Synthesis of 2-(β-D-glucopyranosylamino)-5-substituted-1,3,4-oxadiazoles for inhibition 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 2-(bDD -glucopyranosylamino)-5-substituted-1,3,4-oxadiazoles for inhibition of glycogen phosphorylase Marietta Tóth * , Béla Sz} ocs, Tímea Kaszás, Tibor Docsa, Pál Gergely, László Somsák * O OAc AcO AcO OAc NCO O OAc AcO A cO OAc H N O H NNAr O OH HO HO OH H N O H NNAr Ar = Ph, 4-Me-Ph, 4-NO 2 -Ph, 4-F-Ph, 3-Cl-Ph, 1-Naphthyl, 2-Naphthyl O OH HO HO OH H N NN OAr Best inhibitor: Ar = 4-NO 2 -Ph (K i =4.5µM) Best inhibitor: Ar = 4-Me-Ph (K i =12µM) CAR 6468 No. of Pages 1, Model 5G 1 May 2013 Highlights Preparation of aromatic aldehyde 4-(bDD -glucopyranosyl)semicarbazones. Synthesis of 2-(bDD -glucopyranosylamino)-5-substituted-1,3,4oxadiazoles. Low micromolar inhibitors of glycogen phosphorylase. 1 Synthesis of 2-(b-D-glucopyranosylamino)-5-substituted-1,3,4-oxadiazoles for inhibition of glycogen phosphorylase Marietta Tóth a, ⇑ ,Béla Sz} ocs a ,Tímea Kaszás 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 13 March 2013 Received in revised form 18 April 2013 Accepted 18 April 2013 Available online xxxx 20 Keywords: Semicarbazone 1,3,4-Oxadiazole bD -Glucopyranosyl derivatives Glycogen phosphorylase Inhibitor abstract Aromatic aldehyde 4-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)semicarbazones were synthesized by the addition of different hydrazones onto O-peracetylated b-D-glucopyranosyl isocyanate. Oxidative transformations of these precursors gave O-protected 2-(b-D-glucopyranosylamino)-5-substituted1,3,4-oxadiazoles. Removal of the O-acetyl protecting groups under Zemplén conditions gave test compounds to show low micromolar inhibition against rabbit muscle glycogen phosphorylase b. Best inhibitors of these series were 4-(b-D-glucopyranosyl)semicarbazones of 4-fluorobenzaldehyde (K i = 4.5 l M), 2-naphthaldehyde (K i = 5.5 l M) and 2-(b-D-glucopyranosylamino)-5-(4-methylphenyl)- 1,3,4-oxadiazole (K i =12 l M). Ó2013 Published by Elsevier Ltd. 1. Introduction 40 Inhibition of glycogen phosphorylase (GP) can be a new therapeutic method for the treatment of type 2 diabetes mellitus, and potential application of GP inhibitors (GPIs) in some other diseased states, such as early cardiac and cardiovascular disorders in non-diabetics, cardiac arrhythmias, ischemic injuries, and tumor growth was also proposed. 1–5 A large array of compounds was shown to have inhibitory effect against GP under in vitro conditions. 4,6 Among these molecules glucose derivatives are the most intensively investigated GPIs, and detailed studies can be found in the literature on their structure–activity relationships (SAR). 5,7 50 Some glucose derived GPIs had in vivo hypoglycaemic 8 and other interesting physiological effects. 9 Widely studied glucose analogue GPIs are derivatives of bD -glucopyranosylamine, such as N-acyl-bD -glucopyranosylamines 10–13 (Iin Chart 1,e.g., K i =10–13 l M 11,14 against rabbit muscle GPb(RMGPb) 15 for R = 2-naphthyl), N-aryl-N 0 -bD -glucopyranosyl ureas 4 (II,e.g., K i = 5.2 l M (RMGPb) for R = 2-naphthyl), as well as N-acyl-N 0 -bD -glucopyranosyl urea derivatives 4,16 (III,e.g., K i = 0.35 l M (RMGPb) for R = 2-naphthyl) which inhibited the enzyme in or below the low micromolar range. Micromolar efficiency 60 was reported also for 4-(bD -glucopyranosyl)thiosemicarbazones of aromatic aldehydes 17,18 (IV X=S, e.g., IC 50 = 5.7 l M (RMGPb) for Ar = 4-fluorophenyl). Compounds V, in which the sugar and the aromatic parts are interchanged in comparison to IV, have very recently been shown to be GPIs (K i =29 l M for VX = O, and K i = 300 l M for VX = S against RMGPb). 19 Replacement of the NHCO moiety by 1,2,3-triazole (a nonclassical bioisosteric heterocyclic linker A) in molecules Iresulted in effective GP inhibitors 20 (IA,e.g., K i =16 l M (RMGPb) for R = 2-naphthyl). Enzymatic tests and crystallographic studies have 70 shown high similarity of the amide (I) and the 1,2,3-triazole (IA) type molecules both in binding strength and structural features of the enzyme–inhibitor complexes. 14 Replacement of the NHCO moiety with isomeric oxadiazoles B,C, and Dresulted in inhibitors IB–D with varying efficiency. Among these molecules the 3-aryl-5bD -glucopyranosyl-1,2,4-oxadiazoles IC proved to be the most effective compounds (e.g., K i = 2.4 l M(RMGPb) for R = 2-naphthyl). 21,22 Bioisosteric replacement studies were also carried out with compounds III. 13,19,23 As part of a program to systematically replace NHCO moieties of 80 GPIs I–III by heterocyclic bioisosteres, herein we report on the syntheses and enzymatic tests of a series of 2-(bD -glucopyranosylamino)-5-substituted-1,3,4-oxadiazoles IIB. For the construction of 1,3,4-oxadiazole rings from acyclic precursors acidic treatment of N,N 0 -diacyl- (or N-acyl-N 0 -thioacyl)- hydrazines as well as oxidative cyclization of acylhydrazones are the most frequently used procedures, and both methods were applied for the syntheses of 2-amino-5-substituted-1,3,4-oxadiazoles, too. 24 2-Glycosylamino-5-substituted-1,3,4-oxadiazoles are also known and have been synthesized from glycosyl isothiocya90 nates either by the addition of acid hydrazides followed by ring 0008-6215/$ - see front matter Ó2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.carres.2013.04.025 ⇑ Corresponding authors. Tel.: +36 52512900x22474; fax: +36 52512744 (M.T.); tel.: +36 52512900x22348; fax: +36 52512744 (L.S.). E-mail addresses: [email protected] (M. Tóth), somsak.laszlo@ science.unideb.hu (L. Somsák). Q1 Carbohydrate Research xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDirect Carbohydrate Research journal homepage: www.elsevier.com/locate/carres CAR 6468 No. of Pages 10, Model 5G 2 May 2013 Please cite this article in press as: Tóth, M.