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SYNTHESIS OF 7-SUBSTITUTED 3-[Beta]-D-RIBOFURANOSYL-3H-IMIDAZO[2,1-i]PURINES [Collection of Czechoslovak Chemical Communication](Collection of Czechoslovak Chemical Communications Via Acquire Media NewsEdge) A method for the synthesis of 7-substituted 3-ß-D-ribofuranosyl-3H-imidazo[2,1-i]purines has been devised whereby compounds were prepared in a few steps from a common intermediate, 3-(2',3'-O-isopropylidene-ß-D-ribofuranosyl)-3H-imidazo[2,1-i]purine-7-carbaldehyde, obtained from the reaction of 2',3'-O-isopropylideneadenosine with bromomalonaldehyde. The formyl group of the carbaldehyde was subsequently reductively aminated and the resulting secondary amines were then further derivatized either by acylation, lactamization or reductive alkylation. Keywords: Nucleosides; Heterocycles; Purine bases; NMR spectroscopy. Purine nucleosides and their congeners have found applications in medicinal research as antivirals1,2, antiprotozoal agents3, adenosine receptor agonists4-6, adenosine kinase inhibitors7,8 and in the treatment of lymphoid malignancies9-12 and systemic mastocytosis13, just to name a few examples. Although the structural analogs of purine nucleosides have for this reason been the subject of extensive synthetic studies, there is still room for further modifications. We now report on the preparation of 7-substituted 3-(ß-D-ribofuranosyl)-3H-imidazo[2,1-i]purines to expand the structural diversity of purine-derived compounds. The rationale for the preparation of these compounds is that whilst the structures closely resemble adenosine, the nitrogen atoms analogous to the N6 and N-1 atoms in adenosine are blocked. These moieties of adenosine are usually engaged in H-bonding with the target protein. It is hoped that this blocking will lead to improved selectivity, since it is expected that only a limited number of potential targets are able to bind these compounds efficiently as ligands. As an extension of our previous studies on solid-supported derivatization of 3-substituted 3H-imidazo[2,1-i]purine-7-carbaldehydes14,15, a solutionphase synthesis for related nucleoside analogs, 7-substituted 3-ß-D-ribofuranosyl- 3H-imidazo[2,1-i]purines, was developed. The compounds were prepared in a few steps from a common intermediate, 3-(2',3'-O-isopropylidene- ß-D-ribofuranosyl)-3H-imidazo[2,1-i]purine-7-carbaldehyde (2), obtained by reacting 2',3'-O-isopropylideneadenosine (1) with bromomalonaldehyde (Scheme 1). In this sequence, the formyl group was first reductively aminated with piperidine (3a), morpholine (4a), benzylamine (5) or tert-butyl 4-aminobutanoate (7). The resulting secondary amines (5 and 7) were then derivatized either by acetylation (6), lactamization (8) or reductive alkylation (9) after which the isopropylidene protecting group was removed. RESULTS AND DISCUSSION Formylethenoadenosine (3-ß-D-ribofuranosyl-3H-imidazo[2,1-i]purine- 7-carbaldehyde)16 and its 2'-deoxy analog17 have been previously synthesized by the reaction of bromomalonaldehyde with adenosine in aqueous solution and 2'-deoxyadenosine in DMF, respectively. Both methods, however, only provided low yields of their respective product carbaldehydes. By contrast, ethyl and tert-butyl 2-(7-formyl-3H-imidazo[2,1-i]purin-3-yl)acetates have been obtained by solution-phase synthesis in good yields15 by applying the anhydrous conditions previously14 optimized for solid-support chemistry. However, the reaction of unprotected adenosine under similar conditions produced only a thick dark product mixture that was difficult to fractionate into pure products. Protection of the 2' and 3' hydroxyl groups of the ribose moiety with an isopropylidene group to yield 