SUBSTITUTED NAPHTHOIC ACIDS This invention relates to novel C-substituted naph- thoic acid ureides which may be represented by Formula I:

wherein A is selected from the group consisting of hydrogen, lower (Cχ-C$) alkyl and a pharmaceutically acceptable salt cation; B is selected from the group consisting of hydrogen, lower (Ci-Cg) alkanoyl and a pharmaceutically acceptable sal cation; and R is selected from the group consisting of hydro gen and lower (C1-C3) alkyl. These compounds are useful as inhibitors of connective tissue destruction.

Abnormal destruction of connective tissue by colla- genase and/or neutral proteases causes tissue damage and/or tissue dysfunction. In these conditions an inhibitor of con¬ nective tissue destruction acting directly or indirectly would be useful in preventing, retarding, or reversing tissue damage and/or collagen diseases.

The term connective tissue refers to a matrix of at least three protein molecules, collagen, proteoglycan and.

elastiπ. These molecules play an important role in the structural integrity of normal tissues. Collagen, the most abundant protein in the body occupies a central position in the connective tissue matrix ["Biochemistry of Collagen", E G. N. Ramachandran and A. H. Reddi, Academic Press, New Yor (1976); P. Bornstein, Ann. Rev. Biochem. , 43 , 567 (1974); J. Fessler and L. Fessler, Ann. Rev. Biochem. , 47, 129 (1978)].

Collagen is, for example, the main structural com- ponent of the oral tissue (periodontal, ligament, alveolar bone, gingiva, and cementum) [Fullmer, et al. , J. Dental Research, 48, 646 (1969)]. collagen amounts to 40% of cart lage protein, 90% of bone protein, and over 90% of dry dermi Articular cartilage is the resilient tissue that covers the articulating extremities in synovial joints. It consists o collagen fibres that are intimately meshed in a hydrated gel of proteoglycan.

Prσteoglycan, as it exists in cartilage, is a mole¬ cule in which sulfated polysaccharide chains are covalently linked to a protein backbone ["Dynamics of Connective Tissue acromolecules", Ed. P. M. burleigh and A. R. Poole, North Holland, Amsterdam (1975)].

Elastin is a major connective tissue component of pulmonary structure ["Elastin and Elastic Tissue", Ed. L. B. Sandberg, W. R. Gray, and C. Franzblau, Plenum Press, New

York (1977)]. The breakdown of elastin of pulmonary connec¬ tive tissue is considered the primary event in pulmonary emphysema [A. Janoff in "Proteases and Biological Control", ' Cold Spring Harbor Conference on Cell Proliferation, 2_, 603 (1975)].

Degradation of fibrous collagern is initiated by a combination of neutral proteases and tissue collagenase as a integral part of a complex immunopathological process which results in the loss of collagen from normal tissue. Under normal conditions cellular mechanisms maintain a careful bal ance between the rates of collagen synthesis and degradation However, in certain pathological conditions, the ensuii

elevated levels of neutral proteases and collagenase can re¬ sult in rapid collagen degradation and tissue dysfunction. For example, in periodontal disease, the generated elevated levels of neutral proteases and collagenase in the gingival crevicular fluid rapidly degrade the fibrous collagen suppor ing the teeth. Periodontal pockets result ultimately from collagen degradtaion, and as these pockets deepen, support of tooth is lost and alveolar bone is resorbed [K. Ohlsson, I. Ohlsson, and G. I. Basthall, Acta Odontol. Scand., 32, 51 (1974); L. M. golub, S. Kenneth, H. McEwan, J. B. Curran, and N. S. Ramamurthy, J. Dental Research, 55 , 177 (1976); L. M. Golub, J. E. Stakin and D. L. Singer, J. Dental Re¬ search, 53_, 1501 (1974); L. M. Wahl, S. M. Wahl , S. E. Mer- genhagen, and G. R. Martin, Proc. Natl. Acad. Sci. U.S. , 71, 3598 (1974); Science, 187, 261 (1975)].

In arthritic conditions such as in rheumatoid arthrit septic arthritis, and osteoarthritis elevated degradation of collkagen and proteoglycan initiate rapid destruction of articular tissue [J. M. Evanson, J. J. Jefferey, and S. M Krane, Science, 158, 499 (1967); E. D. Harris, D. R. Dibona and S. M. Krane, J. Clin. Invest. , 48, 2104 (1969); E. D. Harris, Rheumatoid Arthritis, Medcom. Press, N. Y. (1974); Z. Werb, C. L. Mainardi, C. A. Vater and E. D. Harris, New Eng. J. Med. , 296, 1017 (1977); J. M. Dayer, R. G. Russell and S. M. Krane, Science, 195, 181 (1977); E. D. Harris,

C. A. Vater, C. L. Mainardi and Z. Werb, Agents and Actions, 8_, 35 (1978); D. E. woolley, E. D. Harris, C. L. Mainardi an C. E. Brinkerhoff, Science, 200, 773 (1978); Ξ. D. Harris, C. S. faulkner, F. E. Brosn, Clin. Orthoped. , 110, 303 (1975) M. G. Ehrlich, H. J. Mankin, H. Jones, R. Wright and C. Cris J. Bone Jt. Surg. , 57A, 565 (1975); S. gordon, W. Newman and B. Bloom, Agents and Action, 8_, 19 (1978); "Mechanisms of Tissue Injury With Reference to Rheumatoid Arthritis", Ed. R. J. Perper, Ann. N. Y. Acad. Sci. , 256, 1-450 (1975)].

