Discovery of 6-[5-(4-fluorophenyl)-3-methyl-pyrazol-4-yl]-benzoxazin-3-one derivatives as novel selective nonsteroidal mineralocorticoid receptor antagonists

Bioorganic & Medicinal Chemistry 22 (2014) 5428–5445

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Discovery of 6-[5-(4-ﬂuorophenyl)-3-methyl-pyrazol-4-yl]-

benzoxazin-3-one derivatives as novel selective nonsteroidal

mineralocorticoid receptor antagonists

a

Tomoaki Hasui ^a,, Norio Ohyabu , Taiichi Ohra , Koji Fuji , Takahiro Sugimoto , Jun Fujimoto ,

Kouhei Asano ^a, Masato Oosawa ^a, Sachiko Shiotani ^a, Nobuhiro Nishigaki ^a, Keiji Kusumoto ^a,

Hideki Matsui ^a, Atsushi Mizukami ^a, Noriyuki Habuka ^a, Satoshi Sogabe ^a, Satoshi Endo ^a, Midori Ono ^a,

Christopher S. Siedem ^b, Tony P. Tang ^b, Cassandra Gauthier ^b, Lisa A. De Meese ^b, Steven A. Boyd ^b, Shoji

Fukumoto ^a

⇑

^aPharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi, 2-Chome, Fujisawa, Kanagawa 251-8555, Japan

^bArray BioPharma Inc., 3200 Walnut Street, Boulder, CO 80301, United States

a r t i c l e i n f o

a b s t r a c t

Article history:

In the course of our study on selective nonsteroidal mineralocorticoid receptor (MR) antagonists, a series

of novel benzoxazine derivatives possessing an azole ring as the core scaffold was designed for the

purpose of attenuating the partial agonistic activity of the previously reported dihydropyrrol-2-one

derivatives. Screening of alternative azole rings identiﬁed 1,3-dimethyl pyrazole 6a as a lead compound

with reduced partial agonistic activity. Subsequent replacement of the 1-methyl group of the pyrazole

ring with larger lipophilic side chains or polar side chains targeting Arg817 and Gln776 increased MR

binding activity while maintaining the agonistic response at the lower level. Among these compounds,

6-[1-(2,2-diﬂuoro-3-hydroxypropyl)-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-4-yl]-2H-1,4-benzoxazin-

3(4H)-one (37a) showed highly potent in vitro activity, high selectivity versus other steroid hormone

receptors, and good pharmacokinetic proﬁles. Oral administration of 37a in deoxycorticosterone

acetate-salt hypertensive rats showed a signiﬁcant blood pressure-lowering effect with no signs of

antiandrogenic effects.

Received 20 June 2014

Revised 23 July 2014

Accepted 24 July 2014

Available online 12 August 2014

Keywords:

Mineralocorticoid receptor

MR

Nonsteroidal

Aldosterone

MR antagonists

1. Introduction

cardiovascular diseases. It should be noted that the marketed ste-

roidal MR antagonists, spironolactone and eplerenone, have proven

The mineralocorticoid receptor (MR) is a ligand-activated tran-

scription factor that belongs to the steroid receptor subfamily

within the nuclear receptor superfamily and shares high structural

homology with the androgen receptor (AR), progesterone receptor

(PR), and glucocorticoid receptor (GR). The classically perceived

role of MR has been one of blood pressure control through regula-

tion of body ﬂuid and electrolyte balance by binding with aldoste-

rone, a primary endogenous mineralocorticoid, in the kidney.¹

Recently, it was revealed that MR is expressed in various tissues,

including those of the heart, brain, and blood vessels, and abnor-

mal activation of the MR by excessive levels of aldosterone is clo-

sely associated with development of congestive heart failure and

renal dysfunction as well as hypertension.^2,3Thus, MR blockade

has been shown to be a promising therapeutic option for such

their clinical utility for those diseases.⁴Clinical use of these drugs,

however, is limited due to the following issues as well as hyperka-

lemia. Spironolactone is a potent antagonist, but its low selectivity

with respect to AR and PR causes sexual hormone-related side

effects, such as impotence, gynecomastia, and menstrual irregular-

ity.⁵On the other hand, eplerenone shows improved selectivity in

mitigating these side effects but is less potent than spironolac-

tone.⁶Accordingly, new, potent, and selective MR antagonists have

been expected to be a clinically useful drugs. In recent years, there

has been a keen interest in the discovery of such compounds.⁷

We have previously reported the benzoxazin-3-one derivatives,

1 and 2, as novel nonsteroidal MR antagonists (Fig. 1).^8,9Although

compound 1 showed potent MR binding activity (IC₅₀= 41 nM), it

still had moderate PR binding activity (PR binding IC₅₀= 1900 nM).

On the other hand, compound 2 showed potent MR binding activ-

ity (IC₅₀= 43 nM) with improved selectivity for PR (PR binding IC₅₀

>10,000 nM). However, in a functional assay using a reporter gene,

⇑

Corresponding author. Tel.: +81 466 32 1142.

E-mail address: tomoaki.hasui@takeda.com (T. Hasui).

http://dx.doi.org/10.1016/j.bmc.2014.07.038

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

5429

Figure 1. Structures of steroidal and nonsteroidal MR antagonists.

compound 2 showed not only potent MR antagonistic activity

Binding mode analyses of compounds 1 and 2 guided our cen-

tral ring selection. The X-ray co-crystal structure of MR with a

dihydrofuran-2-one derivative, a close analog of compound 2

(PDB code: 3WFG),⁹suggested that the dihydropyrrol-2-one deriv-

atives form a water-mediated hydrogen bonding network involv-

ing the carbonyl group of the central ring with Arg817 and

Gln776. On the other hand, compound 1 and its related derivatives

were considered to have no such network. Therefore, it was

assumed that the hydrogen bonding network has a key role in

the partial agonistic activity and the carbonyl group serves as a

‘trigger’ moiety. Indeed, several carbonyl-containing dihydrofu-

ran-2-one derivatives also showed partial agonistic activity,

although they were less likely to show agonistic activity than the

dihydropyrrol-2-one derivatives. This different propensity for ago-

nistic activity might be explained by the weaker hydrogen bonding

ability of the carbonyl group. Thus, we selected azole rings for the

central ring to avoid formation of such water-mediated hydrogen

bonding networks.

As shown in Figure 2, novel azole derivatives (A) were designed.

Although positions Y and Z of the azole ring were thought to allow

introduction of substituents, the azole derivatives possessing sub-

stituents at position Y (exempliﬁed by compound 1) were unlikely

to achieve potent activities and high selectivity simultaneously. In

addition, increasing the polarity of position Y appeared to strongly

improve selectivity (particularly over PR), based on our earlier

ﬁndings for compound 2 showing that the PR binding afﬁnity

was greatly reduced by polar moieties at the corresponding posi-

tion. Therefore, we focused on azole derivatives bearing substitu-

ents at position Z with unsubstituted heteroatoms (O, N, and NH)

for position Y in this study. Both nonpolar and polar side chains

(IC₅₀= 83 nM) but also moderate partial agonistic activity at a high

concentration (30% activation at 10 l

M).¹⁰It has been reported

that strong partial agonistic activity attenuates antimineralocorti-

coid action in animal models.¹¹In addition, the inﬂuence of the

partial agonistic activity on clinical efﬁcacy and safety remains

unclear. Therefore, we decided to identify new compounds with

reduced partial agonistic activity. Herein, we describe our efforts

to discover compounds with potent binding and attenuated partial

agonistic activity toward MR, and high selectivities versus other

steroid receptors.

Figure 2 illustrates our compound design. From our previous

research on benzoxazin-3-one derivatives, we found that a key

pharmacophore for high MR binding afﬁnity consisted of three

parts: a benzoxazin-3-one ring, a 4-ﬂuorobenzene ring, and a cen-

tral cyclic moiety linking these two aromatic rings at the adjacent

position. Among these parts, we have generally found that the cen-

tral ring is the most tolerant to modiﬁcation without loss of bind-

ing activity. To establish a simple structure–partial agonistic

activity relationship, we evaluated the functional activities of the

related compounds of 1 and 2. Consequently, most of the dihydro-

pyrrol-2-one derivatives, exempliﬁed by compound 2 showed

moderate partial agonistic activity at high concentrations (approx-

imately 20–30% at 10 lM), whereas the pyrazole derivatives,

exempliﬁed by compound 1, showed low agonistic activities. These

results suggested that the partial agonistic activity tended to

depend on the structure of the central ring. Accordingly, selection

of an appropriate central ring followed by overall optimization was

expected to produce a compound with potent activity, high

selectivity, and a reduction in partial agonistic activity.

Figure 2. Compound design of novel azole-based MR antagonists.

5430

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

Scheme 1. Synthesis of compound 3 and 4. Reagents and conditions: (a) hydrazine or hydroxylamine, 2-propanol, 77% (8a) and 95% (8b); (b) NBS, DMF, 94% (9a) and 98%

(9b); (c) 4-ﬂuorophenylboronic acid, Pd(P^tBu₃)₂, K₃PO₄, 1,4-dioxane, 54% (3) and 74% (4).

were investigated to assess the impact of hydrophobic or hydrogen

bonding interactions with Arg817 and Gln776, respectively. We

planned to select a lead scaffold by evaluation of the in vitro

potency, selectivity, and partial agonistic activity of compounds

3–6 bearing a methyl group at position Z and then optimize the

methyl group.

Suzuki coupling reaction with 15a afforded the ﬂuorinated

compound 41. Methyl ketone 39 was converted to silyl enol ether

by reaction with silyl triﬂate. The resulting silyl enol ether reacted

with lead acetate(IV) in the presence of potassium carbonate to

give the 1-acetoxymethyl ketone.¹²Diﬂuorination of the 1-acet-

oxymethyl ketone and the subsequent Suzuki coupling reaction

with 15a yielded diﬂuoride 43.

2. Chemistry

3. Results and discussion

The synthesis of 5-methyl pyrazole 3 and 5-methyl isoxazole 4

is described in Scheme 1. Acid-mediated condensation of diketone

7 with hydrazine or hydroxylamine afforded pyrazole 8a or

isoxazole 8b. Bromination of 8a,b by N-bromosuccinimide (NBS)

followed by Suzuki coupling reaction with 4-ﬂuorophenylboronic

acid gave compounds 3 and 4, respectively.

Scheme 2 depicts the synthesis of compounds 5, 6a–c, and 16a–

j. Pyrazole ring formation by acid-mediated condensation of

ketones 11 and 12 with hydrazines afforded the 1-unsubstituted

pyrazole 13b and the 1-substituted pyrazoles 13a, 13c–o. Bromin-

ation followed by Suzuki coupling reaction with boronic acids

15a,b gave the compounds 5, 6a–c and 16a–f, 16h–j. Hydrogenol-

ysis of the benzyl ether of 16f yielded the alcohol 16g.

The synthesized compounds were evaluated for their inhibitory

activity against the binding of [³H]-aldosterone to MR. The selectiv-

ity over the other steroid hormone receptors was determined by

radioligand binding assays (AR: [³H]-testosterone; PR: [³H]-proges-

terone; and GR: [³H]-dexamethasone). MR antagonistic activity and

partial agonistic activity were evaluated in reporter gene assays

using COS-1 cells by measurement of the inhibitory activity for

MR activation induced by aldosterone or the relative transcription

activity to the activity of aldosterone, respectively. The results are

presented in Tables 1–3 as IC₅₀values for binding activity and

antagonistic activity or relative percentage values (%) for agonistic

activity.

Compounds 24a–c were synthesized as shown in Scheme 3.

Bromination of 13b by NBS gave the 1-unsubstituted pyrazole

22. Subsequent Mitsunobu reaction of 22 with alcohols gave the

1-substituted pyrazoles 23a–c as a mixture of regioisomers. The

structure of these regioisomers was conﬁrmed by NMR analysis,

and the undesired regioisomer was removed by careful silica gel

column chromatography. Suzuki coupling reaction of 23a–c with

boronic acid 15a afforded the pyrazoles 24a–c.

Compounds 27a,b and 30 were synthesized according to

Scheme 4. The hydroxyl group of compound 13j was converted

to the azido group, and reduction of the azido group by triphenyl

phosphine followed by acetylation and bromination afforded the

amide 26a. Sulfonamide 26b was synthesized in a similar manner

to 26a. Suzuki coupling reaction of 26a,b with 15a gave the pyra-

zoles 27a,b. Bromination of 13j by NBS, followed by Mitsunobu

reaction with acetone cyanohydrin gave the cyanoethyl compound

29. Suzuki coupling reaction of 29 with 15a afforded compound 30.

Compounds 36a, 37a,b were synthesized as shown in Scheme 5.

Epoxides 31a–c were converted to the substituted hydrazines, and

subsequent acid-mediated pyrazole ring formation afforded com-

pounds 32a–c. Compounds 32a–c were brominated by NBS, and

then the hydroxyl groups were oxidized to give the ketones 34a–

c. Diﬂuorination of the carbonyl group by using diethylamino sul-

fur triﬂuoride (DAST) followed by the Suzuki coupling reaction

afforded the diﬂuorides 36a–c. Hydrogenolysis of the benzyl ether

of 36b,c yielded the corresponding alcohols 37a,b.

First, with the aim of identifying a new lead scaffold, a series of

azole derivatives bearing a methyl group at position Z were inves-

tigated (Table 1). Of the three initial cores prepared (3, 4, 5), the

1-methyl pyrazole 5 demonstrated the greatest binding activity

(IC₅₀= 290 nM) and was selected for further modiﬁcation. The dif-

ferent binding activities among these cores could be explained by

the preference for hydrophobic functionality at position X, which

is surrounded by lipophilic amino acid residues. Introduction of

additional methyl group into position X of pyrazole 5 increased

the binding activity (6a, IC₅₀= 110 nM). Importantly, dimethyl

compound 6a showed good antagonistic activity (IC₅₀= 160 nM)

and high steroid receptor selectivity (particularly over AR and PR)

as well as greatly reduced partial agonistic activity (3% activation

at 10 lM). On the basis of these results, a 1,3-dimethylpyrazole ring

was selected as a lead scaffold. Subsequently, a ﬂuorine atom and a

methyl group were introduced at the 2-position in the 4-ﬂuoroben-

zene ring and the 5-position in the benzoxazin-3-one ring, respec-

tively (6b, 6c). Although these modiﬁcations enhanced the binding

afﬁnities in our previous research, there was no signiﬁcant increase

in the potency of the 1,3-dimethylpyrazole compounds. We specu-

lated that the 1-methyl group on the pyrazole ring stabilized the

preferred twisted conformation between the 4-ﬂuorobenzene ring

and central ring; consequently, an additional effect caused by the

ﬂuorine atoms was not observed, and the unﬁlled space around

the 5-position of the benzoxazin-3-one ring was slightly smaller

than those of compounds 1 and 2. Compound 6a was the best

among these compounds and was selected as a lead compound

for further optimization. It should be noted that all compounds

listed in Table 1 showed signiﬁcantly weaker afﬁnities for AR and

PR and reduced partial agonistic activities as designed, which

encouraged us to advance our synthetic study to the next process.

Compounds 41, 43 were synthesized as illustrated in Scheme 6.

Compound 14h was oxidized by using a pyridine sulfur trioxide

complex to give aldehyde 38. Reaction of 38 with methyl magne-

sium bromide followed by Dess–Martin oxidation gave the methyl

ketone 39. Diﬂuorination of the carbonyl group of 39 followed by

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

5431

Scheme 2. Synthesis of 1-substituted pyrazole derivatives 5, 6a–c, and 16a–i. Reagents and conditions: (a) DMFDMA, 32%; (b) NaH, EtOAc, THF, 74%; (c) methylhydrazine,

EtOH, 81%; (d) R-NHNH₂, TFA, TEA, 2-PrOH, 19–97%; (e) H₂, Pd/C, MeOH; (f) NBS, CH₃CN, or DMF, 30–99%; (g) boronic acid 15a or 15b, PdCl₂(dppf)ꢀCH₂Cl₂, Cs₂CO₃, THF/H₂O,

5–52%; (h) H₂, Pd/C, EtOH, 88%; (i) tert-butyl carbazate, EtOH, 55–90%; (j) (1) NaBH₃CN, AcOH, (2) TFA; (k) (1) hydrazine hydrate, NaOH, (2) HCl, 83%.

