CA1336960C - Inhibition of serine proteases in the treatment of degenerative disorders - Google Patents

Abstract

The treatment of degenerative disorders resultant from the activity of proteolytic enzymes is disclosed. Inhibition of the proteolytic enzyme activity with compositions containing 2-substituted saccharin derivatives, as well as methods of diagnosis and treatment are disclosed.

Description

INHIBITION OF SERINE PROTEASES IN THE
TREATMENT OF DEGENERATIVE DISORDERS

Technical Field This invention is directed to pharmaceutical compositions, diagnostic methods and treatment methods that utilize compounds which function as inhibitors of serine proteases.
Background The inhibition of proteolytic enzymes by non-toxic reagents is useful in the treatment of degenerative disorders for which proteolysis is a substantive element.
Protease inhibitors are widely utilized in biomedical research. Serine proteases are the most widely distributed class of proteolytic enzymes. Some serine proteases are characterized as chymotrypsin-like or elastase-like, based upon their substrate specificity.
Chymotrypsin and chymotrypsin-like enzymes normally cleave peptide bonds in proteins at a site at which the amino acid residue on the carbonyl side is typically Trp, Tyr, Phe, Met, Leu or another amino acid residue which contains aromatic or large alkyl side chains.
Elastase and elastase-like enzymes normally cleave peptide bonds at a site at which the amino acid residue on the carbonyl side of the bond is typically Ala, Val, Ser, Leu or other similar, smaller amino acids.
Both chymotrypsin-like and elastase-like enzymes are found in leukocytes, mast cells, and pancreatic juice in higher organisms, and are secreted by many types of bacteria, yeast and parasites.

Several compounds are known which are inhibitors of serine protease activity. U.S. Patent No.
4,659,855; U.S. Patent No. 4,623,645; U.S. Patent No.
4,547,371; Teshima et al (1982) J. of Biol. Chem.
257:5085-5091; Cha (1975) Biochem. Pharmacol. 24:2177-2185.
Several substituted saccharin derivatives are known. Some of these derivatives can be utilized as enzyme inhibitors and others are useful as photographic reagents.
Some 2-substituted saccharin derivatives have been disclosed to function as inhibitors for elastase-like and chymotrypsin-like enzymes. U.S. Patent No.
4,195,023 discloses 2-N-acyl derivatives of saccharin as inhibitors of human leukocyte elastase (HLE) activity.
U.S. Patent No. 4,276,298 discloses 2-N-phenyl substituted saccharin derivatives that can function as elastase inhibitors. Powers et al. (1985) Biochemistry 24:2048-2058 discloses N-furoylsaccharin and N-(2,4-dicyanophenyl)saccharin inhibitors for chymotrypsin-like enzymes.
Svoboda et al. (1986) Collect. Czech. Chem.
Commun. 51:1133-9 discloses a saccharin derivative containing an acetyl ester substituent in the 2-position. Similar compounds are disclosed as photographic reagents in U.S. Patent No. 4,350,752, U.S.
Patent No. 4,263,393, U.S. Patent No. 4,410,618, and U.S. Patent No. 4,363,865. There is no disclosure that the therein described 2-substituted saccharin compounds act as enzyme inhibitors.
U.S. Patent No. 3,314,960. discloses a 2-substituted saccharin derivative which may be used as a sedative. The compound has a 2-glutaric acid group attached to the nitrogen in the 2-position of saccharin.

Inhibitors of HLE and chymotrypsin-like enzymes are useful for treating degenerative diseases such as emphysema, rheumatoid arthritis and - pancreatitis. Clinical symptoms found in these diseases are believed to be controlled in large part by uncontrolled elastase in the affected tissues, as well as by uncontrolled chymotrypsin-like enzymes such as cathepsin G and mast cell chymase. Thus the production and utilization of specific inhibitors for these proteolytic enzymes enables the diagnosis and treatment of degenerative diseases. These inhibitors may also be utilized to prevent the degradation of stored peptides and proteins by specific proteolytic enzymes.
Summary The present invention is directed to pharmaceutical compositions, methods of inhibiting proteolytic enzyme activity, diagnostic methods and a method for treatment of degenerative disorders.
This invention is directed to pharmaceutical compositions that contain a 2-substituted saccharin derivative as an active agent together with a pharmaceutically acceptable carrier. The 2-substituted saccharin derivatives present in the pharmaceutical compositions are represented by the general structural formula tI):

~1 \I
- S /

O ~ (I) _4_ 1336960 where R1 is selected from the group consisting of halogen, ester, aryloxy, alkylthio, arylthio, sulfinyl, sulfonyl, amino, amido, imidyl, heterocyclyl and substituted derivatives thereof;
R2 is hydrogen or an electron-withdrawing group; and R3 and R4 are each independently selected from the group consisting of hydrogen, halogen, cyano, nitro, amino, amido and a straight, cyclic or branched-chain carbon-containing group having from 1 to 20 carbon atoms; wherein the -CH- group is always appended to a heteroatom on R1.
This invention is also directed to a method of inhibiting proteolytic enzyme activity in which a liquid sample containing proteolytic enzyme activity is contacted with a 2-substituted saccharin derivative corresponding to Formula I.
This invention is also directed to a diagnostic method in which a body fluid sample is contacted with a composition containing a 2-substituted saccharin derivative corresponding to Formula I for a time period sufficient to permit enzyme inhibition, and then measuring the concentrations of inhibited and uninhibited proteolytic enzyme remaining in the sample.
Preferred proteolytic enzymes monitored are human leukocyte elastase and chymotrypsin-like enzymes. The diagnostic method of the present invention enables the detection of abnormal levels of the inhibited proteolytic enzyme which is indicative of physiological disorders.
This invention is further directed to a method of treatment for degenerative disorders for such disorders as emphysema, adult respiratory distress syndrome, pancreatitis, rheumatoid arthritis and other --5- 1336g 60 inflammatory disorders. In this treatment method, a therapeutically effective amount of a pharmaceutical composition containing a 2-substituted saccharin derivative corresponding to Formula I as an active agent is administered to a mammal with the degenerative disorder.
Description of Preferred Embodiments The present invention is directed to pharmaceutical compositions that contain 2-substituted saccharin derivatives as active ingredients and pharmaceutically acceptable carriers, where the 2-substituted saccharin derivative is a compound represented by the general structural formula (I):

