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. 2020 Dec 2;5(49):31821–31830. doi: 10.1021/acsomega.0c04589

Amino Acid-Based Synthesis and Glycosidase Inhibition of Cyclopropane-Containing Iminosugars

Alejandro Puet †, Gema Domínguez †, F Javier Cañada ‡,§, Javier Pérez-Castells †,*
PMCID: PMC7745444  PMID: 33344836

Abstract

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Synthesis of four iminosugars fused to a cyclopropane ring is described using l-serine as the chiral pool. The key steps are large-scale preparation of an α,β-unsaturated piperidinone followed by completely stereoselective sulfur ylide cyclopropanation. Stereochemistry of compounds has been studied by nuclear Overhauser effect spectroscopy (NOESY) experiments and 1H homonuclear decoupling to measure constant couplings. The activity of these compounds against different glycosidases has been evaluated. Although inhibition activity was low (compound 8a presents a (Ki) of 1.18 mM against β-galactosidase from Escherichia coli), interestingly, we found that compounds 8a and 8b increase the activity of neuraminidase from Vibrio cholerae up to 100%.

Introduction

Iminosugars are azaheterocycles with promising biological activities such as glycosidase and glycosyltransferase inhibition and modulation.1 Many iminosugars are natural or synthetic polyhydroxylated piperidines, which can act as biomimetics of their corresponding pyranose analogues. Some of the most important natural piperidine iminosugars are nojirimycin (Figure 1, I) and its epimers, which, together with their deoxy analogues have turned out to be the lead molecules for drug design. Thus, stereochemical changes and functional group variation have led to iminosugars that can modulate glycosidase enzymes, exhibiting immunosuppressive, antiviral, or anti-inflammatory activities.2 Bicyclic iminosugars, such as swainsonine (II), lentiginosine (III), castanospermine (IV), and their derivatives exhibit antitumor and immunosuppressive activities.3

Figure 1.

Figure 1

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Structures of nojirimycin (I), swainsonine (II), lentiginosine (III), castanospermine (IV), miglitol (V), migalastat (VI), and miglustat (VII).

Several iminosugars, miglitol (Glyset, V),4 migalastat (Galafold, VI),5 and miglustat (Zavesca, VII)6 are commercially available for the treatment of type II diabetes and Fabry disease, and as the first oral treatment for Gaucher disease, respectively. Several other competitive inhibitors of glycosidases are being developed as new drugs and are in different phases of clinical trials.

The mechanism associated with glycosidase activity modulation is generally attributed to structural similarity to the oxacarbenium ion-like transition-state, formed during the hydrolysis of carbohydrates.7 These transition states present diverse conformational pathways for different glycosidases,8 making selective inhibition possible. In this context, designing conformationally restricted inhibitors seems to be an interesting approach. In addition, adequate metabolic stability is needed, which may be achieved with more rigid compounds. Recently, a study on α-mannanases showed how the enzyme surface restricts the conformational landscape of the substrate, rendering the B2,5 conformation the most stable on-enzyme (Figure 2a).9 In another study, a cyclopropane containing a cyclophellitol analogue, was designed as a specific β-glucosidase inhibitor for enzymes reacting through the 4H3 transition-state conformation (Figure 2b).10

Figure 2.

Figure 2

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Transition-state conformations on-enzyme: (a) mannose B2,5 conformation (VIII, reprinted with permission from ref (9)) and (b) cyclophellitol analogue and its 4H3 conformation (IX, ref (10), copyright 2017 American Chemical Society).

With these precedents, we expected that the introduction of a three-membered ring annulated to a piperidine ring would render novel iminosugars with a locked conformation that may be the starting point for finding the therapeutic compounds (Figure 3). The substituted cyclopropane moiety renders a fixed conformation and allows many different configurations that could increase selectivity to specific glycosidases.

Figure 3.

Figure 3

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Structure of the target compound.

The development of efficient routes for the preparation of iminosugars has received much attention from the synthetic community.11 Most of the methods use carbohydrates as the chiral pool, which are transformed using reductive aminations12 or other transformation strategies.13 Alternatively, some asymmetric or biocatalysed approaches have been used.14 But, there are fewer reports on approaches where amino acids are used as the chiral pool for the synthesis of iminosugar derivatives.15

Herein, we envisioned the preparation of novel bicyclic iminosugars that include the cyclopropane motif fused with piperidine starting from the natural amino acid l-serine as the chiral pool. The final compounds present five stereogenic centers, and the synthesis involves the inversion of the configuration of the starting l-serine (S-configuration) into the C5 configuration of d-carbohydrates (R).16 Preliminary glycosidase inhibition evaluation is shown.

Results and Discussion

Our first goal was the synthesis of α,β-unsaturated ketone 5 in which the chiral center has R configuration. This configuration was selected as it corresponds to C5 in natural sugars and iminosugars, which share the R configuration in that position. This compound has already been prepared from d-serine and described.17 In our case, we developed a synthesis approach using cheaper and natural l-serine, as depicted in Scheme 1. From this intermediate, a cyclopropanation reaction and further transformations resulted in a new family of piperidines fused to cyclopropanes. l-Serine was esterified and protected with Boc2O, and the resulting intermediate was further protected and reduced to give desymmetrized alcohol 1 in which the configuration has changed from S to R in a few steps. This compound 1 was transformed into oxazolidinone by reaction with a base followed by allylation to give compound 2. Following the previously reported methodology,182 was de-protected and oxidized into carboxylic acid 3. This was converted into Weinreb amide 4, which was treated with vinylmagnesium bromide and subjected to a ring-closing metathesis, RCM, using second-generation Grubbs’ catalyst (Grubbs Catalyst M204), giving the starting material 5.19 This precursor containing the piperidine core was obtained in 13% global yield after 11 steps. No racemization was observed during the synthesis.

Scheme 1. Synthesis of Compound 5.

