Active oxygen scavenger

Abstract: Pyrrolo quinoline esters, oxazopyrrolo quinolines and esters of oxazopyrrolo quinolines per se have no toxicity against animals and besides have strong active oxygen scavenging effect. Therefore, they are expected to be able to be used as preventive and curing medicines against various diseases caused by active oxygen.

Description: The present invention relates to the use of oxazopyrrolo quinolines and/or oxazopyrrolo quinoline esters for the manufacture of a medicament to treat diseases mediated by active oxygen. Human beings and other aerobic organisms inhale oxygen and utilize it for oxidation of biomaterials and synthesis of ATP, in the course of which a part of oxygen is activated to produce so-called "active oxygen". Active oxygens include O₂⁻ (superoxide), H₂O₂ (hydrogen peroxide), ·OH (hydroxy radical), ¹O₂ (singlet oxygen), lipoperoxy radicals (RCOOH, derivatives of H₂O₂), and lipoalkoxy radicals (RO·, derivatives of ·OH). It has been clear that these active oxygens peroxidize lipid in living bodies and the peroxide lipid produced cause various troubles in living bodies. Examples of the troubles include senescences, geriatric diseases and metabolic diseases such as carcinogenesis, arteriosclerosis, diabetes mellitus, ischemic reperfusion, Parkinson's disease, shock, disseminated intravascular coagulation syndrome, oral diseases, lung diseases, digestive diseases, kidney diseases, cataract, and medicinal poisoning caused by bipyridyl herbicides, halogenated hydrocarbons, benzene, chloroform and the like. Furthermore, it has been known that alloxan, streptozotocin, adriamycin and daunomycin which are anticancer agents, methyldopa and hydralazine which are hypotensive agents, primaquine and pamaquine which are antimalarial agents, paracetamol and phenacetin which are analgesics, and hydrocortisone which is both anti-inflammatory and antiallergic agent cause diabetes mellitus, liver metabolic diseases, hemolysis, kidney diseases and cataract as adverse side effects. These are considered to be troubles caused by radical or active oxygen due to administered medicines. On the other hand, it has been considered that living bodies have defensive mechanisms against harmful active oxygen and there are anti-oxidation enzymes such as glutathione peroxidase, catalase and superoxide dismutase and antioxidation substances for non-enzyme micromolecules such as vitamin E, ascorbic acid and glutathione and it is assumed that they complicatedly correlate to one another to perform functions. Recently, it has been clarified that pyrrolo quinoline quinone (PQQ) found as a novel coenzyme for oxidoreductases has a strong active oxygen scavenging action ("Collection of Summaries of Lectures (5) at the 109th meeting of Japan Pharmacological Society", page 42 (1989)). Furthermore, it has been reported that PQQ inhibits liver diseases caused by carbon tetrachloride, endotoxin shock and cataract caused by hydrocortisone. [Watanabe et al, "Current Therapeutic Research", Vol.44, pp 896-901, 1988; Matsumoto et al, "PQQ and Quinoprotein" edited by Jongejan, Duine, pp 162-164, 1989 (Kluwer Academic Publishers); Nishigoori et al, "Life Science", Vol.45, pp 593-598, 1989]. Besides, recently, elucidation of physiological activity of PQQ in living bodies has been intensively conducted. Thus PQQ exhibits a strong active oxygen scavenging action and is expected as curing medicines for various diseases mentioned-above which are considered to be caused by active oxygen. However, it has been clarified that PQQ has kidney toxicity (Watanabe et al, "Hiroshima J. Med. Sci.", Vol.38, No.1, pp 