Regioselective Synthesis of 5-Substituted 3-(β-d-Glycopyranosyl)isoxazoles and -isoxazolines by 1,3-Dipolar Cycloaddition as Potential Anticancer Agents and Glycogen Phosphorylase Inhibitors

Abstract

Anhydro-aldose oximes were employed to generate in situ nitrile oxides via a halogenation/base-induced elimination sequence in the presence of NCS and Et₃N, which were then used in 1,3-dipolar cycloadditions with alkenes and alkynes to afford 5-substituted 3-(β-d-glycopyranosyl)isoxazole and -isoxazoline derivatives exclusively. These newly synthesized glycomimetics were evaluated for their potential to act as antagonists of A2780 ovarian cancer cells and as inhibitors of glycogen phosphorylase; however, they exhibited no significant activity.

1. Introduction

Carbohydrates have been shown to play pivotal roles in many biological processes, including bacterial and viral infections, cell adhesion, contact inhibition of cell division, immune responses, inflammation, and metastasis.

Therefore, the pharmaceutical potential of carbohydrates is considerable [1,2,3]. Unfortunately, the low hydrolytic stability of natural O-glycosidic bonds limits their use as drug candidates [4]. However, more hydrolytically stable moieties can be synthesized by replacing the glycosidic oxygen with different atoms—most frequently S, N, and C—providing a variety of methods to produce glycomimetic compounds [5]. Multi-atomic replacement of the glycosidic oxygen is also well-known (e.g., S-S, S-Se, SO₂-N, N-CO-N) [6]. These compounds offer several advantages, including simpler synthesis, resistance to hydrolysis and metabolic processes, a wide range of derivatization possibilities, and potential utility as glycobiological tools and leads in drug design [7].

Only a few examples of the synthesis of substituted 3-(C-glycopyranosyl)isoxazoles and -isoxazolines have been reported in the literature. In these papers, glycopyranosyl nitrile oxides were reacted with alkynes (dimethyl acetylenedicarboxylate [8,9], ethyl propiolate [8], propargyl glycosides [10,11], 6-O-propargyl cyclomaltoheptaose derivative [10], ethynyl α-amino-acid derivatives [12], tri- and tetrapropargylated compounds [10,11]) and alkenes (styrene, allyl alcohol, 4-C-vinyl furanose derivative, methylenecyclohexene [8], norbornene [8,13], norbornadiene [13]) in 1,3-dipolar cycloaddition reactions. The glycopyranosyl nitrile oxides were generated in situ from the corresponding oximes either by base-induced dehydrohalogenation of the derived hydroximoyl chloride (Cl₂, Et₃N) [8,9,12,14] or bromide (NBS, Et₃N) [8,9,12,14] or by oxidation with aq. hypochlorite (NaOCl, Et₃N) [8,14]. Direct dehydration of nitromethanes to nitrile oxides by phenyl isocyanate or TDI (toluene-2,4-diisocyanate) in the presence of a catalytic amount of trimethylamine was also reported [10,11,13,14]. In the absence of dipolarophiles, dimerization of carbohydrate nitrile oxides leads to the formation of 3,4-bis-glycopyranosyl-1,2,5-oxadiazole N-oxides (bis-glycopyranosylfuroxans) in high yields [8,9,14,15]. These compounds may also appear as by-products in the above 1,3-dipolar cycloadditions. Another possibility to obtain 3-(2′,3′,4′,6′-tetra-O-benzoyl-β-d-glucopyranosyl)-5-phenylisoxazole involved the cyclization of the corresponding C-glucopyranosyl phenylethynyl ketone with hydroxylamine [15,16]. Anhydro-aldose oximes (C-glycosyl formaldoximes) can easily be prepared either from anhydro-aldoses (C-glycosyl formaldehydes) in a condensation reaction with hydroxylamine [9] or from C-glycosyl nitromethanes by reduction with the complex [Et₃NH][(PhS)₃Sn] generated from SnCl₂/PhSH/Et₃N [8,14]. Our group has developed a simple synthetic method for the preparation of anhydro-aldoximes B by the reduction in the readily accessible C-glycosyl cyanides A and the in situ trapping of the imine intermediate by semicarbazide, followed by a transimination reaction in the presence of hydroxylamine hydrochloride (Scheme 1) [15,17].

Scheme 1. Present study to synthesize isoxazoles D and isoxazolines E.

Herein, we report our findings on the synthesis of novel (3-C-glycosyl)isoxazoles D and -isoxazolines E via 1,3-dipolar cycloaddition reactions of nitrile oxides C. We also describe the biological activity of galactose-derived products as antagonists of the A2780 ovarian cancer cell line and of glucose analogues as glycogen phosphorylase inhibitors.

2. Results and Discussion

2.1. Syntheses

We started our investigations with the 1,3-dipolar cycloaddition reactions of C-(2,3,4,6-tetra-O-benzoyl-β-d-glucopyranosyl)formaldehyde oxime (1a) and styrene (2a) (Table 1). First, the literature conditions [17] were applied using aqueous sodium hypochlorite in THF [18] at room temperature under inert (N₂) atmosphere to give 3-(2′,3′,4′,6′-tetra-O-benzoyl-β-d-glucopyranosyl)-5-phenylisoxazoline (3a) in 60% yield as inseparable mixtures of two diastereoisomers with a 1.1:1 diastereomeric ratio (entry 1). Using KI/oxone (2 KHSO₅•KHSO₄•K₂SO₄) as the oxidizing agent in a mixture of methanol and water [19] resulted in a complex reaction mixture (entry 2). Next, base-induced dehydrohalogenation of the derived hydroximoyl bromide [9] was used. In this reaction NBS (1.1 equiv.) was added to a stirred solution of oxime 1a and 5-fold excess of styrene (2a) in DMF followed by dropwise administration of a solution of Et₃N in DMF to get 3a in 58% yield (entry 3).

Table 1. Optimization reactions of 1,3-dipolar cycloaddition reactions of C-(2,3,4,6-tetra-O-benzoyl-β-d-glucopyranosyl)formaldehyde oxime 1a with styrene 2a.

Entry	Reaction Conditions							Yield (%)
	Styrene (2a) (Equiv.)	Reagent (Equiv.)	Et3N (Equiv.)	Solvent	Temp. (°C)	Time (h)	N2 Atm.	3a	4a
1	3	NaOCl (0.14)	-	THF	rt	24	+	60 a	+ b
2	5	KI (1.1) oxone (1.5)	-	MeOH:H2O = 20:1	25–40	72	-	Complex reaction mixture
3	5	NBS (1.1)	1.1	DMF	rt	4	-	58 a	+ b
4	5	NBS (1.1)	1.1	CH2Cl2	rt	4	-	64 a	+ b
5	5	NCS (1.1)	1.1	CH2Cl2	rt	4	-	65 a	+ b

^a Diastereomeric ratio: 1.1:1. ^b Compound 4a was detected after the work-up in the ¹H NMR spectra.

Replacing DMF with CH₂Cl₂ (entry 4) and/or using NCS instead of NBS slightly increased the yield of 3a to 64% and 65%, respectively.

The dimerization of C-(2,3,4,6-tetra-O-benzoyl-β-d-glucopyranosyl)formaldehyde nitrile oxides resulted in diglucopyranosylfuroxan 4a, which was detected after the work-up in the ¹H NMR spectra (entries 1, 3–5) (see Supplementary Materials). In these cycloadditions, two regioisomers, 5- and 4-substituted isoxazolines, are normally formed, but in our case, only 3-(C-glucosyl)-5-phenylisoxazoline 3a was formed.

Under optimized reaction conditions, digalactopyranosyl furoxan (4b) [14] was obtained as the main product in 61% yield from the reaction of phenylacetylene (5a) with C-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)formaldehyde oxime (1b), whereas the expected 5-phenylisoxazole (6a) was isolated in only 29% yield (Table 2, entry 1). Therefore, further optimization reactions were needed. Using 1 equivalent of oxime 1b and 1.5 equivalents of NCS and adding 1.5 equivalents of Et₃N in CH₂Cl₂ dropwise in 2 h, the conversion was very low (38%) even after 3 days. Phenylisoxazole 6a was isolated in good (52%) yield as a sole 3,5-regioisomer and furoxan 4b in 45% yield (yields were corrected with the conversion) (entry 2). Increasing the dosage time of the Et₃N to 4 h resulted in 68% conversion after 1 day, which provided the corresponding product 6a in low (37%) yield and the furoxan 4b in higher yield (entry 3). Reducing the amount of N-chlorosuccinimide to a 1.1-fold excess and increasing the amount of triethylamine to 2.2 equivalents resulted in a lower conversion of 60% and a yield of 40% for 6a (entry 4). Using a syringe pump to increase the dosage time for the Et₃N-dichloromethane solution to 16 h resulted in better conversion (70%), with a higher yield of compound 6a and a lower yield of 28% of furoxan 4b (entry 5). Next, the reaction was carried out with 1.5 equivalents of NCS to achieve isoxazole 6a in moderate yield with 76% conversion (entry 6). Increasing the equivalents of phenylacetylene (5a) from 1 to 1.5 did not significantly improve the conversion and the yield of the product 6a (entry 7). Performing the reaction in the presence of 2-fold excess of N-chlorosuccinimide resulted in the corresponding isoxazole 6a in good (61%) yield with 85% conversion (entry 8). Using NBS instead of NCS resulted in complete conversion but lower yield of 6a (entry 9). Increasing the amount of dipolarophile 5a to 2 equivalents in the presence of NCS decreased both the conversion and the yield (entry 10). The best result, 68% of 6a, was achieved using 2 equivalents of phenylacetylene (5a) in the presence of 3-fold excess of NCS and 3.3-fold excess of triethylamine in dichloromethane (entry 11). The 3-(C-glycosyl)-4-phenylisoxazole regioisomer was not detected in the reaction mixtures.

Table 2. Optimization reactions of 1,3-dipolar cycloaddition reactions of C-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)formaldehyde oxime 1b with phenylacetylene 5a.

Entry	Reaction Conditions					Conversion (%)	Isolated Yield Corr. with Conv. (%)
	Phenylacetylene 5a (Equiv.)	NXS (Equiv.)	Dry Et3N (Equiv.)	Time (h)	Dosage Time (h)		6a	4b
1	5	NCS (1.1)	1.1	24.5	0.5	100	29	61
2	1	NCS (1.5)	1.5	72	2	38	52	45
3	1	NCS (1.5)	1.5	24	4	68	37	57
4	1	NCS (1.1)	2.2	24	4	60	40	- a
5	1	NCS (1.1)	2.2	16.5	16 b	70	50	28
6	1	NCS (1.5)	2.2	16.5	16 b	76	47	- a
7	1.5	NCS (1.5)	2.2	16.5	16 b	77	51	- a
8	1.5	NCS (2)	2.2	16.5	16 b	85	61	- a
9	1.5	NBS (2)	2.2	16.5	16 b	100	36	- a
10	2	NCS (2)	2.2	16.5	16 b	83	56	- a
11 c	2	NCS (3)	3.3	16.5	16 b	100	68	- a

^a Compound 4b was not detected after work-up. ^b Syringe pump was applied. ^c Optimized reaction conditions.

With the resulting optimized conditions, the scope of the 1,3-dipolar cycloaddition reaction was investigated in the reaction of C-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)formaldehyde oxime 1b with a variety of alkyne dipolarophiles 5a–i (Table 3). 5-(naphth-2-yl)isoxazole 6b and 5-(naphth-1-yl)isoxazole 6c were obtained in good yields (56% and 57%, respectively) with 2- (5b) and 1-ethynylnaphthalene (5c) (entries 2 and 3). 1,3-dipolar cycloaddition with 2-ethynylpyridine (5d) gave the isoxazole 6d in 63% yield (entry 4). The reaction with 1,4-diethynylbenzene (5e) resulted in compound 6e with moderate yield and the disaccharide product was not observed (entry 5). The 5-substituted 3-(galactopyranosyl)isoxazoles 6f and 6g were obtained in moderate yields with propargyl alcohol (5f) and propargyl acetate (5g) (entries 6 and 7). Only furoxan 4b was obtained in the case of 3,7-anhydro-4,5,6,8-tetra-O-benzyl-1,2-dideoxy-d-glycero-d-gulo-oct-1-ynitol (5h) [20] (entry 8). The reaction with diethyl acetylenedicarboxylate (5i) resulted in the corresponding product 6i in moderate yield (entry 9).

Table 3. 1,3-Dipolar cycloaddition reactions of C-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)formaldehyde oxime 1b with alkyne dipolarophiles 5a–i.

Entry		R1	R2	Isolated Yield (%)
				6	4b
1	a		H	68	- a
2	b		H	56	- a
3	c		H	57	- a
4	d		H	63	+ b
5	e		H	41	- a
6	f	CH2OH	H	47	- a
7	g	CH2OAc	H	46	+ b
8	h		H	- a	99
9	i	COOEt	COOEt	44	- a

^a Compounds were not detected after work-up. ^b Compound 4b was detected after work-up in ¹H NMR spectra.

Extending the 1,3-dipolar cycloaddition reactions to C-(2,3,4,6-tetra-O-benzoyl-β-d-glucopyranosyl)formaldehyde oxime 1a, the corresponding 5-substituted 3-(glucopyranosyl)isoxazoles 7a,d–I were isolated in low to good yields (Table 4). The transformation with phenylacetylene (5a), 2-ethynylpyridine (5d), and 1,4-diethynylbenzene (5e) furnished the isoxazoles 7a,d and 7e in low to moderate yields (entries 1–3) but the yield of 7a (57%) was better then in the literature process (49%) [16] (entry 1). The cycloaddition reactions with propargyl alcohol (5f) and propargyl acetate (5g) gave compounds 7f and 7g in good (7f: 67%) and moderate (7g: 47%) yields (entries 4 and 5). When carrying out the reaction with the sugar derivative (5h) [20], 3-(glucopyranosyl)-5-(glucopyranosyl)isoxazole 7h was isolated in low (16%) yield beside diglucosylfuroxan 4a (entry 6). The 3,4,5-trisubstituted isoxazole 7i was obtained in moderate (50%) yield (entry 7).

Table 4. 1,3-Dipolar cycloaddition reactions of C-(2,3,4,6-tetra-O-benzoyl-β-d-glucopyranosyl)formaldehyde oxime 1a with alkyne dipolarophiles 5a,d–i.

Entry		R1	R2	Isolated Yield (%)
				7	4a
1	a		H	57	- a
2	d		H	39	+ b
3	e		H	24	- a
4	f	CH2OH	H	67	- a
5	g	CH2OAc	H	47	4
6	h		H	16	31
7	i	COOEt	COOEt	50	+ b

^a Compound 4a was not detected after work-up. ^b Compound 4a was detected after work-up in ¹H NMR spectra.

