7-Fluoroindazoles as potent and selective inhibitors of factor Xa

282

J. Med. Chem. 2008, 51, 282–297

7-Fluoroindazoles as Potent and Selective Inhibitors of Factor Xa^†

Yu-Kai Lee, Daniel J. Parks, Tianbao Lu, Tho V. Thieu,^‡Thomas Markotan, Wenxi Pan, David F. McComsey,

Karen L. Milkiewicz,^‡Carl S. Crysler, Nisha Ninan, Marta C. Abad, Edward C. Giardino, Bruce E. Maryanoff,

Bruce P. Damiano, and Mark R. Player*

Johnson & Johnson Pharmaceutical Research and DeVelopment, Welsh and McKean Roads, Spring House, PennsylVania 19477-0776

ReceiVed September 26, 2007

We have developed a novel series of potent and selective factor Xa inhibitors that employ a key

7-ﬂuoroindazolyl moiety. The 7-ﬂuoro group on the indazole scaffold replaces the carbonyl group of an

amide that is found in previously reported factor Xa inhibitors. The structure of a factor Xa cocrystal containing

7-ﬂuoroindazole 51a showed the 7-ﬂuoro atom hydrogen-bonding with the N–H of Gly216 (2.9 Å) in the

peptide backbone. Thus, the 7-ﬂuoroindazolyl moiety not only occupied the same space as the carbonyl

group of an amide found in prior factor Xa inhibitors but also maintained a hydrogen bond interaction with

the protein’s ꢀ-sheet domain. The structure–activity relationship for this series was consistent with this

ﬁnding, as the factor Xa inhibitory potencies were about 60-fold greater (∆∆G ≈ 2.4 kcal/mol) for the

7-ﬂuoroindazoles 25a and 25c versus the corresponding indazoles 25b and 25d. Highly convergent synthesis

of these factor Xa inhibitors is also described.

Introduction

Thromboembolic diseases remain a leading cause of

morbidity and mortality in developed countries, such as the

U.S.^1–3Anticoagulants provide effective treatment for venous

or arterial thromboembolism, e.g., prevention of postoperative

deep venous thrombosis (DVT^a)and pulmonary embolism

(PE), prevention of stroke during atrial ﬁbrillation, and

treatment of acute coronary syndrome (ACS). Use of the

current oral anticoagulant, warfarin, is plagued by its slow

vitamin K-dependent antagonism, by drug-drug interactions,

and by drug-food interactions, which results in the need for

careful patient monitoring.^4,5Since fast-acting heparin and

direct thrombin inhibitors currently are only available for

parenteral administration,^4,6the development of new oral

anticoagulants is still an unmet medical need.

Figure 1. Compounds 1 and 2 share a pyrazolyl carboxamide core.

elective knee arthroplasty in a phase II clinical study.²⁰However,

since compound 2 and its analogues contain amide bonds, there

was a concern that the amides would be hydrolyzed under in

vivo conditions to release potentially mutagenic biarylamines

(Figure 1).²¹Indeed, as some of these biarylamines have been

found to be mutagenic,²²much of the lead optimization efforts

have been directed toward modiﬁcations of the carboxamide

into a bicyclic core to reduce the susceptibility of in vivo

hydrolysis.^21–25While these designs have made the molecules

less susceptible to hydrolysis, they did not completely eliminate

the possibility of in vivo hydrolytic cleavage.²³Recently, an

effort to eliminate this potential liability, by replacing the

biarylamines with nonaromatic P4 residues, was reported.²⁶

However, an unambiguous way to eliminate such a risk is to

remove the carboxamide core entirely (Figure 2). In our

thrombin inhibitor program, we have successfully replaced the

carbonyl group of the pyrazinone core in known thrombin

inhibitors with a ﬂuorobenzene moiety. This straightforward

change not only retained inhibitory potency but also improved

metabolic stability.^27–30Similar success has also been reported

in factor VIIa inhibitor programs via replacement of the

pyrazinone core with arenes.³¹In both cases, X-ray crystal-

lographic studies showed that these novel ﬂuorobenzene inhibi-

tors occupied the active sites in the same orientation and

maintained the key hydrogen-bonding interactions of the pyrazi-

none carbonyl groups with Gly216 of the protein’s ꢀ-sheet

domain. In this article, we report results from our studies

involving the application of the same principle to the design of

Factor Xa (EC 3.4.21.6) is a unique serine protease in the

blood coagulation cascade in that it is poised at a common

junction where it is activated by both the intrinsic (contact

activation) and extrinsic (tissue factor) pathways. In contrast to

the multifunctional role that thrombin plays in the cascade, factor

Xa only converts prothrombin to thrombin but does not affect

the existing level of circulating thrombin. It has been proposed

that factor Xa inhibitors may exhibit a reduced bleeding risk

and offer a superior safety/efﬁcacy proﬁle with respect to

thrombin inhibitors.^7–10Therefore, in recent years factor Xa has

emerged as an attractive target for the development of new

anticoagulants.^11–17

Compound 1 (DPC423)¹⁸and 2 (razaxaban)¹⁹were reported

to be highly selective, potent, and orally bioavailable small-

molecule factor Xa inhibitors. In addition, compound 2 showed

good oral efﬁcacy in treatment of venous thromboembolism after

†

The coordinates of 51a in the human factor Xa active have been

deposited (PDB code 2RA0).

* To whom correspondence should be addressed. Phone: 610-458-6980.

Fax: 610-458-8249. E-mail: mplayer@prdus.jnj.com.

‡

Present address: Cephalon Inc., 145 Brandywine Parkway, West

Chester, PA 19380.

^aAbbreviations: DVT, deep venous thrombosis; PE, pulmonary embolism;

ACS, acute coronary syndrome; EC, enzyme commission number; aPTT,

activated partial thromboplastin time; HLM, human liver microsomal.

10.1021/jm701217r CCC: $40.75

2008 American Chemical Society

Published on Web 12/27/2007

7-Fluoroindazoles as FXa Inhibitors

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 283

Figure 2. Fluorobenzenes replace carbonyl-based cores in serine protease inhibitors.

a novel series of factor Xa inhibitors, which possess a 7-ﬂu-

oroindazole unit in place of the pyrazolylcarboxamide unit.

20 were coupled with the boronates 23a,b using Suzuki-Miyaura

reaction conditions to yield 24a-d. Nitriles 24a-d were

reduced with Raney Ni under a pressurized hydrogen atmo-

sphere to yield benzylamines 25a-d.

Chemistry

The factor Xa inhibitors 29a-c in Table 2 were prepared as

outlined in Scheme 4. Boronates 26 were generated from

indazoles 16a using the Miyaura boronylation protocol³⁵and

coupled to lactam 23c using Suzuki-Miyaura conditions to yield

indazole 28a. Indazoles 16b-c were coupled to boronate 23d

using similar conditions to yield intermediates 28b-c. Treatment

of the intermediates 28a-c with acetohydroxamic acid under

basic conditions yielded aminobenzoxazoles 29a-c.

The 7-ﬂuoroindazole scaffolds 16a-d were synthesized as

shown in Scheme 1. Commercially available diﬂuorobenzal-

dehyde 3a was protected with ethylene glycol to form dioxolane

4a. Diﬂuorobenzene 4a was treated with lithium 2,2,6,6-

tetramethylpiperidide at –78 °C and quenched with iodine to

yield diﬂuorobenzene 5.³²The protecting group on the carbonyl

group of 5 was removed under acidic conditions to yield

diﬂuoroiodobenzaldehyde 7. Bromodiﬂuoroacetophenone 8 was

prepared in a similar fashion from the commercially available

acetophenone 3b. Benzaldehyde 7 was reacted with trimethyl-

(triﬂuoromethyl)silane and followed by oxidation with the

Dess-Martin periodinane to yield triﬂuoroacetophenone 10. In

the case of ketocarboxamide 13, benzaldehyde 7 was reacted

with trimethylsilyl cyanide to yield the cyanohydrin 11. The

cyanohydrin 11 was hydrolyzed to the corresponding ꢀ-hy-

droxycarboxamide 12 using strongly acidic conditions and then

oxidized to the desired ketoacetamide 13. The carbonyl moieties

8, 10, and 13 were condensed with arylhydrazine 14a³³to afford

arylhydrazones 15a-c. Acetophenone 8 was also reacted with

arylhydrazine 14b to yield arylhydrazone 15d. The arylhydra-

zones 15a-d were cyclized under basic conditions to yield the

7-ﬂuoroindazoles 16a-d.³⁴The corresponding nonﬂuorinated

indazole analogue 20 was prepared in four steps in a similar

fashion. Aldehyde 17 was treated with methylmagnesium

bromide, followed by the oxidation of the secondary alcohol to

ketone 18. Acetophenone 18 was reacted with arylhydrazine

14b to form arylhydrazone 19, which was then cyclized under

basic conditions to yield indazole 20.

The syntheses of the biaryl P3-P4 moieties found in the

factor Xa inhibitors 51a-l in Table 3 are shown in Schemes 5

and 6. The preparations of P3-P4 biaryls 49b and 49d-g are

outlined in Scheme 5. The pyrazole analogue 49b was prepared

by protection of commercially available pyrazole 36 with SEM-

Cl, followed by Suzuki coupling with 4-bromo-3-ﬂuoroben-

zeneboronic acid 30 and reductive amination with dimethy-

lamine. Commercially available 2-pyridinecarboxaldehyde (31)

was treated with n-butyllithium in the presence of N,N,N′-

trimethylethylenediamine and quenched with iodine to form the

3-iodopyridine 32 as described in the literature.³⁶Suzuki

coupling of 4-bromo-3-ﬂuorobenzeneboronic acid 30 with

iodopyridine 32 followed by reductive amination with dimethy-

lamine afforded the biaryl 49f. Similarly, biaryls 49d, 49e, and

49g were prepared from bromopyridines 35 and 34 and

iodopyridine 33,³⁶respectively. The syntheses of P3-P4 biaryls

49a, 49c, and 49h-l are described in Scheme 6. Reductive

amination of commercially available 2-imidazolecarboxaldehyde

with dimethylamine yielded imidazole 43.¹⁹Ullmann coupling

of imidazole 43 and 1-bromo-2-ﬂuoro-4-iodobenzene 38 af-

forded biaryl 49a. Suzuki coupling between iodobenzene 38

and boronic acid 45 followed by reductive amination of the

product with dimethylamine yielded biphenyldimethylamine

49c. Compound 49h was prepared from Buchwald-Hartwig

amination between 4-(methylamino)pyridine 44 and iodoben-

zene 38. Compound 49i was prepared from Buchwald-Hartwig

The syntheses of the factor Xa inhibitors 25a-d in Table 1

are described in Schemes 2 and 3. Anilines 21 and 22 were

coupled to ω-bromoalkylcarboxylic acid chlorides, and the

amides obtained were cyclized under basic conditions to afford

lactams 23a-c. Bromolactam 23c was converted to boronate

23d using the Miyaura borylation protocol.³⁵Indazoles 16d and

284 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2

Lee et al.

Scheme 1. Synthesis of 7-Fluoroindazole and the Corresponding Indazole Scaffolds^a

^aReagents and conditions: (a) ethylene glycol, PhH, PPTS, reﬂux; (b) 2,2,6,6-tetramethylpiperidine, n-BuLi, THF, -78 °C, then Br₂; (c)

2,2,6,6-tetramethylpiperidine, n-BuLi, THF, -78 °C, then I₂; (d) 2 M HCl, THF, reﬂux; (e) TMSCF₃, catalytic TBAF, THF, 0 °C to room temp, then

aqueous HCl; (f) TMSCN, catalytic TBAF, THF, 0 °C to room temp, then aqueous HCl; (g) Dess-Martin; (h) concentrated HCl, HCl_(g), 1,4-dioxane; (i)

MnO₂, CH₃CN; (j) EtOH, TSA; (k) K₂CO₃, DMF, 100 °C, 2 h; (l) CH₃MgBr, THF; (m) CrO₃, CH₂Cl₂.

Scheme 2. Synthesis of P3-P4 Lactams 23^a

Table 1. Importance of 7-Fluoro Substitution on Factor Xa Potency

^aReagents and conditions: (a) TEA, CH₂Cl₂; (b) NaH, DMF; (c)

bis(pinacolato)diboron, Pd(dppf)Cl₂·CH₂Cl₂, KOAc, 1,4-dioxane, 90 °C.

between the product aniline 41 with iodobenzene 38. The pyridyl

analogue 49j was prepared in a similar fashion using 3-bro-

mopyridine 40 as starting material. Compound 49k and 49l were

prepared via the coupling of 4-chloropyridine 46 with N,N-

dimethylaminoalkyldiamines, followed by Buchwald-Hartwig

amination between the product aniline 47 and 48 with iodo-

benzene 38.

The ﬁnal assembly of factor Xa inhibitors 51a-l in Table 3

is outlined in Scheme 7. The biaryl P4 moieties 49a-l were

^aK_ivalues are averaged from two determinations (n ) 2). ^bK_ivalues of

coupled to boronate 27 under Suzuki-Miyaura conditions to

e10000 nM are averaged from two determinations (n ) 2).

form intermediates 50a-l. Compound 50a and 50c-l were

amination between bromobenzene 39 and N,N-dimethylethyl-

enediamine, followed by another Buchwald-Hartwig amination

treated with acetohydroxamic acid under basic conditions to

yield aminobenzisoxazoles 51a and 51c-l, respectively.¹⁹In

7-Fluoroindazoles as FXa Inhibitors

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 285

Scheme 3. Synthesis of Compounds 25a-d^a

^aReagents and conditions: (a) Pd(PPh₃)₄, 2 M Na₂CO₃, EtOH, PhCH₃, 100 °C; (b) Raney Ni, H₂(50 psi), EtOH.

Table 2. Effect of C-3 Substitution of the Indazole on Factor Xa

Potency

The X-ray crystallographic data of these protein–ligand com-

plexes revealed that the distance between the aromatic ﬂuorine

to the amide nitrogen in Gly216 of the serine protease varied

from 3.4 to 3.2 Å and that the ﬂuorinated ligand adopted a

similar orientation in the protein–ligand complex as did the

carbonyl counterpart. However, in both cases no systematic

study was carried out to determine the nature of the interaction

between the aromatic ﬂuorine and the N–H of Gly216.

