2-oxy-4H-3,1-benzoxazin-4-ones and pharmaceutical use

2-Oxy-4H-3,1-benzoxazin-4-ones represented by the formula: ##STR1## and the pharmaceutically acceptable acid addition salts thereof, wherein: a is an integer of 0-4; PA0 A is a bond, or alkylene having one to eight carbon atoms; PA0 R is hydrogen, phenyl, imidazolyl or cycloalkyl having three to six carbon atoms, wherein the phenyl, imidazolyl or cycloalkyl ring is optionally substituted with 1-3 substituents independently selected from the group consisting of lower alkyl having one to four carbon atoms, lower alkoxy having one to four carbon atoms, --N(R.sup.1).sub.2, --NO.sub.2, halo or lower alkylthio having one to four carbon atoms, and, PA0 each R' is independently selected from the group consisting of lower alkyl having one to six atoms, lower alkenyl having two to six carbon atoms, lower alkoxy having one to six carbon atoms, PA0 lower alkylthio or halo-lower alkyl having one to four carbon atoms, halo, --NO.sub.2, --N(R.sup.1).sub.2, ##STR2## --NR.sup.1 COR.sup.2, and ##STR3## in which each R.sup.1 is independently hydrogen or lower alkyl having one to four carbon atoms, or together form a piperidine or a piperazine ring optionally substituted at the ring nitrogen by lower alkyl having one to four carbon atoms or --CH.sub.2 CH.sub.2 OH, PA1 each R.sup.2 is independently lower alkyl having one to four carbon atoms, PA0 A is an alkylene group if R is hydrogen, and the pharmaceutically acceptable acid addition salts thereof are useful as serine protease inhibitors in humans and animals.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to (i) 2-oxy-4H-3,1-benzoxazin-4-ones and the 
pharmaceutically acceptable acid addition salts thereof; (ii) the use of 
these compounds as serine protease inhibitors in humans and animals; (iii) 
pharmaceutical compositions comprising a compound of this invention and at 
least one pharmaceutical excipient; and (iv) processes for preparing the 
compounds of this invention. 
2. Related Art 
The compounds of this invention are 2-oxy substituted derivatives of 
4H-3,1-benzoxazinones having the following structure: 
##STR4## 
2-Ethoxy-4H-3,1-benzoxazin-4-one is disclosed in British Patent 
Specification No. 1,389,128 and in the corresponding German 
Offenlegungschrift No. 2241012. A few 4H-3,1-benzoxazin-4-ones are known 
to possess enzyme-inhibitory activity. Teshima et al. have disclosed 
various 2-alkyl-4H-3,1-benzoxazin-4-ones reported to be active as enzyme 
inhibitors. (J. Biol. Chem., 257, 5085-5091, 1982). 
4H-3,1-benzoxazin-2,4-dione has been disclosed as having some enzyme 
inhibitory activity (Moorman, A. R., and Abeles, R. H. J. Amer. Chem. Soc. 
104, 6785-6786, 1982). 2-Ethoxy-4H-3,1-benzoxazinone and 
2-(trifluoromethyl)-4H-3,1-benzoxazinone are believed to inhibit 
chymotrypsin (Hedstrom et al, Biochemistry 23, 1753-1759, 1984). 
SUMMARY 
We have discovered that the class of 2-oxy-4H-3,1-benzoxazin-4-ones of 
Formula I, as shown below: 
##STR5## 
and described and claimed herein, are biologically active as inhibitors of 
enzymes, and in particular are inhibitors of serine proteases. Within this 
class, several groups of compounds are novel and preferred. Accordingly, 
the invention described and claimed herein contains the following aspects: 
1. Novel compounds of Formula I wherein: 
a is an integer of 0-4; 
A is a bond, or alkylene having one to eight carbon atoms; 
R is phenyl, imidazolyl, or cycloalkyl having three to six carbon atoms, 
wherein the phenyl or cycloalkyl ring is optionally substituted with 1-3 
substituents independently selected from the group consisting of lower 
alkyl having one to four carbon atoms, lower alkoxy having one to four 
carbon atoms, --N(R.sup.1).sub.2, --NO.sub.2, halo, and lower alkylthio 
having one to four carbon atoms, and 
each R' is independently selected from the group consisting of lower alkyl 
having one to six atoms, lower alkenyl having two to six carbon atoms, 
lower alkoxy having one to six carbon atoms, --NO.sub.2, halo-lower alkyl 
or lower alkylthio having one to six carbon atoms, halo, 
--N(R.sup.1).sub.2, 
##STR6## 
--NR.sup.1 COR.sup.2 and 
##STR7## 
in which each R.sup.1 is independently hydrogen or lower alkyl having one 
to four carbon atoms, or together form a piperidine or piperazine ring 
optionally substituted at the ring nitrogen by lower alkyl having one to 
four carbon atoms or --CH.sub.2 CH.sub.2 OH, and 
each R.sup.2 is independently lower alkyl having one to four carbon atoms, 
and the pharmaceutically acceptable acid addition salt thereof. 
2. Novel compounds of Formula I wherein: 
a is an integer of 1-4; 
A is alkylene having one to eight carbon atoms; 
R is hydrogen; and 
each R' is independently selected from the group consisting of lower alkyl 
having one to six carbon atoms, lower alkenyl having two to six carbon 
atoms, halo-lower alkyl or lower alkylthio having one to six carbon atoms, 
halo, --NO.sub.2, --N(R.sup.1).sub.2, 
##STR8## 
--NR.sup.1 COR.sup.2, and in which 
each R.sup.1 is independently hydrogen or lower alkyl having one to four 
carbon atoms, or together form a piperidine or piperazine ring optionally 
substituted at the ring nitrogen by lower alkyl having one to four carbon 
atoms or --CH.sub.2 CH.sub.2 OH, and 
each R.sup.2 is independently lower alkyl having one to four carbon atoms, 
or a pharmaceutically acceptable acid addition salt thereof. 
3. Novel compounds of Formula I wherein: 
a is an integer of 0-4; 
A is alkylene having four to eight carbon atoms; 
R is hydrogen; and 
each R' is independently selected from the group consisting of lower alkyl 
having one to six carbon atoms, lower alkenyl having two to six carbon 
atoms, halo, lower alkoxy having one to six carbon atoms, lower alkylthio 
or halo-lower alkyl having one to six carbon atoms, --NO.sub.2, 
--N(R.sup.1).sub.2, 
##STR9## 
--NR.sup.1 COR.sup.2, and 
##STR10## 
in which each R.sup.1 is independently hydrogen or lower alkyl having one 
to six carbon atoms, or together form a piperidine or piperazine ring 
optionally substituted at the ring nitrogen by lower alkyl having one to 
four carbon atoms or --CH.sub.2 CH.sub.2 OH, and 
each R.sup.2 is independently lower alkyl having one to four carbon atoms, 
or a pharmaceutically acceptable acid addition salt thereof. 
4. Novel compounds of Formula A: 
##STR11## 
wherein: a is an integer of 0-3; 
A is a bond, or alkylene having one to eight carbon atoms; 
R is hydrogen, imidazolyl, phenyl, or cycloalkyl having three to six carbon 
atoms, wherein the phenyl, imidazolyl or cycloalkyl ring is optionally 
substituted with 1-3 substituents independently selected from the group 
consisting of lower alkyl having one to four carbon atoms, lower alkoxy 
having one to four carbon atoms, --N(R.sup.1).sub.2, --NO.sub.2, halo, and 
lower alkylthio having one to four carbon atoms; and 
each R' and R" are independently selected from the group consisting of 
lower alkyl having one to six carbon atoms, lower alkenyl having two to 
six carbon atoms, lower alkoxy having one to six carbon atoms, lower 
alkylthio or halo-lower alkyl having one to six carbon atoms, --NO.sub.2, 
--N(R.sup.1).sub.2, --NR.sup.1 COR.sup.2, 
##STR12## 
in which each R.sup.1 is independently hydrogen or lower alkyl having one 
to four carbon atoms, or together form a piperidine or piperazine ring 
optionally substituted at the ring nitrogen with lower alkyl having one to 
four carbon atoms or --CH.sub.2 CH.sub.2 OH; 
each R.sup.2 is independently lower alkyl having one to four carbon atoms, 
and 
A is alkylene if R is hydrogen, 
or a pharmaceutically acceptable acid addition salt thereof. 
5. Pharmaceutical compositions which comprise a therapeutically effective 
amount of a compound of Formula I or A chosen from among those described 
in Groups 1-4 above, or a pharmaceutically acceptable acid addition salt 
thereof, in admixture with at least one pharmaceutically acceptable 
excipient. 
6. Methods of inhibiting serine proteases in humans and animals which 
comprise administering to a subject in need of such treatment a 
therapeutically effective amount of a compound of Formula I or A chosen 
from among those described in Groups 1 to 4, above. 
7. A method of inhibiting serine proteases in humans and animals which 
method comprises administering to a subject in need of such treatment a 
therapeutically effective amount of a compound of the formula: 
##STR13## 
wherein: a is an integer of 0-4; 
A is a bond, or alkylene having one to eight carbon atoms; 
R is hydrogen, phenyl, imidazolyl or cycloalkyl having three to six carbon 
atoms, wherein the phenyl or cycloalkyl ring is optionally substituted 
with 1-3 substituents independently selected from the group consisting of 
lower alkyl having one to four carbon atoms, lower alkoxy having one to 
four carbon atoms, --N(R.sup.1).sub.2, --NO.sub.2, halo, and lower 
thioalkyl having one to four carbon atoms; and 
each R' is independently selected from the group consisting of lower alkyl 
having one to six carbon atoms, lower alkenyl having two to six carbon 
atoms, halo, lower alkoxy having one to six carbon atoms, lower alkylthio 
or halo-lower alkyl having one to six carbon atoms, --N(R.sup.1).sub.2, 
##STR14## 
--NR.sup.1 COR.sup.2, and 
##STR15## 
in which each R.sup.1 is independently hydrogen or lower alkyl having one 
to four carbon atoms, or together form a piperidine or piperazine ring 
optionally substituted at the ring nitrogen with lower alkyl having one to 
four carbon atoms or --CH.sub.2 CH.sub.2 OH; 
each R.sup.2 is independently lower alkyl having one to four carbon atoms, 
and 
A is alkylene if R is hydrogen, 
or a pharmaceutically acceptable acid addition salt thereof. 
8. Novel compounds of Formulas I and A useful as intermediates in the 
synthesis of compounds of the invention, having the formula: 
##STR16## 
wherein: a is an integer of 0-3; 
A is a bond, or alkylene having one to eight carbon atoms; 
R is hydrogen, phenyl, imidazolyl or cycloalkyl having three to six carbon 
atoms, wherein the phenyl or cycloalkyl ring is optionally substituted 
with 1-3 substituents independently selected from the group consisting of 
lower alkyl having one to four carbon atoms, lower alkoxy having one to 
four carbon atoms, --N(R.sup.1).sub.2, --NO.sub.2, halo, and lower 
thioalkyl having one to four carbon atoms; 
R"' is lower alkyl having one to six carbon atoms; and 
each R' is independently selected from the group consisting of lower alkyl 
having one to six carbon atoms, lower alkenyl having two to six carbon 
atoms, lower alkoxy having one to six carbon atoms, halo-lower alkyl or 
lower alkylthio having one to six carbon atoms, halo, --N(R.sup.1).sub.2, 
##STR17## 
--NR.sup.1 COR.sup.2, and 
##STR18## 
in which each R.sup.1 is independently hydrogen or lower alkyl having one 
to four carbon atoms, or together form a piperidine or piperazine ring 
optionally substituted at the ring nitrogen with lower alkyl having one to 
four carbon atoms or --CH.sub.2 CH.sub.2 OH; 
each R.sup.2 is independently lower alkyl having one to four carbon atoms, 
and 
A is alkylene if R is hydrogen. 
The foregoing paragraphs numbered 1-8 set forth aspects of the invention 
which are referred to here and subsequently as "Groups" 1 to 8.

DETAILED DESCRIPTION OF THE INVENTION 
As used herein: 
"Alkylene" means a branched or unbranched saturated hydrocarbon bridging 
group having one to eight carbon atoms, including but not limited to, 
methylene, ethylene, propylene, isopropylene, n-propylene, butylene, 
sec-butylene, isobutylene, n-pentylene, hexylene, octylene, and the like. 
"Lower alkyl" means a branched or unbranched saturated hydrocarbon chain 
having, unless otherwise noted, one to six carbon atoms, including but not 
limited to methyl, ethyl, propyl, isopropyl, n-propyl, butyl, sec-butyl, 
isobutyl, n-pentyl, hexyl, octyl and the like. Lower alkyl groups may be 
limited to fewer than six carbon atoms when specifically designated, e.g. 
"R.sup.2 is lower alkyl having one to four carbon atoms." 
"Lower alkenyl" means a branched or unbranched unsaturated hydrocarbon 
chain of 2 to 6 carbon atoms, including but not limited to vinyl, allyl, 
1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, 1,3-butadienyl, 
1-pentenyl, 2-pentenyl, isoprenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 
cis-2-butenyl, trans-2-butenyl, cis-2-pentenyl, trans-2-pentenyl, 
3-methyl-1-butenyl, 2-methyl-2-butenyl and 2,3-dimethyl-2-butenyl. 
"Lower alkoxy" means the group --OR wherein R is lower alkyl as herein 
defined. 
"Lower alkylthio" means the group --SR wherein R is lower alkyl as herein 
defined. 
"Halo" refers to chloro, bromo and iodo. 
"Halo-lower alkyl" means the group --R-halo in which R is lower alkyl, and 
both lower alkyl and halo have the definitions given herein. The alkyl 
group may bear one or two halo substituents; examples include but are not 
limited to bromomethyl, dibromomethyl, chloroethyl, dichloroethyl, and the 
like. 
"Pharmaceutically acceptable acid addition salt" refers to those salts 
which retain the biological effectiveness and properties of the free bases 
and which are not biologically or otherwise undesirable, formed with 
inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric 
acid, nitric acid, phosphoric acid and the like, and organic acids such as 
acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, 
malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, 
tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, 
methanesulfonic acid, ethane sulfonic acid, p-toluenesulfonic acid, 
ascorbic acid, salicylic acid and the like. 
"Optional" or "optionally" means that the subsequently described event or 
circumstances may or may not occur, and that the description includes 
instances where said event or circumstance occurs and instances in which 
it does not. For example, "phenyl . . . optionally substituted" means that 
the phenyl may or may not be substituted and that the description includes 
both unsubstituted phenyl and phenyl wherein there is substitution. 
Certain of the compounds of the invention have chiral centers and exist as 
optical antipodes. The invention described and claimed herein includes 
each of the individual enantiomers as well as their racemic modifications 
and the racemic mixture. 
The compounds of this invention are named as 2-oxy-4H-3,1-benzoxazin-4-ones 
using the numbering system set forth below. 
##STR19## 
For example, the compound of Formula I where AR is isopropyl, a is 1 and R' 
is methyl in the 5-position is named 
2-isopropyloxy-5-methyl-4H-3,1-benzoxazin-4-one. 
The compound where AR is phenethyl, a is 2 and both R's are amino in the 5- 
and 7-positions is named 
2-(phenethyloxy)-5,7-di-amino-4H-3,1-benzoxazin-4-one. 
The compound of Formula I where AR is cyclohexyl, and a is zero is named 
2-cyclohexyloxy-4H-3,1-benzoxazin-4-one. 
The compound of Formula I where AR is n-butyl, a is 1 and R' is methylthio 
in the 5-position is named 
2-n-butoxy-5-methylthio-4H-3,1-benzoxazin-4-one. 
Preferred Embodiments 
Within the several aspects of this invention which are set fourth as Groups 
1 to 4 in the Summary of the Invention, certain subgroups are preferred. 
The metes and bounds of these subgroups and their relative degrees of 
preference are described below. 
Within each of the groups of compounds defined as Groups 1 to 4, preferred 
subgroups are compounds of Formulas I and A in which a is at least one. 
Among these, preferred classes encompass compounds in which the R's are in 
the 5- and/or 7-positions. Within these classes, preferred subclasses 
include compounds of Formula I in which an R' (or R" in group 4) is in the 
5-position; of these, especially preferred are compounds of Formula I in 
which an R' is also in 7-position. 
Preferred R' or R" substituents at the 5-position are lower alkyl having 
one to six carbon atoms and lower alkenyl having two to six carbon atoms. 
Especially preferred R' and R" substituents at the 5-position are lower 
alkyl having one to six carbon atoms, particularly one to three carbon 
atoms, and most particularly methyl or ethyl. Preferred R' substituents at 
the 7-position are lower alkoxy having one to six carbon atoms, 
--N(R.sup.1).sub.2, 
##STR20## 
--NR.sup.1 COR.sup.2 and 
##STR21## 
particularly --N(R.sup.1).sub.2 and --NR.sup.1 COOR.sup.2, especially 
where each R' is independently hydrogen, methyl or ethyl, and R.sup.2 is 
methyl or ethyl. 
