Discovery and biological activity of orally active peptidyl trifluoromethyl ketone inhibitors of human neutrophil elastase

Article abstract of DOI:10.1021/jm960819g

Previously we had shown that tripeptidyl trifluoromethyl ketones (TFMKs) possessing an N-terminal diarylacylsulfonamide, such as ICI 200,880 and ICI 200,355, displayed unparalleled protection against the lung damage induced by human neutrophil elastase (H

Full text of DOI:10.1021/jm960819g

1876
J . Med. Chem. 1997, 40, 1876-1885
Discover y a n d Biologica l Activity of Or a lly Active P ep tid yl Tr iflu or om eth yl
Keton e In h ibitor s of Hu m a n Neu tr op h il Ela sta se
Philip D. Edwards,*,† Donald W. Andisik,† Craig A. Bryant,† Barbara Ewing,‡ Bruce Gomes,§ J oseph J . Lewis,†
Donna Rakiewicz,† Gary Steelman,† Ann Strimpler,§ Diane A. Trainor,† Paul A. Tuthill,† Russell C. Mauger,†
Chris A. Veale,† Richard A. Wildonger,† J oseph C. Williams,§ Donald J . Wolanin,† and Mark Zottola†
Departments of Medicinal Chemistry, Pulmonary Pharmacology, and Drug Disposition and Metabolism,
ZENECA Pharmaceuticals, A Business Unit of ZENECA Inc., 1800 Concord Pike, P.O. Box 15437,
Wilmington, Delaware 19850-5437
Received December 4, 1996X
Previously we had shown that tripeptidyl trifluoromethyl ketones (TFMKs) possessing an
N-terminal diarylacylsulfonamide, such as ICI 200,880 and ICI 200,355, displayed unparalleled
protection against the lung damage induced by human neutrophil elastase (HNE) when the
inhibitors were administered intratracheally. Since the diarylacylsulfonamides were designed
specifically to afford a long residence time in the lung, it was not unexpected that inhibitors
from this class of TFMKs were not active when administered orally. Upon evaluating a large
number of peptidyl TFMKs possessing a variety of N-terminal groups, several compounds were
identified which demonstrated oral activity. Compounds were evaluated for their oral activity
by measuring their ability to inhibit the increase in lung weight relative to body weight (Lw/
Bw), the increase in red blood cells, and the increase in white blood cells induced by
intratracheally administered HNE (100 µg/hamster). A number of tripeptidyl trifluoromethyl
ketones containing neutral N-terminal groups displayed good oral activity, while those
containing basic, acidic, or polar groups did not. Compound 50, possessing an N-terminal
4-(CH3O)C6H4CO group, was particularly effective, reducing Lw/Bw by 77%, red cells by 89%,
and white cells by 91% when dosed at 37.5 mg/kg orally. Thus, by modifying the N-terminal
group of tripeptidyl TFMKs, inhibitors can be designed which are effective in vivo when
administered either orally or intratracheally.
In tr od u ction
ability as a result of poor absorption, extensive metabo-
lism, and/or rapid elimination. Thus, we mounted a
large effort to identify nonpeptidic compounds that
might have a superior in vivo profile to that of peptidic
inhibitors following oral administration. This work led
to the discovery of several series of nonpeptidic trifluo-
romethyl ketones including the pyridones,13-17 pyrimi-
dinones,18 carbolines,19 and pyridopyrimidines.20 Com-
pounds from some of these series demonstrated excellent
oral activity in an animal model of elastase-induced lung
injury.16,18
For more than a decade, we have been developing
inhibitors of the serine protease human neutrophil
elastase (HNE, EC 3.4.21.37).1 HNE has been impli-
cated in initiating or exacerbating a number of patho-
logical conditions such as pulmonary emphysema,2
cystic fibrosis,3 and chronic bronchitis.4 These pulmo-
nary diseases could effectively be treated with drugs
administered via an aerosol. Therefore, a major part
of our research has focused on the development of HNE
inhibitors which had a long duration of action following
intratracheal administration. This effort has resulted
in the identification of three series of peptidyl electro-
philic ketones that displayed excellent in vivo activity
in animal models following intratracheal administra-
tion: trifluoromethyl ketones,5,6 R,R-difluoro-â-keto
amides7, and R-ketobenzoxazoles.8
However, there are a number of elastase-mediated
diseases for which intratracheal administration is either
less than optimal or inappropriate. Included among this
group of conditions are acute respiratory distress syn-
drome,9 myocardial reperfusion injury,10,11 and arthri-
tis.12 Thus, having successfully developed a number of
peptide-based elastase inhibitors suitable for aerosol
administration, we focused our efforts on the identifica-
tion of compounds which could be administered orally.
Frequently, peptides have only limited oral bioavail-
Several classes of orally active, nonpeptidic inhibitors
of HNE have been described including benzisothiazo-
lones21 and â-lactams.22-24 Although peptides generally
suffer from poor oral bioavailability, there are examples
of the successful development of orally active peptides,
for example renin inhibitors.25 Thus, while we pursued
the design of orally active nonpeptidic HNE inhibitors,
we also investigated the identification of peptidic inhibi-
tors which would possess the desired level of in vivo
activity following oral administration. Peptidyl pen-
tafluoroethyl ketones (PFEKs) have been described
which inhibit elastase-induced lung injury following oral
administration.26-28 In this report we describe the
discovery of several peptidyl trifluoromethyl ketones
which possess excellent oral activity. Recently it was
reported that peptidyl TFMKs were not orally active
while the corresponding PFEKs were active.27 Not only
have we identified orally active peptidyl TFMKs, but
have identified ones which have superior activity to that
reported for the pentafluoroethyl ketones. These results
† Department of Medicinal Chemistry.
‡ Drug Disposition and Metabolism Department.
§Department of Pulmonary Pharmacology.
X Abstract published in Advance ACS Abstracts, May 15, 1997.
S0022-2623(96)00819-9 CCC: $14.00 © 1997 American Chemical Society

Inhibitors of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1877
Sch em e 1. Synthesis of Peptidyl Trifluoromethyl Ketones 1-58
highlight a finding we have previously observed with
peptidyl electrophilic ketones: interchanging mecha-
nistic pharmacophores does not lead to predictable
biological activity since the observed in vivo activity is
a result of the physiochemical properties of the entire
molecule.
the trifluoromethyl alcohols III. (See Table 1 for
reagents.) Oxidation of alcohols III gave the corre-
sponding trifluoromethyl ketones. Peptidyl TFMKs
containing N-terminal substituents incorporating esters
or phenolic acetates were converted to the corresponding
acids and phenols via either hydrogenation or base
hydrolysis. Further modification of the keto acids was
achieved by coupling with various nucleophilic nitrogen
compounds. TFMKs with an N-terminal sulfone (III-3
and III-33) were prepared by oxidation of the corre-
sponding sulfide (III-4) or sulfoxide (III-33a ) to the
sulfone and subsequent oxidation of the trifluoromethyl
Ch em istr y
The tripeptidyl TFMKs in this study were prepared
starting from the previously described H-Val-Pro-Val-
trifluoromethyl alcohol II29 (Scheme 1). Amino alcohol
II was coupled with a variety of electrophiles to yield

1878 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Edwards et al.
