Reaction between 2prime;,3prime;,5prime;-tri-O-acetyladenosine and aryl chloroformates. 2prime;,3prime;,5prime;-Tri-O-acetyl-N(6)-phenoxycarbonyladenosine as an intermediate in the synthesis of 6-ureidopurine ribosides

摘要

1978 131 Reaction Between 2',3',5'-Tri-O-acetyladenosine and Aryl Chloroformates. 2',3',5'-Tri -O-acetyi-N(6)-p henoxyca rbonyladenosine as an Intermediate in the Synthesis of 6-Ureidopurine Ribosides By P. Anne Lyon and Colin B. Reese,**tUniversity Chemical Laboratory, Lensfield Road, Cambridge CB2 1 EW Reaction between 2'.3',5'-tri-U-acetyladenosine (6) and p-nitrophenyl chloroformate in pyridine solution at 20 "C or between (6) and phenyl chloroformate in pyridine solution at 70 "C gives the protected symmetrical urea derivative (7b) in moderate or high yield. Deacetylation of (7b) gives the corresponding unprotected urea (7a). Treatment of (6) with an excess of phenyl chloroformate in pyridine solution at 20 "C gives the bisphenoxycarbonyl derivative (10) which, on reaction with morpholine in dioxan solution, is rapidly converted into 2',3'.5'-tri-O-acetyl-N( 6)-phenoxycarbonyladenosine (3c).Compound (3c) readily reacts with cyclohexylamine, ammonia, and glycine methyl ester at 20 "C to give, after deacetylation, the corresponding 6-ureidopurine riboside derivatives (9b, c, and d; R1 = H) in high yields. THE 6-ureidopurine riboside N-9-((3-D-ribofuranosy1)-found that the reaction between (3a) and L-threonine in SH-purin-6-ylcarbamoyl-~-threonine(la) occurs as a hypermodified nucleoside at the 3'-ends of the anticodons in yeast lysyl,2 rnethi~nyl,~ and other transfer ~eryl,~ ribonucleic acids (tRNAs). The related N-(purin-6-y1- carbamoy1)glycine riboside (2) and (lb), the N(6)-methyl derivative of (la), also occur in tRNA.In connection with our work on the synthesis of oligoribo- nucleotides,' we were interested in developing a con-venient general method for the preparation of 6-ureido- purine ribosides such as (1)and (2). CO 2H RAN-LH HO OH (1) a; R = H b; R = Me The most obvious approach to the synthesis of such unsymmetrical ureas (Scheme 1) involves the reaction between a protected adenosine carbamate (3) and a protected or unprotected amino-acid (4). This approach has been favoured by previous workers, who have used the comparatively unreactive carbamate (3a) as the starting material, thereby making relatively drastic reaction conditions necessary. Thus Chheda and Hong Presenl address: Department of Chemistry, King's College, Strand, London WC2R 2LS.G. B. Chheda, R. H. Hall, D. I. Magrath, J. Mozejko, M. P. Schweizer, L. Stasiuk, and P. R. Taylor, Biochemistry, 1969, 8, 3278; M. P. Schweizer, G. B. Chheda, L. Baczynskyj, and R. H. Hall, ibid., p. 3283. J. T. Madison, S. J. Boguslawski, and G. H. Teetor, Science, 1972, 176, 687. M. Simsek and U. L. RajBhandary, Biochem. Biophys. Res. Comm., 1972, 49, 508. H. G. Zachau, D. Dutting, and H. Feldmann, ,4ngew. Chem. Internat. Edn., 1966, 5, 422; 2.physiol. Chem., 1966, 347, 212. pyridine solution required 5 h at 100 "C; these workers obtained (la) in ca. 45 yield after removal of the , protecting groups. Similar results were subsequently obtained -in- another lab~ratory.~ We believed that it would be advantageous to start with a more reactive carbamate than (3a) and therefore investigated the reaction between 2',3',5'-tri-O-acetyladenosine (6) and aryl chloroformates.The reaction between 2',3',5'-tri-O-acetyladenosine (6) and a slight excess of 9-nitrophenyl chloroformate in HO OH pyridine solution proceeded rapidly at 20 "C and gave a product which could not be induced to crystallize. The n.m.r. spectrum of this product, which contained no signals assignable to 9-nitrophenyl protons, was con-sistent with its being the symmetrical urea derivative (7b). However, its U.V. absorption spectrum (see below) was quite unlike that of a simple N(6)-acyl- or -carb-amoyl-adenosine derivative. Treatment of (7b) with an excess of 9-nitrobenzoyl chloride in pyridine solution gave a crystalline bis-9-nitrobenzoyl derivative in 66y0 M.P. Schweizer, K. McGrath, and L. Baczynskyj, Biochem. Biophys. Res. Comm., 1970, 40,1046. F. Kimura-Harada, D. L. von Minden, J. A. McCloskey, and S. Nishirnura, Biochemistry, 1972, 11, 3910. For reviews of this work see C. B. Reese, Colloques Inter- nationaux du C.N.R.S., 1970, No. 182, 319; C. B. Reese, Phos-phorus and Sulfur, 1976, 1, 245. G. B. Chheda and C. I. Hong, J. Medicin. Chem., 1971, 14, 748. 