Synthesis, structure - Activity relationships, and pharmacokinetic profiles of nonpeptidic α-keto heterocycles as novel inhibitors of human chymase

Article abstract of DOI:10.1021/jm000496v

We designed nonpeptidic chymase inhibitors based on the structure of a peptidic compound (1) and demonstrated that the combination of a pyrimidinone skeleton as a P₃-P₂ scaffold and heterocycles as P₁ carbonyl-activating g

Full text of DOI:10.1021/jm000496v

1286
J . Med. Chem. 2001, 44, 1286-1296
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 r-Keto Heter ocycles a s Novel In h ibitor s of Hu m a n Ch ym a se
Fumihiko Akahoshi,*,† Atsuyuki Ashimori,† Hiroshi Sakashita,† Takuya Yoshimura,† 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
We designed nonpeptidic chymase inhibitors based on the structure of a peptidic compound
(1) and demonstrated that the combination of a pyrimidinone skeleton as a P3-P2 scaffold and
heterocycles as P1 carbonyl-activating groups can function as a nonpeptidic chymase inhibitor.
In particular, introduction of heterobicycles such as benzoxazole resulted in more potent
chymase-inhibitory activity. Detailed structure-activity relationship studies on the benzoxazole
moiety and substituents at the 2-position of the pyrimidinone ring revealed that 2r (Y-40079)
had the most potent chymase-inhibitory activity (Ki ) 4.85 nM). This compound was also
effective toward chymases of nonhuman origin and showed good selectivity for chymases over
other proteases. Pharmacokinetic studies in rats indicated that 2r was absorbed slowly after
oral administration and showed satisfactory bioavailability (BA) (Tmax ) 6.0 ( 2.3 h, BA )
19.3 ( 6.6%, t1/2 ) 35.7 ( 13.3 h). In conclusion, 2r is a novel, potent, and orally active chymase
inhibitor which would be a useful tool in elucidating the pathophysiological roles of chymase.
In tr od u ction
boronic acid,11 phosphonate ester,22 R-ketoester,20,23
R-ketoamide,24 and difluoromethylene ketone deriva-
tives.25,26 Despite their high potency in chymase inhibi-
tion, however, their therapeutic use is still limited
because of their peptidic nature. Reports on nonpeptidic
inhibitors,20,27,28 on the other hand, have been very few,
and none contain descriptions of potent, orally active,
nonpeptidic inhibitors.
Our goal has been to develop a nonpeptidic chymase
inhibitor characterized by greater potency, higher en-
zymatic selectivity, and metabolic stability. In seeking
such nonpeptidic derivatives, our aim is to elucidate the
pathophysiological roles of chymase and to develop
therapeutic agents for chymase-induced diseases.
In the study of human leukocyte elastase inhibitors,
X-ray crystallographic analysis has demonstrated that
nonpeptidic compounds with a pyrimidinone moiety
bind to the enzyme in a manner similar to that of
peptidic compounds.29 In chymase, X-ray structure
confirms that the P3-P1 residue of the irreversible
inhibitor succinyl-Ala-Ala-Pro-Phe-CH2Cl forms the
same antiparallel â-sheet binding arrangement as in
peptidic elastase inhibitors.30 We therefore hypothesized
that a pyrimidinone structure, which was successfully
used in the design of elastase inhibitors,29,31 would be
an effective P3-P2 mimetic in peptidic chymase inhibi-
tors of the kind exemplified by compound 1.
Chymase (EC 3.4.21.39), which is classified as a
chymotrypsin-like serine protease, is secreted from mast
cells in response to immunological stimuli.1-3 While the
physiological roles of chymase have not yet been clari-
fied due to uncertainty as to its intrinsic substrates,
reports of its possible involvement in the localized
production of angiotensin II (Ang II) in several species
including humans have provided new insight into the
role of proteases in the cardiovascular system.4-6 In
addition, several recent reports have suggested that
chymase is involved not only in Ang II formation but
also in tissue remodeling in the cardiovascular system7,8
and that it is probably an endothelin-processing en-
zyme.9,10
Meanwhile, other studies have suggested that, since
chymase is localized in mast cells, it may be involved
in arthritis and other chronic inflammatory conditions,
including various skin disorders and allergies.11,12 With
particular reference to skin inflammation, involvement
by chymase in the production of soluble-type stem-cell
factor and of interleukin 1â has been suggested,13-16
while more recently the relation between genetic poly-
morphism of mast-cell chymase and atopic eczema has
been discussed.17-19 Chymase inhibitors have therefore
come to be seen as a potential new type of antiinflam-
matory and antiallergic agent.
In elastase inhibitors, several functional groups have
been used to activate the carbonyl group of peptidyl
ketones toward neucleophilic addition by the active site
Ser-195 hydroxy group of elastase. In addition to these
trifluoromethyl, difluoromethylene, halomethyl, ester,
and keto groups,32 the first examples of heterocycle
activation of the carbonyl group in the design of novel
elastase inhibitors were described in a seminal paper
by Edwards et al.33-35 The X-ray crystal structure of
Thus far several peptidic chymase inhibitors have
been synthesized, including chloromethyl ketone,20,21
* 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/jm000496v CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/09/2001

Nonpeptidic R-Keto Heterocycles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1287
Sch em e 1a
a
Generic groups X, Y, and R1 are defined in Tables 2 and 3. Reagents: (a) acetone cyanohydrin, Et3N, CH2Cl2; (b) HCl, EtOH, CHCl3;
(c) monoethanolamine, Et3N, CH2Cl2; (d) H2, Pd/C, CH3OH; (e) EtOH; (f) o-aminothiophenol, EtOH; (g) CF3SO3H, anisole, CH2Cl2; (h)
2-amino-3-hydroxypyridine, EtOH; (i) EDC, HOBT, DMF; (j) EDC, Cl2CHCO2H, DMSO, toluene.
consideration, shows an analogous function as part of
a chymase inhibitor.
On the basis of the postulation of Kinoshita et al. that
the high substrate specificity of human chymase for Ang
I is due to the unique conformation of the S′ subsite of
chymase,38 we hypothesized that the introduction to the
heterocyclic ring of functional groups able to interact
with the S′ subsite would increase affinity and specific-
ity for chymase over other chymotrypsin-type serine
proteases. We anticipated that a highly effective non-
peptidic chymase inhibitor would be produced by the
combination of a pyrimidinone skeleton acting as a
P3-P2 scaffold with substituted heterocycles capable of
activating the P1 carbonyl group and interacting with
acetyl-Ala-Pro-Val-(2-benzoxazole) complexed with por-
cine pancreatic elastase has thus revealed the covalent
bonding between the ketone carbonyl carbon atom and
the hydroxy group of Ser-195 and, moreover, the forma-
tion of a new hydrogen bond between the benzoxazole
nitrogen and His-57 of the catalytic triad, which con-
tributes to the stabilization of the enzyme-inhibitor
complex. Recently, several reports have described the
utilization of such electron-withdrawing heterocycles to
activate the carbonyl group in the development of
protease inhibitors: for example, the R-keto heterocyclic
inhibitors of prolyl endopeptidase used by Tsutsumi et
al.36 and the peptidic thrombin inhibitors used by
Castanzo et al.37 The R-keto heterocycle moiety, in our
the S′ subsite (general structure 2).
In the present report, we describe the synthesis and
structure-activity relationships (SARs) of a series of
5-amino-6-oxo-1,6-dihydropyrimidine derivatives bear-
ing heterocycles at the position corresponding to the P′
site. We also report on the inhibitory selectivity toward
several representative proteases and on the pharmaco-
kinetic profile of the most potent compound, 2r , which
has a 5-methoxycarbonylbenzoxazole moiety.
Ch em istr y
The general method of synthesis for R-keto heterocycle-
substituted pyrimidinone derivatives is summarized in

1288 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8
Akahoshi et al.
Sch em e 2a
a
Reagents: (a) EDC, HOBT, DMF; (b) EDC, Cl2CHCO2H, DMSO, toluene; (c) 2-trimethylsilylthiazole, CH2Cl2 then Bu4NF; (d) CF3SO3H,
anisole, CH2Cl2.
Sch em e 3a
a
Reagents: (a) BBr3, CH2Cl2.
Sch em e 4a
a
Reagents: (a) Fe, 1 N HCl, THF, CH3OH, H2O; (b) CF3SO3H, anisole, CH2Cl2.
