Identification of benzoxazin-3-one derivatives as novel, potent, and selective nonsteroidal mineralocorticoid receptor antagonists

Article

pubs.acs.org/jmc

Identification of Benzoxazin-3-one Derivatives as Novel, Potent, and

Selective Nonsteroidal Mineralocorticoid Receptor Antagonists^†

Tomoaki Hasui,*^,‡Nobuyuki Matsunaga,^‡Taiichi Ora,^‡Norio Ohyabu,^‡Nobuhiro Nishigaki,^‡

Yoshimi Imura,^‡Yumiko Igata,^‡Hideki Matsui,^‡Takashi Motoyaji,^‡Toshimasa Tanaka,^‡

Noriyuki Habuka,^‡Satoshi Sogabe,^‡Midori Ono,^‡Christopher S. Siedem,^§Tony P. Tang,^§

Cassandra Gauthier,^§Lisa A. De Meese,^§Steven A. Boyd,^§and Shoji Fukumoto^‡

^‡Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd., 26-1, Muraoka-higashi 2-chome, Fujisawa,

Kanagawa 251-8555, Japan

^§Array BioPharma Inc., 3200 Walnut Street, Boulder, Colorado 80301, United States

S

* Supporting Information

ABSTRACT: Mineralocorticoid receptor (MR) blockade has

come into focus as a promising approach for the treatment of

cardiovascular diseases such as hypertension and congestive heart

failure. In order to identify a novel class of nonsteroidal MR

antagonists that exhibit significant potency and good selectivity

over other steroidal hormone receptors, we designed a novel

series of benzoxazin-3-one derivatives and synthesized them from

6-(7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-6-yl)-2H-1,4-ben-

zoxazin-3(4H)-one (1a), high-throughput screening (HTS) hit

compound. Our design was based on a crystal structure of an

MR/compound complex and a docking model. In the course of

lead generation from 1a, a 1,2-diaryl framework was characterized

as a key structure with high binding affinity. On the basis of scaffold hopping and optimization studies, benzoxazin-3-one

derivatives possessing 1-phenyl-3-trifluoromethylpyrazol-5-yl moiety at the 6-position were identified as a novel series of potent

and selective MR antagonists. Among these compounds, 6-[1-(4-fluoro-2-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-

2H-1,4-benzoxazin-3(4H)-one (14n) showed highly potent activity and good selectivity and also exhibited a significant

antihypertensive effect in deoxycorticosterone acetate−salt hypertensive rats. On the basis of these results, compound 14n was

progressed for further pharmacological evaluation.

have proved the utility of MR antagonists in heart failure

therapy.^8,9Furthermore, a recent study revealed that MR

antagonists have a therapeutic potential for diabetic

nephropathy.¹⁰

INTRODUCTION

■

Mineralocorticoid receptor (MR) is a member of the super-

family of nuclear hormone receptors (NHRs), which have struc-

tures that share high homology with those of other steroid

hormone receptors, exemplified by androgen receptor (AR),

progesterone receptor (PR), and glucocorticoid receptor (GR).

Aldosterone, a primary natural ligand for MR, regulates body

fluid and electrolyte balance through binding to MR in kidney

and plays an important role in blood pressure control.¹Recently,

it has been reported that aldosterone is also synthesized in heart

and blood vessels, and an elevated level of aldosterone is closely

associated with the development of congestive heart failure and

renal dysfunction in addition to hypertension.^2,3Thus, aldoste-

rone blocking has attracted great attention as a promising ther-

apeutic option for such diseases.

However, spironolactone treatment has limitations like

sexual adverse effects, such as impotence, gynecomastia, and

menstrual irregularity, caused by nonselective binding to AR

and/or PR.¹¹Meanwhile, eplerenone was a more selective MR

antagonist and had few sexual adverse effects but its MR

antagonistic activity and antihypertensive effect were less

potent than those of spironolactone.¹²It is expected that

an MR antagonist with sufficient potency and selectivity

to prevent sexual adverse effects would be a clinically con-

venient and valuable agent. Although several nonsteroidal

MR antagonists have been reported,¹³their clinical utilities

have not been confirmed. Thus, the goal of our project was

to discover a novel series of potent and selective nonsteroidal

MR antagonists.

Indeed, MR antagonists, such as spironolactone⁴and eplere-

none,⁵have shown antihypertensive effects in patients with

essential hypertension.⁶It was noticeable that the addition of

MR antagonists to standard antihypertensive therapy effectively

reduced blood pressure in refractory hypertension patients.⁷In

addition, the large scale clinical trials (RALES and EPHESUS)

Received: September 2, 2011

Published: November 10, 2011

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We performed competitive radiometric binding high-throughput

screening (HTS) assay and identified benzoxazin-3-one 1a as

an MR antagonist (Chart 1, IC₅₀= 2000 nM in binding assay)

CHEMISTRY

■

Triazolothiadiazines 1a−h were prepared according to Scheme 1.

Acetophenone 3a was commercially available, and the ketones

3b−f were obtained by Friedel−Crafts acylation of 2H-1,

4-benzoxazin-3(4H)-one 2. Condensation of acetophenone 3a

with benzaldehyde and subsequent hydrogenation afforded

phenethyl ketone 3g. Benzyl ketone 3c was converted to the 2-

methyl derivative 3h in two steps by a manner similar to that

described for 3g. Ketones 3a−h were brominated and

subsequently condensed with 4-amino-3-mercapto-4H-1,2,4-

triazole 5 to give triazolothiadiazines 1a−h in good yield.

Compounds 7, 8, 9, and 10 were prepared by condensation

of bromoketone 4c with 1-amino-2-mercaptoimidazole 6, 2-

aminothiophenol, thioacetamide, and acetamidine, respectively

(Scheme 2). Imidazole 6 was prepared in two steps from

commercially available thiadiazole 11.

Chart 1

The synthesis of 3-substituted pyrazoles 14a−o and 16 is

described in Scheme 3. The acetophenone 3a was converted to

the diketones 13a−f in good yields. The acid-mediated

condensation of diketones 13a−f with arylhydrazines afforded

the 3-substituted pyrazoles 14a−o. The 3-cyanopyrazole 16

was obtained in three steps from ester 14o via dehydration of

the carboxamide group of compound 15. The ethyl ester of

compound 14o was reduced by lithium aluminum hydride

to afford 3-hydroxymethylpyrazole 17. Hydrazine 18 was pre-

pared by the reduction of the diazonium salt of 4-fluoro-2-

methylaniline.

The 4-trifluoromethylpyrazole 21 was prepared as shown

in Scheme 4. The acetophenone 3a was reacted with N,N-

dimethylformamide dimethyl acetal (DMFDMA) and sub-

sequently condensed with 4-fluorophenylhydrazine to give 3,

4-unsubstituted pyrazole 19. Iodination at the 4-position by

N-iodosuccinimide and subsequent copper-catalyzed trifluoro-

methylation afforded the 4-trifluoromethylpyrazole 21.

with suitable properties for a hit compound such as low

molecular weight and low lipophilicity. In this paper, we

report the lead generation from 1a and the subsequent opti-

mization by utilizing an X-ray crystal structure, which led to the

identification of 6-[1-(4-fluoro-2-methylphenyl)-3-(trifluoro-

methyl)-1H-pyrazol-5-yl]-2H-1,4-benzoxazin-3(4H)-one (14n)

as a novel nonsteroidal MR antagonist. The compound 14n

showed highly potent in vitro activity, good selectivity over

the other steroid hormone receptors, and significant antihyper-

tensive effect in deoxycorticosterone acetate (DOCA)−salt hyper-

tensive rats and was therefore selected for further pharmacological

evaluation.

a

Scheme 1. Synthesis of Triazolothiadiazines 1a−h

a

Reagents and conditions: (a) RCH₂COCl, AlCl₃, ClCH₂CH₂Cl, 25−88%; (b) PhCHO, NaOMe, MeOH, 60 °C, 87%; (c) H₂, Pd/C, EtOH, 25%;

(d) Me₂NCH₂NMe₂, Ac₂O, CH₂Cl₂, 56%; (e) H₂, Pd/C, THF, 66%; (f) HBr₃·Py, HBr, AcOH, 85−99%; (g) 4-amino-3-mercapto-4H-1,2,4-triazole

5, EtOH/toluene, reflux, 24−97%.

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a

Scheme 2. Synthesis of Imidazothiadiazine 7, Benzothiazine 8, Thiazole 9, and Imidazole 10

a

Reagents and reaction conditions: (a) 6, EtOH, reflux, 17%; (b) 2-aminothiophenol, EtOH, reflux, 19%; (c) thioacetamide, NaOEt, EtOH, reflux,

23%; (d) acetamidine, K₂CO₃, EtOH, reflux, 14%; (e) 45% chloroacetaldehyde, EtOH/toluene, reflux, 23%; (f) hydrazine monohydrate, EtOH,

reflux, 32%.

a

Scheme 3. Synthesis of 3-Substituted Pyrazoles 14a−o, 16, and 17

a

Reagents and conditions: (a) 60% NaH, 2,4-dibenzo-18-crown-6, R¹CO₂Et, THF, reflux, 43−95%; (b) ArNH₂NH₂, 2-propanol, TFA, reflux,

12−86%; (c) 1 N NaOH, MeOH, 75%; (d) 2 M NH₃in MeOH, EDCI, HOBt, DMF, 76%; (e) TFAA, pyridine, dioxane, 38%; (f) LiAlH₄, THF,

34% (g) (1) NaNO₂, HCl; (2) SnCl₂, 48% in two steps.

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Scheme 4. Synthesis of 4-Trifluoromethylpyrazole 21

a

Reagents and conditions: (a) DMFDMA, 80 °C, 73%; (b) 4-fluorophenylhydrazine, EtOH, 40 °C, 44%; (c) NIS, DMF, 55 °C, 50%;

(d) FSO₂CF₂CO₂Et, CuI, DMF, 100 °C, 5%.

a

Scheme 5. Synthesis of 8-Substituted Benzoxazin-3-one Derivatives 29a−c

a

Reagents and conditions: (a) BrCH₂CO₂Et, K₂CO₃, DMF, 65 °C; (b) Zn, AcOH, 100 °C, 34−89% in two steps; (c) 1) 4-(vinyloxy)butan-1-ol,

PdCl₂P(o-tolyl)₃, DMF/H₂O, 80 °C; (2) 2 N HCl, reflux, 72% in two steps; (d) HNO₃, AcOH, room temperature, 59%; (e) NaH, 2,4-dibenzo-18-

crown-6, CF₃CO₂Et, THF, 60 °C; (f) 1-(4-fluoro-2-methylphenyl)hydrazine hydrochloride 18, TFA, 2-propanol, reflux, 16−30% in two steps.

The synthesis of 8-substituted benzoxazin-3-one derivatives

29a−c is described in Scheme 5. Alkylation of nitrophenol 22

and subsequent Zn-mediated reductive cyclization afforded the

benzoxazin-3-one 23. Heck reaction of compound 23 and

subsequent acid hydrolysis gave 8-halobenzoxazin-3-ones 27a,b.

The acetophenone 24 was converted to the nitro derivative 25

by nitric acid. 8-Methylbenzoxazin-3-one 27c was prepared

from 25 by a manner similar to that described for benzoxazin-3-

one 23. The acetophenones 27a−c were converted to

diketones 28a−c and subsequent acid-mediated pyrazole-ring

formation afforded 8-substituted benzoxazin-3-ones 29a−c in

good yield.

on the X-ray cocrystal structure of MR with spironolactone

obtained in-house, is illustrated in Figure 1a. This model

suggested that compound 1a binds to the steroid binding site

of MR similarly to spilonolactone and interacts via hydrogen

bonds with Asn770 and Gln776. Notably, compound 1a did

not fill a hydrophobic pocket that was occupied by the thioester

group of spironolactone. This finding led us to hypothesize that

filling this pocket with a lipophilic substituent would increase

the binding affinity by hydrophobic interaction. Therefore,

several lipophilic substituents were introduced at the 2-position

of the triazolothiadiazine ring (Table 1). Introduction of a

propyl (1b) or a benzyl group (1c) had little influence on

potency. In contrast, the phenyl compound 1d, containing 1,2-

RESULTS AND DISCUSSION

diaryl framework, showed a 4-fold increase in potency (IC₅₀

=

■

510 nM). Moreover, compound 1d showed improved

selectivity against PR. Introduction of an additional methyl

group at the 2-position of compound 1d led to a large decrease

in activity (1e).

Figure 1b shows an X-ray crystal structure of 1d with MR. To

our knowledge, this is the first example of an X-ray structure of

a nonsteroidal compound bound to MR. This crystal structure

revealed the binding mode of compound 1d, in which

interactions between benzoxazin-3-one moiety and Asn770

and among nitrogen atoms of the triazole, Gln776, and Arg817

were observed, and the phenyl group at the 2-position of the

The synthesized compounds were evaluated for their inhibitory

activity against the binding of [³H]aldosterone to MR. Further,

the selectivity of these compounds over other steroid hormone

receptors was determined by radioligand binding assays (AR,

[³H]testosterone; PR, [³H]progesterone; GR, [³H]-

dexamethasone). The results are presented in Tables 1−4 as

IC₅₀values.

