2-(Diethylamino)thieno[1,3]oxazin-4-ones as stable inhibitors of human leukocyte elastase

Article abstract of DOI:10.1021/jm991108w

A series of 2-(diethylamino)thieno[1,3]oxazin-4-ones was synthesized and evaluated in vitro for inhibitory activity toward human leukocyte elastase (HLE). The Gewald thiophene synthesis was utilized to obtain several ethyl 2-aminothiophene-3- carboxylates. These precursors were subjected to a five-step route to obtain thieno[2,3-d][1,3]oxazin-4-ones bearing various substituents at positions 5 and 6. Both thieno[2,3-d] and thieno[3,2-d] fused oxazin-4-ones possess extraordinary chemical stability, which was expressed as rate constants of the alkaline hydrolysis. The kinetic parameters of the HLE inhibition were determined. The most potent compound, 2-(diethylamino)-4H- [1]benzothieno[2,3-d][1,3]oxazin-4-one, exhibited a K(i) value of 5.8 nM. 2-(Diethylamino)thieno[1,3]oxazin-4-ones act as acyl- enzyme inhibitors of HLE, similar to the inhibition of serine proteases by 4H-3,1-benzoxazin-4-ones. The isosteric benzene- thiophene replacement accounts for an enhanced stability of the acyl-enzyme intermediates.

Full text of DOI:10.1021/jm991108w

J . Med. Chem. 1999, 42, 5437-5447
5437
2-(Dieth yla m in o)th ien o[1,3]oxa zin -4-on es a s Sta ble In h ibitor s of Hu m a n
Leu k ocyte Ela sta se
Michael Gu¨tschow,*,† Lars Kuerschner,† Ulf Neumann,‡ Markus Pietsch,† Reik Lo¨ser,† Norman Koglin,† and
Kurt Eger†
Institute of Pharmacy, University of Leipzig, D-04103 Leipzig, Germany, and Novartis Pharma AG,
CH-4002 Basel, Switzerland
Received J une 25, 1999
A series of 2-(diethylamino)thieno[1,3]oxazin-4-ones was synthesized and evaluated in vitro
for inhibitory activity toward human leukocyte elastase (HLE). The Gewald thiophene synthesis
was utilized to obtain several ethyl 2-aminothiophene-3-carboxylates. These precursors were
subjected to a five-step route to obtain thieno[2,3-d][1,3]oxazin-4-ones bearing various sub-
stituents at positions 5 and 6. Both thieno[2,3-d] and thieno[3,2-d] fused oxazin-4-ones possess
extraordinary chemical stability, which was expressed as rate constants of the alkaline
hydrolysis. The kinetic parameters of the HLE inhibition were determined. The most potent
compound, 2-(diethylamino)-4H-[1]benzothieno[2,3-d][1,3]oxazin-4-one, exhibited a Ki value of
5.8 nM. 2-(Diethylamino)thieno[1,3]oxazin-4-ones act as acyl-enzyme inhibitors of HLE, similar
to the inhibition of serine proteases by 4H-3,1-benzoxazin-4-ones. The isosteric benzene-
thiophene replacement accounts for an enhanced stability of the acyl-enzyme intermediates.
In tr od u ction
The inactivation by mechanism-based inhibitors (sui-
cide substrates) is assumed to result from the unmask-
ing of a latent reactive function in the acyl-enzyme
intermediate. This function reacts with an available
nucleophilic amino acid in the active site to irreversibly
inactivate the enzyme. A variety of heterocyclic struc-
tures have been described as suicide substrates of HLE.
They are usually activated enzymatically by an estero-
lytic reaction, as for halo enol and ynenol lactones8 and
isocoumarins,9 or by an amidolytic reaction, as for
cephem sulfones,10 monocyclic â-lactams,11 sulfonyloxy
phthalimides,12 and succinimides,13 1,2,5-thiadiazolidin-
3-ones,14 and benzisothiazolones.15 With other types of
mechanism-based inhibitors, the acylation reaction
leads to stable acyl-enzymes, but a modification reaction
does not occur. The resulting acyl-enzymes, which have
the potential for further conversion to products of the
enzymatic turnover, may be trapped in potential energy
wells.16 For such acyl-enzyme inhibitors (alternate
substrate inhibitors), strong inhibition can be achieved
by increasing the rate of acylation or decreasing the rate
of deacylation, or both. 3,1-Benzoxazin-4-ones represent
a class of heterocyclic acyl-enzyme inhibitors of several
mammalian serine proteases such as HLE,17-23 porcine
pancreatic elastase,24 cathepsin G,25 proteinase 3,22
thrombin,26 C1r serine protease of the complement
system,27-29 and chymotrypsin.30-33 Inhibition of the
viral serine proteases HSV-1 protease34 and human
cytomegalovirus protease35 by benzoxazinones has also
been reported.
Human leukocyte elastase (HLE, EC 3.4.21.37), a
potent serine proteinase, is capable of degradating a
variety of structural proteins, including elastin, col-
lagens, laminin, fibronectin, and cartilage proteoglycans,
as well as immunoglobulins and complement compo-
nents, and can indirectly favor the breakdown of
matrix proteins by proteolytic activation of matrix
metalloproteinases.1 HLE is stored in the azurophilic
granules of polymorphonuclear leukocytes and is re-
leased in response to inflammatory stimuli. Under
normal circumstances, the proteolytic activity of HLE
is effectively controlled by its natural inhibitors such
as R1-proteinase inhibitor, R2-macroglobulin, human
mucus proteinase inhibitor, and elafin. An imbalance
between free HLE and its endogenous inhibitors may
result in several pathological states. HLE is thought to
play a role in various disorders including pulmonary
emphysema, cystic fibrosis, chronic bronchitis, adult
respiratory distress syndrome, rheumatoid arthritis,
and periodontitis.1,2
Several strategies have been pursued for the develop-
ment of HLE inhibition3 and various types of inhibitors
have been reported during the last years, e.g., anionic
oligomers and polymers,4 hydrazinopeptides,5 or an-
thraquinones.6 Most of the potent, low molecular weight
inhibitors form covalent adducts with the active-site
serine. Examples include electrophilic ketone-based
inhibitors and acylating agents. Electrophilic ketones,
such as peptidyltrifluoromethyl or pentafluoroethyl
ketones,7 are assumed to act as transition-state ana-
logue inhibitors. Acylating agents react with the active-
site serine in a substratelike manner by eliminating a
leaving group to form a stable acyl-enzyme.
Efforts in the design of heterocyclic HLE inhibitors
have focused on developing compounds that would react
with the enzyme after binding in the active site but
would be sufficiently stable to hydrolysis by nonspecific
nucleophiles. Krantz et al.19 have examined the effect
of electron-donating groups on hydrolytic stability in a
series of benzoxazinone HLE inhibitors. Another strat-
egy to improve the chemical stability is the replacement
* To whom correspondence should be addressed.
† University of Leipzig.
‡ Novartis Pharma AG.
10.1021/jm991108w CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/03/1999

5438 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Gu¨tschow et al.
Sch em e 1. Reaction of Thieno[2,3-d][1,3]oxazin-4-ones
with HLE: Formation of Acyl-Enzymes and Possible
Ways of Deacylation
Sch em e 2a
a
Reagents: (a) CSCl2, CaCO3, CH2Cl2, H2O, 0 °C; (b) diethyl-
amine, CH2Cl2, room temperature; (c) concd H2SO4, room tem-
perature; (d) NaOH, dioxane-H2O, reflux; (e) HgO, CH2Cl2, room
temperature.
Sch em e 3
of the benzene ring in benzoxazinones by an isosteric
thiophene unit. Recently, the inhibitory efficacy of
thieno[2,3-d][1,3]oxazin-4-ones 1 (Scheme 1) toward
HLE has been demonstrated.36 These heterocycles act
as acyl-enzyme inhibitors, similar to benzoxazinones:
Nucleophilic attack of the active-site serine (E-OH) at
the C-4 atom of the inhibitor and ring cleavage forms
the corresponding acyl-enzymes that deacylate hydro-
lytically. As in the class of benzoxazinones,19,28 substitu-
tion at position 2 is a sensitive parameter that affects
chemical stability of thieno[2,3-d][1,3]oxazinones.36 In-
troduction of an amino substituent at position 2 was
advantageous to improve stability in both classes. With
a NHR group at position 2, an intramolecular deacyla-
tion may occur (Scheme 1, R2 ) NHR). Thus, deacyla-
tion of benzoxazinone-derived acyl-enzymes was ad-
versely accelerated by an intramolecular quinazoline
cyclization.18,25,32,33 Therefore, fused [1,3]-oxazin-4-ones
bearing a disubstituted amino group at position 237
represent an attractive structure for the ongoing efforts
in the design of inhibitors for HLE and other serine
proteases.
On the basis of these considerations and previous
results,36 we have prepared a series of thieno[1,3]-
oxazinones with a 2-diethylamino group as a fixed
structural feature and various substituents at positions
5 and 6. The synthesis and kinetic data of the alkaline
hydrolysis and in vitro inhibition of HLE are reported
herein.
carboxylic acid 6. Subsequent treatment with yellow
mercury(II) oxide furnished the final product 7 in 86%
yield.
