Inhibition of human chymase by 2-amino-3,1-benzoxazin-4-ones

Article abstract of DOI:10.1016/S0968-0896(00)00310-2

A series of 2-sec.amino-4H-3,1-benzoxazin-4-ones was evaluated as acyl-enzyme inhibitors of human recombinant chymase. The compounds were also assayed for inhibition of human cathepsin G, bovine chymotrypsin, and human leukocyte elastase. Introduction of

Full text of DOI:10.1016/S0968-0896(00)00310-2

Bioorganic & Medicinal Chemistry 9 (2001) 947±954
Inhibition of Human Chymase by 2-Amino-3,1-benzoxazin-4-ones
Ulf Neumann,a Norman M. Schechterb and Michael Gutschowc,*
aNovartis Pharma AG, CH-4002 Basel, Switzerland
bDepartment of Dermatology, University of Pennsylvania, Philadelphia, PA 19104, USA
cInstitute of Pharmacy, University of Leipzig, D-04103 Leipzig, Germany
Received 18 October 2000; accepted 16 November 2000
AbstractÐA series of 2-sec.amino-4H-3,1-benzoxazin-4-ones was evaluated as acyl-enzyme inhibitors of human recombinant chy-
mase. The compounds were also assayed for inhibition of human cathepsin G, bovine chymotrypsin, and human leukocyte elastase.
Introduction of an aromatic moiety into the 2-substituent resulted in strong inhibition of chymase, cathepsin G, and chymotrypsin.
Extension of the N(Me)CH2Ph substituent by one methylene unit was unfavourable to inhibit these proteases. Towards chymase,
2-(N-benzyl-N-methylamino)-4H-3,1-benzoxazin-4-one (32) and 2-(N-benzyl-N-methylamino)-6-methyl-4H-3,1-benzoxazin-4-one
(33) were found to exhibit Ki values of 11 and 17 nM, respectively, and form stable acyl-enzymes with half-lives of 53 and 25 min,
respectively. Benzoxazinone 33 also inhibited the human chymase-catalyzed formation of angiotensin II from angiotensin I. # 2001
Elsevier Science Ltd. All rights reserved.
Introduction
fold of the three chymotrypsin-like enzymes.2 They have
quite similar structures around their catalytic sites,
characterized by a deep, hydrophobic S1 pocket that can
accommodate the side chains of aromatic amino acids in
P1 position of the substrate. In contrast to chymotrypsin,
human chymase also has exoproteolytic activity. It is
this dipeptidyl carboxypeptidase activity which makes
chymase so e€ective in generating Ang II from Ang I by
cleaving the Phe8-His9 peptide bond. Docking experiments
done with Ang I have shown that the peptide's C-terminus
can make favourable hydrogen bond interactions with
the Lys40-Phe41 chymase peptide bond and with the free
amino group of Lys40.3
The renin±angiotensin-system plays a central role in
regulation of blood pressure. Pharmacological intervention
in hypertension can occur by inhibiting angiotensin
converting enzyme (ACE) and thereby blocking the
generation of the vasoconstricting octapeptide angio-
tensin II (Ang II) from its precursor angiotensin I (Ang
I). Another successful approach is the prevention of
binding of Ang II to its cell-surface receptor with low
molecular weight receptor antagonists. However, ACE
is not the sole generator of Ang II.1 In human heart
tissue and in blood vessel walls, human chymase (E. C.
3.4.21.39) forms 90% of the Ang II.1a Chymase was ®rst
found in mast cells and seems to be involved in the
degradation of the extracellular matrix (directly and/or
by activation of matrix metalloproteases), stimulation
of submucosal gland cell secretion, and complement-
mediated in¯ammation.1 Given the variety of patho-
physiological processes where chymase is involved,
inhibitors of the enzyme could be potential drugs in
cardiovascular and in¯ammatory diseases. The primary
structure of human chymase is 36% identical to chymo-
trypsin and 52% identical to human cathepsin G.
Recent X-ray analysis results show an overall common
A variety of synthetic inhibitors of human chymase have
been described, including peptides and peptidomimetics,4,5
as well as 3-(phenylsulfonyl)-1-phenylimidazolidine-2,4-
dione1b and 1,2,5-thiadiazolidin-3-one derivatives.6 We
and others have investigated the inhibition of chymo-
trypsin-like and elastase-like serine proteases by 4H-3,1-
benzoxazin-4-ones. Such compounds have recently been
shown to be active in vivo after intratracheal admini-
stration.7 Benzoxazinones temporarily inhibit the catalytic
activity of serine proteases by accumulation of a catalyti-
cally inactive acyl-enzyme intermediate (Scheme 1). The
rates of acylation and deacylation as well as the compound
selectivity is determined both by substituents at the
benzene unit and the 2-substituent. Potent benzoxazinone
inhibitors for human leukocyte elastase (HLE),8 human
*Corresponding author. Tel.: +49-341-973-6810; fax: +49-341-973-
6749; e-mail: guetscho@uni-leipzig.de
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00310-2

948
U. Neumann et al. / Bioorg. Med. Chem. 9 (2001) 947±954
Enzyme assays
The inhibitory e€ects of benzoxazinones 21±36 on the
enzymatic activities of serine proteases were evaluated
in the presence of chromogenic substrates at pH 8.0,
except for HLE, which was assayed at pH 7.8.8 Initially,
the inhibiting activity was determined at a single inhibitor
concentration. Percentage of inhibition was calculated
from the steady-state rates (Table 1). Based on these
data, compounds for full kinetic analysis using di€erent
inhibitor concentrations were selected (Tables 2±4). The
time course of product formation after addition of enzyme
to a solution of chromogenic substrate and inhibitor was
monitored. Progress curves were characterized by an
initial exponential phase, and a second, linear steady-
state turnover of the chromogenic substrate. Such curves
have already been observed when acylating inhibitors
were reacted with serine proteases and the acylation±
deacylation mechanism (according to Scheme 1) for the
reaction of fused oxazin-4-ones with HLE and cathepsin G
was proven by product analyses.8,9,11,15 We postulate that
chymase also reacts with the benzoxazinones described
herein by formation of an acyl-enzyme which hydrolyzes
slowly. As discussed before,11 the potency of acylating
inhibitors can be described by the ratio of their deacyl-
ation and acylation rate constants and is called the
dissociation constant Ki, although this kinetic Ki value
is not identical to the thermodynamic Ki value describing
the binding of a fully reversible inhibitor. Inhibition
parameters were obtained from the slow-binding curves
as described.11,14,15 We did not see an accumulation of a
noncovalent enzyme±inhibitor complex before the onset
of the acylation reaction, since the initial rate of the
progress curve was identical to the control rate in the
absence of inhibitor. This was the case for all inhibitors
and for every inhibitor concentration used. In the
screening experiments with cathepsin G and inhibitors
31 and 33 at 20 mM inhibitor concentration (Table 1),
the steady-state was already reached at the beginning of
the progress curve due to fast acylation.
