Stereoselective Synthesis of 2-Acetamido-1 , 2-dideoxynojirimycin ( DNJNAc ) and Ureido-DNJNAc Derivatives as New Hexosaminidase Inhibitors

2-Acetamido-1,2-dideoxyiminosugars are selective and potent inhibitors of hexosaminidases and therefore show high therapeutic potential for the treatment of various diseases, including several lysosomal storage disorders. A stereoselective synthesis of 2-acetamido-1,2dideoxynojirimycin (DNJNAc), the iminosugar analog of N-acetylglucosamine, with high overall yield is here described. This novel procedure further allowed accessing ureido DNJNAc conjugates through derivatization of the endocyclic amine on a key pivotal intermediate. Remarkably, some of the ureido-DNJNAc representatives behaved as potent and selective inhibitors of β-hexosaminidases, including the human enzyme, being the first examples of neutral sp-iminosugar-type inhibitors reported for these enzymes. Moreover, the amphiphilic character of the new ureido-DNJNAc is expected to confer better drug-like properties.


Introduction.
Since the isolation of nojirimycin in 1966, iminosugars -sugar analogs where the oxygen ring atom has been replaced by a nitrogen-have attracted an exponential interest as mimics of the transition state of the enzymatic hydrolysis of glycosidic substrates. 1,24][5] In fact, some iminosugars are already marketed drugs, such as miglitol (Glyset) and N-butyl-1-deoxynojirimycin (Zavesca), used for the treatment of type II diabetes mellitus and type 1 Gaucher disease respectively. 6minosugars reduced at C-1 and bearing an acetamido group at the position equivalent to C-2 in the parent monosaccharides, namely 2-acetamido-1,2dideoxyiminosugars, have been the focus of considerable attention in recent years.Several representatives of acetamido iminosugars, for instance pochonicine (1) 7 , siastatine B (2), 8 or nagstatine (3) 9,10 have been isolated from natural sources while derivatives from those and other compounds have been obtained by chemical synthesis 11,12 .Most of these representatives are piperidine derivatives, such as 2-acetamido-1,2-dideoxynojirimycin (DNJNAc, 4) [13][14][15][16] and its manno (DMJNAc, 5) 14,17 or galacto epimers (DGJNAc, 6), 18,19 although acetamido iminosugars with five-(e.g.7) 20,21 or seven-membered ring skeletons (e.g.8) 22 have also been described.Several of these compounds have proven to be highly selective inhibitors of hexosaminidases -the enzymes cleaving off aminosugar residues from oligosaccharides and glycoconjugates-with inhibition constant (K i ) values in the low micromolar to nanomolar range.6][37][38][39] This is also the case for acetamido iminosugars, 13,16,40,41 with a few exceptions limited to the stereoselective synthesis of 2-acetamido-1,2dideoxyallonojirimycin (DAJNAc, 9) 42 and the manno diastereomer DMJNAc (5). 17Herein, we report a new stereoselective total synthesis of the gluco counterpart DNJNAc (4) and its regioisomer 3-acetamido-1,3dideoxyaltronojirimycin (29).Moreover, the preparation of a series of ureido-DNJAc derivatives as examples of 2-acetamido sp 2 -iminosugars, has also been accomplished.Characterized by the incorporation of a pseudoamide-type nitrogen atom with high sp 2 hybridation character in the ring [43][44][45] , this subtype of glycomimetics, from which nagstatine 3 can be considered a natural representative, has previously shown an unprecedented potential for fine tuning the inhibitory potency and selectivity towards glycosidases by modulating the basicity of the N-functionality and the nature of the exocyclic moiety. 46In our case, the evaluation of the new ureido-DNJNAc against a panel of glycosidases allowed the identification of hexosaminidase inhibitors with an amphiphilic character and a greatly reduced basicity, features that make these compounds better suited as drug candidates.

