Discovery of a novel acyl-CoA: Cholesterol acyltransferase inhibitor: The synthesis, biological evaluation, and reduced adrenal toxicity of (4-phenylcoumarin)acetanilide derivatives with a carboxylic acid moiety

Article abstract of DOI:10.1248/cpb.59.1369

As a part of our research for novel potent and orally available acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors that can be used as anti-atherosclerotic agents, we recently reported the discovery of the (4-phenylcoumarine) acetanilide derivative 1. However, compound 1 showed adrenal toxicity in animal models. In order to search for safer ACAT inhibitors that do not have adrenal toxicity, we examined the inhibitory activity of ACAT in human macrophage and adrenal cells. The introduction of a carboxylic acid moiety on the pendant phenyl ring and the adjustment of the lipophilicity led to the discovery of (2E)-3-[7-chloro-3-[2-[[4-fluoro-2-(trifluoromethyl) phenyl]amino]-2-oxoethyl]-6-methyl-2-oxo-2H-chromen-4-yl]phenyl]acrylic acid (21e), which showed potent ACAT inhibitory activity in macrophages and a selectivity of around 30-fold over adrenal cells. In addition, compound 21e showed high adrenal safety in guinea pigs.

Full text of DOI:10.1248/cpb.59.1369

November 2011
Regular Article
Chem. Pharm. Bull. 59(11) 1369—1375 (2011)
1369
Discovery of a Novel Acyl-CoA: Cholesterol Acyltransferase Inhibitor:
The Synthesis, Biological Evaluation, and Reduced Adrenal Toxicity of
(4-Phenylcoumarin)acetanilide Derivatives with a Carboxylic Acid Moiety
Masaki OGINO,* Yoshihisa NAKADA, Nobuyuki NEGORO, Shigekazu ITOKAWA, Satoshi NISHIMURA,
Tsukasa SANADA, Tomoko SATOMI, Shunbun KITA, Kazuki KUBO, and Shogo MARUI
Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd.; 2–26–1 Muraoka-higashi, Fujisawa,
Kanagawa 251–8555, Japan. Received July 28, 2011; accepted August 16, 2011; published online August 17, 2011
As a part of our research for novel potent and orally available acyl-CoA: cholesterol acyltransferase (ACAT)
inhibitors that can be used as anti-atherosclerotic agents, we recently reported the discovery of the (4-phenyl-
coumarine)acetanilide derivative 1. However, compound 1 showed adrenal toxicity in animal models. In order to
search for safer ACAT inhibitors that do not have adrenal toxicity, we examined the inhibitory activity of ACAT
in human macrophage and adrenal cells. The introduction of a carboxylic acid moiety on the pendant phenyl
ring and the adjustment of the lipophilicity led to the discovery of (2E)-3-[7-chloro-3-[2-[[4-fluoro-2-(trifluo-
romethyl)phenyl]amino]-2-oxoethyl]-6-methyl-2-oxo-2H-chromen-4-yl]phenyl]acrylic acid (21e), which showed
potent ACAT inhibitory activity in macrophages and a selectivity of around 30-fold over adrenal cells. In addi-
tion, compound 21e showed high adrenal safety in guinea pigs.
Key words acyl-CoA: cholesterol acyltransferase inhibitor; cholesterol; atherosclerosis; adrenal toxicity; macrophage;
(4-phenylcoumarin)acetanilide
Atherosclerosis is one of the risk factors for stroke and
coronary heart diseases. The progression of atherosclerotic
lesions is associated with the accumulation of cholesteryl es-
ters in macrophage-derived foam cells of the arterial wall.1)
Acyl-CoA: cholesterol acyltransferase (ACAT) is an intra-
cellular enzyme that catalyzes the intracellular cholesterol es-
terification. Two ACAT isozymes have been identified to
date. ACAT-1 is expressed ubiquitously in various human tis-
sues and cells, such as various types of macrophages, adrenal
glands, sebaceous glands, liver, and intestine.2—5) ACAT-2 is
expressed exclusively in the small intestine and liver.2—5) The
progression of atherosclerotic lesions is associated with the
accumulation of cholesteryl esters through ACAT-1 in
macrophage foam cells of the arterial wall.5—7)
Approaches that target arterial plaques with ACAT in-
hibitors have been investigated experimentally for two
decades.8) ACAT inhibitors interfere with intracellular cho-
lesterol transport within plaque macrophages and may delay
the formation of foam cells while activating reverse choles-
terol transport. Experimental evidence suggested that ACAT
inhibitors reduced and stabilized atherosclerotic plaques in
animals.9—11) In addition, we have previously reported that
the (4-phenylcoumarin)acetanilide derivative 1 is a potent
ACAT inhibitor, which showed potent regressive effects on
atherosclerotic plaques in apolipoprotein E (apoE)-knockout
mice12) (Table 1). However, compound 1 and its related com-
pounds showed potent adrenal toxicity in guinea pigs (data
not shown).
Table 1. ACAT Inhibitory Activity of Compounds 1 and 2
Cell-based assayb) IC50 (nM)
Enzyme assaya)
IC50 (nM)
Compds.
R
Macrophage
Adrenal Selectivityc)
1
2
–Cl
–CO2H
12
125
5
96
14
560
3
6
a) Enzymatic ACAT inhibitory activity was determined by using homogenates from
human THP-1 macrophages. b) Cellar ACAT inhibitory activity in human macro-
phage and adrenal was determined by using THP-1 and H295R cell-lines, respectively.
c) Selectivity was evaluated by the value of IC50 (adrenal)/IC50 (macrophage).
Fig. 1. Structure of Pactimibe
Although the adrenal toxicity was probably caused by the pounds may show reduced adrenal toxicity. By the screening
acute increase in free cholesterol levels in adrenal cells, the of our compound collection around compound 1, compound
relationship between ACAT inhibition and adrenal toxicity 2 was found to show moderate selectivity for macrophages
has not been fully clarified.13—15) Recently, Takahashi et al. over adrenal cells in ACAT inhibition (Table 1). The com-
reported that pactimibe showed less potent ACAT inhibitory mon feature of both 2 and pactimibe seems to be the car-
activity in adrenal tissue than in other tissues16) (Fig. 1). boxylic acid moiety, and we therefore hypothesized that this
While the cause was not discussed in the paper, we specu- acidic moiety could lead to the identification of selective
lated that it is possible to find compounds possessing a simi- compounds. In this paper, we report a modification of the
lar profile by using cell-based assays and that such com- carboxylic acid moiety of compound 2 in order to improve
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: Ogino_Masaki@takeda.co.jp

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Vol. 59, No. 11
the potency and the selectivity for macrophages in cell-based pot synthesis. The ester 5 was hydrolyzed under acidic condi-
assays. The adrenal toxicity of the optimized compounds was tions to give acetic acid 6. Aldehyde 7 was prepared by an-
also evaluated.
