Synthesis of 2-(β-D-glucopyranosylamino)-5-substituted-1,3,4-oxadiazoles for inhibition of glycogen phosphorylase

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Please check his box i you ha e no co ec ions o make o he PDF ile G aphical abs ac pp xxx–xxxSyn hesis o 2-(b- DD -glucopy anosylamino)-5-subs i u ed-1,3,4-oxadiazoles o inhibi ion o glycogen phospho ylase Ma ie a Tó h * , Béla Sz} ocs, Tímea Kaszás, Tibo Docsa, Pál Ge gely, László Somsák * O OAc AcO AcO OAc NCO O OAc AcO A cO OAc H N O H NNA O OH HO HO OH H N O H NNA A = Ph, 4-Me-Ph, 4-NO 2 -Ph, 4-F-Ph, 3-Cl-Ph, 1-Naph hyl, 2-Naph hyl O OH HO HO OH H N NN OA Bes inhibi o : A = 4-NO 2 -Ph (K i =4.5µM) Bes inhibi o : A = 4-Me-Ph (K i =12µM) CAR 6468 No. o Pages 1, Model 5G 1 May 2013 Highligh s P epa a ion o a oma ic aldehyde 4-(b- DD -glucopy anosyl)semica bazones. Syn hesis o 2-(b- DD -glucopy anosylamino)-5-subs i u ed-1,3,4- oxadiazoles. Low mic omola inhibi o s o glycogen phospho ylase. 1 Syn hesis o 2-(b-D-glucopy anosylamino)-5-subs i u ed-1,3,4-oxadi- azoles o inhibi ion o glycogen phospho ylase Ma ie a Tó h a, ⇑ ,Béla Sz} ocs a ,Tímea Kaszás a ,Tibo Docsa b ,Pál Ge gely b ,László Somsák a, ⇑ a Depa men o O ganic Chemis y, Uni e si y o Deb ecen, POB 20, H-4010 Deb ecen, Hunga y b Depa men o Medical Chemis y, Medical and Heal h Science Cen e, Uni e si y o Deb ecen, Egye em é 1, H-4032 Deb ecen, Hunga y a icle in o A icle his o y: Recei ed 13 Ma ch 2013 Recei ed in e ised o m 18 Ap il 2013 Accep ed 18 Ap il 2013 A ailable online xxxx 20 Keywo ds: Semica bazone 1,3,4-Oxadiazole b- D -Glucopy anosyl de i a i es Glycogen phospho ylase Inhibi o abs ac A oma ic aldehyde 4-(2,3,4,6- e a-O-ace yl-b-D-glucopy anosyl)semica bazones we e syn hesized by he addi ion o di e en hyd azones on o O-pe ace yla ed b-D-glucopy anosyl isocyana e. Oxida i e ans o ma ions o hese p ecu so s ga e O-p o ec ed 2-(b-D-glucopy anosylamino)-5-subs i u ed- 1,3,4-oxadiazoles. Remo al o he O-ace yl p o ec ing g oups unde Zemplén condi ions ga e es compounds o show low mic omola inhibi ion agains abbi muscle glycogen phospho ylase b. Bes inhibi o s o hese se ies we e 4-(b-D-glucopy anosyl)semica bazones o 4-fluo obenzaldehyde (K i = 4.5 l M), 2-naph haldehyde (K i = 5.5 l M) and 2-(b-D-glucopy anosylamino)-5-(4-me hylphenyl)- 1,3,4-oxadiazole (K i =12 l M). Ó2013 Published by Else ie L d. 1. In oduc ion 40 Inhibi ion o glycogen phospho ylase (GP) can be a new he apeu ic me hod o he ea men o ype 2 diabe es melli us, and po en ial applica ion o GP inhibi o s (GPIs) in some o he diseased s a es, such as ea ly ca diac and ca dio ascula diso de s in non-diabe ics, ca diac a hy hmias, ischemic inju ies, and umo g ow h was also p oposed. 1–5 A la ge a ay o compounds was shown o ha e inhibi o y e ec agains GP unde in i o condi- ions. 4,6 Among hese molecules glucose de i a i es a e he mos in ensi ely in es iga ed GPIs, and de ailed s udies can be ound in he li e a u e on hei s uc u e–ac i i y ela ionships (SAR). 5,7 50 Some glucose de i ed GPIs had in i o hypoglycaemic 8 and o he in e es ing physiological e ec s. 9 Widely s udied glucose analogue GPIs a e de i a i es o b- D -glucopy anosylamine, such as N-acyl-b- D -glucopy anosylam- ines 10–13 (Iin Cha 1,e.g., K i =10–13 l M 11,14 agains abbi muscle GPb(RMGPb) 15 o R = 2-naph hyl), N-a yl-N 0 -b- D -glucopy anosyl u eas 4 (II,e.g., K i = 5.2 l M (RMGPb) o R = 2-naph hyl), as well as N-acyl-N 0 -b- D -glucopy anosyl u ea de i a i es 4,16 (III,e.g., K i = 0.35 l M (RMGPb) o R = 2-naph hyl) which inhibi ed he en- zyme in o below he low mic omola ange. Mic omola e ficiency 60 was epo ed also o 4-(b- D -glucopy anosyl) hiosemica bazones o a oma ic aldehydes 17,18 (IV X=S, e.g., IC 50 = 5.7 l M (RMGPb) o A = 4-fluo ophenyl). Compounds V, in which he suga and he a oma ic pa s a e in e changed in compa ison o IV, ha e e y ecen ly been shown o be GPIs (K i =29 l M o VX = O, and K i = 300 l M o VX = S agains RMGPb). 19 Replacemen o he NHCO moie y by 1,2,3- iazole (a non- classical bioisos e ic he e ocyclic linke A) in molecules I esul ed in e ec i e GP inhibi o s 20 (IA,e.g., K i =16 l M (RMGPb) o R = 2-naph hyl). Enzyma ic es s and c ys allog aphic s udies ha e 70 shown high simila i y o he amide (I) and he 1,2,3- iazole (IA) ype molecules bo h in binding s eng h and s uc u al ea u es o he enzyme–inhibi o complexes. 14 Replacemen o he NHCO moie y wi h isome ic oxadiazoles B,C, and D esul ed in inhibi o s IB–D wi h a ying e ficiency. Among hese molecules he 3-a yl-5- b- D -glucopy anosyl-1,2,4-oxadiazoles IC p o ed o be he mos e ec i e compounds (e.g., K i = 2.4 l M(RMGPb) o R = 2-naph- hyl). 