Ta ble 1. Method of Preparation and Physicochemical Properties of Peptidyl TFMKs 1-58
step in Scheme 1
yield
MSd
(M + 1)
compd
R
1
2a
2
3
4
(%)
formulae
1
2
3
4
5
6
7
8
9
1-Npth-SO2NHCOCH2CH2CO
4-(CH3SO2NHCO)C6H4CO
4-(CH3SO2)C6H4CO
4-(CH3S)C6H4CO
4-(CH3CONH)C6H4SO2)
4-(HO2C)C6H4CO
trans-4-(HO2C)C6H4CHdCHCO
3,5-(HO2C)2C6H4CO
HO2CCO
4-(HO2C)C6H4NHCO
3-(HO2C)-2-Npth-CO
HO2CCH2CH2CO
CH3O2CCH2CH2CO
CH3O2CCO
4-(C2H5O2C)C6H4NHCO
3-(PhCH2O2C)-2-Npth-CO
trans-4-(EtO2C)C6H4CHdCHCO
3,5-(CH3O2C)2C6H4CO
3-pyridyl-CO
4-pyridyl-CO
2-pyridyl-CO
12;C
10;C
45
60
86
84
84
93
73
91
72
92
61
87
74
57
78
84
65
94
32
24
38
21
93
46
70
11
26
55
79
76
42
67
40
61
41
37
68
74
66
47
91
ND
ND
548
516
ND
ND
ND
501
438
ND
ND
ND
ND
452
ND
ND
ND
586
471
471
471
460
513
584
650
408
507
555
515
ND
ND
498
ND
506
540
551
536
596
632
ND
ND
ND
ND
ND
502
530
488
504
ND
500
486
ND
520
498
470
530
462
562
C30H37F3N4O7S‚0.5H2O
C25H34F3N5O7‚0.5H2O
C24H32O6N3F3S‚1.0H2O
C24H32O4N3F3Sf
C24H33F3N4O6S‚1.0H2O
C24H30N3F3O6‚1.0H2O
C26H32F3N3O6‚0.35H2O
C25H30N3O8‚0.8H2O
C18H26F3N3O6‚0.75H2O
C24H31F3N4O6‚0.5H2O
C28H32F3N3O6
intermediate
intermediate
C19H28F3N3O6‚0.65H2O
C26H35F3N4O6
C35H38F3N3O6‚0.75H2O
C28H36F3N3O6
C27H34F3N3O8
C22H29F3N4O4‚1.1H2O
C22H29F3N4O4‚0.65H2O
C22H29F3N4O4‚0.33H2O
C20H28F3N5O4‚1.0H2O
C25H35F3N4O4‚0.6H2O
C28H40F3N5O5‚0.5H2O
C34H46F3N3O6‚1.0HCl‚1.0H2O
C18H28F3N3O4
C23H37F3N4O5‚0.9H2O
C27H37N4O5F3‚0.9H2O
C23H29F3N4O6‚0.3H2O
C23H31F3N4O4‚0.75H2Og
C23H30F4N4O4
4;L
N
N
P
A
H
52;S
17;S
18;S
14;S
15;S
16;U
13;S
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
K
H
I
Q
N
P
B
E
A
A
A
A
A
A
O
R
O
N
N
N
N
N
4-imidazole-CO
4-(Me2N)C6H4CO
4-(Me2NCH2CH2NHCO)C6H4CO
(PhCH2CH2CH2)2
CH3CO
CH3CON(nPr)CH2CO
CH3CON(Bn)CH2CO
4-(NO2)C6H4CO
PhNHCO
4-F-C6H4NHCO
CF3SO2
1-Adamantyl-SO2
PhSO2
6;B
J
F
A
A
A
I
P
N
N
N
N
P
I
P
H
H
H
H
H
H
H
H
H
B
D
B
N
Q
N
N
N
N
Q
N
O
O
O
O
C17H25F6N3O5S‚0.25H2O
C26H40N3O5F3S‚0.4H2O
C22H30F3N3O5S‚0.25H2O
C22H29ClF3N3O5S
M
4-Cl-C6H4SO2
4-(NO2)C6H4SO2
4-(CH3O)C6H4SO2
pentafluorophenyl-SO2
2,4,6-(iPr)3C6H4SO2
2-Npth-SO2
4-(CH3CO2)C6H4CO
3,4-(CH3CO2)2C6H4CO
3,5-(CH3CO2)2C6H4CO
4-(HO)C6H4CO
3,4-(HO)2C6H4CO
2,6-(CH3O)2C6H4CO
4-F-C6H4CO
4-F-C6H4OC(O)
Cbz
4-(CH3O)C6H4CO
PhOC(O)
4-(CH3O2C)C6H4CO
2-Npth-CO
3,4-(CH3)2C6H4CO
PhCO
3,4-(CH3O)2C6H4CO
CF3CO
C22H29F3N4O7S
C23H32F3N3O6S‚0.25H2O
C22H25F8N3O5S‚0.5H2O
C31H48F3N3O5S‚0.20H2O
C26H32F3N3O5S‚0.5H2O
C25H32N3O6F3‚0.6H2O
not isolated
C27H34F3N3O8‚0.25H2O
C23H30N3O5F3‚0.55H2O
C23H30F3N3O6‚1H2Oh
C25H34N3O6F3
C23H29F4N3O4‚0.2H2O
C23H29F4N3O5‚0.25H2O
C24H32F3N3O5‚0.5H2O
C24H32F3N3O5‚0.33H2O
C23H30F3N3O5‚0.4H2O
C25H32F3N3O6‚0.5H2O
C27H32F3N3O4‚0.75H2O
C25H34F3N3O4
61
62
32
60
44
67
45
87
80
55
71
65
62
32
62
41
41;T
42;T
B
A
H
N
N
N
I;P
N
N
Q
O
N
N
N
N
N
H
H
C
A
B
A
B
G
A
C23H30F3N3O4‚0.25H2O
C25H34N3O6F3
C18H25F6N3O4‚0.2H2O
C29H34F3N3O5‚0.5H2O
4-(PhO)C6H4CO
a
A, RCO2H, EDAC, with or without DMAP; B, RCO2H (or RNH2 for 24), EDAC, HOBt; C, RNH2 (or RCO2H for 52), DCC, HOBT or
i
DMAP; D, RCO2H, BuOCOCl, NMM; E, RCO2H, CDI, THF; F, Ac2O, TEA; G, CF3CO2Et, TEA; H, active chloride (ROCOCl, RSO2Cl,
RSOCl, or RCOCl), TEA or K2O3; I, RNCO, TEA; J , ROH, Tf2O, Proton Sponge; K, RCOCl, NaOH; L, MCPBA; M, KMnO4; N, EDAC,
d
Cl2CHCO2H, DMSO, toluene; O, DMP; P, (COCl)2, DMSO; Q, Ac2O, DMSO; R, CrO3, pyridine; S, NaOH; T, K2CO3; U, H2, Pd/C. ND )
not determined. e All elemental analyses for C, H, and N agree within (0.4% of calculated values unless otherwise noted. f N: calculated,
g
h
7.42; found, 6.31. N: calculated, 11.25; found, 10.72. N: calculated, 8.08; found, 7.44
alcohol to the ketone. All compounds in this report were
roughly 1:1 mixtures of epimers at the stereogenic
center R to the ketone carbonyl group and thus are
mixtures of diastereomers.
P h a r m a cologica l Eva lu a tion
The oral activity of the TFMK inhibitors was assessed
in two in vivo models. When HNE is administered
intratracheally to hamsters, a hemorrhagic lesion de-

Inhibitors of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1879
set of physicochemical properties before we identified
the unique properties of the acylsulfonamides. Thus we
had a large stable of compounds which could be tested
orally. Table 2 lists representative examples of the
compounds which were tested orally in the ALIM.
Consistent with our findings with the peptidyl acylsul-
fonamides, other acidic compounds were inactive (7-
11). However, the corresponding esters were also
inactive (17-18). A number of basic compounds were
found to be inactive in our oral model as well (19-25).
Finally, a large collection of neutral compounds were
evaluated. These inhibitors contained N-caps possess-
ing a broad range of lipophilicities and functional groups
including amides (26-29), ureas (30 and 31), alkyl and
aryl sulfonamides (32-40), and a number of phenols (44
and 45), phenolic esters (43), and phenolic ethers (46).
While the classification used to group the peptidic
inhibitors above is somewhat ill-defined and overlap-
ping, it serves to highlight the general strategy we
employed: modification of the charge, hydrogen bond
donor/acceptor ability, hydrophobicity/lipophilicity, and
solubility. The lack of activity for compounds 1-5,
7-11, 17-40, and 43-47 indicates that obtaining the
correct physical properties of a compound to promote
oral absorption/activity within this series of peptidyl
TFMKs is not simply a function of the presence of a
particular class of N-terminal group.
F igu r e 1. ICI 200,880 and ICI 200,355.
velops which is characterized by an increase in lung
weight relative to body weight (Lw/Bw) as a result of
edema, an increase in white blood cells, and an increase
in red blood cells. In the acute lung injury model
(ALIM), all three parameters are measured.5 In the
acute hemorrhagic assay (AHA), only the degree of
hemorrhage is determined as measured by the amount
of hemoglobin produced.30 In both assays, the effect of
the test compound is compared to HNE and saline
controls. Compounds showing 100% inhibition are
identical to saline, and those showing 0% are identical
to HNE control. Compounds with a negative value for
per cent inhibition exacerbate the effects of HNE and
produce a greater degree of lung damage than HNE
alone.