Lead Generation. The issues of HTS hit compound 1a

that were initially addressed were potency (IC₅₀= 2000 nM)

and selectivity over PR (IC₅₀= 1600 nM in PR binding assay,

Table 1). A docking model of 1a with MR, constructed based

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a

Table 1. Effects of Introduction of Substituents at the 2-Position of Triazolothiadiazine Ring

selectivity, IC₅₀(nM)

PR binding

MR binding

compd

R¹

R²

H

IC₅₀(nM)

AR binding

>10000

GR binding

>10000

1a

H

2000

1600

(1500−2800)

4100

(1100−2300)

nt

b

1b

1c

1d

n-Pr

Bn

H

nt

(3300−5100)

1300

b

nt

(990−1800)

510

Ph

3600

5600

(340−770)

>10000

(2800−4700)

nt

>10000

(3100−10000)

nt

b

1e

1f

Ph

Me

H

nt

2-F-Ph

600

5900

5500

(500−710)

310

(3300−10000)

4700

>10000

(3100−9600)

>10000

1g

1h

3-F-Ph

4-F-Ph

H

(210−470)

270

(2700−8400)

>10000

(190−400)

a

IC₅₀values are shown as the mean of duplicate experiments. IC₅₀values and 95% confidence limits are calculated from the concentration−response

b

curves generated by GraphPad Prism. Not tested.

a

Table 2. SAR Summary for Central Ring Moiety

a

IC₅₀values are shown as the mean of duplicate experiments. IC₅₀values and 95% confidence limits are calculated from the concentration−response

b

curves generated by GraphPad Prism. Not tested.

triazolothiadiazine ring filled the hydrophobic pocket as

designed. The structure also showed a small space around

the introduced phenyl ring. Therefore, a fluorine atom was

introduced at the ortho-, meta-, or para-position of the benzene

ring with the expectation of an increase in binding affinity.

Among these compounds, the 3-fluoro and 4-fluorophenyl

hormone receptors were improved compared to that of

compound 1d. In particular, the 4-fluorophenyl compound

1h showed significantly improved selectivity (vs AR, PR, and

GR > 35-fold). On the basis of this result, we rationalized that

the corresponding hydrophobic pockets of AR, PR, and GR are

smaller than that of MR.

compounds showed increased binding affinities (1g, IC₅₀

310 nM; 1h, IC₅₀= 270 nM). Interestingly, the selectivities of

all these fluorinated compounds over the other steroid

=

Although compound 1h exhibited good potency and

selectivity, 1h and related analogues showed poor PK profiles

in rats (data not shown), probably because of the metabolically

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hydrogen-bonding interaction with Asn770 and the hydro-

phobic interaction with the phenyl group were more important

for potent binding affinity in this benzoxazin-3-one series. The

reason for the increase in binding affinity of these compounds

could be explained by the increase in lipophilicity of the central

ring. Further, these results suggested that more drastic

modification of the central ring moiety could be tolerated

and encouraged us to explore a new “leadlike” scaffold such as a

five-membered heteroaromatic ring. Replacement of the bicyclic

cores by a thiazole or an imidazole ring led to a significant

decrease in activity (9, 10). In contrast, a pyrazole derivative

14a showed moderate binding affinity. In anticipation of

increasing the binding affinity of 14a, the fluorine atom was

introduced at the para-position of the benzene ring. As

expected, the fluorinated compound 14b showed a 5-fold

improvement in the binding affinity over 14a and importantly

exhibited good selectivity over the other steroid hormone

receptors. While compound 14b was still metabolically unstable

(metabolic clearance in rat microsomes of 164 μL min⁻¹mg⁻¹),

it was presumed that this metabolic instability could be

improved by modifying the methyl group, which was the

putative metabolic site of 14b. On the basis of the advantageous

features such as good selectivity, lower molecular weight,

elimination of the chiral center, and the potential for improved

metabolic clearance, the pyrazole ring was selected as a lead

scaffold for further modification. Thus, the compound 14b was

advanced into lead optimization.

Table 3. SAR Summary for Substituent on Pyrazole Ring

compd

R¹

R²

MR binding IC₅₀(nM)

14c

14d

14e

14f

16

Et

H

960 (670−1400)

260 (220−310)

570 (480−670)

>10000

CF₃

H

CF₂CF₃

i-Pr

H

CN

H

920 (750−1100)

6800 (4000−12000)

2700 (1900−4000)

17

CH₂OH

H

21

CF₃

a

IC₅₀values are shown as the means of duplicate experiments. IC₅₀

values and 95% confidence limits are calculated from the

concentration−response curves generated by GraphPad Prism.

Table 4. SAR Summary for Benzene Ring (A-Ring) at 1-

Position of Pyrazole Ring and Benzene Ring (B-Ring) in the

a

Benzoxazin-3-one Moiety

Lead Optimization. Initially, our effort was focused on

changing the methyl group on the pyrazole ring to increase

the binding affinity and improve the metabolic clearance of

compound 14b (Table 3). The docking model of compound 14b

with MR, shown in Figure 2, suggested that the 3-methylpyrazole

moiety did not fully occupy the space filled by the

triazolothiadiazine ring of compound 1d in the MR binding site.

Accordingly, larger substituents were introduced at the pyrazole 3-

position. Compounds 14c−e, bearing an ethyl, trifluoromethyl, or

pentafluoroethyl group, showed increased potency as predicted. In

particular, 3-trifluoromethylpyrazole 14d showed a 4-fold

improvement in activity over 14b (IC₅₀= 260 nM vs 1100

nM). Furthermore, compound 14d showed improved PK profile

in rats (BA = 28%), likely due to improved metabolic clearance

(metabolic clearance in rat microsomes: 14b, 164 μL min⁻¹mg⁻¹;

14d, 24 μL min⁻¹mg⁻¹). In contrast, isopropyl analogue 14f

showed significantly decreased potency, suggesting that the

branched substituents were too large for this position. The MR

binding model of 3-methylpyrazole 14b further showed that the

3-position substituent of the pyrazole ring was oriented toward

Gln776 and Arg817. Hydrogen bond accepting or donating

groups were introduced with a hope to establish an interac-

tion with these residues. However, replacement of the methyl

group with a cyano or a hydroxymethyl group did not result in

a significant improvement in activity (16, IC₅₀= 920 nM; 17,

IC₅₀= 6800 nM). Transposition of the trifluoromethyl group

from the 3- to the 4-position also led to decreased bind-

ing activity (21, IC₅₀= 2700 nM). Consequently, the 3-tri-

fluoromethylpyrazole was optimal among the substituents

explored here.

compd

X

Y

MR binding IC₅₀(nM)

14g

14h

14i

2-F

3-F

H

440 (350−560)

1100 (880−1300)

430 (350−530)

320 (240−430)

80 (65−100)

590 (400−860)

700 (500−990)

41 (32−53)

H

F

2-Cl

14j

4-Cl

14k

14l

2-Me

4-Me

14m

14n

29a

29b

29c

2-Et

2-Me, 4-F

76 (64−90)

12 (9.4−15)

21 (19−24)

Cl

Me

a

IC₅₀values are shown as the mean of duplicate experiments. IC₅₀

values and 95% confidence limits are calculated from the

concentration−response curves generated by GraphPad Prism.

labile thioether and cyclic imine of triazolothiadiazine ring (1h,

the metabolic clearance in rat microsomes of 60 μL min⁻¹mg⁻¹).

Therefore, we concentrated our efforts to investigate alternative

scaffolds without metabolically labile groups. Table 2 shows

structure−activity relationships (SAR) of the central ring moiety.

Replacement of triazolothiadiazine ring with an imidazothiadi-

azine (7) or a benzothiazine ring (8) was investigated for an

initial SAR, while these compounds still possessed the metabolic

labile groups. Surprisingly, these compounds exhibited

significant binding affinities despite the decrease or lack of

hydrogen-bonding interactions with Gln776 and Arg817, which

we had assumed to be a key interaction for binding to MR

(7, IC₅₀= 36 nM; 8, IC₅₀= 69 nM). These results indicated

that the interaction with Gln776 and Arg817 did not

significantly contribute to binding activity of 1h and that the

Optimization of the substituents on the benzene rings (A and B)

at the 1-position of the pyrazole ring and in the benzoxazin-

3-one moiety was performed within the 3-trifluoromethylpyrazole

series, and the results are shown in Table 4. Among the three

fluorophenyl derivatives (14d, 14g, and 14h), the 4-fluoro

compound 14d showed the most potent binding affinity. The

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Figure 1. (a) Overlay of a compound 1a (yellow) and spironolactone (green) in MR. Compound 1a was docked to MR in the X-ray crystal structure

of MR/spironolactone obtained in-house. (b) X-ray crystal structure of MR/compound 1d complex. The hydrogen bonds are shown as orange or

yellow dotted lines.

potent binding activity (IC₅₀= 41 nM). Compound 14n was

docked to MR, and its binding mode was similar to that of 14b.¹⁴

These docking models showed a small pocket around the 8-

position of the B-ring. In the 8-position of 14n, substitution with a

fluorine atom, chlorine atom, or methyl group was performed in

anticipation of further enhancement of potency. While the

installation of a fluorine atom had little influence on the binding

activity (29a, IC₅₀= 76 nM), introduction of a chlorine atom or a

methyl group led to about 2- or 3-fold more potent activity than

14n (29b, IC₅₀= 12 nM; 29c, IC₅₀= 21 nM).

The five compounds with potent binding activities (14k,

14n, 29a, 29b, and 29c) were evaluated for their selectivity over

the other steroid hormone receptors and MR antagonistic

activity in a reporter gene assay in COS-1 cells (Table 5). The

2-methyl compound 14k showed an approximately 3-fold loss

in functional activity relative to spironolactone. In contrast, the

4-fluoro-2-methyl compound 14n showed equipotent MR

antagonistic activity (IC₅₀= 43 nM) to spironolactone, and it

Figure 2. Overlay of the compound 14b (yellow) and compound 1d

also showed good selectivity over the other steroidal hormone

(green) in MR. Compound 14b was docked to the X-ray crystal

receptors (vs AR, PR, and GR > 40-fold). It was noteworthy

structure of MR/compound 1d complex. The hydrogen bonds are

that the selectivity of 14n over AR and PR was greatly

shown as orange or yellow dotted lines.

improved compared to those of spironolactone (14n, for AR >

240-fold, for PR = 45-fold; spironolactone, for AR = 3-fold, for

PR = 15-fold). An in vivo safety evaluation will be conducted

for an estimation of the sexual side effects in clinical use.

Compound 29a also showed good functional activity and

selectivity, but it was inferior to 14n. Although compounds 29b

and 29c showed potent functional activity, their selectivity over

GR was less than that of spironolactone. Compound 14n

showed the best balance of potency and selectivity and in

incorporation of a chlorine atom gave similar results as that of the

fluorine atom. The 4-chloro compound 14j showed more potent

activity than the 2-chloro compound 14i. Surprisingly, installation

of a methyl group at the 2-position resulted in a marked increase

in binding affinity and its activity was 5-fold more potent (14k,

IC₅₀= 80 nM) than the 2-fluoro and the 2-chloro compounds

(14g and 14i). This effect of high potency unique to the 2-methyl

group could be explained by a hydrophobic interaction with

addition showed a superior PK profile in rats (Table 6). On the

basis of these results, compound 14n was evaluated for MR

Leu769, Leu814, and Met852 around the 2-position of the A-ring

and/or a stabilization effect of preferred twisted conformation

antagonist activity in vivo.

between the pyrazole ring and A ring. In contrast, installation of a

First, the potassium-sparing and natriuretic activity of com-

methyl group at the 4-position (14l) led to a 3-fold decrease in

potency relative to the 4-fluoro and 4-chloro compounds (14d

and 14j). Replacement of the 2-methyl group with an ethyl group

decreased the activity (14m, IC₅₀= 700 nM). A synergistic effect

pound 14n was evaluated in Wistar rats (Figure 3A). Compound

14n (10, 30, or 100 mg/kg) and spironolactone (30 mg/kg)

were orally administered 30 min before the aldosterone injection

(3 μg/kg, sc) and oral saline load (25 mL/kg). Urine (5 h after

saline load) was collected, and urinary Na⁺and K⁺concentrations

were measured. Compound 14n increased the urinary Na⁺/K⁺

ratio in a dose-dependent manner, and the effect at doses of

was observed when combining the optimal 2-methyl (14k, IC₅₀

=

80 nM) and the 4-fluoro (14d, IC₅₀= 260 nM) substituents,

resulting in 2-methyl-4-fluoro compound 14n with significantly

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a

Table 5. Steroid Hormone Receptor Selectivities and MR Antagonistic Activities of Selected Compounds

selectivity, IC₅₀(nM)

b

compd

X

Y

MR binding

80

AR binding

>10000

PR binding

GR binding)

MR antagonistic activity IC₅₀(nM)

14k

14n

29a

29b

29c

2-Me

H

4900

2500

150

(65−100)

41

(3000−8000)

1900

(1600−3700)

1800

(80−280)

43

2-Me, 4-F

H

F

>10000

(32−53)

76

(1400−2700)

1800

(1300−2200)

1700

(24−79)

85

(64−90)

12

(1200−2500)

930

(1200−2400)

200

(42−170)

70

2-Me, 4-F Cl

2-Me, 4-F Me

4500

(9.5−15)

21

(3000−8000)

>10000

(730−1200)

2400

(150−260)

470

(45−110)

15

(19−24)

49

(1600−3700)

650

(370−590)

1400

(8.2−26)

60

spironolactone

eplerenone

120

(36−66)

2600

(100−140)

>10000

(460−920)

>10000

(880−2100)

>10000

(32−110)

1300

(1900−3400)

(700−2400)

a

IC₅₀values are shown as the mean of duplicate or triplicate experiments. IC₅₀values and 95% confidence limits are calculated from the

concentration−response curves generated by GraphPad Prism. Inhibitory activity of transcription of aldosterone (reporter gene assay).

b

a

Table 6. Pharmacokinetic Profiles of Compound 14n in SD Rats

CL (mL h⁻¹kg⁻¹

)

Vd_ss(mL/kg)

AUC_0−24h,po(μg·h/mL)

3.829 ± 1.172

MRT_po(h)

BA (%)

1328 ± 88

4626 ± 315

4.39 ± 0.12

50.7 ± 15.9

a

Rats were administered the drug intravenously at 3 mg/kg and orally at 10 mg/kg (n = 3).

