AU780284B2

AU780284B2 - Immunosurpressive effects of pteridine derivatives

Info

Publication number: AU780284B2
Application number: AU24418/00A
Authority: AU
Inventors: Piet Andre Maurits Maria Herdewijn; Wolfgang Eugen Pfleiderer; Mark Jozef Albert Waer
Original assignee: 4 AZA Bioscience NV
Current assignee: 4-AZA BIOSCIENCE NV
Priority date: 1999-02-02
Filing date: 2000-02-02
Publication date: 2005-03-10
Anticipated expiration: 2020-02-02
Also published as: EP1187615A2; WO2000045800A3; WO2000045800A2; CA2361561A1; JP2002536320A; AU2441800A

Description

IMMUNOSUPPRESSIVE EFFECTS OF PTERIDINE DERIVATIVES FIELD OF THE INVENTION The invention relates to a pharmaceutical composition for the treatment of autoimmuno disorders and/or the treatment or prevention of transplantrejections comprising pteridine derivatives.

The invention further relates to combined pharmaceutical preparations comprising one or more pteridine derivates and one or more known immunosuppressants, and to a group of novel pteridine derivates as such.

Further the invention is also related to a method for the treatment of autoimmuno disorders and/or of transplant-rejections.

BACKGROUND OF THE INVENTION Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Several pteridine derivates are known in nature and used in the preparation of medicines, for example as 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 tetrahydropteridine derivatives as NOsynthase inhibitors.

25 Both above-mentioned WO publications disclose also the use of these specific pteridine derivatives in the treatment of pathologically low blood pressure, in particular septic shock and combined with cytokines in tumor therapy and in transplant-rejection diseases.

S: Although some of these pteridine derivatives are claimed as potentially active 30 for the treatment of transplant-rejection diseases, direct evidence for their 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.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. It is an advantage of the invention in a preferred form to provide a pharmaceutical composition having high immunosuppressive activity. A further advantage of the invention in another preferred form is to provide a combined immunosuppressive preparation which causes a superadditive effect, comprising a pteridine derivative of the invention and other known immunosuppressants.

A further advantage of the invention in yet another preferred form is to 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, substituted xanthines, tacrolimus (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 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 vitro.

2 0 Recently (Lin Y. et al, Transplantation 63 (1997)) it has been found that the comedication of an immunosuppressive compound such as cyclosporine A (CyA) or •FK506 or RPM (rapamycine) with a methylxanthine derivative, in particular A802715 i (7-propyl-1(5-hydroxy-5-methylhexyl)-3-methylxanthine) leads to a superadditive oo "increase in the immunosuppressive action.

25 Likewise, other substituted, in particular substituted 8-phenylxanthines have been found to possess immunosuppressive effects in vitro (application EP 98.201323.7).

SUMMARY OF THE INVENTION The present invention relates in particular to the application of a group pteridine S derivatives and their pharmaceutical salts, possessing unexpectedly desirable pharmaceutical properties, i.e. are highly active immunosuppressive agents.

According to a first aspect of the present invention there is provided a pharmaceutical composition for the treatment of autoimmuno disorders and/or for the -3treatment or prevention of transplant-rejections comprising a pteridine derivative of general formula: 0 RiN N R 3 N0 N N R4 R2 wherein: RI and R 2 are independently selected from the group consisting of hydrogen; aliphatic saturated or unsaturated straight or branched carbon chain with 1 to 7 carbon atoms; unsubstituted aryl or alkylaryl substituents;

R

3 and R 4 are independently selected from the group consisting of hydrogen, halogen, haloalkyl, alkoxy, wherein the alkyl group may be branched or straight and contains one to four carbon atoms, formyl, sulfhydryl, hydroxylamino, acetals, cyano, carboxylic acids, carboxylic acid amides, amino, alkylamino, cycloalkylamino, 15 alkenylamino, alkynylamino, benzylamino, hydroxylalkylamino, morpholinoalkylamino, ***phenylhydrazino, morpholino, piperidino, mercaptobenzyl, mercaptoalkyl, cysteinyl ester, styryl; substituted aromatic ring having as substituents one or more functions chosen from the group comprising halogen, alkoxy, hydroxy, amino, alcohol, ether, nitro, alkyl, alkenyl, haloalkyl and haloalkenyl, cyano, carboxylic acids and derivatives; aromatic or heterocyclic substituent substituted with an aliphatic spacer between the 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 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 and halogen; under the proviso that R 3 and R 4 are not both H and under the proviso that Ri and R 2 are not both H; or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

-4- According to a second aspect there is provided a method for treating autoimmuno disorders or transplant-rejections in a subject by administering an effective amount of a pharmaceutical composition according to the first aspect.

