Non-peptidic inhibitors of human chymase. Synthesis, structure-activity relationships, and pharmacokinetic profiles of a series of 5-amino-6-oxo-1,6-dihydropyrimidine-containing trifluoromethyl ketones

Article abstract of DOI:10.1016/S0968-0896(00)00244-3

Chymase possesses a wide variety of actions, including promotion of angiotensin II production and histamine release from mast cells. However, due to a lack of effective inhibitors featuring both high inhibitory activity and high metabolic stability, the p

Full text of DOI:10.1016/S0968-0896(00)00244-3

Bioorganic & Medicinal Chemistry 9 (2001) 301±315
Non-Peptidic Inhibitors of Human Chymase. Synthesis,
Structure±Activity Relationships, and Pharmacokinetic Pro®les
of a Series of 5-Amino-6-oxo-1,6-dihydropyrimidine-containing
Tri¯uoromethyl Ketones
Fumihiko Akahoshi,a,* Atsuyuki Ashimori,a Takuya Yoshimura,a Teruaki Imada,a
Masahide Nakajima,a Naoko Mitsutomi,a Shigeki Kuwahara,a Tatsuyuki Ohtsuka,a
Chikara Fukaya,a,y Mizuo Miyazakib and Norifumi Nakamuraa,{
aDrug Discovery Laboratories, Wel®de Corporation, 2-25-1 Shodai-Ohtani, Hirakata, Osaka 573-1153, Japan
bDepartment of Pharmacology, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki, Osaka 569-8686, Japan
Received 10 July 2000; accepted 2 September 2000
AbstractÐChymase possesses a wide variety of actions, including promotion of angiotensin II production and histamine release
from mast cells. However, due to a lack of e€ective inhibitors featuring both high inhibitory activity and high metabolic stability,
the pathophysiological role of chymase has not been fully elucidated. We designed non-peptidic inhibitors based on the predicted
binding mode of the peptidic chymase inhibitor Val-Pro-Phe-CF3 and demonstrated that the Val-Pro unit is replaceable with a
(5-amino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl)acetyl moiety. Structure±activity relationship studies revealed that phenyl sub-
stitution at the 2-position of the pyrimidinone ring is indispensable for high activity. The most potent compound 1h
(Ki=0.0506 mM) is superior in potency to the parent peptidic inhibitor Val-Pro-Phe-CF3 and has good selectivity for chymase over
other proteases. The related analogue 1e was orally absorbed and maintained high plasma levels for at least 2 h. These results sug-
gest that the derivatives reported here could be developed as agents for treatment of chymase-induced disease. # 2001 Elsevier
Science Ltd. All rights reserved.
Introduction
pharmacological importance of chymase. Recently,
attention has been focused on the localized production
of angiotensin II (Ang II) by human chymase in the
cardiovascular system:4,5 chymase levels have been
found to increase in balloon-injured blood vessels of
dogs and in the heart of a hamster model of cardio-
myopathy,6,7 while increased chymase-dependent Ang
II formationhas beenobserved inhumanathero-
sclerotic aorta.8 Inaddition, several reports have sug-
gested that chymase is involved not only in Ang II
formationbut also intissue remodeling inthe cardio-
vascular system,9,10 and that chymase is one of the
endothelin-processing enzymes.11,12
Chymase (EC 3.4.21.39) is a chymotrypsin-like serine
protease which is synthesized and stored in mast cells
localized mainly in the heart, blood vessels and skin.
Mast cells secrete chymase with other biologically active
substances in response to immunological stimuli.
Reports onthe invitro activities of chymase have indi-
cated that it degrades IgG and extracellular matrices
such as type IV collagen, ®bronectin and vitronectin,
produces active-type collagenase from procollagenase,
and promotes the conversion of macrophages into foam
cells by degrading apolipoprotein B-100.1À3 However,
few reports have discussed the physiological and
Independently of this, the localization of chymase in
mast cells has provoked study of its roles inallergic and
in¯ammatory diseases. Chymase has been reported to
be involved in the process of histamine release,13 and to
provoke in®ltration of in¯ammatory cells when injec-
ted.14 With particular reference to skin in¯ammation,
involvement of chymase in the production of soluble
*Corresponding author. Tel.: +81-72-856-9283; fax: +81-72-868-
9597; e-mail: akahoshi@wel®de.co.jp
yPresent address: Production Division, Wel®de Corporation, 2-6-9
Hiranomachi, Chuo-ku, Osaka 541-0046, Japan.
{Present address: Strategic Project Development, Wel®de Corpora-
tion, 2-6-9 Hiranomachi, Chuo-Ku, Osaka 541-0046, Japan.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00244-3

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F. Akahoshi et al. / Bioorg. Med. Chem. 9 (2001) 301±315
type stem cell factor and of interleukin 1b has been
suggested,15À18 while more recently the relation between
genetic polymorphism of mast-cell chymase and atopic
range of inhibitors has emerged: peptide types include
chloromethyl ketone,22,23 boronic acid,13 phosphonate
ester,24 a-ketoester,22,25 a-ketoamide26 and di¯uoro-
methylene ketone;27,28 and non-peptide types sulfonyl-
¯uoride29 and imidazolidine.22 However, a non-peptidic
chymase inhibitor which is highly speci®c and has high
metabolic stability has yet to be identi®ed. In seeking
19À21
eczema has beendiscussed.
As the various physiological and pharmacological roles
of chymase have beendiscovered, a structurally varied
Figure 1. Schematic diagrams of the key interactions within the active site model of human leukocyte elastase with a peptide inhibitor (a) and a
pyrimidinone inhibitor (b) found in the crystal structure (refs 31 and 32).
Figure 2. (a) Schematic diagram of the key interactions of a peptide inhibitor complexed to human chymase based on the docking study described in
ref 28; (b) hypothetical binding mode of pyrimidinyl tri¯uoromethyl ketones in human chymase.
Scheme 1. Generic group R1 is de®ned in Table 2. Reagents: (a) EDC, HOBt, DMF; (b) EDC, Cl2CHCO2H, DMSO, toluene; (c) CF3SO3H, ani-
sole, CH2Cl2; (d) HCO2H, Pd/C, MeOH; (e) H2, Pd/C, MeOH, THF, 1N HCl; (f ) H2, Pd/C, HClO4, AcOH; (h) H2, Pd/C, MeOH.

F. Akahoshi et al. / Bioorg. Med. Chem. 9 (2001) 301±315
303
such non-peptidic derivatives, our aim was to elucidate
the pathophysiological role of chymase and to develop
therapeutic agents for chymase-induced disease.
tyrosine. Burzycki et al. have reported on peptidic chy-
mase inhibitors with the typical structure shown in
Figure 2(a).25 We constructed a three-dimensional
model of human chymase utilizing the coordinate data
of rat mast cell protease-II,33 and predicted that the P3±
P1 residue of the peptidic inhibitor would form an anti-
parallel b-sheet binding arrangement with Ser-214 to
Gly-216 of chymase, which corresponds to the residues
of the elastase inhibitor.28 This predictionwas co-n
®rmed by a recent report on the crystal structure of
humanchymase incomplex with peptidic compounds
having a chloromethyl ketone.34 We therefore decided
to use a pyrimidinone skeleton as the basic framework
of the chymase inhibitor.
Initially, we hypothesized that a pyrimidinone structure
would function e€ectively as a sca€old for a non-pep-
tidic chymase inhibitor in the same way as for an elastase
inhibitor. Human leukocyte elastase, which is also a
serine protease, recognizes a-lower alkyl amino acids
such as valine and isoleucine in the S1 site of the sub-
strate-speci®c pocket. Recently, Veale et al. reported the
development of non-peptidic tri¯uoromethyl ketone
(TFMK) derivatives as elastase inhibitors based on the
binding mode of a peptidic inhibitor.30,31 As shownin
Figure 1, the P2 proline and P3 alanine residues could be
replaced with the proposed dipeptide isostere of
pyrimidinone. Indeed, X-ray crystallographic analysis
con®rms that, like a peptidic inhibitor, this pyr-
imidinone derivative forms an anti-parallel b-sheet
binding arrangement with Ser-214 to Val-216 of elas-
tase.31,32 Meanwhile, chymase recognizes aromatic
amino acids at the S1 site such as phenylalanine and
Inthe present report, we describe the structure±activity
relationships of a series of 5-amino-6-oxo-1,6-dihy-
dropyrimidine-containing TFMKs, and enzyme selec-
tivities and pharmacokinetic pro®les of a range of
compounds representative of the series. As a result of
our studies, we discovered a compound with more
potent chymase-inhibitory activity than the peptidic
Scheme 2. Reagents: (a) H2N(CH2)3OH, EtOH; (b) diethyl ethoxymethylenemalonate, EtOH, Á; (c) tert-butyldimethylsilyl tri¯ate, 2,6-lutidine,
.
CH2Cl2; (d) LiI, pyridine, Á; (e) diphenylphosphoryl azide, Et3N, 1,4-dioxane, Á thenbenzyl alcohol; (f ) Bu 4NF, THF; (g) SO3 pyridine, Et3N,
DMSO, CH2Cl2; (h) NaClO2, NaH2PO4, 2-methyl-2-butene, tBuOH, H2O.
Scheme 3. Reagents: (a) H2, Pd/C, HClO4, AcOH.
Scheme 5. Reagents: (a) R3C(O)Cl, Na2CO3, THF; (b) CH3O-
C(O)(CH2)nC(O)Cl, Na2CO3, THF; (c) 0.1N NaOH, THF;
Scheme 4. Reagents: (a) BBr3, CH2Cl2.
(d) R4SO2Cl, pyridine, THF.

304
F. Akahoshi et al. / Bioorg. Med. Chem. 9 (2001) 301±315
inhibitor and another compound which was orally
absorbed and maintained high plasma levels for several
hours.
presence of anisole or by hydrogenolysis furnished the
desired compounds 1 and 10. Compound 1m was
directly formed by hydrogenolysis of the 4-nitrophenyl
derivative 5l using perchloric acid±acetic acid as a sol-
vent (Scheme 3). Removal of the Cbz group from
compound 4a a€orded the tri¯uoromethyl alcohol 6.
Chemistry
The synthetic method of TFMK-substituted 5-amino-
pyrimidine-6-one derivatives used as the basic frame-
work is showninScheme 1. The oxopyrimidinylacetic
and propionic acids 2 and 7 were prepared using the
method reported by Veale et al.,31 as showninScheme 2.
The acids 2 and 7 and the amine 3, prepared by the Peet
method,35 were condensed using EDC and HOBT to
give the amides 4 and 8. Modi®ed P®tzner±Mo€att oxi-
dation36 followed by removal of the carbonylbenzyloxy
(Cbz) group with tri¯uoromethanesulfonic acid in the
The 2-(4-hydroxyphenyl)-substituted pyrimidinone 1k
was obtained by demethylation of 1j with borontri-
bromide (Scheme 4).
Modi®cations of the 5-amino substituent of the pyr-
imidinone ring are shown in Scheme 5. Acylations and
sulfonylations of the amine 1a were achieved by treat-
ment with the corresponding acylating reagents in the
presence of sodium carbonate, or sulfonylating reagents
in the presence of pyridine. After introduction of a
Table 1. Enzyme inhibitory activities of peptide compounds and pyrimidinone analogues
Compd
Structure
Inhibitory activity Ki (mM)a
Chymase
Chymotrypsin
14
0.0821Æ0.0024
4.40Æ0.77
1a
0.389Æ0.032
38.5Æ15.1
84.8Æ40.3
111Æ68
0.296Æ0.074
>100
6
15a
>100
15b
>100
16
21.6Æ6.3
24.1Æ12.2
>100
10
39.9Æ9.1
Chymostatin
0.0131Æ0.0014
0.00936Æ0.00219
aValues are meansÆSEM of three independent experiments.

