wherein R, represents phenylamino group substituted 1 to 3 with a substituent selected from the group consisting of a halogen atom and an aceto group, or Cl-C20 alkylmercapto group; and R2 represents thiocyano group; or C1-C20 alkylmercapto group.

Description of the Prior Art The recent increase of fungal infections has generated a renewed interest in antifungal drugs, including development of new antifungal agents. We focused on developing 6,7-disubstituted-5,8-quinolinediones with noble mode of antifungal activities. As antimetabolites of coenzyme Q, the 6- (substituted)-7-chloro-5, 8-quinolinediones inhibit mitochondrial Co-Q dependent succinoxidase and electron transport in Saccarornyces cerevisine, that may be correlated with antifungal activity (Arch. Biochem. Biophys., 191, 306-315,1978). The 5,8-quinolindiones, as antimetabolites of coenzvme Q,

inhibit cytochrome bc complex due to blockade of mitochondrial electron transport in fungi (1. Med. Clam., 16,206-209,1973). 6/7-Substituted-5,8- quinolinedione derivatives have also been reported fungicide in German Patent Publication No. 4,208,874 (1993), but no antifungal activity against human pathogenic fungi was not tested or mentioned.

SUMMARY OF THE INVENTION The present invention has been completed by synthesizing 6,7- disubstituted-5,8-quinolinedion derivatives and confirming the antifungal activity thereof.

Therefore, an object of the present invention is to provide 6,7- disubstituted-5,8-quinolinedion derivatives expressed in the formula 1, including its preparing method and use as an antifungal agent.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention relates to 6,7-disubstituted-5,8-quinolinedion derivatives expressed in the formula 1, wherein Rl represents phenylamino group substituted 1 to 3 with a substituent selected from the group consisting of a halogen atom and aceto group or Cl- Czo alkylmercapto group; and R2 represents thiocyano group or Cl-C20 alkylmercapto group.

The present invention is described in more detail as set forth hereunder.

Among 6,7-disubstituted-5,8-quinolinedion derivatives expressed in the formula 1, preferable compounds are the following formulas la, lb, 1 c and Id,

wherein Ra, R, and Rc are same or different and a hydrogen atom, a halogen atom or aceto group; and X is a halogen atom;

wherein Ra Ri, and Rc are the same as defined for the compound la; wherein Ra, Rb and Rc are the same as defined for the compound la; and

wherein Rd is Cl-C20 alkyl group; and X is a halogen atom.

The examples of 6, 7-disubstituted-5, 8-quinolindion derivatives of formula 1 are as follows; 6-[(N-4-chlorophenyl) amino]-7-brom o-5,[(N-4-chlorophenyl) amino]-7-brom o-5, 8-quinolindion (compound 1),

6-[(N-3, 5-dichlorophenyl)[(N-3, 5-dichlorophenyl) amino]-7-chloro-5,8-quinolindion (compound 2), 6-[(N-4-bromophenyl) amino]-7-bromo-5,8-quinolindion (compound 3), 6- [(N-4-acetophenyl) amino]-7-chloro-5,8-quinolindion (compound 4), 6- [ (N-2,3,4-trifulorophenyl) amino]-7-methylmercapto-5, 8-quinolindion (compound 5), 6- [ (N-4-chlorophenyl) amino]-7-methvlmercapto-5, 8-quinolindion (compound 6), 6-[(N-4-iodophenyl)amino]-7-methylmercapto-5, 8-quinolindion (compound 7), 6- [ (N-4-iodophenyl)amino]-7-thiocyano-5, 8-quinolindion (compound 8), 6- [(N-4-bromophenyl) amino]-7-thiocyano-5,8-quinolindion (compound 9), 6-[(N-4-chlorophenyl) amino]-7-thiocyano-5,[(N-4-chlorophenyl) amino]-7-thiocyano-5, 8-quinolindion (compound 10), 6-[(N-4-acetophenyl)amino]-7-thiocyano-5, 8-quinolindion (compound 11), 6- [(N-3, 5-difulorophenyl) amino]-7-thiocyano-5,8-quinolindion (compound 12), 6-1l-ethylmercapto-7-chloro-5,8-quinolindion (compound 13), 6-1l-propylmercapto-7-chloro-5, 8-quinolindion (compound 14), 6- «-butylmercapto-7-chloro-5,8-quinolindion (compound 15), 16),6-n-pentylmercapto-7-chloro-5,8-quinolindion(compound 6-n-heoxylmercapto-7-chloro-5, 8-quinolindion (compound 17), 18),6-n-heptylmercapto-7-chloro-5,8-quinolindion(compound 6-n-octylmercapto-7-chloro-5, 8-quinolindion (compound 19), 6-n-nonylmercapto-7-chloro-5, 8-quinolindion (compound 20).