R~

F~4-- 1 \N--CH--R1 ~ S
~J~
O O

where R1 is selected from the group consisting of halogen, ester, aryloxy, alkylthio, arylthio, sulfinyl, sulfonyl, amino, amido, imidyl, heterocyclyl and substituted derivatives thereof;
R2 is hydrogen or an electron-withdrawing group and R3 and R4 are each independently selected from the group consisting of hydrogen, halogen, cyano, nitro, amino, amido and a straight, cyclic or branched-chain carbon-containing group having from 1 to 20 carbon -I

atoms; wherein the -CH- group is always appende~ to a heteroatom on R1.
S R1 groups are preferably leaving groups having a pKa of less than about 7 that carry away an electron pair (March, Advanced Organic Chemistry, 3rd edition, 1985 (Wiley and Sons, N.Y.) page 219).
As used herein "substituted derivatives"
include the attachment to a group of a substituent selected from alkyl, aryl, cycloalkyl alkylamidoaryl, heterocyclyl, arylthio, nitro, amino, amido, sulfinyl and sulfonyl groups.
Exemplary halogens include fluorine, chlorine, bromine and iodine derivatives and exclude astatine derivatives that are radioactive.
As used herein, the terms "alkyl" and "lower alkyl" include C1 to C6 lower aliphatic groups, as for example, methyl, ethyl, propyl, iso-propyl, n-butyl, sec-butyl, t-butyl, n-pentyl, 2-methyl-3-butyl, l-methylbutyl, 2-methylbutyl, neopentyl, n-hexyl, 1-methylpentyl, 3-methylpentyl, l-ethylbutyl, 2-ethylbutyl, 2-hexyl, 3-hexyl, and the like.
As used herein, the term "cycloalkyl" includes C3 to C6 carbocyclic ring groups, as for example, cyclopropyl, cyclopentyl, cyclohexyl and the like.
As used herein, the term "aryl" includes aromatic rings that are fused, unfused or linked and can include one or more heteroatoms, for example, phenyl, naphthyl, biphenylyl, anthracenyl, quinolyl, pyrimidinyl, and the like.
As used herein, the phrase "a straight, cyclic or branched chain carbon-containing group having 1 to 20 carbon atoms" includes alkyl, cycloalkyl and aryl groups that can include one or more heteroatoms, as for 1~36960 example, 3,3-diphenylpropanamido, isopropyl, 1,3,3-trimethylcyclopentyl, and the like.
As used herein, the term "aryloxy" includes fused or unfused aromatic rings, that can include one or more heteroatoms, which form an ether linkage, such as p-nitrophenoxy, biphenylyloxy, 2-indolyloxy, and the like.
As used herein, the term "alkylthio" includes C1 to C6 lower alkylthio groups that form sulfide ethers, as for example, methylsulfide, propylsulfide, hexylsulfide, and the like.
As used herein, the term "arylthio" includes fused or unfused aromatic rings, that can include one or more heteroatoms, that form a thioether linkage through a thio group. Exemplary arylthio groups include tetrazolylthio groups such as:
l-phenyl-lH-tetrazol-5-ylthio; l-methyl-lH-tetrazol-5-ylthio; 1-(3-acetamidophenyl)-lH-tetrazol-5-ylthio;
1-cyclohexyl-lH-tetrazol-5-ylthio;
1-(3-heptanamidophenyl)-1-H-tetrazol-5-ylthio; and phenylthio.
As used herein, the term "ester" includes acyloxy radicals and thioesters, such as acylthio groups that are formed between a mercapto group and a lower alkyl or aryl carboxylic acid. Exemplary acyloxy or acylthio groups are acyclic, cyclic or aromatic. When acyclic, the acyl groups preferably have about 1 to about 20 carbon atoms in the alkyl portion. When cyclic, the ring contains 3 to 6 carbon atoms. When aromatic, the aryl groups preferably are substituted or unsubstituted aryl groups and can be a heterocyclic aromatic group having about 5 to about 10 nuclear carbon -13369fiO