9 R.W. Adamiak and M. Wiewihrowski, Bull. Acad. polon. Sci., Sir. Sci. chim., 1975, 23, 241. P32 yield. The U.V. absorption spectrum of tlie latter was consistent with structure (7c). When (7b) reacted with methanolic ammonia at 20 "C for 48 h, the unprotected symmetrical urea derivative (7a) was obtained crystalline in high yield.Treatment (3) a; R1 = Ac, R2 = Et b; R1 Ac, R2 = 4-02N*CgHr, C; R1 = Ac, R2 = Ph d; R1 = H, R2 = Me J.C.S. Perkin I jsee (b). The latter spectrum is typical for an N(6)-acyl derivative of adenosine. The remarkable U.V. spectrum of (7a), the absence of a molecular ion in its mass spectrum, and several unsuccessful attempts to obtain satisfactory microana.lytica1 data corresponding SCHEME1 of compound (7a) with acetic anhydride in pyridine regenerated (7b). The U.V. absorption spectrum of (7a), which has intense maxima at 283 and 291 nm and Ac 0dN$j0 R1N NR'I I R20 OR2 HN NHI I closely resembles that of (7b), is illustrated in Figure 1(a).For comparison, the spectrum of N(6)-carbamoyl- adenosine (9c; R1 = H) (see below) is also illustrated to anhydrous (7a) led us to seek further confirmation for this structural assignment. The fact that (7a) contains two identical nucleoside residues was confirmed by converting it, via its di-iso- propylidene derivative (see Experimental section) into its analytically pure crystalline monoisopropylidene deriva- tive (8). The di-isopropylidene derivative was readily obtained as a crystalline monohydrate in 92 yield by treating (7a) with 2,2-dimethoxypropane loyll in the presence of toluene-9-sulphonic acid in dioxan solution. 35 uu 220 240 260 280 300 320 200 220 2LO 260 280 300 X/nm Xlnm U.V.absorption spectra of (a) NN'-bis-9-(@-D-ribofuranosyl)-purin-6-yllurea (7a) and (b) N(6)-carbamoyladenosine (9c;R1= H) in 95 ethanol The U.V. spectra of both (8) and the di-isopropylidene derivative closely correspond to that of (7a). Further evidence in support of structure (7a) was adduced from lo A. Hampton, J. Amer. Chem. Soc., 1961, 83, 3640. l1 H. P. M. Fromageot, B. E. Griffin, C. B. Reese, and J. E. Sulston, Tetrahedron, 1967, 23. 2315. hydrolysis studies. When (7a) was heated in aqueous solution, under reflux, for 4 h, adenosine was obtained as the sole product (isolated in 82 yield). Under the same conditions, a related compound, N(6)-phenyl- carbamoyladenosine l2 (9a; R1 = H), prepared by de- acetylation of the product from treating 2lsquo;,3lsquo;,5rsquo;-tri-O- acetyladenosine (6) with phenyl isocyanate, similarly underwent quantitative hydrolysis to give adenosine.When (7a) was heated in aqueous M-sodium hydroxide at 100 ldquo;C for 20 h it was also completely converted into adenosine; however, (7a) was unchanged after 18 h in the same alkaline medium at 20 ldquo;C. Additional support for its structural assignment was provided by the observation that when (7a) was heated, under reflux, with an excess of neat cyclohexylamine for 10 min, it was quantitatively converted into adenosine (92 isolated yield) and N(6)-cyclohexylcarbamoyladenosine (9b; R1 = H); SSyo isolated yield. Compound (9b; R1 = H) was prepared independently by treating 2rsquo;,3rsquo;,5rsquo;-tri-O-acetyladenosine(6) with cyclohexyl iso-cyanate and then deacetylating the product.The remarkable U.V. spectra of (7a) Figure l(a), (7b), the di-isopropylidene derivative of (7a), and (8) are pre-sumably due to base-stacking or to some other inter- action between the two adenine residues in these com- pound~.~~ R10 OR1 AcO OAc After the unsuccessful attempt to prepare 2rsquo;,3rsquo;,5rsquo;-tri-0-acetyl-N(6)-p-nitrophenoxycarbonyladenosine(3b), the preparation of the corresponding phenyl carbamate (3c) was undertaken. Treatment of 2rsquo;,3rsquo;,5rsquo;-tri-O-acetyl- adenosine (6) with 1.2 mol. equiv. of phenyl chloro- formate in pyridine solution at 70 ldquo;C gave (7b) in high yield ; deacetylation of the products with methanolic ammonia gave (7a) as a crystalline solid in 83 overall yield.However, when (6) was treated with a threefold excess of phenyl chloroformate in pyridine solution * By comparison with the structures of the corresponding diaroyl derivatives of (6) P. A. Lyon and C. B. Reese, J.C.S. Perkin I, 1974, 26451, this compound is assumed to be an N(6),N(6)- rather than an N(l),N(g)-derivative.t Since the preparation of the manuscript of this paper, R. W. Adamiak and J. Stawinski (personal communication ; Tetra-hedron Letters, 1977. 