Synthesis, structure - Activity relationships, and pharmacokinetic profiles of nonpeptidic difluoromethylene ketones as novel inhibitors of human chymase

Article abstract of DOI:10.1021/jm000497n

Potent human chymase inhibitors with high enzymatic selectivity and satisfactory metabolic stability were obtained by replacing the Val-Pro (P₃-P₂) dipeptide portion of the previously described inhibitor 1 with a nonpeptidic pyrimidi

Full text of DOI:10.1021/jm000497n

J . Med. Chem. 2001, 44, 1297-1304
1297
Syn th esis, Str u ctu r e-Activity Rela tion sh ip s, a n d P h a r m a cok in etic P r ofiles of
Non p ep tid ic Diflu or om eth ylen e Keton es a s Novel In h ibitor s of Hu m a n Ch ym a se
Fumihiko Akahoshi,*,† Atsuyuki Ashimori,† Hiroshi Sakashita,† Takuya Yoshimura,† Masahiro Eda,†
Teruaki Imada,† Masahide Nakajima,† Naoko Mitsutomi,† Shigeki Kuwahara,† Tatsuyuki Ohtsuka,†
Chikara Fukaya,‡ Mizuo Miyazaki,§ and Norifumi Nakamura#
Drug Discovery Laboratories, Welfide Corporation, 2-25-1, Shodai-Ohtani, Hirakata, Osaka 573-1153, J apan, and
Department of Pharmacology, Osaka Medical College, 2-7, Daigaku-cho, Takatsuki, Osaka 569-8686, J apan
Received November 21, 2000
Potent human chymase inhibitors with high enzymatic selectivity and satisfactory metabolic
stability were obtained by replacing the Val-Pro (P3-P2) dipeptide portion of the previously
described inhibitor 1 with a nonpeptidic pyrimidinone skeleton. The potency of the novel
compounds was further enhanced by the introduction of carbamoyl-substituted difluorometh-
ylene ketone moieties. The most potent chymase inhibitor of the newly created series was 2u
(Y-40018), which had a Ki of 2.62 nM. Compound 2u possessed high selectivity for human
chymase since it lacked significant activity toward other representative human proteolytic
enzymes. Moreover its strict specificity for human chymase suggested that 2u strongly inhibited
human and canine chymases but not rat and mouse ones. Pharmacokinetic studies in rats and
dogs indicated that 2u was absorbed rapidly after oral administration and had satisfactory
bioavailability in these experimental animal species (rat, 17%; dog, 32%). In conclusion, 2u is
a novel, potent, and orally active chymase inhibitor which would prove very useful in revealing
the precise roles of the latter in various pathophysiological processes.
In tr od u ction
mast cell protease-II14 allowed analysis of its docking
behavior with compound 1 and revealed three favorable
interactions of the carboxyphenylamide group with the
S′ subsite. These were (1) a hydrogen bond of the anilide
nitrogen atom with the Phe-41 carbonyl oxygen atom,
(2) an aromatic-aromatic interaction with the Phe-41
side chain, and (3) an electrostatic interaction of the
terminal carboxylic acid moiety with the side chain of
Lys-40.
Chymase (EC 3.4.21.39) is a chymotrypsin-like serine
protease localized mainly in mast cell secretory gran-
ules.1-3 Recent reports suggest that this enzyme might
intervene in numerous important physiological pro-
cesses, such as stimulation of submucosal gland secre-
tion,4 hormone and cytokine processing,5,6 growth factor
release,7 parasite expulsion,8 and tissue remodeling.9,10
However, its precise physiological functions as well as
its intrinsic substrates are still unclear. Although
several chymase inhibitors have been reported,11 one
which is nonpeptidic, specific, and metabolically stable
remains to be found. This is one of the reasons why the
pathophysiological roles of chymase have not yet been
clearly elucidated.
Previously we described a peptidic human chymase
inhibitor incorporating a difluoromethylene ketone
(DFMK) moiety which, by interacting with the S′ subsite
of the enzyme, increases affinity and selectivity for
human chymase compared to other chymotrypsin-like
serine proteases.12,13 In particular compound 1, bearing
a carboxyphenylamide group at the N-terminus, showed
potent chymase-inhibitory activity (Ki ) 5.6 nM) and
high human chymase selectivity and was 10-fold more
potent than the corresponding trifluoromethyl ketone
(TFMK) derivative.
In the search for human chymase inhibitors charac-
terized by greater potency, higher enzymatic selectivity,
and metabolic stability, we designed nonpeptidic com-
pounds based on the DFMK (DIMK) derivative 1. Al-
though it was possible to modify substantially the pep-
tidic portion (P3-P1) of 1, the P1 phenylalanine residue
had to be conserved because it functions as a critical
recognition element for chymase activity. X-ray crystal-
lographic analysis of the irreversible inhibitor succinyl-
Ala-Ala-Pro-Phe-CH2Cl complexed with human chy-
mase indicated that the P3-P1 residue forms an anti-
The construction of a three-dimensional model of
human chymase utilizing the coordinate data of rat
* To whom correspondence should be addressed. Tel: +81-72-856-
9283. Fax: +81-72-868-9597. E-mail: akahoshi@welfide.co.jp.
† Welfide Corp.
§ Osaka Medical College.
‡ Present address: Production Division, Welfide Corp., 2-6-9, Hira-
nomachi, Chuo-ku, Osaka 541-0046, J apan.
#
Present address: Welfide International Corp., 2410 Lillyvale Ave.,
Los Angeles, CA 90032.
10.1021/jm000497n CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/08/2001

1298 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8
Akahoshi et al.
