Synthesis and structure-activity relationships of novel selective factor Xa inhibitors with a tetrahydroisoquinoline ring

3586

J. Med. Chem. 2005, 48, 3586-3604

Synthesis and Structure-Activity Relationships of Novel Selective Factor Xa

Inhibitors with a Tetrahydroisoquinoline Ring

Hiroshi Ueno, Katsuyuki Yokota, Jun-ichi Hoshi, Katsutaka Yasue, Mikio Hayashi, Yasunori Hase,

Itsuo Uchida, Kazuo Aisaka, Susumu Katoh, and Hidetsura Cho*

Central Pharmaceutical Research Institute, Japan Tobacco Inc., 1-1, Murasaki-cho, Takatsuki, Osaka, 569-1125, Japan

Received January 3, 2005

A series of novel 2,7-disubstituted tetrahydroisoquinoline derivatives were designed and

synthesized. Among these derivatives, compounds 1 and 2 exhibited potent inhibitory activity

against factor Xa (FXa) and good selectivity with respect to other serine proteases (thrombin,

plasmin, and trypsin). In addition, compound 2 exhibited potent anti-FXa activity after

intravenous and oral administration to cynomolgus monkeys, showed a dose-dependent

antithrombotic effect at 0.1, 0.3, and 1 mg kg^-1h^-1in a rat model of venous thrombosis, and

significantly reduced the size of brain infarction in a middle cerebral artery occlusion model at

a dose of 0.1 mg kg^-1h^-1. These results suggest that compound 2 (JTV-803) is likely to be

useful as both a venous and arterial antithrombotic agent.

Introduction

inhibitors.⁴As reported previously, we obtained a potent

FXa inhibitor 4, which is a benzimidazole derivative

with the side chain oriented to the prime site of FXa

(see Figure 1).^4a

Activation of the intravascular coagulation system is

involved in a number of cardiovascular diseases such

as deep vein thrombosis (DVT), disseminated intravas-

cular coagulation (DIC), pulmonary embolism, ischemic

stroke, and unstable angina. Interruption of the clotting

cascade has been investigated to inhibit clot formation

for the prevention or treatment of these thrombotic

disorders. Heparin and warfarin are the most widely

used anticoagulants for the prophylaxis and treatment

of thrombotic diseases. However, these anticoagulants

have clinical limitations because of the dependence on

antithrombin III and antagonism of vitamin K, respec-

tively. For instance, heparin administration is some-

times associated with the development of an antibody

to platelet factor (PF4) that causes thrombocytopenia,

while warfarin requires a longer time for the onset of

its action, needs continual monitoring, and sometimes

interacts with food or other drugs.

In the present study, our focus was directed toward

the discovery of other novel inhibitors. From analysis

of the binding of FXa to compound 3,⁵it has been

demonstrated that the basic part of both ends of the

compound is important for inhibition of FXa. We focused

on two basic parts, the S1 and S4 sites, and designed a

fundamental skeleton (compound 5) as follows. First,

we selected an N-amidinotetrahydroisoquinoline ring

that was expected to completely fill the S1 pocket of FXa

and to interact with Asp 189.^4bThen a 4-piperidinyl-

methyloxy group was selected for position 7 of the

tetrahydroisoquinoline ring as a spacer for the introduc-

tion of basic substituents because the nitrogen atom of

the piperidinyl group could be oriented toward the S4

site.⁵

Thus, we designed and synthesized a series of novel

2,7-disubstituted tetrahydroisoquinoline (2,7-THIQ) de-

rivatives and investigated the structure-activity rela-

tionships (SAR) of these compounds by evaluation of

their FXa inhibitory activity. Among these derivatives,

compounds 1 and 2 exhibited potent inhibitory activity

against FXa. Extensive pharmacological examinations

led us choose compound 2 as the most suitable candidate

for clinical evaluation.

Factor Xa (FXa) is a trypsin-like serine protease that

is an important enzyme in the clotting cascade, since it

represents the confluence of the intrinsic and extrinsic

pathways. Factor Xa forms the prothrombinase complex,

with nonenzymatic cofactor Va and Ca²⁺, on the surface

phospholipids of platelets or endothelial cells, and this

complex is responsible for the conversion of prothrombin

to thrombin. Then thrombin catalyzes the cleavage of

fibrinogen to fibrin, initiating a process that ultimately

leads to clot formation. Therefore, we have been inter-

ested in achieving inhibition of FXa by the development

of a novel FXa inhibitor.

Chemistry

The synthesis of compound 5 was initially under-

taken. Then additional modifications of the nitrogen

atom in the piperidinyl group of 5 afforded a series of

2,7-THIQ derivatives 6-22. After comprehensive as-

sessment of structure-activity relationships, further

efforts were made to prepare the 2-benzazepine 23 and

naphthalene 24 as well as compounds 25-28 with a

linker having an extended carbon or a new linker

displacing OCH₂with a SCH₂, SO₂CH₂, or SO₂NH

moiety (see Tables 1-3).

Since the discovery of compound 3 (DX-9065a),¹

a

variety of other compounds have been reported.²The

release of fondaparin sodium, which is a synthetic

pentasaccharide, proved the clinical effectiveness of FXa

inhibitors as anticoagulants.³This situation prompted

us to disclose our experimental results on novel FXa

* To whom correspondence should be addressed. Phone: +81-72-

681-9700. Fax: +81-72-681-9725. E-mail: hidetsura.cho@ims.jti.co.jp.

Published on Web 04/21/2005

Selective Factor Xa Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3587

Figure 1. Chemical structures of key compounds 1-5.

Scheme 1. Synthesis of Compounds 5-18^a

^aReagents and conditions: (a) Boc₂O, NaOH, dioxane/H₂O, room temp. (b) From 31 to 40: (i) NaH, 33, DMF, room temp; (ii) H₂/7.5%

Pd/C, THF/MeOH, room temp. (c) From 31 to 37 or 38: diisopropyl azodicarboxylate (DIPAD), PPh₃, 35 or 36, THF/CH₂Cl₂, room temp;

(d) 4-chloro-3-formylpyridine, Et₃N, EtOH, reflux; (e) MnO₂, NaCN, CHCl₃/MeOH, room temp; (f) triethyl phosphonoacetate, NaH, THF,

room temp. (g) From 37 to 8, from 38 to 9, from 42 to 14, or from 43 to 44: (i) TFA, CHCl₃, room temp; (ii) 1H-pyrazole-1-carboxamidine

hydrochloride, DIPEA, DMF, room temp; (h) 1H-pyrazole-1-(N,N′-bis-tert-butoxycarbonyl)carboxamidine, DMF, room temp; (i) (i) 33, NaOH,

DMSO, room temp; (ii) H₂, 7.5 % Pd/C, THF, EtOH, room temp. (j) From 34 to 5: TFA, CHCl₃, room temp. From 34 to 6: Ac₂O, pyridine,

THF, room temp, then TFA, CHCl₃, room temp. From 34 to 7: MeC(dNH)OEt, Et₃N, THF/EtOH, room temp, then TFA, CHCl₃, room

temp. From 34 to 10 or 11: 4-picolyl chloride or 2-picolyl chloride, NaOH, THF/DMF, 50 °C, then TFA, CHCl₃, room temp. From 34 to

12 or 13: 2-nitrobenzyl bromide or 3-nitrobenzyl bromide, NaOH, DMF/THF, room temp, then TFA, CHCl₃, room temp, then H₂/7.5%

Pd/C, MeOH, room temp; (k) NaOH, MeOH/H₂O, room temp; (l) H₂/7.5% Pd/C, EtOH, room temp.

First, synthesis of compounds 5-22 was achieved in

several ways, as shown in Schemes 1 and 2, using

intermediate 29 and its derivatives 30-32 with differ-

ent substituents at the nitrogen atom of 2,7-THIQ of

29. Synthesis of compounds 5-7 and 10-13 was per-

formed according to these procedures via intermediate

3588 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10

Scheme 2. Synthesis of Compounds 1, 16, and 19-22^a

Ueno et al.

^aReagents and conditions: (a) CbzCl, NaOH(aq), THF, room temp; (b) (i) ClCH₂SMe, NaH, DMF, 0 °C to room temp; (ii) SO₂Cl₂,

CH₂Cl₂, 0 °C; (c) 46 or 48, LDA, THF, -70 °C to room temp. (d) From 47 to 16 and from 55 to 21: (i) 30 % HCl/EtOH, 80 °C; (ii) 1H-

i

pyrazole-1-carboxamidine, Pr₂NEt, DMF, room temp; (e) concentrated HCl, reflux. (f) From 52 to 50: 4-chloro-2-methylpyridine, Et₃N,

EtOH, 150 °C. From 52 to 53: 2,4-dichloropyrimidine, Et₃N, EtOH, 150 °C. (g) From 50 to 51 and from 56 to 22: 25% HBr/AcOH, room

temp, then 1H-pyrazole-1-carboxamidine, ⁱPr₂NEt, DMF, room temp. (h) From 53 to 54: 7.5% Pd/C, HCO₂NH₄, reflux, then 1H-pyrazole-

i

1-carboxamidine, Pr₂NEt, DMF, room temp; (i) (i) 4-fluorobenzonitrile, K₂CO₃, DMSO, 120 °C;(ii) H₂S, Et₃N/pyridine, room temp; (iii)

i

MeI, acetone, reflux; (iv) NH₄OAc, EtOH, 75 °C; (j) TFA, CHCl₃, room temp; (k) MeI, Pr₂NEt, acetone, room temp, then HCl/dioxane.

34. Hydrogenation of 7-methoxyisoquinoline⁶gave com-

pound 29, which was treated with 1H-pyrazol-1-(N,N′-

bis-tert-butoxycarbonyl)carboxyamidine to afford com-

pound 30. Treatment of 30 with benzyl 4-bromomethyl-

piperidine-1-carboxylate 33, followed by cleavage of a

benzyloxycarbonyl (Cbz) group under a hydrogen atmo-

sphere at a pressure of 3 atm provided the crucial

intermediate 34 (see Scheme 1). Compound 34 furnished

a series of compounds 5-7 and 10 under various

reactionconditions;namely,deprotectionoftwotert-butoxy-

carbonyl (Boc) groups with trifluoroacetic acid (TFA)

gave compound 5; acetylation of 34 with acetic anhy-

dride or treatment of 34 with ethyl acetoimidate and

successive treatment with TFA yielded 6 or 7; and

treatment of 34 with 4-picolyl chloride in the presence

of NaOH followed by acidic deprotection gave compound

10.

(DIPAD) and triphenylphosphine or with bromide 33

in the presence of NaH provided 37, 38, or 39, respec-

tively. Removal of the protecting group of 39 under a

hydrogen atmosphere afforded compound 40, which was

reacted with 4-chloro-3-formylpyridine⁷to give com-

pound 41. The resulting aldehyde 41 was treated with

NaCN and MnO₂in MeOH/AcOH to give compound 42.

Condensation of 41 with triethyl phosphonoacetate

provided compound 43. Finally, treatment of 37, 38, 42,

or 43 with TFA, followed by 1H-pyrazole-1-carboxami-

dine, resulted in the 1,2,3,4-tetrahydroisoquinoline-2-

carboxamidine derivative 8, 9, 14, or 44. Compound 15

was obtained after hydrolysis of 14. Hydrogenation of

compound 44, followed by hydrolysis, furnished com-

pound 18. Compounds 1, 16, and 19-22 with a carboxyl

group or an ethoxycarbonyl group at position 4 of the

piperidine ring in the fundamental compound 5 were

synthesized as shown in Scheme 2 (see Table 1).

Namely, compound 45 derived from 29 was coupled with

compound 46 at -70 °C in the presence of lithium

diisopropylamide (LDA) to create the intermediate 47,

which was deprotected with 30% HCl in EtOH at 85

°C, followed by reaction with 1H-pyrazole-1-carboxami-

Compounds 11-13 were obtained in a similar man-

ner.

Alternatively, compounds 8, 9, 14, 15, 17, and 18 were

synthesized as follows. Protection of 29 by a Boc group

gave compound 31. Alkylation of 31 with alcohol 35 or

36 in the presence of diisopropyl azodicarboxylate

Selective Factor Xa Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3589

Table 1. Inhibitory Activity for FXa and FIIa (Thrombin)^1aand Acute Toxicity in Mice (Mortality)

compd^a

R₁

R₂

FXa IC₅₀(µM)^b

FIIa IC₅₀(µM)^b

mortality (10 mg/kg, iv)

5

6

H

3.34 ( 0.25

4.06 ( 0.21

0.51 ( 0.01

0.056 ( 0.003

0.42 ( 0.02

0.80 ( 0.01

1.35 ( 0.11

3.25 ( 0.20

4.06 ( 0.15

0.27 ( 0.04

1.98 ( 0.32

0.074 ( 0.002

0.026 ( 0.003

0.19 ( 0.03

0.16 ( 0.01

0.23 ( 0.02

0.12 ( 0.01

1.13 ( 0.10

0.40 ( 0.02

>10

NT^c

3/3^d

3/3

C(dO)Me

C(dNH)Me

pyridin-4-yl

quinolin-4-yl

7

8

9

3/3

10

11

12

13

14

15

16

1

17

18

19

20

21

22

pyridin-4-ylmethyl

pyridin-2-ylmethyl

2-aminobenzyl

3/3

NT^c

3/3

3-aminobenzyl

(3-CO₂Me)pyridin-4-yl

(3-CO₂H)pyridin-4-yl

pyridin-4-yl

0/3

CO₂Et

CO₂H

H

3/3

pyridin-4-yl

0/3

(3-(CH₂)₂CO₂Et)pyridin-4-yl

(3-(CH₂)₂CO₂H)pyridin-4-yl

2-methylpyridin-4-yl

1,3-pyrimidin-4-yl

4-(1-methylpyridinium)chloride

4-amidinophenyl

3/3

H

0/3

CO₂H

CO₂Et

0/3

NT^c

^aCompounds 1, 5, 7-9, 14-20: two HCl salts. Compounds 10-13 and 22: three HCl salts. Compounds 6 and 21: HCl salt. ^bn ) 3,

mean ( SEM (standard errors). ^cNot tested. ^dNumber of deaths/number of animals tested.

Scheme 3. Synthesis of Compound 57^a

^aReagents and conditions: (a) Boc₂O, NaOH, dioxane/H₂O, room temp; (b) (i) ClCH₂SMe, NaH, DMF, 0 °C to room temp; (ii) SO₂Cl₂,

i

CH₂Cl₂, 0 °C; (c) 46, LDA, THF, -70 °C to room temp; (d) (i) TFA, CHCl₃, room temp; (ii) 1H-pyrazole-1-carboxamidine, Pr₂NEt, DMF,

room temp; (e) concentrated HCl, reflux.

dine to afford compound 16. Hydrolysis of this com-

pound yielded compound 1, which was treated with

methanesulfonic acid in aqueous acetone to provide

compound 2. Treatment of 47 with MeI, followed by

anion exchange with HCl, gave pyridinium salts, from

which compound 21 was obtained in a similar manner

as for compound 16. For preparation of compounds 19,

20, and 22, a lithium enolate of compound 48 generated

in THF at -70 °C was similarly reacted with chloride

45 to give compound 49. Selective deprotection of the

Boc group in 49 with TFA and reaction with 4-chloro-

2-methylpyridine provided 50. After deprotection of the

Cbz group of 50, amidination with 1H-pyrazole-1-

carboxamidine, and successive hydrolysis of 51, com-

pound 19 was obtained. Similarly, treatment of 52 with

2,4-dichloropyrimidine gave 53, followed by hydrogena-

tion with 7.5% Pd/C-HCO₂NH₄to give compound 54.

Hydrolysis of 54 afforded compound 20.

Treatment of compound 52 with 4-fluorobenzonitrile,

followed by successive reactions with H₂S, MeI, and

NH₄OAc, provided compound 56. Deprotection of the

Cbz group of 56 and amidination gave compound 22.

As for 2,6-THIQ derivatives, compound 57 correspond-

ing to 1 was obtained via intermediates 59-62 as

depicted in Scheme 3.

We undertook the synthesis of 2-benzazepine 23 and

naphthalene 24 as shown in Schemes 4 and 5, respec-

tively. Namely, the 2-benzazepine derivative 64 was

prepared by rearrangement of compound 63 with NaN₃/

HCl and by successive reduction with LiAlH₄. De-

methylation and successive protection of the nitrogen

atom of 64 gave compound 65, after which alkylation

with NaH/chloromethyl methyl sulfide and then chlo-

rination with sulfuryl chloride provided chloride 66.

Crucial coupling of 66 with 46 in the presence of LDA

furnished compound 67. Deprotection and amidination

3590 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10

Scheme 4. Synthesis of Compound 23^a

Ueno et al.

^aReagents and conditions: (a) (i) NaN₃, concentrated HCl, 0 °C to room temp; (ii) LiAlH₄, THF/dioxane, reflux; (b) (i) 48% HBr(aq),

reflux; (ii) Boc₂O, NaOH, dioxane/H₂O, room temp; (c) (i) ClCH₂SMe, NaH, DMF, 0 °C to room temp; (ii) SO₂Cl₂, CH₂Cl₂, 0 °C; (d) 46,

i

LDA, THF, -70 °C to room temp; (e) (i) TFA, CHCl₃, room temp; (ii) 1H-pyrazole-1-carboxamidine, Pr₂NEt, DMF, room temp; (h)

concentrated HCl, reflux.

Scheme 5. Synthesis of Compound 24^a

^aReagents and conditions: (a) (i) Tf₂O, Et₃N, CHCl₃, 0 °C; (ii) Zn(CN)₂, Pd(PPh₃)₄, DMF, 80 °C; (iii) AlCl₃, chlorobenzene, reflux; (b) (i)

ClCH₂SMe, NaH, DMF, 0 °C to room temp; (ii) SO₂Cl₂, CH₂Cl₂, 0 °C; (c) 46, LDA, THF, -70 °C to room temp; (d) (i) H₂S, Pyr, Et₃N, room

temp; (ii) HCl/EtOH, then MeI, acetone/MeOH, reflux; (iii) NH₄OAc, EtOH, 75 °C; (e) concentrated HCl, reflux.

Scheme 6. Synthesis of Compound 25^a

gave compound 68, and hydrolysis afforded the desired

compound 23. Synthesis of 24 was undertaken with 69

as the starting material, as depicted in Scheme 5.

Coupling of 7-methoxy-2-naphthol 69 with Zn(CN)₂in

the presence of Pd(PPh₃)₄via the trifluoromethane-

sulfonate derivative yielded compound 70, followed by

the same procedure mentioned above to give compound

71. Conversion of 71 with 46 to 72 was done in a similar

manner. Treatment of 72 with H₂S-pyridine was fol-

lowed by methylation and amination to produce 73,

which was hydrolyzed to afford compound 24. Then

compound 25, with one more carbon inserted in the

linker compared to compound 1, was synthesized as

shown in Scheme 6. This compound was similarly

prepared via intermediates 74-77. Synthesis of sulfide

26, sulfone 27, and sulfonamide 28, which had different

linkers compared with compound 1, was carried out via

intermediates 78-81, 82 and 83, and 84-89, respec-

tively, as depicted in Schemes 7 and 8.

