Synthesis and anticancer profile of novel sphingoid base-like compounds with a quaternary stereocentre

Carbohydrate Research 487 (2020) 107862

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Synthesis and anticancer profile of novel sphingoid base-like compounds with a quaternary stereocentre

T

Dominika Jackováa, Mária Brunderováa, Martin Fábiana, Miroslava Martinkováa,∗, Jozef Gondaa, Martina Bago Pilátováb a b

Institute of Chemical Sciences, Department of Organic Chemistry, P.J. Šafárik University, Moyzesova 11, 040 01, Košice, Slovak Republic Institute of Pharmacology, Faculty of Medicine, P.J. Šafárik University, SNP 1, 040 66, Košice, Slovak Republic

ARTICLE INFO

ABSTRACT

Keywords: Sphingoid base Quaternary stereocentre [3,3]-sigmatropic rearrangements Metathesis Antiproliferative activity

The synthesis of novel sphingoid base-like compounds with a quaternary stereocentre was achieved in a sequence featuring [3,3]-sigmatropic rearrangements and olefin cross-metathesis transformation as the key reaction steps, which were accompanied by the rational selection of suitable functional group transformations. The stereochemistry of the desired tetra-substituted carbon bearing nitrogen functionality was determined via NOESY experiments of the advanced oxazolidine-2-thiones. Cell viability experiments revealed significant antiproliferative/cytotoxic activity of the target compounds 7, ent-7 and 29 against the Jurkat cell line, with the IC50 values of 6.6 μM, 5.6 μM and 6.1 μM, respectively.

1. Introduction α-Substituted α-amino acids, such as myriocin 1, sphingofungins, and mycestericins, the general structures of which are illustrated by sphingofungins E (2), F (3) and mycestericin A (4), respectively, and sulfamisterin 5 (Fig. 1), represent a family of fungal metabolites structurally related to sphingolipids [1]. These amino acid natural products inhibit serine palmitoyltranferase (SPT) [2], a key enzyme that catalyzes the first step in the biosynthesis of sphingolipids in eukaryotic cells [3]. Myriocin 1, mycestericins and sulfamisterin 5 are well-known immunosuppressants [1a], and sphingofungins E and F act as antifungal agents [1a]. It should be noted that Fujita and co-workers [4] used myriocin as a lead compound for the development of the more potent and less toxic immunosuppressant agent FTY720 (6) [5] (Fig. 1), which was approved by the FDA as an orally active agent for the treatment of relapsing forms of multiple sclerosis [6]. From a structural standpoint, the aforementioned SPT inhibitors have an amphiphilic character resulting from the combination of their long hydrocarbon chain and a hydroxylated serine skeleton bearing the quaternary stereocentre. The interesting structural profile of these compounds, coupled with their promising activities, has stimulated the interest of organic community in them for the development of many synthetic strategies [1,7], as well as their analogues [7j,7l,8]. Since Bittmanʼs review in 2006, 15 total syntheses of these SPT inhibitors

∗

have been elaborated. Most of them have relied on the Chiron approach, and substrates, such as L-quebrachitol [7a], chiral dehydroamino acid [7b], L-tartaric acid [7c,7f], L-tartrates [7d,7m], D-glucose [7e], (R)-serine [7h], enantiomerically pure epoxide [7i], D-xylosederived oxazolidinones [7j], D-ribose [7k,7n], diethyl-D-tartrate [7l], were chosen as the starting materials. On the other hand, there are only two examples of a total synthesis, which used an enantioselective approach and achiral material (β-propiolactone [7g] and trishydroxymethylaminomethane [7o]). Given that natural sphingoid bases and their structurally related analogues exhibit significant biological properties [9], the preparation of new derivatives would be useful for further exploration of their structure-activity relationships as well as for the possible discovery of drugs. With this premise in mind, and in a continuation of our interest in the construction of the various sphingosines [10], including sphingolipids cyclic derivatives such as tetrahydrofurans [11a-11d] and pyrrolidines [11e-11f] as well as sphingoid base-like compounds [7e,7j,12], we decided to focus our attention on synthesizing of novel types of isomeric amino alcohols bearing a quaternary stereocentre. The polar heads of our target compounds could constitute the modified analogues of the hydrophilic part of the natural SPT inhibitors outlined in Fig. 1. On the other hand, the proposed final molecules could also be considered as the isomeric derivatives of D-erythro-sphingosine [3] (Scheme 1), the most common sphingoid base in eukaryotic cells, in which a formal permutation of the C-2 and C-3 substituents was made,

Corresponding author. E-mail address: [email protected] (M. Martinková).

https://doi.org/10.1016/j.carres.2019.107862 Received 27 September 2019; Received in revised form 5 November 2019; Accepted 5 November 2019 Available online 11 November 2019 0008-6215/ © 2019 Elsevier Ltd. All rights reserved.

Carbohydrate Research 487 (2020) 107862

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Fig. 1. Structures of the natural SPT inhibitors and the synthetic drug FTY720.

Scheme 1. Retrosynthetic analysis of our final molecules 7 and ent-7 and their design.

and a hydrogen atom at the novel C-3 position was replaced by the hydroxymethyl side chain (Scheme 1).

obtained from commercially available dimethyl L-tartrate in a 59% overall yield (three steps) according to the reported procedure (Scheme 2) [15]. Exposure of 10 to MOMCl and Hünig's base delivered the corresponding methoxymethyl ether 11 (94%). The O-Bn group in 11 was removed by catalytic hydrogenation using a combination of two catalysts to furnish alcohol 12 (91%). IBX-mediated oxidation of 12 gave the stable ketone 13 (94%), which was then submitted to HWE olefination, affording a mixture of the α,β-unsaturated esters 14 (E:Z = 85:15, determined by NMR analysis) with a combined yield of 94%. The geometries of the double bonds were assigned via NOESY experiments, which were conducted on both isomers (see Supporting Information). Subsequent DIBAl-H reduction of the major ester (E)-14 provided the expected synthon 15 (96%, Scheme 2). To build up the quaternary stereocentre, we examined the [3,3]sigmatropic rearrangements of allylic substrates 16 and 17 (Scheme 3). The preparation of the corresponding thiocyanate 16 was accomplished by means of nucleophilic substitution after mesylation of 15 (73% yield, over two steps). On the other hand, treatment of 15 with CCl3CN

2. Results and discussion 2.1. Chemistry As shown in the retrosynthetic strategy outlined in Scheme 1, a well-established olefin cross-metathesis is expected to construct the carbon backbone of our final molecules 7 and ent-7. In the case of sphingoid base 7, the [3,3]-sigmatropic rearrangement would install the quaternary stereogenic centre in amine 8, which could be accessible from dimethyl L-tartrate. On the other hand, we planned to prepare the enantiomeric congener ent-7 from the known furanose 9 [13], whose tetrasubstituted carbon with nitrogen was introduced via an aza-Claisen transformation with excellent stereocontrol (de = 100%). The synthesis of the template 15 started from the cyclohexylidene derivative 10 [14] (see the Supporting Information), which was 2

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Scheme 2. Reagents and conditions: (a) (i) PhCHO, p-TsOH, benzene, 95 °C, 77%; (ii) LiAlH4, AlCl3, Et2O/CH2Cl2, 0 °C→rt→reflux, 83%; (iii) cyclohexanone, p-TsOH, toluene, rt, 93%; (b) MOMCl, DIPEA, CH2Cl2, 0 °C→rt; (c) H2, Pd/C, 20% Pd(OH)2/C, MeOH, rt; (d) IBX, MeCN, reflux; (e) (EtO)2P(O)CH2CO2Et, NaH, THF, 0 °C; (f) DIBAl-H, CH2Cl2, ‒50 °C→(−)30 °C.

in the presence of a catalytic amount of DBU yielded the trichloroacetimidate 17, which was used in its crude form. Construction of two intermediates, 16 and 17, thus set the stage for carrying out the aforementioned rearrangement processes. The aza-Claisen [3,3]-sigmatropic rearrangement of 16 was done in n-heptane at 70 °C, 90 °C and 110 °C using both conventional heating as well as microwave irradiation conditions to provide the corresponding isothiocyanates 18 and 19 in very good yields, though surprisingly without stereocontrol (Table 1). The diastereomeric rearranged products were then readily separated using column chromatography. The microwave assisted synthesis of trichloroacetamides 20 and 21 from imidate 17 took place in o-xylene in the presence of K2CO316 at four different temperatures. As shown in Table 2, the Overman rearrangement proceeded with a moderate selectivity (20:21–1:2), providing both products in good combined yields, but as an inseparable mixture of diastereoisomers due to their identical Rf values. The prepared amides were separated later, in the stage of their amines 8 and 24 (vide infra). The stereochemistry of the installed quaternary stereocentre was confirmed either via NOESY experiments realized on the corresponding oxazolidine-2-thiones 22 (67%) and 23 (65%), which were derived from the aza-Claisen products 18 and 19 by the action of p-TsOH in MeOH, or by the method of chemical correlation in the case of amides 20 and 21 (Scheme 4). The stronger enhancements between the vinyl

Table 1 [3,3]-Sigmatropic rearrangement of thiocyanate 16. Entry

Conditionsa

Time (h)

Ratiob 18:19

Yieldc (%)

Isolated thiocyanate 16 (%)

1 2 3 4 5 6

Δ, 70 °C Δ, 90 °C Δ, 110 °C MW, 70 °C MW, 90 °C MW, 110 °C

7 2.5 2 4 1.5 0.5

46:54 48:52 49:51 44:56 47:53 48:52

82 85 84 66 84 84

17 5 8 21 6 6

a b c

In n-heptane. Ratio in the crude reaction mixtures determined by1H NMR. Isolated combined yield.

