CA1262551A - Inhibitors of mammalian collagenase and elastase - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
There are disclosed [benzothiazolythio]phenylacetyl-L-cysteines having the formula wherein R1 is hydrogen, halo, nitro, amino, lower alkyl, lower alkoxy, trifluoromethyl or hydroxy; R2 is halo, lower alkyl, lower alkoxy, amino, nitro or trifluoromethyl and the pharmaco-logically acceptable salts thereof, a method of inhibiting mammalian elastase and collagenase by using the [benzothiazolyl-thio]phenylacetyl-L-cysteines and the pharmacologically accep-table salts thereof, and mammalian elastase and collagenase inhibiting compositions containing the [benzothiazoylthio]
phenylacetyl-L-cysteines or the pharmacologically acceptable salts thereof.

Description

2551 831 0-2-Nl INHIBITORS OP MAMMALIAN COLLI~GENASl~ AND ELASTASE

Background of the Invention Collagen is the major organic component of the surface tissue found in the cornea, skin7 gastrointestinal viscera, joint mucosa and other areas o~ the body.
The collagen molecule has a molecular weight of 300,000, and is composed of three helical polypeptide chains which are Nound around a common axis forming a coiled chain. In solution collagen molecules exist as long rods about 300 x 15 A, but at a temperature of 37C and a pH of 7, the molecules polymerize into insoluble fibrils.
Thus, it is as fibrils that collagen invariably exists in tissue. The helical structure of 10 undenatured collagen is remarkably resistant to attaclc by proteolytic enzymes;
however, there have been discovered a number of natural enzymesJ i.e., animal collagenases, which are capable of breaking down collagen by cleaving the collagen molecule across the helical backbone yielding 3/4 and 1/4 length fragmentsO
Similarly, elastin is a signlficant component of the elastic fibers of connective tissue, being found particularly in joints, the ligaments of the vertebrae, the wRlls of the large arteries and in the connective tissue of the lungs. The polymeric structure of elastin contains large amounts of glycine, alanine and valine residues, and its elastic properties are brought about by the cross-linking of the amino acids in its structure. Elastin is broken down by the protease elastase, which is 20 ~capable of hydrolyzing proteins as the N-terminal peptide bond of aliphatic amino :
~' acid residues.
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The relationship between collagenase and the destruction of collagen-:
based tissue has been found in a number of disease states affecting various parts of the body9 all of which are basically similar in that collagen constitutes the major organic component, e.g., 3kîn, cornea, gastro-intestinal viscera, ioint mucosaJ etc.
For exampleg in connection with corneal tissue, it has been shown that collagenase is responsible for ulcers appearing a~ter the eye has been burned wlth allcali. Similarly, the relationship exists for other ulcerous conditions of the cornea such as viral ulcers, e.g., herpes simplex, vaccinia, etc.; bacterial ulcers, e.g. E'seudomonas, etc.;

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:, ~L2 6~2S~ 831 0-2-Nl degenerative ulcers and ulcers of unknown origin, e.g., associated with rheumatoid arthritis, Mooren's ulcer, furrow ulcer; and ulcers seco~ndary to dryin~, e.g. erythema multiforme (Stevens-Johnson Syndrome~.
In mam mals, collagenase is one of the key enæymes involved in the cartilage and joint destruction of rheumatoid arthritis; see9 for example, Arthritis and Rhematism, 20 (6):1231 (1977). Further, recent research results have been reported (Pharmacology International, 2, p. 11-16 (1982~) which support the conclusion that the destruction of human articular cartilage proceeds through the joint action of the enzymes elastase and collagenase. Elastase degrades the 10 proteoglycans which along with collagen cross-linking provide a barrier to the action of collagenase on collagen. Elastase also solubilizes collagen, eliminating the cross-linking portion of the collagen fibrils, permitting the collagen to then be morecompletely degraded by both elastase and collagenase~ Thus, both elastase and collagenase are key factors in the joint cartilage destruction folmd in rheumatoid arthritis.
The action of mammalian collagenase has also been implieated as a causative factor in several other diseases in mammals. These diseases inolude periodontal disease, tumor ;nvasiveness, and epidermolysis bullosa; see, for example, ~L~y, 92 (2): 509 (1978) and The New England Journal of 20 Medicine, 291(13): 652 (1974).
The action of mammalian elastase, likewise, has been determined to be a causative factor in other disease states. Thus, the first step in the disease process of emphysema has been shown to be the breakdown of lung elastin into small peptides.
The unchecked destruction of lung connective tissae elastin by elastase result~ in the enlargement of distal air spaees and destruction of alveolar cell walls. The healthy lung is protected from the destructive effects of elastase by normal levels of the naturally occuring elastase inhibitor, c~l-an~itrypsin. If the level of l~antitrypsin falls below about 80mg/dL, such as oc- urs in individuals afflicted with emphysema, elastase begins to destroy the lungs~ Therefore, the exogenous administration of3n elastase inhibitor to supplement the low levels of endogenous c~ l-antitrypsin is - ~ , 5~L. 831n-2-Nl indicated in the treatment of emphysema ~see Journal of the Arnerican Medical Association9 24~ on7 (19~
Accordingly, collagenase/elastase inhibitors can be advantageously used to block pathologies in which destruction of collagen- and elastin-containing connective tissue plays a central role, such as for example9 periodontal diseaseg rheumatoid arthritis, emphysema, corneal ulceration, and so forth.