A method of preparing 6,7-disubstituted-5,8-quinolindion derivatives of formula 1 is shown in the following scheme 1, Scheme 1

wherein Ra, Rb, Ra, Rd and X are the same as defined above.

A method of preparing 6,7-dihalo-5,8-quinolindione as starting material expressed in the following formula 2 in the present invention has been disclosed in J. am. Chenl. Soc., 82,1155 (1960) and An} del-Clielil., 624, 108-119 (1959). It is prepared from 8-hydroxyquinoline through 5-nitroso-8- hydroxyquinoline and 5-amino-8-hydroxyquinoline chloride as intermediates.

The detailed preparing method in scheme 1 is described hereunder.

The compound of formula la is prepared by condensation reaction of 6,7-dihalo-5,8-quinolindione of formula 2 and an arylamine of formula 3. A solvent used in this reaction is Cl-C3 lower alcohol such as methanol, ethanol and isopropanol, acetic acid, dimethylsulfoxide (DMSO) or dioxane, preferably ethanol. The reaction is carried at the temperature of from room temperature to 100°C for 2-6 hours, preferably at reflux temperature for 3-5 hours.

The compound of formula 1b is prepared by reacting compound la dissolved in an alcohol solvent with sodium sulfite (Na2S) at room temperature for 2-5 hours, preferably 2-3 hours.

The compound of formula 1c is prepared by reacting compound la with ammonium thiocyanate (NH4SCN) in acetone. Acetone as a solvent can be replaced to an alcohol such as methanol and ethanol, preferably ethanol.

The compound of formula Id is prepared by reacting 6,7-dihalo-5,8- quinolindione of formula 2 with alkylmercaptane (Rd-SH) at the same reaction condition of the preparing method of compound la.

These compounds of formulas la, lb, 1c and 1d are isolated and purified bv conventional procedures such as recrystallization and chromatography.

The following examples are intended to be illustrative of the present invention and should not be construed as limiting the scope of this invention defined by the appended claims.

Example 1: 5-nitroso-8-hydroxyquinoline hydrochloride To a mixture of 8-hydroxyquinoline (58 g, 0.4 mol) in distilled water, concentrated hydrochloric acid (75 iiiv.) and ice (200 g) was added NaNOz (30 g) in water (100 iiiv.) at 0-4 °C during 1 hr. The reaction mixture was kept at 0oC overnight and then filtered by washing with cold water to give 5-nitroso- 8-hydroxyquinoline hydrochloide (95%).

Example 2: 5-amino-8-hydroxyquinoline dihydrochloride 5-nitroso-8-hydroxyquinoline chloride (40 g) was added to a mixture of water (160mu) and 5N-NaOH (260 mQ) and heated to 40°C. Na2S204 (95 g) was added to the reaction mixture with increasing temperature to 75-80 °C.

The reaction mixture was cooled to 50 °C and 12N-HCl (250 mA) was added to it. Then the reaction mixture was cooled to 0 °C and filtered to give 5-amino- 8-hydroxyquinoline dihydrochloride (34 g, 69%)

