Synthesis of tri- and tetramines containing two 2,3-dihydroxypyrrolidine moieties and their inhibitory activity toward α-mannosidases

Issue in Hono o P o . J. M. Muchowski ARKIVOC 2002 (xii) 23-37 ISSN 1424-6376 Page 23 ©ARKAT USA, Inc Syn hesis o i- and e amines con aining wo 2,3-dihyd oxypy olidine moie ies and hei inhibi o y ac i i y owa d α-mannosidases Sand ine Ge be -Lemai e,*a Flo ence Popowycz, a Eliaza Rod iguez-Ga cía, a Ca he ine Schü z, a Ana T. Ca mona Asenjo, b Inmaculada Robina, b and Pie e Vogel a a Ins i u de chimie moléculai e e biologique, Ecole Poly echnique Fédé ale de Lausanne, BCH, CH-1015 Lausanne, Swi ze land and b Depa amen o de Química O gánica, Facul ad de Química, Uni e sidad de Se illa, E-41071 Se illa, Spain E-mail: [email p o ec ed] Dedica ed o P o esso Jose Muchowski on he occasion o his 65 h bi hday ( ecei ed 20 Feb 03; accep ed 08 Ap 03; published on he web 16 Ap 03) Abs ac Th ough he educ i e amina ion o N-[( e -bu oxy)ca bonyl]-2,5-dideoxy-2,5-imino-3,4-O- isop opylidene-L- ibose wi h e ame hylenediamine, hexame hylenediamine, 2,7- diamino luo ene, 4,4'-diaminodiphenylme hane and 1,4-(diaminome hyl)benzene, i e e amines con aining wo (2R,3R,4S)-2-aminome hylpy olidine-3,4-diol moie ies ha e been p epa ed and assayed o hei inhibi o y ac i i ies owa d 24 glycosidases. Te amines con aining he e ame hylene o benzene-1,4-dime hylene linke s a e mo e po en α- mannosidase inhibi o s han simple (2R,3R,4S)-2-aminome hylpy olidine-3,4-diols. T iamines such as (2S,3R,4S)-bis(3,4-dihyd oxy-py olidin-2-e hyl)amine we e also p epa ed and shown o be be e α-mannosidase inhibi o s han (2S,3R,4S)-2-(2-aminoe hyl)py olidin-3,4-diol. Keywo ds: α-Mannosidase inhibi o s, polyamines con aining hyd oxyla ed py olidines, educ i e amina ion In oduc ion Cell sociology in ol es a language based on molecula ecogni ion be ween cell-su ace ca bohyd a es and p o eins.1 The biosyn hesis o he su ace oligosaccha ides uses glycosyl ans e ases and glycosidases as ca alys s. Inhibi o s o hese enzymes2 a e impo an molecula ools o glycobiology, and can be used o modula e cellula unc ions. They a e also po en ial d ugs in new he apeu ic s a egies.3 Among he mos po en glycosidase inhibi o s a e polyhyd oxypipe idines (1,5-dideoxy-1,5-iminoaldi ols) ha a e mimics o he glycosyl ca ion Issue in Hono o P o . J. M. Muchowski ARKIVOC 2002 (xii) 23-37 ISSN 1424-6376 Page 24 ©ARKAT USA, Inc in e media es libe a ed du ing enzyme-ca alyzed hyd oly ic p ocesses.4,5 De i a i es o 3,4- dihyd oxypy olidines (1,4-dideoxy-1,4-iminoaldi ols) also eme ge as an impo an class o glycosidase4a,5,6 and glycosyl ans e ase7 inhibi o s. Simple meso-3,4-dihyd oxypy olidine 1 is a non-selec i e, weak inhibi o o se e al glycosidases (Figu e 1).8 