Scheme 1. The aldehyde 339 was converted into the
cyanohydrin 4 with acetone cyanohydrin/Et3N.34 Treat-
ment of 4 with anhydrous HCl in ethanol afforded the
imidate 5, which was cyclized with monoethanolamine
in the presence of Et3N to the oxazoline ring. Removal
of the carbonylbenzyloxy (Cbz) group by hydrogenolysis
furnished the amine 6. Reaction of the imidate 5 with
substituted o-aminophenol 7, o-aminothiophenol, or
2-amino-3-hydroxypyridine in refluxing ethanol afforded
the corresponding heterobicycles 8-10. EDC-mediated
coupling of the amines 6 and 8-10 to the pyrimidiny-
lacetic acids 1129 followed by modified Pfitzner-Moffatt
oxidation40 and removal of the Cbz group with trifluo-
romethanesulfonic acid in the presence of anisole af-
forded the desired R-keto heterocycles 2 and 16-18.
A different approach was necessary for the prepara-
tion of R-ketothiazole derivative 23 because of the
difficulty of thiazole cyclization (Scheme 2). Condensa-
tion of the acetic acid 11a and the phenylalaninol 19
followed by DMSO oxidation afforded the aldehyde 21.
Reaction of this aldehyde with 2-trimethylsilylthiazole
using the method described by Dondoni et al.41 gave a
trimethylsilyl ether which on desilylation by fluoride
anion in situ afforded the R-hydroxymethylthiazole 22.
Similarly, oxidation and deprotection provided the
desired R-ketothiazole 23.
Further transformations of the substituents at the
5-position of the benzoxazole ring were achieved by the
routes illustrated in Schemes 3-6. The methoxy 2c was
transformed using boron tribromide to the hydroxy 2d .
The amine 2e was prepared by reduction of the nitro
24 using an Fe-HCl system, as palladium-catalyzed
hydrogenolysis reduced the carbonyl group at the R-po-
sition of the benzoxazole ring. Demethylation of the
methoxycarbonyl 2g using an aluminum tribromide-
dimethyl sulfide system gave the carboxylic acid 2i.42
Hydrolysis of the methoxycarbonyl 13g to the carboxylic
acid 13i followed by condensation with various am-
monium salts, oxidation, and deprotection gave the
desired carbamoyl analogues 2j-l.
Biologica l Assa ys
All compounds were evaluated for in vitro inhibitory
activity using purified chymase from human heart and

Nonpeptidic R-Keto Heterocycles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1289
Sch em e 5a
a
Reagents: (a) AlBr3, (CH3)2S, CH2Cl2.
Sch em e 6a
a
Reagents: (a) 1 N NaOH, DMSO; (b) R3R4NH‚HCl, N-ethylmorpholine, EDC, HOBT, DMF; (c) Dess-Martin periodinate, DMSO or
DMF; (d) CF3SO3H, anisole, CH2Cl2.
Ta ble 1. Enzyme Inhibitory Activities of R-Keto Heterocycles
bovine pancreas R-chymotrypsin purchased from Sigma
Chemical Co. The assay was based on a published
method.25 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.
Resu lts a n d Discu ssion
As it had been successfully used in the design of a
inhibitory activity: Ki (nM)a
human leukocyte elastase inhibitor,29 we hypothesized
compd
R2
chymase
chymotrypsin
that a [5-amino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-
pyrimidinyl]acetyl moiety would be an effective P3-P2
(Val-Pro) mimetic in peptidic chymase inhibitors of the
kind exemplified by the parent compound 1. First of all,
we investigated the effect of a variety of heterocycles
acting as P1 carbonyl-activating groups on chymase- and
chymotrypsin-inhibitory activity (Table 1). The intro-
duction of a monocycle such as oxazoline (16) or thiazole
(23) resulted in reduction of chymase-inhibitory activity,
whereas the introduction of a bicycle such as benzox-
azole (2a ), benzothiazole (17), or oxazolopyridine (18)
led to increased activity toward both chymase and
chymotrypsin. In particular, compound 18 showed
potent activity, with Ki of ca. 60 nM toward both
chymase and chymotrypsin. Edwards et al. reported two
important SARs between elastase and heterocycles of
the inhibitors: (1) the value of the inhibitory constant
Ki tends to be positively correlated to the electron-
withdrawing properties (σI value) of the heterocycle; (2)
in the covalent enzyme-inhibitor adduct, the azole
16
23
2a
17
18
589 ( 10
9320 ( 460
3850 ( 1000
125 ( 58
3500 ( 750
229 ( 63
182 ( 63
166 ( 25
60.6 ( 3.4
60.5 ( 8.1
1
82.1 ( 2.4
13.1 ( 1.4
4400 ( 770
9.36 ( 2.19
chymostatin
a
Values are means ( SEM of three independent experiments.
nitrogen atom of the inhibitor heterocycle participates
in a hydrogen-bonding interaction with the active-site
His-57.33,34 They found that the oxazoline derivative is

1290 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8
Akahoshi et al.
Ta ble 2. Enzyme Inhibitory Activities of 5- or 6-Substituted
Benzoxazole Analogues
Ta ble 3. Enzyme Inhibitory Activities of 2-Substituted
Pyrimidinone Analogues
inhibitory activity: Ki (nM)a
inhibitory activity: Ki (nM)a
compd
R1
chymase
chymotrypsin
compd
X
Y
chymase
chymotrypsin
2g (Y-40613) 4-FC6H4
22.6 ( 8.8
5.57 ( 0.57
14.3 ( 2.4
26.1 ( 3.6
44.2 ( 24.6
4.85 ( 0.82
41.8 ( 1.0
5.60 ( 0.15
77.4 ( 15.2
43.5 ( 11.7
52.9 ( 23.5
46.8 ( 17.0
1770 ( 80
3820 ( 690
47.7 ( 3.3
943 ( 122
11500 ( 2900
855 ( 106
45.1 ( 9.8
467 ( 8
2n
C6H5
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
F
H
H
H
H
H
H
H
H
H
H
H
H
125 ( 58
93.1 ( 0.2
73.3 ( 5.2
2120 ( 1020
183 ( 23
229 ( 63
65.1 ( 1.5
48.0 ( 17.4
2230 ( 90
60.0 ( 13.0
1630 ( 450
52.9 ( 23.5
477 ( 44
2o
3-FC6H4
2p
3-ClC6H4
OCH3
OH
NH2
2q
3-(CH3)C6H4
3-(CH3O)C6H4
3-(NO2)C6H4
3-(NH2)C6H4
3-pyridyl
2r (Y-40079)
2s
2t
2u
2v
C6H5
174 ( 9
CO2CH3
CO2Et
CO2H
CONH2
CONHEt
CONEt2
H
22.6 ( 8.8
30.0 ( 7.5
76.0 ( 8.4
94.0 ( 18.0
68.6 ( 0.2
59.5 ( 6.8
56.3 ( 45.0
4-pyridyl
133 ( 3
a
2j
2k
2l
53.4 ( 20.3
32.6 ( 1.8
23.3 ( 5.2
71.3 ( 21.7
Values are means ( SEM of three independent experiments.
a hydroxy group led to a 10-fold decrease in both
chymase- and chymotrypsin-inhibitory activity (2d ), but
neither an amino nor a phenyl group had any effect on
chymase-inhibitory activity (2e,f). The methoxycarbonyl
analogue 2g showed 5-fold greater inhibitory activity
toward both chymase and chymotrypsin. The replace-
ment of the methoxycarbonyl group of 2g with an
ethoxycarbonyl group did not substantially influence
potency (2h ), while replacement with a carboxyl or
carbamoyl group resulted in loss of inhibitory activity
(2i-l). Shifting the methoxycarbonyl group to the
6-position of the benzoxazole ring did not improve
inhibitory activity (2m ). In our chymase inhibitors, as
in elastase inhibitors, no correlation was observed
between the electronic effect of substituents on the
benzoxazole ring and inhibitory activity. Substitution
on the benzoxazole ring also had no great effect on
selectivity for chymase over chymotrypsin. This suggests
that the substituents screened do not interact specifi-
cally with a part of the S′ subsite but have some
hydrophobic interaction with the S′ subsite of both
enzymes.
