Iminosugars Spiro-Linked with Morpholine-Fused 1,2,3-Triazole: Synthesis, Conformational Analysis, Glycosidase Inhibitory Activity, Antifungal Assay, and Docking Studies.

Synthesis of iminosugars 1, 2, 3a, and 4a and N-alkyl (ethyl, butyl, hexyl, octyl, decyl, and dodecyl) derivatives 3b-g and 4b-g spiro-linked with morpholine-fused 1,2,3-triazole is described. Conformation of the piperidine ring in each spiro-iminosugar was evaluated by 1H NMR spectroscopy, and conformational change in N-alkylated compounds 4b-g with respect to parent spiro-iminosugar 4a is supported by density functional theory calculations. Out of 16 new spiro-iminosugars, the spiro-iminosugars 3a (IC50 = 0.075 μM) and 4a (IC50 = 0.036 μM) were found to be more potent inhibitors of α-glucosidase than the marketed drug miglitol (IC50 = 0.100 μM). In addition, 3a (minimum inhibition concentration (MIC) = 0.85 μM) and 4a (MIC = 0.025 μM) showed more potent antifungal activity against Candida albicans than antifungal drug amphotericin b (MIC = 1.25 μM). In few cases, the N-alkyl derivatives showed increase of α-glucosidase inhibition and enhancement of antifungal activity compare to the respective parent iminosugar. The biological activities were further substantiated by molecular docking studies.

Introduction

Glycosidases play a key role in intestinal digestion, post-translational processing of glycoproteins, endoplasmic reticulum-associated degradation mechanisms, and the lysosomal catabolism of glycoconjugates.[1] Any malfunction in this process leads to discomforts in the body, and in case of long-run processes, one suffers from disease.[2] For example, disorders in functioning of amylase and/or α-glucosidase enzymes results in alten class="Species">ration in the blood glucose level, a condition noted as diabetes mellitus type-II (DM-II).[3] Patients suffering from long-term DM-II develop low immunity[4] and fall as a prey to a variety of comorbidities, such as retinopathy,[5] nephropathy,[6] neuropathy,[7] infections,[8] and many more.[9] In particular, sugar damp in diabetes nurtures the infections of Candida albicans yeast in mouth, skin, nails, and surroundings of urethral openings.[10] In such cases of comorbidity discomforts, an effective treatment is a challenge and prescription of two separate drugs is a common medicinal practice. Among a variety of antidiabetic drugs, an iminosugar (cyclic sugars with the nitrogen atom in place of the ring oxygen atom) class of compound, namely, glycet/miglitol I (Figure ) is consumed for the treatment of DM-II,[11] whereas, for the treatment of fungal infection, 1,2,4-triazole-containing drug fluconazole[12] is practiced. Alternatively, hybrid/chimeric drugs, in which two pharmacophores are present in one molecule and thus showing dual therapeutic mechanism, would have been the most appropriate choice.[13,14] In this direction, a number of hybrid drugs were synthesized and evaluated for biological activities.[14] Among these, iminosugars attached with other pharmacophores have attracted a great deal of interest.[1,15] For example, iminosugar fused with tetrazole II (Figure ) showed antitubercular as well as α-rhamnosidase inhibitory activity.[16] Iminosugar fused with imidazole ring, namely, nagstatin (III) is an N-acetyl-β-glucosaminidase inhibitor and acts as an anticancer agent,[1,17] whereas iminosugar fused with 1,2,4-triazole IV shows inhibition toward α-mannosidase along with anticancer activity.[1] A few examples of the iminosugars spiro-linked with other pharmacophores are also known.a,[18] Laroche et al. reported a titanium-mediated aminocyclopropanation of nitriles to give pyrrolidine iminosugar spiro-linked with cyclopropane V.[19] Vankar and co-workers utilized the RCM strategy in the synthesis of iminosugar spiro-linked with the sugar pyranose VI.[20] Compain and co-workers used C–H amination and RCM methodology to build 5-azaspiro[3,4]octane VII skeleton.[21]

Figure 1

Known iminosugars and target molecules.

Our group has exploited Corey-link approach with 3-oxo-1,2;5,6-diisopropylidene-d-gluco-furanose to give n class="Chemical">C3-azido-3-formyl-substituted d-gluco-furanose that was elaborated toward the synthesis of spiro-iminosugars VIIIa/VIIIb, which showed a promising α-glucosidase inhibitory activity.[22] In the continuation of our work, we now report the synthesis of tricyclic azepine 1, piperidine iminosugars 2, 3a–g, and iminosugars 4a–g spiro-linked with the morpholine-fused 1,2,3-triazol. The conformations 1C4/4C1 were obtained on the basis of the 1H NMR study and substantiated by density functional theory (DFT) studies. The glycosidase inhibitory and antifungal (on Candida albicans yeast cells) activities were evaluated. Among the 16 targeted molecules, iminosugar 3a (IC50 = 0.075 μM) and iminosugar 4a (IC50 = 0.036 μM) were found to be highly potent α-glucosidase inhibitors compared to miglitol I (IC50 = 0.100 μM). In addition, 3a (minimum inhibition concentration (MIC) = 0.85 μM) and 4a (MIC = 0.025 μM) were noticed to be highly potent antifungal agents compared to marketed drug amphotericin b (MIC = 1.25 μM). As the hybrid drugs are known to increase cellular uptake that augment the specific pharmacological activity,b,[23] an analogous trend was noticed with the spiro-iminosugars 1, 2, 3a–g, and 4a–g in the augmentation of the glycosidase inhibitory as well as antifungal activity due to the presence of iminosugar and triazole pharmacophores in the same molecule. Our results toward the synthesis, conformations, glycosidase, and antifungal activity as well as molecular docking studies of spiro-iminosugars are described herein.

Results and Discussion

Synthesis of Spiro-Iminosugars (1, 2, 3a–g, and 4a–g)

Recently, we have studied the role of gem-disubstituent effect in intramolecular alkyne-azide 1,3-dipolar cycloaddition (AAC) reaction with O-propargylated n class="Chemical">azido compounds IXa–d by experimental kinetic data and thermodynamic parameters obtained from computational studies (Figure ).[24] During this study, we noticed that the AAC reaction of 1,2;5,6-di-O-isopropylidene-3-O-propargyl-3-azido-d-glucofuranose IXd in the presence or absence of solvent occurred spontaneously at ambient temperature to afford morpholine-fused 1,2,3,-triazole moiety spirocyclic with d-gluco-furanose 5 in 89% yield. We thought of exploiting the same intermediate 5, by suitable manipulation of 5,6- and 1,2-O-isopropylidene groups of d-gluco-furanose, for the synthesis of targeted spiro-iminosugars.

Figure 2

Catalyst-free AAC reaction in IXa–d.

As shown in Scheme , selective deprotection of 5,6-O-isopropylidene group in 5 with n class="Chemical">AcOH/H2O (3:2) at 60 °C afforded diol 6. The primary hydroxyl group in 6 was protected as −OTs group using Bu2SnO and TsCl in triethylamine to get 7, which on nucleophilic displacement of C6–OTs group with NaN3 in dimethylformamide (DMF) afforded C6– azido compound 8 in 89% yield. In the next step, hydrolysis of 1,2-O-isopropylidene group in 8 with TFA/H2O (3:1) at 0 °C gave an anomeric mixture of hemiacetal (as evident from the 1H NMR of crude product), which on intramolecular reductive aminocyclization under hydrogenation condition, using 10% Pd/C in methanol at 80 psi pressure, gave azepine iminosugar spiro-linked with the morpholine-fused 1,2,3-triazole 1 in 71% yield.

Scheme 1

Synthesis of Spiro-Iminosugars 1 and 2

Reagents and conditions: (i) 60% aq AcOH, 60 °C, 3 h, 92%; (ii) Bu2SnO, TsCI, tetraethylammonium (TEA), room temperature (RT), 1 h, 97%; (iii) NaN3, tetrabutylammonium iodide (TBAI), DMF, 95 °C, 3 h, 89%; (iv) (a) TFA/H2O (3:1), 0 °C, 3 h, (b) 10% Pd/C, H2, 80 psi, 24 h, RT, 71%; (v) BnBr, NaH, THF, 0 °C to RT, 3 h, 75%; (vi) (a) TFA/H2O (3:1), RT, 3 h, (b) NalO4, acetone/water (8:2), 3 h, (c) 10% Pd/C, H2, 120 psi, 30 h, 68%.

Reagents and conditions: (i) 60% aq AcOH, 60 °C, 3 h, 92%; (ii) n class="Chemical">Bu2SnO, TsCI, tetraethylammonium (TEA), room temperature (RT), 1 h, 97%; (iii) NaN3, tetrabutylammonium iodide (TBAI), DMF, 95 °C, 3 h, 89%; (iv) (a) TFA/H2O (3:1), 0 °C, 3 h, (b) 10% Pd/C, H2, 80 psi, 24 h, RT, 71%; (v) BnBr, NaH, THF, 0 °C to RT, 3 h, 75%; (vi) (a) TFA/H2O (3:1), RT, 3 h, (b) NalO4, acetone/water (8:2), 3 h, (c) 10% Pd/C, H2, 120 psi, 30 h, 68%.

For the synthesis of spiro-iminosugar 2, it is necessary to remove anomeric C1 n class="Chemical">carbon atom in compound 8, for which it is necessary to protect C5–OH group with benzyl derivative. Thus, 8 was reacted with NaH and benzyl bromide in THF to get 6-azido 5-benzyloxy derivative 9 (Scheme ). Hydrolysis of 1,2-acetonide in 9 with TFA/H2O (3:1) followed by treatment with NaIO4 in acetone/H2O (8:2) and intramolecular reductive aminocyclization of C6– azido group with C2– formyl group using H2 in 10% Pd/C at 120 psi afforded piperidine iminosugar spiro-linked with morpholine-fused 1,2,3-triazole 2 in 68% yield.