Scheme 1. Reaction of 2-amino-4H-3,1-benzoxazin-4-ones with serine
proteases. Formation of acyl-enzymes and possible ways of deacyl-
ation.
cathepsin G, and bovine chymotrypsin9 have been
described. Introduction of 2-amino substituents is a
successful strategy to improve the chemical stability of
benzoxazinones8À10 and of isosteric thieno[1,3]oxazin-4-
ones.11,12 To prevent rapid deacylation via intramole-
cular cyclization and formation of corresponding quina-
zolines (R=H, see Scheme 1) we focused on benzoxa-
zinones with 2-sec.amino substitution (R, R0ˆH).
The results obtained so far with benzoxazinone-type
compounds as inhibitors of serine proteases suggest that
they may be useful inhibitors of chymase activity. We
therefore investigated in vitro the inhibition of human
chymase by a series of 2-sec.aminobenzoxazinones. In
parallel, the compounds were assayed for inhibition of the
related enzymes bovine chymotrypsin, human cathepsin
G, and human leukocyte elastase. Using angiotensin I as a
substrate, we tested whether a selected benzoxazinone was
able to inhibit also the biologically relevant exopeptidase
activity of chymase.
General pattern of inhibition
The inhibition data in Table 1 clearly show that human
chymase, human cathepsin G and bovine chymotrypsin
were all strongly inhibited by benzoxazinones carrying
aromatic substituents at position 2. In contrast, and in
agreement with published data, the inhibitory pro®le for
HLE was di€erent. The results ®tted to the known
active-site architecture of these enzymes. Chymase,
cathepsin G and chymotrypsin all have large primary
speci®city pockets which can accommodate aromatic
rings. Their preference for benzoxazinones bearing an
aromatic moiety in the 2-substituent indicates that this
substituent binds into the primary speci®city pocket.
While it is obvious that selective inhibition of chymase
over elastase can be achieved, the di€erentiation
between the chymotrypsin-like enzymes is much
more dicult. To obtain a ®rst structure±activity
relationship for the inhibition of chymase, cathepsin
G and chymotrypsin, we determined the individual
kinetic parameters of inhibition for the most potent
compounds.
Results and Discussion
Chemistry
Benzoxazinones 21±35 (Table 1) were prepared by
three alternative routes (Scheme 2). Mesyloxyphthal-
imides 1±3 were reacted with the appropriate secondary
amine to obtain 2-ureidobenzamides 9±11, 17, and
ethyl 2-ureidobenzoates 6, 8, 12, 14, respectively.13
Cyclocondensation of these precursors (method A,
method B, respectively) to the corresponding benzox-
azinones was then performed with concentrated sulfuric
acid. The third route involves transformation of isatoic
anhydrides 4, 5 to corresponding 2-ureidobenzoic
acids which were conveniently cyclized to the ®nal
benzoxazinones upon treatment with acetic anhydride
(method C).

U. Neumann et al. / Bioorg. Med. Chem. 9 (2001) 947±954
Table 1. Inhibition of serine proteases by 4H-3,1-benzoxazin-4-ones
949
% Inhibitiona
Compound
Method
(prepared from)
R5
R6
R7
R8
NRR0
Chymase
[I]=1 mM
Cathepsin G
[I]=20 mM
Chymotrypsin
[I]=1 mM
HLE
[I]=1 mM
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
B (6)
C (7)
B (8)
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
H
H
H
H
H
Me
H
H
H
H
H
Me
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
4-Morpholinyl
4-Morpholinyl
4-Morpholinyl
4-Morpholinyl
1-Pyrrolidinyl
N(Et)2
N(Me)cyclohexyl
N(Me)cyclohexyl
N(Me)cyclohexyl
N(Me)Ph
<15
<15
<15
<15
<15
<15
<15
30
<15
>95*
90*
>95*
>95*
17
53b
<15
<15
<15c
94b
50
31
66
5270
69
75
75
25
38
<15
A (9)
A (10)
A (11)
B (12)
C (13)
B (14)
C (15)
C (16)
A (17)
C (18)
C (19)
C (20)
28
31
65
77b
H
83b
5281
23c
95*
56d
97*
86d
71b
15
95*
Me
Me
H
Me
H
Me
H
Me
H
18
96*
90*
77*
92*
83*
67
<15
37
26
48
42
40
44
50
N(Me)Ph
H
H
H
H
N(Me)CH2Ph
N(Me)CH2Ph
N(Me)CH2CH2Ph
N(Me)CH2CH2Ph
NHCH2Ph
<15
>95*
38
93*
H
aInhibition was measured at a single inhibitor concentration as indicated. Percentage inhibition was determined from the steady-state rate constants.
