Aziridines from alkenyl-β-D-galactopyranoside derivatives: Stereoselective synthesis and in vitro selective anticancer activity

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in European Journal of Medicinal Chemistry on December 2013, available at: https://doi.org/10.1016/j.ejmech.2013.10.020 .” Graphical Abstract for: “Azidirines from alkenyl--D-galactopyranoside derivatives: Stereoselective synthesis and in vitro selective anticancer activity”. José M. Vega-Pérez,* Carlos Palo-Nieto, Margarita Vega-Holm, Purificación Góngora Vargas, José Manuel Calderón-Montaño, Estefanía Burgos-Morón, Miguel López- Lázaro, Fernando Iglesias-Guerra* O O O Ph O OR4 R5O R1 R3 R2 O O O Ph O OR4 R5O R1 R3 R2 NTs R1 = H, alkyl, aryl R2 = H, Me R3 = H, alkyl, aryl R4, R5 = H, Bn 12 Highlights for: “Azidirines from alkenyl--D-galactopyranoside derivatives: Stereoselective synthesis and in vitro selective anticancer activity”. José M. Vega-Pérez,* Carlos Palo-Nieto, Margarita Vega-Holm, Purificación Góngora Vargas, José Manuel Calderón-Montaño, Estefanía Burgos-Morón, Miguel López- Lázaro, Fernando Iglesias-Guerra* Preparation of a series new aziridines -D-galactopyranoside derivatives The synthesis took place through a short and high yielded methodology High selectivity against tumoral cells was found in some of them Structural requirements for improving biological activity are proposed Azidirines from alkenyl--D-galactopyranoside derivatives: Stereoselective synthesis and in vitro selective anticancer activity. José M. Vega-Pérez,a,* Carlos Palo-Nieto,a Margarita Vega-Holm,a Purificación Góngora-Vargas,a José Manuel Calderón-Montaño,b Estefanía Burgos-Morón,b Miguel López-Lázaro,b Fernando Iglesias-Guerraa,* aDepartamento de Química Orgánica y Farmacéutica, Facultad de Farmacia, Universidad de Sevilla, 41071 Sevilla, Spain bDepartamento de Farmacología, Facultad de Farmacia, Universidad de Sevilla, 41071 Sevilla, Spain Abstract A series of new aziridines -D-galactopyranoside derivatives were synthesized from alkenyl -D-galactopyranosides employing Sharpless conditions. The structures of the compounds were established by 1H NMR, 13C NMR, MS, HRMS and elemental analysis. The stereoselectivity of the reaction and the structural requirements of the alkenyl precursor for improving diastereoisomeric excesses of the direct aziridination reaction were also studied. The new compounds were subjected to a preliminary screening for cytotoxic activity against human lung cancer cells vs. human non-malignant lung cells. Terminal aziridine derivatives showed activity and, most notably, selectivity. One of the most active and selective compounds was also evaluated against breast cancer cells, melanoma cells, and non-malignant cells from the same origin. Its cytotoxic activity was similar to that of the positive controls, displaying a highly selective cytotoxic activity against both types of cancer cells. Keywords Cancer, stereoselective synthesis, aziridine carbohydrate derivative, selective cytotoxic activity. *Corresponding author: [email protected], [email protected] 1. Introduction Aziridine moiety, a three-membered heterocycle, analogue to epoxide with a nitrogen group replacing the oxygen, is an important scaffold in organic synthesis 1. The chemistry of aziridines has attracted considerable attention over the last few decades, and it is profoundly dependent on their ability to undergo nucleophilic ring opening, both stereo- and regioselectively 2. This allows the installation of a wide range of functional groups in a 1, 2-relationship to nitrogen, and provides access to an important pool of nitrogen-containing compounds, such as chiral amines, amino acids, - aminosulfonic acids, amino alcohols, alkaloids, -lactams, antibiotics, etc. 3-7. This makes them versatile synthetic intermediates that are used for the synthesis of biologically active products 8-16. Besides their importance as reactive intermediates, many aziridine-containing compounds have demonstrated to possess biological activity, essentially due to the presence of the aziridine ring. An important characteristic en el diseño de un antitumor agents is their specificity for tumor cells, avoinding the death of healthy cells. In this sense bioreductive agents use the cellular environment of tumor cells to provide specificity entering the cell in an inactive, oxidized form and becoming cytotoxic when reduced by enzymes within the cell. This reduction is optimized under the hypoxic conditions that are frequently found in tumor cells. Mitomycin C (1) is an important antitumor antibiotic isolated from Streptomyces caespitosus, that belongs to the family of antitumor quinones, which acts as a bioreductive agent 17. Studies on structural requirements for manifestations of biological activities of mitomycins were especially useful for understanding the role of aziridine moiety. Mitomycins, generally nonreactive in the natural oxidized state, behave as bifunctional alkylating agent upon chemical or enzymatic reduction. This has been studied extensively and used clinically in the treatment of many cancers including those of the bladder 18, colorectal 19 and gastrointestinal tract 20. FR900482, isolated from Streptomyces sandaensis 6897 as a mixture of the two hydroxylamine hemiketal isomers (2), possesses structural similarities to Mitomycin and also shares similar modes of action and medicinal properties. This makes it an appropriate replacement candidate showing superior activity and decreased toxicity 21. FR900482 has been studied extensively, and several synthesis have been described 22, including a methodology for the synthesis for its C-7 epimer, 7-Epi (+)-FR900482 (3)23. (Figure 1). Fig. 1. Aziridinylbenzoquinones, containing aziridine rings that react with DNA upon conversion of the quinone moiety to the hydroquinone have been also studied (4, 5). The reduction to the hydroquinone increases the basicity of the aziridine groups, increasing protonation and improving cross-linking yields at physiological pH. Several of these ligands have been found