2',3'-O-isopropylideneadenosine (1) markedly improved the situation. This approach thereby facilitated the production of the desired target compound, 3-(2',3'- O-isopropylidene-ß-D-ribofuranosyl)-3H-imidazo[2,1-i]purine-7-carbaldehyde (2), in 81% yield after analogous reaction followed by purification using silica gel chromatography. The conditions applied previously14 for the reductive amination of a formyl group on a solid support were found to be too harsh to be used in solution-phase synthesis. For example, treatment with sodium cyanoborohydride in THF containing 1% formic acid was found to give a product mixture where the reduced starting material was the main component (data not presented). The replacement of formic acid with acetic acid reduced the amount of this side product but still did not lead to a generally satisfactory result. Only when using benzylamine as the nucleophile was the obtained yield of the amination product 5 sufficiently acceptable. Sodium triacetoxyborohydride in DCE was found to produce less byproducts and thus it was used for the amination of 2 with piperidine, morpholine and tertbutyl 4-aminobutanoate to obtain, respectively, compounds 3a, 4a and 7. Of the secondary amines, 5 was subjected to acetylation and 7 to lactamization and alkylation. Although the treatment of 5 with acetic anhydride esterified the 5'-hydroxyl group in addition to the secondary nitrogen atom, the 5'-O-acetyl group could be selectively removed by the application of sodium hydride in methanol. Finally, acidolytic removal of the isopropylidene protection then yielded the desired nucleoside 6. NMR spectroscopic examination of 6 revealed that it existed as two interconverting conformations in solution as evidenced by the cross-signals present in an EXSY spectrum between corresponding protons in the major and minor species. Compound 6 contains an acetamide moiety and thus it is expected to have restricted rotation about the amide bond. The structural assignment of these conformers was based on quantum mechanical (QM)- calculated energies and QM-predicted 13C NMR chemical shifts, both of which indicated that the conformer having the carbonyl oxygen trans to the benzyl group (conformer A, the major species) was preferred (dG = 12 kJ mol-1) over the conformer with cis orientation (conformer B), see Fig. 1. Compound 7 was first converted to a carboxylic acid by TFA promoted removal of the tert-butyl group under anhydrous conditions and the exposed ?-aminobutyric acid moiety was then lactamized in dilute solution using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) as an activator and N,N-diisopropylethylamine as the base. Finally, the isopropylidene protecting group was also removed to obtain 8. Alternatively, compound 7 was also subjected to reductive benzylation with benzaldehyde in DCE using zinc-modified NaCNBH3 18 as the reducing agent. Of note, the reaction was found to be very slow using either NaBH(OAc)3 or NaCNBH3 without zinc chloride addition and, furthermore, the addition of acetic or formic acid instead brought about the formation of acylated and lactamized side products19. The product (9) was acidolytically deprotected to yield compound 10. All the synthetic processes described above involved the removal of the 2',3'-O-isopropylidene protection as the last step. Of note, this step, somewhat unexpectedly, was found to be a rather difficult one. Of the numerous methods attempted, including 80% aqueous AcOH at 25 °C 20, I2/MeOH 21, PdCl2(CH3CN)2 22, CAN 23, CAN-SiO2 24, and hydrolysis at pH 2, many were found to be too inefficient whilst harsher treatments such as 80% aqueous AcOH at reflux20 or a 1:1 mixture of 1 M HCl (aq.) and THF 25, resulted in side