Example 3: 6,7-dichloro-5,8-quinolindion of formula 2 After addition of 5-amino-8-hydroxyquinoline dihydrochloride (9 g) to hydrochloric acid (81 g), the reaction mixture was heated to 60 °C and NaClOn (4.5 g) was added. The reaction mixture was stirred for 30 min at 50-60 °C, filtered and recrystallized twice with buthanol to give yellow precipitate of (90%) m. p.: 221-222 °C Example 4: 6,7-dibromo-5,8-quinolindione of formula 2 After addition of 5-amino-8-hydroxyquinoline dihydrochloride (9 g) to hydrobromic acid (81 g), the reaction mixture was heated to 60 °C and NaBrOn (7.5 g) was added. The reaction mixture was stirred for 30 min at 50-60 °C, filtered and recrystallized twice with buthanol to give yellow precipitate of 6,7-dibromo-5,8-quinolindion (67%) m. p.: 243-245°C Example 5 : 6- [(N-4-chlorophenyl) amino]-7-bromo-5, 8-quinolindione of formula 1 CeCl3 (0.1 mmol) as a catalyst and 4 equivalents of 4-chloroaniline (10 mmol) were added to 6,7-dibromo-5,8-quinolindion (10 mmol) dissolved in ethanol (100 nlQ). The reaction mixture was heated at reflux for 8 hrs. After reading the completion of reaction by TLC, it was cooled, filtered, and recrystallized with ethanol to give redish powder of 6- [ (N-4- chlorophenyl) amino]-7-bromo-5, 8-quinolidione.

Example 6: 6-[(N-4-chlorophenyl) amino]-7-methylmercapto-5,8- quinolidione of formula 6

Dimethylsulfate (12 mmol) was added to 6- [ (N-4- chlorophenyl) amino]-7-bromo-5, 8-quinolidione (10 mmol) dissolved in ethanol (100 The reaction mixture was stirred for 2 hrs at room temperature The reaction mixture was cooled, filtered, and recrvstallized with ethanol to give 6-[(N-4-chlorophenyl) amino]-7-methylmercapto-5,8- quinolidione.

Example 7: 6- [ (N-4-chlorophenyl) amino]-7-thiocyano-5,8-quinolidione of formula 10 1 equivalent of ammonium thiocyanate (NH4SCN) was added to 6- [ (N- 4-chlorophenyl) amino]-7-bromo-5,8-quinolidione (1 g). The reaction mixture was heated at reflux for 3 hrs. After reading the completion of reaction by TLC, it was cooled, filtered, and recrvstallized with ethanol to give 6- [ (N-4- chlorophenyl) amino]-7-thiocyano-5,8-quinolidione.

Example 8: 6-ethylcercapto-7-chloro-5,8-quinolindione of formula 13 Cet13 (0. 1 mmol) as a catalyst and 1 equivalent of sodium sulfide (Na2S) were added to 6,7-dichloro-5,8-quinolindion (1 g) dissolved in ethanol (100 m#). The reaction mixture was stirred for 30 min at room temperature. 1 equivalent of ethyl mercaptane was added to the reaction mixture and heated at reflux for 1 hr. The reaction mixture was cooled and filtered to give 6-ethylmercapto-7-chloro-5,8-quinolidione.

Physical properties of the compounds of formula 1 prepared by the above procedure are summarized in table la and table lb.

