Esters and amides of 6-(chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylic acid as inhibitors of α-chymotrypsin: Significance of the "aromatic" nature of the novel ester-type coumarin for strong inhibitory activity

Article abstract of DOI:10.1021/jm960090b

A series of esters and amides of 6-(chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylic acid were synthesized and evaluated in vitro for their inhibitory activity toward bovine α-chymotrypsin and human leukocyte elastase. Both series behaved as time-dependen

Full text of DOI:10.1021/jm960090b

J . Med. Chem. 1996, 39, 2579-2585
2579
Ester s a n d Am id es of 6-(Ch lor om eth yl)-2-oxo-2H-1-ben zop yr a n -3-ca r boxylic Acid
a s In h ibitor s of r-Ch ym otr yp sin : Sign ifica n ce of th e “Ar om a tic” Na tu r e of th e
Novel Ester -Typ e Cou m a r in for Str on g In h ibitor y Activity
Lionel Pochet,† Caroline Doucet,‡ Marc Schynts,† Nicole Thierry,‡ Nicole Boggetto,‡ Bernard Pirotte,*,†
Kai Y. J iang,‡ Bernard Masereel,† Pascal de Tullio,† J acques Delarge,† and Miche`le Reboud-Ravaux‡
Laboratoire de Chimie Pharmaceutique, Universite´ de Lie`ge, 3, rue Fusch, B-4000 Lie`ge, Belgium, and Laboratoire
d’Enzymologie Mole´culaire et Fonctionnelle, De´partement de Biologie Supramole´culaire et Cellulaire, Institut J acques Monod,
Universite´ de Paris VII, tour 43, 2, place J ussieu, F-75251 Paris Cedex 05, France
Received J anuary 31, 1996X
A series of esters and amides of 6-(chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylic acid were
synthesized and evaluated in vitro for their inhibitory activity toward bovine R-chymotrypsin
and human leukocyte elastase. Both series behaved as time-dependent inhibitors of R-chy-
motrypsin, but ester-type coumarins were clearly more efficient than the corresponding amides
in inactivating the serine proteinase. The best inactivations were observed with “aromatic”
esters, in particular with meta-substituted phenyl esters such as m-chlorophenyl 6-(chloro-
methyl)-2-oxo-2H-1-benzopyran-3-carboxylate, which appears to be one of the most powerful
inactivators of R-chymotrypsin yet reported (kinact/KI ) 760 000 M-1 s-1 at pH 7.5 and 25 °C).
Usually, the coumarin derivatives failed to inhibit significantly human leukocyte elastase. As
a result, the reported series of aromatic coumarinic esters behaves as a new chemical family
of selective R-chymotrypsin inhibitors.
In tr od u ction
A variety of chemical structures have been described
as suicide substrates. They are usually activated by an
esterolytic reaction, as for halo enol and ynenol
lactones,7-9 isocoumarins,10,11 and 3,4-dihydro-6-halo-
methylcoumarins,12,13 or by an amidolytic reaction, as
for functionalized cyclopeptides14,15 and â-lactams.16-19
Halomethylated 3,4-dihydrocoumarins (Figure 1, 1)
were the first suicide substrates described for serine
proteinases. They act as efficient inactivators of serine
proteinases lacking selectivity since bovine R-CT,12,20
porcine pancreatic elastase,21 HLE,22 human uroki-
nase,23 human plasmin, t-PA, and thrombin13 are
inhibited. The enzymatic hydrolysis of the ester bond
of the lactone ring leads to the unmasking of a p-
hydroxybenzyl halide. After elimination of the halogen
ion, a very reactive quinone methide is formed24 which
alkylates the active site histidine.20,23 In order to
develop a new class of inactivators of serine proteinases
displaying simpler synthetic pathways, a higher ef-
ficiency and hopefully better selectivity, we prepared a
series of esters (Figure 1, 3) and amides (Figure 1, 4) of
6-(chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylic acid
and evaluated their potency as inactivators of R-CT and
HLE. These novel coumarins differ from 6-(bromo-
methyl)coumarin (Figure 1, 2) previously described as
a very poor inactivator of R-CT (kobs/[I] ) 12.5 M-1 s-1
at 20 °C12) by the presence of a strong electron-
withdrawing group at the 3-position, expected to in-
crease the electrophilicity of the lactonic carbonyl group,
thus facilitating the nucleophilic attack by the serine
hydroxyl residue. Moreover, modification of the nature
of R is assumed to facilitate discrimination between
different classes of serine proteinases.
Serine proteinases have been the focus of extensive
study in terms of their vital roles in biological processes,
their involvement in numerous diseases, and the de-
velopment of suitable therapeutic inhibitors.1,2 Among
them, chymotrypsin-like proteinases are implicated in
a wide variety of pathological states including inflam-
mation for cathepsin G, allergic responses, and psoriasis
for mast cell chymases.1 The multicatalytic proteinase
complex (proteasome) responsible for much of the pro-
teolysis within cells possesses a chymotrypsin-like
activity besides a trypsin-like and a peptidylglutamyl-
peptide hydrolase activity.3 Bovine R-chymotrypsin (R-
CT; EC 3.4.21.1) constitutes a model for the evaluation
of new synthetic inhibitors directed against chymo-
trypsin-like proteinases and for the comprehensive
approach of the biological role of the targeted protease.
Human leukocyte elastase (HLE; EC 3.4.21.37) has been
implicated in the pathogenesis of numerous disease
states such as pulmonary emphysema and rheumatoid
arthritis.4
Various types of synthetic inhibitors of chymotrypsin-
and elastase-like proteinases were reported including
substrate and transition-state analogs, affinity labels,
and mechanism-based inhibitors.1,2,4,5 The inactivation
by mechanism-based inhibitors (also called suicide
substrates) is assumed to result from the unmasking
of a reactive latent group after catalytic action by the
enzyme, which then reacts with an active site residue
leading to enzyme inactivation.6 These inhibitors are
expected to display a maximum selectivity in vitro and
in vivo since their inhibitory activity requires discrimi-
nation in the binding steps, the catalytic activation by
the enzyme, and the irreversible modification of the
active center.
In order to evaluate the importance of a latent
alkylating moiety in the 6-position, the 6-methyl-
substituted coumarinic ester (Scheme 2, 14) was pre-
pared.
* Author to whom correspondence should be addressed.
† Universite´ de Lie`ge.
‡ Universite´ de Paris VII.
X Abstract published in Advance ACS Abstracts, May 1, 1996.
S0022-2623(96)00090-8 CCC: $12.00 © 1996 American Chemical Society

2580 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Pochet et al.
Sch em e 2a
F igu r e 1. Chemical structures of 6-(halomethyl)dihydrocou-
marins (1), 6-(bromomethyl)coumarin (2), and newly designed
6-(halomethyl)coumarin esters (3) and amides (4).
a
Reagents: (i) SOCl2; (ii) C6H5OH/pyridine/dioxane.