proton and the hydrogen at C-5 in 22 suggested that they occupy the same face of the oxazolidine-2-thione skeleton (Scheme 4). In order to obtain the common products 24 and 8, a reduction of the –NCS functionality in 18 and 19 with bis(tributyltin)oxide (TBTO) [17] and the trichloroacetyl group in 20 and 21 with DIBAL-H was required. In the case of an inseparable mixture of amides, the produced diastereomeric amines 24 (32%) and 8 (54%) were easily separated on silica gel. The prepared material had physical and spectroscopic properties in excellent agreement with those found for the same structures previously

Scheme 3. Reagents and conditions: (a) (i) MsCl, Et3N, CH2Cl2, 0 °C; (ii) KSCN, MeCN, 0 °C→rt; (b) n-heptane, MW, 110 °C; (c) CCl3CN, DBU, CH2Cl2, 0 °C; (d) Table 2 (20 and 21 isolated as an inseparable mixture of isomers). 3

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33 (98%) was achieved via a three-step approach, initially involving the acid-promoted cleavage of the 1,2-O-isopropylidene moiety in 33, the oxidative fragmentation of a diol moiety with NaIO4, followed by NaBH4 treatment. Finally, hydrolysis of the O-acetyl group and removal of the N-methylcarbamate protecting functionality delivered the targeted compound ent-7 (82%, Scheme 6). The obtained material had spectroscopic data in excellent agreement with those for the antipode 7. The optical rotation was opposite in sign, but its magnitude was comparable to that reported for 7 (see the Experimental section). Moreover, the stereochemistry of the novel quaternary chiral centre in 7 and 29, as well as in the rearranged products 18–21, has been independently assigned through the construction of ent-7.

Table 2 Overman rearrangement of imidate 17.

a

Entry

Conditions

Time (h)

Ratiob 20:21

Yieldc (%)

1 2 3 4

MW, MW, MW, MW,

17 3 1.5 0.5

33:67 34:66 35:65 35:65

59 71 85 83

150 °C 170 °C 190 °C 210 °C

In o-xylene and in the presence of K2CO3 [16]. b Ratio in the crude reaction mixtures determined by1H NMR. c Isolated combined yield.

derived from isothiocyanates 18 and 19, confirming the (S)-configured quaternary chiral centre in the prevalent amide 21. After benzyloxycarbonylation (CbzCl, NaHCO3) of the amino group in 24 and 8 and formation of the protected compounds 25 (98%) and 26 (98%), our focus was then shifted to the olefin cross-metathesis (Scheme 5). After several trials, we found that the manner of addition of the catalyst and the coupling counterpart tetradec-1-ene was important for the outcome of this transformation. When Grubbsʼ second generation catalyst and the corresponding alkene were added in one portion, we recovered at least 60% of the starting material. If the aforementioned components were added in several doses to 25 and 26, the yields of the desired coupling products 27 (58%) and 28 (48%) were improved. In both cases an excellent (E)-selectivity was observed, and the corresponding (Z)-isomer was not detected in the crude reaction mixture. Completion of the synthesis then relied on the tandem hydrogenation/hydrogenolysis of 27 and 28 to remove the double bond along with the Cbz-group and acid-mediated global deprotection, which provided the novel type of sphingoid bases 7 and 29 in 65% and 77% yields (over two steps), respectively. As outlined in our retrosynthetic plan (Scheme 1), the construction of ent-7 started from the protected 3-amino-3-deoxy-3-C-vinyl-α-D-xylofuranose 9 [13] (Scheme 6). Early introduction of the hydrophobic chain was realized by means of an olefin cross-metathesis (OCM) reaction. The microwave-promoted coupling of 9 with tetradec-1-ene in the presence of Grubbsʼ second generation catalyst resulted in the formation of the (E)-configured alkene 30 (69%). Subsequent hydrogenation of 30, directly followed by desilylation of 31 (82%), furnished the advanced furanose intermediate 32 in 94% yield. After acetylation of the liberated hydroxyl group in 32, the synthesis of 34 (63%) from

2.2. Antiproliferative/cytotoxic activity The target compounds 7, ent-7 and 29 together with the three advanced intermediates of our strategy (compounds 18, 19 and 8) were evaluated for their antiproliferative/cytotoxic activities against four different human cancer cell lines ‒ PaTu (human pancreatic adenocarcinoma), HeLa (human cervical adenocarcinoma), A2058 (human melanoma cells), and Jurkat (human acute T-lymphoblastic leukaemia) ‒ as well as the non-malignant cell line MCF-10 (human mammary epithelial cells), using MTT assay with triplicate experiments [18]. The results are shown in Table 3. To allow comparisons in the case of two cancer cell lines, Table 3 also includes IC50 values for the anticancer agent cisplatin. Notably, the final derivatives 7, ent-7 and 29 demonstrate potent and selective antiproliferative/cytotoxic activities against Jurkat cells, with the IC50 values of 6.6 μM, 5.6 μM and 6.1 μM, respectively. Moreover, these aforementioned compounds are about 2.5–3 times more active than cisplatin on this cell line. In addition, sphingoid bases 7 and 29 exhibit interesting activity on HeLa cells comparable to that of cisplatin (Table 3). On the other hand, compound ent-7 was found to be a potent inhibitor of the growth of human melanoma cells A2058 (IC50 = 9.0 μM). Cell viability experiments revealed that screened intermediates 18 and 19 are not toxic, and for amine 8 the cytotoxicity was significantly reduced. 3. Conclusions In summary, three novel sphingoid base-like compounds 7, 29 and

Scheme 4. Reagents and conditions: (a) p-TsOH, MeOH, rt; (b) TBTO, toluene, 90 °C, 81% for 24, 84% for 8; (c) DIBAL-H, CH2Cl2, ‒50 °C→(−)30 °C, 32% for 24, 54% for 8. 4

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Scheme 5. Reagents and conditions: (a) CbzCl, NaHCO3 THF/H2O (1:1), rt; (b) tetradec-1-ene, Grubbs II, toluene, reflux, (c) (i) H2, 10% Pd/C, 20% Pd(OH)2/C, MeOH, rt; (ii) 6 M HCl, rt.

ent-7 bearing a quaternary stereocentre were synthesized from two different chirons: dimethyl L-tartrate and the protected 3-amino-3deoxy-3-C-vinyl-α-D-xylofuranose, respectively. The developed route relied on the [3,3]-sigmatropic aza-Claisen rearrangements and the olefin cross-metathesis reaction. For the targeted sphingoid bases, their capacity to alter the viability of malignant cells was evaluated. Overall, the final compounds 7, ent-7 and 29 demonstrated higher in vitro potency against leukaemia than on solid tumour cells. This observed cell selectivity has prompted us to conduct further experiments to improve the biological profile of our final molecules. The flexible synthetic plan using Grubbs’ coupling to introduce an alkyl chain unit is effective for further investigations of potent candidates with promising anticancer activities. Continuing studies are underway in our laboratory and progress will be reported in due course.

or to the solvent signals CD3OD (δ = 3.31 or δ = 4.87) and C6D6 (δ = 7.16 ppm) and for 13C relative to CDCl3 (δ = 77.16), CD3OD (δ = 49.00) and C6D6 (δ = 128.06 ppm). The multiplicity of the 13C NMR signals concerning the 13C–1H coupling was determined by the HSQC method. Chemical shifts (in ppm) and coupling constants (in Hz) were obtained by first-order analysis; assignments were derived from COSY and H/C correlation spectra. NOESY experiments were carried out on a Varian Premium COMPACT 600 spectrometer in CDCl3 (esters E-14 and Z-14) and CD3OD (compounds 22 and 23). Infrared (IR) spectra were measured with a Nicolet 6700 FT-IR spectrometer and reported in wavenumber (cm−1). High-resolution mass spectra (HRMS) were recorded on a micrOTOF-Q II quadrupole-time of flight hybrid mass spectrometer (Bruker Daltonics). Optical rotations were determined using a P-2000 Jasco polarimeter. Melting points were recorded on a Kofler hot block and are uncorrected. Microwave reactions were carried out on a focused microwave system (CEM Discover). The contents of the vessel were cooled rapidly using a stream of compressed air. All commercial reagents were purchased from suppliers such as Sigma-Aldrich or Acros Organics. Solvents were dried and purified before use according to standard procedures.

4. Experimental section 4.1. Chemistry (general methods) Column chromatography was run on silica gel Kieselgel 60 (0.040–0.063 mm, 230–400 mesh, Merck) under pressure, with solvents that had been distilled prior to use. Analytical thin layer chromatography was performed on Merck silica gel 60 F254 plates and the visualization utilized either UV light (254 nm) or spraying with a solution of phosphomolybdic acid, with subsequent heating. NMR spectra were recorded on a Varian Mercury Plus 400 FT NMR (400.13 MHz for 1H and 100.61 MHz for 13C) spectrometer. For 1H, δ are given in parts per million (ppm) either relative to TMS (δ = 0.0) as the internal standard

4.1.1. (S)-2-[(S)-1'-(Benzyloxy)-2'-(methoxymethoxy)ethyl]-1,4dioxaspiro[4.5]decane (11) A solution of the known alcohol 10 [14] (5.70 g, 19.5 mmol) in dry CH2Cl2 (200 mL) was gradually treated with MOMCl (5.90 mL, 78.0 mmol) and DIPEA (17.50 mL, 97.5 mmol) at 0 °C. The resulting mixture was stirred at 0 °C for 10 min and then heated at reflux for 2 h.