Scheme 3. Synthesis of 24a–c. Reagents and conditions: (a) NBS, DMF, 98%; (b) R-OH, PPh₃, diisopropyl azodicarboxylate, THF, 11–30%; (c) boronic acid 15a,

PdCl₂(dppf)ꢀCH₂Cl₂, Cs₂CO₃, THF/H₂O, 5–43%.

In the second step of our optimization studies, the 1-methyl

hand, the phenyl compound 16d had greatly reduced afﬁnity, most

group of compound 6a was changed to various nonpolar or polar

likely because of the steric repulsion against the wall of MR. These

side chains to increase the binding activity. First, larger lipophilic

results suggested that at least more than one methylene linker was

needed for incorporation of bulky substituents. Next, ﬂuorine

atoms were introduced into the n-butyl group of compound 24a,

the most potent compound at this point. These ﬂuorinated com-

side chains were investigated in anticipation of the hydrophobic

contact (Table 2). Although the ethyl compound 16a showed

decreased binding activity, the n-propyl and n-butyl compounds

showed increased activity (16b, 24a). Particularly, the n-butyl

pounds 24c, 41, and 36a showed further increased activities.

compound 24a had 5-fold more potent activity (IC₅₀= 22 nM) than

Among them, compounds 24c and 36a showed remarkably potent

the methyl compound 6a. Furthermore, isobutyl compound 24b

single-digit nanomolar binding activity (24c: IC₅₀= 8.4 nM, 36a:

possessing a branched-type side chain also showed potent binding

IC₅₀= 5.8 nM). Thus, replacement of the 1-methyl group with a

activity. These results indicated that there is some space to allow

bulkier lipophilic substituent successfully increased the binding

the introduction of various sizes of substituents at the 1-postion

activity and, importantly, maintained the partial agonistic activi-

of the pyrazole ring. To occupy the space more tightly, benzene

ties at the lower levels. Meanwhile, these compounds also showed

ring and ﬂuorine atoms were introduced onto the side chains.

increased binding activity for the other steroid receptors. In addi-

Notably, a bulky benzyl group was tolerated, and the compound

tion, they tended to have high metabolic clearance in rat micro-

16c showed potent binding activity (IC₅₀= 100 nM). On the other

somes (>100 lL/mg/min). We speculated that this metabolic

5432

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

Scheme 4. Synthesis of 27a,b and 30. Reagents and conditions: (a) (1) TsCl, pyridine; (2) NaN₃, DMF, 83% in 2 steps; (b) (1) PPh₃, THF, H₂O; (2) Ac₂O, pyridine; (3) NBS, DMF,

94% in three steps; (c) (1) NBS, DMF; (2) PPh₃, THF, H₂O; (3) MsCl, pyridine, 82% in 3 steps; (d) boronic acid 15a, PdCl₂(dppf)ꢀCH₂Cl₂, Cs₂CO₃, THF/H₂O, 44% (27a) and 33%

(27b); (e) NBS, DMF, 69%; (f) acetone cyanohydrin, ADDP, PBu₃, toluene, 76%; (g) boronic acid 15a, PdCl₂(dppf)ꢀCH₂Cl₂, Cs₂CO₃, THF/H₂O, 42%.

Scheme 5. Synthesis of 36a, 37a,b, Reagents and conditions: (a) hydrazine hydrate, (b) 12a, HCl, MeOH, 47–66% in 2 steps; (c) NBS, DMF, 4% in three steps (31a), 98% (33b)

and 98% (33c); (d) Dess–Martin periodinane, toluene, 93–99%; (e) DAST, toluene, 39–71%; (f) boronic acid 15a, PdCl₂(dppf)ꢀCH₂Cl₂, Cs₂CO₃, THF/H₂O, 40–61%; (g) H₂, Pd/C,

88% (37a), and 70% (37b).

Scheme 6. Synthesis of compound 41 and 43. Reagents and conditions: (a) SO₃ꢀPy, DMSO, TEA, 70%; (b) (1) MeMgBr, THF; (2) Dess–Martin periodinane, toluene, 77% in two

steps; (c) DAST, toluene, 28%; (d) boronic acid 15a, PdCl₂(dppf)ꢀCH₂Cl₂, Cs₂CO₃, THF/H₂O, 30%; (e) (1) TBSOTf, TEA, toluene; (2) Pb(OAc)₄, K₂CO_3,toluene; (3) TBAF, THF; (4)

DAST, toluene, 10% in four steps; (f) boronic acid 15a, PdCl₂(dppf)ꢀCH₂Cl₂, Cs₂CO₃, THF/H₂O, 34%.

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

5433

Table 1

Exploration of a central ring as a new template^a

R'

O

X

O

N

H

Y

Z

Me

R

F

Compound

No.

Azole core

R

R⁰

MR binding

IC₅₀(nM)

Selectivity

MR antagonistic activity

IC₅₀(nM)

AR binding

IC₅₀(nM)

PR binding

IC₅₀(nM)

GR binding

IC₅₀(nM)

(agonistic activity)^b

N

NH

3

4

5

H

3700

710

>10,000

1600

>10,000

—

N

O

>10,000

1400 (3%)

170 (1%)

N

290

Me

6a

6b

6c

H

F

H

110

190

150

>10,000

9800

3200

2300

2500

160 (3%)

600 (2%)

350 (8%)

N

Me

H

9300

N

Me

H

Me

>10,000

N

a

IC₅₀values are shown as the means of duplicate experiments. IC₅₀values are calculated from the concentration–response curves generated by GraphPad Prism.

Agonistic activity at a concentration of 10 lM.

b

Table 2

Exploration of lipophilic side chains^a

O

Me

O

N

H

N

R

F

Compound

No.

R

MR binding

IC₅₀(nM)

Selectivity

MR antagonistic activity

IC₅₀(nM)

AR binding

IC₅₀(nM)

PR binding

IC₅₀(nM)

GR binding

IC₅₀(nM)

(agonistic activity)^b

6a

Me

Et

n-Pr

n-Bu

Isobutyl

Bn

Ph

CH₂CH₂CH₂CF₃

CH₂CH₂CF₂Me

CH₂CF₂CH₂Me

110

510

81

22

60

100

>10,000

8.4

10

5.8

>10,000

3600

3700

5600

9800

>10,000

5700

4000

4300

2000

—

6800

4000

3200

5600

1600

1400

390

630

—

1100

370

540

160 (3%)

950 (ꢁ1%)

180 (ꢁ1%)

45 (0%)

49 (ꢁ7%)

64 (ꢁ9%)

—

33 (6%)

18 (4%)

27 (11%)

16a

16b

24a

24b

16c

16d

24c

41

—

>10,000

9200

>10,000

36a

a

IC₅₀values are shown as the means of duplicate experiments. IC₅₀values are calculated from the concentration–response curves generated by GraphPad Prism.

Agonistic activity at a concentration of 10 lM.

b

instability was probably due to their high lipophilicity (logD >3),

and thus we focused on less lipophilic derivatives in the second

round of the side chain investigation.^13,14

To increase the binding activity while avoiding high lipophilic-

ity, the 1-methyl group was changed to less lipophilic side chains

substituted with polar groups targeting the two residues Arg817

and Gln776 (Table 3). A range of functional groups including

amide, sulfonamide, cyano, hydroxy, and pyridine ring were inves-

tigated. All of these compounds displayed greatly reduced logD

values, and indeed, many of them tended to have improved meta-

bolic clearance in rat microsomes relative to those of the lipophilic

compounds described above. Acetamide 27a showed signiﬁcantly

5434

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

Table 3

Exploration of polar side chains targeting for Arg817 and/or Gln776^a

O

Me

O

N

H

N

R

F

Compound

No.

R

MR binding

IC₅₀(nM)

Selectivity

MR antagonistic activity

IC₅₀(nM)

LogD (pH7.4)

AR binding

IC₅₀(nM)

PR binding

IC₅₀(nM)

GR binding

IC₅₀(nM)

(agonistic activity)^b

6a

27a

27b

30

16e

16g

16h

16i

16j

37a

37b

43

Spironolactone

Eplerenone

Me

110

>10,000

250

530

440

75

2800

36

520

51

>10,000

—

9800

—

>10,000

5700

>10,000

650

3200

—

>10,000

4600

>10,000

5400

>10,000

1700

>10,000

2400

1300

2500

160 (3%)

—

2.51

—

1.7

CH₂CH₂NHCOMe

CH₂CH₂NHSO₂Me

CH₂CH₂CN

CH₂CH₂CH₂OH

CH₂CH₂CH₂CH₂OH

CH₂-(2-Py)

CH₂-(3-Py)

CH_2-(4-Py)

CH₂CF₂CH₂OH

CH₂CF₂CH₂CH₂OH

CH₂CH₂CF₂CH₂OH

>10,000

120

2000 (5%)

490 (ꢁ1%)

680 (2%)

130 (8%)

990 (ꢁ14%)

36 (ꢁ4%)

430 (ꢁ8%)

71 (1%)

1.89

1.99

2.16

2.45

2.22

2.2

2.05

2.11

2.34

—

38

95

49

2600

540 (1%)

180 (3%)

60 (4%)

1400

>10,000

1300 (5%)

—

a

IC₅₀values are shown as the means of duplicate experiments. IC₅₀values are calculated from the concentration–response curves generated by GraphPad Prism.

Agonistic activity at a concentration of 10 lM.

b

reduced binding activity, and the methanesulfonamide 27b,

2-cyanoethyl 30, and 3-hydroxypropyl 16e showed moderately

decreased activities. On the other hand, the hydroxybutyl com-

pound 16g showed potent binding activity (IC₅₀= 75 nM). Subse-

quently, 2-, 3-, and 4-pyridylmethyl compounds were explored

(16h–j). The position of the nitrogen atom in the pyridine ring

was critical for high afﬁnity, and only the 3-pyridylmethyl com-

pound 16i showed highly potent binding activity among the three

tested molecules (IC₅₀= 36 nM). Noticeably, compound 16i was 3-

fold more potent than the benzyl compound 16c, which indicated

that the pyridine nitrogen atom formed a strong hydrogen bond

with MR. Thus, it was revealed that replacement of the 1-methyl

group with polar side chains could be utilized to increase the

binding activity.

Next, we were interested in the incorporation of ﬂuorine atoms

into the polar side chains. This combination was expected to pro-

vide potent activity with low lipophilicity. 3-Hydroxypropyl and

4-hydroxybutyl groups were selected for the polar side chains,

and the ﬂuorine atoms were introduced at their methylene linkers

(Table 3, 37a,b, 43). These compounds showed increased (37a,b) or

retained (43) binding activities with little increase of lipophilicity,

and compounds 37a and 37b showed highly potent binding activ-

ities (37a: IC₅₀= 51 nM; 37b: IC₅₀= 38 nM). It is notable that com-

pound 37a was 8-fold more potent than the nonﬂuorinated

compound 16e. Consequently, four compounds (16g, 16i, 37a,b)

were identiﬁed as potent and less lipophilic derivatives. They all

showed high selectivity over the other steroid receptors (impor-

tantly, with little afﬁnity for AR and PR) and signiﬁcantly reduced

partial agonistic activities. Although the antagonistic activities of

compounds 16g and 37b were moderate, compounds 16i and

37a showed highly potent antagonistic activities comparable to

that of spironolactone (16i: IC₅₀= 36 nM; 37a: IC₅₀= 71 nM).

Unfortunately, the 3-pyridylmethyl compound 16i showed rapid

metabolic clearance in rat microsomes (>200 l )

L min^ꢁ1mg^ꢁ1

despite its reduced lipophilicity, probably due to the metabolically

labile diarylmethane moiety and/or N-oxidation on the pyridine

ring. On the other hand, the 2,2-diﬂuoro-3-hydroxyl compound

37a showed acceptable metabolic clearance in rat microsomes

(42 l

L min^ꢁ1mg^ꢁ1) and had good PK proﬁles (bioavailability in

rats = 21%), as shown in Table 4. On the basis of these results,

compound 37a was progressed to in vivo efﬁcacy studies in rats.

Figure 3 shows the X-ray crystal structure of the MR/compound

37a complex, which shows that compound 37a binds to the steroid

binding domain in MR in a fashion similar to that of the previously

reported benzoxazin-3-one derivatives. The amide moiety in the

benzoxazin-3-one ring forms hydrogen bonds with Asn770 and

Thr945, and the 4-ﬂuorobenzene ring occupies the hydrophobic

pocket. The side chain at the 1-position of the pyrazole ring, which

was the 2,2-diﬂuoropropyl-3-hydroxy group, orients towards

Arg817 and Gln776, and the hydroxyl group directly forms a

hydrogen bond to Gln776. The two ﬂuorine atoms ﬁll the space

with no speciﬁc hydrogen bonding interactions, which suggests

that the major effect of these ﬂuorine atoms was the hydrophobic

interaction. It was conﬁrmed that compound 37a has no water-

mediated hydrogen bonding network between the MR and the

ligand side chain. This ﬁnding is consistent with our hypothesis

regarding the attenuation of partial agonistic activity in the

benzoxazin-3-one derivatives.

The effects of compound 37a on systolic blood pressure (SBP)

and seminal vesicle weight were evaluated in established

Table 4

Pharmacokinetic proﬁles of compound 37a in Sprague–Dawley Rats^a

CL (mL/h/kg)

990 165

Vd_ss(mL/kg)

1356 59

AUC_{0–24 h,p.o.}(ng h/mL)

662 372

MRT_p.o.(h)

3.77 0.66

B.A. (%)

21.4 12.6

a

Rats were administered the drug intravenously at 1 mg/kg and orally at 3 mg/kg (n = 3).

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

5435

Figure 3. (A) X-ray crystal structure of compound 37a bound to MR. (B) Overlay of compound 37a (green) and a dihydrofuran-2-one derivative (orange) in MR. Yellow or

orange dotted lines mean hydrogen bonds.

Figure 4. Anti-hypertensive (A), and anti-androgenic (B) effects of compound 37a in DOCA-salt rats. (A) DOCA-salt hypertensive rats were treated with vehicle, compound

37a, or spironolactone at a dose of 100 mg/kg for 18 days. SBP and HR were measured by tail-cuff method approximately 24 h after the 7th and 14th administration. Data

were shown as mean SEM (n = 8); ^⁄p 6 0.05 versus vehicle by repeated-measures ANOVA, which indicated signiﬁcant group ꢂ time interaction but not the main effects of

group and time. N.S. means not signiﬁcant. (B) Seminal vesicle weight was measured by autopsy after the measurement of body weight approximately 24 h after the last

administration. Data were shown as mean SEM (n = 7–8); ^⁄p 6 0.05 versus vehicle by Aspin–Welch test.

deoxycorticosterone acetate (DOCA)-salt hypertensive rats, and

the results are shown in Figures 4A and B. Compound 37a

(100 mg/kg, q.d.) and spironolactone (100 mg/kg, q.d.) were orally

administered for 18 days. SBP and heart rate (HR) were measured

by the tail-cuff method approximately 24 h after the 7th and

14th administration, and seminal vesicle weight was measured

by autopsy after the body weight measurement approximately

24 h after the last administration. Both compound 37a and spiro-

nolactone signiﬁcantly lowered SBP, and their antihypertensive

effects were similar. Meanwhile, these compounds did not affect

the HR (data not shown). Furthermore, compound 37a showed

no signiﬁcant reduction of seminal vesicle weight, a sign of an

antiandrogenic effect, probably because of its little binding activity

for AR, whereas spironolactone signiﬁcantly reduced it. Thus, com-

pound 37a exhibited a potent blood pressure-lowering effect with-

out an antiandrogenic effect. These results indicated that

compound 37a was a new, potent, and selective MR antagonist

that appeared to mitigate sexual hormone-related side effects,

which have been reported as issues associated with spironolactone

treatment in the clinic.

the position adjacent to the 4-ﬂuorobenzene ring. Screening of

azole rings in the central ring moiety identiﬁed the 1,3-dimethyl

pyrazole 6a as a lead compound with attenuated partial agonistic

activity. Replacement of the 1-methyl group of 6a with larger lipo-

philic side chains or polar side chains targeting Arg817 and Gln776

increased the binding activity while maintaining the agonistic

response at the lower level. Reduction of the lipophilicity was

effective for improvement of metabolic clearance in rat micro-

somes. Consequently, the combination of ﬂuorine atoms with polar

groups achieved potent binding activity with favorable in vitro

metabolic proﬁles. Thus, we discovered 2,2-diﬂuoro-3-hydroxy-

propyl derivative 37a, which showed highly potent in vitro activ-

ity, high steroidal receptor selectivity, and attenuated partial

agonistic activity as well as good PK proﬁles. Compound 37a

exhibited a signiﬁcant blood pressure lowering effect in DOCA-salt

hypertensive rats with no signs of an antiandrogenic effect. On the

basis of these results, compound 37a was progressed for further

pharmacological evaluations.

