, s). Anal. Calcd for

C₂₇H₃₀ClN₃O₅SÆ0.55H₂O: C, 58.54; H, 5.66; N, 7.59.

Found: C, 58.27; H, 5.77; N, 7.87.

5.1.38. 1-(2,6-Dimethylpyridin-4-yl)piperidin-4-one (20d).

¹H NMR (CDCl₃) d: 2.45 (6H, s), 2.54 (4H, t,

J = 6.2 Hz), 3.72 (4H, t, J = 6.2 Hz), 6.45 (2H, s).

5.1.47. Ethyl N-{3-[(6-chloronaphthalen-2-yl)sulfonyl]pro-

panoyl}-N-[1-(pyridin-4-yl)piperidin-4-yl]-beta-alaninate

(2p). A mixture of 10c (0.30 g, 1.00 mmol) and thionyl

chloride (2 mL) was stirred at 90 ꢁC for 1 h and then

concentrated in vacuo. The residue was dissolved in

THF (5 mL), and the solution was added to an ice-

cooled solution of 21b (0.28 g, 1.00 mmol) and N,N-

diisopropylethylamine (0.40 mL, 2.30 mmol) in THF

(20 mL). After the resulting mixture was stirred at room

temperature for 2 h, it was concentrated in vacuo. The

residue was partitioned between brine and THF. The or-

ganic layer was separated, dried over anhydrous

MgSO₄, and concentrated in vacuo. The residue was

puriﬁed by basic silica gel chromatography (EtOAc/

MeOH = 60/1) to give 2p (45 mg, 8%) as a colorless

amorphous powder. ¹H NMR (CDCl₃) d: 1.22–1.28

(3H, m), 1.50–2.00 (5H, m), 2.35–2.60 (2H, m), 2.72–

5.1.39. Ethyl N-[1-(pyridin-4-yl)piperidin-4-yl]glycinate

(21a). Compound 21a was prepared in a manner similar

to that described for 13. ¹H NMR (CDCl₃) d: 1.33 (3H,

t, J = 7.2 Hz), 1.60–1.95 (2H, m), 2.25–2.40 (2H, m),

3.18–3.40 (2H, m), 3.55–3.80 (1H, m), 4.09 (2H, s),

4.33 (2H, q, J = 7.2 Hz), 4.35–4.55 (2H, m), 7.25 (2H,

d, J = 8.0 Hz), 8.17 (2H, d, J = 8.0 Hz).

5.1.40. Ethyl N-[1-(pyridin-4-yl)piperidin-4-yl]-beta-alan-

inate (21b). Compound 21b was prepared in a manner

1

similar to that described for 13. H NMR (CDCl₃) d:

1.26 (3H, t, J = 7.1 Hz), 1.28–1.55 (2H, m), 1.88–2.08

(2H, m), 2.51 (2H, t, J = 6.4 Hz), 2.64–2.85 (1H, m),

2.85–3.05 (4H, m), 3.50–4.00 (1H, br s), 3.75–3.92 (2H,

m), 4.15 (2H, q, J = 7.2 Hz), 6.66 (2H, d, J = 6.4 Hz),

8.23 (2H, d, J = 6.4 Hz).

2256

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 2243–2260

3.08 (4H, m), 3.32–3.68 (4H, m), 3.70–4.20 (4H, m),

6.58–6.74 (2H, m), 7.61 (1H, dd, J = 2.2 and 8.8 Hz),

7.80–8.05 (4H, m), 8.20–8.40 (2H, m), 8.49 (1H, s). Anal.

Calcd for C₂₈H₃₂ClN₃O₅SÆ0.5H₂O: C, 59.30; H, 5.87; N,

7.41. Found: C, 59.38; H, 5.61; N, 7.51.

(CDCl₃) d: 1.56–1.68 (4H, m), 2.44 (6H, s), 2.83 (3H, s),

2.88–3.04 (4H, m), 3.57 (2H, dd, J = 7.0 and 8.0 Hz),

3.94 (2H, m), 4.60 (1H, m), 6.38 (2H, s), 7.60 (1H, dd,

J = 2.2 and 8.8 Hz), 7.93–7.97 (4H, m), 8.48 (1H, s).