The kinetic parameters of inhibition indicated by an asterisk are outlined in Tables 2±4.
bSlow inhibition with second-order rate constants kobs/[I] ꢀ100 MÀ1 sÀ1
dInhibition was determined from the initial part of the progress curve.
.
cSlight precipitation occurred in the course of the assay.
Chymase inhibition
substrate angiotensin I. Compound 33 was selected
for this study considering the results with chromo-
genic substrates towards chymase (see above) as well
as related serine proteases (see below). The cleavage
of human angiotensin I to angiotensin II by human
chymase was followed by HPLC. The reaction was dis-
tinctly inhibited in the presence of 33 at concentrations
of 100 and 250 nM, respectively (Fig. 4). From the
initial part of the curves, an approximate Ki value of
27 nM could be estimated, being in the range of the Ki
value obtained for 33 with chromogenic substrate
(Table 2).
The kinetic parameters of four 2-sec.amino-3,1-benzoxa-
zinones together with the benzylamino compound 36
are outlined in Table 2. The benzylamino compound 36
(Ki=5 nM) was the most potent compound in the series.
This is mainly due to its very high acylation rate, which
is an order of magnitude higher than for any of the
sec.amino derivatives tested. Introduction of a morpho-
lino, pyrrolidino, or diethylamino residue (21±26) led to
inactive compounds (Table 1). Compounds with cyclo-
hexyl residues also had little inhibitory potency towards
chymase (27±29). We found chymase inhibition to be
strongly in¯uenced by the length of the spacer between
secondary amino group and aryl residue within the 2-sub-
stituent: extension of the N(Me)CH2Ph substituent by
one methylene unit was highly unfavourable in chymase
inhibition (32 versus 34; 33 versus 35). Removal of one
methylene unit of the N(Me)CH2Ph residue resulted in an
acceleration of both the acylation and the deacylation step
(30 versus 32; 31 versus 33, Table 2). 6-Methyl substitu-
tion (31, 33) did not have a big impact on the inhibitory
potency of the compounds. From the present series,
benzoxazinones 32 and 33 appear as the most promising
inhibitors with Ki values around 10 nM; they form
highly stable acyl-enzymes with half-lives of 53 min and
25 min, respectively. As an example, the analysis of the
chymase inhibition kinetics by the benzoxazinone 33 is
illustrated in Figures 1±3.
Inhibition of cathepsin G, chymotrypsin, and HLE
In agreement with previous data,9 cathepsin G was less
potently inhibited by benzoxazinones, compared to
chymase and chymotrypsin. Thus, for the initial inhibition
studies, an inhibitor concentration of 20mM was chosen,
whereas 1 mM was used for the other enzymes. Cathepsin
G also shows lower hydrolysis rates and kcat/KM values
with peptide p-nitroanilide substrates.16 A recent inves-
tigation using peptide substrates spanning the P- and
the P0-side of the substrate-binding region, however,
identi®ed substrates with kcat/KM values comparable to
the other serine proteases.17 Cathepsin G recruits a
considerable fraction of its catalytic power from
enzyme±substrate interactions occurring at subsites
remote from the scissile bond. The benzoxazinones studied
here, however, cannot interact with distant subsites, which
is re¯ected by their low second-order rate constants
We investigated the inhibition of human chymase-
catalyzed conversion of the physiologically relevant

950
U. Neumann et al. / Bioorg. Med. Chem. 9 (2001) 947±954
Table 2. Kinetic data of the inhibition of human chymase by 4H-
3,1-benzoxazin-4-ones
Compound
kon (MÀ1 sÀ1
)
ko€ (10À4 sÀ1
)
Ki (nM)
pKi
30
31
32
33
36
79,000
85,800
19,000
40,300
623,000
11
25
2.2
6.8
31
14
29
11
17
5.0
7.87
7.53
7.94
7.77
8.30
Table 3. Kinetic data of the inhibition of human cathepsin G by 4H-
3,1-benzoxazin-4-ones
Compound
kon (MÀ1 sÀ1
)
ko€ (10À4 sÀ1
)
Ki (nM)
pKi
30
32
36
785
363
2640
9.7
1.6
9.5
1200
440
360
5.91
6.36
6.44
Table 4. Kinetic data of the inhibition of bovine chymotrypsin by
4H-3,1-benzoxazin-4-ones
Compound
kon (MÀ1 sÀ1
)
ko€ (10À4 sÀ1
)
Ki (nM)
pKi
29
30
31
32
33
36
137,000
81,800
91,400
74,300
58,800
810,000
17
9.0
45
17
22
127.92
11
49
23
38
7.97
7.31
7.63
7.43
7.48
270
33
Scheme 2. (a) HNRR0, acetone, re¯ux, 25 min, or N-benzylmethyl-
amine, toluene, 0±25 ꢁC, 7 h, 64±95%; (b) concd H2SO4, rt or 0 ꢁC, 11±
71%, method A; (c) HNRR0, acetone, re¯ux, 25 min, then 0.25 M
ethanolic HCl, re¯ux, 2min, then À15 ꢁC, 66±91%; (d) concd H2SO4,
rt, 86±98%, method B; (e) HNRR0, H2O, rt, or HNRR0, EtOH, H2O,
re¯ux, 10±53%; (f) acetic anhydride, re¯ux, 15min, 52±94%, method C.