and hetero atoms. Exemplary acylthio radicals are acetylthio, propionylthio, hexanoylthio, and the like.
As used herein, the term "thionyl" includes groups containing the sulfinyl group, as for example, methylsulfinyl, phenylsulfinyl, tetrazolylsulfinyl, and the like.
As used herein, the term "heterocyclyl"
includes fused or unfused cyclic organic groups that contain one or more heteroatoms such as N, S, or 0 in the ring structure such as phthalimidyl, benzoxazolyl, benzothiazolyl, and the like.
As used herein, the term "alkylamidoaryl"
includes C1 to C12 lower aliphatic carbonyl groups coupled in an amide bond with arylamine groups, such as, for example, heptanamidophenyl, and the like.
As used herein, the term "sulfonyl" includes alkyl, aryl and heterocyclic groups that contain a sulfonyl group attached thereto, such as phenylsulfonyl, 2-pyrimidinylsulfonyl, and the like.
The R2 group is preferably hydrogen or an electron-withdrawing group such as trifluoromethyl.
As used herein an "electron-withdrawing group"
includes a substituent group that will manifest greater inductive effect to draw electrons to itself than would a hydrogen atom if it occupied the same position on the molecule. Exemplary electron-withdrawing groups include cyano, halo, nitro, carboxy, acyl, arylthio, e.g., phenylthio, and the like.
Suitable R3 and R4 groups are preferably substituents such as hydrogen, halogen, methyl, cyano, nitro, amino, amido, cyanato, thiocyanato, hydroxy, alkoxy, straight, cyclic and branched chain carbon-containing groups having from 1 to 20 carbon atoms and the like. The R3 and R4 groups of the present invention can be unsubstituted or can contain a substituent ` -selected from the group consisting of alkyl, phenyl and nitro.
The present invention provides a - pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, hereinafter referred to as the "active compound" or "agent", in association with a pharmaceutically acceptable carrier.
As used herein, the phrase "pharmaceutically 10 acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a mammal. The physiologically tolerable carrier may take a wide variety of forms depending upon the preparation desired for administration and the intended route of administration. A carrier is a material useful for administering the active compound and must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.
The pharmaceutical compositions are prepared by any of the methods well known in the art of pharmacy all of which involve bringing into association the active compound and the carrier therefor.
For therapeutic use, the agent utilized in the present invention can be administered in the form of conventional pharmaceutical compositions. Such compositions can be formulated so as to be suitable for oral or parenteral administration, or as suppositories.
In these compositions, the agent is typically dissolved or dispersed in a physiologically tolerable carrier.
As an example, the compounds of the present invention can be utilized in liquid compositions such as sterile suspensions or solutions, or as isotonic preparations containing suitable preservatives.
Injectable media containing aqueous injectable isotonic and sterile saline or glucose solutions may be utilized.
Additional liquid forms in which the present compounds may be incorporated for administration include flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, peanut oil, and the like, as well as elixirs and similar pharmaceutical vehicles.
The present agents can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono-or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to the agent of the present invention, stabilizers, preservatives, excipients and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic.
Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y.
(1976), p. 33 et seq.
The present compounds can also be used in compositions such as tablets or pills, preferably containing a unit dose of the compound. To this end, the agent (active ingredient) is mixed with conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, gums, or similar materials as non-toxic, physiologically tolerable carriers. The tablets or pills of the present compositions can be laminated or otherwise compounded to 13369~0 provide unit dosage forms affording prolonged or delayed action.
It should be understood that in addition to the aforementioned carrier ingredients the pharmaceutical formulation described herein can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants), and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
The tablets or pills can also be provided with an enteric layer in the form of an envelope that serves to resist disintegration in the stomach and permits the active ingredient to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, including polymeric acids or mixtures of such acids with such materials as shellac, shellac and cetyl alcohol, cellulose acetate, and the like. A particularly suitable enteric coating comprises a styrene-maleic acid copolymer together with known materials that contribute to the enteric properties of the coating.
The 2-substituted saccharin compounds utilized in the pharmaceutical compositions of the present invention inhibit the activity of serine proteases, specifically human leukocyte elastase and chymotrypsin-like enzymes.
The present invention is further directed to a method of inhibiting proteolytic enzymes, such as human leukocyte elastase and chymotrypsin-like enzymes. A
composition containing an effective amount of a 2-substituted saccharin derivative corresponding to Formula I is contacted with a liquid sample containing such proteolytic enzyme activity.
- The present invention is also directed to a diagnostic method which enables the detection of proteolytic enzymes such as human leukocyte elastase and chymotrypsin-like enzymes which can indicate the presence of physiological disorders. In the diagnostic method, a body fluid sample is contacted with an effective amount of a composition containing a 2-substituted saccharin derivative corresponding to Formula I for a time period sufficient to permit inhibition of appropriate proteolytic activities. The concentration of inhibited proteolytic enzyme in the sample is then measured.
The present invention is further directed to a method of treatment of degenerative disorders produced by the activity of proteolytic enzymes. Such disorders include emphysema, adult respiratory distress syndrome, pancreatitis, rheumatoid arthritis and other inflammatory disorders. In the treatment method, a therapeutically effective amount of a 2-substituted saccharin derivative corresponding to Formula I is administered, preferably in a pharmaceutical composition, to a mammal such as a human with such a degenerative disorder.
Preparation of the compounds of the present invention is carried out by known methods. 5-nitro saccharin derivatives are prepared as described in D'Alelio et al, J. Macromol. Sci-Chem. A3(5):941 (1969) and in Saari et al., J. Heterocyclic Chem. 23:1253 (1986).
4- or 5- Substituted saccharin derivatives are prepared from appropriate 6- or 5- substituted anthranilic acids as described in the above-referenced article by Saari, et al. Basically, a methyl ester is prepared by conventional means from the substit=uted anthranilic acid and then diazotized. The diazonium - salt is then reacted with sulfur dioxide and cupric chloride to produce a sulfonyl chloride which is then reacted with concentrated ammonium hydroxide to produce the substituted saccharin derivative. This reaction is schematically shown for the production of a 4-substituted saccharin from a 6-substituted anthranilic acid as follows:
S~EME I
P

~ - ~
.~ ~

IV nl The present invention is further illustrated by the following examples which are not intended to limit the scope of the invention in any way.

EXAMPLE 1: 2-AcetoxYmethyl-4-methyl saccharin (a) MethYl 6-methylanthranilate Powdered KOH (7.4 g; 132 mmol; 2 equiv.) was admixed with dimethyl sulfoxide (DMSO) (100 mL) and the mixture was stirred for 5 minutes. 6-Methylanthranilic acid (10.0 g; 66 mmol) was then added to the mixture, followed dropwise by iodomethane (4.52 mL; 73 mmol; 1.1 equiv.). The reaction mixture was stirred for 30 minutes at room temperature and then diluted with ether - (250 mL), washed with water (3 x 100 mL), dried (MgSO4) and concentrated. The crude product was filtered through a pad of flash grade (32-63) silica gel and eluted with 1:9 ether:hexanes to afford 4.23 g (3g%) of the anthranilate ester as an oil. lH nmr (300 MHz, CDCl3): 7.078 (lH, t, J=7.67 Hz); 6.529 (2H, d, J=7.79 Hz); 5.111 (2H, br s); 3.887 (3H, s); 2.424 (3H, s). IR
(neat film, cm~1): 3480 (m), 3380 (m), 2950 (w), 1690 (s); 1605 (s).

(b) 4-Methylsaccharin The anthranilate ester, methyl 6-methylanthranilate prepared in (a) (4.23 g; 25.6 mmol) was dissolved in acetic acid (25 mL) and cooled to 0 C.
Concentrated hydrochloric acid (45mL) was added to produce a tan slurry. Sodium nitrite (1.89 g; 27.4 mmol; 1.07 equiv.) dissolved in water (8 mL) was added slowly, dropwise, and the resulting orange solution was stirred at 0 C for 1 hour. This solution was then added in 6 portions to a 0 C mixture of cupric chloride dihydrate (2.18 g; 12.8 mmol; 0.5 equiv.) and sulfur dioxide (6.3 g; excess) in acetic acid (33 mL) and water (6 mL). The dark green solution was stirred at room temperature overnight, poured into ice-water (300 mL), and the solid was collected and dried by suction to 3~ provide 1.11 g of the sulfonyl chloride. This was immediately added to ice cold ammonium hydroxide (100 mL) and stirred at room temperature overnight. The solution was acidified to pH 1 with concentrated HCl and the resulting precipitate was collected and air-dried to provide 729 mg (12%) of 4-methylsaccharin, mp 224-226 C.

-1H nmr (300 MHz, CD3CN): 9.5 (lH, br s); 7.782 (2H, d, J=4.35 Hz); 7.644 (lH, t, J=4.20 Hz); 2.683 (3H, s). IR
(KBr, cm1): 3400 (w); 3100 (s); 3000 (s); 1720 (s);
1580 (m). FDMS: m/e 197 (M ).
s (c) 2-Hydroxymethyl-4-methylsaccharin 4-Methylsaccharin prepared in (b) (500 mg;
2.54 mmol) was dissolved in 2.53 mL of warm ethanol (steam bath). Once a homogeneous solution was achieved, formalin (37% in methanol; 1.76 mL; excess) was added dropwise. The solution was allowed to cool to room temperature and then chilled to 0 C for 4 days. The resulting solid was collected and air-dried to afford 476 mg (82%) of 2-hydroxymethyl-4-methylsaccharin, mp 196-198 C. ~H nmr (300 MHz, CDCl3): 7.767 (lH, t, J=6.75 Hz); 7.732 (lH, d, J=7.72 Hz); 7.600 (lH, d, J=6.64 Hz); 5.361 (2H, d, J=8.00 Hz); 3.296 (lH, t, J=8.16 Hz); 2.793 (3H, s). IR (KBr, cm1): 3505 (s);
3070 (w); 1735 (s); 1580 tm).

