Nonpeptidic inhibitors of human neutrophil elastase. 7. Design, synthesis, and in vitro activity of a series of pyridopyrimidine trifluoromethyl ketones

Article abstract of DOI:10.1021/jm950684z

Using molecular modeling and the information derived from X-ray crystal structures of human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE) complexed to peptidic ligands, we have developed a new series of nonpeptidic inhibitors of HNE, the

Full text of DOI:10.1021/jm950684z

1112
J . Med. Chem. 1996, 39, 1112-1124
Non p ep tid ic In h ibitor s of Hu m a n Neu tr op h il Ela sta se. 7. Design , Syn th esis,
a n d in Vitr o Activity of a Ser ies of P yr id op yr im id in e Tr iflu or om eth yl Keton es†
Philip D. Edwards,*,‡ Donald W. Andisik,‡ Anne M. Strimpler,§ Bruce Gomes,§ and Paul A. Tuthill‡
Departments of Medicinal Chemistry and Pulmonary Pharmacology, ZENECA Pharmaceuticals,
A Business Unit of ZENECA Inc., 1800 Concord Pike, P.O. Box 15437, Wilmington, Delaware 19850-5437
Received September 18, 1995X
Using molecular modeling and the information derived from X-ray crystal structures of human
neutrophil elastase (HNE) and porcine pancreatic elastase (PPE) complexed to peptidic ligands,
we have developed a new series of nonpeptidic inhibitors of HNE, the pyridopyrimidine
trifluoromethyl ketones (TFMKs). These bicyclic inhibitors were designed to extend the concept
of the related pyridone trifluoromethyl ketones by incorporating a rigidly positioned carbonyl
group to participate in a hydrogen bonding interaction with the backbone NH groups of Gly-
218 and Gly-219 of the enzyme. In addition, the pyrimidine ring serves as a scaffold to vector
substituents toward the S5-S4 subsites of the enzyme’s extended binding pocket. Furthermore,
the heteroatoms of the pyrimidine ring generally increase the aqueous solubility of the
pyridopyrimidines relative to pyridone TFMKs. Pyridopyrimidine TFMKs containing a 6-phenyl
substituent afforded potent inhibitors of elastase, and several inhibitors from this class of
compounds possessed aqueous solubilities of >0.1 mg/mL and Ki values of e10 nM.
In tr od u ction
able for administration intratracheally to treat inflam-
matory pulmonary diseases. Many different types of
inhibitors have been evaluated for their ability to inhibit
the pulmonary effects of HNE when administered
intratracheally.12 Several classes of these inhibitors
have been shown to be particularly effective in inhibit-
ing HNE-induced lung injury including fluorinated
ketones,13-15 R-ketoheterocycles,16 boronic acids,17-19
and â-lactams.20-22
For over a quarter century, researchers in both
industry and academics have pursued the identification
of compounds which would prevent the tissue destruc-
tion caused by the proteolytic enzyme human neutrophil
elastase (HNE, EC 3.4.21.37).1 This search has resulted
in the development of numerous strategies to inhibit
HNE and led to the discovery of several classes of potent
HNE inhibitors.2 While no elastase inhibitor has yet
been approved for commercial use, this class of com-
pounds should afford extremely important therapeutic
agents. HNE is one of the most promiscuous proteolytic
enzymes in the body with the ability to degrade a
variety of endogenous and exogenous proteins.3 As a
result, HNE is believed to play a role in either initiating
or contributing to the pathological effects associated
with pulmonary emphysema,4 adult respiratory distress
syndrome,5 rheumatoid arthritis,6 atherosclerosis,7 cys-
tic fibrosis,8 and other inflammatory disorders.9 Under
normal homeostatic conditions, the destructive activity
of elastase is controlled by a number of endogenous
proteinase inhibitors. However, in the disease state, the
balance between HNE and its inhibitors is shifted in
favor of the enzyme. The search for elastase inhibitors
has been aimed at discovering drugs which would
supplement the body’s elastase inhibitory capacity and
restore the enzyme-inhibitor balance.
The elastase inhibition program at ZENECA initially
focused on the development of peptide-based electro-
philic ketone inhibitors of HNE which would be suitable
for aerosol administration. This effort culminated in
the identification of three series of peptidyl ketones
which afford unparalleled protection against the de-
structive effects of HNE following intratracheal admin-
istration: trifluoromethyl ketones (A, Figure 1),11,12,23,24
R,R-difluro-â-keto amides (B),25,26 and R-ketobenzox-
azoles (C).14,27,28 While intratracheal administration of
drugs may be an acceptable, in some cases even pre-
ferred, method of treating pulmonary diseases such as
emphysema and cystic fibrosis, oral dosing is the
method of choice for treating most diseases, both
pulmonary and nonpulmonary. This is due to the
comparative ease of administration, which not only may
improve effectiveness but also increases patient compli-
ance. Unfortunately, the physiochemical properties of
a compound which make it suitable for intratracheal
administration generally render it ineffective following
oral dosing. For example, diarylacylsulfonamide pep-
tidyl trifluoromethyl ketones (e.g. A) have a long
residence time in the lung following intratracheal
administration as a result of their relative inability to
cross the interstitium and penetrate the capillary
membranes perfusing the lung. However, this property
confers low bioavailability following oral administration
as a result of poor absorption from the gastrointestinal
tract. Peptides are often inactive following oral admin-
istration not only due to poor absorption but also as the
result of extensive metabolism and/or rapid elimination.
Therefore, large modifications to the structure of an
One of the first diseases associated with HNE was
emphysema. The seminal studies by Laurell and Eriks-
son led to the hypothesis that emphysema was caused
by the uncontrolled destruction of lung tissue as a result
of a deficiency in serum levels of one the primary
endogenous inhibitors of HNE, R1-proteinase inhibi-
tor.10,11 Consequently, the initial development of HNE
inhibitors was focused on the identification of com-
pounds which possessed physiochemical properties suit-
† For part 6 in this series see ref 34.
‡ Department of Medicinal Chemistry.
§ Department of Pulmonary Pharmacology.
X Abstract published in Advance ACS Abstracts, February 1, 1996.
0022-2623/96/1839-1112$12.00/0 © 1996 American Chemical Society

Nonpeptidic Inhibitors of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1113
F igu r e 1. Peptidic and nonpeptidic inhibitors of HNE discovered at ZENECA Pharmaceuticals.
intratracheally active compound are generally required
to change its profile such that it will be orally active.
Thus, while it has been possible to develop peptidic
fluorinated ketones which possess oral activity,29,30 we
mounted a large effort to identify nonpeptidic com-
pounds that would have an in vivo profile superior to
F igu r e 2. Pyridone and pyridopyrimidine dipeptide isosteres.
that of peptidic inhibitors following oral administration.
This work led to the discovery of the nonpeptidic,
pyridone TFMKs (D, Figure 1).31-33 Structure-activity
studies within this series resulted in nonpeptidic TFMKs
which not only displayed excellent in vitro potency (Ki
<10 nM) but also possessed good in vivo activity
following either intratracheal34 or oral administration.35
In addition, we were able to extend the concept of
pyridone-based peptidomimetics to the design of the
tricyclic â-carbolinone TFMKs (E).36 However, when we
began our investigation of pyridone TFMKs, the early
members of this series were not active following oral
administration. It was determined that these com-
pounds were not well-absorbed from the gastrointestinal
tract, which we ascribed to the fact that they possessed
poor aqueous solubility. One approach taken to improve
aqueous solubility was the incorporation of water solu-
bilizing groups onto the 3-amino group of the pyridone
ring.35 A second approch was to incorporate solubilizing
groups directly into the pyridone ring such as found in
the pyrimidinone TFMKs (F ).37 The third strategy was
to incorporate the solubilizing groups into a second
heterocyclic ring. In this report we describe the in vitro
structure-activity relationships of a new series of
bicyclic pyridone-containing inhibitors, the pyridopy-
rimidine TFMKs.
Ta ble 1. Comparison of Peptidic and Pyridone TFMKs
ability to participate in almost all of the key binding
interactions with the enzyme as observed for peptidic
inhibitors (Figure 3a,b). In particular, the 3-amino
group and the carbonyl of the pyridone ring were able
to form hydrogen bonds to Val-216 of the enzyme.
Structure-activity studies with peptidic inhibitors have
demonstrated that formation of these two hydrogen
bonds is particularly important for maximizing potency.2
Simple ring-unsubstituted pyridone TFMKs were sig-
nificantly less potent than the corresponding peptidyl
TFMKs with Ki values in the 1-0.1 µM range (Table 1,
compare I vs J ). Analysis of the pyridone-based inhibi-
tors modeled into HNE revealed that these inhibitors
were incompletely occupying the S2 subsite as a result
Design Con cep ts
The pyridone TFMKs were designed via the modeling
of peptidic inhibitors into the active site of HNE as
previously described.32 These studies suggested that
the P3 residue of the peptides could be cyclized onto the
P2 nitrogen atom and that the P2 side chain could be
eliminated. These modifications were incorporated into
the pyridone-based P3-P2 dipeptide isostere 2-(3-amino-
2-oxo-1,2-dihydro-1-pyridyl)acetic acid (G, Figure 2).
Minimization of pyridone-containing TFMKs into the
active site of HNE revealed that they retained the