(Table 3; kobs/[I] ꢀ100 MÀ1 sÀ1 for the moderately
potent compounds 21, 25±27, 34, Table 1). Also, for the
most active compound, 36, kon was more than 100-fold
lower, compared to chymase and chymotrypsin. Intro-
duction of methyl groups into the benzene unit was
disadvantageous. The Ki value of 32 was similar to that of
the 2-benzylamino derivative 36. Both compounds were
already part of previous series of benzoxazinones evaluated
at pH 7 as inhibitors for cathepsin G (and chymo-
trypsin).9 Chymotrypsin, in contrast to cathepsin G,
was potently inhibited by the benzoxazinones, especially
those with aromatic 2-substituents. Ki values in the low
nanomolar range were achieved (Table 4). Extension of
the N(Me)CH2Ph substituent by one methylene unit
resulted in less potent compounds (34 versus 32; 35
versus 33, Table 1). However, this e€ect was moderate
for chymotrypsin and cathepsin G, and pronounced
only for chymase.
Figure 1. Slow-binding inhibition of human chymase by compound 33
in 0.1 M Tris±HCl, 1.8 M NaCl, pH 8.0. Substrate Suc-Ala-Ala-Pro-
Phe-pNA. Data were ®tted to eq (1) to obtain the best-®t parameters
for vi, vs, kobs, and o€set. Open circles: [I]=0; full circles: [I]=100 nM;
open squares: [I]=200 nM; full squares: [I]=300 nM; open triangles:
[I]=400 nM; and full triangles: [I]=500 nM.
HLE inhibition by 2-amino-3,1-benzoxazin-4-ones,
including compounds 21, 25, 26, 36, has extensively
been studied by Krantz et al.8 HLE preferentially
cleaves proteins or peptidic substrates with Ala, Leu or
Val at P1 position. Therefore, as expected,8,11 only
moderate inhibition was detected with benzoxazinones
30±36 bearing an aromatic moiety in the 2-substituent
(Table 1). Introduction of methyl groups at positions 6
and 7 was disadvantageous (23 versus 21, 29 versus 27).

U. Neumann et al. / Bioorg. Med. Chem. 9 (2001) 947±954
951
Strong inhibition, on the other hand, was noted by
introducing methyl groups at positions 5 and 8 (24 versus
21). A Ki value of 27 nM (kon=2970 MÀ1 sÀ1, ko€=0.8Â
10À4 sÀ1) was detected for the 5,8-dimethyl-2-morpholino
derivative 24. Strong inhibition by this compound
resulted from a highly stable acyl-enzyme with a half-
life of more than 2h. The decreased deacylation rate is
thought to result from the 5-methyl introduction that leads
to steric hindrance of water attack at the ester carbonyl
of the acyl-enzyme.8 It is noteworthy that the new inhibitor
24 showed speci®city for HLE over both chymase and
cathepsin G.
Conclusions
Figure 2. Plot of kobs versus [I] for the inhibition of human chymase
by compound 33. The values for kobs were obtained from ®ts to the
This report describes, for the ®rst time, the inhibition of
human chymase by benzoxazinones. We restricted our
investigations to conveniently available compounds,
which are known to be suciently stable at physio-
logical pH.8,9 Inhibition of chymase by 4H-3,1-benz-
oxazin-4-ones occurs by an acylation-deacylation
mechanism, as shown before for other serine proteases.
Introduction of an aromatic moiety into the 2-substituent
furnished potent inhibitors of chymase, indicating the
accommodation of the phenyl group at the primary
speci®city site S1 of the enzyme. The introduction of a
disubstituted amino group resulted in relatively stable
acyl-enzyme complexes. This comparison of deacylation
rates revealed a favourable deceleration of the deacylation
step, when intramolecular quinazoline formation is
structurally prevented. Within this set of compounds,
we identi®ed acylating inhibitors of chymase with Ki
values in the low nanomolar range. It was further
demonstrated exemplary that the chymase-catalyzed
conversion of angiotensin I was inhibited by a selected
benzoxazinone at nanomolar concentrations. Selectivity
for chymase over HLE was achieved by incorporation
of aromatic structures into the 2-substituent of the benz-
oxazinones.
data shown in Figure 1. The slope corresponds to a value for kon
(1+[S]/Km)=22,200 MÀ1 sÀ1
/
.
Figure 3. Plot of the steady-state rates versus [I] for the inhibition of
human chymase by compound 33. The data were obtained from ®ts of
the curves shown in Figure 1. The solid line was drawn using the best-
®t parameters from a ®t according to an equation of a competitive
inhibition, which gave Ki (1+[S]/Km)=32.1 Æ 1.3 nM. The insert is a
Dixon plot to show the linearity.
Interestingly, besides ACE and chymase, neutrophil
cathepsin G was found to generate angiotensin II from
angiotensin I and also to convert angiotensinogen
directly to angiotensin II.18,19 However, our data show
that there is a considerable di€erence in terms of acyla-
tion ecacy towards benzoxazinones between human
chymase and cathepsin G. It can be expected that the
concentration required to inhibit chymase is 1±2orders
of magnitude lower than the one required to inhibit
cathepsin G.
