Synthesis and Structureâ Activity Relationships of Novel Selective

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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-1 h-1 in 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-1 h-1. These results suggest that compound 2 (JTV-803) is likely to be useful as both a venous and arterial antithrombotic agent. Introduction Activation of the intravascular coagulation system is involved in a number of cardiovascular diseases such as deep vein thrombosis (DVT), disseminated intravascular 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, respectively. For instance, heparin administration is sometimes 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. 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 interested in achieving inhibition of FXa by the development of a novel FXa inhibitor. Since the discovery of compound 3 (DX-9065a),1 a variety of other compounds have been reported.2 The release of fondaparin sodium, which is a synthetic pentasaccharide, proved the clinical effectiveness of FXa inhibitors as anticoagulants.3 This situation prompted us to disclose our experimental results on novel FXa * To whom correspondence should be addressed. Phone: +81-72681-9700. Fax: +81-72-681-9725. E-mail: [email protected].

inhibitors.4 As 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 In the present study, our focus was directed toward the discovery of other novel inhibitors. From analysis of the binding of FXa to compound 3,5 it 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 189.4b Then a 4-piperidinylmethyloxy group was selected for position 7 of the tetrahydroisoquinoline ring as a spacer for the introduction 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) derivatives and investigated the structure-activity relationships (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. Chemistry The synthesis of compound 5 was initially undertaken. 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 assessment 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 OCH2 with a SCH2, SO2CH2, or SO2NH moiety (see Tables 1-3).

10.1021/jm058160e CCC: $30.25 © 2005 American Chemical Society 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 5-18a

a Reagents and conditions: (a) Boc O, NaOH, dioxane/H O, room temp. (b) From 31 to 40: (i) NaH, 33, DMF, room temp; (ii) H /7.5% 2 2 2 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 performed according to these procedures via intermediate

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

a Reagents and conditions: (a) CbzCl, NaOH(aq), THF, room temp; (b) (i) ClCH SMe, NaH, DMF, 0 °C to room temp; (ii) SO Cl , 2 2 2 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) 1Hi 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-pyrazole1-carboxamidine, iPr2NEt, DMF, room temp; (i) (i) 4-fluorobenzonitrile, K2CO3, DMSO, 120 °C;(ii) H2S, Et3N/pyridine, room temp; (iii) MeI, acetone, reflux; (iv) NH4OAc, EtOH, 75 °C; (j) TFA, CHCl3, room temp; (k) MeI, iPr2NEt, acetone, room temp, then HCl/dioxane.

34. Hydrogenation of 7-methoxyisoquinoline6 gave compound 29, which was treated with 1H-pyrazol-1-(N,N′bis-tert-butoxycarbonyl)carboxyamidine to afford compound 30. Treatment of 30 with benzyl 4-bromomethylpiperidine-1-carboxylate 33, followed by cleavage of a benzyloxycarbonyl (Cbz) group under a hydrogen atmosphere 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-butoxycarbonyl (Boc) groups with trifluoroacetic acid (TFA) gave compound 5; acetylation of 34 with acetic anhydride 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. Compounds 11-13 were obtained in a similar manner. 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

(DIPAD) and triphenylphosphine or with bromide 33 in the presence of NaH provided 37, 38, or 39, respectively. Removal of the protecting group of 39 under a hydrogen atmosphere afforded compound 40, which was reacted with 4-chloro-3-formylpyridine7 to give compound 41. The resulting aldehyde 41 was treated with NaCN and MnO2 in 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-carboxamidine, resulted in the 1,2,3,4-tetrahydroisoquinoline-2carboxamidine derivative 8, 9, 14, or 44. Compound 15 was obtained after hydrolysis of 14. Hydrogenation of compound 44, followed by hydrolysis, furnished compound 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-



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

compda

R1

R2

FXa IC50 (µM)b

FIIa IC50 (µM)b

mortality (10 mg/kg, iv)

