Carbohydrate Research 408 (2015) 25e32

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Synthesis, conformational study, glycosidase inhibitory activity and molecular docking studies of dihydroxylated 4- and 5-aminoiminosugars Vijay M. Kasture a, Navnath B. Kalamkar a, Roopa J. Nair b, Rakesh S. Joshi c, Sushma G. Sabharwal b, Dilip D. Dhavale a, * a b c

Department of Chemistry, Garware Research Centre, Savitribai Phule Pune University (formerly University of Pune), Pune 411007, India Division of Biochemistry, Department of Chemistry, Savitribai Phule Pune University (formerly University of Pune), Pune 411007, India Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University (formerly University of Pune), Pune 411007, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 December 2014 Received in revised form 3 March 2015 Accepted 4 March 2015 Available online 12 March 2015

An efficient methodology for the synthesis of new amino iminosugars 6a, 7a and 8, starting from Dglucose, is reported. The conformational study using 1H NMR data showed that the amino iminosugar 6a exists in the 2C5 while; the 7a and 8 exist in the 5C2 conformation. The inhibition activities with different glycosidases showed that 6a and 7a are poor glycosidase inhibitors. However, amino iminosugar 8 showed selective inhibition against the b-galactosidase (IC50¼43 mM, Ki¼153 mM). These results are substantiated by the molecular docking studies. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Iminosugar Amino piperidine Glycosidase inhibitor Chiron approach Reductive amino-cyclization Molecular docking

1. Introduction Polyhydroxylated cyclic compounds with the nitrogen atom in the ring, commonly called iminosugars, are the most notable carbohydrate mimetics, which comprises the low molecular weight heterocyclic molecules such as azitidines, pyrrolidines, piperidines, azepanes and bi-cyclic (nitrogen atom at the ring fusion) pyrrolizidines, indolizidines and quinolizidines etc.1e7 This class of molecules are valuable for the basic understanding of glycobiology due to their action as inhibitors of glycosidases in the biological processes such as digestion, endoplasm reticulum associated degradations and lysosomal catabolism of glycoconjugates.8e11 As a result, iminosugars are investigated as promising candidates for the treatment of carbohydrate disorder mediated diseases such as diabetes, obesity, cancer and viral infections.12,13 For example, piperidine iminosugars namely N-hydroxyethyl-1deoxynojirimycin (trade name Miglitol) and N-butyl-1-

* Corresponding author. E-mail address: [email protected] (D.D. Dhavale). http://dx.doi.org/10.1016/j.carres.2015.03.004 0008-6215/© 2015 Elsevier Ltd. All rights reserved.

deoxynojirimycin (trade name Zavesca) are being used for the treatment of type-II diabetes and Gaucher diseases, respectively.14 In the search for promising glycosidase inhibitors, modification of natural and unnatural iminosugars with the variation of stereochemistry and replacement of hydroxyl substituent with hydroxyalkyl,15e22 aminoalkyl,23,24 amine,25e31 halogen32e34 alkyl groups35e39 and study of their structureeactivity relationship is the established protocol in synthetic carbohydrate chemistry. Amongst these, mono- and bi-cyclic iminosugars in which hydroxyl group is substituted with amino group-amino iminosugars are known to be selective glycosidase inhibitors.40,41 For example, Pandey et al. reported the synthesis of amino iminosugar 1 (Fig. 1) that showed good a-glucosidase inhibitory activity.42 Bols et al. reported the synthesis of amino dihydroxypiperidine 2-an amino analog of isofagomine, which showed selective inhibition activity against the bglycosidase.43 Fleet. et al. reported the C-2 amino analog of 1-deoxy nojirimycin 3a (R¼H) that showed a potent anticancer activity with 50% inhibition at 6 mM with Ki 0.9 mM and also showed a selective inhibition of b-N-acetylaminoglucosaminidase.44 Tropak et al. and Wong. et al. reported a series of potent amino-piperidine compounds 3aec for Tay-Sachs and Sandhoff diseases.45,46 In addition,

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V.M. Kasture et al. / Carbohydrate Research 408 (2015) 25e32

N

NH3 Cl

HO

OH

OH

1

2

R1

OH

HO

O

NHAc

HO

N

NH2

R2

N

O

NH2

OH

OH

synthesis and glycosidase inhibitory activity of iminosugars (piperidine triols) 6b and 7b.53 The structureeactivity relationship (SAR) activity study presented here that the substitution of C4eOH in 6b and 7b with the amino group having same configuration diminishes the glycosidase inhibitory activity. While, substitution of the C3eOH in 7b with the amino group having inverted orientation led to the potent selective inhibition against the b-Galactosidase. Our results are depicted herein.

H

H2 Cl N

2. Results and discussion

NH

As shown in Scheme 1, D-glucose was converted to a 3-azido-3deoxy-5-O-p-toluenesulphonyl-a-D-xylofuranose 9 using reported method.54 Treatment of 9 with NaN3 in DMF at 90  C afforded a diazido-compound 10 in 93% yield.55,56 Hydrolysis of 1,2-acetonide group in 10 using TFA/H2O provided hemiacetal that was directly subjected for reductive amino-cyclization using 10% Pd/C, H2, (80 psi) in methanol to afford amino-iminosugar 6a in 78% (in two steps). In order to synthesize amino iminosugar 7a, D-glucose was converted to the 1,2:5,6-di-O-isopropylidene-3-deoxy-3benzyloxycarbonylamino-a-D-allofuranose 11 using the reported method (Scheme 1).57 Selective deprotection of 5,6-acetonide functionality with 1% H2SO4 afforded diol 12 in 88% yield.58 Diol 12 on treatment with sodium metaperiodate in acetoneewater followed by reduction with sodium borohydride in methanolewater afforded a primary alcohol 13 in 87% yield (two steps).59 The primary hydroxyl group of 13 was converted to a tosyl derivative 14 (TsCl, Py)59 and then to a C-5 azido compound 15 (NaN3, DMF) in 87% yield. Hydrolysis of the 1,2-acetonide group in 15 using TFA/ H2O followed by reductive amino-cyclization (10% Pd/C, H2, 80 psi) afforded an amino iminosugar 7a in 68% yield (two steps).