None of the chymase inhibitors did exhibit speci®city
for chymase over chymotrypsin. This is not very sur-
prising given the structural and functional similarity
between the two enzymes. This initial study, however,
investigated only a few modi®cations within the benz-
oxazinone system. The recent identi®cation of the
structural basis for the exopeptidase activity, which
chymase does not share with chymotrypsin, o€ers intri-
guing possibilities for the design of selective benzoxa-
zinone-type chymase inhibitors for the treatment of
cardiovascular and in¯ammatory diseases.
Figure 4. Inhibition of the chymase catalyzed cleavage of angiotensin
I (to angiotensin II) by compound 33. Open circles: [I]=0; full circles:
[I]=100 nM; open squares: [I]=250 nM.

952
U. Neumann et al. / Bioorg. Med. Chem. 9 (2001) 947±954
Experimental
with ethyl acetate (1Â100 mL) and acidi®ed with 10%
H2SO4. The precipitate was collected by ®ltration,
washed with water, and dried to yield 13 (2.6 g, 45%):
General methods and materials
mp 154±156 ꢁC; IR (KBr, cmÀ1) 1680, 1636 (CˆO); H
1
Melting points were determined on a Boetius apparatus
and are not corrected. Thin-layer chromatography was
performed on Merck aluminium sheets, silica gel 60
NMR (CDCl3) d 1.10±1.90 (m, 10H, CH2), 2.32 (s, 3H,
5-CH3), 2.94 (s, 3H, NCH3), 4.05±4.20 (m, 1H, CH),
7.37 (dd, J=8.7, 2.2 Hz, 1H, H-4), 7.85 (s, br, 1H, H-6),
8.51 (d, J=8.7 Hz, 1H, H-3), 10.37 (s, 1H, NH). Anal.
calcd for C16H22N2O3: C, 66.18; H, 7.64; N, 9.65.
Found: C, 65.96; H, 7.93; N, 9.94.
1
F254. H NMR spectra (300 MHz) were recorded on a
Varian Gemini 300. IR spectra were measured with a
Perkin-Elmer 16 PC FTIR spectrometer. Mass spectra
(70 eV) were obtained using a Varian MAT CH6 spectro-
meter. Spectrophotometric assays were done on a Varian
Cary 3 Bio spectrophotometer with six-cell holder.
2-(3-Phenyl-3-methylureido)benzoic acid (15). Compound
15 was prepared from 4 and N-methylaniline in 42%
yield: mp >159 ꢁC (dec) (lit.24 mp 161 ꢁC); IR (KBr,
Analytical HPLC was performed on
a Thermo-
SeparationProducts liquid chromatograph with PC1000
software. A Nucleosil C18 2Â250-mm column was used
at a ¯ow rate of 0.2mL/min. Mobile phase A was H 2O,
0.1% TFA, and mobile phase B was H2O/acetonitrile/
TFA 30/70/0.085%. A gradient of 30±57% B in 14 min
was used. Recombinant human mast cell chymase was
puri®ed as described.20 Elastase was prepared from human
leukocytes and puri®ed by anity chromatography using
an immobilized synthetic inhibitor.21 Human cathepsin G
was purchased from Calbiochem. Chymotrypsin was pur-
chased from Worthington, Freehold, USA. Suc-Ala-Ala-
Pro-Phe-pNA, MeOSuc-Ala-Ala-Pro-Val-pNA, angio-
tensin I, and angiotensin II were from Bachem, Bubendorf,
Switzerland.
1
cmÀ1) 1696, 1658 (CˆO); H NMR (CDCl3) d 3.36 (s,
3H, CH3), 6.98±7.04 (m, 1H, H-5), 7.30±7.52(m, 5 ArH),
7.54±7.60 (m, 1H, H-4), 7.99 (dd, J=8.1, 1.6 Hz, 1H,
H-6), 8.71 (d, J=8.5 Hz, 1H, H-3), 9.92(s, 1H, NH).
5-Methyl-2-(3-methyl-3-phenylureido)benzoic acid (16).
Compound 16 was prepared from 5 and N-methylaniline.
The sodium salt that precipitated in the extraction with
0.5 M NaOH was separated and combined with the
material obtained after acidi®cation. Recrystallization
from EtOH/5% AcOH gave 16 in 44% yield: mp >200ꢁC
(dec); IR (KBr, cmÀ1) 1692, 1654 (CˆO); 1H NMR
(CDCl3) d 2.35 (s, 3H, 5-CH3), 3.35 (s, 3H, NCH3),
7.30±7.52(m, 6 ArH), 7.76 (d, J=1.9 Hz, 1H, H-6), 8.58
(d, J=8.8 Hz, 1H, H-3), 9.81 (s, 1H, NH). Anal. calcd
for C16H16N2O3: C, 67.59; H, 5.67; N, 9.85. Found: C,
67.36; H, 5.39; N, 9.84.
2-Ureidobenzamides 9±11,13 17,9 and ethyl 2-ureido-
benzoates 6, 8, 12, 1413 were prepared from corresponding
N-(mesyloxy)phthalimides 1±3 as reported. The following
2-sec.amino-4H-3,1-benzoxazin-ones were prepared as
reported 21, 23±27, 29,13 32.9 2-Benzyl-amino-4H-3,1-
benzoxazin-4-one 368 was synthesized from methyl 2(-3-
benzylureido)benzoate22 and puri®ed on column chro-
matography.
2-(3-Benzyl-3-methylureido)-5-methylbenzoic acid (18).
