Inhibition of human neutrophil elastase. 4. Design, synthesis, X-ray crystallographic analysis, and structure-activity relationships for a series of P2-modified, orally active peptidyl pentafluoroethyl ketones

Article abstract of DOI:10.1021/jm970812e

A series of P₂-modified, orally active peptidic inhibitors of human neutrophil elastase (HNE) are reported. These pentafluoroethyl ketone-based inhibitors were designed using pentafluoroethyl ketone 1 as a model. Rational structural modifications were made at the P₃, P₂, and activating group (A(G)) portions of 1 based on structure-activity relationships (SAR) developed from in vitro (measured K(i)) data and information provided by modeling studies that docked inhibitor 1 into the active site of HNE. The modeling-based design was corroborated with X-ray crystallographic analysis of the complex between 1 and porcine pancreatic elastase (PPE) and subsequently the complex between 1 and HNE.

Full text of DOI:10.1021/jm970812e

J . Med. Chem. 1998, 41, 2461-2480
2461
Articles
In h ibition of Hu m a n Neu tr op h il Ela sta se. 4. Design , Syn th esis, X-r a y
Cr ysta llogr a p h ic An a lysis, a n d Str u ctu r e-Activity Rela tion sh ip s for a Ser ies of
P 2-Mod ified , Or a lly Active P ep tid yl P en ta flu or oeth yl Keton es
Robert J . Cregge, Sherrie L. Durham, Robert A. Farr, Steven L. Gallion,† C. Michelle Hare, Robert V. Hoffman,§
Michael J . J anusz, Hwa-Ok Kim,§ J ack R. Koehl, Shujaath Mehdi, William A. Metz,* Norton P. Peet,
J ohn T. Pelton, Herman A. Schreuder,‡ Shyam Sunder, and Chantal Tardif∇
Hoechst Marion Roussel Inc., 2110 East Galbraith Road, Cincinnati, Ohio 45215
Received December 1, 1997
A series of P2-modified, orally active peptidic inhibitors of human neutrophil elastase (HNE)
are reported. These pentafluoroethyl ketone-based inhibitors were designed using pentafluo-
roethyl ketone 1 as a model. Rational structural modifications were made at the P3, P2, and
activating group (AG) portions of 1 based on structure-activity relationships (SAR) developed
from in vitro (measured Ki) data and information provided by modeling studies that docked
inhibitor 1 into the active site of HNE. The modeling-based design was corroborated with
X-ray crystallographic analysis of the complex between 1 and porcine pancreatic elastase (PPE)
and subsequently the complex between 1 and HNE.
In tr od u ction
Human neutrophil elastase (HNE) is a major granule
proteinase of human neutrophils and one of a variety
of destructive enzymes involved in phagocytosis. Dur-
ing this process, HNE is released and controlled by
endogenous proteinase inhibitors. However, during
some pathological conditions, intense neutrophil infil-
tration results in an imbalance between the amount of
F igu r e 1. Generalized structure of tripeptidyl elastase inhibi-
tors (PG-P3-P2-P1-AG).
HNE and endogenous inhibitors.1,2 The excess HNE
which accumulates can then cause abnormal degrada-
tion of healthy tissue.3,4
clinical trials. With the aid of molecular modeling,
HNE has been implicated in the degradation of a
inhibitor 1 was rationally modified at the P2 and P3 sites
variety of connective tissue proteins, including elastin,
in an attempt to improve its affinity for the catalytic
collagen, laminin, fibronectin, and proteoglycan.5 As a
site of the enzyme.
result, it is thought to have a major role in the
development of diseases such as pulmonary emphy-
sema, rheumatoid arthritis, cystic fibrosis, adult respi-
ratory syndrome (ARDS), and chronic bronchitis.6,7
Therefore, it would be of therapeutic interest to develop
a synthetic inhibitor of HNE which would restore the
balance between the free enzyme and the endogenous
inhibitors. Since HNE has been implicated in a variety
of different clinical ailments, the ideal HNE inhibitor
should be available by oral (po), intravenous (iv), or
In parts 28b and 38a of this series, the effects of varying
the N-protecting group (PG) portion and the P1 portion
of inhibitor 1 (Figure 1) were reported. In an effort to
explore the effects of modifying the P3, P2, and activating
group (AG) portions15 of the inhibitor, computer model-
ing was used to guide the synthetic design.
aerosol administration.
There have been several reports describing both
peptidic8-10 and nonpeptidic11-14 HNE inhibitors. Pep-
tidyl pentafluoroethyl ketone 1 is a potent, orally active
inhibitor of HNE8b and was selected to enter human
† Department of Theoretical Chemistry. Current address: ArQule,
200 Boston Ave., Suite 3600, Medford, MA 02155.
§ Department of Chemistry, New Mexico State University, Las
Cruces, NM 88003-0001.
Ch em istr y
The synthesis of 1 started with commercially avail-
able Boc-Val-OCH3 2a . The HCl salt of Val pentafluo-
roethyl ketone 3, prepared by the alkylation of 2a with
‡ Department of Biophysics, Hoechst Marion Roussel, Frankfurt,
Germany.
∇ Synthe´labo Biomole´culaire, Strasbourg, France.
S0022-2623(97)00812-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/03/1998

2462 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Sch em e 1. Synthesis of 1 (MDL 101,146)a
Cregge et al.
of diastereomeric carboxylic acids 7 as evidenced by the
lack of coalescence of the alanyl R-proton quartets in
the NMR spectrum. Although the site of racemization
is not known, it is speculated to be at the lactam
R-carbon, since proton abstraction at this position and
cyclization to an intermediate spirocyclopropane (which
could proceed by ring opening with iodide followed by
intramolecular cyclization to the lactam) would give a
mixture of diastereoisomers. Coupling of acid 7 with
pentafluoroethyl ketone 3, using the mixed anhydride
method (method I), gave the easily separable diastere-
omeric pentafluoroethyl ketones 10a ,b in 75% yield.
When HOBT/EDC was used as the coupling reagent,
only a 19% yield of mixture 10a ,b was obtained.
Finally, deprotection and acylation of 10a ,b gave the
isomeric products 16 and 17, respectively.
The phenylketomethylene and morpholinoketometh-
ylene isosteres of the PG-Val portion of inhibitor 1 were
prepared as both R and S enantiomers. The corre-
sponding keto acids 19a ,b and 20a ,b were then coupled
respectively to Pro-Val pentafluoroethyl ketone 21 to
afford isosteres of inhibitor 1 (Scheme 3). To accomplish
this as efficiently as possible, pentafluoroethyl ketone
21 was synthesized by coupling Boc-Pro-OH via the
mixed anhydride method (method I) to 3 in 90% yield.
Deprotection and coupling to the appropriate unit
provided the desired isosteric analogues 22 and 23a ,b.
Attempts to couple the R enantiomer 19a with 21
resulted in internal cyclization of the starting keto acid
19a to give a â-enollactone, and none of the desired
coupled product was isolated.
2a
a
Reagents: (a) ICF2CF3, MeLi‚LiBr, -78 °C; (b) HCl(g), EtOAc,
rt; (c) IBCF, NMM, -20 °C; (d) HCl(g), EtOAc, rt; (e) 4-[(4-
morpholinyl)carbonyl]benzoyl chloride, NMM, -20 °C.
pentafluoroethyllithium,16 was coupled to the com-
mercially available protected dipeptide 4 via the mixed
anhydride (method I). Treatment of 5 with HCl (gas)
provided the corresponding salt which was stored in a
refrigerator prior to use. Pentafluoroethyl ketone 5,
following deprotection, was coupled with 4-[(4-morpholi-
nyl)carbonyl]benzoyl chloride to give 1 (Scheme 1). Our
strategy in preparing analogues of 1 was to follow this
route, when possible, substituting the desired peptide/
nonpeptide residue for the Boc-Val-Pro-OH portion of
the molecule in the sequence.
The syntheses of constrained analogues 15-18 were
carried out by coupling the appropriate carboxylic acid
to pentafluoroethyl ketone 3 (Scheme 2). N-Boc-3-
amino-2-oxopiperidineacetic acid 8 was readily synthe-
sized from N-R-(tert-butoxycarbonyl)-L-ornithine via
reductive alkylation with glyoxylic acid followed by
intramolecular cyclization.17 The corresponding γ-lac-
tam was prepared from the sulfonium salt of Boc-L-Met-
Gly-OMe followed by intramolecular cyclization to give
6. Both lactams have the S configuration at the
R-position. Coupling of acids 6 and 8 to the pentafluo-
roethyl ketone 3 gave products 9 and 11, respectively.
Deprotection and acylation with 4-[(4-morpholinyl)-
carbonyl]benzoyl chloride gave the respective con-
strained inhibitors 15 and 18.
To complement this strategy, the P3 D-valine and
dehydrovaline analogues, 32 and 33, were readily
synthesized by coupling the corresponding Boc-protected
D-Val or dehydrovaline amino acids, 24 and 25, to the
methyl ester of proline hydrochloride using BOP (cou-
pling method IV, Scheme 4). Hydrolysis to the corre-
sponding acid gave a dipeptide intermediate analogous
to 4 (in Scheme 1). Coupling to 3, deprotection, and
acylation gave P3 analogues 32 and 33.
The intramolecular cyclization of Boc-D-Met-Ala-OMe,
under similar conditions, gave an inseparable mixture
The P2-modified analogues 54-56 (Scheme 5) were
synthesized using the same sequence of coupling reac-
Sch em e 2. Synthesis of P2/P3-Constrained Analogues 15-18a
a
Reagents: (a) IBCF, NMM, -20 °C; (b) HCl(g), EtOAc, rt; (c) 4-[(4-morpholinyl)carbonyl]benzoyl chloride, NMM, -20 °C.

Inhibition of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2463
Sch em e 3. Synthesis of P3 Isosteres 22 and 23a ,ba
a
Reagents: (a) NaH, THF; (b) LiOH, H2O; (c) H+/PhH, ∆; (d) NaH, DMF, (S)-TfOCH(CH3)2CO2Me, 2,6-lutidine; (e) NaH, DMF, (R)-
TfOCH(CH3)2CO2Me, 2,6-lutidine; (f) EDCI, HOBt, DMF, morpholine; (g) LiOH, H2O; (h) EDCI, HOBt, NMM, CH2Cl2/DMF.
tions shown in Scheme 1. Coupling the P2 unit with
Boc-Val-OSu 2 (method II) gave dipeptides 39-42.
Coupling of these dipeptide units to 3 followed by
deprotection and acylation with 4-[(4-morpholinyl)-
carbonyl]benzoyl chloride gave the corresponding in-
hibitors 52-56. In some cases, different coupling
reagents were used to optimize the yield in the more
difficult coupling reactions such as those incorporating
the hindered homoproline and tetrahydroisoquinolin-
ecarboxylic acids (vide infra). In the case of the 4-sub-
stituted proline analogues, derivatization of the 4-hy-
droxyl group had to precede incorporation of the
hydroxyproline unit into the tripeptide. Attempts to
acylate intermediate 45 directly led to complex mixtures
and incomplete reactions. During the deprotection of
compound 45, a minor product was isolated and identi-
fied as the acetoxy derivative 50. This intermediate was
carried through the same sequence of reactions as
hydroxy and O-benzyl intermediates 49 and 51, respec-
tively, to give 55. Intermediates 49 and 51 gave
products 54 and 56, respectively.
described for the other P2 analogues. Coupling of
L-thiaproline and tetrahydro-1-isoquinolinecarboxylic
acid to Boc-Val-OSu 2 (method II) provided the dipep-
tides 57 and 60, respectively. Coupling of dipeptides
57 and 60 to pentafluoroethyl ketone 3 followed by
deprotection and acylation with 4-[(4-morpholinyl)-
carbonyl]benzoyl chloride gave the corresponding in-
hibitors 59 and 62a ,b.
Positional isomers 69 and 70 were also prepared
following the general synthetic scheme described for
inhibitor 1. Formation of dipetides 63 and 64 using
coupling method II and subsequent coupling to 3
provided pentafluoroethyl ketones 65 and 67. Depro-
tection of these N-tert-butyloxycarbonyl intermediates
and acylation with 4-[(4-morpholinyl)carbonyl]benzoyl
chloride provided 69 and 70 (Scheme 8).
In a previous report8a conditions are described for the
direct nucleophilic perfluoroalkylation of peptide esters
to form the corresponding perfluoroalkyl ketones, which
is the procedure chosen for the preparation of heptafluo-
ropropyl ketone 73. Thus, the dipeptide unit 4 was
coupled with Val-OMe‚HCl to provide 71 in quantitative
yield. Nucleophilic perfluoroalkylation with heptafluo-
The thiaproline analogue 59 (Scheme 6) and isoquino-
line analogues 62a ,b (Scheme 7) were prepared as

2464 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Sch em e 4. Synthesis of P3 Analogues 32 and 33a
Cregge et al.
a
Reagents: (a) ProOMe‚HCl, BOP, Et3N, CH3CN, rt; (b) LiOH, MeOH/H2O, rt; (c) HCl‚ValCF2CF3 (3), IBCF, NMM, -20 °C; (d) HCl(g),
EtOAc, rt; (e) 4-[(4-morpholinyl)carbonyl]benzoyl chloride, NMM, -20 °C.
ropropyllithium gave heptafluoropropyl ketone 72, which
was deprotected and acylated under the usual condi-
tions to give the heptafluoropropyl ketone 73 (Scheme
9).
Resu lts a n d Discu ssion
Con str u ction of th e Com p lex betw een HNE a n d
In h ibitor 1. HNE inhibitor 1 is a reversible, mecha-
nism-based21 inhibitor of elastase. The pentafluoroethyl
functionality activates the carbonyl group of 1, which
corresponds to the scissile amide carbonyl group of
substrate, to promote nucleophilic addition at the elec-
trophilic carbonyl group and afford a stable hemiketal
with the hydroxyl group of Ser 195 at the active site of
the enzyme.22 Since fluorinated ketone inhibitors have
a tendency to epimerize at the chiral center R to the
ketone,23 inhibitor 1 was prepared as approximately a
1:1 mixture of diastereomers. Since the natural sub-
strates of elastase contain only L-peptides and inhibitor
1 is a tripeptide, only the L-stereoisomer was modeled
in the enzyme. Coordinates from the Brookhaven
Protein Data Bank24 (code 1HNE) were used for con-
structing models of the enzyme-inhibitor complex.25
The structure of HNE inhibited by methoxysuccinyl-Ala-
Ala-Pro-Ala chloromethyl ketone as the hemiketal,
covalently bonded to Ser 195, was used to model all
subsequent inhibitors using the corresponding atoms in
the crystal structure to guide placement of analogous
recognition units in our inhibitors. The initial model
was constructed using inhibitor 1. The binding site was
defined by any protein residue having any atom located
within 8 Å from any atom of the inhibitor in its initial
conformation. Protein atoms and crystallographic water
outside the binding site were held fixed during all
calculations. The average structure for the complex was
determined and the final structure was minimized and
used to rationalize modifications to the lead compound
(Figure 2). This model was validated by the X-ray
P h a r m a cologica l Eva lu a tion
The in vitro HNE inhibitory activity (Ki)18 and in vivo
oral potency of selected compounds are listed in Table
1. The hamster HNE-induced pulmonary hemorrhage
model, described previously,19 was used as the primary
screening model to determine if the compounds, when
orally administered, were effective inhibitors of HNE-
induced pulmonary hemorrhage. Intratracheal (i.t.)
instillation of HNE into rodents induced acute pulmo-
nary hemorrhage which was measured by the hemo-
globin content in the bronchoalveolar fluid.20
In a routine screening protocol, 25 and 50 mg/kg of
the test compound were administered, as a suspension
in 20% Emulphor (GAF Corp.), orally by gavage 45 min
prior to i.t. instillation of 100 µg of HNE/animal. One
hour later the animals were sacrificed, the lungs were
lavaged with saline, and the amount of hemoglobin in
the fluid was measured spectrophotometrically at 536-
546 nm. The percent inhibition was calculated as the
ratio of pulmonary hemorrhage (BAL Hgb) in the drug-
treated animals compared to the vehicle-treated ani-
mals. For those compounds with activity at the screen-
ing dose, a dose-response curve was obtained. The
duration of action of selected compounds was deter-
mined by administering compound orally at various
times before i.t. In vivo HNE inhibitory activity, at
various time points, is presented in Table 2.

Inhibition of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2465
Sch em e 7. Synthesis of Isoquinolines 62a ,ba
Sch em e 5. Synthesis of P2 Analogues 54-56a
a
Reagents: (a) HCl‚ValCF2CF3 (3), IBCF, NMM, -20 °C; (b)
HCl(g), EtOAc, rt; (c) 4-[(4-morpholinyl)carbonyl]benzoyl chloride,
NMM, -20 °C.
Sch em e 8. Synthesis of P2-Positional Isomers 69 and
70a
a
Reagents: (a) Boc-Val-OSuc (2), DMF, Et3N, ∆; (b)
HCl‚ValCF2CF3 (3), IBCF, NMM, -20 °C; (c) HCl(g), EtOAc, rt;
(d) 4-[(4-morpholinyl)carbonyl]benzoyl chloride, NMM, -20 °C.
Sch em e 6. Synthesis of Thiazoles 59a
a
Reagents: (a) HCl‚ValCF2CF3 (3), IBCF, NMM, -20 °C; (b)
HCl(g), EtOAc, rt; (c) 4-[(4-morpholinyl)carbonyl]benzoyl chloride,
NMM, -20 °C.
valine or isoleucine.26 While the predominant local
feature of the S2 and S3 subsites of the enzyme is the
presence of hydrophobic pockets, there are several
oxygen atoms that line the interface of these subsites,
namely, HO Tyr 94, O Pro 98, and O Val 99. The side
chains of Asn 61 and Asn 99 may also contribute
carbonyl oxygens to this array. Substitution in the P2
and P3 positions of the inhibitor could exploit interac-
tions with these oxygen atoms as well as with the
neighboring hydrophobic areas.
a
Reagents: (a) HCl‚ValCF2CF3 (3), IBCF, NMM, -20 °C; (b)
HCl(g), EtOAc, rt; (c) 4-[(4-morpholinyl)carbonyl]benzoyl chloride,
NMM, -20 °C.
Conformationally constrained analogues were pre-
pared by substituting five- and six-membered cyclic
lactams for the P3 valine and P2 proline of inhibitor 1.
These were intended to simulate a tethered Val-Pro
serving as a P3-P2 dipeptide surrogate (Figure 3). For
synthetic simplicity the γ-carbon of L-valine was omit-
ted. The use of a lactam in this manner has been shown
to improve metabolic stability of substrate-based inhibi-
tors and improved potency.27
crystal structure of 1 cocrystallized in porcine pancreatic
elastase (PPE) and subsequently complexed with HNE.
The high-resolution (1.8 Å) crystal structure of the PPE
complex showed that only the LLL-diastereomer binds
to the active site, even though cocrystallization was done
with a racemic mixture.
Con for m a tion a lly Con str a in ed An a logu es of In -
h ibitor 1. The primary specificity site of HNE (S3) will
accommodate small lipophilic residues at P3 such as