We subsequently turned our attention to substitution
at the 2-position of the pyrimidinone ring of compounds
bearing 5-methoxycarbonylbenzoxazole, which had shown
the most favorable effect on chymase-inhibitory activity.
The results are summarized in Table 3. Substituents
at this position are thought to interact with the S2
pocket of the enzyme. SAR study of 5-amino-6-oxo-1,6-
dihydropyrimidine derivatives with a trifluoromethyl
ketone group has indicated that an aromatic ring at this
position is essential to maintain potent chymase-inhibi-
tory activity and that the introduction of a substituent
at the 3-position of the phenyl ring increases activity.43
We therefore focused on 3-substituted phenyl groups.
The nonsubstituted phenyl analogue 2n was found to
be more potent (Ki ) 5.57 nM) than compound 2g. The
introduction of various substituents at the 3-position
(2o-t) resulted in maintenance or slight loss of chy-
mase-inhibitory activity; chymotrypsin-inhibitory activ-
ity, on the other hand, was dramatically reduced. The
3-methoxyphenyl analogue 2r showed the most potent
chymase-inhibitory activity (Ki ) 4.85 nM) and high
2m
CO2CH3
a
Values are means ( SEM of three independent experiments.
the most effective elastase inhibitor, since the nitrogen
atom of the oxazoline is the best hydrogen bond acceptor
in a variety of heterocycles.34 In chymase inhibitors with
heteromonocycles, the increase in inhibitory activity for
oxazoline compared to thiazole (7-fold) may have some
hydrogen-bonding interaction with His-57 of chymase.
In contrast to elastase inhibitor, heterobicycles showed
more chyamse-inhibitory activity than heteromono-
cycles. This finding suggests that the aromatic rings of
the heterobicycles interact with the S′ subsite of chy-
mase. In Costanzo’s study, the best thrombin inhibitor
possesses a 2-benzothiazole group.37 This was explained
clearly with reference to novel interactions at the S1′
subsite, where the benzothiazole stacks the Trp-60D of
the insertion loop of thrombin in a face-to-edge manner.
In chymase inhibitors, it seems possible that aromatic
rings of the heterobicycles have such stacking interac-
tions with an aromatic amino acid residue of the
enzyme. For further study, we selected the benzoxazole
ring as the heterocyclic part due to its ease of synthesis.
In elastase inhibitors of the benzoxazole type, SAR
studies have revealed that the electronic effects of
various substituents on the benzoxazole ring do not
greatly influence inhibitory activity.35 Increasing the
electron-withdrawing ability of the benzoxazole ring
substituent does increase the electrophilicity of the
ketone carbonyl carbon atom and increase the strength
of the covalent bond formed with Oγ of Ser-195, but any
increased ketone carbonyl activation by the ring sub-
stituent is counterbalanced by a corresponding decrease
in the hydrogen-bonding ability between the benzox-
azole nitrogen atom and His-57.
The results of our investigations of the effect of
substituents on the benzoxazole ring are shown in Table
2. Substitution of an electron-withdrawing fluorine atom
(2b) or an electron-releasing methoxy group (2c) at the
5-position of the benzoxazole ring resulted in both cases
in ca. 4-fold more potent activity toward chymotrypsin
and slightly more potent activity toward chymase than
in the nonsubstituted analogue 2a . The introduction of

Nonpeptidic R-Keto Heterocycles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1291
F igu r e 2. Plasma concentration profile of 2r in rats.
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 2r . 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 2r .
phenomenon indicated that 2r is a slow-binding inhibi-
tor44 like that of an R-keto heterocycle-bearing peptidic
elastase inhibitor.45 The inhibitory constant Ki, the
association rate constant kon, and the dissociation
constant koff calculated from the progress curve were
1.21 nM, 9.56 × 107 M-1 s-1, and 0.030 s-1, respectively.
This Ki value differed little from the one determined
from the conventional ‘stopped’ method (incubation of
enzyme, inhibitor, and substrate for a given time
followed by discontinuance of the reaction)25 as shown
in Tables 1-3. Due to the exceeding shortness of the
pre-steady-state of the R-keto heterocycle-bearing in-
hibitor in comparison with the total reaction time (2 h),
we regarded the first phase as having virtually no
influence on the determination of Ki.
Compound 2g and the most potent human chymase
inhibitor 2r were measured for their inhibitory activity
toward nonhuman chymases and other representative
proteases (Table 4). Both compounds showed strong
inhibitory activity toward canine chymase and slightly
less potency toward rat and mouse chymases. As
expected, inhibitory activity was shown only toward
chymotrypsin-type serine proteases. Notably, compound
2r had 80-fold stronger activity toward human chymase
than human catepsin G, which has similar substrate
specificity.
Next, we examined plasma levels of compound 2r
after intravenous (iv) and oral (po) administration to
rats. The results are shown in Figure 2 and pharma-
cokinetic parameters summarized in Table 5. The
investigations indicated that 2r was absorbed slowly
after oral administration and had satisfactory bioavail-
ability (BA) (Tmax ) 6.0 ( 2.3 h, BA ) 19.3 ( 6.6%).
Pharmacokinetic half-life and plasma clearance values
were 35.7 ( 13.3 h and 10.1 ( 3.2 mL/h/kg, respectively.
An in vitro degradation study using an S9 function of
rat liver homogenate indicated that the major metabo-
lite was a corresponding alcohol form by reduction of
the active carbonyl group.
Ta ble 4. Enzyme Selectivity Data for Compounds 2g,r
Ki (nM)a
enzyme
2g
2r
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
22.6 ( 8.8
3.68 ( 0.48
103 ( 10
4.85 ( 0.82
1.12 ( 0.15
86.6 ( 8.4
63.9 ( 26.7
943 ( 122
379 ( 126
NI (10 µM)b
NI (10 µM)b
NI (10 µM)b
40.4 ( 6.6
52.9 ( 23.5
720 ( 58
NI (10 µM)b
NI (10 µM)b
NI (10 µM)b
a
Values are means ( SEM of three independent experiments.
NI ) no inhibition at highest concentration (in parentheses)
b
tested.
chymotrypsin/chymase selectivity [Ki(chymotrypsin)/Ki-
(chymase) ) 194]. These findings clearly indicate that
the introduction of substituents at the 3-position of the
phenyl ring is highly effective in enhancing enzymatic
selectivity for chymase. Accordingly, we assumed that
this series of 5-amino-6-oxo-1,6-dihydropyrimidine de-
rivatives utilizes S2-P2 interaction to discriminate
between related enzymes. Finally, pyridyl substitutions
led to a decrease in chymase-inhibitory activity (2u ,v).
In the next step, we investigated the kinetics of
compound 2r . A progress curve for hydrolysis of succi-
nyl-Ala-Ala-Pro-Phe-p-nitroanilide by chymase dis-
played a biphasic curve composed of a rapid initial phase
and a slower steady-state phase (Figure 1). Although
slow-binding inhibition is not a general property of
R-keto heterocyclic inhibitors of serine proteases,32 this
Ta ble 5. Pharmacokinetic Parameters of 2r in Rats
route, dose
na
t1/2b (h)
CLc (mL/h/kg)
Vd(ss)d (mL/kg)
469 ( 60
AUCe (µg‚h/mL)
Cmaxf (µg/mL)
0.396 ( 0.164
Tmaxg (h)
6.0 ( 2.3
BAh (%)
iv, 1 mg/kg
po, 1 mg/kg
4
4
35.7 ( 13.3
10.1 ( 3.2
107.6 ( 34.3
20.8 ( 7.1
19.3 ( 6.6
a
b
c
d
e
n ) number of animals. t1/2 ) pharmacokinetic half-life. CL ) plasma clearance. Vd(ss) ) steady-state volume of distribution. AUC
f
) integrated area under plasma concentration vs time curve from time 0 to time infinity. Cmax ) maximum plasma concentration taken
directly from measured values. Tmax ) time to reach Cmax
g
.
hBA ) oral bioavailability: (AUCpo/AUCiv) × 100.

