Discovery of 1-(3′-aminobenzisoxazol-5′-yl)-3-trifluoromethyl- N-[2-fluoro-4-[(2′-dimethylaminomethyl)imidazol-1-yl]phenyl] -1H-pyrazole-5-carboxyamide hydrochloride (razaxaban), a highly potent, selective, and orally bioavailable factor Xa inhibitor

J. Med. Chem. 2005, 48, 1729-1744

1729

Articles

Discovery of 1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-N-[2-fluoro-4-

[(2′-dimethylaminomethyl)imidazol-1-yl]phenyl]-1H-pyrazole-5-carboxyamide

Hydrochloride (Razaxaban), a Highly Potent, Selective, and Orally Bioavailable

Factor Xa Inhibitor

Mimi L. Quan,*^,§Patrick Y. S. Lam,^§Qi Han,^§Donald J. P. Pinto,^§Ming Y. He,^§Renhua Li,^§

Christopher D. Ellis,^§Charles G. Clark,^§Christopher A. Teleha,^§Jung-Hui Sun,^§Richard S. Alexander,^§

Steve Bai,^†Joseph M. Luettgen,^‡Robert M. Knabb,^‡Pancras C. Wong,^‡and Ruth R. Wexler^§

Discovery Chemistry, Pharmaceutical Research Institute, Bristol-Myers Squibb Co., P.O. Box 5400,

Princeton, New Jersey 08543-5400

Received March 15, 2004

Modification of a series of pyrazole factor Xa inhibitors to incorporate an aminobenzisoxazole

as the P₁ligand resulted in compounds with improved selectivity for factor Xa relative to trypsin

and plasma kallikrein. Further optimization of the P₄moiety led to compounds with enhanced

permeability and reduced protein binding. The SAR and pharmacokinetic profile of this series

of compounds is described herein. These efforts culminated in 1-(3′-aminobenzisoxazol-5′-yl)-

3-trifluoromethyl-N-[2-fluoro-4-[(2′-dimethylaminomethyl)imidazol-1-yl]phenyl]-1H-pyrazole-

5-carboxyamide (11d), a potent, selective, and orally bioavailable inhibitor of factor Xa. On

the basis of its excellent in vitro potency and selectivity profile, high free fraction in human

plasma, good oral bioavailability, and in vivo efficacy in antithrombotic models, the HCl salt

of this compound was selected for clinical development as razaxaban (DPC 906, BMS-561389).

Introduction

cascade than thrombin. Inactivation of factor Xa by

specific inhibitors does not influence preformed throm-

bin but does effectively prevent the generation of

thrombin. Extensive preclinical and clinical proof-of-

principle data show that inhibition of factor Xa is

effective in both venous and arterial thrombosis.²In

animal models a better therapeutic index (antithrom-

botic efficacy vs antihemostatic effects) has been shown

for direct factor Xa inhibitors compared with that for

direct thrombin inhibitors.^4,5On the basis of these data,

a cleaner side effect profile would be expected for a

factor Xa inhibitor relative to a thrombin inhibitor in

the clinical setting.²

Thromboembolic disorders including acute myocardial

infarction, unstable angina, deep vein thrombosis, pul-

monary embolism, and ischemic stroke continue to be

the leading cause of morbidity and mortality in the U.S.

and other Western countries. Current therapies for the

treatment and prevention of thrombotic disorders are

inadequate because they require parenteral administra-

tion or careful monitoring of clotting time to achieve

desired efficacy and dose titration to minimize excessive

bleeding.¹Therefore, there is a significant medical need

for safer and more effective orally active anticoagulants

to combat these diseases.

Factor Xa is a key serine protease in the coagulation

cascade and is a promising target enzyme for the design

of new therapeutic agents with potential for the treat-

ment and prevention of arterial and venous thrombosis.²

Factor Xa is essential in the formation of thrombin, a

key mediator of both fibrin formation and platelet

activation. It plays an important role in the coagulation

network at the common pathway that connects both the

tissue factor-activated extrinsic pathway and the surface-

activated intrinsic pathway.³Factor Xa, together with

calcium and factor Va, forms the prothrombinase com-

plex, which amplifies the procoagulant action of factor

Xa. Factor Xa acts at an earlier level in the coagulation

Previous reports from our laboratories have detailed

several approaches to the design of small-molecule

factor Xa inhibitors.⁶These efforts resulted in the

discovery of clinical candidate DPC423, which showed

potent antithromobotic efficacy in animal models of

thrombosis and excellent pharmacokinetics in preclini-

cal and clinical studies.^5,7,8Simultaneously, we evalu-

ated the pharmacokinetic profile of an extensive series

of compounds containing benzamidine P₁mimics and

found that the aminobenzisoxazole moiety showed an

excellent balance of potency, selectivity, and oral bio-

availability.⁹Therefore, a series of pyrazole derivatives

containing an aminobenzisoxazole in the P₁position was

explored. Optimization of this series of compounds has

culminated in the discovery of clinical candidate raza-

xaban (DPC 906, BMS-561389), a highly potent, selec-

tive, and orally bioavailable factor Xa inhibitor.

* To whom correspondence should be addressed. Phone: (609) 818-

5301. Fax: (609) 818-3331. E-mail: mimi.quan@bms.com.

^§Discovery Chemistry.

^†Clinical Discovery.

^‡Discovery Biology.

Published on Web 07/16/2004

1730 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Quan et al.

Scheme 1. Synthesis of 11d (Razaxaban) and Related Analogues^a

^aReagents: (a) SnCl₂, EtOAc, reflux, 85%; (b) NaNO₂, concentrated HCl; SnCl₂, HCl; (c) HOAc, reflux, 31% for steps b and c; (d)

NaIO₄, RuCl₃, CH₃CN/CCl₄/H₂O, 64%; (e) (COCl)₂, CH₂Cl₂, reflux, quantitative; (f) K₂CO₃, CuI, DMSO, 120 °C, 50-67%; (g) DMAP,

CH₂Cl₂, 15-85%; (h) MeCONHOH, t-BuOK or K₂CO₃, DMF, 36-86%; (i) TFA, reflux, 67%.

Chemistry

complished with TFA at reflux to afford the monomethyl

compound 12. Compounds 38 and 39 were prepared

using 3-fluoro-2′-methanesulfonylbiphenyl-4-ylamine^7,11

and 4′-amino-3′-fluorobiphenyl-2-sulfonic acid amide^7,11

instead of aniline 9 following the procedures described

in Scheme 1. Similarly, compounds 40, 41, 42, 44, and

45 were prepared from anilines 14, 16, 17, 15, and 18,

respectively. The syntheses of these anilines are shown

in Scheme 2.

The pyridyl analogue 23 was prepared as shown in

Scheme 3. Acyl chloride 6 was coupled with 4-iodo-2-

fluoroaniline 8 to give amide 19. Conversion of the iodo

intermediate to boronic ester 20, followed by coupling

with bromide 21, afforded biaryl compound 22. Ami-

nobenzisoxazole formation using the same methods

described for 11d resulted in compound 23.

The synthesis of compound 28 is shown in Scheme 4.

2-Fluoro-4-bromoaniline was protected with a Boc group

and then coupled with tin intermediate 25, which was

prepared via lithiation of 1-methylimidazole with n-

BuLi, followed by reaction with Bu₃SnCl. Removal of

the Boc group with HCl resulted in aniline 26. Acid 5

was esterified with sulfonyl chloride and methanol. The

resulting product was converted to aminobenzisoxazole

The syntheses of 11d and related analogues were

prepared as shown in Scheme 1. Reduction of com-

mercially available 2-fluoro-5-nitrobenzonitrile 1 af-

forded aniline 2. Diazotization and reduction with

stannous chloride gave the hydrazine as the tin salt,

which was reacted with commercially available 4,4,4-

trifluoro-1-(2-furyl)-1,3-butanedione 3 in situ to give

pyrazole 4. The furan ring was then oxidized with

sodium periodate to give acid 5,^5bwhich was converted

to the acyl chloride 6 with oxalyl chloride. Anilines 9a-f

were prepared via Ullmann¹⁰coupling of imidazoles

7a-f with 2-fluoro-4-iodoaniline 8. Imidazoles 7a-c

were obtained commercially, and 7d-e were obtained

from reductive amination of 2-imidazolecarboxaldehyde

with dimethylamine or pyrrolidine. Compound 7f was

prepared similarly by reductive amination of 2-imida-

zolecarboxyaldehyde with monomethylamine, followed

by protection with CbzCl/Et₃N in CH₂Cl₂. Anilines 9a-f

were coupled with acyl chloride 6 with DMAP in

CH₂Cl₂to give amides 10a-f. Reaction of 10a-f with

acetohydroxamic acid and potassium tert-butoxide or

potassium carbonate in DMF gave aminobenzisoxazoles

11a-f.⁹Cleavage of the Cbz group in 11f was ac-

Factor Xa Inhibitor

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1731

Scheme 2. Syntheses of 4-Substituted Anilines^a

^aReagents: (a) K₂Cr₂O₇, 82%; (b) (COCl)₂, CH₂Cl₂; pyrrolidine, Et₃N, CH₂Cl₂; (c) H₂, 5% Pd/C, 55% for steps b and c; (d)

2-methylimidazole, CH₃CN, K₂CO₃, reflux, 90%; (e) H₂, 10% Pd/C, 56%; (f) morpholine, THF, 8%; (g) H₂, 10% Pd/C, 95%; (h) NaCN, DMF,

31%; (i) 2-methylimidazole, CH₃CN, 96%; (j) H₂, 10% Pd/C, 98%; (k) 2-methylimidazole, CH₃CN, 78%; (l) MeI, K₂CO₃, 20%; (m) H₂, 10%

Pd/C, quantitative.

Scheme 3. Synthesis of Compound 23^a

^aReagents: (a) DMAP, CH₂Cl₂, 45%; (b) KOAc, pinacol diborane, PdCl₂(dppf), DMSO, 80 °C; (c) Pd(PPh₃)₄, EtOH, toluene, reflux; (d)

MeCONHOH, K₂CO₃, DMF; (e) TFA, 15% for the steps b-e.

with acetohydroxamic acid and potassium tert-butoxide,

followed by reaction with 18% aqueous HCl in methanol

and dichloromethane. Hydrolysis of the ester group

resulted in acid 27, which coupled with aniline 26 using

DMAP and PyBrop to give compound 28.

The synthesis of aminomethylimidazole 37 is shown

in Scheme 5. 2-Imidazolecarboxaldehyde 29 was re-

duced to the corresponding alcohol 30 with sodium

borohydride. Aniline 31 was prepared via the coupling

of alcohol 30 with 2-fluoro-4-iodoaniline 8 using Ull-

mann conditions as described above. The alcohol group

was protected as the TBDMS ether and then coupled

with acyl chloride 6 in DMAP to afford amide 33.

Compound 33 was converted to aminobenzisoxazole 34

by reaction with acetohydroxamic acid and potassium

carbonate. Removal of the TBDMS group with

1732 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Scheme 4. Synthesis of Compound 28^a

Quan et al.

^aReagents: (a) Pd(PPh₃)₄, THF, reflux, 60%; (b) HCl, EtOAc, 83%; (c) SOCl₂, MeOH, 92%; (d) MeCONHOH, t-BuOK, DMF, 68%; (e)

18% aqueous HCl, MeOH/CH₂Cl₂, reflux, 65%; (f) NaOH, THF, 96%; (g) PyBrop, (i-Pr)₂NEt, DMF, 60 °C, 41%.

Scheme 5. Synthesis of Compound 37^a

^aReagents: (a) NaBH₄, MeOH, 45%; (b) K₂CO₃, CuI, DMSO, 120 °C, 10%; (c) TBDMSCl, Et₃N, DMF, 67%; (d) DMAP, CH₂Cl₂, 53%;

(e) MeCONHOH, K₂CO₃, DMF; (f) Bu₄NF (1 N in THF), THF, 90% for steps e and f; (g) PBr₃, CH₂Cl₂; (h) NaN₃, DMF; (i) SnCl₂, MeOH,

reflux, 15% for steps g-i.

n-Bu₄NF in THF (1 N) afforded the alcohol functionality,

which was then converted to the bromide with PBr₃.

Reaction of the bromide with sodium azide afforded

azide 36, which was reduced with SnCl₂to provide

aminomethylimidazole 37.

Results and Discussion

Our first clinical candidate, DPC423, a potent and

orally active factor Xa inhibitor (Figure 1),^5,7,8showed

excellent selectivity over thrombin and many other

Factor Xa Inhibitor

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1733

Figure 1. Comparison of benzylamine and aminobenzisoxazole P₁.

serine proteases (>10000-fold) with the exception of

trypsin and plasma kallikrein in which selectivity was

approximately 400-fold. We have previously demon-

strated that selectivity for factor Xa over other trypsin-

like serine proteases was significantly improved by

replacement of the benzamidine P₁moiety with a larger

P₁ligand such as aminobenzisoxazole.⁹We believe this

increased selectivity is due to the larger S₁pocket in

factor Xa relative to other serine proteases such as

trypsin (Ala190 at the bottom of the S₁pocket in factor

Xa compared to Ser190 in trypsin). Indeed, replacement

of the benzylamine in DPC423 with an aminobenzisox-

azole P₁moiety (38, Figure 1) greatly improved the

selectivity for factor Xa relative to trypsin and plasma

kallikrein from approximately 400-fold to >25000-fold

while maintaining the factor Xa potency and selectivity

over other key serine proteases. Albeit highly selective,

aminobenzisoxazole 38 had several limitations including

poor permeability (Caco-2 Papp is <0.1 × 10^-6cm/s)¹²

and poor solubility (<0.0001 mg/mL).¹³As predicted by

the poor permeability, compound 38 was found to have

only 2% bioavailability when administered orally to dogs

(0.2 mg/kg, n ) 2). These issues precluded compound

38 from further advancement.

