Synthesis of (5,6 & 6,6)-oxa-oxa annulated sugars as glycosidase inhibitors from 2-formyl galactal using iodocyclization as a key step

Oxa-oxa (5,6 & 6,6) annulated sugars were synthesized from 2-formyl galactal using iodocyclization as a key step. The glycosidase inhibitory activities of the synthesized molecules were tested against commercially available enzymes which showed that the sugar-furan molecules are potent and selective inhibitors


Results and Discussion
We were interested in the synthesis of a new type of structurally modified annulated sugars namely 1,2annulated sugar-pyran and sugar-furan fused molecules. This was planned based on our earlier findings related to the synthesis of a new class of annulated sugars, including compound 13 (Figure 2), in which the ring oxygen of the parent sugar ring was one carbon (anomeric carbon) away from another ring hetero-atom (compounds i-iii Figure 3) of O-C-X type. It was observed that, among these molecules, compound 13 was the best with respect to selectivity and extent of inhibition (IC50 = ~38.8 µM) and compound iv was the worst which is devoid of a hetero-atom X in the second ring. This supported our hypothesis that the presence of the hetero-atom X could lead to the species B and C (Figure 4) wherein the oxocarbenium ion part could easily mimic the accepted transition state The newly designed molecules I-III are shown in Figure 5 in which one of them bears a hydrophobic butyl group. This was planned with a view to minimize the number of hydroxyl groups and increase some amount of hydrophobicity to assess their effects on the selectivity and the extent of inhibitions. The target molecules were envisioned to be obtained via iodocyclization of the corresponding primary alcohols IV and V, followed by dehalogenation. These primary alcohols could be obtained from olefins VI and VII whose side chains can be accessed by addition of vinyl and allyl magnesium bromides on 2-formyl galactal 15. C2-formyl glycals are versatile synthons in organic chemistry 38 as has been illustrated in the synthesis of many biologically important natural products, and synthetic analogues and intermediates. From our group also, we have exploited the potential of C-2-formyl group in the synthesis of sugar -amino acids, 39 iminosugars, 40 hybrid molecules 36 and C-2-methylene glycosides. 41 In the present study, synthesis of the designed 1,2-annulated sugars I-III commenced from 3,4,6-tri-Obenzyl-D-galactal 16 (Scheme 1) which was converted to the vinyl aldehyde 15 using the Vilsmeier-Haack reaction. 42 To this vinyl aldehyde was added vinylmagnesium bromide at 0 o C in dry THF. The resultant hydroxyl group was protected using benzyl bromide and sodium hydride forming the benzyl ether 17 in 58% yield over two steps (Scheme 1). The regioselective dihydroxylation of terminal alkene in 17 was carried out using OsO4/NMO to form a mixture of diols which was subsequently exposed to oxidative cleavage using sodium metaperiodate in THF/H2O (1:1) medium at room temperature to afford the corresponding aldehyde 18a/b. The aldehyde was then reduced with NaBH4 in methanol affording the diastereomeric mixture of alcohols 19 (Scheme 1) which was chromatographically difficult to separate. Thus, this mixture of primary alcohols 19 was subjected to iodocyclization (Scheme 1) using N-Iodosuccinimide (NIS) 43 at room temperature in CH2Cl2 to form 6-endo iodocyclized products 20a and 20b. The acetals 20a (20%) and 20b (58%) were obtained in a 0.35:1 ratio in 78% yield which were easily separable by column chromatography at this stage. The major isomer 20b was then subjected to radical mediated deiodination using a catalytic amount of n-Bu3SnCl and AIBN in presence of NaBH3CN in toluene and t BuOH (1:1) at 80 o C for 1 h resulting in the formation of the desired product 21 in 65% yield. Deprotection of the benzyl groups in 21 with Pd(OH)2/C in MeOH under 1 atm H2 for 4 h afforded furan-fused sugar molecule 22 in 81% yield.  For the synthesis of n-butyl substituted sugar-furan hybrid molecule, a careful column chromatographic separation of aldehyde 18b ( isomer) from the diastereomeric mixture of 18a/b was done to perform the Grignard reaction. A freshly prepared n-butylmagnesium bromide was reacted with aldehyde 18b at -30 o C (Scheme 2) to provide the corresponding alcohol. The crude alcohol was then subjected to iodocyclization to give a furan-fused sugar derivative 23 as a single compound. Compound 23 upon radical deiodination to obtain 24 followed by hydrogenation, as described earlier, afforded the desired annulated sugar derivative 25. The free hydroxyl groups of compound 25 were protected as acetates using a 1:1 mixture of acetic anhydride and Et3N in presence of DMAP over 6 h (Scheme 2) to afford peracetylated compound 26.

