Potent and selective TF/FVIIa inhibitors containing a neutral P1 ligand

Bioorganic & Medicinal Chemistry 14 (2006) 7688–7705

Potent and selective TF/FVIIa inhibitors containing

a neutral P1 ligand

Masanori Miura,^*Norio Seki, Takanori Koike, Tsukasa Ishihara, Tatsuya Niimi,

Fukushi Hirayama, Takeshi Shigenaga, Yumiko Sakai-Moritani, Tomihisa Kawasaki,

Shuichi Sakamoto, Minoru Okada, Mitsuaki Ohta and Shin-ichi Tsukamoto

Institute for Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan

Received 2 June 2006; revised 9 August 2006; accepted 10 August 2006

Available online 30 August 2006

Abstract—Inhibition of tissue factor/factor VIIa complex (TF/FVIIa) is an attractive strategy for antithrombotic therapies. We

began with an investigation of a non-amidine TF/FVIIa inhibitor based on a modiﬁcation of amidine compound 1. Optimization

of the substituents on the P1 phenyl portion of the compound 1 led to a neutral or less basic alternative for the 4-amidinophenyl

moiety. By further optimization of the substituents on the central phenyl ring, a highly potent and selective TF/FVIIa inhibitor 17d

was discovered.

1. Introduction

More recently, much attention has also been directed

toward the inhibitors of the tissue factor/factor VIIa

complex (TF/FVIIa). The TF is an integral membrane

protein not normally in contact with blood, but in a dis-

ease state or during injury it is exposed to the blood and

comes in contact with FVIIa localized on the surface of

subendothelial cells, forming the TF/FVIIa complex.

TF/FVIIa is a serine protease complex that triggers the

cascade of coagulation reactions by activating factors

X and IX to the corresponding active serine protease

forms, ultimately resulting in the generation of thrombin

and a ﬁbrin clot.⁹It is noteworthy that bleeding studies

indicate that inhibition of the TF/FVIIa complex has the

widest safety window with respect to therapeutic eﬀec-

tiveness and bleeding risk of any anticoagulant ap-

proach tested, such as the inhibition of thrombin and

factor Xa.¹⁰Since there is a clear need for safe and eﬀec-

tive anticoagulants to obtain maximum patient compli-

ance, inhibitors of the TF/FVIIa complex have been of

considerable interest to medicinal chemists.

Thromboembolic disorders are the major cause of

morbidity and mortality in the developed world.¹

Although current anticoagulant therapies for these

diseases consist of low molecular weight heparin

(LMWH)²and warfarin³in acute and chronic set-

tings, respectively, the problems associated with these

agents are well recognized.⁴Within the past decade,

research eﬀorts to identify small molecule inhibitors

of blood coagulation enzymes as novel therapies for

thromboembolic disorders have increased. The most

widely studied targets for antithrombotic intervention

have been trypsin-like serine proteases, such as

thrombin⁵and factor Xa (FXa),⁶which play critical

roles in the coagulation cascade, with thrombin initi-

ating clot formation and FXa providing the sole

mechanism for thrombin activation.⁷There is sub-

stantial evidence suggesting that FXa should be a

more attractive target for inhibitor design;⁸however,

there are currently no direct FXa inhibitors marketed

as drugs.

Several groups have reported their eﬀorts toward the de-

sign and synthesis of novel TF/FVIIa inhibitors.¹¹Many

of these inhibitors retain a highly basic amidine group

bound in the S1 pocket of TF/FVIIa in the vicinity of

Asp 189. It is generally accepted that inhibitors contain-

ing highly basic functions such as amidine groups are

often poorly absorbed and/or are associated with unde-

sirable side eﬀects.¹²In addition, it is not easy to develop

Keywords: Thromboembolic disorders; Trypsin-like serine protease;

Tissue factor/factor VIIa; Non-amidine inhibitor.

*

Corresponding author. Tel.: +81 29 863 6691; fax: +81 29 852

5387; e-mail: masanori-miura@jp.astellas.com

doi:10.1016/j.bmc.2006.08.010

M. Miura et al. / Bioorg. Med. Chem. 14 (2006) 7688–7705

7689

Figure 1. Structure of compound 1.

highly selective compounds from inhibitors that possess

amidine moiety, because other serine protease enzymes

such as thrombin, FXa, and trypsin have Asp 189 at

the base of the S1 pocket.

Our strategy was to ﬁrst ﬁnd a less basic or neutral alter-

native for this amidine moiety, and then to convert the

inhibitor to potent and selective TF/FVIIa inhibitors

by exploiting the potential structure based drug design.

With the above considerations in mind, we had initiated

the TF/FVIIa inhibitor development program using a

compound 1 recently reported by Senokuchi and co-

workers as a starting point (Fig. 1).^11aHerein, we wish

to report our progress on the P1 group optimization

of 1 and the identiﬁcation of 3-aminocarbonylphenyl

and 4-aminomethylphenyl moieties as important

replacements for the 4-amidinophenyl moiety at the P1

group. In addition, we describe our progress on the cen-

tral phenyl group optimization, which has led to the

identiﬁcation of 17d as a potent and selective TF/FVIIa

inhibitor (IC₅₀= 0.69 lM).

Scheme 1. Reagents and conditions: (a) triﬂuromethanesulfonic anhy-

dride, pyridine, CH₂Cl₂; (b) NaClO₂, NaH₂PO₄, 2-methyl-2-butene,

tert-butanol, CH₃CN, H₂O; (c) (COCl)₂, isobutylamine, NEt₃,

CH₂Cl₂; (d) (2-formylphenyl)boronic acid, Pd(PPh₃)₄, K₃PO₄, DMF;

(e) NaClO₂, NaH₂PO₄, 2-methyl-2-butene, tert-butanol, CH₃CN,

H₂O; (f) ArNH₂. 1-ethyl-3-(3-dimethylaminopropyl)carbodimide

hydrochloride, 1-hydroxybenzotriazole, DMF; (g) NaOH, MeOH;

(h) HCl, AcOEt for 7g and 7h.

NBS, followed by treatment with AcONa in AcOH,

gave acetoxy compound 19. After hydrolysis of 19,

condensation with amine gave amide 20. The alcohol

group of 20 was converted to an aldehyde with

MnO₂, which was protected by acetal, to aﬀord the

1,3-dioxolane analogue 21. Lithiation of 21 with

n-BuLi and B(OⁱPr)₃followed by quenching with

hydrochloric acid gave arylboronic acid 22. Suzuki

cross-coupling reaction with the boronic acid 22 and

corresponding 2-bromobenzoate analogues produced

biaryls (23 and 24), which were converted to the de-

sired products (17b and 17c) by the same method as

described in Scheme 1.

2. Chemistry

The synthesis of compounds (7a–h) is shown in Scheme

1. Treatment of triﬂuoromethanesulfonic anhydride

with phenol 2 and oxidation of aldehyde 3 by NaClO₂

aﬀorded an acid, which was converted to acyl chloride

with oxalyl chloride and then reacted with 2-methyl-1-

propylamine to aﬀord amide 4. Suzuki cross-coupling

reaction with (2-formylphenyl)boronic acid aﬀorded

biphenyl 5. Oxidation of aldehyde 5 gave compound 6.

After condensation of carboxylic acid 6 with corre-

sponding aniline derivatives, hydrolysis of the methyl

ester under basic conditions (NaOH/water) gave the

desired carboxylic acid (7a–f). The benzylamine

analogues (7g and 7h) were achieved by deprotection

of the Boc group.

The synthesis of compounds 12h–k and 17d, which have

nitrogen atoms at the 4⁰-position, is shown in Scheme 4.

The 4⁰-nitrobiaryl intermediates (26 and 27) were pre-

pared in four steps from methyl 2-bromo-5-nitrobonzo-

ate 25 in the same manner as described in Scheme 2.

Hydrolysis of the methyl ester of 27, followed by con-

densation of the resulting acid with 3-aminobenzamide,

gave amide 28. Deprotection of the tert-butyl group

with TFA aﬀorded the desired compound 12h. Com-

pound 12i was synthesized by the same method as that

for 12h, from 29, which was obtained by hydrogenation

of the nitro group of 28 in the presence of 10% palladi-

um on carbon. Because of a reaction failure from 29 to

33,¹⁴we used a methoxycarbonyl instead of an amino-

carbonyl group at the 3-position of the P1 phenyl ring.

The intermediate 26 was converted to an amine 30 in

the same manner as that for 29, followed by reductive

amination with HCHO, which gave dimethylamino ana-

logue 32. Hydrolysis of the methyl ester of 32, followed

Compounds 12a–f were synthesized in seven steps from

2-bromobenzoic acid derivatives (8a–f) in the same man-

ner as described in Scheme 1 (Scheme 2). In the case of

4⁰-methoxy analogue 12g, biaryl intermediate 10g was

prepared by cross-coupling reaction of methyl 2-bro-

mobenzoate with commercially available 2-formyl-4-

methoxybenzenboronic acid.

The synthesis of compound 17a was accomplished by

the use of bis(pinacolato)diboron;¹³however, with only

unsatisfactory yield (34%), the cross-coupling product

16 was obtained. We thus synthesized the compounds

with other 4⁰-substituents (17b and 17c) by the use of

boronic acid 22 (Scheme 3). Bromination of 18 using

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M. Miura et al. / Bioorg. Med. Chem. 14 (2006) 7688–7705

Scheme 2. Reagents and conditions: (a) BnBr, KHCO₃, DMF for 9a–e, or tert-butanol, MgSO₄, H₂SO₄for 9f; (b) (2-formylphenyl)boronic acid for

10a–f or 2-formyl-4-methoxybezenboronic acid for 10g, Pd(PPh₃)₄, K₃PO₄, DMF; (c) NaClO₂, NaH₂PO₄, 2-methyl-2-butene, tert-butanol, CH₃CN,

H₂O; (d) EtI for 11b or MeI for 11c–g, KHCO₃, DMF; (e) H₂, MeOH for 12b–e or TFA, CH₂Cl₂for 12f; (f) 3-aminobenzamide, 1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide hydrochloride 1-hydroxybenzotriazole, DMF; (g) NaOH, MeOH.

Scheme 3. Reagents and conditions: (a) TMSCH₂CH₂OH, DCC, DMF, pyridine, cat. DMAP; (b) benzyl 5-[(isobutylamino)-carbonyl]-2-

{[(triﬂuromethyl)sulfonyl]oxy}benzoate (15), bis(pinacolato)diboron, PdCl₂(dppf), K₃PO₄, dioxane; (c) TBAF, THF; (d) 3-aminobezamide, 1-ethyl-

3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, DMF; (e) NaOH, MeOH; (f) NBS, AIBN, CCl₄; (g) AcONa,

AcOH; (h) NaOH, MeOH; (i) isobutylamine, WSC, HOBt, DMF; (j) MnO₂, CHCl₃; (k) TsOH, ethyleneglycol, toluene; (l) n-BuLi, B(O-i-Pr)₃, Et₂O,

then HCl aq; (m) benzyl 2-bromo-5-methylbenzoate for 23 or tert-butyl 2-bromo 5-chlorobenzoate for 24, Pd(PPh₃)₄, K₃PO₄, DMF; (n) NaClO₂,

NaH₂PO₄, 2-methyl-2-butene, tert-butanol, CH₃CN, H₂O; (o) Mel, KHCO₃, DMF; (p) H₂, Pd-C, MeOH for 16b or TFA, CH₂Cl₂for 16c.

by condensation of the resulting acid with ammonia,

gave the amide 33, which converted to the desired prod-

uct 12j. Compounds 17d and 12k were synthesized

similarly as described above via preparation of

N-methyltriﬂuoroacetamide analogues (34 and 35) in

three steps from the aniline analogues (30 and 31) under

standard conditions, respectively.

3. Results and discussion

To evaluate the synthesized target compounds, the IC₅₀

values for the inhibition of TF/FVIIa enzymatic activi-

ties were determined using the chromogenic substrate

s-2288. We investigated the alternative structures of

the amidine moiety for the S1 site by the modiﬁcation

M. Miura et al. / Bioorg. Med. Chem. 14 (2006) 7688–7705

7691

Scheme 4. Reagents and conditions: (a) boronic acid 22 for 26 or (2-formylphenyl)boronic acid for 27, Pd(PPh₃)₄, K₃PO₄, DMF; (c) NaClO₂,

NaH₂PO₄, 2-methyl-2-butene, tert-butanol, CH₃CN, H₂O; (d) MgSO₄, H₂SO₄, tert-butanol, CH₂Cl₂; (e) NaOH, MeOH; (f) 3-aminobenzamide, for

28 or methyl 3-aminobenzoate for 30 and 31, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, DMF; (g) H₂,

Pd–C, MeOH; (h) TFA, CH₂Cl₂; (i) HCHO, NaBH(OAc)₃, 1,2-dichloroethane; (j) NaOH, MeOH; (k) NH₄Cl, i-Pr₂NEt, 1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, DMF; (l) TFAA, pyridine, CH₂Cl₂; (m) MeI, K₂CO₃, 2-butanone.

Table 1. In vitro inhibitory activities against TF/FVIIa

of compound 1 shown in Table 1. Removal of the ami-

dine moiety from compound 1 or introduction of sub-

stituents such as MeO and Cl resulted in loss of the

activity (7a–d). In the case of other substituents, though

the 3-acetyl and 3-aminomethyl analogues 7f and 7h,

respectively, did not have potency, the 3-aminocarbonyl

and 4-aminomethyl derivatives 7e and 7g showed mod-

erate inhibitory activities (IC₅₀= 19 and 40 lM,

respectively).

Compound

R

IC₅₀(lM)

1

0.089

>200

In order to understand the SARs for these compounds,

the binding conformation of compound 1 to the TF/

FVIIa complex was studied.¹⁵As shown in Figure 2,

the molecular modeling suggested that the amidine moi-

ety interacts with Asp 189 in the S1 pocket to form a salt

bridge, the central phenyl group lies nearby the S2-pock-

et, and the carboxylic acid ﬁts optimal binding with His

57. We used the molecular modeling of our compounds

7e and 7g. From Figures 3a and b, it is clear that the

proposed overall binding mode for each compound is

generally similar except for the S1 site. For example,

the molecular modeling suggested that the nitrogen of

the central amide group might be capable of hydro-

gen-bonding with the OH of Ser 195, and that the car-

boxylic acid group also forms a salt bridge with His

57. As for the S1 site, the aminocarbonyl group of com-

pound 7e is in the S1 speciﬁcity pocket, and the carbonyl

oxygen of the aminocarbonyl substituent is in close

proximity to the OH group of Ser 190 and forms a

hydrogen-bond. Moreover, the nitrogen of the amino-

carbonyl group forms a hydrogen-bond to the carbonyl

oxygen of Trp 215 and Val 227 through a bridging water

molecule.¹⁶This might contribute to the improved

potency of compound 7e. These interactions are diﬀer-

7a

7b

7c

7d

7e

7f

>200

19

>200

7g

7h

40

>200

ent from those of the amidine analogue, compound 1.