1292 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8
Akahoshi et al.
purified by silica gel column chromatography (66:34 hexanes-
ethyl acetate) to give 4 (5.15 g, 94% yield) as a pale yellow
solid: 1H NMR (500 MHz, DMSO-d6) δ 7.68 (d, J ) 8.8 Hz,
0.5H, NH), 7.54 (d, J ) 9.4 Hz, 0.5H, NH), 7.35-7.17 (m, 10H,
ArH), 6.85 (m, 0.5H,OH), 6.81 (m, 0.5H, OH), 5.00-4.90 (m,
2H, CH2-O), 4.61 (m, 0.5H, CH-O), 4.41 (m, 0.5H, CH-O),
3.91 (m, 1H, CH-N), 2.99 (m, 1H, CHH-Ph), 2.71 (dd, J )
13.5, 11.4 Hz, 0.5H, CHH-Ph), 2.61 (dd, J ) 13.7, 11.2 Hz,
0.5H, CHH-Ph).
Compound 2r , which displayed the most potent hu-
man chymase-inhibitory activity, is also a good inhibitor
of nonhuman chymases. Its high selectivity for chy-
mases over other proteases as well as their distinctive
pharmacokinetic profiles suggest that the nonpeptidic
chymase inhibitors reported here would be useful tools
in elucidating the pathophysiological roles of chymase.
2-(2-Am in o-1-h ydr oxy-3-ph en ylpr opyl)ben zoxazole (8a).
A solution of chloroform (30 mL) and absolute ethanol (28 mL,
0.48 mol) at 0 °C under nitrogen was treated with acetyl
chloride (31 mL, 0.44 mol) dropwise over 20 min. The cyano-
hydrin 4 (4.50 g, 14.5 mmol) in chloroform (30 mL) was added,
the mixture stirred at 0 °C for 3 h, and the solvent evaporated
while maintaining the temperature at 25 °C or below. The
crude imidate in anhydrous ethanol (100 mL) was treated with
o-aminophenol (1.90 g, 17.4 mmol) and heated under reflux
for 6 h and the solvent evaporated. The residue was taken up
in ethyl acetate, washed successively with 0.5 N NaOH, 0.5 N
HCl, saturated NaHCO3 and brine, dried (MgSO4) and con-
centrated. The residue was purified by silica gel column
chromatography (98:2 chloroform-methanol) to give 2-(2-
benzyloxycarbonylamino-1-hydroxy-3-phenylpropyl)benzox-
azole (5.12 g, 88% yield) as a pale brown solid: 1H NMR (500
MHz, DMSO-d6) δ 7.76-7.66 (m, 2H, ArH), 7.42-7.00 (m, 13H,
ArH), 6.43 (d, J ) 6.2 Hz, 0.4H, OH), 6.25 (d, J ) 5.4 Hz, 0.6H,
OH), 4.93-4.71 (m, 3H, CH2-O, CH-O), 4.16 (m, 1H, CH-
N), 3.15 (dd, J ) 13.8, 2.5 Hz, 0.4H, CHH-Ph), 3.01 (dd, J )
13.8, 4.7 Hz, 0.6H, CHH-Ph), 2.80-2.72 (m, 1H, CHH-Ph).
To a solution of the above intermediate (3.63 g, 9.02 mmol)
in methanol (50 mL) was added 10% palladium-carbon (480
mg) under a nitrogen atmosphere. The reaction mixture was
stirred under a hydrogen atmosphere at room temperature for
18 h. Palladium-carbon was removed by filtration and washed
with methanol. The filtrate was concentrated to give 8a (2.43
g, 100% yield) as a pale brown solid: 1H NMR (500 MHz,
DMSO-d6) δ 7.74-7.68 (m, 2H, ArH), 7.41-7.15 (m, 7H, ArH),
6.17 (m, 0.4H, OH), 6.08 (m, 0.6H, OH), 4.61 (m, 0.6H, CH-
O), 4.54 (m, 0.4H, CH-O), 3.34 (m, 1H, CH-N), 3.05 (dd, J )
13.4, 3.8 Hz, 0.4H, CHH-Ph), 2.78 (dd, J ) 13.4, 5.9 Hz, 0.6H,
CHH-Ph), 2.60 (dd, J ) 13.4, 7.9 Hz, 0.6H, CHH-Ph), 2.53 (dd,
J ) 13.4, 8.9 Hz, 0.4H, CHH-Ph), 1.47 (br s, 2H, NH2).
Con clu sion
Starting from the peptidic chymase inhibitor 1, we
synthesized a series of 5-amino-6-oxo-1,6-dihydropyri-
midine derivatives bearing heterocycles and evaluated
their human chymase-inhibitory activity. We found that
compounds with heterobicycles such as benzoxazole
were more potent than those with heteromonocycles
such as oxazoline or thiazole and that the introduction
of a methoxycarbonyl group at the 5-position on the
benzoxazole ring led to a 5-fold increase in chymase-
inhibitory activity. These findings suggest that the S′
subsite of chymase not only has such a stacking interac-
tion with the aromatic ring of the heterobicycles but also
participates in a hydrophobic interaction with the
substituents on the heterobicycles. Further SAR studies
at the 2-position of the pyrimidinone ring revealed that
compound 2r (Y-40079) had the most potent chymase-
inhibitory activity (Ki ) 4.85 nM). This compound was
also effective toward chymases of nonhuman orgins and
showed good selectivity for chymases over other pro-
teases. Pharmacokinetic studies in rat indicated that
2r was absorbed slowly after oral administration and
showed satisfactory bioavailability (BA) (Tmax ) 6.0 (
2.3 h, BA ) 19.3 ( 6.6%, t1/2 ) 35.7 ( 13.3 h). This
biological profile suggests that compound 2r will prove
useful in elucidating the pathophysiological roles of
chymase.
Exp er im en ta l Section
2-[5-Ben zyloxycar bon ylam in o-2-(4-flu or oph en yl)-6-oxo-
1,6-d ih yd r o-1-p yr im id in yl]-N -[1-[(2-b e n zoxa zolyl)h y-
d r oxym eth yl]-2-p h en yleth yl]a ceta m id e (13a ). To a solu-
tion of [5-benzyloxycarbonylamino-2-(4-fluorophenyl)-6-oxo-1,6-
dihydro-1-pyrimidinyl]acetic acid (11a ) (1.78 g, 4.48 mmol) and
2-(2-amino-1-hydroxy-3-phenylpropyl)benzoxazole (8a ) (1.37 g,
5.11 mmol) in DMF (15 mL) were added HOBT (1.21 g, 8.95
mmol) and EDC (1.03 g, 5.37 mmol). After stirring at room
temperature for 4 h, the reaction mixture was poured into 0.5
N HCl (100 mL), and then extracted with ethyl acetate. The
extract was washed with saturated NaHCO3 and brine, dried
(MgSO4) and concentrated. The residue was washed with
diethyl ether and dried in vacuo to give 13a (2.43 g, 84% yield)
as colorless crystals: 1H NMR (500 MHz, DMSO-d6) δ 8.79
(br s, 0.7H, NH), 8.68 (br s, 0.3H, NH), 8.39 (s, 1H, CHdN),
8.34 (d, J ) 8.7 Hz, 0.3H, NH), 8.30 (d, J ) 8.7 Hz, 0.7H, NH),
7.73-7.64 (m, 2H, ArH), 7.47-7.31 (m, 9H, ArH), 7.28-7.07
(m, 7H, ArH), 6.44 (d, J ) 6.0 Hz, 0.3H, OH), 6.38 (d, J ) 5.1
Hz, 0.7H, OH), 5.19 (s, 2H, CH2-O), 4.81 (m, 1H, CH-O),
4.48-4.18 (m, 3H, CH2-N, CH-N), 3.00 (dd, J ) 13.8, 5.9
Hz, 0.7H, CHH-Ph), 2.93 (dd, J ) 14.1, 3.5 Hz, 0.3H, CHH-
Ph), 2.83 (dd, J ) 14.1, 9.6 Hz, 0.3H, CHH-Ph), 2.69 (dd, J )
13.8, 8.4 Hz, 0.7H, CHH-Ph).
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
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.
3-Ben zyloxycar bon ylam in o-2-h ydr oxy-4-ph en ylbu tyln i-
tr ile (4). To a solution of N-(benzyloxycarbonyl)phenylalaninal
(3) (5.00 g, 17.6 mmol) and acetone cyanohydrin (4.8 mL, 53
mmol) in dichloromethane (50 mL) was added triethylamine
(1.5 mL, 11 mmol). The reaction mixture was stirred at room
temperature for 4 h, at which time the solvent was evaporated.
