AU2441800A - Immunosurpressive effects of pteridine derivatives - Google Patents

Description

WO 00/45800 1 PCTiEPOO/00938 IMMUNOSUPPRESSIVE EFFECTS OF PTERIDINE DERIVATIVES The invention relates to a pharmaceutical composition for the treatment of autoimmuno disorders and/or the treatment or prevention of transplant rejections comprising pteridine derivatives. 5 The invention further relates to combined pharmaceutical preparations comprising one or more pteridine derivates and one or more known immunosuppressant, and to a group of novel pteridine derivates as such. 10 Further the invention is also related to a method for the treatment of autoimmuno disorders and/or of transplant-rejections. Several pteridine derivates are known in nature and used in the preparation of medicines, for example as 15 described in EP-A-108 890. Other medical uses of derivatives of pteridine are described in WO 95-31987 as NO-synthase inhibitors, for example for the treatment of diseases caused by a high nitrogen monoxide level. Further, WO-95-32203 describes also the use of 20 tetrahydropteridine derivatives as NO-synthase inhibitors. Both above-mentioned WO publications disclose also the use of these specific pteridine derivatives in the treatment of pathologically low blood pressure, in 25 particular septic shock and combined with cytokines in tumor therapy and in transplant-rejection diseases. Although some of these pteridine derivatives are claimed as potentially active for the treatment of transplant-rejection diseases, direct evidence for their 30 effectiveness is lacking. Thus there still is a need for specific and highly active immunosuppressive compounds, in particular immunosupressive compounds active in the cosignal pathway. A first object of the invention is to provide a 35 pharmaceutical composition having high immunosuppressive activity. Another object of the invention is to provide a CONFIRMATION COPY WO 00/45800 PCT/EP0O/00938 2 combined immunosuppressive preparation which causes a superadditive effect, comprising a pteridine derivative of the invention and other known immunosuppressants. Another further object of the invention is to 5 provide immunosuppressive compounds, which are active in a minor dose, in order to decrease the considerable treatment costs. Known immunosuppressive compounds are for example cyclosporine A, subsituted xanthines, tacrolimus 10 (FK 506), rapamycine (RPM), leflunomide, mofetil, adrenocortical steroids, cytotoxic drugs and antibody preparations. The immunosuppressive effect of cyclosporine A (CyA) is already known since 1972. However, due to its 15 nephrotoxicity and several other side effects CyA has not been able to establish itself as the optimal and final drug of choice. Methylxanthines, for example pentoxifylline (PTX), are known having immunosuppressive effects in 20 vitro. Recently (Lin Y. et al, Transplantation 63 (1997) it has been found that the co-medication of an immunosuppressive compound such as cyclosporine A (CyA) or FK506 or RPM (rapamycine) with a methylxanthine 25 derivative, in particular A802715 (7-propyl-1(5-hydroxy 5-methylhexyl)-3-methylxanthine) leads to a superadditive increase in the immunosuppressive action. Likewise, other substituted, in particular substituted 8-phenylxanthines have been found to possess 30 immunosuppressive effects in vitro (application EP 98.201323.7). The present invention relates in particular to the application of a group pteridine derivatives and their pharmaceutical salts, possessing unexpectedly 35 desirable pharmaceutical properties, i.c. are highly active immunosuppressive agents. The invention demonstrates the WO 00/45800 PCT/EP00/00938 3 immunosuppressive effects of pharmaceutical composition for the treatment of autoimmuno disorders and/or for the treatment or prevention of transplant-rejections comprising a pteridine derivative of general formula: 5 0 N R 3

R

1 -N 0 N N R4 10 R wherein: R. and R, are independently hydrogen; aliphatic 15 saturated or unsaturated; straight or branched carbon chain with 1 to 7 carbon atoms; substituted or unsubstituted aryl or alkylaryl substituents, whereby the carbon atoms may be oxidized represented by alcohol or carbonyl function or carboxylic acids and their esters; 20

R

3 and R 4 are independently hydrogen, hydroxyl, halogen,alkyl, haloalkyl, alkoxy, wherein alkyl and the alkyl group may be branched or straight and contains one or four carbon atoms, formyl and derivatives such as 25 hydroxylamino conjugates and acetals, cyano, carboxylic acids and carboxyl acid derivatives such as esters and amides, sulfhydryl, amino, alkylamino, cycloalkylamino, alkenylamino, alkynylamino, benzylamino, hydroxylalkylamino, morfolinoalkylamino, fenylhydrazino, 30 morfoline, piperidine, mercaptobenzyl, mercaptoalkyl, cysteinyl ester, styryl, substituted or unsubstituted aromatic ring; aromatic or heterocyclic substituent substituted with an aliphatic spacer between the pteridine ring and the aromatic substituent of 1 to 4 35 carbon atoms, whereby said spacer may contain an alcohol function, carbonyl function, halogen, ether, and may be saturated or unsaturated; branched or straight, saturated or unsaturated aliphatic chain of 1 to 7 carbon atoms WO 00/45800 PCT/EPOO/00938 4 which may contain one or more functions chosen from the group comprising carbonyl, alcohol, ether, carboxyester, nitro, thioalkyl, halogen or a pharmaceutically acceptable salt thereof; and 5 X and Y are independently oxygen or sulfur or a pharmaceutical salt thereof, and a pharmaceutically acceptable carrier. Preferred pteridine derivatives comprising 10 compositions are given in claims 2-9. Particularly preferred are the compositions according to claim 10. The invention further relates to a combined preparation having synergetic effects containing 1) cyclosporine A, substituted xanthines, tacrolimus 15 (FK506), Rapamycin (RPM), Leflunomide, Mofetil, adrenocortical steriods, cytotoxic drugs and antibody compositions and 2) at least one pteridine derivative of formula (I) defined above, and optionally a pharmaceutical excipient, for simultaneous, separate or 20 sequential use in (auto)immune disorders and/or in the treatment of transplant-rejections. The invention further relates to a method for treating auto-immuno disorders or transplant-rejections in a subject by administering an effective amount of a 25 pharmaceutical composition of claims 1-11, to the compounds as such as defined above, to the use of these compounds for the treatment of autoimmuno disorders and/or the treatment and/or prevention of transplant rejections, and to a method for selecting potent 30 immunosuppressive agents based on the determination of the three parameters MLC, ACD 3 and ACD 28 . Hereunder the effects of the pteridine derivatives on the lymphocyte activation are elucidated and are compared with standard reference compounds (see 35 table I, compound 4, 6, 7, 11, 13, 19, 20, 21, 22, 25, 26, 28, 30, 34, 35). Table I summarizes the tested compounds. These pteridine derivatives were obtained as follows: WO 00/45800 PCT/EPOO/00938 5 6-Bromomethyl-1,3-dimethyllumazine: To a solution of 1,3,6-trimethyllumazine [1] (2.06 g, 0.01 moles) in glacial AcOH (60 ml) was added dropwise bromine (3.2 g, 0.02 moles) in AcOH (10 ml) and then 5 heated under reflux for 1 hour. After cooling was evaporated, the residue dissolved in CHCl, (100 ml), washed with H 2 0 (3 x 70 ml), the organic layer dried over Na 2