Additionally, certain subgroups within each individual Group have 
preference. Among the compounds of Formula I as set forth in Group 1, a 
preferred subgroup is those compounds in which A is a bond, or alkylene 
having one to four carbon atoms, especially a bond or methylene. Among the 
compounds of Formula I defined by Group 2, a preferred subgroup are those 
compounds in which A is lower alkylene having one to four carbon atoms, 
particularly methylene and ethylene. Among the compounds of Formula I as 
set forth in Group 3, a preferred subgroup encompasses compounds in which 
A is alkylene having five to eight carbon atoms. Among the compounds of 
Formula A as defined in Group 4, a preferred subgroup consists of 
compounds in which a is zero or one and A is lower alkylene having one to 
four carbon atoms, particularly methylene and ethylene. 
At the present time, the most preferred compounds of this invention are: 
2-ethoxy-5-methyl-4H-3,1-benzoxazin-4-one; 
2-ethoxy-5-ethyl-4H-3,1-benzoxazin-4-one; 
7-amino-2-ethoxy-5-methyl-4H-3,1-benzoxazin-4-one, and 
7-amino-2-ethoxy-5-ethyl-4H-3,1-benzoxazin-4-one. 
Methods of Preparation 
In this section the remainder of the specification, the compounds of the 
invention which are referred to in the Summary of the Invention and in the 
Claims as compounds of Formula I and compounds of Formula A will be 
referred to collectively as compounds of Formula I, and further defined as 
compounds of Formula IA, IB, IC, etc. 
A. Compounds of Formula I in Which R' is Lower Alkyl, Lower Alkoxy, Lower 
Alkylthio, Halo, --NO.sub.2 or an Amine Other than NH.sub.2 (Compounds of 
Formula IA). 
The compounds of Formula I in which each R' substituent is lower alkyl, 
lower alkoxy, lower alkylthio, halo, --NO.sub.2 or an amine other than 
--NH.sub.2, (hereinafter defined as compounds of Formula IA), can be 
prepared by the general procedure set forth in Reaction Scheme I, below. 
##STR22## 
As outlined in Reaction Scheme I, the compounds of Formula IA are prepared 
by cyclization of the corresponding appropriately substituted or 
unsubstituted anthranilic acid. Cyclization is preferably achieved by 
reaction of the chosen anthranilic acid with about 3 to about 5, 
preferably about 4 equivalents of the desired phenyl-, cycloalkyl- or 
alkyl chloroformates of the formula ClCOOAR, wherein A and R are defined 
as hereinabove. The reaction takes place in a basic organic solvent such 
as triethylamine, or, preferably, pyridine, and is carried out at room 
temperature over a period of about 0.5 to about 5 hours, preferably about 
1 to about 3 hours. The final product, a compound of Formula IA, is then 
isolated by conventional means. 
Isolation and purification of the final compounds and intermediates 
described herein can be effected, if desired, by any suitable separation 
or purification procedure such as, for example, filtration, extraction, 
crystallization, column chromatography, thin-layer chromatography, thick 
layer chromatography, or a combination of these procedures. Specific 
illustrations of suitable separation and isolation procedures can be had 
by reference to the examples hereinbelow. However, other equivalent 
separation or isolation procedures could, of course, also be used. 
Unsubstituted anthranilic acid is readily commercially available. The 
substituted anthranilic acids (Formula II) used in preparing the compounds 
of this invention are either commercially available, or can be prepared by 
methods well known in the art. The commercially available anthranilic 
acids include, but are not limited to, 3-methyl-anthranilic acid, 4-methyl 
anthranilic acid, 5-methyl-anthranilic acid, 6-methyl-anthranilic acid, 
5-iodo-anthranilic acid, 4-nitro-anthranilic acid, and 
4,5-dimethoxy-anthranilic acid. A list of commercially available 
anthranilic acids is available in Chem. Sources-U.S.A., 24th Ed., 1983, 
Directories Publishing Company, Inc., Ormond Beach, Fla. Appropriately 
substituted anthranilic acids which are not commercially available can be 
readily prepared by methods known in the art. Suitable methods include 
those of B. R. Baker, et al., J. Org. Chem., 17, 141, (1952) and of L. A. 
Paquette, et al., J. Am. Chem. Soc. 99, 3734, (1981). The former method 
involves the preparation of an isatin from a substituted aniline 
derivative. Subsequent oxidation of the isatin gives the anthranilic acid. 
The latter procedure employs the reduction of the corresponding aromatic 
nitro-derivative to the anthranilic acid. These methods are further 
illustrated in Preparation I, below. 
The phenyl-, cycloalkyl- and alkyl chloroformates of Formula III are either 
commercially available, or can be prepared by methods well known or 
readily available in the chemical literature. Commercially available 
chloroformates include, but are not limited to benzyl chloroformate, 
methyl chloroformate, ethyl chloroformate, n-butyl chloroformate, isobutyl 
chloroformate. Suitable chloroformates which are not commercially 
available can be prepared by known methods. Preparative methods include 
those of D. H. R. Barton et al., J. Chem Soc. 18 55-1857 (1968) and K. 
Kurita et al., J. Org Chem. 41, 2070-2071, (1976). The former method 
involves treatment of an appropriate alcohol with phosgene in an inert 
solvent such as anhydrous ether. Further description of this method is 
provided in Preparation II, below. The latter procedure involves the 
reaction of an appropriate alcohol with trichloromethyl chloroformate 
(diphosgene) in dry dioxane at refluxing temperature. 
B. Compounds of Formula I in Which an R' is NH.sub.2 (Compounds of Formula 
IB) 
Compounds of Formula I in which one of the R' substituents is NH.sub.2 
(hereinafter referred to as compounds of Formula IB) can be prepared by 
the procedure set forth in Reaction Scheme II, below. 
##STR23## 
As shown above, a nitro-substituted 2-oxy-4H-3,1-benzoxazin-4-one of 
Formula IA is reduced to the corresponding amino-substituted compound of 
Formula I by transfer hydrogenation. The reaction takes place in benzene 
or THF in the presence of 10% Pd-C as catalyst and cyclohexene as hydrogen 
donor and is carried out at reflux temperature over about one to five, 
preferably about three hours. The final product, a compound of Formula IB, 
is then isolated by conventional means. 
The nitro-substituted benzoxazinone of Formula IA may be commercially 
obtained, or is prepared according to the procedure set forth above in 
Section A. 
C. Compounds in Which an R' is --NR.sup.1 COR.sup.2 (Compounds of Formula 
IC) 
Compounds of the invention in which one of the R' substituents is an amine 
of the formula --NR.sup.1 COR.sup.2, (hereinafter referred to as compounds 
of Formula IC) can be prepared from the corresponding amino-substituted 
compound of Formula IB, as shown in Reaction Scheme III. 
##STR24## 
The amino-substituted compound of Formula IA or IB, prepared as described 
in section A or B, above, is reacted with about 1 to 3 equivalents of an 
appropriate acid anhydride of Formula IV, with or without the presence of 
an inert organic solvent such as dichloromethane or tetrahydrofuran. The 
reaction takes place at room temperature over a period of about 15 min to 
about 3 hrs, usually about 1 hour, and the final product, a compound of 
Formula IC, is then isolated by conventional means. 
The acid anhydrides (Formula IV) used in preparing the compounds of Formula 
IC are either commercially available or can be prepared by methods well 
known in the art. The commercially available acid anhydrides include, but 
are not limited to, acetic anhydride, propionic anhydride, and butyric 
anyhydride. A list of commercially available acid anhydrides is available 
in Chem. Sources-U.S.A., 24th Ed., 1983, Directories Publishing Company, 
Inc., Ormond Beach, Fla. Acid anhydrides which are not commercially 
available can be readily prepared by methods known in the art. Suitable 
methods include the direct removal of water from acids to form acid 
anhydride and from acid chlorides and alkali salts of carboxylic acids by 
mixing the reactants and distilling off the anhydride formed as described 
in Preparative Organic Chemistry, edited by G. Hilgetag and A. Martini, 
page 387-390, John Wiley & Sons, New York-London-Sydney-Toronto (1972), 
and further illustrated in Preparation III, below. 
D. Compounds in Which an R' is 
##STR25## 
(Compounds of Formula ID) 
Compounds of the invention which bear an R' substituent of the formula 
--NR.sup.1 COOR.sup.2 can be prepared by the procedure set forth in 
Section A, above, or from the corresponding amino-substituted compound of 
Formula IA or IB, as shown below. 
##STR26## 
In carrying out the above conversion, the appropriate compound of Formula 
IA or IB (prepared as described in Sections A and B, above) bearing an 
R.sup.1 NH substituted as shown, is reacted with about one to three 
equivalents, preferably about 1.5 equivalents, of an appropriately chosen 
phenoxy-, cycloalkoxy- or alkoxy chloroformate of Formula III. (The 
commercial availability and methods of preparing the various compounds of 
Formula III used in making the compounds of this invention are discussed 
in detail in Section A, hereinabove.) The reaction takes place in an inert 
solvent such as dichloromethane or tetrahydrofuran in the presence of a 
tertiary amine such as triethylamine or, preferably, pyridine, at room 
temperature over a period of about one to about three, usually about two, 
hours. The final product, a compound of Formula ID, is then isolated by 
conventional means. 
E. Compounds in Which an R' is --NR.sup.1 CON(R.sup.1).sub.2 (Formula IE) 
Compounds of the invention which bear an R' substituent of the formula 
--NR.sup.1 CON(R.sup.1).sub.2 (hereinafter referred to as compounds of 
Formula IE) can be prepared from the corresponding amino-substituted 
compounds of Formula IA and IB as shown below in Reaction Scheme V. 
##STR27## 
The amino-substituted compound of Formula IA or IB is reacted in an inert 
solvent such as benzene or tetrahydrofuran with about 0.5 to 1 preferably 
0.75, equivalent of trichloromethyl chloroformate, for a period of about 
15 to 60 minutes, preferably about 30 minutes. Without isolation, the 
resulting carbamyl chloride derivative is then treated with about 5 to 10, 
preferably about 7.5 equivalents of pyridine and about 1 to 3, preferably 
about 2 equivalents of an appropriate primary or secondary alkyl amine for 
a period of about 15 to 60, usually about 30 minutes. The final product, a 
compound of Formula IE, is then isolated by conventional means. 
F. Compounds in which an R' is Halo-Lower Alkyl (Formula IF) 
Compounds of the Formula I which bear an R' substituent in which R' is 
lower alkyl can be mono- or di-halogenated at a benzylic position with an 
N-halo-succinimide such as N-bromosuccinimide, and AIBN 
(2-2'-azobis-isobutyronitrile) to give either the mono-halo compound of 
Formula (IF.sub.1) or the di-halo compound of Formula (IF.sub.2). 
Normally, only a catalytic amount of AIBN is used. This procedure is shown 
n Reaction Scheme VI, below, in which N-bromosuccinimide is used as an 
example. N-chlorosuccinimide or N-iodosuccinimide can also be used. 
##STR28## 
The halogenation reaction is carried out in carbon tetrachloride at reflux 
temperature for 2 to 4 hours, and the product is isolated by conventional 
means. When, for example, one equivalent of N-bromosuccinimide is used, 
the monobromo compound of Formula IF.sub.1 is the major product; likewise, 
when two equivalents of N-bromosuccinimide are used, the dibromo compound 
of Formula IF.sub.2 is the major product. 
G. Compounds in which an R' is lower Alkenyl or Lower Alkyl 
Compounds of Formula I in which an R' is lower alkenyl or lower alkyl can 
be prepared from the corresponding compounds of Formula IF.sub.1 and 
IG.sub.2, as shown in Reaction Scheme VII. Compounds of Formula I in which 
an R' is lower alkyl can also be prepared by the method described in 
Section A, above. 
##STR29## 
In Formulas IF.sub.1, IG.sub.1 and IG.sub.2, R' is hydrogen or lower alkyl 
having one to six carbon atoms. In Formula IG.sub.3, R' is the remaining 
saturated portion of the alkene substituent. For illustrative purposes the 
double bond is depicted at carbons 2-3; however, other straight and 
branched chain lower alkene substituents can be obtained by the method 
shown in Reaction Scheme VII from the appropriate corresponding 
triphenylphosphine intermediate of Formula IG.sub.2. 
As shown in Reaction Scheme VII, a compound of Formula IF.sub.1 reacts with 
lithium dialkyl cuprate at low temperature to give the compound of Formula 
IG.sub.1. The reaction is normally carried out in an ethereal solvent, 
preferably anhydrous diethyl ether, at about -25.degree. C. to -78.degree. 
C., preferably about -40.degree. C. The displacement of benzyl bromide 
with lithium dialkyl cuprate is known in the art, (See Organic Reactions, 
Vol. 13, pp. 252 and 401, (John Wiley & Sons) and G. H. Posner, 
Substitution Reactions using Organocopper Reagents; John Wiley & Sons 
(1980)) and is further illustrated in Example VII, below. 
The monobromo compound of Formula IF.sub.1 can be converted to the Wittig 
salt of Formula IG.sub.2 by reacting with triphenylphosphine in toluene. 
The reaction is preferably carried out between 60.degree. C. to 70.degree. 
C., preferably 65.degree. C., for a period of about 4 to 6, preferably 
about 5 hours. 
A compound of Formula IG.sub.2 reacts with one equivalent of DBU 
(1,8-diazabicyclo[5,4,0]-undec-7-ene) at from about -40.degree. C. to 
-60.degree. C., preferably -50.degree. C., to form the Wittig ylid. 
Subsequent quenching with an alkyl aldehyde gives the compound of Formula 
IG.sub.3. The reaction is normally carried out in dry tetrahydrofuran; and 
the product is recovered by conventional means. The compound of Formula 
IG.sub.3 represents a mixture of both (E) and (Z) enantiomers. 
K. Compounds in which R is Imidazolyl (Formula IH) 
Compounds of Formula I in which R is imidazolyl (compounds of Formula IH) 
can be prepared by the method shown in Reaction Scheme VIII, below: 
##STR30## 
As shown in Reaction Scheme VIII, the compounds of Formula VI are prepared 
from the corresponding methyl anthranilate derivative of Formula V. Methyl 
anthranilate, as well as variously substituted methyl 2-amino benzoates 
(methyl anthranilates) are commercially available, or can be prepared by 
treating the corresponding anthranilic acid with diazomethane in an inert 
organic solvent such as tetrahydrofuran or preferably ether at about 
0.degree. C., a method that is standard for the formation of methyl 
esters. Alternatively, variously substituted methyl 2-amino benzoates can 
be prepared by treating the corresponding isatoic anhydride with methanol 
in the presence of base such as sodium methoxide or dimethylaminopyridine, 
preferably dimethylaminopyridine, according to the literature methods such 
as that reported by M. C. Venuti, Synthesis, 266 (1982), R. P. Straiger 
and E. B. Miller, J. Org. Chem., 24, 1214 (1959). 
The corresponding unsubstituted or appropriately substituted anthranilate 
of Formula V is treated with about 0.5 to 1 equivalent of trichloromethyl 
chloroformate in tetrahydrofuran at room temperature for a period of about 
1 to 2, preferably about 1.5 hours; the resulting carbamyl chloride 
derivative is then refluxed with about 1 to 2 equivalents of 
4(N-triphenylmethyl)imidazolylmethanol, or another appropriate imidazolyl 
alcohol, in the presence of 5 to 10 equivalents of a tertiary amine, 
preferably triethylamine, for a period of about 0.5 to 2 hours. The 
product, a compound of Formula VI, is isolated by conventional means. The 
compound of Formula IH is obtained by base hydrolysis of the compound of 
Formula VI to the corresponding carboxylic acid, followed by cyclization 
with DCC or EDC in an inert solvent such as dichloromethane or 
tetrahydrofuran. The cyclization reaction takes place at room temperature 
over a period of about 1 to 3 hours, and the final product is then 
isolated by conventional means. 
L. Compounds of Formula I in which an R' is Alkyl at the 5-Position and a 
Second R' is Nitro or Amino at the 7-Position (Formulas IA and IB) 
Compounds of Formula I which bear a lower alkyl R' substituent at the 
5-position and a nitro or amino R' substituent at the 7-position 
(compounds of Formulas IA and IB) can be prepared by the procedures set 
forth in Reaction Schemes I and II, or alternatively, by the procedure 
illustrated below in Reaction Scheme IX. 
