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

Issue in Honor of Prof. J. M. Muchowski ARKIVOC 2002 (xii) 23-37 ISSN 1424-6376 Page 23 ©ARKAT USA, Inc Synthesis of triand tetramines containing two 2,3-dihydroxypyrrolidine moieties and their inhibitory activity toward α-mannosidases Sandrine Gerber-Lemaire,*a Florence Popowycz, a Eliazar Rodriguez-García, a Catherine Schütz, a Ana T. Carmona Asenjo, b Inmaculada Robina, b and Pierre Vogel a a Institut de chimie moléculaire et biologique, Ecole Polytechnique Fédérale de Lausanne, BCH, CH-1015 Lausanne, Switzerland and b Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, E-41071 Sevilla, Spain E-mail: [email protected] Dedicated to Professor Josef Muchowski on the occasion of his 65th birthday (received 20 Feb 03; accepted 08 Apr 03; published on the web 16 Apr 03) Abstract Through the reductive amination of N-[(tert-butoxy)carbonyl]-2,5-dideoxy-2,5-imino-3,4-Oisopropylidene-L-ribose with tetramethylenediamine, hexamethylenediamine, 2,7diaminofluorene, 4,4'-diaminodiphenylmethane and 1,4-(diaminomethyl)benzene, five tetramines containing two (2R,3R,4S)-2-aminomethylpyrrolidine-3,4-diol moieties have been prepared and assayed for their inhibitory activities toward 24 glycosidases. Tetramines containing the tetramethylene or benzene-1,4-dimethylene linkers are more potent αmannosidase inhibitors than simple (2R,3R,4S)-2-aminomethylpyrrolidine-3,4-diols. Triamines such as (2S,3R,4S)-bis(3,4-dihydroxy-pyrrolidin-2-ethyl)amine were also prepared and shown to be better α-mannosidase inhibitors than (2S,3R,4S)-2-(2-aminoethyl)pyrrolidin-3,4-diol. Keywords: α-Mannosidase inhibitors, polyamines containing hydroxylated pyrrolidines, reductive amination Introduction Cell sociology involves a language based on molecular recognition between cell-surface carbohydrates and proteins.1 The biosynthesis of the surface oligosaccharides uses glycosyltransferases and glycosidases as catalysts. Inhibitors of these enzymes2 are important molecular tools for glycobiology, and can be used to modulate cellular functions. They are also potential drugs in new therapeutic strategies.3 Among the most potent glycosidase inhibitors are polyhydroxypiperidines (1,5-dideoxy-1,5-iminoalditols) that are mimics of the glycosyl cation Issue in Honor of Prof. J. M. Muchowski ARKIVOC 2002 (xii) 23-37 ISSN 1424-6376 Page 24 ©ARKAT USA, Inc intermediates liberated during enzyme-catalyzed hydrolytic processes.4,5 Derivatives of 3,4dihydroxypyrrolidines (1,4-dideoxy-1,4-iminoalditols) also emerge as an important class of glycosidase4a,5,6 and glycosyltransferase7 inhibitors. Simple meso-3,4-dihydroxypyrrolidine 1 is a non-selective, weak inhibitor of several glycosidases (Figure 1).8 We have found that derivatives 2b with (2R)-aminomethyl side chains can be highly selective and competitive inhibitors of αmannosidases, especially for Ar = phenyl, thiophenyl.8 N HO OH H N HO OH H NHR N HO OH HO 12a R = H 2b R = CH2Ar 3 H Figure 1. Inhibitors of glycosidases and glycosyltransferases. Clinical trials have shown that swainsonine 3, a natural α-mannosidase inhibitor that contains a 4-amino-4-deoxy-mannofuranoside moiety,9,10 reduces solid tumors and hematological malignancies.11 Analogues of 3 have also shown interesting properties.12 Mannosidase inhibitors mediate increased secretion of mutant α1-antitrypsin Z. They are thus leads in the development of drugs for the chemoprophylaxis of liver injury and emphysema in patients with α1-antitrypsin Z deficiency.13 Mannostatin A and B isolated from the soil microorganism Streptoverticillum verticillus14 and a synthetic analogue15 are probably the most potent inhibitors of