Since the donation of a hydrogen to the nearby water

and the acceptance of a hydrogen from Ser 190 may

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M. Miura et al. / Bioorg. Med. Chem. 14 (2006) 7688–7705

Table 2. In vitro inhibitory activities against TF/FVIIa

Compound

R¹

R²

IC₅₀(lM)

7e

12a

12b

12c

12d

12e

12f

12g

12h

12i

CONH-i-Bu

H

19

H

>200

89

H

3⁰-Me

4⁰-Me

5⁰-Me

6⁰-Me

4⁰-Cl

H

>200

102

137

>200

94

H

4⁰-MeO

4⁰-NO₂

4⁰-NH₂

4⁰-NMe₂

4⁰-NHMe

4⁰-MeO

4⁰-Me

4⁰-Cl

H

12j

H

12k

17a

17b

17c

17d

H

23

7.6

Figure 2. Docking model of compound 1 in TF/FVIIa. Compound is

shown in green. H57 and S195 of the catalytic triad are shown in

orange. S190 is shown in pink. Residue numbers of some key residues

are displayed. Predicted position of water molecule in S1 pocket is

indicated by red spheres. The hydrogen-bonds formed by the inhibitor

are shown in dotted white line.

CONH-i-Bu

3.3

3.7

4⁰-NHMe

0.69

stituted compound 12a, the substitution of the methyl

group at the 4⁰-position increased potency, although

the substitutions at other positions did not show the

enhancement of the potency (12b–e). Among 4⁰-substi-

tuted compounds (12f–k), though NO₂and NH₂ana-

logues did not have enhanced activity, Cl, MeO, and

the dimethylamino groups contributed to the binding

aﬃnity (12f, 12g, and 12i) and the methylamino ana-

logue 12k also showed relatively potent activity. From

the result of the SAR of the 5-unsubstituted series of

compounds, selected compounds having 5-isobutylami-

nocarbonyl moiety were prepared (17a–d). Comparing

the series of the 5-unsubstituted compounds (12a–k)

with those containing the 5-isobutylaminocarbonyl

(17a–d), similar results were observed. That is, introduc-

tion of methyl, Cl, and MeO groups at the 4⁰-positio-

n(17a–c) resulted in increased potency compared to the

4⁰-unsubstituted compound7e. In addition, the methyl-

amino analogue 17d was about 20-fold more potent

than the corresponding unsubstituted analogue 7e.

both play important roles in the potency, compound 7f,

which is missing the heteroatom that would bind with

the water, and compound 7h, which does not have a car-

bonyl group that would accept the hydrogen, did not

show potency. In the case of compound 7g, as shown

in Figure 2, the aminomethyl group of 7g forms a salt

bridge with Asp 189 similar to that of amidine com-

pound 1.

From the above modeling study, we considered that

there is a possibility to modify the central phenyl ring

since there is no interaction between its moiety and a

speciﬁc pocket formed by Gly 97, Tyr 94, Thr 98, and

Thr 99, which is known as the S2 site. In order to regain

the potency lost upon replacing the amidine moiety, our

synthetic eﬀorts were focused on the additional substitu-

tions on the phenyl ring (Table 2). From the SAR stud-

ies shown in Table 1, the 3-aminocarbonyl analogue 7e

was identiﬁed as the lead structure for further optimiza-

tion. In regard to the synthetic eﬃciency, we undertook

to understand the SARs by the use of 5-unsubstituted

compounds (12a–k). Substitutions on the central phenyl

ring yielded interesting results. Compared to the unsub-

In order to understand the enhancement of the potency

by the modiﬁcation of the central phenyl ring, we used a

molecular modeling of compound 17d (Fig. 4). The

molecular modeling suggested that the aminocarbonyl

Figure 3. Docking model of (a) 7e and (b) 7g in human IF/FVIIa. Color schemes are the same as those of Figure 2.

M. Miura et al. / Bioorg. Med. Chem. 14 (2006) 7688–7705

7693

12i, which lacks the methyl group of 12k, also decreases

the potency. Although the reasons why the other 4⁰-

substituted analogues (12c, 12f, 12g, and 17a–c) increase

the potency compared with the unsubstituted analogues

(12a and 7e) are not well understood at this point, this

result might be caused by the van der Waals interaction

or hydrophobic interaction between these substituents

and the S2 site.

The non-amidine compound 17d and the amidine ana-

logue, compound 1, were assayed for their inhibitory

potencies against a larger series of trypsin-like protein-

ases, and the resulting IC₅₀values are reported in Table

3. The inhibitors were also tested in standard clotting

assays including prothrombin time (PT) and activated

partial thromboplastin time (APTT) determinations,

which were used as qualitative in vitro indicators of

the potential antithrombotic activity.

From this it can be seen that amidine compound 1, which

was used as the starting compound, is found to inhibit

against FXa and trypsin. On the other hand, the com-

pound 17d has better selectivity for TF/FVIIa over other

serine proteases in comparison to the amidine analogue,

compound 1. As described above, in the S1 site the amino-

carbonyl group of 17d is bound to Ser 190, a speciﬁc res-

idue at FVIIa compared to FXa that may exhibit the

higher degree of speciﬁcity against FXa than that of ami-

dine compound 1. In the case of the selectivity against

trypsin, we suspect that the diﬀerence in the residues at site

S2 between FVIIa and trypsin plays an important role.

The residues at site S2 are Gly 97, Tyr 94, Thr 98, and

Thr 99 for FVIIa, as described above, and the correspond-

ing residues are Lys 97, Tyr 94, Thr 98, and Leu 99 for

trypsin, which causes the diﬀerent formsof the S2 pockets:

that is, the S2 pocket of trypsin is smaller in size than that

of FVIIa (Fig. 5). The molecular modeling suggested that

Figure 4. Docking model of 17d in human TF/FVIIa. Color schemes

are the same as those of Figure 2. Predicted positions of water

molecules in S1 and S2 pockets are displayed.

P1 group of 17d sits in the S1 site in a manner similar to

that portion of 7e when bound to the enzyme. As for the

S2 site, the molecular modeling suggested that the nitro-

gen of the methylamino substituent formed a hydrogen-

bonding network among the phenol oxygen of Tyr 94

and the carbonyl oxygen of Thr 98 through a structural-

ly conserved water molecule.¹⁶Furthermore, the methyl

group of the methylamino moiety contributes to the

binding aﬃnity through the hydrophobic interaction in

the S2 site. This is supported by the fact that the absence

of a hydrogen-bonding network between the enzyme

and the substituents such as MeO and NMe₂results in

loss of potency (12g and 12j) and the 4⁰-NH₂analogue

Table 3. Selectivity proﬁles of TF/VIIa inhibitors

Compound

IC₅₀(lM)

Thrombin

CT₂(lM)

TF/VIIa

FXa

Trypsin

PT

APTT

1

17d

0.090

0.69

0.88

>200

4.8

>200

4.2

150

4.3

>300 (1.0-fold)

PT/CT₂, concentration of inhibitor required to double the prothrombin time in human plasma.

APTT/CT₂, concentration of inhibitor required to double the activated partial thromboplastin time in human plasma.

In bracket, ploidy of the elongation at concentrations of 300 lM.

Figure 5. The active site regions of (a) human TF/FVIIa (PDBcode: 1DAN) and (b) human trypsin (PDB code: ITRN). The proteins are displayed

with Connoly surface. S1/S2 pockets are indicated as dotted circles. Compound 17d is shown in green. Docking pose of 17d with TF/FVIIa is

transported to trypsin for the comparison.

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M. Miura et al. / Bioorg. Med. Chem. 14 (2006) 7688–7705

the interaction of 17d with trypsin was less favorable than

that with TF/FVIIa, due to the lack of hydrophobic inter-

action between methylamino substituent of 17d and the

S2 pocket of trypsin.

less oil: ¹H NMR (400 MHz, DMSO-d₆) d: 3.94 (3H, s),

7.84 (1H, d, J = 8.5 Hz), 8.32 (1H, dd, J = 2.1 Hz,

8.5 Hz), 8.55 (1H, d, J = 2.1 Hz), 10.11 (1H, s); FAB-

MS (m/z): 313 (M+H)⁺.

The PT/CT₂value for compound 17d is 150 lM. Further-

more, as expected for the selective TF/FVIIa inhibitors, it

should be noted that compound 17d does not prolong the

APTT at all, even at concentrations of 300 lM. On the

other hand, the amidine analogue, compound 1, doubled

the APTT at concentrations of 4.3 lM, which may be

caused by the inhibition of FXa. Though the IC₅₀and

PTCT₂values of 17d were reduced compared to that of

compound 1, the relative selectivity was signiﬁcantly

more desirable. This property of high selectivity would

be expected to escape the bleeding risk.

5.3. Methyl 5-[(isobutylamino)carbonyl]-2-{[(triﬂuoro-

methyl)sulfonyl]oxy}benzoate (4)

To a solution of 3 (6.50 g, 20.8 mmol), NaH₂PO₄ÆH₂O

(3.25 g, 20.8 mmol), and 2-methyl-2-butene (11.0 mL,

104 mmol) in t-BuOH/H₂O/CH₃CN mixture (98 mL,

12:2:1 v/v) was added sodium chlorite (9.41 g, 80 wt %,

104 mmol), and the mixture was stirred at room tempera-

ture for 1 h. The mixture was partitioned between AcOEt

and H₂O, and extracted with AcOEt, and the organic

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

ﬁltered, and concentrated in vacuo to give a colorless solid

(6.83 g). To a solution of the compound obtained above

(6.50 g, 19.8 mmol) in CH₂Cl₂(65 mL) were added oxalyl

chloride (2.07 mL, 23.8 mmol) and DMF (0.10 mL), and

the mixture was stirred at room temperature for 2 h. The

mixture was concentrated in vacuo. To a solution of the

compound obtained above and NEt₃(2.76 mL,

19.8 mmol) in CH₂Cl₂(65 mL) at 0 °C was added 2-meth-

yl-1-propanamine (1.45 g, 19.8 mmol), and the mixture

was stirred at 0 °C for 1 h. The mixture was partitioned

between CHCl₃and H₂O and extracted with CHCl₃,

and the organic layer was dried over MgSO₄, ﬁltered,

and concentrated in vacuo. The residue was puriﬁed by

silica gel column chromatography (hexane/AcOEt = 4:1)

4. Conclusion

Optimization of the substituents on the P1 phenyl por-

tion of compound 1 led to a neutral or less basic alterna-

tive for amidine moieties such as the aminocarbonyl and

aminomethyl moieties. By further optimization of the

substituents on the central phenyl ring, a highly potent

and selective TF/VIIa inhibitor, 17d, was discovered.

This non-amidine compound is positioned to be a valu-

able and novel lead in the exploration of selective TF/

FVIIa inhibitors.

1

to give 4 (4.09 g, 54%) as a colorless solid: H NMR

5. Experimental

5.1. Chemistry

(400 MHz, DMSO-d₆) d: 0.90 (6H, d, J = 6.7 Hz), 1.77–

1.93 (1H, m), 3.08-3.14 (2H, m), 3.92 (3H, s), 7.70 (1H,

d, J = 8.6 Hz), 8.24 (1H, dd, J = 2.3 Hz, 8.6 Hz), 8.50

(1H, d, J = 2.3 Hz), 8.83 (1H, t, J = 5.7 Hz); FAB-MS

(m/z): 384 (M+H)⁺.

In general, reagents and solvents were used as pur-

chased, without further puriﬁcation. Melting points

were determined with a Yanaco MP-500D melting point

5.4. Methyl 2⁰-formyl-4-[(isobutylamino)carbonyl]biphe-

nyl-2-carboxylate (5)

1

apparatus and left uncorrected. H NMR spectra were

recorded on a JEOL JNM-LA300 or a JEOL JNM-

EX400 spectrometer. Chemical shifts were expressed in

d (ppm) values with tetramethylsilane as an internal

standard (in NMR description, s = singlet, d = doublet,

t = triplet, m = multiplet, and br = broad peak). Mass

spectra were recorded on a JEOL JMS-LX2000 spec-

trometer. The elemental analyses were performed with

a Yanaco MT-5 microanalyzer (C, H, and N) and

Yokogawa IC-7000S ion chromatographic analyzer

(halogens) and were within 0.4% of theoretical values.

To a solution of 4 (1.00 g, 2.61 mmol), (2-formylphe-

nyl)boronic acid (391 mg, 2.61 mmol), and K₃PO₄

(831 mg, 3.91 mmol) in DMF (10 mL) was added

Pd(PPh₃)₄(90 mg, 0.078 mmol), and the mixture was stir-

red at 100 °C for 5 h. The mixture was partitioned be-

tween AcOEt and H₂O, and extracted with AcOEt, and

the organic layer was washed with H₂O and brine, dried

over MgSO₄, ﬁltered, and concentrated in vacuo. The res-

idue was puriﬁed by silica gel column chromatography

(hexane/AcOEt = 2:1) to give 5 (822 mg, 93%) as a color-

1

5.2. Methyl 5-formyl-2-{[(triﬂuoromethyl)sulfo-

nyl]oxy}benzoate (3)

less oil: H NMR (300 MHz, DMSO-d₆) d: 0.92 (6H, d,

J = 6.8 Hz), 1.86–1.96 (1H, m), 3.10–3.17 (2H, m), 3.57

(3H, s), 7.26–7.31 (1H, m), 7.47 (1H, d, J = 7.9 Hz),

7.57–7.66 (1H, m), 7.68–7.75 (1H, m), 7.94 (1H, dd,

J = 1.3 Hz, 7.7 Hz), 8.12 (1H, dd, J = 1.8 Hz, 7.9 Hz),

8.42 (1H, d, J = 1.8 Hz), 8.74 (1H, t, J = 5.7 Hz), 9.74

(1H, s); FAB-MS (m/z): 340 (M+H)⁺.