N

OH

3a, R1 = R2=H 3b, R1 = alkyl, R2=H 3c, R1=H, R2=OH

H N

N

Cl NH2

N

N

5 Cisapride

4 BMS-690514

6 5

HO

H

OMe H

H

N

N

N

1

2 3

4

OH HO

OH HO

R

NH2

R

OH

7a, R=NH2 7b, R=OH

6a, R=NH2 6b, R=OH

8

Fig. 1. Natural and synthesized iminosugars.

Ref. 54

D-Glucose

TsO O O

N3 Ref. 57

O O HN Cbz

O O 11

9

1% H2SO4, MeOH:Water,

O

NaN3, DMF, 90 oC, 8 h,

rt, 12h, 88%

93%

O O

N3

O

10

HN Cbz

O O O 12

two steps 78 %

2.NaBH4, MeOH:Water, 0 oC-rt , 3 h, two steps 87% Ts-Cl, Py, DCM, rt, 8 h, 93%

NH2 OH N H

O

1. NaIO4, Acetone;Water, rt, 3h

HO HO

1. TFA/H2O 2. H2, Pd/C, MeOH, HO 12 h

N3

6a R O O HN Cbz

O

i. TFA/H2O ii. H2, Pd/C, MeOH, 24 h HO two steps 68 %

13 R = OH 14 R = OTs 15 R = N3

NH2 OH N H 7a

NaN3, DMF,90oC, 12h, 94%

Scheme 1. Syntheses of amino iminosugars 6a, 7a.

the amino iminosugar framework is sometimes also part of drug candidates, such as, BMS-690514 4 (developed for the treatment of non-small cell lung cancer) and Cisapride 5 (a potent gastric prokinetic agent with reduced dopamine D2 receptor antagonist activity).47,48 As a part of our continuous efforts in the area of iminosugars,49e52 we now report a modest approach, in gram scale, for the synthesis of new amino iminosugars 6a, 7a and 8 from Dglucose and study of their glycosidase inhibitory activity along with correlation using molecular docking. Earlier we have reported the

For the synthesis of amino iminosugar 8, D-glucose was transformed to 6-O-tosyl derivative 16 as per reported method (Scheme 2).60 Intermolecular SN2 displacement of a 6-O-tosyl group in 16 with NaN3 in DMF at 90  C afforded an azido-alcohol 17 in 92% yield.61,62 The secondary C-5 hydroxy functionality in 17 was protected as benzyl ether using benzyl bromide and sodium hydride to get compound 18 in 93% yield. Hydrolysis of 1,2-acetonide group in 18 with TFA/H2O provided hemiacetal that was directly subjected for the oxidative cleavage using NaIO4/H2O to afford

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27

Scheme 2. Synthesis of amino iminosugar 8.

unstable aldehyde, which on reductive amino cyclization (10% Pd/C, H2, 80 psi) in methanol afforded amino-iminosugar 8 in 52% yield (in three steps). 2.1. Conformational analysis It is known that configuration and conformation of the substituents in iminosugars are the decisive factors in their specific enzymatic activities.63,64 Therefore, the conformations of amino iminosugars 6a, 7a and 8 were studied based on the 1H NMR data wherein; the assignment of chemical shifts to different protons and coupling constant values were determined using the spindecoupling experiments and are given in Table 1. In case of the amino-iminosugars 6a, 7a and 8 two conformations 2C5 and 5C2 were considered. In conformation 5C2, the J values between axially orientated H2a, H3, H4, H5 and H6a is expected to be large (~8e11 Hz) as compared to the conformation 2C5 in which the H3, H4, H5 and H6 are equatorially oriented and expected to show small J values (~1e4 Hz). In case of a symmetrical aminoiminosugar 6a, the equivalent protons H2a and H6a showed doublet of doublet at 2.36 d with J values 11.9 (J2ae2e, J6ae6e) and 10.9 (J2ae3a, J6ae5a) Hz indicating the axial orientation of H3 and H5 (equivalent protons), respectively. This suggests the 5C2 conformation for a compound 6a. In agreement with this, the H3 (axial proton) showed a doublet of doublet of doublet at 3.35 d with J values 10.9, 9.5 and 4.5 Hz and the H4 (axial proton) showed a triplet at 2.55 d with J value 10.9 Hz thus confirming the 5C2 conformation for 6a (Fig. 2).

Table 1 1 H NMR chemical shifts (d) and coupling constants (J) in compounds 6a, 7a and 8 Compound 6a

Compound 7a

Compound 8

H2a, 2.36 d (dd) H2a-H2e¼11.9 H2a-H3a¼10.9 H6a, 2.36 d (dd) H6a-H6e¼11.9 H6a-H5a¼10.9 H2e, 3.03 d (dd) H2e-H2a¼11.9 H2e-H3a¼4.5 H6e, 3.03 d (dd) H6e-H6a ¼ 11.9 H6e-H5a ¼ 4.5 H3a, 3.35 d (ddd) H3a-H2a¼10.9 H3a-H2e¼4.5 H3a-H4a¼9.5 H5a, 3.35 d (ddd) H5a-H6a ¼ 10.9 H5a-H6e ¼ 4.5 H5a-H4a ¼ 9.5 H4a, 2.55 d (dd) H4a-H3a¼9.5 H4a-H5a¼9.5

H2a, 2.98 d (dd) H2a-H2e¼14.5 H2a-H3e¼1.4 H6a, 2.98 d (dd) H6a-H6e ¼ 14.5 H6a-H5e ¼ 1.4 H2e, 3.17 (dd) H2e-H2a¼14.5 H2e-H3e¼3.5 H6e, 3.17 (dd) H6e-H6a ¼ 14.5 H6e-H5e ¼ 3.5 H3e, 4.02e4.08 d (m) H5e, 4.02e4.08 d (m) H4a, 3.35 d (t) H4a-H3a¼3.5 H4a-H5a¼3.5