Results and discussion
Our approach to DNJNAc, 4 and the ureido-DNJNAc derivatives 10 is shown in Scheme 1.An appropriate protecting group scheme was needed to introduce the urea fragment in the last steps.The protected compounds were prepared by introducing the amino function by nucleophilic ring opening of an epoxide or a cyclic sulfate obtained from the key intermediate 11, which is readily accessible by Sharpless asymmetric epoxidation of 2,4-pentadien-1-ol. 47This intermediate has been widely used for the synthesis of various iminosugars 17,48- 50 including our recent synthesis of DAJNAc (9). 42heme 1. Retrosynthetic analysis for the preparation of DNJNAc (4) and ureido-DNJNAc conjugates (10) from the common bicyclic precursor 11.
Optically pure carbamate 11 was prepared in multigram scale from penta-1,4-dien-3-ol, and the allylic alcohol group was subsequently protected as the corresponding benzyl ether 12. 48 We first considered the epoxidation of the double bond in 12 followed by regioselectively ringopening by azide anion to introduce the amino substituent.Deceivingly, classical methodologies using m-chloroperoxybenzoic acid (MCPBA) or H 2 O 2 proved inefficient while harsher oxidant methods such as CF 3 CO 3 H 51,52 or oxone 49 generated inseparable 1:1 mixtures of the corresponding epoxides in moderate yields.We hypothesized that the rigid bicyclic skeleton of 12 was probably responsible for the low reactivity observed.However, although hydrolysis of the cyclic carbamate by treatment with 6M NaOH at reflux, followed by in situ Cbz-protection of the endocyclic amine afforded the monocylic derivative 13 in satisfactory yield, all attempts at diastereoselective epoxidation of 13 failed, regardless of the epoxidation methodology used.Various combinations of N-carbamate and O-ester/ether protecting groups were also assayed without success.In view of these results, we explored the use of cyclic sulfates as an alternative to epoxides, 53,54 an approach that has been applied successfully in other iminosugar syntheses. 55,56he protection of the primary alcohol of 13 as a benzoate, followed by Sharpless asymmetric dihydroxylation of the intermediate ester (14), yielded a 90:10 mixture (HPLC) of diastereomeric diols, from which the major isomer 15 was isolated in 62% yield (Scheme 2).Treatment of 15 with thionyl chloride gave a mixture of sulfites that was oxidized without further purification with RuCl 3 /NaIO 4 to the corresponding cyclic sulfate 16, which was obtained as a single diasteroisomer in 80% overall yield (two steps).Treatment of 16 with NaN 3 at 50ºC gave an inseparable mixture of the azidoalcohols 17 and 18. Attempts to quantify the relative proportion of the two compounds at this stage by NMR or HPLC failed.The two azidoalcohols were hypothesized to be the result of the nuclephilic attack of the azide anion at C2 (glucoconfiguration) and C3 (altro-configuration) positions.Sequential treatment of this mixture with NaOMe, in order to cleave the benzoate group, and NaH regenerated the 2-oxazolidinone ring, affording a 1:1 mixture of the bicyclic azidoalcohols 19 and 20 (Scheme 2).Although no selectivity was achieved during the cyclic sulfate opening reaction, both carbamates 19 and 20 were easily separated by column chromatography, which yielded crystalline compounds that could be analyzed by X-ray diffraction, † thus confirming the proposed stereochemistry (Figure 2).We envisaged that carbamate 19 would be an excellent precursor in the synthesis of DNJNAc (4) and ureido-DNJAc derivatives 10.The straightforward purification and facile separation of the two regioisomers encouraged us to look for a shorter route to synthetize the mixture of 19 and 20.Direct dihydroxylation of carbamate 12 using K 2 OsO 4 •2H 2 O/NMO afforded 21 in satisfactory yield and diastereoselectivity (78%, 85:15) (Scheme 3).Sharpless asymmetric dihydroxylation conditions increased both the yield and diasteroselectivity, affording diol 21 in 94% yield and nearly complete diasteroselectivity, as observed by 1 H-NMR. 17The corresponding cyclic sulfate 22 was obtained in 80% yield by reaction of diol 21 with SOCl 2 /TEA followed by in situ oxidation with NaIO 4 /RuCl 3 , as in the previous case.Attempts to perform direct sulfation of 21 using SO 2 Cl 2 /TEA 57 also afforded 22 but in lower yields.Scheme 3. Synthesis of cyclic sulfate from 12 followed by ring-opening with sodium azide.
Regioselective ring-opening reactions of the key precursor 22 using NaN 3 as the nucleophile were extensively studied and are summarized in Table 1.We expected that the presence of the benzyl group at the C4 position would sterically hinder approaching of the azide anion nucleophile to C3, directing the attack to the C2 position (iminosugar numbering).The reaction did not take place in acetonitrile (entry 1) but proceeded in N,Ndimethylformamide (entries 2 and 3).Thus, treatment of sulfate 22 with sodium azide in DMF, followed by acidic hydrolysis (to cleave the intermediate residual sulfate), gave a 2:1 mixture of azidoalcohols in 70% yield (entry 2).However, increasing the temperature and the equivalents of NaN 3 , led to a dramatic decrease in yield and a total loss of selectivity (entry 3).In an attempt to improve the regioselectivity, the reaction was performed in acetone/water (entry 4 and 5), observing that fewer equivalents of azide allowed similar ratios.The use of lower temperatures (40ºC), even fewer equivalents of azide (1.2) and a longer reaction time (16 h), afforded higher yields, but also at the expenses of a total loss of regioselectivity (entry 6).Conversely, portion-wise addition of sodium azide increased the regioselectivity, but with a significant decrease in yield (entry 7).According to our objective of obtaining derivatives of 4, conditions of entry 5 were chosen for scaling up purposes.The spectroscopic data of this compound were consistent with previously reported data. 18The total synthesis of DNJNAc (4) from 11 was thus accomplished in 10 synthetic steps achieving a 23% overall yield.Although some 3-acetamido iminosugar derivatives have been reported we could not find precedents of evaluation of their properties as glycosidase inhibitors. 41,58,59We thus considered it of interest to apply the above synthetic sequence to azido alcohol 20, i.e. benzylation (26), azide reduction and acetylation of the resulting amine (27), basic hydrolysis of the cyclic carbamate group (28) and final hydrogenolysis of the benzyl protecting groups.In this manner, 3-acetamido-1,3dideoxyaltronojirimycin 29 was prepared in excellent overall yield (Scheme 5).