One possible explanation for the lack of oral activity
observed for the peptidyl TFMKs described above is that
the physical or chemical properties of the trifluoro-
methyl group itself renders the attached peptide inac-
tive following oral administration. Indeed, it was
recently reported that peptidyl TFMKs that lacked oral
activity could be converted into compounds with good
activity in the AHA following oral dosing by replacing
the trifluoromethyl group with a pentafluoroethyl
Resu lts a n d Discu ssion
The first phase of our elastase program was directed
at the development of elastase inhibitors which would
have the physical properties suitable for administration
as an aerosol. Our primary goal was the identification
of compounds which had a long duration of action in
the ALIM following it administration. Within the series
of trifluoromethyl ketones, ICI 200,880 and ICI 200,355
(Figure 1) emerged as leading compounds with a
superior duration of action. From a structural stand-
point, this is the result of the diarylacylsulfonamide
N-terminal group or N-cap. In physical terms, this long
duration of action is the result of the compound’s
relative inability to cross the lung epithelial membrane,
pass through the interstitial space, and finally cross the
endothelium and enter the blood stream.
Thus, it was no surprise that peptides with acylsul-
fonamide N-caps (ICI 200,355, 1, and 2, Table 2) were
not active in the ALIM following oral administration.
The same properties of the peptidyl acylsulfonamides
which contribute to their long duration of action follow-
ing it administration also retard their absorption from
the gastrointestinal tract into, and migration out of, the
blood stream. Pharmacokinetic studies indicated that
the acylsulfonamides were not absorbed into the sys-
temic circulation from the intestinal tract. One possible
explanation for the lack of oral absorption of these
peptides might be that they are ionized at the pH of
the intestinal tract. However, related compounds in
which the charge of the acylsulfonamide is removed (3-
5) were also inactive.
group.27
Thus, the TFMKs 60 and 62 (Table 3) were
inactive at a dose of 50 mg/kg when administered 30
min prior to a 50 µg it dose of HNE, while the
pentafluoroethyl analogs 59 and 61 significantly re-
duced the lesion at the same dose and displayed
excellent reduction in the HNE (10 µg/animal) induced
hemorrhage when dosed at 25 mg/kg. Thus it would
appear from these results that the pentafluoroethyl
group possesses special properties which render an
attached peptide orally active.
The reported difference in oral activity between the
TFMKs and the PFEKs may reside in the relative
degree of ketone hydration. A number of studies have
shown that the TFMKs are heavily, if not completely,
hydrated in aqueous solution while other electrophilic
peptidyl ketones such as R-keto esters, R,R-difluoro
â-keto amides, and R-keto hetereocycles are not signifi-
cantly hydrated.31 Pentafluoroethyl ketones have been
shown to be much less hydrated than TFMKs, being
only about 80% hydrated. Thus, it has been speculated
that the hydrated form of the TFMKs is either not well
absorbed or very rapidly eliminated.27
However, we have found several peptidyl trifluoro-
methyl ketones that possess excellent oral activity,
exceeding the reported activity of pentafluoroethyl
ketones. Compounds 48-58 (Table 2) are part of a
subset of peptidyl TFMKs we have found that demon-
strate oral activity in the ALIM. For the most part,
these compounds are all structurally similar, possessing
a relatively small aromatic ring attached to the peptide
backbone through an amide or carbamate linkage, and
It was clear, therefore, that a different SAR would
have to be developed for an orally active elastase
inhibitor than that for an inhibitor delivered intratra-
cheally. As part of our aerosol program, we had
prepared a large number of compounds with a diverse

1880 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Edwards et al.
Ta ble 2. Potency and Oral Activity in ALIM Assay of Peptidyl TFMKs 1-58
Lw/Bwb,c
red cellsb,c
white cellsb,c
a
compd
R
Ki (nM)
ICI 200,355
1
2
3
4
5
7
8
9
4-[4-Br-PhSO2NHCO]PhCO
1-Npth-SO2NHCOCH2CH2CO
4-(CH3SO2NHCO)C6H4CO
4-(CH3SO2)C6H4CO
4-(CH3S)C6H4CO
4-(CH3CONH)C6H4SO2
trans-4-(HO2C)C6H4CHdCHCO
3,5-(HO2C)2C6H4CO
HO2CCO
4-(HO2C)C6H4NHCO
3-(HO2C)-2-Npth-CO
trans-4-(EtO2C)C6H4CHdCHCO
3,5-(CH3O2C)2C6H4CO
3-pyridyl-CO
4-pyridyl-CO
2-pyridyl-CO
4-imidazole-CO
4-(Me2N)C6H4CO
4-(Me2NCH2CH2NHCO)C6H4CO
(PhCH2CH2CH2)2
CH3CO
0.62 ( 0.1
6.1 ( 1.1
4.2 ( 0.7
2.0 ( 0.6
2.3 ( 0.7
55 ( 6.5
1.8 ( 0.7
43 ( 8.7
810 ( 170
10 ( 2.0
1.7 ( 0.6
0.43 ( 0.13
11 ( 4.0
7.8 ( 1.7
3.8 ( 1.0
20 ( 4.0
63 ( 8.0
2.5 ( 0.9
5.4 ( 4.2
1.7 ( 0.4
45 ( 12
2.0 ( 0.7
0.96 ( 0.5
1.2 ( 0.4
2.6 ( 0.7
11 ( 2.1
150 ( 40
1.2 ( 0.34
14 ( 6.0
3.3 ( 0.5
5.2 ( 1.6
8.0 ( 2.7
1.6 ( 0.2
3.2 ( 1.0
2.7 ( 0.7
60 ( 30
3.9 ( 0.4
16 ( 4.0
15 ( 5.0
2.9 ( 0.9
0.63 ( 0.2
1.6 ( 0.3
1.9 ( 0.8
1.3 ( 0.4
1.8 ( 0.2
0.87 ( 0.13
1.0 ( 0.4
6.8 ( 1.1
1.1 ( 0.3
15 ( 3.0
0.41 ( 0.2
-0.5 (10)
-80* (10)
-9 (9)
20 (9)
2 (9)
40* (9)
2 (10)
73* (8)
0.3 (8)
-11 (10)
-12 (7)
13 (7)
17 (8)
29 (8)
17 (8)
25 (8)
1 (6)
9 (9)
64* (8)
-27 (8)
39* (10)
5 (7)
30 (9)
-212* (8)
43 (9)
-16 (10)
20 (9)
58 (10)
36 (9)
32 (9)
2 (9)
13 (10)
-45 (8)
2 (7)
-1 (8)
23 (8)
68 (10)
-105 (7)
38 (6)
10
11
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
48 (7)
53 (8)
-122* (9)
-48 (7)
6 (9)
6 (9)
-6 (9)
-15 (9)
8 (*)
-42 (9)
36 (10)
20 (9)
23 (6)
-45 (9)
44 (8)
-267* (8)
0 (10)
71 (7)
-23 (10)
6 (9)
-39 (9)
-120 (9)
78 (10)
36 (10)
4 (10)
8 (10)
51* (8)
53* (9)
16 (9)
70* (10)
-8 (8)
33 (10)
38 (8)
-70 (9)
17 (8)
41 (10)
28 (9)
62 (*10)
28 (10)
-16 (7)
-69 (8)
-21 (7)
21 (7)
CH3CON(nPr)CH2CO
CH3CON(Bn)CH2CO
4-(NO2)C6H4CO
PhNHCO
4-F-C6H4NHCO
CF3SO2
1-adamantyl-SO2
PhSO2
4-Cl-C6H42
4-(NO2)C6H4SO2
4-(CH3O)C6H4SO2
pentafluorophenyl-SO2
2,4,6-(iPr)3C6H4SO2
2-Npth-SO2
3,5-(CH3CO2)2C6H4CO
4-(HO)C6H4CO
3,4-(HO)2C6H4CO
2,6-(CH3O)2C6H4CO
4-F-C6H4CO
4-F-C6H4OC(O)
Cbz
4-(CH3O)C6H4CO
PhOC(O)
4-(CH3O2C)C6H4CO
2-Npth-CO
3,4-(CH3)2C6H4CO
PhCO
3,4-(CH3O)2C6H4CO
CF3CO
24 (10
20 (10)
54 (10)
-2 (8)
-3 (10)
20 (8)
-36 (9)
29 (10)
-41 (9)
-36 (9)
29 (10)
-24 (10)
-17 (10)
-26 (10)
-38 (8)
35 (8)
-184* (8)
8 (10)
17 (8)
61 (9)
-48 (9)
7 (8)
-118* (9)
-42 (10)
27 (10)
-186* (7)
20 (9)
-12 (8)
28 (7)
-73* (10)
20 (8)
-44 (9)
-41 (10)
-177* (6)
22 (10)
-67 (10)
38 (9)
91* (8)
77 (9)
56* (8)
73* (10)
55* (8)
-7 (9)
7 (6)
44 (10)
63* (10)
53* (10)
77* (9)
62* (10)
49* (9)
43* (10)
75* (10)
62* (9)
66* (10)
67 (7)
23 (9)
99* (9)
78* (9)
89* (9)
97* (9)
50* (8)
50* (10)
91* (8)
62 (*9)
91* (9)
77* (6)
61* (9)
46 (10)
91* (6)
78* (10)
4-(PhO)C6H4CO
31 (10)
a
b
Inhibition of HNE-catalyzed hydrolysis of the synthetic substrate MeO-Suc-Ala-Ala-Pro-Val-pNa. Values of wet lung weight (Lw)
relative to body weight (Bw), red cells, and white blood cells are the percent reduction relative to HNE and saline controls: 100% indicates
identical with saline control; 0% indicates identical with HNE control; a negative value indicates parameter was worse than HNE control.
