Synthesis of new glycosyl biuret and urea derivatives as potential glycoenzyme inhibitors

Our reference: CAR 5203 P-authorquery-v7 AUTHOR QUERY FORM Journal: CAR Article Number: 5203 Please e-mail or fax your responses and any corrections to: E-mail: [email protected] Fax: +31 2048 52799 Dear Author, Any queries or remarks that have arisen during the processing of your manuscript are listed below and highlighted by flags in the proof. Please check your proof carefully and mark all corrections at the appropriate place in the proof (e.g., by using on-screen annotation in the PDF file) or compile them in a separate list. For correction or revision of any artwork, please consult http://www.elsevier.com/artworkinstructions. Articles in Special Issues: Please ensure that the words ‘this issue’ are added (in the list and text) to any references to other articles in this Special Issue. Uncited references: References that occur in the reference list but not in the text – please position each reference in the text or delete it from the list. Missing references: References listed below were noted in the text but are missing from the reference list – please make the list complete or remove the references from the text. Location in article Query / remark Please insert your reply or correction at the corresponding line in the proof No Queries Electronic file usage Sometimes we are unable to process the electronic file of your article and/or artwork. If this is the case, we have proceeded by: hScanning (parts of) your article hRekeying (parts of) your article hScanning the artwork Thank you for your assistance. RO OF Graphical abstract pp xxx–xxxSynthesis of new glycosyl biuret and urea derivatives as potential glycoenzyme inhibitors Nóra Felföldi, Marietta Tóth, Evangelia D. Chrysina, Maria-Despoina Charavgi, Kyra-Melinda Alexacou, László Somsák * O OR' R'O R'O OR' H NH N O H N O R O OR' R'O R'O OR' H NH N OO R R=Ph,(R'O) 4 - β - D -Glc p , (R'O) 4 -βD -Gal p , (R'O) 3 -βD -Xyl p R' = Ac, Bz, H The deprotected biuret derivatives showed moderate inhibitory effect against rabbit muscle glycogen phosphorylase band human salivary a -amylase. CAR 5203 No. of Pages 1, Model 5G 12 November 2009 ARTICLE IN PRESS 1 UNCORRECTED PROOF Synthesis of new glycosyl biuret and urea derivatives as potential glycoenzyme inhibitors Nóra Felföldi a , Marietta Tóth a , Evangelia D. Chrysina b , Maria-Despoina Charavgi b , Kyra-Melinda Alexacou b , László Somsák a,* a Department of Organic Chemistry, University of Debrecen, POB 20, H-4010 Debrecen, Hungary b Institute of Organic and Pharmaceutical Chemistry, The National Hellenic Research Foundation, 48, Vas. Constantinou Ave. 116 35 Athens, Greece article info Article history: Received 14 July 2009 Received in revised form 11 October 2009 Accepted 20 October 2009 Available online xxxx Keywords: Glycosyl urea Glycosyl biuret 20 Inhibitor a -Amylase Glycogen phosphorylase abstract O-Peracetylated 1-(b-D-glucopyranosyl)-5-phenylbiuret was prepared in the reaction of O-peracetylated b-D-glucopyranosylisocyanate and phenylurea. The reaction of O-peracetylated N-b-D-glucopyranosylurea with phenylisocyanate furnished the corresponding 1-(b-D-glucopyranosyl)-3,5-diphenylas well as 3-(b-D-glucopyranosyl)-1,5-diphenyl biurets besides 1-(b-D-glucopyranosyl)-3-phenylurea. O-Peracetylated 1-(b-D-glucopyranosyl)-5-(b-D-glycopyranosyl)biurets were obtained in one-pot reactions of O-peracetylated b-D-glucopyranosylamine with OCNCOCl followed by a second glycopyranosylamine of b-D-gluco,b-D-galacto and b-D-xylo configurations. O-Acyl protected 1-(b-D-glucopyranosyl)-3-(b-D-glycopyranosylcarbonyl)ureas were obtained from the reaction of b-D-glucopyranosylisocyanate with C-(glycopyranosyl)formamides of b-D-gluco and b-D-galacto configurations. The O-acyl protecting groups were removed under acidor base-catalyzed transesterification conditions, except for the N-acylurea derivatives where the cleavage of the N-acyl groups was faster than deprotection. Some of the new compounds exhibited moderate inhibition against rabbit muscle glycogen phosphorylase band human salivary a -amylase. Ó2009 Elsevier Ltd. All rights reserved. 