193.5) have reported an alternative synthesis of (3c). at 20 ldquo;C, 2rsquo;,3rsquo;,5rsquo;-tri-O-acetyl-N(6),N(6)-bisphenoxy-carbonyladenosine * (10) was obtained and could be isolated as a crystalline solid in 79 yield. When smaller quantities of phenyl chloroformate were used, t.1.c.did not reveal the presence of the desired phenyl carbamate (3c) but only mixtures of (10) and starting material (6). The bisphenoxycarbonyl compound (10) proved to be a reactive precursor of 6-ureidopurine riboside deriva- tives. Thus treatment of (10) with a five-fold excess of cyclohexylamine in dioxan solution at 20 ldquo;C for 1 h gave 2rsquo;,3rsquo;,5rsquo;-tri-0-acetyl-N(6)-cyclohexylcarbamoyl-adenosine (9b; R1 = Ac) as the sole nucleoside product, which was isolated as a crystalline solid in SOY0 yield. An obvious disadvantage which (10) has as a synthetic intermediate is that an excess of what may be a valuable amino-compound must be used in conjunction with it. Fortunately, however, (10) reacted rapidly with a stoicheiometric quantity of morpholine in dioxan solution at 20 ldquo;C to give the desired 2rsquo;,3rsquo;,5rsquo;-tri-O-acetyl- N(6)-phenoxycarbonyladenosine(3c), which was isolated crystalline in 55 yield.2rsquo;,3rsquo;, 5rsquo;-Tri-O-ace tyl-N (6) -phenox ycarbon yladenosine (3c) had precisely the properties required in a precursor of 6-ureidopurine riboside derivatives. 7 When (3c) was treated with a stoicheiometric quantity of cyclohexyl- amine in dioxan solution at 20 ldquo;C, complete reaction occurred within 1 h. Deacetylation of the products with methanolic ammonia gave N(6)-cyclohexylcarb-amoyladenosine (9b; R1 = H), which was isolated crystalline in 92 overall yield. In contrast to the latter experiment, the reaction between N(6)-methoxy- carbonyladenosine (3d) and 1.5 mol.equiv. of cyclo-hexylamine in boiling pyridine solution was only ca. 75 complete after 4.5h; after 16 h under these conditions, N(6)-cyclohexylcarbamoyladenosine (9b; R1 = H) was obtained in quantitative yield. The value of 2rsquo;,3rsquo;,5rsquo;-tri-O-acetyl-N(6)-phenoxy-carbonyladenosine (3c) as a synthetic intermediate was further demonstrated in two other experiments. First, the reaction between (3c) and a saturated solution of ammonia in dioxan was complete within 2 h at 20 ldquo;C. Deacetylation of the product with methanolic ammonia gave N(6)-carbamoyladenosine (9c; R1 = H) as a crystalline hemihydrate in 92 overall yield. Secondly, (3c) was treated with slight excesses of glycine methyl ester hydrochloride and triethylamine in dioxan at 20 ldquo;C.After 18 h, when the reaction was complete, the products were worked up and treated with sodium methoxide in methanol to give N(6)-methoxycarbonyl-methylcarbamoyiadenosine (9d; R1 = H) as a crystal- line solid in 78 overall yield. Compound (9d; R1 = H) is the methyl ester of (2),the simplest 6-ureidopurine riboside which occurs naturally in tRNA. Although the reaction between (3c) and glycine methyl ester in dimethylformamide solution was complete within 1 h 12 J. J. McDonald, N. J. Leonard, R. Y.Schmitz, and F. Skoog, Phytochemistvy, 1971, 10,1429. 13 J. Temlieka and J. Owens, J. Org. Chem., 1977, 42, 517. at 20 ldquo;C, the above reaction conditions in dioxan were found to be more convenient. AcO OAc (1 1) Finally, it seems likely that the mechanism of the J.C.S.Perkin I bis-9-(2,3,5-tri-O-acetyl-P-~-ribofuranosyl)purin-6-ylurea (0.406 g, 0.5 mmol) in anhydrous pyridine (10 ml) and the reactants were stirred at 20 ldquo;C. After 30 min the products were poured onto ice (60 g) and the mixture was stirred as the ice melted. The precipitate was filtered off and dissolved in chloroform. The solution was extracted with ice-cold saturated aqueous sodium hydrogen carbonate, dried (MgSO,), and evaporated under reduced pressure. The solid was dissolved in CHC1,-MeOH (99.4 : 0.6 v/v)and fractionated by short column chromatography 85 g of silica gel; CHC1,-MeOH (99.4: 0.6 v/v). The appropriate fractions were combined and concentrated under reduced pressure to give a solid which was carefully recrystallized reaction between 2rsquo;,3rsquo;,5rsquo;-tri-O-acetyl-N(6)-phenoxy-from hot ethanol (without boiling the solution) to give the carbonyladenosine (3c) and amino-compounds involves elimination of phenol l4 to give the 6-isocyanato-derivative (11) as an intermediate which then reacts with the amino-compound.EXPERIMENTAL lH N.m.r. spectra were measured at 100 MHz with a Varian HA- 100 spectrometer (tetramethylsilane used as internal standard). Mass spectra were recorded with an A.E.I. MS9 spectrometer, i.r. spectra with a Perkin-Elmer 257 G spectrometer, and U.V. spectra with a Pye Unicani SP 1800 recording spectrophotometer. Thin-layer chromatograms were run on glass plates coated with Merck Kieselgel F,,, in the following solvent systems: (A) CHC1,-MeOH (80 : 20 v/v), (B) CHC1,-MeOH (95 : 5 v/v), (C) CHC1,-MeOH (97.5: 2.5 v/v) ; and on Merck DC-Alufolien Cellulose F254in solvent system (D): butan- l-ol-acetic acid-water (5 : 2 : 3).Reeve Angel Silica Gel/CT was used for short column chromatography. Pyridine was dried by heating under reflux with calcium hydride and then distilled. Reaction between 2lsquo;, 3rsquo;, 5rsquo;-Tri-O-acetyladenosine (6) and p-Nitrophenyl Chloroformate.