The Gewald thiophene synthesis39,40 allows for a facile
synthetic entry to the class of thieno[2,3-d][1,3]oxazin-
4-ones. Ethyl 2-aminothiophene-3-carboxylates 10-17
(Scheme 4) were used as starting materials to prepare
the final thieno[2,3-d][1,3]oxazin-4-ones 50-57. The
requisite aminothiophenes 14-16 were synthesized
from the appropriate ketone, sulfur, and ethyl cyano-
acetate in the presence of an organic base by an one-
pot thiolation-heterocyclization reaction.39 The 4,5-
unsubstituted aminothiophene 10 was obtained by
reacting the mercaptoacetaldehyde dimer (2,5-dihy-
droxy-1,4-dithiane) with ethyl cyanoacetate. To prepare
the benzothiophene 17,41 the corresponding tetrahy-
drobenzo derivative was first acetylated to protect the
amino group and then dehydrogenated with sulfur in
diethyl phthalate, followed by alkaline deprotection. The
synthesis of three further precursors is shown in
Scheme 3. Compound 11 was synthesized by a facile
Resu lts a n d Discu ssion
Syn th esis. The preparation of 2-diethylaminothieno-
[3,2-d][1,3]oxazin-4-one 7 (Scheme 2) was achieved
following a route that has been established for the
synthesis of thieno[2,3-d]oxazinone derivatives.36 Con-
version of the aminothiophene 2 to the isothiocyanate
3, followed by treatment with diethylamine, gave the
thiourea derivative 4. Ring closure to the thiazinone 5
was then performed upon the action of concentrated
sulfuric acid, according to a previously reported proce-
dure.38 To replace the thiazine sulfur atom in 5 by
oxygen, the thiazinone ring was first cleaved hydrolyti-
cally upon the action of sodium hydroxide to afford the

Thieno[1,3]oxazin-4-ones as HLE Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5439
Sch em e 4a
promoted cyclization was performed at room tempera-
ture to furnish the products in 69-83% yield, but more
vigorous conditions were needed to obtain 53 in only
31% yield. This might be attributed to the electron-
withdrawing effect of the carbamoyl group to reduce
nucleophilicity of the thiourea sulfur, which is assumed
to interact with mercury oxide.
Alk a lin e Hyd r olysis. Stability of the compounds
was determined spectrophotometrically in aqueous buffer,
pH 11.25 at 25 °C. Alkaline hydrolysis rate constants
of thieno[1,3]oxazin-4-ones 7 and 50-58 were deter-
mined at 335 nm by the disappearance of the long-
wavelength ultraviolet chromophore (330-346 nm) that
followed the first-order exponential decay for all com-
-
pounds. The second-order rate constants, kOH , were
calculated and are outlined in Table 1. In addition, the
alkaline hydrolysis of the reference 3,1-benzoxazin-4-
one 59, bearing the same 2-diethylamino substituent,
-
was also determined. The kOH value was in accordance
with reported data for comparable amino-substituted
benzoxazinones.19 As described for compound 58,36
aqueous alkaline hydrolysis of the thieno[1,3]oxazinones
7 and 50-57 was assumed to proceed by hydroxide
attack at C-4 to produce the corresponding ureido-
thiophenecarboxylic acids. An analogous hydrolytic ring
opening for 3,1-benzoxazin-4-ones is well estab-
lished.19,33,37,43 Both the alkaline hydrolysis of fused 1,3-
oxazin-4-ones and the process by which they inhibit
HLE involve attack of a nucleophilic oxygen upon the
lactone carbonyl carbon. Therefore, recognition of struc-
tural features promoting enzyme inhibition over chemi-
cal reactivity is crucial in the design of such mechanism-
based inhibitors. The amino-substituted thieno[1,3]-
oxazin-4-ones 7 and 50-58 possessed extraordinary
low susceptibility to alkaline hydrolysis, with values
a
Reagents: (a) CSCl2, CaCO3, CH2Cl2, H2O, 0 °C, or room
temperature; (b) diethylamine, CH2Cl2, room temperature; (c)
concd H2SO4, room temperature, or polyphosphoric acid, 170 °C;
(d) NaOH, dioxane-H2O, reflux; (e) HgO, CH2Cl2, room temper-
ature, or reflux.
two-step route: The thiophene diester 8 was prepared
from tert-butyl acetoacetate, sulfur, and ethyl cyanoace-
tate. Treatment of 8 with hydrochloric acid resulted in
selective deesterification and decarboxylation, to obtain
the desired methylthiophene 11. To afford 12, the
Knoevenagel adduct of 3-methyl-2-butanone and ethyl
cyanoacetate was prepared and subsequently treated
with sulfur. To prepare the 5-carbamoyl thiophene 13,
acetoacetamide was subjected to the conditions of the
Gewald reaction.
kOH e 0.4 M-1 s-1 (Table 1). A comparison of the
-
benzoxazinone 59 with the two 5,6-unsubstituted thieno-
[1,3]oxazinones 7 and 50 revealed the favorable effect
of the benzene-thiophene replacement; the second-
order rate constants of both thieno compounds were 50-
fold lower. Except for 53, 5,6-substituted thieno[2,3-
d][1,3]oxazin-4-ones 51-58 were found to be even more
stable.
The synthesis of a series of thieno[2,3-d] fused [1,3]-
oxazinon-4-ones 50-57 is outlined in Scheme 4. The
route corresponds to the preparation of the thieno[3,2-
d] fused [1,3]-oxazin-4-one 7 described above. Cyclocon-
densation of ethyl 2-thioureidothiophene-3-carboxylates
26-33 was performed either with concentrated sulfuric
acid or upon treatment with polyphosphoric acid to
furnish 34-41. Ring cleavage of 34-41 was carried out
in refluxing NaOH-dioxane to produce the thiophene-
3-carboxylic acids 42-49. A prolonged reaction time,
compared to the standard methodology,36 did improve
the yields in certain cases but was less satisfactory to
prepare 49 due to the formation of byproducts. Finally,
compounds 42-49 were converted to thieno[2,3-d][1,3]-
oxazin-4-ones 50-57 upon treatment with yellow mer-
cury(II) oxide in dichloromethane. The ring closure is
similar to known heterocyclizations in which carbodi-
imides react with nucleophiles.42 Since the formation
of an intermediate carbodiimide is not possible in the
present transformation, the thiourea carbon, activated
by the action of HgO, is believed to be attacked by the
carboxylate oxygen. With the exception of 53, the HgO-
HLE In h ibitin g Activity. HLE inhibition by thieno-
[1,3]oxazin-4-ones 7 and 50-58 and the 3,1-benzoxazin-
4-one 59 was assayed in the presence of Suc-Ala-Ala-
Pro-Val-pNA as a chromogenic substrate. Progress
curves were characterized by an initial exponential
phase, followed by a linear steady-state turnover of the
substrate, and could be analyzed by slow-binding kinet-
ics.44 Progress curves were fitted to
[P] ) vst + (vi - vs) [1 - exp(-kobst)]/kobs + offset
(1)
where vs and vi are the steady-state and the initial
velocity and kobs is the first-order rate constant for the
approach to the steady state. The second-order rate
constant, kon, the first-order rate constant, koff, and the
steady-state inhibition constant, Ki, were obtained as
described.17,36
In the present study on HLE inhibition by compounds
7 and 50-59, except for compounds 51 and 53, a
preassociation complex did not accumulate at the in-
hibitor concentration range used. This was indicated by

5440 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Gu¨tschow et al.
Ta ble 1. Thieno[1,3]oxazin-4-ones Prepared and Kinetic Data of the Alkaline Hydrolysis and HLE Inhibition
-
kOH
log
kOH
kon
koff
Ki
(nM)
-
compd
R5
R6
(M-1 s-1
)
(M-1 s-1
)
(10-4 s-1
)
pKi
7
50
51
52
53
54
55
56
57
58
59
0.11
0.12
-0.95
-0.91
-1.14
-1.27
-0.40
-1.03
-1.42
-1.41
-1.13
-1.49
0.77
330
530
140
10000
4000
3600
990
0.43
1.4
0.64
1.3
130
260
450a
13
6.88
6.59
6.35
7.88
7.04
7.90
7.36
6.69
8.24
7.90c
6.52d
H
H
H
H
Me
i-Pr
Me
-(CH2)3-
-(CH2)5-
-CH(Me)CH2CH2CH2-
-CHdCH-CHdCH-
-(CH2)4-
0.073
0.054
0.40
0.094
0.038
0.039
0.075
0.032
5.9
CONH2
3.6
91b
13
0.45
0.43
0.13
0.55
0.32
7.9
44
63
210
5.8
13
9500
2500
2600
310
a
b
d
Kd ) 5.5 µM. Kd ) 620 nM. c Reference 36: pKi ) 7.79, obtained with MeOSuc-Ala-Ala-Pro-Val-pNA as substrate. Reference 19:
pKi ) 6.43, obtained with MeOSuc-Ala-Ala-Pro-Val-pNA as substrate.
F igu r e 1. Slow-binding inhibition of HLE by compound 52
in 50 mM sodium phosphate and 500 mM NaCl, pH 7.8.
Substrate was Suc-Ala-Ala-Pro-Val-pNA. Data were fitted to
eq 1 to obtain the best-fit parameters for vi, vs, kobs, and offset.
(O) [I] ) 0; (b) [I] ) 100 nM; (0) [I] ) 200 nM; (9) [I] ) 300
nM; (4) [I] ) 400 nM; (2) [I] ) 500 nM.
F igu r e 3. Plot of the steady-state rates versus [I] for the
inhibition of HLE by compound 52. The data were obtained
from fits of the curves shown in Figure 1. The solid line was
drawn by using the best-fit parameters from a fit according
to an equation of a competitive inhibition, which gave Ki (1 +
[S]/Km) ) 31.5 ( 2.6 nM. The inset is a Dixon plot to show the
linearity.
increased inhibitor concentrations, whereas the kobs
values were identical. This indicated the accumulation
of a preassociation complex under the experimental
conditions used. The dissociation constants of a preas-
sociation complex, Kd, and the final inhibition constants,
Ki, as well as kon and koff values, were obtained as
described.36
The kinetic data of the HLE inhibition by compounds
7 and 50-59 are outlined in Table 1. It was concluded
from their kinetic behavior as well as from product
analysis experiments36 that the compounds act as acyl-
enzyme inhibitors of HLE. In this case, the rate con-
stants, kon and koff, reflect the acylation and deacylation
step, respectively, and not the simple association-
dissociation equilibrium of a competitive inhibition.