In an effort to address the permeability issue, we

investigated the replacement of the terminal phenyl ring

with heterocycles (Table 1). Pyridylsulfonamide 23

maintained factor Xa inhibitory potency and improved

Caco-2 permeability slightly compared with phenylsul-

fonamide 39. Incorporation of solubilizing heterocycles

such as 1-methylimidazol-2-yl and 2-methylimidazol-1-

yl groups greatly improved Caco-2 permeability (28 and

11a) but showed a 3-fold decrease in factor Xa inhibitory

potency. Employing less lipophilic neutral moieties such

as pyrrolidinylamide 40 also improved the permeability,

but at a greater expense of factor Xa inhibitory potency

(5-fold loss). Replacement of the terminal ring with a

morpholino group resulted in compound 41 with a 40-

fold reduction in factor Xa inhibitory potency. Although

improved permeability was obtained by replacing the

terminal phenyl ring with solubilizing groups with

reduced lipophilicity, protein binding was high for 11a,

28, and 40 (98.0%, 97.5%, and 97.0% bound to human

plasma, respectively),¹⁴which resulted in low antico-

agulant activity as shown by their aPTT values¹⁵(Table

1). Therefore, reducing protein binding was necessary

in order to improve anticoagulant activity of these

compounds. Since solubility was lower for compound 40

and since the 1-methylimidazol-yl 28 was less selective

for factor Xa versus thrombin (thrombin K_i) 34 nM)

compared to the 2-methylimidazol-1-yl analogue 11a,

further optimization focused on the 2-methylimidazol-

1-yl series.

We have previously observed that substitution at the

2′-position on the proximal phenyl ring favorably affects

both the factor Xa affinity and the pharmacokinetic

profile of the compounds.^6-7,11Therefore, a series of

2-methylimidazol-1-yl containing compounds were ex-

plored with a variety of substituents at the “ortho”

position of the proximal phenyl ring (Table 2). The

2-fluoro analogue 11a provided the best combination of

potency and permeability. Cyano-substituted analogue

42 maintained factor Xa inhibitory potency; however,

the permeability was decreased. Other “ortho”-substi-

tuted compounds such as imidazole 44, methoxy 45, and

amide 46 reduced factor Xa inhibitory affinity by 3-, 5-,

and 13-fold, respectively. All the compounds listed in

Table 2 have a trypsin K_ivalue greater than 2500 nM,

and they are very selective over thrombin except for 45

with a thrombin K_iof 200 nM. After determining that

fluoro substitution improved Caco-2 permeability sig-

nificantly (11a), this substituent was maintained while

1734 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Table 1. Effect of P₄Substitution on Caco-2 Permeability

Quan et al.

^aK_ivalues in nM were obtained using human purified enzymes.²¹

Table 2. Effect of “Ortho” Substitution on Caco-2 Permeability

^aK_ivalues in nM were obtained using human purified enzymes.²¹Trypsin K_i> 2500 nM for all compounds.

further optimization of the heterocyclic portion of the

P₄moiety was investigated.

or an aminomethyl moiety at the 2-position of the

imidazole resulted in decreased Caco-2 permeability (35)

and in decreased potency (37). However, alkylation of

this aminomethyl group resulted in improved factor Xa

inhibitory affinity as observed in compounds 12, 11d,

and 11e. The dialkylated compounds 11d and 11e

showed good Caco-2 permeability relative to the monom-

ethylated compound 12. Furthermore, human plasma

protein binding was reduced for the dimethylated and

the monomethylated compounds 11d and 12 (90.5% and

Further modification of the substituent at the 2-posi-

tion of the imidazole was explored in an attempt to

further improve S₄binding and to decrease protein

binding (Table 3). Replacement of the methyl group with

an ethyl group at the 2-position of the imidazole resulted

in 11b with similar factor Xa activity, while a decrease

in factor Xa inhibitory potency was observed for the

isopropyl analogue 11c. Introduction of a hydroxymethyl

Factor Xa Inhibitor

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1735

Table 3. Effect of 2-Imidazole Substitution on Permeability and Human Plasma Protein Binding

^aK_ivalues in nM were obtained using human purified enzymes.²¹

Table 4. Dog Pharmacokinetics Profile^a

^aCompounds were dosed as TFA salts in an N-in-1 format²⁴at

0.4 mg/kg iv and 0.2 mg/kg po. (n ) 2).

85.6%, respectively).¹⁴The lower protein binding re-

sulted in higher anticoagulation activity observed in the

aPTT assay for 11d and 12 (Table 3).

As a result of the excellent in vitro profiles of

compounds 12 and 11d, the pharmacokinetic profiles

of these compounds were studied in dogs (Table 4).

While the clearance values of the mono- and dimethy-

laminomethylimidazole compounds 12 and 11d were

determined to be similar, dimethylaminomethylimida-

zole 11d showed significantly better oral bioavailability

(F is 84% compared to 27%) as predicted by the observed

Caco-2 permeability for these two compounds.

An X-ray crystal structure of 11d in the human factor

Xa active site was obtained at 1.8 Å resolution with a

crystallographic R factor of 0.207 (Figure 2). The overall

binding mode was found to be similar to what was

observed for DPC423.¹⁶The inhibitor was shown to bind

to the active site through interactions with the S₁and

S₄pockets with no major reorientation of the protein

upon inhibitor binding. As anticipated, the aminoben-

zisoxazole group was bound in the S₁pocket. The amino

group formed hydrogen bonds with Asp189 (2.7 Å) and

the carbonyl group of Gly218 (3.5 Å). As indicated

earlier, the specificity of this series of inhibitors over

trypsin can be attributed to the binding mode in the S₁

Figure 2. X-ray structure of 11d (razaxaban) in the factor

Xa active site. The amino group formed hydrogen bonds with

Asp189 (2.7 Å) and the carbonyl group of Gly218 (3.5 Å). The

pyrazole N-2 nitrogen interacts with the backbone NH of

Gln192 (3.4 Å). The 5-carboxamide carbonyl showed a strong

interaction with the NH of Gly216 (3.0 Å). The N-3 nitrogen

of the imidazole P₄group formed a direct H bond (3.6 Å) and

indirect H bonds through an H₂O molecule (3.0 and 3.1 Å) with

Glu97.

pocket. The aminobenzisoxazole is observed to bind in

close contact with the side chain of Ala190 (3.4 Å) in

factor Xa. This orientation would lead to unfavorable

interactions in trypsin (Ser190) if a similar binding

mode was employed. The pyrazole ring was optimally

oriented for the N-2 nitrogen to interact with the

backbone NH of Gln192 (3.4 Å). The pyrazole 3-triflu-

oromethyl group partially fit in the small lipophilic

pocket above the S₁pocket near the Cys191-220 disul-

1736 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Table 5. Human Enzyme Selectivity Profile for Razaxaban

Quan et al.

Figure 3. Antithrombotic effect of razaxaban in the rabbit

arterio-venous shunt thrombosis model.

However, the resulting compounds were poorly soluble,

poorly permeable, and highly protein-bound. Employing

solubilizing and less lipophilic heterocycles at the

terminal ring of the P₄moiety led to the identification

of phenylimidazole analogues with increased perme-

ability. Substitution of the 2-position of the imidazole

with a dimethylaminomethyl moiety reduced protein

binding and provided the best balance of potency,

selectivity, and pharmacokinetic profile. These efforts

culminated in the discovery of clinical candidate raza-

xaban, a potent, selective, and orally active factor Xa

inhibitor that shows antithrombotic efficacy in the

rabbit arterio-venous shunt thrombosis model.

^aAll K_ivalues were obtained using human purified enzymes.²¹

fide bridge. The 5-carboxamide carbonyl showed a

strong interaction with the NH of Gly216 (3.0 Å). The

orientation of the 5-carboxamide functionality allowed

the phenylimidazole P₄moiety to nicely fit into the S₄

region of the enzyme. The N-3 nitrogen of the imidazole

P₄group formed a direct hydrogen-bonding interaction

with Glu97 (3.6 Å) as well as an indirect hydrogen-

bonding interaction to the same residue through an H₂O

molecule (3.0 and 3.1 Å). It is not clear from the X-ray

structure how the dimethyl group contributes to the

factor Xa potency. Overall, the inhibitor was highly

constrained and fit into the enzyme active site in a

complementary manner.

Experimental Section

All reactions were run under an atmosphere of dry nitrogen

unless otherwise noted. Solvents and reagents were obtained

from commercial vendors in the appropriate grade and used

without further purification unless otherwise indicated. NMR

spectra (¹H, ¹³C, ¹⁹F) were obtained on VXR or Unity 300 MHz

instruments (Varian Instruments, Palo Alto, CA) with chemi-

cal shift in ppm downfield from TMS as an internal reference

standard. ¹H assignment abbreviations are the following:

singlet (s), doublet (d), triplet (t), quartet (q), pentet (p), broad

singlet (bs), doublet of doublets (dd), doublet of triplets (dt),

and multiplet (m). Elemental analyses were performed by

Quantitative Technologies, Inc. (Whitehouse, NJ 08888) and

were within 0.4% of the theoretical values. Mass spectra were

measured with an HP 5988A mass spectrometer with a

particle beam interface using NH₃for chemical ionization or

a Finnigan MAT 8230 mass spectrometer with NH₃-DCI or

VG TRIO 2000 for ESI. High-resolution mass spectra were

measured on a VG 70-VSE instrument with NH₃ionization.

Flash chromatography was done using EM Science silica gel

60 (230-400 mesh). Preparative thin-layer chromatography

was done on EM Science 60 plates F₂₅₄(2 mm; 20 cm × 20

cm). HPLC purification was performed on a Jasco 900 series

instrument or a Rainin Dynamax SD200 using a C18 reverse-

phase column with acetonitrile/H₂O (containing 0.05% TFA)

as a mobile phase. All compounds were found to be >95% pure

by HPLC analysis unless otherwise noted. Melting points were

determined on a Thomas-Hoover melting point apparatus and

are uncorrected.

Compound 11d exhibited excellent selectivity (>5000-

fold) against related serine proteases (Table 5). The

anticoagulant activity of 11d was evaluated in the in

vitro human plasma aPTT and human prothrombin

time (PT) assays.¹⁵In these assays, 11d showed a

doubling of aPTT and PT at 6.1 and 2.1 µM, respectively.

The human and rabbit plasma protein binding were

found to be 90.5% and 93.4%, respectively, using equi-

librium dialysis.¹⁴Compound 11d was found to have

similar affinity in the rabbit factor Xa assay with a K_i

of 0.16 nM. Upon intravenous dosing in the rabbit

arterio-venous shunt thrombosis model,^17acompound

11d inhibited thrombus formation in a dose-dependent

manner with an ID₅₀of 1.6 µmol kg^-1h^-1(Figure 3).^17b

Compound 11d has an ID₅₀comparable to that for

DPC423 (ID₅₀) 1.1 µmol kg^-1h^{-1 5,7}

in this model. On

)

the basis of its factor Xa inhibitory affinity, selectivity

over other enzymes, bioavailability, and in vivo anti-

thrombotic efficacy, the HCl salt of compound 11d

(razaxaban, DPC 906, BMS-562389) was selected for

clinical trials.

The factor Xa-11d crystals were obtained from GLA-

domainless â-factor Xa (Haematologic Technologies) that had

been fractionated on a Pharmacia mono-Q 10/10 column

equilibrated with 50 mM Tris, pH 8.0, 100 mM NaCl, and 1

mM CaCl₂and eluted with a 20-bed volume, 0-500 mM NaCl

gradient. Inhibitor was added to the fractions as they were

collected. The protein eluted at about 100 mM NaCl. This

solution was incubated for 12 h and concentrated to 6 mg/mL

using a Vivaspin 6 mL concentrator with a 5000 molecular

weight cut-off (MWCO) membrane. The crystals were grown

using hanging drop vapor diffusion at 4 °C with 18% PEG 6000

Conclusion

Replacement of the benzylamine P₁moiety of DPC423

with an aminobenzisoxazole afforded compounds with

improved selectivity over trypsin and plasma kallikrein.

Factor Xa Inhibitor

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1737

buffered with 200 mM NaOAc (pH 5.5) in the reservoir. The

drops contain 4 µL of protein solution and 4 µL of reservoir

solution. The drops were microseeded with a crushed crystal

from a previous crystal growth. The cryoprotectant was

introduced to the factor Xa crystals by first transferring the

crystals to a 2 µL drop of the soaking solution. This drop was

then bridged into a 20 µL drop of a solution of 22% PEG with

200 mM sodium acetate, pH 5.5, and 20% ethylene glycol. After

about a minute, the crystals were frozen. Data for the Xa-

11d complex were collected at the DND beamline 4 ID at the

Advanced Photon Source using a marCCD detector. Data were

collected at 100 K using an Oxford cryosystems cooling device

with a wavelength of 1.0 Å. Data frames of 1° rotation were

collected. Data were 99% complete. Raw data were processed

with the program Scalepack/Denzo.¹⁸

The program EPMR¹⁹was used to determine the initial

model for refinement using the PDB coordinates 1fjs (minus

the inhibitor and solvent molecules) as the search model. The

CNX (Accelrys) program was used for crystallographic refine-

ment. Simulated annealing (at a maximum temperature of

3000 °C) was followed by B-factor refinement. The inhibitors

were built with the program QUANTA (Accelrys). Peaks in

the difference electron density map that were greater than 3σ

and were less than 4 Å away from the protein were built in as

solvent molecules. No major adjustments to the protein model

were needed during the course of the refinements. Final R

factors as well as other relevant data collection statistics are

found in the supporting data. Coordinates for the enzyme

inhibitor structure will be deposited with the Protein Data

Bank.²⁰

sodium sulfate, filtered, and concentrated to give 2.4 g of acid

1

5 (64%). H NMR(DMSO-d₆) δ 8.31-8.29 (m, 1H), 8.04-7.99

(m, 1H), 7.67 (t, J ) 3.0 Hz, 1H), 7.47 (s, 1H); MS m/z 298 (M

- H)^-.

2-Dimethylaminomethylimidazole (7d). To a 5 L three-

neck round-bottomed flask equipped with a reflux condenser,

pressure-equalizing addition funnel, and an internal temper-

ature probe was added 2-imidazolecarboxaldehyde (100 g, 1.04

mol) in MeOH (1000 mL). To this suspension (ambient

temperature) was added a solution of dimethylamine (40%

aqueous, 1000 mL) at a fast dropping rate (20 min). After the

addition was complete, solid sodium borohydride (118 g, 3.12

mol, 2.99 equiv) was CAUTIOUSLY added portionwise over

45 min. Foaming occurred after each portion, and the internal

temperature was allowed to reach 54 °C without external

cooling. The reaction mixture was then heated to 65 °C for 3

h and allowed to cool to ambient temperature for 12 h. The

reaction contents were concentrated in vacuo and added to a

12 L separatory funnel containing brine (2 L). The contents

were extracted with EtOAc (2 × 500 mL) and with CHCl₃(4

× 1600 mL). The EtOAc extract was discarded. The CHCl₃

extract was dried (MgSO₄), filtered, and concentrated in vacuo

to give 99.0 g of the desired product 7d as a waxy solid (76%):

1

mp 58-59 °C; H NMR (CDCl₃) δ 6.98 (s, 2H), 3.62 (s, 2H),

2.30 (s, 6H); MS m/z 126 (M + H)⁺.