H2a, 2.68 d (dd) H2a-H2e¼14.3 H2a-H3e¼1.4 H2e, 2.83e3.08 d (m) H5a, 2.83e3.08 d (m) H6e, 2.83e3.08 d (m) H3e, 3.87e3.93 d (m) H4a, 3.38 d (dd) H4a-H3a¼2.8 H4a-H5a¼9.5 H6a, 2.23 d (dd) H6a-H6e¼12.9 H6a-H5a¼10.0

While, in case of the symmetrical amino iminosugar 7a, the H2a and H6a (both protons are equivalent) showed a doublet of doublet at 2.98 d with J values 14.5 (J2ae2e, J6ae6e) and 1.4 (J2ae3e, J6ae5e) Hz, indicating the equatorial orientation of H3 and H5 (equivalent protons), respectively. In accordance with this, the H3 appeared as a narrow multiplet (WH¼9.6 Hz) and the H4 appeared as a triplet at 3.35 d with small coupling constant value of 3.5 Hz indicating their relative equatorial orientation. Based on this data the 2C5 conformation was assigned for compound 7a. In case of amino iminosugar 8, all protons were found to be non-equivalent. The H6a proton appeared as a doublet of doublet at 2.23 d with J values 12.9 (J6ae6e) and 10.0 Hz (J6ae5a), indicating the axial orientation of H5. In analogy with this, H4 showed a doublet of doublet at 3.38 d with J values 9.5 and 2.8 Hz, indicating the axial orientation of H5 and equatorial orientation of H3. This requires the 2C5 conformation for the compound 8. This fact is supported by the appearance of H2a as doublet of doublet at 2.68 d with J values 14.3 (J2ae2e) and 1.4 (J2ae3e) indicates the equatorial orientation of the H3 proton, confirming the 2C5 conformation for the compound 7a. Thus, it is interesting to note that an amino iminosugar 6a exists in 5C2 conformation while, 7a and 8 exist in 2C5. In all the preferred conformations the amino group attains an equatorial position. This fact can be rationalized as follows. In compound 6a, all substituents are equatorial thus stabilizing the 5C2 conformation. In compound 7a the C-4 amino substituent is residing axially in 5C2 conformation that destabilizes the conformation by 1.2 kcal/mol65 however, in case of 2C5 conformation the equatorial amino functionality stabilizes the conformation, which is augmented by the intramolecular hydrogen bonding between the 1,3-diaxial hydroxyl groups as shown in Fig. 2. An analogous observation was noticed in amino iminosugar 8 wherein an equatorial orientation of amino group stabilizes the 2C5 over 5C2 conformation. 2.2. Biological activities 2.2.1. Glycosidase inhibition Synthesized amino-iminosugars 6a, 7a, 8 were screened for the glycosidase inhibitory profile against b-galactosidase, b-glucosidase, a-mannosidase and a-galactosidase (isolated from almond seeds), a-mannosidase and N-acetyl-b-D-glucosaminidase (isolated from Jack bean seeds), a-glucosidase(isolated from Rice seeds), bgalactosidase (isolated from Bovine liver),a-fucosidase and Nacetyl-b-D-glucosaminidase (isolated from Bovine kidney), a-amylase(isolated from Geobacillus JN704808) and a-glucosidase (Yeast -procured from Sigma Chemical Co). The IC50 values and Ki values were determined only for the amino-iminosugars showing high inhibitory activity. Amongst three amino iminosugars 6a, 7a and 8, the 6a, 7a showed no inhibition against any of the enzyme under study. However, the amino iminosugar 8 found to be selective inhibitor of b-galactosidase. These results were compared with the parent iminosugars namely piperidines triols 6a, 7a that were synthesized by us. The SAR study indicated that the substitution of C4eOH group in 6b as

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V.M. Kasture et al. / Carbohydrate Research 408 (2015) 25e32

Fig. 2. 2C5 and 5C2 conformations of amio-iminosugars 6a, 7a and 8.

well as in 7b, with the amino group having same orientation (retention of configuration), giving amino iminosugars 6a and 7a, diminished the glycosidase inhibitory activity. The substitution of C3eOH in 7b, with amino group with inverted orientation giving amino iminosugar 8, showed potent selective inhibition against the b-Galactosidase (Bovine liver) (IC50¼43 mM, Ki¼153 mM). The glycosidase assays were repeated three times. All the compounds were tested for their inhibitory activity initially at 10 mM concentration (the highest concentration used). At this concentration no significant inhibition was observed due to 6a and 7a, although compound 8 showed inhibitory activity. Hence, for 8 inhibitions was studied at different concentration between 20 mM and 10 mM. Even at 20 mM compound 8 showed good inhibitory activity with IC 50 value of 43 mM and Ki value 153 mM.

2.2.2. Molecular docking Interactions of synthesized amino iminosugars 6a, 7a and 8 with the binding pocket of bovine liver b-galactosidase were analyzed and depicted using molecular docking studies. Galactosidase inhibitory activity of compound 8 showed its efficiency as selective b-galactosidase inhibitors. Homology model for the Bovine liver bgalactosidase was predicted and quality of model was assessed using ProSA and Ramchandran plot analysis. Docking studies of the amino-iminosugars 6a, 7a and 8 showed that, 5-amino iminosugar 8 showed strong binding affinity (5.8 kcal/mol) with b-galactosidase. Binding score for the compound 6a and 7a is 5.0 and 5.1 kcal/mol, respectively, indicating that these derivatives have lower binding affinity as compared to the compound 8. Binding free energies are corroborated with inhibition kinetic studies, where the compound 8 was found to be selective competitive inhibitor of bovine liver b-galactosidase. Binding poses of 6a, 7a and 8 are conserved, but there are significant variations in term of molecular interactions with active site of b-galactosidase (Fig. 3). Analysis of polar contacts and other interactions such as electrostatic and pi-interaction displayed that amino iminosugar 8 form multiple hydrogen bonds with GLU128, ASN186, GLU187, GLU267 and TYR269 residues of b-galactosidase. There are also electrostatic interactions with GLU128, GLU187 and GLU267. Strong binding of the compound 8 with active site of b-galactosidase is also contributed by its position in the pi-interaction in space of TRP272 and TYR484. Whereas the compound 6a form hydrogen bond with ASN186, GLU187 and GLU267, and the compound 7a contributes to hydrogen bonds with GLU187 only resulting into weak binding. Although all three compounds are forming hydrogen bonds with binding pocket of b-galactosidase, the higher number of