Therefore, early in our program a pair of the 7-ﬂuoroindazoles

25a and 25c and the corresponding indazole analogues 25b and

25d were prepared to assess the role of the 7-ﬂuoro atom in the

scaffold, and the assay results are summarized in Table 1. The

factor Xa inhibitory activities of 7-ﬂuoroindazoles 25a (fXa K_i

) 223 nM) and 25c (fXa K_i) 124 nM) are about 60 times

more potent (∆∆G ≈ 2.4 kcal/mol) than the corresponding

indazoles 25b (fXa K_i> 14400 nM) and 25d (fXa K_i) 6850

nM). The comparison of these two pairs of analogues demon-

strated the importance of the ﬂuorine at the 7-position on binding

afﬁnity to factor Xa. The differences in the inhibitory potencies

of the ﬂuorinated and nonﬂuorinated compounds are also much

higher than reported (60-fold versus 6-fold). The binding energy

difference derived (∆∆G ≈ 2.4 kcal/ mol) from comparison of

the inhibitory potencies of these two pairs of analogues is in

line with a plausible hydrogen bond of the carbon-bound

ﬂuorine.^38,41

Although 7-ﬂuoroindazoles 25a,c were active against factor

Xa, the potency was not optimal and the selectivity versus

trypsin was only about 4-fold and 10-fold, respectively. Table

2 shows that the factor Xa potency was increased by about 5-fold

and the selectivity against trypsin and thrombin was greatly

improved when the P1 benzylamine was changed to an

aminobenzisoxazole, along with the addition of a ﬂuorine at

the 2-position of P3 as in compound 29a (fXa K_i) 26 nM).

This improvement in potency and selectivity resulted from the

larger size of the aminobenzisoxazole P1, which complemented

more favorably with the larger S1 pocket found in factor Xa as

opposed to other serine proteases.¹⁹While the replacement of

the methyl group at the C-3 position of the indazole with a

triﬂuoromethyl group in 29b (fXa K_i) 22.9 nM) did not

improve the factor Xa potency further, the potency of 29c (fXa

K_i) 6.4 nM) was increased by about 4-fold when the C-3

substitutent was changed to an amide group capable of

hydrogen-bonding to Glu146, which was conﬁrmed later by

X-ray crystallography.

compd

R

fXa K_i(nM)^athrombin K_i(nM)^btrypsin K_i(nM)^b

29a

29b

29c

CH₃

CF₃

CONH₂

26

22.9

6.4

>15200

5486

>10000

^aK_ivalues are averaged from multiple determinations (n g 2), and the

standard deviations are <5% of the mean. ^bK_ivalues are obtained from

single determination (n ) 1).

the case of pyrazole 50b, the SEM protecting group was ﬁrst

removed with TFA before the conversion to aminobenzisoxazole

51b.

Results and Discussion

Fluorinated compounds have become increasingly important

in pharmaceutical research, whether used to improve metabolic

stability, modulate physiochemical properties, or guide protein–

ligand interactions.³⁷While a number of reports have been

published on interactions between proteins and ﬂuorinated

ligands over the past decade,^37,38the extent of ﬂuorine participa-

tion in hydrogen-bonding interactions remains an ongoing

debate.^38–40Until recently, it was believed that C(sp³)-F is a

better hydrogen bond acceptor than C(sp²)-F.^38,41Not surpris-

ingly, the few reported examples of hydrogen bonding of

ﬂuorinated compounds in protein–ligand complexes were mostly

of the C(sp³)-F type.^40,42While many favorable interactions

of aromatic ﬂuorine in protein–ligand complexes have been

described, no systematic study has been carried out to understand

more fully the nature of these interactions. Böhm et al. have

reported a systematic comparison between a pair of aryl

molecules that differ by one ﬂuorine atom and their respective

thrombin inhibitory potency.³⁷In this case, the ﬂuorinated

analogue was a moderately good thrombin inhibitor (K_i) 260

nM) but only 6-fold (∆∆G ≈ 1 kcal/mol) more potent than the

corresponding nonﬂuorinated compound (K_i) 1600 nM). The

distance between the ﬂuorine and the amide nitrogen of Gly216

of thrombin was 3.47 Å. They concluded that the evidence

pointed toward a very weak hydrogen bond or a favorable

dipolar interaction. Replacements of the carbonyl groups of

pyrazinones with ﬂuorobenzenes in inhibitors of thrombin or

factor VIIa have also been recently published.^28,30,31The ﬂuorine

atom in each of these active ﬂuorinated inhibitors certainly

provided favorable interactions in the protein–ligand complex.

The factor Xa inhibitory activity of 29c (fXa K_i) 6.4 nM)

differs from compound 2 (fXa K_i) 1.6, 0.19 nM reported)¹⁹

about 4-fold while also demonstrating good selectivity against

trypsin and thrombin. However, the in vitro anticoagulant

activity of 29c proved disappointing, as shown by the plasma

concentration required to increase aPTT by 2-fold (2-fold

aPTT_EC50) value (Table 3). Because the 7-ﬂuoroindazole

286 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2

Scheme 4. Synthesis of Compounds 29a-c^a

Lee et al.

^aReagents and conditions: (a) Pd(dppf)Cl₂·CH₂Cl₂, 1,4-dioxane, KOAc, 90 °C; (b) 23c, Pd(dppf)Cl₂·CH₂Cl₂, K₂CO₃, DMF, 95 °C; (c) 23d, Pd(PPh₃)₄,

aqueous Na₂CO₃, EtOH, PhCH₃, 80 °C; (d) AcNHOH, K₂CO₃, DMF, H₂O.

scaffold, consisting of an aminobenzisoxazole moiety as P1,

primary carboxamide at the C-3 position of the indazole, and

the 2-ﬂuorophenyl moiety as P3 showed good potency and

selectivity, we redirected our efforts to ﬁnd a suitable P4

group (Table 3). The initial choice was to install a dimethy-

laminomethylimidazole P4 moiety as found in compound 2.

Surprisingly, the factor Xa potency of 51a (fXa K_i) 15.9

nM), a close analogue of compound 2, was lower than 29c

(fXa K_i) 6.4 nM). However, the 2-fold aPTT_EC50for 51a

was 4-fold lower than 29c. In a human liver microsomal

(HLM) stability assay, 29c showed good stability; therefore,

the poor stability of 51a and 2 may be due to the dimethy-

laminoimidazole group at P4, which both compounds share.

We prepared dimethylaminomethylpyrazole 51b and were

surprised to ﬁnd that the rearrangement of the nitrogen atoms

had improved the factor Xa K_iby more than 10-fold to 1.4

nM while also improving the 2-fold aPTT_EC50of 51b (10.1

µM), similar to that observed with compound 2 (13.5 µM).

Despite these improvements, the issues of poor permeability

(Caco-2 P_app) 1.10 × 10^-6cm/s) and fair microsomal

stability (54% parent compound remaining after 10 min)

precluded the advancement of 51b.

An X-ray crystal structure of 51a in the human factor Xa

active site (PDB code 2RA0) was obtained to a resolution

of 2.3 Å (Figure 3). The overall binding mode is similar to

what was reported for 1¹⁸and 2.¹⁹As anticipated, the ﬂuorine

at the 7-position of the indazole forms a hydrogen bond with

the N–H of Gly216 (2.90 Å). This is close to the distance

observed between the 5-carboxamide carbonyl in compound

2 and Gly216 (3.04 Å).¹⁹The aminobenzisoxazole was also

bound in S1 as predicted, and the amino group formed a

hydrogen bond with the carboxylic acid of Asp189 (2.77 and

2.96 Å) and the carbonyl group of the Ala190 (3.37Å). The

N–H of the amide at the C-3 position of the indazole

interacted with the carbonyl group Glu146 (2.56 Å). The

ﬂuorine at the 2-position of the P3 is within hydrogen bond

distance to the hydroxy group of Try99 (3.28 Å). The distance

between the N-3 nitrogen of the imidazole P4 group and

Glu97 was reduced to 2.58 Å compared to 3.66 Å in

compound 2. Overall, these interactions showed that the

7-ﬂuoroindazole 51a bound into the active site in a highly

complementary fashion. The combination of the X-ray

crystallographic structure and the structure–activity relation-

ship data for these 7-ﬂuoroindazoles demonstrates, for the

ﬁrst time unequivocally, hydrogen bonding between a

C(sp²)-F and N–H in a peptide backbone.

We then investigated different dimethylaminoaryl moieties

(51c-g) as P4 substituents and found that the presence and

the position of the pyridyl ring nitrogen atom were also

crucial to the factor Xa activities, as well as to the in vitro

anticoagulation parameters. The nitrogens at the 6- or

5-positions in 51d (fXa K_i) 2255 nM) and 51e (fXa K_i)

147 nM) were not well tolerated. Their factor Xa inhibitory

potencies were poorer than the corresponding phenyl ana-

logue 51c (fXa K_i) 8.1 nM). However, the nitrogens at the

4- or 3-position of the pyridyl system in 51f (fXa K_i) 3.0

nM) and 51 g (fXa K_i) 4.4 nM) afforded better factor Xa

potency than the corresponding phenyl group 51c. In addition,

3-pyridyl analogue 51g showed a signiﬁcantly better in vitro

anticoagulation parameter (2-fold aPTT_EC50) 4.4 µM) when

compared to the other two analogues 51c (2-fold aPTT_EC50

) 47 µM) and 51f (2-fold aPTT_EC50) 55.1 µM). While the

microsomal stabilities of phenyl 51c and 3-pyridyl 51g

analogues were greatly improved over 4-pyridyl analogue

51f, the permeability of 51f was found to be higher than the

phenyl analogue 51c and much higher than the 3-pyridyl

analogue 51g.

In an attempt to optimize the microsomal stability and the in

vitro 2-fold aPTT_EC50, we replaced the P4 group with the

N-alkylaminopyridine moiety found in 51h-l. Compound 51h

(fXa K_i) 4.1 nM) showed that the dimethylaminomethyl groups

in these analogues are not essential to the factor Xa activities

and the in vitro anticoagulation parameter when compared to

51k (fXa K_i) 4.9 nM) and 51l (fXa K_i) 5.2 nM). However

the permeability was improved by about 4-fold with the presence

of the dimethylamino “tail” as in 51l (Caco-2 P_app) 5.5 ×

10^-6cm/s), when it is compared to that of the methylamine

51h (Caco-2 P_app) 1.4 × 10^-6cm/s). The length of the

dimethylamino chain in 51k (fXa K_i) 4.9 nM) and 51l (fXa

K_i) 5.2 nM) did not affect the factor Xa activity. However,

extension of the pendent dimethylamino group improved in vitro

anticoagulation parameters about 2-fold in 511 compared to 51k.

The importance of the presence and location of the nitrogen

atom on the pyridine ring was demonstrated once again in

7-Fluoroindazoles as FXa Inhibitors

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 287

Table 3. Effect of P4 Substitutents on Factor Xa Potency, in Vitro Anticoagulant Activity in Human Plasma, HLM Stability, and Permeability

^aK_ivalues are averaged from multiple determinations (n g 3), and the standard deviations are <14% of the mean. ^bK_ivalues of <10000 nM are

averaged from two determinations (n ) 2). ^cHuman liver microsomal stability: percentage of compound remaining after 10 min in the assay. ^dData in

parentheses were reported in ref 19.

51h-k. The presence of the nitrogen atom accounted for a

3-fold increase in the factor Xa activities as shown in 51i (fXa

K_i) 690 nM) and 51j (fXa K_i) 231 nM). The movement of

the nitrogen atom from the 3-position to the 4-position in 51k

(fXa K_i) 4.9 nM) enhanced the factor Xa activity by 46-fold.

We also found that the potency, anticoagulation parameters, and

permeability of 51l were similar to those of compound 2 and

with improvements in microsomal stability.

Conclusion

Novel factor Xa inhibitors that use a 7-ﬂuoroindazole as a

carbonyl group replacement were developed. Comparison of the

288 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2

Scheme 5. Synthesis of P4 Biaryls 49b and 49d-g^a

Lee et al.

^aReagents and conditions: (a) (i) n-BuLi, N,N,N′-trimethylethylenediamine, -42 °C; (ii) iodine; (b) PdCl₂(PPh₃)₂, DME, 2 M Na₂CO₃, 90 °C; (c) (CH₃)₂NH,

NaBH(OAc)₃, DMF; (d) SEM-Cl, NaH, DMF.

Scheme 6. Synthesis of P4 Biaryls 49a, 49c, and 49h-l^a

^aReagents and conditions: (a) Pd₂(dba)₃, XANTPHOS, t-BuONa, 1,4-dioxane, 140 °C; (b) CuI, Cs₂CO₃, DMF, 125 °C; (c) Pd(PPh₃)₄, aqueous Na₂CO₃,

EtOH/PhCH_3,reﬂux; (d) (CH₃)₂NH, NaBH(OAc)₃, DMF; (e) 1-pentanol, 140 °C.

structure–activity relationship of 7-ﬂuoroindazoles with their

corresponding nonﬂuorinated indazoles unequivocally demon-

strated that the interaction of the 7-ﬂuoro group in the series

(∆∆G ≈ 2.4 kcal/ mol) with factor Xa was beyond a simple

van der Waals complex while X-ray crystallography also

conﬁrmed the interaction between the 7-ﬂuoro group and the

N–H of Gly216 of factor Xa (2.9 Å). The binding orientation

of the 7-ﬂuoroindazole was similar to the carboxamides that it

replaced. Our data also support the notion of ﬂuorobenzene as

a good hydrogen bond acceptor. Several 7-ﬂuoroindazoles, such

as 51g and 51l, were found to be very potent, highly selective

inhibitors of factor Xa.

Experimental Section

Chemistry. Reagents and solvents were obtained from com-

mercial suppliers and used without further puriﬁcation. Flash

chromatography was performed using Fisher Chemical silica gel

sorbent (230–400 mesh, grade 60). Preparative thin-layer chroma-

tography was performed on Analtech silica gel GF plates (1000

µm or 2000 µm, 20 cm × 20 cm). ¹H NMR spectra were recorded

on a Bruker B-ACS-120 (400 MHz) spectrophotometer at room

7-Fluoroindazoles as FXa Inhibitors

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 289

Scheme 7. Syntheses of Compounds 51a-l ^a

^aReagents and conditions: (a) Pd(dppf)Cl₂·CH₂Cl₂, K₂CO₃, DMF, 95 °C; (b) AcNHOH, K₂CO₃, DMF, H₂O; (c) TFA, CH₂Cl₂, EtOH.

2-(2,3-Diﬂuorophenyl)[1,3]dioxolane (4a). A mixture of 2,3-

diﬂuorobenzaldehyde (10 g, 70.4 mmol), ethylene glycol (5.1 mL,

91.5 mmol), and pyridinium p-toluenesulfonate (1.77 g, 7 mmol)

in benzene (60 mL) was reﬂuxed overnight using a Dean–Stark

apparatus. The mixture was allowed to cool to room temperature,

and cold water was added to the mixture. The two layers were

separated, and the aqueous layer was thrice extracted with hexanes.