Figure 3. Potassium-sparing and natriuretic (A) and antihypertensive (B) effects of compound 14n in rats. (A) Wistar rats were administered

vehicle, compound 14n at 10, 30, or 100 mg/kg, or spironolactone at 30 mg/kg 30 min before aldosterone injection (3 μg/kg, sc) and oral saline

load (25 mL/kg). After the saline load, 5 h urine was collected and urinary Na⁺and K⁺were measured: mean ± SEM (n = 5); ∗, p < 0.025 vs vehicle

by one-tailed Williams’ test; †, p < 0.05 vs vehicle by Aspin−Welch test. (B) DOCA−salt hypertensive rats were treated with vehicle, compound 14n,

or spironolactone at a dose of 100 mg/kg for 13 days. SBP and HR were measured by tail-cuff method approximately 24 h after the last

administration. Pretreatment values of SBP in each group were 193−194 mmHg: mean ± SEM (n = 7−8); ‡, p < 0.05 vs vehicle by Student’s t test.

30 and 100 mg/kg was significant compared to vehicle treat-

ment. Similarly, spironolactone (30 mg/kg) increased the urinary

Na⁺/K⁺ratio significantly. The effect of both compounds at

30 mg/kg was comparable. Second, the antihypertensive effect of

14n was evaluated in established DOCA−salt hypertensive rats

(Figure 3B). Compound 14n (100 mg/kg, q.d.) and spironol-

actone (100 mg/kg, q.d.) were administered orally for 13 days, and

systolic blood pressure (SBP) and heart rate (HR) were measured

by tail-cuff method approximately 24 h after the last administration.

Both compounds significantly lowered SBP at a dose of 100 mg/kg,

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mixture was stirred at 80 °C for 3 h, poured into ice-cooled water, and

extracted with dichloromethane. The organic layer was washed with

saturated NaHCO₃solution, dried over anhydrous Na₂SO₄, and

concentrated under reduced pressure. The residue was crystallized

from MeOH to give 3b (12.0 g, 77%) as white crystals. ¹H NMR (300

MHz, DMSO-d₆) δ 0.89 (3H, t, J = 7.4 Hz), 1.24−1.40 (2H m), 1.50−

1.64 (2H, m), 2.91 (2H, t, J = 7.2 Hz), 4.68 (2H, s), 7.03 (1H, d, J =

8.4 Hz), 7.48 (1H, d, J = 2.0 Hz), 7.61 (1H, dd, J = 8.4, 2.0 Hz), 10.85

(1H, brs).

and their antihypertensive effects were similar. In addition,

these compounds did not affect the HR (data not shown).

These results indicated that compound 14n exhibited MR

antagonistic activity and blood pressure-lowering effect in vivo,

comparable to that of spironolactone.

CONCLUSION

■

We have discovered benzoxazin-3-one derivatives as a novel

class of potent and selective nonsteroidal MR antagonists and

described the lead generation and optimization from hit

compound 1a. Installation of a phenyl group at the 2-position

in the triazolothiadiazine ring revealed that a 1,2-diaryl

framework was a key structure for potent activity in this series.

Subsequent scaffold hopping led to a central pyrazole ring in

the lead compound 14b showing selective MR binding activity.

Optimization of the substituents of 14b resulted in the

identification of 6-[1-(4-fluoro-2-methylphenyl)-3-(trifluoro-

methyl)-1H-pyrazol-5-yl]-2H-1,4-benzoxazin-3(4H)-one

(14n). Compound 14n showed excellent in vitro potency and

good selectivity, and it also exhibited significant blood-pressure-

lowering effect in DOCA−salt hypertensive rats. On the basis

of these results, compound 14n was progressed for further

pharmacological evaluation. Additional results of continued

investigation of this benzoxazin-3-one series will be reported in

due course.

Compound 3c−f were prepared in a manner similar to that

described for 3b.

6-(Phenylacetyl)-2H-1,4-benzoxazin-3(4H)-one (3c). Yield,

1

46%. H NMR (300 MHz, CDCl₃) δ 4.22 (2H, s), 4.69 (2H, s),

7.00 (1H, d, J = 8.4 Hz), 7.22−7.36 (5H, m), 7.48−7.49 (1H, d, J =

2.1 Hz), 7.68 (1H, dd, J = 8.4, 2.1 Hz), 8.10 (1H, brs).

6-[(2-Fluorophenyl)acetyl]-2H-1,4-benzoxazin-3(4H)-one

1

(3d). Yield, 88%. H NMR (300 MHz, CDCl₃) δ 4.26 (2H, s), 4.70

(2H, s), 7.02−7.29 (5H, m), 7.51 (1H, d, J = 1.8 Hz), 7.70 (1H, dd,

J = 1.8, 8.4 Hz), 8.15−8.30 (1H, brs).

6-[(3-Fluorophenyl)acetyl]-2H-1,4-benzoxazin-3(4H)-one

1

(3e). Yield, 25%. H NMR (300 MHz, DMSO-d₆) δ 4.35 (2H, s),

4.69 (2H, s), 7.01−7.15 (4H, m), 7.30−7.40 (1H, m), 7.52 (1H, d, J =

2.0 Hz), 7.73 (1H, dd, J = 2.0, 8.5 Hz), 10.89 (1H, brs).

6-[(4-Fluorophenyl)acetyl]-2H-1,4-benzoxazin-3(4H)-one

(3f). Yield, 36%. ¹H NMR (300 MHz, DMSO-d₆) δ 4.31 (2H, s), 4.69

(2H, s), 7.02−7.20 (3H, m), 7.23−7.33 (2H, m), 7.52 (1H, d, J = 1.9

Hz), 7.73 (1H, dd, J = 1.9, 8.3 Hz), 10.88 (1H, brs).

6-(3-Phenylpropanoyl)-2H-1,4-benzoxazin-3(4H)-one (3g). To

a stirred mixture of 3a (4 g, 20.9 mmol) and benzaldehyde (2.7 g,

25.5 mmol) in MeOH (40 mL) was added 28% sodium methoxide in

MeOH (4.4 g, 22.8 mmol) at room temperature. The mixture was

stirred at 50 °C for 24 h and concentrated under reduced pressure.

The residue was treated with water and 10% hydrochloric acid. The

precipitate was collected by filtration and washed with MeOH (40 mL)

to give 6-(3-phenylprop-2-enoyl)-2H-1,4-benzoxazin-3(4H)-one

(5.07 g, 87%) as a white solid. A mixture of 6-(3-phenylprop-2-

enoyl]-2H-1,4-benzoxazin-3(4H)-one (4.0 g, 14.3 mmol), 10%

palladium on carbon (2.0 g) in EtOH (80 mL), and THF (80 mL)

was stirred at room temperature for 2 h and filtered. The filtrate was

concentrated under reduced pressure. The residue was crystallized

EXPERIMENTAL SECTION

■

General. 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.

Abbreviations used are as follows: s = singlet, d = doublet, t = triplet,

q = quartet, m = multiplet, dd = doublets of doublet, brs = broad

singlet. Coupling constants (J) are given in hertz (Hz). The acidic

protons of diketones, carboxylic acids, alcohols, or anilines were not

frequently observed in ¹H NMR spectra. Elemental analyses and high-

resolution mass spectrometry (HRMS) were performed by Takeda

Analytical Laboratories Ltd. Chemical intermediates were charac-

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

systems were assessed as being >95% using analytical high-perform-

ance liquid chromatography (HPLC). The HPLC analyses were

performed using a Shimadzu UFLC instrument equipped with a

L-column 2 ODS (3.0 mm × 50 mm, 2 μm). Elution was with a

gradient of 5−90% 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. Reagents and solvents were

obtained from commercial sources and used without further

purification. Reaction progress was determined by thin layer

chromatography (TLC) analysis on Merck Kieselgel 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 (Chromatorex NH-DM 1020, 100−200

mesh size)] or on Purif-Pack (SI or NH, particle size 60 μm, Fuji

Silysia Chemical, Ltd.). Preparative HPLC purification was performed

by using a Waters Corporation UV purification system equipped with

a Develosil ODS-UG-10 (4.6 mm × 150 mm, 5 μm) column, and

elution was with a gradient of 5−90% solvent B in solvent A (solvent

A was 0.1% TFA in water, and solvent B was 0.1% TFA in acetoni-

trile), at a flow rate of 150 mL/min with UV detection at 220 nm.

Abbreviations of the solvents are used as follows: EtOAc, ethyl acetate;

THF, tetrahydrofuran; MeOH, methanol; EtOH, ethanol; DMF, N,N-

dimethylformamide; CH₃CN, acetonitrile; IPE, diisopropyl ether.

6-Pentanoyl-2H-1,4-benzoxazin-3(4H)-one (3b). To a suspen-

sion of 2H-1,4-benzoxazin-3(4H)-one (10 g, 67 mmol) in 1,2-

dichloroethane (120 mL) were added powdered AlCl₃(20 g, 150 mmol)

and valeryl chloride (9.6 mL, 80.9 mmol) at 0 °C, successively. The

1

from MeOH to give 3g (1.0 g, 25%) as white crystals. H NMR (300

MHz, DMSO-d₆) δ 2.91 (2H, t, J = 7.6 Hz), 3.27 (2H, t, J = 7.6 Hz),

4.68 (2H, s), 7.03 (1H, d, J = 8.3 Hz), 7.12−7.33 (5H, m), 7.49 (1H,

d, J = 2.1 Hz), 7.63 (1H, dd, J = 8.3, 2.1 Hz), 10.84 (1H, brs).

6-(2-Phenylpropanoyl)-2H-1,4-benzoxazin-3(4H)-one (3h). To

a stirred mixture of 3a (7.0 g, 36.6 mmol) and N,N,N′,N′-tetra-

methyldiaminomethane (10.5 mL, 77.0 mmol) in dichloromethane

(14 mL) was added acetic anhydride (10.5 mL, 111 mmol) at 0 °C.

The mixture was stirred at room temperature for 72 h and

concentrated under reduced pressure. The residue was diluted with

water and extracted with EtOAc. The organic layer was washed with

brine, dried over anhydrous Na₂SO₄, and concentrated under reduced

pressure. The residue was suspended in MeOH (70 mL), and the

resulting solid was collected by filtration to give 6-(2-phenylacryloyl)-

2H-1,4-benzoxazin-3(4H)-one (5.75 g, 56%). A mixture of 6-(2-

phenylacryloyl)-2H-1,4-benzoxazin-3(4H)-one (3.0 g, 10.7 mmol) and

10% palladium on carbon (1.0 g) in THF (60 mL) was stirred under

hydrogen atmosphere at room temperature for 1 h and filtered. The

filtrate was concentrated under reduced pressure. The residue was

purified by column chromatography on basic silica gel with hexane/

1

EtOAc (1:1) as eluant to give 3h (1.98 g, 66%) as a white solid. H

NMR (300 MHz, DMSO-d₆) δ 1.38 (3H, d, J = 6.8 Hz), 4.63 (2H, s),

4.81 (1H, q, J = 6.8 Hz), 6.96 (1H, d, J = 8.6 Hz), 7.13−7.34 (5H, m),

7.50 (1H, d, J = 2.2 Hz), 7.64 (1H, dd, J = 8.6, 2.2 Hz), 10.84 (1H, brs).