##STR31## 
As shown in Reaction Scheme IX, the appropriately substituted ethyl 
anthranilate of Formula VII is converted to the carbamoyl chloride 
derivative by treatment with about 0.5 to 1, preferably about 0.75 
equivalents of trichloromethyl chloroformate in ethyl acetate at room 
temperature for a period of about 2 to 3, preferably 2, hours. The 
resulting carbamoyl chloride derivative is then quenched with about a five 
fold excess of an appropriate alcohol of the formula HOAR (in which A and 
R have the definitions given herein), and a base such as pyridine or 
triethylamine. The product of Formula VIII is isolated by conventional 
means. Base hydrolysis of the compound of Formula VIII is then carried out 
in about a 1:1 mixture of aqueous sodium hydroxide and 1,2-dimethoxyethane 
to give the carboxylic acid of Formula IX. The acid of Formula IX is then 
cyclized with DCC or EDC in an inert organic solvent to give the final 
product of Formula IA, which is then isolated by conventional means. 
The amino substituted compound of Formula IB can then be obtained form the 
corresponding nitro substituted compound of Formula IA by the procedure 
outlined in Reaction Scheme II. 
Preparation of the ethyl anthranilate starting materials of Formula VII can 
be accomplished by the method illustrated in Reaction Scheme X, as 
follows: 
##STR32## 
As shown in Reaction Scheme X, above, the di-nitro phenol derivative of 
Formula 2, which is either commercially available of readily prepared by 
standard known methods such as those illustrated in Preparation IV, 
Paragraph A, herein, is converted to the corresponding chloro-compound of 
Formula 3 according to the procedure described by B. Boothroyd and E. R. 
Clark, J. Chem. Soc., p. 1504, London (1953). Details of this reaction may 
also be had by reference to Preparation IV, Paragraph B, below. The 
compound of Formula 3 is then reacted at room temperature with about a 10 
fold excess of pentan-2,4-dione and about a 3-4 fold excess of sodium 
methoxide in the presence of HMPA as solvent, to give the 
(2-alkyl-4,6-dinitrophenyl)-diacetylmethane of Formula 4. The compound of 
Formula 4 is then cyclized in concentrated sulphuric acid at about 
100.degree.-120.degree. C., preferably about 110.degree. C. for a period 
of about 1 to 5, preferably about 3 hours, to give the 4-alkyl-6-nitro 
anthranil of Formula 5. Details of this procedure may be had by reference 
to the method described by I. R. Gambir and S. S. Joshi in the Indian 
Chem. Soc. Journal, V. 41, pp. 43-46 (1964), which is specifically 
illustrated in Preparation IV, Paragraph C, herein. Subsequent ring 
opening by treating the anthril of Formula 5 with potassium carbonate and 
ethanol at reflux temperature gives the ethyl 4-nitro-6-alkyl-2-amino 
benzoate of Formula VII. 
Certain compounds of this invention may be converted to their corresponding 
pharmaceutically acceptable acid addition salts by virtue of the presence 
of a basic amine nitrogen. These compounds may be converted from the free 
base form to various acid addition salts by treating with a stoichiometric 
excess of the appropriate organic or inorganic acid, such as, for example, 
phosphoric, pyruvic, hydrochloric or sulfuric acid and the like. 
Typically, the free base is dissolved in a polar organic solvent such as 
p-dioxane or dimethoxyethane, and the acid added thereto. The temperature 
is maintained between about 0.degree. C. and 50.degree. C. The resulting 
acid addition salt precipitates spontaneously or may be brought out of 
solution with a less polar solvent. 
The acid addition salts of the compounds of Formula may be decomposed to 
the corresponding free base by treating with a stoichiometric amount of a 
suitable base, such as potassium carbonate or sodium hydroxide, typically 
in the presence of aqueous solvent, and at a temperature of between about 
0.degree. C. and 50.degree. C. The free base form is isolated by 
conventional means, such as extraction with an organic solvent. 
Acid addition salts of the compounds of the invention may be interchanged 
by taking advantage of differential solubilities of the salts, 
volatilities or acidities of the acids, or by treating with an 
appropriately loaded ion exchange resin. For example, the interchange is 
effected by the reaction of a salt of the compounds of Formula I with a 
slight stoichiometric excess of an acid of a lower pKa than the acid 
component of the starting salt. This invention is carried out at a 
temperature between about 0.degree. C. and the boiling point of the 
solvent being used as the medium for the procedure. 
In summary, then, the compounds of Formula I can be prepared by the 
following last-step procedures: 
I. Cyclization of a compound of Formula II with a phenoxy-, cycloalkoxy- or 
alkoxy chloroformate to give a compound of Formula IA; 
II. Conversion of a nitro-substituted compound of Formula IA to give a 
compound of Formula IB; 
III. Acylation of an amine-substituted compound of Formula IA or IB to give 
a compound of Formula IC; 
IV. Carboalkoxylation of an amino-substituted compound of Formula IA or IB 
to give a compound of Formula ID; 
V. Conversion of an amino-substituted compound of Formula IA or IB to give 
a compound of Formula IE; 
VI. Halogenation of a compound of Formula I to give a compound of Formula 
IF.sub.1 or IF.sub.2. 
VII. Reaction of a compound of Formula IF.sub.1 with a lithium dialkyl 
cuprate to give a compound of Formula IG.sub.1. 
VIII. Reaction of a compound of Formula IF.sub.1 with triphenylphosphine to 
give a compound of Formula IG.sub.2. 
IX. Reaction of a compound of Formula IG.sub.2 with 
1,8-diazabicyclo[5,4,0']-undec-7-ene and an alkyl aldehyde to give a 
compound of Formula IG.sub.3. 
X. Reaction of a compound of Formula I with a stoichiometric excess of an 
acid to give a pharmaceutically acceptable non-toxic acid addition salt. 
XI. Reaction of a salt of a compound of Formula I with a base to give the 
corresponding free base. 
Utility and Administration 
The compounds of Formulas I and A have been shown in standard laboratory 
tests to inhibit a variety of physiologic enzymes, particularly serine 
proteases, including human leukocyte elastase, human thrombin, human 
urokinase, porcine acrosin, porcine pancreatic elastase, bovine cathepsin 
B, bovine chymotrypsin, and bovine trypsin. Accordingly, the compounds of 
the invention, their salts, esters, and/or pharmaceutical compositions 
thereof, may be used in inhibiting, preventing, or controlling physiologic 
conditions and disease states in animals which are known to involve 
enzymes, or may be used as contraceptives. 
Knowledge of the roles of enzymes in a wide variety of diseases is 
constantly growing. Recent reviews of the state of the art include 
"Protein Degradation in Health and Disease", Ciba Foundation Symposium 75, 
Excerpta Medica, Amsterdam, 1980; "Proteinases in Mammalian Cells and 
Tissues", A. J. Barrett, ed., North Holland Publishing Company, Amsterdam, 
1977; and "Proteases and Biological Control", E. Reich, D. B. Rifkin and 
E. Shaw, eds., Cold Spring Harbor Laboratory, 1975. 
Experimental evidence has revealed the roles of many enzymatic pathways in 
various physiologic conditions and disease states. Plasminogen activator 
(PA), a serine protease, causes the conversion of plasminogen to plasmin 
which in turn is responsible for fibrinolysis. This process is implicated 
in a number of systems requiring controlled local proteolysis, including 
inflammation (J. D. Vassalli, et al. Cell, 8, 271 [1976]), and cell 
migration and tissue remodeling, J. E. Valinski, Cell, 25, 471 (1981). The 
production and secretion of PA is also correlated with certain human 
disorders such as arthritis (Neats, et al., Nature [London], 286, 891, 
1980; Hamilton, et al., J. Exp. Med., 155, 1702 [1982]) and the expression 
of transformed phenotypes, D. B. Rifkin, et al., in Proteases and 
Biological Control, D. Rifkin, E. Reich, E. Shaw, eds., Cold Spring 
Harbor, 1975, pp. 841-847. 
There is considerable evidence that plasminogen activator (such as 
urokinase), leukocyte elastase, and/or related enzymes play a role in 
tumor cell metastasis (Salo,, et al., Int. J. Cancer, 30, 669-673, 1973; 
Kao, et al., Biochem. Biophys., Res. Comm., 105, 383-389, 1982; Powers, J. 
C., in Modification of Proteins, R. E. Feeney and J. R. Whitaker, eds., 
Adv. Chem. Ser. 198, Amer. Chem. Soc., Washington, D.C., pp. 347-367, 
1982), suggesting that compounds of this invention may have 
anti-metastatic activity. 
Other evidence suggests an antiparasitic role for the compounds of this 
invention (Aoki, T., et al., Mol. Biochem., Parasitol, 8, 89-97, 1983). 
Pulmonary emphysema is a disease characterized by a progressive loss of 
lung elasticity due to the destruction of lung elastin and alveoli. It is 
widely held that the destructive changes in lung parenchyma associated 
with pulmonary emphysema are mediated in large part by unrestrained 
proteolytic activity in lung connective tissue. (A. Janoff, Chest, 83, 
54-58 [1983]. A number of proteases have been shown to induce 
emphysematous lesions in animals when instilled in lungs (V. Marco, et 
al., Am. Rev. Respir. Dis., 104, 595-8, 1971; P. D. Kaplan, J. Lab. Clin, 
Med., 82, 349-56 (1973)). In particular, human leukocyte elastase has been 
shown to produce emphysema in animals (A. Janoff, ibid, 115, 461-78 
(1977)). Prophylactic administration of an inhibitor of elastase 
significantly diminishes the extent of elastase induced emphysema in 
hamsters (J. Kleinerman, et al., ibid, Am. Rev. Respir. Dis., 121, 381-7, 
1980). 
Leukocyte elastase and other mediators of inflammation appear to play a 
role in such acute and high-risk diseases as mucocutaneous lymph node 
syndrome (Rieger, et al., Eur. J. Pediatr., 140, 92-97, 1983), and adult 
respiratory distress syndrom (Stockley, R. A., Clinical Science, 64, 
119-126, 1983; Lee, et al., N. Eng. J. Med., 304, 192-196, 1981; Rinaldo, 
ibid, 301, 900-909, 1982. 
Oral anticoagulants are some of the most important drugs for the prevention 
and treatment of a variety of venous and, to a lesser extent, arterial 
thromboembolic disorders (R. A. O'Reilly in "The Pharmacological Basis of 
Therapeutics", 6th Ed., A. G. Goodman, L. S. Goodman, A. Gilman, eds., 
1980). The enzymes that participate in the cascade leading to blood 
coagulation are proteases. The coagulation of blood entails the formation 
of fibrin by the interaction of more than a dozen proteins in a cacading 
series of proteolytic reactions. Inhibition of these proteinases should 
block fibrin formation and hence inhibit coagulation. For example, 
inhibition of thrombin limits the formation of fibrin and is regarded as 
an approach to thromboembolic therapy. 
However, anticoagulants that are in current use and that affect clotting 
factors do not have a direct onset of action. Consequently, prothrombin 
time must be monitored, as the degree of Vitamin K antagonism Varies from 
individual to individual. 
Thus there is a critical need for new anticoagulants which have a direct 
onset of action. Pulmonary embolism (PE), for example, is a common 
complication that usually affects patients who are hospitalized for other 
medical or surgical problems (A. A. Sasahara, et al., JAMA, 249, 2945 
(1983) and references therein). The mortality of undiagnosed and therefore 
untreated PE is relatively high, ranging from about 18% to 35%. Patients 
undergoing total hip or knee replacement are at extremely high risk for 
development of deep vein thrombosis, with a reported incidence of 45% to 
70% in untreated patients S. Sagar, et al., Lancet, 1, 1151 (1978)). 
Pancreatitis is a disease which affects large numbers of people includng 
patients having acute alcoholic, acute biliary traumatic and 
post-operative pancreatitis. Furthermore, with the high incidence of 
alcoholism, 10,000,000 alcoholics in the U.S. alone, acute and chronic 
relapsing pancreatitis are seen with increasing frequency. Geokas, et al. 
has proposed that an effective therapy for acute pancreatitis might be 
achieved by the use of "a combination of a low molecular weight specific 
active-site inhibitors for trypsin, chymotrypsin, and elastase", (Am. J. 
Pathol, 1981, 105, 31-39). 
Enzymes possessing cathepsin B-like activity have also received attention 
because of their extracellular release by neoplastic epithelial cells 
(Pietras, J. Histochem. Cytochem., 29, 440-450 1981), their presence in 
the interstitial fluid (Sylven, et al., Cancer Res., 20, 831-836, 1960; 
Eur. J. Cancer., 463-474, 1968; Virchows Arch. B. Cell Pathol., 17, 
97-112, 1974), and in the invasive zone of malignant tissue (R. R. 
Labrosse, Mol. Cell Biochem., 19, 181-189, 1978) and their enhanced 
secretion by malignant and metastatic tumors (B. F. Sloane, Cancer Res., 
42, 980-986, 1982). 
Proteolytic cleavage of precursors is an essential step in the replication 
of many animal viruses, and there is considerable evidence that protease 
inhibitors can be effective anti-viral agents (Korant, B. D., (1975) in 
"Proteases and Biological Control"). Such viruses include influenza 
(Chirov, O. P. et al. (1981) Vopr. Virusol. 6, 677-687). In Sendai virus, 
for example, a host trypsin-like protease is essential for infectivity 
(Scheid, A., and Choppin, P. (1975) in "Proteases and Biological 
Control"). It is reasonable then that compounds of this invention could 
play a role in amelioration of viral diseases. 
Acrosin is a unique serine proteinase which is present in mammalian sperm 
acrosomes (L. J. D. Zaneveld (1975) in "Proteases and Biological Control", 
pp. 683-706; R. F. Parrish, Int. J. Biochem., 10, 391-395 (1979)). Since 
acrosin activity is required for fertilization, it is a rational target 
for birth control. Further, the inhibition of acrosin is known to prevent 
fertilization (Zaneveld, L. J. D., et al., (1979), Biol. Repr. 20, 
1045-1054), supporting a role for acrosin inhibitors as contraceptives. 
Initial screening tests to determine enzyme-inhibitory potential can be 
performed with commercially available enzyme substrates such as peptidyl 
amides of 4-methyl-7-amino coumarin or 4-nitroaniline. The assays are 
performed by mixing the substrate and enzyme of interest in an appropriate 
buffer, and monitoring the rate of enzyme inhibition 
spectrophotometrically. The reaction rate is monitored continuously either 
by fluorescence (for coumarin substrates) or absorbance (for nitroanilide 
substrates) until a constant reaction rate is established. A solution of 
the compound to be tested in an appropriate solvent, such as a 5 to 20 
millimolar solution in dimethyl sulfoxide, is then added, and the increase 
in fluorescence or absorbance is monitored until a new stable rate is 
achieved. This is repeated for several concentrations of test compound 
solution, and the inhibition constant is calculated by non-linear multiple 
regression fit to the appropriate equation. The compounds of Formula I 
have been tested in assays of this type and have demonstrated marked 
inhibitory activity against human leukocyte elastase, human thrombin, 
human urokinase, porcine acrosin, porcine pancreatic elastase, bovine 
chymotrypsin and bovine and human trypsin. Some of the compounds of 
Formula I have also been tested and shown to be active in inhibiting the 
degradation of basement membrane by macrophages, tumor cells and elastase. 
More detailed descriptions of several of these assays may be found in the 
Examples, below. 
Administration of the active compounds and salts described herein can be 
via any of the accepted modes of administration for systemically active 
therapeutic medicaments. These methods include oral, parenteral and 
otherwise systemic, aerosol or topical forms. 
Depending on the intended mode of administration, the compositions used may 
be in the form of solid, semi-solid or liquid dosage forms, such as, for 
example, tablets, suppositories, pills, capsules, powders, liquids, 
aerosols, suspensions, or the like, preferably in unit dosage forms 
suitable for single administration of precise dosages. The compositions 
will include a conventional pharmaceutical carrier or excipient and an 
active compound of Formula I or A or the pharmaceutically acceptable salts 
thereof and, in addition, may include other medicinal agents, 
pharmaceutical agents, carriers, adjuvants, etc. 
For solid compositions, conventional non-toxic solid carriers includes, for 
example, pharmaceutical grades of mannitol, lactose, starch, magnesium 
stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium 
carbonate, and the like may be used. The active compound as defined above 
may be formulated as suppositories using, for example, polyalkylene 
glycols, for example, propylene glycol, as the carrier. Liquid 
pharmaceutically administerable compositions can, for example, be prepared 
by dissolving, dispersing, etc. an active compound as defined above and 
optional pharmaceutical adjuvants in a carrier, such as, for example, 
water, saline, aqueous dextrose, glycerol, ethanol, and the like, to 
thereby form a solution or suspension. If desired, the pharmaceutical 
composition to be administered may also contain minor amounts of nontoxic 
auxiliary substances such as wetting or emulsifying agents, pH buffering 
agents and the like, for example, sodium acetate, sorbitan monolaurate, 
triethanolamine sodium acetate, triethanolamine oleate, etc. Actual 
methods of preparing such dosage forms are known, or will be apparent, to 
those skilled in this art; for example, see Remington's Pharmaceutical 
Sciences, Mack Publishing Company, Easton, Pa, 15th Edition, 1975. The 
composition or formulation to be administered will, in any event, contain 
a quantity of the active compound(s) in an amount effective to alleviate 
the symptoms of the subject being treated. 