An asterisk (*) indicates value is statistically significant relative to control (p ) 0.05). Number in parentheses is number of animals used
in experiment. Dose of HNE was 100 mg/animal; dose of inhibitor was 37.5 mg/kg po administered 30 min prior to HNE administration.
c NT ) not tested in the particular assay.
incorporating small, relatively nonpolar substituents on
the aromatic ring. Particularly interesting is a com-
parison of the active compounds 48 and 56 with the
inactive analogs 47 and 46, respectively, which il-
lustrates the subtle balance of properties necessary for
obtaining oral activity.
400 µg/animal. This dose of enzyme causes an extensive
hemorrhagic lesion in the lung. Since the inhibitors
were administered directly into the lung in our it
program, compounds such as ICI 200,880 and ICI
200,355 were able to block the HNE-induced lung
damage when predosed up to 36 h prior to administra-
tion of the enzyme. However, this dose of enzyme
presents a very severe challenge for a compound ad-
ministered orally since a significantly larger dose of
We designed the ALIM5 as an in vivo screen to assist
in the development of compounds which would be
suitable for administration by aerosol. When evaluating
compounds intratracheally, we used an enzyme dose of

Inhibitors of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1881
Ta ble 3. Comparative Oral Activity of Peptidyl Trifluoromethyl and Pentafluoroethyl Ketones in AHA
compound must be administered orally to obtain an
equivalent concentration in the lung. Thus we chose a
dose of 100 µg/animal of enzyme when evaluating our
compounds orally.
while the TFMKs 60 and 62 were inactive in this model,
the TFMKs 50 and 51, even at low doses of inhibitor
(10 mg/kg), can significantly block the hemorrhage
induced by a 50 µg/animal insult of HNE. Significantly,
50 and 51 demonstrated a greater degree of protection
with a more severe enzyme challenge (50 µg/animal) and
smaller dose of inhibitor (10 mg/kg) than the PFEKs
59 and 61 at a lower dose of enzyme (10 µg/animal) and
larger dose of inhibitor (25 mg/kg).
Thus, it is not the properties of the electrophilic
ketone activating group per se which are responsible for
the differences in the oral activity between the TFMKs
and PFEKs. Rather, it is the overall physicochemical
properties of the molecules which render them active
or inactive following oral administration. Therefore, if
the extensive hydration of the TFMKs is a factor which
decreases oral activity, this can be compensated for by
the appropriate choice of N-terminal group. Clearly,
hydration of the ketone carbonyl does not preclude oral
activity. On the basis of the results from the current
study, the difference between the TFMKs and PFEKs
may have as much to do with the different lipophilicities
of the pentafluoroethyl and trifluoromethyl groups as
Even a dose of 100 µg/animal represents a fairly
severe challenge for an orally administered compound.
However, it is difficult to obtain a consistent hemor-
rhagic response in all three parameters measured in the
ALIM if a dose much less than 100 µg of HNE is used.
On the other hand, lower doses of HNE do generate a
reproducible response in the degree of hemorrhage. This
is the basis of the acute hemorrhagic assay (AHA), a
model that is similar to the ALIM (see Pharmacological
Evaluation section) except that only the degree of
hemorrhage is determined and quantified by spectro-
photometrically measuring the amount of hemoglobin
in lavage rather than the number of red cells.30
Because of the ease of analysis and the ability to
generate reproducible responses using low doses of
enzyme, the AHA has been widely used for evaluating
the activity of elastase inhibitors following oral admin-
istration. Table 3 lists the activity of several peptidyl
PFEKs and TFMKs in the AHA. It can be seen that

1882 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Edwards et al.
Meth od B: (2RS,3SR)-(4-Acetoxyben zoyl)-L-va lyl-N-[3-
(1,1,1-t r iflu or o-2-h yd r oxy-4-m e t h ylp e n t yl)]-L-p r olin -
a m id e (III-41). A 0 °C solution of amino alcohol II (1.00 g,
2.72 mmol) in THF (15 mL) was treated with 4-acetoxybenzoic
acid (738 mg, 4.08 mmol), HOBt (1.10 g, 8.16 mmol), and
EDAC (783 mg, 4.08 mmol), warmed to room temperature,
stirred for 18 h, and evaporated. The residue was taken up
in ethyl acetate, washed with 1 N HCl, saturated NaHCO3
and brine, dried (MgSO4), and evaporated. Purification by
flash chromatography eluting with diethyl ether afforded
trifluoromethyl alcohol III-41 (1.2 g, 86%) as a white solid:
it does with ketone hydration. As was observed with
the development of TFMKs having a long duration of
action following it administration, the in vivo activity
of peptidyl electrophilic ketones following oral admin-
istration is dependent upon the overall properties of the
entire molecule, and not upon a particular functionality.
Con clu sion
Several tripeptidyl TFMKs have been identified which
display excellent in vivo activity following oral admin-
istration in two different models of HNE-induced lung
injury. Within the series of TFMKs, the in vivo profile
can be controlled by modifications of the N-terminal
group. Thus, compounds containing a diarylacylsul-
fonamide, such as ICI 200,880, that displayed excellent
activity following it administration were not active
orally. By changing the N-terminal group to a small,
noncharged substituent, compounds with excellent orally
activity can be obtained. These results contradict a
previous report which concluded that TFMKs as a class
lacked oral activity as a result of extensive hydration
of the ketone carbonyl. Our results demonstrate that,
with the proper N-terminal group, peptidyl TFMKs
possess excellent oral activity. It should be possible to
extrapolate this finding to other peptidyl electrophilic
ketones. With the proper balance of physicochemical
properties between the ketone activating group and the
N-terminal substituents, oral activity should be obtain-
able in many different series of peptidyl ketones.
1
TLC Rf ) 0.38, diethyl ether; H NMR (250 MHz, DMSO-d6/
TFA) δ 0.86-1.01 (12H, m), 1.72-2.27 (6H, m), 2.29 (3H, s),
3.73 (1H, m), 3.81 (2H, m), 4.10 (1H, m), 4.45 (2H, m), 7.21
(2H, d, J ) 7.5 Hz), 7.96 (2H, d, J ) 7.5 Hz).
Meth od C: (2RS,3SR)-[[4-(Meth oxyca r bon yl)p h en yl]-
ca r bon yl]-L-va lyl-N-[3-(1,1,1-tr iflu or o-2-h yd r oxy-4-m eth -
ylp en tyl)]-L-p r olin a m id e (III-52). A 0 °C solution of amino
alcohol II (4.5 g, 12.2 mmol), 4-carbomethoxybenzoic acid (2.2
g, 12.2 mmol), and HOBt (3.3 g, 24.5 mmol) in THF (250 mL)
was treated with DCC (2.84 g, 13.8 mmol), warmed to room
temperature, stirred for 18 h, and filtered and the filtrate
evaporated. The residue was taken up in chloroform, washed
with 20% aqueous citric acid and saturated NaHCO3, dried
(MgSO4), and evaporated. Purification by flash chromatog-
raphy eluting with chloroform/methanol (97:3) afforded tri-
fluoromethyl alcohol III-52 (5.13 g, 79%) as a pale yellow
foam: TLC Rf ) 0.36 and 0.40, chloroform/methanol (95:5);
1H NMR (250 MHz, DMSO-d6) δ 0.83-1.20 (12H, m), 1.63-
2.23 (6H, m), 3.63 (1H, m), 3.71-3.90 (5H, m), 4.13 (1H, m),
4.30-4.55 (2H, m), 6.46 (0.5H, d, J ) 8.2 Hz), 6.56 (0.5H, d, J
) 8.2 Hz), 7.24 (0.5H, d, J ) 8.2 Hz), 7.67 (0.5H, d, J ) 8.2
Hz), 8.02 (4H, m), 8.75 (1H, t, J ) 8.2 Hz).