2466 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Sch em e 9. Synthesis of AG Analogue 73a
Cregge et al.
model. There was a good overlap of low-energy confor-
mations between the two compounds in the active site
of the enzyme. When 15 was modeled in HNE, a
hydrophobic pocket was present at the P2 glycine site.
To provide a better fit with this pocket, the glycine at
P2 was replaced with an alanine residue to give com-
pound 16. Although 16 showed improved binding
affinity, the binding was 1000-fold less than with 1.
The corresponding γ-lactam, with the R configuration
at the R-carbon, was prepared. An alanine residue was
substituted for glycine at P2, e.g., compound 18, to
accommodate a hydrophobic pocket observed in the
model of this inhibitor docked with the enzyme at this
site. These subunits provided constrained analogues of
1 without replacing the peptide backbone of the original
tripeptide-based structure.
Isost er ic R ep la cem en t of t h e P 3 Va l a n d P 3
An a logu es. The carbonyl oxygen of P3 Val of inhibitor
1 accepts a hydrogen bond from the amide nitrogen of
Val 216 of the enzyme, while the amide nitrogen of P3
Val donates a hydrogen bond to the carbonyl group of
Val 216. The strength of this latter hydrogen bond is
dependent on the orientation of the amide plane be-
tween the P3 Val and the aromatic ring. The dihedral
angle (Ψ1) is -82° (-32° in the X-ray structure of 1 +
PPE, Figure 4). Thus, the internal strain energy of this
angle is more than compensated by the strength of the
resulting hydrogen bond.
a
Reagents: (a) ICF2CF2CF3, MeLi‚LiBr, -78 °C; (b) HCl(g),
EtOAc, rt; (c) 4-[(4-morpholinyl)carbonyl]benzoyl chloride, NMM,
-20 °C.
Ta ble 1. In Vitro and In Vivo HNE Inhibitory Activity of
Selected Compounds in the Hamstera
compd no.
Ki (nM)
% inhibition (n)b
1
20
340
34
150
120
40
74
0
79*
54*
0
33cd
52c
53
54
55
0
56
59
62a
73
20
70
100
18
7
0
NT
47*
To explore the importance of the hydrogen bonding
of the inhibitor Val 216 NH to the carbonyl of Val 216
of the enzyme observed from the model (Figure 5),
ketomethylene isosteres of the PG-Val portion of inhibi-
tor 1 were prepared as both R and S enantiomers.28
These analogues, e.g., 22 (Ki ) 4.2 µM, (S)-ketometh-
ylene), 23a (Ki ) 41 µM, (R)-ketomethylene), and 23b
(Ki ) 9.8 µM, (S)-ketomethylene), while showing better
affinity for the enzyme than the constrained analogues,
clearly demonstrated the importance of the P3 valine
amide group for enzyme affinity. Replacing the P3
valine with the D-stereoisomer 32 (Ki ) 5.5 µM) and
dehydrovaline 33 (Ki ) 340 nM) demonstrated the
importance of the L-stereocenter at P3. Compound 32
showed a 100-fold loss in activity versus inhibitor 1.
Dehydro analogue 33c showed only a 10-fold loss of
binding affinity (Ki ) 340 nM). With the absence of the
stereocenter at P3, the L- and D-diastereomers of 33c at
P1 were separated to give the L-isomer 33a (Ki ) 240
nM) and the D-isomer 33b (Ki ) 1100 nM). These
differences in affinity clearly demonstrate that the
L-stereocenters of the P1 and P3 valines are critical for
optimal binding activity. The observed in vitro results
substantiated the hydrogen-bonding interactions be-
tween the P3 unit of the inhibitor and Val 216 of the
enzyme suggested by the model and also the importance
of the stereochemistry at the P3 valine of the inhibitor.
a
See Pharmacological Evaluation section for methodology. The
compounds (25 mg/kg) were administered orally to hamsters 30
min before i.t. instillation of HNE (25 µg). Percent inhibition of
b
lung hemorrhage is presented as the mean ( SEM of BAL Hgb
from 6-8 hamsters (*p < 0.05 is the criterion for statistical
significance). c Compound given 15 min prior to administration of
d
HNE. 50 mg/kg of compound p.o. NT, not tested.
Ta ble 2. In Vivo Inhibitory Activity of Selected Compounds at
Various Time Points in the Hamster
% inhibitiona
compd no.
dose (mg/kg)
1 h
1.5 h
2 h
4 h
0
52
25
50
25
50
25
50
25
57*
0
39
49*
0
53
43*
73*
52*
0
55
56
73
44
30
25
a
Compounds were administered orally to hamsters at the times
indicated before i.t. instillation of HNE (25 µg). Percent inhibition
of lung hemorrhage is presented as the mean ( SEM of BAL Hgb
from 6-8 hamsters (*p < 0.05 is the criterion for statistical
significance).
The constrained analogues of 1 showed a loss in
binding affinity with respect to 1, e.g., 15 (Ki ) 400 µM),
16 (Ki ) 14 µM), 17 (Ki ) 1100 µM), and 18 (Ki ) 95
µM). The restricted conformation of these compounds
had shifted the backbone of the inhibitor enough that
the 5-amino group and carbonyl function of the lactam
and the Val 216 and Val 214 of the enzyme were now
not within hydrogen-bonding distances. In contrast to
17, with the natural S configuration at P3, 16, with the
unnatural R configuration, did not show a change in
the position of the backbone carbons relative to 1 in the
P 2-P osition a l Isom er s, Azetid in e, Hom op r olin e,
Th ia zolid in e, Isoqu in olin e, a n d 4-Su bstitu ted P r o-
lin e An a logu es. The S2 pocket of the HNE enzyme is
hydrophobic. In particular, the side chain of Leu 99 and
the hydrophobic face of His 57 help to delineate this
region. The upper interface of the pocket is bordered
by the carbonyl oxygen of Pro 98 and the side chain of
Tyr 94. There appears to be a hydrophobic cavity
proximal to the 4-position of the proline residue of the

Inhibition of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2467
F igu r e 2. Stereoview of inhibitor 1. Only the non-hydrogen atoms are shown for clarity. The coordinates of the inhibitor were
taken from the crystal structure of the complex of inhibitor 1 with PPE.
F igu r e 3.
F igu r e 4. Stereodiagram of the electron density of the bound inhibitor 1 and PPE. The protein molecule is drawn in thin lines,
the inhibitor in thick lines. To calculate this map, the inhibitor was omitted from the model and an Fobs - Fcalc difference map was
calculated. This means that not all the information of the bound inhibitor is present in the phases and that the electron density
visible is determined solely by the experimental (reflection) data. The map is contoured at 4σ.
inhibitor. Placement of the appropriate lipophilic group
in this region via substitution or modification of the P3
and/or P2 residues could result in better inhibitor-
enzyme interaction.
The P2 proline of inhibitor 1 was replaced with
nipecotic and isonipecotic acids which provided posi-
tional isomers of inhibitor 1. This introduced an ad-
ditional carbon into the backbone of the molecule which
affected the hydrogen bonding between the inhibitor and
the enzyme. Azetidinone and homoproline analogues
were synthesized to observe the effect of ring size on in
vitro and in vivo activity. A thiazolidamide analogue
was synthesized to observe the effect of a heteroatom
near the S2 enzyme subsite, while L-1,2,3,4-tetrahy-
droisoquinoline and 4-substituted proline analogues
were prepared based on spacial volume and hydropho-
bicity of the S2 subsite of the enzyme.
When proline was replaced by azetidinone and ho-
moproline, to provide inhibitors 52 (Ki ) 34 nM) and
53 (Ki ) 150 nM), respectively, both compounds showed
good binding affinity for the enzyme and oral activity
was similar to that of inhibitor 1. The homoproline
analogue 53 showed a longer duration of activity in vivo
than 1. On the basis of these observations, the thiazo-

2468 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Cregge et al.
F igu r e 6. Connolly surface model of inhibitor 56 and HNE.
The P2 extension of inhibitor 56 fits nicely in the S2 pocket of
HNE. The hydrophobic region is shown in green and white.
The region of positive charge is blue, and the negative charge
is red.
F igu r e 5. Model of inhibitor 1 covalently bonded at Ser 195
of HNE. H-Bonding is apparent between N of Val (P3) and Val
216, the carbonyl of Pro (P2) and Val 216, and N of Val (P1)
and Ser 214 of HNE.
lidine analogue 59 and tetrahydroisoquinoline ana-
logues 62a ,b were prepared. Although these com-
pounds showed improved binding affinity over 53, there
was loss of oral activity when compound 59 was tested
in vivo (Table 1).
The spacial volume and hydrophobicity of the S2
subsite of the enzyme, observed in the model, suggested
that the synthesis of the 4-subtituted proline analogues
54-56 may provide better binding affinity. Substitution
of 4-hydroxyproline for proline afforded compound 54
which had a Ki value of 120 nM. Long aliphatic side
chains terminating with basic groups (such as lysine)
have been shown to bind in this region.29 The acetoxy
derivative of 4-hydroxyproline, 55, had a Ki value of 40
nM, reflecting the lesser hydrophilic and greater lipo-
philic nature of this group. The O-benzyl ether com-
pound 56 bound with a Ki value of 20 nM, showing that
fairly large groups are accommodated at this position
(Figure 6), as has been shown by others.30
Positional isomers 69 (Ki ) 8.5 µM) and 70 (Ki ) 200
µM) of inhibitor 1 showed a decrease in binding activity
as the distance between the ring nitrogen and the amide
carbonyl increased, demonstrating that changes in the
backbone carbonyl and nitrogen positions are not toler-
ated by the enzyme.
Va r ia t ion of t h e Ca r b on yl Act iva t in g Gr ou p
(AG). The oxyanion produced by addition of the Ser 195
hydroxyl group to the pentafluoroethyl ketone of the
inhibitor 1 forms hydrogen bonds with the amide
protons of Gly 193 and Ser 195. The amide NH of the
P1 Val hydrogen-bonds with the carbonyl oxygen of Ser
214. The X-ray crystal structure was consistent with
only the LLL-diastereomer of the inhibitor 1 binding to
the enzyme, although it is known that the inhibitor
quickly racemizes at blood serum pH. The P1 dehy-
drovaline analogue was previously synthesized8a to
explore the effect on binding activity.
To observe the effect of increasing the electrophilicity
of the activating group of the inhibitor 1 on the forma-
tion of the hemiketal in the active site of the enzyme,
the heptafluoro-n-propyl ketone analogue 73 was syn-
thesized. Although binding and oral activity for 73 (Ki
F igu r e 7. Overlay of the X-ray structure of HNE (green; PDB
code 1HNE) with the modeled inhibitor 1 (blue) and the X-ray
structure of the complex of PPE (orange) with inhibitor 1
(white).
) 18 nM) were comparable to those of 1 (Ki ) 20 nM),
the lack of both longer duration of in vivo activity and
significant increase in binding did not warrant further
investigation.
Con clu sion s
A number of P3- and P2-modified peptidyl pentafluo-
roethyl ketones were synthesized based on rational
design. The model of inhibitor 1 in the enzyme, using
the Brookhaven Protein Data Bank coordinates for HNE
(from file entry 1HNE), proved to be very useful in
guiding the synthetic effort. When the X-ray crystal
structures were solved for complex PPE-1 and HNE-
1, there was good correlation between the model and
crystal structure (Figure 7). Through structure modi-
fication, the tolerance of the enzyme to structural
changes to inhibitor 1 could be monitored. Varying the
P2 and P3 regions of inhibitor 1 had a dramatic effect
on the in vitro potency. In general, analogues which

Inhibition of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2469
Ta ble 4. Statistics on the HNE-1 Complex
Ta ble 3. Statistics on the PPE-1 Complex
space group
cell dimensions
resolution
P31
space group
cell dimensions
resolution
P4322
a ) b ) 69.1 Å, c ) 51.1 Å, R ) â ) 90°, γ ) 120°
1.8 Å
a ) b ) 75.8 Å, c ) 108.5 Å, R ) â ) γ ) 90°
3.0 Å
no. of observations 101240
no. of observations 48509
no. of unique
reflections
R-sym
24302
no. of unique
reflections
R-sym
6729
5.0%
9.9%
completeness
R-factor
93.4%
completeness
R-factor
99.1%
0.180 for 24038 reflections between 8.0 and 1.8 Å
0.160 for 5799 reflections between 8.0 and 3.0 Å
be estimated from a Luzzati plot. With the exception of a few
surface loops, which are presumably quite mobile, the electron
density maps were of sufficient quality (1.8 Å) to unambigu-
ously determine the chirality of the bound inhibitor.
varied the position of the nitrogens and carbonyls of
inhibitor showed a loss in binding activity. The con-
strained analogues (15, Ki ) 400 µM; 16, Ki ) 140 µM;
17, Ki ) 1100 µM; and 18, Ki ) 95 µM) and the isosteres
(22, Ki ) 4.2 µM; 23a , Ki ) 41 µM; and 23b, Ki ) 9.8
µM), which did not contain the P3 Val nitrogen, showed
a loss of binding affinity. The latter suggests that the
hydrogen bonds formed between the P3 valine of the
inhibitor and Val 216 of the enzyme, observed in the
X-ray crystal structure, were critical for activity.
Although the homoproline analogue 53 showed ac-
ceptable binding (Ki ) 150 nM) compared with inhibitor
1, and a longer duration of action, the difficulty in
preparing this analogue and reaction yield made this
an unlikely candidate for replacing inhibitor 1 as the
lead compound. This was also the case with the
heptafluoro-n-propyl ketone analogue 73 (Ki ) 18 nM).
Although compound 73 was orally active, there was not
a significant enough increase in the duration of activity
to warrant development of reaction conditions which
would provide adequate amounts of the material for
additional evaluation.
The 4-substituted prolines (analogues 54-56) proved
to be successful modifications of inhibitor 1 which were
based on the model. Substitution with various groups
defined a cavity in the S2 subsite of HNE which is very
hydrophobic. This finding may allow placement of a
lipophilic residue (e.g., aryl) in this region which could
lead to enhanced binding and an increased duration of
action. Further exploration of this pocket may lead to
a more potent, orally active HNE inhibitor.
Superposition of the crystal structure of the PPE-1 complex
with the crystal structure of HNE taken from the Brookhaven
Protein Data Bank (code 1HNE) revealed that the folding of
the porcine and the human enzyme is very similar. The rms
difference is 1.8 Å for 210 equivalent CR atoms, and the rms
difference is 0.5 Å for 141 equivalent CR atoms that differ less
than 3σ. The inhibitor forms a covalent tetrahedral adduct
with the enzyme, mimicking the tetrahedral transition state
that occurs during hydrolysis of peptide substrates. The
carbonyl carbon of the perfluoroethyl ketone group is linked
to the active site Ser 195, and the carbonyl oxygen occupies
the oxyanion hole. As expected, no buried hydrogen bond
donors or acceptors are present which are unable to make at
least one hydrogen bond.
2. Cr ysta l Str u ctu r e of Hu m a n Neu tr op h ile Ela sta se
(HNE) w ith 1. Crystals of the HNE-1 complex were obtained
by the hanging drop method.35 Data were collected to 3.0 Å
resolution using a Siemens multiwire area detector, mounted
on a Siemens rotating anode generator. As with the complex
formed with the porcine enzyme, the complex crystallized in
a crystal form which has not previously been published. The
space group is P4322. Data were processed with XDS,33 and
statistics of the data collection are given in Table 4.
The crystal structure was solved by molecular replacement
using the AMoRe36 package, and refinement was done with
X-plor.34 The structure of HNE inhibited by methoxysuccinyl-
Ala-Ala-Pro-Ala chloromethyl ketone (PDB code 1HNE) was
used as a model. The procedure was performed using data
between 15.0 and 3.5 Å for the two possible enantiomorphic
space groups P4122 and P4322. The correct solution was very
clear with a correlation of 0.346 and an R-factor of 0.471 in
space group P4322. A map calculated at 3.0 Å clearly showed
the bound inhibitor and carbohydrate attachment sites at Asn
109 and 159. Four cycles of map inspection, rebuilding, and
refinement yielded a final model with an R-factor of 0.160 for
5799 reflections between 8.0 and 3.0 Å. A mean coordinate
error of 0.2-0.3 Å can be estimated from a Luzzati plot. The
electron density is well-defined considering the relatively low
resolution of 3.0 Å.
Superposition of the HNE-1 complex with the crystal
structure of HNE taken from the Protein Data Bank (code
1HNE) revealed that the folding of HNE does not change very
much upon binding of the different inhibitors. The rms
difference is 0.6 Å for 217 equivalent CR atoms and 0.2 Å for
175 equivalent CR atoms that differ less than 3σ. The largest
difference in CR position is 4.2 Å for Asn 148 in loop 147-151,
a flexible surface loop. The rms differences between the
HNE-1 complex and the PPE-1 complex are 1.7 Å for all 214
equivalent CR atoms and 0.5 Å for 138 CR atoms deviating less
than 3σ, which are virtually identical to the differences
between PPE-1 and the 1HNE complex. Figure 8 shows that
the binding mode of 1 is identical for the C2F5 unit through
the proline moiety, but the positions of the phenyl and
especially the morpholine ring differ considerably. This is due
to steric hindrance with loop 173-175 in the PPE structure.
It should be noted, however, that the inhibitor bound to HNE
is involved in crystal contacts which may lead to subtle
alterations of the binding mode.
Exp er im en ta l Section
X-r a y Cr ysta l Str u ctu r e. 1. Cr ysta l Str u ctu r e of
P or cin e P a n cr ea tic Ela sta se (P P E) w ith 1. Crystals of
the complex formed with PPE and 1 were prepared in buffer
using the sitting drop method.31 Part of a big needle (dimen-
sions 1.0×0.5×0.2 mm3) was mounted. Data were collected
using a Siemens multiwire area detector mounted on
a
Siemens rotating anode generator. The crystal diffracted to
approximately 1.8 Å to give a completely different space group
(P31) from what has been previously reported (P212121).32 Data
were processed with the XDS package,33 and the statistics of
the data collection are given in Table 3. The crystal form of
the complex differed considerably from the form of native PPE
crystals. Whereas native PPE crystals were cube-shaped,
crystals of this complex had a needle shape.
The crystal structure was solved by molecular replacement
with X-plor34 using the structure of native elastase (PDB code
3EST) as a model and refined also using X-plor. The procedure
was straightforward, yielding clear peaks for the rotation and
translation functions. The starting R-factor for data between
8.0 and 3.0 Å was 0.309 after rigid body refinement. A map
calculated at 3.0 Å clearly showed the bound inhibitor which
was built into the density. Five cycles of map inspection,
rebuilding, and refinement yielded a final model with an
R-factor of 0.180 for all 24 038 measured unique reflections
between 8.0 and 1.8 Å. A mean coordinate error of 0.2 Å can
3. Ch ir a lity of th e Bou n d In h ibitor . At 3.0 Å resolution,
it is not possible to establish unambiguously the chirality of