5. Experimental procedure

4. Conclusion

Proton nuclear magnetic resonance (¹H NMR) spectra were

recorded on Bruker Ultra Shield-300 (300 MHz) or Varian INOVA-

400 (400 MHz) instruments. Chemical shifts are given in parts

per million (ppm) with tetramethylsilane as an internal standard.

Peak multiplicities are expressed as follows: s = singlet, d = dou-

blet, t = triplet, q = quartet, m = multiplet, dd = doublets of doublet,

We have discovered 6-[5-(4-ﬂuorophenyl)-3-methyl-pyrazol-

4-yl]-benzoxazin-3-one derivatives as novel selective nonsteroidal

MR antagonists. To reduce the partial agonistic activity, we

designed a series of azole derivatives possessing side chains at

5436

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

br s = broad singlet. Coupling constants (J values) are given in hertz

(Hz). Elemental analyses were performed by Takeda Analytical

Laboratories Ltd. Chemical intermediates were characterized by

¹H NMR. The purities of all compounds tested in biological systems

were assessed as being >95% using analytical high-performance

liquid chromatography (HPLC). The HPLC analyses were performed

using a Shimadzu UFLC instrument, equipped with a L-column 2

(3H, s), 4.70 (2H, s), 7.15–7.20 (1H, m), 7.55–7.60 (2H, m), 10.95

(1H, br s).

5.5. 6-[4-(4-Fluorophenyl)-5-methyl-1H-pyrazol-3-yl]-2H-1,4-

benzoxazin-3(4H)-one (3)

Under nitrogen atmosphere,

a mixture of 9a (158 mg,

ODS (3.0 ꢂ 50 mm, 2

l

m) column, eluting with a gradient of 5–

0.513 mmol), 4-ﬂuorophenylboronic acid (215 mg, 1.54 mmol),

bis(tri-t-butylphosphine)palladium (0) (13.1 mg, 0.025 mmol)

and tripotassium phosphate (1.05 g, 4.87 mmol) in THF/water

(12 mL/2.4 mL) was stirred at 90 °C for 12 h, diluted with water

and extracted with EtOAc. The organic layer was washed with

brine, dried over anhydrous MgSO₄, and concentrated under

reduced pressure. The residue was suspended in hexane and aceto-

nitrile, and the solid was collected by ﬁltration to give 3 (89 mg,

54%). ¹H NMR (400 MHz, CDCl₃) d 2.30 (3H, s), 4.69 (2H, s), 6.72

(1H, dd, J = 8.6, 2.0 Hz), 6.78 (1H, d, J = 2.0 Hz), 7.02–7.10 (2H, m),

7.16–7.22 (2H, m), 7.26 (1H, s), 7.52 (1H, s), 10.56 (1H, br s). Anal.

Calcd for C₁₈H₁₄N₃O₂F: C, 66.87; H, 4.36; N, 13.00. Found: C, 66.57;

H, 4.52; N, 13.04.

90% solvent B in solvent A (solvent A was 0.1% TFA in water, and

solvent B was 0.1% TFA in acetonitrile), at a ﬂow rate of 1.2 mL/

min, with UV detection at 220 nm. Reaction progress was deter-

mined by thin layer chromatography (TLC) analysis on Merck Kie-

selgel 60 F254 plates or Fuji Silysia NH plates. Chromatographic

puriﬁcation was performed on silica gel columns [(Merck Kieselgel

60, 70–230 mesh size or 230–400 mesh size, Merck) or (Chromat-

orex NH-DM 1020, 100–200 mesh size)] or on Purif-Pack (SI or NH,

particle size: 60 lm, Fuji Silysia Chemical, Ltd). Reagents and sol-

vents were obtained from commercial sources and used without

further puriﬁcation. Abbreviations of solvents are used as follows:

CDCl₃, deuterated chloroform; DMSO-d₆, dimethyl sulfoxide-d₆;

EtOAc, ethyl acetate; DMF, N,N-dimethylformamide; MeOH, meth-

anol; THF, tetrahydrofuran; EtOH, ethanol; IPE, diisopropyl ether.

5.6. 6-[4-(4-Fluorophenyl)-5-methyl-1,2-oxazol-3-yl]-2H-1,4-

benzoxazin-3(4H)-one (4)

5.1. 6-(5-Methyl-1,2-oxazol-3-yl)-2H-1,4-benzoxazin-3(4H)-one

(8b)

The compound 4 was prepared in a manner similar to that

described for 3. Yield (74%). ¹H NMR (400 MHz, DMSO-d₆) d 2.15

(3H, s), 4.62 (2H, s), 6.95–6.99 (2H, m), 7.07 (1H, s), 7.30–7.35 (2H,

m), 7.37–7.42 (2H, m), 10.84 (1H, s). Anal. Calcd for C₁₈H₁₃N₂O₃F:

C, 66.66; H, 4.04; N, 8.64. Found: C, 66.40; H, 4.16; N, 8.55.

A

mixture of hydroxylamine hydrochloride (188 mg,

2.70 mmol) and triethylamine (369 L, 2.65 mmol) in 2-propanol

(15 mL) was stirred at room temperature for 15 min. To the reac-

tion mixture was added 2,2,2-triﬂuoroacetic acid (423 L,

l

5.53 mmol) and the mixture was stirred at room temperature for

15 min. To the resulting mixture was added 1-(3-oxo-3,

5.7. 3-(Dimethylamino)-1-(4-ﬂuorophenyl)prop-2-en-1-one

(11)

4-dihydro-2H-1,4-benzoxazin-6-yl)butane-1,3-dione

(600 mg,

2.57 mmol) at room temperature. The reaction mixture was stirred

at 60 °C for 12 h, and concentrated under reduced pressure. To the

residue was added water, and then 1 N NaOH was added to neu-

tralize the solution. The precipitate was collected by ﬁltration

and washed with ether to give 8b (560 mg, 95%). ¹H NMR

(400 MHz, CDCl₃) d 2.25 (3H, s), 4.64 (2H, s), 6.72 (1H, s),

7.05–7.10 (1H, m), 7.30–7.45 (2H, m), 10.90 (1H, br s).

A mixture of 1-(4-ﬂuorophenyl)ethanone (2.0 g, 14.5 mmol)

and 1,1-dimethoxy-N,N-dimethylmethanamine (4 mL) was

reﬂuxed for 2 h, and concentrated under reduced pressure. The

residue was suspended in hexane, and the solid was collected by

ﬁltration to give 11 (0.89 g, 32%). ¹H NMR (300 MHz, DMSO-d₆) d

2.92 (3H, s), 3.14 (3H, s), 5.82 (1H, d, J = 12.2 Hz), 7.24 (2H, m),

7.71 (1H, d, J = 12.2 Hz), 7.96 (2H, dd, J = 8.9, 5.7 Hz).

5.2. 6-(5-Methyl-1H-pyrazol-3-yl)-2H-1,4-benzoxazin-3(4H)-one

5.8. 1-(4-Fluorophenyl)butane-1,3-dione (12a)

(8a)

To a suspension of 60% sodium hydride (43.4 g, 1.81 mol) in tet-

rahydrofuran (1500 mL) was added 4-ﬂuoroacetophenone (50.0 g,

0.361 mol) at room temperature, and the mixture was stirred at

room temperature for 30 min. To the mixture was added EtOAc

(127.6 g, 1.45 mmol) at room temperature. The mixture was stirred

at 40 °C for 3 h, and then poured into 1 N HCl solution. The organic

solvent was removed under reduced pressure. The residual mix-

ture was extracted with EtOAc, washed with 1 N HCl solution

and saturated brine, dried over anhydrous MgSO₄, and concen-

trated under reduced pressure. The residue was crystallized from

hexane to give 12a (48.3 g, 74%). ¹H NMR (300 MHz, DMSO-d₆) d

2.19 (3H, s), 6.13 (1H, s), 7.01–7.18 (2H, m), 7.82–7.96 (2H, m),

16.15 (1H, br s).

The compound 8a was prepared in a manner similar to that

described for 8b. Yield (77%). ¹H NMR (400 MHz, CDCl₃) d 2.40

(3H, s), 4.72 (2H, s), 6.38 (1H, s), 7.00–7.05 (1H, m), 7.30–7.35

(1H, m), 7.67 (1H, s), 10.82 (1H, br s).

5.3. 6-(4-Bromo-5-methyl-1H-pyrazol-3-yl)-2H-1,4-benzoxazin-

3(4H)-one (9a)

To a stirred solution of 8a (390 mg, 1.66 mmol) in DMF (10 mL)

was added NBS (295 mg, 1.66 mmol) at room temperature. The

mixture was stirred at room temperature for 2 h, and then water

was added. The mixture was cooled to 0 °C, and the precipitate

was collected by ﬁltration and washed with water and ether to give

9a (480 mg, 94%). ¹H NMR (400 MHz, CDCl₃) d 2.39 (3H, s), 4.72

(2H, s), 7.05–7.11 (1H, m), 7.42 (1H, s), 7.77–7.81 (1H, m), 10.20

(1H, br s).

5.9. 5-(4-Fluorophenyl)-1-methyl-1H-pyrazole (13a)

A mixture of 11 (800 mg, 4.14 mmol) and methylhydrazine

(257 lL, 4.88 mmol) in ethanol was reﬂuxed for 4 h. The solvent

5.4. 6-(4-Bromo-5-methyl-1,2-oxazol-3-yl)-2H-1,4-benzoxazin-

3(4H)-one (9b)

was removed, and the residue was puriﬁed by column chromatog-

raphy on NH-silica gel with hexane/ethyl acetate as an eluent to

give 13a (592 mg, 81%). ¹H NMR (300 MHz, DMSO-d₆) d 3.83 (3H,

s), 6.39 (1H, d, J = 1.9 Hz), 7.31–7.40 (2H, m), 7.46 (1H, d,

J = 1.5 Hz), 7.58 (2H, dd, J = 8.9, 5.5 Hz).

The compound 9b was prepared in a manner similar to that

described for 9a. Yield (98%). ¹H NMR (400 MHz, CDCl₃) d 2.30

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

5437

5.10. 5-(4-Fluorophenyl)-1,3-dimethyl-1H-pyrazole (13c)

5.19. 2-{[5-(4-Fluorophenyl)-3-methyl-1H-pyrazol-1-yl]methyl}

pyridine (13m)

A mixture of 12a (12.6 g, 69.9 mmol), methylhydrazine (6.4 g,

139 mmol), triﬂuoroacetic acid (10.7 mL, 144 mmol), and triethyl-

amine (19.4 mL, 139 mmol) in 2-propanol (350 mL) was stirred at

80 °C for 1 h, and then concentrated under reduced pressure. The

residue was diluted with ethyl acetate, and washed with saturated

NaHCO₃solution and brine. The organic layer was dried over anhy-

drous MgSO₄and concentrated under reduced pressure. The resi-

due was puriﬁed by column chromatography on silica gel with

hexane/ethyl acetate as an eluent to give 13c (10.0 g, 75%). ¹H

NMR (DMSO-d₆) d 2.16 (3H, s), 3.74 (3H, s), 6.16 (1H, s), 7.27–

7.40 (2H, m), 7.54 (2H, dd, J = 8.7, 5.5 Hz).

Yield (30%). ¹H NMR (300 MHz, CDCl₃) d 2.34 (3H, s), 5.49 (2H,

s), 6.19 (1H, s), 6.99–7.10 (3H, m), 7.27–7.37 (3H, m), 7.73–7.82

(1H, m), 8.61 (1H, d, J = 4.9 Hz).

5.20. 3-{[5-(4-Fluorophenyl)-3-methyl-1H-pyrazol-1-yl]methyl}

pyridine (13n)

Yield (19%). ¹H NMR (300 MHz, CDCl₃) d 2.32 (3H, s), 5.34 (2H, s),

6.16 (1H, s), 7.07–7.17 (2H, m), 7.22–7.30 (2H, m), 7.48–7.56 (1H,

m), 7.72 (1H, d, J = 8.1 Hz), 8.28–8.40 (1H, m), 8.63 (1H, d, J = 4.0 Hz).

The compound 13b, 13e,f, 13h–o were prepared in a manner

similar to that described for 13c.

5.21. 4-{[5-(4-Fluorophenyl)-3-methyl-1H-pyrazol-1-yl]methyl}

pyridine (13o)

5.11. 5-(4-Fluorophenyl)-3-methyl-1H-pyrazole (13b)

Yield (33%). ¹H NMR (300 MHz, DMSO-d₆) d 2.21 (3H, s), 5.31

(2H, s), 6.26 (1H, s), 6.92–6.94 (2H, m), 7.24–7.43 (4H, m),

8.46–8.48 (2H, m).

Yield (72%). ¹H NMR (300 MHz, CDCl₃) d 2.25 (3H, s), 7.01–7.17

(2H, m), 7.65–7.81 (2H, m), 8.97 (1H, br s).

5.12. 1-Ethyl-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazole (13e)

5.22. 5-(2,4-Diﬂuorophenyl)-1,3-dimethyl-1H-pyrazole (13d)

Yield (90%). ¹H NMR (300 MHz, DMSO-d₆) d 1.26 (3H, t,

J = 7.0 Hz), 2.17 (3H, s), 3.99 (2H, q, J = 7.0 Hz), 6.11 (1H, s), 7.27–

7.37 (2H, m), 7.43–7.54 (2H, m).

To

a

solution of 1-(2,4-diﬂuorophenyl)ethanone (2.0 g,

12.8 mmol) in THF (100 mL) was added dropwise lithium bis(tri-

methylsilyl)amide in THF (1.1 M, 12.8 mL, 14.1 mmol) at ꢁ25 °C.

The mixture was stirred for 1 h at ꢁ25 °C, and then was cooled

to ꢁ78 °C. To the mixture was added acetyl chloride (1.19 mL,

16.8 mmol). The mixture was stirred for 3 h, treated with 1 N

HCl, and extracted with EtOAc. The organic layer was washed with

brine, dried over anhydrous MgSO₄and concentrated under

reduced pressure to give 12b. To the crude 12b was added 2-pro-

panol (100 mL), triﬂuoroacetic acid (1.99 mL, 26.8 mmol), methyl

hydrazine (1.35 mL, 25.6 mmol) and triethylamine (3.61 mL,

25.6 mmol), successively. The mixture was stirred at 80 °C for

30 min, concentrated under reduced pressure, treated with satu-

rated NaHCO₃solution, and extracted with EtOAc. The organic

layer was washed with water, dried over anhydrous MgSO₄and

concentrated under reduced pressure. The residue was puriﬁed

by column chromatography on silica gel with hexane/ethyl acetate

as an eluent to give 13d (0.63 g, 25% in 2 steps). ¹H NMR (300 MHz,

DMSO-d₆) d 2.18 (3H, s), 3.62 (3H, s), 6.17 (1H, s), 7.23 (1H, td,

J = 8.4, 2.5 Hz), 7.43–7.60 (2H, m).

5.13. 1-Allyl-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazole (13f)

Yield (95%). ¹H NMR (300 MHz, DMSO-d₆) d 2.18 (3H, s), 4.57–

4.69 (2H, m), 4.86 (1H, dd, J = 17.0, 1.5 Hz), 5.11 (1H, dd, J = 10.5,

1.5 Hz), 5.85–6.01 (1H, m), 6.19 (1H, s), 7.31 (2H, t, J = 9.0 Hz),

7.43–7.53 (2H, m).

