J. CHEM. SOC. PERKIN TRANS. 1 1992 21 1 A New and Unequivocal Method for Establishing the Position of N-G lycosylation of Unsymmetrically C-Su bst ituted lmidazoles Timothy J. Benson and Brian Robinson Department of Pharmacy, University of Manchester, Manchester, M 73 9PL, UK N-Substitution of an unsymmetrically C-substituted imidazolecan give rise toa pair of structurally isomeric derivatives and to differentiate between such related compounds can be difficult. Two methods, one spectroscopic and one chemical, for ascertaining the orientation of such N-substitutions are described, with particular application to the establishment of the direction of N-ribosidation of a series of halogeno nitroimidazoles. N-Unsubstituted imidazoles exhibit a tautomerism which can become evident upon the N-alkylation of an unsym-metrically C-substituted imidazole.In such circumstances, the reaction conditions and physicochemical properties of the reactants determine the ratio of structurally isomeric products,' whose subsequent separation and substituent-orientation elu- cidation can be potentially problematic. Whilst conducting a study on the N-ribosidation of some such above-mentioned imidazoles, an example of an N-alkylation which could give rise to a pair of structurally isomeric ribosides, it became necessary to assign unambiguously the orientation of N-ribosidation in each nucleoside product. We now wish to report a new chemical/chromatographictechnique, which provides a rigorous confirmation of conclusions reached from UV spectroscopic analysis, by which this was achieved. Results and Discussion The halogeno nitroimidazoles 1-3, each of which is in tautomeric equilibrium with the corresponding 5-halogeno-4- nitroimidazole, were treated with l-O-acetyl-2,3,5-tri-O-benzo-yl-P-D-ribofuranose 10 according to a literature procedure.2 In every case only a single ribosidic product was isolated which was deprotected by treatment with saturated methanolic ammonia to afford the corresponding nucleoside.One of two structures, 11 or 12,13 or 14 and 15 or 16 are possible for each of these products, in all of which the f3-anomeric conformation at C-1' is assumed from earlier observation^.^^^ To permit the differentiation between each of these two possible structures, the isomeric pairs of N-methylimidazoles 4 and 5,6 and 7, and 8 and 9, of known substituent orientation, were synthesised for use as model compounds.4(5)-Chloro-5(4)-nitroimidazole,1, prepared by heating 4( 5)-bromo-5(4)-nitroimidazole 2 (see below) with concentrated hydrochloric acid,5 was N-methyl- ated using dimethyl sulfate to afford a mixture of 4-chloro-l- methyl-5-nitroimidazole 4 '-'and 5-chloro-1 -methyl-4-nitro- imidazole 5 7*8 which were separated by chromatography upon a column of neutral alumina. 4(5)-Bromo-5(4)-nitroimidazole, prepared from imidazole by sequential bromination to 2,4,5- tribromoimidazole, nitrate salt formation and heating with sulfuric acid,4 was N-methylated using dimethyl sulfate to afford a mixture of 4-bromo- l-methyl-5-nitroimidazole6 and 5-bromo-1-methyl-4-nitroimidazole 7 which were separated chromatographically upon a column of neutral alumina.Iodination of imidazole gave 4,5-diiodoimidazole 'O*t which upon reaction with a mixture of fuming nitric acid and t Prepared by an application of unpublished experimental directions kindly supplied by Dr. R. L. Dyer of Searle Research and Development, High Wycombe, Bucks. R' R2 R3 R' $ R3 H NO2 CI 6 Me NO2 Br H NO2 Br 7 Me Br NO2 H NO2 I 8 Me NO2 I Me NO2 CI 9 Me I NO2 Me CI NO2 10 OH R' 11 CI 12 NO2 13 Br 14 NO2 15 I 16 NO2 concentrated sulfuric acid at -20 "C yielded 4(5)-iodo-5(4)- nitroimidazole 3." N-Methylation of this product with di- methyl sulfate in boiling 1,4-dioxane under reflux