Quaternary Indolizidine and Indolizidone Iminosugars as Potential

Article pubs.acs.org/jmc

Quaternary Indolizidine and Indolizidone Iminosugars as Potential Immunostimulating and Glycosidase Inhibitory Agents: Synthesis, Conformational Analysis, Biological Activity, and Molecular Docking Study Nitin J. Pawar,† Vijay Singh Parihar,† Ayesha Khan,‡ Rakesh Joshi,§ and Dilip D. Dhavale*,† †

Department of Chemistry, Garware Research Centre, Savitribai Phule Pune University (formerly University of Pune), Pune 411 007, India ‡ Department of Chemistry, Savitribai Phule Pune University, Pune 411 007, India § Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune 411 007, India S Supporting Information *

ABSTRACT: New quaternary indolizidine iminosugars, with hydroxymethyl group at the ring junction, namely, C-8ahydroxymethyl-1-deoxycastanospermine congeners 1a, 2a, 3a and their 3-oxo analogs 1b, 2b, and 3b were synthesized by using intramolecular reductive aminocyclization/lactamization of D-mannose/D-glucose derived C5-γ-azido esters as a key step wherein both the rings of the indolizidine skeleton were built up in one pot following the cascade reaction pathway. The conformations (5C8 or 8C5) of 1−3 were assigned on the basis of the 1H NMR studies. All compounds were found to be potent inhibitors of various glycosidase enzymes with Ki and IC50 values in the micromolar/nanomolar concentration range and further substantiated by molecular docking studies. The effect of synthesized iminosugars 1−3 on the cytokine secretion of IL-4, IL-6, and IFN-γ was evaluated. All compounds were found to be TH1 bias increasing the TH1/TH2 cytokines ratio (IL-6 and IL-4) indicating their potency as immunostimulating agents. Our study suggests that immunomodulatory activity of indolizidine iminosugars can be tuned by minor structural/stereochemical alterations.

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INTRODUCTION Immunostimulators have aroused enormous interest due to their potential value to enhance the ability of the human immune system. This property renders them to act as adjuvant therapeutic agents for the treatment of infectious diseases, viral infections, and tumor.1 In the literature, macromolecules such as polynucleotides,2 glycoproteins,3 glycolipids,4 and polysaccharides5 as well as small molecules including nucleosides6 and heterocycles7 are known to have immunomodulatory properties. A few heterocyclic compounds used in the clinic as immunomodulating drugs are β-glucan,5 interferon,3 pidotimod,8 and levamisole.9 Among these, iminosugars in which the ring oxygen atom is replaced by the nitrogen atom are found to be potent inhibitors of many carbohydrate-processing enzymes10 as well as immunomodulators.11 In this direction, a variety of monocyclic (azitidine, pyrrolidine, piperdine, and azepane) as well as bicyclic (pyrrolizidine, indolizidine, quinolizidine) iminosugars were either isolated or synthesized and evaluated for their glycosidase inhibitory10 and immunomodulatory11 activities. The usefulness of iminosugars for the treatment of diabetes type II and lysosomal storage disorder is evident from the introduction of miglitol Ia (N-hydroxyethyl-1-deoxynojirimycin) and Zaveska Ib (N-butyl-1-deoxynojirimycin), respectively, as drug candidates in the market (Figure 1).10c Now, it is of © XXXX American Chemical Society

great interest and challenge to explore iminosugars as more effective and less toxic immunostimulants for the treatment of immunological disorders. Applications of iminosugars as immunomodulators have received limited attention, and literature reports indicate that the piperidine, piperidone, pyrrolidine, and indolizidine iminosugars such as castanospermine,12 swainsonine,13 and kifunensine14 are known to have immunosuppressive activity.15 Our recent finding suggested that immunomodulating activity of polyhydroxylated indolizidine iminosugars/castanospermine analogues can be tuned by minor structural/stereochemical alteration.16 For example, castanospermine IIa is known to be an immunosuppressive agent, while replacement of C-1 hydroxyl functionality in IIa with hydroxymethyl substituent having (1S) IIb and (1R) IIc configuration showed significant immunostimulating properties by up-regulation of TH1/TH2 cytokine ratio (IL-6 and IL-4) with their excellent cell proliferating activity without showing any inhibition toward the glycosidase enzymes.16 In the continuation of our interest in this area,17 we now report synthesis of hitherto unknown quaternary indolizidines 1a, 2a, 3a and indolizidone iminosugars 1b, 1c, Received: June 20, 2015

A

DOI: 10.1021/acs.jmedchem.5b00951 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Scheme 1. Synthesis of 1a, 1b, 1c, 2a, 2b, 3a, and 3c

to get target molecules in short steps. Our results are described herein.

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RESULTS AND DISCUSSION Required benzyl-2,3-O-isopropylidene-5-deoxy-5-azido-5-Cbenzyloxymethyl-α-L-gulo-furano-1,6-dialdose 4 was prepared from the D-mannose as reported earlier (Scheme 2).24 The

Figure 1. Iminosugar analogues.

Scheme 2. Synthesis of Indolizidine/Indolizidone Iminosugars 1a, 1b, 1ca

2b, and 3b (Figure 1), wherein hydroxymethyl group is present at the ring junction C8a. These castanospermine analogues showed good immunopotentiating activity as compared to the parent castanospermine IIa as well as moderate to potent glycosidase inhibitory activity against various enzymes under study. In the search for structure−activity relationship (SAR), a variety of hydroxymethyl substituted indolizidine iminosugars/ castanospermine analogues (general structure III) were synthesized and evaluated for biological activities.18 For example, Liu19 and Johnson et al.20 synthesized C-5 hydroxymethyl 1deoxycastanospermine derivative which showed potent αglucosidase inhibitory activity. Pandey et al. reported C-8 hydroxymethyl substituted 1-deoxycastanospermine which showed moderate inhibitory activity against β-glucosidase,21 while only two reports in which hydroxymethyl group is at the ring junction IVa,b are known so far, from Langlois22 and Santos23 groups, which were found to be moderate α-/βgalactosidase inhibitors. In the synthesis of target molecules, the major challenge was the installation of a quaternary carbon center bearing the nitrogen atom and a chemical equivalent to the hydroxymethyl group. To achieve this, Langlois et al. used methodology that involves nucleophilic addition to N-acyliminium ions followed by ring-closure metathesis.22 Santos et al. used 2,3-sigmatropic rearrangement of (S)-allylproline derivative to construct quaternary stereocenter bearing the nitrogen atom.23 Recently, we have developed a methodology for the construction of a quaternary carbon, bearing the nitrogen atom and formyl group (chemical equivalent to −CH2OH), using the Jocic−Reeve and Corey−Link type reaction with C-5 keto hexoses that leads to the formation of sugar α-azidoaldehyde (A) which gives an access for the synthesis of α-geminal dihydroxymethyl piperdine and pyrrolidine iminosugars.24 In the present work, we thought of utilizing the same sugar derived α-azidoaldehyde intermediate (A) for the synthesis of target molecules in which the hydroxymethyl functionality at C-5 of hexoses is retained at the ring junction. Thus, as shown in Scheme 1, the C5-formyl group in A is converted to γ-azido α,β unsaturated ester B by the Wittig olefination, which could be exploited to construct six−five membered fused indolizidine/indolizidone skeleton, in one pot, using intramolecular reductive aminocyclization/lactamization

Reagents and conditions: (a) PPh3CHCOOEt, DCM, 50 °C, 95%; (b) H2, 10%Pd/C, 100 psi, methanol; (c) conc HCl, MeOH, 0 °C to rt, 98%; (d) LiAlH4, THF, reflux, 20 h, 10%; (e) DIBAL-H, toluene, − 78 °C, 68%; (f) Dowex, basic resin, MeOH, 98%. a

Wittig olefination of 4 with PPh3CHCOOEt gave γ-azido-α,β unsaturated ester 5 as a single E-diastereomer in 95% yield. In the next step, hydrogenation of 5 using H2, 10% Pd/C in methanol at 100 psi directly afforded tricyclic indolizidone 6a (R = H) and 6b (R = Bn) in 70% and 21% yield, respectively. This one-pot fivesteps cascade process involves probably reduction of azide to amine, reduction of the double bound, hydrogenolysis of C6- and C1-O-benzyl groups (to get anomeric mixture of hemiacetals) followed by an intramolecular reductive aminocyclization of C5amino functionality with C1 aldehyde (to get six-membered ring) and concomitant lactamization with C-8 ester to give 6. Finally, treatment of 6a and 6b with 10% HCl solution in CH 3 OH gave 8a(R)-hydroxymet hyl-6-epi-1-deoxycastanospermine-3-one 1b and 8a-benzyloxymethyl-1-deoxy6,8a-di-epi-castanospermine-3-one 1c, respectively.25 B



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Targeting toward indolizidine iminosugar 1a, the γ-lactam ring in indolizidone 1b was reduced using LiAlH4 in THF17a that afforded 8a(R)-hydroxymethyl-6-epi-1-deoxycastanospermine 1a only in 10% yield.26 The low yield of 1a prompted us to find an alternative method for the synthesis in good yield. Thus, controlled reduction of γ-azido-α,β unsaturated ester 5 using DIBAL-H (1.05 equiv) in toluene at −78 °C gave γazidoaldehyde 7 in 68% yield. In the next step, double reductive aminocyclization of 7 using H2, 10% Pd/C in methanol afforded tricyclic indolizidine skeleton 8 in 91% yield as a gummy solid. This one-pot, four-steps cascade follows the same pathway as discussed for the conversion of 5 to 6 except the last step, in which intramolecular double reductive aminocyclization of C5amino functionality with C1- and C-8 aldehyde groups directly gave indolizidine skeleton 8 as a single product. Treatment of 8 with 10% HCl solution in CH3OH gave hydrochloride salt 9 as a gummy liquid that was stirred with the Dowex resin (basic) in methanol to give 8a(R)-hydroxymethyl-6-epi-1-deoxycastanospermine 1a. The synthesis of indolizidone iminosugar 2b was started with 5(S)-5-hydroxymethyl-α-D-gluco-hexofuranodialdose 10, which was prepared from D-glucose as a single diastereomer as per the literature procedure in 81% yield (Scheme 3).27 The Wittig

Scheme 4. Synthesis of Indolizidine/Indolizidone Iminosugars 3a and 3ba

a

Reagents and conditions: (a) NaBH4, MeOH, 90%; (b) MOMCl, DIPEA, DMAP, DCM, 92%; (c) n-Bu4NF, THF, 88%; (d) Dess− Martin periodinane, DCM, 85%; (e) PPh3CHCOOEt, DCM, 50 °C, 96%; (f) (i) TFA/H2O; (ii) H2, 10% Pd/C, 100 psi, methanol; (g) DIBAL-H, toluene, −78 °C, 85%.