21,22 Bioisos e ic eplacemen s udies we e also ca ied ou wi h compounds III. 13,19,23 As pa o a p og am o sys ema ically eplace NHCO moie ies o 80 GPIs I–III by he e ocyclic bioisos e es, he ein we epo on he syn heses and enzyma ic es s o a se ies o 2-(b- D -glucopy ano- sylamino)-5-subs i u ed-1,3,4-oxadiazoles IIB. Fo he cons uc ion o 1,3,4-oxadiazole ings om acyclic p e- cu so s acidic ea men o N,N 0 -diacyl- (o N-acyl-N 0 - hioacyl)- hyd azines as well as oxida i e cycliza ion o acylhyd azones a e he mos equen ly used p ocedu es, and bo h me hods we e applied o he syn heses o 2-amino-5-subs i u ed-1,3,4-oxadiaz- oles, oo. 24 2-Glycosylamino-5-subs i u ed-1,3,4-oxadiazoles a e also known and ha e been syn hesized om glycosyl iso hiocya- 90 na es ei he by he addi ion o acid hyd azides ollowed by ing 0008-6215/$ - see on ma e Ó2013 Published by Else ie L d. h p://dx.doi.o g/10.1016/j.ca es.2013.04.025 ⇑ Co esponding au ho s. Tel.: +36 52512900x22474; ax: +36 52512744 (M.T.); el.: +36 52512900x22348; ax: +36 52512744 (L.S.). E-mail add esses: [email p o ec ed] (M. Tó h), somsak.laszlo@ science.unideb.hu (L. Somsák). Q1 Ca bohyd a e Resea ch xxx (2013) xxx–xxx Con en s lis s a ailable a SciVe se ScienceDi ec Ca bohyd a e Resea ch jou nal homepage: www.else ie .com/loca e/ca es CAR 6468 No. o Pages 10, Model 5G 2 May 2013 Please ci e his a icle in p ess as: Tó h, M.; e al. Ca bohyd . Res. (2013), h p://dx.doi.o g/10.1016/j.ca es.2013.04.025 closu e o he esul ing 1-acyl-4-glycosyl- hiosemica bazides 25–27 o by chlo ina ion o gi e in e media e glycosyl isocyanide dichlo- ides o be cyclized wi h acid hyd azides. 28 In his wo k we in es iga ed he syn hesis o compounds IIB om aldehyde 4-glycosyl-semica bazones (c . O-p o ec ed IV X=O in Cha 1), a ou e which, o he bes o ou knowledge, has no ye been applied o ob ain 2-glycosylamino-1,3,4-oxadiaz- oles. On he o he hand, he p epa a ion o semica bazones IV (X = O) ga e he oppo uni y o compa e hei GPI p ope ies o 100 he co esponding hiosemica bazones IV (X = S). 2. Resul s and discussion 2.1. Syn heses Fo he o ma ion o he a ge oxadiazoles oxida i e ing closu e o a oma ic aldehyde 4-(2,3,4,6- e a-O-ace yl-b- D -gluco- py anosyl)semica bazones (simila o ha applied o he syn heses o 2-a yl-5-(2,3,4,6- e a-O-benzoyl-b- D -glucopy ano- syl)-1,3,4-oxadiazoles 22 ) was en isaged as he key s ep. To his end, O-pe ace yla ed 4-(b- D -glucopy anosyl)semica bazide 2was p epa ed (Scheme 1) by he eac ion o glucopy anosyl isocyana e 110 1 29 (ob ained om he co esponding glucopy anosylamine 30 ) wi h hyd azine eagen s unde di e en eac ion condi ions ( eagen s: NH 2 NH 2 HCl, NH 2 NH 2 HOAc; sol en s: d y py idine, d y CH 2 Cl 2 ,and 1 equi E 3 N). The bes yield (75%) was achie ed wi h NH 2 NH 2 HOAc in d y CH 2 Cl 2 in he p esence o E 3 N. In each o he abo e eac ions bis-glucopy anosyl u ea was also isola ed in a ious amoun s which could be due o he p esence o aces o wa e in he mix u es. 31 In o de o ge semica bazones 3–9 condensa ion o semica b- azide 2wi h he p ope aldehydes was planned. Howe e , 120 eac ions o 2wi h 2-nap haldehyde in d y E OH o d y oluene in he p esence o ca aly ic amoun s o ei he AcOH o CF 3 COOH a eflux empe a u e esul ed in complex eac ion mix u es. The e o e, b- D -glucopy anosyl isocyana e 2was eac ed wi h alde- hyde hyd azones 32 in boiling d y dioxane o esul in he a ge molecules 3–9 in good yields. Debenzoyla ions we e pe o med by he Zemplén p o ocol o gi e excellen yields o semica bazones 10–16. Ring closing eac ions o he p ecu so s unde oxida i e condi- ions we e s udied nex . Semica bazones 3,5–7, and 9we e 130 eac ed wi h phenyliodonium diace a e (PIDA) in CH 2 Cl 2 a (Scheme 2) o u nish he co esponding O-pe ace yla ed 2-(b- D - glucopy anosylamino)-5-subs i u ed-1,3,4-oxadiazoles 17,19–21, and 23, espec i ely, in mode a e yields. Applica ion o Pb(OAc) 4 as he oxidizing agen in glacial AcOH a 80 °C esul ed in be e yields o 1,3,4-oxadiazoles 17–22 (compa e yields unde condi- ions aand bin Scheme 2). Debenzoyla ions we e pe o med by he Zemplén p o ocol o gi e mos ly good yields o 1,3,4-oxadiaz- oles 24–30 (Scheme 2). S uc u al elucida ion o he new compounds was based on NMR 140 spec a. The b- D -anome ic configu a ion was indica ed by he 8.1– 9.9 Hz coupling cons an s be ween 1-H and 2-H p o ons in he 4 C 1 con o ma ion o each compound. Coupling o 1-H and N(4)H (9.2–9.8 Hz) could be obse ed in he spec a o semica