Page  95 Nucleosides. Part LXVI.1 Syntheses and properties of pterin ribonucleosides Werner Pfadler and Wolfgang Pfleiderer* Fachbereich Chemie, Universität Konstanz, D-78457 Konstanz, Germany E-mail: [email protected] Dedicated to Professor Harri Lönnberg on the occasion of his 60th birthday Abstract Several pterin derivatives (1-8) have been ribosylated in form of their trimethylsilyl derivatives (9) with 1-bromo-(10) and 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose (11) under the catalysis of HgO/HgBr2, BF3-etherate and trimethylsilyl triflate, respectively. Mixtures of the N-1- (19-25) and N-3-ribofuranosides (12-18) which are difficult to be separated were obtained. Debenzoylation by the Zemplen method led to the free pterin-nucleosides (28-30). A second approach starting from 2-methylthio-4(3H)pteridinones (31-33) gave again mixtures of the N-1-(35-37) and N-3-ribonucleosides (38-40). The 2-methylthio function in 35-37 can easily be substituted by various amines leading after subsequently debenzoylation to the N-2- substituted pterin-ribonucleosides (41-50). The structural assignments were based on comparisons of the UV spectra with the corresponding N-methyl substituted model substances. 1H-NMR-spectra functioned as additional structural proof. Keywords: Pterin ribosylations, silyl methods, UV comparisons, pK -determinations Introduction The synthesis of pteridine nucleosides has been a major subject in our laboratory for many years. Lumazine2-11 and isopterin nucleosides12 can be regarded as structural analogs of the pyrimidine nucleosides whereas the many pteridin-7-one N8-nucleosides13-23 are structurally related to the purine nucleosides. The syntheses could be achieved either by a classical Hilbert-Johnson reaction24, the mercury salt method by Fox and Davoll25, the Hilbert-Johnson-Birkofer silyl procedure26, 27 or the silyl variant by Vorbrüggen28. Pterin (2-amino-4(3H)pteridone) (1) , the basic molecule of most naturally occurring pteridine derivatives, has so far not been included in our investigations. Thin layer chromatographic analysis of the reaction mixture obtained from preliminary experiments with 1 suggested that the reaction is not straightforward; formation of a complex mixture of several reactions products was thereby indicated.

Page  96 Synthesis Starting with 6,7-diphenylpterin (2) silylation with hexamethyldisilazane took 6 days till all starting material had dissolved to form 2-trimethylsilylamino-4-trimethylsilyloxypteridine (9) which was first treated with 1-bromo-2,3,5-tri-O-benzoyl-D-ribofuranose (10) in presence of HgO and HgBr2 in analogy to the conditions of Wittenburg29. After a very tedious chromatographic separation by column, low-pressure and preparative thick layer chromatography three compounds 2-amino-6,7-diphenyl-3-(-(2,3,5-tri-O-benzoyl-ß-D-ribo- furanosyl)-4(3H)-pteridone (12), the corresponding N1-riboside (19) and the 2-imino-6,7- diphenyl-N1,O4-bis-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-1,2-dihydropteridine (26) could be isolated in low yields. An analogous reaction with 1-O-acetyl-2,3,5-tri-O-benzoyl-ß-D- ribofuranose (11) and BF3- etherate as a catalyst gave predominately 2-amino-6,7-diphenyl-1- (2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-4(3H)pteridone (19) in 50% yield whereas the isomeric 12 was obtained in only 1% yield. NNNNOH2NRHR11 H H2 C6H5 C6H53 CH3 CH34 CH3 H5 H CH36 C6H5 H7 H C6H58 H C(CH3)3RR1OBzOOBzBzOXHgO HgBr2NNNNOH2NRR1OOBzBzOBzONNNNOH2NRR1OBzOBzOOBzNNNNHNRR1OBzOBzOOBzOOBzBzOBzO++ O10 Br11 OAcX+ 12 C6H5 C6H513 CH3 CH314 CH3 H15 H CH316 C6H5 H17 H C6H518 H C(CH3)3RR119 C6H5 C6H520 CH3 CH321 CH3 H22 C6H5 H23 H C6H524 H C(CH3)325 H HRR126 C6H5 C6H527 C6H5 HRR1NNNNNHRR1OSi(CH3)3(H3C)3SiHMDS or BF3or CF3SO3Si(CH3)39NNNNOH2NCH3CH3OOHHOHONNNNOH2NRR1OHOHOOH2829 CH330 C6H5RScheme 1.

Page  97 Analogously, BF3-catalysis of 7-phenylpterin (7) and 11 gave small amounts of the N1- (23) and N3-nucleoside (17). Similarly 6-phenylpterin (6) and 11 in presence of trimethyl-silyl trifluorosulfonate gave three components N1-(22), N3-monoriboside (16) and the N1,O4- diriboside (27) that were separated from the complex reaction mixture. Ribosylations of 6,7- dimethylpterin (3) led with the halosugar 10 and HgO/HgBr2 to 3-(2,3,5-tri-O-benzoyl-ß-D- ribofuranosyl)-6,7-dimethylpterin (13) in 14% yield whereas the use of 11 and BF3-catalysis formed the N1-riboside (20) in 14% as the main reaction product besides 8% of the N3-isomer (13). The ribosylation reaction have also been extended to 6-methylpterin (4) yielding with 11 and BF3 small amounts of the N1-(21) and N3-riboside (14), with 7-methyl-pterin (5) the N3- riboside ((15) in 6% yield and with 7-tert.butylpterin (8) again a mixture of N1- (24) and N3- riboside (18). A highly unpleasant reaction was encountered with pterin (1) itself which led after a tedious isolation and purification process only to 10% yield of the 1-(2,3,5.tri-O-benzoyl-ß-D- ribofuranosyl)pterin (25). Debenzoylations of the sugar protecting groups can be achieved by the Zemplen30 method as demonstrated with 13, 19 and 20, respectively, forming the free pterin- nucleosides 28-30. The encountered difficulties during the ribosylations of the pterin derivatives, in general, force us to search for a more convenient synthetic pathway to this class of pteridine nucleosides. The more soluble 2-methylthio-4(3H)pteridione (31) and its 6,7-dimethyl-(32) and 6,7-diphenyl- (33) derivatives have been chosen as the most likely candidates due to the fact that the methylthio group can be displaced by amino functions nucleophilicly. The ribosylations of 31, 32 and 33 via their O4-trimethylsilyl derivatives (34) with 11 and BF3 catalysis led in moderate to good yields in each case to a mixture of the corresponding N1-(35-37), and N3-ribosides (38- 40). Treatment of the 2-methylthio-1-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-4(3H)-pteridones 35-37 with a great variety of amines led under displacement of the methylthio group and subsequent cleavage of the benzoyl groups by sodium methoxide to the corresponding pterin-N1- ribofuranosides 41-51.

Page  98 NNNNOH3CSRHR31 H32 CH333 C6H5RNNNNOH3CSRROOBzBzOBzONNNNOH3CSRROBzOBzOOBz+ 35 H36 CH3 37 C6H5R38 H39 CH3 40 C6H5RNNNNH3CSRROSi(CH3)3HMDS34BF311NNNNOR1RROHOHOOH41 H NHCH342 CH3 NHCH343 CH3 NHC2H544 CH3 NHCH2CH2OH30 C6H5 NH245 C6H5 NHCH346 C6H5 NHC2H547 C6H5 NHCH2CH2OH48 C6H5 NHCH(CH3)249 C6H5 NHCH2CH2CH2CH350 C6H5 NHCH2CH(CH3)2RR1NNNNO(H3C)2NRROBzOBzOOBz51Scheme 2. Similar treatment of 37 with dimethylamine afforded first 2-dimethylamino-6,7-diphenyl-1- (2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-4(3H)-pteridone (51) but debenzoylation by the Zemplen method was not successful since sodium methoxide led to the cleavage of the glycosidic linkage forming 2-dimethylamino-6,7-diphenyl-4-(3H)-pteridone (66) (Fig. 1).

Page  99 80 min40 min20 min10 min5 min2 min1 min0 minNNNNO(H3C)2NC6H5C6H5OBzOOBzBzONNNNO(H3C)2NC6H5C6H5pH 125166 Figure 1. Cleavage of 51 at pH 12 to form 66. Structural assignment The site of attachment of the sugar moiety to the pterin nucleus can best be assigned by comparison of the UV spectra with those of the corresponding model substances 52-66 most of which are already described in literature. We have determined in several cases also the pKa values31 in order to compare the spectra of the cations and the neutral species as an additional structural proof (Tab. 1). NNNNOH3CSRRNNNNOH3CSRH3CRNNNNOH2NRRNNNNOH2NRH3CRCH3CH352 H53 CH354 C6H5R55 H56 CH357 C6H5R58 H59 CH360 C6H5R61 H62 CH363 C6H5RNNNNORC6H5C6H5CH364 NHCH365 N(CH3)2RNNNNO(H3C)2NC6H5HC6H566Scheme 3. Figure 2. UV spectral comparison of 30 and 54 as well as 12 and 57.