Scheme 1

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The cyclopropanation reaction of 5 was performed using sulfur ylide. Interestingly, only one reaction product was observed and isolated in 70% yield. This product was designated as structure 6, as a result of NMR analysis (nuclear Overhauser effect (NOE) and coupling constants) of compounds 7a–b (vide infra). Cyclopropanation occurred on the same side of oxazolidinone (endo attack) and the subsequent ring-closing step exclusively gave exo-cyclopropane. The ylide mediated cyclopropanation is a stepwise reaction in which the formation of the first C–C bond is the rate-determining step.20 The attack of the ylide on 5 is more favored from the opposite face to the nitrogen lone electron pair as depicted in Scheme2; therefore, it occurs through the same face of the oxazolidinone ring (endo). This selectivity was observed previously in one unsaturated γ-lactam cyclopropanation,21 although other precedents have described mixtures of endo and exo attacks.22 Then, the stereoselectivity of cyclopropane is determined in the second step. Studies reported by Aggarwal’s group,23 showed that two intermediate betaines A and B are formed in a 1:1 ratio after the nucleophilic addition. The cyclization of betaine A is faster than that of B. Moreover, B can epimerize to give A before it closes the three-membered ring generally leading to high diastereoselectivity of exo-cyclopropane.

Scheme 2. Stereoselectivity of the Cyclopropanation Reaction of 5(24).

Scheme 2

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With the cyclopropane containing compound 6 in hand, the reduction of the ketone afforded a (3:2) mixture of the two diastereomers 7a–b (Scheme 3). These alcohols were separated, characterized, and separately transformed into the final products. The stereochemistry of 7a and 7b was determined using NOE experiments and coupling constant values. Figure 4 shows the main correlations observed for 7a and 7b that allowed assigning the relative configuration of H6, H7, and H7a. The coupling constants between H7a, H7, and H6a were determined using homonuclear decoupling experiments. Values are shown in Figure 4, and the model agrees with the calculated angles for these couplings.

Scheme 3. Synthesis of Final Compounds from 5.

Scheme 3

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Figure 4.

Figure 4

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NOE signals and constant couplings in products 7a and 7b, respectively.24

In continuation of the synthesis, treatment with trifluoroacetyl (TFA) and further reaction with ethylendiamine gave products 8a and 8b, respectively, in excellent yields. On the other hand, compounds 7a and 7b gave different products on reacting with DIBAL-H, whereas 7b gave the expected alcohol 10 (62%), additionally, the reaction of 7a caused the cleavage of the oxazolidinone ring giving 9 in 58% yield. This behavior has been described previously (Scheme 3).25

Final compounds were screened for glycosidase inhibition activities (α-glucosidase from Bacillus stearothermophilus, β-glucosidase from almonds, α-galactosidase from green coffee beans, β-galactosidase from Escherichia coli, α-mannosidase from Jack beans, β-mannosidase from Helix pomatia, and α-l-fucosidase from Homo sapiens) using p-nitrophenyl monosaccharides as substrates.

Carboxylic containing compounds 8a and 8b, could resemble the zwitterionic form of oseltamivir and zanamivir, well-known inhibitors of neuraminidase from Influenza virus26 with carboxyl-amino and carboxyl-guanidine moieties, respectively. Preliminary docking calculations using AutoDock27 showed that 8a and 8b could fit in the binding site of neuraminidase. Thus they were also evaluated as possible inhibitors of neuraminidase from Vibrio cholerae.

The enzymatic activities were calculated by measuring the absorbance of the phenoxide released in the enzymatic reaction at 405 nm. The compounds were initially screened at 1, 5, and 25 mM concentrations. With compounds 8a and 9, inhibition over 50% was observed with selected enzymes at 5 mM; 8b and 10 did not show any significant inhibition of any glycosidase (Chart 1). Inhibition constants (Ki) were estimated assuming a competitive type inhibition in the cases of compound 8a against β-galactosidase (Ki = 1.18 mM) and 9 against β-glucosidase (Ki = 4.43 mM). These two compounds exhibit some selectivity such that even at 25 mM no significant inhibition was observed against other glycosidases.

Chart 1. Residual Glycosidase Activities in the Presence of 5 mM Synthesized Compounds.

Chart 1

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However, the observed inhibition was very weak compared to other iminosugar-based glycosidase inhibition, for example the measured Ki for deoxinojirimycin (DNJ) is 0.44 μM for α-glucosidase from B. stearothermophilus. The inhibition constant changes depending on the species that is studied, even for the same glycosidase of other species.28 Other iminosugars present great activity against mannosidases.29

On the other hand, we found that compounds 8a and 8b, bearing a carboxylate group, also did not show any inhibition against neuraminidase but unexpectedly produced activation of the enzyme; these two compounds increased neuraminidase activity up to 100%. The possibility that the compounds act as favorable transglycosylation acceptors causing an increase of nitrophenol release was considered. NMR experiments were performed continuously following the reaction, but potential transient transglycosylation products could not be observed. Further research to explain this behavior is needed. Interestingly there are not many precedents on glycosidase activation by iminosugars. Two reports have accounted for this activation behavior. Thus, up to 70-fold activation of some of glycosidases was detected with multivalent iminosugars.30 In another study, thienopyrimidines were found to activate certain glycosidases.31 The activation mechanism could be explained by the stabilization of the active structure of the enzyme by the introduction of a small molecule adjacent/close to the substrate-binding site, locking the reactive form. Alternatively, if the activation is of the allosteric type occurring in a site different from the active site, it could be interesting to check if the activators have any pharmacological chaperone activity but avoid the temporal inhibition of the enzymatic activity, unlike the aforementioned migalastat and other proposed pharmacological chaperones that help maintaining the correct fold of the protein although temporally blocking the active site of the enzyme.

Conclusions

We described a multigram synthesis of an α,β-unsaturated ketone, which upon a stereoselective cyclopropanation reaction and further transformations gave a novel series of bicyclic piperidine-based iminosugars. The final products were studied against different glycosidases. Inhibition in most cases was low, but interestingly, the activation of neuraminidase was observed with products 8. Possible explanations of this behavior, for example, allosteric activation, enzyme stabilization, or transglycosylation acceptor activity can be proposed. Current studies in our lab will provide insight into these possible mechanisms, and their potential applications will be explored/pursued.