We ha e ound ha de i a i es 2b wi h (2R)-aminome hyl side chains can be highly selec i e and compe i i e inhibi o s o α- mannosidases, especially o A = phenyl, hiophenyl.8 N HO OH H N HO OH H NHR N HO OH HO 12a R = H 2b R = CH2A 3 H Figu e 1. Inhibi o s o glycosidases and glycosyl ans e ases. Clinical ials ha e shown ha swainsonine 3, a na u al α-mannosidase inhibi o ha con ains a 4-amino-4-deoxy-manno u anoside moie y,9,10 educes solid umo s and hema ological malignancies.11 Analogues o 3 ha e also shown in e es ing p ope ies.12 Mannosidase inhibi o s media e inc eased sec e ion o mu an α1-an i ypsin Z. They a e hus leads in he de elopmen o d ugs o he chemop ophylaxis o li e inju y and emphysema in pa ien s wi h α1-an i ypsin Z de iciency.13 Mannos a in A and B isola ed om he soil mic oo ganism S ep o e icillum e icillus14 and a syn he ic analogue15 a e p obably he mos po en inhibi o s o α- mannosidases epo ed so a .16 O en α-mannosidase inhibi o s ha a e monosaccha ide mimics4a,17 also inhibi o he ypes o glycosidases,18 in pa icula α-L- ucosidases.4a,19 To become a d ug, a good inhibi o mus sa is y a numbe o condi ions apa om i s low oxici y and enzyme speci ici y.20 We ha e en isioned ha polyamines con aining wo (2R,3R,4S)-2- (aminome hyl)-3,4-dihyd oxypy olidine agmen s could be al e na i e α-mannosidase inhibi o s wi h imp o ed pha macological p ope ies. We epo he e he syn hesis o i e e amines 4 (Figu e 2). We ha e also p epa ed iamine 5 ha con ains wo (2S,3R,4S)-2-(1- aminoe h-2-yl)-3,4-dihyd oxypy olidine moie ies, as well as i s enan iome en -5. These new compounds ha e been assayed o hei inhibi o y ac i i y owa d 24 comme cially a ailable glycosidases, and in pa icula owa d α-mannosidase om jack bean, an enzyme known o be a use ul model o mammalian α-mannosidases such as Golgi α-mannosidase II.21 Whe eas iamine en -5 does no inhibi any o he enzyme es ed (excep o a poo 38% inhibi ion o β- glucosidase om almond a 1 mM concen a ion), i s enan iome 5 is a mode a e inhibi o o α- mannosidase om jack bean (Ki = 74 µM) and om almond (Ki = 92 µM). Among he i e e amines 4, bes inhibi o y ac i i ies owa d hese enzymes we e ound wi h 4a and 4e. Bu con a y o inhibi o s o ype 2b, hese polyamines a e less enzyme selec i e. Issue in Hono o P o . J. M. Muchowski ARKIVOC 2002 (xii) 23-37 ISSN 1424-6376 Page 25 ©ARKAT USA, Inc N HO OH N H H AN HN OHHO H a: A = (CH 2 ) 4 b: A = (CH 2 ) 6 c: A = d: A = e: A = N HO OH H N H N HO OH H 45 N HO OH H N H N HO OH H en -5 Figu e 2. T i- and e amines con aining wo 2,3-dihyd oxypy olidine moie ies. Resul s and Discussion Syn hesis o he polyamines Te amines 4 we e all p epa ed om aldehyde 68 by eac ion wi h he co esponding diamine H2N-A-NH2 (1.8 equi alen ) in he p esence o NaBH(OAc)322 