Meth od D: (2RS,3SR)-(3,4-Dia cetoxyben zoyl)-L-va lyl-
N-[3-(1,1,1-tr iflu or o-2-h yd r oxy-4-m eth ylp en tyl)]-L-p r olin -
a m id e (III-42). A -40 °C solution of 3,4-diacetoxybenzoic acid
(640 mg, 3.00 mmol) and N-methylmorpholine (1.0 g, 0.33 mL,
3.00 mmol) in THF (10 mL) was treated dropwise with isobutyl
chloroformate (0.411 g, 0.390 mL, 3.00 mmol). The resulting
suspension was stirred at -40 °C for 35 min and was treated
with a solution of amino alcohol II (1.00 g, 2.72 mmol) in
CH2Cl2 (2 mL), and the mixture was allowed to warm to room
temperature and stirred for 18 h. The reaction mixture was
diluted with ethyl acetate, washed with 1 N HCl, saturated
NaHCO3, and brine, dried (MgSO4), and evaporated. The
crude material was purified by flash chromatography, eluting
with chloroform/methanol (95:5). The fractions containing the
desired product were combined, dissolved in ethyl acetate,
washed with saturated NaHCO3, dried (MgSO4), and evapo-
rated to afford alcohol III-42 (1.16 g, 76%) as a solid: HPLC
tR ) 2.51 and 3.38, H2O/CH3CN (3:2), flow rate ) 2 mL/min,
Zorbax ODS analytical column (4.6 mm × 25 cm); 1H NMR
(250 MHz, DMSO-d6/TFA) δ 0.83 (6H m), 0.97 (6H, m), 1.72-
2.30 (6H, m), 2.31 (6H, s), 3.55-4.1 (4H, m), 4.32-4.58 (2H,
Exp er im en ta l Section
Gen er a l. Analytical samples were homogeneous by TLC
and afforded spectroscopic results consistent with the assigned
structures. Proton NMR spectra were obtained using either
a Bruker WM-250 or AM-300 spectrometer. Chemical shifts
are reported in parts per million (δ) relative to tetramethyl-
silane as internal standard. Mass spectra (MS) were recorded
on a Kratos MS-80 instrument or a Finnigan MAT-60 operat-
ing in the chemical ionization (DCI) mode (only peaks g10%
of the base peak are reported). Elemental analyses for carbon,
hydrogen, and nitrogen were determined by the Analytical
Section, ZENECA Pharmaceuticals, on a Perkin-Elmer 241
elemental analyzer and are within (0.4% of theory for the
formulas given. Analytical thin-layer chromatography (TLC)
was conducted on prelayered silica gel GHLF plates (Analtech,
Newark, DE). Visualization of the plates was accomplished
using either UV light or phosphomolybdic acid-ethanol or
iodoplatinate charring. Flash chromatography was conducted
on Kieselgel 60, 230-400 mesh (E. Merck, Darmstadt, West
Germany). Solvents were either reagent or HPLC grade.
Reactions were run at ambient temperature and under a
nitrogen atmosphere unless otherwise noted. Solvent mixtures
are expressed as volume:volume ratios. Solutions were evapo-
rated under reduced pressure on a rotary evaporator. All
starting materials were commercially available unless other-
wise indicated.
Gen er a l P r oced u r es for th e P r ep a r a tion of Tr iflu o-
r om eth yl Alcoh ols III. Meth od A: (2RS,3SR)-[[4-(Meth -
ylt h io)p h en yl]ca r b on yl]-L-va lyl-N-[3-(1,1,1-t r iflu or o-2-
h ydr oxy-4-m eth ylpen tyl)]-L-pr olin am ide (III-4). A solution
of amino alcohol II (1.00 g, 2.72 mmol), 4-(methylthio)benzoic
acid (520 mg, 3.09 mmol), and EDAC (650 mg, 3.39 mmol) in
DMF (15 mL) was treated with DMAP (754 mg, 6.17 mmol)
and stirred at room temperature for 18 h. The resulting
solution was diluted with ethyl acetate, washed with 1 N HCl,
saturated NaHCO3, H2O, and brine, dried (MgSO4), and
evaporated. The crude material was purified by flash chro-
matography eluting with chloroform/methanol (95:5) to afford
trifluoromethyl alcohol III-4 (1.16 g, 78%) as a white foam
which was used without further purification: TLC Rf ) 0.70,
chloroform/methanol (9:1).
m), 7.37 (1H, d,
J
) 8.0 Hz), 7.90 (2H, m). Anal.
(C27H36F3N3O8‚0.5H2O) C, H, N.
Meth od E: (2RS,3SR)-[(E)-3-[4-(Eth oxyca r bon yl)p h en -
yl]-1-oxop r op -2-en yl]-L-va lyl-N-[3-(1,1,1-t r iflu or o-2-h y-
d r oxy-4-m eth ylp en tyl)]-L-p r olin a m id e (III-17). A solution
of 4-carbethoxy-(E)-cinnamic acid (6.82 g, 30.9 mmol) and CDI
(5.02 g, 30.9 mmol) in THF (150 mL) was warmed to 40 °C
until CO2 evolution ceased. The resulting solution was cooled
to room temperature, treated with amino alcohol II (735 mg,
2.00 mmol), stirred for 18 h, and evaporated. The residue was
taken up in ethyl acetate, washed with saturated NaHCO3,
1 N HCl, and brine, dried (MgSO4), and evaporated to afford
alcohol III-17 (15.7 g, 89%). The crude material was used
without further purification: TLC Rf ) 0.26, chloroform/
methanol (94:6); MS (DCI) m/ z ) 570 (M + 1, base).
Meth od F: (2RS,3SR)-Acetyl-L-valyl-N-[3-(1,1,1-tr iflu or o-
2-h yd r oxy-4-m eth ylp en tyl)]-L-p r olin a m id e (III-26). A so-
lution of amino alcohol II (4.00 mg, 1.13 mmol) and TEA (445
mg, 0.613 mL, 4.40 mmol) in CH2Cl2 (5 mL) was treated with
acetic anhydride (224 mg, 0.207 mL, 2.20 mmol), stirred at
room temperature for 2 h, and evaporated. The residue was
diluted with ether, filtered, and evaporated. The crude mate-

Inhibitors of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1883
rial was purified by flash chromatography, eluting with
chloroform/methanol (95:5) to afford alcohol III-26 (483 mg,
100%) as a white foam: TLC Rf ) 0.4, chloroform/methanol
tyl)]-L-p r olin a m id e (III-13). A 0 °C solution of amino alcohol
II (10.0 g, 27.2 mmol) and NaOH (27.2 mL, 1.00 N, 27.2 mmol)
in dichloromethane (200 mL) was treated with a solution of
3-carbomethoxypropionyl chloride (4.10 g, 27.2 mmol) in CH2-
Cl2 (20 mL) and stirred at 0 °C for 0.5 h. The reaction mixture
was acidified to pH 2 with 1 N HCl, the organic phase
separated, and the aqueous phase extracted with two portions
of CH2Cl2. The combined organic layers were dried (Na2SO4)
and evaporated. Purification by flash chromatography eluting
with acetone/hexanes (1:1) afforded trifluoromethyl alcohol III-
13 (11.1 g, 84%) as a white foam: TLC Rf ) 0.45, chloroform/
1
(9:1); MS (DCI) m/ z ) 410 (M + 1, base), 269; H NMR (300
MHz, DMSO-d6) δ 0.93 (12H, m), 1.89 (1.5H, s), 1.92 (1.5H,
s), 1.94 (6H, m), 3.55 (1H, m), 3.73 (2H, m), 4.09 (1H, m), 4.42
(2H, m), 7.20 (1H, d, J ) 10 Hz), 7.63 (1H, d, J ) 10 Hz).