to react with DNA 24. Indeed one of these agents (4) have been studied clinically for its antitumor propierties 25. Most recently, it was evaluated for possible use as a chemotherapeutic drug for childhood cancers including neuroblastomas, osteosarcomas, and Ewing’s scarcoma 26. Fig. 2. Aziridinylbenzoquinone derivatives The antitumor agents azinomycins A (6) and B (7) (Figure 3) were isolated in 1986 from cultures of Streptomyces griseofuscus S-42227 27. The azinomycins exhibit potent in vitro cytotoxic activity and significant in vivo antitumor activity against P388 leukemia in mice 28. These agents possess an intricately functionalized structure containing an aziridino[1,2-a]pyrrolidine system The presence of electrophilic epoxide and aziridine rings suggests that the azinomycins act by covalent alkylation and crosslinking of DNA, in a manner similar to that of mitomycin C without prior activation 29. Azinomycins are attractive targets for synthetic efforts particularly due to their unusual structure, complex molecular mechanism of action, and effective antitumor activity. An effective and stereocontrolled synthesis of the crucial aziridino[1,2- a]pyrrolidine core substructure of the azinomycins has been described 30. Fig. 3. Structure of azinomycins Epothilones are cytotoxic molecules identified as metabolites produced by myxobacterium Sorangium cellulosum. This new class of microtubule-stabilizing natural products exhibits the same mechanism of action as paclitaxel. A series of C12 C13-aziridinyl epothilone derivatives were synthesized in an efficient manner from epothilone A. All aziridine analogues were tested for effects on tubulin binding polymerization and cytotoxicity. The results indicate that the aziridine moiety is a viable isosteric replacement for the epoxide in the case of epothilones (8), Figure 4 31. Fig. 4. Aziridinyl epothilone derivatives Other natural and/or synthesized aziridine-containing compounds, with different structures (lipids, steroids, amino acids, as well as their peptide derivatives), have also shown to be promising candidates for the development of new drugs toward several diseases, especially neoplasms 32. It is a fact that the toxicity of the aziridine derivatives will depend on their own structure and activity; due to their reactivity as electrophiles they are subject to attack by endogenous nucleophiles such as nitrogen bases of DNA structure resulting in a potential mutagenic effect. In spite of this, incorporation of an aziridine warhead will undoubtedly allow development of interesting new synthetic and semi-synthetic compounds with clinical utility. The inherent toxic effect of aziridines has promoted intensive studies aimed at obtaining new derivatives and evaluating their pharmacological activity and selectivity, to procure substances that can be used as chemotherapeutic agents. The work presented here is included in a research line of our group aimed at the synthesis of new compounds with potential antitumor activity, possessing different chemical structures. We recently described the synthesis of new isoprenyl-thiourea and urea derivatives designed to act as farnesyl diphosphate analogues, and evaluated their citotoxic activity, with promising results 33. General structure 9, Figure 5. Fig. 5. General backbone for new isoprenyl thiourea and urea derivatives We have also prepared a variety of nuevos glycosylglycerol derivatives and 1-OAcyl-3- O-β-D-glycosyl-sn-glycerol analogues by stereoselective dihydroxylation of a range of alkenyl β-D-hexopyanosides under Donohoe’s conditions, (general structure 10, Figure 6) and presented our preliminary results of cytotoxicity and selectivity assays 34. Among all the tested compounds, the best results for both antitumor activity and selectivity were those of galacto glycosylglycerol derivatives. Fig. 6. General backbone for new glycosylglycerol derivatives Subsequent to these results (synthesis of new compounds derived from carbohydrates with posible antitumor activity) our next objective was to combine in a general scaffold (11) the aziridine ring (crucial for the antitumor activity of various compounds) and the galacto moiety as the carbohydrate template, in order to obtain new potentially active compounds and study their activity-selectivity profile, Figure 7. Fig. 7. General backbone for new aziridine-containing galactopyranoside derivatives This paper therefore has a dual purpose. On one hand, the development of a synthetic methodology for the stereoselective azidirination reaction of alkenyl glycosides (galacto derivatives). Interestingly, this is the first time carbohydrates are used to induce chirality in direct aziridination reactions of alkenes. An on the other hand, the evaluation of antitumor activity and analysis of the structure-activity relationship of these compounds. 2. Results & discussion 2.1. Chemistry From a range of alkenyl galactopyranosides previously described by us 34, 35, we proceeded to the aziridination reaction using the conditions described by Sharpless, who uses TsNClNa as a nitrogen source and a bromine-based catalyst system: phenyltrimethylammonium tribromide, PhNMe3+Br3- (also known as PTAB) 36, and to evaluate the stereoselectivity of the process. Our goal was to make an initial screening to identify the structural requirements that provided greater chemical and stereochemical yields. For this we used as precursors a set of different functionalized alkenes which differ in the substituents of the olefin moiety as well as in the presence of a protective group on the sugar residue. Scheme 1 outlines the direct aziridination reaction of alkenes 12-21. Scheme 1. Synthesis of aziridinyl galactopyranoside derivatives (22-31). Reagent and conditions: Substrate (1 mmol), TsNClNa (1.1 mmol), PTAB (10 mmol %), CH3CN, –20 ºC, 12 h. The new aziridine derivatives 22-31 were isolated in satisfactory chemical yields (60– 90%). Stereochemical yields obtained (de’s) were stablished by 1H NMR and are summarized in Table 1. Fig.12. The percentages of cell viability () SEM in