reaction with MOM-Cl gave 14. The selective deprotection of 6OTBS group in 14 with TBAF afforded azido alcohol 15 that on the Dess−Martin periodinane oxidation gave 16 with “5R” absolute configuration. In the further steps, the same protocol as discussed for the conversion of 11 to 2a and 11 to 2b was followed to get target molecules. Thus, Wittig olefination of 16 gave γ-azido-α,β unsaturated ester 17. Reaction of 17 with TFA−water (to get hemiacetal) followed by hydrogenation using H2, 10% Pd/C in methanol at 100 psi afforded 8a(R)-hydroxymethyl-1-deoxycastanospermine-3-one 3b in 91% yield. Alternatively, partial reduction of ester group in 17 with DIBAL-H gave 18 as a viscous liquid in 85% yield that on reaction with TFA/H2O at 0 °C to rt for 2 h followed by double reductive aminocyclization using H2, 10% Pd/C in methanol at 100 psi afforded 8a(R)-hydroxymethyl-1deoxycastanospermine 3a as a gummy solid in 87% yield. Conformational Assignments of Indolizidine/Indolizidone Iminosugars 1a, 1b, 1c, 2a, 2b, 3a, and 3b. In general, indolizidine iminosugars exist in 8C5 or 5C8 conformation depending on orientation of substituents in a ring. A change in conformation has profound effect on their binding properties with glycosidase enzyme affecting their inhibitory potential.17c,21 The castanospermine IIa is found to be in the 8C5 conformation; however, the newly synthesized castanospermine analogues 1−3 have one hydroxymethyl substituent at the ring junction. Therefore, it is interesting to find their conformations. Thus, we considered two conformations 8C5 and 5C8 for each of the castanospermine analogues 1−3. While deciding the conformations, coupling constant values between the H-5, H-6, H-7, and H-8 protons are decisive. For example, as shown in Figure 2 in the 5C8 conformation for 1a, the H-5 axial proton is expected to show doublet of doublet with large geminal coupling constant value (∼15 Hz) with the H-5 equatorial and vicinal coupling constant value (∼9 Hz) with the H-6 axial, while in 8C5 conformation for 1a the H-5a is expected to show doublet of doublet with large geminal coupling constant value (∼15 Hz) with H-5e and small vicinal coupling constant (∼0−3 Hz) with the H-6e proton. Such type of axial−axial, axial−equatorial, and equatorial− equatorial coupling constants information with other protons at the H-6, H-7, and H-8 were used to decide the conformations. The coupling constant values obtained from the 1 H NMR spectra for compound 1−3 are listed in Table 1. Thus,

Scheme 3. Synthesis of Indolizidine/Indolizidone Iminosugars 2a and 2ba

Reagents and conditions: (a) PPh3CHCOOEt, DCM, 50 °C, 96%; (b) (i) TFA:H2O; (ii) H2, 10% Pd/C, 100 psi, methanol; (c) DIBALH, toluene, −78 °C, 66%. a

olefination of 10 with Ph3PCHCOOEt gave γ-azido-α,β unsaturated ester 11 (as a single E-diastereomer) in 96% yield. Reaction of 11 with TFA−water (to hydrolyze 1,2-acetonide) followed by hydrogenation using H2, 10% Pd/C in methanol at 100 psi directly afforded indolizidone iminosugar 2b in 94% yield via concomitant reductive aminocyclization and lactamization. The synthesis of indolizidine iminosugar 2a was achieved following same protocol, as described (vide infra) for 1a (Scheme 3). Thus, partial reduction of ester group with DIBAL-H gave the γ-azido-α,β unsaturated aldehyde 12. Treatment of γ-azido-α,β unsaturated aldehyde 12 with TFA/ H2O (as in the case of 2b) and subsequent hydrogenation using H2, 10% Pd/C in methanol at 100 psi afforded 8a(S)hydroxymethyl-1-deoxycastanospermine 2a a sticky solid in 90% yield. With the success in synthesis of 8a(S)-hydroxymethyl-1deoxycastanospermine 2a from D-glucose derived C5-azidoaldehyde 10, we thought to synthesize 3a (C8a epimer of 2a). For this it is necessary to invert the stereochemistry at C5 of azidoaldehyde 10 and follow the same reaction sequence as above. To achieve the inversion of configuration at C5 in compound 10, the hydroxymethyl group and formyl group at C5 needs be interchanged, and there exists a distinct possibility to do so by judicious manipulation of oxidation/reduction protocol on compound 10. Thus, as shown in Scheme 4, treatment of azidoaldehyde 10 with NaBH4 afforded azido alcohol 13 that on C



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Figure 2. Conformations of 1a, 1b, 1c, 2a, 2b, 3a, and 3b using 1H NMR.

Table 1. Coupling Values of 1a, 1b, 1c, 2a, 2b, 3a, and 3b compd

H5a

H5e

H6

H7

1a

2.80 (bd) J5a,5e = 13.0 Hz

2.92 (dd) J5e,6e = 4.0 Hz

3.96−4.03 (m)

2a

2.75 (dd) J5a,5e = 13.8 J5a,6a = 11.0 Hz

2.98−3.12 (m, 3H)

3a

3.08 (t) J5a,5e = J5a,6a = 11.5 Hz 2.84−2.98 (m)

3.64−3.75 (m, 3H)

3.78 (ddd) J6a,5a = 11.0 Hz J6a,7a = 9.9 Hz J6a,5e = 5.1 Hz 3.64−3.75 (m, 3H)

3.74−3.90 (m, 2H)

3.74−3.90 (m, 2H)

2.49 (t) J5a,5e = J5a,6a = 10.5 Hz 2.75 (dd) J5a,5e = 13.2 Hz J5a,6a = 12.3 Hz 3.29 (bd) J5a,5e = 13.5 Hz

3.50−3.70 (m, 3H)

3.50−3.70 (m, 3H)

4.07 (dd) J5e,6a = 5.7 Hz

3.42−3.54 (m, 2H)

3.63 (t) J7a,6a = J7a,8a = 9.6 Hz

2.92 (dd) J5e,6e = 4.0 Hz

3.93 (bt) J6e,5e = J6e,7e = 4.0 Hz

3.61 (bd) J7e,6e = 4.0 Hz

1b 1c 2b

3b

H8

3.72 (dd) J7a,8a = 9.0 Hz J7a,6e = 3.0 Hz 3.58 (t) J7a,6a = J7a,8a = 9.9 Hz

4.05 (d) J8a,7a = 9.0 Hz

3.64−3.75 (m, 3H)

3.84 (d) J7a,8a = 9.0 Hz 3.99 (bs) (θ ≈ 90°) 3.82 (bs) (θ ≈ 90°) 3.42−3.54 (m, 2H)

3.71 (bd) J7e,6a = 3.0 Hz 3.50−3.70 (m, 3H)

3.46 (d) J7a,8a = 9.9 Hz

3.90 (bs) (θ ≈ 90°)

Table 2. IC50 (μM) and Ki (μM) Values of 1a, 1b, 1c, 2a, 2b, 3a, 3b, and Standard Miglitola compd 1a 2a 3a 1b 1c 2b 3b miglitol a

IC50 (μM) Ki (μM) IC50 (μM) Ki (μM) IC50 (μM) Ki (μM) IC50 (μM) Ki (μM) IC50 (μM) Ki (μM) IC50 (μM) Ki (μM) IC50 (μM) Ki (μM) IC50 (μM) Ki (μM)

α-glucosidase (rice)

β-glucosidase (bovine liver)

α-galactosidase (Aspergillus niger)

β-galactosidase (bovine liver)

α-mannosidase (Jack bean)

NI NI NI NI NI NI NI NI NI NI NI NI NI NI 0.1 0.06

156 86 116 95 202 102 46 24 342 113 22 11 39 19 342 32

0.63 0.44 0.13 0.09 0.24 0.54 630 68 NI NI 0.42 0.33 130 14 NI NI

1.2 0.5 3.6 1.5 2.3 1 7.2 3.1 NI NI 0.8 0.2 5.6 2.4 200 100

NI NI NI NI NI NI 1.3 0.9 NI NI 0.056 0.035 0.075 0.043 NI NI

NI; no 50% inhibition at 1 mM concentration of inhibitor. Data are the average of five sets of assays performed.

in 1a the H5a appeared as a broad doublet with geminal coupling constant value of 13.0 Hz. The broad doublet indicates small (∼1 Hz) vicinal coupling constant value for the H-5a which requires equatorial orientation of the H-6 vicinal proton. This suggests that compound 1a exists in 8C5 conformation (Figure 2),

whereas in 2a the H-5a (axial proton) appeared as a doublet of doublet with J = 13.8 and 11.0 Hz. The large vicinal coupling constant value of 11.0 Hz for H-5a requires axial orientation of the H6, suggesting the 8C5 conformation for 2a. Similarly, in 3a the H-5a appeared as a triplet with J = 11.5 Hz. The large vicinal D



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compounds as bicyclic analogues of 1-deoxy nojirimycin which is known to be potent glycosidase inhibitor.28 Molecular Docking Studies. Three dimensional (3D) model of rice α-glucosidase, bovine liver β-glucosidase, Aspergillus niger α-galactosidase, bovine β-galactosidase, and Jack bean α-mannosidase were built by comparative modeling using MODELLER 9, V4.29 Crystallographic structures of homologue protein were considered as a template to predict the structures of enzymes which were further assessed by the Ramchandran plot analysis. Structures of enzymes and synthesized molecules were energy minimized using GROMOS 43B1 force field. Receptor and ligand molecules were prepared for docking simulation by adding hydrogens and assigning Kollman charges. Likewise, the ligands were established by assigning the Gasteiger charges and nonpolar hydrogens. Structures of receptor and ligand are converted from PDB to PDBQT format using AutoDock 4.2.30 Grid was set around the active site of enzymes with dimensions of 20 Å × 20 Å × 20 Å using Autogrid module of Autodock program. Binding score was calculated by the Lamarckian genetic algorithm (LGA), which uses a set of 30 structurally known protein ligand complexes with experimentally determined binding constants to calibrate empirical free energy functions. Parameters were set to the LGA calculation of 10 000 runs, whereas energy evaluations were set to 1 500 000 and 27 000 generations (repetition of process). The obtained docked poses and intermolecular interactions were gathered and analyzed using PyMol visualize (The PyMol Molecular Graphics System, version 1.2r3 pre, Schrodinger LLC). Binding scores for all synthesized iminosugars 1−3 are illustrated in Table 3, and binding poses on the active site of selected enzymes are shown in Figure 3. The binding free energies are corroborated with in vitro inhibition kinetic studies. Minute deviation from this correlation might be due to the variation in ligand conformation, in vitro assay condition, solvation of ligand, and ligand charge state. Compounds 2b and 3b were found to be potent inhibitors (lower binding energy) of α-mannosidase (Jack bean) compared to other molecules. Sugar-hydrolysis inhibitory activity of compounds 1a, 2a, and 3a showed its efficiency against Aspergillus niger αgalactosidase and bovine liver β-galactosidase activity. Furthermore, compounds 1b and 3b have broad selectivity toward bovine liver β-glucosidase, bovine liver β-galactosidase, and Jack bean α-mannosidase. Corroboration of binding free energies with the inhibition kinetic studies indicated that among iminosugars 1−3 compounds 2b and 3b are better competitive inhibitors of Jack bean α-mannosidase. Analysis of polar contacts and other weak interactions (electrostatic and π-interaction) of synthesized derivatives showed that the higher binding affinity is presumably attributed by the formation of higher number of stable intermolecular hydrogen bonds between the reactive group of compounds and several amino acids at the binding site of the enzyme (Figure 3). Immunomodulatory Activity. In the cell mediated immunity, the T cells play a central role for which concavalin A (Con A) is specific activator. The T cells contain two classless lymphocytes, T helper (TH) cells and T cytotoxicity (TC) cells. The TH cells are divided into TH1 and TH2, and their ratio affects several human autoimmunity diseases.6a,31 TH1 responses predominate in acute allograft rejection, organ-specific autoimmune disorders and in some chronic inflammatory disorders. In contrast, TH2 responses predominate in transplantation tolerance, chronic graft-versus-host disease, Omann’s syndrome, systemic sclerosis, and allergic diseases. The TH1 cells produce