other method. Anisole 3 was transformed into phenol 8 by
Chemistry (4-Phenylcoumarin)acetanilide derivatives bromination and demethylation. Treatment of compound 8
were synthesized by the construction of the coumarin skele- with n-butyl lithium generated a dilithiated compound, which
ton according to our previous report (Chart 1)12) and the suc- reacted with 3-(1,3-dioxolan-2-yl)benzaldehyde, followed by
cessive introduction of a carboxylic acid moiety (Charts 2— oxidation with manganese(IV) dioxide to afford 2-hydroxy-
6). 2-Hydroxybenzophenone 4, which was prepared from benzophenone 9. Construction of the coumarin ring was per-
anisole 3 and 3-bromobenzoyl chloride by Friedel–Crafts formed by the typical method to give coumarinacetate 10.
acylation, was converted into coumarinacetate 5 by 1,8-diaz- Hydrolysis of both the ester group and the 1,3-dioxolan
abicyclo[5.4.0]undec-7-ene (DBU)-mediated condensation group under acidic conditions resulted in coumarinacetic
with ethyl 4-chloro-4-oxobutanoate and cyclization in one- acid 7. The acids 6 and 7 was converted into acyl chloride,
Reagents and conditions: (a) 3-bromobenzoyl chloride, AlCl3, chlorobenzene, 100 °C, 0.5 h; (b) EtOCOCH2CH2COCl, DBU, CH3CN, 40 °C, 0.5 h; (c) HCl, AcOH, re-
flux, 1 h; (d) Br2, AcOH, rt, 1 h; (e) BBr3, CH2Cl2, ꢀ78 °C to rt, 2 h; (f) n-BuLi in hexane, THF, ꢀ78 °C, 0.5 h; (g) 3-(1,3-dioxolan-2-yl)benzaldehyde, THF, ꢀ78 °C, 1 h;
(h) MnO2, toluene, reflux, 4 h; (i) (COCl)2, DMF (cat.), THF, rt, 0.5 h; (j) 11, THF, rt, overnight.
Chart 1
Reagents and conditions: (a) NaClO2, NaH2PO4, tBuOH, 2-methyl-2-butene, THF, water, rt, 2 h.
Chart 2
Reagents and conditions: (a) CH2ꢁCHCO2Et, Pd(OAc)2, Ph3P, E t 3N, DMF, 100 °C, 5 h; (b) Raney Ni, H2, EtOH, rt, overnight; (c) methyl crotonate, Pd(OAc)2, Ph3P,
Et3N, DMF, 120 °C, 5 h.
Chart 3

November 2011
1371
Reagents and conditions: (a) triethyl 4-phosphonocrotonate, NaH, THF, 0 °C to rt, 0.5 h; (b) Raney Ni, H2, EtOH, rt, overnigh; (c) triethyl 2-phosphonopropionate,
NaH, THF, 0 °C to rt 0.5 h.
Chart 4
and the corresponding acyl chloride was coupled with ani-
lines 11 to afford coumarinacetanilides 12 and 13a, b.
The aldehydes 13a and b were oxidized to give corre-
sponding acids 2 and 14 as shown in Chart 2. The bromide 6
was converted into propenoate 15 or its b-methyl analog 17
by the Heck reaction with ethyl acrylate or methyl crotonate
(Chart 3). The propenoate 15 was transformed into propi-
onate 16 by hydrogenation with Raney Ni. The aldehyde 13a
was reacted with triethyl 4-phosphonopropionate and triethyl
2-phosphonocrotonate to give diene 18 and a-methyl analog
20 (Chart 4). The diene 18 was converted into pentanoate 19
by hydrogenation. These esters 15—20 were hydrolyzed to
give the corresponding acids 21b, d, and e—h (Chart 5).
Acetic acid 21a and butyric acid 21c were synthesized by the
Erundt-ainstert reaction as a key step (Chart 6).17) The acids
14 and 21b were converted into acyl chloride, and the corre-
sponding acyl chloride was reacted with diazomethane,
–Y–CO2H
21b
21d
21e
21f
21g
21h
–CH2CH2CO2H
–CH2CH2CH2CH2CO2H
–CHꢁCHCO2H
–C(Me)ꢁCHCO2H
–CHꢁC(Me)CO2H
–CHꢁCH–CHꢁCHCO2H
Reagents and conditions: (a) NaOH aq., THF, EtOH, rt, overnight.
Chart 5
which was followed by treatment with methanol and than compound 14. Compound 21c was the most potent
silver(II) oxide to afford methyl ester analogs 22 and 23, re- ACAT inhibitor among the synthesized compounds.
spectively. Hydrolysis of the esters 22 and 23 afforded corre-
sponding acids 21a and c.
Compounds 21b—h showed similar potent inhibitory ac-
tivities in macrophage cells despite possessing different en-
zymatic activities. On the other hand, the more lipophilic
compounds, 21c and d, showed more potent ACAT in-
Results and Discussion
In Vitro Study The synthesized compounds were exam- hibitory activities in adrenal cells than the other compounds.
ined for ACAT inhibitory activities in three ways (Table 2). These results show that the ACAT inhibitory activity might
We determined the enzymatic activity by using the human be more susceptible to lipophilicity in adrenal cells than in
macrophage homogenates derived from the THP-1 cell line macrophage cells. The enhancement of adrenal ACAT inhibi-
and the cellular activities by using both of the human tion led to a lower selectivity for macrophages. Compounds
macrophage and adrenal cell lines, THP-1 and H295R, re- 21b and e with log D values ranging from 2.8 to 3.1 showed
spectively. The selectivity for macrophages over adrenal cells potent inhibitory activities to ACAT in macrophages and
was evaluated by the ratio of each IC50 value (IC50 high selectivity for macrophages over adrenal cells (26-, 28-
(adrenal)/IC50 (macrophage)).
fold, respectively). The selectivity was determined by a bal-
First, we replaced the chlorine at the 4-position on the ance between the effects of the carboxyl acid moiety and the
anilide moiety of compound 2 with a fluorine in order to im- lipophilicity.
prove its activity on the basis of our previous structure–activ-
As a result of the in vitro studies, we chose compounds
ities relationship study.12) Compound 14 showed more potent 21b and e as candidates for further investigation in order to
inhibitory activity than compound 2 with respect to enzy- confirm the reduced adrenal toxicity properties. Compound 1
matic activity, as we expected, and its selectivity for was also tested as a reference compound of potent adrenal
macrophage was slightly improved compared with com- toxicity.
pound 2.