o in si u educ ion o he esul ing diimine in e media e (Scheme 1). NCHO OO Boc + H2N-A-NH2Cl(CH2)2Cl NaBH(OAc)3, 50°C N N Boc H AN HN Boc OO OO 6 7(a-e) CF3COOH H2O, 20°C 4(a-e), 50 o 90% yield Scheme 1. Syn hesis o e amines 4. Issue in Hono o P o . J. M. Muchowski ARKIVOC 2002 (xii) 23-37 ISSN 1424-6376 Page 26 ©ARKAT USA, Inc The so- o med semi-p o ec ed e amines we e ea ed wi h aqueous CF3COOH, a oom empe a u e, o clea e he Boc and ace onide moie ies. O e all yields based on 6 anged om 50 o 90%. T iamines 5 and en -5 we e de i ed om aldehydes 8 and en -8, hemsel es de i ed om L- and D-a abinose, espec i ely23,24 (Scheme 2). T ea men o a 1:1.1 mix u e o 8 and benzylamine wi h NaBH(OAc)3 in 1,2-dichlo oe hane esul ed in he o ma ion o 9 and 10 wi h 46% and 18% yield, espec i ely. Using a hal equi alen o benzylamine, 10 was ob ained in 55% yield. Hyd ogenolysis o he benzyl g oup (10% Pd / cha coal, THF/MeOH) ga e 11 in 98% yield. Dep o ec ion unde acidic condi ions p o ided 5 in almos quan i a i e yield. The same eac ions we e applied o en -8 p o iding en -(9–13). Compound en -8 was ob ained om known 1424 a e Boc-p o ec ion and educ ion wi h DIBAL-H. N OO CHO Boc BnNH 2 NaBH(OAc) 3 Cl(CH 2 ) 2 Cl N OO Boc NHR N Boc N R N Boc OO OO + R = Bn, 9R = Bn, 10 8 L-a abinose e . 23 Pd / C, MeOH Pd / C MeOH, THF 98% R = H, 12 100% CF 3 COOH, H 2 O 5 R = H, 11 100% CF 3 COOH, H 2 O N HO OH H NH 2 13 D-a abinose en -5 ; en -13 e . 24 N OO COOE H 14 92% Boc 2 O, py idine N OO COOE Boc 15 DIBAL-H -78 °C 72% N OO CHO Boc en -8 Scheme 2. P epa a ion o iamines 5 and en -5. Issue in Hono o P o . J. M. Muchowski ARKIVOC 2002 (xii) 23-37 ISSN 1424-6376 Page 27 ©ARKAT USA, Inc Glycosidase inhibi o y ac i i ies App op ia e p-ni ophenyl py anosides we e used as subs a es and comme cially a ailable glycosidases (see below and Table) we e used as ca alys s o he bu e ed hyd olysis unde op imal pH.25 A 1 mM concen a ion and unde op imal pH condi ions e amines 4 and iamines 5 and en -5 did no inhibi he ollowing enzymes: α-L- ucosidase om bo ine epididymis, α-D-galac osidases om co ee bean, Aspe gillus nige and E. coli, β-galac osidase om o izae, β-D-mannosidase om Helix poma ia, β-N-ace ylgalac osamidase om jack bean, bo ine epididymis A and B. The inhibi o y ac i i ies owa d o he glycosidases a e epo ed in Table 1. We ha e ound ha (2R,3R,4S)-2-aminome hylpy olidine-3,4-diol 2a is a weak inhibi o o α-mannosidase om jack bean and om almond. This diamine also mode a ely inhibi s β- galac osidases, α-glucosidases and β-glucosidases. De i a i es 2b a e much be e and mo e selec i e α-mannosidase inhibi o s.8 Thus, we expec ed ha compounds 4 and 5 would also show imp o ed inhibi o y ac i i ies owa d α-mannosidases. This is indeed he case o 4a wi h