reactions such as depurination or even pyrimidine ring opening. The best results were obtained using a 1:1 mixture of 0.1 M HCl (aq.) and THF at 55 °C or FeCl3.6H2O in a 1:19 mixture of MeOH and DCM at elevated temperature26. The Brønsted acid-catalyzed reaction, however, was still accompanied by some depurination whilst the Lewis acid-catalyzed reaction, though cleaner, was hampered by a more laborious work up that resulted ultimately in reduced yields. The former method was therefore mainly used since it was more convenient, faster and approximately equally yielding as the latter. In conclusion, a method to produce 7-substituted 3H-imidazo[2,1-i]- purines on solid support was successfully adapted to solution chemistry to produce 7-substituted 3-ß-D-ribofuranosyl-3H-imidazo[2,1-i]purines. Because of the different nature of the reaction media, several modifications to the reaction conditions were necessitated. In addition, the use of a protecting group for the ribose ring of the starting material was required. Unfortunately, the isopropylidene protection was more stable than expected which reduced the yields of the acid labile end products. For further developments, it could be worthwhile to thus evaluate other protection strategies. EXPERIMENTAL Solvents and reagents were dried or tested for dryness before use when appropriate. The purified products existed as transparent glassy solids after rotary evaporation except compound 2 which was a white solid. NMR spectra (d, ppm; J, Hz), at 298.1 K (uncorrected) unless otherwise indicated, were recorded on Bruker Avance spectrometers with field strengths of 14.1, 10.75 and 9.4 T. General NMR experimental details (e.g. standard gradient-selected COSY, HMBC and HSQC) have been described elsewhere27. NMR samples in solvents as indicated consisted of dilute samples (~5 mg in ca. 0.6 ml) up to concentrated samples (~50 mg in ca. 0.6 ml). 1H and 13C chemical shifts were referenced internally to the solvent peaks28 (DMSO: dH 2.50, dC 39.52; MeOH: dH 3.31, dC 49.00 and HOD: dH 4.79) or to TMS in the case of CDCl3 solutions (d 0 for both 1H and 13C). 15N chemical shifts were referenced externally to 90% nitromethane in CD3NO2 (d 0). Assignment of the H-5's 29 (proR and proS) and isopropylidene methyl 1H and 13C nuclei30 (endo and exo) were derived from literature. Direct observations of the 15N nuclei were facilitated by the QDEPT pulse sequence adapted31 from methodology32,33 developed for one-bond 1H-13C interactions. The method of cycling the 1H-selection pulse and the J-dependent interpulse delays, scaled accordingly to span the range 24.1-4.7 Hz, was taken from Jiang33. In the case of dilute samples, indirect 15N observations were taken from HMBC spectra (appropriately only reported to one decimal place). 15N chemical shifts were in accordance with previous examinations27a,27b,34 with shielded resonances for sp3-type nitrogens (N-3, N-6) and deshielded resonances for sp2-type nitrogens (N-1, N-4, N-9). Geometry optimizations, frequency calculations and chemical shielding calculations using GIAO were performed using the Gaussian09W program35 at the restricted B3LYP/6-31G(d,p) level of theory for all calculations with inclusion of methanol as the solvent using the IEF-PCM model. A Bruker ESI-QTOF mass spectrometer was used for the measurement of high resolution mass spectra. 