1. Introduction Carbohydrate processing enzymes (glycoenzymes) catalyze the assembly and degradation of vital oligoand polysaccharides. Discovery of inhibitors of glycoenzymes and revealing their structure–activity relationship (SAR) is a principal trend in the development of carbohydrate-based drugs. 1 During our previous research several inhibitors of glycosidase 2–5 and glycogen phosphorylase 6,7 (GP) enzymes were synthesized and characterized. The first nanomolar glucose-based inhibitor of rabbit muscle GPb(RMGPb) was 50 identified among N-acyl-N 0 -bD -glucopyranosyl ureas B(for selected examples see Table 1, entries 5–7). The strong binding of the 2-naphthyl derivative (entry 7) was attributed to its extensive interactions upon binding with the residues lining the so-called b-pocket of the catalytic channel of the enzyme. 8 GP’s b-pocket is located next to the catalytic site of the enzyme in the direction of the b-anomeric substituent of bound D -glucose derivatives surrounded by both polar and apolar amino acid side chains. 9 In the native RMGPb, this site is occupied by water molecules the positions of which give insights for the design of new glucose 60 analogues with substituents that would optimize the network of interactions with the residues in close vicinity. In order to track down the nature of interactions in the b-pocket, and the role of the linker between the sugar and the aromatic part of the molecule some N-aryl-N 0 -bD -glucopyranosyl ureas A(for selected examples see Table 1, entries 1–3) have been investigated so far. Derivatives Aexhibited weaker binding to RMGPbin comparison to B. To study the effect of a longer linker of similar composition, synthesis of biuret derivatives Cwas envisaged. As the series of compounds B investigated so far contained mainly apolar residues 7 (R = e.g., 70 methyl, cyclohexyl, (substituted)phenyl and naphthyl), an effort to exploit polar interactions in the b-pocket by substituting sugar rings for R in both Band Cwas also planned. O OH HO HO OH H NH NR O A O OH HO HO OH H NH N O O R B O OH HO HO OH H NH N O O H NR C 0008-6215/$ - see front matter Ó2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2009.10.016 *Corresponding author. Tel.: +36 52512900x22348; fax: +36 52453836. E-mail address: [email protected] (L. Somsák). Carbohydrate Research xxx (2009) xxx–xxx Contents lists available at ScienceDirect Carbohydrate Research journal homepage: www.elsevier.com/locate/carres CAR 5203 No. of Pages 7, Model 5G 12 November 2009 ARTICLE IN PRESS Please cite this article in press as: Felföldi, N.; et al. Carbohydr. Res. (2009), doi:10.1016/j.carres.2009.10.016 UNCORRECTED PROOF 2. Results and discussion The synthesis of the protected 1-bD -glucopyranosyl-5-phenyl biuret 4was achieved by a reaction of phenylurea with bD -gluco80 pyranosylisocyanate 3 10 generated in situ from glucosylamine 2 11 obtained by catalytic reduction of glucosylazide 1 12 (Scheme 1). Compound 4was isolated by crystallization from MeOH, and deprotection was effected by acid-catalyzed transesterification to give 5. We have also attempted to produce 4in a somewhat shorter way from bD -glucosyl urea 6 13 and PhNCO. In refluxing EtOAc no reaction occurred between 6and 1.5 equiv of PhNCO. Using the same ratio of the reagents in boiling toluene allowed isolation of 1-bD -glucosyl-3-phenyl urea 7 10 in 41% yield. Performing the reaction in neat, boiling PhNCO gave compound 7as well as biuret 90 derivatives 8and 9in 4%, 24% and 31% isolated yields, respectively. This unexpected result could be explained by the presence of water in the reaction mixture. When 6was reacted with 1 equiv of PhNCO in refluxing toluene in the presence of 1 equiv of H 2 O, the formation of 7could be observed. Urea 7was also formed when 6and 1 equiv of PhNH 2 were reacted in boiling toluene. Finally, heating of 7in neat PhNCO gave biuret 9. All attempts to avoid the formation of these products by carefully drying the solvents and reactants as well as the use of molecular sieves in the reaction mixtures failed. Structural