The 1,3-dipolar cycloaddition reactions of C-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)formaldehyde oxime 1b with alkene dipolarophiles 2a–g were carried out using our optimized conditions (Table 2, entry 11) and the results are summarized in Table 5. The reactions with styrene (2a) and 2-vinylnaphthalene (2b) produced isoxazolines 8a and 8b with good yields with 1.1:1 diastereomeric ratios (entries 1 and 2). The isoxazoline derivative 8c was obtained in the presence of vinyl acetate (2c) in a moderate yield of 56% with a diastereomeric ratio of 1.6:1 (entry 3). However, the transformation with cis-stilbene (2d) furnished only furoxan 4b (entry 4). Cycloaddition with trans-stilbene (2e) gave 8e as the sole isomer, albeit in low yield, and furoxan 4b as the major product (entry 5). Neither 2-methoxyprop-1-ene (2f) nor cyclohexene (2g) gave the expected products, only furoxan 4b was formed in the transformations (entries 6 and 7).

Table 5. 1,3-Dipolar cycloaddition reactions of C-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)formaldehyde oxime 1b with alkene dipolarophiles 2a–g.

Entry		R1	R2	R3	Diastereomeric Ratio 8	Isolated Yield (%)
						8	4b
1	a		H	H	1.1:1	83	- a
2	b		H	H	1.1:1	71	+ b
3	c	OAc	H	H	1.6:1	53	+ b
4	d	H (cis)			-	- a	99
5	e	H (trans)			-	23	76
6	f	OMe	CH3	H	-	- a	23
7	g	H	(CH2)4		-	- a	99

^a Compounds were not detected after work-up. ^b Compound 4b was detected after work-up in ¹H NMR spectra.

Cycloaddition reactions of C-(2,3,4,6-tetra-O-benzoyl-β-d-glucopyranosyl)formaldehyde oxime 1a with alkene dipolarophiles 2a–c, f were also performed (see Table 6). Using the optimized conditions (Table 2, entry 11), the corresponding products 3a–c were isolated in good to excellent yields with diastereomeric ratios of 1.1–1.3:1, respectively (entries 1–3). A complex reaction mixture was obtained with 2-methoxyprop-1-ene (2f) (entry 4).

Table 6. 1,3-Dipolar cycloaddition reactions of C-(2,3,4,6-tetra-O-benzoyl-β-d-glucopyranosyl)formaldehyde oxime 1a with alkene dipolarophiles 2a–c,f.

Entry		R1	R2	Diastereomeric Ratio 3	Isolated Yield (%)
					3	4a
1	a		H	1.3:1	77	- a
2	b		H	1:1	93	- a
3	c	OAc	H	1.1:1	59	+ b
4	f	OMe	CH3	Complex reaction mixture

^a Compounds 4a was not detected after work-up. ^b Compound 4a was detected after work-up in ¹H NMR spectrum.

To obtain (glycopyranosyl)isoxazoles 6 and 7 from (glycopyranosyl)isoxazolines 3 and 8, the literature methods were tested. Bromination of the isoxazoline 8a followed by elimination of hydrogen bromide [21] or oxidation with manganese(IV) oxide (MnO₂) [22] with 8b in dry toluene at reflux temperature using a Dean–Stark apparatus failed. Finally, acetic acid was eliminated from 8c using a literature method [23] to obtain isoxazole 9 in a moderate yield of 49% (Scheme 2). However, a complex reaction mixture formed in the case of the benzoylated 3c.

Scheme 2. Synthesis of 3-(β-d-galactopyranosyl)isoxazole 9 from [3-(2′,3′,4′,6′-tetra-O-acetyl-β-d-galactopyranosyl)isoxazol-5-yl] acetates 8c.

Finally, the deprotection of 5-substituted 3-(2,3,4,6-tetra-O-acyl-β-d-glycopyranosyl)isoxazoles 6a–d and 7a,d,f, -isoxazolines 8a,b and 3a,b, and furoxan 4b was achieved by the Zemplén-method using a catalytic amount of NaOMe in dry MeOH at room temperature (Table 7 and Table 8, Scheme 3). The isoxazole 10a–d and 11a,d,f and isoxazoline 12a,b and 13a,b and furoxan 14 derivatives were isolated in low to excellent yields.

Table 7. Deacylation of 5-substituted 3-(2,3,4,6-tetra-O-acyl-β-d-glycopyranosyl)isoxazoles 6a–d and 7a,d,f.

Entry	Isoxazole	Sugar Configuration	Protecting Group	R	Isoxazole	Isolated Yield (%)
1	6a	Gal	Ac		10a	49
2	6b	Gal	Ac		10b	43
3	6c	Gal	Ac		10c	75
4	6d	Gal	Ac		10d	63
5	7a	Glc	Bz		11a	80
6	7d	Glc	Bz		11d	67
7	7f	Glc	Bz	CH2OH	11f	99

Table 8. Deacylation 5-substituted 3-(2,3,4,6-tetra-O-acyl-β-d-glycopyranosyl)isoxazolines 8a,b and 3a,b.

Entry	Isoxazoline	Sugar Configuration	Protecting Group	R	Diastereomeric Ratio 12 and 13	Isoxazoline	Isolated Yield (%)
1	8a	Gal	Ac		1.1:1	12a	76
2	8b	Gal	Ac		1.1:1	12b	70
3	3a	Glc	Bz		1.3:1	13a	80
4	3b	Glc	Bz		1:1	13b	46

Scheme 3. Deprotection of 3,4-di(β-d-galactopyranosyl)-1,2,5-oxadiazole-2-oxide 4b.

2.2. Glycogen Phosphorylase Assays

The inhibition of hepatic glycogen phosphorylase (GP), the rate-determining enzyme for the degradation of the storage polysaccharide glycogen, can reduce the hepatic glucose output and may directly influence blood glucose levels [24]. Furthermore, glycogen phosphorylase inhibitors can support the proliferation and function of pancreatic beta cells [25,26]. Thus, GP inhibition has become a validated target in finding new therapeutic possibilities for type 2 diabetes [27,28,29,30]. The most populated class of inhibitors of GP is that of the glucose analogues, which primarily bind to the active site of the enzyme.

Phenyl isoxazole 11a was tested as an inhibitor of GP and showed no significant binding [16]. However, the 5-(pyridin-2-yl)isoxazole 11d and isoxazolylmethanol 11f derivatives and the isoxazolines 13a,b were not investigated in GP inhibition.

Some of the newly synthesized glucose derivatives were assayed against rabbit muscle glycogen phosphorylase b (RMGPb) according to previously described protocols [31]. Compared to previous molecules, isoxazoles 11d,f and isoxazolines 13a,b did not inhibit the GP enzyme.

2.3. Cell Proliferation Assays

Toxicity or the inhibition of cell proliferation are pre-requisites for application as cancer chemotherapy agents. A subset of the compounds (6d, 8b, 10a, 12a) was assessed for rapid toxicity (MTT assay) and for the inhibition of cell proliferation (SRB assay), as described previously on human ovarian adenocarcinoma cell model (A2780) [32]. Compounds 8b, 10a, and 12a did not induce rapid toxicity or cytostasis (Figure 1). However, 6d induced cytostasis at 100 µM with a ~50% maximal inhibition without inducing rapid toxicity (Figure 1).

Figure 1. Cytostatic properties of compounds 6d, 8b, 10a, and 12a on A2780 cells.

For MTT assays 4 × 10³ and for SRB assays 2 × 10³, A2780 cells were plated to 96-well plates. The cells were treated with the compounds in the concentrations indicated for either 4 h for an MTT assay or for 48 h for an SRB assay. Data is represented as average ± SD, from three biological replicates, and individual assays were performed in duplicates. Values were normalized for vehicle-treated cells; absorbance for vehicle-treated cells equals to 1. Normality was assessed using the Shapiro–Wilk test. Statistical significance was assessed using one-way ANOVA or Kruskal–Wallis test as a function of normality followed by Holm–Sidak’s, Dunett’s, or Dunn’s post hoc test. For better visibility normality, statistical tests and p values are presented in an excel sheet at https://figshare.com/s/2261964aba80a88bd606, accessed on 9 July 2025. Nonlinear regression was performed on the datasets.

3. Materials and Methods

3.1. Synthesis

3.1.1. General Methods

Optical rotations were determined with a Jasco P-2000 (Easton, MD, USA) polarimeter at room temperature. NMR spectra were recorded with Bruker AM Avance DRX 360 MHz (360/90 MHz for ¹H/¹³C) or Bruker AM Avance I 400 MHz (400/100 MHz for ¹H/¹³C) or Bruker AM Avance II 500 MHz (500/125 MHz for ¹H/¹³C) or Bruker Avance Neo 700 MHz (700/175 MHz for ¹H/¹³C) spectrometers (Bruker, Karlsruhe, Germany). Chemical shifts are referenced to TMS as the internal reference (¹H), or to the residual solvent signals (¹H and ¹³C). The assignments of the ¹H and ¹³C NMR signals of compounds 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, and 14 were performed by their COSY (3a–c, 4a,b, 6a,d,g,i, 7a,d–i, 8a–c,e, 9, 10b, 11a,d,f, 12a,b, 13a,b, and 14), HSQC (3a–c, 4a,b, 6a,d,g,i, 7a,d–i, 8a–c,e, 9, 10b, 11a,d,f, 12a,b, 13a,b, and 14) and HMBC (3a–c, 4a,b, 6a,d,g,i, 7a,d–i, 8a–c,e, 9, 10b, 11a,d,f, 12a,b, 13a,b, and 14) spectra. Mass spectra were recorded with maXis II UHR ESI-QTOF MS (Bruker Daltonik, Bremen, Germany) instruments in positive ion mode with the electrospray ionization technique or Thermo LTQ XL (Thermo Electron Corp., San Jose, CA, USA) mass spectrometers operated in a full scan positive and negative ion ESI mode. TLC was performed on DCAlurolle Kieselgel 60 F254 (Merck & Co., Inc., Rahway, NJ, USA). TLC plates were visualized under UV light and by gentle heating (generally no spray reagent was used but, if more intense charring was necessary, the plate was sprayed with the following solution: abs. EtOH (95 mL), cc. H₂SO₄ (5 mL), anisaldehyde (1 mL)). For column chromatography, Kieselgel 60 (Merck & Co., Inc., Rahway, NJ, USA, particle size (0.063–0.200 mm) was applied.

3.1.2. General Procedure I for Synthesis of 5-Substituted and 4,5-Disubstituted 3-(2′,3′,4′,6′-Tetra-O-acyl-β-d-glycopyranosyl)isoxazoles and -isoxazolines (3, 6, 7, 8)

A C-(2,3,4,6-tetra-O-acyl-β-d-glycopyranosyl)formaldehyde oxime (3,4,5,7-tetra-O-acyl-2,6-anhydro-heptose oxime) (1a and 1b 1 mmol), N-chlorosuccinimide (3 mmol), and dipolarophile (alkene or alkyne (2 mmol)) were added to dry dichloromethane (17 mL). The suspension was stirred for 30 min at room temperature, and then a solution of dry trimethylamine (3.3 mmol) in dry dichloromethane (40 mL) was added dropwise with a syringe pump in 16 h. When TLC (1:2 EtOAc–hexane for 1a, 1:1 EtOAc–hexane for 1b) indicated complete consumption of the starting compound (~16 h), the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography with eluents indicated for the particular compounds to give 5-substituted and 4,5-disubstituted 3-(2′,3′,4′,6′-tetra-O-acyl-β-d-glycopyranosyl)isoxazoles 6 and 7 and -isoxazolines 3 and 8.

3-(2′,3′,4′,6′-Tetra-O-benzoyl-β-d-glucopyranosyl)-5-phenylisoxazolines (3a)

Prepared from oxime 1a (0.10 g, 0.16 mmol) and ethenylbenzene 2a (2 equiv., 36.7 µL, 0.03 g, 0.32 mmol) according to the General Procedure I. Purified by column chromatography (1:3 EtOAc–hexane) to yield 89 mg (77%) of (diastereomeric ratio: 1.3:1) as a white amorphous product. R_f: 0.26 (1:2 EtOAc–hexane). 3a-I ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.14–7.75 (8H, m, Ar), 7.64–7.14 (17H, m, Ar), 6.03 (1H, pseudo t, J_3′,4′ 9.5 Hz, H-3′), 5.72 (1H, pseudo t, J_4′,5′ 9.4 Hz, H-4′), 5.63–5.52 (2H, m, H-5, H-2′), 4.78 (1H, d, J_1′,2′ 9.9 Hz, H-1′), 4.61 (1H, dd, J_6a′,6b′ 12.3 Hz, H-6_a′), 4.47 (1H, dd, H-6_b′), 4.23 (1H, ddd, J_5′,6a′ 2.3, J_5′,6b′ 5.2 Hz, H-5′), 3.68 (1H, dd, J_4a,4b 17.0, J_4a,5 10.9 Hz, H-4_a), 3.13 (1H, dd, J_4b,5 9.2 Hz, H-4_b). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 166.2, 165.9, 165.8, 165.4 (4 × CO), 155.2 (C-3), 140.4–125.9 (Ar), 83.1 (C-5), 76.6 (C-5′), 74.6 (C-1′), 73.7 (C-3′), 70.1 (C-2′), 69.5 (C-4′), 63.2 (C-6′), 40.7 (C-4). 3a-II ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.14–7.75 (8H, m, Ar), 7.64–7.14 (17H, m, Ar), 6.00 (1H, pseudo t, J_3′,4′ 9.5 Hz, H-3′), 5.70 (1H, pseudo t, J_4′,5′ 9.4 Hz, H-4′), 5.63–5.52 (1H, m, H-5), 5.55 (1H, pseudo t, J_2′,3′ 9.7 Hz, H-2′), 4.82 (1H, d, J_1′,2′ 10.0 Hz, H-1′), 4.65 (1H, dd, J_6a′,6b′ 12.3 Hz, H-6_a′), 4.47 (1H, dd, H-6_b′), 4.23 (1H, ddd, J_5′,6a′ 1.9, J_5′,6b′ 5.2 Hz, H-5′), 3.55 (1H, dd, J_4a,4b 17.2, J_4a,5 11.3 Hz, H-4_a), 3.26 (1H, dd, J_4b,5 9.2 Hz, H-4_b). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 166.2, 165.9, 165.8, 165.4 (4 × CO), 154.8 (C-3), 140.4–125.9 (Ar), 83.1 (C-5), 76.7 (C-5′), 74.6 (C-1′), 73.9 (C-3′), 69.6 (C-2′), 69.4 (C-4′), 63.2 (C-6′), 40.7 (C-4). HR-ESI-MS positive mode (m/z): calcd. for C₄₃H₃₅NO₁₀ (725.23) [M + Na]⁺ = 748.2153, found: [M + Na]⁺ = 748.2152.