1114 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Edwards et al.
enzyme and appear to form a hydrogen-bonding pocket
analogous to the oxyanion hole in the catalytic site
(Figure 3b,c). It is possible that the carbonyl group of
a 3-amido substituent could form hydrogen bonds with
the NH groups of Gly-218 and Gly-219 and thereby
increase the stability of the enzyme-inhibitor complex.
However, to participate in this interaction, it would be
necessary for the amide group to adopt an unfavorable
cis conformation about the nitrogen/carbonyl bond.
Thus, our static modeling studies suggested that the
substituent on the 3-amino group of the pyridone
TFNKs would not bind to either the S5-S4 subsite or
the loop formed by Gly-218 and Gly-219. Supporting
this hypothesis, aqueous molecular dynamics simula-
tions indicated that a 3-amido carbonyl group and its
substituents do not interact strongly with the enzyme
surface, either through hydrophobic or hydrogen-bond-
ing interactions.35
We initiated the design of bicyclic pyridone-based
inhibitors both as a means of locking the 3-amido group
into the desired cis conformation that would allow the
carbonyl oxygen atom to form hydrogen bonds to Gly-
218 and Gly-219 and as a means of directing a hydro-
phobic substituent toward the S5-S4 subsite. The
pyridopyrimidine H (Figures 2 and 3c) appeared to
satisfy these design goals. Static modeling of the
pyridopyrimidine TFMKs in the active site of HNE
indicated that not only could this ring system partici-
pate in the hydrogen-bonding interactions with Val-216
in a pyridone-like manner but that the carbonyl group
in the 2-position (pyridopyrimidine numbering) could
also form hydrogen bonds to the NH groups of both Gly-
218 and Gly-219. Furthermore, appropriate substitu-
tions off the nitrogen atom in the 3-position should be
able to access the S5-S4 binding pocket. An additional
perceived advantage of the pyridopyrimidine scaffold
was that the heteroatoms at the 1-, 2-, 3-, and 4-posi-
tions of the pyrimidine ring would increase aqueous
solubility and thereby might help to improve oral
bioavailablitity.
F igu r e 3. Binding interactions between HNE and (a) peptidic,
(b) pyridone, and (c) pyridopyrimidine TFMKs.
Ch em istr y
of the elimination of the P2 proline ring, and that
substituents in the 5- or 6-positions of the pyridone ring
should fill this binding pocket and restore activity. This
proved to be the case, with 6-phenylpyridone TFMKs
displaying potency comparable to the corresponding
peptidic inhibitors (Table 1, compare I vs K).33
The strategy we developed for the preparation of the
pyridopyrimidine bicyclic nucleus involves (a) construc-
tion of a 3,4-disubstituted pyridone (8, Scheme 1) and
(b) cyclization of these substituents to form the bicyclic
ring system (19 and 20, Scheme 2). The preferred
procedure we used for the preparation of 8 is a modi-
fication of that reported by Borch et al.38 (method A,
Scheme 1). Mixed anhydride coupling of phthaloyl
glycine with amino alcohol 139 followed by hydroxyl
group protection and hydrazinolysis afforded intermedi-
ate 4. The other cyclization partner, enamine 7, was
prepared by alkylation of 540 with either dimethyl-
formamide dimethyl acetal to afford 7a or with N,N-
dimethylbenzamide imino chloride 6 in the presence of
sodium hydride to give 7b. Dienamine 7b is somewhat
unstable to the reaction conditions and requires rapid
isolation. However, modest yields (50%) of highly pure
7b could consistently be prepared. Dienamine 7a was
cyclized with amine 4 in refluxing ethanol to afford the
2-pyridone 8a in good yield (70%). Cyclization of the
phenyl-substituted dienamine 7b with 4, on the other
hand, required more forcing conditions (DMF, 120 °C)
and the yields were lower (40-50%). An alternative
procedure used for the preparation of 8a involved
One of the advantages of the pyridone-based inhibi-
tors over the tripeptides is that the pyridone TFMKs
have only a single stereogenic center. Hence pyridone
TFMKs which are epimeric at the stereogenic center R
to the ketone carbonyl are a mixture of enantiomers
rather than diastereomers, a property which could be
of major significance with respect to both regulatory and
manufacturing concerns. However, as a result of the
fact that the 3-amino group of the pyridione is attached
to a trigonal sp2 center rather than a tetrahedral sp3
center, the 3-amino group and its substituents are
vectored away from the S5-S4 subsite and cannot
readily take full advantage of the binding opportunities
afforded by this hydrophobic pocket of the enzyme. On
the other hand, static modeling of the pyridone inhibi-
tors indicated that the 3-amino substituent is vectored
toward a region of the enzyme that possesses two
accessible NH groups. The hydrogen atoms of Gly-218
and Gly-219 point up away from the surface of the

Nonpeptidic Inhibitors of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1115

1116 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Sch em e 2. Synthesis of Pyridopyrimidines 23 and 24
Edwards et al.
cyclization of 7a with p-methoxybenzylamine to afford
the N-(methoxybenzyl)-2-pyridone 9 (method B, Scheme
1). Oxidative debenzylation afforded 10 which was
N-alkylated with iodide 13 to give 8a . This sequence
generally gave lower overall yields than method A.
Introduction of the functionality required for forma-
tion of the bicyclic system hinged on selective dealky-
lation of the methyl ester 8 with LiI (Scheme 2).
Curtius rearrangement of the monoacid 14 in the
presence of benzyl alcohol introduced the 3-amino group.
Hydrolysis of the ethyl ester 15 followed by carbodiimide
or mixed anhydride-mediated amide formation set the
stage for cyclization to the pyridopyrimidine bicyclic
system which was effected by heating 17 or 18 in DMF.
Removal of the silyl group and oxidation afforded the
pyridopyrimidine TFMKs 23 and 24.
q). Aromatic substituents afforded the best potency.
The distance between the aromatic group and the
pyrimidine ring is clearly important (compare 23c, 23h ,
and 23j). We believe this results from the ability of the
extended 3-substituents to partially occupy the S5-S4
subsites (see modeling discussion below). Indeed, the
3-phenyl derivative 23c is slightly less potent than the
3-methyl compound 23b, showing that simply increas-
ing lipophilicity does not necessarily increase potency.
Further support for the binding of the extended 3-sub-
stituents to a specific enzyme subsite (rather than a
nonspecific hydrophobic interaction) comes from modi-
fications that restrict the flexibility of the ethylene
chain: both 23k with a cyclopropyl group and 23l with
a carbonyl group in the two atom linker were signifi-
cantly weaker than 23j. That this decrease in potency
is not due to either increased substitution or the
incorporation of polar groups can be seen from a
comparison with compounds 23m and 23n . In fact, 23n ,
with a Ki of 16 nM, is the most potent of the compounds
which are unsubstituted in the 6-position.
Modifications of the 3-substituent were not sufficient
to push the potency of the pyridopyrimidine TFMKs into
the <10 nM range. A similar phenomenon was ob-
served with the monocyclic pyridone-based TFMKs
where substituents on the 3-amino group did not afford
nanomolar inhibitors when the 4-, 5-, and 6-positions
were unsubstituted.32 However, it was discovered that
a 6-phenyl group was a unique substituent which
afforded nanomolar pyridione TFMKs.33 Neither 4- or
5-substituents nor other 6-substituents (e.g. methyl,
benzyl, or phenethyl) were as effective as the 6-phenyl
substituent. Molecular modeling studies suggested that
Biologica l Eva lu a tion
The compounds listed in Table 2 were tested in vitro
for their ability to inhibit HNE activity. Inhibition
constants (Ki values) were determined for the inhibition
of the enzyme’s ability to hydrolyze the synthetic
substrate MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide as
previously described.27
Resu lts a n d Discu ssion
We have attempted to extend the discovery of non-
peptidic monocyclic pyridone-based TFMKs to bicyclic
scaffolds and found that TFMKs containing a pyridopy-
rimidine ring afford potent inhibitors of HNE. The
parent 3-unsubstituted analog 23a had a Ki of 910 nM.
Incorporation of lipophilic and aromatic groups in the
3-position increased activity significantly (Table 2, 23b-