5.14. 1-Benzyl-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazole (13h)

Yield (88%). ¹H NMR (300 MHz, CDCl₃) d 2.31 (3H, s), 5.22 (2H,

s), 6.09 (1H, s), 6.94–7.07 (4H, m), 7.16–7.33 (5H, m).

5.15. 5-(4-Fluorophenyl)-3-methyl-1-phenyl-1H-pyrazole (13i)

Yield (39%). ¹H NMR (300 MHz, CDCl₃) d 2.37 (3H, s), 6.27 (2H,

s), 6.91–7.00 (2H, m), 7.13–7.21 (2H, m), 7.21–7.34 (5H, m).

5.16. 2-[5-(4-Fluorophenyl)-3-methyl-1H-pyrazol-1-yl]ethanol

5.23. 5-(4-Fluorophenyl)-3-methyl-1-propyl-1H-pyrazole (13g)

(13j)

A suspension of 13f (2.0 g, 9.24 mmol) and 10 wt% Palladium on

carbon (300 mg) in MeOH (50 mL) was stirred at room tempera-

ture for 12 h under hydrogen atmosphere. The insoluble material

was removed by ﬁltration, and the ﬁltrate was concentrated under

reduced pressure to give 13g. The crude 13g was used for the next

reaction without further puriﬁcation.

Yield (97%). ¹H NMR (300 MHz, DMSO-d₆) d 2.18 (3H, s), 3.75

(2H, q, J = 5.5 Hz), 3.98 (2H, t, J = 5.5 Hz), 4.92 (1H, t, J = 5.0 Hz),

6.12 (1H, s), 7.25–7.40 (2H, m), 7.51–7.67 (2H, m).

5.17. 3-[5-(4-Fluorophenyl)-3-methyl-1H-pyrazol-1-yl]propan-

1-ol (13k)

5.24. 4-Bromo-5-(4-ﬂuorophenyl)-1,3-dimethyl-1H-pyrazole

(14b)

Yield (77%). ¹H NMR (DMSO-d₆) d 1.85 (2H, quin, J = 6.7 Hz),

2.17 (3H, s), 3.25–3.39 (2H, m), 4.03 (2H, t, J = 7.2 Hz), 4.50 (1H,

m), 6.11 (1H, s), 7.31 (2H, t, J = 8.7 Hz), 7.49 (2H, t, J = 2.8 Hz).

A mixture of 13c (5 g, 26.3 mmol) and NBS (4.68 g, 26.3 mmol)

in acetonitrile (40 mL) was stirred at room temperature for 10 min.

The mixture was diluted with EtOAc, and washed with saturated

NaHCO₃solution and brine. The organic layer was dried over anhy-

drous MgSO₄and concentrated under reduced pressure. The resi-

due was puriﬁed by column chromatography on NH-silica gel

with hexane/EtOAc as an eluent to give 14b (2.11 g, 30%). ¹H

NMR (DMSO-d₆) d 2.17 (3H, s), 3.69 (3H, s), 7.35–7.40 (2H, m),

7.54 (2H, dd, J = 8.7, 5.7 Hz).

5.18. 1-[4-(Benzyloxy)butyl]-5-(4-ﬂuorophenyl)-3-methyl-1H-

pyrazole (13l)

Yield (49%). ¹H NMR (300 MHz, CDCl₃) d 1.40–1.65 (2H, m),

1.81–1.93 (2H, m), 2.29 (3H, s), 3.37 (2H, t, J = 6.3 Hz), 4.02 (2H,

t, J = 7.2 Hz), 4.41 (2H, s), 6.00 (1H, s), 7.03–7.12 (2H, m),

7.23–7.35 (7H, m).

5438

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

The compound 14a, 14c–l were prepared in a manner similar to

5.35. 4-{[4-Bromo-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-1-

that described for 14b.

yl]methyl}pyridine (14l)

5.25. 4-Bromo-5-(4-ﬂuorophenyl)-1-methyl-1H-pyrazole (14a)

Yield (43%). ¹H NMR (300 MHz, CDCl₃) d 2.33 (3H, s), 5.31 (2H,

s), 7.08–7.32 (6H, m), 8.66 (2H, s).

Yield (39%). ¹H NMR (300 MHz, DMSO-d₆) d 3.76 (3H, s), 7.40

(2H, t, J = 8.9 Hz), 7.56 (2H, dd, J = 8.9, 5.5 Hz), 7.66 (1H, s).

5.36. 6-[5-(4-Fluorophenyl)-1-methyl-1H-pyrazol-4-yl]-2H-1,4-

benzoxazin-3(4H)-one (5)

5.26. 4-Bromo-5-(2,4-diﬂuorophenyl)-1,3-dimethyl-1H-

pyrazole (14c)

Under argon atmosphere, a mixture of 14a (500 mg, 1.96 mmol),

6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-1,4-benzoxazin-

3(4H)-one (647 mg, 2.35 mmol), [1,1⁰-bis(diphenylphosphino)fer-

rocene]dichloropalladium(II) dichloromethane adduct (160 mg,

0.196 mmol) and cesium carbonate (1.92 g, 5.89 mmol) in THF/

H₂O (8 mL/2 mL) was stirred at reﬂux for 18 h. The reaction mixture

was diluted with brine, and extracted with EtOAc. The organic layer

was dried over anhydrous MgSO₄and concentrated under reduced

pressure. The residue was puriﬁed by column chromatography on

NH-silica gel with hexane/EtOAc as an eluent and crystallized from

EtOAc/hexane to give 5 (196 mg, 31%). ¹H NMR (300 MHz, DMSO-d₆)

d 3.68 (3H, s), 4.51 (2H, s), 6.59–6.73 (2H, m), 6.82 (1H, d, J = 8.9 Hz),

7.24–7.49 (4H, m), 7.66 (1H, s), 10.62 (1H, s). Anal. Calcd for

Yield (82%). ¹H NMR (300 MHz, DMSO-d₆) d 2.18 (3H, s), 3.63

(3H, s), 7.30 (1H, td, J = 8.5, 1.9 Hz), 7.43–7.62 (2H, m).

5.27. 4-Bromo-1-ethyl-5-(4-ﬂuorophenyl)-3-methyl-1H-

pyrazole (14d)

Yield (99%). ¹H NMR (300 MHz, DMSO-d₆) d 1.21 (3H, t,

J = 7.0 Hz), 2.18 (3H, s), 3.96 (2H, q, J = 7.0 Hz), 7.34–7.45 (2H, m),

7.45–7.54 (2H, m).

5.28. 4-Bromo-5-(4-ﬂuorophenyl)-3-methyl-1-propyl-1H-

pyrazole (14e)

C

₁₈H₁₄N₃O₂F: C, 66.87; H, 4.36; N, 13.00. Found: C, 66.77; H, 4.34;

N, 12.93.

The compound 6a–c, 16a–e, 16g–i were prepared in a manner

Yield (99% in 2 steps). ¹H NMR (300 MHz, CDCl₃) d 0.69 (3H, t,

J = 7.5 Hz), 1.54–1.70 (2H, m), 2.18 (3 H, s), 3.90 (2H, t, J = 7.0 Hz),

7.39 (2H, t, J = 9.0 Hz), 7.48 (2H, dd, J = 9.0, 5.5 Hz).

similar to that described for 5.

5.37. 6-[5-(4-Fluorophenyl)-1,3-dimethyl-1H-pyrazol-4-yl]-2H-

1,4-benzoxazin-3(4H)-one (6a)

5.29. 1-Benzyl-4-bromo-5-(4-ﬂuorophenyl)-3-methyl-1H-

pyrazole (14f)

Yield (22%). ¹H NMR (DMSO-d₆) d 2.17 (3H, s), 3.64 (3H, s), 4.53

(2H, s), 6.57 (1H, dd, J = 8.1, 2.1 Hz), 6.65 (1H, d, J = 1.9 Hz), 6.83

(1H, d, J = 8.3 Hz), 7.18–7.42 (4H, m), 10.58 (1H, s). Anal. Calcd for

Yield (80%). ¹H NMR (300 MHz, CDCl₃) d 2.30 (3H, s), 5.17 (2H, s),

6.97 (2H, dd, J = 5.4, 1.9 Hz), 7.02–7.12 (2H, m), 7.18–7.27 (5H, m).

C

₁₉H₁₅N₃O₂F₂: C, 64.22; H, 4.25; N, 11.83. Found: C, 64.09; H,

5.30. 4-Bromo-5-(4-ﬂuorophenyl)-3-methyl-1-phenyl-1H-

pyrazole (14g)

4.21; N, 11.75.

5.38. 6-[5-(2,4-Diﬂuorophenyl)-1,3-dimethyl-1H-pyrazol-4-yl]-

2H-1,4-benzoxazin-3(4H)-one (6b)

Yield (88%). ¹H NMR (300 MHz, CDCl₃) d 2.38 (3H, s), 6.97–7.07

(2H, m), 7.15–7.33 (7H, m).

Yield (48%). ¹H NMR (300 MHz, DMSO-d₆) d 2.20 (3H, s), 3.59

(3H, s), 4.53 (2H, s), 6.58 (1H, dd, J = 8.1, 2.1 Hz), 6.64 (1H, d,

J = 2.3 Hz), 6.83 (1H, d, J = 8.3 Hz), 7.18 (1H, td, J = 8.6, 2.1 Hz),

7.31–7.47 (2H, m), 10.59 (1H, s). Anal. Calcd for C₁₉H₁₅N₃O₂F₂: C,

64.22; H, 4.25; N, 11.83. Found: C, 64.09; H, 4.21; N, 11.75.

5.31. 3-[4-Bromo-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-1-

yl]propan-1-ol (14h)

Yield (95%). ¹H NMR (DMSO-d₆) d 1.80 (2H, quin, J = 6.5 Hz),

2.18 (3H, s), 3.22–3.34 (2H, m), 3.99 (2H, t, J = 7.3 Hz), 4.48 (1H,

m), 7.34–7.44 (2H, m), 7.50 (2H, dd, J = 8.7, 5.5 Hz).

5.39. 6-[5-(4-Fluorophenyl)-1,3-dimethyl-1H-pyrazol-4-yl]-8-

methyl-2H-1,4-benzoxazin-3(4H)-one (6c)

5.32. 1-[4-(Benzyloxy)butyl]-4-bromo-5-(4-ﬂuorophenyl)-3-

Yield (12%). ¹H NMR (300 M Hz, DMSO-d₆) d 2.06 (3H, s), 2.16

(3H, s), 3.64 (3H, s), 4.54 (2H, s), 6.45 (1H, d, J = 1.9 Hz), 6.51 (1H,

d), 7.18–7.41 (4H, m), 10.51 (1H, s). Anal. Calcd for C₂₀H₁₈N₃O₂F:

C, 68.36; H, 5.16; N, 11.96. Found: C, 68.23; H, 5.16; N, 11.86.

methyl-1H-pyrazole (14i)

Yield (87%). ¹H NMR (300 MHz, CDCl₃) d 1.42–1.55 (2H, m),

1.75–1.87 (2H, m), 2.28 (3H, s), 3.36 (2H, t, J = 6.3 Hz), 3.90 (2H,

t, J = 7.2 Hz), 4.41 (2H, s), 7.09–7.18 (2H, m), 7.23–7.36 (7H, m).

5.40. 6-(1-Ethyl-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-4-yl)-

2H-1,4-benzoxazin-3(4H)-one (16a)

5.33. 2-{[4-Bromo-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-1-

yl]methyl}pyridine (14j)

Yield (28%). ¹H NMR (300 MHz, DMSO-d₆) d 1.24 (3H, t,

J = 7.0 Hz), 2.19 (3H, s), 3.91 (2H, q, J = 7.0 Hz), 4.53 (2H, s), 6.57

(1H, dd, J = 8.0, 2.0 Hz), 6.65 (1H, d, J = 2.0 Hz), 6.81 (1H, d,

J = 8.0 Hz), 7.19–7.36 (4H, m), 10.60 (1H, s). Anal. Calcd for C₂₀H₁₈N₃O₂F:

C, 68.36; H, 5.16; N, 11.96. Found: C, 68.58; H, 5.39; N, 11.72.

Yield (69%). ¹H NMR (300 MHz, CDCl₃) d 2.32 (3H, s), 5.31 (2H,

s), 6.93 (1H, d, J = 7.6 Hz), 7.05–7.21 (3H, m), 7.31–7.40 (2H, m),

7.58–7.68 (1H, m), 8.52 (1H, d, J = 4.2 Hz).

5.34. 3-{[4-Bromo-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-1-

yl]methyl}pyridine (14k)

5.41. 6-[5-(4-Fluorophenyl)-3-methyl-1-propyl-1H-pyrazol-4-

yl]-2H-1,4-benzoxazin-3(4H)-one (16b)

Yield (55%). ¹H NMR (300 MHz, CDCl₃) d 2.31 (3H, s), 5.26 (2H,

s), 7.12–7.22 (2H, m), 7.22–7.31 (2H, m), 7.41–7.50 (1H, m), 7.64

(1H, d, J = 7.9 Hz), 8.36 (1H, s), 8.63 (1H, s).

Yield (36%). ¹H NMR (300 MHz, DMSO-d₆) d 0.73 (3H, t,

J = 7.5 Hz), 1.57–1.73 (2H, m), 2.19 (3H, s), 3.84 (2H, t, J = 7.5 Hz),

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

5439

4.53 (2H, s), 6.56 (1H, dd, J = 8.5, 2.0 Hz), 6.65 (1H, d, J = 2.0 Hz),

6.81 (1H, d, J = 8.5 Hz), 7.22–7.35 (4H, m), 10.60 (1H, br. s). Anal.

Calcd for C₂₁H₂₀N₃O₂F: C, 69.03; H, 5.52; N, 11.50. Found: C,

69.04; H, 5.51; N, 11.50.

6.83 (1H, d, J = 8.0 Hz), 6.96 (2H, d, J = 6.1 Hz), 7.20–7.25 (4H, m),

8.47 (2H, d, J = 6.1 Hz), 10.60 (1H, s). Anal. Calcd for C₂₄H₁₉N₄O₂F:

C, 69.55; H, 4.62; N, 13.52. Found: C, 69.38; H, 4.75; N, 13.25.

5.49. 6-[5-(4-Fluorophenyl)-1-(4-hydroxybutyl)-3-methyl-1H-

pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (16g)

5.42. 6-[1-Benzyl-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-4-yl]-

2H-1,4-benzoxazin-3(4H)-one (16c)

A mixture of 16f (0.49 g, 1.01 mmol) and 10 wt% palladium on

carbon (0.1 g) in EtOH (30 mL) was stirred at room temperature

for 5 h and at 50 °C for 18 h, under hydrogen atmosphere. The

insoluble material was removed by ﬁltration, and the ﬁltrate was

concentrated under reduced pressure. The residue was crystallized

from EtOAc/hexane to give 16g (0.35 g, 88%). ¹H NMR (300 MHz,

DMSO-d₆) d 1.10–1.37 (2H, m), 1.60–1.73 (2H, m), 2.18 (3H, s),

3.23–3.34 (2H, m), 3.88 (2H, t, J = 7.2 Hz), 4.34 (1H, t, J = 5.1 Hz),

4.52 (2H, s), 6.55 (1H, dd, J = 8.4, 1.8 Hz), 6.64 (1H, d, J = 1.8 Hz),

6.80 (1H, d, J = 8.4 Hz), 7.21–7.32 (4H, m), 10.58 (1H, s). Anal. Calcd

for C₂₂H₂₂N₃O₃F: C, 66.82; H, 5.61; N, 10.63. Found: C, 66.92; H,

5.71; N, 10.50.

Yield (38%). ¹H NMR (300 MHz, DMSO-d₆) d 2.21 (3H, s), 4.53

(2H, s), 5.15 (2H, s), 6.59 (1H, dd, J = 8.3, 2.1 Hz), 6.68 (2H, d,

J = 2.1 Hz), 6.82 (1H, d, J = 8.3 Hz), 6.97 (2H, dd, J = 7.9, 1.5 Hz),

7.19–7.32 (7H, m), 10.60 (1H, s). Anal. Calcd for C₂₅H₂₀N₃O₂F: C,

72.63; H, 4.88; N, 10.16. Found: C, 72.61; H, 4.94; N, 10.07.