Adrenal Toxicological Test in Guinea Pig Adrenal tox-
Thus, a further modification of the carboxylic acid moiety icity was evaluated by a histopathologic examination in adre-
was examined with compound 14 in order to improve the po- nal cortex 24 h after the intravenous administration of com-
tency and the selectivity for macrophages in cell-based as- pounds 1, 21b and e in order to remove differences in their
says. Compound 21a showed a weaker enzymatic activity bioavailability (Table 3). Compound 1 showed adrenal toxic-

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Vol. 59, No. 11
Reagents and conditions: (a) (COCl)2, DMF (cat.), THF, rt, 0.5 h; (b) CH2N2 in Et2O, rt, 2 h; (c) MeOH, Ag2O, reflux, 2 h; (d) NaOH aq., THF, EtOH, rt, overnight.
Chart 6
Table 2. Effect of Introduction of Carboxylic Acid Moiety on ACAT Inhibitory Activity and Lipophylicity
Cell-based assayb) IC50 (nM)
Enzyme assaya)
Log Dd)
Compds.
R
IC50 (nM)
Macrophage
Adrenal
Selectivityc)
14
–CO2H
–CH2CO2H
–CH2CH2CO2H
–CH2CH2CH2CO2H
–CH2CH2CH2CH2CO2H
–CHꢁCHCO2H
–C(Me)ꢁCHCO2H
–CHꢁC(Me)CO2H
–CHꢁCH–CHꢁCHCO2H
57
ꢂ100
40
62
204
9
584
1940
249
63
9
7
28
10
4
26
5
6
2.65
2.73
3.06
3.30
3.66
2.89
3.17
3.20
3.22
21a
21b
21c
21d
21e
21f
21g
21h
7
28
84
44
26
81
7
10
16
41
30
29
39
420
222
193
256
9
a) Enzymatic ACAT inhibitory activity was determined by using homogenates from human THP-1 macrophages. b) Cellar ACAT inhibitory activity in human macrophage
and adrenal was determined by using THP-1 and H295R cell lines, respectively. c) Selectivity was evaluated by the value of IC50 (adrenal)/IC50 (macrophage). d) Log D values
were measured at pH 6.8.
Table 3. Adrenal Toxicity Test in Guinea Pigsa)
Adrenal toxicity testa)
Table 4. Ex Vivo Study in Atherosclerotic Rabbitsa)
Aorta ACAT (% of control)
Compds.
Compds.
5 mg/kg, i.v.
15 mg/kg, i.v.
50 mg/kg, i.v.
1 mg/kg, p.o.
10 mg/kg, p.o.
1
21b
21e
ꢃ
NT
NT
NT
—
NT
ꢃb)
—
1
21e
40ꢄ10*
54ꢄ4*
8ꢄ1*
7ꢄ2*
—
a) Atherosclerotic male New Zealand white rabbits were used. Compounds or vehi-
cle were administrated orally once daily for 3 d. The ACAT activity of thoracic aortas
was determined, which was converted to percentage compared with mean ACAT activ-
ity of vehicle group. Data were represented as the meanꢄS.E.M. ∗ pꢅ0.05 vs. control
(nꢁ5—7).
a) Compounds in dimethylsulfoxide were intravenously administered in guinea pig
(nꢁ3). The degree of adrenal toxicity was determined by histological diagnosis; ꢃ: evi-
dence of adrenal cortical necrosis. —: no evidence of adrenal cortical necrosis. NT: not
tested. b) Modest evidence of adrenal cortical necrosis was observed in one of three
case.
ity at a dose of 5 mg/kg. This toxicity was characterized by ACAT activities in walls of blood vessels were measured
adrenal cortical necrosis. Compound 21b showed no evi- after daily oral administration of compounds 1 and 21e for
dence of toxicity at a dose of 15 mg/kg and moderate necro- 3 d (Table 4). Both compounds 1 and 21e showed similar
sis in only one of three cases at a dose of 50 mg/kg. Surpris- ACAT inhibitory activities at a dose of 1 and 10 mg/kg, re-
ingly, compound 21e showed no adrenal toxicity even at a spectively.
high dose of 50 mg/kg. Thus, compound 21e may show high
adrenal safety in sufficient plasma drug levels that are far in Conclusion
excess of the concentration necessary to inhibit ACAT in the
walls of blood vessels.
We investigated a (4-phenylcoumarin)acetanilide series in
an effort to identify ACAT inhibitors with reduced adrenal
Ex Vivo Study in Atherosclerotic Rabbit In order to toxicity. We hypothesized that the carboxylic acid moiety
predict the anti-atherosclerotic effects in a short period, the may lead to the identification of selective compounds and

November 2011
1373
62.6 mmol) in AcOH (350 ml) was added dropwise bromine (100 g,
62.6 mmol) dissolved in AcOH (150 ml). After the addition, the mixture was
stirred for 1 h. The resulting mixture was treated with Na2S2O3 aq., followed
by stirring for 1 h. After evaporation of the solvent, the obtained residue was
diluted with water and extracted with EtOAc. The organic layer was washed
with sat. NaHCO3 aq. and water, dried over MgSO4, and concentrated. The
resulting oil was distilled under reduced pressure to give 1-bromo-4-chloro-
2-methoxy-5-methylbenzene (130 g, 88%) as an oil. BBr3 (52.5 ml,
555 mmol) was added dropwise to a stirred solution of obtained 1-bromo-4-
chloro-2-methoxy-5-methylbenzene (63.3 g, 269 mmol) in CH2Cl2 (400 ml)
at ꢀ78 °C under N2. The resulting mixture was allowed to warm to room
temperature and stirred for 2 h. The mixture was poured into ice-cold water
and the organic layer was washed with brine, dried over MgSO4 and concen-
evaluated the carboxylic acid analogs in cell-based assays
with macrophages and adrenal cells. Adjustment of the
lipophilicity of the compounds led to the discovery of (2E)-3-
[7-chloro-3-[2-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-2-
oxoethyl]-6-methyl-2-oxo-2H-chromen-4-yl]phenyl]acrylic
acid (21e), which showed potent ACAT inhibitory activities
in macrophages and selectivity of around 30-fold over adre-
nal cells. Compound 21e also showed high adrenal safety in
guinea pigs.