Results and Discussion

As mentioned above, we designed and prepared

^aReagents and conditions: (a) (i) Br(CH₂)₂OH, diisopropyl

compound 5. This compound showed moderate FXa

inhibitory activity (IC₅₀) 3.34 ( 0.25 µM),^1ademon-

azodicarboxylate (DIPAD), PPh₃, THF, room temp; (ii) NaI, DMF,

90 °C; (b) 48, LDA, THF, -70 °C to room temp; (c) (i) TFA, CHCl₃,

strating that it had sufficient potential for further

room temp; (ii) 4-chloropyridine, Et₃N, EtOH, 150 °C; (d) (i) 25%

HBr/AcOH, room temp; (ii) 1H-pyrazole-1-carboxamidine, ⁱPr₂NEt,

studies on structure-activity relationships. Introduction

of basic substituents on the nitrogen atom of the

DMF, room temp; (e) concentrated HCl, reflux.

Selective Factor Xa Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3591

Scheme 7. Synthesis of Compounds 26 and 27^a

^aReagents and conditions: (a) LDA, CH₂I₂, THF, -78 °C to room temp; (b) K₂CO₃, DMF, 100 °C; (c) TFA, room temp; (d) 4-chloropyridine

hydrochloride, Et₃N, EtOH, 150 °C; (e) concentrated HCl, 90 °C; (f) (i) 1H-pyrazole-1-carboxamidine hydrochloride, NaOH, acetone, room

temp; (ii) HCl(aq); (g) m-CPBA, CH₂Cl₂, room temp.

Scheme 8. Synthesis of Compound 28^a

^aReagents and conditions: (a) (i) 4-chloropyridine hydrochloride, Et₃N, EtOH, 150 °C; (ii) concentrated HCl, reflux; (b) (i) NH₄OAc,

NaCN, MeOH, room temp; (ii) HCl, MeOH, room temp, then H₂O; (c) pyridine, CHCl₃, room temp; (d) (i) NaOH, MeOH, room temp; (ii)

i

1H-pyrazole-1-carboxamidine hydrochloride, Pr₂NEt, DMF, room temp; (e) HCl(aq), reflux.

piperidine ring provided compounds 7-13 (Table 1).^4b,c

Among the compounds with a pyridinyl group, quino-

linyl group, pyridinylmethyl group, or aminobenzyl

group, compound 8 (with a pyridin-4-yl group) showed

more potent FXa inhibitory activity (IC₅₀) 0.056 (

0.003 µM). This potent activity of compound 8 might be

explained by interaction of the protonated pyridine ring

with the S4 site, which favors a positive charge,⁸and

by formation of a hydrogen bond between the protonated

nitrogen atom of pyridine and Glu 97, as expected from

the docking simulation of compounds with FXa. Unfor-

tunately, most of these compounds showed fatal acute

toxicity. When these compounds were administered

intravenously at a bolus dose of 10 mg/kg, severe

convulsions occurred immediately and the mice died

after a few minutes.

compound 8. As a result, compound 1, as well as

compounds 15 and 18 with a carboxyl group, showed

good survival rates in mice treated at 10 mg/kg iv, while

the corresponding ester 16 showed a high mortality rate

as well as 14 and 17. Loss of toxicity after introduction

of a carboxyl group at different positions suggested that

one reason for severe toxicity might be the strong

basicity of the compound. Moreover, compound 1 ex-

hibited potent inhibitory activity against FXa (IC₅₀

)

0.026 ( 0.003 µM). This finding was explained by

conformational analysis of compounds 8 and 1. Namely,

introduction of the carboxyl group at the tertiary carbon

of the piperidinyl group did not influence the conforma-

tion of compound 8 and the introduced hydrophilic group

was oriented toward the aqueous area (Figure 2).⁵

On the other hand, the introduction of substituents

at position 3 of the pyridine ring caused a change of the

torsional angle between the pyridine ring and the

piperidine ring. The lower activity of compounds 14, 15,

17, and 18 might be explained in this fashion. On the

Such fatal acute toxicity was also observed in our

previous research on other FXa inhibitors and was

prevented by introduction of an acidic functional group.^4a

Hence, we tried to introduce a carboxyl group to active

3592 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10

Ueno et al.

Figure 2. Complex model of compound 1 (orange) with FXa (green).

Table 2. Enzyme Inhibitory Activities for FXa and FIIa

Table 3. Enzyme Inhibitory Activities for FXa and FIIa

(Thrombin)

compd^a

-R-

FXa IC₅₀(µM)^bthrombin IC₅₀(µM)^b

FXa

thrombin

compd^a

-R

IC₅₀(µM)^b

1

25

26

27

28

-OCH₂-

0.026 ( 0.003

0.23 ( 0.02

0.39 ( 0.01

5.44 ( 0.12

2.90 ( 0.13

>10

-OCH₂CH₂-

-SCH₂-

1

0.026 ( 0.003

>10

-SO₂CH₂-

-SO₂NH-

^aCompounds 1 and 25-28: two HCl salts. ^bn ) 3, mean (

SEM (standard errors).

23

24

to give N-amidino-2-benzazepine 23, but it did not show

any inhibition of FXa (see Table 2).

Next, the naphthalene derivative 24 (synthesized as

shown in Scheme 5) was found to have only /₂₀of the

0.51 ( 0.01

1

activity of 1. Regarding the effect of modifications or

extension of the middle spacer R in Table 3, the

transformation from OCH₂to OCH₂CH₂, SCH₂, SO₂CH₂,

or SO₂NH was undertaken to afford compounds 25-

28, which all had weaker inhibitory activity than 1 (see

Table 3). Then, studies on substitution of a side chain

(2,7-THIQ vs 2,6-THIQ) were carried out but showed

that compound 57 with a 2,6-THIQ ring was totally

inactive in the inhibition assay (IC₅₀g 10 µM), so the

position of substitution of the side chain was found to

be important for potent inhibition. Therefore, a suitable

relative position (distance and angle between the THIQ

ring and the pyridine ring) was found to be crucial for

a high level of activity.

^aCompounds 1, 23, 24: two HCl salts. ^bn ) 3, mean ( SEM

(standard errors).

basis of the computer modeling, the amidino group on

the 2,7-THIQ ring of 1 interacts with the carboxyl

moiety of Asp 189, and the protonated pyridine moiety

interacts with the carboxyl moiety of Glu 97, as shown

in Figure 2. Subsequently, we tested the following

modifications of compound 1, although we suspected

that some attempts (such as (i) modification of the

pyridine ring at the S4 site, (ii) modification of the

piperidine ring on the 2,7-THIQ ring, and (iii) extension

of the middle spacer or replacement of 2,7-THIQ with

2,6-THIQ) would weaken the FXa inhibitory activity

because of a poor fit with the active site of FXa. We

prepared compounds 19-22 with other moieties at the

S4 site, but they all showed lower potency than 1. As

for the S1 site, expansion of the piperidine ring of the

2,7-THIQ ring was performed as depicted in Scheme 4

As mentioned above, we found a series of novel 2,7-

THIQ derivatives, initially with the aid of computer-

associated molecular modeling followed by conventional

SAR studies. Among them, we concluded that compound

1 had sufficient potency for further development. To

Selective Factor Xa Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3593

Table 4. Selectivity of Compound 2 for Serine Protease

Enzymes: Inhibitory Activities (K_iValue, µM) against Factor

Xa, Thrombin, Plasmin, Trypsin

K_i(µM)^a

compd

factor Xa

thrombin

plasmin

trypsin

2

3

0.019 ( 0.001

0.041 ( 0.002

>100

78.2 ( 2.8 13.6 ( 1.8

23.0 ( 0.8 0.62 ( 0.08

^aThe K_ivalues for each enzyme were determined from Dixon’s

plot constructed at two concentrations of the substrate. Data

represent the mean ( SEM (n ) 3).

Figure 4. Effect of compound 2 and LMWH on venous

thrombosis in rats. Data represent the mean ( SEM: (//) P

< 0.01, (/) P < 0.05, Dunnett’s test (n ) 5-7).¹⁸

Figure 3. Percent inhibition of human factor Xa after

intravenous and oral administration of compound 2 in cyno-

molgus monkey. Data represent the mean ( SEM; n ) 6.¹⁰

obtain stable fine crystals, we examined the crystalliza-

tion of 1 with various organic and inorganic acids and

found that methanesulfonate 2 was obtained as fine

crystals with adequate water solubility. Compound 2

exhibited potent inhibition of FXa (IC₅₀) 0.030 ( 0.002

µM) as well as compound 1, showed resistance to

metabolism in blood and liver, and was excreted in the

urine without formation of any metabolites. Moreover,

compound 2 exhibited no toxicity at effective doses.

Therefore, compound 2 was chosen for further pharma-

cological evaluations.

As depicted in Table 4, selectivity of 2 for FXa relative

to other serine proteases (thrombin, plasmin, and

trypsin) was observed. As for anti-FIIa (anti-thrombin)

activity, none of the compounds synthesized in these

studies showed any such activity (IC₅₀g 10 µM) (see

Tables 1-3). Then, the anti-FXa activity of 2 after

intravenous and oral administration to cynomolgus

monkey was evaluated on the basis of inhibition of

human FXa in plasma (see Figure 3). After oral admin-

istration, inhibition increased to 41 ( 4% at 120 min

and then gradually declined. The plasma concentration

(C_max) of 2 at a dose of 10 mg/kg was 0.39 µg/mL at 120

min.⁹Subsequently, the antithrombotic effect of 2 was

examined in a rat venous thrombosis model and a rat

model of middle cerebral artery thrombosis.

As shown in Figure 4, compound 2 showed a dose-

dependent antithrombotic effect when infused at 0.1-1

mg kg^-1h^-1, and the effect was significant at 0.3 and 1

mg kg^-1h^-1. Low molecular weight heparin (LMWH)

also showed a dose-dependent antithrombotic effect in

this model at doses of 30-300 U kg^-1h^-1. As depicted

in Figure 5, the effect of 2 on the size of brain infarction

in the middle cerebral artery occlusion model was also

evaluated. The infarct size was significantly reduced

when compound 2 (0.1 mg kg^-1h^-1) was administered

as a continuous intravenous infusion from 20 min before

occlusion to 24 h after occlusion. These studies sug-

gested that compound 2 is likely to be useful as both a

venous and arterial antithrombotic agent.

Figure 5. Effect of compound 2 on the infarct volume induced

by middle cerebral artery occlusion with PIT methods in rats.

Data represent the mean ( SEM: (/) P < 0.05, (#) P < 0.1,

Dunnett test, n ) 8-10.¹⁹

In conclusion, a series of novel 2,7-disubstituted

tetrahydroisoquinoline derivatives were designed and

synthesized. Among them, compounds 1 and 2 were

found to show the most potent inhibition of FXa. The

selectivity of compound 2 for FXa relative to other serine

proteases (thrombin, plasmin, and trypsin) was dem-

onstrated. Moreover, compound 2 exhibited good efficacy

when pharmacological evaluation was done in a rat

venous thrombosis model, in a rat middle cerebral

artery occlusion model, and an AV shunt model in

monkeys (iv, po).¹⁰After further evaluation of toxicology

and physical properties (crystallization, pharmaceutical

stability, etc.), compound 2 (JTV-803) was finally se-

lected for clinical studies as a potent FXa inhibitor.

Experimental Section

General Methods (Chemistry). Melting points were

determined with a Yanagimoto (Yanako) micromelting point

apparatus (HK-10D) and are uncorrected. ¹H NMR spectra

(ppm, δ) were recorded using a JEOL JNMA 300 (300 MHz)

spectrometer with tetramethylsilane as the internal standard.

IR spectra (cm^-1) were obtained with a Perkin-Elmer FT 1650

infrared spectrometer. HPLC was performed with a Shimazu

LC-6A instrument, and thin-layer chromatography (TLC) was

carried out on Merck silica gel plates (60F-254). Column

chromatography was performed with Merck silica gel (70-230

mesh). Elemental analyses were accomplished on a Perkin-

Elmer 2400II instrument.

7-Hydroxy-1,2,3,4-tetrahydroisoquinoline Hydrobro-

mide (29). A solution of 7-methoxyisoquinoline⁶(1.43 g, 8.96

mmol) in AcOH (20 mL) was stirred for 3 h under hydrogen

atmosphere (3 atm) in the presence of PtO₂(50 mg). The

mixture was filtered through Celite, and the filtrate was

concentrated in vacuo. The residue was dissolved in 48%

aqueous HBr (35 mL) and heated at reflux for 3 h. The mixture

was concentrated in vacuo, and the solid was washed with

3594 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10

Ueno et al.

EtOH/Et₂O to give compound 29 (1.80 g, 87%): ¹H NMR

(DMSO-d₆) δ 2.84-2.90 (2H, m), 3.25-3.38 (2H, m), 4.17 (2H,

brs), 6.58 (1H, d, J ) 2.7 Hz), 6.67 (1H, dd, J ) 2.7, 8.4 Hz),

7.00 (1H, d, J ) 8.4 Hz), 8.96 (2H, brs), 9.40 (1H, s). Anal.

(C₉H₁₂BrNO) C, H, N.

2-tert-Butoxycarbonyl-7-hydroxy-1,2,3,4-tetrahydroiso-

quinoline (31). To a stirred solution of 29 (370 mg, 1.61 mmol)

in 1 N NaOH (4 mL) and 1,4-dioxane (8 mL) was added Boc₂O

(386 mg, 1.77 mmol). After being stirred at room temperature

for 12 h, the reaction mixture was extracted with EtOAc. The

organic layer was washed with brine and dried over anhydrous

Na₂SO₄, and the solvent was removed under reduced pressure

to give compound 31 (385 mg, 93%): ¹H NMR (CDCl₃) δ 1.49

(9H, s), 2.75 (2H, t, J ) 5.7 Hz), 3.62 (2H, t, J ) 5.7 Hz), 4.52

(2H, brs), 6.60-6.70 (2H, m), 6.99 (1H, d, J ) 8.4 Hz). Anal.

(C₁₄H₁₉NO₃) C, H, N.

N,N′-Di-tert-butoxycarbonyl-7-hydroxy-1,2,3,4-tetra-

hydroisoquinoline-2-carboxamidine (30). To a suspension

of 29 (500 mg, 2.17 mmol) in CH₃CN (5 mL) was added Et₃N

(0.3 mL, 2.15 mmol) at 0 °C, and the mixture was stirred at

room temperature for 1 h. The precipitated solid was collected

by filtration. To the solid in DMF (5 mL) was added 1H-

pyrazole-1-(N,N′-di-tert-butoxycarbonyl)carboxamidine¹¹(742

mg, 2.39 mmol). After being stirred at room temperature for

2 h, the reaction mixture was diluted with water and extracted

with EtOAc. The organic layer was washed with brine, dried

over anhydrous Na₂SO₄, and evaporated under reduced pres-

sure to leave the residue, which was purified by SiO₂column

chromatography (n-hexane/EtOAc ) 3:1) to give compound 30

(700 mg, 82%): ¹H NMR (CDCl₃) δ 1.50 (18H, s), 2.87 (2H,

m), 3.73 (2H, m), 4.62 (2H, m), 6.53 (1H, d, J ) 2.5 Hz), 6.66

(1H, dd, J ) 2.5, 8.2 Hz), 6.97 (1H, d, 1H, J ) 8.2 Hz). Anal.

(C₂₀H₂₉N₃O₅) C, H, N.

4-Hydroxymethyl-1-(pyridin-4-yl)piperidine (35). To a

stirred solution of 1-(pyridin-4-yl)piperidine-4-carboxylic acid¹²

(500 mg, 2.42 mmol) in THF (5 mL) was added 1 M BH₃in

THF solution (14.5 mL, 14.5 mmol) at 0 °C. Stirring was

continued at room temperature for 12 h. The reaction mixture

was poured into ice/water and extracted with EtOAc. The

organic layer was washed with water and brine and dried over

anhydrous Na₂SO₄, and the solvent was removed under

reduced pressure. The residue was dissolved in 12% HCl and

stirred at 50 °C for 1 h. The mixture was neutralized with 4

N NaOH and extracted with EtOAc. The organic layer was

washed with brine and dried over anhydrous Na₂SO₄, and the

solvent was removed under reduced pressure to give compound

35 (400 mg, 86%): ¹H NMR (CDCl₃) δ 1.24-1.38 (2H, m),

1.70-1.90 (3H, m), 2.86 (2H, m), 3.53 (2H, d, J ) 6.2 Hz), 3.92

(2H, m), 6.66 (2H, d, J ) 5.1 Hz), 8.22 (2H, d, J ) 5.1 Hz).

Anal. (C₁₁H₁₆N₂O₂) C, H, N.

4-Hydroxymethyl-1-(quinoline-4-yl)piperidine (36). A

solution of 4-chloroquinoline (2.0 g, 12.22 mmol), ethyl iso-

nipecotinate (2.8 mL, 18.16 mmol), and Et₃N (3.4 mL, 24.39

mmol) in EtOH (7.5 mL) was stirred at 150 °C for 5 days in a

sealed tube. Then water was added and extracted with EtOAc.

The organic layer was washed with brine and dried over

anhydrous Na₂SO₄, and the solvent was removed under

reduced pressure. The residue was purified by SiO₂column

chromatography (n-hexane/acetone ) 3:1) to give ethyl 1-(quin-

olin-4-yl)piperidin-4-carboxylate (3.2 g). The compound (3.0 g,

10.55 mmol) was dissolved in THF (30 mL), and the solution

was added dropwise to a suspension of LiAlH₄(800 mg, 21.08

mmol) in THF (30 mL) at 0 °C under argon atmosphere. The

mixture was stirred at 0 °C for 2 h, aqueous Na₂SO₄solution

at 0 °C was added, and the mixture was filtered through Celite.

The solvent was removed under reduced pressure to leave the

residue, which was washed with isopropyl ether (IPE) to give

compound 36 (2.25 g, 88%): ¹H NMR (CDCl₃) δ 1.57-1.90 (3H,

m), 1.93-2.05 (2H, m), 2.85 (2H, m), 3.64-3.68 (4H, m), 6.84

(1H, d, J ) 4.8 Hz), 7.47 (1H, m), 7.65 (1H, m), 8.01 (1H, m),

8.67 (1H, d, J ) 4.8 Hz). Anal. (C₁₅H₁₈N₂O) C, H, N.

was added NaH (230 mg of a 60% dispersion in mineral oil,

5.75 mmol) at 0 °C. After the mixture was stirred at room

temperature for 30 min, compound 33 (1.80 g, 5.77 mmol) was

added to the resulting slurry. The mixture was stirred at room

temperature for 1 h and diluted with water and extracted with

EtOAc. The organic layer was washed with brine, dried over

anhydrous Na₂SO₄, and evaporated under reduced pressure

to leave the residue, which was purified by SiO₂column

chromatography (n-hexane/acetone ) 6:1) to give compound

1

39 (1.82 g, 79%): H NMR (CDCl₃) δ 1.20-1.35 (2H, m), 1.48

(9H, s), 1.75-2.05 (3H, m), 2.70-2.90 (4H, m), 3.62 (2H, m),

3.77 (2H, d, J ) 6.3 Hz), 4.10-4.35 (2H, m), 4.53 (2H, s), 5.14

(2H, s), 6.67 (1H, d, J ) 2.5 Hz), 6.71 (1H, dd, J ) 2.5, 8.3

Hz), 7.29 (1H, d, J ) 8.3 Hz), 7.26-7.38 (5H, m). Anal.