49-51 (1987)). Thus, if medicines which are low in toxicity and kidney toxicity and have active oxygen scavenging action are found, they are expected to be developed as inhibitory and curing medicines for various senescences, geriatric diseases and metabolic diseases caused by active oxygen. The inventors have conducted intensive research on medicines which are low in toxicity and have active oxygen scavenging action. As a result, it has been found that oxazopyrrolo quinolines and/or oxazopyrrolo quinoline esters are low in toxicity and have strong active oxygen scavenging effect and have inhibitory and curing effects against various diseases caused by active oxygen. That is, the present invention relates to active oxygen scavengers containing oxazopyrrolo quinolines and/or oxazopyrrolo quinoline esters as active ingredient. Pyrrolo quinoline quinone esters (hereinafter referred to as "PQQ esters") are esters of PQQ and have the following formulas. [wherein R₁, R₂ and R₃ represent hydrogen, alkyl, alkenyl or benzyl group and may be identical or different] Oxazopyrrolo quinolines (hereinafter referred to as "OPQ") are 2,8,10-tricarboxy-1H-oxazo[5,4-h]-pyrrolo[2,3-f]quinoline and its 5-substitution products and these have the following formula. [wherein R represents hydrogen or a lower alkyl group having 1 to 4 carbon atoms which may be substituted by substituent(s), said substituent(s) being selected from the class consisting of hydroxyl, carboxyl,mercapto, amino, carbamyl, phenyl, hydroxyphenyl, guanidino, imidazolyl and methylmercapto groups, and R₁, R₂ and R₃ represent hydrogen, alkyl, alkenyl or benzyl group and may be identical or different]. The OPQ used in the present invention can be easily produced by allowing PQQ to react with various α-amino acids, methylamine and the like in the presence of oxygen. The OPQ in the present invention includes, for example, OPQ (R=H) obtained from a PQQ and one of glycine, threonine, tryptophan, proline, tyrosine, serine and monomethylamine (Japanese Patent Application No. 1-292459; JP-A-3294281), hydroxymethyl OPQ (R= CH₂OH) obtained from a PQQ and serine (Japanese Patent Application No.1-258791; JP-A-3123782), 1-methylethyl OPQ (R=CH(CH₃)₂) obtained from a PQQ and valine (Japanese Patent Application No. 1-309479; JP-A-3170484), 1-methylpropyl OPQ (R=CH(CH₃)CH₂CH₃) obtained from a PQQ and isoleucine (Japanese Patent Application No.1-309480; JP-A-3170485), 2-methylpropyl OPQ (R=CH₂CH(CH₃)₂) obtained from a PQQ and leucine (Japanese Patent Application No.1-309481; JP-A-3170486), methyl OPQ (R=CH₃) obtained from a PQQ and alanine (Japanese Patent Application No.1-327347; JP-A-3188081), 2-carboxyethyl OPQ (R=CH₂CH₂CO₂H) obtained from a PQQ and glutamic acid (Japanese Patent Application No. 1-327351; JP-A-3190882), 2-carbamoylethyl OPQ (R=CH₂CH₂CONH₂) obtained from a PQQ and glutamine (Japanese Patent Application No.1-327348; JP-A-3188082), 2-methylthioethyl OPQ (R=CH₂CH₂SCH₃) obtained from a PQQ and methionine (Japanese Patent Application No. 1-327349; JP-A-3190880), benzyl OPQ obtained from a PQQ and phenylalanine (Japanese Patent Application No. 1-327350; JP-A-3190881), 4-hydroxyphenylmethyl OPQ obtained from a PQQ and tyrosine (Japanese Patent Application No. 2-107357; JP-A-3188762), 1-carboxymethyl OPQ (R=CH₂CO₂H) obtained from a PQQ and aspartic acid, 1-carbamoylmethyl OPQ (R=CH₂CONH₂) obtained from a PQQ and asparagine, 1-(4-imidazolyl)methyl OPQ obtained from a PQQ and histidine, 4-aminobutyl OPQ (R=(CH₂)₄NH₂) obtained from a PQQ and lysine, 3-guanidinopropyl OPQ obtained from a PQQ and arginine, and 1-mercaptomethyl OPQ (R=CH₂SH) obtained