3-(2,3-O-Isopropylidene-ß-D-ribofuranosyl)-3H-imidazo[2,1-i]purine-7-carbaldehyde (2) 2',3'-O-Isopropylideneadenosine (1; 0.50 g, 1.63 mmol) and bromomalonaldehyde (0.37 g, 2.44 mmol) were dissolved in a mixture of formic acid (0.19 ml, 5.01 mmol), 2,6-lutidine (0.53 ml, 4.57 mmol) and dry DMF (9.3 ml). The reaction mixture was agitated in a stoppered bottle at 60 °C for 6 h during which time the color of the mixture turned a dark orange. The reaction was monitored by TLC (10% MeOH/CH2Cl2) and upon completion, the volatiles were removed by a rotary evaporator and the residue taken up in DCM followed by extraction with saturated NaHCO3. The organic phase was dried over Na2SO4 and then evaporated to dryness after filtration. The crude material was purified by silica gel column chromatography (5% MeOH in DCM) yielding 0.47 g (81%) of 2. Some batches contained minor amounts of a fluorescent intensely orange-colored byproduct. The impurity was not characterized. 1H NMR (500 MHz, DMSO-d6): 10.00 s, 1 H (H-1"); 9.91 s, 1 H (H-5); 8.74 s, 1 H (H-2); 8.58 s, 1 H (H-8); 6.33 d, 1 H, J = 2.6 (H-1'); 5.40 dd, 1 H, J = 6.2, 2.6 (H-2'); 5.12 br, 1 H (OH); 5.03 dd, 1 H, J = 6.2, 2.6 (H-3'); 4.29 dt, 1 H, J = 4.9, 2.6 (H-4'); 3.59 dd, 1 H, J = -11.8, 4.9 (H-5'proS); 3.55 dd, 1 H, J = -11.8, 4.9 (H-5'proR); 1.56 s, 3 H (H-7'endo); 1.34 s, 3 H (H-7'exo). 13C NMR (126 MHz, DMSO-d6): 179.17 (C-1"), 147.83 (C-8), 144.71 (C-9a), 142.22 (C-2), 140.96 (C-3a), 136.76 (C-5), 124.72 (C-7), 122.99 (C-9b), 113.13 (C-6'), 90.12 (C-1'), 87.23 (C-4'), 84.07 (C-2'), 81.36 (C-3'), 61.44 (C-5'), 26.99 (C-7'endo), 25.15 (C-7'exo). 15N NMR (50 MHz, DMSO-d6): -204.00 (N-3), -184.15 (N-6), -145.62 (N-4), -142.61 (N-9), -137.16 (N-1). HRMS: calculated for [M + H+] C16H18N5O5 + 360.1302 amu; found 360.1303 amu. 3-(2,3-O-Isopropylidene-ß-D-ribofuranosyl)-7-(piperidin-1-ylmethyl)- 3H-imidazo[2,1-i]purine (3a) Compound 2 (0.100 g, 0.278 mmol) was suspended in DCE (1 ml) to which piperidine (30.2 µl, 0.306 mmol) and acetic acid (31.8 µl, 0.556 mmol) were added under magnetic stirring. NaBH(OAc)3 (0.295 g, 1.39 mmol) in DCE (1 ml) was added to the reaction mixture in three aliquots with reaction monitoring by TLC (10% H2O/MeCN). After ca. 24 h, the reaction mixture was taken to dryness and the residue taken up twice with water and once with MeOH with solvent removal effecting the co-evaporation of any residual acetic acid. The crude material was purified by silica gel column chromatography (10% H2O in MeCN) yielding 0.083 g (61%) of 3a as an acetate salt. 1H NMR (500 MHz, CDCl3): 9.25 s, 1 H (H-5); 8.48 s, 1 H (H-2); 7.47 s, 1 H (H-8); 6.33 d, 1 H, J = 2.9 (H-1'); 5.37 dd, 1 H, J = 6.2, 2.9 (H-2'); 5.08 dd, 1 H, J = 6.2, 2.8 (H-3'); 4.41-4.35 m, 1 H (H-4'); 3.99 s, 2 H (H-1"); 3.79 dd, 1 H, J = -11.9, 4.1 (H-5'proS); 3.73 dd, 1 H, J = -11.9, 4.7 (H-5'proR); 2.55 s, 4 H (H-3"); 1.94 s, 3 H (AcO-); 1.63 s, 3 H (H-7'endo); 1.62-1.54 m, 4 H (H-4"); 1.54-1.42 m, 2 H (H-5"); 1.40 s, 3 H (H-7'exo). 13C NMR (126 MHz, CDCl3): 178.96 (AcO-), 143.06 (C-9a), 141.93 (C-2), 139.83 (C-3a), 137.51 (C-5), 133.71 (C-8), 124.21 (C-9b), 122.18 (C-7), 115.33 (C-6'), 92.58 (C-1'), 88.62 (C-4'), 86.03 (C-2'), 82.95 (C-3'), 63.34 (C-5'), 55.13 (C-3"), 52.63 (C-1"), 27.58 (C-7'endo), 26.66 (C-4"), 25.55 (C-7'exo), 25.07 (C-5"), 22.79 (AcO-). HRMS: calculated for [M + H+] C21H29N6O4 + 429.2245 amu; found 429.2248 amu. 7-(Piperidin-1-ylmethyl)-3-ß-D-ribofuranosyl-3H-imidazo[2,1-i]purine (3b) Compound 3a (34.2 mg, 70.0 µmol), FeCl3.6H2O (37.8 mg, 0.140 mmol) and MeOH (50 µl) in DCM (950 µl) were refluxed first for 7 h and then allowed to stand at room temperature overnight. One eq. of FeCl3.6H2O was then added and refluxing was continued for a few hours until the reaction was complete as indicated by TLC (10% MeOH in DCM). Aqueous NaHCO3 (1 ml, half-saturated solution) was then added under stirring followed by evaporation to dryness. The residue containing the deprotected product 3b and ferric hydroxide was suspended in aqueous MeOH and mixed with silica gel followed by evaporation of the solvent. The