Functionalized d- and l-Arabino-Pyrrolidines as Potent and Selective Glycosidase Inhibitors

Abstract

The efficient synthesis of enantiomeric pairs of iminosugars including 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) and 1,4-dideoxy-1,4-imino-l-arabinitol (LAB) analogues with an amidine, hydrazide, hydrazide imide, or amide oxime moiety is described. The preparation of DAB and LAB analogues commenced from l-xylose and d-xylose, respectively. The obtained iminosugars are tested against a panel of glycosidases with pharmaceutical relevance, revealing enhanced activity for the DAB analogues in comparison with the LAB analogues. In particular, the d-arabino-configured amidine behaved as a potent (submicromolar range) and selective inhibitor of α-mannosidase.

Iminosugars such as naturally occurring 1,4-dideoxy-1,4-imino-d-arabinitol (d-1)[1] and its unnatural l- arabino-configured mirror image, 1,4-dideoxy-1,4-imino-l-arabinitol (l- 1),[2] (Figure [1]) are monosaccharide analogues with a nitrogen instead of the endocyclic oxygen. More than 100 examples of such alkaloids have been isolated from bacteria and microorganisms.[3] Iminosugars are especially renowned for their glycosidase inhibitory properties,[4] which has made them attractive as lead compounds for the treatment of diseases such as viral infections, cancer, diabetes, HIV, and lysosomal storage disorders.[5] Currently, three iminosugars are approved for the treatment of type 2 diabetes (miglitol), Gaucher’s disease (N-butyldeoxynojirimycin), and Fabry’s disease (1-deoxygalactonojirimycin).[6] [7] [8]

The pharmaceutical potential of iminosugars has triggered extensive work on a large body of synthetic d-iminosugars,[9] [10] whereas much less work has been performed on l-iminosugars. This is presumably due to their rare occurrence in Nature and hence, for a long time, have been expected to lack biological activity.[11] However, some synthetic l-iminosugars have shown much stronger inhibition than their corresponding natural d-enantiomers against the same glycosidase,[12] [13] even though this is not a general feature.[14] For instance, naturally occurring pyrrolidine d- 1 is a ca. 60-fold stronger yeast α-glycosidase inhibitor than its synthetic antipode l- 1,[2] whereas l- 1 is a much stronger inhibitor of mammalian α-glucosidases than d- 1.[15] In this context, it is worth mentioning that d-iminosugars, including d- 1, and l-iminosugars, including l- 1, tend to behave as competitive and non-competitive glycosidase inhibitors, respectively,[13] which implies that l-iminosugars bind to an allosteric site of the glycosidases. Another interesting finding when comparing d-iminosugars with the corresponding synthetic l-iminosugars is that the mirror compounds can exhibit strong inhibition of different glycosidases.[16] [17] [18]

As part of our ongoing iminosugar program,[19] [20] [21] [22] we recently reported the synthesis and glycosidase inhibitory activity of compounds d- 2, d- 3, and d- 4 (Figure [1]).[20] The glycosidase inhibitory tests showed that hydrazide imides d- 2 and d- 3 were selective inhibitors (low micromolar range) against jack bean α-mannosidase, which is a member of the retaining glycoside hydrolase family 38 that hosts α-mannosidases of pharmaceutical relevance.[23] Following this line of research, we herein report: (1) the synthesis of the d-iminosugars d- 5 and d- 6 from commercially available l-xylose in which the hydrazide imide moiety in d- 2 has been replaced by an amidine and amide oxime group, respectively; (2) the synthesis of all the enantiomers of d- 2–6, namely, l- 2–6, from commercially available d-xylose; and (3) the evaluation of d- 5, d- 6 and l- 2–6 as glycosidase inhibitors. The configuration of the iminosugar, together with the modification of the functionality decorating the endocyclic nitrogen atom, can provide valuable structure–activity relationships regarding glycosidase inhibition.

The key steps in the synthetic strategy leading to d- and l- arabino-pyrrolidines 2–6 are presented in Scheme [1]. The synthesis of the d-enantiomers and l-enantiomers commences from l-xylose and d-xylose, respectively. The first key step includes installation of a nitrile moiety at C1 of the benzylated furanose 7. This is followed by a stereospecific inversion of C4 by a suitable N-nucleophile, and a final cyclization with concomitant formation of the desired functional group.

We previously reported nitrile formation at C1 in our synthesis of arabino-pyrrolidines d-2–4,[20] based on the method reported by Ermert and Vasella.[24] In our report, nitrile l-8 was obtained in only 52% yield due to substantial formation of a lactone byproduct (20%) (Scheme [2a]).[20] We later found that the yield of the overall reaction could be enhanced by avoiding water during work-up of the aldoxime intermediate, obtained from condensation of xylose l-7 with hydroxylamine. Instead, the crude oxime from step 1 was simply dried by co-evaporation with toluene prior to subjecting the oxime to Appel conditions,[24] which avoided lactone formation and furnished nitrile l-8 in 73% yield. The mirror image of l-8, namely d-8, was obtained in 67% yield when d-7,[22] the antipode of l-7, was subjected to an identical reaction sequence (Scheme [2b]).

The l- arabino-hydrazides l- 2, l- 3, and l- 4 were prepared by a similar procedure to our reported synthesis of the d- arabino-hydrazides d- 2, d- 3, and d- 4 (Scheme [2b]).[20] 4-Hydroxynitrile d- 8 was first converted into the corresponding triflate intermediate. Stirring tert-butyl carbazate with 4 Å molecular sieves in THF for 24 hours before addition of the triflate intermediate gave hydrazide l- 9 in 66% yield after 4 days. Acid-mediated Boc removal promoted spontaneous cyclization to hydrazide imide l- 10, which underwent palladium-promoted hydrogenolysis of the benzyl groups to afford target compound l- 2. The hydrazide imide l- 2 could be converted into hydrazide amide l- 4 upon microwave irradiation in a methanolic HCl solution. In our previous report, N-ethylated hydrazide imide d- 3 was obtained in 85% yield from the corresponding tribenzylated hydrazide imide upon running the hydrogenolysis reaction in ethanol for 48 hours.[20] When the enantiomeric tribenzylated compound l- 10 was subjected to identical conditions, only minor alkylation occurred and the alkylated product l- 3 was afforded in 17% yield.

The preparation of the arabino-amidines d-5 and l-5 from nitriles l-8 and d-8, respectively, proceeded uneventfully (Scheme [3]). Azides d-11 and l-11 were prepared in a straightforward triflation and azidation sequence. Chemoselective hydrogenation of azides d-11 and l-11 in the presence of 6 mol% palladium on charcoal provided tribenzylated amidines d- 12 and l- 12 in yields of 91% and 96%, respectively, after final treatment with HCl in methanol. Finally, deprotection of the O-benzyl groups with 10 equivalents of 10% palladium on charcoal[25] furnished arabino-amidines d- 5 and l- 5 in excellent yields. These two products are stable as the HCl salt for weeks in water.

Next, we wanted to introduce an amide oxime functionality in place of the amidine moiety in pyrrolidine d-5. The intermediate l-13, obtained by triflation of nitrile l-8, was therefore treated with hydroxylamine in ethanol (Scheme [4]). The hydroxylamine was produced in situ from its corresponding HCl salt by treatment with sodium ethoxide, resulting in the formation of d-14 and epimeric d-ribo-14. The desired d-arabino-amide oxime d-14 could be isolated in 36% yield after careful separation by silica gel column chromatography. In addition, d-ribo-14 was isolated in 2% yield, and a mixture of the isomers d-14 and d-ribo-14 was isolated in 21% combined yield. The formation of the unexpected isomer d-ribo-14 was presumably caused by epimerization of the amide oxime (or nitrile) α-carbon due to the presence of residual sodium ethoxide in the reaction mixture.

The position of the N-hydroxy group on the exocyclic nitrogen atom in amide oxime d- 14 was examined by 2D NMR analysis of the alkylated derivative of d- 14, viz. d- 16 (Scheme [4]). Correlations from the endocyclic NH group into the pyrrolidine ring strongly suggest that the ethoxy group is not attached to the endocyclic nitrogen, but rather to the exocyclic nitrogen in compound d- 16.

Removal of benzyl groups from the tribenzylated pyrrolidine d- 14 was successfully executed with boron trichloride instead of via a palladium-promoted hydrogenolysis to eliminate the possibility of reducing the amide oxime functionality. The d- arabino-amide oxime d- 6 was thus isolated in 67% yield. To validate the ribo-configuration of d- ribo-14, the stereomeric mixture consisting of d- 14 and d- ribo-14 was converted into the corresponding de-O-benzylated mixture consisting of d- 6 and d- ribo-6 (Scheme [4]). The latter has previously been prepared by Ganem and co-workers,[26] and was thus used as a reference for the characterization of compound d- ribo-6 prepared by us.

l- Arabino-amide oxime l- 6 was prepared from nitrile d- 8 by using similar reaction conditions as for the preparation of the enantiomer d- 6. In an attempt to avoid epimerization of the amide oxime (or nitrile) α-carbon, and consequent formation of a ribo-conformer, hydroxylamine was obtained from the corresponding HCl salt upon treatment with triethylamine instead of the sodium ethoxide employed in the preparation of amide oxime d- 6. Indeed, no formation of the ribo-counterpart was observed upon treatment of triflate d- 13 under these conditions. However, a substantial amount of known lactam l- 15 was isolated from the reaction,[27] and amide oxime l- 14 was obtained in 26% yield. The formation of amides in amide oxime synthesis in the presence of Et₃N has been encountered by others and presumably originates from the primary attack on the nitrile group by the hydroxylamine oxygen.[28] [29] The isolated quantities of both amide oxime enantiomers, viz. d- 6 and l- 6, were sufficient for biological testing.

Compounds d- 5, d- 6 and l- 2–6 were screened as glycosidase inhibitors using the commercial enzymes α-glucosidase (Baker’s yeast), β-glucosidase (almonds), and α-mannosidase (jack beans) (Table [1]). The inhibition data indicates that d- arabino-amidine d- 5 is a potent competitive inhibitor of α-mannosidase (K _i = 0.11 ± 0.03 μM) and of α-glucosidase (K _i = 1.5 ± 0.2 μM), with a 14-fold selectivity towards α-mannosidase. When including our previously reported data,[20] it is evident that enhanced selectivity towards α-mannosidase over α-glucosidase is achieved by N-arming of the charged arabino-amidine endocyclic nitrogen. This is indicated by the 22-fold and >400-fold selectivity displayed by hydrazide imides d- 2 and d- 3, respectively. Moreover, the profile of compound d- 6 shows that the inhibitory activity is impaired by hydroxylation of the exocyclic nitrogen. The l-antipodes, viz. l- 2–6, display low or no inhibition of the tested enzymes. Due to the absence of interfering glycosidase inhibitory activity, evaluation of other biological properties for the presented l-series is of great interest, and further studies are ongoing in our group.