Nonpeptidic Inhibitors of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1117
Ta ble 2. In Vitro Inhibitory Activities of Pyridopyrimidine TFMKs
the 6-phenyl group was capable of filling the S2 subsite,
which was occupied by the pyrrolidine ring of the P2
proline in our peptidic inhibitors. Thus the hydrophobic
binding lost by removal of the proline is restored to the
pyridone-based TFMKs by incorporation of a 6-phenyl
group.
excellent inhibitors of HNE. As observed with the
pyridone TFMKs, the 6-phenyl group affords an ap-
proximately 60-fold increase in potency over the unsub-
stituted analogs (compare 23a vs 24a and 23b vs 24b,
Table 2). Several derivatives had Ki values <10 nM
(24c-f, 2k ,l). As with the 6-unsubstituted analogs 23,
the potency of the 6-phenylpyridopyrimidine TFMKs is
increased by substitutions at the 3-position, especially
We applied the same tactic to the pyridopyrimidines
and discovered that 6-phenylpyridopyrimidines are

1118 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Edwards et al.
Ta ble 3. Characterization of Pyridopyrimidine TFMKs 23 and
24
binding pocket from the pyridopyrimidine scaffold but
accesses this pocket by appropriate rotations about the
methylene groups connecting the phenyl group to the
bicyclic ring. This observation provides a possible
explanation of why the 3-phenyl-substituted analog 23c
was no more potent than the methyl-substituted com-
pound 23b and why the phenethyl derivatives afford the
best inhibition constants.
yield
(%)b
MS
(M + 1)
compda
formulac
23a
23b
23c
23d
23e
23f
23g
23h
23i
26
50
37
24
83
53
23
39
49
89
59
49
47
83
20
50
84
37
64
54
77
44
46
55
24
74
23
30
52
389
403
465
445
447
461
d
C15H15N4F3O5‚0.5H2O‚0.5CH3OH
C
C
C
16H17N4F3O5‚0.65H2O
21H19N4F3O5‚0.7H2O‚0.5C2H5OH
18H19N4F3O6‚0.65H2O‚0.2C6H14
C17H17N4F3O7‚1.4H2O‚0.5CH3OH
C
18H19N4F3O7
Our objective at the outset was the identification of a
pyridone-based scaffold which would maintain the
potency of the parent 3-aminopyridone TFMKs while
affording increased aqueous solubility. These objectives
have been realized with the series of pyridopyrimidine
TFMKs. A comparison of pyridopyrimidine TFMKs
with related pyridone TFMKs reveals that the two
series of inhibitors have similar potencies against HNE.
Many of the pyridopyrimidine TFMKs with a 6-phenyl
group have Ki values in the nanomolar range, with one,
24d , having a Ki of 0.95 nM. In fact, 24d is one of only
two pyridone-based TFMKs which has been reported
with a subnanomolar Ki.34,35 As part of our initial
design concepts, we envisioned that the rigidly posi-
tioned 2-carbonyl group of the pyridopyrimidines would
form a hydrogen-bonding interaction with the NH
groups of Gly-218 and Gly-219 (Figure 3), and that this
interaction would increase the potency of the pyridopy-
rimidine TFMKs relative to the pyridone TFMKs. As
seen in Table 4, the pyridopyrimidine TFMKs are only
marginally more potent, at best. However, the pyri-
dopyrimidine TFMKs contain three additional hetero-
atoms capable of forming hydrogen bonds; these atoms
should therefore be strongly solvated in aqueous solu-
tion. Upon binding, these atoms must be partially, if
not completely, desolvated: a process which will con-
tribute to a decrease in binding energy. A corresponding
degree of desolvation is not required for the pyridone
TFMKs. Thus, it is believed that the energetic penalty
which must be paid for desolvation of the pyridopy-
rimidine TFMKs is compensated by the hydrogen-
bonding interactions between the 2-carbonyl group and
the enzyme such that the two series of inhibitors have
similar inhibition constants.
C21H25N4F3O7‚0.2C6H14
C22H21N4F3O5
C23H23N4F3O6
479
509
493
505
507
537
521
502
529
543
465
479
585
597
612
613
552
536
508
553
537
578
23j
23k
23l
C23H23N4F3O5‚0.8H2O
C24H23N4F3O5‚1.0H2O‚0.2C6H14
C23H21N4F3O6‚0.2H2O‚0.4C2H5OH
23m
23n
23o
23p
23q
24a
24b
24c
24d
24e
24f
24g
24h
24i
C
24H23N4F3O7
C25H27N4F3O5
C21H26N5F3O6‚0.7H2O
C
C
C
C
C
C
C
C
C
C
26H23N4F3O5‚1.2H2O
27H25N4F3O5‚0.3H2O‚0.6CH3OH
21H19N4F3O5‚1.0H2O‚0.5CH3OH
22H21N4F3O5‚1.2H2O
29H27N4F3O6‚0.2CH3OH
31H31N4F3O5‚0.5H2O
30H28N5F3O6‚1.0H2O‚1.5CH3OH
30H27N4F3O7‚0.4H2O‚0.4CH3OH
23H22N5F3O6‚2.5H2O
24H24N5F3O6‚2.0H2O‚0.2C6H14
C23H24N5F3O5‚1.4H2O‚0.4C2H6OS
24j
24k
24l
C
C
C
25H27N4F3O7‚1.5H2O
25H27N4F3O6‚1.0H2O
27H30N5F3O6‚1.0H2O
a
All compounds are approximately a 1:1 mixture of epimers at
b
the stereogenic center R to the ketone carbonyl group. Yields are
for final synthetic step. All compounds were purified by flash
chromatography on silica gel except 21e and 21q which were
analytically pure following work up. c All elemental analyses for
d
C, H, and N agree within (0.4% of calculated values. Base peak
was 447 (M + 1 - isobutylene).
by 3-benzyl and 3-phenethyl derivatives (24c-f). Much
of our efforts in the 6-phenyl pyridopyrimidine series
was directed at introducing substituents in the 3-posi-
tion which would impart aqueous solubility (>0.1 mg/
mL) while retaining good in vitro potency (Ki e10 nM).
As can been seen in Table 2, many of the compounds
we prepared satisfied these criteria (23a , 24c, 24g-j,
and 24l).
It was anticipated that the array of heteroatoms
present in the pyrimidine ring of the pyridopyrimidine
TFMKs would confer improved aqueous solubility to this
series of compounds relative to the pyridone TFMKs.
This property was perceived to be important for obtain-
ing oral bioavailability since early studies with the
pyridone TFMKs indicated that their poor absorption
from the gastrointestinal tract was at least in part the
result of poor aqueous solubility.35 Table 4 lists the
aqueous solubility and C log P values for several
pyridopyrimidine TFMKs and structurally related pyr-
idone TFMKs. While it is inappropriate to make rigid
comparisons of the properties of these two series because
of their structural differences, the data in Table 4
suggest that the pyridopyrimidine TFMKs generally
have a greater aqueous solubility and smaller C log P
values than related pyridone TFMKs. Where these
properties are similar, the pyridopyrimidine TFMKs
have better in vitro potency.
Molecular modeling studies have provided further
support for the proposed binding interactions between
the pyridopyrimidines and HNE. A model pyridopy-
rimidine, desdimethyl 24d , was minimized in the ex-
tended binding pocket of HNE in a fashion analogous
to that previously described for pyridone TFMKs.32,33,35
Figure 4 is a view of the minimized enzyme-inhibitor
complex with a solvent accessible surface applied to the
enzyme. The inhibitor interacts with the enzyme in a
substrate-like fashion occupying the S3-S1 subsites as
depicted in Figure 3c. The carbonyl oxygen atom at the
8-position and the NH group in the 1-position form
hydrogen bonds to Val-216 as observed in the crystal
structures of pyridone TFMKs complexed to HNE.35,37
The phenyl group in the 6-position is nestled over the
top of the S2 subsite of the enzyme located in the upper
left quadrant of Figure 4. The carbonyl group in the
2-position is pointed directly into the pocket formed by
the NH groups of Gly-218 and Gly-219 where it can form
hydrogen bonds with these residues. In addition, the
aromatic group of the 3-substituent is located in the
mouth of the S5-S4 subsite. It can be seen that the
3-substituent is not vectored directly toward the S5-S4
One outlier in the above analysis is 24a compared to
27. However, 27 is the only pyridone which is not
acylated on the 3-position aniline nitrogen atom. This
nitrogen atom would be significantly protonated at pH