F. Akahoshi et al. / Bioorg. Med. Chem. 9 (2001) 301±315
305
methyl oxalyl or a methyl succinyl moiety, basic hydroly-
sis a€orded the terminal carboxy derivatives 12a and b.
Suc-Ala-Ala-Pro-Phe-p-nitroanilide. The structurally
optimized peptidic inhibitor 14 had Ki values of 0.0821
and 4.40 mM toward chymase and chymotrypsin,
respectively. The compound 1a, bearing a 2-phenyl-
pyrimidinone unit instead of the Val-Pro unit, was
slightly less potent toward chymase and 10-fold more
potent toward chymotrypsin than the peptidic com-
pound 14. This con®rmed that the Val-Pro unit is
replaceable with 2-phenylpyrimidinone. Compound 6,
which no longer has the carbonyl thought to bind to the
Ser-195 of chymase, was almost 100-fold less potent
than 1a. Removal of either the P1 group and the TFMK
unit or the TFMK unit alone resulted in further loss of
inhibitory activity (15a,b). Similarly, removal of the
pyrimidinone unit required for interaction with the S2
pocket and of the hydrogen bond with Gly-216 a€orded
a 100-fold less potent compound, 16. Insertion of one
methylene between the amide carbonyl and the pyr-
imidinone unit also had a detrimental e€ect on chy-
mase-inhibitory activity (10). (Substitutionof the
m-tolyl group at the 2-position of the pyrimidinone ring
had a better e€ect oninhibitory activity, as described
below.) Accordingly, we decided that 1a is the minimum
essential to inhibit chymase.
1H NMR measurements of TFMK-substituted com-
pounds in DMSO-d6 indicated that the ketone activated
by the tri¯uoromethyl group was immediately converted
to hydrate form by a slight amount of moisture in the
solvent (1H NMR data for the hydrate form are
reported in the Experimental section).
Results and Discussion
First of all, to con®rm that the 5-aminopyrimidinone
unit functions as a substitute for the Val-Pro unit and
that the interaction with the enzyme shown in Figure
2(b) is essential to potent inhibitors, we investigated the
activity of the compounds listed in Table 1. Human heart
chymase- and bovine pancreatic a-chymotrypsin-inhibi-
tory activities (Kis) were determined using previously
described methods27 with the synthetic substrate
Table 2. Enzyme inhibitory activities of 2-substituted pyrimidinone
analogues
Compd
R1
Inhibitory activity Ki (mM)a
We next studied the structure±activity relationship of
compounds with substituents at the 2-position of the
pyrimidinone ring (Table 2). Compared to the phenyl-
substituted compound 1a, the non-substituted com-
pound 1b is 100-fold less potent in both chymase- and
Chymase
Chymotrypsin
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
C6H5
H
CH3
3-FC6H4
4-FC6H4
0.389Æ0.032
61.8Æ22.3
0.296Æ0.074
49.3Æ12.5
>100
>1000
0.141Æ0.009
0.305Æ0.066
0.124Æ0.008
0.471Æ0.058
0.0506Æ0.0142
0.186Æ0.034
0.487Æ0.094
0.479Æ0.069
0.547Æ0.141
0.120Æ0.011
0.658Æ0.105
1.22Æ0.18
4.45Æ0.54
0.463Æ0.074
7.07Æ1.19
1.30Æ0.27
0.455Æ0.117
0.446Æ0.054
0.605Æ0.136
1.02Æ0.13
2.32Æ0.48
0.254Æ0.022
0.877Æ0.124
1.57Æ0.11
0.224Æ0.045
3-ClC6H4
4-ClC6H4
3-(CH3)C6H4
4-(CH3) C6H4
4-(CH3O) C6H4
4-(HO) C6H4
4-(NO2) C6H4
4-(NH2) C6H4
3-Pyridyl
Table 4. Enzyme selectivity data for compound 1h
Enzyme
Ki (mM)a
Humanchymase
Bovine pancreatic chymotrypsin
HumancathepsinG
Humanleukocyte elastase
Humanplasma thrombin
Human angiotensin-converting enzyme
0.0506 Æ0.0142
0.455Æ0.117
6.04 Æ0.66
NI (10 mM)b
NI (10 mM)b
NI (10 mM)b
4-Pyridyl
2-Thienyl
0.377Æ0.025
aValues are meansÆSEM of three independent experiments.
bNI=no inhibition at highest concentration (in parentheses) tested.
aValues are meansÆSEM of three independent experiments.
Table 3. Enzyme inhibitory activities of 5-(substituted amino)pyrimidinone analogues
Compd
R1
R2
Inhibitory activity Ki (mM)a
Chymase
Chymotrypsin
1a
5a
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
4-FC6H4
C6H5
C6H5
H
0.389Æ0.032
0.146Æ0.046
0.105Æ0.011
0.306Æ0.029
0.229 Æ0.041
4.47Æ0.25
0.296Æ0.074
0.101Æ0.029
0.141Æ0.046
0.364Æ0.079
3.60Æ0.50
C6H5CH2OC(O)
CH3C(O)
C6H5C(O)
11a
11b
11c
12a
12b
12c
13a
13b
CH3OC(O) (CH2)2C(O)
HOC(O)C(O)
HOC(O) (CH2)2C(O)
HOC(O) (CH2)2C(O)
CH3SO2
16.2Æ2.9
0.0656Æ0.0073
0.237Æ0.030
1.03Æ0.03
0.0899Æ0.0141
4.29Æ0.57
0.334Æ0.032
0.188Æ0.037
C6H5SO2
0.369Æ0.127
aValues are meansÆSEM of three independent experiments.

306
F. Akahoshi et al. / Bioorg. Med. Chem. 9 (2001) 301±315
chymotrypsin-inhibitory activity, while the methyl-sub-
stituted compound 1c completely lacked chymase-inhibi-
tory activity. These results suggest that a phenyl sub-
stitution at the 2-position of the pyrimidinone ring gen-
erates a lipophilic interaction with the S2 pocket of the
enzyme, as observed with elastase inhibitor.31
substitutions (1k±m) and replacement of phenyl with
heteroaryl (1n±p) did not increase potency.
We next examined the in¯uence of substituents on the
5-amino group of the pyrimidinone ring (Table 3). Sub-
stitution with Cbz, acetyl, benzoyl and methoxysuccinyl
groups did not much a€ect inhibitory activity (5a, 11a±
c). Carboxyl groups introduced at an altered distance
from the pyrimidinone ring showed some e€ect on
activity. Compound 12a with anoxalo group was over
10-fold less potent than 1a, but introduction of a succi-
nyl group resulted in an increase in inhibitory activities
toward both chymase and chymotrypsin (12b). Methane-
sulfonyl and benzenesulfonyl substitution did not sub-
stantially in¯uence potency (13a,b). Bernstein et al.
reported that, in their elastase inhibitor containing a
pyridinone skeleton as a backbone mimetic, substituents
of the corresponding amino group did not settle in a
speci®c pocket of the enzyme but protruded above the
enzyme surface.37 They also noted that the increased
inhibitory activity of their 5-acyl and sulfonyl amino
derivatives was attributable to increased donor activity
of the hydrogenonthe amino group. Such anacyl e€ect
was not observed in our chymase inhibitors, and the
fact that this series of compounds except 12b did not
much increase chymase-inhibitory activity suggests that,
inchymase too, substituents onthis site may be placed
on the solvent-exposed surface. This ®nding was sig-
ni®cant, as it allowed the physicochemical properties of
the inhibitors to be improved without loss of activity.
Next, the e€ect of substituents on this phenyl ring was
investigated. Introduction of ¯uoro, chloro and methyl
at the 4-position of the phenyl ring had no e€ect on
inhibitory activity (1e,g,i), whereas introduction of these
groups at the 3-positionincreased activity ( 1d,f,h). In
particular, the m-tolyl analogue 1h was 10-fold more
potent than 1a, and superior in potency to the parent
peptidic inhibitor 14. Regarding chymotrypsin, on the
other hand, introduction of these substituents at the
3-position only maintained or reduced inhibitory activity.
Substitution with an electron-releasing methoxy group
(1j) and an electron-withdrawing nitro group (1l) at the
4-position of the phenyl ring resulted in similar levels of
chymase-inhibitory activity. This suggests that elec-
tronic factors are not involved in the interaction of the
phenyl group with the enzyme. The 3-substitutent on the
phenyl ring therefore probably acts sterically by improving
chymase-binding through additive interaction. Other
Compound 1h, the most potent inhibitor of chymase,
was measured for its inhibitory activity toward other
representative proteases (Table 4). It inhibited only
chymotrypsintype serine proteases, with activity toward
cathepsinG 100-fold less potent thanthat toward chy-
mase. Data on enzyme speci®city (Tables 2 and 4) sug-
gested that this series of inhibitors utilizes S2±P2
interaction to discriminate between related enzymes.
We examined plasma concentration levels after intrave-
nous (iv) and oral administration to rats of the repre-
sentative compounds 1e and 12c, which has a succinyl
group and is more water-soluble. The results are shown
in Figure 3 and the pharmacokinetic parameters sum-
marized in Tables 5 and 6. Compounds 1e and 12c were
dosed in aqueous solutions containing DMSO and pro-
pylene glycol, respectively. Blood samples were obtained
at 5, 10, 30 and 60 min after iv administration and at 10,
Figure 3. Plasma concentration pro®les of 1e and 12c inrats.
Table 5. Pharmacokinetic parameters of 1 mg/kg intravenous dose in
rats
Table 6. Pharmacokinetic parameters of 10 mg/kg oral dose in rats
b
c
d
Compd
na
Cmax
(mg/mL)
Tmax
(h)
AUCpo
.
BAe
(%)
b
c
e
Compd
na
AUCiv
.
t1/2
(h)
CLd
(L/h/kg)
Vdss
(L/kg)
(mg h/mL)
(mg h/mL)
1e
12c
5
4
2.59Æ0.31
0.18Æ0.05
0.5Æ0.0
0.3Æ0.2
9.79Æ2.39
0.18Æ0.06
14.5Æ3.5
3.4Æ1.1
1e
12c
3
3
6.74Æ2.13
0.54Æ0.11
3.2Æ1.2
0.2Æ0.0
0.16Æ0.06
1.88Æ0.34
0.68Æ0.05
0.40Æ0.03
an=number of animals.
an=number of animals.
bMaximum plasma concentration of unchanged drug in plasma
recorded inthe period 0±2 h post-dose.
bIntegrated area under plasma concentration versus time curve from
time 0 to time in®nity.
cTime of maximum concentration.
cPharmacokinetic half-life.
dIntegrated area under plasma concentration versus time curve from
time 0 to time in®nity.
dPlasma clearance.
eSteady-state volume of distribution.
eOral bioavailability: (AUCpoÂdoseiv)/(AUCivÂdosepo)Â100.