-p-Nitrophenyl chloroformate (0.222 g, 1.1 mmol) was added to a stirred solution of 2rsquo;,3rsquo;,5rsquo;-tri-O-acetyladenosine (0.393 g, 1.O mmol) in an-hydrous pyridine (5 ml) at 20 ldquo;C. After 2 h, water (0.1 ml) was added and, after a further 1 h the products were concentrated under reduced pressure.The resulting gum was dissolved in chloroform (10 ml), and the solution extracted with saturated aqueous sodium hydrogen carbonate (10 ml), dried (MgSO,) and evaporated under reduced pressure. The residual gum was dissolved in dichloromethane and the solution applied to a column of Mallinckrodt SilicAR CC4 (10 g). The column was eluted with (a) CHCl,-CH,Cl, (3 : 1 v/v) and (b) CHC1,-EtOH (99 : 1 v/v). The latter fractions (b) were combined and evaporated under reduced pressure to give NNrsquo;-bis-9-(2,3,5-tri-O-acet~l-~-~-~Zbo~ura~~syl)purin-as a6-ylIurea t.1.c. homogeneous RF 0.33 (system B)glass (0.24 g, 59) ; T(CDC1,) 1.14 (2 H, s), 1.45 (2 H, s), 3.70 (2 H, d, J 5 Hz), 3.94 (2 H, m), 4.28 (2 H, m), 5.57 (6 H, m), 7.86 (6 H, s), 7.90 (6 H, s), and 7.94 (6 H, s); A,,,.(95 ethanol) 291, crystalline NNrsquo;-bis-p-nitrobenzoyl derivative Found (material dried in vucuo over P,O, at 90rdquo;): C, 51.0; H, 3.8; N, 15.1. C,,H,,Nl,Ol, requires C, 50.8; H, 3.8; N, 15.1, m.p. 125-126rdquo;; yield 0.368 g (66); T(CDC1,) 1.28 (2 H, s), 1.55 (4 H, d, J 9 Hz), 1.74 (4 H, d, J 9 Hz), 1.91 (2 H, s), 3.92 (2 H, d, J 5 Hz), 4.23 (2 H, m), 4.40 (2 H, m), 5.58 (6 H, m), 7.87 (6 H, s), and 7.92 (12 H, s); A,,,. (95ethanol) 270 (E 37 loo), Alnin. 234 nm (E 17 100). NNrsquo;-Bis-9-(P-~-ribofuranosyl)purin-6-yZurea (7a).-(a) 2rsquo;,3rsquo;,5rsquo;-Tri-O-acetyladenosine(0.393 g, 1.0 mmol) was treated with p-nitrophenyl chloroformate (0.222 g, 1.1 mmol) in pyridine (5 ml) according to the procedure described above and the products were worked-up in the same way but not chromatographed.The crude material was dissolved in methanol (5 ml) and the stirred solution treated with methanolic ammonia (half-saturated at 0 ldquo;C; 5 ml) at 20 ldquo;C. After 48 h, the products were filtered off and the crystalline residue was washed with ice-cold methanol. Careful crystallization from water gave NNrsquo;-bis-9-(~-~-r~bofurunosyZ)pur~n-6-yZureamonohydrate Found (material dried in vacua over P,O, at 100 ldquo;C): C, 43.6; H, 4.3; N, 24.05. C21H,,Nl,0,,H,0 requires C, 43.6; H, 4.5; N, 24.2 as crystals, m.p. 165-166rsquo;; yield 0.164 g (57); RF0.09 (system A), 0.35 (system D) ; T(CD,),SO-D,O 1.31 (2 H, s), 1.34 (2 H, s), 3.96 (2 H, d, J 5.5 Hz, 5.41 (2 H, m), 5.78 (2 H, m), 5.97 (2 H, m), and 6.32 (4 H, m); Amax, (9574 ethanol) 298, 284, and 266 (amp; 36 200, 36 200, and 22 300), Amin, 289, 269, and 235 nm (E 34 400, 21 300, and 8 000); Amax, (0,1~-HCl)292 and 265 (34 300 and 15 600), Amin.268 and 242 (15 500 and 11 800); Amax. (0.1M-NaOH) 322 and 274 (37 000 and 12 600), Ajnfl. 268 (12 loo), Amin. 286 and 244 (6 000 and 6 700); vmax. (Nujol) 3 600-3 000br,m and 1 720m cm-l; m/e 293 (M -267)rsquo;. (b) Phenyl chloroformate (5.64 g, 36 mmol) was added to a stirred solution of 2rsquo;,3rsquo;,5rsquo;-tri-O-acetyladenosine(1 1.79 g, 30 mmol) in anhydrous pyridine and the reactants were heated to 70 ldquo;C. After 1 h, the products were cooled to room temperature and water (0.6 ml) was added; after a further 30 min the products were concentrated under reduced pressure.The resulting gum was dissolved in chloroform (300 ml) and the solution extracted with aqueous sodium hydrogen carbonate (300 ml), dried (MgSO,), and concentrated under reduced pressure. 284, and 266; Amin. 289, 269, and 235 nm; v,~~~.(CHC~,) 3 360w, 3 020m, 2 990m, 1 7453, 1 6lOs, and 1 590s cm-l; m/e 419 (M-393)+. NNlsquo;-Bis-p-nitrobenzoyl-NNrsquo;-bis-19-(2,3,5-tri-O-ucetyl-P-~-ribof~ranosyZ)purin-6-yZureu(7c) .-p-Nitrobenzoyl chloride (0.928 g, 5.0 mmol) was added to a stirred solution of NNlsquo;-* Care should be taken during the recrystallization of this compound as prolonged heating in aqueous solution leads to its conversion into adenosine (see text and below).The residual gum was dissolved in methanol (150 ml) and the stirred solution treated with methanolic ammonia (half- saturated at 0 ldquo;C; 150 ml) at 20 ldquo;C. After 48 h the products were filtered and the crystalline residue was washed with ice-cold methanol and then recrystallized from hot water * (600 ml) to give NNrsquo;-bis-9-(P-~-ribofuranosyl)-14 M. L. Bender and R. B. Homer, J. Org. Chem., 1965, 30, 3975. purin-6-yllurea monohydrate as crystals, m.p. 165-166' ; were filtered and the residue was carefully crystallized from yield 7.20 g (8304). hot water to give N-9-(2,3-O-~sopropylidene-~-~-ribofurano-asA cetylation of NN'-Bis-9-( P-~-ribofuranosyl)purzn-6-yl-syl)pur~n-6-yl-N'-9-(~-~-ribofuranosyl)purin-6-yZurea urea (7a) .