Experimental Section

General Information

All chemicals were obtained from Aldrich/Merck, VWR, Fluorochem, and ABCR. Thin-layer chromatography (TLC) analyses were performed on Merck silica gel 60 F254 plates using phosphomolybdic acid or anisaldehyde and heat for detection. Silica gel NORMASIL 60 40–63 μm was used for flash chromatography. NMR spectra were recorded on a Bruker spectrometer (400 MHz for 1H and 100 MHz for 13C). Chemical shifts are reported in δ ppm referenced to CDCl3 (δ = 7.26 for 1H and 77.00 for 13C), CD3OD (δ = 3.31 for 1H and 49.00 for 13C), or D2O (δ = 4.79 for 1H). Bidimensional spectra (heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond coherence (HMBC), correlated spectroscopy (COSY), and nuclear Overhauser effect spectroscopy (NOESY)) were recorded in order to carry out the assignment. IR spectra were recorded on a Perkin-Elmer Spectrum 100. Specific optical rotation was measured using a polarimeter Anton Parr MCP 100. Melting points of solid compounds were determined using a Stuart Scientific Melting Point Apparatus SMP3. The absorbance of p-nitrophenoxide released in the enzymatic reactions was measured at 405 nm in a Perkin-Elmer Lamba 25.

Methyl (tert-Butoxycarbonyl)-l-serinate

Thionyl chloride (83 mL, 1.1 mol) was added to methanol (280 mL) at 0 °C, then l-serine (60.00 g, 571 mmol) is added. After 10 min, at 0 °C, the solution is heated at 65 °C for 2 h. The solvent is evaporated in vacuo, and 600 mL of AcOEt and a saturated solution of NaHCO3 (until basic pH) are added. Di-tert-butyl dicarbonate (124.62 g, 0.571 mmol) in 265 mL of AcOEt is added. The reaction is stirred overnight at room temperature. The aqueous layer is extracted with AcOEt (2 × 300 mL). The combined organic layers are washed with brine (200 mL), dried over MgSO4, and evaporated in vacuo. The crude product is filtered through a pad of silica gel using Hex/AcOEt (9:1) to Hex/AcOEt (3:1) as eluents. A colorless oil is obtained (101.6 g, 81% after two steps). 1H NMR (400 MHz, CDCl3) δ 5.46 (brs, 1H, NH), 4.39 (brs, 1H, CH), 3.99–3.89 (m, 2H, CH2O), 3.78 (s, 3H, OMe), 2.47–2.32 (m, 1H, OH), 1.45 (s, 9H, 3 × CH3). 13C NMR (100 MHz, CDCl3) δ 171.6, 155.9, 80.3, 63.3, 55.8, 52.8, 28.3 (3C). IR (NaCl): 3378, 2984, 2868, 1740, 1708 cm–1. [α]D25 (c 0.13 in dichloromethane (DCM)): +4.14. Found: C, 49.1; H, 7.9%. Calc. for C9H17NO5: C, 49.3; H, 7.8%.

tert-Butyl (R)-(1-((tert-Butyldimethylsilyl)oxy)-3-hydroxypropan-2-yl)carbamate (1)

To a solution of methyl (tert-butoxycarbonyl)-l-serinate (101.4 g, 463 mmol) in 400 mL of dimethylformamide (DMF) cooled to 0 °C is added imidazole (37.8 g, 555 mmol) and 4-dimethylaminopyridine (DMAP; 5.6 g, 46 mmol). After 10 min, tert-butyldimethylsilyl chloride (73.2 g, 486 mmol) is added. The reaction is stirred for 30 min at room temperature. AcOEt (400 mL) is added, and the organic layer is washed with water (3 × 1 L) and brine (400 mL), dried over MgSO4, and evaporated in vacuo. To a suspension of NaBH4 (35.0 g, 926 mmol) and LiCl (39.3 g, 926 mmol) in 800 mL of ethanol cooled to 0 °C, a solution of the crude in 190 mL of ethanol is added slowly. The reaction is stirred at 0 °C for 10 min, at room temperature for 30 min, and at 50 °C for 2.5 h. The reaction is cooled to 0 °C, and a saturated solution of NH4Cl is added (until salts are dissolved, 450 mL). The aqueous layer is extracted with AcOEt (3 × 350 mL). The combined organic layers are washed with brine (200 mL), dried over MgSO4, and evaporated in vacuo. The reaction crude is filtered through a pad of silica gel using Hex/AcOEt (19:1) to Hex/AcOEt (1:1) as eluents. A colorless oil is obtained (123.2 g, 85% after two steps). 1H NMR (400 MHz, CDCl3) δ 5.14 (brs, 1H, NH), 3.86–3.66 (m, 5H, 2 × CH2O + CH), 2.70 (brs, 1H, OH), 1.45 (s, 9H, 3 × CH3), 0.90 (s, 9H, 3 × CH3), 0.08 (s, 6H, 2 × CH3Si). 13C NMR (100 MHz, CDCl3) δ 156.1, 79.6, 64.0 (2C), 52.7, 28.5 (3C), 25.9 (3C), 18.3, −5.5 (2C). IR (NaCl): 3371, 2952, 2861, 1707 cm–1. [α]D25 (c 0.28 in DCM): +9.30. Found: C, 55.1, H, 10.4%. Calc. for C14H31NO4Si: C, 55.0; H, 10.2%.

(R)-4-(((tert-Butyldimethylsilyl)oxy)methyl)oxazolidin-2-one

To a suspension of NaH 60% w/w (18.9 g, 472 mmol) in 300 mL of THF cooled to 0 °C is added a solution of 1 (123.2 g, 403 mmol) in 550 mL of THF. The reaction is stirred at 0 °C for 15 min, at room temperature for 25 min, and at 40 °C for 2.5 h. The reaction is cooled down to 0 °C, and a saturated solution of NH4Cl is added until all salts are dissolved (250 mL). The aqueous layer is extracted with AcOEt (2 × 500 mL). The combined organic layers are washed with brine (300 mL), dried over MgSO4, and evaporated in vacuo. A colorless wax is obtained (78.9 g, 72%). 1H NMR (400 MHz, CDCl3) δ 6.27 (brs, 1H, NH), 4.42 (t, J = 8.6 Hz, 1H, CH2O), 4.18 (dd, J = 8.8, 4.8 Hz, 1H, CH2O), 3.94–3.88 (m, 1H, CH), 3.60 (d, J = 5.4 Hz, 2H, CH2OSi), 0.87 (s, 9H, 3 × CH3), 0.05 (s, 6H, 2 × CH3Si). 13C NMR (100 MHz, CDCl3) δ 160.2, 67.3, 64.8, 53.8, 25.9 (3C), 18.3, −5.4 (2C). IR (NaCl): 3315, 2959, 2848, 1745 cm–1. [α]D25 (c 0.32 in DCM): −15.94. Found: C, 52.2, H, 9.0%. Calc. for C10H21NO3Si: C, 51.9; H, 9.2%.