Meth od G: (2RS,3SR)-(Tr iflu or oa cetyl)-L-va lyl-N-[3-
(1,1,1-t r iflu or o-2-h yd r oxy-4-m e t h ylp e n t yl)]-L-p r olin -
a m id e (III-57). To a solution of amino alcohol II (0.79 g, 2.15
mmol) and ethyl trifluoromethyl acetate (0.360 g, 0.301 mL,
2.50 mmol) in methanol (10 mL) was added TEA (0.500 g,
0.685 mL, 4.95 mmol), and the mixture was stirred at room
temperature for 18 h and evaporated. The residue was taken
up in ethyl acetate, washed with 1 N HCl, saturated NaHCO3,
and brine, dried (MgSO4), and evaporated to afford alcohol III-
57 (0.870 g, 87%) as a white foam which was used without
further purification: TLC Rf ) 0.41 and 0.44, chloroform/
methanol (95:5).
Meth od H: (2RS,3SR)-[(4-Meth oxyp h en yl)ca r bon yl]-
L-va lyl-N-[3-(1,1,1-tr iflu or o-2-h yd r oxy-4-m eth ylp en tyl)]-
L-p r olin a m id e (III-50). A 0 °C suspension of amino alcohol
II (175 g, 476 mmol) and K2CO3 (126 g, 1.19 mol) in THF (2.5
L) was treated dropwise over 1 h with p-anisoyl chloride (81.3
g, 476 mmol), maintaining a reaction temperature of e3 °C.
After being stirred at 0 °C for 2 h, the reaction mixture was
filtered and the filter cake washed with THF (500 mL) and
ethyl acetate (1 L). The combined filtrates were washed with
0.15 N HCl, brine, saturated NaHCO3, and brine, dried
(MgSO4), and evaporated. The crude material was recrystal-
lized from ethyl acetate (1 L, two crops) to afford 174 g. The
mother liquors were evaporated, and the residue was purified
by chromatography, eluting with a gradient of dichloromethane/
methanol (100:0, 99:1, 97.5:2.5). The chromatographed mate-
rial was further purified by recrystallization from ethyl acetate
to afford 34.5 g. The material from all crystallizations was
combined to afford alcohol III-50 (209 g, 87%) as a white
solid: TLC Rf ) 0.33, chloroform/methanol (95:5); 1H NMR
(300 MHz, DMSO-d6/TFA) δ 0.94 (12H, m), 1.77-2.31 (6H, m),
3.63 (1H, m), 3.82 (3H, s), 3.86 (1H, m), 4.12 (1H, m), 4.49
(2H, m), 6.98 (2H, d, J ) 8.9 Hz), 7.92 (2H, d, J ) 8.9). Anal.
(C24H34F3N3O5) C, H, N.
Meth od I: (2RS,3SR)-[[[4-(Eth oxyca r bon yl)p h en yl]-
a m in o]ca r bon yl]-L-va lyl-N-[3-(1,1,1-tr iflu or o-2-h yd r oxy-
4-m eth ylp en tyl)]-L-p r olin a m id e (III-15). A solution of
amino alcohol II (2.5 g, 6.80 mmol) in chloroform (25 mL) was
treated with ethyl 4-isocyanatobenzoate (1.33 g, 6.80 mmol),
stirred at room temperature for 4 h, and evaporated to afford
alcohol III-15 (4.49 g, >100%) as a gray foam which was used
without further purification: TLC Rf ) 0.34, methanol/
chloroform (5:95); 1H NMR (250 MHz, DMSO-d6/TFA) δ 0.9
(12H, m), 1.3 (3H, t, J ) 8 Hz), 1.9 (6H, m), 3.7 (3H, m), 4.1
(1H, m), 4.3 (2H, q, J ) 8 Hz), 4.4 (2H, m), 7.5 (2H, d, J ) 10
Hz), 7.8 (2H, d, J ) 10 Hz).
Meth od J : (2RS,3SR)-[Bis(4-p h en ylp r op yl)]-L-va lyl-N-
[3-(1,1,1-t r iflu or o-2-h yd r oxy-4-m et h ylp en t yl)]-L-p r olin -
a m id e (III-25). A 0 °C solution of 3-phenyl-1-propanol (370
mg, 2.72 mmol) and Proton Sponge ((Aldrich; 580 mg, 2.72
mmol) in CH2Cl2 (10 mL) was treated with (CF3SO2)2O (672
mg, 0.450 mL, 2.72 mmol) and stirred for 10 min. The reaction
mixture was treated dropwise with a solution of amino alcohol
II (1.00, 2.72 mmol) and Proton Sponge (580 mg, 2.72 mmol)
in CH2Cl2 (10 mL), stirred at room temperature for 4 d, and
evaporated. The residue was taken up in ethyl acetate, and
filtered, and the filtrate washed with saturated NaHCO3 and
brine, dried (MgSO4), and evaporated. Purification by flash
chromatography on silica gel eluting with hexanes/ethyl
acetate (7:3) afforded trifluoromethyl alcohol III-25 (300 mg,
18%) as a colorless oil: TLC Rf ) 0.43, ethyl acetate/hexanes
(30:70); MS (DCI) m/ z ) 604 (M + 1, base), 308; 1H NMR (250
MHz, DMSO-d6) δ 0.90 (12H, m), 1.73 (10H, m), 2.50 (8H, m),
3.08 (1H, m), 3.27 (1H, m), 3.57 (1H, m), 3.73 (1H, m), 4.09
(1H, m), 4.36 (1H, m), 6.44 (0.25H, d, J ) 7 Hz), 6.57 (0.75H,
d, J ) 7 Hz), 7.18 (10H, m).
1
methanol (97:3); H NMR (250 MHz, DMSO-d6) δ 0.85 (12H,
m), 1.75-2.00 (6H, br m), 2.41 (4H, m), 3.56 (4H, s), 3.71 (2H,
m), 3.85 (1H, m), 4.32 (2H, m), 6.35-8.08 (2H, multiple
fractional peaks due to the NH resonances of the four diaster-
eomers).
Gen er a l P r oced u r e for Oxid a tion of Su lfid es III-4.
Meth od L: (2RS,3SR)-[[4-(Meth ylsu lfon yl)p h en yl]ca r -
b on yl]-L-va lyl-N-[3-(1,1,1-t r iflu or o-2-h yd r oxy-4-m et h yl-
p en tyl)]-L-p r olin a m id e (III-3). A solution of sulfide III-4
(590 mg, 1.14 mmol) and NaHCO3 (925 mg, 1.14 mmol) in
CH2Cl2 (10 mL) was treated with MCPBA (756 mg, 80-85%,
4.40 mmol) and stirred at room temperature for 1 h. The
reaction mixture was treated with an excess of triphenylphos-
phine (>4.4 mmol), diluted with ethyl acetate, washed with 1
N NaOH, H2O, and brine, dried (MgSO4), and evaporated.
Purification by flash chromatography eluting with chloroform/
methanol (95:5) afforded sulfone III-3 (580 mg, 93%) as a white
foam: TLC Rf ) 0.3, dichloromethane/methanol (95:5); MS
(DCI) m/ z ) 546 (M + 1), 267 (base), 99; 1H NMR (300 MHz,
DMSO-d6/TFA) δ 0.91 (12H, m), 1.83 (6H, m), 3.26 (1.5H, s),
3.37 (1.5H, s), 3.65 (1H, m), 3.84 (2H, m), 4.47 (3H, m), 8.01
(2H, d, J ) 8 Hz), 8.12 (2H, d, J ) 8 Hz).
Gen er a l P r oced u r es for Oxid a tion of Su lfoxid es III-
33a . Meth od M: (2RS,3SR)-[(Tr icyclo[3.3.1.13,7]d ec-1-yl)-
su lfon yl]-L-va lyl-N-[3-(1,1,1-tr iflu or o-2-h yd r oxy-4-m eth -
ylp en tyl)]-L-p r olin a m id e (III-33). A refluxing solution of
sulfoxide III-33a (860 mg, 1.56 mmol) in acetone (75 mL) was
treated dropwise with a saturated solution of KMnO4 in
acetone (excess, ca. 150 mL). Following addition of KMnO4,
the reaction mixture was refluxed for an additional 15 min,
cooled to room temperature, filtered through diatomaceous
earth, and evaporated. Purification by flash chromatography
eluting with hexanes/ethyl acetate (3:2) afforded sulfone III-
33 (637 mg, 72%) as a white solid: TLC Rf ) 0.46 and 0.50,
chloroform/methanol (95:5); 1H NMR (300 MHz, DMSO-d6/
TFA) δ 0.76-1.00 (12H, m), 1.50-2.12 (21H, m), 3.58 (2H, m),
3.74 (2H, m), 4.02 (1H, m), 4.39 (1H, m). Anal. (C26H42
-
N3O5F3S‚2.5H2O) C, H, N.