The synthesis of iminosugar 3a started from the n class="Chemical">diol 6. Thus, reaction of 6 with NaIO4 in acetone/H2O (8:2) at 0 °C to RT followed by treatment with NaBH4 in MeOH at 0 °C afforded d-xylofuranose derivative 10 (Scheme ). Compound 10 was treated with TsCl in pyridine to give −OTs derivative 11, which on nucleophilic displacement using NaN3 in DMF afforded C5– azido compound 12. Finally, hydrolysis of 1,2-acetonide group in 12 with TFA/H2O (3:1) followed by intramolecular aminocyclization using H2 in 10% Pd/C at 100 psi afforded 3a in 86% yield. In general, the N-alkylated derivatives of iminosugars showed enhanced glycosidase inhibitory activity compared to their parent iminosugars.[25] Thus, reaction of 3a with different alkyl bromides (alkyl = ethyl, butyl, hexyl, octyl, decyl, and dodecyl) in the presence of K2CO3 in DMF at 80 °C afforded corresponding N-alkyl derivatives 3b–g in good yield (Scheme ).

Scheme 2

Synthesis of Spiro-Iminosugars 3a–g and 4a–g

Reagents and conditions: (i) (a) NalO4, acetone/water (4:1), 0 °C to RT 2 h, (b) NaBH4, MeOH, 0 °C, 1 h, 88% two steps; (ii) TsCI, pyridine, DCM, 0 °C to RT, 4 h, 95%; (iii) NaN3, TBAI, DMF, 95 °C, 6 h 86%; (iv) 10% Pd/C, H2, 100 psi, 30 h, RT, 86%; (v) for b ethylbromide, for c butylbromide, for d hexylbromide, for e octylbromide, for f decylbromide, for g dodecylbromide, K2CO3, DMF, 80 °C, 1 h, and for 3b–g, 60 °C, 2 h for 4b–g; (vi) TBDMSCI, imidazole, DCM, 2 h, RT, 95%; (vii) (a) OTf2, pyridine, DMAP, DCM, 0 °C, 30 min, (b) NaN3, DMF, 65 °C, 1 h, 92% two steps; (viii) (a) TFA/H2O (3:1), 3 h, 0 °C, (b) 10% Pd/C, H2, 120 psi, 24 h, RT, 78%.

Reagents and conditions: (i) (a) NalO4, acetone/n class="Chemical">water (4:1), 0 °C to RT 2 h, (b) NaBH4, MeOH, 0 °C, 1 h, 88% two steps; (ii) TsCI, pyridine, DCM, 0 °C to RT, 4 h, 95%; (iii) NaN3, TBAI, DMF, 95 °C, 6 h 86%; (iv) 10% Pd/C, H2, 100 psi, 30 h, RT, 86%; (v) for b ethylbromide, for c butylbromide, for d hexylbromide, for e octylbromide, for f decylbromide, for g dodecylbromide, K2CO3, DMF, 80 °C, 1 h, and for 3b–g, 60 °C, 2 h for 4b–g; (vi) TBDMSCI, imidazole, DCM, 2 h, RT, 95%; (vii) (a) OTf2, pyridine, DMAP, DCM, 0 °C, 30 min, (b) NaN3, DMF, 65 °C, 1 h, 92% two steps; (viii) (a) TFA/H2O (3:1), 3 h, 0 °C, (b) 10% Pd/C, H2, 120 psi, 24 h, RT, 78%.

Iminosugar 4a was prepared from compound 6 (Scheme ). Thus, selective protection of C6– primary hydroxy functionality in 6 with n class="Chemical">TBDMSCl in the presence of imidazole in dry dichloromethane afforded C6–OTBDMS compound 13. Reaction of 13 with triflic anhydride in Et3N in dry dichloromethane afforded C5–OTf derivative, which was directly treated with NaN3 in DMF to get C5– azido derivative 14 with inversion of configuration at C5, as evident from the 1H NMR spectrum (J4,5 = 1.5 Hz in l-ido derivative against J4,5 = 6.0 Hz in d-gluco derivative in 13). Finally, cleavage of 1,2-acetonide group in 14 with TFA/H2O (3:1) and intramolecular reductive aminocyclization with H2 in 10% Pd/C in MeOH at 120 psi and room temperature afforded 4a in 78% yield. N-Alkylations of 4a with alkyl bromides (alkyl = ethyl, butyl, hexyl, octyl, decyl, and dodecyl) using K2CO3 in DMF at 60 °C afforded N-alkyl iminosugars 4b–g, respectively (Scheme ).

Conformational Analysis for 2, 3a–g, and 4a–g

It is known that six-membered piperidine n class="Chemical">iminosugars exist in 4C1 or 1C4 conformations, depending on the substituents present on the ring.[26] These conformations play a key role in deciding the binding property of iminosugars with enzymes rendering the glycosidase inhibition activity.[27] In view of this fact, conformational aspects of newly synthesized spiro-linked iminosugars 2, 3a–g, and 4a–g were studied using 1H NMR spectroscopy at neutral pH. In case of iminosugar 2, the assignment of H3 and H4 proton signals was difficult as these protons were obscured in the deuterated water (HOD). To resolve the spectra, we studied the temperature-dependent 1H NMR spectrum of 2 in D2O as the HOD signal at δ 4.85 and 22 °C is known to shift upfield with increasing the temperature, and at 72 °C, it appears at δ 4.30.[28] Therefore, the 1H NMR spectrum of 2 in D2O was recorded at different temperatures of 22, 32, 52, and 72 °C (Figure S13). At 72 °C, the doublet at δ 4.72 with J = 3.2 Hz was assigned to H3 and a narrow multiplate at δ 4.20 was assigned to H4 on the basis of the DEPT-135, 1H–1H COSY, and 1H–13C HSQC NMR techniques (Figures S14–S16). The protons appearing at δ 3.10 (dd, J = 14.4, 1.5 Hz, 1H) and δ 2.97 (dd, J = 14.4, 2.0 Hz, 1H) were assigned to H5e and H5a, respectively. Other signals were unchanged, indicating no change in the conformation of 2 even at high temperature. With these 1H NMR data, two conformations 4C1 and 1C4 were considered for compound 2. The coupling constants between H5a (axial) and H4 and between H4 and H3 are decisive in determining the conformation.

In the 1H NMR sn class="Chemical">pectrum of 2, a small coupling constant value between H5a/H5e and H4 (J ≈ 1.7 Hz) indicated the cis-relative stereochemistry with the equatorial orientation of H4 (Figure ). In accordance with this, H3 (axial) appeared as a doublet with a small coupling constant value (J4,5 = 3.2 Hz), showing cis-relative stereochemistry between H3 and H4. On the basis of these data, the 1C4 conformation was assigned to spiro-iminosugar 2 (Figure ). The single crystal for 2 was developed in CHCl3/MeOH, and X-ray crystallographic analysis supported the 1C4 conformation (Figure ).

Figure 3

Conformations of spiro-iminosugars 2, 3a–g, and 4a–g and ORTEP diagram of 2 and 3a.

The spiro-linked n class="Chemical">iminosugar 3a is symmetric. Therefore, H1 and H5 protons (both axial and equatorial) as well as H2 and H4 protons are identical. The equivalent H1a and H5a showed dd at δ 2.84 with J = 13.4 and 10.8 Hz. The large coupling constant of 10.8 Hz between H1a and H2 or between H5a and H4 requires axial orientation of both H2 and H4 protons with trans-relative stereochemistry, thus suggesting the 4C1 conformation to 3a (Figure ). The single crystal of 3a (Figure ) was obtained in CHCl3/MeOH, and X-ray crystallographic study supported the 4C1 conformation. The N-alkyl derivatives of 3a, namely, 3b–g also showed 4C1 conformation analogous to parent compound 3a on the basis of their nearly identical J values (Figure ).

In case of spiro-linked n class="Chemical">iminosugar 4a, the H1a (axial) proton appeared at δ 3.07 as a dd (Figure ) with J = 14.8 and 3.3 Hz and H1e was observed at δ 3.60 as dd with J = 14.4 and 3.3 Hz. Accordingly, H2 appeared at as a triplet with J = 3.3 Hz. The small coupling constant value between H1a and H2 requires equatorial orientation of H2 (cis-relative stereochemistry), thus accounting for the 1C4 conformation of compound 4a. This fact is further supported by H4, which appeared as a doublet at δ 4.11 with small J4,5 = 2.6 Hz, indicating equatorial orientation of H4. We presume that the 1C4 conformation is stabilized due to the equatorial orientation of CH2OH as well as intramolecular H-bonding between the C2 and C4–OH groups, as shown in Figure . In case of N-alkyl derivatives 4b–g, we noticed the change in conformation (Figure ) as H1a appeared as dd with J ≈ 12.9 and ≈ 8.0 Hz. The large coupling constant between H1a and H2 requires relative trans-diaxial orientation suggesting 4C1 conformation.

The conformational preferences were supported by the DFT calculations. Thus, DFT-optimized geometries of conformations 1C4/4C1 for compounds 3a, 3c, 4a, and 4c were subjected for hydrogen-bonding interaction analysis (Table S1) and are represented in Figure .c

Figure 4

Geometrically DFT-optimized conformations of 3a, 3c, 4a, and 4c and their energies.

As shown in Figure a, the adopted 4C1 conformation of 3a that is stabilized by intramolecular O6···H–O4, N3···H–O2, and O2···H–C6 hydrogen-bonding interactions was found to be energetically more stable by −5.64 kcal/mol over its 1C4 conformation. In 4C1 conformation, the O6···H–O4 interaction was found to be strong as compared to 1C4 conformation. Further, the weak interactions between N3···H–C2 and N3···H–C4 provided additional stability to 4C1 conformation. Similarly, N-butyl analogue 3c also adopted the same 4C1 conformation, which is stable by −9.41 kcal/mol as compared to its 1C4 conformation (Figure b, Table S1). This conformation was stabilized by n class="Chemical">hydrogen-bonding interactions between O6···H–O4, N3···H–O2, and N3···H–C2.

In case of compound 4a, the DFT-optimized 1C4 conformation, which is stabilized by O7···H–O4, O4···H–O2, O6···H–N1, and N1···H–O2 interactions (Figure c), was found to be more stable by −4.39 kcal/mol as compared to the 4C1 conformation. The preference of bulkier C6 hydroxy methylene group toward the equatorial orientation (that avoids 1,3-diaxial steric interactions) might be contributing in stability of 1C4 conformation by lowering its energy. However, the optimized geometry of N-butyl derivative 4c was adopted 4C1 conformation, which is more stable by −6.90 kcal/mol over its 1C4 (Figure d). The preference of 4C1 conformation was supported by strong H-bonding between O7···H–O4, O4···H–O6, and N3···H–O2 and by weak N3···H–C2, N3···H–C4, and O2···H–C4 interactions. This leads to axial orientation of hydroxy methylene group, which is stabilized by the O4···H–O6 interaction in 4c.