Nonpeptidic Inhibitors of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1119
F igu r e 4. Two identical stereoviews of desmethyl 24d energy minimized in the active site of HNE. The enzyme grid is the
solvent accessible surface. Top view displays backbone atoms of inhibitor with the hydrogens masked. Inhibitor in bottom view
is displayed with van der Waals surface. See refs 32, 33, and 35 for a description of modeling procedures.
7 and therefore should have increased aqueous solubility
within the pyridone series. Indeed, it is the most soluble
of the pyridones in Table 4. In contrast, the 3-position
nitrogen atom in the pyridopyrimidines, whether or not
it is acylated or alkylated, is nonbasic and not proto-
nated at pH 7. Even so, 24a is only 2-fold less soluble
than 27, further demonstrating the solubilizing power
of the pyridopyrimidine ring system. Thus, the bicyclic
pyridopyrimidine appears to afford a dipeptide mimetic
which imparts good solubility/lipophilicity characteris-
tics while allowing generation of potent inhibitors
through appropriate substituents at the 3- and 6-posi-
tions. One compound, 24l, has a particularly interesting
profile with a Ki of 8.4 nM and an aqueous solubility of
0.3 mg/mL.
enzyme and from the ability of substituents in the
3-position to access the S5-S4 binding site. Further-
more, the array of heteroatoms present in the bicyclic
ring system imparts increased solubility to the pyri-
dopyrimidines such that this series of inhibitors has a
significantly greater aqueous solubility than monocyclic
pyridone TFMKs having similar potency and structure.
It is therefore anticipated that, with the proper sub-
stituents in the 3-position, the pyridopyrimidine TFMKs
will afford orally active inhibitors of HNE. In addition,
this bicyclic dipeptide isostere should find wide ap-
plicability as a scaffold for the design of nonpeptidic
inhibitors of other proteolytic enzymes.
Exp er im en ta l Section
Gen er a l. Analytical samples were homogeneous by TLC
and afforded spectroscopic results consistent with the assigned
structures. Proton NMR spectra were obtained using either
a Bruker WM-250 or AM-300 spectrometer. Chemical shifts
are reported in parts per million (δ) relative to tetramethyl-
silane as internal standard. Mass spectra (MS) were recorded
on a Kratos MS-80 instrument or a Finnigan MAT-60 operat-
ing in the chemical ionization (DCI) mode (only peaks g10%
of the base peak are reported). Elemental analyses for carbon,
hydrogen, and nitrogen were determined by the Analytical
Section, ZENECA Pharmaceuticals, on a Perkin-Elmer 241
elemental analyzer and are within (0.4% of theory for the
formulas given. Analytical thin-layer chromatography (TLC)
was conducted on prelayered silica gel GHLF plates (Analtech,
Newark, DE). Visualization of the plates was accomplished
using UV light, phosphomolybdic acid-ethanol, and/or iodo-
Su m m a r y
Monocyclic and tricyclic pyridone-based TFMKs have
previously been shown to be potent inhibitors of HNE.
When the proper substituents are incorporated into the
monocyclic pyridones, in vivo activity following oral
administration has been achieved. In this report, we
have described a novel series of bicyclic pyridone-based
TFMKs, the pyridopyrimidine TFMKs. This class of
compounds has produced inhibitors with nanomolar and
subnanomolar Ki values. This level of potency is
believed to result from the ability of the carbonyl group
in the 2-position to participate in a unique hydrogen-
bonding interaction with Gly-218 and Gly-219 of the

1120 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Ta ble 4. Comparison of Pyridopyrimidine and Pyridone TFMKs
Edwards et al.
platinate charring. Flash chromatography was conducted on
Kieselgel 60, 230-400 mesh (E. Merck, Darmstadt, West
Germany). Solvents were either reagent or HPLC grade.
Reactions were run at ambient temperature and under a
nitrogen atmosphere unless otherwise noted. Solvent mixtures
are expressed as volume:volume ratios. Solutions were evapo-
rated under reduced pressure on a rotary evaporator. All
starting materials were commercially available unless other-
wise indicated.
2-P h t h a lim id o-N-(3,3,3-t r iflu or o-2-h yd r oxy-1-isop r o-
p ylp r op yl)a ceta m id e (2). A solution of N-phthaloylglycine
(34.0 g, 162 mmol) and NMM (39.1 mL, 36.0 g, 356 mmol) in
THF (680 mL) at -40 °C was treated dropwise with isobutyl
chloroformate (22.1 mL, 23.2 g, 170 mmol) and stirred between
-20 and -40 °C for 1.5 h. 3-Amino-1,1,1-trifluoro-4-methyl-
2-pentanol hydrochloride39 (1) (33.6 g, 162 mmol) was added,
and the resulting mixture was allowed to warm to room
temperature. The solution was stirred for 1.5 h, diluted with
ethyl acetate, washed with 1 N HCl, 1 N NaOH, and brine,
dried (MgSO4), and evaporated to afford crude 2 as a white
solid (47.2 g, 82%) which was used without further purifica-
tion: TLC Rf ) 0.30, chloroform/acetone (97:3); MS (DCI) m/z
) 359 (M + 1, base), 341; 1H NMR (300 MHz, DMSO-d6/TFA)
δ 0.90 (6H, m), 1.83 (1H, heptet, J ) 6.7 Hz), 3.84 (1H, t, J )
7.3 Hz), 4.14 (1H, q, J ) 6.2 Hz), 4.26 (2H, q, J ) 13.9 Hz),
7.89 (4H, m), 8.09 (1H, d, J ) 8.1 Hz, NH).
crude 3 (65.3 g, >100%) as a white solid which was used
without further purification: TLC Rf ) 0.54 hexanes/acetone
(2:1); MS (DCI) m/z ) 473 (M + 1), 147, 134, 133 (base) 115,
108, 91, 89, 75, 73; 1H NMR (250 MHz, DMSO-d6/TFA) δ 0.08
(3H, s), 0.12 (3H, s), 0.86 (15H, m), 1.50 (1H, m), 3.81 (1H, t,
J ) 8.3 Hz), 4.17-4.50 (3H, m), 7.68 (1H, d, J ) 9.7 Hz, NH),
7.86 (4H, m).
2-Am in o-N-[2-[(ter t-b u t yld im et h ylsilyl)oxy]-3,3,3-t r i-
flu or o-1-isop r op ylp r op yl]a ceta m id e (4). A refluxing solu-
tion of phthalimide 3 (62.3 g, 132 mmol) was treated with
hydrazine hydrate (19.2 mL, 19.8 g, 396 mmol). The solution
was refluxed for 3 h, acidified (pH 2) with 1 N HCl, refluxed
for 1 h, cooled to 0 °C, filtered, and evaporated. The residue
was taken up in ethyl acetate, washed with 1 N NaOH until
the pH of the washes exceeded 8, then washed with brine, and
evaporated. The crude amine was purified by flash chroma-
tography on silica gel, eluting with a chloroform/methanol
gradient (98:2, 95:5) to afford 4 (43.8 g, 81% from 1) as a
colorless oil: TLC Rf ) 0.68, hexanes/acetone (3:2); MS (DCI)
1
m/z ) 343 (M + 1, base), 327, 285, 129; H NMR (250 MHz,
DMSO-d6) δ 0.13 (3H, s), 0.16 (3H, s), 0.92 (12H, m), 0.98 (3H,
d, J ) 6.6 Hz), 1.81 (1H, m), 3.55 (1H, AB q, J ) 16.3 Hz),
3.75 (1H, AB q, J ) 16.3 Hz), 3.92 (1H, t, J ) 8.6 Hz), 4.34
(1H, m), 7.92 (1H, d, J ) 9.7 Hz, NH).
Dim eth yl [2-(Eth oxycar bon yl)eth yliden e]m alon ate (5).
A suspension of anhydrous ZnCl2 (258 g, 1.89 mol) in acetic
anhydride was stirred for 2 h. The solution was decanted and
treated dropwise with ethyl pyruvate (120 g, 1.03 mol) followed
by dimethyl malonate (136 g, 1.03 mol). The resulting mixture
was allowed to reflux for 2 h and the product isolated by
vacuum distillation (bp 136 °C, 1 mmHg) to afford 5 (183 g,
77%) as a light yellow liquid: TLC Rf ) 0.25, hexanes/acetone
(9:1); MS (DCI) m/z ) 231 (M + 1), 199 (base), 185, 167, 125;
1H NMR (250 MHz, DMSO-d6) δ 1.22 (3H, t, J ) 7.0 Hz), 2.13
(3H, s), 3.72 (3H, s), 3.79 (3H, s), 4.19 (2H, q, J ) 7.0 Hz).
2-P h th alim ido-N-[2-[(ter t-bu tyldim eth ylsilyl)oxy]-3,3,3-
tr iflu or o-1-isop r op ylp r op yl]a ceta m id e (3). A solution of
alcohol 2 (47.2 g, 132 mmol) and 2,6-lutidine (30.7 mL, 28.3 g,
264 mmol) in THF (600 mL) at 0 °C was treated dropwise with
a solution of tert-butyldimethylsilyl trifluoromethanesulfonate
(45.4 mL, 52.3 g, 198 mmol) in THF (50 mL) and was stirred
for 16 h at room temperature. The resulting mixture was
evaporated, taken up in ethyl acetate, washed with 1 N HCl,
1 N NaOH, and brine, dried (MgSO4), and evaporated to afford