To a solution of methyl 5-formyl-2-hydroxybenzoate

(8.89 g, 49.3 mmol) and pyridine (16.0 mL, 197 mmol)

in CH₂Cl₂(89 mL) was added triﬂuoromethanesulfonic

anhydride (14.9 mL, 88.8 mmol), and the mixture was

stirred at room temperature for 30 min. The mixture

was partitioned between CH₂Cl₂and H₂O, extracted

with CH₂Cl₂, and the organic layer was dried over

MgSO₄, ﬁltered, and concentrated in vacuo. The residue

was puriﬁed by silica gel column chromatography

(hexane/AcOEt = 9:1) to give 3 (7.47 g, 48%) as a color-

5.5. 4⁰-[(Isobutylamino)carbonyl]-2⁰-(methoxycarbon-

yl)biphenyl-2-carboxylic acid (6)

To a solution of 5 (3.00 g, 8.84 mmol), NaH₂PO₄Æ2H₂O

(1.38 g, 8.85 mmol), and 2-methyl-2-butene (4.7 mL,

M. Miura et al. / Bioorg. Med. Chem. 14 (2006) 7688–7705

7695

44.4 mmol) in t-BuOH/H₂O/CH₃CN mixture (30 mL,

6:2:1 v/v) was added sodium chlorite (4.00 g, 80 wt %,

35.4 mmol), and the mixture was stirred at room tem-

perature for 6 h. The mixture was partitioned between

AcOEt and H₂O, and extracted with AcOEt, and the

organic layer was washed with H₂O and brine, dried

over MgSO₄, ﬁltered, and concentrated in vacuo to give

6 (3.14 g, 100%) as a colorless solid: ¹H NMR

(300 MHz, DMSO-d₆) d: 0.91 (6H, d, J = 6.6 Hz),

1.80–1.95 (1H, m), 3.08–3.17 (2H, m), 3.57 (3H, s),

7.16–7.21 (1H, m), 7.31 (1H, d, J = 7.9 Hz), 7.44–7.53

(1H, m), 7.55–7.64 (1H, m), 7.88–7.96 (1H, m), 8.04

(1H, dd, J = 1.8 Hz, 8.1 Hz), 8.35 (1H, d, J = 1.8 Hz),

8.69 (1H, t, J = 5.7 Hz), 12.55 (1H, br s); FAB-MS

(m/z): 356 (M+H)⁺.

70.28; H, 5.94; N, 6.56. Found: C, 70.15; H, 5.92; N,

6.48.

5.8. 4-[(Isobutylamino)carbonyl]-2⁰-{[(4-methoxyphe-

nyl)amino]carbonyl}biphenyl-2-carboxylic acid (7b)

Compound 7b was synthesized from 6 and 4-methox-

yaniline following the same procedure as that for 7e.

Compound 7b was obtained as a colorless solid (60%,

157 mg): mp 149–151 °C (MeOH–H₂O); ¹H NMR

(400 MHz, DMSO-d₆) d: 0.89 (6H, d, J = 6.3 Hz),

1.77–1.92 (1H, m), 3.09 (2H, t, J = 5.8 Hz), 3.68 (3H,

s), 6.80 (2H, d, J = 9.8 Hz), 7.17–7.23 (1H, m), 7.27

(1H, d, J = 7.8 Hz), 7.36 (2H, d, J = 9.8 Hz), 7.44–

7.54(1H, m), 7.64 (1H, dd, J = 1.9 Hz, 6.8 Hz), 7.92

(1H, dd, J = 1.9 Hz, 7.8 Hz), 8.27 (1H, d, J = 1.9 Hz),

8.63 (1H, t, J = 5.8 Hz), 10.12 (1H, br s), 12.94 (1H, br

s); FAB-MS (m/z): 447 (M+H)⁺; Anal. Calcd for

C₂₆H₂₆N₂O₅Æ0.3H₂O: C, 69.10; H, 5.93; N, 6.20. Found:

C, 68.96; H, 6.01; N, 6.02.

5.6. 2⁰-({[3-(Aminocarbonyl)phenyl]amino}carbonyl)-4-

[(isobutylamino)carbonyl]biphenyl-2-carboxylic acid (7e)

To a solution of 6 (355 mg, 0.999 mmol) and 3-aminob-

enzamide (185 mg, 1.20 mmol) in DMF (7.0 mL) were

added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

hydrochloride (WSCÆHCl) (230 mg, 1.20 mmol) and

1-hydroxybenzotriazole (HOBt) (162 mg, 1.20 mmol),

and the mixture was stirred at room temperature for

12 h. The mixture was partitioned between AcOEt and

H₂O, and extracted with AcOEt, and the organic layer

was washed with H₂O and brine, dried over MgSO₄, ﬁl-

tered, and concentrated in vacuo. The residue was puri-

ﬁed by silica gel column chromatography (CHCl₃/

MeOH = 99:1) to give a colorless solid (458 mg). To a

solution of the compound obtained above in MeOH

(8.0 mL) was added 1 M NaOH/H₂O (1.8 mL,

1.8 mmol), and the mixture was reﬂuxed for 2 h. After

cooling, the reaction mixture was acidiﬁed with 1 M

HCl/H₂O (1.8 mL, 1.8 mmol). The resulting precipitate

was ﬁltered, washed with H₂O, and dried in vacuo to

give 7e (124 mg, 29%) as a colorless solid: mp 137–

138 °C (MeOH–H₂O); ¹H NMR (400 MHz, DMSO-

d₆) d: 0.89 (6H, d, J = 6.8 Hz), 1.77–1.92 (1H, m),

3.04–3.13 (2H, m), 7.21–7.26 (1H, m), 7.26–7.35 (3H,

m), 7.47–7.57 (3H, m), 7.61 (1H, d, J = 7.8 Hz), 7.66–

7.72 (1H, m), 7.86 (1H, s), 7.95 (1H, dd, J = 1.9 Hz,

8.3 Hz), 8.01 (1H, s), 8.30 (1H, d, J = 1.9 Hz), 8.63

(1H, t, J = 5.8 Hz), 10.20 (1H, s), 12.88 (1H, br s);

FAB-MS (m/z): 458 (M+H)⁺; Anal. Calcd for

C₂₆H₂₅N₃O₅Æ0.7H₂OÆ0.1AcOEt: C, 65.93; H, 5.70; N,

8.74. Found: C, 66.13; H, 5.53; N, 8.54.

5.9. 2⁰-{[(4-Chlorophenyl)amino]carbonyl}-4- [(isobutyl-

amino)carbonyl]biphenyl-2-carboxylic acid (7c)

Compound 7c was synthesized from 6 and 4-chloroani-

line following the same procedure as that for 7e. Com-

pound 7c was obtained as a colorless solid (26%,

84 mg): mp 117–118 °C (MeOH–H₂O); ¹H NMR

(400 MHz, DMSO-d₆) d: 0.89 (6H, d, J = 6.4 Hz),

1.79–1.93 (1H, m), 3.09 (2H, t, J = 5.8 Hz), 7.22–7.27

(1H, m), 7.27–7.34 (3H, m), 7.47–7.58 (4H, m), 7.68

(1H, dd, J = 1.5 Hz, 7.3 Hz), 7.95 (1H, dd, J = 1.9 Hz,

7.8 Hz), 8.30 (1H, d, J = 1.5 Hz), 8.64 (1H, t,

J = 5.8 Hz), 10.22 (1H, s), 12.81 (1H, br s); FAB-MS

(m/z): 451 (M+H)⁺; HRMS (ESI). Calcd for

C₂₅H₂₃ClN₂O₄: 451.1424. Found: 451.1420.

5.10. 2⁰-{[(3-Chlorophenyl)amino]carbonyl}-4-[(isobutyl-

amino)carbonyl]biphenyl-2-carboxylic acid (7d)

Compound 7d was synthesized from 6 and 3-chloroani-

line following the same procedure as that for 7e. Com-

pound 7d was obtained as a colorless solid (36%,

114 mg): mp 107–109 °C (MeOH–H₂O); ¹H NMR

(400 MHz, DMSO-d₆) d: 0.89 (6H, d, J = 6.4 Hz),

1.79–1.92 (1H, m), 3.09 (2H, t, J = 5.8 Hz), 7.05–7.10

(1H, m), 7.20–7.33 (3H, m), 7.40 (1H, d, J = 8.8 Hz),

7.46–7.58 (3H, m), 7.67 (1H, dd, J = 2.0 Hz, 7.3 Hz),

7.96 (1H, dd, J = 2.0 Hz, 7.8 Hz), 8.30 (1H, d,

J = 2.0 Hz), 8.64 (1H, t, J = 5.8 Hz), 10.30 (1H, s),

12.78 (1H, br s); FAB-MS (m/z): 451 (M+H)⁺; HRMS

(ESI) Calcd for C₂₅H₂₃ClN₂O₄: 451.1424. Found:

451.1416.

5.7. 2⁰-(Anilinocarbonyl)-4-[(isobutylamino)carbonyl]-

biphenyl-2-carboxylic acid (7a)

Compound 7a was synthesized from 6 and aniline fol-

lowing the same procedure as that for 7e. Compound

7a was obtained as a colorless solid (49%, 121 mg): mp

115–117 °C (MeOH–H₂O); ¹H NMR (400 MHz,

DMSO-d₆) d: 0.88 (6H, d, J = 6.3 Hz), 1.76–1.92 (1H,

m), 3.08 (2H, t, J = 5.8 Hz), 7.00 (1H, t, J = 7.3 Hz),

7.15-7.30 (4H, m), 7.40–7.55 (4H, m), 7.61 (1H, dd,

J = 1.9 Hz, 6.8 Hz), 7.92 (1H, dd, J = 1.9 Hz, 8.0 Hz),

8.28 (1H, d, J = 1.9 Hz), 8.63 (1H, t, J = 5.8 Hz), 10.32

(1H, br s), 12.92 (1H, br s); FAB-MS (m/z): 417

(M+H)⁺; Anal. Calcd for C₂₅H₂₄N₂O₄Æ0.6H₂O: C,

5.11. 2⁰-{[(3-Acetylphenyl)amino]carbonyl}-4-[(isobutyl-

amino)carbonyl]biphenyl-2-carboxylic acid (7f)

Compound 7f was synthesized from 6 and 1-(3-amino-

phenyl)ethanol following the same procedure as that

for 7e. Compound 7f was obtained as a colorless solid

(65%, 203 mg): mp 107–108 °C (MeOH–H₂O); ¹H

NMR (400 MHz, DMSO-d₆) d: 0.89 (6H, d,

J = 6.4 Hz), 1.77–1.92 (1H, m), 2.51 (3H, s), 3.09 (2H,

7696

M. Miura et al. / Bioorg. Med. Chem. 14 (2006) 7688–7705

t, J = 6.0 Hz), 7.25 (1H, dd, J = 0.8 Hz, 7.2 Hz), 7.31

(1H, d, J = 8.0 Hz), 7.40 (1H, t, J = 8.0 Hz), 7.48–7.58

(2H, m), 7.63 (1H, d, J = 8.0 Hz), 7.67–7.78 (2H, m),

7.95 (1H, dd, J = 2.0 Hz, 8.0 Hz), 8.09 (1H, s), 8.30

(1H, d, J = 2.0 Hz), 8.63 (1H, t, J = 6.0 Hz), 10.31

(1H, s), 12.85 (1H, br s); FAB-MS (m/z): 459 (M+H)⁺;

HRMS (FAB) Calcd for C₂₇H₂₇N₂O₅: 459.1920. Found:

459.1922.

5.14. Benzyl 2⁰-formyl-4-methylbiphenyl-2-carboxylate

(10c)

To a solution of 8c (3.00 g, 14.0 mmol) in DMF (30 mL)

were added K₂CO₃(2.90 g, 21.0 mmol) and benzylbro-

mide (1.7 mL, 14.3 mmol), and the mixture was stirred

at room temperature for 12 h. The mixture was parti-

tioned between AcOEt and H₂O, and extracted with

AcOEt, and the organic layer was washed with H₂O

and brine, dried over MgSO₄, ﬁltered, and concentrated

in vacuo to give 9c (4.42 g, 100%) as a colorless oil. To a

solution of 9c (2.04 g, 6.68 mmol) and (2-formylphe-

nyl)boronic acid (1.00 g, 6.67 mmol) in DMF (15 mL)

were added Pd(PPh₃)₄(231 mg, 0.200 mmol) and

K₃PO₄(2.12 g, 9.99 mmol), and the mixture was stirred

at 100 °C for 3 h. The mixture was partitioned between

AcOEt and H₂O, and extracted with AcOEt, and the

organic layer was washed with H₂O and brine, dried

over MgSO₄, ﬁltered, and concentrated in vacuo. The

residue was puriﬁed by silica gel column chromatogra-

phy (hexane/AcOEt = 9:1) to give 10c (1.95 g, 88%) as

5.12. 2⁰-({[4-(Aminomethyl)phenyl]amino}carbonyl)-4-

[(isobutylamino)carbonyl]biphenyl-2-carboxylic acid (7g)

To a solution of 6 (320 mg, 0.900 mmol) and tert-butyl

(4-aminobenzyl)carbamate (200 mg, 0.900 mmol) in

DMF (9.0 mL) were added WSCÆHCl (345 mg,

1.80 mmol) and HOBt (276 mg, 1.80 mmol), and the mix-

ture was stirred at 50 °C for 12 h. The mixture was parti-

tioned between AcOEt and H₂O, and extracted with

AcOEt, and the organic layer was washed with H₂O

and brine, dried over MgSO₄, ﬁltered, and concentrated

in vacuo. The residue was puriﬁed by silica gel column

chromatography (CHCl₃/MeOH = 97:3) to give a yellow

amorphous (350 mg). To a solution of the compound ob-

tained above in MeOH (6.0 mL) was added 1 M NaOH/

H₂O (1.3 mL, 1.3 mmol), and the mixture was reﬂuxed

for 2 h. After cooling, the reaction mixture was acidiﬁed

with 1 M HCl/H₂O (2.6 mL, 2.6 mmol) and concentrated

in vacuo. To a solution of the compound obtained above

in 1,4-dioxane (20 mL) was added 1 M HCl/H₂O

(4.0 mL, 4.0 mmol), and the mixture was stirred at

50 °C for 12 h. The reaction mixture was concentrated

in vacuo. The residue was puriﬁed by ODS-gel column

chromatography (CH₃CN/0.001 HCl aq = 23:77) to give

1

a colorless oil: H NMR (300 MHz, DMSO-d₆) d: 2.43

(3H, s), 5.00 (2H, s), 7.05–7.12 (2H,m), 7.21–7.33 (5H,

m), 7.46–7.56 (1H, m), 7.59–7.67 (1H, m), 7.77–7.80

(1H, m), 7.82 (1H, dd, J = 1.5 Hz, 7.5 Hz), 9.69 (1H,

s); FAB-MS (m/z): 331 (M+H)⁺.