It has been described that modifications of the acetamide moiety in DNJNAc lead to a dramatic decrease in the inhibitory activity against hexosaminidases, 40 while modifications at the endocyclic amine are well tolerated.Indeed, the incorporation of hydrophobic N-alkyl substituents has been previously investigated, 18,60 and found to lead to an improvement of the inhibitory potency which is consistent with the presence of a hydrophobic pocket in the vicinity of the active site of the enzyme. 61All DNJNAc analogs reported to date keep the basic character of the piperidine glycone-like skeleton, generally considered a favorable structural feature to promote strong binding to the enzyme.However, it has been demonstrated that higher glycosidase affinities and, especially, improved selectivities can be achieved by the interplay of neutral glycone-type cores and substituents that provide additional non-glycone interactions. 62,635][66][67][68] For instance, N-(N'-butylaminocarbamoyl)-1-deoxynojirimycin, the urea analog of the marketed drug Zavesca, was found to be a very selective inhibitor of bovine liver galactosidase. 67The sp 2 -hybridized character is also observed in some natural products such as kifunensine, a potent inhibitor of class I α-mannosidase. 69,70To check this strategy for the particular case of hexosaminidases, we synthesized a series of ureido-DNJNAc derivatives (10).In addition to a much lower basic character at the endocyclic nitrogen, conversion of an amine into a urea offers flexibility in the choice of substituents, which can be taken advantage of to optimize the inhibitory capacity and the pharmacokinetic behavior.The oxazolidinone ring of azido alcohol 19 was hydrolyzed under the usual conditions and the endocyclic amine was protected in situ using Boc 2 O/NaHCO 3 to give azidoalcohol 30.Concomitant azide reduction and cleavage of the benzyl group were accomplished by hydrogenation in methanol/acetic acid.The resulting vicaminoalcohol was acetylated without further purification by treatment with Ac 2 O in pyridine to afford acetamide 31 in 83% yield (2 steps).Next, the N-Boc group was selectively cleaved using TFA, and the resulting cyclic amine was reacted in situ with n-butyl, n-octyl, phenyl or benzyl isocyanate in the presence of triethylamine (TEA) to give the corresponding urea adducts 32a-d in 70-85% yield.Final deacetylation using a saturated solution of ammonia in MeOH gave the target ureido-DNJNAc derivatives 10a-d (Scheme 6).Scheme 6. Synthesis of ureido-DNJNAc derivatives 10a-d.
Evaluation of the glycosidase inhibitory activity of the DNJNAc regioisomer 29 and the ureido-DNJNAc derivatives 10a-d, in comparison with the parent acetamido iminosugar 4, confirmed their total selectivity towards hexosaminidases among a panel that included the following: β-glucosidases (almonds and bovine liver), α-glucosidase (yeast), α-mannosidase (jack bean), βmannosidase (Helix pomatia), trehalase (pig kidney), amyloglucosidase (Aspergillus niger), α-rhamnosidase (naringinasa; Penicillium decumbens), α-galactosidase (green coffee), β-galactosidase (E.coli), and isomaltase (yeast).Compound 29 was a much weaker inhibitor than DNJNAc, confirming that even when hexosaminidases are relatively promiscuous regarding the configurational pattern of iminosugar-type ligands, the location of the acetamido group next to the anomeric position is critical to ensure strong enzyme binding.Gratifyingly, all ureido-DNJNAc derivatives 10a-d behaved as µM inhibitors of the three hexosaminidases assayed in this work, namely those from human placenta, bovine kidney, and jack beans.N'-alkyl substituents (n-butyl, n-octyl or benzyl; 10a, 10b and 10d) led to a slight decrease in the inhibitory potency as compared with 4, with inhibition constant (K i ) values in the 56-20 µM range for the human enzyme.The N'-phenyl derivative 10c was an about one order of magnitude stronger inhibitor of the hexosaminidases as compred with the N'alkyl counterparts.Notably, the inhibition potency against the human enzyme surpassed that of 4 by over 3-fold.This result is remarkable considering the much lower basicity of 10c as compared with 4. The data suggest the involvement of the urea NH proton in hydrogen bonding in the complex of ureido-DNJNAc with the hexosaminidases, compensating the electrostatic interactions operating in the case of the basic iminosugar, as previously demonstrated for other sp 2iminosugar:glycosidase complexes. 71he higher hydrogen bond donor capability of arylureas as compared with alkylureas, due to the electron withdrawing character of the aromatic ring, is consistent with the observed activity trend.Most interestingly, the amphiphilic character of the compounds is expected to confer better drug-like properties.Altogether, the results reported herein are promising for the further development of therapeutic agents for β-GlcNAcaserelated diseases.