Increasec collagen degradation in bone can result in abnormal bone destruction as in osteoporosis [C. G. Griffith, G. Nichols, J. D. Asher and B. Flannagan,J. Λm. _M e _d . Assoc. _^

193, χ (i965); 3. Gardner, H. Gray and G. Hedyati, Curr. To Surg. Res. , 2, 175 (1970); B. Gardner, S. Wallach, H. Gray and R. K. Baker, Surg. Forum, 2_2, 435 (1971)]. Collagenase activity has also resulted in tissue damage in cholesteatoma [M. Abramson, R. W. S. Schilling, C. C. Huang and R. G. Salome, Ann. Otol. Rhiπol. Faryngol., 81, 158 (1975); M. Abramson and C. C. Huang, Laryngoscope, 77, 1 (1976)1. In corneal ulcerations that progress to loss of corneal inte¬ grity and function, collagenase has been implicated as a direct factor in corneal destruction [S. I. Brown, C. W.

Hook and N. P. Tragakis, Invest. Ophthamol. , 11, 149 (1972); M. B. Ber an, C. H. Dohlman, P.F. Davison, and M. Ghadringer Exptl. Eye Res. , 11, 225 (1971)]. Elevated levels of col¬ lagenase have also been observed in patients with epidermo- lysis bullosa, and a group of related genetic diseases of the dkin [E. A. Bauer, T. G. Dahl, and A. Z. Eisen. J. In¬ vest. Dermatology, 68, 119 (1977).

Increased breakdown of elastin of the lung tissue by neutral proteases (elastase) may contribute to the lesions in pulmonary emphysema [I. Mandel , T. V. Dar le, J. A. Frier ^• er, S. Keller and G. M. Turino, Elastin and Elastic Tissue, Ed. L. B. Sandberg, W. R. Gray and C. Fransblau, Plenum Pres N. Y. , p. 221 (1977)].

A variety of substances, bot naturally occurring and synthetically prepared, have been found to be inhibitors of connective tissue destruction, e.g., inhibitors of colla¬ gen degradation, that is, as collagenase inhibitors. Suich substances include, for example, ethylenediaminetetraacetate,

1,10-phenanthroline, cysteine, dithiothretol and sodium auri thiomalate [D. E. Woolley, R. W. glanville, D. R. Roberts an J. M. Evanson, Biochem J. , 169 265 (1978); S. Seifter and E. Harper, Chap. 18, The Collagenases" in The Enzymes (3rd Edition), 3_, 649-697, Ed. by P. D. Boyer, Academic Press, N. (1971)]. In the eye, -a number of studies using collagenase inhibitors directly applied to corneal ulcerations have been reported. Calcium ethylenediaminetetraacetate and acetyl- cysteine reduce the frequency of ulceration in the alkali

burned rabbit [M. Ber an and C. Dohlman, Arch. Qphthamol . , 3 , 95 (1975)]. Both cysteine and acetylcysteine have been effective in the treatment of acute and chronic corneal ulceration in the human, although the latter compound was preferred because of its greater stability [S. I. Brown, N. P. Tragakis and D. B. Pease, Am. J. Qphthamol. , 74, 316 (1972); M. Berman, Trace Components of Plasma: Isolation and Clinical Significance, 7th Annual Red Cross Symposium, p. 225, Alan R. Liss, Inc., N. Y. (1976)]. Naturarally occurring collagenase inhibitors incl the serum components alpha2-macroglobulin and beta -anti- collagenase [D. E. Woolley, R. W. glanville, D. R. Roberts and J. M. Evanson, Biochem. J. , 169, 265 (1978)].