In the work presented herein, a library of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) and 1,4-dideoxy-1,4-imino-l-arabinitol (LAB) analogues with a functionalized endocyclic amidine moiety has been prepared. d- Arabino-amidine d- 5 was found to be a potent inhibitor of α-glucosidase and α-mannosidase in the submicromolar concentration range. Furthermore, l- arabino-pyrrolidines displayed no or low inhibition of the glycosidases tested.

All chemicals were obtained from Sigma-Aldrich/Merck or VWR and used as supplied. Unless otherwise specified, solvents were dried by storing over 4 Å molecular sieves. For petroleum ether (PE), the fraction boiling at 40–65 °C was used. All reactions were carried out under a nitrogen or argon atmosphere, unless otherwise specified. TLC analyses were performed on Merck silica gel 60 F₂₅₄ plates using UV light or KMnO₄ (with heating) for visualization. Silica gel NORMASIL 60^® 40–63 μm was used for flash column chromatography. Melting points were recorded using a Stuart Scientific SMP3 melting point apparatus and are uncorrected. Infrared spectroscopy (IR) was performed on a Cary 360 FTIR spectrophotometer. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Ascend™ 400 series, operating at 400.13 MHz for ¹H and 100.61 MHz for ¹³C in CDCl₃, CD₃OD or D₂O. The assignment of signals in all NMR spectra was assisted by conducting ¹H–¹H correlation spectroscopy (COSY), ¹H–¹³C/¹H–¹⁵N heteronuclear single-quantum correlation spectroscopy (HSQC) and/or ¹H–¹³C/¹H–¹⁵N heteronuclear multiple bond correlation spectroscopy (HMBC). Chemical shifts (δ) are reported in ppm relative to an internal standard of residual chloroform (δ = 7.26 for ¹H NMR; δ = 77.16 for ¹³C NMR), residual methanol (δ = 3.31 for ¹H NMR; δ = 49.00 for ¹³C NMR), or residual water (δ = 4.79 for ¹H NMR). High-resolution mass spectra were obtained on a JEOL AccuTOF™ T100GC mass spectrometer. The instrument was operated with an orthogonal electrospray ionization source (ESI), an orthogonal accelerated time-of-flight (TOF) single-stage reflectron mass analyzer and a dual micro channel plate (MCP) detector. The microwave-assisted experiments were performed in a CEM Focused Microwave™ Synthesis System, model type Discover, operating at 0–300 W at a temperature of 150 °C, a pressure range of 0–290 psi, and with reactor vial volumes of 10 mL.

2,3,5-Tri-O-benzyl-l-xylononitrile (l-8)[20]

Step 1: To a solution of sodium (270 mg, 11.7 mmol) in absolute EtOH (40 mL) was added NH₂OH·HCl (1.63 g, 23.5 mmol). The reaction mixture was stirred at room temperature for 15 min before a solution of furanose l- 7 (1.23 g, 2.93 mmol) in absolute EtOH (10 mL) was added. The resulting mixture was stirred at room temperature for 1 h before the volatiles were removed under reduced pressure. Toluene was then added to the residue, which was subsequently concentrated under reduced pressure and used directly in the following step without further purification.

Step 2: A solution of PPh₃ (1.21 g, 4.40 mmol, 1.5 equiv) in MeCN (18 mL) at room temperature was added to the dried residue from step 1. The resulting mixture was stirred at room temperature for 20 min before a solution of CBr₄ (2.43 g, 7.33 mmol, 2.5 equiv) in MeCN (6 mL) was added. The reaction mixture was then stirred at room temperature for 20 min before adding a solution of PPh₃ (809 mg, 2.93 mmol, 1.0 equiv) in MeCN (12 mL) and MeOH (37 mL). The mixture was stirred for an additional 20 min before the solvent was removed under reduced pressure. The residue was subjected to flash chromatography (PE/EtOAc, 5:1 → 4:1). Collection of the appropriate fractions (R_f = 0.4, PE/EtOAc, 3:1) provided nitrile l- 8 (893 mg, 73%) as a clear, light-yellow oil.

[α]_D ^26.0 = –29.0 (c 0.5, CHCl₃) [Lit.[20] [α]_D ^26.6 = –28.0 (c 0.5, CHCl₃)].

¹H NMR (400 MHz, CDCl₃): δ = 7.37–7.24 (m, 15 H, Ar-H), 4.88 (d, J = 11.5 Hz, 1 H, CHPh), 4.82 (d, J = 11.2 Hz, 1 H, CHPh), 4.60 (d, J = 11.2 Hz, 1 H, CHPh), 4.55 (d, J = 11.5 Hz, 1 H, CHPh), 4.45 (d, J = 1.5 Hz, 2 H, CHPh), 4.43 (d, J = 6.6 Hz, 1 H, H-2), 4.11 (m, 1 H, H-4), 3.88 (dd, J _3,4 = 2.9 Hz, J _3,2 = 6.6 Hz, 1 H, H-3), 3.45 (ddd, J _5a,4 = 6.0 Hz, J _5a,5b = 9.5 Hz, J _5b,4 = 16.3 Hz, 2 H, H-5), 2.29 (d, J = 7.2 Hz, 1 H, OH).

¹³C NMR (100 MHz, CDCl₃): δ = 137.7 (ArC), 137.3 (ArC), 135.7 (ArC), 128.8–128.0 (ArCH, 7 signals), 116.8 (CN), 78.1 (C-3), 75.2 (CH₂Ph), 73.4 (CH₂Ph), 73.0 (CH₂Ph), 70.4 (C-5), 69.6 (C-4), 69.3 (C-2).

The spectroscopic data were in full accord with the previously reported data.[20]

2,3,5-Tri-O-benzyl-d-xylofuranose (d-7)[22]

Step 1: To a solution of AcCl (0.29 mL, 4 mmol) in MeOH (40 mL) was added d-xylose (3 g, 20 mmol) at room temperature and the mixture was stirred at this temperature for 5.5 h. The reaction mixture was then cooled to 0 °C and the pH was adjusted to ca. 9 with NaOH (1 M). The solvent was removed under reduced pressure and the resulting residue was suspended in toluene (6 × 10 mL) and concentrated to dryness.

Step 2: The residue from step 1 was dissolved in dry DMF (64 mL) and NaH (4.24 g, 5.3 equiv) was added at room temperature. The reaction mixture was then cooled to 0 °C prior to the addition of BnBr (11.9 mL, 5 equiv). The reaction mixture was stirred at room temperature for 23 h before being extracted with EtOAc (2 × 75 mL). The combined extracts were then dried over MgSO₄ and concentrated in vacuo.

Step 3: The residue from step 2 was dissolved in dioxane (60 mL) and HCl (60 mL, 4 M) and stirred at 65 °C for 4 d. After cooling to room temperature, the reaction mixture was extracted with EtOAc (2 × 75 mL) and the combined organic fractions were concentrated and subjected to flash column chromatography (PE/EtOAc, 17:3 → 3:1). Collection of the appropriate fractions (R_f = 0.25, PE/EtOAc, 3:1) provided product d- 7 (5.54 g, 66%) as a clear, light-yellow oil.

[α]_D ^26.6 = +10.0 (c 1.2, CHCl₃) [mirror image: Lit.[20] [α]_D ^26.6 = –10.0 (c 1.2, CHCl₃), Lit.[22] [α]_D ^26.0 = –12 (c 1.2, EtOAc)].

¹H NMR (400 MHz, CDCl₃): δ = 7.32–7.18 (m, 15 H, ArH), 5.43 (d, J = 4.1 Hz, 1 H, H-2a), 5.20 (br s, 1 H, H-2b), 4.64–4.32 (m, 7 H, CHPh + CH), 4.06 (dd, J = 5.4 Hz, J = 3.0 Hz, 1 H, H-4b), 3.99 (dd, J = 4.4 Hz, J = 2.4 Hz, 1 H, H-4a), 3.95 (dd, J = 3.0 Hz, J = 1.0 Hz, 1 H, H-3b), 3.89 (dd, J = 4.2 Hz, J = 2.4 Hz, 1 H, H-3a), 3.74–3.60 (m, 2 H, H-5); a and b refer to the two diastereomeric cyclic isomers in which the hydroxyl group connected to the anomeric carbon is in alpha or beta position.

¹³C NMR (100 MHz, CDCl₃): δ = 138.3 (ArCa), 137.8 (ArCa), 137.7 (ArCb), 137.6 (ArCb), 137.5 (ArCb), 136.9 (ArCa), 128.8–128.4 (ArCH, 6 signals), 128.1–127.7 (ArCH, 8 signals), 101.8 (C-2b), 96.3 (C-2a), 86.7 (C-3b), 81.4 (C3a + C-4b), 81.2 (C-4a), 80.0 (CH), 77.4 (CH), 73.8 (CH₂b), 73.6 (CH₂a), 73.2 (CH₂a), 72.8 (CH₂b), 72.5 (CH₂a), 72.0 (CH₂b), 68.8 (C-5b), 68.4 (C-5a); a and b refer to the two diastereomeric cyclic isomers in which the hydroxyl group connected to the anomeric carbon is in alpha or beta position.

The spectroscopic data were in full accord with the previously reported data.[22]

2,3,5-Tri-O-benzyl-d-xylononitrile (d-8)

Step 1: To a solution of sodium (460 mg, 20 mmol) in absolute EtOH (80 mL) was added NH₂OH·HCl (2.65 g, 38 mmol). The reaction mixture was stirred at room temperature for 10 min before a solution of furanose d- 7 (2.20 g, 5.23 mmol) in absolute EtOH (15 mL) was added. The resulting reaction mixture was stirred at room temperature for 1 h before the volatiles were removed under reduced pressure. Toluene was then added to the residue, which was subsequently concentrated under reduced pressure and used directly in the following step without further purification.

Step 2: To the dried residue from step 1 was added a solution of PPh₃ (2.89 g, 10.46 mmol, 2 equiv) in MeCN (42 mL). The resulting mixture was stirred at room temperature for 20 min before a solution of CBr₄ (4.38 g, 13.08 mmol, 2.5 equiv) in MeCN (18 mL) was added. The reaction mixture was then stirred at room temperature for 20 min before adding a solution of PPh₃ (720 mg, 2.61 mmol, 0.5 equiv) in MeCN (10.5 mL) and MeOH (66 mL). The reaction mixture was stirred for an additional 20 min before the solvent was removed under reduced pressure. The residue was subjected to flash chromatography (PE/EtOAc, 5:1 → 4:1). Collection of the appropriate fractions (R_f = 0.40, PE/EtOAc, 3:1) provided nitrile d- 8 (1.46 g, 67%) as a clear, light-yellow oil.