Table la (DMSO-d6),ppmComp.-1H-NMR State mp (°C) MS (KBr,IR cm-1) No. 7.2#7.4 (4H, m, aromatic 3500(s,NH),3200,1600Redish215,255,282, 223#225ring),8.4#8.9(3H,m,1Powder 362(M+) (s,C=0), 1550,1470 C5H3N), 9.3 (111,s,NH) 3340(s, NH), 3040. Redish 7.0#8.49.21(1H,NH), 228#2301675(s,C=0),1600,2Powder aromaticring).352(M+)(8H,m,2 1575, 1575, 1380, 7.1#7.5(4H,m,aromatic DarkDark218,246,274, C=1620(s, 236#238ring),7.8#9.0(3H,m,3brown Powder 406(M+) 0),1550,1470 C5H3N),9.3(1H,s,NH) 2.55(3H,s,COCH3), 6.57, 3200(s,NH),3025,1650 7.614(4H,dd,2aromaticRedish130,224,311, 239-241(s,C=0),1560,12904brown ring),8.0#9.23(3H,m.Powder326(M+) 1255 C5H3N),9.3(1H,s,NH) 2.18#2.32 (3H,s,SCH3), Dark 78,130,228 3200(NH), 1670(C=0), redish6.97#7.41(4H,m,aromatic 396#400260,316,5plate 1400,1250,1030,750, 8.39#8.50(3H,m,Crystalring), 338,350(M+) 700 C5H3N),9.4(1H,m,NH) 2.2(3H,s,SCHs),7.15# 1650(C=0),111,192,220,3200(NH), Black 7.3(4H,m,aromatic ring), 288#290283,318,1650,1450,1300,1200,6Powder- 7.86#9.1(3H,m,C5H3N), 330(M+) 1000,830 9,39(1H,m,NH) 2.14#2.33(3H,s,SCH3), 3200(NH),1700(C=0)Redish76,128,164, plate 6.99#7.07 (4H,m,aromatic 192,220,2481550,1510,1430,1300,7244#246 7.68#7.92(3H,m,Crystalring), 410,422(M+) 1000, 830 C5H3N),9.30(1H,m,NH) Bla,k 76, 164,192,3200 (NH), 2320 (C=N), 7.03-7.87 (4H, m, aromat i c 3 Po-der-.-254-257 220,248,375,1700 (C=0), 1400,1200, ring), 8.47-9. 07 (3H, m, C5H3N),9.50(1H,s,NH)410,433(M+)1000,850,800 127,192,220, 3200(NH, 2300(C=N) 7.18#7.22(3H,m,aromatic Dark 9 brown 245#248248,283,327, 1660(C=0),1500,1400, ring), 8.46#8.50(3H,m, Powder 364,386(M+)364,386(M+)1310,1200,800,700 C5H3N), 9.45(1H,s,NH) Black 75, 111,164,3200 (NH), 2320 (C=N), (3H, m, aromatic Powder |279#281| 192,220, 283,1700 (C=0), 1500,1400, ring), (3H, m, 318,341(M+) 1200, 1000,800,700 CsHsN), 9.47 (1H, s, NH)

Table lb mp(°C)MS(m/e)1R(KBr,cm-1)1H-NMR(DMSO-d6),ppm@mp.State 2.28#2.32(3H,q,p-COCH3), 3300(NH),2000(C#N), 7,47#7,75(4H,m,aromaticBrown215,291,306, 253#2571500(C=0),1200,11Powder 349(M+) ring), 8.54#8.85(3H,m, 1160,1000 C5H3N),9.54(1H,s,NH) 3200(NH), 2300(C#N), 63,77,125,7,30#7,86(3H,m,aromatic 1600,1400,Redish1700(C=0), 211#213202,257,285,ring),8.45#8.48(3H,m,12Powder 1310,1220,900,850, 9.31(1H,s,NH)343(M+)C5H3N), 700 Dark 1700 (C=0), 1450 (CH2), 1. 15-1.29 (3H, m, 3CH3), brown 91,263,298 328#3301300(CH3),1200,1150,2,63#2,97(2H,t,CH2),13plate Crystal(M+) 8007.96#8.69(3H,m,C5H3N) Dark 1.02#1.12(3H,q,CH3).1450(CH2), brown 91,263,298 1300(CH3),1250,1150,3.42#3.72(4H,m,2CH2),14299#302 plate (M+) 8.44#9.23(3H,m,C5H3N)Crystal800 1680(C=0),1460(CH2),1.06#1.35(3H,q,3CH3), Black 400°C 91,263,298 15 plate 2.59#2.60(6H,m,3CH2), to(M+)CrystalUp 800 8. 44-9. 13 (3H, m, CsHsN) 0.94#0.97(3H,q,CH3),Black1670(C=0),1450(CH2), plate 91,263,298 16 1.15#1.80(8H,m,3CH2),1300(CH3),1250,1000, Crystal (M+) C5H3N)8008.44#8.58(3H,m, Dark 0.93#0.96(3H,q,CH3), 91,263,298 1670(C=0),1150(CH3), redish 17 289#303 2.01#2.59(10H,m,5CH2), Powder 1320(CH3),1130,820 7.82#9.09(3H,m, C5H3N) 1650(C=0),1500(CH2),0.88#0.91(3H,q,CH3),Dark55,134,190, borwn 225,288,3231280(CH3),1140,890,1.33#1.65(12H,m,CH3).18128#131 plate Crystal 7.89#9.09(3H,m,C5H3N)830 Black 0.87#1.28(3H,m,CH3), 1670(C=0),15309CH3),400°C91,263,298 plate to2.58#2.63(14H,m,7CH2),19Up 1300(CH3),1160,780Crystal(M+) 7.74#9.04(3H,m,C5H3N) 55,69,134,1680(C=0),1460(CH2), 0.87#0.92(3H,q,CH3), Yellow 20 Powder142#145 1.30#2.59(10H,m,8CH2), 351(M+) 890 7.94#9. 05 (3H, m, CsHsN)