F. Akahoshi et al. / Bioorg. Med. Chem. 9 (2001) 301±315
307
30, 60 and 120 min after oral administration. Levels of
1e and 12c were determined by HPLC. It was found that
1e is absorbed rapidly following oral administration and
has moderate plasma clearance, while 12c is quickly
eliminated from plasma. Since another experiment
revealed that 59.9% of total iv dosing of 12c is excreted
inbile, anorganic aniontransporter inhepatocytes may
be involved in this elimination.38,39
ambient temperature unless otherwise noted. The fol-
lowing abbreviations are used: THF, tetrahydrofuran;
DMF, N,N-dimethylformamide; DMSO, dimethyl sulf-
oxide; EDC, 1-[3-(dimethylamino)propyl]-3-ethylcarbo-
diimide hydrochloride; HOBT, 3-hydroxybenztriazole
hydrate.
2-(5-Benzyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-
1-pyrimidinyl)-N-(1-benzyl-3,3,3-tri¯uoro-2-hydroxypro-
pyl)acetamide (4a, R1=C6H5). A solutionof (5-benzyl-
oxycarbonylamino - 6 - oxo - 2 - phenyl - 1,6 - dihydro - 1 -
pyrimidinyl)acetic acid31 (2, R1=C6H5) (8.57 g,
22.6 mmol) and 3-amino-1,1,1-tri¯uoro-4-phenyl-2-
butanol35 (3) (5.91 g, 27.2 mmol), HOBT (6.10 g,
45.1 mmol), and EDC (5.20 g, 27.2 mmol) in DMF
(75 mL) was stirred at room temperature for 16 h. The
reactionmixture was poured into 0.5N HCl (500 mL),
and then extracted with ethyl acetate. The extract was
washed with saturated NaHCO3 and brine, dried
(MgSO4), and concentrated. The residue was puri®ed by
silica gel columnchromatography (83:17 chloroform:
ethyl acetate) to give ꢀ4a (11.4 g, 87% yield) as colorless
Our ®ndings suggest that the non-peptidic chymase
inhibitors reported here may prove useful as tools to
elucidate the physiological and pathological roles of
chymase and as therapeutic agents for chymase-induced
disease.
Conclusion
We designed non-peptidic chymase inhibitors based on
the predicted binding mode of the peptidic inhibitor
Val-Pro-Phe-CF3 and demonstrated that the Val-Pro
unit is replaceable with a (5-amino-6-oxo-2-phenyl-1-
pyrimidinyl)acetyl moiety. The aromatic ring at the
2-position of the pyrimidinone ring is essential to main-
tain potent chymase-inhibitory activity. The compound
1h, in particular, has an inhibitory constant of
0.0506 mM toward chymase, anactivity superior in
potency to that of the parent peptidic inhibitor Val-Pro-
Phe-CF3, and has good selectivity for chymase over
other proteases. Pharmacokinetic studies in rats
demonstrated that compound 1e is orally absorbed and
maintains high plasma levels for at least 2 h. The deriva-
tives reported here may thus prove useful as tools to
elucidate the pathophysiological roles of chymase and
as therapeutic agents for chymase-induced disease. Fur-
ther study of these derivatives with a view to develop-
ment of clinical candidates is under way and results will
be presented shortly.
1
crystals: mp 198±202 C; H NMR (500 MHz, DMSO-
d6) d 8.85 (s, 1H, NH), 8.43 (s, 1H, CHˆN), 8.32 (d,
J=8.9 Hz, 1H, NH), 7.54±7.08 (m, 15H, ArH), 6.70 (d,
J=7.1 Hz, 1H, OH), 5.19 (s, 2H, CH2±O), 4.41 (d,
J=16.3 Hz, 1H, CHH±N), 4.25 (d, J=16.3 Hz, 1H,
CHH±N), 4.07 (m, 1H, CH±O), 3.90 (m, 1H, CH±N),
2.92 (dd, J=14.1, 2.6 Hz, 1H, CHH±Ph), 2.75 (dd,
J=14.1, 10.4 Hz, 1H, CHH±Ph).
2-(5-Benzyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihy-
dro-1-pyrimidinyl)-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopro-
pyl)acetamide (5a). To
a
solutionof
4 (2.00 g,
3.44 mmol) in DMSO (15 mL) and toluene (15 mL) were
added EDC (6.60 g, 34.4 mmol) and then dichloroacetic
acid (1.1 mL, 13 mmol). After stirring at room tempera-
ture for 2.5 h, the reactionmixture was poured into 1N
HCl (150 mL), and then extracted with ethyl acetate.
The extract was washed with saturated NaHCO3 and
brine, dried (MgSO4), and concentrated. The residue
was puri®ed by silica gel columnchromatography
(83:17 chloroform:ethyl acetate) to give 5a (1.29 g, 65%
yield) as colorless crystals. Recrystallizationfrom 1:1
chloroform:hexane a€orded 858 mg of colorless crystals:
Experimental
Chemistry
ꢀ
1
mp 186±188 C; H NMR (500 MHz, DMSO-d6+D2O)
d 8.40 (s, 1H, CHˆN), 7.50 (t, J=7.3 Hz, 1H, ArH),
7.44 (d, J=7.1 Hz, 2H, ArH), 7.42±7.30 (m, 7 H, ArH),
7.22±7.10 (m, 5H, ArH), 5.18 (s, 2H, CH2±O), 4.43±4.21
(m, 3H, CH2±N, CH±N), 3.12 (dd, J=14.1, 2.1 Hz, 1H,
CHH±Ph), 2.60 (dd, J=14.1, 11.4 Hz, 1H, CHH±Ph);
MS (CI) m/z 579 (MH+). Anal. calcd for
Melting points were determined with a Yanaco melting
1
point apparatus and are uncorrected. H NMR spectra
were measured oneither a Bruker DPX-300 or AMX-
500 instrument with tetramethylsilane as the internal
standard; chemical shifts are reported in parts per mil-
lion(ppm, d units). Splitting patterns are designated as
s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
br, broad. Mass spectra (MS) were recorded ona Hita-
chi M-2000 instrument operating in the chemical ioni-
zation (CI) mode. Elemental analyses for carbon,
hydrogen and nitrogen were determined with a Yanaco
MT-6, and are within Æ0.4% of theory for formulas
given. Chromatography refers to ¯ash chromatography
conducted on Kieselgel 60 230±400 mesh (E. Merck,
Darmstadt) using the indicated solvents. All chemicals
and solvents are reagent grade unless otherwise speci-
®ed. Reactions were run under a nitrogen atmosphere at
.
C30H25F3N4O5 1.5H2O: C, 59.50; H, 4.66; N, 9.25;
found: C, 59.27; H, 4.68; N, 9.27.
2-(5-Amino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl)-
N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)acetamide (1a).
To a solutionof 5a (7.26 g, 12.5 mmol) and anisole
(4.3 mL, 40 mmol) indichloromethaen (75 mL) was
added tri¯uoromethanesulfonic acid (6.0 mL, 68 mmol)
with ice-water cooling. The reaction mixture was stirred
at 0 ꢀC to room temperature for 1 h and then cooled
with ice-water, at which time saturated NaHCO3

