Design, synthesis, and structure-activity relationships of dihydrofuran-2-one and dihydropyrrol-2-one derivatives as novel benzoxazin-3-one-based mineralocorticoid receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2013.07.043

Dihydrofuran-2-one and dihydropyrrol-2-one derivatives were identified as novel, potent and selective mineralocorticoid receptor (MR) antagonists by the structure-based drug design approach utilizing the crystal structure of MR/compound complex. Introduct

Full text of DOI:10.1016/j.bmc.2013.07.043

Bioorganic & Medicinal Chemistry 21 (2013) 5983–5994
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and structure–activity relationships of
dihydrofuran-2-one and dihydropyrrol-2-one derivatives as novel
benzoxazin-3-one-based mineralocorticoid receptor antagonists
a
a
a
a
a
Tomoaki Hasui a, , Taiichi Ohra , Norio Ohyabu , Kouhei Asano , Hideki Matsui , Atsushi Mizukami ,
Noriyuki Habuka a, Satoshi Sogabe a, Satoshi Endo a, Christopher S. Siedem b, Tony P. Tang b,
Cassandra Gauthier b, Lisa A. De Meese b, Steven A. Boyd b, Shoji Fukumoto a
⇑
a Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi, 2-Chome, Fujisawa, Kanagawa 251-8555, Japan
b Array 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:
Dihydrofuran-2-one and dihydropyrrol-2-one derivatives were identified as novel, potent and selective
mineralocorticoid receptor (MR) antagonists by the structure-based drug design approach utilizing the
crystal structure of MR/compound complex. Introduction of lipophilic substituents directed toward the
unfilled spaces of the MR and identification of a new scaffold, dihydropyrrol-2-one ring, led to potent
in vitro activity. Among the synthesized compounds, dihydropyrrol-2-one 11i showed an excellent
in vitro activity (MR binding IC50 = 43 nM) and high selectivity over closely related steroid receptors such
as the androgen receptor (AR), progesterone receptor (PR) and glucocorticoid receptor (GR) (>200-fold for
AR and PR, 100-fold for GR).
Received 20 June 2013
Revised 19 July 2013
Accepted 22 July 2013
Available online 31 July 2013
Keywords:
Mineralocorticoid receptor
MR
Nonsteroidal
Aldosterone
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
We have already reported benzoxazin-3-one derivatives as no-
vel nonsteroidal MR antagonists.9 Among them, 3-trifluoromethyl
The mineralocorticoid receptor (MR) is a member of the steroid
receptor sub-family within the nuclear hormone receptor super-
family, and it plays an important role in blood pressure control
through the regulation of body fluid and electrolyte balance.1
Abnormal activation of the MR by excessive levels of aldosterone,
a primary natural ligand for the MR, is known to cause cardiovas-
cular diseases such as hypertension and congestive heart failure.2
Accordingly, MR blockade is an attractive therapeutic option for
such diseases. Indeed, available steroidal MR antagonists, spirono-
lactone3 and eplerenone,4 have proven to be clinically useful for
those diseases.5 However, spironolactone treatment is limited by
the sex hormone-related side effect such as impotence, gyneco-
mastia, and menstrual irregularity caused by the lack of selectivity
over androgen receptor (AR) and progesterone receptor (PR).6
Meanwhile, the more selective antagonist, eplerenone, shows few
side effects but its potency is lower than spironolactone.7 Accord-
ingly, a new, potent and selective MR antagonist would be a clini-
cally useful drug. Recently, several nonsteroidal MR antagonists
were disclosed,8 but their clinical utilities have not been proven.
pyrazole 1 showed highly potent in vitro activity (MR binding
IC50 = 41 nM) and good selectivity over the AR, PR, and glucocorti-
coid receptor (GR) (Fig. 1). While compound 1 showed little affinity
for the AR, it still had moderate binding activity for the PR (AR
binding IC50 >10,000 nM, PR binding IC50 = 1900 nM), suggesting
that we should seek to reduce PR binding to minimize the potential
for sex hormone-related side effects. Thus, we aimed to identify
compounds with potent MR binding affinities with little affinities
for the AR and PR.
Initially, we explored a new lead compound (Fig. 2). Our previ-
ous study of benzoxazin-3-one derivatives suggested that a key
pharmacophore for a potent MR binding affinity consisted of three
parts: a benzoxazin-3-one ring, a 4-fluorobenzene ring, and a cen-
tral cycle linking these two aromatic rings at the adjacent
position.9 Among these parts, the central ring moiety tended to al-
low the easiest introduction of structural diversity. In addition to
monocyclic aromatic rings, fused-heterocycles such as triazolothi-
adiazines and benzothiazines also exhibited potent MR antagonis-
tic activity as central rings. Importantly, in the course of our
previous research, we found the modification of central ring moi-
ety affected not only MR binding affinity but also steroid receptor
selectivity. Therefore, our early efforts were focused on the identifi-
cation of new lead scaffolds other than 3-trifluoromethylpyrazole.
⇑
Corresponding author. Tel.: +81 466 32 1142.
E-mail address: tomoaki.hasui@takeda.com (T. Hasui).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.043

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T. Hasui et al. / Bioorg. Med. Chem. 21 (2013) 5983–5994
Figure 1. Structure of steroidal and nonsteroidal MR antagonists
Figure 2. Identification of new lead compound 2a.
Since non-aromatic heteromonocycles had still been poorly-
explored, variety of five- or six-membered non-aromatic
has already stabilized the twisted conformation and thereby the
synergistic effect with the fluorine atom or the methyl group at
the 2-position was not observed. Other substituted benzene rings
were also investigated; however the 4-fluorobenzene was best
among them (data not shown). Accordingly, the 4-fluorobenzene
ring was fixed during the following lead optimization process.
The X-ray co-crystal structure of the MR/compound 2b complex
was utilized for compound design (Fig. 3a). As expected, compound
2b bound to the ligand binding domain of MR in a similar manner
to compound 1. Characteristically, the carbonyl group in the dihy-
drofuran-2-one scaffold formed a hydrogen bond with Arg817 and
Gln776 via two water molecules, and the carbonyl group of the
benzoxazin-3-one moiety newly established an additional hydro-
gen bond interaction with Thr945. These interactions were consid-
ered to be important for the binding affinity of dihydrofuran-2-one
derivatives. Moreover, Figure 3a revealed that compound 2b did
not fully occupy the ligand binding site of the MR, suggesting that
space for lead optimization still remained. On the basis of this find-
ing, we hypothesized that the MR binding affinity could be in-
creased by the introduction of a lipophilic group to fill spaces ‘A’,
‘B’ and/or ‘C’ shown in Figure 3a, or by the introduction of a polar
a
heterocycles were investigated. As a result, we selected the dihy-
drofuran-2-one derivative 2a as a lead compound, because it
showed moderate MR binding activity (IC50 = 500 nM) with low
activity for AR, PR and GR (IC50 >10,000 nM, for each receptor),
and had a ‘leadlike’ profile, such as low molecular weight and
low lipophilicity. In this paper, we describe the lead optimization
of 2a by a structure-based drug design approach utilizing the
X-ray co-crystal structure of the MR/compound complex obtained
in-house.
To establish the initial SAR, the 4-fluorobenzene ring of 2a was
modified (Table 1). In compound 1, introduction of a methyl group
at the 2-position on the 4-fluorobenzene ring significantly en-
hanced the binding affinity, probably due to the stabilization effect
of the preferred twisted conformation between the central ring and
the 4-fluorobenzene ring.9 Therefore, a fluorine atom or methyl
group was introduced into the corresponding position of the lead
compound 2a. However, these compounds (2b, c) showed no
advantage in terms of affinity and selectivity. From this result,
we speculated that the carbonyl group of dihydrofuran-2-one ring
Table 1
Effect of introduction of substituents into the 4-fluorobenzene ringa
Compound No.
R
MR binding IC50 (nM)
Selectivity
AR binding IC50 (nM)
PR binding IC50 (nM)
GR binding IC50 (nM)
2a
2b
2c
H
F
Me
500
660
750
>10,000
>10,000
>10,000
>10,000
4600
1800
>10,000
>10,000
>10,000
a
IC50 values were shown as the means of duplicate experiments. IC50 values are calculated from the concentration–response curves generated by GraphPad Prism.

T. Hasui et al. / Bioorg. Med. Chem. 21 (2013) 5983–5994
5985
Figure 3. (a) X-ray co-crystal structure of the MR/compound 2b complex. Orange dot means hydrogen bond. ‘A’, ‘B’ and ‘C’ means the unfilled spaces available for lead
optimization. (b) Compound design for potent MR binding affinity.
group at space ‘B’ to form a hydrogen bond with Arg817 or Gln776,
directly or via water molecule. Thus, the substituted dihydrofuran-
2-one and dihydropyrrol-2-one derivatives, shown as compound
(I), were designed (Fig. 3b).