Scheme 6. Reagents and conditions: (a) tetradec-1-ene, Grubbs II, toluene, MW, 180 °C; (b) H2, 10% Pd/C, 20% Pd(OH)2/C, MeOH, rt; (c) TBAF, THF, rt; (d) Ac2O, DMAP, pyridine, rt; (e) (i) 80% TFA, 0 °C→rt; (ii) NaIO4, MeOH/H2O (1:1), rt; (iii) NaBH4, MeOH, 0 °C→rt; (f) 6 M HCl, reflux. 5

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Table 3 Antiproliferative/cytotoxic activities of sphingoid bases 7, ent-7 and 29, including the advanced precursors 18, 19 and 8, on four human cancer cell lines (PaTu, HeLa, A2058, Jurkat) and non-malignant human mammary epithelial cells MCF-10A Compd no.

Cell line, IC50a ± SD (μmol × L−1) PaTu

7 ent-7 29 18 19 8 cisplatin

18.3 ± 18.5 ± 20.7 ± > 100 > 100 42.1 ± ‒b

8.4 10.1 11.7 15.1

HeLa

A2058

Jurkat

MCF-10A

11.2 ± 4.9 14.8 ± 11.7 9.4 ± 6.6 86.1 ± 14.1 > 100 44.1 ± 1.0 13.1 ± 0.2

15.3 ± 7.2 9.0 ± 9.1 16.7 ± 11.3 > 100 > 100 69.8 ± 8.2 ‒b

6.6 ± 6.6 5.6 ± 1.5 6.1 ± 1.4 76.1 ± 8.9 > 100 36.4 ± 1.8 16.2 ± 0.6

15.2 ± 16.8 ± 19.1 ± > 100 > 100 43.3 ± ‒b

9.9 10.3 6.4 2.6

a The potency of compounds was determined using MTT assay after 72 h incubation of cells and given as IC50 (concentration of a tested compound that decreased the number of viable cells to 50% relative to untreated control cells, see Section 4.2). b Not tested.

4.47–4.59 (m, 3H, 2 × H-2, H-2′), 4.68–4.71 (m, 2H, CH2); 13C NMR (100 MHz, CDCl3): δ 23.6 (CH2), 23.9 (CH2), 24.9 (CH2), 34.1 (CH2), 35.5 (CH2), 55.7 (OCH3), 66.2 (C-3′), 70.0 (C-2), 78.8 (C-2′), 96.5 (CH2), 111.8 (C-5′), 206.8 (C=O). ESI-HRMS: m/z calcd for C12H21O5 [M + H]+ 245.1384, found 245.1371.

After cooling to room temperature, the mixture was poured into H2O (100 mL) and extracted with CH2Cl2 (2 × 80 mL). The combined organic layers were dried over Na2SO4, the solvent was evaporated, and the residue was subjected to flash chromatography on silica gel (nhexane/EtOAc, 3:1) to afford 6.17 g (94%) of compound 11 as a colourless oil; [α]D21 −12.9 (c 0.31, CHCl3). IR (neat) υ 926, 1035, 1102, 1305, 1449, 1497, 2861, 2931 cm−1; 1H NMR (400 MHz, CDCl3) δ 1.36–1.44 (m, 2H, CH2), 1.54–1.66 (m, 8H, 4 × CH2), 3.35 (s, 3H, CH3), 3.60–3.69 (m, 3H, H-1′, 2 × H-2′), 3.75–3.79 (m, 1H, H-3), 3.98–4.02 (m, 1H, H-3), 4.27 (dd, J = 12.6, 6.5 Hz, 1H, H-2), 4.61 (m, 2H, CH2), 4.74–4.80 (m, 2H, OCH2Ph), 7.25–7.39 (m, 5H, Ph); 13C NMR (100 MHz, CDCl3) δ 23.9 (CH2), 24.0 (CH2), 25.2 (CH2), 34.9 (CH2), 36.1 (CH2), 55.3 (CH3), 65.4 (C-3), 67.5 (C-2′), 72.8 (OCH2Ph), 76.3 (C-2), 78.5 (C-1′), 96.7 (CH2), 109.7 (C-5), 127.5 (CHPh), 127.8 (2 × CHPh), 128.3 (2 × CHPh), 138.5 (Ci). ESI-HRMS: m/z calcd for C19H29O5 [M + H]+ 337.2010, found 337.1989.

4.1.4. Ethyl (R,E)-4-(methoxymethoxy)-3-(1′,4′-dioxaspiro[4.5]decan-2′yl)but-2-enoate (E−14) and ethyl (R,Z)-4-(methoxymethoxy)-3-(1′,4′dioxaspiro[4.5]decan-2′-yl)but-2-enoate (Z-14) To a suspension of NaH (0.756 g, 19.7 mmol, ∼60% emulsion in mineral oil) in dry THF (30 mL) that had been pre-cooled to 0 °C was added (EtO2)P(O)CH2COOEt (4.70 mL, 21.75 mmol). After being stirred for 25 min at the same temperature, the solution of 13 (3.54 g, 14.50 mmol) in dry THF (6.2 mL) was added and the stirring was continued for 30 min at 0 °C. The reaction was quenched with a saturated solution of NH4Cl (40 mL) and the resulting mixture was extracted with EtOAc (2 × 75 mL). The combined organic layers were dried over Na2SO4, the solvent was evaporated, and the residue was purified by flash chromatography on silica gel (n-hexane/EtOAc, 15:1) to afford 3.64 g (80%) of (E)-14 and 0.643 g (14%) of (Z)-14. Ester (E)-14: colourless oil; [α]D21 −57.1 (c 0.42, CHCl3). IR (neat) υ 922, 1031, 1102, 1142, 1651, 1711, 2933 cm−1; 1H NMR (400 MHz, CDCl3) δ 1.29 (t, J = 7.1 Hz, 3H, CH3), 1.39–1.47 (m, 2H, CH2), 1.57–1.74 (m, 8H, 4 × CH2), 3.36 (s, 3H, CH3), 3.66–3.70 (m, 1H, H3′), 4.11–4.23 (m, 2H, CH2), 4.29–4.33 (m, 1H, H-3′), 4.62–4.68 (m, 3H, H-4, CH2), 4.74 (d, J = 14.7 Hz, 1H, H-4), 4.83 (t, J = 7.1 Hz, 1H, H-2′), 6.20–6.29 (m, 1H, H-2); 13C NMR (100 MHz, CDCl3) δ 14.2 (CH3), 23.8 (CH2), 23.9 (CH2), 25.1 (CH2), 35.1 (CH2), 35.7 (CH2), 55.5 (CH3), 60.1 (CH2), 65.2 (C-4), 69.3 (C-3′), 75.7 (C-2′), 96.5 (CH2), 110.0 (C-5′), 115.2 (C-2), 155.7 (C=O), 166.1 (C-3). ESI-HRMS: m/z calcd for C16H26NaO6 [M + Na]+ 337.1622, found 337.1622. Ester (Z)-14: colourless oil; [α]D21 −59.2 (c 0.24, CHCl3). IR (neat) υ 1034, 1108, 1149, 1228, 1366, 1651, 1709, 2858, 2933 cm−1; 1H NMR (400 MHz, CDCl3) δ 1.28 (t, J = 7.1 Hz, 3H, CH3), 1.38–1.44 (m, 2H, CH2), 1.53–1.69 (m, 8H, 4 × CH2), 3.38 (s, 3H, CH3), 3.61 (dd, J = 8.2, 7.0 Hz, 1H, H-3′), 4.13–4.24 (m, 3H, H-4, CH2), 4.38–4.47 (m, 2H, H-4, H-3′), 4.67–4.71 (m, 2H, CH2), 5.70 (t, J = 7.0 Hz, 1H, H-2′), 6.05–6.10 (m, 1H, H-2); 13C NMR (100 MHz, CDCl3) δ 14.2 (CH3), 23.7 (CH2), 24.0 (CH2), 25.1 (CH2), 34.0 (CH2), 35.6 (CH2), 55.5 (CH3), 60.1 (CH2), 65.7 (C-4), 69.1 (C-3′), 73.8 (C-2′), 96.2 (CH2), 110.3 (C-5′), 114.8 (C-2), 157.3 (C=O), 166.0 (C-3). ESI-HRMS: m/z calculated for C16H27O6 [M + H]+ 315.1804, found 315.1794.

4.1.2. (S)-2-(Methoxymethoxy)-1-{(S)-1′,4′-dioxaspiro[4.5]decan-2′-yl} ethan-1-ol (12) To a solution of 11 (6.12 g, 18.2 mmol) in dry MeOH (350 mL) were successively added 10% Pd/C (0.88 g) and 20% Pd(OH)2/C (0.88 g). After being stirred at room temperature under an atmosphere of hydrogen for 45 min, catalysts were removed by filtration through a small pad of Celite and the filtrate was concentrated. The residue was chromatographed on silica gel (n-hexane/EtOAc, 3:1) to furnish 4.08 g (91%) of compound 12 as a colourless oil; [α]D21 +3.2 (c 0.38, CHCl3). IR (neat) υ 1029, 1099, 1152, 1366, 1449, 2360, 2681, 2888, 2932, 3466 cm−1; 1H NMR (400 MHz, CDCl3) δ 1.33–1.47 (m, 2H, CH2), 1.53–1.67 (m, 8H, 4 × CH2), 2.59 (br d, J = 5.3 Hz, 1H, OH), 3.38 (s, 3H, CH3), 3.56–3.64 (m, 2H, 2 × H-2) 3.71–3.77 (m, 1H, H-1), 3.84 (dd, J = 8.1, 6.8 Hz, 1H, H-3′), 4.04 (dd, J = 8.1, 6.7 Hz, 1H, H-3′), 4.15–4.20 (m, 1H, H-2′), 4.66 (m, 2H, CH2); 13C NMR (100 MHz, CDCl3) δ 23.7 (CH2), 24.0 (CH2), 25.1 (CH2), 34.7 (CH2), 36.1 (CH2), 55.4 (CH3), 65.4 (C-3′), 69.3 (C-2), 70.8 (C-1), 75.8 (C-2′), 96.8 (CH2), 109.9 (C-5′). ESI-HRMS: m/z calcd for C12H23O5 [M + H]+ 247.1540, found 247.1539. 4.1.3. (S)-2-(Methoxymethoxy)-1-(1′,4′-dioxaspiro[4.5]decan-2′-yl) ethan-1-one (13) IBX (6.80 g, 24.3 mmol) was added to a solution of 12 (4.0 g, 16.2 mmol) in MeCN (170 mL). The reaction mixture was heated and stirred at reflux for 40 min, then cooled to room temperature. The insoluble parts were removed by filtration and the filtrate was concentrated. The crude product was flash-chromatographed through a short column of silica gel (n-hexane/EtOAc, 5:1) to give 3.7 g (94%) of compound 13 as a colourless oil; [α]D21 −63.3 (c 0.18, CHCl3). IR (neat) υ 920, 1038, 1150, 1734, 2859, 2933 cm−1; 1H NMR (400 MHz, CDCl3) δ 1.37–1.47 (m, 2H, CH2), 1.55–1.73 (m, 8H, 4 × CH2), 3.39 (s, 3H, CH3), 4.04 (dd, J = 8.8, 5.4 Hz, 1H, H-3′), 4.22–4.26 (m, 1H, H-3′),