Sch em e 1a
a
Generic groups X and R1 are defined in Tables 1 and 2. Reagents: (a) BrCF2CO2Et, Zn, THF; (b) (i) 0.1 N NaOH, THF, (ii) R2R3NH,
EDC, HOBT, DMF; (c) R2R3NH, THF; (d) H2, Pd/C, CH3OH; (e) EDC, HOBT, N-ethylmorpholine, DMF; (f) Dess-Martin periodinate,
DMF or EDC, Cl2CHCO2H, DMSO, toluene; (g) H2, Pd/C, CH3OH or CF3SO3H, anisole, CH2Cl2; (h) 0.1 N NaOH, THF.
parallel â-sheet binding arrangement with Ser-214 to
Gly-216.15 This suggested that the corresponding portion
of 1 may bind to chymase in a similar manner. Mean-
while, the X-ray crystal structure of a complex of porcine
pancreatic elastase with the peptidic inhibitor acetyl-
Ala-Pro-Val-CF3 indicated that this binding is charac-
terized by an analogous antiparallel â-sheet hydrogen-
bonding pattern.16 On the basis of these findings then,
we hypothesized that pyrimidinone structures, which
have been successfully used in the design of elastase
inhibitors,17,18 would be effective P3-P2 mimetics in our
chymase inhibitors, and likewise, the same results
allowed us to predict that the combination of nonpep-
tidic pyrimidinone skeletons with DFMK moieties ca-
pable of interacting with the S′ subsite (general struc-
ture 2) would yield orally active chymase inhibitors.
The present report describes the synthesis and struc-
ture-activity relationships (SARs) of a series of 5-amino-
6-oxo-1,6-dihydropyrimidine-containing DFMK moieties
bearing a variety of substituents at the position corre-
sponding to the P′ site. We also describe the pharma-
cokinetic profile and the inhibitory profile against
several representative proteases of the N-benzylcar-
bamoyl-substituted DFMK 2u (Y-40018), the most
potent compound of the series.
of zinc with a small amount of ethyl bromodifluoroace-
tate and subsequent addition of a solution of residuary
acetate and the aldehyde 3 in THF at room temperature,
followed by stirring for a few hours. The desired
compound 4 was produced at 82% yield (cf. Experimen-
tal Section).
The ethyl ester 4 was hydrolyzed and the resulting
carboxylic acid condensed with several amines to give
the amide 5. Alternatively, direct reaction of the ethyl
ester 4 with amines also gave the desired amide 5. The
amines 6 and 7, prepared by hydrogenolysis of 4 and 5,
were condensed with the oxopyrimidinylacetic acids 818
followed by Dess-Martin or modified Pfitzner-Moffatt
oxidation to furnish compounds 11 and 12. Treatment
of 11 with various amines also afforded compound 12.
Removal of the carbonylbenzyloxy group of 11 and 12
by hydrogenolysis or trifluoromethanesulfonic acid in
the presence of anisole afforded the target compounds
13 and 2, respectively. Basic hydrolysis of the ethyl ester
13 gave the carboxylic acid 14.
Biologica l Assa ys
All compounds were evaluated for in vitro inhibitory
activity using purified chymase from human heart and
bovine pancreas R-chymotrypsin purchased from Sigma
Chemical Co. The assay was based on a published
method.12 Hydrolysis of the substrate succinyl-Ala-Ala-
Pro-Phe-p-nitroanilide was monitored spectrophoto-
metrically by measuring the release of p-nitroaniline at
405-nm absorbance.
Ch em istr y
The general method of synthesis for DFMK-substi-
tuted 5-aminopyrimidin-6-one derivatives is summa-
rized in Scheme 1. Introduction of a difluoromethylene
moiety to the phenylalaninal 319 was performed by
Reformatsky reaction, which usually requires some type
of activation, e.g. reflux,20,21 ultrasonic irradiation,22,23
electrolysis,24,25 or addition of various metals.26-28 Al-
though yields were good, except for with reflux, this
method was found to be unsuitable and impractical for
large-scale reaction. We therefore developed a modified
Reformatsky reaction which produces good yields of
difluoromethylene compounds with high reproducibility.
Reformatsky reaction was promoted by initial activation
Resu lts a n d Discu ssion
First of all, using a [5-amino-6-oxo-2-(m-tolyl)-1,6-
dihydro-1-pyrimidinyl]acetyl moiety as the basic frame-
work, we investigated the effect of substitution at the
C-terminus, which is thought to interact with the S′
subsite of the enzyme (Table 1). The nonpeptidic TFMK
compound 15 had Ki values of 50.6 and 455 nM toward
human chymase and bovine pancreatic R-chymotrypsin,
respectively. Introduction of ethoxycarbonyl and car-

Nonpeptidic Difluoromethylene Ketones
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1299
Ta ble 1. Enzyme Inhibitory Activities of DFMK Analogues
inhibitory activity: Ki (nM)a
selectivity:
chymotrypsin/chymase
8.99
compd
X
chymase
chymotrypsin
15
13
14
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
F
50.6 ( 14.2
455 ( 117
27800 ( 5000
17900 ( 8800
12.6 ( 0.3
348 ( 82
CO2Et
CO2H
CONH2
CONH-CH3
CON(CH3)2
CONH-C6H4-m-(CO2H)
CONH-C6H4-p-(CO2H)
CONH-CH2-CO2CH3
CONH-CH2-CO2H
CONH-CH2-CONH2
CONH-CH2-CONHEt
CONH-CH2-CONEt2
CONH-CH2-CO(cNC5H10
CONH-CH2-CO(cNC6H12
CONH-(CH2)2-CO2H
CONH-(CH2)2-CONEt2
CONH-nC5H11
>10000
>10000
13.3 ( 1.3
0.947
3.84
28.0
80.4
17.0
14.4
108
120
182
210
350
90.7 ( 15.4
489 ( 113
9.7 ( 0.3
13700 ( 2300
780 ( 62
16.6 ( 1.6
10.2 ( 1.1
16.9 ( 1.6
12.0 ( 0.0
7.71 ( 1.57
9.32 ( 0.84
10.2 ( 2.2
3.56 ( 0.89
23.0 ( 2.6
33.7 ( 2.6
18.5 ( 1.8
16.9 ( 4.6
28.9 ( 4.1
5.6 ( 1.9
282 ( 21
147 ( 13
1830 ( 320
1440 ( 40
1400 ( 330
1960 ( 120
3570 ( 480
3220 ( 170
1430 ( 112
295 ( 107
451 ( 162
1920 ( 100
1090 ( 50
364 ( 27
)
)
904
2m
2n
2o
2p
2q
1
62.2
8.75
24.4
114
37.7
65
CONH-CH2-C6H5
CONH-CH2-C5H4N
chymostatin
13.1 ( 1.4
9.36 ( 2.19
0.715
a
Values are means ( SEM of three independent experiments.