The residue was poured into water (100 mL) and then
extracted with ethyl acetate. The extract was washed with
brine, dried (MgSO4) and concentrated. The residue was
2-[5-Am in o-2-(4-flu or op h en yl)-6-oxo-1,6-d ih yd r o-1-p y-
r im idin yl]-N-[1-[(2-ben zoxazolyl)car bon yl]-2-ph en yleth yl]-
a ceta m id e (2a ). To a solution of 13a (2.12 g, 3.27 mmol) in
DMSO (20 mL) and toluene (20 mL) were added EDC (3.13 g,
16.3 mmol) and then dichloroacetic acid (0.54 mL, 6.5 mmol).
After stirring at room temperature for 3.5 h, the reaction
mixture was poured into 1 N HCl (100 mL), and then extracted
with ethyl acetate. The extract was washed with saturated

Nonpeptidic R-Keto Heterocycles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1293
NaHCO3 and brine and dried (MgSO4). The solvent was
evaporated and the residue purified by silica gel column
chromatography (83:17 dichloromethane-ethyl acetate) to give
2-[5-benzyloxycarbonylamino-2-(4-fluorophenyl)-6-oxo-1,6-di-
hydro-1-pyrimidinyl]-N-[1-[(2-benzoxazolyl)carbonyl]-2-phenyl-
ethyl]acetamide (1.69 g, 80% yield) as colorless crystals.
Recrystallization from ethyl acetate afforded colorless crystals
(1.43 g): mp 222-225 °C; 1H NMR (500 MHz, DMSO-d6) δ
8.98 (d, J ) 6.9 Hz, 1H, NH), 8.86 (s, 1H, NH), 8.40 (s, 1H,
CHdN), 8.00 (d, J ) 8.0 Hz, 1H, ArH), 7.91 (d, J ) 8.0 Hz,
1H, ArH), 7.66 (t, J ) 8.0 Hz, 1H, ArH), 7.55 (t, J ) 8.0 Hz,
1H, ArH), 7.46-7.40 (m, 4H, ArH), 7.38 (t, J ) 7.1 Hz, 2H,
ArH), 7.33 (t, J ) 7.1 Hz, 1H, ArH), 7.25 (t, J ) 6.7 Hz, 2H,
ArH), 7.22-7.13 (m, 5H, ArH), 5.56 (m, 1H, CH-CO), 5.17 (s,
2H, CH2-O), 4.53 (d, J ) 16.6 Hz, 1H, CHH-N), 4.45 (d, J )
16.6 Hz, 1H, CHH-N), 3.31 (dd, J ) 14.2, 4.8 Hz, 1H, CHH-
Ph), 2.96 (dd, J ) 14.2, 9.0 Hz, 1H, CHH-Ph); MS (SIMS,
positive) m/z 646 (MH+).
To a solution of the above intermediate (581 mg, 0.900
mmol) and anisole (0.31 mL, 2.9 mmol) in dichloromethane (9
mL) was added trifluoromethanesulfonic acid (0.48 mL, 5.4
mmol) with ice-water cooling. The reaction mixture was stirred
at 0 °C to room temperature for 1 h, after which saturated
NaHCO3 (10 mL) was added. After stirring at room temper-
ature for 1.5 h, the reaction mixture was poured into saturated
NaHCO3 (50 mL) and then extracted with ethyl acetate. The
extract was washed with brine and dried (MgSO4). The solvent
was evaporated and the residue purified by silica gel column
chromatography (97:3 chloroform-methanol) to give 2a (437
mg, 95% yield) as colorless crystals: mp 233-236 °C; 1H NMR
(500 MHz, DMSO-d6) δ 8.92 (d, J ) 6.8 Hz, 1H, NH), 8.01 (d,
J ) 8.0 Hz, 1H, ArH), 7.91 (d, J ) 8.0 Hz, 1H, ArH), 7.66 (t,
J ) 8.0 Hz, 1H, ArH), 7.56 (t, J ) 8.0 Hz, 1H, ArH), 7.35 (dd,
J ) 8.6, 5.5 Hz, 2H, ArH), 7.28 (s, 1H, CHdN), 7.26 (t, J )
8.0 Hz, 2H, ArH), 7.23-7.17 (m, 3H, ArH), 7.09 (t, J ) 8.0
Hz, 2H, ArH), 5.55 (m, 1H, CH-CdO), 5.13 (s, 2H, NH2), 4.48
(d, J ) 16.7 Hz, 1H, CHH-N), 4.41 (d, J ) 16.7 Hz, 1H, CHH-
N), 3.30 (dd, J ) 14.1, 4.9 Hz, 1H, CHH-Ph), 2.97 (dd, J )
14.1, 8.9 Hz, 1H, CHH-Ph); MS (SIMS, positive) m/z 512
(MH+). Anal. (C28H22FN5O4) C, H, N.
NaHCO3 and brine, dried (MgSO4) and concentrated. The
residue was purified by silica gel column chromatography (97:3
chloroform-methanol) to give 2-[5-benzyloxycarbonylamino-
2-(4-fluorophenyl)-6-oxo-1,6-dihydro-1-pyrimidinyl]-N-[1-[(5-
aminobenzoxazol-2-yl)carbonyl]-2-phenylethyl]acetamide (1.00
g, 78% yield) as red brown crystals: mp 163-169 °C; 1H NMR
(300 MHz, DMSO-d6) δ 8.93 (s, 1H, NH), 8.92 (d, J ) 6.6 Hz,
1H, NH), 8.41 (s, 1H, CHdN), 7.54 (d, J ) 8.9 Hz, 1H, ArH),
7.48-7.13 (m, 14H, ArH), 6.95 (d, J ) 2.0 Hz, 1H, ArH), 6.92
(dd, J ) 8.9, 2.0 Hz, 1H, ArH), 5.56 (m, 1H, CH-CdO), 5.37
(s, 2H, NH2), 5.17 (s, 2H, NH2), 4.53 (d, J ) 16.7 Hz, 1H, CHH-
N), 4.44 (d, J ) 16.7 Hz, 1H, CHH-N), 3.25 (dd, J ) 14.0, 4.4
Hz, 1H, CHH-Ph), 2.92 (dd, J ) 14.0, 8.9 Hz, 1H, CHHPh).
The above intermediate was deprotected using a method
similar to that described for 2a to give 2e (83% yield) as yellow
crystals: mp 163-169 °C; 1H NMR (500 MHz, DMSO-d6) δ
8.83 (d, J ) 7.0 Hz, 1H, NH), 7.53 (d, J ) 8.8 Hz, 1H, ArH),
7.36 (m, 2H, ArH), 7.28 (s, 1H, ArH), 7.26 (t, J ) 7.3 Hz, 2H,
ArH), 7.23-7.17 (m, 3H, ArH), 7.13 (t, J ) 8.8 Hz, 2H, ArH),
6.96 (d, J ) 2.2 Hz, 1H, ArH), 6.92 (dd, J ) 8.8, 2.2 Hz, 1HV),
5.56 (m, 1H, CH-CdO), 5.35 (s, 2H, NH2), 5.14 (s, 2H, NH2),
4.48 (d, J ) 16.6 Hz, 1H, CHH-N), 4.41 (d, J ) 16.6 Hz, 1H,
CHH-N), 3.25 (dd, J ) 14.1, 4.8 Hz, 1H, CHH-Ph), 2.93 (dd, J
) 14.1, 8.8 Hz, 1H, CHH-Ph); MS (SIMS, positive) m/z 527
(MH+). Anal. (C28H22FN5O5‚0.2H2O) C, H, N.