elucidation of the new biurets 8and 9 100 was straightforward by MS and NMR measurements as follows from the selected characteristic data shown in Scheme 1. Coupling of a glycosylurea and a glycosylisocyanate could give 1,5-bis-glycosyl biuret derivatives. However, the use of commercial OCNCOCl as a bielectrophilic reagent 14,15 ,and glycosylamines as nucleophiles offered a simpler and shorter synthetic pathway towards such target compounds. Thus, reaction of glucopyranosylamine 2 11 with 0.5 equiv of OCNCOCl gave cleanly the expected 1,5-bis-glucosyl biuret 12 (Scheme 2). Asymmetric derivatives could also be obtained in two-step, one-pot reactions from 2by 110 the addition of 1 equiv of OCNCOCl followed by a second glycosylamine 10 16 or 11 17,18 to give 14 or 16, respectively. Deacetylation was performed by the Zemplén protocol to result in high yields of biurets 13,15 and 17. To obtain N-acyl-N 0 -bD -glucopyranosyl ureas with a sugar part in the acyl group the reaction of isocyanate 3 10 with O-peracetylated anhydro-aldonamide 18 19 was investigated first (Scheme 3). When 3and 18 were reacted in refluxing EtOAc in equimolar amounts the conversion of 18 was 25%, and the expected acylurea 20 could be isolated in 32% yield. Raising the temperature to the 120 boiling point of toluene gave a 56% conversion of 18 and 50% isolated yield for 20. A satisfactory result was achieved by applying 3in a twofold excess for a full conversion of 18, and the yield of 20 increased to 89%. From a reaction of 3and O-perbenzoylated anhydro-aldonamide 19 20 (molar ratio 2:1) in boiling toluene 23 was obtained in a 98% yield. In each of the above reactions bis-glucopyranosyl urea 21 was also isolated in various amounts which could be due to the presence of traces of water in the mixtures. 21 Attempted deprotection of acyl ureas 20 and 23 was successful under neither basic nor acidic transesterification conditions because 130 cleavage of the N-acyl moiety was faster than removal of the Oacyl-protecting groups. Bis-glucopyranosyl urea 21 was deprotected under Zemplén conditions to give 22 22 in satisfactory yield. The deprotected compounds were tested for their potency to inhibit rabbit muscle glycogen phosphorylase bactivity according to the protocol described earlier, 23,24 and the results are summarized in Table 1. Compounds with two sugar moieties attached to the terminal nitrogens of either urea 22 (entry 4) or biurets 13,15 and 17 (entries 9–11) showed very low inhibition of the enzyme activity. Comparison with the inhibition shown by the phenylbi140 uret derivative 5(entry 8) this may reveal that the highly polar sugar residues opposite to the bD -glucopyranosyl part of the compounds are unfavourable for the binding. Among biurets the bD -xylopyranosyl derivative 17 proved the most efficient, and this may be in accord with the less polar character of this residue with three OH groups compared to four ones in the bD -glucoand galactopyranosyl parts of 13 and 15, respectively. A comparison of the phenyl substituted derivatives (entries 1, 5and 8) allows to conclude that the acyl urea linker is superior to the urea and the biuret type ones. 150 Compounds 13 and 22 were also tested against human salivary a -amylase according to the method reported earlier, 3 and exhibited inhibition in the low millimolar range (IC 50 10.7 and 8.3 mM, respectively). In conclusion, synthesis of 1-(bD -glucopyranosyl) biurets with an aromatic and several bD -glycopyranosyl residues in the 5-position allowed to extend structure–activity relationships of glucose analogue inhibitors of glycogen phosphorylase. Introduction of the highly polar sugar moieties resulted in weak binding. The length of the linker composed of NHCO elements between the b160 D -glucopyranosyl and the aromatic parts of the inhibitors proved to be optimal in the acyl urea series. Table 1 Inhibition of rabbit muscle glycogen phosphorylase b(RMGPb) by selected glucose derivatives and the new compounds O OH HO HO OH H NH NR O A O OH HO HO OH H NH N O R O B Entry Inhibition ( l M) R Entry Inhibition (K i ( l M)) 1K i 18 7 5 4.6 8 2IC 50 350 7 6 15.2 6,7 3K i 5.2 7 7 0.35 6,7 4IC 50 1209 ± 8 K ia 725 ± 5 O OH HO HO OH 22 O OH HO HO OH H NH N O H N O R C Inhibition IC 50 ( l M) K ia ( l M) 8 5 41.6 ± 4 20.8 ± 2 9 O OH HO HO OH 13 37% at 1 mM 10 O OH HO HO OH 15 32% at 1 mM 11 O OH HO HO 17 987 ± 127 592 ± 76 a K i values were calculated for comparison purposes by the Cheng–Prusoff equation: 25 K i =IC 50 /(1 + [S]/K m ). 