Experimental
1
All melting points were determined on a Yanagimoto micro melting point
apparatus and are uncorrected. Proton nuclear magnetic resonance (1H-
NMR) spectra were recorded on a Varian Gemini 200 (200 MHz) or Varian
Mercury 300 (300 MHz) spectrometer. Chemical shifts are reported as d val-
ues (ppm) downfield from internal tetramethylsilane of the indicated organic
solution. Peak multiplicities are expressed as follow: s, singlet; d, doublet; t,
triplet; q, quartet; dd, doublet of doublet; dt doublet of triplet; br s, broad
singlet; m, multiplet; br, broad. Coupling constants (J values) are given in
hertz (Hz). Elemental analyses were carried out by Takeda Analytical Re-
search Laboratories Ltd. Reaction progress was determined by thin layer
chromatography (TLC) analysis on silica gel 60 F254 plate (Merck). Chro-
matographic purification was carried out on silica gel columns 60 (0.063—
0.200 mm, Merck). Commercial reagents and solvents were used without ad-
ditional purification. Abbreviations 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; CH3CN, acetonitrile; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene;
AcOH, acetic acid.
(3-Bromophenyl)(4-chloro-2-hydroxy-5-methylphenyl)methanone (4)
AlCl3 (37.0 g, 277 mmol) was added portionwise to a stirred solution of 3-
chloro-4-methylanisole (3) (37.6 g, 240 mmol) in chlorobenzene (100 ml) at
0 °C. Following the addition, 3-bromobenzoyl chloride (50.8 g, 231 mmol)
was added dropwise, and the mixture was stirred at 100 °C for 30 min. After
cooling to 0 °C, EtOAc (220 ml), MeOH (35 ml), and 4 N HCl aq. (150 ml)
were successively added dropwise. The mixture was diluted with THF and
extracted with EtOAc. The organic layer was separated, washed with 2 N HCl
aq. and brine, dried over MgSO4 and concentrated to give 4 (66.8 g, 89%) as
pale yellow crystals; mp 115—117 °C; 1H-NMR (CDCl3) d: 2.28 (3H, s),
7.12 (1H, s), 7.36—7.42 (2H, m), 7.54—7.57 (1H, m), 7.72—7. 80 (2H, m),
11.70 (1H, s).
[4-(3-Bromophenyl)-7-chloro-6-methyl-2-oxo-2H-chromen-3-yl]acetic
Acid (6) To a suspension of 4 (186 g, 571 mmol) in CH3CN (400 ml) was
added DBU (230 ml, 1530 mmol). A solution of ethyl succinyl chloride
(157 g, 954 mmol) was added dropwise to the mixture at 40 °C for 1 h. After
stirring for 0.5 h, water (450 ml) was added and the precipitate was collected
by filteration and washed with EtOH to give crude ethyl [4-(3-bro-
mophenyl)-7-chloro-6-methyl-2-oxo-2H-chromen-3-yl]acetate (5) (182 g).
To the crude 5, AcOH (1600 ml) and conc. HCl (600 ml) were added and the
mixture was refluxed for 1 h. After concentration of the solvent, the residue
was washed with water and dried. The obtained solid was crystallized from
EtOAc to give 6 (166 g, 71%) as colorless crystals: mp ꢂ270 °C (decomp.);
1H-NMR (CDCl3) d: 2.30 (3H, s), 3.33 (2H, s), 6.81 (1H, s), 7.25 (1H, d,
Jꢁ7.6 Hz), 7.39 (1H, s), 7.45 (2H, m), 7.67 (1H, d, Jꢁ8.0 Hz); Anal. Calcd
for C18H12BrClO4: C, 53.03; H, 2.97. Found: C, 52.96; H, 3.00.
2-[4-(3-Bromophenyl)-7-chloro-6-methyl-2-oxo-2H-chromen-3-yl]-N-
[4-fluoro-2-(trifluoro-methyl)phenyl]acetamide (12) To a solution of 6
(42.0 g, 103 mmol) in THF (400 ml) and DMF (5 drops) was added oxalyl
chloride (11.0 ml, 124 mmol). The mixture was stirred at room temperature
for 0.5 h. After concentration of the solvent, the residue was dissolved with
THF (400 ml). To the solution was added 4-fluoro-2-(trifluoromethyl)aniline
(11a) (14.7 ml, 120 mmol) and the mixture was stirred at room temperature
overnight. The mixture was diluted with water and extracted with EtOAc.
The organic layer was separated, washed with 1 N HCl aq., sat. NaHCO3 aq
and water successively, dried over MgSO4 and concentrated in vacuo. The
residue was crystallized from EtOAc-THF to give 12 (60.0 g, 77%) as color-
less crystals: mp 222—223 °C; 1H-NMR (CDCl3) d: 2.32 (3H, s), 3.38 (1H,
d, Jꢁ14.0 Hz), 3.52 (1H, d, Jꢁ14.0 Hz), 6.86 (1H, s), 7.29 (3H, m), 7.45
(3H, m), 7.69 (1H, m), 7.98 (1H, m), 8.13 (1H, br s); Anal. Calcd for
C25H15BrClF4NO3: C, 52.80; H, 2.66; N, 2.46. Found: C, 52.87; H, 2.80; N,
2.29.
trated to give 8 (59.4 g, 99%) as a pale yellow solid: H-NMR (CDCl3) d:
2.28 (3H, s), 5.38 (1H, s), 7.04 (1H, s), 7.31 (1H, s).
(4-Chloro-2-hydroxy-5-methylphenyl)[3-(1,3-dioxolan-2-yl)phenyl]
methanone (9) n-Buthyl lithium (1.6 M in hexane, 300 ml, 480 mmol) was
added dropwise to a solution of 8 (48.7 g, 220 mmol) in THF (400 ml) at
ꢀ78 °C. After 0.5 h,
a solution of 3-(1,3-dioxolan-2-yl)benzaldehyde
(39.2 g, 220 mmol) in THF (100 ml) was added dropwise and the resulting
mixture was stirred for 1 h, then allowed to warm to room temperature. The
mixture was quenched with sat. NH4Cl aq. and extracted with EtOAc. The
organic layer was washed with brine, dried over MgSO4 and concentrated to
give the oily product, which was used for the next reaction without further
purification. A mixture of this product and MnO2 (52.2 g, 600 mmol) in
toluene (600 ml) was stirred at reflux with a Dean–Stark apparatus to remove
water azeotropically. After 4 h, the mixture was filtered through a Celite pad
and the filtrate was concentrated. The residue was purified by silica gel col-
umn chromatography (hexane–EtOAc) to give 9 (28.3 g, 44%) as an oil: 1H-
NMR (CDCl3) d: 2.26 (3H, s), 4.04—4.17 (4H, m), 5.88 (1H, s), 7.10 (1H,
s), 7.39 (1H, s), 7.53 (1H, t, Jꢁ7.8 Hz), 7.63 (1H, dt, Jꢁ7.8, 1.5 Hz), 7.72
(1H, dt, Jꢁ7.8, 1.5 Hz), 7.77 (1H, t, Jꢁ1.5 Hz).