[α]_D ^26.0 = +32.0 (c 1.2, CHCl₃).

IR (ATR): 3470, 3065, 3032, 2920, 2870, 1955, 1882, 1812, 1554, 1497, 1455, 1398, 1353, 1250, 1210, 1096, 1028, 1002, 913, 821, 738 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ = 7.38–7.26 (m, 15 H, ArH), 4.89 (d, J = 11.6 Hz, 1 H, CHPh), 4.83 (d, J = 11.2 Hz, 1 H, CHPh), 4.61 (d, J = 11.2 Hz, 1 H, CHPh), 4.56 (d, J = 11.6 Hz, 1 H, CHPh), 4.52–4.43 (m, 3 H, CHPh and H-2), 4.13 (ddd, J = 13.0 Hz, J = 6.1 Hz, J = 2.9 Hz, 1 H, H-4), 3.89 (dd, J = 6.6 Hz, J = 2.9 Hz, 1 H, H-3), 3.49 (dd, J = 9.5 Hz, J = 6.0 Hz, 1 H, H-5a), 3.43 (dd, J = 9.5 Hz, J = 6.0 Hz, 1 H, H-5b), 2.37 (d, J = 7.2 Hz, 1 H, OH).

¹³C NMR (100 MHz, CDCl₃): δ = 137.8 (ArC), 137.3 (ArC), 135.7 (ArC), 128.9–127–9 (ArCH, 7 signals), 116.8 (CN), 78.2 (C-3), 75.3 (CH₂Ph), 73.5 (CH₂Ph), 73.1 (CH₂Ph), 70.5 (C-5), 69.7 (C-4), 69.4 (C-2).

HRMS (ESI): m/z [M + Na]⁺ calcd for C₂₆H₂₇O₄NNa: 440.1838; found: 440.1835.

The spectroscopic data were in full accord with the previously reported data of its enantiomer.[20]

2,3,5-Tri-O-benzyl-4-(2-tert-butoxycarbonyl)hydrazinyl-4-deoxy-l-arabinonitrile (l-9)

Step 1: To a solution of alcohol d- 8 (334 mg, 0.8 mmol) in DCM (7 mL) at 0 °C was added pyridine (0.16 mL, 2.0 mmol, 2.5 equiv). The reaction mixture was stirred for 10 min prior to the dropwise addition of triflic anhydride (0.17 mL, 1.0 mmol, 1.25 equiv). The reaction mixture was further stirred at 0 °C for 15 min before being diluted with DCM (60 mL), washed with ice-cold HCl (15 mL, 1 M) and sat. aq NaHCO₃ (20 mL), dried over Na₂SO₄, and filtered. The residue (R_f = 0.71, PE/EtOAc, 7:3) was concentrated under reduced pressure and used directly in the following step.

Step 2: A suspension of tert-butyl carbazate (1.06 g, 8.0 mmol, 10 equiv) and 4 Å MS in THF (4.5 mL) was stirred for 24 h. To the obtained suspension was added a solution of the triflate from step 1 in THF (4.5 mL). The resulting mixture was stirred at room temperature for 4 d before the volatiles were removed under reduced pressure. The residue was subjected to flash column chromatography (PE/EtOAc, 23:2 → 17:3). Collection of the appropriate fractions (R_f = 0.39, PE/EtOAc, 3:1) provided hydrazide l- 9 (277 mg, 66%) as a clear, slightly white oil.

[α]_D ^25.4 = +8.0 (c 1.0, CHCl₃).

IR (ATR): 3339, 3014, 2979, 2929, 2870, 1718, 1497, 1454, 1393, 1367, 1249, 1216, 1157, 1088, 1073, 1028 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ = 7.37–7.29 (m, 15 H), 5.83 (m, 1 H, NH), 4.89 (d, J = 11.5 Hz, 1 H, CHPh), 4.85 (d, J = 11.1 Hz, 1 H, CHPh), 4.76 (d, J _2,3 = 3.7 Hz, 1 H, H-2), 4.66 (d, J = 11.1 Hz, 1 H, CHPh), 4.58 (d, J = 11.5 Hz, 1 H, CHPh), 4.54 (d, J = 11.8 Hz, 1 H, CHPh), 4.46 (d, J = 11.8 Hz, 1 H, CHPh), 4.21 (br s, 1 H, NH), 3.84 (dd, J _2,3 = 3.7 Hz, J _3,4 = 7.0 Hz, 1 H, H-3), 3.73 (dd, J _5a,4 = 4.4 Hz, J _5a,5b = 9.7 Hz, 1 H, H-5a), 3.62 (dd, J _5b,4 = 5.7 Hz, J _5b,5a = 9.7 Hz, 1 H, H-5b), 3.30 (ddd, J _4,5a = 4.4 Hz, J _4,5b = 5.7 Hz, J _4,3 = 7.0 Hz, 1 H, H-4), 1.45 (2 br s, 9 H).

¹³C NMR (100 MHz, CDCl₃): δ = 156.7 (C=O), 137.9 (ArC), 137.5 (ArC), 136.0 (ArC), 128.8–128.0 (ArCH, 9 signals), 117.8 (CN), 80.6 (C), 78.1 (C-3), 74.9 (CH₂Ph), 73.5 (CH₂Ph), 72.9 (CH₂Ph), 68.3 (C-2), 67.8 (C-5), 60.5 (C-4), 28.4 (CH₃).

HRMS (ESI): m/z [M + Na]⁺ calcd for C₃₁H₃₇O₅N₃Na: 554.2630; found: 554.2626.

The spectroscopic data were in full accord with the previously reported data for its enantiomer.[20]

(2S,3S,4S)-1-Amino-3,4-bis(benzyloxy)-2-[(benzyloxy)methyl]-5-iminopyrrolidine Hydrochloride (l-10)

To a solution of compound l- 9 (165 mg, 0.31 mmol) in DCM (10 mL) at room temperature under argon was added TFA (2 mL) dropwise. The reaction was kept stirring for 2 h before the volatiles were removed under reduced pressure. The residue was dissolved in 0.5 M methanolic HCl (10 mL) and evaporated to afford the HCl salt. The residue was subjected to flash column chromatography [CHCl₃/MeOH (0.1 M HCl), 9:1]. Collection of the appropriate fractions [R_f = 0.25, CHCl₃/MeOH (0.1 M HCl), 9:1] provided compound l- 10 (128 mg, 87%) as a clear, slightly yellow oil.

[α]_D ^27.0 = +10.0 (c 0.20, MeOH) [mirror image: Lit.[20] [α]_D ^27.2 = –11.0 (c 0.2, MeOH)].

IR (ATR): 3029, 2929, 2110, 1953, 1882, 1811, 1702, 1624, 1495, 1453, 1362, 1261, 1209, 1121, 1099, 1063, 1028, 971, 915 cm^–1.

¹H NMR (400 MHz, CD₃OD): δ = 7.36–7.25 (m, 15 H, ArH), 4.82 (d, J _4,3 = 3.8 Hz, 1 H, H-4), 4.73 (d, J = 11.5 Hz, 1 H, CHPh), 4.63 (d, J = 11.5 Hz, 1 H, CHPh), 4.59 (d, J = 11.7 Hz, 1 H, CHPh), 4.48 (d, J = 11.7 Hz, 1 H, CHPh), 4.48 (br s, 2 H, CHPh), 4.25 (t, J = 3.8 Hz, 1 H, H-3), 3.97 (m, 1 H, H-2), 3.89 (dd, J _6a,2 = 3.5 Hz, J _6a,6b = 10.8 Hz, 1 H, H-6a), 3.63 (dd, J _6b,2 = 2.8, J _6a,6b = 10.8 Hz, 1 H, H-6b).

¹³C NMR (100 MHz, CD₃OD): δ = 166.6 (C=N), 138.8 (ArC), 138.4 (ArC), 138.1 (ArC), 129.5–129.0 (ArCH, 7 signals), 82.2 (C-4), 79.5 (C-3), 74.2 (CH₂Ph), 74.0 (CH₂Ph), 73.3 (CH₂Ph), 70.9 (C-2), 66.3 (C-6).

HRMS (ESI): m/z [M + H – HCl]⁺ calcd for C₂₆H₃₀O₃N₃: 432.2287; found: 432.2283.

The spectroscopic data were in full accord with the previously reported data for its enantiomer.[20]

(2S,3S,4S)-1-Amino-3,4-dihydroxy-2-(hydroxymethyl)-5-iminopyrrolidine Hydrochloride (l-2)

A suspension of compound l- 10 (30 mg, 0.064 mmol) and 10% Pd/C (84 mg, 0.79 mmol) in EtOH/TFA (9:1, 2.5 mL) was hydrogenated at room temperature for 24 h. The reaction mixture was then filtered through Celite with the aid of methanol (10 mL). The filtrate was added to 0.5 M methanolic HCl (10 mL) and evaporated to afford the HCl salt. The residue was subjected to gravity column chromatography (MeCN/H₂O, 97:3). Collection of the appropriate fractions [R_f = 0.29, MeCN/H₂O (0.1 M HCl), 8:2] provided compound l- 2 (12 mg, 0.061 mmol, 95%) as a yellow solid.

Mp 156.0–156.5 °C (dec.); [α]_D ^26.2 = +5.4 (c 0.37, MeOH) [mirror image: Lit.[20] [α]_D ^27.2 = –5.7 (c 0.35, MeOH)].

IR (ATR): 3369, 2125, 1724, 1624, 1205, 1109, 1063 cm^–1.

¹H NMR (400 MHz, D₂O): δ = 4.83 (d, J _4,3 = 7.0 Hz, 1 H, H-4), 4.27 (t, J = 7.0 Hz, 1 H, H-3), 4.13 (dd, J _2′a,2 = 2.6 Hz, J _2′a,2′b = 13.3 Hz, 1 H, H-2′a), 3.87 (dd, J _2′b,2 = 2.5 Hz, J _2′b,2′a = 13.3 Hz, 1 H, H-2′b), 3.72 (app dt, J _2,2′ = 2.5 Hz, J _2,3 = 7.0 Hz, 1 H, H-2).

¹³C NMR (100 MHz, D₂O): δ = 167.4 (C=N), 74.1 (C-4), 72.7 (C-3), 69.0 (C-2), 55.6 (C-2′).

HRMS (ESI): m/z [M + H – HCl]⁺ calcd for C₅H₁₂O₃N₃: 162.0878; found: 162.0874.