(C₂₈H₃₆N₂O₅) C, H, N.

tert-Butyl 7-(Piperidin-4-ylmethoxy)-1,2,3,4-tetrahydro-

isoquinolin-2-carboxylate (40). A solution of 39 (1.3 g, 2.70

mmol) in THF (10 mL) and MeOH (20 mL) was stirred for 3 h

under hydrogen atmosphere (3 atm) in the presence of 7.5%

Pd/C (300 mg). The mixture was filtered through Celite, and

the filtrate was concentrated in vacuo to give compound 40

(815 mg, 87%): ¹H NMR (CDCl₃) δ 1.20-1.35 (2H, m), 1.49

(9H, s), 1.75-2.00 (4H, m), 2.62-2.76 (4H, m), 3.10-3.20 (2H,

m), 3.62 (2H, m), 3.76 (2H, d, J ) 6.0 Hz), 4.53 (2H, s), 6.63

(1H, s), 6.71 (1H, d, J ) 8.4 Hz), 7.03 (1H, d, J ) 8.4 Hz).

Anal. (C₂₀H₃₀N₂O₃) C, H, N.

tert-Butyl 7-(Piperidin-4-ylmethoxy)-1,2,3,4-tetrahydro-

isoquinoline-2-carboxylate (37). To a stirred solution of 31

(389 mg, 1.56 mmol) and 35 (300 mg, 1.56 mmol) in THF (15

mL) and CH₂Cl₂(4 mL) were added PPh₃(450 mg, 1.72 mmol)

and diisopropyl azodicarboxylate (DIPAD) (0.34 mL, 1.73

mmol). After 24 h at room temperature, the reaction mixture

was concentrated under reduced pressure to leave the residue,

which was purified by SiO₂column chromatography (n-hexane/

acetone ) 3:2 to 1:1) to give compound 37 (500 mg, 76%): ¹H

NMR (CDCl₃) δ 1.36-1.50 (11H, m), 1.92-2.15 (3H, m), 2.76

(2H, brt), 2.90 (2H, brt), 3.62 (2H, brt), 3.80 (2H, d, J ) 6.3

Hz), 3.90-3.96 (2H, m), 4.53 (1H, s), 6.62-6.74 (4H, m), 7.04

(1H, d, J ) 8.4 Hz), 8.20-8.25 (2H, m). Anal. (C₂₅H₃₃N₃O₃) C,

H, N.

tert-Butyl 7-[1-(Quinolin-4-yl)piperidin-4-ylmethoxy]-

1,2,3,4-tetrahydroisoquinoline-2-carboxylate (38). Com-

pound 38 (280 mg, 71%) was obtained from 31 (206 mg, 0.83

mmol), 36 (200 mg, 0.83 mmol), DIPAD (0.18 mL, 0.91 mmol),

and PPh₃(238 mg, 0.91 mmol) as described for 37: ¹H NMR

(CDCl₃) δ 1.50 (9H, s), 1.65-1.85 (2H, m), 2.00-2.15 (3H, m),

2.78 (2H, t, J ) 6.0 Hz), 2.85-2.95 (2H, m), 3.60-3.70 (4H,

m), 3.92 (2H, d, J ) 6.0 Hz), 4.56 (2H, s), 6.68 (1H, d, J ) 2.7

Hz), 6.77 (1H, dd, J ) 2.7, 8.4 Hz), 6.86 (1H, d, J ) 5.1 Hz),

7.06 (1H, d, J ) 8.4 Hz), 7.45-7.50 (1H, m), 7.62-7.68 (1H,

m), 8.00-8.06 (2H, m), 8.72 (1H, d, J ) 5.1 Hz). Anal.

(C₂₉H₃₅N₃O₃) C, H, N.

tert-Butyl 7-[1-(3-Methoxycarbonylpyridin-4-yl)piperi-

din-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinoline-2-car-

boxylate (42). To a stirred solution of 40 (442 mg, 1.28 mmol)

in EtOH (3 mL) were added 4-chloro-3-formylpyridine⁷(150

mg, 1.06 mmol) and Et₃N (0.3 mL, 2.15 mmol). After the

mixture was heated at reflux for 27 h, THF and Et₂O were

added and insoluble material was removed. The solvent was

evaporated under reduced pressure to leave the residue, which

was purified by SiO₂column chromatography (CHCl₃/MeOH

) 100:1) to give compound 41 (430 mg, 90%): ¹H NMR (CDCl₃)

δ 1.49 (9H, m), 1.60-1.70 (2H, m), 1.95-2.15 (3H, m), 2.76

(2H, m), 3.07 (2H, m), 3.60-3.65 (4H, m), 3.86 (2H, d, J ) 6.3

Hz), 4.54 (2H, s), 6.65 (1H, s), 6.73 (1H, d, J ) 8.4 Hz), 6.84

(1H, d, J ) 6.0 Hz), 7.05 (1H, d, J ) 8.4 Hz), 8.42 (1H, d, J )

6.0 Hz), 8.74 (1H, s), 10.02 (1H, s). To a stirred solution of 41

(400 mg, 0.89 mmol) in CHCl₃(4 mL) and MeOH (1.5 mL) were

added NaCN (65 mg, 1.33 mmol), MnO₂(2 g, 11.5 mmol), and

AcOH (0.025 mL, 0.44 mmol). After 12 h at room temperature,

the solvent was evaporated under reduced pressure to leave

the residue, which was purified by SiO₂column chromatog-

raphy (CHCl₃/MeOH ) 15:1) to give compound 42 (400 mg,

93 mmol): ¹H NMR (CDCl₃) δ 1.49-1.70 (11H, m), 1.90-2.10

Benzyl 4-(2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroiso-

quinolin-7-yloxymethyl)piperidin-1-carboxylate (39). To

a stirred solution of 31 (1.2 g, 4.81 mmol) in DMF (15 mL)

Selective Factor Xa Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3595

(3H, m), 2.76 (2H, m), 2.97 (2H, m), 3.50-3.68 (4H, m), 3.83

(2H, d, J ) 6.3 Hz), 3.91 (3H, s), 4.54 (2H, s), 6.64 (1H, s),

6.72 (1H, d, J ) 8.4 Hz), 6.78 (1H, d, J ) 6.0 Hz), 7.04 (1H, d,

J ) 8.4 Hz), 8.35 (1H, d, J ) 6.0 Hz), 8.73 (1H, s). Anal.

(C₂₇H₃₅N₃O₅) C, H, N.

NMR (CDCl₃) δ 1.20-1.40 (2H, m), 1.56 (18H, s), 1.75-2.00

(3H, m), 2.60-2.70 (2H, m), 2.85-2.95 (2H, m), 3.10-3.20 (2H,

m), 3.65-3.85 (4H, m), 4.67 (2H, s), 6.62 (1H, d, J ) 2.7 Hz),

6.72 (1H, dd, J ) 2.7, 8.4 Hz), 7.03 (1H, J ) 8.4 Hz). Anal.

(C₂₆H₄₀N₄O₅) C, H, N.

7-(Piperidin-4-ylmethoxy)-1,2,3,4-tetrahydroisoquino-

lin-2-carboxamidine (5). To a stirred solution of 34 (70 mg,

0.14 mmol) in CHCl₃(0.7 mL) was added TFA (0.35 mL).

Stirring was continued at room temperature for 5 h. The

solvent was removed under reduced pressure to leave the

residue, which was treated with HCl/EtOH to give compound

5 (50 mg, 95%) as a dihydrochloride: ¹H NMR (DMSO-d₆) δ

1.40-1.55 (2H, m), 1.75-1.90 (2H, m), 1.90-2.10 (1H, m), 2.82

(2H, t, J ) 5.7 Hz), 2.85-2.95 (2H, m), 3.25-3.30 (2H, m),

3.57 (2H, t, J ) 5.7 Hz), 3.83 (2H, d, J ) 6.2 Hz), 4.54 (2H, s),

6.71 (1H, d, J ) 2.2 Hz), 6.83 (1H, dd, J ) 2.2, 8.4 Hz), 7.14

(1H, d, J ) 8.4 Hz), 7.60 (4H, brs). Anal. (C₁₆H₂₄N₄O‚2.1HCl‚

2.3H₂O) C, H, N.

Ethyl 3-[4-[4-(2-tert-Butoxycarbonyl-1,2,3,4-tetrahydro-

isoquinolin-7-yloxymethyl)piperidin-1-yl]pyridin-3-yl]-

2-propenate (43). To a stirred slurry of triethyl phosphono-

acetate (0.05 mL, 0.252 mmol) in THF (1 mL) and NaH (12

mg of 60% dispersion in mineral oil, 0.300 mmol) at room

temperature was added dropwise a solution of 41 (95 mg, 0.210

mmol) in THF (1 mL). The mixture was stirred at room

temperature for 2 h. The reaction mixture was quenched with

aqueous NH₄Cl solution and extracted with EtOAc. The

organic layer was washed with brine, dried over anhydrous

Na₂SO₄, and evaporated under reduced pressure to leave the

residue, which was purified by SiO₂column chromatography

(CHCl₃/MeOH ) 20:1) to give compound 43 (100 mg, 91%):

¹H NMR (CDCl₃) δ 1.35 (3H, t, J ) 6.4 Hz), 1.49 (9H, s), 1.50-

1.80 (2H, m), 1.95-2.10 (3H, m), 2.75-2.95 (4H, m), 3.40-

3.50 (2H, m), 3.63 (2H, m), 3.86 (2H, d, J ) 6.3 Hz), 4.27 (2H,

q, J ) 6.4 Hz), 4.54 (2H, s), 6.45 (1H, d, J ) 15.9 Hz), 6.65

(1H, s), 6.74 (1H, d, J ) 8.4 Hz), 6.83 (1H, d, J ) 6.0 Hz), 7.05

(1H, d, J ) 8.4 Hz), 7.76 (1H, d, J ) 15.9 Hz), 8.38 (1H, d, J

) 6.0 Hz), 8.55 (1H, s). Anal. (C₃₀H₃₉N₃O₅) C, H, N.

Benzyl 4-Bromomethylpiperidine-1-carboxylate (33).

To a stirred solution of 1-benzyloxycarbonylpiperidine-4-

carboxylic acid¹³(10.0 g, 37.98 mmol) in THF (100 mL) were

added dropwise Et₃N (5.56 mL, 39.89 mmol) and isobutyl

chloroformate (IBCF) (5.1 mL, 39.32 mmol) at -15 °C under

argon atmosphere. After being stirred at -15 °C for 20 min,

the reaction mixture was filtered, and the filtrate was added

dropwise to an aqueous NaBH₄(4.3 g, 0.11 mmol) solution at

0 °C. The mixture was stirred at 0 °C for 15 min, then at room

temperature for 2 h, and quenched with water. After removal

of insoluble material, the solvent was removed under reduced

pressure to leave the residue, which was extracted with EtOAc.

The organic layer was washed with 1 N NaOH and dried over

anhydrous Na₂SO₄, and the solvent was removed under

reduced pressure to give benzyl 4-hydroxymethylpiperidine-

1-carboxylate (7.2 g, 76%): ¹H NMR (CDCl₃) δ 1.05-1.30 (2H,

m), 1.55-1.80 (3H, m), 2.70-2.85 (2H, m), 3.50 (2H, d, J )

6.3 Hz), 4.10-4.30 (2H, m), 5.13 (2H, s), 7.26-7.40 (5H, m).

7-(1-Acetylpiperidin-4-ylmethoxy)-1,2,3,4-tetrahydroiso-

quinolin-2-carboxyamidine (6). Step 1. To a stirred solu-

tion of 34 (100 mg, 0.21 mmol) in THF (1.5 mL) were added

Ac₂O (0.021 mL, 0.22 mmol) and pyridine (0.025 mL, 0.31

mmol). Stirring was continued at room temperature for 2 h.

The reaction mixture was diluted with water and extracted

with EtOAc. The organic layer was washed with water and

brine, dried over anhydrous Na₂SO₄, and evaporated under

reduced pressure to give N,N′-di-tert-butoxycarbonyl-7-(1-

acetylpiperidin-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinolin-2-

1

carboxamidine (100 mg, 90%): H NMR (CDCl₃) δ 1.20-1.40

(2H, m), 1.45-1.65 (20H, m), 1.80-2.10 (6H, m), 2.50-2.65

(1H, m), 2.85-2.95 (2H, m), 3.00-3.20 (2H, m), 3.85-3.90 (4H,

m), 4.67 (2H, brs), 6.62 (1H, d, J ) 2.4 Hz), 6.72 (1H, dd, J )

2.4, 8.4 Hz), 7.04 (1H, d, J ) 8.4 Hz).

Step 2. To a stirred solution of the compound (94 mg, 0.18

mmol) in CHCl₃(1.0 mL) was added TFA (0.5 mL). Stirring

was continued at room temperature for 5 h. The solvent was

removed under reduced pressure to leave the residue, which

was treated with HCl/EtOH to give compound 6 (60 mg, 91%)

as a hydrochloride: ¹H NMR (DMSO-d₆) δ 1.00-1.30 (2H, m),

1.75-1.85 (2H, m), 1.98 (4H, m), 2.52 (1H, m), 2.81 (2H, t, J

) 6.0 Hz), 3.18-3.25 (1H, m), 3.57 (2H, t, J ) 6.0 Hz), 3.57-

3.90 (3H, m), 4.53 (2H, s), 6.69 (1H, d, J ) 2.4 Hz), 6.82 (1H,

dd, J ) 2.4, 8,4 Hz), 7.13 (1H, d, J ) 8.4 Hz), 7.57 (4H, brs).

Anal. (C₁₈H₂₆N₄O₂‚HCl‚2.2H₂O) C, H, N.

7-(1-Acetoimidoylpiperidin-4-ylmethoxy)-1,2,3,4-tetra-

hydroisoquinolin-2-carboxamidine (7). To a stirred solu-

tion of 34 (100 mg, 0.21 mmol) in THF (1.5 mL) and EtOH

(1.5 mL) were added Et₃N (0.086 mL, 0.62 mmol) and ethyl

acetoimidate hydrochloride (38 mg, 0.31 mmol). After being

stirred at room temperature for 18 h, the reaction mixture was

diluted with EtOAc and Et₂O. Insoluble material was removed,

and the solvent was evaporated under reduced pressure to

give N,N′-di-tert-butoxycarbonyl-7-(1-acetoimidoylpiperidin-4-

ylmethoxy)-1,2,3,4-tetrahydroisoquinolin-2-carboxyamidine (105

mg, 94%): ¹H NMR (CDCl₃) δ 1.43-1.54 (20H, m), 2.00-2.20

(3H, m), 2.46 (3H, s), 2.85-2.95 (2H, m), 3.05-3.15 (2H, m),

3.70-3.95 (5H, m), 4.67 (2H, brs), 4.80-4.90 (1H, m), 6.60 (1H,

s), 6.70 (1H, d, J ) 8.4 Hz), 7.04 (1H, d, J ) 8.4 Hz). The

compound (100 mg, 0.19 mmol) was treated as described in

step 2 for 6 to give compound 7 (67 mg, 88%) as a dihydro-

chloride: ¹H NMR (DMSO-d₆) δ 1.28-1.48 (2H, m), 1.86-1.90

(2H, m), 2.12 (1H, m), 2.29 (3H, s), 2.83 (2H, t, J ) 4.4 Hz),

3.00-3.29 (2H, m), 3.59 (2H, t, J ) 4.4 Hz), 3.85 (2H, m), 3.92-

4.20 (2H, m), 4.56 (2H, s), 6.72 (1H, d, J ) 1.7 Hz), 6.84 (1H,

dd, J ) 1.7, 6.3 Hz), 7.15 (1H, d, J ) 6.3 Hz), 7.65 (4H, brs),

8.77 (1H, brs), 9.33 (1H, brs). Anal. (C₁₈H₂₇N₅O‚2.1HCl‚

2.4H₂O) C, H, N.

To a stirred solution of the compound (5.5 g, 22.06 mmol)

in CH₂Cl₂(55 mL) were added CBr₄(8.85 g, 26.68 mmol) and

PPh₃(7.0 g, 26.68 mmol). After the mixture was stirred at room

temperature for 5 h, the solvent was removed under reduced

pressure to leave the residue, which was purified by SiO₂

column chromatography (n-hexane/acetone ) 10:1) to give

compound 33 (6.25 g, 91%): ¹H NMR (CDCl₃) δ 1.05-1.30 (2H,

m), 1.70-1.90 (3H, m), 2.70-2.90 (2H, m), 3.29 (2H, d, J )

6.0 Hz), 4.10-4.30 (2H, m), 5.13 (2H, s), 7.26-7.38 (5H, m).

Anal. (C₁₄H₁₈BrNO₂) C, H, N.

N,N′-Di-tert-butoxycarbonyl-7-(piperidin-4-ylmethoxy)-

1,2,3,4-tetrahydroisoquinolin-2-carboxamidine (34). To a

stirred solution of 30 (50 mg, 0.13 mmol) and 33 (120 mg, 0.38

mmol) in DMSO (1 mL) was added 4 N NaOH (0.13 mL).

Stirring was continued at room temperature for 17 h. The

reaction mixture was diluted with water and extracted with

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

dried over anhydrous Na₂SO₄, and evaporated under reduced

pressure. The residue was purified by SiO₂column chroma-

tography (n-hexane/acetone ) 5:1) to afford benzyl 4-[2-(N,N′-

di-tert-butoxycarbonylamidin)-1,2,3,4-tetrahydroisoquinolin-7-

1

yloxymethyl]piperidine-1-carboxylate (55 mg, 68%): H NMR

(CDCl₃) δ 1.20-1.35 (2H, m), 1.51 (18H, s), 1.75-2.05 (3H,

m), 2.70-2.95 (4H, m), 3.65-3.80 (4H, m), 4.15-4.35 (2H, m),

4.67 (2H, brs), 5.14 (2H, s), 6.61 (1H, d, J ) 2.7 Hz), 6.71 (1H,

dd, J ) 2.7, 8.4 Hz), 7.03 (1H, d, J ) 8.4 Hz), 7.30-7.40 (5H,

m). A solution of the compound (50 mg, 0.081 mmol) in THF

(0.5 mL) and EtOH (10 mL) was stirred for 3 h under hydrogen

atmosphere (3 atm) in the presence of 7.5% Pd/C (15 mg). The

mixture was filtered through Celite, and the filtrate was

concentrated in vacuo to give compound 34 (38 mg, 96%): ¹H

7-[1-(Pyridin-4-yl)piperidin-4-ylmethoxy]-1,2,3,4-tetra-

hydroisoquinolin-2-carboxamidine (8). To a stirred solu-

tion of 37 (500 mg, 1.18 mmol) in CHCl₃(5 mL) was added

TFA (2.5 mL). After the mixture was stirred at room temper-

ature for 1 h, the solvent was removed under reduced pressure.

Aqueous NaHCO₃solution was added, and the mixture was

extracted with EtOAc. The organic layer was washed with

3596 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10

Ueno et al.

water and brine and dried over anhydrous Na₂SO₄, and the

solvent was removed under reduced pressure to give 7-[1-

(pyridin-4-yl)piperidin-4-ylmethoxy]-1,2,3,4-tetrahydroisoquin-

oline (350 mg, 92%): ¹H NMR (CDCl₃) δ 1.30-1.49 (2H, m),

1.91-2.15 (3H, m), 2.72 (2H, t, J ) 6.0 Hz), 2.85-2.95 (2H,

m), 3.11 (2H, t, J ) 6.0 Hz), 3.79 (2H, d, J ) 6.6 Hz), 3.90-

3.98 (4H, m), 6.54 (1H, d, J ) 2.4 Hz), 6.66-6.72 (3H, m), 6.99

(1H, d, J ) 8.4 Hz), 8.25 (2H, m).