Parenteral administration is generally characterized by injection, either 
subcutaneously, intramuscularly or intravenously. Injectables can be 
prepared in conventional forms, either as liquid solutions or suspensions, 
solid forms suitable for solution or suspension in liquid prior to 
injection, or as emulsions. Suitable excipients are, for example, water, 
saline, dextrose, glycerol, ethanol or the like. In addition, if desired, 
the pharmaceutical compositions to be administered may also contain minor 
amounts of non-toxic auxiliary substances such as wetting or emulsifying 
agents, pH buffering agents and the like, such as for example, sodium 
acetate, sorbitan monolaurate, triethanolamine oleate, etc. 
For the compounds of Formula I, either oral or nasal (bronchial) 
administration is preferred, depending on the nature of the disorder being 
treated. 
For oral administration, a pharmaceutically acceptable non-toxic 
composition is formed by the incorporation of any of the normally employed 
excipients, such as, for example pharmaceutical grades of mannitol, 
lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, 
glucose, sucrose, magnesium, carbonate, and the like. Such compositions 
take the form of solutions, suspensions, tablets, pills, capsules, 
powders, sustained release formulations and the like. Such compositions 
may contain 1%-95% active ingredient, preferably 25-70%. 
Oral and nasal administration to the lungs can also be effected by aerosol 
delivery forms. For aerosol administration, the active ingredient is 
preferably supplied in finely divided form along with a surfactant and a 
propellant. Typical percentages of active ingredients are 0.01 to 20% by 
weight, preferably 0.04 to 1.0%. 
Surfactants must, of course, be non-toxic, and preferably soluble in the 
propellant. Representative of such agents are the esters or partial esters 
of fatty acids containing from 6 to 22 carbon atoms, such as caproic, 
octanoic, lauric, palmitic, stearic, linoleic, linolenic, olestearic and 
oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride 
such as, for example, ethylene glycol, glycerol, erythritol, arabitol, 
mannitol, sorbitol, the hexitol anhydrides derived from sorbitol (the 
sorbitan esters sold under the trademark "Spans") and the polyoxyethylene 
and polyoxypropylene derivatives of these esters. Mixed esters, such as 
mixed or natural glycerides may be employed. The preferred surface-active 
agents are the oleates or sorbitan, e.g., those sold under the trademarks 
"Arlacel C" (Sorbitan sesquioleate), "Span 80" (sorbitan monooleate) and 
"Span 85" (sorbitan trioleate). The surfactant may constitute 0.1-20% by 
weight of the composition, preferably 0.25-5%. 
The balance of the composition is ordinarily propellant. Liquefied 
propellants are typically gases at ambient conditions, and are condensed 
under pressure. Among suitable liquefied propellants are the lower alkanes 
containing up to five carbons, such as butane and propane; and preferably 
fluorinated or fluorochlorinated alkanes, such as are sold under the 
trademark "Freon." Mixtures of the above may also be employed. 
In producing the aerosol, a container equipped with a suitable valve is 
filled with the appropriate propellant, containing the finely divided 
active ingredient and surfactant. The ingredients are thus maintained at 
an elevated pressure until released by action of the valve. 
For topical administration, these compositions comprise an effective amount 
of a compound of this class in admixture with a pharmaceutically 
acceptable non-toxic carrier. A suitable range of composition would be 
0.1%-10% active ingredient, and the balance carrier, preferably 1-2% 
active ingredient. The concentration of active ingredient in 
pharmaceutical compositions suitable for topical application will vary 
depending upon the particular activity of the compound used in conjunction 
with the condition and subject to be treated. Suitable carriers or 
medicament vehicles for topical application of these compounds include 
creams, ointments, lotions, emulsions, solutions and the like. 
For example, a suitable ointment for topical application of compounds of 
the invention contains 15 to 45 percent of a saturated fatty alcohol 
having 16 to 24 carbon atoms such as cetyl alcohol, stearyl alcohol, 
behenyl alcohol, and the like and 45 and 85 wt. percent of a glycol 
solvent such as propylene glycol, polyethylene glycol, dipropylene glycol, 
and mixtures thereof. The ointment can also contain 0 to 15 wt. percent of 
a plasticizer such as polyethylene glycol, 1,2,6-hexanetriol, sorbitol, 
glycerol, and the like; 0 to 15 wt. percent of a coupling agent such as a 
saturated fatty acid having from 16 to 24 carbon atoms, e.g., stearic 
acid, palmitic acid, behenic acid, a fatty acid amide e.g., oleamide, 
palmitamide, stearamide, behenamide and an ester of a fatty acid having 
from 16 to 24 carbon atoms such as sorbitol monostearate, polyethylene 
glycol monostearate, polypropylene glycol or the corresponding mono-ester 
of other fatty acids such as oleic acid and palmitic acid; and 0 to 20 
wt. percent of a penetrant such as dimethyl sulfoxide or 
dimethylacetamide. 
The amount of active compound administered will of course, be dependent on 
the subject being treated, the severity of the affliction, the manner of 
administration and the judgement of the prescribing physician. However, an 
effective dosage is in the range of 1-100 mg/kg/day, preferably about 25 
mg/kg/day. For an average 70 kg human, this would amount to 70 mg-7 g per 
day, or preferably about 1.5 g/day. 
The following examples serve to illustrate the invention. They should not 
be construed as narrowing or limiting its scope. 
PREATION I 
Preparation of Substituted anthranilic acids of Formula II 
A. Preparation of 4-ethyl anthranilic acid and 6-ethyl-anthranilic acid 
4-ethyl anthranilic acid and 6-ethyl anthranilic acid were prepared 
according to Baker's procedure, as described in J. Org. Chem. 17, 141, 
(1952) and further detailed below. 
(i) Preparation of m-ethyl-alpha-isonitrosoacetanilide. 
In a 5 liter round-bottom flask equipped with overhead stirrer and 
condensers were placed 74.2 gm. of chloral dihydrate and 900 ml of water. 
To this solution was then added, sequentially, 107.2 gm of anhydrous 
sodium sulfate, a solution of 50 gm of m-ethyl aniline dissolved in 248 ml 
of water and 42 ml of concentrated hydrochloric acid, and lastly, a 
solution of 90.8 gm of hydroxylamine hydrochloride in 412 ml of water. The 
mixture was slowly heated over a period of 45 minutes to a temperature of 
95.degree. C. The heating mantle was then removed and the flask rapidly 
cooled to room temperature by immersion in an ice-bath. The crude 
isonitrosoacetanilide was collected by suction filtration and washed with 
water. The product was then further purified by the following procedure: 
the crude isonitrosoacetanilide was dissolved in 500 ml of a 4M sodium 
hydroxide solution, transferred to a separatory funnel and washed with 
ether (3.times.300 ml). The alkaline phase was then treated with charcoal, 
filtered through Celite and strongly acidified with concentrated 
hydrochloric acid. The precipitated m-ethyl-alpha-isonitrosoacetanilide 
was collected by filtration and dried under vacuum, mp. 
140.degree.-142.degree. C. 
(ii) Preparation of 4-ethyl and 6-ethyl isatin. 
A 1 liter round-bottom flask containing 370 ml of concentrated sulfuric 
acid and 30 ml of water was heated to 60.degree. C. 
m-Ethyl-alpha-isonitrosoacetanilide (64 gm) was added at such a rate as to 
maintain the temperature between 60.degree. and 65.degree. C. After the 
addition was completed, the mixture was heated to 80.degree. C. for 10 
minutes. The flask was then cooled to room temperature and poured onto 8 
to 10 times its volume of ice. After standing for one-half hour, the crude 
isatin mixture was collected by filtration and washed well with water. The 
crude extract was then dissolved in about 300 ml of a 3M sodium hydroxide 
solution by heating on a steam bath, treated with charcoal and filtered 
through Celite. On acidification to pH 6-7 with concentrated hydrochloric 
acid, a gummy material appeared and was removed by filtration through 
Celite. The solution was then acidified to pH 4 and the 4-ethyl isatin was 
collected by filtration and washed with water: Yield 14.6 gm, mp 
128.degree.-136.degree. C. The cooled filtrate was then strongly acidified 
with concentrated hydrochloric acid and collected by filtration to give 
the 6-ethyl isatin: Yield 16.4 gm (28%), mp. 171.degree.-173.degree. C. 
(iii) Preparation of 4-ethyl-anthranilic acid 
In a 500 ml flask, was placed 16.84 gm of 6-ethyl isatin which was covered 
with 216 ml of 1.5M sodium hydroxide solution. With stirring, the mixture 
was warmed to 50.degree. C. Heating was discontinued and the solution was 
treated with a 30% solution of hydrogen peroxide (24 ml) which was added 
at such a rate to maintain the temperature at between 50.degree. to 
65.degree. C. The mixture was left to slowly cool to room temperature and 
was then acidified to pH 4 with concentrated hydrochloric acid. The 
precipitated product was then collected by filtration: mp. 
117.degree.-120.degree. C.; yield 8.93 gm. 
(iv) Preparation of 6-ethyl-anthranilic acid. 
Oxidation of 9.6 gm of 4-ethyl isatin according to the method described in 
(iii), above, gave 7.3 gm of the title compound: mp. 
99.degree.-104.degree. C. 
B. In a similar manner, but replacing m-ethyl aniline with other anilines, 
the following exemplary compounds of Formula VII are prepared. 
6-butyl-anthranilic acid; 
4-iodo-anthranilic acid; 
6-methyl anthranilic acid; 
4,6-dimethyl-anthranilic acid; 
3,5-dimethyl-anthranilic acid; 
3,5.6-trimethylanthranilic acid; 
3,6-dimethylanthranilic acid; 
5-butyl-anthranilic acid; 
4-methoxy-6-methyl-anthranilic acid; and 
4,6-dichloro-anthranilic acid. 
C. Preparation of 6-methoxyanthranilic acid by reduction of the 
corresponding aromatic nitro compounds was carried out in accordance with 
Paquette's procedure, J. Am. Chem. Soc., 99, 3734, (1981), mp. 
71.degree.-75.degree. C., which can also be used to prepare other 6-alkoxy 
anthranilic acids. 
D. Preparation of 4-nitro-6-iodo-anthranilic acid and 
4-nitro-6-bromo-anthranilic acid are prepared according to the procedure 
of I. R. Gambir and S. S. Joshi, Indian Chem. Soc., Journal 43-46, (1964). 
In a similar manner, the following compounds can be prepared: 
4-nitro-6-ethyl-anthranilic acid; 
4-nitro-6-methyl-anthranilic acid; and 
4-nitro-6-propyl-anthranilic acid. 
E. Preparation of 5-Alkoxyanthranilic Acid and 5-Alkylthioanthranilic acid 
5-Methoxyanthranilic acid and 4-methylthioanthranilic acid were prepared 
according to the procedure of J. W. Tilley, J. Kudless, R. W. Kierstead, 
Organic Preparations Procedure Int. 13(3-4), 189-196 (1981). 
In a similar manner the following cmpounds are prepared: 
5-ethoxy-anthranilic acid; 
5-butoxy-anthranilic acid; 
5-hexoxy-anthranilic acid; 
5-isopropoxy-anthranilic acid, 
5-ethylthio-anthranilic acid. 
5-methylthio-anthranilic acid; 
5-n-pentylthio-anthranilic acid; 
5-n-propylthio-anthranilic acid. 
F. Preparation of 4-N,N-Dimethylamino-Anthranilic Acid 
4-N,N-dimethylamino-anthranilic acid was prepared according to the 
procedure of D. H. Klaubert, J. H. Sellstedt, C. J. Guinosso, R. J. 
Capetola, J. S. C. Bell, J. Med. Chem., 1981, 24, 742-748. 
In a similar manner the following compounds are prepared: 
4-N,N-diethylamino-anthranilic acid; and 
4-N,N-dipropylamino-anthranilic acid. 
PREATION II 
Phenyl-, Cycloalkyl- and Alkyl-Chloroformates of Formula III 
A. Preparation of N-Butyl Chloroformate 
Phosgene was passed into dry ether until saturated (15-20% w/v). n-Butyl 
alcohol (10 gm) in dry ether (50 ml) was treated with ethereal phosgene 
(1.1 mol.) at room temperature until the reaction was complete. Removal of 
the solvent in vacuo gave n-butyl chloroformate in quantitative yield. In 
representative cases these compounds were purified by distillation, but 
this is not necessary for the subsequent cyclization. For the preparation 
of the chloroformates of hindered alcohols, quinoline can be added as a 
catalyst, quinoline hydrochloride being subsequently removed by 
filtration. 
B. Thus, in a similar manner, but replacing the n-butyl alcohol with 
n-hexyl alcohol, s-butyl alcohol, isobutyl alcohol, n-pentyl alcohol, 
benzyl alcohol, phenol, phenethyl alcohol, 1-phenyl-2-methyl-propanol, 
1-phenyl-3-methyl-2-butanol, 1-phenyl-pentanol, salicyl alcohol, 
procatechol, citronehol, (-)-menthol, cholestanol, and the like, the 
following compounds of Formula III are prepared: 
methyl chloroformate; 
ethyl chloroformate; 
n-propyl chloroformate; 
isopropyl chloroformate; 
n-butyl chloroformate; 
s-butyl chloroforomate; 
isobutyl chlorormate; 
n-pentyl chlorformate; 
n-hexyl chloroformate; 
n-octyl chloroformate; 
phenyl chloroformate; 
benzyl chloroformate; 
phenethyl chloroformate; 
1-phenylpentyl chloroformate; 
1-phenyloctyl chloroformate; 
1-phenyl-2-methyl-propyl chloroformate; 
1-phenyl-3-methyl-2-butyl chloroformate; 1; 
1-(3-methoxyphenyl)-octyl chloroformate; 
1-(4-isobutoxyphenyl)-heptyl chloroformate; 
2-carbomethoxyphenyl chloroformate; 
(-)-menthyl chloroformate; 
cholesteryl chloroformate; 
cyclopropyl chloroformate; 
cyclobutyl chloroformate; 
cyclohexyl chloroformate; 
1-cyclopropyl-ethyl chloroformate; 
1-cyclobutyl-methyl chloroformate; 
1-cyclohexyl-propyl chloroformate; 
1-(2-ethylcyclohexyl)methyl chloroformate; 
1-(4-nitrocyclohexyl)-pentyl chloroformate; and 
1-(2-dimethylaminobenzyl)chloroformate. 
PREATION III 
Acid Anhydrides of Formula IV 
A. Preparation of butyric anhydride 
Molar quantities of butyric acid and butyryl chloride are heated together 
on a water-bath for 1 hour and then boiled for 7 hours in an oil-bath. 
Butyric anhydride, b.p. 198.degree.-199.degree. C./765 mm, is obtained on 
distillation of the resulting mixture. 
B. In a similar manner, but starting instead with acetyl chloride and 
sodium acetate, acetic anhydride is obtained. 
C. In like manner, other symmetrical and mixed acid anhydrides are obtained 
from the corresponding free acids and acid chlorides, or alternatively 
from the acid chlorides and alkali salts of the carboxylic acids, 
including; 
hexanoic anhydride; and 
acetic propionic anhydride. 
PREATION IV 
Ethyl 2-amino-6-ethyl-4-nitrobenzoate and Related Compounds of Formula VII 
A. Preparation of 2,4-dinitro-6-ethylphenol 
(i) Concentrated sulfuric acid (25 g) was added to 2-ethylphenol (25 g, 
Aldrich) with swirling. The solution was heated on a steam bath for one 
hour, cooled and 25 ml water added. The solution was placed in a dropping 
funnel and added dropwise to 70% nitric acid (40 g), with cooling provided 
by an ice-salt bath cooled by glycol. The solution in the dropping funnel 
was added over 1.5 to 2 hours, with the temperature kept below 0.degree. 
C. The resulting mixture was stirred at 0.degree. C. for three hours, the 
ice bath removed, and the mixture further stirred overnight at room 
temperature. The mixture was then heated on a steam bath for one hour, 
cooled and 50 ml of water added. Following extracted with diethyl ether, 
the ether layer was washed with brine, dried over MgSO.sub.4 and 
evaporated to a dark oil which was columned on silica gel using 10% ethyl 
acetate-petroleum ether. The combined filtrates gave a yellow-orange oil, 
which solidified upon being pumped dry to give 2,4-dinitro-6-ethylphenol, 
yield 34 gm. 
(ii) Proceeding in a similar matter but starting instead with 
2-propylphenol, 2-4-dinitro-6-propylphenol was obtained as an oil. 
(iii) Similarly prepared are the following compounds of Formula 2: 
2,4-dinitro-6-isopropylphenol; 
2,4-dinitro-6-butylphenol; and 
2,4-dinitro-6-isobutylphenol. 