5 6 7 8 9 10 11 12 13 14 15 16 1 17 18 19 20 21 22

H C(dO)Me C(dNH)Me pyridin-4-yl quinolin-4-yl pyridin-4-ylmethyl pyridin-2-ylmethyl 2-aminobenzyl 3-aminobenzyl (3-CO2Me)pyridin-4-yl (3-CO2H)pyridin-4-yl pyridin-4-yl pyridin-4-yl (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

H H H H H H H H H H H CO2Et CO2H H H CO2H CO2H CO2Et CO2Et

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 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10

NTc NTc 3/3d 3/3 3/3 3/3 NTc NTc NTc 3/3 0/3 3/3 0/3 3/3 0/3 0/3 0/3 NTc NTc

a Compounds 1, 5, 7-9, 14-20: two HCl salts. Compounds 10-13 and 22: three HCl salts. Compounds 6 and 21: HCl salt. b n ) 3, mean ( SEM (standard errors). c Not tested. d Number of deaths/number of animals tested.

Scheme 3. Synthesis of Compound 57a

a Reagents and conditions: (a) Boc O, NaOH, dioxane/H O, room temp; (b) (i) ClCH SMe, NaH, DMF, 0 °C to room temp; (ii) SO Cl , 2 2 2 2 2 CH2Cl2, 0 °C; (c) 46, LDA, THF, -70 °C to room temp; (d) (i) TFA, CHCl3, room temp; (ii) 1H-pyrazole-1-carboxamidine, iPr2NEt, DMF, room temp; (e) concentrated HCl, reflux.

dine to afford compound 16. Hydrolysis of this compound 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-chloro2-methylpyridine provided 50. After deprotection of the Cbz group of 50, amidination with 1H-pyrazole-1carboxamidine, and successive hydrolysis of 51, compound 19 was obtained. Similarly, treatment of 52 with 2,4-dichloropyrimidine gave 53, followed by hydrogenation with 7.5% Pd/C-HCO2NH4 to 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 corresponding 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, respectively. Namely, the 2-benzazepine derivative 64 was prepared by rearrangement of compound 63 with NaN3/ HCl and by successive reduction with LiAlH4. Demethylation and successive protection of the nitrogen atom of 64 gave compound 65, after which alkylation with NaH/chloromethyl methyl sulfide and then chlorination with sulfuryl chloride provided chloride 66. Crucial coupling of 66 with 46 in the presence of LDA furnished compound 67. Deprotection and amidination

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a Reagents and conditions: (a) (i) NaN , concentrated HCl, 0 °C to room temp; (ii) LiAlH , THF/dioxane, reflux; (b) (i) 48% HBr(aq), 3 4 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, LDA, THF, -70 °C to room temp; (e) (i) TFA, CHCl3, room temp; (ii) 1H-pyrazole-1-carboxamidine, iPr2NEt, DMF, room temp; (h) concentrated HCl, reflux.


aReagents and conditions: (a) (i) Tf O, Et N, CHCl , 0 °C; (ii) Zn(CN) , Pd(PPh ) , DMF, 80 °C; (iii) AlCl , chlorobenzene, reflux; (b) (i) 2 3 3 2 3 4 3 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.

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)2 in the presence of Pd(PPh3)4 via the trifluoromethanesulfonate 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 followed 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, respectively, as depicted in Schemes 7 and 8.


Results and Discussion As mentioned above, we designed and prepared compound 5. This compound showed moderate FXa inhibitory activity (IC50 ) 3.34 ( 0.25 µM),1a demonstrating that it had sufficient potential for further studies on structure-activity relationships. Introduction of basic substituents on the nitrogen atom of the

a Reagents and conditions: (a) (i) Br(CH ) OH, diisopropyl 2 2 azodicarboxylate (DIPAD), PPh3, THF, room temp; (ii) NaI, DMF, 90 °C; (b) 48, LDA, THF, -70 °C to room temp; (c) (i) TFA, CHCl3, room temp; (ii) 4-chloropyridine, Et3N, EtOH, 150 °C; (d) (i) 25% HBr/AcOH, room temp; (ii) 1H-pyrazole-1-carboxamidine, iPr2NEt, DMF, room temp; (e) concentrated HCl, reflux.