5.43. 6-[5-(4-Fluorophenyl)-3-methyl-1-phenyl-1H-pyrazol-

4-yl]-2H-1,4-benzoxazin-3(4H)-one (16d)

Yield (16%). ¹H NMR (300 MHz, DMSO-d₆) d 2.26 (3H, s), 4.56

(2H, s), 6.59–6.75 (2H, m), 6.68 (1H, d, J = 8.3 Hz), 7.10–7.23 (6H,

m), 7.27–7.39 (3H, m), 10.65 (1H, s). Anal. Calcd for C₂₄H₁₈N₃O₂F:

C, 72.17; H, 4.54; N, 10.52. Found: C, 72.18; H, 4.58; N, 10.48.

5.50. 8-Methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-

2H-1,4-benzoxazin-3(4H)-one (15b)

5.44. 6-[5-(4-Fluorophenyl)-1-(3-hydroxypropyl)-3-methyl-1H-

pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (16e)

A

mixture of 4-bromo-2-methyl-6-nitrophenol (9.4 g,

40.5 mmol), methyl bromoacetate (6.5 g, 42.5 mmol) and potas-

sium carbonate (8.4 g, 60.8 mmol) in DMSO (50 mL) was stirred

at room temperature for 48 h, diluted with water and 10% hydro-

chloric acid, and extracted with EtOAc. The organic layer was

washed with water and brine, dried over Na₂SO₄and concentrated

in vacuo. The resulting solid was washed with hexane to give

methyl (4-bromo-2-methyl-6-nitrophenoxy)acetate (9.86 g, 80%).

A mixture of methyl (4-bromo-2-methyl-6-nitrophenoxy)acetate

(10.9 g, 35.8 mmol) and zinc dust (35 g, 535 mmol) in acetic acid

(100 mL) and THF (200 mL) was stirred at 45 °C for 30 min and at

reﬂux for 1 h, and ﬁltered. The ﬁltrate was concentrated in vacuo,

and the residue was diluted with EtOAc. The organic layer was

washed with aqueous NaHCO₃solution, water and brine, dried

over Na₂SO₄and concentrated in vacuo. The residue was washed

with hexane, suspended in MeOH, and collected by ﬁltration to

Yield (17%). ¹H NMR (DMSO-d₆) d 1.73–1.90 (2H, m), 2.18 (3H,

s), 3.27–3.40 (2H, m), 3.94 (2H, t, J = 7.2 Hz), 4.47 (1H, t,

J = 4.9 Hz), 4.53 (2H, s), 6.56 (1H, dd, J = 8.1, 2.1 Hz), 6.64 (1H, d,

J = 1.9 Hz), 6.81 (1H, d, J = 8.0 Hz), 7.17–7.41 (4H, m), 10.60 (1H,

s). Anal. Calcd for C₂₁H₂₀N₃O₃F: C, 66.13; H, 5.29; N, 11.02. Found:

C, 65.89; H, 5.16; N, 10.80.

5.45. 6-{1-[4-(Benzyloxy)butyl]-5-(4-ﬂuorophenyl)-3-methyl-

1H-pyrazol-4-yl}-2H-1,4-benzoxazin-3(4H)-one (16f)

Yield (44%). ¹H NMR (300 MHz, CDCl₃) d 1.47–1.59 (2H, m),

1.81–1.92 (2H, m), 2.30 (3H, s), 3.38 (2H, t, J = 6.3 Hz), 3.99 (2H,

t, J = 7.5 Hz), 4.22 (2H, s), 4.57 (2H, s), 6.43 (1H, d, J = 2.1 Hz),

6.65 (1H, dd, J = 8.4, 2.1 Hz), 6.84 (1H, d, J = 8.4 Hz), 6.98–7.08

(2H, m), 7.11–7.18 (2H, m), 7.22–7.36 (5H, m), 7.97 (1H, s).

give

6-bromo-8-methyl-2H-l,4-benzoxazin-3(4H)-one

(5.8 g,

70%). ¹H NMR (300 MHz, DMSO-d₆) d 2.14 (s, 3H), 4.60 (s, 2H),

6.87 (d, J = 2.3 Hz, 1H), 7.00 (d, J = 2.3 Hz, 1H), 10.72 (s, 1H). A mix-

ture of 6-bromo-8-methyl-2H-1,4-benzoxazin-3(4H)- one (2.0 g,

8.3 mmol), bis(pinacolato)diboron (2.3 g, 9.1 mmol), potassium

acetate (2.9 g, 29.5 mmol) [1,1-bis(diphenylphosphino)ferro-

cene]dichloropalladium(II) dichloromethane complex (0.34 g,

0.42 mmol) in 1,4-dioxane (50 mL) was stirred at 90 °C for 12 h

under argon atmosphere, diluted with water and extracted with

EtOAc. The organic layer was dried over Na₂SO₄and concentrated

in vacuo. The residue was suspended in IPE, and collected by

ﬁltration to give 15b (2.36 g, 98%) as a solid. ¹H NMR (300 MHz,

DMSO-d₆) d 1.27 (s, 12H), 2.15 (s, 3H), 4.61 (s, 2H), 7.06 (s, 1H),

7.13 (s, 1H), 10.61 (s, 1H).

5.46. 6-[5-(4-Fluorophenyl)-3-methyl-1-(pyridin-2-ylmethyl)-

1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (16h)

Yield (52%). ¹H NMR (300 MHz, DMSO-d₆) d 2.20 (3H, s), 4.54

(2H, s), 5.22 (2H, s), 6.61 (1H, d, J = 8.1 Hz), 6.69 (1H, s), 6.83 (1H,

d, J = 8.1 Hz), 7.02 (1H, d, J = 7.7 Hz), 7.16–7.37 (5H, m), 7.68–

7.82 (1H, m), 8.49 (1H, d, J = 4.5 Hz), 10.60 (1H, s). Anal. Calcd for

C₂₄H₁₉N₄O₂F: C, 69.55; H, 4.62; N, 13.52. Found: C, 69.28; H,

4.66; N, 13.13.

5.47. 6-[5-(4-Fluorophenyl)-3-methyl-1-(pyridin-3-ylmethyl)-

1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (16i)

Yield (21%). ¹H NMR (300 MHz, DMSO-d₆) d 2.21 (3H, s), 4.53

(2H, s), 5.20 (2H, s), 6.60 (1H, dd, J = 8.3, 1.9 Hz), 6.68 (1H, d,

J = 1.9 Hz), 6.82 (1H, d, J = 8.3 Hz), 7.21–7.35 (5H, m), 7.37–7.44

(1H, m), 8.19 (1H, s), 8.45 (1H, d, J = 3.8 Hz), 10.60 (1H, s). Anal.

Calcd for C₂₄H₁₉N₄O₂F 0.1(EtOAc): C, 69.24; H, 4.72; N, 13.24.

Found: C, 68.92; H, 4.69; N, 13.16.

5.51. tert-Butyl-2-(pyridin-2-ylmethylene)hydrazinecarboxylate

(18a)

A mixture of pyridine-2-carbaldehyde (5.0 g, 46.7 mmol) and

tert-butyl carbazate (7.4 g, 56.0 mmol) in EtOH (50 mL) was stirred

at room temperature for 3 h, and then concentrated under reduced

pressure. The residue was suspended in IPE and collected by ﬁltra-

tion to give 18a (5.6 g, 55%). ¹H NMR (300 MHz, CDCl₃) d 1.55 (9H,

s), 7.21–7.28 (1H, m), 7.65–7.73 (1H, m), 7.94 (1H, s), 8.07 (1H, d,

J = 8.1 Hz), 8.36 (1H, s), 8.53–8.59 (1H, m).

5.48. 6-[5-(4-Fluorophenyl)-3-methyl-1-(pyridin-4-ylmethyl)-

1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (16j)

Yield (5%). ¹H NMR (300 MHz, DMSO-d₆) d 2.22 (3H, s), 4.54 (2H,

s), 5.20 (2H, s), 6.62 (1H, dd, J = 8.3, 2.3 H), 6.69 (1H, d, J = 1.9 Hz),

The compound 18b,c were prepared in a manner similar to that

described for 18a.

5440

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

5.52. tert-Butyl-2-(pyridin-3-ylmethylene)hydrazinecarboxylate

(18b)

under reduced pressure, and the residue was puriﬁed by column

chromatography on silica gel with hexane/EtOAc as an eluent to

give 23a (211 mg, 20%). ¹H NMR (300 MHz, DMSO-d₆) d 0.73 (3H,

t, J = 7.2 Hz), 1.03–1.17 (2H, m), 1.52–1.63 (2H, m), 2.18 (3H, s),

3.94 (2H, t, J = 7.0 Hz), 7.33–7.43 (2H, m), 7.45–7.54 (2H, m).

The compound 23b,c were prepared in a manner similar to that

described for 23a.

Yield (90%). ¹H NMR (300 MHz, CDCl₃) d 1.54 (9H, s), 7.27–7.34

(1H, m), 7.95 (1H, s), 8.09–8.16 (1H, m), 8.57 (1H, dd, J = 4.9,

1.7 Hz), 8.74 (1H, d, J = 1.9 Hz).

5.53. tert-butyl-2-(pyridin-4-ylmethylene)hydrazinecarboxylate

(18c)

5.60. 4-Bromo-5-(4-ﬂuorophenyl)-1-isobutyl-3-methyl-1H-

pyrazole (23b)

Yield (62%). ¹H NMR (300 MHz, CDCl₃) d: 1.54 (9H, s), 7.51–7.57

(2H, m), 7.87 (1H, s), 8.59–8.64 (2H, m), 8.74 (1H, s).

Yield (11%). ¹H NMR (300 MHz, DMSO-d₆) d 0.67 (6H, d,

J = 6.8 Hz), 1.87–2.02 (1H, m), 2.19 (3H, s), 3.77 (2H, d, J = 7.3 Hz),

7.31–7.43 (2H, m), 7.43–7.53 (2H, m).

5.54. 2-(Hydrazinomethyl)pyridine triﬂuoroacetate (19a)

A mixture of 18a (3.0 g, 13.6 mmol), sodium cyanoborohydride

(0.85 g, 13. mmol) and acetic acid/H₂O (15 mL/15 mL) was stirred

at room temperature for 2 h, neutralized with 1 N NaOH, and

extracted with EtOAc. The organic layer was washed with water

and brine, dried over anhydrous MgSO₄and concentrated under

reduced pressure. The residue was diluted with dichloromethane,

and then triﬂuoroacetic acid (10 mL, 136 mmol) was added drop-

wise. The mixture was stirred at room temperature for 2 h, and

concentrated under reduced pressure to give 19a. The crude 19a

was used for the next reaction.

5.61. 4-Bromo-5-(4-ﬂuorophenyl)-3-methyl-1-(4,4,4-

triﬂuorobutyl)-1H-pyrazole (23c)

Yield (30%). ¹H NMR (300 MHz, DMSO-d₆) d 1.86 (2H, quin,

J = 7.5 Hz), 2.07–2.18 (2H, m), 2.19 (3H, s), 4.02 (2H, t, J = 7.1 Hz),

7.28–7.45 (2H, m), 7.46–7.56 (2H, m).

5.62. 6-[1-Butyl-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-4-yl]-

2H-1,4-benzoxazin-3(4H)-one (24a)

The compound 24a were prepared in a manner similar to that

described for 5. Yield (43%). ¹H NMR (300 MHz, DMSO-d₆) d 0.75

(3H, t, J = 7.3 Hz), 1.04–1.25 (2H, m), 1.52–1.69 (2H, m), 2.19 (3H,

s), 3.88 (2H, t, J = 7.2 Hz), 4.53 (2H, s), 6.56 (1H, dd, J = 8.3,

2.1 Hz), 6.65 (1H, d, J = 2.1 Hz), 6.81 (1H, d, J = 8.3 Hz), 7.21–7.34

(4H, m), 10.60 (1H, s). Anal. Calcd for C₂₂H₂₂FN₃O₂: C, 69.64; H,

5.84; N, 11.07. Found: C, 69.63; H, 5.72; N, 11.1.

The compound 19b,c were prepared in a manner similar to that

described for 19a.

5.55. 3-(Hydrazinomethyl)pyridine triﬂuoroacetate (19b)

The crude product was used for the next reaction.

5.56. 4-(Hydrazinomethyl)pyridine triﬂuoroacetate (19c)

The crude product was used for the next reaction.

5.63. 6-[5-(4-Fluorophenyl)-1-isobutyl-3-methyl-1H-pyrazol-4-

yl]-2H-1,4-benzoxazin-3(4H)-one (24b)

The compound 24b were prepared in a manner similar to that

described for 5. Yield (43%). ¹H NMR (300 MHz, DMSO-d₆) d 0.70

(6H, d, J = 6.8 Hz), 1.88–2.16 (1H, m), 2.19 (3H, s), 3.71 (2H, d,

J = 7.2 Hz), 4.53 (2H, s), 6.56 (1H, dd, J = 8.3, 1.9 Hz), 6.65 (1H, d,

J = 1.9 Hz), 6.81 (1H, d, J = 8.3 Hz), 7.24–7.32 (4H, m), 10.60 (1H,

s). Anal. Calcd for C₂₂H₂₂FN₃O₂: C, 69.64; H, 5.84; N, 11.07. Found:

C, 69.45; H, 5.84; N, 11.05.

5.57. [4-(Benzyloxy)butyl]hydrazine hydrochloride (21)

To a mixture of hydrazine hydrate (11.6 g, 231 mmol) and

sodium hydroxide (2.01 g, 50 mmol) was added [(4-chlorobut-

oxy)methyl]benzene (10 g, 50 mmol) at 95 °C. The mixture was

stirred for 2 h at the temperature, and concentrated under reduced

pressure. To the residue was added concentrated HCl (5 mL) and

water (120 mL). The mixture was extracted with diethyl ether.

The organic layer was concentrated under reduced pressure to give

21 (9.6 g, 83%). ¹H NMR (300 MHz, DMSO-d₆) d 1.50–1.77 (4H, m),

2.86–3.05 (2H, m), 3.43 (2H, t, J = 5.4 Hz), 4.45 (2H, s), 6.60 (3H, br

s), 7.23–7.37 (5H, m).

5.64. 6-[5-(4-Fluorophenyl)-3-methyl-1-(4,4,4-triﬂuorobutyl)-

1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (24c)

The compound 24c were prepared in a manner similar to that

described for 5. Yield (5%). ¹H NMR (300 MHz, DMSO-d₆) d 1.89

(2H, m), 2.10–2.33 (5H, m), 3.96 (2H, t, J = 7.0 Hz), 4.53 (2H, s),

6.58 (1H, dd, J = 8.3, 1.9 Hz), 6.66 (1H, d, J = 1.9 Hz), 6.82 (1H, d,

J = 8.3 Hz), 7.22–7.40 (4H, m), 10.60 (1H, s). Anal. Calcd for

5.58. 4-Bromo-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazole (22)

The compound 22 were prepared in a manner similar to that

described for 14b. Yield (98%). ¹H NMR (300 MHz, CDCl₃) d 2.25

(3H, s), 7.01–7.17 (2H, m), 7.65–7.81 (2H, m), 8.97 (1H, br s).

C₂₂H₁₉N₃O₂F₄: C, 60.97; H, 4.42; N, 9.70. Found: C, 60.59; H,

4.16; N, 9.65.

5.65. 1-(2-Azidoethyl)-5-(4-ﬂuorophenyl)-3-methyl-1H-

pyrazole (25)

5.59. 4-Bromo-1-butyl-5-(4-ﬂuorophenyl)-3-methyl-1H-

pyrazole (23a)

A mixture of 13j (5.35 g, 24.3 mmol) and p-toluenesulfonyl

chloride (6.0 g, 31.5 mmol) in pyridine (30 mL) was stirred at room

temperature for 12 h, diluted with 1 N HCl, and extracted with

EtOAc. The organic layer was washed with 1 N HCl and brine, dried

over anhydrous Na₂SO₄and concentrated under reduced pressure.