3-(2′,3′,4′,6′-Tetra-O-benzoyl-β-d-glucopyranosyl)-5-(naphth-2-yl)isoxazolines (3b)

Prepared from oxime 1a (0.10 g, 0.16 mmol) and 2-ethenylnaphthalene 2b (2 equiv., 0.05 g, 0.32 mmol) according to the General Procedure I. Purified by column chromatography (1:3 EtOAc–hexane) to yield 116 mg (93%) of 3b (diastereomeric ratio: 1:1) as a yellow amorphous product. R_f: 0.23 (1:2 EtOAc–hexane). 3b-I ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.15–7.63 (12H, m, Ar), 7.62–7.16 (15H, m, Ar), 6.05 (1H, pseudo t, J_3′,4′ 9.6 Hz, H-3′), 5.81–5.73 (1H, m, H-5), 5.73 (1H, pseudo t, J_4′,5′ 10.0 Hz, H-4′), 5.62 (1H, pseudo t, J_2′,3′ 9.9 Hz, H-2′), 4.81 (1H, d, J_1′,2′ 9.9 Hz, H-1′), 4.61 (1H, dd, J_6a′,6b′ 12.3 Hz, H-6_a′), 4.48 (1H, dd, H-6_b′), 4.25 (1H, ddd, J_5′,6a′ 2.4, J_5′,6b′ 4.9 Hz, H-5′), 3.75 (1H, dd, J_4a,4b 17.1, J_4a,5 11.0 Hz, H-4_a), 3.21 (1H, dd, J_4b,5 9.2 Hz, H-4_b). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 166.2, 165.9, 165.8, 165.4 (4 × CO), 155.3 (C-3), 137.6–123.7 (Ar), 83.2 (C-5), 76.6 (C-5′), 74.6 (C-1′), 73.7 (C-3′), 70.2 (C-2′), 69.5 (C-4′), 63.1 (C-6′), 40.8 (C-4). 3b-II ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.15–7.63 (12H, m, Ar), 7.62–7.16 (15H, m, Ar), 6.01 (1H, pseudo t, J_3′,4′ 9.6 Hz, H-3′), 5.81–5.73 (1H, m, H-5), 5.71 (1H, pseudo t, J_4′,5′ 10.0 Hz, H-4′), 5.57 (1H, pseudo t, J_2′,3′ 9.7 Hz, H-2′), 4.85 (1H, d, J_1′,2′ 10.0 Hz, H-1′), 4.66 (1H, dd, J_6a′,6b′ 12.2 Hz, H-6_a′), 4.46 (1H, dd, H-6_b′), 4.25 (1H, ddd, J_5′,6a′ 2.1, J_5′,6b′ 4.9 Hz, H-5′), 3.63 (1H, dd, J_4a,4b 17.1, J_4a,5 11.2 Hz, H-4_a), 3.37 (1H, dd, J_4b,5 9.0 Hz, H-4_b). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 166.3, 165.9, 165.4, 165.3 (4 × CO), 154.9 (C-3), 137.6–123.7 (Ar), 83.1 (C-5), 76.7 (C-5′), 74.6 (C-1′), 73.8 (C-3′), 69.6 (C-2′), 69.4 (C-4′), 63.1 (C-6′), 40.8 (C-4). HR-ESI-MS positive mode (m/z): calcd. for C₄₇H₃₇NO₁₀ (775.24) [M + Na]⁺ = 798.2310, found: [M + Na]⁺ = 798.2310.

[3-(2′,3′,4′,6′-Tetra-O-benzoyl-β-d-glucopyranosyl)isoxazol-5-yl] acetates (3c)

Prepared from oxime 1a (0.10 g, 0.16 mmol) and ethenyl acetate 2c (2 equiv., 29.6 µL, 0.03 g, 0.32 mmol) according to the General Procedure I. Purified by column chromatography (1:3 EtOAc–hexane) to yield 67 mg (59%) of 3c (diastereomeric ratio: 1.1:1) as a white amorphous product. R_f: 0.36 (1:1 EtOAc–hexane). 3c-I ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.08–7.74 (8H, m, Ar), 7.62–7.12 (12H, m, Ar), 6.72 (1H, d, H-5), 6.03 (1H, pseudo t, J_3′,4′ 9.6 Hz, H-3′), 5.76 (1H, pseudo t, J_4′,5′ 9.9 Hz, H-4′), 5.49 (1H, pseudo t, J_2′,3′ 9.7 Hz, H-2′), 4.81 (1H, d, J_1′,2′ 9.9 Hz, H-1′), 4.66 (1H, dd, H-6_a′), 4.51 (1H, dd, J_6a′,6b′ 12.3 Hz, H-6_b′), 4.25 (1H, ddd, J_5′,6a′ 2.4, J_5′,6b′ 4.8 Hz, H-5′), 3.56 (1H, dd, J_4a,4b 18.3, J_4a,5 7.2 Hz, H-4_a), 3.13 (1H, dd, J_4b,5 < 1.0 Hz, H-4_b), 2.03 (3H, s, CH₃). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 169.7 (OCOCH₃), 166.2, 165.8, 165.3 (4 × CO), 156.3 (C-3), 134.1–128.1 (Ar), 95.8 (C-5), 76.7 (C-5′), 74.2 (C-1′), 73.5 (C-3′), 69.9 (C-2′), 69.3 (C-4′), 63.0 (C-6′), 39.3 (C-4), 21.1 (CH₃). 3c-II ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.08–7.74 (8H, m, Ar), 7.62–7.12 (12H, m, Ar), 6.63 (1H, d, H-5), 5.99 (1H, pseudo t, J_3′,4′ 9.6 Hz, H-3′), 5.74 (1H, pseudo t, J_4′,5′ 9.9 Hz, H-4′), 5.67 (1H, pseudo t, J_2′,3′ 9.7 Hz, H-2′), 4.87 (1H, d, J_1′,2′ 10.0 Hz, H-1′), 4.66 (1H, dd, H-6_a′), 4.46 (1H, dd, J_6a′,6b′ 12.3 Hz, H-6_b′), 4.25 (1H, ddd, J_5′,6a′ 2.4, J_5′,6b′ 5.2 Hz, H-5′), 3.35 (1H, dd, J_4a,4b 18.2, J_4a,5 6.7 Hz, H-4_a), 3.20 (1H, dd, J_4b,5 < 1.0 Hz, H-4_b), 1.82 (3H, s, CH₃). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 169.7 (OCOCH₃), 166.2, 165.8, 165.3 (4 × CO), 155.7 (C-3), 134.1–128.1 (Ar), 95.7 (C-5), 76.9 (C-5′), 74.0 (C-1′), 73.8 (C-3′), 69.7 (C-2′), 69.4 (C-4′), 63.0 (C-6′), 39.3 (C-4), 20.7 (CH₃). HR-ESI-MS positive mode (m/z): calcd. for C₃₉H₃₃NO₁₂ (707.20) [M + Na]⁺ = 730.1895, found: [M + Na]⁺ = 730.1895.

3-(2′,3′,4′,6′-Tetra-O-acetyl-β-d-galactopyranosyl)-5-phenylisoxazole (6a)

Prepared from oxime 1b (0.10 g, 0.27 mmol) and ethynylbenzene 5a (2 equiv., 59.7 µL, 0.06 g, 0.53 mmol) according to the General Procedure I. Purified by column chromatography (1:2 EtOAc–hexane) to yield 86 mg (68%) of 6a as a yellow amorphous product. R_f: 0.59 (1:1 EtOAc–hexane); [α]_D−16 (c 0.18, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.86–7.73 (2H, m, Ar), 7.52–7.39 (3H, m, Ar), 6.68 (1H, s, H-4), 5.55 (1H, dd, J_4′,5′ 0.9 Hz, H-4′), 5.47 (1H, pseudo t, J_2′,3′ 10.1 Hz, H-2′), 5.21 (1H, dd, J_3′,4′ 3.4 Hz, H-3′), 4.69 (1H, d, J_1′,2′ 9.9 Hz, H-1′), 4.24–4.14 (2H, m, H-6_a′, H-6_b′), 4.12 (1H, ddd, J_5′,6a′ 6.1, J_5′,6b′ 6.7 Hz, H-5′), 2.21, 2.05, 2.01, 1.97 (12H, 4s, 4 × CH₃). ¹³C NMR (90 MHz, CDCl₃) δ (ppm) 170.7, 170.3, 170.2, 169.6 (4 × CO), 170.6 (C-5), 161.5 (C-3), 130.9–125.6 (Ar), 97.9 (C-4), 75.1 (C-5′), 73.5 (C-1′), 71.9 (C-3′), 67.8 (C-2′), 67.7 (C-4′), 61.8 (C-6′), 20.82, 20.78, 20.72, 20.68 (4 × CH₃). HR-ESI-MS positive mode (m/z): calcd. for C₂₃H₂₅NO₁₀ (475.15) [M + Na]⁺ = 498.1371, found: [M + Na]⁺ = 498.1370.

3-(2′,3′,4′,6′-Tetra-O-acetyl-β-d-galactopyranosyl)-5-(naphth-2-yl)isoxazole (6b)

Prepared from oxime 1b (0.10 g, 0.27 mmol) and 2-ethynylnaphthalene 5b (2 equiv., 0.08 g, 0.53 mmol) according to the General Procedure I. Purified by column chromatography (1:1 EtOAc–hexane) to yield 78 mg (56%) of 6b as a yellow amorphous product. R_f: 0.35 (1:1 EtOAc–hexane); [α]_D−23 (c 0.23, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.31 (1H, bs, Ar), 7.91 (2H, d, J 8.1 Hz, Ar), 7.88–7.79 (2H, m, Ar), 7.57–7.51 (2H, m, Ar), 6.80 (1H, s, H-4), 5.57 (1H, dd, J_4′,5′ 0.5 Hz, H-4′), 5.50 (1H, pseudo t, J_2′,3′ 10.1 Hz, H-2′), 5.23 (1H, dd, J_3′,4′ 3.3 Hz, H-3′), 4.72 (1H, d, J_1′,2′ 9.9 Hz, H-1′), 4.25–4.07 (3H, m, H-5′, H-6_a′, H-6_b′), 2.23, 2.06, 2.01, 1.99 (12H, 4s, 4 × CH₃). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 170.7, 170.5, 170.3, 170.2, 169.6 (4 × CO, C-5), 161.6 (C-3), 134.2–122.8 (Ar), 98.3 (C-4), 75.1 (C-5′), 73.5 (C-1′), 71.9 (C-3′), 67.8 (C-2′), 67.7 (C-4′), 61.8 (C-6′), 20.82, 20.81, 20.80, 20.71 (4 × CH₃). HR-ESI-MS positive mode (m/z): calcd. for C₂₇H₂₇NO₁₀ (525.16) [M + H]⁺ = 526.1708, found: [M + H]⁺ = 526.1702.

3-(2′,3′,4′,6′-Tetra-O-acetyl-β-d-galactopyranosyl)-5-(naphth-1-yl)isoxazole (6c)

Prepared from oxime 1b (0.10 g, 0.27 mmol) and 2-ethynylnaphthalene 5c (2 equiv., 0.08 g, 0.53 mmol) according to the General Procedure I. Purified by column chromatography (1:2 EtOAc–hexane) to yield 80 mg (57%) of 6c as a pale yellow amorphous product. R_f: 0.41 (1:1 EtOAc–hexane); [α]_D−17 (c 0.11, CH₂Cl₂). ¹H NMR (700 MHz, CDCl₃) δ (ppm) 8.28 (1H, d, J 8.4 Hz, Ar), 7.97 (1H, d, J 8.2 Hz, Ar), 7.92 (1H, d, J 8.1 Hz, Ar), 7.81 (1H, dd, J 1.0, 7.1 Hz, Ar), 7.61 (1H, ddd, J 1.2, 6.8, 8.3 Hz, Ar), 7.59–7.48 (2H, m, Ar), 6.77 (1H, s, H-4), 5.57 (1H, dd, J_4′,5′ 0.8 Hz, H-4′), 5.55 (1H, pseudo t, J_2′,3′ 10.1 Hz, H-2′), 5.25 (1H, dd, J_3′,4′ 3.5 Hz, H-3′), 4.76 (1H, d, J_1′,2′ 9.9 Hz, H-1′), 4.23–4.17 (2H, m, H-6_a′, H-6_b′), 4.17–4.13 (1H, m, H-5′), 2.21, 2.06, 2.02, 2.01 (12H, 4s, 4 × CH₃). ¹³C NMR (175 MHz, CDCl₃) δ (ppm) 170.7, 170.6, 170.4, 170.2, 169.7 (4 × CO, C-5), 161.1 (C-3), 134.0–124.9 (Ar), 102.1 (C-4), 75.2 (C-5′), 73.6 (C-1′), 71.9 (C-3′), 67.9 (C-2′), 67.7 (C-4′), 61.8 (C-6′), 20.87, 20.84, 20.83, 20.75 (4 × CH₃). HR-ESI-MS positive mode (m/z): calcd. for C₂₇H₂₇NO₁₀ (525.16) [M + H]⁺ = 526.1708, found: [M + H]⁺ = 526.1713.

3-(2′,3′,4′,6′-Tetra-O-acetyl-β-d-galactopyranosyl)-5-(pyridin-2-yl)isoxazole (6d)

Prepared from oxime 1b (0.10 g, 0.27 mmol) and 2-ethynylpyridine 5d (2 equiv., 56.1 µL, 0.05 g, 0.53 mmol) according to the General Procedure I. Purified by column chromatography (1:1 EtOAc–hexane) to yield 80 mg (63%) of 6d as a pale orange amorphous product. R_f: 0.57 (1:1 EtOAc–hexane); [α]_D−21 (c 0.19, CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃) δ (ppm) 8.75–8.64 (1H, m, Ar), 7.93–7.77 (2H, m, Ar), 7.41–7.30 (1H, m, Ar), 7.09 (1H, s, H-4), 5.54 (1H, dd, J_4′,5′ 0.9 Hz, H-4′), 5.46 (1H, pseudo t, J_2′,3′ 10.1 Hz, H-2′), 5.21 (1H, dd, J_3′,4′ 3.3 Hz, H-3′), 4.71 (1H, d, J_1′,2′ 9.9 Hz, H-1′), 4.21–4.07 (3H, m, H-5′, H-6_a′, H-6_b′), 2.20, 2.05, 2.01, 1.98 (12H, 4s, 4 × CH₃). ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 170.6, 170.4, 170.2, 169.5 (4 × CO), 169.7 (C-5), 162.0 (C-3), 177.9–120.6 (Ar), 101.0 (C-4), 74.9 (C-5′), 73.2 (C-1′), 71.8 (C-3′), 67.8 (C-2′), 67.6 (C-4′), 61.9 (C-6′), 20.77, 20.76, 20.73, 20.69 (4 × CH₃). HR-ESI-MS positive mode (m/z): calcd. for C₂₂H₂₄N₂O₁₀ (476.14) [M + H]⁺ = 477.1504, found: [M + H]⁺ = 477.1504.