According to a third aspect there is provided a pteridine derivative having the formula: 0 R N R 3 O N N R4

I

R2 wherein: SRI, R 2

R

3 and R 4 are as defined according to the first aspect.

According to a fourth aspect there is provided the use of a pteridine S• "derivative according to the first aspect in the manufacture of a medicament for the treatment of autoimmuno disorders and/or the treatment and/or prevention of transplant rejections.

15According to a fifth aspect there is provided a method for selecting potent immunosuppressive agents from pteridine derivatives having the formula: 0

O

R1.N N R 3 O 'N N R4

I

R2 wherein: RI, R 2

R

3 and R 4 are as defined according to the first aspect by a combination of at least three test systems based on MLC, ACD 3 and ACD 28 The invention demonstrates the 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 as described above.

4a Preferred pteridine derivatives comprising compositions are defined in the claims.

The invention further relates to a combined preparation having synergetic effects containing 1) cyclosporine A, substituted xanthines, tacrolimus (FK506), Rapamycin (RPM), Leflunomide, Mofetil, adrenocortical steriods, cytotoxic drugs and antibody compositions and 2) at least one pteridine derivative of formula defined above, and optionally a pharmaceutical excipient, for simultaneous, separate or 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 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 immunosuppressive agents based on the determination of the three parameters MLC, ACD 3 and ACD 2 8 Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

Although the invention will be described with reference to specific examples it will be appreciated by those skilled in the art that the invention may be embodied in many other 25 forms.

DESCRIPTION OF THE INVENTION S EXAMPLES 30 Hereunder the effects of the pteridine derivatives on the lymphocyte activation *g F" are elucidated and are compared with standard reference compounds (see table I, compound 4, 6, 7, 11, 13, 19, 20, 21, 22, 25, 26, 28, 30, 34, Table I (see Figures) summarizes the tested compounds. These pteridine derivatives were obtained as follows: WO 00/45800 PCT/EP00/00938 6-Bromomethyl-l,3-dimethyllumazine: To a solution of 1,3,6-trimethyllumazine (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 heated under reflux for 1 hour. After cooling was evaporated, the residue dissolved in CHC1 3 (100 ml), washed with H 2 O (3 x 70 ml), the organic layer dried over Na 2 SO and again evaporated. The residue was purified by silica gel column chromatography starting with toluene/EtOAc 9/1 to elute first 6-dibromomethyl-l,3dimethyllumazine (1.49 g, 41%) and followed by toluene/EtOAc 4/1 to get 6-bromomethyl-1.3dimethyllumazine. Yield: 1.2 g M.p. 228 0

C

(decomp.). UV (MeOH) 244 [264 337 (3.86).

7-Bromomethyl-1,3-dimethyllumazine: Analogous to the preceding procedure with 1,3,7trimethyllumazine (2.06 g, 0.01 moles) and bromine (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-l,3-dimethyllumazine (2.07 g, 57%) and second 7-bromomethyl-l,3-dimethyllumazine.

Yield: 0.97 g M.p.165-166 0 C. UV (MeOH): 241 338 (4.02).

1,3-Dimethyllumazin-6-ylmethyl-triphenylphosphonium bromide: To a suspension of 6-bromomethyl-l,3-dimethyllumazine (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 of a colorless powder of m.p.

2890C. UV (MeOH): 204 227 [243 [262 338 (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 heating under reflux for 1 day. Yield: 1.86 g M.p.

261 0 C. UV (MeOH) 204 [221 342 (4.09); 414 (4.38).

General synthesis of 1,3-dimethyl-6-(E)-styryllumazines 1, 2, 3, To a solution of 1,3-dimethyllumazin-6-ylmethyltriphenylphosphonium bromide and 1,3-dimethyllumazin-7ylmethyl-triphenylphosphonium bromide (0.547 g, 1 mmole), respectively, in MeOH (5 ml) was added sodium methoxide (0.108 g, 2 mmoles) and stirred at room temperature for 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 give a yellowish powder.

1,3-Dimethyl-6-(E)-styryllumazine According to the general procedure with benzaldehyde (0.16 Yield: 0.124 g M.p. 238 0 C. UV (MeOH): [220 308 372 (4.03).

1,3-Dimethyl-6- (E)-2-(pyrid-3-yl)vinyl]lumazine According to the general procedure with pyridine-3carboxaldehyde (0.162 Yield: 0.195 g M.p.

210 0 C. UV (MeOH) [236 308 370 (3.97).