2-(Pyrrolidin-1-ylmethyl)imidazole (7e). This compound

was prepared following the same procedures described for 7d

using pyrrolidine instead of dimethylamine (56%). ¹H NMR

(CDCl₃) δ 6.96 (s, 2H), 6.33 (bs, 2H), 3.78 (s, 2H), 2.60 (m, 4H),

1.81 (m, 4H); MS m/z 152.2 (M + H)⁺.

2-Fluoro-5-aminobenzonitrile (2). To a solution of 2-fluoro-

5-nitrobenzonitrile (2.0 g, 12 mmol) in ethyl acetate (50 mL)

was added stannous chloride dihydrate (27.0 g). The mixture

was brought to reflux for 1.5 h and allowed to cool. The mixture

was partitioned between EtOAc and saturated sodium bicar-

bonate. The aqueous phase was extracted with EtOAc four

times. The combined organic phases were washed with 4 ×

H₂O, dried over sodium sulfate, filtered, and concentrated to

Benzyl 1H-Imidazol-2-ylmethyl(methyl)carbamate (7f).

2-Imidazolecarboxyaldehyde (5.0 g, 52.0 mmol) was suspended

in 200 mL of methanol. Methylamine (20 mL of 33% solution

in methanol) was added. After the mixture was stirred for 15

min, NaBH₄(3.95 g, 0.10 mol) was added portionwise. The

reaction mixture was then heated at 50 °C for 2 h under N₂.

The solvent was removed. The solid was washed with CH₂Cl₂

and filtered. The CH₂Cl₂solution was dried over MgSO₄,

filtered, concentrated, and dried under vacuum to give 2-me-

thylaminomethylimidazole as a yellow oil. This oil was dis-

solved in a 1:1 solution of CH₂Cl₂and THF. To it was added

Et₃N (7.94 mL, 57.0 mmol) and benzyl chloroformate (7.4 mL,

52.0 mmol). The mixture was stirred at ambient temperature

under N₂for 1 h. The solvent was removed, and the residue

was partitioned between EtOAc and H₂O. The EtOAc layer

was washed with brine, dried over MgSO₄, filtered, and

concentrated. The mixture was heated to reflux with 15 mL

of TFA for 30 min to convert most of the bis-acylated byproduct

to the desired product. The TFA was removed. The mixture

was dissolved in EtOAc and washed with saturated aqueous

NaHCO₃and brine. The organic layer was dried over MgSO₄,

filtered, concentrated, and chromatographed on silica gel,

eluting with 1:1 EtOAc/hexane to give 6.56 g of benzyl 1H-

imidazol-2-ylmethyl(methyl)carbamate 7f (51.4% yield). ¹H

NMR (CDCl₃) δ 7.35 (s, 6H), 6.90 (s, 1H), 5.14 (s, 2H), 4.48 (s,

2H), 3.00 (s, 3H); MS m/z 246.3 (M + H)⁺.

2-Fluoro-4-[(2-dimethylaminomethyl)imidazol-1-yl]a-

niline (9d). To a 500 mL round-bottomed flask equipped with

a reflux condenser and an internal temperature probe was

added 2-fluoro-4-iodoaniline 8 (9.8 g, 0.042 mol), K₂CO₃(11.5

g, 0.083 mol, 2 equiv), Cu(I) (1.58 g, 0.0083 mol, 0.2 equiv),

and 7d (7.8 g, 0.023 mol, 1.5 equiv), followed by DMSO (300

mL). The reaction contents were heated to 120 °C for 12 h.

After cooling to ambient temperature, the reaction mixture

was partitioned between brine (1 L) and EtOAc (2 × 500 mL).

The aqueous layer was extracted with additional CHCl₃(500

mL) and kept separate. Both organic extracts were washed

separately with brine, dried (MgSO₄), and filtered. They were

then combined and concentrated. The crude black oil was

purified on silica gel, eluting first with (a) CHCl₃, then (b)

CHCl₃(1 L with 8 drops of concentrated ammonia), (c) 5%

MeOH in CHCl₃(1 L with 8 drops of concentrated ammonia),

and (d) 5% MeOH in CHCl₃(5 L with 40 drops concentrated

ammonia). The product was isolated (5.6 g) as a tan waxy solid

1

give 1.4 g (85%) of 2. H NMR (DMSO-d₆) δ 7.16 (t, J ) 3.1

Hz, 1H), 6.87 (m, 2H), 5.49 (bs, 2H); MS m/z 137 (M + H)⁺.

1-(4′-Fluoro-3′-cyanophenyl)-3-trifluoromethyl-5-furyl-

1H-pyrazole (4). 2-Fluoro-5-aminobenzonitrile 2 (1.4 g, 10

mmol) was added to 10 mL of concentrated hydrochloric acid

at 0 °C. Sodium nitrite (0.71 g) was dissolved in H₂O (3 mL),

cooled to 0 °C, and added dropwise. The reaction mixture was

stirred at 0 °C for 30 min. Stannous chloride dihydrate (6.95

g) was dissolved in HCl (concentrated, 4 mL) and cooled to 0

°C. It was then added dropwise to the reaction mixture. The

reaction mixture was placed in the refrigerator for 12 h. The

precipitate was isolated by filtration and washed with ice-cold

brine (30 mL) followed by a 2:1 petroleum ether/ethyl ether

(30 mL) solution. The yellow solid was dried under vacuum

for 12 h to give 4-fluoro-3-cyanophenylhydrazine tin chloride

(2.5 g).

To a suspension of 4-fluoro-3-cyanophenylhydrazine tin

chloride (17 g, 50 mmol) in acetic acid (200 mL) was added

4,4,4-trifluoro-1-(2-furyl)-2,4-butanedione 3 (10.3 g, 50 mmol).

The reaction mixture was heated to reflux for 12 h. The acetic

acid was evaporated, and the residue was partitioned between

EtOAc and H₂O. The organic phase was washed with 1 N HCl,

H₂O, and brine. It was then dried over sodium sulfate, filtered,

and concentrated. Flash chromatography gave 7.0 g (44%) of

1

4. H NMR (CDCl₃) δ 7.49 (m, 2H), 7.44 (d, J ) 0.5 Hz, 1H),

7.34 (t, J ) 2.8 Hz, 1H), 6.90 (s, 1H), 6.46 (m, 1H), 6.32 (d, J

) 1.1 Hz, 1H); MS m/z 322 (M + H)⁺.

1-(4′-Fluoro-3′-cyanophenyl)-3-trifluoromethyl-1H-pyra-

zole-5-carboxylic Acid (5). To a solution of 4 (4.0 g, 12.5

mmol) in acetonitrile (30 mL) was added carbon tetrachloride

(30 mL), ruthenium chloride (0.4 g), and a solution of sodium

periodate (11.9 g, 56.1 mmol) in H₂O (45 mL). The reaction

mixture was stirred at ambient temperature for 12 h. The

reaction mixture was filtered through Celite. The filtrate was

concentrated and partitioned between EtOAc and 1 N aqueous

HCl. The organic phase was washed with H₂O, dried over

1738 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Quan et al.

(58%): mp 121-122 °C; ¹H NMR (CDCl₃) δ 7.32 (dd, J ) 11.7,

2.2 Hz, 1H), 7.09-7.01 (m, 2H), 6.81 (t, J ) 8.6 Hz, 1H), 3.92

(bs, 2H), 3.36 (s, 2H), 2.25 (s, 6H); MS m/z 235 (M + H)⁺.

3H), 7.05 (d, J ) 2.5 Hz, 3H), 6.92 (d, J ) 2.5 Hz, 3H), 2.96

(m, J ) 6.9 Hz, 2H), 1.23 (d, J ) 6.9 Hz, 3H); MS m/z 501.2

(M + H)⁺.

2-Fluoro-4-[2-methylimidazol-1-yl]aniline (9a). This com-

pound was prepared following the same procedures described

for 9d using 2-methylimidazole instead of 2-dimethylaminom-

1-(3′-Cyano-4′-fluorophenyl)-3-trifluoromethyl-N-[2-

fluoro-4-[(2′-pyrrolidin-1-ylmethyl)imidazol-1-yl]phenyl]-

1H-pyrazole-5-carboxyamide (10e). This compound was

prepared following the same procedures described for 10d

1

ethylimidazole (53%). H NMR (CDCl₃) δ 6.99-6.80 (m, 5H),

3.92 (bs, 2H), 2.33 (s, 3H); MS m/z 192.0 (M + H)⁺.

using 9e instead of 9d (44%). H NMR (CDCl₃) δ 8.28 (t, J )

1

8.8 Hz, 1H), 8.12 (s, 1H), 7.82 (m, 2H), 7.72 (dd, J ) 2.6, 12.1

Hz, 1H), 7.40 (m, 2H), 7.20 (s, 1H), 7.08 (s, 2H), 3.04 (s, 2H),

2.57 (bs, 4H), 1.77 (bs, 4H); MS m/z 542.2 (M + H)⁺.

2-Fluoro-4-[2-ethylimidazol-1-yl]aniline (9b). This com-

pound was prepared following the same procedures described

for 9d using 2-ethylimidazole instead of 2-dimethylaminom-

1

ethylimidazole (55%). H NMR (CDCl₃) δ 7.02-6.96 (m, 5H),

Benzyl {1-[4-({[1-(3-Cyano-4-fluorophenyl)-3-(triflu-

oromethyl)-1H-pyrazol-5-yl]carbonyl}amino)-3-fluo-

rophen-yl]-1H-imidazol-2-yl}methyl(methyl)carbamate

(10f). This compound was prepared following the same pro-

cedures described for 10d using 9f instead of 9d (52%). ¹H

NMR (CDCl₃) δ 7.85-7.76 (m, 2H), 7.30 (m, 8H), 7.15 (m, 2H),

7.00 (m, 2H), 5.02 (m, 2H), 4.56 (s, 2H), 2.93 (s, 3H); MS m/z

636.1 (M + H)⁺.

3.98 (bs, 2H), 2.64 (q, J ) 7.6 Hz, 2H), 1.24 (t, J ) 7.6 Hz,

3H); MS m/z 206.1 (M + H)⁺.

2-Fluoro-4-[2-isopropylimidazol-1-yl]aniline (9c). This

compound was prepared following the same procedures de-

scribed for 9d using 2-isopropylimidazole instead of 2-dim-

1

ethylaminomethylimidazole (50%). H NMR (CDCl₃) δ 7.04-

6.81 (m, 5H), 4.10 (bs, 2H), 2.95 (m, 1H), 1.25 (d, J ) 6.9 Hz,

6H); MS m/z 220.2 (M + H)⁺.

1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-N-[2-

fluoro-4-[(2′-dimethylaminomethyl)imidazol-1-yl]phenyl]-

1H-pyrazole-5-carboxyamide (11d), Hydrochloride Salt

(Razaxaban). Into a 2 L flask with mechanical stirrer was

added potassium tert-butoxide (19.5 g, 0.157 mol), acetohy-

droxamic acid (11.77 g, 0.157 mol), and 500 mL of DMF. This

mixture rapidly produced a thick white precipitate. After 40

min, 10d (26.93 g, 0.052 mol) was added all at once as a solid.

The mixture turned brown and became very fluid. Stirring was

continued at ambient temperature for 12 h. The mixture was

poured into a separatory funnel containing 500 mL of aqueous

ammonium chloride and 500 mL of EtOAc. After the mixture

was shaken, the lower aqueous phase separated rapidly. This

was drawn off, and the organic phase was washed again with

500 mL of H₂O. A solid formed at the interface, which was

filtered off to give 8.5 g of the desired product 11d. The organic

solution was evaporated to 100 mL, and another 6.5 g of

product was precipitated. The aqueous washing was extracted

again with EtOAc. The EtOAc solution was washed with H₂O

and was then evaporated to give 8.6 g of the desired product.

All fractions were combined to give 23.6 g (86%) of the free

base (11d) with 99% HPLC purity. ¹H NMR (DMSO-d₆) δ 8.10

(d, J ) 1.8 Hz, 1H), 7.73-7.68 (m, 4H), 7.59 (d, J ) 8.4, 1H),

7.49 (d, J ) 1.5 Hz, 1H), 7.46 (d, J ) 1.1 Hz, 1H), 7.00 (d, J )

1.1 Hz, 1H), 6.59 (s, 2H), 3.34 (s, 2H), 2.13 (s, 6H); ¹³C NMR

(75 MHz, DMSO-d₆) δ 162.00, 159.14, 157.11, 156.64, 153.35,

144.87, 141.63, 141.14, 138.86, 136.51, 136.38, 134.24, 128.01,

126.74, 124.25, 124.14, 123.23, 122.02, 120.93, 119.50, 117.50,

1132.23, 112.91, 110.05, 108.23, 54.89, 44.92; ¹⁹F NMR (282

MHz, DMSO-d₆) δ -61.243 (s), -119.571 (t, J ) 6.8 Hz); MS

m/z 529.2 (M + H)⁺. The free base (20.78 g, 0.039 mol) was

dissolved in 105 mL of methanol with gentle heating. Once

dissolved, the solution was cooled in an ice bath, with stirring.

To this was added 1 N HCl/ether (85 mL, 0.085 mol, 2.18 equiv)

all at once. After the mixture was stirred for 10 min, 25 mL of

ether was added. The hydrochloride salt crystallized out over

the next 10 min. Cooling was continued for an additional 90

min. Product was collected on a filter, washed with ether (2 ×

50 mL), and dried under house vacuum for 12 h to give 20.67

g (92%) of a crystalline solid of the HCl salt (razaxaban) with

2-Fluoro-4-(2-pyrrolidin-1-ylmethylimidazol-1-yl)-

aniline (9e). This compound was prepared following the same

procedures described for 9d using 7e instead of 2-dimethy-

laminomethylimidazole (55%). ¹H NMR (CDCl₃) δ 7.23 (dd, J

) 12.6, 2.2 Hz, 2H), 7.05-6.68 (m, 4H), 3.89 (bs, 2H), 3.49 (s,

2H), 2.50 (m, 4H), 1.66 (m, 4H); MS m/z 261.2 (M + H)⁺.

[1-(4-Amino-3-fluorophenyl)-1H-imidazol-2-ylmethyl]-

methylcarbamic Acid Benzyl Ester (9f). This compound

was prepared following the same procedures described for 9d

using 7f instead of 2-dimethylaminomethylimidazole (67%).

¹H NMR (CDCl₃): δ 7.40-7.10 (m, 5H), 7.05 (s, 1H), 7.00-

6.70 (m, 4H), 5.00 (m, 2H), 4.68 (m, 2H), 3.83 (s, 2H), 2.89 (s,

3H); MS m/z 355.2, (M + H)⁺.