Inhibition constants, Ki, are equal to the deacylation
rate constant, koff, divided by the acylation rate constant,
kon. Since a steady-state condition is reached, and as
long as the inhibitor is not depleted due to its turn-
over by HLE, Ki values will reflect the dissociation of
all enzyme-bound inhibitor complexes that accumu-
late in the steady state17 and are used to express the
potency of acyl-enzyme inhibitors of serine pro-
teases.18-20,22,25,36
F igu r e 2. Plot of kobs versus [I] for the inhibition of HLE by
compound 52. The values for kobs were obtained from fits to
the data shown in Figure 1. The slope corresponds to a value
for kon/(1 + [S]/Km) ) 4640 ( 430 M-1 s-1
.
similar initial velocities, vi, that equaled the velocity in
the absence of inhibitor, v0, as well as by a linear
dependence of kobs on [I]. As an example, the analysis
of the HLE inhibition kinetics by the thieno[2,3-d][1,3]-
oxazinone 52 is illustrated in Figures 1-3.
HLE inhibition by compounds 51 and 53 was char-
acterized by initial velocities, vi, that decreased with

Thieno[1,3]oxazin-4-ones as HLE Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5441
The present thieno[1,3]oxazinones 7 and 50-58 ex-
hibited Ki values in the nanomolar range (Table 1).
Considering their extraordinary stability in alkaline
hydrolysis, 2-(diethylamino)thieno[1,3]oxazin-4-ones con-
stitute an interesting class of HLE inhibitors. Studies
on a series of 2-alkoxy-, 2-alkylthio-, and 2-aminothieno-
[2,3-d][1,3]oxazin-4-ones36 have demonstrated the mode
of action as acyl-enzyme inhibitors of HLE (Scheme 1),
similar to benzoxazinones. Likely, the inhibitors of the
present series act in the same manner, by acylating the
active-site serine to form an acyl-enzyme intermediate
that deacylates slowly. The replacement of the benzene
ring in 2-(diethylamino)-3,1-benzoxazinone by an un-
substituted thiophene (59 versus 7 and 50) resulted in
around a 10-fold deceleration of both the acylation and
deacylation step. These results support a general trend36
in which the kinetic parameters of the HLE inhibition
were affected by the isosteric benzene-thiophene re-
placement. The reduced susceptibility to be attacked by
an oxygen nucleophile, as it was demonstrated in
alkaline hydrolysis, is assumed to account for the
diminished acylation rates. On the other hand, in the
thiophene-derived acyl-enzymes, the ester bond, as a
part of a heteroaromatic â-enamino ester,45 is expectedly
less reactive toward nucleophiles, leading to a reduced
deacylation rate and an enhanced stability of the acyl-
enzyme intermediates.
The thieno[1,3]thiazin-4-ones 5 (Scheme 2) and 34-
41 (Scheme 4) were also evaluated as inhibitors of HLE.
Due to limits of solubility, assays were performed only
at a single inhibitor concentration (2 µM). For none of
the compounds was enzyme inhibition detected, prob-
ably as a result of the further decreased reactivity of
the sulfur analogues. As described,33 replacement of the
ring oxygen of 3,1-benzoxazin-4-ones by sulfur affected
both the intrinsic stability and the parameters of the
chymotrypsin inhibition. Resonance stabilization was
assumed to account for the enhanced stability of the
resulting 3,1-benzothiazin-4-ones. However, benzothi-
azinones were found to acylate chymotrypsin, but with
much lower rate constants. Toward complement C1r
protease, the oxygen-sulfur exchange in benzenesulfona-
mide-derived 3,1-benzoxazin-4-ones led to inactive com-
pounds.29 HLE inhibition with micromolar Ki values was
reported for some 2-amino-substituted indolo[1,3]thiazin-
4-ones.46
Con clu sion s
A series of thieno[1,3]oxazin-4-ones was prepared and
evaluated in vitro as inhibitors of human leukocyte
elastase. The present study demonstrates the versatility
of the Gewald thiophene synthesis to provide ethyl
2-aminothiophene-3-carboxylates as precursors to thieno-
[2,3-d][1,3]oxazin-4-ones with various substituents at
positions 5 and 6. On the basis of the inhibitory efficacy
of 3,1-benzoxazin-4-ones toward serine proteases, the
title compounds were designed as a result of an isosteric
benzene-thiophene replacement. Both thieno[2,3-d] and
thieno[3,2-d] fused oxazin-4-ones exhibit remarkable
chemical stability of the oxazinone ring as a result of
the enhanced electron density at the thiophene carbons.
A 2-diethylamino group provided further improvement
of the stability, compared to 2-alkoxy- or 2-(alkylthio)-
thieno[2,3-d][1,3]oxazin-4-ones.36 From the data of the
alkaline hydrolysis at 25 °C, pH 11.25, half-lives at pH
8 can be calculated, being in the range of 2-8 months,
for the most potent inhibitors 52, 54, 57, and 58. The
design of stable compounds is a special challenge in
the development of covalently reacting inhibitors for
serine proteases. It has been addressed by many
authors7a,10a,11a,15c,47 that hydrolytic stability is a pre-
requisite for stability in biological fluids.
Substitutions at position 5 and 6 of the parent thieno-
[2,3-d][1,3]oxazin-4-one 50 provided inhibitors with
lower Ki values. Remarkably, replacement of the 5-
methyl group by isopropyl (51 versus 52) strongly
increased the acylation rate. A comparable effect has
been reported in a series of highly potent benzisothia-
zolones:15a,b An isopropyl group in the peri position to
the carbonyl of the scissile lactam bond led to rapid
inactivation and a hydrophobic interaction with the S1
pocket of HLE was proposed. The tetramethylene
derivative 5836 was already part of a previous series of
thieno compounds investigated as HLE inhibitors. The
potency of 58 was retained by removing one methylene
unit from the cycloaliphatic chain (58 versus 54), and
was slightly diminished by extending to the pentam-
ethylene bridge (58 versus 55). Introduction of a 5-m-
ethyl group was disadvantageous and resulted in a 40-
fold decelaration of the acylation step [58 versus (R,S)-
56]. This might be attributed to a restricted confor-
mation of the methyl group relative to the thieno-
[2,3-d][1,3]oxazinone skeleton. When the C(7)-C(8)
ethylene unit was removed to allow for a free rota-
tion of the isopropyl group around the C-CH bond, a
150-fold increased kon value was observed (56 versus
52). Annellation of a benzene ring in 57 provided the
most potent inhibitor of the present series. Formally,
in this compound, a thiophene unit is placed between
the two rings of the benzoxazinone 59. Introduction of
a carbamoyl moiety at the 6-position in 53 resulted in
an increased affinity of 53 to HLE as detected by an
accumulation of a preassociation complex (Kd ) 620
nM). However, although influenced by Kd,36 the kon
value of 53 was in the range obtained for the po-
tent compounds of the present series. Both acylation
and deacylation rates might also be increased due to
the electron-withdrawing effect of the carbamoyl sub-
stituent.
In summary, 2-(diethylamino)thieno[1,3]oxazin-4-
ones represent an attractive new class of acyl-enzyme
inhibitors for human leukocyte elastase. Their acylation
rates were moderate, but the acyl-enzyme intermediates
were stable with half-lives of 1-6 h at 25 °C, pH 7.8,
for the most potent compounds 52, 54, 57, and 58. Con-
sequently, the consumption of a given inhibitor concen-
tration by the enzyme will occur slowly. This appears
as a favorable feature for acyl-enzyme inhibitors.
The strategy of the benzene-thiophene replacement
was also followed to develop thiophene-derived inhibi-
tors for viral serine proteases. Thieno[1,3]oxazin-4-ones
with -CH(Me)NHCOR substitution at position 2 were
found to strongly inhibit HSV-1, HSV-2, VZV, and CMV
herpes proteases.48 Thus, the thieno[1,3]oxazin-4-one
systems are suitable scaffolds that might find further
applicability for the design of nonpeptidic inhibitors of
other serine proteases.

5442 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Gu¨tschow et al.
Eth yl 2-Am in o-5-ca r ba m oyl-4-m eth ylth iop h en e-3-ca r -
boxyla te (13). A mixture of sulfur (2.24 g, 70 mmol), aceto-
acetamide (7.08 g, 70 mmol), ethyl cyanoacetate (7.9 g, 70
mmol), and EtOH (20 mL) was treated dropwise with mor-
pholine (6.1 g, 70 mmol) at 45 °C over 15 min. After being
stirred for 5 h at 45 °C, the precipitate was collected by
filtration. The filtrate was diluted with H2O to obtain an
additional amount of product. The combined product was
washed with 50% EtOH, dried, and recrystallized from ethyl
acetate to obtain 13 (11 g, 69%): mp 181-182 °C; IR (KBr,
Exp er im en ta l Section
Gen er a l Meth od s a n d Ma ter ia ls. Melting points were
determined on a Boetius apparatus and are not corrected.Thin-
layer chromatography was performed on Merck aluminum
sheets, silica gel 60 F254. Preparative column chromatography
was performed on silica gel 60 (Merck) 70-230 mesh, with an
ethyl acetate/hexane mixture (1:4). 13C NMR spectra (75 MHz)
and 1H NMR spectra (300 MHz) were recorded on a Varian
Gemini 300. 13C NMR signals were assigned on the basis of
13C/1H correlation experiments. IR spectra were measured with
a Perkin-Elmer 16 PC FTIR spectrometer. UV spectra were
recorded on a Shimadzu UV-vis spectrophotometer UV-160A.
Mass spectra (70 eV) were obtained on a Varian MAT CH6
spectrometer. Spectrophotometric assays were done on a
Perkin-Elmer Lambda 16 UV/VIS spectrophotometer. Elastase
was prepared from human leukocytes and purified by affinity
chromatography using an immobilized synthetic inhibitor.49
The substrate Suc-Ala-Ala-Pro-Val-pNA was from Bachem,
Heidelberg, Germany.
1
cm-1) 1652, 1638 (CdO); H NMR (DMSO-d6) δ 1.27 (t, J )
7.1 Hz, 3H), 2.47 (s, 3H), 4.20 (q, J ) 7.1 Hz, 2H), 7.06 (s,
2H), 7.63 (s, 2H). Anal. (C9H12N2O3S) C, H, N; S, calcd, 14.05;
found, 14.68.