The compounds of 6,7-disubstituted-5,8-quinolindione derivatives of formula 1 and its pharmaceutically acceptable salts have excellent antifungal activity. A pharmaceutical composition containing the compounds as an effective ingredient is disclosed in the present invention. Said pharmaceutically acceptable salts include hydrochloric acid salt, sulfuric acid

salt and bisulfite salt.

A pharmaceutical composition comprises a compound of formula 1, pharmaceutically acceptable carriers, fillers, and other additives. The carrier (s) must be'acceptable'in the sense of being compatible with other ingredients of the formulation and not deleterious to the recipient thereof.

The compositions of the invention include those in a foam especiallv formulated for a) oral administration such as tablets, capsules, troches, solutions, emulsions and suspensions, b) parenteral administration bv injection presented in such forms as suspension, solutions, emulsions and powder for constitution with a suitable vehicle, e. g. sterile water, before use, and c) topical administration in the form of ointments, creams, gels, or lotions.

Pharmaceutically acceptable carriers in use for suitable formulations are: binding agents, lubricants, diluents, solubilizers, disintegrants, stabilizers, emulsifying agents, colorants and aromatics for oral administration; preservatives, solubilizers, and stabilizers for parenteral administration; and bases, diluents, lubricants, thickeners, and preservatives for topical administration. These pharmaceutically acceptable formulations are administered orally, parenterally by intravenous, subcutaneous and intraperitoneal injection/infusion, or topically. In oral administration, antacids can be combined in a pharmaceutical composition to prevent from degradation by gastric acid and tablets may be coated to give enteric-coated tablet.

The dose varies depending on age, body weight, sex, symptom and the like of patients, and types of administration but it is usually 0.01-200 mg/kg/day, which is administered in several doses a day, preferably in a single dose or several doses.

Examples for formulation comprising 6,7-disubstituted-5,8-

quinolindion derivatives of formula 1 and its pharmaceutically acceptable salts as an effective ingredient are described but it should be noted that the present invention is not limited to these exemplifications.

Formulation Example 1: Capsule for oral administration 100 mg of an effective compound, 10 mg of anhydrous silicic acid, 190 mg of microcrystalline cellulose, 5 mg of magnesium stearate, 60 mg of starch and sodium gluconate and 135 mg of anhydrous calcium hydrogenphosphate were mixed and stirred. The mixture was put into hard gelatin capsules to give a capsule drug.

Formulation 2: Injection for parental administration 100 mg of an effective compound, 180 mg of mannitol, 26 mg of sodium hydrogenphosphate were dissolved in 2,974 mg of sterile water to give an injection solution.

Formulation 3: Ointment for topical administration 2 g of an effective compound, 7 g of lanoline tablets and effective amount of white ointment to give 10 g of total weight.

Antifungal activities of the compounds of formula 1 according to the present invention are determined by the following Experimental Examples.

Experimental Example 1 : Determination of Minimum Inhibitory Concentration (MIC) Values The minimum inhibitory concentration (MIC), which indicates the lowest concentration of the active ingredient in the growth medium, was determined by solid medium dilution method. Pathogens strains used are

Candida albicans ATCC10231, Candida glabrata ATCC2001, Caitdlda tnopicnlis ATCC28775, ATCC22019,Cryptococussneoformansparasilosis KCTC7223.

4 mg of a test compound was dissolved in 2 m I of dimethylfulfoxide (DMSO) to produce solutions having a concentration of 2 mg/ml for use and serial two-fold dilution was prepared. The test medium was Sabouraud's agar. 501lP aliquot of serial two-fold dilution was added to 1 ml of agar medium in 10 x 1.2 cm petri dishes to be 12.5 6.3,1.6, and 0.8/g/ml of test compound concentration and the slant was prepared by tilting the petri dishes. The MIC value without containing a test compound was determined in order to test an effect of a solvent to an antifungal activity.