1-(3′-Cyano-4′-fluorophenyl)-3-trifluoromethyl-N-[2-

fluoro-4-[(2′-dimethylaminomethyl)imidazol-1-yl]phenyl]-

1H-pyrazole-5-carboxyamide (10d). To a suspension of the

acid 5 (38.31 g, 0.128 mol) in 1 L of dichloromethane was added

oxalyl chloride (32.5 g, 0.256 mol) and DMF (0.5 mL). The

mixture was stirred at ambient temperature for 1 h and then

heated to reflux for 1 h. The solvent and excess reagent in the

solution were evaporated to give the acid chloride 6 as a yellow

solid. The solid was dissolved in 1 L of dichloromethane and

cooled with an ice bath. To this was added aniline 9d (30 g,

0.128 mol) and (dimethylamino)pyridine (31.3 g, 0.256 mol).

The solution was stirred at ambient temperature for 12 h. The

solution was washed with H₂O (3 × 200 mL) and brine (200

mL), dried (Na₂SO₄), filtered, and concentrated. Column

chromatography (gradient of 0-5% MeOH in CH₂Cl₂) gave

1

56.1 g (85%) of 10d. H NMR (CDCl₃) δ 8.27 (t, J ) 8.5 Hz,

1H), 8.17 (bs, 1H), 7.86-7.79 (m, 2H), 7.74 (dd, J ) 9.5, 2.5

Hz, 1H), 7.40-7.34 (m, 2H), 7.20 (s, 1H), 7.10-7.08 (m, 2H),

3.37 (s, 2H), 2.26 (s, 6H); MS m/z 561.2 (M + H)⁺.

1-(3′-Cyano-4′-fluorophenyl)-3-trifluoromethyl-N-[2-

fluoro-4-[(2′-methyl)imidazol-1-yl]phenyl]-1H-pyrazole-

5-carboxyamide (10a). This compound was prepared follow-

ing the same procedures described for 10d using 9a instead

1

of 9d (64%). H NMR (CDCl₃) δ 8.31 (m, 1H), 8.17 (bs, 1H),

7.82 (m, 2H), 7.38 (t, J ) 8.0 Hz, 1H), 7.18 (m, 2H), 7.20 (s,

1H), 7.00 (m, 2H), 2.38 (s, 3H); MS m/z 473.0 (M + H)⁺.

1

99% purity by HPLC. H NMR (DMSO-d₆) δ 8.12 (d, J ) 1.8

1-(3′′-Cyano-4′-fluorophenyl)-3-trifluoromethyl-N-[2-

fluoro-4-[(2′-ethyl)imidazol-1-yl]phenyl]-1H-pyrazole-5-

carboxyamide (10b). This compound was prepared following

the same procedures described for 10d using 9b instead of 9d

(15%). ¹H NMR (CDCl₃) δ 9.22 (s, 1H), 8.17 (t, J ) 8.5 Hz,

1H), 7.80 (m, 2H), 7.38 (m, 1H), 7.12 (m, 2H), 7.06-6.83 (m,

3H), 2.64 (q, J ) 7.3 Hz, 2H), 1.24 (t, J ) 7.3 Hz, 3H); MS m/z

487.2 (M + H)⁺.

1-(3′-Cyano-4′-fluorophenyl)-3-trifluoromethyl-N-[2-

fluoro-4-[(2′-isopropyl)imidazol-1-yl]phenyl]-1H-pyrazole-

5-carboxyamide (10c). This compound was prepared follow-

ing the same procedures described for 10d using 9c instead

of 9d (35%). ¹H NMR (CDCl₃) δ 8.47 (d, J ) 2.2 Hz, 1H), 8.27

(t, J ) 8.4 Hz, 1H), 7.82 (m, 2H), 7.38 (m, 1H), 7.26-7.08 (m,

Hz, 1H), 7.83-7.78 (m, 2H), 7.70-7.67 (m, 1H), 7.61-7.57 (m,

2H), 7.741 (dd, J ) 8.8, 1.8 Hz, 1H), 7.25 (d, J ) 1.5 Hz, 1H),

4.41 (s, 2H), 2.78 (s, 6H); ¹³C NMR (75 MHz, DMSO-d₆) δ

161.97, 159.14, 157.03, 156.64, 153.30, 141.53, 141.03, 138.68,

137.84, 134.28, 133.27, 133.14, 128.11, 127.15, 126.66, 126.49,

125.01, 123.23, 122.99, 119.75, 117.49, 115.38, 115.07, 111.06,

109.97, 108.57, 49.66, 48.98, 43.04; ¹⁹F NMR (282 MHz,

DMSO-d₆) δ -56.458 (s), -113.218 (t, J ) 9.6 Hz); IR (KBr)

1687,1625,1528,1235,1172,1140,975cm^-1.Anal.(C₂₄H₂₀N₈F₄O₂‚

HCl) C, H, N, Cl, F. HRMS calcd for (C₂₄H₂₁N₈F₄O₂) (M + H)⁺

529.1724, found 529.1715.

1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-5-[4′-

(2′′-methylimidazol-1′′-yl)-2′-fluorophenyl)aminocarbo-

nyl]pyrazole, Bis(trifluoroacetic acid) Salt (11a). This

Factor Xa Inhibitor

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1739

compound was prepared following the same procedures de-

scribed for 11d using 10a instead of 10d. The final product

was purified by reverse-phase HPLC with acetonitrile/H₂O

(containing 0.05% TFA) as a mobile phase and isolated as a

2H), 2.92 (s, 3H); ¹⁹F NMR (acetone-d₆) δ -63.16, -76.72

(TFA), -123.07; HRMS calcd for C₂₃H₁₉F₄O₂N₈(M + H)

515.1567, found 515.1577.

(4-Amino-3-fluorophenyl)pyrrolidin-1-ylmethanone

(14). To a suspension of potassium dichromate (128 g, 0.430

mol) in glacial acetic acid (400 mL) was added 2-fluoro-4-

methyl-1-nitrobenzene (50.0 g, 0.323 mol) portionwise followed

by stirring for 15 min. Concentrated sulfuric acid (340 g) was

added slowly (CAUTION: strong exothermic reaction occurs!)

followed by heating to 120 °C for 1.5 h. The flask was allowed

to cool to ambient temperature, and 1.5 L of H₂O was added

with vigorous stirring to precipitate straw-colored crystals. The

solid was isolated by filtration and air-dried to give 3-fluoro-

1

TFA salt (54%). H NMR (CD₃OD) δ 8.09 (t, J ) 8.4 Hz, 1H),

7.97 (d, J ) 1.9 Hz, 1H), 7.66 (dd, J ) 8.8, 2.2 Hz, 1H), 7.67

(d, J ) 2.1 Hz, 1H), 7.58 (d, J ) 2.2 Hz, 1H), 7.55 (dd, J ) 9.1,

2.1 Hz, 1H), 7.50 (d, J ) 9.1 Hz, 1H), 7.46 (s, 1H), 7.40 (d, J

) 8.8 Hz, 1H), 2.56 (s, 3H); ¹³C NMR (CD₃OD) δ 163.76, 160.43,

159.05, 157.17, 154.67, 146.80, 143.78, 139.61, 135.74, 129.10,

127.30, 124.48, 123.35, 121.03, 120.08, 119.77, 118.23, 115.47,

115.23, 110.92, 108.65, 11.33; ¹⁹F NMR (CD₃OD) δ -64.21,

-77.62 (TFA), -121.45; MS m/z 486.2 (M + H)⁺. Anal.

(C₂₂H₁₅N₇O₂F₄‚1.3TFA‚1H₂O) C, H, N, F.

1

4-nitrobenzoic acid (49 g, 82%). H NMR (DMSO-d₆) δ 13.93

1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-5-[[4-

(2′-ethylimidazol-1′-yl)-2-fluorophenyl]aminocarbonyl]-

pyrazole, Bis(trifluoroacetic acid) Salt (11b). This com-

pound was prepared following the same procedures described

for 11d using 10b instead of 10d. The final product was

purified by reverse-phase HPLC with acetonitrile/H₂O (con-

taining 0.05% TFA) as a mobile phase and isolated as a TFA

(bs, 1H), 8.23 (t, J ) 7.7 Hz, 1H), 7.99-7.94 (m, 2H). The above

acid (18.8 g, 0.10 mol) was dissolved in 200 mL of dichlo-

romethane. The mixture was cooled in an ice bath, and to it

was added oxalyl chloride (18.2 mL, 0.20 mol), followed by a

few drops of DMF. The mixture was stirred at 0 °C for 5 h,

and it was evaporated to dryness. After the mixture was dried

under vacuum, the solid was dissolved in 200 mL of dichlo-

romethane. Triethylamine (21.3 mL, 0.15 mol) was added,

followed by pyrrolidine (9.3 mL, 0.20 mol). The reaction

mixture was stirred at ambient temperature under N₂for 48

h. The mixture was washed with H₂O and brine, dried over

MgSO₄, filtered, and concentrated to 24.5 g of yellow solid. The

solid (15.0 g) was dissolved in 200 mL of methanol, and Pd/C

(1.5 g of 5%) was added. The mixture was hydrogenated at 30

psi for 30 min. It was filtered through Celite, concentrated,

and chromatographed with EtOAc to give 7.0 g of 14 (55%).

¹H NMR (CDCl₃) δ 7.23 (m, 2H), 6.74 (t, J ) 8.8 Hz, 1H), 3.93

(bs, 2H), 3.59 (m, 4H), 1.90 (m, 4H); MS m/z 209 (M + H)⁺.

2,4-Bis-(2-methylimidazol-1-yl)phenylamine (15). 2,4-

Difluoronitrobenzene (5.0 g, 31.4 mmol) was dissolved in 50

mL of acetonitrile. 2-Methylimidazole (5.15 g, 62.8 mmol) and

K₂CO₃(8.69 g, 63.0 mmol) were added. The mixture was

heated to reflux for 12 h. It was cooled and poured into 500

mL of H₂O and then extracted with EtOAc. The organic

solution was washed with H₂O and brine, dried over MgSO₄,

filtered, and concentrated. It was dissolved in 20 mL of

dichloromethane, and hexane (200 mL) was added. The

precipitate was filtered and dried to give 7.98 g of 2,4-bis(2-

methylimidazol-1-yl)nitrobenzene (90%). ¹H NMR (CDCl₃) δ

8.21 (d, J ) 8.8 Hz, 1H), 7.60 (dd, J ) 2.2, 8.2 Hz, 1H), 7.40

(d, J ) 2.2, 8.2 Hz, 1H), 7.10 (m, 3H), 6.95 (d, J ) 1.5 Hz, 1H),

2.49 (s, 3H), 2.29 (s, 3H); MS m/z 284.2 (M + H)⁺. The nitro

intermediate (1.12 g) was dissolved in 80 mL of MeOH. Pd/C

(42 mg of 10%) was added. The mixture was hydrogenated at

40 psi for 12 h. It was filtered through Celite and concentrated

to give 0.60 g of 15 (56%). ¹H NMR (DMSO-d₆) δ 8.62 (s, 1H),

8.01(s, 1H), 7.36 (t, J ) 8.4 Hz, 1H), 7.26 (d, J ) 1.5 Hz, 1H),

7.12 (d, J ) 1.5 Hz, 1H), 6.93 (d, J ) 1.5 Hz, 1H), 6.88 (d, J )

1.5 Hz, 1H), 3.37 (bs, 2H), 2.27 (s, 3H), 2.17 (s, 3H); MS m/z

254.2 (M + H)⁺.

1

salt (67%). H NMR (acetone-d₆) δ 9.99 (s, 1H), 8.27 (t, J )

8.8 Hz, 1H), 8.10 (d, J ) 2.2 Hz, 1H), 7.80-7.57 (m, 7H), 3.04

(q, J ) 7.7 Hz, 2H), 1.30 (t, J ) 7.7 Hz, 3H); ¹⁹F NMR (acetone-

d₆) δ -63.17, -76.65 (TFA), -122.91; HRMS calcd for

C₂₃H₁₈F₄O₂N₇(M + H) 500.1458, found 500.1471.

1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-5-[[4-

(2′-isopropylimidazol-1′-yl)-2-fluorophenyl]aminocarbo-

nyl]pyrazole, Bis(trifluoroacetic acid) Salt (11c). This

compound was prepared following the same procedures de-

scribed for 11d using 10c instead of 10d. The final product

was purified by reverse-phase HPLC with acetonitrile/H₂O

(containing 0.05% TFA) as a mobile phase and isolated as a

1

TFA salt (36%). H NMR (acetone-d₆) δ 10.02 (s, 1H), 8.28 (t,

J ) 8.5 Hz, 1H), 8.11 (d, J ) 1.9 Hz, 1H), 7.82-7.56 (m, 7H),

3.33 (m, 1H), 1.40 (d, J ) 7.3 Hz, 6H); ¹⁹F NMR (acetone-d₆)

δ -63.16, -76.72 (TFA), -122.56; HRMS calcd for C₂₃H₁₉F₃O₃N₇

(M + H) 514.1615, found 514.1634.

1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-N-[2-

fluoro-4-[(2′-pyrrolidinylmethyl)imidazol-1-yl]phenyl]-

1H-pyrazole-5-carboxyamide, Bis(trifluoroacetic acid)

Salt (11e). This compound was prepared following the same

procedures described for 11d using 10e instead of 10d. The

final product was purified by reverse-phase HPLC with

acetonitrile/H₂O (containing 0.05% TFA) as a mobile phase and

isolated as a TFA salt (50%). ¹H NMR (acetone-d₆) δ 9.42 (bs,

1H), 8.15 (t, J ) 8.8 Hz, 1H), 8.11 (d, J ) 1.8 Hz, 1H), 7.78

(dd, J ) 2.2, 8.8 Hz, 1H), 7.58 (m, 3H), 7.48 (s, 1H), 7.42 (d, J

) 8.8 Hz, 1H), 7.25 (s, 1H), 4.68 (s, 2H), 3.58 (m, 4H), 2.08 (m,

4H); ¹⁹F NMR (acetone-d₆) δ -63.16, -76.772 (TFA), -123.08;

HRMS calcd for C₂₆H₂₃F₄O₂N₈(M + H) 555.1880, found

555.1898.