Sch em e 1a
F igu r e 2. Inactivation of R-chymotrypsin by 3f at pH 7.5 and
25 °C. The enzyme (12.5 nM) was incubated in 0.025 M
sodium phosphate buffer (0.05 M KCl, 10% v/v DMSO) with
the chromogenic substrate Suc-A2-P-F-pNA (40 µM) in the
presence of different concentrations of coumarin 3f ranging
from 75 to 600 nM. The changing slopes ∆A/∆t (or velocities
v) of the progress curves for the hydrolysis of the chromogenic
substrate were obtained from computer-assisted spectropho-
tometer (A was the absorbance at 405 nm). The kinetic
parameters KI and kinact were determined by fitting the
experimental data to the equation π ) kinact[I](1 - R)/[KI +
[I](1 - R)] where -π is the slope of the linear plot of ln v versus
time at a given inhibitor concentration, R the ratio [S]/[KM
+
[S]], [S] the substrate concentration, [I] the inhibitor concen-
tration, and KM the Michaelis constant for the substrate.
-1/KI and 1/kinact are also the x- and y-intercept, respectively,
of the plot of 1/π versus 1/[I](1 - R).
a
Reagents: (i) HCHO/HCl; (ii) diethyl malonate/piperidine/
EtOH/HOAc; (iii) SOCl2; (iv) HCl/H2O/EtOH; (v) SOCl2; (vi) H2O;
(vii) ROH/pyridine/dioxane; (viii) RNH2/pyridine/dioxane.
Ch em istr y
Resu lts a n d Discu ssion
5-(Hydroxymethyl)salicyl aldehyde (6) obtained from
salicyl aldehyde (5) following the Stoerner and Behn
process25 was engaged in a Knoevenagel-type reaction
with diethyl malonate and piperidine as described for
the preparation of ethyl 2-oxo-2H-1-benzopyran-3-car-
boxylate26 to give the corresponding 6-(hydroxymethyl)-
coumarin homologue 7 (Scheme 1). Treatment with
thionyl chloride provided the 6-chloromethyl ethyl ester
derivative 8. Hydrolysis of the ester function of 7 in
acidic hydroalcoholic solution led to the corresponding
carboxylic acid 9, which was converted with thionyl
chloride to the 6-chloromethyl acid chloride 10, the
starting material for the preparation of the coumarinic
esters 3 and amides 4. The 6-chloromethyl carboxylic
acid 11 was obtained by treatment of the acid chloride
10 with water.
R-CT and HLE were exposed to the novel coumarins
using either the preincubation method, in which the
enzyme and the inhibitor were incubated before the
determination of remaining activity at intervals of time,
or a method in which the reagent competed with the
chromogenic substrate (Figure 2). Time-dependent
inhibitions of R-CT were observed for all compounds
except for 4a -c and 11 (Tables 1 and 2). After 24 h of
incubation at 4 °C, the enzyme inactivated by 3f
regained less than 2% of its initial activity. In the same
conditions, the spontaneous loss of activity of control
samples was less than 5%. No noticeable reactivation
resulted from the treatment of the inactivated enzyme
by 0.66 M hydroxylamine (pH 7.5; 30 min; 25 °C). This
excluded inactivation simply due to the formation of a
stable acyl enzyme. Increasing amounts of the chro-
mogenic substrate (Suc-A2-P-F-pNA) at a fixed coumarin
3f concentration protected R-CT against inactivation,
suggesting that a chemical modification occurred in the
active site.
Starting from 6-methyl-2-oxo-2H-1-benzopyran-3-car-
boxylic acid (Scheme 2, 12)27 treatment with thionyl
chloride led to the corresponding acid chloride 13, which
in turn was converted to the phenyl ester 14.

Inhibitors of R-Chymotrypsin
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2581
Ta ble 1. Kinetic Parameters for the Inactivation of R-CT (pH
7.5 and 25 °C) and HLE (pH 8.0 and 25 °C) by Esters and
Amides of 6-(Chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylic
Acida
inactivation was detected with 11 (charged carboxylate).
Conversely, small (3a , 8, 3b) or large (3f-r ) hydrophobic
groups favored the inactivation. However, a too long
aliphatic chain (3c and 3d versus 3b or 8) or a benzylic
chain (3g versus 3f) led to a decrease of kinact/KI. For
3f versus 3b, kinact was increased by a factor of ∼3
whereas KI was lowered by a factor of ∼50, suggesting
that for these compounds the inactivation potency
appeared to be rather governed by the recognition step
(KI) than by the inactivation step (kinact).
4
3, 8, 11, 14
R-CT
HLE
A peptide mimic at the 3-position in an aliphatic (4i)
or an aromatic (4j) amide did not enhance the inhibitory
activity (Table 1).
compd
no.
kinact
(s-1
kinact/KI
k
obs/[I]
R
X
)
KI (M)
(M-1 s-1
)
(M-1 s-1
)
11
3a
8
3b
3c
3d
3e
3f
3g
H
CH3
CH2CH3
CH(CH3)2
CH2(CH2)3CH3 Cl
CH2(CH2)6CH3 Cl
CH2CHdCH2
C6H5
Cl
Cl
nib
ni
ni
ni
ni
ni
ni
ni
23
ni
110c
The influence of the nature and the position of
substituents on the aromatic ring is reported in Table
2. Interestingly, ortho and meta substitutions were
always found to be more favorable than para ones.
Among those, the meta-substituted derivative 3l (m-
chlorophenyl 6-(chloromethyl)-2H-1-benzopyran-3-car-
boxylate) appeared to be the most powerful R-CT
inactivator of the series (kinact/KI ) 762 700 M-1 s-1).
Phenyl 6-methyl-2-oxo-2H-1-benzopyran-3-carboxy-
late (14), which corresponded to the nonhalogenated
analog of 3f and therefore was devoid of latent alkylat-
ing function, was found to be essentially inactive on
R-CT. These results indicated that the halomethyl
moiety in the 6-position was required to lead to an
irreversible inhibition probably through the formation
of a covalent bond between the benzylic carbon atom
and a nucleophilic residue located within the enzyme
recognition site, i.e. a histidine residue. Nevertheless,
an alkylation of the Met-192 residue, which caps the
primary specificity pocket, could not be excluded.
When human leukocyte elastase was used as the
target serine proteinase, no significant inhibition of the
serine enzyme was observed except with 3l and 3q
(Table 2). Interestingly, compound 14 also inhibited
HLE. As for 3l and 3q, this inactivation was transient
and the enzyme slowly recovered its activity, suggesting
the formation of a stable acyl enzyme for the three
compounds. The reported values of kobs/[I] in Tables 1
and 2 correspond to the acylation step.