(R,S)-Eth yl 2-Am in o-4-m eth yl-4,5,6,7-tetr a h yd r oben zo-
[b]th iop h en e-3-ca r boxyla te (16). A mixture of sulfur (6.4
g, 200 mmol), (R,S)-2-methylcyclohexanone (22.4 g, 200 mmol),
ethyl cyanoacetate (22.6 g, 200 mmol), and EtOH (57 mL) was
treated dropwise with morpholine (17.4 g, 200 mmol) at 45 °C
over 15 min. The mixture was stirred for 5 h at 45 °C and 24
h at room temperature. Unreacted sulfur was removed by
filtration, and the filtrate was evaporated under reduced
pressure. Compound 16 (20.5 g, 43%) was obtained by column
chromatography: mp 68-70 °C (MeOH) (lit.52 mp 73 °C); IR
Methyl 3-aminothiophene-2-carboxylate (2) was purchased
from Aldrich, Steinheim, Germany. 2-(Diethylamino)-5,6,7,8-
tetrahydro-4H-[1]benzothieno[2,3-d][1,3]oxazin-4-one (58) was
prepared as reported.36 2-(Diethylamino)-4H-3,1-benzoxazin-
4-one (59)19 was prepared as reported.37
1
(KBr, cm-1) 1643 (CdO); H NMR (CDCl3) δ 1.16 (d, J ) 6.8
Eth yl 2-Am in o-4-m eth yl-5-(ter t-bu toxyca r bon yl)th io-
p h en e-3-ca r boxyla te (8). A mixture of tert-butyl acetoacetate
(15.8 g, 100 mmol), ethyl cyanoacetate (11.3 g, 100 mmol),
sulfur (3.5 g, 110 mmol), and EtOH (25 mL) was stirred at 45
°C. Morpholine (10 g, 115 mmol) was added dropwise over 15
min. The mixture was stirred at 60 °C for 5 h and filtered.
The filtrate was diluted with water (50 mL) and cooled. The
precipitate was collected by filtration, washed with 30% EtOH,
and dried to obtain 8 (21.2 g, 74%): mp 116-117 °C (cyclo-
Hz, 3H), 1.35 (t, J ) 7.1 Hz, 3H), 1.58-1.92 (m, 4H), 2.46-
2.52 (m, 2H), 3.20-3.30 (m, 1H), 4.18-4.38 (m, 2H), 5.99 (s,
2H).
Eth yl 2-(Isoth iocya n a to)th iop h en e-3-ca r boxyla te (18):
Gen er a l P r oced u r e for Isoth iocya n a tes 18-25. A mixture
prepared from thiophosgene (4.6 g, 40 mmol), CaCO3 (4 g, 40
mmol), CH2Cl2 (20 mL), and H2O (40 mL) was stirred at 0 °C.
A solution of compound 10 (6.84 g, 40 mmol) in CH2Cl2 (70
mL) was added dropwise over a period of 40 min. The mixture
was stirred for additional 3 h at 0 °C. The organic layer was
washed with water and dried (Na2SO4). After removal of the
solvent, the residue was triturated with silica gel (2 g) and
boiling hexane (2 × 125 mL). The combined filtrates were
concentrated to 50 mL and cooled. The precipitate was
collected by filtration to obtain 18 (2.5 g, 29%): mp 40-42 °C
(hexane) (lit.53 mp 43-44 °C); IR (KBr, cm-1) 2143 (NCS), 1699
(CdO); 1H NMR (CDCl3) δ 1.40 (t, J ) 7.1 Hz, 3H), 4.37 (q, J
) 7.1 Hz, 2H), 6.92 (d, J ) 5.9 Hz, 1H), 7.29 (d, J ) 5.5 Hz,
1H).
1
hexane/hexane); IR (KBr, cm-1) 1670, 1588 (CdO); H NMR
(CDCl3) δ 1.37 (t, J ) 7.1 Hz, 3H), 1.53 (s, 9H), 2.67 (s, 3H),
4.31 (q, J ) 7.1 Hz, 2H), 6.47 (s, 2H). Anal. (C13H19NO4S) C,
H, N, S.
Eth yl 2-Am in oth iop h en e-3-ca r boxyla te (10). Triethyl-
amine (3.6 g, 50 mmol) was added dropwise over 10 min to a
mixture of 2,5-dihydroxy-1,4-dithiane (7.6 g, 50 mmol), ethyl
cyanoacetate (11.3 g, 100 mmol), and DMF (40 mL). The
mixture was stirred at 45 °C for 30 min, diluted with 0.4 M
acetic acid (200 mL), extracted with ether (4 × 40 mL), washed
with water (2 × 40 mL), and dried (Na2SO4). After removal of
the solvent, the residue was cooled and washed with cold
hexane to obtain 10 (13.6 g, 80%): mp 47-48 °C (ethyl acetate/
Eth yl 2-(Isoth iocya n a to)-4-m eth ylth iop h en e-3-ca r box-
yla te (19). Yield 22%; mp 50.5-51 °C; IR (KBr, cm-1) 2126
1
(NCS), 1694 (CdO); H NMR (CDCl3) δ 1.42 (t, J ) 7.1 Hz,
1
hexane) (lit.50 mp 46-48 °C); H NMR (CDCl3) δ 1.33 (t, J )
3H), 2.38 (s, 3H), 4.38 (q, J ) 7.1 Hz, 2H), 6.59 (s, 1H). Anal.
7.1 Hz, 3H), 4.27 (q, J ) 7.1 Hz, 2H), 6.17 (d, J ) 5.8 Hz, 1H),
6.70 (d, J ) 5.8 Hz, 1H), 5.86 (br s, 2H); 13C NMR (CDCl3) δ
14.53, 59.77, 106.91, 107.17, 125.93, 162.74, 165.50.
(C9H9NO2S2) C, H, N, S.
Eth yl 2-(Isoth iocya n a to)-4-isop r op ylth iop h en e-3-ca r -
boxyla te (20). Compound 20 was prepared from 12. The
combined filtrates were evaporated under reduced pressure.
The residue was purified by column chromatography to give
20 as an oil (1.53 g, 15%): IR (KBr, cm-1) 2113 (NCS), 1716
(CdO); 1H NMR (CDCl3) δ 1.21 (d, J ) 6.8 Hz, 6H), 1.43 (t, J
) 7.1 Hz, 3H), 3.50 (septet, J ) 6.8 Hz, 1H), 4.34 (q, J ) 7.1
Hz, 2H), 6.66 (s, 1H).
Eth yl 2-Am in o-4-m eth ylth iop h en e-3-ca r boxyla te (11).
A mixture of compound 8 (14.3 g, 50 mmol) and EtOH (100
mL) was treated with 1 M HCl (100 mL). It was refluxed for
1 h, cooled, and neutralized with 1 M NaOH. The mixture was
cooled for 48 h and the precipitate was collected by filtration
to obtain 11 (7.9 g, 85%): mp 76-78 °C (hexane); IR (KBr,
cm-1) 1646 (CdO); 1H NMR (CDCl3) δ 1.36 (t, J ) 7.1 Hz, 3H),
2.29 (s, 3H), 4.30 (q, J ) 7.1 Hz, 2H), 5.84 (s, 1H), 6.02 (s,
2H). Anal. (C8H11NO2S) H, N, S; C: calcd, 51.87; found, 51.45.
Eth yl 2-Am in o-4-isopr opylth ioph en e-3-car boxylate (12).
A mixture of (E,Z)-2-cyano-3,4-dimethyl-2-pentenoic acid ethyl
ester 9,51 (18.1 g, 100 mmol), sulfur (3.2 g, 100 mmol), and
EtOH (25 mL) was stirred at 45 °C. Morpholine (8.7 g, 100
mmol) was added dropwise over 15 min. The mixture was
stirred at 45 °C for 4 h, cooled, poured into 0.4 M acetic acid
(400 mL), extracted with ether (4 × 60 mL), washed with water
(2 × 60 mL), dried (Na2SO4), and evaporated under reduced
pressure. The residue was purified by column chromatography
to obtain 12 (7.9 g, 37%): mp 54-55 °C (cyclohexane); IR (KBr,
cm-1) 1650 (br, CdO); 1H NMR (CDCl3) δ 1.19 (d, J ) 6.8 Hz,
6H), 1.36 (t, J ) 7.1 Hz, 3H), 3.42 (septet, J ) 6.8 Hz, 1H),
4.31 (q, J ) 7.1 Hz, 2H), 5.89 (s, 1H), 6.06 (br s, 2H). Anal.
(C10H15NO2S) C, H, N, S.
Eth yl 5-Ca r ba m oyl-2-(isoth iocya n a to)-4-m eth ylth io-
p h en e-3-ca r boxyla te (21). Compound 13 was reacted with
thiophosgene. The reaction mixture was stirred for 3 h at 0
°C and for an additional 48 h at room temperature. The
precipitate was collected by filtration and thoroughly washed
with H2O to obtain 21 in 55% yield: mp 171-173 °C (CH3-
CN); IR (KBr, cm-1) 2122 (NCS), 1700, 1668 (CdO); 1H NMR
(DMSO-d6) δ 1.34 (t, J ) 7.1 Hz, 3H), 2.49 (s, 3H), 4.31 (q, J
) 7.1 Hz, 2H), 7.72 (s, 2H). Anal. (C10H10N2O3S2) C, H, N, S.
Eth yl 5,6-Dih yd r o-2-(isoth iocya n a to)-4H-cyclop en ta -
[4,5]th iop h en e-3-ca r boxyla te (22). Compound 22 was pre-
pared from 1439 in 15% yield: mp 55-56 °C; IR (KBr, cm-1
)
1
2102 (NCS), 1710 (CdO); H NMR (CDCl3) δ 1.39 (t, J ) 7.1
Hz, 3H), 2.32-2.44 (m, 2H), 2.81-2.96 (m, 4H) 4.34 (q, J )
7.1 Hz, 2H). Anal. (C11H11NO2S2) C, H, N; S: calcd, 25.31;
found, 24.24.