3-(2′,3′,4′,6′-Tetra-O-acetyl-β-d-galactopyranosyl)-5-(4-ethynylphenyl)isoxazole (6e)

Prepared from oxime 1b (0.10 g, 0.27 mmol) and 1,4-diethynylbenzene 5e (2 equiv., 0.07 g, 0.53 mmol) according to the General Procedure I. Purified by column chromatography (1.2:1 EtOAc–hexane) to yield 55 mg (41%) of 6e as a yellow amorphous product. R_f: 0.58 (1:1 EtOAc–hexane); [α]_D−37 (c 0.18, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.75 (2H, d, J 8.3 Hz, Ar), 7.58 (2H, d, J 8.4 Hz, Ar), 6.70 (1H, s, H-4), 5.55 (1H, dd, J_4′,5′ 0.9 Hz, H-4′), 5.45 (1H, pseudo t, J_2′,3′ 10.1 Hz, H-2′), 5.20 (1H, dd, J_3′,4′ 3.4 Hz, H-3′), 4.68 (1H, d, J_1′,2′ 10.0 Hz, H-1′), 4.21–4.15 (2H, m, H-6_a′, H-6_b′), 4.11 (1H, ddd, J_5′,6a′ 6.7, J_5′,6b′ 6.6 Hz, H-5′), 3.21 (1H, s, CCH), 2.21, 2.05, 2.01, 1.98 (12H, 4s, 4 × CH₃). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 170.6, 170.3, 170.2, 169.7 (4 × CO), 169.7 (C-5), 161.7 (C-3), 133.0–124.2 (Ar), 98.7 (C-4), 83.0 (CCH), 79.6 (CCH), 75.1 (C-5′), 73.5 (C-1′), 71.8 (C-3′), 67.7 (C-2′, C-4′), 61.8 (C-6′), 20.85 (2), 20.81, 20.75 (4 × CH₃). HR-ESI-MS positive mode (m/z): calcd. for C₂₅H₂₅NO₁₀ (499.47). [M + H]⁺ = 500.1551, found: [M + H]⁺ = 500.1552.

[3-(2′,3′,4′,6′-Tetra-O-acetyl-β-d-galactopyranosyl)isoxazol-5-yl]methanol (6f)

Prepared from oxime 1b (0.10 g, 0.27 mmol) and prop-2-yn-1-ol 5f (2 equiv., 28.8 µL, 0.03 g, 0.53 mmol) according to the General Procedure I. Purified by column chromatography (from 1:3 to 1:1 EtOAc–hexane) to yield 54 mg (47%) of 6f as a pale orange amorphous product. R_f: 0.15 (1:1 EtOAc–hexane); [α]_D−0.1 (c 0.15, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 6.42 (1H, s, H-4), 5.52 (1H, dd, J_4′,5′ 0.6 Hz, H-4′), 5.38 (1H, pseudo t, J_2′,3′ 10.2 Hz, H-2′), 5.18 (1H, dd, J_3′,4′ 2.8 Hz, H-3′), 4.76 (2H, s, CH₂OH), 4.63 (1H, d, J_1′,2′ 9.9 Hz, H-1′), 4.18–4.12 (2H, m, H-6_a′, H-6_b′), 4.08 (1H, ddd, J_5′,6a′ 6.1, J_5′,6b′ 6.2 Hz, H-5′), 2.38 (1H, bs, CH₂OH), 2.19, 2.05, 2.00, 1.96 (12H, 4s, 4 × CH₃). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 172.1 (C-5), 170.6, 170.3, 170.2, 169.8 (4 × CO), 161.0 (C-3), 100.5 (C-4), 75.0 (C-5′), 73.3 (C-1′), 71.8 (C-3′), 67.8 (C-2′), 67.6 (C-4′), 61.8 (C-6′), 56.7 (CH₂OH), 20.83, 20.81, 20.79, 20.73 (4 × CH₃). HR-ESI-MS positive mode (m/z): calcd. for C₁₈H₂₃NO₁₁ (429.13) [M + H]⁺ = 430.1344, found: [M + H]⁺ = 430.1343.

[3-(2′,3′,4′,6′-Tetra-O-acetyl-β-d-galactopyranosyl)isoxazol-5-yl]methyl acetate (6g)

Prepared from oxime 1b (0.10 g, 0.27 mmol) and prop-2-yn-1-yl acetate 5g (2 equiv., 52.9 µL, 0.05 g, 0.53 mmol) according to the General Procedure I. Purified by column chromatography (from 1:2 to 1:0 EtOAc–hexane) to yield 58 mg (46%) of 6g as a pale yellow amorphous product. R_f: 0.55 (2:1 EtOAc–hexane); [α]_D−5 (c 0.22, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 6.46 (1H, s, H-4), 5.52 (1H, dd, J_4′,5′ 0.4 Hz, H-4′), 5.37 (1H, pseudo t, J_2′,3′ 10.1 Hz, H-2′), 5.19 (1H, d, J_CHa,CHb 13.3 Hz, CH_aOAc), 5.18 (1H, dd, J_3′,4′ 3.2 Hz, H-3′), 5.15 (1H, d, CH_bOAc), 4.64 (1H, d, J_1′,2′ 9.9 Hz, H-1′), 4.19–4.11 (2H, m, H-6_a′, H-6_b′), 4.08 (1H, ddd, J_5′,6a′ 6.1, J_5′,6b′ 6.2 Hz, H-5′), 2.14 (3H, s, CH₂OCOCH₃), 2.19, 2.05, 2.00, 1.96 (12H, 4s, 4 × CH₃). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 170.3 (CH₂OCOCH₃), 170.5, 170.2, 170.1, 169.9 (4 × CO), 167.4 (C-5), 161.1 (C-3), 102.6 (C-4), 75.0 (C-5′), 73.2 (C-1′), 71.7 (C-3′), 67.7 (C-2′), 67.5 (C-4′), 61.7 (C-6′), 56.4 (CH₂OAc), 20.78, 20.77, 20.72, 20.69, 20.68 (5×CH₃). HR-ESI-MS positive mode (m/z): calcd. for C₂₀H₂₅NO₁₂ (471.42) [M + Na]⁺ = 494.1269, found: [M + Na]⁺ = 494.1268.

Diethyl 3-(2′,3′,4′,6′-Tetra-O-acetyl-β-d-galactopyranosyl)isoxazole-4,5-dicarboxylate (6i)

Prepared from oxime 1b (0.10 g, 0.27 mmol) and diethyl but-2-ynedioate 5i (2 equiv., 85.3 µL, 0.09 g, 0.53 mmol) according to the General Procedure I. Purified by column chromatography (from 1:2 to 2:1 EtOAc–hexane) to yield 64 mg (44%) of 6i as a pale yellow amorphous product. R_f: 0.30 (1:1 EtOAc–hexane); [α]_D+2 (c 0.33, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 5.77 (1H, pseudo t, J_2′,3′ 10.2 Hz, H-2′), 5.50 (1H, dd, J_4′,5′ 0.6 Hz, H-4′), 5.17 (1H, dd, J_3′,4′ 3.3 Hz, H-3′), 4.88 (1H, d, J_1′,2′ 10.0 Hz, H-1′), 4.49–4.32 (4H, m, 2 × CH₂CH₃) 4.15–4.04 (3H, m, H-5′, H-6_a′, H-6_b′), 2.19, 2.04, 2.01, 1.96 (12H, 4s, 4 × CH₃), 1.40 (6H, 2 × t, J 7.1 Hz, 2 × CH₂CH₃). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 170.5, 170.4, 170.3, 169.2 (4 × CO), 160.1 (C-5, COOEt), 159.0 (C-3), 156.0 (COOEt), 115.7 (C-4), 75.2 (C-5′), 72.7 (C-1′), 72.0 (C-3′), 67.5 (C-4′), 67.2 (C-2′), 63.1, 62.2 (2 × CH₂CH₃), 61.6 (C-6′), 20.78, 20.75, 20.74 (2) (4 × CH₃), 14.2, 14.1 (2 × CH₂CH₃). HR-ESI-MS positive mode (m/z): calcd. for C₂₃H₂₉NO₁₄ (543.16) [M + H]⁺ = 544.1661, found: [M + H]⁺ = 544.1664.

3-(2′,3′,4′,6′-Tetra-O-benzoyl-β-d-glucopyranosyl)-5-phenylisoxazole (7a)

Prepared from oxime 1a (0.10 g, 0.16 mmol) and ethynylbenzene 5a (2 equiv., 35.2 µL, 0.03 g, 0.32 mmol) according to the General Procedure I. Purified by column chromatography (from 1:3 to 1:2 EtOAc–hexane) to yield 67 mg (57%) of 7a as a yellow amorphous product. R_f: 0.34 (1:2 EtOAc–hexane). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.14–7.68 (10H, m, Ar), 7.64–7.20 (15H, m, Ar), 6.72 (1H, s, H-4), 6.05 (1H, pseudo t, J_3′,4′ 9.5 Hz, H-3′), 5.84 (1H, pseudo t, J_4′,5′ 9.5 Hz, H-4′), 5.82 (1H, pseudo t, J_2′,3′ 9.4 Hz, H-2′), 5.10 (1H, d, J_1′,2′ 10.0 Hz, H-1′), 4.69 (1H, dd, J_6a′,6b′ 12.3 Hz, H-6_a′), 4.53 (1H, dd, H-6_b′), 4.35 (1H, ddd, J_5′,6a′ 2.6, J_5′,6b′ 5.1 Hz, H-5′). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 170.8 (C-5), 166.3, 165.9, 165.4, 165.2 (4 × CO), 161.2 (C-3), 134.9–125.6 (Ar), 97.8 (C-4), 76.9 (C-5′), 74.3 (C-3′), 73.5 (C-1′), 71.3 (C-2′), 69.6 (C-4′), 63.3 (C-6′). C₄₃H₃₃NO₁₀ (723.21). NMR spectra are identical with those reported [16].

3-(2′,3′,4′,6′-Tetra-O-benzoyl-β-d-glucopyranosyl)-5-(pyridin-2-yl)isoxazole (7d)

Prepared from oxime 1a (0.10 g, 0.16 mmol) and 2-ethynylpyridine 5d (2 equiv., 32.4 µL, 0.03 g, 0.32 mmol) according to the General Procedure I. Purified by column chromatography (1:3 EtOAc–hexane) to yield 46 mg (39%) of 7d as a pale orange amorphous product. R_f: 0.16 (1:2 EtOAc–hexane); [α]_D−57 (c 0.22, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.67 (1H, d, J 4.8 Hz, Ar), 8.08–7.71 (10H, m, Ar), 7.59–7.22 (13H, m, Ar), 7.12 (1H, s, H-4), 6.05 (1H, pseudo t, J_3′,4′ 9.5 Hz, H-3′), 5.84 (1H, pseudo t, J_4′,5′ 9.4 Hz, H-4′), 5.82 (1H, pseudo t, J_2′,3′ 9.6 Hz, H-2′), 5.12 (1H, d, J_1′,2′ 10.0 Hz, H-1′), 4.67 (1H, dd, J_6a′,6b′ 12.3 Hz, H-6_a′), 4.53 (1H, dd, H-6_b′), 4.35 (1H, ddd, J_5′,6a′ 2.5, J_5′,6b′ 5.1 Hz, H-5′). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 170.0 (C-5), 166.3, 166.0, 165.4, 165.1 (4 × CO), 161.6 (C-3), 150.3–120.7 (Ar), 100.8 (C-4), 76.9 (C-5′), 74.3 (C-3′), 73.5 (C-1′), 71.3 (C-2′), 69.6 (C-4′), 63.4 (C-6′). HR-ESI-MS positive mode (m/z): calcd. for C₄₂H₃₂N₂O₁₀ (724.21) [M + Na]⁺ = 747.1949, found: [M + Na]⁺ = 747.1946.

3-(2′,3′,4′,6′-Tetra-O-benzoyl-β-d-glucopyranosyl)-5-(4-ethynylphenyl)isoxazole (7e)

Prepared from oxime 1a (0.10 g, 0.16 mmol) and 1,4-diethynylbenzene 5e (2 equiv., 0.04 g, 0.32 mmol) according to the General Procedure I. Purified by column chromatography (1:3 EtOAc–hexane) to yield 29 mg (24%) of 7e as a yellow amorphous product. R_f: 0.36 (1:2 EtOAc–hexane); [α]_D−96 (c 0.13, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.08–7.78 (8H, m, Ar), 7.68 (1H, d, J 8.3 Hz, Ar), 7.60–7.24 (14H, m, Ar), 6.73 (1H, s, H-4), 6.04 (1H, pseudo t, J_3′,4′ 9.7 Hz, H-3′), 5.83 (1H, pseudo t, J_4′,5′ 9.6 Hz, H-4′), 5.80 (1H, pseudo t, J_2′,3′ 9.7 Hz, H-2′), 5.09 (1H, d, J_1′,2′ 10.0 Hz, H-1′), 4.69 (1H, dd, J_6a′,6b′ 12.3 Hz, H-6_a′), 4.52 (1H, dd, H-6_b′), 4.34 (1H, ddd, J_5′,6a′ 2.8, J_5′,6b′ 5.1 Hz, H-5′), 3.19 (1H, s, CCH). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 169.8 (C-5), 166.3, 165.9, 165.4, 165.2 (4 × CO), 161.3 (C-3), 135.5–124.0 (Ar), 98.5 (C-4), 83.0 (CCH), 79.5 (CCH), 77.0 (C-5′), 74.2 (C-3′), 73.4 (C-1′), 71.2 (C-2′), 69.6 (C-4′), 63.3 (C-6′). HR-ESI-MS positive mode (m/z): calcd. for C₄₂H₃₂N₂O₁₀ (747.21) [M + Na]⁺ = 770.1997, found: [M + Na]⁺ = 770.1998.