2470 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Cregge et al.
F igu r e 8. Superposition of the X-ray crystal structure of HNE-1 and PPE-1 complexes. Shown are the backbones of HNE (thin
green lines) and PPE (thin orange lines) and inhibitor 1 as bound to HNE (thick green lines) and PPE (thick orange lines). Also
indicated is loop 173-175 of PPE, which hinders entrance of the morpholine ring of 1 into the active site cleft, resulting in a
different binding mode for this part of the inhibitor.
the bound inhibitor in HNE. However, inhibitor 1 was used
(the LLL-diastereomer) for cocrystallation, and this diastere-
omer fits well to the electron density maps. Since the binding
mode of this part of the molecule is identical in the PPE
complex and the stereochemistry was determined unambigu-
ously as being LLL in PPE, it is almost certain that this is the
diastereomer which binds to HNE as well.
accordance with NMR data.44 Adjustments to the parameters
(V2 ) 7.0 kcal/mol and V4 ) 0.1 kcal/mol) for the C-C-C-N
torsion were made in order to reproduce the semiempirical
curve.
Gen er a l Meth od s. All reactions were performed in oven-
dried, magnetically stirred 50-, 100-, or 250-mL, round-
bottomed flasks under an atmosphere of nitrogen. Except
where noted otherwise, reagents and starting materials were
obtained from common commercial sources and used as
received. Anhydrous solvents were purchased from Aldrich
Chemical Co., Inc. in Sure/Seal bottles. Other reaction
solvents and all chromatographic, recrystallization, and work-
up solvents were spectroscopic grade and used as received. The
organic extracts were dried over anhydrous MgSO4 or Na2-
SO4 prior to solvent evaporation. Thin-layer chromatograms
were visualized first with UV light and then by I2, unless
specified otherwise.45 Flash chromatography46 was performed
on Merck silica gel 60 (230-400 mesh ASTM, EM Science).
Melting points were determined on a Thomas-Hoover capil-
lary melting point apparatus and are uncorrected. NMR
spectra were recorded on a Varian VXR-300, Unity 300, Unity
400, or Gemini-300 spectrometer in CDCl3, unless otherwise
stated. 1H NMR chemical shifts (δ) are reported in ppm
relative to TMS (0.00 ppm) or chloroform (7.26 ppm) as a
reference, 19F NMR (282 MHz unless otherwise indicated)
signals are reported in ppm from CDCl3 with an external
reference standard of CFCl3, and coupling constants (J ) are
reported in hertz (Hz). Signals were designated as s (singlet),
d (doublet), t (triplet), q (quartet), p (pentet), m (multiplet),
br (broad), etc. First-order analysis of spectra were attempted
when possible; consequently, chemical shifts and coupling
constants for multiplets may only be approximate. 13C NMR
spectra were recorded on the Varian Gemini instrument (75
MHz) with chemical shifts (δ) reported in ppm relative to
CDCl3 (77.0 ppm), unless stated otherwise. IR spectra were
recorded on a Perkin-Elmer model 1800 or a Mattson Galaxy
5020 FT-IR spectrophotometer. Mass spectra (MS) data were
collected at 70 eV on a Finnigan MAT 4600, MAT TSQ-700,
or VG Analytical Limited ZAB2-SE mass spectrophotometer
using electron impact (EI) or chemical ionization (CI) with the
molecular ion designated as M+ and the relative peak height
in percent given in parentheses. Computerized peak matching
with perfluorokerosene as the reference was used for HRMS.
Combustion analysis was performed using a Perkin-Elmer
model 2400 elemental analyzer, and analysis fell within (0.4%
of the calculated values.
The inhibitor forms a covalent tetrahedral adduct with the
enzyme, mimicking the tetrahedral transition state that occurs
during hydrolysis of peptide substrates. The carbonyl carbon
atom of the pentafluoroethyl ketone is linked to the active site
Ser 195 and the carbonyl oxygen occupies the oxyanion hole.
As was observed for the PPE-1 complex, the fluorine atoms
F75 and F76 hydrogen-bond with the active site His 57.
4. Mod elin g P a r a m eter Estim a tion . After placement
of the inhibitor in the binding site, all water molecules were
removed and the inhibitor geometry was optimized for 100
steps of steepest descent, holding all enzyme atoms fixed. A
distance-dependent dielectric constant was used in conjunction
with a 12 Å cutoff in all calculations. The inhibitor was then
subjected to 20 ps of molecular dynamics (MD) at 300 K and
reoptimized using the conjugate gradient algorithm until the
gradient norm fell below 0.01 kcal/mol/Å. Two solvent layers
of thickness 8 and 14 Å were added to the binding region,
keeping crystallographic positions where possible. Hydrogen
positions were established by placing harmonic positional
restraints on oxygen atoms in the second solvent cap and all
crystallographic waters, fixing the position of protein heavy
atoms during a 30-ps MD simulation. This was followed by
relaxation of all protein and solvent positions for 30-ps and a
50 ps production run during which coordinates were saved
every picosecond.
The conformation of the terminal N-[4-[(4-morpholinyl)-
carbonyl]benzoyl] group is largely governed by the values of
the dihedral angles between the aromatic ring and the
adjacent amide groups. N-Methylbenzamide is an appropriate
template by which to judge the quality of the molecular
mechanics parameters used for this torsion potential. The
default CVFF force field37 of DISCOVER 2.938 yields a 2-fold
rotational barrier in excess of 14 kcal/mol for N-methylben-
zamide. Similiar results were obtained with the CFF91 force
field.39 This steep barrier tends to keep the amide group
coplanar with the aromatic ring and will affect the capability
of this group to form proper hydrogen-bonding geometries with
atoms at the receptor. The energy profile for this dihedral as
measured by the semiempirical AM140 Hamiltonian in MOPAC
6.041 is 4-fold, with the minimum at about 45° and barriers of
0.6 and 1.1 kcal/mol to bring the amide group in-plane and
perpendicular to the aromatic ring, respectively.42 Similiar
results have recently been noted by others43 and are in
Starting materials were purchased from Aldrich with the
exception of some specified amino acids which were purchased
from Bachem Bioscience Inc. and Advanced Chem Tech.
Hydrogen chloride gas was purchased from AGA Burdox, and

Inhibition of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2471
the pentafluoroethyl iodide (CF3CF2I) used was purchased
from Aldrich Chemical Co. (lot #04512HZ, purity 97%).
The reaction was then capped with a drying tube and stirred
for 1 h at room temperature. The reaction was conc and then
triturated with Et2O to afford 3 as white solid: 1H NMR (300
MHz, CDCl3) δ 9.10 (br s, 3H, NH3), 4.75 (br s, 1H, CH), 2.70-
2.51 (m, 1H, CH), 1.35 (d, 3H, CH3), 1.09 (d, 3H, CH3).
The coupling reactions between the amino acids were
performed by one of four methods: method I, using a mixed
carbonic-carboxylic acid anhydride (isobutyl chloroformate,
IBCF); method II, generating the N-hydroxysuccinic imido
ester of valine 2; method III, using a carbodiimide (dicyclo-
hexylcarbodiimide, diisopropylcarbodiimide, or water-soluble
carbodiimide, EDCI); or method IV, using phosphorus reagents
such as (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate (BOP). Each method is initially de-
scribed in detail where appropriate and then referred to as
general method I, II, III, or IV for subsequent experimental
procedures. Also, repetitious experimental procedures are
described initially in detail and subsequently referred to as
general procedures.
Abbr evia tion s: Boc, tert-butyloxycarbonyl; NMM, N-meth-
ylmorpholine; IBCF, isobutyl chloroformate; DMF, N,N-di-
methylformamide; HOBT, 1-hydroxybenzotriazole hydrate;
HMPA, hexamethylphosphoramide; DCC, 1,3-dicyclohexylcar-
bodiimide; EDCI, 1-[3-(dimethylamino)propyl]-3-ethylcarbodi-
imide‚HCl; HOSu, N-hydroxysuccinamide; BOP, (benzotriazol-
1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate;
DME, ethylene glycol dimethyl ether; TMS, tetramethylsilane;
PhMe, toluene; RB, round-bottom; rt, room temperature; TFA,
trifluoroacetic acid; conc, concentrated.
(ter t-Bu tyloxyca r bon yl)-3-a m in o-2-oxo-1-p yr r olid in e-
a cetic Acid (6). A solution of Boc-R-Met-Gly methyl ester17
(5.0 g, 15.6 mmol) and methyl iodide (30 mL, 0.48 mol) was
stirred at room temperature for 16 h. The supernatant was
drawn off, and the residue was dried under high vacuum to
give the methyl sulfonium salt as an amorphous solid. To this,
dissolved in DMF/CH2Cl2 (1:1, 312 mL) under N2, was added
NaH (1.25 g, 31.5 mmol, as a 60% oil dispersion), and the
reaction was stirred at 0 °C for 2.5 h. EtOAc (104 mL) was
then added followed by H2O (24 mL), and the resulting solution
was left at room temperature overnight, then conc in vacuo,
and partitioned beween H2O (50 mL) and CH2Cl2 (50 mL). The
aqueous phase was then acidified to pH 4 with 0.5 M citric
acid and continuously extracted with CH2Cl2. Conc in vacuo
yielded a solid which was crystallized from CH2Cl2 to give 6
1
(1.0 g, 3.8 mmol) as a white solid: mp 168-169 °C; H NMR
(DMSO-d6) δ 12.80 (br s, 1H), 7.13 (d, 1H, J ) 8.8 Hz), 4.10
(br q, 1H, J ) 9.6 Hz), 4.02 (d, 1H, J ) 17.6 Hz), 3.82 (d, 1H,
J ) 17.6 Hz), 3.40-3.23 (m, 2H), 2.30-2.19 (m, 1H), 1.90-
1.75 (m, 1H); MS (CI/CH4) m/z 259 (MH+), 243, 231, 217, 203
(base peak), 185, 173, 159, 141, 114, 88, 74.
(ter t-Bu tyloxycar bon yl)-3-am in o-r-m eth yl-2-oxo-1-pyr -
r olid in ea cetic Acid (7). Boc-D-Met-L-Ala-methyl ester17
(6.08 g, 18.2 mmol) was dissolved with stirring in CH3I (25
mL, 0.40 mol) in a stoppered flask wrapped in aluminum foil.
After 6 days, the solution was concentrated in vacuo; CH2Cl2
was added, and the solution was concentrated in vacuo three
times to give 8.10 g of an amorphous solid, which was dissolved
in CH2Cl2 (125 mL). This solution (and a CH2Cl2 (25 mL)
rinse) was added rapidly to a vigorously stirred suspension of
pentane-washed NaH (1.82 g of 60% dispersion, 45.5 mmol,
2.5 equiv) in DMF (150 mL) at 0 °C under nitrogen. The
reaction mixture was allowed to stir at 0 °C for 3 h, then
poured into cold dilute HCl, and extracted twice with ether.
The combined extracts were washed twice with water and
brine and dried (MgSO4). Concentration in vacuo gave 2.94 g
of pale-yellow oil. Cystallization from cyclohexane/EtOAc gave
819 mg (17%) of fine white crystals. The analytical sample
was obtained after a second recystallization from cyclohexane/
EtOAc to give acid 7 as a pale-yellow cystalline powder.
Variable temperature NMR showed a coalescence of the methyl
doublets, but not of the coupled R-proton quartets, indicating
that the sample was a mixture of diastereomers: mp 143-
151 °C dec; IR (KBr) 3387, 2982, 1744, 1701, 1669, 1518, 1246,
1167 cm-1; 1H NMR (DMSO-d6) δ 7.10 (m, 1H), 4.54 and 4.48
(two overlapping q, 1H, J ) 7.4 Hz), 4.24-4.01 (m, 1H), 3.37-
3.15 (m, 4H - H2O peak + 3H), 2.25 (m, 1H), 1.79 (m, 1H),
1.39 (s, 9H), 1.31 and 1.30 (2d in 3:2 ratio, 3H, J ) 7.4 Hz);
MS (CI/CH4) m/z (rel intensity) 310 (M+ + 29), 273 (MH+),
245, 218, 217 (100), 216, 199, 173. Anal. (C12H20N2O5) C, H,
N.
(ter t-Bu tyloxyca r bon yl)-3-L-a m in o-2-oxo-1-p ip er id in e-
a cetic Acid (8). A solution of N-R-(tert-butyloxycarbonyl)-N-
â-(carboxymethyl)ornithine17 (1.0 g, 3.45 mmol) in DMF (30
mL) was heated at 55 °C for 16 h and then conc in vacuo to
provide a yellow oil which crystallized from EtOAc-hexane
affording 8 (800 mg, 85%) as a white solid: 1H NMR (CDCl3)
δ 7.38 (br m, 1H), 5.4 (m, 1H), 4.34-3.15 (series of m, 5H),
2.35 (m, 1H), 2.0-1.54 (m, 3H), 1.44 (s, 9H, tBu).
(ter t-Bu tyloxycar bon yl)-3-am in o-2-oxo-N-(1,1,1,2,2-pen -
ta flu or o -5-m eth yl-3-oxo -4-h exa n yl)-1-p yr r olid in ea cet-
a m id e (9). Cou p lin g Meth od I. To a stirred solution of 6
(225 mg, 0.872 mmol) and NMM (0.10 mL, 0.872 mmol) in CH2-
Cl2 (10 mL) at -20 °C was added IBCF (0.11 mL, 0.872 mmol).
The reaction was stirred at -20 °C for 20 min when additional
NMM (0.10 mL, 0.872 mmol) was added followed by addition
of acid 3 (223 mg, 0.87 mmol) as a solid in one portion. After
stirring at -15 °C for 1 h the reaction was allowed to warm to
room temperature, diluted with additional CH2Cl2, extracted
(ter t-Bu tyloxyca r bon yl)-L-va lylsu ccin im id e (2). To a
cooled (ice bath) stirred solution of N-Boc-L-valine (4.56 g, 21
mmol) and N-hydroxysuccinimide (2.41 g, 21 mmol) in DME
(50 mL) was added DCC (4.75 g, 23 mmol). The reaction
mixture was stirred for 6 h at 5 °C and then left to stand in
the refrigerator overnight. The reaction mixture was then cold-
filtered, the solid washed with Et2O, and the combined filtrate
conc to yield a solid, which was crystallized from EtOAc/hexane
to afford 2 (4.59 g, 69.5%) as a white crystalline solid: mp 123-
1
124 °C; H NMR (300 MHz, CDCl3) δ 5.00-4.95 (d, 1H, J )
9.3 Hz), 4.62 (dd, 1H, J ) 4.97 Hz), 2.85 (s, 4H), 2.75-2.44
(m, 1H), 1.45 (s, 9H), 1.25-0.90 (m, 6H); 13C NMR (CDCl3) δ
168.6, 167.9, 155.1, 155.0, 80.4, 77.4, 77.2, 77.0, 76.6, 76.5, 57.0,
31.6, 31.1, 28.2, 28.1, 28.0, 27.99, 27.93, 25.5, 18.6, 17.3; MS
(CI/CH4) m/z 315 (MH+), 299, 287, 259, 241, 215 (base peak),
173, 172, 145, 144, 116, 100, 72. Anal. (C14H22N2O6) C, H, N.
4-Am in o-1,1,1,2,2-pen taflu or o-5-m eth yl-3-h exan on e Hy-
d r och lor id e (3). A solution of N-Boc-L-valine methyl ester
2a (2.27 g, 9.81 mmol) in Et2O (14 mL)/toluene (11.3 mL) was
cooled to -50 °C and treated with CF3CF2I (3.7 mL, 31.1 mmol,
3.2 equiv) and then further cooled to -60 °C and treated
dropwise with MeLi‚LiBr (1.5 M in Et2O, 20 mL, 30.0 mmol,
3.1 equiv) in 55 min, -60 to -50 °C. The resulting reaction
mixture was stirred for 1 h and then treated dropwise with
2-propanol (10 mL, 20 min; < -50 °C). After stirring for 30
min, the reaction mixture was allowed to warm to 0 °C and
then poured into 1 M KHSO4 (60 mL). Phases were separated,
and the aqueous phase was extracted with Et2O (50 mL). The
organic phases were combined, dried (MgSO4), and filtered,
and the filtrate was conc in vacuo (room temperature, 15
mmHg) to provide a white solid. This solid was chromato-
graphed on SiO2 [40 g, eluting with hexane (400 mL) and then
10% EtOAc/hexane (400 mL)] to provide 4-[(tert-butyloxycar-
bonyl)amino]-1,1,1,2,2-pentafluoro-5-methylhexan-3-one (2.22
g) as a white solid. This solid was crystallized from hexane
(40 mL, reflux and then cooled to 0 °C) to provide an
analytically pure sample (1.62 g, 57%) (first crop; remaining
material in the mother liquor): Rf ) 0.77 in 20% EtOAc/
hexane; mp 69-70 °C; IR (CHCl3) νmax 3443, 2976, 1753, 1716,
1500, 1369, 1234, 1197, 1163 cm-1; 1H NMR (300 MHz, CDCl3)
δ 5.00 (m, 1H), 4.80 (m, 1H), 2.30 (m, 1H), 1.44 (s, 9H), 1.10
(d, 3H, J ) 6.8 Hz), 0.84 (d, 3H, J ) 6.9 Hz); 19F NMR (282
MHz, CDCl3) δ -82.1 (s), -121.4 (d, J ) 297 Hz), -122.8 (d,
J ) 297 Hz); UV (MeOH) λmax 225 nm (ꢀ ) 754); MS (CI/CH4)
m/z (rel intensity) 320 (MH+, 100). Anal. (C12H18F5NO3) C,
H, N. To a stirred, ice-cooled solution of 4-[(tert-butyloxycar-
bonyl)amino]-1,1,1,2,2-pentafluoro-5-methylhexan-3-one (1.6 g,
5.0 mmol) in EtOAc (30 mL) was bubbled HCl gas for 3 min.

