Dual inhibition of human leukocyte elastase and lipid peroxidation: In vitro and in vivo activities of azabicyclo[2.2.2]octane and perhydroindole derivatives

Article abstract of DOI:10.1021/jm960772z

A series of potent and selective human leukocyte elastase (HLE) inhibitors of the Val-Pro-Val type has been developed. Initially, the central proline residue was replaced by nonnatural amino acids Phi ((2S,3aS,7aS)- perhydroindole-2-carboxylic acid) and A

Full text of DOI:10.1021/jm960772z

1906
J . Med. Chem. 1997, 40, 1906-1918
Du a l In h ibition of Hu m a n Leu k ocyte Ela sta se a n d Lip id P er oxid a tion : In Vitr o
a n d in Vivo Activities of Aza bicyclo[2.2.2]octa n e a n d P er h yd r oin d ole
Der iva tives
Bernard Portevin,† Michel Lonchampt,‡ Emmanuel Canet,‡ and Guillaume De Nanteuil*,†
Division D of Medicinal Chemistry and Division of Respiratory Pharmacology, Institut de Recherche Servier,
11 rue des Moulineaux, 92150 Suresnes, France
Received November 8, 1996X
A series of potent and selective human leukocyte elastase (HLE) inhibitors of the Val-Pro-Val
type has been developed. Initially, the central proline residue was replaced by nonnatural
amino acids Phi ((2S,3aS,7aS)-perhydroindole-2-carboxylic acid) and Abo ((3S)-2-azabicyclo-
[2.2.2]octane-3-carboxylic acid), and secondly several groups able to confer antioxidant properties
to the molecule were introduced at the lipophilic N-terminal side chain. When compared to
reference inhibitors, in vitro HLE inhibitory potency was maintained (10-100 nM) both with
compounds containing the antioxidant moiety at the end of the N-terminal side chain and with
compounds in which the N-terminal valine of the tripeptidic sequence had been replaced by a
ꢀ-substituted lysine. The lipidic peroxidation inhibitory potency of this series of inhibitors was
found to be similar to that of the reference antioxidant compounds (around 1 µM). Moreover,
HLE-induced hemorrhage in the hamster lung was effectively prevented (40-60% at 15 µg/
kg) by most of the inhibitors tested when administered intratracheally 3 h before instillation
of elastase. Among the most active analogs, compounds 11a ,c,g were still active when
administered 18 h before elastase. Interestingly, compound 14a was able to prevent HLE-
mediated lung damage when administered 72 h prior to enzymatic challenge, indicating
exceptional stability and retention in the lung. Finally, in a 14-day chronic model of emphysema
in the hamster, 14a significantly conserved alveolar spaces, a marker of lung tissue destruction,
and was more potent than reference inhibitor ICI 200 880. This indicates that addition of
peroxidation inhibitory properties to an HLE inhibitor can provide a powerful in vivo inhibitor
of pulmonary tissue destruction.
In tr od u ction
reported to inactivate R1-PI and ceruloplasmine fer-
roxidase, an important oxygen radical scavenger.
When we started our investigations in the early
1990s, the tripeptidic trifluoromethyl ketone ICI 200
880 (or ZD 200 880) (1) was already described as a
potent and selective HLE inhibitor.6 Moreover, this
compound presented a strong in vivo activity in several
animal models of pulmonary lesions.7
Human leukocyte elastase (HLE, EC 3.4.21.37) has
been considered for over 2 decades as one of the most
interesting biological targets in the research field of
proteolytic destruction of lung tissue.1 Subsequent to
its secretion by polymorphonuclear neutrophils, one of
the main actions of HLE is the degradation of structural
proteins, including elastin, fibronectin, and collagen.
Elastin is a highly cross-linked and flexible protein,
mainly present in the macroscopic structure of pulmo-
nary parenchyma. Under physiological conditions, the
lung is protected from elastolytic activity by endogenous
inhibitors such as R1-protease inhibitor (R1-PI); how-
ever, in the course of pathologies such as emphysema,
cystic fibrosis, or adult respiratory distress syndrome
(ARDS), the balance between the natural inhibitor and
HLE is displaced in favor of the enzyme2,3 (Scheme 1).
Moreover, oxygen radical species (ORS) have been
shown to be implicated in the development of emphy-
sema and ARDS in two ways:4 firstly, through a direct
degradative action of cell structure and, secondly,
through an indirect oxidation, by inactivation of anti-
proteases such R1-PI. In fact, ORS is contributing to
lung destruction by increasing HLE degradative activ-
ity, suggesting that a synergestic action could exist
between HLE and ORS. Furthermore, it is known that
cigarette smoke is a source of active ORS:5 it was
Since then, several other pharmaceutical companies
involved in research in the respiratory field have
patented similar structures to the ICI compound,8,9 all
of them being derived from the tripeptidic sequence Val-
Pro-Val. In our ongoing search for new and therapeuti-
cally useful low molecular weight inhibitors of serine
proteinases, we have recently described the replacement
of a proline residue by one of our original nonnatural
amino acids in the generic structure of a known inhibi-
tory peptidic sequence.10,11 ICI 200 880 appeared at this
time to be a suitable reference molecule in order to
validate our structural hypothesis. Moreover, we were
interested by introducing in the same molecule an anti-
HLE activity and an antioxidant activity. With this
aim, our chemical approach consisted of coupling modi-
fied Val-Pro-Val sequences with different groups able
to confer an antioxidant property to the respective
molecules.
† Division D of Medicinal Chemistry.
‡ Division of Respiratory Pharmacology.
X Abstract published in Advance ACS Abstracts, May 15, 1997.
S0022-2623(96)00772-8 CCC: $14.00 © 1997 American Chemical Society

Dual Inhibition of HLE and Lipid Peroxidation
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1907
Sch em e 1
I
Sch em e 2a
methodology. In contrast to previous papers where
oxidation of the alcohol was performed at the very last
step of the synthesis of the inhibitors, we were able to
prepare the dipeptidic trifluoromethyl ketone iv in
satisfactory yields using Moffat’s reagent (Ac2O-
DMSO). This reagent proved to be superior to other
oxidants such as the Swern reagent or Dess-Martin
periodinane. Deprotection of the N-terminal amino
group of iv to give the dipeptide v was effected by
treatment with dioxane, previously saturated with dry
hydrogen chloride gas. A second coupling reaction with
Boc-valine in the presence of DCC and HOBT in DMF
followed, and the free tripeptidyl trifluoromethyl ketone
vii was effectively isolated after treatment with dioxane
previously saturated with hydrogen chloride gas. This
ketone was obtained as a mixture of diastereomers.
Isomer separation was not performed, because of a noted
rapid epimerization observed in vitro as well as in vivo,
most probably due to the propensity of the trifluoro-
methyl ketone to enolize and exist in a hydrated
form.18,19
The suitable antioxidant functionalities were intro-
duced through the use of different carboxylic acids 6,
which were either commercially available or prepared
by standard methods according to Scheme 3. One of
these acids (6d ) necessitated the BOP, DIEA peptidic
coupling methodology to react with tripeptide vii-b
yielding inhibitor 7 (Scheme 4). However, the majority
of the carboxylic acids 6 were reacted with sulfonamides
8a ,b via standard peptidic coupling conditions (DCC,
DMAP) in DMF, to give esters of general formula 9 as
illustrated in Scheme 4.
a
a : AA ) Phi. b: AA ) Abo. Reagents: (a) isobutyl chlorofor-
mate, NMM; (b) Ac2O, DMSO; (c) gaseous HCl, dioxane; (d) Boc-
Val-OH, HOBT, DCC, DMF.
Few compounds appear in the literature with this
dual inhibitory capacity. CT-1037 (2) is described as a
potent elastase inhibitor (IC50 ) 3 nM) embedded with
an oxidative activation feature,12 and P 1517 (3) is given
as an elastase inhibitor with an IC50 on HLE around
0.1 mM but with in vivo radical scavenger properties
superior to N-acetylcysteine.13
Ch em istr y
Nonnatural amino acids H-Phi-OH (Phi) (4) and
H-Abo-OH (Abo) (5), containing a perhydroindole and
an azabicyclo[2.2.2]octane ring, respectively, were pre-
pared according to published procedures.14-16
Ester 9c was obtained as a secondary product during
the preparation of ester 9b. Both were used directly
as a mixture and separated at the next step using
reverse phase HPLC. Esters of generic formula 9 were
converted to the corresponding carboxylic acids 10 in
the presence of NaOH in EtOH followed by acidic
workup (Scheme 4). In the case of X being hydrogen
on generic formula 10 and the carboxylic acid function
being para with respect to the sulfonamide (compound
8a ), compounds 10 were coupled directly with tripeptidic
trifluoromethyl ketones viia ,b using the BOP, DIEA
methodology to give inhibitors 11 (route 1). The latter
were generally purified by column chromatography on
silica gel followed by trituration with ether or pentane.
In the case of X being a chlorine atom and the carboxylic
The compounds described in Tables 1 and 2 were
prepared according to Schemes 2-5. The synthesis of
the tripeptidic moiety is described in Scheme 2 and
relies essentially on previously reported procedures:
amino trifluoro alcohol ii was obtained in five steps from
N-benzoylvaline as described by Peet.17 Compound ii
was conveniently reacted with either one of our Boc-
protected amino acids i using mixed anhydride coupling