To a stirred solution of the compound (300 mg, 0.92 mmol)

in DMF (0.5 mL) and diisopropylethylamine (DIPEA, 0.16 mL,

0.92 mmol) was added 1H-pyrazole-1-carboxamidine hydro-

chloride (140 mg, 0.96 mmol). After the mixture was stirred

at room temperature for 1 h, the reaction mixture was diluted

with Et₂O and insoluble material was removed. The solvent

was evaporated under reduced pressure to leave the residue,

which was treated with HCl/EtOH to give compound 8 (265

mg, 66%) as a dihydrochloride: ¹H NMR (DMSO-d₆) δ 1.20-

1.45 (2H, m), 1.88-1.93 (2H, m), 2.17 (1H, m), 2.81 (2H, t, J

) 6.0 Hz), 3.20 (2H, m), 3.57 (2H, t, J ) 6.0 Hz), 3.85 (2H, m),

4.23-4.28 (2H, m), 4.54 (2H, s), 6.70 (1H, d, J ) 2.7 Hz), 6.82

(1H, dd, J ) 2.7, 8.4 Hz), 7.13 (1H, d, J ) 8.4 Hz), 7.19 (2H,

d, J ) 7.8 Hz), 7.64 (4H, brs), 8.19 (2H, d, J ) 7.8 Hz), 13.68

(1H, brs). Anal. (C₂₁H₂₇N₅O‚2 HCl‚2.6 H₂O) C, H, N.

7-[1-(Quinolin-4-yl)piperidin-4-ylmethoxy]-1,2,3,4-tetra-

hydroisoquinolin-2-carboxamidine (9). Compound 9 (65

mg, 89%) was obtained as a dihydrochloride from 38 (70 mg,

0.15 mmol), 1H-pyrazole-1-carboxamidine hydrochloride (28

mg, 0.19 mmol), and DIPEA (0.033 mL, 0.19 mmol) as

described for 8: ¹H NMR (DMSO-d₆) δ 1.55-1.66 (2H, m),

1.96-2.02 (2H, m), 2.22 (1H, m), 2.82 (2H, t, J ) 6.0 Hz), 3.49

(2H, m), 3.58 (2H, t, J ) 6.0 Hz), 3.92 (2H, m), 4.17-4.23 (2H,

m), 4.56 (2H, s), 6.73 (1H, d, J ) 2.4 Hz), 6.85 (1H, dd, J )

2.4, 8.1 Hz), 7.15 (1H, d, J ) 8.4 Hz), 7.20 (1H, d, J ) 6.9 Hz),

7.64 (4H, brs), 7.69 (1H, m), 7.96 (1H, m), 8.12 (2H, m), 8.63

(1H, d, J ) 6.9 Hz). Anal. (C₂₅H₂₉N₅O‚2.1HCl‚2.5H₂O) C, H,

N.

7-[1-(2-Aminobenzyl)piperidin-4-ylmethoxy]-1,2,3,4-

tetrahydroisoquinolin-2-carboxamidine (12). To a stirred

solution of 34 (100 mg, 0.205 mmol) in DMF (1 mL) and THF

(1 mL) were added 9 N NaOH (0.027 mL, 0.246 mmol) and

2-nitrobenzyl bromide (53 mg, 0.246 mmol). The mixture was

stirred at room temperature for 1 h, quenched with water, and

extracted with EtOAc. The organic layer was washed with

brine, dried over anhydrous Na₂SO₄, and evaporated under

reduced pressure to leave the residue, which was purified by

SiO₂column chromatography (n-hexane/acetone ) 7:1) to give

N,N′-di-tert-butoxycarbonyl-7-[1-(2-nitrophenylmethyl)piperi-

din-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinolin-2-carboxami-

dine (90 mg, 70%). Successively, the compound (90 mg, 0.144

mmol) was treated with TFA as described in step 2 for 6 to

give 7-[1-(2-nitrophenylmethyl)piperidin-4-ylmethoxy]-1,2,3,4-

tetrahydroisoquinolin-2-carboxamidine (70 mg, 98%) as a

dihydrochloride. Without purification, a solution of the com-

pound (70 mg, 0.141 mmol) in 1 N HCl (1 mL) and MeOH (1

mL) was stirred for 2 h under hydrogen atmosphere (3 atm)

in the presence of 7.5% Pd/C (15 mg). The mixture was filtered

through Celite, and the filtrate was concentrated in vacuo to

give compound 12 (70 mg, 99%) as a trihydrochloride: ¹H NMR

(DMSO-d₆) δ 1.71 (2H, m), 1.97 (3H, m), 2.81 (2H, m), 3.12

(2H, m), 3.57 (2H, m), 3.83 (2H, m), 4.38 (2H, s), 4.54 (2H, s),

6.71 (1H, s), 6.83 (1H, d, J ) 8.4 Hz), 7.02 (1H, m), 7.13 (2H,

m), 7.34 (1H, m), 7.51 (1H, m), 7.64 (4H, brs). Anal. (C₂₃H₃₁N₅O‚

3HCl‚2.5H₂O) C, H, N.

7-[1-(3-Aminobenzyl)piperidin-4-ylmethoxy]-1,2,3,4-

tetrahydroisoquinolin-2-carboxamidine (13). Compound

13 (60 mg, 58%) was obtained as a trihydrochloride from 34

(100 mg, 0.205 mmol), 3-nitrobenzyl bromide (53 mg, 0.246

mmol), and 9 N NaOH (0.027 mL, 0.246 mmol) as described

for 12: ¹H NMR (DMSO-d₆) δ 1.67(2H, m), 1.95 (3H, m), 2.81

(2H, m), 2.96 (2H, m), 3.34 (2H, m), 3.56 (2H, m), 3.86 (2H,

m), 6.70 (1H, s), 6.81 (1H, d, J ) 8.4 Hz), 7.07-7.37 (5H, m),

7.56 (4H, brs), 10.52 (1H, brs). Anal. (C₂₃H₃₁N₅O‚3HCl‚2.4H₂O)

C, H, N.

7-[1-(Pyridin-4-ylmethyl)piperidin-4-ylmethoxy]-1,2,3,4-

tetrahydroisoquinolin-2-carboxamidine (10). To a stirred

solution of 34 (100 mg, 0.20 mmol) in THF (1 mL) and DMF

(1 mL) were successively added 9.1 N NaOH (0.068 mL, 0.62

mmol) and 4-picolyl chloride hydrochloride (51 mg, 0.31 mmol).

After being stirred at 50 °C for 5 h, the reaction mixture was

quenched with water and extracted with EtOAc. The organic

layer was washed with water and brine, dried over anhydrous

Na₂SO₄, and evaporated under reduced pressure to leave the

residue, which was purified by SiO₂preparative TLC (CHCl₃/

MeOH ) 20:1) to give N,N′-di-tert-butoxycarbonyl-7-[1-(pyri-

din-4-ylmethyl)piperidin-4-ylmethoxy]-1,2,3,4-tetrahydroiso-

quinolin-2-carboxamidine (93 mg, 80%): ¹H NMR (CDCl₃) δ

1.50 (18H, s), 1.40-1.90 (5H, m), 2.00-2.10 (2H, m), 2.85-

2.95 (4H, m), 3.51 (2H, s), 3.70-3.85 (4H, m), 4.67 (2H, brs),

6.62 (1H, s), 6.72 (1H, d, J ) 8.4 Hz), 7.03 (1H, d, J ) 8.4 Hz),

7.28 (2H, d, J ) 6.0 Hz), 8.54 (2H, d, J ) 6.0 Hz). The

compound (88 mg, 0.15 mmol) was treated as described in step

2 for 6 to give compound 10 (65 mg, 89%) as a trihydrochlo-

ride: ¹H NMR (DMSO-d₆) δ 1.70-2.00 (5H, m), 2.81 (2H, m),

3.00 (2H, m), 3.38 (2H, m), 3.57 (2H, m), 3.81 (2H, m), 4.48-

4.54 (4H, m), 6.71 (1H, s), 6.82 (1H, d, J ) 8.4 Hz), 7.13 (1H,

d, J ) 8.4 Hz), 7.60 (4H, brs), 8.19 (2H, d, J ) 6.0 Hz), 8.88

(2H, d, J ) 6.0 Hz), 11.63 (1H, brs). Anal. (C₂₂H₂₉N₅O‚3.1HCl‚

2.2H₂O) C, H, N.

7-[1-(Pyridin-2-ylmethyl)piperidin-4-ylmethoxy]-1,2,3,4-

tetrahydroisoquinolin-2-carboxamidine (11). Compound

11 (65 mg, 63%) was obtained as a trihydrochloride from 34

(110 mg, 0.225 mmol), 2-picolyl chloride hydrochloride (44 mg,

0.270 mmol), and 9 N NaOH (0.060 mL, 0.541 mmol), followed

by deprotection of the Boc groups with TFA as described for

10. ¹H NMR (DMSO-d₆) δ 1.75 (2H, m), 2.00 (2H, m), 2.10 (1H,

m), 2.88 (2H, m), 3.16 (2H, m), 3.49 (2H, m), 3.63 (2H, m),

3.91 (2H, m), 4.51 (2H, s), 4.60 (2H, s), 6.77 (1H, d, J ) 2.3

Hz), 6.89 (1H, dd, J ) 2.3, 8.4 Hz), 7.21 (1H, d, J ) 8.4 Hz),

7.55 (1H, m), 7.63 (4H, brs), 7.69 (1H, m), 7.99 (1H, m), 8.74

(1H, m), 10.35 (1H, brs). Anal. (C₂₂H₂₉N₅O‚3.1HCl‚2.5H₂O) C,

H, N.

Methyl 4-[4-(1,2,3,4-Tetrahydroisoquinolin-7-yloxy-

methyl)piperidin-1-yl]pyridin-3-carboxylate (14). Com-

pound 14 (15 mg, 39%) was obtained as a dihydrochloride from

42 (37 mg, 0.077 mmol), 1H-pyrazole-1-carboxamidine hydro-

chloride (13 mg, 0.092 mmol), and DIPEA (0.016 mL, 0.092

mmol) as described for 8: ¹H NMR (DMSO-d₆) δ 1.34-1.54

(2H, m), 1.87-1.93 (2H, m), 2.15 (1H, m), 2.82 (2H, m), 3.29

(2H, m), 3.57 (2H, m), 3.76-3.87 (7H, m), 4.54 (2H, s), 6.71

(1H, s), 6.82 (1H, d, J ) 8.4 Hz), 7.14 (1H, d, J ) 8.4 Hz), 7.40

(1H, d, J ) 7.2 Hz), 7.59 (4H, brs), 8.30 (1H, d, J ) 7.2 Hz),

8.58 (1H, s). Anal. (C₂₃H₂₉N₅O₃‚2HCl‚2.8H₂O) C, H, N.

4-[4-(1,2,3,4-Tetrahydroisoquinolin-7-yloxymethyl)-

piperidin-1-yl]pyridin-3-carboxylic Acid (15). To a stirred

solution of 14 (43 mg, 0.087 mmol) in MeOH (0.2 mL) was

added 1 N NaOH (0.3 mL), and the mixture was heated at

reflux for 3 h. After neutralization with 1 N HCl, the solvent

was removed under reduced pressure to leave the residue,

which was purified by reversed-phase HPLC (0.1% aqueous

TFA/CH₃CN ) 6:4). The fraction containing the desired

product was treated with HCl aqueous solution to give

compound 15 (15 mg, 36%) as a dihydrochloride. ¹H NMR

(DMSO-d₆) δ 1.38-1.58 (2H, m), 1.85-1.95 (2H, m), 2.15 (1H,

m), 2.82 (2H, m), 3.56 (2H, m), 3.80-3.95 (4H, m), 4.51 (2H,

s), 6.70 (1H, m), 6.82 (1H, m), 7.14 (1H, d, J ) 8.4 Hz), 7.34

(1H, d, J ) 7.4 Hz), 7.44 (4H, brs), 8.27 (1H, d, J ) 7.4 Hz),

8.59 (1H, s). Anal. (C₂₂H₂₇N₅O₃‚2HCl‚2.6H₂O) C, H, N.

Ethyl 3-[4-[4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-

7-yloxymethyl)piperidin-1-yl]piperidin-3-yl]-2-pro-

penate (44). Compound 44 (50 mg, 49%) was obtained as a

dihydrochloride from 43 (100 mg, 0.191 mmol), 1H-pyrazole-

1-carboxamidine hydrochloride (27 mg, 0.184 mmol), and

DIPEA (0.032 mL, 0.184 mmol) as described for 8: ¹H NMR

(DMSO-d₆) δ 1.25 (3H, t, J ) 7.3 Hz), 1.40-1.55 (2H, m), 1.82-

2.05 (3H, m), 2.76-2.96 (4H, m), 3.42 (2H, m), 3.57 (2H, m),

3.90 (2H, m), 4.18 (2H, q, J ) 7.3 Hz), 4.53 (2H, s), 6.63 (1H,

d, J ) 16.1 Hz), 6.73 (1H, s), 6.85 (1H, d, J ) 8.4 Hz), 7.04

(1H, d, J ) 6.0 Hz), 7.14 (1H, d, J ) 8.4 Hz), 7.51 (4H, brs),

Selective Factor Xa Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3597

7.60 (1H, d, J ) 16.1 Hz), 8.35 (1H, d, J ) 6.0 Hz), 8.64 (1H,

s). Anal. (C₂₆H₃₃N₅O₃‚2HCl‚2.4H₂O) C, H, N.

residue, which was purified by SiO₂column chromatography

(CHCl₃/MeOH ) 20:1 to 10:1) to give compound 47 (10.87 g,

92%): ¹H NMR (CDCl₃) δ 1.25 (3H, t, J ) 7.3 Hz), 1.65-1.80

(2H, m), 2.30-2.40 (2H, m), 2.77 (2H, m), 3.10-3.20 (2H, m),

3.65-3.80 (4H, m), 3.96 (2H, s), 4.22 (2H, q, J ) 7.3 Hz), 4.60

(2H, s), 5.17 (2H, s), 6.61 (1H, brs), 6.65-6.71 (3H, m), 7.02

(1H, d, J ) 8.0 Hz), 7.26-7.40 (5H, m), 8.25 (2H, m). Anal.

(C₃₁H₃₅N₃O₅) C, H, N.

4-[4-(2-Benzyloxycarbonyl-1,2,3,4-tetrahydroisoquino-

lin-7-oxymethyl)-4-ethoxycarbonylpiperidin-1-yl]-1-

methylpyridinium Chloride (55). To a stirred solution of

compound 47 (80 mg, 0.151 mmol) in acetone (3 mL) were

added MeI (0.20 mL, 3.210 mmol) and DIPEA (0.026 mL, 0.151

mmol). After the mixture was stirred at room temperature for

12 h, the solvent was removed under reduced pressure to leave

the residue, which was diluted with water and CHCl₃. The

organic layer was separated, washed with brine, dried over

anhydrous Na₂SO₄, and evaporated under reduced pressure

to leave the residue, which was treated with HCl/dioxane to

give compound 55 (95 mg, 97%). The crude product was used

for the next reaction without further purification.

Benzyl 7-(1-tert-Butoxylcarbonyl-4-ethoxycarbonyl-

piperidin-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinolin-

2-carboxylate (49). To a stirred solution of 48 (300 mg, 1.17

mmol) in THF (3 mL) was added dropwise LDA (0.97 mL of

1.5 M solution in THF, 1.46 mmol) at -70 °C under argon

atmosphere. After the mixture was stirred at -70 °C for 50

min, a solution of 45 (213 mg, 0.64 mmol) in THF (3 mL) was

added dropwise to the solution at -70 °C. The mixture was

allowed to warm to room temperature and was stirred for 2

h. The reaction mixture was quenched with aqueous NH₄Cl

solution and extracted with EtOAc. The organic layer was

washed with brine, dried over anhydrous Na₂SO₄, and evapo-

rated under reduced pressure to leave the residue, which was

purified by SiO₂column chromatography (n-hexane/acetone

) 7:1) to give compound 49 (170 mg, 48%): ¹H NMR (CDCl₃)

δ 1.23 (3H, t, J ) 7.2 Hz), 1.49 (9H, s), 1.52-1.62 (2H, m),

2.16-2.21 (2H, m), 2.77 (2H, m), 3.04 (2H, m), 3.69 (2H, m),

3.80-4.00 (4H, m), 4.19 (2H, q, J ) 7.2 Hz), 4.60 (2H, s), 5.17

(2H, s), 6.60 (1H, brs), 6.69 (1H, dd, J ) 2.4, 8.4 Hz), 7.02 (1H,

d, J ) 8.4 Hz), 7.32-7.38 (5H, m). Anal. (C₃₁H₄₀N₂O₇) C, H,

N.

Ethyl 4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-

yloxymethyl)-1-(pyridin-4-yl)piperidin-4-carboxylate (16).

A stirred solution of 47 (12.1 g, 22.85 mmol) in 30% HCl/EtOH

(200 mL) was heated at 80 °C for 1 h. The solvent was removed

under reduced pressure, and the residue was dissolved in DMF

(50 mL). To the solution were added 1H-pyrazole-1-carbox-

amidine hydrochloride (3.69 g, 25.17 mmol) and diisopropyl-

ethylamine (15.4 mL, 88.38 mmol), and the mixture was

stirred at room temperature for 12 h. The solvent was

evaporated under reduced pressure to leave the residue, which

was purified by reversed-phase HPLC (0.05% TFA in H₂O/

MeOH ) 6:4). The fraction containing desired product was

treated with HCl/EtOH to give compound 16 (9.2 g, 92%) as a

dihydrochloride: ¹H NMR (DMSO-d₆) δ 1.15 (3H, t, J ) 7.1

Hz), 1.65-1.80 (2H, m), 2.81 (2H, t, J ) 6.0 Hz), 3.30-3.50

(2H, m), 3.56 (2H, t, J ) 6.0 Hz), 3.95-4.18 (6H, m), 4.53 (2H,

s), 6.68 (1H, d, J ) 2.1 Hz), 6.81 (1H, dd, J ) 2.1, 8.4 Hz),

7.14 (1H, d, J ) 8.4 Hz), 7.19 (2H, d, J ) 7.5 Hz), 7.58 (4H,

brs), 8.22 (2H, d, J ) 7.5 Hz). Anal. (C₂₄H₃₁N₅O₃‚2.1HCl‚

2.2H₂O) C, H, N.

