Abstract:
An epoxysuccinyl amino acid derivative of the formula ##STR1## wherein R 1  is hydrogen or alkali metal, R 2  is isobutyl, R 3  is hydrogen or benzyloxycarbonyl, and n is an integer of 5 to 7. The subject compounds exhibit inhibitory activity towards thiol protease, especially, calcium-activated neutral thiol protease which exists in excess in muscle tissues of mammals afflicted with muscular dystrophy.

Description:
BACKGROUND AND BRIEF SUMMARY OF THE INVENTION 
     This is a continuation-in-part of Ser. No. 149,312, filed May 13, 1980. 
    
    
     The present invention relates to a novel epoxysuccinyl amino acid derivative of the formula ##STR2## wherein R 1  is hydrogen or alkali metal, R 2  is isobutyl, R 3  is hydrogen or benzyloxycarbonyl and n is an integer of 5 to 7. 
     The prior art discloses E-64, ie., N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine (U.S. Pat. No. 3,911,111), its intermediates [Chemical Abstracts, 87, 202108y (1977), ibid., 87, 85238c (1977), ibid., 87, 202125b (1977), ibid., 87, 68128z (1977)], and epoxysuccinic acid derivatives of German Patent Laid Open Application No. P 28 09 036 and Chemical Abstracts, 87, 68129a (1977) prepared by several of the present inventors. 
     The compounds of the present invention are distinguished from the prior art compounds by inhibitory activity to thiol protease, especially, calcium-activated neutral thiol protease (hereinafter referred to as CANP) which exists in excess in muscle tissues of mammals afflicted with muscular dystrophy, and by good absorption and distribution in tissues after administration to such mammals without acceleration of vascular permeability. 
     DETAILED DESCRIPTION OF THE INVENTION 
     In the present specification, unless otherwise noted, the epoxysuccinic acid derivatives are the trans isomers, namely two carbonyl groups on the oxirane ring are in the trans configuration. 
     The compounds of the present invention are the compounds of formula(I). 
     A compound of the present invention may be prepared, for example, in accordance with the following process: An epoxysuccinic acid monoester of the formula ##STR3## wherein R 4  is hydrogen or alkali metal, and R 5  is a carboxylic acid protecting group, may be treated with a chlorinating agent such as oxalyl chloride, thionyl chloride or the like to give the corresponding acid chloride. To the acid chloride, an amino acid compound of the formula ##STR4## wherein R 2 , R 3  and n are as defined above, may be added dropwise under ice cooling to give the intermediate, ie., the compound of formula(I) wherein R 1  is R 5 . In this amidation, the compound of formula(III) may be added with a base such as triethylamine, pyridine, methylmorphorine or the like. When the compound of formula(III) is employed directly in the form of an acid-addition salt, it may be provided for the reaction after removing the acid with a base such as an alkali hydroxide, triethylamine, pyridine or methylmorpholine; alternatively it may be allowed to react in the presence of the base mentioned above. Examples of the carboxylic acid protecting groups in R 5  are benzyl, cycloalkyl having 5 to 6 carbon atoms, alkyl having 1 to 10 carbon atoms, and like ester forming moieties. 
     The compound of formula(II) wherein R 4  is hydrogen also can be directly converted to the compound of formula(I) wherein R 1  is R 5  without chlorinating. In this case, the compound of formula(II) wherein R 4  is hydrogen may be reacted with the compound of formula(III) in the presence of a condensing agent such as N,N&#39;-dicyclohexylcarbodiimide, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide or the like. Also preferred in this reaction is the addition of an N-hydroxy compound such as N-hydroxysuccinimide, 1-hydroxy-benzotirazole or the like. 
     Alternatively, the compound of formula(I) wherein R 1  is R 5  may be prepared by the amidation of the compound of the formula ##STR5## wherein R 2  and R 5  are as defined above, with an amine of the formula 
     