5.15. Benzyl 2⁰-formylbiphenyl-2-carboxylate (10a)

Compound 10a was synthesized from 8a following the

same procedure as that for 10c. Compound 10a was ob-

tained as a colorless oil (81%, 1.71 g): ¹H NMR

(300 MHz, DMSO-d₆) d: 5.02 (2H, s), 7.06–7.14 (2H,

m), 7.24–7.33 (4H, m), 7.82 (1H, dd, J = 1.3 Hz,

7.5 Hz), 7.50–7.72 (4H, m), 7.84 (1H, dd, J = 1.5 Hz,

7.5 Hz), 7.99 (1H, dd, J = 1.5 Hz, 7.5 Hz), 9.69 (1H, s);

FAB-MS (m/z): 317 (M+H)⁺.

1

7g (150 mg, 95%) as a colorless amorphous powder: H

NMR (400 MHz, DMSO-d₆) d: 0.89 (6H, d,

J = 6.8 Hz), 1.81–1.88 (1H, m), 3.08 (2H, t, J = 6.0 Hz),

3.92 (2H, br s), 7.24 (1H, dd, J = 1.6 Hz, 7.2 Hz), 7.29–

7.34 (3H, m), 7.49–7.54 (4H, m), 7.66 (1H, dd,

J = 1.6 Hz, 7.2 Hz), 7.95 (1H, dd, J = 1.6 Hz, 7.6 Hz),

8.19 (3H, br s), 8.29 (1H, d, J = 1.6 Hz), 8.66 (1H, t,

J = 6.0 Hz), 10.19 (1H, br s); FAB-MS (m/z): 446

(M+H)⁺Anal. Calcd for C₂₆H₂₇N₃O₄Æ1.1HClÆ1.5H₂O:

C, 60.92; H, 6.11; N, 8.20; Cl, 7.61. Found: C, 60.69; H,

6.11; N, 8.31; Cl, 7.33.

5.16. Benzyl 2⁰-formyl-3-methylbiphenyl-2-carboxylate

(10b)

Compound 10b was synthesized from 8b following the

same procedure as that for 10c. Compound 10b was ob-

tained as a colorless oil (72%, 2.94 g): ¹H NMR

(300 MHz, DMSO-d₆) d: 2.34 (3H, s), 4.87 (1H, d,

J = 12.2 Hz), 4.95 (1H, d, J = 12.2 Hz), 6.95–7.03

(2H,m), 7.20–7.31 (4H, m), 7.38–7.51 (2H, m), 7.53–

7.60 (1H, m), 7.65 (1H, dt, J = 1.7 Hz, 7.5 Hz), 7.86

(1H, dd, J = 1.3 Hz, 7.5 Hz), 9.65 (1H, s); FAB-MS

(m/z): 331 (M+H)⁺.

5.13. 2⁰-({[4-(Aminomethyl)phenyl]amino}carbonyl)-4-

[(isobutylamino)carbonyl]biphenyl-2-carboxylic acid (7h)

Compound 7h was synthesized from 6 and tert-butyl (3-

aminobenzyl)carbamate following the same procedure

as that for 7g. Compound 7h was obtained as a colorless

amorphous powder (39%, 143 mg): ¹H NMR (400 MHz,

DMSO-d₆) d: 0.89 (6H, d, J = 6.0 Hz), 1.79–1.92 (1H,

m), 3.09 (2H, t, J = 4.8 Hz), 3.92 (2H, br s), 7.15 (1H,

d, J = 7.6 Hz), 7.24 (1H, dd, J = 2.0 Hz, 6.8 Hz), 7.26–

7.38 (3H, m), 7.47–7.57 (2H, m), 7.66 (1H, dd,

J = 2.0 Hz, 7.2 Hz), 7.76 (1H, s), 7.96 (1H, dd,

J = 2.0 Hz, 8.4 Hz), 8.25 (3H, br s), 8.31 (1H, d,

J = 2.0 Hz), 8.67 (1H, t, J = 6.0 Hz), 10.22 (1H, s),

12.88 (1H, br s); FAB-MS (m/z): 446 (M+H)⁺; Anal.

Calcd for C₂₆H₂₇N₃O₄Æ1.0HClÆ1.5H₂O: C, 61.35; H,

6.14; N, 8.26; Cl, 6.97. Found: C, 61.30; H, 6.10; N,

8.18; Cl, 6.87.

5.17. Benzyl 2⁰-formyl-5-methylbiphenyl-2-carboxylate

(10d)

Compound 10d was synthesized from 8d following the

same procedure as that for 10c. Compound 10d was ob-

tained as a colorless oil (83%, 1.84 g): ¹H NMR

(300 MHz, DMSO-d₆) d: d 2.40 (3H, s), 5.00 (2H, s),

7.07–7.13 (2H,m), 7.18 (1H, br s), 7.23–7.32 (4H, m),

7.38–7.43 (1H, m), 7.49–7.56 (1H, m), 7.64 (1H, dt,

J = 1.5 Hz, 7.5 Hz), 7.83 (1H, dd, J = 1.3 Hz, 7.7 Hz),

7.91 (1H, d, J = 8.0 Hz), 9.69 (1H, s); FAB-MS (m/z):

331 (M+H)⁺.

M. Miura et al. / Bioorg. Med. Chem. 14 (2006) 7688–7705

7697

5.18. Benzyl 2⁰-formyl-6-methylbiphenyl-2-carboxylate

(10e)

7.06–7.12 (3H,m), 7.18 (1H, dd, J = 1.5 Hz, 7.7 Hz),

7.26–7.32 (3H, m), 7.38–7.46 (2H, m), 7.55 (1H, dt,

J = 1.5 Hz, 7.5 Hz), 7.72 (1H, d, J = 1.5 Hz), 7.81 (1H,

dd, J = 1.5 Hz, 7.7 Hz); FAB-MS (m/z): 361 (M+H)⁺.

Compound 10e was synthesized from 8b following the

same procedure as that for 10c. Compound 10e was ob-

tained as a colorless oil (31%, 675 mg): ¹H NMR

(300 MHz, DMSO-d₆) d: 1.91 (3H, s), 4.95 (2H, s),

7.08–7.13 (2H, m), 7.16 (1H, dd, J = 0.8 Hz, 7.5 Hz),

7.27–7.33 (3H, m), 7.44–7.60 (3H, m), 7.66 (1H, dt,

J = 1.5 Hz, 7.5 Hz), 7.78 (1H, d, J = 7.7 Hz), 7.85 (1H,

dd, J = 1.1 Hz, 7.7 Hz), 9.60 (1H, s); FAB-MS (m/z):

331 (M+H)⁺.

5.22. 2⁰-[(Benzyloxy)carbonyl]biphenyl-2-carboxylic acid

(11a)

Compound 11a was synthesized from 10a following the

same procedure as that for 11c without protection of the

acid by methyl group. Compound 11a was obtained as a

1

colorless amorphous powder (100%, 1.80 g): H NMR

(300 MHz, DMSO-d₆) d: 5.01 (2H, s), 7.08–7.26 (5H,

m), 7.26–7.32 (3H, m), 7.38–7.56 (2H, m), 7.58 (1H,

dt, J = 1.5 Hz, 7.5 Hz), 7.85 (1H, dd, J = 1.2 Hz,

7.7 Hz), 7.92 (1H, dd, J = 1.2 Hz, 7.7 Hz), 12.45 (1H,

br s); FAB-MS (m/z): 333 (M+H)⁺.

5.19. tert-Butyl 4-chloro-2⁰-formylbiphenyl-2-carboxylate

(10f)

Compound 10f was synthesized from 8f following the

same procedure as that for 10c except for the protection

with the acid group by tert-butyl group.¹⁷Compound

5.23. 2-Benzyl 2⁰-ethyl 3-methylbiphenyl-2,2⁰-dicarboxy-

late (11b)

1

10f was obtained as a colorless oil (97%, 3.25 g): H

NMR (300 MHz, DMSO-d₆) d: 1.10 (9H, s), 7.30 (1H,

d, J = 7.8 Hz), 7.39 (1H, d, J = 8.0 Hz), 7.60-7.78 (3H,

m), 7.84 (1H, d, J = 2.2 Hz), 7.95 (1H, dd, J = 1.3 Hz,

7.7 Hz), 9.75 (1H, s); FAB-MS (m/z): 317 (M+H)⁺.

Compound 11b was synthesized from 10b following the

same procedure as that for 11c except for the protection

of the acid by the ethyl group. Compound 11b was ob-

tained as a colorless oil (89%, 2.95 g): ¹H NMR

(300 MHz, DMSO-d₆) d: 0.86 (3H, t, J = 7.1 Hz), 2.29

(3H, s), 3.84–4.00 (2H, m), 4.81–4.98 (2H, m), 6.99–

7.07 (3H,m), 7.21 (1H, dd, J = 1.1 Hz, 7.3 Hz), 7.24–

7.32 (4H, m), 7.34–7.41 (1H, m), 7.44–7.58 (2H, m),

7.84 (1H, dt, J = 1.7 Hz, 7.5 Hz); FAB-MS (m/z): 375

(M+H)⁺.

5.20. Methyl 2⁰-formyl-4⁰-methoxybiphenyl-2-carboxyl-

ate (10g)

Compound 10g was synthesized from 8g following the

same method of that for 10c except for cross-coupling

between 9g and 2-formyl-4-methoxyboronic acid. Com-

pound 10g was obtained as a colorless oil (55%,

1

410 mg): H NMR (300 MHz, DMSO-d₆) d: 3.55 (3H,

5.24. 2-Benzyl 2⁰-methyl 5-methylbiphenyl-2,2⁰-dicarb-

oxylate (11d)

s), 3.87 (3H, s), 7.21 (1H, d, J = 8.4 Hz), 7.27 (1H, dd,

J = 2.7 Hz, 8.4 Hz), 7.36 (1H, dd, J = 1.3 Hz, 7.5 Hz),

7.39 (1H, d, J = 2.7 Hz), 7.57 (1H, dt, J = 1.3 Hz,

7.7 Hz), 7.66 (1H, dt, J = 1.5 Hz, 7.7 Hz), 7.60–7.78

(3H, m), 7.84 (1H, d, J = 2.2 Hz), 7.95 (1H, dd,

J = 1.3 Hz, 7.5 Hz), 7.93 (1H, dd, J = 1.5 Hz, 7.7 Hz),

9.65 (1H, s); FAB-MS (m/z): 271 (M+H)⁺.

Compound 11d was synthesized from 10d following the

same procedure as that for 11c. Compound 11d was ob-

tained as a colorless oil (100%, 1.04 g): ¹H NMR

(300 MHz, DMSO-d₆) d: 2.37 (3H, s), 3.49 (3H, s),

4.97–5.02 (2H, m), 7.02 (1H, br s), 7.06–7.13 (2H, m),

7.20 (1H, dd, J = 1.1 Hz, 7.7 Hz), 7.26–7.32 (4H, m),

7.40–7.47 (1H, m), 7.56 (1H, dt, J = 1.5 Hz, 7.5 Hz),

7.82 (1H, dd, J = 1.3 Hz, 7.7 Hz), 7.84 (1H, d,

J = 7.7 Hz); FAB-MS (m/z): 361 (M+H)⁺.

5.21. 2-Benzyl 2⁰-methyl 4-methylbiphenyl-2,2⁰-dicarb-

oxylate (11c)

To a solution of 10c (1.95 g, 5.90 mmol), NaH₂PO₄Æ2H₂O

(920 mg, 5.90 mmol), and 2-methyl-2-butene (3.2 mL,

30.2 mmol) in t-BuOH/H₂O/CH₃CN mixture (27 mL,

6:2:1 v/v) was added sodium chlorite (2.67 g, 80 wt %,

23.6 mmol), and the mixture was stirred at room temper-

ature for 9 h. The mixture was partitioned between

AcOEt and H₂O, and extracted with AcOEt, and the

organic layer was washed with H₂O and brine, dried over

MgSO₄, ﬁltered, and concentrated in vacuo to give as a

colorless solid (2.04 g). To a solution of compound ob-

tained above (1.00 g, 2.89 mmol) in DMF (10 mL) were

added K₂CO₃(798 mg, 5.77 mmol) and MeI (0.54 mL,

8.67 mmol), and the mixture was stirred at room temper-

ature for 3 h. The mixture was partitioned between

AcOEt and H₂O, and extracted with AcOEt, and the

organic layer was washed with H₂O and brine, dried over

MgSO₄, ﬁltered, and concentrated in vacuo to give 11c

5.25. 2-Benzyl 2⁰-methyl 6-methylbiphenyl-2,2⁰-dicarb-

oxylate (11e)

Compound 11e was synthesized from 10e following the

same procedure as that for 11c. Compound 11e was ob-

tained as a colorless oil (100%, 468 mg): ¹H NMR

(300 MHz, DMSO-d₆) d: 1.87 (3H, s), 3.50 (3H, s),

4.92 (1H, d, J = 16.3 Hz), 4.96 (1H, d, J = 16.3 Hz),

7.07 (1H, d, J = 7.7 Hz), 7.09–7.15 (3H, m), 7.27–7.51

(3H, m), 7.57 (1H, dt, J = 1.3 Hz, 7.5 Hz), 7.73 (1H, d,

J = 6.8 Hz), 7.89 (1H, dd, J = 1.3 Hz, 7.7 Hz); FAB-

MS (m/z): 361 (M+H)⁺.

5.26. 2-tert-Butyl 2⁰-methyl 4-chlorobiphenyl-2,2⁰-dicarb-

oxylate (11f)

1

(1.04 g, 100%) as a colorless oil: H NMR (300 MHz,

DMSO-d₆) d: 2.40 (3H, s), 3.50 (3H, s), 5.00 (2H, br s),

Compound 11f was synthesized from 10f following the

same procedure as that for 11c. Compound 11f was

7698

M. Miura et al. / Bioorg. Med. Chem. 14 (2006) 7688–7705

obtained as a colorless oil (94%, 3.32 g): ¹H NMR

(300 MHz, DMSO-d₆) d: 1.12 (9H, s), 3.56 (3H, s),

7.19–7.26 (2H, m), 7.49–7.56 (1H, m), 7.60–7.68 (2H,

m), 7.78 (1H, d, J = 2.4 Hz), 7.95 (1H, d, J = 7.7 Hz);

FAB-MS (m/z): 347 (M+H)⁺.

obtained as a colorless solid (8.0%, 53 mg): mp 130–

132 °C (MeOH–H₂O); ¹H NMR (400 MHz, DMSO-

d₆) d: 2.38 (3H, s), 7.02 (1H, d, J = 6.8 Hz), 7.24–7.40

(6H, m), 7.42–7.51 (3H, m), 7.76 (1H, dd, J = 1.0 Hz,

6.9 Hz), 7.85 (1H, br s), 7.87 (1H, br s), 9.99 (1H, s),

12.87 (1H, br s); FAB-MS (m/z): 375 (M+H)⁺. Anal.