Scheme 7. Synthesis of Compounds 26 and 27a

a Reagents and conditions: (a) LDA, CH I , THF, -78 °C to room temp; (b) K CO , DMF, 100 °C; (c) TFA, room temp; (d) 4-chloropyridine 2 2 2 3 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.


a Reagents and conditions: (a) (i) 4-chloropyridine hydrochloride, Et N, EtOH, 150 °C; (ii) concentrated HCl, reflux; (b) (i) NH OAc, 3 4 NaCN, MeOH, room temp; (ii) HCl, MeOH, room temp, then H2O; (c) pyridine, CHCl3, room temp; (d) (i) NaOH, MeOH, room temp; (ii) 1H-pyrazole-1-carboxamidine hydrochloride, iPr2NEt, DMF, room temp; (e) HCl(aq), reflux.

piperidine ring provided compounds 7-13 (Table 1).4b,c Among the compounds with a pyridinyl group, quinolinyl 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,8 and 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. Unfortunately, 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. 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

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 exhibited 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 conformation 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

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Figure 2. Complex model of compound 1 (orange) with FXa (green). Table 2. Enzyme Inhibitory Activities for FXa and FIIa (Thrombin)

compda

1

23

24

-R

FXa IC50 (µM)b

thrombin IC50 (µM)b

0.026 ( 0.003

>10

>10

0.51 ( 0.01

>10

>10

a Compounds 1, 23, 24: two HCl salts. b n ) 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

Table 3. Enzyme Inhibitory Activities for FXa and FIIa (Thrombin)

compda

-R-

FXa IC50 (µM)b

thrombin IC50 (µM)b

1 25 26 27 28

-OCH2-OCH2CH2-SCH2-SO2CH2-SO2NH-

0.026 ( 0.003 0.23 ( 0.02 0.39 ( 0.01 5.44 ( 0.12 2.90 ( 0.13

>10 >10 >10 >10 >10

a Compounds 1 and 25-28: two HCl salts. b n ) 3, mean ( SEM (standard errors).

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 1/20 of the activity of 1. Regarding the effect of modifications or extension of the middle spacer R in Table 3, the transformation from OCH2 to OCH2CH2, SCH2, SO2CH2, or SO2NH was undertaken to afford compounds 2528, 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 (IC50 g 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. As mentioned above, we found a series of novel 2,7THIQ derivatives, initially with the aid of computerassociated molecular modeling followed by conventional SAR studies. Among them, we concluded that compound 1 had sufficient potency for further development. To



Table 4. Selectivity of Compound 2 for Serine Protease Enzymes: Inhibitory Activities (Ki Value, µM) against Factor Xa, Thrombin, Plasmin, Trypsin Ki (µM)a compd

factor Xa

thrombin

plasmin

trypsin

2 3

0.019 ( 0.001 0.041 ( 0.002

>100 >100

78.2 ( 2.8 23.0 ( 0.8

13.6 ( 1.8 0.62 ( 0.08

a The K values for each enzyme were determined from Dixon’s i 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 cynomolgus monkey. Data represent the mean ( SEM; n ) 6.10

obtain stable fine crystals, we examined the crystallization 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 pharmacological 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 (IC50 g 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 administration, inhibition increased to 41 ( 4% at 120 min and then gradually declined. The plasma concentration (Cmax) of 2 at a dose of 10 mg/kg was 0.39 µg/mL at 120 min.9 Subsequently, 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 dosedependent antithrombotic effect when infused at 0.1-1 mg kg-1 h-1, and the effect was significant at 0.3 and 1 mg kg-1 h-1. Low molecular weight heparin (LMWH) also showed a dose-dependent antithrombotic effect in this model at doses of 30-300 U kg-1 h-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-1 h-1) was administered as a continuous intravenous infusion from 20 min before occlusion to 24 h after occlusion. These studies suggested 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 demonstrated. 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).10 After further evaluation of toxicology and physical properties (crystallization, pharmaceutical stability, etc.), compound 2 (JTV-803) was finally selected 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 PerkinElmer 2400II instrument. 7-Hydroxy-1,2,3,4-tetrahydroisoquinoline Hydrobromide (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