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2013.04.025 closure of the resulting 1-acyl-4-glycosyl-thiosemicarbazides 25–27 or by chlorination to give intermediate glycosyl isocyanide dichlorides to be cyclized with acid hydrazides. 28 In this work we investigated the synthesis of compounds IIB from aldehyde 4-glycosyl-semicarbazones (cf. O-protected IV X=O in Chart 1), a route which, to the best of our knowledge, has not yet been applied to obtain 2-glycosylamino-1,3,4-oxadiazoles. On the other hand, the preparation of semicarbazones IV (X = O) gave the opportunity to compare their GPI properties to 100 the corresponding thiosemicarbazones IV (X = S). 2. Results and discussion 2.1. Syntheses For the formation of the target oxadiazoles oxidative ring closure of aromatic aldehyde 4-(2,3,4,6-tetra-O-acetyl-bD -glucopyranosyl)semicarbazones (similar to that applied for the syntheses of 2-aryl-5-(2,3,4,6-tetra-O-benzoyl-bD -glucopyranosyl)-1,3,4-oxadiazoles 22 ) was envisaged as the key step. To this end, O-peracetylated 4-(bD -glucopyranosyl)semicarbazide 2was prepared (Scheme 1) by the reaction of glucopyranosyl isocyanate 110 1 29 (obtained from the corresponding glucopyranosylamine 30 ) with hydrazine reagents under different reaction conditions (reagents: NH 2 NH 2 HCl, NH 2 NH 2 HOAc; solvents: dry pyridine, dry CH 2 Cl 2 ,and 1 equiv Et 3 N). The best yield (75%) was achieved with NH 2 NH 2 HOAc in dry CH 2 Cl 2 in the presence of Et 3 N. In each of the above reactions bis-glucopyranosyl urea was also isolated in various amounts which could be due to the presence of traces of water in the mixtures. 31 In order to get semicarbazones 3–9 condensation of semicarbazide 2with the proper aldehydes was planned. However, 120 reactions of 2with 2-napthaldehyde in dry EtOH or dry toluene in the presence of catalytic amounts of either AcOH or CF 3 COOH at reflux temperature resulted in complex reaction mixtures. Therefore, bD -glucopyranosyl isocyanate 2was reacted with aldehyde hydrazones 32 in boiling dry dioxane to result in the target molecules 3–9 in good yields. Debenzoylations were performed by the Zemplén protocol to give excellent yields of semicarbazones 10–16. Ring closing reactions of the precursors under oxidative conditions were studied next. Semicarbazones 3,5–7, and 9were 130 reacted with phenyliodonium diacetate (PIDA) in CH 2 Cl 2 at rt (Scheme 2) to furnish the corresponding O-peracetylated 2-(bD - glucopyranosylamino)-5-substituted-1,3,4-oxadiazoles 17,19–21, and 23, respectively, in moderate yields. Application of Pb(OAc) 4 as the oxidizing agent in glacial AcOH at 80 °C resulted in better yields for 1,3,4-oxadiazoles 17–22 (compare yields under conditions aand bin Scheme 2). Debenzoylations were performed by the Zemplén protocol to give mostly good yields of 1,3,4-oxadiazoles 24–30 (Scheme 2). Structural elucidation of the new compounds was based on NMR 140 spectra. The bD -anomeric configuration was indicated by the 8.1– 9.9 Hz coupling constants between 1-H and 2-H protons in the 4 C 1 conformation of each compound. Coupling of 1-H and N(4)H (9.2–9.8 Hz) could be observed in the spectra of semicarbazones 3–9 only. For the oxadiazoles 17–23 1-H and NH appeared as doublets and singlets, respectively, probably because of a specific dihedral angle between these protons resulting in a very small coupling constant. A 1 H spectrum of semicarbazone 5recorded immediately (5 min) after dissolution showed the presence of one compound only. A NOE difference spectrum obtained by irradi150 ation of the N(2)H signal gave positive NOE for the signal of CH@N (7.86 ppm) and N(4)H (7.25 ppm) indicating Econfiguration of the C@Ndouble bond. After 72 h the 1 H spectrum of 5exhibited two sets of signals as a result of E/Z isomerisation (1:1 ratio, characteristic chemical shifts (d,ppm) for the Eisomer: 10.10 (N(2)H), 7.86 (CH@N), 7.25 (N(4)H); for the Zisomer: 8.62 (N(2)H), 7.41 (CH@N), O HO HO HO OH H NH N X IV N O HO HO HO OH H NR O linker I O HO HO HO OH H NH N O linker II R linkers N N NN O NN N OO N N ABC O HO HO HO OH H NH N O O R III D X = O, S O HO HO HO OH H CN H N V H N X H CAr Ph Chart 1. O OAc AcO AcO OAc NCO O OAc AcO A cO OAc H N O H NNH 2 O OAc AcO AcO O Ac H N O H NNAr 2 (75 %) b 1 O OH HO HO O H H N O H NNAr c Ph 4-Me-Ph 4-NO 2 -Ph 4-F-Ph 3-Cl-Ph 1-Naphthyl 2-Naphthyl Ar 3 (83%) 4 (72%) 5 (66%) 6 (76%) 7 (71%) 8 (89%) 9 (80%) 10 (99%) 11 (98%) 12 (93%) 13 (98%) 14 (94%) 15 (80%) 16 (98%) 61-019-3 a Scheme 1. Reagents and conditions: (a) NH 2 NH 2 HOAc, dry CH 2 Cl 2 ,Et 3 N, rt; (b) ArCH@N–NH 2 , dry dioxane, reflux; (c) NaOMe, dry MeOH, rt. 2M. Tóth et al. / Carbohydrate Research xxx (2013) xxx–xxx CAR 6468 No. of Pages 10, Model 5G 2 May 2013 Please cite this article in press as: Tóth, M.