(d) 2-Acetoxymethyl-4-methylsaccharin 2-Hydroxymethyl-4-methylsaccharin produced in (c) (76 mg; 0.33 mL) was admixed into acetic anhydride (1 mL; excess) and concentrated sulfuric acid (2 drops) was added. The reaction mixture was stirred for 2 hours at room temperature, at which time a non-polar spot was observed by thin-layer chromatography (tlc) analysis.
The reaction mixture was diluted with dichloromethane (50 mL) and washed with saturated NaHCO3 (2 x 15 mL).
After drying (Na2SO4), the solvent was removed to afford 64 mg (72%) of 2-acetoxy-4-methylsaccharin, mp 198-205 C
(decomp.) 1H nmr (300 MHz, CDCl3): 7.8 (2H, m); 7.64 (lH, d, J=6.18 Hz); 5.84 (2H, s); 2.82 (3H, s); 2.15 -(3H, s). IR (KBr, cm1): 2920 (w); 1745 (s); 1735 (s);
1630 (w). FDMS: m/e 269 (M).

- EXAMPLE 2: 2-Acetoxymethyl-4-chlorosaccharin (a) Methyl 6-chloroanthranilate This compound was prepared by the same method as used for preparing methyl-6-methylanthranilate in EXAMPLE 1, (a), using powdered KOH (4.08 g; 72.7 mmol;
2.5 equiv.), 6-chloroanthranilic acid (5.00 g; 29.2 mmol), and iodomethane (2.75 mL; 44 mmol; 1.5 equiv) to give 4.22 g (78%) of the compound as an oil. 1H nmr (300 MHz, CDCl3): 7.077 (lH, t, J=8.06 Hz); 6.744 (lH, d, J=6.7 Hz); 6.575 (lH, d, J=8.25 Hz); 4.871 (lH, br s); 3.929 (3H, s). IR (neat film, cm1): 3480 (m); 3380 (m); 2950 (w); 1705 (s); 1610 (s).

(b) 4-Chlorosaccharin 4-Chlorosaccharin was prepared by the same method as used for preparing 4-methylsaccharin using methyl 6-chloroanthranilate ((a) above) (4.22 g; 22.7 mmol) in AcOH (22 mL) and conc. HCl (40 mL) and sodium nitrite (1.68 g; 24.3 mmol) in water (7 mL) to prepare the diazonium salt. This was added to cupric chloride dihydrate (1.93 g: 11.4 mmol; 0.5 equiv) and sulfur dioxide (6.5 g; excess) in AcOH (30 mL)/water (5 mL).
The resulting sulfonyl chloride was treated with ammonium hydroxide (150 mL) as previously described to afford 3.07 g (62%) of 4-chlorosaccharin as a pale yellow solid, mp 245-246 C. 1H nmr (300 MHz, CD3CN):
7.918 (lH, dd, J=7.39, 1.91 Hz); 7.865 (lH, t, J=7.52 Hz); 7.829 (lH, br d, J=7.30 Hz). IR (KBr, cm1): 3570 (s); 3520 (s); 2950 (s,b); 1735 (s); 1630 (m). FDMS:
m/e 217 (M).

(c) 2-Hydroxymethyl-4-chlorosaccharin This compound was prepared in the same manner as 2-hydroxymethyl-4-methylsaccharin, in EXAMPLE 1, (c), from 4-chlorosaccharin (1.00 g; 4.60 mmol) and formalin (37%; 3.22 mL; excess). Unfortunately, this product did not crystallize from solution, but rather formed a separate layer of viscous oil. This oil was not characterized, but was used as is, since any attempt to isolate 2-hydroxymethyl-4-chlorosaccharin resulted in its reversion to 4-chlorosaccharin.
(d) 2-Acetoxymethyl-4-chlorosaccharin Acetoxymethyl-4-chlorosaccharin was prepared in the same manner as used for 2-acetoxymethyl-4-methylsaccharin, in EXAMPLE 1, (d), from the crude hydroxymethyl compound of (c) (0.34 g max; 1.4 mmol max) and acetic anhydride (2.5 mL) with 2 drops of sulfuric acid. In this case, after isolation, the product was purified by filtration through a pad of silica gel and elution with 1:1 ether:hexanes to afford 2-acetoxy-4-chlorosaccharin (35 mg, 9% yield) as a white solid, mp 138-142 C. 1H nmr (300 MHz, CDCl3): 7.921 (lH dd, J=6.54, 2.63 Hz); 7.874 (lH, t, J=7.98 Hz); 7.842 (lH, dd, J-6.70, 2.20 Hz); 5.869 (2H, s); 2.172 (3H, s). IR
(KBr, cm1): 1745 (s); 1735 (m, shoulder); 1575 (w).
Comb. anal.:
Theor C, 41.46; H, 2.78; N, 4.83;
Found C, 41.17; H, 2.81; N, 4.75.

