Synthesis of substituted 2-(β-d-glucopyranosyl)-benzimidazoles and their evaluation as inhibitors of glycogen phosphorylase

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Please check this box if you have no corrections to make to the PDF file Graphical abstract pp xxx–xxxSynthesis of substituted 2-(bDD -glucopyranosyl)-benzimidazoles and their evaluation as inhibitors of glycogen phosphorylase Éva Bokor, Enik} o Szilágyi, Tibor Docsa, Pa ´l Gergely, La ´szlo ´Somsa ´k * CAR 6384 No. of Pages 1, Model 5G 5 February 2013 Highlights "Synthesis of C-(bDD -glucopyranosyl)formimidates and -thioformimidates. "New synthesis of C-(bDD -glucopyranosyl)-benzimidazoles. "Low micromolar inhibitors of glycogen phosphorylase. X=O,S O OR 1 R 1 O R 1 O OR 1 N H N O OBz BzO BzO COOH OBz O OBz BzO BzO CXNH 2 OBz O OBz BzO BzO C OBz NH XEt H 2 N H 2 N R 2 Et 3 OBF 4 from X=O R 2 R 1 =Bz H NO 2 O OH HO HO OH N H N best inhibitor K i =2.1μM(rabbitmu s R 2 =5-F,5-Cl,5-Br,5N 4-Me, 5-Me, 5,6-di 1 Synthesis of substituted 2-(b-D-glucopyranosyl)-benzimidazoles and their evaluation as inhibitors of glycogen phosphorylase Éva Bokor a ,Enik} oSzilágyi a ,Tibor Docsa b ,Pál Gergely b ,László Somsák a, ⇑ a Department of Organic Chemistry, University of Debrecen, POB 20, H-4010 Debrecen, Hungary b Department of Medical Chemistry, Medical and Health Science Centre, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary article info Article history: Received 3 November 2012 Received in revised form 15 January 2013 Accepted 16 January 2013 Available online xxxx Keywords: C-Glycopyranosyl-formimidate C-Glycopyranosyl-thioformimidate 2-C-Glycopyranosyl benzimidazole Glycogen phosphorylase Inhibitor abstract Microwave assisted condensation of O-perbenzoylated C-(b-D-glucopyranosyl)formic acid with 1,2-diaminobenzenes in the presence of triphenylphosphite gave the corresponding O-protected 2-(b-D-glucopyranosyl)-benzimidazoles in moderate yields. O-Perbenzoylated C-(b-D-glucopyranosyl) formamide and -thioformamide were transformed into the corresponding ethyl C-(b-D-glucopyranosyl) formimidate and -thioformimidate, respectively, by Et 3 OBF 4 . Treatment of the formimidate with 1,2-diaminobenzenes afforded O-protected 2-(b-D-glucopyranosyl)-benzimidazoles-in good to excellent yields. Similar reaction of the thioformimidate gave these compounds in lower yields. The O-benzoyl protecting groups were removed by the Zemplén protocol. These test compounds were assayed against rabbit muscle glycogen phosphorylase (GP) b,the prototype of liver GP, the rate limiting enzyme of glycogen degradation. The best inhibitors were 2-(b-D-glucopyranosyl)-4-methyl-benzimidazole (K i = 2.8 l M) and 2-(b-D-glucopyranosyl)-naphtho[2,3-d]imidazole (K i = 2.1 l M) exhibiting a 3–4 times stronger binding than the unsubstituted parent compound. Ó2013 Published by Elsevier Ltd. 1. Introduction Inhibition of glycogen phosphorylase (GP) has been considered as an effective therapeutic approach in combating type 2 diabetes (for the biochemical and pharmacological background of targeting liver GP as a validated concept for lowering blood glucose levels, please survey recent review articles 1–6 ). Furthermore, the pharmaceutical utility of GP inhibitors in the intervention of other diseased states associated with GP activity (e.g., cardiovascular dis50 orders, 7–9 ischaemic lesions, 10–13 and tumorous growth 11,14–16 ) has also been under investigation. A large array of compounds were shown to have an inhibitory effect against this enzyme 17–19 including glucose derivatives 20,21 which primarily bind to the catalytic site of the enzyme. In the course of searching for potent glucose based inhibitors, several C-bD -glucopyranosyl heterocycles (Chart 1), such as tetrazole 22 1A, 1,3,4-oxadiazoles 22–24 1B, 1,2,4-oxadiazoles 23,25,26 1C,D, benzothiazole 22 1E and benzimidazole 22 1F have been synthesized and some of them proved to be efficient against rabbit muscle glycogen 60 phosphorylase b(RMGPb, the prototype of GP enzymes for enzymatic tests 2 ). In this class, 2-(bD -glucopyranosyl)-benzimidazole (1F) was the first compound to have a K i value in the low micromolar range. 22,27 As evidenced by X-ray crystallography, the strong binding in the catalytic centre is the result of direct and water mediated H-bonds between the protein and the heteroaromatic ring, and van der Waals interactions of the large aromatic part in the so-called b-channel of the enzyme. 27 The higher affinity of 1F in comparison with its thio counterpart 1E can be attributed to the direct H-bond of the imidazole NH with the His377 main chain 70 carbonyl group of the enzyme which is certainly absent for benzothiazole 1E. 27 Analogous NH-(His377)CO interactions were identified in other GP enzyme-inhibitor complexes (e.g., in cases of spiro(thio)hydantoin 28,29 and N-acyl-bD -glucopyranosylamine type inhibitors 30 ) indicating the prominent importance of this special H-bridge, as well. Additionally, X-ray crystallographic investigation of 1F in complex with RMGPbrevealed that besides the active site the compound also occupied the new allosteric site, and a new binding cleft called the benzimidazole site was also discovered. 