afforded 4- iodo-1-methyl-5-nitroimidazole 8,' whereas when the reaction was effected in hot aqueous sodium hydroxide a mixture of the previous product, which was removed by washing the total reaction mixture with acetone, and the isomeric 5-iodo-1- methyl-4-nitroimidazole 9 was obtained.lo Various spectroscopic techniques have been investigated as possible means of distinguishing between structurally isomeric pairs of N-substituted imidazoles. IR spectroscopy and mass spectrometry have been found to be of no significant diagnostic value in differentiating between isomeric pairs of 1-alkyl-4 (or 5)-monosubstituted or unsymmetrically C-substituted imida- zoles ' and, likewise, the IR and mass spectra of the present 212 J.CHEM. SOC. PERKIN TRANS. I 1992 Table 1 UV spectroscopic details of compounds4-9 303 237 263 220 4 (9700) 307 (4550) 242 (2990) 268 (4050) 223 6 (6750)322 (3600) 259 (2350)285 (3400) 228 8 (6050) (3850) (3000) (3100) All spectra obtained in 96 EtOH. Table 2 UV spectroscopic details of compounds 11, 13 and 15 294 255 225 11 (6400) (2 100) (5900)299 258 223 13 (5940) (1 550) (5250) 314 269 238 15 (6950) (2350) (5200) All spectra obtained in 96 EtOH. three isomeric pairs of model compounds were all markedly similar with no distinguishing features. 'H NMR spectroscopy has been employed to distinguish 1,4- from 1,5-disubstituted imidazoles by noting differences between the cross-ring coupling constants of 2-H and 5-H or 4-H, respectively," but this technique is not applicable in the present study because the imidazoles under investigation possessed only one ring-proton.I3C NMR spectroscopy has been successfully utilised to distinguish between some 1-alkyl-4 (or 5)-nitroimidazoles but the feasibility of the technique has not been investigated in this study. However, significant differences between the UV spectra of isomeric pairs of N-alkylnitroimidazoles have been and, in relation to the present study, compounds 8 and 9 have previously been reported" to have A,,, 323 and 258 nm (E 6870 and 4080),and A,,, 315 and hinf,240 nm (E 7600 and SOOO), respectively.These data have been largely confirmed, and extended, in the present study in which similar differences have also been discerned between the UV spectra of the isomeric pairs of compounds 4 and 5, and 6 and 7 (Table 1). The orientation of each of the present N-ribosidations was, therefore, determined, after the necessary removal of the three UV-absorbing benzoyl moieties, by comparison of the UV spectrum of the product with those of the above corresponding model isomeric pair (Table 2). Consequently, the three deprotected nucleosides were clearly assignable as the 5-halo- geno-4-nitro-ribofuranosylimidazoles11, 13 and 15, respec-tively. However, since only one nucleosidic product could be isolated from each of the current ribosidations, it was not possible to effect a direct UV comparison between structurally isomeric pairs of such products.Furthermore, in the cases of the initially formed protected nucleosides, the orientation of N-ribosidation could not be ascertained because the UV absorption of the protecting benzoyl moieties completely masked that of the imidazoyl moiety. It was, therefore, considered desirable to establish rigorously the orientation of the above N-ribosidations by an alternative method and the following chemical/chromatographic procedure was, accord- ingly, developed. Quaternisation of each nucleosidic product using dimethyl sulfate could be effected without the need to deprotect, although this permitted the use of milder reaction conditions in the subsequent quaternisations.In both situations, acid-catalysed 295 254 224 5 (8100) 298 (2350)255 (5800)223 7 (6450)3 14 (1500)268 (5700)238 9 (9650) (2400) (6400) hydrolysis