While some compounds may inhibit the destructive feet of collagenase on connective tissue by acting directly on collagenase itself, other compounds may inhibit such destruction by coating, binding or competing with sights on the connective tissue in such a manner as to prevent colla¬ genase from attacking it. The present invetion, however, is not to be restricted or limited to any particular mech¬ anism or mode of action. Suffice it to say, that the com¬ pounds of this invention have utility as inhibitors of connective tissue destructioin albeit in whatever manner or mode. U. S. Patent No. 2,687,436 discloses substituted

3-(2-naphthyl)-cyclohkexanes useful in the treatment of collagen diseases. British Patent Nos. 856,357 and 1,246,14 disclose 2-aryl-hexahydro-quinolizines and 1-hydroxypraline derivatives, respectively, useful for treating diseases af- fecting connective tissue. The closest known structurally related compound to those of the present invention and dis¬ closed as having collagenase inhibiting activity is found in Thromb. Res. , 10(4), 605-11 (1977), wherein the trypanocidal agent trypan blue is reported as inhibiting the activity of collagenase, or a proteinase contaminant in the collagenase preparation. It is interesting, however, that in this same article, the ureide Suramin is reported as not hibiting _.

the action of collagenase. The closest known ureides to those of the present invention, and not disclosed as inhibi¬ tors of connective tissue destruction or as collagenase inhi bitors are those ureides found in Journal of the Chemical ^" . Society, 3069 (1927) , and in U. S. Patent Nos. 1,218,654 and 1,308,071. The genric disclosure of the "071 patent encompasses a vast number of ureides and with proper selec¬ tion, among the many possible variables, some of the com¬ pounds of this invetion may be encompassed within this broad generic disclosure. However, such disclosure by itself does not anticipate or render obvious the invention claimed herei

Of particular interest are the group of compounds encompassed within Formula I and illustrated by Formulas II and III:

(II)

(III)

^JREA,

wherein A, B and R are aε defined with reference to Formul I.

By pharmaceutically acceptable salt cation is meant an alkali metal; and alkaline earth metal; ammonium; primary amine, e.g. ethyl amine; secondary amine, e.g. di- ethylamine or diethanolamine; tertiary amine, e.g. pyridine triethylamine or 2-dimethylaminomethyldibenzofuran; aliphat amine, e.g. decamethylenediamine; or an aromatic amine.

Representative compounds encompassed within this invention include, for example:

6,6 '-[Ureylenebis(m-phenylenecarbonylimino) ]bis[4-hydroxy-2 -naphthoic acid] diethyl ester diacetate

6,6 '-[Ureylenebis-m-phenylenecarbonylimino) ]bis[ -hydroxy-2 -naphthoic acid

6,6 '-[Ureylenebis-m-phenylenecarbonylimino) ]bis[4-hydroxy-2 -naphthoic acid] diethyl ester

This invention is also concerned with C-substitut aminobenzamido naphthoic acids which are intermediates for the preparation of the biologically active compounds of For mula I and which may be represented by Formula IV:

(IV)

wherein A, B and R are as defined with reference to FOrmula I.

Of particular interest are the group of interme¬ diate compounds encomp-assed within formula IV and illustrate by Formulas V and VI:

(V)

(VI)

wherein A , B and R are as de f : ^" ned wi th reference to Formul a

I .

Representative compounds encompassed by Formula

IV include, for example:

6-(m-Aminobenzamido)-4-hydroxy-2-naphthoic acid ethyl ester acetate

This invetion is also concerned with a method of inhibiting connective tissue destruction in a warm-blooded animal which comprises administering to said animal an effec tive inhibiting amount of a compound encompassed within For¬ mula I. Moreover, this invention is concerned with a etHod of inhibiting the degradation sequelae of collagenase activi ty in a body fluid, such as crevicular fluid, synovial fluid and the like, which comprises subjecting body fluid collagen ase to the action of an effective collagenase inhibiting am¬ ount of a compound encompassed within the above formula.

Body fluid can include blood, plasma, serum, synovial fluid crevicular fluid, ocular fluid, etc., containing collagen¬ ase. The method of use aspect of this invention is further concerned with a method of inhibiting the action of colla- genase in a warm-blooded animal which comprises internally administering to said animal an effective collagenase inhi¬ biting amount of a compound encompassed within the above formula.

Sincer the compounds of the present invention find utility as inhibitors of connective tissue destruction or as collagenase inhibitors in body fluids, as such they may be useful in ameliorating or preventing those patholo¬ gical reactions resulting from the functioning of collagen¬ ase, and in the therapeutic treatment of warm-blooded ani- mals having connective tissue disorders such as periodontal diseases and diseases of the teeth, osteoporosis, Paget's disease, hyperparathyroidism or renal failure, rheumatoid arthritis, septic arthritis, osteoarthritis, gout, acute synovitis, scleroderma, psoriasis, epidermolysis bullosa, keloids, blisters, cholesteatoma of the ear, and corneal ulceration. The compounds of the present invention may also be useful in those pathological states where excessive activity of neutral proteases causes tissue damage.

The compounds of the present invention may be pre- pared according to the following Flowchart A.