Cl 0.013 5.9 × 10-5 220
Cl 0.011 1.6 × 10-5 687
12c
190c
Cl 0.014 3.3 × 10-5 424
Cl 0.034 3.4 × 10-7 100 000
CH2C6H5
Cl
H
186c
14
C6H5
ni
330
4a
4b
4c
4d
4e
4f
4g
4h
4i
CH3
CH(CH3)2
CH2(CH2)3CH3
CH2(CH2)8CH3
CH2CHdCH2
C6H5
ni
ni
ni
ni
ni
ni
ni
ni
ni
58
ni
ni
ni
36c
80c
32c
24c
6c
CH2C6H5
CH2CH2C6H5
CH2CO2CH3
CH(CH2C6H5)
CO2CH3
12c
80c
4j
a
b
Standard errors are less than 15%. No inhibition. c Obtained
as kobs/[I] at low inhibitor concentration.
Ta ble 2. Kinetic Parameters for the Inactivation of R-CT (pH
7.5 and 25 °C) and HLE (pH 8.0 and 25 °C) by “Phenolic”
Esters of 6-(Chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylic
Acida
R-CT
HLE
compd no.
R
kinact/KI (M-1 s-1
)
kobs/[I] (M-1 s-1
)
The present results led to the identification of aryl
6-(chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylate
esters as a new generic class of R-CT inactivators.
Structure-activity relationships strongly suggest that
the position of the substituent on the phenyl ring
dramatically influenced the inhibitory potency on the
serine proteinase since the meta or the ortho position
appear to be the most favorable. This core coumarinic
structure may be of interest in the design of inactivators
targeting serine proteinases of other specificities by
modifying the nature of the substituent at the 3-posi-
tion.
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
o-CH3
114 000b
71 750
685d
nic
ni
ni
23
630
ni
85
85
ni
m-CH3
p-CH3
o-Cl
m-Cl
p-Cl
o-I
m-I
p-I
m-NO2
48 100b
762 700
330d
80 000b
72 120
1 775d
11 300d
500
3r
m-CH3-p-Cl
7 550d
90
Standard errors are less than 15%. 3h : kinact ) 0.043 s-1
;
a
b
KI ) 3.8 × 10-7 M. 3k : kinact ) 0.038 s-1; KI ) 7.9 × 10-7 M. 3n :
kinact ) 0.020 s-1; KI ) 2.5 × 10-7 M. c No inhibition. Obtained
d
as kobs/[I] at low inhibitor concentration.
Exp er im en ta l Section
Noticeable differences in the inactivation potency
towards R-CT were obtained (Tables 1 and 2). The
potency dramatically depended upon the nature of the
substituent at the 3-position. Better inactivations were
observed with the esters than with the corresponding
amides (table 1). In both series, the presence of an
aromatic group enhanced the inhibition potency when
the aryl side chain was attached directly to the oxy and
aminocarbonyl moiety. As expected from the known
primary specificity of R-CT (hydrophobic pocket), no
Ch em istr y. Melting points were determined with a Bu¨chi-
Tottoli apparatus in open capillary tubes and are uncorrected.
Analysis (C, H, N, S) were within (0.4% of the theoretical
values. The IR spectra were recorded in KBr on a Perkin-
Elmer 1750 spectrophotometer. The 1H-NMR spectra were
recorded on a Brucker AW 80 (80 MHz) in CDCl3 (or DMSO-
d6) with TMS (or HMDS) as an internal standard; chemical
shifts are reported in δ values (ppm) relative to internal TMS
(or HMDS).
E t h yl 6-(H yd r oxym et h yl)-2-oxo-2H -1-b en zop yr a n -3-
ca r b oxyla t e H em ih yd r a t e (7). A mixture of (hydroxy-

2582 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Pochet et al.
methyl)salicyl aldehyde25 (30.0 g, 197 mmol), diethyl malonate
(34.8 g, 217 mmol), piperidine (2.5 mL), and acetic acid (1.0
mL) in absolute ethanol (100 mL) was heated under reflux for
17 h. Boiling distilled water (250 mL) was then added with
stirring to the hot mixture, and the resulting suspension was
placed at +4 °C overnight. The crystalline precipitate so
obtained was collected by filtration, washed 2-fold with etha-
nol:water (6:4, v/v, 200 mL), and dried. Recrystallization in
boiling ethanol (4 mL/g) gave 7 (43,1 g, 85%): mp 109-110
°C; IR (KBr) 3548, 3508, 3392, 3239 (O-H), 3057 (C-H arom),
2981, 2914 (C-H aliph), 1746 (CdO ester), 1698 (CdO
lactone), 1625, 1578, 1259 cm-1; 1H NMR (CDCl3, TMS) δ 1.35
(t, 3H, OCH2CH3), 2.20 (s, 2H, CH2OH, HOH), 4.35 (q, 2H,
OCH2CH3), 4.75 (s, 2H, CH2OH), 7.25 (d, 1H, 8-H), 7.60 (s,
1H, 5-H), 7.65 (d, 1H, 7-H), 8.45 (s, 1H, 4-H). Anal.
(C13H12O5‚0.5H2O) C, H.
solvent was removed by evaporation under reduced pressure.
The residue was partitioned between CHCl3 (30-60 mL) and
0.1 N HCl (30 mL), and the decanted organic layer was dried
over MgSO4, filtered, and evaporated to dryness under reduced
pressure. Crude 3 or 4 was then recrystallized in the ap-
propriate solvent. Yields were 40-80%.
Met h yl 6-(Ch lor om et h yl)-2-oxo-2H -1-b en zop yr a n -3-
ca r boxyla te (3a ). The title compound was obtained as
described above after the reaction of 10 with methanol and
was recrystallized in ethyl acetate:petroleum ether, 40-60
°C: mp 152-155 °C; IR 3068 (C-H arom), 2947 (C-H aliph),
1758 (CdO ester), 1714 (CdO lactone), 1622, 1576, 1378, 1251
cm-1; 1H NMR (CDCl3, TMS) δ 3.95 (s, 3H, OCH3), 4.65 (s,2H,
CH2Cl), 7.35 (d, 1H, 8-H), 7.60 (s, 1H, 5-H), 7.65 (d, 1H, 7-H),
8.45 (s, 1H, 4-H). Anal. (C12H9O4Cl) C, H.
Isop r op yl 6-(Ch lor om eth yl)-2-oxo-2H-1-ben zop yr a n -3-
ca r boxyla te (3b). The title compound was obtained as
described above after the reaction of 10 with 2-propanol and
was recrystallized in ethyl acetate:petroleum ether, 40-60
°C: mp 185-189 °C; IR 3063 (C-H arom), 2980 (C-H aliph),
Eth yl 6-(Ch lor om eth yl)-2-oxo-2H-1-ben zop yr a n -3-ca r -
boxyla te (8). Ethyl 6-(hydroxymethyl)-2H-1-benzopyran-3-
carboxylate (7) (0.5 g, 1.94 mmol) was heated in thionyl
chloride (10 mL) for 3 h. Most of the excess reagent was
evaporated under reduced pressure, and the residue was
suspended in water (30 mL) and extracted 2-fold with CHCl3
(30 mL). The combined organic layers were dried over MgSO4,
filtered, and evaporated to dryness under reduced pressure.