Scheme 2. Synthesis of compound 26.
The absolute configuration of the newly generated stereocenters was determined with the help of 1 H NMR, COSY, and nOe experiments. 44  Hz. Based on these data, the structure of 26 was deduced to be as shown in Figure 7. In a similar manner, sugar-pyran annulated molecule 32 was obtained by reacting 2-formyl galactal 15 with allylmagnesium bromide, followed by hydroxyl group protections as benzyl ethers to form 27 as a 1:0.9 ( and ). This mixture of 27 was subjected to the same sequence of reactions as described earlier viz.
dihydroxylation, oxidative cleavage, NaBH4 reduction etc., to provide a diastereomeric mixture 28a/b (Scheme 3) of the primary alcohols. From this mixture of 28a/b was separated the isomer 28a in pure form which was subjected to a similar set of reactions such as iodocyclization, radical deiodination and hydrogenation reactions to afford sugar-pyran fused molecule 31 (Scheme 3).

Scheme 3. Synthesis of sugar-pyran fused molecule 32.
The structure and stereochemistry of compound 31 was determined from the spectral data of the corresponding acetate 32 by using 1 H NMR, COSY, and nOe experiments. 44 Thus, in nOe experiments, irradiation of proton H-3 at δ 5.53, no enhancement was observed for H-7 at δ 5.07 indicating that H-3 and H-7 are in trans oriented with respect to each other. Further, irradiation of H-1 at δ 4.82 led to the enhancement of the signal for proton H-7 at δ 5.07 and no enhancement was observed for H-3 and H-5 protons suggesting that H-1 and H-7 are in cis orientation. On the other hand, when H-2 at δ 2.54 was irradiated no correlation was observed with H-4 indicating that H-2 and H-4 are in trans orientation. In addition, the coupling between H-1 with H-2 led to a doublet at δ 4.83 with J = 3.50 Hz. Based on these spectral data, the structure of compound 32 is assigned to be as shown in Figure 8.  The hence obtained annulated molecules were then examined for their glycosidase inhibitory behaviour. 32,45 They were tested against 8 commercially available enzymes and the results are summarized in Table 1 wherein the IC50 values are indicated in  The n-butyl substituted sugar-furan molecule 25 was found to be quite potent and highly selective against −galactosidase (bovine liver source) with IC50 value being 8.1 . In addition, sugar-furan fused molecule 22 showed good and selective inhibition against mannosidase (Jack beans), with IC50 value to be 8.8  On the other hand, sugar-pyran fused molecule 31 showed non-specific moderate inhibition against -glucosidase, -galactosidase and −mannosidase, with IC50 values of 12.1  26.2  and 16.0  respectively. From these studies it is clear that furan-fused sugar molecules (smaller in ring size) showed stronger inhibitory properties and presence of hydrophobic butyl group (cf. 25) appeared to influence the activity in a positive way, making it to be equally good inhibitor as the corresponding parent molecule namely 22. On comparing these results with our earlier work 32 (cf. compounds 13 and i-iii), it is obvious that our hypothesis as shown in Figure 4 is valid for the present set of compounds. Interestingly, even O-C-O type of molecules having pyran-furan fused rings are better than pyran-piperidine fused compound namely 13, suggesting that decreasing the size of the ring and thereby decreasing the number of carbon atoms, and also decreasing the number of -OH groups influence the selectivity as well as the IC50 value irrespective of whether or not 'X' in O-C-X type arrangement is 'N'. Although it is not very clear how does the hydrophobic side chain affect the selectivity, but it is obvious that species of type B and C ( Figure 4) that should form with both 22 and 25, as well as with 31 do influence the inhibition data. It would be of interest to synthesize molecules of similar type but with an arrangement of O-C-N type and study their inhibition behavior. Work towards this direction will be a subject of future studies.

