Synthesis of the Caffeine Metabolites 5-Acetylamino-6-formylamino- 3-methyluracil (AFMU) and 5-Acetylamino-6-amino-3-methyluracil (AAMU) on a Preparative Scale R. Röhrkasten3, P. Raatz3, R. P. Kreher3*, M. Blaszkewiczb a Lehrstuhl für Organische Chemie II, Fachbereich Chemie, Universität Dortmund. D-44227 Dortmund b Institut für Arbeitsphysiologie an der Universität Dortmund, ZWE Analytische Chemie, Ardeystraße 67, D-44139 Dortmund Z. Naturforsch. 52b, 1526-1532 (1997); received April 7, 1995 Pyrimidine-diones, Caffeine Metabolites, Synthesis 5-Acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3- methyluracil (AFMU) have been prepared by simple chemical transformations starting from thiourea and ethyl cyanoacetate. These compounds AAMU and AFMU are required as standard materials for qualitative identification and quantitative determination in connection with the metabolism of caffeine.

Introduction procedures are applied to obtain weighable amounts. The first method consisted in the con­ The N-acetyltransferase plays an important role sumption of a caffeinated beverage and the extrac­ in the metabolism of many xenobiotics; the pro­ tion of the metabolites from the urine; but the duction of the enzyme is genetically controlled. In­ yields are low [10]. A very expensive synthetic dividuals differ in the amount of the acetylated procedure is based on a procedure of Khmelevskii metabolites and can be classified as slow or rapid et al. [3] modified by Tang et al. [10] using 1-MU acetylators. The acetylator status is connected with instead of uric acid. After acylation with formic some diseases such as diabetes mellitus and blad­ acid/acetic anhydride the yield was only 19% pro­ der cancer and the knowledge of it is for the bene­ ducing 2 mg of AFMU 9. fit of therapeutics. In recent years, caffeine has been used as a non- invasive probe for the phenotyping of the acetyla­ Synthesis tor status. The metabolism of caffeine comprises a Pfleiderer and co-workers [1,6,7] have estab­ variety of reactions, such as oxidative demethyla- lished a synthesis of eight steps for AAMU 8 with tion, ring hydroxylation, ring-opening and acetyla­ an overall yield of 10%. While working on pyrimi­ tion. The ratios of the molar concentrations of uri­ dines, we have recognized a general interest in nary caffeine metabolites, like 5-acetylamino-6- some of our pyrimidine derivatives; AAMU 8 was formylamino-3-methvluracil (AFMU) (9), 5-ace- one of it. After analyzing the fundamental synthe­ tylamino-6-amino-3-methyluracil (AAMU) (8), 1- sis of Pfleiderer and co-workers [7] we were suc­ methylxanthine (1-MX) or 1-methyluric acid (1- cessful in shortening the sequence to six steps and MU) enable the determination of the acetylator in increasing the yield by modification of the reac­ status. tion conditions up to 42% ( cf. Scheme 1). For quantitative analytical determinations, Cyclization of thiourea (1) (NCN-compound) AFMU 9 or AAMU 8 are required as standard and ethyl cyanoacetate (2) (CCC-compound) - materials. Both metabolites are not available com­ based on a modified procedure of Taylor and mercially. Therefore biochemical and synthetic Cheng [12] and of Traube [13] - affords 99% of 6-amino-2-thiouracil (3) after treatment with base (NaOEt/EtOH/3h/reflux). The procedure is very simple and using an optimized work-up method it * Reprint requests to Prof. Dr. R. P. Kreher. is possible after complete removal of solvents to

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Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. R. Röhrkasten et al. ■ Synthesis of the Caffeine Metabolites 1527

h3c h3c N N N T A O'A, N O ^N nh2 I H

Scheme 1. rt: NaOEt / EtOH (3 h; reflux) - 99%; r2: (H3C0)2S02 / 2N NaOH (1 h; H3Cv 40 °C) - 62%; r3; 1 N NaOH (2 h; reflux) - HsC'NJ Y NH 91%; r4; NaOAc / HOAc / H.O / NaN02 (65 min; 100 °C) - 95%; r5: Pd;C / H2 / NaOH NI NH2 H A, (6-12 h; 20 °C) - 82%; r6: NaOAc / Xc20 (3 h; reflux) - 97%; r7: H C O -O -C O C H , / HCOOH (7 d; 20 °C) - 60-80%.