Description of the Invention \
The present invention is directed to novel compounds having the formula ~R2 S Cll2s~I
R~ r~CH- ONilbElCOOII

wherein Rl is hydrogen, halo, nitro, amino, lower alkyl, lower alkoxy, trifluoromethyl 10 or hydroxy; R2 is halo, lower alkyl, lower alkoxy, amino, nitro or trifluoromethyl; and the pharmacologically acceptable salts thereof. The invention further is directed to collagenase/elastase inhibiting compositions comprising said compounds and a pharmacologically acceptable carrier. And there is also disclosed a method of inhibiting mammalian collagenase/elastase in mammals afflicted with a disease state in which collagen- or elastin-containing tissue Is broken down by elastase and collagenase which comprises administering to such an afflicted mammal an amount sufficient to inhibit said elastase/collagenase-induced collagen breakdown of an elastase/collagenase inhibitor having the formula:

~ .
, ' ~R2 ~ ~r ~CHCoNH8HCOOH
Rl -- ¦
~' . -~

3lZ~25~ 5L 8310-2-Nl wherein Rl is hydrogen, halo, nitro, amino, lower alkyl, lower alkoxy, trifluoromethyl or hydroxy, R~ iS halo~ -1QW9r alkiyl lower all~o~.y9 ~mino9 nitro or ~rifluoromethyl; and the pharmacologically acceptable salts thereof~
The term "lower alkyl" when used herein includes straight and branched chain hydrocarbon radicals having from 1 to about 6 carbon atoms. The term "lower alkoxy" designates radicals in which the hydrocarbon portion has 1 to about 6 carbon atoms.
The term "halo" when used herein refers to radicals of the elements fluorine, chlorine and bromine.
The term pharmacologically acceptable salts includes the salts of pharmacologically-acceptable organic and inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, methanesulfonic1 benzenesulfonic and the like, alkali metal carboxylates and carboxylates of a pharmacologically acceptable cation derived from ammonia or a basic amine.
The compounds of the invention are most convenienltly prepared by reacting the tricyclic nnesonionic didehydro compounds disclosed in U.S. Patent No.
4,275,065 with appropriate nucleophilic reactants. This reaction, in~lolving the ring cleavage of the terminal thiazole ring, is as follows:

R 1~\1/ s~s \~I~R2 CH2SH
~L--N ~:O NH2C~ICOOH

- ~2 i~l i~ SCllCONilCElCOOH

AHP 7501-3-C3t 83 1 û-2-N 1 ~26~

wherein Rl and R2 are as described hereinbefore. The reaction is carried out in a . su~t~e ocganic solvent, such as for example methylene chloride, and over a range of temperatures, such as room temperature as well as under reElux conditions.
The term "pharmacologically acceptable carrier'~ contemplates usual and customary substances employed to formulate solid, oral unit dosages for pharmacological purposes, including in its broadest form animal feedstuff. It also includes those employed to formulate either in unit dose or multidose form, oral and injectable suspensions and solutions, either directly or ~or reconstitution before administration.
To Iormulate dosages for administration according to this invention the compounds of formula I can be compounded into oral dosage forms such as tablets, capsules and the like. This is done by combining the compounds with conventional carriers, such as magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, low melting wax, cocoa butter, and the like. Diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, tablet~isintegrating agents and the like may be employed. The active ingredient may be encapsulated with or without other carriers. The compounds may also be injected parenterally, in which case they are used in the form of a sterile solution containing other solutes, for 20 example, enough saline or glucose to make the solution isotonlc. In all cases the proportion of active ingredients in said compositions both solid and liquid will be at least sufficient to imp~rt collagenase inhibitory activity thereto on oral or parenteral administration.
In practicing the method of t~le invention, the instant compositions can be administered to warm-blooded anirnals, in a Yariety of dosage forms, alone or in combination with pharmacologically effective carriers, preferably orally or by - injection.
~;~ The dosage requirements vary with the particular compositions employed, the route of administration, the severity of the symptoms presented and the 30 particular subject being treated. Treatment will generally be initiated with small dosages less than the optimum dose of the compound. Thereafter, the dosage is ,

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increased until the optimum effect under the circumstances is reached. In general, the compounds of the invention are most desirably administered at a concentration level that will generally afford effective results without causing any harmful or deleterious side effects. Y1ith large animals (about 70 kg. body weight), for injection administration the dose is from about 25 milligrams to about 50 milligrams and for oral administration the dose is from about 50 milligrams to about 20û milligrams and preferably from about S0 milligrams to about 100 milligrams per day either as a single unit dose9 or if desired, the dosage may be divided into convenient subunits administered at convenient times throughtout the day.
The ability of the compounds of the invention to inhibit elastase and collagenase is demonstrated by testing in an enzyme assay using collagenase produced by normal human leukocytes or by normal human fibroblasts in culture and in an enzyme assay using human leukocyte elastase.
The following examples show the preparation and testing of a compound used in the invention.

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831n 2-Nl i25~
~xample 1 N-~[(5 Chlor~2 benzothiazolyl~thio]phenylacet~l]-I~ysteine A. c~-(5-Chlorobenzothiazol-2-ylthio)benzene acetic acid 50.Q g. (0.248 m) 5-chloro-2-mercflptobenzothi~zole and 53.0 g (0.24 m) a-bromopheylacetic acid are dissolved in 1.5 1. acetone and the solution is heated for 4 hours in the presence of 50 ml. glacial acetic acid. The solution i5 concentrated to a smaller volume ~about 200 ml) and the residual solid (90 g.) is collected. The resulting salt is suspended in 1 1. of water and the mixture is stirred at room temperature overnight. The collected solid is recrystallized from 2.51. acetonitrile to give a total of 64 g. (85% yield) of title compound melting at 190-2C.
Cl ~Hl oclNO2s2 Calculated: C~ 53.64; H, 3.00; N, 4.17 Found: C, 53.83; H, 3.13; N, 4.13 B. Chloro-2-phenylthiazolo[2,3-b ]benzothiazol-3(2H~-one mesoionic didehydro derivative 31 g. (0.092 m) of the compound of A. above is suspended in 3.5 1.
methylene chloride and the mixture is heated to gentle reflux in the presence of 25 ml. acetic anhydride. The solid is gradually dissolved~ the solution turning reddish.
After heating overnight, the solution is filtered and concentrated to 200 ml. The 20 residual orange solld, weighing 29.5 g. (quantitative yield~, is collected and has a melting point of 215-6C.