2N. Felföldi et al. / Carbohydrate Research xxx (2009) xxx–xxx CAR 5203 No. of Pages 7, Model 5G 12 November 2009 ARTICLE IN PRESS Please cite this article in press as: Felföldi, N.; et al. Carbohydr. Res. (2009), doi:10.1016/j.carres.2009.10.016 UNCORRECTED PROOF 3. Experimental 3.1. General methods 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 rt. 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) or Avance DRX 500 (500/125 MHz for 1 H/ 13 C) spectrometers. Chemical shifts are referenced to inter170 nal TMS ( 1 H), or to the residual solvent signals ( 13 C). 1 HNMR assignments were established on the basis of gradient enhanced DQF-COSY spectra. 26 Proton chemical shifts and scalar coupling constants were extracted from the resolution enhanced 1D proton spectra. COSY spectra were recorded with 512 2 k data points, spectral widths 4000 Hz, number of transients 4 and recycle delay of 1.8 s. Microanalyses were performed on a Carlo-Erba analyser Type 1106. ESIMS were recorded with a Bruker micrOTOF-Q instrument. TLC was performed on DC-Alurolle Kieselgel 60 F 254 (Merck), and the plates were visualized under UV light and by gentle heat180 ing. For column chromatography Kieselgel 60 (Merck, particle size 0.063–0.200 mm) was used. Flasks were flame-dried before performing the reactions. Organic solutions were dried over anhydrous MgSO 4 ,and concentrated under diminished pressure at 40–50 °C (water bath). 3.2. 1-(2,3,4,6-Tetra-O-acetyl-bD -glucopyranosyl)-5-phenyl biuret (4) 2,3,4,6-Tetra-O-acetyl-bD -glucopyranosylisocyanate (3) prepared in situ by Ichikawa’s method 10 from glucosylamine 2 11 (0.6 g, 1.73 mmol) was dissolved in toluene (12 mL) and then trea190 ted with phenyl urea (0.47 g, 3.46 mmol). The mixture was refluxed and monitored by TLC (2:1 EtOAc–hexane). When the reaction was complete, the solvent was evaporated, and the residue was crystallized from MeOH to give 0.37 g (42%) of 4.Mp: 207–209 °C; [ a ] D 23 (c1.68, acetone); 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 9.42 (s, 1H, NH), 7.44–7.12 (m, 6H, Ar, NH), 5.33 (t, 1H, 3 J 3,4 = 10.0 Hz, H-3), 5.23 (t, 1H, H-1), 5.10 (t, 1H, 3 J 4,5 = 9.5 Hz, H-4), 4.98 (t, 1H, 3 J 2,3 = 9.2 Hz, H-2), 4.33 (dd, 1H, 2 J 6,6 0 = 12.6 Hz, H-6), 4.13 (dd, 1H, 3 J 5,6 0 = 2.1 Hz, H-6 0 ), 3.87 (ddd, 1H, 3 J 5,6 = 5.0 Hz, H-5), 2.44 (s, 1H, NH), 2.10, 2.08, 2.05 (s, 12H, 200 4OCOCH 3 ). 13 C NMR (CDCl 3 , 90 MHz) d(ppm) 170.7, 170.4, 170.0, 169.5 (CO), 155.0, 152.3 (NHCONH), 136.8, 129.4, 129.1, 124.4, 120.5 (Ar), 78.9 (C-1), 73.3, 72.9, 70.0, 68.0 (C-2–C-5), 61.7 (C-6), 20.7, 20.6, 20.56, 20.5 (CH 3 ). ESIMS: [M+Na] + calcd 532.46, O OAc H NH N O O OAc NH N O O OAc H NN O Ph Ph Ph OH NPh OH NPh O OAc AcO AcO OAc N3O OR RO RO OR H NH N O O OAc NH2O OAc NCO H NNH2 O H N O Ph 608.56 10.24 (s); 6.44 (d, 3 J = 8.8 Hz) 5.11 (pseudo t, 3 J = 9.6 Hz) 155.8; 152.1 608.56 10.73 (s); 6.32 (d, 3 J = 10.0 Hz) 151.8; 151.1 489.14 7.69 (s); 6.24 (d, 3 J = 9.2 Hz) 5.28 (pseudo t, 3 J = 9.2 Hz) 154.6 [M+Na]+ NH (δ, ppm) H-1 (δ, ppm) C=O (δ, ppm) 8.75 (s) ++ 4321 R = Ac (42%) 5 R = H (91%) 76 (4%) 8 (24%) 9 (31%) abc e d f O OAc AcO AcO OAc Scheme 1. Reagents and conditions: (a) H 2 , Raney-Ni, EtOAc, rt; (b) (Cl 3 CO) 2 CO, NaHCO 3 ,CH 2 Cl 2 ,H 2 O, rt; (c) PhNHCONH 2 , toluene, reflux; (d) AcCl, CHCl 3 –MeOH, rt; (e) PPh 3 , EtOAc, NH 3 ,CO 2 , rt; and (f) neat PhNCO, reflux. O OAc AcO AcO OAc NH 2 O R R R RH NH N O H N O O R R R 3 R 1 R 2 NH 2 1 . a to 12 b to 14 and 16 2. c to 14 and 16 2 10-11 12-17 OR 3 R R 2 R 1 R R R1 R 2 R 3 Yield 12 OAc CH2OAc H OAc 91% d 13 OH CH2OH H OH 96% 10, 14 OAc CH2OAc OAc H 83% d 15 OH CH2OH OH H 98% 11, 16 OAc H H OAc 86% d 17 OH H H OH 96% Scheme 2. Reagents and conditions: (a) OCNCOCl, Et 3 N, dry THF, N 2 atm, rt; (b) OCNCOCl, dry THF, N 2 atm, 26 °C; (c) 10 or 11,Et 3 N, dry THF, N 2 atm, 0–25 °C; and (d) cat. NaOMe, abs MeOH, rt. N. Felföldi et al. / Carbohydrate Research xxx (2009) xxx–xxx 3 CAR 5203 No. of Pages 7, Model 5G 12 November 2009 ARTICLE IN PRESS Please cite this article in press as: Felföldi, N.; et al. Carbohydr. Res. (2009), doi:10.1016/j.carres.2009.10.016 UNCORRECTED PROOF found: 532.16. Anal. Calcd for C 22 H 27 N 3 O 11 (509.47): C, 51.87; H, 5.22; N, 7.51. Found: C, 51.97; H, 5.24; N, 7.50. 