bazones 3–9 only. Fo he oxadiazoles 17–23 1-H and NH appea ed as double s and single s, espec i ely, p obably because o a specific dihed al angle be ween hese p o ons esul ing in a e y small coupling cons an . A 1 H spec um o semica bazone 5 eco ded immedia ely (5 min) a e dissolu ion showed he p esence o one compound only. A NOE di e ence spec um ob ained by i adi- 150 a ion o he N(2)H signal ga e posi i e NOE o he signal o CH@N (7.86 ppm) and N(4)H (7.25 ppm) indica ing Econfigu a ion o he C@Ndouble bond. A e 72 h he 1 H spec um o 5exhibi ed wo se s o signals as a esul o E/Z isome isa ion (1:1 a io, cha ac e - is ic chemical shi s (d,ppm) o he Eisome : 10.10 (N(2)H), 7.86 (CH@N), 7.25 (N(4)H); o he Zisome : 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 linke I O HO HO HO OH H NH N O linke II R linke s 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 CA Ph Cha 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 NNA 2 (75 %) b 1 O OH HO HO O H H N O H NNA c Ph 4-Me-Ph 4-NO 2 -Ph 4-F-Ph 3-Cl-Ph 1-Naph hyl 2-Naph hyl A 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. Reagen s and condi ions: (a) NH 2 NH 2 HOAc, d y CH 2 Cl 2 ,E 3 N, ; (b) A CH@N–NH 2 , d y dioxane, eflux; (c) NaOMe, d y MeOH, . 2M. Tó h e al. / Ca bohyd a e Resea ch xxx (2013) xxx–xxx CAR 6468 No. o Pages 10, Model 5G 2 May 2013 Please ci e his a icle in p ess as: Tó h, M.; e al. Ca bohyd . Res. (2013), h p://dx.doi.o g/10.1016/j.ca es.2013.04.025 7.15 (N(4)H). These upfield shi s o he Zisome a e in acco d wi h li e a u e expe iences o semica bazones, 33 howe e , con as hose o O-pe ace yla ed 4-(b- D -glucopy anosyl) hiosemica ba- zones whe e he N(2)Hsignals we e epo ed o appea a 10.1 160 (E) and 14.6 (Z) ppm. 17 2.2. Enzyme inhibi ion s udies The kine ic pa ame e s (inhibi ion po ency agains abbi mus- cle glycogen phospho ylase b(RMGPb)) o he dep o ec ed com- pounds we e de e mined acco ding o he p o ocol desc ibed ea lie . 34 The esul s a e summa ized in Table 1 showing he inhib- i o y e ficiency o some ele an e e ence compounds, as well. Type IV semica bazones 10–16, he ‘open chain’ p ecu so s o he a ge compounds o his wo k, p o ed low mic omola inhib- i o s wi h he p ac ically equipo en 4-ni ophenyl (12) and 170 2-naph hyl (16) compounds as he bes ones. The s ong binding o 16 fi s he gene al end obse ed wi h se e al glucose de i a- i es exhibi ing mos e ficien inhibi ion wi h la ge hyd ophobic g oups in he aglycons. 4 A compa ison o he s uc u ally ela ed hiosemica bazone coun e pa s would be possible o he pai s 12–31,13–32, and 14–33, howe e , he epo ed inhibi ion da a 18 o he sul u con aining compounds 31–33 a e IC 50 alues no di ec ly compa able wi h he inhibi o cons an s. Ne e heless, i is in e es ing o no e ha he endency is di e en in he wo se - ies: wi hin semica bazones he 4-ni ophenyl (12) compound 180 while wi hin he hiosemica bazones he 4-fluo ophenyl (32) de i a i e p o ed 4 o6 imes s onge inhibi o s han he nex bes ones 14 and 33, espec i ely. A compa ison o inhibi ion o 10 o ha o he biu e ype com- pound 37, 4 ha ing he same numbe o a oms be ween he suga and he a oma ic ing, indica es he la e o be a  wice be e inhibi o han he o me one. This may unde line he impo ance o bo h ca bonyl uni s in he binding. On he o he hand, 10 binds 15- old s onge han 38 4 wi h wo C@Omoie ies bu also ha ing a o a able CH 2 uni in place o NH, he eby emphasizing ha he 190 igidi y o 10 makes an impo an con ibu ion o he binding. A u he compa ison o 10 o ype Vcompound 39, 19 whe ein he semica bazone linke is o mally e e sed be ween he ca bohyd a e ing and he phenyl g oup, shows a mode a e s eng hening o he inhibi ion ha may e e o he highe con ibu ion o he second ca bonyl uni o he binding. This is co - obo a ed by he smalle dec ease caused by he change 37?39 han ha o 37?10. A o mal e e sal o he hiosemica bazone linke as in 40 19 makes a e y la ge dec ease in he binding s eng h mos p obably because he hioca bonyl in he posi ion 200 o he ‘second amide’ moie y is much less sui able o make s ong in e ac ions o he enzyme. Replacemen o he NHCO moie y in compounds ype II by he 1,3,4-oxadiazole ing (compounds ype IIB:24–30) esul ed in mode a e GPIs. Fo he phenyl and 1-naph hyl subs i u ed pai s 34–24 and 35–29 he e is no significan change in he binding s eng h. Subs i u ion in he 4-posi ion o he a oma ic ing (25– 27) esul ed in a small and unspecific inc ease o he e ficiency, while he 3-chlo o eplacemen (28) made a somewha weake inhibi o . Based on hese esul s one could a gue ha he 1,3,4-oxa- 210 diazole is an accep able bioisos e ic eplacemen o he NHCO moi- e y in his sys em. Howe e , his a gumen is weakened by he 5 imes wo se inhibi ion o 30 as compa ed o 36 o which a specu- la i e explana ion can be ha he la ge and igid aglycon is unable o find an accomoda able posi ion in he binding si e o he enzyme. 