1908 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Portevin et al.
Sch em e 3a
a
Reagents: (a) CH3COOtBu, 70% HClO4; (b) EtOH, H2SO4; (c) NaH, MEM-Cl, DMF; (d) NaOH, EtOH; (e) tBubromoacetate, K2CO3;
(f) TFA, CH2Cl2; (g) EtONa, 4-(2-chloroethyl)benzoic acid; (h) ethyl 4-bromobutyrate, K2CO3; (i) CH3COCH2COOEt, NH4OH, EtOH.
Sch em e 4a
Compounds in which the N-terminal valine was
replaced by an ꢀ-substituted lysine were prepared as
outlined in Scheme 5. The hydrochloride of ꢀ-N-(ben-
zyloxycarbonyl)lysine methyl ester (15) was reacted with
sulfonamide A-OH, already used by others,6 to give
intermediate 16. Removal of the ꢀ-amino protecting
group was effected by treatment with HBr in AcOH.
This was followed by condensation with carboxylic acids
6a ,e under classical peptidic coupling conditions (DCC,
HOBT in DMF) to give intermediates 18 and 21.
Saponification was performed to provide acids 19 and
22 which were condensed with dipeptidic trifluoro-
methyl ketone Phi-Val-CF3 (v-a ) in the presence of DCC,
HOBT, and triethylamine to give the desired inhibitors
20 and 23, respectively.
Finally, as illustrated in Scheme 6, the commercially
available L-serine methyl ester (24) was converted into
its chloro analog by treatment with PCl5 in chloroform
to give chloromethyl amino acid 25 as the hydrochloride
salt.20 After protection of the amino group (Boc2O,
Et3N), condensation with 4-hydroxy-3,5-di-tert-butyl-
thiophenol in the presence of potassium carbonate in
acetonitrile was performed to provide cysteine derivative
27. The required free amino group was obtained by
deprotection with gaseous HCl in dioxane as described
previously. Subsequent condensation with sulfonami-
docarbonyl derivative A-OH, again using BOP, DIEA
coupling methodology, gave intermediate 29. Saponi-
fication of the ester function under usual conditions was
followed by coupling with dipeptide Phi-Val-CF3 (v-a )
(DCC, HOBT, Et3N) to give the final derivative 31.
a
Reagents: (a) vii-b, BOP, DIEA, DMF; (b) 8a or 8b, DCC,
DMAP, DMF; (c) NaOH, EtOH; (d) 8a , DCC, DMAP, DMF; (e)
vii-a , BOP, DIEA, DMF.
acid moiety being meta to the sulfonamide (compound
8b), a second coupling reaction was performed with 8a ,
this time under DCC, DMAP coupling conditions, to give
esters 12 (Route 2). Saponification followed by coupling
with the tripeptidic entity vii-a as described above
provided the two inhibitors 14a ,b.

Dual Inhibition of HLE and Lipid Peroxidation
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1909
Sch em e 5a
enzymatic preparation compared to their proline coun-
terpart (in our hands, IC50 for ICI compound was 34
nM).
The aim of our ongoing investigations was to add a
complementary activity to the strict elastase inhibition.
Among several possible synergistic activities (mucolytic,
antiinflammatory, etc.), we turned to the hypothesis
that it could be beneficial to embody our inhibitors with
antioxidant properties.
As briefly mentioned by Stein et al.,21 the minimum
structural sequence necessary to obtain a strong in vitro
anti-HLE inhibitory potency was derived from
H-Val-AA-Val-CF3: in our hands, the tripeptidic ketone
vii-b (H-Val-Abo-Val-CF3) was found to be moderately
active (IC50 ) 210 nM), which was in contrast to
intermediate vi-b (Boc-Val-Abo-Val-CF3) (IC50 ) 32
nM), thus suggesting the possible importance of inhibi-
tor interaction at the P3 region of the enzyme to obtain
a good activity. The first inhibitor incorporating an
antioxidant moiety (7) was quite atypical, mainly be-
cause the phenol ring was directly linked to the tri-
peptidic sequence through an alkylaryl chain (Table 1).
Surprisingly this compound retained some in vitro
inhibitory potency with an IC50 of 24 nM.
a
Reagents: (a) BOP, DIEA, DMF; (b) HBr, CH3COOH; (c) 6a
or 6c, DCC, HOBT, DMF; (d) NaOH, CH3OH; (e) v-a , HOBT, DCC,
ET3N.
Sch em e 6a
On the other hand, the (acylamino)sulfonyl derivative
34, with an IC50 value of 24 nM, was equipotent to the
reference inhibitor 1. This observation opened up the
field of introducing different carboxylic acids via cou-
pling with p-sulfonamidobenzoic acid or a substituted
analog.
a
Reagents: (a) PCl5, CHCl3; (b) Boc2O, Et3N; (c) 4-hydroxy-
3,5-di-tert-butylthiophenol, K2CO3, CH3CN; (d) gaseous HCl, di-
oxane; (e) A-OH, BOP, DIEA, DMF; (f) NaOH, CH3OH; (g) v-a ,
DCC, HOBT, Et3N.
When carboxylic acid 6a was adjoined to the tripep-
tide through the p-sulfonamidophenyl moiety, the po-
tencies of the corresponding Phi- and Abo-containing
inhibitors 11a ,b were in the same range of potency (38
and 30 nM, respectively) as those of the reference
inhibitors. If the methylene linker was removed to give
11c, inhibition was still potent (IC50 ) 36 nM), as was
also observed with the bis di-tert-butylphenol derivative
11d (IC50 ) 46 nM). The vinyl analogs 11e,f had similar
potencies with an IC50 around 40 nM. The inhibitory
potency was still robust when a 4-mercaptodi-tert-
butylphenol group was linked to the acylsulfonamido
En zym e In h ibitor y Activities
Previous studies in our laboratories had documented
that it was possible to replace the central proline (Pro)
of the tripeptidic backbone Val-Pro-Val found in ICI 200
880 (1) by our nonnatural amino acids Phi (4) and Abo
(5) and obtain potent and specific HLE inhibitors.10,11
This was exemplified herein by compounds 32 and 33
which displayed similar IC50 values against an HLE

1910 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Portevin et al.
Ta ble 1

Dual Inhibition of HLE and Lipid Peroxidation
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1911
Ta ble 1 (Continued)
a
b
Compounds gave satisfactory analyses ((0.4%) unless otherwise indicated. ND: not determined, but satisfactory results by high-
resolution MS analysis were obtained. c C: found, 60.84; calcd 60.42. S: found, 3.73; calcd, 3.00. e S: found, 7.93; calcd, 7.28. f S: found,
d
g
h
i
5.49; calcd. 6.60. C: found, 57.86; calcd, 58.53. H: found, 6.45; calcd, 5.95. IC50 values are concentrations of compounds required to
achieve 50% inhibition against HLE from human sputum. j IC50 values are concentrations of compounds required to achieve 50% inhibition
against Fe3+ ascorbate-induced lipid peroxidation from liver microsomal preparation. in: inactive.
k
moiety either by a methylene group as in 11g (IC50
)
acids. These two compounds appeared to be slightly less
potent against HLE than their related valine-containing
counterparts, displaying IC50s around 70-80 nM. Fi-
nally, the P3 valine was replaced by a S-substituted
cysteine to give compound 31 which presented a potency
similar to that of the reference inhibitors (IC50 ) 59 nM).
From these preliminary in vitro studies, it appears
that (1) Phi and Abo are effective proline mimics in
order to obtain potent HLE inhibitors in vitro. (2) The
[(p-chlorophenyl)sulfonamido]carbonyl moiety used in
the standard inhibitors can effectively be replaced by
entities in which a chemical entity able to bring some
additionnal antioxidant properties is incorporated: all
the compounds tested presented in vitro anti-HLE
potencies between 10 and 100 nM, indicating that the
P4-P5 region of the enzyme has a high degree of
tolerance with regard to the substituent group.
43 nM) or by an ethylphenyl group as in 11h (IC50 ) 40
nM). Compound 11i which embodied a 4-chloro-3-
sulfonamidobenzoic acid entity also retained a strong
in vitro activity, having an IC50 value of 46 nM.
At this point, we selected the trolox group22 as well
as a 4-phenyldihydropyridyl entity as potential antioxi-
dant functions, giving rise to inhibitors 11j (IC50 ) 20
nM) and 11k (IC50 ) 41 nM), respectively. It has been
hypothesized that the sulfonamide functionality confers
high in vitro and in vivo activity, probably through
improved interactions with the S4-S5 enzymatic sites
and also due to the acidic nature of this acylsulfonamide
function which could prevent the rapid clearance of the
compounds from the lungs.23 We took this reasoning
one step further by adding a second sulfonamide func-
tion to give the bis sulfonamide derivatives 14a ,b. Both
compounds displayed potent in vitro inhibitory activity
against HLE with IC50s of 26 and 34 nM, respectively.
The preparation of an HLE inhibitor in which the P3
valine is replaced by a lysine has been documented in
1990 by Peet et al.17 This compound was reported to
have a Ki value of 0.58 nM on an HLE enzymatic
preparation. As illustrated in Table 2, inhibitors 20 and
23 incorporated the tripeptidic trifluoromethyl ketone
Lys-Phi-Val-CF3. On the R-amino group of the lysine
residue was appended the p-[p-Cl(C6H4)SO2NHCO]-
(C6H4)CO system (A), while the ꢀ-amino group on the
lysine side chain was derivatized with two different
4-mercaptodi-tert-butylphenol-containing carboxylic
Selectivity Stu d ies
Selectivity of inhibitor 14a was studied on a panel of
22 receptors24 where Ki values were higher than 10-5
M. Affinities for sodium (Veratridine) and calcium
(Nifedipine) channels were superior to 10-4 and 2.4 ×
10-6 M, respectively. Moreover, compound 14a gave an
IC50 of 1 µM against pig pancreatic elastase (PPE) and
2 µM against prolyl endopeptidase. No inhibitory
activity was detected up to 33 µM on several factors of
the coagulation and the fibrinolysis cascades, including
thrombin, plasmin, factor Xa, kallikrein, activated
protein C, urokinase, tPa, and trypsin, as well as on