Page  100 Table 1. UV-data of pterin nucleosides and model substances -pterin pKa .max (nm) log e pH1,6,7-Trimethyl- (53)42 3.25 217 254 320 (333) 4.28 3.95 4.00 (3.92) 0 13.24 241 (315) 329 (342) 4.24 (3.92) 3.95 (3.98) 7 20 230 272 320 (335) 4.69 4.03 4.02 (3.93) MeOH1-§.D-Ribofuranosyl- 2.68 218 (250) 316 (330) 4.28 (3.95) 4.00 (3.91) 06,7-dimethyl- (29) 12.58 238 (310) 322 (335) 4.23 (3.96) 4.02 (3.96) 7 21 230 275 315 4.74 4.13 3.97 MeOH 24 230 274 (282) 312 4.60 3.84 (3.82) 3.93 MeOH 25 230 272 315 4.61 3.89 3.91 MeOH3,6,7-Trimethyl- (56)43 2.34 218 250 322 390 4.17 3.81 3.89 2.61 0 241 276 352 4.16 4.09 3.83 5 13 (222) 242 282 355 (4.28) 4.18 4.31 3.88 MeOH 14 227 275 360 4.68 4.20 3.67 MeOH 15 228 280 350 4.80 4.31 3.91 MeOH 18 230 278 348 4.70 4.20 3.84 MeOH 3-§-D-Ribofuranosyl- 2.14 218 255 322 390 4.11 4.00 3.83 2.98 06,7-dimethyl- (28) 247 273 352 4.35 4.74 3.76 71-Methyl-6,7-diphenyl- 2.95 222 280 362 4.42 4.13 4.15 0 (54)43 12.85 222 265 362 4.37 4.30 4.20 7 19 228 269 357 4.75 4.34 4.14 MeOH 51 228 273 363 4.81 4.37 4.20 MeOH1-§-D-Ribofuranosyl- 2.60 228 280 350 4.41 4.13 4.14 06,7-diphenyl- (30) 12.22 226 266 359 4.39 4.30 4.17 7 22 230 275 (283) 361 4.71 4.22 (4.20) 4.10 MeOH 23 228 273 (281) 345 4.76 4.12 (4.06) 4.28 MeOH3-Methyl-6,7-diphenyl- 2.34 229 278 362 4.46 4.13 4.18 0 (57)44 13.02 224 (250) 292 380 4.42 (4.27) 4.35 4.10 7 12 227 (250) 297 380 4.69 (4.35) 4.35 3.98 MeOH 16 230 305 374 4.75 4.41 3.93 MeOH 17 227 304 375 4.76 4.48 3.95 MeOH

Page  101 Table 2. UV-data of 2-methylthio-lumazine nucleosides and model substances - 2-methylthio-lumazine .max (nm) log e pH1-Methyl- (58)40 231 258 289 333 344 4.13 4.28 3.80 4.12 4.08 41,6,7-Trimethyl- (59)40 231 258 289 333 344 4.13 4.28 3.80 4.12 4.08 41-Methyl-6,7-diphenyl- (60)40 253 277 370 4.29 4.40 4.30 43-Methyl- (61)40 243 264 283 337 4.06 4.08 4.11 3.81 53,6,7-Trimethyl- (62)40 246 286 335 4.19 4.16 3.91 53-Methyl-6,7-diphenyl- (63)40 263 300 367 4.30 4.31 4.15 5-(2,3,5-tri.O-benzoyl-§-D- ribofuranosyl)lumazine2-Methylthio-1- (35) 229 (252) 282 323 (340) 4.68 (4.08) 3.98 4.05 (3.80) MeOH6,7-Dimethyl-2-methyl- 228 (255) 282 324 (340) 4.70 (4.10) 3.98 4.09 (4.00) MeOHthio-1- (36) 2-Methylthio-6.7- 229 275 366 4.78 4.43 4.24 MeOH diphenyl-1- (37) 2-Methyl-3- (38) 230 (275) 282 330 4.64 (4.15) 4.19 3.85 MeOH6,7-Dimethyl-2-methyl- 228 (274) 282 (293) 330 4.72 (4.19) 4.25 (4.18) 3.92 MeOHthio-3- (39) 2-Methylthio-6,7- 230 266 307 367 4.77 4.28 4.27 4.13 MeOHdiphenyl-3- (40)

Page  102 Table 3. UV-data of N2-substituted pterin nucleosides 1-ß-D-ribofuranosylpterin pKa .max (nm) log e pHN2-Methyl- (41) 1.34 210 239 280 324 4.25 4.11 3.68 3.86 0 12.97 210 239 324 4.30 4.11 3.86 7N2,6,7-Trimethyl- (42) 1.84 222 253 324 4.30 4.04 4.01 0 13.78 241 280 330 (343) 4.26 3.65 4.06 (4.02) 7N2-Ethyl-6,7-dimethyl- 1.87 222 (252) 322 (334) 4.28 (4.03) 3.99 (3.91) 0 (43) 14.22 242 280 331 (343) 4.27 3.68 4.05 (4.00) 5N2-§-Hydroxyethyl- 1.48 220 252 (293) 332 4.25 4.04 (3.68) 3.93 0 6,7-dimethyl- (44) 13.59 240 278 328 (342) 4.26 3.68 4.06 (4.01) 56,7-Diphenyl- (30) 2.60 228 280 350 4.41 4.13 4.14 0 12.22 226 266 359 4.39 4.30 4.17 7N2-Methyl-6,7- 1.55 233 280 364 4.43 4.18 4.18 0diphenyl- (45) 13,14 225 269 364 4.37 4.34 4.23 5N2-Ethyl-6,7-diphenyl- 1.88 236 279 364 4.43 4.17 4.17 0 (46) 13.76 270 364 4.37 4.23 5N2-Isopropyl-6,7- 1.69 233 280 364 4.43 4.18 4.18 0diphenyl- (47) 14.04 (225) 269 364 (4.46) 4.35 4.24 5N2-§-Hydroxyethyl- 1.27 235 280 364 4.43 4.18 4.17 06,7-diphenyl- (48) 12.88 (234) 270 364 (4.47) 4.34 4.24 7N2-n-Butyl-6,7- 1.58 236 279 364 4.44 4.19 4.19 0diphenyl. (49) 13.98 (226) 270 364 (4.45) 4.36 4.25 5N2-Isobutyl-6,7- 1.74 235 280 364 4.61 4.36 4.33 0diphenyl- (50) 14.05 270 365 4.51 4.47 5 The 1H-NMR spectra (experimental part) of the benzoyl protected ribonucleosides have not been very informative since overlapping signals make accurate assignments difficult. The free ß- D-ribofuranosylpterin nucleosides (28-30, 41-50) on the other hand showed well separated proton signals of the sugar moieties which are in good agreement with the pattern of the

Page  103 ribonucleosides, in general. The H-C(1') appears always as doublet at lowest field followed by the 5'-OH, 2'-OH, 3'OH, H-C(2'), H-C(3'), H-C(4') and H-C(5') towards higher fields. Experimental Section General Procedures. Products were dried under high vacuum. TLC: precoated cellulose thin- layer sheets F 1440b LS 254 and silica gel thin-layer sheets F 1500 LS 254 from Schleicher and Schüll. Preparative TLC: plates 20 x 20 x 0.2 cm with silica gel 60 PF 254 from Merck. Column chromatograhy (CC): silica gel 60, 70 - 230 mesh from Merck. Low pressure chromatography32 (LPC): LiChroprep Si 60 from Merck according to 33 under 8-10 atm.. Short column chromatography (SCC): silica gel 60 H from Merck. UV/VIS: Perkin-Elmer Lambda 5; max in nm (log ( ). 1H-NMR: Bruker AC 250; in CDCl3 or ((D6)DMSO), in ppm rel. to SiMe4 as internal standard. M.p.: Büchi apparatus, model Dr. Tottuli; no corrections. The pKa measurements were performed by the spectrophotometric method31. Products were dried under high vacuum. 2-Amino-3-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-6,7-diphenyl-4(3H)pteridinone (12), 2-Amino-1-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-6,7-diphenyl-4(3H)pteridinone (19) and 2-Imino-1,O4-bis-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)1,2-dihydropteridine (26). A mixture of 6,7-diphenylpterin (2)34 (0.945 g, 3 mmol) and (NH4)2SO4 (0.1 g) was heated in hexamethyldisilazane (HMDS) (15 ml) 6 days under reflux till a clear solution was obtained. The excess of HMDS was removed in vacuum and the resulting 9 dissolved in abs. benzene (15 ml). A solution of 1-bromo-2,3,5-tri-O-benzoyl-D-ribofuranose (10)35 (1.575 g, 3 mmol) in benzene (15 ml) and each 0.75 g of HgO and HgBr2 were added. The mixture was refluxed for 4 h, evaporated and the residue treated with CHCl3 (100 ml). The mercury salts were filtered off and the filtrate shaken with a KJ solution (15%). The organic phase was tried over Na2SO4, evaporated to a smaller volume, put onto a silica gel column (7 x 35 cm) and first developed with CHCl3 (4 l). Evaporation of this fraction 1 gave a mixture of 3 nucleosides (1.26 g). The solvents system was changed to CHCl3/MeOH (19:1, 1 l) followed by (9:1, 1l) and gave on evaporation fraction 2 (0.15 g). Fraction 3 resulted from the elution with (CHCl3/MeOH 4:1 (500 ml) and (1:1, 1.5 l) to give 0.3 g. Fraction 1 was further separated by low pressure chromatography on a column type C30 (3 x 50 cm, silica gel Lichroprep Si 60) and a pressure of 10 atm. The eluents n-hexane/CHCl3 (7/3) separated first unreacted sugar and with 6/4 next 26 (0.21 g, 9%) and followed by 12 (0.675 g, 30%). Fraction 2 was separated by preparative thick-layer chromatography on plates (40 x 20 x 0.2 cm) with CHCl3/MeOH (19:1). The main band was eluted with CHCl3/MeOH (9:1) to give 12 (0.12 g, 15%). Fraction 3 gave on chromatography on thick-layer plates with CHCl3/MeOH (9:1) 19 (0.165 g, 7%). 12. Yield: 0.795 g (35%). M.p. 154-158°C. 1H-NMR (CDCl3): 8.08 (d, 2 H, arom. H); 7.96-7.90