Anal. Calcd for C₂₆H₃₀ClN₃O₃SÆ1.5H₂O: C, 59.25; H,

6.31; N, 7.97. Found: C, 59.34; H, 6.19; N, 8.33.

5.1.48. 3-[(6-Chloronaphthalen-2-yl)sulfonyl]-N-methyl-

N-[1-(2-methylpyridin-4-yl)piperidin-4-yl]propanamide (2s).

To a stirred solution of 10c (0.45 g, 1.50 mmol) and 21d

(0.31 g, 1.50 mmol) in THF (50 mL) was added

DMTMM (0.56 g, 2.00 mmol) and the resulting mixture

was stirred at room temperature for 16 h. The reaction

mixture was concentrated in vacuo and the residue

was partitioned between THF and aqueous K₂CO₃.

The organic layer was separated, dried over anhydrous

Na₂SO₄, and concentrated in vacuo. The residue was

puriﬁed by basic silica gel chromatography (EtOAc/

MeOH = 40/1) and crystallized from EtOAc to give 2s

(0.34 g, 70%) as a colorless powder. Mp 186 ꢁC. ¹H

NMR (CDCl₃) d: 1.52–1.95 (4H, m), 2.50 (3H, s),

2.75–3.15 (4H, m), 2.84 (3H, s), 3.50–3.65 (2H, m),

4.45–4.80 (1H, m), 6.50–6.65 (2H, m), 7.60 (1H, dd,

J = 2.2 and 8.8 Hz), 7.90–8.00 (4H, m), 8.20 (1H, d,

J = 6.4 Hz), 8.45 (1H, s). Anal. Calcd for

C₂₅H₂₈ClN₃O₂S: C, 61.78; H, 5.81; N, 8.65. Found: C,

61.51; H, 5.82; N, 8.44.

5.1.53. N-(2-Aminoethyl)-3-[(6-chloronaphthalen-2-yl)sul-

fonyl]-N-[1-(pyridin-4-yl)piperidin-4-yl]propanamide ditri-

ﬂuoroacetate (2r). To

a solution of 2q (0.14 g,

0.24 mmol) in toluene (2 mL) was added triﬂuoroacetic

acid (2 mL) at room temperature and the resulting mix-

ture was stirred at room temperature for 1 h. The reac-

tion mixture was concentrated in vacuo and the residual

solid was collected by ﬁltration to give 2r (0.14 g,

quant.) as a colorless amorphous powder. ¹H NMR

(CD₃OD) d: 1.64–2.05 (4H, m), 2.97 (2H, t, 6.3 Hz),

3.05 (2H, t, J = 7.2 Hz), 3.15–3.40 (2H, m), 3.47 (2H,

t, J = 6.3 Hz), 3.70 (2H, t, J = 7.2 Hz), 4.10–4.48 (3H,

m), 7.18 (2H, d, J = 7.8 Hz), 7.66 (1H, dd, J = 2.2 and

8.8 Hz), 7.90–8.20 (6H, m), 8.59 (1H, s). Anal. Calcd

for C₂₅H₂₉ClN₄O₃SÆ2CF₃CO₂HÆ2H₂O: C, 45.52; H,

4.61; N, 7.32. Found: C, 45.67; H, 4.60; N, 7.32.

5.1.54.

3-[(7-Chloronaphthalen-2-yl)sulfonyl]propanoic

acid (24a). To a solution of sodium sulﬁte (0.54 g,

4.20 mmol) and sodium bicarbonate (0.66 g, 7.86 mmol)

in water (20 mL) was added 22a²³(1.00 g, 3.83 mmol) at

75 ꢁC and the mixture was stirred at 75 ꢁC for 1.5 h. A

solution of sodium hydroxide (0.31 g, 7.75 mmol) in

water (1 mL) and bromosuccinic acid (1.54 g,

8.65 mmol) were successively added and the reaction

mixture was stirred at 110 ꢁC for 20 h. The resulting pre-

cipitate was collected by ﬁltration, washed with water,

and dried to give 24a (0.61 g, 53%) as a colorless solid.