3. Conclusion Reac ion o O-pe ace yla ed b- D -glucopy anosyl isocyana e wi h di e en hyd azones ga e he co esponding aldehyde 4-(2, 3,4,6- e a-O-ace yl-b- D -glucopy anosyl)semica bazones in good yields. Oxida ion o hese compounds by PIDA o mo e ad an a- 220 geously by Pb(OAc) 4 esul ed in 2-(b- D -glucopy anosylamino)-5- subs i u ed-1,3,4-oxadiazoles. Zemplén deace yla ion u nished es compounds which p o ed low mic omola inhibi o s agains RMGPb.S uc u e–ac i i y conside a ions allow o es ima e he ela i e con ibu ion o he binding by ca bonyl and hioca bonyl g oups o N-acyl-N 0 -glucopy anosyl u ea ype GPIs, and aise he possibili y o applying 1,3,4-oxadiazoles as bioisos e ic eplace- men s o NHCO moie ies. 4. Expe imen al 4.1. Gene al me hods 230 Mel ing poin s we e measu ed in open capilla y ubes o on a Kofle ho -s age and a e unco ec ed. Op ical o a ions we e de e mined wi h a Pe kin–Elme 241 pola ime e a oom empe - a u e. NMR spec a we e eco ded wi h B uke 360 (360/90 MHz o 1 H/ 13 C) o B uke 400 (400/100 MHz o 1 H/ 13 C) spec ome e s. Chemical shi s a e e e enced o TMS as he in e nal e e ence ( 1 H), o o he esidual sol en signals ( 13 C). Mic oanalyses we e pe o med on an Elemen a a io Mic o cube. ESIMS we e eco ded wi h a B uke mic OTOF-Q ins umen . TLC was pe o med on DC- Alu olle Kieselgel 60 F 254 (Me ck). TLC pla es we e isualized unde 240 UV ligh , and by gen le hea ing. Fo column ch oma og aphy Kieselgel 60 (Me ck, pa icle size (0.063–0.200 mm) was applied. O ganic solu ions we e d ied o e anhyd ous MgSO 4 , and concen- a ed unde diminished p essu e a 40–50 °C (wa e ba h). Alde- hyde hyd azones we e ob ained om he co esponding aldehydes and H 2 NNH 2 H 2 O acco ding o a li e a u e p ocedu e. 32 4.2. Gene al p ocedu e I o he syn hesis o a oma ic aldehyde 4-(2,3,4,6- e a-O-ace yl-b- D -glucopy anosyl)semica bazones (3–9) 2,3,4,6-Te a-O-ace yl-b- D -glucopy anosyl isocyana e 29 (1, 250 0.22 g, 0.59 mmol) was added o he solu ion o an aldehyde hyd a- zone (0.27 g, 1.80 mmol) in d y dioxane (5 mL). The eac ion mix- u e was s i ed a eflux empe a u e o 24 h. When he eac ion O OR RO RO OR H N NN OA O OAc AcO AcO O Ac H N O H NNA a o b 3-9 c Ph 4-Me-Ph 4-NO 2 -Ph 4-F-Ph 3-Cl-Ph 1-Naph hyl 2-Naph hyl A 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. Reagen s and condi ions: (a) PIDA, CH 2 Cl 2, ; (b) Pb(OAc) 4 , AcOH, 80 °C; (c) NaOMe, d y MeOH, . M. Tó h e al. / Ca bohyd a e Resea ch xxx (2013) xxx–xxx 3 CAR 6468 No. o Pages 10, Model 5G 2 May 2013 Please ci e his a icle in p ess as: Tó h, M.; e al. Ca bohyd . Res. (2013), h p://dx.doi.o g/10.1016/j.ca es.2013.04.025 was comple e (TLC 1:1 E OAc/hexane) he eac ion mix u e was fil e ed wi h suc ion, and he sol en was e apo a ed unde educed p essu e. The c ude p oduc was pu ified by column ch oma og aphy. 4.3. Gene al p ocedu e II o he syn hesis o 5-a yl-2-(2,3,4,6- e a-O-ace yl-b- D -glucopy anosylamino)-1,3,4-oxadiazoles (17–23) 260 An aldehyde 4-(2,3,4,6- e a-O-ace yl-b- D -glucopy anosyl) semica bazone (3–9, 0.03 mmol/mL) was dissol ed in CH 2 Cl 2 , hen PIDA (1.1 equi ) was added, and he mix u e was s i ed a . When he eac ion was comple e (TLC, 1:1 E OAc/hexane) he sol en was e apo a ed unde educed p essu e, and he esidue was pu ified by column ch oma og aphy. 4.4. Gene al p ocedu e III o he syn hesis o 5-a yl-2-(2,3,4,6- e a-O-ace yl-b- D -glucopy anosylamino)-1,3,4-oxadiazoles (17–23) An aldehyde 4-(2,3,4,6- e a-O-ace yl-b- D -glucopy ano- 270 syl)semica bazone (3–9, 0.03 mmol/mL) was dissol ed in glacial AcOH, hen Pb(OAc) 4 (1 equi ) was added, and he mix u e was s i ed a 80 °C. The eac ion was moni o ed by TLC (1:1 E OAc/ hexane). When he eac ion was comple e, he eac ion mix u e was dilu ed wi h H 2 O (10 mL), and washed wi h E OAc (3  6 mL). The o ganic laye was sepa a ed, d ied, and he sol en was e apo a ed unde educed p essu e. The esidue was pu ified by column ch oma og aphy. 4.5. Gene al p ocedu e IV o he emo al o O-ace yl p o ec ing g oups 280 An O-pe ace yla ed compound (100 mg) was dissol ed in d y MeOH (1 mL) and a solu ion o NaOMe (0.1 M in MeOH) was added o he solu ion in a ca aly ic amoun . The eac ion mix u e was s i ed a . When he eac ion was comple e (TLC, 3:1 CHCl 3 / MeOH) he solu ion was neu alized wi h a ca ion exchange esin Ambe lys 15 (H + o m). The esin was fil e ed o wi h suc ion and he fil a e was e apo a ed unde educed p essu e. The c ude p oduc was pu ified by column ch oma og aphy. 4.6. 