Compound 18 was prepared from 5 and N-benzyl-
methylamine in 10% yield: mp 144±146 ꢁC; IR (KBr,
1
cmÀ1) 1700, 1644 (CˆO); H NMR (CDCl3) d 2.32 (s,
3H, 5-CH3), 3.04 (s, 3H, NCH3), 4.65 (s, 2H, CH2),
7.25±7.41 (m, 6 ArH), 7.84 (d, J=1.7 Hz, 1H, H-6), 8.55
(d, J=8.7 Hz, 1H, H-3), 10.64 (s, 1H, NH). Anal. calcd
for C17H18N2O3: C, 68.44; H, 6.08; N, 9.39. Found: C,
68.39; H, 6.23; N, 9.18.
5-Methyl-2-[(morpholinocarbonyl)amino]benzoic acid (7).
A mixture of 5-methylisatoic anhydride 523 (3.54 g,
20 mmol) and 50 mL of an aqueous solution of morpholine
(4 M) was stirred at room temperature for 15min, diluted
with water (200 mL) and extracted with ethyl acetate
(1Â50 mL). The organic layer was extracted with 0.5 M
NaOH (1Â50 mL). The combined aqueous layers were
washed with ethyl acetate (1Â100 mL) and acidi®ed with
10% H2SO4. The precipitate was collected by ®ltration,
washed with water, and dried to yield 7 (1.9 g, 36%): mp
173±175 ꢁC; IR (KBr, cmÀ1) 1686, 1645 (CˆO); 1H
NMR (CDCl3) d 2.33 (s, 3H, CH3), 3.54±3.60 (m, 4H,
NCH2), 3.74±3.80 (m, 4H, OCH2), 7.39 (dd, J=8.6,
2.0 Hz, 1H, H-4), 7.86 (s, br, 1H, H-6), 8.46 (d,
J=8.6 Hz, 1H, H-3), 10.49 (s, 1H, NH). Anal. calcd for
C13H16N2O4: C, 59.08; H, 6.10; N, 10.60. Found: C,
59.04; H, 6.05; N 10.40.
2-[3-Methyl-3-(2-phenylethyl)ureido]benzoic acid (19).
Compound 19 was prepared from 4 and N-benzyl-
methylamine in 36% yield: mp 139±141 ꢁC; IR (KBr,
1
cmÀ1) 1696, 1640 (CˆO); H NMR (CDCl3) d 2.94 (t,
J=7.6 Hz, 2H, CH2), 3.00 (s, 3H, CH3), 3.64 (t,
J=7.6 Hz, 2H, NCH2), 6.96±7.05 (m, 1H, H-5), 7.14±
7.32(m, 5 ArH), 7.5±27.60 (m, 1H, H-4), 8.07 (dd,
J=8.1, 1.6 Hz, 1H, H-6), 8.63 (d, J=8.8 Hz, 1H, H-3),
10.55 (s, 1H, NH). Anal. calcd for C17H18N2O3: C,
68.44; H, 6.08; N, 9.39. Found: C, 68.65; H, 6.29; N,
9.50.
5-Methyl-2-[3-methyl-3-(2-phenylethyl)ureido]benzoic acid
(20). Compound 20 was prepared from 5 and N-benzyl-
methylamine in 53% yield: mp 173±175 ꢁC; IR (KBr,
2-(3-Cyclohexyl-3-methylureido)-5-methylbenzoic
acid
(13): General procedure for 2-ureidobenzoic acids 13,
15, 16, 18±20. A mixture of 5 (3.54 g, 20 mmol) and
50 mL of an ethanolic solution of N-methylcyclohexyl-
amine (4 M) was re¯uxed for 15 min, diluted with water
(200 mL) and extracted with ethyl acetate (1Â50 mL).
The organic layer was extracted with 0.5 M NaOH
(1Â50 mL). The combined aqueous layers were washed
1
cmÀ1) 1692, 1636 (CˆO); H NMR (CDCl3) d 2.33 (s,
3H, 5-CH3), 2.94 (t, J=7.7 Hz, 2H, CH2), 3.00 (s, 3H,
NCH3), 3.63 (t, J=7.7 Hz, 2H, NCH2), 7.20±7.41 (m, 6
ArH), 7.86 (s, br, 1H, H-6), 8.52(d, J=8.6, 1H, H-3),
10.40 (s, 1H, NH). Anal. calcd for C15H20N2O3: C,
69.21; H, 6.45; N, 8.97. Found: C, 69.26; H, 6.65; N, 8.78.

U. Neumann et al. / Bioorg. Med. Chem. 9 (2001) 947±954
953
6-Methyl-2-morpholino-4H-3,1-benzoxazin-4-one (22).
General procedure for the preparation of 4H-3,1-benzox-
azin-4-ones 22, 28, 30, 31, 33±35 from 2-ureidobenzoic
acids. A mixture of compound 7 (529 mg, 2 mmol) and
acetic anhydride (7 mL) was re¯uxed for 15 min. After
cooling, it was poured onto ice±water (100 mL). The
precipitate was collected by ®ltration, washed with
water, and dried to yield 22 (390 mg, 79%): mp 135±
138 ꢁC; IR (KBr, cmÀ1) 1752(C ˆO); 1H NMR (CDCl3)
d 2.39 (s, 3H, CH3), 3.70±3.80 (m, 8H, CH2), 7.16 (d,
J=8.4 Hz, 1H, H-8), 7.46 (d, J=8.4 Hz, 1H, H-7), 7.82
(s, br, 1H, H-5); MS (EI) m/z (rel intensity) 246 (M+, 71),
160 (M+ÀNRR0, 100). Anal. calcd for C13H14N2O3: C,
63.40; H, 5.73; N, 11.38. Found: C, 63.16; H, 6.02, N,
11.13.
(rel intensity) 280 (M+, 25), 146 (M+ÀNRR0, 100).