(R)-3-Allyl-4-(((tert-butyldimethylsilyl)oxy)methyl)oxazolidin-2-one (2)

To a suspension of NaH 60% w/w (16.4 g, 409.1 mmol) in 500 mL of THF at 0 °C, a solution of (R)-4-(((tert-butyldimethylsilyl)oxy)methyl)oxazolidin-2-one (78.9 g, 340.9 mmol) in 500 mL of THF is added slowly. Allyl bromide (29.5 mL, 340.9 mmol) is added and stirred for 15 min at 0 °C, 30 min at room temperature, and 2 h at 50 °C. A saturated solution of NH4Cl is added until the salts are dissolved. The aqueous layer is extracted with AcOEt (3 × 200 mL). The combined organic layers are washed with brine (150 mL) and dried over MgSO4. The solvent is evaporated in vacuo, and the residue is filtered through a pad of silica gel using Hex/AcOEt (19:1) to Hex/AcOEt (1:1) as eluents. A yellow oil is obtained (64.8 g, 70%). 1H NMR (400 MHz, CDCl3) δ 5.84–5.74 (m, 1H, HC=), 5.27–5.21 (m, 2H, H2C=), 4.33 (t, J = 8.7 Hz, 1H, CH2O), 4.18–4.13 (m, 2H, CH2O + CH2N), 3.86–3.80 (m, 1H, CH), 3.69–3.62 (m, 3H, CH2OSi + CH2N), 0.89 (s, 9H, 3 × CH3), 0.06 (s, 6H, 2 × CH3Si). 13C NMR (100 MHz, CDCl3) δ 158.4, 132.7, 118.5, 65.0, 62.2, 56.0, 45.3, 25.9 (3C), 18.3, −5.4 (2C). IR (NaCl): 3084, 2948, 2866, 1744 cm–1. [α]D25 (c 0.21 in CHCl3): −11.84. Found: C, 57.1, H, 9.1%. Calc. for C13H25NO3Si: C, 57.5; H,9.3%.

(S)-3-Allyl-4-(hydroxymethyl)oxazolidin-2-one

To a solution of 2 (55.5 g, 204.6 mmol) in 220 mL of THF is added TBAF·3H2O (58.8 g, 225.0 mmol). The mixture is stirred for 30 min at room temperature. The solvent is evaporated in vacuo and filtered through a pad of silica gel using Hex/AcOEt (2:1) to Hex/AcOEt (1:2) as eluents. A colorless oil is obtained (28.9 g, 90%). 1H NMR (400 MHz, CDCl3) δ 5.86–5.76 (m, 1H, HC=), 5.30–5.24 (m, 2H, H2C=), 4.36 (t, J = 8.8 Hz, 1H, CH2O), 4.25 (dd, J = 8.7, 6.0 Hz, 1H, CH2O), 4.09 (ddt, J = 15.7, 5.3, 1.6 Hz, 1H, CH2N), 3.90–3.84 (m, 1H, CHN), 3.80–3.73 (m, 2H, CH2N + CH2OH), 3.65 (dd, J = 11.9, 3.3 Hz, 1H, CH2OH). 13C NMR (100 MHz, CDCl3) δ 158.7, 132.5, 118.9, 64.7, 60.9, 56.3, 45.4. IR (NaCl): 3427, 3048, 2975, 2851, 1753 cm–1. [α]D25 (0.25 in CHCl3): −44.28. Found: C, 53.8; H, 7.3%. Calc. for C7H11NO3: C, 53.5; H, 7.1%.

(R)-3-Allyl-2-oxo-oxazolidine-4-carboxylic Acid (3)

A solution of (S)-3-allyl-4-(hydroxymethyl)oxazolidin-2-one (28.9 g, 184.1 mmol) in 1 L of acetone is cooled to 0 °C, and 92 mL of Jones’ reagent is added slowly. The reaction is stirred for 1.5 h at 0 °C. Isopropanol is added until the solution turns blue. The mixture is filtered through a pad of celite. The solvent is evaporated in vacuo and the residue is filtered through a pad of silica gel using Hex/AcOEt (1:1) to AcOEt 100% as eluents. A pale yellow oil is obtained (23.0 g, 73%). 1H NMR (400 MHz, CDCl3) δ 9.83 (brs, 1H, OH), 5.80–5.70 (m, 1H, HC=), 5.27–5.24 (m, 2H, H2C=), 4.52 (t, J = 9.4 Hz, 1H, CH2O), 4.42 (dd, J = 9.0, 4.5 Hz, 1H, CH2O), 4.36 (dd, J = 9.7, 4.6 Hz, 1H, CHN), 4.27 (dd, J = 15.4, 4.8 Hz, 1H, CH2N), 3.75 (dd, J = 15.4, 8.0 Hz, 1H, CH2N). 13C NMR (100 MHz, CDCl3) δ 172.3, 158.5, 131.0, 120.1, 65.1, 56.0, 46.0. IR (NaCl): 3454, 2933, 2839, 1731 cm–1. [α]D25 (0.30 in CHCl3): +8.98. Found: C, 48.8; H, 5.4%. Calc. for C7H9NO4: C, 49.1; H, 5.3%.