The spectroscopic data were in full accord with the previously reported data for its enantiomer.[20]

(2S,3S,4S)-1-(Ethylamino)-3,4-dihydroxy-2-(hydroxymethyl)-5-iminopyrrolidine Hydrochloride (l-3)

A suspension of compound l -10 (20 mg, 0.043 mmol) and 10% Pd/C (70 mg, 0.65 mmol) in EtOH/TFA (9:1, 2.5 mL) was hydrogenated at room temperature for 48 h. The reaction mixture was then filtered through Celite with the aid of methanol (10 mL). The filtrate was added to 0.5 M methanolic HCl (10 mL) and evaporated to afford the HCl salt. The residue was subjected to gravity column chromatography (MeCN/H₂O, 97:3). Collection of the appropriate fractions [R_f = 0.45, MeCN/H₂O (0.1 M HCl), 4:1] provided compound l -3 (1.7 mg, 7.5 μmol, 17%) as a yellow solid.

Mp 130.0–131.0 °C (dec.); [α]_D ^25.2 = –12.0 (c 0.10, MeOH) [mirror image: Lit.[20] [α]_D ^25.2 = +12.0 (c 0.17, MeOH)].

IR (ATR): 3215, 2977, 2934, 2878, 2110, 1702, 1637, 1454, 1383, 1332, 1276, 1201, 1103, 1063, 905 cm^–1.

¹H NMR (400 MHz, D₂O): δ = 4.90 (d, J _4,3 = 6.9 Hz, 1 H, H-4), 4.31 (app t, J = 6.8 Hz, 1 H, H-3), 4.07 (dd, J _2′a,2′b = 13.0 Hz, J _2′a,2 = 2.6 Hz, 1 H, 2′a), 4.03 (dt, J _2,3 = 6.7 Hz, J _2,2′b = 2.7 Hz, J _2,2′a = 2.6 Hz, 1 H, H-2), 3.90 (dd, J _2′b,2′a = 13.0 Hz, J _2′b,2 = 2.7 Hz, 1 H, 2′b), 3.04 (dq, J _6a,6b = 11.7 Hz, J _6a,7 = 7.3 Hz, 1 H, H-6a), 2.91 (dq, J _6b,6a = 11.7 Hz, J _6b,7 = 7.1 Hz, 1 H, H-6b), 1.09 (app t, J = 7.2 Hz, 3 H, H-7).

¹³C NMR (100 MHz, D₂O): δ = 167.9 (C=N), 73.9 (C-4), 73.0 (C-3), 64.8 (C-2), 56.0 (C-2′), 42.0 (C-6), 11.8 (C-7).

HRMS (ESI): m/z [M + H – HCl]⁺ calcd for C₇H₁₆O₃N₃: 190.1191; found: 190.1186.

The spectroscopic data were in full accord with the previously reported data for its enantiomer.[20]

(3R,4S,5S)-1-Amino-3,4-dihydroxy-5-(hydroxymethyl)-2-pyrrolidinone Hydrochloride (l-4)

A solution of compound l- 2 (13 mg, 0.07 mmol) in methanolic HCl (0.5 M, 1 mL) was subjected to microwave irradiation (150 °C) for 90 min. The volatiles were removed under reduced pressure. The resulting black residue was subjected to gravity column chromatography (MeCN/H₂O, 97:3). Collection of the appropriate fractions (R_f = 0.37, MeCN/H₂O, 8:2) provided compound l- 4 (10 mg, 0.05 mmol, 72%) as a brown wax.

[α]_D ^26.2 = +5.0 (c 0.4, MeOH) [mirror image: Lit.[20] [α]_D ^25.5 = –5.1 (c 0.39, MeOH)].

IR (ATR): 3295, 2926, 2855, 2358, 1699, 1455, 1349, 1261, 1200, 1095, 909 cm^–1.

¹H NMR (400 MHz, D₂O): δ = 4.31 (d, J _4,3 = 7.1 Hz, 1 H, H-3), 4.10 (t, J = 7.1 Hz, 1 H, H-4), 4.05 (dd, J _6a,5 = 2.7 Hz, J _6a,6b = 12.9 Hz, 1 H, H-6a), 3.79 (dd, J _6b,5 = 2.5 Hz, J _6b,6a = 12.9 Hz, 1 H, H-6b), 3.47 (m, 1 H, H-5).

¹³C NMR (100 MHz, D₂O): δ = 172.5 (C=O), 75.2 (C-3), 72.3 (C-4), 65.3 (C-5), 57.2 (C-6).

HRMS (ESI): m/z [M + H – HCl]⁺ calcd for C₅H₁₁O₄N₂: 163.0719; found: 163.072.

The spectroscopic data were in full accord with the previously reported data for its enantiomer.[20]

2,3,5-Tri-O-benzyl-4-azido-4-deoxy-d-arabinonitrile (d-11)

Step 1: To a solution of compound l- 8 (676 mg, 1.62 mmol) in DCM (15 mL) at 0 °C was added pyridine (0.30 mL, 4.05 mmol, 2.5 equiv). The reaction mixture was stirred for 10 min prior to the dropwise addition of triflic anhydride (0.33 mL, 2.03 mmol, 1.25 equiv). The reaction mixture was further stirred at 0 °C for 15 min before being diluted with DCM (150 mL) and washed with ice-cold HCl (45 mL, 1 M) and sat. aq NaHCO₃ (60 mL). After drying over Na₂SO₄ and filtration, the filtrate (R_f = 0.71, PE/EtOAc, 7:3) was concentrated under reduced pressure and used directly in the following step.

Step 2: To a solution of the residue from step 1 in DMF (10 mL) at 0 °C was added NaN₃ (160 mg, 2.46 mmol). The reaction mixture was stirred at 0 °C for 5 h and then water (100 mL) was added. The mixture was extracted with EtOAc (150 mL) and the organic fraction was concentrated under reduced pressure. The residue was subjected to flash column chromatography (PE/EtOAc, 95:5). Collection of the appropriate fractions (R_f = 0.37, PE/EtOAc, 9:1) provided azide d- 11 (620 mg, 1.40 mmol, 87%) as a clear, colorless oil.

[α]_D ^25.4 = –56.0 (c 2.0, CHCl₃).

IR (ATR): 3065, 3030, 2923, 2870, 2101, 1497, 1455, 1398, 1362, 1315, 1269, 1210, 1094, 1028, 915, 820, 737 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ = 7.41–7.27 (m, 15 H, ArH), 4.92 (d, J = 11.7 Hz, 1 H, CHPh), 4.87 (d, J = 10.9 Hz, 1 H, CHPh), 4.61 (d, J = 10.9 Hz, 1 H, CHPh), 4.57 (d, J = 11.7 Hz, 1 H, CHPh), 4.52 (br s, 2 H, CHPh), 4.41 (d, J = 2.7 Hz, 1 H, H-2), 3.81–3.79 (m, 3 H), 3.70–3.66 (m, 1 H).

¹³C NMR (100 MHz, CDCl₃): δ = 137.5 (ArC), 136.9 (ArC), 135.3 (ArC), 128.9–127.9 (ArCH, 9 signals), 117.0 (CN), 77.9 (C-3), 75.3 (CH₂Ph), 73.6 (CH₂Ph), 72.9 (CH₂Ph), 68.8 (C-2), 67.4 (C-5), 60.2 (C-4).

HRMS (ESI): m/z [M + Na]⁺ calcd for C₂₆H₂₆O₃N₄Na: 465.1902; found: 465.1898.

2,3,5-Tri-O-benzyl-4-azido-4-deoxy-l-arabinonitrile (l-11)

Step 1: To a solution of alcohol d- 8 (222 mg, 0.53 mmol) in DCM (5 mL) at 0 °C was added pyridine (0.10 mL, 1.33 mmol, 2.5 equiv). The reaction mixture was stirred for 10 min prior to the dropwise addition of triflic anhydride (0.11 mL, 0.66 mmol, 1.25 equiv). The reaction mixture was further stirred at 0 °C for 15 min before being diluted with DCM (50 mL) and washed with ice-cold HCl (15 mL, 1 M) and sat. aq NaHCO₃ (20 mL). After drying over Na₂SO₄ and filtration, the filtrate (R_f = 0.71, PE/EtOAc, 7:3) was concentrated under reduced pressure and used directly in the following step.

Step 2: To a solution of the residue from step 1 in DMF (5 mL) at 0 °C was added NaN₃ (58 mg, 0.89 mmol). The reaction mixture was stirred at 0 °C for 5 h and then water (50 mL) was added. The mixture was extracted with EtOAc (100 mL) and the organic fraction was concentrated under reduced pressure. The residue was subjected to flash column chromatography (PE/EtOAc, 9:1). Collection of the appropriate fractions (R_f = 0.37, PE/EtOAc, 9:1) provided azide l- 11 (182 mg, 78%) as a clear, colorless oil.

[α]_D ^25.6 = +53.0 (c 2.0, CHCl₃).

IR (ATR): 3065, 3030, 2923, 2870, 2101, 1497, 1455, 1398, 1362, 1315, 1269, 1210, 1094, 1028, 915, 820, 737 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ = 7.41–7.27 (m, 15 H, ArH), 4.92 (d, J = 11.7 Hz, 1 H, CHPh), 4.87 (d, J = 10.9 Hz, 1 H, CHPh), 4.61 (d, J = 10.9 Hz, 1 H, CHPh), 4.57 (d, J = 11.7 Hz, 1 H, CHPh), 4.52 (br s, 2 H, CHPh), 4.41 (d, J = 2.7 Hz, 1 H, H-2), 3.81–3.79 (m, 3 H), 3.70–3.66 (m, 1 H).

¹³C NMR (100 MHz, CDCl₃): δ = 137.5 (ArC), 136.9 (ArC), 135.3 (ArC), 128.9–127.9 (ArCH, 9 signals), 117.0 (CN), 77.9 (C-3), 75.3 (CH₂Ph), 73.6 (CH₂Ph), 72.9 (CH₂Ph), 68.8 (C-2), 67.4 (C-5), 60.2 (C-4).

HRMS (ESI): m/z [M + Na]⁺ calcd for C₂₆H₂₆O₃N₄Na: 465.1902; found: 465.1900.