Conclusions
We have synthesized 1,2-linearly fused (5,6& 6,6)-oxa-oxa annulated sugars. The synthesis of these annulated molecules was achieved from 2-formyl galactal in short sequences and good yields via Grignard addition and halocyclization as key steps. Glycosidase inhibition studies revealed that the sugar-furan and n-butyl substituted sugar-furan molecules were most active and highly selective inhibitors.

Experimental Section
General. All experiments were performed in oven-dried apparatus and under nitrogen atmosphere in dry solvents, unless indicated otherwise. Commercial grade solvents were dried by known methods, and dry solvents were stored over 4 Å molecular sieves. IR spectra were recorded as a thin film and expressed in cm −1 . Mass spectra were obtained using Q-TOF apparatus from high resolution ESI mass spectrometer. 1 H NMR (400 or 500 MHz) and 13 C NMR (100 or 125 MHz) spectra were recorded using CDCl3 or D2O as a solvent. Chemical shifts have been reported in ppm downfield to tetramethylsilane and coupling constants expressed in Hertz (Hz); splitting patterns have been assigned as s (singlet), d (doublet), dd (doublet of doublet), td (triplet of doublet), q (quartet), m (multiplet), br (broad), etc. Optical rotations were measured at 25 °C in indicated solvents. TLC plates were prepared using thin layers of silica gel on microscopic slides, and visualization of spots was effected by exposure to iodine or spraying with 10% H2SO4 and charring. Column chromatography was performed over silica gel (100−200 Mesh) using hexane and ethyl acetate as eluent.
Compound 15 (400 mg, 0.91 mmol) was dissolved in THF (4 mL) and cooled to 0 o C. The solution was treated with commercially available vinylmagnesium bromide solution (1M in THF, 4.5 mL, 4.5 mmol) and the resulting solution was stirred for 1 h with gradual warming to room temperature. Saturated NH4Cl (10 mL) was added carefully and the contents were extracted using EtOAc (3 × 15 mL). The combined extracts were dried and concentrated using rotary evaporator. The crude alcohol was used for the next step without further purification. Rf = 0.5 (hexane/EtOAc = 4:1). The crude alcohol was dissolved in dry DMF (5 mL) and NaH (47 mg, 1.5 mmol) was added to it at 0 o C. The mixture was stirred at the same temperature for 15 min and then benzyl bromide (0.12 mL, 1.2 mmol) was added dropwise to it. The solution was subsequently stirred at room temperature for 3 h and then quenched with ice and extracted with ether (3 × 5 mL). The combined extracts were dried over Na2SO4 and concentrated in vacuo and the crude product was purified by silica gel chromatography to obtain 17 (296 mg, 58% over 2 steps) as a colourless oil: Rf = 0.6 (hexane/EtOAc = 9:1); IR