Page  104 (dd, 4 H. arom. H); 7.60-7.26 (m, 20 H, arom. H); 7.17 (d, 1 H, H-C(1')); 6.19 (pt, 1 H, H-C(2')); 6.12 (bs, 2 H, NH2); 6.02 (m, 1 H, H-C(3')); 4.88 (d, 2 H, H-C( 5'); 4.47 (m, 1 H, H-C(4')). Anal. Calc. for C44H33N5O8 (759.6): C, 69.57; H, 4.38; N, 9.22. Found: C, 68.99; H, 4.49; N, 9.19. 26. Yield: 0.21 g (9%). M.p. 132-136°C. 1H-NMR (CDCl3): 8.10-7.85 (m, 14 H, arom. H), 7.56- 7.28 (m, 28 H. 26 arom. H, H-N. H-C(1'))); 6.42 (d, 1 H, H-C(1')); 6.26-6.19 (m, 2 H, H-C(2')); 5.99 (m, 2 H, H-C(3')); 4.73 (m, 4 H, H-C( 5')); 4.57 (m, 2 H, H-C(4')). Anal. Calc. for C70H53N5O15 x 2 H2O (1240.2): C, 67.79; H, 4.63; N, 5.64. Found: C, 67.38; H, 4.87; N, 5.35. 2-Amino-1-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-6,7-diphenyl-4(3H)pteridinone (19). Silylation analogous to the preceding procedure with 2 (1.265 g, 4 mmol) . The intermediate 9 was dissolved in CH2Cl2 (30 ml), 1-O-acetyl-2,3,5-tri-O-benzoyl-ß-D-ribofuranose (11)35 (2.01 g, 4.1 mmol) and BF3-etherate (4 ml) were added and stirred at rt for 4 h. Dilution with CH2Cl2 (50 ml), treatment with saturated NaHCO3 solution, drying of the organic layer with Na2SO4 and evaporation to give a crude mixture (3.2 g). Separation by CC (7 x 30 cm) first with CHCl3 (1.5 l), then with CHCl3/MeOH (19:1, 500 ml; 13:1, 500 ml and 9:1, 1.5 l) to give the main fraction on evaporation. The mixture was further purified by chromatography on 10 thick-layer plates (40 x 20 x 0.2 cm) with CHCl3/MeOH (19:1). The main band was cut out, eluted with CHCl3/MeOH (12:1), evaporated to give 1.535 g of 19. Recrystallization from isopropanol/H2O (1:1, 40 ml) gave 1.47 g (50%) of pure 19 of m.p. 152°C. 1H-NMR (CDCl3): 8.00-7.20 (m, 28 H, NH2, 25 arom. H, H-C(1')); 6.41 (m, 1H, H.C(2')); 5.49 (pt, 1 H, H-C(3')); 4.70-4.50 (m, 3 H, H-C(4'), H- C(5')). Anal. Calc. for C44H33N5O8 x H2O (777.6): C, 67.96; H, 4.53; N, 9.00. Found: C, 67.82; H, 4.43; N, 8.91. 2-Amino-3-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-6,7-dimethyl-4(3H)pteridinone (13) and 2-Amino-1-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-6,7-dimethyl-4(3H)pteridinone (20). A mixture of 6,7-dimethylpterin (3) (0.955 g, 5 mmo) and (NH4)2SO4 (0.1 g) in hexa- methyldisilazane (10 ml) was heated under reflux for 5 h to form a clear solution. The excess of HMDS was distilled off, the residue dissolved in CH2Cl2 (15 ml), 11 (2.52 g, 5.1 mmol) in CH2Cl2 (15 ml) and BF3-etherate (5 ml) added. After stirring at rt for 4 h the reaction solution was treated with saturated aqueous NaHCO3 solution, the organic layer separated, dried over Na2SO4 and evaporated to dryness. The residue was dissolved in CHCl3, put onto a silica gel column (7 x 30 cm) and developed first with CHCl3 (1,5 l) to give unreacted sugar and followed by CHCl3/MeOH (19:1, 500 ml; 12: 1, 1000 ml and 9:1, 1000 ml) to give a mixture of 3 substances (1.1 g). This fraction was separated on preparative silica gel plates (40 x 20 x 0.2 cm) with CHCl3/MeOH (9:1). The lower band (Rf 0.28) was eluted with CHCl3/MeOH (9:1) and gave on evaporation pure 20 (0.425 g, 14%). The upper band was still a mixture of two substances and had to be rechromatographed on plates with CHCl3/MeOH (19:1) to get partial separation. The band (Rf 0.51) gave after elution with CHCl3/MeOH (9:1), evaporation and recrystallization from i-PrOH/H2O (1:1) 13 (0.25 g, 8%). 20. Yield: 0.425 g, (14%).M.p. 152-154°C. 1H-NMR (CDCl3): 7.99-7.26 (m, 17 H, 15 arom. H, NH2); 7.05 (d, 1 H, H-C(1')); 6.51-6.30 (m, 2 H, H-C(2', 3')); 4.92-4.71 (d, 3 H, H-C(4', 5'); 2.61,