separate water insoluble compounds easily. Neu­ o c h 3 tralization with acetic acid instead of mineral acids N seems to be more effective, because the sodium JL + 4 h3cs n nh2 h3cs L n nh2 acetate formed a buffer and therefore organic salts aren’t formed. 2-Thio-4-amino-6-hydroxy-pyrimi- 10 dine is alkylated by means of methyl iodide or di­ Scheme 2. methyl sulphate at the sulfur atom [2], Regioselec- tive S- and N-alkylation of 3 at 1-position can be readily achieved with dimethyl sulfate in NaOH. the 2-methylthio group; with a 2-methoxy group The first step is the formation of a thiolate anion; O-alkylation did not occur. its enhanced reactivity is responsible for the for­ The methylthio group in 2-position represents a mation of 6-amino-2-methylthio-uracil (10). The favorite center for nucleophilic attack of hydrox­ rate of this step and the following reaction are al­ ide anions. 6-Amino-2-methylthio-3-methyluracil most the same. If not enough alkylating reagent is (4) reacts with 1 N NaOH (2 h/reflux) to give 91% used, starting material 3 is isolated beside of 6- of 6-amino-3-methyluracil (5). After neutraliza­ amino-2-methylthio-3-methyluracil (4) and 6- tion with acetic acid 5 can be isolated as colorless amino-4-methoxy-2-methylthio-uracil (11). Using solid. a 2.5fold excess of the alkylating reagent the py­ The nitrosation of 6-amino-3-methyluracil (5) rim idines 4 (62% ) and 11 (17%) can be synthe­ was carried out in diluted acetic acid with sodium sized (cf. Scheme 2). A direct synthesis of 10 can­ nitrite (65 min/100 °C). To increase yield and pro­ not be realized in this way. Alkylation of duct quality (microcrystals) the use of sodium ace­ independently synthesized 10 leads to the result tate as buffer seems to be advantageous. The that 4 and 11 were generated in the same ratio. higher reaction temperature is necessary because The unusual O-alkylation must be dependent on the starting material is almost insoluble. The insol- 1528 R. Röhrkasten et al. • Synthesis of the Caffeine Metabolites

üble violet 6-amino-5-nitroso-3-methyluracil (6) Formylation of AAMU 8 to prepare 6-ace- was collected and washed extensively with water tylamino-5-formylamino-3-methyluracil (AFMU) and carefully dried (48 h over P20 5; yield 95% (9) was achieved by Tang and co-workers [10] with with m.p. >350 °C). The main advantage of func- a mixture of formic acid/acetic anhydride (4:1). tionalizing with a nitroso group is the effective This method (yield: 19%) was not effective on a work-up procedure as well as the considerable preparative scale; therefore it seemed to be advan­ yield and the remarkable regioselectivity; the ena- tageous to use the mixed anhydride of formic and mine structure permits only nitrosation at 5- acetic acid, which can be synthesized as an reagent position. on a preparative scale [4], In earlier literature Raney-Nickel [5,11] was AAMU 8 was suspended and stirred in a 1:1 used to perform catalytic hydrogenation of the ni­ mixture of formic acid and acetic formic anhydride tro group at 5 position. Remarkable amounts of (7 d/20 °C). Thereby the turnover of AAMU 8 is the catalyst are required and lower yields can be not complete, because the resulting AFMU 9 is caused by adsorption of the product on surface. decomposed to AAMU 8, which can be reused. Using palladium [8] only small amounts of catalyst An indication for the increased reactivity of are required. Because