         H.sub.2 N(CH.sub.2).sub.n NHR.sup.3                        (V) 
    
     wherein R 3  and n are as defined above. This amidation can be carried out by following the same procedure as that of the reaction of the compound of formula(II) wherein R 4  is hydrogen with the compound of formula(III). 
     The compound of formula(I) wherein R 1  is alkali metal may be prepared by a process which comprises treating the compound of formula(I) wherein R 1  is R 5  with an alkali hydroxide such as sodium hydroxide, potassium hydroxide or the like, and then, if necessary, following by addition of an organic solvent such as ethanol, acetone, ethyl ether, petroleum ether or the like. 
     The compound of formula(I) wherein R 1  is hydrogen may be prepared by a process which comprises acidifying the compound of formula(I) wherein R 1  is alkali metal with an inorganic acid such as hydrochloric acid or sulfuric acid, or an organic acid such as formic acid or acetic acid, and then, extracting with a suitable organic solvent such as ethyl acetate, ethyl ether, benzene or chloroform. 
     In the case where the compound of formula(I) has amino protected with benzyloxycarbonyl, such a protecting group may be removed by catalytic reduction using palladium-on-charcoal or palladium black. 
     The compounds of formulae(II) and (IV) can be prepared by the method as described in German Patent Laid Open Application No. P 28 09 036 or the like. 
     The compound of formula(III) can be prepared as follows: The compound for the formula ##STR6## wherein R 2  is as defined above and R 6  is a protecting group, may be reacted with the compound of formula(V), followed by removal of the protecting group to give the desired compound. Examples of the protecting groups are conventional groups in the field of peptide syntheses such as t-butoxycarbonyl, carbobenzoxy or methylbenzyloxycarbonyl. The amidation of the compound of formula(VI) with the compound of formula(V) can be carried out by following the same procedure as that of the reaction of the compound of formula(II) wherein R 4  is hydrogen with the compound of formula(III). Removal of the protecting group can be carried out according to the conventional manner in the field of peptide chemistry. 
    