boxyl groups to the DFMK moiety completely canceled
chymase-inhibitory activity (13, 14). Since an electron-
withdrawing property in the functional group adjacent
to the P1 carbonyl group would normally increase
reactivity toward the Ser-195 of the enzyme, and since
the ethoxycarbonyldifluoromethyl group has a similar
electron-withdrawing ability to the trifluoromethyl group,
the reason for the complete loss of activity is unclear.
The carbamoyl analogue 2a showed strong activity, with
a Ki of ca. 13 nM toward both chymase and chymo-
trypsin, and is superior in potency to the TFMK
compound 15. We propose that this carbamoyl group
interacts with the carbonyl of the Phe-41 of chymase
as observed in the corresponding peptidic inhibitor.13
To improve enzymatic selectivity (selectivity against
chymotrypsin), we next explored further substitution on
this carbamoyl nitrogen atom. Introduction of a methyl
group resulted in a 7-fold decrease in chymase-inhibi-
tory activity but a 4-fold increase in selectivity against
chymotrypsin (2b). Introduction of a dimethyl group
induced a further decrease in inhibitory activity (2c).
These results suggest that at least one hydrogen atom
on this carbamoyl nitrogen is essential to maintain
potent chymase-inhibitory activity. Next, we carried out
substitutions on this carbamoyl nitrogen atom with
carboxyphenyl and carboxymethyl moieties, which in
our previous study had provided potent peptidic chy-
mase inhibitors with high selectivity against chymo-
trypsin.12 The m- and p-carboxylphenyl analogues 2d ,e
did not greatly affect chymase-inhibitory activity. How-
ever, as in the corresponding series of peptidic ana-
logues,12 they improved selectivity; in particular the
meta-substituted analogue 2d showed a comparable
chymotrypsin/chymase selectivity ratio to the peptidic
compound 1. Meanwhile, the glycine methyl ester,
glycine, and glycine amide derivatives 2f-l retained
potent chymase-inhibitory activity, with the N-homopi-
peridinylglycine analogue 2l being the most potent (Ki
) 3.56 nM). On the other hand, these glycyl derivatives
had considerably decreased chymotrypsin-inhibitory
activity compared with 2a , with a chymase/chymot-
rypsin selectivity ratio of almost 1000 in compound 2l.
These results suggest that chymase has some space
where a large hydrophobic group can comfortably settle
but that chymotrypsin does not. Incorporation of an
additional methylene in the glycine moiety resulted in
a decrease in activity (2m ,n ). Substitution with a benzyl
group resulted in unchanged chymase-inhibitory activ-
ity but significant loss of chymotrypsin-inhibitory activ-
ity (2p , chymotrypsin/chymase selectivity ratio 114).
Replacement of the benzyl group with a pyridylmethyl
group (2q) had little effect on either potency. It is
noteworthy that chymotrypsin/chymase selectivity can
be enhanced by increasing the interaction between the
chymase S′ subsite and either a polar carboxylic acid
or a nonpolar hydrophobic moiety. Due to its high
intrinsic stability in plasma, the benzyl amide analogue
2p was selected for further SAR studies.
Next, we focused on study of SAR at the 2-position of
the pyrimidinone ring of the selected N-benzylcarbam-
oyldifluoromethyl derivative 2p , which had both high
chymase-inhibitory activity and high selectivity against
chymotrypsin. The results are summarized in Table 2.
The nonsubstituted phenyl analogue 2r was 2-fold less

1300 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8
Akahoshi et al.
Ta ble 3. Enzyme Selectivity Data for Compound 2u
Ta ble 2. Enzyme Inhibitory Activities of 2-Substituted
Pyrimidinone Analogues
enzyme
Ki (nM)a
human heart chymase
canine skin chymase
rat peritoneal chymase
mouse peritoneal chymase
bovine pancreatic chymotrypsin
human cathepsin G
human leukocyte elastase
human plasma thrombin
human angiotensin-converting enzyme
2.62 ( 0.39
28.0 ( 1.6
4240 ( 560
1260 ( 300
458 ( 19
1390 ( 230
NI (10 µM)b
NI (10 µM)b
NI (10 µM)b
selectivity:
inhibitory activity:
Ki (nM)a
chymotrypsin/
chymase
a
Values are means ( SEM of three independent experiments.
NI ) no inhibition at highest concentration (in parentheses)
tested.
compd
R1
chymase chymotrypsin
b
2p
2r
2s
2t
2u
2v
3-(CH3)C6H4 16.9 ( 4.6
1920 ( 100
177 ( 10
306 ( 20
3280 ( 346
458 ( 19
109 ( 21
171 ( 32
240 ( 46
114
C6H5
32.5 ( 3.4
15.7 ( 1.4
440 ( 36
5.45
19.5
7.45
175
7.90
2.30
2.83
3-FC6H4
4-FC6H4
3-ClC6H4
2.62 ( 0.39
3-(CH3O)C6H4 13.8 ( 1.7
2w 3-pyridyl
2x
74.3 ( 0.9
84.7 ( 3.7
4-pyridyl
a
Values are means ( SEM of three independent experiments.