Page  105 2.58 (2 s, 6H, 2 CH3). Anal. Calc. for C34H29N5O8 x 0.5 H2O (644.6): C, 63.35; H, 4.69; N, 10.86. Found: C, 63.24; H, 4.53; N, 10.27. 2-Amino-3-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-6,7-dimethyl-4(3H)pteridinone (13). Silylation of 3 (0.955 g, 5 mmol) was performed analogous to the preceding procedure. The silylated intermediate 9 was dissolved in abs. C6H6 (35 ml), then 10 (2.27 g, 4.5 mmol), HgO (1.25 g) and HgBr2 (1.25 g) added. The mixture was heated under reflux for 4 h. After cooling MeOH (2 ml) was added, the mixture evaporated to dryness, the residue dissolved in CHCl3 (50 ml) and then shaken several times with 15% aqueous KJ solution to remove the mercury salts. The organic layer was separated, dried over Na2SO4 and again evaporated. The residue was dissolved in CHCl3, put onto a silica gel column (4.5 x 45 cm) and developed with CHCl3/MeOH (19:1). After 1.5 l elution the next fraction (400 ml) was collected, evaporated to give 0.6 g. This mixture was further purified on 5 preparative thick layer plates (40 x 20 x 0.2 cm) with CHCl3, MeOH (19:1). The main band was cut out, eluted by CHCl3/MeOH (15:1) to give after evaporation 13 (0.425 g, 14%) of m.p. 168°C (decomp.). 1H-NMR (CDCl3): 8.06-7.92 (m, 5 H, arom. H); 7.55-7.20 (10 H, arom. H); 7.18 (d, 1 H, H-C(1')); 6.20 (bs, 2 H, NH2); 6.18 (m, 1 H, H-C(2')); 6.08 (m, 1 H, H-C(3')); 4.91 (m, 2 H, H-C(5'); 4.72 (m, 1 H, H-C(4')); 2.61 (s, 3 H, CH3); 2.57 (s, 3 H, CH3), Anal. Calc. for C34H29N5O8 x 0.5 H2O (644.6): C, 63.35; H, 4.69; N, 10.86. Found: C, 63.15; H, 4.78; N, 10.71. 2-Amino-3-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-6-methyl-4(3H)pteridinone (14) and 2- Amino-1-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-6-methyl-4(3H)pteridinone (21). A mixture of 6-methylpterin (4)36 (0.709 g, 4 mmol) and (NH4)2SO4 (0.1 g) in HMDS (30 ml) was refluxed for 24 h, then evaporate, the residue dissolved in CH2Cl2 (40 ml), 11 (2.01 g, 4 mmol) and BF3-etherate (4 ml) added and then stirred at rt for 4 h. The reaction solution was treated with saturated aqueous NaHCO3 solution, the organic phase separated and dried over Na2SO4 and evaporated. The residue was dissolved in CHCl3, put onto a silica gel column (8 x 30 cm) and developed first with CHCl3 (2 l) to give unreacted sugar. Extended elution with CHCl3/MeOH (19:1, 2.5 l) and CHCl3/MeOH (14:1, 500 ml) gave on evaporation a mixture of 14 and 21 (0.745 g). Its difficult separation was performed on preparative thick-layer silica gel plates (40 x 20 x 0.2 cm) by repeated development with CHCl3/MeOH (19:1) to get separation of 2 main bands. Elution of the faster moving band yielded after evaporation 0.18 g (6%) of 14 and from the lower moving band 0.27 g (9%) of 21. 14: 1H-NMR (CDCl3): 8.30 (s, 1 H, H-C(7)); 8.20-7.20 (m, 15 H, arom. H); 7.15 (d, 1 H, H- C(1')); 6.17 (bs, 2 H, NH2); 6.15-5.82 (m, 2 H, H-C(2', 3')); 4.93-4.62 (m, 3 H, H-C(4', 5'); 2.65 (s, 3 H, CH3). Anal. Calc. for C33H27N5O8 x 0.5 H2O (630.6): C, 62.85; H, 4.32; N, 11.10. Found: C, 62.85; H, 4.31; N, 10.57. 21: 1H-NMR (CDCl3): 8.53 (s, 1 H, H-C(7)); 8.10-7.20 (m, 17 H, 15 arom. H, NH2); 7.05 (d, 1 H, H-C(1')); 6.30-6.05 (m, 2 H, H-C(2', 3')); 4.93-4.44 (m, 3 H, H-C(4', 5'); 2.62 (s, 3 H, CH3). Anal. Calc. for C33H27N5O8 x 0.5 H2O (630.6): C, 62.85; H, 4.32; N, 11.10. Found: C, 62.63; H, 4.20; N, 11.27.

Page  106 2-Amino-3-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-7-methyl-4(3H)pteridinone (15). Analogous to the preceding procedure with 7-methylpterin (5)37 (0.886 g, 5 mmol) and 11 (2.52 g, 5 mmol) and BF3-etherate (4 ml). The reaction product was purified by short column chromatography with CHCl3/n-hexane (4:1). The main fraction was collected, evaporated and the residue recrystallized from EtOH/H2O to give 0.186 g (6%) of 15. M.p. 122-124°C. 1H-NMR (CDCl3): 8.39 (s, 1 H, H-C(6)); 8.20-7.22 (m, 15 H, arom. H,); 7.20 (d, 1 H, H-C(1')); 6.11 (bs, 2 H, NH2); 6.30-5.90 (m, 2 H, H-C(2', 3')); 5.00-4.44 (m, 3 H, H-C(4', 5'); 2.62 (s, 3 H, CH3). Anal. Calc. for C33H27N5O8 (621.6): C, 63.76; H, 4.38; N, 11.27. Found: C, 63.78; H, 4.27; N, 10.93. 2-Amino-3-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-6-phenyl-4(3H)pteridinone (16), 2- Amino-1-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-6-phenyl-4(3H)pteridinone (22) and 2- Imino-1,O4-bis-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)1,2-dihydropteridine (27). Analogous to the preceding procedure with 6-phenylpterin (6)38 (0.957 g, 4 mmol), 11 (2.01 g, 4 mmol) and trimethylsilyl trifluormethanesulfonate (2.6 g, 1.2 mmol) as catalyst. After stirring at rt for 24 h was diluted with CH2Cl2 (20 ml) , treated with cold aqueous Na2HPO4 solution, the organic phase separated, dried with Na2SO4 and evaporated to give a crude mixture (2.46 g). This mixture was put onto a silica gel column (9 x 15 cm) for chromatography with CHCl3 (3 l, 1. fraction, unreacted sugar), then with CHCl3/MeOH (19:1, 2 l, 2. fraction). The second fraction was further separated on preparative thick layer silica gel plates (40 x 20 x 0.2 cm) by repeated development with CHCl3/MeOH (19:1) to give 3 main bands which were cut out and eluted separately with CHCl3(MeOH (12:1). The fastest moving band (Rf 0.82) gave 27 (0.08 g, (8%), the middle band 16 (0.09 g, 6%) and the lowest band 22 (0.04 g, 2%). 16. 1H-NMR (CDCl3): 9.14 (s, 1 H, H-C(7)); 8.20-7.20 (m, 22 H, 20 arom. H, NH2); 7.18 (d, 1 H, H-C(1')); 6.24 (bs, 2 H, NH2); 6.30-6.00 (m, 2 H, H-C(2', 3')); 5.00-4.60 (m, 3 H, H-C(4', 5'). Anal. Calc. for C38H29N5O8 (683.7): C, 66.76; H, 4.28; N, 10.24. Found: C, 66.53; H, 4.28; N, 10.00. 22. 1H-NMR (CDCl3): 8.96 (s, 1 H, H-C(7)); 8.20-7.20 (m, 22 H, 20 arom. H, NH2); 6.82 (d, 1 H, H-C(1')); 6.50-6.25 (m, 2 H, H-C(2', 3')); 5.40-5.00 (m, 3 H, H-C(4', 5')). Anal. Calc. for C38H29N5O8 x H2O (701.7):C, 64.47; H, 4.45; N, 9.98. Found: C, 64.47; H, 4.23; N, 9.91. 27: 1H-NMR (CDCl3): 8.51 (s, 1 H, H-C(7)); 8.20-7.20 (m, 37 H, 35 arom. H, NH2); 7.00-6.30 (m, 6 H, H-C(1', 2', 3'); 4.90-4.50 (m, 6 H, H-C(4', 5'). Anal. Calc. for C64H49N5O15 (1128.1): C, 68.14; H, 4.38; N, 6.21. Found: C, 67.89; H, 4.48; N, 5.89. 2-Amino-3-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-7-phenyl-4(3H)pteridinone(17) and 2- Amino-1-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-7-phenyl-4(3H)pteridinone (23). Analogous to the preceding procedure with 7-phenylpterin (7)38 (0.957 g, 4 mmol), 11 (2.01 g, 4 mmol) and BF3-etherate (4 ml) as catalyst. After stirring at rt for 4 h was diluted with CH2Cl2 (20 ml) , treated with cold aqueous Na2HPO4 solution, the organic phase separated, dried with Na2SO4 and evaporated to give a crude mixture (2.35 g). This mixture was put onto a silica gel column (9 x 15 cm) for chromatography with CHCl3 (3 l, 1. fraction, unreacted sugar), then with CHCl3/MeOH (19:1, 3.5 l). This fraction was evaporated and separated on 6 preparative thick-