4-(2,3,4,6-Te a-O-ace yl-b- D -glucopy anosyl)semica bazide (2) 290 NH 2 NH 2 HOAc (97%, 26.6 mg, 0.28 mmol) was added o he solu ion o 2,3,4,6- e a-O-ace yl-b- D -glucopy anosyl isocyana e 29 (1, 0.10 g, 0.27 mmol) and E 3 N (39 l L, 0.28 mmol) in d y CH 2 Cl 2 (2 mL). The eac ion mix u e was s i ed a . When he eac ion was comple e (TLC 5:1 E OAc/hexane) he sol en was e apo a ed unde educed p essu e. The c ude p oduc was pu ified by column ch oma og aphy (eluen : 5:1 E OAc/hexane) o gi e 81 mg (75%) o 2as a whi e amo phous p oduc . [ a ] D 4(c0.50, DMSO); R = 0.27 (5:1 E OAc/hexane); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 9.28, 6.82, Table 1 Inhibi ion o abbi muscle glycogen phospho ylase b(RMGPb) by selec ed glucose de i a i es and he new compounds (K i [ l M]) A O HO HO HO OH H NH N X N H CA O HO HO HO OH H NH N O A O OH HO HO OH H N NN OA 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. Tó h e al. / Ca bohyd a e Resea ch xxx (2013) xxx–xxx CAR 6468 No. o Pages 10, Model 5G 2 May 2013 Please ci e his a icle in p ess as: Tó h, M.; e al. Ca bohyd . Res. (2013), h p://dx.doi.o g/10.1016/j.ca es.2013.04.025 6.53, 6.27 (4H, 4 s, NH), 5.32, 5.14, 5.07, 4.97 (4H, 4 p , 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, H- 6b), 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 o C-5), 61.7 (C-6), 20.5, 20.3 (CH 3 ). Anal. Calcd o 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- e a-O-ace yl-b- D -glucopy anosyl) semica bazone (3) F om isocyana e 1(0.10 g, 0.27 mmol) and benzaldehyde hyd azone (0.064 g, 0.54 mmol) acco ding o Gene al p ocedu e I 310 (Sec ion 4.2). Pu ified by column ch oma og aphy (1:1 E OAc/ hexane) o yield 110 mg (83%) o 3as a whi e amo phous p oduc . [ a ] D –61 (c0.99, CHCl 3 ); R : 0.58 (5:1 E OAc/hexane); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 9.47 (1H, b s, NH), 7.77 (1H, s, CH@N), 7.70–7.66 (2H, m, A ), 7.43–7.40 (3H, m, A ), 7.16 (1H, d, J 1,NH = 9.5 Hz, NH), 5.39, 5.28, 5.15, 5.13 (4H, 4 p , 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 (A ), 79.3, 73.2, 72.8, 70.3, 68.2 (C-1 o C-5), 61.6 (C-6), 20.7, 20.6 (CH 3 ). Anal. Calcd o 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-Me hylbenzaldehyde 4-(2,3,4,6- e a-O-ace yl-b- D - glucopy anosyl)semica bazone (4) F om isocyana e 1(0.40 g, 1.07 mmol) and 4-me hylbenzalde- hyde hyd azone (0.29 g, 2.14 mmol) acco ding o Gene al p oce- du e I (Sec ion 4.2). Pu ified by column ch oma og aphy (1:1 E OAc/hexane) o yield 390 mg (72%) o 4as a whi e amo phous 330 p oduc . [ a ] D –50 (c0.86, CHCl 3 ); R : 0.66 (5:1 E OAc/hexane); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 9.21 (1H, b s, NH), 7.71 (1H, s, CH@N), 7.56 (2H, d, J= 8.0 Hz, A ), 7.27–7.20 (2H, m, A ), 7.13 (1H, d, J 1,NH = 9.5 Hz, NH), 5.37, 5.26, 5.14, 5.12 (4H, 4 p , 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 (A ), 79.4, 73.2, 72.9, 70.3, 68.2 (C-1 o C-5), 61.7 (C-6), 340 21.5, 20.7 (CH 3 ). Anal. Calcd o 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-Ni obenzaldehyde 4-(2,3,4,6- e a-O-ace yl-b- D - glucopy anosyl)semica bazone (5) F om isocyana e 1(0.40 g, 1.07 mmol) and 4-ni obenzaldehyde hyd azone (0.37 g, 2.14 mmol) acco ding o Gene al p ocedu e I (Sec ion 4.2). Pu ified by column ch oma og aphy (1:1 E OAc/hex- ane) o yield 380 mg (66%) o 5as an o ange amo phous p oduc . [ a ] D 71 (c0.35, CHCl 3 ); R : 0.18 (1:1 E OAc/hexane); 1 H NMR (CDCl 3 , 400 MHz) d(ppm) 10.10 (1H, b s, NH), 8.29 (2H, d, 350 J= 8.8 Hz, A ), 7.87 (2H, d, J= 8.8 Hz, A ), 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 p , 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 (A ), 79.3, 73.2, 72.5, 70.4, 68.1 (C-1 o C-5), 61.6 (C-6), 20.5 (CH 3 ). Anal. Calcd o 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-Fluo obenzaldehyde 4-(2,3,4,6- e a-O-ace yl-b- D - glucopy anosyl)semica bazone (6) F om isocyana e 1(0.25 g, 0.67 mmol) and 4-fluo obenzalde- hyde hyd azone (0.19 g, 1.23 mmol) acco ding o Gene al p oce- du e I (Sec ion 4.2). Pu ified by column ch oma og aphy (1:1 E OAc/hexane) o yield 260 mg (76%) o 6as a whi e amo phous p oduc . [ a ] D –54 (c0.27, CHCl 3 ); R : 0.22 (1:1 E OAc/hexane); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 10.09 (1H, b s, NH) 7.78 (1H, s, CH@N), 7.68 (2H, dd, J= 5.6 Hz, J= 7.9 Hz, A ), 7.21–7.07 (3H, m, A , NH), 5.41, 5.29, 5.15, 5.12 (4H, 4 p , J= 9.3, 9.9 Hz in each, H- 370 1, H-2, H-3, H-4), 4.38 (1H, dd, J 5,6a = 3.7 Hz, J 6a,6b = 12.5 Hz, H- 6a), 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, A ), 155.8 (NHCONH), 141.4 (CH@N), 129.9 (A ), 128.9 (d, J= 7.3 Hz, A ), 115.8 (d, J= 22.0 Hz, A ), 79.3, 73.1, 72.7, 70.3, 68.1 (C-1 o C-5), 61.6 (C- 6), 20.5 (CH 3 ). Anal. Calcd o 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-Chlo obenzaldehyde 4-(2,3,4,6- e a-O-ace yl-b- D - glucopy anosyl)semica bazone (7) 380 F om isocyana e 1(0.22 g, 0.59 mmol) and 3-chlo obenzalde- hyde hyd azone (0.18 g, 1.18 mmol) acco ding o Gene al p oce- du e I (Sec ion 4.2). Pu ified by column ch oma og aphy (1:1 E OAc/hexane) o yield 222 mg (71%) o 7as a whi e amo phous p oduc . [ a ] D –65 (c1.06, CHCl 3 ); R : 0.61 (5:1 E OAc/hexane); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 9.60 (1H, b s, NH), 7.64–7.46 (3H, m, A , CH@N), 7.31–7.27 (2H, m, A ), 7.12 (1H, d, J 1,NH = 9.2 Hz, NH), 5.31, 5.16, 5.07, 5.05 (4H, 4 p , 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 (A ), 79.5, 73.3, 72.7, 70.4, 68.3 (C-1 o C-5), 61.7 (C-6), 20.7, 20.6 (CH 3 ). Anal. Calcd o 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-Naph haldehyde) 4-(2,3,4,6- e a-O-ace yl-b- D - glucopy anosyl)semica bazone (8) F om isocyana e 1(0.40 g, 1.07 mmol) and 1-naph aldehyde hyd azone (0.37 g, 2.14 mmol) acco ding o Gene al p ocedu e I 400 (Sec ion 4.2). Pu ified by column ch oma og aphy (1:1 E OAc/hex- ane) o yield 520 mg (89%) o 8as a yellow amo phous p oduc . [ a ] D 53 (c0.96, CHCl 3 ); R : 0.63 (5:1 E OAc/hexane); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 9.63 (1H, b s, NH), 8.52 (1H, d, J= 8.5 Hz, A ), 8.44 (1H, s, CH@N), 7.93–7.90 (3H, m, A ), 7.68 (1H, p , J= 8.1, 8.3 Hz, A ), 7.58–7.51 (2H, m, A ), 7.17 (1H, d, J 1,NH = 9.6 Hz, NH), 5.42, 5.34, 5.17, 5.16 (4H, 4 p , J= 9.5, 9.7 Hz in each, H-1, H-2, H- 3, 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 (A ), 79.4, 73.3, 72.9, 70.3, 68.2 (C-1 o C-5), 61.6 (C-6), 20.7, 20.6 (CH 3 ). Anal. Calcd o 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. Tó h e al. / Ca bohyd a e Resea ch xxx (2013) xxx–xxx 5 CAR 6468 No. o Pages 10, Model 5G 2 May 2013 Please ci e his a icle in p ess as: Tó h, M.; e al. Ca bohyd . Res. (2013), h p://dx.doi.o g/10.1016/j.ca es.2013.04.025 4.13. 4-(2-Naph haldehyde) 4-(2,3,4,6- e a-O-ace yl-b- D -gluco- py anosyl)semica bazone (9) F om isocyana e 1(0.27 g, 0.71 mmol) and 2-naph aldehyde hyd azone (0.24 g, 1.42 mmol) acco ding o Gene al p ocedu e I (Sec ion 4.2). Pu ified by column ch oma og aphy (1:1 E OAc/hex- 420 ane) o yield 310 mg (80%) o 9as a yellow amo phous p oduc . [ a ] D 73 (c0.93, CHCl 3 ); R : 0.64 (5:1 E OAc/hexane); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 9.83 (1H, s, NH), 7.99–7.83 (6H, m, A , CH@N), 7.53–7.50 (2H, m, A ), 7.26 (1H, d, J 1,NH = 9.8 Hz, NH), 5.42, 5.32, 5.17, (4H, 3 p , 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 (A ), 79.5, 73.3, 72.9, 70.4, 68.3 430 (C-1 o C-5), 61.7 (C-6), 20.6 (CH 3 ). Anal. Calcd o 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-(b- D -glucopy anosyl)semica bazone (10) F om 3(0.15 g, 0.30 mmol) acco ding o Gene al p ocedu e IV (Sec ion 4.5). Pu ified by column ch oma og aphy (4:1 CHCl 3 / MeOH) o yield 98 mg (99%) o 10 as a whi e amo phous p oduc . [ a ] D +33 (c0.54, CH 3 OH); R : 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, A ), 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 d op CH 3 OH, 360 MHz) d(ppm) 158.4 (NHCONH), 144.1 (CH@N), 135.7, 130.9, 129.7, 128.1 (A ), 82.4, 79.4, 78.8, 73.9, 71.4 (C-1 o C-5), 62.7 (C- 6). Anal. Calcd o 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-Me hylbenzaldehyde 4-(b- D - glucopy anosyl)semica bazone (11) F om 4(0.09 g, 0.18 mmol) acco ding o Gene al p ocedu e IV (Sec ion 4.5). Pu ified by column ch oma og aphy (4:1 CHCl 3 / MeOH) o yield 60 mg (98%) o 11 as a whi e amo phous p oduc . 