F igu r e 2. Plasma concentration profile of 2u in rats and dogs.
due to a time-dependent inactivation of chymase and
ending in a steady-state phase. This phenomenon is
typical of slow-binding kinetics30 and suggests that 2u
binds to the predicted site of chymase in a manner
similar to that of peptidic compounds.15 The inhibitory
constant Ki, the association rate constant kon, and the
dissociation constant koff were calculated from the
progress curve as 1.53 nM, 8.80 × 107 M-1 s-1, and 0.050
s-1, respectively. This Ki value differed little from one
determined from the conventional ‘stopped’ method
(incubation of enzyme with inhibitor and substrate for
a given time followed by discontinuance of the reaction12
as used in Tables 1 and 2).
F igu r e 1. Progress curve of succinyl-Ala-Ala-Pro-Phe-p-
nitroanilide hydrolysis by human chymase in the presence of
various concentrations of 2u . Measurement was done in 20
mM Tris-HCl (pH 7.5) containing 0.1 mM aprotinin and 2 M
KCl in the presence of 2.5 mM substrate. The temperature
was maintained at 37 °C, and reactions were initiated by the
addition of enzyme. Values are shown corrected for background
absorbance. Numbers on the right correspond to the molar
concentration of 2u .
potent than 2p . Introduction of a fluoro atom at the
4-position of the phenyl ring resulted in a significant
decrease in chymase-inhibitory activity (2t), whereas
introduction at the 3-position gave unchanged activity
(2s). The 3-chlorophenyl analogue 2u showed potent
chymase-inhibitory activity (Ki ) 2.62 nM) and a high
chymotrypsin/chymase selectivity ratio of 175. The
3-methoxy analogue 2v did not significantly improve
inhibitory activity, and the pyridyl analogues 2w ,x were
inferior to 2r . Clearly, introduction of substituents at
the 3-position of the phenyl ring enhances selectivity
for chymase as observed in heterocyclic inhibitors.29 The
results suggest that this series of inhibitors utilizes S2-
P2 interaction to discriminate between these enzymes.
To further investigate chymase-inhibitory activity, we
performed kinetic studies on compound 2u and obtained
a progress curve for the hydrolysis of succinyl-Ala-Ala-
Pro-Phe-p-nitroanilide by human chymase29 as shown
in Figure 1. A biphasic curve was observed at various
concentrations of 2u with a first phase (pre-steady-state)
Compound 2u , the most potent inhibitor of chymase,
was measured for its inhibitory activity toward nonhu-
man origin (canine, rat, and mouse) chymases and other
representative human proteolytic enzymes (Table 3).
Only chymotrypsin-type serine proteases were inhibited
by 2u , which showed 10-fold weaker activity toward
canine chymase and drastically reduced potency toward
rat and mouse chymases and human cathepsin G, thus
demonstrating very high selectivity for human chymase.
Finally, we examined plasma levels of 2u after
intravenous (iv, 1 mg/kg) and oral administration (po,
10 mg/kg) to rats and dogs. Changes in plasma concen-
tration are shown in Figure 2 and pharmacokinetic
parameters summarized in Tables 4 and 5. This com-
pound was absorbed rapidly after oral administration
and had moderate plasma clearance. Although the
pharmacokinetic half-life was relatively short and plasma
levels were below the quantification limit at 24 h after
dosing, the high values for maximum plasma concentra-
tion and oral bioavailability (BA) allow investigation of

Nonpeptidic Difluoromethylene Ketones
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1301
Ta ble 4. Pharmacokinetic Parameters after Intravenous
Administration of 2u to Rats and Dogs at 1 mg/kg
chemicals and solvents were reagent grade unless otherwise
specified. Reactions were run under a nitrogen atmosphere at
ambient temperature unless otherwise noted. The following
abbreviations are used: THF, tetrahydrofuran; DMF, N,N-
dimethylformamide; DMSO, dimethyl sulfoxide; EDC, 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride;
HOBT, 3-hydroxybenztriazole hydrate.
b
c
e
AUCiv
(µg‚h/mL)
t1/2
(h)
CLd
(mL/h/kg)
Vd(ss)
(mL/kg)
species na
rat
dog
3
3
7.33 ( 0.61 2.1 ( 0.7 137 ( 11
5.72 ( 2.17 1.4 ( 0.5 195 ( 82
369 ( 94
257 ( 114
a
b
n ) number of animals. AUCiv ) integrated area under
Eth yl 4-Ben zyl-4-ben zyloxycar bon ylam in o-2,2-diflu or o-
3-h yd r oxybu tyla te (4). To a suspension activated zinc (1.51
g, 23.1 mmol) in THF (2.5 mL) was added dropwise ethyl
bromodifluoroacetate (0.50 mL, 3.9 mmol). After the reaction
was started, a solution of residuary ethyl bromodifluoroacetate
(2.50 mL, 19.5 mmol) and phenylalaninal (3) (2.18 g, 7.70
mmol) in THF (12 mL) added dropwise for 2 min. After stirring
at room temperature for 30 min, the reaction mixture was
cooled by ice-water and treated with saturated NH4Cl (10
mL). The mixture was poured into saturated NH4Cl (40 mL),
and then extracted with ethyl acetate. The extract was washed
with brine and dried (MgSO4). The solvent was removed and
the residue purified by silica gel column chromatography (67:
33 hexane-ethyl acetate) to give 4 (2.57 g, 82% yield) as a
white solid: 1H NMR (300 MHz, DMSO-d6) δ 7.50-6.93 (m,
11H, ArH, NH), 6.39 (d, J ) 7.7 Hz, 0.4H, OH), 6.29 (d, J )
7.5 Hz, 0.6H, OH), 5.02-4.77 (m, 2H, CH2-O), 4.28-4.15 (m,
2H, CH2-O), 4.10-3.80 (m, 2H, N-CH-CH-O), 3.07 (dd, J
) 13.8, 2.6 Hz, 0.4H, CHH-Ph), 2.90-2.70 (m, 1.2H, CH2-Ph
× 0.6), 2.62 (m, 0.4H, CHH-Ph), 1.30-1.14 (m, 3H, CH3).
plasma concentration vs time curve from time 0 to time infinity.
c t1/2 ) half-life. CL ) plasma clearance. Vd(ss) ) steady-state
d
e
volume of distribution.