13~6960 EXAMPLE 3: 2-Chloromethyl-4-chlorosaccharin Crude 2-hydroxymethyl-4-chlorosaccharin, from EXAMPLE 2, (c), (609 mg; 2.46 mmol max) was admixed into diethyl ether (5 mL), and thionyl chloride (3 mL;
excess) was added. The resulting mixture was heated until homogeneity was achieved and then stirred at room temperature overnight. It was then diluted with ether (20 mL) and filtered through a pad of celite topped with sand and eluted with ether. Removal of the solvent afforded 430 mg of crude chloromethyl derivative. A
portion (225 mg) was removed for further reactions. The remainder (205 mg) was chromatographed on flash silica gel and eluted with 40% ether/pentane to provide 137 mg, mp 135-136 C. 1H nmr (300 MHz, CDC13): 7.925 (lH, dd, J=6.62, 2.26 Hz); 7.882 (lH, t, J=8.18 Hz); 7.846 (lH, dd, J=7.42, 2.36 Hz); 5.561 (2H, s). IR (KBr, cm1):
3090 (w); 3050 (w); 1750 (s); 1575 (m). FDMS: m/e 265 (M).
EXAMPLE 4: 4-Chloro-2-(1-phenyl-lH-tetrazol-5-ylthiomethyl)saccharin The chloromethyl derivative prepared in EXAMPLE 3 (225 mg; 0.85 mmol) and sodium N-phenylmercaptotetrazole (200 mg; 1.01 mmol; 1.2 equiv) were dissolved in acetone (5 mL) to give a tan solution.
After about 10 minutes a precipitate was observed, and after stirring overnight at room temperature no 2-chloromethyl-4-chlorosaccharin was present by tlc analysis. The reaction mixture was poured into water and extracted with dichloromethane (3 x 25 mL). The combined extracts were dried (Na2SO4), concentrated and the residue was chromatographed on flash silica gel and eluted with 1:1 ether:hexanes. The major spot was -collected to afford 122 mg of product as a white solid, mp 175-177 C. lH nmr (300 MHz, CDCl3): 7.813 (3H, m);
7.515 (5H, s); 5.710 (2H, s). IR (KBr, cm1): 3080 (w);
1740 (s); 1590 (w). FDMS: m/e 407 (M); 230 (M -PMT);
178 (1%, PMT).

EXAMPLE 5: 4-Chloro-2-(4-phenyl-5-thioxo-2-tetrazolin-1-ylmethyl)saccharin The chloromethyl derivative prepared as in EXAMPLE 3, (337 mg crude; maximum 1.27 mmol) was dissolved (as much as possible) in acetone (10 mL).
Sodium N-phenylmercaptotetrazole (304 mg; 1.52 mmol; 1.2 equiv) was added and the reaction mixture was stirred at room temperature for 3 days. The mixture was diluted with dichloromethane (50 mL), washed with water (3 x 25 mL), dried (Na2S04), concentrated and filtered through a pad of silica gel (1:1 ether:hexanes elution). The material thus obtained was chromatographed on flash silica gel and eluted with 1:1 ether:hexanes to afford 44 mg (8.5~) of the nitrogen-linked PMT derivative, mp 158-162 C. lH nmr (300 MHz, CDCl3): 7.981 (lH, d, J=7.12Hz); 7.95 (2H, m); 7.887 (lH, t, J=6.74 Hz); 7.864 (lH, d, J=7.32Hz); 7.567 (3H, m); 6.392 (2H, s). IR
(KBr, cm1): 1745 (s); 1185 (s). FDMS: m/e 407 (M); 230 (M -PMT).

EXAMPLE 6: 2-[1-(3-Acetamidophenyl)-lH-tetrazol-5-ylthiomethyl~saccharin A mixture of 2-(chloromethyl)saccharin (0.98 g, 4.2 mmol), 1-(m-acetamidophenyl)-5-mercaptotetrazole (1 g, 4.2 mmol) and potassium bicarbonate (0.84 g, 8.4 mmol) in methyl ethyl ketone (50 mL) was heated at 50 C
under N2 overnight. The reaction mixture was cooled and -poured into dilute HCl/ice water (300 mL). The water was decanted from the semi-solid which was solidified by stirring in hot EtOAc. The resultant white solid isolated by filtration was treated with NoritR, filtered and then was recrystallized from acetonitrile (MeCN), to afford 0.82 g of the desired product as small white needles, mp 195-196 C decomp. 1H nmr (90 MHz, CDCl3) 2.05 (3H, s); 5.65 (2H, s). FDMS: m/e 430 (M).
Theor C, 47.43; H, 3.28; N, l9.S2;
Found C, 47.02; H, 3.27; N, 19.53.

EXAMPLE 7: 2-[1-(3-Heptanamidophenyl)-lH-tetrazol-5-ylthiomethyl~saccharin A mixture of 2-(bromomethyl)saccharin (2.7 g, 9.8 mmol), 1-(m-heptanamidophenyl)-5-mercaptotetrazole (3 g, 9.8 mmol) and potassium carbonate (3.4 g, 24.5 mmol) was heated at reflux in methyl ethyl ketone (50 mL) under nitrogen for 1 hour. The mixture was cooled and poured into a NaHCO3/ice solution. The water layer was decanted from the resultant white semi-solid. The semi-solid was washed with water then dissolved in hot MeCN, treated with NoritR and filtered. The filtrate was freed of solvent under vacuum and the resultant solid chromatographed (silica gel-95:5 CH2Cl2:acetone) to give a clear oil. The oil was crystallized from hot EtOH to afford 1.6 g of the desired product as a white solid, mp 146-147.5 C. 1H nmr (90 MHz, CDCl3) 5.65 (2H, s). FDMS: m/e 500 (M).
Theor C, 52.79; H, 4.83; N, 16.79;
Found C, 52.44; H, 4.75; N, 16.64.

EXAMPLE 8: 2-(1-Methyl-lH-tetrazol-5-ylthiomethyl)-saccharin A mixture of 2-(bromomethyl)saccharin (3 g, 10.8 mmol) and 5-mercapto-1-methyltetrazole, sodium salt (1.49 g, 10.8 mmol) was heated at reflux in methyl ethyl ketone (75 mL) for 2 hours. The reaction mixture was cooled, poured into dilute sodium bicarbonate/ice solution and extracted with methylene chloride (2x's).
The combined organic extracts were dried (Na2SO4) and freed of solvent under vacuum. The crude product was chromatographed (silica gel-95:5 CH2Cl2:ether) and the resultant oil was crystallized from hot isopropanol to afford 2.7 g (80%) of the desired product as a white solid, mp 106-110 C. 1H nmr (90 MHz, CDCl3) 5.55 (2H, s).
FDMS: m/e 311 (M).
Theor C, 38.58; H, 2.91; N, 22.49;
Found C, 38.58; H, 2.79; N, 22.60.

EXAMPLE 9: 2-(1-Cyclohexyl-lH-tetrazol-5-ylthiomethyl)saccharin A mixture of 2-(chloromethyl)saccharin (3 g, 12.9 mmol), 1-cyclohexyl-5 mercaptotetrazole (2.37 g, 12.9 mmol) and potassium carbonate (4.45 g, 32.2 mmol) was heated at reflux in methyl ethyl ketone (50 mL) for 1 hour. The reaction mixture was cooled, poured into dilute sodium bicarbonate/ice solution and extracted with EtOAc (2x's). The combined organic layers were washed with water, dried (Na2SO4) and freed of solvent under vacuum. Chromatography (silica gel; CH2Cl2) afforded 2 g of the desired product as a white foam that could be crystallized from hot cyclohexane, mp 103-105 C. lH nmr (90 MHz, CDC13) 5.65 (2H, s). FDMS: m/e 379 (M ).