1912 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Portevin et al.
Ta ble 2
a
b
Compounds gave satisfactory analyses ((0.4%) unless otherwise indicated. The four compounds were amorphous solids. c ND: not
d
determined, but satisfactory results by high-resolution MS analysis were obtained. C: found, 59.37; calcd, 60.26. N: found, 6.43; calcd,
6.89. e IC50 values are concentrations of compounds required to achieve 50% inhibition against HLE from human sputum. f IC50 values
are concentrations of compounds required to achieve 50% inhibition against Fe3+ ascorbate-induced lipid peroxidation from liver microsomal
g
preparation. in: inactive.
acetylcholinesterase and cathepsin G. Finally, matrix
metalloproteinases 1, 2, 3, and 9 were inhibited by
compound 14a with IC50 values of 58, 19, 38, and 24
µM, respectively.
standard antioxidant compounds probucol and BHT
gave IC50s of 3 µM in this assay.
In Vivo Stu d ies
The main goal of our investigations was to identify
compounds affording a strong and long lasting in vivo
activity after intratracheal administration. The animal
model used along these studies was based on the fact
that HLE induces an acute hemorrhage in the hamster
lung when administered intratracheally (it).25 This
hemorrhage can be quantitated 3 h later by evaluation
of the hemoglobin content in bronchial alveolar lavage
fluid. All the compounds tested were administered it
at various times prior to the HLE challenge at the dose
of 15 nmol/animal. The results of this time-effect study
are disclosed in Table 3.
In this assay, ICI 200 880 exhibited a potent activity,
since 78% of the HLE-induced lung injury was inhibited
at 3 h; this activity was maintained for at least 24 h.
Several novel inhibitors with strong in vitro potencies
were partially or even totally devoid of in vivo activity:
compound 11j in which the trolox group is appended
on the (acylamino)sulfonyl linker showed no meaningful
in vivo activity, as well as the bis di-tert-butylphenol
derivative 11d . As expected, compound 7 exhibited a
modest activity when administered 3 h before the
inflammatory challenge, probably due to the lack of
acylsulfonamide function within its structure; more
curiously, its extented analog 11h was also poorly
active. Inhibitor 11k gave a good inhibition of the HLE-
induced edema at 3 h but was devoid of activity at 6 h.
More generally, incorporation of the (acylamino)sulfonyl
functionality within the structure of the inhibitors
An tioxid a n t P r op er ties
In vitro antioxidant potential of our inhibitors was
estimated by inhibition of the Fe3+ ascorbate-induced
lipid peroxidation on a rat liver microsomal preparation.
As expected, ICI 200 880 was completely devoid of
activity in this assay. Inhibitor 11k containing a
dihydropyridine entity was virtually inactive on the
peroxidation test. In contrast, the trolox group contain-
ing inhibitor 11j gave potent inhibition with an IC50 of
1.2-2.5 µM. However, for the di-tert-butylphenol moiety-
containing compounds, we found that the antioxidant
potency depended on the link between the phenol ring
and the acylsulfonamido group; as expected, the pro-
tected phenol 11e was completely devoid of activity, and
an ester function such as in 11d was also deleterious
for activity. Middle range potency was obtained with
direct bonding (11c, 10-20 µM), a methylene spacer
(11a ,b or 14b, 2.5-10 µM), or the vinylogous 11f (2.5-5
µM). The strongest inhibition of peroxidation was
obtained when the 4-mercaptodi-tert-butylphenol was
used: it gave rise to IC50 values between 2.5 and 5 µM
for inhibitors 20, 23, and 31. Potency was further
increased for inhibitors 7 and 11g,h (IC50 ) 1.2-2.5 µM)
and culminated with compounds 11i and 14a with IC50s
ranging between 0.6 and 1.2 µM.
Furthermore, compound 14a protected the purified
human LDL from copper sulfate-induced oxidative
modifications with an IC50 of 0.38 µM. In our hands,

Dual Inhibition of HLE and Lipid Peroxidation
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1913
Ta ble 3.
% inhibition of HLE-induced hemorrhage
in the hamster lung at
compd no.
3 h
6 h
63
18 h
47
24 h
37
48 h
18
72 h
ICI 200 880
7
78
24
66
28
53
0
19
63
60
20
41
0
11a
11b
11c
11d
11e
11f
11g
11h
11i
65
0
71
26
40
3
11
0
58
60
49
11
0
0
52
23
0
11j
F igu r e 1. Effects of ICI 200 880 and 14a on 14-day elastase-
induced emphysema in the hamster. C ) no treatment. PPE
) 60 IU + 0.2 mL of saline of pig pancreatic elastase. ICI 200
880 and 14a were administered it at 100 nmol in a 0.1 mL
volume of saline, 3 h prior to PPE challenge.
11k
14a
14b
20
56
68
41
40
3
0
66
40
41
65
52
45
76
62
14
73
0
0
50
23
31
42
49
12
6
replaced by another substituted polar amino acid.
However, the length of the chain appended on the
ꢀ-nitrogen of the lysine residue seemed to be of prime
importance since in vivo activity was completely lost
when a CH2 chain (as in 20) was replaced by a (CH2)3
chain (as in 23).
Furthermore, ICI 200 880 and compound 14a were
compared in a model of elastase-induced chronic lung
injury in the hamster, where instillation of PPE caused
microvascular damage and subsequent alveolar space
enlargment, i.e. emphysema.27 This was determined by
quantifying the number of alveolar intercepts (mean
linear intercept) on lung histological sections 14 days
after the challenge (Figure 1). Intratracheal adminis-
tration of ICI 200 880 just before HLE at the single dose
of 100 nmol had no significant action on the increase of
the alveolar spaces. In contrast, 14a significantly
inhibited pulmonary lesions associated with increased
alveolar spaces.
afforded, as anticipated, potent compounds in vivo. For
example, compound 11a was found to be very potent
on the reduction of the HLE-induced hemorrhage when
predosed 3 and 6 h before injection of elastase; however,
its activity decreased when it was administered 18 h
before HLE, and no activity subsisted when adminis-
tered 24 h before HLE. Noteworthy, replacement of Phi
by Abo (11b) in the tripeptidic sequence resulted in
almost total loss of activity. This could perhaps be
explained by a difference in lipophilicity between the
two central amino acids (log P(Phi) ) -1.19, log P(Abo)
) -1.75), hence between the two corresponding inhibi-
tors, indicating that lipophilicity could also be of im-
portance for absorption or retention of the compounds
in the lungs. Compounds 11c,f,g,i presented an activity
profile comparable to that of 11a with more or less
potency remaining at 18 h but without any activity left
when dosed at 24 or 48 h before challenge. (Note that
the protected analog of 11f, 11e, was very poorly active
at 3 h.) All these compounds have in common the
presence of two aryl rings linked together by an X-
(acylamino)sulfonyl group, X being respectively a direct
bond, CHdCH, CH2, or SCH2.
A similar profile of activity was observed with the bis
acylsulfonamide containing inhibitor 14b. More inter-
estingly, analog 14a , with only one more sulfur atom
on the phenol ring, gave a strong and very long lasting
in vivo potency: since at a fixed dose of 15 nmol it, the
first administration time giving 50% protection against
elastase-induced lung injury was 72 h for 14a and only
18 h for ICI compound. As mentioned earlier, this long
duration of action in the lung could at least in part be
explained by the inability of the inhibitors to be cleared
from the lung, although this was not investigated.26 This
property seems to be conferred to the molecule by the
presence of the acylsulfonamido moiety and is probably
exacerbated if two such groups are present, as in 14a .
The longer duration of action observed with 14a as
compared to 14b can probably be rationalized in terms
of differences in their in vivo metabolism.
Discu ssion a n d Su m m a r y
Among the many HLE inhibitors described in the
recent literature, the tripeptidic trifluoromethyl ketone
derivatives remain one of the most promising series for
aerosol administration in man. We decided to extend
the field of structural variations in this class of inhibi-
tors, and several conclusions can be drawn from the
foregoing results:
(1) Our proline analog amino acids have proven to be
useful tools in order to obtain potent inhibitors as active
as the reference compound ICI 200 880 in several assays
of in vitro and in vivo reduction of HLE activity.
(2) We then hypothesized that it could be worthwhile
to add to the strict antielastase property of our com-
pounds a second activity, if possible acting synergisti-
cally. We decided to embody our compounds with
antioxidant properties, since several studies had re-
cently demonstrated that the destructive potential of
HLE in the lung was enhanced by active ORS. This
hypothesis was chemically expressed with the use of
antioxidant groups such as the tert-butylphenol ring,
which was appended in different ways on the modified
tripeptidic sequence. This gave rise to inhibitors that
were effective HLE inhibitors in vitro with IC50 values
in the 10-100 nM range. Moreover, these compounds
exhibited a real antioxidant potential, since they inhib-
Finally, the lysine and cysteine derivative-containing
inhibitors 20 and 31 diplayed similar potencies as
inhibitors 11c,f suggesting that in vitro as well as in
vivo activity can be obtained with compounds where the
N-terminal valine of the tripeptidic sequence has been