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2013.04.025 7.15 (N(4)H). These upfield shifts for the Zisomer are in accord with literature experiences for semicarbazones, 33 however, contrast those for O-peracetylated 4-(bD -glucopyranosyl)thiosemicarbazones where the N(2)Hsignals were reported to appear at 10.1 160 (E) and 14.6 (Z) ppm. 17 2.2. Enzyme inhibition studies The kinetic parameters (inhibition potency against rabbit muscle glycogen phosphorylase b(RMGPb)) of the deprotected compounds were determined according to the protocol described earlier. 34 The results are summarized in Table 1 showing the inhibitory efficiency of some relevant reference compounds, as well. Type IV semicarbazones 10–16, the ‘open chain’ precursors of the target compounds of this work, proved low micromolar inhibitors with the practically equipotent 4-nitrophenyl (12) and 170 2-naphthyl (16) compounds as the best ones. The strong binding of 16 fits the general trend observed with several glucose derivatives exhibiting most efficient inhibition with large hydrophobic groups in the aglycons. 4 A comparison to the structurally related thiosemicarbazone counterparts would be possible for the pairs 12–31,13–32, and 14–33, however, the reported inhibition data 18 for the sulfur containing compounds 31–33 are IC 50 values not directly comparable with the inhibitor constants. Nevertheless, it is interesting to note that the tendency is different in the two series: within semicarbazones the 4-nitrophenyl (12) compound 180 while within the thiosemicarbazones the 4-fluorophenyl (32) derivative proved 4to6times stronger inhibitors than the next best ones 14 and 33, respectively. A comparison of inhibition of 10 to that of the biuret type compound 37, 4 having the same number of atoms between the sugar and the aromatic ring, indicates the latter to be a twice better inhibitor than the former one. This may underline the importance of both carbonyl units in the binding. On the other hand, 10 binds 15-fold stronger than 38 4 with two C@Omoieties but also having a rotatable CH 2 unit in place of NH, thereby emphasizing that the 190 rigidity of 10 makes an important contribution to the binding. A further comparison of 10 to type Vcompound 39, 19 wherein the semicarbazone linker is formally reversed between the carbohydrate ring and the phenyl group, shows a moderate strengthening of the inhibition that may refer to the higher contribution of the second carbonyl unit to the binding. This is corroborated by the smaller decrease caused by the change 37?39 than that of 37?10. A formal reversal of the thiosemicarbazone linker as in 40 19 makes a very large decrease in the binding strength most probably because the thiocarbonyl in the position 200 of the ‘second amide’ moiety is much less suitable to make strong interactions to the enzyme. Replacement of the NHCO moiety in compounds type II by the 1,3,4-oxadiazole ring (compounds type IIB:24–30) resulted in moderate GPIs. For the phenyl and 1-naphthyl substituted pairs 34–24 and 35–29 there is no significant change in the binding strength. Substitution in the 4-position of the aromatic ring (25– 27) resulted in a small and unspecific increase of the efficiency, while the 3-chloro replacement (28) made a somewhat weaker inhibitor. Based on these results one could argue that the 1,3,4-oxa210 diazole is an acceptable bioisosteric replacement of the NHCO moiety in this system. However, this argument is weakened by the 5 times worse inhibition of 30 as compared to 36 for which a speculative explanation can be that the large and rigid aglycon is unable to find an accomodatable position in the binding site of the enzyme. 3. Conclusion Reaction of O-peracetylated bD -glucopyranosyl isocyanate with different hydrazones gave the corresponding aldehyde 4-(2, 3,4,6-tetra-O-acetyl-bD -glucopyranosyl)semicarbazones in good yields. Oxidation of these compounds by PIDA or more advanta220 geously by Pb(OAc) 4 resulted in 2-(bD -glucopyranosylamino)-5substituted-1,3,4-oxadiazoles. Zemplén deacetylation furnished test compounds which proved low micromolar inhibitors against RMGPb.Structure–activity considerations allow to estimate the relative contribution to the binding by carbonyl and thiocarbonyl groups of N-acyl-N 0 -glucopyranosyl urea type GPIs, and raise the possibility of applying 1,3,4-oxadiazoles as bioisosteric replacements of NHCO moieties. 4. Experimental 4.1. General methods 230 Melting points were measured in open capillary tubes or on a Kofler hot-stage and are uncorrected. Optical rotations were determined with a Perkin–Elmer 241 polarimeter at room temperature. NMR spectra were recorded with Bruker 360 (360/90 MHz for 1 H/ 13 C) or Bruker 400 (400/100 MHz for 1 H/ 13 C) spectrometers. Chemical shifts are referenced to TMS as the internal reference ( 1 H), or to the residual solvent signals ( 13 C). Microanalyses were performed on an Elementar vario Micro cube. ESIMS were recorded with a Bruker micrOTOF-Q instrument. TLC was performed on DCAlurolle Kieselgel 60 F 254 (Merck). TLC plates were visualized under 240 UV light, and by gentle heating. For column chromatography Kieselgel 60 (Merck, particle size (0.063–0.200 mm) was applied. Organic solutions were dried over anhydrous MgSO 4 , and concentrated under diminished pressure at 40–50 °C (water bath). Aldehyde hydrazones were obtained from the corresponding aldehydes and H 2 NNH 2 H 2 O according to a literature procedure. 