308
F. Akahoshi et al. / Bioorg. Med. Chem. 9 (2001) 301±315
(200 mL) was added. The resulting mixture was extrac-
ted with ethyl acetate. The extract was washed with
brine, dried (MgSO4), and concentrated. The residue
was crystallized with diethyl ether to give 1a (5.16 g,
2-[5-Amino-2-(4-¯uorophenyl)-6-oxo-1,6-dihydro-1-pyri-
midinyl]-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)aceta-
mide (1e). Compound 5e (R1=4-F±C6H4, 2.02 g,
3.39 mmol) was deprotected as described for 1a to give
1d (1.18 g, 75% yield) as colorless crystals: mp 133±
135 ꢀC; 1H NMR (500 MHz, DMSO-d6) d 8.10 (d,
J=9.7 Hz, 1H, NH), 7.30 (dd, J=8.7, 5.5 Hz, 2H), 7.26
(s, 1H, CHˆN), 7.20±7.13 (m, 5H, ArH), 7.12±7.05 (m,
4H, ArHÂ2, OHÂ2), 5.12 (s, 2H, NH2), 4.35 (d,
J=16.6 Hz, 1H, CHH±N), 4.27±4.18 (m, 2H, CHH±N,
CH±N), 3.11 (dd, J=14.0, 2.2 Hz, 1H, CHH±Ph), 2.60
92% yield) as pale yellow crystals: mp 208±211 ꢀC; H
1
NMR (500 MHz, DMSO-d6+D2O) d 7.45 (t, J=7.3 Hz,
1H, ArH), 7.35±7.09 (m, 10H, ArH), 4.36±4.18 (m, 3H,
CH2±N, CH±N), 3.12 (dd, J=14.1, 2.2 Hz, 1H, CHH±
Ph), 2.61 (dd, J=14.1, 11.5 Hz, 1H, CHH±Ph); MS (CI)
+
.
m/z 445 (MH ). Anal. calcd for C22H19F3N4O3 0.5
H2O: C, 58.28; H, 4.45; N, 12.36; found: C, 57.94; H,
4.44; N, 12.22.
(dd, J=14.0, 11.4 Hz, 1H, CHH±Ph); MS (CI) m/z 481
+
.
(MH ). Anal. calcd for C22H18F4N4O3 1.2H2O: C,
2-(5-Amino-6-oxo-1,6-dihydro-1-pyrimidinyl)-N-(1-benzyl-
3,3,3-tri¯uoro-2-oxopropyl)acetamide (1b). To a solu-
tionof 5b (R1=H, 385 mg, 0.766 mmol) inmethanol
(6 mL) and formic acid (0.3 mL) was added 10% palla-
dium/carbon (165 mg) under a nitrogen atmosphere.
The reactionmixture was stirred at room temperature
for 22 h. Palladium/carbonwas removed by ®ltration
and washed with ethyl acetate. The ®ltrate was poured
into saturated NaHCO3 (50 mL) and extracted with
ethyl acetate. The extract was washed with brine, dried
(MgSO4), and concentrated. The residue was puri®ed by
silica gel columnchromatography (91:9 chloroform:
methanol) to give 1b (100 mg, 35% yield) as colorless
crystals. Recrystallizationfrom chloroform a€orded
54.59; H, 4.25; N, 11.58; found: C, 54.22; H, 4.06; N,
11.36.
2-[5-Amino-2-(3-chlorophenyl)-6-oxo-1,6-dihydro-1-pyri-
midinyl]-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)aceta-
mide (1f). Compound 5f (R1=3-Cl±C6H4, 2.22 g,
3.62 mmol) was deprotected as described for 1a to give
1f (1.18 g, 68% yield) as colorless crystals: mp 127±
130 ꢀC; 1H NMR (300 MHz, DMSO-d6) d 8.14 (d,
J=9.7 Hz, 1H, NH), 7.70±6.90 (m, 12H, ArHÂ10,
OHÂ2), 5.19 (br s, 2H, NH2), 4.50±4.10 (m, 3H, CH2±
N, CH±N), 3.20±2.45 (m, 2H, CH2±Ph). Anal. calcd for
.
C22H18ClF3N4O3 0.8H2O: C, 53.57; H, 4.01; N, 11.36;
found: C, 53.92; H, 4.11; N, 11.21.
65 mg of colorless crystals: mp 208±211 ꢀC; H NMR
1
(500 MHz, DMSO-d6+D2O) d 7.28 (s, 1H, CHˆN),
7.27±7.21 (m, 4H, ArH), 7.18±7.14 (m, 2H, ArH), 4.53
(d, J=15.9 Hz, 1H, CHH±N), 4.37 (d, J=15.9 Hz, 1H,
CHH±N), 4.23 (dd, J=11.5, 2.4 Hz, 1H, CH±N), 3.12
(dd, J=13.7, 2.4 Hz, 1H, CHH±Ph), 2.65 (dd, J=13.7,
11.5 Hz, 1H, CHH±Ph); MS (CI) m/z 369 (MH+). Anal.
2-[5-Amino-2-(4-chlorophenyl)-6-oxo-1,6-dihydro-1-pyri-
midinyl]-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)acetam-
ide (1g). Compound 5g (R1=4-Cl±C6H4, 561 mg,
0.915 mmol) was deprotected as described for 1a to give
1g (372 mg, 85% yield) as colorless crystals: mp 197±
200 ꢀC; H NMR (500 MHz, DMSO-d6+D2O) d 7.33
1
.
calcd for C16H15F3N4O3 1.0H2O: C, 49.74; H, 4.44; N,
14.50; found: C, 49.87; H, 4.54; N, 14.35.
(d, J=8.5 Hz, 2H, ArH), 7.29 (s, 1H. CHˆN), 7.26 (d,
J=8.5 Hz, 2H, ArH), 7.20±7.13 (m, 5H, ArH), 4.38 (d,
J=16.4 Hz, 1H, CHH±N), 4.27±4.17 (m, 2H, CHH±N,
CH±N), 3.11 (dd, J=14.0, 2.4 Hz, 1H, CHH±Ph), 2.60
(dd, J=14.0, 11.6 Hz, 1H, CHH±Ph); MS (CI) m/z 479,
2-(5-Amino-2-methyl-6-oxo-1,6-dihydro-1-pyrimidinyl)-
N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)acetamide (1c).
Compound 5c (R1=CH3, 1.42 g, 2.75 mmol) was
deprotected as described for 1b to give 1cꢀ(435 mg, 41%
+
.
481 (MH ). Anal. calcd for C22H18ClF3N4O3 0.7H2O:
C, 53.77; H, 3.98; N, 11.40; found: C, 53.67; H, 4.09; N,
11.13.
1
yield) as colorless crystals: mp 117±120 C; H NMR
(500 MHz, DMSO-d6+D2O) d 7.27±7.21 (m, 4H, ArH),
7.17 (m, 1H, ArH), 7.10 (s, 1H, CHˆN), 4.77 (d,
J=16.8 Hz, 1H, CHH±N), 4.36±4.28 (m, 2H, CHH±N,
CH±N), 3.15 (dd, J=13.6, 2.7 Hz, 1H, CHH±Ph), 2.64
(dd, J=13.6, 12.1 Hz, 1H, CHH±Ph), 1.78 (s, 3H,
CH3); MS (CI) m/z 383 (MH+). Anal. calcd for
2-[5-Amino-6-oxo-2-(m-tolyl)-1,6-dihydro-1-pyrimidinyl]-
N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)acetamide (1h).
To
a
solutionof
5h (R1=3-CH3±C6H4, 1.28 g,
2.16 mmol) in methanol (20 mL) and THF (20 mL) were
added 1N HCl (0.4 mL), followed by 10% palladium/
carbon (460 mg) under a nitrogen atmosphere. The
resulting mixture was stirred at room temperature for
6 h under a hydrogen atmosphere. Palladium/carbon
was removed by ®ltrationand washed with methanol.
The ®ltrate was concentrated, and the residue was puri-
®ed by silica gel columnchromatography (95:5 chloro-
form:methanol) to give 1h (330 mg, 33% yield) as pale
yellow crystals. Recrystallizationfrom 4:1 chloroform:
hexane a€orded 149 mg of colorless crystals: mp 177±
.
C17H17F3N4O3 1.0H2O: C, 51.00; H, 4.78; N, 13.99;
found: C, 51.03; H, 4.77; N, 13.87.
2-[5-Amino-2-(3-¯uorophenyl)-6-oxo-1,6-dihydro-1-pyri-
midinyl]-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)aceta-
mide (1d). Compound 5d (R1=3-F±C6H4, 4.02 g,
6.74 mmol) was deprotected as described for 1a to give
1d (1.72 g, 55% yield) as colorless crystals: mp 198±
201 ꢀC; 1H NMR (300 MHz, DMSO-d6) d 8.14 (d,
J=9.7 Hz, 1H, NH), 7.40±7.00 (m, 12H, ArHÂ10,
OHÂ2), 5.18 (br s, 2H, NH2), 4.45±4.10 (m, 3H, CH2±
N, CH±N), 3.18±3.00 (m, 1H, CHH±Ph), 2.70±2.48 (m,
181 ꢀC; H NMR (500 MHz, DMSO-d6+D2O) d 7.34
1
(s, 1H, CHˆN), 7.30 (d, J=7.7 Hz, 1H, ArH), 7.22 (t,
J=7.7 Hz, 1H, ArH), 7.10±7.21 (m, 6H, ArH), 7.02 (d,
J=7.7 Hz, 1H, ArH), 4.39 (d, J=16.2 Hz, 1H, CHH±
N), 4.31 (d, J=16.2 Hz, 1H, CHH±N), 4.21 (dd,
J=11.3, 2.3 Hz, 1H, CH±N), 3.12 (dd, J=14.1, 2.3 Hz,
.
1H, CHH±Ph). Anal. calcd for C22H18F4N4O3 1.0H2O:
C, 55.00; H, 4.20; N, 11.66; found: C, 55.13; H, 4.27; N,
11.65.