-Acetic anhydride ( 1.63 g, 16 mmol), NN'-bis- 9-(~-~-ribofuranosyl)purin-6-ylureamonohydrate (1.16 g, 2.0 mmol) and anhydrous pyridine (10 ml) were stirred together at 20 "C.After 16 h, methanol (2 ml) was added and, after a further 1 h, the products were concentrated under reduced pressure. The resulting gum was dissolved in chloroform (40 ml) and the solution extracted with saturated aqueous sodium hydrogen carbonate (40 ml) , dried (MgSO,), and evaporated under reduced pressure. The glass obtained was identical RF(system B); n.m.r., i.r., u.v., and mass spectra with the NNr-bis-9-(2,3,5-tri-0-acetyl-~-~-ribofuranosy~)purin-6-y~ureaobtained from the reaction between 2', 3', 5'-tri-O-acetyladenosine and p-nitro- phenyl chloroformate.NN'-Bis-9-(2,3-O-isopropylidene-P-~-ribofuranosyl)puran-6-ylurea.-2,2-Dimethoxypropane (4.16 g, 40 mmol) was added to a stirred suspension of NN'-bis-9-( P-D-ribo- furanosyl)purin-6-ylurea monohydrate (1.16 g, 2.0 mmol) and toluene-p-sulphonic acid monohydrate (0.84 g, 4.4 mniol) in anhydrous dioxan (40 ml) at 20 "C. After 2 h, water (0.6 ml) was added, and after a further 30 min the products were neutralized with methanolic ammonia (half- saturated at 0 "C; ca. 1 ml) and then filtered. The filtrdte was evaporated under reduced pressure and the residue crystallized from water to give NN'-bis-9-(2,3-O-iso-propylidene-P-u-ribofuranosyl)purin-6-ylureamonohydrate as needles (Found: C, 48.9; H, 4.9; N, 21.1.C,,H,,Nl,Og,-H,O requires C, 49.2; H, 5.2; N, 21.3y0), m.p. 152-153"; yield 1.21 g (92); RF (system A) 0.66; T(CD,),SO -1.82br (2 H, s), 1.32 (4 H, s), 3.73 (2 H, d, J 2.5 Hz), 4.64 (2 H, dd, J 2.5 and 6 Hz), 4.99 (dd, J 2.5 and 6 Hz), 5.69 (2 H, m), 6.39 (4 H, m), 8.41 (6 H, s), and 8.64 (6 H, s); A,,,, (95 ethanol) 292, 285, and 267 (E 33 800, 35 300, and 20 200), Amin. 289, 269, and 235 nm (E 33 100, 19 600, and 7 000); m/e 333 (M -307)+. N- 9- (2,3-0- Isopropylidene-P-~-ribo furanosyl)purin-6-yZ-N'-9-(P-D-rzbofuranosyl)purin-6-yZ urea (8).-Acetic an-crystals Found (material dried in vacuo over P,O, at 85 "C) : C, 47.7; H, 4.85; N, 23.5. C,,H,,N,,Og requires C, 48.0; H, 4.7; N, 23.3y0, m.p. 184-185"; yield 0.135 g (22 based on the di-isopropylidene derivative) ; RF (system A) 0.37; T(CD,),SO -1.85br (1 HI s) and -1.79br (1 H, s); T(CD,),SO-D,O 1.30 (4 H, m), 3.73 (1HI d, J 3 Hz), 3.92 (1H, d, J 5 Hz), 4.63 (1H, m), 4.98 (1 H, m), 5.38 (1 H, m), 5.71 (2 H, m), 5.95 (1H, m), 8.41 (3 H, s),and 8.62 (3 H, s); A,,,.(95 ethanol) 292, 285, and 267 (E 37 700, 39 200, and 21 loo), Amin. 289, 269, and 235 nm (E 37 600, 20 900, and 7 500); m/e 307 (M -293)+. Hydrolysis of NN'-Bis- 9-( P-~-r~bofuranosyl)fiurzn-6-yl-urea (7a) under (a) Neutral and (b) Alkaline Conditions.- (a) The substrate (0.05 g) and water (3 ml) were heated together, under reflux. After 4 h, t.1.c. (system A) revealed that no substrate (RF0.09) was present but solely a product with RF 0.25.The aqueous solution was concentrated under reduced pressure and the resulting gum crystallized from water (1ml) to give adenosine (0.038 g, say0),identical with authentic material m.p. (235 "C), mixed.m.p., and U.V. and n.m.r. spectroscopy. (b) The substrate (0.025 g) was dissolved in aqueous M-sodium hydroxide (1 ml) and the solution stirred at 20 "C. After 18 h, t.1.c. (system A) revealed the presence of sub- strate only. The solution was then heated on a steam-bath. After a further 20 h, t.1.c. (system A) revealed a single product (RF 0.25) and no substrate. The solution was cooled and treated with an excess of Dowex 50 (pyr-idinium form) cation-exchange resin, and the mixture was filtered. Concentration of the filtrate under reduced pressure and crystallization of the residual solid from methanol gave a crystalline compound characterized (see above) as adenosine.N(6)-~henylcarbamoyladenosine(9a; R1= H).-Phenyl isocyanate (0.952 g, 8.0 mmol) was added to a stirred solution of 2',3',5'-tri-O-acetyladenosine( 1.572 g, 4.0 mmol) and pyridine (2 ml) in anhydrous dioxan (30 ml). The solution was heated, under reflux, for 1 h, cooled and then treated with water (0.3 ml). After 30 min, the products were concentrated under reduced pressure ; the resulting gum was dissolved in methanolic ammonia (half-saturated at 0 "C; 40 ml). After 48 h, the crystalline precipitate was collected, washed with ice-cold methanol, and recrystallized from methanol (400 ml) to give N(6)-phenylcarbamoyl- adenosine l2 as crystals Found (material dried in vacuo over P,O, at 90 "C): C, 52.9; H, 4.7; N, 21.7.Calc. for Cl,H,,hT,O,: C, 52.8; H, 4.7; N, 21.75y0; m.p. 190-191"; yield 1.06 g (69); RF (system A) 0.48; "(CD,),SO-D,O * 1.31 (1 H, s), 1.33 (1H, s), 2.3-3.05 (5H, m), 3.96 (1 H, d, J 5.5 Hz), 5.40 (1H, m), 5.77 (1 H, m), 5.96 (1 H, m), and 6.32 (2 H, m) ; Amax. (95 ethanol) 280 (E 26 goo), Amin. 243 nm (E 8 700); m/e 312 (M -74)+. Hydrolysis of N(6)-Phenylcarbamoyladenosine.