Theor C, 47.48; H, 4.52; N, 18.46;
Found C, 47.84; H, 4.61; N, 18.36.

- EXAMPLE 10: 2-(1-Phenyl-lH-tetrazol-5-ylsulfinylmethyl)saccharin A mixture of m-chloroperbenzoic acid (0.43 g, 2.67 mmol) and 2-(1-phenyl-5-tetrazolyl-thiomethyl)saccharin(l g, 2.67 mmol) in methylene chloride was stirred at room temperature (RT) for 24 hours. TLC (95:5 CH2Cl2:ether) revealed the presence of starting sulfide. Additional peracid (0.2 g) was added and the mixture stirred for an additional 2 days. The reaction mixture was washed with sodium bicarbonate solution, dried (Na2S04) and freed of solvent under vacuum. Chromatography (silica gel-95:5 CH2Cl2:ether) afforded a foam that was crystallized from ether to afford 0.52 g of the desired product as a white solid, mp 161-162 C. 1H nmr (90 MHz, CDCl3) 5.5-6.0 (2H, q).
FDMS: m/e 196 (M -PMT), 389 (M).
Theor C, 46.27; H, 2.85; N, 17.98;
Found C, 46.00; H, 2.83; N, 17.76.

EXAMPLE 11: 5-Nitro-2-(1-phenyl-lH-tetrazol-5-Ylthiomethyl)saccharin A mixture of 2-bromomethyl-5-nitrosaccharin (2 g,6.2 mmol) and 1-phenyl-5-mercaptotetrazole, sodium salt in methyl ethyl ketone (40 mL)/DMF (10 mL) was heated at reflux for 2 hours. The reaction mixture was cooled and poured into a dilute sodium bicarbonate/ice solution. The resultant white solid, isolated by filtration, was washed with water and air dried. The compound was sonicated with 50:50 CH2Cl2:acetone and filtered to remove soluble impurities. The remaining solid was recrystallized from 2:1 MeCN:EtOH to afford 1.5 g of the desired product as an off white solid, mp 189-190 C. 1H nmr (90 MHz, DMSO-d6) 5.75 (2H, s). FDMS:
m/e 418 (M ).
Theor C, 43.06; H, 2.41; N, 20.09;
Found C, 42.29; H, 2.43; N, 20.13.

EXAMPLE 12: 2-(Phenylsulfonylmethyl)saccharin A mixture of m-chloroperbenzoic acid (2.2 g, 12.8 mmol) and 2-(phenylsulfinylmethyl)saccharin (3.75 g, 11.6 mmol) in methylene chloride (50 mL) was stirred at room temperature for 2 hours. An additional spatula of peracid was added and stirring was continued for an additional 1 hour. m-Chlorobenzoic acid was removed by filtration and the solid was washed with a small amount of methylene chloride. The filtrate was washed with sodium bicarbonate solution, dried (Na2SO4) and freed of solvent under vacuum. The resultant solid was recrystallized from 50:50 EtOH:MeCN to afford the desired product as a white solid, mp 169-171 C. 1H nmr (90 MHz, DMSO-d6, CDCl3) 5.15 (2H, s). FDMS: m/e 196 (M).
Theor C, 49.84; H, 3.29; N, 4.15;
Found C, 49.92; H, 3.24; N, 4.13.

EXAMPLE 13: 2-(2-PYrimidylsulfinylmethyl)saccharin A mixture of m-chloroperbenzoic acid (0.9 g, 5.37 mmol) and 2-(2-pyrimidylthiomethyl)saccharin prepared by procedures similar to those of Examples 9 and 11 (1.5 g, 4.8 mmol) in methylene chloride (75 mL) was stirred overnight at room temperature. The reaction mixture was washed with sodium bicarbonate solution, dried (Na2SO~) and freed of solvent under vacuum. Part of this crude product (0.5 g) was saved for direct conversion to the sulfone; the remaining material was chromatographed (silica gel-95:5 CH2Cl2:acetone).
Recrystallization (EtOH/MeCN) afforded 0.95 g of white crystals, mp 197-198 C. decomp. 1H nmr (90 MHz, CDCl3, DMSO-d6) 5.1-5.5 (2H, q).
Theor C, 44.57; H, 2.81; N, 13.00;
Found C, 44.67; H, 2.84; N, 12.97.

EXAMPLE 14: 2-(2-Pyrimidylsulfonylmethyl)saccharin A mixture of m-chloroperbenzoic acid (0.4 g, 2.3 mmol) and the sulfoxide prepared in EXAMPLE 13 (0.75 g, 2.3 mmol) in methylene chloride (50 mL) was stirred at room temperature with TLC (95:5 CH2Cl2:acetone) monitoring. After 2 hours, some of the starting sulfoxide still remained; an additional spatula of peracid was added and the reaction was stirred overnight. Methylene chloride (100 mL) was added and the mixture was washed with sodium bicarbonate solution.
The organic layer was dried (Na2SO4) and solvent was removed under vacuum. Recrystallization (MeCN/EtOH) afforded 0.95 g of white solid, mp 225-227 C decomp. 1H
nmr (90 MHz, DMSO-d6) 5.78 (2H, s). FDMS: m/e 196 (M -PMT), 339 (M ).
Theor C, 42.47; H, 2.67; N, 12.38;
Found C, 42.20; H, 2.62; N, 12.46.

EXAMPLE 15: 2-(4-Nitrophenoxymethyl)saccharin A mixture of 2-(chloromethyl)saccharin (3 g, 12.9 mmol) and sodium p-nitrophenoxide (2.55 g, 12.9 mmol) in tetrahydrofuran (THF) (50 mL) was heated overnight at 50C and then was refluxed for 45 minutes.
The reaction mixture was cooled, poured into a dilute sodium bicarbonate/ice solution and extracted with EtOAc (2x's). The combined organic layers were washed with sodium bicarbonate solution and water, dried (Na2SO4) and then solvent was removed under vacuum.
Chromatography (silica gel; CH2Cl2) afforded an oil that was crystallized from hot cyclohexane/ether. The resultant solid was recrystallized from EtOH to afford 0.92 g of the desired product as white shiny platlets, mp 162-164 C. H nmr (90 MHz, CDCl3, DMSO-d6) 5.95 (2H, s). FDMS: m/e 334 (M).
Theor C, 50.30; H, 3.02; N, 8.38;
Found C, 50.06; H, 2.91; N, 8.28.