Calcd for C₂₂H₁₈N₂O₄Æ0.7H₂O: C, 68.28; H, 5.05; N,

7.24. Found: C, 68.31; H, 5.14; N, 7.21.

5.27. 2⁰-({[3-(Aminocarbonyl)phenyl]amino}carbonyl)-4⁰-

methylbiphenyl-2-carboxylic acid (12c)

To a solution of 11c (1.03 g, 2.86 mmol) in MeOH

(20 mL) was added 10% Pd/C powder (100 mg), and

the mixture was stirred in a hydrogen atmosphere at

room temperature for 12 h. The catalyst was ﬁltered

on Celite and the ﬁltrate was concentrated in vacuo to

give a solid (773 mg). To a solution of the compound

obtained above and tert-butyl (4-aminobenzyl)carba-

mate (879 mg, 5.70 mmol) in DMF (10 mL) were added

WSCÆHCl (820 mg, 4.28 mmol) and HOBt (578 mg,

4.28 mmol), and the mixture was stirred at room tem-

perature for 12 h. The mixture was partitioned between

AcOEt and H₂O, and extracted with AcOEt, and the

organic layer was washed with H₂O and brine, dried

over MgSO₄, ﬁltered, and concentrated in vacuo. The

residue was puriﬁed by silica gel column chromatogra-

phy (CHCl₃/MeOH = 97:3) to give a colorless solid

(892 mg). To a solution of the residual solid (840 mg,

2.16 mmol) in MeOH (10 mL) was added 1 M NaOH/

H₂O (3.4 mL, 3.4 mmol), and the mixture was reﬂuxed

for 12 h. After cooling, the reaction mixture was acidi-

ﬁed with 1 M HCl/H₂O (1.8 mL, 1.8 mmol). The result-

ing precipitate was ﬁltered, washed with H₂O, and dried

in vacuo to give 12c (812 mg, 100%) as a colorless solid:

mp 200–201 °C (MeOH–H₂O); ¹H NMR (400 MHz,

DMSO-d₆) d: 2.42 (3H, s), 3.34 (3H, br s), 4.08 (1H,

br s), 7.10 (1H, d, J = 7.8 Hz), 7.20 (1H, d,

J = 7.3 Hz), 7.25–7.35 (3H, m), 7.35–7.43 (1H, m),

7.43–7.53 (3H, m), 7.57 (1H, d, J = 7.8 Hz), 7.80 (1H,

dd, J = 1.5 Hz, 7.3 Hz), 7.88 (1H, br s), 7.94 (1H, br

s), 10.02 (1H, s), 12.80 (1H, br s); FAB-MS (m/z): 375

(M+H)⁺. Anal. Calcd for C₂₂H₁₈N₂O₄Æ1.0H₂OÆ1.0-

MeOH: C, 65.08; H, 5.70; N, 6.60. Found: C, 65.23;

H, 5.71; N, 6.65.

5.30. 2⁰-({[3-(Aminocarbonyl)phenyl]amino}carbonyl)-5⁰-

methylbiphenyl-2-carboxylic acid (12d)

Compound 12d was synthesized from 11d following the

same procedure as that for 12c. Compound 12d was ob-

tained as a colorless solid (84%, 929 mg): mp 212–214 °C

1

(MeOH–H₂O); H NMR (400 MHz, DMSO-d₆) d: 2.37

(3H, s), 7.02 (1H, s), 7.20 (1H, dd, J = 0.9 Hz, 7.8 Hz),

7.26–7.32 (3H, m), 7.37–7.43 (1H, m), 7.46–7.60 (2H,

m), 7.81 (1H, dd, J = 1.5 Hz, 7.8 Hz), 7.86 (1H, br s),

7.93 (1H, br s), 9.94 (1H, s), 12.84 (1H, br s); FAB-

MS (m/z): 375 (M+H)⁺; HRMS (ESI). Calcd. for

C₂₂H₁₈N₂O₄: 375.1345. Found: 375.1349.

5.31. 2⁰-({[3-(Aminocarbonyl)phenyl]amino}carbonyl)-6⁰-

methylbiphenyl-2-carboxylic acid (12e)

Compound 12e was synthesized from 11e following the

same procedure as that for 12c. Compound 12e was ob-

tained as a colorless solid (80%, 361 mg): mp 236–238 °C

1

(MeOH–H₂O); H NMR (400 MHz, DMSO-d₆) d: 1.93

(3H, s), 7.12 (1H, d, J = 7.3 Hz), 7.24–7.32 (2H, m),

7.39–7.46 (4H, m), 7.46–7.55 (3H, m), 7.83–7.91 (3H,

m), 10.03 (1H, s), 12.86 (1H, br s); FAB-MS (m/z):

375 (M+H)⁺. Anal. Calcd for C₂₂H₁₈N₂O₄: C, 70.58;

H, 4.85; N, 7.48. Found: C, 70.53; H, 4.84; N, 7.39.

5.32. 2⁰-({[3-(Aminocarbonyl)phenyl]amino}carbonyl)-4⁰-

chlorobiphenyl-2-carboxylic acid (12f)

Compound 12f was synthesized from 11f following the

same procedure as that for 12c. Compound 12f was ob-

tained as a colorless solid (25%, 367 mg): mp 132–135 °C

(MeOH–H₂O); ¹H NMR (400 MHz, DMSO-d₆) d: 7.20–

7.36 (4H, m), 7.39–7.45 (1H,m), 7.48–7.55 (2H, m),

7.55–7.62 (2H, m), 7.69 (1H, d, J = 2.0 Hz), 7.82–7.89

(2H, m), 7.96 (1H, br s), 10.27 (1H, s), 12.72 (1H, br

s); FAB-MS (m/z): 395 (M+H)⁺. Anal. Calcd for

C₂₁H₁₅ClN₂O₄Æ0.5H₂O: C, 62.46; H, 3.99; N, 6.94; Cl,

8.78. Found: C, 62.13; H, 3.86; N, 6.91; Cl, 8.84.

5.28. 2⁰-({[3- (Aminocarbonyl)phenyl]amino}carbonyl)-

biphenyl-2-carboxylic acid (12a)

Compound 12a was synthesized from 11a following the

same procedure as that for 12c without deprotection of

the benzyl group. Compound 12a was obtained as a color-

less solid (72%, 702 mg): mp 135–136 °C (MeOH–H₂O);

¹H NMR (400 MHz, DMSO-d₆) d: 7.20–7.25 (2H, m),

7.27–7.35 (2H,m), 7.37–7.43 (1H, m), 7.45–7.58 (5H, m),

7.63–7.68 (1H, m), 7.82 (1H, dd, J = 1.5 Hz, 7.8 Hz),

7.86 (1H, br s), 7.94 (1H, br s), 10.04 (1H, s), 12.81 (1H,

br s); FAB-MS (m/z): 361 (M+H)⁺. Anal. Calcd for

C₂₁H₁₆N₂O₄Æ0.5AcOEt: C, 68.31; H, 4.98; N, 6.93.

Found: C, 68.14; H, 4.88; N, 6.88.

5.33. 2⁰-({[3-(Aminocarbonyl)phenyl]amino}carbonyl)-4⁰-

methoxybiphenyl-2-carboxylic acid (12g)

Compound 12g was synthesized from 10g following

the same procedure as that for 12c. Compound 12g

was obtained as a colorless solid (81%, 432 mg): mp

125–127 °C (MeOH–H₂O); ¹H NMR (300 MHz,

DMSO-d₆) d: 3.87 (3H, s), 7.07 (1H, dd, J = 2.4 Hz,

8.4 Hz), 7.13 (1H, d, J = 8.4 Hz), 7.17–7.23 (2H, m),

7.26–7.34 (2H, m), 7.38 (1H, dt, J = 1.3 Hz, 7.5 Hz),

7.44–7.53 (2H, m), 7.57 (1H, d, J = 8.4 Hz), 7.78

(1H, dd, J = 1.3 Hz, 7.5 Hz), 7.87 (1H, br s), 7.93

(1H, br s), 10.05 (1H, s), 12.77 (1H, s); ESI-MS

5.29. 2⁰-({[3-(Aminocarbonyl)phenyl]amino}carbonyl)-3⁰-

methylbiphenyl-2-carboxylic acid (12b)

Compound 12b was synthesized from 11b following the

same procedure as that for 12c. Compound 12b was

M. Miura et al. / Bioorg. Med. Chem. 14 (2006) 7688–7705

7699

(m/z): 389 (MꢀH)^ꢀ; HRMS (ESI) Calcd for

layer was washed with H₂O and brine, dried over

MgSO₄, ﬁltered, and concentrated in vacuo. The residue

was puriﬁed by silica gel column chromatography

(CHCl₃/MeOH = 98:2) to give a pale yellow amorphous

(219 mg). To a solution of the compound obtained

above and tert-butyl (4-aminobenzyl)carbamate

(110 mg, 0.713 mmol) in DMF (4.0 mL) were added

WSCÆHCl (137 mg, 0.715 mmol) and HOBt (96 mg,

0.710 mmol), and the mixture was stirred at room tem-

perature for 12 h. The mixture was partitioned between

AcOEt and H₂O and extracted with AcOEt, and the

organic layer was washed with H₂O and brine, dried

over MgSO₄, ﬁltered, and concentrated in vacuo. The

residue was puriﬁed by silica gel column chromatogra-

phy (CHCl₃/MeOH = 98:2) to give a colorless solid

(128 mg). To a solution of the residual solid in MeOH

(2.0 mL) was added 1 M NaOH/H₂O (0.50 mL,

0.50 mmol), and the mixture was reﬂuxed for 2 h. After

cooling, the reaction mixture was acidiﬁed with 1 M

HCl/H₂O (0.60 mL, 0.60 mmol). The resulting precipi-

tate was ﬁltered, washed with H₂O, and dried in vacuo

to give 17a (60 mg, 57%) as a colorless solid: mp 135–

137 °C (MeOH–H₂O); ¹H NMR (400 MHz, DMSO-

d₆) d: 0.88 (6H, d, J = 6.3 Hz), 1.78–1.92 (1H, m),

3.04-3.12 (2H, m), 3.88 (3H, s), 7.10 (1H, dd,

J = 2.5 Hz, 8.3 Hz), 7.18 (1H, d, J = 8.3 Hz), 7.23 (1H,

d, J = 2.5 Hz), 7.26–7.34 (3H, m), 7.51 (1H, d,

J = 7.8 Hz), 7.63 (1H, d, J = 8.3 Hz), 7.88 (1H, s), 7.92

(1H, dd, J = 1.9 Hz, 7.8 Hz), 8.00 (1H, s), 8.26 (1H, d,

J = 1.9 Hz), 8.61 (1H, t, J = 5.8 Hz), 10.19 (1H, s),

12.84 (1H, br s); FAB-MS (m/z): 490 (M+H)⁺; Anal.

Calcd for C₂₇H₂₇N₃O₆Æ1.0H₂O: C, 63.90; H, 5.76; N,

8.28. Found: C, 63.74; H, 5.64; N, 8.08.

C₂₂H₁₈N₂O₅: 391.1294. Found: 391.1296.

5.34. 2-(Trimethylsilyl)ethyl 2-bromo-5-methoxybenzoate

(14)

To a solution of 13 (2.50 g, 10.8 mmol) and 2-trimethyl-

silylethanol (1.53 g, 12.9 mmol) in DMF (5.0 mL) and

pyridine (2.5 mL) were added DCC (2.45 g, 11.9 mmol)

and DMAP (2 mg), and the mixture was stirred at room

temperature for 12 h. The resulting precipitate was

ﬁltered and concentrated in vacuo. The residue was

puriﬁed by silica gel column chromatography (hexane/

AcOEt = 9:1) to give 14 (2.00 g, 56%) as a colorless

oil: ¹H NMR (400 MHz, DMSO-d₆) d: 0.06 (9H, s),

0.99–1.09 (2H, m), 3.73 (3H, s), 4.26–4.35 (2H, m),

7.00 (1H, dd, J = 3.1 Hz, 8.8 Hz), 7.18 (1H, d,

J = 3.1 Hz), 7.55 (1H, d, J = 8.8 Hz).

5.35. Benzyl 5-[(isobutylamino)carbonyl]-2-{[(triﬂuoro-

methyl)sulfonyl]oxy}benzoate (15)

Compound 15 was synthesized from benzyl 5-formyl-2-

hydroxybenzoate following the same procedure as that

for 4. Compound 15 was obtained as a colorless solid

1

(85%, 2.89 g): H NMR (300 MHz, DMSO-d₆) d: 0.89

(6H, d, J = 6.6 Hz), 1.75–1.92 (1H, m), 3.06–3.13 (2H,

m), 5.42 (2H, s), 7.33–7.45 (3H, m), 7.45–7.52 (2H, m),

7.71 (1H, d, J = 8.6 Hz), 8.23 (1H, dd, J = 2.4 Hz,

8.6 Hz), 8.48 (1H, d, J = 2.4 Hz), 8.82 (1H, t,

J = 5.9 Hz); FAB-MS (m/z): 460 (M+H)⁺.

5.36. 2-Benzyl 2⁰-[2-(trimethylsilyl)ethyl] 4-[(isobutylami-

no)carbonyl]-4⁰-methoxybiphenyl-2,2⁰-dicarboxylate (16)

5.38. Methyl 3-[(acetyloxy)methyl]-4-bromobenzoate (19)

To a solution of 14 (1.40 g, 4.23 mmol) and 15 (971 mg,

2.11 mmol) in DMF (10 mL) were added bis(pinacola-

to)diboron (1.07 g, 4.53 mmol), PdCl₂(dppf)ÆCH₂Cl₂

(86 mg, 0.105 mmol), and K₃PO₄(1.35 g, 6.35 mmol),

the mixture was stirred at 120 °C for 12 h. The mixture

was partitioned between AcOEt and H₂O, and extracted

with AcOEt, and the organic layer was washed with

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

trated in vacuo. The residue was puriﬁed by silica gel

column chromatography (hexane/AcOEt = 4:1) to give

16 (401 mg, 34%) as a colorless amorphous powder:

¹H NMR (300 MHz, DMSO-d₆) d: ꢀ0.07 (9H, s),

0.63–0.73 (2H, m), 0.97 (6H, d, J = 6.9 Hz), 1.88–2.00

(1H, m), 3.15–3.23 (2H, m), 3.89 (3H, s), 3.98–4.07

(2H, m), 5.08–5.13 (2H, m), 7.15–7.23 (4H, m), 7.33–

7.39 (5H, m), 8.11 (1H, dd, J = 1.9 Hz, 7.9 Hz), 8.43

(1H, d, J = 1.9 Hz), 8.74 (1H, t, J = 5.9 Hz); FAB-MS

(m/z): 562 (M+H)⁺.