of the quaternary product yielded the corresponding N-methylhalogenonitroimidazolehaving the opposite C-sub- stituent orientation to that of the original nucleoside. The imidazolic product could be readily identified since it was found that a significant difference in R, values exists between the isomers in each of the three pairs of model compounds 4 and 5, 6 and 7, and 8 and 9 (Table 3). Indeed, this is not surprising since each isomeric pair was preparatively separable by column chromatography. Furthermore, the imidazolic component re- sulting from hydrolysis was isolable by TLC and characterised by UV spectroscopy and melting point determination.Each of the three nucleosides yielded the corresponding 4-halogeno- 1-methyl-5-nitroimidazoles 4, 6 and 8, thereby confirming their structures as 11,13 and 15, respectively. This procedure provides a facile general method by which the substituent orientation(s) in the product(s) resulting from the N-glycosylation of an unsymmetrically C-substituted imida- zole may be unequivocally established, provided the corres- ponding pair of N-methylated imidazoles of known substituent orientation are available. Such an approach would be of value where the various spectral techniques are either inapplicable or give results which are open to doubt.Experimenta1 M.p.s were determined on a Kofler hot stage apparatus and are corrected. Microanalyses were carried out on a Perkin-Elmer 240 analyser. UV spectra were recorded on a Pye-Unicam SP8-500 spectrophotometer and wavelengths are expressed in nm. Mass spectral data were obtained using an AEU-M530 mass spectrometer equipped with a DS-55 data system. Qualitative TLC was performed on either pre-coated silica gel (UVZs4) or neutral aluminium oxide (UV, 54) microplates (Camlab: polygram SIL G-UV,,, and polygram ALOX N/UV,,,) and RFvalues, as appropriate, are quoted in the experimental text. Spots were detected by fluorescence quenching at 254 nm. Column chromatography was carried out using either neutral aluminium oxide (Merck: neutral aluminium oxide 90, activity I, 70-230 mesh) or silica gel (Fisons, 60-120 mesh) as stationary phases and columns were prepared by wet packing. 'H NMR spectra were recorded on a Bruker WP80 spectrometer operating at 80 MHz using tetramethylsilane (TMS) as an internal standard.All evaporations were carried out at less than 30 "C. All starting materials were purchased from the Aldrich Chemical Co. Anhydrous solvents were prepared according to standard procedures. 4(5)-Chloro-, bromo- and iodo-5(4)-nitroimidazoles (1,5 2 and 3 lo), 4-iodo-1-methyl-5-nitro-imidazole 8 lo and 5-iodo- 1 -methyl-4-nitroimidazole 9 lo were all prepared according to the established literature procedures. Ch-omatogruphic Systems.-Neutral aluminium oxide: (A) chloroform-diethyl ether ( 1:3) and (B) chloroform-diethyl ether (I :1).Silica gel: (C) diethyl ether-light petroleum (b.p. 3040"C) (35:65), (D) diethyl ether-chloroform (3: l), (E) diethyl ether- J. CHEM. SOC. PERKIN TRANS. I 1992 Table 3 RF Values of compounds 4-9 Compd. R, Compd. RF Chromatography system 4 0.47 5 0.28 A 6 0.33 7 0.18 B 8 0.77 9 0.44 A chloroform (1: l), (F) diethyl ether-light petroleum (b.p. 30- 40 "C) (85 :15) and (G)chloroform. 4-Chloro- 1 -methyl-5-nitroimidazole4 and 5-Chloro- 1 -methyl- 4-nitroimidazole 5.-These compounds were prepared by application of the literature procedure for the preparation of the corresponding bromo analogues,' but with modification of the work-up procedure to involve product separation by column chromatography (system A) instead of by fractional recrystallisation.Thus, treatment of4(5)-chloro-5(4)-nitroimida-zole (4.15 g, 28.1 mmol) with dimethyl sulfate (2.7 cm3) afforded, after work-up, a brownish oil from which was initially eluted 4-chloro-1-methyl-5-nitroimidazole4 (250 mg, 5.573, m.p. 74-75 "C (from EtOH) 76-77, 77-78, 78 "C), followed by 5-chloro-1-methyl-4-nitroimidazole 5 (321 mg, 6.9), m.p. 144-145 "C (from EtOH) 148, 147- 148 "C). 