(R)-3-Allyl-N-methoxy-N-methyl-2-oxo-oxazolidine-4-carboxamide (4)

A solution of 3 (23.0 g, 134.4 mmol) in 500 mL of DCM is cooled to 0 °C. Diisopropylethylamine (DIPEA; 23.5 mL, 134.4 mmol), EDCI (25.8 g, 134.4 mmol), and N,O-dimethylhydroxylamine hydrochloride (13.1 g, 134.4 mmol) are added. The reaction is stirred for 2 h at 0 °C. The solvent is evaporated in vacuo and filtered through a pad of silica gel using Hex/AcOEt (1:4) as the eluent. A yellow oil is obtained (25.3 g, 88%). 1H NMR (400 MHz, CDCl3) δ 5.83–5.73 (m, 1H, HC=), 5.25–5.21 (m, 2H, H2C=), 4.66 (dd, J = 9.8, 5.6 Hz, 1H, CHN), 4.50 (t, J = 9.3 Hz, 1H, CH2O), 4.32 (ddt, J = 15.4, 4.7, 1.7 Hz, 1H, CH2N), 4.18 (dd, J = 8.8, 5.6 Hz, 1H, CH2O), 3.69 (s, 3H, OCH3), 3.68 (dd, J = 15.4, 8.3 Hz, 1H, CH2N), 3.22 (s, 3H, NCH3). 13C NMR (100 MHz, CDCl3) δ 169.6, 158.0, 132.2, 119.3, 64.5, 61.7, 54.8, 45.9, 32.7. IR (NaCl): 3088, 2979, 2928, 1754, 1672 cm–1. [α]D25 (c 0.37 in DCM): +31.11. Found: C, 50.4; H, 6.9%. Calc. for C9H14N2O4: C, 50.5; H, 6.6%.

(R)-4-Acryloyl-3-allyloxazolidin-2-one

To a solution of 4 (12.0 g, 56.0 mmol) in 270 mL of THF cooled to −30 °C, 0.7 M vinylmagnesium bromide (200 mL) is added slowly, keeping the temperature below −25 °C. When the addition is finished, the reaction is stirred for another 30 min at −30 °C. The reaction mixture is poured into a mixture of 200 mL of HCl 10% and 100 mL of MeOH cooled in a bath at −15 °C. This mixture is stirred for another 15 min. The aqueous layer is extracted with AcOEt (3 × 150 mL). The combined organic layers are washed with a solution of 1 M HCl (200 mL), with a saturated solution of NaHCO3 (150 mL) and brine (150 mL), dried over MgSO4, and evaporated in vacuo. The crude is used without further purification. 1H NMR (400 MHz, CDCl3) δ 6.50 (dd, J = 17.5, 10.4 Hz, 1H, =CHCO), 6.39 (d, J = 17.4 Hz, 1H, H2C = CHCO, trans), 6.01 (d, J = 10.4 Hz, 1H, H2C = CHCO, cis), 5.78–5.68 (m, 1H, HC=), 5.24–5.16 (m, 2H, H2C=), 4.59 (dd, J = 10.0, 5.2 Hz, 1H, CHN), 4.52 (t, J = 9.3 Hz, 1H, CH2O), 4.26 (dd, J = 15.3, 4.6 Hz, 1H, CH2N), 4.15 (dd, J = 8.6, 5.2 Hz, 1H, CH2O), 3.59 (dd, J = 15.3, 8.0 Hz, 1H, CH2N). 13C NMR (100 MHz, CDCl3) δ 195.1, 157.7, 132.3, 131.8, 131.4, 120.1, 63.8, 60.5, 46.2.

(R)-1,8a-Dihydro-3H-oxazolo[3,4-a]pyridine-3,8(5H)-dione (5)

The crude of the previous reaction is dissolved in 180 mL of DCM and heated to reflux. Grubb’s second-generation catalyst is added (1.2 g, 1.4 mmol). The reaction is stirred under reflux for 1.5 h. The solvent is evaporated in vacuo. The crude is purified in silica gel in Hex/AcOEt (1:1). A brown oil is obtained (5.6 g, 65% after two steps). 1H NMR (400 MHz, CDCl3) δ 7.06 (ddd, J = 10.5, 4.4, 2.0 Hz, 1H, =CHCH2), 6.26 (dt, J = 10.5, 2.3 Hz, 1H, =CHCO), 4.69 (dd, J = 9.0, 4.4 Hz, 1H, CHN), 4.61–4.50 (m, 2H, CH2O + CH2N), 4.27 (ddd, J = 9.4, 4.4, 2.1 Hz, 1H, CH2O), 4.02 (dq, J = 20.5, 2.3 Hz, 1H, CH2N). 13C NMR (100 MHz, CDCl3) δ 192.1, 157.4, 146.5, 127.7, 64.2, 57.9, 41.8. IR (NaCl): 3081, 2959, 2920, 2854, 1752, 1748 cm–1. [α]D25 (c 0.09 in DCM): +65.02. Found: C, 55.1; H, 4.5%. Calc. for C7H7NO3: C, 54.9; H, 4.6%.

tert-Butyl (5aR,6S,6aS,7aR)-3,7-Dioxohexahydro-1H,3H-cyclopropa[d]oxazolo[3,4-a]pyridine-6-carboxylate (6)

To a solution of 5 (2.4 g, 15.9 mmol) in 13 mL of DCM at 0 °C is added a solution of tert-butyl (tetrahydrothiophenylidene)acetate (9.6 g, 47.6 mmol) in 207 mL of DCM slowly. The reaction is stirred at room temperature for 30 min. Deionized water (20 mL) is added. The aqueous layer is extracted with DCM (2 × 30 mL). The combined organic phases are washed with brine (20 mL), dried over MgSO4, and evaporated in vacuo. The crude is purified in silica gel using Hex/AcOEt (2:1) as the eluent. A yellow wax is obtained (3.0 g, 70%). 1H NMR (400 MHz, CDCl3) δ 4.53 (t, J = 9.7 Hz, 1H, CH2O), 4.32 (dd, J = 9.3, 5.7 Hz, 1H, CH2O), 4.23 (d, J = 14.1 Hz, 1H, CH2N), 4.04 (dd, J = 10.3, 5.7 Hz, 1H, CHN), 3.53 (d, J = 14.0 Hz, 1H, CH2N), 2.39 (dd, J = 7.9, 4.2 Hz, 1H, CHCO), 2.21–2.18 (m, 1H, CHCH2N) 2.10 (t, J = 4.5 Hz, 1H, CHCO2), 1.44 (s, 9H, 3 × CH3). 13C NMR (100 MHz, CDCl3) δ 200.0, 168.9, 156.9, 82.8, 64.2, 58.8, 37.2, 31.6, 28.1, 24.5, 22.7. IR (NaCl): 2975, 2863, 1748, 1736, 1719 cm–1. [α]D25 (c 0.11 in DCM): +38.02. Found: C, 58.0; H, 4.9%. Calc. for C13H17NO5: C, 58.4; H, 4.6%.