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

a stirred mixture of 3b (10 g, 42.9 mmol), acetic acid (80 mL), and

25% hydrogen bromide in acetic acid (20 mL) was added portionwise

pyridinium hydrobromide perbromide (14.4 g, 45.0 mmol) at room

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

poured into water (300 mL). The precipitate was collected by filtration

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to give 4b (13.1 g, 98%) as a white solid. ¹H NMR (300 MHz, CDCl₃)

δ 0.99 (3H, t, J = 7.4 Hz), 1.32−1.70 (2H, m), 2.01−2.26 (2H, m),

4.73 (2H, s), 5.07 (1H, dd, J = 7.6, 6.7 Hz), 7.04 (1H, d, J = 8.5 Hz),

7.56 (1H, d, J = 2.0 Hz), 7.67 (1H, dd, J = 8.5, 2.0 Hz), 8.56 (1H, s).

Compounds 4a, 4c, and 4e−h were prepared in a manner similar to

that described for 4b.

6-(7-Benzyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-6-yl)-

2H-1,4-benzoxazin-3(4H)-one (1c). Yield, 97%. ¹H NMR (300

MHz, DMSO-d₆) δ 2.77 (1H, dd, J = 14.1, 9.2 Hz), 3.01 (1H, dd, J =

14.1, 5.7 Hz), 4.69 (2H, s), 5.14 (1H, dd, J = 9.2, 5.7 Hz), 7.06 (1H, d,

J = 8.6 Hz), 7.11−7.31 (5H, m), 7.51 (1H, dd, J = 8.6, 2.2 Hz), 7.58

(1H, d, J = 2.2 Hz), 9.12 (1H, s), 10.95 (1H, brs). Anal. Calcd for

C₁₉H₁₅N₅O₂S: C, 60.46; H, 4.01; N, 18.56. Found: C, 60.43; H, 4.66;

N, 16.55.

6-(Bromoacetyl)-2H-1,4-benzoxazin-3(4H)-one (4a). Yield,

1

92%. H NMR (300 MHz, DMSO-d₆) δ 4.71 (2H, s), 4.81 (2H, s),

6-(7-Phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-6-yl)-

2H-1,4-benzoxazin-3(4H)-one (1d). Yield, 24%. ¹H NMR (300

MHz, DMSO-d₆) δ 4.67 (2H, s), 6.32 (1H, s), 7.03−7.22 (3H, m),

7.26−7.41 (3H, m), 7.47 (1H, dd, J = 8.7, 2.3 Hz), 7.59 (1H, d, J =

2.3 Hz), 9.25 (1H, s), 10.93 (1H, s). Anal. Calcd for C₁₈H₁₃N₅O₂S·-

0.3EtOAc: C, 59.16; H, 3.98; N, 17.97. Found: C, 58.83; H, 4.18;

N, 17.61.

6-[7-(2-Fluorophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]-

thiadiazin-6-yl]-2H-1,4-benzoxazin-3(4H)-one (1f). Yield, 37%.

¹H NMR (300 MHz, DMSO-d₆) δ 4.66 (2H, s), 6.44 (1H, s), 6.76−

6.81 (1H, m), 7.04−7.10 (2H, m), 7.30−7.43 (2H, m), 7.47−7.50

(1H, m), 7.53−7.54 (1H, m), 9.28 (1H, s), 10.92 (1H, brs). ESI-

HRMS calcd for C₁₈H₁₂FN₅O₂S m/z 382.0769 (M + H), found

382.0754 (M + H).

6-[7-(3-Fluorophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]-

thiadiazin-6-yl]-2H-1,4-benzoxazin-3(4H)-one (1g). Yield, 73%.

¹H NMR (300 MHz, DMSO-d₆) δ 4.68 (2H, s), 6.35 (1H, s), 6.87−

6.94 (1H, m), 7.04−7.12 (2H, m), 7.12−7.22 (1H, m), 7.32−7.42

(1H, m), 7.47 (1H, dd, J = 2.5, 8.5 Hz), 7.58 (1H, d, J = 2.5 Hz), 9.28

(1H, s), 10.96 (1H, brs). ESI-HRMS calcd for C₁₈H₁₂FN₅O₂S m/z

382.0769 (M + H), found 382.0741 (M + H).

6-[7-(4-Fluorophenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]-

thiadiazin-6-yl]-2H-1,4-benzoxazin-3(4H)-one (1h). Yield, 69%.

¹H NMR (300 MHz, DMSO-d₆) δ 4.68 (2H, s), 6.35 (1H s), 7.09

(1H, d, J = 8.5 Hz), 7.12−7.26 (4H, m), 7.46 (1H, dd, J = 2.3, 8.5 Hz),

7.58 (1H, d, J = 2.3 Hz), 9.27 (1H, s), 10.95 (1H, brs). Anal. Calcd for

C₁₈H₁₂FN₅O₂S·0.1EtOAc: C, 56.64; H, 3.31; N, 17.95. Found: C,

56.25; H, 3.27; N, 17.79.

7.07 (1H, d, J = 8.3 Hz), 7.49 (1H, d, J = 2.3 Hz), 7.66 (1H, dd, J =

8.3, 2.3 Hz), 10.91 (1H, brs).

6-(2-Bromo-3-phenylpropanoyl)-2H-1,4-benzoxazin-3(4H)-

1

one (4c). Yield, 95%. H NMR (300 MHz, DMSO-d₆) δ 3.23 (1H,

dd, J = 14.3, 7.3 Hz), 3.52 (1H, dd, J = 14.3, 7.3 Hz), 4.70 (2H, s),

5.82 (1H, t, J = 7.3 Hz), 7.05 (1H, d, J = 8.4 Hz), 7.16−7.39 (5H, m),

7.52 (1H, d, J = 2.0 Hz), 7.74 (1H, dd, J = 8.4, 2.0 Hz), 10.87 (1H, brs).

6-(2-Bromo-2-phenylpropanoyl)-2H-1,4-benzoxazin-3(4H)-

one (4e). Yield, 97%. ¹H NMR (300 MHz, DMSO-d₆) δ 2.14 (3H, s),

4.63 (2H, s), 6.82 (1H, d, J = 8.4 Hz), 7.15 (1H, dd, J = 8.4, 2.1 Hz),

7.29−7.50 (6H, m), 10.88 (1H, brs).

6-[Bromo(2-fluorophenyl)acetyl]-2H-1,4-benzoxazin-3(4H)-

1

one (4f). Yield, 85%. H NMR (300 MHz, CDCl₃) δ 4.71 (2H, s),

6.66 (1H, s), 6.99−7.36 (4H, m), 7.55−7.65 (3H, m), 8.79 (1H, brs).

6-[Bromo(3-fluorophenyl)acetyl]-2H-1,4-benzoxazin-3(4H)-

1

one (4g). Yield, 99%. H NMR (300 MHz, DMSO-d₆) δ 4.69 (2H,

s), 6.97−7.11 (2H, m), 7.13−7.26 (1H, m), 7.29−7.57 (4H, m), 7.78

(1H, dd, J = 8.5, 2.1 Hz), 10.91 (1H, brs).

6-[Bromo(4-fluorophenyl)acetyl]-2H-1,4-benzoxazin-3(4H)-

one (4h). Yield, 96%. ¹H NMR (300 MHz, DMSO-d₆) δ 4.69 (2H, s),

7.01−7.12 (2H, m), 7.23 (2H, t, J = 8.90 Hz), 7.54 (1H, d, J = 2.0 Hz),

7.56−7.65 (2H, m), 7.78 (1H, dd, J = 2.0, 8.5 Hz), 10.93 (1H, s).

6-[Bromo(phenyl)acetyl]-2H-1,4-benzoxazin-3(4H)-one

(4d). To a stirred mixture of 3c (25 g, 93.5 mmol), AcOH (280 mL),

and 25% hydrogen bromide in acetic acid (70 mL) was added portion-

wise pyridinium hydrobromide perbromide (30.4 g, 95.1 mmol) at

room temperature. The mixture was stirred at room temperature for

30 min. Aqueous Na₂S₂O₃solution was added, and the supernatant

was decanted. The residue was diluted with 10% aqueous citric acid

and extracted with EtOAc. The organic layer was washed with

saturated NaHCO₃solution, dried over anhydrous MgSO₄, passed

through silica gel, and concentrated under reduced pressure. The

residue was suspended in EtOAc/IPE and the solid was collected by

6-(7-Methyl-7-phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]-

thiadiazin-6-yl)-2H-1,4-benzoxazin-3(4H)-one (1e). A mixture of

4e (0.3 g, 0.83 mmol), 4-amino-4H-1,2,4-triazole-3-thiol (0.29 g,

2.50 mmol), and triethylamine (3 mL) in EtOH (3 mL) was stirred at

80 °C for 6 h and then concentrated under reduced pressure. The

residue was diluted with saturated NaHCO₃solution and extracted

with EtOAc. The organic layer was dried over anhydrous MgSO₄and

concentrated under reduced pressure. The residue was purified by

column chromatography on silica gel with EtOAc/MeOH (20:1) as

1

filtration to give 4d (28.2 g, 87%) as a white solid. H NMR (300

MHz, CDCl₃) δ 4.70 (2H, s), 6.30 (1H, s), 6.98 (1H, d, J = 8.7 Hz),

7.29−7.53 (6H, m), 7.62 (1H, dd, J = 8.7, 2.1 Hz), 8.64 (1H, brs).

6-(7H-[1,2,4]Triazolo[3,4-b][1,3,4]thiadiazin-6-yl)-2H-1,4-

benzoxazin-3(4H)-one (1a). A mixture of 4a (0.2 g, 0.74 mmol), 4-

amino-3-mercapto-4H-1,2,4-triazole (0.10 g, 0.89 mmol) in EtOH

(4 mL), and toluene (2 mL) was stirred at reflux for 24 h and

concentrated under reduced pressure. To the residue were added

MeOH (30 mL) and 10% aqueous potassium carbonate (10 mL). The

mixture was stirred at room temperature for 3 h, and the resulting solid

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

1a (180 mg, 85%) as white crystals. ¹H NMR (300 MHz, DMSO-d₆) δ

4.38 (2H, s), 4.70 (2H, s), 7.12 (1H, d, J = 9.0 Hz), 7.51−7.59 (2H,

m), 9.14 (1H, s), 10.95 (1H, brs). Anal. Calcd for C₁₂H₉N₅O₂S: C,

50.17; H, 3.16; N, 24.38. Found: C, 49.80; H, 3.19; N, 24.24.

1

eluant to give 1e (0.2 g, 64%) as a white solid. H NMR (300 MHz,

DMSO-d₆) δ 2.02 (3H, s), 4.62 (2H, s), 6.86−6.94 (2H, m), 7.05

(1H, s), 7.28−7.49 (5H, m), 9.26 (1H, s), 10.75 (1H, brs). ESI-HRMS

calcd for C₁₉H₁₅N₅O₂S m/z 378.1019 (M + H), found 378.0989

(M + H).

2-(Benzylthio)imidazo[2,1-b][1,3,4]thiadiazole (12). A mixture

of 5-(benzylthio)-1,3,4-thiadiazol-2-amine (5.0 g, 20.2 mmol) and

45% chloroacetaldehyde (3.9 g, 22.3 mmol) in EtOH (20 mL) and

toluene (10 mL) was refluxed for 12 h and concentrated under

reduced pressure. The residue was diluted with saturated NaHCO₃

solution and extracted with EtOAc. The organic layer was dried over

anhydrous MgSO₄and concentrated under reduced pressure. The

residue was purified by column chromatography on silica gel with

hexane/EtOAc (1:1) as eluant to give 12 (1.27 g, 23%) as a white

6-(7-Propyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-6-yl)-

2H-1,4-benzoxazin-3(4H)-one (1b). A mixture of 3b (0.50 g, 1.60

mmol), 4-amino-4H-1,2,4-triazole-3-thiol (0.19 g, 1.63 mmol) in

EtOH (10 mL), and toluene (5 mL) was stirred at reflux for 12 h and

then concentrated under reduced pressure. The residue was diluted

with saturated NaHCO₃solution and extracted with EtOAc. The

organic layer was dried over anhydrous MgSO₄and concentrated

under reduced pressure. The residue was crystallized from MeOH to

1

solid. H NMR (300 MHz, CDCl₃) δ 4.44 (2H, s), 7.30−7.40 (6H,

m), 7.68 (1H, m).

1-Amino-1H-imidazole-2-thiol (6). A mixture of 12 (1.2 g,

5.1 mmol) and hydrazine monohydrate (2.4 g, 47.94 mmol) in EtOH

(20 mL) was stirred under reflux for 50 h and concentrated under

reduced pressure. The residue was purified by column chromatography on

silica gel with EtOAc as eluant to give 6 (0.19 g, 32%) as a white solid.

¹H NMR (300 MHz, DMSO-d₆) δ 5.62 (2H, s), 6.80 (1H, d, J = 2.3

Hz), 7.04 (1H, d, J = 2.3 Hz), 12.06 (1H, brs).