coupling constant value of 11.5 Hz for H-5a requires axial orientation of H-6, indicating the 8C5 conformation for 3a. However, in the case of indolizidone iminosugar 1b, the H-5 and H-6 protons appeared as a multiplet and the H-7 appeared as a broad doublet with small vicinal coupling constant value of 3.0 Hz and the H-8 appeared as a broad singlet. This indicates that J7,8 is small (∼1 Hz) and the H-7 has vicinal coupling constant value of 3.0 Hz with the H-6. As the H-6 is axially oriented, the small coupling constant of 3.0 Hz could be explained only if the H-7 is equatorial. This indicates the 5C8 coformation to 1b (in 8 C5 conformation the H-7 is expected to show large diaxial coupling constant). Similarly, in 1c the H-5a appeared as a triplet with J = 10.5 Hz. The large vicinal coupling constant value of 10.5 Hz for H-5a requires axial orientation of the H-6, suggesting the 5 C8 conformation for 1c. In 2b, the H-5a appeared as a doublet of doublet with coupling constant values of 13.2 and 12.3 Hz. The large vicinal coupling constant value for the H-5a requires axial orientation of the H-6. This suggests that compound 2b exists in 8 C5 conformation, whereas in 3b the H-5a appeared as a broad doublet with J = 13.5 Hz. The small coupling constant (less than ∼1 Hz) for the H-6 requires its equatorial orientation, indicating the 5C8 conformation for 3b. In the case of indolizidone iminosugars 1b, 1c, and 3b, the H-8e appeared as a broad singlet with small (∼1) vicinal coupling constant with the H-7e, indicating the dihedral angle between H-8e and H-7e close to 90°. This indicates that these compounds exist in little distorted 5 C8 conformation due to presence of sp2 hybridized C-3 carbonyl functionality. Biological Activity of Indolizidine/Indolizidone Iminosugars. Glycosidase Inhibitory Activity. Glycosidase inhibitory activity of compounds 1−3 was studied against αglucosidase (rice), β-glucosidase (bovine liver), α-galactosidase (Aspergillus niger), β-galactosidase (bovine liver), and αmannosidase (Jack bean) enzymes, with reference to the known standard N-hydroxyethyl-1-deoxynojirimycin (trade name miglitol). The IC50 and Ki (determined from Lineweaver−Burk plots) values of all iminosugar derivatives against selected enzymes are summarized in Table 2. The compounds 1a, 2a, and 3a exhibited potent activity with IC50 in the micromolar concentration range against α-galactosidase (Aspergillus niger) and β-galactosidase (bovine liver), whereas compounds 1b and 3b showed moderate activity against βglucosidase (bovine liver) and potent activity against βgalactosidase (bovine liver) and α-mannosidase (Jack bean), respectively. Compound 1c (benzyl protected analogue of 1b) showed moderate activity against β-glucosidase (bovine liver), suggesting necessity of free hydroxymethyl group for biological activity. Among all compounds, the indolizidone iminosugar 2b showed wide range of inhibitory activity against β-glucosidase, α-/β-galactosidase and α-mannosidase. The D-glucose derived indolizidone iminosugars 2b and 3b were found to be potent against α-mannosidase (Jack bean) with IC50 and Ki values in the nanomolar concentration range. The castanospermine IIa exhibited inhibition against rice α-galactosidase (Ki = 15 nM) and almonds β-galactosidase (Ki = 1.5 μM);21 however, 1hydroxymethyl-1-deoxycastanospermine analogues IIa,b showed no inhibition against tested enzymes.16 On the other hand, 8a-hydroxymethyl-1-deoxycastanospermine analogues 1− 3 showed moderate to potent inhibition against tested enzymes. This inhibitory activity could be attributed to the presence of hydroxymethyl group at the ring junction making these E


−5.5

ASN186, ILE125, TYR82, TYR269, GLU267 GLU187, ASN186, GLU128, GLU267, TYR269 −5.8

TYR269, GLU267, GLU187 −5.3

−7.1

IL-2 and IL-6 cytokines which are responsible for the body’s natural response to microbial infection and stimulate immune response leading to inflammation, respectively.32 In addition, γ interferon (IFN-γ) is the featured cytokine of the TH1 cells, active for migration of immune cells to the site of inflammation,33 while the cytokine IL-4- an anti-inflammatory cytokine secreted by the TH2 cells is a key regulator for humoral and adaptive immunity, including stimulation of the activated β cell as well as proliferation and differentiation of the T cells.31 To evaluate the cytokine secretion ratio TH1/TH2, the effect of the indolizidine/indolizidone iminosugars 1−3 on the concavalin A (Con A) induced proliferation of splenocytes isolated from Swiss albino mice was assessed by the MTT assay. The cells were harvested after 72 h of incubation at 37 °C. The studies revealed that treatment of the mice splenocytes with Con A at 10 μM concentration enhanced cell proliferation by 4-fold (p < 0.01) as compared to the untreated cells (Figure 4). Therefore, the subsequent experiments were performed with the same concentration (10 μM) of Con A. After the administration of the compounds 1a, 1b, 1c, 2a, 2b, 3a, and 3b at four different concentrations (0.5, 1, 2.5, 5 μM), we observed increase in proliferation of the Con A-treated cells.34 Thus, compared to the Con A-treated control sample, treatment of Con A + 2b increased the cell proliferation by 13 (p < 0.05), 80 (p < 0.05), 130 (p < 0.01), and 103%, respectively. Compounds 1a, 2a, and 3a increased the T-cell proliferation concentration dependently up to 5 μM, whereas the compounds 1b, 1c, 2b, and 3b increased the T-cell proliferation concentration dependently up to 2.5 μM. This indicated the increase in immunostimulating activity in the presence of lactam moiety of the indolizidone iminosugars. In addition, γ-lactam 2b with α-hydroxymethyl group at the quaternary center showed better T-cell proliferation than the compounds 1a, 1b, 1c, 2a, 3a, and 3b. In the absence of Con A, all indolizidine/indolizidone iminosugars 1, 2, and 3 did not induce cell proliferation, suggesting the compounds are nontoxic and showed synergistic effects to the Con A-induced immune response. Further, the effects of these compounds on the secretion of various cytokines from the mouse splenocytes were assayed. To achieve this, the spleen cells were induced by 10 μM Con A in combination with iminosugars 1−3, and secretion levels of cytokines, such as interleukin-4 (IL-4), IL-6, and interferon-γ (IFN-γ), were measured from the supernatant of spleen cells using a mice ELISA kit. Assays were carried out for compounds 1a, 2a, and 3a at their optimized effective concentrations of 5 μM and for 1b, 1c, 2b, and 3b of 2.5 μM. As shown in Figure 5, the Con A (10 μM) treated cells showed significant increase in the level of IL-4 secretion (35%), compared to the untreated cells. However, treatment of the Con A-treated cells with 1a, 2a, 3a, 1b, 1c, 2b, and 3b reduced the levels of IL-4 secretion by 31% (p < 0.05), 31% (p < 0.001), 34% (p < 0.01), 34% (p < 0.05), 40% (p < 0.01), 57% (p < 0.01) and 47% (p < 0.05), respectively. This suggests that compounds moderately inhibit the anti-inflammatory cytokines IL-4 secretion by the TH2 cells. In the next experiment, treatment of the mouse splenocytes with the Con A did not show significant change in IL-6 secretion (Figure 6), whereas treatment of the Con A-treated cells in conjugation with 1a, 2a, 3a, 1b, 1c, 2b, and 3b increased IL-6 secretion by 3.7- (p < 0.01), 4.1- (p < 0.01), 4.4- (p < 0.05), 8.2(p < 0.01), 4.9- (p < 0.05), 12.1- (p < 0.01), and 9.0-fold (p < 0.01), respectively. The increase of IL-6 by indolizidone iminosugars 1b, 2b, and 3b was significantly higher than that of indolizidine iminosugars 1a, 2a, and 3a.

−5.5 3b

a

−6.7 GLU211, TRP399, TRP485

−6.8 2b

−7.0

−7.3 1c

−6.6

−7.3

−6.1

GLN64, TRP485, GLU484, GLU211, GLU427, TRP399 ASN209, ARG47, ASP204

−6.3 1b

−5.1

ASP204, ILE205, LYS478 ASN209, ARG473, LYS478 ASP204, ASN209, ARG473 SER203, ASP204, LYS478

−6.2 3a

AMBER’95 force field present in AUTODOCK 4.2 was used for total binding energy determination.