Nonpeptidic Inhibitors of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1121
(r-Ch lor ob en zylid in e)d im et h yla m m on iu m Ch lor id e
(6). A solution of N,N-dimethylbenzamide (20.0 g, 134 mmol)
in CH2Cl2 (150 mL) was treated with oxalyl chloride (30.0 mL,
43.6 g, 344 mmol) and stirred at room temperature for 3 h.
The solution was evaporated, diluted with hexanes, and stirred
vigorously until a solid formed, and the solvent was decanted.
The residue was triturated with hexanes and then ether and
dried under vacuum to afford (R-chlorobenzylidine)dimethyl-
ammonium chloride 6 as a mositure-sensitive white solid
which was stored under vacuum and used without further
purification.
silyl)oxy]-3,3,3-tr iflu or o-1-isopr opylpr opyl]acetam ide (8b).
A mixture of the amine 4 (18 g, 70 mmol) and diene 7b (18.0
g, 50.0 mmol) was heated under vacuum (1 mmHg) at 120 °C
for 8 h, treated with additional 4 (6 g, 17.5 mmol), heated
under vacuum at 120 °C for 16 h, and cooled to room
temperature. The residue was taken up in ether, washed with
1 N HCl, 1 N NaOH, and brine, dried [50% (w/w) K2CO3/Na2-
SO4], and evaporated. The crude product was purified by flash
chromatography eluting with a gradient of ether/hexanes (40:
60, 50:50, 55:45, 60:40, 70:30, 100:0). The impure fractions
were purified by a second flash chromatography eluting with
a gradient of ether/hexanes (50:50, 55:45, 60:40, 70:30, 80:20,
100:0). The material from both chromatographies was com-
bined to afford 8b (14.9 g, 48%) as a solid: TLC Rf ) 0.41,
hexanes/acetone (7:3); MS (DCI) m/z ) 627 (M + 1), 105, 97
(base), 79; 1H NMR (300 MHz, DMSO-d6/TFA) δ 0.10 (3H, s),
0.11 (3H, s), 0.80 (3H, d, J ) 6.6 Hz), 0.88 (9H, s), 0.93 (3H, d,
J ) 6.8 Hz), 1.77 (1H, m), 3.80 (1H, m), 3.82 (3H, s), 4.29 (4H,
m), 4.67 (1H, m), 6.45 (1H, s), 7.50 (5H, m), 7.71 (1H, d, J )
9.9 Hz, NH).
4-(Eth oxyca r bon yl)-1-(4-m eth oxyben zyl)-3-(m eth oxy-
ca r bon yl)-2-p yr id on e (9). A solution of diene 7a (37.2 g,
130 mmol) in ethanol (186 mL) was treated dropwise with
p-methoxybenzylamine (18.8 mL, 19.7 g, 143 mmol) and
heated at reflux for 1.1 h. The solution was evaporated, and
the residue was taken up in ethyl acetate, washed with 1 N
HCl and brine, dried [50% (w/w) K2CO3/Na2SO4], and evapo-
rated to afford 9 (45.6 g, >100%) as a brown gum which was
used without further purification: TLC Rf ) 0.42, chloroform/
acetone (15:1); MS (DCI) m/z ) 346 (M + 1, base), 121; 1H
NMR (250 MHz, DMSO-d6) δ 1.25 (3H, t, J ) 7.0 Hz), 3.72
(3H, s), 3.75 (3H, s), 4.26 (2H, q, J ) 7.0 Hz), 5.07 (2H, s),
6.58 (1H, d, J ) 6.9 Hz), 6.91 (2H, d, J ) 6.8 Hz), 7.30 (2H, d,
J ) 8.6 Hz), 8.10 (1H, d, J ) 6.9 Hz).
Dim et h yl [3-(Dim et h yla m in o)-2-(et h oxyca r b on yl)-2-
p r op en ylid en e]m a lon a te (7a ). A solution of 5 (38.0 g, 165
mmol) and N,N-dimethylformamide diethyl acetal (29.1 mL,
25.0 g, 170 mmol) in DMF was heated at 80 °C for 3 h and
partitioned between brine and ethyl acetate. The ethyl acetate
layer was washed with brine, and the combined aqueous
washes were extracted with ethyl acetate. The combined
organic layers were dried (Na2SO4) and evaporated. The crude
product was crystallized from CCl4 (550 mL) to afford diene
7a (37.5 g, 80%) as a yellow solid: TLC Rf ) 0.54, chloroform/
acetone (15:1); MS (DCI) m/z ) 286 (M + 1), 285, 255, 254
1
(base); H NMR (250 MHz, DMSO-d6) δ 1.24 (3H, t, J ) 7.0
Hz), 2.84 (3H, br s), 3.09 (3H, br s), 3.55 (3H, s), 3.67 (3H, s),
4.23 (2H, q, J ) 7.0 Hz), 5.48 (1H, d, J ) 13 Hz), 6.90 (1H, d,
J ) 13 Hz).
Dim et h yl [2-(E t h oxyca r b on yl)-3-(d im et h yla m in o)-3-
p h en yl-2-p r op en ylid en e]m a lon a te (7b). A suspension of
hexanes-washed NaH (8.0 g, 60% oil dispersion, 20.0 mmol)
in THF (300 mL) at 0 °C was treated with 5 (40.0 g, 174 mmol),
stirred at room temperature for 4.5 h, cooled to 0 °C, and
treated with a suspension of 6 (crude product isolated as
described above) in THF (100 mL). The mixture was stirred
at room temperature for 1 h. The resulting material was
filtered through silica gel (1000 mL), eluting with ether until
the filtrate ran colorless. The filtrate was evaporated, and the
residue was triturated with ether and filtered to afford diene
7b (13.1 g) as yellow crystals. The filtrate was evaporated
and the residue purified by dry-column flash chromatography41
on silica gel, eluting with a gradient of ether/hexanes (1:1, 100:
0). The fractions containing the product were combined and
evaporated on a rotorary evaporator without external heat
until additional 7b (8.5 g) crystallized as a yellow solid. Total
yield was 21.6 g (45%): TLC Rf ) 0.29, ether/hexanes (7:3);
4-(E t h oxyca r b on yl)-3-(m et h oxyca r bon yl)-2-p yr id on e
(10). A solution of 9 (24.0 g, 69.5 mmol) in CH3CN/H2O (275
mL, 10:1) was treated with cerric ammonium nitrate (190 g,
347 mmol) and stirred at room temperature for 0.5 h. The
solution was diluted with chloroform (1 L) and filtered, and
the organic filtrate was washed with 1 N HCl and brine, dried
(MgSO4), and evaporated. The crude product was purified by
flash chromatography on silica gel eluting with a gradient of
chloroform/methanol (100:0, 95:5) to afford pyridone 10 (6.3
g, 40%) as a white solid: TLC Rf ) 0.49, chloroform/methanol
1
1
MS (DCI) m/z ) 362 (M + 1), 330 (base), 316; H NMR (300
(9:1); MS (DCI) m/z ) 266 (M + 1), 194 (base), 180, 166; H
MHz, DMSO-d6) δ 0.93 (3H, t, J ) 7.1 Hz), 2.81 (6H, br s),
3.13 (2H, q, J ) 7.1 Hz), 3.48 (3H, s), 3.71 (3H, s), 5.66 (1H,
s), 7.15 (2H, m), 7.40 (3H, m).
NMR (250 MHz, DMSO-d6/TFA) δ 1.28 (3H, t, J ) 7.1 Hz),
3.78 (3H, s), 4.27 (2H, q, J ) 7.1 Hz), 6.51 (1H, d, J ) 6.8 Hz),
7.64 (1H, d, J ) 6.8 Hz).
2-[4-(Eth oxyca r bon yl)-3-(m eth oxyca r bon yl)-2-oxo-1,2-
d ih yd r o-1-p yr id yl]-N-[2-[(ter t-b u t yld im et h ylsilyl)oxy]-
3,3,3-tr iflu or o-1-isop r op ylp r op yl]a ceta m id e (8a ). A so-
lution of 4 (30.1 g, 87.9 mmol) and 7a (27.6 g, 96.7 mmol) in
ethanol (140 mL) was heated at reflux for 16 h and evaporated.
The residue was taken up in ethyl acetate, washed with 1 N
HCl and brine, dried [50% (w/w) K2CO3/Na2SO4], and evapo-
rated. The residue was taken up in ether, washed with 1 N
HCl, and brine, dried [50% (w/w) K2CO3/Na2SO4], and evapo-
rated to afford pyridone 8a (34.5 g, 71%) as a light yellow
solid: TLC Rf ) 0.68, hexanes/acetone (1:1); MS (DCI) m/z )
551 (M + 1, base), 535, 519, 493, 266; 1H NMR (250 MHz,
DMSO-d6/TFA) δ 0.13 (3H, s), 0.17 (3H, s), 0.95 (15H, m), 1.29
(3H, t, J ) 6.0 Hz), 1.81 (1H, m), 3.78 (3H, s), 3.84 (1H, t, J )
8.4 Hz), 4.30 (3H, m), 4.59 (1H, d, J ) 13.1 Hz), 4.89 (1H, d,
J ) 13.1 Hz), 6.59 (1H, d, J ) 5.8 Hz), 7.88 (2H, m, CH, NH).
Alter n a tive P r oced u r e for P r ep a r a tion of 8a (Meth od
B, Sch em e 1). A solution of pyridone 10 (6.2 g, 27.5 mmol)
and iodide 13 (18.1 g, 40.0 mmol) in DMF (80 mL) was treated
with DBU (6.47 mL, 6.59 g, 43.3 mmol) and stirred at room
temperature for 24 h. The reaction mixture was diluted with
ethyl acetate, washed with 1 N HCl, brine, 1 N NaOH, and
brine, dried [50% (w/w) K2CO3/Na2SO4], and evaporated. The
crude product was purified by flash chromatography eluting
with acetone/hexanes (1:4) to afford 8a (8.82 g, 58%) as a light
yellow solid.
2-Ch lor o-N-(3,3,3-tr iflu or o-2-h yd r oxy-1-isop r op ylp r o-
p yl)a ceta m id e (11). A solution of 3-amino-1,1,1-trifluoro-4-
methyl-2-pentanol hydrochloride39 (1) (10.0 g, 48.2 mmol) and
NMM (10.9 mL, 10.0 g, 98.8 mmol) in THF (240 mL) at 0 °C
was treated dropwise with a solution of chloroacetyl chloride
(3.85 mL, 10.0 g, 48.2 mmol) in THF (20 mL). The solution
was stirred at 0 °C for 3 h and evaporated. The residue was
taken up in ethyl acetate, washed with 1 N HCl, 1 N NaOH,
and brine, dried (MgSO4), and evaporated to afford 11 (12.2
g, >100% as a yellow oil which was used without further
purification: TLC Rf ) 0.75, chloroform/methanol (95:5); MS
(DCI) m/z ) 248 (M + 1 for 35Cl, base), 75; 1H NMR (250 MHz,
DMSO-d6/TFA) δ 0.87 (3H, d, J ) 6.8 Hz), 0.93 (3H, d, J ) 6.8
Hz), 1.85 (1H, m), 3.87 (1H, m), 4.03-4.18 (1H, m), 4.12 (2H,
s), 7.95 (1H, d, J ) 9.7 Hz, NH).
2-Ch lor o-N-[2-[(ter t-b u t yld im et h ylsilyl)oxy]-3,3,3-t r i-
flu or o-1-isop r op ylp r op yl]a ceta m id e (12). A solution of
alcohol 11 (11.4 g, 46.0 mmol) and 2,6-lutidine (10.7 mL, 9.86
g, 92 mmol) in dichloromethane (46 mL) was treated dropwise
with tert-butyldimethylsilyl trifluoromethanesulfonate (15.9
mL, 18.3 g, 69.0 mmol). The solution was stirred for 3 h at
room temperature, diluted with ethyl acetate, washed with 1
N HCl, 1 N NaOH, and brine, dried (MgSO4), and evaporated.
The crude material was purified by flash chromatography
eluting with a gradient of hexanes/ethyl acetate (100:0, 90:
10, 80:20) to afford silyl ether 12 (10.3 g, 62%) as a white
solid: TLC Rf ) 0.62, hexanes/ethyl acetate (4:1); MS (DCI)
m/z ) 362 (M + 1 for 35Cl, base), 346, 326, 304, 148; 1H NMR
2-[4-(E t h oxyca r b on yl)-3-(m et h oxyca r b on yl)-2-oxo-6-
p h en yl-1,2-d ih yd r o-1-p yr id yl]-N-[2-[(ter t-bu tyld im eth yl-