1,3-Dimethyl-6- (pyrid-4-yl) vinyl] lumazine According to the general procedure with pyridine-4carboxaldehyde (0.162 Yield: 0.156 g M.p.

262 0 C. UV (MeOH) 202 [238 307 (4.51); 370 (4.20).

6-(1,2-Dibromo-2-phenylethyl) -1,3-dimethyllumazine 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, (20 ml) was added bromine (0.8 g, 5 mmoles) dissolved in CHC1, (5 ml) and then the mixture stirred at room temperature for 4 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: 1.067 g M.p. 176 0 C. UV (MeOH): 245 [260 341 (3.83).

1,3-Dimethyl-6-[(E)-4-(phenyl)butadienyl]lumazine According to the general procedure with cinnamaldehyde (0.2 Yield: 0.138 g M.p. 252 0 C (decomp.). UV (MeOH) 228 [244 330 389 (4.23).

6-[(E)-2-methoxycarbonylethenyl]lumazine: To a suspension of methoxycarbonylmethyltriphenylphosphonium bromide (0.415 g, 1 mmole) in dioxane (3 ml) was added DBU (0.23 g, 1.5 mmoles) and stirred at room temperature for 30 min. Then 1,3dimethyllumazine-6-carboxaldehyde (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 M.p. 211-213 0 C (decomp.). UV (MeOH): 202 [256 286 348 (4.08).

6-(1,2-Dibromo-2-(methoxycarbonyl)ethyl)-1,3dimethyllumazine To a solution of 6-[(E)-2-methoxycarbonylethenyl]lumazine (0.7 g, 2.53 mmoles) in CHC1, (20 ml) was added bromine (0.64 g, 4 mmoles) dissolved in CHC1 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 to give a colorless precipitate. The solid was collected, washed with MeOH and dried in a vacuum desiccator. Yield: 0.97 g M.p. 163°C. UV (MeOH): 247 [260 339 (3.88).

WO 00/45800 PCT/EP00/00938 8 6-(2-Bromo-2-methoxycarbonyl-ethenyl)-1,3dimethyllumazine To a solution of 6 (0.1 g, 0.23 mmoles) in dioxane ml) was added DBU (70 MG, 0.43 g, 0.43 mmoles) and then stirred at room temperatur for 2 hours. It was diluted with ethyl acetate (100 ml), washed with H 2 (3 x 50 ml), the organic layer separated, dried over Na 2

SO

4 and then evaporated. The residue was treated with MeOH, the solid collected and purified by recrystallization from DMF to give a yellowish powder. Yield: 0.055 g M.p.

2040C. UV (MeOH): [254 285 360 (4.01).

6-Chlorocarbonyl-1,3-dimethyllumazine: A suspension of 1,3-dimethyllumazine-6-carboxylic acid (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.

Yield: 3.13 g M.p. 262-264°C. UV (dioxane): 256 [280 333 (4.03).

6-[(2-Acetyl-2-ethoxycarbonyl)acetyl]-1,3dimethyllumazine A solution of ethyl acetoacetate ethoxy-magnesium salt (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 and the residue treated with cold 1 N HCl (20 ml, 0-50C).

The precipitate was collected, washed with H.O and dried in a vacuum desiccator. Purification was achieved by column chromatography (silica gel 3.5 x 12 cm) with CHC1/MeOH 95/5 and the first main fraction collected.

After evaporation was recrystallized from toluene (12 ml) to give colorless crystals. Yield: 0.247 g M.p.

153-1560C. UV (pH 251 293 330 (4.11).

WO 00/45800 PCT/EPO/00938 9 6-[2,2-(Diethoxycarbonyl)acetyl]-1,3-dimethyllumazine To a solution of ethylmalonate ethoxy-magnesium salt [41 (0.685 g, 3 mmoles) in THF (12 ml) was added 6chlorocarbonyl-1,3-dimethyllumazine (0.51 g, 2 mmoles) and then the mixture stirred at room temperature for hours. It was evaporated, the residue treated with 1 N HC1 (20 ml) and the resulting solid collected.

Recyrstallization from EtOH (40 ml) gave yellowish crystals. Yield: 0.585 g M.p. 124-1260C. UV (pH 253 (4.05) 291 332 (4.04).

6-(l-Methoxy-2-methoxycarbonyl)ethenyl)-1,3dimethyllumazine 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 min. Then NH 4 Cl (0.1 g) and HO0 (10 ml) were added and the mixture extracted with CHC1, (2 x 50 ml). The organic layer was dried over Na SO 4 evaporated and the residue crystallized from CHCl/n-hexane. Yield: 0.085 g M.p. 160 0 C. UV (MeOH): 204 245 288 350 (3.99).