of increased reactivity, the AFMU 9 is the formation of 5,6-diacetylamino-3- decision for an appropiate solvent is important: methyluracil (DAMU) (12) ( cf. Scheme 3). This The conversion of nitroso-pyrimidines in water transformation can be described as a transamida- leads presumably to the hydration of the CC- tion of the 6-formylamino-group. An analytical double bond, in methanol no transformation takes pure sample of DAMU 12 was isolated by semi­ place, because of the poor solubility of the nitroso preparative HPLC, so that characterization and compound 6. The sodium salt of the starting identification were possible. material is more polar then the free base and therefore soluble in methanol. For this reason so­ dium hydroxide was added with 10% excess to the mixture. Hydrogenation is successful with 10% palladium on charcoal in a mixture of methanol/ aqueous sodium hydroxide at 20 °C in 6-12 h; the 12 reaction is completed after the red solid has disap­ Scheme 3. peared. Work up must be performed rapidly and under an argon atmosphere. The neutralization AAMU 8 could be excluded as a source for the with hydrochloric acid have to be carried out ex­ formation of DAMU 12, because it cannot be de­ actly to pH = 7.0 (pH-electrode; otherwise the pre­ tected during the synthesis of AAMU 8 with acetic cipitate is very unstable); after this the precipitate anhydride under forced conditions. was filtered off and dried. The isolated 5,6-dia- Attempts to suppress the competitive reaction mino-3-methyluracil (7) is relative stable and can are not successful, because the turnover of be stored for a longer period at 20 °C without A A M U 8 is negligible below -5 °C. O therw ise the argon. consecutive reaction takes place with measurable Acylation of 7 at 5-position can be achieved re- amounts at T > 0 °C. At 40 °C the reaction can be giospecifically and with excellent yield. During stopped after 3-8 h, but the yield of AFMU 9 is acylation with acetyl chloride hydrochloric acid is unsatisfactory, because of the increased amount of developed, but bases cannot be used [9], Acylation DAM U 12 (-30%). The temperature of 20 °C of 5,6-diamino-uracil with acid chlorides in the seemes to be a good compromise: on the one hand presence of pyridine furnished the bisacylamino the turnover is 70-80% and on the other side the derivative; therefore an alternative process was formylating reagent is relatively stable under these applied to generate the free base. The diamino conditions and DAMU 12 is formed in only 5- com pound 7 was treated with an equimolar 10% yield. amount of acetic anhydride and dry sodium ace­ In solution AFMU 9 is relative unstable and the tate in glacial acetic acid (3 h/reflux); colorless quantitative separation is extremely difficult. AAMU 8 was separated in 97% yield. Chromatographic methods are not applicable, be- R. Röhrkasten et al. • Synthesis of the Caffeine Metabolites 1529 cause the separation is incomplete; deformylation or alumina F254 (E. Merck). HPLC was carried out of AFMU 9 on the column material seems to be with a Shimadzu LC-6A liquid chromatograph the main reason. Flash chromatography carried equipped with a photo diode array detector (SPD- out on silica gel with ice-cold trichloromethane/ M6A). Chromatography was performed with Su- methanol (9:1) at 20 °C gives rise to the generation perspher 100 RP-18 EC-4//m column (E. Merck); of -10-15% AAMU 