tert-Butyl (5aR,6S,6aS,7S,7aR) and tert-butyl (5aR,6S,6aS,7R,7aR)-7-Hydroxy-3-oxohexahydro-1H,3H-cyclopropa[d]oxazolo[3,4-a]pyridine-6-carboxylate (7a and 7b)

To a solution of 6 (1.1 g, 4.1 mmol) in 35 mL of absolute ethanol at 0 °C is added NaBH4 (312 mg, 8.2 mmol). The reaction is stirred for 1 h at room temperature. A solution of saturated NH4Cl (20 mL) and water (until salts dissolve) is added. The aqueous phase is extracted with AcOEt (3 × 60 mL). The combined organic layers are washed with brine (50 mL), dried over MgSO4, and evaporated in vacuo. The crude contained a 3:2 mixture of isomers 7a/7b as determined by the integration of signals in the 1H NMR spectrum of the reaction crude. This mixture was separated by silica gel chromatography using Hex/AcOEt (1:1) to Hex/AcOEt (1:2) as eluents. A yellow wax is obtained for isomer 7a (463 mg, 42%). A yellow solid is obtained for isomer 7b (330 mg, 30%).

Spectroscopic data for tert-butyl (5aR,6S,6aS,7S,7aR)-7-hydroxy-3-oxohexahydro-1H,3H-cyclopropa[d]oxazolo[3,4-a]pyridine-6-carboxylate 7a: 1H NMR (400 MHz, CDCl3) δ 4.52 (t, J = 8.6 Hz, 1H, CH2O), 4.12 (dd, J = 9.1, 4.8 Hz, 1H, CH2O), 4.00 (d, J = 13.6 Hz, 1H, CH2N), 3.80 (dd, J = 8.5, 4.5 Hz, 1H, CHOH), 3.40 (dd, J = 13.6, 4.1 Hz, 1H, CH2N), 3.33 (td, J = 8.3, 4.9 Hz, 1H, CHN), 2.41 (d, J = 5.0 Hz, 1H, OH), 1.76–1.70 (m, 1H, CHCH2N), 1.67 (dd, J = 9.1, 5.0 Hz, 1H, CHCHOH), 1.44 (s, 9H, 3 × CH3), 1.40 (t, J = 4.9 Hz, 1H, CHCO2). 13C NMR (100 MHz, CDCl3) δ 172.2, 157.5, 81.4, 69.0, 68.3, 56.8, 38.5, 28.2 (3C), 27.3, 24.5, 20.3. IR (NaCl): 3361, 2975, 2863, 1748, 1736 cm–1. [α]D25 (c 0.14 in DCM): −2.20. Found: C, 58.2; H, 6.8%. Calc. for C13H19NO5: C, 58.0; H, 7.1%.

Spectroscopic data for tert-butyl (5aR,6S,6aS,7R,7aR)-7-hydroxy-3-oxohexahydro-1H,3H-cyclopropa[d]oxazolo[3,4-a]pyridine-6-carboxylate 7b: 1H NMR (400 MHz, CDCl3) δ 4.54 (dd, J 0 8.5, 6.4 Hz, 1H, CH2O), 4.29 (t, J = 8.8 Hz, 1H, CH2O), 4.24–4.20 (m, 1H, CHOH), 4.00 (d, J = 13.4 Hz, 1H, CH2N), 3.61–3.55 (m, 1H, CHN), 3.29 (dd, J = 13.4, 4.0 Hz, 1H, CH2N), 2.11 (td, J = 8.4, 5.0 Hz, 1H, CHCHOH), 1.97 (d, J = 4.1 Hz, 1H, OH), 1.84–1.74 (m, 2H, 2 × CH cyclopropane), 1.44 (s, 9H, 3 × CH3). 13C NMR (100 MHz, CDCl3) δ 172.1, 157.8, 81.5, 63.1, 60.4, 55.7, 38.8, 28.3 (3C), 25.9, 21.2, 20.5. IR (KBr): 3361, 2975, 2863, 1748, 1736 cm–1. [α]D25 (c 0.04 in DCM): −10.46. Found: C, 58.3; H, 7.0%. Calc. for C13H19NO5: C, 58.0; H, 7.1%. Mp > 180.0 °C, dec.

(5aR,6S,6aS,7S,7aR)-7-Hydroxy-3-oxohexahydro-1H,3H-cyclopropa[d]oxazolo[3,4-a]pyridine-6-carboxylic Acid

To a solution of 7a (250 mg, 0.9 mmol) in 1.5 mL of DCM is added 9.3 mL of TFA at room temperature. The reaction is stirred for 30 min. The solvent is evaporated in vacuo. Toluene is added until all TFA is evaporated. The crude is purified in silica gel in 10% DCM/MeOH. A brown wax is obtained (187 mg, 94%). 1H NMR (400 MHz, MeOD) δ 4.54 (t, J = 8.5 Hz, 1H, CH2O), 4.14 (dd, J = 8.9, 5.2 Hz, 1H, CH2O), 3.87 (d, J = 13.6 Hz, 1H, CH2N), 3.82 (d, J = 8.7 Hz, 1H, CHOH), 3.44 (dd, J = 13.5, 4.2 Hz, 1H, CH2N), 3.37 (td, J = 8.5, 5.2 Hz, 1H, CHN), 1.84–1.75 (m, 1H, CHCH2N), 1.70 (dd, J = 9.3, 4.7 Hz, 1H, CHCHOH), 1.38 (brs, 1H, CHCO2). 13C NMR (100 MHz, D2O) δ 176.6, 159.5, 69.8, 69.6, 58.2, 39.5, 29.0, 24.5, 21.9. IR (NaCl): 3396, 2984, 2851, 1748, 1729 cm–1. [α]D25 (c 0.05 in MeOH): −3.68. Found: C, 51.1; H, 5.3%. Calc. for C9H11NO5: C, 50.7; H, 5.2%.