3.3. 1-(bD -Glucopyranosyl)-5-phenyl biuret (5) Biuret 4(250 mg, 0.49 mmol) was dissolved in a mixture of MeOH and CHCl 3 (1:1). A catalytic amount of AcCl was added and the mixture was stirred at rt. The reaction was monitored by 210 TLC (1:1 CHCl 3 –MeOH). When the reaction was complete, it was neutralized with solid NaHCO 3 ,and then filtered and the solvent was evaporated. The residue was purified by column chromatography (9:1 CHCl 3 –MeOH) to give 162 mg (97%) of 5as a white powder. Mp: 191–193 °C; [ a ] D +4 (c0.68, MeOH);. 1 H NMR (DMSOd 6 +D 2 O, 360 MHz) d(ppm) 7.41 (d, 2H, Ar), 7.29 (t, 2H, Ar), 7.04 (t, 1H, Ar), 4.68 (d, 1H, 3 J 1,2 = 8.9 Hz, H-1), 3.63 (dd, 1H, 3 J 5,6 0 = 1.6 Hz, H-6 0 ), 3.42 (dd, 1H, 2 J 6,6 0 = 12.1 Hz, H-6), 3.19–3.14 (m, 1H, 3 J 5,6 = 5.8 Hz, H-5), 3.22, 4.26, 4.25 (t, 1H, 3 J 2,3 = 3 J 3,4 = 3 J 4,5 = 8.9 Hz, H-2,3,4). 13 C NMR (DMSO-d 6 , 90 MHz) d(ppm) 154.6, 152.2 220 (NHCONH), 138.2, 128.9, 123.1, 119.0 (Ar), 80.3 (C-1), 78.5, 77.3, 72.9, 69.8 (C-2–C-5), 60.8 (C-6). Anal. Calcd for C 14 H 19 N 3 O 7 (341.32): C, 49.27; H, 5.61; N, 12.31. Found: C, 49.35; H, 5.66; N, 12.30. 3.4. Reaction of 2,3,4,6-tetra-O-acetyl-bD -glucopyranosyl urea 13 (6) with phenylisocyanate Urea 6(100 mg, 0.26 mmol) was refluxed in neat phenylisocyanate(1 mL). The reaction was monitored by TLC (2:1 EtOAc–hexane). When the reaction was complete, the excess amount of phenylisocyanate was removed by diluting the mixture with hex230 ane. The formed precipitate was filtered, dissolved in CH 2 Cl 2 and washed with satd aq NaHCO 3 . The organic layer was separated, dried and the solvent was evaporated. The residue was separated by column chromatography (50:1 CH 2 Cl 2 –acetone) to give, in the order of elution, compounds 8,9and 7 10 (4%). 3.4.1. 3-(2,3,4,6-Tetra-O-acetyl-bD -glucopyranosyl)-1,5diphenyl biuret (8) Yield: 36 mg (24%), colourless syrup. R f = 0.89 (25:1 CHCl 3 –acetone) [ a ] D +0.2 (c1.71, DMSO). 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 10.73 (s, 1H, N(CONHC 6 H 5 ) 2 ), 8.75 (s, 1H, N(CONHC 6 H 5 ) 2 ), 7.51– 240 7.09 (m, 10H, Ar), 6.32 (d, 1H, 3 J 1,2 = 10.0 Hz, H-1), 5.66 (t, 1H, 3 J 3,4 = 9.5 Hz, H-3), 5.39 (t, 1H, 3 J 4,5 = 9.5 Hz, H-4), 5.16 (t, 1H, 3 J 2,3 = 10.0 Hz, H-2), 4.52 (dd, 1H, 2 J 6,6 0 = 12.6 Hz, H-6), 4.18 (dd, 1H, 3 J 5,6 0 = 2.1 Hz, H-6 0 ), 4.05 (ddd, 1H, 3 J 5,6 = 3.7 Hz, H-5), 2.13, 2.06, 2.04, 2.00 (s, 12H, 4OCOCH 3 ). 13 C NMR (CDCl 3 + DMSO-d 6 , 90 MHz) d(ppm) 168.6, 167.9, 167.8, 167.7 (CO), 151.8, 151.1 (NCON), 138.3–116.0 (Ar), 79.0 (C-1), 73.1, 71.4, 66.3, 66.1 (C-2– C-5), 60.1 (C-6), 19.2, 19.1, 19.0, 18.9 (CH 3 ). ESIMS: [M+Na] + calcd for C 28 H 31 N 3 O 11 (585.57): 608.56, found: 608.19. 3.4.2. 1-(2,3,4,6-Tetra-O-acetyl-bD -glucopyranosyl)-3,5250 diphenyl biuret (9) Yield: 46 mg (31%), white powder. R f = 0.80 (25:1 CHCl 3 –acetone). Mp: 226–229 °C; [ a ] D 4(c0.64, DMSO). 1 H NMR (CDCl 3 , 360 MHz) d(ppm) 10.24 (s, 1H, NHCON(C 6 H 5 )CONHC 6 H 5 ), 7.56– 7.06 (m, 10H, Ar), 6.44 (t, 1H, 3 J H-1,NH = 8.8 Hz, NHCON(C 6 H 5 )CONHC 6 H 5 ), 5.28 (t, 1H, 3 J 3,4 = 9.6 Hz, H-3), 5.11 (t, 1H, 3 J 1,2 = 9.6 Hz, H1), 5.02 (t, 1H, 3 J 4,5 = 9.6 Hz, H-4), 4.78 (t, 1H, 3 J 2,3 = 9.6 Hz, H-2), 4.31 (dd, 1H, 2 J 6,6 0 = 12.3 Hz, H-6), 4.10 (dd, 1H, 3 J 5,6 0 = 1.8 Hz, H6 0 ), 3.81 (ddd, 1H, 3 J 5,6 = 4.4 Hz, H-5), 2.09, 2.024, 2.019, 1.98 (s, 12H, 4OCOCH 3 ). 13 C NMR (CDCl 3 , 90 MHz) d(ppm) 170.6, 260 170.1, 169.8, 169.4 (CO), 155.8, 152.1 (NCON), 137.4, 135.6, 130.3, 129.9, 129.4, 128.9, 124.1, 120.1 (Ar), 79.8 (C-1), 73.5, 72.5, 69.8, 67.9 (C-2–C-5), 61.5 (C-6), 20.7, 20.5 (CH 3 ). ESIMS: [M+Na] + calcd for C 28 H 31 N 3 O 11 (585.57): 608.56, found: 608.18. 