6-(2-Phenyl-2H-imidazo[2,1-b][1,3,4]thiadiazin-3-yl)-2H-1,4-

benzoxazin-3(4H)-one (7). Compound 7 was prepared in a manner

1

give 1b (0.34 g, 65%) as white crystals. H NMR (300 MHz, DMSO-

d₆) δ 0.85 (3H, t, J = 7.0 Hz), 1.20−1.66 (4H, m), 4.70 (2H, s), 4.87

(1H, dd, J = 9.1, 5.0 Hz), 7.13 (1H, d, J = 9.2 Hz), 7.54−7.62 (2H, m),

9.17 (1H, s),10.96 (1H, brs). Anal. Calcd for C₁₅H₁₅N₅O₂S: C, 54.70;

H, 4.59; N, 21.26. Found: C, 54.67; H, 4.48; N, 21.19.

Compounds 1c−d and 1f−g were prepared in a manner similar to

that described for 1b.

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1

similar to that described for 1e. Yield, 17%. H NMR (300 MHz,

DMSO-d₆) δ 4.65 (2H, s), 6.14 (1H, s), 6.99−7.00 (1H, m), 7.04−

7.07 (1H, m), 7.13−7.16 (2H, m), 7.27−7.34 (3H, m), 7.41−7.45

(1H, m), 7.57−7.58 (1H, m), 7.77−7.79 (1H, m), 10.92 (1H, brs).

ESI-HRMS calcd for C₁₉H₁₄N₄O₂S m/z 363.0910 (M + H), found

363.0877 (M + H).

MHz, DMSO-d₆) δ 4.59 (2H, s), 5.80 (1H, s), 6.91 (1H, d, J = 8.2

Hz), 7.34 (1H, dd, J = 8.2, 2.0 Hz), 7.38 (1H, d, J = 2.0 Hz), 10.71

(1H, s).

Ethyl 2,4-Dioxo-4-(3-oxo-3,4-dihydro-2H-1,4-benzoxazin-6-

1

yl)butanoate (13f). Yield, 95%. H NMR (400 MHz, DMSO-d₆)

δ 1.18−1.25 (3H, m), 4.02−4.13 (2H, m), 4.58 (2H, s), 5.18 (1H, s),

6.80−6.82 (1H, m), 7.20−7.40 (2H, m), 10.68 (1H, brs).

6-(2-Phenyl-2H-1,4-benzothiazin-3-yl)-2H-1,4-benzoxazin-

3(4H)-one (8). A suspension of 4c (0.50 g, 1.44 mmol) and 2-

amionothiophenol (0.18 g, 1.44 mmol) in EtOH (10 mL) was refluxed

for 6 h and concentrated under reduced pressure. The residue was

dissolved in CHCl₃, and the solution was washed with water, dried

over anhydrous MgSO₄, and concentrated under reduced pressure.

The residue was purified by column chromatography on silica gel with

hexane/EtOAc (4:1) as eluant and crystallized from EtOAc/IPE to

give 8 (0.10 g, 19%) as white crystals. ¹H NMR (300 MHz, CDCl₃) δ

4.67 (2H, s), 5.17 (1H, s), 6.98 (1H, d, J = 8.4 Hz), 7.03−7.32 (8H,

m), 7.44−7.57 (2H, m), 7.63 (1H, s), 7.98 (1H, brs). Anal. Calcd for

C₂₂H₁₆N₂O₂S·0.1EtOAc: C,70.95; H, 4.33; N, 7.52. Found: C, 70.14;

H, 4.55; N, 7.24.

6-(2-Methyl-5-phenyl-1,3-thiazol-4-yl)-2H-1,4-benzoxazin-

3(4H)-one (9). Compound 9 was prepared in a manner similar to

that described for 8. Yield, 23%. H NMR (300 MHz, CDCl₃) δ 2.74

(3H, s), 4.61 (2H, s), 6.83 (1H, d, J = 8.4 Hz), 7.00−7.07 (2H, m),

7.33 (5H, s), 7.63 (1H, brs). ESI-HRMS calcd for C₁₈H₁₄N₂O₂S m/z

323.0849 (M + H), found 323.0833 (M + H).

6-(2-Methyl-5-phenyl-1H-imidazol-4-yl)-2H-1,4-benzoxazin-

3(4H)-one (10). A mixture of 4c (200 mg, 0.58 mmol), acetamidine

hydrochloride (273 mg, 2.89 mmol), and K₂CO₃(399 mg, 2.89 mmol)

in 2-propanol (5 mL) was stirred at reflux for 12 h and concentrated

under reduced pressure. The residue was diluted with water and

extracted with EtOAc. The organic layer was washed with water, dried

over anhydrous MgSO₄, and concentrated under reduced pressure.

The residue was purified by column chromatography on basic silica

gel with EtOAc/MeOH (10:1) as eluant and crystallized from

6-(3-Methyl-1-phenyl-1H-pyrazol-5-yl)-2H-1,4-benzoxazin-

3(4H)-one (14a). Compound 14a was prepared in a manner similar

to that described for 14b. Yield, 61%. ¹H NMR (400 MHz, CDCl₃) δ

2.38 (3H, s), 4.70 (2H, s), 6.24 (1H, s), 6.63 (1H, s), 6.80−6.92

(2H, m), 7.25−7.40 (5H, m), 7.83 (1H, brs). Anal. Calcd for

C₁₈H₁₅N₃O₂: C, 70.81; H, 4.95; N, 13.76. Found: C, 70.72; H, 5.09;

N, 13.60.

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

benzoxazin-3(4H)-one (14b). To a stirred mixture of (4-

fluorophenyl)hydrazine hydrochloride (105 mg, 0.64 mmol) and

triethylamine (90 μL, 0.64 mmol) in 2-propanol (5 mL) were added

2,2,2-trifluoroacetic acid (99 μL, 1.29 mmol) and 13a (150 mg,

0.64 mmol) at room temperature, successively. The reaction mixture

was stirred at 80 °C for 12 h and concentrated under reduced pressure.

The residue was diluted with saturated NaHCO₃solution and

extracted with EtOAc. The organic layer was dried over MgSO₄and

concentrated under reduced pressure. The residue was purified by

column chromatography on silica gel with EtOAc/hexane (1:1) as

eluant and crystallized from EtOAc/hexane to give 14b (70 mg, 34%)

1

as white crystals. H NMR (400 MHz, CDCl₃) δ 2.35 (3H, s), 4.63

(2H, s), 6.25 (1H, s), 6.64 (1H, d, J = 1.9 Hz), 6.78 (1H, dd, J = 8.2,

1.9 Hz), 6.89 (1H, d, J = 8.2 Hz), 7.00−7.08 (2H, m), 7.20−7.28 (2H,

m), 8.35 (1H, brs). Anal. Calcd for C₁₈H₁₄N₃O₂F: C, 66.87; H, 4.36;

N, 13.00. Found: C, 66.75; H, 4.35; N, 12.90.

6-[3-Ethyl-1-(4-fluorophenyl)-1H-pyrazol-5-yl]-2H-1,4-ben-

zoxazin-3(4H)-one (14c). To a stirred mixture of (4-fluorophenyl)-

hydrazine hydrochloride (99 mg, 0.61 mmol) and triethylamine (85 μL,

0.61 mmol) in 2-propanol (5 mL) were added 2,2,2-trifluoroacetic acid

(93 μL, 1.21 mmol) and 13b (150 mg, 0.61 mmol), successively. The

reaction mixture was stirred at 80 °C for 12 h and concentrated under

reduced pressure. The residue was diluted with saturated NaHCO₃

solution and extracted with EtOAc. The organic layer was dried over

MgSO₄and concentrated under reduced pressure. The residue was

purified by preparative HPLC and crystallized from EtOAc/hexane to

give 14c (51 mg, 25%) as white crystals. ¹H NMR (400 MHz, CDCl₃)

δ 1.32 (3H, t, J = 7.6 Hz), 2.74 (2H, q, J = 7.6 Hz), 4.63 (2H, s), 6.29

(1H, s), 6.67 (1H, d, J = 2.0 Hz), 6.79 (1H, dd, J = 8.4, 2.0 Hz), 6.98

(1H, d, J = 8.4 Hz), 7.00−7.08 (2H, m), 7.22−7.30 (2H, m), 8.72

(1H, brs). Anal. Calcd for C₁₉H₁₆N₃O₂F: C, 67.65; H, 4.78; N, 12.46.

Found: C, 67.74; H, 4.84; N, 12.44.

1

EtOAc/hexane to give 10 (24 mg, 14%) as white crystals. H NMR

(300 MHz, DMSO-d₆) δ 2.32 (3H, s), 4.57 (2H, brs), 6.76−7.54

(8H, m), 10.58−10.77 (1H, m), 11.99 (1H, brs). ESI-HRMS calcd

for C₁₈H₁₅N₃O₂m/z 306.1237 (M + H), found 306.1212

(M + H).

4,4,4-Trifluoro-1-(3-oxo-3,4-dihydro-2H-1,4-benzoxazin-6-

yl)butane-1,3-dione (13d). To a stirred mixture of 60% NaH

(2.51 g, 105 mmol) in THF (100 mL) were added ethyl 2,2,2-

trifluoroacetate (12.5 mL, 105 mmol), 3a (5.00 g, 26.2 mmol), EtOH

(2.5 mL), and a solution of 2,4-dibenzo-18-crown-6 (150 mg,

0.418 mmol) in THF (50.0 mL), successively. The mixture was

stirred at reflux for 16 h, treated with 10% H₂SO₄(200 mL), and

extracted with EtOAc (200 mL). The organic layer was washed with

water (200 mL) and saturated NaHCO₃solution (200 mL), dried over

anhydrous Na₂SO₄, and concentrated under reduced pressure. The

residue was crystallized from diethyl ether to give 13c (6.67 g, 80%)

6-[1-(4-Fluorophenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-

2H-1,4-benzoxazin-3(4H)-one (14d). To a stirred mixture of (4-

fluorophenyl)hydrazine hydrochloride (133 mg, 0.82 mmol) and

triethylamine (113 μL, 0.82 mmol) in 2-propanol (4.6 mL) were

added 2,2,2-trifluoroacetic acid (129 μL, 1.68 mmol) and 13c (225

mg, 0.74 mmol), successively. The reaction mixture was stirred at

60 °C for 12 h and concentrated under reduced pressure. The residue

was treated with water, and 1 N NaOH was added to the solution to

adjust the pH to 5−6. The precipitate was collected by filtration and

suspended in diethyl ether. The resulting crystals were collected by

1

as a yellow solid. H NMR (300 MHz, CDCl₃) δ 4.74 (2H, s), 6.50

(1H, s), 7.08 (1H, d, J = 8.7 Hz), 7.44 (1H, d, J = 1.9 Hz), 7.60 (1H,

dd, J = 8.7, 1.9 Hz), 8.08 (1H, brs).

Compound 13a−c, 13e, and 13f were prepared in a manner similar

to that described for 13d.

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

dione (13a). Yield, 51%. H NMR (400 MHz, CDCl₃) δ 2.19 (3H,

1

s), 4.70 (2H, s), 6.10 (1H, s), 7.01 (1H, d, J = 8.2 Hz), 7.51 (1H, dd,

J = 8.2, 2.0 Hz), 7.40 (1H, d, J = 2.0 Hz), 8.26 (1H, brs).

1-(3-Oxo-3,4-dihydro-2H-1,4-benzoxazin-6-yl)pentane-1,3-

dione (13b). Yield, 80%. ¹H NMR (300 MHz, CDCl₃) δ 1.22 (4H, t,

J = 7.6 Hz), 2.46 (3H, d, J = 7.6 Hz), 4.70 (2H, s), 6.10 (1H, s), 7.02

(1H, d, J = 8.7 Hz), 7.40 (1H, d, J = 1.9 Hz), 7.52 (1H, dd, J = 8.7 Hz,

1.9 Hz), 8.23 (1H, brs).

4-Methyl-1-(3-oxo-3,4-dihydro-2H-1,4-benzoxazin-6-yl)-

pentane-1,3-dione (13c). Yield, 43%. ¹H NMR (300 MHz, CDCl₃)

δ 1.22 (6H, d, J = 7.2 Hz), 2.61 (1H, sep, J = 7.2 Hz), 4.70 (2H, s),

6.10 (1H, s), 7.02 (1H, d, J = 8.5 Hz), 7.42 (1H, d, J = 1.9 Hz), 7.53

(1H, dd, J = 8.5, 1.9 Hz), 8.64 (1H, brs).

1

filtration to give 14d (198 mg, 67%) as a pale yellow solid. H NMR

(400 MHz, CDCl₃) δ 4.65 (2H, s), 6.65 (1H, d, J = 2.0 Hz), 6.71 (1H,

s), 6.80 (1H, dd, J = 8.4, 2.0 Hz), 6.93 (1H, d, J = 8.4 Hz), 7.05−7.16

(2H, m), 7.25−7.38 (2H, m), 8.28 (1H, brs). Anal. Calcd for

C₁₈H₁₁N₃O₂F₄: C, 57.30; H, 2.94; N, 11.14. Found: C, 57.31; H, 2.90;

N,11.10.