1,3-Dimethyl-6- [(2-nitro)ethenyl]lumazine (11): A solution of 12 (0.562 g, 2 mmoles) in pyridine (10 ml) was cooled to 0°C and then acetic anhydride ml) dropwise added. Cooling was removed and the mixture stirred at room temperature for 3 hours. The resulting precipitate was collected, washed with H,0 and dried in a vacuum desiccator to give a chromatographically pure product. Yield: 0.515 g Crystallization from CHC1 3 M.p. 232-234 0 C. UV (MeOH): [239 309 365 (3.95).

(l-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/EP00/00938 added 6-formyl-1,3-dimethyllumazine (2.0 g, 9 mmoles) and then the mixture stirred at room temperature for hours. The precipitate was collected, washed with MeOH and ether and dried. Yield: 2.22 g Recrystallization from CHC1,. M.p. 166-1670C. UV (MeOH): 240 336 [347 6-[(1-Ethylthio-2-nitro)ethyl]-1,3-dimethyllumazine (13): To a suspension of compound 11 (0.263 g, 1 mmole) in MeOH (5 ml) and HO 2 (5 ml) was added ethylmercaptane (0.093 g, 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 M.p.

880C. UV (MeOH) 203 240 [262 341 (3.89).

6-Hydroxymethyl-1,3-dimethyllumazine (14) 1,3-Dimethyl-7- [(E)-2-(pyrid-2-yl)vinyl]lumazine According to the general procedure with pyridine-2carboxaldehyde (0.162 Yield: 0.233 g M.p. 282- 283 0 C. UV (MeOH): 203 238 312 375 (4.36).

1,3-Dimethyl-7- [(E)-2-(pyrid-3-yl)vinyl]lumazine (16): According to the general procedure with pyridine-3carboxaldehyde (0.162 Yield: 0.195 g M.p. 264- 2650C. UV (MeOH): 208 234 [274 307 375 (4.48).

1,3-Dimethyl-7-[(E)-2-(pyrid-4-yl)vinyl]lumazine (17): According to the general procedure with pyridine-4carboxaldehyde (0.162 Yield: 0.215 g M.p. 307- 3100C. UV (MeOH): 207 229 282 (3.79); [296 372 (4.00).

1,3-Dimethyl-7-[(E)-4-(phenyl)butadienyl]lumazine (18): WO 00/45800 PCT/EPOO/00938 11 According to the general procedure with cinnamaldehyde (0.2 Yield: 0.195 g M.p. 277-287 0 C (decomp.).

UV (MeOH): 239 299 402 (4.15).

7-[1,2-Dibromo-2- (methoxycarbonyl)ethyl)-1,3dimethyllumazine (19): To a suspension of 7-[(E)-2-methoxycarbonylethenyl]lumazine (1.79 g, 6.5 mmoles) in CHC1, (70 ml) was added bromine (0.7 g, 14 mmoles) dissolved in CHC1, (10 ml) and 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 Crystallization from EtOAc/n-hexane. M.p. 144-1450C. UV (MeOH) 240 (4.14); 343 (3.90).

7-[(E)-2-methoxycarbonylethenyl]lumazine To a suspension of methoxycarbonylmethyltriphenylphosphonium 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 1,3dimethyllumazine-7-carboxaldehyde (0.2 g, 0.91 mmoles) was added and stirring continued for 20 hours.

The precipitate was collected, washed with MeOH and dried to give a colorless crystal powder. Recrystallization from DMF. Yield: 0.15 g M.p. 242-245 0 C (decomp.) UV (MeOH): 201 225 252 364 (4.11).

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, (5 ml) and 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: WO 00/45800 PCT/EP00/00938 12 1.08 g M.p. 187-188 0 C. UV (MeOH) 241 341 (4.06).

7-(l-Bromo-2-phenyl)ethenyl-1,3-dimethyllumazine (22): 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 Yellowish powder from DMF. M.p. 245-246 0 C. UV (MeOH): 243 372 (4.15).

7-Benzyl-1,3-dimethyllumazine: A solution of 5,6-diamino-1,3-dimethyluracil monohydrochloride (2.06 g, 0.01 mole) in HO (50 ml) was treated with benzylglyoxal (2.22 g, 0.015 moles) in EtOH (20 ml) and heated under reflux for 1 hour. It was diluted with H2O (50 ml) and then extracted with CHC1, x 100 ml). The organic layer was dried over Na2SO 4 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 M.p. 147-148 0 C. UV (MeOH): 238 332 (4.07).