Scheme 2, and then converted to triflate 18 as a key precursor
for a coupling reaction. Suzuki coupling of 18 with the boronic acid
ester 19 gave compound 20 in good yields, and the subsequent
deprotection of the 4-methoxybenzyl group afforded the N-unsub-
stituted dihydropyrrol-2-ones 21a, b.
2. Chemistry
Dihydrofuran-2-ones 2a–g were prepared according to
3. Results and discussion
Scheme 1. Condensation of the a-haloketone 5 with the carboxylic
acid 6 followed by base-mediated cyclization–dehydration affor-
ded the dihydrofuran-2-ones 2a–e, g. Electrophilic fluorination of
2e with N-fluorobenzenesulfonimide gave the 5-fluoro compound
2f.
In our first synthetic studies, several alkyl substituents were
introduced at the 5-position on the dihydrofuran-2-one ring to fill
space ‘A’ of the ligand binding site (Table 2). The 5,50-dimethyl ana-
log 2d showed decreased activity, most likely because the dimethyl
group might be too bulky for this position. In contrast, the mono-
methyl derivative showed a fourfold increase in binding inhibition,
as expected (2e). Introduction of a fluorine atom onto compound
2e and a replacement of the methyl group with an ethyl group re-
sulted in decreased binding activity (2f, 2g). The X-ray co-crystal
structure obtained with the MR and compound 2e (racemate)
showed that the (5S)-methyl group occupied space ‘A’ with little
conformational change of the ligand as designed (Fig. 4), and also
explained the SAR described above. Moreover, the (5R)-methyl
group appeared to clash with the wall of the MR and/or to cause
The dihydropyrrol-2-ones 11a–i were synthesized as shown in
Scheme 2. Condensation of the
a-haloketone 5 or 8 with primary
amines followed by acylation with 4-fluorophenylacetyl chloride
gave the amides 10a–f. The subsequent base-mediated cycliza-
tion-dehydration afforded the dihydropyrrol-2-ones 11a–d, f, h.
Acid-mediated deprotection of the tetrahydropyranyl group or of
the 4-methoxybenzyl group gave the hydroxyl ethyl compound
11e or the NH compounds 11g, i, respectively.
Synthesis of compounds 21a, b is shown in Scheme 3. Com-
pound 17 was prepared by a method similar to that described in
Scheme 1. Synthesis of dihydrofuran-2-one derivatives 2a–g. Reagents and conditions: (a) AlCl3, ClCOCRR0Cl, ClCH2CH2Cl, 37–99%; (b) AlCl3, ClCOCRR0H, ClCH2CH2Cl, 16%; (c)
HBr3ꢀPy, HBr, AcOH, 69–92%; (d) (1) carboxylic acid 6, TEA, DMF; (2) DBU, under nitrogen atmosphere, 15–65% in 2 steps; (e) 6, K2CO3, BnNEt3Cl, DMA, 11%; (f) N-
fluorobenzenesulfonimide, DBU, THF, 24%.

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T. Hasui et al. / Bioorg. Med. Chem. 21 (2013) 5983–5994
Scheme 2. Synthesis of dihydropyrrol-2-ones 11a–d, 11g, 11i. Reagents and conditions: (a) RNH2, THF/H2O, 20–90%; (b) 4-fluorophenylacetyl chloride, K2CO3, THF/H2O, 25–
86%; (c) KOtBu, tBuOH, under nitrogen atmosphere, 28–86%; (d) PTSA, MeOH, 61%; (e) TFA, anisole, 64–68%; (f) AlCl3, ClCOCH(CH3)Br, CH2Cl2, 89%; (g) NaNO2, H2SO4,AcOH,
51%; (h) Zn, AcOH; (i) (1) ClCOCH2Cl, K2CO3; (2) reflux, 62% (2 steps); (j) HBr3ꢀPy, HBr, AcOH, 84%
Scheme 3. Synthesis of dihydropyrrol-2-one 21a,b. Reagents and conditions: (a) PMBNH2, THF/H2O, 90%; (b) 4-F-BnCOCl, K2CO3, THF/H2O, 81%; (c) NaH, DMF, under nitrogen
atmosphere, 93%; (d) Tf2O, TEA, CH2Cl2, 71%; (e) boronic acid ester 19, 20 mol % PdCl2(dppf)ꢀCH2Cl2, Cs2CO3, THF/H2O, 42–64%; (f) TFA, anisole, 57–78%; (g)
bis(pinacolato)diboron, PdCl2(dppf)ꢀCH2Cl2, KOAc, dioxane, 99%.
significant change in the conformation of the ligand. These obser-
vations suggested that the (S)-enantiomer should be more potent
than the (R)-enantiomer, and that chiral separation of the racemate
would increase the binding affinity. This hypothesis was further
supported by the finding that dimethyl compound 2d was less po-
tent than the monomethyl compound 2e. As for selectivity towards
the other steroidal receptors, the 5-methyl group also increased
the binding activity for PR and GR. On the basis of the increase in
MR binding, 5-methyl dihydrofuran-2-one ring was selected as
the template for further optimization.
In our second synthetic studies, we switched the central scaf-
fold to a dihydropyrrol-2-one ring and introduced various substit-
uents directed toward space ‘B’, as listed in Table 3. First, a methyl,
ethyl or cyclopropyl group was introduced at the 1-position to fill
space ‘B’ (11a–c). Whereas the methyl derivative 11a was slightly
less potent than 2e, the bulkier ethyl and cyclopropyl derivatives
11b, c showed increased binding activity as expected. Next, a
hydroxyethyl group was introduced with the aim of forming a
hydrogen bond interaction with Arg817, Gln776, and/or a water
molecule (11e). However, 11e showed significantly decreased MR

T. Hasui et al. / Bioorg. Med. Chem. 21 (2013) 5983–5994
5987
Table 2
SAR summary for space ‘A’a
Compound No.
R1
R2
MR binding IC50 (nM)
AR binding IC50 (nM)
Selectivity
PR binding IC50 (nM)
GR binding IC50 (nM)
2d
2e
2f
Me
Me
Me
Et
Me
H
F
1200
120
550
>10,000
>10,000
>10,000
>10,000
1000
740
5000
2200
4700
3800
>10,000
2000
2g
H
1200
a
IC50 values were shown as the means of duplicate experiments. IC50 values are calculated from the concentration–response curves generated by GraphPad Prism.
In our third round of synthetic studies, we sought to further in-
crease the binding affinity by filling space ‘C’ with small substitu-
ents (F, Cl and Me) introduced at the 5-position of the
benzoxazin-3-one ring (Table 4). While introduction of a fluorine
atom had little impact on potency (21a), incorporation of a chlo-
rine atom (21b) or a methyl group (11i) increased the binding
activity as expected. Notably, their activities were equipotent to
that of the spironolactone. Whereas the chlorine atom also in-
creased PR binding activity (21b: PR binding IC50 = 1600 nM), the
methyl group decreased PR binding activity (11i: PR binding IC50
>10,000 nM). These results indicated that space ‘C’ in the PR might
be slightly narrower than that in the MR. Consequently, compound
11i showed highly potent MR binding activity (MR binding
IC50 = 43 nM) and high selectivity over AR, PR, and GR (> 200-fold
for AR and PR, 100-fold for GR), suggesting that the potential for
sex hormone-related side effects with 11i may be significantly re-
duced compared with that of compound 1.
Functional activity was evaluated in a reporter gene assay in COS-
1 cells, and compound 11i exhibited potent MR antagonistic activity
(IC50 = 83 nM) comparable to spironolactone (IC50 = 60 nM).
Figure 4. Overlay of a compound 2b (green) and compound 2e (yellow) in MR.
Cocrystallization of MR and compound 2e provided only the crystal of MR/(S)-
enantiomer complex.