Biological Activities

Glycosidase Inhibitory Activity

The newly synthesized spiro-iminosugars (1, 2, 3a–g, and 4a–g) were evaluated for their inhibitory activity against a number of commercially available glycosidases, such as α-n class="Chemical">glucosidase (rice) [E.C.3.2.1.20], β-glucosidase (rice) [E.C.3.2.1.21], α-galactosidase (bovine liver) [E.C.3.2.1.22], β-galactosidase (bovine liver, cytosolic) [E.C.3.2.1.23], and α-mannosidase (Jack beans) [E.C.3.2.1.24], with reference to known standards, namely, miglitol (N-hydroxyethyl 1-deoxynojirimycin, trade name Glycet) and 1-deoxynojirimycin (DNJ). The corresponding IC50 values and inhibition constants (Ki) were determined from Lineweaver–Burk plots (Figures S55–S59), and the results are summarized in Table . The six-membered (piperidine) and seven-membered (azepine) iminosugars are known to be α-glucosidase inhibitors among various glycosidases, and the same trend was noticed with 1, 2, 3a–g, and 4a–g that showed good to potent inhibition of α-glucosidase. In addition, compounds 1, 2, 3a–g, and 4a–g were found to be selective inhibitors of α-glucosidase (Table ), wherein the spiro-iminosugar 2 (IC50 = 0.159 and Ki = 0.129 μM) was found to be a weak inhibitor of α-glucosidase, whereas 4a (IC50 = 0.036 and Ki = 0.033 μM) was found to a better inhibitor of α-glucosidase than miglitol (IC50 = 0.1 and Ki = 0.081 μM). This fact could be attributed to the synergetic effect of two pharmacophores, namely, iminosugar and 1,2,3-triazole (known to be α-glucosidase inhibitor).[29]

Table 1

IC50 and Ki (in μM) Values for Synthesized Compounds and Standards Miglitol and DNJ

compound		α-glucosidase (rice)	β-glucosidase (rice)	α-galactosidase (bovine)	β-galactosidase (bovine)	α-mannosidase (Jack bean)
miglitol	IC50	0.100	342	NI	NI	NI
	Ki	0.081	320	NI	NI	NI
DNJ	IC50	0.05	327	890	NI	NI
	Ki	0.012	450	1030	NI	NI
1	IC50	0.069	336	695	NI	NI
	Ki	0.012	628	896	NI	NI
2	IC50	0.159	428	993	NI	NI
	Ki	0.129	456	932	NI	NI
3a	IC50	0.075	285	632	NI	NI
	Ki	0.098	240	1300	NI	NI
3b	IC50	0.032	84	84	81	79
	Ki	0.025	14	14	23	80
3c	IC50	0.033	89	73	81	66
	Ki	0.028	59	58	33	90
3d	IC50	0.035	251	84	113	73
	Ki	0.028	152	68	134	43
3e	IC50	0.037	286	86	73	91
	Ki	0.022	191	75	68	56
3f	IC50	0.038	291	12	64	98
	Ki	0.031	93	04	38	61
3g	IC50	0.049	286	88	86	101
	Ki	0.042	135	71	38	95
4a	IC50	0.036	120	578	NI	NI
	Ki	0.033	798	1650	NI	NI
4b	IC50	0.030	295	93	191	63
	Ki	0.024	214	82	69	33
4c	IC50	0.032	298	86	58	74
	Ki	0.025	171	58	39	65
4d	IC50	0.032	287	87	96	78
	Ki	0.027	215	55	84	58
4e	IC50	0.032	241	82	89	91
	Ki	0.026	ND	61	79	61
4f	IC50	0.037	>1000	88	74	86
	Ki	0.024	ND	67	52	58
4g	IC50	0.034	>1000	87	76	68
	Ki	0.019	ND	54	54	69

From Table , it is evident that the α-glucosidase inhibitory activity of N-n class="Chemical">alkylated iminosugars 3b–g and 4b–g was significantly improved as compared to their parent compounds 3a and 4a, respectively. At the same time, N-alkylated compounds 3b–g/4b–g were found to inhibit other enzymes in a micromolar range (Table ) and suggest fall in selectivity of enzyme inhibition for them in contrast to 3a and 4a, respectively. Elongation of alkyl chain from ethyl to dodecyl, however, showed decrease in α-glucosidase inhibition with each higher homologue of alkyl chain.

Antifungal Activity

The use of 1,2,3-triazole moiety in advanced antifungal drug designing provided a platform to synthesize new antifungal agents containing n class="Chemical">1,2,3-triazole pharmacophore.[30] In view of this, we have investigated the spiro-iminosugars 1, 2, 3a–g, and 4a–g for their antifungal activities against C. albicans in comparison to a well-known marketed antifungal drug, namely, amphotericine b, recommended for infection of C. albicans (Table ). The MIC value of 3a (0.85 μg/mL) is found to be less than that of amphotericine b (1.25 μg/mL), whereas the value of 4a (0.025 μg/mL) was found to be ∼50 times higher active than that of amphotericine b. Compounds 1 (2.5 μg/mL) and 2 (1.25 μg/mL) are, respectively, either weak or comparatively active with the marketed drug.

Table 2

Antifungal Activities of 1, 2, 3a–g, and 4a–ga

compound	MIC	compound	MIC	compound	MIC
amphotericin b	1.25	1	2.5	2	1.25
3a	0.85	3b	>1.25	3c	>2.5
3d	>2.5	3e	>2.5	3f	>1.25
3g	1.25	4a	0.025	4b	>1.25
4c	0.025	4d	0.020	4e	>1.25
4f	>1.25	4g	>1.25

Minimum inhibition concentration (MIC) values in μg/mL.

Minimum inhibition concentn class="Species">ration (MIC) values in μg/mL.

Among N-alkylated derivatives, compound 4c (MIC = 0.025 μg/mL) was found to be comparably active to parent molecule 4a, whereas compound 4d (MIC = 0.020 μg/mL) showed higher activity than 4a. The MIC values of compounds 3b–g (Table ) are higher than those of amphotericine b (1.25 μg/mL), whereas for 4b–g, various MIC values are found. Compounds 4b and 4e–g (MIC > 1.25 μg/mL) are weakly or less active than 4a. Thus, all other N-n class="Chemical">alkyl derivatives (except 4b and 4c) were found to be less antifungal than their parent compounds. The confocal images of all screened compounds showed aggregation of the molecules at the fungal cell surface and exhibited cell wall disruption (Figure ).

Figure 5

Confocal images of compounds 1, 2, 3a–g, and 4a–g with yeast cell. The red arrows show site of cell wall disruption.

Molecular Docking Studies

Glycosidases and Ligand Interactions

The observed biological activities were supported by molecular docking studies. Thus, molecular interaction of inhibitor molecules with rice α-n class="Chemical">glucosidase was analyzed by docking studies. Binding scores of synthesized spiro-iminosugars (1, 2, 3a–g, and 4a–g) are illustrated in Table S3 and binding poses on the active site of α-glucosidase (rice) enzyme are shown in Figure . Corroboration of binding free energies with the inhibition kinetic studies indicated that among synthesized compounds, molecules 1, 2, 3a, and 4a are potent competitive inhibitors of rice α-glucosidase. All screened molecules with their respective conformations (1C4 or 4C1 for piperidine iminosugars) form favorable contacts with binding pocket of α-glucosidase. Analysis of polar contacts and other weak interactions (electrostatic and π-interaction) of these compounds showed that the higher binding affinity is presumably attributed to the formation of higher number of stable intermolecular hydrogen bonds between the reactive group of compounds and ASP204, ILE205, SER469, ARG473, ASN477, and LYS478 at the binding site of the enzyme (Figure ). However, several π-alkyl interactions observed between ligand aromatic ring and interacting residues of enzyme assist polar interaction. In case of N-alkyl derivatives 3b–g and 4b–g, it has been observed that binding of these ligands with enzyme was unfavorable as compared to the parental compounds (Table S3). This suggests that addition of N-alkyl chain to 3a and 4a reduces the number of interactions with binding site residues. As a result, changes in functional group lead to differential interactions with binding pocket of enzyme. Minute deviation in biological activity with respect to molecular docking observations might be due to variations in ligand conformation, in vitro assay condition, solvation of ligand, and ligand charge state.

Figure 6

Docking images and binding energies (B.E.) of iminosugars with α-glucosidase (rice).

Docking images and binding energies (B.E.) of iminosugars with α-n class="Chemical">glucosidase (rice).

Molecular Docking Studies for Antifungal Activity

Antifungal potential of spiro-iminosugars (1, 2, 3a, 4a, 4c, and 4d) was analyzed using a docking simulation in relation with amphotericine b as a positive control. The positive binding energy (10.5 kcal/mol) for n class="Chemical">amphotericine b suggests that its interaction with ergosterol is predominated by unfavorable interaction over favorable contacts (Figure ). Analogously, we have predicted binding of spiro-iminosugars with ergosterol, a component of fungal cell membranes, which might result in the disruption of the fungal cell wall and lead to fungal cell death. Compounds 1, 2, and 3a showed approximately equal binding energies (−1.9, −1.8, and −1.8 kcal/mol) with ergosterol, whereas the highest binding energy of −2.2 kcal/mol was noticed for compound 4a. All compounds exhibited π-alkyl interaction as shown in Figure . The N-alkylated derivatives 4c (B.E. −2.4 kcal/mol) and 4d (B.E. −2.6 kcal/mol) (Figure ) exhibit strong binding with ergosterol as compared to their parent compound 4a. This increase in binding energy in derivatives 4c and 4d than their parent iminosugar 4a is in agreement with the in vitro antifungal data (Table ).

Figure 7

Docking of 1, 2, 3a, 4a, 4c, and 4d with ergosterol component of cell wall of C. albicans (binding energy (B.E.) is in kcal/mol).