he e ame hylene linke , and o 4e wi h he p-benzenedime hylene space . Bo h a e compe i i e inhibi o s. The bad su p ise is ha hese e amines also inhibi o he glycosidases, mode a ely hough, excep o 4a which is a good, non-compe i i e inhibi o o β-glucosidase om almond. This esul sugges s ha 4a "s icks" o his enzyme and inhibi s i o allos e ic easons, a mechanism di e en om ha making 4a a compe i i e inhibi o o α-mannosidases. Te amine 4b wi h he hexame hylene linke and analogues 4c and 4d wi h diphenylme hane linke s a e poo inhibi o s in e ms o bo h po ency and selec i i y. They a e e en wo se han simple diamine 2a. As (2S,3R,4S)-2-(2-aminoe hyl)py olidine-3,4-diol 13 is a weak inhibi o o α-mannosidase, al hough he side chain is in a β-con igu a ion a he han α, we en isioned ha iamine 5 migh ha e imp o ed inhibi o y ac i i y. In e es ingly, we ind 5 o be a mo e po en α-mannosidase inhibi o han 13. Un o una ely, i is no a mo e selec i e inhibi o han 13 because i inhibi s mode a ely a ew α-glucosidases, β-glucosidases and α-N-ace yl- galac osamidase om chicken li e (Table 1). As expec ed, iamine en -5, which does no sha e he con igu a ion o any o he hexoses libe a ed du ing he hyd oly ical p ocess ca alyzed by he enzymes used in his s udy, igno es all hese glycosidases. Conclusions The conjuga ion o wo (2R,3R,4S)-2-(2-aminome hyl)py olidine-3,4-diols by hei p ima y amines o alkane o a ene linke s can gene a e po en α-mannosidase inhibi o s. This wo k opens a new oad in he sea ch o new glycosidase inhibi o s. Analogues o e amines 4a and 4e ha will be mo e enzyme selec i e emain o be made. Issue in Hono o P o . J. M. Muchowski ARKIVOC 2002 (xii) 23-37 ISSN 1424-6376 Page 28 ©ARKAT USA, Inc Table 1. Inhibi o y ac i i ies o diamines 2a, 2b, iamines 5 and en -5 and e amines 4a-4e. Pe cen age o inhibi ion a 1mM concen a ion, IC50 (in pa en hesis) and Ki in µM, op imal pH, 35°C25,26 Enzyme / inhibi o 2a 2b 4a 4b 4c 4d 4e 5 en -5 β-galac osidase om E-coli bo ine li e Aspe gillus nige jack bean 92% ni 24% 76% 24% 26% ni ni 95% ni ni 45% 43% 24% ni 23% 47% 95% ni ni ni 82% ni ni 37% 41% 40% 39% ni ni 22% 31% ni ni ni ni α-glucosidase om yeas (mal ase) ice (mal ase) bake yeas (isomal ase) Aspe gillus nige (amyloglucosidase) Rhyzopus mold (amyloglucosidase) 24% 53% 98% ni ni ni ni ni ni ni 88% ni ni ni ni 37% ni 69% ni ni ni 26% ni ni 26% ni ni ni ni ni 55% ni 86% 28% 39% ni ni 50% 26% ni ni ni ni ni ni β-glucosidase om almonds Ki = caldocellum sacch. 