Page  107 layer silica gel plates (40 x 20 x 0.2 cm) by repeated development with CHCl3. The faster moving main band was cut out, eluted with CHCl3, MeOH (9:1) to give 60 mg (2%) of 17. The slower moving band gave 0.12 g (4%) of 23. 17. 1H-NMR (CDCl3): 9.16 (s, 1 H, H-C(7)); 8.20-7.20 (m, 21 H, 20 arom. H, H-C(1')); 6.18 (m, 1 H, H-C(2')); 6.06 (m, 1 H, H-C(3')); 6.04 (bs, 2 H, NH2); 4.75(m, 2 H, H-C(5')); 4.88 (m, 1 H, H-C(4')). Anal. Calc. for C38H29N5O8 x H2O (701.7): C, 65.04; H, 4.45; N, 9.98. Found: C, 64.85; H, 3.97; N, 9.96. 23. 1H-NMR (CDCl3): 9.14 (s, 1 H, H-C(7)); 8.20-7.20 (m, 21 H, 20 arom. H, H-C(1')); 6.42 (bs, 2 H, NH2); 6.17 (m, 1 H, H-C(2')); 5.99 (m, 1 H, H-C(3')); 6.04 (bs, 2 H, NH2); 5.00-4.70 (m, 3 H, H-C(4',5')). Anal. Calc. for C38H29N5O8 x 0.5 H2O (692.7): C, 65.89; H, 4.36; N, 10.11. Found: C, 65.74; H, 4.17; N, 9.89. 2-Amino-3-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-7-tert.butyl-4(3H)pteridinone (18) and 2-Amino-1-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-7-tert.butyl-4(3H)pteridinone (24). Silylation and ribosylation was performed analogous to the preceding procedures with 7- tert.butyl-pterin (8)39 (0.877 g, 4 mmol), 11 (2.01 g, 4 mmol) and BF3-etherate (4 ml). Work-up was done after 5 days stirring at rt. The crude product mixture was separated on 10 preparative thick-layer plates with CHCl3/MeOH (11:1) to give two main bands. The faster moving band gave after elution, evaporation and recrystallization from i-PrOH/H2O 0.24 g (10%) of 18 and from the slower moving band were 0.363 g (14%) of 24 isolated. 18. 1H-NMR (CDCl3): 8.62 (s, 1 H, H-C(6)); 8.20-7.20 (m, 16 H, 15 arom. H, H-C(1')); 5.95 (bs, 2 H, NH2); 6.16 (m, 1 H, H-C(2')); 5.93 (m, 1 H, H-C(3')); 5.00-4.70 (m, 3 H, H-C(4', 5')); 1.43 (s, 9 H, (CH3)3). Anal. Calc. for C36H33N5O8 (663.7): C, 65.15; H, 5.01; N, 10.55. Found: C, 65.24; H, 5.00; N, 10.19. 24. 1H-NMR (CDCl3): 8.76 (s, 1 H, H-C(6)); 8.20-7.10 (m, 16 H, 15 arom. H, H-C(1')); 6.15 (m, 1 H, H-C(2')); 6.08 (bs, 2 H, NH2); 4.93-4.44 (m, 3 H, H-C(4', 5')); 1.39 (s, 9 H, (CH3)3). Anal. Calc. for C36H33N5O8 x 0.5 H2O (672.7): C, 64.34; H, 5.10; N, 10.41. Found: C, 64.35; H, 5.04; N, 10.26. 2-Amino-1-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-4(3H)pteridinone (25). A mixture of pterin (1) (1.63 g, 10 mmol) and (NH4)2SO4 (0.1 g) in HMDS (50 ml) was heated under reflux for 20 h. The excess of HMDS was distilled off under high vacuum and the residue dissolved in abs. CH2Cl2 (30 ml). To the solution was added 11 (5.5 g, 11 mmol) and trimethylsilyl triflate (2.6 g, 12 mmol) and then stirred at rt for 24 h. The solution was diluted with CH2Cl2 (30 ml), treated with saturated aqueous NaHCO3, the layers separated and the organic phase dried over Na2SO4. After evaporated the resulting residue was dissolved in CHCl3 and put onto a silica gel column (3.5 x 29 cm) for elution first with CHCl3 (1 l), followed by CHCl3/MeOH (100:1, 500 ml), (100:3, 500 ml), (100:4, 500 ml) and (100:5, 1 l). These 4 fractions were evaporated to give 3.2 g crude product. Recrystallization from i-PrOH/H2O gave 1.2 g (24%) of 25. 1H-NMR (CDCl3): 8.84-8.66 (m, 6 H, arom. H); 8.29 (d, 1 H, H-C(7)); 8.07 (d, 1 H, H-C(6)); 8.00-7.26 (m, 11 H, arom. H, NH2); 7.05 (d, 1 H, H-C(1')); 6.11 (m, 1 H, H-C(2')); 4.99-4.58 (m, 3 H, H- C(4', 5')). Anal. Calc. for C32H25N5O8 (607.6): C, 63.26; H, 4.15; N, 11.53. Found: C, 63.11; H,

Page  108 4.25; N, 11.36. 2-Amino-6,7-dimethyl-3-ß-D-ribofuranosyl-4(3H)pteridinone (28). A solution of 13 (0.2 g, 0.3 mmol) in abs. MeOH (100 ml) was treated with 0.2% CH3ONa solution (2 ml) with stirring for 18 h. Little DOWEX 50 x 4 (H+-form, 200-400 mesh) and H2O (5 ml) was added to bring the pH to 5. After filtration was evaporated, the residue dissolved in CHCl3/MeOH (4:1, 10 ml), ether (10 ml) added and after cooling for 2 h the precipitate collected by centrifugation to give 44 mg (43%) of 28. M.p. 161°C (decomp.). 1H-NMR ((D6)DMSO): 7.49 (bs, 2 H, NH2); 6.51 (d, 1 H, H-C(1')); 5.62(t, 1 H, 5'-OH)); 5.28 (d, 1 H, 2'OH)); 5.15 (d, 1 H, 3'-OH)); 4.47 (dd, 1 H; H-C(2')); 4.10-3.90 (m, 2 H, H-C(3', 4')); 3.64 (m, 2 H, H-C(5')); 2.51 (2 s, 6 H, 2 CH3). Anal. Calc. for C13H17N5O5 x 2 H2O (359.3): C, 43.45; H, 5.89; N, 19.49. Found: C, 43.89; H, 5.82; N, 19.09. 2-Amino-6,7-dimethyl-1-ß-D-ribofuranosyl-4(3H)pteridinone (29). To a solution of 20 (0.15 g, 0.24 mmol) in abs. MeOH (5 ml) was added 1 M CH3ONa (0.25 ml) and stirred for 2 h. The pH was brought to 5 by AcOH and the solution kept overnight in the icebox. The precipitate was collected and recrystallized from i-PrOH/H2O (4:1, 15 ml) to give 53 mg (69%) of 29. M.p. 170°C (decomp.). 1H-NMR ((D6)DMSO): 7.70 (bs, 2 H, NH2); 6.68 (d, 1 H, H-C(1')); 5.78 (t, 1 H, 5’-OH); 5.35 (d, 1 H, 2’-OH); 5.18 (d, 1 H, 3’-OH); 4.56 (dd, 1 H; H-C(2')); 4.10-3.90 (m, 2 H, H-C(3', 4')); 3.65 (m, 2 H, H-C(5')); 2.55 (2 s, 6 H, 2 CH3). Anal. Calc. for C13H17N5O5 x 0.5 H2O (332.3): C, 46.98; H, 5.45; N, 21.07. Found: C, 46.64; H, 5.54; N, 20.48. 2-Amino-6,7-diphenyl-1-ß-D-ribofuranosyl-4(3H)pteridinone (30). (a). Analogous to the preceding procedure with 19 (0.15 g, 0.2 mmol). After stirring for 2 h, H2O (3 ml) was added and the pH adjusted to 5 by AcOH. The precipitate was collected after cooling and recrystallized from i-PrOH/H2O (1:1, 15 ml) to give 53 mg (60%) of 30. M.p. 180°C (decomp.). (b). A solution of 2-methylthio-6,7-diphenyl-1-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-4(3H)pteridinone (37) (0.158 g, 0.2 mmol) in abs. dioxane (5 ml) was treated with conc. NH3 (5 ml) for 3 days stirring in a closed flask. After evaporation the residue was dissolved in little H2O and acidified by AcOH. On cooling the precipitate was collected and recrystallized from EtOH/H2O (1:1) to give 60 mg (70%) of 30. M.p. 180°C. 1H-NMR ((D6)DMSO): 7.90 (bs, 2 H, NH2); 7.50-7.30 (m, 10 H, arom. H); 6.99 (d, 1 H, H-C(1')); 5.98 (t, 1 H, 5’-OH); 5.42 (d, 1 H, 2’-OH); 5.25 (d, 1 H, 3’- OH); 4.58 (dd, 1 H; H-C(2')); 4.12 (m, 1 H, H-C(3')); 4.00 (m, 1 NH, H-C(4')); 3.67 (m, 2 H, H- C(5')). Anal. Calc. for C23H21N5O5 x 0.5 H2O (456.4): C, 60.52; H, 4.95; N, 15.34. Found: C, 60.75; H, 4.73; N, 15.46. 2-Methylthio-1-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-4(3H)pteridinone (35) and 2-Methylthio-3-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-4(3H)pteridinone (38). A mixture of 2-methylthio-4(3H)pteridinone (31)40 (1.36 g, 7 mmol) and (NH4)2SO4 (0.1 g) in hexamethyldisilazane (HMDS) (30 ml) was refluxed for 2 h. (30 ml). The excess of HMDS was distilled off, the residue dissolved in dry CH2Cl2 (20 ml), then 11 (3.52 g, 7 mmol) and BF3- etherate (7 ml) added. The reaction solution was stirred at rt for 1 day, then treated with a mixture of CHCl3/H2O/NEt3 (5:5:1, 40 ml), the organic phase separated, dried over Na2SO4 and evaporated to give 3.3 g crude product. The mixture was separated by short column