27 80 To get an insight into the structure–activity relationship of this type of inhibitor the aim of our present work has been to synthesize a series of substituted 2-(bD -glucopyranosyl)-benzimidazoles and to evaluate their effect on RMGPb. For the formation of 2-C-glycosyl-benzimidazoles several synthetic methods are known from the literature most of which have been described for furanose based derivatives. Furanosyl benzimidazoles were prepared (a) by acid catalysed condensation of C-glycofuranosyl formic acids and o-phenylenediamine (OPD), 31,32 (b) by coupling of C-glycofuranosyl formic acids or their chlorides with 90 1,2-diaminobenzenes, followed by acid or POCl 3 mediated ring closure of the resulting amide type intermediates, 33,34 (c) in the 0008-6215/$ - see front matter Ó2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.carres.2013.01.011 ⇑ Corresponding author. Tel.: +36 52512900x22348; fax: +36 52512744. E-mail address: [email protected] (L. Somsák). Q1 Q2 Q3 Carbohydrate Research xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDirect Carbohydrate Research journal homepage: www.elsevier.com/locate/carres CAR 6384 No. of Pages 9, Model 5G 5 February 2013 Please cite this article in press as: Bokor,É.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2013.01.011 reaction of C-glycofuranosyl formaldoximoyl chlorides (actually the nitrile oxide obtained by base induced dehydrochlorination) with OPD, 35 (d) by intramolecular ring closure of suitably protected 2-(pentitol-1 0 -yl)-benzimidazoles obtained from 2-lithiated benzimidazoles and sugar lactone or acyclic pentose derivatives, 36–38 and (e) by acid induced cyclodehydration of unprotected 2-(tetritol-1 0 -yl)-benzimidazoles. 39 For the synthesis of 2-C-glycopyranosyl-benzimidazoles three 100 procedures were reported: (a) acid catalysed condensation of unprotected C-glycopyranosylmethanals (generated in situ from the appropriate dimethyl acetals) with OPD, followed by spontaneous oxidation of the intermediate benzimidazolines, 40 (b) reaction of O-perbenzoylated ethyl (C-bD -glucopyranosyl)thioformimidate hydrochloride (prepared from the corresponding glycosyl cyanide) with OPD, 22 and (c) treatment of O-peracetylated C-glycopyranosyl formaldoximoyl chlorides with OPD. 41 In addition, a 2-benzimidazolyl moiety was also attached to C-1 of D -galactal in the reaction of methyl C-(2-deoxyD -lyxo-hex-1-enopyranosyl)formimidate and 110 the dihydrochloride salt of OPD. 42 In this paper, we disclose further synthetic possibilities to construct benzimidazole at the anomeric centre of the pyranose unit. 2. Results and discussion 2.1. Syntheses C-(bD -Glucopyranosyl)formic acid 5, prepared from cyanide 2 via amide 3 43 by the literature protocol 44 (Table 1), was reacted with 1,2-diaminobenzenes (a,f,h) in the presence of triphenylphosphite in pyridine under microwave irradiation (conditions were adapted from a published procedure applied for non-sugar based com120 pounds 45 ). Although the conversions were complete at 140 °Cin 20 min, the desired products could be isolated only in moderate yields (8a: 45%, 8f: 53%, 8h: 43%). Nevertheless, it has to be noted, that earlier attempts to transform 5into benzimidazole 8a by the classical acid catalysed procedure with conventional heating brought about no reaction even at elevated temperatures. 22 Therefore, application of the more reactive iminoesters 6and 7 was envisaged for the construction of benzimidazoles. Previously, hydrochloride salt of O-perbenzoylated ethyl C-(bD -glucopyranosyl)thioformimidate obtained from glucopyr130 anosyl cyanide 2by an acid catalysed addition of EtSH to the nitrile group was used for this purpose. 22 Since these conditions were rather unpleasant and subsequent transformation of the salt gave benzimidazole 8a in a 34% yield only, we set out to produce and use the free form of this type of precursor. Free thioimidate 7 was obtained from thioamide 4by Et 3 OBF 4 . The preparation of 4 46 was also modified to avoid the use of H 2 S; thus, cyanide 2 was reacted with P 4 S 10 in refluxing EtOH as described for the synthesis of aliphatic and aromatic thioamides. 47 Similarly to 7, imidate 6was obtained from amide 3by Et 3 OBF 4 . 140 Next, the preparation of benzimidazole 8a from either 6or 7 was compared in preliminary experiments. Reaction of 6or 7with OPD gave 8a in 89% and 62% yields, respectively. Therefore, also taking into account the instability of 7(decomposition was observed on storage at rt after a few days), further reactions to get the desired benzimidazoles were performed with 6. Similarly to 8a, high yields were also achieved in reactions of 6 with methyl-substituted 1,2-diaminobenzenes f–h and 2,3-diaminonaphthalene i, respectively (Table 1). A comparison of the yields for 8a,f,h obtained from acid 5under conditions vand from imi150 date 6under conditions vi, respectively, showed the ring closure of 6with 1,2-diaminobenzenes to be superior to that of 5. On treatment of imidate 6with 1,2-diaminobenzenes containing electron withdrawing substituents (b–e) benzoic acid elimination was also observed, and beside the 2-glucopyranosyl benzimidazoles 8b–e 4-substituted-2-(3 0 ,4 0 ,6 0 -tri-O-benzoyl-2 0 - deoxyD -arabino-hex-1-enopyranosyl)-benzimidazoles (1-C-benzimidazolyl glucals, 9b–e) were also isolated. Furthermore, total consumption of the starting material 6in reaction with 4-nitro-1,2-diaminobenzene (e) required higher temperature, thus 160 cyclization was performed in boiling ethanol to yield benzimidazole 8e together with 9e. This tendency for elimination might be attributed to an increased acidity of the C-1–H proton in the 2-glucosyl-benzimidazoles probably due to the presence of the electron withdrawing substituent in the aromatic ring system. For enzymatic studies removal of the benzoyl protecting groups of 8b–i was effected by the Zemplén method to give test compounds 10b–i in good yields. The structure of the new compounds was determined by 1 H and 13 C NMR spectroscopy. The presence of a bD -configured glucopyr170 anosyl moiety in the 4 C 1 conformation was confirmed by the vicinal proton–proton coupling constants for compounds 6,7,8b–i and 10b–i, respectively. In the 1 H NMR spectra of 1-C-hetaryl glucals 9b–e the observed small couplings between the vicinal ring protons (3–6 Hz) suggested a conformational change of the pyranose ring (from chair to half chair). 