97% 93% 68% ni 97%(160) 8(NC) 90% 87%(110) 110 (C) 76% 35% 36% 52% 29% 85%(99) 65(C) 67% 37% 26% 38% ni α-mannosidase om jack bean Ki = almonds Ki = 81% 53(C) 51% 92% 7.4(C) 69% 7(C) 76%(330) 21 (C) 85%(92) 10 (C) 72% 70% ni 39% 47% ni 95%(50) 12 (C) 81%(145) 48 (C) 71%(300) 74 (C) 65%(280) 92 (C) ni ni β-xylosidase om Aspe gillus nige ni ni ni ni ni ni 26% ni ni α− Ν −ace ylgalac osamidase chicken li e Ki = ni ni ni 92%(100) 43 (C) ni ni 91%(53) 31 (C) ni ni ni = no inhibi ion, C = compe i i e, NC = non-compe i i e Expe imen al Sec ion Gene al P ocedu es. All comme cially a ailable eagen s (Fluka, Ald ich) we e used wi hou u he pu i ica ion. Sol en s we e d ied by s anda d me hods. Ligh pe oleum e he used e e s o he ac ion boiling a 40–60 °C. Solu ions a e eac ions and ex ac ions we e e apo a ed in a Issue in Hono o P o . J. M. Muchowski ARKIVOC 2002 (xii) 23-37 ISSN 1424-6376 Page 29 ©ARKAT USA, Inc o a o y e apo a o unde educed p essu e. Liquid/solid lash ch oma og aphy (FC): columns o silica gel (Me ck No.9385 silica gel 60, 240–400 mesh). TLC o eac ion moni o ing: Me ck silica gel 60F254 pla es; de ec ion by UV ligh , Pancaldi eagen [(NH4)6MoO4, Ce(SO4)2, H2SO4, H2O] o KMnO4. IR spec a: Pe kin-Elme -1420 spec ome e . Op ical o a ions we e de e mined a oom empe a u e on a Jasco DIP-370 pola ime e . [α]D alues a e gi en in uni s o 10–1 deg cm2 g–1. 1H NMR spec a: B uke -ARX-400 spec ome e (400 MHz), B uke AMX-300 spec ome e (300 MHz); δ(H) in ppm ela i e o he sol en ’s esidual 1H signal [CHCl3, δ(H) 7.27; CH3OD, δ(H) 3.31; D2O, δ(H) 4.79; DMSO-d6, δ(H) 2.54] as in e nal e e ence; all 1H assigmen s we e con i med by 2D-COSY-45 and 2D-NOESY spec a. 13C NMR spec a: same ins umen as abo e (100.6 MHz and 75.4 MHz); δ(C) in ppm ela i e o he sol en ’s C-signal [CDCl3, δ(C) 77.0; CD3OD, δ(C) 49.8; DMSO-d6 δ(C) 39.7] as in e nal e e ence; all 13C assigmen s we e con i med by 2D-HMQC; coupling cons an s J in Hz. MS: Ne mag R 10-10C, chemical ioniza ion (NH3) mode m/z (amu) [% ela i e o base peak (100%)]. High esolu ion mass spec ome y: Mic omass Au oSpecQ, esolu ion o 10000 (5% alley de ini ion). Elemen al analyses: Ilse Bee z, D-96301 K onach, Ge many. Glycosidase inhibi ions. A known p o ocol was applied.25,26 We e i ied ha he delay o inhibi o /enzyme incuba ion did no a ec he inhibi ion measu emen s. Unde s anda d condi ions, op imal inhibi o y ac i i ies we e measu ed a e i e minu es o incuba ion. Reduc i e amina ion. Gene al p ocedu e A. To a solu ion o N-[( -bu oxy)ca bonyl]-2,5- dideoxy-2,5-imino-3,4-O-isop opylidene-L- ibose (200 mg, 0.737 mmol) in anhyd ous 1,2-di- chlo oe hane (7 mL) we e added he diamine (0.6 eq, 0.442 mmol) and NaBH(OAc)3 (1.8 eq, 281 mg, 1.327 mmol). The solu ion was s i ed a 50 °C o 12 h and hen pou ed in o a sa . aq solu ion o NaHCO3 (20 mL). The mix u e was ex ac ed wi h E OAc (3 x 20 mL). The combined o ganic ex ac s we e d ied (MgSO4) and concen a ed unde educed p essu e. The esidue was di ec ly used in he dep o ec ion s ep. Reduc i e amina ion. Gene al p ocedu e