ιR£

Flowchart A

With reference to Flowchart A, a substituted- -amino-naphthoic acid 1_ is dissolved in pyridine, cooled and reacted with an excess substituted nitrobenzoylchlorid 2_, giving a substituted nitrobenzamido-substi uted πaph- thoic acid 3_. which is hydrogenated in the presence of a suitable catalyst to give the corresponding amine derivati 4_. The amine 4_ is dissolved in pyridine and phosgenated to give the final ureide product 5_ which is isolated by conventional procedures.

OMPI

Example 1 6-(m-Aminobenzamido)-4-hydτoxy-

-2-naphthoic acid, ethyl ester, acetate A solution of 20 g. or 4-hydroxy-6-nitro-2-naρh- thoic acid [W. F. Beech and N. Legg, J. Chem. Soc. , 1887 (1949)], 385 ml. of absolute ethanol and 20 ml. of con¬ centrated sulfuric acid is refluxed for 4 hours, concentra- ated and diluted with water. The solid is collected by filtration, washed with water until neutral and crystallize from 250 ml. of acetonitrile, giving 16.5 g. of 4-hydroxy- -6-nltro-2-naphthoic acid ethyl ester as yellow crystals.

To a mixture of 22.47 g. of 4-hydroxy-6-nitro-2- -naphthoic acid ethyl ester in 150 ml. of pyridine is added 8.5 ml. of acetic anhydride. The mixture is stirred for 5 minutes, warmed on a steam bath until solution is complete and then allowed to stand for 10 minutes. The solution is poured into one liter of ice water and then filtered. The solid is dissolved in 500 ml. of ethylene chloride, dried over sodium sulfate, filtered and concentrated to about 200 ml. A 300 ml. portion of ethanol is added and the produce is allowed to crystallize, giving 25.1 g. of 4-hydroxy-6- -nitro-2-naphthoic acid ethyl ester acetate as pale yellow crystals.

A mixture of 26.3 g. of 4-hydroxy-6-nitro-2-naph- thoic acid ethyl ester acetate, 250 ml. of tetrahydrofuran and 2.5 g. of 10% palladium on carbon is hydrogenated on a Parr shaker at 40-20 psi over 45 minutes. The mixture if filtered through diatomaceous earth and evaporated _in vacuo to an oil. This oil is crystallized from 200 ml. of ether, giving 21.8 g. of 6-amino-4-hydroxy-2-naphthoic acid ethyl ester acetate as beige crystals.

To a cooled (ice bath) solution of 9.02 g. of 6- -amino-4-hydroxy-2-naphthoic acid ethyl ester acetate in 50 ml. of dry pyridine is added 6.74 g. of m-nitrobenzoyl chloride. After 5 minutes the ice bath is removed and stirring is continued at room temperature for 30 minutes. The solution is poured into 500 ml. of water and stir:

until the precipitate solidifies. The solid is collected by filtration, washed with water, dried and crystallized from 250 ml. of acetonitrile at 5°C. , giving 13.0 g. of 4-hydroxy-6-m-nitrobenzamido-2-naphthoic acid ethyl ester acetate as beige crystals.

A mixture of 13.0 g. of 4-hydroxy-6-m-nitrobenz mido-2-naphthoic acid ethyl ester acetate, 125 ml. of tetrahydrofuran and 1.25 g. of 10% palladium on carbon is hydrogenated in a Parr shaker at 45-37 psi for one hour. The mixture is filtered through diatomaceous earth and the filtrate is evaporated _in vacuo to a pale yellow glass This glass is crystallized by trituration with ether and the solid is recrystallized from 100 ml. of acetonitrile a 5°C. , giving 10.35 g. of the desired product as colorless crystals, m.p. 185-187°C.

Example 2 6,6'-[Uτeylenebis( -phenylenecarbonylimino) ]bis-

[4-hydroxy-2-naphthoic acid]diethyl ester diacetate

To a solution of 10.23 g. of 6-(m-aminobenzamido -4-hydroxy-2-naphthoic acid ethyl ester acetate in 60 ml. of dry pyridine is added a solution of 1.3 g. of phosgene 5 ml. of dry ethylene glycol, dimethyl ether, dropwise, with stirring and cooling, during 2-3 minutes. Stirring is continued at room temperature for 2 hours, then the solution is poured into 800 ml. of water. The gummy pre¬ cipitate is triturated with water, giving a red solid. This solid is stirred and refluxed in 300 ml. of ethanol, cooled, filtered and the solid is washed with ethanol, the ether. This solid is dissolved in 120 ml. of hot dimethyl formamide, treated with charcoal and filtered through dia¬ tomaceous earth. The filtrate is warmed to 80°C. and dilu¬ ted slowly, with stirring with 60 ml. of water. The mixtur is cooled to room temperature and the solid is collected b filtration, washed with 67% aqueous dimethylformamide, ethanol, then ether and dried overnight at 110°C. , giving 8.45 g. of the desired product as a pale tan powder, m.p. 285-287 ⁰ C. ^^