48 In the 13 C NMR spectra of 9b–e characteristic resonances appeared for C-1 0 (143–145 ppm) and C-2 0 (99–101 ppm) providing evidence for the unsaturated nature of the pyranoid ring. 48 Among 13 C NMR signals of the benzimidazole units only those for C-2 appeared as sharp peaks at 146– 180 158 ppm, the others usually gave broad signals. These observations are in accord with the literature experiences, 35,41 and may be explained by the rapid proton exchange between N-1 and N-3 of the benzimidazole moiety. 35,41,49 Additionally, in the 13 CNMR spectra duplication of certain carbon peaks was also observed (primarily for the benzimidazole carbon signals for example, in 8b,9c, 9e and 10b), which may refer to the coexistence of the tautomeric pairs. For the fluoro-benzimidazole 8b this phenomenon seemed quite clear as 14 resonances were observed in the 13 C NMR spectrum which could be tentatively assigned on the basis of the char190 acteristic C–F couplings 50 as illustrated in Figure 1. O OH HO HO C OH 1 Het R Het R K i [μM] A NN NHN - No inhibition 22 B O NN R CH 3 145 27 212 22 2-naphthyl 10 % at 625 μM 23 C N ON R 2-naphthyl 38 25 D N NO R 2-naphthyl 2.4 23 E S N -76 27 229 22 F N H N -8.6 27 11 22 Chart 1. Inhibitory potency (K i )ofC-glucosyl heterocyclic derivatives against rabbit muscle glycogen phosphorylase b(RMGPb). 2Éva Bokor et al. /Carbohydrate Research xxx (2013) xxx–xxx CAR 6384 No. of Pages 9, Model 5G 5 February 2013 Please cite this article in press as: Bokor,É.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2013.01.011 2.2. Enzyme kinetic studies Enzyme kinetic measurements with rabbit muscle glycogen phosphorylase bwere performed as described previously, 51,52 and the obtained inhibitor constants are given in Table 2. The new compounds showed inhibitory effects in the micromolar range and some of them proved equivalent or somewhat better inhibitors than 10a (=1F in Chart 1). Among benzimidazoles with a halogen substituent in the 5-position fluoro derivative 10b exhibited modest activity, while the 200 chloro (10c) as well as bromo (10d) compounds were as good inhibitors as 10a. Substitution of the benzimidazole ring in the same position by a nitro group (10e) brought about a significant decrease of the inhibition. Among the alkyl substituted derivatives the 5-methyl- (10f) and 4-methyl-benzimidazoles (10g) displayed similar and slightly better (3-fold increase) potency than 10a, respectively. However, introduction of two methyl groups into the 5and 6-positions of the heterocycle (10h) resulted in a remarkable weakening of the inhibition. Finally, the naphtho[2,3d]imidazole 10i showed 4 times stronger binding than 10a and 210 proved to be the most effective inhibitor of this series. To elucidate the nature of the binding modes of the new compounds to GP enzyme X-ray crystallographic studies are in progress and will be reported elsewhere. Table 1 Synthesis of 2-(b-D-glucopyranosyl)-benzimidazoles 3X=O(94%) 4X=S(66%) + 6X=O(96%) 7X=S(90%) 8a-i R 1 =Bz 9a-i a-i 5(76 %) iii iv v vi O OR 1 R 1 O R 1 O OR 1 N H N R 2 R 3 R 4 O OBz BzO BzO COOH OBz O OBz BzO BzO CXNH 2 OBz O OBz BzO BzO C OBz NH XEt O BzO BzO OBz N H N R 2 R 3 R 4 10a-i R 1 =H vii 2 O OBz BzO BzO CN OBz H 2 N H 2 N R 2 R 3 R 4 ito 3 ii to 4 i) HBr-AcOH, rt;43 ii) P4S10, abs. EtOH, reflux; iii) NO2, abs. CH2Cl2, rt;44 iv) Et3O·BF4, abs. CH2Cl2, Ar, rt; v) P(OPh)3 (1.2 equiv.), a or f or h (1.0 equiv), abs. pyridine, μW, 140 ºC, 20 min; vi) a-i (2 equiv.), abs. CH2Cl2, reflux; vii) ~1 M NaOMe in abs. MeOH, rt. Reagent R 2 R 3 R 4 Conditions and yields (%) 8910 aHH Hv45 — vii — c,d vi 89 (from 6)— vi 62 a (from 7) bHF Hvi 46 35 vii 54 cHCl Hvi 76 24 vii 89 dHBrHvi 54 29 vii 58 eHNO 2 Hvi b 46 20 vii 53 fHMeHv53 — vii 87 vi 83 — gMe H H vi 79 — vii 73 hHMeMev43 — vii 78 vi 81 — iHvi 79 — vii 77 a Reported yield of 8a by the earlier method from perbenzoylated bD -glucopyranosyl cyanide via the thioimidate hydrochloride salt: 34%. 22 b Modified reaction conditions were necessary for complete consumption of the starting material 6: abs. EtOH, reflux. c Debenzoylation of 8a by the Zemplén protocol was carried out earlier. Reported yield of 10a: 84%. 22 d Reported yield of 10a by a different method: 54%. 40 N N H F Bz 4 -β-D-Glc p (H) 1(3) 3(1) 3a(7a) 4(7) 5(6) 6(5) 7(4) 7a(3a) 2(2) 151.1 (s) 150.3 (s) 158.9 (d, 1 J C-F =237Hz) 158.2 (d, 1 J C-F =237Hz) 104.5 (d, 2 J C-F =24Hz) 98.0 (d, 2 J C-F =27Hz) 142.8 (d, 3 J C-F ~9Hz) 134.2 (d, 3 J C-F ~9Hz) 139.0 (s) 130.8 (s) 110.9 (d, 2 J C-F =23Hz) 109.7 (d, 2 J C-F =23Hz) 120.0 (d, 3 J C-F ~7Hz) 112.3 (d, 3 J C-F ~6Hz) interchangable assignments Figure 1. Tentative assignment of 13 C NMR signals for tautomers of compound 8b. Éva Bokor et al. /Carbohydrate Research xxx (2013) xxx–xxx 3 CAR 6384 No. of Pages 9, Model 5G 5 February 2013 Please cite this article in press as: Bokor,É.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2013.01.011 3. Conclusion New synthetic pathways were elaborated for the formation of substituted 2-(bD -glucopyranosyl)-benzimidazoles by heterocyclizations of 2,6-anhydro-aldonic acid derivatives as precursors. Condensation of O-perbenzoylated C-(bD -glucopyranosyl)formic acid with 1,2-diaminobenzenes in the presence of P(OPh) 3 under 220 microwave irradiation gave the desired benzimidazoles in moderate yields. Ring closure of O-perbenzoylated ethyl C-(bD -glucopyranosyl)formimidate, prepared from the corresponding formamide by Et 3 OBF 4 , with the 1,2-diaminobenzenes afforded a more efficient procedure. With electron depleted aromatic diamines undesirable b-elimination of benzoic acid from the 1,2-positions of the glucopyranosyl moiety also took place to yield 1-C-benzimidazolyl glucals besides the target compounds. Enzyme kinetic studies with the new 2-(bD -glucopyranosyl)-benzimidazoles showed the compounds to inhibit RMGPbin the low micromolar 230 range. Substitution of the benzimidazole moiety by a methyl group in the 4-position or further annelation of a benzene ring provided more efficient inhibitors than the unsubstituted parent compound. 