Ta ble 5. Pharmacokinetic Parameters after Oral
Administration of 2u to Rats and Dogs at 10 mg/kg
Cmax
Tmax
AUCpo
BAe
(%)
b
c
d
species
na
(µg/mL)
(h)
(µg‚h/mL)
rat
dog
3
3
1.52 ( 0.09
7.32 ( 2.20
1.3 ( 0.6
0.7 ( 0.3
12.1 ( 4.9
18.5 ( 4.8
17 ( 7
32 ( 8
a
b
n ) number of animals. Cmax ) maximum plasma concentra-
c
tion taken directly from measured values. Tmax ) time to reach
d
Cmax
.
AUCpo ) integrated area under plasma concentration vs
time curve from time 0 to time infinity. e BA ) oral bioavailability:
(AUCpo × doseiv)/(AUCiv × dosepo) × 100.
the function of chymase in vivo (t1/2 ) 1.4 ( 0.5 h, Cmax
) 7.32 ( 2.20 µg/mL, BA ) 32 ( 8%).
4-Ben zyl-4-ben zyloxyca r bon yla m in o-2,2-d iflu or o-3-h y-
d r oxy-N-m eth ylbu ta n a m id e (5b). To a solution of 4 (10.2
g, 25.0 mmol) in THF (100 mL) and methanol (150 mL) was
added a solution of K2CO3 (13.8 g, 100 mmol) in water (100
mL). The resulting mixture was stirred at room temperature
for overnight, at which time organic solvents were evaporated.
The residual solution was poured into water (100 mL) and
washed with diethyl ether. The aqueous layer was acidified
with concentrated HCl to pH 3 and extracted with ethyl
acetate. The extract was washed with brine, dried (MgSO4)
and concentrated. The residue was crystallized from ethyl
acetate-hexane to give 4-benzyl-4-benzyloxycarbonylamino-
2,2-difluoro-3-hydroxybutanoic acid (8.21 g, 86% yield) as a
pale yellow solid. To a solution of this intermediate (733 mg,
1.93 mmol), methylamine hydrochloride (141 mg, 2.09 mmol)
and N-ethylmorpholine (0.27 mL, 2.1 mmol) in DMF (7 mL)
were added HOBT (600 mg, 3.92 mmol) and EDC (419 g, 2.19
mmol). After stirring at room temperature for 18 h, the
reaction mixture was poured into water (30 mL), and then
extracted with ethyl acetate. The extract was washed succes-
sively with 10% citric acid, saturated NaHCO3 and brine, and
dried (MgSO4). The solvent was removed and the residue
purified by silica gel column chromatography (ethyl acetate)
to give 5b (294 mg, 39% yield) as a white solid: 1H NMR (300
MHz, DMSO-d6) δ 8.55 (m, 1H, NH), 7.40-7.06 (m, 10H, ArH),
6.13 (d, J ) 7.1 Hz, 0.3H, OH), 6.05 (d, J ) 7.2 Hz, 0.7H, OH),
5.02-4.75 (m, 2H, CH2-O), 4.12-3.80 (m, 2H, N-CH-CH-
O), 3.07-2.55 (m, 2H, CH2-Ph), 2.63 (s, 3H, CH3-N).
4-Am in o-4-ben zyl-2,2-d iflu or o-3-h yd r oxy-N-m eth ylbu -
ta n a m id e Hyd r och lor id e (7b). To a solution of 5b (290 mg,
0.739 mmol) in methanol (16 mL) were added 1 N HCl (0.77
mL) followed by 10% palladium-carbon (100 mg) under a
nitrogen atmosphere. The resulting mixture was stirred at
room temperature for 15 h under a hydrogen atmosphere.
Palladium-carbon was removed by filtration and washed with
methanol. The filtrate was concentrated to give 7b (217 mg,
quant.) as a white solid: 1H NMR (500 MHz, DMSO-d6) δ 8.95
(m, 0.7H, NH), 8.89 (m, 0.3H, NH), 8.11 (br s, 3H NH3), 7.40-
7.26 (m, 5H, ArH), 7.20 (m, 0.7H, OH), 6.76 (m, 0.3H, OH),
4.45 (m, 0.3H, CH-N), 3.94 (m, 0.7H, CH-N), 3.60 (m, 0.7H,
CH-O), 3.54 (m, 0.3H, CH-O), 3.08 (dd, J ) 13.8, 4.0 Hz,
1H, CHH-Ph), 2.92-2.84 (m, 1H, CHH-Ph), 2.71 (d, J ) 4.5
Hz, 0.9H, CH3-N × 0.3), 2.66 (d, J ) 4.5 Hz, 2.1H, CH3-N ×
0.7).
We generated the nonpeptidic chymase inhibitor 2u ,
which showed potent inhibitory activity, high selectivity
for chymase, and a satisfactory pharmacokinetic profile
in rats and dogs. This orally active, specific chymase
inhibitor will help further our understanding of the roles
of chymase in numerous pathophysiological processes.
Con clu sion
Starting from the peptidic DFMK chymase inhibitor
1 reported previously, we synthesized a series of the
nonpeptidic compounds 2 and evaluated their human
chymase-inhibitory activity. We discovered that car-
bamoyl-substituted DFMK derivatives brought about
enhanced chymase-inhibitory activity, while introduc-
tion of hydrophobic groups at the N-terminus led to
increased selectivity for chymase over chymotrypsin.
The compound 2u (Y-40018), in particular, had a Ki of
2.62 nM toward human chymase, a Ki of 28.0 nM toward
canine chymase, and high selectivity against chymase
of other species and other representative human pro-
teolytic enzymes. Pharmacokinetic studies in rat and
dog indicated that 2u was absorbed rapidly following
oral administration and had satisfactory bioavailability.
Given its favorable biological profile, this compound was
selected for further evaluation to determine its thera-
peutic potential against chymase-induced diseases.