Page  109 chromatography (SCC)41 with CHCl3/n-hexane (3:2) to give as the first fraction 38 and followed by 35. The fractions were evaporated and the residues recrystallized from EtOH/H2O. 38. Yield: 1.95 g (44%). M.p. 105-108°C. 1H-NMR (CDCl3): 8.89 (d, 1 H, H-C(7)); 8.76 (d, 1 H, H-C(6)); 8.10-7.20 (m, 15 H, arom. H); 6.40 (d, 1 H, H-C(1')); 6.32-6.20 (m, 2 H, H-C(2', 3')); 4.90-4.60 (m, 3 H, H-C(4', 5')); 2.74 (s, 3 H, S-CH3). Anal. Calc. for C33H26N4O8S (638.6): C, 62.06; H, 4.10; N, 8.77. Found: C, 61.75; H, 4.15; N, 8.62. 35. Yield: 0.95 g (24%). M.p. 135°C. 1H-NMR (CDCl3): 8.75 (d, 1 H, H-C(7)); 8.48 (d, 1 H, H- C(6)); 8.00-7.20 (m, 15 H, arom. H); 6.64 (d, 1 H, H-C(1')); 6.39 (m, 1 H, H-C(2')); 6.26 (m, 1 H, H-C(3')); 5.00-4.60 (m, 3 H, H-C(4', 5')); 2.68 (s, 3 H, S-CH3).Anal. Calc. for C33H26N4O8S x 0.5 H2O (647.6): C, 61.20; H, 4.20; N, 8.65. Found: C, 61.49; H, 3.91; N, 8.68. 6,7-Dimethyl-2-methylthio-1-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-4(3H)pteridinone (36) and 6,7-Dimethyl-2-methylthio-3-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-4(3H)- pteridinone (39). Analogous to the preceding procedure with 6,7-dimethyl-2-methylthio- 4(3H)pteridinone (32)40 (1.11 g, 6 mmol), 11 (3.02 g, 6 mmol) and BF3-etherate (6 ml) for 3 days. After work-up the crude material (3.85 g) was separated and purified by SSC with CHCl3/n-hexane (4:1). The first main fraction gave 39 followed by 36. Recrystallizsation from EtOH/H2O. 39. Yield: 1.82 g (42%). M.p. 113°C. 1H-NMR (CDCl3): 8.10-7.20 (m, 15 H, arom. H); 6.33 (d, 1 H, H-C(1')); 6.50-6.40 (m, 2 H, H-C(2', 3')); 4.90-4.70 (m, 3 H, H-C(4', 5')); 2.69 (s, 3 H, S- CH3); 2.75 (s, 3 H, CH3(7)); 2.73 (s, 3 H, CH3(6)). Anal. Calc. for C35H30N4O8S (666.7): C, 63.05; H, 4.54; N, 8.40. Found: C, 63.26; H, 4.49; N, 8.35. 36. Yield: 1.08 g (32%). M.p. 110°C. 1H-NMR (CDCl3): 8.00-7.20 (m, 15 H, arom. H); 6.59 (d, 1 H, H-C(1')); 6.50-6.40 (m, 2 H, H-C(2', 3')); 4.90-4.70 (m, 3 H, H-C(4', 5')); 2.69 (s, H, S- CH3); 2.68 (2 s, 6 H, 2 CH3). Anal. Calc. for C35H30N4O8S (666.7): C, 63.05; H, 4.54; N, 8.40. Found: C, 63.01; H, 4.66; N, 8.37. 2-Methylthio-6,7-diphenyl-1-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-4(3H)pteridinone (37) and 2-Methylthio-6,7-diphenyl-1-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-4(3H)- pteridinone (40). Analogous to the preceding procedure first silylation with 2-methylthio- 6,7.diphenyl-4(3H)pteridinone (33)40 (1.386 g, 4 mmol) and HMDS under reflux for 1 day. After evaporation and solution in CH2Cl2 (10 ml) 11 (2.01 g, 4 mmol) and BF3-etherate (5 ml) were added and stirred for day. After work-up the crude material (3.36 g) was separated and purified by SSC with CHCl3/n-hexane (3:2). The first main fraction gave 39 followed by 37. Recrystallizsation from EtOH/H2O. 40. Yield: 1.636 g (52%). M.p. 213°C. 1H-NMR (CDCl3): 8.20-7.20 (m, 25 H, arom. H); 6.50- 6.30 (m, 3 H, H-C(1', 2', 3')); 5.00-4.70 (m, 3 H, H-C(4', 5')); 2.77 (s, 3 H, S-CH3). Anal. Calc. for C45H34N4O8S (790.8): C, 68.35; H, 4.33; N, 7.08. Found: C, 68.05; H, 4.36; N, 7.02. 37. Yield: 0.81 g (26%). M.p. 169-171°C. 1H-NMR (CDCl3): 8.10-7.20 (m, 15 H, arom. H); 6.77 (d, 1 H, H-C(1')); 6.60 (dd, 1 H, H-C(2')); 5.97 (m, 1 H, H-C(3')); 4.72 (m, 1 H, H-C(4')); 4.35 (m, 2 H, H.C(5')); 2.69 (s, H, S-CH3). Anal. Calc. for C45H34N4O8S (790.8): C, 68.35; H, 4.33; N, 7.08. Found: C, 68.17; H, 4.41; N, 7.04.

Page  110 2-Methylamino-1-ß-D-ribofuranosyl-4(3H)pteridinone (41). A solution of 35 (0.128 g, 0.2 mmol) in abs. tetrahydrofurane (THF) (5 ml) was treated with methanolic CH3NH2-solution (20%, 3 ml) in a closed flask for 2 days. It was evaporated, the residue dissolved in abs. MeOH (5 ml), 1 N-CH3ONa (0.2 ml) added and 1 day stirred at rt. The solution was acidified by AcOH to pH 5, evaporated and the residue purified by chromatography on a preparative thick layer plate (40 x 20 x 0.2 cm) with CHCl3/MeOH (4:1) to give after recrystallization from EtOH/H2O 46 mg (74%) of 41. M.p. 170°C (decomp.). 1H-NMR ((D6)DMSO): 8.67 (d, 1 H, H-C(7)); 8.63 (d, 1 H, H-C(6)); 7.99 (bs, H, NH); 6.92 (d, 1 H, H-C(1')); 5.95 (bs, 1 H, 5'-OH); 5.41 (d, 1 H, 2'-OH); 5.21 (d, 1 H, 3'-OH); 4.49 (dd, 1 H; H-C(2')); 4.11 (m, 1 H, H-C(3')); 4.07 (m, 1 H, H- C(4')); 3.71 (m, 2 H, H-C(5')); 2.85 (s, 3 H, H3C-NH). Anal. Calc. for C12H15N5O5 x H2O (327.3): C, 44.03; H, 5.23; N, 21.40. Found: C, 44.09; H, 5.25; N, 21.29. 2-Methylamino-6,7-dimethyl-1-ß-D-ribofuranosyl-4(3H)pteridinone (42). Analogous to the preceding procedure with 36 (0.133 g, 0.2 nmmol) to give after recrystallization from EtOH/H2O (7:3) 59 mg (88%) of 42. M.p. 205°C. 1H-NMR ((D6)DMSO): 7.85 (bs, H, NH); 6.87 (d, 1 H, H-C(1')); 5.95 (bs, 1 H, 5'-OH); 5.45 (d, 1 H, 2'-OH); 5.30 (bs, 1 H, 3'-OH); 4.49 (dd, 1 H; H- C(2')); 4.10 (m, 1 H, H-C(3')); 4.02 (m, 1 H, H-C(4')); 3.70 (m, 2 H, H-C(5')); 2.84 (s, 3 H, H3C- NH); 2.55, 2.53 (2 s, 6 H, 2 CH3). Anal. Calc. for C14H19N5O5 x 0.5 H2O (346.3): C, 48.66; H, 5.82; N, 20.22. Found: C, 48.95; H, 5.88; N, 20.30. 2-Ethylamino-6,7-dimethyl-1-ß-D-ribofuranosyl-4(3H)pteridinone (43). A solution of 36 (0.133 g, 0.2 mmol) in abs. THF (5 ml) was cooled to -15°C, then saturated with C2H5NH2-gas and kept in the icebox for 2 days. It was evaporated, the residue dissolved in abs. MeOH (10 ml), 1 N CH3ONa (0.2 ml) added and stirred for 1 day. The solution was acidified by AcOH to pH 5, again evaporated and the residue purified by chromatography on a preparative thick layer silica gel plate (40 x 20 x 0.2 cm) with CHCl3/MeOH (4:1). The main band was cut out, eluted, evaporated and the residue recrystallized from little from H2O to give 64 mg (91%) of 43. M.p. 167-168°C. 1H-NMR ((D6)DMSO): 7.85 (bs, H, NH); 6.82 (d, 1 H, H-C(1')); 5.82 (t, 3 H, 5’- OH); 5.40 (d, 1 H, 2’-OH); 5.20 (d, 1 H, 3’-OH); 4.48 (dd, 1 H; H-C(2')); 4.11 (m, 1 H, H-C(3')); 4.04 (m, 1 H, H-C(4')); 3.69 (m, 2 H, H-C(5')); 3.40 (q, 2-H, HNCH2CH3); 2.55 (2 s, 6 H, 2 CH3); 1.18 (t, 3 H, CH3CH2NH). Anal. Calc. for C15H21N5O5 (351.4): C, 51.27; H, 6.08; N, 19.93. Found: C, 50.94; H, 5.88; N, 19.52. 2-Ethanolamino-6,7-dimethyl-1-ß-D-ribofuranosyl-4(3H)pteridinone (44). Analogous to the preceding procedure with 36 (0.133 g, 0.2 nmmol) and methanolic ethanolamine (10%, 3 ml) and keeping in the icebox for 1 day. It was evaporated, the residue dissolved in abs. MeOH (10 ml), 1 N CH3ONa (0.2 ml) added and stirred at rt for 1 day. Again evaporation and purification on a preparative thick layer silica gel plate (40 x 20 x 0.2 cm) with CHCl3, MeOH (4:1) to give 25 mg (34%) of 44. M.p. 161°C. 1H-NMR ((D6)DMSO): 7.79 (bs, H, NH); 6.84 (d, 1 H, H-C(1')); 5.79 (t, 1 H, 5’-OH); 5.45 (d, 1 H, 2’-OH); 5.21 (d, 1 H, 3’-OH); 4.83 (t, 1 H, CH2CH2OH); 4.50 (dd, 1 H; H-C(2')); 4.12 (m, 1 H, H-C(3')); 3.98 (m, 1 H, H-C(4')); 3.71 (m, 2 H, H-C(5')); 3.55 (bs, 2 H. CH2CH2OH); 3.40 (m, 2 H, HOCH2CH2NH); 2.55, 2.53 (2 s, 6 H, 2 CH3). Anal. Calc. for C15H21N5O6 x H2O (385.4): C, 46.74; H, 6.02; N, 18.17. Found: C, 46.95; H, 5.63; N, 17.82.