    
     The following examples show representative compounds encompassed within the scope of the present invention and the manner in which such compounds are prepared. However, it is to be understood that the examples are for purposes of illustration only. 
     EXAMPLE 1 
     (a) In 80 ml of the tetrahydrofuran were dissolved 1.00 g of DL-trans-benzylhydrogenepoxysuccinate, 1.70 g of N-L-leucyl-N&#39;-benzyloxycarbonyl-1,7-diaminoheptane, 0.73 g of 1-hydroxybenzotriazole and 0.55 g of N-methylmorpholine. To the solution was slowly added 0.95 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride under ice-cooling with stirring. The mixture was stirred for 2 hours under ice-cooling and then for 2 hours at room temperature. The solution was concentrated under reduced pressure and admixed with 100 ml of ethyl acetate and 100 ml of water and the mixture was shaken vigorously. The ethyl acetate layer was separated and washed successively with a 10% aqueous hydrochloric acid solution, an aqueous saturated bicarbonate solution and an aqueous saturated sodium chloride solution and concentrated to dryness. The resulting residue was purified by column chromatography on silica gel (chloroform:acetone=10:1) and recrystallized from ethyl acetate-n-hexane to give 1.50 g of N-[N-DL-3-trans-benzyloxycarboxyloxirane-2-carbonyl)-L-leucyl]-N&#39;-benzyloxycarbonyl-1,7-diaminoheptane m.p. 134° C. Yield: 67%. 
     In 20 ml of ethanol was dissolved 2.32 g of N-[N-(DL-3-trans-benzyloxycarbonyloxirane-2-carbonyl)-L-leucyl]-N&#39;-benzyloxycarbonyl-1,7-diaminoheptane prepared by the method of Example 1(a). To the solution was added dropwise 10 ml of an ethanol solution of 0.24 g of potassium hydroxide with stirring. After stirring for 2 hours at room temperature, petroleum ether was added to the solution and the produced white precipitates of N-[N-(DL-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]-N&#39;-benzyloxycarbonyl-1,7-diaminoheptane potassium salt were collected on a filter. The precipitates were dissolved in 50 ml of water, made acidic with hydrochloric acid and extracted with ethyl acetate (50 ml×3). The ethyl acetate layers were combined, washed with saturated aqueous sodium chloride, dried on magnesium sulfate and concentrated to dryness. The resulting powders were recrystallized from chloroform-petroleum ether to obtain 0.968 g of N-[N-(DL-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]-N&#39;-benzyloxycarbonyl-1,7-diaminoheptane (compound 3). m.p. 60° C. Yield: 68%. 
     EXAMPLE 2 
     (a) Following the procedure of Example 1(a) and using 1.30 g of DL-trans-benzylhydrogenepoxysuccinate and 1.75 g of N-L-leucyl-N&#39;-benzyloxycarbonyl-1,5-diaminopentane, there was obtained 1.80 g of N-[N-(DL-3-trans-benzyloxycarbonyloxirane-2-carbonyl)-L-leucyl]-N&#39;-benzyloxycarbonyl-1,5-diaminopentane, m.p. 145°-146° C. Yield: 66%. 
     (b) In 40 ml of the mixture of methanol-acetic acid-water(8:2:1) was dissolved 1 g of N-[N-(DL-3-trans-benzyloxycarbonyloxirane-2-carbonyl)-L-leucyl]-N&#39;-benzyloxycarbonyl-1,5-diaminopentane and followed by addition of 0.3 g of 5% palladium carbon. The mixture was stirred vigorously under a weak stream of hydrogen at room temperature for 6 hours. The catalyst was filtered off and the filtrate was evaporated to dryness. The residue was purified by active charcoal column chromatography to give 0.42 g of N-[N-(DL-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]-1,5-diaminopentane (compound 2). m.p. 149°-152° C. (with decomposition). Yield: 72%. 
     EXAMPLE 3 
     In 12 ml of ethanol and 2.7 ml of dimethylformamide was dissolved 1.7 g of N-[N-(L-3-trans-ethyloxycarbonyloxirane-2-carbonyl)-L-leucyl]-N&#39;-benzyloxycarbonyl-1,7-diaminoheptane prepared by the method similar to that of Example 1(a). To the solution was added dropwise 5.5 ml of 10% aqueous ethanol solution of 0.2 g of sodium hydroxide with stirring. After stirring for 4 hours at room temperature, the reaction mixture was poured into 40 ml of water and then extracted with 50 ml of ethyl acetate. The aqueous layer was acidified with hydrochloric acid and extracted with ethyl acetate (50 ml×2). The organic layers were combined, washed with saturated aqueous sodium chloride, dried on magnesium sulfate and concentrated to dryness. The resulting powder was suspended in 36 ml of the mixture of methanol-acetic acid-water (4:2:1) and followed by addition of 0.36 g of 5% palladium carbon. The mixture was stirred vigorously under a weak stream of hydrogen at room temperature for 5 hours. The catalyst was filtered off and the filtrate was concentrated in vacuo. The residue was purified by active charcoal column chromatography and crystallized from water-acetone to gove 0.618 g of N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]-1,7-diaminoheptane (compound 1), m.p. 270°-280° C. (with decomposition). Yield: 53%. 
     The compounds of the present invention have superior inhibitory activity to CANP which exists in excess in muscle of mammals having muscular dystrophy, and superior absorption and distribution in tissues after administration to mammals as compared with the epoxysuccinic acid derivatives of German Patent Laid Open Application No. P 28 09 036 and Chemical Abstracts, 87, 68129a (1977). Their muscular dystrophy inhibitory activity was assayed by the method of Ishiura et al., [J. Biochem., 84, 225 (1978)] using CANP prepared from a muscle of hereditary muscular dystrophy chicken, and the resulting value for 50% inhibition in the molar ratios for the enzyme expressed as ID 50  (mole/mole) is shown in Table 1 below. 
     The compounds of the present invention are better absorbed by the subcutaneous route in mammals such as rats or rabbits, as compared with the epoxysuccinic acid derivatives of the German Patent Laid Open Application described above and Chemical Abstracts, 87, 68129a (1977). To determine absorption, these compounds were administered subcutaneously to rats at a dose of 50 mg/kg, and the concentration of the test compound in rat plasma at an hour after the administration is shown in following Table 1. 
     