4- Bromo- 1 -methyl-5-nitroimidazole6 and 5-Bromo- 1 -methyl- 4-nitroimidazole 7.-These compounds were prepared from 4(5)-bromo-5(4)-nitroimidazole according to the literature method 'but again employing column chromatography (sys- tem B) in place of fractional recrystallisation to separate the products.4-Bromo- 1-methyl-5-nitroimidazole 6 was eluted first (8.5 mg, 0.1), m.p. 98-100 "C (from water) (lit.,8 101-102 "C), followed by 5-bromo- 1-methyl-4-nitroimidazole 7 (21.5 mg, 0.3), m.p. 187-189 "C (from water) (lit.,' 188 "C). Preparation of Benzoylated Nucleosides: General Method.- All such nucleosides were prepared by application of a general literature procedure involving the stirring together, at room temperature for 2 h with exclusion of moisture, of the appropriate N-unsubstituted nitroimidazole (5 mmol), 1-0- acetyl-2,3,5-tri-O-benzoyl-~-~-ribofuranose(2.52 g, 5 mmol), hexamethyldisilazane (0.85 ml, 4 mmol), trimethylchlorosilane (0.5 ml, 4 mmol) and tin(iv) chloride (0.7 ml, 6 mmol) in absolute acetonitrile (75 cm3) under dry nitrogen gas., Deprotection of Benzoylated Nuc1eoside.-The starting material was dissolved in a minimum volume of dry saturated methanolic ammonia and the solution stirred for 2 h at room temperature, with exclusion of moisture; after this time, the solvent was removed under reduced pressure to afford the crude deprotected nucleoside. 5-Chloro-4-nitro-1-P-D-ribofuranosylimidazole 11.The ben- zoylated nucleoside was prepared as above, using 4(5)-chloro- 5(4)-nitroimidazole 1 (750 mg, 5 mmol) as the imidazolic component. Column chromatography of the crude product (systems C, RF 0.05, and D, RF 0.85, respectively) yielded the pure acylated nucleoside as a foam (2.29 g, 77.3) (Found: C, 58.6; H, 3.6; CI, 16.2; N, 6.9.C,,H,,ClN30g requires C, 58.85; H, 3.75; CI, 16.0; N, 7.1). Deprotection furnished a yellowish syrup and column chromatography of this (system E, R, 0.35) yielded the title compound as a pale yellow foam which could not be crystallised (405 mg, 43); dH(CD3SOCD3) 5.60-5.82 (2 H, m, exchangeable-collapsed to symmetrical d, 1 H, 5.74, J,,.t,, 4.2 Hz, anomeric-H and 2'-OH), 8.36 (1 H, s, imidazoyl 2-H); (Found: m/z 280.0334 C,H loCIN30, requires 280.0337 forM' + 1). 21 3 5-Bromo-4-nitro-1-P-D-ribofuranosylinziduzole13. The benzo- ylated nucleoside was prepared as above, using 4(5)-bromo- 5(4)-nitroimidazole 2 (958 mg, 5 mmol) as the imidazolic component. Column chromatography of the crude product (systems C, RF 0.05, and E, RF 0.8, respectively) yielded the pure acylated nucleoside as a foam (2.66 g, 85) (Found: C, 55.0; H, 3.55; Br, 12.7; N, 6.5.C2,H,,BrN30, requires C, 54.7; H, 3.48; Br, 12.55; N, 6.6). Deprotection afforded the title compound as a brownish solid (612 mg, 50),m.p. 172-173 "C (from EtOH) (1it.,l3 174-175 "C) (Found: C, 29.7; H, 3.1; Br, 24.4; N, 13.0. C,H,,BrN,O, requires C, 29.6; H, 3.1; Br, 24.7; N, 13.0); G,(CD,SOCD,) 5.565.79 (2 H, m, exchange-able--collapsed to a symmetrical d, 1 H, 5.71, J1,,,,,4.7, anomeric-H and 2'-OH), 8.4 (1 H, s, imidazoyl 2-H); m/z 324 (M+, 2 and exhibiting a typical bromine isotopic peak arrangement). 