32 4.2. General procedure I for the synthesis of aromatic aldehyde 4-(2,3,4,6-tetra-O-acetyl-bD -glucopyranosyl)semicarbazones (3–9) 2,3,4,6-Tetra-O-acetyl-bD -glucopyranosyl isocyanate 29 (1, 250 0.22 g, 0.59 mmol) was added to the solution of an aldehyde hydrazone (0.27 g, 1.80 mmol) in dry dioxane (5 mL). The reaction mixture was stirred at reflux temperature for 24 h. When the reaction O OR RO RO OR H N NN OAr O OAc AcO AcO O Ac H N O H NNAr a or b 3-9 c Ph 4-Me-Ph 4-NO 2 -Ph 4-F-Ph 3-Cl-Ph 1-Naphthyl 2-Naphthyl Ar 17 (45%) 18 - 19 (43%) 20 (24%) 21 (52%) 22 - 23 (49%) 24 (70%) 25 (33%) 26 (79%) 27 (83%) 28 (74%) 29 (84%) 30 (84%) 17-23 (R = Ac) ab (85%) (66%) (53%) (88%) (64%) (89%) - 24-30 (R = H) 3 4 5 6 7 8 9 Scheme 2. Reagents and conditions: (a) PIDA, CH 2 Cl 2, rt; (b) Pb(OAc) 4 , AcOH, 80 °C; (c) NaOMe, dry MeOH, rt. M. Tóth et al. / Carbohydrate Research xxx (2013) xxx–xxx 3 CAR 6468 No. of Pages 10, Model 5G 2 May 2013 Please cite this article in press as: Tóth, M.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2013.04.025 was complete (TLC 1:1 EtOAc/hexane) the reaction mixture was filtered with suction, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography. 4.3. General procedure II for the synthesis of 5-aryl-2-(2,3,4,6tetra-O-acetyl-bD -glucopyranosylamino)-1,3,4-oxadiazoles (17–23) 260 An aldehyde 4-(2,3,4,6-tetra-O-acetyl-bD -glucopyranosyl) semicarbazone (3–9, 0.03 mmol/mL) was dissolved in CH 2 Cl 2 , then PIDA (1.1 equiv) was added, and the mixture was stirred at rt. When the reaction was complete (TLC, 1:1 EtOAc/hexane) the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography. 4.4. General procedure III for the synthesis of 5-aryl-2-(2,3,4,6tetra-O-acetyl-bD -glucopyranosylamino)-1,3,4-oxadiazoles (17–23) An aldehyde 4-(2,3,4,6-tetra-O-acetyl-bD -glucopyrano270 syl)semicarbazone (3–9, 0.03 mmol/mL) was dissolved in glacial AcOH, then Pb(OAc) 4 (1 equiv) was added, and the mixture was stirred at 80 °C. The reaction was monitored by TLC (1:1 EtOAc/ hexane). When the reaction was complete, the reaction mixture was diluted with H 2 O (10 mL), and washed with EtOAc (3  6 mL). The organic layer was separated, dried, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography. 4.5. General procedure IV for the removal of O-acetyl protecting groups 280 An O-peracetylated compound (100 mg) was dissolved in dry MeOH (1 mL) and a solution of NaOMe (0.1 M in MeOH) was added to the solution in a catalytic amount. The reaction mixture was stirred at rt. When the reaction was complete (TLC, 3:1 CHCl 3 / MeOH) the solution was neutralized with a cation exchange resin Amberlyst 15 (H + form). The resin was filtered off with suction and the filtrate was evaporated under reduced pressure. The crude product was purified by column chromatography. 4.6. 4-(2,3,4,6-Tetra-O-acetyl-bD -glucopyranosyl)semicarbazide (2) 290 NH 2 NH 2 HOAc (97%, 26.6 mg, 0.28 mmol) was added to the solution of 2,3,4,6-tetra-O-acetyl-bD -glucopyranosyl isocyanate 29 (1, 0.10 g, 0.27 mmol) and Et 3 N (39 l L, 0.28 mmol) in dry CH 2 Cl 2 (2 mL). The reaction mixture was stirred at rt. When the reaction was complete (TLC 5:1 EtOAc/hexane) the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography (eluent: 5:1 EtOAc/hexane) to give 81 mg (75%) of 2as a white amorphous product. [ a ] D 4(c0.50, DMSO); R f = 0.27 (5:1 EtOAc/hexane); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 9.28, 6.82, Table 1 Inhibition of rabbit muscle glycogen phosphorylase b(RMGPb) by selected glucose derivatives and the new compounds (K i [ l M]) Ar O HO HO HO OH H NH N X N H CA r O HO HO HO OH H NH N O Ar O OH HO HO OH H N NN OA r X=O X=S 10 38 — 34 18 4 24 20 Me 11 136 — — 25 12 O 2 N 12 4.5 31 25.7 18 (IC 50 )— 26 15 F 13 48 32 5.7 18 (IC 50 )— 27 14 Cl 14 30 33 23.2 18 (IC 50 )— 28 33 15 124 — 35 350 4 (IC 50 )29 315 (IC 50 ) 16 5.5 — 36 5.2 4 30 27 O HO HO HO OH H NH N O X O O HO HO HO OH H CN H NH N X 37 X = NH 21 4 39 X=O 29 19 38 X=CH 2 600 4 40 X = S 300 19 4M. Tóth et al. / Carbohydrate Research xxx (2013) xxx–xxx CAR 6468 No. of Pages 10, Model 5G 2 May 2013 Please cite this article in press as: Tóth, M.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2013.04.025 6.53, 6.27 (4H, 4 s, NH), 5.32, 5.14, 5.07, 4.97 (4H, 4 pt, J= 8.9, 300 9.6 Hz in each, H-1, H-2, H-3, H-4), 4.31–4.12 (2H, m, H-6a, H6b), 3.85–3.83 (1H, m, H-5), 2.08, 2.07, 2.04 (12H, 3 s, CH 3 ). 13 C NMR (CDCl 3 , 360 MHz) d(ppm) 170.0, 169.4, 169.3, 169.2 (CO), 157.2 (NHCONH), 78.5, 72.8, 71.7, 70.4, 67.9 (C-1 to C-5), 61.7 (C-6), 20.5, 20.3 (CH 3 ). Anal. Calcd for C 15 H 23 N 3 O 10 (405.36): C, 44.44; H, 5.72; N, 10.37. Found: C, 44.58; H, 5.84; N, 10.26. 4.7. Benzaldehyde 4-(2,3,4,6-tetra-O-acetyl-bD -glucopyranosyl) semicarbazone (3) From isocyanate 1(0.10 g, 0.27 mmol) and benzaldehyde hydrazone (0.064 g, 0.54 mmol) according to General procedure I 310 (Section 4.2). Purified by column chromatography (1:1 EtOAc/ hexane) to yield 110 mg (83%) of 3as a white amorphous product. [ a ] D –61 (c0.99, CHCl 3 ); R f : 0.58 (5:1 EtOAc/hexane); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 9.47 (1H, br s, NH), 7.77 (1H, s, CH@N), 7.70–7.66 (2H, m, Ar), 7.43–7.40 (3H, m, Ar), 7.16 (1H, d, J 1,NH = 9.5 Hz, NH), 5.39, 5.28, 5.15, 5.13 (4H, 4 pt, J= 9.5, 9.7 Hz in each, H-1, H-2, H-3, H-4), 4.38 (1H, dd, J 6a,6b = 12.5 Hz, H-6a), 4.12 (1H, dd, H-6b), 3.92 (1H, ddd, J 5,6a = 4.1 Hz, J 5,6b = 1.8 Hz, J 4,5 = 10.1 Hz, H-5), 2.08, 2.05, 2.04, 2.03 (12H, 4 s, CH 3 ). 