intermolecular hydrogen bonds and presence of electrostatic piinteraction augments the stronger binding of the compound 8 to the active site of beta-galactosidase. Binding energy is calculated after multiple interactions using Lamarckian Genetic Algorithm. It is well accepted method for binding energy calculation. Although, the difference among the binding energy is low, the density of intermolecular hydrogen bonds is higher in compound 8. According to the thermodynamics of binding, hydrogen bond mainly contributes to the strong binding. This is supported by docking studies. In case of compound 8, axial eOH makes polar contact with GLU267 and thus projects remaining equatorial functional groups to establish the maximum contact. In compound 8, axial eOH and other equatorial groups lead to synergistic effect on binding. Furthermore, in case 6a all of the groups are equatorial, giving to least number of contacts. Similarly, 7a makes polar contact via axial eOH group, thus resulted ineNH2 equatorial unreactive in terms of polar contact. 3. Conclusion An efficient process from D-glucose that can be scaled up to grams quantity is reported for the synthesis of amino iminosugars 6a, 7a and 8 in overall yields of 23%, 16% and 14% from D-glucose, respectively. The conformational study indicates that the aminoiminosugar 6a exists in 2C5 conformation while; 7a and 8 prefer 5 C2 conformation. It has been noticed that the amino group in amino iminosugar dictates the conformation and the stable conformation is the one in which the amino group is equatorially orientated. Out of all three amino iminosugars, compound 8 showed selective b-galactosidase inhibitory activity, while; compounds 6a and 7a were found to be poor inhibitors of glycosidases. 4. Experimental section 4.1. General experimental methods Melting points were recorded with melting point apparatus and are uncorrected. IR spectra were recorded with an FTIR as a thin film or using KBr pellets and are expressed in cm1. 1H (500/ 300 MHz) and 13C (125/75 MHz) and NMR spectra were recorded using CDCl3, D2O as solvent. Chemical shifts were reported in d units (ppm) with reference to TMS as an internal standard and J values are given in Hz. Elemental analyses were carried out with a C, H-analyzer. Thin layer chromatography was performed on pre-

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Fig. 3. Molecular docking study of bovine liver b-galactosidase with derivatives of amino iminosugar (6a, 7a and 8) (A), (C) and (E) binding poses of compound 6a, 7a and 8, while (B), (D) and (F). Intermolecular hydrogen bonding and other interactions between b-galactosidase and amino iminosugar derivatives complex.

coated plates. Column chromatography was carried out with silica gel (100e200 mesh). The reactions were carried out in oven-dried glassware under dry N2. Methanol and THF were purified and dried before use. Distilled n-hexane and ethyl acetate were used for column chromatography. After quenching of the reaction with water, the work-up involves washing of combined organic layers with water, brine, drying over anhydrous sodium sulphate and evaporation of the solvent at reduced pressure. We have used AR grade methanol and tetrahydrofuran. For drying THF: AR grade THF was stirred for 12 h on LAH. Then it was refluxed over sodium wire and benzophenone (indicator) and distilled whenever needed in presence of nitrogen atmosphere. Methanol was refluxed over CaCO3 for 6 h, distilled out. Then refluxed with magnesium in the presence of catalytic amount of iodine and distilled out. The iminosugar was dissolved in double distilled water and filtered through the syringe filters (NY0.45 mm, make-ALLPURE).

4.1.2. (3R, 4r, 5S)-4-Amino-3,5-dihydroxy-piperidine (6a) An ice-cold solution of 10 (0.20 g, 0.83 mmol) in TFA-H2O (2 mL, 3:2) was stirred for 15 min at 0  C and at 25  C for 3 h. Trifluoroacetic acid (TFA) was co-evaporated with toluene at rotary evaporator using high vacuum to furnish hemiacetal as a thick liquid (crude wt¼0.150 g). A solution of hemiacetal (0.150 g, 0.75 mmol) and 10% Pd/C (0.030 g) in methanol (12 mL) was hydrogenolyzed at 80 psi for 12 h at 25  C. The catalyst was filtered through Celite. Evaporation of solvent and column purification (MeOH/30% NH4OH¼20/1) afforded 6a (0.086 g, 78%) as a pale yellow solid. Mp 146  C (decomposition); ½a26 D 0.0 (c 1.0, H2O); Rf 0.2 (MeOH/30% NH4OH): 10/1); IR (KBr): 3347e2989 (broad), 1110, cm1; 1H NMR (300 MHz, D2O): 3.35 (ddd, J¼10.9, 9.5, 4.5 Hz, 2H), 3.03 (dd, J¼10.9, 4.3 Hz, 2H), 2.55 (t, J¼9.5 Hz, 1H), 2.35 (dd, J¼11.9, 10.9 Hz, 2H); 13C NMR (75 MHz, D2O): 71.0, 60.4, 49.4; Anal. Calcd. For C5H12N2O2: C, 45.44; H, 9.15; Found: C, 45.40; H, 9.11.

4.1.1. 3,5-Diazido-3,5-dideoxy-1,2-O-isopropylidene-a-Dxylofuranose (10) To a stirred solution of compound 9 (2.00 g, 5.42 mmol) in DMF (20 mL), NaN3 (0.53 g, 8.13 mmol) was added. Reaction mixture was stirred at 90  C for 8 h. Reaction mixture was cooled, diluted with ethyl acetate (20 mL) and reaction mixture was extracted with water. Evaporation of solvent and column purification (n-hexane/ ethyl acetate¼8.0/2.0) afforded 10 (1.20 g, 93%) as pale yellow oil. 56 ½a26 ½aD 60 (c 1.0, CHCl3); Rf 0.4 (ethyl D 55 (c 1.0, CHCl3); [lit acetate/n-hexane: 4/6); IR (neat): 2989, 2105, 1083 cm1; 1H NMR (300 MHz, CDCl3): 5.91 (d, J¼3.8 Hz, 1H), 4.69 (d, J¼3.8 Hz, 1H), 4.34 (dt, J¼6.6, 3.4 Hz, 1H), 4.01 (d, J¼3.4 Hz, 1H), 3.62 (dd, J¼12.4, 6.6 Hz, 1H), 3.48(dd, J¼12.4, 6.6 Hz, 1H), 1.52 (s, 3H), 1.35 (s, 3H); 13C NMR (75 MHz, CDCl3):112.4, 104.6, 83.3, 77.5, 65.9, 49.5, 26.5, 26.11; Anal. Calcd. For C8H12N6O3: C, 40.00; H, 5.04; Found: C, 40.03; H, 5.08.