[3-(2′,3′,4′,6′-Tetra-O-benzoyl-β-d-glucopyranosyl)isoxazol-5-yl]methanol (7f)

Prepared from oxime 1a (0.10 g, 0.16 mmol) and prop-2-yn-1-ol 5f (2 equiv., 18.5 µL, 0.02 g, 0.32 mmol) according to the General Procedure I. Purified by column chromatography (1:2 EtOAc–hexane) to yield 73 mg (67%) of 7f as a white amorphous product. R_f: 0.18 (1:2 EtOAc–hexane); [α]_D−3 (c 0.34, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.10–7.77 (8H, m, Ar), 7.59–7.22 (12H, m, Ar), 6.45 (1H, s, H-4), 6.03 (1H, pseudo t, J_3′,4′ 9.5 Hz, H-3′), 5.81 (1H, pseudo t, J_4′,5′ 9.8 Hz, H-4′), 5.74 (1H, pseudo t, J_2′,3′ 9.6 Hz, H-2′), 5.05 (1H, d, J_1′,2′ 10.0 Hz, H-1′), 4.67 (2H, s, CH₂OH), 4.66 (1H, dd, J_6a′,6b′ 12.3 Hz, H-6_a′), 4.50 (1H, dd, H-6_b′), 4.32 (1H, ddd, J_5′,6a′ 2.7, J_5′,6b′ 5.1 Hz, H-5′), 2.57 (1H, bs, CH₂OH). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 172.4 (C-5), 166.3, 165.9, 165.4, 165.3 (4 × CO), 160.6 (C-3), 133.9–128.0 (Ar), 100.3 (C-4), 76.9 (C-5′), 74.2 (C-3′), 73.3 (C-1′), 71.3 (C-2′), 69.5 (C-4′), 63.2 (C-6′), 56.6 (CH₂OH). HR-ESI-MS positive mode (m/z): calcd. for C₃₈H₃₁NO₁₁ (677.19) [M + Na]⁺ = 700.1789, found: [M + Na]⁺ = 700.1789.

[3-(2′,3′,4′,6′-Tetra-O-benzoyl-β-d-glucopyranosyl)isoxazol-5-yl]methyl acetate (7g)

Prepared from oxime 1a (0.10 g, 0.16 mmol) and prop-2-yn-1-yl acetate 5g (2 equiv., 31.8 µL, 0.03 g, 0.32 mmol) according to the General Procedure I. Purified by column chromatography (1:3 EtOAc–hexane) to yield 54 mg (47%) of 7g as a white amorphous product. R_f: 0.18 (1:2 EtOAc–hexane); [α]_D−11 (c 0.20, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.10–7.74 (8H, m, Ar), 7.60–7.13 (12H, m, Ar), 6.51 (1H, s, H-4), 6.02 (1H, pseudo t, J_3′,4′ 9.5 Hz, H-3′), 5.80 (1H, pseudo t, J_4′,5′ 9.8 Hz, H-4′), 5.73 (1H, pseudo t, J_2′,3′ 9.6 Hz, H-2′), 5.12 (2H, s, CH₂OAc), 5.05 (1H, d, J_1′,2′ 10.0 Hz, H-1′), 4.66 (1H, dd, J_6a′,6b′ 12.3 Hz, H-6_a′), 4.50 (1H, dd, H-6_b′), 4.32 (1H, ddd, J_5′,6a′ 2.8, J_5′,6b′ 5.1 Hz, H-5′), 2.08 (3H, s, CH₃). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 170.2 (CH₂OCOCH₃), 167.6 (C-5), 166.3, 165.9, 165.3, 165.1 (4 × CO), 160.8 (C-3), 134.0–128.0 (Ar), 102.4 (C-4), 76.9 (C-5′), 74.2 (C-3′), 73.3 (C-1′), 71.2 (C-2′), 69.5 (C-4′), 63.2 (C-6′), 56.4 (CH₂OAc), 20.7 (CH₃). HR-ESI-MS positive mode (m/z): calcd. for C₄₀H₃₃NO₁₂ (719.20) [M + Na]⁺ = 742.1895, found: [M + Na]⁺ = 742.1892.

3-(2′,3′,4′,6′-Tetra-O-benzoyl-β-d-glucopyranosyl)-5-(2″,3″,4″,6″-tetra-O-benzyl-β-d-glucopyranosyl)isoxazole (7h)

Prepared from oxime 1a (0.05 g, 0.08 mmol) and 2-C-(2′,3′,4′,6′-tetra-O-benzyl-β-d-glucopyranosyl)ethyne (3,7-anhydro-4,5,6,8-tetra-O-benzyl-1,2-dideoxy-d-glycero-d-gulo-oct-1-ynitol) 5h [20] (2 equiv., 0.09 g, 0.16 mmol) according to the General Procedure I. Purified by column chromatography (from 1:4 to 1:2 EtOAc–hexane) to yield 15 mg (16%) of 7h as a white amorphous product. R_f: 0.35 (1:2 EtOAc–hexane); [α]_D−5 (c 0.10, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.09–7.69 (8H, m, Ar), 7.61–6.99 (32H, m, Ar), 6.57 (1H, s, H-4), 6.02 (1H, pseudo t, J_3′,4′ 9.6 Hz, H-3′), 5.79 (1H, pseudo t, J_4′,5′ 9.7 Hz, H-4′), 5.73 (1H, pseudo t, J_2′,3′ 9.7 Hz, H-2′), 5.07 (1H, d, J_1′,2′ 10.0 Hz, H-1′), 4.87–4.77 (3H, m, CH₂), 4.65 (1H, dd, J_6a′,6b′ 12.3 Hz, H-6_a′), 4.61–4.47 (3H, m, CH₂), 4.51 (1H, dd, H-6_b′), 4.44 (1H, d, J_1″,2″ 8.9 Hz, H-1″), 4.32 (1H, ddd, J_5′,6a′ 2.7, J_5′,6b′ 4.6 Hz, H-5′), 4.31 (1H, d, J 10.5 Hz, CH₂), 4.14 (1H, d, J 10.5 Hz, CH₂), 3.79–3.60 (5H, m, H-2″, H-3″, H-4″, H-6_a″, H-6_b″), 3.54 (1H, ddd, J_4′,5′ 9.6, J_5″,6a″ 2.0, J_5″,6b″ 3.6 Hz, H-5″). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 170.3 (C-5), 166.3, 165.9, 165.4, 165.0 (4 × CO), 160.7 (C-3), 138.7–127.4 (Ar), 102.1 (C-4), 86.4 (C-3″), 81.2 (C-2″), 79.9 (C-5″), 77.8 (C-4″), 76.7 (C-5′), 75.7, 75.3, 75.0 (3×CH₂), 74.3 (C-3′), 73.7 (C-1″, CH₂), 73.5 (C-1′), 71.3 (C-2′), 69.6 (C-4′), 68.9 (C-6″), 63.4 (C-6′). HR-ESI-MS positive mode (m/z): calcd. for C₇₁H₆₃NO₁₅ (1169.42) [M + Na]⁺ = 1192.4090, found: [M + Na]⁺ = 1192.4093.

3,4-Di(2,3,4,6-Tetra-O-benzoyl-β-d-glucopyranosyl)-1,2,5-oxadiazole-2-oxide (4a)

Prepared from the previous reaction mixture from oxime 1a (0.05 g, 0.08 mmol) and 2-C-(2′,3′,4′,6′-tetra-O-benzyl-β-d-glucopyranosyl)ethyne (3,7-anhydro-4,5,6,8-tetra-O-benzyl-1,2-dideoxy-d-glycero-d-gulo-oct-1-ynitol) 5h [20] (2 equiv., 0.09 g, 0.16 mmol) according to the General Procedure I. Purified by column chromatography (from 1:4 to 1:2 EtOAc–hexane) to yield 15 mg (31%) of 4a as a white amorphous product. R_f: 0.29 (1:2 EtOAc–hexane); [α]_D−18 (c 0.14, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.12–7.89 (8H, m, Ar), 7.87–7.70 (8H, m, Ar), 7.59–7.09 (24H, m, Ar), 6.18–6.09 (3H, m, H-2′, H-3′, H-3″), 6.07 (1H, pseudo t, J_2″,3″ 9.5 Hz, H-2″), 5.79 (1H, pseudo t, J_3′,4′ 9.7, J_4′,5′ 10.2 Hz, H-4′), 5.77 (1H, pseudo t, J_3″,4″ 9.7, J_4″,5″ 9.9 Hz, H-4″), 5.26 (1H, d, J_1′,2′ 10.1 Hz, H-1′), 5.17 (1H, d, J_1″,2″ 9.4 Hz, H-1″), 4.87–4.81 (2H, m, H-6_a′, H-6_a″), 4.81 (1H, dd, H-6_b′), 4.75 (1H, dd, J_6a″,6b″ 12.8 Hz, H-6_b″), 4.47 (1H, ddd, J_5′,6a′ 2.8, J_5′,6b′ 6.9 Hz, H-5′), 4.43 (1H, ddd, J_5″,6a″ 2.7, J_5″,6b″ 7.4 Hz, H-5″). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 166.2, 165.9, 165.8, 165.4, 165.0, 164.9 (8×CO), 153.7 (C-4), 134.0–128.1 (Ar), 112.9 (C-3), 77.6 (C-5′, C-5″), 74.4 (C-1″), 73.7 (C-3′), 73.5 (C-3″), 72.0 (C-1′), 71.2 (C-2′), 71.0 (C-2″), 70.0 (C-4′), 69.9 (C-4″), 63.8 (C-6′, C-6″). HR-ESI-MS positive mode (m/z): calcd. for C₇₀H₅₄N₂O₂₀ (1242.33). [M + Na]⁺ = 1265.3162, found: [M + Na]⁺ = 1265.3161.

Diethyl 3-(2′,3′,4′,6′-Tetra-O-benzoyl-β-d-glucopyranosyl)isoxazole-4,5-dicarboxylate (7i)

Prepared from oxime 1a (0.10 g, 0.16 mmol) and diethyl but-2-ynedioate 5i (2 equiv., 51.3 µL, 0.05 g, 0.32 mmol) according to the General Procedure I. Purified by column chromatography (1:2 EtOAc–hexane) to yield 64 mg (50%) of 7i as a pale yellow amorphous product. R_f: 0.42 (1:1 EtOAc–hexane); [α]_D−3 (c 0.58, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.11–7.74 (8H, m, Ar), 7.58–7.16 (12H, m, Ar), 6.22 (1H, pseudo t, J_2′,3′ 9.7 Hz, H-2′), 6.02 (1H, pseudo t, J_3′,4′ 9.5 Hz, H-3′), 5.82 (1H, pseudo t, J_4′,5′ 9.7 Hz, H-4′), 5.27 (1H, d, J_1′,2′ 10.0 Hz, H-1′), 4.62 (1H, dd, J_6a′,6b′ 12.4 Hz, H-6_a′), 4.45 (1H, dd, H-6_b′), 4.41 (2H, q, J 7.2 Hz, CH₂CH₃), 4.30 (1H, ddd, J_5′,6a′ 2.5, J_5′,6b′ 5.1 Hz, H-5′), 4.29–4.18 (2H, m, CH₂CH₃), 1.37, 1.30 (6H, 2 × t, J 7.1 Hz, 2 × CH₂CH₃). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 166.3, 166.0, 165.3, 164.9 (4 × CO), 160.8 (C-5), 160.2 (COOEt), 159.0 (C-3), 156.1 (COOEt), 133.7–128.4 (Ar), 115.1 (C-4), 77.0 (C-5′), 74.3 (C-3′), 72.9 (C-1′), 70.5 (C-2′), 69.2 (C-4′), 63.1 (C-6′), 63.1, 62.3 (2 × CH₂CH₃), 14.1 (2 × CH₂CH₃). HR-ESI-MS positive mode (m/z): calcd. for C₄₇H₃₇NO₁₄ (791.22) [M + H]⁺ = 814.2106, found: [M + H]⁺ = 814.2098.

3-(2′,3′,4′,6′-Tetra-O-acetyl-β-d-galactopyranosyl)-5-phenylisoxazolines (8a)

Prepared from oxime 1b (0.10 g, 0.27 mmol) and ethenylbenzene 2a (2 equiv., 61.1 µL, 0.06 g, 0.53 mmol) according to the General Procedure I. Purified by column chromatography (1:1 EtOAc–hexane) to yield 106 mg (83%) of 8a (diastereomeric ratio: 1.1:1) as a yellow amorphous product. R_f: 0.44 (1:1 EtOAc–hexane). 8a-I ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.43–7.25 (5H, m, Ar), 5.59 (1H, dd, H-5), 5.49 (1H, dd, J_4′,5′ 0.5 Hz, H-4′), 5.27 (1H, pseudo t, J_2′,3′ 10.7 Hz, H-2′), 5.17 (1H, dd, J_3′,4′ 3.3 Hz, H-3′), 4.39 (1H, d, J_1′,2′ 9.6 Hz, H-1′), 4.17–4.06 (2H, m, H-6_a′, H-6_b′), 4.05–3.96 (1H, m, H-5′), 3.57 (1H, dd, J_4a,4b 17.2, J_4a,5 11.0 Hz, H-4_a), 3.08 (1H, dd, J_4b,5 9.3 Hz, H-4_b), 2.15, 2.08, 2.03, 2.01 (12H, 4s, 4 × CH₃). ¹³C NMR (90 MHz, CDCl₃) δ (ppm) 170.4, 170.1, 170.0 (4 × CO), 155.4 (C-3), 140.6–125.6 (Ar), 82.7 (C-5), 74.6 (C-5′), 74.4 (C-1′), 71.2 (C-3′), 67.4 (C-4′), 66.4 (C-2′), 61.5 (C-6′), 40.9 (C-4), 20.77, 20.68, 20.64 (2) (4 × CH₃). 8a-II ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.43–7.25 (5H, m, Ar), 5.57 (1H, dd, H-5), 5.48 (1H, dd, J_4′,5′ 0.5 Hz, H-4′), 5.22 (1H, pseudo t, J_2′,3′ 10.5 Hz, H-2′), 5.14 (1H, dd, J_3′,4′ 3.3 Hz, H-3′), 4.41 (1H, d, J_1′,2′ 9.3 Hz, H-1′), 4.17–4.06 (2H, m, H-6_a′, H-6_b′), 4.05–3.96 (1H, m, H-5′), 3.49 (1H, dd, J_4a,4b 17.3, J_4a,5 11.2 Hz, H-4_a), 3.16 (1H, dd, J_4b,5 10.2 Hz, H-4_b), 2.13, 2.06, 2.01, 1.98 (12H, 4s, 4 × CH₃). ¹³C NMR (90 MHz, CDCl₃) δ (ppm) 170.4, 170.1, 170.0, 169.9 (4 × CO), 155.4 (C-3), 140.6–125.6 (Ar), 83.0 (C-5), 74.6 (C-5′), 74.5 (C-1′), 71.4 (C-3′), 67.5 (C-4′), 66.0 (C-2′), 61.7 (C-6′), 40.9 (C-4), 20.73, 20.64 (2), 20.59 (4 × CH₃). HR-ESI-MS positive mode (m/z): calcd. for C₂₃H₂₇NO₁₀ (477.16) [M + H]⁺ = 478.1708, found: [M + H]⁺ = 478.1710.