(3R,4R,5R)-3,4-Bis(benzyloxy)-5-[(benzyloxy)methyl]-2-iminopyrrolidine Hydrochloride (d-12)

A suspension of azide d- 11 (100 mg, 0.23 mmol), 10% Pd/C (1.4 mg, 6 mol%) and acetic acid (2 drops) in EtOH (3 mL) was hydrogenated for 16 h at room temperature. The mixture was then filtered through Celite and rinsed with MeOH. The filtrate was concentrated under reduced pressure and then 0.5 M methanolic HCl (30 mL) was added. The volatiles were removed under reduced pressure. The resulting HCl salt was subjected to flash column chromatography [CHCl₃/MeOH (0.1 M HCl), 95:5]. Collection of the appropriate fractions [R_f = 0.35, CHCl₃/MeOH (0.1 M HCl), 9:1] provided amidine d- 12 (95 mg, 0.21 mmol, 91%) as a clear, slightly yellow oil.

[α]_D ^26.1 = +6.0 (c 1.0, MeOH).

IR (ATR): 3029, 2929, 2866, 1701, 1496, 1453, 1395, 1359, 1204, 1092, 1027, 911, 732 cm^–1.

¹H NMR (400 MHz, CD₃OD): δ = 7.34–7.26 (m, 15 H), 4.83 (d, J _3,4 = 4.6 Hz, 1 H, H-3), 4.76 (d, J = 11.5 Hz, 1 H, CHPh), 4.67 (d, J = 11.5 Hz, 1 H, CHPh), 4.56–4.48 (m, 4 H, CHPh), 4.26 (t, J = 4.4 Hz, 1 H, H-4), 3.98 (app q, J = 4.5 Hz, 1 H, H-5), 3.64 (dd, J = 4.2 Hz, J = 10.3 Hz, 1 H, H-6), 3.53 (dd, J = 5.0 Hz, J = 10.3 Hz, 1 H, H-6).

¹³C NMR (100 MHz, CD₃OD): δ = 168.8 (C=N), 138.9 (ArC), 138.6 (ArC), 138.1 (ArC), 129.6–129.0 (ArCH, 8 signals), 84.0 (C-3), 81.7 (C-4), 74.39 (CH₂Ph), 74.36 (CH₂Ph), 73.5 (CH₂Ph), 69.8 (C-6), 64.5 (C-5).

HRMS (ESI): m/z [M + H – HCl]⁺ calcd for C₂₆H₂₉O₃N₂: 417.2178; found: 417.2172.

(3S,4S,5S)-3,4-Bis(benzyloxy)-5-[(benzyloxy)methyl]-2-iminopyrrolidine Hydrochloride (l-12)

To a suspension of 10% Pd/C (1 mg, 6 mol%) and azide l- 11 (62 mg, 0.14 mmol) in EtOH (2 mL) was added acetic acid (2 drops) and the reaction mixture was hydrogenated at room temperature for 16 h. The mixture was then filtered through Celite with the aid of MeOH. After concentration under reduced pressure, the residue was converted into the HCl salt by evaporation from 0.5 M methanolic HCl (20 mL). The obtained HCl salt was subjected to flash column chromatography [CHCl₃/MeOH (0.1 M HCl), 195:5]. Collection of the appropriate fractions [R_f = 0.35, CHCl₃/MeOH (0.1 M HCl), 9:1] provided amidine l- 12 (61 mg, 0.134 mmol, 96%) as a clear, slightly yellow oil.

[α]_D ^26.1 = –6.0 (c 1.0, MeOH).

IR (ATR): 3029, 2929, 2866, 1701, 1496, 1453, 1395, 1359, 1204, 1092, 1027, 911, 732 cm^–1.

¹H NMR (400 MHz, CD₃OD): δ = 7.34–7.26 (m, 15 H), 4.83 (d, J _3,4 = 4.6 Hz, 1 H, H-3), 4.76 (d, J = 11.5 Hz, 1 H, CHPh), 4.67 (d, J = 11.5 Hz, 1 H, CHPh), 4.56–4.48 (m, 4 H, CHPh), 4.26 (t, J = 4.4 Hz, 1 H, H-4), 3.98 (app q, J = 4.5 Hz, 1 H, H-5), 3.64 (dd, J = 4.2 Hz, J = 10.3 Hz, 1 H, H-6), 3.53 (dd, J = 5.0 Hz, J = 10.3 Hz, 1 H, H-6).

¹³C NMR (100 MHz, D₂O): δ = 168.8 (C=N), 138.9 (ArC), 138.6 (ArC), 138.1 (ArC), 129.6–129.0 (ArCH, 8 signals), 84.0 (C-3), 81.7 (C-4), 74.39 (CH₂Ph), 74.36 (CH₂Ph), 73.5 (CH₂Ph), 69.8 (C-6), 64.5 (C-5).

HRMS (ESI): m/z [M + H – HCl]⁺ calcd for C₂₆H₂₉O₃N₂: 417.2178; found: 417.2174.

(2R,3R,4R)-3,4-Dihydroxy-2-(hydroxymethyl)-5-iminopyrrolidine Hydrochloride (d-5)

A solution of amidine d- 12 (65 mg, 0.16 mmol) and 10% Pd/C (170 mg, 10 equiv) in EtOH/TFA (5 mL, 9:1) was hydrogenated at room temperature for 20 h. The mixture was filtered through Celite with the aid of MeOH and the filtrate was concentrated under reduced pressure. The residue was treated with 0.5 M methanolic HCl (10 mL) and was concentrated under reduced pressure. The resulting HCl salt was subjected to gravity column chromatography (MeCN/H₂O, 97:3). Collection of the appropriate fractions (R_f = 0.22, MeCN/H₂O, 8:2) provided amidine d- 5 (23.1 mg, 0.158, 99%) as a white wax.

[α]_D ^26.2 = +15.1 (c 0.53, MeOH).

IR (ATR): 3153, 1699, 1405, 1333, 1271, 1200, 1096, 1042, 990, 922, 886 cm^–1.

¹H NMR (400 MHz, D₂O): δ = 4.88 (d, J _4,3 = 7.8 Hz, 1 H, H-4), 4.23 (dd, J _3,4 = 7.8 Hz, J _3,2 = 6.6 Hz, 1 H, H-3), 3.95–3.91 (m, 1 H, H-2′a), 3.78–3.73 (m, 2 H, H-2′b + H-2).

¹³C NMR (100 MHz, D₂O): δ = 168.7 (C=N), 75.9 (C-4), 74.8 (C-3), 62.3 (C-2), 59.2 (C-2′).

HRMS (ESI): m/z [M + H – HCl]⁺ calcd for C₅H₁₁O₃N₂: 147.0769; found: 147.0764.

(2S,3S,4S)-3,4-Dihydroxy-2-(hydroxymethyl)-5-iminopyrrolidine Hydrochloride (l-5)

A solution of compound l- 12 (59 mg, 0.13 mmol) and 10% Pd/C (138 mg, 10 equiv) in EtOH/TFA (5 mL, 9:1) was hydrogenated at room temperature for 20 h. The mixture was filtered through Celite with the aid of MeOH and the filtrate was treated with 0.5 M methanolic HCl (10 mL) and concentrated under reduced pressure. The resulting HCl salt was subjected to gravity column chromatography (MeCN/H₂O, 97:3). Collection of the appropriate fractions (R_f = 0.30, MeCN/H₂O, 8:2) provided compound l- 5 (21.4 mg, 90%) as a white wax.

[α]_D ^26.2 = –16 (c 0.50, MeOH).

IR (ATR): 3153, 1699, 1405, 1333, 1271, 1200, 1096, 1042, 990, 922, 886 cm^–1.

¹H NMR (400 MHz, D₂O): δ = 4.89 (d, J _4,3 = 7.8 Hz, 1 H, H-4), 4.24 (dd, J _3,2 = 6.6 Hz, J _3,4 = 7.8 Hz, 1 H, H-3), 3.94 (ddd, J _2,2′ = 4.7 Hz, J _2,3 = 6.6 Hz, J = 12.6 Hz, 1 H, H-2), 3.76 (ddd, J _2′a2′b = 1.3 Hz, J _2′2 = 4.7 Hz, J = 12.6 Hz, 2 H, H-2′).

¹³C NMR (100 MHz, D₂O): δ = 168.6 (C=N), 75.9 (C-4), 74.7 (C-3), 62.2 (C-2), 59.2 (C-2′).

HRMS (ESI): m/z [M + H – HCl]⁺ calcd for C₅H₁₁O₃N₂: 147.0769; found: 147.0766.

(3R,4R,5R)-3,4-Bis(benzyloxy)-5-[(benzyloxy)methyl]pyrrolidin-2-one Oxime (d-14) and (3S,4R,5R)-3,4-Bis(benzyloxy)-5-[(benzyl­oxy)methyl]pyrrolidin-2-one Oxime (d- ribo-14)

Step 1: To a solution of alcohol l- 8 (271 mg, 0.65 mmol) in DCM (10 mL) at 0 °C was added pyridine (0.13 mL, 1.63 mmol, 2.5 equiv). The reaction mixture was stirred for 10 min prior to the dropwise addition of triflic anhydride (0.13 mL, 0.81 mmol, 1.25 equiv). The reaction mixture was further stirred at 0 °C for 15 min before being diluted with DCM (60 mL), washed with ice-cold HCl (20 mL, 1 M) and sat. aq NaHCO₃ (30 mL), dried over Na₂SO₄, and filtered. The residue (R_f = 0.71, PE/EtOAc, 7:3) was concentrated under reduced pressure and used directly in the following step.

Step 2: To a solution of sodium (58 mg, 2.52 mmol) in EtOH (3 mL) was added NH₂OH·HCl (275 mg, 3.96 mmol) and 4 Å MS (5 mg) at 0 °C. The heterogeneous mixture was stirred for 30 min before adding a solution of the residue from step 1 in EtOH (3 mL) at 0 °C. The resulting reaction mixture was stirred at room temperature for 17 h. The volatiles were then removed under reduced pressure and the residue was subjected to flash column chromatography (CHCl₃/MeOH, 95:5). Collection of the appropriate fractions (R_f = 0.40, CHCl₃/MeOH, 95:5) provided the products d- ribo-14 (6.3 mg, 2%), d- 14 (103 mg, 36%), and a mixture of d- ribo-14 and d- 14 (57.5 mg, 21%) as clear, colorless oils.

Data for d-14

[α]_D ^24.0 = –14 (c 1.0, CHCl₃).

IR (ATR): 3152, 3032, 2868, 1707, 1496, 1454, 1364, 1281, 1223, 1164, 1103, 913, 735 cm^–1.