4. Experimental 4.1. General methods Melting points were measured on a Kofler hot-stage and are uncorrected. Optical rotations were determined with a Perkin–Elmer 241 polarimeter at rt. NMR spectra were recorded with Bruker 360 (360/90 MHz for 1 H/ 13 C) spectrometer. Chemical shifts are referenced to Me 4 Si ( 1 H), or to the residual solvent signals ( 13 C). 240 Microwave assisted procedures were performed in a CEM-Discover Focused Microwave Synthesis System (2450 MHz) with a built-in infrared temperature sensor and a CEM-Explorer computer controlled robotic sampler. TLC was performed on DC-Alurolle Kieselgel 60 F 254 (Merck), and the plates were visualized under UV light and by gentle heating. For column chromatography Kieselgel 60 (Merck, particle size 0.063–0.200 mm) was used. Dichloromethane was distiled from P 4 O 10 and stored over 4 Å molecular sieves. 2,3,4,6-Tetra-O-benzoyl-bD -glucopyranosyl cyanide, 43 C-(2,3,4, 250 6-tetra-O-benzoyl-bD -glucopyranosyl)formamide 43 and C-(2,3,4, 6-tetra-O-benzoyl-bD -glucopyranosyl)formic acid 44 were synthesized according to published procedures. 4.2. C-(2,3,4,6-Tetra-O-benzoyl-bD -glucopyranosyl) thioformamide (4) A solution of phosphorus pentasulfide (2.23 g, 10 mmol) in anhydrous EtOH (20 mL) was stirred at rt for 1.5 h, then 2,3,4,6-tetra-O-benzoyl-bD -glucopyranosyl cyanide 43 (2, 3.02 g, 5 mmol) was added and the mixture was heated at reflux temperature for 2h.After cooling the heterogenous mixture to rt, the precipitate 260 was filtered off and washed with EtOH to yield 2.1 g (66%) of 4 as a pale yellowish solid. Mp: 199–201 °C (Lit. 46 mp: 198– 200 °C). 1 H and 13 C NMR data correspond to the reported spectra. 46 4.3. Ethyl C-(2,3,4,6-tetra-O-benzoyl-bD -glucopyranosyl) formimidate (6) C-(2,3,4,6-Tetra-O-benzoyl-bD -glucopyranosyl)formamide 43 (3, 1.0 g, 1.60 mmol) and Et 3 OBF 4 (0.91 g, 4.80 mmol) were dissolved in anhydrous CH 2 Cl 2 (15 mL), the mixture was stirred at rt under Ar and monitored by TLC (1:1 EtOAc–hexane). After completion of the reaction (2 days), the mixture was diluted with CH 2 Cl 2 270 (30 mL), extracted with satd aq NaHCO 3 solution (30 mL) and then with water (30 mL). The organic phase was dried over MgSO 4 ,filtered and the solvent was evaporated. The crude pale yellow amorphous product (1.0 g, 96%) was used without further purification. R f : 0.55 (1:1 EtOAc–hexane); 1 H NMR (CDCl 3 )d(ppm): 8.06–7.25 (21H, m, aromatics, NH), 5.99, 5.72, 5.55 (3 1H, 3 pseudo t, J= 10.6, 9.2 Hz in each, H-2, H-3, H-4), 4.70 (1H, dd, J= 11.9, 2.6 Hz, H-6a), 4.52 (1H, dd, J= 11.9, 5.3 Hz, H-6b), 4.25 (1H, ddd, J= 9.2, 5.3, 2.6 Hz, H-5), 4.21 (1H, d, J= 9.2 Hz, H-1), 4.12–3.91 (2H, m, CH 2 ), 0.84 (3H, t, 6.6 Hz, CH 3 ); 13 C NMR (CDCl 3 )d(ppm): 280 167.5, 166.1, 165.7, 165.1 (2) (CO, CNH), 133.5–128.2 (aromatics), 75.8, 74.6, 73.6, 70.7, 69.1 (C-1–C-5), 62.8, 62.1 (C-6, CH 2 ), 13.4 (CH 3 ). 4.4. Ethyl C-(2,3,4,6-tetra-O-benzoyl-bD -glucopyranosyl) thioformimidate (7) C-(2,3,4,6-Tetra-O-benzoyl-bD -glucopyranosyl)thioformamide (4, 0.2 g, 0.31 mmol) and Et 3 OBF 4 (0.18 g, 0.94 mmol) were dissolved in anhydrous CH 2 Cl 2 (4 mL), the mixture was stirred at rt under Ar and monitored by TLC (1:1 EtOAc–hexane). After completion of the reaction (2 days), the mixture was diluted with CH 2 Cl 2 290 (10 mL), extracted with satd aq NaHCO 3 solution (15 mL), then with water (15 mL). The organic phase was dried over MgSO 4 ,filtered and the solvent was evaporated. The crude pale yellow amorphous product (0.19 g, 90%) was used without further purification. R f : 0.62 (1:1 EtOAc–hexane); 1 H NMR (CDCl 3 )d(ppm): 8.06–7.24 (21H, m, aromatics, NH), 5.95, 5.74, 5.70 (3 1H, 3 pseudo t, J= 9.8, 9.1 Hz in each, H-2, H-3, H-4), 4.68 (1H, dd, J= 11.9, 2.8 Hz, H-6a), 4.51 (1H, dd, J= 11.9, 4.9 Hz, H-6b), 4.39 (1H, d, J= 9.1 Hz, H-1), 4.22 (1H, ddd, J= 9.1, 4.9, 2.8 Hz, H-5), 2.88–2.71 (2H, m, CH 2 ), 1.17 (3H, pseudo t, 7.7, 7.0 Hz, CH 3 ); 13 C NMR (CDCl 3 ) d(ppm): 172.7 (CNH), 166.1, 165.7, 165.1, 164.9 (CO), 133.4–128.2 (aromatics), 80.6, 76.1, 73.9, 70.9, 69.1 (C-1–C-5), 62.8 (C-6), 23.1 (CH 2 ), 13.1 (CH 3 ). 4.5. General procedure I for the synthesis of substituted 2-(2 0 ,3 0 ,4 0 ,6 0 -tetra-O-benzoyl-bD -glucopyranosyl)- benzimidazoles from O-perbenzoylated C-(bD -glucopyranosyl)formic acid (5) C-(2,3,4,6-Tetra-O-benzoyl-bD -glucopyranosyl)formic acid 44 (5, 0.1 g, 0.16 mmol), a 1,2-diaminobenzene (aor for h, 0.16 mmol, 1equiv) and triphenyl phosphite (50 l l, 0.19 mmol, 1.2 equiv) 310 were dissolved in anhydrous pyridine (2 mL). The closed vial was irradiated by microwaves for 20 min at 140 °C (maximum presTable 2 Kinetic data on the inhibition of rabbit muscle glycogen phosphorylase bby the new compounds 10b–i O OH HO HO OH N H N R 2 R 3 R 4 10 R 2 R 3 R 4 K i ( l M) aH H H 8.6 27 11 22 bHF H55 cH Cl H 9.7 dH Br H 7.5 eHNO 2 H 179 fHMeH12 gMe H H 2.8 hH Me Me 152 iH2.1 Q4 4Éva Bokor et al. /Carbohydrate Research xxx (2013) xxx–xxx CAR 6384 No. of Pages 9, Model 5G 5 February 2013 Please cite this article in press as: Bokor,É.