product 3b was eluted from the silica gel with 20% H2O in MeCN by suctionfiltration and then further purified by HPLC (0-50% MeOH/H2O, 0.1 TFA, 250 × 21.2 mm, 5 µm Hypersil ODS2, UV detection at ? = 220 nm). Yield 23 mg (65%) of 3b as a TFA salt. 1H NMR (500 MHz, DMSO-d6): 9.21 s, 1 H (H-5); 8.57 s, 1 H (H-2); 7.44 s, 1 H (H-8); 6.05 d, 1 H, J = 5.7 (H-1'); 5.60 br, 1 H (OH); 5.33 br, 1 H (OH); 5.12 br, 1 H (OH); 4.60-4.62 m, 1 H (H-2'); 4.18-4.22 m, 1 H (H-3'); 3.98-4.00 m, 1 H (H-4'); 3.87 s, 2 H (H-1"); 3.69-3.72 m, 1 H (H-5'proS); 3.57-3.60 m, 1 H (H-5'proR); 2.37 br, 4 H (H-3"); 1.43-1.49 m, 4 H (H-4"); 1.33-1.41 m, 2 H (H-5"). 13C NMR (126 MHz, DMSO-d6): 141.23 (C-9a), 140.10 (C-2), 138.32 (C-3a), 136.22 (C-5), 132.52 (C-8), 123.09 (C-9b), 121.00 (C-7), 87.95 (C-1'), 85.63 (C-4'), 74.14 (C-2'), 70.33 (C-3'), 61.34 (C-5'), 53.55 (C-3"), 51.45 (C-1"), 25.56 (C-4"), 23.98 (C-5"). 15N NMR (50 MHz, DMSO-d6): -206.90 (N-3), -179.15 (N-6), -152.42 (N-4), -150.82 (N-9), -137.77 (N-1), N-1" not observed. HRMS: calculated for [M + H+] C18H25N6O4 + 389.1932 amu; found 389.1924 amu. 3-(2,3-O-Isopropylidene-ß-D-ribofuranosyl)-7-(morpholinomethyl)- 3H-imidazo[2,1-i]purine (4a) The synthesis was performed as described for 3a except that morpholine (48.4 µl, 0.556 mmol) was substituted for piperidine. The reaction was monitored by TLC (10% MeOH in DCM) and upon completion, the reaction mixture was stirred with aqueous NaHCO3 prior to evaporation. The crude material was purified by silica gel column chromatography (10% MeOH/ CH2Cl2) yielding 0.116 g (97%) of 4a. 1H NMR (600 MHz, CDCl3): 9.16 s, 1 H (H-5); 8.18 s, 1 H (H-2); 7.50 s, 1 H (H-8); 6.07 d, 1 H, J = 4.5 (H-1'); 5.24 dd, 1 H, J = 5.9, 4.5 (H-2'); 5.16 dd, 1 H, J = 6.0, 1.6 (H-3'); 4.60-4.56 m, 1 H, J = 1.9 (H-4'); 4.05 dd, J = -12.4, 2.1 (H-5'proS); 3.89 dd, 1 H, J = -12.4, 2.0 (H-5'proR); 3.88 d, 1 H, J = 14.1 (H-1"); 3.84 d, 1 H, J = 14.1 (H-1"); 3.68 t, 4 H, J = 4.3 (H-4"); 2.49-2.42 m, 4 H (H-3"); 1.68 s, 3 H (H-7'endo); 1.40 s, 3 H (H-7'exo). 13C NMR (151 MHz, CDCl3): 142.23 (C-9a), 140.45 (C-2), 137.62 (C-3a), 135.71 (C-5), 133.99 (C-8), 125.31 (C-9b), 119.88 (C-7), 114.35 (C-6'), 93.71 (C-1'), 86.15 (C-4'), 83.95 (C-2'), 81.66 (C-3'), 66.84 (C-4"), 63.20 (C-5'), 53.40 (C-3"), 52.60 (C-1"), 27.60 (C-7'endo), 25.34 (C-7'exo). 15N NMR (50 MHz, DMSO-d6): -206.05 (N-3), -179.18 (N-6), -152.39 (N-4), -150.7 (N-9), -137.86 (N-1), N-1" not observed. HRMS: calculated for [M + H+] C20H27N6O5 + 431.2037 amu; found 431.2033 amu. 7-(Morpholinomethyl)-3-ß-D-ribofuranosyl-3H-imidazo[2,1-i]purine (4b) Compound 4a (23.8 mg, 55.2 µmol) was dissolved in 0.1 M HCl in aqueous (53%) THF (2 ml) and the reaction mixture was stirred on an oil bath at 50 °C for 4.5 h and monitored by TLC (20% H2O in MeCN). Upon completion, the solution was cooled to room temperature, the pH adjusted to 8 with saturated NaHCO3 followed by evaporation to dryness. The crude material was purified by silica gel column chromatography (10-12.5% H2O in MeCN) yielding 17 mg (80%) of 4b. 1H NMR (400 MHz, D2O): 9.03 s, 1 H (H-5); 8.41 s, 1 H (H-2); 7.45 s, 1 H (H-8); 6.15 d, 1 H, J = 5.6 (H-1'); 4.84 t, 1 H, J = 5.4 (H-2'); 4.49 dd, 1 H, J = 5.2, 4.0 (H-3'); 4.30 q, 1 H, J = 3.8 (H-4'); 3.96 dd, 1 H, J = -12.8, 3.2 (H-5'proS); 3.94 s, 2 H (H-1"); 3.89 dd, 1 H, J = -12.8, 4.2 (H-5'proR); 3.73 t, 4 H, J = 4.6 (H-4"); 2.69-2.52 m, 4 H (H-3"). 13C NMR (101 MHz, D2O): 142.40 (C-9a), 142.08 (C-2), 139.61 (C-3a), 137.45 (C-5), 134.33 (C-8), 123.65 (C-9b), 121.55 (C-7), 90.14 (C-1'), 86.96 (C-4'), 75.50 (C-2'), 71.90 (C-3'), 67.56 (C-4"), 62.88 (C-5'), 53.81 (C-3"), 51.49 (C-1"). 