Hyphae with a diameter of 2 mm were inoculated with 2 x 105 CFU. After the medium was cultured for 3 days at 30 °C, the MIC values were determined.

Control drugs were fluconazole and ketoconazole. The datas are shown in table 2.

Table 2 MIC (g/ml) Compound No. C. albícans C. glabrata C. tropicallis C. parapsilosis C. neoformans Compound No. 6.3 12.5 12.5 25.0 12.5 Compound No. 2 12. 5 12.5 25.0 6.3 12.5 Compound No. 3 6.3 6.3 12.5 25.0 6. 3 Compound 1.63.23.26.312.5No.4 Compound No. 5 1.6 3.2 3.2 25.0 6.3 Compound 12.56.3<0.825.012.5No.6 Compound 6. 7 6. 3 3.2 3.2 25.0 3.2 Compound 12.512.512.5100.012.5No.8 Compound <0.86.3<0.8<0.83.2No.9 Compound No. 10 12.5 12.5 12.5 100.0 6. 3 Compound No. 11 6.3 25 3.2 3.2 <0.8 Compound No. 12 12.5 12.5 3.2 1.6 <0.8 Compound No. 13 12.5 50 100 3.2 6. 3 Compound No. 14 12.5 50 50 12.5 3.2 Compound No. 15 25 12. 5 100 25 12.5 Compound 3.212.5506.325No.16 Compound No. 17 25 50 50 25 12. 5 Compound No. i8 6. 3 50 12.5 6.3 12. 5 Compound 12.52512.53.212.5No.19 Compound 50506.32525No.20 Fluconazole 25.0 25.0 100. 0 12.5 25.0 12.512.512.512.5Ketoconazole12.5 As shown in table 2, the compounds of the present invention show excellent inhibitory activity against Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, Cryptococuss neoformans and the like and their activities are superior or similar to those of the control drugs, fluconazole and ketoconazole.

Especially, the growth of fungus was completely inhibited against 5 types of strains when the concentration of the compound was 50/g/ml.

Experimental Example 2: C. nlbica1is (1 x 107) isolated was inoculated to Sabouraud's agar, suspended in a physiological saline solution, and injected into tail vein of a rat.

Strains to kill a rat within 48 hours were selected. C. albicans (2 x 10"which is concentration to kill a rat within 48 hours) suspended in a physiological saline solution was injected and infected into tail veins of 125 rats. Each concentration (10,2,0.5,0.1,0.025 mg/kg) of the compounds of table 3 was administered to each 10 rats. After infecting with Candida strain, each compound was administered to each 10 rats. Fluconazol as a control drug was also administered with 10,2,0.5 and 0.1 mg/kg of concentrations. A physiological saline solution containing 0.2 % tween 20 was administered as a control group. The datas are shown in table 3.

Table 3 Compound No. ED5oSD (n=5), (mg/kg) Compound No. 1 0. 060. 03 Compound No. G 0.06#0.03CompoundNo.3 Compound0.40#0.25 Fluconazole6.00#1.70

According to the table 3, while EDso of a control drug, fluconazole, was 6.00 mg/kg of the concentration to a rat infected with C. albicmis, each EDso of

the compounds 1,2,3, and 4 was 0.06,0.07,0.06 and 0.04 mg/kg of the concentration.

Experimental Example: Survival prolongation effect of a rat infected with C. albicarzs 2 x 104/0.1 ml of C. (zlbicni1s suspended in physiological saline was injected into 49 rats via tail vein to infect and divided to 4 groups of 7 rats.

The concentration of effective dose (EDso) was injected intraperitoneallv once daily for 14 days after 4 days of infection. In other words, 0.06,0.07,0.06 and 0.04 mg/kg of the test compounds 1,2,3, and 4 and 6.00 mg/kg of the control drug, ketoconazole were injected to determine the survival rate. The results were shown in Figs. 1 through 4.

According to Figs. 1 through 4, the compounds of the present invention show similar inhibitory activity at lower concentration than that of ketoconazole which is the control drug. Therefore, 6,7-disubstituted-5,8- quinolindione derivatives of formula 1 have excellent inhibitory activity against various fungus.

Based on foregoing, the compounds of formula 1 of the present invention are useful for the treatment of a variety of fungal infections in human and animals.