Ethyl {7-Chloro-4-[3-(1,3-dioxolan-2-yl)phenyl]-6-methyl-2-oxo-2H-
chromen-3-yl}acetate (10) Ethyl succinyl chloride (12.7 ml, 88.8 mmol)
was added dropwise to a solution of 9 (28.3 g, 88.8 mmol) and Et3N
(12.4 ml, 88.8 mmol) in THF (200 ml) at 0 °C, and the mixture was stirred
for 0.5 h. After evaporation of the solvent, the residue was partitioned be-
tween water and EtOAc. The organic layer was washed with brine dried over
MgSO4 and concentrated. The residue was purified by silica gel column
chromatography (hexane–EtOAc) to give a yellow oil (27.4 g, 69%). The ob-
tained product was dissolved in toluene (300 ml), and DBU (4.49 ml,
30.0 mmol) was added. The resulting mixture was stirred under reflux with a
Dean–Stark apparatus to remove water azeotropically. After 2 h, the mixture
was partitioned between water and EtOAc. The organic layer was washed
with brine dried over MgSO4 and concentrated. The residue was purified by
silica gel column chromatography (hexane–EtOAc) to give 10 (21.3 g, 81%)
as a pale yellow oil: 1H-NMR (CDCl3) d: 1.22 (3H, t, Jꢁ7.0 Hz), 2.27 (3H,
s), 3.34 (2H, s), 4.03—4.17 (6H, m), 5.83 (1H, s), 6.82 (1H, s), 7.25—7.29
(1H, m), 7.38—7.40 (2H, m), 7.55 (1H, t, Jꢁ7.8 Hz), 7.63 (1H, dt, Jꢁ7.8,
1.2 Hz).
2-[7-Chloro-4-(3-formylphenyl)-6-methyl-2-oxo-2H-chromen-3-yl]-N-
[4-fluoro-2-(trifluoro-methyl)phenyl]acetamide (13a) A solution of 10
(2.80 g, 8.86 mmol) in AcOH (150 ml) and conc. HCl (75 ml) was refluxed
for 1 h. After concentration of the solvent, the residue was washed with
water and dried to give crude [7-chloro-4-(3-formylphenyl)-6-methyl-2-oxo-
2H-chromen-3-yl]acetic acid (7) (2.50 g) as a solid, which was used for the
next reaction without further purification. The crude 7 was converted into
13a in a manner similar to that described for 12.
Colorless crystals: mp 214—215 °C; 1H-NMR (CDCl3) d: 2.29 (3H, s),
3.36 (1H, d, Jꢁ14.0 Hz), 3.50 (1H, d, Jꢁ14.0 Hz), 6,80 (1H, s), 7.31 (2H,
m), 7.48 (1H, s), 7.68 (1H, m), 7.77 (1H, t, Jꢁ7.7 Hz), 7.87 (1H, s), 7.98
(1H, m), 8.09 (1H, d, Jꢁ7.6 Hz), 8.19 (1H, br s), 10.11 (1H, s); Anal. Calcd
for C26H16ClF4NO4·H2O: C, 59.68; H, 3.20; N, 2.68. Found: C, 59.45; H,
3.01; N, 2.63.
2-[7-Chloro-4-(3-formylphenyl)-6-methyl-2-oxo-2H-chromen-3-yl]-N-
[4-chloro-2-(trifluoro-methyl)phenyl]acetamide (13b) The compound 13b
was prepared in a manner similar to that described for 13a.
Colorless crystals: mp 232—233 °C; 1H-NMR (CDCl3) d: 2.29 (3H, s),
3.37 (1H, d, Jꢁ14.0 Hz), 3.51 (1H, d, Jꢁ14.0 Hz), 6,79 (1H, s), 7.47 (2H,
m), 7.58 (1H, s), 7.66 (1H, d, Jꢁ7.4 Hz), 7.78 (1H, t, Jꢁ7.4 Hz), 7.87 (1H,
s), 8.07 (2H, m), 8.28 (1H, br s), 10.11 (1H, s); Anal. Calcd for
C26H16Cl2F3NO4·H2O: C, 58.45; H, 3.20; N, 2.62. Found: C, 58.41; H, 3.03;
N, 2.39.
2-Bromo-5-chloro-4-methylphenol (8) To
a solution of 3 (98 g,
3-[7-Chloro-3-[2-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-2-oxoethyl]-

1374
Vol. 59, No. 11
6-methyl-2-oxo-2H-chromen-4-yl]benzoic Acid (14) Sodium chlorite (0.48
Ethyl (2E,4E)-5-{3-[7-Chloro-3-(2-{[4-fluoro-2-(trifluoromethyl)-
g, 5.31 mmol) was added to a solution of 13a (0.78g, 1.51mmol), 2-methyl-2- phenyl]amino}-2-oxoethyl)-6-methyl-2-oxo-2H-chromen-4-yl]phenyl}-
butene (0.72 ml, 6.80 mmol) and sodium phosphate monobasic (0.18 g, 1.50
mmol) in tert-butanol (15 ml), THF (5 ml) and water. The mixture was
stirred at room temperature for 2 h. The mixture was quenched with 1 N HCl
aq., extracted with EtOAc. The organic layer was separated, washed with
2,4-pentadienoate (18) The compound 18 was prepared in a manner simi-
lar to that described for 20.