15N NMR (50 MHz, DMSO-d6): -206.90 (N-3), -179.31 (N-6), -152.27 (N-4), -150.88 (N-9), -137.77 (N-1), N-1" not observed. HRMS: calculated for [M + H+] C17H23N6O5 + 391.1724 amu; found 391.1728 amu. 7-[(Benzylamino)methyl]-3-(2,3-O-isopropylidene-ß-D-ribofuranosyl)- 3H-imidazo[2,1-i]purine (5) Compound 2 (0.108 g, 0.300 mmol), benzylamine (32.8 µl, 0.300 mmol), NaCNBH3 (85%, 23.0 mg, 0.311 mmol) and acetic acid (34.4 µl, 0.601 mmol) were stirred in dry THF (2.0 ml) for 24 h. The reaction mixture was evaporated to dryness and the residue taken up in DCM and extracted with saturated NaHCO3. The organic phase was dried over Na2SO4 and again evaporated to dryness after filtration. The crude material was purified by silica gel column chromatography (10% MeOH/CH2Cl2) yielding 0.11 g (81%) of 5. 1H NMR (500 MHz, CDCl3): 9.18 s, 1 H (H-5); 8.16 s, 1 H (H-2); 7.44 s, 1 H (H-8); 7.34-7.22 m, 5 H (Ph); 6.07 d, 1 H, J = 4.3 (H-1'); 5.43 br, 1 H (OH); 5.24 dd, 1 H, J = 6.0, 4.3 (H-2'); 5.16 dd, 1 H, J = 6.0, 1.7 (H-3'); 4.56-5.54 m, 1 H (H-4'); 4.15 s, 2 H (H-1"); 4.03 dd, 1 H, J = -12.4, 2.1 (H-5'proS); 3.88 dd, 1 H, J = -12.4, 1.6 (H-5'proR); 3.80 s, 2 H (H-1"'); 1.67 s, 3 H (H-7'endo); 1.40 s, 3 H (H-7'exo). 13C NMR (101 MHz, CDCl3): 141.99 (C-9a), 140.35 (C-2), 139.47 (C-2"'), 137.61 (C-3a), 135.99 (C-5), 132.84 (C-8), 128.66 (C-4"'), 128.32 (C-3"'), 127.38 (C-5"'), 125.17 (C-9b), 122.13 (C-7), 114.32 (C-6'), 93.57 (C-1'), 86.30 (C-4'), 84.03 (C-2'), 81.69 (C-3'), 63.20 (C-5'), 53.17 (C-1"'), 42.60 (C-1"), 27.64 (C-7'endo), 25.40 (C-7'exo). HRMS: calculated for [M + H+] C23H27N6O4 + 451.2088 amu; found 451.2087 amu. 7-[(N-Acetyl-N-benzyl)aminomethyl]-3-ß-D-ribofuranosyl-3H-imidazo[2,1-i]purine (6) Compound 5 (20.5 mg, 45.5 µmol), acetic anhydride (0.500 ml, 5.29 mmol) and pyridine (0.200 ml, 2.48 mmol) were stirred in DCM (4.5 ml) until all of the starting material had been consumed according to TLC monitoring (10% MeOH in DCM). The reaction mixture was then evaporated to dryness and the residue taken up in DCM and extracted with saturated NaHCO3. The organic phase was dried over Na2SO4 and again evaporated to dryness after filtration. The remaining mixture of acetylated products was dissolved in dry MeOH (1.0 ml) containing sodium hydride (60% dispersion in mineral oil, 3.0 mg, 75 µmol). When deacetylation of the hydroxyl groups was complete, the mixture was diluted with DCM and extracted with 3% aq. KH2PO4. The organic phase was dried over Na2SO4 and the solvent was removed after filtration. The product was purified by silica gel column chromatography (5% MeOH in DCM) yielding 16.5 mg (74%) of 6. The isopropylidene protection was removed as described for 4b and the deprotected material was purified by silica gel column chromatography (10% H2O/MeCN) yielding 11 mg (53% from 5) of 6. Conformers A and B denoted by demarkations A and B, respectively. 1H NMR (500 MHz, CD3OD): 9.48 s, 0.9 H (H-5A); 8.98 s, 0.1 H (H-5B); 8.85 s, 0.9 H (H-2A); 8.81 s, 0.1 H (H-2B); 7.84 s, 0.1 H (H-8B); 7.81 s, 0.9 H (H-8A); 7.32-7.24 m, 1.8 H (H-4"'A); 7.23-7.16 m, 2.7 H (H-3"A, H-5"'A); 7.11-7.03 m, 1.8 H (H-4"'B); 7.01-6.94 m, 2.7 H (H-3"'B, H-5"'B); 6.26 d, 0.9 H, J = 5.1 (H-1'A); 6.23 d, 0.1 H, J = 5.0 (H-1'B); 5.15 s, 2 H (1"); 4.72 s, 2 H (H-1"'); 4.70-4.68 m, 1 H (H-2'); 4.40-4.38 m, 1 H (H-3'); 4.20-4.18 m, 1 H (H-4'); 3.92 dd, 1 H, J = -12.2, 3.2 (H-5'proS); 3.82 dd, 1 H, J = -12.2, 3.5 (H-5'proR); 2.40 s, 0.3 H (H-4"B); 2.28 s, 2.7 H (H-4"A). 