Ethyl 3-[4-[4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-

7-yloxymethyl)piperidin-1-yl]pyridin-3-yl]-2-propi-

onate (17). A solution of 44 (35 mg, 0.056 mmol) in EtOH (2

mL) was stirred for 3 h under hydrogen atmosphere (3 atm)

in the presence of 7.5% Pd/C (10 mg). The mixture was filtered

through Celite, and the filtrate was concentrated in vacuo to

give compound 17 (30 mg, 86%) as a dihydrochloride: ¹H NMR

(DMSO-d₆) δ 1.14 (3H, t, J ) 7.2 Hz), 1.44 (2H, m), 1.91 (2H,

m), 2.09 (1H, m), 2.72-2.84 (4H, m), 2.95 (2H, m), 3.15 (2H,

m), 3.57 (2H, m), 3.76 (2H, m), 3.88 (2H, m), 4.04 (2H, q, J )

7.2 Hz), 4.54 (2H, s), 6.72 (1H, s), 6.83 (1H, d, J ) 8.4 Hz),

7.14 (1H, d, J ) 8.4 Hz), 7.26 (1H, m), 7.60 (4H, brs), 8.33

(2H, m). Anal. (C₂₆H₃₅N₅O₃‚2HCl‚2.1H₂O) C, H, N.

3-[4-[4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-

yloxymethyl)piperidin-1-yl]pyridin-3-yl]-2-propionic acid

(18). Compound 18 (20 mg, 85%) was obtained as a dihydro-

chloride from 17 (25 mg, 0.046 mmol) and 1 N NaOH (0.2 mL)

as described for compound 15: ¹H NMR (DMSO-d₆) δ 1.46 (2H,

m), 1.90 (3H, m), 2.59-2.87 (8H, m), 3.57 (2H, m), 3.88 (2H,

m), 4.53 (2H, s), 6.72 (1H, s), 6.84 (1H, d, J ) 8.4 Hz), 6.97

(1H, m), 7.14 (1H, d, J ) 8.4 Hz), 7.51 (4H, brs), 8.25 (2H, m).

Anal. (C₂₄H₃₁N₅O₃‚2.1HCl‚2.5H₂O) C, H, N.

Benzyl 7-Chloromethoxy-1,2,3,4-tetrahydroisoquino-

lin-2-carboxylate (45).¹⁴To a stirred solution of 32 (2.5 g,

8.82 mmol) in DMF (25 mL) was added NaH (390 mg of a 60%

dispersion in mineral oil, 9.75 mmol) at 0 °C. After the mixture

was stirred at 0 °C for 30 min, MeSCH₂Cl (0.96 mL, 11.46

mmol) was added. The mixture was stirred at room temper-

ature for 12 h. The reaction mixture was quenched with water

and extracted with EtOAc. The organic layer was washed with

brine, dried over anhydrous Na₂SO₄, and evaporated under

reduced pressure to leave the residue, which was purified by

SiO₂column chromatography (n-hexane/EtOAc ) 8:1) to give

benzyl 7-methylthiomethoxy-1,2,3,4-tetrahydroisoquinolin-2-

carboxylate (2.7 g, 89%): ¹H NMR (CDCl₃) δ 2.24 (3H, s), 2.79

(2H, brs), 3.71 (2H, m), 4.62 (2H, s), 5,11 (2H, s), 5.18 (2H,s),

6.69 (1H, brs), 6.79 (1H, dd, J ) 2.7, 8.4 Hz), 7.06 (1H, d, J )

8.4 Hz), 7.26-7.38 (5H, m). Without purification, to a stirred

solution of the compound (2.0 g, 5.82 mmol) in CH₂Cl₂(40 mL)

was added SO₂Cl₂(0.52 mL, 6.47 mmol) at 0 °C. After the

mixture was stirred at 0 °C for 1 h, the solvent was removed

under reduced pressure to give compound 45 (1.85 g, 96%) in

sufficient purity to be used without purification: ¹H NMR

(CDCl₃) δ 2.82 (2H, m), 3.72 (2H, m), 4.65 (2H, s), 5.18 (2H,

s), 5.87 (2H, s), 6.84 (1H, brs), 6.91 (1H, d, J ) 8.4 Hz), 7.11

(1H, d, J ) 8.4 Hz), 7.26-7.39 (5H, m). Anal. (C₁₈H₁₈ClNO₃)

C, H, N.

Ethyl 1-tert-Butoxycarbonylpiperidin-4-carboxylate

(48). To a stirred solution of ethyl isonipecotinate (25.5 g, 0.16

mmol) in 1 N NaOH (160 mL) and 1,4-dioxane (100 mL) was

added Boc₂O (36.5 g, 0.17 mmol). The mixture was stirred at

room temperature for 12 h and then extracted with EtOAc.

The organic layer was washed with brine and dried over

anhydrous Na₂SO₄, and the solvent was removed under

reduced pressure to give quantitatively compound 48 (41.4 g)

in sufficient purity to be used without purification: ¹H NMR

(CDCl₃) δ 1.27 (3H, t, J ) 7.1 Hz), 1.60-1.70 (2H, m), 1.80-

1.92 (2H, m), 2.38-2.50 (1H, m), 2.78-2.90 (2H, m), 3.90-

4.10 (2H, m), 4.14 (2H, q, J ) 7.1 Hz). Anal. (C₁₃H₂₃NO₄) C,

H, N.

Benzyl 7-[4-Ethoxycarbonyl-1-(pyridin-4-yl)piperidin-

4-ylmethoxy]-1,2,3,4-tetrahydroisoquinolin-2-carbox-

ylate (47). To a stirred solution of 46¹¹(13.4 g, 57.19 mmol)

in THF (300 mL) was added dropwise LDA (31 mL of 2 M

solution in THF, 62 mmol) at -70 °C under argon atmosphere.

After the mixture was stirred at -70 °C for 45 min, a solution

of 45 (7.4 g, 22.30 mmol) in THF (80 mL) was added dropwise

to the solution at -70 °C. The mixture was allowed to warm

to room temperature and stirred for 5.5 h, then quenched with

aqueous NH₄Cl solution and extracted with EtOAc. The

organic layer was washed with brine, dried over anhydrous

Na₂SO₄, and evaporated under reduced pressure to leave the

4-[4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-oxy-

methyl)-4-ethoxycarbonylpiperidin-1-yl]-1-methylpyri-

dinium Chloride (21). Compound 21 (37 mg, 52%) was

obtained as a hydrochloride from 55 (99 mg, 0.147 mmol), 1H-

pyrazole-1-carboxamidine hydrochloride (45 mg, 0.308 mmol),

and diisopropylethylamine (0.054 mL, 0.312 mmol) as de-

scribed for 16: ¹H NMR (DMSO-d₆) δ 1.15 (3H, t, J ) 7.2 Hz),

1.65-1.80 (2H, m), 2.10-2.20 (2H, m), 2.78-2.85 (2H, m),

3.35-3.45 (2H, m), 3.55-3.60 (2H, m), 3.89 (3H, s), 4.00-4.10

(4H, m), 4.14 (2H, q, J ) 7.2 Hz), 4.53 (2H, s), 6.68 (1H, d, J

) 2.1 Hz), 6.80 (1H, dd, J ) 2.7, 8.4 Hz), 7.13 (1H, d, J ) 8.4

3598 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10

Ueno et al.

Hz), 7.24 (2H, d, J ) 7.8 Hz), 7.64 (4H, brs), 8.25 (2H, d, J )

7.8 Hz). Anal. (C₂₅H₃₄ClN₅O₃‚HCl‚1.8H₂O) C, H, N.

Benzyl 7-[1-(1-Amidinophenyl-4-yl)-4-ethoxycarbonyl-

piperidin-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinolin-2-

carboxylate (56). To a stirred solution of 52 (120 mg, 0.265

mmol) in DMSO (1 mL) were added K₂CO₃(100 mg, 0.723

mmol) and 4-fluorobenzonitrile (68 mg, 0.562 mmol). After

heating at 120 °C for 12 h, the reaction mixture was diluted

with water and extracted with AcOEt. The organic layer was

washed with brine and dried over anhydrous Na₂SO₄, and the

solvent was removed under reduced pressure to give the

residue, which was dissolved in pyridine (2.5 mL) and Et₃N

(0.5 mL). To the stirred solution was bubbled H₂S gas for 15

min. The mixture was stirred at room temperature for 12 h.

After removal of the solvent, acetone (2 mL) and MeI (1.0 mL,

16.1 mmol) were added to the residue. The mixture was heated

at reflux for 2 h. The solvent was removed under reduced

pressure to give the residue, which was dissolved in EtOH (2

mL). To the solution was added NH₄OAc (25 mg, 0.324 mmol),

and the mixture was heated at 75 °C for 2 h. The solvent was

removed under reduced pressure to afford compound 56 (56

mg, 36%). This compound was used for the next step without

purification.

Ethyl 4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-

yloxymethyl)-1-(2-methylpyridin-4-yl)piperidin-4-car-

boxylate (51). Compound 50 (100 mg, 0.18 mmol) was

dissolved in 25% HBr/AcOH (2 mL), and the mixture was

stirred at room temperature for 10 min. After addition of IPE

(diisopropyl ether), the resulting insoluble solid was collected.

To a stirred solution of the solid dissolved in DMF (1 mL) were

added 1H-pyrazole-1-carboxamidine hydrochloride (55 mg, 0.38

mmol) and diisopropylethylamine (0.16 mL, 0.92 mmol). The

mixture was stirred at room temperature for 12 h. The solvent

was evaporated under reduced pressure to leave the residue,

which was washed with THF and purified by reversed-phase

HPLC (0.05% TFA in H₂O/MeOH ) 6:4). The fraction contain-

ing desired product was treated with HCl/EtOH to give

compound 51 (85 mg, 90%) as a dihydrochloride. ¹H NMR

(DMSO-d₆) δ 1.16 (3H, t, J ) 6.9 Hz), 1.73 (2H, m), 2.14-2.22

(2H, m), 2.83 (2H, m), 3.35-3.60 (4H, m), 4.00-4.10 (4H, m),

4.16 (2H, q, J ) 6.9 Hz), 4.54 (2H, s), 6.70 (1H, m), 6.80-6.85

(1H, m), 7.05-7.17 (3H, m), 7.58 (4H, brs), 8.13 (1H, m), 13.64

(1H, brs). Anal. (C₂₅H₃₃N₅O₃‚2HCl‚2.2H₂O) C, H, N.

4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-yloxy-

methyl)-1-(2-methylpyridin-4-yl)piperidin-4-carboxylic

Acid (19). A solution of 51 (63 mg, 0.12 mmol) in 6 N HCl (1

mL) was heated at reflux for 3 h. After removal of an insoluble

material, the filtrate was concentrated under reduced pressure

to give compound 19 (53 mg, 89 %) as a dihydrochloride: ¹H

NMR (DMSO-d₆) δ 1.63-1.75 (2H, m), 2.05-2.25 (2H, m), 2.47

(3H, s), 2.83 (2H, m), 3.30-3.70 (4H, m), 3.95-4.10 (4H, m),

4.55 (2H, s), 6.71 (1H, m), 6.83 (1H, m), 7.07-7.16 (3H, m),

7.63 (4H, brs), 8.12 (1H, m), 13.74 (1H, brs). Anal. (C₂₃H₂₉N₅O₃‚

2HCl‚2.7H₂O) C, H, N.

Ethyl 4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-

yloxymethyl)-1-(pyrimidin-4-yl)piperidin-4-carbox-

ylate (54) and 4-(2-Amidino-1,2,3,4-tetrahydroisoquino-

lin-7-yloxymethyl)-1-(1,3-pyrimidin-4-yl)piperidin-4-car-

boxylate (20). To a stirred solution of 53 (300 mg, 0.53 mmol)

in EtOH (5 mL) were added 7.5% Pd/C (120 mg) and HCO₂NH₄

(200 mg, 3.17 mmol), and the mixture was heated at reflux

for 30 min. After removal of an insoluble material, the fil-

trate was concentrated under reduced pressure to give ethyl

1-(pyrimidin-4-yl)-4-(1,2,3,4-tetrahydroisoquinolin-7-yloxy-

methyl)piperidin-4-carboxylate (210 mg, 99%). Without puri-

fication, to a stirred solution of the compound (200 mg, 0.50

mmol) in DMF (1 mL) were added 1H-pyrazole-1-carboxami-

dine hydrochloride (154 mg, 1.05 mmol) and DIPEA (0.18 mL,

1.03 mmol). After the mixture was stirred at room temperature

for 2 h, the solvent was evaporated under reduced pressure.

The residue was purified by reversed-phase HPLC (0.05% TFA

in H₂O/MeOH ) 6:4). The fraction containing the desired

product was collected, and MeOH was evaporated under

reduced pressure to give an aqueous solution of 54. To the

resulting aqueous solution was added concentrated HCl (5

mL), and the mixture was heated at reflux for 2 h. Then the

4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-yloxy-

methyl)-1-(pyridin-4-yl)piperidin-4-carboxylic Acid (1).

A solution of 16 (9.0 g, 20.57 mmol) in concentrated HCl (50

mL) was heated at reflux for 3 h. The insoluble material was

removed, and the filtrate was evaporated under reduced

pressure to give compound 1 (9.0 g, 91%) as a dihydrochlo-

ride: ¹H NMR (DMSO-d₆) δ 1.65-1.75 (2H, m, Ar-NCH₂CH₂),

2.14-2.20 (2H, m, Ar-NCH₂CH₂), 2.81 (2H, t, J ) 5.7 Hz,

NCH₂CH₂), 3.30-3.50 (2H, m, Ar-NCH₂CH₂), 3.57 (2H, t, J

) 5.7 Hz, NCH₂CH₂), 4.00-4.15 (4H, m, Ar-NCH₂CH₂and

NCH₂Ar), 4.54(2H, s, OCH₂), 6.70 (1H, d, J ) 2.4 Hz, ArH),

6.81 (1H, dd, J ) 2.4, 8.1 Hz, ArH), 7.15 (3H, m, ArH), 7.63

(4H, brs, C(dNH)-N⁺H₃), 8.22 (2H, m, ArH), 13.71 (1H, brs).

Anal. (C₂₂H₂₇N₅O₃‚2HCl‚2.8H₂O) C, H, N.

4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-yloxy-

methyl)-1-(pyridin-4-yl)piperidine-4-carboxylicAcidMono-

methanesulfonate (2) (JTV-803). To a stirred solution of 1

(10 g, 24.42 mmol) in water (100 mL) was added 1 N NaOH

(75 mL), and the precipitated solid was collected. To a stirred

suspension of the solid in water (50 mL) was added methane-

sulfonic acid (1.6 mL, 24.64 mmol), and the mixture was

dissolved at 50 °C. To the resulting solution was added acetone

(150 mL), and stirring was continued over 1 h to room

temperature. Filtration of the precipitates afforded compound

2 (11.5 g, 93%): ¹H NMR (D₂O) δ 1.60 (2H, m, Ar-NCH₂CH₂),

2.19 (2H, m, Ar-NCH₂CH₂), 2.72 (3H, s, CH₃SO₃H), 2.77 (2H,

t, J ) 5.8 Hz, NCH₂CH₂), 3.34 (2H, m, Ar-NCH₂CH₂), 3.45

(2H, t, J ) 5.8 Hz, NCH₂CH₂), 3.84-3.92 (4H, m, Ar-

NCH₂CH₂and NCH₂Ar), 4.38 (2H, s, OCH₂), 6.72 (1H, s, ArH),

6.79 (1H, d, J ) 8.4 Hz, ArH), 6.92 (2H, d, J ) 7.8 Hz, ArH),

7.09 (1H, d, J ) 8.4 Hz, ArH), 7.89 (2H, d, J ) 7.8 Hz, ArH);

IR (KBr) 2918 cm^-1, 1639 cm^-1, 1547 cm^-1, 1209 cm^-1. Anal.

(C₂₂H₂₇N₅O₃‚CH₃SO₃H‚3.0H₂O) C, H, N.

Benzyl 7-[4-Ethoxycarbonyl-1-(2-methylpyridin-4-yl)-

piperidin-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinolin-2-

carboxylate (50). To a stirred solution of 49 (165 mg, 0.30

mmol) in CHCl₃(1.5 mL) was added TFA (0.5 mL), and the

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

was removed under reduced pressure to leave the residue,

which was diluted with aqueous NaHCO₃solution. The

mixture was extracted with EtOAc, and the organic layer was

washed with brine, dried over anhydrous Na₂SO₄, and evapo-

rated under reduced pressure to give compound 52 (135 mg,

99%): ¹H NMR (CDCl₃) δ 1.23 (3H, t, J ) 7.5 Hz), 1.50-1.65

(2H, m), 2.15-2.25 (2H, m), 2.65-2.90 (4H, m), 2.95-3.05 (2H,

m), 3.69 (2H, m), 3.93 (2H, s), 4.19 (2H, q, J ) 7.5 Hz), 4.60

(2H, s), 5.17 (2H, s), 6.60 (1H, brs), 6.69 (1H, d, J ) 8.4 Hz),

7.02 (1H, d, J ) 8.4 Hz), 7.26-7.38 (5H, m). To a stirred

solution of 52 (100 mg, 0.22 mmol) in EtOH (3 mL) were added

Et₃N (0.05 mL, 0.36 mmol) and 4-chloro-2-methylpyridine

hydrochloride¹⁵(40 mg, 0.24 mmol). The mixture was heated

at 150 °C for 25 h in a sealed tube. The solvent was removed

under reduced pressure to leave the residue, which was

purified by SiO₂column chromatography (CHCl₃/MeOH ) 4:1)

to give compound 50 (105 mg, 88%): ¹H NMR (CDCl₃) δ 1.26

(3H, t, J ) 6.6 Hz), 1.70-1.85 (2H, m), 2.35-2.45 (2H, m),

2.64 (3H, s), 2.78 (2H, m), 3.28-3.37 (2H, m), 3.70 (2H, m),

3.80-3.90 (2H, m), 3.97 (2H, s), 4.24 (2H, q, J ) 6.6 Hz), 4.60

(2H, s), 5.17 (2H, s), 6.45 (1H, m), 6.60-6.69 (2H, m), 7.04 (1H,

m), 7.34-7.38 (5H, m), 8.25 (1H, d, J ) 7.2 Hz). Anal.

(C₃₂H₃₇N₃O₅) C, H, N.

Benzyl 7-[1-(2-Chloropyrimidin-4-yl)-4-ethoxycarbonyl-

piperidin-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinolin-2-

carboxylate (53). Compound 53 (263 mg, 47%) was obtained

from 49 (550 mg, 1.00 mmol), 2,4-dichloropyrimidine (150 mg,

1.01 mmol), and Et₃N (0.15 mL, 1.08 mmol) as described for

50: ¹H NMR (CDCl₃) δ 1.25 (3H, t, J ) 6.9 Hz), 1.50-1.70

(2H, m), 2.25-2.40 (2H, m), 2.78 (2H, m), 3.18-3.30 (2H, m),

3.70 (2H, m), 3.95 (2H, s), 4.09-4.26 (4H, m), 4.60 (2H, s), 5.17

(2H, s), 6.40 (1H, d, J ) 6.1 Hz), 6.60 (1H, m), 6.69 (1H, m),

7.03 (1H, m), 7.26-7.38 (5H, m), 8.03 (1H, d, J ) 6.1 Hz). Anal.

(C₃₀H₃₃ClN₄O₅) C, H, N.

Selective Factor Xa Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3599

solvent was evaporated under reduced pressure to give com-

pound 20 (51 mg, 21%) as a dihydrochloride: ¹H NMR (DMSO-

d₆) δ 1.60-1.80 (2H, m), 2.10-2.25 (2H, m), 2.81 (2H, m),

3.40-3.60 (4H, m), 4.05 (2H, s), 4.05-4.80 (2H, m), 4.54 (2H,

s), 6.69 (1H, d, J ) 2.4 Hz), 6.81 (1H, dd, J ) 2.4, 8.4 Hz),

7.13 (1H, d, J ) 8.4 Hz), 7.23 (1H, d, J ) 7.8 Hz), 7.66 (4H,

brs), 8.32 (1H, d, J ) 7.8 Hz), 8.81 (1H, s). Anal. (C₂₁H₂₆N₆O₃‚

2.1HCl‚2.8H₂O) C, H, N.