450 [ a ] D +30 (c0.48, CH 3 OH); R : 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, A ), 7.20 (2H, d, J= 8.5 Hz, A ), 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 (A ), 82.4, 79.5, 78.9, 74.0, 71.4 (C-1 o C-5), 62.7 (C-6), 21.5 (CH 3 ). Anal. Calcd o 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-Ni obenzaldehyde 4-(b- D -glucopy anosyl) semica bazone (12) F om 5(0.15 g, 0.28 mmol) acco ding o Gene al p ocedu e IV (Sec ion 4.5). Pu ified by column ch oma og aphy (1:1 CHCl 3 / MeOH) o yield 107 mg (93%) o 12 as an o ange amo phous p od- uc . [ a ] D +30 (c0.29, DMSO); R : 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, A ), 8.00 (2H, d, J= 9.0 Hz, A ), 7.98 (1H, s, CH@N), 4.94 (1H, d, J 1,2 = 8.5 Hz, H- 1), 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 o H-3 470 o H-4, H-5), 3.40–3.36 (2H, m, H-2 and/o H-3, and/o 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 (A ), 80.9, 78.4, 77.4, 71.9, 69.9 (C-1 o C-5), 61.0 (C-6). Anal. Calcd o 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-Fluo obenzaldehyde 4-(b- D -glucopy anosyl) semica bazone (13) F om 6(0.12 g, 0.24 mmol) acco ding o Gene al p ocedu e IV (Sec ion 4.5). Pu ified by column ch oma og aphy (4:1 CHCl 3 / 480 MeOH) o yield 80 mg (98%) o 13 as a b own amo phous p oduc . [ a ] D +42 (c0.28, DMSO); R : 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, A ), 7.15 (2H, p , J= 8.7 Hz, A ), 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, H- 6b), 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, A ), 155.4 (NHCOHN), 138.0 (CH@N), 131.2, 129.2 (d, J= 6.4 Hz, A ), 115.8 (d, J= 21.6 Hz, A ), 80.9, 78.4, 77.5, 72.1, 70.1 (C-1 o C-5), 61.1 (C-6). Anal. Calcd o 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-Chlo obenzaldehyde 4-(b- D -glucopy anosyl) semica bazone (14) F om 7(0.15 g, 0.28 mmol) acco ding o Gene al p ocedu e IV (Sec ion 4.5). Pu ified by column ch oma og aphy (4:1 CHCl 3 / MeOH) o yield 96 mg (94%) o 14 as a yellow amo phous p oduc . [ a ] D +30 (c0.54, CH 3 OH); R : 0.40 (4:1 CHCl 3 /MeOH); 1 HNMR (CD 3 OD, 360 MHz) d(ppm) 7.83–7.32 (5H, m, A , 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, H- 500 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 (A ), 82.4, 79.4, 78.8, 73.8, 71.3 (C-1 o C-5), 62.6 (C-6). Anal. Calcd o 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-Naph haldehyde 4-(b- D -glucopy anosyl)semica bazone (15) F om 8(0.17 g, 0.18 mmol) acco ding o Gene al p ocedu e IV (Sec ion 4.5). Pu ified by column ch oma og aphy (4:1 CHCl 3 / MeOH) o yield 93 mg (80%) o 15 as a b own amo phous p oduc . 510 [ a ] D +32 (c0.48, CH 3 OH); R : 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, A ), 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 (A ), 82.4, 79.5, 79.0, 74.1, 71.4 (C-1 o C-5), 62.7 (C-6). Anal. Calcd o 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-Naph haldehyde 4-(b- D -glucopy anosyl)semica bazone 520 (16) F om 9(0.14 g, 0.26 mmol) acco ding o Gene al p ocedu e IV (Sec ion 4.5). Pu ified by column ch oma og aphy (4:1 CHCl 3 / MeOH) o yield 96 mg (98%) o 16 as a whi e amo phous p oduc . [ a ] D +39 (c0.47, CH 3 OH); R : 0.43 (4:1 CHCl 3 /MeOH); 1 HNMR (CD 3 OD, 360 MHz) d(ppm) 8.08–7.46 (8H, m, A , 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, H- 3, 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 (A ), 82.4, 79.5, 78.9, 74.0, 71.4 (C-1 o 530 C-5), 62.7 (C-6). Anal. Calcd o 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. Tó h e al. / Ca bohyd a e Resea ch xxx (2013) xxx–xxx CAR 6468 No. o Pages 10, Model 5G 2 May 2013 Please ci e his a icle in p ess as: Tó h, M.; e al. Ca bohyd . Res. (2013), h p://dx.doi.o g/10.1016/j.ca es.2013.04.025 4.21. 5-Phenyl-2-(2,3,4,6- e a-O-ace yl-b- D - glucopy anosylamino)-1,3,4-oxadiazole (17) (a) F om 3(0.11 g, 0.23 mmol) acco ding o Gene al p ocedu e II (Sec ion 4.3). Pu ified by column ch oma og aphy (1:1 E OAc/hex- ane) o yield 50 mg (45%) o 17 as a whi e amo phous p oduc . (b) F om 3(0.25 g, 0.51 mmol) acco ding o Gene al p ocedu e III (Sec ion 4.4). Pu ified by column ch oma og aphy (1:1 E OAc/ hexane) o yield 212 mg (85%) o 17 as a whi e amo phous p oduc . 540 [ a ] D –20 (c0.28, CHCl 3 ); R : 0.23 (1:1 E OAc/hexane);1H NMR (CDCl 3 , 360 MHz) d(ppm) 7.96–7.83 (2H, m, A ), 7.54–7.42 (3H, m, A ), 6.22 (1H, b s, NH), 5.39 (1H, p , J= 9.4 Hz, H-2 o H-3 o H-4), 5.21 (1H, d, J 1,2 = 9.0 Hz, H-1), 5.14, 5.11 (2H, 2 p , J= 9.4, 9.9 Hz in each, H-2 and/o H-3, and/o 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, H- 6b), 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 (A ), 82.7, 73.4, 72.6, 70.6, 68.1 (C-1 o C-5), 61.6 (C-6), 20.6, 20.5 (CH 3 ). Anal. 550 Calcd o 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-Me hylphenyl)-2-(2,3,4,6- e a-O-ace yl-b- D - glucopy anosylamino)-1,3,4-oxadiazole (18) F om 4(0.29 g, 0.57 mmol) acco ding o Gene al p ocedu e III (Sec ion 4.4). Pu ified by column ch oma og aphy (1:1 E OAc/hex- ane) o yield 190 mg (66%) o 18 as a whi e amo phous p oduc . [ a ] D –14 (c0.27, CHCl 3 ); R : 0.31 (1:1 E OAc/hexane);1H NMR (CDCl 3 , 360 MHz) d(ppm) 