3.5. 1,5-Bis-(2,3,4,6-tetra-O-acetyl-bD -glucopyranosyl)biuret (12) Glucosylamine 2 11 (400 mg, 1.15 mmol) was dissolved in dry THF (5 mL), then Et 3 N (80 l L, 0.58 mmol) and OCNCOCl (46 l L, 0.58 mmol) were added. The mixture was stirred at rt under nitrogen atmosphere. After the reaction was complete (TLC, 10:1 EtOAc–hexane) the mixture was diluted with water (5 mL), and 270 washed with EtOAc (3 5 mL). The organic phase was dried and the solvent was evaporated under reduced pressure to yield 402 mg (91%) colourless syrup. R f = 0.83 (10:1 EtOAc–hexane); [ a ] D 19 (c0.97, CHCl 3 ); 1 H NMR (DMSO-d 6 ): d(ppm) 9.11 (s, 1H, NH), 8.02 (d, 2H, J=9.5 Hz, 2 NH), 5.42, 5.34, 4.92, 4.86 (4 pseudo t, 8H, J=9.5, 9.6 Hz in each, 2 H-1, 2 H-2, 2 H-3, 2H-4), 4.16–3.94 (m, 6H, 2 H-5, 2 H-6, 2 H-6 0 ), 2.00, 1.99, 1.98, 1.95 (4s, 24H, 8 CY 3 ); 13 C NMR (CDCl 3 ): d(ppm) 170.6, 170.3, 169.9, 169.4 (COCH 3 ), 154.3 (2 NHCO), 78.7 (C-1), 73.1, 72.8, 70.0, 67.9 (C-2 to C-5), 61.5 (C-6), 20.6, 20.5 (CH 3 ). Anal. O OAc AcO AcO OAc NCO 3 O OAc AcO AcO OAc CONH 2 O OBz BzO BzO OBz CONH 2 O OAc AcO AcO OAc H N O O OAc AcO AcO OAc H N O 18 19 20 R = Ac (89%) PhCH 3 , Δ PhCH 3 , Δ O OR RO RO OR H NH N O 21 R = Ac 22 R = H (79%) + + NaOMe, MeOH, rt O RO OR OR RO O BzO OBz OBz BzO H N O H N O O AcO OAc AcO AcO 23 R=Ac,R'= Bz (98%) Scheme 3. 4N. Felföldi et al. / Carbohydrate Research xxx (2009) xxx–xxx CAR 5203 No. of Pages 7, Model 5G 12 November 2009 ARTICLE IN PRESS Please cite this article in press as: Felföldi, N.; et al. Carbohydr. Res. (2009), doi:10.1016/j.carres.2009.10.016 UNCORRECTED PROOF 280 Calcd for C 30 H 41 N 3 O 20 (763.65): C, 47.18; H, 5.41; N, 5.50. Found: C, 47.23; H, 5.50; N, 5.58. 3.6. General procedure I for the synthesis of 1-(2,3,4,6-tetra-Oacetyl-bD -glucopyranosyl)-5-(per-O-acetyl-bD - glycopyranosyl)biurets 14 and 16 Glucosylamine2 11 (100 mg,0.29 mmol)wasdissolvedindryTHF (2 mL), and some freshly heated molecular sieves were added. The mixture was cooled to 20 °C, OCNCOCl (23 l L, 0.29 mmol) was added, and stirred at 26 °C under nitrogen atmosphere for a day. Then a solution of 2,3,4,6-tetra-O-acetyl-bD -galactopyranosyl290 amine 16 (10, 100 mg, 0.29 mmol) or 2,3,5-tri-O-acetyl-bD -xylopyranosylamine 17,18 (11, 80 mg, 0.29 mmol) in dry THF (2 mL) and Et 3 N (40 l L, 0.29 mmol) were added, and the mixture was allowed to warm up to rt. When the reaction was complete (TLC, 10:1 EtOAc– hexane) the insoluble materials were filtered off with suction, and thesolventwasremovedunderreducedpressure.Thecrudeproduct was purified by column chromatography (7:1 EtOAc–hexane). 3.6.1. 1-(2,3,4,6-Tetra-O-acetyl-bD -galactopyranosyl)-5- (2,3,4,6-tetra-O-acetyl-bD -glucopyranosyl)biuret (14) Prepared according to General procedure I (Section 3.6) from 300 glucosylamine 2(100 mg, 0.29 mmol) and galactosylamine 10 (100 mg, 0.29 mmol). Yield: 183 mg (83%) colourless syrup. R f = 0.58 (10:1 EtOAc–hexane); [ a ] D 15 (c0.98, CHCl 3 ); 1 H NMR (CD 3 CN): d(ppm) 7.83 (br s, 1H, NH), 7.60–7.41 (m, 2H, 2 NH), 5.37 (pseudo d, 1H, J=3.0 Hz, H-4-Gal), 5.34 (pseudo t, J=9.6 Hz H-3-Glc), 5.23 (pseudo t, 1H, J=9.3, 9.4 Hz, H-1-Glc), 5.20–5.15 (m, 2H, H-1-Gal, H-3-Gal), 5.09 (pseudo t, 1H, J=9.3 Hz, H-2-Gal), 5.07–4.98 (m, 2H, H-2-Glc, H-4-Glc), 4.18 (dd, 1H, J=4.5, 12.4 Hz, H-6a-Glc), 4.13–4.09 (m, 1H, H-5-Gal), 4.09–4.00 (m, 3H, H-6aGal, H-6b-Gal, H-6b-Glc), 3.94 (m, 1H, H-5-Glc), 2.21, 2.11, 2.01, 310 1.99, 1.98, 1.96 (6br s, 24H, 8 CH 3 ); 13 C NMR (CDCl 3 ): d(ppm) 170.6, 170.3, 169.9, 169.4 (COCH 3 ), 154.3 (2 NHCO), 79.1, 78.9 (C-1-Glc, C-1-Gal), 73.2, 72.8, 71.9, 71.0, 70.1, 68.0, 67.8, 67.2 (C-2Glc to C-5-Glc, C-2-Gal to C-5-Gal), 61.6, 61.0 (C-6-Glc, C-6-Gal), 20.6, 20.5 (CH 3 ). Anal. Calcd for C 30 H 41 N 3 O 20 (763.65): C, 47.18; H, 5.41; N, 5.50. Found: C, 47.29; H, 5.54; N, 5.61. 3.6.2. 