(5aR,6S,6aS,7R,7aR)-7-Hydroxy-3-oxohexahydro-1H,3H-cyclopropa[d]oxazolo[3,4-a]pyridine-6-carboxylic Acid

To a solution of 7b (86 mg, 0.3 mmol) in 1 mL of DCM is added 3.2 mL of TFA at room temperature. The reaction is stirred for 30 min. The solvent is evaporated in vacuo. Toluene is added until all TFA is evaporated. The crude is purified in silica gel in 10% DCM/MeOH. A brown wax is obtained (60 mg, 90%). 1H NMR (400 MHz, D2O) δ 4.47 (dd, J = 8.5, 5.8 Hz, 1H, CH2O), 4.31 (t, J = 8.8 Hz, 1H, CH2O), 4.17 (dd, J = 8.0, 3.8 Hz, 1H, CHOH), 3.86 (d, J = 13.4 Hz, 1H, CH2N), 3.71 (ddd, J = 9.5, 5.9, 3.9 Hz, 1H, CHN), 3.38 (dd, J = 13.4, 4.4 Hz, 1H, CH2N), 2.09 (td, J = 8.4, 4.7 Hz, 1H, CHCHOH), 1.82 (t, J = 4.9 Hz, 1H, CHCO2), 1.80–1.75 (m, 1H, CHCH2N). 13C NMR (100 MHz, D2O) δ: 177.7, 159.6, 64.2, 59.8, 55.4, 38.4, 26.0, 21.2, 20.0. IR (KBr): 3388, 2991, 2867, 1740, 1732 cm–1. [α]D25 (c 0.02 in MeOH): −48.87. Found: C, 51.0; H, 4.9%. Calc. for C9H11NO5: C, 50.7; H, 5.2%. Mp > 205.4 °C, dec.

(1R,4R,5S,6S,7S)-5-Hydroxy-4-(hydroxymethyl)-3-azabicyclo[4.1.0]heptane-7-carboxylic Acid (8a)

To a solution of (5aR,6S,6aS,7S,7aR)-7-hydroxy-3-oxohexahydro-1H,3H-cyclopropa[d]oxazolo[3,4-a]pyridine-6-carboxylic acid (187 mg, 0.9 mmol) in 2 mL of MeOH is added ethylenediamine (0.18 mL, 2.6 mmol) at room temperature and heated at 60 °C for 1.5 h. The solvent is evaporated in vacuo and methanol is added to evaporate excess amine. A solution of HCl 4 N in dioxane (5 mL) is added and stirred for 30 min. A yellow wax is obtained (150 mg, 91%).1H NMR (400 MHz, MeOD) δ 4.54 (t, J = 8.5 Hz, 1H, CH2O), 4.14 (dd, J = 8.9, 5.3 Hz, 1H, CH2O), 3.86 (d, J = 13.5 Hz, 1H, CH2N), 3.72 (d, J = 8.7 Hz, 1H, CHOH), 3.44 (dd, J = 13.5, 4.3 Hz, 1H, CH2N), 3.36 (td, J = 8.4, 5.2 Hz, 1H, CHN), 1.78–1.70 (m, 1H, CHCH2N), 1.67 (dd, J = 9.2, 4.9 Hz, 1H, CHCHOH), 1.32 (t, J = 4.9 Hz, 1H, CHCO2). 13C NMR (100 MHz, MeOD) δ 159.5, 70.1, 69.6, 58.2, 39.6, 28.5, 25.7, 21.3. IR (NaCl): 3405, 2996, 2895, 1736 cm–1. [α]D25 (c 0.05 in MeOH): −5.41. Found: C, 51.0; H, 7.2%. Calc. for C8H13NO4: C, 51.3; H, 7.0%.

(1R,4R,5R,6S,7S)-5-Hydroxy-4-(hydroxymethyl)-3-azabicyclo [4.1.0]heptane-7-carboxylic Acid (8b)

To a solution of (5aR,6S,6aS,7R,7aR)-7-hydroxy-3-oxohexahydro-1H,3H-cyclopropa[d]oxazolo[3,4-a]pyridine-6-carboxylic acid (75 mg, 0.4 mmol) in 1 mL of MeOH is added ethylenediamine (0.07 mL, 1.1 mmol) and heated at 60 °C for 1.5 h. The solvent is evaporated in vacuo and methanol is added to evaporate excess amine. A solution of HCl 4 N in dioxane (1 mL) is added and stirred for 30 min. A yellow wax is obtained (60 mg, 90%). 1H NMR (400 MHz, MeOD) δ 4.47 (dd, J = 8.5, 6.0 Hz, 1H, CH2O), 4.30 (t, J = 8.8 Hz, 1H, CH2O), 4.16 (dd, J = 8.0, 3.9 Hz, 1H, CHOH), 3.84 (d, J = 13.3 Hz, 1H, CH2N), 3.75–3.62 (m, 1H, CHN), 3.37 (dd, J = 13.2, 4.4 Hz, 1H, CH2N), 2.11–1.93 (m, 1H, CHCHOH), 1.79–1.64 (m, 2H, 2 × CH cyclopropane). 13C NMR (100 MHz, MeOD) δ 158.80, 63.53, 59.64, 55.65, 38.46, 25.01, 21.50, 19.60. IR (NaCl): 3402, 2984, 2890, 1733 cm–1. [α]D25 (c 0.02 in MeOH): −32.0. Found: C, 51.4; H, 7.2%. Calc. for C8H13NO4: C, 51.3; H, 7.0%.