from a PQQ and cystine. Furthermore, salts of respective OPQ include, for example, alkali metal salts, alkaline earth metal salts, ammonium salts and substituted ammonium salts and these salts are also effective as active oxygen scavengers. Typical examples thereof are sodium salts, potassium salts, magnesium salts, calcium salts, ammonium salts, trimethylammonium salts, triethylammonium salts, and triethanolammonium salts. R₁, R₂ and R₃ in the esters of OPQ represented by the formula [I] may be identical or different and represent hydrogen atom or alkyl, alkenyl or benzyl group and they form monoesters, diesters and triesters. These esters of OPQ can be easily produced by allowing an OPQ or salts thereof to react with alcohols by conventional process. The alkyl group includes, for example, methyl group and ethyl group and the alkenyl group includes, for example, allyl group. It is also possible to produce the desired esters of OPQ by allowing PQQ or PQQ salts to react with alcohols by conventional process to obtain esters of PQQ and then allowing the esters to react with various amino acids or methylamine. The active ingredient of the present invention is formulated using optionally surface active agent, vehicle, colorant, preservative, coating aid and the like. Moreover, it may be used in combination with other medicines. Dosage of the active ingredient may vary depending on kind of desease, condition of desease, kind of the active ingredient and manner of administration, normally, 1 - 100 mg, preferably 5 - 50 mg/kg (body weight/day). The agents of the above dosage are administered once or with divided administration in twice or three times a day. [Acute toxicity test and kidney toxicity test of OPQ] Acute toxicity test (1): To male mice SPF-ICR five weeks old (supplied by Japan Charles River Co.) were intraperitoneally administered PQQ·2Na, OPQ, 1-methylpropyl OPQ, 2-methylthioethyl OPQ and benzyl OPQ at dosages of 40, 80, 160 and 200 mg per kg of body weight of mouce, respectively and they were bred for 14 days at 25°C. One group consisted of 8 mice. For administration, PQQ . 2Na and OPQ and esters thereof were dissolved in physiological saline. No mice died with administration of 40 mg/kg-mouse of PQQ·2Na, but five mice died with administration of 80 mg and eight mice all died with administration of 160 mg and 200 mg. LD₅₀ of PQQ·2Na was about 70 mg/kg-mouse. On the other hand, no mice died with administration OPQ and esters thereof. Thus, it can be seen that OPQ and esters thereof are conspicuously low in toxicity as compared with PQQ. Acute toxicity test (2): To male mice SPF-ICR five weeks old (supplied by Japan Charles River Co.) was intraperitoneally administered OPQ at dosages of 0.1, 0.2, 0.4, 0.8, and 1.2 g per kg of body weight of mouse, respectively and they were bred for 14 days at 25°C. One group consisted of 8 mice. No mice died with administration of 0.1 - 0.4 g and two mice died with administration of 0.8 g, three mice died with administration of 1.0 g and six mice died with administration of 1.2 g. LD₅₀ was about 1.0 g/kg-mouse. Acute toxicity test (3): To male mice SPF-ICR five weeks old (supplied by Japan Charles River Co.) was orally administered OPQ at dosages of 1.0, 1.5 and 2.0 g per kg of body weight of mouse, respectively and they were bred for 14 days at 25°C. One group consisted of 8 mice. No mice died. Kidney toxicity determined by urine test PQQ·2Na, OPQ and esters thereof were intraperitoneally administered to mice and they were bred in the same manner as in the above acute toxicity test. Urine of the mice was taken every day and glucose concentration