B. To a solu ion o N-[( -bu oxy)ca bonyl]-2,3,6- ideoxy-3,6-imino-4,5-O-isop opylidene-L- (o D-) a abino-hexose (1 mmol) in anhyd ous 1,2- dihlo oe hane (3 mL) we e added benzylamine (118 mg, 1.1 mmol) and NaBH(OAc)3 (276 mg, 1.3 mmol). The solu ion was s i ed a . . o 3 h and hen pou ed in o a sa . aq solu ion o NaHCO3 (20 mL). The mix u e was ex ac ed wi h E OAc (3 x 20 mL) and he combined o ganic ex ac s we e d ied (MgSO4). A e sol en e apo a ion unde educed p essu e he esidue was pu i ied by lash ch oma og aphy on silica gel (CH2Cl2/MeOH 60:1 o 5:1). Dep o ec ion. Gene al p ocedu e C. A solu ion o bis (py olidine) de i a i es in CF3COOH / H2O (4:1; 5–10%) was s i ed a 20 °C o 2 h. A e sol en e apo a ion in acuo, he esidue was pu i ied by lash ch oma og aphy on silica gel (MeCN/aq NH3). Issue in Hono o P o . J. M. Muchowski ARKIVOC 2002 (xii) 23-37 ISSN 1424-6376 Page 30 ©ARKAT USA, Inc Dep o ec ion. Gene al p ocedu e D. A solu ion o he p o ec ed py olidine de i a i e (0.1 mmol) in CF3COOH H2O (4:1; 3 mL) was s i ed a 20 °C o 2 h. The mix u e was passed h ough a Dowex 50WX8 (100–200 mesh) column and elu ed, successi ely wi h MeOH (30 mL), H2O (30 mL) and NH4OH (10%, 50 mL). The ac ions con aining he unp o ec ed p oduc we e concen a ed o yield he co esponding py olidine de i a i e. (2R,3R,4S)-2-[[4-[[[(2R,3R,4S)-3,4-Dihyd oxypy olidin-2-yl]me hyl]amino]bu yl]amino- me hyl]py olidine-3,4-diol (4a). P ocedu e A was applied o 1,4-diaminobu ane (45 µL, 0.442 mmol) o a o d c ude 7a (180 mg). Dep o ec ion acco ding o p ocedu e C ga e 4a (127 mg, 90%, 2 s eps) as a pale o ange oil. R = 0.15 (MeCN / NH4OH 1:1). [α]589 25 = –106, [α]577 25 = –262, [α]546 25 = –409, [α]435 25 = –690, [α]405 25 = –1114 (c = 0.25, H2O). IR ( ilm): ~ 3500– 2900, 1440, 1200, 1140, 840, 800, 710, 695 cm-1. UV (MeCN): λmax (ε) 195 (1360). 1H NMR (D2O): δ 4.11 (m, 2H, H-4, H-4IV), 3.94 (dd, 2H, 3J = 10.7, 3J = 3.9 Hz, H-3, H-3IV), 3.75 (ddd, 2H, 3J = 10.7, 3J = 5.1, 3J = 3.3 Hz, H-2, H-2IV), 3.23 (dd, 2H, 2J = 9.3, 3J = 2.1, H-5, H-5IV), 3.13 (dd, 2H, 2J = 12.6, 3J = 5.1, H-1', H-1'''), 3.09−3.01 (m, 2H, H-5, H-5IV), 2.81 (dd, 2H, 2J = 12.6, 3J = 3.3, H-1', H-1'''), 2.72–2.64 (m, 4H, H-1'', H-4''), 1.71−1.56 (m, 4H, H-2'', H-3''). 13C NMR (D2O): δ 76.9 (d, C-3, C-3IV), 73.5 (d, C-4, C-4IV), 60.7 (d, C-2, C-2IV), 53.9 ( , C-5, C- 5IV), 52.6 ( , C-1, C-1'''), 50.4 ( , C-1'', C-4''), 26.3 ( , C-2'', C-3''). CI-MS: m/z 319 (100, M + H+), 293 (74), 204 (33), 133 (35), 102 (36), 84 (55). Anal. calcd o C14H30N4O4 (318.42): C, 52.81; H, 9.50. Found: C, 52.79; H, 9.32. (2R,3R,4S)-2-[[6-[[[(2R,3R,4S)-3,4-Dihyd oxypy olidin-2-yl]me hyl]aminohexyl]amino- me hyl]py olidine-3,4-diol (4b). P ocedu e A was applied o 1,6-diaminohexane (51 mg, 0.442 mmol) o a o d c ude 7b (155 mg). Dep o ec ion acco ding o p ocedu e C ga e 4b (86 mg, 56% yield, 2 s eps) as a colo less oil. R = 0.1 (MeCN, NH4OH 1/1). [α]589 25 = −54 (c = 0.5, H2O). IR ( ilm): ~ 3500−2900, 1450, 1195, 1150, 840, 800, 705, 700 cm-1. UV (MeCN): λmax (ε) 197 (1450). 