C. N-[~(5-Chloro-2-benzothiazolyl)thio ]phenylacetyl-[~cysteine 6.36 g. (0.02 m) ~of the compound of B. above, 2.4 g. (0.0~ m) L-(-) cysteine and 2.5 g. (0.025 m) ;trlethylamine are suspended in methylene chloride (800 ml) and the mixture is heated for 56 hours. After filtering off some insoluble material~ the filtrate is washed with dilute hydrochloric acid solution and then dried . ~
over anhydrous magnesium sulfate. The oily residue left after solvent removal is -' ~ X~i25~1 8310-2-Nl triturated with ether and the solid is collected. The ether solution, upon standing, yields more solid. The combined solids are recrystallized from acetonitrile to give 1.5 g. (17% yield) of the title compound, which has a melting point of 155-8C.
Analysis for: ClgHlsClN~O3S3 Calculated: C9 49.25; H, 3.44; N, 6.38 Found- C, 49.36; H, 3.45; N, 6.0 Example 2 The compound N-[[(5-chloro-2-benzothiazolyl)thio~-phenylacetyl]-L-cysteine is tested for collagenase inhibition in an in vitro assay based on the 10 procedure described by A. Sellers and J. J. Reynolds, Biochem. J., 167 (19~7) pp. 353-60.
Collagenase produced by normal human leukocytes or by normal human skin fibroblasts in cell cuItures is purified by adsorption onto a collagen Sepharose 4B column. Prior to use in the assay, the zymogen is activated with trypsin, while the trypsin in turn, is inactivated with an excess of soybean trypsin inhibitor.
According to the assay procedure, mierofuge tubes are prepared containing a total of about 150 ~. o solution containing: 25 ~1. collagen tl 4C-acetylated collagen -2 mg/ml in 0.01% acetic acid); 25 ~1. of 0.15 M tris/0.015 M
20 CaC12, ph 7.4; 75 1ll. collagenase in tris buffer (0.05 M tris/0.005 M CaC12, pH 7.4);
and 25 ~1. of collagenase inhibitor in tris buffer. Samples and controls are incubated at 35C for one to five hours depending upon potency of the enzyme. At the end of the reaction period, the tubes are spun down In a Beckman Microfuge. A 25 ~
aliquot of each tube is then assayed in a scintillation counter. Since native collagen forms insoluble fibrils under these conditions7 radioactivity detected in the supernate is a measure of collagen hydrolysis.
IJ1 a set of experiments7 the compound of Example 1 is tested in the assay to determine its collagenase inhibition activity. In the first experiment, Collagen I (most predominant form of collagen, found in skin) is used as the substrate.

AHP-750 1 ~3-C 3/
~255i~ 8~10-2-~l The second e~cperiment used Collagen II ( which is found in cartilage) as the substrate.
The results are sumrnarized below:

Experiment Number IC50l1M

5.7 2 12.5 The results show that the compound tested is a potent inhibitor of collagenase, whose inhibi~ory activity shows no significant difference when tested with different collagen substrates.

, Example 3 The compound N-[[(5-chloro-2-benzolthiazolyl)thio ~-phenylacetyl ]-L-cysteine is tested for human elastase inhibition in an in vitro assay which is carried out as follows:
A crude elastase preparation is made by homogeni~ing human leukocyte granules [prepared according to the method of R. J. Baryk and J. Travis~
, 15, (4), 837 (1976~], spinning and dialyzing the supernates against cold 0~05M tris-HCl/0.05M NaCl at ph 8Ø
This elastase preparation is used in two assay methods to test for elastase inhibition.
Method l 400 ~1 of the elastase preparation9 ln 111 (0.0625 mg) of succinyl-L-alanyl-_-alanyl-L-alanyl-p-nitroanilide (the enzyme substrate), 207 111 of buffer and varying amounts of the elastase inhibitor under testing are incubated at 37C for lS

hours. The hydrolysis is measured by use of hlgh pressure liquid chromatography (HPLC) or by the spectrophotometric measurement of the release of ~nitroaniline at `~ a wavelength of 410 nm. In this method, l:he compound of Example 1 demonstrated an ICso of 2.1 ~g/ml.
. ~ .

_ g _ ; ~ .

AHP-7~01-3-C3/
83 l 0-2-N 1 ;255~
Method 2 In this method, the substrate used is an elastin-Congo red eomplex.
Hydrolysis of this substrate by elastase release~; Congo red, which is assayed by HPLC. The assay system also contains buffer and either 20 ,ug of the compound of Example l, or no compound. The system is incubated at 37C for 18 hours. The standard sample represents the complete hydrolys;s of the elastin-Congo red complex by hog pancreatic elastase.
Sample ContentRelative Release of Con~o Red Standard l.0 No Inhibitor 0.lO4 Compound of Example l 0.000 The results of these assays show the strong inhibitory effect the compound of Example l exerts on the enzymatic activity of human leukocyte elastase.

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Claims (2)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A compound having the formula wherein R1 is hydrogen, halo, nitro, amino, lower alkyl, lower alkoxy, trifluoromethyl or hydroxy; R2 is halo, lower alkyl, lower alkoxy, amino, nitro or trifluoromethyl; and the pharmacologically acceptable salts thereof.

2. The compound of Claim 1, having the name N-[[(5-chloro-2-benzothia-zolyl)thio]phenylacetyl]-L-cysteine.