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2013.01.011 sure: 20 bar, power 220 W). The completion of the reaction was monitored by TLC (1:1 EtOAc–hexane). The reaction mixture was concentrated under reduced pressure, traces of pyridine were removed by repeated co-evaporations with toluene. The crude product was purified by column chromatography (2:3 EtOAc– hexane). 4.6. General procedure II for the synthesis of substituted 2-(2 0 ,3 0 ,4 0 ,6 0 -tetra-O-benzoyl-bD -glucopyranosyl)- 320 benzimidazoles from O-perbenzoylated ethyl (C-bD -glycopyranosyl) formimidate (6) Ethyl C-(2,3,4,6-tetra-O-benzoyl-bD -glycopyranosyl)formimidate (6, 0.1 g, 0.15 mmol) and a 1,2-diaminobenzene (a–i, 0.30 mmol, 2 equiv) were dissolved in anhydrous CH 2 Cl 2 (3 mL). The mixture was refluxed and monitored by TLC (2:3 EtOAc–hexane). After completion of the reaction the solvent was evaporated, and the residue was purified by column chromatography. 4.7. General procedure III for the Zemplén-deacylation A benzoylated compound was dissolved in dry MeOH (5 mL/ 330 100 mg, a few drops of CHCl 3 were added in case of incomplete dissolution) and a catalytic amount of a NaOMe solution (1M in MeOH) was added. The mixture was kept at rt and monitored by TLC (7:3 CHCl 3 –MeOH). When the starting material was consumed the mixture was neutralised with a cation exchange resin Amberlyst 15 (H + form), then the resin was filtered off and the solvent removed. The residue was purified by column chromatography. 4.8. 2-(2 0 ,3 0 ,4 0 ,6 0 -Tetra-O-benzoyl-bD -glucopyranosyl)- benzimidazole (8a) A: From acid 5(0.1 g, 0.16 mmol) and o-phenylenediamine (a, 340 0.02 g, 0.16 mmol) according to General procedure I. Yield: 0.05 g (45%). B: From imidate 6(0.10 g, 0.15 mmol) and o-phenylenediamine (a, 0.04 g, 0.30 mmol) according to General Procedure II. Reaction time: 3 h. Purified by column chromatography (2:3 EtOAc–hexane) to yield 0.09 g (89%) of white solid. Mp: 122–124 °C (Lit. 22 mp: 120–123 °C). 1 H and 13 C NMR data correspond to the reported spectra. 22 4.9. 5(6)-Fluoro-2-(2 0 ,3 0 ,4 0 ,6 0 -tetra-O-benzoyl-bD - glucopyranosyl)-benzimidazole (8b) and 5(6)-fluoro-2-(3 0 ,4 0 ,6 0 - 350 tri-O-benzoyl-2 0 -deoxyD -arabino-hex-1-enopyranosyl)- benzimidazole (9b) From imidate 6(0.10 g, 0.15 mmol) and 1,2-diamino-4-fluorobenzene (b, 0.04 g, 0.30 mmol) according to General Procedure II. Reaction time: 5 h. Purified by column chromatography (1:2, then 2:3 EtOAc–hexane) to give 9b as the first than 8b as the second fraction. Compound 8b: Yield: 0.051 g (46%) pale yellow solid; R f : 0.31 (2:3 EtOAc–hexane); mp: 124–126 °C; [ a ] D =39 (c0.5, DMSO); 1 H NMR (CDCl 3 )d(ppm): 11.58 (1H, br s, benzimidazole NH), 360 7.96–6.76 (23H, m, aromatics), 6.30–6.21, 6.02 (3 1H, 3 pseudo t, J= 9.2, 9.2 Hz in each, H-2 0 , H-3 0 , H-4 0 ), 5.36 (1H, d, J= 9.2 Hz, H-1 0 ), 4.69 (1H, dd, J= 12.3, <1 Hz, H-6 0 a), 4.57 (1H, dd, J= 12.3, 5.3 Hz, H-6 0 b), 4.52 (1H, ddd, J= 9.2, 5.3, <1 Hz, H-5 0 ); 13 C NMR (DMSO-d 6 )d(ppm): 165.4, 165.1, 164.8, 164.3 (CO), 158.9 (d, 1 J C-F = 237 Hz, benzimidazole), 158.2 (d, 1 J C-F = 237 Hz, benzimidazole), 151.1, 150.3 (benzimidazole), 142.8 (d, 3 J C-F = 9 Hz, benzimidazole), 139.0 (br s, benzimidazole), 134.2 (d, 3 J C-F = 9 Hz, benzimidazole), 133.7–133.3 (aromatics), 130.9 (br s, benzimidazole), 129.3–128.5 (aromatics), 120.0 (d, 3 J C-F = 7 Hz, benzimid370 azole), 112.3 (d, 3 J C-F = 6 Hz, benzimidazole), 110.9 (d, 2 J C-F = 23 Hz, benzimidazole), 109.7 ( 2 J C-F = 23 Hz, benzimidazole), 104.5 (d, 2 J C-F = 24 Hz, benzimidazole), 98.0 (d, 2 J C-F = 27 Hz, benzimidazole), 79.1, 74.8, 74.2, 73.4, 71.5, 69.0 (C-1 0 –C-5 0 ), 62.8 (C-6 0 ). Anal. Calcd for C 41 H 31 FN 2 O 9 (714.69): C, 68.90; H, 4.37; N, 3.92. Found: C, 68.84; H, 4.29; N, 4.03. Compound 9b: Yield: 0.032 g (35%), pale yellow syrup; R f : 0.58 (2:3 EtOAc–hexane); [ a ] D =7(c0.5, CHCl 3 ); 1 H NMR (CDCl 3 )d (ppm): 10.41 (1H, br s, benzimidazole NH), 8.02–6.94 (18H, m, aromatics), 6.43 (1H, br s, H-2 0 ), 5.95–5.92 (2H, m, H-3 0 , H-4 0 ), 4.88 380 (1H, ddd, J= 4.0, <1 Hz, H-5 0 ), 4.79 (2H, s, H-6 0 a, H-6 0 b); 13 CNMR (CDCl 3 )d(ppm): 166.2, 165.6, 165.0 (CO), 159.8 (d, 1 J C-F = 239 Hz, benzimidazole C-5), 146.8 (benzimidazole C-2), 145.0 (C-1 0 ), 143.1, 139.8 (2 br s, benzimidazole C-3a, C-7a), 133.5–128.4 (aromatics), 120.2 (br s, benzimidazole C-4 or C-7), 111.9 (d, 2 J C-F = 25 Hz, benzimidazole C-6), 104.4 (br s, benzimidazole C-4 or C-7), 99.4 (C-2 0 ), 75.2, 67.9, 67.3 (C-3 0 –C-5 0 ), 61.7 (C-6 0 ). Analysis: C 34 H 25 FN 2 O 7 (592.57), ESI-MS (positive mode) m/z: 615.157 [M+Na] + , 1207.327 [2 M+Na] + . 4.10. 5(6)-Chloro-2-(2 0 ,3 0 ,4 0 ,6 0 -tetra-O-benzoyl-bD - 390 glucopyranosyl)-benzimidazole (8c) and 5(6)-chloro-2-(3 0 ,4 0 ,6 0 - tri-O-benzoyl-2 0 -deoxyD -arabino-hex-1-enopyranosyl)- benzimidazole (9c) From imidate 6(0.10 g, 0.15 mmol) and 1,2-diamino-4-chlorobenzene (c, 0.04 g, 0.30 mmol) according to General Procedure II. Reaction time: 5 h. Purified by column chromatography (2:3 EtOAc–hexane) to give 9c as the first and 8c as the second fraction. Compound 8c: Yield: 0.085 g (76%), pale yellow solid; R f : 0.29 (2:3 EtOAc–hexane); mp: 123–125 °C; [ a ] D =61 (c0.5, CHCl 3 ); 1 H NMR (CDCl 3 )d(ppm): 11.33 (1H, br s, benzimidazole NH), 400 7.94–6.95 (23H, m, aromatics), 6.21, 6.14, 6.00 (3 1H, 3 pseudo t, J= 9.4, 9.4 Hz in each, H-2 0 , H-3 0 , H-4 0 ), 5.31 (1H, d, J= 9.4 Hz, H-1 0 ), 4.72 (1H, dd, J= 12.3, <1 Hz, H-6 0 a), 4.60 (1H, dd, J= 12.3, 4.4 Hz, H-6 0 b), 4.50 (1H, ddd, J= 9.4, 4.4, <1 Hz, H-5 0 ); 13 CNMR (CDCl 3 )d(ppm): 166.5, 166.0, 165.1 (2) (CO), 149.8 (benzimidazole C-2), 142.0, 137.8 (2 br s, benzimidazole C-3a, C-7a), 133.3–127.8 (aromatics), 126.9, 123.3, 119.3 (br s), 112.4 (br s) (benzimidazole C-4–C-7), 77.0, 75.5, 74.1, 71.7, 69.5 (C-1 0 –C-5 0 ), 63.4 (C-6 0 ). Anal. Calcd for C 41 H 31 ClN 2 O 9 (731.15): C, 67.35; H, 4.27; N, 3.84. Found: C, 67.47; H, 4.13; N, 3.90. 