The organic layers were combined and washed with brine. The

solution was dried with anhydrous sodium sulfate, ﬁltered, and

concentrated under reduced pressure to yield a colorless oil (12.9

g, 99%) as the desired product. ¹H NMR (400 MHz, CDCl₃) δ

7.28 (tdd, J ) 1.5, 7.6, 5.9 Hz, 1H), 7.17 (dddd, J ) 9.3, 8.2, 7.5,

1.8 Hz, 1H), 7.09 (ddt, J ) 4.8, 1.5, 8.2 Hz, 1H), 6.09 (s, 1H),

4.21–4.11 (m, 2H), 4.10–4.04 (m, 2H).

2-(2,3-Diﬂuoro-4-iodophenyl)[1,3]dioxolane (5). To a solution

of 2,2,6,6-tetramethylpiperidine (16.6 mL, 98.5 mmol) in anhydrous

THF (100 mL) under argon at –78 °C was added n-butyllithium

(33.3 mL, 83.4 mmol, 2.5 M in hexanes) dropwise at a rate

maintaining the internal temperature below –60 °C. After the

mixture was held at –78 °C for 15 min, a solution of 2-(2,3-

diﬂuorophenyl)[1,3]dioxolane (4a) (14.1 g, 75.8 mmol) in anhy-

drous THF (50 mL) was added dropwise to the mixture to maintain

the internal temperature below –65 °C. After being held at –78 °C

for 15 min, the lithiated diﬂuorobenzene solution was then

transferred to a solution of iodine (28.9 g, 113.7 mmol) in anhydrous

THF (80 mL) at –78 °C using a cannula. The resulting mixture

was stirred at –78 °C for 1 h and then allowed to warm to room

temperature slowly. The mixture was quenched with water and then

poured into a solution of sodium thiosulfate (100 mL, 30% w/v).

The aqueous layer was extracted with ethyl acetate (3 × 50 mL).

The organic layers were combined and dried with anhydrous

magnesium sulfate, ﬁltered, and concentrated under reduced pres-

sure to yield a pale-orange liquid. This was used in the next step

Figure 3. X-ray crystal structure of compound 51a in the factor Xa

active site (PDB code 2RA0).

temperature. Chemical shifts are given in ppm (δ), coupling

constants (J) are in hertz (Hz), and signals are designed as follows:

(s) singlet, (d) doublet, (t) triplet, (q) quartet, (m) multiplet, (br s)

broad singlet, (dd) doublet of doublet, (dt) doublet of triplet, (dq)

doublet of quartet, (tm) triplet of multiplet, (ddd) doublet of doublet

of doublet, (ddt) doublet of doublet of triplet, (dddd) doublet of

doublet of double of doublet. LC-MS was performed on a system

consisting of an electrospray source on a Finnigan LCQ ion trap

mass spectrometer, a SEDEX 75C evaporative light scattering

detector, a Shimadzu LC-10ADvp binary gradient pumping system,

a Gilson 215 conﬁgured as an autosampler, and a Princeton

Chromatography HTS HPLC column (5 µm, 50 mm × 3.0 mm).

Accurate mass measurements were performed using either of two

methods: (1) One method is a Micromass (Manchester, U.K.)

Autospec E OA-TOF high-resolution magnetic sector mass spec-

trometer tuned to a resolution of >5000 using the 5% valley

deﬁnition. The ions were produced in a fast atom bombardment

(FAB) ion source at an accelerating voltage of 8kV. Linear voltage

scans at 33 Da/s were collected to include the protonated sample

ion and two polyethylene glycol (PEG) or PEG monomethyl ether

ions, which were used as internal reference standards. The molecular

mass was calculated using a linear extrapolation method. Reported

mass values are averages over 100 s. (2) Positive ion electrospray

(ESI) high-resolution mass spectrometry was carried out with a

Micromass Q-Tof API US hybrid quadrupole/time-of-ﬂight mass

spectrometer. The mass spectrometer was externally calibrated using

cesium iodide. PEG 400 was added to the samples as internal

calibrant. The purities of the key target compounds were determined

on a Varian HPLC system equipped with two Varian PrepStar

delivery pumps (model 218) and a Varian UV–vis detector (model

345). The HPLC method used was as follows: column, Princeton-

SPHER-100 phenyl, 4.6 mm × 150 mm, 5 µm; column temperature,

ambient; ﬂow rate, 1.0 mL/min; gradient, 20% acetonitrile (0.01%

TFA and 0.2% formic acid) in water (0.01% TFA and 0.2% formic

acid) to 80% acetonitrile (0.01% TFA and 0.2% formic acid) in

water (0.01% TFA and 0.2% formic acid) in 30 min. All of the

ﬁnal compounds had 98% or greater purity.

1

without further puriﬁcation. H NMR (400 MHz, CDCl₃) δ 7.47

(ddd, J ) 8.3, 5.3, 1.9 Hz, 1H), 7.03 (ddd, J ) 8.1, 6.4, 1.7 Hz,

1H), 6.00 (s, 1H), 4.12–3.97 (m,4H).

2,3-Diﬂuoro-4-iodobenzaldehyde (7). A mixture of 5 obtained

above in THF (60 mL) and 3.6 N hydrochloric acid (60 mL) was

heated to reﬂux for 18 h. The mixture was allowed to cool to room

temperature and concentrated under reduced pressure. Yellow

precipitate formed in the remaining aqueous solution. The precipi-

tate was then ﬁltered, rinsed with cold water, and dried under

vacuum. The dried solid was then triturated with hexanes (100 mL)

for 15 min, ﬁltered, and dried under vacuum to yield 14.7 g (80%

1

over two steps) of the title compound as a yellow solid. H NMR

(400 MHz, CDCl₃) δ 10.33 (d, J ) 0.5 Hz, 1H), 7.69 (dddd, J )

0.6, 1.8, 5.0, 9.0 Hz, 1H), 7.42 (ddd, J ) 1.7, 6.1, 8.4 Hz, 1H).

2-(2,3-Diﬂuorophenyl)-2-methyl[1,3]dioxolane (4b). 2′,3′-Di-

ﬂuoroacetophenone was converted to the title compound using a

similar procedure as described in the preparation of compound 4a.

¹H NMR (400 MHz, CDCl₃) δ 7.15 (ddt, J ) 7.99, 6.40, 1.71 Hz,

1H), 7.01 (m, 1H), 6.93 (tdd, J ) 8.12, 4.86, 1.56 Hz, 1H), 3.99

(dt, J ) 4.12, 5.96 Hz, 2H), 3.75 (dt, J ) 4.12, 6.29 Hz, 2H), 1.66

(d, J ) 0.94 Hz, 3H).

290 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2

Lee et al.

2-(4-Bromo-2,3-diﬂuorophenyl)-2-methyl[1,3]dioxolane (6).

Compound 4b was converted into the title compound using a similar

procedure as described in the preparation of compound 5 except

that neat bromine was used instead of solution of iodine in THF.

¹H NMR (400 MHz, CDCl₃) δ 7.28 (m, 1H), 7.19 (ddd, J ) 8.65,

6.92, 1.96 Hz, 1H), 4.10 (m, 2H), 3.85 (m, 2H), 1.75 (d, J ) 0.93

Hz, 3H).

1-(2,3-Diﬂuoro-4-bromophenyl)ethanone (8). Compound 6 was

then converted into the title compound using a similar procedure

as described in the preparation of compound 7. ¹H NMR (400 MHz,

CDCl₃) δ 7.54 (ddd, J ) 8.73, 6.78, 2.01 Hz, 1H), 7.40 (ddd, J )

8.69, 5.69, 1.85 Hz, 1H), 2.64 (d, J ) 4.95 Hz, 3H).

1-(2,3-Diﬂuoro-4-iodophenyl)-2,2,2-triﬂuoroethanol (9). To a

solution of aldehyde 7 (16.1 g, 60 mmol) in THF (60 mL) under

argon at 0 °C was added trimethyl(triﬂuoromethyl)silane (10.6 mL,

72 mmol) slowly, followed by a catalytic amount of tetrabutylam-

monium ﬂuoride hydrate (150 mg, 0.6 mmol). The resulting mixture

was allowed to warm to room temperature. After the mixture was

stirred at room temperature for an additional 1 h, dilute hydrochloric

acid (30 mL, 3 M) was added to the mixture. The aqueous layer

was extracted with dichloromethane (3 × 30 mL). The organic

layers were combined, washed with saturated sodium bicarbonate

solution, dried with anhydrous magnesium sulfate, ﬁltered, and

concentrated under reduced pressure to yield the title compound.

This was used in the next step without further puriﬁcation. ¹H NMR

(400 MHz, CDCl₃) δ 7.59 (ddd, J ) 8.35, 5.37, 1.99 Hz, 1H),

7.18 (tm, J ) 7.12 Hz, 1H), 5.40 (q, J ) 6.26 Hz, 1H), 3.11 (s,

1H).

1-(2,3-Diﬂuoro-4-iodophenyl)-2,2,2-triﬂuoroacetone (10). To

a solution of alcohol 9 (21 g, 60 mmol) in dichloromethane (140

mL) at 0 °C was added Dess-Martin periodinane (25.7 g, 61 mmol)

in small portions. After the addition, the mixture was allowed to

warm to room temperature and stirred for an additional 1 h.

The mixture was then poured into a solution of potassium carbonate

(10% w/v) and sodium hydroxide (10% w/v). The pH of the mixture

was adjusted to about pH 8 using a saturated sodium bicarbonate

solution. The aqueous layer was extracted with ethyl acetate (3 ×

150 mL). The organic layers were combined, dried with anhydrous

magnesium sulfate, ﬁltered, and concentrated under reduced pres-

sure to yield 17.1 g (85% over two steps) of the title compound.

¹H NMR (400 MHz, CDCl₃) δ 7.72 (ddd, J ) 8.58, 5.12, 1.90 Hz,

1H), 7.42 (tm, J ) 7.84 Hz, 1H).

(2,3-Diﬂuoro-4-iodophenyl)hydroxyacetonitrile (11). Trimeth-

ylsilyl cyanide (3 mL, 22.3 mmol) was added slowly to a solution

of benzaldehyde 7 (5 g, 18.7 mmol) in anhydrous THF (30 mL) at

0 °C. After the addition was completed, a catalytic amount of

tetrabutylammonium ﬂuoride hydrate (50 mg, 0.2 mmol) was added

to the mixture. The mixture was allowed to warm to room

temperature over a period of 2 h and concentrated under reduced

pressure to yield an orange oil. ¹H NMR (400 MHz, CDCl₃) δ

7.63 (ddd, J ) 2.06, 5.33, 8.44 Hz, 1H), 7.22 (ddd, J ) 1.84, 6.41,

8.30 Hz, 1H), 5.72 (s, 1H), 0.28 (s, 9H).

The oil was dissolved in THF (15 mL) to which was added

diluted hydrochloric acid (3 N, 3 mL). After being heated to 65 °C

for 1 h, the mixture was allowed to cool to room temperature and

diluted with 10 mL of water. The two layers were separated, and

the aqueous layer was extracted with ethyl acetate thrice. The

organic layers were combined, washed with brine, dried with

anhydrous sodium sulfate, ﬁltered, and concentrated under reduced

pressure to yield 5.76 g (quantative over two steps) of the title

compound as a yellowish brown solid. ¹H NMR (400 MHz, CDCl₃)

δ 7.66 (ddd, J ) 2.04, 5.31, 8.35 Hz, 1H), 7.23 (ddd, J ) 1.75,

6.43, 8.39 Hz, 1H), 5.81 (s, 1H), 3.03 (br s, 1H).

(5 N, cooled in ice–water) was added slowly to the mixture at 0

°C until the pH of the solution was 8. The pink solution was then

thrice extracted with ethyl acetate. The organic layers were

combined, washed with brine, dried with anhydrous sodium sulfate,

ﬁltered, and concentrated under reduced pressure to yield 1.95 g

(36%) of pink solid as the title product. ¹H NMR (400 MHz,

DMSO-d₆) δ 7.65 (ddd, J ) 1.61, 5.76, 8.29 Hz, 1H), 7.52 (br s,

1H), 7.42 (br s, 1H), 7.07 (ddd, J ) 1.38, 6.63, 8.29 Hz, 1H), 6.42

(d, J ) 5.15 Hz, 1H), 5.11 (d, J ) 5.12 Hz, 1H).

2-(2,3-Diﬂuoro-4-iodophenyl)-2-oxoacetamide (13). To a solu-

tion of acetamide 12 (5.15 g, 16.4 mmol) in 300 mL of anhydrous

acetonitrile was added activated manganese(IV) oxide (20 g, 230

mmol) in one portion. The reaction was monitored with TLC

and stirred at room temperature for 1 h. The mixture was then

ﬁltered through a pad of Celite. The pale-yellow ﬁltrate was

concentrated under reduced pressure to yield 3.43 g (67%) of pale-

1

yellow solid as the desired product. H NMR (400 MHz, CDCl₃)

δ 7.65 (ddd, J ) 1.86, 5.10, 8.44 Hz, 1H), 7.50 (ddd, J ) 1.80,

6.02, 8.43, 1H), 6.84 (br s, 1H), 5.75 (br s, 1H).

2-[(3-Cyano-4-ﬂuorophenyl)hydrazono]-2-(2,3-diﬂuoro-4-io-

dophenyl)acetamide (15c). A mixture of acetamide 13 (3.43 g,

11.1 mmol), 2-ﬂuoro-5-hydrazinobenzonitrile¹⁹(14a) (1.67 g, 11.1

mmol), and p-toluenesulfonic acid monohydrate (110 mg, 0.55

mmol) in 100 mL of anhydrous ethanol was reﬂuxed for 4 h. Yellow

precipitate was formed, and the mixture was allowed to cool to

room temperature. The orange suspension was then cooled to 0 °C

in an ice–water bath. The mixture was ﬁltered, and the pale-yellow

solid was rinsed with cold ethanol. The ﬁltrate was concentrated

under reduced pressure, and the residue was triturated with cold

ethanol. The mixture was ﬁltered and the yellow solid obtained

was combined with the ﬁrst crop of solid to give 3.63 g (74%) of

pale-yellow solid as the desired product. The NMR indicated the

yellow solid is a mixture of cis/trans isomers in a ratio of 1:2. The

trans isomer: ¹H NMR (400 MHz, DMSO-d₆) δ 12.35 (s, 1H), 7.92

(br s, 1H), 7.72–7.62 (m, 4H), 7.44 (t, J ) 9.07 Hz, 1H), 7.25

(ddd, J ) 1.52, 6.82, 8.34 Hz, 1H). The cis and trans isomers: ¹H

NMR (400 MHz, DMSO-d₆) δ 12.35 (s), 9.98 (s), 8.11 (dd, J )

2.87, 5.55 Hz), 8.02 (br s), 7.92 (br s), 7.77–7.58 (m), 7.44 (t, J )

9.10 Hz), 7.40 (t, J ) 9.10 Hz), 7.31 (br s), 7.25 (ddd, J ) 1.59,

7.07, 8.42 Hz), 6.95 (ddd, J ) 1.56, 6.09, 8.14 Hz).