Example 3 6,6'-[Ureylenebis(m-phenyl necarbonylimino) ]- bis[4-hydroxy-2-naphthoic acid To a cooled (water bath) solution of 4.06 g. of 6,6 '-5-[ureylenebis(m-phenylenecarbonylimino) ]bis[4-hydroxy -2-naρhthoic acid]diethyl ester diacetate in 60 ml. of di- methylsulfoxide is added 60 ml. of 2N sodium hydroxide, por tionwise, with stirring, in a nitrogen atmosphere. The mixture is stirred under nitrogen at room temperature for 2 hours, then poured into 300 ml. of water and filtered. The filtrate is acidified to pH 2 with the addition of 10 ml. o concentrated hydrochloric acid and 50 g. of sodium acetate trihydrate are added. The gel is filtered and washed with water, then further washed with water in a centrifuge and dried by co-evaporation with 750 ml. of n-propanol , giving a red-brown powder. This powder is dissolved in 25 ml. of hot dimethylformamide, diluted slowly with 15 ml. of water and cooled in a refrigerator. The precipitate is collected by filtration, washed successively with 8 ml. of 50% aqueous dimethylformamide, ethanol:ether (1:1) and finally ether, then dried overnight at 110°C. , giving 1.6 g. of the desired product as a pale tan powder, m.p. 297-300°C. (dec)

Example 4 6,6 ^t -[Ureylenebis(a-phenylenecarbonyIimino) ]- bis[4-hydroxy-2-naphthoic acid] diethyl ester

To a cooled (water bath) solution of 2.0 g. of 6,6 '-[ureylenebis( -ρhenylenecarbonylimino) ]bis[4-hydroxy-2- -naphthoic acid] diethyl ester diacetate in 60 ml. of dimeth ylformamide is added 40 ml. of 0.25N sodium hydroxide, drop- wise with stirring over 10 minutes. The solution is stirred for an additional 10 minutes, 80 ml. of pyridine is added and the solution is poured with cooling into a mixture of 800 ml. of water and 85 ml. of concentrated hydrochloric acid. The solid is collected by filtration, washed with water and dried at room temperature. This solid is dissolve

in hot 2-methoxyethanol at a concentration of 4% (w/v) . This solution is then distilled with 1/2 its volume of water and then cooled to room temperature. The precipitate is collected by filtration and washed with 50% aqueous 2-methoxyethanol . ethanol, then ether. This solid is dis- solved in a mixture of 2-methoxyethanol .-dimethylformamide (6:5:1) giving an approximate 4% (w/v) solution, diluted with water and filtered. The solid is washed as described above, then with acetone and dried at 110°C. , overnight giving 417 mg. of the desired product as a tan powder, m.p. 265-280°C. (dec).

Example 5 Preparation of Compressed Tablet Ingredient mg. /Tablet

Active Compound 0.5-500 Dibasic Calcium Phosphate N.F qs

Starch U.S. P 40

Modified Starch 10

Magnesium Stearate U.S.P 1-5

Example 6 Preparation of Compressed Tablet - Sustained Action

Ingredient mε . /Tablet

Active Compound as Aluminum 0.5-500 (as acid

Lake*, Micronized

Dibasic Calcium Phosphate N.F qs Alginic Acid 20

Starch U.S.P 35

Magnesium Stearate U.S.P 1-10

"Collagenase inhibitor plus aluminum sulfate yields aluminum collagenase inhibitor. Collagenase inhibitor content in aluminum lake ranges from 5-30%.

Example 7 Prsπarafinn of Hard Shell Caosule Ingredferit: ^" mg. /Capsule

Active Compound 0.5-500

Lactose, Spray Dried qs

Magnesium Stearate 1-10

OMPI

Example 8 Preparation of Oral Liquid (Syrup) Ingredient % W/V

Active Compound 0 . 05 -5

Liquid Sugar 75 .0

Methyl Paraben U.S.P 0 . 18' Propyl Paraben U.S.P 0 . 02

Flavoring Agent qs

Purified Water qs ad 100 . 0

Example 9

Preparation of Oral Liαuid (Elixir)

Ingredient % W/V

Active Compound 0.05-5

Alcohol U.S.P 12.5

Glycerin U.S.P 45.0

Syrup U.S.P 20.0

Flavoring Agent σs

Purified Water qs ad 100.0

Example 10

Preparation of Oral Suspension (Syrup)

Ingredient % W/V

Active Compound as Aluminum 0.05-5

Lake, Micronized (acid equivalent)

Polysorbatε 80 U.S.P 0.1

Magnesium Aluminum Silicate, Colloidal 0.3

Flavoring Agent qs

Methyl Paraben U.S.P 0.18 Propyl Paraben U.S.P 0.02

Liquid Sugar 75.0

Purified Water qs ad 100.0

Example 11 Preparation of Injectable Solution

Ingredient % W/V

Active Compound 0.05-5

Benzyl Alcohol N.F 0.9

Water for Injection 100.0

O PI

Example 12 Preparation of Injectable Oil Ingredient % W/V

Active Compound 0.05-5 Benzyl Alcohol 1.5

Sesame Oil qs ad 100.0

Example 13

Preparation of Intra-Articular Product

Ingredient Amount Active Compound 2-20 mg.