8. Methanol (10%) is re­ 250 mm long and 4.6 mm i.d. for analytical appli­ sponsible for this effect, but this polar solvent is cations or Partisil 5 ODS 3 (Whatman); 250 mm always necessary for chromatography. Therefore long and 8.0 mm i.d. for preparative applications. the use of longer columns is not effective. High The elution mixture is water containing 28 /Al\ of quantities of AAMU 8 could be separated by re- formic acid and 6% of methanol (only analytical fluxing the raw solid for 10 min in dioxane con­ column). taining 3% of water. The diacetyl compound 12 could be largely separated by washing with small Experimental amounts of ice-cold trichloromethane/methanol The used chemicals are commercially available (9:1). Unfortunately, this operation produces again and were only purified if it was specially noted. small amounts of AAMU 8. Digestion with ace­ Acetic formic anhydride was synthesized accord­ tone (30 min/5 °C) gives AFMU 9 in -91-95% ing to the procedure of Krim en [4], purity on a preparative scale. AFMU 9 reacts at T > 35 °C easily with 6-Amino-2-methylthio-3-methyluracil (4) electrophiles; by this way AAMU 8 and DAMU 38.1 g (500 mmol) of thiourea (1) and 50.9 g 12 are formed. AFMU 9 reacts as well with nu­ (450 mmol) of ethyl cyanoacetate (2) were re­ cleophiles under moderate conditions (T < 20 °C) fluxed for 3 h in a solution of 20.7 g (900 mg-atom) and produces AAMU 8. The ambiphilic property of sodium and 300 ml of dry ethanol. After re­ of AFMU 9 is the main problem for separating moval of the solvent the residual 6-amino-2-thio- uracil (3) was dissolved in 500 ml (1013 mmol) of this urinary m etabolite [10]. 2 N NaOH and 171 ml (1800 mmol) of dimethyl sulfate were added and the mixture was vigorously Instrumentation and Procedures stirred for 1 h at 40 °C (exothermic reaction). Af­ ter cooling (16 h at 0 °C) the precipitate was col­ Melting points were determined on a Kofler- lected by filtration and washed neutral with 250 ml heatplate-microscope (Reichert) and are uncor­ of ice water and dried in vacuo. The raw product rected. The microanalyses were performed in the was suspended in 150 ml dry acetone and the solid micro chemical laboratory of the Institut für was collected by filtration and dried in vacuo ; Chemie der Universität Dortmund. The sub­ yield: 47.0 g (61%) of 4, m.p. 257 °C (ref. [7]; m.p. stances were usually dried at 20 °C in vacuo for 24 257 °C) and RF = 0.80 (alumina; methanol). An analytical sample was recrystallized from water. h over P20 5. Mass spectral data were collected The organic (acetone) filtrate gave 17% of 4- from a model CH-7 (Atlas Mat) or a 8230 (Finni- amino-6-methoxy-2-methylthio-pyrimidine (11), gan Mat) mass spectrometer. IR spectra were reg­ m.p. 194 °C (ref. [2]; m.p. 144 °C, yield 20% ), RF = istered with a Shimadzu IR-470 spectrometer as 0.88 (silica gel; methanol). KBr-disc. UV-Vis spectroscopic data were mea­ sured with a Varian Cary 17 D spectrometer. 6-Amino-2-thiouracil (3) NMR spectra were determined on a Varian EM C4H5N3OS (143.16) 360A (60 MHz) or a Bruker AM 300 (300 MHz). The chemical shifts are given in ppm as (3 values ’H NMR ([D6]-DMSO, 60 MHz): Ö (ppm) = referred to tetramethylsilane (TMS) with TMS or 4.70 (s, 1H, H-5); 3.50-4.90 (s(broad), 1H, H-l); 6.33 (s(broad), 2H, NH2); 10.60-11.60 (s(broad), [D6]-DMSO as internal standard. 