4.1.5. (R,E)-4-(Methoxymethoxy)-3-(1′,4′-dioxaspiro[4.5]decan-2′-yl) but-2-en-1-ol (15) DIBAL-H (28.80 mL, 34.5 mmol, ∼1.2 M solution in toluene) was added to a solution of (E)-14 (3.63 g, 11.5 mmol) in dry CH2Cl2 (60 mL) that had been pre-cooled to −50 °C. After stirring for 30 min at −30 °C, the reaction was quenched with MeOH (9.5 mL), then poured into 30% 6

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aqueous solution of potassium sodium tartrate (200 mL) and stirred for 1 h at room temperature. The whole mixture was then extracted with CH2Cl2 (2 × 200 mL), the combined organic layers were dried over Na2SO4 and concentrated. The residue was subjected to flash chromatography on silica gel (n-hexane/EtOAc, 2:1) to afford 3.0 g (96%) of compound 15 as a colourless oil; [α]D21 −23.3 (c 0.60, CHCl3). IR (neat) υ 1029, 1096, 1147, 2861, 2932, 3422 cm−1; 1H NMR (400 MHz, CDCl3) δ 1.37–1.45 (m, 2H, CH2), 1.55–1.70 (m, 8H, 4 × CH2), 2.06 (br s, 1H, OH), 3.39 (s, 3H, CH3), 3.60–3.70 (m, 1H, H-3′), 4.11–4.14 (m, 3H, 2 × H-4, H-3′), 4.24–4.26 (m, 2H, 2 × H-1), 4.57 (t, J = 7.1 Hz, 1H, H-2′), 4.61–4.65 (m, 2H, CH2), 6.10 (t, J = 6.8 Hz, 1H, H-2); 13C NMR (100 MHz, CDCl3) δ 23.8 (CH2), 23.9 (CH2), 25.1 (CH2), 35.1 (CH2), 35.9 (CH2), 55.5 (CH3), 58.4 (C-1), 62.0 (C-4), 68.5 (C-3′), 77.9 (C-2′), 95.6 (CH2), 109.8 (C-5′), 131.4 (C-2), 135.9 (C-3). ESI-HRMS: m/ z found for C14H24NaO5 [M + Na]+ 295.1516, found 295.1517.

(400 MHz, CD3OD) δ 1.38–1.45 (m, 2H, CH2), 1.53–1.67 (m, 8H, 4 × CH2), 3.39 (s, 3H, CH3), 3.55 (d, J = 10.2 Hz, 1H, H-1′), 3.76 (d, J = 10.2 Hz, 1H, H-1′), 3.87 (dd, J = 8.7, 6.1 Hz, 1H, H-3), 4.09–4.13 (m, 1H, H-3), 4.40–4.44 (m, 1H, H-2), 4.65–4.69 (m, 2H, CH2), 5.40 (d, J = 10.6 Hz, 1H, H-4′), 5.50 (d, J = 17.0 Hz, 1H, H-4′), 5.89 (dd, J = 17.0, 10.6 Hz, 1H, H-3′); 13C NMR (100 MHz, CD3OD) δ 24.8 (CH2), 25.0 (CH2), 26.3 (CH2), 35.3 (CH2), 36.7 (CH2), 55.9 (CH3), 66.0 (C-3), 71.2 (C-2′), 71.6 (C-1′), 77.8 (C-2), 97.6 (CH2), 111.8 (C-5), 118.6 (C4′), 134.0 (C-3′), 138.2 (NCS). ESI-HRMS: m/z calcd for C15H24NO4S [M + H]+ 314.1421, found 314.1423. Diastereoisomer 19: colourless oil; [α]D21 −72.5 (c 0.24, CHCl3 IR (neat) υ 1043, 1110, 1448, 2044, 2886, 2934 cm−1; 1H NMR (400 MHz, CD3OD) δ 1.43–1.50 (m, 2H, CH2), 1.58–1.85 (m, 8H, 4 × CH2), 3.41 (s, 3H, CH3), 3.80 (d, J = 9.4 Hz, 1H, H-1′), 3.85 (dd, J = 8.5, 6.5 Hz, 1H, H-3), 3.89 (d, J = 9.4 Hz, 1H, H-1′), 4.01–4.05 (m, 1H, H-3), 4.35 (t, J = 6.6 Hz, 1H, H-2), 4.67–4.71 (m, 2H, CH2), 5.37 (d, J = 10.6 Hz, 1H, H-4′), 5.52 (d, J = 17.0 Hz, 1H, H-4′), 5.88 (dd, J = 17.0, 10.6 Hz, 1H, H-3′); 13C NMR (100 MHz, CD3OD) δ 24.9 (CH2), 25.1 (CH2), 26.2 (CH2), 35.5 (CH2), 37.0 (CH2), 55.8 (CH3), 65.7 (C-3), 71.5 (C-2′), 72.3 (C-1′), 77.2 (C-2), 97.7 (CH2), 112.5 (C-5), 117.9 (C-4′), 135.0 (C-3′), 137.7 (NCS). ESI-HRMS: m/z calcd for C15H24NO4S [M + H]+ 314.1421, found 314.1419.

4.1.6. (R,E)-2-[1'-(Methoxymethoxy)-4′-thiocyanatobut-2′-en-2′-yl]-1,4dioxaspiro[4.5]decane (16) To a solution of 15 (1.50 g, 5.50 mmol) in dry CH2Cl2 (37 mL) that had been pre-cooled to 0 °C was gradually added Et3N (1.55 mL, 11.0 mmol) and MsCl (0.85 mL, 11.0 mmol). The mixture was stirred at the same temperature for 30 min, then concentrated and treated with Et2O. The insoluble material was filtered off and the solvent was evaporated. The crude mesylate (1.93 g, 5.50 mmol) dissolved in MeCN (65 mL) was cooled to 0 °C, and the solid KSCN (1.07 g, 11.0 mmol) was added. After stirring at room temperature for 3.5 h, acetonitrile was removed, and the residue was diluted with Et2O. The solid parts were separated by filtration, the filtrate was concentrated, and the crude product was chromatographed on silica gel (n-hexane/EtOAc, 5:1) to furnish 1.26 g (73%) of compound 16 as a colourless oil; [α]D21 −41.0 (c 0.40, CHCl3). IR (neat) υ 1279, 1448, 2153, 2801, 2933 cm−1; 1H NMR (400 MHz, CDCl3) δ 1.39–1.45 (m, 2H, CH2), 1.56–1.71 (m, 8H, 4 × CH2), 3.38 (s, 3H, CH3), 3.67 (t, J = 8.0 Hz, 1H, H-3), 3.76–3.78 (m, 2H, 2 × H-4′), 4.15 (m, 2H, 2 × H-1′), 4.17–4.21 (m, 1H, H-3), 4.59–4.65 (m, 3H, H-2, CH2), 6.03 (t, J = 8.3 Hz, 1H, H-3′); 13C NMR (100 MHz, CDCl3) δ 23.8 (CH2), 23.9 (CH2), 25.1 (CH2), 30.9 (C-4′), 35.2 (CH2), 35.8 (CH2), 55.6 (CH3), 61.9 (C-1′), 69.0 (C-3), 77.2 (C-2), 95.7 (CH2), 110.2 (C-5), 111.7 (SCN), 122.3 (C-3′), 141.1 (C-2′). ESIHRMS: m/z calcd for C15H24NO4S [M + H]+ 314.1421, found 314.1417.

4.1.8. 2,2,2-Trichloro-N-{(R)-1-(methoxymethoxy)-2-[(R)-1′,4′dioxaspiro[4.5]decan-2′-yl]but-3-en-2-yl}acetamide (20) and 2,2,2trichloro-N-{(S)-1-(methoxymethoxy)-2-[(R)-1′,4′-dioxaspiro[4.5]decan2′-yl]but-3-en-2-yl}acetamide (21) To a solution of 15 (68 mg, 0.25 mmol) in dry CH2Cl2 (1.3 mL) were gradually added DBU (3.7 μL, 25 μmol) and CCl3CN (0.05 mL, 0.5 mmol) at 0 °C. After stirring at the same temperature for 15 min, the mixture was filtered through a small pad of Celite and concentrated. This procedure yielded imidate 17, which was used to the subsequent reaction without purification. The crude imidate 17 (104 mg, 0.25 mmol) was transferred to a 10 mL microwave tube equipped with a magnetic stirbar. o-Xylene (3.4 mL) followed by K2CO3 (39 mg, 285 μmol) were added and the mixture was subjected to microwave irradiation conditions (for the temperatures and reaction times, see Table 2). After filtration, the solvent was evaporated, and the residue was chromatographed on silica gel (n-hexane/EtOAc, 11:1) to give an inseparable mixture of trichloroacetamides 20 and 21 (for the combined yields, see Table 2). Both rearranged compounds were characterized as the corresponding amines 24 and 8, respectively (vide infra). Requiring greater amounts of amides, they were prepared via a procedure at 210 °C (see Table 2).