−4.6

−4.8

−3.2

ARG597, THR688, ARG334, GLU330 GLU330, ALA396, ALA460, GLU689, THR688, GLU599 GLU663, THR688, GLU467, SER464 ARG394, GLU663, GLU689 −4.2

SER201, LYS160, TYR126, CYS134, ASP234 TYR205, ALA202, SER201, CYS164, ASP162, CYS134 TRP49, ASP271, ASP234, LYS160, ASP162 ARG80, ASP271, ALA297, TRP292, ASN325 −6.6 ILE205, ASP204, LYS478

−5.3 −6.5 LYS478, ILE205, SER469

−6.6 2a

−5.2 ILE205, SER469, LYS478

−6.4

−5.7

interacting residues

ARG473, SER469, ILE205, LYS478 ILE205

ΔGbind

1a

−6.1

−6.4

GLU128, ILE125, ALA127, TYR62, TRP272, GLU267 GLU128, ALA127, GLU187, ASN186, TYR269, TRP272, TYR332 GLU187, GLU267, GLU128 −6.4 −5.5 −6.3

interacting residues

GLU128, CYS126, TYR484, GLU187 −5.9

ΔGbind interacting residues

ASP463, SER464, TRP688, GLU599 GLU689, THR688, GLU663, GLU602, LEU525 SER464, THR688, GLU599, ASP463, SER464 −4.9


ASP162, ASP84, ASP234, TRP49 ARG230, LYS160, ASP162, CYS134, ASP234, TRP45 LYS169, ASP84, TRP49, ASP234, ASP162, SER201 −5.4


GLU211, TRY354, TRP399, TRP477, PHE493, TRP485, GLN225 TRP477, TYR354, GLU211, TRP399

ΔGbind

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compd

β-galactosidase (bovine liver) α-galactosidase (Aspergillus niger) β-glucosidase (bovine liver) α-glucosidase (rice)

Table 3. Total Binding Energy (ΔGbind) in kcal mol−1 and Interacting Amino Acid Residues between Enzymes and Tested Ligandsa

−6

α-mannosidase (Jack bean)


F



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Figure 3. Binding of ligands 1a, 1b, 1c, 2a, 2b, 3a, and 3b with active pocket of α-glucosidase (rice), β-glucosidase (bovine liver), α-galactosidase (Aspergillus niger), β-galactosidase (bovine liver), and α-mannosidase (Jack bean).

reported to be an immunosuppressive agent.12 Our previously studied castanospermine congeners IIb/IIc (Figure 1) showed significant immunopotentiating property by increasing the TH1/ TH2 cytokines ratio and being active at 5 and 10 μM concentration, respectively. The comparative study revealed that incorporation of hydroxymethyl group at the C8a position made compounds 1a, 2a, and 3a comparably potent to IIb (5 μM concentration for both) and more potent than IIc (5 μM against 10 μM for IIc). However, their 3-oxo analogs 1b, 2b, and 3b were found to be more potent immunostimulating agents being active at 2.5 μM concentration and thus may have potential in controlling infection as well as treatment of autoimmune diseases. These studies help to establish the role C-8a hydroxymethyl group, its stereochemistry, and lactam functionality of the indolizidine/indolizidone for the immunomodulatory activity.

While considering IFN-γ, the Con A treated cells showed significant increase (5.27-fold, p < 0.01) in secretion of the interferon-γ compared to control cells. Compound 2a was found to be increasing the cell proliferation by 17% (p < 0.05), while the effect of other compounds was comparatively insignificant, indicating 2a as a better inducer of IFN-γ (Figure 7). In the present study, compounds 1a, 1b, 1c, 2a, 2b, 3a, and 3b showed their ability to increase the ratio of TH1/TH2 cytokines (IL-6 and IL-4) in a dose dependent manner. The increase in the TH1/TH2 ratio could be corroborated with their cell proliferating activity. Thus, the compounds can be treated as potent immunostimulator. Compounds 1b, 2b, and 3b are able to enhance the secretion of IL-6 remarkably and secretion of IFN-γ moderately. All the compounds exhibit a moderate inhibitory effect on the secretion of IL-4. Hence, they may have stronger immune-potentiating efficiency via both TH1 and TH2 mediated cellular as well as humoral immune reactions. With regard to the immunomodulatory activity, castanospermine has been earlier G



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Figure 4. Proliferation (%) of mouse splenocytes by Con A (10 μM) alone and in conjunction with various concentrations of compounds 1a, 1b, 1c, 2a, 2b, 3a, and 3b. The cell proliferation was monitored by the MTT assay, and the values (mean ± SEM, n = 5) are calculated considering that of the Con Atreated cells as 100%: (#) p < 0.01 with respect to the untreated cells; (∗) p < 0.05, (∗∗) p < 0.01 with respect to Con A-treated cells.

Figure 6. Effects of Con A (10 μM) alone and in conjunction with compounds 1a (5 μM), 2a (5 μM), 3a (5 μM), 1b (2.5 μM), 1c (2.5 μM), 2b (2.5 μM), and 3b (2.5 μM) on the secretion of the mouse IL-6 from normal mouse splenocytes (2 × 106 cells/well). The values are mean ± SEM (n = 5): (#) p < 0.05, (∗) p < 0.05, (∗∗) p < 0.01.

Figure 5. Effects of Con A (10 μM) alone and in conjunction with compounds 1a (5 μM), 2a (5 μM), 3a (5 μM), 1b (2.5 μM), 1c (2.5 μM), 2b (2.5 μM), and 3b (2.5 μM) on the secretion of the mouse IL-4 from normal mouse splenocytes (2 × 106 cells/well). The values are mean ± SEM (n = 5): (#) p < 0.01, (∗) p < 0.05, (∗∗) p < 0.01, (∗∗ ∗) p < 0.001.

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CONCLUSION We have explored the D-gluco- and D-manno-hexoses derived azidoaldehyde with required formyl and amino functionalities for the short synthesis of 8a(S)-hydroxymethyl-1-deoxycastanospermine 2b and its analogues 1a, 1b, 1c, 2a, 3a, and 3b. These iminosugars exhibited differential potency and specificity against β-glucosidase (bovine liver), α-galactosidase (Aspergillus niger) and β-galactosidase (bovine liver), and α-mannosidase (Jack bean). The indolizidone iminosugars 2b and 3b were found to be more potent against α-mannosidase (Jack bean) with IC50 and Ki values in the nanomolar concentration range and further substantiated with theoretical binding energy calculations. Interestingly, in contrast to castanospermine IIa, its C-8ahydroxymethyl congeners 1, 2, and 3 act synergistically with Con A and showed significant immunostimulating activity. The indilizidone iminosugars 1b, 2b, and 3b showed higher potency than that of indilizidine iminosugars 1a, 2a, and 3a. It is important to note that all compounds (1−3) augmented

Figure 7. Effects of Con A (10 μM) alone and in conjunction with compounds 1a (5 μM), 2a (5 μM), 3a (5 μM), 1b (2.5 μM), 1c (2.5 μM), 2b (2.5 μM), and 3b (2.5 μM) on the secretion of the mouse IFNγ from normal mouse splenocytes (2 × 106 cells/well). The values are mean ± SEM (n = 5): (#) p < 0.01, (∗) p < 0.05, (∗∗) p < 0.01.

H



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Na]+, 370.1630; found, 370.1630. Further elution with (dichloromethane/MeOH 96:04 to 94:06) gave 6a (171 mg, 70%) as gummy solid. Rf = 0.40 (dichloromethane/MeOH, 9:1). [α]D29 +4 (c 0.63, CH3OH). IR (neat, ν, cm−1): 1665, 3200−3300 (br). 1H NMR (300 MHz, CDCl3) δ 1.35 (s, 3H), 1.53 (s, 3H), 1.95−2.08 (m, 1H), 2.12− 2.26 (m, 1H), 2.29−2.42 (m, 1H), 2.42−2.57 (m, 1H), 2.82 (dd, J = 13.5, 9.0 Hz, 1H), 2.90−3.14 (bs, exchangeable with D2O, 1H), 3.24− 3.44 (bs, exchangeable with D2O, 1H,), 3.64 (d, J = 11.4 Hz, 1H), 3.92 (d, J = 6.3 Hz, 1H), 3.95 (d, J = 11.4 Hz, 1H), 4.15 (t, J = 6.3 Hz, 1H), 4.26 (dd, J = 13.5, 8.0 Hz, 1H), 4.35−4.50 (m, 1H). 13C NMR (75 MHz, CDCl3) δ 22.9, 24.7, 26.9, 30.7, 38.3, 61.8, 62.2, 67.0, 67.7, 68.9, 110.8, 178.2. Anal. Calcd for C12H19NO5: C, 56.02; H, 7.44; N, 5.44. Found: C, 56.17; H, 7.59; N, 5.27. HR-MS (ESI): m/z calculated [M + H]+, 258.1341; found, 258.1338. 8a(R)-Hydroxymethyl-6-epi-1-deoxycastanospermine-3-one (1b). To an ice-cooled solution of 6a (50 mg, 0.19 mmol) in MeOH (1 mL) was added concentrated HCl (0.1 mL), and reaction mixture was stirred for 2 h. The solvent was evaporated to dryness, and the residue was dissolved in distilled water (2 mL) and extracted with chloroform (1 mL × 3). The aqueous layer was concentrated to afford 1b (49 mg, 98%) as a gummy solid: [α]D29 −6 (c 2.10, CH3OH). IR (neat, ν, cm−1): 1665, 3200−3300 (br). 1H NMR (300 MHz, D2O) δ 1.90−2.04 (m, 1H), 2.12−2.28 (m, 1H), 2.30−2.54 (m, 2H), 2.84−2.98 (m, 1H), 3.46 (d, J = 12.3 Hz, 1H), 3.71 (d, J = 3.0 Hz, 1H), 3.74−3.90 (m, 2H), 3.99 (s, 1H), 4.30 (d, J = 12.3 Hz, 1H). 13C NMR (75 MHz, D2O) δ 22.1, 30.7, 37.1, 61.3, 63.1, 67.9, 70.0, 71.1, 177.9. Anal. Calcd for C9H15NO5: C, 49.76; H, 6.96; N, 6.45. Found: C, 49.87; H, 6.99; N, 6.54. HR-MS (ESI): m/z calculated [M + H]+, 218.1028; found, 218.1024. U-HPLC−MS: tR (min) 2.7, purity 97.65%. 8a(R)-Benzyloxymethyl-6-epi-1-deoxycastanospermine-3one (1c). Reaction of 6b (50 mg, 0.14 mmol) with concentrated HCl as described for 1b gave 1c (49 mg, 98%) as a gummy solid: [α]D29 +14.3 (c 1.40, CH3OH). IR (neat, ν, cm−1): 1665, 3200−3300 (br). 1H NMR (300 MHz, D2O) δ 1.74−1.90 (m, 1H), 1.96−2.10 (m, 1H), 2.12−2.38 (m, 2H), 2.49 (t, J = 10.5 Hz, 1H), 3.37 (d, J = 10.5 Hz, 1H), 3.50−3.70 (m, 3H), 3.82 (bs, 1H), 3.98 (d, J = 10.5 Hz, 1H), 4.29 (d, J = 12.0 Hz, 1H) 4.41 (d, J = 12.0 Hz, 1H), 7.10−7.35 (m, 5H). 13C NMR (75 MHz, D2O) δ 22.7, 30.8, 37.2, 63.1, 66.9, 69.5, 70.1, 71.1, 73.2, 128.3, 128.4, 128.7, 137.3, 177.7 (CO). Anal. Calcd for C16H21NO5: C, 62.53; H, 6.89; N, 4.56. Found: C, 62.69; H, 6.98; N, 4.44. HR-MS (ESI): m/z calculated [M + H]+, 308.1498; found, 308.1495; [M + Na]+, 330.1317; found, 330.1312. U-HPLC−MS: tR (min) 2.9, purity 97.01%. Benzyl-2,3-O-isopropylidene-5-deoxy-5-azido-5-C-benzyloxymethyl-6,7-dideoxy-6(E)-ene-α-L-gulo-furano-1,8-dialdose (7). To a stirred solution of 5 (0.40 g, 0.76 mmol) in dry toluene (10 mL) at −78 °C was added 1 M solution of diisobutylaluminum hydride in toluene (0.80 mL, 0.80 mmol). The reaction mixture was stirred at −78 °C for 1 h, and saturated solution of NH4Cl (10 mL) was added slowly. The mixture was diluted with EtOAc and stirred for 3 h. Reaction mixture was filtered through Celite, washed with EtOAc, and solvent was evaporated at reduced pressure. Purification by column chromatography (petroleum ether/EtOAc, 9:1) gave 7 (0.26 g, 68%) as a viscous liquid. Rf = 0.47 (petroleum ether/EtOAc, 9:1). [α]D29 +46 (c 1.40, CH3OH). IR (neat, ν, cm−1): 2111, 1719, 1691. 1H NMR (300 MHz, CDCl3) δ 1.26 (s, 3H), 1.36 (s, 3H), 3.68 (d, J = 10.0 Hz, 1H), 3.84 (d, J = 10.0 Hz, 1H), 4.44 (d, J = 11.4 Hz, 2H), 4.58−4.68 (m, 4H), 4.80 (dd, J = 5.7, 3.3 Hz, 1H), 5.13 (s, 1H), 6.37 (dd, J = 15.6, 7.5 Hz, 1H), 6.99 (d, J = 15.6 Hz, 1H), 7.24−7.40 (m,10H) 9.56 (d, J = 7.5 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 24.1, 25.4, 66.7, 69.2, 72.5, 73.8, 79.4, 79.6, 84.8, 105.0, 113.0, 127.7, 128.0, 128.1, 128.5, 131.8, 136.8, 137.1, 153.2, 193.1. Anal. Calcd for C26H29N3O6: C, 65.12; H, 6.10; N, 8.76. Found: C, 65.22; H, 6.29; N, 8.66. HR-MS (ESI): m/z calculated [M + Na]+, 502.1953; found, 502.1949. 6,7-O-Isopropylidene-8a(R)-hydroxymethyl-6-epi-1deoxycastanospermine (8). Reaction of 7 (200 mg, 0.42 mmol) with 10% Pd/C (40 mg) as described for 6a gave 8 (92 mg, 91%) as a gummy solid: Rf = 0.35 (dichloromethane/MeOH, 9;1). [α]D29 −34 (c 0.30, CH3OH). IR (neat, ν, cm−1): 3200−3300 (br). 1H NMR (300 MHz, CDCl3 + D2O) δ 1.35 (s, 3H), 1.49 (s, 3H), 1.62−1.76 (m, 3H), 1.82− 1.95 (m, 1H), 2.70−2.85 (m, 2H), 3.05−3.18 (m, 1H), 3.27 (dd, J =