¹H NMR (DMSO-d₆) d: 2.58 (2H, t, J = 7.3 Hz), 3.62

(2H, d, J = 7.3 Hz), 7.78 (1H, dd, J = 2.2 and 8.8 Hz),

7.95 (1H, dd, J = 2.0 and 8.8 Hz), 8.16 (1H, d,

J = 8.8 Hz), 8.25 (1H, d, J = 8.8 Hz), 8.40 (1H, d,

J = 2.0 Hz), 8.59 (1H, s).

The following compounds 2q and 2t–v were prepared in

a manner similar to that described for 2s.

5.1.49. tert-Butyl[2-({3-[(6-chloronaphthalen-2-yl)sulfonyl]-

propanoyl}[1-(pyridin-4-yl)piperidin-4-yl]amino)ethyl]car-

bamate (2q). Yield 56%, colorless amorphous powder.

¹H NMR (CDCl₃) d: 1.40 (4.5H, s), 1.45 (4.5H, s),

1.55–2.00 (4H, m), 2.70–3.10 (5H, m), 3.10–3.40 (4H,

m), 3.50–3.70 (2H, m), 3.80–4.10 (2H, m), 4.80–5.05

(1H, m), 6.60–6.75 (2H, m), 7.55–7.65 (1H, m), 7.90–

8.00 (4H, m), 8.20–8.35 (2H, m), 8.50 (1H, s).

5.1.50. 3-[(6-Chloronaphthalen-2-yl)sulfonyl]-N-{1-[2-

(hydroxymethyl)pyridin-4-yl]piperidin-4-yl}-N-methylpro-

panamide (2t). Yield 54%, colorless amorphous powder.

The following compounds 24b–e were prepared in a

manner similar to that described for 24a.

¹H NMR (CDCl₃) d: 1.56–1.79 (4H, m), 2.83 (3H, s),

2.86–2.99 (4H, m), 3.57 (2H, dd, J = 3.2 and 8.2 Hz),

3.95 (2H, m), 4.63 (2H, s), 4.65 (1H, m), 6.56–6.63

(2H, m), 7.60 (dd, J = 2.2 and 8.2 Hz), 7.93–7.97 (4H,

m), 8.20 (1H, d, J = 6.6 Hz), 8.49 (1H, s). Anal. Calcd

for C₂₅H₂₈ClN₃O₄SÆ0.5H₂O: C, 58.76; H, 5.72; N,

8.22. Found: C, 58.89; H, 5.92; N, 8.02.

5.1.55.

3-[(5-Chloronaphthalen-2-yl)sulfonyl]propanoic

acid (24b). ¹H NMR (DMSO-d₆) d: 2.59 (2H, t,

J = 7.3 Hz), 3.64 (2H, d, J = 7.3 Hz), 7.71 (1H, t,

J = 8.0 Hz), 7.96 (1H, d, J = 7.8 Hz), 8.09 (1H, dd,

J = 2.0 and 8.8 Hz), 8.28 (1H, d, J = 8.4 Hz), 8.42 (1H,

d, J = 8.8 Hz), 8.71 (1H, d, J = 2.0 Hz).

5.1.51. N-[1-(2-Aminopyridin-4-yl)piperidin-4-yl]-3-[(6-

chloronaphthalen-2-yl)sulfonyl]-N-methylpropanamide (2u).

Yield 50%, colorless amorphous powder. ¹H NMR

(CDCl₃) d: 1.56–1.75 (4H, m), 2.82 (3H, s), 2.82–2.96

(4H, m), 3.56 (2H, m), 3.81 (2H, m), 4.24 (2H, br s), 4.57

(1H, m), 5.84 (1H, d, J = 2.6 Hz), 6.16 (1H, dd, J = 2.6

and 6.6 Hz), 7.60 (1H, m), 7.78–7.94 (5H, m), 8.48 (1H,

s). Anal. Calcd for C₂₄H₂₇ClN₄O₃SÆ0.5H₂O: C, 58.11;

H, 5.69; N, 11.30. Found: C, 58.38; H, 5.91; N, 11.56.