Conclusions
Here we have described a new stereoselective synthesis of 2-acetamido-1,2-dideoxynojirimycin (DNJNAc), the iminosugar analog of N-acetylglucosamine, with high overall yield.The strategy is based on the stereoselective ring-opening of cyclic sulfates derived from the key intermediate 11, which was conveniently prepared by a multigram procedure based on Sharpless epoxidation.This novel procedure gave access to the advanced intermediate 19 which provided us with the necessary protecting group arrangement to synthesize sp 2iminosugar conjugates through derivatization of the endocyclic amine by reaction with isocyanates.These new ureido-DNJNAc derivatives are the first neutral inhibitors of hexosaminidases described to date.These compounds were potent inhibitors of β-GlcNAcase and, given their amphiphlic character, they are expected to show acceptable drug-like properties.

Table 2.
Inhibition constants (K i, µM) a against commercial β-N-acetylglucosaminidases 10a-d and 29 determined from thee slope of Lineweaver-Burk plots and double reciprocal analysis compared with previously reported values for DNJNAc (4). 18zyme origin

Experimental General
All commercial reagents were used without further purification.Non-aqueous reactions were performed out under nitrogen atmosphere.Dry tetrahydrofuran, dichloromethane, and diethyl ether were obtained using a Solvent Purification System (SPS).Other solvents were used with no further purification.All reactions were monitored by TLC analysis using Merck 60 F254 silica gel on aluminum sheets.Silica gel chromatography was performed by using 35-70 mm silica or an automated chromatography system.NMR spectra were recorded at room temperature on a 400 MHz instrument. 1 H and 13 C-NMR spectra were referenced to the residual peaks of the deuterated solvent.The following abbreviations were used to define the multiplicities: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; and br, broad signal.The chemical shifts (δ) are expressed in ppm and the coupling constants (J), in Hertz (Hz).IR spectra were recorded either by preparing a KBr pastille or by depositing a film of the product on a NaCl window.Absorptions are given in wavenumbers (cm -1 ).Melting points were recorded in a Büchi M-540 apparatus without recrystallization of the final solids.Optical rotations were measured at room temperature (25°C).Concentration is expressed in g/100 mL and solvent is expressed for each case in brackets.The cell was 10 cm long and had 1 mL of capacity.
Measuring λ was 589 nm, which corresponds to a sodium lamp.High Resolution Mass Spectrometry were conducted using nanoelectrospray technique.. Preparation of 11 47 , 12 48 and 21 17,48 was done following literature procedures.Starting material 11 was 99% ee.Syntheses and characterizations of compounds in Scheme 5, and derivatives 32b-d and 10b-d can be found in the supporting information.

2-Azido-4-O-benzyl-5-N-benzyloxycarbonyl-1,2dideoxynojirimycin (30)
NaOH 6M (2 mL, 11.89 mmol) was added to a solution of compound 19 (301 mg, 0.99 mmol) in MeOH:H 2 O 9:1 (20 mL) and the reaction was heated at reflux for 15 h.Solvent was then removed under low pressure and the crude was redissolved in EtOAc:NaHCO 3 saturated aqueous 1:1 (14 mL).After 30 min of stirring, Boc 2 O (436 mg, 1.91 mmol) was added and the crude was allowed to stir for 24 h.The crude was treated with water (6 mL), extracted with EtOAc (3x5 mL), dried with MgSO 4 , and purified by chromatography on silica gel using hexane/ethyl acetate to give 30 (334 mg, 90%) as a colorless oil.(8 mL) and the reaction was stirred at r.t. until no starting material was observed by TLC.Solvent was removed under reduced pressure and the resulting oil was dissolved in CH 2 Cl 2 (8 mL).TEA (0.23 mL, 1.64 mmol) and butyl isocyanate (71 µl, 0.63mmol) were added and the reaction was heated at reflux for 4h.H 2 O (5 mL) was then added and the reaction was extracted with CH 2 Cl 2 (3x 5 mL), dried over MgSO 4 , and purified by chromatography on silica gel using CH 2 Cl 2 /MeOH to give 32a (73 mg, 81%) as a colorless oil.

Table 1 .
Optimization of the ring-opening reaction of sulfate 22 with sodium azide. Bn a Inhibition was competitive in all cases.