5-Iodo-4-nitro-1-P-D-ribofuranosylimidazole 15. The benzo- ylated nucleoside was prepared as above, using 4(5)-iodo-5(4)- nitroimidazole 3 (1.2 g, 5 mmol) as the imidazolic component.Column chromatography of the crude product (systems F, RF 0.6, C, RF 0.0, and G, RF 0.9, respectively) yielded the pure acylated nucleoside as a yellow foam (2.07 g, 61) (Found: C, 51.3; H, 3.4; I, 18.2; N, 6.3. C,,H,,IN,O, requires C, 50.9; H, 3.25; I, 18.6; N, 6.15). Deprotection afforded the title com- pound as a cream coloured solid (256 mg, 27), m.p. 178 "C (decomp.) (from acetone-water) (Found: C, 26.1; H, 2.7; I, 34.4; N, 11.1. C8HloIN306 requires C, 25.85; H, 2.7; I, 34.3; N, 11.3); d~(CD3soCD3) 5.48-5.79 (2 H, m, exchangeable- collapsed to symmetrical d, 1 H, 5.72, J1,,,,, 4.7 Hz, anomeric -H and 2'-OH), 8.46 (1 H, s, imidazoyl 2-H); m/z (CI) 372 (M' + 1, 5.6). General Quaternisation Procedures.-Of benzoylated (pro-tected) nucleosides. The starting material (10 mg) was placed in a 5 cm3 ampoule and methyl iodide (2 cm3) added.After freezing by immersion in liquid nitrogen, the ampoule was immediately sealed and then heated in a steam-bath for 8 h (no reaction occurred after an earlier period of 2 days at room temperature). Subsequent removal of the excess of methyl iodide (allowed to evaporate at room temperature in an efficient fume cupboard) yielded the quaternary derivative. Ofdeprotected nucleosides. The starting material (5 mg) was dissolved in anhydrous methanol (5 cm3) and dimethyl sulfate (1 cm3) added, care being taken to exclude moisture.The reaction mixture was stirred overnight (ca. 18 h) at room temperature after which time the solvent and excess of dimethyl sulfate were removed the latter by co-evaporation with absolute EtOH (3 x 10 cm3) to yield the quaternary derivative. General Procedure for Acid-catalysed Hydrolysis of Quater-nary Derivatives.-To each quaternary derivative, hydrochlo- ric acid (1 mol dm-3; 5 cm3) was added and the reaction mixture heated over a steam-bath for 6 h. After cooling, the crude product was made slightly alkaline (pH 8.5) by careful addition of aqueous sodium carbonate (10) and shaken with chloroform (2 x 15 cm3). The organic phases were pooled, dried, filtered and evaporated to furnish the hydrolysis product. The imidazolic component of this was initially identified by comparative TLC and UV absorption spectros- copy.Preparative TLC in the appropriate system followed by recrystallisation from the appropriate solvent yielded pure imidazolic material which was identified by its melting point and mixed melting point with authentic material. Acknowledgements We thank the SERC for a studentship (to T. J. B.). References 1 J. H. Ridd and B. V. Smith, J. Chem. Soc., 1960, 1363; A. Grimison, J. H. Ridd and B. V. Smith, J. Chem. Soc., 1960,1352, 1357. 2 H. Vorbruggen and B. Bennua, Chem. Ber., 1981,114, 1279. 3 V. Niedballa and H. Vorbruggen, J.Org. Chem., 1974,39,3654; 1976, 41, 2084; H. Vorbruggen, K. Krolikiewicz and B. Bennua, Chem. Ber., 198 1,114,1234; H.Vorbruggen and G. Home, Chem. Ber., 1981, 114,1256. 4 I. E. Balaban and F. L. Pyman, J.Chem. Soc., 1922, 121,947. 5 G. P. Sharnin, R. Kh. Fassakhov and T. A. Enenkina, Chem. Heterocycl. Compd. (Engl. Transl.), 1977, 1332. 6 J. Baddiley, J. G. Buchanan, F. E. Hardy and J. Stewart,J.Chem.Soc., 1959,2893. 7 C. Gallo, C. R. Pasqualucci, P. Radaelli and G. C. Lancini, J. Org. Chem., 1964,29,862. J. CHEM. SOC. PERKJNTRANS. I 1992 8 J. Sarasin and E. Wegman, Helv. Chim. Acta, 1924,7, 7 13. 9 I. E. Balaban and F. L. Pyman, J. Chem. Soc., 1924,125,1564. 10 M. Hoffer, V. Toome and A. Brossi, J. Heterocycl. Chem., 1966, 3, 454; J. P. Dickens, R. L. Dyer, B. J. Hamill, T. R. Harrow, R.H. Bible, P. M. Finnegan, K. Henrick and P. G. Owston, J. Org. Chem., 1981, 46, 1781. 11 H. R. Matthews and H. Rapaport, J.Am. Chem. Soc., 1973,95,2297. 12 A. McKillop, D. E. Wright, M. L. Podmore and R. K. Chambers, Tetrahedron, 1983,39,3797. 13 R. J. Rousseau, R. K. Robins and L. B. Townsend, J. Am. Chem. Soc., 1968,90,266 1. Paper 1/04904F Received 23rd September 1991 Accepted 3rd October 1991
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