7.78 (2H, d, J= 8.1 Hz, A ), 7.24 (2H, d, J= 8.0 Hz, A ), 6.40 (1H, b s, NH), 5.38 (1H, p , J= 9.4 Hz, H-2 o 560 H-3 o H-4), 5.21 (1H, d, J 1,2 = 9.3 Hz, H-1), 5.14, 5.12 (2H, 2 p , J= 9.3, 9.7 Hz in each, H-2 and/o H-3, and/o 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 (A ), 82.6, 73.2, 72.7, 70.5, 68.0 (C-1 o C-5), 61.6 (C-6), 21.4 (CH 3 ), 20.6, 20.5 (CH 3 ). Anal. Calcd o 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-Ni ophenyl)-2-(2,3,4,6- e a-O-ace yl-b- D - 570 glucopy anosylamino)-1,3,4-oxadiazole (19) (a) F om 5(0.19 g, 0.35 mmol) acco ding o Gene al p oce- du e II (Sec ion 4.3). Pu ified by column ch oma og aphy (1:1 E OAc/hexane) o yield 80 mg (43%) o 19 as a whi e amo phous p oduc . (b) F om 5(0.25 g, 0.46 mmol) acco ding o Gene al p ocedu e III (Sec ion 4.4). Pu ified by column ch oma og aphy (1:1 E OAc/ hexane) o yield 130 mg (53%) o 19 as a whi e amo phous p oduc . [ a ] D –21 (c0.23, CHCl 3 ); R : 0.27 (1:1 E OAc/hexane); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 8.34 (2H, d, J= 8.6 Hz, A ), 8.10 (2H, d, 580 J= 8.6 Hz, A ), 6.45 (1H, b s, NH), 5.41 (1H, p , J= 9.4 Hz, H-2 o H-3 o H-4), 5.24 (1H, d, J 1,2 = 9.0 Hz, H-1), 5.16, 5.12 (2H, 2 p , J= 9.7, 10.0 Hz in each, H-2 and/o H-3, and/o 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 (A ), 82.6, 73.4, 72.5, 70.6, 68.0 (C-1 o C-5), 61.5 (C-6), 20.6, 20.5 (CH 3 ). Anal. Calcd o 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-Fluo ophenyl)-2-(2,3,4,6- e a-O-ace yl-b- D - glucopy anosylamino)-1,3,4-oxadiazole (20) (a) F om 6(0.20 g, 0.39 mmol) acco ding o Gene al p ocedu e II (4.3). Pu ified by column ch oma og aphy (1:1 E OAc/hexane) o yield 48 mg (24%) o 20 as a whi e amo phous p oduc . (b) F om 6(0.1 g, 0.19 mmol) acco ding o Gene al p ocedu e III (4.4). Pu ified by column ch oma og aphy (1:1 E OAc/hexane) o yield 85 mg (88%) o 20 as a whi e amo phous p oduc . [ a ] D =–4 (c0.30, CHCl 3 ); R : 0.33 (1:1 E OAc/hexane); 1H NMR (CDCl 3 , 360 MHz) d(ppm) 7.91 (2H, dd, J= 5.4 Hz, J= 8.2 Hz, A ), 7.16 600 (2H, p , J= 8.4 Hz A ), 6.27 (1H, b s, NH), 5.39 (1H, p , J= 9.4 Hz, H-2 o H-3 o H-4), 5.21 (1H, d, J 1,2 = 9.2 Hz, H-1), 5.14, 5.10 (2H, 2 p , J= 9.3, 9.8 Hz in each, H-2 and/o H-3, and/o 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, A ), 161.4, 159.1 (C-oxadiazole), 128.2 (d, J= 8.0 Hz, A ), 120.1, 116.2 (d, J= 22.3 Hz, A ), 82.6, 73.3, 72.6, 70.5, 68.0 (C-1 o C-5), 61.5 (C-6), 20.6, 20.5 (CH 3 ). Anal. Calcd o 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-Chlo ophenyl)-2-(2,3,4,6- e a-O-ace yl-b- D - glucopy anosylamino)-1,3,4-oxadiazole (21) (a) F om 7(0.13 g, 0.24 mmol) acco ding o Gene al p ocedu e II (Sec ion 4.3). Pu ified by column ch oma og aphy (1:1 E OAc/hex- ane) o yield 65 mg (52%) o 21 as a whi e amo phous p oduc . (b) F om 7(0.17 g, 0.33 mmol) acco ding o Gene al p ocedu e III (Sec ion 4.4). Pu ified by column ch oma og aphy (1:1 E OAc/ hexane) o yield 110 mg (64%) o 21 as a whi e amo phous p oduc . [ a ] D –13 (c0.32, CHCl 3 ); R : 0.29 (1:1 E OAc/hexane); 1 HNMR 620 (CDCl 3 , 360 MHz) d(ppm) 7.89 (1H, b s, A ), 7.80 (1H, d, J= 7.3 Hz, A ), 7.51–7.36 (2H, m, A ), 6.22 (1H, b s, NH), 5.39 (1H, p , J= 9.4 Hz, H-2 o H-3 o H-4), 5.21 (1H, d, J 1,2 = 9.2 Hz, H- 1), 5.14, 5.10 (2H, 2 p , J= 9.4, 9.8 Hz in each, H-2 and/o H-3, and/o 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 (A ), 82.6, 73.4, 72.6, 70.6, 68.1 (C-1 o C-5), 61.6 (C-6), 20.6, 20.5 (CH 3 ). Anal. Calcd o 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-Naph hyl)-2-(2,3,4,6- e a-O-ace yl-b- D - glucopy anosylamino)-1,3,4-oxadiazole (22) F om 8(0.34 g, 0.63 mmol) acco ding o Gene al p ocedu e III (Sec ion 4.4). Pu ified by column ch oma og aphy (1:1 E OAc/hex- ane) o yield 305 mg (89%) o 22 as a whi e amo phous p oduc . [ a ] D –10 (c0.29, CHCl 3 ); R : 0.32 (1:1 E OAc/hexane); 1 HNMR (CDCl 3 , 360 MHz) d(ppm) 9.01 (1H, d, J= 8.4 Hz, A ), 7.86–7.69 (3H, m, A ), 7.52–7.26 (3H, m, A ), 6.94 (1H, b s, NH), 5.32 (1H, 640 p , J= 9.2 Hz, H-2 o H-3 o H-4), 5.19 (1H, d, J 1,2 = 9.0 Hz, H-1), 5.09, 5.06 (2H, 2 p , J= 9.5, 9.8 Hz in each, H-2 and/o H-3, and/o 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 (A ), 82.4, 73.1, 72.7, 70.4, 67.9 (C-1 o C-5), 61.5 (C-6), 20.3 (CH 3 ). Anal. Calcd o 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. Tó h e al. / Ca bohyd a e Resea ch xxx (2013) xxx–xxx 7 CAR 6468 No. o Pages 10, Model 5G 2 May 2013 Please ci e his a icle in p ess as: Tó h, M.; e al. Ca bohyd . Res. (2013), h p://dx.doi.o g/10.1016/j.ca es.2013.04.025