B. Preparation of 1-chloro-2,4-dinitro-6-ethylbenzene 
(i) 2,4-Dinitro-6-ethylphenol (10 g) was placed in a 250 ml round bottom 
flask, and phosphorous oxychloride (60 ml, Fisher) was added. 
N,N-diethylamiline (15 ml, Aldrich was added portionwise, and the mixture 
became hot. The flask was placed under a condenser equiped with a drying 
tube heated on a steam bath for two hours and cooled. The mixture was then 
carefully poured onto ice, with stirring and extracted with ethyl acetate. 
The ethyl acetate layer was washed with brine, dried over MgSO.sub.4 and 
evaporated in a dark oil which was columned on silica gel using 10% ethyl 
acetate petroleum ether. The evaporated filtrates gave a reddish oil, 
which solidified upon being pumped dry. The solid was recrystallized from 
ethyl acetate-petroleum ether to give pale yellow needles of 
1-chloro-2,4-dinitro-6-ethylbenzane. 
Yield: 8.1 g, m.p. 41.degree.-44.degree. C. 
IR: 3400 cm, 3090 cm, 2980 cm, 1800 cm (w), 1540 cm, 1345 cm 
(ii) Proceeding in a similar manner, the following compounds of Formula 3 
were prepared: 
1-chloro-2,4-dinitro-6-methylbenzene mp; 
1-chloro-2,4-dinitro-6-propylbenzene oil. 
(iii) Similarly, but starting instead with other appropriate corresponding 
compounds of Formula 2, the following compounds of Formula 3 are prepared: 
1-chloro-2,4-dinitro-6-isopropylbenzene; 
1-chloro-2,4-dinitro-6-butylbenzene; and 
1-chloro-2,4-dinitro-6-isobutylbenzene. 
C. Preparation of (6-ethyl-2,4-dinitrophenyl)-diacetylmethane 
(i) Sodium methoxide (8.16 g) was placed in a flask contain 
hexamethylphosphoramide (50 ml, Aldrich). 2,4-pentanedione (50 ml, 
Aldrich) was added and the mixture stirred while gently heated with a 
heating mantle (Variac 25/140) for thirty minutes. 
6-Ethyl-2,4-dinitrochlorobenzene (10 g) in some dry tetrahydrofuran was 
added, and the mixture heated at the same setting for two more hours. The 
reaction mixture was cooled and partitioned between ethyl acetate and 5N 
hydroclhoric acid. 
The ethyl acetate layer was washed with 5N HCl, water and brine, dried over 
MgSO.sub.4 and evaporated to a dark oil. 350 ml of 10% ethyl 
acetate-petroleum ether was added, and a pale ywellow solid precipitated 
out. This was colummed on silica gel using 20% ethyl acetate-petroleum 
ether to give 5.4 g of (2,4-dinitro-6-ethyl)-diacetylmethane, 
m.p.: 126.degree.-128.degree. C., 
IR: 3100 cm, 2980 cm, 1525 cm, 1345 cm. 
(ii) Proceeding in the same manner, the following compounds of Formula 4 
were prepared: 
(6-methyl-2,4-dinitrophenyl)-diacetulmethane, m.p. 145.degree.-147.degree. 
C.; and 
(6-propyl-2,4-dinitrophenyl)-diacetylmethane, m.p. 
147.degree.-147.5.degree. C. 
(iii) In like manner, but substituting other corresponding compounds of 
Formula 3, the following compounds of Formula 4 are prepared: 
(6-isopropyl-2,4-dinitrophenyl)-diacetylmenthane; 
(6-butyl-2,4-dinitrophenyl)-diacetylmenthane; and 
(6-isobutyl-2,4-dinitrophenyl)-diacetylmethane. 
B. Preparation of 4-ethyl-6-nitro-anthranil 
(i) (6-ethyl-2,4-dinitrophenyl)-diacetylmethane (5 g) was dissolved in 
concentrated sulfuric acid and heated to 90.degree.-110.degree. C. in an 
oil bath for three hours. The mixture was poured onto ice, with stirring, 
and extracted with methylene chloride. The resulting emulsion was filtered 
through Celite to separate the layers. The methylene chloride layer was 
dried over MgSO.sub.4 and evaporated to a dark solid which was 
re-dissolved in some methylene chloride and colummed on silica using 10% 
ethyl acetate-petroleum ether. Evaporation of the filtrate 2.57 g of 
4-ethyl-6-nitro-anthranil as an orange solid, 
m.p 69.degree.-72.degree. C., 
IR: 3140 cm, 3100 cm, 2970 cm, 1550 cm, 740 cm. 
(ii) Proceeding in the same manner, but starting with other appropriate 
compounds of Formula 4, the following compounds of Formula 5 were 
prepared: 
4-methyl-6-nitro-anthranil, m.p. 158.degree.-160.degree. C.; and 
4-propyl-6-nitro-anthranil, m.p. 82.degree.-84.degree. C. 
(iii) In like manner, the following compounds of Formula 5 are prepared: 
4-isopropyl-6-nitro anthranil; 
4-butyl-6-nitro anthranil; and 
4-isobutyl-6-nitro anthranil 
E. Preparation of Ethyl-2-amino-6-ethyl-4-nitrobenzoate 
(i) 4-ethyl-6-nitro anthranil (2 g) was refluxed for three hours in ethanol 
with potassium carbonate. The reaction mixture was cooled, filtered and 
evaporated to a dark oily solid which was dissolved in ethyl acetate and 
dried. The residue was re-dissolved in methylene chloride and columned on 
silica gel using 15% ethyl acetate-petroleum ether. The residue from the 
filtrate was recrystallized from methylene chloride-petroleum ether to 
give 1.9 g ethyl-2-amino-6-ethyl-4-nitrobenzoate, m.p. 
68.degree.-70.degree. C., IR: 3490 cm, 3380 cm, 3080 cm, 2980 cm, 1690 cm, 
1620 cm, 1515 cm, 1350 cm. 
(ii) Proceeding in the same manner, but starting instead with other 
appropriate, 4-alkyl-6-nitro anthranils of Formula 5, the following 
compounds of Formula VII were prepared: 
ethyl 2-amino-6-methyl-4-nitrobenzoate, m.p. 67.degree.-68.degree. C.; and 
ethyl 2-amino-6-propyl-4-nitrobenzoate, m.p. 78.degree.-79.degree. C. 
(iii) In like manner, the following compounds of Formula VII are prepared: 
ethyl-2-amino-6-isopropyl-4-nitrobenzoate; 
ethyl-2-amino-6-butyl-4-nitrobenzoate; and 
ethyl-2-amino-6-isobutyl-4-nitrobenzoate. 
PREATION V 
A. Preparation of Ethyl 2-carbethoxyamino-6-ethyl-4-nitro-benzoate and 
Related Compounds of Formula VIII 
A solution of ethyl-2-amino-6-ethyl-4-nitro-benzoate (400 mg) in ethyl 
acetate (10 ml) was added dropwise to a solution of trichloromethyl 
chloroformate in ethylacetate (10 ml). A precipitate was formed 
immediately which dissolved on prolonged sitrring for 21/2 hours. A 
solution of ethanol (5 ml) and triethylamine (5 ml) was added. A yellowish 
precipitate was formed. After 1 hour the solution was partitioned between 
ethylacetate and water. The ethylacetate layer was washed with brine 
solution, dried over magnesium sulfate and evaporated to a reddish oil. 
The material was further purified by column chromatography on silica gel 
(10% EtOAc:pet.ether) to give a solid material, 
m.p. 47.degree.-48.degree. C., 
IR: 1530, 1710, 1740, 1610 cm. 
B. Proceeding in a similar manner but replacing the 
2-amino-6-ethyl-4-nitro-benzoate with other appropriate corresponding 
compounds of Formula VII, prepared as described in Preparation IV and 
replacing the ethanol with other appropriate alcohols, as desired, the 
follwoing compounds of Formula VIII are prepared: 
ethyl 2-carbobenzyloxyamino-6-ethyl-4-nitro-benzoate; 
ethyl 2-carboethoxyamino-6-methyl-4-nitro-benzoate; 
ethyl 2-carboisopropoxyamino-6-propyl-4-nitro-benzoate; and 
ethyl 2-carbocyclopropyloxyamino-6-isobutyl-4-nitro-benzoate. 
PREATION VI 
A. Preparation of 2-carboethoxyamino-4-nitro-6-ethyl benzoic acid and 
Related Compounds of Formula IX 
A solution of ethyl 2-carboethoxyamino-4-nitro-6-ethyl-benzoate in 
tetrahydrofuran (10 ml) and sodium hydroxide (20 ml, 10%) was stirred at 
room temperature for 20 hours. The solution was extracted with ethyl 
acetate. The aqueous layer was acidified to pH=1 with 6M HCl, and then 
immediately extracted with ethylacetate. The ethyl acetate extract was 
washed with water and dried over magnesium sulphate. Solvent evaporation 
gave a solid which was further recrystallized from methylene 
chloride:petroleum ether to yield 2-carboethoxyamino-4-nitro-6-ethyl 
benzoic acid, 
m.p. 121.degree.-123.degree. C. as orange crystals; 
IR: 1665, 1720, 1620, 1510, 2500-3200(br), 3500 cm. 
B. Proceeding in a similar manner, but replacing the 
2-carboethoxyamino-4-nitro-6-ethyl-benzoate with other compounds of 
Formula VIII, the preparation of which is described in Preparation V 
above, the following compounds of Formula IX are prepared: 
2-carbobenzyloxyamino-6-ethyl-4-nitro-benzoic acid; 
2-carboethoxyamino-6-methyl-4-nitro-benzoic acid; 
2-carboisopropoxy-amino-6-propyl-4-nitro-benzoic acid; 
2-carboisopropoxyamino-6-butyl-4-nitrobenzoic acid; and 
2-carbocyclopropyloxyamino-6-isobutyl-4-nitrobenzoate. 
EXAMPLE I 
A. Synthesis of 2-ethoxy-4H-3,1-benzoxazin-4-one, and Related Compounds of 
Formula IA 
To a solution of anthranilic acid (0.1 mol., 13.71 gm) in dry pyridine (100 
ml) at room temperature under anhydrous conditions was added ethyl 
chloroformate (4 equiv., 38.25 ml) in a dropwise manner over 15 minutes. 
After stirring for 2 hours, excess pyridine was removed under reduced 
pressure at 40.degree. C. (bath temperature) and the residue was stirred 
vigorously in ice cold water (250 ml) for 15 minutes. The pale yellow 
powder was collected by filtration, washed with water (100 ml), and 
air-dried to give 18.6 gm of crude product. The crude product was treated 
with active charcoal (2 gm) in ethyl acetate (150 ml) to afford a white 
solid after removal of solvent. Recrystallization from EtOAc-ether gave 
17.02 (89%) of the title compound, 2-ethoxy-4H-3,1-benzoxazin-4-one, as 
colorless crystals; m.p. 88.degree.-90.5.degree. C.; IR (KBr): 
.nu..sub.max 1760 cm.sup.-1 (C.dbd.O), 1630 cm.sup.-1 (C.dbd.N); H'NMR 
(CDCl.sub.3); .delta.1.46 ppm (t, J=7.1 Hz, 3H,)CH.sub.2 CH.sub.3), 4.53 
ppm (q, J=7.1 Hz, 2H, OCH.sub.2 CH.sub.3), 7.30-8.20 ppm (m, 4H, aromatic 
protons). 
B. In a similar manner, but substituting other appropriately substituted 
anthranilic acids (which may be commerially obtained or prepared as 
described in Preparation I, above), for the anthranilic acid, the 
following substituted compounds of Formula IA were prepared: 
2-ethoxy-5-methyl-4H-3,1-benzoxazin-4-one, m.p. 104.degree.-105.degree. C.; 
2-ethoxy-5-ethyl-4H-3,1-benzoxazin-4-one, m.p. 89.degree.-91.degree. C.; 
2-ethoxy-6-methylthio-4H-3,1-benzoxzazin-4-one, m.p. 
67.degree.-68.5.degree. C.; 
6-dimethylamino-2-ethoxy-4H-3,1-benzoxazin-4-one, m.p. 
93.degree.-95.degree. C.; 
7-carboethoxyamino-2-ethoxy-4H-3,1-benzoxazin-4-one, m.p. 
191.degree.-193.degree. C.; 
6,7-dimethoxy-2-ethoxy-4H-3,1-benzoxazin-4-one, m.p. 
169.degree.-170.degree. C.; 
2-ethoxy-7-nitro-4H-3,1-benzoxazin-4-one, m.p. 106.degree.-109.degree. C.; 
7-dimethylamino-2-ethoxy-4H-3,1-benzoxazin-4-one, m.p. 
196.degree.-198.degree. C.; 
2-benzyloxy-4H-3,1-benzoxazin-4-one; m.p. 88.degree.-89.5.degree. C.; and 
2-benzyloxy-6,7-dimethoxy-4H-3,1-benzoxazin-4-one, m.p. 
152.degree.-154.degree. C. 
C. In like manner, but replacing the ethyl chloroformate in Paragraph A, 
above, with other corresponding chloroformates of Formula III, (which may 
be commercially obtained, or prepared as described in Preparation II, 
above), the following compounds of Formula IA are prepared: 
2-methoxy-4H-3,1-benzoxazin-4-one; 
2-n-propoxy-4H-3,1-benzoxazin-4-one; 
2-isopropoxy-4H-3,1-benzoxazin-4-one; 
2-n-butoxy-4H-3,1-benzoxazin-4-one; 
2-s-butoxy-4H-3,1-benzoxazin-4-one; 
2-isobutoxy-4H-3,1-benzoxazin-4-one; 
2-n-pentoxy-4H-3,1-benzoxazin-4-one; 
2-n-hexoxy-4H-3,1-benzoxazin-4-one; 
2-n-octyloxy-4H-3,1-benzoxazin-4-one; 
2-phenyloxy-4H-3,1-benzoxazin-4-one; 
2-benzyloxy-4H-3,1-benzoxazin-4-one; 
2-phenethyloxy-4H-3,1-benzoxazin-4-one; 
2-phenylbutyloxy-4H-3,1-benzoxazin-4-one; 
2-phenyloctyloxy-4H-3,1-benzoxazin-4-one; 
2-(1-phenyl-2-methyl-propyl)oxy-4H-3,1-benzoxazin-4-one; 
2-(1-phenyl-3-methyl-2-butyl)oxy-4H-3,1-benzoxazin-4-one; 
2-(1-(3-methoxyphenyl)octyloxy-4H-3,1-benzoxazin-4-one; 
2-(1-(4-isobutoxyphenyl)heptyloxy-4H-3,1-benzoxazin-4-one 
2-(4-ethylbenzyl)oxy-4H-3,1-benzoxazin-4-one; 
2-(3,5-dimethoxybenzyl)oxy-4H-3,1-benzoxazin-4-one; 
2-carbomethoxyphenyloxy-4H-3,1-benzoxazin-4-one; 
2-citronyloxy-4H-3,1-benzoxazin-4-one; 
2-(-)-methyloxy-4H-3,-benzoxazin-4-one; 
2-cholesteryloxy-4H-3,1-benzoxazin-4-one; 
2-cyclopropyloxy-4H-3,1-benzoxazin-4-one; 
2-cyclobutyloxy-4H-3,1-benzoxazin-4-one; 
2-cyclohexyloxy-4H-3,1-benzoxazin-4-one; 
2-cyclopropylbutyloxy-4H-3,1-benzoxazin-4-one; 
2-(4-methylthiocyclohexyl)butyloxy-4H-3,1-benzoxazin-4-one; 
2-(3-methylpentoxy)-4H-3,1-benzoxazin-4-one; 
2-5-methylhexyloxy)-4H-3,1-benzoxazin-4-one; 
2-(3-methyl-4-ethylpentyl)oxy-4H-3,1-benzoxazin-4-one; 
2-(2-methylpentyl)oxy-4H-3,1-benzoxazin-4-one; 
2-(1-[2-ethylcyclohexyl]methyl)oxy-4H-3,1-benzoxazin-4-one 
2-(1-[4-nitrocyclohexyl]pentyl)oxy-4H-3,1-benzoxazin-4-one 
2-(1-[2-methylthiocyclopentyl]ethyl)oxy-4H-3,1-benzoxazin-4-one; and 
2-(1-dimethylaminobenzyl)oxy-4H-3,1-benzoxazin-4-one; 
D. Similarly, but further replacing the unsubstituted anthranilic acid with 
appropriate substituted anthranilic acids of Formula II, (which may be 
commercially obtained, or can be prepared as described in Preparation I, 
above), the following compounds of Formula IA are prepared: 
2-methoxy-5-methyl-4H-3,1-benzoxazin-4-one; 
2-isopropoxy-5-ethyl-4H-3,1-benzoxazin-4-one; 
2-n-butoxy-5-methyl-7-methoxy-4H-3,1-benzoxazin-4-one; 
2-s-butoxy-5-methylthio-4H-3,1-benzoxazin-4-one; 
2-isobutoxy-5-chloro-4H-3,1-benzoxazin-4-one; 
2-n-hexoxy-7-ethoxy-4H-3,1-benzoxazin-4-one; 
2-n-octyloxy-5-methyl-7-nitro-4H-3,1-benzoxazin-4-one; 
2-phenyloxy-5-ethyl-4H-3,1-benzoxazin-4-one; 
2-benzyloxy-5-methyl-4H-3,1-benzoxazin-4-one; 
2-phenethyloxy-5,7-dimethoxy-4H-3,1-benzoxazin-4-one; 
2-phenylbutyloxy-6-methylthio-4H-3,1-benzoxazin-4-one; 
2-phenyloctyloxy-5-methyl-6,7-dichloro-4H-3,1-benzoxazin-4-one; 
2-(1-phenyl-2-methyl-propyl)oxy-5-isopropoxy-4H-3,1-benzoxazin-4-one; 
2-(1-phenyl-3-methyl-2-butyl)oxy-5-methyl-4H-3,1-benzoxazin-4-one; 
2-(1-(4-isobutoxyphenyl)heptyloxy-5-methyl-7-methoxy-4H-3,1-benzoxazin-4-on 
e 
2-(4-ethylbenzyl)oxy-4H-3,1-benzoxazin-4-one; 
2-(3,5-dimethoxybenzyl)oxy-4H-3,1-benzoxazin-4-one; 
2-cyclopropyloxy-5-ethyl-4H-3,1-benzoxazin-4-one; 
2-cyclobutyloxy-6-methylthio-4H-3,1-benzoxazin-4-one; 
2-cyclohexyloxy-5-isopropyl-4H-3,1-benzoxazin-4-one; 
2-(5-methylhexyloxy)-4H-3,1-benzoxazin-4-one; 
2-(3-methyl-4-ethylpentyl)oxy-4H-3,1-benzoxazin-4-one; 
2-(2-methylpentyl)oxy-7-nitro-4H-3,1-benzoxazin-4-one; 
2-(1-[4-aminocyclohexyl]methyl)oxy-6-iodo-4H-3,1-benzoxazin-4-one; 
2-(1-N,N-dimethylaminobenzyl)oxy-6-N,N-dimethylamino-4H-3,1-benzoxazin-4-on 
e; 
E. Similarly, the following nitro-substituted 
2-oxy-4H-3,1-benzoxazin-4-ones of Formula IA are prepared from the 
correspondingly substituted nitro-substituted anthranilic acids. 