Exp er im en ta l Section
Ch em istr y. Melting points were determined with a Yanaco
melting point apparatus and are uncorrected. 1H NMR spectra
were measured on either a Bruker DPX-300 or AMX-500
instrument with tetramethylsilane as the internal standard;
chemical shifts are reported in parts per million (ppm, δ units).
Splitting patterns are designated as s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; or br, broad. Mass spectra
(MS) were recorded on a Hitachi M-2000 or PE Sciex API-165
instrument operating in the secondary ionization mass spec-
trometry (SIMS) and electrospray ionization (ESI) modes,
respectively. Elemental analyses for carbon, hydrogen, and
nitrogen were conducted with a Yanaco MT-6 and are within
(0.4% of theory for formulas given. Chromatography refers
to flash chromatography conducted on Kieselgel 60 230-400
mesh (E. Merck, Darmstadt) using the indicated solvents. All
2-[5-Ben zyloxyca r bon yla m in o-6-oxo-2-(m -tolyl)-1,6-d i-
h ydr o-1-pyr im idin yl]-N-[1-ben zyl-3,3-diflu or o-2-h ydr oxy-
3-(N-m eth ylca r ba m oyl)p r op yl]a ceta m id e (10b). To a so-

1302 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8
Akahoshi et al.
lution of [5-benzyloxycarbonylamino-6-oxo-2-(m-tolyl)-1,6-
dihydro-1-pyrimidinyl]acetic acid (8a ) (290 mg, 0.737 mmol),
7b (217 mg, 0.736 mmol) and N-ethylmorpholine (0.10 mL,
0.79 mmol) in DMF (2.5 mL) were added HOBT (227 mg, 1.48
mmol) and EDC (159 mg, 0.828 mmol). After stirring at room
temperature for 16 h, the reaction mixture was poured into
water (10 mL), and then extracted with ethyl acetate. The
extract was washed successively with 10% citric acid, satu-
rated NaHCO3 and brine, and dried (MgSO4). The solvent was
removed and the residue purified by recrystallization (2:5 ethyl
acetate-hexane) to give 10b (383 mg, 82% yield) as a white
solid: 1H NMR (300 MHz, DMSO-d6) δ 8.95 (m, 0.7H, NH),
8.86 (m, 0.3H, NH), 8.62 (m, 0.3H, NH), 8.45 (s, 0.7H, CHd
N), 8.41 (m, 0.3H, CHdN), 8.33 (m, 0.7H, NH), 8.23 (d, J )
8.9 Hz, 0.3H, NH), 8.15 (d, J ) 9.0 Hz, 0.7H, NH), 7.49-7.03
(m, 14H, ArH), 6.33 (d, J ) 7.1 Hz, 0.7H, OH), 6.20 (d, J ) 7.1
Hz, 0.3H, OH), 5.19 (s, 1H, CH2-O), 4.55-4.35 (m, 2H, CH2-
N), 4.29-3.86 (m, 2H, N-CH-CH-O), 2.94-2.60 (m, 2H,
CH2-Ph), 2.57 (s, 0.9H, CH3-N × 0.3), 2.56 (s, 2.1H, CH3-N
× 0.7), 2.31 (s, 2.1H, CH3-Ar × 0.7), 2.29 (s, 0.9H, CH3-Ar ×
0.3).
5.18 (s, 2H, CH2-O), 5.05 (m, 1H, CH-N), 4.43 (s, 2H, CH2-
N), 3.19 (dd, J ) 14.0, 3.6 Hz, 1H, CHH-Ph), 2.78 (dd, J )
14.0, 10.1 Hz, 1H, CHH-Ph), 2.29 (s, 3H, CH3-Ar); MS (SIMS)
m/z 738 (MH+).
2-[5-Am in o-6-oxo-2-(m-tolyl)-1,6-dih ydr o-1-pyr im idin yl]-
N-[1-b en zyl-3-[N-(3-ca r b oxyp h en yl)ca r b a m oyl]-3,3-d if-
lu or o-2-oxop r op yl]a ceta m id e (2d ). Compound 12d was
deprotected using method similar to that described for 2b to
1
give 2d as pale yellow crystals: mp >177 °C; H NMR (300
MHz, DMSO-d6) δ 11.01 (br s, 1H, NH), 8.88 (d, J ) 7.2 Hz,
1H, NH), 8.26 (s, 1H, ArH), 7.75 (d, J ) 7.7 Hz, 1H, ArH),
7.72 (d, J ) 8.7 Hz, 1H, ArH), 7.38 (t, J ) 7.9 Hz, 1H, ArH),
7.32-7.06 (m, 10H, ArH, CHdN), 5.15 (s, 2H, NH2), 5.05 (m,
1H, CH-N), 4.40 (s, 2H, CH2-N), 3.19 (m, 1H, CHH-Ph), 2.79
(dd, J ) 14.2, 9.5 Hz, 1H, CHH-Ph), 2.28 (s, 3H, CH3-Ar);
MS (SIMS) m/z 604 (MH+). Anal. (C31H27F2N5O6‚0.8CHCl3‚
1.0H2O) C, H, N.
Other analogues 2 were prepared using the same procedures
as described above.