Colorless crystals (29%): mp 199—201 °C; 1H-NMR (CDCl3) d: 1.32
(3H, t, Jꢁ7.2 Hz), 2.30 (3H, s), 3.46 (2H, s), 4.23 (2H, q, Jꢁ7.2 Hz), 5.98
water, dried over MgSO4 and concentrated in vacuo to give 14 (0.69 g, 86%) (1H, d, Jꢁ15.4 Hz), 6.90—6.95 (3H, m), 7.19—7.66 (8H, m), 7.99 (1H, dd,
as colorless solid: mp 278—280 °C; 1H-NMR (CDCl3) d: 2.28 (3H, s), 3.35 Jꢁ9.2, 5.0 Hz), 8.17 (1H, s); Anal. Calcd for C32H24ClF4NO5: C, 62.60; H,
(1H, d, Jꢁ16.0 Hz), 3.59 (1H, d, Jꢁ16.0 Hz), 6.82 (1H, s), 7.29 (2H, m),
7.45 (1H, d, Jꢁ9.2 Hz), 7.55 (2H, m), 7.75 (1H, m), 7.98 (1H, s), 8.22 (1H, d,
Jꢁ7.6 Hz), 8.77 (1H, br s); Anal. Calcd for C26H16ClF4NO5: C, 58.49; H, 3.02;
N, 2.62. Found: C, 58.41; H, 3.25; N, 2.43.
3.94; N, 2.28. Found: C, 62.71; H, 3.90; N, 2.26.
Ethyl 5-{3-[7-Chloro-3-(2-{[4-fluoro-2-(trifluoromethyl)phenyl]-
amino}-2-oxoethyl)-6-methyl-2-oxo-2H-chromen-4-yl]phenyl}pentanoate
(19) The compound 19 was prepared in a manner similar to that described
for 16.
3-[7-Chloro-3-[2-[[4-chloro-2-(trifluoromethyl)phenyl]amino]-2-oxo-
ethyl]-6-methyl-2-oxo-2H-chromen-4-yl]benzoic Acid (2) The com-
pound 2 was prepared in a manner similar to that described for 14.
Colorless crystals (65%): mp 141—142 °C; 1H-NMR (CDCl3) d: 1.23
(3H, t, Jꢁ7.2 Hz), 1.65—1.70 (4H, m), 2.29—2.33 (5H, m), 2.69—2.74
Colorless crystals (92%): mp 270—272 °C; 1H-NMR (CDCl3) d: 2.28 (2H, m), 3.43 (1H, d, Jꢁ13.8 Hz), 3.50 (1H, d, Jꢁ13.8 Hz), 4.10 (2H, q,
(3H, s), 3.34 (1H, d, Jꢁ14.8 Hz), 3.57 (1H, d, Jꢁ14.8 Hz), 6.82 (1H, s),
7.40—7.70 (5H, m), 7.99 (2H, m), 8.23 (1H, d, Jꢁ7.8 Hz), 8.38 (1H, br s);
Jꢁ7.2 Hz), 6.91 (1H, s), 7.13—7.35 (5H, m), 7.43—7.48 (2H, m), 7.98 (1H,
dd, Jꢁ8.7, 4.8 Hz), 8.16 (1H, s); Anal. Calcd for C32H28ClF4NO5: C, 62.19;
Anal. Calcd for C26H16Cl2F3NO5: C, 56.75; H, 2.93; N, 2.55. Found: C, H, 4.57; N, 2.27. Found: C, 62.10; H, 4.44; N, 2.22.
56.46; H, 3.02; N, 2.58.
(2E)-3-[7-Chloro-3-[2-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-2-
Ethyl (2E)-3-[7-Chloro-3-[2-[[4-fluoro-2-(trifluoromethyl)phenyl]-
amino]-2-oxoethyl]-6-methyl-2-oxo-2H-chromen-4-yl]phenyl]acrylate
(15) Ethyl acrylate (5.8 ml, 53.5 mmol), triethylamine (7.9 ml, 56.7 mmol),
palladium acetate(II) (0.60 g, 2.67 mmol) and triphenylphosphine (1.30 g,
5.00 mmol) were added to a solution of 6 (30.0 g, 52.8 mmol) in DMF
(300 ml) under N2 atmosphere. The mixture was stirred at 100 °C for 5 h.
The mixture was quenched with water, extracted with EtOAc. The organic
layer was separated, washed with water, dried over MgSO4 and concentrated
in vacuo. The residue was purified by silica gel column chromatography
oxoethyl]-6-methyl-2-oxo-2H-chromen-4-yl]phenyl]acrylic Acid (21e)
To a solution of 15 (575 mg, 0.98 mmol) in THF (10 ml) and EtOH (5 ml)
was added 1 N NaOH aq. (5 ml). The mixture was stirred at room tempera-
ture overnight. The mixture was neutralized with 1 N HCl aq. and extracted
with EtOAc. The organic layer was separated, washed with water, dried over
MgSO4 and concentrated in vacuo. The residue was crystallized from EtOAc
to give 21e (346 mg, 61%) as colorless crystals: mp 260—262 °C; 1H-NMR
(CDCl3) d: 2.30 (3H, m), 3.46 (2H, s), 6.52 (1H, d, Jꢁ15.8 Hz), 6.86 (1H,
s), 7.20—7.40 (4H, m), 7.60—7.80 (3H, m), 7.82 (1H, d, Jꢁ15.8 Hz), 7.98
(hexane–EtOAc) and the product was crystallized from EtOAc to give 15 (1H, m), 8.23 (1H, br s); Anal. Calcd for C28H18ClF4NO5: C, 60.06; H, 3.24;
1
(16.7 g, 55%) as colorless crystals: mp 193—196 °C; H-NMR (CDCl3) d:
1.30 (3H, t, Jꢁ7.2 Hz), 2.30 (3H, m), 3.45 (1H, s), 4.26 (2H, q, Jꢁ7.2 Hz),
6.51 (1H, d, Jꢁ16.0 Hz), 6.86 (1H, s), 7.20—7.40 (3H, m), 7.48 (2H, m),
7.60 (1H, m), 7.73 (2H, m), 8.00 (1H, m), 8.18 (1H, br s); Anal. Calcd for
C30H22ClF4NO5: C, 61.28; H, 3.77; N, 2.38. Found: C, 61.35; H, 3.89; N, 2.33.
Ethyl 3-[3-[7-Chloro-3-[2-[[4-fluoro-2-(trifluoromethyl)phenyl]-
N, 2.50. Found: C, 59.94; H, 3.19; N, 2.45.
Compounds 21b, d and f—h were prepared in a manner similar to that de-
scribed for 21e.