Gen er a l P r oced u r es for th e Oxid a tion of Tr iflu or o-
m eth yl Alcoh ols III. Meth od N: (3RS)-[[4-(Meth ylsu lfo-
n yl)ph en yl]car bon yl]-L-valyl-N-[3-(1,1,1-tr iflu or o-4-m eth yl-
2-oxopen tyl)]-L-pr olin am ide (3). A solution of trifluoromethyl
alcohol III-3 (650 mg, 1.18 mmol) and EDAC (2.30 g, 12.0
mmol) in DMSO/toluene (1:1, 0.8 mL) was treated with
dichloroacetic acid (400 mg, 4.8 mmol) and stirred at room
temperature for 18 h. The reaction mixture was diluted with
ethyl acetate, washed with 1 N HCl, H2O, and brine, dried
(MgSO4), and evaporated. Purification was by flash chroma-
tography eluting with a gradient of hexanes/acetone (40:60,
60:40) to afford trifluoromethyl ketone 3 (560 mg, 86%) as a
white foam: TLC Rf ) 0.3, acetone/hexane (2:3); MS (DCI) m/ z
) 548 (M + 1), 282, 267 (base), 99; 1H NMR (300 MHz, DMSO-
d6/TFA) δ 0.84 (3H, m), 0.91 (9H, m), 1.99 (4H, m), 2.19 (2H,
m), 3.31 (3H, s), 3.41 (1H, m), 3.64 (2H, m), 4.45 (2H, m), 8.02
(2H, d, J ) 8 Hz), 8.13 (2H, d, J ) 8 Hz). Anal. (C24H32O6-
N3F3S‚1.0 H2O) C, H, N.
Meth od O: (3RS)-(4-Acetoxyben zoyl)-L-valyl-N-[3-(1,1,1-
t r iflu or o-4-m et h yl-2-oxop en t yl)]-L-p r olin a m id e (41).
A
suspension of amino alcohol III-41 (903 mg, 1.70 mmol) and
DMP (2.16 g 5.1 mmol) in dichloromethane (6 mL) was treated
with TFA (584 mg, 395 mL, 5.1 mmol) and stirred at room
temperature for 18 h. The reaction mixture was partitioned
between a solution of saturated Na2S2O3/saturated NaHCO3
Meth od K: 2(RS,3SR)-[[2-(Meth oxyca r bon yl)eth yl]ca r -
bon yl]va lyl-N-[3-(1,1,1-tr iflu or o-2-h yd r oxy-4-m eth ylp en -

1884 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Edwards et al.
(1:1) and ethyl acetate, the ethyl acetate layer was separated,
washed with saturated Na2S2O3/saturated NaHCO3 (1:1) and
brine, dried (MgSO4), and evaporated to afford analytically
pure trifluoromethyl ketone 41 (820 mg, 91%) as a white
combined ethyl acetate extracts were dried (Na2SO4) and
evaporated to afford analytically pure acid 6 (482 mg, 71%)
as a white foam: TLC Rf ) 0.35, methanol/chloroform/acetic
acid (5:95:0.1); 1H NMR (250 MHz, DMSO-d6) δ 0.93 (12H, m),
1.7-2.3 (6H, m), 3.65 (1H, m), 3.96 (1H, m), 4.06 (0.4H, m),
4.58 (2 H, m), 4.63 (0.6H, t, J ) 8.2 Hz), 6.91 (0.2H, m), 7.40
(0.2H, m), 7.99 (4H, m), 8.61 (0.6H, m), 8.73 (1H, d, J ) 7.7
Hz). Anal. (C24H30N3F3O6‚1.0H2O) C, H, N.
Met h od T: (3RS)-(4-H yd r oxyb en zoyl)-L-va lyl-N-[3-
(1,1,1-tr iflu or o-4-m eth yl-2-oxopen tyl)]-L-pr olin am ide (44).
A solution of acetate 41 (820 mg, 1.56 mmol) and H2O (2 mL)
in methanol (12 mL) was treated with K2CO3 (excess) stirred
at room temperature for 18 h and evaporated. The residue
was acidified with 1 N HCl and extracted with ethyl acetate.
The combined ethyl acetate extracts were washed with brine,
dried (MgSO4), and evaporated to afford analytically pure
phenol 44 (462 mg, 62%) as a white foam: TLC Rf ) 0.64 and
0.72, diethyl ether/ethyl acetate (1:1); 1H NMR (250 MHz,
DMSO-d6/TFA) δ 0.95 (12Η, m), 1.72-2.38 (6H, m), 3.65 (1H,
m), 3.96 (1H, m), 4.08 (0.4H, m, CH R to CF3 carbonyl for
hydrate), 4.48 (2.3H, m), 4.64 (0.3H, d, J ) 6.0 Hz, CH R to
CF3 carbonyl for one of two ketone diastereomers), 6.82 (2H,
1
solid: TLC Rf ) 0.32 and 0.38, diethyl ether; H NMR (300
MHz, DMSO-d6/TFA) δ 0.94 (12H, m), 1.71-2.33 (6H, m), 2.29
(3H, s), 3.60 (1H, m), 3.87 (1H, m), 4.03 (0.5 H, m, CH R to
CF3 carbonyl for hydrate), 4.48 (2.2H, m), 4.57 (0.3H, d, J )
6.7 Hz, CH R to CF3 carbonyl for ketone), 7.21 (2H, d, J ) 8.5
Hz), 7.98 (2H, d, J ) 8.5 Hz). Anal. (C25H32N3O6F3‚0.6H2O)
C, H, N.
Meth od P : (3RS)-[[[4-(Eth oxyca r bon yl)p h en yl]a m in o]-
ca r bon yl]-L-va lyl-N-[3-(1,1,1-tr iflu or o-4-m eth yl-2-oxop en -
tyl)]-L-p r olin a m id e (15). A -60 °C solution of DMSO (42.4
g, 38.7 mL, 544 mmol) and oxalyl chloride (34.4 g, 23.3 mL,
272 mmol) in dichloromethane (275 mL) was stirred for 15 min
and treated with a solution of trifluoromethyl alcohol III-15
(7.80 g, 13.6 mmol) in CH2Cl2 (50 mL). The mixture was
stirred for 1 h, treated dropwise with diisopropyl ethylamine
(70.3 g, 94.8 mL, 544 mmol), and allowed to warm to room
temperature. The reaction mixture was washed with 1 N HCl
and brine, dried (Na2SO4), and evaporated. The crude mixture
was purified by flash chromatography eluting with a gradient
of ether/hexanes (50:50, 70:30, 80:20) and then chloroform/
methanol (95:5). The impure fractions were purified by a
second chromatography eluting with chloroform/methanol (97:
3). The material from both chromatographies was dissolved
in dichloromethane and evaporated to afford trifluoromethyl
ketone 15 (6.09 g, 80%) as an off-white solid: TLC Rf ) 0.40,
d,
J ) 8.60 Hz), 7.81 (2H, d, J ) 8.60 Hz). Anal.
(C23H30N3F3O5‚0.55 H2O) C, H, N.
Meth od U: (3RS)-[[2-[3-(Hyd r oxyca r bon yl)n a p h th yl]]-
ca r bon yl]-L-va lyl-N-[3-(1,1,1-tr iflu or o-4-m eth yl-2-oxop en -
tyl)]-L-p r olin a m id e (11). A solution of benzyl ester 16 (420
mg, 0.64 mmol) in ethanol (20 mL) was hydrogenated over 10%
Pd-C (50 mg, dry) at 50 psi for 8 h, filtered, and evaporated
to afford acid 11 (255 mg, 70%) as an analytically pure white
solid: HPLC tR ) 7.82 and 8.86, H2O/CH3CN/THF/TFA (55:
35:15:0.1), flow rate ) 1 mL/min, Phenomenex Zorbax C-8
analytical column (4.6 mm × 35 cm); 1H NMR (300 MHz,
DMSO-d6/TFA) δ 1.02 (12H, m) 1.77-2.37 (6H, m), 3.69 (1H,
m), 3.97 (1H, m), 4.11 (0.4H, m, CH R to CF3 carbonyl for
hydrate), 4.57 (2.6H, m), 7.67 (2H, m), 7.98 (1H, s), 8.07 (2H,
m), 8.39 (1H, s). Anal. (C28H32F3N3O6) C, H, N.