Docking of 1, 2, 3a, 4a, 4c, and 4d with ergosterol component of cell wall of n class="Species">C. albicans (binding energy (B.E.) is in kcal/mol).

Conclusions

In conclusion, the AAC reaction of 3-O-propargyl-3-azido-d-glucofuranose derivative (IXd) gave adduct 5 that was used as a common intermediate, by judicious n class="Species">manipulation of the 1,2- and 5,6-O-isopropylidene groups, in the synthesis of library of spiro-iminosugars (1, 2, 3a–g, and 4a–g) linked with the 1,2,3-triazole-fused morpholine. The presence of two pharmacophores, such as iminosugar and triazole, showed dual biological activity. In addition, the synergetic effect of two pharmacophores led to augmentation of the glycosidase inhibitory as well as antifungal activity. Among 16 examples, the spiro-iminosugars 3a and 4a were found to be more potent inhibitors of α-glucosidase than miglitol as well as showed high antifungal activity against C. albicans than amphotericin b. The N-alkyl derivatives showed increase of α-glucosidase inhibition and enhancement of antifungal activity as compared to their respective parent iminosugar. Thus, the synthesized spiro-iminosugars may open a new era of potential chimeric drugs in comorbidity treatments.

Experimental Section

General Methods for Synthesis

Melting points were recorded using Thomas–Hoover melting point apparatus and are uncorrected. IR sn class="Chemical">pectra were recorded with an FTIR spectroscope as a thin film or using KBr pellets and are expressed in cm–1. 1H NMR (300, 400, or 500 MHz) and 13C NMR (100 or 125 MHz) spectra were recorded using CDCl3, CD3OD, or D2O as solvent(s). Chemical shifts were reported in δ unit (parts per million) with reference to TMS as an internal standard, and coupling constant (J) values were given in Hertz. Optical rotations were measured using a polarimeter at 22 °C. High-resolution mass spectra (HRMS) were obtained in positive-ion electrospray ionization (ESI) mode using 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). Reactions were carried out in oven-dried glassware under dry N2 atmosphere. Methanol, dichloromethane, and THF were purified and dried according to reported methods. Petroleum ether (PE) was a distillation fraction between 40 and 60 °C. The 10% Pd/C was purchased from Aldrich. After neutralization, workup involves washing of combined organic layer with water and brine, drying over anhydrous sodium sulfate, and evaporating the solvent under reduced pressure, followed by vacuum drying.

Synthesis of 6

Compound 5 (2.5 g, 7.07 mmol) was dissolved in 60% aq AcOH (15 mL) and heated at 60 °C for 3 h. RM was directly concentrated in a rotary evaporator under vacuum, and the thick residue obtained was dissolved in ethyl acetate (EtOAc) and washed with saturated aq NaHCO3 and distilled water. The organic layer was evaporated to dryness to give a thick syrup. Column purification of the syrup was done with eluting of a 7:3 mixture of ethyl acetate and petroleum ether to ease the synthesis of 6 (2.04 g, 92%) as a solid. R = 0.15, EtOAc; mp = 66–68 °C; [α]D22 = −21.7 (c 0.108, methanol); IR (KBr, ν, cm–1) 3433, 2987, 2937, 1639, 1554, 1458, 1379, 1091, 869, 732; 1H NMR (300 MHz, CDCl3) δ 7.56 (s, 1H), 6.26 (d, J = 3.6 Hz, 1H), 4.98 (ABq, J = 15.1 Hz, 2H), 4.65 (d, J = 3.6 Hz, 1H), 4.39 (ABq, J = 12.6 Hz, 2H), 4.28 (d, J = 8.8 Hz, 1H), 3.74 (dd, J = 11.6, 3.2 Hz, 1H), 3.64 (dd, J = 11.6, 5.1 Hz, 1H), 3.12 (ddd, J = 8.8, 5.1, 3.2 Hz, 1H), 1.60 (s, 3H), 1.35 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 131.6, 128.3, 113.5, 105.7, 85.2, 81.1, 69.9, 68.9, 65.9, 64.1, 62.7, 26.9, 26.5; HRMS calculated for C13H19N3O6Na [M + Na]+ 336.1169, Found: 336.1167.

Synthesis of 7

Compound 6 (0.800 g, 2.55 mmol) was dissolved in dry DCM (10 mL), to which n class="Chemical">Bu2SnO (0.063 g, 10% mol) was added and stirred for 10 min, followed by pinchwise addition of TsCl (0.535 g, 2.81 mmol) and dropwise addition of TEA (1.07 mL, 7.66 mmol). Reactions were maintained at RT for 1 h and then quenched by 1 M aq HCl solution and extracted by dichloromethane (2 × 10 mL). The dichloromethane layer was concentrated, and the obtained thick residue was purified by column chromatography using EtOAc/PE (3:2) as mobile phase 7 (1.16 g, 97%) observed as a white solid. R = 0.56, EtOAc/PE (4:1); mp = 86–88 °C; [α]D22 = −23.2 (c 0.102, methanol); IR (KBr, ν, cm–1) 3433, 2985, 2933, 1597, 1494, 1452, 1357, 1176, 1095, 985, 819, 667, 553; 1H NMR (500 MHz, CDCl3) δ 7.75 (d, J = 8.1 Hz, 2H), 7.53 (s, 1H), 7.33 (d, J = 8.1 Hz, 2H), 6.16 (d, J = 3.5 Hz, 1H), 4.94 (s, 2H), 4.56 (d, J = 3.5 Hz, 1H), 4.38 (ABq, J = 12.6 Hz, 2H), 4.22 (d, J = 8.9 Hz, 1H), 4.15 (dd, J = 10.4, 2.4 Hz, 1H), 4.06 (dd, J = 10.4, 5.8 Hz, 1H), 3.42 (ddd, J = 10.4. 5.8, 2.4 Hz, 1H), 2.44 (s, 3H), 1.57 (s, 3H), 1.34 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 145.0, 132.4, 131.5, 129.9 (2C), 128.4, 128.0 (2C), 113.7, 105.8, 85.2, 80.4, 71.8, 69.7, 67.0, 65.9, 62.7, 27.1, 26.5, 21.7; HRMS calculated for C20H25N3O8SNa [M + Na]+ 490.1255, Found: 490.1254.

Synthesis of 8

Under N2 atmosphere, into a mixture of 7 (1.0 g, 2.14 mmol), n class="Chemical">NaN3 (0.166 g, 2.57 mmol), and TABI (0.079 g, 10% mol), dry DMF (10 mL) was added and heated to 95 °C for 3 h. RM was concentrated directly, water (5 mL) was added, and extraction was carried out by EtOAc (2 × 10 mL). A thick residue was obtained, which was further purified by EtOAc/PE (3:2) as mobile phase for column chromatography. A white solid 8 was observed (0.644 g, 89%). R = 0.65, EtOAc/PE (4:1); mp = 151–153 °C; [α]D22 = −32.0 (c 0.104, methanol); IR (KBr, ν, cm–1) 3400, 2989, 2935, 2104, 1664, 1440, 1381, 1220, 1093, 929, 869, 771, 553; 1H NMR (500 MHz, CDCl3) δ 7.53 (s, 1H), 6.23 (d, J = 3.5 Hz, 1H), 5.03 (d, J = 15.2 Hz, 1H), 4.93 (d, J = 15.2 Hz, 1H), 4.65 (d, J = 3.5 Hz, 1H), 4.43 (d, J = 12.7 Hz, 1H), 4.33 (d, J = 12.7 Hz, 1H), 4.29 (d, J = 8.5 Hz, 1H), 3.46 (dd, J = 10.5, 3.2 Hz, 1H), 3.42 (dd, J = 10.5, 5.2 Hz, 1H) 3.17 (ddd, J = 8.5, 5.2, 3.2 Hz, 1H), 1.60 (s, 3H), 1.35 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 131.0, 128.4, 113.8, 105.6, 85.4, 82.1, 69.6, 68.0, 66.4, 62.8, 54.0, 27.0, 26.5; HRMS calculated for C13H18N6O5Na [M + Na]+ 361.1231, Found: 361.1237.

Synthesis of 1

To compound 8 (200 mg, 0.591 mmol), 75% aq TFA (5 mL, precooled to 0 °C) was added and stirred for 3 h. After concentn class="Species">ration under vacuum, saturated aq NaHCO3 (5 mL) was added and extracted with EtOAc (2 × 5 mL). The organic layer was filtered through a 60–120 mesh silica bed with eluting EtOAc. A thick residue was observed on concentration, which was directly dissolved in distilled methanol (5 mL) and hydrogenated using 10% Pd/C (catalytic) at RT and 80 psi pressure of H2 for 24 h and filtered through a celite bed. MeOH was concentrated and purified by column chromatography to obtain a syrup with MeOH/CHCl3 (1:4) as mobile phase. Amorphous solid (0.107 g, 71% over two steps) of 1 was observed. R = 0.50, MeOH/CHCl3 (1:4); mp = 235 °C (dec.); [α]D22 = −42.8 (c 0.101, methanol); IR (KBr, ν, cm–1); 2968, 2106, 1639, 1450, 1377, 1217, 1166, 1095, 916, 868, 748, 700; 1H NMR (500 MHz, D2O) δ 7.60 (s, 1H), 4.93 (ABq, J = 15.8 Hz, 2H), 4.52 (ddd, J = 8.1, 3.4, 1.7 Hz, 1H), 4.47 (dd, J = 8.1, 2.4 Hz, 1H), 4.47 (d, J = 8.1, 2.5 Hz, 1H), 4.43 (d, J = 1.7 Hz, 1H), 4.39 (d, J = 12.8 Hz, 1H), 4.32 (d, J = 12.8 Hz, 1H), 3.70 (dd, J = 14.2, 2.5 Hz, 1H), 3.52–3.38 (m, 3H); 13C NMR (125 MHz, D2O) δ 133.2, 128.8, 75.1, 68.6, 67.1, 66.3, 65.9, 62.0, 46.5, 45.7; HRMS calculated for C10H17N4O4 [M + H]+ 257.1249, Found: 257.1255.