2-[5-Am in o-2-(4-flu or op h en yl)-6-oxo-1,6-d ih yd r o-1-p y-
r im id in yl]-N-[1-[(5-ca r boxyben zoxa zol-2-yl)ca r bon yl]-2-
p h en yleth yl]a ceta m id e (2i). To a solution of 2g (180 mg,
0.316 mmol) in dimethyl sulfide (5 mL) and dichloromethane
(5 mL) was added aluminum bromide (680 mg, 2.55 mmol)
with ice-water cooling. The reaction mixture was stirred at 0
°C-room temperature for 5 h, after which water (10 mL) and
1 N HCl (10 mL) were added. After stirring at room temper-
ature for 1 h, the resulting suspension was filtrated. The solid
was washed with water and chloroform and purified by silica
gel column chromatography (67:33 chloroform-methanol) to
give 2i (146 mg, 83% yield) as yellow crystals: mp 207-214
1
°C; H NMR (500 MHz, DMSO-d6) δ 8.94 (d, J ) 6.8 Hz, 1H,
NH), 8.46 (s, 1H, ArH), 8.24 (d, J ) 8.6 Hz, 1H, ArH), 7.87 (d,
J ) 8.6 Hz, 1H, ArH), 7.35 (dd, J ) 8.7, 5.5 Hz, 2H, ArH),
7.29-7.18 (m, 6H, ArH), 7.11 (t, J ) 8.7 Hz, 2H, ArH), 5.55
(m, 1H, CH-CdO), 5.13 (s, 2H, NH2), 4.48 (d, J ) 16.6 Hz,
1H, CHH-N), 4.42 (d, J ) 16.6 Hz, 1H, CHH-N), 3.30 (m, 1H,
CHH-Ph), 2.97 (dd, J ) 14.1, 8.8 Hz, 1H, CHH-Ph); MS
(SIMS, positive) m/z 556 (MH+). Anal. (C29H22FN5O6‚2.3H2O)
C, H, N.
The compounds 2b,c,f-h ,m -v were prepared using the
same procedures as described above.
2-[5-Am in o-2-(4-flu or op h en yl)-6-oxo-1,6-d ih yd r o-1-p y-
r im id in yl]-N-[1-[(5-h yd r oxyben zoxa zol-2-yl)ca r bon yl]-2-
p h en yleth yl]a ceta m id e (2d ). To a solution of 2c (452 mg,
0.835 mmol) in dichloromethane (10 mL) was added a solution
of boron tribromide in dichloromethane (1.0 M, 8.4 mL, 8.4
mmol) with ice-water cooling. The resulting mixture was
stirred at 0 °C-room temperature for 4 h, at which time
methanol (1.5 mL) was added. After stirring for 10 min, the
reaction mixture was poured into saturated NaHCO3 (50 mL),
and then extracted with ethyl acetate. The extract was washed
with brine, dried (MgSO4) and concentrated. The residue was
purified by silica gel column chromatography (91:9 chloroform-
methanol) to give 2d (340 mg, 77% yield) as a yellow solid:
1H NMR (500 MHz, DMSO-d6) δ 9.95 (brs, 1H, OH), 8.89 (d, J
) 6.9 Hz, 1H, NH), 7.69 (d, J ) 8.9 Hz, 1H, ArH), 7.35 (dd, J
) 8.5, 5.6 Hz, 2H, ArH), 7.29-7.07 (m, 10H, ArH), 5.54 (m,
1H, CH-CdO), 5.15 (s, 2H, NH2), 4.48 (d, J ) 16.5 Hz, 1H,
CHH-N), 4.41 (d, J ) 16.5 Hz, 1H, CHH-N), 3.27 (dd, J ) 14.1,
4.7 Hz, 1H, CHH-Ph), 2.94 (dd, J ) 14.1, 9.0 Hz, 1H, CHHPh);
MS (SIMS, positive) m/z 528 (MH+). Anal. (C28H22FN5O5‚
0.2H2O) C, H, N.
2-[5-Am in o-2-(4-flu or op h en yl)-6-oxo-1,6-d ih yd r o-1-p y-
r im id in yl]-N-[1-[(5-a m in ob en zoxa zol-2-yl)ca r b on yl]-2-
p h en yleth yl]a ceta m id e (2e). To a solution of 2-[5-benzylox-
yca r bon yla m in o-2-(4-flu or oph en yl)-6-oxo-1,6-dih ydr o-1-
pyr im idin yl]-N-[1-[(5-n it r oben zoxa zol-2-yl)ca r bon yl]-2-
phenylethyl]acetamide (24) (1.34 g, 1.94 mmol) in THF (40
mL), water (7 mL) and methanol (7 mL) were added succes-
sively iron powder (2.73 g, 49.0 mmol) and 1 N HCl (1.64 mL).
The resulting mixture was stirred at room temperature for
18 h. The iron powder was removed by filtration and washed
with chloroform. The filtrate was washed with saturated
2-[5-Ben zyloxycar bon ylam in o-2-(4-flu or oph en yl)-6-oxo-
1,6-d ih yd r o-1-p yr im id in yl]-N-[1-[(5-ca r boxylben zoxa zol-
2-yl)h yd r oxym eth yl]-2-p h en yleth yl]a ceta m id e (13i). To
a solution of 2-[5-benzyloxycarbonylamino-2-(4-fluorophenyl)-
6-oxo-1,6-dihydro-1-pyrimidinyl]-N-[1-[[5-(methoxycarbonyl)-
benzoxazol-2-yl]hydroxymethyl]-2-phenylethyl]acetamide (13g)
(2.00 g, 2.83 mmol) in DMSO (250 mL) was added 1 N NaOH
(30 mL). After stirring at room temperature for 1 h, the
reaction mixture was poured into 0.1 N HCl (1000 mL) and
then extracted with ethyl acetate. The extract was washed
with brine, dried (MgSO4) and concentrated. The residue was
washed with diethyl ether and dried in vacuo to give 13i (1.58
g, 81% yield) as a colorless solid: 1H NMR (500 MHz, DMSO-
d6) δ 13.0 (br s, 1H, OH), 8.79 (s, 0.6H, NH), 8.65 (s, 0.4H,
NH), 8.39-8.32 (m, 2H, CHdN, NH), 8.24 (d, J ) 1.2 Hz, 0.4H,
NH), 8.21 (d, J ) 1.2 Hz, 0.6H, NH), 8.04-7.97 (m, 1H, ArH),
7.82-7.77 (m, 1H, ArH), 7.46-7.07 (m, 14H, ArH), 6.53 (d, J
) 6.0 Hz, 0.4H, OH), 6.46 (d, J ) 4.9 Hz, 0.6H, OH), 5.19 (s,
2H, CH2-O), 4.87-4.80 (m, 1H, CH-O), 4.52-4.18 (m, 3H,
CH2-N, CH-N), 3.02 (dd, J ) 13.8, 6.0 Hz, 0.6H, CHH-Ph),
2.96 (dd, J ) 14.0, 3.7 Hz, 0.4H, CHH-Ph), 2.82 (dd, J ) 14.0,
9.5 Hz, 0.4H, CHH-Ph), 2.71 (dd, J ) 13.8, 8.6 Hz, 0.6H,
CHH-Ph).
2-[5-Ben zyloxycar bon ylam in o-2-(4-flu or oph en yl)-6-oxo-
1,6-dih ydr o-1-pyr im idin yl]-N-[1-[(5-car bam oylben zoxazol-
2-yl)h yd r oxym eth yl]-2-p h en yleth yl]a ceta m id e (13j). To
a solution of 13i (1.23 g, 1.77 mmol), NH4Cl (190 mg, 3.55
mmol) and N-ethylmorpholine (0.45 mL, 3.5 mmol) in DMF

1294 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8
Akahoshi et al.
(30 mL) were added HOBT (479 mg, 3.55 mmol) and EDC (408
mg, 2.13 mmol). After stirring at room temperature for 15 h,
the reaction mixture was poured into 1 N HCl (100 mL), and
then extracted with ethyl acetate. The extract was washed
with saturated NaHCO3 and brine, dried (MgSO4) and con-
centrated. The residue was washed with diethyl ether and
dried in vacuo to give 13j (952 mg, 78% yield) as a pale brown
solid: 1H NMR (300 MHz, DMSO-d6) δ 8.84 (br s, 0.6H, NH),
8.75 (br s, 0.4H, NH), 8.42-8.31 (m, 2H, CHdN, NH), 8.26 (s,
0.4H, NH), 8.22 (s, 0.6H, NH), 8.08 (br s, 1H, NHH), 7.94-
7.92 (m, 1H, ArH), 7.77 (d, J ) 8.6 Hz, 0.4H, ArH), 7.73 (d, J
) 8.6 Hz, 0.6H, ArH), 7.47-7.07 (m, 15H, ArH, NHH), 6.52
(br s, 0.4H, OH), 6.47 (br s, 0.6H, OH), 5.19 (s, 2H, CH2-O),
4.82 (br s, 1H, CH-O), 4.52-4.18 (m, 3H, CH2-N, CH-N),
3.03-2.63 (m, 2H, CH2-Ph).