F. Akahoshi et al. / Bioorg. Med. Chem. 9 (2001) 301±315
309
1H, CHH±Ph), 2.61 (dd, J=14.1, 11.3 Hz, 1H, CHH±
Ph), 2.29 (s, 3H, CH3); MS (CI) m/z 459 (MH+). Anal.
.
calcd for C23H21F3N4O3 0.5H2O: C, 59.10; H, 4.74; N,
11.99; found: C, 58.99; H, 4.72; N, 11.89.
1l (287 mg, 62% yield) as pale browncrystals: mp 130±
134 ꢀC; H NMR (500 MHz, DMSO-d6+D2O) d 8.12
(d, J=8.8 Hz, 2H, ArH), 7.52 (d, J=8.8 Hz, 2H, ArH),
7.33 (s, 1H, CHˆN), 7.18±7.08 (m, 5H, ArH), 4.45 (d,
J=16.4 Hz, 1H, CHH±N), 4.27±4.18 (m, 2H, CHH±N,
CH±N), 3.10 (dd, J=14.1, 2.5 Hz, 1H, CHH±Ph), 2.57
(dd, J=14.1, 11.7 Hz, 1H, CHH±Ph); MS (CI) m/z 490
1
2-[5-Amino-6-oxo-2-(p-tolyl)-1,6-dihydro-1-pyrimidinyl]-
N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)acetamide (1i).
Compound 5i (R1=4-CH3±C6H4, 500 mg, 0.844 mmol)
was deprotected as described for 1b to give 1i (235 mg,
61% yield) as colorless crystals: mp 200±203 ꢀC; 1H
NMR (500 MHz, DMSO-d6+D2O) d 7.31 (s, 1H,
CHˆN), 7.22±7.09 (m, 9H, ArH), 4.36 (d, J=16.3 Hz,
1H, CHH±N), 4.28±4.20 (m, 2H, CHH±N, CH±N), 3.13
(dd, J=14.2, 2.4 Hz, 1H, CHH±Ph), 2.62 (dd, J=14.2,
+
.
(MH ). Anal. calcd for C22H18F3N5O5 0.5H2O: C,
53.02; H, 3.84; N, 14.05; found: C, 52.94; H, 3.68; N,
14.02.
2-[5-Amino-2-(4-aminophenyl)-6-oxo-1,6-dihydro-1-pyri-
midinyl]-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)aceta-
mide (1m). To a solutionof 5l (196 mg, 0.314 mmol) in
acetic acid (3 mL) and perchloric acid (70%, 3 drops)
was added 10% palladium/carbon(100 mg) udner a
nitrogen atmosphere. The resulting mixture was stirred
at room temperature for 65 h under a hydrogen atmos-
phere. Palladium/carbonwas removed by ®ltrationand
washed with ethyl acetate. The ®ltrate was poured into
saturated NaHCO3 and extracted with ethyl acetate.
The extract was washed with brine, dried (Na2SO4), and
concentrated. The residue was puri®ed by silica gel col-
umnchromatography (97.5:2.5 ethyl acetate:methanol)
and recrystallized from chloroform:ethyl acetate:hexane
to give 1m (68 mg, 44% yield) as pale yellow ®ne crys-
11.4 Hz, 1H, CHH±Ph), 2.36 (s, 3H, CH3); MS (CI) m/z
+
.
459 (MH ). Anal. calcd for C23H21F3N4O3 0.2H2O: C,
59.79; H, 4.67; N, 12.13; found: C, 59.63; H, 4.54; N,
12.08.
2-[5-Amino-2-(4-methoxyphenyl)-6-oxo-1,6-dihydro-1-
pyrimidinyl]-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)-
acetamide (1j). Compound 5j (R1=4-CH3O±C6H4,
497 mg, 0.817 mmol) was deprotected as described for
1a to give 1j (377 mg, 97% yield) as colorless crystals:
mp 137±140 ꢀC; H NMR (500 MHz, DMSO-d6+D2O)
1
d 7.30 (s, 1H, CHˆN), 7.24±7.15 (m, 7H, ArH), 6.82 (d,
J=8.8 Hz, 2H, ArH), 4.37 (d, J=16.3 Hz, 1H, CHH±
N), 4.31±4.23 (m, 2H, CHH±N, CH±N), 3.81 (s, 3H,
CH3±O), 3.13 (dd, J=14.2, 2.2 Hz, 1H, CHH±Ph), 2.63
tals: mp >210 ꢀC dec.; H NMR (300 MHz, DMSO-d6)
1
d 8.17 (d, J=9.6 Hz, 1H, NH), 7.35±7.15 (m, 6H,
ArHÂ5, CHˆN), 7.15 (s, 1H, OH), 7.11 (s, 1H, OH),
6.94 (d, J=8.5 Hz, 2H, ArH), 6.45 (d, J=8.5 Hz, 2H,
ArH), 5.46 (s, 2H, NH2), 4.92 (s, 2H, NH2), 4.38 (d,
J=16.1 Hz, 1H, CHH±N), 4.31 (d, J=16.1 Hz, 1H,
CHH±N), 4.23 (br t, J=9.7 Hz, 1H, CH±N), 3.14 (dd,
J=14.1, 2.0 Hz, 1H, CHH±Ph), 2.65 (dd, J=14.1,
11.2 Hz, 1H, CHH±Ph); MS (CI) m/z 460 (MH+). Anal.
(dd, J=14.2, 11.5 Hz, 1H, CHH±Ph); MS (CI) m/z 475
+
.
(MH ). Anal. calcd for C23H21F3N4O4 1.0H2O: C, 56.10;
H, 4.71; N, 11.38; found: C, 56.25; H, 4.58; N, 11.51.
2-[5-Amino-2-(4-hydroxyphenyl)-6-oxo-1,6-dihydro-1-
pyrimidinyl]-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)-
.
acetamide (1k). To
a
solutionof
1j (375 mg,
calcd for C22H20F3N5O3 1.2H2O: C, 54.93; H, 4.69; N,
14.56; found: C, 54.71; H, 4.64; N, 14.57.
0.790 mmol) indichloromethane (10 mL) was added a
solutionof borontribromide indichloromethane (1.0M,
16 mL, 16 mmol). The resulting mixture was stirred at
room temperature for 24 h, at which time methanol
(3 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 puri®ed by silica gel columnchroma-
tography (91:9 chloroform:methanol) and further pur-
i®ed by reverse phase columnchromatography (67:33
water:acetonitrile) to give 1k (168 mg, 46% yield) as a
2-[5-Amino-6-oxo-2-(3-pyridyl)-1,6-dihydro-1-pyrimidi-
nyl]-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)acetamide (1n).
Compound 5n (R1=3-pyridyl, 200 mg, 0.345 mmol) was
deprotected as described for 1h to give 1n (111 mg, 72%
yield) as colorless crystals: mp 88±91 ꢀC; 1H NMR
(500 MHz, DMSO-d6+D2O) d 8.63 (dd, J=4.8, 1.5 Hz,
1H, ArH), 8.50 (d, J=1.8 Hz, 1H, ArH), 7.61 (m, 1H,
ArH), 7.33 (s, 1H, CHˆN), 7.32 (dd, J=7.9, 4.8 Hz,
1H, ArH), 7.20±7.11 (m, 5H, ArH), 4.38 (d, J=16.9 Hz,
1H, CHH±N), 4.32 (d, J=16.9 Hz, 1H, CHH±N), 4.20
(dd, J=11.3, 2.3 Hz, 1H, CH±N), 3.10 (dd, J=14.0,
2.3 Hz, 1H, CHH±Ph), 2.58 (dd, J=14.0, 11.3 Hz, 1H,
CHH±Ph); MS (CI) m/z 446 (MH+). Anal. calcd for
pale brownsolid:
1H NMR (500 MHz, DMSO-
d6+D2O) d 7.31 (s, 1H, CHˆN), 7.25±7.12 (m, 5H,
ArH), 7.08 (d, J=8.6 Hz, 2H, ArH), 6.72 (d, J=8.6 Hz,
2H, ArH), 4.38 (d, J=16.2 Hz, 1H, CHH±N), 4.31 (d,
J=16.2 Hz, 1H, CHH±N), 4.25 (dd, J=11.3, 2.1 Hz,
1H, CH±N), 3.13 (dd, J=13.8, 2.1 Hz, 1H, CHH±Ph),
.
C21H18F3N5O3 1.0H2O: C, 54.43; H, 4.35; N, 15.11;
found: C, 54.13; H, 4.19; N, 15.06.
2.62 (dd, J=13.8, 11.3 Hz, 1H, CHH±Ph); MS (CI) m/z
+
2-[5-Amino-6-oxo-2-(4-pyridyl)-1,6-dihydro-1-pyrimidi-
nyl]-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)acetamide
(1o). Compound 5o (R1=4-pyridyl, 250 mg, 0.431 mmol)
was deprotected as described for 1h to give 1o (161 mg,
.
461 (MH ). Anal. calcd for C22H19F3N4O4 1.0H2O: C,
55.23; H, 4.42; N, 11.71; found: C, 55.34; H, 4.51; N,
11.51.
1
84% yield) as a pale yellow solid: H NMR (500 MHz,
2-[5-Amino-2-(4-nitrophenyl)-6-oxo-1,6-dihydro-1-pyrimi-
dinyl]-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)acetamide
(1l). Compound 5l (R1=4-NO2±C6H4, 587 mg,
0.941 mmol) was deprotected as described for 1a to give
DMSO-d6+D2O) d 8.52 (d, J=6.0 Hz, 2H, ArH), 7.32
(s, 1H, CHˆN), 7.24 (d, J=6.0 Hz, 2H, ArH), 7.19±
7.13 (m, 5H, ArH), 4.38 (d, J=16.9 Hz, 1H, CHH±N),
4.29 (m, 1H, CHH±N), 4.25 (dd, J=11.5, 2.5 Hz, 1H,

310
F. Akahoshi et al. / Bioorg. Med. Chem. 9 (2001) 301±315
CH±N), 3.11 (dd, J=14.1, 2.5 Hz, 1H, CHH±Ph), 2.59
(dd, J=14.1, 11.5 Hz, 1H, CHH±Ph); MS (CI) m/z 446
To a solutionof the above itnermediate (23.1 g,
73.0 mmol) and 2,6-lutidine (13 mL, 110 mmol) in di-
chloromethane (250 mL) was added tert-butyldi-
methylsilyl tri¯ate (20 mL, 87 mmol) with ice-water
cooling. After stirring at 0 ꢀC to room temperature for 5 h,
the reactionmixture was poured into 1N HCl (400 mL),
an d then extracted with chloroform. The extract was
washed with saturated NaHCO3 and brine, dried
(MgSO4), and concentrated. The residue was puri®ed by
silica gel columnchromatography (99:1 chloroform:
methanol) to give ethyl 1-{3-[(tert-butyldimethylsilyl)
oxy]propyl}-2-(m-tolyl)pyrimidin-6(1H)-one-5-carboxyl-
+
.
(MH ). Anal. calcd for C21H18F3N5O3 1.0H2O: C,
54.43; H, 4.35; N, 15.11; found: C, 54.34; H, 4.33; N,
15.04.
2-[5-Amino-6-oxo-2-(2-thienyl)-1,6-dihydro-1-pyrimidinyl]-
N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)acetamide (1p).
Compound 5p (R1=2-thienyl, 760 mg, 1.30 mmol) was
deprotected as described for 1a to give 1p (415 mg, 71%
yield) as pale yellow crystals: mp 198±201 ꢀC; H NMR
1
(500 MHz, DMSO-d6+D2O) d 7.60 (d, J=5.1 Hz, 1H,
ArH), 7.28±7.22 (m, 6H, ArHÂ5, CHˆN), 6.91 (dd,
J=5.1, 3.7 Hz, 1H, ArH), 6.64 (br s, 1H, ArH), 4.68 (d,
J=16.5 Hz, 1H, CHH±N), 4.43 (d, J=16.5 Hz, 1H,
CHH±N), 4.37 (dd, J=11.6, 2.4 Hz, 1H, CH±N), 3.18
(dd, J=13.9, 2.4 Hz, 1H, CHH±Ph), 2.65 (dd, J=13.9,
11.6 Hz, 1H, CHH±Ph); MS (CI) m/z 481 (MH+). Anal.
1
ate (29.6 g, 94% yield) as a pale yellow oil: H NMR
(500 MHz, DMSO-d6) d 8.49 (s, 1H, CHˆN), 7.43±7.35
(m, 4H, ArH), 4.26 (q, J=7.1 Hz, 2H, CH2±O), 3.94 (m,
2H, CH2±N), 3.46 (t, J=5.6 Hz, 2H, CH2±O), 2.36 (s,
3H, CH3), 1.70 (m, 2H, CH2), 1.28 (t, J=7.1 Hz, 3H,
CH3), 0.72 (s, 9H, CH3Â3), À0.11 (s, 6H, CH3±SiÂ2).
.
calcd for C20H17F3N4O3S 0.8H2O: C, 51.68; H, 4.03; N,
12.05; found: C, 51.75; H, 4.09; N, 11.95.
A solutionof the above intermediate (29.3 g, 68.0 mmol)
and lithium iodide (22.0 g, 164 mmol) in pyridine
(120 mL) was heated at re¯ux temperature for 10 h, at
which time organic solvents were evaporated. The resi-
due was poured into 2N HCl (450 mL) and then extrac-
ted with ethyl acetate. The extract was washed with
brine, dried (MgSO4), and concentrated. The residue
was crystallized from 1:3 diethyl ether:hexane to give 1-
{3-[(tert-butyldimethylsilyl)oxy]propyl}-2-(m-tolyl)pyr-
imidin-6(1H)-one-5-carboxylic acid (14.6 g, 53% yield)
2-(5-Amino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl)-
N-(1-benzyl-3,3,3-tri¯uoro-2-hydroxypropyl)acetamide (6).
To a solutionof 4a (290 mg, 0.500 mmol) inmethanol
(15 mL) was added 10% palladium/carbon(106 mg)
under a nitrogen atmosphere. The resulting mixture was
stirred at room temperature for 2.5 h under a hydrogen
atmosphere. Palladium/carbonwas removed by ®ltra-
tionand washed with methanol. The ®ltrate was con-
centrated, and then the residue was puri®ed by silica gel
columnchromatography (91:9 chloroform:methanol) to
give 6 (202 mg, 90% yield) as a colorless solid: 1H NMR
(300 MHz, DMSO-d6) d 8.29 (d, J=8.5 Hz, 0.6H, NH),
8.19 (d, J=9.2 Hz, 0.4H, NH), 7.48±7.09 (m, 11H,
ArH), 6.73 (br s, 1H, OH), 5.14 (s, 2H, NH2), 4.47±3.82
(m, 4H, CH±O, CH2±N, CH±N), 2.98±2.61 (m, 2H,
CH2±Ph). Anal. calcd for C22H21F3N4O3: C, 59.19; H,
4.74; N, 12.55; found: C, 59.30; H, 4.78; N, 12.29.
as colorless crystals: mp 65±66 ꢀC; H NMR (500 MHz,
1
DMSO-d6) d 13.15 (br s, 1H, OH), 8.66 (s, 1H,
CHˆN), 7.46±7.37 (m, 4H, ArH), 4.01 (m, 2H, CH2±
N), 3.48 (t, J=5.7 Hz, 2H, CH2±O), 2.37 (s, 3H, CH3),
1.74 (m, 2H, CH2), 0.73 (s, 9H, CH3Â3), À0.10 (s, 6H,
CH3±SiÂ2).
To a mixture of the above intermediate (14.3 g,
35.5 mmol), 1,4-dioxane (150 mL), and triethylamine
(10 mL, 72 mmol) was added dropwise diphenylphos-
phoryl azide (9.6 mL, 43 mmol). The resulting mixture
was heated at re¯ux temperature for 2 h, at which time
benzyl alcohol (4.8 mL, 46 mmol) was added. The mix-
ture was further heated at re¯ux temperature for 12 h,
cooled to room temperature, and then concentrated
under reduced pressure. The residue was poured into
saturated NH4Cl (300 mL) and extracted with ethyl
acetate. The extract was washed with 1N NaOH
(300 mL) and brine, dried (MgSO4), and concentrated.
The residue was puri®ed by silica gel columnchromato-
graphy (83:17 hexane:ethyl acetate) to give 1-[5-benzyl-
oxycarbonylamino-6-oxo-2-(m-tolyl)-1,6-dihydro-1-pyr-
imidinyl]-3-[(tert-butyldimethylsilyl)oxy]propane (12.4 g,
61% yield) as a colorless oil: 1H NMR (500 MHz,
DMSO-d6) d 8.78 (s, 1H, NH), 8.39 (s, 1H, CHˆN),
7.44 (d, J=7.2 Hz, 2H, ArH), 7.42±7.30 (m, 7H, ArH),
5.18 (s, 2H, CH2±O), 3.95 (m, 2H, CH2±N), 3.45
(t, J=5.8 Hz, 2H, CH2±O), 2.36 (s, 3H, CH3), 1.69 (m,
2H, CH2), 0.73 (s, 9H, CH3Â3), À0.10 (s, 6H, CH3±
SiÂ2).
3-[5-Benzyloxycarbonylamino-6-oxo-2-(m-tolyl)-1,6-dihy-
dro-1-pyrimidinyl]propionic acid (7). To a solutionof
ethyl 3-methylbenzimidate hydrochloride (15.0 g,
75.1 mmol) in ethanol (75 mL) was added 3-aminopro-
panol (6.9 mL, 90 mmol) with ice-water cooling. After
stirring at room temperature for 16 h, the reaction mix-
ture was concentrated under reduced pressure. The
residue was poured into 1N NaOH (200 mL) and
extracted with chloroform. The extract was dried
(MgSO4) and concentrated to give N-(3-hydroxy-
propyl)-3-methylbenzamidine (17.1 g) as a colorless oil.
To a solution of this amidine in ethanol (300 mL) was
added dropwise diethyl ethoxymethylenemalonate
(18.0 mL, 89.1 mmol). The reactionmixture was heated
at 90 ꢀC for 3 h, and then concentrated under reduced
pressure. The residue was puri®ed by silica gel column
chromatography (95:5 chloroform:methanol) to give
ethyl 1-(3-hydroxypropyl)-2-(m-tolyl)pyrimidin-6(1H)-
one-5-carboxylate (23.5 g, 99% yield) as a pale yellow
oil: 1H NMR (500 MHz, DMSO-d6) d 8.50 (s, 1H,
CHˆN), 7.45±7.35 (m, 4H, ArH), 4.39 (t, J=5.3 Hz,
1H, OH), 4.26 (q, J=7.1 Hz, 2H, CH2±O), 3.91 (m, 2H,
CH2±N), 3.25 (t, J=5.3 Hz, 2H, CH2±O), 2.38 (s, 3H,
CH3), 1.68 (m, 2H, CH2), 1.29 (t, J=7.1 Hz, 3H, CH3).
To a solutionof the above itnermediate (12.0 g,
21.0 mmol) inTHF (100 mL) was added tetra-
butylammoiunm ¯uoride (1.0M solutioninTHF,