was measured using clinical examination reagent (trademark: URISTICKS 2 manufactured by Miles·Sankyo Co.). As shown in Table 2, glucose was detected in urine of mice to which PQQ·2Na was administered while no glucose was detected in urine of all mice to which OPQ or esters thereof were administered. That is, PQQ showed kidney toxicity, but OPQ and esters thereof did not show kidney toxicity. Kidney toxicity determined by blood examination (1): PQQ·2Na, OPQ and esters thereof were administered to mice and they were bred in the same manner as in the acute toxicity test. After lapse of one day from the administration, the mice were fasted (with feeding only water) and then after 18 hours, blood was collected and serum was obtained therefrom. Glucose, urea nitrogen and creatinine in the serum were examined by clinical examination reagent (trademark: FUJI DRYCHEMSLIDE manufactured by Fuji Photo Film Co., Ltd.). The results in average value of eight mice are shown in Table 3. Administration of compound (mg/kg) Glucose (mg/dl) Urea nitrogen (mg/dl) Creatinine (mg/dl) No administration 79 ± 10 23 ± 3 0.82 ± 0.08 PQQ·2Na 20 70 ± 13 25 ± 3 0.8 ± 0.1 40 60 ± 12 110 ± 38 2.3 ± 1.0 80 57 ± 15 130 ± 18 3.4 ± 0.4 160 35 ± 7 139 ± 3 4.1 ± 0.2 OPQ 40 90 ± 18 25 ± 3 0.70 ± 0.10 80 86 ± 10 23 ± 4 0.80 ± 0.10 160 90 ± 23 27 ± 6 0.73 ± 0.09 200 85 ± 24 28 ± 4 0.81 ± 0.08 1-methylpropyl OPQ 40 87 ± 20 26 ± 1 0.80 ± 0.08 80 104 ± 19 22 ± 3 0.78 ± 0.04 160 87 ± 22 27 ± 4 0.78 ± 0.04 200 66 ± 11 27 ± 6 0.83 ± 0.17 2-methylthioethyl OPQ 40 96 ± 26 26 ± 4 0.76 ± 0.05 80 88 ± 29 26 ± 5 0.68 ± 0.08 160 97 ± 22 26 ± 3 0.64 ± 0.05 200 72 ± 16 26 ± 2 0.73 ± 0.06 Benzyl OPQ 40 90 ± 24 23 ± 3 0.68 ± 0.05 80 87 ± 17 23 ± 2 0.68 ± 0.04 160 87 ± 21 26 ± 3 0.68 ± 0.08 200 84 ± 18 25 ± 3 0.73 ± 0.05 Sharp reduction of glucose and much increase of urea nitrogen and creatinine were seen and kidney toxicity was recognized when PQQ·2Na was administered. On the other hand, when OPQ and esters thereof were administered, contents of glucose, urea nitrogen and creatinine were nearly the same as those in the case of administration of no compounds and kidney toxicity was not seen. Kidney toxicity determined by blood examination (2): OPQ was intraperitoneally administered to mice at dosages of 150, 300, 400 and 600 mg/kg-mouse and they were bred for one day in the same manner as in the acute toxicity test. Thereafter, the mice were fasted (with feeding only water) and then after 18 hours, blood was collected and serum was obtained therefrom. Glucose, urea nitrogen and creatinine in the serum were examined by clinical examination reagent (trademark: FUJI DRYCHEMSLIDE manufactured by Fuji Photo Film Co., Ltd.). The results in average value of eight mice are shown in Table 4. Administration of compound (mg/kg·mouse) Glucose (mg/dl) Urea nitrogen (mg/dl) Creatinine (mg/dl) No administration 105 ± 29 28 ± 4 0.78 ± 0.04 OPQ 150 106 ± 21 32 ± 3 0.75 ± 0.05 300 80 ± 15 29 ± 2 0.78 ± 0.04 400 104 ± 18 29 ± 3 0.78 ± 0.04 600 74 ± 11 32 ± 5 0.78 ± 0.11 For any dosages, contents of glucose, urea nitrogen and creatinine were nearly the same as those when no compounds were administered. From the results of the urea and blood examinations, PQQ showed kidney toxicity, but OPQ and esters thereof showed no kidney toxicity. The following nonlimiting examples show active oxygen scavenging action and inhibitory and curing activities of the OPQ and esters thereof of the present invention against various diseases caused by active oxygen.420 Example 1 Superoxide scavenging activity of OPQ and esters thereof: Activity of OPQ and esters thereof to scavenge