1-(3-Cyano-4-ﬂuorophenyl)-7-ﬂuoro-6-iodo-1H-indazole-3-

carboxylic Acid Amide (16c). A mixture of hydrazone 15c (3.63

g, 8.2 mmol) and potassium carbonate (1.3 g, 9.4 mmol) in 60 mL

of anhydrous DMF was heated to 100 °C for 2 h. The mixture was

cooled to room temperature, ﬁltered through a pad of Celite, and

concentrated under reduced pressure. The residue was triturated

with dichloromethane overnight and ﬁltered to yield 2.92 g of light-

brown solid. The red ﬁltrate was concentrated under reduced

pressure. The residue was then triturated with cold ethyl acetate

for 1 h and ﬁltered to yield another 0.37 g of pale-yellow solid.

The solid was combined to give 3.28 g (95%) of pale-yellow solid

as the desired product. ¹H NMR (400 MHz, DMSO-d₆) δ 8.47 (dt,

J ) 5.62, 2.67 Hz, 1H), 8.22 (ddt, J ) 4.70, 9.04, 2.87 Hz, 1H),

8.09 (br s, 1H), 7.90 (d, J ) 8.45 Hz, 1H), 7.81 (t, J ) 9.01 Hz,

1H), 7.72 (dd, J ) 5.00, 8.50 Hz, 1H), 7.70 (br s, 1H).

5-(6-Bromo-7-ﬂuoro-3-methylindazol-1-yl)-2-ﬂuorobenzoni-

trile (16a). Compound 8 was converted into hydrazone 15a using a

similar procedure as described in the preparation of compound 15c.

Compound 15a was then converted into the title compound using

a similar procedure as described in the preparation compound 16c.

¹H NMR (400 MHz, CDCl₃) δ 7.87 (td, J ) 5.44, 2.75, 2.75 Hz,

1H), 7.83 (m, 1H), 7.38–7.33 (m, 3H), 2.62 (s, 3H).

2-(2,3-Diﬂuoro-4-iodophenyl)-2-hydroxyacetamide (12). To a

solution of acetonitrile 11 (5.12 g, 17.4 mmol) in anhydrous 1,4-

dioxane (35 mL) at 0 °C was added cold concentrated HCl (3.5

mL), and anhydrous hydrogen chloride gas was then bubbled into

the mixture at 0 °C for 30 min. The mixture was then allowed to

stand without stirring and warm to room temperature overnight.

(Caution: pressure developed as the solution was warming up.) The

orange solution was then poured into ice. Sodium hydroxide solution

2-Fluoro-5-(7-ﬂuoro-6-iodo-3-triﬂuoromethylindazol-1-yl)ben-

zonitrile (16b). Compound 10 was converted into hydrazone 15b

using a similar procedure as described in the preparation of

compound 15c. Compound 15b was then converted into the title

compound using a similar procedure as described in the preparation

of compound 16c. H NMR (400 MHz, CDCl₃) δ 7.92 (td, J )

2.94, 8.36 Hz, 1H), 7.88 (m, 1H), 7.69 (dd, J ) 8.61, 4.90 Hz,

1H), 7.48 (dq, J ) 8.60, 0.93 Hz, 1H), 7.42 (t, J ) 8.40, 1H).

1

7-Fluoroindazoles as FXa Inhibitors

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 291

3-(6-Bromo-7-ﬂuoro-3-methylindazol-1-yl)benzonitrile (16d).

Compound 8 was converted into hydrazone 15d using 3-hydrazi-

nobenzonitrile (14b) and a similar procedure as described in the

preparation of compound 15c. Compound 15d was then converted

into the title compound using a similar procedure as described in

preparation of compound 23a. ¹H NMR (400 MHz, CDCl₃) δ 7.54

(dd, J ) 8.42, 7.82 Hz, 1H), 7.11 (dd, J ) 9.64, 2.38 Hz, 1H),

6.98 (ddd, J ) 8.58, 2.36, 0.96 Hz, 1H), 3.63 (t, J ) 5.91 Hz, 2H),

2.56 (t, J ) 6.32 Hz, 2H), 1.94 (m, 4H).

1-[3-Fluoro-4-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)phe-

nyl]piperidin-2-one (23d). A mixture of lactam 23c (50 mg, 0.184

mmol), Pd(dppf)Cl₂·CH₂Cl₂, (20 mg, 0.024 mmol), bis(pinacola-

to)diboron (70 mg, 0.276 mmol), and potassium acetate (54 mg,

0.550 mmol) under argon in 4 mL of anhydrous 1,4-dioxane was

heated to 90 °C for 18 h. Upon cooling to room temperature, the

mixture was ﬁltered through a pad of Celite and concentrated under

reduced pressure. The residue was puriﬁed using preparative TLC

plates (silica gel, 20 cm × 20 cm, 1000 µm, ethyl acetate/hexanes,

1:1 v/v) to give 35.2 mg (60%) of off-white solid as the title

compound. ¹H NMR (400 MHz, CDCl₃) δ 7.74 (dd, J ) 7.99,

6.83 Hz, 1H), 7.08 (dd, J ) 8.03, 1.88 Hz, 1H), 7.00 (dd, J )

10.49, 1.82 Hz, 1H), 3.63 (t, J ) 5.88 Hz, 2H), 2.55 (t, J ) 6.32

Hz, 2H), 2.03–1.82 (m, 4H), 1.34 (s, 12H).

1

the preparation of compound 16c. H NMR (400 MHz, CDCl₃) δ

7.91 (m, 1H), 7.84 (dm, J ) 7.90 Hz, 1H), 7.65 (td, J ) 1.39,

7.72, Hz, 1H), 7.60 (t, J ) 7.84 Hz, 1H), 7.39–7.35 (m, 2H), 2.62

(s, 3H).

1-(4-Bromo-2-ﬂuorophenyl)ethanone (18). Methylmagnesium

bromide (3.0 M) in ether (2.1 mL, 6.3 mmol) was added dropwise

to a solution of 4-bromo-2-ﬂuorobenzaldehyde (17) (1.1 g, 5.4

mmol) in anhydrous THF (20 mL) at –78 °C. The mixture was

allowed to warm to room temperature and then stirred for 1.5 h.

Additional methylmagnesium bromide (0.3 mL, 0.9 mmol, 3.0 M

in ether) was added. After being stirred at room temperature for

18 h, the mixture was quenched with ammonium chloride solution.

The layers were separated, and the aqueous layer was twice

extracted with ethyl acetate. The organic layers were combined,

dried with anhydrous sodium sulfate, and concentrated under

reduced pressure to yield an oil. The oil was dissolved in

dichloromethane (50 mL), and pyridinium dichromate (3.2 g, 8.4

mmol) was added. After being stirred at room temperature for 3 d,

the mixture was ﬁltered through a pad of Celite and concentrated

under reduced pressure. The residue was puriﬁed by silica gel ﬂash

chromatography (eluted with ethyl acetate/hexanes, 0:1 to 1:9, v/v)

to yield 1.1 g (91% over two steps) of the title compound. ¹H NMR

(400 MHz, CDCl₃) δ 7.76 (t, J ) 8.13 Hz, 1H), 7.36 (m, 2H), 2.62

(d, J ) 5.04 Hz, 3H).

3-{3-Methyl-6-[4-(2-oxopiperidin-1-yl)phenyl]indazol-1-

yl}benzonitrile (24d). A mixture of boronic acid ester 23a (58 mg,

0.192 mmol), indazole 20 (50 mg, 0.160 mmol), tetrakis(triph-

enylphosphine)palladium(0) (19 mg, 0.016 mmol), sodium carbon-

ate solution (0.64 mL, 1.28 mmol), and 3 mL of ethanol/toluene

(1:2, v/v) was heated to 100 °C for 18 h. Upon cooling to room

temperature, the mixture was concentrated under reduced pressure.

The residue was partitioned between ethyl acetate and water. The

aqueous layer was extracted with ethyl acetate twice. The organic

layers were combined, dried with anhydrous sodium sulfate, and

concentrated under reduced pressure. The residue was puriﬁed using

sequential preparative TLC (silica gel, 20 cm × 20 cm, 1000 µm,

ethyl acetate/hexanes, 7:3 v/v, and then ethyl acetate/dichlo-

3-(6-Bromo-3-methylindazol-1-yl)benzonitrile (20). Compound

18 was converted into hydrazone 19 using 3-hydrazinobenzonitrile

(14b) and a similar procedure as described in the preparation of

compound 15c. Compound 19 was then converted into the title

compound using a similar procedure as described in the preparation

1

romethane, 3:7 v/v) to give the title compound (48 mg, 70%). H

NMR (400 MHz, CDCl₃) δ 8.07 (m, 1H), 8.01 (dm, J ) 8.03 Hz,

1H), 7.82 (s, 1H), 7.78 (d, J ) 8.32 Hz, 1H), 7.66–7.64 (m, 3H),

7.58 (dm, J ) 7.70 Hz, 1H), 7.47 (dd, J ) 8.15, 0.84 Hz, 1H),

7.38 (d, J ) 8.40 Hz, 2H), 3.71 (t, J ) 5.55 Hz, 2H), 2.67 (s, 3H),

2.60 (t, J ) 6.24 Hz, 2H), 1.98 (m, 4H).

1

of compound 16c. H NMR (400 MHz, CDCl₃) δ 8.00 (m, 1H),

7.93 (dm, J ) 7.76 Hz, 1H), 7.85 (d, J ) 1.00 Hz, 1H), 7.67–7.55

(m, 3H), 7.35 (dd, J ) 8.50, 1.46 Hz, 1H), 2.61 (s, 3H).

1-[4-(4,4,5,5-Tetramethyl[1,3,2]dioxaborolan-2-yl)phenyl]pi-

peridin-2-one (23a). 5-Bromovaleryl chloride (0.65 mL, 4.85

mmol) was added slowly to a mixture of 4-aminophenylboronic

acid pinacol ester (1.0 g, 4.6 mmol) and triethylamine (0.75 mL,

5.4 mmol) in dichloromethane (30 mL). After the mixture was

stirred at room temperature for 15 min, water was added to the

mixture. The two layers were separated, and the organic layer was

washed with saturated sodium bicarbonate solution and brine, dried

with anhydrous sodium sulfate, and concentrated under reduced

pressure. The residue was dissolved in 40 mL of anhydrous DMF

and cooled to 0 °C, and sodium hydride (0.23 g, 5.75 mmol, 60%

dispersion in mineral oil) was added in small portions. After being

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

under reduced pressure. The residue was dissolved in dichlo-

romethane and washed with 10% aqueous citric acid solution. The

aqueous layer was extracted with dichloromethane three times. The

organic layers were combined, washed with brine, dried with

anhydrous sodium sulfate, ﬁltered, and concentrated under reduced

pressure. The yellow solid was triturated with ether/hexanes (1:1,

v/v). After ﬁltration and drying under vacuum, 0.77 g (52% over

two steps) of off-white solid was obtained as the title compound.

¹H NMR (400 MHz, CDCl₃) δ 7.83 (dm, J ) 8.41 Hz, 2H), 7.27

(m, J ) 8.41 Hz, 2H), 3.64 (m, 2H), 2.56 (m, 2H), 1.94 (m, 4H),

1.33 (s, 12H).

1-{4-[1-(3-Aminomethylphenyl)-3-methyl-1H-indazol-6-

yl]phenyl}piperidin-2-one (25d). A mixture of benzonitrile 24d

(47.8 mg, 0.118 mmol) and Raney nickel (∼0.5 g) in 15 mL of

ethanol was hydrogenated in a Parr apparatus at 50 psi and room

temperature for 18 h. The mixture was ﬁltered through a pad of

Celite and concentrated under reduced pressure. The residue was

puriﬁed by silica gel ﬂash chromatography (eluted with 7 N

ammonia in methanol/dichloromethane, 0:1 v/v to 5:95 v/v) to yield

1

20 mg (39%) of white solid as the title compound. H NMR (400

MHz, CDCl₃) δ 7.84 (br s, 1H), 7.76–7.74 (m, 2H), 7.63 (d, J )

8.59 Hz, 2H), 7.60 (ddd, J ) 2.65, 2.06, 1.07 Hz, 1H), 7.46 (t, J

) 7.77 Hz, 1H), 7.42 (dd, J ) 8.34, 1.35 Hz, 1H), 7.35 (br d, J )

7.51 Hz, 1H), 7.32 (d, J ) 8.57 Hz, 2H), 3.96 (s, 2H), 3.66 (t, J )

5.72 Hz, 2H), 2.66 (s, 3H), 2.58 (t, J ) 6.24 Hz, 2H), 1.95 (m,

4H). HRMS (FAB+) m/z calcd for C₂₆H₂₅N₄O [M - H]⁺,

409.2028; found, 409.2034.

Compounds 25a-c were prepared using the same procedures

as for the synthesis of compound 25d using appropriate starting

materials.

1-{4-[1-(3-Aminomethylphenyl)-7-ﬂuoro-3-methyl-1H-inda-

zol-6-yl]phenyl}pyrrolidin-2-one (25a). ¹H NMR (400 MHz,

CD₃OD/CDCl₃) δ 7.66 (d, J ) 8.75 Hz, 2H), 7.57–7.54 (m, 2H),

7.52–7.48 (m, 2H), 7.46–7.40 (m, 2H), 7.30 (br d, J ) 6.90 Hz,

1H), 7.21 (dd, J ) 8.20, 5.98 Hz, 1H), 3.90–3.87 (m, 4H), 2.63–2.59

(m, 5H), 2.17 (m, 2H). HRMS (FAB+) m/z calcd for C₂₅H₂₂N₄OF

[M - H]⁺, 413.1778; found, 413.1773.

1-[4-(4,4,5,5-Tetramethyl[1,3,2]dioxaborolan-2-yl)phenyl]pyr-

rolidin-2-one (23b). Compound 21 was converted into boronic acid

ester 23b using 4-bromobutyryl chloride and a similar procedure

1

as described in the preparation of compound 23a. H NMR (400

1-{4-[1-(3-Aminomethylphenyl)-3-methyl-1H-indazol-6-

MHz, CDCl₃) δ 7.80 (d, J ) 8.65 Hz, 2H), 7.64 (d, J ) 8.67 Hz,

2H), 3.88 (t, J ) 7.04 Hz, 2H), 2.62 (t, J ) 8.08 Hz, 2H), 2.16 (m,

2H), 1.34 (s, 12H).