(4-nitro-anthranilic acid is commercially available. Alternatively, these, 
as well as the 5- and 6-nitro anthranilic acids can be prepared according 
to the method set forth in Preparation I, above.): 
2-ethoxy-7-nitro-4H-3,1-benzoxazin-4-one; 
2-benzyloxy-7-nitro-4H-3,1-benzoxazin-4-one; 
2-phenethyloxy-5-ethyl-7-nitro-4H-3,1-benzoxazin-4-one; 
2-cyclopropyloxy-5-propyl-7-nitro-4H-3,1-benzoxazin-4-one; 
2-cyclobutyloxy-5-thiomethyl-7-nitro-4H-3,1-benzoxazin-4-one; 
2-cyclohexylyoxy-6-nitro-4H-3,1-benzoxazin-4-one; 
2-phenylbutyloxy-7-nitro-4H-3,1-benzoxazin-4-one; 
2-cyclopropylbutyloxy-5-methyl-7-nitro-4H-3,1-benzoxazin-4-one; 
2-(4-ethylbenzyl)oxy-5-methyl-7-nitro-4H-3,1-benzoxazin-4-one; 
2-(3,5-dimethoxybenzyl)oxy-5-iodo-7-nitro-4H-3,1-benzoxazin-4-one; 
2-(4-thiomethylcyclohexyl)butyloxy-7-nitro-4H-3,1-benzoxazin-4-one; 
2-methoxy-5-nitro-4H-3,1-benzoxazin-4-one; 
6-nitro-2-propoxy-4H-3,1-benzoxazin-4-one; 
5-methyl-7-nitro-2-pentoxy-4H-3,1-benzoxazin-4-one; 
2-hexoxy-6-nitro-4H-3,1-benzoxazin-4-one; 
8-nitro-2-(3-methylpentoxy)-4H-3,1-benzoxazin-4-one; 
2-isobutoxy-5-bromo-7-nitro-4H-3,1-benzoxazin-4-one; 
EXAMPLE II 
A. Synthesis of 7-Amino-2-ethoxy-4H-3,1-benzoxazin-4-one, and Related 
Compounds of Formula IB 
7-Nitro-2-ethoxy-4H-3,1-benzoxazin-4-one (1 gm, 4.2 m mol.), 10% Pd-C (1 
gm), and cyclohexene (2.5 ml) were refluxed in dry benzene (50 ml) under 
anhydrous conditions for 3 hours. The hot reaction mixture was filtered 
immediately through celite and the catalyst on celite was washed with hot 
benzene (25 ml). The combined filtrate was evaporated to dryness under 
reduced pressure. The residue was stirred in anhydrous ether (30 ml) to 
give 410 mg (47%) of the title compound, 
7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one, as a pale yellow powder, after 
filtration. The product can be further purified by chromatography on 
silica gel column, if necessary (silica gel 60; EtOAc:pet. ether=1:2); 
m.p. 185.degree.-187.degree. C.; IR (KBr): .nu..sub.max 3425, 3330 
cm.sup.-1 (NH.sub.2), 1740 cm.sup.-1 (C.dbd.O), 1640 cm.sup.-1 (C.dbd.N); 
N'NMR (CDCl.sub.3): .delta. 1.43 ppm (t, J=7.1 Hz, 3H, OCH.sub.2 
CH.sub.3), 4.30 ppm (b, 2H, NH.sub.2), 4.75 ppm (q, J=7.1 Hz, 2H, 
OCH.sub.2 CH.sub.3), 6.5-7.98 ppm (m, 3H, aromatic protons). 
B. In a similar manner, but replacing the 2-ethoxy-7-nitro-benzoxazin-4-one 
with other corresponding nitro-substituted compounds of Formula IA, (which 
are prepared as described in Example I, paragraphs D and E, above), and 
following compounds of Formula IB are prepared: 
7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one; 
7-amino-2-benzyloxy-4H-3,1-benzoxazin-4-one; 
8-amino-2-ethoxy-4H-3,1-benzoxazin-4-one; 
6-amino-2-benzyloxy-4H-3,1-benzoxazin-4-one; 
2-cyclopropyloxy-6,8-diamino-4H-3,1-benzoxazin-4-one; 
5-amino-2-cyclobutyloxy-7-thiomethyl-4H-3,1-benzoxazin-4-one; 
6-amino-2-cyclohexyloxy-4H-3,1-benzoxazin-4-one; 
7-amino-2-phenylbutyloxy-4H-3,1-benzoxazin-4-one; 
7-amino-2-(4-thiomethylcyclohexyl)butyloxy-4H-3,1-benzoxazin-4-one; 
5-amino-2-methoxy-4H-3,1-benzoxazin-4-one; 
6-amino-2-propoxy-4H-3,1-benzoxazin-4-one; 
6,7-diamino-2-butoxy-4H-3,1-benzoxazin-4-one; 
7-amino-5-methyl-2-pentoxy-4H-3,1-benzoxazin-4-one; 
7-amino-2-hexoxy-4H-3,1-benzoxazin-4-one; 
8-amino-2-(3-methylpentoxy)-4H-3,1-benzoxazin-4-one; and 
7-amino-2-isobutoxy-6-chloro-4H-3,1-benzoxazin-4-one; 
7-ethyl-7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one 
5-propyl-7-amino-2-benzyloxy-4H-3,1-benzoxazin-4-one 
C. In a similar manner, but replacing the 
2-ethoxy-7-nitro-4H-3,1-benzoxazin-4-one with 
2-ethoxy-5-ethyl-7-nitro-4H-3,1-benzoxazin-4-one the following compound of 
Formula IB was prepared: 
2-ethoxy-5-ethyl-7-amino-4H-3,-benzoxazin-4-one, IR 3440, 3360, 3240, 1735, 
1660, 1640, 1610 cm 
D. Similarly, but starting with other corresponding 
5-alkyl-7-nitro-substituted compounds of Formula IA, (which are prepared 
as described in Example XII, Paragraphs A, B and C), the following 
compounds of Formula IB are prepared: 
2-ethoxy-5-methyl-7-amino-4H-3,1-benzoxazin-4-one; 
2-ethoxy-5-propyl-7-amino-4H-3,1-benzoxazin-4-one 
2-ethoxy-5-isopropyl-7-amino-4H-3,1-benzoxazin-4-one; 
2-ethoxy-5-butyl-7-amino-4H-3,1-benzoxazin-4-one; and 
2-ethoxy-5-isobutyl-7-amino-4H-benzoxazin-4-one. 
EXAMPLE III 
A. 7-acetylamino-2-ethoxy-4H-3,1-benzoxazin-4-one, and Related Compounds of 
Formula IC 
7-Amino-2-ethoxy-4H-3,1-benzoxazin-4-one (0.48 m mol., 100 mg) was stirred 
in acetic anhydride (5 ml) at room temperature under anhydrous conditions 
for 30 minutes. The excess acetic anhydride was removed under reduced 
pressure at 35.degree. C. (bath temperature). The residue was dissolved in 
EtOAc (20 ml) and washed with saturated sodium bicarbonate solution (15 
ml), water (15 ml). The organic layer was dried (MgSO.sub.4) and 
evaporated to give the crude product. Chromatography on silica gel 60 
(EtOAc:pet. ether=3:2) gave 85 mg (71%) of the title compound, 
7-acetylamino-2-ethoxy-4H-3,1-benzoxazin-4-one as a white powder; m.p. 
245.degree.-246.degree. C.; IR (KRr): .nu..sub.max 3360 cm.sup.-1 (NH), 
1740 cm.sup.-1 (C.dbd.O), 1620 cm.sup.-1 (C.dbd.N), 1590 cm.sup.-1 
(aromatic amide); H'NMR (CDCl.sub.3): .delta.1.44 ppm (t, J=711 Hz, 3H, 
OCH.sub.2 CH.sub.3), 2.24 ppm (s, 3H, CH.sub.3 CO), 4.51 ppm (q, J=7.1 Hz, 
2H, OCH.sub.2 CH.sub.3), 7.30 ppm (b, 1H, NH), 7.28-8.10 ppm (m, 3H, 
aromatic protons). 
B. In a similar manner, but substituting other appropriate acid anhydrides 
(which are commercially available, or can be prepared as set forth in 
Preparation III, above) for the acetic anhydride, and other corresponding 
nitro-substituted compounds of Formula IB (prepared as described in 
Example I, paragraph E) the following compounds of Formula IC are 
prepared: 
5-acetylamino-2-ethoxy-4H-3,1-benzoxazin-4-one; 
5-acryloylamino-2-ethoxy-4H-3,1-benzoxazin-4-one; 
6-acetylamino-2-benzyloxy-4H-3,1-benzoxazin-4-one; 
2-benzyloxy-6-butyrylamino-4H-3,1-benzoxazin-4-one; 
5-crotonoylamino-2-cyclobutyloxy-7-thiomethyl-4H-3,1-benzoxazin-4-one; 
6-acetylamino-2-cyclohexylyoxy-4H-3,1-benzoxazin-4-one; 
2-cyclohexylyoxy-6-hexanoylamino-4H-3,1-benzoxazin-4-one; 
7-acetylamino-2-phenylbutyloxy-4H-3,1-benzoxazin-4-one; 
8-acetylamino-2-cyclopropylbutyloxy-5-methyl-4H-3,1-benzoxazin-4-one; 
8-acryloylamino-2-cyclopropylbutyloxy-5-methyl-4H-3,1-benzoxazin-4-one; 
7-acetylamino-2-(4-thiomethylcyclohexyl)butyloxy-4H-3,1-benzoxazin-4-one; 
5-acetylamino-2-methoxy-4H-3,1-benzoxazin-4-one; 
6-acetylamino-2-propoxy-4H-3,1-benzoxazin-4-one; 
6-butyrylamino-2-propoxy-4H-3,1-benzoxazin-4-one; 
7-acetylamino-5-methyl-2-pentoxy-4H-3,1-benzoxazin-4-one; 
6-acetylamino-2-hexoxy-4H-3,1-benzoxazin-4-one; 
8-acetylamino-2-(3-methylpentoxy)-4H-3,1-benzoxazin-4-one; 
EXAMPLE IV 
A. 6-Carboethoxyamino-2-ethoxy-4H-3,1-benzoxazin-4-one and Related 
Compounds of Formula ID 
To a solution of 7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one (0.2 m mol., 41 
mg) and dry pyridine (0.1 ml) in dry dichloromethane (4 ml) at room 
temperature under anhydrous conditions was added ethyl chloroformate (0.05 
ml, 0.52 m mol.) in one portion. After stirring for one hour, the reaction 
mixture was diluted with dichloromethane (16 ml), washed successively with 
water (15 ml), 5% CuSO.sub.4) and evaporated to dryness. The residue was 
stirred in anhydrous ether (2 ml) and filtered to afford 53 mg (95%) of 
the title compound, 7-carboethoxyamino-2-ethoxy-4H-3,1-benzoxazin-4-one as 
a colorless powder; m.p. 191.degree.-193.degree. C.; IR (KBr): 
.nu..sub.max 3310 cm.sup.-1 (NH), 1735 cm.sup.-1 (ester, carbamyl); 1640 
cm.sup.-1 (C.dbd.N); H'NMR (CDCl.sub.3): .delta.1.34 ppm (t, J=7.1 Hz, 3H, 
CH.sub.3 CH.sub.2 OCON), 1.44 ppm (t, J=7.1 Hz, 3H, CH) 
B. In a similar manner, but replacing the 
2-ethoxy-7-nitro-4H-3,1-benzoxazin-4-one with other 7-nitro-substituted 
compounds of Formula IA (which can be prepared according to the method of 
Example I, paragraph E,) and substituting other suitable acid halides of 
Formula III for the chloroformate, the following compounds of Formula ID 
are prepared: 
5-carbomethoxyamino-2-ethoxy-4H-3,1-benzoxazin-4-one; 
5-carboethoxyamino-2-ethoxy-4H-3,1-benzoxazin-4-one; 
2-benzyloxy-6-carboethoxyamino-4H-3,1-benzoxazin-4-one; 
2-benzyloxy-6-carbopropoxyamino-4H-3,1-benzoxazin-4-one; 
6-carbobutoxyamino-2-cyclohexyloxy-4H-3,1-benzoxazin-4-one; 
6-carboethoxyamino-2-cyclohexylyoxy-4H-3,1-benzoxazin-4-one; 
7-carbomethoxyamino-2-phenylbutyloxy-4H-3,1-benzoxazin-4-one; 
8-carboethoxyamino-2-cyclopropylbutyloxy-5-methyl-4H-3,1-benzoxazin-4-one; 
8-carbopropoxyamino-2-cyclopropylbutyloxy-5-methyl-4H-3,1-benzoxazin-4-one; 
7-carboethoxyamino-2-(4-thiomethylcyclohexyl)butyloxy-4H-3,1-benzoxazin-4-o 
ne; 
5-carbomethoxyamino-2-methoxy-4H-3,1-benzoxazin-4-one; 
6-carboethoxyamino-2-propoxy-4H-3,1-benzoxazin-4-one; 
6-carbomethoxyamino-2-propoxy-4H-3,1-benzoxazin-4-one. 
EXAMPLE V 
A. 7-(3-Diethylureido)-2-ethoxy-4H-3,1-benzoxazin-4-one and Related 
Compounds of Formula IE 
Trichloromethyl chloroformate (0.1 m mol., 13.3 .mu.l) was added to a 
solution of 7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one (21 mg, 0.1 m mol) in 
dry THF (3 ml) at room temperature under argon. After stirring for 30 
minutes dry pyridine (0.1 ml) and diethylamine (0.1 ml) were added to the 
reaction mixture and the stirring was continued for an additional 15 
minutes. The mixure was diluted with dichloromethane (20 ml) and washed 
successively with water (15 ml), 5% CuSO.sub.4 solution (2.times.10 ml), 
water (15 ml). The organic layer was dried (MgSO.sub.4) and evaporated to 
give a semi-solid residue. The residue was stirred in anhydrous ether (2 
ml) and filtered to afford 12 mg (39%) of the expected carbamate 
derivative; m.p. 163.degree.-164.5.degree. C. IR (KBr): .nu..sub.max 3380 
cm.sup.-1 (NH), 1740 and 1730 cm.sup.-1 (C.dbd.O), 1670 cm.sup.-1 (urea), 
1640 cm.sup.-1 (C--N); H'NMR (CDCl.sub.3): 1.26 ppm (t, J=7.2 Hz, 6H, 
(CH.sub.3 CH.sub.2).sub.2 N), 1.43 ppm (t, J=7.2 Hz, 3H, CH.sub.3 CH.sub.2 
O), 3.41 ppm (q, J=7.2 Hz, 4H, (CH.sub.3 CH.sub.2).sub.2 N), 4.50 ppm (q, 
J=7.2 Hz, 2H, CH.sub.3 CH.sub.2 O), 6.59 ppm (b, 1H, NH), 7.29-8.06 ppm 
(m, 3H, aromatic protons). 