superoxide (O₂⁻) produced in the system of hypoxanthine-xanthine oxidase was examined by spin trapping method [using 5,5-dimethyl-1-pyrroline-N-oxide (hereinafter referred to as "DMPO") as a spin trapping agent] where electron spin resonance (hereinafter referred to as "ESR") was used. Hypoxanthine + 20₂ → 20₂⁻ + uric acid (xanthine oxidase) The following reagents were successively put in a test tube and stirred after addition of the hypoxanthine 7 ○ to initiate the reaction and the reaction mixture was transferred to a quartz cell used for measurement of ESR. After 1 minute from initiation of the reaction, ESR was measured and comparison was made on concentration of the test compound required to reduce the produced superoxide to 1/2 (IC₅₀ value). As test compounds, OPQ and esters thereof and ascorbic acid (control) were used and scavenging activity thereof for superoxide was determined at five kinds of concentrations and IC₅₀ was calculated. Reaction liquid 1 ○ 50 mM sodium phosphate buffer solution (pH 7.3) 50 µl 2 ○ 10 mM diethylenetriaminepentaacetic acid (hereinafter referred to as "DTPA") 10 µl 3 ○ Xanthine oxidase (2 U/ml) 20 µl 4 ○ Distilled water 50 µl 5 ○ Test compound (2 % DMSO solution) 50 µl 6 ○ 900 mM DMPO 10 µl 7 ○ 7.3 mM hypoxanthine 10 µl Conditions for measurement of ESR Magnetic field 236.1 ± 5 mM Output 10 mW Modulation 100 kHz, 0.079 mT Response time 0.3 second Sweeping time 2 minutes (interval of 10 mT) Amplification ratio 500 The results are shown in Table 5. IC₅₀ of OPQ and esters thereof was 1/5 - 1/20 of IC₅₀ of ascorbic acid. OPQ and esters thereof had strong superoxide scavenging activity of 5 - 20 times that of ascorbic acid. Compound O₂⁻ scavenging activity IC₅₀ (x 10⁻⁶M) Ascorbic acid 24.5 OPQ 1.22 Methyl OPQ 1.64 2-Carboxyethyl OPQ 2.96 Benzyl OPQ 3.01 1-Methylethyl OPQ 3.14 2-Carbamoylethyl OPQ 3.20 Hydroxymethyl OPQ 3.52 1-Methylpropyl OPQ 3.53 2-Methylthioethyl OPQ 3.94 4-Hydroxyphenylmethyl OPQ 4.12 2-Methylpropyl OPQ 4.88 OPQ triallyl ester 2.39 Example 2 Hydroxy radical scavenging activity of OPQ and esters thereof: Activity of OPQ and esters thereof to scavenge hydroxy radical ( . OH) produced in the system of H₂O₂-(Fe²⁺-DTPA) was examined by spin trapping method [ using 5,5-dimethyl-1-pyrroline-N-oxide (hereinafter referred to as "DMPO") as a spin trapping agent] where electron spin resonance (hereinafter referred to as "ESR") was used. DTPA means diethylenetriaminepentaacetic acid. H₂O₂ + Fe²-DTPA complex → ·OH + OH⁻+ Fe³⁺-DTPA complex The following reagents were successively put in a test tube and stirred after addition of H₂O₂ 6 ○ to initiate the reaction and the reaction mixture was transferred to a quartz cell used for measurement of ESR. After 1 minute from initiation of the reaction, ESR was measured and comparison was made on concentration of the test compound required to reduce the concentration of the produced hydroxy radical to 1/2 (IC₅₀ value). As test compounds, OPQ and esters thereof and ascorbic acid (control) were used and scavenging activity thereof for hydroxy radical was determined at five kinds of concentrations and IC₅₀ was calculated. Reaction liquid 1 ○ 50 mM sodium phosphate buffer solution (pH 7.3) 50 µl 2 ○ 1 mM Fe²⁺-DTPA complex 10 µl 3 ○ Distilled water 70 µl 4 ○ Test compound ( 2% DMSO solution) 50 µl 5 900 mM DMPO 10 µl 6 10 mM H₂O₂ 10 µl Conditions for measurement of ESR Magnetic field 336 ± 5 mT Output 10 mW Modulation 100 kHz, 0.1 mT Response time 0.1 second Sweeping time 1 minute (interval of 10 mT) Amplification ratio 125 The results are shown in Table 6. Compound · OH scavenging activity IC₅₀ (x 10⁻⁴ M) Ascorbic acid 