To the residue was added DMF (30 ml) and NaN₃(2.05 g,

31.5 mmol) at room temperature, successively. The mixture was

stirred at 50 °C for 6 h and at room temperature for 2 days, diluted

saturated NaHCO₃solution, and extracted with EtOAc. The organic

To a stirred mixture of 22 (600 mg, 3.41 mmol), butan-1-ol

(523 mg, 7.06 mmol) and triphenylphosphine (1.8 g, 6.86 mmol)

in THF (6 mL) was added dropwise diisopropyl azodicarboxylate

in toluene (1.9 M, 3.7 mL, 7.03 mmol) at 60 °C. The mixture was

stirred for 30 min, diluted with water, and extracted with EtOAc.

The organic layer was dried over anhydrous MgSO₄and concen-

trated under reduced pressure. The residue was suspended in

IPE, and the solid was ﬁltered off. The ﬁltrate was concentrated

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

5441

layer was washed with brine, dried over anhydrous Na₂SO₄and

concentrated under reduced pressure. The residue was puriﬁed

by column chromatography on silica gel with hexane/EtOAc as

an eluent to give 25 (4.94 g, 83%). ¹H NMR (300 MHz, DMSO-

5.68. N-{2-[5-(4-Fluorophenyl)-3-methyl-4-(3-oxo-3,4-dihydro-

2H-1,4-benzoxazin-6-yl)-1H-pyrazol-1-yl]ethyl}acetamide (27a)

The compound 27a were prepared in a manner similar to that

described for 5. Yield (44%). ¹H NMR (300 MHz, DMSO-d₆) d 1.69

(3H, s), 2.20 (3H, s), 3.32 (2H, q, J = 6.0 Hz), 3.90 (2H, t,

J = 6.0 Hz), 4.53 (2H, s), 6.56 (1H, dd, J = 8.5, 2.0 Hz), 6.66

(1H, d, J = 2.0 Hz), 6.81 (1H, d, J = 8.5 Hz), 7.20–7.34 (4H, m), 7.94

(1H, t, J = 6.0 Hz), 10.61 (1H, br s). Anal. Calcd for C₂₂H₂₁N₄O₃F:

C, 64.70; H, 5.18; N, 13.72. Found: C, 65.05; H, 5.53; N, 13.38.

d₆)

d 2.20 (3H, s), 3.66 (2H, t, J = 5.5 Hz), 4.14 (2H, t,

J = 5.5 Hz), 6.18 (1H, s), 7.34 (2H, t, J = 9.0 Hz), 7.51 (2H, dd,

J = 9.0, 5.5 Hz).

5.66. N-{2-[4-Bromo-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-

1-yl]ethyl}acetamide (26a)

To a solution of 25 (1.00 g, 4.08 mmol) in THF (25 mL) was

added triphenylphosphine (1.28 g, 4.88 mmol) at room tempera-

ture. The mixture was stirred at room temperature for 3 h, and

5.69. N-{2-[5-(4-Fluorophenyl)-3-methyl-4-(3-oxo-3,4-dihydro-

2H-1,4-benzoxazin-6-yl)-1H-pyrazol-1-

yl]ethyl}methanesulfonamide (27b)

then water (360 lL, 20.0 mmol) was added. The resulting mix-

ture was stirred at 50 °C for 12 h, and diluted with 1 N HCl

and EtOAc. The aqueous layer was separated, basiﬁed with 8 N

NaOH, and extracted with EtOAc. The organic layer was washed

with brine, dried over anhydrous Na₂SO₄and concentrated

under reduced pressure. The residue was dissolved in pyridine

(15 mL), and then acetic anhydride (10 mL), and the mixture

was stirred at room temperature for 3 h. The solvent was

removed under reduced pressure, and the residue was dissolved

in DMF (30 mL). To the resulting solution was added NBS

(871 mg, 4.89 mmol) at 0 °C. The mixture was stirred at room

temperature for 30 min, diluted with aqueous Na₂S₂O₃solution,

and extracted with EtOAc. The organic layer was washed with

brine, dried over anhydrous Na₂SO₄and concentrated under

reduced pressure. The residue was puriﬁed by column chroma-

tography on silica gel with hexane/EtOAc as an eluent to give

26a (1.30 g, 94% in 3 steps). ¹H NMR (300 MHz, DMSO-d₆) d

1.65 (3H, s), 2.20 (3H, s), 3.27 (2H, q, J = 6.0 Hz), 3.97 (2H, t,

J = 6.0 Hz), 7.37 (2H, t, J = 9.0 Hz), 7.45 (2H, dd, J = 9.0, 5.5 Hz),

7.87 (1H, t, J = 6.0 Hz).

The compound 27b were prepared in a manner similar to that

described for 5. Yield (33%). ¹H NMR (300 MHz, DMSO-d₆) d 2.20

(3H, s), 2.81 (3H, s), 3.25–3.33 (2H, m), 3.97 (2H, t, J = 6.5 Hz),

4.53 (2H, s), 6.57 (1H, dd, J = 8.5, 2.0 Hz), 6.61–6.68 (1H, m), 6.82

(1H, d, J = 8.5 Hz), 7.18 (1H, t, J = 6.0 Hz), 7.22–7.41 (4H, m),

10.60 (1H, br. s). Anal. Calcd for C₂₁H₂₁N₄O₄SF: C, 56.75; H, 4.76;

N, 12.61. Found: C, 56.46; H, 4.70; N, 12.29.

5.70. 3-[5-(4-Fluorophenyl)-3-methyl-1H-pyrazol-1-yl]propane-

nitrile (28)

The compound 28 were prepared in a manner similar to that

described for 14b. Yield (69%). ¹H NMR (300 MHz, DMSO-d₆) d

2.19 (3H, s), 3.68 (2H, q, J = 5.5 Hz), 3.965 (2H, t, J = 5.5 Hz), 4.89

(1H, t, J = 5.5 Hz), 7.32–7.43 (2H, m), 7.50–7.61 (2H, m).

5.71. 3-[4-Bromo-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-1-

yl]propanenitrile (29)

To

a solution of 28 (1.0 g, 3.34 mmol), tributylphosphine

5.67. N-{2-[4-Bromo-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-

1-yl]ethyl}methanesulfonamide (26b)

(1.25 mL, 5.01 mmol), 90% acetone cyanohydrin (0.44 mL,

4.34 mmol) in toluene (25 mL) was added 1,1⁰-(azodicarbonyl)dip-

iperidine (1.3 g, 5.01 mmol) at 0 °C. The mixture was stirred at

room temperature for 12 h, diluted with water, and extracted with

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

drous Na₂SO₄, and concentrated under reduced pressure. The resi-

due was puriﬁed by column chromatography on silica gel with

hexane/EtOAc to give 29 (781 mg, 76%). ¹H NMR (300 MHz,

DMSO-d₆) d 2.21 (3H, s), 2.98 (2H, t, J = 6.5 Hz), 4.18 (2H, t,

J = 6.5 Hz), 7.37–7.46 (2H, m), 7.47–7.56 (2H, m).

A

mixture of 25 (3.84 g, 15.7 mmol) and NBS (3.35 g,

18.8 mmol) in DMF (50 mL) was stirred at room temperature

for 30 min, diluted with aqueous Na₂S₂O₃solution, and extracted

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

anhydrous Na₂SO₄and concentrated under reduced pressure. The

residue was puriﬁed by column chromatography on silica gel

with hexane/EtOAc as an eluent to give 1-(2-azidoethyl)-

4-bromo-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazole (4.94 g, 97%).

To a solution of 1-(2-azidoethyl)-4-bromo-5-(4-ﬂuorophenyl)-3-

methyl-1H-pyrazole (800 mg, 2.46 mmol) in THF (20 mL) was

added triphenylphosphine (774 mg, 2.95 mmol) at room temper-

ature. The mixture was stirred at room temperature for 3 h, and

5.72. 3-[5-(4-Fluorophenyl)-3-methyl-4-(3-oxo-3,4-dihydro-2H-

1,4-benzoxazin-6-yl)-1H-pyrazol-1-yl]propanenitrile (30)

The compound 30 were prepared in a manner similar to that

described for 5. Yield (43%). ¹H NMR (300 MHz, DMSO-d₆) d 2.21

(3H, s), 3.01 (2H, t, J = 6.5 Hz), 4.12 (2H, t, J = 6.5 Hz), 4.54 (2H, s),

6.59 (1H, dd, J = 8.5, 2.0 Hz), 6.66 (1H, d, J = 2.0 Hz), 6.83 (1H, d,

J = 8.5 Hz), 7.23–7.40 (4H, m), 10.60 (1H, br s). Anal. Calcd for

then water (220 lL, 12.2 mmol) was added. The resulting mixture

was stirred at 50 °C for 12 h, diluted with ethyl acetate and 1 N

HCl. The aqueous layer was separated, basiﬁed with 8 N NaOH,

and extracted with EtOAc. The organic layer was washed with

brine, dried over anhydrous Na₂SO₄and concentrated under

reduced pressure. The residue was dissolved in pyridine (5 mL),

C₂₁H₁₇N₄O₂F: C, 67.01; H, 4.55; N, 14.89. Found: C, 66.67; H,

4.68; N, 14.49.

and then methanesulfonyl chloride (285 lL, 3.68 mmol) was

added. The mixture was stirred at room temperature for 12 h,

diluted with 1 N HCl, and extracted with EtOAc. The organic layer

was separated, washed with brine, dried over anhydrous Na₂SO₄

and concentrated under reduced pressure. The residue was

puriﬁed by column chromatography on silica gel with hexane/

EtOAc as an eluent to give 26b (760 mg, 82%). ¹H NMR

(300 MHz, DMSO-d₆) d 2.20 (3H, s), 2.79 (3H, s), 3.27 (2H, t,

J = 7.0 Hz), 4.02 (2H, t, J = 7.0 Hz), 7.14 (1H, br s.), 7.38 (2H, t,

J = 9.0 Hz), 7.51 (2H, dd, J = 9.0, 5.5 Hz).

5.73. 1-(Benzyloxy)-3-[5-(4-ﬂuorophenyl)-3-methyl-1H-

pyrazol-1-yl]propan-2-ol (32b)

To hydrazine monohydrate (100 g) was added dropwise benzyl

glycidyl ether (25.0 g, 152 mmol) at 60 °C. The mixture was stirred

at 60 °C for 3 h, and then concentrated under reduced pressure to

give 1-(benzyloxy)-3-hydrazinopropan-2-ol (29.6 g). The crude

1-(benzyloxy)-3-hydrazinopropan-2-ol (6.60 g, 33.6 mmol) was

added to a solution of 12a (5.00 g, 27.8 mmol) in methanol

5442

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

(50 mL) and conc. hydrochloric acid (2.80 mL) under ice-cooling.

The mixture was stirred at room temperature for 12 h, and concen-

trated under reduced pressure. To the residue was added water

and EtOAc. The aqueous layer was basiﬁed with potassium carbon-

ate, and then organic layer was separated. The organic layer was

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

trated under reduced pressure. The residue was puriﬁed by column

chromatography on silica gel with hexane/EtOAc to give 32b as an

oil (6.21 g, 66%). ¹H NMR (300 MHz, CDCl₃) d 2.29 (3H, s), 3.29–3.38

(1H, m, J = 9.5, 6.7 Hz), 3.48–3.56 (1H, m), 4.06–4.25 (3H, m), 4.45

(2H, s), 4.53 (1H, s), 6.07 (1H, s), 7.00–7.10 (2H, m), 7.15–7.22 (2H,

m), 7.25–7.41 (5H, m).

NMR (300 MHz, CDCl₃) d 2.29 (3H, s), 4.05 (2H, s), 4.51 (2H, s), 4.98

(2H, s), 7.08–7.17 (2H, m), 7.22–7.43 (7H, m).

The compound 34a,c were prepared in a manner similar to that

described for 34b.

5.80. 1-[4-Bromo-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-1-

yl]butan-2-one (34a)

Yield (94%). ¹H NMR (300 MHz, DMSO-d₆) d 0.85 (3H, t,

J = 7.2 Hz), 2.18 (3H, s), 2.37 (2H, q, J = 7.2 Hz), 4.99 (2H, s), 7.31–

7.47 (4H, m).

The compound 32a,c were prepared in a manner similar to that

described for 32b.

5.81. 4-(Benzyloxy)-1-[4-bromo-5-(4-ﬂuorophenyl)-3-methyl-

1H-pyrazol-1-yl]butan-2-one (34c)

5.74. 1-(Benzyloxy)-3-[5-(4-ﬂuorophenyl)-3-methyl-1H-

pyrazol-1-yl]propan-2-ol (32a)

Yield (99%). ¹H NMR (300 MHz, CDCl₃) d 2.30 (3H, s), 2.61 (2H, t,

J = 6.0 Hz), 3.69 (2H, t, J = 6.0 Hz), 4.43 (2H, s), 4.82 (2H, s), 6.99–

7.10 (2H, m), 7.19–7.38 (7H, m).

The crude product was used for the next reaction.

5.82. 1-[3-(Benzyloxy)-2,2-diﬂuoropropyl]-4-bromo-5-(4-

5.75. 4-(Benzyloxy)-1-[5-(4-ﬂuorophenyl)-3-methyl-1H-

ﬂuorophenyl)-3-methyl-1H-pyrazole (35b)

pyrazol-1-yl]butan-2-ol (32c)

To a solution of 34b (890 mg, 2.13 mmol) in toluene (20 mL)

was added dropwise diethylamino sulfur triﬂuoride (700 mg,

4.34 mmol) under ice-cooling. The mixture was stirred at 40 °C

for 6 h, and then diethylamino sulfur triﬂuoride (350 mg,

2.17 mmol) was further added. The mixture was stirred at 40 °C

for 24 h, treated with saturated NaHCO₃solution with ice-cooling,

and extracted with EtOAc. The organic layer was washed with

water, brine, dried over anhydrous Na₂SO₄, and concentrated

under reduced pressure. The residue was puriﬁed by column

chromatography on silica gel with hexane/EtOAc as an elutant to

give 35b (622 mg, 66%). ¹H NMR (300 MHz, CDCl₃) d 2.30 (3H, s),

3.68 (2H, t, J = 12.5 Hz), 4.44 (2H, t, J = 12.7 Hz), 4.58 (2H, s),

7.09–7.19 (2H, m), 7.23–7.40 (7H, m).

Yield (47%). ¹H NMR (300 MHz, DMSO-d₆) d 1.62–1.73 (2H, m),

2.29 (3H, s), 3.53–3.69 (2H, m), 3.91–4.01 (1H, m), 4.10 (1H, dd,

J = 14.0, 3.0 Hz), 4.15–4.26 (1H, m), 4.45 (2H, s), 4.48 (1H, d,

J = 3.4 Hz), 6.06 (1H, s), 7.03–7.14 (2H, m), 7.22–7.41 (7H, m).

The compound 33a–c were prepared in a manner similar to that

described for 14b.

5.76. 1-[4-Bromo-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-1-

yl]butan-2-ol (33a)

Yield (4% in 2 steps).¹H NMR (300 MHz, DMSO-d₆) d 0.79 (3H, t,

J = 7.3 Hz), 1.11–1.41 (2H, m), 2.19 (3H, s), 3.69–3.88 (3H, m), 4.87

(1H, d, J = 5.1 Hz), 7.37 (2H, t, J = 8.9 Hz), 7.56 (2H, dd, J = 8.9,

5.6 Hz).

The compound 35a,c were prepared in a manner similar to that

described for 35b.

5.83. 4-Bromo-1-(2,2-diﬂuorobutyl)-5-(4-ﬂuorophenyl)-3-

methyl-1H-pyrazole (35a)

5.77. 1-(Benzyloxy)-3-[4-bromo-5-(4-ﬂuorophenyl)-3-methyl-

1H-pyrazol-1-yl]propan-2-ol (33b)

Yield (71%). ¹H NMR (300 MHz, DMSO-d₆) d 0.84 (3H, t,

J = 7.4 Hz), 1.79 (2H, tt, J = 17.7, 7.5 Hz), 2.21 (3H, s), 4.45 (2H, t,

J = 13.4 Hz), 7.34–7.43 (2H, m), 7.44–7.51 (2H, m).