1914 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Portevin et al.
layer was washed with a saturated sodium bicarbonate solu-
tion and then water. After drying on calcium sulfate and
evaporation of the solvent, 15.1 g (96%) of an amorphous solid
was obtained: mp 110 °C; IR (Nujol) 1764, 1680 cm-1; 1H NMR
(DMSO-d6) δ 0.95 (6H, m), 1.3-2.0 (9H, m), 1.35 (9H, s), 2.3
(3H, m), 3.75 (1H, m), 4.2 (1H, m), 4.4 (1H, m), 8.4 (1H, br s).
ited lipid peroxidation on a rat liver microsomal prepa-
ration as well as on purified human LDL with an IC50
similar to or even better than IC50s of reference anti-
oxidant compounds. The elastase-induced acute lung
injury was very discriminant for the selection of a leader
compound in terms of duration of action in the lung:
one of the most active compounds of the series, 14a ,
showed significant activity in this assay, reducing the
hemorrhage by 50% when predosed 72 h prior to HLE
challenge. Furthermore, in a 14-day model of elastase-
induced emphysema in the hamster, 14a significantly
inhibited alveolar space enlargement, when adminis-
tered once just before instillation of HLE. In this model,
ICI 200 880 was devoid of significant activity, indicating
that the addition of an antioxidant moiety in the
structure of an HLE inhibitor could be relevant in order
to gain some in vivo potency in a chronic model of lung
injury. Although promising, these results warrant some
more in vivo confirmation: to adress the importance of
the antioxidant effect of our compounds, it would be
interesting to compare 14a with a close analog devoid
of antioxidant properties on an animal model of em-
physema involving both elastase and reactive oxygen
species.
Boc-Abo-Va l-CF 3 (iv-b): IR (Nujol) 3311, 1761, 1670 cm-1
;
1H NMR (DMSO-d6) δ 0.95 (6H, m), 1.35 (9H, s), 1.4-1.75 (7H,
m), 1.9 (1H, m), 2.1 (1H, m), 2.2 (1H, m), 3.9 (1H, m), 4.15
(1H, dd), 4.6-4.7 (1H, 2m).
P h i-Va l-CF 3 (v-a ). Gen er a l Meth od 1. The protected
dipeptide obtained above (15.1 g, 0.036 mol) was dissolved in
350 mL of dioxane. Gaseous HCl was bubbled at 10 °C until
saturation of the solution was obtained. Stirring was contin-
ued overnight at room temperature. After evaporation of the
solvent, the crude solid was taken up with pentane to give 12
g of the desired hydrochloride (94%): mp 134 °C; IR (Nujol)
3600-2400, 1764, 1680, 1171 cm-1; 1H NMR (DMSO-d6) δ 0.8-
1.0 (6H, 2d), 1.2-1.9 (9H, m), 2.3 (3H, m), 3.6 (1H, m), 4.15
(1H, m), 4.4 (1H, m), 8.4 (1H, br s), 10.5 (1H, br s).
Abo-Va l-CF 3 (v-b): mp 165-170 °C sublimation; IR (Nujol)
3600-2400, 1765, 1677, 1173 cm-1; 1H NMR (DMSO-d6) δ 0.8-
1.0 (6H, 2d), 1.3-2.0 (8H, m), 2.2 (1H, m), 2.3 (1H, m), 3.4
(1H, br s), 4.0-4.2 (2H, m), 8.5 (1H, br s), 9.8 (1H, br s).
Boc-Va l-P h i-Va l-CF 3 (vi-a ). Gen er a l Meth od 2. To 6 g
(0.0168 mol) of Phi-Val-CF3 in 5 mL of DMF were added 2.34
mL (0.0168 mol) of triethylamine, 3.65 g (0.0168 mol) of Boc-
Val-OH in 3 mL of DMF, 2.57 g of HOBT (0.0168 mol) in 2
mL of DMF, and 3.46 g (0.0168 mol) of DCC in 3 mL of DMF.
Stirring was continued for 20 h. DCU was filtered and DMF
evaporated. The gummy residue was taken up with ethyl
acetate; the organic solution was washed consecutively with
brine, a saturated sodium bicarbonate solution, brine, a 10%
citric acid solution, brine, and water. Solution was dried over
calcium sulfate, filtered, and evaporated to give 9.1 g of a crude
solid. This product was purified by chromatography (CH2Cl2-
EtOH, 98:2) to give 4.4 g of the protected tripeptide: IR (Nujol)
3276, 1765, 1687, 1207-1150 cm-1; 1H NMR (DMSO-d6) δ 0.8-
1.0 (12H, m), 1.15-1.9 (8H, m), 1.4 (9H, s), 1.9 (1H, m), 2.2
(2H, m), 3.75 (1H, m), 4.0-4.4 (2H, m), 4.45-4.65 (1H, 2m).
Boc-Va l-Abo-Va l-CF 3 (vi-b): IR (Nujol) 3500-3300, 1793,
1697, 1624 cm-1; 1H NMR (CDCl3) δ 0.8-1.1 (12H, m), 1.4 (9H,
s), 1.5-1.7 (4H, m), 1.8-2.0 (4H, m), 2.4 (1H, m), 2.6 (2H, m),
3.95 (1H, m), 4.4 (1H, m), 5.02 (2H, m), 6.45 (1H, br s), 6.7
(1H, br s).
Exp er im en ta l Section
Melting points were determined on a Tottoli apparatus and
were not corrected. Elemental analyses were carried out by
the analytical department of the Institut de Recherches
Servier; results obtained for specified elements are within
(0.4% of the theoretical values. IR spectra were recorded on
a Bruker IFS 28 spectrophotometer. 1H NMR spectra of
deuteriochloroform or DMSO-d6 solutions were recorded on a
Bruker AC 200 or AM 300 spectrometer. Chemical shifts are
given in ppm with TMS as the internal standard. Optical
rotations were recorded with a 241 Perkin Elmer polarimeter.
Abbreviations: DCC ) N, N ′-dicyclohexylcarbodiimide; DCU
) N, N ′-dicyclohexylurea; HOBT ) 1-hydroxybenzotriazole;
BOP ) (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate; DMAP ) 4-(dimethylamino)pyridine;
DIEA ) N,N-diisopropylethylamine; NMM ) N-methylmor-
pholine.
(2S,3aS,7aS)-1-(ter t-Bu tyloxycar bon yl)-2-[[(1-isopr opyl-
2-h y d r o x y -3,3,3-t r i flu o r o p r o p y l)a m i n o ]c a r b o n y l]-
p er h yd r oin d ole (iii-a ). To a mixture of 12.4 g (0.046 mol)
of Boc-Phi-OH (i)9-11 and 5 mL (0.046 mol) of N-methylmor-
pholine in 80 mL of THF was added dropwise at -10 °C a
solution of 5.95 mL (0.046 mol) of isobutyl chloroformate in
10 mL of THF. After 10 min at -10 °C, a solution of the amino
alcohol ii (9.5 g, 0.046 mol) and N-methylmorpholine (5 mL)
in 60 mL of DMF was added dropwise at -10 °C. Stirring
was continued overnight and temperature slowly raised to
ambient. Solvents were evaporated, and the residue was
taken up with isopropyl ether (150 mL); after filtration of an
insoluble, the organic phase was washed with water and dried
over calcium sulfate. Evaporation of the solvent was followed
by trituration with pentane to give 16.8 g (83%) of the desired
Va l-P h i-Va l-CF 3 (vii-a ): see General Method 1; IR (Nujol)
1763, 1695 cm-1; 1H NMR (DMSO-d6) δ 0.7-1.1 (12H, m), 1.1-
2.5 (13H, m), 3.5-4.8 (4H, m).
Va l-Abo-Va l-CF 3 (vii-b): IR (Nujol) 1761, 1693, 1631 cm-1
;
1H NMR (DMSO-d6) δ 1.0 (12H, m), 1.3-1.8 (8H, m), 1.8-2.2
(3H, m), 4.0 (1H, m), 4.1 (1H, m), 4.4 (1H, dt), 4.6 (1H, 2m).
4-[2-(4-Hydr oxy-3,5-di-ter t-bu tylph en yl)th io]eth yl]ben -
zoyl-(S)-Va l-(S)-Abo-(R,S)-Va l-CF 3 (7). Gen er a l Meth od
3. Tripeptide vii-b (662 mg, 0.015 mol), carboxylic acid 6d
(580 mg, 0.015 mol), BOP (668 mg, 0.015 mol), and DIEA (0.79
mL, 0.045 mol) were stirred at room temperature for 18 h in
50 mL of DMF. After evaporation of the solvent, the residue
was taken up with 100 mL of ethyl acetate. The solution was
washed with water, dried over calcium sulfate, and evaporated.
Purification by chromatography (CH2Cl2-AcOEt, 90:10) gave
0.7 g (60%) of the title compound: IR (Nujol) 1761, 1640-1700
cm-1; 1H NMR (DMSO-d6) δ 0.95 (12H, m), 1.4 (18H, s), 1.5-
2.0 (9H, m), 2.15 (2H, m), 2.9 (2H, t), 3.1 (2H, t), 4.1-4.7 (4H,
m), 7.15 (2H, s), 7.25 (2H, d), 7.75 (2H, d).
intermediate: mp 193 °C; IR (Nujol) 3600-3300, 1662 cm-1
;
1H NMR (DMSO-d6) δ 0.95 (6H, m), 1.3-2.0 (9H, m), 1.4 (9H,
s), 2.3 (3H, m), 3.75-4.4 (4H, m), 6.5 (1H, br s), 8.4 (1H, br s).
(3S)-2-Aza -2-(ter t-bu tyloxyca r bon yl)-3-[[(1-isop r op yl-
2-h yd r oxy-3,3,3-tr iflu or op r op yl)a m in o]ca r bon yl]bicyclo-
[2.2.2]octa n e (iii-b): IR (Nujol) 3600-3300, 1662, 1165, 1138,
Eth yl 4-[[[(4-Hyd r oxy-3,5-d i-ter t-bu tylp h en yl)a cetyl]-
a m in o]su lfon yl]ben zoa te (9a ). Gen er a l Meth od 4. To a
solution of 5 g (0.019 mol) of 4-hydroxy-3,5-di-tert-butylphen-
ylacetic acid (6a ) in 60 mL of DMF were successively added
4.35 g (0.019 mol) of ethyl 4-sulfonamidobenzoate (8a ) in 10
mL of DMF, 2.32 g (0.019 mol) of DMAP in 10 mL of DMF,
and DCC (3.92 g, 0.019 mol). The resulting solution was
stirred at room temperature for 2 days. DCU was filtered and
DMF was concentrated under reduced pressure. The residue
was treated with ethyl acetate. The organic layer was washed
consecutively with saturated aqueous NaCl, 10% aqueous citric
acid, and again brine. The EtOAc was dried (CaSO4) and
1
1105 cm-1; H NMR (DMSO-d6) δ 0.8 (6H, m), 1.35-2.0 (9H,
m), 1.4 (9H, s), 2.15 (1H, m), 3.7-4.15 (4H, m), 6.35 (1H, br
s), 7.2 and 7.6 (1H, br s).
Boc-P h i-Va l-CF 3 (iv-a ). Alcohol obtained at the previous
step (15.8 g, 0.037 mol) was oxidized by the DMSO-Ac2O
mixture (160 and 130 mL, respectively). After stirring at room
temperature during 48 h, 300 mL of water was added; stirring
was continued for 3 more h and the resulting solid filtrated.
This solid was taken up with dichloromethane; the organic