To a solution of methyl 4-bromo-3-methylbenzoate (18)

(13.5 g, 58.9 mmol) in CCl₄(300 mL) were added NBS

(12.5 g, 70.3 mmol) and AIBN (5 mg). The mixture

was stirred at 70 °C for 2 h, then partitioned between

CHCl₃and H₂O, and the organic layer was washed with

5% NaHCO₃in H₂O and brine, dried over MgSO₄, ﬁl-

tered, and concentrated in vacuo to give a colorless sol-

id. To a solution of the compound obtained above in

AcOH (60 mL) was added AcONa (4.81 g, 117 mmol),

and the mixture was stirred at 100 °C for 12 h. After

concentrated in vacuo, the mixture was partitioned be-

tween AcOEt and H₂O, and extracted with AcOEt,

and the organic layer was washed with 5% NaHCO₃

in H₂O and brine, dried over MgSO₄, ﬁltered, and con-

centrated in vacuo. The residue was puriﬁed by silica gel

column chromatography (hexane/AcOEt = 5:1) to give

19 (10.8 g, 64%) as a colorless solid: ¹H NMR

(300 MHz, CDCl₃) d: 2.17 (3H, s), 3.93 (3H, s), 5.22

(2H, s), 7.66 (1H, d, J = 8.2 Hz), 7.85 (1H, dd,

J = 2.0 Hz, 8.2 Hz), 8.06 (1H, d, J = 2.0 Hz).

5.37. 2⁰-({[3-(Aminocarbonyl)phenyl]amino}carbonyl)-4-

[(isobutylamino)carbonyl]-4⁰-methoxybiphenyl-2-carbox-

ylic acid (17a)

5.39. 4-Bromo-3-(hydroxymethyl)-N-isobutylbenzamide

(20)

To a solution of 16 (480 mg, 0.850 mmol) in DMF

(5.0 mL) was added TBAF (1.0 M solution in THF,

1.0 mL, 1.0 mmol). The mixture was stirred at room

temperature for 8 days, then partitioned between AcOEt

and H₂O, and extracted with AcOEt, and the organic

To a solution of 19 (10.7 g, 37.3 mmol) in MeOH

(140 mL) was added 1 M NaOH/H₂O (112 mL,

112 mmol), and the mixture was stirred at room

7700

M. Miura et al. / Bioorg. Med. Chem. 14 (2006) 7688–7705

temperature for 2 h. After cooling, the reaction mix-

ture was acidiﬁed with 1 M HCl/H₂O (112 mL,

112 mmol). The resulting precipitate was ﬁltered,

washed with H₂O, and dried in vacuo to give a color-

less solid (8.29 g). To a solution of the compound ob-

tained above (4.30 g, 18.6 mmol) and 2-methylpropan-

1-amine (4.08 g, 55.8 mmol) in DMF (40 mL) were

added WSCÆHCl (5.33 g, 27.9 mmol) and HOBt

(4.18 g, 27.9 mmol), and the mixture was stirred at

room temperature for 12 h. After adding H₂O to the

reaction mixture, the resulting precipitate was ﬁltered,

washed with H₂O, and dried in vacuo to give 20

5.42. 2⁰-Benzyl 2-methyl 4-[(isobutylamino)carbonyl]-4⁰-

methylbiphenyl-2,2⁰-dicarboxylate (23)

To a solution of 22 (280 mg, 1.08 mmol) and 9c (395 mg,

1.3 mmol) in DMF (11 mL) were added K₃PO₄(344 mg,

1.62 mmol) and Pd(PPh₃)₄(62 mg, 0.054 mmol), and the

mixture was stirred at 120 °C for 24 h. After cooling, the

mixture was partitioned between AcOEt and H₂O, and

extracted with AcOEt, and the organic layer was washed

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

concentrated in vacuo. The residue was puriﬁed by silica

gel column chromatography (hexane/AcOEt = 2:1) to

give a colorless oil. To a solution of the compound

obtained above, NaH₂PO₄Æ2H₂O (115 mg, 0.955 mmol),

and 2-methyl-2-butene (0.50 mL, 4.78 mmol) in

t-BuOH/H₂O/CH₃CN mixture (18 mL, 6:2:1 v/v) was

added sodium chlorite (432 mg, 80 wt %, 3.82 mmol),

and the mixture was stirred at room temperature for

12 h. The mixture was partitioned between AcOEt and

H₂O, and extracted with AcOEt, and the organic layer

was washed with H₂O and brine, dried over MgSO₄, ﬁl-

tered, and concentrated in vacuo to give a colorless

amorphous. To a solution of the acid and MeI

(0.089 mL, 1.43 mmol) in DMF (10 mL) was added

K₂CO₃(197 mg, 1.43 mmol), and the mixture was stirred

at room temperature for 12 h. The mixture was parti-

tioned between AcOEt and H₂O, and extracted with

AcOEt, and the organic layer was washed with H₂O

and brine, dried over MgSO₄, ﬁltered, and concentrated

in vacuo to give 23 (0.44 g, 52%) as a colorless solid: ¹H

NMR (300 MHz, DMSO-d₆) d: 0.92 (6H, d, J = 6.8 Hz),

1.82–1.97 (1H, m), 3.09–3.17 (2H, m), 3.53 (3H, s), 4.98–

5.04 (2H, m), 7.03–7.08 (2H, m), 7.10 (1H, d,

J = 7.9 Hz), 7.23–7.31 (4H, m), 7.44 (1H, d,

J = 7.9 Hz), 7.75 (1H, s), 8.01 (1H, dd, J = 2.0 Hz,

7.9 Hz), 8.29 (1H, d, J = 2.0 Hz), 8.65 (1H, t,

J = 6.0 Hz); FAB-MS(m/z): 474 (M+H)⁺.

(4.27 g, 80%) as

a

colorless solid: ¹H NMR

(300 MHz, DMSO-d₆) d: 0.88 (6H, d, J = 6.6 Hz),

1.76–1.92 (1H, m), 3.04-3.12 (2H, m), 4.54 (1H, d,

J = 4.2 Hz), 5.53 (1H, br s), 7.64–7.67 (2H, m), 8.02

(1H, s), 8.55 (1H, t, J = 5.5 Hz).

5.40. 4-Bromo-3-(1,3-dioxolan-2-yl)-N-isobutylbenzamide

(21)

To a solution of 20 (3.21 g, 11.2 mmol) in CHCl₃

(55 mL) was added MnO₂(9.74 g, 112 mmol), and the

mixture was stirred at 70 °C for 1 h. After cooling, the

reaction mixture was ﬁltered through a pad of Celite

and concentrated in vacuo to give a colorless solid. To

a solution of the solid obtained above and ethylene gly-

col (1.39 g, 22.4 mmol) in toluene (110 mL) was added

4-methylbenzenesulfonic acid monohydrate (20 mg,

0.116 mmol), and the mixture was reﬂuxed for 12 h.

After cooling, to the reaction mixture was added 5%

NaHCO₃in H₂O. The organic layer was washed with

brine, dried over MgSO₄, ﬁltered, and concentrated in

vacuo. The residue was washed with diisopropyl ether

1

to give 21 (3.53 g, 96%) as a colorless solid: H NMR

(300 MHz, DMSO-d₆) d: 0.88 (6H, d, J = 6.6 Hz),

1.76–1.92 (1H, m), 3.04–3.11 (2H, m), 3.97–4.06 (2H,

m), 4.06–4.16 (2H, m), 5.97 (1H, s), 7.71–7.82 (2H, m),

8.00 (1H, s), 8.62 (1H, t, J = 5.9 Hz).

5.43. 2-tert-Butyl 2⁰-methyl 4-chloro-4⁰-[(isobutylami-

no)carbonyl]biphenyl-2,2⁰-dicarboxylate (24)

5.41. {2-Formyl-4-[(isobutylamino)carbonyl]phen-

yl}boronic acid (22)

Compound 24 was synthesized from 22 and 9f following

the same procedure as that for 23. Compound 24 was

obtained as a colorless solid (21%, 185 mg): H NMR

(300 MHz, DMSO-d₆) d: 0.90 (6H, d, J = 6.0 Hz), 1.14

(9H, s), 1.80–1.95 (1H, m), 3.08–3.16 (2H, m), 3.60

(3H, s), 7.24 (1H, d, J = 8.3 Hz), 7.34 (1H, d,

J = 7.9 Hz), 7.67 (1H, dd, J = 2.2 Hz, 8.3 Hz), 7.82

(1H, d, J = 2.2 Hz), 8.10 (1H, dd, J = 1.5 Hz, 7.9 Hz),

8.43 (1H, d, J = 1.5 Hz), 8.72 (1H, t, J = 5.9 Hz);

FAB-MS (m/z): 446 (M+H)⁺.

1

To the solution of 21 (630 mg, 1.92 mmol) in THF

(19 mL) was added at -78 °C 1.59 M n-butyllithium/hex-

ane (2.66 mL, 4.23 mmol), and the mixture was stirred

at ꢀ78 °C for 2 h. To the reaction mixture was added

triisopropyl borate (0.89 mL, 3.84 mmol), allowed to

warm to room temperature over 12 h. To the reaction

mixture was added 1 M HCl/H₂O (9.0 mL, 9.0 mmol)

and the mixture was stirred at room temperature for

3 h. After adding 1 M NaOH/H₂O (18 mL, 18 mmol),

the mixture was partitioned between Et₂O and H₂O,

and after the water layer was acidiﬁed with 1 M HCl/

H₂O (10 mL, 10 mmol), the water layer was partitioned

between AcOEt and H₂O, and extracted with AcOEt,

and the organic layer was washed with brine, dried

over MgSO₄, ﬁltered, and concentrated in vacuo to

5.44. 2⁰-({[3-(Aminocarbonyl)phenyl]amino}carbonyl)-4-

[(isobutylamino)carbonyl]-4⁰-methylbiphenyl-2-carboxylic

acid (17b)

To a solution of 23 (440 mg, 0.955 mmol) in MeOH

(10 mL) was added 10% Pd/C powder (44 mg), and the

mixture was stirred under hydrogen pressure (3.0 kg/

cm²) at room temperature for 12 h. The catalyst was ﬁ-

lered on Celite and the ﬁltrate was concentrated in

vacuo. The residue was puriﬁed by silica gel column

1

give 22 (280 mg, 59%) as a colorless solid: H NMR

(300 MHz, DMSO-d₆) d: 0.90 (6H, d, J = 6.8 Hz),

1.81–1.92 (1H, m), 3.11 (2H, t, J = 6.4 Hz), 7.66 (1H,

d, J = 7.7 Hz), 8.07 (1H, dd, J = 2.6 Hz, 7.7 Hz), 8.31–

8.35 (3H, br), 8.65 (1H, t, J = 6.0 Hz), 10.17 (1H, s).

chromatography (CHCl₃/MeOH = 30:1) to give

a

M. Miura et al. / Bioorg. Med. Chem. 14 (2006) 7688–7705

7701

colorless oil (350 mg). To a solution of the compound

5.47. 2⁰-tert-Butyl 2-methyl 4-nitrobiphenyl-2,2⁰-dicarb-

oxylate (27)

obtained above and 3-aminobenzamide (339 mg,

1.49 mmol) in DMF (11 mL) were added WSCÆHCl

(316 mg, 1.65 mmol) and HOBt (282 mg, 1.65 mmol),

and the mixture was stirred at 60 °C for 12 h. The mix-

ture was partitioned between AcOEt and H₂O, and

extracted with AcOEt, and the organic layer was washed

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

concentrated in vacuo. The residue was puriﬁed by silica

gel column chromatography (CHCl₃/MeOH = 97:3) to

give a colorless solid (150 mg). To a solution of the

residual solid in MeOH (6.0 mL) was added 1 M

NaOH/H₂O (0.46 mL, 0.46 mmol), and the mixture

was stirred at room temperature for 12 h. After cooling,

the reaction mixture was acidiﬁed with 1 M HCl/H₂O

(0.46 mL, 0.46 mmol). The resulting precipitate was ﬁl-

tered, washed with H₂O, and dried in vacuo to give

17b (71 mg, 49%) as a colorless solid: mp 143–144 °C

(MeOH–H₂O); ¹H NMR (400 MHz, DMSO-d₆) d:

0.88 (6H, d, J = 6.8 Hz), 1.79–1.89 (1H, m), 2.43 (3H,

s), 3.08 (2H, t, J = 6.4 Hz), 7.12 (1H, d, J = 7.6 Hz),

7.27–7.35 (4H, m), 7.49–7.51 (2H, m), 7.62 (1H, d,

J = 8.4 Hz), 7.86 (1H, br s), 7.93 (1H, dd, J = 2.0 Hz,

8.0 Hz), 8.00 (1H, br s), 8.28 (1H, d, J = 2.0 Hz), 8.62

(1H, t, J = 6.0 Hz), 10.18 (1H, br s), 12.85 (1H, br s);

FAB-MS(m/z): 474 (M+H)⁺; Anal. Calcd for

C₂₇H₂₇N₃O₅Æ1.3H₂O: C, 65.26; H, 6.00; N, 8.46. Found:

C, 65.30; H, 5.73; N, 8.19.

Compound 27 was synthesized from 25 following the

same procedure as that for 22. Compound 27 was ob-

tained as a yellow solid (30%, 4.32 g): ¹H NMR

(300 MHz, DMSO-d₆) d: 1.17 (9H, s), 3.61 (3H, s),

7.22 (1H, d, J = 7.3 Hz), 7.51–7.60 (2H, m), 7.61–7.68

(1H, m), 7.93 (1H, d, J = 7.7 Hz), 8.46 (1H, dd,

J = 2.5 Hz, 8.4 Hz), 8.66 (1H, d, J = 2.5 Hz); FAB-MS

(m/z): 358 (M+H)⁺.