4. Conclusion
In summary, we have discovered dihydrofuran-2-one and dihy-
dropyrrol-2-one derivatives as novel, potent and selective MR
binding activity, suggesting that the hydroxyethyl group failed to
antagonists. Exploration of heterocycles in the central ring moiety
establish the desired interaction. Unexpectedly, the unsubstituted
NH compound 11g showed a highly potent binding activity
(IC50 = 94 nM); indeed, it was more potent than the corresponding
dihydrofuran-2-one compound 2e. This result might be explained
resulted in the identification of a new lead scaffold, dihydrofuran-
2-one ring. X-ray co-crystal structure of the MR/compound
complex revealed the binding mode of the dihydrofuran-2-one
derivatives and also guided lead optimization. Introduction of lipo-
by reinforcement of the hydrogen-bond with Arg817 and/or
philic substituents directed toward the unfilled space of the MR
Gln776 via a water molecule due to the increased electron density
into the central core and into the benzoxazin-3-one ring moiety,
of the carbonyl group. These studies provided important SAR data
as well as scaffold replacement with a dihydropyrrol-2-one ring in-
in relation to the PR binding affinity. While the derivatives substi-
creased the MR binding activity. The N-unsubstituted dihydropyr-
rol-2-one was identified as a key scaffold for high selectivity
tuted with small alkyl groups (11a–c) showed moderate to potent
PR binding (IC50 = 61–770 nM), the NH compound 11g exhibited a
against steroid receptors. Thus, we discovered compound 11i,
weak activity (IC50 = 5600 nM). These results suggested that the PR
which showed excellent in vitro activity (MR binding IC50 = 43 nM)
tends to prefer a lipophilic group at space ‘B’, not to a polar one.
and high selectivity over AR, PR, and GR (>200-fold for AR and PR,
Interestingly, we observed a ‘selectivity switch’ between com-
100-fold for GR). Further optimization of the benzoxazin-3-one
derivatives will be reported in due course.
pounds 11a and 11g (11a: MR binding IC50 = 170 nM, PR binding
IC50 = 61 nM vs 11g: MR binding IC50 = 94 nM, PR binding
IC50 = 5600 nM), suggesting that the N-methyl derivative 11a
5. Experimental procedure
might serve as a new lead compound for a selective PR modulator
program.10 Overall, compound 11g showed the best balance of MR
binding activity and selectivity over AR, PR, and GR among com-
pounds 11a–g, and was therefore selected for further optimization.
Proton nuclear magnetic resonance (1H NMR) spectra were re-
corded on Bruker Ultra Shield-300 (300 MHz) or Varian INOVA-

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T. Hasui et al. / Bioorg. Med. Chem. 21 (2013) 5983–5994
Table 3
SAR summary for space ‘B’a
Compound No.
R
MR binding IC50 (nM)
Selectivity
AR binding IC50 (nM)
PR binding IC50 (nM)
GR binding IC50 (nM)
11a
11b
11c
11e
11g
Me
Et
cyclopropyl
CH2CH2OH
H
170
33
68
1600
94
>10,000
>10,000
>10,000
>10,000
>10,000
61
770
400
>10,000
5600
2100
1600
2200
>10,000
9100
a
IC50 values were shown as the means of duplicate experiments. IC50 values are calculated from the concentration–response curves generated by GraphPad Prism.
Table 4
SAR summary for space ‘C’a
Compound No.
R
MR binding IC50 (nM)
Selectivity
AR binding IC50 (nM)
PR binding IC50 (nM)
GR binding IC50 (nM)
11g
21a
21b
11i
H
F
94
92
23
43
41
49
2600
>10,000
>10,000
>10,000
>10,000
>10,000
120
5600
3200
1600
>10,000
1900
9100
5000
1100
4900
1800
1400
>10,000
Cl
Me
—
—
—
1
Spironolactone
Eplerenone
650
>10,000
>10,000
a
IC50 values were shown as the means of duplicate experiments. IC50 values are calculated from the concentration–response curves generated by GraphPad Prism.
400 (400 MHz) instruments. Chemical shifts are given in parts per
million (ppm) with tetramethylsilane as an internal standard. Peak
multi-plicities are expressed as follows: s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet, dd = doublets of doublet, 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 li-
quid chromatography (HPLC). The HPLC analyses were performed
using a Shimadzu UFLC instrument, equipped with a L-column 2
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 150 mL/
min, with UV detection at 220 nm. Reagents and solvents were ob-
tained from commercial sources and used without further purifica-
tion. Abbreviations of solvents are used as follows: CDCl3,
deuterated chloroform; DMSO-d6, dimethyl sulfoxide-d6; EtOAc,
ethyl acetate; DMF, N,N-dimethylformamide; MeOH, methanol;
THF, tetrahydrofuran; EtOH, ethanol; DMSO, dimethyl sulfoxide;
DMA, N,N-dimethylactamide.
5.1. 6-Isobutyryl-2H-1,4-benzoxazin-3(4H)-one (4b)
ODS (3.0 ꢁ 50 mm, 2
lm) column, eluting 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. 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 (Chromat-
orex NH-DM 1020, 100–200 mesh size)] or on Purif-Pack (SI or NH,
To a stirred suspension of 2H-1,4-benzoxazin-3(4H)-one (15 g,
100 mmol) in 1,2-dichloroethane (180 mL) was added powdered
AlCl3 (30 g, 225 mmol) and isobutyryl chloride (12.6 mL,
120.3 mmol) at 0 °C, successively. The mixture was stirred at
80 °C for 3 h, diluted with water and extracted with dichlorometh-
ane. The organic layer was dried over anhydrous MgSO4, and con-
centrated under reduced pressure. The residue was crystallized
from EtOAc/hexane to give 4b (3.6 g, 16%). 1H NMR (300 MHz,
CDCl3) d 1.21 (6H, d, J = 7.2 Hz), 3.49 (1H. sep, J = 7.2 Hz), 4.70
(2H, s), 7.03 (1H, d, J = 9.0 Hz), 7.50 (1H, s), 7.61 (1H, d,
J = 9.0 Hz), 8.16 (1H, br s).
particle size: 60
purification was performed by using a Waters Corporation UV
purification system equipped with Develosil ODS-UG-10
(4.6 ꢁ 150 mm, 5 m) column, and eluted with a gradient of 5–
lm, Fuji Silysia Chemical, Ltd). Preparative HPLC
a
l

T. Hasui et al. / Bioorg. Med. Chem. 21 (2013) 5983–5994
5989
5.2. 6-(Bromoacetyl)-2H-1,4-benzoxazin-3(4H)-one (5a)
7.60 (2H, m), 10.82 (1H, s). Anal. Calcd for C18H12FNO4: C, 66.46;
H, 3.72; N, 4.31. Found: C, 66.40; H, 3.75; N, 4.35.
To a stirred mixture of 4a (20 g, 104.6 mmol), acetic acid
(180 mL) and 25% hydrogen bromide in acetic acid (45 mL) was
added portionwise pyridinium hydrobromide perbromide (35 g,
109.8 mmol) at room temperature. The mixture was stirred at
room temperature for 3 h, and then the solvent was removed in va-
cuo. The residue was suspended in EtOAc. The solid was collected
by filtration, and washed with water and EtOAc to give 5a (26.1 g,
92%). 1H NMR (300 MHz, DMSO-d6) d 4.71 (2H, s), 4.81 (2H, s), 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, s).
5.8. 6-[4-(4-Fluoro-2-methylphenyl)-5-oxo-2,5-dihydrofuran-3-
yl]-2H-1,4-benzoxazin-3(4H)-one (2c)
Yield (15%). 1H NMR (300 MHz, DMSO-d6) d 2.13 (3H, s), 4.54
(2H, s), 5.29 (2H, d, J = 2.1 Hz), 6.77–6.86 (3H, m), 6.95–7.18 (3H,
m), 10.75 (1H, s). Anal. Calcd for C19H14FNO4: C, 67.25; H, 4.16;
N, 4.13. Found: C, 66.97; H, 4.22; N, 4.08.
5.9. 6-[4-(4-Fluorophenyl)-2-methyl-5-oxo-2,5-dihydrofuran-3-
yl]-2H-1,4-benzoxazin-3(4H)-one (2e)
5.3. 6-(2-Chloropropanoyl)-2H-1,4-benzoxazin-3(4H)-one (5b)
Yield (42%). 1H NMR (300 MHz, DMSO-d6) d 1.34 (3H, t,
J = 6.8 Hz), 4.63 (2H, s), 5.76 (1H, q,J = 6.8 Hz), 6.80 (1H, d,
J = 1.9 Hz), 6.90–7.07 (2H, m), 7.18–7.20 (2H, m), 7.33–7.42 (2H,
m), 10.75 (1H, s). Anal. Calcd for C19H14FNO4: C, 67.25; H, 4.16;
N, 4.13. Found: C, 67.10; H, 4.29; N, 4.06.
To a suspension of2H-1,4-benzoxazin-3(4H)-one(5 g, 33.5 mmol)
in 1,2-dichloroethane (50 mL) was added powdered AlCl3 (9.8 g,
73.7 mmol) and 2-chloropropionyl chloride (3.9 mL, 40.2 mmol) at
0 °C, successively. The mixture was stirred at room temperature for
12 h, poured into ice-cooled water. The precipitate was collected by
filtration and washed with 1 N HCl, water and IPE to give 5b (8.0 g,
99%). 1H NMR (300 MHz, DMSO-d6) d 1.60 (3H, d, J = 6.6 Hz), 4.71
(2H, s), 5.65 (1H, q, J = 6.6 Hz), 7.08 (1H, d, J = 8.7 Hz), 7.54 (1H, d,
J = 2.3 Hz), 7.70 (1H, dd, J = 8.7, 2.3 Hz), 10.91 (1H, s).