NaCl (physiological saline) 0.9%

Benzyl Alcohol 0.9%

Sodium Carboxymethylcellulose 1.5% pH adjusted to 5.0-7.5 Water for Injection qs ad 100%

Example 14

Preparation of Injectable Deoo Suspension

Ingredient % W/V

Active Compound 0.05-5 (acid equivalent)

Polysorbate 80 U ^' _.S_P 0.2

Polyethylene Glycol 400 U.S.P 3.0

Sodium chloride U.S.P 0.8

Benzyl Alcohol N.F 0.9 HC1 to pH 6-8 qs

Water for Injection qs ad 100.0

Example 15

Preparation of Dental Paste

Ingredient % W/V Active Compound 0.05-5

Zinc Oxide 15

Polyethylene Glycol 4000 U.S.P. .. 50 Distilled Water qs 100

Example 16 Preparation of Dental Ointment Ingredient % W/V

Active Compound 0.05-5

Petrolatum, White U.S.P. qs 100

Example.17 Preparation of Dental Cream Ingredient % W/V

5 Active Compound 0.05-5

Mineral Oil 50

Beeswax 15

Sorbitan Monostearate 2

Polyoxyethylene 20 Sorbitan

10 Monostearate

Methyl Paraben U.S.P 0.18

Propyl Paraben U.S.P 0.02

Distilled Water qs 100

Example 18 ι ς Preparation of Topical Cream

Ingredient % W/V

Active Compound 0.05-5-

Sodium Lauyl Sulfate 1

Propylene Glycol 12

2 _Q Stearyl Alcohol 25

Petrolatum, White U.S.P 25

Methyl Paraben U.S.P. 0.18

Propyl Paraben U.S.P 0.02

Purified Water qs 100

25 Example 19

Preparation of Topical Ointment Ingredient % W/W

Active Compound 0.05-5

Cholesterol 3

30 Stearyl Alcohol 3

White Wax 8

Petrolatum, White U.S.P. qs 100

Example 20 Preparation of Spray Lotion (Non-Aerosol) Ingredient % W/W

Active Compund 0.05-5

Isopropyl Myristate 20

Alcohol (Denatured) qs

Example 21 Preparation of Buccal Tablet

Ingredient /Tablet

Active Ingredient 0.00325

6 x Sugar 0.29060

Acacia 0.01453

Soluble Starch 0.01453

F. D. & C. Yellow No. 6 Dye 0.00049

Magnesium Stearate 0.00160

0.32500

The final tablet will weigh about 325 mg. and may be compressed into buccal tablets in flat faced or any other tooling shape convenient for buccal administration.

Example 22

Preparation of Lozenge

Ingredient g. /Lozenge

Active Ingredient 0.0140

Kompact ^® Sugar (Sucrest Co.) 0.7138

6 x Sugar 0.4802

Sorbitol (U.S.P. Crystalline) 0.1038

Flavor 0.08^0

Magnesium Stearate 0.0021

Dye qs

Stearic Acid 0.0021

1.4000

The ingredients are compressed into 5/8" flat base lozenge tooling. Other shapes may also be utilized.