3-[3-[7-Chloro-3-[2-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-2-
oxoethyl]-6-methyl-2-oxo-2H-chromen-4-yl]phenyl]propionic Acid (21b)
1
Colorless crystals (91%): mp 212—214 °C; H-NMR (CDCl3) d: 2.29 (3H,
amino]-2-oxoethyl]-6-methyl-2-oxo-2H-chromen-4-yl]phenyl]propionate s), 2.68 (2H, t, Jꢁ7.2 Hz), 3.01 (2H, t, Jꢁ7.2 Hz), 3.37 (1H, d, Jꢁ13.5 Hz),
(16) To a solution of 15 (1.00 g, 1.70 mmol) in THF (50 ml) and EtOH
3.54 (1H, d, Jꢁ13.5 Hz), 6.89 (1H, s), 7.10—7.50 (7H, m), 7.90 (1H, m),
(50 ml) was added Raney Ni (ca. 1.0 g). The mixture was stirred at room 8.46 (1H, br s); Anal. Calcd for C28H20ClF4NO5: C, 59.88; H, 3.88; N, 2.52.
temperature under H2 atmosphere overnight. The mixture was filtered
through a Celite pad and the filterate was concentrated in vacuo. The residue
was crystallized from EtOAc to give 16 (0.70 g, 69%) as colorless crystals:
mp 121—123 °C; 1H-NMR (CDCl3) d: 1.21 (3H, t, Jꢁ7.0 Hz), 2.30 (3H, s),
Found: C, 59.85; H, 3.59; N, 2.49.
5-{3-[7-Chloro-3-(2-{[4-fluoro-2-(trifluoromethyl)phenyl]amino}-2-
oxoethyl)-6-methyl-2-oxo-2H-chromen-4-yl]phenyl}pentanoic Acid (21d)
Colorless crystals (70%): mp 179—181 °C; 1H-NMR (CDCl3) d: 1.60—
2.67 (2H, t, Jꢁ7.5 Hz), 3.03 (2H, t, Jꢁ7.5 Hz), 3.45 (2H, s), 4.11 (2H, q, 1.76 (4H, m), 2.29 (3H, s), 2.35 (2H, t, Jꢁ6.6 Hz), 2.72 (2H, t, Jꢁ6.6 Hz),
Jꢁ7.0 Hz), 6.91 (1H, s), 7.20—7.50 (7H, m), 7.99 (1H, m), 8.18 (1H, br s); 3.47 (2H, s), 6.91 (1H, s), 7.13—7.36 (5H, m), 7.43—7.51 (2H, m), 7.97
Anal. Calcd for C30H24ClF3NO5: C, 61.08; H, 4.10; N, 2.37. Found: C, (1H, dd, Jꢁ9.0, 5.0 Hz), 8.22 (1H, s); Anal. Calcd for C30H24ClF4NO5: C,
61.03; H, 4.16; N, 2.41.
61.08; H, 4.10; N, 2.37. Found: C, 60.86; H, 4.05; N, 2.29.
Methyl (2E)-3-{3-[7-Chloro-3-(2-{[4-fluoro-2-(trifluoromethyl)-
(2E)-3-{3-[7-Chloro-3-(2-{[4-fluoro-2-(trifluoromethyl)phenyl]amino}-
phenyl]amino}-2-oxoethyl)-6-methyl-2-oxo-2H-chromen-4-yl]phenyl}-2- 2-oxoethyl)-6-methyl-2-oxo-2H-chromen-4-yl]phenyl}-2-butenoic Acid
butenoate (17) The compound 17 was prepared in a manner similar to that
described for 15.
(21f) Colorless crystals (66%): mp 252—255 °C; 1H-NMR (DMSO-d6) d:
2.27 (3H, s), 2.50 (3H, s), 3.34 (2H, s), 6.17 (1H, s), 6.93 (1H, s), 7.34—
7.80 (8H, m), 9.67 (1H, s), 12.23 (1H, s); Anal. Calcd for C29H20ClF4NO5:
Colorless crystals (27%): mp 164—165 °C; 1H-NMR (CDCl3) d: 2.30
(3H, s), 2.60 (3H, d, Jꢁ1.2 Hz), 3.46 (2H, s), 3.75 (3H, s), 6.23 (1H, d, C, 60.69; H, 3.51; N, 2.44. Found: C, 60.66; H, 3.56; N, 2.35.
Jꢁ1.2 Hz), 6.88 (1H, s), 7.20—7.27 (1H, m), 7.30—7.37 (2H, m), 7.45—
(2E)-3-{3-[7-Chloro-3-(2-{[4-fluoro-2-(trifluoromethyl)phenyl]amino}-
7.46 (2H, m), 7.59 (1H, t, Jꢁ7.8 Hz), 7.64—7.68 (1H, m), 8.01 (1H, d, 2-oxoethyl)-6-methyl-2-oxo-2H-chromen-4-yl]phenyl}-2-methylacrylic
Jꢁ1.2 Hz), 8.22 (1H, s); Anal. Calcd for C30H22ClF4NO5: C, 61.28; H, 3.77; Acid (21g) Colorless crystals (72%): mp 268—271 °C; 1H-NMR (DMSO-
N, 2.38. Found: C, 61.36; H, 3.85; N, 2.33.
d6) d: 2.00 (3H, d, Jꢁ1.2 Hz), 2.26 (3H, s), 3.27—3.43 (2H, m), 6.94 (1H,
s), 7.30—7.41 (3H, m), 7.48—7.55 (1H, m), 7.60 (1H, dd, Jꢁ9.0, 2.7 Hz),
7.64—7.68 (3H, m), 7.70 (1H, s), 9.65 (1H, s); Anal. Calcd for
C29H20ClF4NO5: C, 60.69; H, 3.51; N, 2.44. Found: C, 60.54; H, 3.68; N,
2.27.
Ethyl (2E)-3-{3-[7-Chloro-3-(2-{[4-fluoro-2-(trifluoromethyl)-
phenyl]amino}-2-oxoethyl)-6-methyl-2-oxo-2H-chromen-4-yl]phenyl}-2-
methylacrylate (20) NaH (65.5 mg, 1.80 mmol) was added portionwise to
a solution of triethyl 2-phosphonopropionate (0.536 g, 2.25 mmol) in THF
(10 ml) at 0 °C and the mixture was stirred for 30 min. 13a (0.777 g,
1.50 mmol) was added and the resulting mixture was stirred at the same tem-
perature for 30 min and at room temperature for 2 h. The mixture was di-
luted with water and extracted with AcOEt. The extract was washed with
brine, dried over MgSO4, and concentrated. The residue was purified by sil-
ica gel column chromatography (hexane–EtOAc–CHCl3) to give 20 (346 mg,
61%) as colorless crystals (0.84 g, 93%): mp 172—174 °C; 1H-NMR
(CDCl3) d: 1.34 (3H, t, Jꢁ7.2 Hz), 2.11 (3H, d, Jꢁ1.5 Hz), 2.29 (3H, s),
3.42 (1H, d, Jꢁ13.8 Hz), 3.52 (1H, d, Jꢁ13.8 Hz), 4.27 (2H, q, Jꢁ7.2 Hz),
(2E,4E)-5-{3-[7-Chloro-3-(2-{[4-fluoro-2-(trifluoromethyl)-
phenyl]amino}-2-oxoethyl)-6-methyl-2-oxo-2H-chromen-4-yl]phenyl}-
1
2,4-pentadienoic Acid (21h) Colorless crystals (87%): mp ꢂ300 °C; H-
NMR (DMSO-d6) d: 2.26 (3H, s), 3.41 (2H, s), 5.98 (1H, d, Jꢁ14.6 Hz),
6.92 (1H, s), 7.12—7.18 (2H, m), 7.24—7.41 (3H, m), 7.47—7.67 (4H, m),
7.71—7.81 (2H, m), 9.65 (1H, s), 12.30 (1H, s); Anal. Calcd for
C30H20ClF4NO5: C, 61.50; H, 3.44; N, 2.39. Found: C, 61.61; H, 3.68; N,
2.26.