1H NMR (D2O): δ 4.18 (m, 2H, H-4, H-4IV), 3.95 (dd, 2H, 3J = 5.4, 3J = 2.7 Hz, H-3, H-3IV), 3.75 (m, 2H, H-2, H-2IV), 3.29 (m, 2H, H-5, H-5IV), 3.15 2H, (2H, dd, 2J = 13.2, 3J = 4.8, H-1', H-1'''), 3.09 (m, 2H, H-5, H-5IV), 2.94 (dd, 2H, 2J = 13.2, 3J = 3.1, H-1', H-1'''), 2.73 (m, 4H, H-1'', H-6''), 1.65-1.54 (m, 4H, H-2'', H-5''), 1.38 (m, 4H, H-3'', H-4''). 13C NMR (D2O): δ 77.0 (d, C-3, C-3IV), 71.7 (d, C-4, C-4IV), 60.7 (d, C-2, C-2IV), 54.2 ( , C-5, C-5IV), 52.6 ( , C-1, C-1'''), 50.9 ( , C-1'', C-6''), 29.7 ( , C-2'', C-5''), 26.3 ( , C-3'', C-4''). CI-MS: m/z 347 (28, M + H+), 274 (9), 232 (12), 117 (100), 98 (85), 86 (63). Anal. calcd o C16H34N4O4 (346.47): C, 55.47; H, 9.89; N, 16.17. Found: C, 55.18; H, 9.70; N, 16.01. (2R,3R,4S)-2-[4-[4-[[[(2R,3R,4S)-3,4-Dihyd oxypy olidin-2-yl]me hyl]amino]benzyl]phenyl- aminome hyl]py olidine-3,4-diol (4c). P ocedu e A was applied o 4,4'-diaminodiphenyl- me hane (88 mg, 0.442 mmol) o a o d c ude 7c (150 mg). Dep o ec ion acco ding o p ocedu e C ga e 4c (113 mg, 60% yield, 2 s eps) as a pale yellow oil. R = 0.10 (MeCN/NH4OH 4:1). [α]589 25 = +27, [α]577 25 = +34, [α]546 25 = +41 (c = 0.9, MeOH). IR ( ilm): ~ 3400−3200, 2950, 1675, 1515, 1450, 1205, 1140, 1025, 725 cm-1. UV (MeCN): λmax (ε) 260 (7250), 207 (13980). 1H NMR (MeOD): δ 6.94, 6.68 (2d, 8H, 3J = 8.5 Hz, H-2'', H-6'', H-3VI, H-5VI), 4.29 (m, 2H, H-4, H-4VI), 4.08 (dd, 2H, 3J = 8.6, 3J = 4.0 Hz, H-3, H-3VI), 3.80 (bs, 2H, 2H-1'''), 3.76 (ddd, 2H, 3J = Issue in Hono o P o . J. M. Muchowski ARKIVOC 2002 (xii) 23-37 ISSN 1424-6376 Page 31 ©ARKAT USA, Inc 9.1, 3J = 8.6, 3J = 3.7 Hz, H-2, H-2VI), 3.60 (dd, 2H, 2J = 14.4, 3J = 3.7 Hz, H-1', H-1V), 3.48 (dd, 2H, 2J = 14.4, 3J = 4.0 Hz, H-5, H-5VI), 3.45 (dd, 2H, 2J = 14.4, 3J = 3.7 Hz, H-1', H-1V), 3.27 (dd, 2H, 2J = 14.4, 3J = 1.9 Hz, H-5, H-5VI). 13C NMR (MeOD): δ 133.3 (s, C-1'', C-4IV), 133.2 (d, C-2'', C-6'', C-3IV, C-5IV), 122.3 (s, C-4'', C-1IV), 116.9 (d, C-3'', C-5'', C-2IV, C-6IV), 77.3 (d, C-3, C-3VI), 73.5 (d, C-4, C-4VI), 64.0 (d, C-2, C-2VI), 53.3 ( , C-5, C-5VI), 47.1 ( , C-1', C-1V), 43.8 ( , C-1'''). Anal. calcd o C23H30N4O4 (426.51): C, 64.77; H, 7.09; N, 13.14. Found: C, 64.34; H, 7.28; N, 12.99. (2R,3R,4S)-2-[[7-[[[(2R,3R,4S)-3,4-Dihyd oxypy olidin-2-yl]me hyl]amino]-9H- luo en-2- yl]aminome hyl]py olidine-3,4-diol (4d). P ocedu e A was applied wi h 2,7-diamino luo ene (87 mg, 0.442 mmol) o a o d c ude 7d (153 mg). Dep o ec ion acco ding o p ocedu e C ga e 4d (94 mg, 50% yield, 2 s eps) as a pale yellow oil. R = 0.09 (MeCN/NH4OH 2/1). [α]589 25 = −62, [α]577 25 = −74, [α]546 25 = −103, [α]435 25 = −107, [α]405 25 = −130 (c = 1, MeOH). IR ( ilm): ~ 3400− 3200, 2960, 1675, 1520, 1455, 1210, 1135, 125, 880, 765 cm-1. UV (MeCN): λmax (ε) 308 (5820), 215 (5680), 203 (6200). 