2472 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Cregge et al.
with 10% HCl (3 × 20 mL), saturated NaHCO3 (2 × 20 mL),
and brine (1 × 20 mL), dried (Na2SO4), and conc to afford 9
(400 mg, 100%) as an amorphous solid: 1H NMR (CDCl3) δ
7.45 (d, 1H, J ) 4.5 Hz), 5.15-4.90 (m, 1H), 4.65-4.30 (br d,
1H), 4.10-3.40 (m, 4H), 2.60-2.10 (m, 3H), 1.45 (s, 9H), 1.16-
0.85 (dd, 6H, J ) 7 Hz); 13C NMR (CDCl3) δ 192.4, 172.4, 168.7,
168.36, 168.32, 155.2, 115.6, 78.0, 59.2, 51.0, 50.9, 50.8, 44.9,
44.8, 44.79, 44.77, 43.8, 43.7, 43.6; 19F NMR (CDCl3) δ -194.3
(s, 3F), -234 (s, 2F).
titative) which was used without further purification. In a
separate RB flask, a stirred solution of 12 (1.0 g, 2.50 mmol)
in CH2Cl2 (15 mL) was cooled to -20 °C. NMM (0.33 mL, 3.0
mmol) was added and immediately followed by the dropwise
addition of the acid chloride in CH2Cl2 (5 mL) at such a rate
as to maintain the internal reaction temperature at -10 °C
or less. After the addition was complete, the reaction mixture
was allowed to warm to room temperature. After 2 h at room
temperature, the reaction mixture was diluted with CH2Cl2
(20 mL) and washed with 1 N HCl (2 × 20 mL), saturated
NaHCO3 (2 × 20 mL), and brine (1 × 20 mL). Drying (MgSO4)
and conc in vacuo afforded a crude yellow oil which was
immediately flash chromatographed (2- × 15-cm column eluted
with 1:27 MeOH-CH2Cl2) to give 15 (876 mg, 60%) as an
(ter t-Bu t yloxyca r b on yl)-3-a m in o-r-m et h yl-2-oxo-N-
(1,1,1,2,2-p en ta flu or o-5-m eth yl-3-oxo-4-h exa n yl)-1-p yr r o-
lid in ea ceta m id e (10a ,b). A stirred solution of acid 7 (274
mg, 1.01 mmol) was coupled to 3 (269 mg, 1.05 mmol) by
method I to give, after flash chromatography, a less polar
diastereomer of pentafluoroethyl ketone 10a (248 mg, 41%)
as a colorless oil and the more polar diastereomer of pentafluo-
roethyl ketone 10b (163 mg, 34%) as white crystals. For the
less polar diastereomer 10a : 1H NMR (CDCl3) δ 7.1-6.98 (m,
1H), 5.23 (m, 1H), 5.02-4.65 (m, 2H), 4.23 (m, 1H), 3.52-3.1
(m, 2H), 2.61 (m, 1H), 2.45 (m, 1H), 1.94 (m, 1H), 1.45 (2s)
and 1.44 (m) and 1.39 (d, J ) 7.2 Hz) [12H total], 1.07 (d, J )
6.9 Hz) and 0.99 (d, J ) 6.8 Hz) and 0.85 (d, J ) 6.9 Hz) [6H
total]; 19F NMR (CDCl3) δ -79.44 (s) and -82.11 (s) in 1:12
ratio, -121.25 (d, J ) 296 Hz), -121.86 and -122.17 (inner
peaks of an AB pattern), -122.80 (d, J ) 296 Hz). Recrys-
tallization from cyclohexane gave the analytical sample with
mp 152-154 °C, for the more polar diastereomer 10b: IR
(KBr) 3395, 3295, 2976, 1684, 1532, 1505, 1285, 1235, 1200,
1167 cm-1; 1H NMR (CDCl3) δ 7.3 (m, 1H), 5.25 (m, 1H), 4.97-
4.8 (m, 2H), 4.09-3.94 (m, 1H), 3.4-3.24 (m, 2H), 2.6-2.33
(m, 2H), 2.3-2.04 (m, 1H), 1.43 (s, 9H), 1.40 and 1.38 (2d in
1:2 ratio, 3H, J ) 7.2 Hz), 1.05, 1.00, and 0.93 (3d, in a 2:1:3
ratio, 6H, J ) 6.8, 6.7, 6.8 Hz, respectively); 19F NMR (CDCl3)
δ -82.05 (s, 3F), -121.73, -121.76, and -121.81 (3s in 5:4:18
ratio, 2F); MS (CI/CH4) m/z (rel intensity) 514 (M+ + 41), 502
(M+ + 29), 474 (MH+), 446, 419, 418 (100), 400; [R]D +90.4° (c
0.52, CHCl3). Anal. (C19H28F5N3O5) C, H, N.
(ter t-Bu t yloxyca r b on yl)-3-L-a m in o-2-oxo-N-(1,1,1,2,2-
p en ta flu or o-5-m eth yl-3-oxo-4-h exa n yl)-1-p ip er id in ea cet-
a m id e (11). A stirred solution of acid 8 (750 mg, 2.75 mmol)
was coupled to 3 (702 mg, 2.75 mmol) via method I to give 11
(1.10 g, 85%) as an amorphous solid: 1H NMR (CDCl3) δ 7.37
(m, 1H), 4.97 (m, 1H), 4.1-3.3 (series of m, 6H), 2.35 (m, 2H),
2.1-1.33 (series of m, 3H), 1.44 (s, 9H, tBu), 0.97 (m, 6H, 2 ×
CH3).
Gen er a l P r oced u r e I: Rep r esen tin g Rem ova l of a n
N-t-Boc P r ot ect in g Gr ou p . 3-Am in o-2-oxo-N-(1,1,1,2,
2-p en ta flu or o-5-m eth yl-3-oxo-4-h exa n yl)-1-p yr r olid in e-
a ceta m id e Hyd r och lor id e Sa lt (12). A stirred solution of
9 (400 mg, 0.87 mmol) in EtOAc (10 mL) at ice bath temper-
ature was treated with HCl gas for 4 min; then the reaction
was stoppered with a drying tube and stirred at room tem-
perature for 1 h. Evaporation of the solvent in vacuo yielded
12 (300 mg, 87.3%) as an amorphous solid: 19F NMR (CDCl3)
δ -52.5 (s, 3F), -92.1 (s, 2F).
1
amorphous solid: H NMR (300 MHz, DMSO-d6) δ 9.05-8.90
(t, 1H, J ) 8.8 Hz), 8.62-7.60 (t, 1H, J ) 8.8 Hz), 7.93 (d, 2H,
J ) 8.5 Hz), 7.50 (d, 2H, J ) 7.9 Hz), 4.79-4.69 (m, 2H), 4.16-
3.92 (m, 2H), 3.80-3.10 (m, 10H), 2.26-2.20 (m, 2H), 2.00 (m,
1H), 0.98 (m, 3H), 0.91 (m, 3H); 13C NMR (DMSO-d6) δ 172.08,
172.0, 168.3, 165.4, 138.4, 138.3, 134.6, 134.5, 127.3, 126.9,
65.9, 59.3, 59.2, 50.3, 50.2, 44.9, 44.2, 44.1, 40.3, 40.0, 39.9,
39.7, 39.5, 39.2, 38.9, 38.6, 27.9, 25.2, 19.2, 19.1, 17.1, 17.0;
MS (CI/CH4) m/z 577 (MH+), 533, 490, 429, 404, 358, 330, 272,
228, 220, 200 (base peak), 180, 152, 91, 70. Anal.
(C25H29F5N4O6‚1H2O) C, H, N.
[3R-[1(rS*),3R*]]-r-Met h yl-3-[[4-[(4-m or p h olin yl)ca r -
b on yl]b en zoyl]a m in o]-2-oxo-N-(1,1,1,2,2-p en t a flu or o-5-
m eth yl-3-oxo-4-h exa n yl)-1-p yr r olid in ea ceta m id e (16). A
flask containing the less polar diastereomer of pentafluoroethyl
ketone 10a (241 mg, 0.509 mmol) was treated as described in
general procedure I to give 13a . 13a was then treated
according to general procedure II to afford 16 (174 mg, 58%)
as an amorphous solid: IR (KBr) 3422, 3376, 3322, 1682, 1645,
1
1539, 1281, 1223, 1202 cm-1; H NMR (CDCl3) δ 7.85 (d, 2H,
J ) 8.2 Hz), 7.47 and 7.46 (2d, 2H, J ) 8.3, 8.2 Hz), 6.90 (m,
1.3H), 6.78 (d, 0.7H, J ) 8.1 Hz), 5.03 (ddd, 0.3H, J ) 8.5, 4.6,
0.9 Hz), 4.93 (ddd, 0.7H, J ) 8.1, 3.9, 1.0 Hz), 4.79 (q, 0.7H, J
) 7.1 Hz), 4.64-4.44 (m, 1.3H), 3.88-3.17 (m, 11H), 2.83 (m,
1H), 2.38 (m, 1H), 2.12-1.94 (m, 1H), 1.50 and 1.44 (2d in a
1:2 ratio, 3H, J ) 7.2 Hz), 1.09 and 1.01 (2d in a 1:2 ratio, 3H,
J ) 6.8 Hz), 0.085 (d, 3H, J ) 6.8 Hz); 19F NMR (CDCl3) δ
major -82.11 (s), -121.23 (d, J ) 295 Hz), -122.91 (d, J )
295 Hz); minor -82.09 (s), -121.25 (d, J ) 295 Hz), -122.71
(d, J ) 295 Hz); MS (CI/CH4) m/z (rel intensity) 631 (M+
+
41), 619 (M+ + 29), 592, 591 (MH+, 100), 305, 139, 99. Anal.
(C26H31F5N4O6) C, H, N.
[3S-[1(rR*),3R*]]-r-Met h yl-3-[[4-[(4-m or p h olin yl)ca r -
b on yl]b en zoyl]a m in o]-2-oxo-N-(1,1,1,2,2-p en t a flu or o-5-
m eth yl-3-oxo-4-h exan yl)-1-pyr r olidin eacetam ide (17). The
more polar diastereomer of pentafluoroethyl ketone 10b (147
mg, 0.310 mmol) was deprotected as described in general
procedure I to give 13b. This was then treated as in general
procedure II to provide 17 (99 mg, 54%) as an amorphous
solid: IR (KBr) 3310, 1684, 1645, 1541, 1281, 1223, 1200 cm-1
;
1H NMR (CDCl3) δ 7.99 (d, <0.5H, J ) 6.1 Hz), 7.76 and 7.72
(2d in 2:1 ratio, 2H, J ) 8.3 Hz), 7.63 (d, <1H, J ) 8.2 Hz),
7.57 (d, <1H, J ) 6.5 Hz), 7.40 and 7.35 (2d in 2:1 ratio, 2H,
J ) 8.3 Hz), 5.00 (m, 1H), 4.92 (q, 1H, J ) 7.3 Hz), 4.44-4.35
and 4.28-4.17 (2m in 2:1 ratio), 3.85-3.25 (m, 10H), 2.63 (m,
1H), 2.47 (m, 1H), 2.27 (m, 1H), 1.48 and 1.44 (2d in a 1:2
ratio, 3H, J ) 7.3 Hz), 1.10 and 1.06 (2d in a 2:1 ratio, 3H, J
L-3-Am in o-2-oxo-N-(1,1,1,2,2-p en t a flu or o-5-m et h yl-3-
oxo-4-h exan yl)-1-piper idin eacetam ide Hydr och lor ide Salt
(14). A stirred solution of 11 (900 mg, 1.90 mmol), treated as
in general procedure I, afforded 14 (770 mg, 99%) as an
amorphous solid: 1H NMR (CDCl3) δ 7.98 (m, 0.5H, NH), 7.35
(m, 1H), 4.92 (m, 1H), 4.28-3.2 (series of m, 6H), 2.41 (m, 2H),
2.29-1.54 (series of m, 3H), 1.01 (m, 6H, 2 × CH3).
) 6.8 Hz), 1.01 and 0.096 (2d in 1:2 ratio, 3H, J ) 6.9 Hz); 19
F
NMR (CDCl3) δ -81.96 and -82.03 (2s in 1:2 ratio, 3F),
Gen er a l P r oced u r e II: Rep r esen ta tive Cou p lin g w ith
4-[(4-Mor p h olin yl)ca r bon yl]ben zoyl Ch lor id e. N-[4-[(4-
Mor ph olin yl)car bon yl]ben zoyl]-3-am in o-2-oxo-N-(1,1,1,2,2-
p en ta flu or o-5-m eth yl-3-oxo-4-h exa n yl)-1-p yr r olid in a m -
id e (15). To a stirred suspension of 4-[(4-morpholinyl)-
carbonyl]benzoic acid (0.717 g, 3.1 mmol) and benzyltriethy-
lammonium chloride (5 mg, 0.02 mmol) in 1,2-dichloroethane
(20 mL) was added thionyl chloride (0.225 mL, 3.1 mmol), and
the reaction was heated at reflux. After 2.5 h, the reaction
was allowed to cool to room temperature and conc in vacuo.
The residue was then azeotroped with CCl4 and placed under
vacuum to give the acid chloride as a light-orange oil (quan-
-121.70 (apparent d, inner peaks of AB pattern), -121.81 (s
in 2:1 ratio, 2F); MS (CI/CH4) m/z (rel intensity) 631 (M+
+
41), 619 (M+ + 29), 592, 591 (MH+, 100), 99. Anal.
(C26H31F5N4O6) C, H, N.
N-[4-[(4-Mor p h olin yl)ca r b on yl]b en zoyl]-L-3-a m in o-2-
oxo-N-(1,1,1,2,2-p en ta flu or o-5-m eth yl-3-oxo-4-h exa n yl)-
1-p ip er id in ea ceta m id e (18). A stirred solution of 14 (690
mg, 1.68 mmol) was treated as described in general procedure
II to give 18 (160 mg, 16%) as an amorphous solid: IR (KBr)
3059, 3423, 3381, 3373, 3362, 3310, 2966, 2931, 2862, 1753,
1639, 1541, 1496, 1464, 1438 cm-1; 1H NMR (300 MHz, CDCl3)

Inhibition of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2473
δ 7.84 (d, 2H, J ) 8.5 Hz, aryl), 7.46 (d, 2H, J ) 7.9 Hz), 7.32
(m, 1H, NH), 7.05 (d, 1H, J ) 8.5 Hz, NH), 5.02 (m, 1H), 4.75
and 4.44 (2d, 1H, J ) 18 Hz), 4.29 (m, 1H), 3.9-3.25 (series of
m, 11H), 2.60-2.32 (m, 2H), 1.98 (m, 3H), 1.04 (d, 3H, J ) 9
Hz, CH3), 0.91 (dd, 3H, J ) 12, 9 Hz, CH3); 13C NMR (CDCl3)
δ 168.6, 138.56, 134.9, 127.47, 127.41, 127.3, 127.2, 77.5, 77.4,
77.3, 77.2, 77.1, 77.0, 76.8, 76.79, 76.72, 76.5, 66.8, 59.6, 59.3,
52.1, 51.9, 51.8, 49.37, 49.33, 29.6, 28.9, 28.7, 27.4, 27.2, 21.1,
21.0, 19.86, 19.81, 16.8, 16.7; 19F NMR (CDCl3) δ -82.04 (d, J
) 5.6 Hz, CF3), -121.76 and -122.01 (d, J ) 11.3 Hz, CF2);
MS (CI/CH4) m/z 631, 619, 591 (MH+, 100), 571, 504, 443, 416,
372, 357, 301, 218, 153, 104, 71. Anal. (C26H31F5N4O6) C, H,
N.
chromatography (hexane/EtOAc, 90:10, 80:20, 60:40): [R]25
D
-14.0 (c 0.4, MeOH); IR (neat) 3500-2500 (br), 2977, 1745,
1716, 1442, 1377, 1242, 1183 cm-1; 1H NMR (CDCl3, 400 MHz)
δ 3.70 (s, 3H, OCH3), 2.76 (m, 2H, B and X for ABX, CH2CH),
2.46 (dd, 1H, J ) 16, 2.8 Hz, A of ABX, CH2CH), 2.00 (septet,
1H, J ) 6.8 Hz, CH(CH3)2), 0.94 (dd, 6H, J ) 6.8, 7.2 Hz, CH-
(CH3)2); 13C NMR (CDCl3, 100 MHz) δ 178.5, 175.0, 51.8, 47.2,
32.9, 30.2, 20.0, 19.6.
To a stirred solution of the above oil (2.02 g, 13.8 mmol) in
DMF (50 mL) were added HOBt (2.16 g, 15.5 mmol) and
morpholine (1.4 mL, 16 mmol), followed by EDCI (3.0 g, 16
mmol) at 0 °C. The resulting solution was stirred at 0 °C for
3 h and then allowed to warm to room temperature and stir
overnight; 1 N HCl (100 mL) and H2O (100 mL) were added,
and the reaction was extracted with EtOAc (3 × 100 mL). The
combined extracts were washed with saturated NaHCO3 (100
mL) and brine (100 mL), passed through a short pad of MgSO4/
silica gel, and conc to provide a colorless oil (2.15 g, 64%).
Without further purification this oil (2.15 g, 8.8 mmol) was
dissolved in THF and H2O (50 mL/50 mL) and treated with
LiOH‚H2O (420 mg, 10 mmol) at room temperature overnight.
After 16 h the reaction was washed with Et2O (100 mL) and
the remaining aqueous phase acidified with 6 N HCl (pH 3).
The aqueous phase was then extracted with EtOAc (3 × 100
mL), and the combined extracts were dried (MgSO4), conc, and
flash chromatographed (hexane/EtOAc, 60:40, 20:80) to provide
20a as a colorless oil (1.3 g, 65%). Crystallization from hexane
L-2-Isop r op yl-4-oxo-5-p h en ylbu ta n oic Acid (19a ).47 To
a stirred, cold (0 °C) solution of l-methylhydroxyisovaleric acid
(1.32 g, 10 mmol) in CH2Cl2 (15 mL) was added triflic
anhydride (1.84 mL, 11 mmol) followed by 2,6-lutidine (1.28
mL, 1 mmol) under N2. The resulting solution was stirred for
10 min and used for the next reaction without further
purification. Meanwhile, to a stirred cold (0 °C) suspension
of NaH (1.0 g of 60% in oil, 22 mmol) in dry THF (50 mL) was
added, dropwise, a solution of ethyl benzoylacetate (3.84 g, 20
mmol) in THF (50 mL) under N2. After 10 min of stirring,
the above triflate was diluted with CH2Cl2 (10 mL) and added
dropwise to the gray suspension. The resulting solution was
stirred at reflux for 13 h. Upon cooling, 1 N HCl solution (60
mL) was added and the reaction mixture was extracted with
EtOAc (3 × 80 mL). The organic extracts were combined,
washed with brine (100 mL), dried (MgSO4), passed through
a short pad of silica gel, and conc by rotary evaporator (bath
temperature 30 °C) to provide a yellow oil. This oil was
dissolved in H2O (70 mL) and THF (70 mL), treated with
LiOH‚H2O (2.5 g), and stirred at room temperature for 5 h.
The resulting solution was acidified by 6 N HCl (pH 5) and
heated at reflux for 12 h. After the reaction cooled to room
temperature, the solution was basified by solid KOH (pH 10)
and washed with Et2O (70 mL). The aqueous phase was
acidified by 6 N HCl (pH 3) and extracted with EtOAc (3 × 80
mL). The combined EtOAc extracts were dried (MgSO4),
passed through a short pad of silica gel, and conc to give 19a
as an oil (920 mg, 42%) after purification by radial chroma-
tography (hexane/EtOAc, 95:5, 90:10, 80:20). The oily product
solidified upon addition of hexane/EtOAc (1:1) to give a white
solid: [R]25D -24.06 (c 0.64, MeOH); mp 103-105 °C; IR (neat)
3600-2500 (br), 2996, 1802, 1708, 1598, 1582, 1450, 1214
and EtOAc gave a white solid: [R]25 -24.9 (c 0.45, MeOH);
D
mp 79-28 °C; 1H NMR (CDCl3, 400 MHz) δ 10.47 (br, 1H,
C00H), 3.66 (set of m, 8H, CH2CH2), 2.82 (m, 2H, B and X for
ABX, CH2CH), 2.32 (dd, 1H, J ) 15.6, 2.8 Hz, A of ABX, CH2-
CH), 2.04 (m, 1H, CH(CH3)2), 0.98 (dd, 6H, J ) 6.8, 7.2 Hz,
CH(CH3)2); 13C NMR (CDCl3, 100 MHz) δ 179.0, 170.6, 66.8,
66.5, 47.5, 46.0, 42.2, 31.4, 30.0, 20.2, 19.8.
D-2-Isopr opyl-4-oxo-5-m or ph olin ylbu tan oic Acid (20b).47
The corresponding D-isomer was prepared from D-2-methyl-
hydroxyisovaleric acid ester in the same manner as described
above in 62% yield: [R]25 +26.36 (c 0.33, MeOH).
D
N-(1,1,1,2,2-P en t a flu or o-5-m et h yl-3-oxo-4-h exa n yl)-L-
p r olin a m id e (21). Boc-proline (2.15 g, 0.01 mol) and 3 (2.26
g, 0.01 mol) were coupled by method I to yield Boc-Pro-Val-
C2F5 (3.30 g, 79%) as an amorphous solid: 1H NMR (CDCl3) δ
7.85 (m, 1H, NH), 4.98 (m, 1H), 4.38 (m, 1H, R-CH), 3.43 (m,
2H), 2.36 (m, 1H, â-CH Val), 2.3-1.71 (m, 4H), 1.44 (s, 9H,
tBu), 1.06, (d, 3H, J ) 6.6 Hz, CH3), 0.87 (d, 3H, J ) 6.6 Hz,
CH3); MS (CI/CH4) m/z (rel intensity) 457, 445 (M+ + 29), 417
(MH+), 389, 361, 341, 317 (100), 269, 213, 186, 170, 114, 84,
70.
cm-1 1H NMR (CDCl3, 400 MHz) δ 7.96 and 7.46 (m, 5H,
;
phenyl), 3.48 (dd, 1H, B of ABX, CH2CH), 3.30 (m, 2H, A and
X for ABX, CH2CH), 2.10 (m, 1H, CH(CH3)2), 1.02 (d, 6H, J )
8 Hz, CH(CH3)2). Anal. (C13H16O3) C, H, N.
Treating this Boc-Pro-Val-C2F5 (624 mg, 1.50 mmol) as
described in general procedure I gave HCl salt 21 (544 mg) as
a very hygroscopic white foam.
D-2-Isopr opyl-4-oxo-5-ph en ylbu tan oic Acid (19b).47 The
corresponding D-isomer was prepared in the same manner
from D-methylhydroisovaleric acid, as described above, in 45%
yield: [R]25 +32.36 (c 0.72, MeOH).
[2S-[1(R*),2R*]]-1-[2-(1-Meth yleth yl)-4-p h en yl-1,4-d iox-
obu tyl]-N-(1,1,1,2,2-pen taflu or o-5-m eth yl-3-oxo-4-h exan yl)-
p r olin a m id e (22). Amine hydrochloride 21 (620 mg, 1.77
mmol) was coupled to 19b (380 mg, 1.77 mmol) using coupling
method III affording 22 (140 mg, 15%) as a colorless glass:
1H NMR (CDCl3) δ 8.19 (d, 1H, J ) 7.5 Hz), 7.98-7.89 (m,
3H), 7.6-7.3 (m, 5H), 4.91 (m, 1H), 4.65 and 4.60 (2dd, 1H, J
) 8.0, 1.4 Hz and J ) 8.1, 2.0 Hz), 4.14 and 3.98 (q and m,
1H, J ) 7.0 Hz), 3.75-3.60 (m, 2H), 3.13-3.07 (m, 2H), 2.97
(m, 1H), 2.55-2.23 (m, 2H), 2.13-1.78 (m, 4H), 1.30 (d, 1H, J
) 7.0 Hz), 1.17-1.00 (m, 2H), 0.89-0.85 (m, 12H, d at 0.88, J
) 6.9 Hz, and d at 0.86, J ) 6.9 Hz, discernible); 19F NMR
(CDCl3) mixture of isomers (6:4) A:B δ A -82.20 (s, CF3),
-121.45 and -122.86 (AB quartet, J ) 296 Hz, CF2); B -82.17
(s, CF3), -121.37 and -122.71 (AB quartet, J ) 296 Hz, CF2);
MS (CI/CH4) m/z (rel intensity) 519 (MH+), 499, 399, 317, 300,
272, 203 (100), 105, 84, 70. Anal. (C25H31F5N2O4) C, H, N.
D
L-2-Isopr opyl-4-oxo-5-m or ph olin ylbu tan oic Acid (20a).47
To a stirred suspension of NaH (2.4 g, 60% oil dispersion, 60
mmol) in DMF (50 mL) was added di-tert-butylmalonate (4.32
g, 20 mmol). The reaction mixture was stirred at 0 °C for 3 h.
Meanwhile to a stirred solution of L-2-methylhydroxyisovaleric
acid ester (2.64 g, 20 mmol) in CH2Cl2 (20 mL) was added triflic
anhydride (4 mL, 22 mmol), followed by 2,6-lutidine (2.8 mL,
24 mmol) at 0 °C, and the solution was stirred for 10 min.
This triflate was diluted with CH2Cl2 (10 mL) and added to
the above enolate. The resulting mixture was then stirred at
0 °C for 3 h. The reaction was then quenched with H2O (100
mL) and extracted with EtOAc (3 × 80 mL), and the combined
extracts were washed successively with 1 N HCl (100 mL) and
brine (100 mL), dried (MgSO4), passed through a short pad of
silica gel, and conc to a yellow oil. This oil was dissolved in
toluene (50 mL), treated with TFA (5 mL) at room temperature
for 1 h, and heated at reflux for an additional 5 h. After cooling
to room temperature the reaction was concentrated to a
residue which was dissolved in EtOAc (150 mL), washed with
H2O (100 mL) and brine (100 mL), passed through a short pad
of MgSO4 and silica gel, and conc to provide 2-isopropyl methyl
succinate as a sticky oil (2.35 g, 71%) after purification by flash
[2S-[1(S*),2R*]]-1-[2-(1-Meth yleth yl)-4-(4-m or p h olin yl)-
1,4-d ioxobu tyl]-N-(1,1,1,2,2-p en ta flu or o-5-m eth yl-3-oxo-
4-h exa n yl)-2-p yr r olid in eca r boxa m id e (23a ). Cou p lin g
Meth od III. Coupling of the amine hydrochloride 21 (370 mg,
1.05 mmol) with acid 20a (232 mg, 1.01 mmol) using HOBT
(159 mg, 1.04 mmol), EDCI (256 mg, 1.34 mmol), and NMM