secretion of IL-6 cytokines. Among these compounds, 2b showed augmentation of IL-6 by 12-fold so that it could be exploited for clinical application in cancer therapy. Thus, our findings open a new avenue for the usefulness of iminosugars in the treatment of immunological disorders.

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EXPERIMENTAL SECTION

General Experimental Methods. Melting points were recorded with Thomas−Hoover melting point apparatus and are uncorrected. IR spectra were recorded on a FTIR-ATR spectrometer and are expressed in cm−1. 1H NMR (300/500 MHz) and 13C NMR (75/125 MHz) spectra were recorded using CDCl3 or D2O as solvent. Chemical shifts were reported in δ unit (parts per million) with reference to TMS as an internal standard, and J values are given in hertz. Elemental analyses were carried out with C, H analyzer. Optical rotations were measured using polarimeter at 25 °C. High resolution mass spectra (HRMS) were obtained in positive ion electrospray ionization (ESI) mode using TOF (time-of-flight) analyzer. Thin-layer chromatography was performed on precoated plates (0.25 mm, silica gel 60 F254). Column chromatography was carried out with silica gel (100−200 mesh). The reactions were carried out in oven-dried glassware under dry N2 atmosphere. Petroleum ether (PE) that was used is a distillation fraction between 40 and 60 °C. After neutralization, workup involves washing of combined organic layer with water, brine, drying over anhydrous sodium sulfate, and evaporation of solvent under reduced pressure. The purity of final compounds was determined using U-HPLC−MS technique. The reverse phase C18 column (Thermoscientific Acclaim 120, 4.6 mm × 250 mm) and mobile phase acetonitrile/water/formic acid (9:0.9:0.1) was used along with the Q-TOF mass detector and +ve mode electron spray ionization. All compounds showed purity more than 95%. Ethyl (Benzyl-2,3-O-isopropylidene-5-deoxy-5-azido-5-Cbenzyloxymethyl-6,7-dideoxy-6(E)-ene-α-L-gulo)octofuranouronate (5). To a stirred solution of 4 (1.74 g, 3.84 mmol) in dry CH2Cl2 (20 mL) was added PPh3CHCOOEt (1.74 g, 4.99 mmol). The reaction mixture was refluxed for 2 h. Solvent evaporation followed by chromatographic purification by eluting with petroleum ether/EtOAc, (9:1) gave 5 (1.91 g, 95%) as a viscous liquid. Rf = 0.63 (petroleum ether/EtOAc, 9:1). [α]D29 +43 (c 0.77, CH3OH). IR (neat, ν, cm−1): 2111, 1716, 1656. 1H NMR (300 MHz, CDCl3) δ 1.25 (s, 3H), 1.28 (t, J = 7.2 Hz, 3H), 1.37 (s, 3H), 3.64 (d, J = 10.2 Hz, 1H), 3.82 (d, J = 10.2 Hz, 1H), 4.20 (q, J = 7.2 Hz, 2H), 4.42 (d, J = 11.4 Hz, 1H), 4.44 (d, J = 3.3 Hz, 1H), 4.58−4.65 (m, 4H), 4.79 (dd, J = 5.7, 3.3 Hz, 1H), 5.12 (s, 1H), 6.12 (d, J = 15.9 Hz, 1H), 7.12 (d, J = 15.9 Hz, 1H), 7.20− 7.40 (m,10H). 13C NMR (75 MHz, CDCl3) δ 14.1, 24.0, 25.3, 60.3, 66.4, 68.8, 72.5, 73.5, 79.3, 79.4, 84.7, 104.6, 112.7, 121.5, 127.5, 127.6, 127.7, 128.0, 128.3, 128.6, 136.7, 137.2, 144.4, 165.8. Anal. Calcd for C28H33N3O7: C, 64.23; H, 6.35; N, 8.03. Found: C, 64.41; H, 6.46; N, 7.97. HR-MS (ESI): m/z calculated [M + Na]+, 546.2216; found, 546.2216. 8a(R)-Hydroxymethyl-6,7-O-isopropylidene-6-epi-1-deoxycastanospermine-3-one (6a) and 8a(R)-benzyloxymethyl-6,7O-isopropylidene-6-epi-1-deoxycastanospermine-3-one (6b). To a solution of 5 (0.50 g, 0.95 mmol) in methanol (15 mL) was added 10% Pd/C (50 mg), and reaction mixture was hydrogenated at 100 psi for 24 h. Reaction mixture was filtered through Celite, washed with methanol, and solvent was evaporated at reduced pressure. Purification by column chromatography first elution using dichloromethane/MeOH 98:03 gave partially debenzylated compound 6b (69 mg, 21%) as gummy solid: Rf = 0.57 (dichloromethane/MeOH, 9;1). [α]D29 +10 (c 1.80, CH3OH). IR (neat, ν, cm−1): 1665, 3200−3300 (br). 1H NMR (300 MHz, CDCl3 + D2O) δ 1.32 (s, 3H), 1.39 (s, 3H), 1.94−2.07 (m, 1H), 2.12−2.38 (m, 2H), 2.42−2.57 (m, 1H), 2.68 (dd, J = 13.2, 9.3 Hz, 1H), 3.46 (d, J = 9.3 Hz, 1H), 3.72 (d, J = 9.3 Hz, 1H), 3.92 (d, J = 6.0 Hz, 1H), 4.10 (t, J = 6.0 Hz, 1H), 4.29 (dd, J = 13.2, 7.8 Hz, 1H), 4.36−4.64 (m, 3H), 7.24−742 (m, 5H). 13C NMR (75 MHz, CDCl3) δ 23.9, 25.3, 27.6, 30.6, 38.8, 64.5, 69.6, 70.1, 71.3, 73.5, 77.2, 110.1, 127.6, 127.9, 128.5, 137.6, 175.4. Anal. Calcd for C19H25NO5: C, 65.69; H, 7.25; N, 4.03. Found: C, 65.61; H, 7.06; N, 4.12. HR-MS (ESI): m/z calculated [M + H]+, 348.1811; found, 348.1807; [M + I