A549 and MRC5 cells exposed to compound 24 for 48 h were determined with the MTT assay. Fig.13. The percentages of cell viability () SEM in A549 and MRC5 cells exposed to compound 25 for 48 h were determined with the MTT assay. Fig.14. The percentages of cell viability () SEM in A549 and MRC5 cells exposed to compound 28 for 48 h were determined with the MTT assay. Fig.15. The percentages of cell viability () SEM in A549 and MRC5 cells exposed to compound 29 for 48 h were determined with the MTT assay. Fig.16. The percentages of cell viability () SEM in A549 and MRC5 cells exposed to compound 30 for 48 h were determined with the MTT assay. Fig.17. The percentages of cell viability () SEM in A549 and MRC5 cells exposed to compound 31 for 48 h were determined with the MTT assay. Fig.18. The percentages of cell viability () SEM in A549 and MRC5 cells exposed to compound 47 for 48 h were determined with the MTT assay. Fig.19. The percentages of cell viability () SEM in A549 and MRC5 cells exposed to compound 48 for 48 h were determined with the MTT assay. Fig.20. The percentages of cell viability () SEM in A549 and MRC5 cells exposed to compound 49 for 48 h were determined with the MTT assay. Fig.21. The percentages of cell viability () SEM in A549 and MRC5 cells exposed to compound 50 for 48 h were determined with the MTT assay. From the data collected in Table 5 we see that compound 22 was, for these tumor cells (breast and melanona), more active than the positive control used in each case (cisplatin and 5-fluorouracil respectively) and clearly more selective than the positive control against cells of breast cancer. 3. Conclusions Focused on the design of new compounds with cytotoxic activity and based on the fact that many aziridine-containing compounds have demonstrated to possess biological activity, essentially due to the presence of the aziridine ring, our first objective was to prepare new aziridinic compounds derived from sugar. We performed direct aziridination reaction on alkenes using different alkenyl -D-galactopyranosides (acting as chiral templates) through an easy and high yielded reaction. A range of aziridinyl derivatives differently substituted in positions 1 and 2 of the aziridine ring and with positions 2 and 3 of the sugar either free or di-O-protected were synthesized. Those terminal aziridines with protected sugar and the 1-position of the aziridine ring substituted were obtained as a single diastereoisomer, independently of the nature of the substituent. Our next aim was to assess the cytotoxic activity of these compounds, for which they were tested in vitro against human A549 lung cancer cells by using the MTT method.. To evaluate their selectivity they were also tested in an MTT assay against MRC5 human non-malignant lung fibroblasts. The best compounds (higher activity and selectivity) were terminal aziridines with the sugar moiety di-O-protected, with R: H, Methyl and Nonyl at 1-position of aziridine ring, being the activity higher when R: H. The presence of a Ph or a Propyl groups in the aziridine considerably decreases activity. In general, nonterminal aziridines showed higher IC50 values and yet the activity of these products was higher for unprotected derivatives than for dibencilated ones. Aziridines were submitted to ring opening reaction with N-nucleophiles and the obtained compounds were subjected to biological assays. They showed higher citotoxicity against non-malignant cells than to human lung cancer cells. We selected compound 22 (one of the most active against lung cancer cells, and one of the most selective) and subjected it to similar tests of antitumor activity against both breast and melanona cancer cells. The results showed that compound 22 is very active and selective against these types of cancer. To sum up, compound 22 has been shown to possess a high selectivity against 3 different types of cancer cells: lung, breast and skin. These are initial results that are still under study, in order to understand the mechanism of action of this substance, besides trying to get other sugar-derived aziridines with antitumor activity. 4. Experimental Section 4.1. Chemistry 4.1.1. General All reagents, solvents, and starting materials were obtained from commercial suppliers and used without further purification. Evaporations were conducted under reduced pressure. Reactions were monitored by thin layer chromatography (TLC) using Kieselgel 60 F254 (E. Merck) plates and UV detector for visualization. Flash column chromatography was performed on Silica Gel 60 (E. Merck). Yields are of purified products. Melting points were obtained on a Stuart Melting Point Apparatus SMP 10 and are uncorrected. Optical rotations were obtained on a Perkin Elmer Polarimeter Model 341 at 25 ºC. Mass spectra were recorded on a Micromass AUTOSPECQ mass spectrometer: EI at 70 eV and CI at 150 eV, HR mass measurements with resolutions of 10,000. NMR spectra were recorded at 25 ºC on a Bruker AMX500 spectrometer and on a Bruker AV500 spectrometer at 500 MHz for 1H and 125 MHz for 13C. The chemical shifts are reported in ppm on the  scale relative to TMS. COSY, DEPT, HSQC, and NOESY experiments were performed to assign the signals in the NMR spectra. 4.1.2. Methodology for the synthesis of alkenyl  -D-galactopyranoside derivatives (43- 46) 4.1.2.1. 2-Oxoethyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-β-D-galactopyranoside (33) To a solution of olefin (1.0 mmol) and trimethylamine-N-oxide (2.0 mmol) in dichloromethane (20 mL), was added catalytic OsO4, and the reaction mixture was stirred at room temperature for 2 h. After this time sodium bisulphite was added and stirring for 15 min and washed with water. The organic phase was concentrated under reduced pressure. To a solution of the resultant diol in ethanol (40 mL) a solution of sodium periodate (2.0 mmol) in distilled water was added and stirring for 2 h. The ethanol was evaporated and the aqueous phase was extracted with dichloromethane and the organic phase was concentrated to dryness. The solid was purified by column chromatography using hexane-ethyl acetate (1:1) as eluent (0.416 g , 85% yield). Mp 122–123 ºC; [α]D = +43.5 (c 1.0, CH2Cl2); MS (FAB): m/z 513 (50%) [M+Na]+; 1H NMR (500 MHz, CDCl3,  ppm): 9.67 (s, 1H, OCH2COH), 7.8–7.3 (m, 15H, Ph), 5.49 (s, 1H, PhCH), 4.93 (d, 1H, Jgem = 11.0 Hz, 2-PhCHAHBO), 4.85 (d, 1H, Jgem =11.0 Hz, 2-PhCHAHBO), 4.80 (d, 1H, Jgem = 11.0 Hz, 3-PhCHAHBO), 4.75 (d, 1H, Jgem = 11.0 Hz, 3-PhCHAHBO), 4.46 (d, 1H, J1,2 = 7.7 Hz, H-1), 4.29–4.20 (m, 3H, H-6e, OCH2CHO), 4.13 (dd, 1H, J3,4 = 3.7 Hz, J4,5 = 1.0 Hz, H-4), 4.01 (dd, 1H, J5,6a = 1.7 Hz, J6e,6a = 12.3 Hz, H-6a), 3.94 (dd, 1H, J1,2 = 7.7 Hz, J2,3 = 9.6 Hz, H-2), 3.58 (m, 1H, H-3), 3.33 (s, 1H, H-5). 