N-[4-(2-{[Methylamino]methyl}-1H-imidazol-1-yl)-2-

fluorophenyl]-1-(3-amino-1,2-benzisoxazol-5-yl)-3-(tri-

fluoromethyl)-1H-pyrazole-5-carboxamide, Bis(trifluo-

roacetic acid) Salt (12). Acetohydroxamic acid (0.40 g, 5.34

mmol) was dissolved in 10 mL of DMF. K₂CO₃(0.98 g, 7.12

mmol) was added, followed by 1 mL of H₂O. The mixture was

stirred at room temperature under N₂for 30 min, and a

solution of benzyl {1-[4-({[1-(3-cyano-4-fluorophenyl)-3-(triflu-

oromethyl)-1H-pyrazol-5-yl]carbonyl}amino)-3-fluorophenyl]-

1H-imidazol-2-yl}methyl(methyl)carbamate 10f (1.13 g, 1.78

mmol) in 10 mL of DMF was added. The resulting mixture

was stirred at ambient temperature under N₂for 12 h. H₂O

was added to the reaction mixture. The precipitate formed was

filtered and dried to give 11f. MS (ES⁺) m/z 647.1, (M - H)^-.

2-Fluoro-4-morpholinoaniline (16). A solution of 2,4-

difluoronitrobenzene (10.0 mL) and morpholine (17.4 mL) in

THF (100 mL) was stirred at ambient temperature under N₂

for 2 h. The solvent was removed, and the residue was

partitioned between EtOAc and H₂O. The organic layer was

washed with brine, dried over MgSO₄, filtered, and concen-

trated. The resulting solid was purified by chromatography

on silica gel with 20-50% EtOAc in hexane to give 18.1 g of

4-fluoro-2-morpholinonitrobenzene and 1.81 g of 2-fluoro-4-

morpholinonitrobenzene. 2-Fluoro-4-morpholinonitrobenzene

(1.80 g) was dissolved in methanol (100 mL), and 10% Pd/C

(94 mg) was added. The mixture was placed in a hydrogenator

(45 psi) for 2.5 h. The reaction mixture was filtered through

Celite and washed with methanol. The filtrate was concen-

The above solid was heated to reflux with 20 mL of TFA

under N₂for 30 min. The TFA was removed. The residue was

purified by reverse-phase HPLC (C18 reverse-phase column,

eluted with a H₂O/CH₃CN gradient with 0.05% TFA) to give

0.61 g of 12 as the bis-TFA salt (67%). ¹H NMR (acetone-d₆) δ

8.16 (t, J ) 8.4 Hz, 1H), 8.07 (d, J ) 1.8 Hz, 1H), 7.74 (dd, J

) 8.8 Hz, 2.2 Hz, 1H), 7.55 (m, 4H), 7.40 (m, 2H), 4.64 (bs,

1

trated to give 1.51 g of 16 (8.4%). H NMR (CDCl₃) δ 6.76-

6.54 (m, 3H), 3.84 (t, J ) 4.7 Hz, 4H), 3.45 (bs, 2H), 3.02 (t, J

) 4.7 Hz, 4H); MS m/z 197.1 (M + H)⁺.

2-Amino-5-(2-methylimdazol-1-yl)benzylnitrile (17). To

a solution of 2,4-difluoronitrobenzene (1.98 g, 12.6 mmol) in

1740 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Quan et al.

20 mL of DMF was added NaCN (0.63 g, 12.9 mmol). The

mixture was stirred at ambient temperature for 12 h. It was

poured into H₂O and extracted with EtOAc. The organic

solution was washed with H₂O and brine, dried over MgSO₄,

filtered, and concentrated. The crude product was purified by

flash chromatography with 0-20% EtOAc in hexane to give

0.65 g of 5-fluoro-2-nitrobenzylnitrile (31%). ¹H NMR (CDCl₃)

δ 8.41 (m, 1H), 7.62 (m, 1H), 7.53 (m, 1H); ¹⁹F NMR (CDCl₃)

δ -100 (s); MS m/z 167.0 (M + H)⁺.

5-Fluoro-2-nitrobenzylnitrile(0.64 g, 3.85 mmol) and 2-me-

thylimidazole (0.66 g, 8.04 mmol) were dissolved in CH₃CN

(25 mL) and heated to reflux under N₂for 12 h. The solvent

was removed, and the residue was purified by chromatography

on silica gel with 2-5% MeOH in CH₂Cl₂to give 0.84 g (96%)

of 5-(2-methylimdazol-1-yl)-2-nitrobenzylnitrile. ¹H NMR

(CDCl₃) δ 8.50 (d, J ) 8.7 Hz, 1H), 7.87 (d, J ) 2.2 Hz, 1H),

7.76 (dd, J ) 2.6, 8.8 Hz, 1H), 7.11 (dd, J ) 1.4, 12.1 Hz, 2H),

2.49 (s, 3H); MS m/z 246.2 (M + NH₄)⁺.

To a suspension 5-(2-methylimdazol-1-yl)-2-nitrobenzylni-

trile (0.40 g, 1.75 mmol) in methanol (50 mL) was added 10%

Pd/C (50 mg). The mixture was placed on a hydrogenator at

47 psi for 3 h. It was filtered through Celite and concentrated

to give 0.34 g of 17 (98%). ¹H NMR (CDCl₃) δ 7.32 (d, J ) 2.2

Hz, 1H), 7.14 (dd, J ) 2.6, 8.8 Hz, 1H), 6.99 (d, J ) 1.5 Hz,

1H), 6.92 (d, J ) 1.5 Hz, 1H), 6.75 (d, J ) 8.8 Hz, 1H), 5.93

(br s, 2H), 2.26 (s, 3H).

2-Methoxy-4-(2-methylimidazol-1-yl)aniline (18). A so-

lution of 5-fluoro-2-nitrophenol (2.03 g) and 2-methylimidazole

(2.14 g) in CH₃CN (50 mL) was stirred at reflux under N₂for

16 h. The solvent was removed, and the residue was purified

by chromatography on silica gel with 0-10% MeOH in CH₂-

Cl₂to give 2.21 g (78%) of 5-(2-methylimdazol-1-yl)-2-nitro-

phenol. ¹H NMR (CDCl₃) δ 10.78 (bs, 2H), 8.26 (d, J ) 8.8 Hz,

1H), 7.13 (d, J ) 2.2 Hz, 1H), 7.07 (dd, J ) 1.5, 4.3 Hz, 2H),

6.97 (dd, J ) 2.2, 9.1 Hz, 1H), 5.30 (s, 1H), 2.48 (s, 3H); MS

m/z 220.1 (M + H)⁺.

1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-5-[[4-

(2′-aminosulfonyl-4-pyridinyl)-3-fluorophenyl]aminocar-

bonyl]pyrazole, Trifluoroacetic Acid Salt (23). The iodo

intermediate 19 obtained above (0.18 g, 0.35 mmol) was

dissolved in dry and degassed DMSO (5 mL). To this solution

was added KOAc (0.10 g, 1.05 mmol) and pinacol diborane (0.1

g, 0.38 mmol), followed by PdCl₂(dppf) (9 mg, 0.01 mmol). The

reaction mixture was heated at 80 °C for 2 h, and then the

reaction was quenched with H₂O (100 mL). The organic

mixture was extracted with EtOAc (2 × 50 mL), dried over

MgSO₄, filtered, and concentrated to a brownish oil (0.26 g).

The oil was redissolved in an ethanol/toluene mixture (4:1, 50

mL), and to this solution was added 4-bromopyridine-3-sulfonic

acid tert-butylamide 21 (0.168 g, 0.49 mmol), followed by a

catalytic amount of tetrakistriphenylphosphine palladium

(spatula, tip). The reaction mixture was heated to reflux for

12 h. It was cooled, filtered over Celite, and concentrated in

vacuo. The crude mixture without purification was redissolved

in dry DMF (1 mL) and converted to the desired compound

23 via reaction with acetohydroxamic acid and K₂CO₃(see

compound 35 for detailed procedure) followed by deprotection

with TFA. The final product was purified by reverse-phase

HPLC (C18 reverse-phase column), eluted with an H₂O/CH₃-

CN gradient with 0.05% TFA, and isolated as the TFA salt

1

(15%). H NMR (DMSO-d₆) δ 10.7 (s, 1H), 9.14 (s, 1H), 8.80

(d, J ) 5.1 Hz, 1H), 8.10 (d, J ) 1.8 Hz, 1H), 7.75-7.65 (m,

4H), 7.60 (d, J ) 8.8 Hz, 1H), 7.48-7.40 (m, 2H), 7.42-7.27

(dd, J ) 8.8 Hz, 2H), 3.80 (bs, 2H); ¹⁹F NMR (DMSO-d₆) δ

-61.21, -75.19; HRMS calcd for C₂₃H₁₆F₄N₇O₄S 562.0921,

found 562.0911.

2-Fluoro-4-(1-methylimidazol-2-yl)aniline (26). To a

solution of 4-bromo-2-fluoroaniline (19.2 g, 100 mmol) in THF

(100 mL) at 0 °C was slowly added LiN(TMS) (1 M in THF,

2

200 mL) over 30 min. After the resulting solution was warmed

to ambient temperature, a solution of di-tert-butyl dicarbonate

(21.8 g, 100 mmol) in THF (50 mL) was slowly added. The

mixture was stirred for 15 min and filtered through a pad of

silica gel. The filtrate was concentrated and recrystallized from

hexane to give 4-bromo-2-fluoro-1-tert-butoxycarbonylaniline

5-(2-Methylimdazol-1-yl)-2-nitrophenol (1.16 g) was dis-

solved in DMF (30 mL). To this solution was added K₂CO₃

(0.92 g) and iodomethane (0.33 mL). The reaction mixture was

stirred at ambient temperature under N₂for 6 h. The reaction

mixture was poured into 100 mL of H₂O and extracted with

EtOAc (4 × 50 mL), dried over MgSO₄, filtered, and concen-

trated to give 0.25 g (20%) of 2-methoxy-4-(2-methylimidazol-

1-yl)nitrobenzene. ¹H NMR (CDCl₃) δ 8.00 (d, J ) 9.2 Hz, 1H),

7.06 (d, J ) 8.7 Hz, 2H), 7.00 (s, 1H), 6.97 (d, J ) 2.2 Hz, 1H),

4.01 (s, 3H), 2.44 (s, 3H); MS m/z 234.2 (M + H)⁺.

1

24 (27.7 g, 95%). H NMR (CDCl₃) δ 8.00 (t, J ) 8.8 Hz, 1H),

7.25-7.20 (m, 2H), 6.66 (bs, 1H), 1.52 (s, 9H); ¹⁹F NMR (CDCl₃)

δ -130.42; MS m/z 290/292 (M + H)⁺.

To a solution of 1N-methylimidazole (1.64 g, 20 mmol) in

THF (40 mL) at -78 °C was added n-BuLi (2.5 M, 9.6 mL),

and the resulting solution was stirred at -78 °C for 30 min.

After Bu₃SnCl (7.18 g, 22 mmol) was added, the resulting

mixture was slowly warmed to ambient temperature over 2 h

and was stirred for an additional 16 h. To the reaction mixture

was added 4-bromo-2-fluoro-1-tert-butoxycarbonylaniline 24

(0.58 g, 2 mmol) and Pd(PPh₃)₄(92 mg, 0.08 mmol). The

resulting mixture was degassed and filled with nitrogen three

times. The mixture was heated to reflux under N₂for 18 h

and then cooled to ambient temperature. After saturated

aqueous KF (10 mL) was added, the resulting mixture was

stirred for 1 h and filtered through a pad of Celite. The filtrate

was washed with H₂O and brine, dried over MgSO₄, filtered,

concentrated, and purified by silica gel chromatography with

EtOAc to give 2-fluoro-4-(1′-methylimidazol-2′-yl)-1-tert-bu-

toxycarbonylaniline (0.35 g, 60%) as a white solid. ¹H NMR

(CDCl₃) δ 8.19 (t, J ) 8.0 Hz, 1H), 7.42 (dd, J ) 12.1, 1.8 Hz,

1H), 7.36 (d, J ) 9.1 Hz, 1H), 7.10 (d, J ) 1.1 Hz, 1H), 6.96 (s,

1H), 6.80 (bs, 1H), 3.75 (s, 3H), 1.54 (s, 9H); ¹⁹F NMR (CDCl₃)

δ -132.59; MS m/z 292.2 (M + H)⁺.

2-Methoxy-4-(2-methylimidazol-1-yl)nitrobenzene (0.25 g)

was dissolved in methanol (20 mL), and 10% Pd/C (29.3 mg)

was added. The mixture was placed on a hydrogenator (40 psi)

for 4 h. The reaction mixture was filtered and washed with

methanol. The filtrate was concentrated to give 0.27 g (quan-

1

titative) of 18. H NMR (CDCl₃) δ 2.32 (s, 3H), 3.86 (s, 3H),

3.95 (bs, 2H, NH₂), 6.68 (t, 1H, J ) 1.8 Hz, 1H), 6.72 (m, 2H),

6.95 (d, J ) 1.4 Hz, 1H), 6.99 (d, J ) 1.1 Hz, 1H); MS m/z

+

204.2 (M + H)

.

2-(3-Cyano-4-fluorophenyl)-5-trifluoromethyl-2H-pyra-

zole-3-carboxylic Acid (2-Fluoro-4-iodophenyl)amide (19).

To a dichloromethane suspension of 1-(4′-fluoro-3′-cyanophe-

nyl)-3-trifluoromethyl-1H-pyrazole-5-carboxylic acid (5) (3.21

g, 11.27 mmol) was added oxalyl chloride (1.48 mL, 16.90

mmol) and a catalytic amount of DMF. The reaction mixture

was stirred at ambient temperature for 3 h and then concen-

trated to a pale-yellow solid. The crude mixture was redis-

solved in dichloromethane, and to this solution was added

2-fluoro-4-iodoaniline 8 (2.67 g, 11.27 mmol), followed by

DMAP (3.44 g, 28.17 mmol). The reaction mixture was stirred

at ambient temperature for 12 h and concentrated. The crude

product was taken in EtOAc(100 mL), and the reaction was

quenched with HCl (3 N, 50 mL). The mixture was shaken in

a separatory funnel and the organic layer was separated, dried

over MgSO₄, filtered, and concentrated to afford pure 19 (2.61

To a solution of 2-fluoro-4-(1′-methylimidazol-2′-yl)-1-tert-

butoxycarbonylaniline (0.33 g, 1.13 mmol) in EtOAc (10 mL)

was added 3 M HCl (5 mL), and the resulting solution was

stirred at ambient temperature for 30 min. The solution was

cooled to 0 °C, neutralized with 50% NaOH to pH 8, and

extracted with EtOAc (50 mL × 3). The EtOAc layer was

concentrated and purified by silica gel chromatography and

eluted with 5% MeOH in EtOAc to give 2-fluoro-4-(1′-meth-

1

g, 45%). H NMR (CDCl₃) δ 7.15 (s, 1H), 7.23 (t, J ) 8.5 Hz,

ylimidazol-2′-yl)aniline 26 (0.18 g, 83%). H NMR (CD₃OD) δ

1H), 7.49-7.58 (m, 2H), 7.75-7.99 (m, 4H); MS m/z 517 (M -

7.54 (d, J ) 2.2 Hz, 1H), 7.51 (d, J ) 2.2 Hz, 1H), 7.37 (dd, J

) 11.8, 2.2 Hz, 1H), 7.27 (dd, J ) 8.4, 2.2 Hz, 1H), 6.97 (t, J

H)^-.