13 C NMR (CDCl 3 , 360 MHz) d(ppm) 170.7, 170.5, 170.0, 169.5 (CO), 155.7 320 (NHCONH), 142.6 (CH@N), 133.6, 130.0, 128.7, 127.1 (Ar), 79.3, 73.2, 72.8, 70.3, 68.2 (C-1 to C-5), 61.6 (C-6), 20.7, 20.6 (CH 3 ). Anal. Calcd for C 22 H 27 N 3 O 10 (493.46): C, 53.55; H, 5.51; N, 8.52. Found: C, 53.46; H, 5.61; N, 8.60. 4.8. 4-Methylbenzaldehyde 4-(2,3,4,6-tetra-O-acetyl-bD - glucopyranosyl)semicarbazone (4) From isocyanate 1(0.40 g, 1.07 mmol) and 4-methylbenzaldehyde hydrazone (0.29 g, 2.14 mmol) according to General procedure I (Section 4.2). Purified by column chromatography (1:1 EtOAc/hexane) to yield 390 mg (72%) of 4as a white amorphous 330 product. [ a ] D –50 (c0.86, CHCl 3 ); R f : 0.66 (5:1 EtOAc/hexane); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 9.21 (1H, br s, NH), 7.71 (1H, s, CH@N), 7.56 (2H, d, J= 8.0 Hz, Ar), 7.27–7.20 (2H, m, Ar), 7.13 (1H, d, J 1,NH = 9.5 Hz, NH), 5.37, 5.26, 5.14, 5.12 (4H, 4 pt, J= 9.5 Hz in each, H-1, H-2, H-3, H-4), 4.36 (1H, dd, J 6a,6b = 12.5 Hz, H-6a), 4.11 (1H, dd, H-6b), 3.89 (1H, ddd, J 5,6a = 4.1 Hz, J 5,6b = 1.9 Hz, J 4,5 = 10.1 Hz, H-5), 2.39, 2.08, 2.05, 2.04, 2.02 (16H, 5s,CH 3 ). 13 C NMR (CDCl 3 , 360 MHz) d(ppm) 170.7, 170.5, 170.0, 169.5 (CO), 155.7 (NHCONH), 142.6 (CH@N), 140.3, 130.8, 129.5, 127.0 (Ar), 79.4, 73.2, 72.9, 70.3, 68.2 (C-1 to C-5), 61.7 (C-6), 340 21.5, 20.7 (CH 3 ). Anal. Calcd for C 23 H 29 N 3 O 10 (507.49): C, 54.43; H, 5.76; N, 8.28. Found: C, 54.32; H, 5.64; N, 8.38. 4.9. 4-Nitrobenzaldehyde 4-(2,3,4,6-tetra-O-acetyl-bD - glucopyranosyl)semicarbazone (5) From isocyanate 1(0.40 g, 1.07 mmol) and 4-nitrobenzaldehyde hydrazone (0.37 g, 2.14 mmol) according to General procedure I (Section 4.2). Purified by column chromatography (1:1 EtOAc/hexane) to yield 380 mg (66%) of 5as an orange amorphous product. [ a ] D 71 (c0.35, CHCl 3 ); R f : 0.18 (1:1 EtOAc/hexane); 1 H NMR (CDCl 3 , 400 MHz) d(ppm) 10.10 (1H, br s, NH), 8.29 (2H, d, 350 J= 8.8 Hz, Ar), 7.87 (2H, d, J= 8.8 Hz, Ar), 7.86 (1H, s, CH@N), 7.25 (1H, d, J 1,NH = 9.2 Hz, NH), 5.41, 5.24, 5.14, 5.12 (4H, 4 pt, J= 9.3, 9.8 Hz in each, H-1, H-2, H-3, H-4), 4.38 (1H, dd, J 6a,6b = 12.5 Hz, H-6a), 4.13 (1H, dd, H-6b), 3.91 (1H, J 5,6a = 4.1 Hz, J 5,6b = 2.1 Hz, J 4,5 = 9.9 Hz, m, H-5), 2.09, 2.06, 2.04 (12H, 3 s, CH 3 ). 13 C NMR (CDCl 3 , 360 MHz) d(ppm) 170.7, 170.5, 169.9, 169.4 (CO), 155.7 (NHCONH), 148.2 (C–NO 2 ), 139.8 (CH@N), 139.6, 127.5, 124.0 (Ar), 79.3, 73.2, 72.5, 70.4, 68.1 (C-1 to C-5), 61.6 (C-6), 20.5 (CH 3 ). Anal. Calcd for C 22 H 26 N 4 O 12 (538.46): C, 49.07; H, 4.87; N, 10.40. Found: C, 48.98; H, 4.80; N, 10.52. 360 4.10. 4-Fluorobenzaldehyde 4-(2,3,4,6-tetra-O-acetyl-bD - glucopyranosyl)semicarbazone (6) From isocyanate 1(0.25 g, 0.67 mmol) and 4-fluorobenzaldehyde hydrazone (0.19 g, 1.23 mmol) according to General procedure I (Section 4.2). Purified by column chromatography (1:1 EtOAc/hexane) to yield 260 mg (76%) of 6as a white amorphous product. [ a ] D –54 (c0.27, CHCl 3 ); R f : 0.22 (1:1 EtOAc/hexane); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 10.09 (1H, br s, NH) 7.78 (1H, s, CH@N), 7.68 (2H, dd, J= 5.6 Hz, J= 7.9 Hz, Ar), 7.21–7.07 (3H, m, Ar, NH), 5.41, 5.29, 5.15, 5.12 (4H, 4 pt, J= 9.3, 9.9 Hz in each, H370 1, H-2, H-3, H-4), 4.38 (1H, dd, J 5,6a = 3.7 Hz, J 6a,6b = 12.5 Hz, H6a), 4.12 (1H, dd, J 5,6b < 1 Hz, H-6b), 3.97–3.89 (1H, m, H-5), 2.05 (12H, s, CH 3 ). 13 C NMR (CDCl 3 , 360 MHz) d(ppm) 170.6, 170.5, 169.9, 169.4 (CO), 163.7 (d, J= 250.0 Hz, Ar), 155.8 (NHCONH), 141.4 (CH@N), 129.9 (Ar), 128.9 (d, J= 7.3 Hz, Ar), 115.8 (d, J= 22.0 Hz, Ar), 79.3, 73.1, 72.7, 70.3, 68.1 (C-1 to C-5), 61.6 (C6), 20.5 (CH 3 ). Anal. Calcd for C 22 H 26 FN 3 O 10 (511.45): C, 51.66; H, 5.12; N, 8.22. Found: C, 51.54; H, 5.23; N, 8.33. 4.11. 3-Chlorobenzaldehyde 4-(2,3,4,6-tetra-O-acetyl-bD - glucopyranosyl)semicarbazone (7) 380 From isocyanate 1(0.22 g, 0.59 mmol) and 3-chlorobenzaldehyde hydrazone (0.18 g, 1.18 mmol) according to General procedure I (Section 4.2). Purified by column chromatography (1:1 EtOAc/hexane) to yield 222 mg (71%) of 7as a white amorphous product. [ a ] D –65 (c1.06, CHCl 3 ); R f : 0.61 (5:1 EtOAc/hexane); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 9.60 (1H, br s, NH), 7.64–7.46 (3H, m, Ar, CH@N), 7.31–7.27 (2H, m, Ar), 7.12 (1H, d, J 1,NH = 9.2 Hz, NH), 5.31, 5.16, 5.07, 5.05 (4H, 4 pt, J= 9.2, 9.7 Hz in each, H-1, H-2, H-3, H-4), 4.29 (1H, dd, J 5,6a = 3.9 Hz, J 6a,6b = 12.4 Hz, H-6a), 4.06 (1H, dd, J 5,6b = 1.0 Hz, H-6b), 3.87–3.80 (1H, m, H-5), 2.02, 1.97 390 (12H, 2 s, CH 3 ). 13 C NMR (CDCl 3 , 360 MHz) d(ppm) 170.7, 170.5, 170.0, 169.5 (CO), 155.7 (NHCONH), 141.0 (CH@N), 135.4, 134.9, 130.0, 126.9, 125.3 (Ar), 79.5, 73.3, 72.7, 70.4, 68.3 (C-1 to C-5), 61.7 (C-6), 20.7, 20.6 (CH 3 ). Anal. Calcd for C 22 H 26 ClN 3 O 10 (527.91): C, 50.05; H, 4.96; N, 7.96. Found: C, 50.16; H, 4.84; N, 7.85. 4.12. 