αmannosidases reported so far.16 Often α-mannosidase inhibitors that are monosaccharide mimics4a,17 also inhibit other types of glycosidases,18 in particular α-L-fucosidases.4a,19 To become a drug, a good inhibitor must satisfy a number of conditions apart from its low toxicity and enzyme specificity.20 We have envisioned that polyamines containing two (2R,3R,4S)-2- (aminomethyl)-3,4-dihydroxypyrrolidine fragments could be alternative α-mannosidase inhibitors with improved pharmacological properties. We report here the synthesis of five tetramines 4 (Figure 2). We have also prepared triamine 5 that contains two (2S,3R,4S)-2-(1aminoeth-2-yl)-3,4-dihydroxypyrrolidine moieties, as well as its enantiomer ent-5. These new compounds have been assayed for their inhibitory activity toward 24 commercially available glycosidases, and in particular toward α-mannosidase from jack bean, an enzyme known to be a useful model for mammalian α-mannosidases such as Golgi α-mannosidase II.21 Whereas triamine ent-5 does not inhibit any of the enzyme tested (except for a poor 38% inhibition of βglucosidase from almond at 1 mM concentration), its enantiomer 5 is a moderate inhibitor of αmannosidase from jack bean (Ki = 74 µM) and from almond (Ki = 92 µM). Among the five tetramines 4, best inhibitory activities toward these enzymes were found with 4a and 4e. But contrary to inhibitors of type 2b, these polyamines are less enzyme selective. Issue in Honor of Prof. 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 ent-5 Figure 2. Triand tetramines containing two 2,3-dihydroxypyrrolidine moieties. Results and Discussion Synthesis of the polyamines Tetramines 4 were all prepared from aldehyde 68 by reaction with the corresponding diamine H2N-A-NH2 (1.8 equivalent) in the presence of NaBH(OAc)322 for in situ reduction of the resulting diimine intermediate (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 to 90% yield Scheme 1. Synthesis of tetramines 4. Issue in Honor of Prof. J. M. Muchowski ARKIVOC 2002 (xii) 23-37 ISSN 1424-6376 Page 26 ©ARKAT USA, Inc The so-formed semi-protected tetramines were treated with aqueous CF3COOH, at room temperature, to cleave the Boc and acetonide moieties. Overall yields based on 6 ranged from 50 to 90%. Triamines 5 and ent-5 were derived from aldehydes 8 and ent-8, themselves derived from Land D-arabinose, respectively23,24 (Scheme 2). Treatment of a 1:1.1 mixture of 8 and benzylamine with NaBH(OAc)3 in 1,2-dichloroethane resulted in the formation of 9 and 10 with 46% and 18% yield, respectively. Using a half equivalent of benzylamine, 10 was obtained in 55% yield. Hydrogenolysis of the benzyl group (10% Pd / charcoal, THF/MeOH) gave 11 in 98% yield. Deprotection under acidic conditions provided 5 in almost quantitative yield. The same reactions were applied to ent-8 providing ent-(9–13). Compound ent-8 was obtained from known 1424 after Boc-protection and reduction with 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-arabinose ref. 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-arabinose ent-5 ; ent-13 ref. 24 N OO COOEt H 14 92% Boc 2 O, pyridine N OO COOEt Boc 15 DIBAL-H -78 °C 72% N OO CHO Boc ent-8 Scheme 2. Preparation of triamines 5 and ent-5. Issue in Honor of Prof. J. M. Muchowski ARKIVOC 2002 (xii) 23-37 ISSN 1424-6376 Page 27 ©ARKAT USA, Inc Glycosidase inhibitory activities Appropriate p-nitrophenyl pyranosides were used as substrates and commercially available glycosidases (see below and Table) were used as catalysts of the buffered hydrolysis under optimal pH.25 At 1 mM concentration and under optimal pH conditions tetramines 4 and triamines 5 and ent-5 did not inhibit the following