2-[5-Am in o-2-(4-flu or op h en yl)-6-oxo-1,6-d ih yd r o-1-p y-
r im id in yl]-N-[1-[(5-ca r ba m oylben zoxa zol-2-yl)ca r bon yl]-
2-p h en yleth yl]a ceta m id e (2j). To a solution of 13j (687 mg,
0.995 mmol) in DMF (10 mL) was added Dess-Martin perio-
dinate (1.06 g, 2.49 mmol). The reaction mixture was stirred
at room temperature for 4 h, after which saturated NaHCO3
(12 mL) containing sodium thiosulfate (2.64 g) was added
with ice-water cooling. After stirring at 0 °C for 30 min, the
reaction mixture was poured into saturated NaHCO3 (100 mL)
and extracted with ethyl acetate. The extract was washed with
10% citric acid aqueous solution, saturated NaHCO3 and brine,
dried (MgSO4) and concentrated. The residue was purified by
silica gel column chromatography (95:5 chloroform-ethanol)
and recrystallization from chloroform-diethyl ether to give
2-[5-benzyloxycarbonylamino-2-(4-fluorophenyl)-6-oxo-1,6-di-
hydro-1-pyrimidinyl]-N-[1-[(5-carbamoylbenzoxazol-2-yl)carbo-
nyl]-2-phenylethyl]acetamide (295 mg, 43% yield) as pale
yellow crystals: mp 264-266 °C; 1H NMR (300 MHz, DMSO-
d6) δ 9.01 (d, J ) 7.1 Hz, 1H, NH), 8.89 (s, 1H, NH), 8.50 (s,
1H, ArH), 8.40 (s, 1H, CHdN), 8.22-8.10 (m, 2H, ArH), 7.98
(d, J ) 8.7 Hz, 1H, ArH), 7.62-7.10 (m, 16H, ArH, NH2), 5.60-
5.42 (m, 1H, CH-CdO), 5.17 (s, 2H, CH2-O), 4.60-4.38 (m,
2H, CH2-N), 3.45-2.85 (m, 2H, CH2-Ph).
and phenylalaninol (19) (2.51 g, 16.6 mmol) were coupled using
a similar procedure as described for 13a to give 20 (6.51 g,
81% yield) as colorless crystals: mp 232-235 °C; 1H NMR (500
MHz, DMSO-d6) δ 8.89 (s, 1H, NH), 8.43 (s, 1H, CHdN), 8.05
(d, J ) 8.2 Hz, 1H, NH), 7.51 (dd, J ) 8.6, 5.5 Hz, 1H, ArH),
7.44 (d, J ) 7.1 Hz, 2H, ArH), 7.39 (t, J ) 7.1 Hz, 2H, ArH),
7.34 (t, J ) 7.1 Hz, 1H, ArH), 7.28-7.21 (m, 4H, ArH), 7.18 (t,
J ) 7.1 Hz, 1H, ArH), 5.19 (s, 2H, CH2-O), 4.78 (br s, 1H,
OH), 4.47 (d, J ) 16.4 Hz, 1H, CHH-N), 4.35 (d, J ) 16.6 Hz,
1H, CHH-N), 3.87 (m, 1H, CH-N), 3.28 (m, 2H, CH2-O), 2.79
(dd, J ) 13.7, 5.8 Hz, 1H, CHH-Ph), 2.58 (dd, J ) 13.7, 7.9
Hz, 1H, CHH-Ph).
2-[5-Ben zyloxycar bon ylam in o-2-(4-flu or oph en yl)-6-oxo-
1,6-d ih yd r o-1-p yr im id in yl]-N-[1-for m yl-2-p h en ylet h yl]-
a ceta m id e (21). Compound 20 (5.89 g, 11.1 mmol) was
oxidized using a similar procedure as described for 2a to give
21 (5.45 g, 93% yield) as colorless crystals: mp 138-141 °C;
1H NMR (500 MHz, CDCl3) δ 9.62 (s, 1H, CHdO), 8.77 (br s,
1H, CHdN), 7.53 (dd, J ) 8.7, 5.2 Hz, 2H, ArH), 7.48 (s, 1H,
ArH), 7.43-7.33 (m, 5H, ArH), 7.28-7.08 (m, 7H, ArH), 6.52
(d, J ) 6.8 Hz, 1H, ArH), 5.24 (s, 2H, CH2-O), 4.77 (q, J )
6.6 Hz, 1H, CH-N), 4.52 (AB-q, J ) 15.4 Hz, 2H, CHH-N),
3.17 (m, 2H, CH2-Ph); MS (SIMS, positive) m/z 529 (MH+).
2-[5-Ben zyloxycar bon ylam in o-2-(4-flu or oph en yl)-6-oxo-
1,6-d ih yd r o-1-p yr im id in yl]-N-[2-p h en yl-1-[(2-th ia zolyl)-
h yd r oxym eth yl]eth yl]a ceta m id e (22). To a solution of 21
(724 mg, 1.37 mmol) in dichloromethane (15 mL) was added
2-trimethylsilylthiazole (0.23 mL, 1.4 mmol), and the mixture
stirred at room temperature for 1 day. Tetrabutylammonium
fluoride (1.0 M solution in THF, 2.5 mL, 2.5 mmol) was then
added. After stirring at room temperature for 30 min, the
reaction mixture was poured into saturated NaHCO3 (50 mL),
and then extracted with chloroform. The extract was washed
with brine, dried (MgSO4) and concentrated. The residue was
purified by silica gel column chromatography (98:2 chloroform-
methanol) to give 22 (665 mg, 79% yield) as a colorless solid:
1H NMR (500 MHz, DMSO-d6) δ 8.88 (s, 1H, NH), 8.42 (s,
0.25H, CHdN), 8.40(s, 0.75H, CHdN), 8.33 (d, J ) 10.7 Hz,
0.25H, NH), 8.21 (d, J ) 9.0 Hz, 0.75H, NH), 7.78 (d, J ) 3.2
Hz, 0.25H, ArH), 7.68 (d, J ) 3.2 Hz, 0.75H, ArH), 7.65 (d, J
) 3.2 Hz, 0.25H, ArH), 7.56 (d, J ) 3.2 Hz, 0.75H, ArH), 7.46-
7.12 (m, 13.5H, ArH), 7.04 (d, J ) 6.8 Hz, 0.5H, ArH), 6.64 (d,
J ) 5.1 Hz, 0.75H, OH), 6.61 (d, J ) 5.6 Hz, 0.25H, OH), 5.18
(s, 2H, CH2-O), 4.85 (dd, J ) 5.6, 3.8 Hz, 0.25H, CH-O), 4.78
(dd, J ) 5.1, 2.6 Hz, 0.75H, CH-O), 4.57-4.22 (m, 3H, CH2-
N, CH-N), 2.89 (dd, J ) 13.5, 7.4 Hz, 0.75H, CHH-Ph), 2.68
(dd, J ) 14.4, 10.4 Hz, 0.25H, CHH-Ph), 2.58 (dd, J ) 13.5,
7.1 Hz, 0.75H, CHH-Ph), 2.5 (m, 0.25H, CHH-Ph).
The above intermediate was deprotected using a method
similar to that described for 2a to give 2j (58% yield) as
colorless crystals: mp 270 °C dec; 1H NMR (300 MHz, DMSO-
d6) δ 8.96 (d, J ) 6.8 Hz, 1H, NH), 8.51 (s, 1H, ArH), 8.24-
8.11 (m, 2H, ArH), 7.98 (d, J ) 8.7 Hz, 1H, ArH), 7.56 (br s,
1H NHH), 7.35 (dd, J ) 8.4, 5.6 Hz, 2H, ArH), 7.30-7.15 (m,
5H, ArH, NHH), 7.10 (t, J ) 8.7 Hz, 2H, ArH), 5.60-5.40 (m,
1H, CH-CdO), 5.14 (s, 2H, NH2), 4.58-4.30 (m, 2H, CH2-
N), 3.40-3.20 (m, 1H, CHH-Ph), 3.00-2.80 (m, 1H, CHH-Ph).
Anal. (C29H23FN6O5‚0.8H2O) C, H, N.
The compounds 2k ,l were prepared using the same proce-
dures as described above.