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+2 (c 0.43, CH3OH). IR (neat, ν, cm−1): 2113, 1722, 1695. 1H NMR (300 MHz, CDCl3) δ 0.00 (s, 3H), 0.18 (s, 3H), 0.87 (s, 9H), 1.33 (s, 3H), 1.48 (s, 3H), 3.79 (s, 2H), 4.05 (d, J = 3.3 Hz, 1H), 4.42 (d, J = 11.4 Hz, 1H), 4.47 (d, J = 3.3 Hz, 1H), 4.63 (d, J = 3.9 Hz, 1H), 4.69 (d, J = 11.4 Hz, 1H), 5.96 (d, J = 3.9 Hz, 1H), 6.33 (dd, J = 16.2, 8.1 Hz, 1H), 6.90 (d, J = 16.2 Hz, 1H), 7.22−7.40 (m, 5H), 9.42 (d, J = 8.1 Hz, 1H). 13 C NMR (75 MHz, CDCl3) δ −5.7, −5.8, 18.0, 25.6 (strong, 3C), 26.3, 26.7, 66.1, 67.4, 71.9, 80.5, 81.4, 82.0, 104.6, 112.1, 128.1, 128.3, 128.6, 133.3, 136.4, 152.7, 193.3. Anal. Calcd for C25H37N3O6Si: C, 59.62; H, 7.40; N, 8.34. Found: C, 59.69; H, 7.57; N, 8.23. HR-MS (ESI): m/z calculated [M + Na]+, 526.2349; found, 526.2341. 8a(S)-Hydroxymethyl-1-deoxycastanospermine (2a). The reaction of 12 (200 mg, 0.36 mmol) with TFA−water followed by hydrogenated with 10% Pd/C as described for 2b gave 2a (72 mg, 90%) as a gummy solid: Rf = 0.26 (dichloromethane/MeOH, 8:2). [α]D29 +10 (c 1.00, CH3OH). IR (neat, ν, cm−1): 3200−3300 (br). 1H NMR (300 MHz, D2O) δ 1.70−1.96 (m, 4H), 2.75 (dd, J = 13.8, 11.0 Hz, 1H), 2.98−3.12 (m, 3H), 3.46 (d, J = 9.9 Hz, 1H), 3.58 (t, J = 9.9 Hz, 1H), 3.68 (s, 2H), 3.78 (ddd, J = 11.1, 9.9, 5.1 Hz, 1H). 13C NMR (75 MHz, D2O) δ 19.7, 30.8, 47.4, 51.1, 59.1, 66.6, 68.0, 72.5, 74.3. Anal. Calcd for C9H17NO4: C, 53.19; H, 8.43; N, 6.89. Found: C, 53.32; H, 8.56; N, 6.76. HR-MS (ESI): m/z calculated [M + H]+, 204.1236; found, 204.1236. U-HPLC−MS: tR (min) 2.0, purity 98.71%. 1,2-O-Isopropylidene-3-O-benzyl-5S-deoxy-5-azido-5-C-hydroxymethyl-6-O-(tert-butyldimethylsilyloxy)-α-L-ido-furanose (13). To an ice-cooled solution of 10 (3.30 g, 6.91 mmol) in MeOH (20 mL) was added sodium borohydride (0.53 g, 13.82 mmol) in two portions. Reaction mixture was stirred for 3 h and quenched by adding saturated aq NH4Cl solution (5 mL). Methanol was evaporated under reduced pressure and residue extracted with EtOAc (50 mL × 3) and concentrated in vacuo. Then, purification by column chromatography (petroleum ether/ethyl acetate, 8:2) gave 13 (2.98g, 90%) as a thick liquid: Rf = 0.40 (petroleum ether/ethyl acetate, 8:2). [α]D27 −33.7 (c 2.20, CH3OH). IR (neat, ν, cm−1): 3300−3500 (br), 2114 (st). 1H NMR (300 MHz, CDCl3) δ 0.0.00 (s, 3H), 0.03 (s, 3H), 0.06 (s, 9H), 1.31 (s, 3H), 1.47 (s, 3H), 2.65 (t, J = 6.6 Hz, exchangeable with D2O, 1H), 3.72 (dd, J = 11.7, 6.6 Hz; after D2O exchange appear as d, J = 11.7 Hz, 1H), 3.82 (dd, J = 11.7, 6.6 Hz; after D2O exchange appear as d, d, J = 11.7 Hz, 1H), 3.86 (s, 2H), 4.05 (d, J = 3.0 Hz, 1H), 4.38 (d, J = 3.0 Hz, 1H), 4.48 (d, J = 11.7 Hz, 1H), 4.61 (d, J = 3.9 Hz, 1H), 4.69 (d, J = 11.7 Hz, 1H), 5.93 (d, J = 3.9 Hz, 1H), 7.28−7.38 (m, 5H). 13C NMR (75 MHz, CDCl3) δ −5.8 (strong), 18.0, 25.7 (strong), 26.3, 26.7, 63.9, 65.1, 66.8, 71.9, 79.6, 81.5, 82.3, 104.2, 111.9, 128.0, 128.2, 128.6, 136.5. Anal. Calcd for C23H37N3O6Si: C, 57.59; H, 7.78; N, 8.76. Found: C, 57.45; H, 7.86; N, 8.82. HR-MS (ESI): m/z calculated [M + Na]+, 502.2349; found, 502.2356. 1,2-O-Isopropylidene-3-O-benzyl-5S-deoxy-5-azido-5-C(methoxymethylene)methyl)-6-O-(tert-butyldimethylsilyloxy)α-L-ido-furanoside (14). To a stirred solution of 13 (2.21 g, 4.61 mmol), diisopropylethylamine (6.36 mL, 36.8 mmol), and NaI (69 mg, 0.46 mmol) in dry CH2Cl2 (20 mL) was added MOMCl (2.10 mL g, 27.66 mmol) under nitrogen atmosphere at 0 °C, and the mixture was warmed to room temperature. After being stirred for 4 h, the reaction mixture was quenched with cold water (30 mL) and extracted with CH2Cl2 (3 × 50 mL). The combined organic extracts were washed with water (3 × 50 mL), dried over anhydrous Na2SO4, and concentrated by reducing pressure. The residue was purified by silica gel column chromatography using petroleum ether/EtOAc (9:1) to give compound 14 (2.21 g, 92% yield) as a colorless gummy liquid: Rf = 0.60 (petroleum ether/ethyl acetate, 8:2). [α]D27 −25.0 (c 2.50, CH3OH). IR (neat, ν, cm−1): 2118 (st). 1H NMR (500 MHz, CDCl3) δ 0.0.04 (s, 3H), 0.05 (s, 3H), 0.89 (s, 9H), 1.34 (s, 3H), 1.49 (s, 3H), 3.36 (s, 3H), 3.74 (d, J = 10.5 Hz, 1H), 3.84 (d, J = 11.0 Hz, 1H), 3.87 (d, J = 11.0 Hz, 1H), 4.01 (d, J = 10.5 Hz, 1H), 4.05 (d, J = 3.0 Hz, 1H), 4.41 (d, J = 3.0 Hz, 1H), 4.53 (d, J = 11.5 Hz, 1H), 4.58−4.65 (m, 3H), 4.72 (d, J = 11.5 Hz, 1H), 5.94 (d, J = 4.0 Hz, 1H), 7.32−7.42 (m, 5H). 13C NMR (125 MHz, CDCl3) δ −5.7 (strong), 18.1, 26.0 (strong), 26.3, 26.7, 55.3, 63.6, 66.5, 71.9, 77.2, 78.5, 81.7, 82.3, 96.7, 104.4, 111.7, 128.1, 128.2, 128.5, 137.0. Anal. Calcd for C25H41N3O7Si: C, 57.34; H, 7.89; N, 8.02. Found: C,

14.4, 6.6 Hz, 1H), 3.28 (d, J = 10.5 Hz, 1H), 3.54 (d, J = 10.5 Hz, 1H), 3.83 (d, J = 9.0 Hz, 1H), 4.17 (t, J = 9.0 Hz, 1H), 4.55−4.70 (m, 1H). 13C NMR (75 MHz, CDCl3) δ 24.2, 24.7, 27.2, 29.6, 48.3, 54.5, 65.5, 67.3, 70.1, 72.0, 76.3, 109.1. Anal. Calcd for C12H21NO4: C, 59.24; H, 8.70; N, 5.76. Found: C, 59.14; H, 8.65; N, 5.83. HR-MS (ESI): m/z calculated [M + H]+, 244.1549; found, 244.1543. 8a(R)-Hydroxymethyl-1-deoxy-6-epi-castanospermine Hydrochloride (9). Reaction of 8 (50 mg, 0.21 mmol) with concentrated HCl (0.1 mL) as described for 1b afforded 9 (48 mg, 98%) as a gummy solid: [α]D28 −4 (c 1.8, CH3OH). IR (neat, ν, cm−1): 3200−3300 (br). 1 H NMR (300 MHz, D2O) δ 1.62−1.78 (m, 1H), 1.92−2.12 (m, 3H), 3.14−3.24 (m, 2H), 3.28 (dd, J = 13.5, 3.0 Hz, 1H), 3.41 (d, J = 12.6 Hz, 1H), 3.62−3.47 (m, 1H), 3.69 (dd, J = 9.6, 3.0 Hz, 1H), 3.73 (d, J = 12.6 Hz, 1H), 4.03 (d, J = 9.6 Hz, 1H), 3.99−4.06 (m, 1H). 13C NMR (75 MHz, D2O) δ 17.6, 23.8, 52.8 (strong, 2C), 59.0, 65.9, 66.2, 68.8, 75.3. Anal. Calcd for C9H18ClNO4: C, 45.10; H, 7.57; N, 5.84. Found: C, 45.38; H, 7.71; N, 5.74. HR-MS (ESI): m/z calculated (C9H17NO4-salt free) [M + H]+, 204.1236; found, 204.1240. 8a(R)-Hydroxymethyl-1-deoxy-6-epi-castanospermine (1a). To a solution of 9 (40 mg, 0.17 mmol) in MeOH (1 mL) was added Dowex (strongly basic anion exchange resin) (0.1 g), and reaction mixture was stirred for 2 h. Reaction mixture was filtered through Celite, washed with methanol, and solvent was evaporated at reduced pressure to afford 1a (33 mg, 97%) as a gummy solid: [α]D28 −2 (c 1.0, CH3OH). IR (neat, ν, cm−1): 3200−3300 (br). 1H NMR (500 MHz, D2O) δ 1.46−1.56 (m, 1H), 1.70−1.82 (m, 1H), 1.84−2.05 (m, 2H), 2.80 (bd, J = 13.0, Hz, 1H), 2.85−2.88 (m, 1H), 2.92 (dd, J = 13.0, 4.0 Hz, 1H), 3.05−3.16 (m, 1H), 3.45 (d, J = 12.0 Hz, 1H), 3.55 (d, J = 12.0 Hz, 1H), 3.72 (dd, J = 9.0, 3.0 Hz, 1H), 3.96−4.02 (m, 1H), 4.05 (d, J = 9.0 Hz, 1H). 13C NMR (125 MHz, D2O) δ 19.3, 24.9, 51.9, 52.2, 61.9, 66.9, 68.4, 70.2, 71.5. Anal. Calcd for C9H17NO4: C, 53.19; H, 8.43; N, 6.89. Found: C, 53.37; H, 8.26; N, 6.94. HR-MS (ESI): m/z calculated [M + H]+: 204.1236; found, 204.1235. U-HPLC−MS: tR (min) 2.1, purity 97.60%. Ethyl (1,2-O-Isopropylidene-3-O-benzyl-5R-deoxy-5-azido5-C-(tert-butyldimethylsilyloxy)methyl-6,7-dideoxy-6(E)-eneβ-D-gluco-octofurano)uronate (11). Reaction of 10 (1.49 g, 3.12 mmol) with PPh3CHCOOEt (1.41 g, 4.05 mmol) as described for 5 afforded 11 (1.65 g, 96%) as a viscous liquid. Rf = 0.35 (petroleum ether/ EtOAc, 9:1). [α]D29 +1 (c 0.54, CH3OH). IR (neat, ν, cm−1): 2113, 1718, 1658. 1H NMR (300 MHz, CDCl3) δ −0.04 (s, 3H), 0.00 (s, 3H), 0.82 (s, 9H), 1.22 (t, J = 7.2 Hz, 3H), 1.28 (s, 3H), 1.43 (s, 3H), 3.71 (ABq, J = 10.2 Hz, 2H), 3.99 (d, J = 3.3 Hz, 1H), 4.13 (q, J = 7.2 Hz, 2H), 4.41 (d, J = 12.0 Hz, 1H), 4.45 (d, J = 3.3 Hz, 1H), 4.56 (d, J = 3.9 Hz, 1H), 4.64 (d, J = 12.0 Hz, 1H), 5.92 (d, J = 3.9 Hz, 1H), 6.08 (d, J = 15.6 Hz, 1H), 7.01 (d, J = 15.6 Hz, 1H), 7.20−7.35 (m, 5H). 13C NMR (75 MHz, CDCl3) δ −5.8, −5.9, 14.0, 17.9, 25.5 (strong, 3C), 26.2, 26.6, 60.3, 66.3, 67.0, 71.7, 80.1, 81.4, 82.0, 104.5, 111.8, 123.2, 127.7, 127.9, 128.4, 136.6, 143.6, 165.8. Anal. Calcd for C27H41N3O7Si: C, 59.21; H, 7.55; N, 7.67. Found: C, 59.40; H, 7.65; N, 7.74. HR-MS (ESI): m/z calculated [M + Na]+, 570.2611; found, 570.2602. 8a(S)-Hydroxymethyl-1-deoxycastanospermine-3-one (2b). A solution of 11 (0.48 mg, 0.88 mmol) in TFA−water (4 mL, 3:1) was stirred for 3 h at 0 °C. TFA coevaporated with toluene at reduced pressure gave crude hemiacetal. That was hydrogenated with H2/Pd/C as described for 6a and gave 2b (178 mg, 94%) as a gummy solid: Rf = 0.36 (dichloromethane/MeOH, 8:2). [α]D29 +30 (c 1.08, CH3OH). IR (neat, ν, cm−1): 3200−3300 (br), 1641. 1H NMR (300 MHz, D2O) δ 1.96−2.16 (m, 1H), 2.17−2.30 (m, 1H), 2.38−2.64 (m, 2H), 2.75 (dd, J = 13.2, 12.3 Hz, 1H), 3.42−3.54 (m, 2H), 3.63 (t, J = 9.6 Hz, 1H), 3.73 (d, J = 12.3 Hz, 1H), 3.96 (d, J = 12.3 Hz, 1H), 4.07 (dd, J = 13.2, 5.7 Hz, 1H). 13C NMR (75 MHz, D2O) δ 26.1, 30.1, 40.1, 59.3, 67.5, 69.1, 74.1, 76.6, 177.5. Anal. Calcd for C9H15NO5: C, 49.76; H, 6.96; N, 6.45. Found: C, 49.88; H, 6.99; N, 6.32. HR-MS (ESI): m/z calculated [M + H]+, 218.1028; found, 218.1024; [M + Na]+, 240.0848; found, 240.0835. U-HPLC−MS: tR (min) 2.7, purity 98.06%. 1,2-O-Isopropylidene-3-O-benzyl-5R-deoxy-5-azido-5-C(tert-butyldimethylsilyloxymethyl)-6,7-dideoxy-6(E)-ene-β-Dgluco-furano-1,8-dialdose (12). Reaction of 11 (0.53 g, 0.97 mmol) with diisobutylaluminum hydride as described for 7 gave 12 (0.32 g, 66%) as a viscous liquid. Rf = 0.40 (petroleum ether/EtOAc, 9:1). [α]D29 J