1122 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Edwards et al.
(300 MHz, DMSO-d6) δ 0.13 (3H, s), 0.16 (3H, s), 0.87 (3H, d,
J ) 6.7 Hz), 0.91 (9H, s), 0.97 (3H, d, J ) 6.6 Hz), 1.77 (1H,
m), 3.84 (1H, t, J ) 8.0 Hz), 4.18 (2H, s), 4.38 (1H, m), 7.56
(1H, d, J ) 9.6 Hz, NH).
Gen er a l P r oced u r e for th e P r ep a r a tion of 3-Am id o-
p yr id op yr im id in es 19 a n d 20 Usin g EDAC. 2-[3-(4-
Meth oxyben zyl)-2,4,8-tr ioxo-6-p h en yl-1,2,3,4,7,8-h exa h y-
d r o p y r id o [3,4-d ]p y r im id in -7-y l]-N -[2-[(t er t -b u t y ld i-
m eth ylsilyl)oxy]-3,3,3-tr iflu or o-1-isop r op ylp r op yl]a ceta -
m id e (20c). A solution of acid 16b (1.50 g, 2.18 mmol),
4-methoxybenzylamine (0.57 mL, 597 mg, 4.35 mmol), and
HOBt (588 mg, 4.35 mmol) in DMF (15 mL) was treated with
EDAC (501 mg, 2.61 mmol) and stirred at room temperature
for 16 h. The reaction mixture was dissolved in ethyl acetate,
washed with 1 N HCl, brine, saturated NaHCO3, and brine,
dried (MgSO4), and evaporated to afford 18c. Crude 18c was
dissolved in DMF (15 mL), heated at 120 °C for 2.5 h, taken
up in ethyl acetate, washed with 1 N HCl, brine, saturated
NaHCO3, and brine, dried (MgSO4), and evaporated. The
crude product was purified by flash chromatography on silica
gel eluting with hexanes/acetone (4:1) to afford 20c (940 mg,
62%) as a light yellow foam: TLC Rf ) 0.35, hexanes/acetone
(4:1); MS (DCI) m/z ) 701 (M + 1), 190, 169, 150, 121, 107,
2-Iodo-N-[2-[(ter t-bu tyldim eth ylsilyl)oxy]-3,3,3-tr iflu or o-
1-isop r op ylp r op yl]a ceta m id e (13). A solution of NaI (8.45
g, 56.4 mmol) and chloride 12 (5.10 g, 14.1 mmol) in acetone
(56 mL) was stirred at room temperature for 16 h and was
partitioned between ethyl acetate and water. The organic
layer was washed with saturated Na2S2O3 and brine, dried
(MgSO4), and evaporated to afford iodide 13 (6.12 g, 96%) as
a white solid: TLC Rf ) 0.37, hexanes/ethyl acetate (9:1); MS
(DCI) m/z ) 454 (M + 1, base), 396, 326, 326, 240, 85; 1H NMR
(300 MHz, DMSO-d6/TFA) δ 0.13 (3H, s), 0.16 (3H, s), 0.89
(3H, m), 0.91 (9H, s), 0.96 (3H, d, J ) 6.6 Hz), 1.77 (1H, m),
3.77 (3H, m), 4.30 (1H, m), 7.79 (1H, d, J ) 9.7 Hz, NH).
Gen er a l P r oced u r e for th e P r ep a r a tion of P yr id on es
14. 2-[3-Ca r boxy-4-(eth oxyca r bon yl)-2-oxo-6-p h en yl-1,2-
d ih yd r o-1-p yr id yl]-N-[2-[(ter t-b u t yld im et h ylsilyl)oxy]-
3,3,3-tr iflu or o-1-isop r op ylp r op yl]a ceta m id e (14b). A so-
lution of 8b (14.9 g, 22.4 mmol) and anhydrous LiI (7.49 g,
55.9 mmol) in pyridine (250 mL) was heated at reflux for 4 h,
diluted with ether, washed with 1 N HCl and brine, dried
(MgSO4), and evaporated. The crude product was purified by
dry-column flash chromatography41 eluting with chloroform/
methanol (100:0, 95:5, 90:10, 85:15, 80:20, 70:30, 50:50) to
afford acid 14b (13.1 g, 96%) as a gray solid: TLC Rf ) 0.55,
chloroform/methanol/acetic acid (95:5:1); MS (DCI) m/z ) 631
1
97, 91, 79 (base); H NMR (250 MHz, DMSO-d6/TFA) δ 0.11
(3H, s), 0.12 (3H, s), 0.85 (3H, d, J ) 8.3 Hz), 0.88 (9H, s),
0.95 (3H, d, J ) 6.5 Hz), 1.77 (1H, m), 3.73 (3H, s), 3.85 (1H,
t, J ) 9.0 Hz), 4.25 (1H, m), 4.44 (1H, m), 4.65 (1H, m), 5.04
(2H, s), 6.50 (1H, s), 6.68 (2H, d, J ) 8.6 Hz), 7.31 (1H, d, J )
8.6 Hz), 7.44 (5H, m), 7.66 (2H, d, J ) 9.6 Hz, CH, NH).
Gen er a l P r oced u r e for th e P r ep a r a tion of 3-Am id o-
p yr id op yr im id in es 19 a n d 20 Usin g Mixed An h yd r id e
Cou p lin g. 2-[3-[2-(4-Mor p h olin o)et h yl]-2,4,8 t r ioxo-6-
p h en yl-1,2,3,4,7,8-h exa h yd r op yr id o[3,4-d ]p yr im id in -7-
yl]-N-[2-[(ter t-b u t yld im et h ylsilyl)oxy]-3,3,3-t r iflu or o-1-
isop r op ylp r op yl]a ceta m id e (20l). A solution of acid 16b
(603 mg, 0.875 mmol) and NMM (0.125 mL, 115 mg, 1.14
mmol) in THF (10 mL) at -40 °C was treated with isobutyl
chloroformate (0.150 mL, 0.155 mg, 1.14 mmol) and stirred at
-40 to -30 °C for 1 h. 4-(2-Aminoethyl)morpholine (0.150 mL,
0.148 g, 1.14 mmol) was added and the resulting mixture
stirred at room temperature for 16 h. The reaction solution
was taken up in ethyl acetate, washed with saturated NaHCO3
and brine, dried (MgSO4), and evaporated. Crude 18l was
dissolved in DMF and heated at 120 °C for 16 h and
evaporated. Purification by flash chromatography on silica gel
eluting with a gradient of chloroform/methanol (97:3, 95:5)
afforded 20l (350 mg, 58%) as a white solid: TLC Rf ) 0.47,
chloroform/methanol (95:5); MS (DCI) m/z ) 694 (M + 1), 231,
100, 74 (base); 1H NMR (300 MHz, DMSO-d6/TFA) δ 0.07 (3H,
s), 0.08 (3H, s), 0.80-0.91 (6H, m), 0.85 (9H, s), 1.73 (1H, m),
3.17 (2H, m), 3.48 (2H, m), 3.66 (4H, m), 3.98 (2H, m), 4.25