Example 23

Preparation of Gelled Vehicles

Ingredient % W/W

Active Compound 9-11

Sodium Chloride 0.9-1.2

Buffer and Flavor qs

Purified Water qs ad 100

Ingredient % W/W

Active Compound 0.005-9

Sodium Alginate 0.5-2

Buffer and Flavor qs

Purified Water qs ad 100

Ingredient % W/W

Active Compound 0.005-9

Hydroxypropyl Cellulose 0.5-2 Buffer and Flavor qs

Purified Water qs ad 100

Ingredient % W/W

Active Compund 0.005-9

Guar Gum 0.5-2 Buffer and Flavor qs

Purified Water .qs ad 100

Examp -le 24

Preparation of Oral Mouth Rinse Ingredient % W/V Active Compound 0.05-20

Alcohol U.S.P 0.20

Sorbitol 1-30

Buffer and Flavor qs

Polysorbate 80 0.1-3 Cetyl Pyridinium Chloride 0.025-0.20

Purified Water qs ad 100

Example 25 Preparation of Tooth Paste Ingredient % W/W Active Compound 0.05-15

Glycerin 5-15

Sorbitol 5-15

Sodium Carboxymethylcellulose 0.5-2

Magnesium Aluminum Silicate 0.1-1 Carrageenin 0.25-2

Preservative .qs

Sodium Lauryl Sulfate 0.1-3

Calcium Carbonate 25-45

Flavor qs

Purified Water... qs ad.... 100

Example 26 Preparation of Dental Paste

Ingredient % W/W

Active Compound 0.05-20

Carboxymethylcellulose 5-20

Pectin 5-20 Plastibase ^® 20-70

Gelatin 5-20

Example 27

Preparation of Dental Ointment

Ingredient % W/W Active Compound 0.05-20

Polyethylene Glycol 4000 50-80

Polyethylene Glycol 400 10-40

Example 28

Preparation of Dental Powder for Brushing or for Use in Water Spray (e.g. Water Pik ^® )

Ingredient % W/W

Active Compound 0.05-10

Flavor qs

Wetting Agents....qs Dextrin qs ad 100

Example 29

Preparation of Stick for Application to Gums

Ingredient % W/W

Active Compound 0.05-10 Glycerin 5-10

Propylene Glycol ^' 40-80

Sodium Stearate 6-10

Flavor qs

Water 0-10 Example 30

Preparation of Periodontal Packing Paste

Paste Part A

Ingredient % W/W

Active Compound 0.05-20

Caprylic Acid 9.0

Laurie Acid 27.0

Ethylcellulose (100 cps.) 2.0

O PI

Polypale Resin- 39.0

Gum Elemi 4.0

Bro inol** 4.0

Mica (Powdered) 7.5 Chlorothymol 1.0

Zinc Acetate 2.0

Bay Oil (Essential Oil) 1.0

- Ethanol 1.5

Paste Part B Magnesium Oxide 43.0

Zinc Oxide 21.0

Calcium Hydroxide 3.5

Copper Oxide 2.0

Mineral Oil, Heavy 26.0 Rosin Oil 3.0

Chlorothymol 1.4

Cumarin (Flavor) 0.1

-Partially polymerized rosin (i.e. modified rosin) **Brominated olive oil When equal parts of A and B are mixed together at

25°C. a hard mass is formed in about 3 minutes.

Example 31 Preparation of Periodontal Packing Paste Part A (Powder) Ingredient % W/W

Active Compound 0.05-20

Canada Balsam, Neutral 8.5

Rosin NF 8.5

Calcium Hydroxide 34.4 Zinc Oxide U.S.P 46.6

Part B (Liquid Hardener)

Eugenol 85.0

Turpentine Oil, Rectified 15.0

A mixture of three drops of Part B added to 130 mg of Part A produces a hard mass in about 2-3 minutes at 30°C

The compounds of this invention may be administere internally to a warm-blooded animal to inhibit connective

tisue destruction or collagenase, such inhibition being useful in the amelioration or prevention of those reac¬ tions causing connective tissue damage. A range of doses may be employed depending on the mode of administration, the condition being treated and the particular compound being used. For example, for intravenous or subcutaneous use from about 5 to about 50 mg./kg./day, or every six hours for more rapidly excreted salts, may be used. For intra-articular use for large joints such as the knee, from about 2 to about 20 mg. /joint per week may be used, with proportionally smaller doses for smaller joints. The dosage range is to be adjusted to provide optimum thera¬ peutic response in the warm-balooded animal being treated. In general, the amount of compound administerred can vary over a wide range to provide from about 1.5 mg./kg. to ab¬ out 100 mg./kg. of body weight of animal per day. The usual daily dosage for a 70 kg. subject may vary from abou 100 mg. to about 3.5 g. Unit doses can contain from about 0.5 mg. to about 500 mg. While in general the sodium salts of the acids of the invetion are suitable for parenteral use, other salts may also be prepared, such as those of primary amines, e.g., ethyla ine; secondary amines, e.g., diethylamine or diethanolamine; tertiary amines, e.g., pyridine or triethy amine or 2-dimethylaminomethyldibenzofuran; aliphatic di- amines, e.g., decamethylenediamine; and aromatic diamines, can be prepared. Some of these are soluble in water, others are soluble in saline solution, and still others are insoluble and can be used for purposes of preparing suspensions for injection. Furthermore, as well as the sodium salt, those of the alkali metals, such as potassium and lithium; of ammonia; and of the alkaline earth metals, such as calcium or magnesium, may be employed. It will be apparent, therefore, that these salts embrace, in general, derivatives of salt-forming cations.