The residue of crude 8 was recrystallized in CHCl3:petroleum
ether, 40-60 °C (1:3, v/v), to give the title compound (0.42 g,
81%): mp 182-186 °C; IR 3061 (C-H arom), 2978 (C-H
aliph), 1777 (CdO ester), 1757 (CdO lactone), 1622, 1574,
1746 (CdO ester), 1709 (CdO lactone), 1628, 1580, 1254 cm-1
;
1H NMR (CDCl3, TMS) δ 1.30 (d, 6H, CH(CH3)2), 4.60 (s, 2H,
CH2Cl), 5.25 (m, 1H, CH(CH3)2), 7.30 (d, 1H, 8-H), 7.55 (s, 1H,
5-H), 7.65 (d, 1H, 7-H), 8.40 (s, 1H, 4-H). Anal. (C14H13O4Cl)
C, H.
n -P en tyl 6-(Ch lor om eth yl)-2-oxo-2H-1-ben zop yr a n -3-
ca r boxyla te (3c). The title compound was obtained as
described above after the reaction of 10 with 1-pentanol and
was recrystallized in ethyl acetate:petroleum ether, 40-60
°C: mp 120-124 °C; IR 3060 (C-H arom), 2957, 2929, 2859
(C-H aliph), 1751 (CdO ester), 1703 (CdO lactone), 1627,
1380, 1252 cm-1 1H NMR (CDCl3, TMS) δ 1.35 (t, 3H,
;
OCH2CH3), 4.35 (q, 2H, OCH2CH3), 4.65 (s, 2H, CH2Cl), 7.30
(d, 1H, 8-H), 7.60 (s, 1H, 5-H), 7.65 (d, 1H, 7-H), 8.45 (s, 1H,
4-H). Anal. (C13H11O4Cl) C, H.
1
1579, 1253 cm-1; H NMR (CDCl3, TMS) δ 0.95 (t, 3H, CH2-
6-(Hyd r oxym eth yl)-2-oxo-2H-1-ben zop yr a n -3-ca r boxy-
lic Acid (9). Ethyl 6-(hydroxymethyl)-2H-1-benzopyran-3-
carboxylate (7) (20.0 g, 78 mmol) was dissolved in boiling
ethanol (350 mL). Ebullition was maintained during the slow
and progressive addition of 3 N HCl (2000 mL). After heating
for 3 h in an open vessel, the mixture was cooled and the white
precipitate of crude 9 was collected by filtration, washed with
water and recrystallized in ethanol:water (1:5, v/v) (14.5 g,
85%), mp 238-240 °C; IR 3388 (O-H), 1735 (CdO carboxylic
(CH2)3CH3), 1.25-1.85 (m, 6H, CH2(CH3)2CH3), 4.30 (t, 2H,
CH2(CH3)2CH3), 4.65 (s, 2H, CH2Cl), 7.30 (d, 1H, 8-H), 7.60
(s, 1H, 5-H), 7.65 (d, 1H, 7-H), 8.45 (s, 1H, 4-H). Anal.
(C16H17O4Cl) C, H.
n -Oct yl 6-(Ch lor om et h yl)-2-oxo-2H -1-b en zop yr a n -3-
ca r boxyla te (3d ). The title compound was obtained as
described above after the reaction of 10 with 1-octanol and
was recrystallized in ethyl acetate:petroleum ether, 40-60
°C: mp 118-121 °C; IR 3062 (C-H arom), 2955, 2921, 2852
(C-H aliph), 1748 (CdO ester), 1703 (CdO lactone), 1626,
acid), 1703 (CdO lactone), 1620, 1575, 1247 cm-1 1H NMR
;
(CDCl3, DMSO-d6, TMS) δ 4.65 (s, 2H, CH2OH), 7.35 (d, 1H,
8-H), 7.65 (d, 1H, 7-H), 7.70 (s, 1H, 5-H), 8.65 (s, 1H, 4-H).
Anal. (C11H8O5) C, H.
1
1579, 1308, 1252 cm-1; H NMR (CDCl3, TMS) δ 0.90 (t, 3H,
CH2(CH2)6CH3), 1.25-1.95 (m, 12H, CH2(CH2)6CH3), 4.25 (t,
2H, CH2(CH2)6CH3), 4.60 (s, 2H, CH2Cl), 7.30 (d, 1H, 8-H), 7.60
(s, 1H, 5-H), 7.65 (d, 1H, 7-H), 8.50 (s, 1H, 4-H). Anal.
(C19H23O4Cl) C, H.
6-(Ch lor om et h yl)-2-oxo-2H -1-b en zop yr a n -3-ca r b oxy-
lic Acid (11). The suspension of 6-(hydroxymethyl)-2H-1-
benzopyran-3-carboxylic acid (9) (1.0 g, 4.54 mmol) in thionyl
chloride (10 mL) was refluxed for 3 h. The resulting solution
was evaporated to dryness under reduced pressure, and the
residue of crude 10 was dispersed in dry toluene (10 mL). After
elimination of the solvent under reduced pressure, the residue
was suspended in distilled water (10 mL) and supplemented
with pyridine (0.76 mL, 9.40 mmol). After 10 min of stirring
at room temperature, the suspension was ajusted to pH 3 and
the precipitate of crude 11 was collected by filtration, washed
with water, and recrystallized in ethanol (0.89 g, 82%): mp
158-162 °C; IR 3046 (C-H arom), 1741 (CdO carboxylic acid),
1694 (CdO lactone), 1623, 1576 cm-1; 1H NMR (CDCl3, TMS)
δ 4.65 (s, 2H, CH2Cl), 7.50 (d, 1H, 8-H), 7.75 (s, 1H, 5-H), 7.80
(d, 1H, 7-H), 8.90 (s, 1H, 4-H). Anal. (C11H7O4Cl) C, H.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e Ester s
(3) a n d th e Am id es (4) of 6-(Hyd r oxym eth yl)-2-oxo-2H-
1-ben zop yr a n -3-ca r boxylic Acid . The suspension of 6-(hy-
droxymethyl)-(2H-1-benzopyran-3-carboxylic acid (9) (1.0 g,
4.54 mmol) in thionyl chloride (10 mL) was refluxed for 3 h.