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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-tetrahydroisoquinoline (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-tetrahydroisoquinoline-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 1Hpyrazole-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 pressure to leave the residue, which was purified by SiO2 column 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 BH3 in 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 isonipecotinate (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 SiO2 column chromatography (n-hexane/acetone ) 3:1) to give ethyl 1-(quinolin-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 Na2SO4 solution 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. Benzyl 4-(2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquinolin-7-yloxymethyl)piperidin-1-carboxylate (39). To a stirred solution of 31 (1.2 g, 4.81 mmol) in DMF (15 mL)

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 SiO2 column chromatography (n-hexane/acetone ) 6:1) to give compound 39 (1.82 g, 79%): 1H 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-tetrahydroisoquinolin-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-tetrahydroisoquinoline-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 SiO2 column 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). Compound 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)piperidin-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinoline-2-carboxylate (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 SiO2 column 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 SiO2 column chromatography (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



(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. Ethyl 3-[4-[4-(2-tert-Butoxycarbonyl-1,2,3,4-tetrahydroisoquinolin-7-yloxymethyl)piperidin-1-yl]pyridin-3-yl]2-propenate (43). To a stirred slurry of triethyl phosphonoacetate (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 SiO2 column chromatography (CHCl3/MeOH ) 20:1) to give compound 43 (100 mg, 91%): 1 H NMR (CDCl3) δ 1.35 (3H, t, J ) 6.4 Hz), 1.49 (9H, s), 1.501.80 (2H, m), 1.95-2.10 (3H, m), 2.75-2.95 (4H, m), 3.403.50 (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-4carboxylic 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-hydroxymethylpiperidine1-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). 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 SiO2 column chromatography (n-hexane/acetone ) 5:1) to afford benzyl 4-[2-(N,N′di-tert-butoxycarbonylamidin)-1,2,3,4-tetrahydroisoquinolin-7yloxymethyl]piperidine-1-carboxylate (55 mg, 68%): 1H 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

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-tetrahydroisoquinolin-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. 7-(1-Acetylpiperidin-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinolin-2-carboxyamidine (6). Step 1. To a stirred solution 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-(1acetylpiperidin-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinolin-2carboxamidine (100 mg, 90%): 1H 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), 3.573.90 (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-tetrahydroisoquinolin-2-carboxamidine (7). To a stirred solution 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-4ylmethoxy)-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 dihydrochloride: 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), 3.924.20 (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. 7-[1-(Pyridin-4-yl)piperidin-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinolin-2-carboxamidine (8). To a stirred solution of 37 (500 mg, 1.18 mmol) in CHCl3 (5 mL) was added TFA (2.5 mL). After the mixture was stirred at room temperature for 1 h, the solvent was removed under reduced pressure. Aqueous NaHCO3 solution was added, and the mixture was extracted with EtOAc. The organic layer was washed with

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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-tetrahydroisoquinoline (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), 3.903.98 (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 hydrochloride (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 1.201.45 (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-tetrahydroisoquinolin-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-(Pyridin-4-ylmethyl)piperidin-4-ylmethoxy]-1,2,3,4tetrahydroisoquinolin-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 SiO2 preparative TLC (CHCl3/ MeOH ) 20:1) to give N,N′-di-tert-butoxycarbonyl-7-[1-(pyridin-4-ylmethyl)piperidin-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinolin-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), 2.852.95 (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 trihydrochloride: 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), 4.484.54 (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]-1,2,3,4tetrahydroisoquinolin-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.