6-[1-(4-Fluorophenyl)-3-(perfluoroethyl)-1H-pyrazol-5-yl]-

2H-1,4-benzoxazin-3(4H)-one (14e). Compound 14e was pre-

1

pared in a manner similar to that described for 14d. Yield, 65%. H

NMR (400 MHz, CDCl₃) δ 4.66 (2H, s), 6.66 (1H, d, J = 2.0 Hz),

6.72 (1H, s), 6.81 (1H, dd, J = 8.2, 2.0 Hz), 6.93 (1H, d, J = 8.2 Hz),

7.09−7.15 (2H, m), 7.31−7.38 (2H, m), 8.26 (1H, brs). Anal. Calcd

4,4,5,5,5-Pentafluoro-1-(3-oxo-3,4-dihydro-2H-1,4-benzoxa-

zin-6-yl)pentane-1,3-dione (13e). Yield, 87%. ¹H NMR (400

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for C₁₉H₁₁N₃O₂F₆: C, 53.41; H, 2.59; N, 9.83. Found: C, 53.48; H,

2.74; N, 9.52.

Calcd for C₁₉H₁₃N₃O₂F₄: C, 58.32; H, 3.35; N, 10.74. Found: C,

58.25; H, 3.23; N, 10.80.

Ethyl 1-(4-Fluorophenyl)-5-(3-oxo-3,4-dihydro-2H-1,4-ben-

zoxazin-6-yl)-1H-pyrazole-3-carboxylate (14o). Compound 14o

was prepared in a manner similar to that described for 14d. Yield, 80%.

¹H NMR (400 MHz, DMSO-d₆) δ 1.32 (3H, t, J = 7.2 Hz), 4.33 (2H,

q, J = 7.2 Hz), 4.60 (2H, s), 6.76 (1H, d, J = 2.0 Hz), 6.80 (1H, dd, J =

8.2, 2.0 Hz), 6.93 (1H, d, J = 8.2 Hz), 7.02 (1H, s), 7.30−7.37 (2H,

m), 7.37−7.41 (2H, m), 10.76 (1H, s).

1-(4-Fluorophenyl)-5-(3-oxo-3,4-dihydro-2H-1,4-benzoxa-

zin-6-yl)-1H-pyrazole-3-carboxamide (15). To a stirred solution

of 14o (1.26 g, 3.30 mmol) in THF (25 mL) was added 1 N NaOH

(8.26 mL). The mixture was stirred at reflux for 12 h and acidified with

1 N HCl. The precipitate was collected by filtration and recrystallized

from EtOH to give 1-(4-fluorophenyl)-5-(3-oxo-3,4-dihydro-2H-1,4-

benzoxazin-6-yl)-1H-pyrazole-3-carboxylic acid (877 mg, 75%) as

white crystals. To a stirred mixture of 1-(4-fluorophenyl)-5-(3-oxo-3,4-

dihydro-2H-1,4-benzoxazin-6-yl)-1H-pyrazole-3-carboxylic acid (170 mg,

0.48 mmol), 1-hydroxybenzotriazole monohydrate (74.4 mg, 0.48 mmol),

and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

(100 mg, 0.52 mmol) in DMF (5 mL) was added 2 M ammonia in

MeOH (253 μL, 0.51 mmol) at room temperature. The mixture was

stirred for 12 h, diluted with water, and extracted with EtOAc. The

organic layer was washed with water and brine, dried over anhydrous

MgSO₄, and concentrated under reduced pressure. The residue was

suspended in EtOAc, and the solid was collected by filtration to give

15 (130 mg, 76%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ

4.60 (2H, s), 6.77−6.80 (2H, m), 6.89 (1H, s), 6.93 (1H, d, J = 8.9

Hz), 7.31−7.42 (5H, m), 7.69 (1H, s), 10.76 (1H, brs).

6-[1-(4-Fluorophenyl)-3-(1-methylethyl)-1H-pyrazol-5-yl]-

2H-1,4-benzoxazin-3(4H)-one (14f). Compound 14f was prepared

1

in a manner similar to that described for 14c. Yield, 12%. H NMR

(300 MHz, DMSO-d₆) δ 1.16 (6H, d, J = 6.8 Hz), 2.90−3.05 (1H, sep,

J = 6.8 Hz), 4.59 (2H, s), 6.66 (1H, s), 6.97 (1H, d, J = 8.3 Hz), 7.28−

7.46 (4H, m), 7.51−7.64 (2H, m), 10.69 (1H, brs). ESI-HRMS calcd

for C₂₀H₁₈N₃O₂F m/z 352.1456 (M + H), found 352.1434 (M + H).

Compounds 14g−m were prepared in a manner similar to that

described for 14d.

6-[1-(2-Fluorophenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-

1

2H-1,4-benzoxazin-3(4H)-one (14g). Yield, 85%. H NMR (400

MHz, CDCl₃) δ 4.63 (2H, s), 6.65 (1H, d, J = 2.0 Hz), 6.73 (1H, s),

6.80 (1H, dd, J = 8.4, 2.0 Hz), 6.90 (1H, d, J = 8.4 Hz), 7.08−7.18

(1H, m), 7.23−7.30 (1H, m), 7.45−7.58 (2H, m), 7.62 (1H, brs).

Anal. Calcd for C₁₈H₁₁N₃O₂F₄: C, 57.30; H, 2.94; N, 11.14. Found: C,

57.24; H, 2.99; N, 11.08.

6-[1-(3-Fluorophenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-

1

2H-1,4-benzoxazin-3(4H)-one (14h). Yield, 86%. H NMR (400

MHz, CDCl₃) δ 4.67 (2H, s), 6.66 (1H, d, J = 1.9 Hz), 6.71 (1H, s),

6.84 (1H, dd, J = 8.4, 1.9 Hz), 6.96 (1H, d, J = 8.4 Hz), 7.08−7.18

(3H, m), 7.35−7.40 (1H, m), 7.81 (1H, brs). Anal. Calcd for

C₁₈H₁₁N₃O₂F₄: C, 57.30; H, 2.94; N, 11.14. Found: C, 57.33; H, 3.02;

N, 11.12.

6-[1-(2-Chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-

2H-1,4-benzoxazin-3(4H)-one (14i). Yield, 76%. ¹H NMR (400

MHz, CDCl₃) δ 4.62 (2H, s), 6.63 (1H, d, J = 2.0 Hz), 6.79 (1H, dd,

J = 8.4, 2.0 Hz), 6.88 (1H, d, J = 8.4 Hz), 6.97 (1H, d, J = 2.0 Hz),

7.40−7.47 (4H, m), 7.66 (1H, brs). ESI-HRMS calcd for

C₁₈H₁₁N₃O₂F₃Cl m/z 392.0419 (M + H), found 392.0415 (M + H).

6-[1-(4-Chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-

2H-1,4-benzoxazin-3(4H)-one (14j). Yield, 66%. ¹H NMR (400

MHz, CDCl₃) δ 4.66 (2H, s), 6.65 (1H, d, J = 2.0 Hz), 6.70 (1H, s),

6.82 (1H, dd, J = 8.4, 2.0 Hz), 6.95 (1H, d, J = 8.4 Hz), 7.27−7.30

(2H, m), 7.37−7.40 (2H, m), 7.86 (1H, brs). Anal. Calcd for

C₁₈H₁₁N₃O₂ClF₃: C, 54.91; H, 2.82; N, 10.67. Found: C, 54.70; H,

2.87; N, 10.56.

1-(4-Fluorophenyl)-5-(3-oxo-3,4-dihydro-2H-1,4-benzoxa-

zin-6-yl)-1H-pyrazole-3-carbonitrile (16). To a stirred solution of

15 (25 mg, 0.07 mmol) in dioxane (1 mL) and pyridine (0.1 mL) was

added trifluoroacetic anhydryde (25 μL, 0.18 mmol) at 0 °C. The

mixture was stirred for 30 min, quenched with 1 N HCl, and extracted

with EtOAc. The organic layer was dried over anhydrous MgSO₄and

concentrated under reduced pressure. The residue was purified by

column chromatography on silica gel with hexane/EtOAc (2:1) as

1

eluant to give 16 (9 mg, 38%) as a white solid. H NMR (400 MHz,

CDCl₃) δ 4.60 (2H, s), 6.75 (1H, d, J = 2.0 Hz), 6.80 (1H, dd, J = 8.6,

2.0 Hz), 6.95 (1H, d, J = 8.6 Hz), 7.08−7.15 (3H, m), 7.23−7.44 (2H,

m), 7.60 (1H, brs). ESI-HRMS calcd for C₁₈H₁₁N₄O₂F m/z 392.0419

(M + H), found 392.0415 (M + H).

6-[1-(2-Methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-

1

2H-1,4-benzoxazin-3(4H)-one (14k). Yield, 73%. H NMR (400

MHz, CDCl₃) δ 1.97 (3H, s), 4.61 (2H, s), 6.53 (1H, d, J = 2.0 Hz),

6.75−6.93 (3H, m), 7.26−7.34 (3H, m), 7.34−7.40 (1H, m), 7.69

(1H, brs). Anal. Calcd for C₁₉H₁₄N₃O₂F₃: C, 61.13; H, 3.78; N, 11.26.

Found: C, 61.00; H, 3.82; N, 11.18.

6-[1-(4-Methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-

2H-1,4-benzoxazin-3(4H)-one (14l). Yield, 83%. ¹H NMR (400

MHz, CDCl₃) δ 2.39 (3H, s), 4.64 (2H, s), 6.61 (1H, d, J = 2.0 Hz),

6.69 (1H, s), 6.84 (1H, dd, J = 8.4, 2.0 Hz), 6.93 (1H, d, J = 8.4 Hz),

7.18−7.20 (1H, 2 m), 7.23−7.27 (3H, m), 7.73 (1H, brs). Anal. Calcd

for C₁₉H₁₄N₃O₂F₃: C, 61.13; H, 3.78; N, 11.26. Found: C, 61.22; H,

3.84; N, 11.23.

6-[1-(2-Ethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-

2H-1,4-benzoxazin-3(4H)-one (14m). Yield, 47%. ¹H NMR

(300 MHz, DMSO-d₆) δ 0.94 (3H, t, J = 7.6 Hz), 2.22 (2H, q, J =

7.6 Hz), 4.57 (2H, s), 6.66−6.81 (2H, m), 6.79−6.94 (1H, m), 7.11

(1H, s), 7.26−7.38 (2H, m), 7.34−7.59 (2H, m), 10.77 (1H, brs).

Anal. Calcd for C₂₀H₁₆N₃O₂F₃: C, 62.01; H, 4.16; N, 10.85.Found: C,

62.28; H, 4.31; N,10.65.

6-[1-(4-Fluoro-2-methylphenyl)-3-(trifluoromethyl)-1H-pyr-

azol-5-yl]-2H-1,4-benzoxazin-3(4H)-one (14n). To a stirred mix-

ture of 18 (29.0 g, 164 mmol) and triethylamine (22.9 mL, 164 mmol)

in 2-propanol (350 mL) were added trifluoroacetic acid (12.6 mL, 164

mmol) and 13d (47.1 g, 164 mmol), successively. The resulting

mixture was stirred at 80 °C for 3 h and then poured into water. The

precipitate was collected by filtration and purified by column

chromatography on silica gel with hexane/EtOAc (1:1) as eluant to

give 14n (31.4 g, 54%) as a white solid. ¹H NMR (400 MHz, DMSO-

d₆) δ 1.90 (3H, s), 4.58 (2H, s), 6.70 (1H, d, J = 2.1 Hz), 6.82 (1H, dd,

J = 8.4, 2.1 Hz), 6.92 (1H, d, J = 8.4 Hz), 7.13 (1H, s), 7.11−7.23 (1H,

m), 7.28−7.32 (1H, m), 7.41−7.45 (1H, m), 10.78 (1H, brs). Anal.

6-[1-(4-Fluorophenyl)-3-(hydroxymethyl)-lH-pyrazol-5-yl]-

2H-1,4-benzoxazin-3(4H)-one (17). To a stirred solution of 14o

(100 mg, 0.26 mmol) in THF (1 mL) was added 1 M lithium

aluminum hydride in THF (0.26 mL, 0.26 mmol) at 0 °C. The mixture

was stirred at room temperature for 1 h. To the mixture was added

1 M lithium aluminum hydride in THF (0.10 mL, 0.10 mmol) at room

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

diluted with THF, and quenched with sodium sulfate decahydrate. The

resulting mixture was stirred at room temperature for 12 h and filtered.

The filtrate was treated with 1 N NaOH and extracted with EtOAc.

The organic layer was dried over Na₂SO₄and concentrated under

reduced pressure. The residue was purified by column chromatog-

raphy on silica gel with dichloromethane/MeOH (20:1) as eluant to

give 17 (30 mg, 34%) as a white solid.¹H NMR (300 MHz, DMSO-d₆)

δ 4.49 (2H, d, J = 6.0 Hz), 4.59 (2H, s), 5.17 (1H, t, J = 6.0 Hz), 6.49

(1H, s), 6.65−6.81 (2H, m), 6.92 (1H, d, J = 8.3 Hz), 7.16−7.33 (4H,

m), 10.73 (1H, brs). ESI-HRMS calcd for C₁₈H₁₄N₃O₃F m/z 340.1092

(M + H), found 340.1066 (M + H).