Synthesis of 9

Compound 8 (0.300 g, 0.886 mmol) in THF (5 mL) was added dropwise to a precooled (0 °C) susn class="Chemical">pension of NaH (0.042 g, 1.06 mmol) in THF (5 mL), followed by drowise addition of BnBr (0.120 mL, 0.975 mmol). The reaction was maintained initially at 0 °C for 30 min and then at RT for 2.5 h. The reaction was quenched with saturated aq ammonium chloride, extracted with EtOAc (2 × 5 mL), and concentrated in vacuo at reduced pressure, which gave a thick residue. Purification of this residue was done by column chromatography using EtOAc/PE (2:3) as mobile phase. A white solid 9 (0.284 g, 75%) was observed. R = 0.40 EtOAc/PE (2:3); mp = 108–110 °C; [α]D22 = −80.6 (c 0.105, methanol); IR (KBr, ν, cm–1); 2968, 2935, 2106, 1550, 1450, 1494, 1095, 1018, 868, 748; 1H NMR (500 MHz, CDCl3) δ 7.55 (s, 1H), 7.35–7.29 (m, 3H), 7.07 (dd, J = 6.3, 2.9 Hz, 2H), 6.21 (d, J = 3.4 Hz, 1H), 4.83 (d, J = 14.8 Hz, 1H), 4.57 (d, J = 12.1 Hz, 1H), 4.45 (d, J = 10.8 Hz, 1H), 4.42 (d, J = 3.4 Hz, 1H), 4.38 (d, J = 8.9 Hz, 1H), 4.12 (d, J = 14.8 Hz, 1H), 4.05 (d, J = 12.1 Hz, 1H), 3.85 (dt, J = 8.9, 2.8 Hz, 1H), 3.73 (dd, J = 13.5, 2.8 Hz, 1H), 3.58 (d, J = 10.8 Hz, 1H), 3.50 (dd, J = 13.5, 2.8 Hz, 1H), 1.62 (s, 3H), 1.37 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 136.6, 132.0, 128.6(2C), 128.5, 128.2, 128.0(2C), 113.8, 105.9, 85.4, 77.8, 71.9, 70.0, 65.6, 62.4, 50.4, 27.4, 26.8; HRMS calculated for C20H24N6O5Na [M + Na]+ 451.1705, Found: 451.1711.

Synthesis of 2

Compound 9 (0.200 g, 0.591 mmol) was dissolved in 75% aq TFA (5 mL, precooled to 0 °C), stirred for 3 h, and concentrated under vacuum. EtOAc (5 mL) was added and organic layer was washed with saturated aq NaHCO3. The EtOAc layer was filtered through a 60–120 mesh silica bed with eluting more EtOAc and concentrated. The thick residue obtained was directly dissolved in acetone/water (8:2, 15 mL) and cooled to 0 °C. NaIO4 (0.573 g, 2.68 mmol) was pinchwise added to this mixture over 30 min, and the reaction was maintained at 0 °C for 1 h and then at RT for another 1 h. The reaction was quenched by cooling to 0 °C and adding ethylene glycol and then concentrated on a rotary evaporator. Water was added and extracted twice by EtOAc (6 × 5 mL each). A concentrated and thick residue obtained was directly dissolved in MeOH (5 mL) and hydrogenated using 10% Pd/C (catalytic) and stirred at 120 psi pressure for 30 h. RM was filtered through celite, concentrated, and purified by MeOH/CHCl3 (1:4) as mobile phase. Compound 2 was obtained as a white solid (0.071 g, 68% over two steps). R = 0.54, MeOH/CHCl3 (1:4); mp = 185–187 °C; [α]D22 = −78.5 (c 0.101, methanol); IR (KBr, ν, cm–1); 3308, 2928, 1674, 1450, 1246, 1093, 833, 758; 1H NMR (500 MHz, D2O, 72 °C) δ 7.67 (s, 1H), 5.08 (d, J = 15.0 Hz, 1H), 4.90 (d, J = 15.0 Hz, 1H), 4.72 (d, J = 3.2 Hz, 1H), 4.53 (d, J = 13.0 Hz, 1H), 4.20 (bs, 1H), 4.19 (d, J = 13.0 Hz, 1H), 3.32 (d, J = 13.8 Hz, 1H), 3.10 (dd, J = 14.4, 1.5 Hz, 1H) 2.97 (dd, J = 14.4, 2.0 Hz, 1H), 2.93 (d, J = 13.8 Hz, 1H); 13C NMR (125 MHz, D2O) δ 133.5, 128.7, 71.3, 68.1, 64.8, 63.4, 61.8, 50.0, 48.5; DEPT-135, down (48.5, 50.0, 61.8, 64.8), up (68.2, 71.3, 128.8); HRMS calculated for C9H15N4O3 [M + H]+ 227.1144, Found: 227.1142.

Synthesis of 10

Acetone/n class="Chemical">water (8:2, 15 mL) was added to 6 (0.700 g, 2.23 mmol) and cooled to 0 °C. NaIO4 (0.573 g, 2.68 mmol) was added pinchwise to this mixture over 30 min and stirred at 0 °C for 1 h and then at RT for another 1 h. RM was cooled to 0 °C and ethylene glycol was added to quench the reaction and then RM was concentrated on a rotary evaporator. Water (5 mL) was added and extracted by ethyl acetate (2 × 10 mL). Concentrated organic layer gave a syrup that was dissolved in MeOH (10 mL), cooled to 0 °C, and NaBH4 (0.101 g, 2.68 mmol) was added portionwise over a period of 20 min. The reaction was maintained at 0 °C for another 30 min and then quenched by saturated aq NH4Cl and extracted by EtOAc (2 × 10 mL). The EtOAc layer was concentrated and purified by column chromatography EtOAc/PE (1:1) as mobile phase. A white solid 10 was observed (0.556 mg, 88% over two steps). R = 0.26 in ethyl EtOAc; mp = 122–124 °C [α]D22 = +44.3 (c 0.101 methanol); IR (KBr, ν, cm–1); 3232, 3130, 2949, 1545, 1448, 1379, 1242, 1168, 1101, 991, 871, 759; 1H NMR (500 MHz, CDCl3) δ 7.58 (s, 1H), 6.36 (d, J = 3.6 Hz, 1H), 5.09 (d, J = 15.2 Hz, 1H), 4.87 (d, J = 15.2 Hz, 1H), 4.84 (d, J = 3.6 Hz, 1H), 4.53 (t, J = 5.4 Hz, 1H), 4.38 (d, J = 12.7 Hz, 1H), 4.14 (d, J = 12.7 Hz, 1H), 3.38–3.30 (m, 2H), 1.62 (s, 4H), 1.38 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 130.6, 128.3, 113.5, 105.7, 85.3, 83.4, 68.4, 66.9, 62.7, 60.3, 26.8, 26.4; HRMS calculated for C12H17N3O5Na [M + Na]+ 306.1068, Found: 306.1074.

Synthesis of 11

Compound 10 (20.500 g, 1.77 mmol) was dissolved in dry dichloromethane (10 mL) and cooled to 0 °C, to which dry n class="Chemical">pyridine (0.426 mL, 5.30 mmol) was added dropwise followed by addition of TsCl (0.403 g, 2.12 mmol) pinchwise over 15 min. The reaction was maintained at 0 °C for 30 min and then at RT for 3 h. The reaction was quenched by 1 M aq HCl solution and extracted using dichloromethane (2 × 10 mL). The organic layer was concentrated, and the thick residue obtained was purified by column chromatography as mobile phase. A white solid 11 was observed (0.733 g, 95%). R = 0.50 in EtOAc/PE (2:3); mp = 165–167 °C; [α]D22 = +14.12 (c 0.103 methanol); IR (KBr, ν, cm–1); 3373, 2991, 2924, 1595, 1492, 1440, 1373, 1178, 1097, 981, 868, 763, 665, 555; 1H NMR (500 MHz, CDCl3) δ 7.66 (d, J = 8.3 Hz, 2H), 7.53 (s, 1H), 7.31 (d, J = 8.3 Hz, 2H), 6.25 (d, J = 3.5 Hz, 1H), 4.96 (d, J = 15.1 Hz, 1H), 4.87 (d, J = 15.1 Hz, 1H), 4.66 (d, J = 3.5 Hz, 1H), 4.56 (t, J = 5.5 Hz, 1H), 4.24 (ABq, J = 12.7 Hz, 1H), 4.00 (dd, J = 10.8, 5.5 Hz, 1H), 3.69 (dd, J = 10.8, 5.5 Hz, 1H), 2.44 (s, 3H), 1.56 (s, 3H), 1.34 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 145.3, 132.2, 131.2, 130.0 (2C), 128.5, 128.0 (2C), 113.8, 106.1, 85.0, 79.9, 68.7, 67.0, 66.2, 62.6, 27.0, 26.5, 21.7; HRMS calculated for C19H24N3O7S [M + H]+ 438.1335, Found: 438.1338.

Synthesis of 12

To a mixture of 11 (0.600 g, 1.37 mmol), NaN3 (0.107 g, 1.65 mmol), and TBAI (0.051 g, 10% mol) dry n class="Chemical">DMF (10 mL) was added under N2 environment and heated at 95 °C for 6 h. The mixture was concentrated directly on a rotary evaporator, added with water (5 mL), extracted by EtOAc (2 × 10 mL), concentrated, and purified by EtOAc/PE (1:1) as mobile phase using column chromatography. A white solid 12 was observed (0.363 g, 86%). R = 0.55, EtOAc/PE (1:1); mp = 191–193 °C; IR [α]D22 = +70.5 (c 0.104 methanol); IR (KBr, ν, cm–1); 2991, 2941, 2094, 1654, 1546, 1448, 1379, 1230, 1166, 1107, 991, 854, 763, 665, 549; 1H NMR (500 MHz, CDCl3) δ 7.58 (s, 1H), 6.37 (d, J = 3.6 Hz, 1H), 5.03 (d, J = 15.2 Hz, 1H), 4.84 (d, J = 15.2 Hz, 1H), 4.78 (d, J = 3.6 Hz, 1H), 4.53 (dd, J = 7.7, 3.8 Hz, 1H), 4.31 (d, J = 12.8 Hz, 1H), 4.15 (d, J = 12.8 Hz, 1H), 3.25 (dd, J = 13.0, 3.8 Hz, 1H), 2.84 (dd, J = 13.0, 7.7 Hz, 1H), 1.61 (s, 3H), 1.37 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 130.9, 128.5, 113.7, 106.1, 85.2, 82.3, 68.6, 66.9, 62.7, 50.3, 27.1, 26.5; HRMS calculated for C12H16N6O4Na [M + Na]+ 331.1130, Found: 331.1138.