Anal. calcd for C17H16N2O2: C, 72.84; H, 5.75; N, 9.99.
Found: C, 72.96; H, 6.15; N, 10.38.
6-Methyl-2-[N-methyl-N-(2-phenylethyl)amino]-4H-3,1-
benzoxazin-4-one (35). Compound 35 was prepared
from 20 in 87% yield: mp 96±97 ꢁC; IR (KBr, cmÀ1
)
1
1750 (CˆO); H NMR (CDCl3) d 2.37 (s, 3H, 6-CH3),
2.97 (t, J=7.4 Hz, 2H, CH2), 3.06 (s, 3H, CH3), 3.76 (t,
J=7.4 Hz, 2H, NCH2), 7.18 (d, J=8.3 Hz, 1H, H-8),
7.20±7.33 (m, 5 ArH), 7.44 (dd, J=8.3, 2.1 Hz, 1H, H-
7), 7.80 (s, br, 1H, H-5); MS (EI) m/z (rel intensity) 294
(M+, 28), 160 (M+ÀNRR0, 100). Anal. calcd for
C18H18N2O2: C, 73.45; H, 6.16; N, 9.52. Found: C,
73.64; H, 6.42; N, 9.33.
2-(N-Cyclohexyl-N-methylamino)-6-methyl-4H-3,1-benz-
oxazin-4-one (28). Compoundꢁ28 was synthesized from
13 in 90% yield: mp 111±112 C; IR (KBr, cmÀ1) 1762
Enzyme inhibition assays
Inhibition of serine proteases by compounds 21±36 was
assayed spectrophotometrically by the progress curve
method. All experiments were carried out at 25 ꢁC with
a ®nal DMSO concentration of 1.5%. Progress curves
were ®tted to eq (1):
1
(CˆO); H NMR (CDCl3) d 1.06±1.90 (m, 10H, CH2),
2.36 (s, 3H, 6-CH3), 3.01 (s, 3H, NCH3), 4.24±4.40 (m,
1H, CH), 7.15 (d, J=8.4 Hz, 1H, H-8), 7.41 (d,
J=8.4 Hz, 1H, H-7), 7.79 (s, br, 1H, H-5); MS (EI) m/z
(rel intensity) 272 (M+, 50), 190 (M+ÀC6H10, 100), 160
(M+ÀNRR0, 70). Anal. cald for C16H20N2O3: C, 70.56;
H, 7.40; N, 10.29. Found: C, 70.26; H, 7.61; N, 10.23.
‰PŠ ˆ vs t ‡ ꢀvi À vs† ‰1 À expꢀÀkobs t†Š=kobs ‡ offset ꢀ1†
where kobs is the ®rst-order rate constant for the
approach to the steady-state, vs and vi are the steady-
state and the initial velocity, respectively. Except for
cathepsin G inhibition by 31 and 33 (Table 1), vi values
equalled to the velocity in the absence of inhibitor, v0. In
the screening experiments, inhibitors were evaluated at a
single concentration (Table 1). To estimate Ki values
from these assays, eq (2) was used.
2-(N-Methyl-N-phenylamino)-4H-3,1-benzoxazin-4-one
(30). Compound 30 was prepared from 15 in 52%
yield: mp 127±128 ꢁC (acetone/hexane) (lit.25 mp 130±
131 ꢁC); IR (KBr, cmÀ1) 1768 (CˆO); 1H NMR
(CDCl3) d 3.55 (s, 3H, CH3), 7.15±7.21 (m, 1H, H-6),
7.28±7.47 (m, 6 ArH), 7.60±7.67 (m, 1H, H-7), 8.03 (dd,
J=8.0 Hz, 1H, H-5); MS (EI) m/z (rel intensity) 252
(M+, 39), 146 (M+ÀNRR0, 100).
Ki ˆ Km‰IŠ=‰ꢀKm ‡ ‰SŠ† ꢀv0=vs À 1†Š
ꢀ2†
6-Methyl-2-(N-methyl-N-phenylamino)-4H-3,1-benzoxazin-
4-one (31). Compound 31 was synthesized from 16 in
92% yield: mp 117±119 (ethyl acetate/hexane), IR (KBr,
cmÀ1) 1759 (br, CˆO); 1H NMR (CDCl3) d 2.38 (s, 1H,
6-CH3), 3.53 (s, 3H, NCH3), 7.23 (d, J=8.4 Hz, 1H,
H-8), 7.26±7.48 (m, 6 ArH), 7.82 (s, br, 1H, H-5); MS (EI)
m/z (rel intensity) 266 (M+, 60), 160 (M+ÀNRR0, 100).
Anal. calcd for C15H14N2O2: C, 72.17; H, 5.30; N,
10.52. Found: C, 71.83; H, 5.27; N, 10.46.
Values from these calculations were found to be in a
sucient agreement with the Ki values outlined in
Tables 2±4. To determine the kinetic parameters of
inhibition (Tables 2±4), at least ®ve di€erent inhibitor
concentrations were used.