(1S,4R,5S,6S,7S)-4,7-bis(Hydroxymethyl)-3-methyl-3-azabicyclo[4.1.0]heptan-5-ol (9)

To a solution of 7a (120 mg, 0.5 mmol) in 3 mL of DCM is added 1.86 mL of DIBAL-H 1,2M in toluene at 0 °C. The reaction is stirred for 4 h at room temperature. Methanol is added (10 mL). The salts are filtered and rinsed with methanol (2 × 10 mL). The solvent is evaporated in vacuo. The crude is purified in silica gel using MeCN/H2O (9:1) as the eluent. A yellow wax is obtained (49 mg, 58%). 1H NMR (400 MHz, MeOD) δ 3.81 (dd, J = 11.7, 3.2 Hz, 1H, CH2O), 3.77–3.71 (m, 2H, CH2O + CHOH), 3.45 (dd, J = 11.3, 6.7 Hz, 1H; HOCH2Ccyclopropane), 3.37 (dd, J = 11.3, 6.8 Hz, 1H, HOCH2Ccyclopropane), 3.07 (d, J = 11.7 Hz, 1H, CH2N), 2.63 (dd, J = 11.6, 3.9 Hz, 1H, CH2N), 2.38 (s, 3H, NCH3), 1.75 (dt, J = 7.9, 2.9 Hz, 1H, CHN), 1.23–1.15 (m, 1H, HOCH2CHcyclopropane), 1.12–1.03 (m, 1H, CHCH2N), 0.99 (dd, J = 9.0, 4.6 Hz, 1H, CHCHOH). 13C NMR (100 MHz, MeOD) δ 70.2, 67.1, 65.9, 60.1, 55.6, 43.0, 23.24, 23.22, 17.3. IR (NaCl): 3357, 2993, 2892 cm–1. [α]D25 (c 0.03 in MeOH): −4.65. Found: C, 57.5, H, 9.5%. Calc. for C9H17NO3: C, 57.7; H, 9.2%.

(5aS,6S,6aS,7R,7aR)-7-Hydroxy-6-(hydroxymethyl)hexahydro-1H,3H-cyclopropa[d]oxazolo[3,4-a]pyridin-3-one (10)

To a solution of 7b (117 mg, 0.4 mmol) in 3 mL of DCM at 0 °C is added 1.80 mL of 1,2M DIBAL-H in toluene. The reaction is stirred for 4 h at room temperature. Methanol is added (10 mL). The salts are filtered and rinsed with methanol (2 × 10 mL). The solvent is evaporated in vacuo. The crude is purified in silica gel using MeCN as the eluent. A yellow wax is obtained (54 mg, 62%). 1H NMR (400 MHz, MeOD) δ 4.48 (dd, J = 8.5, 5.8 Hz, 1H, CH2O), 4.32 (t, J = 8.8 Hz, 1H, CH2O), 4.16 (dd, J = 8.1, 3.8 Hz, 1H, CHOH), 3.83 (d, J = 12.9 Hz, 1H, CH2N), 3.69 (ddd, J = 9.4, 5.8, 3.8 Hz, 1H, CHN), 3.54 (dd, J = 11.2, 6.4 Hz, 1H, HOCH2Ccyclopropane), 3.42 (dd, J = 11.3, 6.7 Hz, 1H, HOCH2Ccyclopropane), 3.36 (dd, 12.9, 4.7 Hz, 1H, CH2N), 1.42 (td, J = 8.4, 4.9 Hz, 1H, CHCHOH), 1.30–1.20 (m, 1H, HOCH2CH cyclopropane), 1.19–1.09 (m, 1H, CHCH2N). 13C NMR (100 MHz, MeOD) δ 160.4, 65.6, 65.0, 61.4, 57.2, 40.2, 21.9, 21.0, 16.5. IR (NaCl): 3384, 2991, 2888, 1705 cm–1. [α]D25 (c 0.01 in MeOH): −35.3. Found: C, 54.5; H, 6.5%. Calc. for C9H13NO4: C, 54.3; H, 6.6%.

General Procedure for Enzymatic Reactions

Glycosidase activities were assessed in 80 μL reaction volumes in Eppendorf vials. Buffer composition and enzyme concentration were adjusted depending on the enzyme assayed: 20 mM Na2HPO4 at pH 7.3 for β-glucosidase (3 μg/mL) and β-galactosidase (1 μg/mL); 20 mM Na2HPO4 at pH 6.8 for α-glucosidase (1 μg/mL) and α-galactosidase (20 μM); 20 mM NaH2PO4 at pH 5.5 for α- and β-mannosidase (7 and 2 μM respectively); 0.1 M NaOAc at pH 4.0 with 1 mg/mL of bovine serum albumin (BSA) for α-l-fucosidase (2 μM); and 50 mM NaOAc at pH 5.0 for neuraminidase (6 μM). The inhibitors were tested at 1, 5, and 25 mM final concentrations in the assays. Each enzyme mixture and inhibitor were homogenized and preincubated for 10 min at 37 or 40 °C (α-l-fucosidase). Each reaction was initiated and brought to a final volume of 80 μL, by addition of an aliquot of the corresponding p-nitrophenyl glycoside substrate to obtain the following final concentrations in the reaction mixtures: p-nitrophenyl α- and β-d-glucopyranoside (1 mM), p-nitrophenyl α- and β-d-galactopyranoside (0.5 mM), p-nitrophenyl α- and β-d-mannopyranoside (1 mM), p-nitrophenyl α-l-fucopyranoside (1 mM), or p-nitrophenyl neuraminic acid (1 mM). After 10 min of incubation time at the same temperature, each reaction was quenched with 400 μL of 1.0 M Na2CO3, and the absorbance at 405 nm was measured. Assays were repeated twice and data were averaged.

The residual activity of each enzyme was calculated by the ratio of the absorbance measured after 10 min of reaction in the presence and absence of synthesized compounds. The equation used to calculate Ki was derived from Michaelis–Menten, where Vi is the absorbance measured in the absence of the synthesized compounds; V is the absorbance when the compounds were added to the enzymatic reaction; Km indicates the Michaelis–Menten constant for each enzyme; and [I] is the concentration of the synthesized compounds (5 mM) and [S] is the concentration of the substrate (eq 1).

Calculated Ki for compounds 8a and 9 graphic file with name ao0c04589_m001.jpg 1

Acknowledgments

Funding of this project by the Spanish MINECO, Grant no. RTI2018–095588-B-I00 and RTI2018-094751-B-C22 (Co-funded by European Regional Development Fund/European Social Fund, “Investing in your future”), FUSP-CEU (PC17/17) and CIBERES, an initiative from the Spanish Institute of Health Carlos III are acknowledged.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c04589.

  • 1H NMR, 13C NMR, and IR spectra of synthesized compounds (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao0c04589_si_001.pdf (2.4MB, pdf)

References

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