Yield (98%). ¹H NMR (300 MHz, CDCl₃) d 2.28 (3H, s), 3.33–3.42

(1H, m), 3.45–3.53 (1H, m), 3.90–4.22 (4H, m), 4.46 (2H, s), 7.04–

7.44 (9H, m).

5.84. 1-[4-(Benzyloxy)-2,2-diﬂuorobutyl]-4-bromo-5-(4-ﬂuoro-

phenyl)-3-methyl-1H-pyrazole (35c)

5.78. 4-(Benzyloxy)-1-[4-bromo-5-(4-ﬂuorophenyl)-3-methyl-

1H-pyrazol-1-yl]butan-2-ol (33c)

Yield (39%). ¹H NMR (300 MHz, CDCl₃) d 2.20 (2H, tt, J = 16.3,

6.3 Hz), 2.30 (3H, s), 3.60 (2H, t, J = 6.3 Hz), 4.31–4.47 (4H, m),

7.09–7.20 (2H, m), 7.22–7.39 (7H, m).

Yield (97%). ¹H NMR (300 MHz, CDCl₃) d 1.62–1.72 (2H, m), 2.28

(3H, s), 3.53–3.68 (2H, m), 3.88–3.98 (1H, m), 4.03 (1H, dd, J = 13.8,

3.2 Hz), 4.14–4.26 (1H, m), 4.44 (2H, s), 7.09–7.19 (2H, m),

7.21–7.44 (7H, m).

5.85. 6-{1-[3-(Benzyloxy)-2,2-diﬂuoropropyl]-5-(4-ﬂuorophen-

yl)-3-methyl-1H-pyrazol-4-yl}-2H-1,4-benzoxazin-3(4H)-one

(36b)

5.79. 1-(Benzyloxy)-3-[4-bromo-5-(4-ﬂuorophenyl)-3-methyl-

1H-pyrazol-1-yl]acetone (34b)

A

mixture of 35b (600 mg, 1.37 mmol), 15a (490 mg,

To a solution of 33b (1.50 g, 3.58 mmol) in toluene (30 mL) was

added Dess–Martin periodinane (2.00 g, 4.72 mmol) under

ice-cooling. The mixture was stirred at room temperature for

12 h, diluted with EtOAc and a solution of sodium thiosulfate

pentahydrate (6.90 g) in saturated NaHCO_3,and then stirred for

30 min. The organic layer was separated, washed with water and

brine, dried over anhydrous Na₂SO₄and concentrated under

reduced pressure. The residue was puriﬁed by column chromatog-

raphy on silica gel with hexane/EtOAc to give 34b (1.39 g, 93%). ¹H

1.78 mmol), [1,1’-bis(diphenylphosphino)ferrocene]dichloropalla-

dium(II) dichloromethane adduct (220 mg, 0.27 mmol), cesium

carbonate (1.20 g, 3.69 mmol) in THF/water (25 mL/5 mL) was stir-

red at reﬂux for 24 h under argon atmosphere, diluted with water

and EtOAc, and then ﬁltered. The organic layer was separated,

washed with brine, dried over anhydrous Na₂SO₄and concentrated

under reduced pressure. The residue was puriﬁed by column

chromatography on silica gel with hexane/EtOAc as an elutant,

and crystallized from IPE/hexane to give 36b (420 mg, 61%). ¹H

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

5443

NMR (300 MHz, DMSO-d₆) d 2.20 (3H, s), 3.74 (2H, t, J = 13.3 Hz),

4.41–4.57 (6H, m), 6.58 (1H, dd, J = 8.2, 2.0 Hz), 6.66 (1H, d,

J = 2.0 Hz), 6.82 (1H, d, J = 8.2 Hz), 7.18–7.40 (9H, m), 10.61 (1H, s).

The compound 36a,c were prepared in a manner similar to that

described for 36b.

5.91. 4-[4-Bromo-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-1-yl]

butan-2-one (39)

To a solution of 38 (1.00 g, 3.21 mmol) in THF (10 mL) was

added dropwise methylmagnesium bromide in diethyl ether

(3 M, 1.60 mL, 4.8 mmol) at room temperature. The mixture was

stirred at room temperature for 30 min, and then pored into the

ice water. The aqueous layer was treated with conc. HCl, and then

basiﬁed with 28% ammonia solution. The mixture was extracted

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

dried over anhydrous Na₂SO₄, and concentrated under reduced

pressure to give 4-[4-bromo-5-(4-ﬂuorophenyl)-3-methyl-1H-

pyrazol-1-yl]butan-2-ol. To a solution of the crude 4-[4-bromo-5-

(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-1-yl]butan-2-ol in toluene

(20 mL) was added Dess–Martin periodinane (1.80 g, 4.24 mmol)

at 0 °C. The mixture was stirred at room temperature for 12 h,

diluted with EtOAc, treated with a solution of sodium thiosulfate

pentahydrate (5.80 g) in saturated NaHCO₃solution, and then stir-

red at room temperature for 30 min. The organic layer was sepa-

rated, washed with water and brine, dried over anhydrous

Na₂SO_4,and concentrated under reduced pressure. The residue

was puriﬁed by column chromatography on silica gel with hex-

ane/EtOAc to give 39 (0.8 g, 77%). ¹H NMR (300 MHz, CDCl₃) d

2.13 (3H, s), 2.27 (3H, s), 3.00 (2H, t, J = 6.9 Hz), 4.19 (2H, t,

J = 6.9 Hz), 7.14–7.24 (2H, m), 7.36–7.44 (2H, m).

5.86. 6-[1-(2,2-Diﬂuorobutyl)-5-(4-ﬂuorophenyl)-3-methyl-1H-

pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (36a)

Yield (40%). ¹H NMR (300 MHz, DMSO-d₆) d 0.86 (3H, t,

J = 7.4 Hz), 1.84 (2H, m), 2.20 (3H, s), 4.38 (2H, t, J = 13.3 Hz),

4.53 (2H, s), 6.58 (1H, dd, J = 8.1, 2.1 Hz), 6.65 (1H, d, J = 1.9 Hz),

6.82 (1H, d, J = 8.3 Hz), 7.21–7.35 (4H, m), 10.61 (1H, s). Anal. Calcd

for C₂₂H₂₀N₃O₂F₃: C, 63.61; H, 4.85; N, 10.12. Found: C, 63.44; H,

4.83; N, 10.03.

5.87. 6-{1-[4-(Benzyloxy)-2,2-diﬂuorobutyl]-5-(4-ﬂuoro-

phenyl)-3-methyl-1H-pyrazol-4-yl}-2H-1,4-benzoxazin-3(4H)-

one (36c)

Yield (55%). ¹H NMR (300 MHz, DMSO-d₆) d 2.11–2.31 (5H, m),

3.51 (2H, t, J = 6.4 Hz), 4.36–4.56 (6H, m), 6.58 (1H, dd, J = 8.3,

1.9 Hz), 6.65 (1H, d, J = 1.9 Hz), 6.82 (1H, d, J = 8.3 Hz), 7.18–7.39

(9H, m), 10.61 (1H, s).

5.88. 6-[1-(2,2-Diﬂuoro-3-hydroxypropyl)-5-(4-ﬂuorophenyl)-

3-methyl-1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (37a)

5.92. 4-Bromo-1-(3,3-diﬂuorobutyl)-5-(4-ﬂuorophenyl)-3-

methyl-1H-pyrazole (40)

A mixture of 36b (380 mg, 0.75 mmol), 10wt% palladium on

carbon (400 mg) in MeOH/THF (8 mL/8 mL) was stirred at 50 °C

for 12 h under hydrogen atmosphere, and then ﬁltered. The ﬁltrate

was concentrated under reduced pressure, and the residue was

suspended in IPE. The crystals were collected by ﬁltration to give

37a (275 mg, 88%). ¹H NMR (300 MHz, DMSO-d₆) d 2.20 (3H, s),

3.60 (2H, td, J = 13.9, 6.3 Hz), 4.42 (2H, t, J = 14.0 Hz), 4.53 (2H, s),

5.53 (1H, t, J = 6.3 Hz), 6.58 (1H, dd, J = 8.3, 2.1 Hz), 6.65 (1H, d,

J = 2.1 Hz), 6.82 (1H, d, J = 8.3 Hz), 7.21–7.35 (4H, m), 10.60 (1H,

s). Anal. Calcd for C₂₁H₁₈N₃O₃F₃: C, 60.43; H, 4.35; N, 10.07. Found:

C, 60.51; H, 4.32; N, 10.02,

The compound 40 were prepared in a manner similar to that

described for 35. Yield (28%). ¹H NMR (300 MHz, DMSO-d₆) d

1.51 (3H, t, J = 19.2 Hz), 2.19 (3H, s), 2.26–2.46 (2H, m), 4.10 (2H,

dd, J = 8.4, 6.7 Hz), 7.40 (2H, t, J = 8.9 Hz), 7.46–7.56 (2H, m).

5.93. 6-[1-(3,3-Diﬂuorobutyl)-5-(4-ﬂuorophenyl)-3-methyl-1H-

pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (41)

The compound 41 were prepared in a manner similar to that

described for 36b. Yield (30%). ¹H NMR (300 MHz, DMSO-d₆) d

1.53 (3H, t, J = 19.2 Hz), 2.19 (3H, s), 2.38 (2H, m), 4.00–4.11 (2H,

m), 4.53 (2H, s), 6.57 (1H, dd, J = 8.3, 2.1 Hz), 6.65 (1H, d,

J = 2.1 Hz), 6.82 (1H, d, J = 8.3 Hz), 7.24–7.39 (4H, m), 10.59 (1H,

s). Anal. Calcd for C₂₂H₂₀N₃O₂F₃: C, 63.61; H, 4.85; N, 10.12. Found:

C, 63.49; H, 4.77; N, 9.96.

5.89. 6-[1-(2,2-Diﬂuoro-4-hydroxybutyl)-5-(4-ﬂuorophenyl)-3-

methyl-1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (37b)

The compound 37b were prepared in a manner similar to that

described for 37a. Yield (70%). ¹H NMR (300 MHz, DMSO-d₆) d

1.93–2.14 (2H, m), 2.21 (3H, s), 3.45–3.55 (2H, m), 4.43 (2H, t,

J = 14.2 Hz), 4.53 (2H, s), 4.69 (1H, t, J = 5.1 Hz), 6.58 (1H, dd,

J = 8.2, 2.2 Hz), 6.66 (1H, d, J = 2.2 Hz), 6.82 (1H, d, J = 8.2 Hz),

7.21–7.33 (4H, m), 10.61 (1H, s). Anal. Calcd for C₂₂H₂₀N₃O₃F₃: C,

61.25; H, 4.67; N, 9.74. Found: C, 61.34; H, 4.64; N, 9.62.

5.94. 4-[4-Bromo-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-1-

yl]-2,2-diﬂuorobutyl acetate (42)

To a mixture of 39 (470 mg, 1.45 mmol) and triethylamine

(4 mL) in toluene (4 mL) was added tert-butyldimethylsilyl triﬂuo-

romethanesulfonate (535 mg, 2.02 mmol) at 0 °C. The mixture was

stirred at 0 °C for 30 min, diluted with saturated NaHCO₃solution,

and extracted with EtOAc. The organic layer was dried over anhy-

drous MgSO_4,and concentrated under reduced pressure. To the

residue was added toluene (4 mL), lead(IV) acetate (962 mg,

2.96 mmol) and potassium bicarbonate (579 mg, 5.78 mmol) at

room temperature, successively. The mixture was stirred at room

temperature for 3 h, diluted with saturated NaHCO₃solution, and

extracted with EtOAc. The organic layer was dried over anhydrous

MgSO_4,and concentrated under reduced pressure. The residue was

dissolved in THF (4 mL), and then N,N,N-tributylbutan-1-aminium

ﬂuoride in THF (1 M, 2.9 mL, 2.90 mmol) was added. The mixture

was stirred at room temperature for 1 h, diluted with saturated

NaHCO₃solution, and extracted with EtOAc. The organic layer

was dried over anhydrous MgSO₄and concentrated under reduced

pressure. To the residue was added toluene (3 mL) and

5.90. 3-[4-Bromo-5-(4-ﬂuorophenyl)-3-methyl-1H-pyrazol-1-

yl]propanal (38)

To a solution of 14h (4.20 g, 13.4 mmol) in DMSO (42 mL) was

added triethylamine (30 mL) at room temperature, and then pyri-

dine sulfur trioxide complex (17.1 g, 107 mmol) was added to the

mixture at room temperature. The mixture was stirred at room

temperature for 1 h, poured into ice water, basiﬁed with potassium

carbonate, and then extracted with EtOAc. The organic layer was

washed with water and brine, dried over anhydride Na₂SO₄, and

concentrated under reduced pressure. The residue was puriﬁed

by column chromatography on silica gel with hexane/EtOAc as

an elutant to give 38 (2.95 g, 70%). ¹H NMR (300 MHz, CDCl₃) d

2.26 (3H, s), 2.99 (2H, td, J = 6.7, 0.9 Hz), 4.27 (2H, t, J = 6.7 Hz),

7.15–7.25 (2H, m), 7.35–7.44 (2H, m), 9.74 (1H, t, J = 0.9 Hz).

5444

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

diethylamino sulfur triﬂuoride (436 mg, 2.70 mmol) at room tem-

perature, successively. The mixture was stirred at 40 °C for 24 h,

poured into iced-water, and extracted with EtOAc. The organic

layer was washed with saturated NaHCO₃solution and brine, dried

over anhydrous MgSO₄and concentrated under reduced pressure.

The residue was by column chromatography on silica gel with hex-

ane/EtOAc to give 42 (60 mg, 10% in 4 steps). ¹H NMR (300 MHz,

DMSO-d₆) d 2.05 (3H, s), 2.19 (3H, s), 2.33–2.49 (2H, m), 4.13

(2H, t, J = 7.3 Hz), 4.23 (2H, t, J = 13.8 Hz), 7.35–7.46 (2H, m),

7.47–7.56 (2H, m).

substitution of culture media with Opti-MEM, the mixture was

added to the cells. After 3 h incubation, 25 mL D-MEM (low glu-

cose) supplemented with 0.1% BSA and 50 lg/mL gentamicin was

added and then the cells were incubated at 37 °C in 5% CO₂for

1 day. The transfected cells were harvested and re-suspended at

3.3 ꢂ 10⁵cells/mL in D-MEM (low glucose) supplemented with

0.1% BSA and 50 lg/mL gentamicin. Then, in agonist assay, 45 lL

of the cell suspension was transferred in 96-well plate (corn-

ing#3688). After incubation at 37 °C in 5% CO₂for 3 h, 5 L/well

of test compound at various concentrations was added to the cells.

In antagonist assay, 40 L of the cell suspension was transferred in

96-well plate (corning#3688). After incubation, 5 L/well of test

compound at various concentrations and 5 L/well of aldosterone

(ﬁnal concentration: 1 nM) were added to the cells. After 1 day

incubation at 37 °C in 5% CO_2,the medium was removed. 20 L/

l

5.95. 6-[1-(3,3-Diﬂuoro-4-hydroxybutyl)-5-(4-ﬂuorophenyl)-3-

methyl-1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (43)

l

The compound 43 were prepared in a manner similar to that

described for 36b. Yield (34%). ¹H NMR (300 MHz, DMSO-d₆) d

2.19 (3H, s), 2.25–2.48 (2H, m), 3.51 (2H, td, J = 13.7, 6.3 Hz),

4.00–4.14 (2H, m), 4.53 (2H, s), 5.51 (1H, t, J = 6.2 Hz), 6.57 (1H,

dd, J = 8.3, 2.1 Hz), 6.65 (1H, d, J = 2.1 Hz), 6.82 (1H, d, J = 8.3 Hz),

7.22–7.42 (4H, m), 10.60 (1H, s). Anal. Calcd for C₂₂H₂₀N₃O₃F₃: C,

61.25; H, 4.67; N, 9.74. Found: C, 61.40; H, 4.65; N, 9.55.

l

well of two-fold diluted pikkagene (NIPPON GENE CO., Ltd) solu-

tion with HBSS was added to each well and the luciferase activity

was measured. The data were nonlinear ﬁtted to a sigmoidal equa-

tion to calculate the IC₅₀and its 95% conﬁdence interval (CI).