1H NMR (MeOD): δ 7.43 (d, 2H, 3J = 8.1 Hz, H-3'', H-6''), 6.92 (bs, 2H, H-1'', H-8''), 6.71 (d, 2H, 3J = 8.1 Hz, H-4'', H-5''), 4.32 (m, 2H, H-4, H-4IV), 4.13 (dd, 2H, 3J = 8.5, 3J = 4.0 Hz, H-3, H-3IV), 3.82 (ddd,2H, 3J = 8.5, 3J = 8.4, 3J = 3.8 Hz, H-2, H-2IV), 3.73 (bs, 2H, H-9''), 3.67 (dm, 2H, 2J = 13.3 Hz, H-5, H-5IV), 3.52 (m, 2H, H-5, H-5IV), 3.51 (dd, 2H, 2J = 12.6, 3J = 4.0 Hz, H-1', H-1'''), 3.30 (dd, 2H, 2J = 12.6, 3J = 1.8 Hz, H-1', H-1'''). 13C NMR (MeOD): δ 148.2, 146.2 (2s, C-4a'', C-4b'', C-8a'', C-9a''), 121.0 (s, C-2'', C-7''), 120.5 (d, C-3'', C-6''), 114.2 (d, C-4'', C-5''), 111.9 (d, C-1'', C-8''), 75.7 (d, C-3, C-3IV), 71.9 (d, C-4, C- 4IV), 62.5 (d, C-2, C-2IV), 51.7 ( , C-1', C-1'''), 45.7 (d, C-5, C-5IV), 42.1 ( , C-9''). CI-MS: m/z 427 (21, M + H+), 370 (14), 311 (50), 197 (28), 98 (100), 80 (57). Anal. calcd o C23H30N4O4 (426.51): C, 64.77; H, 7.09. Found: C, 64.88; H, 7.23. (2R,3R,4S)-2-[[4-[[[(2R,3R,4S)-3,4-Dihyd oxypy olidin-2-yl]me hyl]aminome hyl]benzyl]- aminome hyl]py olidine-3,4-diol (4e). P ocedu e A was applied wi h 1,4-(diamino- me hyl)benzene (60 mg, 0.442 mmmol) o a o d c ude 7e (157 mg). Dep o ec ion acco ding o p ocedu e C ga e 4e (89 mg, 55% yield, 2 s eps) as a colo less oil. R = 0.14 (MeCN/NH4OH 1:1). [α]589 25 = + 57, [α]577 25 = + 77, [α]546 25 = + 83, [α]435 25 = + 93, [α]405 25 = + 110 (c = 0.65, H2O). IR ( ilm): ~ 3500−3000, 1675, 1425, 1200, 1130, 835, 800, 740, 700 cm-1. UV (MeCN): λmax (ε) 197 (5600). 1H NMR (D2O): δ 7.50 (bs, 4H, Ha om), 4.26 (m, 2H, H-4, H-4VI), 3.94 (s, 4H, 2H-1'', 2H-1IV), 3.87 (dd, 2H, 3J = 7.6, 5.0 Hz, H-3, H-3VI), 3.30−3.25 (m, 4H, H-2, H-2VI, H-5, H-5VI), 2.99-2.94 (m, 4H, H-1', H-1V, H-5, H-5VI), 2.79 (dd, 2H, 2J = 12.5, 3J = 8.6 Hz, H-1', H-1V). 13C NMR (D2O): δ 137.4 (s, C-1''', C-4'''), 129.2 (d, C-2''', C-3''', C-5''', C-6'''), 75.2 (d, C-3, C-3VI), 70.9 (d, C-4, C-4VI), 59.9 ( , C-1'', C-1IV), 52.1 ( , C-5, C-5VI), 50.6 (d, C-2, C-2VI), 50.0 ( , C-1', C-1V). CI-MS : m/z 368 (24, M+), 252 (7), 133 (100), 117 (59). Anal. calcd o C18H32N4O4 (368.48): C 58.67; H 8.75; N 15.21. Found: C 58.42, H 8.60, N 15.12. N-( e -Bu oxyca bonyl)-(2S,3R,4S)-2-[2-(benzylamino)e hyl]-3,4-O-isop opylidenepy ol- idine-3,4-diol (9) and N,N-bis[N-( e -bu oxyca bonyl)-[(2S,3R,4S)-3,4-O-isop opylidenoxy- py olidinyl]e hyl]benzylamine (10). P ocedu e B was applied o ca baldehyde 823 (298 mg, 1.05 mmol) a o ding 9 (178.8 mg, 46%) as oil and 10 (123.4 mg, 18%) as whi e solid.