The resulting solution was evaporated to dryness under
reduced pressure, and the residue of crude 10 was dispersed
in dry toluene (10 mL). The solvent was eliminated under
reduced pressure. Dispersion in dry toluene and solvent
elimination was repeated twice. The residue was dissolved
in anhydrous dioxane (3 mL) and added with stirring to a
solution of the appropriate alcohol (5.0 mmol) [or amine (5.0
mmol)] and of anhydrous pyridine (5.0 mmol) in anhydrous
dioxane (7 mL). After 30-90 min at room temperature,
depending upon the nature of the alcohol or the amine, the
Allyl 6-(Ch lor om eth yl)-2-oxo-2H-1-ben zop yr a n -3-ca r -
boxyla te (3e). The title compound was obtained as described
above after the reaction of 10 with allyl alcohol and was
recrystallized in ethyl acetate:petroleum ether, 40-60 °C: mp
135-139 °C; IR 3050 (C-H arom), 1746 (CdO ester), 1708
(CdO lactone), 1623, 1575, 1253 cm-1; 1H NMR (CDCl3, TMS)
δ 4.60 (s, 2H, CH2Cl), 4.85 (d, 2H, CH2CHdCH2), 5.30-5.65
(m, 2H, CH2CHdCH2), 6.00 (m, 1H, CH2CHdCH2), 7.30 (d,
1H, 8-H), 7.60 (s, 1H, 5-H), 7.65 (d, 1H, 7-H), 8.50 (s, 1H, 4-H).
Anal. (C14H11O4Cl) C, H.
P h en yl 6-(Ch lor om et h yl)-2-oxo-2H -1-b en zop yr a n -3-
ca r boxyla te (3f). The title compound was obtained as
described above after the reaction of 10 with phenol and was
recrystallized in ethyl acetate:petroleum ether, 40-60 °C: mp
187-191 °C; IR 3068 (C-H arom), 1772 (CdO ester), 1733
(CdO lactone), 1624, 1577, 1245 cm-1; 1H NMR (CDCl3, TMS)
δ 4.65 (s, 2H, CH2Cl), 7.10-7.50 (m, 6H, 8-H, C6H5), 7.60 (s,
1H, 5-H), 7.65 (d, 1H, 7-H), 8.65 (s, 1H, 4-H). Anal. (C17H11O4-
Cl) C, H.
Ben zyl 6-(Ch lor om et h yl)-2-oxo-2H -1-b en zop yr a n -3-
ca r boxyla te (3g). The title compound was obtained as
described above after the reaction of 10 with benzyl alcohol
and was recrystallized in ethyl acetate:petroleum ether, 40-
60 °C: mp 164-166 °C; IR 3057 (C-H arom), 1746 (CdO
1
ester), 1709 (CdO lactone), 1629, 1581, 1252 cm-1; H NMR
(CDCl3, TMS) δ 4.60 (s, 2H, CH2Cl), 5.40 (s, 2H, CH2C6H5),
7.30 (d, 1H, 8-H), 7.35 (s, 5H, CH2C6H5), 7.60 (s, 1H, 5-H), 7.65
(d, 1H, 7-H), 8.45 (s, 1H, 4-H). Anal. (C18H13O4Cl) C, H.

Inhibitors of R-Chymotrypsin
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2583
3082, 3062 (C-H arom), 1773 (CdO ester), 1717 (CdO
o-Tolyl 6-(Ch lor om et h yl)-2-oxo-2H -1-b en zop yr a n -3-
ca r boxyla te (3h ). The title compound was obtained as
described above after the reaction of 10 with o-methylphenol,
and was recrystallized in ethyl acetate:petroleum ether: mp
179-182 °C; IR 3069 (C-H arom), 1732 (CdO ester and
lactone), 1625, 1578, 1489, 1246 cm-1; 1H NMR (CDCl3, TMS)
δ 2.25 (s, 3H, CH3), 4.60 (s, 2H, CH2Cl), 7.00-7.35 (m, 4H,
C6H4), 7.40 (d, 1H, 8-H), 7.70 (s + d, 2H, 5-H, 7-H), 8.65 (s,
1H, 4-H). Anal. (C18H13O4Cl) C, H.
m -Tolyl 6-(Ch lor om et h yl)-2-oxo-2H -1-b en zop yr a n -3-
ca r boxyla te (3i). The title compound was obtained as
described above after the reaction of 10 with m-methylphenol
and was recrystallized in ethyl acetate:petroleum ether: mp
163-167 °C; IR 3065 (C-H arom), 2955 (C-H aliph), 1729
(CdO ester and lactone), 1626, 1578, 1487, 1250 cm-1; 1H NMR
(CDCl3, TMS) δ 2.40 (s, 3H, CH3), 4.60 (s, 2H, CH2Cl), 7.00-
7.30 (m, 4H, C6H4), 7.35 (d, 1H, 8-H), 7.70 (s + d, 2H, 5-H,
7-H), 8.65 (s, 1H, 4-H). Anal. (C18H13O4Cl) C, H.
p -Tolyl 6-(Ch lor om et h yl)-2-oxo-2H -1-b en zop yr a n -3-
ca r boxyla te (3j). The title compound was obtained as
described above after the reaction of 10 with p-methylphenol
and was recrystallized in chloroform:petroleum ether: mp
191-193 °C; IR 3065 (C-H arom), 1776 (CdO ester), 1734
(CdO lactone), 1627, 1579, 1507, 1247 cm-1; 1H NMR (CDCl3,
HMDS) δ 2.30 (s, 3H, CH3), 4.50 (s, 2H, CH2Cl), 6.90-7.30
(m, 4H, C6H4), 7.35 (d, 1H, 8-H), 7.70 (s + d, 2H, 5-H, 7-H),
8.45 (s, 1H, 4-H). Anal. (C18H13O4Cl) C, H.
o-Ch lor op h en yl 6-(Ch lor om eth yl)-2-oxo-2H-1-ben zop y-
r a n -3-ca r boxyla te (3k ). The title compound was obtained
as described above after the reaction of 10 with o-chlorophenol
and was recrystallized in ethyl acetate:petroleum ether: mp
153-156 °C; IR 3063 (C-H arom), 1735 (CdO ester and
lactone), 1623, 1576, 1475, 1244, 1209 cm-1; 1H NMR (CDCl3,
TMS) δ 4.65 (s, 2H, CH2Cl), 7.00-7.60 (m, 5H, C6H4, 8-H),
7.70 (s + d, 2H, 5-H, 7-H), 8.75 (s, 1H, 4-H). Anal. (C17H10O4-
Cl2) C, H.
1
lactone), 1624, 1577, 1478, 1249, 1223, 1199 cm-1; H NMR
(DMSO-d6, HMDS) δ 4.80 (s, 2H, CH2Cl), 6.90-8.20 (m, 7H,
C6H4, 8-H, 5-H, 7-H), 9.00 (s, 1H, 4-H). Anal. (C17H10O4ClI)
C, H.
m -Nitr op h en yl 6-(Ch lor om eth yl)-2-oxo-2H-1-ben zop y-
r a n -3-ca r boxyla te (3q). The title compound was obtained
as described above after the reaction of 10 with m-nitrophenol
and was recrystallized in ethyl acetate: mp 208-210 °C; IR
3113, 3086, 3055 (C-H arom), 1772 (CdO ester), 1723 (CdO
1
lactone), 1620, 1573, 1530, 1241, 1223, 1212 cm-1; H NMR
(CDCl3, HMDS) δ 4.60 (s, 2H, CH2Cl), 7.20-7.80 (m, 5H, 2H
of C6H4, 8-H, 5-H, 7-H), 8.05 (m, 2H, 2H of C6H4), 8.65 (s, 1H,
4-H). Anal. (C17H10NO6Cl) C, H.