2-[5-Ben zyloxyca r bon yla m in o-6-oxo-2-(m -tolyl)-1,6-d i-
h yd r o-1-p yr im id in yl]-N-[1-b en zyl-3-(et h oxyca r b on yl)-
3,3-d iflu or o-2-h yd r oxyp r op yl]a cet a m id e (9). Compound
8a (3.52 g, 8.95 mmol) and ethyl 4-benzyl-4-benzyloxycarbo-
nylamino-2,2-difluoro-3-hydroxybutylate hydrochloride (6) (2.59
g, 8.36 mmol) were coupled using a method to that described
for 10b to give 9 (4.28 g, 73% yield) as a pale brown solid: 1H
NMR (500 MHz, DMSO-d6) δ 8.86 (s, 1H, NH), 8.43 (s, 1H,
CHdN), 8.16 (d, J ) 8.9 Hz, 1H, NH), 7.44 (d, J ) 7.1 Hz, 2H,
ArH), 7.39 (t, J ) 7.1 Hz, 2H, ArH), 7.36-7.18 (m, 10H, ArH),
6.50 (d, J ) 7.0 Hz, 1H, OH), 5.19 (s, 2H, CH2-O), 4.55 (d, J
) 16.5 Hz, 1H, CHH-N), 4.40 (d, J ) 16.5 Hz, 1H, CHH-N),
4.23 (m, 1H, CH-O), 4.18 (q, J ) 7.0 Hz, 2H, CH2-O), 3.87
(m, 1H, CH-N), 2.78 (dd, J ) 13.4, 8.6 Hz, 1H, CHH-Ph), 2.63
(dd, J ) 13.4, 6.2 Hz, 1H, CHH-Ph), 2.31 (s, 3H, CH3-Ar),
1.17 (t, J ) 7.0 Hz, 3H, CH3).
2-[5-Ben zyloxyca r b on yla m in o-6-oxo-2-(m -t olyl)-1,6-
d ih yd r o-1-p yr im id in yl]-N-[1-b en zyl-3,3-d iflu or o-3-(N-
m eth ylca r ba m oyl)-2-oxop r op yl]a ceta m id e (12b). To a
solution of 10b (360 mg, 0.568 mmol) in DMF (2.5 mL) was
added Dess-Martin periodinate (370 mg, 0.872 mmol). The
reaction mixture was stirred at room temperature for 14 h,
after which saturated NaHCO3 (10 mL) containing sodium
thiosulfate (2.5 g) and ethyl acetate were added. After stirring
for 2 h, the mixture was poured into water (10 mL) and
extracted with ethyl acetate. The extract was washed with
saturated NaHCO3 and brine, dried (MgSO4) and concentrated.
The residue was purified by silica gel column chromatography
(60:40 dichloromethane-ethyl acetate) and recrystallization
(1:5 ethyl acetate-hexane) to give 12b (248 mg, 69% yield) as
1
a white solid: mp 157-162 °C; H NMR (300 MHz, DMSO-
2-[5-Ben zyloxyca r bon yla m in o-6-oxo-2-(m -tolyl)-1,6-d i-
h yd r o-1-p yr im id in yl]-N-[1-b en zyl-3-(et h oxyca r b on yl)-
3,3-d iflu or o-2-oxop r op yl]a ceta m id e (11). To a solution of
9 (4.04 g, 5.72 mmol) in DMSO (30 mL) and toluene (30 mL)
were added EDC (5.48 g, 28.6 mmol) and then dichloroacetic
acid (0.90 mL, 11 mmol) with ice-water cooling. After stirring
at 0 °C for 3 h, the reaction mixture was poured into 1 N HCl
(120 mL), and then extracted with ethyl acetate. The extract
was washed with saturated NaHCO3 and brine, and dried
(MgSO4). The solvent removed and the residue purified by
silica gel column chromatography (80:20 dichloromethane-
ethyl acetate) to give pale yellow crystals. Recrystallization
from 1:1 ethyl acetate-hexane afforded 11 (1.97 g, 53% yield)
as white crystals: mp 186-188 °C; 1H NMR (500 MHz, DMSO-
d6) δ 8.95 (d, J ) 6.8 Hz, 1H, NH), 8.88 (s, 1H, NH), 8.42 (s,
1H, CHdN), 7.44 (d, J ) 7.1 Hz, 2H, ArH), 7.39 (t, J ) 7.1
Hz, 2H, ArH), 7.36-7.17 (m, 10H, ArH), 5.19 (s, 2H, CH2-O),
4.86 (m, 1H, CH-N), 4.48 (d, J ) 16.6 Hz, 1H, CHH-N), 4.41
(d, J ) 16.6 Hz, 1H, CHH-N), 4.25 (q, J ) 7.1 Hz, 2H, CH2-
O), 3.09 (dd, J ) 14.2, 5.0 Hz, 1H, CHH-Ph), 2.79 (dd, J )
14.2, 9.0 Hz, 1H, CHH-Ph), 2.32 (s, 3H, CH3-Ar), 1.16 (t, J )
7.1 Hz, 3H, CH3).
d6) δ 9.07 (q, J ) 4.4 Hz, 1H, NH), 8.94 (s, 1H, NH), 8.84 (d,
J ) 7.4 Hz, 1H, NH), 8.43 (s, 1H, CHdN), 7.49-7.08 (m, 14H,
ArH), 5.18 (s, 1H, CH2-O), 4.96 (m, 1H, CH-N), 4.41 (m, 2H,
CH2-N), 3.15 (dd, J ) 14.4, 3.9 Hz, 1H, CHH-Ph), 2.79-2.60
(m, 4H, CH3-N, CHH-Ph), 2.31 (s, 3H, CH3-Ar).
2-[5-Am in o-6-oxo-2-(m-tolyl)-1,6-dih ydr o-1-pyr im idin yl]-
N-[1-b en zyl-3,3-d iflu or o-3-(N-m et h ylca r b a m oyl)-2-oxo-
p r op yl]a ceta m id e (2b). To a solution of 12b (209 mg, 0.331
mmol) in methanol (15 mL) was added 10% palladium-carbon
(62 mg) under a nitrogen atmosphere. The resulting mixture
was stirred at room temperature for 16 h under a hydrogen
atmosphere. Palladium-carbon was removed by filtration and
washed with methanol. The filtrate was concentrated and the
residue purified by recrystallization (1:2 ethyl acetate-hexane)
to give 2b (139 mg, 84% yield) as white crystals: mp 199-
201 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.07 (m, 1H, NH),
8.78 (d, J ) 7.3 Hz, 1H, NH), 7.38-7.05 (m, 10H, ArH,
CHdN), 5.15 (br s, 1H, NH2), 4.95 (m, 1H, CH-N), 4.38 (m,
2H, CH2-N), 3.15 (dd, J ) 14.3, 4.0 Hz, 1H, CHH-Ph), 2.79-
2.61 (m, 4H, CH3-N, CHH-Ph), 2.29 (s, 3H, CH3-Ar); MS
(SIMS) m/z 498 (MH+). Anal. (C25H25F2N5O4‚0.7H2O) C, H, N.