1.01 2-Carboxyethyl OPQ 1.10 Methyl OPQ 1.53 2-Carbamoylethyl OPQ 1.94 OPQ 1.98 1-Methylethyl OPQ 2.00 2-Methylthioethyl OPQ 2.00 Benzyl OPQ 2.11 1-Methylpropyl OPQ 2.52 Hydroxymethyl OPQ 3.05 4-Hydroxyphenylmethyl OPQ 3.14 2-Methylpropyl OPQ 4.21 IC₅₀ of OPQ and esters thereof was nearly the same as that of ascorbic acid. OPQ and esters thereof had strong hydroxy radical scavenging activity like ascorbic acid. Example 3 Pharmacological effect of OPQ and esters thereof on hydrocortisone-induced cataract of hen's eggs Fertilized eggs of white leghorn were incubated in an incubator at a temperature of 37°C and 70 % in humidity. The day on which the incubation was started was taken to be one day old. Twenty-five eggs of 15 day old were prepared and divided into 5 groups, each of which included 5 eggs (A-E). A small hole was bored through the eggshell above air chamber of the eggs of groups B-E and 0.25 µmole (as 0.2 ml solution) of hydrocortisone sodium succinate was administered into the air chamber through the hole and then, the hole was sealed with a cellophane tape. To the eggs of groups C-E was administered 1 µmole of the following compounds after 3, 10 and 20 hours from administration of hydrocortisone sodium succinate. That is, 0.2 ml of a solution of PQQ·2Na in distilled water was administered to the eggs of group C, 0.2 ml of a solution of OPQ in 10 mM NaCO₃ solution was administered to the eggs of group D, and 0.2 ml of a solution of methyl OPQ in 10 mM NaCO₃ solution was administered to the eggs of group E. Crystal lens was taken out after 48 hours from administration of hydrocortisone sodium succinate and degree of cataract was judged by the criteria according to the Nishigori et al method (Nishigori et al, "Investigative Ophthalmology & Visual Science", Vol.25, page 1051, 1984). Criteria for judgement 1 ○ No haze was seen in crystal lens. 2 ○ Slight opaque ring was seen in crystal lens. 3 ○ Clear ring of white haze was seen in crystal lens. 4 ○ White haze of pinhole size was seen in nucleus. 5 ○ The whole nucleus was hazed in white. The results are shown in Table 7. Group Judgement 1 ○ 2 ○ 3 ○ 4 ○ ∼ 5 ○ A (Blank) 5/5 B (Control) 5/5 C (PQQ·2Na was administered) 1/5 2/5 2/5 D (OPQ was administered) 1/5 1/5 3/5 E Methyl OPQ was administered) 1/5 2/5 2/5 OPQ and esters thereof showed clear control action on cataract like PQQ. It is considered that OPQ and esters thereof scavenged active oxygen caused by hydrocortisone.

Claims: The use of at least one compound selected from the group consisting of the compounds represented by the following formula (I): (wherein R represents hydrogen or a lower alkyl group having 1 to 4 carbon atoms which may be substituted by substituent(s), said substituent(s) being selected from the class consisting of hydroxyl, carboxyl, mercapto, amino, carbamyl, phenyl, hydroxyphenyl, guanidino, imidazolyl and methylmercapto groups, and R₁, R₂ and R₃ represent hydrogen, alkyl, alkenyl or benzyl group and may be identical or different). for the manufacture of a medicament to treat diseases mediated by active oxygen. The use of said compounds according to claim 1 for the manufacture of a medicament to treat cataract mediated by active oxygen.

Inventors: AKAIKE TAKAAKI, MAEDA HIROSHI, NISHIGOORI HIDEO, URAKAMI TEIZI, YOSHIDA CHIEKO, AKAIKE, TAKAAKI, MAEDA, HIROSHI, NISHIGOORI, HIDEO, URAKAMI, TEIZI, YOSHIDA, CHIEKO, Urakami, Teizi, c/o MITSUBISHI GAS CHEMICAL CO.INC, Yoshida, Chieko, c/o Niigata Research Laboratory
Applicants: MITSUBISHI GAS CHEMICAL CO, MITSUBISHI GAS CHEMICAL COMPANY, INC.
Application year: 1992
Main classification: A61K31
IPC codes: A61K31