enzymes: α-L-fucosidase from bovine epididymis, α-D-galactosidases from coffee bean, Aspergillus niger and E. coli, β-galactosidase from orizae, β-D-mannosidase from Helix pomatia, β-N-acetylgalactosamidase from jack bean, bovine epididymis A and B. The inhibitory activities toward other glycosidases are reported in Table 1. We have found that (2R,3R,4S)-2-aminomethylpyrrolidine-3,4-diol 2a is a weak inhibitor of α-mannosidase from jack bean and from almond. This diamine also moderately inhibits βgalactosidases, α-glucosidases and β-glucosidases. Derivatives 2b are much better and more selective α-mannosidase inhibitors.8 Thus, we expected that compounds 4 and 5 would also show improved inhibitory activities toward α-mannosidases. This is indeed the case for 4a with the tetramethylene linker, and for 4e with the p-benzenedimethylene spacer. Both are competitive inhibitors. The bad surprise is that these tetramines also inhibit other glycosidases, moderately though, except for 4a which is a good, non-competitive inhibitor of β-glucosidase from almond. This result suggests that 4a "sticks" to this enzyme and inhibits it for allosteric reasons, a mechanism different from that making 4a a competitive inhibitor of α-mannosidases. Tetramine 4b with the hexamethylene linker and analogues 4c and 4d with diphenylmethane linkers are poor inhibitors in terms of both potency and selectivity. They are even worse than simple diamine 2a. As (2S,3R,4S)-2-(2-aminoethyl)pyrrolidine-3,4-diol 13 is a weak inhibitor of α-mannosidase, although the side chain is in a β-configuration rather than α, we envisioned that triamine 5 might have improved inhibitory activity. Interestingly, we find 5 to be a more potent α-mannosidase inhibitor than 13. Unfortunately, it is not a more selective inhibitor than 13 because it inhibits moderately a few α-glucosidases, β-glucosidases and α-N-acetylgalactosamidase from chicken liver (Table 1). As expected, triamine ent-5, which does not share the configuration of any of the hexoses liberated during the hydrolytical process catalyzed by the enzymes used in this study, ignores all these glycosidases. Conclusions The conjugation of two (2R,3R,4S)-2-(2-aminomethyl)pyrrolidine-3,4-diols by their primary amines to alkane or arene linkers can generate potent α-mannosidase inhibitors. This work opens a new road in the search for new glycosidase inhibitors. Analogues of tetramines 4a and 4e that will be more enzyme selective remain to be made. Issue in Honor of Prof. J. M. Muchowski ARKIVOC 2002 (xii) 23-37 ISSN 1424-6376 Page 28 ©ARKAT USA, Inc Table 1. Inhibitory activities of diamines 2a, 2b, triamines 5 and ent-5 and tetramines 4a-4e. Percentage of inhibition at 1mM concentration, IC50 (in parenthesis) and Ki in µM, optimal pH, 35°C25,26 Enzyme / inhibitor 2a 2b 4a 4b 4c 4d 4e 5 ent-5 β-galactosidase from E-coli bovine liver Aspergillus niger 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 from yeast (maltase) rice (maltase) baker yeast (isomaltase) Aspergillus niger (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 from 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 from 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 from Aspergillus niger ni ni ni ni ni ni 26% ni ni α− Ν −acetylgalactosamidase chicken liver Ki = ni ni ni 92%(100) 43 (C) ni ni 91%(53) 31 (C) ni ni ni = no inhibition, C = competitive, NC = non-competitive Experimental Section General Procedures. All commercially available reagents (Fluka, Aldrich) were used without further purification. Solvents were dried by standard methods. Light petroleum ether used refers to the fraction boiling at 40–60 °C. Solutions after reactions and