¹H NMR (400 MHz, CD₃OD): δ = 7.33–7.26 (m, 15 H, ArH), 4.77 (d, J _3,4 = 3.5 Hz, 1 H, H-3), 4.70 (d, J = 11.4 Hz, 1 H, CHPh), 4.60 (d, J = 11.4 Hz, 1 H, CHPh), 4.53 (d, J = 12.0 Hz, 2 H, CHPh), 4.49 (d, J = 12.0 Hz, 2 H, CHPh), 4.25 (t, J = 3.5 Hz, 1 H, H-4), 3.96 (m, 1 H, H-5), 3.62 (dd, J _6a,6b = 10.2 Hz, J _6a,5 = 5.2 Hz, 1 H, H-6a), 3.56 (dd, J _6b,6a = 10.2 Hz, J _6b,2 = 4.7 Hz, 1 H, H-6b).

¹³C NMR (100 MHz, CD₃OD): δ = 162.0 (C=N), 139.0 (ArC), 138.6 (ArC), 138.1 (ArC), 129.5–128.9 (ArCH, 7 signals), 82.5 (C-4), 82.2 (C-3), 74.3 (CH₂Ph), 73.9 (CH₂Ph), 73.1 (CH₂Ph), 70.0 (C-6), 64.3 (C-5).

HRMS (ESI): m/z [M + Na]⁺ calcd for C₂₆H₂₈O₄N₂Na: 455.1946; found: 455.1940.

Data for d- ribo-14

[α]_D ^25.0 = +4 (c 0.5, CHCl₃).

IR (ATR): 3230, 3062, 3031, 2924, 2866, 1706, 1496, 1454, 1363, 1262, 1208, 1096, 1027, 736, 697 cm^–1.

¹H NMR (400 MHz, CD₃OD): δ = 7.32–7.27 (m, 15 H, ArCH), 4.70 (d, J = 11.5 Hz, 1 H, CHPh), 4.56–4.50 (m, 6 H, CHPh + H-3), 4.12 (t, J = 2.8 Hz, 1 H, H-4), 3.85 (ddd, J _5,4 = 2.8 Hz, J _5,6b = 5.5 Hz, J _5,6a = 5.9 Hz, 1 H, H-5), 3.56 (dd, J _6a,5 = 6.0 Hz, J _6a,6b = 9.8 Hz, 1 H, H-6a), 3.53 (dd, J _6b,5 = 5.6 Hz, J _6b,6a = 9.8 Hz, 1 H, H-6b).

¹³C NMR (100 MHz, CD₃OD): δ = 160.0 (C=N), 139.2 (ArC), 138.8 (ArC), 138.6 (ArC), 129.5–128.8 (ArCH, 6 signals), 82.6 (C-4), 82.1 (C-3), 74.3 (CH₂Ph), 73.3 (CH₂Ph), 72.8 (CH₂Ph), 71.2 (C-6), 63.3 (C-5).

HRMS (ESI): m/z [M + Na]⁺ calcd for C₂₆H₂₈O₄N₂Na: 455.1946; found: 455.1941.

(3S,4S,5S,)-3,4-Bis(benzyloxy)-5-[(benzyloxy)methyl]pyrrolidin-2-one Oxime (l-14) + 2,3,5-Tri-O-benzyl-l-arabino-γ-lactam (l-15)

Step 1: To a solution of alcohol d- 8 (125 mg, 0.3 mmol) in DCM (5 mL) at 0 °C was added pyridine (0.06 mL, 0.75 mmol, 2.5 equiv). The reaction mixture was stirred for 10 min prior to the dropwise addition of triflic anhydride (0.06 mL, 0.38 mmol, 1.25 equiv). The reaction mixture was further stirred at 0 °C for 15 min before being diluted with DCM (30 mL), washed with ice-cold HCl (10 mL, 1 M) and sat. aq NaHCO₃ (15 mL), dried over Na₂SO₄, and filtered. The residue (R_f = 0.71, PE/EtOAc, 7:3) was concentrated under reduced pressure and used directly in the following step.

Step 2: To a suspension of NH₂OH·HCl (112.5 mg, 1.62 mmol) in EtOH (2.0 mL) was added Et₃N (0.23 mL, 1.62 mmol) dropwise at 0 °C under argon. The resulting mixture was stirred for 30 min at room temperature before adding a solution of the residue from step 1 in EtOH (2.5 mL) at 0 °C under argon. The resulting reaction mixture was stirred for 17 h at room temperature. The volatiles were then removed under reduced pressure and the residue was subjected to flash column chromatography (CHCl₃/MeOH, 99:1 → 96:4). Collection of the appropriate fractions (R_f = 0.34, CHCl₃/MeOH, 9:1) provided the products l- 14 (34 mg, 26%) and l- 15 (22 mg, 17%) as clear, colorless oils.

Data for l-14

[α]_D ^24.5 = +14 (c 1.0, CHCl₃).

IR (ATR): 3029, 2928, 2883, 2799, 1702, 1496, 1453, 1396, 1364, 1285, 1239, 1209, 1090, 1027, 911, 819, 733 cm^–1.

¹H NMR (400 MHz, CD₃OD): δ = 7.35–7.26 (m, 15 H, ArH), 4.86 (d, J _3,4 = 3.7 Hz, 1 H, H-3), 4.69 (d, J = 11.4 Hz, 1 H, CHPh), 4.61 (d, J = 11.4 Hz, 1 H, CHPh), 4.56–4.49 (m, 4 H, CHPh), 4.32 (t, J = 3.7 Hz, 1 H, H-4), 4.01 (app dd, J = 4.5 Hz, J = 8.3 Hz, 1 H, H-5) 3.65 (dd, J _6a,5 = 4.5 Hz, J _6a,6b = 10.3 Hz, 1 H, H-6a), 3.58 (dd, J _6b,5 = 4.5 Hz, J _6b,6a = 10.3 Hz, 1 H, H-6b).

¹³C NMR (100 MHz, CD₃OD): δ = 162.8 (N=C), 138.9 (ArC), 138.5 (ArC), 137.9 (ArC), 129.8–128.9 (ArCH, 7 signals), 82.7 (C-3), 82.1 (C-4), 74.4 (CH₂Ph), 74.2 (CH₂Ph), 73.3 (CH₂Ph), 69.6 (C-6), 64.7 (C-5).

HRMS (ESI): m/z [M + Na]⁺ calcd for C₂₆H₂₈O₄N₂Na: 455.1946; found: 455.1941.

Data for l-15[27]

¹H NMR (400 MHz, CDCl₃): δ = 7.42–7.22 (m, 15 H, Ar-H), 5.83 (br s, 1 H, NH), 5.12 (d, J = 11.5 Hz, 1 H, CHPh), 4.80 (d, J = 11.5 Hz, 1 H, CHPh), 4.61 (d, J = 11.7 Hz, 1 H, CHPh), 4.51–4.49 (m, 3 H, CHPh), 4.22 (d, J = 5.8 Hz, 1 H, H-2), 3.89 (t, J = 5.8 Hz, 1 H, H-3), 3.70–3.66 (m, 1 H, H-4), 3.64–3.60 (m, 1 H, H-5a), 3.34–3.29 (m, 1 H, H-5b).

¹³C NMR (100 MHz, CDCl₃): δ = 173.0 (C=N), 137.7 (ArC), 137.51 (ArC), 137.50 (ArC), 128.7–127.9 (ArCH, 9 signals), 81.3 (C-3), 80.9 (C-2), 73.6 (CH₂Ph), 72.7 (CH₂Ph), 72.4 (CH₂Ph), 71.6 (C-5), 56.1 (C-4).

The spectroscopic data were in full accord with the previously reported data.[27]

(3R,4R,5R)-3,4-Dihydroxy-5-(hydroxymethyl)pyrrolidin-2-one Oxime (d-6)

To a solution of compound d- 14 (57 mg, 0.13 mmol) in DCM (10 mL) at –78 °C under argon was added a solution of BCl₃ (0.78 mL, 1 M in heptane, 0.78 mmol). The reaction was stirred at –78 °C for 3 h and then slowly allowed to warm to 0 °C over 15 h. The reaction was quenched with EtOH (4 mL) at 0 °C. The volatiles were removed under reduced pressure and the residue was subjected to gravity column chromatography (MeCN/H₂O, 98:2). Collection of the appropriate fractions (R_f = 0.37, MeCN/H₂O, 8:2) provided compound d- 6 (14.1 mg, 67%) as a clear, white wax.

[α]_D ^24.0 = –25.8 (c 0.5, MeOH).

IR (ATR): 3162, 1701, 1405, 1333, 1271, 1200, 1096, 1042, 990, 922, 886 cm^–1.

¹H NMR (400 MHz, D₂O): δ = 4.93 (d, J _3,4 = 7.4 Hz, 1 H, H-3), 4.24 (t, J = 7.2 Hz, 1 H, H-4), 3.91–3.87 (m, 1 H, H-6a), 3.77–3.73 (m, 2 H, H-5 + H-6b).

¹³C NMR (100 MHz, D₂O): δ = 162.7 (C=N), 75.2 (C-4), 74.9 (C-3), 62.4 (C-5), 59.0 (C-6).

HRMS (ESI): m/z [M + H]⁺ calcd for C₅H₁₁N₂O₄: 163.0718; found: 163.0713.

(3S,4S,5S)-3,4-Dihydroxy-5-(hydroxymethyl)pyrrolidin-2-one Oxime (l-6)

To a solution of l- 14 (27 mg, 0.06 mmol) in DCM (6 mL) at –78 °C under argon was added a solution of BCl₃ (0.36 mL, 1 M in heptane, 2 equiv per benzyl unit). The reaction was stirred at –78 °C for 3 h and then slowly allowed to warm to 0 °C over 15 h. The reaction was quenched with EtOH (1 mL) at 0 °C before the volatiles were removed under reduced pressure. The residue was subjected to gravity column chromatography [MeCN/H₂O (0.05 M HCl), 97:3]. Collection of the appropriate fractions (R_f = 0.37, MeCN/H₂O, 8:2) provided l- 6 (5.0 mg, 50%) as a yellow wax.

[α]_D ^23.9 = +26.4 (c 0.53, MeOH).

IR (ATR): 3160, 1700, 1405, 1333, 1270, 1200, 1096, 1042, 990 cm^–1.

¹H NMR (400 MHz, D₂O): δ = 4.93 (d, J _3,4 = 7.4 Hz, 1 H, H-3), 4.24 (t, J = 7.2 Hz, 1 H, H-4), 3.91–3.87 (m, 1 H, H-6a), 3.77–3.73 (m, 2 H, H-5 + H-6b).

¹³C NMR (100 MHz, D₂O): δ = 162.7 (C=N), 75.2 (C-4), 74.9 (C-3), 62.4 (C-5), 59.0 (C-6).

HRMS (ESI): m/z [M + H]⁺ calcd For C₅H₁₁N₂O₄: 163.0718; found: 163.0712.