4-(1-Naphthaldehyde) 4-(2,3,4,6-tetra-O-acetyl-bD - glucopyranosyl)semicarbazone (8) From isocyanate 1(0.40 g, 1.07 mmol) and 1-naphtaldehyde hydrazone (0.37 g, 2.14 mmol) according to General procedure I 400 (Section 4.2). Purified by column chromatography (1:1 EtOAc/hexane) to yield 520 mg (89%) of 8as a yellow amorphous product. [ a ] D 53 (c0.96, CHCl 3 ); R f : 0.63 (5:1 EtOAc/hexane); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 9.63 (1H, br s, NH), 8.52 (1H, d, J= 8.5 Hz, Ar), 8.44 (1H, s, CH@N), 7.93–7.90 (3H, m, Ar), 7.68 (1H, pt, J= 8.1, 8.3 Hz, Ar), 7.58–7.51 (2H, m, Ar), 7.17 (1H, d, J 1,NH = 9.6 Hz, NH), 5.42, 5.34, 5.17, 5.16 (4H, 4 pt, J= 9.5, 9.7 Hz in each, H-1, H-2, H3, H-4), 4.37 (1H, dd, J 6a,6b = 12.5 Hz, H-6a), 4.11 (1H, dd, H-6b), 3.94 (1H, ddd, J 5,6a = 3.9 Hz, J 5,6b = 2.0 Hz, J 4,5 = 10.2 Hz, H-5), 2.07, 2.06, 2.05 (12H, 3 s, CH 3 ). 13 C NMR (CDCl 3 , 360 MHz) d(ppm) 410 170.7, 170.6, 170.0, 169.5 (CO), 155.7 (NHCONH), 142.1 (CH@N), 133.8, 130.5, 130.7, 128.9, 128.8, 127.3, 126.2, 125.3, 123.7 (Ar), 79.4, 73.3, 72.9, 70.3, 68.2 (C-1 to C-5), 61.6 (C-6), 20.7, 20.6 (CH 3 ). Anal. Calcd for C 26 H 29 N 3 O 10 (543.52): C, 57.45; H, 5.38; N, 7.73. Found: C, 57.56; H, 5.27; N, 7.85. M. Tóth et al. / Carbohydrate Research xxx (2013) xxx–xxx 5 CAR 6468 No. of Pages 10, Model 5G 2 May 2013 Please cite this article in press as: Tóth, M.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2013.04.025 4.13. 4-(2-Naphthaldehyde) 4-(2,3,4,6-tetra-O-acetyl-bD -glucopyranosyl)semicarbazone (9) From isocyanate 1(0.27 g, 0.71 mmol) and 2-naphtaldehyde hydrazone (0.24 g, 1.42 mmol) according to General procedure I (Section 4.2). Purified by column chromatography (1:1 EtOAc/hex420 ane) to yield 310 mg (80%) of 9as a yellow amorphous product. [ a ] D 73 (c0.93, CHCl 3 ); R f : 0.64 (5:1 EtOAc/hexane); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 9.83 (1H, s, NH), 7.99–7.83 (6H, m, Ar, CH@N), 7.53–7.50 (2H, m, Ar), 7.26 (1H, d, J 1,NH = 9.8 Hz, NH), 5.42, 5.32, 5.17, (4H, 3 pt, J= 9.3, 9.6 Hz in each, H-1, H-2, H-3, H-4), 4.39 (1H, dd, J 5,6a = 3.8 Hz, J 6a,6b = 12.5 Hz, H-6a), 4.14 (1H, dd, J 5,6b < 1.0 Hz, H-6b), 3.97–3.94 (1H, m, H-5), 2.06 (12H, s, CH 3 ). 13 C NMR (CDCl 3 , 360 MHz) d(ppm) 170.7, 170.6, 170.0, 169.5 (CO), 155.7 (NHCONH), 142.1 (CH@N), 134.2, 133.1, 131.4, 128.7, 128.3, 127.9, 127.0, 126.6, 122.7 (Ar), 79.5, 73.3, 72.9, 70.4, 68.3 430 (C-1 to C-5), 61.7 (C-6), 20.6 (CH 3 ). Anal. Calcd for C 26 H 29 N 3 O 10 (543.52): C, 57.45; H, 5.38; N, 7.73. Found: C, 57.57; H, 5.49; N, 7.63. 4.14. Benzaldehyde 4-(bD -glucopyranosyl)semicarbazone (10) From 3(0.15 g, 0.30 mmol) according to General procedure IV (Section 4.5). Purified by column chromatography (4:1 CHCl 3 / MeOH) to yield 98 mg (99%) of 10 as a white amorphous product. [ a ] D +33 (c0.54, CH 3 OH); R f : 0.34 (4:1 CHCl 3 /MeOH); 1 H NMR (D 2 O, 360 MHz) d(ppm) 7.82 (1H, s, CH@N), 7.72–7.68 (5H, m, Ar), 4.97 (1H, d, J 1,2 = 8.3 Hz, H-1), 3.90 (1H, dd, J 5,6b = 1.5 Hz, J 6a,6b = 11.7 Hz, H-6b), 3.75 (1H, dd, J 5,6a = 5.2 Hz, H-6a), 3.62–3.48 440 (4H, m, H-2, H-3, H-4, H-5). 13 C NMR (D 2 O+1 drop CH 3 OH, 360 MHz) d(ppm) 158.4 (NHCONH), 144.1 (CH@N), 135.7, 130.9, 129.7, 128.1 (Ar), 82.4, 79.4, 78.8, 73.9, 71.4 (C-1 to C-5), 62.7 (C6). Anal. Calcd for C 14 H 19 N 3 O 6 (325.32): C, 51.69; H, 5.89; N, 12.92. Found: C, 51.57; H, 5.99; N, 12.81. 4.15. 4-Methylbenzaldehyde 4-(bD - glucopyranosyl)semicarbazone (11) From 4(0.09 g, 0.18 mmol) according to General procedure IV (Section 4.5). Purified by column chromatography (4:1 CHCl 3 / MeOH) to yield 60 mg (98%) of 11 as a white amorphous product. 450 [ a ] D +30 (c0.48, CH 3 OH); R f : 0.48 (3:1 CHCl 3 /MeOH); 1 H NMR (CD 3 OD, 360 MHz) d(ppm) 7.85 (1H, s, CH@N), 7.59 (2H, d, J= 8.5 Hz, Ar), 7.20 (2H, d, J= 8.5 Hz, Ar), 4.97 (1H, d, J 1,2 = 8.4 Hz, H-1), 3.89 (1H, dd, J 5,6b < 1.0 Hz, J 6a,6b = 11.7 Hz, H-6b), 3.73 (1H, dd, J 5,6a = 3.8 Hz, H-6a), 3.48–3.35 (4H, m, H-2, H-3, H-4, H-5), 2.35 (3H, s, CH 3 ). 13 C NMR (CD 3 OD, 360 MHz) d(ppm) 158.4 (NHCONH), 144.1 (CH@N), 141.3, 133.0, 130.4, 128.1 (Ar), 82.4, 79.5, 78.9, 74.0, 71.4 (C-1 to C-5), 62.7 (C-6), 21.5 (CH 3 ). Anal. Calcd for C 15 H 21 N 3 O 6 (339.34): C, 53.09; H, 6.24; N, 12.38. Found: C, 52.98; H, 6.35; N, 12.26. 460 4.16. 4-Nitrobenzaldehyde 4-(bD -glucopyranosyl) semicarbazone (12) From 5(0.15 g, 0.28 mmol) according to General procedure IV (Section 4.5). Purified by column chromatography (1:1 CHCl 3 / MeOH) to yield 107 mg (93%) of 12 as an orange amorphous product. [ a ] D +30 (c0.29, DMSO); R f : 0.70 (2:3 CHCl 3 /MeOH); 1 H NMR (CD 3 OD, 400 MHz) d(ppm) 8.28 (2H, d, J= 8.7 Hz, Ar), 8.00 (2H, d, J= 9.0 Hz, Ar), 7.98 (1H, s, CH@N), 4.94 (1H, d, J 1,2 = 8.5 Hz, H1), 3.84 (1H, dd, J 5,6b = 1.0 Hz, J 6a,6b = 12.3 Hz, H-6b), 3.70 (1H, dd, J 5,6a = 4.5 Hz, J 6a,6b = 11.9 Hz, H-6a), 3.51–3.43 (2H, m, H-2 or H-3 470 or H-4, H-5), 3.40–3.36 (2H, m, H-2 and/or H-3, and/or H-4). 13 C NMR (DMSO-d 6 ,360 MHz) d(ppm) 155.0 (NHCONH), 147.3 (C– NO 2 ), 138.0 (CH@N), 141.0, 127.8, 129.8 (Ar), 80.9, 78.4, 77.4, 71.9, 69.9 (C-1 to C-5), 61.0 (C-6). Anal. Calcd for C 14 H 18 N 4 O 8 (370.31): C, 45.41; H, 4.90; N, 15.13. Found: C, 45.54; H, 4.79; N, 15.24. 4.17. 4-Fluorobenzaldehyde 4-(bD -glucopyranosyl) semicarbazone (13) From 6(0.12 g, 0.24 mmol) according to General procedure IV (Section 4.5). Purified by column chromatography (4:1 CHCl 3 / 480 MeOH) to yield 80 mg (98%) of 13 as a brown amorphous product. [ a ] D +42 (c0.28, DMSO); R f : 0.20 (4:1 CHCl 3 /MeOH); 1 HNMR (CD 3 OD, 400 MHz) d(ppm) 7.89 (1H, s, CH@N), 7.78 (2H, dd, J= 5.6 Hz, J= 8.5 Hz, Ar), 7.15 (2H, pt, J= 8.7 Hz, Ar), 4.95 (1H, d, J 1,2 = 8.4 Hz, H-1), 3.86 (1H, dd, J 5,6b < 1.0 Hz, J 6a,6b = 11.7 Hz, H6b), 3.70 (1H, dd, J 5,6a = 4.0 Hz, H-6a), 3.48–3.29 (4H, m, H-2, H-3, H-4, H-5). 