Methyl 3-[7-Chloro-3-[2-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-
6.87 (1H, s), 7.19—7.26 (1H, m), 7.29—7.32 (3H, m), 7.45 (1H, s), 7.55— 2-oxoethyl]-6-methyl-2-oxo-2H-chromen-4-yl]phenyl]acetate (22) To a
7.62 (2H, m), 7.70 (1H, d, Jꢁ1.5 Hz), 7.97 (1H, dd, Jꢁ9.0, 5.2 Hz), 8.13 solution of 14 (2.0 g, 3.75 mmol) in THF (100 ml) and DMF (3 drops) was
(1H, s); Anal. Calcd for C31H24ClF4NO5: C, 61.85; H, 4.02; N, 2.33. Found: added oxalyl chloride (0.40 ml, 4.59 mmol). The mixture was stirred at room
C, 61.84; H, 4.22; N, 2.40.
temperature for 0.5 h. After concentration of the solvent, the residue was dis-

November 2011
1375
solved with THF (50 ml). This solution was added to a solution of dia-
zomethane in diethylether (50 ml), prepared from N-methyl-Nꢆ-nitroso-N-ni-
troguanizine (3.0 g, 20.4 mmol), at room temperature. After stirring at room
temperature for 2 h, the mixture was concentrated in vacuo. To the residue
was added MeOH (100 ml), followed by silver oxide (1.2 g, 5.18 mmol). The
mixture was refluxed for 2 h. The mixture was filtered through a Celite pad
and the filterate was concentrated in vacuo. The residue was purified by sil-
Ex Vivo Study in Atherosclerotic Rabbits Atherosclerotic male New
Zealand white rabbits (14-week-old) were used. Animals were fed a chow
diet supplemented with 0.5% cholesterol, 3% peanut oil, and 3% coconut oil
to induce atherosclerotic lesions for 8 weeks followed by a chow diet for 6
weeks. Animals were orally given compounds or vehicle once daily for 3 d
(1: solution in Gelucire,12) 21e: suspension in 0.5% methylcellulose). About
2 h after the last administration, animals were sacrificed under the anesthesia
ica gel column chromatography (hexane–EtOAc) to give 22 as a pale yellow and thoracic aortas were dissected and frozen. The ACAT activity of these
1
solid, which was used for the next reaction without further purification: H- tissue homogenates were determined by incorporation of [3H] oleoyl-CoA
NMR (CDCl3) d: 2.30 (3H, s), 3.42 (1H, d, Jꢁ10.2 Hz), 3.47 (1H, d, into cholesteryl esters. All homogenate samples were determined ACAT
Jꢁ10.2 Hz), 3.70 (3H, s), 3.72 (3H, s), 6.94 (1H, s), 7.25—7.33 (4H, m), activity by incorporated [3H] oleoyl-CoA per protein concentration. ACAT
7.43—7.52 (4H, m), 7.97 (1H, m), 8.16 (1H, br s).
activity of each sample was converted to percentage compared with mean
3-[7-Chloro-3-[2-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-2-ox-
oethyl]-6-methyl-2-oxo-2H-chromen-4-yl]phenyl]acetic Acid (21a) The
ACAT activity of vehicle group. Data were represented as the meanꢄS.E.M.
compound 21a was prepared in a manner similar to that described for 21e.
Acknowledgement We would like to thank Dr. K. Kato, Dr. Y. Imura
1
Colorless crystals (8% from 14): mp 173—175 °C; H-NMR (DMSO-d6) for their encouragement and help advice. We would like to thank Dr. T.
d: 2.25 (3H, s), 3.38 (2H, s), 3.62 (2H, s), 6.97 (1H, s), 7.25 (2H, m), 7.47— Watanabe for toxicological test. We would like to thank Mr. M. Nakamura
7.63 (5H, m), 7.70 (1H, s), 9.69 (1H, br s); Anal. Calcd for for pharmacological directions.
C27H18ClF4NO5·H2O: C, 58.23; H, 3.44; N, 2.52. Found: C, 58.11; H, 3.61;
N, 2.45.
Methyl 4-[3-[7-Chloro-3-[2-[[4-fluoro-2-(trifluoromethyl)phenyl]-
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amino]-2-oxoethyl]-6-methyl-2-oxo-2H-chromen-4-yl]phenyl]butanoate
(23) The compound 23 was prepared in a manner similar to that described
for 22.
A pale yellow solid: 1H-NMR (CDCl3) d: 2.00 (2H, m), 2.30 (3H, s), 2.36
(2H, t, Jꢁ7.6 Hz), 2.74 (2H, t, Jꢁ7.7 Hz), 3.46 (2H, s), 3.64 (3H, s), 6.92
(1H, s), 7.15—7.37 (5H, m), 7.48 (2H, m), 8.00 (1H, m), 8.19 (1H, br s);
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60.95; H, 3.98; N, 2.24.
4-[3-[7-Chloro-3-[2-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-2-
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The compound 21c was prepared in a manner similar to that described for
21a.
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1
Colorless crystals (19% from 21b): mp 195—196 °C; H-NMR (DMSO-
d6) d: 1.84 (2H, m), 2.24 (2H, m), 2.49 (3H, s), 2.68 (2H, m), 3.33 (2H, s),
6.92 (1H, s), 7.18 (2H, m), 7.39—7.64 (5H, m), 7.71 (1H, s), 9.65 (1H, br s),
12.06 (1H, br); Anal. Calcd for C29H22ClF4NO5: C, 60.48; H, 3.85; N, 2.43.
Found: C, 60.16; H, 3.75; N, 2.28.
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