SO

4 and again evaporated. The residue was purified by silica gel column chromatography starting with 10 toluene/EtOAc 9/1 to elute first 6-dibromomethyl-1,3 dimethyllumazine (1.49 g, 41%) and followed by toluene/EtOAc 4/1 to get 6-bromomethyl-1.3 dimethyllumazine. Yield: 1.2 g (42%). M.p. 228 0 C (decomp.). UV (MeOH) : 244 (4.16); [264 (400)1; 337 15 (3.86). 7-Bromomethyl-1,3-dimethyllumazine: Analogous to the preceding procedure with 1,3,7 trimethyllumazine [11 (2.06 g, 0.01 moles) and bromine 20 (3.2 g, 0.02 moles) by heating for 2 hours. Isolation by silica gel column chromatography with toluene/EtOAc 9/1 to elute first 7-dibromomethyl-1,3-dimethyllumazine (2.07 g, 57%) and second 7-bromomethyl-1,3-dimethyllumazine. Yield: 0.97 g (34%). M.p.165-166 0 C. UV (MeOH): 241 25 (4.23); 338 (4.02). 1,3-Dimethyllumazin-6-ylmethy1-triphenylphosphonium bromide: To a suspension of 6-bromomethyl-1,3-dimethyllumazine 30 (1.0 g, 3.5 mmoles) in toluene (20 ml) triphenylphosphane (1.1 g, 4.2 mmoles) was added and then heated at 80 0 C in an oilbath with stirring for 8 hours. After cooling the precipitate was collected, washed with EtOAc and dried at 100 0 C to give 1.8 g (94%) of a colorless powder of m.p. 35 289 0 C. UV (MeOH): 204 (4.74); 227 (4.52); [243 (4.42)]; (262 (4.21)]; 338 (3.88).

WO 00/45800 PCT/EPOO/00938 6 1, 3 -Dimethyllumazin-7-ylmethyl-triphenylphosphonium bromide: Analogous to the preceding procedure from 7-bromomethyl 1,3-dimethyllumazine and triphenylphosphane in toluene by 5 heating under reflux for 1 day. Yield: 1.86 g (97) . M.p. 261 0 C. UV (MeOH) : 204 (4.76); [221 (4.54)]; 342 (4.09); 414 (4.38). General synthesis of 1,3-dimethyl-6- (E) -styryllumazines 10 1, 2, 3, 5: To a solution of 1,3-dimethyllumazin-6-ylmethyl triphenylphosphonium bromide and 1,3 -dimethyllumazin-7 ylmethyl-triphenylphosphonium bromide (0.547 g, 1 mmole), respectively, in MeOH (5 ml) was added sodium methoxide 15 (0.108 g, 2 mmoles) and stirred at room temperature for 30 min. Then 1.5 mmoles of the aromatic or heteroaromatic aldehyde were added and stirring continued for 5 hours. The resulting precipitate was filtered off, washed with MeOH and purified by recrystallization from DMF/H 2 0 to 20 give a yellowish powder. 1,3-Dimethyl-6-(E)-styryllumazine (1): According to the general procedure with benzaldehyde (0.16 g). Yield: 0.124 g (42%). M.p. 238 0 C. UV (MeOH): 25 [220 (4.17)]; 308 (4.42); 372 (4.03). 1,3-Dimethyl-6-[(E)-2-(pyrid-3-yl)vinyl]lumazine (2): According to the general procedure with pyridine-3 carboxaldehyde (0.162 g). Yield: 0.195 g (66%). M.p. 30 210 0 C. UV (MeOH): [236 (3.92)]; 308 (4.29); 370 (3.97). 1,3-Dimethyl-6-[(E)-2-(pyrid-4-yl)vinyl]lumazine (3): According to the general procedure with pyridine-4 carboxaldehyde (0.162 g). Yield: 0.156 g (53%) . M.p. 35 262 0 C. UV (MeOH): 202 (4.20); [238 (3.92)]; 307 (4.51); 370 (4.20). 6- (1,2-Dibromo-2-phenylethyl) -1,3-dimethyllumazine (4): WO 00/45800 PCT/EP00/00938 7 To a solution of 1,3-dimethyl-6-(E)-styryllumazine (1) (0.735 g, 2.5 mmoles) in CHC1 3 (20 ml) was added bromine (0.8 g, 5 mmoles) dissolved in CHCl 3 (5 ml) and then the mixture stirred at room temperature for 4 hours. It was 5 evaporated to dryness and the residue treated with MeOH to give a colorless precipitate. The solid was collected, washed with MeOH and dried in a vacuum desiccator. Yield: 1.067 g (94%). M.p. 176 0 C. UV (MeOH): 245 (4.18); [260 (4.10)]; 341 (3.83). 10 1,3-Dimethyl-6-[(E)-4-(phenyl)butadienyl]lumazine (5): According to the general procedure with cinnamaldehyde (0.2 g). Yield: 0.138 g (43%). M.p. 252*C (decomp.). UV (MeCH): 228 (4.02); [244 (3.97)]; 330 (4.66); 389 (4.23). 15 6-[(E)-2-methoxycarbonylethenyl]lumazine: To a suspension of methoxycarbonylmethyl triphenylphosphonium bromide (0.415 g, 1 mmole) in dioxane (3 ml) was added DBU (0.23 g, 1.5 mmoles) and 20 stirred at room temperature for 30 min. Then 1,3 dimethyllumazine-6-carboxaldehyde [1] (0.2 g, 0.91 mmoles) was added and stirring continued for 5 hours. The precipitate was collected, washed with MeOH and dried to give a colorless crystal powder. Yield: 0.158 g (63%.). 25 M.p. 211-213 0 C (decomp.). UV (MeOH): 202 (4.46); [256 (4.14)]; 286 (4.21); 348 (4.08). 6-(1,2-Dibromo-2-(methoxycarbonyl)ethyl)-1,3 dimethyllumazine (6): 30 To a solution of 6-[(E)-2-methoxycarbonylethenyl]lumazine (0.7 g, 2.53 mmoles) in CHCl 3 (20 ml) was added bromine (0.64 g, 4 mmoles) dissolved in CHCl 3 (5 ml) and then the mixture stirred at room temperature for 6 hours. It was evaporated to dryness and the residue treated with MeOH 35 to give a colorless precipitate. The solid was collected, washed with MeOH and dried in a vacuum desiccator. Yield: 0.97 g (88%). M.p. 1630C. UV (MeOH): 247 (4.16); [260 (4.08)]; 339 (3.88).