1,3-Dimethyl-6-(E)-styryllumazine (23): According to the general procedure with benzaldehyde (0.16 Yield: 0.223 g M.p. 259-2600C. UV (MeOH): 203 237 379 (4.29).

7-Benzoyl-1,3-dimethyllumazine (24): A suspension of 7-benzyl-1,3-dimethyllumazine (0.56 g, 2 mmoled) in H 0 (30 ml) was treated with KMnO 4 (0.6 g) and heated under reflux for 30 min. After cooling was extracted with CHC1, (3 x 100 ml), the organic layer dried over Na SO filtered and evaporated to dryness.

Crystallization from dioxane/HO0. Yield: 0.5 g M.p.

190-191 0 C. UV (MeOH) 233 [255 347 (3.97).

WO 00/45800 PCT/EPOO/00938 13 7-Chloro-1,3-dimethyllumazine (25) 1,3-Dimethyl-7-mercaptolumazine (26) 1,3-Dimethyl-6,7-diphenyllumazine (27) 1,3-Dimethyl-6-phenyl-7-mercaptolumazine (28): A mixture of 7-hydroxy-1,3-dimethyl-6-phenyllumazine (2.84 g, 0.01 mole) and PS, (3.3 g) was heated in pyridine (75 ml) under reflux for 1 hour. After cooling was diluted with HO2 (50 ml) and after standing for several hours the yellow precipiptate (28-pyridinium salt, 3.3 g, The salt was dissolved in hot H O (100 ml) and acidified by HC1 to pH 0. The resulting yellow crystals were collected, washed and dried in the oven.

Yield: 2.22 g M.p. 145 0 C (decomp.). UV (MeOH): 203 (4.37) 227 [283 370 (4.05).

7-Methoxy-1,3-dimethyl-6-phenyllumazine (29): A solution 7-hydroxy-1,3-dimethyl-6-phenyllumazine (1.42 g, 0.005 moles) in 0.5 N NaOH (20 ml) and MeOH 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 g M.p. 194 0 C. UV (MeOH): 205 [240 281 343 (4.23).

7-Chloro-1,3-dimethyl-6-phenyllumazine A mixture of 7-hydroxy-1,3-dimethyl-6-phenyllumazine (2.84 g, 0.01 mole) and NH 4 Cl (1 g) was heated in POC1, 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,0, dried and then recrystallized from MeOH. Yield: 2.36 g M.p. 1800C. UV (MeOH): 204 249 273 350 (4.05).

WO 00/45800 PCT/EP00/00938 14 6-Benzoyl-7,8-dihydro-1,3-dimethyl-7-(4-methoxyphenyl)lumazine (31a): A solution of 6-benzoyl-l,3-dimethyllumazine (0.2 g, 0.68 mmoles) in dry 1,2-dichloroethane (20 ml) was treated with A1C1, (0.4 g, 3 mmoles) and freshly distilled anisol ml, 92 mmoles) at room temperature and stirred for 24 hours. Then ice (50 g) was added, the aquous phase extracted with CHCl (3 x 50 ml), the organic phase washed with 2%-NaHCO 3 solution (50 ml) and H,0 (50 ml), dried over Na 2 SO 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 M.p. 240-244 0 C (decomp.). UV (MeOH): 254 [270 406 (4.08).

6-Benzoyl-1,3-dimethyl-7-(4-methoxyphenyl)lumazine (31): A suspension of 6-benzoyl-7,8-dihydro-1,3-dimethyl-7-(4methoxyphenyl)lumazine (31a) (0.3 g, 0.74 mmoles) in dioxane (40 ml) was treated at room temperature with 1%- 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.

The residue was purified by silica gel chromatography with CHC 3 /MeOH The main fraction was collected, evaporated and the solid recrystallized from EtOAc with charcoal. Yield: 0.175 g M.p. 255-257 0 C. UV (MeOH) 253 367 (4.10).

6-Benzoyl-7,8-dihydro-l,3-dimethyl-7-phenyllumazine (32a): Analogous to procedure 31a from 6-benzoyl-1,3dimethyllumazine (0.2 g, 0.68 mmoles) and benzene ml). Yield: 0.21 g UV (MeOH): 254 407 (4.12).

6-Benzoyl-l,3-dimethyl-7-phenyllumazine (32): WO 00/45800 PCT/EP00/00938 Analogous to procedure 31 from 6-benzoyl-7,8-dihydro-1,3dimethyl-7-phenyllumazine (32a)(0.3 g, 0.78 mmoles).

Yield: 0.18 g M.p. 185-187 0 C. UV (MeOH): 252 [290 349 (4.16).

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 was filtered off, washed with MeOH and dried in a vacuum desiccator. Yield: 0.134 Crystallization from DMF.