7-[1-(2-Aminobenzyl)piperidin-4-ylmethoxy]-1,2,3,4tetrahydroisoquinolin-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 SiO2 column chromatography (n-hexane/acetone ) 7:1) to give N,N′-di-tert-butoxycarbonyl-7-[1-(2-nitrophenylmethyl)piperidin-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinolin-2-carboxamidine (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]-1,2,3,4tetrahydroisoquinolin-2-carboxamidine (70 mg, 98%) as a dihydrochloride. Without purification, a solution of the compound (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]-1,2,3,4tetrahydroisoquinolin-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. Methyl 4-[4-(1,2,3,4-Tetrahydroisoquinolin-7-yloxymethyl)piperidin-1-yl]pyridin-3-carboxylate (14). Compound 14 (15 mg, 39%) was obtained as a dihydrochloride from 42 (37 mg, 0.077 mmol), 1H-pyrazole-1-carboxamidine hydrochloride (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-tetrahydroisoquinolin7-yloxymethyl)piperidin-1-yl]piperidin-3-yl]-2-propenate (44). Compound 44 (50 mg, 49%) was obtained as a dihydrochloride from 43 (100 mg, 0.191 mmol), 1H-pyrazole1-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), 1.822.05 (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),



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. Ethyl 3-[4-[4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin7-yloxymethyl)piperidin-1-yl]pyridin-3-yl]-2-propionate (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-7yloxymethyl)piperidin-1-yl]pyridin-3-yl]-2-propionic acid (18). Compound 18 (20 mg, 85%) was obtained as a dihydrochloride 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-tetrahydroisoquinolin-2-carboxylate (45).14 To 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 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 SiO2 column chromatography (n-hexane/EtOAc ) 8:1) to give benzyl 7-methylthiomethoxy-1,2,3,4-tetrahydroisoquinolin-2carboxylate (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), 1.801.92 (2H, m), 2.38-2.50 (1H, m), 2.78-2.90 (2H, m), 3.904.10 (2H, m), 4.14 (2H, q, J ) 7.1 Hz). Anal. (C13H23NO4) C, H, N. Benzyl 7-[4-Ethoxycarbonyl-1-(pyridin-4-yl)piperidin4-ylmethoxy]-1,2,3,4-tetrahydroisoquinolin-2-carboxylate (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

residue, which was purified by SiO2 column 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-tetrahydroisoquinolin-7-oxymethyl)-4-ethoxycarbonylpiperidin-1-yl]-1methylpyridinium 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-ethoxycarbonylpiperidin-4-ylmethoxy)-1,2,3,4-tetrahydroisoquinolin2-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 evaporated under reduced pressure to leave the residue, which was purified by SiO2 column 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-7yloxymethyl)-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-carboxamidine hydrochloride (3.69 g, 25.17 mmol) and diisopropylethylamine (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. 4-[4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-oxymethyl)-4-ethoxycarbonylpiperidin-1-yl]-1-methylpyridinium Chloride (21). Compound 21 (37 mg, 52%) was obtained as a hydrochloride from 55 (99 mg, 0.147 mmol), 1Hpyrazole-1-carboxamidine hydrochloride (45 mg, 0.308 mmol), and diisopropylethylamine (0.054 mL, 0.312 mmol) as described 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

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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. 4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-yloxymethyl)-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 dihydrochloride: 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-NCH2CH2 and 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-yloxymethyl)-1-(pyridin-4-yl)piperidine-4-carboxylicAcidMonomethanesulfonate (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 methanesulfonic 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, ArNCH2CH2 and 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-2carboxylate (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 NaHCO3 solution. The mixture was extracted with EtOAc, and the organic layer was washed with brine, dried over anhydrous Na2SO4, and evaporated 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 SiO2 column 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-ethoxycarbonylpiperidin-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinolin-2carboxylate (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.

Benzyl 7-[1-(1-Amidinophenyl-4-yl)-4-ethoxycarbonylpiperidin-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinolin-2carboxylate (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-7yloxymethyl)-1-(2-methylpyridin-4-yl)piperidin-4-carboxylate (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 containing 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-yloxymethyl)-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-7yloxymethyl)-1-(pyrimidin-4-yl)piperidin-4-carboxylate (54) and 4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-yloxymethyl)-1-(1,3-pyrimidin-4-yl)piperidin-4-carboxylate (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 filtrate was concentrated under reduced pressure to give ethyl 1-(pyrimidin-4-yl)-4-(1,2,3,4-tetrahydroisoquinolin-7-yloxymethyl)piperidin-4-carboxylate (210 mg, 99%). Without purification, to a stirred solution of the compound (200 mg, 0.50 mmol) in DMF (1 mL) were added 1H-pyrazole-1-carboxamidine 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