Dual Inhibition of HLE and Lipid Peroxidation
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1915
concentrated in vacuo to give the crude title compound as a
dark solid. This compound was purified by chromatography
over silica gel (CH2Cl2-CH3OH-NH4OH, 90:10:1) to give a
colorless oil (6.8 g, 75.5%) that crystallized upon trituration
with pentane: mp 150 °C; IR (Nujol) 3600, 3300-3000, 1726,
1711-1685, 1360, 1144 cm-1; 1H NMR (DMSO-d6) δ 1.3 (21H,
t + s), 3.4 (2H, s), 4.35 (2H, q), 6.85 (2H, s), 6.9 (1H, br s), 8.0
(2H, d), 8.15 (2H, d), 12.45 (1H, br s).
E t h yl 4-[[[(4-H yd r oxy-3,5-d i-ter t-b u t ylp h en yl)ca r b o-
n yl]a m in o]su lfon yl]ben zoa te (9b) a n d Eth yl 4-[[[[4-[(4-
Hyd r oxy-3,5-d i-ter t-bu tylp h en yl)ca r boxy]-3,5-d i-ter t-bu -
tylp h en yl]ca r bon yl]a m in o]su lfon yl]ben zoa te (9c). To a
100 mL toluene solution of the sulfonamide 3 (3.45 g, 0.015
mol) was added 2.1 mL (0.015 mol) of triethylamine; then at
80 °C the acid chloride of 4-hydroxy-3,5-di-tert-butylbenzoic
acid (4.05 g, 0.015 mol) (itself prepared with 2 mol equiv of
oxalyl chloride in toluene at 80 °C) was added. The reaction
mixture was refluxed overnight and cooled down to room
temperature, and water was added. The remaining starting
sulfonamide was filtered, and the organic layer was washed
several times with water. Drying (CaSO4) and evaporation
gave an amorphous solid (5.5 g). This compound was purified
by chromatography (CH2Cl2-CH3OH-NH4OH, 90:10:0.5) to
give 3.3 g of a mixture of both 9b,c.
Eth yl 4-[[[4-(eth oxym eth oxy)-3,5-d i-ter t-bu tylcin n a m -
oyl]a m in o]su lfon yl]ben zoa te (9d ): IR (Nujol) 3217, 1726,
1622, 1275, 1109 cm-1; 1H NMR (DMSO-d6) δ 1.2 (3H, t), 1.35
(3h, t), 1.4 (18H, s), 3.8 (2H, q), 4.35 (2H, q), 4.9 (2H, s), 6.55
(1H, d), 7.5 (1H, d), 7.45 (2H, s), 8.15 (4H, dd), 12.3 (1H, br s).
E t h yl 4-[[[[(4-h yd r oxy-3,5-d i-ter t-b u t ylp h en yl)t h io]-
a cetyl]a m in o]su lfon yl]ben zoa te (9e): mp 188 °C; IR (Nujol)
3600, 3245, 1722, 1699 cm-1; 1H NMR (DMSO-d6) δ 1.3 (18H,
s), 1.35 (3H, t), 3.55 (2H, s), 4.35 (2H, q), 6.95 (2H, s), 7.10
(1H, br s), 7.55 (1H, br s), 8.00-8.2 (4H, m).
E t h yl 3-[[[[(4-h yd r oxy-3,5-d i-ter t-b u t ylp h en yl)t h io]-
a cetyl]a m in o]su lfon yl]-4-ch lor oben zoa te (9g): IR (Nujol)
3500-3000, 1718, 1593 cm-1; 1H NMR (DMSO-d6) δ 1.3 (21H,
m), 3.35 (2H, s), 4.35 (2H, q), 6.9 (1H, s), 7.05 (2H, s), 7.55
(1H, d), 7.95 (1H, dd), 8.5 (1H, d).
3,5-d i-ter t-bu tylph en yl)ca r boxy]-3,5-di-ter t-bu tylp h en yl]-
ca r bon yl]a m in o]su lfon yl]ben zoic Acid (10c). Separation
of the two compounds (on a 2 g scale) was performed by reverse
phase HPLC on a RP 18 15/25 column (5 mL/min) with CH3-
CN-H2O-CH3COOH (70:30:1) as eluent. After evaporation
of the eluent, the crude powder was triturated in pentane to
give 450 mg of 10b and 700 mg of 10c: mp 272 °C; IR (Nujol)
3626, 3298, 3000-2400, 1685 cm-1; 1H NMR (DMSO-d6) δ 1.35
(18H, s), 7.2 (1H, br s), 7.5 (1H, br s), 7.7 (2H, s), 7.95 (4H, m),
12.7 (1H, br s); IR (Nujol) 3570, 3257, 1716, 1685 cm-1 1H
;
NMR (DMSO-d6) δ 1.25 (18H, s), 1.4 (18H, s), 7.8 (2H, s), 7.9
(2H, s), 8.15 (4H, m), 13.5 (1H, br s).
4-[[[4-(Eth oxym eth oxy)-3,5-di-ter t-bu tylcin n am oyl]am i-
n o]su lfon yl]ben zoic a cid (10d ): IR (Nujol) 3101, 1687, 1612
1
cm-1; H NMR (DMSO-d6) δ 1.2 (3H, t), 1.4 (18H, s), 3.8 (2H,
q), 4.9 (2H, s), 6.55 (1H, d), 7.5 (2H, s), 7.55 (1H, d), 8.15 (4H,
dd).
4-[[[[(4-H yd r oxy-3,5-d i-ter t-b u t ylp h en yl)t h io]a cet yl]-
a m in o]su lfon yl]ben zoic a cid (10e): mp 240°C; IR (Nujol)
3607, 3279, 3200-2450, 1703 cm-1; 1H NMR (DMSO-d6) δ 1.3
(18H, s), 3.55 (2H, s), 7.0 (2H, s), 7.1 (1H, br s), 7.95-8.2 (4H,
2d), 13-13.5 (1H, br s).
4-[[[4-[2-[(4-Hyd r oxy-3,5-d i-ter t-bu tylp h en yl)th io]eth -
yl]ben zoyl]a m in o]su lfon yl]ben zoic a cid (10f): IR (Nujol)
3610, 3500-2400, 1703, 1360, 1171 cm-1 1H NMR (DMSO-
;
d6) δ 1.35 (18H, s), 2.85 (2H, t), 3.15 (2H, t), 7.0 (1H, br s),
7.05 (2H, s), 7.35 (2H, d), 7.8 (2H, d), 8.15 (4H, m).
3-[[[[(4-H yd r oxy-3,5-d i-ter t-b u t ylp h en yl)t h io]a cet yl]-
a m in o]su lfon yl]-4-ch lor oben zoic a cid (10g): IR (Nujol)
3600, 3500-3000, 1709 cm-1; 1H NMR (DMSO-d6) δ 1.3 (18H,
s), 3.55 (2H, s), 7.0 (2H, s), 7.1 (1H, s), 7.8 (1H, d), 8.15 (1H,
dd), 8.55 (1H, d), 12.5 (1H, br s).
3-[[[(4-Hyd r oxy-3,5-d i-ter t-bu tylp h en yl)a cetyl]a m in o]-
su lfon yl]-4-ch lor oben zoic a cid (10h ): IR (Nujol) 3641,
3350-2500, 1709, 1682, 1377, 1109 cm-1 1H NMR (DMSO-
;
d6) δ 1.3 (18H, s), 3.45 (2H, s), 6.8 (1H, br s), 6.85 (2H, s), 7.75
(1H, d), 8.15 (1H, dd), 8.55 (1H, d), 12.8 (1H, br s), 13.6 (1H,
br s).
4-[[[(2(R,S)-6-Hyd r oxy-2,5,7,8-tetr a m eth ylch r om a n -2-
yl)ca r bon yl]a m in o]su lfon yl]ben zoic a cid (10i): IR (Nujol)
Eth yl 3-[[[(4-h yd r oxy-3,5-d i-ter t-bu tylp h en yl)a cetyl]-
a m in o]su lfon yl]-4-ch lor oben zoa te (9h ): IR (Nujol) 3610-
3583, 3097, 1726, 1684 cm-1; 1H NMR (CDCl3) δ 1.45 (3H, t),
1.45 (18H, s), 3.55 (2H, s), 4.4 (2H, q), 5.3 (1H, br s), 6.95 (2H,
s), 7.55 (1H, d), 8.1 (1H, br s), 8.2 (1H, dd), 8.9 (1H, d).
Eth yl 4-[[[(2(R,S)-6-h yd r oxy-2,5,7,8-tetr a m eth ylch r o-
m a n -2-yl)ca r bon yl]a m in o]su lfon yl]ben zoa te (9i): IR (Nu-
1
3650-2300, 1724-1700, 1354, 1184 cm-1; H NMR (DMSO-
d6) δ 1.35 (3H, s), 1.65 (1H, m), 1.85 (3H, s), 2.0-2.4 (3H, m),
2.1 (6H, 2s), 7.5 (1H, br s), 7.80 (2H, d), 8.05 (2H, d), 11.9 (1H,
br s), 13.5 (1H, br s).
4-[[[(2,6-Dim eth yl-3,5-dicar beth oxy-1,4-dih ydr opyr idin -
4-yl)ca r bon yl]a m in o]su lfon yl]ben zoic a cid (10j): IR (Nu-
jol) 3600-2500, 1685, 1655, 1336, 1128 cm-1; 1H NMR (DMSO-
d6) δ 1.1 (6H, m), 2.25 (6H, s), 3.95 (4H, m), 4.85 (1H, s), 7.05
(2H, d), 7.7 (2H, d), 7.75-7.9 (4H, 2d), 8.8 (1H, br s).
E t h yl 4-[[[[4-ch lor o-3-[[[(4-h yd r oxy-3,5-d i-ter t-b u t yl-
p h en yl)a cetyl]a m in o]su lfon yl]p h en yl]ca r bon yl]a m in o]-
jol) 3700-2500, 1707, 1649, 1375, 1141 cm-1 1H NMR
;
(CH3OH-d4) δ 1.4 (3H, t), 1.45 (3H, s), 1.65 (1H, m), 1.9-2.15
(9H, 3s), 2.35 (3H, m), 4.35 (2H, q), 7.65 (2H, d), 7.9 (2H, d).
Eth yl 4-[[[(2,6-d im eth yl-3,5-d ica r beth oxy-1,4-d ih yd r o-
p yr id in -4-yl)ca r bon yl]a m in o]su lfon yl]ben zoa te (9j): mp
202 °C; IR (Nujol) 3500-3000, 1737-1676, 1136, 1130 cm-1
;
su lfon yl]ben zoa te (12b): IR (Nujol) 3627, 3232, 1705 cm-1
;
1H NMR (DMSO-d6) δ 1.1 (6H, t), 1.3 (3H, t), 2.25 (6H, s), 4.0
(4H, m), 4.3 (2H, q), 4.85 (1H, s), 7.05 (2H, d), 7.7 (2H, d), 7.9
(4H, m), 8.75 (1H, br s).
1H NMR (DMSO-d6) δ 1.3 (3H, t), 1.3 (18H, s), 3.45 (2H, s),
4.35 (2H, q), 6.8 (1H, br s), 6.85 (2H, s), 7.7 (1H, d), 8.1 (5H,
m), 8.5 (1H, d), 12.8 (1H, br s).
4-[[[(4-Hyd r oxy-3,5-d i-ter t-bu tylp h en yl)a cetyl]a m in o]-
su lfon yl]ben zoic Acid (10a ). Gen er a l Meth od 5. A 5 g
(0.0105 mol) portion of ester 9a was dissolved in 125 mL of
ethanol. Nitrogen was bubbled through the solution for 1 h;
then 22 mL (0.021 mol) of 1 N NaOH was added. The reaction
mixture was stirred at room temperature for 2 h, followed by
addition of 25 mL of water. The reaction mixture was then
heated at 60 °C for 20 h under nitrogen. After cooling, the
solvents were evaporated under reduced pressure, and the
residue was taken up with water. The aqueous layer was
extracted with ethyl acetate, acidified with 1 N HCl (30 mL),
and extracted with ethyl acetate. The organic layer was
washed with water to neutral pH, dried over calcium sulfate,
and evaporated to give 4.2 g of a crude solid. This material
was crystallized from pentane to give 4 g (70%) of the title
4-[[[[4-Ch lor o-3-[[[[(4-h yd r oxy-3,5-d i-ter t-bu tylp h en yl)-
t h io]a ce t yl]a m in o]su lfon yl]p h e n yl]ca r b on yl]a m in o]-
su lfon yl]ben zoic a cid (13a ): IR (Nujol) 3500-3000, 3500-
2500, 1705 cm-1; 1H NMR (DMSO-d6) δ 1.25 (18H, s), 3.55 (2H,
s), 7.0 (2H, s), 7.1 (1H, br s), 7.75 (1H, s), 7.8 (1H, d), 8.15
(5H, m), 8.55 (1H, d), 12.8 (1H, br s).
4-[[[[4-Ch lor o-3-[[[(4-h yd r oxy-3,5-d i-ter t-bu tylp h en yl)-
acetyl]am in o]su lfon yl]ph en yl]car bon yl]am in o]su lfon yl]-
ben zoic a cid (13b): IR (Nujol) 3627-3221, 2600, 1703 cm-1
;
1H NMR (DMSO-d6) δ 1.3 (18H, s), 3.5 (2H, s), 6.9 (2H, 2s),
7.8 (1H, d), 8.15 (5H, m), 8.5 (1H, d), 12.9 (1H, br s).
4-[[[(4-Hyd r oxy-3,5-d i-ter t-bu tylp h en yl)a cetyl]a m in o]-
su lfon yl]b e n zoyl-(S )-Va l-(2S ,3a S ,7a S )-P h i-(R ,S )-Va l-
CF 3 (11a ): prepared according to General Method 3 from 10a
and tripeptide vii-a ; IR (Nujol) 3639, 3400-3100, 1763-1650,
compound: mp 228 °C; IR (Nujol) 3635, 3300-2400, 1707 cm-1
;
1H NMR (DMSO-d6) 1.3 (18H, s), 3.4 (2H, s), 6.85 (2H, s), 7.5
(1H, br s), 7.95 (2H, d), 8.1 (2H, d), 12.4 (1H, br s), 13.3 (1H,
br s).
1360, 1673 cm-1 1H NMR (DMSO-d6) δ 0.8-0.95 (12H, m),
;
1.2-1.8 (8H, m), 1.3 (18H, s), 1.8-2.0 (2H, m), 2.15-2.3 (3H,
m), 3.35 (2H, s), 4.2-4.5 (3H, m), 4.7 (1H, m), 6.8 (1H, br s),
6.85 (2H, s), 7.9-8.05 (4H, d), 8.5-8.7 (1H, 2d), 8.9 (1H, br s),
12.3 (1H, br s).
4-[[[(4-Hyd r oxy-3,5-d i-ter t-bu tylp h en yl)ca r bon yl]a m i-
n o]su lfon yl]ben zoic Acid (10b) a n d 4-[[[[4-[(4-Hyd r oxy-