                       TABLE 1______________________________________    CANP Inhibitory Activity                     Concentration inCompound ID.sub.50 (mole/mole)                     rat plasma (μg/ml)______________________________________1        33               152        54               203        20               11A        5,400            below 1.0B        410              below 1.0C        580              below 1.0D        574              below 1.0E        4,600            below 1.0F        480              below 1.0______________________________________ (Note) The compounds 1-3 in Table 1 are as defined in Examples as described above. The compounds A-F in Table 1 refer to the following known compounds: A: Ethyl hydrogen epoxysuccinate B: Benzyl hydrogen epoxysuccinate C: N--(3ethoxy-carbonyloxirane-2-carbonyl)-L-leucine benzyl ester D: N--(3carboxyoxirane-2-carbonyl)-L-phenylalanine benzyl ester E: N--(3carboxyoxirane-2-carbonyl)-L-leucine F: N--[N--(DL3-trans-ethoxycarbonyloxirane-2-carbonyl)-L-leucyl] N&#39;--benzyloxycarbonyl1,4-diaminobutane 
    
     The compounds of the present invention also inhibit effectively and specifically thiol proteases such as papain, bromelains and some kinds of cathepsin in which some sulfhydryl groups are essential for activity. On the other hand, they have neither inhibitory activity against proteolysis of casein by trypsin, chymotrypsin, pepsin, an acid protease of Paecilomyces varioti or Nagase (trademark of Nagase Industry), against esterolysis of benzoylarginine ethyl ester by kallikrein nor against fibrinolysis by human plasmin. 
     Papain inhibitory activity of the compounds of the present invention was assayed by the method of K. Hanada et al [Argic. Biol. Chem., 42, (3)523(1978)] using papain (80 μg/ml, Sigma Chem. Co.) after twice crystallization. The amounts of inhibitor for 50% inhibition was expressed as ID 50  and shown in Table 2. 
     
                       TABLE 2______________________________________  Compound          ID.sub.50 (μg)______________________________________  1       0.103  2       0.105  3       0.203______________________________________ (Note) The compounds 1-3 in Table 2 are as defined with reference to Table 1. 
    
     The compounds of the present invention show no side-effects such as acceleration of vascular permeability. 
     No acceleration of vascular permeability of the compounds of the present invention is established by the following procedure: 12.5 μg of each test compound in 0.05 ml of saline and control vehicle(0.05 ml of saline) were injected intradermally into the depilated dorsal skin of male Hartley strain guinia-pig in groups of six each, immediately before the intravenous injection of 0.4 ml of 2% evans blue solution. The response was evaluated by the blueing spot at 15 minutes after the dye injection. As the results, the blueing spots with E-64 and control vehicle were 43.0 mm 2  and 19.0 mm 2  in area, respectively. However, since there was no difference between control vehicle and each compound of the invention in terms of size of the blueing spots, the procedure given above was further repeated using 100 μg of each compound of the present invention, and relative effect(%) to E-64 of the compounds was measured by the following expression: ##EQU1## wherein A is the blueing spot(mm 2 ) when administered with the test compound. The results are shown in the following Table 3. On the other hand, relative effect(%) to E-64 of histamine was established as 10000 by repeating the procedure given above using 1 μg of histamine. 
     
                       TABLE 3______________________________________Acceleration of vascular permeabilityCompound    Relative effect (%) to E-64______________________________________1           2.82           2.33           2.4E-64        100.0______________________________________ (Note) The compounds 1-3 in Table 3 are as defined with reference to Table 1. 
    
     The pharmaceutical forms contemplated by the present invention include pharmaceutical compositions suited for oral, parenteral, and rectal use, eg., tablets, powder packets, cachets, dragees, capsules, solutions, suspensions, sterile injectable forms, suppositories, bougies, and the like. The carrier employed may be, for example, either a solid or liquid. Examples of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Examples of liquid carriers are syrup, peanut oil, olive oil, water and the like. Similarly, the carrier or diluent can include any time delay material well known to the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax. 
     The compounds of the present invention can be used to inhibit CANP which exists in excess in muscle of muscular dystrophy mammals, by the administration of about 5 to 400 mg/kg/day in single or two to four divided doses in oral or injectable preparations as described above. 
     The compounds of the present invention are of extremely low toxicity. That is, they show hardly any oral acute toxicity to mice at the dosages less than 2 g/kg of body weight. Moreover, no side-effects were observed after administration of 1 g/kg/day orally for 30 days to laboratory mice.