Ethyl 1-(1-Amidinophenyl-4-yl)-4-(2-amidino-1,2,3,4-

tetrahydroisoquinolin-7-yloxymethyl)piperidin-4-car-

boxylate (22). Compound 22 (47 mg, 81%) was obtained as a

dihydrochloride from 56 (56 mg, 0.098 mmol) by deprotection

of the Cbz group with 25% HBr/AcOH, followed by 1H-

pyrazole-1-carboxamidine hydrochloride (30 mg, 0.205 mmol)

and diisopropylethylamine (0.090 mL, 0.517 mmol) as de-

scribed for 51: ¹H NMR (DMSO-d₆) δ 1.14 (3H, t, J ) 6.9 Hz),

1.60-1.75 (2H, m), 2.10-2.20 (2H, m), 2.78-2.85 (2H, m),

3.10-3.20 (2H, m), 3.52-3.60 (2H, m), 3.70-3.90 (2H, m), 4.05

(2H, s), 4.14 (2H, q, J ) 6.9 Hz), 4.52 (2H, s), 6.74 (1H, s),

6.81 (1H, m), 7.07 (2H, d, J ) 9.0 Hz), 7.13 (1H, d, J ) 8.4

Hz), 7.56 (4H, brs), 7.75 (2H, d, J ) 9.0 Hz), 8.71 (2H, brs),

8.97 (2H, brs). Anal. (C₂₆H₃₄N₆O₃‚3HCl‚3.1H₂O) C, H, N.

2-tert-Butoxycarbonyl-6-hydroxy-1,2,3,4-tetrahydroiso-

quinoline (59). To a stirred solution of 58¹⁶(350 mg, 1.52

mmol) in 1 N NaOH (4 mL) and 1,4-dioxane (8 mL) was added

Boc₂O (365 mg, 1.64 mmol). After being stirred at room

temperature for 12 h, the reaction mixture was extracted with

EtOAc. The organic layer was washed with brine, dried over

anhydrous Na₂SO₄, and evaporated under reduced pressure

to leave the residue, which was purified by SiO₂column

chromatography (n-hexane/acetone ) 5:1) to give compound

59 (325 mg, 86%): ¹H NMR (CDCl₃) δ 1.49 (9H, s), 2.76 (2H,

t, J ) 6.0 Hz), 3.61 (2H, t, J ) 6.0 Hz), 4.49 (2H, s), 5.33 (1H,

brs), 6.63 (1H, s), 6.67 (1H, d, J ) 8.4 Hz), 6.95 (1H, d, J ) 8.4

Hz). Anal. (C₁₄H₁₉NO₃) C, H, N.

tert-Butyl 6-Chloromethoxy-1,2,3,4-tetrahydroisoquin-

olin-2-carboxylate (60). To a stirred solution of 59 (320 mg,

1.29 mmol) in DMF (5 mL) was added NaH (62 mg of a 60%

dispersion in mineral oil, 1.54 mmol) at 0 °C. After the mixture

was stirred at 0 °C for 30 min, MeSCH₂Cl (0.16 mL, 1.93 mmol)

was added. The mixture was stirred at room temperature for

12 h. The reaction mixture was quenched with water and

extracted with EtOAc. The organic layer was washed with

brine, dried over anhydrous Na₂SO₄, and evaporated under

reduced pressure to leave the residue, which was purified by

SiO₂column chromatography (n-hexane/EtOAc ) 8:1) to give

tert-butyl 6-methylthiomethoxy-1,2,3,4-tetrahydroisoquinolin-

2-carboxylate (330 mg, 83%): ¹H NMR (CDCl₃) δ 1.49 (9H, s),

2.25 (3H, s), 2.78 (2H, t, J ) 5.7 Hz), 3.61 (2H, t, J ) 5.7 Hz),

4.51 (2H, s), 5.13 (2H, s), 6.73 (1H, s), 6.78 (1H, d, J ) 8.4

Hz), 7.02 (1H, d, J ) 8.4 Hz). Without purification, to a stirred

solution of the compound (320 mg, 1.03 mmol) in CH₂Cl₂(5

mL) was added SO₂Cl₂(0.091 mL, 1.14 mmol) at 0 °C. After

the mixture was stirred at 0 °C for 1 h, the solvent was

removed under reduced pressure to give compound 60 (290

mg, 95%) in sufficient purity to be used without purification:

¹H NMR (CDCl₃) δ 1.49 (9H, s), 2.80 (2H, t, J ) 6.3 Hz), 3.63

(2H, t, J ) 6.3 Hz), 4.54 (2H, s), 5.88 (2H, s), 6.88 (1H, s), 6.91

(1H, d, J ) 8.4 Hz), 7.07 (1H, d, J ) 8.4 Hz). Anal. (C₁₅H₂₀-

ClNO₃) C, H, N.

pound 61 (390 mg, 81%): ¹H NMR (CDCl₃) δ 1.25 (3H, t, J )

6.9 Hz), 1.48 (9H, s), 1.70-1.80 (2H, m), 2.25-2.40 (2H, m),

2.78 (2H, t, J ) 5.7 Hz), 3.10-3.20 (2H, m), 3.61 (2H, t, J )

5.7 Hz), 3.65-3.80 (2H, m), 3.98 (2H, s), 4.22 (2H, q, J ) 6.9

Hz), 4.49 (2H, s), 6.61 (1H, s), 6.65-6.75 (3H, m), 6.99 (1H, d,

J ) 8.1 Hz), 8.26 (2H, d, J ) 6.3 Hz). Anal. (C₂₈H₃₇N₃O₅) C,

H, N.

Ethyl 4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-6-

yloxymethyl)-1-(pyridin-4-yl)piperidin-4-carboxylate (62).

To a stirred solution of compound 61 (350 mg, 0.71 mmol) in

CHCl₃(5 mL) was added TFA (2.5 mL). Stirring was continued

at room temperature for 5 h. The solvent was removed under

reduced pressure to leave the residue. To a stirred solution in

DMF (5 mL) were added 1H-pyrazole-1-carboxamidine hydro-

chloride (155 mg, 1.06 mmol) and diisopropylethylamine (0.62

mL, 3.53 mmol), and the mixture was stirred at room tem-

perature for 12 h. The solvent was evaporated under reduced

pressure to leave the residue, which was purified by reversed-

phase HPLC (0.05% TFA in H₂O/MeOH ) 6:4). The fraction

containing desired product was treated with HCl/EtOH to give

compound 62 (320 mg, 90%) as a dihydrochloride: ¹H NMR

(DMSO-d₆) δ 1.15 (3H, t, J ) 7.1 Hz), 1.65-1.80 (2H, m), 2.15-

2.25 (2H, m), 2.84 (2H, t, J ) 6.0 Hz), 3.30-3.50 (2H, m), 3.56

(2H, t, J ) 6.0 Hz), 3.95-4.20 (6H, m), 4.50 (2H, s), 6.68 (1H,

s), 6.81 (1H, d, J ) 8.4 Hz), 7.11 (1H, d, J ) 8.4 Hz), 7.19 (2H,

d, J ) 7.5 Hz), 7.58 (4H, brs), 8.22 (2H, d, J ) 7.5 Hz). Anal.

(C₂₄H₃₁N₅O₃‚2.2HCl‚2.5H₂O) C, H, N.

4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-6-yloxy-

methyl)-1-(pyridin-4-yl)piperidin-4-carboxylic Acid (57).

A solution of 62 (300 mg, 0.59 mmol) in concentrated HCl (5

mL) was heated at reflux for 3 h. The insoluble material was

removed, and the filtrate was evaporated under reduced

pressure to give compound 57 (255 mg, 91%) as a dihydro-

chloride: ¹H NMR (DMSO-d₆) δ 1.65-1.75 (2H, m), 2.15-2.20

(2H, m), 2.84 (2H, t, J ) 6.0 Hz), 3.30-3.50 (2H, m), 3.57 (2H,

t, J ) 6.0 Hz), 4.00-4.15 (4H, m), 4.51 (2H, s), 6.70 (1H, s),

6.81 (1H, d, J ) 8.1 Hz), 7.10-7.20 (3H, m), 7.63 (4H, brs),

8.22 (2H, m), 13.75 (1H, brs). Anal. (C₂₂H₂₇N₅O₃‚2.1HCl‚

2.8H₂O) C, H, N.

8-Methoxy-2,3,4,5-tetrahydro-1H-benz[c]azepine (64).

To a stirred solution of 7-methoxy-1,2,3,4-tetrahydronaphtha-

len-1-one (63, 5.21 g, 29.56 mmol) in concentrated HCl (26 mL)

was added NaN₃(1.98 g, 30.46 mmol) in small portions at 0

°C. After being stirred at room temperature for 2 h, the

reaction mixture was poured into aqueous K₂CO₃solution at

0 °C. The mixture was extracted with CHCl₃, and the organic

layer was washed with brine, dried over anhydrous Na₂SO₄,

and evaporated under reduced pressure to leave the residue,

which was purified by SiO₂column chromatography (EtOAc)

to give 8-methoxy-2,3,4,5-tetrahydrobenz[c]azepin-1-one (4.7

g, 83%): ¹H NMR (CDCl₃) δ 1.98 (2H, q, J ) 6.9 Hz), 2.80

(2H, q, J ) 6.9 Hz), 3.13 (2H, q, J ) 6.9 Hz), 3.83 (3H, s), 6.96

(1H, dd, J ) 2.7, 8.4 Hz), 7.09 (1H, d, J ) 8.4 Hz), 7.26 (1H,

d, J ) 2.7 Hz).

A solution of the compound (4.42 g, 23.11 mmol) in 1,4-

dioxane (30 mL) was added dropwise to a stirred suspension

of LiAlH₄(3.07 g, 80.90 mmol) in THF (60 mL) at 0 °C. The

mixture was heated at reflux for 20 h. After the mixture was

cooled to room temperature, 20% NaOH solution was added

dropwise. An insoluble material was filtered, and the filtrate

was concentrated under reduced pressure to give compound

64 (4.05 g, 98%): ¹H NMR (CDCl₃) δ 1.68 (2H, m), 2.82-2.90

(2H, m), 3.10-3.25 (2H, m), 3.77 (3H, s), 3.88 (2H, s), 6.60-

6.70 (2H, m), 7.05 (1H, d, J ) 8.4 Hz). Anal. (C₁₁H₁₅NO) C, H,

N.

tert-Butyl 6-[4-Ethoxycarbonyl-1-(pyridin-4-yl)piperi-

din-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinolin-2-carbox-

ylate (61). To a stirred solution of 46¹¹(570 mg, 2.43 mmol)

in THF (10 mL) was added dropwise LDA (1.34 mL of 2 M

solution in THF, 2.68 mmol) at -70 °C under argon atmo-

sphere. After the mixture was stirred at -70 °C for 45 min, a

solution of 60 (290 mg, 0.97 mmol) in THF (3 mL) was added

dropwise to the solution at -70 °C. The mixture was allowed

to warm to room temperature and was stirred for 5.5 h, then

quenched with aqueous NH₄Cl solution and extracted with

EtOAc. The organic layer was washed with brine, dried over

anhydrous Na₂SO₄, and evaporated under reduced pressure

to leave the residue, which was purified by SiO₂column

chromatography (CHCl₃/MeOH ) 20:1 to 10:1) to give com-

2-tert-Butoxycarbonyl-8-hydroxy-2,3,4,5-tetrahydro-

1H-benz[c]azepine (65). A solution of 64 (3.65 g, 20.59 mmol)

in concentrated HBr (36 mL) was heated at reflux for 2 h. The

solvent was evaporated under reduced pressure to leave the

residue, which was washed with EtOH and Et₂O to give

8-hydroxy-2,3,4,5-tetrahydro-1H-benz[c]azepine (4.56 g, 91%)

as a hydrobromide: ¹H NMR (DMSO-d₆) δ 1.80-1.83 (2H, m),

2.85-2.88 (2H, m), 3.22 (2H, m), 4.20-4.22 (2H, m), 6.69 (1H,

dd, J ) 2.5, 8.1 Hz), 6.82 (1H, d, J ) 2.5 Hz), 7.05 (1H, d, J )

3600 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10

Ueno et al.

8.1 Hz), 8.80 (2H, brs), 9.42 (1H, brs). Compound 65 (2.64 g,

82%) was obtained from the compound (3.00 g, 12.29 mmol),

Boc₂O (3.20 g, 14.66 mmol), and 2 N NaOH (30 mL, 15 mmol)

as described for 31: ¹H NMR (CDCl₃) δ 1.40 (9H, s), 1.68-

1.80 (2H, m), 2.84-2.88 (2H, m), 3.66 (2H, m), 4.28-4.37 (2H,

m), 5.19-5.91 (1H, m), 6.63 (1H, m), 6.70-6.85 (1H, m), 6.98

(1H, m). Anal. (C₁₅H₂₁NO₃) C, H, N.

tert-Butyl 8-Chloromethoxy-2,3,4,5-tetrahydro-1H-benz-

[c]azepin-2-carboxylate (66). Compound 66 (1.07 g, 85%)

was obtained from 65 (1.06 g, 4.03 mmol), MeSCH₂Cl (0.41

mL, 4.89 mmol), and NaH (195 mg of 60% dispersion in

mineral oil, 4.88 mmol) as described for 45: ¹H NMR (CDCl₃)

δ 1.40 (9H, s), 1.75 (2H, m), 2.89-2.93 (2H, m), 3.68 (2H, m),

4.36 (2H, m), 5.88 (2H, s), 6.87-6.95 (1H, m), 7.10-7.20 (2H,

m). Anal. (C₁₆H₂₂ClNO₃) C, H, N.

tert-Butyl 8-[4-Ethoxycarbonyl-1-(pyridin-4-yl)piperi-

din-4-ylmethoxy]-2,3,4,5-tetrahydro-1H-benz[c]azepin-2-

carboxylate (67). Compound 67 (755 mg, 54%) was obtained

from 66 (850 mg, 2.73 mmol) and 46 (1.60 g, 6.82 mmol) as

described for 47: ¹H NMR (CDCl₃) δ 1.27 (3H, t, J ) 6.9 Hz),

1.39 (9H, s), 1.64-1.78 (4H, m), 2.30-2.36 (2H, m), 2.85-2.88

(2H, m), 3.01-3.18 (2H, m), 3.62-3.75 (4H, m), 3.98 (2H, s),

4.21 (2H, q, J ) 6.9 Hz), 4.30-4.38 (2H, m), 6.62-6.67 (3H,

m), 6.72-6.84 (1H, m), 7.02 (1H, m), 8.25 (2H, d, J ) 6.3 Hz).

Anal. (C₂₉H₃₉N₃O₅) C, H, N.

Ethyl 4-(2-Amidino-2,3,4,5-tetrahydro-1H-benz[c]azepin-

8-yloxymethyl)-1-(pyridin-4-yl)piperidin-4-carboxylate

(68). Compound 68 (200 mg, 78%) was obtained as a dihydro-

chloride from 67 (250 mg, 0.49 mmol), 1H-pyrazole-1-carbox-

amidine hydrochloride (90 mg, 0.61 mmol), and DIPEA (0.11

mL, 0.63 mmol) as described for 8: ¹H NMR (DMSO-d₆) δ 1.16

(3H, t, J ) 7.2 Hz), 1.75 (4H, m), 2.20 (2H, m), 2.89 (2H, m),

3.41 (2H, m), 4.04 (4H, m), 4.16 (2H, q, J ) 7.2 Hz), 4.58 (2H,

s), 6.76 (1H, dd, J ) 2.6, 8.1 Hz), 7.11-7.22 (4H, m), 7.47 (4H,

brs), 8.24 (1H, d, J ) 6.6 Hz), 13.60 (1H, brs). Anal. (C₂₅H₃₃N₅O₃‚

2HCl‚2.6H₂O) C, H, N.

mixture was cooled to 0 °C, 1 N HCl was added and the

mixture was extracted with EtOAc. The organic layer was

washed with brine, dried over anhydrous Na₂SO₄, and evapo-

rated under reduced pressure to leave the residue, which was

washed with n-hexane to give compound 70 (3.02 g, 82%): ¹H

NMR (CDCl₃) δ 5.42 (1H, brs), 7.20 (1H, s), 7.25-7.28 (1H,

m), 7.46 (1H, d, J ) 8.4 Hz), 7.79-7.85 (2H, m), 8.07 (1H, s).

Anal. (C₁₁H₇NO) C, H, N.

7-Chloromethoxynaphthalen-2-carbonitrile (71). Com-

pound 71 (567 mg, 39%) was obtained from 70 (1.12 g, 6.62

mmol), MeSCH₂Cl (0.72 mL, 8.61 mmol), and NaH (318 mg of

a 60% dispersion in mineral oil, 7.94 mmol) as described for

45: ¹H NMR (CDCl₃) δ 6.01 (3H, s), 7.39 (1H, dd, J ) 2.5, 9.0

Hz), 7.51 (1H, d, J ) 2.5 Hz), 7.56 (1H, dd, J ) 1.5, 8.5 Hz),

7.87 (1H, d, J ) 9.0 Hz), 7.89 (1H, d, J ) 8.5 Hz), 8.20 (1H, d,

J ) 1.5 Hz). Anal. (C₁₂H₈ClNO) C, H, N.

Ethyl 4-(2-Cyanonaphthalen-7-yloxymethyl)-1-(pyri-

din-4-yl)piperidin-4-carboxylate (72). Compound 72 (430

mg, 72%) was obtained from 71 (314 mg, 1.44 mmol) and 46

(743 mg, 3.17 mmol) as described for 47: ¹H NMR (CDCl₃) δ

1.25 (3H, t, J ) 7.1 Hz), 1.75-1.85 (2H, m), 2.35-2.45 (2H,

m), 3.10-3.25 (2H, m), 3.70-3.82 (2H, m), 4.14 (2H, s), 4.25

(2H, q, J ) 7.1 Hz), 6.69 (2H, d, J ) 6.6 Hz), 7.13 (1H, d, J )

2.5 Hz), 7.24-7.28 (1H, m), 7.49 (1H, dd, J ) 1.5, 8.4 Hz), 7.78

(1H, d, J ) 9.0 Hz), 7.84 (1H, d, J ) 8.4 Hz), 8.08 (1H, d, J )

1.5 Hz). Anal. (C₂₅H₂₅N₃O₃) C, H, N.

Ethyl 4-(2-Amidinonaphthalen-7-yloxymethyl)-1-(pyri-

din-4-yl)piperidin-4-carboxylate (73). To a stirred solution

of 72 (51 mg, 0.12 mmol) in pyridine (1.5 mL) and Et₃N (0.3

mL) was bubbled H₂S gas for 15 min. The mixture was stirred

at room temperature for 12 h. The solvent was evaporated

under reduced pressure to leave the residue, which was treated

with HCl/EtOH. To the residue were added acetone (1 mL),

MeOH (1 mL), and MeI (0.6 mL, 9.63 mmol), and the mixture

was heated at reflux for 2 h. The solvent was evaporated under

reduced pressure to leave the residue. To a stirred solution of

the residue in EtOH (2 mL) was added NH₄OAc (19 mg, 0.24

mmol), and the mixture was heated at 75 °C for 2 h. The

solvent was evaporated under reduced pressure to leave the

residue, which was purified by SiO₂column chromatography

(CHCl₃/MeOH/concentrated aqueous NH₃) 100:10:1), followed

by treatment with HCl/EtOH to give compound 73 (36 mg,

69%) as a dihydrochloride: ¹H NMR (DMSO-d₆) δ 1.14 (3H, t,

J ) 7.1 Hz), 1.75-1.88 (2H, m), 2.19-2.31 (2H, m), 3.44 (2H,

m), 4.00-4.20 (4H, m), 4.27 (2H, s), 7.21 (2H, d, J ) 7.0 Hz),

7.34 (1H, d, J ) 8.9 Hz), 7.48 (1H, s), 7.66 (1H, d, J ) 8.9 Hz),

7.98 (1H, d, J ) 8.9 Hz), 8.07 (1H, d, J ) 8.9 Hz), 8.24 (2H, d,

J ) 7.0 Hz), 8.32 (1H, s), 9.19 (2H, brs), 9.45 (2H, brs), 13.53

(1H, brs). Anal. (C₂₅H₂₈N₄O₃‚2HCl‚2.3H₂O) C, H, N.