(3R,4R,5R)-3,4-Dihydroxy-5-(hydroxymethyl)pyrrolidin-2-one Oxime (d-6) and (3S,4R,5R)-3,4-Dihydroxy-5-(hydroxymethyl)pyrrolidin-2-one Oxime (d- ribo-6)

To a mixture of compounds d- 14 + d- ribo-14 (50 mg, 0.11 mmol, ca. 1:1) in DCM (10 mL) at –78 °C under argon was added a solution of BCl₃ (0.66 mL, 1 M in heptane, 0.66 mmol). The reaction was stirred at –78 °C for 3 h and then slowly allowed to warm to 0 °C over 22 h. The reaction was then quenched with EtOH (4 mL) at 0 °C. The volatiles were removed under reduced pressure and the residue was subjected to gravity column chromatography (MeCN/H₂O, 98:2). Collection of the appropriate fractions (R_f = 0.37, MeCN/H₂O, 8:2) provided a mixture of isomers d- 6 and d- ribo-6 (11.0 mg, 62%) as a clear, white wax.

IR (ATR): 3163, 1698, 1405, 1333, 1271, 1202, 1096, 1042, 990, 923, 886 cm^–1.

¹H NMR (400 MHz, D₂O): δ = 5.13 (d, J _3,4 = 5.3 Hz, 1 H, H-3, d- ribo-6), 4.93 (d, J _3,4 = 7.4 Hz, 1 H, H-3, d- 6), 4.39 (dd, J _4,3 = 5.3 Hz, J _4,5 = 1.0 Hz, 1 H, H-4, d- ribo-6), 4.25 (t, J = 7.2 Hz, 1 H, H-4, d- 6), 3.94 (td, J _5,4 = 1.0 Hz, J _5,6 = 3.8 Hz, 1 H, H-5, d- ribo-6), 3.93–3.88 (m, 1 H, H-6a, d- 6), 3.77–3.73 (m, 2 H, H-5 + H-6b, d- 6 and 2 H, H-6, d- ribo-6).

¹³C NMR (100 MHz, D₂O): δ = 164.0 (C=N, d- ribo-6), 162.7 (C=N, d- 6), 75.3 (C-4, d- 6), 74.9 (C-3, d- 6), 71.1 (C-4, d- ribo-6), 70.9 (C-3, d- ribo-6), 67.0 (C-5, d- ribo-6), 62.5 (C-5, d- 6), 60.6 (C-6, d- ribo-6), 59.0 (C-6, d- 6).

HRMS (ESI): m/z [M + H]⁺ calcd for C₅H₁₁N₂O₄: 163.0718; found: 163.0712.

(3R,4R,5R)-3,4-Bis(benzyloxy)-5-[(benzyloxy)methyl]pyrrolidin-2-one O-Ethyl Oxime (d-16)

A solution of compound d- 14 (10 mg, 0.023 mmol), iodoethane (0.4 mL, 5 mmol) and Cs₂CO₃ (7.5 mg, 1 equiv) in MeCN (1 mL) was stirred for 3 d at room temperature before the volatiles were removed under reduced pressure. The residue was subjected to flash column chromatography (PE/EtOAc, 5:1). Collection of the appropriate fractions (R_f = 0.52, PE/EtOAc, 3:1) provided compound d- 16 (8.7 mg, 82%) as a clear, colorless oil.

IR (ATR): 3062, 3032, 2925, 2857, 1735, 1668, 1496, 1454, 1377, 1362, 1314, 1287, 1207, 1090, 1055, 910, 869, 735, 697 cm^–1.

¹H NMR (400 MHz, CDCl₃): δ = 7.34–7.25 (m, 15 H, ArH), 5.40 (br s, 1 H, NH), 4.93 (d, J = 11.7 Hz, 1 H, CHPh), 4.60 (d, J = 11.7 Hz, 1 H, CHPh), 4.57 (d, J = 11.8 Hz, 1 H, CHPh), 4.52 (d, J = 11.9 Hz, 1 H, CHPh), 4.51 (d, J = 11.8 Hz, 1 H, CHPh), 4.48 (d, J = 11.9 Hz, 1 H, CHPh), 4.36 (d, J _3,4 = 3.0 Hz, 1 H, H-3), 4.03 (dq, J = 3.1 Hz, J = 7.0 Hz, 2 H, CH₂), 3.87 (t, J = 3.0 Hz, 1 H, H-4), 3.75 (m, 1 H, H-5), 3.56 (dd, J _5′a,5 = 5.2 Hz, J _5′a,5′b = 9.2 Hz, 1 H, H-5′a), 3.42 (dd, J _5′b,5 = 8.2 Hz, J _5′b,5′a = 9.2 Hz, 1 H, H-5′b), 1.26 (t, J = 7.0 Hz, 3 H, CH₃).

¹³C NMR (100 MHz, CDCl₃): δ = 154.3 (C=N), 137.89 (ArC), 137.85 (ArC), 137.5 (ArC), 128.5–127.7 (ArCH, 9 signals), 82.3 (C-4), 80.0 (C-3), 73.4 (CHPh), 72.2 (C-5′), 71.8 (CHPh), 71.6 (CHPh), 69.0 (CH₂), 60.2 (C-5), 14.8 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₈H₃₃O₄N₂: 461.2440; found: 461.2438.

Glycosidase Inhibition Assays

Measurement of the inhibition activity against glycosidases was accomplished using minor modifications[20] of the procedure previously reported by Bols and co-workers.[30] The following glycosidases were used: α-glucosidase (Baker’s yeast), β-glucosidase (almonds) and α-mannosidase (jack beans); the corresponding p-nitrophenyl α- or β-glycopyranosides were used as model substrates. For screening the activity and IC₅₀ calculations, substrate concentrations were fixed at 0.25 mM, 4.0 mM and 1.53 mM for α-glucosidase, β-glucosidase and α-mannosidase, respectively. For the calculation of percentage inhibition, the inhibitor concentrations were fixed at 100 μM for α- and β-glucosidases, and at 50 μM for α-mannosidase.

Monitoring of the reactions was carried out using a Hitachi U-2900 spectrophotometer equipped with a 6-cuvette holder (α- and β-glucosidase) or with a Thermo Scientific™ Varioskan™ LUX microplate reader (for α-mannosidase).

For glucosidases, PS cuvettes were used containing 0.1 M phosphate buffer (pH 6.8, 0.6 mL), substrate, DMSO (5%, control or inhibitor solution) and water (up to 1.14 mL). Reactions were started by the addition of 60 μL of appropriately diluted solutions of the enzyme, and were monitored at 25 °C at 400 nm for 125 s.

For α-mannosidase, PS Greiner F-bottom 96-wells were employed (total volume = 100 μL), using 0.1 M phosphate–citrate buffer (pH 5.6, 50 μL). Reaction mixtures were incubated at 35 °C for 4 min, after which 1 M Na₂CO₃ solution (150 μL) was added and the resulting absorbance was measured at 400 nm.

The percentage of inhibition was calculated from the following expression (Equation 1), using the initial rates of the enzyme- and inhibitor-containing solutions (v is calculated from the slope of the plot of Abs vs t).

Where v_o is the rate of the enzyme solution used as the control, and v is the rate of the inhibitor-containing solution.

IC₅₀ values were obtained by plotting %I vs log[I] (6–8 inhibitor concentrations), and adjusting to a second-order equation.

For the most potent inhibitors, the mode of inhibition and the inhibition constants were obtained. The mode of inhibition is deduced from the Cornish-Bowden plots (1/v vs [I] (Dixon plot) and [S]/v vs [I]).[31] The kinetic parameters (K _M and V_max) were obtained via nonlinear regression analysis (Prism 8.0 software) using substrate concentrations ranging from 0.25 K _M to 4 × K _M, and 3–6 inhibitor concentrations were used.

The mode of inhibition for all the most active compounds turned out to be competitive (binding only the free enzyme); inhibition constants (K _ia) were calculated using Equation 2.

Where K _M is the Michaelis constant for the enzyme, and K _M,app is the Michaelis constant in the presence of the inhibitor.

The Cornish-Bowden plots for compound d- 5 against α-glucosidase and α-mannosidase are included in the Supporting Information.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgment

Dr. Holmelid, University of Bergen, is thanked for recording HRMS analysis. Dr. K. Jørgensen, University of Stavanger, is thanked for his help in operating and maintaining the NMR instrument.