M.p. 271-272 0 C. UV (MeOH) [232 306 375 (4.38).

1-Methyl-6,7-diphenyllumazine (34) 7-Hydroxy-3-methyl-6-phenyllumazine (35) 7-Hydroxy-1,6-diphenyllumazine (36).

To a suspension of [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 filtrate treated with ethyl phenylglyoxylate (2.5 g, 0.014 mmoles) by heating under reflux for 30 min. The warm solution was acidified by HC1 to pH 0 and the resulting precipitate collected after cooling.

Recrystallization from DMF. Yield: 2.59 M.p.

330 0 C. UV (MeOH) 204 [222 284 (4.17); 346 (4.25).

7-Hydroxy-3,6-dimethyl-l-phenyllumazine (37) [121: 7-Hydroxy-6-phenyl-1, 3-di-n-propyllumazine (38): A suspension of 5,6-diamino-l,3-di-n-propyluracil (1.13 g, 0.005 moles) in H 2 0 (30 ml), EtOH (5 ml) and AcOH (2 ml) was treated with ethyl phenylglyoxylate (1.25 g, WO 00/45800 PCT/EPOO/00938 16 0.007 tnrroles) and heated under ref lux for 30 min forming a brownish oil. After cooling was acidified by FIC1 to pH 0 whereby the oil solidified. Filtration and recrystallization from EtOH/H 2 0 gave yellowish needles.

Yield: 1.28 g M.p. 245 0 C. UiV (MeOH) 212 (4.30); [243 01)) 284 (4.02) 349 (4.19).

References Y. Kang, R. Soyka, W. Pfleiderer, J. HeterocVcl.

Chem.' 1987, 24, 597.

R. Eisele, K. Aritomo, W. Pfleiderer, Pteridines 1993, 4, 178.

M. Viscontini, K. Adank, Helv. Chim. Acta 1952, 1342.

R.E. Bowman, J. Chem. Soc. 1950, 324.

W. Pfleiderer, W. Hutzenlaub, Chem. Ber. 1973, 106, 3149.

H.D. Dakin, H.W. Dudley, J. Biol. Chem. 1914, ia8, 42.

H. Steppan, J. Hammer, R. Baur, R. Gottlieb, W.

Pfleiderer, Liebigs Ann. Chem. 1982, 2135.

Z. Kazimierczuk, W. pfleiferer, Chem. Ber. 1979, 112,~ 1499.

F.F. Blicke, H.C. Godt, J. Am. Chem. Soc. 1954, .76, 2798.

H. Fink, W. Pfleiderer,. Chem. Ber. 1963,.,96, 2950.

[11] J. Litschitz, Ber. Deut. Chem. Ges. 1922, 15, 1619.

[12) W. Hutzenlaub, H. Yamamoto, G.B. Barlin, W.

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 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 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 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.

CD3 assay: here there is an activation of the Tlymphocytes 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 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 Tlymphocyte goes also via an exogenously added antibody 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.

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-164 0

FCS.

Mixed Lymphocvte Reaction 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 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 37 0 C, 1 ACi 3 H]-thymidine was added to each cup. After a further 16 hours incubation, cells were harvested and counted in a S-counter.

The percent suppression of proliferation by drugs was counted using the formula: Percent inhibition (cpm+drugs) cpm Cult. Med x 100 (cpm-drugs) cpm Cult. Med T cell purification T cells were purified by removing non-T cells.

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 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.

Measurements of T cell proliferations induced by anti-CD3 mAb PMA or anti-CD28 mAb PMA WO 00/45800 PCT/EP00/00938 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 incubating the wells with 50 gl 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, l PMA (Sigma, St. Louis, MO, USA) solution (final concentration: 0.5 ng/ml) was added. Subsequently, 20 p 1 of immunosuppressants were added by serial dilution in triplicate wells. Finally 100 1l of the T cell suspension 6 /ml) was added. After 48-hour incubation at 37 0 C in

CO

2 20 il BrdU (100 pM solution) (Cell Proliferation 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 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: Percent inhibition (OD+drugs) (OD Cult. Med.) x 100 (OD-drugs) (OD Cult. Med.) In vitro immunosuppressive effect of Pteridine Derivatiyes as measured with the MLR and with tests involving polvclonal 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 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 anti-CD28 mAb PMA pathway.

As a comparison the values of other immunosuppressants: CsA, FK506, Rapamycin, Leflunomide and Mycophenolic acid methatroxate (MTX) and 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 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.

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 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 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 g/ml of concanavalin A (Con A) in triplicate wells in 96-well microtiter plates at 37 OC and 5% CO 2 After 96-h culture, proliferation was determined by measuring the incorporation (cpm) of ['H]-thymidine.