410 Compound 9c: Yield: 0.022 g (24%), pale yellow syrup; R f : 0.59 (2:3 EtOAc–hexane); [ a ] D =5(c0.5, CHCl 3 ); 1 H NMR (CDCl 3 )d (ppm): 11.40 (1H, br s, benzimidazole NH), 8.00–7.07 (18H, m, aromatics), 6.45 (1H, d, J= 4.0 Hz, H-2 0 ), 5.98 (1H, pseudo t, J= 6.6, 5.9 Hz, H-3 0 or H-4 0 ), 5.94 (1H, pseudo t, J= 5.9, 4.0 Hz, H-3 0 or H-4 0 ), 4.81 (1H, ddd, J= 6.6, 4.6, 4.0 Hz, H-5 0 ), 4.71–4.70 (2H, m, H-6 0 a, H-6 0 b); 13 C NMR (CDCl 3 )d(ppm): 166.1, 165.6, 164.9 (CO), 146.8 (br s, benzimidazole C-2), 145.0 (C-1 0 ), 144.1, 141.8 (2 br s, benzimidazole C-3a, C-7a), 133.6–133.2 (aromatics), 132.0 (br s, benzimidazole C-5), 129.8–128.3 (aromatics), 123.9, 120.3, 119.1, 420 112.2, 111.3 (br s for each, benzimidazole C-4, C-6, C-7), 99.8 (C-2 0 ), 75.2, 68.1, 67.3 (C-3 0 –C-5 0 ), 61.7 (C-6 0 ). Analysis: C 34 H 25 ClN 2 O 7 (609.02), ESI-MS (positive mode) m/z:631.123 [M+Na] + , 1239.257 [2M+Na] + . 4.11. 5(6)-Bromo-2-(2 0 ,3 0 ,4 0 ,6 0 -tetra-O-benzoyl-bD - glucopyranosyl)-benzimidazole (8d) and 5(6)-bromo-2-(3 0 ,4 0 ,6 0 - tri-O-benzoyl-2 0 -deoxyD -arabino-hex-1-enopyranosyl)- benzimidazole (9d) From imidate 6(0.10 g, 0.15 mmol) and 1,2-diamino-4-bromobenzene (d, 0.06 g, 0.30 mmol) according to General Procedure II. 430 Reaction time: 3 h. Purified by column chromatography (2:3 EtOAc–hexane) to give 9d as the first and 8d as the second fraction. Q5 Éva Bokor et al. /Carbohydrate Research xxx (2013) xxx–xxx 5 CAR 6384 No. of Pages 9, Model 5G 5 February 2013 Please cite this article in press as: Bokor,É.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2013.01.011 Compound 8d: Yield: 0.064 g (54%) pale yellow solid; R f : 0.31 (2:3 EtOAc–hexane); mp: 126–128 °C; [ a ] D =62 (c0.5, CHCl 3 ); 1 H NMR (CDCl 3 )d(ppm): 8.02–6.76 (23H, m, aromatics), 6.42, 6.29, 6.14 (3 1H, 3 pseudo t, J= 9.6, 9.6 Hz in each, H-2 0 , H-3 0 , H-4 0 ), 5.39 (1H, d, J= 9.6 Hz, H-1 0 ), 4.72 (1H, dd, J= 12.3, 2.2 Hz, H-6 0 a), 4.63 (1H, dd, J= 12.3, 4.9 Hz, H-6 0 b), 4.56 (1H, ddd, J= 9.6, 4.9, 2.2 Hz, H-5 0 ); 13 C NMR (CDCl 3 )d(ppm): 166.4, 166.0, 165.0 (2) (CO), 149.6 (benzimidazole C-2), 140.9, 138.1 (2 br s, benzimid440 azole C-3a, C-7a), 133.3–127.7 (aromatics), 125.9, 119.0 (br s), 117.3 (br s), 115.9 (benzimidazole C-4, C-5, C-6, C-7), 77.0, 75.5, 74.1, 71.7, 69.4 (C-1 0 –C-5 0 ), 63.4 (C-6 0 ). Analysis: C 41 H 31 BrN 2 O 9 (775.60), ESI-MS (positive mode) m/z:799.112 [M+Na] + , 1573.242 [2M+Na] + . Compound 9d: Yield: 0.029 g (29%) pale yellow syrup; R f : 0.61 (2:3 EtOAc–hexane); [ a ] D =4(c0.25, CHCl 3 ); 1 H NMR (CDCl 3 )d (ppm): 10.06 (1H, br s, imidazole NH), 8.04–7.33 (18H, m, aromatics), 6.46 (1H, br s, H-2 0 ), 5.97–5.93 (2H, m, H-3 0 , H-4 0 ), 4.91 (1H, ddd, J= 4.0, <1 Hz, H-5 0 ), 4.81 (2H, s, H-6 0 a, H-6 0 b). Analysis: 450 C 34 H 25 BrN 2 O 7 (653.48), ESI-MS (positive mode) m/z:677.074 [M+Na] + , 1329.197 [2M+Na] + . 4.12. 5(6)-Nitro-2-(2 0 ,3 0 ,4 0 ,6 0 -tetra-O-benzoyl-bD - glucopyranosyl)-benzimidazole (8e) and 5(6)-nitro-2-(3 0 ,4 0 ,6 0 - tri-O-benzoyl-2 0 -deoxyD -arabino-hex-1-enopyranosyl)- benzimidazole (9e) From imidate 6(0.10 g, 0.15 mmol) and 1,2-diamino-4-nitrobenzene (e, 0.05 g, 0.30 mmol) according to General procedure II. in anhydrous ethanol. Reaction time: 1 day. Purified by column chromatography (1:2 EtOAc–hexane) to give 9e as the first and 460 8e as the second fraction. Compound 8e: Yield: 0.052 g (46%) yellow solid; R f : 0.25 (2:3 EtOAc–hexane); mp: 134–136 °C; [ a ] D =65 (c0.5, CHCl 3 ); 1 H NMR (CDCl 3 )d(ppm): 12.87 (1H, br s, benzimidazole NH), 8.45– 6.96 (23H, m, aromatics), 6.20, 6.12, 6.01 (3 1H, 3 pseudo t, J= 9.4, 9.4 Hz in each, H-2 0 , H-3 0 , H-4 0 ), 5.33 (1H, d, J= 9.4 Hz, H-1 0 ), 4.77 (1H, dd, J= 11.8, <1 Hz, H-6 0 a), 4.63 (1H, dd, J= 11.8, <1 Hz, H-6 0 b), 4.51 (1H, ddd, J=J= 9.4, <1 Hz, H-5 0 ); 13 C NMR (CDCl 3 )d(ppm): 166.3, 165.8, 165.1, 164.8 (CO), 152.7 (benzimidazole C-2), 143.3 (benzimidazole C-5), 141.6, 138.0 (2 br s, benz470 imidazole C-3a, C-7a), 133.5–127.6 (aromatics), 118.9, 116.1, 111.6 (br s for each, benzimidazole C-4, C-6, C-7), 77.1, 75.4, 74.0, 71.7, 69.0 (C-1 0 –C-5 0 ), 63.2 (C-6 0 ). Anal. Calcd for C 41 H 31 N 3 O 11 (741.70): C, 66.39; H, 4.21; N, 5.67. Found: C, 66.26; H, 4.13; N, 5.79. Compound 9e:Yield: 0.019 g (20%) yellow syrup; R f : 0.39 (2:3 EtOAc–hexane); [ a ] D =+1 (c0.5, CHCl 3 ); 1 H NMR (DMSO-d 6 )d (ppm): 13.51 (1H, br s, benzimidazole NH), 8.13–7.48 (18H, m, aromatics), 6.40 (1H, br s, H-2 0 ), 6.02 (1H, pseudo t, J= 5.3, 4.0 Hz, H-3 0 or H-4 0 ), 5.93 (1H, pseudo t, J= 6.6, 5.9 Hz, H-3 0 or H-4 0 ), 5.23 (1H, 480 ddd, J= 5.9, 5.3, 3.3 Hz, H-5 0 ), 4.89 (1H, dd, J= 12.6, 5.3 Hz, H-6 0 a), 4.78 (1H, dd, J= 12.6, 3.3 Hz, H-6 0 b); 13 C NMR (DMSO-d 6 )d (ppm): 165.3, 165.0, 164.5 (CO), 150.5 (br s), 149.7 (br s), 147.5 (br s), 145.0, 143.0, 142.3 (br s), 138.7 (br s) (benzimidazole, C-1 0 ), 133.8–128.7 (aromatics), 119.2, 118.5, 117.8, 115.2, 112.3, 108.4 (br s for each, benzimidazole), 100.5 (C-2 0 ), 74.6, 67.9, 67.0 (C-3 0 –C-5 0 ), 61.5 (C-6 0 ). Analysis: C 34 H 25 N 3 O 9 (619.58), ESI-MS (positive mode) m/z:642.148 [M+Na] + , 1261.309 [2M+Na] + . 4.13. 