5.98. Metabolic stability assay

5.96. Radioligand Binding Assay

In vitro oxidative metabolic studies of tested compounds were

carried out using hepatic microsomes obtained from rats. The reac-

tion mixture with a ﬁnal volume of 0.1 mL consists of 0.2 mg/mL of

hepatic microsome in 50 mmol/L KH₂PO₄-K₂HPO₄phosphate buf-

Binding displacement assays were carried out in 96-well v-bot-

tom polypropylene plates with a ﬁnal volume of 50.5 lL of TEGM

buffer (10 mM Tris–HCl (pH 7.2), 1 mM EDTA, 10% glycerol,

1 mM DTT, 1 mM 2-mercaptoethanol, 10 mM sodium molybdate,

Protease inhibitor Cocktail (Roche)) containing [³H]-Aldosterone

(ﬁnal concentration, 10 nM), serially diluted test compounds, and

0.75–1.5 mg/mL of cytosolic protein prepared from human MR

transiently transfected FreeStyle™ 293 cells (Invitrogen). Each con-

centration was run in duplicate. The cytosols were incubated for

16 h at 4 °C. Unbound radioactivity was removed by the addition

fer (pH 7.4) and 1 lmol/L test compound. After 5-min preincuba-

tion at 37 °C, the reaction was initiated by addition of an

NADPH-generating system containing 50 mmol/L MgCl₂,

50 mmol/L gulucose-6-phosphate, 5 mmol/L b-NADP+ and

15 unit/mL glucose-6-phosphate dehydrogenase at 10% volume of

reaction mixture. After the addition of NADPH-generating system,

the mixture was incubated at 37ꢀC for 0 and 60 min. The reaction

was terminated by addition of equivalent volume of acetonitrile.

After the samples were mixed and centrifuged, the supernatant

fractions were subjected to high performance liquid chromatogra-

phy equipped with UV detector. Test compound in the reaction

mixture was measured by HPLC system equipped with a UV

detector. For metabolic stability determinations, chromatograms

were analyzed for parent compound disappearance rate from the

reaction mixtures. All incubations were made in duplicate.

of 35 lL of dextran/gelatin coated charcoal suspension (5% char-

coal, 0.5% dextran T-70 (GE Healthcare UK Ltd), 0.1% gelatin

(Sigma–Aldrich Co.), and 10 mM Tris–HCl (pH 7.2), 1 mM EDTA).

The mixture was incubated for 10 min at 4 °C and centrifuged at

910 ꢂ g for 10 min at 4 °C. Then 30

l

L of the supernatant from each

well was transferred to a 96-well white plate with 150

lL of scin-

tillation ﬂuid and the radioactivity was measured by TopCount™

(PerkinElmer Inc.). For the determination of non-speciﬁc binding,

cold aldosterone instead of drug was added to reaction mixture

5.99. Pharmacokinetic analysis in rats

at 100 lM. Speciﬁc binding was determined by subtracting the

value of non-speciﬁc binding component from the total binding

value. The raw data for the speciﬁcally bound counts were normal-

ized between 0% and 100% activity and nonlinear ﬁtted to a sigmoi-

dal equation to calculate the IC₅₀values using PRISM 3.0 (GraphPad

Software Inc.). Other steroid receptor (GR, AR, and PR) binding

assays were carried out by a method similar to MR binding assay

except for ligands. [³H]-Dexamethasone, [³H]-Testosterone or

[³H]-Progesterone was used as a ligand in GR, AR or PR binding

assay, respectively. In the case of PR binding assay, ligand concen-

tration was 5 nM.

Compounds 37a was administered to rats (non-fasted, 8-week

old) intravenously at 1 mg/kg and orally at 3 mg/kg. After intrave-

nous and oral administration, blood samples were collected at des-

ignated time points. The blood samples were centrifuged to obtain

the plasma fraction. The plasma samples were deproteinized with

acetonitrile containing an internal standard. After centrifugation,

the supernatant was diluted with 0.01 mol/L ammonium acetate.

The compound concentrations were measured by a liquid chroma-

tography equipped with tandem mass spectrometry.

5.100. Anti-hypertensive effect and anti-androgenic effect in

DOCA-salt hypertensive rats

5.97. MR agonist/antagonist assay (luciferase reporter gene

assay)

Male 5-week-old Wistar rats were obtained from CLEA Japan,

Inc. (Tokyo, Japan). At 6 weeks of age, rats were anesthetized by

injection of pentobarbiturate (50 mg/kg body wt, i.p.) for subcuta-

neous implantation of a 25 mg pellet of DOCA. After recovery from

anesthesia, rats were housed in standard cages and maintained on

standard chow and ad libitum access to both tap water and a 1%

NaCl drinking solution. After 3 weeks, SBP and HR were measured

by a tail-cuff method (Softron BP-98A, Softron Co.) and develop-

ment of hypertension was conﬁrmed. Hypertensive rats were

COS-1 cells were inoculated at 5 ꢂ 10⁶cells/F150 in D-MEM

(low glucose) supplemented with 10% FBS and 50 mg/mL gentami-

cin and then cultured at 37 °C in 5% CO₂for 1 day. To prepare

DNA:transfection reagent complexes, a solution of 2.5 mL Opti-

MEM, 100

g pMAM-Luc and 1

2.5 mL Opti-MEM and 125

The mixture was maintained at room temperature for 15 min. After

l

L Plus Reagent (Invitrogen), 9

g pRL-TK was mixed with a solution of

L Lipofectamine Reagent (Invitrogen).

lg pMCMYneo-hMR,

5

l

T. Hasui et al. / Bioorg. Med. Chem. 22 (2014) 5428–5445

5445

Table 5

model. The dictionary ﬁle for compound 37a was prepared using

AFITT (OpenEye Scientiﬁc Software, USA). The ﬁnal model was val-

idated using Molprobity.¹⁸All structural ﬁgures were generated

using PyMOL (Schrödinger, USA). Crystallographic processing and

reﬁnement statistics are summarized in Table 5. The coordinates

and structure factors have been deposited in PDB with accession

codes 4PF3.

Data collection and reﬁnement statistics

Crystal

MR-LBD/compound 37a

P2₁2₁2₁

Data collection

Space group

Unit cell dimensions

a, b, c (Å)

Resolution range (Å)

Observed reﬂections

Unique reﬂections

Redundancy

58.1, 66.4, 74.9

50–1.1 (1.14–1.10)

514,047

111,992

4.6 (2.1)

Acknowledgment

Completeness (%)

95.1 (73.6)

12.3 (2.1)

0.052 (0.377)

1

The authors thank Keiji Kubo and Takanobu Kuroita for helpful

discussions.

I/

R_sym

r

a

Molecules in ASU

References and notes

Reﬁnement

Resolution (Å)

Reﬂections

R_work/R_free

Number of atoms

Protein

Ligand/ion

40–1.10 (1.13–1.10)

106,388

0.154/0.177 (0.342/0.326)

1. Fardella, C. E.; Miller, W. L. Annu. Rev. Nutr. 1996 , 16 , 443.

2. Young, M. J.; Clyne, C. D.; Cole, T. J.; Funder, J. W. Clin. Endocrinol. Metab. 2001 ,

86 , 5121.

3. Del Vecchio, L.; Procaccio, M.; Vigano, S.; Cusi, D. Nat. Clin. Pract. Nephrol. 2007 ,

3 , 42.

4. (a) Pitt, B.; Zannad, F.; Remme, W. J.; Cody, R.; Castaigne, A.; Perez, A.; Palensky,

J.; Wittes, J. N. Engl. J. Med. 1999 , 341 , 709; (b) Pitt, B.; Remme, W.; Zannad, F.;

Neaton, J.; Martinez, F.; Roniker, B.; Bittman, R.; Hurley, S.; Kleiman, J.; Gatlin,

M. N. Engl. J. Med. 2003 , 348 , 1309; (c) Zannad, F.; Mcmurray, J. J.; Krum, H.; van

veldhuisen, D. J.; Swedberg, K.; Shi, H.; Vincent, J.; Pocock, S. J.; Pitt, B. N. Engl. J.

Med. 2011 , 364 , 11.

b

2118

62

224

18.2

Water

Average B value (Å²)

Rms deviation from ideal geometry

Bond lengths (Å)

Bond angles (°)

Ramachandran^cplot (%)

Preferred regions

Allowed regions

Outliers

0.009

1.379

5. De Gasparo, M.; Whitebread, S. E.; Preiswerk, G.; Jeunemaitre, X.; Corvol, P.;

Menard, J. Steroid Biochem. 1989 , 32 , 223.

6. Sica, D. A. Heart Fail. Rev. 2005 , 10 , 23.

97.4

2.6

0

7. (a) Meyers, M. J.; Hu, X. Expert Opin. Ther. Pat. 2007 , 17 , 17. and references

therein; (b) Meyers, M. J.; Arhancet, G. B.; Hockerman, S. L.; Chen, X.; Long, S.

A.; Mahoney, M. W.; Rico, J. R.; Garland, D. J.; Blinn, J. R.; Collins, J. T.; Yang, S.;

Huang, H. C.; McGee, K. F.; Wendling, J. M.; Dietz, J. D.; Payne, M. A.; Homer, B.

L.; Heron, M. I.; Reitz, D. B.; Hu, X. J. Med. Chem. 2010 , 53 , 5979; (c) Nariai, T.;

Fujita, K.; Mori, M.; Katayama, S.; Hori, S.; Matsui, K. J. Pharmacol. Sci. 2011 , 115 ,

346; (d) Bärfacker, L.; Kuhl, A.; Hillisch, A.; Grosser, R.; Figueroa-Pérez, S.;

Heckroth, H.; Nitsche, A.; Ergüden, J. K.; Gielen-Haertwig, H.; Schlemmer, K. H.;

Mittendorf, J.; Paulsen, H.; Platzek, J.; Kolkhof, P. Chem. Med. Chem. 2012 , 8 ,

1385; (e) Futatsugi, K.; Pitrowski, D. W.; Casimiro-Garcia, A.; Robinson, S.;

Sammons, M.; Loria, P. M.; Banker, M. E.; Petersen, D. N.; Schmidt, N. J. Bioorg.

Med. Chem. Lett. 2013 , 23 , 6239; (f) Cox, J. M.; Chu, H. D.; Yang, C.; Shen, H. C.;

Wu, Z.; Balsells, J.; Crespo, A.; Brown, P.; Zamlynny, B.; Wiltsie, J.; Clemas, J.;

Gibson, J.; Contino, L.; Lisnock, J.; Zhou, G.; Garcia-Calvo, M.; Bateman, T.; Xu,

L.; Tong, X.; Crook, M.; Sinclair, P. Bioorg. Med. Chem. Lett. 2014 , 24 , 1681; (g)

Casimiro-Garcia, A.; Pitrowski, D. W.; Ambler, C.; Arhancet, G. B.; Banker, M. E.;

Banks, T.; Boustany-Kari, C. M.; Cai, C.; Chen, X.; Eudy, R.; Hepworth, D.;

Hulford, C. A.; Junnings, S. M.; Loria, P. M.; Meyers, M. J.; Petersen, D. N.; Raheja,

N. K.; Sammons, M.; She, L.; Song, K.; Vrieze, D.; Wei, L. J. Med. Chem. 2014 , 57 ,

4273.

8. Hasui, T.; Matsunaga, N.; Ora, T.; Ohyabu, N.; Nishigaki, N.; Imura, Y.; Igata, Y.;

Matsui, H.; Motoyaji, T.; Tanaka, T.; Habuka, N.; Sogabe, S.; Ono, M.; Siedem, C.

S.; Tang, T. P.; Gauthier, C.; De Meese, L. A.; Boyd, S. A.; Fukumoto, S. J. Med.

Chem. 2011 , 54 , 8616.

9. Hasui, T.; Ora, T.; Ohyabu, N.; Matsui, H.; Mizukami, A.; Habuka, N.; Sogabe, S.;

Endo, S.; Siedem, C. S.; Tang, T. P.; Gauthier, C.; De Meese, L. A.; Boyd, S. A.;

Fukumoto, S. Bioorg. Med. Chem. 2013 , 21 , 5983.

10. Partial agonistic activity means relative transcription activity to the activity of

1 nM aldosterone.

11. Nariai, T.; Fujita, K.; Mori, M.; Katayama, S.; Hori, S.; Matsui, K. J. Cardiovasc.

Pharmacol. 2012 , 59 , 458.

PDB code

4PF3

Values in parentheses are for the highest resolution shell.

a

R_sym

=

R_hR_i|I(h)_iꢁ hI(h)i|/R_hR_ihI(h)i, where hI(h)i is the mean intensity of sym-

metry-related reﬂections.

b

R_work

=

R

||F_obs|_iꢁ |F_calc||/

R

|F_obs|. R_freewas calculated for randomly chosen 5% of

reﬂections excluded from reﬁnement.

c

Calculated with Coot.

divided into groups with equalization of body weight, SBP and HR.

The groups of rats were treated with vehicle (0.5 w/v % methylcel-

lulose, n = 8), compound 37a (n = 8) and spironolactone (n = 8) at a

dose of 100 mg/kg. Drugs were administrated orally once a day.

After 7 days and 14 days of treatment, SBP and HR were measured

approximately 24 h after the last dosing. After 18 days of treat-

ment, rats were sacriﬁced under anesthesia and seminal vesicle

weight was measured by autopsy after the measurement of body

weight approximately 24 h after the last administration. Data are

shown as mean SEM. The care and use of the animals and the

experimental protocols used in the studies were reviewed and

approved by the Experimental Animal Care and Use Committee

of Takeda Pharmaceutical Company (Osaka, Japan).

5.101. Crystallization and structure determination

12. Rubottom, G. M.; Gruber, J. M.; Kincaid, K. Synth. Commun. 1976 , 6 , 59.

13. The log D values were measured at pH 7.4 with the relative retention time over

the standard compounds of HPLC analysis.^19,20

The human MR-LBD triple mutant, 712–984 (C808S/S810L/

A976V), with compound 37a was prepared as described previ-

ously.⁸Crystals of the MR-LBD/compound 37a complex were

obtained at 20 °C by sitting-drop vapor diffusion method with

the reservoir solution contained 0.1 M Tris pH 8.0, 23% ethanol.

The crystals were immersed in the reservoir solution containing

25% ethylene glycol and ﬂash-frozen with liquid nitrogen. Diffrac-

tion data were collected at the SPring-8 beamline BL41XU and pro-

cessed using d*trek.¹⁵The structure was reﬁned with Refmac¹⁶and

Coot¹⁷using the MR-LBD structure (PDB code: 3WFG) as a start

14. Waring, M. J. Expert Opin. Drug Disc. 2010 , 5 , 235.

15. Pﬂugrath, J. W. Acta Crystallogr. 1999 , D55 , 1718.

16. Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Crystallogr. 1997 , D53 , 240.

17. Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Acta Crystallogr. 2010 , D66 ,

486.

18. Chen, V. B.; Arendall, W. B., 3rd; Headd, J. J.; Keedy, D. A.; Immormino, R. M.;

Kapral, G. J.; Murray, L. W.; Richardson, J. S.; Richardson, D. C. Acta Crystallogr.

2010 , D66 , 12.

19. Nakashima, S.; Yamamoto, K.; Arai, Y.; Ikeda, Y. Chem. Pharm. Bull. 2013 , 12 ,

1228.

20. Masumoto, K.; Takeyasu, A.; Oizumi, K.; Kobayashi, T. Yakugaku Zasshi 1995 ,

115 , 213.

Article Doi

Discovery of 6-[5-(4-fluorophenyl)-3-methyl-pyrazol-4-yl]-benzoxazin-3-one derivatives as novel selective nonsteroidal mineralocorticoid receptor antagonists

DOI: 10.1016/j.bmc.2014.07.038

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2014.07.038

Full text of DOI:10.1016/j.bmc.2014.07.038

Products guided by the article

R&D Labs maybe for 1221503-40-7

Relevant to this article

Hot Product