13C NMR (125 MHz, CDCl3,  ppm):δ 201.2 (OCH2CHO), 129.0–126.4 (3Ph), 103.9 (C-1), 101.3 (PhCH), 79.1 (C-3), 78.2 (C-2), 75.4 (2- PhCH2O), 74.4 (OCH2CHO), 73.7 (C-4), 72.0 (3-PhCH2O), 69.0 (C-6), 66.7 (C-5). HRMS (FAB): calcd. for C29H30O7Na 513.1889; found, 513.1885 [M+Na]+. Anal. Calcd. for C29H30O7: C, 71.00; H, 6.16. Found: C, 70.93; H, 6.09. 4.1.2.2. Reaction of the aldehyde 33 with Grignard reagents (34-38) To a cooled solution (–78 °C) of the aldehyde 33 (1.0 mmol) in THF (30 mL) under argon atmosphere, the appropiate RMgBr (2.0 mmol) was added and the mixture was stirred at this temperature for 12 h. The reaction mixture was diluted in dichloromethane and washed successively with an aqueous saturated solution of ammonium chloride and with water, dried over MgSO4, filtered and the filtrate was evaporated to dryness under reduced pressure. The resultant crude was purified by column chromatography. 4.1.2.2.1 2-Hydroxy-2-phenylethyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-β-D- galactopyranoside (34) Two stereoisomers were obtained in a 1.7:1 ratio (26% de). The pure diastereomeric mixture was obtained as a solid by column chromatography using hexane-ethyl acetate (2:1) as eluent (0.465 g, 82% yield). Mp 143–144 ºC; [α]D = +25.5 (c 1.0, CH2Cl2); MS (FAB): m/z 591 (100%) [M+Na]+. 1H NMR (500 MHz, CDCl3,  ppm): 7.6–7.3 (m, 20H, Ph), 5.51 (s, 0.37H, PhCH minor), 5.50 (s, 0.63H, PhCH major), 4.93–4.76 [m, 5H, 2-PhCH2O, 3-PhCH2O, OCH2CH(OH)Ph], 4.53 (d, 0.63H, J1,2 = 7.6 Hz, H-1 major), 4.48 (d, 0.37H, J1,2 = 7.6 Hz, H-1 minor), 4.32 (dd, 0.37H, J5,6e = 1.5 Hz, J6e,6a = 12.4 Hz, H-6e minor), 4.28 (dd, 0.63H, J5,6e = 1.5 Hz, J6e,6a = 12.4 Hz, H-6e major), 4.14 (m, 1H, H-4), 4.10–4.00 [m, 1.63H, H-6a, OCHAHBCH(OH)Ph major], 3.96–3.88 (m, 1.37H, H-2, OCHAHBCH(OH)Ph minor ], 3.80 [m, 0.37 H, OCHAHBCH(OH)Ph minor], 3.60 [m, 2H, H-3, OCHAHBCH(OH)Ph major, OCH2CH(OH)Ph minor], 3.39 (s, 0.37H, H-5 minor), 3.35 (s, 0.63H, H-5 major), 3.30 [d, 0.63H, JH,OH = 2.7 Hz, OCH2CH(OH)Ph major]. 13C NMR (125 MHz, CDCl3): δ 128.9–126.1 (4Ph), 104.1 (C- 1), 101.4 (PhCH major), 101.3 (PhCH minor), 79.6 (C-3 major), 79.3 (C-3 minor), 78.4 (C-2 major), 78.3 (C-2 minor), 77.1 [OCH2CH(OH)Ph major], 76.4 [OCH2CH(OH)Ph minor], 75.6 (2-PhCH2O major), 75.4 (2-PhCH2O minor), 73.7 (C-4 major), 73.6 (C-4 minor), 73.1 [OCH2CH(OH)Ph major], 72.9 [OCH2CH(OH)Ph minor], 71.9 (3- PhCH2O minor), 71.8 (3-PhCH2O major), 69.2 (C-6 major), 69.0 (C-6 minor), 66.7 (C- 5 minor), 66.6 (C-5 major). HRMS (FAB): calcd. for C35H36O7Na 591.2359; found, 591.2365 [M+Na]+. Anal. Calcd. for C35H36O7: C, 73.92; H, 6.38. Found: C, 74.01; H, 6.34. 4.1.2.2.2. 2-Hydroxypentyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-β-D- galactopyranoside (35) Two stereoisomers were obtained in a 1.3:1 ratio (13% de). The pure diastereomeric mixture was obtained as a solid by column chromatography using hexane-ethyl acetate (2:1) as eluent (0.485 g, 91% yield). Mp 121–122 ºC; [α]D = +37.5 (c 1.0, CH2Cl2); MS (FAB): m/z 557 (90%) [M+Na]+. 1H NMR (500 MHz, CDCl3,  ppm): 7.6–7.2 (m, 15H, Ph), 5.49 (s, 1H, PhCH), 4.90–4.84 (m, 2H, 2-PhCH2O), 4.79–4.73 (m, 2H, 3-PhCH2O), 4.45 (d, 0.43H, J1,2 = 7.8 Hz, H-1 minor), 4.42 (dd, 0.57H, J1,2 = 7.8 Hz, H-1 major), 4.29 (m, 1H, H-6e), 4.13 (m, 1H, H-4), 4.01 (m, 1H, H-6a), 3.94 [dd, 0.43H, Jgem = 10.6 Hz, J = 2.7 Hz, OCHAHBCH(OH)CH2CH2CH3 minor], 3.88 (m, 1H, H-2), 3.82–3.78 [m, 1.57H, OCHAHBCH(OH)CH2CH2CH3 major, OCH2CH(OH)CH2CH2CH3], 3.65 [dd, 0.57H, Jgem = 11.4 Hz, J = 8.1 Hz, OCHAHBCH(OH)CH2CH2CH3 major], 3.58 (m, 1H, H-3), 3.46 [dd, 0.45 H, Jgem = 10.6 Hz, J = 8.1 Hz, OCHAHBCH(OH)CH2CH2CH3 minor], 3.36 (s, 0.57H, H-5 major), 3.34 (s, 0.43H, H-5 minor), 3.00 [s, 0.57H, OCH2CH(OH)CH2CH2CH3 major], 2.76 [s, 0.43H, OCH2CH(OH)CH2CH2CH3 minor], 1.51–1.33 [m, 4H, OCH2CH(OH)CH2CH2CH3], 0.93 [m, 3H, OCH2CH(OH)CH2CH2CH3].13C NMR (125 MHz, CDCl3,  ppm): 138.6–126.4 (3Ph), 104.24 (C-1 major), 104.22 (C-1 minor), 101.30 (PhCH minor), 101.27 (PhCH major), 79.6 (C-3 minor), 79.5 (C-3 major), 78.5 (C-2 minor), 78.4 (C-2 major), 76.1 [OCH2CH(OH)CH2CH2CH3 major], 75.6 (2-PhCH2O minor), 75.5 (2-PhCH2O major), 75.4 [OCH2CH(OH)CH2CH2CH3 minor], 73.7 (C-4), 71.9 (3-PhCH2O major), 71.8 (3- PhCH2O minor), 70.4 [OCH2CH(OH)CH2CH2CH3 minor], 76.2 [OCH2CH(OH)CH2CH2CH3 major], 69.2 (C-6 minor), 69.1 (C-6 major), 66.6 (C-5 major), 66.5 (C-5 minor), 35.0 [OCH2CH(OH)CH2CH2CH3 major], 34.9 [OCH2CH(OH)CH2CH2CH3 minor], 18.7 [OCH2CH(OH)CH2CH2CH3], 14.0 [OCH2CH(OH)CH2CH2CH3]. HRMS (FAB): calcd. for C32H38O7Na 557.2515; found, 557.2512 [M+Na]+. Anal. Calcd. for C32H38O7: C, 71.89; H, 7.16. Found: C, 71.74; H, 7.08. 4.1.2.2.3. 2-Hydroxy-3,3-dimethylbutyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-β-D- galactopyranoside (36) Two stereoisomers were obtained in a 1.5:1 ratio (20% de). The pure diastereomeric mixture was obtained as a syrup by column chromatography using hexane-ethyl acetate (2.5:1) as eluent (0.411 g, 75% yield). Mp 144–145 ºC; [α]D = +23.3 (c 1.0, CH2Cl2); MS (FAB): m/z 571 (100%) [M+Na]+. 