1916 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Portevin et al.
4-[[[(4-Hyd r oxy-3,5-d i-ter t-bu tylp h en yl)a cetyl]a m in o]-
su lfon yl]ben zoyl-(S)-Va l-(S)-Abo-(R,S)-Va l-CF 3 (11b): IR
4.4 (4H, m), 7.0 (2H, s), 7.1 (1H, s), 7.8 (1H, d), 8.1-8.2 (5H,
m), 8.6 (1H, d).
(Nujol) 3700-2500, 1761, 1700-1620, 1370, 1173 cm-1 1H
;
4-[[[[4-Ch lor o-3-[[[(4-h yd r oxy-3,5-d i-ter t-bu tylp h en yl)-
acetyl]am in o]su lfon yl]ph en yl]car bon yl]am in o]su lfon yl]-
ben zoyl-(S)-Va l-(2S,3a S,7a S)-P h i-(R,S)-Va l-CF 3 (14b): IR
NMR (DMSO-d6) δ 0.9 (12H, m), 1.3 (9H, s), 1.4-1.8 (8H, m),
2.0-2.25 (3H, m), 3.4 (2H, s), 4.0-4.7 (4H, m), 6.8 (1H, br s),
6.85 (2H, s), 7.5 (1H, d), 7.9 (2H, d), 8.05 (2H, d), 8.4 (1H, d),
8.7 (1H, d), 12.4 (1H, br s).
(Nujol) 3500-3000, 1763-1622, 1377, 1173 cm-1 1H NMR
;
(DMSO-d6) δ 0.85 (12H, m), 1.1-2.2 (8H, m), 1.3 (18H, s), 1.75
(2H, dd), 2.15 (2H, m), 2.25 (1H, m), 3.45 (2H, s), 4.15-4.7
(4H, m), 6.8 (2H, s), 7.7 (1H, d), 8.0 (5H, m), 8.1 (1H, br s),
8.45 (1H, d), 8.8 (1H, br s), 12.8 (1H, br s).
4-[[[(4-Hyd r oxy-3,5-d i-ter t-bu tylp h en yl)ca r bon yl]a m i-
n o]su lfon yl]ben zoyl-(S)-Va l-(2S,3a S,7a S)-P h i-(R,S)-Va l-
1
CF 3 (11c): IR (Nujol) 3624, 3278, 1763-1622 cm-1; H NMR
4-[[[(4-Ch lor oph en yl)su lfon yl]am in o]car bon yl]ben zoyl-
N-E-(b en zyloxyca r b on yl)-(S)-Lys-OCH 3, h yd r och lor id e
(16): was prepared according to General Method 3 and directly
treated without any further purification.
(DMSO-d6) δ 0.9 (12H, m), 1.1-2.0 (8H, m), 1.4 (18H, s), 1.95
(2H, m), 2.2 (3H, m), 4.25-4.5 (4H, m), 7.55 (2H, s), 8.0 (4H,
q), 8.4 (1H, br s), 8.9 (1H, br s), 12.5 (1H, br s).
4-[[[[4-[(4-H yd r oxy-3,5-d i-ter t-b u t ylp h en yl)ca r b oxy]-
3,5-di-ter t-bu tylph en yl]car bon yl]am in o]su lfon yl]ben zoyl-
(S)-Va l-(2S,3a S,7a S)-P h i-(R,S)-Va l-CF 3 (11d ): IR (Nujol)
3626, 3359, 1763-1620 cm-1; 1H NMR (DMSO-d6) δ 0.95 (12H,
m), 1.25 (4H, m), 1.3 (18H, s), 1.4 (18H, s), 1.7 (4H, m), 2.0
(3H, m), 2.2 (3H, m), 4.2-4.5 (3H, m), 7.8 (2H, s), 7.95 (2H, s),
8.1 (4H, dd), 8.5 (2H, br s), 8.9 (2H, br s).
4-[[[(4-Ch lor oph en yl)su lfon yl]am in o]car bon yl]ben zoyl-
(S)-Lys-OCH3, Hyd r och lor id e (17). A 1.2 g (0.0019 mol)
sample of the hydrochloride obtained in the previous step was
dissolved in 50 mL of acetic acid. A 33% HBr in acetic acid
solution (50 mL) was added, and the reaction was continued
for 4 h. After evaporation of the solvent under reduced
pressure, the residue was taken up with 80 mL of ethyl
acetate. The organic layer was washed with a saturated
sodium bicarbonate solution to neutral pH and evaporated.
Treatment of the residue with dry methanol to eliminate traces
of inorganic salts was followed by column chromatography
(CH2Cl2-CH3OH, 60:40) to give 1 g of the deprotected lysine
derivative, probably containing a small amount of inorganic
salts: IR (Nujol) 3600-3163, 1736, 1645, 1333, 1132 cm-1; 1H
NMR (DMSO-d6) δ 1.2-1.85 (6H, m), 2.75 (2H, t), 3.6 (3H, s),
4.45 (1H, t), 7.2 (1H, br s), 7.8 (4 + 1H, 2d + br s), 7.9 (2H, d),
8.7 (1H, br s).
4-[[[(4-Ch lor oph en yl)su lfon yl]am in o]car bon yl]ben zoyl-
N-E-[(4-h yd r oxy-3,5-d i-ter t-b u t ylp h en yl)a cet yl]-(S)-Lys-
OCH 3 (18): prepared from carboxylic acid 6a and lysine
derivative 17 described above according to General Method 2
and directly treated without any further purification.
4-[[[(4-Ch lor oph en yl)su lfon yl]am in o]car bon yl]ben zoyl-
N-E-[[(4-h yd r oxy-3,5-d i-ter t-bu tylp h en yl)th io]bu tyr oyl]-
(S)-Lys-OCH3 (21): IR (Nujol) 3636, 3329, 1740, 1639, 1339,
1129 cm-1; 1H NMR (DMSO-d6) δ 1.3-1.8 (8H, m), 1.35 (18H,
s), 2.2 (2H, t), 2.8 (2H, t), 3.05 (2H, m), 3.65 (3H, s), 4.4 (1H,
t), 5.55 (1H, br s), 7.0 (1H, br s), 7.05 (2H, s), 7.45 (2H, d),
7.85 (4H, m), 7.95 (2H, d), 8.7 (1H, d).
4-[[[4-(Eth oxym eth oxy)-3,5-di-ter t-bu tylcin n am oyl]am i-
n o]su lfon yl]ben zoyl-(S)-Va l-(2S,3a S,7a S)-P h i-(R,S)-Va l-
1
CF 3 (11e): IR (Nujol) 3269, 1763, 1668-1624 cm-1; H NMR
(DMSO-d6) δ 0.9 (12H, m), 1.25 (3H, t), 1.4 (18H, s), 1.7-1.9
(11H, m), 2.2 (2H, m), 3.8 (2H, q), 4.35-4.8 (4H, m), 4.9 (2H,
s), 6.6-7.5 (2H, d), 7.5 (2H, s), 7.7 (1H, d), 8.1 (4H, dd), 8.8
(1H, d), 12.2 (1H, br s).
4-[[(4-Hyd r oxy-3,5-d i-ter t-bu tylcin n a m oyl)a m in o]su l-
fon yl]ben zoyl-(S)-Val-(2S,3aS,7aS)-P h i-(R,S)-Val-CF3 (11f):
prepared according to General Method 1 from 11e; IR (Nujol)
3627, 3276, 1763-1622 cm-1; 1H NMR (DMSO-d6) δ 0.9 (12H,
m), 1.2-2.3 (13H, m), 1.4 (18H, s), 4.3-4.7 (4H, m), 6.5 (1H,