2474 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Cregge et al.
(115 mL, 1.05 mmol) in 2:1 CH2Cl2/DMF (4.5 mL) gave 479
mg of yellow oil which was combined with 89 mg of crude
material from a similar experiment and purified by flash
chromatography (10% acetone/EtOAc) to give 333 mg (47%)
of 23a as a very pale-yellow tacky glass: IR (CHCl3) 2967,
a-CH of Val), 4.3 (m, 1H), 3.92 (m, 1H), 3.57 (m, 1H), 2.3-1.9
(series of m, 5H), 1.44 (s, 9H, tBu), 0.95 (m, 6H, 2 × CH3).
Boc-d eh yd r ova lyl-L-p r olin a m id e (27). 25 (592 mg, 2.75
mmol) and L-proline methyl ester hydrochloride (455 mg, 2.75
mmol) were coupled using method IV to give the dipeptide
methyl ester as an amorphous solid (406 mg, 45%): 1H NMR
(CDCl3) δ 6.07-5.80 (br s, 1H), 4.57 (br t, 1H, J ) 6.1 Hz),
3.94-3.60 (m, 4H), 3.60-3.42 (m, 1H), 2.39-2.19 (m, 1H),
2.12-1.84 (m, 6H), 1.72 (s, 3H), 1.44 (s, 9H); 13C NMR (CDCl3)
δ 172.6, 166.8, 153.1, 127.9, 124.4, 79.9, 58.3, 52.0, 47.9, 29.6,
28.2, 24.9, 19.5, 18.9. Hydrolysis in CH3OH/H2O (4:1, 20 mL)
and LiOH‚H2O (78.3 mg, 1.8 mmol) and heating at 50 °C for
2.5 h provided 27 (281 mg, 79%) as an amorphous solid: 1H
NMR (CDCl3) δ 6.17 (br s, 1H), 4.70-4.56 (br m, 1H), 3.79-
3.48 (m, 2H), 2.41-2.07 (br m, 2H), 2.04-1.90 (m, 2H), 1.79
(s, 3H), 1.74 (s, 3H), 1.44 (s, 9H); 13C NMR (CDCl3) δ 172.7,
168.3, 153.4, 125.9, 124.0, 80.9, 59.6, 48.5, 28.8, 28.2, 24.6, 19.8,
18.6.
1674, 1634, 1466, 1439, 1227, 1198, 1117 cm-1 1H NMR
;
(CDCl3) δ 7.60 (d, 0.5H, J ) 8.7 Hz), 7.57 (d, 0.5H, J ) 9.1
Hz), 4.67 (m, 1H), 4.06-3.89 (m, 1H), 3.76-3.37 (m, 10H),
2.96-2.65 (m, 2H), 2.51 and 2.50 (2dd, 1H, J ) 16.3, 6.8 Hz
and J ) 16.3, 6.6 Hz), 2.38-2.23 (m, 1H), 2.14-1.79 (m, 4H),
1.01-0.85 (m, 12H, d at 0.99, J ) 6.6 Hz), 0.92 (d, J ) 6.9
Hz), 0.87 (J ) 6.9 Hz, discernible); 19F NMR (CDCl3) δ -79.29
and -79.45 (2s, trace), -82.01 and -82.13 (2s, 3F), -120.97
(d, 0.5F, J ) 293 Hz), -121.25 (d, 0.5F, J ) 297 Hz), -123.09
(d, 0.5F, J ) 297 Hz), -123.32 (d, 0.5F, J ) 293 Hz); MS (CI/
CH4) m/z (rel intensity) 568 (M+ + 41), 556 (M+ + 29), 528
(MH+), 441, 213, 212 (100). Anal. (C23H34F5N3O5) C, H, N.
[2S-[1(R*),2R*]]-1-[2-(1-Meth yleth yl)-4-(4-m or ph olin yl)-
1,4-d ioxobu tyl]-N-(1,1,1,2,2-p en ta flu or o-5-m eth yl-3-oxo-
4-h exa n yl)-2-p yr r olid in eca r boxa m id e (23b). Coupling of
the amine hydrochloride 21 (402 mg, 1.14 mmol) with acid 20b
(229 mg, 1.00 mmol) using coupling method III gave pen-
tafluoroethyl ketone 23b (303 mg, 57%) as a colorless glass. A
second chromatography (3.5-6.5% i-PrOH/ CH2Cl2) taking
only the center fractions failed to remove an impurity (pre-
sumably a third diastereomer), giving 23b (225 mg) as a
colorless glass: IR (film) 2969, 1686, 1643, 1437, 1225, 1200,
1117 cm-1; 1H NMR (CDCl3) δ 9.25, 8.16, and 7.93 (3d in 1:7:5
ratio, 1H, J ) 8.8, 7.5, 7.3 Hz), 4.97-4.88 (m, 1H), 4.79-4.43
(m, 1H), 3.96-3.84 (m, 1H), 3.74-3.43 (m, 10H), 3.00-2.84
(m, 2H), 2.70-2.23 (m, 3H), 2.11-1.75 (m, 4H), 1.09-0.85 (m,
12H, d at 1.07, J ) 6.8 Hz, d at 0.88, J ) 6.9 Hz, and d at
0.87, J ) 6.9 Hz, discernible); 19F NMR (CDCl3) 53:34:8
mixture of isomers A:B:C δ A -82.18 (s), 121.45 (d, J ) 295
Hz), -122.84 (d, J ) 295 Hz); B -82.14 (s), -121.33 (d, J )
295 Hz), -122.69 (d, J ) 295 Hz); C -82.22 (s), -120.60 (d, J
) 292 Hz), -123.57 (d, J ) 292 Hz); MS (CI/CH4) m/z (rel
intensity) 556 (M+ + 29), 529, 528 (MH+, 100), 212. Anal.
(C23H34F5N3O5) C, H, N.
(ter t-Bu tyloxycar bon yl)-D-valyl-N-(1,1,1,2,2-pen taflu or o-
5-m eth yl-3-oxo-4-h exa n yl)-L-p r olin a m id e (28). Acid 26
(1.0 g, 3.18 mmol) was coupled to 3 (816 mg, 3.18 mmol) via
method I to yield 28 (1.4 g, 85%) as a white solid: 1H NMR
(300 MHz, CDCl3) δ 8.02 (d, 1H, J ) 8 Hz, NH), 7.8 (br d,
0.5H, NH), 5.3 (br d, 0.5H, NH), 5.18 (dd, 0.5H, J ) 8.6, 4.6
Hz), 4.91 (dd, 1 H, J ) 7.6, 4.3 Hz), 4.68 (dd, 0.5H, J ) 7.8,
6.2 Hz), 4.26 (m, 1H), 3.85 and 3.65 (pr m, 2H, CH2N), 2.55-
1.7 (series of m, 6H, â-CH of Val, CH2CH2), 1.44 (s, 9H, tBu),
1.1-0.82 (m, 12H, 4 × CH3); 19F NMR (CDCl3) δ -82.12 (s,
CF3), -121.31 and -122.75 (AB quartet, J ) 296 Hz, CF2).
(ter t-Bu tyloxyca r bon yl)d eh yd r ova lyl-N-(1,1,1,2,2-p en -
taflu or o-5-m eth yl-3-oxo-4-h exan yl)-L-pr olin am ide (29a,b).
A stirred solution of 27 (280 mg, 0.90 mmol) and 3 (229 mg,
0.90 mmol) were coupled using method I to provide, after flash
chromatography, diastereomers 29b (higher Rf, 84.7 mg, 18%)
and 29a (lower Rf, 67.7 mg, 15%) as amorphous solids. 29a :
1H NMR (CDCl3) δ 7.99 (d, 1H, J ) 6.9 Hz), 6.12 (s, 1H), 4.97
(t, 1H, J ) 7.6 Hz), 4.67 (dd, 1H, J ) 8.2, 4.4 Hz), 3.58-3.38
(m, 2H), 2.46-2.05 (m, 4H), 1.97-1.83 (m, 2H), 1.74 (s, 3H),
1.72 (s, 3H), 1.44 (s, 9H), 1.00 (d, 3H, J ) 6.8 Hz), 0.97 (d, 3H,
J ) 6.9 Hz); 19F NMR (CDCl3) δ -79.25, -82.13, -121.90,
-122.11. 29b: 1H NMR (CDCl3) δ 8.00 (d, 1H, J ) 6.9 Hz),
6.05 (s, 1H), 4.82 (t, 1H, J ) 7.0 Hz), 4.68 (dd, 1H, J ) 11.9,
3.8 Hz), 3.68-3.55 (m, 1H), 3.55-3.42 (m, 1H), 2.48-2.25 (m,
2H), 2.14-1.86 (m, 3H), 1.72 (s, 3H), 1.71 (s, 3H), 1.44 (s, 9H),
1.01 (d, 3H, J ) 6.7 Hz), 0.93 (d, 3H, J ) 6.9 Hz); 19F NMR
(CDCl3) δ -79.35, -82.09, -121.99, -122.03.
2-[(ter t-Bu tyloxyca r bon yl)a m in o]-3-m eth yl-2-bu ten o-
ic Acid (25). N-Boc-dehyrovaline methyl ester (730 mg, 3.20
mmol), prepared from the dehydrovaline methyl ester hydro-
chloride,48 was hydrolyzed in MeOH/H2O (4:1, 20 mL) with
LiOH‚H2O (2.13 mg, 51 mmol). The reaction mixture was
heated at 50 °C for 24 h, then allowed to cool to room
temperature, and conc in vacuo to a residue which was
dissolved in H2O and washed with EtOAc. The aqueous
solution was then acidified with 3 M HCl, with ice bath cooling,
to pH 2 and extracted with EtOAc. The organic extract was
then washed with brine, dried (Na2SO4), and conc in vacuo to
afford 25 (637 mg, 92%) as a white glass. This was used in
the next step without further purification.
D-Va lyl-N-(1,1,1,2,2-p en ta flu or o-5-m eth yl-3-oxo-4-h exa -
n yl)-L-p r olin a m id e Hyd r och lor id e Sa lt (30). Treatment
of 28 (1.3 g, 2.5 mmol) as described in general procedure I
afforded 30 (964 mg, 85%) as a white foam.
Deh yd r ova lyl-N-(1,1,1,2,2-p en ta flu or o-5-m eth yl-3-oxo-
4-h exa n yl)-L-p r olin a m id e Hyd r och lor id e Sa lt (31a ,b). A
solution of 29b (67.7 mg, 0.132 mmol) was treated as described
in general procedure I to afford 31b. The synthesis of 31a
was carried out in the same fashion starting with 29a .
Boc-D-va lyl-L-p r olin e (26). Cou p lin g Meth od IV. To a
stirred solution of Boc-D-valine (24) (from Novabiochem; 2.4
g, 0.011 mol), L-proline methyl ester HCl (1.8 g, 0.011 mol),
and BOP (Novabiochem; 4.9 g, 0.011 mol) in CH3CN (100 mL)
at room temperature was added Et3N (3.1 mL, 0.022 mmol).
After stirring at room temperature for 2 h, the reaction was
treated with brine (100 mL) and extracted with EtOAc (3 ×
100 mL). The combined extracts were washed with 1 N HCl
(100 mL) and then saturated NaHCO3 (100 mL) and dried
(MgSO4). The solvent was removed in vacuo, and the crude
oil was immediately flashed chromatographed (3- × 21-cm
column eluted with 2:3 Et2O-hexane) to give the dipeptide
methyl ester (1.97 g, 55%) as a colorless oil: 1H NMR (300
MHz, CDCl3) δ 5.27 (d, 1H, J ) 8 Hz, NH), 4.44 (m, 1H, R-CH
of Val), 4.35 (m, 1H), 3.9 (m, 1H), 3.73 (s, 3H, OMe), 3.6 (m,
1H), 2.27-1.9 (pr m, 5H), 1.44 (s, 9H, t-Bu), 1.00 (d, 3H, J )
6.8 Hz, CH3), 0.92 (d, 3H, J ) 6.6 Hz, CH3). Saponification of
the ester (1.63 g, 4.96 mmol) in MeOH (30 mL) at room
temperature with 1 N NaOH (4.0 mL) at room temperature
for 3 h gave 26 (1.0 g, 64%) as an amorphous solid: 1H NMR
(300 MHz, CDCl3) δ 5.38 (d, 1H, J ) 8 Hz, NH), 4.51 (m, 1H,
N -[4-[(4 -Mor p h olin yl)ca r b on yl]b en zoyl]-D -va lyl-N-
(1,1,1,2,2-p en ta flu or o -5-m eth yl-3-oxo-4-h exa n yl)-L-p r o-
lin a m id e (32). A solution of 30 (960 mg, 2.12 mmol) in
CH2Cl2 (20 mL) was treated as described in general procedure
II to afford 32 (900 mg, 67%) as an amorphous solid: IR (KBr)
3427, 3313, 3053, 2968, 2935, 2897, 2877, 1751, 1635, 1533,
1435, 1394, 1327, 1302, 1280, 1261, 1222, 1197, 1161, 1114,
1068, 1026, 1014, 918, 896, 862, 842, 785, 738, 655, 597 cm-1
;
1H NMR (300 MHz, CDCl3) δ 7.9-7.7 and 7.52-7.40 (pr m,
5H, aryl and NH), 6.83 (m, 1H, NH), 4.93 (m, 1H, R-CH of
Val), 4.7 (m, 2H, R-CH of Val and CH of Pro), 4.08-3.25 (series
of m, 10 H), 2.55-1.82 (series of m, 6H), 1.16-0.70 (m, 12H,
4 × CH3); 13C NMR (CDCl3) δ 172.1, 170.8, 170.6, 169.3, 169.2,
166.6, 166.4, 138.6, 138.5, 134.7, 127.4, 127.36, 127.34, 127.2,
66.83, 66.80, 59.9, 59.7, 59.5, 59.3, 58.9, 57.1, 56.8, 55.93, 55.90,
52.2, 48.1, 48.0, 47.6, 47.2, 42.5, 31.7, 31.1, 31.0, 29.1, 28.8,
28.7, 27.5, 27.3, 24.67, 24.61, 24.4, 19.7, 19.5, 19.4, 18.5, 18.3,
17.6, 17.5, 16.4, 16.3; 19F NMR (CDCl3) δ -82.10, -82.15 (2s,
CF3), -121.39, -122.69 (AB quartet, J ) 298 Hz, CF2) also