4-Ch lor o-3-m eth ylp h en yl 6-(Ch lor om eth yl)-2-oxo-2H-
1-ben zop yr a n -3-ca r boxyla te (3r ). The title compound was
obtained as described above after the reaction of 10 with
4-chloro-3-methylphenol and was recrystallized in ethyl ac-
etate: mp 188-192 °C; IR 3065 (C-H arom), 1774 (CdO
ester), 1757 (CdO lactone), 1620, 1573, 1479, 1245, 1221 cm-1
;
1H NMR (CDCl3, TMS) δ 2.35 (s, 3H, CH3), 4.60 (s, 2H, CH2-
Cl), 6.80-7.20 (m, 3H, C6H3), 7.40 (d, 1H, 8-H), 7.65 (s + d,
2H, 5-H, 7-H), 8.60 (s, 1H, 4-H). Anal. (C18H12O4Cl2) C, H.
N-Meth yl-6-(ch lor om eth yl)-2-oxo-2H-1-ben zop yr a n -3-
ca r boxa m id e Hem ih yd r a te (4a ). The title compound was
obtained as described above after the reaction of 10 with
methylamine and was recrystallized in ethyl acetate: mp 215-
218 °C; IR 3360 (N-H), 3068 (C-H arom), 1702 (CdO lactone),
1
1657 (CdO amide), 1618, 1577, 1536 cm-1; H NMR (CDCl3,
TMS) δ 3.00 (d, 3H, NHCH3), 4.60 (s, 2H, CH2Cl), 7.35 (d, 1H,
8-H), 7.65 (d, 1H, 7-H), 7.70 (s, 1H, 5-H), 8.70 (m, 1H, NHCH3),
8.90 (s, 1H, 4-H). Anal. (C12H10NO3Cl‚0.5H2O) C, H, N.
N-Isop r op yl-6-(ch lor om eth yl)-2-oxo-2H-1-ben zop yr a n -
3-ca r boxa m id e (4b). The title compound was obtained as
described above after the reaction of 10 with isopropylamine
and was recrystallized in ethyl acetate: mp 202-208 °C; IR
3321 (N-H), 2970, 2917, 2849 (C-H aliph), 1731 (CdO
lactone), 1649 (CdO amide), 1618, 1580, 1545 cm-1; 1H NMR
(CDCl3, TMS) δ 1.25 (d, 6H, CH(CH3)2), 4.30 (m, 1H, CH(CH3)2),
4.65 (s, 2H, CH2Cl), 7.30 (d, 1H, 8-H), 7.60-7.75 (m, 2H, 5-H,
7-H), 8.55 (m, 1H, NH), 8.85 (s, 1H, 4-H). Anal. (C14H14NO3-
Cl) C, H, N.
N-P en t yl-6-(ch lor om et h yl)-2-oxo-2H-1-b en zop yr a n -3-
ca r boxa m id e (4c). The title compound was obtained as
described above after the reaction of 10 with n-pentylamine
and was recrystallized in ethyl acetate: mp 167-170 °C; IR
3322 (N-H), 3047 (C-H arom), 2935, 2856 (C-H aliph), 1707
(CdO lactone), 1656 (CdO amide), 1618, 1575, 1540 cm-1; 1H
NMR (CDCl3, TMS) δ 0.95 (t, 3H, CH2(CH2)3CH3), 1.10-1.80
(m, 6H, CH2(CH2)3CH3), 3.45 (m, 2H, CH2(CH2)3CH3), 4.60 (s,
2H, CH2Cl), 7.35 (d, 1H, 8-H), 7.65 (d, 1H, 7-H), 7.75 (s, 1H,
5-H), 8.80 (m, 1H, NH), 8.90 (s, 1H, 4-H). Anal. (C16H18NO3-
Cl) C, H, N.
m-Ch lor oph en yl 6-(Ch lor om eth yl)-2-oxo-2H-1-ben zopy-
r a n -3-ca r boxyla te (3l). The title compound was obtained as
described above after the reaction of 10 with m-chlorophenol
and was recrystallized in ethyl acetate:petroleum ether: mp
176-179 °C; IR 3087 (C-H arom), 1772 (CdO ester), 1727
(CdO lactone), 1621, 1591, 1576, 1472, 1244, 1224, 1206 cm-1
;
1H NMR (CDCl3, HMDS) δ 4.60 (s, 2H, CH2Cl), 7.00-7.40 (m,
5H, C6H4, 8-H), 7.60 (s + d, 2H, 5-H, 7-H), 8.60 (s, 1H, 4-H).
Anal. (C17H10O4Cl2) C, H.
p-Ch lor oph en yl 6-(Ch lor om eth yl)-2-oxo-2H-1-ben zop y-
r a n -3-ca r boxyla te (3m ). The title compound was obtained
as described above after the reaction of 10 with p-chlorophenol
and was recrystallized in chloroform:petroleum ether: mp
223-226 °C; IR 3091, 3067 (C-H arom), 1768 (CdO ester),
1718 (CdO lactone), 1623, 1577, 1485, 1250, 1223, 1200 cm-1
;
1H NMR (CDCl3 + DMSO-d6, HMDS) δ 4.65 (s, 2H, CH2Cl),
7.05-7.50 (m, 5H, C6H4, 8-H), 7.65-7.95 (m, 2H, 5-H, 7-H),
8.90 (s, 1H, 4-H). Anal. (C17H10O4Cl2) C, H.
N-Dod ecyl-6-(ch lor om eth yl)-2-oxo-2H-1-ben zop yr a n -3-
ca r boxa m id e (4d ). The title compound was obtained as
described above after the reaction of 10 with n-dodecylamine
and was recrystallized in ethyl acetate: mp 142-145 °C; IR
3312 (N-H), 3046 (C-H arom), 2919, 2852 (C-H aliph), 1704
(CdO lactone), 1654 (CdO amide), 1618, 1575, 1533 cm-1; 1H
NMR (CDCl3, TMS) δ 0.85 (t, 3H, CH2(CH2)8CH3), 1.20-1.80
(m, 16H, CH2(CH2)8CH3), 3.45 (m, 2H, CH2(CH2)8CH3), 4.60
(s, 2H, CH2Cl), 7.35 (d, 1H, 8-H), 7.65 (d, 1H, 7-H), 7.75 (s,
o-Iod op h en yl 6-(Ch lor om eth yl)-2-oxo-2H-1-ben zop y-
r a n -3-ca r boxyla te (3n ). The title compound was obtained
as described above after the reaction of 10 with o-iodophenol
and was recrystallized in ethyl acetate:petroleum ether: mp
162-164 °C; IR 3060 (C-H arom), 1772 (CdO ester), 1733
(CdO lactone), 1623, 1576, 1465, 1241, 1221, 1199 cm-1; H
1
NMR (CDCl3, TMS) δ 4.60 (s, 2H, CH2Cl), 6.80-7.50 (m, 4H,
3H of C6H4, 8-H), 7.60-7.95 (m, 3H, 1H of C6H4, 5-H, 7-H),
8.85 (s, 1H, 4-H). Anal. (C17H10O4ClI) C, H.