2-[5-Am in o-6-oxo-2-(m -t olyl)-1,6-d ih yd r o-1-p yr im id i-
n yl]-N-[1-b en zyl-3-(et h oxyca r b on yl)-3,3-d iflu or o-2-oxo-
p r op yl]a ceta m id e (13). Compound 11 was deprotected using
method similar to that described for 2b to give 13 (682 mg,
2-[5-Ben zyloxyca r bon yla m in o-6-oxo-2-(m -tolyl)-1,6-d i-
h yd r o-1-p yr im id in yl]-N-[1-ben zyl-3-[N-(3-ca r boxyp h en -
yl)ca r ba m oyl]-3,3-d iflu or o-2-oxop r op yl]a ceta m id e (12d ).
To a solution of 2-[5-benzyloxycarbonylamino-6-oxo-2-(m-tol-
yl)-1,6-dihydro-1-pyrimidinyl]-N-[1-benzyl-3-[N-(3-tert-butyl-
oxycarbonylphenyl)carbamoyl]-3,3-difluoro-2-oxopropyl]acet-
amide (352 mg, 0.443 mmol) in dichloromethane (7 mL) was
added trifluoroacetic acid (3.4 mL). After stirring at room
temperature for 5.5 h, the reaction mixture was concentrated.
The residue was dissolved in ethyl acetate, washed with water
and brine, and concentrated. The residue was purified by silica
gel column chromatography (70:30 ethyl acetate-methanol)
and crystallization (diethyl ether-hexane) to give 12d (282
1
92% yield) as white crystals: mp 146-147 °C; H NMR (500
MHz, DMSO-d6) δ 8.89 (d, J ) 6.8 Hz, 1H, NH), 7.31-7.09
(m, 10H, ArH, CHdN), 5.12 (s, 2H, NH2), 4.85 (m, 1H, CH-
N), 4.44 (d, J ) 16.5 Hz, 1H, CHH-N), 4.36 (d, J ) 16.5 Hz,
1H, CHH-N), 4.25 (q, J ) 7.1 Hz, 2H, CH2-O), 3.09 (dd, J )
14.1, 5.0 Hz, 1H, CHH-Ph), 2.81 (dd, J ) 14.1, 9.0 Hz, 1H,
CHH-Ph), 2.30 (s, 3H, CH3-Ar), 1.18 (t, J ) 7.1 Hz, 3H, CH3);
MS (SIMS) m/z 513 (MH+). Anal. (C26H26F2N4O5‚0.5H2O) C,
H, N.
1
mg, 86% yield) as white crystals: mp 157-165 °C; H NMR
2-[5-Am in o-6-oxo-2-(m -t olyl)-1,6-d ih yd r o-1-p yr im id i-
n yl]-N-[1-b e n zyl-3-ca r b oxy-3,3-d iflu or o-2-oxop r op yl]-
a ceta m id e (14). To a solution of 13 (90.2 mg, 0.176 mmol) in
THF (2.5 mL) was added 0.1 N NaOH (2.5 mL). The resulting
(300 MHz, DMSO-d6) δ 10.99 (br s, 1H, NH), 8.94 (d, J ) 6.6
Hz, 1H, NH), 8.93 (s, 1H, NH), 8.43 (s, 1H, CHdN), 8.24 (s,
1H, ArH), 7.78-7.65 (m, 2H, ArH), 7.47-7.10 (m, 15H, ArH),

Nonpeptidic Difluoromethylene Ketones
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1303
mixture was stirred at room temperature for 1.5 h, at which
time THF was evaporated and 0.1 N HCl (5 mL) and ethyl
acetate (10 mL) were added. After the reaction mixture was
stirred at room temperature for 0.5 h, the precipitated solid
was collected by filtration, washed with water (20 mL) and
dried in vacuo to give 14 (71 mg, 83% yield) as white crystals:
mp 118-121 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.79 (d, J )
7.3 Hz, 1H, NH), 7.31-7.10 (m, 10H, ArH, CHdN), 4.94 (m,
1H, CH-N), 4.42 (d, J ) 16.5 Hz, 1H, CHH-N), 4.37 (d, J )
16.5 Hz, 1H, CHH-N), 3.13 (dd, J ) 14.2, 4.1 Hz, 1H, CHH-
Ph), 2.75 (dd, J ) 14.2, 9.3 Hz, 1H, CHH-Ph), 2.30 (s, 3H,
CH3-Ar); MS (SIMS, negative) m/z 483 (M-H+). Anal.
(C24H22F2N4O5‚2.0H2O) C, H, N.
P r otea se In h ibition Assa y. Human heart chymase was
purified according to the method of Urata et al.31 Bovine
pancreas R-chymotrypsin and human cathepsin G were pur-
chased from Sigma Chemical Co., St. Louis, MO, and Athens
Research and Technologies Inc., respectively. Human throm-
bin, elastase and angiotensin-converting enzyme (ACE) were
from Welfide Corp., Osaka, J apan, Elastins Products Co., and
Nippon Zoki Pharmaceutical Co., Osaka, J apan, respectively.
Ack n ow led gm en t. The authors thank Masahiro
Takeuchi for his support in analytical chemistry and
Dr. Yoshihisa Inoue and Mikio Tanaka for helpful
discussions throughout the course of this work.
Su p p or tin g In for m a tion Ava ila ble: Experimental data
and elemental analyses. This material is available free of
charge via the Internet at http://pubs.acs.org.
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P h a r m a cok in etic Mea su r em en ts. Male Sprague-Daw-
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the cutaneous vein of dogs) or 10 mg/kg orally (via gavage to
the stomach). For intravenous administration the test com-
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