-The sub-strate (0.03 g) and water (4 ml) were heated together under reflux. After 4 h, t.1.c. (system A) revealed a product with RF0.25 and no substrate (RP0.48). The mixture was concentrated under reduced pressure to give a gum.Crystallization from methanol gave adenosine, m.p. 235O, identical with an authentic specimen. * The n.m.r. spectrum of (9a; R1 = H) in anhydrous (CD,),SO has additional signals at T -1.82 (1 H, s), -0.04 (1 H, s), 4.4-4.6 (1 H, m), and 4.7-5.0 (2 H, m). hydride (1.0 g, 10 mmol), NN'-bis-9-(2,3-O-isopropylidene-P-~-ribofuranosyl)purin-6-ylurea monohydrate ( 1.32 g, 2.0 mmol) and anhydrous pyridine (20 ml) were stirred together at 20 "C. After 16 h, methanol (3 ml) was added, and after a further 1 h the products were concentrated under reduced pressure. The resulting gum was dissolved in ethanol and the solution evaporated. This process was repeated and the residual gum triturated with ether to give a t.1.c. homogeneous RF(system B) 0.341 solid (1.30 g).A portion of the latter was dissolved in 30 aqueous formic acid (30 ml) and the solution stirred at 20 "C. After 22 h, the products were concentrated under reduced pressure, the residual gum was dissolved in ethanol, and the solution was re-evaporated. This process was repeated and the resulting gum triturated with ether to give a solid. This partially dissolved in CHC1,-MeOH (94 : 6 v/v; 10 ml), and the soluble fraction was separated into its pure com- ponents by short column chromatography 85 g of silica gel ; CHC1,-MeOH (94 : 6 v/v). The appropriate fractions were combined and concentrated under reduced pressure to give a t.1.c. homogeneous RF(system A) 0.601 glass (0.247 g). Methanolic ammonia (half-saturated at 0 "C; 5 ml) was added to a solution of the glass in methanol (15 ml) and the resulting solution stirred at 20 "C.After 48 h the products J.C.S. Perkin I Reaction between NNrsquo;-Bis-9-(P-~-ribofuranosyZ)pur~n-6-2.25-2.95 (10 H, m), 3.54 (1 H, d, J 5.5 Hz), 3.86 (1 H, t, yZurea (7a) and CycZohexyZamine.-NNrsquo;-Bis-9-(P-o-ribo-J 5.5 Hz), 4.22 (1 H, t, J 5.5 Hz), 5.4-5.75 (3 H, m), and furanosyl)purin-6-ylurea(0.578g, 1.O mmol) was dissolved in anhydrous pyridine (20 ml) and the solution evaporated. After this process had been repeated a further four times, anhydrous cyclohexylamine (20 ml) was added to the solid residue and the mixture heated under reflux. After 10 min, when t.1.c. (system A) revealed that no substrate remained, the products were cooled and concentrated under reduced pressure.The residue was triturated with ether (2 x 20 ml) and the resulting powder stirred with water (20 ml). After 1 h, the mixture was filtered and the residue crystallized from water to give colourless crystals of a compound, m.p. 179-180rdquo;, which was identical t.l.c. (system A) ; mixed m.p. ; u.v., i.r., and n.m.r. spectra with authentic (see below) N(6) -cyclohexylcarbamoyl- adenosine; yield 0.346 g (88). The filtrate obtained after the crude products had been stirred with water was concentrated under reduced pressure. Crystallization of the residue from methanol gave adenosine (0.247 g, 92), m.p. 235rdquo;, identical with authentic material. N(6)-CycZohexyZcarbamoyZadenos~ne (9b; R1= H).-Cyclohexyl isocyanate (0.75 g, 6.0 mmol) was added to a stirred solution of 2rsquo;,3rsquo;,5rsquo;-tri-O-acetyIadenosine(1.179 g, 3.0 mmol) and pyridine (2 ml) in anhydrous dioxan (30 ml).The solution was heated, under reflux, for 24 h, cooled, and then treated with water (0.2 ml). After 30 min, the products were concentrated under reduced pressure ; the resulting gum was dissolved in methanolic ammonia (half saturated at 0 ldquo;C; 30 ml) and the solution was stirred at 20 ldquo;C. After 48 h the crystalline precipitate was collected, washed with ice-cold water, and recrystallized from methanol to give N( 6) -cycZohexyZcarbamoyZadenosine as crystals Found (material dried in vacuo over P205at 90 ldquo;C): C, 51.8; H, 6.1; N, 21.2. C,,H2,N,05 requires C, 52.0; H, 6.1; N, 21.4y0, m.p.179-180rdquo;; yield 1.10 g (93); RF (system A) 0.49; r(CD,),SO * 0.45-0.75 (2 H, m), 1.35 (1 H, s), 1.46 (1 H, s), 3.99 (1 H, d, J 5 Hz), 4.52 (1 H, d, J 6 Hz), 4.75-5.0 (2 H, m), 5.42 (1 H, m), 5.80 (1 H, m), 6.00 (1 H, m), 6.15-6.5 (2 H, m), and 7.9- 9.0 (11H, m); hinfl.