extractions were evaporated in a Issue in Honor of Prof. J. M. Muchowski ARKIVOC 2002 (xii) 23-37 ISSN 1424-6376 Page 29 ©ARKAT USA, Inc rotatory evaporator under reduced pressure. Liquid/solid flash chromatography (FC): columns of silica gel (Merck No.9385 silica gel 60, 240–400 mesh). TLC for reaction monitoring: Merck silica gel 60F254 plates; detection by UV light, Pancaldi reagent [(NH4)6MoO4, Ce(SO4)2, H2SO4, H2O] or KMnO4. IR spectra: Perkin-Elmer-1420 spectrometer. Optical rotations were determined at room temperature on a Jasco DIP-370 polarimeter. [α]D values are given in units of 10–1 deg cm2 g–1. 1H NMR spectra: Bruker-ARX-400 spectrometer (400 MHz), Bruker AMX-300 spectrometer (300 MHz); δ(H) in ppm relative to the solvent’s residual 1H signal [CHCl3, δ(H) 7.27; CH3OD, δ(H) 3.31; D2O, δ(H) 4.79; DMSO-d6, δ(H) 2.54] as internal reference; all 1H assigments were confirmed by 2D-COSY-45 and 2D-NOESY spectra. 13C NMR spectra: same instrument as above (100.6 MHz and 75.4 MHz); δ(C) in ppm relative to the solvent’s C-signal [CDCl3, δ(C) 77.0; CD3OD, δ(C) 49.8; DMSO-d6 δ(C) 39.7] as internal reference; all 13C assigments were confirmed by 2D-HMQC; coupling constants J in Hz. MS: Nermag R 10-10C, chemical ionization (NH3) mode m/z (amu) [% relative to base peak (100%)]. High resolution mass spectrometry: Micromass AutoSpecQ, resolution of 10000 (5% valley definition). Elemental analyses: Ilse Beetz, D-96301 Kronach, Germany. Glycosidase inhibitions. A known protocol was applied.25,26 We verified that the delay of inhibitor/enzyme incubation did not affect the inhibition measurements. Under standard conditions, optimal inhibitory activities were measured after five minutes of incubation. Reductive amination. General procedure A. To a solution of N-[(t-butoxy)carbonyl]-2,5dideoxy-2,5-imino-3,4-O-isopropylidene-L-ribose (200 mg, 0.737 mmol) in anhydrous 1,2-dichloroethane (7 mL) were added the diamine (0.6 eq, 0.442 mmol) and NaBH(OAc)3 (1.8 eq, 281 mg, 1.327 mmol). The solution was stirred at 50 °C for 12 h and then poured into a sat. aq solution of NaHCO3 (20 mL). The mixture was extracted with EtOAc (3 x 20 mL). The combined organic extracts were dried (MgSO4) and concentrated under reduced pressure. The residue was directly used in the deprotection step. Reductive amination. General procedure B. To a solution of N-[(t-butoxy)carbonyl]-2,3,6trideoxy-3,6-imino-4,5-O-isopropylidene-L- (or D-) arabino-hexose (1 mmol) in anhydrous 1,2dihloroethane (3 mL) were added benzylamine (118 mg, 1.1 mmol) and NaBH(OAc)3 (276 mg, 1.3 mmol). The solution was stirred at r.t. for 3 h and then poured into a sat. aq solution of NaHCO3 (20 mL). The mixture was extracted with EtOAc (3 x 20 mL) and the combined organic extracts were dried (MgSO4). After solvent evaporation under reduced pressure the residue was purified by flash chromatography on silica gel (CH2Cl2/MeOH 60:1 to 5:1). Deprotection. General procedure C. A solution of bis (pyrrolidine) derivatives in CF3COOH / H2O (4:1; 5–10%) was stirred at 20 °C for 2 h. After solvent evaporation in vacuo, the residue was purified by flash chromatography on silica gel (MeCN/aq NH3). Issue in Honor of Prof. J. M. Muchowski ARKIVOC 2002 (xii) 23-37 ISSN 1424-6376 Page 30 ©ARKAT USA, Inc Deprotection. General procedure D. A solution of the protected pyrrolidine derivative (0.1 mmol) in CF3COOH H2O (4:1; 3 mL) was stirred at 20 °C for 2 h. The mixture was passed through a Dowex 50WX8 (100–200 mesh) column and eluted, successively with MeOH (30 mL), H2O (30 mL) and NH4OH (10%, 50 mL). The fractions containing the unprotected product were concentrated to yield the corresponding pyrrolidine