5.1.56. 3-[(6-Methylnaphthalen-2-yl)sulfonyl]propanoic

1

acid (24c). H NMR (DMSO-d₆) d: 2.54 (3H, s), 2.56

(2H, t, J = 7.4 Hz), 3.59 (2H, d, J = 7.4 Hz), 7.56 (1H,

dd, J = 1.4 and 8.4 Hz), 7.86 (1H, dd, J = 2.0 and

8.6 Hz), 7.87 (1H, s), 8.08 (1H, d, J = 8.6 Hz), 8.12

(1H, d, J = 8.4 Hz), 8.52 (1H, s).

5.1.57. 3-[(5-Chloro-1-benzothien-2-yl)sulfonyl]propanoic

acid (24d). ¹H NMR (CDCl₃+ DMSO-d₆) d: 2.75 (2H, t,

J = 7.7 Hz), 3.55 (2H, t, J = 7.7 Hz), 7.47 (1H, dd,

J = 2.2 and 8.4 Hz), 7.87 (1H, d, J = 8.4 Hz), 8.25 (1H,

d, J = 2.2 Hz), 8.42 (1H, s).

5.1.52. 3-[(6-Chloronaphthalen-2-yl)sulfonyl]-N-[1-(2,6-

dimethylpyridin-4-yl)piperidin-4-yl]-N-methylpropanamide

(2v). Yield 61%, colorless amorphous powder. ¹H NMR

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 2243–2260

2257

5.1.58. 3-[(6-Chloro-1-benzothien-2-yl)sulfonyl]propanoic

to that described for 2s in 50% yield as a colorless amor-

phous powder. H NMR (CDCl₃) d: 1.45–2.00 (5H, m),

1

acid (24e). ¹H NMR (CDCl₃+ DMSO-d₆) d: 2.74 (2H, t,

J = 7.5 Hz), 3.54 (2H, t, J = 7.5 Hz), 7.52 (1H, dd,

J = 1.8 and 8.8 Hz), 7.94 (1H, d, J = 1.8 Hz), 8.18 (1H,

d, J = 8.8 Hz), 8.33 (1H, s).

2.45–2.47 (3H, m), 2.70–3.10 (4H, m), 2.77–2.83 (3H,

m), 3.50–3.70 (2H, m), 3.70–4.10 (2.25H, m), 4.48–4.80

(0.75H, m), 6.40–6.60 (2H, m), 7.52 (1H, dd, J = 1.8

and 8.8 Hz), 7.93 (1H, d, J = 1.8 Hz), 8.10–8.20 (1H,

m), 8.19 (1H, d, J = 8.8 Hz), 8.34–8.35 (1H, m). Anal.

Calcd for C₂₃H₂₆ClN₃O₃S₂Æ0.25H₂O: C, 55.56; H, 5.38;

N, 8.46. Found: C, 55.58; H, 5.41; N, 8.35.

5.1.59. 3-[(7-Chloronaphthalen-2-yl)sulfonyl]-N-methyl-

N-[1-(2-methylpyridin-4-yl)piperidin-4-yl]propanamide (2w).

Compound 2w was prepared in a manner similar to that

described for 2s in 58% yield as colorless crystals. Mp

96–98 ꢁC. ¹H NMR (CDCl₃) d: 1.50–1.95 (4H, m), 2.45–

2.48 (3H, m), 2.76–2.83 (3H, m), 2.80–3.05 (4H, m), 3.57

(2H, t, J = 7.7 Hz), 3.93 (2H, m), 4.59 (1H, m), 6.45–

6.60 (2H, m), 7.64 (1H, dd, J = 2.2 and 8.8 Hz), 7.85–

8.05 (4H, m), 8.15 (1H, d, J = 6.0 Hz), 8.42 (1H, s). Anal.