F. Akahoshi et al. / Bioorg. Med. Chem. 9 (2001) 301±315
311
crystals: mp 119±123 ꢀC; H NMR (500 MHz, DMSO-
d6+D2O) d 8.78 (s, 1H, NH), 8.38 (s, 1H, CHˆN), 7.80
(d, J=9.8 Hz, 1H, NH), 7.44 (d, J=7.1 Hz, 2H, ArH),
7.40 (t, J=7.1 Hz, 2H, ArH), 7.37±7.32 (m, 3H, ArH),
7.29±7.25 (m, 2H, ArH), 7.11±7.05 (m, 7H, ArHÂ5,
OHÂ2), 5.19 (s, 2H, CH2±O), 4.12 (m, 1H, CH±N), 3.86
(m, 1H, CHH±N), 3.71 (m, 1H, CHH±N), 3.04 (dd,
J=13.5, 2.5 Hz, 1H, CHH±Ph), 2.56 (dd, J=13.5,
11.8 Hz, 1H, CHH±Ph), 2.47±2.30 (m, 5H, CH3, CH2±
CˆO); MS (CI) m/z 607 (MH+).
1
24 mL, 24 mmol). After stirring for 1.5 h, the reaction
mixture was poured into water (150 mL) and extracted
with ethyl acetate. The extract was washed with water
and brine, dried (MgSO4), and concentrated. The resi-
due was puri®ed by silica gel columnchromatography
(50:50 hexane:ethyl acetate) to give 3-[5-benzyloxy-
carbonylamino-6-oxo-2-(m-tolyl)-1,6-dihydro-1-pyr-
imidinyl]propanol (8.19 g, 99% yield) as a colorless oil:
1H NMR (500 MHz, DMSO-d6) d 8.79 (s, 1H, NH),
8.40 (s, 1H, CHˆN), 7.44 (d, J=7.0 Hz, 2H, ArH),
7.42±7.32 (m, 7H, ArH), 5.19 (s, 2H, CH2±O), 4.40 (t,
J=5.3 Hz, OH), 3.91 (m, 2H, CH2±N), 3.24 (t,
J=5.3 Hz, 2H, CH2±O), 2.37 (s, 3H, CH3), 1.67 (m, 2H,
CH2).
3-[5-Amino-6-oxo-2-(m-tolyl)-1,6-dihydro-1-pyrimidinyl]-
N - (1 - benzyl - 3,3,3 - tri¯uoro - 2 - oxopropyl)propanamide
(10). Compound 9 (500 mg, 0.824 mmol) was depro-
tected using conditions described for the preparation of
1
To a solutionof the above itnermediate (7.99 g,
20.3 mmol) indichloromethaen (60 mL) were added
successively triethylamine (8.5 mL, 61 mmol) and a
solutionof sulfur trioxide±pyridien complex (9.70 g,
60.9 mmol) inDMSO (60 mL) with ice-water cooling.
After stirring at 0 ꢀC for 3 h, the reactionmixture was
poured into a mixture of ice and brine, and then
extracted with ethyl acetate. The extract was washed
successively with 0.5N HCl and brine, dried (MgSO4),
and concentrated to give a pale brown oil (7.95 g). To a
mixture of this oil, 2-methyl-2-propanol (130 mL) and
2-methyl-2-butene (22mL, 0.21mol) was added a solution
of sodium dihydrogenphosphate dihydrate (22.2 g,
0.142 mol) and sodium chlorite (85%, 15.8 g, 0.148 mol)
inwater (60 mL). The resulting mixture was stirred at
room temperature for 3 h, at which time organic sol-
vents were evaporated. The residue was acidi®ed with
2N HCl to pH 3 and extracted with ethyl acetate. The
extract was washed with brine, dried (MgSO4), and
concentrated. The residue was puri®ed by silica gel col-
umnchromatography (95:5 chloroform:methaonl) to
1a to give 10 (1.57 g, 79% yield) as a colorless solid: H
NMR (500 MHz, DMSO-d6) d 7.78 (d, J=9.8 Hz, 1H,
NH), 7.36±7.04 (m, 12H, ArHÂ9, CHˆN, OHÂ2), 5.11
(s, 2H, NH2), 4.11 (m, 1H, CH±N), 3.84 (m, 1H, CHH±
N), 3.68 (m, 1H, CHH±N), 3.04 (dd, J=13.5, 2.6 Hz,
1H, CHH±Ph), 2.56 (dd, J=13.5, 11.9 Hz, 1H, CHH±
Ph), 2.45±2.27 (m, 5H, CH3, CH2±CˆO); MS (CI) m/z
+
.
473 (MH ). Anal. calcd for C24H23F3N4O3 0.6H2O: C,
59.65; H, 5.05; N, 11.59; found: C, 59.60; H, 5.10; N,
11.54.
2-(5-Acetylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidi-
nyl)-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)acetamide
(11a). To a solutionof 1a (559 mg, 1.26 mmol) and
sodium carbonate (372 mg, 3.51 mmol) in THF (10 mL)
was added acetyl chloride (0.10 mL, 1.4 mmol) with ice-
water cooling. After stirring at 0 ꢀC for 2 h, the resulting
mixture was poured into saturated NaHCO3, and then
extracted with ethyl acetate. The organic layer was
washed with brine and concentrated. The residue was
puri®ed by silica gel columnchromatography (95:5
chloroform:methanol) to give 11a (509 mg, 83% yield)
as colorless crystals. Recrystallizationfrom 5:2 chlor-
oform:hexane a€orded 414 mg of colorless crystals: mp
110±112 ꢀC; 1H NMR (500 MHz, DMSO-d6+D2O) d
8.73 (s, 1H, CHˆN), 7.53 (t, J=7.5 Hz, 1H, ArH), 7.38
(t, J=7.5 Hz, 2H, ArH), 7.29 (d, J=7.5 Hz, 2H, ArH),
7.23±7.13 (m, 5H, ArH), 4.42 (d, J=15.9 Hz, 1H,
CHH±N), 4.33 (d, J=15.9 Hz, 1H, CHH±N), 4.26 (dd,
J=11.5, 2.2 Hz, 1H, CH±N), 3.13 (dd, J=13.9, 2.2 Hz,
1H, CHH±Ph), 2.59 (dd, J=13.9, 11.5 Hz, 1H, CHH±
Ph), 2.15 (s, 3H, CH3); MS (CI) m/z 487 (MH+). Anal.
1
give 7 (8.16 g, 99% yield) as a colorless solid: H NMR
(500 MHz, DMSO-d6) d 12.3 (br s, 1H, OH), 8.85 (s,
1H, NH), 8.41 (s, 1H, CHˆN), 7.44 (d, J=7.2 Hz, 2H,
ArH), 7.32±7.42 (m, 7H, ArH), 5.19 (s, 2H, CH2±O),
4.03 (t, J=7.8 Hz, 2H, CH2±N), 2.59 (t, J=7.8 Hz, 2H,
CH2±CˆO), 2.37 (s, 3H, CH3).
3-[5-Benzyloxycarbonylamino-6-oxo-2-(m-tolyl)-1,6-dihy-
dro-1-pyrimidinyl]-N-(1-benzyl-3,3,3-tri¯uoro-2-hydroxy-
propyl)propanamide (8). Compound
7
(2.37 g,
5.82 mmol) was coupled with 3 using a method similar
to that described for 4a to give 8 (3.05 g, 86% yield) as
.
calcd for C24H21F3N4O4 1.3H2O: C, 56.54; H, 4.67; N,
10.99; found: C, 56.43; H, 4.51; N, 10.88.
ꢀ
1
colorless crystals: mp 165±168 C; H NMR (500 MHz,
DMSO-d6) d 8.78 (s, 1H, NH), 8.39 (s, 1H, CHˆN),
7.98 (d, J=8.8 Hz, 1H, NH), 7.44 (d, J=7.1 Hz, 2H,
ArH), 7.40 (t, J=7.1 Hz, 2H, ArH), 7.37±7.06 (m, 10H,
ArH), 6.58 (d, J=7.3 Hz, 1H, OH), 5.19 (s, 2H, CH2±
O), 4.01 (m, 1H, CH±N), 3.96±3.78 (m, 3H, CH2±N,
CH±O), 2.93 (dd, J=13.9, 2.7 Hz, 1H, CHH±Ph), 2.62
(dd, J=13.9, 11.0 Hz, 1H, CHH±Ph), 2.43±2.28 (m, 5H,
CH3, CH2±CˆO).
2-(5-Benzoylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimi-
dinyl)-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)acetamide
(11b). Compound 1a (300 mg, 0.678 mmol) was reacted
with benzoyl choride (126 mg, 0.908 mmol) using condi-
tions described for the preparation of 11a to give 11b
(271 mg, 73% yield) as colorless crystals: mp 225±
227 ꢀC; H NMR (300 MHz, DMSO-d6) d 9.42 (s, 1H,
1
NH), 8.75 (s, 1H, CHˆN), 8.21 (d, J=9.7 Hz, 1H, NH),
7.96 (m, 2H, ArH), 7.67±7.49 (m, 4H, ArH), 7.38±7.36
(m, 4H, ArH), 7.23±7.18 (m, 5H, ArH), 7.16 (s, 1H,
OH), 7.14 (s, 1H, OH), 4.44 (d, J=16.6 Hz, 1H, CHH±
N), 4.35 (d, J=16.6 Hz, 1H, CHH±N), 4.27 (m, 1H,
CH±N), 3.14 (dd, J=14.1, 2.1 Hz, 1H, CHH±Ph), 2.61
3-[5-Benzyloxycarbonylamino-6-oxo-2-(m-tolyl)-1,6-dihy-
dro-1-pyrimidinyl]-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopro-
pyl)propanamide (9). Compound 8 (2.00 g, 3.29 mmol)
was oxidized using conditions described for the pre-
parationof 5a to give 9 (1.57 g, 79% yield) as colorless