WO 00/45800 PCT/EP00/00938 8 6-( 2 -Bromo-2-methoxycarbonyl-ethenyl)-1,3 dimethyllumazine (7): To a solution of 6 (0.1 g, 0.23 mmoles) in dioxane (20 ml) was added DBU (70 MG, 0.43 g, 0.43 mmoles) and then 5 stirred at room temperatur for 2 hours. It was diluted with ethyl acetate (100 ml), washed with H 2 0 (3 x 50 ml), the organic layer separated, dried over Na 2SO4 and then evaporated. The residue was treated with MeOH, the solid collected and purified by recrystallization from DMF to 10 give a yellowish powder. Yield: 0.055 g (68%). M.p. 2040C. UV (MeOH): [254 (4.08))] ; 285 (4.25); 360 (4.01). 6-Chlorocarbonyl-1,3-dimethyllumazine: A suspension of 1,3-dimethyllumazine-6-carboxylic acid 15 [2] (3.0 g, 12.7 mmoles) in dry toluene (80 ml) was treated with freshly destilled thionyl chloride (50 ml) under reflux for 3 hours. It was evaporated to dryness, the residue treated with dry ether, the solid collected, washed with ether and dried in a vaccuum desiccator. 20 Yield: 3.13 g (93%). M.p. 262-264 0 C. UV (dioxane): 256 (4.08); [280 (4.00)]; 333 (4.03). 6-[(2-Acetyl-2-ethoxycarbonyl)acetyl]-1,3 dimethyllumazine (8): 25 A solution of ethyl acetoacetate ethoxy-magnesium salt [3] (0.8 g, 4 mmoles) in THF (8 ml) was added dropwise to a suspension of 6-chlorocarbonyl-1,3-dimethyllumazine (0.51 g, 2 mmoles) in THF (10 ml) and then the mixture stirred at room temperature for 3 days. It was evaporated 30 and the residue treated with cold 1 N HCl (20 ml, 0-5*C). The precipitate was collected, washed with H 2 0 and dried in a vacuum desiccator. Purification was achieved by column chromatography (silica gel 3.5 x 12 cm) with CHCl 3 /MeOH 95/5 and the first main fraction collected. 35 After evaporation was recrystallized from toluene (12 ml) to give colorless crystals. Yield: 0.247 g (360). M.P. 153-156 0 C. UV (pH 2.0): 251 (4.09); 293 (4.10); 330 (4.11).

WO 00/45800 PCT/EPOO/00938 9 6-[2,2-(Diethoxycarbonyl)acetyl]-1,3-dimethyllumazine (9): To a solution of ethylmalonate ethoxy-magnesium salt [4] (0.685 g, 3 mmoles) in THF (12 ml) was added 6 5 chlorocarbonyl-1,3-dimethyllumazine (0.51 g, 2 mmoles) and then the mixture stirred at room temperature for 20 hours. It was evaporated, the residue treated with 1 N HCl (20 ml) and the resulting solid collected. Recyrstallization from EtOH (40 ml) gave yellowish 10 crystals. Yield: 0.585 g (78%). M.p. 124-126 0 C. UV (pH 2.0): 253 (4.05); 291 (4.08); 332 (4.04). 6-(l-Methoxy-2-methoxycarbonyl)ethenyl)-1,3 dimethyllumazine (10): 15 A suspension of 6 (0.2 g, 0.46 mmoles) in dry MeOH (8 ml) was treated with a solution of sodium (0.046 g, 2 mmoles) in MeOH (2 ml) at room temperature with stirring for 15 min. Then NH 4 Cl (0.1 g) and H 2 0 (10 ml) were added and the mixture extracted with CHC1 3 (2 x 50 ml). The organic 20 layer was dried over Na 2

SO

4 , evaporated and the residue crystallized from CHCl,/n-hexane. Yield: 0.085 g (60%). M.p. 160 0 C. UV (MeOH): 204 (4.20); 245 (4.15); 288 (4.23); 350 (3.99). 25 1,3-Dimethyl-6-[(2-nitro)ethenylllumazine (11): A solution of 12 (0.562 g, 2 mmoles) in pyridine (10 ml) was cooled to OC and then acetic anhydride (4 ml) dropwise added. Cooling was removed and the mixture stirred at room temperature for 3 hours. The resulting 30 precipitate was collected, washed with H 2 0 and dried in a vacuum desiccator to give a chromatographically pure product. Yield: 0.515 g (98%). Crystallization from CHCl,. M.p. 232-234 0 C. UV (MeOH) : [239 (3.63)]; 309 (3.87); 365 (3.95). 35 6-[(1-Hydroxy-2-nitro)ethyl]-1,3-dimethyllumazine (12): To a solution of nitromethane (0.61 g, 10 mmoles) and triethylamine (1.44 g, 10 mmoles) in MeOH (20 ml) was WO 00/45800 PCT/EPOO/00938 10 added 6-formyl-1,3-dimethyllumazine [1] (2.0 g, 9 mmoles) and then the mixture stirred at room temperature for 5 hours. The precipitate was collected, washed with MeOH and ether and dried. Yield: 2.22 g (78%). 5 Recrystallization from CHCl,. M.p. 166-167 0 C. UV (MeOH) 240 (4.38); 336 (3.98); [347 (3.89)). 6-[(1-Ethylthio-2-nitro)ethyl]-1,3-dimethyllumazine (13): To a suspension of compound 11 (0.263 g, 1 mmole) in MeOH 10 (5 ml) and H 2 0 (5 ml) was added ethylmercaptane (0.093 g, 1.5 mmoles) and DBU (0.2 g) and then the mixture stirred at room temperature for 1 hour. The precipitate was collected, washed and dried. Yield: 0.25 g (77%). M.p. 88 0 C. UV (MeOH): 203 (4.28); 240 (4.26); [262 (4.05)]; 15 341 (3.89). 6-Hydroxymethyl-1,3-dimethyllumazine (14) [1]: 1,3-Dimethyl-7-[(E)-2-(pyrid-2-yl)vinyl]lumazine (15): 20 According to the general procedure with pyridine-2 carboxaldehyde (0.162 g). Yield: 0.233 g (79%) . M.p. 282 283 0 C. UV (MeOH): 203 (4.14); 238 (4.23); 312 (3.95); 375 (4.36). 25 1,3-Dimethyl-7-[(E)-2-(pyrid-3-yl)vinyl]lumazine (16): According to the general procedure with pyridine-3 carboxaldehyde (0.162 g). Yield: 0.195 g (66%). M.p. 264 265 0 C. UV (MeOH): 208 (4.45); 234 (4.43); [274 (4.09)]; 307 (4.08); 375 (4.48). 30 1,3-Dimethyl-7-[(E)-2-(pyrid-4-yl)vinyl]lumazine (17): According to the general procedure with pyridine-4 carboxaldehyde (0.162 g). Yield: 0.215 g (73%). M.p. 307 310 0 C. UV (MeOH): 207 (4.12); 229 (4.01); 282 (3.79); 35 [296 (3.76)]; 372 (4.00). 1,3-Dimethyl-7-[(E)-4-(phenyl)butadienyl]lumazine (18): WO 00/45800 PCT/EP00/00938 11 According to the general procedure with cinnamaldehyde (0.2 g). Yield: 0.195 g (61%). M.p. 277-287 0 C (decomp.). UV (MeOH): 239 (3.79); 299 (3.66); 402 (4.15). 5 7-[1,2-Dibromo-2-(methoxycarbonyl)ethyl)-1,3 dimethyllumazine (19): To a suspension of 7-[(E)-2-methoxycarbonylethenyl]luma zine (1.79 g, 6.5 mmoles) in CHC1 3 (70 ml) was added bromine (0.7 g, 14 mmoles) dissolved in CHCl 3 (10 ml) and 10 then the mixture stirred at room temperature for 3 hours. It was evaporated to dryness and the residue treated with MeOH to give a colorless precipitate. The solid was collected, washed with MeOH and dried in a vacuum desiccator. Yield: 2.34 g (77%). Crystallization from 15 EtOAc/n-hexane. M.p. 144-145*C. UV (MeOH): 240 (4.14); 343 (3.90). 7-[(E)-2-methoxycarbonylethenyl]lumazine (20): To a suspension of methoxycarbonylmethyl 20 triphenylphosphonium bromide (0.415 g, 1 mmole) in dioxane (5 ml) was added DBU (0.23 g, 1.5 mmoles) and stirred at room temperature for 30 min. Then 1,3 dimethyllumazine-7-carboxaldehyde [1] (0.2 g, 0.91 mmoles) was added and stirring continued for 20 hours. 25 The precipitate was collected, washed with MeOH and dried to give a colorless crystal powder. Recrystallization from DMF. Yield: 0.15 g (60%)-. M.p. 242-2450C (decomp.). UV (MeOH): 201 (4.21); 225 (4.29); 252 (4.20); 364 (4.11). 30 7-(1,2-Dibromo-2-phenylethyl)-1,3-dimethyllumazine (21): To a solution of 1,3-dimethyl-7-(E)-styryllumazine (23) (0.735 g, 2.5 mmoles) in CHC1 3 (20 ml) was added bromine (0.48 g, 3 mmoles) dissolved in CHC1 3 (5 ml) and then the 35 mixture stirred at room temperature for 3 hours. It was evaporated to dryness and the residue treated with MeOH to give a colorless precipitate. The solid was collected, washed with MeOH and dried in a vacuum desiccator. Yield: WO 00/45800 PCT/EPOO/00938 12 1.08 g (951). M.p. 187-188 0 C. UV (MeOH): 241 (4.25); 341 (4.06). 7-(1-Bromo-2-phenyl)ethenyl-1,3-dimethyllumazine (22): 5 To a suspension of 21 (0.2 g, 0.44 mmoles) in dry MeOH (4 ml) was added a solution of sodium (0.05 g, 2.2 mmoles) in MeOH (1 ml) and then the mixture stirred at room temperature for 3 hours. The precipitate was collected, washed with MeOH and dried in vacuum. Yield: 0.117 g 10 (710). Yellowish powder from DMF. M.p. 245-246 0 C. UV (MeOH): 243 (4.15); 372 (4.15). 7-Benzyl-1,3-dimethyllumazine: A solution of 5,6-diamino-1,3-dimethyluracil 15 monohydrochloride [5] (2.06 g, 0.01 mole) in H.0 (50 ml) was treated with benzylglyoxal [6] (2.22 g, 0.015 moles) in EtOH (20 ml) and heated under reflux for 1 hour. It was diluted with H 2 0 (50 ml) and then extracted with CHC1, (5 x 100 ml). The organic layer was dried over Na 2