Thieno[1,3]oxazin-4-ones as HLE Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5443
Eth yl 2-(Isoth iocya n a to)-5,6,7,8-tetr a h yd r o-4H-cyclo-
h ep ta [4,5]th iop h en e-3-ca r boxyla te (23). Compound 23 was
prepared from 15.39 The combined filtrates were evaporated
under reduced pressure. The residue was purified by column
chromatography to give 23 in 35% yield: mp 49-50 °C
2.99-3.05 (m, 2H), 3.80 (q, J ) 7.1 Hz, 4H), 4.34 (q, J ) 7.1
Hz, 2H), 12.21 (s, 1H). Anal. (C17H26N2O2S2) C, H, N, S.
(R,S)-Eth yl 2-(3,3-Dieth ylth iou r eid o)-4-m eth yl-4,5,6,7-
tetr a h yd r oben zo[b]th iop h en e-3-ca r boxyla te (32). Yield
81%; mp 72-73 °C; IR (KBr, cm-1) 1643 (CdO); 1H NMR
(CDCl3) δ 1.16 (d, J ) 6.6 Hz, 3H), 1.31 (t, J ) 7.1 Hz, 6H),
1.39 (t, J ) 7.1 Hz, 3H), 1.62-1.92 (m, 4H), 2.50-2.72 (m, 2H),
3.28-3.39 (m, 1H), 3.81 (q, J ) 7.1 Hz, 4H), 4.25-4.44 (m,
2H), 12.38 (s, 1H). Anal. (C17H26N2O2S2) C, H, N, S.
1
(hexane); IR (KBr, cm-1) 2132 (NCS), 1708 (CdO); H NMR
(CDCl3) δ 1.42 (t, J ) 7.1 Hz, 3H), 1.56-1.67 (m, 4H), 1.81-
1.91 (m, 2H), 2.69-2.74 (m, 2H), 2.93-2.98 (m, 2H), 4.36 (q,
J ) 7.1 Hz, 2H). Anal. (C13H15NO2S2) C, H, N; S, calcd, 22.79;
found, 21.95.
(R,S)-Eth yl 2-(Isoth iocyan ato)-4-m eth yl-4,5,6,7-tetr ah y-
d r oben zo[b]th iop h en e-3-ca r boxyla te (24). Compound 24
was prepared from 16 and purified by column chromatography
to obtain an oil in 37% yield; IR (KBr, cm-1) 2121 (NCS), 1716
(CdO); 1H NMR (CDCl3) δ 1.15 (d, J ) 6.9 Hz, 3H), 1.41 (t, J
) 7.1 Hz, 3H), 1.62-1.92 (m, 4H), 2.55-2.72 (m, 2H), 3.32-
3.42 (m, 1H), 4.32-4.40 (m, 2H).
Eth yl 2-(Isoth iocya n a to)ben zo[b]th iop h en e-3-ca r box-
yla te (25). Compound 1741 dissolved in CH2Cl2 (100 mL) was
reacted with thiophosgene to obtain 25 in 45% yield. An
analytical sample was purified by column chromatography:
mp 70-71 °C; IR (KBr, cm-1) 2130 (NCS), 1706 (CdO); 1H
NMR (CDCl3) δ 1.49 (t, J ) 7.1 Hz, 3H), 4.49 (q, J ) 7.1 Hz,
2H), 7.41-7.49 (m, 2H), 7.66-7.70 and 8.44-8.49 (each m,
1H). Anal. (C12H9NO2S2) C, H, N, S.
E t h yl 2-(3,3-Diet h ylt h iou r eid o)ben zo[b]t h iop h en e-3-
ca r boxyla te (33). Yield 96%; mp 137-138 °C (EtOH); IR
1
(KBr, cm-1) 1654 (CdO); H NMR (CDCl3) δ 1.37 (t, J ) 7.1
Hz, 6H), 1.52 (t, J ) 7.1 Hz, 3H), 3.80-3.95 (m, 4H), 4.50 (q,
J ) 7.1 Hz, 2H), 7.24-7.31 and 7.36-7.42 (each m, 1H), 7.70-
7.75 and 8.23-8.28 (each m, 1H), 12.80 (s, 1H). Anal.
(C16H20N2O2S2) C, H, N; S, calcd, 19.06; found, 18.45.
2-(Dieth ylam in o)-4H-th ien o[3,2-d][1,3]th iazin -4-on e (5):
Gen er a l P r oced u r e for Th ien o[1,3]th ia zin -4-on es 5, 37,
39, a n d 40. A mixture of compound 4 (4.1 g, 15 mmol) and
concentrated H2SO4 (30 mL) was kept at room temperature
for 3 days and poured into ice-water (800 mL). The precipitate
was collected by filtration, washed with H2O, and dried to give
5 (3.2 g, 89%): mp 76-78 °C; IR (KBr, cm-1) 1648 (CdO); 1H
NMR (CDCl3) δ 1.26 (t, J ) 7.1 Hz, 6H), 3.62 (q, J ) 7.1 Hz,
4H), 7.07 (d, J ) 5.3 Hz, 1H), 7.72 (d, J ) 5.3 Hz, 1H). Anal.
(C10H12N2OS2) C, H, N, S.
Meth yl 3-(3,3-Dieth ylth iou r eid o)th iop h en e-2-ca r boxy-
la te (4): Gen er a l P r oced u r e for Th iou r ea s 4 a n d 26-33.
Diethylamine (2.55 g, 22.5 mmol) was added dropwise to a
solution of compound 354 (2.36 g, 15 mmol) in CH2Cl2 (15 mL).
The mixture was stirred at room temperature for 5 h and
acidified with 1 M HCl/EtOH, and the precipitate was collected
by filtration to yield 4 (4.0 g, 98%): mp 81-82 °C (EtOH/H2O);
6-Ca r ba m oyl-2-(dieth yla m in o)-5-m eth yl-4H-th ien o[2,3-
d ][1,3]th ia zin -4-on e (37). Yield 89%; mp 193-194 °C (EtOH);
1
IR (KBr, cm-1) 1660, 1654 (CdO); H NMR (CDCl3) δ 1.27 (t,
J ) 7.1 Hz, 6H), 2.78 (s, 3H), 3.55-3.65 (m, 4H), 5.65 (br s,
2H). Anal. (C12H15N3O2S2) C, H, N, S.
2-(Dieth yla m in o)-6,7,8,9-tetr a h ydr o-4H,5H-cycloh ep ta -
[4,5]th ien o[2,3-d ][1,3]th ia zin -4-on e (39). Yield 99%; mp
128-129 °C (EtOH); IR (KBr, cm-1) 1652 (CdO); 1H NMR
(CDCl3) δ 1.23 (t, J ) 7.1 Hz, 6H), 1.60-1.73 (m, 4H), 1.82-
1.91 (m, 2H), 2.70-2.74 (m, 2H), 3.17-3.21 (m, 2H), 3.56 (q,
J ) 7.1 Hz, 4H). Anal. (C15H20N2OS2) C, H, N; S, calcd, 20.79;
found, 20.12.
(R,S)-2-(Dieth yla m in o)-5-m eth yl-5,6,7,8-tetr a h yd r o-4H-
[1]ben zoth ien o[2,3-d ][1,3]th ia zin -4-on e (40). Yield 90%;
mp 97.5-98 °C (EtOH); IR (KBr, cm-1) 1636 (CdO); 1H NMR
(CDCl3) δ 1.22 (d, J ) 6.8 Hz, 3H), 1.24 (t, J ) 7.1 Hz, 6H),
1.62-1.72 (m, 4H), 2.54-2.70 (m, 2H), 3.26-3.38 (m, 1H),
3.53-3.62 (m, 4H). Anal. (C15H20N2OS2) C, H, N, S.
1
IR (KBr, cm-1) 1678 (CdO); H NMR (CDCl3) δ 1.36 (t, J )
7.1 Hz, 6H), 3.84 (q, J ) 7.1 Hz, 4H), 3.88 (s, 3H), 7.43 (d, J )
5.6 Hz, 1H), 8.84 (d, J ) 5.6 Hz, 1H), 10.55 (s, 1H). Anal.
(C11H16N2O2S2) C, H, N, S.
Eth yl 2-(3,3-Dieth ylth iou r eid o)th iop h en e-3-ca r boxy-
la te (26). Yield 70%; mp 80.5-82.5 °C (hexane); IR (KBr, cm-1
)
1667 (CdO); 1H NMR (CDCl3) δ 1.34 (t, J ) 7.1 Hz, 6H), 1.36
(t, J ) 7.1 Hz, 3H), 3.83 (q, J ) 7.1 Hz, 4H), 4.34 (q, J ) 7.1
Hz, 2H), 6.59 (d, J ) 5.9 Hz, 1H), 7.22 (d, J ) 5.9 Hz, 1H),
11.95 (s, 1H). Anal. (C12H18N2O2S2) C, H, N, S.
Eth yl 2-(3,3-Dieth ylth iou r eid o)-4-m eth ylth iop h en e-3-
ca r boxyla te (27). Yield 90%; mp 114-116 °C; IR (KBr, cm-1
)
1657 (CdO); 1H NMR (CDCl3) δ 1.33 (t, J ) 7.1 Hz, 6H), 1.40
(t, J ) 7.1 Hz, 3H), 2.36 (s, 3H), 3.82 (q, J ) 7.1 Hz, 4H), 4.36
(q, J ) 7.1 Hz, 2H), 6.26 (s, 1H), 12.32 (s, 1H). Anal.
(C13H20N2O2S2) C, H, N, S.
2-(Dieth ylam in o)-4H-th ien o[2,3-d][1,3]th iazin -4-on e (34):
Gen er a l P r oced u r e for Th ien o[2,3-d ][1,3]th ia zin -4-on es
34, 35, 36, 38, a n d 41. A mixture of compound 26 (2.86 g, 10
mmol) and polyphosphoric acid (100 g) was stirred at 170 °C
for 20 min. It was then poured into a mixture of ice (200 g)
and EtOH (200 mL) and stirred to obtain a solid precipitate,
which was filtered off. The precipitate was thoroughly washed
with H2O and dried to give 34 (2.1 g, 87%): mp 78-82 °C; IR
Eth yl 2-(3,3-Dieth ylth iou r eid o)-4-isop r op ylth iop h en e-
3-ca r boxyla te (28). Yield 94%; mp 67.5-69.5 °C; IR (KBr,
cm-1) 1653 (CdO); 1H NMR (CDCl3) δ 1.21 (d, J ) 6.8 Hz,
6H), 1.33 (t, J ) 7.1 Hz, 6H), 1.40 (t, J ) 7.1 Hz, 3H), 3.49
(septet, J ) 6.8 Hz, 1H), 3.82 (q, J ) 7.1 Hz, 4H), 4.38 (q, J )
7.1 Hz, 2H), 6.32 (s, 1H), 12.43 (s, 1H). Anal. (C15H24N2O2S2)
C, H, N, S.