1-(2,3,4,6-Tetra-O-acetyl-bD -glucopyranosyl)-5-(2,3,5-triO-acetyl-bD -xylopyranosyl)biuret (16) Prepared according to General procedure I (Section 3.6) from glucosylamine 2(100 mg, 0.29 mmol) and xylosylamine 11 320 (80 mg, 0.29 mmol). Yield: 171 mg (86%) colourless syrup. R f = 0.58 (10:1 EtOAc–hexane); [ a ] D 28 (c0.58, CHCl 3 ); 1 H NMR (CD 3 CN): d(ppm) 7.75 (br s, 1H, NH), 7.49 (br s, 2H, 2 NH), 5.35 (pseudo t, 1H, J=9.4 Hz, H-3-Glc), 5.29 (pseudo t, 1H, J=9.1 Hz, H-3-Xyl), 5.23 (pseudo t, 1H, J=9.4 Hz, H-1-Glc), 5.13 (pseudo t, 1H, J=9.1 Hz, H-1-Xyl), 5.06–4.88 (m, 4 H, H-4-Glc, H2-Glc, H-2-Xyl, H-4-Xyl), 4.17 (dd, J=4.8, 12.4 Hz, H-6a-Glc), 4.04 (dd, 1H, J=2.0, 12.4 Hz, H-6b-Glc), 3.98 (dd, J=5.4, 11.5 Hz, H5a-Xyl), 3.93–3.88 (m, 1 H, H-5-Glc), 3.51–3.44 (ddd, 1H, J=1.3 Hz, 11.5 Hz, H-5b-Xyl), 2.18, 2.01, 2.00, 1.99, 1.98, 1.96 330 (5br s, 21 H, 7 CY 3 ); 13 C NMR (CDCl 3 ): d(ppm) 170.6, 170.3, 169.9, 169.8, 169.4 (COCH 3 ), 154.4 (2 NHCO), 79.0, 78.7 (C-1Glc, C-1-Xyl), 73.1, 72.8, 71.8, 70.0, 69.9, 68.6, 68.0 (C-2-Glc to C5-Glc, C-2-Xyl to C-4-Xyl), 63.7, 61.5 (C-6-Glc, C-5-Xyl), 20.5, 20.4 (CH 3 ). Anal. Calcd for C 27 H 37 N 3 O 18 (691.59): C, 46.89; H, 5.39; N, 6.08. Found: C, 46.99; H, 5.31; N, 6.15. 3.7. General procedure II for the removal of O-acyl protecting groups An O-peracetylated compound (100 mg) was dissolved in dry MeOH (1 mL), and a solution of NaOMe (1 M in MeOH) was added 340 to the solution in a catalytic amount. The reaction mixture was kept at rt. When the reaction was complete (TLC, 7:3 CHCl 3 –MeOH) the solution was neutralized with a cation exchange resin Amberlyst 15 (H + form). Filtration and removal of the solvent resulted in the corresponding deacetylated sugar derivatives. 3.7.1. 1,5-Bis-(bD -glucopyranosyl)biuret (13) Prepared according to General procedure II (Section 3.7) from biuret 12 (100 mg, 0.13 mmol). Yield: 54 mg (96%) colourless syrup. R f = 0.45 (1:3 CHCl 3 –methanol); [ a ] D 4(c0.51, MeOH); 1 H NMR (D 2 O): d(ppm) 4.91 (d, 2H, J=9.3 Hz, 2 H-1), 3.86 (dd, 350 2H, J=1.6, 12.3 Hz, 2 H-6a), 3.70 (dd, 2H, J=5.3, 12.3 Hz, 2H-6b), 3.52, 3.43, 3.40 (3 pseudo t, 6H, J=9.0, 9.3 Hz in each 2H-2, 2 H-3, 2 H-4), 3.51–3.47 (m, 2H, 2 H-5); 13 CNMR (Me 2 SO-d 6 ): d(ppm) 154.2 (NHCO), 80.3, 78.4, 77.3, 72.8, 69.7 (C-1-C-5), 60.8 (C-6). Anal. Calcd for C 14 H 25 N 3 O 12 (427.36): C, 39.35; H, 5.90; N, 9.83. Found: C, 39.46; H, 5.99; N, 9.90. 3.7.2. 1-(bD -Galactopyranosyl)-5-(bD -glucopyranosyl)biuret (15) Prepared according to General procedure II (Section 3.7) from biuret 14 (100 mg, 0.13 mmol). Yield: 53 mg (98%) colourless syr360 up. R f = 0.35 (1:3 CHCl 3 –MeOH); [ a ] D 7(c0.54, H 2 O); 1 HNMR (DMSO-d 6 ): d(ppm) 4.65–4.56 (m, 2H), 3.69–3.57 (m, 2H), 3.47– 3.00 (m, 9H), 2.95 (t, 1H); 13 C NMR (D 2 O): d(ppm) 159.6 (2 NHCO), 81.5 (C-1-Glc, C-1-Gal), 77.9, 77.2, 72.6, 70.0 (C-2Glc to C-5-Glc, C-2-Gal to C-5-Gal), 61.3 (C-6-Glc, C-6-Gal). Anal. Calcd for C 14 H 25 N 3 O 12 (427.36): C, 39.35; H, 5.90; N, 9.83. Found: C, 39.44; H, 5.98; N, 9.89. 3.7.3. 1-(bD -Glucopyranosyl)-5-(bD -xylopyranosyl)biuret (17) Prepared according to General procedure II (Section 3.7) from biuret 16 (100 mg, 0.14 mmol). Yield: 55 mg (96%) colourless syr370 up. R f = 0.63 (1:3 CHCl 3 –MeOH); [ a ] D 12 (c0.52, H 2 O); 1 HNMR (D 2 O): d(ppm) 4.86 (d, 1 H, J=9.3 Hz), 3.81 (dd, 1H, J=2.1, 12.6 Hz), 3.65 (dd, 1H, J=5.1 Hz, 12.6 Hz), 3.51–3.25 (m, 10 H); 13 C NMR (D 2 O): d(ppm) 156.3 (2 NHCO), 81.6, 80.9 (C-1-Glc, C-1-Xyl), 78.1, 77.1, 72.6, 72.4, 69.9, 69.7 (C-2-Glc to C-5-Glc, C2-Xyl to C-4-Xyl), 67.3, 61.2 (C-6-Glc, C-5-Xyl). Anal. Calcd for C 13 H 23 N 3 O 11 (397.34): C, 39.30; H, 5.83; N, 10.58. Found: C, 39.39; H, 5.90; N, 10.65. 3.8. 