• References

• 1 Nash RJ, Bell EA, Williams JM. Phytochemistry 1985; 24: 1620
  CrossrefSearch in Google Scholar
• 2 Fleet GW. J, Nicholas SJ, Smith PW, Evans SV, Fellows LE, Nash RJ. Tetrahedron Lett. 1985; 26: 3127
  CrossrefSearch in Google Scholar
• 3 Watson AA, Fleet GW. J, Asano N, Molyneux RJ, Nash RJ. Phytochemistry 2001; 56: 265
  CrossrefPubMedSearch in Google Scholar
• 4 Stütz AE, Wrodnigg TM. Adv. Carbohydr. Chem. Biochem. 2011; 66: 187
  CrossrefPubMedSearch in Google Scholar
• 5 Asano N, Nash RJ, Molyneux RJ, Fleet GW. J. Tetrahedron: Asymmetry 2000; 11: 1645
  CrossrefSearch in Google Scholar
• 6 Campbell LK, Baker DE, Campbell RK. Ann. Pharmacother. 2000; 34: 1291
  CrossrefPubMedSearch in Google Scholar
• 7 Cox T, Lachmann R, Hollak C, Aerts J, Van Weely S, Hrebícek M, Platt F, Butters T, Dwek R, Moyses C, Gow I. Lancet 2000; 355: 1481
  CrossrefPubMedSearch in Google Scholar
• 8 Germain DP, Hughes DA, Nicholls K, Bichet DG, Giugliani R, Wilcox WR, Feliciani C, Shankar SP, Ezgu F, Amartino H, Bratkovic D. N. Engl. J. Med. 2016; 375: 545
  CrossrefPubMedSearch in Google Scholar
• 9 Lillelund VH, Jensen HH, Liang X, Bols M. Chem. Rev. 2002; 102: 515
  CrossrefPubMedSearch in Google Scholar
• 10 López Ó, Merino-Montiel P, Martos S, González-Benjumea A. In Carbohydrate Chemistry: Chemical and Biological Approaches, Vol. 38. Rauter AP, Lindhorst T. The Royal Society of Chemistry; Cambridge: 2012: 215
  CrossrefSearch in Google Scholar
• 11 D’Alonzo D, Guaragna A, Palumbo G. Curr. Med. Chem. 2009; 16: 473
  CrossrefPubMedSearch in Google Scholar
• 12 Yu C.-Y, Asano N, Ikeda K, Wang M.-X, Butters TD, Wormald MR, Dwek RA, Winters AL, Nash RJ, Fleet GW. J. Chem. Commun. 2004; 1936
  CrossrefPubMed
• 13 Asano N, Ikeda K, Yu L, Kato A, Takebayashi K, Adachi I, Kato I, Ouchi H, Takahata H, Fleet GW. J. Tetrahedron: Asymmetry 2005; 16: 223
  CrossrefSearch in Google Scholar
• 14 Kato A, Kato N, Kano E, Adachi I, Ikeda K, Yu L, Okamoto T, Banba Y, Ouchi H, Takahata H, Asano N. J. Med. Chem. 2005; 48: 2036
  CrossrefPubMedSearch in Google Scholar
• 15 Scofield AM, Fellows LE, Nash RJ, Fleet GW. J. Life Sci. 1986; 39: 645
  CrossrefPubMedSearch in Google Scholar
• 16 Hakansson AE, van Ameijde J, Guglielmini L, Horne G, Nash RJ, Evinson EL, Kato A, Fleet GW. J. Tetrahedron: Asymmetry 2007; 18: 282
  CrossrefSearch in Google Scholar
• 17 Lundt I, Madsen R, Al Daher S, Winchester B. Tetrahedron 1994; 50: 7513
  CrossrefSearch in Google Scholar
• 18 Ayers BJ, Ngo N, Jenkinson SF, Martínez RF, Shimada Y, Adachi I, Weymouth-Wilson AC, Kato A, Fleet GW. J. J. Org. Chem. 2012; 77: 7777
  CrossrefPubMedSearch in Google Scholar
• 19 Lindbäck E, Lopez Ó, Tobiesen A, Fernández-Bolaños JG, Sydnes MO. Org. Biomol. Chem. 2017; 15: 8709
  CrossrefPubMedSearch in Google Scholar
• 20 Haarr MB, Lopéz Ó, Pejov L, Fernández-Bolaños JG, Lindbäck E, Sydnes MO. ACS Omega 2020; 5: 18507
  CrossrefPubMedSearch in Google Scholar
• 21 de Santana QL. O, Evangelista TC. S, Imhof P, Ferreira SB, Fernández-Bolaños JG, Sydnes MO, Lopéz Ó, Lindbäck E. Org. Biomol. Chem. 2021; 19: 2322
  CrossrefPubMedSearch in Google Scholar
• 22 Evangelista TC. S, Lopéz Ó, Sydnes MO, Fernández-Bolaños JG, Ferreira SB, Lindbäck E. Synthesis 2019; 51: 4066
  Thieme ConnectSearch in Google Scholar
• 23 Gnanesh Kumar BS, Pohlentz G, Schulte M, Mormann M, Siva Kumar N. Glycobiology 2014; 24: 252
  CrossrefPubMedSearch in Google Scholar
• 24 Ermert P, Vasella A. Helv. Chim. Acta 1991; 74: 2043
  CrossrefSearch in Google Scholar
• 25 Kanso R, Striegler S. Carbohydr. Res. 2011; 346: 897
  CrossrefPubMedSearch in Google Scholar
• 26 Boutellier M, Horenstein BA, Semenyaka A, Schramm VL, Ganem B. Biochemistry 1994; 33: 3994
  CrossrefPubMedSearch in Google Scholar
• 27 Overkleeft HS, van Wiltenburg J, Pandit UK. Tetrahedron 1994; 50: 4215
  CrossrefSearch in Google Scholar
• 28 Stephenson L, Warburton WK, Wilson MJ. J. Chem. Soc. C 1969; 861
  Crossref
• 29 Vörös A, Mucsi Z, Baán Z, Timári G, Hermecz I, Mizsey P, Finta Z. Org. Biomol. Chem. 2014; 12: 8036
  CrossrefPubMedSearch in Google Scholar
• 30 Bols M, Hazell RG, Thomsen IB. Chem. Eur. J. 1997; 3: 940
  CrossrefSearch in Google Scholar
• 31 Cornish-Bowden A. Biochem. J. 1974; 137: 143
  CrossrefPubMedSearch in Google Scholar

Corresponding Authors

Publication History

Received: 12 January 2022

Accepted after revision: 07 February 2022

Accepted Manuscript online:
07 February 2022

Article published online:
05 April 2022

© 2022. Thieme. All rights reserved

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 Nash RJ, Bell EA, Williams JM. Phytochemistry 1985; 24: 1620
  CrossrefSearch in Google Scholar
• 2 Fleet GW. J, Nicholas SJ, Smith PW, Evans SV, Fellows LE, Nash RJ. Tetrahedron Lett. 1985; 26: 3127
  CrossrefSearch in Google Scholar
• 3 Watson AA, Fleet GW. J, Asano N, Molyneux RJ, Nash RJ. Phytochemistry 2001; 56: 265
  CrossrefPubMedSearch in Google Scholar
• 4 Stütz AE, Wrodnigg TM. Adv. Carbohydr. Chem. Biochem. 2011; 66: 187
  CrossrefPubMedSearch in Google Scholar
• 5 Asano N, Nash RJ, Molyneux RJ, Fleet GW. J. Tetrahedron: Asymmetry 2000; 11: 1645
  CrossrefSearch in Google Scholar
• 6 Campbell LK, Baker DE, Campbell RK. Ann. Pharmacother. 2000; 34: 1291
  CrossrefPubMedSearch in Google Scholar
• 7 Cox T, Lachmann R, Hollak C, Aerts J, Van Weely S, Hrebícek M, Platt F, Butters T, Dwek R, Moyses C, Gow I. Lancet 2000; 355: 1481
  CrossrefPubMedSearch in Google Scholar
• 8 Germain DP, Hughes DA, Nicholls K, Bichet DG, Giugliani R, Wilcox WR, Feliciani C, Shankar SP, Ezgu F, Amartino H, Bratkovic D. N. Engl. J. Med. 2016; 375: 545
  CrossrefPubMedSearch in Google Scholar
• 9 Lillelund VH, Jensen HH, Liang X, Bols M. Chem. Rev. 2002; 102: 515
  CrossrefPubMedSearch in Google Scholar
• 10 López Ó, Merino-Montiel P, Martos S, González-Benjumea A. In Carbohydrate Chemistry: Chemical and Biological Approaches, Vol. 38. Rauter AP, Lindhorst T. The Royal Society of Chemistry; Cambridge: 2012: 215
  CrossrefSearch in Google Scholar
• 11 D’Alonzo D, Guaragna A, Palumbo G. Curr. Med. Chem. 2009; 16: 473
  CrossrefPubMedSearch in Google Scholar
• 12 Yu C.-Y, Asano N, Ikeda K, Wang M.-X, Butters TD, Wormald MR, Dwek RA, Winters AL, Nash RJ, Fleet GW. J. Chem. Commun. 2004; 1936
  CrossrefPubMed
• 13 Asano N, Ikeda K, Yu L, Kato A, Takebayashi K, Adachi I, Kato I, Ouchi H, Takahata H, Fleet GW. J. Tetrahedron: Asymmetry 2005; 16: 223
  CrossrefSearch in Google Scholar
• 14 Kato A, Kato N, Kano E, Adachi I, Ikeda K, Yu L, Okamoto T, Banba Y, Ouchi H, Takahata H, Asano N. J. Med. Chem. 2005; 48: 2036
  CrossrefPubMedSearch in Google Scholar
• 15 Scofield AM, Fellows LE, Nash RJ, Fleet GW. J. Life Sci. 1986; 39: 645
  CrossrefPubMedSearch in Google Scholar
• 16 Hakansson AE, van Ameijde J, Guglielmini L, Horne G, Nash RJ, Evinson EL, Kato A, Fleet GW. J. Tetrahedron: Asymmetry 2007; 18: 282
  CrossrefSearch in Google Scholar
• 17 Lundt I, Madsen R, Al Daher S, Winchester B. Tetrahedron 1994; 50: 7513
  CrossrefSearch in Google Scholar
• 18 Ayers BJ, Ngo N, Jenkinson SF, Martínez RF, Shimada Y, Adachi I, Weymouth-Wilson AC, Kato A, Fleet GW. J. J. Org. Chem. 2012; 77: 7777
  CrossrefPubMedSearch in Google Scholar
• 19 Lindbäck E, Lopez Ó, Tobiesen A, Fernández-Bolaños JG, Sydnes MO. Org. Biomol. Chem. 2017; 15: 8709
  CrossrefPubMedSearch in Google Scholar
• 20 Haarr MB, Lopéz Ó, Pejov L, Fernández-Bolaños JG, Lindbäck E, Sydnes MO. ACS Omega 2020; 5: 18507
  CrossrefPubMedSearch in Google Scholar
• 21 de Santana QL. O, Evangelista TC. S, Imhof P, Ferreira SB, Fernández-Bolaños JG, Sydnes MO, Lopéz Ó, Lindbäck E. Org. Biomol. Chem. 2021; 19: 2322
  CrossrefPubMedSearch in Google Scholar
• 22 Evangelista TC. S, Lopéz Ó, Sydnes MO, Fernández-Bolaños JG, Ferreira SB, Lindbäck E. Synthesis 2019; 51: 4066
  Thieme ConnectSearch in Google Scholar
• 23 Gnanesh Kumar BS, Pohlentz G, Schulte M, Mormann M, Siva Kumar N. Glycobiology 2014; 24: 252
  CrossrefPubMedSearch in Google Scholar
• 24 Ermert P, Vasella A. Helv. Chim. Acta 1991; 74: 2043
  CrossrefSearch in Google Scholar
• 25 Kanso R, Striegler S. Carbohydr. Res. 2011; 346: 897
  CrossrefPubMedSearch in Google Scholar
• 26 Boutellier M, Horenstein BA, Semenyaka A, Schramm VL, Ganem B. Biochemistry 1994; 33: 3994
  CrossrefPubMedSearch in Google Scholar
• 27 Overkleeft HS, van Wiltenburg J, Pandit UK. Tetrahedron 1994; 50: 4215
  CrossrefSearch in Google Scholar
• 28 Stephenson L, Warburton WK, Wilson MJ. J. Chem. Soc. C 1969; 861
  Crossref
• 29 Vörös A, Mucsi Z, Baán Z, Timári G, Hermecz I, Mizsey P, Finta Z. Org. Biomol. Chem. 2014; 12: 8036
  CrossrefPubMedSearch in Google Scholar
• 30 Bols M, Hazell RG, Thomsen IB. Chem. Eur. J. 1997; 3: 940
  CrossrefSearch in Google Scholar
• 31 Cornish-Bowden A. Biochem. J. 1974; 137: 143
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material