Inhibition of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2475
-121.96 (s); MS (CI/CH4) m/z (rel intensity) 661 (M+ + 29),
634, 633 (MH+), 446, 333, 317 (100), 305, 237, 218, 193, 144,
130. Anal. (C29H37F5N4O6) C, H, N.
3.25 (m, 1H, Pec), 2.31 (m, 1H, Pec), 2.20 (m, 0.5H, Val of
D-compd), 2.01 (m, 0.5H, Val), 1.78-1.33 (m, 5H), 1.44 (s, 9H,
tBu), 1.01-0.86 (m, 6H, 2 × CH3).
(ter t-Bu t yloxyca r b on yl)-L-va lyl-tr a n s-4-h yd r oxyp r o-
lin e (41). trans-4-Hydroxy-L-proline (1.31 g, 10 mmol) was
treated as described in method II to give 41 (1.85 g, 56%) as
an amorphous solid: 1H NMR (300 MHz, CDCl3) δ 5.70-5.25
(m, 1H, NH), 5.05 (m, 1H), 4.80-3.95 (m, 4H), 3.85-2.80
(series of m, 3H), 2.35-1.80 (m, 2H), 1.44 (s, 9H, tBu), 1.01-
0.95 (m, 6H, 2 × CH3); MS (CI/CH4) m/z (rel intensity) 331
(MH+), 303, 275 (100), 259, 231, 217, 172, 162, 144, 132, 116,
86, 72.
(ter t-Bu tyloxyca r bon yl)-L-va lyl-tr a n s-4-ben zyloxyp r o-
lin e (42). trans-4-Benzyloxy-L-proline, hydrochloride salt (38)
(2.57 g, 10 mmol) was coupled to 2 (3.14 g, 10 mmol) as
described in method II to give 42 (1.8 g, 42%). Crystallization
(EtOAc/hexane) afforded a white solid: mp 125-128 °C; 1H
NMR (300 MHz, CDCl3) δ 8.0 (br s, 1H), 7.4-7.2 (m, 5H), 5.35
(d, 1H, J ) 9.2 Hz, NH), 4.65 (m, 1H), 4.52 (m, 2H), 3.70 (m,
1H), 2.5-1.8 (series of m, 3H), 1.44 (s, 9H, tBu), 0.98 (d, 3H,
J ) 6.8 Hz, CH3), 0.92 (d,, 3H, J ) 6.8 Hz, CH3).
N-[4-[(4-Mor p h olin yl)ca r bon yl]ben zoyl]d eh yd r ova lyl-
N-(1,1,1,2,2-p en ta flu or o-5-m eth yl-3-oxo-4-h exa n yl)-L-p r o-
lin a m id e (33a ,b). A solution of 31b (0.132 mmol) was treated
as described in general procedure II to give 33b (38.1 mg, 47%)
as a colorless glass: IR (KBr) 3435, 3298, 3048, 2972, 1753,
1638, 1528, 1497, 1437, 1319, 1302, 1281, 1260, 1223, 1198,
1117 cm-1; 1H NMR (CDCl3) δ 8.90 (s, 1H), 8.05-7.75 (m, 3H),
7.54-7.33 (m, 2H), 4.97 (t, 1H, J ) 7.5 Hz), 4.66 (dd, 1H, J )
8.1, 4.0 Hz), 4.03-3.18 (br m, 4H), 2.46-2.09 (m, 3H), 2.09-
1.87 (m, 2H), 1.77 (s, 3H), 1.66 (s, 3H), 0.96 (d, 3H, J ) 6.6
Hz), 0.84 (d, 3H, J ) 6.7 Hz); 19F NMR (CDCl3) δ -82.02,
-121.91, -121.95; MS (CI/CH4) m/z 631 (MH+), 317 (base
peak). Anal. (C29H35F5N4O6‚0.2 H2O) C, H, N.
The same procedure as above, using 31a as the starting
material, provided 33a (53.6 mg, 52%) as a colorless glass: IR
(KBr) 3435, 3295, 2972, 1753, 1640, 1528, 1497, 1437, 1302,
1280, 1223, 1198, 1117 cm-1; 1H NMR (CDCl3) δ 9.12 (s, 1H),
7.87 (d, 2H, J ) 8.4 Hz), 7.60 (d, 1H, J ) 9.0 Hz), 7.44 (d, 2H,
J ) 8.4 Hz), 5.00 (dd, 1H, J ) 8.3, 5.6 Hz), 4.61 (dd, 1H, J )
8.1, 4.5 Hz), 4.02-3.18 (br m, 4H), 2.51-2.08 (m, 3H), 2.85-
2.08 (m, 2H), 1.74 (s, 3H), 1.58 (s, 3H), 1.00 (d, 3H, J ) 6.8
Hz), 0.94 (d, 3H, J ) 6.9 Hz); 19F NMR (CDCl3) δ -81.97,
-121.89; MS (CI/CH4) m/z 631 (MH+), 317 (base peak). Anal.
(C29H35F5N4O6‚0.4 H2O) C, H, N.
N-[4-[(4-Mor p h olin yl)ca r bon yl]ben zoyl]d eh yd r ova lyl-
N-(1,1,1,2,2-p en ta flu or o-5-m eth yl-3-oxo-4-h exa n yl)-L-p r o-
lin a m id e (33c). The D,L-mixture at P1, 33, was carried
through the same sequence of reactions to provide 33c (600
mg, 54%) as an amorphous solid after flash chromatography
(eluted with 1:27 MeOH/CH2Cl2): 1H NMR (CDCl3) δ 8.19 (s,
0.5H, NH), 8.02-7.93 (m, 1H, NH), 7.87 (m, 2H, aryl), 7.68
(d, 0.5H, J ) 8 Hz, NH), 4.97 (dd, 1H, J ) 9, 7 Hz), 4.68 (2dd,
1H, ratio 3:2, J ) 8, 4 Hz), 3.9-3.3 (series of m, 10H), 2.4-1.9
(series of m, 5H),1.77 (m, 3H, CH3), 1.66 (d, 3H, J ) 9 Hz,
CH3), 0.97 (m, 3H, CH3), 0.85 (d, 3H, J ) 7 Hz); 19F NMR
(CDCl3) δ -82.10 (s, CF3), -121.99 (d, J ) 8.5 Hz), -122.03
(overlapping s); MS (CI/CH4) m/z 659, 631 (MH+), 357, 345,
317 (base peak), 297, 84, 70. Anal. (C29H35F5N4O6‚0.6H2O)
C, H, N.
tr a n s-4-Ben zyloxyp r olin e H yd r och lor id e Sa lt (38).
HCl (g) was bubbled for 2 min into an ice-cooled solution of
(tert-butyloxycarbonyl)-O-benzyl-L-hydroxyproline (5.0 g, 15.6
mmol) in EtOAc (30 mL). The reaction was then stoppered,
stirred at room temperature for 1 h, and conc in vacuo to
provide a white solid which was triturated with Et2O and dried
under vacuum to yield 38 (3.92, 98%): mp 188-190 °C; 1H
NMR (300 MHz, CDCl3) δ 9.1 (br s, 1H), 7.3 (m, 5H), 4.56 (s,
2H), 4.4-4.2 (m, 2H), 3.6-3.38 (m, 3H), 2.6-2.58 (m, 1H),
2.26-2.1 (m, 1H); MS m/z (rel intensity) 262 (M+ + C3H5),250
(M+ + C2H5), 222 (MH+, 100), 176, 130, 107, 91, 85, 69.
(ter t-Bu tyloxycar bon yl)-L-valyl-N-(1,1,1,2,2-pen taflu or o-
5-m eth yl-3-oxo-4-h exa n yl)-L-a zetid in a m id e (43). Acid 39
(1.80 g, 6.0 mmol) was couple to 3 (1.54 g, 6.0 mmol) via
1
method I to give 44 (2.20 g, 73%) as a colorless oil: H NMR
(300 MHz, CDCl3) δ 8.4 (m, 1H, NH), 5.12-4.90 (m, 2H), 4.38
(m, 1H), 4.11 (m, 1H), 4.00 (m, 1H), 2.73 (m, 1H), 2.42 (2m,
2H), 1.92 (m, 2H), 1.45 (s, 9H, tBu), 1.11-0.85 (m, 12H, 4 ×
CH3); 19F NMR (CDCl3) δ -82.14 (s, CF3), -120.99 and
-123.09, -121.23 and -122.84 (2AB quartets, J ) 296 Hz,
CF2).
(ter t-Bu tyloxycar bon yl)-L-valyl-N-(1,1,1,2,2-pen taflu or o-
5-m eth yl-3-oxo-4-h exa n yl)-D,L-p ip ecolin a m id e (44). Pre-
pared from 40 (450 mg, 1.37 mmol) by method II to afford 44
(580 mg, 80%) as an amorphous solid: 1H NMR (300 MHz,
CDCl3) δ 6.98 (m, 1H, NH), 5.36-4.88 (m, 3H), 4.60 (m, 1H),
4.43 (m, 1H), 3.92 (m, 1H), 3.10 (m, 1H, CH of Pec), 2.36-
1.92 (series of m, 3H, Pec and CH of Val), 1.79-1.24 (m, 4H),
1.45 (s, 9H, tBu), 1.05-0.83 (m, 12H, 4 × CH3); 19F NMR
(CDCl3) δ -82.10 (s, CF3), -120.74, -123.40 (m, CF2).
(ter t-Bu tyloxycar bon yl)-L-valyl-N-(1,1,1,2,2-pen taflu or o-
5-m et h yl-3-oxo-4-h exa n yl)-tr a n s -4-h yd r oxyp r olin a m -
id e (45). Acid 41 (1.80 g, 5.60 mmol) was coupled by method
II to yield 45 (1.44 g, 48%) as an amorphous solid: 1H NMR
(300 MHz, CDCl3) δ 7.98 (br d, 0.5H, J ) 7.6 Hz, NH), 7.73
(br d, 0.5H, J ) 7.6 Hz, NH), 5.47 (br d, 1H, J ) 8.6 Hz, NH),
5.04-4.95 (m, 1H), 4.76-4.67 (m 1H), 4.49 (br s, 1H), 4.23 (m,
1H), 3.94 (br d, 1H, J ) 10.8 Hz), 3.66-3.59 (m, 1H), 2.50-
1.98 (series of m, 4H), 1.42 (s, 9H, tBu), 1.09-0.88 (m, 12H, 4
× CH3); 13C NMR (CDCl3) δ 173.9, 173.2, 170.6, 170.4, 156.3,
156.2, 80.5, 80.4, 77.4, 77.2, 77.0, 76.5, 70.0, 69.8, 59.7, 59.5,
58.2, 57.7, 57.6, 55.7, 55.5, 35.7, 34.7, 31.0, 30.9, 29.0, 28.6,
28.3, 28.2, 20.2, 19.9, 19.3, 19.7, 18.3, 18.2, 16.4, 16.1; 19F NMR
(CDCl3) δ -82.17 (s, CF3), -82.18 (s, CF3), -121.37 and
-122.92 (AB quartet, J ) 296 Hz, CF2), -121.6 and -122.8
(AB quartet, J ) 296 Hz, CF2); MS (CI/CH4) m/z (rel intensity)
549 (MNH4+, 12), 532 (MH+, 54), 482 (11), 330 (15), 245 (100),
189 (15).
(ter t-Bu t yloxyca r b on yl)-L-va lyl-L-a zet id in eca r b oxyl-
ic Acid (39). Cou p lin g Meth od II. To a stirred solution of
(S)-(-)-2-azetidinecarboxylic acid (34) (1.0 g, 10 mmol) and
Et3N (1.5 mL, 11 mmol) in DMF (30 mL) was added 2 (2.8 g,
9.0 mmol), and the reaction mixture was heated to 120 °C for
2.5 h. Upon cooling the reaction mixture was conc in vacuo,
and the oily residue was dissolved in EtOAc, washed with 1
N HCl (2 × 30 mL), dried (MgSO4), and conc to afford 39 (1.88
g, 65%) as an amorphous solid: 1H NMR (300 MHz, CDCl3) δ
9.15 (br s, 1H), 5.09 (d, 1H, J ) 8.8 Hz, NH), 5.00 (dd, 1H, J
) 9.2, 7.2 Hz, R-CH of Val), 4.43 (dd, 1H, J ) 7.0, 1.49 Hz),
4.17 (m, 1H), 3.94 (app t, 1H, J ) 8 Hz), 2.62 (m, 3H), 1.44 (s,
9H, tBu), 0.97 (d, 6H, J ) 6.7 Hz, 2 × CH3).
(ter t-Bu tyloxycar bon yl)-L-valyl-N-(1,1,1,2,2-pen taflu or o-
5-m et h yl-3-oxo-4-h exa n yl)-tr a n s-4-b en zyloxyp r olin a m -
id e (46). Acid 22 (1.40 g, 3.30 mmol) was coupled to 3 (840
mg, 3.30 mmol) via method I to yield 46 (2.0 g, 95%) as a white
solid: mp 91-93 °C; 1H NMR (300 MHz, CDCl3) δ 7.96 (0.5H,
NH), 7.33 (m, 5H), 5.28 (m, 1H, NH), 4.95 (m, 1H), 4.83 (m,
1H), 4.70 (t, 0.5H), 4.54 (q, 2H, CH2Ph), 4.30 (m, 2H), 4.07-
3.9 (m, 1H), 3.60 (m, 1 H), 2.68 (dt, 0.5H), 2.49 (dt, 0.5H), 2.33
(m, 1H), 2.2-1.88 (m, 3H), 1.44 (s, 9H, tBu), 1.09-0.86 (m,
12H, 4 × CH3).
(ter t-Bu t yloxyca r b on yl)-L-va lyl-D,L-p ip ecolin ic Acid
(40). Prepared by method II using d,L-pipecolinic acid (35)
(1.30 g, 10 mmol) to afford 40 (579 mg, 29%), a mixture of two
enantiomers, as an amorphous solid: 1H NMR (300 MHz,
CDCl3) δ 8.94 (br s, 1H), 5.62 (d, 1H, J ) 9.8 Hz, NH), 5.45 (d,
0.5H, J ) 3.98 Hz), 5.07 (d, 0.5H, J ) 9.25, D-Pec), 4.60 (dd,
0.5H, J ) 8.83, 5.06 Hz, R-CH of Val), 4.25 (dd, 0.5H, J ) 8.69,
4.66 Hz, Val of D-compd), 3.93 (app d, 1H, J ) 12.47 Hz, Pec),
L-Va lyl-N-(1,1,1,2,2-p en ta flu or o-5-m eth yl-3-oxo-4-h exa -
n yl)-L-a zeta m id e Hyd r och lor id e Sa lt (47). Pentafluoro-
ethyl ketone 43 (2.0 g, 3.98 mmol) was treated as described in
general procedure I to afford 47 (1.63 g, 96%) as an amorphous
1
solid: IR (film) 3196, 2972, 2937, 2883, 2636, 1755, 1655; H
NMR (300 MHz, CDCl3) δ 8.09 (d, 1H, J ) 8.2 Hz, NH), 8.02