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57.42; H, 7.82; N, 8.12. HR-MS (ESI): m/z calculated [M + Na]+, 546.2611; found, 546.2615. 1,2-O-Isopropylidene-3-O-benzyl-5R-deoxy-5-azido-5-C(methoxymethylene)methyl)-L-ido-furanoside (15). The solution of TBS ether 14 (2.20 g, 4.20 mmol) in THF (30 mL) was treated with TBAF (8.40 mL, 1 M in THF, 8.40 mmol) at 0 °C and stirred for 1 h at room temperature. The reaction mixture was diluted with water and extracted with ethyl acetate (2 × 50 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, and concentrated in vacuo, then purified by column chromatography using petroleum ether/EtOAc (7:3) to give 15 (1.51 g, 88% yield) as a thick liquid: Rf = 0.31 (petroleum ether/ethyl acetate, 8:2). [α]D27 −25.3 (c 1.90, CH3OH). IR (neat, ν, cm−1): 3300−3500 (br), 2118 (st). 1H NMR (300 MHz, CDCl3) δ 1.32 (s, 3H), 1.47 (s, 3H), 2.77 (t, J = 6.6 Hz, exchangeable with D2O, 1H), 3.35 (s, 3H), 3.79 (ABq, J = 10.5 Hz, 2H), 3.89 (d, J = 6.3 Hz, became singlet after D2O, 2H), 4.07 (d, J = 3.3 Hz, 1H), 4.27 (d, J = 3.3 Hz, 1H), 4.52−4.64 (m, 4H), 4.70 (d, J = 11.7 Hz, 1H), 5.93 (d, J = 3.6 Hz, 1H), 7.30−7.43 (m, 5H). 13C NMR (125 MHz, CDCl3) δ 26.3, 26.8, 55.5, 63.2, 65.8, 68.7, 72.2, 80.0, 81.6, 82.2, 96.7, 104.4, 112.0, 128.3 (strong), 128.4, 136.4. Anal. Calcd for C19H27N3O7: C, 55.74; H, 6.65; N, 10.26. Found: C, 55.86; H, 6.57; N, 10.40. HR-MS (ESI): m/z calculated [M + Na]+: 432.1747; found, 432.1750. 1,2-O-Isopropylidene-3-O-benzyl-5R-deoxy-5-azido-5-C((methoxymethylene)methyl)-L-ido-furano-1,6-dialdose (16). To stirred solution of alcohol 15 (1.50 g, 3.66 mmol) and NaHCO3 (3.08 g, 36.65 mmol) in CH2Cl2 (20 mL) Dess−Martin periodinane (3.10 g, 3.72 mmol) was added, and reaction mixture was stirred for 20 min at 0 °C. The reaction mixture was quenched with saturated aqueous NaHCO3 (5 mL) and saturated aqueous sodium metabisulfite (5 mL). The resulting cloudy mixture was stirred vigorously for 15 min at 0 °C. The layers were separated, and the aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine (1 × 30 mL), concentrated in vacuo, and purified by column chromatography with petroleum ether/ethyl acetate (9:1) to afford aldehyde 16 (1.27 g, 85% yield) as a thick liquid: Rf = 0.53 (petroleum ether/ethyl acetate, 8:2). [α]D27 +1.1 (c 1.70, CH3OH). IR (neat, ν, cm−1): 2127 (st), 1736. 1H NMR (300 MHz, CDCl3) δ 1.29 (s, 3H), 1.45 (s, 3H), 3.37 (s, 3H), 3.93 (d, J = 10.8 Hz, 1H), 4.02 (d, J = 3.0 Hz, 1H), 4.09 (d, J = 10.8 Hz, 1H), 4.25 (d, J = 3.0 Hz, 1H), 4.47 (d, J = 3.6 Hz, 1H), 4.52 (ABq, J = 11.7 Hz, 2H), 4.64 (ABq, J = 6.6 Hz, 2H), 5.91 (d, J = 3.6 Hz, 1H), 7.23−7.42 (m, 5H), 9.68 (s, 1H). 13C NMR (125 MHz, CDCl3) δ 26.2, 26.8, 55.6, 67.8, 69.5, 72.8, 81.1, 81.2, 81.9, 96.6, 105.2, 112.3, 128.4, 128.5, 136.3, 196.0. Anal. Calcd for C19H25N3O7: C, 56.01; H, 6.18; N, 10.31. Found: C, 56.15; H, 6.28; N, 10.40. HR-MS (ESI): m/z calculated [M + Na]+, 430.1590; found, 430.1594. Ethyl (1,2-O-Isopropylidene-3-O-benzyl-5S-deoxy-5-azido-5C-((methoxymethylene)methyl)-6,7-dideoxy-6(E)-ene-α-L-idooctofurano)uronate (17). Reaction of 16 (1.26 g, 3.09 mmol) with PPh3CHCOOEt (1.51 g, 4.33 mmol) as described for 5 afforded 17 (1.41 g, 96%) as a viscous liquid. Rf = 0.53 (petroleum ether/ethyl acetate, 8:2). [α]D27 −7.8 (c 0.70, CH3OH). IR (neat, ν, cm−1): 2118, 1715, 1651. 1H NMR (300 MHz, CDCl3) δ 1.22 (t, J = 7.2 Hz, 3H), 1.30 (s, 3H), 1.47 (s, 3H), 3.33 (s, 3H), 3.64 (d, J = 10.5 Hz, 1H), 3.84 (d, J = 10.5 Hz, 1H), 4.03 (d, J = 3.0 Hz, 1H), 4.10 (q, J = 7.2 Hz, 2H), 4.43− 4.67 (m, 6H), 5.94 (d, J = 3.6 Hz, 1H), 6.05 (d, J = 15.9 Hz, 1H), 7.08 (d, J = 15.9 Hz, 1H), 7.18−7.32 (m, 5H). 13C NMR (125 MHz, CDCl3) δ 14.2, 26.4, 26.9, 55.5, 60.5, 66.3, 70.1, 72.2, 80.4, 82.0, 82.3, 96.6, 104.9, 112.1, 121.8, 127.7, 127.8, 128.3, 136.8, 144.0, 165.9. Anal. Calcd for C23H31N3O8: C, 57.85; H, 6.54; N, 8.80. Found: C, 57.94; H, 6.60; N, 8.92. HR-MS (ESI): m/z calculated [M + Na]+, 500.2009; found, 500.2015. 8a(R)-Hydroxymethyl-1-deoxycastanospermine-3-one (3b). Reaction of 17 (0.43 g, 0.90 mmol) with TFA−water followed by hydrogenation with 10% Pd/C as described for 2b gave 3b (0.18 g, 91%) as a gummy solid: Rf = 0.48 (dichloromethane/MeOH, 8:2). [α]D27 +3.5 (c 3.50, CH3OH). IR (neat, ν, cm−1): 3200−3300 (br), 1642. 1H NMR (500 MHz, D2O) δ 1.95−2.15 (m, 1H), 2.35−2.40 (m, 1H), 2.42−2.58 (m, 2H), 3.29 (bd, J = 13.5 Hz, 1H), 3.52 (d, J = 12.5 Hz, 1H), 3.61 (d, J = 4.0 Hz, 1H), 3.90 (bs, 1H), 3.92 (dd, J = 13.5, 3.5 Hz, 1H), 3.93 (bt, J = 4.0 Hz, 1H), 4.19 (d, J = 12.5 Hz, 1H). 13C NMR (125 MHz, D2O) δ