5.10. 6-[2-Ethyl-4-(4-fluorophenyl)-5-oxo-2,5-dihydrofuran-3-
yl]-2H-1,4-benzoxazin-3(4H)-one (2g)
Yield (65%). 1H NMR (300 MHz, DMSO-d6) d 0.80 (3H, t,
J = 7.3 Hz), 1.41–1.56 (1H, m), 1.80–1.96 (1H, m), 4.63 (2H, s),
5.72 (1H, dd, J = 7.3, 3.6 Hz), 6.79–6.80 (1H, m), 6.89–7.11 (2H,
m), 7.17–7.31 (2H, m), 7.31–7.55 (2H, m), 10.74 (1H, s). Anal. Calcd
for C20H16FNO4ꢀ0.2H2O: C, 67.30; H, 4.63; N, 3.92. Found: C, 67.15;
H, 4.75; N, 3.60.
5.4. 6-(2-Chlorobutanoyl)-2H-1,4-benzoxazin-3(4H)-one (5c)
The compound 5c was prepared in a manner similar to that de-
scribed for 5b. Yield (37%). 1H NMR (300 MHz, DMSO-d6) d 0.98
(3H, t, J = 7.2 Hz), 1.78–2.13 (2H, m), 4.71 (2H, s), 5.50 (1H, dd,
J = 7.8, 5.5 Hz), 7.08 (1H, d, J = 8.3 Hz), 7.53 (1H, d, J = 2.3 Hz),
7.71 (1H, dd, J = 8.3, 2.3 Hz), 10.91 (1H, s).
5.11. 6-[4-(4-Fluorophenyl)-2,2-dimethyl-5-oxo-2,5-
dihydrofuran-3-yl]-2H-1,4-benzoxazin-3(4H)-one (2d)
5.5. 6-(2-Bromo-2-methylpropanoyl)-2H-1,4-benzoxazin-3(4H)-
one (5d)
A mixture of 5d (300 mg, 1.01 mmol), 4-fluorophenylacetic acid
(155 mg, 1.01 mmol), potassium carbonate (277 mg, 2.01 mmol)
and benzyltriethylammonium chloride (11.4 mg, 0.05 mmol) in
DMA (5 mL) was stirred at 50 °C for 2 days and at 100 °C for 3 days,
diluted with 1 N HCl and extracted with EtOAc. The organic layer
was dried over anhydrous MgSO4, and concentrated under reduced
pressure. The residue was purified by column chromatography on
silica gel with hexane/EtOAc (5:2) as an elutant to give 2d (38 mg,
11%). 1H NMR (300 MHz, DMSO-d6) d 1.54 (6H, s), 4.63 (2H, s),
6.76–6.89 (2H, m), 7.00 (1H, d, J = 8.7 Hz), 7.11–7.22 (2H, m),
7.29–7.48 (2H, m), 10.72 (1H, br s). Anal. Calcd for C20H16FNO4:
C, 67.98; H, 4.56; N, 3.96. Found: C, 67.64; H, 4.64; N, 4.04.
The compound 5d was prepared in a manner similar to that de-
scribed for 5a. Yield (69%). 1H NMR (300 MHz, CDCl3) d 2.03 (6H, d,
J = 7.2 Hz), 4.70 (2H, s), 7.00 (1H, d, J = 8.7 Hz), 7.64 (1H, d,
J = 1.8 Hz), 7.96 (1H, dd, J = 1.8, 8.7 Hz), 8.10 (1H, br s).
5.6. 6-[4-(2,4-Difluorophenyl)-5-oxo-2,5-dihydrofuran-3-yl]-
2H-1,4-benzoxazin-3(4H)-one (2b)
To a stirred solution of 5a (300 mg, 1.11 mmol) and triethyl-
amine (281 lL, 2.02 mmol) in DMF (5 mL) was added 2.4-difuluor-
ophenylacetic acid (173 mg, 1.01 mmol) at room temperature. The
5.12. 6-[2-Fluoro-4-(4-fluorophenyl)-2-methyl-5-oxo-2,5-
mixture was stirred at room temperature for 3 h. To the mixture
dihydrofuran-3-yl]-2H-1,4-benzoxazin-3(4H)-one (2f)
was added DBU (304 lL, 2.02 mmol). The mixture was stirred at
room temperature for 12 h under nitrogen atmosphere, diluted
with water, and extracted with EtOAc. The organic layer was dried
over anhydrous MgSO4, and concentrated under reduced pressure.
The residue was purified by column chromatography on silica gel
with hexane/EtOAc (1:1) as an elutant, and crystallized from hex-
ane/EtOAc to give 2b (74 mg, 19%). 1H NMR (300 MHz, DMSO-d6) d
4.62 (2H, s), 5.43 (2H, s), 6.83 (1H, s), 7.01 (2H, s), 7.12–7.29 (1H,
m), 7.30–7.60 (2H, m), 10.82 (1H, br s). Anal. Calcd for C18H11F2-
NO4: C, 62.98; H, 3.23; N, 4.08. Found: C, 62.81; H, 3.29; N, 3.98.
The compound 2a, 2c–e, 2g were prepared in a manner similar
to that described for 2b.
Under argon atmosphere, to a stirred solution of 2e (500 mg,
1.47 mmol) in THF (10 mL) were added DBU (666
and solution of N-fluorobenzenesulfonimide (700 mg,
2.21 mmol) in THF (4 mL) at ꢂ78 °C, successively. The mixture
was stirred for 1 h, and then DBU (666 L, 4.33 mmol) and a solu-
lL, 4.33 mmol)
a
l
tion of N-fluorobenzenesulfonimide (700 mg, 2.21 mmol) in THF
(4 mL) were added. The mixture was stirred at ꢂ78 °C for 30 min,
quenched with ammonium chloride solution and 1 N HCl and ex-
tracted with EtOAc. The organic layer was dried over anhydrous
MgSO4, and concentrated under reduced pressure. The residue
was purified by column chromatography on silica gel with hex-
ane/EtOAc as an elutant and by preparative HPLC to give 2f
(129 mg, 24%). 1H NMR (300 MHz, DMSO-d6) d 1.82 (3H, d,
J = 18.9 Hz), 4.65 (2H, s), 6.84–7.06 (2H, m), 7.21–7.36 (2H, m),
7.37–7.47 (2H, m), 10.80 (1H, br s). Anal. Calcd for C19H13F2NO4:
C, 63.87; H, 3.67; N, 3.92. Found: C, 63.70; H, 3.62; N, 4.08.
5.7. 6-[4-(4-Fluorophenyl)-5-oxo-2,5-dihydrofuran-3-yl]-2H-
1,4-benzoxazin-3(4H)-one (2a)
Yield (56%). 1H NMR (300 MHz, DMSO-d6) d 4.62 (2H, s), 5.43
(2H, s), 6.83 (1H, s), 6.98–7.02 (2H, m), 7.12–7.29 (1H, m), 7.30–

5990
T. Hasui et al. / Bioorg. Med. Chem. 21 (2013) 5983–5994
5.13. 6-(2-Bromopropanoyl)-2H-1,4-benzoxazin-3(4H)-one (8a)
5.20. 6-{2-[(4-Mthoxybenzyl)amino]propanoyl}-8-methyl-2H-
1,4-benzoxazin-3(4H)-one (9f)
The compound 8a was prepared in a manner similar to that de-
scribed for 5b. Yield (89%). 1H NMR (300 MHz, DMSO-d6) d 1.76
(3H, d, J = 6.8 Hz), 4.71 (2H, s), 5.69 (1H, q, J = 6.8 Hz), 7.07 (1H,
d, J = 8.3 Hz), 7.54 (1H, d, J = 2.3 Hz), 7.70 (1H, dd, J = 8.3, 2.3 Hz),
10.90 (1H, s).
A
mixture of 8b (1 g, 3.35 mmol), 4-methoxybenzylamine
(1.38 g, 10.1 mmol) and triethylamine (0.89 mL, 6.71 mmol) in
THF/water (10 mL/5 mL) was stirred at 50 °C for 2 h, diluted
with water and extracted with EtOAc. The organic layer was
dried over anhydrous MgSO4, and concentrated under reduced
pressure. The residue was crystallized form EtOAc/hexane to give
9e (0.86 g, 72%). 1H NMR (300 MHz, DMSO-d6) d 1.18 (3H, d,
J = 6.8 Hz), 2.13–2.46 (4H, m), 3.47–3.67 (2H, m), 3.72 (3H, s),
4.21 (1H, q, J = 6.8 Hz), 4.70 (2H, s), 6.84–6.85 (2H, m), 7.19–
7.23 (2H, m), 7.36 (1H, d, J = 1.9 Hz), 7.47–7.48 (1H, m), 10.56
(1H. br s).