4.1.7. (R)-2-[(R)-2′-Isothiocyanato-1'-(methoxymethoxy)but-3′-en-2′-yl]1,4-dioxaspiro[4.5]decane (18) and (R)-2-[(S)-2′-isothiocyanato-1'(methoxymethoxy)but-3′-en-2′-yl]-1,4-dioxaspiro[4.5]decane (19) 4.1.7.1. Conventional method (general procedure). A solution of thiocyanate 16 (85 mg, 0.31 mmol) in n-heptane (2 mL) was heated and stirred under a nitrogen atmosphere (for the temperatures and reaction times, see Table 1). After being cooled to room temperature, the mixture was concentrated, and the residue was flashchromatographed through a short column of silica gel (n-hexane/ EtOAc, 18:1 → 5:1) to afford a mixture of isothiocyanates 18 and 19 (for the combined yields, see Table 1).

4.1.9. (4R,5R)-5-(Hydroxymethyl)-4-[(methoxymethoxy)methyl]-4vinyloxazolidine-2-thione (22) A solution of 18 (49 mg, 0.18 mmol) in MeOH (3.5 mL) was treated with p-TsOH (6.8 mg, 36 μmol). After being stirred at room temperature for 24 h, the mixture was neutralized with Et3N, the solvent was evaporated, and the residue was submitted to flash chromatography on silica gel (n-hexane/EtOAc, 1:1) to give 28 mg (67%) of compound 22 as a colourless oil; [α]D21 +36.5 (c 0.34, CHCl3). IR (neat) υ 1032, 1106, 1145, 1494, 2932, 3196 cm−1; 1H NMR (400 MHz, CD3OD) δ 3.38 (s, 3H, CH3), 3.62 (d, J = 10.5 Hz, 1H, H-1″), 3.76 (d, J = 10.5 Hz, 1H, H-1″), 3.91–4.00 (m, 2H, 2 × H-1‴), 4.53–4.56 (m, 1H, H-5), 4.61–4.65 (m, 2H, CH2), 5.33 (d, J = 10.8 Hz, 1H, H-2′), 5.39 (d, J = 17.4 Hz, 1H, H-2′), 5.99 (dd, J = 17.4, 10.8 Hz, 1H, H-1′); 13C NMR (100 MHz, CD3OD) δ 56.0 (CH3), 60.2 (C-1‴), 69.1 (C-4), 69.3 (C-1″), 91.1 (C-5), 97.9 (CH2), 117.0 (C-2′), 137.7 (C-1′), 190.8 (C=S). ESIHRMS: m/z calcd for C9H16NO4S [M + H]+ 234.0795, found 234.0813.

4.1.7.2. Microwave-assisted synthesis (general procedure). Thiocyanate 16 (85 mg, 0.31 mmol) was transferred into a 10-mL microwave tube equipped with a magnetic stirbar. n-Heptane (2 mL) was added, the tube was closed with a silicon septum, and the resulting solution was submitted to microwave irradiation (for the temperatures and reaction times, see Table 1). The mixture was cooled to room temperature, concentrated, and the residue was purified by flash chromatography on silica gel (n-hexane/EtOAc, 18:1 → 5:1) to provide a mixture of diastereoisomers 18 and 19 (for the combined yields, see Table 1). Requiring greater amounts of the rearranged products, they were prepared by the microwave-assisted synthesis at 110 °C. Diastereoisomer 18: colourless oil; [α]D21 −40.1 (c 0.46, CHCl3). IR (neat) υ 1039, 1094, 1448, 2036, 2862, 2882, 2934 cm−1; 1H NMR

4.1.10. (4S,5R)-5-(Hydroxymethyl)-4-[(methoxymethoxy)methyl]-4vinyloxazolidine-2-thione (23) Using the same procedure as described for the preparation of 22, isothiocyanate 19 (49 mg, 0.18 mmol) was converted into derivative 23 7

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(colourless oil, 27 mg, 65%, n-hexane/EtOAc, 1:1); [α]D20 −127.6 (c 0.34, CHCl3). IR (neat) υ 1030, 1107, 1207, 1498, 2935, 3203 cm−1; 1H NMR (400 MHz, CD3OD) δ 3.38 (s, 3H, CH3), 3.58 (d, J = 10.1 Hz, 1H, H-1″), 3.63–3.65 (m, 3H, H-1″, 2 × H-1‴), 4.65–4.69 (m, 2H, CH2), 4.78 (dd, J = 6.7, 5.5 Hz, 1H, H-5) 5.36–5.42 (m, 2H, 2 × H-2′), 5.90 (dd, J = 17.3, 10.8 Hz, 1H, H-1′); 13C NMR (100 MHz, CD3OD) δ 55.9 (CH3), 61.8 (C-1‴), 69.3 (C-4), 71.9 (C-1″), 88.0 (C-5), 97.7 (CH2), 118.6 (C-2′), 133.8 (C-1′), 190.5 (C=S). ESI-HRMS: m/z calcd for C9H16NO4S [M + H]+ 234.0795, found 234.0813.

residue was flash-chromatographed through a short column of silica gel (n-hexane/EtOAc, 7:1) to afford 0.366 g (98%) of compound 25 as a colourless oil; [α]D21 −3.4 (c 0.65, CHCl3). IR (neat) υ 1036, 1240, 1498, 1728, 2933, 3419 cm−1; 1H NMR (400 MHz, CDCl3) δ 1.34–1.44 (m, 2H, CH2), 1.50–1.65 (m, 8H, 4 × CH2), 3.33 (s, 3H, CH3), 3.77 (d, J = 10.0 Hz, 1H, H-1), 3.95–4.04 (m, 3H, H-1, 2 × H-3′), 4.46 (t, J = 6.7 Hz, 1H, H-2′), 4.58–4.62 (m, 2H, CH2), 5.03–5.09 (m, 2H, CH2), 5.24–5.32 (m, 3H, 2 × H-4, NH), 6.00 (dd, J = 17.6, 11.0 Hz, 1H, H-3), 7.29–7.38 (m, 5H, Ph); 13C NMR (100 MHz, CDCl3) δ 23.7 (CH2), 23.9 (CH2), 25.2 (CH2), 34.3 (CH2), 35.9 (CH2), 55.4 (CH3), 60.3 (C-2), 65.2 (C-3′), 66.5 (CH2), 69.7 (C-1), 77.2 (C-2′), 96.8 (CH2), 109.5 (C-5′), 116.0 (C-4), 128.1 (3 × CHPh), 128.5 (2 × CHPh), 135.6 (C-3), 136.4 (Ci), 155.1 (C=O). ESI-HRMS: m/z calcd for pre C22H31NaNO6 [M + Na]+ 428.2044, found 428.2048.

4.1.11. (R)-1-(Methoxymethoxy)-2-{(R)-1′,4′-dioxaspiro[4.5]decan-2′yl}but-3-en-2-amine (24) and (S)-1-(methoxymethoxy)-2-{(R)-1′,4′dioxaspiro[4.5]decan-2′-yl}but-3-en-2-amine (8) Modification of 18 into 24. To a solution of 18 (0.25 g, 0.80 mmol) in dry toluene (3.7 mL) was added TBTO (0.82 mL, 1.60 mmol). After being heated and stirred at 90 °C for 7 h, the solvent was evaporated, and the residue was subjected to flash chromatography on silica gel (n-hexane/EtOAc, 1:2) to afford 0.175 g (81%) of compound 24 as a colourless oil. Modification of 19 into 8. According to the same procedure described for the construction of 24, compound 19 (0. 25 g, 0.80 mmol) was transformed to amine 8 (colourless oil, 0.182 g, 84%, n-hexane/EtOAc, 1:2). Modification of a mixture of amides 20 and 21 into amines 24 and 8. To a solution of the mixture of 20 and 21 (0.50 g, 1.20 mmol) in dry CH2Cl2 (6.5 mL) that had been pre-cooled to −50 °C was added DIBALH (2.0 mL, 2.40 mmol, ∼1.2 M solution in toluene). After stirring at −30 °C for 40 min, the reaction was quenched with MeOH (0.5 mL), the whole mixture was poured into 30% aqueous solution of potassium sodium tartrate (18 mL), and stirring was continued at room temperature for 45 min. Then, the mixture was extracted with CH2Cl2 (2 × 25 mL), the combined organic layers were dried over Na2SO4, striped solvent, and the residue was chromatographed on silica gel (nhexane/EtOAc, 1:2) to furnish 90 mg (32%) and 0.151 g (54%) of compounds 24 and 8, respectively. Amine 24: colourless oil; [α]D21 −3.5 (c 0.5, CHCl3). IR (neat) υ 1038, 1102, 1148, 1448, 2861, 2932 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.34–1.62 (m, 10H, 5 × CH2), 3.37–3.39 (m, 4H, H-1, CH3), 3.63 (d, J = 9.5 Hz, 1H, H-1), 3.80–3.83 (m, 1H, H-3′), 3.97–4.01 (m, 1H, H-3′), 4.14 (t, J = 6.9 Hz, 1H, H-2′), 4.63–4.67 (m, 2H, CH2), 5.29 (d, J = 10.9 Hz, 1H, H-4), 5.36 (d, J = 17.5, 1H, H-4), 5.96 (dd, J = 17.5, 10.9 Hz, 1H, H-3); 13C NMR (100 MHz, CDCl3) δ 23.8 (CH2), 23.9 (CH2), 25.2 (CH2), 34.5 (CH2), 35.8 (CH2), 55.3 (CH3), 58.1 (C-2), 64.9 (C-3′), 72.6 (C-1), 78.1 (C-2′), 96.7 (CH2), 109.6 (C-5′), 115.7 (C-4), 138.6 (C-3). ESI-HRMS: m/z calcd for C14H26NO4 [M + H]+ 272.1856, found 272.1862. Amine 8: colourless oil; [α]D21 +6.2 (c 0.42, CHCl3). IR (neat) υ 918, 1039, 1103, 1448, 2861, 2932 cm−1; 1H NMR (400 MHz, CDCl3) δ 1.36–1.66 (m, 10H, 5 × CH2), 3.36 (s, 3H, CH3), 3.42 (d, J = 9.2 Hz, 1H, H-1), 3.61 (d, J = 9.2 Hz, 1H, H-1), 3.82–3.90 (m, 2H, 2 × H-3′), 4.18 (t, J = 7.0 Hz, 1H, H-2′), 4.63–4.66 (m, 2H, CH2), 5.21 (d, J = 10.9 Hz, 1H, H-4), 5.36 (d, J = 17.5 Hz, 1H, H-4), 5.85 (dd, J = 17.5, 10.9 Hz, 1H, H-3); 13C NMR (100 MHz, CDCl3) δ 23.7 (CH2), 23.9 (CH2), 25.2 (CH2), 34.5 (CH2), 36.0 (CH2), 55.2 (CH3), 57.4 (C-2), 64.4 (C-3′), 73.8 (C-1), 77.1 (C-2′), 96.7 (CH2), 109.9 (C-5′), 115.3 (C4), 138.9 (C-3). ESI-HRMS: m/z calcd for C14H26NO4 [M + H]+ 272.1856, found 272.1876.