1-(4-Fluoro-2-methylphenyl)hydrazine Hydrochloride (18). To

a solution of 4-fluoro-2-methylaniline 17 (125 g, 1.00 mol) in

concentrated HCl (1000 mL) was added sodium nitrite (137 g, 2.00

mol) at 0 °C. The mixture was stirred at 0 °C for 2 h. To the mixture

was added SnCl₂(474 g, 2.50 mol) at 0 °C. The mixture was stirred at

room temperature for 12 h and diluted with diethyl ether (250 mL).

The aqueous layer was separated, basified with NaOH, and extracted

with EtOAc. The organic layer was dried over anhydrous MgSO₄and

concentrated under reduced pressure. The residue was dissolved in

diethyl ether. To the solution was added 4 N HCl in dioxane. The

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precipitate was collected by filtration to give 18 (85.0 g, 48%) as a

white solid. H NMR (300 MHz, DMSO-d₆) δ 2.20 (3H, s), 6.89−

7.12 (3H, m), 7.75 (1H, brs), 10.16 (3H, brs).

acetone-d₆) δ 2.38 (3H, s), 2.62 (3H, s), 8.18 (1H, s), 8.57 (1H, s),

11.05 (1H, brs).

1

6-Acetyl-8-fluoro-2H-1,4-benzoxazin-3(4H)-one (27a). Under

nitrogen atmosphere, a mixture of 23a (93.7 g, 381 mmol), 4-

(vinyloxy)butan-1-ol (156 mL, 1.26 mol), dichlorobis(tri-o-

tolylphosphine)palladium(II) (8.98 g, 11.4 mmol), and K₂CO₃

(105 g, 762 mmol) in DMF (635 mL) and H₂O (38.1 mL) was

stirred at 80 °C for 12 h and then poured into 2 N HCl. The resulting

mixture was stirred for 1 h and extracted with dichloromethane. The

organic layer was dried over anhydrous MgSO₄and concentrated

under reduced pressure. The residue was purified by column

chromatography on silica gel with hexane/EtOAc (2:1) as eluant to

give 27a (58.0 g, 72%) as a tan solid. ¹H NMR (400 MHz, DMSO-d₆)

δ 2.53 (3H, s), 4.78 (2H, s), 7.33 (1H, s), 7.64 (1H, d, J = 10.8 Hz),

11.02 (1H, brs).

6-[1-(4-Fluorophenyl)-1H-pyrazol-5-yl]-2H-1,4-benzoxazin-

3(4H)-one (19). A mixture of 6-acetyl-2H-1,4-benzoxazin-3(4H)-one

(4.0 g, 20.9 mmol) and N,N-dimethylformamide dimethyl acetal (4.46

mL, 33.5 mmol) in EtOH (50 mL) was stirred at 80 °C for 16 h and

then cooled to room temperature. The precipitate was collected by

filtration and washed with EtOH to give 6-(3-(dimethylamino)-

acryloyl)-2H-1,4-benzoxazin-3(4H)-one as a yellow solid (3.8 g, 73%).

To a stirred suspension of 1-(4-fluorophenyl)hydrazine hydrochloride

(0.58 g, 3.54 mmol) in MeOH (14 mL) were added 6 N HCl (3.16

mL, 19.0 mmol) and 6-[3-(dimethylamino)acryloyl]-2H-1,4-benzox-

azin-3(4H)-one (0.78 g, 3.16 mmol), successively. The mixture was

stirred at 40 °C for 16 h and concentrated under reduced pressure.

The residue was washed with diethyl ether and recrystallized from

EtOH to give 19 (0.43 g, 44%) as white crystals. ¹H NMR (400 MHz,

CDCl₃) δ 4.64 (2H, s), 6.46 (1H, d, J = 2.0 Hz), 6.67 (1H, d, J = 2.0

Hz), 6.80 (1H, J = dd, 8.6. 2.0 Hz), 6.91 (1H, d, J = 8.6 Hz), 7.05−

7.18 (2H, m), 7.28−7.38 (2H, m), 7.69 (1H, d, J = 2.0 Hz), 8.54 (1H,

brs).

6-Acetyl-8-chloro-2H-1,4-benzoxazin-3(4H)-one (27b). Com-

pound 27b was prepared in a manner similar to that described for

27a. Yield, 34%. ¹H NMR (400 MHz, DMSO-d₆) δ 2.52 (3H, s), 4.80

(2H, s), 7.40 (1H, s), 7.71 (1H, s), 11.02 (1H, brs).

6-Acetyl-8-methyl-2H-1,4-benzoxazin-3(4H)-one (27c). Com-

pound 27c was prepared in a manner similar to that described for 23a.

1

Yield, 89%. H NMR (400 MHz, DMSO-d₆) δ 2.23 (3H, s), 2.52

6-[1-(4-Fluorophenyl)-4-iodo-1H-pyrazol-5-yl]-2H-1,4-ben-

zoxazin-3(4H)-one (20). To a stirred solution of 19 (1.0 g, 3.2

mmol) in DMF (10 mL) was added NIS (0.73 g, 3.2 mmol) at 0 °C.

The mixture was stirred at 55 °C for 60 h. The resulting mixture was

diluted with water and allowed to cool to 0 °C. The precipitate was

collected by filtration and washed with dichloromethane to give 20

(3H, s), 4.68 (2H, s), 7.34 (1H, s), 7.52 (1H, s), 10.78 (1H, brs).

8-Fluoro-6-[1-(4-fluoro-2-methylphenyl)-3-(trifluoromethyl)-

1H-pyrazol-5-yl]-2H-1,4-benzoxazin-3(4H)-one (29a). To a

slurry of 60% NaH (44.4 g, 1.10 mol) in THF (4.0 L) was added

ethyl 2,2,2-trifluoroacetate (146 mL, 1.10 mol), 27a (58 g, 0.28 mol),

EtOH (1.5 mL), and 2,4-dibenzo-18-crown-6 (1.60 g, 4.43 mmol),

successively. The resulting mixture was stirred at 60 °C for 12 h,

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

was washed with water, dried over anhydrous MgSO₄, and

concentrated under reduced pressure. The residue was suspended in

diethyl ether. The solid was collected by filtration to give 4,4,4-

trifluoro-1-(8-fluoro-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-6-yl)-

butane-1,3-dione (28a) (33.0 g, 39%) as a tan solid. To a stirred

mixture of 18 (19.1 g, 108 mmol) and triethylamine (15.1 mL, 108

mmol) in 2-propanol (500 mL) were added trifluoroacetic acid

(8.33 mL, 108 mmol) and 28a (33.0 g, 108 mmol), successively. The

resulting mixture was stirred at 80 °C for 3 h and poured into water.

The precipitate was collected by filtration and purified by column

chromatography on silica gel with EtOAc/hexane (1:3) as eluant

1

(0.70 g, 50%) as a white solid. H NMR (400 MHz, CDCl₃) δ 4.67

(2H, s), 6.69 (1H, d, J = 2.0 Hz), 6.83 (1H, dd, J = 8.2, 2.0 Hz), 6.97

(1H, d, J = 8.2 Hz), 6.98−7.10 (2H, m), 7.19−7.22 (2H, m), 7.76

(1H, s), 7.80 (1H, brs).

6-[1-(4-Fluorophenyl)-4-(trifluoromethyl)-1H-pyrazol-5-yl]-

2H-1,4-benzoxazin-3(4H)-one (21). Under nitrogen atmosphere, a

mixture of 20 (200 mg, 0.46 mmol), methyl 2,2-difluoro-2-

(fluorosulfonyl)acetate (0.39 mL, 3.03 mmol)m and CuI (96.3 mg,

0.51 mmol) in DMF(3 mL) was stirred at 100 °C for 16 h. The

reaction mixture was diluted with water and extracted with EtOAc.

The organic layer was washed with water and brine, dried over

anhydrous MgSO₄, and concentrated under reduced pressure. The

residue was purified by column chromatography on silica gel with

dichloromethane/EtOAc (10:1) as eluant to give 21 (8.2 mg, 5%) as a

white solid. ¹H NMR (400 MHz, CDCl₃) δ 4.67 (2H, s,), 6.66 (1H, d,

J = 2.0 Hz), 6.80−6.83 (1H, m), 6.93−7.10 (3H, m), 7.18−7.30 (2H,

m), 7.83 (1H, brs), 7.95 (1H, s). ESI-HRMS calcd for C₁₈H₁₁N₃O₂F₄

m/z 376.0715 (M + H), found 376.0715 (M + H).

1

to give 29a (35.2 g, 79%) as a white solid. H NMR (400 MHz,

DMSO-d₆) δ 1.91 (3H, s), 4.67 (2H, s), 6.47 (1H, s), 6.91 (1H, dd, J =

11.3, 2.0 Hz), 7.15−7.28 (2H, m), 7.30−7.34 (1H, m), 7.46−7.50

(1H, m), 10.98 (1H, brs). Anal. Calcd for C₁₉H₁₂N₃O₂F₅: C, 55.75; H,

2.96; N, 10.27. Found: C, 55.85; H, 3.03; N, 10.21.

6-Bromo-8-fluoro-2H-1,4-benzoxazin-3(4H)-one (23a). A

mixture of 4-bromo-2-fluoro-6-nitrophenol (216 g, 917 mmol), methyl

2-bromoacetate (104 mL, 1.10 mol), and K₂CO₃(633 g, 4.58 mol) in

DMF (500 mL) was stirred at 65 °C for 12 h and poured into water.

The precipitate was collected by filtration to give methyl (4-bromo-2-

nitrophenoxy)acetate (282 g) as a yellow solid. To a solution of

methyl (4-bromo-2-nitrophenoxy)acetate (282 g) in acetic acid

(1.5 L) was added Zn dust (209 g, 320 mmol) slowly. The mixture was

stirred at 100 °C for 12 h and filtered. The filter cake was suspended in

DMF and filtered. The filtrates were combined and poured into water.

The precipitate was collected by filtration to give 23a (130 g, 57%) as

8-Chloro-6-[1-(4-fluoro-2-methylphenyl)-3-(trifluoromethyl)-

1H-pyrazol-5-yl]-2H-1,4-benzoxazin-3(4H)-one (29b). Compound

29b was prepared in a manner similar to that described for 29a. Yield,

1

28% in two steps. H NMR (400 MHz, DMSO-d₆) δ 1.91 (3H, s),

4.71 (2H, s), 6.58 (1H, d, J = 1.9 Hz), 7.07 (1H, d, J = 1.9 Hz), 7.13−

7.36 (3H, m), 7.44−7.49 (1H, m), 10.95 (1H, brs). Anal. Calcd for

C₁₉H₁₂N₃O₂ClF₄: C, 53.60; H, 2.84; N, 9.87. Found: C, 53.58; H,

2.89; N, 9.76.

6-[1-(4-Fluoro-2-methylphenyl)-3-(trifluoromethyl)-1H-pyr-

azol-5-yl]-8-methyl-2H-1,4-benzoxazin-3(4H)-one (29c). Com-

pound 29c was prepared in a manner similar to that described for 29a.

Yield, 16% in two steps. ¹H NMR (400 MHz, CDCl₃) δ 1.96 (3H, s),

2.15 (3H, s), 4.64 (2H, s), 6.39 (1H, d, J = 2.0 Hz), 6.70 (1H, m), 6.74

(1H, s), 6.97−7.00 (2H, m), 7.25−7.30 (1H, m), 9.00 (1H, brs). ESI-

HRMS calcd for C₂₀H₁₅N₃O₂F₄m/z 406.1173 (M + H), found

406.1146 (M + H).

1

a white solid. H NMR (300 MHz, DMSO-d₆) δ 4.68 (2 H, s), 6.87

(1 H, t, J = 2.0 Hz), 7.21 (1 H, dd, J = 10.0, 2.0 Hz), 10.99 (1 H, brs).

6-Bromo-8-chloro-2H-1,4-benzoxazin-3(4H)-one (23b).

Compound 23b was prepared in a manner similar to that described

1

for 23a. Yield, 41% in two steps. H NMR (300 MHz, DMSO-d₆) δ

4.72 (2H, s), 6.99 (1H, d, J = 2.5 Hz), 7.30 (1H, d, J = 2.5 Hz), 10.98

(1H, brs).

Radioligand Binding Assay. Binding displacement assays were

carried out in 96-well v-bottom polypropylene plates with a final

volume of 50.5 μL 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 cytosolic protein

prepared from human MR transiently transfected FreeStyle 293 cells

4-Hydroxy-3-methyl-5-nitroacetophenone (25). To a solu-

tion of 4-hydroxy-3-methylacetophenone (100 g, 666 mmol) in acetic

acid (444 mL) was added 70% nitric acid (31.0 mL, 732 mmol) at

room temperature. The mixture was stirred at room temperature for

24 h and poured into water. The precipitate was collected by filtration

to give 25 (77.0 g, 59%) as a yellow solid. ¹H NMR (400 MHz,

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(Invitrogen). Each concentration was run in duplicate. The cytosols

were incubated for 16 h at 4 °C. Unbound radioactivity was removed

by the addition of 35 μL of dextran/gelatin coated charcoal suspension

(5% charcoal, 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 910g for 10 min at 4 °C. Then 30 μL of the supernatant

from each well was transferred to a 96-well white plate with 150 μL of

scintillation fluid and the radioactivity was measured by TopCount

(PerkinElmer Inc.). For the determination of nonspecific binding, cold

aldosterone instead of drug was added to the reaction mixture at

100 μM. Specific binding was determined by subtracting the value of

the nonspecific binding component from the total binding value. The

raw data for the specifically bound counts were normalized between

0% and 100% activity and nonlinear fitted to a sigmoidal equation to

calculate the IC₅₀and its 95% confidence interval (CI) 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, the

ligand concentration was 5 nM.