Synthesis of 3a

Compound 12 (0.300 g, 0.591 mmol) was dissolved in 75% aq TFA/n class="Chemical">H2O (5 mL, precooled to 0 °C), stirred at RT for 3 h, concentrated on a rotary evaporator under vaccum followed by EtOAc addition, and the organic layer was washed with 10% aq NaHCO3 solution. Finally, the organic layer was filtered through a 60–120 mesh silica bed with eluting EtOAc and concentrated to obtain a thick residue. The thick residue was directly dissolved in methanol (5 mL), hydrogenated using 10% Pd/C (catalytic), and stirred at 100 psi pressure for 30 h. The reaction mass was filtered through celite, concentrated, and purified by MeOH/CHCl3 (1:4) as mobile phase. A white solid 3a (0.189 g, 86% over two steps) was observed. R = 0.45 MeOH/CHCl3 (1:4); mp = 229–231 °C (dec.); IR (KBr, ν, cm–1) 3311, 3117, 2960, 2893, 1688, 1599, 1450, 1247, 1111, 1066, 999, 858, 761, 596; 1H NMR (500 MHz, D2O) δ 7.71 (s, 1H), 4.93 (s, 2H), 4.48 (dd, J = 10.8, 4.8 Hz, 1H), 4.43 (s, 2H), 3.29 (dd, J = 13.4, 4.8 Hz, 2H), 2.84 (dd, J = 13.4, 10.8 Hz, 2H); 13C NMR (125 MHz, D2O) δ 135.1, 128.9, 70.9(2C), 65.6, 61.9, 61.5, 46.0(2C); HRMS calculated for C9H15N4O3 [M + H]+ 227.1139, Found: 227.1145.

Synthesis of 13

Dry dichloromethane (10 mL) and 6 (0.500 g, 1.60 mmol) were mixed together and cooled to 0 °C, to which n class="Chemical">imidazole (0.239 g, 3.51 mmol) and TBDMSCl (0.264 g, 1.76 mmol) were added and the reaction was maintained at RT for 2 h. The reaction was quenched by 1 M aq HCl solution and extracted by dichloromethane (4 × 10 mL); organic layers were mixed and concentrated, followed by purification of the obtained thick syrup by column chromatography using EtOAc/PE (1:4) as mobile phase to obtain a colorless thick syrup 13 (0.648 g, 95%). R = 0.60, EtOAc; [α]D22 = −21.2 (c 0.101, methanol); IR (KBr, ν, cm–1); 3462, 2929, 1464, 1379, 1249, 1165, 1095, 1006, 837, 777, 669; 1H NMR (500 MHz, CDCl3) δ 7.56 (s, 1H), 6.22 (d, J = 3.5 Hz, 1H), 5.00 (d, J = 14.7 Hz, 16H), 4.85 (d, J = 14.7 Hz, 1H), 4.54 (d, J = 12.3 Hz, 1H), 4.51 (d, J = 3.5 Hz, 1H), 4.34 (d, J = 12.3 Hz, 1H), 4.14 (d, J = 9.0 Hz, 1H), 3.77 (dd, J = 10.2, 3.1 Hz, 1H), 3.72 (dd, J = 10.2, 5.1 Hz, 1H), 3.51–3.44 (m, 1H, after D2O ex., it appears as ddd J = 9.0, 5.1, 3.1 Hz), 2.68 (d, J = 4.8 Hz, 1H, ex. with D2O), 1.58 (s, 3H), 1.35 (s, 3H), 0.88 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 131.9, 128.4, 113.4, 105.9, 85.3, 79.7, 70.1, 68.9, 65.4, 64.3, 62.7, 27.1, 26.6, 25.9 (3C), 18.3, −5.40, −5.45; HRMS calculated for C19H33N3O6SiNa [M + Na]+ 450.2021, Found: 450.2022.

Synthesis of 14

Compound 13 (0.600 g, 1.40 mmol) was dissolved in dry dichloromethane (10 mL) and cooled to 0 °C, to which dry n class="Chemical">pyridine (0.283 mL, 3.51 mmol) was added slowly, followed by addition of triflic anhydride (0.259 mL, 1.54 mmol, diluted in 2 mL DCM) dropwise over 10 min and catalytic DMAP. The reaction was maintained at 0 °C for 1 h, quenched by 1 M aq HCl solution, and extracted twice with 5 mL of dichloromethane each time and concentrated. A thick syrup (0.810 g) was obtained, to which, without purification, NaN3 (0.129 g, 2.00 mmol, considering 95% yield of OTf intermediate) and dry DMF (5 mL) were added. The reaction mass was heated to 65 °C for 1 h, concentrated directly on a rotary evaporator, water (5 mL) was added, extracted with ethyl acetate (2 × 5 mL), and evaporated. The obtained thick syrup was purified by column chromatography using EtOAc/PE (1:4) as mobile phase, and an off-white solid 14 (0.584 g, 92% over two steps) was obtained. R = 0.37 in EtOAc/PE (1:1); mp = 119–121 °C; [α]D22 = +50.5 (c 0.103 methanol); IR (KBr, ν, cm–1); 2987, 2933, 2858, 2112, 1643, 1464, 1379, 1249, 1170, 1095, 1037, 837, 775; 1H NMR (500 MHz, CDCl3) δ 7.60 (s, 1H), 6.43 (d, J = 3.6 Hz, 1H), 5.14 (d, J = 15.2 Hz, 1H), 4.88 (d, J = 15.2 Hz, 1H), 4.85 (d, J = 3.6 Hz, 1H), 4.57 (d, J = 2.0 Hz, 1H), 4.35 (d, J = 12.7 Hz, 1H), 4.11 (d, J = 12.7 Hz, 1H), 3.83 (dd, J = 10.1, 6.7 Hz, 1H), 3.77 (dd, J = 10.1, 6.7 Hz, 1H), 3.55 (td, J = 6.7, 2.0 Hz, 2H), 1.60 (s, 3H), 1.38 (s, 3H), 0.91 (s, 12H), 0.09 (s, 3H), 0.08 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 130.3, 128.4, 113.3, 105.6, 85.0, 80.5, 67.7, 67.1, 63.2, 62.5, 60.6, 26.8, 26.3, 25.8 (3C), 18.1, −5.51, −5.55; HRMS calculated for C19H33N6O5Si [M + H]+ 453.2276, Found: 453.2286.

Synthesis of 4a

TFA (75% n class="Chemical">aq, 5 mL, precooled to 0 °C) was added to compound 14 (0.500 mg, 1.10 mmol), stirred at 0 °C for 3 h, and concentrated under vacuum and saturated aq NaHCO3 (5 mL), followed by EtOAC (5 mL) addition. The reaction was extracted twice with EtOAc (2 × 5 mL) and the separated organic layer was filtered through a 60–120 mesh silica bed with eluting ethyl acetate and concentrated. A thick residue was obtained, which was directly dissolved in distilled MeOH (10 mL), hydrogenated using 10% Pd/C (cat.), stirred at 120 psi pressure for 24 h, filtered through celite, concentrated, and purified by MeOH/CHCl3 (2:3) as mobile phase. Compound 4a was obtained as a white solid (0.220 g, 78% over two steps). R = 0.35, MeOH/CHCl3 (2:3); mp = 114–116 °C; [α]D22 = −27.5 (c 0.102, methanol); IR (KBr, ν, cm–1); 3308, 2928, 1674, 1450, 1093, 939, 758; 1H NMR (500 MHz, D2O) δ 7.65 (s, 1H), 5.02 (ABq, J = 15.5 Hz, 2H), 4.32 (ABq, J = 12.5 Hz, 2H), 4.11 (d, J = 2.6 Hz, 1H), 4.05 (t, J = 3.3 Hz, 1H), 3.71 (dd, J = 11.0, 6.5 Hz, 1H), 3.68 (dd, J = 11.0, 6.5 Hz, 1H), 3.65–3.62 (m, 1H), 3.60 (dd, J = 14.4, 3.3 Hz, 1H), 3.07 (dd, J = 14.4, 3.3 Hz, 1H); 13C NMR (125 MHz, D2O) δ 133.0, 128.4, 68.2, 68.1, 66.6, 63.3, 62.1, 60.4, 56.0, 46.5; HRMS calculated for C10H17N4O4 [M + H]+ 257.1249, Found: 257.1255.

General Method for N-Alkylation

Iminosugar 3a/4a (1.0 equiv) was dissolved in dry n class="Chemical">DMF (0.5 mL), followed by addition of K2CO3 (2.2 equiv) and alkyl halide (1.1 equiv). The reaction mass was heated under N2 atmosphere and concentrated on a rotary evaporator under high vaccum, followed by column purification using CHCl3 and MeOH system as a mobile phase on silica gel 100–200 mesh size.

Synthesis of 3b

The reaction of 3a (0.020 g, 88.40 μmol), K2CO3 (0.027 g, 194.49 μmol), and n class="Chemical">bromoethane (0.007 mL, 97.25 μmol) in DMF was maintained at 80 °C for 1 h. Usual workup and purification by column chromatography using CHCl3/MeOH (1:9) as mobile phase gave 3b (0.021 g, 95%) as a white solid. R = 0.52, CHCl3/MeOH (1:4); mp = 147–149 °C; 1H NMR (400 MHz, CD3OD) δ 7.47 (s, 1H), 4.72 (s, 2H), 4.44 (dd, J = 11.2, 4.9 Hz, 2H), 4.25 (s, 2H), 2.91 (dd, J = 11.2, 4.9 Hz, 2H), 2.47 (q, J = 7.2 Hz, 2H), 2.09 (t, J = 11.2 Hz, 2H), 1.04 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 135.0, 127.4, 71.0 (2C), 65.9, 61.3, 61.2, 55.1 (2C), 51.1, 10.7; HRMS (ESI) m/z calculated for C11H19N4O3 (M + H)+ 255.1452, Found: 255.1451.