(2H, m), 4.44 (1H, m), 4.63 (1H, m), 6.46 (1H, s), 7.41 (5H, m),
7.68 (1H, d, J ) 9.9 Hz, NH).
1
(M + 1), 123, 117, 97 (base), 91; H NMR (250 MHz, DMSO-
d6/TFA) δ 0.10 (3H, s), 0.12 (3H, s), 0.82 (3H, d, J ) 6.5 Hz),
0.88 (9H, s), 0.94 (3H, d, J ) 6.5 Hz), 1.30 (3H, t, J ) 7.2 Hz)
1.74 (1H, m), 3.82 (1H, t, J ) 9.5 Hz), 4.34 (4H, m), 4.67 (1H,
d, J ) 15.8 Hz), 6.58 (1H, s), 7.55 (5H, m), 7.79 (1H, d, J ) 9.7
Hz, NH).
Gen er a l P r oced u r e for t h e P r ep a r a t ion of Am in o-
pyr idon es 15. 2-[3-[(Ben zyloxycar bon yl)am in o]-4-(eth oxy-
ca r b on yl)-2-oxo-6-p h en yl-1,2-d ih yd r o-1-p yr id yl]-N-[2-
[(ter t-bu tyld im eth ylsilyl)oxy]-3,3,3-tr iflu or o-1-isop r op yl-
p r op yl]a ceta m id e (15b). A solution of acid 14b (13.1 g, 21.4
mmol), diphenyl phosphorazidate (6.91 mL, 8.82 g, 32.1 mmol),
TEA (4.74 mL, 3.46 g, 34.2 mmol), and benzyl alcohol (3.77
mL, 3.94 g, 36.4 mmol) in p-dioxane (200 mL) was heated at
reflux for 4 h and evaporated. The residue was taken up in
ether, washed with 1 N HCl, brine, 1 N NaOH, and brine, dried
[50% (w/w) K2CO3/Na2SO4], and evaporated. The crude prod-
uct was purified by flash chromatography eluting with hex-
anes/ethyl acetate (85:15) to afford aminopyridone 15b (12.8
g, 84%) as a tan semisolid: TLC Rf ) 0.36, hexanes/ethyl
acetate (7:3); MS (DCI) m/z ) 718 (M + 1, base), 91; 1H NMR
(250 MHz, DMSO-d6/TFA) δ 0.11 (3H, s), 0.13 (3H, s), 0.84
(3H, d, J ) 6.5 Hz), 0.89 (9H, s), 0.95 (3H, d, J ) 6.6 Hz), 1.20
(3H, t, J ) 7.0 Hz), 1.77 (1H, m), 3.84 (1H, t, J ) 9.7 Hz), 4.22
(4H, m), 4.65 (1H, m), 5.16 (2H, s), 6.32 (1H, s), 7.43 (10H, m),
7.69 (1H, d, J ) 9.6 Hz, NH).
Gen er a l P r oced u r e for th e P r ep a r a tion of 4-Ca r boxy-
p yr id on es 16. 2-(3-[(Ben zyloxyca r bon yl)a m in o]-4-ca r -
b oxy-2-oxo-6-p h en yl-1,2-d ih yd r o-1-p yr id yl)-N-[2-[(ter t-
b u t y ld im e t h y ls ily l)o x y ]-3,3,3-t r iflu o r o -1-is o p r o p y l-
p r op yl]a ceta m id e (16b). A solution of ester 15b (11.0 g, 15.3
mmol) in ethanol/water (270 mL, 100:35) at 0 °C was treated
with LiOH‚H2O (0.674 g, 16.1 mmol), stirred for 4 h, treated
with additional LiOH‚H2O (0.674 g, 16.1 mmol), stirred for 3
h, diluted with ether, washed with 1 N HCl and brine, dried
(MgSO4), and evaporated. The crude material was purified
by flash chromatography on silica gel eluting with a gradient
of chloroform/methanol/acetic acid (100:0:0, 97.5:2.5:0, 95:5:
0, 95:5:1) to afford acid 16b (5.6 g, 53%) as an off-white foam.
Additional material could be recovered by purification of the
mixed fractions from the chromatography: TLC Rf ) 0.30,
chloroform/methanol (9:1); MS (DCI) m/z ) 690 (M + 1), 672,
582, 92 (base); 1H NMR (250 MHz, DMSO-d6/TFA) δ 0.12 (3H,
s), 0.14 (3H, s), 0.85 (3H, d, J ) 6.4 Hz), 0.90 (9H, s), 0.96
(3H, d, J ) 6.5 Hz), 1.77 (1H, m), 3.85 (1H, t, J ) 9.6 Hz),
4.30 (2H, m), 4.67 (1H, d, J ) 15.4 Hz), 5.18 (2H, s), 6.32 (1H,
s), 7.47 (10H, m), 7.70 (1H, d, J ) 9.6 Hz, NH).
Gen er a l P r oced u r e for th e P r ep a r a tion of 3-Am id o-
p yr id op yr im id in es 21 a n d 22 Usin g TBAF . 2-[3-(4-Meth -
oxyb en zyl)-2,4,8-t r ioxo-6-p h en yl-1,2,3,4,7,8-h exa h yd r o-
pyr ido[3,4-d]pyr im idin -7-yl]-N-(3,3,3-tr iflu or o-2-h ydr oxy-
1-isop r op ylp r op yl)a ceta m id e (22c). A solution of 20c (920
mg, 1.31 mmol) in THF (15 mL) was treated with TBAF (1.58
mL, 1.0 M, 1.58 mmol), stirred at room temperature for 2 h,
and evaporated. The crude material was purified by flash
chromatography eluting with
a gradient of chloroform/
methanol (97:3, 95:5) to afford 22c (730 mg, 95%) as a white
solid: TLC Rf ) 0.20, chloroform/methanol (95:5); MS (DCI)
1
m/z ) 587 (M + 1), 416, 149, 121 (base); H NMR (300 MHz,
DMSO-d6/TFA) δ 0.84 (3H, d, J ) 6.6 Hz), 0.91 (3H, d, J ) 6.6
Hz), 1.78 (1H, m), 3.73 (3H, s), 3.87 (1H, t, J ) 8.6 Hz), 4.10
(1H, m), 4.42 (1H, m), 4.56 (1H, m), 5.04 (2H, s), 6.48 (1H, s),
6.88 (2H, d, J ) 8.7 Hz), 7.31 (2H, d, J ) 8.6 Hz), 7.46 (5H,
m), 7.94 (1H, d, J ) 9.7 Hz, NH).
Gen er a l P r oced u r e for th e P r ep a r a tion of 3-Am id o-
p yr id op yr im id in es 21 a n d 22 Usin g HF . 2-(2,4,8-Tr ioxo-
6-p h en yl-1,2,3,4,7,8-h exa h yd r op yr id o[3,4-d ]p yr im id in -7-
y l)-I -(3,3,3-t r iflu o r o -2-h y d r o x y -1-is o p r o p y lp r o p y l)-
a ceta m id e (22a ). A solution of 20a (300 mg, 0.517 mmol)
and aqueous HF (5.2 mL, 5%) in CH3
CN (46.8 mL) was stirred
at room temperature for 16 h. Methanol was added to dissolve
the precipatated solid, the mixture was preabsorbed onto silica
gel (10 mL), and the product was isolated by flash chroma-
tography on silica gel (100 mL) eluting with chloroform/