Factor Xa Inhibitor

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1741

) 8.8 Hz, 1H), 3.88 (s, 3H); ¹⁹F NMR (CD₃OD) δ -136.77 (dd,

J ) 90.1 Hz, J ) 9.1 Hz); MS m/z 192 (M + H)⁺.

7.52 (dd, J ) 8.8, 0.5 Hz, 1H), 7.48 (s, 1H), 3.93 (s, 3H); ¹³C

NMR (CD₃OD) δ 163.78, 160.43, 159.02, 156.71, 154.22,

144.84, 143.78 (CF₃), 139.64, 135.73, 129.09, 127.05, 126.51,

126.08, 120.52, 120.08, 118.23, 117.99, 110.93, 108.71, 36.16;

¹⁹F NMR (CD₃OD) δ -64.21, -77.58 (TFA), -123.46; HRMS

calcd. for C₂₂H₁₆F₄N₇O₂486.1302, found 486.1323.

2-Hydroxymethyl-1H-imidazole (30). 2-Imidazolecar-

boxyaldehyde (5.0 g, 52.0 mmol) was suspended in 200 mL of

methanol, and NaBH₄(3.95 g, 0.10 mol) was added portion-

wise. The reaction mixture was stirred at ambient temperature

for 1 h under N₂. It was quenched with 10 mL of brine, and

the solvent was removed. The solid was washed with 5%

MeOH in CH₂Cl₂, and the inorganic solid was filtered off. The

filtrate was concentrated and chromatographed with 5%

MeOH in CH₂Cl₂to give 2.32 g of 2-hydroxymethyl-1H-

imidazole 30 (45.2%). ¹H NMR (DMSO-d₆) δ 6.86 (s, 2H), 4.40

(s, 2H).

1-(3′-Aminobenzisoxozol-5-yl)-3-trifluoromethyl-5-pyra-

zolecarboxylic Acid (27). To a solution of 1-(4′-fluoro-3′-

cyanophenyl)-3-trifluoromethyl-5-pyrazolecarboxylic acid 5 (2.0

g, 6.39 mmol) in CH₃CN (30 mL) was added SOCl₂(5.1 g, 42.8

mmol), and the resulting solution was heated to reflux for 2

h. The mixture was concentrated on an evaporator, and the

residue was dissolved in MeOH (20 mL). The resulting solution

was heated to reflux for 30 min and then concentrated and

purified by silica gel chromatography with CH₂Cl₂to give

methyl 1-(4′-fluoro-3′-cyanophenyl)-3-trifluoromethyl-5-pyra-

zolecarboxylic ester (1.93 g, 92%). ¹H NMR (CDCl₃) δ 7.78 (dd,

J ) 5.6, 2.6 Hz, 1H), 7.73 (dd, J ) 8.4, 3.4 Hz, 1H), 7.36 (t, J

) 8.4 Hz, 1H), 7.30 (s, 1H), 3.88 (s, 3H); ¹⁹F NMR (CDCl₃) δ

-63.01, -104.60; MS m/z 331 (M + NH₄)⁺.

To a solution of acetone oxime (0.67 g, 9.2 mmol) in DMF

(20 mL) was added potassium tert-butoxide (1.0 M in THF,

9.2 mL), and the mixture was stirred at ambient temperature

for 15 min. To it was added a solution of methyl 1-(4′-fluoro-

3′-cyanophenyl)-3-trifluoromethyl-5-pyrazolecarboxylic ester

(1.92 g, 6.15 mmol) in DMF (20 mL), and the resulting mixture

was stirred at ambient temperature for 20 h. The reaction was

then quenched with H₂O (10 mL), and the mixture was

extracted with EtOAc (100 mL). The EtOAc layer was washed

with brine (10 mL × 5), dried over MgSO₄, filtered, concen-

trated, and purified by silica gel chromatography, eluting with

80% CH₂Cl₂in hexane to give methyl 1-(4′-isopropylideneami-

nooxy-3′-cyanophenyl)-3-trifluoromethyl-5-pyrazolecarboxyl-

ic ester (1.53 g, 68%) as a white solid. ¹H NMR (CDCl₃) δ 7.69

(d, J ) 9.1 Hz, 1H), 7.66 (d, J ) 2.2 Hz, 1H), 7.60 (dd, J ) 9.1,

2.5 Hz, 1H), 7.26 (s, 1H), 3.85 (s, 3H), 2.19 (s, 3H), 2.08 (s,

3H); ¹⁹F NMR (CDCl₃) δ -62.88; MS m/z 367 (M + H)⁺. To a

solution of methyl 1-(4′-isopropylideneaminooxy-3′-cyanophen-

yl)-3-trifluoromethyl-5-pyrazolecarboxylic ester (1.53 g, 4.18

mmol) in MeOH (13 mL) and CH₂Cl₂(6 mL) was added 18%

HCl (13 mL), and the mixture was heated to reflux for 3 h. It

was then concentrated to remove organic solvents. The result-

ing aqueous solution was neutralized with 2 N NaOH to pH 7

and extracted with EtOAc. The EtOAc layer was washed with

brine, dried over MgSO₄, filtered, and concentrated to give

methyl 1-(3′-aminobenzisoxozol-5-yl)-3-trifluoromethyl-5-pyra-

zolecarboxylic ester (1.32 g, 65%) as a white solid. ¹H NMR

(CD₃OD) δ 7.89 (d, J ) 2.1 Hz, 1H), 7.63 (dd, J ) 8.8, 2.2 Hz,

1H), 7.50 (d, J ) 8.8 Hz, 1H), 7.40 (s, 1H), 3.79 (s, 3H); ¹⁹F

NMR (CD₃OD) δ -64.36; MS m/z 327 (M + H)⁺.

A solution of methyl 1-(3′-aminobenzisoxozol-5-yl)-3-triflu-

oromethyl-5-pyrazolecarboxylic ester (260 mg, 0.8 mmol) in

THF (10 mL) was treated with 2 N NaOH (10 mL) at ambient

temperature for 16 h. The mixture was acidified with concen-

trated HCl to pH 3 and extracted with EtOAc. The EtOAc layer

was dried over Na₂SO₄and concentrated to give 1-(3′-ami-

nobenzisoxozol-5-yl)-3-trifluoromethyl-5-pyrazolecarboxylic acid

27 (240 mg, 96%). ¹H NMR (CD₃OD) δ 7.90 (d, J ) 1.9 Hz,

1H), 7.62 (dd, J ) 8.8, 2.4 Hz, 1H), 7.49 (d, J ) 8.8 Hz, 1H),

7.35 (s, 1H); ¹⁹F NMR (CD₃OD) δ -64.32; MS m/z 311 (M -

H)^-.

1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-5-[4′-

(1-methylimidazol-2-yl)-2′-fluorophenyl)aminocarbonyl]-

pyrazole, Bis(trifluoroacetic acid) Salt (28). To a solution

of 1-(3′-aminobenzisoxozole-5-yl)-3-trifluoromethyl-5-pyrazole-

carboxylic acid 27 (30 mg, 0.096 mmol) in DMF (2 mL) was

added 2-fluoro-4-(1-methylimidazol-2-yl)aniline 26 (20.4 mg,

0.106 mmol), diisopropylethylamine (0.2 mL), and PyBrop

(49.4 mg, 0.106 mmol). The resulting mixture was stirred at

60 °C for 16 h and quenched with EtOAc (75 mL) and H₂O (5

mL). The EtOAc layer was washed with 1 N HCl (5 mL), 1 N

NaOH (5 mL), and brine (5 mL × 4), dried over MgSO₄,

filtered, and concentrated. The residue was purified on silica

gel TLC plates with 10% MeOH in EtOAc, followed by further

purification by HPLC (CH₃CN/H₂O/0.05% TFA) to give 28 as

the TFA salt (19 mg, 40.8%). ¹H NMR (CD₃OD) δ 8.21 (t, J )

8.1 Hz, 1H), 7.99 (dd, J ) 2.2, 0.6 Hz, 1H), 7.70-7.66 (m, 3H),

7.64 (d, J ) 2.2 Hz, 1H), 7.57 (dt, J ) 8.3 Hz, 1.0 Hz, 1H),

1-(4-Amino-3-fluorophenyl)-2-hydroxymethylimida-

zole (31). 2-Hydroxymethyl-1H-imidazole 30 (2.30 g, 23.47

mmol), 2-fluoro-4-iodoaniline 8 (5.56 g, 23.47 mmol), K₂CO₃

(1.56 g, 25.82 mmol), 1,10-phenanthroline (0.42 g, 2.35 mmol),

CuI (0.45 g, 2.35 mmol), and DMSO (50 mL) were added

together and degassed. The mixture was then heated at 130

°C under N₂for 12 h. The mixture was cooled, and 14%

aqueous NH₄OH (200 mL) and EtOAC (200 mL) were added.

The mixture was filtered through Celite and washed with

EtOAc. The filtrate was extracted with EtOAc. The combined

organic solution was washed with brine and dried over MgSO₄.

It was filtered, concentrated, and purified by chromatography

on silica gel with 5% MeOH in CH₂Cl₂to give 0.48 g of 1-(4-

amino-3-fluorophenyl)-2-hydroxymethylimidazole 31 (10%).

1

MS m/z 208.2 (M + H)⁺; H NMR (DMSO-d₆) δ 7.27 (m, 2H),

7.06 (dd, J ) 2.2, 8.5 Hz, 1H), 6.94 (s, 1H), 6.83 (t, J ) 8.4 Hz,

1H), 5.41 (s, 2H), 5.34 (t, J ) 5.5 Hz, 1H), 4.36 (d, J ) 5.5 Hz,

2H).

1-(4-Amino-3-fluorophenyl)-2-(tert-butyldimethylsilyl-

oxymethyl)imidazole (32). 1-(4-Amino-3-fluorophenyl)-2-

hydroxymethylimidazole 31 (0.48 g, 2.32 mmol), TBDMSCl

(0.52 g, 3.48 mmol), and Et₃N (0.65 mL, 4.64 mmol) were

dissolved in 20 mL of DMF. The mixture was stirred at

ambient temperature under N₂for 12 h. It was diluted with

H₂O and extracted with EtOAc, and the combined organic

solution was washed with brine and dried over MgSO₄. It was

filtered, concentrated, and purified by chromatography on

silica gel with 5% MeOH in CH₂Cl₂to give 0.50 g of 1-(4-amino-

3-fluorophenyl)-2-(tert-butyldimethylsilyloxymethyl)im-

1

idazole 32 (67%). H NMR (CDCl₃) δ 7.20 (dd, J ) 2.6, 11.8

Hz, 1H), 7.02 (m, 3H), 6.78 (t, J ) 9.1 Hz, 1H), 4.56 (s, 2H),

3.84 (bs, 2H), 0.82 (s, 9H), 0.00 (s, 6H).

N-[4-[(2-tert-Butyldimethylsilyloxymethyl)-1H-imida-

zol-1-yl]-2-fluorophenyl]-1-(3-cyano-4-fluorophenyl)-3-

(trifluoromethyl)-1H-pyrazole-5-carboxamide (33). 1-(3-

Cyano-4-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-

carboxylic 5 (0.45 g, 1.50 mmol) was stirred in 20 mL of CH₂Cl₂

at ambient temperature under N₂. Oxalyl chloride (0.20 mL,

2.25 mmol) was added, followed by a few drops of DMF. The

mixture was stirred for 2 h. The solvent was removed, and

the resulting solid (6) was dried under vacuum. This solid was

then dissolved in 20 mL of CH₂Cl₂, and 1-(4-amino-3-fluo-

rophenyl)-2-(tert-butyldimethylsilyloxymethyl)imidazole 32 (0.48

g, 1.50 mmol) was added followed by DMAP (0.50 g, 4.25

mmol). The mixture was stirred at ambient temperature under

N₂for 12 h. It was diluted with CH₂Cl₂, washed with H₂O and

brine, dried over MgSO₄, filtered, and concentrated. The crude

product was purified by chromatography on silica gel with 50%

hexane in EtOAc to give 0.48 g of N-[4-[(2-tert-butyldimeth-

ylsilyloxymethyl)-1H-imidazol-1-yl]-2-fluorophenyl]-1-(3-cyano-

4-fluoropheyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxam-

1

ide 33 (53%). H NMR (CDCl₃) δ 8.54 (s, 1H), 8.18 (m, 1H),

7.78 (m, 2H), 7.50, 4.56 (s, 2H), 3.84 (bs, 2H), 0.82 (s, 9H),

0.00 (s, 6H); MS m/z 603.31 (M + H)⁺.

N-[4-(2-[Hydroxymethyl]-1H-imidazol-1-yl)-2-fluoro-

phenyl]-1-(3-amino-1,2-benzisoxazol-5-yl)-3-(trifluoro-

methyl)-1H-pyrazole-5-carboxamide (35). Acetohydrox-

1742 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Quan et al.

amic acid (0.18 g, 1.80 mmol) was dissolved in 5 mL of DMF.

K₂CO₃(0.44 g, 3.20 mmol) was added, followed by a few drops

of H₂O. The mixture was stirred at ambient temperature under

N₂for 30 min, and a solution of N-[4-[(2-tert-butyldimethyl-

silyloxymethyl)-1H-imidazol-1-yl]-2-fluorophenyl]-1-(3-cyano-

4-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5-carboxam-

ide 33 (0.48 g, 0.80 mmol) was added. The resulting mixture

was stirred at ambient temperature under N₂for 12 h, and

then H₂O was added to the reaction mixture. The precipitate

formed was filtered and dried to give 34. MS (ES⁺) m/z 616.3

(M + H)⁺. This solid was dissolved in 20 mL of THF.