solvent was evaporated under reduced pressure to give compound 20 (51 mg, 21%) as a dihydrochloride: 1H NMR (DMSOd6) 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-1,2,3,4tetrahydroisoquinolin-7-yloxymethyl)piperidin-4-carboxylate (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 1Hpyrazole-1-carboxamidine hydrochloride (30 mg, 0.205 mmol) and diisopropylethylamine (0.090 mL, 0.517 mmol) as described 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-tetrahydroisoquinoline (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 SiO2 column 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-tetrahydroisoquinolin-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 SiO2 column chromatography (n-hexane/EtOAc ) 8:1) to give tert-butyl 6-methylthiomethoxy-1,2,3,4-tetrahydroisoquinolin2-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 (CDCl ) δ 1.49 (9H, s), 2.80 (2H, t, J ) 6.3 Hz), 3.63 3 (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. (C15H20ClNO3) C, H, N. tert-Butyl 6-[4-Ethoxycarbonyl-1-(pyridin-4-yl)piperidin-4-ylmethoxy]-1,2,3,4-tetrahydroisoquinolin-2-carboxylate (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 atmosphere. 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 SiO2 column chromatography (CHCl3/MeOH ) 20:1 to 10:1) to give com-

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-6yloxymethyl)-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 hydrochloride (155 mg, 1.06 mmol) and diisopropylethylamine (0.62 mL, 3.53 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 reversedphase 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), 2.152.25 (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-yloxymethyl)-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 dihydrochloride: 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-tetrahydronaphthalen-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 K2CO3 solution 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 SiO2 column 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 1,4dioxane (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), 6.606.70 (2H, m), 7.05 (1H, d, J ) 8.4 Hz). Anal. (C11H15NO) C, H, N. 2-tert-Butoxycarbonyl-8-hydroxy-2,3,4,5-tetrahydro1H-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 )

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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), 1.681.80 (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)piperidin-4-ylmethoxy]-2,3,4,5-tetrahydro-1H-benz[c]azepin-2carboxylate (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]azepin8-yloxymethyl)-1-(pyridin-4-yl)piperidin-4-carboxylate (68). Compound 68 (200 mg, 78%) was obtained as a dihydrochloride from 67 (250 mg, 0.49 mmol), 1H-pyrazole-1-carboxamidine 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. 4-(2-Amidino-2,3,4,5-tetrahydro-1H-benz[c]azepin-8yloxymethyl)-1-(pyridin-4-yl)piperidin-4-carboxylic Acid (23). Compound 23 (140 mg, 97%) was obtained as a dihydrochloride from 68 (150 mg, 0.29 mmol) and 6 N HCl (2 mL, 12 mmol) as described for 19: 1H 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 NaHCO3 solution was added 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 SiO2 column chromatography (n-hexane/EtOAc ) 9:1) to give 7-methoxy2-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 SiO2 column 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

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 evaporated 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). Compound 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-(pyridin-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-(pyridin-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 SiO2 column 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-4yl)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-tetrahydroisoquinolin2-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 azodicarboxylate (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 SiO2 column 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),



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. Benzyl 7-[2-(1-tert-Butoxycarbonyl-4-ethoxycarbonylpiperidin-4-yl)ethoxy]-1,2,3,4-tetrahydroisoquinolin-2carboxylate (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)piperidin-4-yl]ethoxy]-1,2,3,4-tetrahydroisoquinolin-2-carboxylate (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-7yloxy)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 hydrochloride (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), 2.002.10 (2H, m), 2.10-2.25 (2H, m), 2.80-2.88 (2H, m), 3.203.35 (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-iodomethylpiperidin1-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 SiO2 column chromatography (n-hexane/EtOAc ) 9:1) to give compound 78 (8.68 g, 79%): 1H NMR (CDCl3) δ 1.27-1.33 (3H, m), 1.381.45 (10H, m), 1.59-1.64 (1H, m), 2.10-2.22 (2H, m), 2.903.10 (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-7ylthiomethyl)-4-ethoxycarbonylpiperidin-1-carboxylate (80). To a stirred solution of 2-acetyl-1,2,3,4-tetrahydroisoquinolin-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 SiO2 column 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. Ethyl 4-(2-Acetyl-1,2,3,4-tetrahydroisoquinolin-7-ylthiomethyl)-1-(pyridin-4-yl)piperidin-4-carboxylate (81). Com-