13C NMR 1H, H-3). - MS (70 eV, 250 °C): m /z (% ) = 143 spectra were determined on a Bruker AM 300 (79) (M +); 127 (100); 115 (23); 99 (19); 84 (33); 68 (75.5 MHz) and the multiplicity of the signals was (86); 55 (16); 43 (89). - IR (KBr): v (cm“1) = 3450 established by the relative phase of the corre­ (m); 3340 (m); 3120 (m); 2990 (m); 2920 (m); 1640 sponding signal in the DEPT spectra. Thin layer- (vs); 1605 (s); 1555 (s); 1445 (s); 1310 (m); 1195 chromatography was performed on silica gel F254 (s); 800 (m); 620 (m). 1530 R. Röhrkasten et al. ■ Synthesis of the Caffeine Metabolites

6-Amino-2-methylthio-3-methyluracil (4) and 12.6 ml (220 mmol) of glacial acetic acid and 500 ml of water. During a period of 35 min a solu­ C6H9N3OS (171.22) tion of 16.56 g (240 mmol) of sodium nitrite in Calcd C 42.09 H 5.30 N 24.54%, 100 ml of water was dropped in. A fter 30 min stir­ Found C 42.22 H 5.47 N 24.30%. ring the resulting suspension was cooled to 20 °C *H NMR ([D6]-DMSO, 300 MHz): ö (ppm ) = and the violet microcrystalline precipitate was col­ 2.50 (s, 3H, SCH,); 3.27 (s, 3H, N C H 3); 4.92 (s, 1H, lected by filtration and washed twice with 50 ml of H-5); 6.43 (s(broad), 2H, NH,). - 13C NMR ([D6]- water and dried over P?05 for at least 48 h; yield: DMSO, 75.5 MHz): (3 (ppm ) = 14.18 (q, SCH 3); 35.56 g (95% ) 6, m.p. >350 °C (ref. [6]; m.p. 28.61 (q, N C H 3); 80.43 (d, C-5); 161.11 (s, C-6); >350 °C), R f = 0.00 (alumina; methanol). - 161.34 (s, C-4); 161.65 (s, C-2). - MS (70 eV, Recrystallization or chromatography was impossi­ 100 °C): m /z (% ) = 171 (91) (M +); 156 (16); 125 ble and 'H NMR- and 13C NMR spectra could not (89); 110 (22); 95 (20); 86 (15); 83 (36); 74 (17); 68 be recorded, because of the very poor solubility in (100); 57 (65): 43 (79). - IR (KBr): v (cm *1) = usual solvents. 3410 (s); 3335 (s); 3210 (s); 1670-1600 (s); 1574 C5H6N40 3 (170.13) (s); 1510 (s); 1501 (s); 1461 (s); 1451 (s); 1410 (s); Calcd C 35.30 H 3.55 N 32.93%, 1334 (m); 1284 (s); 1235 (m); 1098 (s); 809 (s); 758 Found C 34.90 H 3.48 N 32.24%. (m); 637 (m). - UV (methanol): 2max(nrn) (lg e) = 218 (4.41); 230 (4.39); 275 (3.85). MS (70 eV, 200 °C): m /z (% ) = 170 (84) (M +); 153 (93); 141 (20); 112 (47); 110 (39); 96 (100); 70 (36); 69 (36); 58 (56); 53 (46); 44 (20). - IR (KBr): 6-Amino-3-methyluracil (5) v (cm“1) = 3240 (s); 3015 (m); 1727 (vs); 1698 (s); 42.81 g (250 mmol) of 6-amino-2-methylthio-3- 1656 (vs); 1533 (m); 1462 (s); 1444 (s); 1334 (s); methyluracil (4) in 700 ml (600 mmol) of 1324 (s); 1308 (m); 1288 (s); 1280 (s); 1207 (m); 1 N NaOH were refluxed for 2 h. To the boiling 1090 (m); 791 (m); 770 (s); 704 (m); 644 (m). - solution 46 ml (800 mmol) of glacial acetic acid UV (methanol): 2max(nm) (lg e) = 224 (3.94); 245 were added and then the mixture was cooled for (sh, 3.64); 319 nm (4.20). 16 h at 0 °C. The crystalline precipitate was col­ lected by filtration and washed three times with 5,6-Diamino-3-methyluracil-semihydrate (7) 50 ml of ice water and dried in vacuo; yield: 32.0 g 32.32 g (190 mmol) of 6-amino-5-nitroso-3- (91%) of 5 as colorless needles, m.p. 342-346 °C methyluracil (6) were hydrogenated with shaking (ref. [6]; m.p. 327 °C), RF = 0.75 (silica gel; at 20 °C for 6-12 h at an initial hydrogen pressure m ethanol). of 3.5 bar in a suspension of 8.00 g (200 mmol) of C5H7N30 2 (141.13) sodium hydroxide and 940 mg (10 mmol) of Pd- Calcd C 42.55 H 5.00 N 29.77%, catalyst (10% on charcoal) in 300 ml of methanol/ Found C 42.81 H 