Synthesis of 3c

The reaction of 3a (0.020 g, 88.40 μmol), K2CO3 (0.027 g, 194.49 μmol), and n class="Chemical">n-bromobutane (0.010 mL, 97.25 μmol) in DMF was carried out at 80 °C for 1 h. Usual workup and purification by column chromatography using CHCl3/MeOH (1:9) as mobile phase gave 3c (0.024 g, 98%) as a white solid. R = 0.55, CHCl3/MeOH (1:4); mp = 156–158 °C; 1H NMR (400 MHz, CD3OD) δ 7.47 (s, 1H), 4.72 (s, 2H), 4.43 (dd, J = 11.2, 4.9 Hz, 2H), 4.25 (s, 2H), 2.90 (dd, J = 11.2, 4.9 Hz, 2H), 2.39 (q, J = 7.2 Hz, 2H), 2.09 (t, J = 11.2 Hz, 2H), 1.49–1.37 (m, 2H), 1.28 (sextate, J = 7.2 Hz, 2H), 0.87 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 135.0, 127.3, 71.0 (2C), 65.9, 61.3, 61.2, 57.1, 55.6 (2C), 28.6, 20.2, 12.9; HRMS (ESI) m/z calculated for C13H23N4O3 (M + H)+: 283.1765, Found: 283.1769.

Synthesis of 3d

The reaction of 3a (0.020 g, 88.40 μmol), K2CO3 (0.027 g, 194.49 μmol), and n-bromohexane (0.014 mL, 97.25 μmol) in DMF was carried out at 80 °C for 1 h. Usual workup and purification by column chromatography using CHCl3/MeOH (1:9) as mobile phase gave 3d (0.024 g, 89%) as a white solid. R = 0.56, CHCl3/MeOH (1:4); mp = 153–155 °C; 1H NMR (400 MHz, CD3OD) δ 7.47 (s, 1H), 4.72 (s, 2H), 4.43 (dd, J = 11.2, 4.9 Hz, 2H), 4.25 (s, 2H), 2.90 (dd, J = 11.2, 4.9 Hz, 2H), 2.38 (q, J = 7.2 Hz, 2H), 2.09 (t, J = 11.2 Hz, 2H), 1.49–1.37 (m, 2H), 1.30–1.21 (m, 6H), 0.82 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 135.0, 127.5, 70.9 (2C), 65.9, 61.3, 61.2, 57.4, 55.5 (2C), 31.5, 26.8, 26.3, 22.3, 13.0; HRMS (ESI) m/z calculated for C15H27N4O3 (M + H)+: 311.2084, Found: 311.2078.

Synthesis of 3e

The reaction of 3a (0.020 g, 88.40 μmol), K2CO3 (0.027 g, 194.49 μmol), and n class="Chemical">n-bromooctane (0.017 mL, 97.25 μmol) in DMF was carried out at 80 °C for 1 h. Usual workup and purification by column chromatography using CHCl3/MeOH (1:9) as mobile phase gave 3e (0.028 g, 94%) as a white solid. R = 0.58, CHCl3/MeOH (1:4); mp = 141–143 °C; 1H NMR (400 MHz, CD3OD) δ 7.48 (s, 1H), 4.73 (s, 2H), 4.44 (dd, J = 11.5, 4.9 Hz, 2H), 4.26 (s, 2H), 2.91 (dd, J = 11.5, 4.9 Hz, 2H), 2.42–2.36 (m, 2H), 2.10 (t, J = 11.5 Hz, 2H), 1.49–1.41 (m, 2H), 1.30–1.18 (m, 10H), 0.82 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, D2O) δ 135.0, 127.4, 71.0 (2C), 65.9, 61.3, 61.2, 57.4, 55.6 (2C), 31.6, 29.2, 29.0, 27.1, 26.4, 22.3, 13.0; HRMS (ESI) m/z calculated for C17H31N4O3 (M + H)+: 339.2391, Found: 339.2390.

Synthesis of 3f

The reaction of 3a (0.020 g, 88.40 μmol), K2CO3 (0.027 g, 194.49 μmol), and n class="Chemical">n-bromodecane (0.020 mL, 97.25 μmol) in DMF was carried out at 60 °C for 1 h. Usual workup and purification by column chromatography using CHCl3/MeOH (1:9) as mobile phase gave 3f (0.029 g, 92%) as a white solid. R = 0.60, CHCl3/MeOH (1:4); mp = 147–149 °C; 1H NMR (400 MHz, CD3OD) δ 7.47 (s, 1H), 4.72 (s, 2H), 4.43 (dd, J = 11.5, 4.4 Hz, 2H), 4.25 (s, 2H), 2.90 (dd, J = 11.5, 4.4 Hz, 2H), 2.39 (App. triplet, J = 7.7 Hz, 2H), 2.09 (t, J = 11.5 Hz, 2H), 1.50–1.40 (m, 2H), 1.31–1.12 (m, 14H), 0.81 (t, J = 7.7 Hz, 3H); 13C NMR (125 MHz, D2O) δ 135.0, 127.4, 71.0 (2C), 66.0, 61.3, 61.2, 57.4, 55.6 (2C), 31.7, 29.33, 29.30, 29.2, 29.1, 27.1, 26.4, 22.3, 13.0; HRMS (ESI) m/z calculated for C19H35N4O3 (M + H)+: 367.2704, Found: 367.2703.

Synthesis of 3g

The reaction of 3a (0.020 g, 88.40 μmol), K2CO3 (0.027 g, 194.49 μmol), and n class="Chemical">n-bromododecane (0.023 mL, 97.25 μmol) in DMF was carried out at 60 °C for 1 h. Usual workup and purification by column chromatography using CHCl3/MeOH (1:9) as mobile phase gave 3g (0.031 g, 90%) as a white solid. R = 0.66, CHCl3/MeOH (1:4); mp = 214–216 °C; 1H NMR (400 MHz, CD3OD) δ 7.47 (s, 1H), 4.72 (s, 2H), 4.43 (dd, J = 11.2, 4.8 Hz, 2H), 4.25 (s, 2H), 2.90 (dd, J = 11.2, 4.8 Hz, 2H), 2.38 (App. t, J = 7.7 Hz, 2H), 2.09 (t, J = 11.2 Hz, 2H), 1.50–1.40 (m, 2H), 1.30–1.13 (m, 18H), 0.80 (t, J = 7.7 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 135.0, 127.3, 71.0 (2C), 66.0, 61.3, 61.2, 57.4, 55.6 (2C), 31.7, 29.4, 29.32, 29.30, 29.2, 29.1, 27.1, 26.4, 22.3, 13.0; HRMS (ESI) m/z calculated for C21H39N4O3 (M + H)+: 395.3017, Found: 395.3017.

Synthesis of 4b

The reaction of 4a (0.020 g, 78.05 μmol), K2CO3 (0.024 g, 171.70 μmol), and n class="Chemical">bromoethane (0.006 mL, 85.85 μmol) in DMF was carried out at 60 °C for 2 h. Usual workup and purification by column chromatography using CHCl3/MeOH (2:3) as mobile phase gave 4b (0.020 g, 93%) as a thick syrup. R = 0.46, CHCl3/MeOH (2:3); [α]D22 = +0.5 (c 0.15, methanol); 1H NMR (400 MHz, CD3OD) δ 7.45 (s, 1H), 4.85 (d, J = 15.0 Hz, 1H), 4.74 (d, J = 15.0 Hz, 1H), 4.24 (d, J = 4.7 Hz, 1H), 4.16 (ABq, J = 12.2 Hz, 1H), 4.10 (dd, J = 8.0, 3.6 Hz, 1H), 3.71 (dd, J = 11.0, 6.0 Hz, 1H), 3.67 (dd, J = 11.0, 4.7 Hz, 1H), 3.30 (App. q, J = 4.7 Hz, 1H), 3.21 (dd, J = 12.8, 3.6 Hz, 1H), 2.85–2.71 (m, 3H), 1.06 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 133.6, 127.3, 78.1, 70.5, 69.8, 65.5, 64.9, 62.0, 61.7, 58.3, 51.6, 10.2; HRMS (ESI) m/z calculated for C12H21N4O4 (M + H)+: 285.1557, Found: 285.1557.

Synthesis of 4c

The reaction of 4a (0.020 g, 78.05 μmol), K2CO3 (0.024 g, 171.70 μmol), n class="Chemical">n-bromobutane (0.010 mL, 85.85 μmol) in DMF at 60 °C for 2 h. Usual workup and purification by column chromatography using CHCl3/MeOH (2:3) as mobile phase gave 4c (0.021 g, 89%) as a thick syrup. R = 0.50, CHCl3/MeOH (2:3); [α]D22 = +6.4 (c 0.13, methanol); 1H NMR (400 MHz, CD3OD) δ 7.45 (s, 1H), 4.84 (d, J = 15.0 Hz, 1H), 4.72 (d, J = 15.0 Hz, 1H), 4.28 (d, J = 4.3 Hz, 1H), 4.18 (d, J = 12.2 Hz, 1H), 4.13 (dd, J = 8.1, 3.9 Hz, 1H), 4.11 (d, J = 12.2 Hz, 1H), 3.70 (dd, J = 12.0, 4.3 Hz, 1H), 3.68 (dd, J = 12.0, 4.3 Hz, 1H), 3.25 (dd, J = 12.9, 3.9 Hz, 1H), 3.13 (dd, J = 6.9, 3.9 Hz, 1H) 2.76 (dd, J = 12.9, 7.3 Hz, 1H) (s, 3H), 2.74–2.61 (m, 2H), 1.55–1.41 (m, 2H), 1.28 (sextate, J = 7.4 Hz), 0.87 (t, J = 7.3 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 136.1, 129.5, 72.9, 72.2, 67.5, 67.4, 65.0, 63.9, 60.4, 55.6, 54.0, 30.6, 22.4, 15.2; HRMS (ESI) m/z calculated for C14H25N4O4 (M + H)+: 313.1870, Found: 313.1870.