5(6)-Methyl-2-(2 0 ,3 0 ,4 0 ,6 0 -tetra-O-benzoyl-bD - glucopyranosyl)-benzimidazole (8f) 490 A: From acid 5(0.1 g, 0.16 mmol) and 3,4-diaminotoluene (f, 0.02 g, 0.16 mmol) according to General procedure I. Yield: 0.06 g (53%). B: From imidate 6(0.10 g, 0.15 mmol) and 3,4-diaminotoluene (f, 0.04 g, 0.30 mmol) according to General Procedure II. Reaction time: 2 h. Purified by column chromatography (2:3 EtOAc–hexane) to yield 0.09 g (83%) of pale yellow syrup. R f : 0.41 (1:1 EtOAc–hexane); [ a ] D =65 (c0.5, CHCl 3 ); 1 H NMR (CDCl 3 )d(ppm): 10.73 (1H, br s, benzimidazole NH), 7.92–6.90 (23H, m, aromatics), 6.19, 6.11, 5.96 (3 1H, 3 pseudo t, J= 9.2, 9.2 Hz in each, H-2 0 , H-3 0 , H-4 0 ), 500 5.32 (1H, d, J= 9.2 Hz, H-1 0 ), 4.68 (1H, dd, J= 12.2, 2.6 Hz, H-6 0 a), 4.54 (1H, dd, J= 12.2, 5.3 Hz, H-6 0 b), 4.44 (1H, ddd, J= 9.2, 5.3, 2.6 Hz, H-5 0 ), 2.32 (3H, s, CH 3 ); 13 C NMR (CDCl 3 )d(ppm): 166.4, 165.9, 165.2 (2) (CO), 148.1 (benzimidazole C-2), 142.4, 138.8 (2 br s, benzimidazole C-3a, C-7a), 133.4–128.0 (aromatics, benzimidazole C-5), 124.4, 118.9, 111.1 (br s for each, benzimidazole C-4, C-6, C-7), 76.8, (C-1 0 ), 75.3, 74.1, 71.5, 69.5 (C-1 0 –C-5 0 ), 63.4 (C-6 0 ), 21.5 (CH 3 ). Anal. Calcd for C 42 H 34 N 2 O 9 (710.73): C, 70.98; H, 4.82; N, 3.94. Found: C, 70.83; H, 4.96; N, 4.09. 4.14. 4(7)-Methyl-2-(2 0 ,3 0 ,4 0 ,6 0 -tetra-O-benzoyl-bD - 510 glucopyranosyl)-benzimidazole (8g) From imidate 6(0.10 g, 0.15 mmol) and 2,3-diaminotoluene (g, 0.04 g, 0.30 mmol) according to General Procedure II. Reaction time: 5 h. Purified by column chromatography (1:2 EtOAc–hexane) to yield 0.087 g (79%) of yellow solid. Mp: 117–119 °C; [ a ] D =43 (c0.5, CHCl 3 ); 1 H NMR (CDCl 3 )d(ppm): 10.88 (1H, br s, benzimidazole NH), 7.96–6.88 (23H, m, aromatics), 6.18, 6.06, 5.92 (3 1H, 3 pseudo t, J= 9.2, 9.2 Hz in each, H-2 0 , H-3 0 , H-4 0 ), 5.38 (1H, d, J= 9.2 Hz, H-1 0 ), 4.69 (1H, dd, J= 11.7, <1 Hz, H-6 0 a), 4.51 (1H, dd, J= 11.7, 5.3 Hz, H-6 0 b), 4.44 (1H, ddd, J= 9.2, 5.3, <1 Hz, H-5 0 ), 520 2.33 (3H, s, CH 3 ); 13 C NMR (CDCl 3 )d(ppm): 166.3, 165.8, 165.3, 165.2 (CO), 147.7 (benzimidazole C-2), 142.1 (br s, benzimidazole C-3a, C-7a), 133.4–128.0 (aromatics), 123.1, 122.4, 116.8, 108.8 (br s for each, benzimidazole C-4–C-7), 76.8, 75.2, 73.9, 71.4, 69.5 (C-1 0 –C-5 0 ), 63.3 (C-6 0 ), 16.5 (CH 3 ). Anal. Calcd for C 42 H 34 N 2 O 9 (710.73): C, 70.98; H, 4.82; N, 3.94. Found: C, 70.87; H, 4.90; N, 3.85. 4.15. 5,6-Dimethyl-2-(2 0 ,3 0 ,4 0 ,6 0 -tetra-O-benzoyl-bD - glucopyranosyl)-benzimidazole (8h) A: From acid 5(0.1 g, 0.16 mmol) and 1,2-diamino-4,5-dimeth530 ylbenzene (h, 0.02 g, 0.16 mmol) according to General procedure I. Yield: 0.05 g (43%). B: From imidate 6(0.10 g, 0.15 mmol) and 1,2-diamino-4,5dimethylbenzene (h, 0.04 g, 0.30 mmol) according to General procedure II. Reaction time: 3 h. Purified by column chromatography (2:3 EtOAc–hexane) to yield 0.09 g (81%) of pale yellow solid. Mp: 207–209 °C; [ a ] D =64 (c0.5, CHCl 3 ); 1 H NMR (CDCl 3 )d (ppm): 11.00 (1H, br s, benzimidazole NH), 7.98–6.92 (22H, m, aromatics), 6.27, 6.21, 6.05 (3 1H, 3 pseudo t, J= 9.2, 9.2 Hz in each, H-2 0 , H-3 0 , H-4 0 ), 5.34 (1H, d, J= 9.2 Hz, H-1 0 ), 4.67 (1H, dd, J= 11.9, 540 2.6 Hz, H-6 0 a), 4.54 (1H, dd, J= 11.9, 5.3 Hz, H-6 0 b), 4.48 (1H, ddd, J= 9.2, 5.3, 2.6 Hz, H-5 0 ), 2.17 (6H, s, CH 3 ); 13 C NMR (CDCl 3 )d (ppm): 166.3, 165.9, 165.1, 165.0 (CO), 147.5 (benzimidazole C-2), 141.1 (br s, benzimidazole C-3a, C-7a), 133.2–127.9 (aromatics, benzimidazole C-5, C-6), 119.5, 111.6 (2 br s, benzimidazole C4, C-7), 76.8, 75.6, 74.3, 71.7, 69.5 (C-1 0 –C-5 0 ), 63.3 (C-6 0 ), 20.2 (2 CH 3 ). Anal: Calcd for C 43 H 36 N 2 O 9 (724.75): C, 71.26; H, 5.01; N, 3.87. Found: C, 71.15; H, 4.92; N, 3.99. 4.16. 2-(2 0 ,3 0 ,4 0 ,6 0 -Tetra-O-benzoyl-bD -glucopyranosyl)- naphtho[2,3-d]imidazole (8i) 550 From imidate 6(0.10 g, 0.15 mmol) and 2,3-diamino-naphthalene (i, 0.05 g, 0.30 mmol) according to General procedure II. 6Éva Bokor et al. /Carbohydrate Research xxx (2013) xxx–xxx CAR 6384 No. of Pages 9, Model 5G 5 February 2013 Please cite this article in press as: Bokor,É.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2013.01.011 Reaction time: 4 h. Purified by column chromatography (4:5 EtOAc–hexane) to yield 0.091 g (79%) of pale brown solid. Mp: 184–186 °C; [ a ] D =113 (c0.25, CHCl 3 ); 1 H NMR (CDCl 3 )d (ppm): 10.64 (1H, br s, naphthoimidazole NH), 7.81–6.92 (26H, m, aromatics), 6.14, 6.05, 5.90 (3 1H, 3 pseudo t, J= 9.6, 9.6 Hz in each, H-2 0 , H-3 0 , H-4 0 ), 5.29 (1H, d, J= 9.6 Hz, H-1 0 ), 4.64 (1H, dd, J= 12.3, <1 Hz, H-6 0 a), 4.49 (1H, dd, J= 12.3, 5.3 Hz, H-6 0 b), 4.39 (1H, ddd, J= 9.6, 5.3, <1 Hz, H-5 0 ); 13 C NMR (CDCl 3 )d(ppm): 560 166.2, 165.9, 165.1 (2) (CO), 152.9 (naphthoimidazole C-2), 142.7 (br s, naphthoimidazole C-3a, C-9a), 133.2–127.8, 123.6 (aromatics), 116.2, 107.8 (2 br s, naphthoimidazole C-4, C-9), 76.9, 75.6, 74.2, 71.7, 69.5 (C-1 0 –C-5 0 ), 63.4 (C-6 0 ). Analysis: C 45 H 34 N 2 O 9 (746.76), ESI-MS (positive mode) m/z:769.219 [M+Na] + , 1516.457 [2M+Na] + . 4.17. 5(6)-Fluoro-2-(bD -glucopyranosyl)-benzimidazole (10b) From 8b (0.22 g, 0.30 mmol) according to General Procedure III. Reaction time: 3.5 h. Purified by column chromatography (4:1 CHCl 3 –MeOH) to yield 0.048 g (54%) of pale yellow syrup. R f : 570 0.19 (10:3 CHCl 3 –MeOH); [ a ] D =+9 (c0.2, DMSO); 1 H NMR (CD 3 OD) d(ppm): 7.48 (1H, s, benzimidazole H-4), 7.21 (1H, d, J= 8.7 Hz, benzimidazole H-7), 6.97 (1H, t, J= 8.7 Hz, benzimidazole H-6), 4.46 (1H, d, J= 9.4 Hz, H-1 0 ), 3.88 (1H, dd, J= 11.8, <1 Hz, H-6 0 a), 3.73 (1H, dd, J= 11.8, <1 Hz, H-6 0 b), 3.61 (1H, pseudo t, J= 9.8, 9.8 Hz, H-2 0 or H-3 0 or H-4 0 ), 3.52–3.47 (3H, m, H-2 0 and/or H-3 0 and/or H-4 0 , H-5 0 ); 13 C NMR (DMSO-d 6 )d(ppm): 158.3 (d, 1 J C-F = 236 Hz, benzimidazole C-5,), 153.9, 143.2, 138.9, 133.7, 130.6, 119.7, 112.0, 109.7, 104.0, 98.0 (br s for each, benzimidazole), 81.4, 77.8, 75.9, 72.7, 70.0 (C-1 0 –C-5 0 ), 61.3 (C-6 0 ). Anal. 580 Calcd for C 13 H 15 FN 2 O 5 (298.27): C, 52.35; H, 5.07; N, 9.39. Found: C, 52.26; H, 5.21; N, 9.33. 4.18. 