2476 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Cregge et al.
(d, 1H, J ) 8.5 Hz, NH), 5.15 (m, 1H), 5.00 (m, 1H), 4.55 (m,
1H), 4.13 (m, 1H), 3.90 (m, 1H), 2.56 (m, 2H), 2.30 (m, 3H),
1.12-0.84 (m, 12H, 4 × CH3); 13C NMR (CDCl3) δ 193.2, 170.7,
170.4, 170.3, 169.9, 62.2, 61.5, 59.7, 59.5, 55.1, 55.0, 50.1, 30.0,
29.9, 28.9, 28.7, 19.8, 19.7, 18.8, 18.5, 18.2, 18.1, 17.8, 16.6,
16.3; 19F NMR (CDCl3) δ -82.06 and -82.14 (2s, CF3), -121.16
and -122.76, -121.33 and -122.88 (2AB quartets, J ) 296
Hz, CF2); MS (CI/CH4) m/z (rel intensity) 442 (6), 430 (18),
402 (MH+, 100), 303 (9), 72 (42). Anal. (C16H24F5N3O3‚HCl)
C, H, N.
L-Va lyl-N-(1,1,1,2,2-p en ta flu or o-5-m eth yl-3-oxo-4-h exa -
n yl)-D,L-p ip ecolin a m id e Hyd r och lor id e Sa lt (48). Pre-
pared from 44 (530 mg, 1.0 mmol) as described in general
procedure I to afford 48 (460 mg, 99%) as an amorphous solid.
N-L-Valyl-N-(1,1,1,2,2-pen taflu or o-5-m eth yl-3-oxo-4-h ex-
a n yl)-tr a n s-4-h yd r oxyp r olin a m id e Hyd r och lor id e Sa lt
(49) a n d N-L-Va lyl-N-(1,1,1,2,2-p en ta flu or o-5-m eth yl-3-
oxo-4-h exa n yl)-tr a n s-4-a cetoxyp r olin a m id e Hyd r och lo-
r id e Sa lt (50). Pentafluoroethyl ketone 45 (1.44 g, 2.70 mmol)
was treated as described in general procedure I to afford 49
and the acetoxy derivative 50 as a minor product (1.27 g, 100%)
as an amorphous solid.
3H, CH3); 13C NMR (CDCl3) δ 173.2, 172.5, 170.7, 170.4, 169.2,
167.1, 138.8, 134.9, 127.52, 127.50, 127.4, 127.3, 77.55, 77.52,
77.51, 77.46, 77.44, 77.3, 77.2, 77.1, 77.0, 76.88, 76.85, 76.6,
76.56, 76.54, 70.1, 69.9, 66.8, 59.6, 59.5, 58.5, 57.9, 57.0, 56.0,
56.0, 55.7, 36.0, 34.9, 31.42, 31.40, 31.3, 29.2, 28.8, 20.0, 19.85,
19.83, 19.4, 19.2, 18.4, 16.5, 16.4, 16.3, 16.2; 19F NMR (CDCl3)
δ -82.1, -82.15 (s, CF3), -121.31, -123.02 (AB quartet, J )
293 Hz, CF2), -121.35, -122.82 (AB quartet, J ) 298 Hz, CF2);
MS (CI/CH4) m/z (rel intensity) 649 (MH+), 361, 334, 333 (100),
317, 289, 218, 200, 111, 86. Anal. (C29H37F5N4O7) C, H, N.
55: 1H NMR (400 MHz, CDCl3) δ 7.84 (d, 2H, J ) 8.4 Hz,
aryl), 7.72 (0.5H, J ) 8.4 Hz, NH), 7.47 (d, 2H, J ) 8.3 Hz,
aryl), 6.74 (d, 1H, J ) 8.6 Hz, NH), 5.36 (m, 1H), 5.00 (m, 1H),
4.83 (dd, 0.5H, J ) 8.6, 7.2 Hz), 4.74 (dd, 1H, J ) 8.0, 7.2 Hz),
4.69 (t, 0.5H), 4.08 (br d, 1H), 3.9-3.3 (br s overlapping m,
9H), 2.81 (m, 0.5H), 2.64 (m, 0.5H), 2.41-2.06 (series of m,
3H), 2.04 (s, 3H, OCH3), 1.02 (m, 9H, 3 × CH3), 0.94 (m, 3H,
CH3); 13C NMR (CDCl3) δ 173.0, 172.3, 170.4, 170.1, 169.9,
169.2, 166.3, 138.7, 127.4, 127.3, 72.5, 72.4, 66.8, 59.6, 59.5,
58.5, 57.8, 56.3, 56.2, 53.2, 52.9, 34.0, 32.8, 31.8, 31.7, 31.6,
29.3, 29.2, 28.8, 24.9, 20.9, 20.0, 19.8, 19.5, 19.3, 17.95, 17.92,
17.7, 16.4, 16.2; 19F NMR (CDCl3) δ -82.1, -82.13 (s, CF3),
-121.22, -123.06 (AB quartet, J ) 296 Hz, CF2), -121.28,
-122.86 (AB quartet, J ) 301 Hz, CF2); MS (CI/CH4) m/z (rel
intensity) 691 (MH+), 631, 472, 444, 389, 375 (100), 349, 318,
317, 289, 264, 225, 218, 128, 100. Anal. (C31H39F5N4O8) C,
H, N.
N-L-Valyl-N-(1,1,1,2,2-pen taflu or o-5-m eth yl-3-oxo-4-h ex-
a n yl)-tr a n s-4-ben zyloxyp r olin a m id e Hyd r och lor id e Sa lt
(51). Pentafluoroethyl ketone 46 (330 mg, 0.53 mmol) was
treated as described in general procedure I to afford 51 (240
mg, 81%) as an amorphous solid: 1H NMR (300 MHz, CDCl3)
δ 8.22 (m, 2H, NH2), 7.33 (m, 5H), 5.04 (app q, 1H), 4.88 (dq,
1H), 4.52 (q, 2H, CH2Ph), 4.33 (m, 1H), 4.0-3.8 (series of m,
4H), 2.42-2.1 (m, 4 H), 1.09-0.86 (m, 12H, 4 × CH3).
N -[4-[(4-Mor p h olin yl)ca r b on yl]b en zoyl]-L -va lyl-N -
(1,1,1,2,2-p en ta flu or o-5-m eth yl-3-oxo-4-h exa n yl)-tr a n s-4-
ben zyloxyp r olin a m id e (56). Prepared from 51 (558 mg, 1.0
mmol) as described in general procedure II to afford 56 (520
mg, 70%) as an amorphous solid: 1H NMR (300 MHz, CDCl3)
δ 7.84 (dd, 2H, aryl), 7.78 (d, 0.5H, NH), 7.45 (dd, 2H, aryl),
7.29 (m, 5H), 7.21 (d, 0.5H, NH), 6.87 (d, 1H, NH), 4.98 (m,
1H), 4.82 (m, 1H), 4.69 (t, 0.5H), 4.54 (dq, 2H, CH2Ph), 4.31
(br s, 1H), 4.08 (dq, 1H), 3.9-3.25 (series of m, 9H), 2.69 (dt,
0.5H), 2.46 (dt, 0.5H), 2.34 (m, 1H), 2.17 (m, 1H), 1.02 (m, 9H,
3 × CH3), 0.89 (m, 3H, CH3); 13C NMR (CDCl3) δ 193.1, 172.9,
172.2, 170.7, 170.4, 169.3, 166.2, 138.4, 137.5, 137.4, 135.2,
128.5, 128.4, 128.2, 127.9, 127.8, 127.78, 127.73, 127.71, 127.5,
127.4, 127.3, 119.5, 115.76, 107.1, 106.6, 71.4, 71.2, 66.7, 59.6,
59.4, 58.7, 58.0, 56.0, 52.6, 52.3, 48.14, 48.11, 48.10, 48.0, 42.6,
42.57, 42.52, 42.4, 33.5, 32.4, 31.8, 29.1, 28.6, 20.0, 19.8, 19.4,
19.3, 17.8, 17.6, 16.3, 16.1; 19F NMR (CDCl3) δ -82.10, -82.13
(s, CF3), -121.3, -122.9 and -121.4, -122.8 (AB quartet, J
N -[4-[(4-Mor p h olin yl)ca r b on yl]b en zoyl]-L-va lyl -N-
(1,1,1,2,2-pen ta flu or o -5-m eth yl-3-oxo -4-h exa n yl)-L -a zet-
a m id e (52). Pentafluoroethyl ketone 47 (1.5 g, 3.43 mmol)
was treated as described in general procedure II to afford 52
(335 mg, 16%) as an amorphous solid: IR (KBr) 3690, 3678,
3429, 3271, 3011, 2972, 2931, 2899, 2876, 2862, 1755, 1680,
1631; 1H NMR (300 MHz, CDCl3) δ 8.32 (m, 1H, NH), 7.86 (d,
2H, J ) 8.5 Hz, aryl), 7.49 (d, 2H, J ) 7.9 Hz, aryl), 6.70 (m,
1H, NH), 5.00 (m, 2H), 4.50 (m, 2H), 4.19 (m, 1H), 4.86-3.30
(series of m, 8H), 2.82-1.95 (series of m, 4H), 1.05 (m, 9H, 3
× CH3), 0.88 (d, 3H, J ) 6.9 Hz, CH3); 13C NMR (CDCl3) δ
174.07, 174.05, 170.6, 169.2, 166.4, 138.66, 138.62, 135.0,
134.9, 127.4, 127.3, 66.8, 66.7, 62.0, 61.7, 59.7, 59.6, 54.1, 54.0,
49.3, 31.5, 31.4, 28.7, 28.5, 20.0, 19.0, 18.8, 18.2, 18.1, 18.09,
18.05, 16.1, 16.0; 19F NMR (CDCl3) δ -82.12 (s, CF3), -120.98
and -123.12, -121.20 and -122.86 (2AB quartets, J ) 296
Hz, CF2); MS (CI/CH4) m/z (rel intensity) 647, 619 (MH+), 303
(100), 289, 218. Anal. (C28H35F5N4O6‚0.3 H2O) C, H, N.
N -[4-[(4-Mor p h olin yl)ca r b on yl]b en zoyl]-L -va ly l -N-
(1,1,1,2,2-pen ta flu or o-5-m eth yl-3-oxo-4-h exa n yl)-D,L-p ip e-
colin a m id e (53). Prepared from 48 (450 mg, 10.0 mmol) as
described in general procedure II to afford 53 (270 mg, 42%)
as an amorphous solid: 1H NMR (300 MHz, CDCl3) δ 7.95-
7.76 (m, 2H, aryl), 7.58-7.39 (m, 2H), 7.20-6.86 (m, 2H, aryl),
5.40-4.30 (m, 4H), 4.20-3.20 (m, 10H, 2 × NCH2CH2O and
NCH2 of Pro), 2.60-1.95 (m, 3H), 1.90-1.82 (m, 4H), 1.25-
0.75 (m, 12H); 19F NMR (CDCl3) δ -81.97 (m, CF3), -121.82
and -119.87 (m, CF2); MS (CI/CH4) m/z (rel intensity) 647
(MH+), 564, 536, 474, 428, 363, 331 (100), 317, 289, 246, 218,
186, 158, 104, 84, 72.
N -[4-[(4-Mor p h olin yl)ca r b on yl]b en zoyl]-L -va ly l -N-
(1,1,1,2,2-p en ta flu or o-5-m eth yl-3-oxo-4-h exa n yl)-tr a n s-4-
h yd r oxyp r olin a m id e (54) a n d N-[4-[(4-Mor p h olin yl)-
ca r b on yl]b en zoyl]-L-va lyl-N -(1,1,1,2,2-p en t a flu or o -5-
m eth yl-3-oxo-4-h exan yl)-tr a n s-4-acetoxypr olin am ide (55).
Prepared from 49 and 50 (800 mg, 1.71 mmol) as described in
general procedure II to afford 55 (higher Rf fractions, 90 mg,
7.6%) as an amorphous solid and 54 (lower Rf fractions, 160
mg, 14.5%) also as an amorphous solid. 54: 1H NMR (400
MHz, CDCl3) δ 7.82 (m, 2H, aryl), 7.45 (d, 2H, J ) 8.3 Hz,
aryl), 7.35 (d, 0.5H, J ) 8.4 Hz, NH), 6.91 (d, 1H, J ) 8.7 Hz,
NH), 4.99 (m, 1H), 4.84 (t, 1H), 4.72 (t, 1H), 4.68 (m, 1H), 4.55
(br s, 1H), 4.18 (m, 1H), 3.8-3.4 (br s overlapping m, 9H),
2.86-2.03 (series of m, 4H), 1.26 (m, 9H, 3 × CH3), 0.96 (m,
) 296 Hz, CF2); MS (CI/CH4) m/z (rel intensity) 767 (M+
+
29), 740 (10), 739 (MH+, 27), 632 (11), 520 (7), 492 (5), 424
(18), 423 (100), 403 (3), 345 (5), 317 (30), 289 (4), 218 (3), 176
(11), 91 (2). Anal. (C36H43F5N4O7‚0.4 H2O) C, H, N.
(ter t-Bu t yloxyca r b on yl)-L-va lyl-L-t h ia zolid in e-4-ca r -
boxylic Acid (57). Prepared from l-thiazolidine-4-carboxylic
acid (1.3 g, 10 mmol) using coupling method II to afford 57
(2.27 g, 68%) as an amorphous solid: 1H NMR (300 MHz,
CDCl3) δ 7.95 (br s, 1H), 5.57 (app t, 0.5H), 5.39 (d, 0.5H, J )
9.5 Hz), 5.11 (t, 1H, J ) 5.6 Hz), 4.94 (d, 1H, J ) 8.6 Hz), 4.55
(d, 1H, J ) 8.3 Hz), 4.35 (m, 1H), 3.29 (d, 1H, J ) 5.6 Hz),
2.30-1.54 (series of m, 2H), 1.43 (s, 9H, tBu), 1.04-0.92 (m,
6H, 2 × CH3).
(ter t-Bu tyloxycar bon yl)-L-valyl-N-(1,1,1,2,2-pen taflu or o-
5-m eth yl-3-oxo-4-h exa n yl)-L-th ia zolid a m id e (58). Pre-
pared from 57 (2.27 g, 6.8 mmol) using coupling method I to
give 58 (3.28 g, 92%) as an amorphous solid: 1H NMR (300
MHz, CDCl3) δ 7.52 (br d, 1H, J ) 8 Hz, NH), 5.27 (m, 1H,
NH), 5.00 (m, 2H), 4.51 (m, 1H), 4.38 (m, 1H), 3.64-3.38 (pr
m, 1H), 3.12 (m, 1H), 2.40-1.84 (series of m, 3H), 1.44 (s, 9H,
tBu), 1.15-0.84 (m, 12H, 4 × CH3).
N -[4-[(4-Mor p h olin yl)ca r b on yl]b en zoyl]-L -va lyl-N-
(1,1,1,2,2-p en t a flu or o-5-m et h yl-3-oxo-4-h exa n yl)-L-t h ia -
zolid a m id e (59). The HCl salt was prepared from 58 (3.2 g,
6.0 mmol) using general procedure I to afford an amorphous
solid (2.68 g, 96%): 1H NMR (300 MHz, CDCl3) δ 8.18 (m, 2H,
NH2), 5.36 (m, 1H), 5.12 (m, 1H), 4.95 (m, 1H), 4.58 (app t,
1H), 4.40 (m, 1H), 3.42 (m, 1H), 3.20 (m, 1H), 2.39 (m, 3H),
1.30-0.84 (m, 12H, 4 × CH3); 19F NMR (CDCl3) δ -82.0 (2s,

Inhibition of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2477
ratio 1:3, CF3), -120.0 and -123.8 (overlapping AB quartets,
ratio 1:3, CF2).
mmol) via method II to give 63 (2.96 g, 90%), a mixture of
diastereomers, as an amorphous solid: 1H NMR (300 MHz,
CDCl3) δ 6.10 (br s, 1H), 5.61 (m, 1H, NH), 5.48 (m, 1H), 4.65
(m, 1H), 4.48 (m, 1H), 4.30-2.80 (m, 1H), 3.42-2.35 (series of
m, 2H), 2.16 (m, 1H), 2.00-1.30 (m, 4H), 1.44 (s, 9H, tBu),
1.01-0.84 (m, 6H, 2 × CH3).
The HCl salt was treated as described in general procedure
II to afford 59 (275 mg, 10%) as an amorphous solid: 1H NMR
(300 MHz, CDCl3) δ 7.85 (m, 2H, aryl), 7.49 (d, 2H, J ) 7.9
Hz, aryl), 7.04 (m, 1H, NH), 5.08 (m, 2H), 4.92 (m, 1H), 4.60
(pr d, 1H, J ) 9 Hz), 4.25 (br d, 0.5H, J ) 11 Hz), 3.88-3.30
(series of m, 7H), 3.14 (m, 1H), 2.34 (m, 1H), 2.22 (m, 1H),
1.92 (pr m, 1H), 1.64 (m, 1H), 1.32 (m, 1H), 1.05 (m, 9H, 3 ×
CH3), 0.87 (m, 3H, CH3); 13C NMR (CDCl3) δ 172.1, 171.7,
169.3, 169.25, 169.20, 166.5, 156.6, 138.6, 134.97, 134.94,
127.4, 127.3, 127.2, 66.8, 62.5, 61.7, 59.5, 59.4, 58.7, 56.3, 56.2,
50.2, 49.9, 49.1, 48.17, 48.15, 48.12, 48.08, 48.05, 48.02, 47.9,
33.9, 33.8, 31.86, 31.81, 31.1, 30.9, 30.5, 29.1, 29.0, 25.6, 24.9,
20.0, 19.9, 19.7, 19.6, 19.5, 19.4, 19.0, 18.1, 17.8, 17.7, 16.3,
16.2, 16.1; 19F NMR (CDCl3) δ -82.10 (s, CF3), -121.22 and
-122.88 (AB quartets, J ) 296 Hz, CF2); MS (CI/CH4) m/z (rel
intensity) 679, 651 (MH+), 536, 416, 335, 317 (100), 225, 214.
(ter t-Bu tyloxyca r bon yl)-L-va lyl-D,L-tetr a h yd r oisoqu in -
olin e-1-ca r boxylic Acid (60). A stirred solution of D,L-
tetrahydro-1-isoquinolinecarboxylic acid hydrochloride salt (2.1
g, 10 mmol) was coupled to 2 (2.0 g, 6 mmol) via method II to
give 60 (484 mg, 13%) as an isomeric mixture amorphous
solid: 1H NMR (300 MHz, CDCl3) δ 7.2 (m, 4H), 5.6 (d, 0.25H,
J ) 8 Hz, NH), 5.4 (d, 0.75H, J ) 8 Hz, NH), 5.38 (m, 1H),
5.1-4.4 (series of m, 3H), 3.5-3.05 (series of m, 2H), 2.08 and
1.93 (pr of m, 1H, ratio 2:1, l/d), 1.4 (s, 9H, tBu), 1.1-0.8 (m,
6H, 2 × CH3).
(ter t-Bu tyloxyca r bon yl)-L-va lylison ip ecotic Acid (64).
Isonipecotic acid (1.29 g, 10 mmol) was coupled to 2 (3.14 g,
10 mmol) via method II to give 64 (1.2 g, 36%), a mixture of
diastereomers, as a yellow oil: 1H NMR (300 MHz, CDCl3) δ
5.54 (d, 0.75H, J ) 9 Hz, NH), 5.04 (d, 0.25H, J ) 9 Hz, NH),
4.55-4.20 (series of m, 2H), 3.95 (m, 1H), 3.22 (m, 1H), 2.93
(m, 1H), 2.61 (m, 1H), 2.3-1.57 (series of m, 5H), 1.44 (s, 9H,
tBu), 1.02-0.87 (m, 6H, 2 × CH3).
(ter t-Bu tyloxycar bon yl)-L-valyl-N-(1,1,1,2,2-pen taflu or o-
5-m eth yl-3-oxo-4-h exa n yl)n ip ecota m id e (65). Acid 63
(820 mg, 2.50 mmol) was coupled to 3 (640 mg, 2.50 mmol)
via method I to give 65 (920 mg, 71%) as an amorphous solid:
1H NMR (300 MHz, CDCl3) δ 7.25 (m, 1H, NH), 5.45-4.85 (m,
2H), 4.75-4.15 (m, 4H), 3.80-3.05 (m, 4H), 2.60-2.20 (series
of m, 4H), 2.10-1.65 (m, 3H), 1.45 (s, 9H, tBu), 1.12-0.75 (m,
12H, 4 × CH3).
N-L-Valyl-N-(1,1,1,2,2-pen taflu or o-5-m eth yl-3-oxo-4-h ex-
a n yl)n ip ecota m id e Hyd r och lor id e Sa lt (66). Pentafluo-
roethyl ketone 65 (920 mg, 1.74 mmol) was treated as
described in general procedure I to afford 66 (800 mg, 99%)
as an amorphous solid.
(ter t-Bu tyloxycar bon yl)-L-valyl-N-(1,1,1,2,2-pen taflu or o-
5-m eth yl-3-oxo-4-h exa n yl)ison ip ecota m id e (67). Acid 64
(950 mg, 2.9 mmol) and NMM (0.32 mL, 2.9 mmol) were
coupled via method I to hydrochloride 3 (737 mg, 2.9 mmol)
to give 67 (1.32 g, 86%) as an amorphous solid: 1H NMR (300
MHz, CDCl3) δ 5.98 (m, 1H), 5.34 (m, 1H), 5.13 (m, 1H), 4.75-
4.25 (m, 2H), 4.12-3.82 (m, 2H), 3.55-2.25 (series of m, 2H),
2.1-1.55 (series of m, 6H), 1.44 (s, 9H, tBu), 1.12-0.75 (m,
12H, 4 × CH3); 19F NMR (CDCl3) δ -82.02 (s, CF3), -121.1
and -122.8 (AB quartet, J ) 310 Hz, CF2).
(ter t-Bu tyloxycar bon yl)-L-valyl-N-(1,1,1,2,2-pen taflu or o-
5-m et h yl-3-oxo-4-h exa n yl)-D,L-t et r a h yd r oisoq u in olin e-
ca r boxa m id e (61). Prepared from 60 (450 mg, 1.2 mmol) by
method I to give 61 (641 mg, 93%) as a colorless oil: 1H NMR
(300 MHz, CDCl3) δ 7.23 (m, 4H), 5.5 (m, 0.25H, NH), 5.45-
4.15 (series of m, 6H), 4.1-3.75 (series of m, 1H), 3.5-2.9
(series of m, 2H), 2.4-1.7 (m, 2H), 1.43 (s, 9H, tBu), 1.12-
0.63 (m, 12H, 4 × CH3); 19F NMR (CDCl3) δ -82.01 (s, CF3),
-120.5 to -123.6 (overlapping AB quartets, CF2).
N -[4-[(4-Mor p h olin yl)ca r b on yl]b en zoyl]-L -va lyl-N-
(1,1,1,2,2,-p en t a flu or o -5-m et h yl-3-oxo-4-h exa n yl)-D,L-
1,2,3,4-tetr a h yd r oisoqu in olin eca r boxa m id e (62a ) a n d -L-
1,2,3,4-t et r a h yd r oisoq u in olin eca r b oxa m id e (62b). The
hydrochloride salt of the deprotected amine was prepared from
61 (641 mg, 1.11 mmol) using general procedure I to give an
amorphous solid (385 mg, 68%): 1H NMR (300 MHz, CDCl3)
δ 8.26 (br m, 2H), 7.18 (m, 4H), 5.5-4.5 (series of m, 6H), 4.1-
3.7 (series of m, 1H), 3.5-2.8 (series of m, 2H), 2.3 (m, 1H),
1.9 (m, 1H), 1.2-0.7 (m, 12H, 4 × CH3); 19F NMR (CDCl3) δ
-82.0 (overlapping s, CF3), -120.5 to -123.7 (overlapping AB
quartets, CF2).
Using general procedure II the hydrochloride salt of the
deprotected amine from 61 (385 mg, 0.749 mmol) gave 62a
(95 mg, 18%) as an amorphous solid: 1H NMR (300 MHz,
CDCl3) δ 7.84 (m, 2H, aryl), 7.46 (m, 2H, aryl), 7.2 (m, 4H),
7.1 (m, 1H, NH), 6.83 (d, 0.5H, J ) 9 Hz, NH), 6.54 (d, 0.5H,
J ) 9 Hz, NH), 5.45-4.6 (series of m, 5H), 3.9-2.9 (series of
m, 10H), 2.4-2.1 (pr of overlapping m, 2H), 1.2-0.65 (m, 12H,
4 × CH3); 19F NMR (CDCl3) δ -82.13 (app t, CF3), -121.3 and
-123.1 (AB quartet, J ) 296 Hz, overlapping m, CF2); MS (CI/
CH4) m/z 723, 695 (MH+), 578, 550, 549, 476, 379, 317, 289,
259, 234 (100), 220, 200.
The L-analogue was prepared using the same sequence to
yield 62b (77 mg, 40%) as an amorphous solid: 1H NMR (300
MHz, CDCl3) δ 7.87 (m, 2H, aryl), 7.47 (m, 2H, aryl), 7.22 (m,
4H), 7.06 (m, 1H, NH), 6.82 (d, 0.5H, J ) 9 Hz, NH), 6.54 (d,
0.5H, J ) 9 Hz, NH), 5.5-4.54 (series of m, 5H), 3.9-2.98
(series of m, 10H), 2.4-2.1 (pr of overlapping m, 2H), 1.2-
0.65 (m, 12H, 4 × CH3); 19F NMR (CDCl3) δ -82.13 (app t,
CF3), -121.3 and -123.1, -121.34 and -122.99 (overlapping
AB quartets, J ) 296 Hz, CF2); MS (CI/CH4) m/z 723, 695
(MH+), 562, 550, 536, 522, 476, 393, 379, 363, 331, 317, 289,
262, 246, 234, 218, 206 (100), 186, 149, 139, 132. Anal.
(C34H39F5N4O6) C, H, N.
N-L-Valyl-N-(1,1,1,2,2-pen taflu or o-5-m eth yl-3-oxo-4-h ex-
a n yl)ison ip ecota m id e Hyd r och lor id e Sa lt (68). A stirred
solution of 67 (1.3 g, 2.45 mmol) was treated as in general
procedure I to afford 68 (1.11 g, 97%) as an amorphous solid.
N -[4-[(4-Mor p h olin yl)ca r b on yl]b en zoyl]-L -va lyl-N-
(1,1,1,2,2-p en ta flu or o-5-m eth yl-3-oxo-4-h exa n yl)n ip eco-
ta m id e (69). A solution of 66 (800 mg, 1.77 mmol) was treated
as described in general procedure II to afford 69 (300 mg, 26%)
as an amorphous solid: 1H NMR (300 MHz, CDCl3) δ 7.92-
7.70 (m, 2H, aryl), 7.55-7.39 (m, 2H), 5.18-4.82 (m, 2H),
4.76-4.20 (m, 1H), 4.00-2.90 (m, 11H), 2.60-1.50 (m, 9H),
1.18-0.80 (m, 12H); 19F NMR (CDCl3) δ -82.07, -82.11 (m,
CF3), -120.46 and -123.92 (m, CF2); MS (CI/CH4) m/z 647
(MH+), 576, 526, 465, 447, 430, 412, 331 (100), 305, 301, 289,
273, 262, 218, 200.
N -[4-[(4-Mor p h olin yl)ca r b on yl]b en zoyl]-L -va lyl-N-
(1,1,1,2,2-p en ta flu or o-5-m eth yl-3-oxo-4-h exa n yl)ison ip e-
cota m id e (70). A stirred solution of 68 (800 mg, 1.77 mmol)
was treated as in general procedure II to afford 70 (300 mg,
26%) as an amorphous solid: IR (KBr) 3427, 3323, 3049, 3034,
2968, 2933, 2874, 1753, 1631, 1527, 1454 cm-1; 1H NMR (300
MHz, CDCl3) δ 7.87 (d, 2H, J ) 7.8 Hz, aryl), 7.47 (d, 2H, J )
8.1 Hz, aryl), 7.12 (dd, 1H, J ) 8.5, 3.9 Hz, NH), 6.09 (d
overlapping t, 1H, J ) 8.6, 5.3 Hz), 5.13 (dd, 1H, J ) 8.3, 4.1
Hz), 5.05 (dd, 1H, J ) 8.6, 5.3 Hz), 4.53 (m, 1H), 4.12 (br t,
1H, J ) 16.4 Hz), 3.86-3.10 (series of m, 10H), 2.78 (m, 1H),
2.55-2.27 (pr of overlapping m, 2H), 2.16-1.55 (series of m,
4H), 1.12-0.8 (m, 12H, 4 × CH3); 13C NMR (CDCl3) δ 173.9,
173.6, 170.1, 170.0, 169.96, 169.91, 169.3, 166.4, 166.3, 138.3,
135.6, 135.5, 127.4, 127.2, 127.1, 66.8, 58.8, 58.7, 48.16, 48.12,
48.09, 48.05, 48.02, 45.3, 44.8, 42.8, 42.7, 42.6, 42.5, 42.4, 42.3,
41.6, 41.2, 31.9, 29.3, 29.2, 29.1, 28.9, 28.7, 28.6, 28.4, 28.3,
28.1, 19.9, 19.8, 19.7, 17.4, 17.2, 17.2, 16.3; 19F NMR (CDCl3)
δ -82.08 (s, CF3), -121.14 and -122.9, -121.17 and -122.85
(overlapping AB quartet, J ) 293, 296 Hz, CF2); MS (CI/CH4)
m/z 687, 675, 647 (MH+), 412, 332, 331 (100), 289, 231, 220,
218, 200, 178. Anal. (C30H39F5N4O6‚0.3H2O) C, H, N.
(ter t-Bu t yloxyca r b on yl)-L-va lyln ip ecot ic Acid (63).
Nipecotic acid (1.29 g, 10 mmol) was coupled to 2 (3.14 g, 10