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-ylthiomethyl)-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 temperature for 12 h. The solvent was evaporated under reduced pressure to leave the residue, which was purified by reversedphase 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 dihydrochloride: 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), 3.954.10 (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-7ylsulfonylmethyl)-4-ethoxycarbonylpiperidin-1-carboxylate (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 Na2S2O5 solution 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 SiO2 column 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-ylsulfonylmethyl)-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. 4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-ylsulfonylmethyl)-1-(pyridin-4-yl)piperidin-4-carboxylic Acid (27). Compound 27 (120 mg, 55%) was obtained as a dihydrochloride 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

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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 SiO2 column 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-carboxylate (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 temperature 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 NaHCO3 and extracted with CHCl3. The organic layer was washed with brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure 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-tetrahydroisoquinolin-7-ylsulfonylamino)piperidin-4-carboxylate (88). To a stirred solution of 86 (330 mg, 1.40 mmol) in CHCl3 (5 mL) were added 7-chlorosulfonyl-2-trifluoroacetyl1,2,3,4-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 evaporated 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), 2.943.08 (2H, m), 3.39-3.58 (5H, m), 3.62-3.76 (2H, m), 3.803.96 (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-7ylsulfonylamino)-1-(pyridin-4-yl)piperidin-4-carboxylate (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 temperature 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-1carboxamidine 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. 4-(2-Amidino-1,2,3,4-tetrahydroisoquinolin-7-ylsulfonylamino)-1-(pyridin-4-yl)piperidin-4-carboxylic Acid (28). Compound 28 (360 mg, 90%) was obtained as a dihydrochloride 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,

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 administrated 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 administration, 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.1 The 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 incubated 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 Thrombin.1 The 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 incubated 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.1 The 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 Plasmin.1 The 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

Selective Factor Xa Inhibitors substrate was measured by reading absorbance at 405 nm with a microplate reader. Determination of Ki Values.1 Factor Xa inhibition mechanisms of compound 2 were determined from a LineweaverBurk plot of the initial velocity [OD at 405 nm] and the concentration of the substrate [µM]. The Ki values 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% FeCl3 was 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-1 h-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.19 PE50 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 introduction; 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 intravenous 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 administration 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-triphenyltetrazolium chloride) in saline solution. “Total infarction volume” was calculated by multiplying slice thickness (2 mm) by the sum of infarction size. Supporting Information Available: Elemental analysis data of new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Hara, T.; Yokoyama, A, Ishihara, H.; Yokoyama, Y.; Nagahara, T. DX-9065a, a new synthetic, potent anticoagulant and selective inhibitor for factor Xa. Thromb. Haemostasis 1994, 71, 314319. (2) (a) Betz, A. Recent advances in factor Xa inhibitors. Expert Opin. Ther. Pat. 2001, 11, 1007-1017. (b) Rai, R.; Sprengeler, P. A.; Elrod, K. C.; Young, W. B. Perspectives on factor Xa inhibition. Curr. Med. Chem. 2001, 8, 101-119. (c) Ewing, W. R.; Pauls,