4-(2-Amidinonaphthalen-7-yloxymethyl)-1-(pyridin-4-

yl)piperidin-4-carboxylic Acid (24). Compound 24 (85 mg,

51%) was obtained as a dihydrochloride from 70 (150 mg, 0.35

mmol) and 6 N HCl (3 mL, 18 mmol) as described for 19: ¹H

NMR (DMSO-d₆) δ 1.75-1.90 (2H, m), 2.20-2.35 (2H, m), 3.47

(2H, m), 4.00-4.15 (2H, m), 4.27 (2H, s), 7.25 (2H, d, J ) 7.0

Hz), 7.34 (1H, d, J ) 8.9 Hz), 7.48 (1H, s), 7.66 (1H, d, J ) 8.9

Hz), 7.98 (1H, d, J ) 8.9 Hz), 8.07 (1H, d, J ) 8.9 Hz), 8.24

(2H, d, J ) 7.0 Hz), 8.32 (1H, s), 9.25 (2H, brs), 9.45 (2H, brs),

13.6 (1H, brs). Anal. (C₂₃H₂₄N₄O₃‚2.1HCl‚2.3H₂O) C, H, N.

Benzyl 7-(2-Iodoethoxy)-1,2,3,4-tetrahydroisoquinolin-

2-carboxylate (74). To a stirred solution of 32 (1.21 g, 4.27

mmol) in THF (30 mL) were added 2-bromoethanol (0.55 mL,

7.76 mmol), PPh₃(2.01 g, 7.76 mmol), and diethyl azodicar-

boxylate (3.36 mL of 40% solution in toluene, 7.72 mmol). The

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

was evaporated under reduced pressure to leave the residue.

To a stirred solution of the residue in DMF (30 mL) was added

NaI (0.96 g, 6.40 mmol), and the mixture was stirred at 90 °C

for 15 h. The solvent was evaporated under reduced pressure

to leave the residue, which was purified by SiO₂column

chromatography (n-hexane/EtOAc ) 4:1) to give compound 74

(1.19 g, 64%): ¹H NMR (CDCl₃) δ 2.79 (2H, m), 3.40 (2H, t, J

) 6.9 Hz), 3.70 (2H, m), 4.21 (2H, t, J ) 6.9 Hz), 4.61 (2H, s),

4-(2-Amidino-2,3,4,5-tetrahydro-1H-benz[c]azepin-8-

yloxymethyl)-1-(pyridin-4-yl)piperidin-4-carboxylic Acid

(23). Compound 23 (140 mg, 97%) was obtained as a dihydro-

chloride from 68 (150 mg, 0.29 mmol) and 6 N HCl (2 mL, 12

1

mmol) as described for 19: H NMR (DMSO-d₆) δ 1.56-1.81

(4H, m), 2.12-2.25 (2H, m), 2.88-3.00 (2H, m), 3.42 (2H, m),

4.00-4.13 (4H, m), 4.59 (2H, s), 6.76 (1H, dd, J ) 2.6, 8.1 Hz),

7.12 (1H, d, J ) 8.1 Hz), 7.19-7.23 (3H, m), 7.54 (4H, brs),

8.23 (1H, m), 13.70 (1H, brs). Anal. (C₂₃H₂₉N₅O₃‚2HCl‚2.8H₂O)

C, H, N.

7-Hydroxynaphthalen-2-carbonitrile (70). To a stirred

solution of 2-hydroxy-7-methoxynaphthalene (69, commercially

available; 12.34 g, 70.84 mmol) in CHCl₃(185 mL) were added

Et₃N (9.87 mL, 70.85 mmol) and Tf₂O (20 g, 70.84 mmol) at 0

°C. After the mixture was stirred at 0 °C for 30 min, aqueous

NaHCO₃solution was added and the mixture was extracted

with CHCl₃. The organic layer was washed with brine, dried

over anhydrous Na₂SO₄, and evaporated under reduced pres-

sure to leave the residue, which was purified by SiO₂column

chromatography (n-hexane/EtOAc ) 9:1) to give 7-methoxy-

2-trifluoromethanesulfonyloxynaphthalene (15.36 g, 71%): ¹H

NMR (CDCl₃) δ 3.96 (3H, s), 7.14 (1H, d, J ) 2.4 Hz), 7.21

(2H, dd, J ) 2.4, 8.9 Hz), 7.64 (1H, d, J ) 2.4 Hz), 7.83 (1H,

d, J ) 8.9 Hz). To a solution of the compound (11.15 g, 7.28

mmol) in DMF (8.9 mL) were added Zn(CN)₂(2.99 g, 5.09

mmol) and Pd(PPh₃)₄(1.68 g, 0.29 mmol). After being stirred

at 80 °C for 1 h, the reaction mixture was quenched with water

and extracted with EtOAc. The organic layer was washed with

brine, dried over anhydrous Na₂SO₄, and evaporated under

reduced pressure to leave the residue, which was purified by

SiO₂column chromatography (n-hexane/EtOAc ) 19:1) to give

7-methoxynaphthalen-2-carbonitrile (4.82 g, 72%): ¹H NMR

(CDCl₃) δ 3.95 (3H, s), 7.15 (1H, d, J ) 2.5 Hz), 7.29 (1H, dd,

J ) 2.5, 9.0 Hz), 7.47 (1H, dd, J ) 1.6, 8.4 Hz), 7.78 (1H, d, J

) 9.0 Hz), 7.83 (1H, d, J ) 8.4 Hz), 8.11 (1H, d, J ) 1.6 Hz).

To a stirred solution of the compound (4.0 g, 21.83 mmol) in

chlorobenzene (40 mL) was added AlCl₃(10.2 g, 76.50 mmol),

and the mixture was heated at reflux for 30 min. After the

Selective Factor Xa Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3601

5.18 (2H, s), 6.63 (1H, m), 6.74 (1H, dd, J ) 2.6, 8.3 Hz), 7.05

(1H, d, J ) 8.3 Hz), 7.26-7.38 (5H, m). Anal. (C₁₉H₂₀INO₃) C,

H, N.

pound 81 (63 mg, 30%) was obtained from 80 (221 mg, 0.46

mmol), 4-chloropyridine hydrochloride (70 mg, 0.47 mmol), and

Et₃N (0.39 mL, 2.80 mmol) as described for 50: ¹H NMR

(CDCl₃) δ 1.21-1.26 (3H, m), 1.58-1.70 (2H, m), 2.17 (3H, s),

2.25-2.40 (2H, m), 2.78-2.90 (2H, m), 3.05-3.22 (4H, m),

3.64-3.85 (4H, m), 4.07-4.15 (2H, m), 4.57-4.69 (2H, m), 6.69

(2H, d, J ) 5.4 Hz), 7.04-7.20 (3H, m), 8.25 (2H, d, J ) 5.4

Hz). Anal. (C₂₅H₃₁N₃O₃S) C, H, N.

4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-ylthio-

methyl)-1-(pyridin-4-yl)piperidin-4-carboxylic Acid (26).

A solution of 81 (63 mg, 0.14 mmol) in concentrated HCl (2

mL) was heated at 90 °C for 21 h. The solvent was evaporated

under reduced pressure to leave the residue. To a stirred

solution of the residue in acetone (2 mL) and 1 N NaOH (0.7

mL) was added 1H-pyrazole-1-carboxamidine hydrochloride

(61 mg, 0.42 mmol). The mixture was stirred at room temper-

ature for 12 h. The solvent was evaporated under reduced

pressure to leave the residue, which was purified by reversed-

phase HPLC (0.05% TFA in H₂O/MeOH ) 6:4). The fraction

containing desired product was treated with HCl aqueous

solution to give compound 26 (47 mg, 67%) as a dihydrochlo-

ride: ¹H NMR (DMSO-d₆) δ 1.57-1.70 (2H, m), 2.08-2.20 (2H,

m), 2.84-2.92 (2H, m), 3.20-3.40 (4H, m), 3.60 (2H, m), 3.95-

4.10 (2H, m), 4.57 (2H, s), 7.14-7.20 (5H, m), 7.66 (4H, brs),

8.22 (2H, m). Anal. (C₂₂H₂₇N₅O₂S‚2HCl‚2.5H₂O) C, H, N.

tert-Butyl 4-(2-Acetyl-1,2,3,4-tetrahydroisoquinolin-7-

ylsulfonylmethyl)-4-ethoxycarbonylpiperidin-1-carbox-

ylate (82). To a stirred solution of 80 (252 mg, 0.53 mmol) in

CH₂Cl₂(5 mL) was added m-chloroperbenzoic acid (m-CPBA)

(260 mg of 70% grade, 1.05 mmol). The mixture was stirred

at room temperature for 12 h. The reaction mixture was

diluted with aqueous Na₂S₂O₅solution and aqueous NaHCO₃

solution, and the mixture was extracted with CHCl₃. The

organic layer was washed with brine, dried over anhydrous

Na₂SO₄, and evaporated under reduced pressure to leave the

residue, which was purified by SiO₂column chromatography

(EtOAc) to give compound 82 (203 mg, 75%): ¹H NMR (CDCl₃)

δ 1.32 (3H, t, J ) 7.1 Hz), 1.49 (9H, s), 1.55-1.75 (2H, m),

2.10-2.30(5H, m), 2.88-3.04 (2H, m), 3.23 (2H, brs), 3.43 (2H,

m), 3.65-3.78 (3H, m), 3.85 (1H, m), 4.22 (2H, q, J ) 7.1 Hz),

4.69-4.81 (2H, m), 7.32-7.37 (1H, m), 7.65-7.75 (2H, m).

Anal. (C₂₅H₃₆N₂O₇S) C, H, N.

Ethyl 4-(2-Acetyl-1,2,3,4-tetrahydroisoquinolin-7-ylsul-

fonylmethyl)-1-(pyridin-4-yl)piperidin-4-carboxylate (83).

Compound 83 (120 mg, 62%) was obtained from 82 (203 mg,

0.40 mmol), TFA (1 mL), 4-chloropyridine hydrochloride (72

mg, 0.48 mmol), and Et₃N (0.32 mL, 2.30 mmol) as described

for 50: ¹H NMR (CDCl₃) δ 1.35 (3H, t, J ) 7.5 Hz), 1.80-2.00

(2H, m), 2.20 (3H, s), 2.40-2.55 (2H, m), 2.90-3.05 (2H, m),

3.40-3.55 (4H, m), 3.70-3.90 (4H, m), 4.20-4.32 (2H, m),

4.70-4.81 (2H, m), 6.80 (2H, d, J ) 6.9 Hz), 7.30-7.40 (1H,

m), 7.65-7.75 (2H, m), 8.28 (2H, d, J ) 6.9 Hz). Anal.

(C₂₅H₃₁N₃O₅S) C, H, N.

Benzyl 7-[2-(1-tert-Butoxycarbonyl-4-ethoxycarbon-

ylpiperidin-4-yl)ethoxy]-1,2,3,4-tetrahydroisoquinolin-2-

carboxylate (75). Compound 75 (935 mg, 52%) was obtained

from 74 (1.4 g, 3.20 mmol) and 48 (1.81 g, 7.03 mmol) as

described for 49: ¹H NMR (CDCl₃) δ 1.25 (3H, t, J ) 6.9 Hz),

1.60-1.66 (2H, m), 2.01-2.05 (2H, m), 2.10-2.20 (2H, m),

2.73-2.80 (2H, m), 2.85-3.00 (2H, m), 3.65-3.75 (2H, m),

3.80-3.95 (4H, m), 4.18 (2H, q, J ) 6.9 Hz), 4.60 (2H, s), 5.18

(2H, s), 6.56 (1H, m), 6.67 (1H, dd, J ) 2.7, 8.4 Hz), 7.02 (1H,

d, J ) 8.4 Hz), 7.30-7.40 (5H, m). Anal. (C₃₂H₄₂N₂O₇) C, H,

N.

Benzyl 7-[2-[4-Ethoxycarbonyl-1-(pyridin-4-yl)piperi-

din-4-yl]ethoxy]-1,2,3,4-tetrahydroisoquinolin-2-carbox-

ylate (76). Compound 76 (680 mg, 76%) was obtained from

75 (935 mg, 1.65 mmol), 4-chloropyridine hydrochloride (248

mg, 1.65 mmol), and Et₃N (1.38 mL, 9.90 mmol) as described

for 50. This compound was used for the next reaction without

further purification.

Ethyl 4-[2-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-

yloxy)ethyl]-1-(pyridin-4-yl)piperidin-4-carboxylate (77).

Compound 77 (208 mg, 63%) was obtained as a dihydrochloride

from 76 (397 mg, 0.73 mmol), 25% HBr/AcOH (3 mL), DIPEA

(0.64 mL, 3.67 mmol), and 1H-pyrazole-1-carboxamidine hy-

drochloride (214 mg, 1.46 mmol) as described for 51: ¹H NMR

(DMSO-d₆) δ 1.19 (3H, t, J ) 7.1 Hz), 1.55-1.70 (2H, m), 2.00-

2.10 (2H, m), 2.10-2.25 (2H, m), 2.80-2.88 (2H, m), 3.20-

3.35 (2H, m), 3.58 (2H, m), 3.97 (2H, m), 4.00-4.10 (2H, m),

4.15 (2H, q, J ) 7.1 Hz), 4.55 (2H, s), 6.65 (1H, s), 6.75 (1H,

m), 7.14 (1H, d, J ) 8.4 Hz), 7.19 (2H, d, J ) 7.6 Hz), 7.60

(4H, brs), 8.20 (2H, d, J ) 7.6 Hz). Anal. (C₂₅H₃₃N₅O₃‚2HCl‚

2.5H₂O) C, H, N.

4-[2-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-yloxy)-

ethyl]-1-(pyridin-4-yl)piperidin-4-carboxylic Acid (25).

Compound 25 (29 mg, 13%) was obtained as a dihydrochloride

from 77 (200 mg, 0.44 mmol) as described for 19: ¹H NMR

(DMSO-d₆) δ 1.54-1.68 (2H, m), 1.95-2.22 (4H, m), 2.82 (2H,

m), 3.58 (2H, m), 3.92-4.12 (4H, m), 4.55 (2H, s), 6.67 (1H, s),

6.78 (1H, m), 7.14 (1H, d, J ) 8.4 Hz), 7.19 (2H, d, J ) 7.4

Hz), 7.61 (4H, brs), 8.21 (2H, d, J ) 7.4 Hz). Anal. (C₂₃H₂₉N₅O₃‚

2HCl‚2.2H₂O) C, H, N.

tert-Butyl 4-Ethoxycarbonyl-4-iodomethylpiperidin-

1-carboxylate (78). To a stirred solution of 48 (7.14 g, 27.75

mmol) in THF (140 mL) were added LDA (16.6 mL of 2 M

solution in THF, 33.2 mmol) and CH₂I₂(2.7 mL, 33.52 mmol)

at -70 °C. The mixture was allowed to warm to room

temperature and stirred for 15 h. An aqueous solution of 10%

citric acid was added, and the mixture was extracted with

EtOAc. The organic layer was washed with brine, dried over

anhydrous Na₂SO₄, and evaporated under reduced pressure

to leave the residue, which was purified by SiO₂column

chromatography (n-hexane/EtOAc ) 9:1) to give compound 78

(8.68 g, 79%): ¹H NMR (CDCl₃) δ 1.27-1.33 (3H, m), 1.38-

1.45 (10H, m), 1.59-1.64 (1H, m), 2.10-2.22 (2H, m), 2.90-

3.10 (2H, m), 3.29 (2H, s), 3.65-3.95 (2H, m), 4.18-4.26 (2H,

m). Anal. (C₁₄H₂₄INO₄) C, H, N.

tert-Butyl 4-(2-Acetyl-1,2,3,4-tetrahydroisoquinolin-7-

ylthiomethyl)-4-ethoxycarbonylpiperidin-1-carbox-

ylate (80). To a stirred solution of 2-acetyl-1,2,3,4-tetrahydro-

isoquinolin-7-thiol¹⁷(79, 1.94 g, 9.36 mmol) in DMF (120 mL)

were added K₂CO₃(4.27 g, 30.90 mmol) and compound 78 (3.71

g, 9.34 mmol). The mixture was stirred at 100 °C for 1 h and

filtered through Celite. The filtrate was concentrated under

reduced pressure to leave the residue, which was purified by

SiO₂column chromatography (n-hexane/EtOAc ) 2:8) to give

compound 80 (3.92 g, 88%): ¹H NMR (CDCl₃) δ 1.18-1.24 (3H,

m), 1.44-1.51 (11H, m), 2.12-2.18 (5H, m), 2.80-3.05 (4H,

m), 3.12 (2H, brs), 3.66 (1H, m), 3.72-3.95 (3H, m), 4.01-4.11

(2H, m), 4.57-4.69 (2H, m), 7.03-7.22 (3H, m). Anal.

(C₂₅H₃₆N₂O₅S) C, H, N.

4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-ylsul-

fonylmethyl)-1-(pyridin-4-yl)piperidin-4-carboxylic Acid

(27). Compound 27 (120 mg, 55%) was obtained as a dihydro-

chloride from 83 (200 mg, 0.41 mmol), concentrated HCl (10

mL), 1H-pyrazole-1-carboxamidine hydrochloride (181 mg, 1.23

mmol), and 1 N NaOH (2.06 mL, 2.06 mmol) as described for

26: ¹H NMR (DMSO-d₆) δ 1.70-1.90 (2H, m), 2.08-2.20 (2H,

m), 3.03 (2H, m), 3.51 (2H, m), 3.67 (2H, m), 3.77 (2H, s), 3.92

(2H, m), 4.72 (2H, s), 7.19 (2H, d, J ) 7.5 Hz), 7.54 (1H, d, J

) 8.1 Hz), 7.65 (1H, s), 7.72-7.76 (5H, m), 8.23 (2H, d, J )

7.5 Hz). Anal. (C₂₂H₂₇N₅O₄S‚2.2HCl‚2.8H₂O) C, H, N.

1-(Pyridin-4-yl)piperidin-4-one (85). To a stirred solution

of 1,4-dioxa-8-azaspiro[4,5]decane 84 (10 g, 69.8 mmol) in

EtOH (10 mL) were added 4-chloropyridine hydrochloride (10

g, 66.4 mmol) and Et₃N (29 mL, 210 mmol). The mixture was

heated at 150 °C for 22 h in a sealed tube. After cooling to

room temperature, the reaction mixture was diluted with 5 N

NaOH (25 mL) and extracted with CHCl₃. The organic layer

was washed with brine, dried over anhydrous Na₂SO₄, and

evaporated under reduced pressure. The residue was dissolved

in concentrated HCl (30 mL), and the solution was stirred at

Ethyl 4-(2-Acetyl-1,2,3,4-tetrahydroisoquinolin-7-ylthio-

methyl)-1-(pyridin-4-yl)piperidin-4-carboxylate (81). Com-

3602 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10

Ueno et al.