-Bis(benzyloxy)-5-(1-(benzyloxy)but-3-en-1-yl)-2-((benzyloxy)methyl)-3,4-dihydro-2H-pyran (27).
The aldehyde 15 (350 mg, 1.05 mmol) was dissolved in THF (4 mL) and cooled to 0 o C. The solution was treated with freshly prepared excess allylmagnesium chloride and the resulting solution was stirred 1 h with gradual warming to room temperature. Saturated NH4Cl (10 mL) was added carefully and the contents were extracted using EtOAc (3 × 15 mL). The extracts were dried and concentrated using rotary evaporator. The crude compound was used for the next step without further purification. Rf = 0.5 (hexane/EtOAc = 4:1). The crude alcohol was dissolved in dry DMF (5 mL) and NaH (47 mg, 1.5 mmol) was added at 0 o C. The mixture was stirred at same temperature for 15 min and then benzyl bromide (0.12 mL, 1.2 mmol) was added dropwise to it. The solution was stirred at room temperature for 3 h and then quenched with ice and extraction was done with ether (3 × 5 mL). The extracts were dried over Na2SO4 and concentrated in vacuo and the crude was purified by silica gel chromatography to obtain 27 (309 mg, 68% over 2 steps) as a colourless oil: Rf = 0.5 (hexane/EtOAc  The diene 27 (500 mg, 0.868 mmol) was dissolved in acetone/ t BuOH/H2O solvent system (3:1:1,8 mL) and Nmethyl morpholine N-oxide (120 mg, 1.04 mmol) followed by catalytic amount of OsO4 were added in succession and the resulting mixture was stirred at room temperature for 4 h. Then saturated Na2S2O5 solution (8 mL) was added and the mixture stirred for 1 h. The compound was extracted using EtOAc (3 × 8 mL) and the extracts were dried (Na2SO4) and concentrated. The crude alcohol was cooled in THF/H2O (4:1) mixture (10 mL) at 0 o C and sodium metaperiodate (557 mg, 2.60 mmol) was added to the vigorous stirred solution in portions over 1 h at same temperature followed by stirring for another 1 h. The reaction mixture was then filtered and the filtrate was extracted with CH2Cl2 (3 × 8 mL). Combined organic extracts were washed once with brine (1 × 20 mL), dried over Na2SO4 and concentrated in vacuo. The crude aldehyde was dissolved in dry MeOH (8 mL) and cooled to 0 o C. Then, NaBH4 (98 mg, 2.60 mmol) was added to the reaction mixture in portions over 5 min and stirring continued for 10 min. Subsequently, aq. NH4Cl (10 mL) was added drop wise to the reaction mixture till the effervescence ceased. Extraction was done using CH2Cl2 (3 × 8 mL) and extracts were washed with brine (1 × 20 mL) and dried over Na2SO4.The removal of solvent under vacuum furnished a crude residue, which was separable by column chromatography to give 28a (190 mg, and other isomer 28b, 171 mg, combined yield 72% over 3 steps) as colourless oil: Rf   2R,3S,4S,5S,8aS)

General procedure for enzyme inhibition studies
All the enzymes and their corresponding substrates were procured from Sigma-Aldrich Chemical Co. The inhibition studies of compounds (22, 25 and 31) were determined by measuring residual hydrolytic activities of the glycosidases. The substrates and enzymes were prepared as 0.025 M solutions in the respective pH buffer solutions of the corresponding enzyme. In all cases, the substrates used were the corresponding p-nitrophenyl glycopyranosides. The incubation mixture consisted of 100 μL of enzyme solution, 200 μL of 1 mg mL−1 aqueous solution of the test compound, and 100 μL of the appropriate buffer solution of the optimum pH for the enzyme. After incubation at 37 °C for 1 h, 100 μL of the substrate solution was added and allowed to react for 10 min. The reaction mixture was quenched using 2.5 mL of 0.05 M borate buffer (pH = 9.8). In all cases, control experiments were run simultaneously in the absence of test compound. A series of blank experiments for the substrate were also carried out in the respective buffer solutions without the enzyme or test compounds. The absorbance of the liberated p-nitrophenol in each reaction (both test and control reactions) was recorded using spectrophotometer at 405 nm. Percentage inhibition was calculated as the ratio of the difference in the observed absorbances of the control and test reactions to the observed absorbance of the control reaction. Results have thus been reported as IC50 values, which is the concentration of the test compound that causes 50% inhibition of the enzyme.