In therapeutic use the compounds of this inven- tion may be administered in the form of conventiona ^ ^RE -

OMPI

pharmaceutical compositions. Such compositions may be formulated so as to be suitable for oral or parenteral administration. The active ingredient may be combined in admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending o the form of preparation desired for administration, i.e., oral or parenteral. The compounds can be used in composi¬ tions such as tablets. Here, the principal _^ active ingre¬ dient is mixed with conventional tabletting ingredients su as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, gums, or similar materials as non-toxic pharmaceutically acceptable diluents or carriers. The tablets or pills of the novel compositions can be laminated or otherwise compounded to provide a dosage form affording the advantage of prolonged or delayed action or predetermined successive action of th enclosed medication. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the for- er. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of mater¬ ials can be used for such enteric layers or coatings, such materials including a number of polymeric acids or mixtures of polymeric acids with such materials as shellac, shellac and cetyl alcohol, cellulose acetate and the like. A par¬ ticularly advantageous enteric coating comprises a styrene aleic acid copolymer together with known materials contri- buting to the enteric properties of the coating. The tablet or pill may be colored through the use of an appro¬ priate non-toxic dye, so as to provide a pleasing appear¬ ance.

The liquid forms in which the novel compositions of the present invetion may be incorporated for adminis¬ tration include suitable 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. Sterile suspensions or solution can be prepared for parenteral use. Isotonic preparation containing suitable preservatives are also desirable for injection use.

The compounds of the present invention may also be administered topically in the form of ointments cream lotions and the like, suitable for the treatment of con¬ nective tissue dependent dermatological disorders. Moreover, the compounds of the present invention may be administered in the form of dental pastes, ointmen buccal tablets and other compositions suitable for applic tion periodontally for the treatment of periodontitis and related diseases of the oral cavity. The term dosage form as described herein refers to physically discrete units suitable as unitary dosage for warm-blooded animal subjects, each unit containing a predetermined quantity of active component calculated to produce the desired therapeutic effect in association with the required pharmaceutical diluent, carrier or vehicle. The speci ication for the novel dosage forms of this in¬ vention are indicated by characteristics of the active com ponent and the particular therapeutic effect to be achie¬ ved or the limitations inherent in the art of compounding such an active component for therapeutic use in warm-blood ed animals as disclosed in this specification. Examples of suitable oral dosage ^' forms in accord with this inven¬ tion are tablets, capsules, pills, powder packets, gran¬ ules, wafers, cachets, teaspoonfuls, dropperfuls, ampules, vials, segregated multiples of any of the foregoing and other forms as herein described.

The inhibiting activity of representative com¬ pounds of the invention on the destruction of connective tissue has been demonstrated by one or more of the follow- ing identified tests: (i)Collagen Assay, Test Code 006 - This test measures the ability of human skin fibroblast collagenase to degrade radiolabeled native collagen fi-

brils. An active inhibitor inhibits the degradation of the collagen fibril; (ii) Crevicular fluid Assay - In an analogous test, collagenase present in the crevicular flu¬ id of inflamed gingival tissue was used to measure its ability to degrade radiolabeled native collagen fibrils. An active inhibitor would inhibit the degradation of the collagen fibril; (lii) Leukocyte Neutral Proteases Inhibit Assay - This test measures the ability of neutral protea¬ ses derived from human leukocytes to degrade radiolabeled proteoglycans entrapped in polyacrylamide beads. An activ Inhibitor inhibits the degradation of proteoglycans. (i) Collagenase Assay - Test Code 006

Collagenase assays were performed by a modifica¬ tion of the method of Harper, e_t al _, . , Biochem. , 10, 3035 (1971). In a typical assay (total volume of 0.45 ml.),

100 ul. of the activated enzyme was added to the 1^ C-label collagen fibrils (250 ul.) followed by 100 ul. of 50 mM cacocylate, pH 7.4, containing 5 mM calcium chloride. Aft incubation at 37°C. for 16 hours, the tubes were centri- fuged in a Beckman microfuge for five minutes at full spee An aliquot (200 ul.) of the supernatant, representing collagenase digestion products of the fibril, was assayed for radioactivity. The effect of the test compound on col¬ lagen degradation by collagenase was examined as follows: The test compound (in distilled water) was added at a test concentration of 30 ug./ml. to the assay tubes containing active collagenase (total volume 450 ul.) and after 16 hours the amount of radioactivity in the superna¬ tant was determined. Appropriate blanks and trypsin contro were run in parallel.

Table I shows that representative compounds of th invention possess collagenase inhibitory activity. The act vities are expressed as % inhibition (lowering) of collagen ase activity, i.e. based on the 0% value for the enzyme con trol.

Table I Biological Activities

■% Inhibition

Compound of Collagenase

6,6 '-[Ureylenebis(m-phenylenecar- bonylimino) ]bis[4-hydroxy-2-naph- thoic acid] diethyl ester diacetate

6,6 '-[Ureylenebis(m-phenylenecar- bonylimino) ]bis[4-h~ydroxy-2-naph- 92 thoic acid]

6,6 '-[Ureylenebis(m-phenylenecar- bonylimino) ]bis[4-hydroxy-2-naph- 57 thoic acid] diethyl ester

iϋ.

Q PI