4.1.3. 3-Benzyloxycarbonylamino-3-deoxy-1,2-O-isopropylidene-aD-allofuranose (12) A solution of compound 11 (5.00 g, 12.72 mmol) in MeOH (50 mL) and 1% H2SO4 in water (2 mL) was stirred at room temperature for 12h. Reaction was quenched with saturated potassium carbonate. Methanol was evaporated and reaction mixture was extracted with chloroform (320 mL). Evaporation of solvent and column purification (n-hexane/ethyl acetate¼7.0/3.0) afforded 12 as thick liquid (3.92 g, 88%). ½a24 D þ29.3(c¼2.3, CH2Cl2); Rf 0.5 (ethyl acetate); IR (neat): 3440e3190, 1724, 1521, 1379, 875 cm1; 1H NMR (500 MHz, CDCl3þdrop of D2O) 7.31e7.51 (m, 5H), 5.84 (d, J¼3.8 Hz, 1H), 5.15 (ABq, J¼12.1 Hz, 2H), 4.65 (dd, J¼5.1, 4.0 Hz, 1H), 4.07e3.96 (m, 1H), 3.80 (dd, J¼9.5, 4.3 Hz, 3H), 3.66 (dd, J¼11.2, 5.9 Hz, 1H), 1.55 (s, 3H), 1.35 (s, 3H); 13C NMR (75 MHz, CDCl3): 26.4, 26.5, 55.2, 63.3, 67.6, 72.3, 79.3, 79.9, 103.8, 112.7, 128.3, 128.4 (str), 128.6 (str),

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135.8, 156.9; Anal. Calcd. For C17H23NO7: C, 57.78; H, 6.56; Found: C, 57.81; H, 5.59. 4.1.4. 3-Benzyloxycarbonylamino-3-deoxy-1,2-O-isopropylidene-aD-ribofuranose (13) To an ice-cooled solution of 12 (3.00 g, 8.4 mmol) in acetoneewater (30 mL, 5:1) was added sodium metaperiodate (2.73 g, 12.74 mmol) and the solution was stirred for 3h at 25  C. Solvent was evaporated on rotary evaporator and the residue was extracted with chloroform (10 mL3). Solvent was evaporated to get crude aldehyde (2.52 g). To an ice cooled solution of crude aldehyde (2.52 g, 7.8 mmol) in MeOH-water (25 mL, 4:1), sodium borohydride (0.44 g, 11.8 mmol) was added in portions. Reaction was stirred at room temperature for 3h. Reaction was quenched with saturated ammonium chloride. Methanol was evaporated and reaction mixture was extracted with chloroform (315 mL). Evaporation of solvent and column purification (ethyl acetate/n-hexane: 4/6) afforded 13 as 26 59 liquid (2.38 g, 87%). ½a26 D þ116 (c 1.0, MeOH); [lit ½aD þ109.4 (c 1.0, MeOH); Rf 0.4 (ethyl acetate/n-hexane; 5:5); IR (neat): 3456, 3279, 1712, 1529, 1186 cm1; 1H NMR (300 MHz, CDCl3) 7.28e7.48 (m, 5H), 5.85 (d, J¼3.8 Hz, 1H), 5.38 (bd, J¼8.6 Hz, 1H), 5.14 (s, 2H), 4.62 (t, J¼4.7 Hz, 1H), 4.09 (dt, J¼9.1, 5.3 Hz, 1H), 3.60e3.98 (m, 3H), 2.96 (s, exchangeable with D2O, 1H), 1.46 (s, 3H), 1.28 (s, 3H); 13C NMR (75 MHz, CDCl3): 26.3, 26.4, 52.9, 60.5, 67.5, 78.9, 80.2, 104.1, 112.6, 128.3, 128.4 (str), 128.6 (str), 135.9, 156.6 Anal. Calcd. For C16H21NO6: C, 59.43; H, 6.55; Found: C, 59.47; H, 6.58. 4.1.5. 3-Benzyloxycarbonylamino-3-deoxy-5-O-ptoluenesulphonyl-1,2-O-isopropylidenea-D-ribofuranose (14) To a stirred solution of compound 13 (2.0 g, 6.19 mmol) in dry dichloromethane (20 mL) and pyridine (2 mL), p-toluenesulphonyl chloride (1.53 g, 8.05 mmol) was added at 0  C. Reaction mixture was stirred for 8 h. Evaporation of solvent and column purification (n-hexane/ethyl acetate¼8/2) afforded tosyl derivative 14 as a thick colorless liquid (2.75 g, 93%); ½a28 D þ55.3 (c 0.57, CH2Cl2); Rf 0.5 (ethyl acetate/n-hexane:4/6); IR (neat): 3389, 1739, 1353, 1186 cm1; 1H NMR (300 MHz, CDCl3) 7.81 (d, J¼8.2 Hz, 2H), 7.32 (d, J¼8.2 Hz, 2H), 7.39 (s, 5H), 5.73 (d, J¼3.3 Hz, 1H), 5.22 (bd, J¼8.6 Hz, 1H), 5.10 (s, 2H), 4.55 (t, J¼3.8 Hz, 1H), 4.38 (bd, J¼11.0 Hz, 1H), 3.82e4.15 (m, 3H), 2.22 (s, 3H), 1.26 (s, 3H), 1.15 (s, 3H); 13C NMR (125 MHz, CDCl3): 21.6, 26.4, 26.5, 53.8, 67.4, 68.8, 77.7, 78.5, 104.2, 112.8, 128.1, 128.4, 128.5, 128.7, 129.8, 132.8, 136.0, 144.8, 155.7 Anal. Calcd. For C23H27NO8S: C, 57.85; H, 5.70; Found: C, 57.83; H, 5.68. 4.1.6. 5-Azido-5-deoxy-3-benzyloxycarbonylamino-3-deoxy-1,2-Oisopropylidene a-D-ribofuranose (15) To a stirred solution of compound 14 (2.50 g, 5.23 mmol) in DMF (25 mL), NaN3 (0.51 g, 7.84 mmol) was added. Reaction mixture was stirred at 90  C for 12 h. Reaction mixture was cooled, diluted with ethyl acetate (20 mL) and combined organic layer was extracted with water (310 mL). Evaporation of solvent and column purification (n-hexane/ethyl acetate¼8/2) afforded compound 15 as a thick liquid (1.72 g, 94%). ½a28 D þ74.5¼(c 0.55, CH2Cl2); Rf 0.4 (ethylacetate/n-hexane: 30/70); IR (neat): 3300e3190 (broad), 2987, 2102, 1728, 1518, 1379, 873 cm1; 1H NMR (300 MHz, CDCl3): 7.18 (s, 5H), 5.78 (d, J¼3.3 Hz, 1H), 5.42 (bd, J¼8.6 Hz, 1H), 5.10 (s, 2H), 4.52 (d, J¼3.5 Hz, 1H), 4.18e3.88 (m, 1H), 3.88e3.74 (m, 1H), 3.64e3.52 (m, 1H), 3.38e3.24 (m, 1H), 1.25 (s, 3H), 1.45 (s, 3H); 13C NMR (75 MHz, CDCl3): 26.3, 26.5, 51.15, 54.2, 67.3, 78.8, 78.9, 104.2, 112.6, 128.4 (str), 128.6 (str), 136.0, 155.9; Anal. Calcd. For C16H20N4O5: C, 55.17; H, 5.79; Found: C, 55.14; H, 5.77. 4.1.7. (3R, 4s, 5S)-4-Amino-3,5-dihydroxy-piperidine (7a) An ice-cold solution of 15 (0.30 g, 0.86 mmol) in TFA-H2O (3 mL, 3:2) was stirred for 15 min at 0  C and at 25  C for 3 h.