1
methanol/chloroform (5:95); H NMR (250 MHz, DMSO-d6) δ
0.9 (12H, m), 1.3 (3H, t, J ) 7 Hz), 2.0 (6H, m), 3.6 (1H, m),
3.7 (1H, m), 4.3 (2H, q, J ) 7 Hz), 4.4 (1H, m), 4.5 (1H, m), 4.6
(1H, m), 6.6 (1H, m), 7.5 (2H, d, J ) 9 Hz), 7.8 (2H, d, J ) 9
Hz), 8.62 (1H, m), 9.10 (1H, s). Anal. (C26H35N4O6F3) C, H,
N.
Meth od Q: (3RS)-[[4-(Meth oxyca r bon yl)p h en yl]ca r -
bon yl]-L-valyl-N-[3-(1,1,1-tr iflu or o-4-m eth yl-2-oxopen tyl)]-
L-p r olin a m id e (52). A solution of amino alcohol III-52 (4.00
g, 7.56 mmol), acetic anhydride (30.3 g, 28 mL, 300 mmol),
and DMSO (35.2 g 32 mL, 451 mmol) was stirred at room
temperature for 18 h. The reaction mixture was washed with
saturated NaHCO3 and brine, dried (MgSO4), and evaporated.
Purification by flash chromatography eluting with chloroform/
methanol (98:2) afforded trifluoromethyl ketone 52 (2.21 g,
55%) as a white foam: TLC Rf ) 0.44 and 0.50, methanol/
chloroform (5:95); 1H NMR (250 MHz, DMSO-d6) δ 0.69-1.03
(12H, m), 1.75-2.26 (6H, m), 3.63 (1H, m), 3.84-4.0 (4H, m),
4.1 (0.6H, m), 4.50 (2H, m), 4.59 (0.4H, t, J ) 8.2 Hz), 6.67-
6.95 (0.4H, m), 7.35-7.59 (0.3H, m), 8.01 (4H, s), 8.59 (0.3H,
m), 8.77 (1H, m). Anal. (C25H32F3N3O6‚0.5H2O) C, H, N.
Meth od R: (3RS)-[(E)-3-[4-(Eth oxyca r bon yl)p h en yl]-
1-oxop r op -2-en yl]-L-va lyl-N-[3-(1,1,1-tr iflu or o-4-m eth yl-
2-oxop en tyl)]-L-p r olin a m id e (17). A suspension of CrO3
(5.00 g, 50.0 mmol) in CH2Cl2 (20 mL) was stirred for 15 min,
treated with diatomaceous earth (5 g), stirred an additional
15 min, and treated with trifluoromethyl alcohol III-17 (1.00
g, 1.75 mmol). The reaction mixture was stirred at room
temperature for 2.5 h, filtered, diluted with ethyl acetate,
washed with 1 N HCl, saturated NaHCO3, and brine, dried
(MgSO4), and evaporated. Purification by flash chromatog-
raphy eluting with dichloromethane/methanol (98:2) afforded
trifluoromethyl ketone 17 (650 mg, 65%) as a white foam: TLC
In Vivo Assa ysAcu te Lu n g In ju r y Mod el (ALIM). The
ALIM has previously been described in detail.5 Male Syrian
hamsters (90-110 g) were anesthetized with Brevital sodium
(30 mg/kg, ip) and the trachea surgically exposed. A dose of
HNE (100 µg) in phosphate-buffered saline (0.3 mL, 0.01 M)
1
was injected into the exposed trachea via a /2 in., 23 ga. needle.
The incision was closed with stainless steel surgical staples,
and the animals were allowed to recover. Twenty-four hours
after the injection of HNE, the animals were killed with an
overdose of pentobarbital sodium. The lungs and heart were
resected and the lungs and trachea carefully cleaned of
extraneous material. Following measurement of wet lung
weight, the tracheas were cannulated and lavaged three times
with PBS (2 mL). The recovered lavages were pooled for each
animal, and the volume was recorded. Total red and white
cells were determined using a Coulter counter. The data are
expressed as lung weight/100 g of body weight and total cells
recovered (white or red, cells/mL × volume recovered). The
values for wet lung weights, total lavagable red cells, and total
lavagable white cells are elevated in a dose-dependent manner
following administration of HNE. Test compounds are evalu-
ated in this model for their ability to reduce this effect of HNE
when they are administered as a solution orally (solution in
10% PEG/PBS) 30 min prior to administration of HNE.
1
Rf ) 0.37, methanol/chloroform/acetic acid (3:97:1); H NMR
(250 MHz, DMSO-d6/TFA) δ 0.82-1.01 (12H, m), 1.35 (3H, t,
J ) 7.2 Hz), 1.70-2.42 (6H, m), 3.60 (1H, m), 3.86 (1H, m),
4.12 (0.6H, m), 4.33 (2H, q, J ) 7.2 Hz), 4.52 (2H, m), 4.65
(0.4H, m), 6.95 (1H, d, J ) 15.8 Hz), 7.50 (1H, J ) 15.8 Hz),
7.70 (2H, d, J ) 8.3 Hz), 8.02 (2H, d, J ) 8.3 Hz). Anal.
(C28H36F3N3O6) C, H, N.
Meth od S: (3RS)-[[4-(Hyd r oxyca r bon yl)p h en yl]ca r -
bon yl]-L-valyl-N-[3-(1,1,1-tr iflu or o-4-m eth yl-2-oxopen tyl)]-
L-p r olin a m id e (6). A solution of ester 52 (700 mg, 1.32 mmol)
in methanol/H2O (1:1, 16 mL) was treated with NaOH (1 N,
2.99 mL, 2.99 mmol) and stirred at room temperature for 18
h. The reaction mixture was treated with H2O, made acidic
with 1 N HCl, and extracted with ethyl acetate and the
In Vivo Assa ysAcu te Hem or r h a gic Assa y (AHA). The
AHA used was a minor modification of a published procedure30
that measures protection against the lung hemorrhage induced
in anesthetized hamsters by 50 mg of intratracheally admin-
istered HNE, as determined by spectrophotometrically mea-
suring the amount of hemoglobin present in lung lavage after
4 h. Test compounds were administered orally (solution in
10% PEG/PBS) 30 min prior to administration of HNE.
Ack n ow led gm en t. We thank Mr. J . M. Hulsizer
and Mr. G. Moore for synthesizing large-scale interme-
diates.

Inhibitors of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1885
Leukocyte Elastase. 3. Design, Synthesis, X-ray Crystallographic
Analysis, and Structure-Activity Relationships for a Series of
Orally Active 3-Amino-6-phenylpyridin-2-one Trifluoromethyl
Ketones. J . Med. Chem. 1994, 37, 3313-3326.
Refer en ces
(1) Abbreviations: AHA, acute hemorrhagic assay; ALIM, acute
lung injury model; HNE, human neutrophil elastase; PPE,
porcine pancreatic elastase; Ac, acetyl; Cbz, benzyloxycarbonyl;
TFMK, trifluoromethyl ketone; PFEK, perfluoroethyl ketone;
CAN, ceric ammonium nitrate; TEA, triethylamine; THF, tet-
rahydrofuran; DMSO, dimethyl sulfoxide; DMAP, (dimethylami-
no)pyridine; DCC, dicyclohexylcarbodiimide; MCPBA, m-chlo-
roperbenzoic acid; EDAC, 1-ethyl-3-[3-(dimethylamino)propyl]-
carbodiimide hydrochloride; HOBt, 1-hydroxybenzotriazole mono-
hydrate; TBAF, tetra-n-butylammonium fluoride; TFA, trifluo-
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pholine; DMP, Dess-Martin periodinane, 1,1,1-triacetoxy-1,1-
dihydro-1,2-benziodoxol-3(1H)-one; MeO, methoxy; Suc, succinyl;
pNa, p-nitroanilide; PEG, polyethylene glycol; PBS, phosphate-
buffered saline; ip, intraperitoneal; po, oral; it, intratracheal.
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