The Con A induced proliferation of lymphocytes taken from rats receiving the test substances (exp) was compared with that from rats receiving only the solvent (con). The percent suppression was calculated as follows: WO 00/45800 21 Results No. %suppress Adinistration Blood taken ion of drugs after: 28 57% 80 ng/kg/d 8 h 34 63% 2x/d 2d 8 h ng/kg/d 2x/d 2d PCT/EP00/00938 First, most of the pteridine classes 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 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 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 treatment of both rejection and the occurrence of graftversus-host-disease after BM transplantation; the prevention and/or treatment of autoimmune diseases including diabetes mellitus, multiple sclerosis, glomerulonephritis, rheumatoid arthritis, psoriasis 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 PCTEPO/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 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 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) 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 (hostversus-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 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 rejection reactions.

Auto-immune disorders include, inter alia, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, pemphigus, atopic dermatitis, myositis, multiple sclerosis, nephrotic syndrome (in particular glomerulonephritis), ulcerative colitis or juvenile diabetes.

An additive or synergetic effect of pteridine derivatives and other immunosuppressants may be WO 00/45800 PCT/EPOO/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 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 the replication of viruses such as picorna-, toga-, bunya-, orthomyxo-, paramyxo-, rhabdo-, retro-, arena-, hepatitis hepatitis hepatitis adeno-, vaccinia-, papilloma-, herpes-, varicella-zoster-virus or human immunodeficiency virus (HIV); or for treating of 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 cyclosporin A or FK506 or rapamycin and at least one pteridine derivative of the general formula for the production of a pharmaceutical composition for the treatment of human after organ transplantation or of (auto)immune disorders.

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 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.

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 AM concentration range 16-150 AM concentration range 1-15 AM concentration range 1 AM The skilled person will appreciate that the preparation according to the invention may contain the 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 preferably within the range of 0.01-10 more preferably within the range of 0.1-10 wt.%, 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 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 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 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 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/EP00/00938 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

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: 0 R Nl.N N R3 O N N R4 I R2 wherein: RI and R 2 are independently selected from the group consisting of hydrogen; aliphatic saturated or unsaturated straight or branched carbon chain with 1 to 7 carbon atoms; unsubstituted aryl or alkylaryl substituents; R 3 and R 4 are independently selected from the group consisting of hydrogen, halogen, haloalkyl, alkoxy, wherein the alkyl group may be branched or straight and contains one to four carbon atoms, formyl, sulfhydryl, hydroxylamino, acetals, cyano, carboxylic acids, carboxylic acid amides, amino, alkylamino, cycloalkylamino, alkenylamino, alkynylamino, benzylamino, hydroxylalkylamino, "morpholinoalkylamino, phenylhydrazino, morpholino, piperidino, mercaptobenzyl, S mercaptoalkyl, cysteinyl ester, styryl; substituted aromatic ring having as substituents one or more functions chosen from the group comprising halogen, alkoxy, hydroxy, amino, alcohol, ether, nitro, alkyl, alkenyl, haloalkyl and haloalkenyl, cyano, carboxylic acids and derivatives; aromatic or heterocyclic substituent substituted with an aliphatic spacer between the 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 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 and halogen; under the proviso that R 3 and R 4 are not both H and under the proviso that Ri and R 2 are not both H; or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. -27-

2. Pharmaceutical composition according to claim 1, wherein R 1 and R 2 are independently selected from the group consisting of hydrogen, methyl, ethyl, propyl, isopropyl, cyclopropyl allyl.

3. Pharmaceutical composition according to any one of the preceding claims, wherein R 1 and R 2 are methyl.

4. Pharmaceutical composition according to claim 3, wherein R 4 is hydrogen. Pharmaceutical composition according to claim 4, wherein R 3 is haloethyl or haloethylene substituted by phenyl or carboxylic acid ester; or is ethyl or ethylene substituted by nitro.

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

7. Pharmaceutical composition according to claim 6, wherein R4 is haloethyl or haloethylene substituted by phenyl or carboxylic acid ester; or is chloro. se.; 8. Pharmaceutical composition according to claim 3, wherein R 3 is selected from cyano, carboxylic acid ester or dihalo methyl; and R 4 is selected from chloro, amino C 4 -alkyl, aminophenyl, thiomethylphenyl, thiomethyl-carboxylic acid ester.