4.1.13. Benzyl {(S)-1-(methoxymethoxy)-2-[(R)-1′,4′-dioxaspiro[4.5] decan-2′-yl]but-3-en-2yl} carbamate (26) The same reaction conditions as employed for the conversion of 24 into 25 were applied to 8 (0.326 g, 1.20 mmol) to give 0.477 g (98%) of compound 26 as a colourless oil (n-hexane/EtOAc, 7:1); [α]D21 −6.9 (c 0.70, CHCl3). IR (neat) υ 1038, 1235, 1496, 1728, 2934, 3414 cm−1; 1H NMR (400 MHz, CDCl3) δ 1.36–1.43 (m, 2H, CH2), 1.50–1.65 (m, 8H, 4 × CH2), 3.34 (s, 3H, CH3), 3.83–3.92 (m, 3H, 2 × H-1, H-3′), 3.95–3.99 (m, 1H, H-3′), 4.57 (t, J = 6.5 Hz, 1H, H-2′), 4.62 (m, 2H, CH2), 5.06 (m, 2H, CH2), 5.27–5.31 (m, 2H, 2 × H-4), 5.44 (br s, 1H, NH), 6.00 (dd, J = 17.8, 10.9 Hz, 1H, H-3), 7.29–7.38 (m, 5H, Ph); 13C NMR (100 MHz, CDCl3) δ 23.7 (CH2), 23.9 (CH2), 25.1 (CH2), 34.0 (CH2), 35.9 (CH2), 55.4 (CH3), 60.1 (C-2), 64.5 (C-3′), 66.5 (CH2), 68.5 (C-1), 76.4 (C-2′), 96.8 (CH2), 110.1 (C-5′), 116.4 (C-4), 128.1 (3 × CHPh), 128.5 (2 × CHPh), 135.5 (C-3), 136.5 (Ci), 155.1 (C=O). ESI-HRMS: m/z calcd for C22H31NaNO6 [M + Na]+ 428.204, found 428.205. 4.1.14. Benzyl {(R,E)-1-(methoxymethoxy)-2-[(R)-1′,4′-dioxaspiro[4.5] decan-2′-yl]hexadec-3-en-2-yl}carbamate (27) To a solution of 25 (53 mg, 0.13 mmol) in dry toluene (0.5 mL) were added tetradec-1-ene (66 μL, 0.26 mmol) and second generation Grubbs catalyst (2.2 mg, 2.6 μmol). The mixture was refluxed for 4 h and then another portion of tetradec-1-ene (66 μL, 0.26 mmol) and catalyst (2.2 mg, 2.6 μmol) was added. This procedure was then repeated again after 4 h. Four hours after the last dose, only catalyst (6.6 mg, 7.8 μmol) was added in three portions at 4 h intervals. The total reaction time was 30 h. The mixture was cooled to room temperature, concentrated, and the residue was chromatographed on silica gel (n-hexane/EtOAc, 9:1) to furnish 43 mg (58%) of compound 27 as a colourless oil; [α]D21 −10.7 (c 0.28, CHCl3). IR (neat) υ 1036, 1101, 1235, 1498, 1732, 2852, 2923 cm−1; 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 6.6 Hz, 3H, CH3), 1.25–1.39 (m, 22H, 11 × CH2), 1.56–1.61 (m, 8H, 4 × CH2), 2.03–2.08 (m, 2H, CH2), 3.32 (s, 3H, CH3), 3.74 (d, J = 9.9 Hz, 1H, H1), 3.92–4.02 (m, 3H, H-1, 2 × H-3′), 4.44 (t, J = 6.5 Hz, 1H, H-2′), 4.58–4.61 (m, 2H, CH2), 5.02–5.08 (m, 2H, CH2), 5.22 (br s, 1H, NH), 5.57 (d, J = 16.0 Hz, 1H, H-3), 5.61–5.68 (m, 1H, H-4), 7.29–7.38 (m, 5H, Ph); 13C NMR (100 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 23.7 (CH2), 23.9 (CH2), 25.2 (CH2), 29.1 (CH2), 29.2 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (2 × CH2), 29.7 (2 × CH2), 31.9 (CH2), 32.6 (CH2), 34.4 (CH2), 35.9 (CH2), 55.4 (CH3), 59.7 (C-2), 65.3 (C-3′), 66.4 (CH2), 70.0 (C-1), 77.4 (C-2′), 96.8 (CH2), 109.3 (C-5′), 127.0 (C-3), 128.1 (3 × CHPh), 128.5 (2 × CHPh), 132.3 (C-4), 136.5 (Ci), 155.0 (C=O). ESI-HRMS: m/z calcd for C34H56NO6 [M + H]+ 574.4102, found 574.4097.

4.1.12. Benzyl {(R)-1-(methoxymethoxy)-2-[(R)-1′,4′-dioxaspiro[4.5] decan-2′-yl]but-3-en-2yl} carbamate (25) To a solution of 24 (0.25 g, 0.92 mmol) in a mixture of THF/H2O (9 mL, 1:1) were gradually added CbzCl (0.18 mL, 1.29 mmol) and NaHCO3 (108 mg, 1.29 mmol). After being stirred at room temperature for 40 min, the mixture was extracted with EtOAc (2 × 20 mL). The combined organic layers were dried over Na2SO4, concentrated, and the

4.1.15. Benzyl {(S,E)-1-(methoxymethoxy)-2-[(R)-1′,4′-dioxaspiro[4.5] decan-2′-yl]hexadec-3-en-2-yl}carbamate (28) Using the same protocol as described for the preparation of 27, compound 26 (57 mg, 0.14 mmol) was converted into alkene 28 (colourless oil, 39 mg, 48%, n-hexane/EtOAc, 9:1); [α]D21 −15.5 (c 0.40, 8

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CHCl3). IR (neat) υ 1097, 1233, 1453, 1496, 1732, 2852, 2923 cm−1; 1 H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 6.8 Hz, 3H, CH3), 1.25–1.41 (m, 22H, 11 × CH2), 1.51–1.62 (m, 8H, 4 × CH2), 2.02–2.07 (m, 2H, CH2), 3.34 (s, 3H, CH3), 3.82–3.87 (m, 3H, 2 × H-1, H-3′), 3.93–3.97 (m, 1H, H-3′), 4.52 (t, J = 6.3 Hz, 1H, H-2′), 4.62 (m, 2H, CH2), 5.06 (m, 2H, CH2), 5.38 (br s, 1H, NH), 5.57 (d, J = 16.2 Hz, 1H, H-3), 5.63–5.70 (m, 1H, H-4), 7.28–7.38 (m, 5H, Ph); 13C NMR (100 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 23.7 (CH2), 23.9 (CH2), 25.2 (CH2), 29.1 (CH2), 29.2 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (2 × CH2), 29.7 (2 × CH2), 31.9 (CH2), 32.7 (CH2), 34.1 (CH2), 35.9 (CH2), 55.3 (CH3), 59.5 (C-2), 64.7 (C-3′), 66.3 (CH2), 68.8 (C-1), 76.8 (C-2′), 96.8 (CH2), 110.0 (C-5′), 126.7 (C-3), 128.0 (CHPh), 128.1 (CHPh), 128.4 (3 × CHPh), 132.6 (C-4), 136.6 (Ci), 155.0 (C=O). ESI-HRMS: m/z calcd for C34H56NO6 [M + H]+ 574.4102, found 574.4114.