2478 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Cregge et al.
Meth yl (ter t-Bu tyloxyca r bon yl)-L-va lyl-L-p r olyl-L-va li-
n a te (71). N-Boc-L-valyl-L-proline (from Advanced ChemTech;
3.1 g, 0.01 mol) was coupled to L-valine methyl ester hydro-
chloride (1.67 g, 0.01 mol; Aldrich) via method I to afford 71
(4.27 g, 100%) as an amorphous solid: Rf ) 0.33 in 3:1 Et2O/
hexane; IR (KBr) νmax 3553, 3537, 3520, 3510, 3310, 2968,
2935, 2876, 1741, 1687, 1631, 1527, 1440, 1390, 1367, 1338,
1309, 1244, 1203, 1172, 1114 cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.22 (br d, 1H, J ) 8.4 Hz, NH), 5.24 (br d, 1H, J ) 11.0 Hz,
NH), 4.62 (dd, 1H, J ) 8.2, 2.9 Hz, CH of Pro), 4.43 (app dd,
1H, J ) 8.6, 5.1 Hz, R-CH of Val), 4.30 (dd, 1H, J ) 9.5, 6.4
Hz, CH of Val) 3.75-3.70 and 3.63-3.59 (pr m, 2H, CH2N),
3.70 (s, 3H, OMe), 2.40 (m, 1H, â-CH of Val), 2.17-1.91 (m,
5H, CH2CH2 and â-CH of Val), 1.43 (s, 9H, tBu), 1.00 (d, 3H,
J ) 6.7 Hz, CH3), 0.95-0.90 (m, 9H, 3 × CH3); 13C NMR
(CDCl3) δ 172.5, 172.1, 170.9, 155.8, 79.5, 77.4, 77.1, 76.9, 76.8,
76.5, 59.9, 57.5, 56.7, 52.0, 47.6, 31.4, 31.0, 28.3, 28.2, 27.1,
25.1, 19.5, 18.9, 17.8, 17.3; MS (CI/CH4) m/z (rel intensity) 428
(MH+, 22), 372 (68), 328 (100). Anal. (C21H37N3O6) C, H, N.
29.1, 27.0, 25.1, 19.8, 19.5, 17.8, 16.2; 19F NMR (470.2 MHz,
CDCl3) δ -80.24 (t, J ) 9 Hz, CF3), -118.39 and -119.87 (dq,
J ) 295, 9 Hz, COCF2), -125.99 (AB m, CF2); MS (CI/CH4)
m/z (rel intensity) 683 (MH+, 59), 367 (100). Anal. (C30H37F7-
N4O6‚1.3H2O) C, H, N.
Ack n ow led gm en t. We wish to thank the Analytical
and Structural Sciences department for their assistance
in characterization and analysis of these compounds.
The authors extend their gratitude to Dr. D. Friedrich
and Dr. E. Huber for 19F-{19F} NOE difference spectra
and homonuclear 19F decoupled spectra.
Su p p or tin g In for m a tion Ava ila ble: Nomenclature (Fig-
ure 9) and modeling coordinates (Tables 5 and 6) for HNE
inhibitor 1 (4 pages). Ordering information is given on any
current masthead page.
Refer en ces
(ter t-Bu tyloxycar bon yl)-L-valyl-N-(1,1,1,2,2,3,3-h eptaflu -
or o-6-m eth yl-4-oxo-5-h ep ta n yl)p r olin a m id e (72). To a
solution of methyl ester 71 (3.8 g, 9.0 mmol) in anhydrous Et2O
(100 mL) at -78 °C was added, dropwise, under N2, CF3CF2-
CF2I (6.6 mL, 48.0 mmol; from Aldrich; stabilized with Cu).
To this mixture was added dropwise MeLi‚LiBr (1.5 M in Et2O,
28.5 mL, 42.0 mmol) at a rate which maintained an internal
reaction temperature below -70 °C. The reaction mixture was
stirred at -78 °C for 1 h, the cold bath removed, and stirring
continued for 5 min. The mixture was then poured into H2O
(100 mL), the layers were separated, and the aqueous phase
was acidified with 1 N HCl. The aqueous phase was then
extracted with additional Et2O (100 mL), and the combined
ethereal extracts were dried (MgSO4). The solvent was
removed in vacuo to yield a crude yellow foam which was flash
chromatographed (3:1 Et2O-hexane) to give 72 (654 mg, 13%)
as an amorphous solid: IR (KBr) 3423, 3292, 2972, 2937, 2879,
2823, 2771, 2739, 2253, 1755, 1687, 1635, 1525, 1444, 1392,
1367, 1348, 1313, 1232, 1178, 1126 cm-1; 1H NMR (400 MHz,
CDCl3) δ 7.63 (d, 1H, J ) 8.2 Hz, NH), 5.44 (d, 1H, J ) 9.2 Hz
NH), 5.02 (dd, 1 H, J ) 7.8, 4.5 Hz, CH of Val), 4.64 (dd, 1H,
J ) 8.0, 3.0 Hz, CH of Pro), 4.30 (dd, 1H, J ) 9.2, 6.8 Hz,
R-CH of Val), 3.80-3.74 and 3.66-3.60 (pr of m, 2H, CH2N),
2.31-1.92 (series of m, 6H, â-CH of Val, CH2CH2), 1.44 (s, 9H,
tBu), 1.02 (d, 3H, J ) 7.0 Hz, CH3), 0.98 (d, 3H, J ) 6.9 Hz,
CH3), 0.94 (d, 3H, J ) 6.7 Hz, CH3), 0.88 (d, 3H, J ) 6.9 Hz,
CH3); 13C NMR (100 MHz, CDCl3) δ 193.3, 193.0, 192.7, 172.9,
171.1, 155.7, 118.7, 115.8, 111.3, 108.9, 108.6, 108.2, 105.9,
79.6, 77.3, 77.2, 76.9, 76.6, 59.7, 59.3, 56.8, 47.8, 31.4, 29.0,
28.3, 26.9, 25.1, 19.9, 19.8, 19.7, 19.5, 19.4, 17.5, 17.4, 16.3,
16.1; 19F NMR (376.3 MHz, CDCl3) δ -80.91 (t, CF3), -119.03
and -120.43 (AB quartet, J ) 297 Hz, CF2), -126.62 (s, CF2);
MS (CI/CH4) m/z (rel intensity) 566 (MH+, 100); HRMS
(C23H34F7N3O5) (M+) calcd 566.2492, obsd 566.2475.
(1) Garver, R. I.; Mornex, J . F.; Nukiwa, T.; Brantly, M.; Courtney,
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Enol Acetate Prodrug. J . Med. Chem. 1995, 38, 223-233. (b)
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T.-M.; Durham, S. L.; Hare, C. M.; Huber, E. W.; J anusz, M. J .;
Koehl, J . R.; Marquart, A. L.; Mehdi, S.; Peet, N. P. Inhibition
of Human Neutrophil Elastase with Peptidyl Electrophilic
Ketones. 2. Orally Active PG-Val-Pro-Val Pentafluoroethyl Ke-
tones. J . Med. Chem. 1994, 37, 4538-4554. (c) Durham, S. L.;
Hare, C. M.; Angelastro, M. R.; Burkhart, J . P.; Koehl, J . R.;
Marquart, A. L.; Mehdi, S.; Peet, N. P.; J anusz, M. J . Pharma-
cology of N-[4-(4- Morpholinylcarbonyl)benzoyl]-L-Valyl-N-
[3,3,4,4-pentafluoro-1-(1-methylethyl)-2-oxobutyl]-L-Prolina-
mide (MDL 101,146); A Potent Orally Active Inhibitor of Human
Neutrophil Elastase. J . Pharmacol. Exp. Ther. 1994, 270, 185-
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(9) (a) Edwards, P. D.; Anisik, D. W.; Bryant, C. A.; Ewing, B.;
Gomes, B.; Lewis, J . J .; Rakiewicz, D.; Steelman, G.; Strimpler,
A.; Trainor, D. A.; Tuthill, P. A.; Mauger, R. C.; Veale, C. A.;
Wildonger, R. A.; Williams, J . C.; Wolanin, D. J .; Zottola, M.
Discovery and Biological Activity of Orally Active Peptidyl
Trifluoromethyl Ketone Inhibitors of Human Neutrophil Elastase.
J . Med. Chem. 1997, 40, 1876-1885. (b) Edwards, P. D.;
Wolanin, D. J .; Anisik, D. W.; Davis, M. W. Peptidyl R-Ketohet-
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Varying the Heterocyclic Ring on In Vitro Potency. J . Med.
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Vijayalakshmi, J .; Tuthill, P. A.; Andisik, D. A.; Gomes, B.;
Strimpler, A. Design, Synthesis and Kinetic Evaluation of a
Unique Class of Elastase Inhibitors, the Peptidyl R-Ketoben-
zoxazoles, and the X-ray Crystal Structure of the Covalent
Complex Between Porcine Pancreatic Elastase and the Ac-Ala-
Pro-Val-2-Benzoxazole. J . Am. Chem. Soc. 1992, 114, 1854-
1863.
(10) Williams, J . C.; Falcone, R. C.; Knee, C.; Stein, R. L.; Strimpler,
A. M.; Reaves, B.; Giles, R. E.; Krell, R. D. Biologic Character-
ization of ICI 200,880 and ICI 200, 355, Novel Inhibitors of
Human Neutrophil Elastase. Am. Rev. Respir. Dis. 1991, 144,
875-883.
(11) (a) Bernsein, P. R.; Gomes, B. C.; Kosmider, B. J .; Vavek, E. P.;
Williams, J . C. Nonpeptidic Inhibitors of Human Leukocyte
Elastase. 6. Design of a Potent, Intratracheally Active, Pyridone-
Based Trifluoromethyl Ketone. J . Med. Chem. 1995, 38, 212-
N -[4-[(4-Mor p h olin yl)ca r b on yl]b en zoyl]-L -va lyl-N -
(1,1,1,2,2,3,3-h ep ta flu or o-6-m eth yl-4-oxo-5-h ep ta n yl)p r o-
lin a m id e (73). The Boc protection was removed from 72
using general procedure I to give the HCl salt (185 mg, 0.37
mmol), and 4-[(4-morpholinyl)carbonyl]benzoyl chloride was
added as described in general procedure II to afford crude 73
(260 mg) as a white foam. This was flash chromatographed
(2- × 15-cm column eluted with 1:27 MeOH-CH2Cl2) to give
73 (162 mg, 64%) as an amorphous solid: IR (KBr) 3431, 3323,
3049, 2970, 2935, 2877, 1755, 1693, 1631, 1529, 1437, 1394,
1346, 1300, 1278, 1259, 1232, 1161, 1118, 1068 cm-1; 1H NMR
(300 MHz, CDCl3) δ 7.86 (d, 2H, J ) 8.4 Hz, aryl), 7.52 (d,
1H, J ) 8.4 Hz, NH), 7.46 (d, 2H, J ) 8.3 Hz, aryl), 7.12 (d,
1H, J ) 8.7 Hz, NH), 5.04 (dd, 1H, J ) 8.2, 4.2 Hz, R-CH of
Val), 4.84 (dd, 1H, J ) 8.6, 7.3 Hz, R-CH of Val), 4.62 (dd, 1H,
J ) 7.9, 2.9 Hz, CH of Pro), 3.94-3.37 (m, 10H, 2 × NCH2-
CH2O and NCH2 of Pro), 2.29-1.97 (series of m, 6H, 2 × â-CH
of Val and CH2CH2), 1.06 (d, 3H, J ) 6.8 Hz, CH3), 1.01 (d,
6H, J ) 6.7 Hz, 2 × CH3), 0.86 (d, 3H, J ) 6.9 Hz, CH3); 13C
NMR (CDCl3) δ 172.2, 170.9, 169.2, 166.3, 138.5, 135.1, 127.4,
127.3, 77.4, 77.1, 76.9, 76.5, 66.7, 59.9, 59.3, 55.9, 47.9, 31.8,

Inhibition of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2479
215. (b) Veale, C. A.; Bernstein, P. R.; Byrant, C.; Ceccarelli, C.;
Damewood, J . R.; Earley, R.; Feeney, S. W.; Gomes, B.; Ko-
smider, B. J .; Steelman, G. B.; Thomas, R. M.; Vacek, E. P.;
Williams, J . C.; Wolanin, D. J .; Wollson, S. Nonpeptidic Inhibi-
tors of Human Leukocyte Elastase. 5. Design, Synthesis and
X-ray Crystallography of a Series of Orally Active 5-Aminopy-
rimidin-6-one-Containing Trifluoromethyl Ketones. J . Med.
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Steelman, G. B.; Byrant, C.; Gomes, B.; Williams, J . Nonpeptidic
Inhibitors of Human Leukocyte Elastase. 4. Design, Synthesis
and In Vitro and In Vivo Activity of a Series of â-Carboline-
Containing Trifluoromethyl Ketones. J . Med. Chem. 1995, 38,
86-97. (d) Bernsein, P. R.; Andisik, D.; Bradley, P. K.; Bryant,
C. B.; Ceccarelli, C.; Damewood, J . R.; Earley, R.; Edwards, P.
D.; Fenney, S.; Gomes, B. C.; Kosmider, B. J .; Steelman, G. B.;
Thomas, R. M.; Vacek, E. P.; Veale, C. A.; Williams, J . C.;
Wolanin, D. J .; Woolson, S. A. Nonpeptidic Inhibitors of Human
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 and preceding
articles on pyridinones in this series.
(21) The term mechanism-based has often been used to describe
specific inhibitors which are irreversible suicide inactivators of
enzymes. Here it is used in a more general context to define
inhibitors whose mode of action is based on the mechanism of
enzymatic substrate hydrolysis and which inactivate an enzyme
by interacting with the catalytic residues within the active site.
(22) Navia, M. A.; McKeever, B. M.; Springer, J . P.; Lin, T. Y.;
Williams, H. R.; Fluder, E. M.; Dorn, C. P.; Hoogsteen, K.
Structure of Human Neutrophil Elastase in Complex with a
Peptide Chloromethyl Ketone Inhibitor at 1.84-Å Resolution.
Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 7-11.
(23) (a) Skiles, J . W.; Fuchs, V.; Miao, C.; Sorcek, R.; Grozinger, K.
G.; Maulgin, S. C.; Vitous, J .; Mui, P. W.; J acober, S.; Chow, G.;
Matteo, M.; Skoog, M.; Weldon, S. M.; Possanza, G.; Keirns, J .;
Letts, G.; Rosenthal, A. S. Inhibition of Human Leukocyte
Elastase (HLE) by N-Substituted Peptidyl Trifluoromethyl
Ketones. J . Med. Chem. 1992, 35, 641-662. (b) Doherty, A. M.;
Sircar, I.; Kornberg, B. L.; Rapundalo, S. R.; Ryan, M. J .;
Painchaud, C. S. Design and synthesis of Potent, Selective and
Orally Active Fluorine-Containing Renin Inhibitors. J . Med.
Chem. 1992, 35, 2-14. (c) Williams, J . C.; Falcone, R. C.; Knee,
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J M970812E