1H NMR (500 MHz, CDCl3,  ppm): 7.6–7.2 (m, 15H, Ph), 5.49 (s, 1H, PhCH), 4.92–4.83 (m, 2H, 2-PhCH2O), 4.80–4.73 (m, 2H, 3- PhCH2O), 4.46 (d, 0.4H, J1,2 = 7.8 Hz, H-1 minor), 4.41 (dd, 0.6H, J1,2 = 7.8 Hz, H-1 major), 4.31 (m, 1H, H-6e), 4.13 (m, 1H, H-4), 4.09 [m, 0.4H, OCHAHBCH(OH)C(CH3)3 minor], 4.01 (m, 1H, H-6a), 3.93–3.83 [m, 1.6H, H-2, OCHAHBCH(OH)C(CH3)3 major], 3.70 [dd, 0.6H, Jgem = 10.5 Hz, J = 9.1 Hz, OCHAHBCH(OH)C(CH3)3 major], 3.58 (m, 1H, H-3), 3.51–3.44 [m, 1.4H, OCHAHBCH(OH)C(CH3)3 minor, OCH2CH(OH)C(CH3)3], 3.36 (s, 0.6H, H-5 major), 3.34 (s, 0.4H, H-5 minor), 3.03 [s, 0.6H, OCH2CH(OH)C(CH3)3 major], 2.81 [s, 0.4H, OCH2CH(OH)C(CH3)3 minor], 0.94 [s, 5.4H, OCH2CH(OH)C(CH3)3 major], 0.92 [s, 3.6H, OCH2CH(OH)C(CH3)3 minor].13C NMR (125 MHz, CDCl3,  ppm): 138.7–126.4 (3Ph), 104.2 (C-1 minor), 104.1 (C-1 major), 101.4 (PhCH minor), 101.3 (PhCH major), 79.6 (C-3 minor), 79.4 (C-3 major), 78.5 (C-2 minor), 78.4 (C-2 major), 77.8 [OCH2CH(OH)C(CH3)3 minor], 77.4 [OCH2CH(OH)C(CH3)3 major], 75.6 (2-PhCH2O minor), 75.4 (2-PhCH2O major), 73.72 (C-4 major), 73.67 (C-4 major), 72.9 [OCH2CH(OH)C(CH3)3 major], 72.4 [OCH2CH(OH)C(CH3)3 minor], 71.9 (3-PhCH2O major), 71.8 (3-PhCH2O minor), 69.2 (C-6 minor), 69.1 (C-6 major), 66.6 (C-5 major), 66.5 (C-5 minor), 33.31 [OCH2CH(OH)C(CH3)3 major], 33.29 [OCH2CH(OH)C(CH3)3 minor], 26.1 [OCH2CH(OH)C(CH3)3]. HRMS (FAB): calcd. for C33H40O7Na 571.2672; found, 571.2695 [M+Na]+. Anal. Calcd. for C33H40O7: C, 72.24; H, 7.35. Found: C, 72.32; H, 7.11. 4.1.2.2.4. 2-Hydroxyundecyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-β-D- galactopyranoside (37) Two stereoisomers were obtained in a 1.6:1 ratio (23% de). The pure diastereomeric mixture was obtained as a syrup by column chromatography using hexane-ethyl acetate (2.5:1) as eluent (0.494 g, 80% yield); [α]D = +12.6 (c 1.0, CH2Cl2); MS (FAB): m/z 641 (100%) [M+Na]+. 1H NMR (500 MHz, CDCl3,  ppm): 7.6–7.2 (m, 15H, Ph), 5.49 (s, 1H, PhCH), 4.86 (m, 2H, 2-PhCH2O), 4.76 (m, 2H, 3-PhCH2O), 4.45 (d, 0.38H, J1,2 = 7.8 Hz, H-1 minor), 4.42 (dd, 0.62H, J1,2 = 7.8 Hz, H-1 major), 4.29 (d, 1H, J6e,6a = 12.4 Hz, H-6e), 4.12 (m, 1H, H-4), 4.01 (m, 1H, H-6a), 3.93 [dd, 0.38H, Jgem = 10.5 Hz, J = 2.6 Hz, OCHAHBCH(OH)CH2(CH2)7CH3 minor], 3.87 (m, 1H, H-2), 3.82–3.77 [m, 1.62H, OCHAHBCH(OH)CH2(CH2)7CH3 major, OCH2CH(OH)CH2(CH2)7CH3], 3.64 [dd, 0.62H, Jgem = 11.3 Hz, J = 8.1 Hz, OCHAHBCH(OH)CH2(CH2)7CH3 major], 3.58 (m, 1H, H-3), 3.46 [dd, 0.38 H, Jgem = 10.6 Hz, J = 8.1 Hz, OCHAHBCH(OH)CH2(CH2)7CH3 minor], 3.36 (s, 0.62H, H-5 major), 3.34 (s, 0.38H, H- 5 minor), 3.00 [s, 0.62H, OCH2CH(OH)CH2(CH2)7CH3 major], 2.75 [s, 0.38H, OCH2CH(OH)CH2(CH2)7CH3 minor], 1.50–1.20 [m, 16H, OCH2CH(OH)CH2(CH2)7CH3], 0.88 [t, 3H, J = 7.0 Hz, OCH2CH(OH)CH2(CH2)7CH3].13C NMR (125 MHz, CDCl3,  ppm): 138.6–126.5 (3Ph), 104.3 (C-1 major), 104.2 (C-1 minor), 101.32 (PhCH minor), 101.28 (PhCH major), 79.6 (C-3 minor), 79.5 (C-3 major), 78.5 (C-2 minor), 78.4 (C-2 major), 76.1 [OCH2CH(OH)CH2(CH2)7CH3 minor], 75.6 (2-PhCH2O minor), 75.5 (2-PhCH2O major), 75.4 [OCH2CH(OH)CH2(CH2)7CH3 major], 73.7 (C-4), 71.9 (3-PhCH2O major), 71.8 (3-PhCH2O minor), 70.7 [OCH2C(OH)CH2(CH2)7CH3 minor], 70.4 [OCH2C(OH)CH2(CH2)7CH3 major], 69.2 (C-6 minor), 69.1 (C-6 major), 66.6 (C-5 major), 66.5 (C-5 minor), 32.9–22.7 [OCH2C(OH)CH2(CH2)7CH3], 14.1 [OCH2C(OH)CH2(CH2)7CH3]. HRMS (FAB): calcd. for C38H50O7Na 641.3454; found, 641.3431 [M+Na]+. 4.1.2.2.5. 2-Hydroxyhexadecyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-β-D- galactopyranoside (38) Two stereoisomers were obtained in a 1.2:1 ratio (9% de). The pure diastereomeric mixture was obtained as a syrup by column chromatography using hexane-ethyl acetate (2.5:1) as eluent (0.357 g, 52% yield); [α]D = +35.2 (c 1.0, CH2Cl2); MS (FAB): m/z 711 (50%) [M+Na]+. 1H NMR (500 MHz, CDCl3,  ppm): 7.6–7.2 (m, 15H, Ph), 5.49 (s, 1H, PhCH), 4.86 (m, 2H, 2-PhCH2O), 4.76 (m, 2H, 3-PhCH2O), 4.45 (d, 0.45H, J1,2 = 7.7 Hz, H-1 minor), 4.42 (dd, 0.55H, J1,2 = 7.7 Hz, H-1 major), 4.29 (d, 1H, J6e,6a = 12.4 Hz, H-6e), 4.12 (m, 1H, H-4), 4.01 (d, 1H, J6e,6a = 12.4 Hz, H-6a), 3.93 [dd, 0.45H, Jgem = 10.6 Hz, J = 2.6 Hz, OCHAHBCH(OH)CH2(CH2)12CH3 minor], 3.88 (m, 1H, H-2), 3.82–3.76 [m, 1.55H, OCHAHBCH(OH)CH2(CH2)12CH3 major, OCH2CH(OH)CH2(CH2)12CH3], 3.64 [dd, 0.55H, Jgem = 11.3 Hz, J = 8.2 Hz, OCHAHBCH(OH)CH2(CH2)12CH3 major], 3.58 (m, 1H, H-3), 3.46 [dd, 0.45 H, Jgem = 10.6 Hz, J = 8.2 Hz, OCHAHBCH(OH)CH2(CH2)12CH3 minor], 3.36 (s, 0.55H, H-5 major), 3.34 (s, 0.45H, H-5 minor), 3.01 [s, 0.55H, OCH2CH(OH)CH2(CH2)12CH3 major], 2.75 [s, 0.45H, OCH2CH(OH)CH2(CH2)12CH3 minor], 1.22 [m, 2H, OCH2CH(OH)CH2(CH2)12CH3], 1.34–1.24 [m, 24H, OCH2CH(OH)CH2(CH2)12CH3], 0.88 [t, 3H, J 7.0 Hz, OCH2CH(OH)CH2(CH2)12CH3].13C NMR (125 MHz, CDCl3, PPM): 128.9–126.4 (3Ph), 104.3 (C-1 major), 104.2 (C-1 minor), 101.3 (PhCH minor), 101.2 (PhCH major), 79.6 (C-3 minor), 79.5 (C-3 major), 78.5 (C-2 minor), 78.4 (C-2 major), 76.1 [OCH2CH(OH)CH2(CH2)12CH3 minor], 75.6 (2-PhCH2O minor), 75.5 (2- PhCH2O major), 75.4 [OCH2CH(OH)CH2(CH2)12CH3 major], 73.7 (C-4), 71.9 (3- PhCH2O major), 71.8 (3-PhCH2O minor), 70.7 [OCH2C(OH)CH2(CH2)12CH3 minor], 70.4 [OCH2C(OH)CH2(CH2)12CH3 major], 69.2 (C-6 minor), 69.1 (C-6 major), 66.6 (C- 5 major), 66.5 (C-5 minor), 32.9 [OCH2C(OH)CH2(CH2)12CH3 major], 32.9 [OCH2C(OH)CH2(CH2)12CH3 minor], 31.9–22.7 [OCH2C(OH)CH2(CH2)12CH3], 14.1 [OCH2C(OH)CH2(CH2)12CH3]. HRMS (FAB): calcd. for C43H60O7Na 711.4237; found, 711.4222 [M+Na]+. 4.1.2.3. Oxidation reaction with PCC (39-42) To a solution of the appropriate 2-hydroxyalkyl derivative (1.0 mmol) in dichloromethane (20 mL), molecular sieve 4Å and PCC (2.0 mmol) were added and the mixture was stirred at room temperature overnight. The resultant crude was filtered by silica flash column using diethylether as solvent and the resultant crude was purified by column chromatography. 4.1.2.3.1 2-Oxo-2-phenylethyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-β-D- galactopyranoside (39) The solid was purified by column chromatography using hexane-ethyl acetate (2.5:1) as eluent (0.441 g, 78% yield). Mp 154–155 ºC; [α]D = +15.5 (c 0.5, CH2Cl2); MS (FAB): m/z 589 (30%) [M+Na]+. 