2-[5-Am in o-2-(4-flu or op h en yl)-6-oxo-1,6-d ih yd r o-1-p y-
r im id in yl]-N-[2-p h en yl-1-[(2-th ia zolyl)ca r bon yl]eth yl]a c-
eta m id e (23). Compound 22 was oxidized using a similar
procedure as described for 2a to give 23 as colorless crystals:
mp 199-203 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.79 (d, J )
7.5 Hz, 1H, NH), 8.30 (d, J ) 2.9 Hz, 1H, ArH), 8.21 (d, J )
2.9 Hz, 1H, ArH), 7.36 (dd, J ) 8.5, 5.6 Hz, 2H, ArH), 7.29-
7.14 (m, 8H, ArH), 5.67 (m, 1H, CH-CdO), 5.15 (s, 2H, NH2),
4.50 (d, J ) 16.6 Hz, 1H, CHH-N), 4.39 (d, J ) 16.6 Hz, 1H,
CHH-N), 3.19 (dd, J ) 14.0, 4.3 Hz, 1H, CHH-Ph), 2.89 (dd, J
) 14.0, 9.2 Hz, 1H, CHH-Ph); MS (SIMS, positive) m/z 478
(MH+). Anal. (C24H20FN5O3S) 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.5 Bovine
pancreatic R-chymotrypsin and human cathepsin G were
purchased from Sigma Chemical Co., St. Louis, MO, and
Athens Research and Technologies Inc., respectively. Human
thrombin, elastase, and angiotensin-converting enzyme (ACE)
were from Welfide Corp., Osaka, J apan, Elastins Products Co.
Inc., Owensville, MO, and Nippon Zoki Pharmaceutical Co.,
Osaka, J apan, respectively.
2-(2-Am in o-1-h yd r oxy-3-p h en ylp r op yl)oxa zolin e (6).
To the crude imidate 5 [prepared from the cyanohidrin 4 (1.33
g, 4.27 mmol)] in dichloromethane (18 mL) were added
monoethanolamine (0.51 mL, 8.5 mmol) and triethylamine (1.2
mL, 8.6 mmol). After stirring at room temperature for 18 h,
the reaction mixture was poured into 1 N NaOH (50 mL), and
then extracted with ethyl acetate. The extract was washed
with brine, dried (MgSO4) and concentrated. The residue was
purified by silica gel column chromatography (97:3 chloroform-
methanol) to give 2-(2-benzyloxycarbonylamino-1-hydroxy-3-
phenylpropyl)oxazoline (509 mg, 33% yield) as a colorless solid.
A solution of this intermediate (505 mg, 1.42 mmol) was
deprotected using a method similar to that described for 8a
to give 6 (284 mg, 91% yield) as a colorless solid: 1H NMR
(300 MHz, DMSO-d6) δ 7.33-7.12 (m, 5H, ArH), 4.20 (m, 2H,
CH2-O), 3.90 (dd, J ) 8.4, 4.6 Hz, 1H, CH-O), 3.72 (m, 2H,
CH2-N), 3.04 (m, 1H, CH-N), 2.90 (dd, J ) 13.4, 4.1 Hz, 0.4H,
CHH-Ph), 2.80-2.65 (m, 1.2H, CH2-Ph), 2.42 (dd, J ) 13.4,
8.8 Hz, 0.4H, CHH-Ph).
Compounds 16-18 were prepared using a similar procedure
as described for 2a .
2-[5-Ben zyloxycar bon ylam in o-2-(4-flu or oph en yl)-6-oxo-
1,6-d ih yd r o-1-p yr im id in yl]-N-[1-h yd r oxym et h yl-2-p h e-
n yleth yl]a ceta m id e (20). Compound 11a (6.00 g, 15.1 mmol)
Inhibitory activities for chymase, chymotrypsin, and cathe-
psin G were measured using previously described methods25
through measurement at 405-nm absorbance of p-nitroaniline
release from the synthetic substrate succinyl-Ala-Ala-Pro-Phe-
p-nitroanilide (Sigma Chemical Co.). In the case of thrombin

Nonpeptidic R-Keto Heterocycles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1295
and elastase, buffer conditions previously described46 were
used with the synthetic substrates S-2238 (Chromogenix AB,
Mo¨lndal, Sweden) and CH3O-Suc-Ala-Ala-Pro-Val-p-nitroa-
niline (Sigma Chemical Co.), respectively. Enzymatic activity
of ACE was measured using the methods described by Miyaza-
ki et al.47 Calculations of Ki’s were performed using previously
described methods.25
internal standard solution (0.6 µM/mL 2n , 50 µL). The sample
was extracted with ethyl acetate (5 mL). After phase separa-
tion through centrifugation, the organic layer was transferred
and evaporated to dryness under nitrogen steam. The residue
was reconstituted in 100 µL of 10 mM ammonium acetate (pH
6.8)-acetonitrile (50:50, v/v), and 50 µL was injected onto an
LC-MS/MS system. Chromatographic separation was per-
formed using a Develosil ODS-MG-3 (50 mm × 2 mm i.d., 3
µm, Nomura Chemical Co., J apan) at a flow rate of 0.2 mL/
min. The mobile phase consisted of 10 mM ammonium acetate
(pH 6.8) (A) and acetonitrile (B). The following gradient system
was used: 20% B to 80% B linearly over the first 2.5 min;
isocratic 80% B for 2.5 min. Protonated analyte ions were
generated using electron spray ionization. For quantitation,
the ion signal was recorded by selective reaction monitoring.
Pharmacokinetic parameters were determined by noncom-
partmental methods.
In Vitr o Degr a d a tion Stu d y by Ra t Liver Hom oge-
n a te. The S9 protein (2.5 mg) of rat liver was incubated with
2r (final concentration, 50 µM; added as 5 µL in DMSO) in
100 mM phosphate buffer (pH 7.4, final volume; 0.5 mL)
consisting of 4 mM NADPH, 4 mM NADH, 8 mM MgCl2, 5
mM glucose-6-phosphate, and 1 U/mL glucose-6-phosphate
dehydrogenase at 37 °C for 15 min. The reaction was termi-
nated by the addition of acetonitrile (2 mL). After centrifuga-
tion the supernatant was analyzed under the above HPLC
conditions.
Kin etic Stu d ies for Rep r esen ta tive Com p ou n d by
P r ogr ess Cu r ve Meth od . The progress of human chymase
inhibition by 2r was measured under a pseudo-first-order
inhibition condition, i.e., [I0] g 10[E0], by reacting chymase
with a mixture of inhibitor and substrate and recording the
liberation of free p-nitroaniline at 405-nm absorbance. Reac-
tions were conducted in 20 mM Tris-HCl (pH 7.5) containing
0.1 mM aprotinin and 2 M KCl at 37 °C. Under these
conditions, Km (Michaelis-Menton constant) for succinyl-Ala-
Ala-Pro-Phe-p-nitroanilide was 741 nM. Reaction progress was
monitored in a 96-well fluorimetric plate using a fluorimetric
plate reader (Spectrafluor Tecan J apan; Tokyo, J apan). Reac-
tion was preformed in 200 µL of final volume and initiated by
the addition of a 20 µL aliquot of enzyme stock. Readings were
taken every 30 s for a total of 1800 s, and initial absorbance
values were subtracted from the data prior to subsequent
calculations. Data were fitted to the integrated rate equation
for slow-binding inhibition (eq 1) according to the method
described by Williams and Morrison,48 by nonlinear regression
analysis:
A ) vst + (v0 - vs)(1 - e-k’t)/k′ + A0
(1)
Ack n ow led gm en t. The authors thank Masahiro
Takeuchi for his support in analytical chemistry and
Dr. Masahiro Eda, Yoshihisa Inoue, and Mikio Tanaka
for helpful discussions throughout the course of this
work.
Values for v0 (initial rate), vs (final steady-state rate), k′
(apparent rate constant for the transition from v0 to vs), and
A0 (the initial absorbance at 405 nm) were obtained for each
progress curve. Mean values for k′, obtained from three
independent assays, were plotted against the inhibitor con-
centration, [I0]. A linear dependency between [I0] and k′ was
observed and fitted to eq 2 to estimate kon′ (apparent associa-
tion rate constant) and koff (dissociation rate constant). Sub-
sequently a value for kon (association rate constant) was
determined from the correction of kon′ for the competition of
the substrate using eq 3:
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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