derivative. (2R,3R,4S)-2-[[4-[[[(2R,3R,4S)-3,4-Dihydroxypyrrolidin-2-yl]methyl]amino]butyl]aminomethyl]pyrrolidine-3,4-diol (4a). Procedure A was applied to 1,4-diaminobutane (45 µL, 0.442 mmol) to afford crude 7a (180 mg). Deprotection according to procedure C gave 4a (127 mg, 90%, 2 steps) as a pale orange oil. Rf = 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 (film): v ~ 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 (t, C-5, C5IV), 52.6 (t, C-1, C-1'''), 50.4 (t, C-1'', C-4''), 26.3 (t, C-2'', C-3''). CI-MS: m/z 319 (100, M + H+), 293 (74), 204 (33), 133 (35), 102 (36), 84 (55). Anal. calcd for 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-Dihydroxypyrrolidin-2-yl]methyl]aminohexyl]aminomethyl]pyrrolidine-3,4-diol (4b). Procedure A was applied to 1,6-diaminohexane (51 mg, 0.442 mmol) to afford crude 7b (155 mg). Deprotection according to procedure C gave 4b (86 mg, 56% yield, 2 steps) as a colorless oil. Rf = 0.1 (MeCN, NH4OH 1/1). [α]589 25 = −54 (c = 0.5, H2O). IR (film): v ~ 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 (t, C-5, C-5IV), 52.6 (t, C-1, C-1'''), 50.9 (t, C-1'', C-6''), 29.7 (t, C-2'', C-5''), 26.3 (t, C-3'', C-4''). CI-MS: m/z 347 (28, M + H+), 274 (9), 232 (12), 117 (100), 98 (85), 86 (63). Anal. calcd for 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-Dihydroxypyrrolidin-2-yl]methyl]amino]benzyl]phenylaminomethyl]pyrrolidine-3,4-diol (4c). Procedure A was applied to 4,4'-diaminodiphenylmethane (88 mg, 0.442 mmol) to afford crude 7c (150 mg). Deprotection according to procedure C gave 4c (113 mg, 60% yield, 2 steps) as a pale yellow oil. Rf = 0.10 (MeCN/NH4OH 4:1). [α]589 25 = +27, [α]577 25 = +34, [α]546 25 = +41 (c = 0.9, MeOH). IR (film): v ~ 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 Honor of Prof. 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 (t, C-5, C-5VI), 47.1 (t, C-1', C-1V), 43.8 (t, C-1'''). Anal. calcd for 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-Dihydroxypyrrolidin-2-yl]methyl]amino]-9H-fluoren-2yl]aminomethyl]pyrrolidine-3,4-diol (4d). Procedure A was applied with 2,7-diaminofluorene (87 mg, 0.442 mmol) to afford crude 7d (153 mg). Deprotection according to procedure C gave 4d (94 mg, 50% yield, 2 steps) as a pale yellow oil. Rf = 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 (film): v ~ 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, C4IV), 62.5 (d, C-2, C-2IV), 51.7 (t, C-1', C-1'''), 45.7 (d, C-5, C-5IV), 42.1 (t, C-9''). CI-MS: m/z 427 (21, M + H+), 370 (14), 311 (50), 197 (28), 98 (100), 80 (57). Anal. calcd for 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-Dihydroxypyrrolidin-2-yl]methyl]aminomethyl]benzyl]- aminomethyl]pyrrolidine-3,4-diol (4e). Procedure A was applied with 1,4-(diaminomethyl)benzene (60 mg, 0.442 mmmol) to afford crude 7e (157 mg). Deprotection according to procedure C gave 4e (89 mg, 55% yield, 2 steps) as a colorless oil. Rf = 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 (film): v ~ 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, Harom), 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 (t, C-1'', C-1IV), 52.1 (t, C-5, C-5VI), 50.6 (d, C-2, C-2VI), 50.0 (t, C-1', C-1V). CI-MS : m/z 368 (24, M+), 252 (7), 133 (100), 117 (59). Anal. calcd for C18H32N4O4 (368.48): C 58.67; H 8.75; N 15.21. Found: C 58.42, H 8.60, N 15.12. N-(tert-Butoxycarbonyl)-(2S,3R,4S)-2-[2-(benzylamino)ethyl]-3,4-O-isopropylidenepyrrolidine-3,4-diol (9) and N,N-bis[N-(tert-butoxycarbonyl)-[(2S,3R,4S)-3,4-O-isopropylidenoxypyrrolidinyl]ethyl]benzylamine (10). Procedure B was applied to carbaldehyde 823 (298 mg, 1.05 mmol) affording 9 (178.8 mg, 46%) as oil and 10 (123.4 mg, 18%) as white solid.