3-(2′,3′,4′,6′-Tetra-O-acetyl-β-d-galactopyranosyl)-5-(naphth-2-yl)isoxazolines (8b)

Prepared from oxime 1b (0.10 g, 0.27 mmol) and 2-ethenylnaphthalene 2b (2 equiv., 0.08 g, 0.53 mmol) according to the General Procedure I. Purified by column chromatography (1:1 EtOAc–hexane) to yield 106 mg (83%) of 8b (diastereomeric ratio: 1.1:1) as a pale yellow amorphous product. R_f: 0.38 (1:1 EtOAc–hexane). 8b-I ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.92–7.76 (4H, m, Ar), 7.54–7.38 (3H, m, Ar), 5.74 (1H, dd, H-5), 5.49 (1H, dd, J_4′,5′ 0.5 Hz, H-4′), 5.30 (1H, pseudo t, J_2′,3′ 10.5 Hz, H-2′), 5.17 (1H, dd, J_3′,4′ 3.5 Hz, H-3′), 4.41 (1H, d, J_1′,2′ 9.4 Hz, H-1′), 4.17–4.05 (2H, m, H-6_a′, H-6_b′), 4.04–3.96 (1H, m, H-5′), 3.64 (1H, dd, J_4a,4b 17.2, J_4a,5 10.9 Hz, H-4_a), 3.16 (1H, dd, J_4b,5 9.2 Hz, H-4_b), 2.15, 2.10, 2.01, 1.97 (12H, 4s, 4 × CH₃). ¹³C NMR (90 MHz, CDCl₃) δ (ppm) 170.5, 170.2, 170.0 (4 × CO), 155.6 (C-3), 137.8–123.3 (Ar), 82.9 (C-5), 74.6 (C-5′), 74.5 (C-1′), 71.3 (C-3′), 67.5 (C-4′), 66.5 (C-2′), 61.5 (C-6′), 41.0 (C-4), 20.83, 20.70, 20.67, 20.64 (4 × CH₃). 8b-II ¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.92–7.76 (4H, m, Ar), 7.54–7.38 (3H, m, Ar), 5.77 (1H, dd, H-5), 5.48 (1H, dd, J_4′,5′ 0.5 Hz, H-4′), 5.24 (1H, pseudo t, J_2′,3′ 10.3 Hz, H-2′), 5.14 (1H, dd, J_3′,4′ 3.4 Hz, H-3′), 4.44 (1H, d, J_1′,2′ 8.9 Hz, H-1′), 4.17–4.05 (2H, m, H-6_a′, H-6_b′), 4.04–3.96 (1H, m, H-5′), 3.56 (1H, dd, J_4a,4b 17.4, J_4a,5 11.2 Hz, H-4_a), 3.26 (1H, dd, J_4b,5 9.8 Hz, H-4_b), 2.12, 2.06, 2.01, 1.97 (12H, 4s, 4 × CH₃). ¹³C NMR (90 MHz, CDCl₃) δ (ppm) 170.5, 170.4, 170.0, 169.9 (4 × CO), 155.5 (C-3), 137.8–123.3 (Ar), 83.1 (C-5), 74.7 (C-5′), 74.6 (C-1′), 71.5 (C-3′), 67.5 (C-4′), 66.1 (C-2′), 61.7 (C-6′), 40.9 (C-4), 20.77, 20.70, 20.67, 20.61 (4 × CH₃). HR-ESI-MS positive mode (m/z): calcd. for C₂₇H₂₉NO₁₀ (527.18) [M + H]⁺ = 528.1684, found: [M + H]⁺ = 528.1682.

[3-(2′,3′,4′,6′-Tetra-O-acetyl-β-d-galactopyranosyl)isoxazol-5-yl] acetates (8c)

Prepared from oxime 1b (0.10 g, 0.27 mmol) and ethenyl acetate 2c (2 equiv., 49.1 µL, 0.05 g, 0.53 mmol) according to the General Procedure I. Purified by column chromatography (from 1:2 to 2:1 EtOAc–hexane) to yield 65 mg (53%) of 8c (diastereomeric ratio: 1.6:1) as a yellow amorphous product. R_f: 0.42 (2:1 EtOAc–hexane). 8c-I ¹H NMR (500 MHz, CDCl₃) δ (ppm) 6.70 (1H, dd, H-5), 5.52–5.48 (1H, m, H-4′), 5.20–5.12 (2H, m, H-2′, H-3′), 4.45–4.39 (1H, m, H-1′), 4.19–4.08 (2H, m, H-6_a′, H-6_b′), 4.04 (1H, ddd, J_5′,6a′ 6.1, J_5′,6b′ 6.3 Hz, H-5′), 3.43 (1H, dd, J_4a,4b 18.3, J_4a,5 7.1 Hz, H-4_a), 3.09 (1H, dd, J_4b,5 < 1.0 Hz, H-4_b), 2.08 (3H, s, CH₂OCOCH₃), 2.17, 2.06, 2.02, 2.00 (12H, 4s, 4 × CH₃). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 170.5, 170.3, 170.1, 170.0 (4 × CO), 169.7 (OCOCH₃), 156.6 (C-3), 95.8 (C-5), 74.7 (C-5′), 74.0 (C-1′), 71.1 (C-3′), 67.3 (C-4′), 66.2 (C-2′), 61.4 (C-6′), 39.5 (C-4), 21.07, 20.96, 20.74, 20.69, 20.62 (5 × CH₃). 8c-II ¹H NMR (500 MHz, CDCl₃) δ (ppm) 6.65 (1H, dd, H-5), 5.52–5.48 (1H, m, H-4′), 5.32 (1H, pseudo t, J_2′,3′ 10.1 Hz, H-2′), 5.15 (1H, dd, J_3′,4′ 3.3 Hz, H-3′), 4.46 (1H, d, J_1′,2′ 9.8 Hz, H-1′), 4.19–4.08 (2H, m, H-6_a′, H-6_b′), 4.03 (1H, ddd, J_5′,6a′ 6.2, J_5′,6b′ 6.0 Hz, H-5′), 3.32 (1H, dd, J_4a,4b 18.5, J_4a,5 6.9 Hz, H-4_a), 3.13 (1H, dd, J_4b,5 < 1.0 Hz, H-4_b), 2.08 (3H, s, CH₂OCOCH₃), 2.18, 2.05, 2.04, 2.00 (12H, 4s, 4 × CH₃). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 170.5, 170.1, 170.0, 169.5 (4 × CO), 169.8 (OCOCH₃), 155.9 (C-3), 95.9 (C-5), 74.9 (C-5′), 73.9 (C-1′), 71.3 (C-3′), 67.5 (C-4′), 66.1 (C-2′), 61.6 (C-6′), 39.8 (C-4), 21.07, 20.96, 20.74, 20.69, 20.62 (5×CH₃). HR-ESI-MS positive mode (m/z): calcd. for C₁₉H₂₅NO₁₂ (459.40) [M + Na]⁺ = 482.1269, found: [M + Na]⁺ = 482.1269.

3-(2′,3′,4′,6′-Tetra-O-acetyl-β-d-galactopyranosyl)-4,5-diphenylisoxazoline (8e)

Prepared from oxime 1b (0.10 g, 0.27 mmol) and (E)-1,2-diphenylethene 2e (2 equiv., 0.10 g, 0.53 mmol) according to the General Procedure I. Purified by column chromatography (from 1:3 to 1:1 EtOAc–hexane) to yield 33 mg (23%) of 8e as a colourless amorphous product. R_f: 0.60 (2:1 EtOAc–hexane); [α]_D+76 (c 0.59, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.39–7.29 (6H, m, Ar), 7.29–7.23 (4H, m, Ar), 5.54 (1H, d, H-5), 5.42 (1H, pseudo t, J_2′,3′ 10.0 Hz, H-2′), 5.29 (1H, dd, J_4′,5′ 0.4 Hz, H-4′), 4.97 (1H, dd, J_3′,4′ 3.3 Hz, H-3′), 4.46 (1H, d, J_4,5 7.9 Hz, H-4), 4.27 (1H, d, J_1′,2′ 10.0 Hz, H-1′), 3.76–3.67 (3H, m, H-5′, H-6_a′, H-6_b′), 2.05, 2.01, 1.98, 1.95 (12H, 4s, 4 × CH₃). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 170.4, 170.2, 169.6 (4 × CO), 155.9 (C-3), 139.9–125.5 (Ar), 91.7 (C-5), 74.2 (C-5′), 73.6 (C-1′), 72.0 (C-3′), 67.4 (C-4′), 66.1 (C-2′), 62.4 (C-4), 61.4 (C-6′), 20.86, 20.78, 20.73, 20.72 (4 × CH₃). HR-ESI-MS positive mode (m/z): calcd. for C₂₉H₃₁NO₁₀ (553.56) [M + Na]⁺ = 576.1840, found: [M + Na]⁺ = 576.1841.

3,4-Di(2,3,4,6-Tetra-O-acetyl-β-d-galactopyranosyl)-1,2,5-oxadiazole-2-oxide (4b)

Prepared from the previous reaction mixture from oxime 1b (0.10 g, 0.27 mmol) and (E)-1,2-diphenylethene 2e (2 equiv., 0.10 g, 0.53 mmol) according to the General Procedure I. Purified by column chromatography (from 1:3 to 1:1 EtOAc–hexane) to yield 76 mg (76%) of 4b as a yellow amorphous product. R_f: 0.47 (2:1 EtOAc–hexane). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 5.65 (1H, pseudo t, J_2′,3′ 10.0 Hz, H-2′), 5.58 (1H, pseudo t, J_2″,3″ 10.0 Hz, H-2″), 5.55 (1H, dd, J_4′,5′ 0.7 Hz, H-4′), 5.51 (1H, dd, J_4″,5″ 0.7 Hz, H-4″), 5.19 (1H, dd, J_3′,4′ 3.3 Hz, H-3′), 5.17 (1H, dd, J_3″,4″ 3.3 Hz, H-3″), 4.84 (1H, d, J_1′,2′ 10.1 Hz, H-1′), 4.82 (1H, d, J_1″,2″ 10.1 Hz, H-1″), 4.31–4.01 (6H, m, H-5′, H-5″, H-6_a′, H-6_a″, H-6_b′, H-6_b″), 2.24, 2.06, 2.05, 2.02, 2.00, 1.99 (24H, 8s, 8 × CH₃). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 170.4, 170.2, 170.1, 170.0, 169.5, 169.2 (8 × CO), 153.3 (C-4), 111.7 (C-3), 75.4 (C-5″), 75.2 (C-5′), 72.9 (C-1″), 71.7 (C-3′, C-3″), 70.8 (C-1′), 67.3 (C-4′, C-4″), 66.9 (C-2″), 65.9 (C-2′), 61.4 (C-6′, C-6″), 20.77, 20.74, 20.70 (2), 20.61 (2), 20.59, 20.49 (8 × CH₃). NMR spectra are identical with those reported [14].

3.1.3. Synthesis of 3-(β-d-Galactopyranosyl)isoxazole (9)

[3-(2′,3′,4′,6′-Tetra-O-acetyl-β-d-galactopyranosyl)isoxazol-5-yl] acetates 8c (0.04 g, 0.09 mmol) were dissolved in dichloromethane (0.5 mL) and ethanol (0.5 mL). Then cc. HCl (36 µL) was added to the mixture. The solution was stirred and heated to reflux temperature. When TLC (5:1 CHCl₃–MeOH) indicated complete consumption of the starting compound (~3 h), the mixture was cooled and the solvent was removed under reduced pressure, and the residue was purified by column chromatography (5:1 CHCl₃–MeOH) to yield 10 mg (49%) of 9 as a colourless amorphous product. R_f: 0.17 (4:1 CHCl₃–MeOH); [α]_D+42 (c 0.05, MeOH). ¹H NMR (500 MHz, CD₃OD) δ (ppm) 8.62 (1H, s, H-5), 6.67 (1H, s, H-4), 4.36 (1H, d, J_1′,2′ 9.7 Hz, H-1′), 3.96 (1H, bs, H-4′), 3.81 (1H, pseudo t, J_2′,3′ 9.5 Hz, H-2′), 3.77 (1H, dd, J_5′,6b′ 7.3, J_6a′,6b′ 11.1 Hz, H-6_a′), 3.73–3.64 (2H, m, H-5′, H-6_b′), 3.59 (1H, dd, J_3′,4′ 3.4 Hz, H-3′). ¹³C NMR (125 MHz, CD₃OD) δ (ppm) 163.3 (C-3), 160.3 (C-5), 104.4 (C-4), 81.1 (C-5′), 76.3 (C-1′), 76.1 (C-3′), 71.5 (C-2′), 70.8 (C-4′), 62.8 (C-6′). ESI-MS negative mode (m/z): calcd. for C₉H₁₃NO₆ (231.07) [M − H]⁻ = 230.0670, found: [M − H]⁻ = 229.99.

3.1.4. General Procedure II for Removal of O-Acyl Protecting Groups (10, 11, 12, 13)

5-Substituted 3-(2′,3′,4′,6′-tetra-O-acyl-β-d-glycopyranosyl)isoxazole or -isoxazolines 3, 6, 7, 8 (100 mg) were dissolved in dry MeOH (5 mL) and dry chloroform (3 mL) then a solution of NaOMe (1 M in MeOH) was added to the solution in a catalytic amount. The reaction mixture was stirred at room temperature. When the reaction was complete (TLC, 7:3 CHCl₃–MeOH) (1–3 h), the solution was neutralized with a cation exchange resin Amberlyst 15 (H⁺ form). The resin was filtered off with suction and the filtrate was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography with eluents indicated for the particular compounds to give 5-substituted 3-(β-d-glycopyranosyl)isoxazoles 10 and 11 and -isoxazolines 12 and 13.