Tetrabutylammonium fluoride (1.66 mL of a 1 M solution) was

added, and the mixture was stirred at ambient temperature

under N₂for 1 h. The THF was removed. The residue was

partitioned between EtOAc and H₂O. The organic solution was

washed with H₂O and brine, dried over MgSO₄, filtered, and

concentrated to give 0.36 g of a light-yellow solid (90%). This

solid was approximately 95% pure by analytical HPLC and

was taken into the next step without further purification. A

small amount was purified by reverse-phase HPLC (C18

reverse-phase column, eluted with a H₂O/CH₃CN gradient with

0.05% TFA) to give N-[4-(2-[hydroxymethyl]-1H-imidazol-1-yl)-

2-fluorophenyl]-1-(3-amino-1,2-benzisoxazol-5-yl)-3-(trifluoro-

(s, 2H), 2.91 (s, 3H); ¹⁹F NMR (DMSO-d₆) δ -60.67, -121.90.

HRMS calcd for C₂₅H₁₈F₄N₅O₄S 560.1016, found 560.1020.

1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-5-[(2′-

aminosulfonyl-3-fluoro[1,1′]biphen-4-yl)aminocarbonyl]-

pyrazole, Trifluoroacetic Acid Salt (39). 1-(3′-Aminoben-

zisoxozole-5-yl)-3-trifluoromethyl-5-pyrazolecarboxylic acid 27

was coupled with 4′-amino-3′-fluorobiphenyl-2-sulfonic acid

amide^7,8,11following the same procedures described in the

synthesis of compound 28 (20%). The coupling product was

heated to reflux in TFA for 0.5 h to remove the tert-butyl group.

The final product was purified by reverse-phase HPLC with

acetonitrile/H₂O (containing 0.05% TFA) as a mobile phase and

isolated as a TFA salt (47%). ¹H NMR (CDCl₃) δ 8.09 (dd, J )

7.8, 1.2 Hz, 1H), 7.91 (t, J ) 8.1 Hz, 1H), 7.82 (d, J ) 2.1 Hz,

1H), 7.67-7.45 (m, 4H), 7.31-7.16 (m, 3H); ¹⁹F NMR (CDCl₃)

δ -62.79, -126.65; HRMS calcd for C₂₄H₁₇F₄N₆O₄S 561.0968,

found 561.0978.

1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-5-[2-

fluoro-4-(N-pyrrolidinocarbonyl)phenylaminocarbonyl]-

pyrazole Trifluoroacetate, Trifluoroacetic Acid Salt (40).

This compound was prepared following the same procedures

described for 11d using aniline 14 to couple with acid 5 (87%).

The resulting amide was converted to the aminobenzisoxazole

by reaction with acetohydroxamic acid and potassium tert-

butoxide, followed by heating in HCl/EtOH at 80 °C. The final

product was purified by reverse-phase HPLC with acetonitrile/

H₂O (containing 0.05% TFA) as a mobile phase and isolated

1

methyl)-1H-pyrazole-5-carboxamide as the TFA salt (35). H

NMR (DMSO-d₆) δ 10.86 (s, 1H), 8.10 (d, J ) 1.9 Hz, 1H), 7.90

(m, 2H), 7.70 (m, 4H), 7.58 (d, J ) 9.1 Hz, 1H), 7.50 (d, J )

8.4 Hz, 1H), 6.62 (bs, 1H), 4.68 (s, 2H); MS m/z 502.2 (M +

H)⁺.

1

as a TFA salt (72%). H NMR (CDCl₃) δ 9.59 (s, 1H), 7.69-

7.40 (m, 5H), 7.07-6.99 (m, 2H), 4.71 (bs, 2H), 3.62 (t, J ) 6.6

Hz, 2H), 3.32 (t, J ) 6.6 Hz, 2H), 2.00-1.84 (m, 4H); ¹⁹F NMR

(CDCl₃) δ -62.68, -123.82; HRMS calcd for C₂₃H₁₉F₄N₆O₃

503.1455, found 503.1469.

N-[4-(2-[Aminomethyl]-1H-imidazol-1-yl)-2-fluorophen-

yl]-1-(3-amino-1,2-benzisoxazol-5-yl)-3-(trifluoromethyl)-

1H-pyrazole-5-carboxamide, Bis(trifluoroacetic acid) Salt

(37). N-[4-(2-[Hydroxymethyl]-1H-imidazol-1-yl)-2-fluorophen-

yl]-1-(3-amino-1,2-benzisoxazol-5-yl)-3-(trifluoromethyl)-1H-

pyrazole-5-carboxamide 35 (0.21 g, 0.42 mmol) was dissolved

in 20 mL of CH₂Cl₂, and PBr₃(0.18 mL, 1.76 mmol) was added.

The mixture was stirred at ambient temperature under N₂for

12 h. The reaction was quenched with H₂O, and the mixture

was extracted with CHCl₃. The organic solution was washed

with H₂O and brine, dried over MgSO₄, filtered, and concen-

trated to give 0.18 g of the desired bromide. MS (ES⁺) m/z

567.3 (M + H)⁺. The bromide was dissolved in 5 mL of DMF,

and NaN₃(62.0 mg, 1.26 mmol) was added. The mixture was

heated at 50 °C under N₂for 2 h. The reaction mixture was

cooled, and H₂O was added. It was extracted with EtOAc. The

organic solution was washed with brine, dried over MgSO₄,

filtered, and concentrated to give 0.08 g of the desired azide

36 as colorless oil. MS (ES⁺) m/z 529.4 (M + H)⁺. This oil was

heated to reflux with SnCl₂(240 mg) in 10 mL of MeOH for 1

h. It was cooled, the reaction was quenched with saturated

sodium bicarbonate, and the mixture was filtered through

Celite and washed with EtOAc. The filtrate was concentrated

and purified by reverse-phase HPLC (C18 reverse-phase

column, eluted with a H₂O/CH₃CN gradient with 0.05% TFA)

to give the desired product 37 as the TFA salt (38.2 mg, 15%).

¹H NMR (DMSO-d₆) δ 10.80 (s, 1H), 8.38 (bs, 2H), 8.05 (s, 1H),

7.76 (t, J ) 8.4 Hz, 1H), 7.68-7.50 (m, 5H), 7.32 (d, J ) 8.4

Hz, 1H), 7.16 (s, J ) 1.4 Hz, 1H), 4.10 (s, 2H); HRMS calcd for

C₂₂H₁₇F₄O₂N₈(M + H) 501.1404, found 501.1410.

1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-5-[(2′-

methylsulfonyl-3-fluoro[1,1′]biphen-4-yl)aminocarbonyl]-

pyrazole, Trifluoroacetic Acid Salt (38). This compound

was prepared following the same procedures described for 11d

using 3-fluoro-2′-methanesulfonylbiphenyl-4-ylamine^7,8,11to

couple with acid 5 (87%). The resulting amide was converted

to the aminobenzisoxazole by reaction with acetohydroxamic

acid and potassium tert-butoxide, followed by heating in HCl/

EtOH at 80 °C. The final product was purified by reverse-

phase HPLC with acetonitrile/H₂O (containing 0.05% TFA) as

a mobile phase and isolated as a TFA salt (64%). ¹H NMR

(DMSO-d₆) δ 10.66 (s, 1H), 8.09 (d, J ) 1.8 Hz, 1H), 8.07 (d, J

) 1.3 Hz, 1H), 7.76 (td, J ) 7.5, 1.8, 1H), 7.71-7.66 (m, 4H),

7.58 (d, J ) 8.8 Hz, 1H), 7.41 (dd, J ) 7.9, 1.3 Hz, 1H), 7.36

(dd, J ) 11, 1.8 Hz, 1H), 7.22 (dd, J ) 8.4, 1.8 Hz, 1H), 6.58

1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-5-[(2-

fluoro-4-morpholinophenyl)aminocarbonyl]pyrazole, Tri-

fluoroacetic Acid Salt (41). This compound was prepared

following the same procedures described for 11d using aniline

16 to couple with acid 5 (45%). The resulting amide was

converted to the aminobenzisoxazole by reaction with aceto-

hydroxamic acid and potassium tert-butoxide, followed by

heating in HCl/EtOH at 80 °C. The final product was purified

by reverse-phase HPLC with acetonitrile/H₂O (containing

0.05% TFA) as a mobile phase and isolated as a TFA salt

(22%). ¹H NMR (DMSO-d₆) δ 9.39 (s, 1H), 8.06 (d, 1H), 7.77-

7.48 (m, 4H), 6.81-6.75 (m, 2H), 3.77 (t, 4H), 3.15 (t, 4H);

HRMS calcd for C₂₂H₁₉F₄N₆O₃491.1455, found 491.1454.

1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-5-[[(2-

cyano-4-(2′-methylimidazol-1′-yl)phenyl]aminocarbonyl]-

pyrazole, Bis(trifluoroacetic acid) Salt (42). This com-

pound was prepared following the same procedures described

for 11d using aniline 17 to couple with acid 5 (31%). The

resulting amide was converted to the aminobenzisoxazole by

reaction with acetohydroxamic acid and potassium tert-butox-

ide, followed by heating in HCl/EtOH at 80 °C. The final

product was purified by reverse-phase HPLC with acetonitrile/

H₂O (containing 0.05% TFA) as a mobile phase and isolated

as a TFA salt (20%). ¹H NMR (DMSO-d₆) δ 11.32 (bs, 1H),

8.23 (d, J ) 2.6 Hz, 1H), 8.12 (d, J ) 2.6 Hz, 1H), 7.95 (dd, J

) 8.8, 2.6 Hz, 1H), 7.88 (d, J ) 2.6 Hz, 1H), 7.81 (s, 1H), 7.75

(s, 1H), 7.71 (m, 2H), 7.55 (d, J ) 8.8 Hz, 1H), 6.57 (bs, 2H),

2.53 (s, 3H); HRMS calcd for C₂₃H₁₆F₃N₈O₂493.1348, found

493.1342.

1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-5-[4′-

(2′′-methylimidazol-1′′-yl)phenyl)aminocarbonyl]pyra-

zole, Bis(trifluoroacetic acid) Salt (43). To a suspension

of NaH (4.8 g, 120 mmol, prewashed with THF) in THF (100

mL) was added a solution of 1-fluoro-4-nitrobenzene (14.1 g,

100 mmol) and 2-methylimidazole (8.2 g, 100 mmol) in THF

(50 mL) at 0 °C. The mixture was heated to reflux for 16 h

and cooled to ambient temperature. To it were added EtOAc

(200 mL) and H₂O (100 mL). The organic layer was separated,

washed with H₂O and brine, dried over MgSO₄, filtered, and

concentrated to give the crude nitro compound. A solution of

the nitro intermediate in MeOH (200 mL) was placed under a

balloon filled with hydrogen gas in the presence of 5% Pd on

Factor Xa Inhibitor

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1743

mendations Based on Internal Normalized Ratio. Postgrad. Med.

J. 1994, 70 (Suppl. 1), S72-S83. (b) Hirsh, J.; Poller, L. The

International Normalized Ratio. A Guide to Understanding and

Correcting Its Problems. Arch. Intern. Med. 1994, 154, 282-

288.

carbon (1.5 g) at ambient temperature for 24 h. The mixture

was filtered through Celite, and the filtrate was concentrated

to give 4-(2-methylimidazol-1-yl)aniline (16.5 g, 95.4% for the

two steps) as a pale-yellow solid. ¹H NMR (CDCl₃) δ 7.05 (dd,

J ) 6.4, 2.1 Hz, 2H), 6.98 (d, J ) 1.1 Hz, 1H), 6.93 (d, J ) 1.1

Hz, 1H), 6.73 (dd, J ) 6.4, 2.1 Hz, 2H), 3.85 (bs, 2H), 2.31 (s,

3H); MS m/z 174 (M + H)⁺.

To a solution of 1-(3′-aminobenzisoxozol-5-yl)-3-trifluoro-

methyl-5-pyrazolecarboxylic acid 27 (240 mg, 0.77 mmol) in

DMF (5 mL) was added 4-(2′-methylimidazol-1′-yl)aniline (133

mg, 0.77 mmol), DMAP (99.5 mg, 0.79 mmol), and PyBrop (372

mg, 0.79 mmol). The resulting mixture was stirred at 60 °C

for 16 h, and then the reaction was quenched with EtOAc (100

mL) and H₂O (20 mL). The EtOAc layer was washed with 1 N

HCl (10 mL), 1 N NaOH (10 mL), H₂O (10 mL), and brine (10

mL × 3). It was dried over MgSO₄, filtered, and concentrated.

The final product 43 was purified by reverse-phase HPLC with

acetonitrile/H₂O (containing 0.05% TFA) as a mobile phase and

isolated as a TFA salt (281 mg, 63%). ¹H NMR (CD₃OD) δ 7.97

(d, J ) 0.8 Hz, 1H), 7.89 (d, J ) 9.1 Hz, 2H), 7.65 (dd, J ) 9.1,

2.2 Hz, 1H), 7.64 (d, J ) 2.2 Hz, 1H), 7.58 (d, J ) 2.2 Hz, 1H),

7.52 (d, J ) 8.8 Hz, 2H), 7.50 (d, J ) 8.4 Hz, 1H), 7.45 (s, 1H),

2.54 (s, 3H); ¹⁹F NMR (CD₃OD) δ -64.21, -77.51 (TFA); HRMS

calcd 468.1396, found 468.1381.

(2) (a) Walenga, J. M.; Jeske, W. P.; Hoppensteadt, D.; Fareed, J.

Factor Xa Inhibitors: Today and Beyond. Curr. Opin. Invest.

Drugs 2003, 4 (3), 272-281. (b) Samama, M. M. Synthetic Direct

and Indirect Factor Xa Inhibitors. Thromb. Res. 2002, 106,

V267-V273. (c) Kaiser, B. Visions & Reflections. Factor XasA

Promising Target for Drug Development. Cell. Mol. Life Sci.

2002, 59, 189-192.

(3) Mann, K. G.; Butenas, S.; Brummel, K. The Dynamics of

Thrombin Formation. Arterioscler., Thromb., Vasc. Biol. 2003,

23, 17-25.

(4) (a) Leadley, R. J., Jr. Coagulation Factor Xa Inhibition: Biologi-

cal Background and Rationale. Curr. Top. Med. Chem. 2001, 1,

151-159. (b) Hauptmann, J.; Stu¨rzebecher, J. Synthetic Inhibi-

tors of Thrombin and Factor Xa: From Bench to Bedside.

Thromb. Res. 1999, 93, 203-241.

(5) (a) Wong, P. C.; Crain, E. J.; Watson, C. A.; Zaspel, A. M.; Wright,

M. R.; Lam, P. Y. S.; Pinto, D. J.; Wexler, R. R.; Knabb, R. M.

Nonpeptide Factor Xa Inhibitors III: Effects of DPC423, An

Orally-Active Pyrazole Antithrombotic Agent, on Arterial Throm-

bosis in Rabbits. J. Pharmacol. Exp. Ther. 2002, 303, 993-1000.