9. Pharmaceutical composition according to any one of the preceding claims, wherein the pteridine derivative is a compound selected from the group comprising: 1,3-Dimethyl-6-(E)-styryllumazine (1) 1,3-Dimethyl-6-[(E)-2-(pyrid-3-yl)vinyl]lumazine (2) 1,3-Dimethyl-6-[(E)-2-(pyrid-4-yl)vinyl]lumazine (3) 6-(1,2-Dibromo-2-phenylethyl)-1,3-dimethyllumazine (4) 1,3-Dimethyl-6-[(E)-4-(phenyl)butadienyl]lumazine 6-(1,2-Dibromo-2-(methoxycarbonyl)ethyl)-1,3-dimethyllumazine (6) 6-(2-Bromo-2-methoxycarbonyl-ethenyl)-1,3-dimethyllumazine (7) 6-[(2-Acetyl-2-ethoxycarbonyl)acetyl] -1,3-dimethyllumazine (8) 28 6- [2,2-(Diethoxycarbonyl)acetyl] -1 ,3-dimethyllumazine (9) 1-Methoxy-2-methoxycarbonyl)ethenyl)- 1,3 -dimethyllumazine 1,3-Dimethyl-6-[(2-nitro)ethenyl]lumazine (11) 6- -Hydroxy-2-nitro)ethyl]-1I,3-dimethyllumazine (12) 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 1 ,3-Dimethyl-7- [(E)-2-(pyrid-3 -yl)vinyl] lumazine (16) 1,3 -Dimethyl-7- [(E)-2-(pyrid-4-yl)vinyl] lumazine (17) 1 ,3 -Dimethyl-7- [(E)-4-(phenyl)butadienyl] lumazine (18) 7-[1 ,2-Dibromo-2-(methoxycarbonyl)ethyl)- 1,3 -dimethyllumazine (19) 7- [(E)-2-methoxycarbonylethenyl]- 1,3-dimethyllumazine 7-(1 ,2-Dibromo-2-phenylethyl)-1 ,3-dimethyllumazine (21) I-Bromo-2-phenyl)ethenyl- 1,3 -dimethyllumazine (22) I ,3-Dimethyl-6-(E)-styryllumazine (23) 7-Benzoyl- 1,3 -dimethyllumazine (24) -7-Chioro- 1,3-dimethyllumazine 1 ,3-Dimethyl-7-mercaptolumazine (26) 6-Benzoyl-7,8-dihydro- 1,3 -dimethyl-7-(4-methoxyphenyl)lumazine (3 1 a) 6-Benzoyl- 1 ,3-dimethyl-7-(4-methoxyphenyl)lumazine (3 1) 6-Benzoyl-7,8-dihydro- 1,3 -dimethyl-7-phenyllumazine (3 2a) 6-Benzoyl-1 ,3-dimethyl-7-phenyllumazine (32) and 7-Methoxy- 1,3 -dimethyl-6-styryllumazine (33). Pharmaceutical composition for the treatment of auto-immune disorders and/or for the treatment or prevention of transplant-rejections comprising a pteridine derivative selected from the group comprising: 1 ,3-Dimethyl-6,7-diphenyllumazine (27) 1 ,3-Dimethyl-6-phenyl-7-mercaptolumazine (28) 7-Methoxy- 1,3 -dimethyl-6-phenyllumazine (29) 7-Chioro- 1,3-dimethyl-6-phenyllumazine (30) and I -Methyl-6,7-diphenyllumazine (34). -29-

11. Pharmaceutical composition containing a pteridine derivative according to any one of the preceding claims and a compound selected from the group comprising cyclosporine A, tacrolimus (FK506), Rapamycin (RPM), Leflunomide, Mofetil, and cytotoxic drugs as a combined preparation for simultaneous or sequential use in the treatment of auto-immuno disorders or of the prevention and/or treatment of transplant-rejection.

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

13. A pteridine derivative having the formula: O *0 R 1 N R3 N N N R4 R2 wherein: 15 RI, R 2 R 3 and R4 are as defined in claim 1. Use of a pteridine derivative as defined according to any one of claims 1 to 10 in the manufacture of a medicament for the treatment of autoimmuno disorders and/or the treatment and/or prevention of transplant rejections. Method for selecting potent immunosuppressive agents from pteridine derivatives according to claim 13 by a combination of at least three test systems based on MLC, ACD 3 and ACD 28

16. A pharmaceutical composition substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying drawings and/or examples. A method for treating autoimmune disorders and/or transplant rejections substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying drawings and/or examples.

18. Use of a pteridine compound substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying drawings and/or examples.

19. A pteridine derivative substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying drawings and/or examples. DATED this 7th day of January, 2005 Shelston IP S. 15 Attorneys for 4-AZA Bioscience nv V. 0@ V