Trifluoroacetic acid was co-evaporated with toluene at rotary evaporator using high vacuum to furnish hemiacetal as a thick liquid (crude wt¼0.24 g). A solution of hemiacetal (0. 24 g, 0.78 mmol) and 10% Pd/C (0.040 g) in methanol (12 mL) was hydrogenolyzed at 80 psi for 24 h at 25  C. The catalyst was filtered through Celite. Evaporation of solvent and column purification (MeOH/CHCl3/30%NH4OH¼20/5/1) afforded 7a (0.077 g, 68%) as a off white solid. Mp 245  C (decomposition); ½a26 D 0.0 (c 1.1, H2O); Rf 0.2 (MeOH/30% NH4OH): 10/1); IR (KBr): 3447e2992 (broad), 1198, 1005 cm1; 1H NMR (300 MHz, D2O): 4.02e4.08 (m, 2H), 3.35 (t, J¼3.5, 1H), 3.17 (dd, J¼14.5, 3.5 Hz, 2H), 2.98 (dd, J¼14.5, 1.4, 2H); 13 C NMR (75 MHz, D2O): 65.3, 50.6, 48.1; Anal. Calcd. For C5H12N2O2: C, 45.44; H, 9.15; Found: C, 45.49; H, 9.20. 4.1.8. 3,6-Diazido-3,6-dideoxy-1,2-O-isopropylidene-a-Dglucofuranose (17) To a stirred solution of compound 16 (5.0 g, 12.53 mmol) in DMF (50 mL), NaN3 (1.22 g, 18.79 mmol) was added. Reaction mixture was stirred at 90  C for 12 h. Reaction mixture was cooled, diluted with ethyl acetate (30 mL) and combined organic layer was extracted with water (310 mL). Evaporation of solvent and column purification (n-hexane/ethyl acetate¼7/3) afforded compound 17 as a thick liquid (3.11 g, 92%). ½a24 D 35.6 (c 0.7, CH2Cl2): Rf 0.4 (ethyl acetate/n-hexane: 3/7); IR (neat): 3400, 2106, 1163 cm1; 1H NMR (300 MHz, CDCl3þdrop of D2O) 5.86 (d, J¼3.4 Hz, 1H), 4.64 (d, J¼3.4 Hz, 1H), 4.09e4.22 (m, 2H), 3.98 (m, 1H), 3.65 (dd, J¼12.6, 2.4 Hz, 1H), 3.5 (dd, J¼12.6, 6.2 Hz, 1H), 1.52 (s, 3H), 1.32 (s, 3H); 13C NMR (75 MHz, CDCl3): 26.2, 26.8, 54.8, 66.6, 68.6, 79.0, 83.2, 104.9, 112.4 Anal. Calcd. For C9H14N6O4: C, 40.00; H, 5.22; Found: C, 40.03; H, 5.24. 4.1.9. 5-O-Benzyl-3,6-diazido-3,6-dideoxy-1,2-O-isopropylidene-aD-glucofuranose (18) To an ice cooled suspension of NaH (0.33 g, 13.88 mmol) in dry THF (20 mL) compound 17 (2.50 g, 9.25 mmol) was added in THF (20 mL) under nitrogen atmosphere over 10 min. Benzyl bromide (1.6 ml, 13.88 mmol) was added dropwise to the reaction mixture. The reaction was stirred at room temperature for 12h. The reaction mixture was quenched by saturated aqueous ammonium chloride, THF was evaporated and reaction mixture was extracted with ethyl acetate (315 mL). Evaporation of solvent and column chromatography (ethyl acetate/n-hexane: 20/80) afforded benzyl ether 18 as a white solid (3.10 g, 93%). m. p.73e74  C; ½a28 D 48.9 (c 1.6, CH2Cl2); Rf 0.5 (ethyl acetate/n-hexane: 30/70); IR (KBr): 2985, 2100, 1452, 1377, 873 cm1; 1H NMR (300 MHz, CDCl3): 7.23e7.45 (m, 5H), 5.84 (d, J¼3.6, 1H), 4.77 (d, J¼10.9 Hz, 1H), 4.64 (d, J¼3.6 Hz, 1H), 4.58 (d, J¼10.9 Hz, 1H), 4.29 (dd, J¼9.1, 2.9 Hz, 1H), 4.09 (d, J¼2.9 Hz, 1H), 3.77e3.82 (m, 1H), 3.73 (dd, J¼13.2, 2.6 Hz, 1H), 3.41 (dd, J¼13.2, 4.1 Hz, 1H), 1.45 (s, 3H), 1.26 (s, 3H); 13C NMR (75 MHz, CDCl3): 26.8, 26.1, 50.7, 65.5, 72.0, 75.2, 77.4, 82.7, 104.1, 112.0, 127.6 (str), 128.0 (str), 136.8; Anal. Calcd. For C16H20N6O4: C, 53.33; H, 5.59; Found: C, 53.38; H, 5.64. 4.1.10. (3R, 4S, 5R)-5-Amino-3,4-dihydroxy-piperidine (8) An ice-cold solution of 18 (0.30 g, 0.86 mmol) in TFA-H2O (3 mL, 3:2) was stirred for 15 min at 0  C and at 25  C for 3 h. Trifluoroacetic acid was co-evaporated with toluene at rotary evaporator using high vacuum to furnish hemiacetal as a thick liquid (crude wt¼0.24 g). To an ice-cooled solution of hemiacetal (0.24 g, 0.75 mmol) in acetoneewater (5 mL, 5:1) was added sodium metaperiodate (0.24 g, 1.12 mmol) and the solution was stirred for 1 h at 25  C. Solvent was evaporated on rotary evaporator and the residue was extracted with chloroform (10 mL3). Usual workup afforded a thick liquid (0.20 g, 95%). Asolution of aldehyde (0.20 g, 0.68 mmol) and 10% Pd/C (0.040 g) in methanol