Synthesis of 4d

The reaction of 4a (0.020 g, 78.05 μmol), K2CO3 (0.024 g, 171.70 μmol), and n class="Chemical">n-bromohexane (0.012 mL, 85.85 μmol) in DMF was carried out at 60 °C for 2 h. Usual workup and purification by column chromatography using CHCl3/MeOH (2:3) as mobile phase gave 4d (0.024 g, 90%) as a thick syrup. R = 0.52, CHCl3/MeOH (2:3); [α]D22 = +1.7 (c 0.15, methanol); 1H NMR (400 MHz, CD3OD) δ 7.45 (s, 1H), 4.84 (d, J = 15.0 Hz, 1H), 4.72 (d, J = 15.0 Hz, 1H), 4.28 (d, J = 4.3 Hz, 1H), 4.18 (d, J = 12.1 Hz, 1H), 4.12 (dd, J = 7.9, 3.7 Hz, 1H), 4.11 (d, J = 12.1 Hz, 1H), 3.69 (dd, J = 11.2, 4.3 Hz, 1H), 3.67 (dd, J = 11.2, 4.3 Hz, 1H), 3.25 (App. q, J = 4.3 Hz, 1H), 3.13 (dd, J = 12.9, 3.7 Hz, 1H), 2.80–2.60 (m, 3H), 1.55–1.42 (m, 2H), 1.32–1.20 (m, 6H), 0.82 (t, J = 6.7 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 136.0, 129.5, 72.9, 72.2, 67.5, 67.4, 64.9, 63.9, 60.4, 55.9, 54.0, 33.8, 29.0, 28.3, 24.6, 15.3; HRMS (ESI) m/z calculated for C16H29N4O4 (M + H)+: 341.2183, Found: 341.2193.

Synthesis of 4e

The reaction of 4a (0.020 g, 78.05 μmol), K2CO3 (0.024 g, 171.70 μmol), and n class="Chemical">n-bromooctane (0.015 mL, 85.85 μmol) in DMF was carried out at 60 °C for 2 h. Usual workup and purification by column chromatography using CHCl3/MeOH (2:3) as mobile phase gave 4e (0.026 g, 92%) as a thick syrup. R = 0.53, CHCl3/MeOH (2:3); [α]D22 = +7.5 (c 0.14, methanol); 1H NMR (400 MHz, CD3OD) δ 7.45 (s, 1H), 4.84 (d, J = 15.0 Hz, 1H), 4.73 (d, J = 15.0 Hz, 1H), 4.28 (d, J = 4.9 Hz, 1H), 4.16 (d, J = 12.1 Hz, 1H), 4.13 (dd, J = 8.1, 3.9 Hz, 1H), 4.11 (d, J = 12.1 Hz, 1H), 3.68 (dd, J = 11.3, 6.2 Hz, 1H), 3.66 (dd, J = 11.3, 4.9 Hz, 1H), 3.25 (App. q, J = 4.9 Hz, 1H), 3.13 (dd, J = 12.9, 3.9 Hz, 1H), 2.80–2.60 (m, 3H), 1.58–1.43 (m, 2H), 1.32–1.15 (m, 10H), 0.81 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 136.0, 129.5, 72.9, 72.2, 67.5, 67.4, 64.9, 63.9, 60.4, 55.9, 54.0, 33.9, 31.5, 31.6, 31.3, 29.3, 28.3, 24.6, 15.3; HRMS (ESI) m/z calculated for C18H33N4O4 (M + H)+: 369.2496, Found: 369.2502.

Synthesis of 4f

The reaction of 4a (0.020 g, 78.05 μmol), K2CO3 (0.024 g, 171.70 μmol), and n class="Chemical">n-bromodecane (0.018 mL, 85.85 μmol) in DMF was carried out at 60 °C for 2 h. Usual workup and purification by column chromatography using CHCl3/MeOH (2:3) as mobile phase gave 4f (0.28 g, 90%) as a thick syrup. R = 0.55, CHCl3/MeOH (2:3); [α]D22 = +6.6 (c 0.16, methanol); 1H NMR (400 MHz, CD3OD) δ 7.45 (s, 1H), 4.84 (d, J = 15.0 Hz, 1H), 4.73 (d, J = 15.0 Hz, 1H), 4.28 (d, J = 4.9 Hz, 1H), 4.18 (d, J = 12.1 Hz, 1H), 4.13 (dd, J = 8.5, 3.9 Hz, 1H), 4.11 (d, J = 12.1 Hz, 1H), 3.70 (dd, J = 11.2, 6.2 Hz, 1H), 3.66 (dd, J = 11.2, 4.9 Hz, 1H), 3.25 (App. q, J = 4.9 Hz, 1H), 3.13 (dd, J = 12.9, 3.9 Hz, 1H), 2.74–2.61 (m, 2H), 2.76 (dd, J = 12.9, 8.5 Hz, 1H), 1.55–1.42 (m, 2H), 1.30–1.12 (m, 14H), 0.80 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 136.0, 129.5, 72.9, 72.2, 67.5, 67.4, 64.9, 63.9, 60.4, 55.9, 54.0, 33.9, 31.7, 31.6, 31.55, 31.3, 29.3, 24.6, 15.3; HRMS (ESI) m/z calculated for C20H37N4O4 (M + H)+: 397.2809, Found: 397.2820.

Synthesis of 4g

The reaction of 4a (0.020 g, 78.05 μmol), K2CO3 (0.024 g, 171.70 μmol), and n class="Chemical">n-bromododecane (0.021 mL, 85.85 μmol) in DMF was carried out at 60 °C for 2 h. Usual workup and purification by column chromatography using CHCl3/MeOH (2:3) as mobile phase gave 4g (0.029 g, 89%) as a thick syrup. R = 0.59, CHCl3/MeOH (2:3); [α]D22 = +18.8 (c 0.10, methanol); 1H NMR (400 MHz, CD3OD) δ 7.45 (s, 1H), 4.84 (d, J = 15.0 Hz, 1H), 4.73 (d, J = 15.0 Hz, 1H), 4.29 (d, J = 4.3 Hz, 1H), 4.18 (d, J = 12.1 Hz, 1H), 4.13 (dd, J = 8.0, 3.9 Hz, 1H), 4.11 (d, J = 12.1 Hz, 1H), 3.70 (dd, J = 11.2, 5.2 Hz, 1H), 3.66 (dd, J = 11.2, 4.9 Hz, 1H), 3.25 (App. q, J = 4.9 Hz, 1H), 3.13 (dd, J = 12.9, 3.9 Hz, 1H), 2.76 (dd, J = 12.9, 8.0 Hz, 1H), 2.74–2.60 (m, 2H), 1.55–1.45 (m, 2H), 1.30–1.13 (m, 18H), 0.80 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 133.8, 127.3, 70.6, 70.0, 65.3, 65.1, 62.7, 61.6, 58.2, 53.6, 51.8, 31.7, 29.4, 29.35, 29.3, 29.1, 27.1, 26.0, 22.3, 13.0; HRMS (ESI) m/z calculated for C22H41N4O4 (M + H)+: 425.3122, Found: 425.3135.

Conformational Analysis Protocol for DFT Method

To investigate conformational preferences of compounds 3a, 3c, 4a, and 4c and to corroborate our NMR exn class="Chemical">perimental observations, we have performed quantum chemical calculations using the density functional theory (DFT) method. The initial geometries (2C5 and 5C2 conformations) for compounds 3a and 3c and (1C4 and 4C1 conformations) for compounds 4a and 4c have been generated using molecular modeling Spartan’14 software (wavefunction.in). Automated complete geometry optimizations were performed using the DFT method by employing B3LYP/6-31 G** basic set function on Spartan’14 software. Pictorial presentations of DFT-optimized geometries of compounds have been made using Mercury 3.7 software.

Methodology Used for Biological Activities

Glycosidase inhibition assay for compounds 1, 2, 3a–g, and 4a–g was carried out by mixing 0.1 U cm–3 each of α-,β-glucosidase, α-,β-galactosidase, and α-n class="Species">mannosidase, and the samples were incubated for 1 h at 37 °C. Enzyme action for α/β-galactosidase was initiated by addition of 10 mM p-nitrophenyl-α-/β-d-galactopyranoside (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. 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. 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 of p-nitrophenyl pyranoside per minute under assay condition.

Inhibition constant (Ki) values were determined by spectrophotometrically measuring the residual hydrolytic activities of the glycosidases in the presence of compounds. Each assay was n class="Chemical">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. 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.

Antifungal Activity

Minimal Inhibition Concentration (MIC) Assay

Fungal strains Candida albicans NCIM 3557 were procured from National Chemical Lab, Repository. Using the multipin class="Chemical">pette dispense, 100 μL of LB medium was dispensed in all wells of microliter plate. The plate and lid were labeled, and 100 μL of 2× antibiotic solutions was pipette into wells in column 1. Using the multipipette set at 100 μL, the antibiotics were mixed into the wells of column 1 by sucking up and down six to seven times. A volume of 100 μL from column 1 was withdrawn and added to column 2. This made column 2 a twofold dilution of column 1, column 3 a twofold dilution of column 3, and so on. Similar procedure was repeated for the test compounds 1, 2, 3a–g, and 4a–g. The plates were incubated at 30 °C for 24 h and scanned with an ELISA plate reader. All experiments were run in triplicate, and standard deviations were determined.

Bright-Field Images

Candida albicans

SC5314 strain obtained from MTCC (National Chemical Lab, Pune) was inoculated in six-well sterile plates. The glass slides were made grease-free thoroughly. Each compound (5 μL) in its subinhibitory concentn class="Species">ration was added to each well along with control. Compounds 1, 2, 3a–g, and 4a–g were added and incubated at 37 °C for 24 h. The cells were treated and fixed using 2% paraformaldehyde. The glass slides were washed with 1× PBS buffer and imaged using confocal microscopy.

Methodology Used for Docking Studies

Three dimensional (3D) models of rice α/β n class="Chemical">glucosidase, rice α/β galactosidase, and Jack bean α-mannosidase were built by comparative modeling using MODELER 9 v4.[31] Crystallographic structures of homologous protein were considered as a template to predict the structures of enzymes, which were further assessed by the Ramachandran plot analysis. Structures of enzymes and synthesized molecules were energy-minimized using GROMOS43B1 energy 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.[32] Grid was set around the active site of enzymes with a dimension of 20 × 20 × 20 Å3 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 (PyMol Molecular Graphics System, version 1.2r3 pre, Schrodinger LLC).