1-(4-Bromo-3-ﬂuorophenyl)piperidin-2-one (23c). 4-Bromo-

3-ﬂuoroaniline (22) was converted into lactam 23c using 4-bro-

movaleryl chloride and a similar procedure as described in the

1

yl]phenyl}pyrrolidin-2-one (25b). H NMR (400 MHz, CDCl₃)

δ 7.85 (s, 1H), 7.78 (d, J ) 8.34 Hz, 1H), 7.75–7.70 (m, 3H), 7.67

(d, J ) 8.80 Hz, 2H), 7.63 (br d, J ) 8.32 Hz, 1H), 7.51 (t, J )

7.78 Hz, 1H), 7.46 (dd, J ) 1.33, 8.35 Hz, 1H), 7.33 (br d, J )

7.57 Hz, 1H), 4.00 (s, 2H), 3.94 (t, J ) 7.03 Hz, 2H), 2.70 (s, 3H),

292 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2

Lee et al.

2.67 (t, J ) 8.11 Hz, 2H), 2.22 (quintet, J ) 7.59 Hz, 2H). HRMS

(FAB+) m/z calcd for C₂₅H₂₃N₄O [M - H]⁺, 395.1872; found,

395.1879.

1-{4-[1-(3-Aminomethylphenyl)-7-ﬂuoro-3-methyl-1H-inda-

zol-6-yl]phenyl}piperidin-2-one (25c). ¹H NMR (400 MHz,

CDCl₃) δ 7.60–7.59 (m, 3H), 7.50–7.48 (m, 2H), 7.42 (t, J ) 7.70

Hz, 1H), 7.36–7.32 (m, 3H), 7.22 (dd, J ) 8.24, 5.92 Hz, 1H),

3.98 (br s, 1H), 3.68 (t, J ) 5.38 Hz, 2H), 2.61 (s, 3H), 2.57 (t, J

) 6.18 Hz, 2H), 1.96 (m, 4H). HRMS (ESI+) m/z calcd for

C₂₆H₂₆N₄OF [M + H]⁺, 429.2091; found, 429.2085.

2-Fluoro-5-[7-ﬂuoro-3-methyl-6-(4,4,5,5-tetramethyl[1,3,2]dioxabo-

rolan-2-yl)indazol-1-yl]benzonitrile (26). A mixture of bromoinda-

zole 16a (398 mg, 1.14 mmol), Pd(dppf)Cl₂·CH₂Cl₂, (94 mg, 0.114

mmol), bis(pinacolato)diboron (436 mg, 1.72 mmol), and potassium

acetate (337 mg, 3.43 mmol) under argon in 35 mL of anhydrous

1,4-dioxane was heated to 90 °C for 18 h. Upon cooling to room

temperature, the mixture was ﬁltered through a pad of Celite and

concentrated under reduced pressure. The residue was puriﬁed by

silica gel ﬂash chromatography (eluted with dichloromethane) to

yield 433 mg (95%) of white solid as the title compound. ¹H NMR

(400 MHz, CDCl₃) δ 7.90–7.83 (m, 2H), 7.53 (dd, J ) 8.04, 4.09

Hz, 1H), 7.48 (dd, J ) 8.02, 0.65 Hz, 1H), 7.31 (t, J ) 8.58 Hz,

1H), 2.63 (s, 3H), 1.39 (s, 12H).

(186 mg, 2.48 mmol), and potassium carbonate (686 mg, 4.96

mmol) in 14 mL of DMF and 2.0 mL of water was stirred at room

temperature for 3 d. The reaction was then quenched with water.

The off-white precipitate was ﬁltered and washed with ethyl acetate.

The solid was then puriﬁed by silica gel ﬂash chromatography

(eluted with ethyl acetate/hexanes, 7:3 v/v) to yield 298 mg (76%)

of white solid as the title compound. ¹H NMR (400 MHz, CDCl₃)

δ 7.79 (ddd, J ) 8.90, 3.01, 2.30 Hz, 1H), 7.73 (m, 1H), 7.56 (d,

J ) 8.23 Hz, 1H), 7.51 (d, J ) 8.87 Hz, 1H), 7.44 (t, J ) 8.25 Hz,

1H), 7.21–7.14 (m, 3H), 4.40 (br s, 2H), 3.69 (t, J ) 5.35 Hz, 2H),

2.68 (s, 3H), 2.60 (t, J ) 6.20 Hz, 2H), 1.97 (m, 4H). HRMS

(FAB+) m/z calcd for C₂₆H₂₂N₅O₂F₂[M + H]⁺, 474.1742; found,

474.1752.

1-{4-[1-(3-Aminobenzo[d]isoxazol-5-yl)-7-ﬂuoro-3-triﬂuoro-

methyl-1H-indazol-6-yl]-3-ﬂuorophenyl}piperidin-2-one (29b).

Compound 28b was converted into the title compound using the

1

procedure as described in the preparation of compound 29a. H

NMR (400 MHz, CDCl₃) δ 7.83 (t, J ) 2.20 Hz, 1H), 7.81–7.75

(m, 2H), 7.54 (d, J ) 8.88 Hz, 1H), 7.40 (t, J ) 8.30 Hz, 1H),

7.35 (dd, J ) 8.37, 5.62 Hz, 1H), 7.20–7.15 (m, 2H), 4.56 (br s,

1H), 3.69 (t, J ) 5.53 Hz, 2H), 2.59 (t, J ) 6.35 Hz, 2H), 1.96 (m,

4H). HRMS (FAB+) m/z calcd for C₂₆H₁₉N₅O₂F₅[M + H]⁺,

528.1459; found, 528.1465.

1-(3-Cyano-4-ﬂuorophenyl)-7-ﬂuoro-6-(4,4,5,5-tetramethyl[1,3,2]-

dioxaborolan-2-yl)-1H-indazole-3-carboxylic Acid Amide (27). A

mixture of 1-(3-cyano-4-ﬂuorophenyl)-7-ﬂuoro-6-iodo-1H-indazole-

3-carboxylic acid amide 16c (105 mg, 0.24 mmol), Pd(dppf)Cl₂

(20 mg, 0.024 mmol), bis(pinacolato)diboron (90 mg, 0.35 mmol),

potassium acetate (70 mg, 0.71 mmol) under argon in 5 mL of

anhydrous 1,4-dioxane was heated at 110 °C for 2 d. After cooling

to room temperature, the mixture was ﬁltered through a pad of Celite

and concentrated under reduced pressure. The residue was then

puriﬁed using silica gel ﬂash chromatography (ethyl acetate/

dichloromethane, 1:1 v/v) to yield 92 mg (88%) of light-yellow

1-(3-Aminobenzo[d]isoxazol-5-yl)-7-ﬂuoro-6-[2-ﬂuoro-4-(2-oxo-

piperidin-1-yl)phenyl]-1H-indazole-3-carboxamide (29c). Com-

pound 16c was converted into compound 28c using the procedure

as described in the preparation of compound 28b. Compound 28b

was then converted into the title compound using the procedure as

described in the preparation of compound 29a. ¹H NMR (400 MHz,

CDCl₃) δ 8.31 (d, J ) 8.4 Hz, 1H), 7.84–7.78 (m, 2H), 7.57 (d, J

) 8.8 Hz, 1H), 7.43 (t, J ) 8.3 Hz, 1H), 7.34 (dd, J ) 5.8, 8.4 Hz,

1H), 7.21–7.14 (m, 2H), 6.96 (br s, 1H), 5.55 (br s, 1H), 4.49 (br

s, 2H), 3.69 (t, J ) 5.4 Hz, 2H), 2.59 (t, J ) 6.3 Hz, 2H), 1.97 (m,

4H). HRMS (FAB+) m/z calcd for C₂₆H₂₀N₆O₃F₂[M]⁺, 502.1565;

found, 502.1562.

1

solid as the title product. H NMR (400 MHz, DMSO-d₆) δ 8.47

3-Iodo-2-pyridinecarboxaldehyde (32)...³⁶To a solution of

N,N,N′-trimethylethylenediamine (1.63 mL, 12.6 mmol) in anhy-

drous THF (30 mL) under argon at –78 °C was added 1.65 M

n-butyllithium in hexanes (7 mL, 21 mmol). After the mixture was

held for 15 min, 2-pyridinecarboxaldehyde (1 mL, 10.5 mmol) was

added to the solution. After the mixture was stirred at –78 °C for

an additional 15 min, another aliquot of 1.65 M n-butyllithium in

hexanes (12.7 mL, 21 mmol) was added to the mixture. After being

stirred at –78 °C for another 1 h and at –42 °C (dry ice/acetonitrile

bath) for 4 h, the solution was transferred to a solution of iodine

(5.6 g, 22 mmol) in anhydrous THF (30 mL) using a cannula. The

mixture was allowed to warm to room temperature and stirred for

18 h. The mixture was quenched with brine, and the two layers

were separated. The aqueous layers was extracted with ether twice.

The organic layers were combined, dried with anhydrous sodium

sulfate, ﬁltered, and concentrated under reduced pressure. The

residue was puriﬁed using preparative TLC (silica gel, 20 cm ×

20 cm, 2000 µm, ethyl acetate/dichloromethane, 15:85 v/v) to give

314 mg (13%) of the title compound. ¹H NMR (400 MHz, CDCl₃)

δ 10.02 (s, 1H), 8.76 (dd, J ) 4.46, 1.33 Hz, 1H), 8.32 (dm, J )

8.02 Hz, 1H), 7.19 (dd, J ) 8.02, 4.49 Hz, 1H).

(dt, J ) 5.59, 2.49 Hz, 1H), 8.21 (ddt, J ) 4.70, 9.06, 2.70 Hz,

1H), 8.10 (d, J ) 8.16 Hz, 1H), 8.07 (br s, 1H), 7.81 (t, J ) 9.01

Hz, 1H), 7.69 (br s, 1H), 7.54 (dd, J ) 4.33, 8.15 Hz, 1H), 1.32 (s,

12H).

2-Fluoro-5-{7-ﬂuoro-6-[2-ﬂuoro-4-(2-oxo-piperidin-1-yl)phe-

nyl]-3-methylindazol-1-yl}benzonitrile (28a). A mixture of in-

dazole boronate 26 (433 mg, 1.26 mmol), lactam 23c (378 mg,

1.38 mmol), Pd(dppf)Cl₂·CH₂Cl₂(103 mg, 0.126 mmol), and

potassium carbonate (698 mg, 5.1 mmol) in 30 mL of anhydrous

DMF was heated to 95 °C for 18 h. Upon cooling to room

temperature, the mixture was ﬁltered through a pad of Celite and

concentrated under reduced pressure. The residue was puriﬁed by

silica gel ﬂash chromatography (eluted with ethyl acetate/hexanes,

7:3 v/v) to yield 382 mg (66%) of white solid as the title compound.

¹H NMR (400 MHz, CDCl₃) δ 7.89 (td, J ) 2.75, 5.57 Hz, 1H),

7.85 (dd, J ) 8.56, 4.24 Hz, 1H), 7.55 (d, J ) 8.22 Hz, 1H), 7.43

(t, J ) 8.28 Hz, 1H), 7.31 (t, J ) 8.61 Hz, 1H), 7.26–7.16 (m,

4H), 3.70 (t, J ) 5.41 Hz, 2H), 2.64 (s, 3H), 2.59 (t, J ) 6.27 Hz,

2H), 1.97 (m, 4H).

2-Fluoro-5-{7-ﬂuoro-6-[2-ﬂuoro-4-(2-oxopiperidin-1-yl)phe-

nyl]-3-triﬂuoromethylindazol-1-yl}benzonitrile (28b). A mixture

of iodoindazole 16b (15 mg, 0.033 mmol), lactam boronate 23d

(16 mg, 0.050 mmol), tetrakis(triphenylphosphine)palladium(0) (2

mg, 0.002 mmol), aqueous sodium carbonate solution (0.1 mL, 0.2

mmol, 2 M), and 0.7 mL of ethanol/toluene (1:6, v/v) was heated

to 80 °C for 20 h. Upon cooling to room temperature, the mixture

was diluted with ethyl acetate. The mixture was puriﬁed using

preparative TLC (silica gel, 20 cm × 20 cm, 250 µm, ethyl acetate/

dichloromethane, 1:9 v/v) to give the title compound. ¹H NMR

(400 MHz, CDCl₃) δ 7.95 (td, J ) 2.65, 5.17, Hz, 1H), 7.91 (m,

1H), 7.77 (dd, J ) 8.42, 0.80 Hz, 1H), 7.45–7.37 (m, 3H), 7.24–7.18

(m, 2H), 3.72 (t, J ) 5.52 Hz, 2H), 2.60 (t, J ) 6.35 Hz, 2H), 1.99

(m, 4H).

4-Iodo-3-pyridinecarboxaldehyde (33)...³⁶3-Pyridinecarbox-

aldehyde was converted into compound 33 using the procedure as

described in the preparation of compound 32. ¹H NMR (400 MHz,

CDCl₃) δ 10.06 (s, 1H), 8.86 (s, 1H), 8.31 (d, J ) 5.25 Hz, 1H),

7.90 (d, J ) 5.26 Hz, 1H).

4-Bromo-2-(2-trimethylsilanylethoxymethyl)-2H-pyrazole-3-

carboxaldehyde (37). To a suspension of sodium hydride (150 mg,

3.6 mmol, 60% dispersion in mineral oil) in anhydrous DMF (30

mL) was added a solution of 4-bromo-1H-pyrazole-5-carboxalde-

hyde (36) (500 mg, 2.9 mmol) in DMF (5 mL) and then

(chloromethyl)methyldiethoxysilane (0.66 mL, 3.6 mmol). After

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

concentrated under reduced pressure. The residue was partitioned

between ethyl acetate and water. The aqueous layer was extracted

with ethyl acetate twice. The organic layers were combined, washed

1-{4-[1-(3-Aminobenzo[d]isoxazol-5-yl)-7-ﬂuoro-3-methyl-1H-

indazol-6-yl]-3-ﬂuorophenyl}piperidin-2-one (29a). A mixture of

lactam indazole 28a (381 mg, 0.83 mmol), acetohydroxamic acid

7-Fluoroindazoles as FXa Inhibitors

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 293

with brine, dried with anhydrous sodium sulfate, ﬁltered, and

concentrated under reduced pressure. The residue was puriﬁed using

preparative TLC (silica gel, 20 cm × 20 cm, 2000 µm, ×3, ethyl

acetate/dichloromethane, 15:85 v/v) to give 331 mg (39%) of the

title compound. ¹H NMR (400 MHz, CDCl₃) δ 9.92 (s, 1H), 7.59

(s, 1H), 5.76 (s, 2H), 3.61–3.53 (m, 2H), 0.94–0.83 (m, 2H), -0.05

(s, 9H).

with anhydrous sodium sulfate, ﬁltered, and concentrated under

reduced pressure. The residue was puriﬁed by preparative TLC

(silica gel, 20 cm × 20 cm, 2000 µm, ethyl acetate/hexanes, 1:9

v/v) to give 285 mg (45%) of 4-(4-bromo-3-ﬂuorophenyl)-2-(2-

1

trimethylsilanylethoxymethyl)-2H-pyrazole-3-carboxaldehyde. H

NMR (400 MHz, CDCl₃) δ 9.93 (s, 1H), 7.69 (s, 1H), 7.64 (dd, J

) 8.14, 7.20 Hz, 1H), 7.24 (dd, J ) 9.15, 1.99 Hz, 1H), 7.13 (dd,

J ) 7.93, 1.72 Hz, 1H), 5.85 (s, 2H), 3.64 (m, 2H), 0.94 (m, 2H),

-0.03 (s, 9H).