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(12 mL) was hydrogenolyzed at 80 psi for 24 h at 25  C. The catalyst was filtered through Celite. Evaporation of solvent and column purification (MeOH/30%NH4OH¼20/1) afforded 8 (0.057 g, 52%) as a pale yellow syrup. ½a28 D 9.2 (c 1.3, H2O); Rf 0.2 (MeOH/ CHCl3/30% NH4OH) 10/2/1); IR (neat): 3347, 2989, 1110, 1005 cm1; 1H NMR (300 MHz, D2O): 3.87e3.97 (m, 1H), 3.38 (dd, J¼9.5, 2.8 Hz, 1H), 2.83e3.08 (m, 3H), 2.68 (dd, J¼14.3, 1.4 Hz, 1H), 2.23 (dd, J¼12.9, 10.0 Hz, 1H); 13C NMR (75 MHz, D2O): 74.9, 68.2, 49.2, 48.9, 48.9; Anal. Calcd. For C5H12N2O2: C, 45.44; H, 9.15; Found: C, 45.49; H, 9.21. 4.2. Glycosidase inhibition assay The substrates p-nitrophenyl-a-D-glucopyranoside, p-nitrophenyl-b-D-glucopyranoside, p-nitrophenyl-a-D-galactopyranoside, p-nitrophenyl-b-D-galactopyranoside, p-nitrophenyl-a-Dmannopyranoside, p-nitrophenyl-a-D-fucopyranoside and p-nitrophenyl N-acetyl-b-D-glucosaminide were procured from sigma chemicals. The inhibition assay with the test compounds was performed by measuring the residual hydrolytic activities of the glycosidases with 2 mM concentration of p-nitrophenylglycopyranoside prepared in citrate buffer (0.025 M, pH 4.0) and used for assay. The test compound was pre-incubated with the enzyme, buffered at its optimal pH, for 1 h at 37  C (for Geobacillus a-amylase at 60  C). The enzyme reaction was initiated by the addition of 100 mL of substrate. Reaction was terminated after 90 min by adding 1.1.mL of Borate buffer (pH 9.8, 0.05 M) and absorbance of the liberated p-nitrophenol was measured at 405 nm with a UV-visible Spectrophotometer. Controls were run simultaneously in the absence of test compound. One unit of glycosidase activity is defined as the amount of enzyme that hydrolyzed 1 mmol of p-nitrophenol per minute under assay condition. 4.3. Molecular docking studies Three dimensional structure of Bovine liver alpha-galactosidase was predicted using Swiss-model server (http://swissmodel. expasy.org/), crystal structure of human b-galactosidase (PDB ID: 3THC) was used as template. Predicted model was energy minimized using CHARRM force field in Chimera 1.8 software.66 AutoDock 4.2 was used for docking simulation, which employs the preparation of receptor by adding hydrogens and assigning Kollman charges, followed by conversion of .pdb file to.pdbqt.67 Molecular structure of ligands was constructed using ChemAxon MarvinSketch software (http://www.chemaxon.com/products/ marvin/marvinsketch/) and geometrically optimized by quantum chemical semi-empirical RM1 method. Ligands were assigned with Gasteiger charges and polar hydrogen. Docking simulations were run using Lamarckian Genetic algorithm (LGA), which is known to be the most efficient and reliable method of Auto Dock.68 The grid points for Autogrid calculations were set to be around active site of alpha-galactosidase (GLU187 and GLU267) with dimension of 202020 Å. The docking parameters were set to a LGA calculation of 10,000 runs. The energy evaluations were set to 1,500,000 and 27,000 generations. Population size was set to 150 and the rate of gene mutation and the rate of gene crossover were set to 0.02 and 0.8, respectively. The obtained conformations were then summarized, collected and extracted by using Autodock Tool. Structure of protein-ligand docked complex was visualised and analyzed using PyMol visualization tool (The PyMol Molecular Graphics System, Version 1.2r3 pre, Schrodinger LLC). Accelerys Discovery studio visualizer 3.1 (downloaded from www.accelerys. com) was used for analysis of hydrogen bonding and other intermolecular interactions from galactosidase-5-amino iminosugar complex.