EXAMPLE 16: 5-(3,3-Diphenylpropionamido)-2-(1-phenyl-lH-tetrazol-5-ylthiomethyl)-saccharin 5-Nitro-2-(1-phenyl-lH-tetrazol-5-ylthiomethyl)saccharin (4 g, 9.56 mmol) was dissolved in THF (250 ml) and placed in a Parr shaker bottle. 10~
Pd/C (2 spatulas) was added under N2and the mixture was shaken under hydrogen (55 psi) for 2.5 days. The reaction mixture was filtered through Celite diatomaceous earth. The filtrate was added to water and extracted with methylene chloride. The organic layer was dried (Na2SO4) and freed of solvent under vacuum.
The resultant yellow foam was sonicated with warm ethanol, cooled, and filtered. The desired 5-aminosaccharin derivative, 0.5 g, was isolated as a cream colored solid. FDMS: m/e 388 (M ).
A mixture of the above 5-aminosaccharin derivative (0.5 g, 1.29 mmol) and 3,3-diphenylpropanoyl chloride (0.315 g, 1.29 mmol) in acetonitrile (50 ml) was heated at reflux for 2.5 hours. TLC (95:5 methylene chloride:acetone) analysis revealed the presence of some -starting amine. A small additional amount of acid chloride was added and reflux was continued for 1.5 hours. The reaction mixture was cooled and poured into ice water (400 ml). After 30 minutes, the mixture was filtered and the resultant tan colored solid was washed with water and air dried. Chromatography on silica gel (95:5 methylene chloride:ether) produced a foam that was crystallized from hot ethanol to yield 0.68 g of a white solid, mp 92-93 C decomp. FDMS: m/e 596(M ). 1H nmr (90 MHz, CDCl3); 3.25 (lH, d); 4.8 (2H, t); 5.6 (2H, s);

6.9-8.2 (m, Ar). NMR also revealed approximately two ethanol molecules of crystallization: 1.25 (t); 3.7 (q). Comb. anal.:
Theor for C3oH24N6o4s2+ 2C2HsH: C~
H, 5.27; N, 12.2;
Found: C, 58.09; H, 5.15; N, 12.09.

EXAMPLE 17: Methyl 2-(1-Phenyl-lH-tetrazol-5-ylthio)-2-(2-saccharinyl)acetate Methyl l-chloro-l-thiophenylacetate was prepared as reported in the literature: I. Fleming and J. Iqbal, Tetra. Lett., 24, 327 (1983); M. Campbell, et al., Tetra. Lett., 21, 3305 (1980).
Saccharin (10 g, 54.6 mmol) was dissolved in EtOH (500 ml) with slight warming. Thallous ethoxide (13.6 g, S4.6 mmol) was added dropwise and the resultant heterogeneous mixture was stirred at room temperature for 2 hours. The mixture was cooled and filtered and the solid was washed with cold ethanol. The grey, white, crystalline solid was dried under vacuum in a desiccator to yield 19.4 g (92%) of the thallium salt of saccharin.
A mixture of the thallium salt of saccharin (1.78 g, 4.6 mmol) and methyl 2-chloro-2-thiophenyl 13~6960 acetate (1 g, 4.6 mmol) in DMF (25 ml) was stirred at 60 C for 7 hours. The mixture was cooled and poured into ice water (400 ml). After 30 minutes, the mixture was filtered and the solid was washed with water and air-dried. Chromatography on silica (methylene chloride) afforded a clear oil that was crystallized from hot EtOH to yield 0.87 g (51%) of white needles of methyl 2-phenylthio-2-(2-saccharinyl)acetate, mp 144-146 C. 1H nmr (90 MHz, CDCl3): 3.8 (3H, s); 5.95 (lH, s);

7.2-8.15 (9H, m). FDMS: m/e 363(M).
A solution of methyl 2-phenylthio-2-(2-saccharinyl)acetate (2 g, 5.5 mmol) and sulfuryl chloride (0.74 g, 5.5 mmol) in methylene chloride (50 ml) was stirred at room temperature for 2 hours.
Solvent was removed under vacuum and the yellow oil was crystallized from warm ethanol to give 0.94 g of product. NMR revealed greater than 50% starting material. An additional amount of starting material (1 g, 2.75 mmol) was added to the crude product mixture and it was redissolved in methylene chloride. Sulfuryl chloride (0.5 ml) was again added and the mixture was stirred at room temperature for about 12 hours. Work up as above afforded 0.66 g of crude chloride that was used immediately in the next step.
A mixture of this chloride (0.66 g crude mixture) and l-phenyl-5-mercapto-lH-tetrazole, sodium salt (0.44 g, 2.2 mmol) was heated at reflux in methyl ethyl ketone (25 ml) for 4 hours. After stirring at room temperature for 2 days, the reaction mixture was poured into ice water. The tan solid isolated by filtration was washed with water and air-dried.
Chromatography on silica gel (methylene chloride) yielded an off-white foam that was crystallized from ethanol to yield 0.36 g of white crystalline solid, mp -160-162 C. lH nmr (9o MHz, CDCl3); 3.8 (3H, s); 7.05 (lH, s); 7.4-8.1 (9H, m). FDMS: m/e 431 (M ).
Theor for C17H~3N5O5S2: C~ 47-33;
- N, 16.23;
Found: C, 47.15; H, 3.09; N, 16.30.
Other compounds of the present invention are prepared by syntheses similar to those described above.
Table 1 lists the compounds corresponding to the above-described examples, together with other exemplary compounds of the present invention.
Measurement of the inhibition constant, Kj, of a HLE-inhibitor complex has been described for "truly reversible inhibition constants" usually concerning competitive inhibitors. The compounds of the present invention, however, do not form truly reversible inhibitor complexes. The compounds of the present invention are consumed by the enzyme to some extent.
Thus, instead of measuring a Kj, a Kj* is calculated which is defined as the ratio of the koff/kon, the rate of reactivation of the enzyme to the rate of inactivation of the enzyme. The values of koff and kon are measured and Kj* is then calculated.
The rate of inactivation, kon, of enzymatic activity was determined for the compounds tested by measuring the enzyme activity of an aliquot of the respective enzyme as a function of time after addition of the test compound. By plotting the log of the enzyme activity against time, an observed rate of inactivation, kobs~ is obtained which can be represented as kobS =
ln2/t~/z where t1/Z is the time required for the enzyme activity to drop by 50%. The rate of inactivation is then equal to -kobs kon [I]

where [I] is the concentration of the inhibiting compound.
The reactivation constant, koff, is similarly determined and the inhibition constant, Kj*, is then calculated as Kj* = koff/kon The values obtained for kon and koff/kon for specific substituted saccharin derivatives are shown in TABLE 1.