SO

4 , 20 evaporated and the residue purified by silica gel column chromatography with toluene/EtOAc 10/1. The main fraction was collected, evaporated and crystallized from EtOH. Yield: 1.7 g (61%). M.p. 147-148 0 C. UV (MeOH): 238 (4.22); 332 (4.07). 25 1,3-Dimethyl-6-(E)-styryllumazine (23): According to the general procedure with benzaldehyde (0.16 g). Yield: 0.223 g (76%). M.p. 259-260 0 C. UV (MeOH): 203 (4.17); 237 (4.11); 379 (4.29). 30 7-Benzoyl-1,3-dimethyllumazine (24): A suspension of 7-benzyl-1,3-dimethyllumazine (0.56 g, 2 mmoled) in H 2 0 (30 ml) was treated with KMnO 4 (0.6 g) and heated under reflux for 30 min. After cooling was 35 extracted with CHCl 3 (3 x 100 ml), the organic layer dried over Na 2

SO

4 , filtered and evaporated to dryness. Crystallization from dioxane/H 2 0. Yield: 0.5 g (84%). M.p. 190-191 0 C. UV (MeOH): 233 (4.23); [255 (4.10)]; 347 (3.97).

WO 00/45800 PCT/EP00/00938 13 7-Chloro-1,3-dimethyllumazine (25) [7]. 1,3-Dimethyl-7-mercaptolumazine (26) [8]. 5 1, 3 -Dimethyl-6,7-diphenyllumazine (27) [9]. 1, 3 -Dimethyl-6-phenyl-7-mercaptolumazine (28): A mixture of 7-hydroxy-1,3-dimethyl-6-phenyllumazine [51 (2.84 g, 0.01 mole) and PS1 (3.3 g) was heated in 10 pyridine (75 ml) under reflux for 1 hour. After cooling was diluted with H 2 0 (50 ml) and after standing for several hours the yellow precipiptate (28-pyridinium salt, 3.3 g, 87%). The salt was dissolved in hot H0 (100 ml) and acidified by HCl to pH 0. The resulting yellow 15 crystals were collected, washed and dried in the oven. Yield: 2.22 g (74%). M.p. 145 0 C (decomp.). UV (MeOH): 203 (4.37); 227 (4.36); [283 (3.86)1; 370 (4.05). 7-Methoxy-1,3-dimethyl-6-phenyllumazine (29): 20 A solution 7-hydroxy-1,3-dimethyl-6-phenyllumazine [5] (1.42 g, 0.005 moles) in 0.5 N NaOH (20 ml) and MeOH (10 ml) was treated with dimethyl sulfate (1 ml) and stirred for 1 hour at room temperature. The resulting precipitate was collected, washed and dried in the oven. Yield: 1.26 25 g (81%). M.p. 194 0 C. UV (MeOH): 205 (4.53); [240 (4.08)]; 281 (4.22); 343 (4.23). 7-Chloro-1,3-dimethyl-6-phenyllumazine (30): A mixture of 7-hydroxy-1,3-dimethyl-6-phenyllumazine [5] 30 (2.84 g, 0.01 mole) and NH 4 Cl (1 g) was heated in POC1 3 under reflux for 36 hours. It was evaporated to a syrup, ice was added and stirred with a glasrod till a precipitate was formed. The solid was collected, washed with H 2 0, dried and then recrystallized from MeOH. Yield: 35 2.36 g (78%) . M.p. 180*C. UV (MeOH) : 204 (4.47); 249 (4.23); 273 (4.24); 350 (4.05).