B. In a similar manner, but starting with the same or other 
amino-substituted compounds of Formulas IA and IB, and replacing the 
ethylamine where appropriate with other alkylamines, the following 
compounds of Formula IE are prepared: 
7-(3-dimethylureido)-2-ethoxy-4H-3,1-benzoxazin-4-one; 
7-(3-methylureido)-5-methyl-2-ethoxy-4H-3,1-benzoxazin-4-one. 
EXAMPLE VI 
A. Preparation of 5-Bromomethyl-2-ethoxy-4H,3,1-benzoxazin-4-one and 
Related Compounds of Formula IF.sub.1 
A solution of 2-ethoxy-5-methyl-4H-3,1-benzoxazin-4-one (330 mg), 
N-bromosuccinimide (340 mg) and AIBN (10 mg, 
2,2'-azobis-iso-butyronitrile) was refluxed for 21/2 hours. The solution 
was evaporated to dryness. The residue was purified by column 
chromatography (silica gel) 10% ethyl acetate:petroleum ether 30-60). This 
afforded the title compound, 
5-bromomethyl-2-ethoxy-4H-3,1-benzoxazin-4-one, m.p. 
112.degree.-114.degree. C.; IR: 1760, 1630, 1590 cm.sup.-1. 
B. In a similar manner, but starting with other appropriate compounds of 
Formula I in which an R' is lower alkyl, the following compounds of 
Formula IF.sub.1 are obtained: 
5-bromoethyl-2-ethoxy-4H-3,1-benzoxazin-4-one; 
6-bromomethyl-2-ethoxy-4H-3,1-benzoxazin-4-one; 
7-bromomethyl-2-ethoxy-4H-3,1-benzoxazin-4-one; 
8-bromomethyl-2-ethoxy-4H-3,1-benzoxazin-4-one; 
5-(5-bromopentyl)-2-isobutoxy-4H-3,1-benzoxazin-4-one; 
5-bromomethyl-2-(1-cyclopropylethyl)oxy-4H-3,1-benzoxazin-4-one; 
7-(iodoethyl-2-imidazolyloxy-4H-3,1-benzoxazin-4-one; 
7-amino-5-(3-chloropropyl)-2-benzyloxy-4H-3,1-benzoxazin-4-one; and 
5-(2-bromobutyl)-2-ethoxy-4H-3,1-benzoxazin-4-one. 
EXAMPLE VII 
A. Preparation of 5-(Dibromomethyl)-2-ethoxy-4H,3,1,-benzoxazin-4-one and 
Related Compounds of Formula IF.sub.2 
Proceeding in the same manner as Example VI, a solution of 
2-ethoxy-5-methyl-4H,3,1-benzoxazin-4-one (2 gm), N-bromosuccinimide (4.05 
gm) and AIBN (25 mg) in carbon tetrachloride was heated for 4 hours at 
reflux. The solvent was evaporated and the residue was purified by column 
chromatography (20% ethyl acetate: petroleum ether) to give 
5-(dibromomethyl)-2-ethoxy-4H-3,1-benzoxazin-4-one, m.p. 
98.degree.-99.degree. C.; IR: 1750, 1640 cm.sup.-1. 
B. In in the same manner, but replacing the 
2-ethoxy-5-methyl-4H-3,1-benzoxazin-4-one with other compounds of Formula 
I, the following compounds of Formula IF.sub.2 are prepared: 
6-dibromomethyl-2-ethoxy-4H-3,1-benzoxazin-4-one; 
7-dibromomethyl-2-ethoxy-4H-3,1-benzoxazin-4-one; 
8-dibromomethyl-2-ethoxy-4H-3,1-benzoxazin-4-one; 
5-(5-dibromopentyl)-2-isobutoxy-4H-3,1-benzoxazin-4-one; 
5-dibromomethyl-2-(1-cyclopropylethyl)oxy-4H-3,1-benzoxazin-4-one; and 
5-(dibromobutyl)-2-ethoxy-4H-3,1-benzoxazin-4-one. 
EXAMPLE VIII 
A. Preparation of 2-Ethoxy-5-ethyl-4H,3,1-benzoxazin-4-one and Related 
Compounds of Formula IG.sub.1 
Methyl lithium (4.69 ml, 1.4M, Aldrich) was added to a suspension of 
cuprous iodide (0.62 gm) in anhydrous ether under argon at -78.degree. C. 
The solution was stirred at -25.degree. C. for 25 min. This solution was 
added to a solution of 2-ethoxy-5-bromomethyl-4H-3,1-benzoxazin-4-one (200 
mg) in 15 ml anhydrous ether and 3 ml dry tetrahydrofuran at -60.degree. 
C. Reaction was monitored by TLC until reaction completion. The reaction 
was quenched with saturated ammonium chloride solution and filtered. The 
filtrate was extracted in the usual manner. The ethereal layer was washed 
with water, dried over magnesium sulphate and evaporated to give an oil. 
The material was purified by thick layer chromatography (R.sub.f =0.75, 
3-% ethyl acetate: petroleum ether), to give 
2-ethoxy-5-ethyl-4H-3,1-benzoxazin-4-one, m.p. 89.degree.-91.degree. C., H 
NMR: 1.3, 1.5 (2t, 6H, 2CH.sub.3), 3.2 (q, 2H, CH.sub.2), 4.5 (q, 2H, 
OCH.sub.2 CH.sub.3), 7.2 (m, 2H, ArH), 7.6 (t, 1H, ArH). 
B. Proceeding in the same manner, but replacing the lithium dimethyl 
cuprate, where desired, with other lithium dialkyl cuprates, and starting 
with other appropriate compounds of Formula IF.sub.1, the following 
compounds of Formula IG.sub.1 are prepared: 
2-ethoxy-5-propyl-4H-3,1-benzoxazin-4-one; 
2-ethoxy-5-butyl-4H-3,1-benzoxazin-4-one; 
2-ethoxy-5-pentyl-4H-3,1-benzoxazin-4-one; 
2-benzyloxy-5-methyl-4H-3,1-benzoxazin-4-one; 
2-phenethyloxy-6-ethyl-4H-3,1-benzoxazin-4-one; 
2-cyclopropyloxy-5-n-propyl-4H-3,1-benzoxazin-4-one; and 
2-(1-cyclohexylethyl)oxy-5-ethyl-3,1-benzoxazin-4-one. 
EXAMPLE IX 
A. Preparation of 
1-(2-Ethoxy-4H-3,1-benzoxazin-4-on-5-yl)-methyl-triphenylphosphonium 
bromide and Related Compounds of Formula IG.sub.2 
A solution of 5-bromomethyl-2-ethoxy-4H-3,1-benzoxazin-4-one (3.15 gm), 
prepared as described in Example VI above, and triphenylphosphine (5.44 
gm) in toluene was heated at 60.degree. C. for 6 hours. The insoluble 
precipitate was filtered, and the mother liquor was reduced to half of its 
original volume and refiltered, yielding 9 gm of 
1-(2-ethoxy-4H-3,1-benzoxazin-4-on-5-yl)-methyl-triphenylphosphonium 
bromide, m.p. (turns yellow at) 125.degree. C. IR: 1740, 1640 cm.sup.-1 ;, 
decom. 135.degree.-140.degree. C. 
B. In a similar manner, but starting instead with other desired compounds 
of Formula IF.sub.1, the preparation of which is described in Example VI, 
the following compounds of Formula IG.sub.2 are prepared: 
1-(2-ethoxy-4H-3,1-benzoxazin-4-on-5-yl)ethyl-triphenylphosphonium bromide; 
1-(2-ethoxy-4H-3,1-benzoxazin-4-on-6-yl)methyl-triphenylphosphonium 
bromide; 
1-(2-ethoxy-4H-3,1-benzoxazin-4-on-7-yl)methyl-triphosphonium bromide; 
1-(2-ethoxy-4H-3,1-benzoxazin-4-on-8-yl)methyl-triphenylphosphonium 
bromide; 
1-(2-ethoxy-4H-3,1-benzoxazin-4-on-5-yl)butyl-triphenylphosphonium bromide. 
1-(2-isopropoxy-7-nitro-4H-3,1-benzoxazin-4-on-5-yl)ethyl-triphenylphosphon 
ium bromide; 
1-[2-(4-methylcyclohexyl)oxy-4H-3,1-benzoxazin-4-on-6-yl]-methyl-triphenylp 
hosphonium bromide. 
EXAMPLE X 
A. Preparation of (2-Ethoxy-1-propenyl)-4H-3,1-benzoxazin-4-one and Related 
Compounds of Formula IG.sub.3 
To a well-stirred suspension of 
(2-ethoxy-4H-3,1-benzoxazin-4-on-5-yl)methyltriphenylphosphonium bromide, 
prepared as described in Example IX, at -60.degree. C. under argon, was 
added DBu (1-8-diazabicyclo[5,4,0]-undec-7-ene), 0.85 ml. After 30 min. at 
-40.degree. C., 2 ml of acetaldehyde was added. The solution was stirred 
for 2 hours and warmed to room temperature. Solvent evaporation gave a 
residual oily solid which was chromatographed (silica gel 30% ethyl 
acetate:petroleum ether) to give the title compound, 
2-ethoxy-5-(propenyl)-4H-3,1-benzoxazin-4-one, (R.sub.f =0.77); IR: 1620, 
1745 cm.sup.-1. 
B. Proceeding in a similar manner, but replacing acetaldehyde with other 
alkylaldehydes, the following compounds of Formula IG.sub.3 are prepared: 
2-ethoxy-5-(1-butenyl)-4H-3,1-benzoxazin-4-one; 
2-ethoxy-5-(1-hexenyl)-4H-3,1-benzoxazin-4-one; 
2-ethoxy-5-(1-pentenyl)-4H-3,1-benzoxazin-4-one 
2-methoxy-5-(1-butenyl)-4H-3,1-benzoxazin-4-one; 
2-n-propoxy-5-(1-propenyl)-7-methylamino-4H-3,1-benzoxazin-4-one; 
2-ethoxy-6-(1-butenyl)-4H-3,1-benzoxazin-4-one; 
5-(1-hexenyl)-2-isopropoxy-7-nitro-4H-3,1-benzoxazin-4-one; 
6-(1-butenyl)-2-(4-methylcyclohexyl)oxy-4H-3,1-benzoxazin-4-one; 
2-n-butoxy-5-(isopropenyl)-7-methoxy-4H-3,1-benzoxazin-4-one; 
2-s-butoxy-5-(isopentenyl)-4H-3,1-benzoxazin-4-one; 
2-n-octyloxy-(5-pentenyl)-7-nitro-4H-3,1-benzoxazin-4-one. 
EXAMPLE XI 
Compounds of Formula IH 
A. Preparation of Methyl 
4,5-dimethoxy-2-[4-(N-triphenylmethyl)-imidazolyl)carbomethyloxy]amino-ben 
zoate and Related compounds of Formula VI 
Trichloromethyl chloroformate (0.16 ml, 1.2 mmol.) was added to a solution 
of methyl 6,7-dimethoxyanthranilate (422 mg, 2 mmol.) in dry THF (30 ml) 
at room temperature under argon. After stirring for 90 minutes, anhydrous 
triethylamine (2 ml, 14.4 mmol.) and 4(N-triphenylmethyl)imidazolymethanol 
(749 mg, 2.2 mmol.) were added and the mixture was refluxed for one hour. 
The solvent was removed under reduced pressure. The residue was shaken 
with a mixture of ether/water (40 ml/40 ml) and the insoluble solid was 
collected by filtration to afford 936 mg (81%) of methyl 
4,5-dimethoxy-2-[4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]amino 
benzoate as a fine powder; m.p. 190.degree.-192.degree. C.; IR (KBr): 
.nu..sub.max 32670 cm.sup.-1 (NH), 1730 cm.sup.-1 (carbamate), 1690 
(COOCH.sub.3); H'NMR (CDCl.sub.3): .delta.3.88 ppm (s, 6H, OCH.sub.3), 
5.13 ppm (s, 2H, OCH.sub.2), 6.95-7.44 ppm (m, 18H, aromatic protons and 
imidazolyl C --H), 8.16 (s, 1H, imidazolyl C.sub.2 --H). 10.49 ppm (s, 1H, 
NH). 
B. In like manner, but starting instead with other appropriately 
substituted anthranilates, the following representative compounds of 
Formula VI are obtained: 
methyl 6-methyl-2-[(4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]-amino 
benzoate; 
methyl 6-ethyl-2-[(4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]-amino 
benzoate; 
methyl 
6-chloro-4-nitro-2-[(4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]-amino 
benzoate; 
methyl 
6-ethylthio-4-bromomethyl-2-[(4-(N-triphenylmethyl)imidazolyl)carbomethylo 
xy]-amino benzoate; 
methyl 
4-amino-6-ethyl-2-[(4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]-amino 
benzoate, and 
methyl 
4-amino-6-ethyl-2-[(4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]-amino 
benzoate. 
C. Preparation of 
6,7-Dimethoxy-2-(4-(N-triphenylmethyl)imidazolyl)methyloxy-4H-3,1-benzoxaz 
in-4-one and Related Compounds of Formula IH 
Methyl 
4,5-dimethoxy-2-[(4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]amino-ben 
zoate (400 mg, 0.72 mmol.), prepared as described in Paragraph A of this 
Example, was stirred in a solution of 1N NaOH (10 ml), THF (20 ml), and 
methanol (20 ml) at room temperature for 3 hours. The organic solvent was 
removed under reduced pressure at 35.degree. C. (bath temperature). The 
aqueous residue was diluted with water (15 ml) and acidified to pH 4 with 
1N HCl. The white precipitate was collected by filtration to yield 73 mg 
(18%) of the expected product. The acidic filtrate was saturated with 
sodium chloride and extracted with ethyl acetate (2.times.20 ml). The 
combined ethyl acetate extract was droed (MgSO.sub.4), evaporated to 
dryness. The residue was stirred in anhydrous ether (5 ml) and filtered to 
afford 173 mg (42%) of 
4,5-dimethoxy-2-[(4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]aminobenz 
oic acid. The combined yield of the expected acid was 246 mg (60%); mp. 
200.degree. C. (dec); IR (KBr): .nu..sub.max 3600-2800 cm.sup.-1 (b, 
COOH), 1730 cm.sup.-1 (carbamate), 1670 cm.sup.-1 (COOH); H'NMR 
(DMSO-d.sub.6): .delta.3.85 ppm (s, 6H, (OCH.sub.2), 6.85-7.65 ppm (m, 
18H, aromatic protons and imidazolyl C.sub.5 --H), 7.90 (s, 1H, imidazolyl 
C.sub.2 --H), 11.07 ppm (b, 1H, NH). 
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (21 mg, 0.11 
mmol.) was added in one portion to a suspension of the 
4,5-dimethoxy-2-[(4-triphenylmethyl)imidazolyl)carbomethyloxy]aminobenzoic 
acid (49 mg, 0.09 mmol.) in anhydrous dichloromethane (2.5 ml) at room 
temperature under anhyrous conditions. After stirring for 30 minutes the 
clear reaction mixture was diluted with dichloromethane (17.5 ml) and 
washed with water (2.times.20 ml). The organic layer was dried with 
magnesium sulfate and evaporated to give a colorless syrup which was 
triturated with 50% pentane in anhydrous ether (3 ml) to give 35 mg (71%) 
of 
6,7-dimethoxy-2-(4(N-triphenylmethyl)imidazolyl)methyloxy-4H-3,1-benzoxazi 
n-4-one as a white powder; m.p. 130.degree.-133.degree. C.; IR (KBr): 
.nu..sub.max 11760 cm.sup.-1 (C.dbd.O), 1630 cm.sup.-1 (C.dbd.N). 