Calcd for C₂₅H₂₈ClN₃O₃SÆ0.5H₂O: C, 60.66; H, 5.90; N,

8.49. Found: C, 60.82; H, 5.72; N, 8.53.

5.2. Biology

5.2.1. In vitro assays for the inhibition of human FXa. Hu-

man factor Xa (0.3 U/mL) was obtained from Roche

Diagnostics. Chromogenic substrate, S-2765 (Chromog-

enix-Instrumentation Laboratory), was used for the

measurement of the inhibition of FXa. Anti-FXa activ-

ity was assayed in a buﬀer containing 50 mM Tris–HCl,

145 mM NaCl, and 2 mM CaCl₂at pH 8.3. Enzyme as-

say was carried out in 96-well microtiter plates. Test

compounds were diluted in DMSO. Compound dilu-

tions and 10 lL of enzyme solution were added to the

well containing buﬀer, and preincubated. The enzymatic

reactions were initiated with the addition of 10 lL of

3 mM substrate and the mixture was incubated for

10 min at 37 ꢁC. The reaction was terminated with the

addition of 25 lL of 50% acetic acid. The color develop-

ment from the release of p-nitroanilide from each chro-

mogenic substrate was measured at 405 nm on a

microtiter plate reader (MTP32, Corona Electric Co.).

Each absorbance [T] was calculated by subtracting the

absorbance measured without the substrate. The control

[C] was performed using DMSO solution in place of test

compound. Inhibitory eﬀect (%) was calculated accord-

ing to the equation (1 À [T]/[C]) · 100. IC₅₀values were

calculated from the regression line based on the method

of least squares between inhibitory eﬀect and concentra-

tion. Data for new compounds were compared to posi-

tive control DX-9065a (FXa IC₅₀= 0.13 lM). The

95% conﬁdence interval for the IC₅₀of DX-9065a was

0.12–0.15 lM (duplicate).

5.1.60. 3-[(5-Chloronaphthalen-2-yl)sulfonyl]-N-methyl-N-

[1-(2-methylpyridin-4-yl)piperidin-4-yl]propanamide (2x).

Compound 2x was prepared in a manner similar to that

described for 2s in 64% yield as a colorless amorphous

1

solid. H NMR (CDCl₃) d: 1.50–1.95 (4H, m), 2.45–

2.47 (3H, m), 2.76–2.82 (3H, m), 2.80–3.05 (4H, m),

3.60 (2H, t, J = 7.5 Hz), 3.93 (2H, m), 4.58 (1H, m),

6.45–6.60 (2H, m), 7.58 (1H, t, J = 8.0 Hz), 7.80 (1H,

d, J = 7.6 Hz), 7.95 (1H, d, J = 8.4 Hz), 8.01 (1H, dd,

J = 1.8 and 8.8 Hz), 8.15 (1H, d, J = 5.8 Hz), 8.48 (1H,

d, J = 8.8 Hz), 8.53 (1H, d, J = 1.8 Hz). Anal. Calcd

for C₂₅H₂₈ClN₃O₃SÆ0.5H₂O: C, 60.66; H, 5.90; N,

8.49. Found: C, 60.54; H, 6.15; N, 8.56.

5.1.61. 3-[(6-Methylnaphthalen-2-yl)sulfonyl]-N-methyl-N-

[1-(2-methylpyridin-4-yl)piperidin-4-yl]propanamide (2y).

Compound 2y was prepared in a manner similar to that

described for 2s in 58% yield as colorless crystals. Mp

1

129–131 ꢁC. H NMR (CDCl₃) d: 1.50–1.95 (4H, m),

2.44–2.46 (3H, m), 2.57 (3H, s), 2.73–2.80 (3H, m),

2.80–3.03 (4H, m), 3.57 (2H, t, J = 7.7 Hz), 3.91 (2H,

m), 4.58 (1H, m), 6.45–6.60 (2H, m), 7.48 (1H, dd,

J = 1.6 and 8.4 Hz), 7.71 (1H, s), 7.80–7.95 (3H, m),

8.14 (1H, d, J = 6.2 Hz), 8.45 (1H, s). Anal. Calcd for

C₂₆H₃₁N₃O₃S: C, 67.07; H, 6.71; N, 9.02. Found: C,

66.77; H, 6.64; N, 8.97.