d), 7.3 (2H, s), 7.5 (1H, d), 7.6 (1H, br s), 8.05 (4H, dd), 8.6-
8.9 (1H, br s).
4-[[[[(4-H yd r oxy-3,5-d i-ter t-b u t ylp h en yl)t h io]a cet yl]-
a m in o]su lfon yl]b en zoyl-(S)-Va l-(2S,3a S,7a S)-P h i-(R,S)-
Va l-CF 3 (11g): IR (Nujol) 3633, 3300, 1763, 1720, 1700, 1660,
1622, 1527 cm-1; 1H NMR (DMSO-d6) δ 0.9 (12H, m), 1.0-2.3
(13H, m), 1.80 (18H, s), 3.1 (1H, m), 3.5 (2H, s), 3.9 (3H, m),
4.7 (1H, t), 6.7 (1H, d), 7.0 (2H, s), 7.05 (1H, s), 8.0 (4H, m),
8.60 (1H, d).
4-[[[4-[2-[(4-Hyd r oxy-3,5-d i-ter t-bu tylp h en yl)th io]eth -
yl]ben zoyl]a m in o]su lfon yl]ben zoyl-(S)-Va l-(2S,3a S,7a S)-
P h i-(R,S)-Va l-CF 3 (11h ): IR (Nujol) 3633, 3500-3200, 1761,
1720-1650, 1350, 1159 cm-1; 1H NMR (DMSO-d6) δ 0.9 (12H,
m), 1.2-1.7 (8H, m), 1.4 (18H, s), 1.8-2.3 (5H, m), 2.8 (2H, t),
3.1 (2H, t), 4.0-4.5 (3H, m), 4.3-4.7 (1H, 2m), 7.05 (1H, br s),
7.1 (2H, s), 7.15 (2H, s), 7.8 (2H, d), 7.9 (4H, d), 8.5-8.7 (1H,
2d).
4-[[[(4-Ch lor oph en yl)su lfon yl]am in o]car bon yl]ben zoyl-
N-E-[(4-h yd r oxy-3,5-d i-ter t-b u t ylp h en yl)a cet yl]-(S)-Lys-
OH (19): prepared according to General Method 5 and directly
treated without any further purification.
4-[[[(4-Ch lor oph en yl)su lfon yl]am in o]car bon yl]ben zoyl-
N-E-[(4-h yd r oxy-3,5-d i-ter t-b u t ylp h en yl)t h io]b u t yr oyl]-
(S)-Lys-OH (22): IR (Nujol) 1705, 1647, 1541, 1350, 1169
3-[[[[(4-H yd r oxy-3,5-d i-ter t-b u t ylp h en yl)t h io]a cet yl]-
a m in o]su lfon yl]-4-ch lor ob en zoyl-(S)-Va l-(2S,3a S,7a S)-
P h i-(R,S)-Va l-CF 3 (11i): IR (Nujol) 3633, 3307, 1763-1620
1
cm-1; H NMR (DMSO-d6) δ 1.35 (18H, s), 1.40 (4H, m), 1.7
(2H, q), 1.8 (2H, m), 2.15 (2H, t), 2.75 (2H, t), 3.0 (2H, q), 4.35
(1H, m), 6.95 (1H, s), 7.05 (2H, s), 7.75 (2H, d), 7.95 (4H, s),
8.05 (2H, d), 8.70 (1H, d), 12.6 (1H, br s).
1
cm-1; H NMR (DMSO-d6) δ 0.9 (12H, m), 1.3 (18H, s), 1.5-
2.3 (12H, m), 3.6 (2H, s), 4.5 (4H, m), 7.0 (2H, s), 7.1 (1H, s),
7.8 (1H, d), 8.25 (1H, d), 8.6 (1H, d).
4-[[[(4-Ch lor oph en yl)su lfon yl]am in o]car bon yl]ben zoyl-
N-E-[(4-h yd r oxy-3,5-d i-ter t-b u t ylp h en yl)a cet yl]-(S)-Lys-
(2S,3a S,7a S)-P h i-(R,S)-Va l-CF 3 (20): prepared according to
General Method 2, using the intermediate dipeptide Phi-Val-
CF3 (v-a ) described above; IR (Nujol) 3639, 3315, 1741, 1645
cm-1; 1H NMR (DMSO-d6) δ 1.0-2.0 (16H, m), 1.1 (6H, d), 1.4
(18H, s), 2.3 (2H, m), 3.0 (2H, m), 3.2 (2H, s), 6.6 (1H, s), 6.9
(2H, s), 7.4 (2H, d), 8.0 (6H, m).
4-[[[(2-(R,S)-6-Hyd r oxy-2,5,7,8-tetr a m eth ylch r om a n -2-
yl)car bon yl]am in o]su lfon yl]ben zoyl-(S)-Val-(S)-Abo-(R,S)-
Va l-CF 3 (11j): IR (Nujol) 3700-3000, 1761, 1730-1626, 1354,
1150 cm-1; 1H NMR (DMSO-d6) δ 0.8-1.0 (12H, m), 1.3-1.75
(8H, m), 1.35 (3H, s), 1.8-2.3 (6H, m), 1.9 (3H, s), 2.1 (6H,
2s), 2.4 (1H, m), 4.0-4.7 (4H, 4m), 6.6-6.9 and 7.1-7.4 (1H,
m), 7.4 (1H, br s), 7.8-7.95 (4H, 2d), 8.3-8.6 (1H, 2d), 11.8
(1H, br s).
4-[[[(2,6-Dim eth yl-3,5-dicar beth oxy-1,4-dih ydr opyr idin -
4-yl)ca r b on yl]a m in o]su lfon yl]b en zoyl-(S)-Va l-(S)-Ab o-
(R,S)-Va l-CF 3 (11k ): IR (Nujol) 3330, 1730-1650, 1350, 1122
cm-1; 1H NMR (DMSO-d6) δ 0.8-1.0 (12H, n), 1.1 (6H, t), 1.2-
1.7 (8H, m), 1.8-2.2 (3H, m), 2.25 (6H, s), 3.95 (4H, m), 4.0-
4.4 (3H, m), 4.65 (1H, m), 4.85 (1H, s), 7.1 (2H, d), 7.3-7.5
(2H, m), 7.65 (2H, d), 7.80 (4H, m), 8.5 (1H, 2m), 8.75 (1H, s).
4-[[[[4-Ch lor o-3-[[[[(4-h yd r oxy-3,5-d i-ter t-bu tylp h en yl)-
t h io]a ce t yl]a m in o]su lfon yl]p h e n yl]ca r b on yl]a m in o]-
su lfon yl]b e n zoyl-(S )-Va l-(2S ,3a S ,7a S )-P h i-(R ,S )-Va l-
CF 3 (14a ): IR (Nujol) 3340, 1761-1622 cm-1; 1H NMR (DMSO-
d6) δ 0.9 (12H, m), 1.2-2.3 (8H, m), 1.3 (18H, s), 3.6 (2H, s),
4-[[[(4-Ch lor oph en yl)su lfon yl]am in o]car bon yl]ben zoyl-
N-E-[[(4-h yd r oxy-3,5-d i-ter t-bu tylp h en yl)th io]bu tyr oyl]-
(S)-Lys-(2S,3a S,7a S)-P h i-(R,S)-Va l-CF 3 (23): IR (Nujol)
1
3600-3000, 1639, 1541 cm-1; H NMR (DMSO-d6) δ 0.7-1.0
(6H, m), 1.1-1.8 (19H, m), 1.35 (18H, s), 2.15 (3H, m), 2.75
(3H, m), 3.0 (2H, m), 4.0 (1H, m), 4.40 (1H, m), 4.55 (1H, m),
7.05 (2H, s), 7.50 (2H, d), 7.85 (4H, m), 7.92 (2H, d).
N-(ter t-Bu tyloxyca r bon yl)-S-(4-h yd r oxy-3,5-d i-ter t-bu -
tylp h en yl)-(S)-Cys-OCH3 (27). A 8.2 g (0.039 mol) portion
of R-[(tert-butyloxycarbonyl)amino]-â-chloropropionic acid (26)
was reacted under nitrogen with thiophenol xii (11.9 g, 0.05
mol) and potassium carbonate (5.53 g, 0.04 mol) in dry
acetonitrile (120 mL) at 65 °C for 20 h. After filtration and
concentration under reduced pressure, the residue was dis-