5.14. 6-(2-Bromopropanoyl)-8-methyl-2H-1,4-benzoxazin-
3(4H)-one (8b)
The compound 8b was prepared in a manner similar to that de-
scribed for 5a. Yield (84%). 1H NMR (300 MHz, DMSO-d6) d 1.75
(3H, d, J = 6.4 Hz), 2.22 (3H, s), 4.72 (2H, s), 5.68 (1H, q,
J = 6.4 Hz), 7.40 (1H, d, J = 1.9 Hz), 7.62 (1H, d, J = 1.9 Hz), 10.85
(1H, s).
5.21. 2-(4-Fluorophenyl)-N-methyl-N-[1-oxo-1-(3-oxo-3,4-
dihydro-2H-1,4-benzoxazin-6-yl)propan-2-yl]acetamide (10a)
5.15. 6-[2-(Methylamino)propanoyl]-2H-1,4-benzoxazin-3(4H)-
one (9a)
To a stirred solution of 9a (200 mg, 0.85 mmol) in THF (2 mL)
were added
a solution of potassium carbonate (354 mg,
2.56 mmol) in water (2 mL) and 4-fluorophenylacetyl chloride
(162 mg, 0.94 mmol) at 0 °C, successively. The mixture was stirred
at room temperature for 30 min, diluted with water, and extracted
with EtOAc. The organic layer was dried over anhydrous MgSO4,
and concentrated under reduced pressure. The residue was sus-
pended in diisopropyl ether and the solid was collected by filtra-
tion to give 10a (240 mg, 76%). 1H NMR (300 MHz, DMSO-d6) d
1.24 (3H, d, J = 6.8 Hz), 2.86 (3H, s), 3.61–3.67 (2H, m), 4.67 (2H,
s), 5.51 (1H, q, J = 6.8 Hz), 6.93–7.15 (5H, m), 7.32–7.44 (2H, m),
10.88 (1H, s).
A mixture of 5b (1 g, 4.17 mmol) and 40% methylamine (15 mL)
in THF (15 mL) was stirred at 40 °C for 12 h, diluted with water and
extracted with EtOAc. The organic layer was dried over anhydrous
MgSO4, and concentrated under reduced pressure. The residue was
purified by column chromatography on NH-silica gel with EtOAc as
an elutant to give 9a (200 mg, 20%). 1H NMR (300 MHz, DMSO-d6) d
1.14 (3H, d, J = 6.8 Hz), 2.20 (3H, s), 4.12 (1H, q, J = 6.8 Hz), 4.68
(2H, s), 7.04 (1H, d, J = 8.3 Hz), 7.53 (1H, d, J = 1.9 Hz), 7.67 (1H,
dd, J = 8.3, 1.9 Hz), 10.85 (1H, br s).
The compound 9b, c was prepared in a manner similar to that
described for 9a.
The compound 10b–f was prepared in a manner similar to that
described for 10a.
5.16. 6-[2-(Ethylamino)propanoyl]-2H-1,4-benzoxazin-3(4H)-
one (9b)
5.22. N-Ethyl-2-(4-fluorophenyl)-N-[1-oxo-1-(3-oxo-3,4-
dihydro-2H-1,4-benzoxazin-6-yl)propan-2-yl]acetamide (10b)
Yield (51%). 1H NMR (300 MHz, DMSO-d6) d 0.98 (3H, t,
J = 7.0 Hz), 1.14 (3H, d, J = 6.8 Hz), 2.35–2.49 (2H, m), 4.22 (2H, q,
J = 6.8 Hz), 4.69 (2H, s), 7.05 (1H, d, J = 8.5 Hz), 7.53 (1H, d,
J = 1.9 Hz), 7.68 (1H, dd, J = 8.5, 1.9 Hz), 10.85 (1H, br s).
Yield (53%). 1H NMR (300 MHz, CDCl3) d 1.13 (3H, t, J = 7.0 Hz),
1.41 (2H, d, J = 6.8 Hz), 3.17–3.49 (2H, m), 3.69 (2H, s), 4.67 (2H, s),
5.97 (1H, q, J = 6.8 Hz), 6.84–6.99 (3H, m), 7.04–7.20 (2H, m), 7.49
(1H, d, J = 2.3 Hz), 7.54–7.67 (1H, m), 9.39 (1H, s).
5.17. 6-[2-(Cyclopropylamino)propanoyl]-2H-1,4-benzoxazin-
3(4H)-one (9c)
5.23. N-Cyclopropyl-2-(4-fluorophenyl)-N-[1-oxo-1-(3-oxo-3,4-
dihydro-2H-1,4-benzoxazin-6-yl)propan-2-yl]acetamide (10c)
The crude product was used for the next reaction.
Yield (25% in 2 steps). 1H NMR (300 MHz, DMSO-d6) d 0.80–1.00
(4H, m), 1.32 (3H, d, J = 6.8 Hz), 2.67–2.77 (1H, m), 3.57–3.93 (2H,
m), 4.65 (2H, s), 4.99 (1H, q, J = 6.8 Hz), 6.87–7.03 (5H, m), 7.16–
7.32 (2H, m), 10.84 (1H, s).
5.18. 2-(4-Fluorophenyl)-N-[1-oxo-1-(3-oxo-3,4-dihydro-2H-
1,4-benzoxazin-6-yl)propan-2-yl]-N-[2-(tetrahydro-2H-pyran-
2-yloxy)ethyl]acetamide (9d)
The crude product was used for the next reaction.
5.24. 6-(2-{[2-(Tetrahydro-2H-pyran-2-
yloxy)ethyl]amino}propanoyl)-2H-1,4-benzoxazin-3(4H)-one
5.19. 6-{2-[(4-Methoxybenzyl)amino]propanoyl}-2H-1,4-
benzoxazin-3(4H)-one (9e)
(10d)
Yield (71% in 2 steps). 1H NMR (300 MHz, CDCl3) d 1.40–1.80
(9H, m), 3.36–3.83 (8H, m), 4.26–4.54 (1H, m), 4.63 (2H, s), 5.49–
5.80 (1H, m), 6.85–7.12 (5H, m), 7.26–7.60 (2H, m), 9.80 (1H, br s).
A mixture of 8a (2 g, 7.04 mmol), 4-methoxybenzylamine (2.9 g,
21.1 mmol) and triethylamine (1.9 mL, 14.08 mmol) in THF/water
(20 mL/5 mL) was stirred at 50 °C for 2 h, diluted with water and
extracted with EtOAc. The organic layer was dried over anhydrous
MgSO4, and concentrated under reduced pressure. The residue was
suspended in IPE and the solid was collected by filtration to give 9d
5.25. 2-(4-Fluorophenyl)-N-(4-methoxybenzyl)-N-[1-oxo-1-(3-
oxo-3,4-dihydro-2H-1,4-benzoxazin-6-yl)propan-2-
yl]acetamide (10e)
(2.15 g, 90%). 1H NMR (300 MHz, DMSO-d6)
d 1.18 (3H, d,
J = 7.0 Hz), 3.43–3.68 (2H, m), 3.72 (3H, s), 4.22 (1H, d, J = 7.0 Hz),
4.69 (2H, s), 6.80–6.91 (2H, m), 7.03 (1H, d, J = 8.3 Hz), 7.18–7.21
(2H, m), 7.51 (1H, d, J = 1.9 Hz), 7.57–7.66 (1H, m). 10.34 (1H, br s).
Yield (70%). 1H NMR (300 MHz, DMSO-d6) d 1.08–1.30 (3H, m),
3.52–3.78 (5H, m), 4.63 (2H, s), 4.67 (2H, s), 5.29 (1H, q, J = 6.8 Hz),
6.84–7.04 (6H, m), 7.09–7.39 (5H, m), 10.84 (1H, s).

T. Hasui et al. / Bioorg. Med. Chem. 21 (2013) 5983–5994
5991
5.26. 2-(4-Fluorophenyl)-N-(4-methoxybenzyl)-N-[1-(8-methyl-
3-oxo-3,4-dihydro-2H-1,4-benzoxazin-6-yl)-1-oxopropan-2-
yl]acetamide (10f)
by column chromatography on silica gel with EtOAc/hexane as
an elutant and crystallized form EtOAc to give 11e (256 mg, 61%).
1H NMR (300 MHz, DMSO-d6) d 1.13 (3H, d, J = 6.6 Hz), 3.19–3.35
(1H, m), 3.53–3.65 (2H, m), 3.75–3.86 (1H, m), 4.59 (2H, s), 4.78–
4.95 (2H, m), 6.62–6.85 (2H, m), 6.88–6.97 (1H, m), 7.09–7.22
(2H, m), 7.28–7.37 (2H, m), 10.66 (1H, s). Anal. Calcd for C21H19FN2O4:
C, 65.96; H, 5.01; N, 7.33. Found: C, 65.72; H, 5.07; N, 7.31.
Yield (86%). 1H NMR (300 MHz, DMSO-d6) d 1.05–1.32 (3H, m),
2.09–2.17 (3H, m), 3.48–3.70 (2H, m), 3.70–3.83 (3H, m), 4.54–4.76
(4H, m), 5.26 (1H, q, J = 6.8 Hz), 6.69–6.95 (2H, m), 6.98–7.03 (4H,
m), 7.09–7.34 (4H, m), 10.78 (1H, br s).