5.2.2. In vitro PT assays. The assay of plasma clotting

time was performed using an automatic coagulometer

(STA Compact, Diagnostica Stago). PT was measured

with PT-Test Wako (Wako Pure Chemical). Compound

dilutions of 1.5 lL in DMSO were added to 48.5 lL of

human normal plasma (fresh human plasma: FFP, Seki-

sui Chemical Co.), and the mixture was preincubated at

37 ꢁC for 4 min. Coagulation was initiated with the

addition of 100 lL of thromboplastin, and coagulation

time was measured. Coagulation time prolonging ratio

(%) was calculated based on coagulation time when

DMSO was added instead of test compound. The plas-

ma clotting time doubling concentration (PT₂) was cal-

culated from the regression line based on the method

of least squares. Data for new compounds were com-

pared to positive control DX-9065a (PT₂= 0.69 lM).

The 95% conﬁdence interval for the PT₂of DX-9065a

was 0.61–0.79 lM (n = 3).

5.1.62. 3-[(5-Chloro-1-benzothien-2-yl)sulfonyl]-N-methyl-

N-[1-(2-methylpyridin-4-yl)piperidin-4-yl]propanamide (2z).

Compound 2z was prepared in a manner similar to

that described for 2s in 50% yield as colorless crystals.

1

Mp 122–124 ꢁC. H NMR (CDCl₃) d: 1.40–2.00 (5H,

m), 2.44 (2.25H, s), 2.47 (0.75H, s), 2.76 (0.75H, s),

2.78–3.10 (4H, m), 2.84 (2.25H, s), 3.52–3.76 (2H,

m), 3.72–4.10 (2.25H, m), 4.48–4.80 (0.25H, m),

6.40–6.60 (2H, m), 7.47 (1H, dd, J = 1.8 and

8.8 Hz), 7.86 (1H, d, J = 8.8 Hz), 8.16 (1H, d,

J = 5.8 Hz), 8.25 (1H, d, J = 1.8 Hz), 8.40 (0.75H, s),

8.42 (0.25H, s). Anal. Calcd for C₂₃H₂₆ClN₃O_3-

S₂Æ0.25EtOHÆ0.25H₂O: C, 55.55; H, 5.55; N, 8.12.

Found: C, 55.58; H, 5.41; N, 8.12.

5.1.63. 3-[(6-Chloro-1-benzothien-2-yl)sulfonyl]-N-methyl-

N-[1-(2-methylpyridin-4-yl)piperidin-4-yl]propanamide

(2aa). Compound 2aa was prepared in a manner similar

5.2.3. Measurement of CYP inhibition activity. Inhibition

activity of test compounds of CYP3A4 was evaluated by

2258

Y. Imaeda et al. / Bioorg. Med. Chem. 16 (2008) 2243–2260

incubating 40 lM 7-benzyloxyquinoline with micro-

somes derived from CYP3A4-expressing insect cell

(BD Biosciences) in the presence of 1 lM test com-

pound. The concentration of 7-benzyloxyquinoline

metabolite was measured with a spectroﬂuorometer.

zyme reactions were initiated by the addition of

substrate and the color developed from the release of

p-nitroanilide from each chromogenic substrate was

monitored continuously for 5 min at 405 nm on a micro-

titer plate reader. The K_ivalues were determined from a

Lineweaver–Burk plot, when the optical densities were

measured with diﬀerent concentrations of substrates.

5.2.4. Measurement of ex vivo PT in mouse after oral

administration. Male ICR mice (30–35 g, Clea Japan

Inc.) fasting for more than 12 h were employed. Test

compounds were orally administered to these animals.

An hour after administration, blood (800 lL) was col-

lected from artery abdominal aorta using 3.8% sodium

citrate (whole blood: sodium citrate solution = 9:1, v/

v), under anesthesia with pentobarbital (50 mg/kg, ip).