0 °C for 10 min. Then NaOH (15 g, 375 mmol) was added to

the solution, and the mixture was extracted with CHCl₃. The

organic layer was washed with brine, dried over anhydrous

Na₂SO₄, and evaporated under reduced pressure to leave the

residue, which was purified by SiO₂column chromatography

(CHCl₃/MeOH/concentrated NH₃) 90:10:1) to give compound

85 (6.1 g, 52 %): ¹H NMR (CDCl₃) δ 2.57 (4H, t, J ) 6.2 Hz),

3.74 (4H, t, J ) 6.2 Hz), 6.71 (2H, dd, J ) 1.5, 4.5 Hz), 8.33

(2H, dd, J ) 1.5, 4.5 Hz). Anal. (C₁₀H₁₂N₂O) C, H, N.

Methy 4-Amino-1-(pyridin-4-yl)piperidin-4-carbox-

ylate (86). To a stirred solution of 85 (1 g, 5.67 mmol) in

MeOH (10 mL) were added NH₄OAc (880 mg, 11.42 mmol),

NaCN (560 mg, 11.43 mmol), concentrated NH₄OH (5 mL),

and AcOH (2 mL). The mixture was stirred at room temper-

ature for 12 h. After the reaction mixture was evaporated

under reduced pressure, the residue was diluted with water,

and the mixture was extracted with CHCl₃. The organic layer

was washed with brine, dried over anhydrous Na₂SO₄, and

evaporated under reduced pressure to leave the residue, which

was dissolved in MeOH (25 mL). To the solution was bubbled

HCl gas at 0 °C for 30 min. After being stirred at room

temperature for 12 h, the solution was concentrated under

reduced pressure. Then 2 N HCl (25 mL) was added to the

residue, and the mixture was stirred at room temperature for

2 h. The mixture was neutralized with NaHCO₃and extracted

with CHCl₃. The organic layer was washed with brine, dried

over anhydrous Na₂SO₄, and evaporated under reduced pres-

sure to give compound 86 (760 mg, 57%) in a sufficient purity

to be used without purification: ¹H NMR (CDCl₃) δ 1.55-1.65

(2H, m), 2.08-2.17 (2H, m), 3.46-3.50 (4H, m), 3.74 (3H, s),

6.66 (2H, dd, J ) 1.5, 4.5 Hz), 8.25 (2H, dd, J ) 1.5, 4.5 Hz).

Methyl 1-(Pyridin-4-yl)-4-(2-trifluoroacetyl-1,2,3,4-tetra-

hydroisoquinolin-7-ylsulfonylamino)piperidin-4-carbox-

ylate (88). To a stirred solution of 86 (330 mg, 1.40 mmol) in

CHCl₃(5 mL) were added 7-chlorosulfonyl-2-trifluoroacetyl-

1,2,3,4-tetrahydroisoquinoline¹⁷(87, 500 mg, 1.53 mmol) and

pyridine (0.23 mL, 2.84 mmol). The mixture was stirred at

room temperature for 12 h. The reaction mixture was diluted

with water and extracted with CHCl₃. The organic layer was

washed with brine, dried over anhydrous Na₂SO₄, and evapo-

rated under reduced pressure to give compound 88 (627 mg,

74%) in a sufficient purity to be used without purification: ¹H

NMR (CDCl₃) δ 2.02-2.16 (2H, m), 2.22-2.35 (2H, m), 2.94-

3.08 (2H, m), 3.39-3.58 (5H, m), 3.62-3.76 (2H, m), 3.80-

3.96 (2H, m), 4.77-4.82 (2H, m), 6.80 (2H, d, J ) 7.0 Hz),

7.16-7.32 (1H, m), 7.68-7.82 (2H, m), 8.20 (2H, m).

Methyl 4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-

ylsulfonylamino)-1-(pyridin-4-yl)piperidin-4-carbox-

ylate (89). To a stirred solution of 88 (627 mg, 1.04 mmol) in

CHCl₃(5 mL) and MeOH (1 mL) was added 2 N NaOH (1.1

mL, 2.2 mmol), and the mixture was stirred at room temper-

ature for 2 h. The reaction mixture was diluted with 2 N HCl

(1.1 mL, 2.2 mmol), and the solvent was evaporated under

reduced pressure to leave the residue, which was dissolved in

DMF (3 mL). To the solution were added 1H-pyrazole-1-

carboxamidine hydrochloride (305 mg, 2.08 mmol) and DIPEA

(0.19 mL, 5.22 mmol). After the mixture was stirred at room

temperature for 12 h, insoluble material was removed and the

filtrate was concentrated under reduced pressure. The residue

was purified by reversed-phase HPLC (0.05% TFA in H₂O/

MeOH ) 6:4), and the fraction containing the desired product

was treated with HCl/EtOH to give compound 89 (480 mg,

88%) as a dihydrochloride: ¹H NMR (DMSO-d₆) δ 1.86-2.16

(4H, m), 3.00 (2H, m), 3.35-3.42 (5H, m), 3.67 (2H, m), 3.84

(2H, m), 4.74 (2H, s), 7.17 (2H, d, J ) 7.1 Hz), 7.47 (1H, d, J

) 8.1 Hz), 7.56 (1H, s), 7.62 (1H, d, J ) 8.1 Hz), 7.82 (4H,

brs), 8.22 (2H, d, J ) 7.1 Hz), 8.56 (1H, s), 13.89 (1H, brs).

Anal. (C₂₂H₃₀Cl₂N₆O₄S) C, H, N.

J ) 7.3 Hz), 7.44 (1H, d, J ) 8.1 Hz), 7.56 (1H, s), 7.67 (1H, d,

J ) 8.1 Hz), 7.80 (4H, brs), 8.20 (2H, d, J ) 7.3 Hz), 8.35 (1H,

s). Anal. (C₂₁H₂₆N₆O₄S‚2HCl‚2.3H₂O) C, H, N.

Pharmacological Experiments. Ex Vivo Assessment of

Anti-Human Factor Xa in Cynomolgus Monkey Plasma.¹⁰

Compound 2 dissolved in physiological saline was adminis-

trated intravenously to male cynomolgus monkeys at a dose

of 1 mg/kg. Before administration and at 5, 10, 15, 30, 60, and

120 min following administration, a total of 1500 µL of blood

samples was collected from the saphenous vein of each monkey

into a syringe containing 300 µL of 3.8% citric acid. Plasma of

each sample was prepared by centrifugation at 2000g for 10

min at 4 °C. Compound 2 was dissolved in deionized distilled

water and administered orally to fasted male cynomolgus

monkeys at a dose of 10 mg/kg. Before administration and at

15, 30, 60, 120, 240, 360, and 480 min following oral admin-

istration, an amount of 1500 µL of blood samples was collected

and plasma was obtained as described above.

Forty microliters of human factor Xa (0.5 U/mL) and 40 µL

of a 4-fold-diluted plasma sample were incubated in 40 µL of

0.1 M Tris/0.2 M NaCl buffer (pH 8.4) at 37 °C for 10 min.

Then, a total of 40 µL of a synthetic substrate, S-2222, adjusted

to 0.8 mM was added and the mixture was incubated at 37 °C

for 3 min. The reaction was stopped by addition of 60% acetic

acid, and the absorbance at 405 nm was measured by a

spectrometer (model 3550, BIO-RAD, Hercules). For the

control, plasma obtained prior to administration of compound

2 was used for measurement. Human factor Xa inhibitory

activity was calculated as percent inhibition relative to control.

Assessment of the Inhibitory Effect on Human Factor

Xa.¹The reaction mixture consisting of 40 µL of human factor

Xa (0.16 U/mL), 40 µL of buffer, and 40 µL of vehicle or

compound solution with each final concentration was incu-

bated for 10 min at 37 °C. To the reaction mixture, samples of

40 µL of S-2222 solution with various final concentrations were

added, and further incubation at 37 °C for 10 min was done.

A total of 40 µL of acetic acid solution (60%) was added to the

reaction mixture to stop the enzyme reaction. p-Nitroaniline

(p-NA) released from substrate was measured by reading

absorbance at 405 nm with a microplate reader.

Assessment of the Inhibitory Effect on Human Throm-

bin.¹The reaction mixture consisting of 40 µL of human

thrombin (0.4 U/mL), 40 µL of buffer, and 40 µL of vehicle or

compound solution with each final concentration was incu-

bated for 10 min at 37 °C. To the reaction mixture, samples of

40 µL of S-2238 solution with various final concentrations were

added, and further incubation at 37 °C for 10 min was done.

A total of 40 µL of acetic acid solution (60%) was added to

reaction mixture to stop the enzyme reaction. p-Nitroaniline

released from substrate was measured by reading absorbance

at 405 nm with a microplate reader.

Assessment of the Inhibitory Effect on Human

Trypsin.¹The reaction mixture consisting of 40 µL of human

trypsin (5 U/mL), 40 µL of buffer, and 40 µL of vehicle or

compound 2 solutions with final concentrations of 50, 100, and

200 µM were incubated for 10 min at 37 °C. To the reaction

mixture, samples of 40 µL of S-2222 solution with final

concentrations of 0.125 and 0.25 mM were added, and further

incubation at 37 °C for 10 min was done. A total of 40 µL of

acetic acid solution (60%) was added to the reaction mixture

to stop the enzyme reaction. p-Nitroaniline released from

substrate was measured by reading absorbance at 405 nm with

a microplate reader.

Assessment of the Inhibitory Effect on Human Plas-

min.¹The reaction mixture consisting of 40 µL of human

plasmin (0.02 U/mL), 40 µL of buffer, and 40 µL of vehicle or

compound 2 solutions with final concentrations of 12.5, 25, and

50 µM were incubated for 10 min at 37 °C. To the reaction

mixture, samples of 40 µL of S-2403 solution with final

concentrations of 0.125 and 0.25 mM were added, and further

incubation at 37 °C for 10 min was done. A total of 40 µL of

acetic acid solution (60%) was added to the reaction mixture

to stop the enzyme reaction. p-Nitroaniline released from

4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-ylsul-

fonylamino)-1-(pyridin-4-yl)piperidin-4-carboxylic Acid

(28). Compound 28 (360 mg, 90%) was obtained as a dihydro-

chloride from 89 (415 mg, 0.878 mmol) as described for 1: ¹H

NMR (DMSO-d₆) δ 1.85-2.10 (4H, m), 2.99 (2H, m), 3.30 (2H,

m), 3.67 (2H, m), 3.80-3.95 (2H, m), 4.71 (2H, s), 7.16 (2H, d,

Selective Factor Xa Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3603

H. W.; Spada, A. P. Progress in the design of inhibitors of

coagulation factor Xa. Drugs Future 1999, 24, 771-787. (d) Zhu,

B. Z.; Scarborough, R. M. Factor Xa inhibitors: Recent advances

in anticoagulant agents. Annu. Rep. Med. Chem 2000, 35, 83-

102. (e) Kucznierz, R.; Grams, F.; Leinert, H.; Marzenell, K.;

Engh, R. A.; von der Saal, W. Tetrahydro-isoquinoline-based

factor Xa inhibitors. J. Med. Chem. 1998, 41, 4983-4994. (f)

Hirayama, F.; Koshio, H.; Ishihara, T.; Watanuki, S.; Hachiya,

S.; Kaizawa, H.; Kuramochi, T.; Katayama, N.; Kurihara, H,

Taniuchi, Y.; Sato, K.; Sakai-Moritani, Y.; Kaku, S.; Kawasaki,

T.; Matsumoto, Y.; Sakamoto, S.; Tsukamoto, S. Design, syn-

thesis and biological activity of YM-60828 derivatives: potent

and orally-bioavailable factor Xa inhibitors based on naphtho-

anilide and naphthalensulfonanilide templates. Bioorg. Med.

Chem. 2002, 10, 2597-2610. (g) Quan, M. L.; Ellis, C. D.; He,

M. Y.; Liauw, A. Y.; Woerner, F. J.; Alexander, R. S.; Knabb, R.

M.; Lam, P. Y. S.; Luettgen, J. M.; Wong, P. C.; Wright, M. R.;

Wexler, R. R. Nonbenzamidine tetrazole derivatives as factor

Xa inhibitors. Bioorg. Med. Chem. Lett. 2003, 13, 369-373.

(3) Hobbelen, P. M.; van Dinther, T. G.; Vogel, G. M. T.; van Boeckel,

C. A. A.; Moelker, H. C. T.; Meuleman, D. G. Pharmacological

profile of the chemically synthesized antithrombin III binding

fragment of heparin (pentasaccharide) in rats. Thromb. Hae-

mostasis 1990, 63, 265-270.

(4) (a) Ueno, H.; Kato, S.; Yokota, K.; Hoshi, J.; Hayashi, M.; Uchida,

I.; Aisaka, K.; Hase, Y.; Cho, H. Structure-activity relationships

of potent and selective factor Xa inhibitors: benzimidazole

derivatives with the side chain oriented to the prime site of factor

Xa. Bioorg. Med. Chem. Lett. 2004, 14, 4281-4286. (b) Ueno,

H.; Yokota, K.; Hoshi, J.; Yasue, K.; Hayashi, M.; Uchida, I.;

Aisaka, K.; Hase, Y.; Kato, S.; Cho, H. Discovery of novel

tetrahydroisoquinoline derivatives as potent and selective factor

Xa inhibitors. Bioorg. Med. Chem. Lett. 2005, 15, 185-189. (c)

Katoh, S.; Yokota, K.; Hayashi, M. PCT Int. Appl. WO9952895,

1999.

substrate was measured by reading absorbance at 405 nm with

a microplate reader.

Determination of K_iValues.¹Factor Xa inhibition mech-

anisms of compound 2 were determined from a Lineweaver-

Burk plot of the initial velocity [OD at 405 nm] and the

concentration of the substrate [µM]. The K_ivalues of compound

2 for factor Xa and other enzymes were determined from a

Dixon plot.

Survival Test Used Mice. Male ICR mice were used in

the study. Each compound at 10 mg/kg was injected from tail

vein, and mice were observed for 1 day. At the next day after

the injection, survival rate was counted.

Effects of Compound 2 in Animal Models. Animals.

Male SD rats were purchased from CRJ (Yokohama,Japan)

and used in the study at the age of 7-9 weeks.

Assessment of the Effect on Rat Venous Thrombosis.¹⁸

Rats were anesthetized with urethane (1.3 g/kg ip). About 1

cm length of an abdominal vein was carefully dissected at a

site below the left renal veins, and Parafilm (Parafilm M, ANC)

was placed on the dorsal aspect of the abdominal vein. A 2

mm × 3 mm piece of filter paper (no. 1, Whattman) containing

25% FeCl₃was applied to the detached vein and removed after

20 min. Immediately after removing the filter paper, a 5 mm

length of the abdominal vein was resected and weighed. The

thrombus weight was calculated by subtracting the weight of

the venal vessel walls from the total measured weight.

Compound 2 (0.1, 0.3, 1 mg kg^-1h^-1) was administered by

continuous intravenous infusion from 1 h prior to placement

of the filter paper.

Assessment of the Effect on Rat Middle Cerebral

Arterial Thrombosis.¹⁹PE50 catheters were inserted into

animals through the right and left femoral veins (for rose

bengal and drug administration) under anesthesia with a

mixture of gases halothane, nitrous oxide, and oxygen (nitrous

oxide/oxygen ) 7:3; halothane concentration, 4% at introduc-

tion; 1.5% at maintenance phase). During cathetherization, the

rectal temperature of the animals was maintained above 37

°C with an insulating pad. A hole approximately 2.5 mm in

diameter was made with a dental drill in the basal part of the

temporal bone, and the intersection of the major trunk of the

middle cerebral artery and the olfactory tract was exposed

without incising the dura. In addition, a hole approximately

2 mm in diameter was made in the skull, peripheral to the

middle cerebral artery. Twenty minutes after starting intra-

venous infusion of saline or compound 2 (0.03, 0.1, 0.3 mg kg^-1

h^-1), rose bengal (Sigma) was intravenously administered at

a dose of 20 mg/kg. The animals were then irradiated with

light for 10 min. After completion of irradiation, the surgical

site was closed and the animals were aroused. Drug admin-

istration under freely moving conditions was continuously

performed until 24 h after occlusion. The study was blinded

through all processes including operation, drug administration,

and imaging analysis by encoding the drug sample labels.

Following decapitation 24 h after occlusion, whole brains were

isolated and cut into six coronal slices at 2 mm intervals

(bregma: +4, +2, 0, -2, -4, -6 mm). Specimens were

measured for infarction size using an imaging analyzer after

staining (37 °C, 20 min) with 2% TTC (2,3,5-triphenyltetra-

zolium chloride) in saline solution. “Total infarction volume”

was calculated by multiplying slice thickness (2 mm) by the

sum of infarction size.

(5) The complex model of FXa-compound 3 (DX9065a) was built

by docking compound 3 into the S1 and aryl binding sites of the

FXa crystal structure (PDB code 1hcg). The Discover and Insight

II programs from Accerlys were used for energy calculation and

graphical displays, respectively. This model was similar to the

subsequently reported crystal structure of FXa-compound 3

(PDB code 1fax) (ref 5b). The complex model of FXa-compound

1 was built by docking compound 1 into the S1 and the aryl

binding site on FXa crystal structure (PDB code 1fax). The

Discover and Insight II programs from Accerlys were used for

energy calculation and graphical displays, respectively. (a)

Katakura, S.; Nagahara, T.; Hara, T.; Kunitada, S.; Iwamoto,

M. Molecular model of an interaction between factor Xa and DX-

9065a, a novel factor Xa inhibitor: contribution of the acet-

imidoylpyrrolidine moiety of the inhibitor to potency and selec-

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914-924.

(7) Marsais, F.; Trecourt, F.; Breant, P.; Queguiner, G. Directed

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,

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A

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(9) Pharmacokinetic parameters in cynomolgus monkeys: C_max

)

0.39 µg/mL; T_1/2) 3.6 h; bioavailability (BA) of 10.7% (10 mg/

kg po); clearance of 0.25 L h^-1kg^-1; volume of distribution of

0.32 L/kg (1 mg/kg iv).

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Supporting Information Available: Elemental analysis

data of new compounds. This material is available free of

charge via the Internet at http://pubs.acs.org.

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JM058160E

Article Doi

Synthesis and structure-activity relationships of novel selective factor Xa inhibitors with a tetrahydroisoquinoline ring

DOI: 10.1021/jm058160e

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Article abstract of DOI:10.1021/jm058160e

Full text of DOI:10.1021/jm058160e

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