J = 6.8 Hz, 3H, CH3), 0.93 (s, 9H, 3 × CH3), 1.26–1.40 (m, 23H, 10 × CH2, CH3), 1.52 (s, 3H, CH3), 2.04–2.19 (m, 2H, CH2), 3.61 (s, 3H, CH3), 3.71–3.76 (m, 1H, H-4), 3.92 (d, J = 12.3 Hz, 1H, H-5), 4.05 (dd, J = 12.3, 2.7 Hz, 1H, H-5), 5.06 (m, 1H, H-2), 5.60–5.73 (m, 2H, H-1′, H-2′), 5.86 (d, J = 3.2 Hz, 1H, H-1), 7.49 (br s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ −5.7 (CH3), −5.5 (CH3), 14.1 (CH3), 18.2 (Cq), 22.7 (CH2), 25.7 (3 × CH3), 26.3 (CH3), 26.8 (CH3), 29.2 (CH2), 29.3 (2 × CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (2 × CH2), 31.9 (CH2), 32.5 (CH2), 51.6 (CH3), 59.6 (C-5), 68.0 (C-3), 79.2 (C-4), 83.2 (C-2), 104.4 (C-1), 111.9 (Cq), 123.7 (C-1′), 133.1 (C-2′), 155.7 (C=O). ESI-HRMS: m/z calcd for C30H57NNaO6Si [M + Na]+ 578.3847, found 578.3847. 4.1.19. 5-O-tert-butyldimethylsilyl-3-deoxy-1,2-O-isopropylidene-3(methoxycarbonyl)amino-3-C-(tetradecyl)-α-D-xylofuranose (31) To a solution of 30 (0.195 g, 0.35 mmol) in dry MeOH (9 mL) were successively added 10% Pd/C (44 mg) and 20% Pd(OH)2/C (44 mg) at room temperature. The resulting suspension was degassed tree times and was stirred under an atmosphere of hydrogen for 1 h. Then, the mixture was filtered through a small pad of Celite, the filtrate was concentrated, and the crude product was purified by flash chromatography on silica gel (n-hexane/EtOAc, 11:1). This procedure yielded 0.16 g (82%) of compound 31 as a colourless oil; [α]D21 +33.3 (c 0.36, CHCl3). IR (neat) υ 1254, 1514,1728, 2854, 2925, 3356 cm−1; 1H NMR (400 MHz, CDCl3) δ 0.10 (s, 3H, CH3), 0.11 (s, 3H, CH3), 0.88 (t, J = 6.6 Hz, 3H, CH3), 0.92 (s, 9H, 3 × CH3), 1.26–1.46 (m, 27H, 12 × CH2, CH3), 1.50 (s, 3H, CH3), 1.57–1.65 (m, 1H, H–CH2), 2.31–2.38 (m, 1H, H–CH2), 3.60 (s, 3H, CH3), 3.79 (m, 1H, H-4), 4.00–4.03 (m, 1H, H-5), 4.14 (dd, J = 12.3, 2.7 Hz, 1H, H-5), 5.05–5.06 (m, 1H, H-2), 5.81 (d, J = 3.6 Hz, 1H, H-1), 7.13 (br s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ −5.8 (CH3), −5.6 (CH3), 14.1 (CH3), 18.1 (Cq), 22.7 (CH2), 23.7 (CH2), 25.7 (3 × CH3), 26.2 (CH3), 26.8 (CH3), 29.3 (CH2), 29.5 (CH2), 29.6 (4 × CH2), 29.7 (3 × CH2), 30.2 (CH2), 31.9 (CH2), 51.6 (CH3), 62.4 (C-5), 67.2 (C-3), 80.9 (C-4), 84.1 (C-2), 103.8 (C-1), 111.7 (Cq), 156.0 (C=O). ESI-HRMS: m/z calcd for C30H59NNaO6Si [M + Na]+ 580.4004, found 580.4006.

4.1.16. (2R,3R)-3-amino-3-tetradecylbutane-1,2,4-triol hydrochloride (29) To a solution of 27 (30 mg, 52 μmol) in dry MeOH (2 mL) were added two catalysts 10% Pd/C/20% Pd(OH)2/C (7 mg, 1:1) at room temperature. The mixture was degassed three times and stirred under an atmosphere of hydrogen for 1 h. After filtration through a small pad of Celite, the solvent was evaporated, and the residue was chromatographed on silica gel (n-hexane/EtOAc, 2:1) to provide the saturated amine (22 mg, 97%), which was submitted to the subsequent reaction without spectral characterisation. The obtained amine (21 mg, 48 μmol) was treated with 6 M HCl (2.3 mL) at room temperature. After stirring for 3 h, the resulting solution was concentrated to dryness under reduced pressure. The crude product was washed three times with Et2O, and then dried under high vacuum for 10 h. This procedure yielded 13 mg (79%) of compound 29 as a pale yellow hygroscopic solid; [α]D21 −6.4 (c 0.28, MeOH). IR (neat) υ 1047, 1499, 1604, 2851, 2920, 3330 cm−1; 1H NMR (400 MHz, CD3OD) δ 0.90 (t, J = 6.8 Hz, 3H, CH3), 1.29–1.44 (m, 24H, 12 × CH2), 1.65–1.73 (m, 1H, H–CH2), 1.82–1.89 (m, 1H, H–CH2), 3.65 (d, J = 11.4 Hz, 1H, H-4), 3.69–3.79 (m, 4H, 2 × H-1, H-2, H-4); 13C NMR (100 MHz, CD3OD) δ 14.4 (CH3), 23.7 (2 × CH2), 30.5 (2 × CH2), 30.7 (CH2), 30.8 (5 × CH2), 31.2 (CH2), 33.0 (CH2), 33.1 (CH2), 62.1 (C-4), 63.7 (C-1), 63.8 (C-3), 71.3 (C-2). ESI-HRMS: m/z calcd for C18H40NO3 [M + H]+ 318.3003, found 318.3007.

4.1.20. 3-Deoxy-1,2-O-isopropylidene-3-(methoxycarbonyl)amino-3-C(tetradecyl)-α-D-xylofuranose (32) Tetrabutylammonium fluoride (0.27 mL, 0.27 mmol, 1 M solution in THF) was added to a solution of 31 (0.151 g, 0.27 mmol) in THF (2.7 mL) at room temperature. After being stirred for 1 h, the mixture was diluted with H2O (5 mL) and extracted with EtOAc (3 × 7 mL). The combined organic layers were dried over Na2SO4, concentrated, and the residue was subjected to flash chromatography on silica gel (n-hexane/ EtOAc, 3:1) to afford 0.113 g (94%) of compound 32 as a colourless oil; [α]D21 +15.2 (c 0.28, CHCl3). IR (neat) υ 1004, 1064, 1522, 1703, 2361, 2852, 2922, 3323 cm−1; 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 6.7 Hz, 3H, CH3), 1.26–1.43 (m, 27H, 12 × CH2, CH3), 1.52 (s, 3H, CH3), 1.72–1.79 (m, 1H, H–CH2), 2.26–2.32 (m, 1H, H–CH2), 2.47–2.48 (m, 1H, OH), 3.64 (s, 3H, CH3), 3.92–3.97 (m, 2H, H-4, H-5), 4.07–4.10 (m, 1H, H-5), 4.93 (m, 1H, H-2), 5.85 (d, J = 3.5 Hz, 1H, H-1), 6.50 (br s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ 14.1 (CH3), 22.7 (CH2), 23.8 (CH2), 26.3 (CH3), 26.9 (CH3), 29.3 (CH2), 29.4 (CH2), 29.6 (7 × CH2), 30.1 (CH2), 31.9 (CH2), 51.8 (CH3), 61.1 (C-5), 67.2 (C-3), 81.5 (C-4), 84.6 (C-2), 103.9 (C-1), 112.2 (Cq), 156.1 (C=O). ESI-HRMS: m/z calcd for C24H45NNaO6 [M + Na]+ 466.3139, found 466.3119.

4.1.17. (2R,3S)-3-amino-3-tetradecylbutane-1,2,4-triol hydrochloride (7) The same procedure as employed for the conversion of 27 to 29 was applied to 28 (35 mg, 61 μmol) to deliver 23 mg (86%) of an amine, which was then treated with 6 M HCl to provide 14 mg (75%) of compound 7 as a pale yellow hygroscopic solid; [α]D21 −5.6 (c 0.18, MeOH). IR (neat) υ 1067, 1465, 1591, 2851, 2920, 3232 cm−1; 1H NMR (400 MHz, CD3OD) δ 0.90 (t, J = 6.8 Hz, 3H, CH3), 1.29–1.47 (m, 24H, 12 × CH2), 1.58–1.65 (m, 1H, H–CH2), 1.81–1.88 (m, 1H, H–CH2), 3.67 (d, J = 11.5 Hz, 1H, H-4), 3.71–3.79 (m, 4H, 2 × H-1, H-2, H-4); 13C NMR (100 MHz, CD3OD) δ 14.4 (CH3), 23.7 (CH2), 23.9 (CH2), 30.5 (2 × CH2), 30.7 (CH2), 30.8 (5 × CH2), 31.2 (CH2), 31.5 (CH2), 33.1 (CH2), 62.7 (C-4), 63.8 (C-1, C-3), 71.1 (C-2). ESI-HRMS: m/z calcd for C18H40NO3 [M + H]+ 318.3003, found 318.3008. 4.1.18. 5-O-tert-butyldimethylsilyl-3-deoxy-1,2-O-isopropylidene-3(methoxycarbonyl)amino-3-C-[(E)-tetradec-1′-en-1′-yl]-α- -xylofuranose (30) A solution of 9 (0.213 g, 0.55 mmol) in dry toluene (1.4 mL) was treated with tetradec-1-ene (0.7 mL, 2.75 mmol) and second generation Grubbs catalyst (47 mg, 55 μmol), which was added in five portions at 30 min intervals. The mixture was submitted to microwave irradiation conditions at 180 °C for 2.5 h. After the solvent was evaporated, the residue was chromatographed on silica gel (n-hexane/EtOAc, 15:1) to give 0.211 g (69%) of compound 30 as a colourless oil; [α]D21 +67.0 (c 0.46, CHCl3). IR (neat) υ 1252, 1510, 1732, 2854, 2925, 3360 cm−1; 1H NMR (400 MHz, CDCl3) δ 0.11 (s, 3H, CH3), 0.12 (s, 3H, CH3), 0.88 (t,