1
(KBr, cm-1) 1653 (CdO); H NMR (CDCl3) δ 1.27 (t, J ) 7.1
Hz, 6H), 3.61 (q, J ) 7.1 Hz, 4H), 6.77 (d, J ) 6.0 Hz, 1H),
7.25 (d, J ) 6.0 Hz, 1H). Anal. (C10H12N2OS2) C, H, N, S.
Eth yl 5-Ca r ba m oyl-2-(3,3-d ieth ylth iou r eid o)-4-m eth -
ylth iop h en e-3-ca r boxyla te (29). Yield 80%; mp 206-207 °C
2-(Dieth ylam in o)-5-m eth yl-4H-th ien o[2,3-d][1,3]th iazin -
4-on e (35). Yield 79%; mp 94-95 °C; IR (KBr, cm-1) 1659 (Cd
1
(EtOH); IR (KBr, cm-1) 1660 (br, CdO); H NMR (CDCl3) δ
1
O); H NMR (CDCl3) δ 1.25 (t, J ) 7.1 Hz, 6H), 2.45 (s, 3H),
1.33 (t, J ) 7.1 Hz, 6H), 1.41 (t, J ) 7.1 Hz, 3H), 2.70 (s, 3H),
3.77-3.85 (m, 4H), 4.38 (q, J ) 7.1 Hz, 2H), 5.66 (s, 2H), 12.55
(s, 1H); MS (EI) m/z (rel intensity) 343 (53, M+), 116 (100).
Anal. (C14H21N3O3S2) H, N, S; C, calcd, 48.96; found, 48.35.
3.59 (q, J ) 7.1 Hz, 4H), 6.34 (s, 1H). Anal. (C11H14N2OS2‚
0.5H2O) C, H, N, S.
2-(Die t h yla m in o)-5-isop r op yl-4H -t h ie n o[2,3-d ][1,3]-
th ia zin -4-on e (36). Yield 86%; mp 71-73 °C (EtOH); IR (KBr,
cm-1) 1659 (CdO); 1H NMR (CDCl3) δ 1.23 (d, J ) 6.8 Hz,
6H), 1.25 (t, J ) 7.2 Hz, 6H), 3.50-3.65 (m, 5H), 6.44 (s, 1H).
Anal. (C13H18N2OS2) C, H, N, S.
E t h yl
2-(3,3-Die t h ylt h iou r e id o)-5,6-d ih yd r o-4H -
cyclop en ta [4,5]th iop h en e-3-ca r boxyla te (30). Yield 92%;
mp 93-94 °C; IR (KBr, cm-1) 1662 (CdO); 1H NMR (CDCl3) δ
1.33 (t, J ) 7.1 Hz, 6H), 1.36 (t, J ) 7.1 Hz, 3H), 2.28-2.41
(m, 2H), 2.78-2.93 (m, 4H), 3.82 (q, J ) 7.1 Hz, 4H), 4.31 (q,
J ) 7.1 Hz, 2H), 11.92 (s, 1H). Anal. (C15H22N2O2S2) H, N, S;
C: calcd, 55.19; found, 54.31.
2-(Diet h yla m in o)-6,7-d ih yd r o-4H,5H-cyclop en t a [4,5]-
th ien o[2,3-d ][1,3]th ia zin -4-on e (38). Compound 38 was
prepared from 30. The crude product was recrystallized from
EtOH with charcoal to give 38 in 33% yield: mp 147-149 °C;
Eth yl 2-(3,3-Dieth ylth iou r eid o)-5,6,7,8-tetr a h yd r o-4H-
cycloh ep ta [4,5]th iop h en e-3-ca r boxyla te (31). Yield 86%;
1
IR (KBr, cm-1) 1662 (CdO); H NMR (CDCl3) δ 1.25 (t, J )
1
mp 93-95 °C (EtOH); IR (KBr, cm-1) 1650 (CdO); H NMR
7.1 Hz, 6H), 2.33-2.44 (m, 2H), 2.80-3.05 (m, 4H), 3.58 (q, J
) 7.1 Hz, 4H). Anal. (C13H16N2OS2) H, N, S; C: calcd, 55.69;
found, 55.10.
(CDCl3) δ 1.31 (t, J ) 7.1 Hz, 6H), 1.36 (t, J ) 7.1 Hz, 3H),
1.57-1.70 (m, 4H), 1.78-1.88 (m, 2H), 2.68-2.73 (m, 2H),

5444 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
2-(Dieth ylam in o)-4H-[1]ben zoth ien o[2,3-d][1,3]th iazin -
Gu¨tschow et al.
The crude product was partitioned between ethyl acetate (100
mL) and 0.25 M NaOH (200 mL). The aqueous layer was cooled
and acidified, and the precipitate was filtered off to give 49 in
4-on e (41). Yield 86%; mp 123-125 °C (EtOH); IR (KBr, cm-1
)
1654 (CdO); 1H NMR (CDCl3) δ 1.27 (t, J ) 7.1 Hz, 6H), 3.54-
3.68 (m, 4H), 7.28-7.35 and 7.39-7.45 (each m, 1H), 7.65-
7.69 and 8.43-8.46 (each m, 1H); 13C NMR (CDCl3) δ 12.74,
44.42, 107.73, 121.35, 123.82, 124.78, 125.90, 131.41, 134.32,
163.37, 173.58, 174.15. Anal. (C14H14N2OS2) H, N, S; C: calcd,
57.90; found, 57.37.
1
15% yield: mp 142-144 °C; IR (KBr, cm-1) 1630 (CdO); H
NMR (DMSO-d6) δ 1.26 (t, J ) 6.8 Hz, 6H), 3.70-3.90 (m, 4H),
7.22-7.29 and 7.30-7.46 (each m, 1H), 7.82-7.90 and 8.21-
8.30 (each m, 1H), 12.13 (s, 1H). Anal. (C14H16N2O2S2) C, H,
N, S.
3-(3,3-Dieth ylth iou r eid o)th iop h en e-2-ca r boxylic Acid
(6): Gen er a l P r oced u r e for Th iop h en eca r boxylic Acid s
6 a n d 42-49. A mixture of compound 5 (2.4 g, 10 mmol), 3 M
NaOH (40 mL), and dioxane (80 mL) was stirred and refluxed
for 80 min. After cooling, it was poured onto ice-water (133
mL). Insoluble material was removed by filtration, and 3 M
HCl (67 mL) was added at 0 °C to the stirred filtrate. The
precipitate was collected by filtration to yield 6 (2.2 g, 85%):
mp 119-128 °C; IR (KBr, cm-1) 1640 (CdO); 1H NMR (CDCl3)
δ 1.36 (t, J ) 7.1 Hz, 6H), 3.84 (q, J ) 7.1 Hz, 4H), 7.56 (d, J
) 5.6 Hz, 1H), 8.90 (d, J ) 5.6 Hz, 1H), 10.42 (s, 1H). Anal.
(C10H14N2O2S2) C, H, N; S: calcd, 24.82; found, 24.31.
2-(3,3-Dieth ylth iou r eid o)th iop h en e-3-ca r boxylic Acid
(42). Yield 87%; mp 142-147 °C (EtOH/hexane); IR (KBr,
cm-1) 1637 (CdO); 1H NMR (CDCl3) δ 1.36 (t, J ) 7.1 Hz, 6H),
3.83 (q, J ) 7.1 Hz, 4H), 6.63 (d, J ) 5.9 Hz, 1H), 7.27 (d, J )
5.9 Hz, 1H), 11.74 (s, 1H). Anal. (C10H14N2O2S2) C, H, N, S.
2-(Dieth ylam in o)-4H-th ien o[3,2-d][1,3]oxazin -4-on e (7):
Gen er a l P r oced u r e for Th ien o[1,3]oxa zin -4-on es 7 a n d
50-57. A mixture of compound 6 (646 mg, 2.5 mmol), yellow
HgO (866 mg, 4 mmol), and CH2Cl2 (50 mL) was stirred at
room temperature for 48 h. Silica gel (1.8 g) was added and
the mixture was stirred for 2 min. The inorganic material was
filtered from the solution and washed with CH2Cl2 (25 mL).
The solvent was removed in vacuo to yield 7 (480 mg, 86%):
mp 36-37 °C (hexane); IR (KBr, cm-1) 1759 (br, CdO); UV
(EtOH) λmax (nm) (log ꢀ) 239 (4.42), 340 (4.03); 1H NMR (CDCl3)
δ 1.23 (t, J ) 7.1 Hz, 6H, CH3), 3.54 (q, J ) 7.1 Hz, 4H, CH2),
6.93 (d, J ) 5.2 Hz, 1H, H-7), 7.72 (d, J ) 5.2 Hz, 1H, H-6);
13C NMR (CDCl3) δ 13.26 (CH3), 42.60 (CH2), 106.39 (C-4a),
123.62 (C-7), 137.28 (C-6), 155.79 and 157.19 (C-2 and C-7a),
161.96 (C-4); MS (EI) m/z (rel intensity) 224 (68, M+), 152 (100).
Anal. (C10H12N2O2S) C, H, N, S.
2-(Dieth ylam in o)-4H-th ien o[2,3-d][1,3]oxazin -4-on e (50).
Yield 73%; mp 77-79 °C (hexane); IR (KBr, cm-1) 1744 (Cd
O); UV (EtOH) λmax (nm) (log ꢀ) 232 (4.31), 266 (3.85), 330
2-(3,3-Dieth ylth iou r eido)-4-m eth ylth ioph en e-3-car box-
ylic Acid (43). Yield 88%; mp 146-148 °C; IR (KBr, cm-1
)
1632 (CdO); 1H NMR (CDCl3) δ 1.34 (t, J ) 7.1 Hz, 6H), 2.39
(s, 3H), 3.82 (q, J ) 7.1 Hz, 4H), 6.29 (s, 1H), 12.13 (s, 1H).