312
F. Akahoshi et al. / Bioorg. Med. Chem. 9 (2001) 301±315
(dd, J=14.1, 11.4 Hz, 1H, CHH±Ph); MS (CI) m/z 549
+
was hydrolyzed using conditions described for the pre-
parationof 12a to give 12b (311 mg, 88% yield) as col-
orless crystals: mp 100±101 ꢀC; 1H NMR (500 MHz,
DMSO-d6) d 13.0±11.5 (br, 1H, OH), 9.46 (s, 1H, NH),
8.76 (s, 1H, CHˆN), 8.14 (d, J=9.7 Hz, 1H, NH), 7.49
(m, 1H, ArH), 7.35 (t, J=8.0 Hz, 2H, ArH), 7.29 (d,
J=7.1 Hz, 2H, ArH), 7.21±7.14 (m, 5H, ArH), 7.13 (s,
1H, OH), 7.11 (s, 1H, OH), 4.42 (d, J=16.0 Hz, 1H,
CHH±N), 4.30 (d, J=16.0 Hz, 1H, CHH±N), 4.26 (m,
1H, CH±N), 3.13 (d, J=14.1, 2.2 Hz, 1H, CHH±Ph),
2.71 (t, J=7.2 Hz, 2H, CH2±CˆO), 2.60 (d, J=14.1,
11.4 Hz, 1H, CHH±Ph), 2.51±2.48 (m, 2H, CH2±CˆO);
MS (CI) m/z 545 (MH+). Anal. calcd for
C26H23F3N4O6: C, 57.35; H, 4.26; N, 10.29; found: C,
57.09; H, 4.52; N, 10.00.
.
(MH ). Anal. calcd for C29H23F3N4O4 1.2H2O: C,
61.09; H, 4.49; N, 9.83; found: C, 60.92; H, 4.47; N, 9.64.
2-(5-Methoxysuccinylamino-6-oxo-2-phenyl-1,6-dihydro-
1-pyrimidinyl)-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)-
acetamide (11c). Compound 1a (600mg, 1.36mmol) was
reacted with methyl succinyl chloride (0.22 mL,
1.8 mmol) using conditions described for the prepara-
tionof 11a to give 11c (576 mg, 76% yield) as colorless
crystals: mp 78.5±79.5 ꢀC; 1H NMR (500 MHz, DMSO-
d6) d 9.52 (s, 1H, NH), 8.75 (s, 1H, CHˆN), 8.15 (d,
J=9.7 Hz, 1H, NH), 7.49 (m, 1H, ArH), 7.34 (t,
J=7.8 Hz, 2H, ArH), 7.29 (d, J=7.3 Hz, 2H, ArH),
7.21±7.14 (m, 5H, ArH), 7.12 (s, 1H, OH), 7.11 (s, 1H,
OH), 4.42 (d, J=16.1 Hz, 1H, CHH±N), 4.31±4.24 (m,
2H, CHH±N, CH±N), 3.59 (s, 3H, CH3±O), 3.14±3.11
(m, 1H, CHH±Ph), 2.77 (t, J=5.6 Hz, 2H, CH2±CˆO),
2-[2-(4-Fluorophenyl)-5-hydroxysuccinylamino-6- oxo-
1,6-dihydro-1-pyrimidinyl]-N-(1-benzyl-3,3,3-tri¯uoro-2-
oxopropyl)acetamide (12c). Compound 1e (600 mg,
1.30 mmol) was reacted with methyl succinyl chloride
(0.18 mL, 1.5 mmol) using conditions described for the
2.62±2.56 (m, 3H, CH2±CˆO, CHH±Ph); MS (CI) m/z
+
.
559 (MH ). Anal. calcd for C27H25F3N4O6 0.5H2O: C,
57.14; H, 4.62; N, 9.87; found: C, 57.34; H, 5.01; N,
9.65.
preparationof
11a to give 2-[2-(4-¯uorophenyl)-5-
methoxysuccinylamino-6-oxo-1,6-dihydro-1-pyrimidinyl]-
N - (1 - benzyl - 3,3,3 - tri¯uoro - 2 - oxopropyl)acetamide
(736 mg, 98% yield) as colorless solid. This intermediate
(347 mg, 0.602 mmol) was hydrolyzed using conditions
described for the preparationof 12a to give 12c (295 mg,
87% yield) as colorless crystals: mp 140±147 ꢀC; 1H
NMR (500 MHz, DMSO-d6) d 12.11 (br s, 1H, OH),
9.50 (s, 1H, NH), 8.76 (s, 1H, CHˆN), 8.16 (d,
J=9.8 Hz, 1H, NH), 7.35 (dd, J=8.5, 5.5 Hz, 2H,
ArH), 7.21±7.11 (m, 9H, ArH, OHÂ2), 4.44 (d,
J=15.8 Hz, 1H, CHH±N), 4.26 (m, 2H, CHH±N, CH±
N), 3.12 (d, J=14.1, 2.2 Hz, 1H, CHH±Ph), 2.71 (t,
J=6.5 Hz, 2H, CH2±CˆO), 2.59 (d, J=14.1, 11.6 Hz,
1H, CHH±Ph), 2.48 (t, J=6.5 Hz, 2H, CH2±CˆO);
MS (CI) m/z 561 (MH+). Anal. calcd for
2-(5-Oxaloamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidi-
nyl)-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)acetamide
(12a). Compound 1a (600 mg, 1.36 mmol) was reacted
with methyl oxalyl chloride (0.17 mL, 1.8 mmol) using
conditions described for the preparation of 11a to give
2-(5-methoxalylamino-6-oxo-2-phenyl-1,6-dihydro-1-
pyrimidinyl)-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)-
acetamide (467 mg, 65% yield) as pale yellow crystals:
mp 210±211ꢀC; H NMR (500 MHz, DMSO-d6) d 9.66
1
(s, 1H, NH), 8.73 (s, 1H, CHˆN), 8.19 (d, J=9.8 Hz, 1H,
NH), 7.52 (m, 1H, ArH), 7.39±7.32 (m, 4H, ArH), 7.21±
7.14 (m, 5H, ArH), 7.13 (s, 1H, OH), 7.11 (s, 1H, H), 4.41
(d, J=16.3 Hz, 1H, CHH±N), 4.33 (d, J=16.3Hz, 1H,
CHH±N), 4.26 (m, 1H, CH±N), 3.86 (s, 3H, CH3±O), 3.13
(dd, J=14.2, 2.4 Hz, 1H, CHH±Ph), 2.60 (dd, J=14.2,
11.4Hz, 1H, CHH±Ph); MS (CI) m/z 531 (MH+).
.
C26H22F4N4O6 0.9H2O: C, 53.96; H, 4.15; N, 9.68;
found: C, 53.63; H, 4.14; N, 9.48.
A
solutionof the above tienrmediate (350 mg,
2-(5-Methanesulfonylamino-6-oxo-2-phenyl-1,6-dihydro-
1-pyrimidinyl)-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)-
0.660 mmol) and 0.1N NaOH (6.6 mL) in THF (20 mL)
was stirred at room temperature for 2 h, at which time
1N HCl (0.7 mL) was added. The organic solvents were
evaporated, after which the suspension was extracted
with ethyl acetate. The extract was washed with brine,
dried (MgSO4) and concentrated. The residue was
recrystallized from 1:5 ethyl acetate:hexane to give 12a
(313 mg, 92% yield) as pale yellow crystals: mp 193±
acetamide (13a). To
a
solutionof
1a (400 mg,
0.900 mmol) and pyridine (0.36 mL, 4.5 mmol) in THF
(30 mL) was added methanesulfonyl chloride (0.14 mL,
1.8 mmol). After stirring at room temperature for 17 h,
the reactionmixture was extracted with ethyl acetate.
The organic solvents were washed with saturated
potassium dihydrogen phosphate and brine, and dried
(MgSO4). Concentration followed by silica gel column
chromatography (4:3 dichloromethane:ethyl acetate)
gave a colorless oil, which was crystallized from 1:20
ethyl acetate:hexane to a€ord 13a (307 mg, 65% yield)
195 ꢀC; H NMR (500 MHz, DMSO-d6) d 9.54 (s, 1H,
1
NH), 8.76 (s, 1H, CHˆN), 8.19 (d, J=9.8 Hz, 1H, NH),
7.52 (m, 1H, ArH), 7.39±7.32 (m, 4H, ArH), 7.20±7.14
(m, 5H, ArH), 7.13 (s, 1H, OH), 7.11 (s, 1H, OH), 4.40
(d, J=16.4 Hz, 1H, CHH±N), 4.34 (d, J=16.4 Hz, 1H,
CHH±N), 4.25 (m, 1H, CH±N), 3.13 (dd, J=14.2,
2.4 Hz, 1H, CHH±Ph), 2.60 (dd, J=14.2, 11.4 Hz, 1H,
CHH±Ph); MS (CI) m/z 517 (MH+). Anal. calcd for
as colorless crystals: mp 93±96 ꢀC; H NMR (300 MHz,
1
DMSO-d6) d 9.27 (s, 1H, NH), 8.19 (d, J=9.8 Hz, 1H,
NH), 7.95 (s, 1H, CHˆN), 7.54 (m, 1H, ArH), 7.40 (m,
2H, ArH), 7.33 (m, 2H, ArH), 7.24±7.15 (m, 5H, ArH),
7.13 (s, 1H, OH), 7.10 (s, 1H, OH), 4.37 (s, 2H, CH2±
N), 4.28 (m, 1H, CH±N), 3.14 (dd, J=14.0, 2.0 Hz, 1H,
CHH±Ph), 3.06 (s, 3H, CH3±S), 2.61 (dd, J=14.2, 11.6
Hz, 1H, CHH±Ph); MS (CI) m/z 523 (MH+). Anal.
.
C24H19F3N4O6 1.0H2O: C, 53.94; H, 3.96; N, 10.48;
found: C, 53.95; H, 3.95; N, 10.26.
2-(5-Hydroxysuccinylamino-6-oxo-2-phenyl-1,6-dihydro-
1-pyrimidinyl)-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)-
acetamide (12b). Compound 11c (360 mg, 0.645 mmol)
.
calcd for C23H21F3N4O5S 1.0H2O: C, 51.11; H, 4.29; N,
10.37; found: C, 50.85; H, 4.31; N, 10.27.