1H, 5-H), 8.75 (m, 1H, NH), 8.85 (s, 1H, 4-H). Anal. (C21H28
NO3Cl) C, H, N.
-
m -Iod op h en yl 6-(Ch lor om eth yl)-2-oxo-2H-1-ben zop y-
r a n -3-ca r boxyla te (3o). The title compound was obtained
as described above after the reaction of 10 with m-iodophenol
and was recrystallized in ethyl acetate:petroleum ether: mp
161-163 °C; IR 3082, 3048 (C-H arom), 1771 (CdO ester),
N-Allyl-6-(ch lor om eth yl)-2-oxo-2H-1-ben zopyr an -3-car -
boxa m id e (4e). The title compound was obtained as de-
scribed above after the reaction of 10 with allylamine and was
recrystallized in ethyl acetate: mp 216-219 °C; IR 3332 (N-
H), 3050 (C-H arom), 1704 (CdO lactone), 1658 (CdO amide),
1618, 1576, 1530 cm-1; 1H NMR (CDCl3, TMS) δ 4.05 (dd, 2H,
CH2CHdCH2), 4.60 (s, 2H, CH2Cl), 5.05-5.55 (m, 2H, CH2-
CHdCH2), 5.95 (m, 1H, CH2CHdCH2), 7.30 (d, 1H, 8-H), 7.65
(d, 1H, 7-H), 7.70 (s, 1H, 5-H), 8.80 (m, 1H, NH), 8.90 (s, 1H,
4-H). Anal. (C14H12NO3Cl) C, H, N.
1720 (CdO lactone), 1619, 1573, 1466, 1246, 1224, 1183 cm-1
;
1H NMR (CDCl3, TMS) δ 4.65 (s, 2H, CH2Cl), 7.05-7.80 (m,
7H, C6H4, 8-H, 5-H, 7-H), 8.65 (s, 1H, 4-H). Anal. (C17H10O4-
ClI) C, H.
p-Iod op h en yl 6-(Ch lor om eth yl)-2-oxo-2H-1-ben zop y-
r a n -3-ca r boxyla te (3p ). The title compound was obtained
as described above after the reaction of 10 with p-iodophenol
and was recrystallized in ethyl acetate: mp 262-263 °C; IR
N-P h en yl-6-(ch lor om eth yl)-2-oxo-2H-1-ben zop yr a n -3-
ca r boxa m id e (4f). The title compound was obtained as

2584 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Pochet et al.
method. In both cases, the inactivation constants kobs, kinact
described above after the reaction of 10 with aniline and was
recrystallized in ethyl acetate: mp 213-216 °C; IR 1704 (CdO
lactone), 1664 (CdO amide), 1615, 1597, 1574, 1557 cm-1; 1H
NMR (CDCl3, TMS) δ 4.65 (s, 2H, CH2Cl), 6.95 (m, 1H, NH),
7.25-7.85 (m, 8H, 5-H, 7-H, 8-H, C6H5), 9.05 (s, 1H, 4-H). Anal.
(C17H12NO3Cl) C, H, N.
N-Ben zyl-6-(ch lor om eth yl)-2-oxo-2H-1-ben zop yr a n -3-
ca r boxa m id e (4g). The title compound was obtained as
described above after the reaction of 10 with benzylamine, and
was recrystallized in ethyl acetate: mp 186-189 °C; IR 3339
(N-H), 3049 (C-H arom), 2917 (C-H aliph), 1707 (CdO
lactone), 1658 (CdO amide), 1621, 1577, 1536 cm-1; 1H NMR
(CDCl3, TMS) δ 4.65 (s, 2H, CH2Cl), 4.70 (s, 2H, CH2C6H5),
7.25-7.50 (m, 6H, 8-H, C6H5), 7.60-7.80 (m, 2H, 5-H, 7-H),
8.90 (s, 1H, 4-H), 9.15 (bs, 1H, NH). Anal. (C18H14NO3Cl) C,
H, N.
N-(P h en yleth yl)-6-(ch lor om eth yl)-2-oxo-2H-1-ben zop y-
r a n -3-ca r boxa m id e (4h ). The title compound was obtained
as described above after the reaction of 10 with phenethy-
lamine and was recrystallized in ethyl acetate: mp 190-193
°C; IR 3341 (N-H), 3047 (C-H arom), 2943 (C-H aliph), 1704
(CdO lactone), 1657 (CdO amide), 1620, 1576, 1543 cm-1; 1H
NMR (CDCl3, TMS) δ 2.90 (t, 2H, CH2CH2C6H5), 3.75 (m, 2H,
CH2CH2C6H5), 4.60 (s, 2H, CH2Cl), 7.20-7.45 (m, 6H, 8-H,
C6H5), 7.60-7.80 (m, 2H, 5-H, 7-H), 8.80 (m, 1H, NH), 8.85 (s,
1H, 4-H). Anal. (C19H16NO3Cl) C, H, N.
N-[(Meth oxyca r bon yl)m eth yl]-6-(ch lor om eth yl)-2-oxo-
2H-1-ben zop yr a n -3-ca r boxa m id e (4i). The title compound
was obtained as described above after the reaction of 10 with
glycine methyl ester and was recrystallized in ethyl acetate:
mp 200-205 °C; IR 3302 (N-H), 3046 (C-H arom), 1741 (CdO
ester), 1708 (CdO lactone), 1656 (CdO amide), 1617, 1575,
1530 cm-1; 1H NMR (CDCl3, HMDS) δ 3.70 (s, 3H, CH3), 4.15
(d, 2H, CH2NH), 4.55 (s, 2H, CH2Cl), 7.25 (d, 1H, 8-H), 7.40
(s, 1H, 5-H), 7.60 (m, 1H, 7-H), 8.80 (s, 1H, 4-H), 9.10 (bt, 1H,
NH). Anal. (C14H12NO5Cl) C, H, N.