Anal. (C11H16N2O2S2) C, H, N, S.
(3.94); H NMR (CDCl3) δ 1.25 (t, J ) 7.0 Hz, 6H, CH3), 3.56
1
(q, J ) 7.0 Hz, 4H, CH2), 6.72 (d, J ) 5.9 Hz, 1H, H-6), 7.18
(d, J ) 5.9 Hz, 1H, H-5); 13C NMR (CDCl3) δ 13.21, 13.26 (CH3),
42.58 (CH2), 109.34 (C-4a), 116.49 (C-6), 122.22 (C-5), 155.65
and 155.81 (C-2 and C-7a), 171.19 (C-4); MS (EI) m/z (rel
intensity) 224 (M+, 82), 152 (100). Anal. (C10H12N2O2S) C, H,
N, S.
2-(3,3-Dieth ylth iou r eid o)-4-isop r op ylth iop h en e-3-ca r -
boxylic Acid (44). Compound 36 was refluxed in NaOH/
dioxane for 120 min. The crude product was partitioned
between ethyl acetate (100 mL) and 0.25 M NaOH (200 mL).
The aqueous layer was cooled and acidified, and the precipitate
was filtered off to give 44 in 30% yield: mp 142-145 °C; IR
(KBr, cm-1) 1631 (CdO); 1H NMR (CDCl3) δ 1.22-1.37 (m,
12H), 3.40-3.60 (m, 1H), 3.70-3.90 (m, 4H), 6.36 (s, 1H), 12.29
(s, 1H); MS (EI) m/z (rel intensity) 300 (38, M+), 117 (100).
Anal. (C13H20N2O2S2‚H2O) C, H, N, S.
2-(Dieth ylam in o)-5-m eth yl-4H-th ien o[2,3-d][1,3]oxazin -
4-on e (51). Yield 77%; mp 102-103 °C (hexane); IR (KBr,
cm-1) 1752 (CdO); UV (EtOH) λmax (nm) (log ꢀ) 238 (4.34), 268
1
(3.88), 332 (4.03); H NMR (CDCl3) δ 1.24 (t, J ) 7.1 Hz, 6H,
CH3CH2), 2.41 (s, 3H, 5-CH3), 3.55 (q, J ) 7.1 Hz, 4H, CH2),
6.30 (s, 1H, H-6);13C NMR (CDCl3) δ 13.29, 13.23 (CH3CH2),
16.17 (5-CH3), 42.46 (CH2), 108.64 (C-4a), 110.99 (C-6), 134.41
(C-5), 155.69 and 155.86 (C-2 and C-7a), 171.48 (C-4); MS (EI)
m/z (rel intensity) 238 (M+, 74), 166 (100). Anal. (C11H14N2O2S)
C, H, N; S, calcd, 13.46; found, 14.09.
5-Ca r b a m oyl-2-(3,3-d iet h ylt h iou r eid o)-4-m et h ylt h io-
p h en e-3-ca r boxylic Acid (45). A mixture of compound 37
(2.97 g, 10 mmol), 3 M NaOH (80 mL), and dioxane (160 mL)
was refluxed and then acidified with 3 M HCl (134 mL) to
yield 45 (3.1 g, 93%): mp 197-200 °C (EtOH); IR (KBr, cm-1
)
2-(Die t h yla m in o)-5-isop r op yl-4H -t h ie n o[2,3-d ][1,3]-
oxa zin -4-on e (52). Yield 73%; mp 38-40 °C (hexane); IR
(KBr, cm-1) 1743 (CdO); UV (EtOH) λmax (nm) (log ꢀ) 239
1
1647 (CdO); H NMR (DMSO-d6) δ 1.22 (t, J ) 7.0 Hz, 6H),
2.53 (s, 3H), 3.70-3.80 (m, 4H), 7.35 (s, 2H), 12.73 (s, 1H, NH);
MS (EI) m/z (rel intensity) 315 (5, M+), 58 (100). Anal.
(C12H17N3O3S2‚H2O) C, H, N, S; C: calcd, 48.96; found, 48.35.
1
(4.36), 268 (3.85), 333 (4.06); H NMR (CDCl3) δ 1.25 (t, J )
7.1 Hz, 6H, CH3CH2), 1.26 (d, J ) 6.7 Hz, 6H, CH3CH), 3.42
(septet, J ) 6.7 Hz, 1H, CH), 3.55 (q, J ) 7.1 Hz, 4H, CH2),
6.36 (s, 1H, H-6); 13C NMR (CDCl3) δ 13.32 (CH3CH2), 22.64
(CH3CH), 28.88 (CH), 42.38 (CH2), 107.73 (C-4b), 108.31 (C-
6), 146.29 (C-5), 155.14 and 155.74 (C-2 and C-7a), 171.99 (C-
4); MS (EI) m/z (rel intensity) 266 (M+, 100), 194 (41). Anal.
(C13H18N2O2S) C, H, N, S.
2-(3,3-Dieth ylth iou r eid o)-5,6-d ih yd r o-4H-cyclop en ta -
[4,5]th iop h en e-3-ca r boxylic Acid (46). Yield 90%; mp >149
°C (dec, EtOH); IR (KBr, cm-1) 1637 (CdO); 1H NMR (CDCl3)
δ 1.34 (t, J ) 7.1 Hz, 6H), 2.32-2.43 (m, 2H), 2.80-2.98 (m,
4H), 3.81 (q, J ) 7.1 Hz, 4H), 11.76 (s, 1H). Anal. (C13H18N2O2S2)
C, H, N, S.
2-(3,3-Dieth ylth iou r eid o)-5,6,7,8-tetr a h yd r o-4H-cyclo-
h ep ta [4,5]th iop h en e-3-ca r boxylic Acid (47). Compound 39
was refluxed in NaOH/dioxane for 170 min to give 47 in 60%
yield: mp 141-145 °C (EtOH); IR (KBr, cm-1) 1628 (CdO);
1H NMR (CDCl3) δ 1.32 (t, J ) 7.1 Hz, 6H), 1.57-1.70 (m,
4H), 1.80-1.90 (m, 2H), 2.68-2.77 (m, 2H), 3.04-3.10 (m, 2H),
3.80 (q, J ) 7.1 Hz, 4H), 12.09 (s, 1H). Anal. (C15H22N2O2S2)
H, N, S; C: calcd, 55.19; found, 54.60.
(R,S)-2-(3,3-Dieth ylth iou r eido)-4-m eth yl-4,5,6,7-tetr ah y-
d r oben zo[b]th iop h en e-3-ca r boxylic Acid (48). Compound
40 was refluxed in NaOH/dioxane for 4 h to give 48 in 63%
yield: mp 142-145 °C (EtOH); IR (KBr, cm-1) 1620 (CdO);
1H NMR (CDCl3) δ 1.20 (d, J ) 6.7 Hz, 3H), 1.33 (t, J ) 7.1
Hz, 6H), 1.63-1.92 (m, 4H), 2.52-2.74 (m, 2H), 3.30-3.38 (m,
1H), 3.81 (q, J ) 7.1 Hz, 4H), 12.24 (s, 1H). Anal. (C15H22N2O2S2)
C, H, N, S.
6-Ca r ba m oyl-2-(dieth yla m in o)-5-m eth yl-4H-th ien o[2,3-
d ][1,3]oxa zin -4-on e (53). A mixture of compound 45 (834 mg,
2.5 mmol), yellow HgO (1.08 g, 5 mmol), CH2Cl2 (100 mL), and
DMF (5 mL) was stirred and refluxed for 30 h. The mixture
was stirred for an additional 60 h at room temperature. The
crude product was recrystallized from ethyl acetate to give 53
(220 mg, 31%). An analytical sample was obtained after a
second recrystallization from acetone: mp 185-190 °C; IR
(KBr, cm-1) 1750, 1650 (CdO); UV (EtOH) λmax (nm) (log ꢀ)
1
242 (4.26), 331 (4.31); H NMR (CDCl3) δ 1.26 (t, J ) 7.1 Hz,
6H, CH3CH2), 2.75 (s, 3H, 5-CH3), 3.45-3.60 (m, 4H, CH2),
5.79 (br s, 2H, NH2);13C NMR (CDCl3) δ 12.75, 13.63 (CH3-
CH2), 14.90 (5-CH3), 42.20, 43.06 (CH2), 109.97 (C-4a), 121.15
(C-6), 139.33 (C-5), 155.20 and 155.41 (C-2 and C-7a), 164.48
(CdO), 171.51 (C-4); MS (EI) m/z (rel intensity) 281 (M+, 100),
209 (85). Anal. (C12H15N3O3S) C, H, N, S.
2-(3,3-Dieth ylth iou r eid o)ben zo[b]th iop h en e-3-ca r box-
ylic Acid (49). Compound 41 was treated with NaOH/dioxane.
2-(Diet h yla m in o)-6,7-d ih yd r o-4H,5H-cyclop en t a [4,5]-
th ien o[2,3-d ][1,3]oxa zin -4-on e (54). Yield 72%; mp 92-93

Thieno[1,3]oxazin-4-ones as HLE Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5445
°C (hexane); IR (KBr, cm-1) 1753 (CdO); UV (EtOH) λmax (nm)
(log ꢀ) 242 (4.22), 269 (3.92), 346 (4.02); 1H NMR (CDCl3) δ
1.24 (t, J ) 7.1 Hz, 6H, CH3), 2.36-2.46 (m, 2H, CH2CH2CH2),
2.80-2.96 (m, 4H, CH2CH2CH2), 3.55 (q, J ) 7.1 Hz, 4H,
NCH2); 13C NMR (CDCl3) δ 13.33 (CH3), 27.67, 28.91, and
29.42 (C-5, C-6, and C-7), 42.44 (NCH2), 105.32 (C-4a), 131.12
(C-7a), 139.60 (C-4b), 155.43, 155.53 (C-2, C-8a), 174.91 (C-
4); MS (EI) m/z (rel intensity) 264 (M+, 100), 192 (83). Anal.