22.5, 30.4, 39.7, 62.0, 68.2, 68.3, 69.6, 70.5, 178.8. Anal. Calcd for C9H15NO5: C, 49.76; H, 6.96; N, 6.45. Found: C, 49.88; H, 6.99; N, 6.32. HR-MS (ESI): m/z calculated [M + H]+, 218.1028; found, 218.1022; [M + Na]+, 240.0848; found, 240.0834. U-HPLC−MS: tR (min) 2.7, purity 97.67%. 1,2-O-Isopropylidene-3-O-benzyl-5S-deoxy-5-azido-5-C((methoxymethylene)methyl)-6,7-dideoxy-6(E)-ene-α-L-idofurano-1,8-dialdose (18). Reaction of 17 (0.48 g, 1.00 mmol) with diisobutylaluminum hydride as described for 7 gave 18 (0.37 g, 85%) as a viscous liquid. Rf = 0.40 (petroleum ether/EtOAc, 8:2). [α]D27 −10.4 (c 2.00, CH3OH). IR (neat, ν, cm−1): 2111, 1719, 1691. 1H NMR (300 MHz, CDCl3) δ 1.33 (s, 3H), 1.49 (s, 3H), 3.37 (s, 3H), 3.65 (d, J = 10.5 Hz, 1H), 3.87 (d, J = 10.5 Hz, 1H), 4.08 (d, J = 3.0 Hz, 1H), 4.45 (d, J = 11.1 Hz, 1H), 4.52 (d, J = 3.0 Hz, 1H), 4.57 (d, J = 11.1 Hz, 1H), 4.60 (d, J = 3.6 Hz, 1H), 4.65 (ABq, J = 6.6 Hz, 2H), 5.96 (d, J = 3.6 Hz, 1H), 6.27 (dd, J = 15.9, 7.8 Hz, 1H), 6.89 (d, J = 15.9 Hz, 1H), 7.15−7.40 (m, 5H), 9.27 (d, J = 7.8 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ, 26.4, 26.9, 55.6, 66.3, 69.9, 72.3, 80.0, 81.8, 81.9, 96.6, 105.0, 112.3, 128.0, 128.1, 128.3, 131.5, 136.4, 152.8, 193.2. Anal. Calcd for C21H27N3O7: C, 58.19; H, 6.28; N, 9.69. Found: C, 58.04; H, 6.47; N, 9.77. HR-MS (ESI): m/z calculated [M + Na]+, 456.1747; found, 456.1741. 8a(R)-Hydroxymethyl-1-deoxycastanospermine (3a). Reaction of 18 (0.35 g, 0.81 mmol) with TFA−water followed by hydrogenation with 10% Pd/C as described for 2a gave 3a (0.14 g, 87%) as a gummy solid: Rf = 0.17 (dichloromethane/MeOH, 3:1). [α]D27 +18.7 (c 0.45, CH3OH). IR (neat, ν, cm−1): 3200−3300 (br). 1H NMR (500 MHz, D2O) δ 1.85−1.95 (m, 1H), 2.15−2.35 (m, 3H), 3.08 (t, J = 11.5 Hz, 1H), 3.39−3.47 (m, 2H), 3.57 (d, J = 12.5 Hz, 1H), 3.64−3.76 (m, 3H), 3.84 (d, J = 9.0 Hz, 1H), 3.89 (d, J = 12.5 Hz, 1H). 13 C NMR (125 MHz, D2O) δ 17.7, 24.0, 51.6, 53.2, 58.8, 66.3, 68.7, 72.4, 75.4. Anal. Calcd for C9H17NO4: C, 53.19; H, 8.43; N, 6.89. Found: C, 53.30; H, 8.51; N, 6.79. HR-MS (ESI): m/z calculated [M + H]+, 204.1236; found, 204.1235. U-HPLC−MS: tR (min) 2.1, purity 95.99%. General Experimental of Glycosidase Inhibitory Activity. Glycosidase inhibition assay for the compounds 1a, 1b, 1c, 2a, 2b, 3a, and 3b was carried out by mixing 0.1 U cm−3 each of α- and βglucosidase, α- and β-galactosidase, and α-mannosidase, and the samples were incubated for 1 h at 37 °C. Initiation of α-glucosidase activity was done by addition of 10 mM p-nitrophenyl-α-D-glucopyranoside in 100 mM phosphate buffer of pH 6.8 and stopped by adding 2 cm3 of 0.1 M Na2CO3 after an incubation of 10 min at 37 °C. Enzyme action for α-/βgalactosidase was initiated by addition of 10 mM p-nitrophenyl-α-/β-Dgalactopyranoside (pNPG) as a substrate in 200 mM sodium acetate buffer. The reaction was incubated at 37 °C for 10 min and stopped by adding 2 cm3 of 200 mM borate buffer of pH 9.8. α-mannosidase activity was initiated by addition of 10 mM p-nitrophenyl-α-D-mannopyranoside as a substrate in 100 mM citrate buffer of pH 4.5. The reaction was incubated at 37 °C for 10 min and stopped by adding 2 cm3 of 200 mM borate buffer of pH 9.8. Glycosidase activity was determined by measuring absorbance of the p-nitrophenol released from pNPG at 420 nm using Molecular Devices Spectramax M5 mulitmode plate reader. One unit of glycosidase activity is defined as the amount of enzyme that hydrolyzed 1 mM p-nitrophenylpyranoside per minute under assay conditions. Inhibition constant (Ki) values were determined spectrophotometrically by measuring the residual hydrolytic activities of the glycosidases in the presence of compounds. Each assay was performed in the appropriate buffer at the optimal pH for the enzymes. The reactions were initiated by addition of enzyme to a solution of the substrate in the absence or presence of various concentrations of inhibitor. Reaction times were appropriate to obtain 10−20% conversion of the substrate in order to achieve linear rates. Approximate values of Ki were determined using a fixed concentration of substrate (around the KM value for the different glycosidases) and various concentrations of inhibitor. Full Ki determinations and enzyme inhibition mode were determined from the slope of Lineweaver−Burk plots and double reciprocal analysis. General Experimental of Immunomodulatory Activity. Lab reared (National Centre for Cell Science, Pune) inbred Swiss albino mice, approximately 8- to 10-week-old (20−25 g) were used for the experiments. We have strictly followed guidelines issued by the Institutional Animal Ethics Committee of National Centre for cell K



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Science, Pune, Government of India, during the maintenance and dissections of small animals. For the biochemical assay, Con A (Calbiochem, USA), RPMI1640 (Sigma Chemical Company, USA), fetal calf serum (FCS, GIBCO BRL.), IL-4 and IL-6 specific ELISA kits (Pierce Biotech) were used. Protocols for IL-4, IL-6, and IFN-γ estimation assay were as described in an earlier report.16 The data were statistically analyzed using a paired “t” test for the paired data or one way analysis of variance (ANOVA) followed by a Dunnet multiple comparisons post-test. A probability value of p < 0.05 was considered significant. Cell Proliferation Assay. The proliferation of the mouse splenocytes was assayed using the MTT assay. Mouse splenocytes (2 × 106 cells per well), pretreated with Con A alone (final concentration 10 μg/mL) or along with the test samples (final concentrations 0.5, 1, 2.5, and 5 μM) were incubated at 37 °C for 72 h under 5% CO2 in a RPMI medium containing 10% fetal calf serum (FCS). IL-4 Assay. The supernatants from the above experiments carried out with Con A alone and in conjunction with the compounds 1a, 1b, 1c, 2a, 2b, 3a, and 3b were collected and centrifuged at 2000g for 5 min and stored at −70 °C until further assay. The levels of IL-4 secreted from mice splenocytes were assayed using the cytokine specific ELISA kit (Pierce Biotech). Briefly, following addition of 50 μL of standard (different concentrations) samples to each well, the plate was incubated at 37 °C for 2 h. The control well contained only the medium containing FCS at the same final concentration. Subsequent to washing with buffer, 100 μL of the peroxidase conjugate was added to each well and incubated for 1 h. The plates were allowed to develop under dark after the addition of 100 μL of the substrate. The absorbance of the chromogen was read at 550 nm using Spectramax multimode plate reader with corrections at 450 nm. For the estimation of IL-4 in the samples, standard curve was plotted using known concentrations of the IL-4. According to the standard curve, the amount in the samples was determined. IL-6 Assay. The levels of IL-6 secreted from mice splenocytes were also detected using the cytokine specific ELISA kit (Pierce Biotech). Briefly, following addition of 50 μL of standards samples to each well, the plate was incubated at 37 °C for 2 h. Subsequent to washing 50 μL of biotinylated antibody reagent was added to each well and incubated for 1 h. The plate was allowed to develop in the dark after the addition of 100 μL of streptavidin−HRP solution followed by the addition of 100 μL of substrate. The absorbance at 550 nm was measured using Spectramax multimode plate reader with corrections at 450 nm. For the estimation of IL-6 in the samples, standard curve was prepared using known concentrations of the IL-6. According to the standard curve, the amount of the cytokine in the samples was calculated. IFN-γ Assay. IFN-γ was detected by sensitive ELISA kit procured from Life Technologies (USA). The concentrations of IFN-γ in the supernatant of control unstimulated cells and cells stimulated with Con A alone as well as Con A + the test samples for 24 h were estimated. The supernatant obtained from the Con A stimulated cells was used as the positive control.

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ACKNOWLEDGMENTS We are thankful to Department of Science and Technology, New Delhi (Project File SR/S1/OC-20/2010) for providing financial support. Author N.J.P. is thankful to UGC, New Delhi, for providing the Senior Research Fellowship. We thank Centre Instrumentation Facility of SPPU for analytical support.

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REFERENCES

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00951. 1 H and 13C NMR spectra of compounds 1a−c, 2a,b, 3a,b, 5, 6a,b, and 7−18; U-HPLC−MS chromatogram of compounds 1a−c, 2a,b, and 3a,b (PDF) Molecular formula strings (CSV)

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Quaternary Indolizidine and Indolizidone Iminosugars as Potential

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