N,N-Dimethyl-N′-phenylethane-1,2-diamine (41). A mixture

of bromobenzene (0.26 mL, 2.5 mmol), N,N-dimethylethylenedi-

amine (0.3 mL, 2.75 mmol), Pd₂(dba)₃(114 mg, 0.125 mmol),

sodium tert-butoxide (504 mg, 5.25 mmol), and XANTPHOS (174

mg, 0.3 mmol) under argon in 1,4-dioxane was heated to 140 °C

in a Biotage Initiator microwave synthesizer for 30 min. Upon

cooling, the mixture was diluted with ethyl acetate and ﬁltered,

and the ﬁltrate was concentrated under reduced pressure. The reside

was puriﬁed using silica gel ﬂash chromatography (eluted with

methanol/dichloromethane, 5:95 v/v) followed by preparative TLC

(silica gel, 20 cm × 20 cm, 2000 µm, ethyl acetate) to give 150

A mixture of 4-(4-bromo-3-ﬂuorophenyl)-2-(2-trimethylsilanyl-

ethoxymethyl)-2H-pyrazole-3-carboxaldehyde (285 mg, 0.71 mmol),

dimethylamine (1.8 mL, 3.6 mmol, 2 M in THF), and 4 Å molecular

sieves in 30 mL of anhydrous DMF was stirred at room temperature

for 18 h. Sodium triacetoxyborohydride (230 mg, 1.1 mmol) was

added to the mixture and stirred for additional 3 d. The mixture

was concentrated under reduced pressure and partitioned between

dichloromethane and water. The aqueous layer was extracted with

dichloromethane twice. The organic layers were combined, dried

with anhydrous sodium sulfate, ﬁltered, and concentrated under

reduced pressure. The residue was puriﬁed by preparative TLC

(silica gel, 20 cm × 20 cm, 2000 µm, ethyl acetate/hexanes, 1:1

1

mg (37%) of the title compound. H NMR (400 MHz, CDCl₃) δ

7.20–7.15 (m, 2H), 6.72–6.67 (m, 1H), 6.65–6.62 (m, 2H), 4.25

(br s, 1H), 3.17–3.13 (m, 2H), 2.58–2.54 (m, 2H), 2.26 (s, 6H).

N,N-Dimethyl-N′-pyridin-3-ylethane-1,2-diamine (42). 3-Bro-

mopyridine was converted into compound 42 using the procedure

1

v/v) to give 152 mg (92%) of the title compound. H NMR (400

MHz, CDCl₃) δ 7.59 (s, 1H), 7.54 (dd, J ) 7.62, 7.49 Hz, 1H),

7.32 (dd, J ) 9.96, 1.87 Hz, 1H), 7.13 (dd, J ) 8.25, 1.86 Hz,

1H), 5.65 (s, 2H), 3.62–3.58 (m, 4H), 2.19 (s, 6H), 0.91 (m, 2H),

-0.03 (s, 9H).

1

as described in the preparation of compound 41. H NMR (400

MHz, CDCl₃) δ 8.05 (d, J ) 2.8 Hz, 1H), 7.95 (dd, J ) 1.3, 4.7

Hz, 1H), 7.08 (ddd, J ) 0.4, 4.7, 8.3 Hz, 1H), 6.88 (ddd, J ) 1.3,

2.9, 8.3 Hz, 1H), 4.39 (br s, 1H), 3.19–3.11 (br m, 2H), 2.60–2.56

(m, 2H), 2.26 (s, 6H).

(4′-Bromo-3′-ﬂuorobiphenyl-2-ylmethyl)dimethylamine (49c).

A mixture of 2-formylphenylboronic acid (500 mg, 3.3 mmol),

1-bromo-2-ﬂuoro-4-iodobenzene (1.1 g, 3.7 mmol), aqueous sodium

carbonate solution (27 mL, 14 mmol, 2M), tetrakis(triphenylphos-

phine)palladium(0) (200 mg, 0.17 mmol) in ethanol/toluene (12

mL, 1:1 v/v) was heated to 100 °C for 16 h. The mixture was

allowed to cool and then partitioned between ethyl acetate and

water. The aqueous layer was then extracted twice with ethyl

acetate. The organic layers were combined, washed with brine, dried

with anhydrous sodium sulfate, and concentrated under reduced

pressure. The residue was puriﬁed using silica gel ﬂash chroma-

tography (eluted with ethyl acetate/hexanes, 0:1 v/v to 1:9 v/v) to

give 733 mg (79%) of 4′-bromo-3′-ﬂuorobiphenyl-2-carboxaldehyde

as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 10.00 (s, 1H),

8.04 (dd, J ) 1.24, 7.78 Hz, 1H), 7.70–7.64 (m, 2H), 7.56 (t, J )

7.57, 1H), 7.42 (dd, J ) 0.69, 7.67 Hz, 1H), 7.19 (dd, J ) 2.01,

9.02 Hz, 1H), 7.06 (dd, J ) 1.98, 8.15 Hz, 1H).

N,N-Dimethyl-N′-pyridin-4-ylethane-1,2-diamine (47). A mix-

ture of 4-chloropyridine hydrochloride (150 mg, 1.0 mmol) and

N,N-dimethylethylenediamine (0.33 mL, 3 mmol) in anhydrous

1-pentanol (1 mL) under argon was heated to 140 °C for 24 h.

After cooling to room temperature, the reaction mixture was

concentrated under reduced pressure. The residue was puriﬁed by

preparative TLC (silica gel, 20 cm × 20 cm, 2000 µm, methanol/

dichloromethane, 1:9 v/v, followed by 10% 7 N ammonia in

methanol/dichloromethane, 1:9 v/v) to give 152 mg (92%) of the

title compound. ¹H NMR (400 MHz, CDCl₃) δ 8.20–8.17 (m, 2H),

6.46–6.43 (m, 2H), 4.86 (br s, 1H), 3.19–3.13 (m, 2H), 2.58–2.53

(m, 2H), 2.26 (s, 6H).

N,N-Dimethyl-N′-pyridin-4-ylpropane-1,3-diamine (48). 4-Chlo-

ropyridine hydrochloride and N,N-dimethyl-1,3-propanediamine

were coupled into compound 48 using the procedure as described

1

in the preparation of compound 47. H NMR (400 MHz, CDCl₃)

To a mixture of 4′-bromo-3′-ﬂuorobiphenyl-2-carboxaldehyde

(100 mg, 0.36 mmol) and 2.0 M dimethylamine in tetrahydrofuran

solution (0.36 mL, 0.72 mmol) in 10 mL of anhydrous DMF was

added sodium triacetoxyborohydride (114 mg, 0.54 mmol) and

stirred at room temperature for 18 h. The mixture was concentrated

under reduced pressure. The residue was partitioned between water

and ethyl acetate. The aqueous layer was extracted with ethyl acetate

twice. The organic layers were combined, washed with brine, dried

with anhydrous sodium sulfate, and concentrated under reduced

pressure to yield 113 mg (100%) of colorless residue as the title

compound. The mixture was used in the next step without further

δ 8.18–8.14 (m, 2H), 6.43–6.39 (m, 2H), 5.42 (br s, 1H), 3.25–3.20

(m, 2H), 2.42 (t, J ) 6.4 Hz, 2H), 2.25 (s, 6H), 1.78 (quintet, J )

6.4 Hz, 2H).

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

thylamine (49a). A mixture of imidazole 43¹⁹(300 mg, 2.4 mmol),

1-bromo-2-ﬂuoro-4-iodobenzene (720 mg, 2.4 mmol), cesium

carbonate (1.56 g, 4.8 mmol), and copper(I) iodide (91 mg, 0.48

mmol) in 15 mL of anhydrous DMF was heated to 125 °C for 18 h.

After cooling to room temperature, the mixture was ﬁltered through

a pad of Celite and concentrated under reduced pressure. The residue

was triturated with ethyl acetate and methanol and then ﬁltered.

The ﬁltrate was concentrated under reduced pressure and the residue

was puriﬁed by silica gel ﬂash chromatography (eluted with

methanol/ethyl acetate, 0:1 v/v to 1:9 v/v) to yield 236 mg (33%)

1

puriﬁcation. H NMR (400 MHz, CDCl₃) δ 7.66 (dd, J ) 7.95,

7.57 Hz, 1H), 7.58 (dd, J ) 7.42, 1.00 Hz, 1H), 7.46 (ddd, J )

1.52, 7.44, 7.52 Hz, 1H), 7.43–7.39 (m, 2H), 7.32 (dd, J ) 1.40,

7.49 Hz, 1H), 7.19 (dd, J ) 1.84, 8.18 Hz, 1H), 3.38 (s, 2H), 2.27

(s, 6H).

1

of pale-yellow solid as the title compound. H NMR (400 MHz,

CDCl₃) δ 7.64 (dd, J ) 9.48, 2.35 Hz, 1H), 7.61 (dd, J ) 8.49,

7.51 Hz, 1H), 7.27 (ddd, J ) 8.55, 2.39, 0.99 Hz, 1H), 7.06 (dd, J

) 2.92, 1.23 Hz, 2H), 3.34 (s, 2H), 2.24 (s, 6H).

[2-(4-Bromo-3-ﬂuorophenyl)pyridin-3-ylmethyl]dimethy-

lamine (49d). A mixture of 2-bromo-3-pyridinecarboxaldehyde

(256 mg, 1.4 mmol), 4-bromo-3-ﬂuorobenzeneboronic acid (100

mg, 0.46 mmol), PdCl₂(PPh₃)₂(32 mg, 0.05 mmol), and aqueous

sodium carbonate solution (2 mL, 3.7 mmol, 2 M) in 15 mL of

1,2-dimethoxyethane was heated to 90 °C for 4 h. The mixture

was partitioned between ethyl acetate and water, and the aqueous

layer was then extracted with ethyl acetate twice. The organic layers

were combined, dried with sodium sulfate, ﬁltered, and concentrated

under reduced pressure. The residue was puriﬁed by preparative

TLC (silica gel, 20 cm × 20 cm, 2000 µm, ethyl acetate/hexanes,

1:4 v/v) to yield 72 mg (18%) of 2-(4-bromo-3-ﬂuorophenyl)py-

[4-(4-Bromo-3-ﬂuorophenyl)-2-(2-trimethylsilanylethoxym-

ethyl)-2H-pyrazol-3-ylmethyl]dimethylamine (49b). A mixture

of pyrazole 37 (480 mg, 1.57 mmol), 4-bromo-3-ﬂuorobenzenebo-

ronic acid (413 mg, 1.88 mmol), PdCl₂(PPh₃)₂(110 mg, 0.16 mmol),

and aqueous sodium carbonate solution (6.3 mL, 13.6 mmol, 2 M)

in 50 mL of 1,2-dimethoxyethane was heated to 90 °C for 18 h.

Upon cooling, the mixture was partitioned between brine and ethyl

acetate, and the aqueous layer was extracted thrice with ethyl

acetate. The organic layers were combined, washed with brine, dried

294 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2

Lee et al.

ridine-3-carboxaldehyde. ¹H NMR (400 MHz, CDCl₃) δ 10.07 (s,

1H), 8.88 (dd, J ) 4.73, 1.79 Hz, 1H), 8.32 (dd, J ) 7.88, 1.79

Hz, 1H), 7.71 (dd, J ) 8.10, 6.98 Hz, 1H), 7.50 (ddd, J ) 7.87,

4.74, 0.50 Hz, 1H), 7.45 (dd, J ) 8.96, 1.99 Hz, 1H), 7.22 (dd, J

) 8.17, 1.81 Hz, 1H).

1H), 3.83–3.78 (m, 2H), 2.57–2.52 (m, 2H), 2.26 (s, 6H). Isolated

in 49% yield.

N-(4-Bromo-3-ﬂuorophenyl)-N′,N′-dimethyl-N-pyridin-4-yle-

thane-1,2-diamine (49k). ¹H NMR (400 MHz, CDCl₃) δ 8.17 (d,

J ) 6.4 Hz, 2H), 7.66 (t, J ) 8.1 Hz, 1H), 7.11 (dd, J ) 2.4, 9.3

Hz, 1H), 7.00 (dd, J ) 1.8, 8.5 Hz, 1H), 6.67 (d, J ) 6.5 Hz, 2H),

3.91–3.86 (m, 2H), 2.68–2.63 (m, 2H), 2.34 (s, 6H). Isolated in

45% yield.

To a mixture of 2-(4-bromo-3-ﬂuorophenyl)pyridine-3-carboxal-

dehyde (72 mg, 0.26 mmol) and dimethylamine (0.26 mL, 0.51 mmol,

2 M in THF) in 10 mL of anhydrous DMF was added sodium

triacetoxyborohydride (82 mg, 0.39 mmol). After being stirred at room

temperature for 18 h, the mixture was concentrated under reduced

pressure. The residue was partitioned between water and ethyl acetate.

The aqueous layer was extracted with ethyl acetate twice. The organic

layers were combined, washed with brine, dried with anhydrous sodium

sulfate, and concentrated under reduced pressure to yield 78.3 mg

(99%) of pale-yellow oil as the title compound. The mixture was used

N-(4-Bromo-3-ﬂuorophenyl)-N′,N′-dimethyl-N-pyridin-4-yl-

1

propane-1,3-diamine (49l). H NMR (400 MHz, CDCl₃) δ 8.23

(d, J ) 6.2 Hz, 2H), 7.58 (t, J ) 8.1 Hz, 1H), 7.05 (dd, J ) 2.4,

9.7 Hz, 1H), 6.94 (dd, J ) 1.8, 8.6 Hz, 1H), 6.64 (d, J ) 6.5 Hz,

2H), 3.79–3.74 (m, 2H), 2.32 (t, J ) 6.8 Hz, 2H), 2.23 (s, 6H),

1.84–1.76 (m, 2H). Isolated in 39% yield.