WO 00/45800 PCT/EP0O/00938 14 6-Benzoyl-7,8-dihydro-1,3-dimethyl-7-(4-methoxyphenyl)lu mazine (31a): A solution of 6-benzoyl-1,3-dimethyllumazine (0.2 g, 0.68 mmoles) in dry 1,2-dichloroethane (20 ml) was treated 5 with AlCl 3 (0.4 g, 3 mmoles) and freshly distilled anisol (10 ml, 92 mmoles) at room temperature and stirred for 24 hours. Then ice (50 g) was added, the aquous phase extracted with CHCl 3 (3 x 50 ml), the organic phase washed with 2%-NaHCO 3 solution (50 ml) and H 2 0 (50 ml), dried 10 over Na 2 So 4 and evaporated in high vacuum to remove excess of anisol. The residue was treated with toluene (50 ml) to obtain a yellow precipitate. Recrystallization from EtOH/H 2 0 1/1 gave yellow crystals. Yield: 0.176 g (65%). M.p. 240-244 0 C (decomp.). UV (MeOH): 254 (4.25); [270 15 (4.21)1; 406 (4.08). 6-Benzoyl-1,3-dimethyl-7-(4-methoxyphenyl)lumazine (31): A suspension of 6-benzoyl-7,8-dihydro-1,3-dimethyl-7-(4 methoxyphenyl)lumazine (31a) (0.3 g, 0.74 mmoles) in 20 dioxane (40 ml) was treated at room temperature with l% KMnO 4 solution (10 ml) by dropwise addition with stirring. After 30 min the excess of KMnO 4 was reduced by NaHSO,, the MnO 2 filtered off, washed with warm EtOH (3 x 20 ml) and then the united organic phases evaporated to dryness. 25 The residue was purified by silica gel chromatography with CHCl 3 /MeOH (25/1). The main fraction was collected, evaporated and the solid recrystallized from EtOAc with charcoal. Yield: 0.175 g (59%). M.p. 255-257*C. UV (MeOH) : 253 (4.24) ; 367 (4.10) 30 6-Benzoyl-7,8-dihydro-1,3-dimethyl-7-phenyllumazine (32a): Analogous to procedure 31a from 6-benzoyl-1,3 dimethyllumazine (0.2 g, 0.68 mmoles) and benzene (15 35 ml). Yield: 0.21 g (83%). UV (MeOH): 254 (4.26); 407 (4.12). 6-Benzoyl-1,3-dimethyl-7-phenyllumazine (32): WO 00/45800 PCT/EPOO/00938 15 Analogous to procedure 31 from 6-benzoyl-7,8-dihydro-1,3 dimethyl-7-phenyllumazine (32a) (0.3 g, 0.78 mmoles). Yield: 0.18 g (62%). M.p. 185-187 0 C. UV (MeOH): 252 (4.39); [290 (4.08)]; 349 (4.16). 5 7-Methoxy-1,3-dimethyl-6-styryllumazine (33): To a suspension of compound 4 (0.2 g, 0.44 mmoles) in dry MeOH (6 ml) was added DBU (0.2 ml, 1.34 mmoles) and then stirred at room temperature for 2 hours. The precipitate 10 was filtered off, washed with MeOH and dried in a vacuum desiccator. Yield: 0.134 (94%). Crystallization from DMF. M.p. 271-272 0 C. UV (MeOH) : [232 (4.11)]; 306 (4.36); 375 (4.38). 15 1-Methyl-6,7-diphenyllumazine (34) [10]. 7-Hydroxy-3-methyl-6-phenyllumazine (35) [5]. 7-Hydroxy-1,6-diphenyllumazine (36). 20 To a suspension of 6-diamino-5-nitroso-l-phenyluracil [11] (2.32 g, 0.01 moles) in HO (50 ml) and EtOH (20 ml) was reduced catalytically with PtO 2

/H

2 in a shaking apparatus till about 450 ml of hydrogen was consumed. The mixture was heated, the catalyst filtered off and the 25 filtrate treated with ethyl phenylglyoxylate (2.5 g, 0.014 mmoles) by heating under reflux for 30 min. The warm solution was acidified by HCl to pH 0 and the resulting precipitate collected after cooling. Recrystallization from DMF. Yield: 2.59 (78%). M.p. 30 330 0 C. UV (MeOH): 204 (4.54); [222 (4.37)1; 284 (4.17); 346 (4.25). 7-Hydroxy-3,6-dimethyl-l-phenyllumazine (37) [121: 35 7-Hydroxy-6-phenyl-1,3-di-n-propyllumazine (38): A suspension of 5,6-diamino-1,3-di-n-propyluracil (1.13 g, 0.005 moles) in H.0 (30 ml), EtOH (5 ml) and AcOH (2 ml) was treated with ethyl phenylglyoxylate (1.25 g, WO 00/45800 PCT/EP0O/00938 16 0.007 mmoles) and heated under reflux for 30 min forming a brownish oil. After cooling was acidified by HCl to pH 0 whereby the oil solidified. Filtration and recrystallization from EtOH/H 2 0 gave yellowish needles. 5 Yield: 1.28 g (75%). M.p. 245 0 C. UV (MeOH): 212 (4.30); [243 (4.01)]; 284 (4.02); 349 (4.19). References 10 [1] Y. Kang, R. Soyka, W. Pfleiderer, J. Heterocycl. Chem. 1987, 24, 597. [2] R. Eisele, K. Aritomo, W. Pfleiderer, Pteridines 1993, 4, 178. [3] M. Viscontini, K. Adank, Helv. Chim. Acta 1952, 35, 15 1342. [4] R.E. Bowman, J. Chem. Soc. 1950, 324. [5] W. Pfleiderer, W. Hutzenlaub, Chem. Ber. 1973, 106, 3149. [6] H.D. Dakin, H.W. Dudley, J. Biol. Chem. 1914, 18, 20 42. [7] H. Steppan, J. Hammer, R. Baur, R. Gottlieb, W. Pfleiderer, Liebigs Ann. Chem. 1982, 2135. [8] Z. Kazimierczuk, W. Pfleiferer, Chem. Ber. 1979, 112, 1499. 25 [91 F.F. Blicke, H.C. Godt, J. Am. Chem. Soc. 1954, 76, 2798. [10] H. Fink, W. Pfleiderer, Chem. Ber. 1963, -96, 2950. [11] J. Litschitz, Ber. Deut. Chem. Ges. 1922, 55, 1619. [12] W. Hutzenlaub, H. Yamamoto, G.B. Barlin, W. 30 Pfleiderer, Chem. Ber. 1973, 106, 3203.

WO 00/45800 PCT/EPOO/00938 17 Materials and methods Various models may be used for testing an immunosuppressive effect. In vivo, for example, different transplantation models are available. They are strongly 5 influenced by different immunogenicities, depending on the donor and recipient species used and depending on the nature of the transplanted organ. The survival time of transplanted organs can thus be used to measure the suppression of the immune response. In vitro, there exist 10 also various models. The most used are lymphocyte activation tests. Usually activation is measured via lymphocyte proliferation. Inhibition of proliferation thus always means immunosuppression under the experimental conditions applied. There exist different 15 stimuli for lymphocyte activation: - coculture of lymphocytes of different species (MLR = mixed lymphocyte reaction): lymphocytes expressing different minor and major antigens of the HLA-DR type (= alloantigens) activate each other non-specifically. 20 - CD3 assay: here there is an activation of the T lymphocytes via an exogenously added antibody (OKT3). This antibody reacts against the CD3 molecule located on the lymphocyte membrane. This molecule has a costimulatory function. The interaction anti-CD3 (= 25 OKT3)-CD3 results in T-cell activation which proceeds via the Ca2+/calmodulin/calcineurin system and can be inhibited by CyA. - CD28 assay: here specific activation of the T lymphocyte goes also via an exogenously added antibody 30 against the CD28 molecule. This molecule is also located on the lymphocyte membrane, and delivers strong costimulatory signals. This activation is Ca 2 +-independent and thus cannot be inhibited by CyA. 35 Reagents All derivatives were dissolved in 0.5 ml DMSO and further diluted in culture medium before use in in vitro WO 00/45800 PCT/EPOO/00938 18 experiments. The culture medium consisted of RPMI-1640 + 10% FCS. Mixed Lymphocyte Reaction 5 Peripheral blood mononuclear cells (PBMC) were isolated from heparinized peripheral blood by density gradient centrifugation over Lymphoprep (Nycomed, Maorstua, Norway). Allogeneic PBMC or EBV-transformed human B cells [RPMI1788 (ATCC name CCL156)] which 10 strongly express B7-1 and B7-2 were used as stimulator cells after irradiation with 30 Gy. MLR was performed in triplicate wells. After 5 days incubation at 370C, 1 pCi