Dual Inhibition of HLE and Lipid Peroxidation
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1917
solved in ethyl acetate. The organic layer was washed with
water to neutral pH and then with brine. Drying (CaSO4) and
evaporation gave a crude solid, which was purified by column
chromatography (cyclohexane-EtOAc, 90:10). The resulting
solid was triturated with pentane to give 9.4 g (55%) of the
percent inhibition of pulmonary hemorrhage in treated ani-
mals as compared to control.
F ou r teen -Da y Em p h ysem a Mod el.27 Six male golden
Syrian hamsters weighing 100-120 g were used in each group.
Anesthesia and drug administration were performed as de-
scribed above. Drugs were solubilized in water and injected
it at a dose of 100 nmol in a 0.1 mL volume. Three hours later,
purified pig pancreatic elastase (Elastin Products Co.), 60 IU
in 0.2 mL of saline, was administered it. After 14 days, the
animals were killed by administration of a nembutal overdose.
Lungs were inflated with 4% neutral buffered formalin under
a 22 cm formalin pressure. Lungs were embedded in paraffin
(5 µm thick sections, stained with hematoxylineosin). Cross
sections of the intact lungs were performed on each animal.
Signs of pulmonary emphysema were estimated by measure
of mean linear intercepts according to Dunnill.31
title intermediate: IR (Nujol) 3600, 3336, 1732, 1695 cm-1
;
1H NMR (DMSO-d6) δ 1.4 (18H, s), 2.95-3.2 (2H, dd), 3.6 (3H,
s), 4.1 (1H, m), 7.15 (2H, s), 7.3 (1H, d).
S-(4-Hydr oxy-3,5-di-ter t-bu tylph en yl)-(S)-Cys-OCH3, h y-
d r och lor id e (28): prepared according to General Method 1;
1
IR (Nujol) 3633, 3400-1980, 1751 cm-1; H NMR (DMSO-d6)
δ 1.4 (18H, s), 3.4 (3H, s), 3.6 (2H, m), 4.4 (1H, m), 5.3 (1H, s),
7.3 (2H, s).
4-[[[(4-Ch lor oph en yl)su lfon yl]am in o]car bon yl]ben zoyl-
S-(4-h yd r oxy-3,5-d i-ter t-bu tylp h en yl)-(S)-Cys-OCH3 (29):
prepared according to General Method 3 and directly treated
without any further purification.
Ack n ow led gm en t. The authors wish to thank Dr.
J ean-Paul Vilaine for evaluation of compound 14a on
the human LDL peroxidation assay, Dr. J ean-Paul
Volland and his group for analytical and spectral
studies, and Mrs. Danielle Pommier for expert technical
assistance.
4-[[[(4-Ch lor oph en yl)su lfon yl]am in o]car bon yl]ben zoyl-
S-(4-h ydr oxy-3,5-di-ter t-bu tylph en yl)-(S)-Cys-OH (30): pre-
pared according to General Method 5; IR (Nujol) 3629, 3400-
1
2500, 1707, 1653 cm-1; H NMR (DMSO-d6) δ 1.35 (18H, s),
3.1-3.4 (2H, m), 4.5 (1H, m), 7.1 (3H, m), 7.6-8.0 (8H, m),
8.8 (1H, d).
4-[[[(4-Ch lor oph en yl)su lfon yl]am in o]car bon yl]ben zoyl-
S-(4-h yd r oxy-3,5-d i-ter t-b u t ylp h en yl)-(S)-Cys-(2S,3a S,-
7a S)-P h i-(R,S)-Va l-CF 3 (31): prepared according to General
Refer en ces
1
Method 2; IR (Nujol) 3650-2500, 1761, 1643, 1539 cm-1; H
(1) For a complete review on HLE and its synthetic inhibitors, see:
Edwards, P. D.; Berstein, P. R. Synthetic Inhibitors of Elastase.
Med. Res. Rev. 1994, 14, 127-194.
(2) J anoff, A. Elastase and Emphysema. Current Assessment of the
Protease-Antiprotease Hypothesis. Am. Rev. Respir. Dis. 1985,
132, 417-433.
(3) Merritt, T. A.; Cochrane, C. G.; Holcomb, K.; Bohl, B.; Hallman,
M.; Strayer, D.; Edwards, D.; Gluck, L. Elastase and R1-PI
Proteinase Inhibitor Activity in Tracheal Aspirates during
Respiratory Distress Syndrome. J . Clin. Invest. 1983, 72, 656-
666.
NMR (DMSO-d6) δ 0.6-0.95 (6H, m), 1.1-2.5 (13H, m), 1.35
(18H, 2s), 3.10 (2H, m), 4.0 (1H, m), 3.35 (1H, m), 4.5 (1H, m),
7.15 (2H, m), 7.45 (2H, d), 7.85 (4H, m), 7.90 (2H, t).
En zym a tic In h ibition of HLE.28 Inhibition of HLE was
assayed spectrophotometrically at 37°C by continuous moni-
toring of the release of p-nitroaniline at 410 nm from the
substrate methoxysuccinyl-Ala-Ala-Pro-Ala-p-nitroanilide. Re-
sults are listed in Tables 1 and 2 and are expressed in terms
(4) Hanley, M. E.; Repine, J . E. Elastase and Oxygen Radicals:
synergistic Interactions. Agents Action 1993, Suppl. 42, 39-47.
(5) Ishizaki, T.; Kishi, Y.; Sasaki, F.; Takahashi, H.; Ameshima, S.;
Onishi, T.; Sakai, T.; Kasamatsu, A.; Nakai, T.; Miyabo, S.
Probucol, an oral hypocholesterolemic agent, inhibits acute
tobacco smoking induced decrease in plasma antielastase activity
and ferroxydase activity in rats. Oxygen Radicals; Elsevier: New
York, 1992; pp 573-576. Kim Seow, W.; Thong, Y. H.; Nelson,
R. D.; Macfarlane, G. D.; Herzberg, M. C. Nicotine-induced
Release of Elastase and Eicosanoids by Human Neutrophils.
Inflammation 1994, 18, 119-127.
of IC50
.
Lip id P er oxid a tion Assa y: (a ) Hep a tic Micr osom es.
Rat livers were homogenized in phosphate buffer (0.01 M, pH
7.4). After differential centrifugation, microsomal proteins
were used at 0.5 mg/mL for the lipid peroxidation assay and
incubated with FeCl3 (100 µM) and ascorbate (10 µM) for 30
min.
Lipid peroxidation was measured as thiobarbituric acid-
reactive substances (TBARS) by mixing 200 µL of the sample
with 2 mL of a 0.3% thiobarbituric acid solution in 15%
trichloroacetic acid and 0.02% tert-butylhydroxytoluene. The
mixture was kept at 95°C for 25 min, and the assessment of
MDA-thiobarbituric acid complex was determined by measur-
ing the absorbance of the solution at 532 nm.29
(b) Hu m a n LDL Oxid a tion . LDL were prepared from
fresh normal human plasma by sequential ultracentrifugation
according to Havel et al.30 Oxidation was performed by
incubating 0.2 mg of LDLwith 5 µM CuSO4 in 1 mL of serum-
free HamF10 medium for 24 h at 37°C with or without test
products. The extent of lipid peroxidation was assessed by
measuring the TBARS according to the same method as
described for hepatic microsomes.
Ela sta se-In d u ced Hem or r h a ge in th e Ha m ster Lu n g.25
Ten male golden Syrian hamsters 10 weeks of age, weighing
100-120 g, were used in each group. Hamsters were anes-
thetized by ip injection of nembutal, and the trachea were
surgically exposed. Drugs solubilized in 20% DMSO were
injected intratracheally (it) at a dose of 15 nmol in a 0.1 mL
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of BAL (bronchoalveolar lavage) fluid from each animal.
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J M960772Z