1H NMR (500 MHz, CDCl3,  ppm): 7.5–7.2 (m, 20H, Ph), 5.49 (s, 1H, PhCH), 5.10 (d, 1H, Jgem = 16.0 Hz, OCHAHBCOPh), 5.02 (d, 1H, Jgem = 11.0 Hz, 2-PhCHAHBO), 4.86 (d, 1H, Jgem = 16.0 Hz, OCHAHBCOPh), 4.79–4.73 (m, 3H, 2-PhCHAHBO, 3-PhCH2O), 4.60 (d, 1H, J1,2 = 7.7 Hz, H-1), 4.29 (dd, 1H, J5,6e = 1.5 Hz, J6e,6a = 12.4 Hz, H-6e), 4.10 (dd, 1H, J3,4 = 3.7 Hz, J4,5 = 1.0 Hz, H-4), 4.01 (dd, 1H, J5,6a = 1.7 Hz, J6e,6a = 12.3 Hz, H-6a), 3.94 (dd, 1H, J1,2 = 7.7 Hz, J2,3 = 9.6 Hz, H-2), 3.58 (dd, 1H, J2,3 = 9.6 Hz, J3,4 = 3.7 Hz, H-3), 3.34 (s, 1H, H-5). 13C NMR (125 MHz, CDCl3): δ 195.4 (OCH2COPh), 138.8–126.5 (4Ph), 103.2 (C-1), 101.3 (PhCH), 79.0 (C- 3), 78.2 (C-2), 75.2 (2-PhCH2O), 73.9 (C-4), 72.2 (3-PhCH2O), 71.1 (OCH2COPh), 69.0 (C-6), 66.6 (C-5). HRMS (FAB): calcd. for C35H34O7Na 589.2200; found 589.2184 [M+Na]+. Anal. Calcd. for C35H34O7: C, 74.19; H, 6.05. Found: C, 74.05; H, 6.20. 4.1.2.3.2. 2-Oxopentyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-β-D-galactopyranoside (40) The solid was purified by column chromatography using hexane-ethyl acetate (2:1) as eluent (0.441 g, 83% yield). Mp 132–133 ºC; [α]D = +29.0 (c 0.5, CH2Cl2); MS (FAB): m/z 555 (80%) [M+Na]+. 1H NMR (500 MHz, CDCl3,  ppm): 7.6–7.3 (m, 15H, Ph), 5.52 (s, 1H, PhCH), 4.98 (d, 1H, Jgem = 11.0 Hz, 2-PhCHAHBO), 4.95 (d, 1H, Jgem = 11.0 Hz, 2-PhCHAHBO), 4.77 (d, 1H, Jgem = 12.0 Hz, 3-PhCHAHBO), 4.75 (d, 1H, Jgem = 12.0 Hz, 3-PhCHAHBO), 4.44 (d, 1H, J1,2 = 7.7 Hz, H-1), 4.33 (d, 1H, Jgem = 16.5 Hz, OCHAHBCOCH2CH2CH3), 4.25 (dd, 1H, J5,6e = 1.5 Hz, J6e,6a = 12.4 Hz, H-6e), 4.14 (d, 1H, Jgem = 16.5 Hz, OCHAHBCOCH2CH2CH3), 4.11 (dd, 1H, J3,4 = 3.7 Hz, J4,5 = 1.0 Hz, H-4), 4.00 (dd, 1H, J5,6a = 1.7 Hz, J6e,6a = 12.3 Hz, H-6a), 3.90 (dd, 1H, J1,2 = 7.7 Hz, J2,3 = 9.6 Hz, H-2), 3.58 (m, 1H, H-3), 3.32 (s, 1H, H-5), 2.68 (m, 1H, OCH2COCHAHBCH2CH3), 2.56 (m, 1H, OCH2COCHAHBCH2CH3), 1.64 (m, 2H, OCH2COCH2CH2CH3), 0.93 (t, 3H, J = 7.40 Hz, OCH2COCH2CH2CH3). 13C NMR (125 MHz, CDCl3,  ppm): 209.1 (OCH2COCH2CH2CH3), 128.9–126.4 (3Ph), 103.4 (C-1), 101.3 (PhCH), 79.2 (C-3), 78.2 (C-2), 75.3 (2-PhCH2O), 74.1 (OCH2COCH2CH2CH3), 73.7 (C-4), 72.0 (3-PhCH2O), 69.0 (C-6), 66.6 (C-5), 41.0 (OCH2COCH2CH2CH3), 16.4 (OCH2COCH2CH2CH3), 13.7 (OCH2COCH2CH2CH3). HRMS (FAB): calcd. for C32H36O7Na 555.2359; found, 555.2371 [M+Na]+. Anal. Calcd. for C32H36O7: C, 72.16; H, 6.81. Found: C, 72.05; H, 6.73. 4.1.2.3.3. 2-Oxoundecyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-β-D-galactopyranoside (41) The solid was purified by column chromatography using hexane-ethyl acetate (3:1) as eluent ( 0.498 g, 82% yield). Mp 122–123 ºC; [α]D = +21.2 (c 1.0, CH2Cl2); MS (FAB): m/z 639 (100%) [M+Na]+. 1H NMR (500 MHz, CDCl3,  ppm): 7.6–7.2 (m, 15H, Ph), 5.49 (s, 1H, PhCH), 4.97 (d, 1H, Jgem = 11.0 Hz, 2-PhCHAHBO), 4.84 (d, 1H, Jgem = 11.0 Hz, 2-PhCHAHBO), 4.76 (d, 1H, Jgem = 12.0 Hz, 3-PhCHAHBO), 4.75 (d, 1H, Jgem = 12.0 Hz, 3-PhCHAHBO), 4.43 (d, 1H, J1,2 = 7.7 Hz, H-1), 4.32 [d, 1H, Jgem = 16.5 Hz, OCHAHBCOCH2(CH2)7CH3], 4.26 (dd, 1H, J5,6e = 1.5 Hz, J6e,6a = 12.4 Hz, H-6e), 4.24 [d, 1H, Jgem = 16.5 Hz, OCHAHBCOCH2(CH2)7CH3], 4.12 (dd, 1H, J3,4 = 3.7 Hz, J4,5 = 1.0 Hz, H-4), 4.01 (dd, 1H, J5,6a = 1.7 Hz, J6e,6a = 12.3 Hz, H-6a), 3.92 (dd, 1H, J1,2 = 7.7 Hz, J2,3 = 9.6 Hz, H-2), 3.57 (m, 1H, H-3), 3.32 (s, 1H, H-5), 2.63 [m, 1H, OCH2COCHAHB(CH2)7CH3], 2.55 [m, 1H, OCH2COCHAHB(CH2)7CH3], 1.24 [m, 14H, OCH2COCH2(CH2)7CH3], 0.88 [t, 3H, J = 7.4 Hz, OCH2COCH2(CH2)7CH3]. 13C NMR (125 MHz, CDCl3,  ppm): 209.2 [OCH2COCH2(CH2)7CH3], 128.9–126.4 (3Ph), 103.3 (C-1), 101.3 (PhCH), 79.2 (C-3), 78.2 (C-2), 75.3 (2-PhCH2O), 74.1 [OCH2COCH2(CH2)7CH3], 73.7 (C-4), 72.0 (3-PhCH2O), 69.1 (C-6), 66.6 (C-5), 39.2 [OCH2COCH2(CH2)7CH3], 31.8–22.6 [OCH2COCH2(CH2)7CH3], 14.1 [OCH2COCH2(CH2)7CH3]. HRMS (FAB): calcd. for C38H48O7Na 639.3298; found, 639.3281 [M+Na]+. Anal. Calcd. for C38H48O7: C, 74.00; H, 7.84. Found: C, 74.04; H, 7.80. 4.1.2.3.4. 2-Oxohexadecyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-β-D- galactopyranoside (42) The solid was purified by column chromatography using hexane-ethyl acetate (2.5:1) as eluent (0.470 g , 75% yield). Mp 136–137 ºC; [α]D = +12.2 (c 1, CH2Cl2); MS (FAB): m/z 709 (80%) [M+Na]+. 1H NMR (500 MHz, CDCl3,  ppm): 7.6–7.2 (m, 15H, Ph), 5.49 (s, 1H, PhCH), 4.97 (d, 1H, Jgem = 10.9 Hz, 2-PhCHAHBO), 4.85 (d, 1H, Jgem = 10.9 Hz, 2-PhCHAHBO), 4.78 (d, 1H, Jgem = 12.3 Hz, 3-PhCHAHBO), 4.75 (d, 1H, Jgem = 12.3 Hz, 3-PhCHAHBO), 4.44 (d, 1H, J1,2 = 7.7 Hz, H-1), 4.34 [d, 1H, Jgem = 16.5 Hz, OCHAHBCOCH2(CH2)12CH3], 4.26 (dd, 1H, J5,6e = 1.2 Hz, J6e,6a = 12.3 Hz, H-6e), 4.15 [d, 1H, Jgem = 16.5 Hz, OCHAHBCOCH2(CH2)12CH3], 4.12 (d, 1H, J3,4 = 3.6 Hz, H-4), 4.00 (dd, 1H, J5,6a = 1.4 Hz, J6e,6a = 12.3 Hz, H-6a), 3.93 (dd, 1H, J1,2 = 7.7 Hz, J2,3 = 9.7 Hz, H-2), 3.58 (dd, 1H, J2,3 = 9.7 Hz, J3,4 = 3.6 Hz, H-3), 3.32 (s, 1H, H-5), 2.65 [m, 1H, OCH2COCHAHB(CH2)12CH3], 2.55 [m, 1H, OCH2COCHAHB(CH2)12CH3], 1.6–1.2 [m, 24H, OCH2COCH2(CH2)12CH3], 0.88 [t, 3H, J = 7.0 Hz, OCH2COCH2(CH2)12CH3]. 13C NMR (125 MHz, CDCl3,  ppm): 209.3 [OCH2COCH2(CH2)12CH3], 138.7–126.4 (3Ph), 103.3 (C-1), 101.3 (PhCH), 79.2 (C-3), 78.2 (C-2), 75.3 (2-PhCH2O), 74.1 [OCH2COCH2(CH2)12CH3], 73.7 (C-4), 72.0 (3-PhCH2O), 69.1 (C-6), 66.6 (C-5), 39.2 [OCH2COCH2(CH2)12CH3], 31.9–22.7 [OCH2COCH2(CH2)12CH3], 14.1 [OCH2COCH2(CH2)12CH3]. HRMS (FAB): calcd. for C43H58O7Na 709.4080; found, 709.4070 [M+Na]+. Anal. Calcd. for C43H58O7: C, 75.19; H, 8.51. Found: C, 75.07; H, 8.35. 4.1.2.4. Wittig reaction (43-46) A suspension of methyl (triphenyl)phosphonium bromide (1.2 mmol) in dry THF (40 mL) was cooled to –70 °C, then a 2.5 M solution of n-butyllithium in hexane (2.3 mmol) was added dropwise under argon atmosphere, maintaining the temperature at – 70 °C. The mixture was stirred until it became bright red (ylide formation, ~1 h) and allowed to slowly warm up to –30 °C, the keto derivate (1.0 mmol) was added, and the mixture was stirred for 1 h at –30 °C and left to stand for 12 h at 20 °C. The reaction mixture was concentrated and the resultant crude was purified by column chromatography. 4.1.2.4.1. 2-Phenylallyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-β-D-galactopyranoside (43) The solid was purified by column chromatography using hexane-ethyl acetate (4:1) as eluent(0.372 g , 65% yield). Mp 155–156 ºC; [α]D = +8.3 (c 0.5, CH2Cl2); MS (FAB): m/z 587 (20%) [M+Na]+. 