(95 ethanol) 276 (E 18 800), Alllax. 270 (E 22 ZOO), A,,,i,, 234 nm (E 3 800); m/e 392 (Mrsquo;). 2rsquo;,3rsquo;,5rsquo;-Tri-O-acetyZ-N(6),N(6)-bisphenoxycarbonyZadeno-sine (lo).-Phenyl chloroformate (11.8 g, 75 mmol) was added dropwise over 2 min to a stirred solution of 2rsquo;,3rsquo;,5rsquo;- tri-O-acetyladenosine (9.825 g, 25 mmol) in anhydrous pyridine (100 ml) at 20 ldquo;C. The reactants were stirred for a further 16 h and then concentrated under reduced pressure. The gum obtained was dissolved in chloroform (400 ml) ; the solution was washed with saturated aqueous sodium hydrogen carbonate (400 ml), dried (MgSO,), and concentrated under reduced pressure.The resulting yellow glass was fractionated by short column chromatography 600 g of silica gel; CHC1,-MeOH (99.25 : 0.75 v/v). The appropriate fractions were combined and concentrated under reduced pressure to give a glass which was kept in vacuo over P,O, at 20 ldquo;C for 16 h. Crystallization from methanol-water (3 1; 1 : 2 v/v) gave 2rsquo;,3rsquo;,5rsquo;-tri-O-acetyZ- N(6),N(6)-bisphenoxycarbonyZadenosineas crystals (Found : C, 56.8; H, 4.4; N, 11.2. C,,H,,N,O,, requires C, 56.9; H, 4.3; N, ll.l),m.p. 66-67rdquo;; yield 12.43 g (79); RF (system C) 0.54; T(CD,),CO 0.96 (1 H, s), 1.18 (1 H, s), * Addition of D,O causes the signals at T 0.45-0.75, 4.52, and 4.75-5.0 to disa.ppear.8.0 (s)and 8.04 (s)(9 H); Amax, (95 ethanol) 269 (E 10 loo), Ajnfl. 256 (E 7 500), Amin. 236 nm (E 4 700); m/e 496 (M -137)lsquo;3 . Reaction between 2rsquo;, 3rsquo;, 5rsquo;-Tri-O-acetyl-N( 6), N( 6)-bisphen- oxycarbonyZadenosine ( 10) and CycZohexyZarnine.-Cyclo-hexylamine (2.47 g, 25 mmol) was added to a stirred solution of crude, unchromatographed 2rsquo;,3rsquo;,5rsquo;-tri-O-acetyl-N(6) ,N(6)- bisphenoxycarbonyladenosine prepared as above from 2rsquo;,3lsquo;,5rsquo;-tri-O-acetyladenosine( 1.965 g, 5.0 mmol) and phenyl chloroformate (2.35 g, 15 mmol) in anhydrous dioxan at 20 ldquo;C. After 1 h, the products were concentrated under reduced pressure; the gum obtained was dissolved in chloroform (50 ml) and the resulting solution extracted with 3hl-hydrochloric acid (50 ml).The dried (MqSO,) organic layer was concentrated under reduced pressure and the residue fractionated by short column chromatography 200 g of silica gel; CHC1,-MeOH (98 : 2 v/v). The appropriate fractions were concentrated under reduced pressure, the resulting gum was redissolved in ethanol, and the solution was evaporated to give a solid. Recrystalliz-ation of this material from ethanol gave 2rsquo;, 3rsquo;,5rsquo;-tri-O-acetyZ-N(6)-cycZohexyZcarbamoyZadenosine as needles Found (ma- terial dried in vacuo over P,O, at 90 ldquo;C): C, 53.0; H, 5.7; N, 16.3. C,,H,,N,O, requires C, 53.3; H, 5.8; N, 16.2, m.p. 138-139rdquo;; yield 2.08 g (80y0,based on 2rsquo;,3rsquo;,5rsquo;-tri- O-acetyladenosine) ; RP (system B) 0.47; $ldquo;CD,),SO 0.40 (1 H, d, J 7 Hz), 0.84 (1 H, s), 1.26 (1 H, s), 1.44 (1 H, s), 3.64 (1 H, d, J 5.5 Hz), 3.94 (1 H, t, J 5.5 Hz), 4.28 (1 H, dd, J 4 and 5.5 Hz),5.45-5.8 (3 H, m), 7.90 (s),7.97 (s), and 8.00 (s) (9 H), and ca.8.0-9.0 (11 H, m); Ainfl. (9574, ethanol) 276 (E 18 600), Amax. 270 (E 21 800), Amin, 232 nm (c 3 000); m/e 518 (Mrsquo;). 2rsquo;, 3rsquo;, 5rsquo;-Tri-O-acetyl-N( 6) -phenoxycarbonyZadenosine (3c). -Morpholine (0.131 g, 1.5 mmol) was added to a stirred solution of 2rsquo;, 3rsquo;,5lsquo;-tri-O-acetyl-N(6),N(6) -bisphenoxycarb- onyladenosine (0.950 g, 1.5 mmol) in anhydrous dioxan (10 ml) at 20 ldquo;C. After 4 min, t.1.c. (system C) revealed no substrate (RP0.54) and essentially one product (RF 0.33).The products were concentrated under reduced pressure and the glass obtained was fractionated by short column chromatography 85 g of silica gel; CHC1,-MeOH (98.5: 1.5 v/v). The appropriate fractions were combined and con- centrated under reduced pressure to give a glass which was kept in vacuo over P205at 20 ldquo;C for 16 h. Crystallization from di-isopropyl ether-ethyl acetate (10 : 1 v/v) gave 2lsquo;, 3lsquo;, 5lsquo;-tvi-O-acetyZ-N (6) -phenoxycarbonyladenosane as crystals Found: C, 54.0; H, 4.7; N, 13.4. C,,H,,N,O, requires C, 53.8; H, 4.5; hrsquo;, 13.65y0, m.p. 58-60rsquo;; yield 0.423 g (55) ; RF (system C) 0.33; T(CD,),SO -1.09br (1H, s), 1.33 (s) and 1.36 (s) (2 H), 2.4-2.9 (5 H, m), 3.67 (1 H, d, J 6 Hz), 3.94 (1 H, m), 4.33 (1 H, m), 5.4-5.8 (3 H, m), and 7.88 (s), 7.96 (s), and 8.00 (s) (9 H); Amax.(95 ethanol) 268 (E 19 000), Amin. 231 nm (E 4 900); m/e 419 (A4-94)+. Preparation of N(6)-CycZohexyZcarbamoyZadenosine (9b; R1= H) from 2rsquo;, 3rsquo;, 5rsquo;-Tri-O-acetyl-N( 6) -phenoxycarbonyZ- adenosine (3c) .