Nonpeptidic Inhibitors of Human Neutrophil Elastase
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1123
(8) Nadel, J . A. Role of Mast Cell and Neutrophil Proteases in
Airway Secretion. Am. Rev. Respir. Dis. 1991, 144, S48-S51.
(9) Stein, R. L.; Trainor, D. A.; Wildonger, R. A. Neutrophil Elastase.
Annu. Rep. Med. Chem. 1985, 20, 237-246.
methanol (9:1) to afford 22a (140 mg, 58%) as a solid: TLC Rf
) 0.45, chloroform/methanol (9:1); MS (DCI) m/z ) 467 (M +
1
1, base); H NMR (300 MHz, DMSO-d6/TFA) δ 0.88 (6H, m),
1.78 (1H, heptet, J ) 8.5 Hz), 3.85 (1H, m), 4.09 (1H, m), 4.23-
4.60 (2H, m), 6.42 (1H, s), 7.47 (5H, m), 7.94 (1H, d, J ) 9.4
Hz, NH).
(10) Laurell, C.-B.; Eriksson, S. The Electrophoretic R1-Globulin
Pattern of Serum in R1-Antitrypsin Deficiency. Scand. J . Clin.
Lab. Invest. 1963, 15, 132-140.
(11) Eriksson, S. Studies in R1-Antitrypsin Deficiency. Acta Med.
Scand. 1965, 177 (Suppl. 432), 1-85.
(12) Bernstein, P. R.; Edwards, P. D.; Williams, J . C. Inhibitors of
Human Leukocyte Elastase. Prog. Med. Chem. 1994, 31, 59-
120.
Gen er a l P r oced u r e for th e P r ep a r a tion of Keton es 23
a n d 24. 2-[3-(4-Met h oxyb en zyl)-2,4,8-t r ioxo-6-p h en yl-
1,2,3,4,7,8-h exah ydr opyr ido[3,4-d]pyr im idin -7-yl]-N-(3,3,3-
tr iflu or o-1-isop r op yl-2-oxop r op yl)a ceta m id e (24c). A so-
lution of alcohol 22c (710 mg, 1.21 mmol) and EDAC (2.32 g,
12.1 mmol) in DMSO/toluene (30 mL, 1:1) at 0 °C was treated
with 2,2-dichloroacetic acid (0.400 mL, 620 mg, 4.84 mmol).
The reaction mixture was stirred at room temperature for 16
h and partitioned between ethyl acetate and 1 N HCl. The
organic layer was washed with 1 N HCl, brine, saturated
NaHCO3, and brine, dried (MgSO4), and evaporated. The
crude product was purified by flash chromatography on silica
gel eluting with a gradient of chloroform/methanol (99:1, 97:
3) to afford 24c (380 mg, 54%) as a white solid: TLC Rf )
0.38, chloroform/methanol (97:3); MS (DCI) m/z ) 585 (M +
1), 416, 149, 121 (base); 1H NMR (250 MHz, DMSO-d6/TFA) δ
0.81 (3H, d, J ) 6.8 Hz), 0.96 (3H, d, J ) 6.6 Hz), 2.26 (1H,
m), 3.75 (3H, s), 4.10 (1H, d, J ) 2.8 Hz), 4.59 (2H, m), 5.06
(2H, s), 6.52 (1H, s), 6.90 (2H, d, J ) 8.6 Hz), 7.30 (2H, d, J )
8.6 Hz), 7.46 (5H, m). Anal. (C29H27N4F3O6‚0.CH3OH) C, H,
N.
2-[3-(Ca r boxym eth yl)-2,4,8-tr ioxo-6-p h en yl-1,2,3,4,7,8-
h exah ydr opyr ido[3,4-d]pyr im idin -7-yl]-N-(3,3,3-tr iflu or o-
1-isop r op yl-2-oxop r op yl)a ceta m id e (23e). A suspension
of benzyl ester 23m (160 mg, 0.298 mmol, prepared according
the general precedure described above for ketones 23 and 24),
and 10% Pd-C (100 mg) in methanol (29 mL) was hydroge-
nated at 50 psi for 3 h, filtered, and evaporated to afford 23e
(110 mg, 83%) as an analytically pure white solid: TLC Rf )
0.15, chloroform/methanol (95:5); MS (DCI) m/z ) 447 (M +
1, base), 429, 150; 1H NMR (250 MHz, DMSO-d6/TFA) δ 0.84-
1.01 (6H, m), 2.25 (1H, m), 4.57 (2H, br s), 4.58-5.00 (3H, m),
6.62 (1H, m), 7.40 (1H, m), 7.93 (1H, d, J ) 10 Hz, NH). Anal.
(C17H17N4F3O7‚1.4H2O‚0.5CH3OH) C, H, N.
(13) 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.
(14) Sommerhoff, C. P.; Krell, R. D.; Williams, J . L.; Gomes, B. C.;
Strimpler, A. M.; Nadel, J . A. Inhibition of Human Neutrophil
Elastase by ICI 200,355. Eur. J . Pharm. 1991, 193, 153-158.
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Mauldin, 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 Ke-
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P. A. Peptidyl R-Ketoheterocyclic Inhibitors of Human Neutro-
phil Elastase. 3. In Vitro and in Vivo Potency of a Series of
Peptidyl R-Ketobenzoxazoles. J . Med. Chem. 1995, 38, 3972-
3982.
(17) Soskel, N. T.; Watanabe, S.; Hardie, R.; Shenvi, A. B.; Punt, J .
A.; Kettner, C. Effects of Dosage and Timing of Administration
of a Peptide Boronic Acid Inhibitor on Lung Mechanics and
Morphometrics in Elastase-Induced Emphysema in Hamsters.
Am. Rev. Respir. Dis. 1986, 133, 635-638.
(18) Soskel, N. T.; Watanabe, S.; Hardie, R.; Shenvi, A. B.; Punt, J .
A.; Kettner, C. A New Peptide Boronic Acid Inhibitor of Elastase-
Induced Lung Injury in Hamsters. Am. Rev. Respir. Dis. 1986,
133, 639-642.
(19) Stone, P. J .; Lucey, E. C.; Snider, G. L. Induction and Exacerba-
tion of Emphysema in Hamsters with Human Neutrophil
Elastase Inactivated Reversibly by a Peptide Boronic Acid. Am.
Rev. Respir. Dis. 1990, 141, 47-52.
(20) Bonney, R. J .; Ashe, B.; Maycock, A.; Dellea, P.; Hand, K.;
Osinga, D.; Fletcher, D.; Mumford, R.; Davies, P.; Frankenfield,
D.; Nolan, T.; Schaeffer, L.; Hagmann, W.; Finke, P.; Shah, S.;
Dorn, C.; Doherty, J . Pharmacological Profile of the Substituted
Beta-Lactam L-569,286: A Member of a New Class of Human
PMN Elastase Inhibitors. J . Cell. Biochem. 1989, 39, 47-53.
(21) Hagmann, W. K.; Shah, S. K.; Dorn, C. P.; O’Grady, L. A.; Hale,
J . J .; Finke, P. E.; Thompson, K. R.; Brause, K. A.; Ashe, B. M.;
Weston, H.; Dahlgren, M. E.; Maycock, A. L.; Dellea, P. S.; Hand,
K. M.; Osinga, D. G.; Bonney, R. J .; Davies, P.; Fletcher, D. S.;
Doherty, J . B. Prevention of Human Leukocyte Elastase-
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Ack n ow led gm en t. We thank Mr. J im Hulsizer and
Mr. Gary Moore for large-scale synthesis of intermedi-
ates and Dr. Prudence Bradley for the determination
of aqueous solubilities.
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(1) Abbreviations: HNE, human neutrophil elastase; PPE, porcine
pancreatic elastase; Ac, acetyl; Cbz, benzyloxycarbonyl; TFMK,
trifluoromethyl ketone; CAN, ceric ammonium nitrate; TEA,
triethylamine; THF, tetrahydrofuran; DMSO, dimethyl sulfox-
ide; EDAC, 1-ethyl-[3-[3-(dimethylamino)propyl]]carbodiimide
hydrochloride; HOBt, 1-hydroxybenzotriazole monohydrate; TBAF,
tetra-n-butylammonium fluoride; TFA, trifluoroacetic acid; DMF,
dimethylformamide; NMM, N-methylmorpholine; DMP, Dess-
Martin periodinane, 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-
3(1H)-one; MeO, methoxy; Suc, succinyl; pNa, p-nitroanilide;
PBS, phosphate-buffered saline; it, intratracheal; DPPA, diphen-
yl phosphorazidate.
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