5.48. tert-Butyl 4⁰-amino-4-[(isobutylamino)carbonyl]-2⁰-

({[3-(methoxycarbonyl)phenyl]amino}carbonyl)biphenyl-

2-carboxylate (30)

To a solution of 27 (1.30 g, 2.85 mmol) in MeOH (20 mL)

was added 1 M NaOH/H₂O (3.0 mL, 3.0 mmol), and the

mixture was reﬂuxed for 2 h. After cooling and concen-

trating, the reaction mixture was acidiﬁed with 1 M

HCl/H₂O (1.8 mL, 1.8 mmol). The mixture was parti-

tioned between AcOEt and H₂O and extracted with

AcOEt, and the organic layer was washed with H₂O and

brine, dried over MgSO₄, ﬁltered, and concentrated in

vacuo to give a pale yellow solid (1.23 g). To a solution

of the compound obtained above (960 mg) and methyl

3-aminobenzoate (656 mg, 4.34 mmol) in DMF (20 mL)

was added WSCÆHCl (623 mg, 3.25 mmol) and HOBt

(439 mg, 3.25 mmol), and the mixture was stirred at

80 °C for 12 h. The mixture was partitioned between

AcOEt and H₂O, and extracted with AcOEt, and the

organic layer was washed with H₂O and brine, dried over

MgSO₄, ﬁltered, and concentrated in vacuo. The residue

was puriﬁed by silica gel column chromatography

(CHCl₃/MeOH = 98:2) to give a yellow solid (1.28 g).

To a solution of the residual solid in MeOH (20 mL) were

added 10% Pd/C powder (130 mg), and the mixture was

stirred in a hydrogen atmosphere at room temperature

for 12 h. The catalyst was ﬁltrated on Celite and the ﬁl-

trate was concentrated in vacuo. The residue was puriﬁed

by silica gel column chromatography (CHCl₃/

MeOH = 30:1) to give 30 (1.00 g, 85%) as a yellow solid:

¹H NMR (300 MHz, DMSO-d₆) d: 0.87 (6H, d,

J = 6.6 Hz), 1.27 (9H, s), 1.78–1.89 (1H, m), 3.07 (2H, t,

J = 6.2 Hz), 3.82 (3H, s), 5.46 (2H, br s), 6.72 (1H, dd,

J = 2.0 Hz, 8.3 Hz), 6.84 (1H, s), 6.86–6.89 (1H, m), 7.26

(1H, d, J = 8.1 Hz), 7.39 (1H, t, J = 7.9 Hz), 7.60 (1H, d,

J = 7.7 Hz), 7.69 (1H, d, J = 7.9 Hz), 7.86 (1H, dd,

J = 1.8 Hz, 8.0 Hz), 8.12 (1H, d, J = 2.0 Hz), 8.22 (1H,

br s), 8.57 (1H, t, J = 5.7 Hz), 10.09 (1H, br s); FAB-MS

(m/z): 546 (M+H)⁺.

5.45. 2⁰-({[3-(Aminocarbonyl)phenyl]amino}carbonyl)- 4⁰-

chloro-4-[(isobutylamino)carbonyl]biphenyl-2-carboxylic

acid (17c)

Compound 17c was synthesized from 24 following the

same procedure as that for 17b except for deprotection

of the tert-butyl group under acidic conditions. Com-

pound 17c was obtained as a colorless solid (49%,

80 mg): mp 155–158 °C (MeOH–H₂O); ¹H NMR

(400 MHz, DMSO-d₆) d: 0.88 (6H, d, J = 6.3 Hz),

1.80–1.90 (1H, m), 3.09 (2H, t, J = 6.3 Hz), 7.27–7.33

(4H, m), 7.52 (1H, d, J = 7.9 Hz), 7.59–7.64 (2H, m),

7.75 (1H, d, J = 2.5 Hz), 7.87 (1H, br s), 7.96 (1H, dd,

J = 1.9 Hz, 7.8 Hz), 8.00 (1H, br s), 8.32 (1H, d,

J = 2.0 Hz), 8.65 (1H, t, J = 5.3 Hz), 10.36 (1H, br s),

12.85 (1H, br s); FAB-MS (m/z): 494 (M+H)⁺; Anal.

Calcd for C₂₆H₂₄ClN₃O₅Æ1.0H₂O: C, 61.00; H, 5.12; N,

8.21; Cl, 6.92. Found: C, 61.24; H, 4.98; N, 8.23; Cl,

7.08.

5.46. 2-tert-Butyl 2⁰-methyl 4-[(isobutylamino)carbonyl]-

4⁰-nitrobiphenyl-2,2⁰-dicarboxylate (26)

Compound 26 was synthesized from 21 and methyl 2-

bromo-5-nitrobenzoate (25) following the same proce-

dure as that for 22. Compound 26 was obtained as a

yellow solid (40%, 2.15 g): ¹H NMR (300 MHz,

DMSO-d₆) d: 0.92 (6H, d, J = 6.8 Hz), 1.16 (9H, s),

1.83–1.94 (1H, m), 3.13 (2H, t, J = 6.4 Hz), 3.64 (3H,

s), 7.33 (1H, d, J = 8.0 Hz), 7.58 (1H, d, J = 8.4 Hz),

8.07 (1H, dd, J = 1.8 Hz, 7.9 Hz), 8.37 (1H, d,

J = 1.7 Hz), 8.49 (1H, dd, J = 2.4 Hz, 8.4 Hz), 8.69

(1H, d, J = 2.4 Hz), 8.74 (1H, t, J = 6.2 Hz); FAB-MS

(m/z): 446 (M+H)⁺.

5.49. tert-Butyl 2⁰-({[3-(aminocarbonyl)phenyl]ami-

no}carbonyl)-4⁰- nitrobiphenyl-2-carboxylate (28)

Compound 28 was synthesized from 26 and 3-aminob-

enzamide following the same procedure as that for 30

without reduction of the nitro group. Compound 28

1

was obtained as a yellow solid (87%, 3.84 g): H NMR

(300 MHz, DMSO-d₆) d: 1.21 (9H, s), 7.25–7.38 (3H,

m), 7.45–7.66 (4H, m), 7.85–8.02 (4H, m), 8.40 (1H,

dd, J = 2.4 Hz, 8.4 Hz), 8.53 (1H, d, J = 2.4 Hz), 10.43

(1H, s).

7702

M. Miura et al. / Bioorg. Med. Chem. 14 (2006) 7688–7705

5.50. tert-Butyl 4⁰-amino-2⁰-({[3- (aminocarbonyl)phen-

yl]amino}carbonyl)biphenyl-2-carboxylate (29)

with AcOEt, and the organic layer was washed with

brine, dried over MgSO₄, ﬁltered, and concentrated in

vacuo to give 34 (190 mg, 53%) as a colorless solid: H

1

Compound 29 was synthesized from 28 with reduction

of the nitro group following the same procedure of as

that for 30. Compound 29 was obtained as a yellow sol-

id (69%, 2.13 g): ¹H NMR (300 MHz, DMSO-d₆) d: 1.27

(9H, s), 5.39 (2H, br s), 6.69 (1H, dd, J = 2.4 Hz,

8.4 Hz), 6.82 (1H, d, J = 8.2 Hz), 6.85 (1H, d,

J = 2.4 Hz), 7.19 (1H, dd, J = 1.1 Hz, 7.7 Hz), 7.24–

7.35 (3H, m), 7.38–7.45 (1H, m), 7.45–7.52 (2H, m),

7.66 (1H, dd, J = 1.3 Hz, 7.7 Hz), 7.85 (1H, br s), 7.95

(1H, br s), 9.65 (1H, s); FAB-MS (m/z): 432 (M+H)⁺.

NMR (300 MHz, DMSO-d₆) d: 0.89 (6H, d,

J = 6.6 Hz), 1.22 (9H, s), 1.78–1.92 (1H, m), 3.09 (2H,

t, J = 6.6 Hz), 3.40 (3H, s), 3.83 (3H, s), 7.33–7.46

(3H, m), 7.60–7.70 (2H, m), 7.75 (1H, d, J = 7.3 Hz),

7.88 (1H, br s), 7.95–8.01 (1H, m), 8.15 (1H, br s),

8.24 (1H, br s), 8.67 (1H, t, J = 5.5 Hz), 10.21 (1H, br

s); FAB-MS (m/z): 656 (M+H)⁺.

5.54. tert-Butyl 2⁰-({[3-(methoxycarbonyl)phenyl]ami-

no}carbonyl)-4⁰-[methyl(triﬂuoroacetyl)amino]biphenyl-2-

carboxylate (35)

5.51. tert-Butyl 4⁰-amino-2⁰-({[3- (methoxycarbon-

yl)phenyl]amino}carbonyl)biphenyl-2-carboxylate (31)

Compound 35 was synthesized from 31 following the

same procedure as that for 34. Compound 35 was

obtained as a colorless solid (83%, 254 mg): H NMR

1

Compound 31 was synthesized from 27 following the

same procedure as that for 30. Compound 31 was ob-

tained as a colorless solid (73%, 783 mg): ¹H NMR

(300 MHz, DMSO-d₆) d: 1.27 (9H, s), 3.82 (3H, s),

5.39 (2H, br s), 6.70 (1H, d, J = 8.2 Hz), 6.81–6.87

(2H, m), 7.21 (1H, d, J = 7.5 Hz), 7.27–7.46 (3H, m),

7.56–7.69 (3H, m), 8.17 (1H, s), 9.87 (1H, s); FAB-MS

(m/z): 447 (M+H)⁺.

(300 MHz, DMSO-d₆) d: 1.22 (9H, s), 3.83 (3H, s),

3.83 (3H, s), 7.25–7.38 (2H, m), 7.38–7.48 (2H, m),

7.50–7.58 (1H, m), 7.58–7.66 (2H, m), 7.70 (1H, d,

J = 7.9 Hz), 7.74–7.84 (2H, m), 8.12 (1H, s), 10.05

(1H, s); FAB-MS (m/z): 557 (M+H)⁺.

5.55. tert-Butyl 2⁰-({[3-(aminocarbonyl)phenyl]ami-

no}carbonyl)-4- [(isobutylamino)carbonyl]-4⁰-(methylami-

no)biphenyl-2-carboxylate (36)

5.52. tert-Butyl 4⁰-(dimethylamino)-2⁰-({[3-(methoxycar-

bonyl)phenyl]amino}carbonyl)biphenyl-2-carboxylate (32)

To a solution of 35 (175 mg, 0.267 mmol) obtained

above in MeOH (7.0 mL) was added 1 M NaOH/H₂O

(0.8 mL, 0.8 mmol), and the mixture was reﬂuxed for

2 h. After cooling, the reaction mixture was acidiﬁed

with 5% citric acid in H₂O. The resulting precipitate

was ﬁltered, washed with H₂O, and dried in vacuo to

give a pale yellow solid (115 mg). To a solution of the

compound obtained above and NH₄Cl (41 mg,

0.766 mmol) in DMF (11 mL) were added WSCÆHCl

(55 mg, 0.288 mmol), HOBt (39 mg, 0.289 mmol), and

N-ethyl-N-isopropylpropan-2-amine (i-Pr₂NEt) (99 mg,

0.766 mmol). The mixture was stirred at room tempera-

ture for 12 h, then partitioned between AcOEt and H₂O,

and extracted with AcOEt, and the organic layer was

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

and concentrated in vacuo. The residue was puriﬁed

by silica gel column chromatography (CHCl₃/

MeOH = 50:1) to give 36 (78 mg, 75%) as a colorless

amorphous powder: ¹H NMR (300 MHz, CDCl₃) d:

0.96 (6H, d, J = 6.6 Hz), 1.83–1.96 (1H, m), 2.77 (3H,

s), 3.25 (2H, t, J = 6.6 Hz), 5.68 (1H, br), 6.28 (1H,

br), 6.54 (1H, t, J = 5.7 Hz), 6.68 (1H, dd, J = 2.6 Hz,

8.4 Hz), 6.93 (1H, d, J = 8.3 Hz), 7.00 (1H, d,

J = 2.6 Hz), 7.22–7.26 (2H, m), 7.32–7.35 (1H, m),

7.42–7.45 (1H, m), 7.58 (1H, t, J = 1.8 Hz), 7.69 (1H,

dd, J = 2.0 Hz, 8.0 Hz), 8.12 (1H, d, J = 1.8 Hz), 8.56

(1H, s); FAB-MS (m/z): 545 (M+H)⁺.

To a solution of 30 (243 mg, 0.544 mmol) and HCHO

(37% solution in H₂O, 1.3 mL, 1.63 mmol) in 1,2-dichlo-

roethane (5.0 mL) and AcOH (326 mg, 5.44 mmol) was

added NaBH(OAc)₃(346 mg, 1.63 mmol), and the mix-

ture was stirred at room temperature for 10 h. The mix-

ture was partitioned between AcOEt and 5% NaHCO₃

in H₂O, and the organic layer was washed with brine,

dried over MgSO₄, ﬁltered, and concentrated in vacuo.

The residue was puriﬁed by silica gel column chroma-

tography (hexane/AcOEt = 4:1) to give 32 (155 mg,

60%) as a colorless solid: ¹H NMR (300 MHz,

DMSO-d₆) d: 1.26 (9H, s), 3.00 (6H, s), 3.83 (3H, s),

6.88 (1H, d, J = 8.0 Hz), 6.95–7.04 (2H, m), 7.22 (1H,

d, J = 7.5 Hz), 7.30–7.49 (3H, m), 7.58–7.72 (3H, m),

8.18 (1H, s), 9.96 (1H, s); FAB-MS (m/z): 475 (M+H)⁺.

5.53. tert-Butyl 4-[(isobutylamino)carbonyl]-2⁰-({[3-

(methoxycarbonyl)phenyl]amino}carbonyl)-4⁰-[methyl(tri-

ﬂuoroacetyl)amino]biphenyl-2-carboxylate (34)

To a solution of 30 (300 mg, 0.550 mmol) and pyridine

(0.06 mL, 0.715 mmol) in CH₂Cl₂(6.0 mL) was added

TFAA (0.09 mL, 0.660 mmol), and the mixture was stir-

red at room temperature for 3 days. The mixture was

partitioned between AcOEt and 5% NaHCO₃in H₂O,

and the organic layer was washed with H₂O and brine,

dried over MgSO₄, ﬁltered, and concentrated in vacuo.

The residue was puriﬁed by silica gel column chroma-

tography (hexane/AcOEt = 4:1) to give a colorless solid.