1H NMR (500 MHz, CDCl3,  ppm): 7.6–7.2 (m, 20H, Ph), 5.56 [s, 1H, OCH2C(Ph)=CHAHB], 5.51 (s, 1H, PhCH), 5.45 [s, 1H, OCH2C(Ph)=CHAHB], 4.87 [d, 1H, Jgem 12.6 Hz, OCHAHBC(Ph)=CH2], 4.78–4.70 (m, 3H, 2-PhCHAHBO, 3-PhCH2O), 4.67 (d, 1H, Jgem = 10.8 Hz, 2-PhCHAHBO), 4.54 (d, 1H, J1,2 = 7.7 Hz, H-1), 4.51 [d, 1H, Jgem = 12.6 Hz, OCHAHBC(Ph)=CH2], 4.33 (dd, 1H, J5,6e = 1.3 Hz, J6e,6a = 12.3 Hz, H-6e), 4.11 (d, 1H, J3,4 = 3.6 Hz, H-4), 4.04 (dd, 1H, J5,6a = 1.7 Hz, J6e,6a = 12.3 Hz, H-6a), 3.89 (dd, 1H, J1,2 = 7.7 Hz, J2,3 = 9.6 Hz, H-2), 3.57 (dd, 1H, J2,3 = 9.6 Hz, J3,4 = 3.6 Hz, H-3), 3.34 (s, 1H, H-5). 13C NMR (125 MHz, CDCl3): δ 143.5 [OCH2C(Ph)=CH2], 129.0–126.2 (4Ph), 114.9 [OCH2C(Ph)=CH2], 102.3 (C-1), 101.3 (PhCH), 79.4 (C-3), 78.4 (C-2), 75.2 (2-PhCH2O), 74.0 (C-4), 72.0 (3-PhCH2O), 70.4 [OCH2C(Ph)=CH2], 69.2 (C-6), 66.5 (C-5). HRMS (FAB): calcd. for C36H36O6Na 587.2410; found, 587.2399 [M+Na]+. Anal. Calcd. for C36H36O6: C, 76.57; H, 6.43. Found: C, 76.53; H, 6.52. 4.1.2.4.2. 2-Propylallyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-β-D-galactopyranoside (44) The solid was purified by column chromatography using hexane-ethyl acetate (3.5:1) as eluent (0.344 g, 65% yield). Mp 138–139 ºC; [α]D = +18.6 (c 0.5, CH2Cl2); MS (FAB): m/z 553 (20%) [M+Na]+. 1H NMR (500 MHz, CDCl3,  ppm): 7.6–7.2 (m, 15H, Ph), 5.50 (s, 1H, PhCH), 5.09 [s, 1H, OCH2C(CH2CH2CH3)=CHAHB], 4.95 (d, 1H, Jgem = 10.8 Hz, 2-PhCHAHBO), 4.91 [s, 1H, OCH2C(CH2CH2CH3)=CHAHB,], 4.81–4.73 (m, 3H, 2-PhCHAHBO, 3-PhCH2O), 4.42 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.37 [d, 1H, Jgem = 12.6 Hz, OCHAHBC(CH2CH2CH3)=CH2], 4.31 (dd, 1H, J5,6e = 1.5 Hz, J6e,6a = 12.4 Hz, H- 6e), 4.11 (d, 1H, J = 3.7 Hz, H-4), 4.07 [d, 1H, Jgem = 12.6 Hz, OCHAHBC(CH2CH2CH3)=CH2], 4.02 (dd, 1H, J5,6a = 1.7 Hz, J6e,6a = 12.3 Hz, H-6a), 3.88 (dd, 1H, J1,2 = 7.8 Hz, J2,3 = 9.6 Hz, H-2), 3.57 (dd, 1H, J2,3 = 9.6 Hz, J3,4 = 3.7 Hz, H-3), 3.31 (s, 1H, H-5), 2.08 [m, 2H, OCH2C(CH2CH2CH3)=CH2], 1.48 [m, 2H, OCH2C(CH2CH2CH3)=CH2], 0.88 [t, 3H, J = 7.40 Hz, OCH2C(CH2CH2CH3)=CH2]. 13C NMR (125 MHz, CDCl3,  ppm): 145.3 [OCH2C(CH2CH2CH3)=CH2], 128.8–126.5 (3Ph), 111.9 [OCH2C(CH2CH2CH3)=CH2], 102.2 (C-1), 101.3 (PhCH), 79.4 (C-3), 78.4 (C-2), 75.3 (2-PhCH2O), 74.1 (C-4), 72.0 (3-PhCH2O), 71.6 [OCH2C(CH2CH2CH3)=CH2], 69.2 (C-6), 66.4 (C-5), 35.2 [OCH2C(CH2CH2CH3)=CH2], 20.6 [OCH2C(CH2CH2CH3)=CH2], 13.8 [OCH2C(CH2CH2CH3)=CH2]. HRMS (FAB): calcd. for C33H38O6Na 553.2566; found, 553.2579 [M+Na]+. Anal. Calcd. for C33H38O6: C, 74.69; H, 7.22. Found: C, 74.78; H, 7.10. 4.1.2.4.3. 2-Nonylallyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-β-D-galactopyranoside (45) The solid was purified by column chromatography using hexane-ethyl acetate (4:1) as eluent (0.456 g, 74% yield). Mp 126–127 ºC; [α]D = +32.1 (c 0.5, CH2Cl2); MS (FAB): m/z 639 (60%) [M+Na]+. 1H NMR (500 MHz, CDCl3,  ppm): 7.6–7.2 (m, 15H, Ph), 5.49 (s, 1H, PhCH), 5.08 [s, 1H, OCH2C(CH2(CH2)7CH3)=CHAHB], 4.94 (d, 1H, Jgem = 11.0 Hz, 2-PhCHAHBO), 4.90 [s, 1H, OCH2C(CH2(CH2)7CH3)=CHAHB], 4.79 (d, 1H, Jgem = 11.0 Hz, 2-PhCHAHBO), 4.76 (d, 1H, Jgem = 12.0 Hz, 3-PhCHAHBO), 4.74 (d, 1H, Jgem = 12.0 Hz, 3-PhCHAHBO), 4.42 (d, 1H, J1,2 = 7.7 Hz, H-1), 4.34 (d, 1H, Jgem = 16.5 Hz, OCHAHBC(CH2(CH2)7CH3)=CH2, 4.29 (dd, 1H, J5,6e = 1.5 Hz, J6e,6a = 12.4 Hz, H-6e), 4.11 (dd, 1H, J3,4 = 3.7 Hz, J4,5 = 1.0 Hz, H-4), 4.06 [d, 1H, Jgem = 16.5 Hz, OCHAHBC(CH2(CH2)7CH3)=CH2], 4.00 (dd, 1H, J5,6a = 1.7 Hz, J6e,6a = 12.3 Hz, H-6a), 3.88 (dd, 1H, J1,2 = 7.7 Hz, J2,3 = 9.6 Hz, H-2), 3.56 (m, 1H, H-3), 3.30 (s, 1H, H-5), 2.08 [m, 2H, OCH2C(CH2(CH2)7CH3)=CH2], 1.32 [m, 14H, OCH2C(CH2(CH2)7CH3)=CH2], 0.89 [t, 3H, J = 7.40 Hz, OCH2C(CH2(CH2)7CH3)=CH2]. 13C NMR (125 MHz, CDCl3,  ppm): 145.6 [OCH2C(CH2(CH2)7CH3)=CH2], 125.9–122.4 (3Ph), 111.7 [OCH2C(CH2(CH2)7CH3)=CH2], 102.2 (C-1), 101.3 (PhCH), 79.4 (C-3), 78.4 (C-2), 75.3 (2-PhCH2O), 74.0 (C-4), 72.0 (3-PhCH2O), 71.6 [OCH2C(CH2(CH2)7CH3)=CH2], 69.2 (C-6), 66.4 (C-5), 33.1 [OCH2C(CH2(CH2)7CH3)=CH2], 31.9–22.6 [OCH2C(CH2(CH2)7CH3)=CH2], 14.1 [OCH2C(CH2(CH2)7CH3)=CH2]. HRMS (FAB): calcd. for C39H52O6Na 639.3662; found, 639.3644 [M+Na]+. Anal. Calcd. for C39H52O6: C, 76.19; H, 8.20. Found: C, 76.12; H, 8.20. 4.1.2.4.4. 2-Tetradecylallyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-β-D- galactopyranoside (46) The solid was purified by column chromatography using hexane-ethyl acetate (3.5:1) as eluent (0.480 g, 77% yield). Mp 123–124 ºC; [α]D = +28.3 (c 0.5, CH2Cl2); MS (FAB): m/z 707 (20%) [M+Na]+. 1H NMR (500 MHz, CDCl3,  ppm): 7.6–7.2 (m, 15H, Ph), 5.51 (s, 1H, PhCH), 5.10 [s, 1H, OCH2C(CH2(CH2)12CH3)=CHAHB], 4.97 (d, 1H, Jgem = 10.8 Hz, 2-PhCHAHBO), 4.92 [s, 1H, OCH2C(CH2(CH2)12CH3)=CHAHB,], 4.82–4.74 (m, 3H, 2-PhCHAHBO, 3-PhCH2O), 4.43 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.38 [d, 1H, Jgem = 12.6 Hz, OCHAHBC(CH2(CH2)12CH3)=CH2], 4.32 (dd, 1H, J5,6e = 1.5 Hz, J6e,6a = 12.4 Hz, H-6e), 4.11 (d, 1H, J3,4 = 3.7 Hz, H-4), 4.09 [d, 1H, Jgem = 12.6 Hz, OCHAHBC(CH2(CH2)12CH3)=CH2], 4.02 (dd, 1H, J5,6a = 1.7 Hz, J6e,6a = 12.3 Hz, H-6a), 3.89 (dd, 1H, J1,2 = 7.8 Hz, J2,3 = 9.6 Hz, H-2), 3.58 (dd, 1H, J2,3 = 9.6 Hz, J3,4 = 3.7 Hz, H-3), 3.31 (s, 1H, H-5), 2.10 [m, 2H, OCH2C(CH2(CH2)12CH3)=CH2], 1.5–1.2 [m, 24H, OCH2C(CH2(CH2)12CH3)=CH2], 0.90 [t, 3H, J = 7.0 Hz, OCH2C(CH2(CH2)12CH3)=CH2]. 13C NMR (125 MHz, CDCl3,  ppm): 145.6 [OCH2C(CH2(CH2)12CH3)=CH2], 138.8–126.5 (3Ph), 111.7 [OCH2C(CH2(CH2)12CH3)=CH2], 102.2 (C-1), 101.3 (PhCH), 79.4 (C-3), 78.4 (C-2), 75.3 (2-PhCH2O), 74.0 (C-4), 72.0 (3-PhCH2O), 71.7 [OCH2C(CH2(CH2)12CH3)=CH2], 69.2 (C-6), 66.4 (C-5), 33.1 [OCH2C(CH2(CH2)12CH3)=CH2], 31.9–22.7 [OCH2C(CH2(CH2)12CH3)=CH2], 14.1 [OCH2C(CH2(CH2)12CH3)=CH2]. HRMS (FAB): calcd. for C44H60O6Na 707.4288; found, 707.4268 [M+Na]+. Anal. Calcd. for C44H60O6: C, 77.16; H, 8.83. Found: C, 77.11; H, 8.69. 4.1.3. Aziridination reaction (22–25, 28–31, 47-50) To a mixture of the olefin (1.0 mmol) and Chloramine-T·3H2O (1.5 mmol) in CH3CN (15 mL), PTAB (0.1 mmol) was added, and the reaction mixture was vigorously stirred at –20 °C for 12 h. Then the mixture was concentrated to dryness and the resultant crude was purified by column chromatography. 