F. Akahoshi et al. / Bioorg. Med. Chem. 9 (2001) 301±315
313
2-(5-Benzenesulfonylamino-6-oxo-2-phenyl-1,6-dihydro-
1-pyrimidinyl)-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)-
acetamide (13b). Compound 1a (400mg, 0.900 mmol)
was reacted with benzenesulfonyl chloride (0.28 mL,
4.3 mmol) using conditions described for the prepara-
tionof 13a to give 13b (396 mg, 75% yield) as colorless
conditions described for the preparation of 1b to give
15b (449 mg, 86% yield) as colorless crystals: mp 198±
200 ꢀC; 1H NMR (300 MHz, DMSO-d6) d 8.16 (t,
J=5.5 Hz, 1H, NH), 7.50±7.40 (m, 5H, ArH), 7.33 (s,
1H, CHˆN), 7.30±7.12 (m, 5H, ArH), 5.16 (s, 2H,
NH2), 4.37 (s, 2H, CH2±N), 3.25 (m, 2H, CH2±N), 2.66
(t, J=7.3 Hz, 2H, CH2±Ph). Anal. calcd for
C20H20N4O2: C, 8.95; H, 5.79; N, 16.08; found: C,
69.23; H, 5.77; N, 16.03.
crystals: mp 92±94 ꢀC; H NMR (300 MHz, DMSO-d6)
1
d 10.05 (s, 1H, NH), 8.13 (d, J=9.7 Hz, 1H, NH), 7.94±
7.90 (m, 2H, ArH), 7.87 (s, 1H, CHˆN), 7.68±7.54 (m,
3H, ArH), 7.51±7.46 (m, 1H, ArH), 7.42±7.23 (m, 4H,
ArH), 7.20±7.12 (m, 5H, ArH), 7.11 (s, 1H, OH), 7.10
(s, 1H, OH), 4.35±4.19 (m, 3H, CH2±N, CH±N), 3.12
(dd, J=14.0, 2.2 Hz, 1H, CHH±Ph), 2.57 (dd, J=14.0,
11.4 Hz, 1H, CHH±Ph); MS (CI) m/z 585 (MH+). Anal.
N-(1-Benzyl-3,3,3-tri¯uoro-2-oxopropyl)acetamide (16).
Compound 3 (438 mg, 2.00 mmol) was coupled with
acetic acid (0.12 mL, 2.1 mmol) using a method similar
to that described for 4a to give N-(1-benzyl-3,3,3-tri-
¯uoro-2-hydroxypropyl)acetamide (503 mg, 96% yield)
as colorless crystals. This intermediate (459 mg,
1.77 mmol) was oxidized using conditions described for
the preparationof 5a to give 1ꢀ6 (292 mg, 64% yield) as
.
calcd for C28H23F3N4O5S 0.8H2O: C, 56.15; H, 4.14; N,
9.35; found: C, 56.11; H, 3.88; N, 9.14.
L-Valyl-N-(1-benzyl-3,3,3-tri¯uoro-2-oxopropyl)-L-proli-
namide hydrochloride (14). To a solutionof tert-butoxy-
carbonyl - l - valyl - N - (1 - benzyl - 3,3,3 - tri¯uoro - 2 -
oxopropyl)-l-prolinamide (143 mg, 0.278 mmol) in 1,4-
dioxane (2 mL) was added hydrogen chloride (4N
solutionin1,4-dioxane, 5 mL, 20 mmol) with ice-water
cooling. The resulting mixture was stirred at 0 ꢀC±room
temperature for 2 h, and then concentrated under
reduced pressure. To the residue was added 1:1 ethyl
acetate-hexane and then the mixture was concentrated
under reduced pressure to give a colorless oil, which was
crystallized from 1:1 ethyl acetate:hexane to a€ord 14
(117 mg, 94% yield) as colorless crystals: mp 125±
1
colorless crystals: mp 121±122 C; H NMR (300 MHz,
DMSO-d6) d 8.73 (d, J=5.9 Hz, 1H, NH), 7.33±7.10 (m,
5H, ArH), 4.77 (m, 1H, CH±N), 3.06 (dd, J=13.8,
5.9 Hz, 1H, CHH±Ph), 2.94 (dd, J=13.8, 8.8 Hz, 1H,
CHH±Ph), 1.81 (s, 3H, CH3). Anal. calcd for
.
C12H12F3NO2 0.2H2O: C, 54.84; H, 4.76; N, 5.33;
found: C, 54.73; H, 4.77; N, 5.24.
Protease inhibition assay
Humanheart chymase was puri®ed according to the
method of Urata et al.5 Bovine pancreatic a-chymo-
trypsinand humancathepsinG 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 Wel®de Corporation, Osaka, Japan,
Elastins Products Co. Inc., Owensville, MO and Nippon
Zoki Pharmaceutical Co., Osaka, Japan, respectively.
140 ꢀC; H NMR (300 MHz, DMSO-d6+D2O) d 7.35±
1
7.08 (m, 5H, ArH), 4.50±4.15 (m, 2H, CH±NÂ2), 3.90
(m, 1H, CH±N), 3.70±3.07 (m, 3H, CH2±N, CHH±Ph),
2.75±2.60 (m, 1H, CHH±Ph), 2.15±1.50 (m, 4H, CH2±
CHH, CH±CH3), 1.15±0.80 (m, 7H, CH3Â2, CHH);
MS (CI) m/z 414 (MH+). Anal. calcd for
.
.
C20H26F3N3O3 HCl 1.0H2O: C, 51.34; H, 6.25; N, 8.98;
found: C, 51.03; H, 6.17; N, 8.78.
Chymase-, chymotrypsin- and cathepsin G-inhibitory
activities were determined using previously described
methods27 through measurement at 405 nm absorbance
of p-nitroaniline release from the synthetic substrate
MeO-Suc-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma Chem-
ical Co.). Inthe case of thrombinand elastase, pre-
viously described bu€er conditions40 were used invol-
ving the synthetic substrates S-2238 (Chromogenix AB)
and MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (Sigma
Chemical Co.), respectively. Enzymatic activity of ACE
was measured using previously described methods.41
Calculations of Kis were performed using previously
described methods.27 Since protease inhibitors contain-
ing tri¯uoromethyl ketone are known to need more than
10 min for interaction with enzymes (slow binding inhi-
bition),42,43 two-hour incubation was used to measure
inhibition in steady state.
2-(5-Amino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl)-
N-methylacetamide (15a). Compound 2a (759 mg,
2.00 mmol) was coupled with methylamine using a
method similar to that described for 4a to give 2-(5-
benzyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-
pyrimidinyl)-N-methylacetamide (704 mg, 90% yield) as
colorless crystals. This intermediate (589 mg, 1.50 mmol)
was deprotected using conditions described for the pre-
parationof 1b to give 15a (352 mg, 91% yield) as col-
orless crystals: mp 252±254 ꢀC; 1H NMR (300 MHz,
DMSO-d6) d 7.99 (q, J=4.5 Hz, 1H, NH), 7.45 (s, 5H,
ArH), 7.33 (s, 1H, CHˆN), 5.14 (s, 2H, NH2), 4.36 (s,
2H, CH2±N), 2.57 (d, J=4.5 Hz, 3H, CH3±N). Anal.
.
calcd for C13H14N4O2 0.2H2O: C, 59.62; H, 5.54; N,
21.39; found: C, 59.39; H, 5.22; N, 21.37.
2-(5-Amino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl)-
N-(2-phenethyl)acetamide (15b). Compound 2a (759 mg,
2.00 mmol) was coupled with phenethylamine (0.30 mL,
2.4 mmol) using a method similar to that described for
4a to give 2-(5-benzyloxycarbonylamino-6-oxo-2-phenyl-
1,6-dihydro-1-pyrimidinyl)-N-(2-phenethyl)acetamide
(859 mg, 89% yield) as colorless crystals. This inter-
mediate (724 mg, 1.50 mmol) was deprotected using
Pharmacokinetic determinations
The test compound 1e was formulated as a solution
(1 mg/mL) inDMSO and aqueous solution(84 mM HCl
containing 34 mM NaCl) cosolvent (5:95 v/v). The test
compound 12c was formulated as a solution(1 mg/mL)
in propylene glycol and 2M Na2CO3 aqueous solution

314
F. Akahoshi et al. / Bioorg. Med. Chem. 9 (2001) 301±315
(50:50 v/v) cosolvent. Male Sprague±Dawley rats
(approximate weight 250 g) were giveneither a 1 mg/kg
dose of the test compound iv or a 10 mg/kg dose orally.
Serial blood samples were collected from the jugular
vein at several time points up to 2 h after dosing. Plasma
was harvested from the blood samples by centrifugation,
and the samples were stored at À20 ꢀC until analysis.
15. Mizutani, H.; Schechter, N.; Lazarus, G.; Black, R. A.;
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To each plasma sample (100 mL) was added acetonitrile
(500 mL) to precipitate protein. After centrifugation, the
supernatant was evaporated to dryness under N2. The
residue was reconstituted in 200 mL of HPLC mobile
phase (1e: 20 mM phosphate bu€er pH 6.8:acetonitrile
68:32 v/v; 12c: water:acetonitrile 65:35 v/v containing
5 mM tetrabutylammonium phosphate). HPLC analysis
was carried out ona Waters Symmetry octyl column
(150 mmÂ4.6 mm) at 40 ꢀC with a ¯ow rate of 1.0 mL/
min and an injection volume of 150 mL. Detectionof
test compounds was achieved by monitoring UV
absorption at 305 nm. All pharmacokinetic parameters
were calculated using non-compartment methods.
22. Powers, J. C.; Tanaka, T.; Harper, J. W.; Minematsu, Y.;
Barker, L.; Lincoln, D.; Crumley, K. V.; Fraki, J. E.; Schech-
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Acknowledgements
The authors thank Hiroshi Sakashita for the syntheses
of compounds 1d and 1f, 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.
28. Eda, M.; Ashimori, A.; Akahoshi, F.; Yoshimura, T.;
Inoue, Y.; Fukaya, C.; Nakajima, M.; Fukuyama, H.; Imada,
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