5.01 N 29.46%. water (2:1). When the red solid is consumed, the 'H NMR ([D6]-DMSO, 300 MHz): Ö (ppm ) = reaction mixture was filtered under argon and the 3.01 (s, 3H, N CH 3); 4.56 (s, 1H, H-5); 6.24 solid residue was washed twice with 50 ml of (s(broad), 2H, NH2); 10.10-10.50 (s(broad), 1H, water. The combined extracts were concentrated H-l). - 13C NMR ([D6]-DMSO, 75.5 MHz): d to 100 ml, neutralized with conc. hydrochloric acid (ppm) - 26.09 (q, N C H 3); 74.23 (d, C-5); 151.40 (s, exactly to pH = 7.0 (definitely recommended; pH- C-2); 153.83 (s, C-6); 163.34 (s, C-4). - MS (70 eV, electrode) and the solid was filtered off immedi­ 280 °C): m /z (%) = 141 (100) (M+); 111 (22); 84 ately under argon and dried in vacuo; yield: (48); 68 (68); 44 (19); 43 (59). - IR (KBr): v (cm “ 1) = 25.73 g (82%) of 7. m.p. 280 °C (dec.) (ref. [6]; m.p. 3425 (s); 3320 (m); 3205 (m); 1682 (vs); 1652 (vs); >340 °C), Rf? = 0.00 (alumina; methanol). An ana­ 1631 (vs); 1596 (vs); 1554 (m); 1456 (s); 1386 (m); lytical sample was recrystallized from water with a 974 (m); 792 (m); 764 (m); 657 (m); 559 (m). - small amount of charcoal. UV (methanol): l max(nm) (lg e) = 220 (3.45); 264 C,H8N40 ,0 .5 H ,0 (165.15) (4.25). Calcd C 36.36 H 5.49 N 33.92%, Found C 36.33 H 5.32 N 33.83%. 6-Amino-5-nitroso-3-methyluracil (6) 'H NMR ([D6]-DMSO. 300 MHz): (3 (ppm) = 31.05 g (220 mmol) of 6-amino-3-methyluracil 3.06 (s, 3H, N CH 3); 3.40-4.40 (s(broad), 3H, 5- (5) were suspended at 100 °C in a solution of NH2, 0.5 H?0); 5.72 (s(broad), 2H, 6-NH2). - 13C 29.94 g (220 mmol) of sodium acetate-trihydrate NMR ([D6]-DMSO, 75.5 MHz): (3 (ppm) = 26.78 R. Röhrkasten et al. • Synthesis of the Caffeine Metabolites 1531

(q, N C H 3); 95.50 (s, C-5); 142.94 (s, C-2); 149.67 of formic acid/acetic formic anhydride (1:1) and (s, C-6); 160.67 (s, C-4). - MS (70 eV, 215 °C): m / stirred for 7 d at 20 °C with exclusion of moisture; z (% ) = 156 (100) (M +-0.5 • H20 ) ; 71 (43); 44 0.5 ml of acetic formic anhydride were added ev­ (61). - IR (KBr): v (cm-1) = 3490 (m); 3390 (s); ery day (after 2 d 8 was almost dissolved and gen­ 3205 (s); 1700-1610 (s); 1590-1510 (s); 1456 (s); erally a colorless solid precipitates). The solvents 1385 (m); 1366 (m); 1284 (m); 1258 (m); 1181 (m); were removed and the solid (-2.4 g; >100%) was 1109 (m); 755 (m); 723 (m); 702 (m); 655 (m). - suspended in 247 ml of dioxane/water (97:3), re­ UV (methanol): Amax(nm) (lg e) = 219 (sh, 3.32); fluxed for 10 min and insoluble 8 was removed by 260 (3.88); 285 (2.96). filtration. After the filtrate was cooled to 20 °C the precipitate was filtered off; concentration of the 5-Acetylamino-6-amino-3-methyluracil-hydrate filtrate to 20 ml under reduced pressure (T < (AAMU) (8) 40 °C) gives another fraction. The residues (1.6- 2.0 g) were suspended in 40 ml of acetone (30 min/ 16.52 g (100 mmol) of 5,6-diamino-3-methylura- 4 °C) and than washed three times with 5 ml of cil-semihydrate (7) in 150 ml of glacial acetic acid ice-cold trichloromethane/methanol (9:1) and were refluxed for 3 h with a mixture of 9.02 g dried over P20 5; yield: 1.35-1.80 g (60-80%) col­ (110 mmol) of dry sodium acetate and 11.23 g orless 9, mp. 227-229 °C (dec), R F = 0.20 (silica (110 mmol) of absol. acetic anhydride. The mix­ gel; ethyl acetate/acetic acid (9:1)); tHPLC = ture was cooled to 20 °C, and the solid was filtered 6.70 min. - HPLC-purity: -9 1 -9 5 % 9, - 3 - 7 % 8, off and washed three times with 50 ml of water -1 -3 % 12. and dried in vacuo\ yield: 19.24 g (89%) of 8 as colorless microcrystals. The filtrate was evapo­ C8H 10N4O4 (226.19) rated to dryness in vacuo and the yellow residue Calcd C 42.48 H 4.46 N 24.77%, recrystallized from 100 ml of water and dried over Found C 41.4 H 4.2 N24.1%. P20 5; further 1.70 g (8% ) of colorless 8 is ob­ *H NMR ([D6]-DMSO, 300 MHz): <3 (ppm) = tained. Overall yield: 20.94 g (97%), subl.