13 C NMR (DMSO-d 6 ,360 MHz) d(ppm) 162.8 (d, J= 246.4 Hz, Ar), 155.4 (NHCOHN), 138.0 (CH@N), 131.2, 129.2 (d, J= 6.4 Hz, Ar), 115.8 (d, J= 21.6 Hz, Ar), 80.9, 78.4, 77.5, 72.1, 70.1 (C-1 to C-5), 61.1 (C-6). Anal. Calcd for C 14 H 18 FN 3 O 6 (343.31): C, 490 48.98; H, 5.28; N, 12.24. Found: C, 49.10; H, 5.41; N, 12.13. 4.18. 3-Chlorobenzaldehyde 4-(bD -glucopyranosyl) semicarbazone (14) From 7(0.15 g, 0.28 mmol) according to General procedure IV (Section 4.5). Purified by column chromatography (4:1 CHCl 3 / MeOH) to yield 96 mg (94%) of 14 as a yellow amorphous product. [ a ] D +30 (c0.54, CH 3 OH); R f : 0.40 (4:1 CHCl 3 /MeOH); 1 HNMR (CD 3 OD, 360 MHz) d(ppm) 7.83–7.32 (5H, m, Ar, CH@N), 4.97 (1H, d, J 1,2 = 8.3 Hz, H-1), 3.85 (1H, dd, J 5,6b < 1.0 Hz, J 6a,6b = 11.8 Hz, H-6b), 3.71–3.67 (1H, m, H-6a), 3.52–3.39 (4H, m, H-2, H-3, H-4, H500 5). 13 C NMR (CD 3 OD, 360 MHz) d(ppm) 158.2 (NHCONH), 142.4 (CH@N), 137.8, 135.8, 131.2, 130.6, 127.3, 126.9 (Ar), 82.4, 79.4, 78.8, 73.8, 71.3 (C-1 to C-5), 62.6 (C-6). Anal. Calcd for C 14 H 18 ClN 3 O 6 (359.76): C, 46.74; H, 5.04; N, 11.68. Found: C, 46.62; H, 5.13; N, 11.80. 4.19. 1-Naphthaldehyde 4-(bD -glucopyranosyl)semicarbazone (15) From 8(0.17 g, 0.18 mmol) according to General procedure IV (Section 4.5). Purified by column chromatography (4:1 CHCl 3 / MeOH) to yield 93 mg (80%) of 15 as a brown amorphous product. 510 [ a ] D +32 (c0.48, CH 3 OH); R f : 0.41 (4:1 CHCl 3 /MeOH); 1 HNMR (CD 3 OD, 360 MHz) d(ppm) 8.66 (1H, s, CH@N), 8.41–7.48 (7H, m, Ar), 4.98 (1H, d, J 1,2 = 8.7 Hz, H-1), 3.87 (1H, dd, J 5,6b = 1.5 Hz, J 6a,6b = 12.0 Hz, H-6b), 3.71 (1H, dd, J 5,6a = 4.6 Hz, H-6a), 3.52–3.38 (4H, m, H-2, H-3, H-4, H-5). 13 C NMR (CD 3 OD, 360 MHz) d(ppm) 158.3 (NHCONH), 142.3 (CH@N), 135.3–124.1 (Ar), 82.4, 79.5, 79.0, 74.1, 71.4 (C-1 to C-5), 62.7 (C-6). Anal. Calcd for C 18 H 21 N 3 O 6 (375.38): C, 57.59; H, 5.64; N, 11.19. Found: C, 57.70; H, 5.52; N, 11.29. 4.20. 2-Naphthaldehyde 4-(bD -glucopyranosyl)semicarbazone 520 (16) From 9(0.14 g, 0.26 mmol) according to General procedure IV (Section 4.5). Purified by column chromatography (4:1 CHCl 3 / MeOH) to yield 96 mg (98%) of 16 as a white amorphous product. [ a ] D +39 (c0.47, CH 3 OH); R f : 0.43 (4:1 CHCl 3 /MeOH); 1 HNMR (CD 3 OD, 360 MHz) d(ppm) 8.08–7.46 (8H, m, Ar, CH@N), 4.97 (1H, d, J 1,2 = 8.6 Hz, H-1), 3.87 (1H, dd, J 5,6b < 1.0 Hz, J 6a,6b = 12.0 Hz, H-6b), 3.71 (1H, dd, J 5,6a = 4.3 Hz, H-6a), 3.51–3.38 (4H, m, H-2, H3, H-4, H-5). 13 C NMR (CD 3 OD, 360 MHz) d(ppm) 157.9 (NHCONH), 143.9 (CH@N), 135.6–123.9 (Ar), 82.4, 79.5, 78.9, 74.0, 71.4 (C-1 to 530 C-5), 62.7 (C-6). Anal. Calcd for C 18 H 21 N 3 O 6 (375.38): C, 57.59; H, 5.64; N, 11.19. Found: C, 57.46; H, 5.75; N, 11.08. 6M. Tóth et al. / Carbohydrate Research xxx (2013) xxx–xxx CAR 6468 No. of Pages 10, Model 5G 2 May 2013 Please cite this article in press as: Tóth, M.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2013.04.025 4.21. 5-Phenyl-2-(2,3,4,6-tetra-O-acetyl-bD - glucopyranosylamino)-1,3,4-oxadiazole (17) (a) From 3(0.11 g, 0.23 mmol) according to General procedure II (Section 4.3). Purified by column chromatography (1:1 EtOAc/hexane) to yield 50 mg (45%) of 17 as a white amorphous product. (b) From 3(0.25 g, 0.51 mmol) according to General procedure III (Section 4.4). Purified by column chromatography (1:1 EtOAc/ hexane) to yield 212 mg (85%) of 17 as a white amorphous product. 540 [ a ] D –20 (c0.28, CHCl 3 ); R f : 0.23 (1:1 EtOAc/hexane);1H NMR (CDCl 3 , 360 MHz) d(ppm) 7.96–7.83 (2H, m, Ar), 7.54–7.42 (3H, m, Ar), 6.22 (1H, br s, NH), 5.39 (1H, pt, J= 9.4 Hz, H-2 or H-3 or H-4), 5.21 (1H, d, J 1,2 = 9.0 Hz, H-1), 5.14, 5.11 (2H, 2 pt, J= 9.4, 9.9 Hz in each, H-2 and/or H-3, and/or H-4), 4.33 (1H, dd, J 5,6a = 4.5 Hz, J 6a,6b = 12.5 Hz, H-6a), 4.11 (1H, dd, J 5,6b = 1.2 Hz, H6b), 3.98–3.88 (1H, m, H-5), 2.09, 2.06, 2.05 (12H, 3 s, CH 3 ). 13 C NMR (CDCl 3 , 360 MHz) d(ppm) 170.8, 170.5, 169.8, 169.5 (CO), 161.4, 160.0 (C-oxadiazole), 131.0, 128.9, 125.0, 123.8 (Ar), 82.7, 73.4, 72.6, 70.6, 68.1 (C-1 to C-5), 61.6 (C-6), 20.6, 20.5 (CH 3 ). Anal. 550 Calcd for C 22 H 25 N 3 O 10 (491.45): C, 53.77; H, 5.13; N, 8.55. Found: C, 53.89; H, 5.24; N, 8.64. 4.22. 5-(4-Methylphenyl)-2-(2,3,4,6-tetra-O-acetyl-bD - glucopyranosylamino)-1,3,4-oxadiazole (18) From 4(0.29 g, 0.57 mmol) according to General procedure III (Section 4.4). Purified by column chromatography (1:1 EtOAc/hexane) to yield 190 mg (66%) of 18 as a white amorphous product. [ a ] D –14 (c0.27, CHCl 3 ); R f : 0.31 (1:1 EtOAc/hexane);1H NMR (CDCl 3 , 360 MHz) d(ppm) 7.78 (2H, d, J= 8.1 Hz, Ar), 7.24 (2H, d, J= 8.0 Hz, Ar), 6.40 (1H, br s, NH), 5.38 (1H, pt, J= 9.4 Hz, H-2 or 560 H-3 or H-4), 5.21 (1H, d, J 1,2 = 9.3 Hz, H-1), 5.14, 5.12 (2H, 2 pt, J= 9.3, 9.7 Hz in each, H-2 and/or H-3, and/or H-4), 4.31 (1H, dd, J 6a,6b = 12.5 Hz, H-6a), 4.09 (1H, dd, H-6b), 3.92 (1H, ddd, J 5,6a = 4.2 Hz, J 5,6b = 1.8 Hz, J 4,5 = 9.8 Hz, H-5), 2.39, 2.07, 2.04, 2.03 (16H, 4 s, CH 3 ). 13 C NMR (CDCl 3 , 360 MHz) d(ppm) 170.6, 170.5, 169.8, 169.4 (CO), 161.2, 160.0 (C-oxadiazole), 141.3, 129.5, 125.9, 121.0 (Ar), 82.6, 73.2, 72.7, 70.5, 68.0 (C-1 to C-5), 61.6 (C-6), 21.4 (CH 3 ), 20.6, 20.5 (CH 3 ). Anal. Calcd for C 23 H 27 N 3 O 10 (505.47): C, 54.65; H, 5.38; N, 8.31. Found: C, 54.78; H, 5.49; N, 8.42. 