,
and KI were obtained by linear or nonlinear regression to the
equations developed in ref 15 (see also legend to Figure 2)
using Kaleidagraph version 2.1.3 from Abelbeck Software. At
low inhibitor concentrations, the ratio kinact/KI was obtained
as kobs/[I]. For the preincubation method, enzyme and cou-
marinic derivative concentrations were [R-CT]0 ) 12.5 nM or
7 nM (3r ), [inhibitor concentrations] ) 0.5-100 µM; [HLE]0
) 20 or 30 nM, [inhibitor concentrations] ) 5-100 µM. For
the progress curve method, enzyme, substrate, and coumarinic
derivative concentrations were [S]0 ) 40 µM, [R-CT]0 ) 12.5
nM, [8] ) 60-170 µM, [3b] ) 10-90 µM, [3e] ) 10-150 µM,
[3f] ) 0.05-0.6 µM, [3h ] ) 0.08-0.5 µM, [3i] ) 0.04-1 µM,
[3k ] ) 0.15-1 µM, [3l] ) 0.01-0.08 µM, [3n ] ) 0.01-0.065
µM, [3o] ) 0.1-0.6 µM.
Hydroxylamine reactivation assays were performed by
treatment of inactivated R-CT solutions with the nucleophile
hydroxylamine (0.66 M) at pH 7.5 and 25 °C during 30 min.
Enzyme activity of aliquots versus a control was monitored.
Ack n ow led gm en t. We thank Dr. I. Vergely for
helpful discussion and Mrs. M.-L. Pirard, P. Neven, and
D. Dewalque for technical assistance. This work was
supported in part by grants from the National Fund for
Scientific Research (F. N. R. S., Belgium) from which
B. Pirotte is a Senior Research Associate.
Refer en ces
(1) Powers, J . C.; Zimmerman, M. Inhibitors of Elastases, Chymases
and Cathepsin G. In Design of Enzyme Inhibitors as Drugs;
Sandler, M., Smith, H. J ., Eds.; Oxford University Press: Oxford,
1989; pp 596-619.
(2) Demuth, H.-U. Recent Developments in Inhibiting Cysteine and
Serine Proteases. J . Enzyme Inhib. 1990, 3, 249-278.
(3) Rivett, A. J . Proteasomes: Multicatalytic Proteinase Complexes.
Biochem. J . 1993, 291, 1-10.
(4) Hlasta, D. J .; Pagani, E. D. Human Leukocyte Elastase Inhibi-
tors. Annu. Rep. Med. Chem. 1994, 29, 195-204.
(5) Bernstein, P. R.; Edwards, P. D.; Williams, J . C. Inhibitors of
Human Leukocyte Elastase. In Progress in Medicinal Chemistry,
Vol. 31; Ellis, G. P., Luscombe, D. K., Eds.; Elsevier: New York,
1994; pp 59-120.
(6) Silverman, R. B. Mechanism-based Enzyme Inactivation: Chem-
istry and Enzymology; CRC Press: Boca Raton, FL, 1988; Vol.1,
pp 95-156.
(7) Daniels, S. B.; Cooney, E.; Sofia, M. J .; Chakravarty, P. K.;
Katzenellenbogen, J . A. Haloenol Lactones: Potent Enzyme-
Activated Irreversible Inhibitors for R-Chymotrypsin. J . Biol.
Chem. 1983, 258, 15046-15053.
(8) Katzenellenbogen, J . A.; Rai, R.; Dai, W. Enol Lactone Deriva-
tives as Inhibitors of Human Neutrophil Elastase and Trypsin-
like Proteases. Bioorg. Med. Chem. Lett. 1992, 2, 1399-1404.
(9) Tam, T. F.; Spencer, R. W.; Thomas, E. M.; Copp, L. J .; Krantz,
A. Novel Suicide Inhibitors of Serine Proteinases. Inactivation
of Human Leukocyte Elastase by Ynenol Lactones. J . Am. Chem.
Soc. 1984, 106, 6849-6851.
N-[1-Ben zyl-1-(m et h oxyca r b on yl)m et h yl]-6-(ch lor o-
m eth yl)-2-oxo-2H-1-ben zop yr a n -3-ca r boxa m id e (4j). The
title compound was obtained as described above after the
reaction of 10 with phenylalanine methyl ester and was
recrystallized in ethyl acetate: mp 122-126 °C; IR 3327, 3290
(N-H), 3030 (C-H arom), 2952 (C-H aliph), 1742 (CdO
ester), 1731 (CdO lactone), 1652 (CdO amide), 1618, 1577,
1528 cm-1; 1H NMR (CDCl3, HMDS) δ 3.15 (d, 2H, CH2C6H5),
3.65 (s, 3H, CH3), 4.55 (s, 2H, CH2Cl), 4.90 (q, 1H, CHNH),
7.20 (s, 5H, C6H5), 7.30 (d, 1H, 8-H), 7.55 (s, 1H, 5-H), 7.60 (d,
1H, 7-H), 8.70 (s, 1H, 4-H), 9.10 (bd, 1H, NH). Anal. (C21H18
NO5Cl) C, H, N.
-
P h en yl 6-Met h yl-2-oxo-2H -1-b en zop yr a n -3-ca r b oxy-
la te (14). 6-Methyl-2-oxo-2H-1-benzopyran-3-carboxylic acid
(12)27 was converted into the corresponding acid chloride 13
and then into the title ester 14 following the general procedure
described for the preparation of the esters 3 of 6-(hydroxym-
ethyl)-2-oxo-2H-1-benzopyran-3-carboxylic acid. The title com-
pound 14 was recrystallized in ethyl acetate:petroleum ether,
40-60 °C: mp 178-183 °C; IR 3066 (C-H arom), 1734 (CdO
ester and lactone), 1627, 1577, 1491, 1243, 1195 cm-1; 1H NMR
(CDCl3, HMDS) δ 2.35 (s, 3H, CH3), 7.05-7.60 (m, 8H, C6H5,
8-H, 5-H, 7-H), 8.55 (s, 1H, 4-H). Anal. (C17H12O4) C, H.
En zym a tic Stu d ies. Bovine R-CT and HLE were pur-
chased from Sigma and Elastase Products Co., respectively.
Enzyme concentrations were determined by active-site titra-
tions as described in ref 19. The enzymes were assayed
spectrophotometrically with the appropriate p-nitroanilide
substrate (Sigma): succinylalanylalanylprolylphenylalanyl p-
nitroanilide for R-CT and methoxysuccinylalanylalanyl-
prolylvalyl p-nitroanilide for HLE. The enzymatic reactions
were followed in 0.025 M sodium phosphate, 0.05 M KCl, pH
7.5, 10% (v/v) DMSO for R-CT and in 0.1 M Hepes, 0.5 M NaCl,
0.01% (v/v) Tween 80, pH 8.0, 10% (v/v) DMSO for HLE.
Assays were run at 25 °C in a Perkin-Elmer Lambda 5
spectrophotometer equipped with a thermostated cell holder.
Enzyme inhibitions were analyzed either by the preincu-
bation method or by the progress curve method as described
in ref 15. The progress curve method was used when the
inhibition was too fast to be observed by the preincubation
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