To a solution of the compound obtained above and

K₂CO₃(53 mg, 0.383 mmol) in 2-butanone (5.0 mL)

was added MeI (136 mg, 0.958 mmol), and the mixture

was stirred at 40 °C for 4 h. After cooling, the mixture

was partitioned between AcOEt and H₂O, and extracted

5.56. tert-Butyl 2⁰-({[3-(aminocarbonyl)phenyl]ami-

no}carbonyl)-4⁰- (dimethylamino)biphenyl-2-carboxylate

(33)

Compound 33 was synthesized from 32 following the

same procedure as that for 36. Compound 33 was ob-

tained as a colorless solid (50%, 125 mg): ¹H NMR

M. Miura et al. / Bioorg. Med. Chem. 14 (2006) 7688–7705

7703

(300 MHz, DMSO-d₆) d: 1.25 (9H, s), 2.99 (3H, s), 6.88

5.60. 4⁰-Amino-2⁰-({[3- (aminocarbonyl)phenyl]ami-

no}carbonyl)biphenyl-2-carboxylic acid (12i)

(1H, dd, J = 2.6 Hz, 8.4 Hz), 6.96 (1H, d, J = 2.6 Hz),

6.99 (1H, d, J = 8.4 Hz), 7.21 (1H, d, J = 7.7 Hz),

7.26–7.37 (3H, m), 7.43 (1H, dt, J = 1.4 Hz, 7.5 Hz),

7.46–7.58 (3H, m), 7.69 (1H, dd, J = 1.3 Hz, 7.7 Hz),

7.87 (1H, br s), 9.77 (1H, s); FAB-MS (m/z): 458

(MꢀH)^ꢀ.

5.57. tert-Butyl 2⁰-({[3-(aminocarbonyl)phenyl]ami-

no}carbonyl)-4⁰-(methylamino)biphenyl-2-carboxylate

(37)

Compound 12i was synthesized from 29 following the

same procedure as that for 17d. Compound 12i was ob-

tained as a colorless amorphous powder (88%, 150 mg):

¹H NMR (400 MHz, DMSO-d₆): d 5.64 (2H, br s), 6.93

(1H, dd, J = 1.5 Hz, 8.3 Hz), 7.01 (1H, d, J = 8.3 Hz),

7.07 (1H, d, J = 1.5 Hz), 7.10–7.22 (1H, m), 7.22–7.34

(2H, m), 7.34–7.40 (1H, m), 7.42–7.55 (3H, m), 7.75

(1H, dd, J = 1.5 Hz, 7.9 Hz), 7.86 (1H, br s), 7.91 (1H,

br s), 9.97 (1H, s); FAB-MS (m/z): 376 (M+H)⁺; HRMS

(ESI) Calcd for C₂₁H₁₇N₃O₄: 376.1297. Found:

376.1290.

Compound 37 was synthesized from 35 following the

same procedure as that for 36. Compound 37 was ob-

tained as a colorless solid (58%, 119 mg): ¹H NMR

(300 MHz, DMSO-d₆) d: 1.27 (9H, s), 2.76 (3H, d,

J = 4.6 Hz), 5.92–6.00 (1H, m), 6.68 (1H, dd,

J = 2.4 Hz, 8.2 Hz), 6.79 (1H, d, J = 2.4 Hz), 6.89 (1H,

d, J = 8.2 Hz), 7.21 (1H, d, J = 7.5 Hz), 7.24–7.36 (3H,

m), 7.42 (1H, dt, J = 1.3 Hz, 7.5 Hz), 7.46–7.54 (2H,

m), 7.67 (1H, dd, J = 1.3 Hz, 7.7 Hz), 7.86 (1H, br s),

7.95 (1H, br s), 9.69 (1H, s); FAB-MS (m/z): 444 (M-

H)^ꢀ.

5.61. 2⁰-({[3-(Aminocarbonyl)phenyl]amino}carbonyl)-4⁰-

(dimethylamino)biphenyl-2-carboxylic acid (12j)

Compound 12j was synthesized from 33 following the

same procedure as that for 17d. Compound 12j was ob-

tained as a colorless solid (40%, 40 mg): mp 141–144 °C

1

(H₂O); H NMR (400 MHz, DMSO-d₆) d: 3.00 (6H, s),

6.85 (1H, dd, J = 2.9 Hz, 8.3 Hz), 6.93 (1H, d,

J = 2.9 Hz), 7.01 (1H, d, J = 8.3 Hz), 7.18 (1H, d,

J = 7.9 Hz), 7.24–7.37 (3H, m), 7.44 (1H, dt,

J = 1.5 Hz, 7.4 Hz), 7.49 (1H, d, J = 7.9 Hz), 7.56 (1H,

d, J = 8.3 Hz), 7.72 (1H, dd, J = 1.5 Hz, 7.9 Hz), 7.86

(1H, br s), 7.92 (1H, br s), 9.96 (1H, s), 12.81 (1H, br

s); FAB-MS (m/z): 404 (M+H)⁺; Anal. Calcd for

C₂₃H₂₁N₃O₄Æ0.1H₂OÆ0.5AcOEt: C, 66.83; H, 5.65; N,

9.35. Found: C, 66.94; H, 5.25; N, 9.48.

5.58. 2⁰-({[3-(Aminocarbonyl)phenyl]amino}carbonyl)-4-

[(isobutylamino)carbonyl]-4⁰-(methylamino)biphenyl-2-

carboxylic acid (17d)

To a solution of 36 (75 mg, 0.143 mmol) in CH₂Cl₂

(5 mL) was added triﬂuoroacetic acid (2 mL), and

the mixture was stirred at room temperature for

12 h. The mixture was concentrated in vacuo. To the

residue was added 1 M NaOH in H₂O, and the solu-

tion was then acidiﬁed with 5% citric acid in H₂O.

The resulting precipitate was ﬁltered, washed with

H₂O, and dried in vacuo to give 17d (36 mg, 52%)

5.62. 2⁰-({[3-(Aminocarbonyl)phenyl]amino}carbonyl)-4⁰-

(methylamino)biphenyl-2-carboxylic acid (12k)

Compound 12k was synthesized from 37 following the

same procedure as that for 17d. Compound 12k was ob-

tained as a colorless solid (49%, 48 mg): mp 135–137 °C

1

as a colorless solid: mp 156–159 °C (H₂O); H NMR

(400 MHz, DMSO-d₆) d: 0.87 (6H, d, J = 6.8 Hz),

1.78–1.91 (1H, m), 2.43 (3H, s), 3.67 (2H, t,

J = 6.4 Hz), 6.03 (1H, br s), 6.67 (1H, dd,

J = 2.4 Hz, 8.3 Hz), 6.79 (1H, d, J = 2.4 Hz), 6.94

(1H, d, J = 8.3 Hz), 7.18–7.32 (3H, m), 7.49 (1H, d,

J = 7.8 Hz), 7.59 (1H, d, J = 9.3 Hz), 7.85–7.88 (2H,

m), 7.99 (1H, br s), 8.19 (1H, d, J = 1.9 Hz), 8.90

(1H, d, J = 1.9 Hz), 10.09 (1H, s), 12.87 (1H, br s);

FAB-MS (m/z): 489 (M+H)⁺; Anal. Calcd for

C₂₇H₂₈N₄O₅Æ1.5H₂O: C, 62.90, H, 6.06; N, 10.87.

Found: C, 63.00; H, 5.82; N, 10.66.

1

(H₂O); H NMR (400 MHz, DMSO-d₆) d: 2.75 (3H, s),

5.98 (1H, br s), 6.65 (1H, dd, J = 2.5 Hz, 8.3 Hz), 6.77

(1H, d, J = 2.5 Hz), 6.91 (1H, d, J = 8.3 Hz), 7.16 (1H,

d, J = 7.3 Hz), 7.25–7.36 (3H, m), 7.42 (1H, dt,

J = 1.4 Hz, 7.4 Hz), 7.46–7.55 (2H, m), 7.70 (1H, dd,

J = 1.0 Hz, 7.8 Hz), 7.86 (1H, br s), 7.92 (1H, br s),

9.95 (1H, s); FAB-MS (m/z): 390 (M+H)⁺; HRMS

(ESI) Calcd for C₂₂H₁₉N₃O₄: 390.1454. Found:

390.1464.

5.63. Docking study

5.59. 2⁰-({[3-(Aminocarbonyl)phenyl]amino}carbonyl)-4⁰-

nitrobiphenyl-2-carboxylic acid (12h)

Docking simulation was carried out using GOLD

(CCDC, Cambridge, UK) software at the active site of

TF/FVIIa complex (PDB code: 1DAN). After docking,

energy minimization was performed based on the

MMFF94s force ﬁeld using MOE 2004.03 (Chemical

Computing Group Inc., Montreal, CA).

Compound 12h was synthesized from 28 following the

same procedure as that for 17d. Compound 12h was ob-

tained as a colorless solid (56%, 112 mg): mp 230–233 °C

(H₂O); ¹H NMR (400 MHz, DMSO-d₆) d: 7.24–7.36

(3H, m), 7.45–7.51 (1H, m), 7.52–7.65 (4H, m), 7.88

(1H, br s), 7.92 (1H, dd, J = 1.0 Hz, 7.8 Hz), 7.97 (1H,

br s), 8.36 (1H, dd, J = 2.4 Hz, 8.3 Hz), 8.48 (1H, d,

J = 2.4 Hz), 10.45 (1H, s), 12.77 (1H, br s); FAB-MS

(m/z): 404 (MꢀH)^ꢀ; Anal. Calcd for C₂₁H₁₅N₃O₆Æ0.8-

H₂O: C, 60.09; H, 3.99; N, 10.01. Found: C, 60.33; H,

3.82; N, 9.66.

5.64. Biology

5.64.1. Chromogenic assay. The hydrolysis rates of syn-

thetic substrates were assayed by continuously measur-

ing absorbance at 405 nm at 37 °C with a microplate

spectrophotometer (Spectramax 340PC, Molecular

7704

M. Miura et al. / Bioorg. Med. Chem. 14 (2006) 7688–7705

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Devices Co. California, USA). Reaction mixtures

(40 lL) were prepared in 96-well plates containing chro-

mogenic substrates and an inhibitor in either 20 mM

HEPES, 0.01% BSA, 5 mM CaCl₂, pH 7.4, or 0.15 M

NaCl. Reactions were initiated with 10 lL portions of

the enzyme solution. Enzymes and substrates were used

as follows: TF/FVIIa and S-2288; factor Xa and s-2222;

thrombin and S-2238; and trypsin and S-2222. The con-

centration of an inhibitor required to inhibit enzyme

activity by 50% (IC₅₀) was calculated from concentra-

tion-response curves in which the logit transformation

of residual activity was plotted against the logarithm

of inhibitor concentration.

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W. B.; Kolesnikov, A.; Rai, R.; Sprengeler, P. A.; Leahy, E.

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5.64.2. Plasma clotting time assay. Citrated blood sam-

ples from human were collected in accordance with

Astellas Research Ethics Committee. Platelet-poor

plasma was centrifuged at 3000 rpm for 10 min and

stored at ꢀ40 °C until use. Plasma clotting times were

recorded using a KC10A coagulometer (Amelung Co.,

Lehbringsweg, Germany) at 37 °C. Prothrombin time

(PT) and activated partial thromboplastin time

(APTT) were measured using Hemos Recombiplastin

and Hemoliance (Instrumentation Laboratory Compa-

ny Lexington, MA, USA), respectively. Coagulation

times for each test sample were compared with coag-

ulation times measured using 4% DMSO in water as

a control. The concentration required to double clot-

ting time (CT₂) was estimated from each individual

concentration–response curve. Each measurement was

performed three times and represented as the mean

value.

Acknowledgments

The authors deeply thank Dr. Toshio Okazaki for his

helpful support in preparing this manuscript, and are

also grateful to the staﬀ of the Division of Analytical

Science Laboratories for the elemental analysis and

spectral measurements.

References and notes

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14. A substantial amount of tert-butyl 4⁰-(dimethylamino)-2⁰-

{[(3-{[(hydroxymethyl)amino]carbonyl}phenyl)amino]car-

bonyl}biphenyl-2- carboxylate, which was obtained from

reaction between HCHO and the carboxamide group of 29,

was obtained in 60% yield: ¹H NMR (300 MHz, DMSO-d₆)

d: 1.25 (9H, s), 2.99 (6H, s), 4.67 (2H, t, J = 6.2 Hz), 5.59–

5.67 (1H, m), 6.87 (1H, d, J = 8.4 Hz), 6.96 (1H, s), 6.99

(1H, d, J = 8.4 Hz), 7.21 (1H, d, J = 7.8 Hz), 7.26–7.38 (2H,

m), 7.40–7.51 (2H, m), 7.57 (1H, d, J = 8.0 Hz), 7.69 (1H, d,

J = 7.8 Hz), 7.97 (1H, br s), 8.98–9.06 (1H, m), 9.80 (1H, s);

FAB-MS (m/z): 488 (MꢀH)^ꢀ.

15. In the course of our research, the X-ray crystal structure of

compound 1 bound to the Des-Gla-FVIIa/sTF complex

was reported by Granberg and co-workers: Granberg, K.

L; Petersen, J. F. W.; Anderson, M.; Nardi, F.; Darby, N.;

Lindskog, P.; Slater, T.; Zetterberg, F. J.; Stocker, A.;

Caulkett, P.; Preston, J.; Walker, R.; Gordon, C.;

Fahlander, U. E. The 226th ACS National Meeting, Poster

85, New York, September 7–11, 2003. The binding mode

of compound 1 is generally similar between the X-ray

crystal structure and our molecular modeling.

16. Before docking simulation, we have checked the conser-

vation of water molecules in S1 and S2 sites referred in this

text for 24 structures of FVIIa deposited in Protein Data

Bank. As for S1 site, the water molecule is conserved

among 19 out of 24 structures. (Note that this water

molecule is also well conserved in trypsin with similar

hydrogen-bonding networks as FVIIa). As for S2 site, the

corresponding position is occupied with hydrophilic

groups of ligand molecule in 5 entries and the water

molecule is conserved among 10 out of remaining 19

structures.

17. Wright, S. W.; Hageman, D. L.; Wright, A. S.; McClure,

L. D. Tetrahedron Lett. 1997, 38, 7345.

Article Doi

Potent and selective TF/FVIIa inhibitors containing a neutral P1 ligand

DOI: 10.1016/j.bmc.2006.08.010

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Authors:

Article abstract of DOI:10.1016/j.bmc.2006.08.010

Full text of DOI:10.1016/j.bmc.2006.08.010

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