5(6)-Chloro-2-(bD -glucopyranosyl)-benzimidazole (10c) From 8c (0.24 g, 0.33 mmol) according to general procedure III. Reaction time: 5 h. Purified by column chromatography (4:1 CHCl 3 –MeOH) to yield 0.092 g (89%) of colourless syrup. R f : 0.23 (4:1 CHCl 3 –MeOH); [ a ] D = +23 (c0.5, DMSO); 1 H NMR (CD 3 OD) d (ppm): 7.50 (1H, d, J= 1.4 Hz, benzimidazole H-4), 7.45 (1H, d, J= 8.9 Hz, benzimidazole H-7), 7.15 (1H, dd, J= 8.9, 1.4 Hz, benzimidazole H-6), 4.47 (1H, d, J= 8.9 Hz, H-1 0 ), 3.88 (1H, dd, J= 11.6, 590 <1 Hz, H-6 0 a), 3.73 (1H, dd, J= 11.6, 4.8 Hz, H-6 0 b), 3.61 (1H, pseudo t, J= 8.9, 8.9 Hz, H-2 0 or H-3 0 or H-4 0 ), 3.55–3.45 (3H, m, H-2 0 and/or H-3 0 and/or H-4 0 , H-5 0 ); 13 C NMR (CD 3 OD) d(ppm): 155.2 (benzimidazole C-2), 140.3, 138.1 (2 br s, benzimidazole C-3a, C-7a), 129.2 (benzimidazole C-5), 124.1, 117.1 (br s), 115.9(br s) (benzimidazole C-4, C-6, C-7), 82.2, 79.2, 77.3, 74.8, 71.2 (C-1 0 –C-5 0 ), 62.8 (C-6 0 ). Anal. Calcd for C 13 H 15 ClN 2 O 5 (314.72): C, 49.61; H, 4.80; N, 8.90. Found: C, 49.72; H, 4.87; N, 8.75. 4.19. 5(6)-Bromo-2-(bD -glucopyranosyl)-benzimidazole (10d) From 8d (0.27 g, 0.35 mmol) according to General procedure III. 600 Reaction time: 6 h. Purified by column chromatography (8:1 CHCl 3 –MeOH) to yield 0.073 g (58%) of colourless syrup. R f : 0.23 (10:3 CHCl 3 –MeOH); [ a ] D = +21 (c0.43, DMSO); 1 H NMR (CD 3 OD) d(ppm): 7.65 (1H, s, benzimidazole H-4), 7.41, 7.29 (2 1H, 2d, J= 8.4 Hz, benzimidazole H-6, H-7), 4.47 (1H, d, J= 9.4 Hz, H-1 0 ), 3.88 (1H, dd, J= 11.8, <1 Hz, H-6 0 a), 3.74 (1H, dd, J= 11.8, <1 Hz, H-6 0 b), 3.61 (1H, pseudo t, J= 9.4, 8.4 Hz, H-2 0 or H-3 0 or H-4 0 ), 3.53–3.46 (3H, m, H-2 0 and/or H-3 0 and/or H-4 0 , H-5 0 ); 13 C NMR (DMSO-d 6 )d(ppm): 153.8 (benzimidazole C-2), 141.5, 134.8 (2 br s, C-3a, C-7a), 124.5, 121.0, 113.9, 113.8 (br s for each, benzimid610 azole C-4–C-7), 81.5, 77.8, 75.9, 72.8, 70.0 (C-1 0 –C-5 0 ), 61.3 (C-6 0 ). Anal. Calcd for C 13 H 15 BrN 2 O 5 (359.17): C, 43.47; H, 4.21; N, 7.80. Found: C, 43.43; H, 4.34; N, 7.93. 4.20. 2-(bD -Glucopyranosyl)-5(6)-nitro-benzimidazole (10e) From 8e (0.30 g, 0.40 mmol) according to General procedure III. Reaction time: 1 day. Purified by column chromatography (85:15 CHCl 3 –MeOH) to yield 0.07 g (53%) of yellow syrup. R f : 0.27 (4:1 CHCl 3 –MeOH); 1 H NMR (DMSO-d 6 )d(ppm): 13.03 (1H, br s, benzimidazole NH), 8.44 (1H, d, J= 1.3 Hz, benzimidazole H-4), 8.11 (1H, dd, J= 9.2, 1.3 Hz, benzimidazole H-6), 7.70 (1H, d, J= 9.2 Hz, 620 benzimidazole H-7), 5.14 (3H, br s, 3 OH), 4.60 (1H, br s, OH), 4.42 (1H, d, J= 9.2 Hz, H-1 0 ), 3.74 (1H, dd, J= 11.9, <1 Hz, H-6 0 a), 3.63 (1H, pseudo t, J= 9.2, 9.2 Hz, H-2 0 or H-3 0 or H-4 0 ), 3.49 (1H, dd, J= 11.9, 5.3 Hz, H-6 0 b), 3.39–3.34 (2H, m, H-2 0 or H-3 0 or H-4 0 , H-5 0 ), 3.24 (1H, pseudo t, J= 9.2, 9.2 Hz, H-2 0 or H-3 0 or H-4 0 ); 13 C NMR (DMSO-d 6 )d(ppm): 157.3 (benzimidazole C-2), 142.5 (benzimidazole C-5), 141.4, 138.8 (2 br s, benzimidazole C-3a, C-7a), 117.7, 114.3 (br s), 112.6 (br s) (benzimidazole C-4, C-6, C-7), 81.6, 77.7, 76.0, 72.8, 70.0 (C-1 0 –C-5 0 ), 61.2 (C-6 0 ). Anal. Calcd for C 13 H 15 N 3 O 7 (325.27): C, 48.00; H, 4.65; N, 12.92. Found: C, 47.87; 630 H, 4.56; N, 12.96. 4.21. 2-(bD -Glucopyranosyl)-5(6)-methyl-benzimidazole (10f) From 8e (0.27 g, 0.38 mmol) according to General procedure III. Reaction time: 3 h. Purified by column chromatography (85:15 CHCl 3 –MeOH) to yield 0.10 g (87%) of colourless syrup. R f : 0.42 (7:3 CHCl 3 –MeOH); [ a ] D = +18 (c0.5, MeOH); 1 H NMR (CD 3 OD) d (ppm): 7.41 (1H, d, J= 7.9 Hz, benzimidazole H-6 or H-7), 7.32 (1H, s, benzimidazole H-4), 7.03 (1H, d, J= 7.9 Hz, benzimidazole H-6 or H-7), 4.49 (1H, d, J= 9.2 Hz, H-1 0 ), 3.91 (1H, dd, J= 11.9, <1 Hz, H-6 0 a), 3.76 (1H, dd, J= 11.9, 4.0 Hz, H-6 0 b), 3.66 (1H, pseudo 640 t, J= 9.2, 9.2 Hz, H-2 0 or H-3 0 or H-4 0 ), 3.61–3.49 (3H, m, H-2 0 and/or H-3 0 and/or H-4 0 , H-5 0 ), 2.42 (3H, s, CH 3 ); 13 C NMR (CD 3 OD) d (ppm): 153.4 (benzimidazole C-2), 139.0, 137.6 (benzimidazole C-3a, C-7a), 133.6 (benzimidazole C-5), 125.2, 115.9, 115.4 (benzimidazole C-4, C-6, C-7), 82.1, 79.3, 77.3, 74.8, 71.2 (C-1 0 –C-5 0 ), 62.7 (C-6 0 ), 21.7 (CH 3 ). Anal. Calcd for C 14 H 18 N 2 O 5 (294.30): C, 57.13; H, 6.16; N, 9.52. Found: C, 57.29; H, 6.07; N, 9.39. 4.22. 2-(bD -Glucopyranosyl)-4(7)-methyl-benzimidazole (10g) From 8g (0.25 g, 0.35 mmol) according to General Procedure III. Reaction time: 4 h. Purified by column chromatography (4:1 650 CHCl 3 –MeOH) to give 0.075 g (73%) of colourless syrup. R f : 0.20 (4:1 CHCl 3 –MeOH); [ a ] D = +13 (c0.5, DMSO); 1 H NMR (DMSO-d 6 ) d(ppm): 12.46 (1H, br s, benzimidazole NH), 7.32, 7.04, 6.95 (3 1H, benzimidazole), 5.14 (3H, br s, 3 OH), 4.64 (1H, br s, OH), 4.36 (1H, d, J= 10.2 Hz, H-1 0 ), 3.73–3.24 (6H, m, H-2 0 , H-3 0 , H-4 0 , H-5 0 , H-6 0 a, H-6 0 b), 2.50 (3H, s, CH 3 ); 13 C NMR (DMSO-d 6 )d (ppm): 151.9 (benzimidazole C-2), 141.8, 133.5, 128.4, 121.6, 115.6, 109.0 (br s for each, benzimidazole C-3a, C-7a, C-4–C-7), 81.3, 77.9, 75.9, 72.7, 70.0 (C-1 0 –C-5 0 ), 61.2 (C-6 0 ). Anal. Calcd for C 14 H 18 N 2 O 5 (294.30): C, 57.13; H, 6.16; N, 9.52. Found: C, 57.18; 660 H, 6.05; N, 9.64. 4.23. 5,6-Dimethyl-2-(bD -glucopyranosyl)-benzimidazole (10h) From 8h (0.27 g, 0.37 mmol) according to General procedure III. Reaction time: 3 h. Purified by column chromatography (85:15 CHCl 3 –MeOH) to yield 0.09 g (78%) of colourless syrup. R f : 0.42 (7:3 CHCl 3 –MeOH); [ a ] D = +24 (c0.5, MeOH); 1 H NMR (CD 3 OD) d (ppm): 7.28 (2H, s, benzimidazole H-4, H-7), 4.47 (1H, d, J= 9.2 Hz, H-1 0 ), 3.91 (1H, dd, J= 11.9, <1 Hz, H-6 0 a), 3.76 (1H, dd, Éva Bokor et al. /Carbohydrate Research xxx (2013) xxx–xxx 7 CAR 6384 No. of Pages 9, Model 5G 5 February 2013 Please cite this article in press as: Bokor,É.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2013.01.011