Inhibitors, dissolved in DMSO (5 mL), were added into
a cuvette containing 845 mL of the corresponding assay
bu€er and 100 mL of the corresponding chromogenic
substrate. After thermal equilibration, the reaction was
initiated by addition of 50mL of enzyme solution. Progress
curves were monitored at 405 nm over 10±70 min. The
kinetics were analyzed as described.11 The following
substrates were used: Suc-Ala-Ala-Pro-Phe-pNA for chy-
mase (®nal concentration 200mM=0.92ÂKm), cathepsin
G (®nal concentration 500 mMꢂKm), and chymotrypsin
(®nal concentration 200 mM=3.46ÂKm), and MeOSuc-
Ala-Ala-Pro-Val-pNA for HLE (®nal concentration
200 mM=3.8ÂKm). Stock solutions of the substrates were
prepared in DMSO and diluted with the corresponding
assay bu€er. Assay bu€ers were as follows: 0.1 M Tris±
HCl, 1.8 M NaCl, pH 8.0 for chymase, 0.1 M Tris±HCl,
0.5 M NaCl, pH 8.0 for cathepsin G and chymotrypsin,
50 mM sodium phosphate, 500 mM NaCl, pH 7.8 for
HLE. Enzyme stock solutions of chymase (approxi-
mately 800 nM in 0.1 M Tris±HCl, 1.8 M NaCl, pH
2-(N-Benzyl-N-methylamino)-6-methyl-4H-3,1-benzoxazin-
4-one (33). Compound 33 was prepared from 18 in 84%
ꢁ
1
yield: mp 96±97 C, IR (KBr, cmÀ1) 1766 (CˆO); H
NMR (CDCl3) d 2.38 (s, 1H, 6-CH3), 3.11 (s, 3H,
NCH3), 4.77 (s, 2H, CH2), 7.19 (d, J=8.3 Hz, 1H, H-8),
7.25±7.39 (m, 5 ArH), 7.44 (dd, J=8.3, 1.8 Hz, H-7),
7.83 (s, br, 1H, H-5); MS (EI) m/z (rel intensity) 280
(M+, 100). Anal. cald for C17H16N2O2: C, 72.84; H,
5.75; N, 9.99. Found: C, 73.10; H, 6.13; N, 9.78.
2-[N-Methyl-N-(2-phenylethyl)amino]-4H-3,1-benzoxazin-
4-one (34). Compoundꢁ 34 was synthesized from 19 in
94% yield: mp 68.5±69 C; IR (KBr, cmÀ1) 1764 (CˆO);
1H NMR (CDCl3) d 2.96 (t, J=7.5 Hz, 2H, CH2), 3.07
(s, 3H, CH3), 3.77 (t, J=7.5 Hz, 2H, NCH2), 7.07±7.17
(m, 1H, H-6), 7.17±7.36 (m, 6 ArH), 7.56±7.65 (m, 1H,
H-7), 8.00 (dd, J=7.9, 1.7 Hz, 1H, H-5); MS (EI) m/z

954
U. Neumann et al. / Bioorg. Med. Chem. 9 (2001) 947±954
8.0), cathepsin G (500mU/mL in 50mM sodium acetate
bu€er, 150 mM NaCl, pH 5.5), HLE (1.17 mM in 100 mM
sodium acetate bu€er, pH 5.5), and chymotrypsin
(100 mg/mL in 1 mM HCl) were freshly diluted with the
corresponding assay bu€er. Assays were typically per-
formed with ®nal enzyme concentrations of chymase
(approximately 2nM), cathepsin G (2.5 mU/mL), HLE
(2.9 nM), and chymotrypsin (25 ng/mL).
5. (a) Eda, M.; Ashimori, A.; Akahoshi, F.; Yoshimura, T.;
Inoue, Y.; Fukaya, C.; Nakajima, M.; Fukuyama, H.; Imada,
T.; Nakamura, N. Bioorg. Med. Chem. Lett. 1998, 8, 913. (b)
Eda, M.; Ashimori, A.; Akahoshi, F.; Yoshimura, T.; Inoue,
Y.; Fukaya, C.; Nakajima, M.; Fukuyama, H.; Imada, T.;
Nakamura, N. Bioorg. Med. Chem. Lett. 1998, 8, 919.
6. Groutas, W. C.; Schechter, N. M.; He, S.; Yu, H.; Huang,
P.; Tu, J. Bioorg. Med. Chem. Lett. 1999, 9, 2199.
7. Mitsuhashi, H.; Nonaka, T.; Hamamura, I.; Kishimoto, T.;
Muratani, E.; Fujii, K. Br. J. Pharmacol. 1999, 126, 1147.
8. Krantz, A.; Spencer, R. W.; Tam, T. F.; Liak, T. J.; Copp, L. J.;
Thomas, E. M.; Ra€erty, S. P. J. Med. Chem. 1990, 33, 464.
9. Gutschow, M.; Neumann, U. Bioorg. Med. Chem. 1997, 5,
1935.
10. Hays, S. J.; Caprathe, B. W.; Gilmore, J. L.; Amin, N.;
Emmerling, M. R.; Michael, W.; Nadimpalli, R.; Nath, R.;
Raser, K. J.; Sta€ord, D.; Watson, D.; Wang, K.; Jaen, J. C.
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11. Gutschow, M.; Neumann, U. J. Med. Chem. 1998, 41, 1729.
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Chymase catalyzed conversion of angiotensin I (77 mM)
was analyzed by HPLC using conditions described under
General Methods. Retention times were as follows:
angiotensin I, 13.5 min; angiotensin II, 11.3 min. The
reaction was performed at 25 ꢁC in 0.1 M Tris±HCl, 1.8 M
NaCl, pH 8.0 with a ®nal enzyme concentration of approx-
imately 2nM. A Km value of 40 mM1b was considered to
estimate Ki for the inhibition by compound 33.
Acknowledgements
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The authors wish to thank Rene Hersperger, Novartis
Pharma, for critically reading the manuscript. M. G.
would like to thank the `Fonds der Chemischen Industrie'
for ®nancial support.
17. Rehault, S.; Brillard-Bourdet, M.; Juliano, M. A.; Juliano,
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