5.32. 6-[4-(4-Fluorophenyl)-1-(4-methoxybenzyl)-2-methyl-5-
oxo-2,5-dihydro-1H-pyrrol-3-yl]-2H-1,4-benzoxazin-3(4H)-one
(11f)
5.27. 6-[4-(4-Fluorophenyl)-1,2-dimethyl-5-oxo-2,5-dihydro-
1H-pyrrol-3-yl]-2H-1,4-benzoxazin-3(4H)-one (11a)
Yield (86%). 1H NMR (300 MHz, DMSO-d6) d 1.10 (3H, d,
J = 6.8 Hz), 3.74 (3H, s), 4.35 (1H, d, J = 15.1 Hz), 4.47 (1H, q,
J = 6.8 Hz), 4.59 (2H, s), 4.89 (1H, d, J = 15.1 Hz), 6.67–6.73 (1H,
m), 6.76–6.83 (1H, m), 6.87–6.97 (3H, m), 7.14–7.29 (4H, m),
7.31–7.42 (2H, m), 10.60 (1H, s).
Under argon atmosphere, to a stirred solution of 10a (240 mg,
0.65 mmol) in THF (5 mL) was added dropwise a solution of potas-
sium tert-buthoxide (181 mg, 1.62 mmol) in tert-buthanol (4 mL)
at 0 °C. The mixture was stirred at room temperature for 30 min,
diluted with water, and extracted with EtOAc. The organic layer
was dried over anhydrous MgSO4, and concentrated under reduced
pressure. The residue was purified by column chromatography on
silica gel with EtOAc/hexane as an elutant and crystallized form
EtOAc/hexane to give 11a (123 mg, 54%). 1H NMR (300 MHz,
DMSO-d6) d 1.13 (3H, d, J = 7.0 Hz), 2.99 (3H, s), 4.58–4.68 (3H,
m), 6.73 (1H, d, J = 1.9 Hz), 6.78–6.88 (1H, m), 6.92–7.00 (1H, m),
7.13–7.24 (2H, m), 7.26–7.38 (2H, m), 10.70 (1H, s). Anal. Calcd
for C20H17FN2O3: C, 68.17; H, 4.86; N, 7.95. Found: C, 68.07; H,
4.85; N, 8.07.
5.33. 6-[4-(4-Fluorophenyl)-1-(4-methoxybenzyl)-2-methyl-5-
oxo-2,5-dihydro-1H-pyrrol-3-yl]-8-methyl-2H-1,4-benzoxazin-
3(4H)-one (11h)
Yield (40%). 1H NMR (300 MHz, DMSO-d6) d 1.10 (3H, d,
J = 6.8 Hz), 2.08 (3H, s), 3.74 (3H, s), 4.33 (1H, d, J = 15.1 Hz), 4.46
(1H, q, J = 6.8 Hz), 4.60 (2H, s), 4.90 (1H, d, J = 15.1 Hz), 6.53 (1H,
d, J = 1.9 Hz), 6.73 (1H, d, J = 1.9 Hz), 6.86–6.97 (2H, m), 7.12–
7.30 (4H, m), 7.30–7.45 (2H, m), 10.53 (1H, s).
The compound 11b–e, 11g was prepared in a manner similar to
that described for 11a.
5.34. 6-[4-(4-Fluorophenyl)-2-methyl-5-oxo-2,5-dihydro-1H-
pyrrol-3-yl]-2H-1,4-benzoxazin-3(4H)-one (11g)
5.28. 6-[1-Ethyl-4-(4-fluorophenyl)-2-methyl-5-oxo-2,5-
dihydro-1H-pyrrol-3-yl]-2H-1,4-benzoxazin-3(4H)-one (11b)
A
mixture of 11e (2 g, 4.36 mmol) and anisole (470 mg,
Yield (28%). 1H NMR (300 MHz, DMSO-d6) d 1.05–1.21 (6H, m),
3.16–3.29 (1H, m), 3.63–3.81 (1H, m), 4.60 (2H, s), 4.68–4.77 (1H,
m), 6.73 (1H, d, J = 1.9 Hz), 6.83–6.91 (1H, m), 6.93–6.99 (1H, m),
7.09–7.22 (2H, m), 7.26–7.37 (2H, m), 10.68 (1H, s). Anal. Calcd
for C21H19FN2O3: C, 68.84; H, 5.23; N, 7.65. Found: C, 68.79; H,
5.25; N, 7.63.
4.36 mmol) in trifluoroacetic acid (15 mL) was stirred at 65 °C for
12 h, and concentrated under reduced pressure. The residue was
diluted with water, and extracted with EtOAc. The organic layer
was separated, and stirred at room temperature. The precipitate
was collected by filtration and purified by recrystallization from
ethanol to give 11g (950 mg, 64%). 1H NMR (300 MHz, DMSO-d6)
d 1.09 (3H, d, J = 6.8 Hz), 4.60 (2H, s), 4.67 (1H, q, J = 6.8 Hz), 6.73
(1H, d, J = 1.9 Hz), 6.81–6.90 (2H, m), 6.91–6.99 (1H, m), 7.10–
7.29 (2H, m), 7.27–7.37 (2H, m), 8.61 (1H, s), 10.67 (1H, s). Anal.
Calcd for C19H15FN2O3: C, 67.45; H, 4.47; N, 8.28. Found: C,
67.11; H, 4.63; N, 8.26.
5.29. 6-[1-Cyclopropyl-4-(4-fluorophenyl)-2-methyl-5-oxo-2,5-
dihydro-1H-pyrrol-3-yl]-2H-1,4-benzoxazin-3(4H)-one (11c)
Yield (78%). 1H NMR (300 MHz, DMSO-d6) d 0.67–0.80 (2H, m),
0.81–0.93 (2H, m), 1.20 (3H, d, J = 6.8 Hz), 2.55–2.65 (1H, m), 4.53–
4.68 (3H, m), 6.72 (1H, d, J = 1.9 Hz), 6.81–6.89 (1H, m), 6.92–7.02
(1H, m), 7.08–7.24 (2H, m), 7.24–7.36 (2H, m), 10.67 (1H, br s).
5.35. 6-[4-(4-Fluorophenyl)-2-methyl-5-oxo-2,5-dihydro-1H-
pyrrol-3-yl]-8-methyl-2H-1,4-benzoxazin-3(4H)-one (11i)
5.30. 6-{4-(4-fluorophenyl)-2-methyl-5-oxo-1-[2-(tetrahydro-
2H-pyran-2-yloxy)ethyl]-2,5-dihydro-1H-pyrrol-3-yl}-2H-1,4-
benzoxazin-3(4H)-one (11d)
The compound 11i was prepared in a manner similar to that de-
scribed for 11g. Yield (68%). 1H NMR (300 MHz, DMSO-d6) d 1.09
(3H, d, J = 6.8 Hz), 2.12 (3H,s), 4.49–4.72 (3H, m), 6.55 (1H, d,
J = 1.9 Hz), 6.79 (1H, d, J = 1.9 Hz), 7.09–7.23 (2H, m), 7.21–7.45
(2H, m), 8.59 (1H, s), 10.59 (1H, s). Anal. Calcd for C20H17FN2O3-
0.1 (H2O): C, 67.83; H, 4.90; N, 7.91. Found: C, 67.46; H, 5.18; N,
7.53.
Yield (62%). 1H NMR (300 MHz, CDCl3) d 1.24 (3H, d, J = 6.9Hz),
1.40–1.90 (6H, m), 3.35–3.58 (2H, m), 3.62–3.72 (1H, m), 3.75–4.02
(2H, m), 4.04–4.22 (1H, m), 4.57–4.78 (2H, m), 4.63 (2H, s), 6.57
(1H, d, J = 1.8 Hz), 6.80 (1H, dd, J = 1.8, 8.1 Hz), 6.91–7.03 (3H, m),
7.32–7.41 (2H, m), 8.13 (1H, s).
5.36. 1-(4-Hydroxy-3-methyl-5-nitrophenyl)propan-1-one (13)
5.31. 6-[4-(4-Fluorophenyl)-1-(2-hydroxyethyl)-2-methyl-5-
oxo-2,5-dihydro-1H-pyrrol-3-yl]-2H-1,4-benzoxazin-3(4H)-one
(11e)
To a stirred solution of 12 (18.3 g, 111.4 mmol) were added a
solution of sulfuric acid (10 mL) in water (20 mL) and a solution
of sodium nitrite (23.1 g, 334.4 mmol) in water (40 mL) at 0 °C,
successively. The mixture was stirred at 0 °C for 3 h, and poured
into water. The precipitate was collected by filtration and washed
with water to give 13 (11.8 g, 51%). 1H NMR (300 MHz, DMSO-d6) d
1.07 (3H, t, J = 7.2 Hz), 2.31 (3H, s), 3.03 (2H, q, J = 7.2 Hz), 8.11 (1H,
d, J = 1.9 Hz), 8.35 (1H, d, J = 1.9 Hz), 11.05 (1H, s).