(C13H16N2O2S) C, H, N, S.
Refer en ces
(1) Vender, R. L. Therapeutic Potential of Neutrophil-Elastase
Inhibition in Pulmonary Disease. J . Invest. Med. 1996, 44, 531-
539.
(2) (a) Nakagawa, T.; Momohara, S.; Fujita, K.; Kodama, T.;
Yamada, H.; Nagai, Y. Serine Proteinase in Articular Cartilage,
Subchondral Bone Marrow and Synovial Fluid in Human
Osteoarthritis and Rheumatoid Arthritis. Biomed. Res. 1995, 16,
11-20. (b) J in, L. J .; Soeder, P.-O.; Aaasman, B.; Bergstroem,
K. Granulocyte Elastase in Gingival Crevicular Fluid: Improved
Monitoring of the Site-Specific Response to Treatment in
Patients with Destructive Periodontitis. J . Clin. Periodontol.
1995, 22, 240-246.
(3) Reviews: (a) Hlasta, D. J .; Pagini, E. D. Human Leukocyte
Elastase Inhibitors. Annu. Rep. Med. Chem. 1994, 29, 195-204.
(b) Edwards, P. D.; Bernstein, P. R. Synthetic Inhibitors of
Elastase. Med. Res. Rev. 1994, 14, 127-194. (c) Bernstein, P.
R.; Edwards, P. D.; Williams, J . C. Inhibitors of Human
Leukocyte Elastase. Prog. Med. Chem. 1994, 13, 59-120.
(4) (a) J anusz, M. J .; Hare, M. Inhibition of Human Neutrophil
2-(Dieth yla m in o)-6,7,8,9-tetr a h yd r o-4H,5H-cycloh epta -
[4,5]th ien o[2,3-d ][1,3]oxa zin -4-on e (55). Yield 78%; mp
103-104 °C (hexane); IR (KBr, cm-1) 1761 (CdO); UV (EtOH)
λmax (nm) (log ꢀ) 241 (4.35), 272 (4.01), 341 (4.05); 1H NMR
(CDCl3) δ 1.23 (t, J ) 7.1 Hz, 6H, CH3), 1.62-1.72 (m, 4H,
CH2), 1.82-1.93 (m, 2H, CH2), 2.68-2.74 (m, 2H, CH2), 3.06-
3.11 (m, 2H, CH2), 3.53 (q, J ) 7.1 Hz, 4H, NCH2); 13C NMR
(CDCl3) δ 13.34 (CH3), 27.25, 27.91, 28.03, 29.48, and 32.38
(C-5, C-6, C-7, C-8, and C-9), 42.32 (NCH2), 109.25 (C-4a),
130.04 (C-9a), 135.91 (C-4b), 155.43 and 155.71 (C-2 and
C-10a), 167.84 (C-4); MS (EI) m/z (rel intensity) 292 (M+, 100),
220 (45). Anal. (C15H20N2O2S) C, H, N, S.
Elastase and Cathepsin
G by a Biphenyl Disulfonic Acid
Copolymer. Int. J . Immunopharmacol. 1994, 16, 623. (b) Regan,
J .; McGarry, D.; Bruno, J .; Green, D.; Newman, J .; Hsu, C.-Y.;
Kline, J .; Barton J .; Travis, J .; Choi, Y. M.; Volz, F.; Pauls, H.;
Harrison, R.; Zilberstein, A.; Ben-Sasson, S. A.; Chang, M.
Anionic- and Lipophilic-Mediated Surface Binding Inhibitors of
Human Leukocyte Elastase. J . Med. Chem. 1997, 40, 3408.
(5) Guy, L.; Vidal, J .; Collet, A.; Amour, A.; Reboud-Ravaux, M.
Design and Synthesis of Hydrazinopeptides and Their Evalua-
tion as Human Leukocyte Elastase Inhibitors. J . Med. Chem.
1998, 41, 4833-4848.
(R,S)-2-(Dieth yla m in o)-5-m eth yl-5,6,7,8-tetr a h ydr o-4H-
[1]ben zoth ien o[2,3-d ][1,3]oxa zin -4-on e (56). Yield 83%; mp
78-79 °C (hexane); IR (KBr, cm-1) 1742 (CdO); UV (EtOH)
λmax (nm) (log ꢀ) 240 (4.35), 267 (3.95), 340 (4.07); 1H NMR
(CDCl3) δ 1.23 (t, J ) 7.1 Hz, 6H, CH3CH2), 1.26 (d, J ) 6.9
Hz, 3H, 5-CH3), 1.65-1.74 (m, 4H, CH2), 2.56-2.66 (m, 2H,
CH2), 3.17-3.25 (m, 1H, CH), 3.53 (q, J ) 7.1 Hz, 4H, CH2-
CH3); 13C NMR (CDCl3) δ 13.32 (CH3CH2), 21.19 (5-CH3),
18.67, 24.93, and 29.54 (C-6, C-7, and C-8), 28.92 (C-5), 42.35
(NCH2), 107.58 (C-4a), 125.96 (C-8a), 135.86 (C-4b), 154.76 and
155.71 (C-2 and C-9a), 170.17 (C-4); MS (EI) m/z (rel intensity)
292 (M+, 100), 220 (32). Anal. (C15H20N2O2S) C, H, N; S: calcd,
10.96; found, 11.50.
(6) Zembower, D. E.; Kam, C.-M.; Powers, J . C.; Zalkow, L. H. Novel
Anthraquinone Inhibitors of Human Leukocyte Elastase and
Cathepsin G. J . Med. Chem. 1992, 35, 1597.
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Bryant, C.; Damewood, J . R., J r.; Earley, R.; Feeney, S. W.;
Edwards, P. D.; Gomes, B.; Hulsizer, J . M.; Kosmider, B. J .;
Krell, R. D.; Moore, G.; Salcedo, T. W.; Shaw, A.; Silberstein, D.
S.; Steelman, G. B.; Strimpler, A. M.; Thomas, R. M.; Vacek, E.
P.; Williams, J . C.; Wolanin, D. J .; Woolson, S. Orally Active
Trifluoromethyl Ketone Inhibitors of Human Leukocyte Elastase.
J . Med. Chem. 1997, 40, 3173-3181. (b) Cregge, R. J .; Durham,
S. L.; Farr, R. A.; Gallion, S. L.; Hare, C. M.; Hoffmann, R. V.;
J anusz, M. J .; Kim, H.-O.; Koehl, J . R.; Mehdi, S.; Metz, W. A.;
Peet, N. P.; Pelton, J . T.; Schreuder, H. A.; Sunder, S.; Tardif,
C. Inhibition of Human Neutrophil Elastase. 4. Design, Syn-
thesis, X-ray Crystallographic Analysis, and Structure-Activity
Relationships of a Series of P2-Modified, Orally Active Peptidyl
Pentafluoroethyl Ketones. J . Med. Chem. 1998, 41, 2461-2480.
(8) (a) Katzenellenbogen, J . A.; Rai, R.; Dai, W. Enol Lactone
Derivatives as Inhibitors of Human Neutrophil Elastase and
Trypsin-Like Proteases. Bioorg. Med. Chem. Lett. 1992, 2, 1399.
(b) Tam, T. F.; Spencer, R. W.; Thomas, E. M.; Copp, J . J .;
Krantz, A. Novel Suicide Inhibitors of Serine Proteases. Inac-
tivation of Human Leukocyte Elastase by Ynenol Lactones. J .
Am. Chem. Soc. 1984, 106, 6849-6851.
2-(Dieth yla m in o)-4H-[1]ben zoth ien o[2,3-d ][1,3]oxa zin -
4-on e (57). Yield 69%; mp 114-116 °C (hexane); IR (KBr,
cm-1) 1748 (CdO); UV (EtOH) λmax (nm) (log ꢀ) 234 (4.38), 257
1
(4.63), 344 (4.25); H NMR (CDCl3) δ 1.30 (t, J ) 7.1 Hz, 6H,
CH3), 3.52-3.69 (m, 4H, CH2), 7.27-7.33 and 7.40-7.55 (each
m, 1H, H-6 and H-7), 7.65-7.70 and 8.16-8.21 (each m, 1H,
H-5 and H-8); 13C NMR (CDCl3) δ 12.83, 13.62 (CH3), 42.37,
43.02 (CH2), 102.59 (C-4a), 122.07 and 122.44 (C-5 and C-8),
124.54 and 125.90 (C-6 and C-7), 132.83 and 134.27 (C-4b and
C-8a), 154.61 and 156.87 (C-2 and C-9a), 173.29 (C-4); MS (EI)
m/z (rel intensity) 274 (M+, 96), 202 (100). Anal. (C14H14N2O2S)
C, H, N, S.
Deter m in a tion of th e Kin etic P a r a m eter s of th e Al-
k a lin e Hyd r olysis. Alkaline hydrolysis was followed spec-
trophotometrically at 335 nm in 50 mM CAPS, pH 11.25, at
25 °C by monitoring the disappearance of the thieno[1,3]-
oxazin-4-ones 7 and 50-58 and the 3,1-benzoxazin-4-one 59
for at least 1.5 half-lives. Stock solutions of the compounds
were prepared in DMSO; the final inhibitor concentration was
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7, 34-41, and 50-59 was assayed spectrophotometrically by
the progress curve method at 25 °C. Assay buffer was 50 mM
sodium phosphate and 500 mM NaCl, pH 7.8. A stock solution
of Suc-Ala-Ala-Pro-Val-pNA (20 mM in DMSO) was diluted
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curves were fitted, and data were analyzed as described.36
Inhibitors, dissolved in DMSO (10 µL), were added into a
cuvette containing 890 µL of assay buffer and 50 µL of Suc-
Ala-Ala-Pro-Val-pNA (final concentration 100 µM ) 1.3 Km).
After thermal equilibration, the reaction was initiated by
addition of 50 µL of HLE solution. Progress curves were
monitored at 405 nm over 33-66 min.

5446 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
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