[

3 H]-thymidine was added to each cup. After a further 16 hours incubation, cells were harvested and counted in a 15 f-counter. The percent suppression of proliferation by drugs was counted using the formula: Percent inhibition = (cpm+drugs) - cpm Cult. Med 20 --------------------------- X 100 (cpm-drugs) - cpm Cult. Med T cell purification T cells were purified by removing non-T cells. 25 Briefly, monocytes were removed by cold agglutination. The resulting lymphoid cells were further purified by a cell enrichment immunocolumn [Cellect Human T (Biotex, Edmonton, Alberta, Canada)] by a process of negative selection. More than 95% of the B cells were removed with 30 this procedure. After depletion, the resulting T cell preparation was highly purified explaining these cells could not be activated by PHA or rIL-2 alone at concentrations capable of stimulating RBMC prior to deletion. 35 Measurements of T cell proliferations induced by anti-CD3 mAb + PMA or anti-CD28 mAb + PMA WO 00/45800 PCT/EPOO/00938 19 Highly purified T cells (10'/ml) were stimulated by immobilized anti-CD3 or anti-CD28 mAb in the presence of PMA. Anti-CD3 mAb (CLB-CD3; CLB, Amsterdam, The Netherlands) were fixed on the 96-microwell plates by 5 incubating the wells with 50 pl of mAb solution (1/800 dilution in culture medium). Anti-CD28 mAb (CLB-CD28; CLB, Amsterdam, The Netherlands) 50 pl (1/650 dilution in culture medium) was added directly to the wells. Further, 20 Al PMA (Sigma, St. Louis, MO, USA) solution (final 10 concentration: 0.5 ng/ml) was added. Subsequently, 20 yl of immunosuppressants were added by serial dilution in triplicate wells. Finally 100 pl of the T cell suspension (10'/ml) was added. After 48-hour incubation at 37 0 C in 5% CO2 20 pl BrdU (100 pM solution) (Cell Proliferation 15 Elisa, Boehringer-Mannheim Belgium) was added to each well. After a further overnight incubation the T cell proliferation was measured using a colorimetric immunoassay for qualification of cell proliferation based on measurements of the incorporation of BrdU during DNA 20 synthesis. The optical density (OD) was measured by a Behring EL311 plate reader at 450 nm (reference wavelength: 690 nm). The percent suppression of proliferation by drugs was counted using the formula: 25 Percent inhibition = (OD+drugs) - (OD Cult. Med.) ---------------------------- x 100 (OD-drugs) - (OD Cult. Med.) In vitro immunosuvpressive effect of Pteridine Derivati 30 ves as measured with the MLR and with tests involving polyclonal T cell proliferation induced by anti-CD3 mAb + PMA or anti-CD28 mAb + PMA (table II) - In the table II column II shows the IC50 values of the various substances in the MLR. The IC50 value 35 represents the lowest concentration of the substances that resulted in a 50% suppression of the MLR. - Column III shows the IC50 value of the various substances for the anti-CD3 mAb + PMA pathway and row IV the IC50 values of the various substances for the WO 00/45800 PCT/EP00/00938 20 anti-CD28 mAb + PMA pathway. - As a comparison the values of other immunosuppressants: CsA, FK506, Rapamycin, Leflunomide and Mycophenolic acid methatroxate (MTX) and 5 5 Fluoro-uracil (5-FU) in table III are given as well. Whole Blood Assay (WBA) WBA is a lymphoproliferation assay performed in vitro but using lymphocytes present in whole blood, taken from 10 animals that were previously given test substances in vivo. Hence it reflects the in vivo effect of substances as assessed with an in vitro read-out assay. Rats: inbred, male 6- to 8-weeks old R/A rats weighing ± 200 g were used as recipients. 15 Drug administration: Pteridine derivatives were dissolved in DMSO and further diluted with PBS. Products were given orally in different concentrations 2 times a day for 2 days. To perform the experiments, 6-8 hours after the last administration 1 ml of blood is taken by 20 heart puncture after ether anesthesia and anticoagulated with 100 U/ml of preservative free heparine. Whole Blood Assay: This assay was performed as we described previously [Use of the Methylxanthine Derivatives A802715 in Transplantation Immunology. II In 25 vitro Experiments. (Yuan Lin, et al., Transplantation 1997, 63, No. 12, 1734-1738)]. Heparinized whole blood was diluted (1:25) with complete RPMI medium and stimulated with 15 pg/ml of concanavalin A (Con A) in triplicate wells in 96-well 30 microtiter plates at 37 *C and 5*1 CO 2 . After 96-h culture, proliferation was determined by measuring the incorporation (cpm) of [ 3 H]-thymidine. The Con A induced proliferation of lymphocytes taken from rats receiving the test substances (exp) was 35 compared with that from rats receiving only the solvent (con). The percent suppression was calculated as follows: WO 00/45800 PCT/EP0O/00938 21 5 Results No. %suppress Akninistration Blood taken ion of drugs after: 28 57% 80 ng/kg/d 8 h 34 63% 2x/d 2d 8 h 40 ng/kg/d 2x/d 2d 10 First, most of the pteridine classes (I) according to the invention contain compounds with a clear suppressive effect in the MLR (mixed lymphocyte reaction). The MLR is considered as an in vitro analogue of the transplant rejection as it is based on the recognition of allogeneic 15 MHC (major histocompatibility antigens) on the stimulator leucotyes, by responding lymphocytes. Various established immunosuppressive drugs are known to suppress the MLR, and were also shown in this description. From these data it can be deduced that the 20 pteridine derivatives are effective in clinical situations where other immunosuppressants are active as well. These include the prevention and/or treatment of organ transplant rejection, the prevention and/or 25 treatment of both rejection and the occurrence of graft versus-host-disease after BM transplantation; the prevention and/or treatment of autoimmune diseases including diabetes mellitus, multiple sclerosis, glomerulonephritis, rheumatoid arthritis, psoriasis 30 systemic diseases such as vasculitis; scleroderma, polymyositis, autoimmune endocrine disorders (thyroiditis), ocular diseases (uveitis), inflammatory bowel diseases (Crohn's disease, colitis ulcerosa), autoimmune liver diseases (autoimmune hepatitis, primary WO 00/45800 PCT/EPOO/00938 22 biliary cirrhosis) autoimmune pneumonitis and auto-immune carditis. Whereas cyclosporine A and FK506 are only active in the anti-CD3 + PMA test, the pteridine derivatives 5 according to the invention were active, not only in the anti-CD3 + PMA but also in the anti-CD28 + PMA test. It has been shown that the latter is Ca-calmodulin resistant, and resistant to CsA and FK506. The anti-CD28 + PMA pathway has also been called the cosignal pathway 10 and is important to induce energy and even tolerance in T cells. Moreover, representative compounds have been found to be active in an whole blood assay. Under the term "organ" in the description is understood all organs or parts of organs (even several) 15 in mammals, in particular humans, for example kidney, heart, skin, liver, muscle, cornea, bone, bone marrow, lung, pancreas, intestine or stomach. After organ transplantation, rejection of the transplanted organ by the recipient occurs (host 20 versus-graft reaction). After bone marrow transplantation, also rejection of the host by the grafted cell may occur (graft-versus-host reaction). Rejection reactions mean all reactions of the recipient body or of the transplanted organ which in the end lead 25 to cell or tissue death in the transplanted organ or adversely affect the functional ability and viability of the transplanted organ or adversely affect the functional ability and viability of the transplanted organ or the recipient. In particular, this means acute and chronic 30 rejection reactions. Auto-immune disorders include, inter alia, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, pemphigus, atopic dermatitis, myositis, multiple sclerosis, nephrotic syndrome (in particular 35 glomerulonephritis), ulcerative colitis or juvenile diabetes. An additive or synergetic effect of pteridine derivatives and other immunosuppressants may be WO 00/45800 PCT/EP00/00938 23 anticipated. This may be especially, although not exclusively the case for combinations with CyA or FK 506 as the latter are not suppressive in the aCD28 pathway of T cell activation (table III) whereas most pteridine 5 derivatives are. The invention further relates to the use of cyclosporin A or FK506 or Rapamycine and at least one pteridine derivative according to the invention for the production of a pharmaceutical composition for inhibiting 10 the replication of viruses such as picorna-, toga-, bunya-, orthomyxo-, paramyxo-, rhabdo-, retro-, arena-, hepatitis B-, hepatitis C-, hepatitis D-, adeno-, vaccinia-, papilloma-, herpes-, varicella-zoster-virus or human immunodeficiency virus (HIV); or for treating of 15 cancer such as lung cancers, leukaemia, ovarian cancers, sarcoma, Kaposi's sarcoma, meningioma, colon cancers, lymp node tumors, glioblastoma multiforme, prostate cancers or skin carcinoses. The invention further relates to the use of 20 cyclosporin A or FK506 or rapamycin and at least one pteridine derivative of the general formula (I) for the production of a pharmaceutical composition for the treatment of human after organ transplantation or of (auto)immune disorders. 25 Hence, the advantage to associate pteridine with other immunosuppressants may be that, first, the therapeutic spectrum of action of the individual components is quantitatively and qualitatively broadened. Secondly that it allows, by means of a dose reduction 30 without reduced efficacy but with increased safety, that the treatment of immune disorders which were hitherto no indication for immunosuppressive therapy as a result of side effects may be considered. At the same time, the therapy costs can be decreased to an appreciable extent. 35 As a comparison, known pteridine derivatives are submitted to the same test conditions as the pteridine derivatives of the invention. These compounds and the results thereof are given in table IV and show no WO 00/45800 PCT/EPOO/00938 24 particular immunosuppressive activity. In all tables 0: concentration > 151 pM +: concentration range 16-150 pM 5 ++: concentration range 1-15 pM +++: concentration range < 1 PM The skilled person will appreciate that the preparation according to the invention may contain the 10 pteridine compounds over a broad content range depending on the contemplated use of the preparation. Generally, the content of the preparation is within the range of 0.01-50 wt.%, preferably within the range of 0.01-10 wt.%, more preferably within the range of 0.1-10 wt.%, 15 and most preferably within the range of 0.1-5 wt.%. Accordingly, the preparation may be used in a dosing regime which is suitable for most contemplated pharmaceutical utilities. The preparation according to the invention may be 20 used as such or in combination with any acceptable carrier material, excipient or diluent. The preparation according to the invention may be administared orally or in any other suitable fashion. Oral administration is preferred and the preparation may 25 have the form of a tablet, aqueous dispersion, dispersable powder or granule, emulsion, hard or soft capsule, syrup-, elixir or gel. The dosing forms may be prepared using any method known in the art for manufacturing these pharmaceutical compositions and may 30 comprise as additives sweeteners, flavoring agents, coloring agents, preservatives and the like. Carrier materials and excipients may include calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, binding 35 agents and the like. The preparation may be included in a gelatin capsule mixed with any inert solid diluent or carrier material, or has the form of a soft gelatin capsule, in which the ingredient is mixed with a water or WO 00/45800 PCT/EPOO/00938 25 oil medium. Aqueous dispersions may comprise the preparation in combination with a suspending agent, dispersing agent or wetting agent. Oil dispersions may comprise suspending agents such as a vegetable oil.