(b) Wong, P. C.; Pinto, D. J.; Knabb, R. M. Nonpeptide Factor

Xa Inhibitors: DPC423, a Highly Potent and Orally Bioavailable

Pyrazole Antithrombotic Agent. Cardiovasc. Drug Rev. 2002, 20

(2), 137-152.

1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-5-[[(2,4-

bis-(2′-methylimidazol-1′-yl)phenyl]aminocarbonyl]pyra-

zole, Bis(trifluoroacetic acid) Salt (44). The title compound

was prepared in a two-step sequence by coupling 2,4-bis-(2-

methylimidazol-1-yl)phenylamine 15 with pyrazole-5-carboxyl-

ic acid 5 followed by aminobenzisoxazole formation via the

methods described for the synthesis of 11d. The final product

44 was purified by reverse-phase HPLC with acetonitrile/H₂O

(containing 0.05% TFA) as a mobile phase and isolated as a

(6) Quan, M. L.; Wexler, R. R. The Design and Synthesis of

Noncovalent Factor Xa Inhibitors. Curr. Top. Med. Chem. 2001,

1, 131-149.

(7) Pinto, D. J.; Orwat, M. J.; Wang, S.; Fevig, J. M.; Quan, M. L.;

Amparo, E.; Cacciola, J.; Rossi, K. A.; Alexander, R. S.; Wong,

P. C.; Knabb, R. M.; Luettgen, J. M.; Aungst, B. J.; Li, L.; Wright,

M.; Jona, J. A.; Wexler, R. R.; Lam, P. Y. S. The discovery of

1-[3-aminomethyl)phenyl]-N-[3-fluoro-2′-(methylsulfonyl)-1,1′-bi-

phenyl-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC-

423), a highly potent, selective and orally bioavailable inhibitor

of blood coagulation factor Xa. J. Med. Chem. 2001, 44, 566-

578.

(8) Barrett, J. S.; Davidson, A. F.; Jiao, Q. T.; Mosqueda-Garcia,

R.; Kornhauser, D. M.; Gangrade, N. K.; Jona, J. A.; Pieniaszek,

H. J., Jr. The Effect of Food, Formulation, and Dosing Duration

on the Pharmacokinetics of DPC423, a Potent Factor Xa Inhibi-

tor. J. Clin. Pharmacol. 2001, 41 (9), 42.

1

TFA salt (5%). H NMR (DMSO-d₆) δ 11.15 (bs, 1H), 8.08 (s,

1H), 8.05 (d, J ) 4.5 Hz, 1H), 7.96-7.93 (m, 4H), 7.79 (dd, J

) 2.7, 8.9 Hz, 3H), 7.61-7.55 (m, 2H), 7.48 (dd, J ) 2.5, 9 Hz,

1H), 6.60 (bs, 1H), 2.89 (s, 3H), 2.60 (s, 3H); ¹⁹F NMR (DMSO-

d₆) δ -60.75, -60.94; HRMS calcd for C₂₆H₂₁F₃O₂N₉(M + H)

548.1770, found 548.1790.

(9) Lam, P. Y. S.; Clark, C. G.; Li, R.; Pinto, D. J.; Orwat, M. J.;

Galemmo, R. A.; Fevig, J. M.; Teleha, C. A.; Alexander, R. A.;

Smallwood, A. M.; Rossi, K. A.; Wright, M. R.; Bai, S. A.; He,

K.; Luettgen, J. M.; Wong, P. C.; Knabb, R. M.; Wexler, R. R.

Structure-Based Design of Novel Guanidine/Benzamidine Mim-

ics: Potent and Orally Bioavailable Factor Xa Inhibitors as

Novel Anticoagulants. J. Med. Chem. 2003, 46, 4405-4418.

(10) Smith, K.; Jones, D. A Superior Synthesis of Diaryl Ethers by

the Use of Ultrasound in the Ullmann Reaction. J. Chem. Soc.,

Perkin Trans. 1 1992, 407-408.

(11) (a) Quan, M. L.; Pruitt, J. R.; Ellis, C. D.; Liauw, A. Y.; Galemmo,

R. A.; Stouten, P. F. W.; Wityak, J.; Knabb, R. M.; Thoolen, M.

J.; Wong, P. C.; Wexler, R. R. Design and Synthesis of Isoxazo-

line Derivatives as Factor Xa Inhibitors. Bioorg. Med. Chem.

Lett. 1997, 7, 2813-2818. (b) Quan, M. J.; Liauw, A. Y.; Ellis,

C. D.; Pruitt, J. R.; Bostrom, L. L.; Carini, D. J.; Huang, P. P.;

Harrison, K.; Knabb, R. M.; Thoolen, M. J.; Wong, P. C.; Wexler,

R. R. Design and Synthesis of Isoxazoline Derivatives as Factor

Xa Inhibitors 1. J. Med. Chem. 1999, 42, 2752-2759. (c) Quan,

M. J.; Ellis, C. D.; Liauw, A. Y.; Alexander, R.; Knabb, R. M.;

Lam, G. N.; Wong, P. C.; Wexler, R. R. Design and Synthesis of

Isoxazoline Derivatives as Factor Xa Inhibitors 2. J. Med. Chem.

1999, 42, 2760-2773.

1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-5-[[(2-

methoxy-4-(2′-methylimidazol-1′-yl)phenyl]aminocarbo-

nyl]pyrazole, Bis(trifluoroacetic acid) Salt (45). This

compound was prepared following the same procedures de-

scribed for 11d using aniline 18 to couple with acid 5 (34%).

The resulting amide was converted to the aminobenzisoxazole

by reaction with acetohydroxamic acid and potassium tert-

butoxide, followed by heating in HCl/EtOH at 80 °C. The final

product was purified by reverse-phase HPLC with acetonitrile/

H₂O (containing 0.05% TFA) as a mobile phase and isolated

as a TFA salt (46%). ¹H NMR (DMSO-d₆) δ 10.15 (bs, 1H, CF₃-

CO₂H), 8.11 (d, J ) 1.4 Hz, 1H), 7.90 (bs, 1H), 7.87 (d, J ) 1.8

Hz, 1H), 7.76 (d, J ) 1.8 Hz, 1H), 7.65 (d, J ) 1.5 Hz, 1H),

7.60 (s, 1H), 7.58 (d, J ) 8.8 Hz, 1H), 7.35 (d, J ) 1.4 Hz, 1H),

7.17 (dd, J ) 10.0, 1.5 Hz, 1H), 3.82 (s, 3H), 2.53 (s, 3H); HRMS

calcd for C₂₃H₁₉F₃O₃N₇(M + H) 498.1501, found 498.1505.

1-(3′-Aminobenzisoxazol-5′-yl)-3-trifluoromethyl-5-[[(2-

aminocarbonyl-4-(2′-methylimidazol-1′-yl)phenyl]ami-

nocarbonyl]pyrazole, Bis(trifluoroacetic acid) Salt (46).

This compound was isolated as a byproduct during the

(12) Hilgers, A. R.; Conradi, R. A.; Burton, S. Caco-2 Cell Monolayers

as a Model for Drug Transport across the Intestinal Mucosa.

Pharm. Res. 1990, 7, 902-910.

(13) Thermodynamic equilibrium aqueous solubility was measured

at ambient temperature in 0.9% saline solution.

(14) Pacific, G. M.; Viani, A. Methods of Determining Plasma and

Tissue Binding of Drugs. Clin. Pharmacokinet., 1992, 23, 449-

468.

1

preparation of 42. H NMR (DMSO-d₆) δ 12.91 (bs, 1H), 8.51

(bs, 1H), 8.49 (d, J ) 8.8 Hz, 1H), 8.15 (bs, 1H), 8.09 (dd, J )

2.2, 9.2 Hz, 2H), 7.86 (d, J ) 1.8 Hz, 1H), 7.79 (m, 1H), 7.77

(m, 2H), 7.59 (d, J ) 8.8 Hz, 1H), 7.51 (s, 1H), 6.57 (bs, 2H),

2.55 (s, 3H); HRMS calcd for C₂₃H₁₈F₃N₈O₃511.1454, found

511.1464.

(15) For in vitro coagulation assays, standard clotting assays were

performed in a temperature-controlled automated coagulation

device (Sysmex 6000, Dade-Behring). Blood was obtained from

healthy volunteers by venipuncture and anticoagulated with one-

tenth volume of 0.11 M buffered sodium citrate (Vacutainer,

Becton Dickinson). Plasma was obtained after centrifugation at

2000g for 10 min and kept on ice prior to use. An initial stock

solution of BMS-561389 at 10 mM was prepared in DMSO.

Subsequent dilutions were done in plasma. Plasma solutions

containing inhibitor were kept on ice prior to assay. Clotting

time was determined on control plasma and plasma containing

Acknowledgment. We thank Earl Crain, Gerry

Everlof, Karen Rossi, Angela Smallwood, and Carol

Watson for technical assistance.

References

(1) (a) Stein, P. D.; Grandison, D.; Hua, T. A. Therapeutic Level of

Anticoagulation with Warfarin in Patients with Mechanical

Prosthetic Heart Valves; Review of Literature and Recom-

1744 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Quan et al.

five to seven different concentrations of inhibitor. Determina-

tions at each plasma concentration were done in duplicate. The

clotting time at each concentration was compared with the

control clotting time for each pooled plasma. The prothrombin

time test was performed using Dade Thromboplastin C Plus

according to the reagent instructions. Plasma (50 µL) was

warmed to 37 °C for 3 min before adding thromboplastin (100

µL). The activated partial thromboplastin time (aPTT) was

performed using AlexinTM (Sigma Diagnostics) according to the

reagent instructions. Plasma (50 µL) was warmed to 37 °C for 1

min before adding aPTT reagent (50 µL). Three minutes later,

calcium chloride (50 µL) was added.

A. Macromolecular Crystallography; Carter, C. W., Jr., Sweet,

R. M., Eds.; Academic Press: New York, 1997; Vol. 276, pp 307-

326.

(19) Kissinger, C. R.; Gehlhaar, D. K.; Fogel, D. B. Rapid Automated

Molecular Replacement by Evolutionary Search. Acta Crystal-

logr. 1999, D55, 484-491.

(20) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.

N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data

Bank. Nucleic Acids Res. 2000, 28, 235-242.

(21) For enzyme affinity assays, all enzyme K_ivalues were obtained

from human purified enzymes. All assays were run in microtiter

plates using a total volume of 250 µL in 0.1 M sodium phosphate

buffer containing 0.2 M NaCl and 0.5% poly(ethylene glycol)

6000 at pH 7.0. The compounds were run at 10, 3.16, 1.0, 0.316,

0.1, 0.0316, 0.01, and 0.00316 µM. Plates were read for 30 min

at 405 nm. Rates were determined for the controls (no inhibitor)

and for the inhibitors. Percent enzyme activity was determined

from these rates and used in the following formula to determine

K_i: K_i) (1000)(inhibitor concentration)/{[(K_m+ S) - (S)(ACT)]/

[(ACT)(K_m)] - 1}, where S is the substrate concentration and

ACT is the % enzyme activity for inhibitor. All compounds were

tested in duplicate studies and were compared with the same

internal standards. The intraassay and interassay variabilities

are 5% and 20%, respectively. These assays are described in

detail in refs 22 and 23.

(16) Pruitt, J. R.; Pinto, D. J.; Galemmo, R. A.; Alexander, R. S.; Rossi,

K. A.; Wells, B. L.; Drummond, S.; Bostrom, L. L.; Burdick, D.;

Bruckner, R. Chen, H.; Smallwood, A.; Wong, P. C.; Wright, M.

R.; Bai, S.; Luettgen, J. M.; Knabb, R. M.; Lam, P. Y. S.; Wexler,

R. R. Discovery of 1-(2-Aminomethylphenyl)-3-trifluoromethyl-

N-[3-fluoro-2′-(aminosulfonyl)[1,1′-biphenyl)]-4-yl]-1H-pyrazole-

5-carboxyamide (DPC 602), a Potent, Selective, and Orally

Bioavailable Factor Xa Inhibitor. J. Med. Chem. 2003, 46, 5298-

5315.

(17) (a) Wong, P. C.; Quan, M. L.; Crain, E. J.; Watson, C. A.; Wexler,

R. R.; Knabb, R. M. Nonpeptide Factor Xa Inhibitors: I. Studies

with SF303 and SK549, a New Class of Potent Antithrombotics.

J. Pharmacol. Exp. Ther. 2000, 292, 351-357. (b) Wong, P. C.;

Crain, E. J.; Knabb, R. M.; Meade, R. P.; Quan, M. L.; Watson,

C. A.; Wexler, R. R.; Wright, M. R.; Slee, A. M. Nonpeptide Factor

Xa Inhibitors: II. Antithrombotic Evaluation in a Rabbit Model

of Electrically Induced Carotid Artery Thrombosis. J. Pharmacol.

Exp. Ther. 2000, 295, 212-218. (c) Wong, P. C.; Watson, C. A.;

Crain, E. J.; Zaspel, A. M.; Luettgen, J. M.; Bai, S. M.; Lam, P.

Y.; Quan, M. L.; Wexler, R. R.; Knabb, R. M. Antithrombotic

Effects of Razaxaban, an Orally-Active Factor Xa Inhibitor, in

Rabbit Models of Thrombosis. Blood 2003, 102, 813a.

(22) Knabb, R. M.; Kettner, C. A.; Timmermans, P. B. M. W. M.;

Reilly, T. M. In vivo characterization of a new synthetic thrombin

inhibitor. Thromb. Haemostasis 1992, 67, 56-59.

(23) Kettner, C. A.; Mersinger, L. J.; Knabb, R. M. The selective

inhibition of thrombin by peptides of boroarginine. J. Biol. Chem.

1990, 265, 18289-18297.

(24) These two compounds were studied in two separate N-in-1 dog

pharmacokinetics experiments in which cassettes of 10 com-

pounds were dosed together to each dog (n ) 2).

(18) Otwinowski, Z.; Minor, W. Processing of X-ray Diffraction Data

Collected in Oscillation Mode. In Methods in Enzymology. Part

JM0497949

Article Doi

Discovery of 1-(3′-aminobenzisoxazol-5′-yl)-3-trifluoromethyl- N-[2-fluoro-4-[(2′-dimethylaminomethyl)imidazol-1-yl]phenyl] -1H-pyrazole-5-carboxyamide hydrochloride (razaxaban), a highly potent, selective, and orally bioavailable factor Xa inhibitor

DOI: 10.1021/jm0497949

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Full text of DOI:10.1021/jm0497949

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