4.1.3.1. 2-Methyl-2,3-[N-(4-methylbenzenesulfonyl)imino]propyl 2,3-di-O-benzyl-4,6- O-(S)-benzylidene-  -D-galactopyranoside (22) Only one stereoisomer was obtained (>99% de). The solid was purified by column chromatography using hexane-ethyl acetate (2:1) as eluent (0.450 g, 67% yield). Mp 67–68 ºC; [α]D = +52.3 (c 1.0, CH2Cl2); MS (FAB): m/z 694 (50%) [M+Na]+. 1H NMR (500 MHz, CDCl3,  ppm): 7.9–7.2 (m, 19H, Ar), 5.49 (s, 1H, PhCH), 4.75 (d, 1H, Jgem = 11.0 Hz, 2-PhCHAHBO), 4.7–4.6 (m, 3H, 2-PhCHAHBO, 3-PhCH2O), 4.41 (d, 1H, J1,2 = 7.7 Hz, H-1), 4.29 (dd, 1H, J5,6e = 1.1 Hz, J6e,6a = 12.3 Hz, H-6e), 4.11 (dd, 1H, J3,4 = 3.7 Hz, J4,5 = 1.0 Hz, H-4), 4.06 [d, 1H, Jgem = 10.8 Hz, OCHAHBC(CH3)(NTs)CH2], 4.01 (dd, 1H, J5,6a = 1.5 Hz, J6e,6a = 12.3 Hz, H-6a), 3.77 (dd, 1H, J1,2 = 7.8 Hz, J2,3 = 9.6 Hz, H-2), 3.62 [d, 1H, Jgem = 10.8 Hz, OCHAHBC(CH3)(NTs)CH2], 3.53 (m, 1H, H-3), 3.31 (s, 1H, H-5), 2.59 [s, 1H, OCH2C(CH3)(NTs)CHAHB], 2.50 [s, 1H, OCH2C(CH3)(NTs)CHAHB], 2.36 [s, 3H, OCH2C(CH3)(NSO2C6H4CH3)CH2], 1.71 [s, 3H, OCHAHBC(CH3)(NTs)CH2]. 13C NMR (125 MHz, CDCl3,  ppm): 143.8–126.4 (4Ar), 103.3 (C-1), 101.2 (PhCH), 79.0 (C-3), 78.2 (C-2), 75.1 (2-PhCH2O), 73.8 (C-4), 71.9 (3-PhCH2O), 71.8 [OCH2C(CH3)(NTs)CH2], 69.1 (C-6), 66.5 (C-5), 48.6 [OCH2C(CH3)(NTs)CH2], 38.6 [OCH2C(CH3)(NTs)CH2], 21.5 [OCH2C(CH3)(NSO2C6H4CH3)CH2], 16.4 [OCH2C(CH3)(NTs)CH2]. HRMS (FAB): ): calcd. for C38H41NO8SNa 694.2451; found, 694.2454 [M+Na]+. Anal. Calcd. for C38H42NO8S: C, 67.84; H, 6.29; N, 2.08; S, 4.77. Found: C, 67.97; H, 6.07; N, 2.01; S, 4.41. 4.1.3.2. 2,3-[N-(4-methylbenzenesulfonyl)imino]propyl 2,3-di-O-benzyl-4,6-O-(S)- benzylidene-  -D-galactopyranoside (23) Two stereoisomers were obtained in a 7.7:1 ratio (77% de). The major diastereoisomer was isolated as a syrup by column chromatography using hexane-ethyl acetate (1.5:1) as eluent (0.486 g , 74% yield); D = +24.6 (c 0.5, CH2Cl2); MS (FAB): m/z 680 (1%) M+Na+. 1H NMR (500 MHz, CDCl3,  ppm): 7.97.2 (m, 19H, Ar), 5.50 (s, 1H, PhCH), 4.98 (d, 1H, Jgem = 10.8 Hz, 2-PhCHAHBO), 4.8–4.7 (m, 3H, 2-PhCHAHBO, 3- PhCH2O), 4.48 (d, 1H, J1,2 = 7.7 Hz, H-1), 4.37–4.25 [m, 3H, H-6e, OCHAHBCH(NTs)CH2], 4.12 (m, 1H, H-4), 4.02 (m, 1H, H-6a), 3.95 [m, 1H OCHAHBCH(NTs)CH2], 3.90–3.79 [m, 3H, H-2, OCH2CH(NTs)CH2], 3.58 (m, 1H, H- 3), 3.34 (m, 1H, H-5), 1.55 [s, 3H, OCH2CH(NSO2C6H4CH3)CH2]. 13C NMR (125 MHz, CDCl3,  ppm): 143.6–126.4 (Ar), 103.8, 103.7 (C-1), 101.3 (PhCH), 79.3, 79.2 (C-3), 78.3, 78.2 (C-2), 75.4 (2-PhCH2O), 73.8 (C-4), 72.1 (3-PhCH2O), 70.9, 70.5 [OCH2CH(NTs)CH2], 69.1 (C-6), 66.6 (C-5), 49.2, 49.1 [OCH2CH(NTs)CH2], 34.2, 33.2 [OCH2CH(NTs)CH2], 21.6 [OCH2CH(NSO2C6H4CH3)CH2]. HRMS (FAB): calcd. for C37H39NO8SNa 680.2294; found; 680.2271 M+Na+. 4.1.3.3. 2-Methyl-2,3-[N-(4-methylbenzenesulfonyl)imino]propyl 4,6-O-(S)- benzylidene-  -D-galactopyranoside (24) Two stereoisomers were obtained in a 7.6:1 ratio (77% de). The pure diastereomeric mixture was obtained as a solid by column chromatography using hexane-ethyl acetate (1:3) as eluent (0.319 g , 65% yield). Mp 95–96 ºC; [α]D = +4.1 (c 1.0, CH2Cl2); MS (CI): m/z 492 (20%) [M+H]+. 1H NMR (500 MHz, CDCl3,  ppm): 7.9–7.3 (m, 9H, Ar), 5.56 (s, 0.11H, PhCH minor), 5.55 (s, 0.89H, PhCH major), 4.41 (d, 0.11H, J1,2 = 7.6 Hz, H-1 minor), 4.37(d, 0.89H, J1,2 = 7.6 Hz, H-1 major), 4.32 (dd, 1H, J5,6e = 1.5 Hz, J6e,6a = 12.4 Hz, H-6e), 4.27 (dd, 1H, J3,4 = 3.7 Hz, J4,5 1.0 Hz, H-4), 4.13–4.06 [m, 2H, H-6a, OCHAHBC(CH3)(NTs)CH2], 3.81 [d, 1H, Jgem = 11.4 Hz, OCHAHBC(CH3)(NTs)CH2], 3.77 (dd, 1H, J1,2 = 7.8 Hz, J2,3 9.6 Hz, H-2), 3.69 (m, 1H, H-3), 3.50 (s, 1H, H-5), 2.67 [s, 1H, OCH2C(CH3)(NTs)CHAHB], 2.54 [s, 1H, OCH2C(CH3)(NTs)CHAHB], 2.41 [s, 3H, OCH2C(CH3)(NSO2C6H4CH3)CH2], 1.73 [s, 0.33H, OCH2C(CH3)(NTs)CH2 minor], 1.73 [s, 2.67H, OCH2C(CH3)(NTs)CH2 major]. 13C NMR (125 MHz, CDCl3, δ ppm): 129.6–126.4 (2Ar), 103.2 (C-1), 101.3 (PhCH), 75.1 (C-4), 72.7 [OCH2C(CH3)(NTs)CH2], 71.3 (C-3), 70.4 (C-2), 69.1 (C-6), 66.8 (C- 5), 49.4 [OCH2C(CH3)(NTs)CH2], 38.4 [OCH2C(CH3)(NTs)CH2], 21.5 [OCH2C(CH3)(NSO2C6H4CH3)CH2], 17.1 [OCH2C(CH3)(NTs)CH2]. HRMS (CI): calcd. for C24H30NO8S 492.169; 2 found, 492.1699 [M+H]+. Anal. Calcd. for 4.2. Citotoxicity assays 4.2.1. Materials and cell culture conditions The human A549 lung cancer cell line, the human lung fibroblastic MRC-5 cell line, the human MCF7 breast adenocarcinoma cell line, the human VH-10 fibroblastic foreskin cell line and the human UACC-62 melanoma cell line were maintained in DMEM supplemented with 2 mM glutamine, 50 μg/ml penicillin, 50 μg/ml streptomycin and 10% foetal bovine serum. The human MCF10 breast epithelial cell line was maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium supplemented with 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 10 μg/ml insulin and 500 ng/ml of hydrocortisone (95%) and horse serum (5%). Cell lines were cultured at 37 °C in a humidified atmosphere containing 5% CO2. Cell culture reagents were obtained from Life Technologies. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] salt, 5-Fluorouracil, cisplatin and were purchased from Sigma Chemical Co. 4.2.3.2. Cell Proliferation Assays The MTT assay is a colorimetric technique that allows the quantitative determination of cell viability. It is based on the capability of viable cells to transform the MTT salt [3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] into a formazan dye. Exponentially growing cells were seeded into 96-well plates and drugs were added 24 h later. Following an incubation period of 48 h, the medium was removed and MTT (125 μL, 1 mg/mL in medium) was added to each well for 5 h. Then 20% sodium dodecyl sulfate (80 μL) in 0.02 m HCl was added, plates were incubated for 10 h at 37 °C and optical densities were measured at 540 nm on a multi-well plate spectrophotometer reader. Cell viability was expressed as a percentage relative to controls. 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