Claims (15)

1. Pharmaceutical composition for the treatment of autoimmuno disorders and/or for the treatment or prevention of transplant-rejections comprising a pteridine derivative of general formula: 5 0 N R 3 R1-N 0 N N R4 10 2 wherein: R and R 2 are independently hydrogen; aliphatic 15 saturated or unsaturated; straight or branched carbon chain with 1 to 7 carbon atoms; substituted or unsubstituted aryl or alkylaryl substituents, whereby the carbon atoms may be oxidized represented by alcohol or carbonyl function or carboxylic acids and their esters; 20 R 3 and R 4 are independently hydrogen, hydroxyl, halogen,alkyl, haloalkyl, alkoxy, wherein alkyl and the alkyl group may be branched cr straight and contains one or four carbon atoms, formyl and derivatives such as 25 hydroxylamino conjugates and acetals, cyano, carboxylic acids and carboxyl acid derivatives such as esters and amides, sulfhydryl, amino, alkylamino, cycloalkylamino, alkenylamino, alkynylamino, benzylamino, hydroxylalkylamino, morfolinoalkylamino, fenylhydrazino, 30 morfoline, piperidine, mercaptobenzyl, mercaptoalkyl, cysteinyl ester, styryl, substituted or unsubstituted aromatic ring; aromatic or heterocyclic substituent substituted with an aliphatic spacer between the WO 00/45800 PCT/EP0O/00938 27 pteridine ring and the aromatic substituent of 1 to 4 carbon atoms, whereby said spacer may contain an alcohol function, carbonyl function, halogen, ether, and may be saturated or unsaturated; branched or straight, saturated 5 or unsaturated aliphatic chain of 1 to 7 carbon atoms which may contain one or more functions chosen from the group comprising carbonyl, alcohol, ether, carboxyester, nitro, thioalkyl, halogen or a pharmaceutically acceptable salt thereof; and 10 X and Y are independently oxygen or sulfur or a pharmaceutical salt thereof, and a pharmaceutically acceptable carrier. 15

2. Pharmaceutical composition according to claim 1, wherein: RI and R2 are independently hydrogen, methyl, ethyl, propyl, isopropyl, cyclopropyl allyl and other 20 alifatic and alicyclic saturated and unsaturated substituents with 1 to 5 carbon atoms;