13C NMR (126 MHz, CD3OD): 174.44 (C-3"A), 174.08 (C-3"B), 143.20 (C-9a), 142.03 (C-2A), 141.81 (C-2B), 140.20 (C-3a), 137.49 (C-2"'), 137.30 (C-5A), 135.96 (C-5B), 134.41 (C-8A), 133.38 (C-8B), 129.92 (C-4"'A), 129.24 (C-4"'B), 128.66 (C-5"'A), 128.37 (C-3"'B), 127.91 (C-5"B), 127.47 (C-3"'A), 124.25 (C-9b), 121.94 (C-7), 90.78 (C-1'A), 90.87 (C-1'B), 87.30 (C-4'), 76.20 (C-2'), 72.03 (C-3'), 62.94 (C-5'), 52.15 (C-1"'A), 49.73 (C-1"'B), 43.86 (C-1"B), 39.00 (C-1"A), 21.88 (C-4"B), 21.81 (C-4"A). HRMS: calculated for [M + H+] C22H25N6O5 + 453.2881 amu; found 453.2883 amu. 3-(2,3-O-Isopropylidene-ß-D-ribofuranosyl)-7-[N-(4-tert-butoxy-4-oxobutyl)aminomethyl]- 3H-imidazo[2,1-i]purine (7) Compound 2 (0.300 g, 0.835 mmol) and tert-butyl 4-aminobutanoate hydrochloride (0.196 g, 1.00 mmol) were suspended in DCE (6 ml) to which NaBH(OAc)3 (0.505 g, 2.38 mmol) was added in three batches. The reaction mixture was stirred at room temperature for 24 h followed by refrigeration over a weekend and then evaporation to dryness. To remove any residual acetic acid, the residue was taken up twice with water (2 × 10 ml) to effect co-removal via solvent evaporation. The crude material was purified by silica gel column chromatography (10-15% H2O/MeCN) yielding 0.224 g (53%) of 7. 1H NMR (500 MHz, CD3OD): 9.18 s, 1 H (H-5); 8.44 s, 1 H (H-2); 7.53 s, 1 H (H-8); 6.25 d, 1 H, J = 2.9 (H-1'); 5.29 dd, 1 H, J = 6.1, 2.9 (H-2'); 5.01 dd, H 1, J = 6.1, 2.7 (H-3'); 4.46 s, 2 H (H-1"); 4.37-4.30 m, 1 H (H-4'); 3.74 dd, 1 H, J = -11.9, 4.0 (H-5'proS); 3.69 dd, 1 H, J = -12.0, 4.7 (H-5'proR); 2.96-2.88 m, 2 H (H-3"); 2.31 t, 2 H, J = 7.2 (H-5"); 1.85 p, 2 H, J = 7.2 (H-4"); 1.58 s, 3 H (H-7'endo); 1.35 s, 12 H (H-7'exo, H-9"). 13C NMR (126 MHz, CD3OD): 173.21 (C-6"), 142.20 (C-9a), 141.26 (C-2), 139.04 (C-3a), 136.17 (C-5), 133.68 (C-8), 123.06 (C-9b), 119.99 (C-7), 114.45 (C-6'), 91.66 (C-1'), 87.76 (C-4'), 85.15 (C-2'), 82.07 (C-3'), 80.90 (C-8"), 62.48 (C-5'), 47.67 (C-3"), 41.11 (C-1"), 32.68 (C-5"), 27.43 (C-9"), 26.73 (C-7'endo), 24.72 (C-7'exo), 23.56 (C-4"). HRMS: calculated for [M + H+] C24H35N6O6 + 503.2613 amu; found 503.2611 amu. 7-[(2-Oxopyrrolidin-1-yl)methyl]-3-ß-D-ribofuranosyl-3H-imidazo[2,1-i]purine (8) Compound 7 (23.0 mg, 45.8 µmol) was stirred for 1 h in a 1:1 mixture of TFA and DCM after which the volatiles were removed under vacuum. To the residue, HBTU (19.1 mg, 50.3 µmol) and DIEA (8.9 µl, 50.3 µmol) dissolved in dry DMF (3.0 ml) were added. After two days, an additional one-half equivalent of HBTU was added to the incomplete reaction and stirring was continued for an additional day. After completion of the reaction, the DMF was evaporated off and the isopropylidene protection removed as described for 4b. The deprotected material was purified by silica gel column chromatography (10-12.5% H2O/MeCN) yielding 7.7 mg (43% from 7) of 8. 1H NMR (500 MHz, CD3OD): 9.15 s, 1 H (H-5); 8.53 s, 1 H (H-2); 7.58 s, 1 H (H-8); 6.16 d, 1 H, J = 5.3 (H-1'); 4.93 s, 1 H (H-1"); 4.71 t, 1 H, J = 5.2 (H-2'); 4.38 t, 1 H, J = 4.6 (H-3'); 4.16 q, 1 H, J = 3.5 (H-4'); 3.90 dd, 1 H, J = -12.2, 3.1 (H-5'proS); 3.79 dd, 1 H, J = -12.2, 3.6 (H-5'proR); 3.39 t, 2 H, J = 7.1 (H-5"); 2.43 t, 2 H, J = 8.1 (H-3"); 2.05-1.95 m, 2 H, J = 8.1, 7.1 (H-4"). 13C NMR (126 MHz, CD3OD): 178.03 (C-3"), 143.19 (C-9a), 142.01 (C-2), 140.27 (C-3a), 136.88 (C-5), 133.98 (C-8), 124.32 (C-9b), 121.28 (C-7), 90.75 (C-1'), 87.23 (C-4'), 76.19 (C-2'), 71.97 (C-3'), 62.87 (C-5'), 47.90 (C-4"), 36.56 (C-1"), 31.77 (C-6"), 18.49 (C-5"). 15N NMR (50 MHz, DMSO-d6): -206.70 (N-3), -180.00 (N-6), -151.62 (N-4), -150.36 (N-9), -137.7 (N-1), N-1" not observed. HRMS: calculated for [M + H+] C17H21N6O5 + 389.1568 amu; found 389.1564 amu. 3-(2,3-O-Isopropylidene-ß-D-ribofuranosyl)-7-{[N-(4-tert-butoxy-4-oxobutyl)- N-benzyl]aminomethyl}-3H-imidazo[2,1-i]purine (9) Compound 7 (35.2 mg, 70.0 µmol) and benzaldehyde (14.3 µl, 0.140 mmol) were dissolved in dry MeOH (0.40 ml) to which ZnCl2 (9.5 mg, 70.0 µmol) and NaCNBH3 (85%, 10.4 mg, 0.140 mmol) in dry MeOH (0.20 ml) were added followed by stirring for 28 h. The reaction was monitored by TLC (10% H2O/MeCN) and by ESI-MS analysis. Upon completion of the reaction, saturated aq. NaHCO3 (0.20 ml) and water (0.3 ml) were added and the mixture was extracted with DCM, after which the water phase was concentrated to near dryness and some MeCN was added. The volatiles were removed and the product was purified by silica gel column chromatography (2.5% H2O/MeCN) yielding 37.4 mg (90%) of 9. 