4.1.21. 5-O-acetyl-3-deoxy-1,2-O-isopropylidene-3-(methoxycarbonyl) amino-3-C-(tetradecyl)-α-D-xylofuranose (33) A solution of 32 (98 mg, 0.22 mmol) in pyridine (1.7 mL) was gradually treated with Ac2O (31 μL, 0.33 mmol) and DMAP (2.7 mg, 22 μmol) at room temperature. After stirring for 30 min, the solvent was evaporated to dryness, and the residue was partitioned between water (5 mL) and CH2Cl2 (5 mL). The aqueous phase was then extracted with another portion of CH2Cl2 (5 mL). The combined organic extracts were 9

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dried over Na2SO4, concentrated, and the crude product was chromatographed through a short column of silica gel (n-hexane/EtOAc, 3:1) to give 100 mg (98%) of compound 33 as a colourless oil; [α]D21 +35.7 (c 0.28, CHCl3). IR (neat) υ 1019, 1238, 1540,1735, 2852, 2922, 3350 cm−1; 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 6.7 Hz, 3H, CH3), 1.26–1.46 (m, 27H, 12 × CH2, CH3), 1.52 (s, 3H, CH3), 1.64–1.70 (m, 1H, H–CH2), 2.03–2.11 (m, 4H, CH3, H–CH2), 3.64 (s, 3H, CH3), 4.06–4.08 (m, 1H, H-4), 4.17 (dd, J = 12.3, 6.0 Hz, 1H, H-5), 4.46 (dd, J = 12.3, 3.3 Hz, 1H, H-5), 4.86–4.87 (m, 1H, H-2), 4.99 (br s, 1H, NH), 5.95 (d, J = 3.5 Hz, 1H, H-1); 13C NMR (100 MHz, CDCl3) δ 14.1 (CH3), 20.9 (CH3), 22.7 (CH2), 23.8 (CH2), 26.2 (CH3), 26.7 (CH3), 29.3 (CH2), 29.4 (CH2), 29.6 (3 × CH2), 29.7 (3 × CH2), 30.0 (CH2), 30.2 (CH2), 31.9 (CH2), 52.1 (CH3), 62.7 (C-5), 67.1 (C-3), 80.8 (C-4), 84.3 (C-2), 104.6 (C-1), 111.9 (Cq), 155.6 (C=O), 170.6 (C=O). ESI-HRMS: m/z calcd for C26H47NNaO7 [M + Na]+ 508.3245, found 508.3271.

4.2. Antiproliferative/cytotoxic activity 4.2.1. Cell culture The following human cancer cell lines were used for this study: HeLa (human cervical adenocarcinoma) and Jurkat (acute T-lymphoblastic leukaemia) cells were maintained in RPMI 1640 medium, MCF10A (human mammary epithelial cells) maintained in DMEM/F-12 supplemented with insulin (5 mg/mL) EGF (10 ng/mL) and cholera toxin (1 ng/mL), A2058 (human melanoma cells) maintained in DMEM + sodium pyruvate (1.5 g/L), and PaTu (human pancreatic adenocarcinoma) maintained in DMEM + sodium pyruvate (1.5 g/L) and Hepes (25 mM). Media were supplemented with Glutamax, and with 10% (V/V) foetal calf serum, penicillin (100 IU × mL−1), and streptomycin (100 mg × mL−1) (all from Invitrogen, Carlsbad, CA USA), in the atmosphere of 5% CO2 in humidified air at 37 °C. Cell viability, estimated by the trypan blue exclusion, was greater than 95% before each experiment.

4.1.22. (2S,3R)-2-hydroxy-3-(hydroxymethyl)-3-[(methoxycarbonyl) amino]heptadecyl acetate (34) The protected derivative 33 (92 mg, 0.19 mmol) was treated with 80% TFA (1.6 mL) at 0 °C After being stirred for 1 h at the same temperature, the mixture was neutralized with NaHCO3 aqueous solution, diluted with water (15 mL) and extracted with EtOAc (3 × 15 mL). The combined organic layers were dried over Na2SO4, concentrated, and the residue was passed through a short column of silica gel (n-hexane/ EtOAc, 1:1) to furnish 72 mg (85%) of a mixture of anomers, which was immediately used in the next step without spectral characterisation. NaIO4 (96 mg, 0.45 mmol) dissolved in H2O (0.36 mL) was added to a solution of the obtained furanoses (69 mg, 0.15 mmol) in MeOH (0.36 mL) at room temperature. The resulting suspension was stirred for 24 h at room temperature. The insoluble parts were filtered off, the filtrate was concentrated, and the crude product was submitted to the reduction step. NaBH4 (8.5 mg, 225 μmol) was added to a solution of the crude aldehyde (66 mg, 0.15 mmol) in MeOH (3.5 mL) at 0 °C. After stirring for 4 h at room temperature, the mixture was neutralized with Amberlite IR 120 H+ form. The insoluble parts were removed by filtration, concentrated, and the residue was flash-chromatographed on silica gel (n-hexane/EtOAc, 1:1) to provide 46 mg (74%) of compound 34 as a colourless oil; [α]D21 −6.7 (c 0.30, CHCl3). IR (neat) υ 1038, 1233, 1702, 2852, 2921, 3356 cm−1; 1H NMR (400 MHz, CDCl3) δ 0.88 (t, 3H, J = 6.8 Hz, 3H, CH3), 1.25–1.34 (m, 24H, 12 × CH2), 1.53–1.70 (m, 2H, CH2), 2.11 (s, 3H, CH3), 3.66–3.69 (m, 4H, CH3, H-1′), 4.00–4.12 (m, 3H, H-1, H-2, H-1′), 4.36 (dd, J = 11.2, 1.3 Hz, 1H, H-1), 5.10 (br s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ 14.1 (CH3), 20.9 (CH3), 22.7 (CH2), 23.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (2 × CH2), 29.7 (3 × CH2), 30.1 (CH2), 31.9 (CH2), 33.4 (CH2), 52.5 (CH3), 60.8 (C-3), 64.4 (C-1′), 66.0 (C-1), 73.5 (C-2), 157.5 (C=O), 172.2 (C=O). ESI-HRMS: m/z calcd for C22H43NNaO6 [M + Na]+ 440.2983, found 440.3001.

4.2.2. Cytotoxicity assay The cytotoxic effect of the tested compounds was studied using the colorimetric microculture assay with the MTT endpoint.26 The amount of MTT reduced to formazan was proportional to the number of viable cells. Briefly, 5 × 103 cells were plated per well in 96-well polystyrene microplates (Sarstedt, Germany) in the culture medium containing tested chemicals at final concentrations 10−4–10−6 mol × L−1. After 72 h incubation, 10 μL of MTT (5 mg × mL−1) were added into each well. After an additional 4 h, during which insoluble formazan was formed, 100 μL of 10% (m/m) sodium dodecylsulfate were added into each well and another 12 h were allowed for the formazan to be dissolved. The absorbance was measured at 540 nm using the automated uQuant™ Universal Microplate Spectrophotometer (Biotek Instruments Inc., Winooski, VT USA). The blank corrected absorbance of the control wells was taken as 100% and the results were expressed as a percentage of the control. Declaration of competing interest The authors declare no conflicts of interest. Acknowledgements The present work was supported by the Grant Agency (no. 1/0047/ 18 and no. 1/0375/19) of the Ministry of Education, Slovak Republic. We thank Dr. Mária Vilková for her assistance in NOESY experiments. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.carres.2019.107862. References

4.1.23. (2S,3R)-3-amino-3-tetradecylbutane-1,2,4-triol hydrochloride (ent-7) The obtained derivative 34 (34 mg, 81 μmol) was treated with 6 M HCl (4 mL) at room temperature. After stirring at reflux for 3 h, the resulting solution was concentrated to dryness under reduced pressure. The crude product was washed three times with Et2O and then dried under high vacuum for 10 h. This procedure yielded 29 mg (82%) of compound ent-7 as a pale yellow hygroscopic solid; [α]D21 +8.8 (c 0.32, MeOH). IR (neat) υ 1051, 1466, 1607, 2851, 2920, 3326 cm−1; 1H NMR (400 MHz, CD3OD) δ 0.90 (t, J = 6.8 Hz, 3H, CH3), 1.29–1.47 (m, 24H, 12 × CH2), 1.58–1.65 (m, 1H, H–CH2), 1.81–1.88 (m, 1H, H–CH2), 3.67 (d, J = 11.5 Hz, 1H, H-4), 3.71–3.79 (m, 4H, 2 × H-1, H2, H-4); 13C NMR (100 MHz, CD3OD) δ 14.4 (CH3), 23.7 (CH2), 23.9 (CH2), 30.5 (2 × CH2), 30.7 (CH2), 30.8 (5 × CH2), 31.2 (CH2), 31.5 (CH2), 33.1 (CH2), 62.7 (C-4), 63.8 (C-1, C-3), 71.1 (C-2). ESI-HRMS: m/z calcd for C18H40NO3 [M + H]+ 318.3003, found 318.3020.

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