A mixture of 11d (0.51 g, 1.1 mmol) and p-toluenesulfonic acid
(22.5 mg, 0.13 mmol) in methanol (50 mL) was stirred at room
temperature for 2 h, diluted with sat. NaHCO3 and extracted with
EtOAc. The organic layer was dried over anhydrous MgSO4 and
concentrated under reduced pressure. The residue was purified

5992
T. Hasui et al. / Bioorg. Med. Chem. 21 (2013) 5983–5994
5.37. 8-Methyl-6-propionyl-2H-1,4-benzoxazin-3(4H)-one (14)
tate (11.2 g, 114 mmol) in 1,4-dioxane (320 mL) was stirred at
90 °C for 13 h under an argon atmosphere, diluted with water
and extracted with EtOAc. The organic layer was dried over anhy-
drous Na2SO4 and concentrated under reduced pressure. The resi-
due was purified by column chromatography on silica gel with
EtOAc/hexane as an elutant and crystallized from EtOAc/hexane
to give 19a (9.48 g, 99%). 1H NMR (300 MHz, DMSO-d6) d 1.28
(12H, s), 4.70 (2H, s), 6.99–7.08 (2H, m), 10.90 (1H, s).
To a stirred suspension of 13 (11.5 g, 54.9 mmol) was added
portionwise zinc powder (35.9 g, 549.7 mmol) under water bath.
The mixture was stirred for 10 min and the insoluble material
was removed by filtration. The filtrate was concentrated under re-
duced pressure. To the residue were added 4-methy-2-pentanone
(100 mL) and a solution of sodium carbonate (17.4 g, 164.9 mmol)
in water (100 mL) and chloroacetyl chloride (6.2 g, 54.9 mmol) at
0 °C, successively. The mixture was stirred at 0 °C for 2 h and at
50 °C for 12 h, diluted with water and extracted with EtOAc.
The organic layer was dried over anhydrous MgSO4, and concen-
trated under reduced pressure. The residue was crystallized form
EtOAc to give 14 (7.5 g, 62% in 2 steps). 1H NMR (300 MHz,
DMSO-d6) d 1.06 (3H, t, J = 7.2 Hz), 2.21 (3H, s), 2.93 (2H, q,
J = 7.2 Hz), 4.68 (2H, s), 7.33 (1H, d, J = 1.9 Hz), 7.50 (1H, d,
J = 1.9 Hz), 10.79 (1H, s).
5.42. 8-Chloro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
2H-1,4-benzoxazin-3(4H)-one (19b)
The compound 19b was prepared in a manner similar to that
described for 19a. Yield (99%). 1H NMR (300 MHz, DMSO-d6) d
1.28 (12H, s), 4.74 (2H, s), 7.14 (1H, d, J = 1.5 Hz), 7.23 (1H, d,
J = 1.5 Hz), 10.89 (1H, s).
5.43. 8-Fluoro-6-[4-(4-fluorophenyl)-1-(4-methoxybenzyl)-2-
methyl-5-oxo-2,5-dihydro-1H-pyrrol-3-yl]-2H-1,4-benzoxazin-
3(4H)-one (20a)
5.38. Ethyl N-[(4-fluorophenyl)acetyl]-N-(4-
methoxybenzyl)alaninate (16)
The compound 16 was prepared in a manner similar to that de-
scribed for 9e and 10a. Yield (81% in 2 steps). 1H NMR (300 MHz,
DMSO-d6) d 1.06–1.16 (3H, m), 1.17–1.26 (3H, m), 3.65–3.81 (5H,
m), 3.93–4.06 (2H, m), 4.10–4.20 (1H, m), 4.49–4.99 (2H, m),
6.79–6.96 (2H, m), 7.06–7.35 (6H, m).
Under argon atmosphere,
a
mixture of 19a (500 mg,
1.71 mmol), 18 (784 mg, 1.71 mmol), 1,10-bis(diphenylphos-
phino)ferrocene-palldium(II)dichloride dichloromethane complex
(278 mg, 0.34 mmol) and cesium carbonate (1.67 g, 5.21 mmol)
in THF/water (20 mL/5 mL) was stirred at 100 °C for 12 h, diluted
with 1 N HCl and extracted with EtOAc. The organic layer was
washed with brine, dried over anhydrous MgSO4, and concentrated
under reduced pressure. The residue was purified by column chro-
matography on silica gel with EtOAc/hexane as an elutant and
crystallized form EtOAc/hexane to give 20a (522 mg, 64%). 1H
NMR (300 MHz, DMSO-d6) d 1.11 (3H, d, J = 6.8 Hz), 3.74 (3H, s),
4.35 (1H, d,J = 15.1 Hz), 4.52 (1H, q, J = 6.8 Hz), 4.68 (2H, s), 4.88
(1H, d, J = 15.1 Hz), 6.51 (1H, d, J = 1.5 Hz), 6.82–6.98 (3H, m),
7.16–7.45 (6H, m), 10.80 (1H, s).
5.39. 3-(4-Fluorophenyl)-4-hydroxy-1-(4-methoxybenzyl)-5-
methyl-1,5-dihydro-2H-pyrrol-2-one (17)
To a stirred suspension of 60% sodium hydride (2.16 g, 53.
91 mmol), in DMF (90 mL) was added dropwise a solution of 16
(18.3 g, 49. 0 mmol) in DMF (90 mL) at 0 °C. The mixture was stir-
red at room temperature for 12 h under nitrogen atmosphere, di-
luted with 1 N HCl and extracted with EtOAc. The organic layer
was washed with brine, dried over anhydrous MgSO4, and concen-
trated under reduced pressure. The residue was crystallized from
methanol to give 17 (15 g, 93%). 1H NMR (300 MHz, DMSO-d6) d
1.30 (3H, d, J = 6.8 Hz), 3.68–3.83 (4H, m), 4.14 (1H, d,
J = 15.3 Hz), 4.82 (1H, d, J = 15.3 Hz), 6.80–6.96 (2H, m), 7.08–
7.25 (4H, m), 7.98–8.15 (2H, m), 11.46 (1H, s).
5.44. 8-Chloro-6-[4-(4-fluorophenyl)-1-(4-methoxybenzyl)-2-
methyl-5-oxo-2,5-dihydro-1H-pyrrol-3-yl]-2H-1,4-benzoxazin-
3(4H)-one (20b)
The compound 20b was prepared in a manner similar to that
described for 20a. Yield (42%). 1H NMR (300 MHz, DMSO-d6) d
1.10 (3H, d, J = 6.8 Hz), 3.32 (3H, s), 4.35 (1H, d,J = 15.1 Hz), 4.55
(1H, q, J = 6.8 Hz), 4.72 (2H, s), 4.88 (1H, d, J = 15.1 Hz), 6.63 (1H,
d, J = 1.9 Hz), 6.92–7.03 (3H, m), 7.15–7.44 (6H, m), 10.78 (1H, s).
5.40. 4-(4-Fluorophenyl)-1-(4-methoxybenzyl)-2-methyl-5-oxo-
2,5-dihydro-1H-pyrrol-3-yl trifluoromethanesulfonate (18)
Under nitrogen atmosphere, to a stirred solution of 17 (15 g,
45.82 mmol) and triethylamine (6.7 mL, 50.40 mmol) in dichloro-
methane (150 mL) was added dropwise trifluoromethanesulfonic
anhydride (14.2 g, 50.40 mmol) at 0 °C. The mixture was stirred
for 3 h, diluted with water. The organic layer was washed with
brine, dried over anhydrous MgSO4, and concentrated under re-
duced pressure. The residue was purified by column chromatogra-
phy on silica gel with EtOAc/hexane as an elutant and crystallized
form EtOH/water to give 18 (15 g, 71%). 1H NMR (300 MHz, DMSO-
d6) d 1.41 (3H, d, J = 6.8 Hz), 3.74 (3H, s), 4.33–4.51 (2H, m), 4.79
(1H, d, J = 15.5 Hz), 6.87–7.00 (2H, m), 7.27 (2H, d, J = 8.7 Hz),
7.31–7.45 (2H, m), 7.70–7.82 (2H, m).
5.45. 8-Fluoro-6-[4-(4-fluorophenyl)-2-methyl-5-oxo-2,5-
dihydro-1H-pyrrol-3-yl]-2H-1,4-benzoxazin-3(4H)-one (21a)
The compound 21a was prepared in a manner similar to that
described for 11g. Yield (78%). 1H NMR (300 MHz, DMSO-d6) d
1.10 (3H, d, J = 6.4 Hz), 4.62–4.77 (3H, m), 6.52 (1H, s), 6.89–6.94
(1H, m), 7.07–7.24 (2H, m), 7.27–7.41 (2H, m), 8.67 (1H, s), 10.85
(1H, s). Anal. Calcd for C19H14F2N2O3: C, 64.04; H, 3.96; N, 7.86.