MR Antagonist Assay (Luciferase Reporter Gene Assay). COS-1

cells were inoculated at 5 × 10⁶cells/F150 in D-MEM (low glucose)

supplemented with 10% FBS and 50 mg/mL gentamicin 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 μL of PLUS

reagent (Invitrogen), 9 μg of pMCMYneo-hMR, 5 μg of pMAM-Luc,

and 1 μg of pRL-TK was mixed with a solution of 2.5 mL of Opti-

MEM and 125 μL of Lipofectamin reagent (Invitrogen). The mixture

was maintained at room temperature for 15 min. After substitution of

culture medium with Opti-MEM, the mixture was added to the cells.

After 3 h of incubation, 25 mL of D-MEM (low glucose) supplemented

with 0.1% BSA and 50 μg/mL gentamicin was added and then the cells

were incubated at 37 °C in 5% CO₂for 1 day. The transfected cells

were harvested and resuspended at 3.3 × 10⁵cells/mL in D-MEM (low

glucose) supplemented with 0.1% BSA and 50 μg/mL gentamicin. Then

40 μL of the cell suspension was transferred in 96-well plate (Corning

no. 3688). After incubation, 5 μL/well of test compounds at various

concentrations and 5 μL/well of aldosterone (final concentration,

1 nM) were added to the cells. After 1 day of incubation at 37 °C in

5% CO₂, the medium was removed. Then 20 μL/well of 2-fold diluted

pikkagene (NIPPON GENE CO., Ltd.) solution with HBSS was

added to each well and the luciferase activity was measured. The data

were nonlinear fitted to a sigmoidal equation to calculate the IC₅₀and

its 95% confidence interval (CI).

After 3 weeks, SBP and HR were measured by a tail-cuff method

(Softron BP-98A, Softron Co.) and development of hypertension

was confirmed. Hypertensive rats were divided into groups with

equalization of body weight, SBP, and HR. The groups of rats were

treated with vehicle (0.5 w/v% methylcellulose, n = 8), compound 14n

(n = 8), and spironolactone (n = 8) at a dose of 100 mg/kg. Drugs

were administrated orally once a day. After 13 days of treatment, SBP

and HR were measured approximately 24 h after the last dosing. One

rat in vehicle group was excluded from analysis because unexpected

large fluctuation in SBP values was observed. Data are shown as the

mean ± SEM.

Pharmacokinetic Analysis in Rats. Compound 14d or 14n

was administered to rats (fasted, 8-week-old) intravenously at 0.1 or

3 mg/kg and orally at 1.0 or 10 mg/kg. After intravenous and oral

administration, blood samples were collected at designated 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 and centrifuged

again. The compound concentrations in the supernatant were

measured by a liquid chromatograph equipped with tandem mass

spectrometer.

Metabolic Clearance Assay. In vitro oxidative metabolic studies

of the tested compounds were carried out using hepatic microsomes

obtained from rats. The reaction mixture with a final volume of 0.1 mL

consists of 0.2 mg/mL hepatic microsome in 50 mmol/L KH₂PO₄−

K₂HPO₄phosphate buffer (pH 7.4) and 1 μmol/L test compound.

After 5 min of preincubation 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 β-NADP⁺, and

15 unit/mL glucose 6-phosphate dehydrogenase at 10% volume of

reaction mixture. After the addition of the NADPH-generating system,

the mixture was incubated at 37 °C for 0 and 60 min. The reaction was

terminated by addition of an equivalent volume of acetonitrile. After

the samples were mixed and centrifuged, the supernatant fractions

were subjected to high performance liquid chromatography with UV

detection. Test compound in the reaction mixture was measured by an

HPLC system equipped with a UV detector. For metabolic clearance

determinations, chromatograms were analyzed for parent compound

disappearance rate from the reaction mixtures. All incubations were

made in duplicate.

Docking Study. The atomic coordinates of the protein structures

analyzed in-house were used for docking. To both protein structures

were added hydrogens, and then only hydrogens were energetically

optimized in MOE (version 2005.06, Chemical Computing Group

Inc.) using MMFF94s force field. Each compound was docked into the

MR-LBD using the GOLD program (version 3.0, the Cambridge

Crystallographic Data Centre) with the default parameter set.

Obtained docking poses showing high score (Gold score) were

subjected to energy minimization using MMFF94s force field in MOE.

During the energy minimization procedure, amino acid residues within

5.0 Å from the each ligand were relaxed and the dielectric constant was

set to 2r, where r is the distance between two interacting atoms.

Crystallography. Several mutants of human MR-LBD were

prepared as described previously.¹⁶The proteins (residues 712−984

or residues 727−984) were expressed in a BL21(DE3) cells (Invitrogen)

with the 6×His tag at the C terminus, which was removed by a TEV

protease cleavage. Ligands were added with a final concentration of

10−100 μM prior to the IPTG induction of expression. The proteins

were then purified using Ni-affinity and size-exclusion chromatog-

raphy. All buffers were supplemented with 10−100 μM ligand. The

purified proteins were concentrated to a protein concentration of

7−10 mg/mL in buffer containing 25 mM HEPES, pH 7.2, 0.2 M

sodium chloride, 10 mM DTT, 10% glycerol, 0.05% β-octyl glucoside,

and 100 μM ligand.

Pharmacological Evaluation in Rats. Male 5-week-old Wistar

rats were obtained from CREA Japan, Inc. (Tokyo, Japan) and

acclimated for 1 week before experiments. 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).

Potassium-Sparing and Natriuretic Effects in Wistar Rats. Pot-

assium-sparing and natriuretic effect was examined using the method

of Kagawa¹⁵with some modifications. In brief, rats aged 6 weeks were

used. On the day before the experiment, rats were deprived of food

and water overnight for approximately 16 h. Compound 14n and

spironolactone were suspended in 0.5% methylcellulose solution and

orally administered 30 min before aldosterone (3 μg/kg, sc) and saline

(25 mL/kg, po) administration. Urine was collected over the next 5 h.

Urinary Na⁺and K⁺concentrations were measured using an electrolyte

analyzer EX-2000 (Jokoh Company Ltd.). Urinary Na⁺/K⁺ratio was

calculated and normalized by the value for the vehicle group as 1.0.

Data are shown as the mean ± SEM.

Antihypertensive Effect in DOCA−Salt Hypertensive Rats. Briefly,

6-week-old Wistar rats were anesthetized by injection of pentobarbi-

turate (50 mg/kg body weight, ip) for subcutaneous implantation of a

25 mg pellet of DOCA. After recovery from anesthesia, rats were

housed in standard cages and maintained on standard chow and with

ad libitum access to both tap water and a 1% NaCl drinking solution.

The crystals of the MR-LBD complexed with spironolactone or

compound 1d were grown at 20 °C using the vapor diffusion method

in sitting drops by mixing 50 nL of protein solution with 50 nL of

reservoir solution. The reservoir solution for the MR C808S/S810L−

spironolactone complex contained 0.1 M MES, pH 7.0, 1.26 M lithium

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sulfate, and 6% PEG 2000 monomethyl ether, and that for the MR

C808S/S810L/A976V−1d complex contained 0.1 M HEPES, pH 7.4,

0.88 M potassium/sodium tartrate, and 5% ethylene glycol. Prior to

data collection, crystals were immersed in mother liquor solution

containing 25% ethylene glycol and flash frozen in liquid nitrogen.

Diffraction data were collected from a single crystal at Advance Light

Source on beamline 5.0.3 using a Quantum210 CCD detector

(ADSC) under a 100 K nitrogen cryostream. The data were reduced

and scaled using HKL2000.¹⁷The structures were solved by molecular

replacement with MOLREP¹⁸from the CCP4 suites¹⁹using the

coordinates of the MRS810L-LBD associated with spironolactone

(PDB code 2AB2) as a search model. The structures were refined

through an iterative procedure utilizing REFMAC²⁰followed by model

building in COOT.²¹The dictionary files for the ligands were prepared

using AFITT (OpenEye Scientific Software). The final models were

validated using Molprobity.²²Crystallographic processing and refine-

ment statistics are summarized in Supporting Information (Table S1).

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Eplerenone, a Selective Aldosterone Blocker, in Patients with Left

Ventricular Dysfunction after Myocardial Infarction. N. Engl. J. Med.

2003, 348, 1309−1321.

(10) (a) Mehdi, U. F.; Adams-Huet, B.; Raskin, P.; Vega, G. L.; Toto,

R. D. Addition of Angiotensin Receptor Blockade or Mineralocorticoid

Antagonism to Maximal Angiotensin-Converting Enzyme Inhibition in

Diabetic Nephropathy. J. Am. Soc. Nephrol. 2009, 20, 2641−2650.

(b) Sato, A.; Hayashi, K.; Naruse, M; Saruta, T. Effectiveness of

Aldosterone Blockade in Patients with Diabetic Nephropathy.

Hypertension 2003, 41, 64−68.

All structural figures were generated using PyMOL (Schrodinger,

̈

LLC).

ASSOCIATED CONTENT

■

S

* Supporting Information

Crystallographic data and details of refinement for compound

1d and spironolactone; docking model of compound 14n. This

material is available free of charge via the Internet at http://

pubs.acs.org.

Accession Codes

^†Atomic coordinates and structure factors have been deposited

in the Protein Data Bank with codes 3VHU and 3VHV for

spironolactone and compound 1d, respectively.

AUTHOR INFORMATION

■

Corresponding Author

*Phone: +81-466-32-1142. Fax: +81-466-29-4468;. E-mail:

Tomoaki_Hasui@takeda.co.jp.

(11) de Gasparo, M.; Whitebread, S. E.; Preiswerk., G.; Jeunemaitre,

X.; Corvol, P.; Menard, J. Antialdosterones: Incidence and Prevention

of Sexual Side Effects. J. Steroid Biochem. 1989, 32, 223−227.

(12) Sica, D. A. Pharmacokinetics and Pharmacodynamics of

Mineralocorticoid Blocking Agents and Their Effects on Potassium

Homeostasis. Heart Failure Rev. 2005, 10, 23−29.

ACKNOWLEDGMENTS

■

The authors thank Keiji Kubo, Masatoshi Hazama, and Keiji

Kusumoto for helpful discussions, and Maori Kouno for sup-

porting the in vitro experiments.

(13) (a) Meyers, M. J.; Hu, X. Non-Steroidal Mineralocorticoid

Receptor Antagonists. Expert Opin. Ther. Pat. 2007, 17, 17−23, and

references therein. (b) Bell, M. G.; Gernert, D. L.; Grese, T. A.; Belvo,

M. D.; Borromeo, P. S.; Kelley, S. A.; Kennedy, J. H.; Kolis, S. P.;

Lander, P. A.; Richey, R.; Sharp, V. S.; Stephenson, G. A.; Williams,

J. D.; Yu, H.; Zimmerman, K. M.; Steinberg, M. I.; Jadhav, P. K. (S)-N-

{3-[1-Cyclopropyl-1-(2,4-difluoro-phenyl)-ethyl]-1H-indol-7-yl}-

methanesulfonamide: A Potent, Nonsteroidal, Functional Antagonist

of the Mineralocorticoid Receptor. J. Med. Chem. 2007, 50, 6443−

6445. (c) 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. Discovery of (3S,3aR)-2-(3-Chloro-4-cyanophenyl)-3-cyclopentyl-

3,3a,4,5-tetrahydro-2H-benzo[g]indazole-7-carboxylic Acid (PF-

3882845), an Orally Efficacious Mineralocorticoid Receptor (MR)

Antagonist for Hypertension and Nephropathy. J. Med. Chem. 2010,

53, 5979−6002. (d) Jiang, C. S.; Zhou, R.; Gong, J. X.; Chen, L. L.;

ABBREVIATIONS USED

■

MR, mineralocorticoid receptor; HTS, high-throughput screen-

ing; AR, androgen receptor; PR, progesterone receptor; GR,

glucocorticoid receptor; DOCA, deoxycorticosterone acetate;

SBP, systolic blood pressure; HR, heart rate; EtOAc, ethyl

acetate; THF, tetrahydrofuran; MeOH, methanol; EtOH,

ethanol; DMF, N,N-dimethylformamide; CH₃CN, acetonitrile;

IPE, diisopropyl ether

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Identification of benzoxazin-3-one derivatives as novel, potent, and selective nonsteroidal mineralocorticoid receptor antagonists

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