4.23. 5-(4-Nitrophenyl)-2-(2,3,4,6-tetra-O-acetyl-bD - 570 glucopyranosylamino)-1,3,4-oxadiazole (19) (a) From 5(0.19 g, 0.35 mmol) according to General procedure II (Section 4.3). Purified by column chromatography (1:1 EtOAc/hexane) to yield 80 mg (43%) of 19 as a white amorphous product. (b) From 5(0.25 g, 0.46 mmol) according to General procedure III (Section 4.4). Purified by column chromatography (1:1 EtOAc/ hexane) to yield 130 mg (53%) of 19 as a white amorphous product. [ a ] D –21 (c0.23, CHCl 3 ); R f : 0.27 (1:1 EtOAc/hexane); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 8.34 (2H, d, J= 8.6 Hz, Ar), 8.10 (2H, d, 580 J= 8.6 Hz, Ar), 6.45 (1H, br s, NH), 5.41 (1H, pt, J= 9.4 Hz, H-2 or H-3 or H-4), 5.24 (1H, d, J 1,2 = 9.0 Hz, H-1), 5.16, 5.12 (2H, 2 pt, J= 9.7, 10.0 Hz in each, H-2 and/or H-3, and/or H-4), 4.34 (1H, dd, J 5,6a = 4.3 Hz, J 6a,6b = 12.5 Hz, H-6a), 4.14 (1H, dd, J 5,6b < 1.0 Hz, H-6b), 4.00–3.91 (1H, m, H-5), 2.10, 2.07 (12H, 2 s, CH 3 ). 13 C NMR (CDCl 3 , 360 MHz) d(ppm) 170.9, 170.5, 169.8, 169.5 (CO), 162.1, 158.2 (C-oxadiazole), 148.9 (C–NO 2 ), 129.3, 126.7, 124.3 (Ar), 82.6, 73.4, 72.5, 70.6, 68.0 (C-1 to C-5), 61.5 (C-6), 20.6, 20.5 (CH 3 ). Anal. Calcd for C 22 H 24 N 4 O 12 (536.45): C, 49.26; H, 4.51; N, 10.44. Found: C, 49.37; H, 4.42; N, 10.33. 590 4.24. 5-(4-Fluorophenyl)-2-(2,3,4,6-tetra-O-acetyl-bD - glucopyranosylamino)-1,3,4-oxadiazole (20) (a) From 6(0.20 g, 0.39 mmol) according to General procedure II (4.3). Purified by column chromatography (1:1 EtOAc/hexane) to yield 48 mg (24%) of 20 as a white amorphous product. (b) From 6(0.1 g, 0.19 mmol) according to General procedure III (4.4). Purified by column chromatography (1:1 EtOAc/hexane) to yield 85 mg (88%) of 20 as a white amorphous product. [ a ] D =–4 (c0.30, CHCl 3 ); R f : 0.33 (1:1 EtOAc/hexane); 1H NMR (CDCl 3 , 360 MHz) d(ppm) 7.91 (2H, dd, J= 5.4 Hz, J= 8.2 Hz, Ar), 7.16 600 (2H, pt, J= 8.4 Hz Ar), 6.27 (1H, br s, NH), 5.39 (1H, pt, J= 9.4 Hz, H-2 or H-3 or H-4), 5.21 (1H, d, J 1,2 = 9.2 Hz, H-1), 5.14, 5.10 (2H, 2 pt, J= 9.3, 9.8 Hz in each, H-2 and/or H-3, and/or H-4), 4.34 (1H, dd, J 5,6a = 4.3 Hz, J 6a,6b = 12.3 Hz,H-6a), 4.12 (1H, dd, J 5,6b < 1.0 Hz, H-6b), 3.98–3.88 (1H, m, H-5), 2.09, 2.06, 2.05 (12H, 3s, CH 3 ). 13 C NMR (CDCl 3 , 360 MHz) d(ppm) 170.8, 170.5, 169.8, 169.5 (CO), 164.1 (d, J= 252.0 Hz, Ar), 161.4, 159.1 (C-oxadiazole), 128.2 (d, J= 8.0 Hz, Ar), 120.1, 116.2 (d, J= 22.3 Hz, Ar), 82.6, 73.3, 72.6, 70.5, 68.0 (C-1 to C-5), 61.5 (C-6), 20.6, 20.5 (CH 3 ). Anal. Calcd for C 22 H 24 FN 3 O 10 (509.44): C, 51.87; H, 4.75; N, 8.25. Found: C, 610 51.76; H, 4.87; N, 8.38. 4.25. 5-(3-Chlorophenyl)-2-(2,3,4,6-tetra-O-acetyl-bD - glucopyranosylamino)-1,3,4-oxadiazole (21) (a) From 7(0.13 g, 0.24 mmol) according to General procedure II (Section 4.3). Purified by column chromatography (1:1 EtOAc/hexane) to yield 65 mg (52%) of 21 as a white amorphous product. (b) From 7(0.17 g, 0.33 mmol) according to General procedure III (Section 4.4). Purified by column chromatography (1:1 EtOAc/ hexane) to yield 110 mg (64%) of 21 as a white amorphous product. [ a ] D –13 (c0.32, CHCl 3 ); R f : 0.29 (1:1 EtOAc/hexane); 1 HNMR 620 (CDCl 3 , 360 MHz) d(ppm) 7.89 (1H, br s, Ar), 7.80 (1H, d, J= 7.3 Hz, Ar), 7.51–7.36 (2H, m, Ar), 6.22 (1H, br s, NH), 5.39 (1H, pt, J= 9.4 Hz, H-2 or H-3 or H-4), 5.21 (1H, d, J 1,2 = 9.2 Hz, H1), 5.14, 5.10 (2H, 2 pt, J= 9.4, 9.8 Hz in each, H-2 and/or H-3, and/or H-4), 4.33 (1H, dd, J 5,6a = 4.4 Hz, J 6a,6b = 12.4 Hz, H-6a), 4.13 (1H, dd, J 5,6b < 1.0 Hz, H-6b), 3.99–3.90 (1H, m, H-5), 2.09, 2.06, 2.05 (12H, 3 s, CH 3 ). 13 C NMR (CDCl 3 , 360 MHz) d(ppm) 170.9, 170.5, 169.8, 169.5 (CO), 161.5, 158.8 (C-oxadiazole), 135.0, 131.0, 130.3, 125.9, 125.4, 124.1 (Ar), 82.6, 73.4, 72.6, 70.6, 68.1 (C-1 to C-5), 61.6 (C-6), 20.6, 20.5 (CH 3 ). Anal. Calcd for 630 C 22 H 24 ClN 3 O 10 (525.89): C, 50.25; H, 4.60; N, 7.99. Found: C, 50.38; H, 4.71; N, 7.89. 4.26. 5-(1-Naphthyl)-2-(2,3,4,6-tetra-O-acetyl-bD - glucopyranosylamino)-1,3,4-oxadiazole (22) From 8(0.34 g, 0.63 mmol) according to General procedure III (Section 4.4). Purified by column chromatography (1:1 EtOAc/hexane) to yield 305 mg (89%) of 22 as a white amorphous product. [ a ] D –10 (c0.29, CHCl 3 ); R f : 0.32 (1:1 EtOAc/hexane); 1 HNMR (CDCl 3 , 360 MHz) d(ppm) 9.01 (1H, d, J= 8.4 Hz, Ar), 7.86–7.69 (3H, m, Ar), 7.52–7.26 (3H, m, Ar), 6.94 (1H, br s, NH), 5.32 (1H, 640 pt, J= 9.2 Hz, H-2 or H-3 or H-4), 5.19 (1H, d, J 1,2 = 9.0 Hz, H-1), 5.09, 5.06 (2H, 2 pt, J= 9.5, 9.8 Hz in each, H-2 and/or H-3, and/or H-4), 4.22 (1H, dd, J 5,6a = 4.0 Hz, J 6a,6b = 12.7 Hz, H-6a), 4.02 (1H, dd, J 5,6b < 1.0 Hz, H-6b), 3.90–3.80 (1H, m, H-5), 1.98, 1.93, 1.90 (12H, 3 s, CH 3 ). 13 C NMR (CDCl 3 , 360 MHz) d(ppm) 170.3, 170.2, 169.7, 169.3 (CO), 161.2, 159.5 (C-oxadiazole), 133.4–120.1 (Ar), 82.4, 73.1, 72.7, 70.4, 67.9 (C-1 to C-5), 61.5 (C-6), 20.3 (CH 3 ). Anal. Calcd for C 26 H 27 N 3 O 10 (541.51): C, 57.67; H, 5.03; N, 7.76. Found: C, 57.55; H, 5.12; N, 7.63. M. Tóth et al. / Carbohydrate Research xxx (2013) xxx–xxx 7 CAR 6468 No. of Pages 10, Model 5G 2 May 2013 Please cite this article in press as: Tóth, M.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2013.04.025