Page  111 2-Methylamino-6,7-diphenyl-1-ß-D-ribofuranosyl-4(3H)pteridinone (45). A solution of 37 (0.158 mg, 0.2 mmol) in abs. THF was cooled to -15°C, then methanolic CH3NH2 (5%, 2 ml) added and kept in the icebox for 1 day. It was evaporated, the residue dissolved in abs. MeOH (10 ml), 1 N CH3ONa (0.2 ml) added and stirred at rt for 1 day. The solution was acidified by AcOH to pH 5, evaporated and purified on a preparative thick layer silica gel plate (40 x 20 x 0.2 cm) with CHCl3/MeOH (4:1) to give after recrystallization from EtOH/H2O (2:3) 62 mg (70%) of 45. M.p. 189°C (decomp.). 1H-NMR ((D6)DMSO): 8.02 (bs, 1 H, NH); 7.40-7.20 (m, 10 H, arom. H); 6.98 (d, 1 H, H-C(1')); 5.02 (bs, 1 H, 5'-OH); 5.40 (d, ,1 H, 2'-OH); 5.21 (d, 1 H, 3'-OH); 4.52 (dd, 1 H; H-C(2')); 4.15 (m, 1 H, H-C(3')); 4.05 (m, 1 NH, H-C(4')); 3.72 (m, 2 H, H-C(5')); 2.91 (s, 3 H, H3C-NH). Anal. Calc. for C24H23N5O5 x 0.5 H2O (470.4): C, 61.16; H, 5.14; N, 14.88. Found: C, 60.84; H, 5.02; N, 14.82. 2-Ethylamino-6,7-diphenyl-1-ß-D-ribofuranosyl-4(3H)pteridinone (46). Analogous to the preceding procedure with 37 (0.158 g, 0.2 mmol) and C2H5NH2-gas for 3 days in the icebox. Treatment with CH3ONa and purification on a silica gel plate to give after recrystallization from EtOH/H2O (2:3) 65 mg (78%) of 46. Mp. 177°C. 1H-NMR ((D6)DMSO): 8.07 (bs, 1 H, NH); 7.50-7.30 (m, 10 H, arom. H); 6.95 (d, 1 H, H-C(1')); 5.89 (t, 1 H, 5’-OH); 5.46 (d, 1 H, 2’-OH); 5.24 (d, 1 H, 3’-OH); 4.52 (dd, 1 H; H-C(2')); 4.14 (m, 1 H, H-C(3')); 4.03 (m, 1 NH, H-C(4')); 3.71 (m, 2 H, H-C(5')); 3.45 (q, 2 H, NCH2CH3);); 1.29 (t, 3 H, NCH2CH3). Anal. Calc. for C25H25N5O5 x H2O (493.5): C, 60.84; H, 5.51; N, 14.09. Found: C, 60.58; H, 5.32; N, 13.61. 2-Ethanolamino-6,7-diphenyl-1-ß-D-ribofuranosyl-4(3H)pteridinone (47). Analogous to the preceding procedure with 37 (0.158 g, 0.2 mmol) and methanolic ethanolamine (10%, 3 ml) for 2 days in the icebox. Treatment with CH3ONa and purification on a silica gel plate gave after recrystallization from EtOH/H2O (2:3) 65 mg (66%) of 47. M.p. 177°. 1H-NMR ((D6)DMSO): 7.98 (bs, 1 H, NH); 7.50-7.30 (m, 10 H, arom. H); 6.96 (d, 1 H, H-C(1')); 5.78 (t, 1 OH, 5’-OH); 5.38 (d, 1 H,2’-OH); 5.21 (d, 1 H, 3’-OH); 4.87 (t, 1 H, CH2CH2OH); 4.56 (dd, 1 H; H-C(2')); 4.14 (m, 1 H, H-C(3')); 4.01 (m, 1 NH, H-C(4')); 3.71 (m, 2 H, H-C(5')); 3.55 (bs, 2 H, CH2CH2OH); 3.41 (m, 2 H, HOCH2CH2N). Anal. Calc. for C25H25N5O6 (491.5): C, 61.09; H, 5.13; N, 14.26. Found: C, 60.60; H, 5.36; N, 14.16. 2-Isopropylamino-6,7-diphenyl-1-ß-D-ribofuranosyl-4(3H)pteridinone (48). Analogous to the preceding procedure with 37 (0.158 g, 0.2 mmol) and methanolic isopropylamine (10%, 3 ml) for 2 days in the icebox. Treatment with CH3ONa and purification on a silica gel plate gave after recrystallization from EtOH/H2O (2:3) 31 mg (32%) of 48. M.p. 190°. 1H-NMR ((D6)DMSO): 7.85 (bs, 1 H, NH); 7.50-7.30 (m, 10 H, arom. H); 6.93 (d, 1 H, H-C(1')); 5.75 (t, 1 H, 5’-OH); 5.45 (d, 1 H, 2’-OH); 5.23 (d, 1 H, 3’-OH); 4.46 (m, 2 H; H-C(2'), Me2CH); 4.15 (m, 1 H, H-C(3')); 3.98 (m, 1 NH, H-C(4')); 3.71 (bs, 2 H, H-C(5')); 1.25 (d, 6 H, (H3C)2CH). Anal. Calc. for C26H27N5O5 x H2O (507.5): C, 61.43; H, 5.41; N, 13.45. Found: C, 61.04; H, 5.75; N, 13.79. 2-n-Butylamino-6,7-diphenyl-1-ß-D-ribofuranosyl-4(3H)pteridinone (49). Analogous to the preceding procedure with 37 (0.158 g, 0.2 mmol) and methanolic n-butylamine (10%, 3 ml) for 2 days in the icebox. Treatment with CH3ONa and purification on a silica gel plate gave after