-A solution of cyclohexylamine (0.049 g, 0.5 mmol) and 2rsquo;,3rsquo;,5rsquo;-tri-O-acetyl-N(6)-phenoxycarbonyl-adenosine (0.257 g, 0.5 mmol) in anhydrous dioxan (5 ml) was stirred at 20 ldquo;C. After 1 h, t.1.c. (system C) revealed no starting material (RF0.33) and a single product (RF 0.27). The solution was then concentrated under reduced pressure, the gum obtained dissolved in methanolic ammonia (half-saturated at 0 "C; 5 ml), and the resulting solution stirred at 20 "C.After 16 h, the crystalline precipitate was collected, washed with ice-cold water, and recrystallized from methanol-water (1 : 1 v/v) to give N(6)-cyclohexylcarbamoyladenosine (0.180 g, 92y0), m.p. 179-180", identical RF (system A); mixed m.p.; u.v., n.m.r., and mass spectra with authentic material. Reaction between N (6) -MethoxycarbonyZadenosine (3d) and CycZohexyZawzine.-Cyclohexylamine (0.025 g, 0.25 mmol) was added to a solution of N(6)-methoxycarbonyl-adenosine l5 (0.054 g, 0.17 mmol) in anhydrous pyridine (2 ml) and the resulting solution heated under reflux. After 4.5 11, t.1.c. (system A) revealed N(6)-methoxy-carbonyladenosine (RF0.34; ca. 25) and a single product (RF 0.49; ca. 75). After 16 h, when no starting material remained, the products were cooled and concentrated under reduced pressure and the resulting gum was dissolved in chloroform (2 ml).The solution was washed with 3~- hydrochloric acid (2 ml), dried (MgSO,), and evaporated to a gum. This was dissolved in ethanol and the solution re- evaporated. Crystallization of the residue from methanol gave N(6)-cyclohexylcarbamoyladenosine,m.p. 179-180", identical Rv (system A), mixed m.p., and u.v., n.m.r., and mass spectra with authentic material. N( 6) -CarbaunoyZadenosine (9c; R1 = H).-2', 3', 5'-Tri-O- acetyl-N(6)-phenoxycarbonyladenosine(0.257 g, 0.5mmol) was dissolved in a stirred, saturated solution of ammonia in dioxan (5 ml) at 20 "C. After 2 h, t.1.c. (system B) revealed no starting material and a single product (RP0.25).The solution was concentrated under reduced pressure, the residual gum dissolved in methanolic ammonia (half-saturated at 0 "C; 5 ml), and the resulting solution stirred at 20 "C. After 18 h the crystalline precipitate was col- lected, washed with ice-cold methanol, and recrystallized from methanol-water (9 : 1 v/v) to give N(6)-carbaunoyl-adenosine hemihydrate as crystals Found (material dried in vacuo over P,O, at 90 "C): C, 41.1; H, 4.5; N, 26.1. C,,H,,N605,0.5H20requires C, 41.4; H, 4.7; N, 26.3, m.p. 128-129"; yield 0.142 g (92); RF (system A) 0.25; T(CD,)~SO--D~O1.24 (1 H, s), 1.33 (1 H, s), 3.88 (1 H, d, 137 J 5 Hz), 5.31 (1 H, m), and 5.69 (1 H, m); vmax. (Nujol) 3500m, 3290m, 1700s, and 1615s cm-l; Ainfl.(95 ethanol) 275 (E 15 800), A,,,. 268 (E 19 goo), Amin. 231 nm (E 3 100). N ( 6)-Methoxycarbonylmethy lcarbarnoyladenosine (9d;R1= H).-2',3',6'-Tri-O-acetyl-N(6)-phenoxycarbonyl-adenosine (0.256 g, 0.5 mmol) and glycine methyl ester hydrochloride (0.069 g, 0.55 mmol) were stirred together in anhydrous dioxan (5 ml) at 20 "C while triethylamine (0.055 g, 0.55 mmol) was added. After the reactants had been stirred for a further 18 h, t.1.c. (system C) revealed no starting material (RF 0.33) and a single product * (RF 0.26). The product was then filtered and the filtrate concentrated under reduced pressure. The gum obtained was fractionated by short column chromatography 50 g of silica gel; CHC1,MeOH (98 : 2 v/v). The appropriate fractions were combined and concentrated under reduced pressure to give a gum which was dissolved in methanolic sodium methoxide (0.5~;5 ml), and the solution was stirred at 20 "C. After 10 min, the products were neutral- ized with Amberlite IR 120 cation-exchange resin (Hf form); the mixture was filtered and the filtrate concen- trated under reduced pressure. Crystallization of the solid obtained gave N(6)-methoxycarbonylmethyZcarbamoyZadeno-sine as crystals Found (material dried in vacuo over P,O, at 90 "C): C, 43.7; H, 4.85; N, 22.0. C,,H1,N60, requires C, 44.0; H, 4.7; N, 22.0, m.p. 178-179"; yield 0.149 g (78); RF(system A) 0.45; -r(CD,),SO 0.1-0.4 (2 H, m), 1.36 (1 H, s), 1.47 (1 H, s), 3.96 (1 H, d, J 5.5 Hz), 4.4-5.1 (3H, m), 5.39 (1 H,m), 5.7-6.05 (4H,m), and 6.1-6.5 (5 H, m); Ajilfl. (95 ethanol) 275 (E 17 500), I,,,,. 269 (E 20 SOO), Amin. 233 nm (E 3 600); m/e 293 (M -89)+. One of us (P.A. L.) thanks the S.R.C.and Girton College, Cambridge, for a Research Studentship and a Fellowship, respectively. 7/1128 Received, 28th June, 19771 * If the reaction is carried out in dimethylformamideit goes to completion in less than 1 h at 20 "C. l5 T. Ravindranathan and C. B. Reese, unpublished results,