1-(3-Cyano-4-ﬂuorophenyl)-6-(2′-dimethylaminomethyl-3-

ﬂuorobiphenyl-4-yl)-7-ﬂuoro-1H-indazole-3-carboxylic Acid

Amide (50c ). A mixture of 1-(3-cyano-4-ﬂuorophenyl)-7-ﬂuoro-

6-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)-1H-indazole-3-car-

boxylic acid amide (27) (38 mg, 0.09 mmol), (4′-bromo-3′-

ﬂuorobiphenyl-2-ylmethyl)dimethylamine (49c) (27.6 mg, 0.09

mmol), dichlorobis(triphenylphosphine)palladium(II) (7 mg, 0.01

mmol), and aqueous sodium carbonate solution (0.36 mL, 0.72 mmol,

2.0 M) was stirred under argon in 2 mL of anhydrous DME. The

mixture was heated to 90 °C for 18 h. Upon cooling to room

temperature, the mixture was ﬁltered through a pad of Celite and

concentrated under reduced pressure. The residue was then parti-

tioned between water and ethyl acetate. The aqueous layer was then

extracted with ethyl acetate twice. The organic layers were

combined, washed with brine, and dried with anhydrous sodium

sulfate. The residue was puriﬁed by preparative TLC (silica gel,

20 cm × 20 cm, 2000 µm, 7 N ammonia in methanol solution/

ethyl acetate, 1:200 v/v) to give 27.2 mg (58%) of the title

1

in the next step without further puriﬁcation. H NMR (400 MHz,

CDCl₃) δ 8.59 (dd, J ) 4.72, 1.69 Hz, 1H), 7.82 (dd, J ) 7.78, 1.65

Hz, 1H), 7.66–7.54 (m, 1H), 7.37 (dd, J ) 8.23, 1.79 Hz, 1H), 7.29

(dd, J ) 7.77, 4.73 Hz, 1H), 3.34 (s, 2H), 2.20 (s, 6H).

[3-(4-Bromo-3-ﬂuorophenyl)pyridin-4-ylmethyl]dimethy-

lamine (49e). Commercially available 3-bromopyridine-4-carbox-

aldehyde (34) was converted into the title compound 49e using the

1

procedures as described in the preparation of compound 49d. H

NMR (400 MHz, CDCl₃) δ 8.57 (d, J ) 5.07 Hz, 1H), 8.45 (s,

1H), 7.62 (dd, J ) 8.02, 7.33 Hz, 1H), 7.48 (d, J ) 5.07 Hz, 1H),

7.25 (dd, J ) 9.38, 1.94 Hz, 1H), 7.05 (dd, J ) 8.18, 1.89 Hz,

1H), 3.30 (s, 2H), 2.18 (s, 6H).

[3-(4-Bromo-3-ﬂuorophenyl)pyridin-2-ylmethyl]dimethy-

lamine (49f). 3-Iodo-2-pyridinecarboxaldehyde (32) was converted

into the title compound 49f using the procedures as described in

the preparation of compound 49d. ¹H NMR (400 MHz, CDCl₃) δ

8.63 (dd, J ) 4.78, 1.70 Hz, 1H), 7.59 (dd, J ) 8.13, 7.28 Hz,

1H), 7.55 (dd, J ) 7.73, 1.72 Hz, 1H), 7.39 (dd, J ) 9.64, 1.98

Hz, 1H), 7.26 (dd, J ) 7.70, 4.82 Hz, 1H), 7.13 (dd, J ) 8.20,

1.53 Hz, 1H), 3.42 (s, 2H), 2.22 (s, 6H).

[4-(4-Bromo-3-ﬂuorophenyl)pyridin-3-ylmethyl]dimethy-

lamine (49g). 4-Iodo-3-pyridinecarboxaldehyde (33) was converted

into the title compound 49g using the procedures as described in

the preparation of compound 49d. ¹H NMR (400 MHz, CDCl₃) δ

8.65 (s, 1H), 8.56 (d, J ) 5.06 Hz, 1H), 7.61 (dd, J ) 8.15, 7.18

Hz, 1H), 7.43 (dd, J ) 9.54, 1.97 Hz, 1H), 7.19 (d, J ) 5.04 Hz,

1H), 7.15 (dd, J ) 8.21, 1.58 Hz, 1H), 3.35 (s, 2H), 2.20 (s, 6H).

N-(4-Bromo-3-ﬂuorophenyl)-N′,N′-dimethyl-N-phenylethane-

1,2-diamine (49i). A mixture of 1-bromo-2-ﬂuoro-4-iodobenzene

(301 mg, 1.0 mmol), N,N-dimethyl-N′-phenylethane-1,2-diamine

(41) (150 mg, 0.91 mmol), Pd₂(dba)₃(42 mg, 0.046 mmol), sodium

tert-butoxide (184 mg, 1.92 mmol), and XANTPHOS (63 mg, 0.11

mmol) under argon in 1,4-dioxane was heated to 140 °C in a

Biotage Initiator microwave synthesizer for 30 min. Upon cooling,

the mixture was diluted with ethyl acetate and ﬁltered and the ﬁltrate

was concentrated under reduced pressure. The reside was puriﬁed

using preparative TLC plates (silica gel, 20 cm × 20 cm, 2000

µm, methanol/dichloromethane, 1:9 v/v) to give 243 mg (79%) of

the title compound. ¹H NMR (400 MHz, CDCl₃) δ 7.37–7.32 (m,

2H), 7.26 (dd, J ) 8.3, 8.6 Hz, 1H), 7.16–7.12 (m, 3H), 6.58 (dd,

J ) 2.8, 11.8 Hz, 1H), 6.49 (dd, J ) 2.7, 8.8 Hz, 1H), 3.80–3.75

(m, 2H), 2.56–2.51 (m, 2H), 2.25 (s, 6H).

1

compound. H NMR (400 MHz, CDCl₃) δ 8.35 (d, J ) 8.37 Hz,

1H), (dt, J ) 5.31, 2.63 Hz, 1H), 7.94 (m, 1H), 7.68 (d, J ) 12.01

Hz, 1H), 7.66 (dd, J ) 1.43, 11.99 Hz, 1H), 7.54 (d, J ) 7.26 Hz,

1H), 7.49–7.44 (m, 2H), 7.43–7.29 (m, 4H), 6.95 (s, 1H), 5.71 (s,

1H), 3.38 (s, 2H), 2.20 (s, 6H). Mass spectrum (LCMS, ESI+)

m/z calcd for C₃₀H₂₂F₃N₅O {M + H]⁺526.2; found, 526.0.

1-(3-Aminobenzo[d]isoxazol-5-yl)-6-(2′-dimethylaminomethyl-

3-ﬂuorobiphenyl-4-yl)-7-ﬂuoro-1H-indazole-3-carboxylic Acid

Amide (51c). A mixture of 1-(3-cyano-4-ﬂuorophenyl)-6-(2′-

dimethylaminomethyl-3-ﬂuorobiphenyl-4-yl)-7-ﬂuoro-1H-indazole-

3-carboxylic acid amide (50c) (37.8 mg, 0.072 mmol), acetohy-

droxamic acid (16 mg, 0.22 mmol), and potassium carbonate (60

mg, 0.43 mmol) in 4.9 mL of DMF and 0.7 mL of water was stirred

at room temperature overnight. The reaction was then quenched

with water. The mixture was extracted with ethyl acetate thrice.

The organic layers were combined, washed with brine, dried with

anhydrous sodium sulfate, and concentrated under reduced pressure.

The residue was puriﬁed by preparative TLC (silica gel, 20 cm ×

20 cm, 2000 µm, 7 N ammonia in methanol/ethyl acetate/

dichloromethane, 1:5:5 v/v/v) to yield 21.6 mg (56%) of the title

1

compound as an off-white solid. H NMR (400 MHz, DMSO-d₆)

δ 8.21 (d, J ) 8.42 Hz, 1H), 8.20 (t, J ) 2.00 Hz, 1H), 8.05 (br s,

1H) 7.93 (dt, J ) 8.87, 2.16 Hz, 1H), 7.65 (d, J ) 8.76 Hz, 1H)

7.65 (br s, 1H), 7.59 (t, J ) 7.90 Hz, 1H), 7.52 (dd, J ) 1.44,

11.49 Hz, 1H), 7.48 (dd, J ) 2.32, 13.89 Hz), 1H), 7.46 (d, J )

13.97 Hz, 1H), 7.41–7.31 (m, 4H), 6.57 (s, 2H) 3.32 (s, 2H), 2.09

(s, 6H). HRMS (FAB+) m/z calcd for C₃₀H₂₅N₆O₂F₂[M + H]⁺,

539.2007; found, 539.2000.

Compounds 50a and 50d-l were prepared using the same

procedure as for the preparation of compound 50c using the

corresponding biarylbromide 49a and 49d-l and were converted

to compounds 51a and 51d-l using the same procedure as for the

preparation of compound 51c. Compound 50b was prepared using

the same procedure as for the preparation of compound 50c using

biaryl bromide 49b.

Compounds 49h, 49j, 49k, and 49l were prepared using the same

procedure as for the preparation of compound 49i using anilines

44, 42, 47, and 48, respectively.

(4-Bromo-3-ﬂuorophenyl)methylpyridin-4-ylamine (49h). ¹H

NMR (400 MHz, CDCl₃) δ 8.28 (br s, 2H), 7.57 (dd, J ) 8.0, 8.4

Hz, 1H), 7.02 (dd, J ) 2.5, 9.7 Hz, 1H), 6.93 (ddd, J ) 0.9, 2.5,

8.6 Hz, 1H), 6.65 (m, 2H), 3.33 (s, 3H). Isolated in quantitative

yield.

N-(4-Bromo-3-ﬂuorophenyl)-N′,N′-dimethyl-N-pyridin-3-yle-

1

thane-1,2-diamine (49j). H NMR (400 MHz, CDCl₃) δ 8.44 (d,

1-(3-Aminobenzo[d]isoxazol-5-yl)-6-[4-(2-dimethylaminome-

J ) 2.4 Hz, 1H), 8.33 (d, J ) 3.9 Hz, 1H), 7.44 (ddd, J ) 1.2, 2.4,

8.2 Hz, 1H), 7.35 (t, J ) 8.3 Hz, 1H), 7.26 (dd, J ) 4.7, 8.3 Hz,

1H), 6.70 (dd, J ) 2.7, 11.1 Hz, 1H), 6.59 (dd, J ) 2.4, 8.8 Hz,

thylimidazol-1-yl)-2-ﬂuorophenyl]-7-ﬂuoro-1H-indazole-3-car-

1

boxylic Acid Amide (51a). H NMR (400 MHz, CD₃OD) δ 8.28

(d, J ) 8.40 Hz, 1H), 8.13 (t, J ) 2.28 Hz, 1H), 7.91 (dt, J ) 8.89,

7-Fluoroindazoles as FXa Inhibitors

Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2 295

2.32 Hz, 1H), 7.74 (dd, J ) 2.00, 10.78 Hz, 1H), 7.69 (t, J ) 8.06

Hz, 1H), 7.60 (d, J ) 8.79 Hz, 1H), 7.57 (td, J ) 6.44, 1.97 Hz,

1H), 7.44 (dd, J ) 8.15, 5.46 Hz, 1H), 4.23 (s, 1H), 3.52 (s, 2H),

2.25 (s, 6H). HRMS (ESI+) m/z calcd for C₂₇H₂₃N₈O₂F₂[M +

H]⁺, 529.1912; found, 529.1915.

11.7 Hz, 1H), 7.26 (dd, J ) 2.2, 8.3 Hz, 1H), 6.83–6.80 (m, 2H),

6.59 (br s, 2H), 3.36 (s, 3H). HRMS (FAB+) m/z calcd for

C₂₇H₂₀N₇O₂F₂[M + H]⁺, 512.1647; found, 512.1662.

1-(3-Aminobenzo[d]isoxazol-5-yl)-6-{4-[(2-dimethylaminoet-

hyl)phenylamino]-2-ﬂuorophenyl}-7-ﬂuoro-1H-indazole-3-car-

boxamide (51i). ¹H NMR (400 MHz, DMSO-d₆) δ 9.48 (br s, 1H),

8.19 (br s, 1H), 8.17 (d, J )8.4 Hz, 1H), 8.03 (br s 1H), 7.91 (dt,

J ) 2.1, 8.8 Hz, 1H), 7.66 (d, J ) 8.9 Hz, 1H), 7.64 (br s, 1H),

7.50–7.45 (m, 2H), 7.38–7.32 (m, 2H), 7.29 (d, J )7.8 Hz, 2H),

7.27 (t, J ) 7.8 Hz, 1H), 6.81 (dd, J ) 2.2, 13.3 Hz, 1H), 6.67 (dd,

J ) 2.3, 8.6 Hz, 1H), 6.58 (br s, 2H), 4.12–4.05 (m, 2H), 3.37–3.31

(m, 2H), 2.85 (br s, 3H), 2.84 (br s, 3H), 2.30 (s, 3H). HRMS

(FAB+) m/z calcd for C₃₁H₂₈N₇O₂F₂[M + H]⁺, 568.2273; found,

568.2270.

1-(3-Aminobenzo[d]isoxazol-5-yl)-6-{4-[(2-dimethylaminoet-

hyl)pyridin-3-ylamino]-2-ﬂuorophenyl}-7-ﬂuoro-1H-indazole-3-

carboxamide (51j). ¹H NMR (400 MHz, DMSO-d₆) δ 9.64 (br s,

1H), 8.55 (d, J )2.0 Hz, 1H), 8.43 (d, J ) 4.8 Hz, 1H), 8.22 (d,

J ) 8.3 Hz, 1H), 8.21 (br s, 1H), 8.06 (br s, 1H), 7.94–7.89 (m,

2H), 7.71 (dd, J ) 5.2, 8.4 Hz, 1H), 7.67 (d, J ) 8.7 Hz, 1H), 7.66

(br s, 1H), 7.56 (t, J ) 8.5 Hz, 1H), 7.39 (dd, J ) 6.0, 8.3 Hz,

1H), 7.23 (d, J ) 12.8 Hz, 1H), 7.07 (d, J ) 8.3 Hz, 1H), 6.58 (br

s, 2H), 4.22–4.16 (m, 2H), 3.42–3.36 (m, 2H), 2.85 (br s, 3H),

2.84 (br s, 3H), 2.33 (s, 6H). HRMS (FAB+) m/z calcd for

C₃₀H₂₇N₈O₂F₂[M + H]⁺, 569.2225; found, 569.2226.