-p. 1.96 (s, 3H, C O C H 3); 3.12 (s, 3H, N C H 3); 8.35 276 °C; m.p. >350 °C (ref. [1]; m.p. >350 °C); R F = (s(broad), 1H, CHO); 8.83 (s, 1H, NH); 10.53; 0.16 (alumina; methanol).- RF = 0.12 (silica gel; 11.03 (s(broad), 2 • 1H, 2 • NH). - 13C NM R ([D 6]- ethyl acetate/acetic acid (9:1)); tHPLC = 4.52 min. DMSO, 75.5 MHz): (3 (ppm) = 22.87 (q, COCH3); C7H 10N4Or H,O (216.20) 27.25 (q, N C H 3); 96.15 (s, C-5); 142.64 (s, C-2); Calcd C 38.89 H 5.59 N 25.92%, 149.23 (s, C-6); 161.26 (s, C-4); 162.79 (d, CH O ); Found C 39.13 H 5.85 N 26.21%. 170.10 (s, C O C H 3). - MS (70 eV, 170 °C): m /z (% ) = 226 (10) (M +); 184 (33); 156 (29); 71 (27); NMR ([D6]-DMSO, 300 MHz): (3 (ppm) = 44 (24); 43 (100); 42 (18). - IR (KBr): v (cm “1) = I.91 (s, 3H, C O C H 3); 3.04 (s, 3H, N C H 3); 6.06 3425 (m); 3245 (s); 3065 (m); 2935 (m); 1739 (s); (s(broad), 2H, N H 2); 8.37 (s, 1H, N H C O ); 10.22- 1715 (s); 1678 (s); 1641 (vs); 1561 (m); 1524 (m); II.03 (s(broad), 1H, H-l). - 13C NMR (5% 1508 (m); 1301 (m); 1204 (m); 752 (m). - UV Na0D/D20/[D6]-DMS0 as int. standard, 75.5 (methanol, <5 min after preparation of the solu­ MHz): <3 (ppm) = 19.21 (q, COCH3); 25.09 (q, tion): Amax(nm) (lg e) = 210 (4.32); 286 (4.13). - N C H 3); 85.60 (s, C-5); 156.79 (s, C-2); 158.33 (s, C- UV (water): Amax(nm) (lg e) = 207.5 (4.24); 285 6); 161.30 (s, C-4); 172.40 (s, C O C H 3). - MS (70 (4.15). eV, 170 °C): m /z (% ) = 198 (26) (M +-H 20 ); 157 (11); 156 (100) ([M+- H 70 ]-C 0 C H 2); 155 (20); 71 5,6-Diacetylamino-3-methyluracil (DAMU) (12) (34); 70 (16); 44 (15); 43 (66). - IR (KBr): v (cm -1) = 3545 (s); 3435 (m); 3365 (m); 3165 (s); 3055 (m); DAMU 12 was separated by semi-preparative 1711 (s); 1667 (s); 1640 (s); 1580-1530 (s); 1470 HPLC; subl.-p. 160 °C (needles developed) and (s); 1377 (m); 1318 (m); 969 (m); 778 (m); 750 (m); m.p. 257 °C.- Rf - 0.61 (alumina; methanol); 713 (m); 638 (m). - UV (methanol): 2max(nrn) (lg ^hplc — 11-20 min. e) = 218 (3.82); 264 (4.25). - UV (water): C9H 12N40 4 (240.22) l mas(nm) (lg e) = 223.5 (3.84); 264 (4.27). 'H NMR ([D6]-DMSO, 300 MHz): (3 (ppm) = 1.97 (s, 3H, 5-COCH3); 2.14 (s, 3H, 4-CO CH 3); 5-Acetylamino-6-formylamino-3-methyluracil 3.12 (s, 3H, N CH 3); 8.75; 10.06; 11.32 (s, 3-1H , (AFMU) (9) 3-NH). - 13C NMR ([D6]-DMSO, 75.5 MHz): (3 2.16 g (10.0 mmol) of 5-acetylamino-6-amino-3- (ppm) = 23.04; 24.20 (q, 2COCH3); 27.30 (q, methyluracil-hydrate (8) were suspended in 6 ml N C H 3); 96.16 (s, C-5); 143.34 (s, C-2); 148.91 (s, C- 1532 R. Röhrkasten et al. • Synthesis of the Caffeine Metabolites

6); 161.31 (s, C-4); 169.99 (s, 6-CO CH 3); 172.46 (s, (cm-1) = 3435 (m); 3250 (m); 1737 (m); 1719 (m); 5-COCH,). - MS (70 eV, 110 °C): m/z (% ) = 241 1685 (m); 1634 (vs); 1526 (m); 1298 (m); 1230 (m); (6) (M ++ l); 240 (47) (M +); 198 (54); 156 (100) 750 (m). - UV (methanol): Amax(nrn) (lg e) = 206 (M+-2CH?CO); 155 (30); 43 (44). - IR (KBr): v (4.23); 282 (4.15).

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