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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, 83102. (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, synthesis and biological activity of YM-60828 derivatives: potent and orally-bioavailable factor Xa inhibitors based on naphthoanilide 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. 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. Haemostasis 1990, 63, 265-270. (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. 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 DX9065a, a novel factor Xa inhibitor: contribution of the acetimidoylpyrrolidine moiety of the inhibitor to potency and selectivity for serine proteases. Eur. J. Med. Chem. 1995, 30, 387394. (b) Brandstetter, H.; Kuhne, A.; Bode, W.; Huber, R.; von der Saal, W.; Wirthensohn, K.; Engh, R. A. X-ray structure of active site-inhibited clotting factor Xa. Implications for drug design and substrate recognition. J. Biol. Chem. 1996, 271, 29988-29992. Schlittler, E.; Muller, J. Eine neue modification der isochinolinsynthese nach Pomeranz-Fritsch. Helv. Chim. Acta. 1948, 31, 914-924. Marsais, F.; Trecourt, F.; Breant, P.; Queguiner, G. Directed lithiation of 4-halopyridines: Chemoselectivity, regioselectivity and application to synthesis. J. Heterocyl. Chem. 1988, 25, 8187. The S4 site consists of three aromatic residues (Phe174, Trp215, Tyr99) that favorably interact with a positive charge. Dougherty, D. A. Cation-π interactions in chemistry and biology. A new view of benzene, Phe, Tyr, and Trp. Science 1996, 271, 163. Pharmacokinetic parameters in cynomolgus monkeys: Cmax ) 0.39 µg/mL; T1/2 ) 3.6 h; bioavailability (BA) of 10.7% (10 mg/ kg po); clearance of 0.25 L h-1 kg-1; volume of distribution of 0.32 L/kg (1 mg/kg iv). Hayashi, M.; Hamada, A.; Okaya, Y.; Wakitani, K.; Aisaka, K. Inhibitory effect of JTV-803, a new cyclic guanidine derivative, on factor Xa in vitro and in vivo. Eur. J. Pharmacol. 2001, 428, 163-168. Drake, B.; Patek, M; Lebl, M. A convenient preparation of monosubstituted N,N′-di(Boc)-protected guanidines. Synthesis 1994, 579-582. Guendouz, F.; Jacquier, R.; Verducci, J. Polymer bound 4-dialkylamino pyridines: Synthesis, characterization and catalytic efficiency. Tetrahedron 1988, 44, 7095-7108. Gueremy, C.; Audiau, F.; Champseix, A. 3-(4-Piperidinylalkyl)indoles, selective inhibitors of neuronal 5-hydroxytryptamine uptake. J. Med. Chem. 1980, 23, 1306-1310. Benneche, T.; Undheim, K. Synthesis of alpha-haloalkyl aryl ethers from O,S-acetals. Acta Chem. Scand. 1983, 37, 93-96.

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(15) Singh, B.; Lesher, G. Y.; Pennock, P. O. A convenient large scale synthesis of 2,6-dimethyl-4-(trimethylstannyl)pyridine. J. Heterocycl. Chem. 1990, 27, 1841-1842. (16) Selnick, H. G.; Smith, G. R.; Tebben, A. J. An improvement procedure for the cyanation of aryl triflates: A convenient synthesis of 6-cyano-1,2,3,4-tetrahydroisoquinoline. Synth. Commun. 1995, 25, 3255-3261. (17) Blank, B.; Krog, A. J.; Weiner, G.; Pendleton, R. G. Inhibitors of phenylethanolamine N-methyltransferase and epinephrine biosynthesis. 2. 1,2,3,4-Tetrahydroisoquinoline-7-sulfonanilides. J. Med. Chem. 1980, 23, 837-840.

(18) Lyle, E. M.; Lewis, S. D.; Lehman, E. D.; Gardell, S. J.; Motzel, S. L.; Lynch, J. J., Jr. Assessment of thrombin inhibitor efficacy in a novel rabbit model of simultaneous arterial and venous thrombosis. Thromb. Haemostasis 1998, 79, 656-662. (19) Umemura, K.; Toshima, Y.; Nakashima, M. Thrombolytic efficacy of a modified tissue-type plasminogen activator, SUN 9216, in the rat middle cerebral artery thrombosis model. Eur. J. Pharmacol. 1994, 262, 27-31.

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Synthesis and Structureâ Activity Relationships of Novel Selective

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