1-(3-Aminobenzo[d]isoxazol-5-yl)-6-{4-[(2-dimethylaminoet-

hyl)pyridin-4-ylamino]-2-ﬂuorophenyl}-7-ﬂuoro-1H-indazole-3-

carboxamide (51k). ¹H NMR (400 MHz, DMSO-d₆) δ 8.27 (d, J

) 6.9 Hz, 2H), 8.28 (d, J ) 8.4 Hz, 1H), 8.11 (t, J )2.2 Hz, 1H),

7.89 (dt, J ) 2.3, 8.9 Hz, 1H), 7.79 (t, J )8.1 Hz, 1H), 7.59 (d, J

) 8.9 Hz, 1H), 7.49–7.38 (m, 3H), 7.09 (d, J ) 5.9 Hz, 2H),

4.36–4.29 (m, 2H), 3.40–3.34 (m, 2H), 2.84 (br s, 6H), 2.70 (s,

6H). HRMS (ESI+) m/z calcd for C₃₀H₂₇N₈O₂F₂[M + H]⁺,

569.2225; found, 569.2225.

1-(3-Cyano-4-ﬂuorophenyl)-6-[4-(3-dimethylaminomethyl-

1H-pyrazol-4-yl)-2-ﬂuorophenyl]-7-ﬂuoro-1H-indazole-3-car-

boxylic Acid Amide (51b). To a mixture of amide 50b (49.4 mg,

0.077 mmol) in dichloromethane (3 mL) and ethanol (0.1 mL) was

added triﬂuoroacetic acid (1 mL, 13.5 mmol) slowly at 0 °C. After

being stirred at 0 °C for 6 h, the mixture was kept in a freezer at

–4 °C without stirring for 18 h. The mixture was stirred and then

allowed to warm to room temperature for 0.5-1 h while the reaction

was monitored by LC-MS. The mixture was then concentrated

under reduced pressure. To the residue was added acetohydroxamic

acid (23 mg, 0.31 mmol), potassium carbonate (85 mg, 0.61 mmol),

4.9 mL of DMF, and 0.7 mL of water. After being stirred at room

temperature for 18 h, the reaction was then quenched with water.

The mixture was thrice extracted with ethyl acetate. The organic

layers were combined, washed with brine, dried with anhydrous

sodium sulfate, and concentrated under reduced pressure. The

residue was puriﬁed by preparative TLC (silica gel, 20 cm × 20

cm, 2000 µm, 7 N ammonia in methanol/ethyl acetate, 1:9 v/v) to

1

yield 39.2 mg (97%) of the title compound. H NMR (400 MHz,

DMSO-d₆) δ 8.23–8.20 (m, 2H), 8.06 (s, 1H), 7.93 (m, J ) 8.89

Hz, 1H), 7.68–7.66 (m, 3H), 7.59–7.53 (m, 2H), 7.44 (dd, J )

6.29, 8.00 Hz, 1H), 7.29 (br s, 1H), 6.69 (br s, 1H), 6.59 (s, 2H),

3.35 (s, 2H), 1.76 (s, 6H). HRMS (FAB+) m/z calcd for

C₂₇H₂₃N₈O₂F₂[M + H]⁺, 529.1912; found, 529.1903.

1-(3-Aminobenzo[d]isoxazol-5-yl)-6-[4-(3-dimethylaminome-

thylpyridin-2-yl)-2-ﬂuorophenyl]-7-ﬂuoro-1H-indazole-3-car-

boxylic Acid Amide (51d). ¹H NMR (400 MHz, DMSO-d₆) δ 8.59

(dd, J ) 1.6, 4.7 Hz, 1H), 8.22 (d, J ) 8.4 Hz, 1H), 8.20 (t, J )

1.9 Hz, 1H), 8.06 (br s, 1H), 7.93 (dt, J ) 8.75, 2.28 Hz, 1H), 7.89

(dd, J ) 1.53, 7.78 Hz, 1H), 7.75 (dd, J ) 10.55, 1.29 Hz, 1H),

7.66–7.61 (m, 4H), 7.47 (dd, J ) 5.83, 8.23 Hz, 1H), 7.42 (dd, J

) 4.71, 7.75 Hz, 1H), 6.57 (s, 2H), 3.40 (s, 2H), 2.13 (s, 6H).

HRMS (FAB+) m/z calcd for C₂₉H₂₄N₇O₂F₂[M + H]⁺, 540.1960;

found, 540.1952.

1-(3-Aminobenzo[d]isoxazol-5-yl)-6-[4-(4-dimethylaminome-

thylpyridin-3-yl)-2-ﬂuorophenyl]-7-ﬂuoro-1H-indazole-3-car-

boxylic Acid Amide (51e). ¹H NMR (400 MHz, DMSO-d₆) δ 8.57

(d, J ) 5.04 Hz, 1H), 8.49 (s, 1H), 8.22 (d, J ) 8.38 Hz, 1H), 8.20

(t, J ) 1.96 Hz, 1H), 8.06 (br s, 1H), 7.93 (dt, J ) 8.86, 2.28 Hz,

1H), 7.66–7.62 (m, 3H), 7.54 (m, 2H), 7.47 (dd, J ) 5.80, 8.23

Hz, 1H), 7.42 (dd, J ) 1.56, 7.91 Hz, 1H), 6.57 (s, 2H), 3.40 (s,

2H), 2.11 (s, 6H). HRMS (ESI+) m/z calcd for C₂₉H₂₄N₇O₂F₂[M

+ H]⁺, 540.1960; found, 540.1959.

1-(3-Aminobenzo[d]isoxazol-5-yl)-6-[4-(3-dimethylaminome-

thylpyridin-4-yl)-2-ﬂuorophenyl]-7-ﬂuoro-1H-indazole-3-car-

boxylic Acid Amide (51f). ¹H NMR (400 MHz, DMSO-d₆) δ 8.64

(br s, 1H), 8.56 (d, J ) 5.00 Hz, 1H), 8.24 (d, J ) 8.39 Hz, 1H),

8.22 (m, 1H), 8.07 (s, 1H), 7.95 (dt, J ) 8.92, 2.26 Hz), 7.72–7.66

(m, 4H), 7.53 (dd, J ) 1.52, 7.95 Hz, 2H), 7.48 (dd, J ) 5.81,

8.23 Hz, 1H), 7.41 (d, J ) 5.03 Hz, 1H), 6.59 (br s, 2H), 3.39 (s,

2H), 2.13 (s, 6H). HRMS (FAB+) m/z calcd for C₂₉H₂₄N₇O₂F₂[M

+ H]⁺, 540.1960; found, 540.1954.

1-(3-Aminobenzo[d]isoxazol-5-yl)-6-[4-(2-dimethylaminome-

thylpyridin-3-yl)-2-ﬂuorophenyl]-7-ﬂuoro-1H-indazole-3-car-

boxylic Acid Amide (51g). ¹H NMR (400 MHz, DMSO-d₆) δ 8.59

(dt, J ) 4.75, 1.58 Hz), 8.25–8.20 (m, 2H), 8.07 (br s, 1H), 7.95

(dt, J ) 8.84, 1.71 Hz, 1H), 7.83 (dt J ) 7.76, 1.54 Hz, 1H), 7.74

(m, J ) 11.51 Hz, 1H), 7.69–7.64 (m, 3H), 7.56 (dt, J ) 7.94,

1.47 Hz, 1H), 7.50–7.44 (m, 2H), 6.59 (br s, 2H), 3.44 (s, 2H),

2.17 (s, 6H). HRMS (FAB+) m/z calcd for C₂₉H₂₄N₇O₂F₂[M +

H]⁺, 540.1960; found, 540.1955.

1-(3-Aminobenzo[d]isoxazol-5-yl)-7-ﬂuoro-6-[2-ﬂuoro-4-(me-

thylpyridin-4-ylamino)phenyl]-1H-indazole-3-carboxamide (51h).

¹H NMR (400 MHz, DMSO-d₆) δ 8.24–8.19 (m, 4H), 8.07 (br s,

1H), 7.94 (dt, J ) 2.2, 8.9 Hz, 1H), 7.69–7.65 (m, 2H), 7.60 (t, J

) 8.4 Hz, 1H), 7.44 (dd, J ) 5.6, 8.0 Hz, 1H), 7.34 (dd, J ) 2.1,

1-(3-Aminobenzo[d]isoxazol-5-yl)-6-{4-[(3-dimethylaminopro-

pyl)pyridin-4-ylamino]-2-ﬂuorophenyl}-7-ﬂuoro-1H-indazole-3-

1

carboxamide (51l). H NMR (400 MHz, DMSO-d₆) δ 8.28 (d, J

) 8.4 Hz, 1H), 8.24 (d, J ) 7.3 Hz, 2H), 8.11 (t, J )2.2 Hz, 1H),

7.89 (dt, J ) 2.4, 8.9 Hz, 1H), 7.79 (t, J ) 8.1 Hz, 1H), 7.59 (d,

J ) 8.9 Hz, 1H), 7.47 (dd, J ) 2.2, 10.0 Hz, 1H), 7.44–7.38 (m,

2H), 7.09 (br s, 2H), 4.09–4.03 (m, 2H), 3.27–3.21 (m, 2H), 2.90

(s, 6H), 2.70 (s, 6H), 2.23–2.13 (m, 2H). HRMS (FAB+) m/z calcd

for C₃₁H₂₉N₈O₂F₂[M + H]⁺, 583.2382; found, 583.2382.

Enzymology. All buffer salts were obtained from Sigma Chemi-

cal Company (St. Louis, MO) and were of the highest purity

available. The enzyme-substrate S-2765 (Z-D-Arg-Gly-Arg-p-

nitroanilide) was obtained from DiaPharma (West Chester, OH).

N-Succinyl-Ala-Ala-Pro-Arg-p-nitroanilide (Bachem L-1720) was

obtained from Bachem Bioscience (King of Prussia, PA). Human

R-thrombin and human factor Xa were obtained from Enzyme

Research Laboratories (South Bend, IN). Human trypsin was

obtained from Calbiochem (La Jolla, CA).

Compounds were assessed for their inhibitory activity toward

factor Xa, thrombin, and trypsin by kinetic analysis using p-

nitroaniline chromogenic substrates monitored at 405 nm. The assay

buffer employed was 50 mM HEPES, pH 7.5, 200 mM NaCl, and

fresh 0.05% n-octyl ꢀ-D-glucopyranoside. DMSO was present at a

ﬁnal concentration of 4%, derived from the substrate and inhibitory

compound stock solutions. In a 96-well low-binding polystyrene

plate, 280 µL of substrate in assay buffer was preincubated at 37

°C for 15 min with 10 µL of test compound in DMSO to obtain

ﬁnal test compound concentrations that bracketed the K_i. Reactions

were initiated by addition of 10 µL of protease, and increase in

absorbance due to proteolytic cleavage of substrate was kinetically

monitored at 37 °C and 405 nm with a Molecular Devices

Spectramax 340 plate reader. Initial velocities were determined by

analysis of the initial linear portion of the reactions. Plots of V_o/V_i

vs inhibitor concentration, where V_ois the velocity without inhibitor

and V_iis the inhibited velocity, were ﬁt to a linear regression line,

and IC₅₀was determined from the reciprocal of the slope. The

dissociation constant (K_i) was calculated using the equation K_i)

296 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 2

Lee et al.

IC₅₀/(1 + S/K_m) where S is the substrate concentration.⁴³K_mfor

the enzyme–substrate pair was determined from Hanes-Wolf plots

using the same ﬁnal buffer conditions as K_ideterminations.

Respective substrate, substrate concentration, K_m, and enzyme

concentrations for human factor Xa were S-2765 (Z-D-Arg-Gly-

Arg-p-nitroanilide), S ) 100 µM, K_m) 260 µM, fXa ) 0.5 nM.

For human R-thrombin, they were L-1720 (succinyl-Ala-Ala-Pro-

Arg-p-nitroanilide), S ) 100 µM, K_m) 320 µM, thrombin ) 1

nM. For human trypsin, they were S-2765, S ) 60 µM, K_m) 61

µM, trypsin ) 0.3 nM. Within-run assay coefﬁcient of variation

(CV) was generally <10%; between-run the CV was <20%.

In Vitro Coagulation Assays. The plasma concentrations of

factor Xa inhibitors needed to produce a 2-fold prolongation of

activated partial thromboplastin time (aPTT) were determined in

human plasma. Fresh whole human blood was centrifuged to obtain

plasma, and factor Xa inhibitors were added to yield concentrations

from 0.001 to 100 µM. After a short stabilization period, the samples

were placed in an ACL-100 microsampler coagulation analyzer

(Instrument Laboratory, Milano, Italy) to determine clotting times

in seconds.

Human Liver Microsomal (HLM) Stability Assay. Pooled

human liver microsomes (1 mg/mL protein) were preincubated with

the test compound at 37 °C for 3 min in the absence of NADPH as

described below. The enzymatic reaction was initiated by addition

of NADPH and then incubated under the same conditions. One

aliquot (1 mL) of the incubation mixture was withdrawn at 0, 15,

30, and 60 min and combined immediately with 0.5 mL of ACN.

After vortex-mixing, the sample was centrifuged at approximately

10000g for 5 min. The supernatant (0.7 mL) was transferred into

a 1 mL vial and was analyzed for metabolites using an Agilent

1100 LC system equipped with a Zorbax SB-C18 column (150 mm

× 2.1 mm i.d., 3.5 µm) interfaced with a Micromass Quattro Micro

mass spectrometer that was operated with the electrospray ionization

in positive ion mode.

carried out using CNX. The ﬁnal structure was reﬁned to an R_factor

of 24.6 and R_freeof 31.3.

Acknowledgment. The authors thank John Masucci and

William J. Jones for HRMS assistance, Karen A. Diloreto and

Rolanda. L. Reed for ADME analytical assistance, Michael X.

Kolpak for NMR assistance, and Kenneth Singleton, Diane

Maguire, and Shariff Bayoumy for the cloning, expression, and

puriﬁcation of factor Xa.

Supporting Information Available: HPLC analysis results for

the target compounds 25a-d, 29a-c, and 51a-l. This material is

available free of charge via the Internet at http://pubs.acs.org.

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JM701217R

Article Doi

7-Fluoroindazoles as potent and selective inhibitors of factor Xa

DOI: 10.1021/jm701217r

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Article abstract of DOI:10.1021/jm701217r

Full text of DOI:10.1021/jm701217r

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