D. In a similar manner, the following representative compounds of Formula 
IH are prepared from the corresponding amino-benzoates of Formula VI, the 
preparation of which is described in Paragrapghs A and B of this Example: 
6-methyl-2-(4-(N-triphenylmethyl)imidazolyl)methyloxy-4H-3,1-benzoxazin-4-o 
ne; 
6-ethyl-2-(4-(N-triphenylmethyl)imidazolyl)methyloxy-4H-3,1-benzoxazin-4-on 
e; 
6-ethylthio-4-bromomethyl-2-(4-(N-triphenylmethyl)imidazolyl)methyloxy-4H-3 
,1-benzoxazin-4-one; and 
7-amino-5-ethyl-2-(4-(4-triphenylmethyl)imidazolyl)methyloxy-4H-3,1-benzoxa 
zin-4-one. 
EXAMPLE XII 
A. Preparation of 2-ethoxy-5-ethyl-7-nitro-H-3,1-benzoxazin-4-one and 
Related Compounds of Formula IA 
A solution of 2-carboethoxyamino-4-nitro-6-ethyl-benzoic acid and 
1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride in anhydrous 
THF (25 ml) was stirred at room temperature for 21/2 hrs. The solution was 
evaporated by dryness and the residue partitioned between ethyl acetate 
and water. The ethyl acetate layer was dried over magnesium sulphate and 
evaporated to a solid. The solid was recrystallized from methylene 
chloride: petroleum ether to give the title compound, 
2-ethoxy-5-ethyl-7-nitro-4H-3,1-benzoxazin-4-one, m.p. 
106.degree.-107.degree. C., Ir. 1770, 1660, 1600, 1595, 1535, 1515 cm. 
B. In a similar manner, the following representative compounds of Formula 
IA are prepared from the corresponding amino-benzoic acids of Formula VII, 
the preparation of which is described in Preparation VI: 
2-ethoxy-5-methyl-7-nitro-4H-3,1-benzoxazin-4-one; 
2-benzyloxy-5-ethyl-7-nitro-4H-3,1-benzoxazin-4-one; 
2-isopropyloxy-5-propyl-7-nitro-4H-3,1-benzoxazin-4-one; 
2-isopropyloxy-5-butyl-4-nitro-4H-3,1-benzoxazin-4-one; and 
2-cyclopropyloxy-5-iso-butyl-4-nitro-4H-3,1-benzoxazin-4-one. 
EXAMPLE XIII 
Conversion of Free Base to Acid Addition Salt 
A stoichiometric amount of 3% hydrogen chloride in dioxane is added to a 
solution of 1.0 g. of 7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one in 20 ml 
dioxane. Diethyl ether is added until precipitation is complete. The 
product is filtered, washed with ether, air dried and recrystallized to 
give 7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one hydrochloride. 
In a similar manner, other compounds of Formula I in free base form may be 
converted to the acid addition salts by treatment with the appropriate 
acid, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, 
nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, 
pyruvic acid, oxalic acid, malonic acid, succinic acid, malic acid, maleic 
acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic 
acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 
p-toluenesulfonic acid, and the like. 
EXAMPLE XIV 
Conversion of Salt to Free Base 
1.0 g of 7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one HCl suspended in 50 ml of 
ether is stirred with a twofold stoichiometric excess of dilute aqueous 
potassium carbonate solution until the salt is completely dissolved. The 
organic layer is then separated, washed twice with water, dried over 
magnesium sulfate and evaporated to yield 
7-amino-2-ethoxy-5-methyl-4H-3,1-benzoxazin-4-one as the free base. 
EXAMPLE XV 
Direct Interchange of Acid Addition Salts 
7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one acetate (1.0 g) is dissolved in 50 
ml water containing a stoichiometric equivalent of sulfuric acid, and the 
solution evaporated to dryness. The product is suspended in ether and 
filtered, air dried and recrystallized from methanol/acetone to yield 
2-ethoxy-5-ethyl-4H-3,1-benzoxazin-4-one sulfate. 
In Examples XIII through XX, the active ingredient is 
7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one. Other compounds of Formula I and 
the pharmaceutically acceptable salts thereof may, of course, be 
substituted. 
EXAMPLE XVI 
______________________________________ 
Quantity per 
Ingredients tablet, mgs. 
______________________________________ 
Active ingredient 
25 
cornstarch 20 
lactose, spray-dried 
153 
magnesium stearate 
2 
______________________________________ 
The above ingredients are thoroughly mixed and pressed into single scored 
tablets. 
EXAMPLE XVII 
______________________________________ 
Quantity per 
Ingredients tablet, mgs. 
______________________________________ 
Active ingredient 
100 
lactose, spray-dried 
148 
magnesium stearate 
2 
______________________________________ 
The above ingredients are mixed and introduced into a hard-shell gelatin 
capsule. 
EXAMPLE XVIII 
______________________________________ 
Quantity per 
Ingredients tablet, mgs. 
______________________________________ 
Active ingredient 
200 
cornstarch 50 
lactose 145 
magnesium stearate 
5 
______________________________________ 
The above ingredients are mixed intimately and pressed into single scored 
tablets. 
EXAMPLE XIX 
______________________________________ 
Quantity per 
Ingredients tablet, mgs. 
______________________________________ 
Active ingredient 
108 
lactose 15 
cornstarch 25 
magnesium stearate 
2 
______________________________________ 
The above ingredients are mixed and introduced into a hard-shell gelatin 
capsule. 
EXAMPLE XX 
______________________________________ 
Quantity per 
Ingredients tablet, mgs. 
______________________________________ 
Active ingredient 
150 
lactose 92 
______________________________________ 
The above ingredients are mixed and introduced into a hard-shell gelatin 
capsule. 
EXAMPLE XXI 
An injectable preparation buffered to a pH of 7 is prepared having the 
following composition: 
______________________________________ 
Ingredients 
______________________________________ 
Active ingredient 0.2 g 
KH.sub.2 PO.sub.4 buffer (0.4 M solution) 
2 ml 
KOH (1 N) q.s. to pH 7 
water (distilled, sterile) 
q.s. to 20 
ml 
______________________________________ 
EXAMPLE XXII 
An oral suspension is prepared having the following composition: 
______________________________________ 
Ingredients 
______________________________________ 
Active ingredient 0.1 g 
fumaric acid 0.5 g 
sodium chloride 2.0 g 
methyl paraben 0.1 g 
granulated sugar 25.5 g 
sorbitol (70% solution) 
12.85 g 
Veegum K (Vanderbilt Co.) 
1.0 g 
flavoring 0.035 ml 
colorings 0.5 mg 
distilled water q.s. to 100 
ml 
______________________________________ 
EXAMPLE XXIII 
Topical Formulation 
______________________________________ 
Ingredients grams 
______________________________________ 
Active compound 0.2-2 
Span 60 2 
Tween 60 2 
Mineral oil 5 
Petrolatum 10 
Methyl paraben 0.15 
Propyl paraben 0.05 
BHA (butylated hydroxy anisole) 
0.01 
Water q.s. 100 
______________________________________ 
All of the above ingredients, except water, are combined and heated to 
60.degree. C. with stirring. A sufficient quantity of water at 60.degree. 
C. is then added with vigorous stirring to emulsify the ingredients, and 
water then added q.s. 100 g. 
EXAMPLE XXIV 
Human Leukocyte Elastase Inhibition Assay 
1. Enzyme 
References: 
Barrett, A. J. (1981), Methods in Enzymology, 80C, 581-588. 
Engelbrecht, et al., (1982), Z. Physiol. Chem., 363, 305-315. 
Fresh human leukocytes were obtained from a healthy donor, frozen and kept 
at -75.degree. C. until use. Enzyme preparation followed the above 
referenced methods: cells were washed in saline, homogenized in the 
presence of 1M NaCl and 0.1% Brij 35 (Sigma Chemical C., No. P-1254). 
After centrifugation and concentration by dialysis against polyethylene 
glycol (MW 20,000), the material was chromatographed on Sephacryl S-300 
(Pharmacia). Active fractions were combined, concentrated as before, and 
chromatographed on an affinity gel of bovine lung trypsin inhibitor 
attached to Sepharose CL-4B. Active fractions were combined, concentrated 
as before to approximately 0.3 micromolar in active elastase, and frozen 
in 1 ml aliquots at -75.degree. C. until use. 
2. Substrate 
Methoxysuccinyl-L-alanyl-L-alanyl-L-prolyl-L-valyl-N-methyl-coumarinamide 
was obtained from Peninsula Laboratories, San Carlos, Calif. Solutions of 
1 mM in dimethylsulfoxide were made and kept at 4.degree. C. until use. 
3. Inhibitors 
The compounds of Formula I to be assayed were dissolved in 
dimethylsulfoxide to give 5, 10, or 20 mM stock solutions, which may be 
further diluted as required. 
4. Assay Buffer 
The buffer consisted of 25 mM N-2-hydroxyethylpiperazine-N-2-ethane 
sulfonic acid, 1M sodium chloride, 0.1% w/v Brij 35, pH 7.8. 
5. Procedure 
A Perkin-Elmer Model 650-40 fluorescence spectrophotometer is set up as 
follows: ratio mode, excitation 370 nm, emission 460 nm, full scale output 
1, 5, or 10 units, cell compartment thermostatted at 25.degree. C. For 
those compounds of Formula I which are themselves fluorescent, the 
excitation wavelength may be optionally 390 nm to minimize interference. 
To 2.0 ml of assay buffer in a fluorescence cuvette is added 5 microliters 
substrate and 20 microliters enzyme, with mixing. The change in 
fluorescence is recorded on a strip chart recorder to measure the initial, 
uninhibited rate, typically 0.8 units per minute. After approximately two 
minutes of such recording, inhibitor (between 0.5 and 20 microliters of 
the stock solution) is added with mixing, and recording continued. The 
reaction is recorded until a new constant rate is achieved. This procedure 
is repeated for several (4-6) inhibitor concentrations. The data--a table 
of substrate concentration, inhibitor concentration, and observed reaction 
velocities--are fit to the appropriate equation by non-linear least 
squares multiple regression. 
EXAMPLE XXV 
Human Thrombin Inhibition Assay 
1. Enzyme 
Human thrombin number T-8885 was obtained from Sigma Chemical Company, St. 
Louis, Mo., and reconstituted with water to approximately 2.5 NIH 
units/ml. 
2. Substrate 
BOC-L-Valyl-L-prolyl-L-arginyl-N-methylcoumarinamide was obtained from 
Peninsula Laboratories, San Carlos, Calif. Solutions were made to 1 mM in 
dimethyl sulfoxide. 
3. Inhibitors 
As Example XXI. 
4. Assay Buffer 
The assay buffer consisted of 25 mM N-2-hydroxy ethylpiperazine-N-2-ethane 
sulfonic acid, 0.5M sodium chloride, 0.1% w/v polyethylene glycol 8000, pH 
7.8. 
5. Procedure 
The procedure was as in Example XXI, except that 5 microliters of substrate 
and 2.5 microliters enzyme solution were used. 
EXAMPLE XXVI 
Human Urokinase Inhibition Assay 
1. Enzyme 
Human Urokinase was obtained from Leo Laboratories, Pickering, Ontario, and 
made to approximately 2.5 mg/ml in 0.10M sodium citrate, 50 mM sodium 
chloride, pH 3. 
2. Substrate 
Glutaryl-glycyl-L-arginyl-methyl coumarin amide (Peninsula Laboratories, 
vide supra) was made to approximately 1 mM in 1:1 water:dimethylsulfoxide. 
3. Inhibitors 
As Example XXI. 
4. Assay Buffer 
The assay buffer consisted of 50 mM tris(hydroxymethyl)amino methane, 0.10M 
sodium chloride, 10 mM calcium chloride, pH 8.0. 
5. Procedure 
The procedure was as in Example XXI, with 5 microliters enzyme used. 
EXAMPLE XXVII 
Bovine Chymotrypsin Inhibition Assay 
1. Enzyme 
Chymotrypsin type II was obtained from Sigma Chemical Company and made to 
0.25 mg/ml in 1 mM hydrochloric acid and kept at 4.degree. C. until use. 
2. Substrate 
7-(Glutaryl-L-phenylalaninamido)-4-methyl coumarin was obtained from Sigma 
and made to 10 mM in 1:1 acetonitrile:dimethylsulfoxide. 
3. Inhibitors 
As Example XXI. 
4. Assay Buffer 
The assay buffer consisted of 25 mM N-2-hydroxy ethyl piperazine-N-2-ethane 
sulfonic acid, 0.1M potassium chloride, pH 7.8. 
5. Procedure 
As Example XXI. 
EXAMPLE XXVIII 
Boar Acrosin Inhibition Assay 
1. Enzyme 
Boar acrosin was a gift of Professor W. Muller-Esterl, as purified in 
Muller-Esterl, et al., Hoppe-Seyler's Z. Physiol. Chem., 361, 1811-1821, 
1980, and was made to approximately 0.1 mg/ml in 1 mM HCl and kept at 
4.degree. C. until use. 
2. Substrate 
7-(N-benzoyl-L-argininamido)-4-methyl coumarin HCl was obtained from Sigma 
Chemical Company and made up to 2 mM in dimethyl sulfoxide. 
3. Inhibitors 
As Example XXI. 
4. Assay Buffer 
The assay buffer consisted of 0.10M N-2-hydroxy ethylpiperazine-N-2-ethane 
sulfonic acid, 50 mM calcium chloride, 0.01% v/v Triton X-100, pH 7.8. 
5. Procedure 
The procedure was as Example XXI, with 5 microliters substrate and 2.5 to 
15 microliters enzyme as required to obtain approximately 0.5 fluorescent 
unit/minute uninhibited rate. 
EXAMPLE XXIX 
Assay for Stability of Compounds in Whole Plasma 
Whole, citrated human plasma was obtained from a local blook bank and kept 
frozen at -70.degree. C. until use. Benzoxazinone (from a 10 mM stock 
solution in dimethylsulfoxide) was added to plasma at 37.degree. C. to a 
final concentration of 50 mM, and incubation was continued at 37.degree.. 
At various times thereafter, aliquots were withdrawn and diluted 5-fold 
into 20 mM potassium phosphate, 0.14M sodium chloride, 3% w/v Brij 35 
(Sigma Chemical Company), pH 7.4, and the fluorescence of this solution 
was monitored at 345 nm (excitation) and 429 nm (emission). The 
fluorescence intensity is proportional to the concentration of 
benzoaxazinone remaining. These data were fit by interative non-linear 
techniques to first-order exponentials to obtain the half-times in plasma. 
Alternatively, for benzoxazininones which are weakly or non-fluorescent, 
high pressure liquid chromatography (HPLC) was used. From plasma 
incubations as above, aliquots were withdrawn and diluted 1:1 (v/v) with 
acetonitrile, mixed on a vortex stirrer, and centrifuged. Ten microliters 
of the supernatant was injected into the HPLC and chromatographed on a 5 
micron RP-18 (reverse phase) column, in 9% acetonitrile, 10% water (v/v), 
with detection by absorbance at 340 nm. Retention times and concentrations 
were determined by comparison to standards. The integrated areas of the 
benzoxazinone peaks vs. incubation time were treated as above to obtain 
half-times. 
EXAMPLE XXX 
Assay for Inhibition of Endothelial Cell Basement Membrane Degredation 
Principle 
Various serine proteases, including elastase, are secreted by murine 
activated macrophages, and cause the degredation of endothelial cell 
basement membrane. This assay tests the inhibitory potency of a test 
compound to as a measure of its ability to inhibit enzyme induced 
degredation of the endothelial cell basement membrane. PG,86 
Methods 
LE II murine lung capillary endothelial cells were grown to confluence in 
24 well cluster dishes. Cells were lageled with 10 .mu.Ci [.sup.35 
S]-methionine per well, in medium deficient in methionine and supplemented 
with 10% fetal calf serum and 10 .mu.g/ml ascorbate for two days. 
Biosynthetically labeled basement membranes were prepared by lysing the 
cells with 0.5% Nonidet P40 for 5 minutes at room temperature and removing 
cytoskeletal degris with 0.25N NH.sub.4 OH, followed by several washes 
with phosphate buffer. 
Activate murine macrophages were obtained by peritoneal lavage of female 
C3H/He mice injected intraperitoneally with corynebacterium parvum 7 days 
prior to the experiment. 
Macrophages were either layere on the basement membrane (5.times.10.sup.5 
cells/well) or used to prepare conditioned medium in separate dishes that 
were then incubated with the basement membrane. 
Test compound was dissolved in DMSO at 10.sup.-2 M-10.sup.-3 M, diluted 
with medium containing 1 mg/ml bovine serum albumin, and tested at 
10.sup.-5 M. 
Machrophages, conditioned medium, or 5 .mu./ml porcine pancreatic elastase 
(Sigma) were incubated with the basement membrane at 37.degree. C. for 4 
hours in the presence or absence of test compound. Aliquots of the medium 
were counted for .sup.3 H and .sup.35 S in a liquid scintillation counter. 
Results may be expressed as % inhibition of the release of radioactivity 
after correction for spontaneous release of radioactivity determined in 
control wells. Compounds of Formula I which were tested in this assay 
exhibited significant inhibitory potency against degredation of the 
basement membrane.