3-(β-d-Galactopyranosyl)-5-phenylisoxazole (10a)

Prepared from isoxazole 6a (0.06 g, 0.12 mmol) according to the General Procedure II. Purified by column chromatography (4:1 CHCl₃–MeOH) to yield 18 mg (49%) of 10a as a pale yellow amorphous product. R_f: 0.56 (7:3 CHCl₃–MeOH); [α]_D+18 (c 0.12, MeOH). ¹H NMR (700 MHz, CD₃OD) δ (ppm) 7.83 (2H, d, J 7.2 Hz, Ar), 7.53–7.45 (3H, m, Ar), 6.96 (1H, s, H-4), 4.35 (1H, d, J_1′,2′ 9.7 Hz, H-1′), 3.99 (1H, dd, J_4′,5′ 0.6 Hz, H-4′), 3.87 (1H, pseudo t, J_2′,3′ 9.4 Hz, H-2′), 3.80 (1H, dd, J_6a′,6b′ 11.3 Hz, H-6_a′), 3.73 (1H, dd, H-6_b′), 3.70 (1H, ddd, J_5′,6b′ 4.9, J_5′,6a′ 6.9 Hz, H-5′), 3.62 (1H, dd, J_3′,4′ 3.3 Hz, H-3′). ¹³C NMR (90 MHz, CD₃OD) δ (ppm) 171.2 (C-5), 165.1 (C-3), 131.5–126.7 (Ar), 99.7 (C-4), 81.1 (C-5′), 76.5 (C-1′), 76.1 (C-3′), 71.5 (C-2′), 70.8 (C-4′), 62.9 (C-6′). HR-ESI-MS positive mode (m/z): calcd. for C₁₅H₁₇NO₆ (307.11) [M + H]⁺ = 308.1129, found: [M + H]⁺ = 308.1129.

3-(β-d-Galactopyranosyl)-5-(naphth-2-yl)isoxazole (10b)

Prepared from isoxazole 6b (0.06 g, 0.12 mmol) according to the General Procedure II. Purified by column chromatography (6:1 CHCl₃–MeOH) to yield 19 mg (43%) of 10b as a white amorphous product. R_f: 0.43 (1:1 CHCl₃–MeOH); [α]_D+32 (c 0.10, MeOH). ¹H NMR (700 MHz, CD₃OD) δ (ppm) 8.38 (1H, bs, Ar), 8.01–7.96 (1H, m, Ar), 7.99 (1H, d, J 8.4 Hz, Ar), 7.93–7.88 (1H, m, Ar), 7.90 (1H, dd, J 1.7, 8.5 Hz, Ar), 7.60–7.54 (2H, m, Ar), 7.08 (1H, s, H-4), 4.38 (1H, d, J_1′,2′ 9.7 Hz, H-1′), 4.00 (1H, dd, J_4′,5′ 0.4 Hz, H-4′), 3.90 (1H, pseudo t, J_2′,3′ 9.4 Hz, H-2′), 3.81 (1H, dd, J_6a′,6b′ 11.3 Hz, H-6_a′), 3.74 (1H, dd, H-6_b′), 3.72 (1H, ddd, J_5′,6a′ 4.9, J_5′,6b′ 6.8 Hz, H-5′), 3.63 (1H, dd, J_3′,4′ 3.3 Hz, H-3′). ¹³C NMR (175 MHz, CD₃OD) δ (ppm) 171.3 (C-5), 165.2 (C-3), 135.5–123.8 (Ar), 100.2 (C-4), 81.2 (C-5′), 76.6 (C-1′), 76.1 (C-3′), 71.6 (C-2′), 70.9 (C-4′), 62.9 (C-6′). HR-ESI-MS positive mode (m/z): calcd. for C₁₉H₁₉NO₆ (357.12) [M + Na]⁺ = 380.1105, found: [M + Na]⁺ = 380.1106.

3-(β-d-Galactopyranosyl)-5-(naphth-1-yl)isoxazole (10c)

Prepared from isoxazole 6c (0.05 g, 0.10 mmol) according to the General Procedure II. Purified by column chromatography (3.5:1 CHCl₃–MeOH) to yield 26 mg (75%) of 10c as a white amorphous product. R_f: 0.46 (7:3 CHCl₃–MeOH); [α]_D+21 (c 0.10, MeOH). ¹H NMR (500 MHz, CD₃OD) δ (ppm) 8.31 (1H, d, J 8.1 Hz, Ar), 8.02 (1H, d, J 8.2 Hz, Ar), 7.97 (1H, dd, J 1.9, 7.5 Hz, Ar), 7.83 (1H, dd, J 1.1, 7.2 Hz, Ar), 7.63–7.54 (3H, m, Ar), 7.02 (1H, s, H-4), 4.44 (1H, d, J_1′,2′ 9.7 Hz, H-1′), 4.01 (1H, dd, J_4′,5′ 0.5 Hz, H-4′), 3.95 (1H, pseudo t, J_2′,3′ 9.4 Hz, H-2′), 3.83 (1H, dd, J_5′,6a′ 8.3, J_6a′,6b′ 12.6 Hz, H-6_a′), 3.79–3.71 (2H, m, H-5′, H-6_b′), 3.66 (1H, dd, J_3′,4′ 3.3 Hz, H-3′). ¹³C NMR (125 MHz, CD₃OD) δ (ppm) 171.2 (C-5), 164.8 (C-3), 135.5–125.7 (Ar), 103.8 (C-4), 81.2 (C-5′), 76.6 (C-1′), 76.1 (C-3′), 71.6 (C-2′), 70.9 (C-4′), 62.9 (C-6′). HR-ESI-MS positive mode (m/z): calcd. for C₁₉H₁₉NO₆ (357.12) [M + Na]⁺ = 380.1105, found: [M + Na]⁺ = 380.1104.

3-(β-d-Galactopyranosyl)-5-(pyridin-2-yl)isoxazole (10d)

Prepared from isoxazole 6d (0.11 g, 0.23 mmol) according to the General Procedure II. Purified by column chromatography (7:3 CHCl₃–MeOH) to yield 45 mg (63%) of 10d as a white amorphous product. R_f: 0.36 (4:1 CHCl₃–MeOH); [α]_D+28 (c 0.20, MeOH). ¹H NMR (700 MHz, CD₃OD) δ (ppm) 8.68–8.65 (1H, m, Ar), 8.00–7.95 (2H, m, Ar), 7.49 (1H, ddd, J 1.6, 4.9, 6.7 Hz, Ar), 7.19 (1H, s, H-4), 4.39 (1H, d, J_1′,2′ 9.7 Hz, H-1′), 3.99 (1H, dd, J_4′,5′ 0.6 Hz, H-4′), 3.89 (1H, pseudo t, J_2′,3′ 9.4 Hz, H-2′), 3.80 (1H, dd, J_6a′,6b′ 11.1 Hz, H-6_a′), 3.72 (1H, dd, H-6_b′), 3.70 (1H, ddd, J_5′,6b′ 4.9, J_5′,6a′ 6.6 Hz, H-5′), 3.62 (1H, dd, J_3′,4′ 3.3 Hz, H-3′). ¹³C NMR (100 MHz, CD₃OD) δ (ppm) 170.0 (C-5), 165.2 (C-3), 151.6–121.5 (Ar), 102.6 (d, J 15.7 Hz, C-4), 81.2 (C-5′), 76.5 (d, J 10.1 Hz, C-1′), 76.1 (C-3′), 71.5 (C-2′), 70.8 (d, J 6.2 Hz, C-4′), 62.9 (C-6′). HR-ESI-MS positive mode (m/z): calcd. for C₁₄H₁₆N₂O₆ (308.10) [M + H]⁺ = 309.1081, found: [M + H]⁺ = 309.1081.

3-(β-d-Glucopyranosyl)-5-phenylisoxazole (11a)

Prepared from isoxazole 7a (0.06 g, 0.08 mmol) according to the General Procedure II. Purified by column chromatography (4:1 CHCl₃–MeOH) to yield 19 mg (80%) of 11a as a pale orange amorphous product. R_f: 0.44 (4:1 CHCl₃–MeOH). ¹H NMR (500 MHz, CD₃OD) δ (ppm) 7.83 (2H, d, J 7.7 Hz, Ar), 7.50 (2H, d, J 7.9 Hz, Ar), 7.54–7.42 (1H, m, Ar), 6.91 (1H, s, H-4), 4.42 (1H, d, J_1′,2′ 8.9 Hz, H-1′), 3.90 (1H, dd, J_6a′,6b′ 12.1 Hz, H-6_a′), 3.72 (1H, dd, H-6_b′), 3.54 (1H, pseudo t, J_2′,3′ 9.0 Hz, H-2′), 3.51 (1H, pseudo t, J_3′,4′ 8.7 Hz, H-3′), 3.45 (1H, pseudo t, J_4′,5′ 8.7 Hz, H-4′), 3.45 (1H, ddd, J_5′,6a′ < 1.0, J_5′,6b′ 2.9 Hz, H-5′). ¹³C NMR (125 MHz, CD₃OD) δ (ppm) 171.2 (C-5), 165.0 (C-3), 131.6–126.5 (Ar), 99.8 (C-4), 82.4 (C-5′), 79.5 (C-3′), 76.1 (C-1′), 74.7 (C-2′), 71.4 (C-4′), 62.9 (C-6′). C₁₅H₁₇NO₆ (307.11). NMR spectra are identical with those reported [16].

3-(β-d-Glucopyranosyl)-5-(pyridin-2-yl)isoxazole (11d)

Prepared from isoxazole 7d (0.03 g, 0.04 mmol) according to the General Procedure II. Purified by column chromatography (4:1 CHCl₃–MeOH) to yield 9 mg (67%) of 11d as a colourless amorphous product. R_f: 0.32 (4:1 CHCl₃–MeOH); [α]_D+21 (c 0.15, MeOH). ¹H NMR (500 MHz, CD₃OD) δ (ppm) 8.66 (1H, dt, J 4.9, 1.2 Hz, Ar), 8.02–7.95 (2H, m, Ar), 7.49 (1H, ddd, J 6.9, 4.9, 2.3 Hz, Ar), 7.13 (1H, s, H-4), 4.46 (1H, d, J_1′,2′ 9.3 Hz, H-1′), 3.90 (1H, dd, J_6a′,6b′ 12.2 Hz, H-6_a′), 3.72 (1H, strongly coupled, H-6_b′), 3.54 (1H, pseudo t, J_2′,3′ 8.9 Hz, H-2′), 3.51 (1H, pseudo t, J_3′,4′ 8.9 Hz, H-3′), 3.45 (1H, pseudo t, J_4′,5′ 9.5 Hz, H-4′), 3.45 (1H, ddd, J_5′,6a′ 1.4, J_5′,6b′ 5.1 Hz, H-5′). ¹³C NMR (125 MHz, CD₃OD) δ (ppm) 170.0 (C-5), 165.2 (C-3), 151.3–122.2 (Ar), 102.5 (C-4), 82.5 (C-5′), 79.5 (C-3′), 76.0 (C-1′), 74.7 (C-2′), 71.5 (C-4′), 62.9 (C-6′). HR-ESI-MS positive mode (m/z): calcd. for C₁₄H₁₆N₂O₆ (308.10) [M + Na]⁺ = 331.0901, found: [M + Na]⁺ = 331.0900.

3-(β-d-Glucopyranosylisoxazol-5-yl)methanol (11f)

Prepared from isoxazole 7f (0.05 g, 0.08 mmol) according to the General Procedure II. Purified by column chromatography (4:1 CHCl₃–MeOH) to yield 21 mg (99%) of 11f as a colourless amorphous product. R_f: 0.07 (4:1 CHCl₃–MeOH); [α]_D+17 (c 0.26, MeOH). ¹H NMR (500 MHz, CD₃OD) δ (ppm) 6.45 (1H, s, H-4), 4.66 (2H, s, CH₂OH), 4.36 (1H, d, J_1′,2′ 9.3 Hz, H-1′), 3.87 (1H, dd, J_6a′,6b′ 12.2 Hz, H-6_a′), 3.69 (1H, strongly coupled, H-6_b′), 3.50–3.43 (2H, m, H-2′, H-3′), 3.41 (1H, pseudo t, J_4′,5′ 9.4 Hz, H-4′), 3.40 (1H, ddd, J_5′,6a′ 1.2, J_5′,6b′ 5.2 Hz, H-5′). ¹³C NMR (125 MHz, CD₃OD) δ (ppm) 173.8 (C-5), 164.2 (C-3), 101.7 (C-4), 82.4 (C-5′), 79.5 (C-3′), 76.0 (C-1′), 74.7 (C-2′), 71.4 (C-4′), 62.8 (C-6′), 56.4 (CH₂OH). HR-ESI-MS positive mode (m/z): calcd. for C₁₀H₁₅NO₇ (261.08) [M + Na]⁺ = 284.0741, found: [M + Na]⁺ = 284.0738.

3-(β-d-Galactopyranosyl)-5-phenylisoxazolines (12a)

Prepared from isoxazolines 8a (0.09 g, 0.18 mmol) according to the General Procedure II. Purified by column chromatography (4:1 CHCl₃–MeOH) to yield 18 mg (76%) of 12a (diastereomeric ratio: 1.1:1) as a white amorphous product. R_f: 0.52 (7:3 CHCl₃–MeOH). 12a-I ¹H NMR (400 MHz, CD₃OD) δ (ppm) 7.41–7.27 (5H, m, Ar), 5.61 (1H, dd, J_4a,5 11.0, J_4b,5 8.4 Hz, H-5), 4.08 (1H, dd, J_1′,2′ 9.7 Hz, H-1′), 3.92 (1H, dd, J_3′,4′ 3.3, J_4′,5′ 0.6 Hz, H-4′), 3.81–3.51 (6H, m, H-2′, H-3′, H-5′, H-6_a′, H-6_b′, H-4_a), 3.15 (1H, dd, J_4a,4b 17.6 Hz, H-4_b). ¹³C NMR (90 MHz, CD₃OD) δ (ppm) 158.8 (C-3), 142.6–126.7 (Ar), 83.4 (C-5), 80.9 (C-5′), 77.1 (C-1′), 75.9 (C-3′), 70.8 (C-4′), 69.8 (C-2′), 62.8 (C-6′), 42.5 (C-4). 12a-II ¹H NMR (400 MHz, CD₃OD) δ (ppm) 7.41–7.27 (5H, m, Ar), 5.57 (1H, dd, J_4a,5 10.9, J_4b,5 9.3 Hz, H-5), 4.09 (1H, dd, J_1′,2′ 9.7 Hz, H-1′), 3.92 (1H, dd, J_3′,4′ 3.3, J_4′,5′ 0.6 Hz, H-4′), 3.81–3.51 (6H, m, H-2′, H-3′, H-5′, H-6_a′, H-6_b′, H-4_a), 3.09 (1H, dd, J_4a,4b 17.5 Hz, H-4_b). ¹³C NMR (90 MHz, CD₃OD) δ (ppm) 159.1 (C-3), 142.6–126.7 (Ar), 83.7 (C-5), 80.9 (C-5′), 77.1 (C-1′), 76.1 (C-3′), 70.8 (C-4′), 69.8 (C-2′), 62.8 (C-6′), 42.6 (C-4). HR-ESI-MS positive mode (m/z): calcd. for C₁₅H₁₉NO₆ (309.12) [M + H]⁺ = 310.1285, found: [M + H]⁺ = 310.1286.