1H NMR (400 MHz, CDCl3): 8.87 s, 1 H (H-5); 8.26 s, 1 H (H-2); 7.48 s, 1 H (H-8); 7.36-7.29 m, 2 H (H-4"'); 7.29-7.19 m, 3 H (H-2"', H-3"'); 6.12 d, 1 H, J = 4.1 (H-1'); 5.24 dd, 1 H, J = 6.0, 4.1 (H-2'); 5.17 dd, 1 H, J = 6.0, 1.5 (H-3'); 4.58-4.55 m, 1 H, J = 2.1 (H-4'); 4.05 dd, 1 H, J = -12.3, 2.2 (H-5'proS); 3.91 dd, 1 H, J = -12.3, 2.5 (H-5'proR); 3.90 d, 1 H, J = 14.3 (H-1"); 3.86 d, 1 H, J = 14.3 (H-1"); 3.63 d, 1 H, J = 13.3 (H-1"'); 3.58 d, 1 H, J = 13.3 (H-1"'); 2.54-2.42 m, 2 H (H-3"); 2.16-2.06 m, 2 H (H-5"); 1.87-1.75 m, 2 H (H-4"); 1.68 s, 3 H (H-7'endo); 1.41 s, 3 H (H-7'exo); 1.36 s, 9 H (H-9"). 13C NMR (101 MHz, CDCl3): 172.61 (C-6"), 141.95 (C-9a), 140.36 (C-2), 138.25 (C-2"'), 137.78 (C-3a), 135.71 (C-5), 133.66 (C-8), 129.23 (C-3"'), 128.60 (C-4"'), 127.49 (C-5"'), 124.76 (C-9b), 121.18 (C-7), 114.21 (C-6'), 93.27 (C-1'), 86.48 (C-4'), 84.27 (C-2'), 81.79 (C-3'), 80.42 (C-8"), 63.09 (C-5'), 58.43 (C-1"'), 53.01 (C-3"), 48.06 (C-1"), 33.17 (C-5"), 28.10 (C-9"), 27.60 (C-7'endo), 25.40 (C-7'exo), 22.13 (C-4"). HRMS: calculated for [M + H+] C31H41N6O6 + 593.3082 amu; found 593.3071 amu. 3-(ß-D-Ribofuranosyl)-7-{[N-(4-tert-butoxy-4-oxobutyl)-N-benzyl]aminomethyl}- 3H-imidazo[2,1-i]purine (10) Compound 9 (26.5 mg, 44.7 µmol) was deprotected as described for 4b and the product was purified by silica gel column chromatography (10% MeOH/CH2Cl2) yielding 12.6 mg (51%) of 10. 1H NMR (500 MHz, 2:5 CD3OD/CDCl3): 8.87 s, 1 H (H-5); 8.33 s, 1 H (H-2); 7.44 s, 1 H (H-8); 7.36-7.30 m, 2 H (H-4"'); 7.30-7.26 m, 2 H (H-3"'); 7.26-7.21 m, 1 H (H-5"'); 6.10 d, 1 H, J = 6.1 (H-1'); 4.73 dd, 1 H, J = 6.1, 5.1 (H-2'); 4.44 dd, 1 H, J = 5.1, 2.9 (H-3'); 4.29 ddd, 1 H, J = 2.9, 2.4 (H-4'); 3.99 dd, 1 H, J = -12.6, 2.4 (H-5'proS); 3.92 s, 2 H (H-1"); 3.84 dd, 1 H, J = -12.6, 2.4 (H-5'proS); 3.63 s, 2 H (H-1"'); 2.57-2.46 m, 2 H (H-3"); 2.15 t, 2 H, J = 7.3 (H-5"); 1.83 p, 2 H, J = 7.3 (H-4"); 1.36 s, 9 H (C-9"). 13C NMR (126 MHz, 2:5 CD3OD/CDCl3): 173.41 (C-6"), 141.91 (C-9a), 140.90 (C-2), 138.48 (C-2"'), 138.43 (C-3a), 135.90 (C-5), 132.93 (C-8), 129.40 (C-3"'), 128.77 (C-4"'), 127.70 (C-5"'), 124.12 (C-9b), 121.84 (C-7), 90.45 (C-1'), 86.76 (C-4'), 81.01 (C-8"), 75.05 (C-2'), 71.33 (C-3'), 62.51 (C-5'), 58.64 (C-1"'), 53.22 (C-3"), 48.09 (C-1"), 33.35 (C-5"), 28.04 (C-9"), 22.34 (C-4"). 15N NMR (50 MHz, DMSO-d6): -206.79 (N-3), -179.50 (N-6), -152.49 (N-4), -150.64 (N-9), -137.58 (N-1), N-1" not observed. 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J., Cammi R., Pomelli C., Ochterski J. W., Martin R. L., Morokuma K., Zakrzewski V. G., Voth G. A., Salvador P., Dannenberg J. J., Dapprich S., Daniels A. D., Farkas Ö., Foresman J. B., Ortiz J. V., Cioslowski J., Fox D. J.: Gaussian 09, Revision A.2. Gaussian, Inc., Wallingford (CT) 2009. Tuomas KARSKELA1,*, Karel D. KLIKA2 and Harri LÖNNBERG3 Department of Chemistry, University of Turku, Vatselankatu 2, FIN-20014 Turku, Finland; e-mail: 1 [email protected], 2 [email protected], 3 [email protected] Received April 8, 2011 Accepted May 2, 2011 Published online July 21, 2011 Dedicated to Professor Antonín Holý on the occasion of his 75th birthday in recognition of his outstanding contributions to the area of nucleic acid chemistry. (c) 2011 Institute of Organic Chemistry and Biochemistry |