Page  112 recrystallization from EtOH/H2O (3:7) 68 mg (68%) of 49. M.p. 193°. 1H-NMR ((D6)DMSO): 8.03 (bs, 1 H, NH); 7.50-7.30 (m, 10 H, arom. H); 6.98 (d, 1 H, H-C(1')); 5.88 (t, 1 H, 5#-OH); 5.46 (d, 1 H, 2’-OH); 5.25 (d, 1 H, 3’-OH); 4.52 (dd, 1 H; H-C(2')); 4.14 (m, 1 H, H-C(3')); 4.03 (m, 1 NH, H-C(4')); 3.70 (m, 2 H, H-C(5')); 3.42 (m, 2 H, CH3CH2CH2CH2NH); 1.60 (m, 2H, CH3CH2CH2CH2NH); 1.35 (m, 2H, CH3CH2CH2CH2NH); 0.95 (t, 3 H, CH3CH2CH2CH2NH). Anal. Calc. for C27H29N5O5 (503.5): C, 64.40; H, 5.80; N, 13.90. Found: C, 64.21; H, 5.82; N, 13.65. 2-Isobutylamino-6,7-diphenyl-1-ß-D-ribofuranosyl-4(3H)pteridinone (50). Analogous to the preceding procedure with 37 (0.158 g, 0.2 mmol) and methanolic isobutylamine (10%, 3 ml) for 3 days in the icebox. Treatment with CH3ONa and purification on a silica gel plate gave after recrystallization from EtOH/H2O (3:7) 63 mg (63%) of 50. M.p. 140-143°. 1H-NMR ((D6)DMSO): 8.08 (bs, 1 H, NH); 7.50-7.30 (m, 10 H, arom. H); 7.03 (d, 1 H, H-C(1')); 5.85 (t, 1 H, 5’-OH); 5.51 (d, 1 H, 2’-OH); 5.23 (d, 1 H, 3’-OH); 4.56 (dd, 1 H; H-C(2')); 4.14 (m, 1 H, H- C(3')); 4.05 (m, 1 NH, H-C(4')); 3.73 (m, 2 H, H-C(5')); 3.25 (m, 2 H, HNCH2); 2.05 (m, 1 H, Me2CH); 0.92 (HC(CH3)2). Anal. Calc. for C27H29N5O5 x C2H5OH (549.5): C, 63.39; H, 6.20; N, 12.74. Found: C, 63.57; H, 5.70; N, 12.44. 2-Dimethylamino-6,7-diphenyl-1-(2,3,5-tri-O-benzoyl-ß-D-ribofuranosyl)-4(3H)- pteridinone (51). A solution of 37 (0.158 g, 0.2 mmol) in abs. THF (5 ml) was cooled to -15°C and then methanolic dimethylamine (115%, 2 ml) added. After storage in the icebox for 2 days was evaporated and the residue purified on a preparative silica gel plate with CHCl3/MeOH (19:1). The main band was eluted, evaporated and the residue recrystallized from EtOH/H2O (1:1) to give 0.135 g (86%) of 51. M.p. 130°C. 1H-NMR (CDCl3): 7.93-7.22 (m, 25 H, arom. H); 6.70 (d, 1 H, H-C(1')); 6.11 (dd, 1 H, H-C(2')); 5.83 (m, 1 H, H-C(3')); 4.64 (m, 1 H, H-C(4')); 4.25 (m, 2 H, H.C(5')); 4.10 (s, 6 H, N(CH3)2). Anal. Calc. for C46H37N5O8 x 0.5 H2O (796.8): C, 69.34; H, 4.81; N, 8.79. Found: C, 69.08; H, 5.03; N, 8.80. 1-Methyl-2-methylamino-6,7-diphenyl-4(3H)pteridinone (64). To a solution of ethanolic methylamine (50%, 30 ml) was added 1-methyl-2-methyltio-6.7-diphenyl-4(3H)pteridone (61)40 (0.12 g, 0.33 mmol) and the mixture stirred for 1 h and then evaportated. The residue was purified by preparative thick layer chromatography on a silica gel plate (40 x 20 x 02 cm) with CHCl3/MeOH (9:1), The main band was eluted, evaporated and the solid recry-stallized from EtOH/H2O to give 98 mg (86%) of 65. M.p. 299°C. Anal. Calc. for C20H17N5O x C2H5OH (389.4): C, 67.85; H, 5.85; N, 17.98. Found: C, 67.67; H, 5.56; N, 18.28. 1-Methyl-2-dimethylamino-6,7-diphenyl-4(3H)pteridinone (65). Analogous to the preceding procedure with 61 (0.12 g, 0.33 mmol) in methanolic dimethylamine solution (20 ml) and stirring for 1 day. Work-up and recrystallization from EtOH/H2O gave 57 mg (71%) of 66. M.p. 229°C. Anal. Calc. for C21H19N5O (357.4): C, 70.57; H, 5.36; N, 19.59. Found: C, 70.43; H, 5.29; N, 19.34.

Page  113 References 1. LCV: Matysiak, S.; Waldscheck, B.; Pfleiderer, W. Nucleosides, Nucleotides & Nucleic Acids 2004, 23, 51. 2. Ritzmann, G.; Pfleiderer, W. Chem. Ber. 1973, 106, 1401. 3. Pfleiderer, W.; Ritzmann, G.; Harzer, K.; Jochims, J. C. Chem. Ber. 1973, 106, 2982. 4. Hutzenlaub, W.; Kobayashi, K.; Pfleiderer, W. Chem. Ber. 1976, 109, 3217. 5. Ritzmann, G.; Ienaga, K.; Pfleiderer, W. Liebigs Ann. Chem. 1977, 1217. 6. Southon, I. W.; Pfleiderer, W. Chem. Ber. 1978, 111, 2571. 7. Al-Msoudi, N. A.; Pfleiderer, W. Nucleosides & Nucleotides 1989, 8, 1485. 8. Al-Masoudi, N. A.; Pfleiderer, W. Pteridines 1990, 2, 9. 9. Al-Masoudi N. A.; Pfleiderer, W. Pteridines, 1993, 4, 119. 10. Cao, X.; Pfleiderer, W. Nucleosides & Nucleotides 1994, 13, 773. 11. Maurinsh, Y.; Pfleiderer, W. Nucleosides & Nucleotides 1996, 15, 431. 12. Harzer, K.; Pfleiderer, W. Helv. Chim. Acta 1973, 56, 1225. 13. Pfleiderer, W.; Autenrieth, D.; Schranner, M. Chem. Ber. 1973, 106, 317. 14. Schmid, H.; Schranner, M.; Pfleiderer, W. Chem. Ber. 1973, 106, 1952. 15. Ott, M.; Pfleiderer, W. Chem. Ber. 1974, 107, 339. 16. Itoh, T.; Pfleiderer, W. Chem. Ber. 1976, 109, 3228. 17. Ritzmann, G.; Kiriasis, L.; Pfleiderer, W. Chem. Ber. 1980, 113, 1524. 18. Ritzmann, G.; Ienaga, K.; Kiriasis, L.; Pfleiderer, W. Chem. Ber. 1980, 113, 1535. 19. Harris, R.; Pfleiderer, W. Liebigs Ann. Chem. 1981, 1457. 20. Kiriasis, L.; Pfleiderer, W. Nucleosides & Nucleotides 1989, 8, 1345. 21. Jungmann, O.; Pfleiderer, W. Tetrahedron Lett. 1996, 37, 8355. 22. Melguizo, M.; Gottlieb, M.; Charubala, R.; Pfleiderer, W. Nucleosides & Nucleotides 1998, 17, 175. 23. Lehbauer, J.; Pfleiderer, W. Helv. Chim. Acta 2001, 84, 2330. 24. Hilbert, G. E.; Johnson, T. B. J. Am. Chem. Soc. 1930, 52, 4489. 25. Fox, J. J.; Yung, J.; Davoll, J.; Brown, G. J. Am. Chem. Soc. 1956, 78, 2117. 26. Birkofer, L.; Kühltau, H. P.; Ritter, A. Chem. Ber. 1964, 93, 2810. 27. Birkofer, L.; Ritter, A. Angew. Chem. 1965, 77, 414. 28. Niedballa, U.; Vorbrüggen, H. J. Org. Chem. 1974, 39, 3654. 29. Wittenburg, E. W. Chem. Ber. 1968, 101, 1095, 1614, 2132. 30. Zemplen, G.; Geres, A.; Hadadcsy, J. Ber. Deut. Chem. Ges. 1936, 69, 1827. 31. Albert, A.; Serjeant, E. P. The Determination of Ionization Constants, Chapman and Hall, Ltd. London, 1971. 32. Helmchen, G.; Nill, G.; Flockerzi, D.; Schühle, W.; Youssef, M. S. K. Angew. Chem. 1979, 91, 64. 33. Master Thesis Flockerzi D, University of Stuttgart, 1977. 34. Cain, F.; Mallette, M. F.; Taylor, E. C. J. Am. Chem. Soc. 1946, 68, 1996.

Page  114 35. Stevens, J. D.; Fletcher Jr, R. G.; Ness, H. G. J. Org. Chem. 1968, 33, 1806. 36. Boothe, J. H., Waller, C. W., Stokstad, E. L. R., Hutchings, B. L., Mowat, J. H., Angier, R. B., Semb, J., Subba Row, Y., Cosulich, D. B., Fahrenbach, M. J., Hultquist, M. E., Kuh, E., Northey, E. H., Seeger, D. R., Sickels, J. P., Smith Jr., J. M. J. Am. Chem. Soc. 1948, 70, 27. 37. Pfleiderer, W., Zondler, H., Mengel, R. Liebigs Ann. Chem. 1970, 741, 64. 38. Storm, C. B., Shiman, R., Kaufman, S. J. Org. Chem. 1971, 36, 3925. 39. Yamamoto, H., Hutzenlaub, W., Pfleiderer, W. Chem. Ber. 1973, 106, 3175. 40. Schneider, H. J., Pfleiderer, W. Chem. Ber. 1974, 107, 3377. 41. Hunt, B. J., Rigby, W. Chem. and Ind. London 1967, 1868. 42. Pfleiderer, W. Pteridines 1993, 4, 11. 43. Angier, R. B., Curran, W. V. J. Org. Chem. 1961, 26, 2129. 44. Angier, R. B. J. Org. Chem. 1963, 28, 1509.