3-(β-d-Galactopyranosyl)-5-(naphth-2-yl)isoxazolines (12b)

Prepared from isoxazolines 8b (0.06 g, 0.10 mmol) according to the General Procedure II. Purified by column chromatography (4:1 CHCl₃–MeOH) to yield 26 mg (70%) of 12b (diastereomeric ratio: 1.1:1) as a brownish amorphous product. R_f: 0.59 (7:3 CHCl₃–MeOH). 12b-I ¹H NMR (400 MHz, CD₃OD) δ (ppm) 7.91–7.80 (4H, m, Ar), 7.56–7.42 (3H, m, Ar), 5.78 (1H, dd, J_4a,5 10.9, J_4b,5 8.3 Hz, H-5), 4.12 (1H, dd, J_1′,2′ 9.7 Hz, H-1′), 3.92 (1H, dd, J_3′,4′ 3.2, J_4′,5′ 0.9 Hz, H-4′), 3.81 (1H, dd, J_2′,3′ 9.5 Hz, H-2′), 3.80–3.59 (4H, m, H-5′, H-6_a′, H-6_b′, H-4_a), 3.56 (1H, dd, H-3′), 3.26 (1H, dd, J_4a,4b 17.6 Hz, H-4_b). ¹³C NMR (90 MHz, CD₃OD) δ (ppm) 158.9 (C-3), 139.9–124.7 (Ar), 83.5 (C-5), 80.9 (C-5′), 77.2 (C-1′), 76.1 (C-3′), 70.8 (C-4′), 69.8 (C-2′), 62.8 (C-6′), 42.5 (C-4). 12b-II ¹H NMR (400 MHz, CD₃OD) δ (ppm) 7.91–7.80 (4H, m, Ar), 7.56–7.42 (3H, m, Ar), 5.73 (1H, dd, J_4a,5 10.8, J_4b,5 9.1 Hz, H-5), 4.13 (1H, dd, J_1′,2′ 9.7 Hz, H-1′), 3.92 (1H, dd, J_3′,4′ 3.2, J_4′,5′ 0.9 Hz, H-4′), 3.80–3.59 (5H, m, H-2′, H-5′, H-6_a′, H-6_b′, H-4_a), 3.55 (1H, dd, H-3′), 3.19 (1H, dd, J_4a,4b 17.4 Hz, H-4_b). ¹³C NMR (90 MHz, CD₃OD) δ (ppm) 159.1 (C-3), 139.9–124.7 (Ar), 83.8 (C-5), 80.9 (C-5′), 77.1 (C-1′), 75.9 (C-3′), 70.8 (C-4′), 69.8 (C-2′), 62.8 (C-6′), 42.6 (C-4). HR-ESI-MS positive mode (m/z): calcd. for C₁₉H₂₁NO₆ (359.14) [M + H]⁺ = 360.1442, found: [M + H]⁺ = 360.1444.

3-(β-d-Glucopyranosyl)-5-phenylisoxazolines (13a)

Prepared from isoxazolines 3a (0.07 g, 0.10 mmol) according to the General Procedure II. Purified by column chromatography (4:1 CHCl₃–MeOH) to yield 24 mg (80%) of 13a (diastereomeric ratio: 1.3:1) as a pale orange amorphous product. R_f: 0.44 (4:1 CHCl₃–MeOH). 13a-I ¹H NMR (500 MHz, CD₃OD) δ (ppm) 7.42–7.26 (5H, m, Ar), 5.61 (1H, dd, J_4a,5 11.1, J_4b,5 8.6 Hz, H-5), 4.14 (1H, dd, J_1′,2′ 9.2 Hz, H-1′), 3.85 (1H, dd, J_5′,6a′ 1.3, J_6a′,6b′ 11.7 Hz, H-6_a′), 3.65 (1H, dd, J_5′,6b′ 4.9 Hz, H-6_b′), 3.54 (1H, dd, J_4a,4b 17.5, J_4a,5 11.4 Hz, H-4_a), 3.47–3.27 (3H, m, H-3′, H-4′, H-5′), 3.41 (1H, pseudo t, J_2′,3′ 8.7 Hz, H-2′), 3.15 (1H, dd, J_4b,5 8.5 Hz, H-4_b). ¹³C NMR (125 MHz, CD₃OD) δ (ppm) 158.8 (C-3), 142.5–126.9 (Ar), 83.4 (C-5), 82.3 (C-5′), 79.3 (C-3′), 76.7 (C-1′), 73.0 (C-2′), 71.4 (C-4′), 62.7 (C-6′), 42.6 (C-4). 13a-II ¹H NMR (500 MHz, CD₃OD) δ (ppm) 7.42–7.26 (5H, m, Ar), 5.57 (1H, dd, J_4a,5 10.8, J_4b,5 10.0 Hz, H-5), 4.13 (1H, dd, J_1′,2′ 9.3 Hz, H-1′), 3.85 (1H, dd, J_5′,6a′ 1.3, J_6a′,6b′ 11.7 Hz, H-6_a′), 3.67 (1H, dd, J_5′,6b′ 5.4 Hz, H-6_b′), 3.60 (1H, dd, J_4a,4b 17.4, J_4a,5 11.1 Hz, H-4_a), 3.47–3.27 (3H, m, H-3′, H-4′, H-5′), 3.41 (1H, pseudo t, J_2′,3′ 8.7 Hz, H-2′), 3.06 (1H, dd, J_4b,5 9.2 Hz, H-4_b). ¹³C NMR (125 MHz, CD₃OD) δ (ppm) 159.0 (C-3), 142.5–126.9 (Ar), 83.7 (C-5), 82.3 (C-5′), 79.5 (C-3′), 76.6 (C-1′), 73.1 (C-2′), 71.4 (C-4′), 62.8 (C-6′), 42.7 (C-4). HR-ESI-MS positive mode (m/z): calcd. for C₁₅H₁₉NO₆ (309.12) [M + Na]⁺ = 332.1105, found: [M + Na]⁺ = 332.1104.

3-(β-d-Glucopyranosyl)-5-(naphth-2-yl)isoxazolines (13b)

Prepared from isoxazolines 3b (0.08 g, 0.10 mmol) according to the General Procedure II. Purified by column chromatography (4:1 CHCl₃–MeOH) to yield 16 mg (46%) of 13b (diastereomeric ratio: 1:1) as a white amorphous product. R_f: 0.40 (4:1 CHCl₃–MeOH). 13b-I ¹H NMR (500 MHz, CD₃OD) δ (ppm) 8.11–7.77 (4H, m, Ar), 7.65–7.39 (3H, m, Ar), 5.79 (1H, dd, J_4a,5 11.2, J_4b,5 8.7 Hz, H-5), 4.16 (1H, dd, J_1′,2′ 9.1 Hz, H-1′), 3.84 (1H, dd, J_5′,6a′ 1.6, J_6a′,6b′ 12.0 Hz, H-6_a′), 3.71–3.54 (2H, m, H-4_a, H-6_b′), 3.50–3.41 (2H, m, H-2′, H-3′), 3.41–3.29 (2H, m, H-4′, H-5′), 3.25 (1H, dd, J_4a,4b 17.5, J_4b,5 8.4 Hz, H-4_b). ¹³C NMR (125 MHz, CD₃OD) δ 158.9 (C-3), 139.8–124.7 (Ar), 83.6 (C-5), 82.3 (C-5′), 79.4 (C-3′), 76.7 (C-1′), 73.1 (C-2′), 71.4 (C-4′), 62.7 (C-6′), 42.6 (C-4). 13b-II ¹H NMR (500 MHz, CD₃OD) δ (ppm) 8.11–7.77 (4H, m, Ar), 7.65–7.39 (3H, m, Ar), 5.75 (1H, dd, J_4a,5 11.3, J_4b,5 9.7 Hz, H-5), 4.17 (1H, dd, J_1′,2′ 9.3 Hz, H-1′), 3.88 (1H, dd, J_5′,6a′ 2.0, J_6a′,6b′ 12.1 Hz, H-6_a′), 3.71–3.54 (2H, m, H-4_a, H-6_b′), 3.50–3.41 (2H, m, H-2′, H-3′), 3.41–3.29 (2H, m, H-4′, H-5′), 3.17 (1H, dd, J_4a,4b 17.4, J_4b,5 9.2 Hz, H-4_b). ¹³C NMR (125 MHz, CD₃OD) δ (ppm) 159.1 (C-3), 139.8–124.7 (Ar), 83.9 (C-5), 82.3 (C-5′), 79.5 (C-3′), 76.7 (C-1′), 73.1 (C-2′), 71.4 (C-4′), 62.8 (C-6′), 42.6 (C-4). HR-ESI-MS positive mode (m/z): calcd. for C₁₉H₂₁NO₆ (359.14) [M + Na]⁺ = 382.1261, found: [M + Na]⁺ = 382.1258.

3,4-Di(β-d-Galactopyranosyl)-1,2,5-oxadiazole-2-oxide (14)

Prepared from isoxazole 4b (0.10 g, 0.13 mmol) according to the General Procedure II. Purified by column chromatography (4:1 CHCl₃–MeOH) to yield 19 mg (34%) of 14 as a white amorphous product. R_f: 0.16 (1:1 CHCl₃–MeOH); [α]_D+82 (c 0.08, DMSO). ¹H NMR (500 MHz, CD₃OD) δ (ppm) 4.44 (1H, d, J_1′,2′ 9.9 Hz, H-1′), 4.42 (1H, d, J_1″,2″ 9.8 Hz, H-1″), 4.31 (1H, pseudo t, J_2′,3′ 9.6 Hz, H-2′), 4.29 (1H, pseudo t, J_2″,3″ 9.6 Hz, H-2″), 3.92 (1H, dd, J_4″,5″ 0.5 Hz, H-4″), 3.89 (1H, dd, J_4′,5′ 0.5 Hz, H-4′), 3.91–3.83 (2H, m, H-6_a′, H-6_a″), 3.75–3.66 (4H, m, H-5′, H-5″, H-6_b′, H-6_b″), 3.56 (1H, dd, J_3″,4″ 3.3 Hz, H-3″), 3.55 (1H, dd, J_3′,4′ 3.4 Hz, H-3′). ¹³C NMR (125 MHz, CD₃OD) δ (ppm) 157.2 (C-4), 115.6 (C-3), 82.2 (C-5′), 81.8 (C-5″), 77.9 (C-1″), 76.2 (C-3″), 76.1 (C-3′), 75.3 (C-1′), 71.0 (C-4′, C-4″), 70.2 (C-2′), 69.9 (C-2″), 62.7 (C-6″), 62.6 (C-6′). HR-ESI-MS positive mode (m/z): calcd. for C₁₄H₂₂N₂O₁₁ (410.33) [M + H]⁺ = 411.1246, found: [M + H]⁺ = 411.1246.

3.2. Chemicals for Cell Proliferation Experiments

All chemicals used in the cell biology and biochemistry assays were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. The compounds were dissolved in dimethyl-sulfoxide for biology experiments, and 0.1% dimethylsulfoxide was used as a vehicle control.

3.3. Cell Culture

The cells were cultured under standard cell culture conditions: 37 °C, 5% CO₂, humidified atmosphere.

A2780 cells were cultured in RMPI 1640 medium, supplemented with 10% fetal calf serum, 2 mM glutamine, and 1% penicillin-streptomycin.

3.4. Methylthiazolyldiphenyl-Tetrazolium Bromide (MTT) Reduction Assay

An MTT reduction assay measures the activity of mitochondrial complex I and can be used to detect toxicity [33]. The assay was performed in a manner similar to that described in [32]. Briefly, the cells were plated in 96-well plates the day before the assay. The cells were treated with the compounds for 4 h; then, MTT was added to a 0.5 mg/mL final concentration, and the cells were incubated at 37 °C in a cell incubator for 40–60 min as a function of the cell line being assessed. Culture medium was removed, the reduced MTT dye was dissolved in dimethyl-sulfoxide, and plates were measured in a plate photometer (Thermo Scientific Multiscan GO spectrophotometer, Waltham, MA, USA) at 540 nm. On each plate, the wells were designed to contain vehicle-treated cells. In calculations, the readings for these wells were considered to be 1, and all readings were expressed relative to these values.

3.5. Sulforhodamine B (SRB) Binding Assay

An SRB assay measures the protein content of cells in correlation with the cell number in an assay well and can therefore be used to assess cell proliferation or long-term cytostasis [34]. The cells were seeded in 96-well plates the day before the treatment for the assay. The cells were treated with the compounds for 48 h. Then the medium was removed and the cells were fixed with 10% trichloroacetic acid. The fixed cells were washed in distilled water 3 times, followed by staining with SRB (0.4 m/V% dissolved in 1% acetic acid) for 10 min. The stained cells were washed in 1% acetic acid 5 times before the acetic acid was removed and the cells were left to dry. Protein-bound SRB was released by adding 100 µL of 10 mM Tris base. The plates were measured in a plate photometer (Thermo Scientific Multiscan GO spectrophotometer, Waltham, MA, USA) at 540 nm. On each plate, the wells were designed to contain vehicle-treated cells. In calculations, the readings for these wells were considered to be 1, and all readings were expressed relative to these values.

4. Conclusions

Nitrile oxides were generated in situ from anhydro-aldose oximes in a two-step halogenation–elimination sequence using NCS/Et₃N/dry CH₂Cl₂ conditions. The resulting nitrile oxides were then used in 1,3-dipolar cycloaddition reactions with alkynes and alkenes. These cycloadditions resulted exclusively in 5-substituted-3-(C-glycopyranosyl)isoxazoles 6a–g,i and -isoxazolines 3a–c in low to good yields. Debenzoylated 3-(β-d-glucopyranosyl)isoxazoles 11d,f and -isoxazolines 13a,b gave no significant inhibition against rabbit muscle glycogen phosphorylase b, and neither the protected nor unprotected galactopyranosyl derivatives 6d, 8b, 10a, and 12a exhibited significant activity against A2780 ovarian cancer cells.