Found: C, 63.66; H, 4.05; N, 7.79.
5.46. 8-Chloro-6-[4-(4-fluorophenyl)-2-methyl-5-oxo-2,5-
dihydro-1H-pyrrol-3-yl]-2H-1,4-benzoxazin-3(4H)-one (21b)
5.41. 8-Fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
2H-1,4-benzoxazin-3(4H)-one (19a)
The compound 21b was prepared in a manner similar to that
described for 11g. Yield (57%). 1H NMR (300 MHz, DMSO-d6) d
1.09 (3H, d, J = 6.8 Hz), 4.62–4.79 (3H, m), 6..64 (1H, d, J = 1.9 Hz),
7.05 (1H, d, J = 1.9 Hz), 7.14–7.23 (2H, m), 7.27–7.39 (2H, m),
8.67 (1H, s), 10.84 (1H, br s). Anal. Calcd for C19H14ClFN2O3: C,
61.22; H, 3.79; N, 7.51. Found: C, 60.98; H, 4.03; N, 7.23.
A mixture of 6-bromo-8-fluoro-2H-1,4-benzoxazin-3(4H)-one
(8.0 g, 32.5 mmol), bis(pinacolato)diboron (9.08 g, 35.8 mmol),
[1,1-1,10-bis(diphenylphosphino)ferrocene-palldium(II)dichloride
dichloromethane complex (1.33 g, 1.63 mmol) and potassium ace-

T. Hasui et al. / Bioorg. Med. Chem. 21 (2013) 5983–5994
5993
5.47. Radioligand binding assay
pH 7.2, 0.2 M sodium chloride, 10 mM DTT, 10% glycerol, 0.05%
b-octyl glucoside, 100 M ligand. Crystals of the MR-LBD com-
l
Binding displacement assays were carried out in 96-well v-bot-
tom polypropylene plates with a final volume of 50.5 lL of TEGM
plexed with compound 2b or compound 2e were obtained at
20 °C by sitting-drop vapor diffusion method. The reservoir solu-
tion contained 0.04 M potassium dihydrogen phosphate, 16% PEG
8000, 20% glycerol for the MR-LBD/2b complex, and contained
0.1 M Tris pH 7.8, 23% ethanol for the MR-LBD/2e complex. Crystals
were immersed in the reservoir solution containing 25% ethylene
glycol and flash-frozen with liquid nitrogen. Diffraction data were
collected at the Advanced Light Source beamline 5.0.3. The data
were processed using HKL200011 and evaluated using SCALA12 of
the CCP4 program suite.13 The structures were solved by molecular
replacement with MOLREP14 of the CCP4 program suite13 using the
MR-LBD structure (PDB code: 3VHV) as a search model. Several cy-
cles of model building with Coot15 and refinement with Refmac16
were performed for improving the quality of the model. The dictio-
nary files for the ligands were prepared using AFITT (OpenEye Sci-
entific Software, USA). The final models were validated using
Molprobity.17 All structural figures were generated using PyMOL
(Schrödinger, USA). Crystallographic processing and refinement
statistics are summarized in Table 5. The coordinates and structure
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 FreeStyleTM 293 cells (Invitrogen). Each
concentration was run in duplicate. The cytosols were incubated
for 16 h at 4 °C. Unbound radioactivity was removed by the addi-
tion of 35 lL 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
910 ꢁ 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 TopCountTM
(PerkinElmer Inc.). For the determination of non-specific binding,
cold aldosterone instead of drug was added to reaction mixture
at 100 lM. Specific binding was determined by subtracting the va-
lue of non-specific binding component from the total binding va-
lue. 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 IC50 values using PRISM 3.0
(GraphPadSoftware 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.
Table 5
Data collection and refinement statistics
Crystal
MR-LBD/compound
2b
MR-LBD/compound
2e
Data collection
Space group
Unit cell dimensions
a, b, c (Å)
P3121
P212121
51.9, 51.9, 206
90, 90, 120
58.4, 66.8, 75.3
90, 90, 90
a
, b, c (°)
Resolution range (Å)
Observed reflections
Unique reflections
Redundancy
50–2.05 (2.10–20.5) 50–1.40 (1.43–1.40)
156,531
269,767
5.48. MR antagonist assay (luciferase reporter gene assay)
21,157
56,402
7.4 (6.6)
4.8 (3.4)
COS-1 cells were inoculated at 5 ꢁ 106 cells/F150 in D-MEM
(low glucose) supplemented with 10% FBS and 50 mg/mL gentami-
cin and then cultured at 37 °C in 5% CO2 for 1 day. To prepare
DNA:transfection reagent complexes, a solution of 2.5 mL Opti-
Completeness (%)
99.9 (99.4)
23.1 (3.4)
0.079 (0.484)
0.084 (0.523)
0.030 (0.197)
0.999 (0.868)
1
96.4 (74.7)
22.9 (2.6)
0.058 (0.369)
0.066 (0.426)
0.029 (0.208)
0.998 (0.886)
1
I/
Rsym
r
a
b
Rmeas
Rpim
CC1/2
b
MEM, 100
g pMAM-Luc and 1
2.5 mL Opti-MEM and 125
l
L Plus Reagent (Invitrogen), 9
g pRL-TK was mixed with a solution of
L Lipofectamin Reagent (Invitrogen).
lg pMCMYneo-hMR,
c
5
l
l
Molecules in ASU
l
Refinement
The mixture was maintained at room temperature for 15 min. After
substitution of culture media with Opti-MEM, the mixture was
added to the cells. After 3 h incubation, 25 mL D-MEM (low glu-
Resolution (Å)
Reflections
40–2.05 (2.10–20.5) 40–1.40 (1.44–1.40)
20,007
53,493
d
Rwork
Rfree
0.186 (0.232)
0.216 (0.248)
0.164 (0.262)
0.179 (0.265)
d
cose) supplemented with 0.1% BSA and 50 lg/mL gentamicin was
Number of atoms
Protein
Ligand/ion
added and then the cells were incubated at 37 °C in 5% CO2 for
1 day. The transfected cells were harvested and re-suspended at
3.3 ꢁ 105 cells/mL in D-MEM (low glucose) supplemented with
2128
30
2089
77
Water
94
38.8
200
15
Average B value (Å2)e
RMS deviation from ideal
geometry
0.1% BSA and 50 lg/mL gentamicin. Then 40 lL of the cell suspen-
sion was transferred in 96-well plate (Corning#3688). After incu-
bation, 5 L/well of test compounds 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% CO2, the
medium was removed. 20 L/well of twofold diluted pikkagene
l
Bond lengths (Å)
Bond angles (°)
Ramachandran plot (%)f
Preferred regions
Allowed regions
Outliers
0.009
1.404
0.01
1.452
l
96.8
2.4
97.5
2.5
l
(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 IC50 values.
0.8
0
PDB code
3WFF
3WFG
a
Rsym
=
RhRi|I(h)i ꢂ <I(h)>|/RhRi<I(h)>, where <I(h)> is the mean intensity of
symmetry-related reflections.
b
Rh[N/(N ꢂ 1)]1/2Ri|I(h)i ꢂ <I(h)>|/RhRi<I(h)>, Rpim
=
Rh[1/(N ꢂ 1)]1/2Ri|-
5.49. Crystallization and structure determination
Rmeas
=
i
I(h) ꢂ <I(h)>|/RhRi<I(h)>.
c
CC1/2, Pearson correlation coefficient between independently merged halves of
the data set.
The human MR-LBD triple mutant, 712–984 (C808S/S810L/
A976V), was prepared as described previously.9 The ligands were
added with a final concentration of 100 lM in all steps of sample
preparation. The purified proteins were concentrated to a protein
concentration of 7–10 mg/mL in buffer containing 25 mM HEPES
d
Rwork
=
R
||Fobs| ꢂ |Fcalc||/
R|Fobs|. Rfree was calculated for randomly chosen 5% of
reflections excluded from refinement.
e
B-Factor includes contributions from TLS parameters.
Calculated with Coot. Values in parentheses are for the highest resolution shell.
f

5994
T. Hasui et al. / Bioorg. Med. Chem. 21 (2013) 5983–5994
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.
factors have been deposited in PDB with accession codes 3WFF and
3WFG for 2b and 2e, respectively.
References and notes
9. Hasui, T.; Matsunaga, N.; Ora, T.; Ohyabu, N.; Nishigaki, N.; Imura, Y.; Igata, Y.;
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10. Winneker, R. C.; Fensome, A.; Wrobel, J. E.; Zhang, Z.; Zhang, P. Semin. Reprod.
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11. Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307.
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