1-(2,3,4,6-Tetra-O-acetyl-bD -galactopyranosylcarbonyl)-3- (2,3,4,6-tetra-O-acetyl-bD -glucopyranosyl)urea (20) 380 C-(2,3,4,6-Tetra-O-acetyl-bD -galactopyranosyl)formamide 19 (18, 100 mg, 0.27 mmol) was dissolved in dry toluene (3 mL). Then some molecular sieves and crystalline isocyanate 3 10 (202 mg, 0.54 mmol) were added. The reaction was stirred at reflux temperature. After one day the reaction mixture was worked up: the molecular sieves were filtered off with suction and the solution was concentrated under reduced pressure. The residue was purified by column chromatography (100:1 CHCl 3 –MeOH). Two products were isolated: 20 (177 mg, 89%) and 21 21 (52 mg). Characterization of 20: colourless syrup; R f = 0.55 (5:1 EtOAc–hexane); 390 [ a ] D +18 (c0.84, CHCl 3 ); 1 H NMR (CD 3 CN): d(ppm) 8.66 (br s, 1H, NH), 8.64 (d, 1H, J=9.3 Hz, NH), 5.41 (pseudo d, J=2.2 Hz, H-4Gal), 5.35 (pseudo t, J=9.6 Hz, H-3-Glc), 5.28 (pseudo t J=9.3 Hz, H-1-Glc), 5.22–5.15 (m, 2H, H-2-Gal, H-3-Gal), 5.04, 5.03 (2 pseudo t, J=9.3, 9.6 Hz in both, H-4-Glc, H-2-Glc), 4.24–4.02 (m, 6 H, H-6aGlc, H-6b-Glc, H-1-Gal, H-5-Gal, H-6a-Gal, H-6b-Gal), 3.91 (ddd, 1H, J=2.3, 4.8, 9.9 Hz, H-5-Glc), 2.12, 2.03, 2.02, 2.01, 1.99, 1.98, 1.96, 1.93 (8br s, 24H,8 CH 3 ). 13 C NMR (CDCl 3 ): d(ppm) 170.6, 170.3, 170.1, 169.9, 169.7, 169.4 (COCH 3 ), 168.0, 152.1 (2 CONH), 78.8, 76.7 (C-1-Glc, C-1-Gal), 74.9, 73.5, 73.0, 70.6, 69.7, 68.2, 67.0, 400 65.9 (C-2-Glc to C-5-Glac, C-2-Gal to C-5-Gal), 61.7, 61.4 (C-6-Glc, N. Felföldi et al. / Carbohydrate Research xxx (2009) xxx–xxx 5 CAR 5203 No. of Pages 7, Model 5G 12 November 2009 ARTICLE IN PRESS Please cite this article in press as: Felföldi, N.; et al. Carbohydr. Res. (2009), doi:10.1016/j.carres.2009.10.016 UNCORRECTED PROOF C-6-Gal), 20.7, 20.6, 20.5 (CH 3 ). Anal. Calcd for C 30 H 40 N 2 O 20 (748.64): C, 48.13; H, 5.39; N, 3.74. Found: C, 48.20; H, 5.43; N, 3.79. 3.9. 1,3-Bis-(bD -glucopyranosyl)urea (22) Prepared according to General procedure II (Section 3.7) from urea 21 (190 mg, 0.26 mmol). Yield 80 mg (79%) amorphous solid. Lit. 27 Mp: 207 °C (dec.); R f = 0.45 (1:3 CHCl 3 -methanol); [ a ] D +23 (c 0.59, DMSO), lit. 27 [ a ] D 32.8 (c2, water); 1 H NMR (D 2 O): d(ppm) 4.86 (d, 1H, J=9.3 Hz, H-1), 3.86 (dd, J=1.5, 12.3 Hz, H-6a), 3.69 410 (dd, 1H, J=5.2, 12.3 Hz, H-6b), 3.53, 3.38, 3.37 (3 pseudo t, 3H, J=9.2, 9.7 Hz in each, H-2, H-3 ,H-4), 3.51–3.48 (m, 1H, H-5). 13 C NMR (D 2 O): d(ppm) 159.6 (CO), 81.5 (C-1), 77.9, 77.2, 72.6, 70.0 (C-2 to C-5), 61.3 (C-6). 3.10. 1-(2,3,4,6-Tetra-O-acetyl-bD -glucopyranosyl)-3-(2,3,4,6tetra-O-benzoyl-bD -glucopyranosylcarbonyl)-urea (23) C-(2,3,4,6-Tetra-O-benzoyl-bD -glucopyranosyl)formamide 20 (19, 54 mg, 0.086 mmol) was dissolved in dry toluene (1 mL), and some molecular sieves were added followed by crystalline isocyanate 3 10 (64 mg, 0.172 mmol) mmol). The reaction mixture was 420 heated to reflux temperature. When the reaction was complete (TLC, 5:1 EtOAc–hexane) the molecular sieves were filtered off with suction and the residue was concentrated under reduced pressure. The crude product was purified by column chromatography (1:1 EtOAc–hexane). Two products were isolated: 23 (84 mg, 98%) and 21 21 (23 mg). Characterization of 23: colourless syrup; R f = 0.53 (1:1 EtOAc–hexane); [ a ] D 6(c0.61, CHCl 3 ); 1 H NMR (CD 3 CN): d(ppm) 9.01 (br s, 1H, NH), 8.56 (d, 1H, J=9.2 Hz, NH), 8.05, 7.94, 7.90, 7.78 (4d, 8H, Ar), 7.63–7.32 (m, 12H, Ar), 6.02 (pseudo t, 1H, J=9.4 Hz, H-3-GlcBz), 5.77 (pseudo t, 1H, 430 J=10.1 Hz, H-4-GlcBz), 5.75 (pseudo t, 1H, J=9.8 Hz, H-2-GlcBz), 5.38 (pseudo t, 1H, J=9.6 Hz, H-3-GlcAc), 5.34 (pseudo t, 1H, J=9.4 Hz, H-1-GlcAc), 5.00, 4.99 (2 pseudo t, 2H, J=9.4, 9.8 Hz in both, H-4-GlcAc, H-2-GlcAc), 4.65 (dd, 1H, J=2.3, 12.3 Hz, H-6aGlcBz), 4.57 (dd, 1H, J=4.6, 12.3 Hz, H-6b-GlcBz), 4.54 (pseudo t, 1H, J=9.9 Hz, H-1-GlcBz), 4.43–4.39 (m, 1H, H-5-GlcBz), 4.14 (dd, 1H, J=4.8, 12.3 Hz, H-6a-GlcAc), 4.01 (dd, 1H, J=1.7, 12.3 Hz, H-6b-GlcAc), 3.94 (ddd, 1H, J=1.9, 4.4, 9.9 Hz), 2.19, 1.96, 1.94 (3br s, 12H, 4CH 3 ). 13 C NMR (CDCl 3 ): d(ppm) 170.5, 170.0, 169.5, 169.3, 166.0, 165.5, 165.2, 165.1 (COCH 3 ), 168.2, 440 152.6 (2 CONH), 133.6, 133.3, 133.2, 129.8, 129.7, 129.6, 128.2, 128.3 (CH–Ar), 129.1, 128.7 (C–Ar), 78.4, 76.6 (2 C-1), 76.1, 73.1, 72.8, 69.8, 69.6, 68.8, 68.1(2 C-2 to C-5), 62.8, 61.6 (2 C6), 20.5, 20.4 (CH 3 ). 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J.; Barofsky, D. F. Carbohydr. Res. 1989,189, 103–112. 6N. Felföldi et al. / Carbohydrate Research xxx (2009) xxx–xxx CAR 5203 No. of Pages 7, Model 5G 12 November 2009 ARTICLE IN PRESS Please cite this article in press as: Felföldi, N.; et al. Carbohydr. Res. (2009), doi:10.1016/j.carres.2009.10.016