The citrated blood was centrifuged at 1000g for 10 min

to obtain platelet poor plasma (PPP). PT was measured

with an automatic coagulometer. Fifty microliters of

PPP was preincubated at 37 ꢁC for 4 min. The PPP

was mixed with 100 lL of thromboplastin solution,

and then its coagulation time was measured. Test com-

pounds were suspended in 0.5% methyl cellulose (Meto-

lose cp100, Shin-Etsu Chemical), and as control, 0.5%

methylcellulose was administered instead of test com-

pounds. Activity of each test compound was shown in

ratio (%) determined by comparing the clotting time of

compound-treated mice with that of the control group.

We have validated the ex vivo PT assay for the evalua-

tion of oral bioavailability in mice by use of the positive

control compound DX-9065a. DX-9065a prolonged

mouse PT by 1.1-fold at a dose of 30 mg/kg and by

1.4-fold at a dose of 100 mg/kg.

5.2.7. Pharmacokinetic analysis in cynomolgus monkeys.

Test compound was administered to fasted cynomolgus

monkeys (male, n = 3) intravenously (1 mg/kg, DMA/

PEG400) or orally (1 mg/kg, 0.5% methylcellulose sus-

pension). Before and 5, 10, 15, 30 min, 1, 2, 4, 8, 24 h

after intravenous administration, or before and 15,

30 min, 1, 2, 4, 8, 24 h after oral administration, blood

samples were collected from femoral vein. The blood

samples were centrifuged to obtain the plasma fraction.

The plasma samples were deproteinized with acetoni-

trile. After centrifugation, the supernatant obtained

was diluted with the same volume of 0.01 M HCO₂NH₄

(adjusted to pH 3.0 with HCO₂H) and centrifuged

again. The compound concentration in the supernatant

was measured by LC/MS/MS with an API3000 triple

quadruple mass spectrometer (Perkin-Elmer Sciex).

The mass spectrometer was equipped with a turbo ion-

spray source and operated in positive ion mode. The

HPLC conditions were as follows: column, an L-column

ODS (2.1 · 150 mm); mobile phase, 0.01 M HCO₂NH₄

(adjusted to pH 3.0 with HCO₂H)/acetonitrile = 5/5;

ﬂow rate, 0.2 mL/min; column temperature, 40 ꢁC.

5.2.5. Measurement of ex vivo PT in monkey after oral

administration. Male cynomolgus monkeys (3.5–4 kg,

NAFOVANNY) fasting for more than 12 h were em-

ployed. Test compounds were orally administered to

these animals. After administration, blood (1.5 mL)

was collected from femoral vein using 3.8% sodium cit-

rate (whole blood: sodium citrate solution = 9:1, v/v) at

several time points. The citrated blood was centrifuged

at 1000g for 10 min to obtain PPP. Plasma clotting time

and anticoagulating activity of drug were determined as

described above.

Acknowledgments

The authors would like to thank Mr. Terufumi Takagi

for his docking model study of the compound 2s and

helpful discussions, and the DMPK group at Takeda

Pharmaceutical Company for carrying out CYP3A4

inhibition measurements for the compounds highlighted

in Table 3 and obtaining monkey pharmacokinetic data

for compound 2s in Table 6.

References and notes

5.2.6. Enzyme inhibition assays. Enzyme assays using

chromogenic substrates were performed as follows. Hu-

man factor Xa was obtained from Roche diagnostics.

Human thrombin was obtained from Sigma Chemical

Co. Human trypsin was obtained from Atheus Research

and Technology, Inc. Human kallikrein and plasmin

were purchased from BioPur AG. Human t-PA was ob-

tained from Kyowa Hakko Kogyo Co. The chromo-

genic substrates used were S-2222, S-2366, S-2222, S-

2366, S-2302, and S-2288 for FXa, thrombin, trypsin,

plasmin, kallikrein, and t-PA, respectively, and obtained

from Chromogenix-Instrumentation Laboratory. All

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sin, plasmin, kallikrein, and t-PA, respectively. Com-

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Article Doi

Discovery of sulfonylalkylamides: A new class of orally active factor Xa inhibitors

DOI: 10.1016/j.bmc.2007.11.073

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