_30-TABLE I

Example Elastase Alpha-ChymotrYpsin Structure10 3 X kon koff/kon 10 3 X kon koff/kon (M1 sec1) (nM) (Ml sec~) (nM) . 1 0.63 102 1.2 917 u O O
~ oJ~
2 4.9 45 2.9 51 3 450 0.5 5.8 26 ~b "=,, 44 6 6.0 25 6 5.5 lS 3.7 100 7 5.2 15 1n TABLE I (Continued~

Example Elastase Alpha-Chymotrypsin Structure 103 x kon koff/kon 10-3 x k koff/kon sec1) (nM) (M1 sec1) (nM) 8 1.0 81 2.1 523 o ~_11 o~ o ~ 9 2.5 32 7.0 11 7.0 157 ~

-~ 11 4.6 ~ r 12 0.97 82 1.1 1000 O~O ~ ~ 13 0-3 285 2.6 423 14 0.6 138 2.8 392 TABLE I (Continued) Example Elastase Alpha-Chymotrypsin Structure 10-3 x k kff/k~ 103 x kon koff/kon (M1 secl) (nM) (M1 secl) (nM) 3.2 69 16 2,600 " _".
17 2.9 270 18 1.0 100 0.21 620 19 50 4.4 '~, f~
~ ~ ~ 10,500 -TABLE I (Continued) ExamPle Elastase Alpha-Chymotrypsin $tructure lO 3 X kon koff/kon 10 3 X kon kOff/kon (M1 sec1) (nM) (M1 sec1) (nM) /~ 8,500 o 23 Z.5 17 '~ ~ ~ 24 7.4 250 TABLE I (Continued) Example Elastase AlDha-chymotrypsin Structure 10-3 x kon koff/kon 103 x k koff/kon (M1 secl) (nM) (M1 sec1) (nM) ~-o ~ ~ 26 .02 40,000 o 27 44 1.8 6.0 180 28 5.6 lS 4.3 254 -~35~ 1 33 6g60 The foregoing description and examples are intended as illustrative and are not to be taken as limiting. Still other variables within the spirit and scope of this invention are possible and will readily present themselves to those skilled in the art.

Claims (19)

1. A pharmaceutical composition comprising a

2-substituted saccharin derivative represented by the general structural formula:

where R1 is selected from the group consisting of halogen, ester, aryloxy, alkylthio, arylthio, sulfinyl, sulfonyl, amino, amido, imidyl, heterocyclyl and substituted derivatives thereof;
R2 is hydrogen, aryl or an electron-withdrawing group; and R3 and R4 are each independently selected from the group consisting of hydrogen, halogen, cyano, nitro, amino, amido and a straight, cyclic or branched-chain carbon-containing group having from 1 to 20 carbon atoms;
wherein the group is always appended to a heteroatom on R1; and a pharmaceutically acceptable carrier.
2. The composition according to claim 1 wherein R3 and R4 are not both hydrogen.

3. The composition according to claim 1, wherein R1 contains a substituent selected from the group consisting of alkyl, cycloalkyl, aryl, cycloalkyl, alkylamidoaryl, heterocyclyl, arylthio, nitro, amino, amido, sulfinyl and sulfonyl radicals.

4. The composition according to claim 1, wherein R1 is chloro.

5. The composition according to claim 1, wherein R3 is chloro.

6. The composition according to claim 1, wherein R3 is a lower alkyl group.

7. A method of inhibiting proteolytic enzyme activity comprising contacting an enzyme-containing liquid sample having proteolytic enzyme activity with an effective amount of a 2-substituted saccharin derivative represented by the general structural formula:

where R1 is selected from the group consisting of halogen, ester, aryloxy, alkylthio, arylthio, sulfinyl, sulfonyl, amino, amido, imidyl, heterocyclyl and substituted derivatives thereof;
R2 is hydrogen, aryl or an electron-withdrawing group; and R3 and R4 are each independently selected from the group consisting of hydrogen, halogen, cyano, nitro, amino, amido, and a straight, a cyclic or a branched-chain carbon-containing group having 1 to 20 carbon atoms;
wherein the group is always appended to a heteroatom on R1.

8. The method according to claim 7 wherein said enzyme is human leukocyte elastase.

9. The method according to claim 7 wherein said enzyme is a chymotrypsin-like enzyme.

10. The method according to claim 7 wherein said liquid sample is a body fluid sample.

11. A diagnostic method comprising:
(a) contacting a body fluid sample with an amount effective to inhibit proteolytic enzyme activity of a 2-substituted saccharin derivative represented by the general structural formula:

where R1 is selected from the group consisting of halogen, ester, aryloxy, alkylthio, arylthio, sulfinyl, sulfonyl, amino, amido, imidyl, heterocyclyl and substituted derivatives thereof;
R2 is hydrogen, aryl or an electron-withdrawing group; and R3 and R4 are each independently selected from the group consisting of hydrogen, halogen, cyano, nitro, amido, amino, and a straight, cyclic or branched-chain carbon-containing group having from 1 to 20 carbon atoms;
wherein the group is always appended to a heteroatom on R1; and (b) measuring the concentration of inhibited proteolytic enzyme in said sample.

12. The diagnostic method according to claim 11 wherein said proteolytic enzyme activity results from human leukocyte elastase.

13. The diagnostic method according to claim 11 wherein said proteolytic enzyme activity results from chymotrypsin-like enzymes.

14. The use of a 2-substituted saccharin derivative represented by the general structural formula:

where R1 is selected from the group consisting of halogen, ester, aryloxy, alkylthio, arylthio, sulfinyl, sulfonyl, amino, amido, imidyl, heterocyclyl and substituted derivatives thereof;
R2 is hydrogen, aryl or an electron-withdrawing group; and R3 and R4 are each independently selected from the group consisting of hydrogen, halogen, cyano, nitro, amino, amido and a straight, cyclic or branched-chain carbon-containing group having from 1 to 20 carbon atoms, wherein the group is always appended to a heteroatom on R1, for treatment of degenerative disorders in a mammal, or for preparation of a medicament therefor.

15. The use according to claim 14 wherein said degenerative disorder is emphysema.

16. The use according to claim 14 wherein said degenerative disorder is rheumatoid arthritis.

17. The use according to claim 14 wherein said degenerative disorder is pancreatitis.

18. The use according to claim 14 wherein said degenerative disorder is adult respiratory distress syndrome.

19. The use according to claim 14 wherein said degenerative disorder is an inflammatory disease.