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Acknowledgements We are grateful to Prof. M. S. Wadia for helpful discussions. V. M. K. and N. B. K. are thankful to the CSIR, New Delhi for the senior research fellowship. We are thankful to the Department of Science and Technology, New Delhi (Project File No. SR/S1/OC-20/2010) for the financial support. Supplementary data Supplementary data (copies of 1H and 13C NMR spectra of compounds 10, 12, 13, 14, 15, 17, 18, 6a, 7a, 8) associated with this article can be found at http://dx.doi.org/10.1016/j.carres.2015.03. 004. References 1. Hughes AB, Rudge A. J Nat Prod Reports 1994:135e62. and references cited there in. 2. Sears P, Wong CH. Angew Chem Int Ed 1999;38:2300e24. 3. Heightman TD, Vasella AT. Angew Chem Int Ed 1999;38:750e70. 4. Stutz AE. Iminosugars as glycosidase inhibitors, Nojirimycin and beyond. Weinheim: Wiley-VCH; 1999. 5. Compain P, Martin OR. Iminosugars: from synthesis to therapeutic applications. New York: Wiley; 2007. 6. Jespersen TM, Dong W, Skrydstrup T, Sierks MR, Lundt I, Bols M. Angew Chem Int Ed Engl 1994;33:1778e9. 7. Compain P, Martin OR. Bioorg Med Chem 2001;9:3077e92. 8. Winchester B, Fleet GWJ. Glycobiology 1992;2:199e210. 9. Look GC, Fotsch CH, Wong CH. Acc Chem Res 1993;26:182e90. 10. Nishimura Y. In: tta-ur-Rahman A, editor. Studies in natural products chemistry, Vol. 10. Amsterdam: Elsevier; 1992. p. 495e583. 11. Raadt DA, Eckhart CW, Ebner M, Stütz AE. Top Curr Chem 1997;187:157e86. 12. Martin OR, Compain P. Curr Top Med Chem 2003;3(5). Bentham: Hilversum, The Netherlands. 13. Nash RJ, Watson AA, Asano N. In: Pelletier SW, editor. Alkaloids Chemical and Biological Perspectives, Vol. 10. Oxford: Elsevier Science Ltd; 1996. p. 345. 14. Horne G, Wilson FX, Tinsley J, Williams DH, Storer R. Drug Discov Today 2011;16:107e18. 15. Asano N, Kato A, Miyauchi M, Kizu H, Kameda Y, Watson AA, et al. J Nat Prod 1998;61:625e8. 16. Karpas A, Fleet GWJ, Dwek RA, Petursson S, Namgoong SK, Ramsden NG, et al. Proc Natl Acad Sci U S A 1988;85:9229e33. 17. Merror YL, Poitout L, Deepazy J, Dosbaa I, Geoffroy S, Foglietti M. Bioorg Med Chem 1997;5:519e33. 18. Bols M, Lillelund VH, Jensen HH, Liang X. Chem Rev 2002;102:515e53. and references cited there in. 19. Goujon JY, Gueyrard D, Philippe C, Olivier MR, Asano N. Tetrahedron: Asymm 2003;14:1969e72. 20. Jensen HH, Bols M. Acc Chem Res 2006;39:259e65. 21. Wicki J, Williams SJ, Withers SG. J Am Chem Soc 2007;129:4530e1. 22. Gloster TM, Meloncelli P, Stick RV, Zechel D, Vasella A, Davies GJ. J Am Chem Soc 2007;129:2345e54. 23. Carmona AT, Popowycz F, Gerber LS, Rodrıguez GE, Schutz C, Vogel P, et al. Bioorg Med Chem 2003;11:4897e911. 24. Kilond A, Compernolle F, Peeters K, Joly GJ, Toppet S, Hoornaert GJ. Tetrahedron 2000;56:1005e12. 25. Pandey G, Dumbre SG, Khan MI, Shabab M. J Org Chem 2006;71:8481e8. 26. Bulow A, Plesner IW, Bols M. J Am Chem Soc 2000;122:8567e8. 27. Li H, Bleriot Y, Mallet JM, Rodriguez GE, Vogel P, Zhang Y, et al. Tetrahedron: Asymm 2005;16:313e9. 28. Curtis KL, Fawcett J, Handa S. Tetrahedron Lett 2005;46:5297e300. 29. Davies SG, Figuccia ALA, Fletcher AM, Roberts PM, Thomson JE. Tetrahedron 2014;70:3601e7. 30. Kim DK, Kim G, Kim YW. J Chem Soc Perkin Trans 1996;1:803e8. 31. Soengas RG, Silva AMS. Tetrahedron Lett 2013;54:2156e9. 32. Wang RW, Qiu XL, Bols M, Caballero FO, Qing FL. J Med Chem 2006;49: 2989e97. 33. Li R, Bols M, Rousseau C, Zhang X, Wang R, Qing F. Tetrahedron 2009;65: 3717e27. 34. Li YX, Huang MH, Yamashita M, Kato YA, Jia YM, Wang WB, et al. Org Biomol Chem 2011;9:3405e14. 35. Pandey G, Kapur M, Khan MI, Gaikwad SM. Org Biomol Chem 2003;1:3321e6. 36. Natori Y, Imahori T, Murakami K, Yoshimura Y, Nakagawa S, Kato A, et al. Bioorg Med Chem Lett 2011;21:738e41. €nemann W, Gallienne E, Compain P, Ikeda K, Asano N, Martin OR. Bioorg 37. Scho Med Chem 2010;18. 2645e2645. 38. Markad SD, Karanjule NS, Sharma T, Sabharwal SG, Dhavale DD. Org Biomol Chem 2006;4:3675e80. 39. Ouchi H, Mihara Y, Takahata H. J Org Chem 2005;70:5207e14.

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