3. Pharmaceutical composition according to claim 1-2, 25 wherein: R and R are independently hydrogen; hydroxyl; halogen; alkoxy whereby the alkyl group being one to four carbon atoms; cyano; carboxyl acids and carboxyl acid 30 derivatives such as esters and amides; sulfhydryl; substituted or unsubstituted aromatic or heterocyclic ring having as substituents one or more of following functions chosen from the group comprising: halogen, alkoxy, hydroxy, amino, alcohol, ether, nitro, alkyl, 35 alkenyl, haloalkyl and haloalkenyl, cyano, carboxyl acids and derivatives; aromatic or heterocyclic substituent with an aliphatic spacer between the pteridine ring and the aromatic substituent of 1 to 4 carbon atoms, this WO 00/45800 PCT/EPOO/00938 28 spacer may contain an alcohol function, carbonyl function, carboxyl, nitro, halgen, ether and may be saturated or unsaturated, branched or unbranched; branched or straight, saturated or unsaturated aliphatic 5 chain of 1 to 7 carbon atoms which may contain one or more of following functions chosen from the group comprising: carbonyl, alcohol, ether, acetal, amino, imino, amino acid, carboxylester, cyano, nitro, thioalkyl, halogen; or a pharmaceutically acceptable salt 10 thereof.

4. Pharmaceutical composition according to claim 1-3, wherein: 15 R. and R 2 are methyl.

5. Pharmaceutical composition according to claim 4, wherein R 4 is hydrogen.

6. Pharmaceutical composition according to claim 5, wherein R 2 is haloethyl or haloethylene substituted by 20 phenyl or carboxylic acid ester; or is ethyl or ethylene substituted by nitro.

7. Pharmaceutical composition according to claim 4, wherein R 3 is hydrogen.

8. Pharmaceutical composition according to claim 25 7, wherein R 4 is haloethyl or haloethylene substituted by phenyl or carboxylic acid ester; or is chloro.

9. Pharmaceutical composition according to claim 4, wherein R 3 is selected from cyano, carboxylic acid ester or dihalo methyl; and R 4 is selected from chloro, 30 amino C 4 -alkyl, aminophenyl, thiomethylphenyl, thiomethyl-carboxylic acid ester.

10. Pharmaceutical composition according to any of the previous claims 1-9, wherein the pteridine derivative is a compound selected from the group comprising: 35 - 1,3-Dimethyl-6-(E)-styryllumazine (1) - 1,3-Dimethyl-6-[(E)-2-(pyrid-3-yl)vinyl]lumazine (2) WO 00/45800 PCT/EPOO/00938 29 - 1,3-Dimethyl-6-[(E)-2-(pyrid-4-yl)vinyl lumazine (3) - 6-(1,2-Dibromo-2-phenylethyl)-1,3-dimethyllumazine (4) 5 - 1,3-Dimethyl-6-[(E)-4-(phenyl)butadienyl]lumazine (5) - 6-(1,2-Dibromo-2-(methoxycarbonyl)ethyl)-1,3 dimethyllumazine (6) - 6-(2-Bromo-2-methoxycarbonyl-ethenyl)-1,3 10 dimethyllumazine (7) - 6-[(2-Acetyl-2-ethoxycarbonyl)acetyl]-1,3 dimethyllumazine (8) - 6-[2,2-(Diethoxycarbonyl)acetyll-1,3 dimethyllumazine (9) 15 - 6-(1-Methoxy-2-methoxycarbonyl)ethenyl)-1,3 dimethyllumazine (10) - 1,3-Dimethyl-6-[(2-nitro)ethenylllumazine (11) - 6-[(1-Hydroxy-2-nitro)ethyl]-1,3-dimethyllumazine (12) 20 - 6-[(1-Ethylthio-2-nitro)ethyl]-1,3 dimethyllumazine (13) - 6-Hydroxymethyl-1,3-dimethyllumazine (14) - 1, 3 -Dimethyl-7-[(E)-2-(pyrid-2-yl)vinyl]lumazine (15) 25 - 1,3-Dimethyl-7-[(E)-2-(pyrid-3-yl)vinylllumazine (16) - 1,3-Dimethyl-7-[(E)-2-(pyrid-4-yl)vinyl]lumazine (17) - 1,3-Dimethyl-7-[(E)-4-(phenyl)butadienyl]lumazine 30 (18) - 7-[1,2-Dibromo-2-(methoxycarbonyl)ethyl)-1,3 dimethyllumazine (19) - 7-[(E)-2-methoxycarbonylethenyl]lumazine (20) - 7-(1,2-Dibromo-2-phenylethyl)-1,3-dimethyllumazine 35 (21) - 7-(1-Bromo-2-phenyl)ethenyl-1,3-dimethyllumazine (22) - 1,3-Dimethyl-6-(E)-styryllumazine (23) WO 00/45800 PCT/EPOO/00938 30 - 7-Benzoyl-1,3-dimethyllumazine (24) - 7-Chloro-1,3-dimethyllumazine (25) - 1,3-Dimethyl-7-mercaptolumazine (26) - 1,3-Dimethyl-6,7-diphenyllumazine (27) 5 - 1,3-Dimethyl-6-phenyl-7-mercaptolumazine (28) - 7-Methoxy-1,3-dimethyl-6-phenyllumazine (29) - 7-Chloro-1,3-dimethyl-6-phenyllumazine (30) - 6-Benzoyl-7,8-dihydro-1,3-dimethyl-7-(4 methoxyphenyl)lumazine (31a) 10 - 6-Benzoyl-1,3-dimethyl-7-(4-methoxyphenyl)lumazine (31) - 6-Benzoyl-7,8-dihydro-1,3-dimethyl-7 phenyllumazine (32a) - 6-Benzoyl-1,3-dimethyl-7-phenyllumazine (32) 15 - 7-Methoxy-1,3-dimethyl-6-styryllumazine (33) - 1-Methyl-6,7-diphenyllumazine (34) - 7-Hydroxy-3-methyl-6-phenyllumazine (35) - 7-Hydroxy-1,6-diphenyllumazine (36) - 7-Hydroxy-3,6-dimethyl-1-phenyllumazine (37) 20 - 7-Hydroxy-6-phenyl-1,3-di-n-propyllumazine (38).

11. Pharmaceutical composition containing a pteridine derivative according to claim 1-10 and a compound selected from the group comprising cyclosporine A, substituted xanthines, tacrolimus (FK506), Rapamycin 25 (RPM), Leflunomide, Mofetil, adrenocortical steriods, cytotoxic drugs and antibody compositions as a combined preparation for simultaneous, separate or sequential use in the treatment of auto-immuno disorders or of the prevention and/or treatment of transplant-rejection. 30

12. Method for treating auto-immuno disorders or transplant-rejections in a subject by administering an effective amount of a pharmaceutical composition of claims 1-11.

13. Compound having the formula: 35 WO 00/45800 PCT/EP00/00938 31 0 N R 3 R 1 -- N 5 0 N N R4 R2 wherein: R, R 2 , R 3 and R, are defined in claims 1-10. 10

14. Use of a compound as defined in claim 10 for the treatment of autoimmuno disorders and/or the treatment and/or prevention of transplant rejections.

15. Method for selecting potent immunosuppressive agents based on the determination of the three parameters 15 MLC, ACD 3 and ACD 28