A new synthetic access to 2-N-(glycosyl)thiosemicarbazides from 3-N-(glycosyl)oxadiazolinethiones and the regioselectivity of the glycosylation of their oxadiazolinethione precursors

Glycosylations of 5-(1H-indol-2-yl)-1,3,4-oxadiazoline-2(3H)-thione delivered various degrees of S- and/or N-glycosides depending on the reaction conditions. S-Glycosides were obtained regiospecifically by grinding oxadiazolinethiones with acylated α-D-glycosyl halides in basic alumina, whereas 3-N-(glycosyl)oxadiazolinethiones were selectively obtained by reaction with HgCl2 followed by heating the resultant chloromercuric salt with α-D-glycosyl halides in toluene under reflux. On using Et3N or K2CO3 as a base, mixtures of S- (major degree) and N-glycosides (minor degree) were obtained. Pure 3-N-(glycosyl)oxadiazolinethiones can also be selectively obtained from glycosylsulfanyloxadiazoles by the thermal S→N migration of the glycosyl moiety, which is proposed to occur by a tight-ion-pair mechanism. Thermal S→N migration of the glycosyl moiety can be used for purification of mixtures of S- or N-glycosides to obtain the pure N-glycosides. The aminolysis of the respective S- or N-glycosides with ammonia in aqueous methanol served as further confirmation of their structures. While in S-glycosides the glycosyl moiety was cleaved off again, 3-N-(glycosyl)oxadiazolinethiones showed a ring opening of the oxadiazoline ring (without affecting the glycosyl moiety) to give N-(glycosyl)thiosemicarbazides. Herewith, a new synthetic access to one of the four classes of glycosylthiosemicarbazides was found. The ultimate confirmation of new structures was achieved by X-ray crystallography. Finally, action of ammonia on benzylated 3-N-(galactosyl)oxadiazolinethione unexpectedly yielded 3-N-(galactosyl)triazolinethione. This represents a new path to the conversion of glycosyloxadiazolinethiones to new glycosyltriazolinethione nucleosides, which was until now unknown.

was cleaved off again, 3-N-(glycosyl)oxadiazolinethiones showed a ring opening of the oxadiazoline ring (without affecting the glycosyl moiety) to give N-(glycosyl)thiosemicarbazides. Herewith, a new synthetic access to one of the four classes of glycosylthiosemicarbazides was found. The ultimate confirmation of new structures was achieved by X-ray crystallography. Finally, action of ammonia on benzylated 3-N-(galactosyl)oxadiazolinethione unexpectedly yielded 3-N-(galactosyl)triazolinethione. This represents a new path to the conversion of glycosyloxadiazolinethiones to new glycosyltriazolinethione nucleosides, which was until now unknown.

Introduction
Modified nucleosides are versatile motifs for studying the relationship between the structure and functions of nucleic acids and problems of metabolism, besides their main potential in curing viral infections and cancer diseases [1]. The 1,3,4-oxadiazolines and 1,2,4-triazolines are potential inhibitors of physiologically relevant isoforms of the zinc enzyme carbonic anhydrase (CA, EC 4.2.2.1), i.e., cytosolic CA I and CA II, the tumor-associated transmembrane isoenzyme CA IX with inhibition constants in the low micromolar range [2]. Coupling of aglycones with relevant glycosyl donors is the common approach involved in the synthesis of most nucleosides. Another strategy for the synthesis of nucleoside analogues is the use of glycosylamines and related N-bonded glycosides [3][4][5][6][7], such as glycosylisothiocyanates, which afford glycosylthiosemicarbazides [8] or glycosyl 3-thioureidothiourea derivatives [9]. These 4-N-(glycosyl)thiosemicarbazides [8] were used for the synthesis of Schiff-like bases [10] and 4-N-glycosyl(thiosemicarbazido)phosphorothionates as precursors for the synthesis of the herbicidal and fungicidal agents thiazolidine-4-ones [11]. Thiosemicarbazide (TSC) and related amines were used to prepare modified amylase and amylopectin for biological studies [12]. Moreover, glycosylthiosemicarbazides are formed in vivo to modify cell-surface sialic acid. This is known as a metabolic cell-surface-engineering technique for cell-surface interactions and consequently shows the potential of these compounds for the development of anticancer agents [13][14][15][16]. Antituberculosis effects of glycosylthiosemicarbazides were also reviewed [17]. Glycosylamines are used also as enzyme inhibitors and vaccine precursors [18][19][20][21], and in glycopeptide synthesis [22,23] and in glycodendrimers and glycoclusters [24,25].
Anomeric β-configurations of the S-linked 5-7 and N-linked 8-10 glycosides were deduced from the 1 H NMR spectra, which revealed large J 1,2 values of 10.3-10.6 and 9.2-9.5 Hz, respectively, for the anomeric protons. The chemical shifts of the anomeric protons of S-glycosides were at lower values (δ 5.45-5.56 ppm) than those of N-glycosides (δ 5.92-6.10 ppm). The differentiation between Sand N-glycosides was supported by the presence or absence of the signal of the carbon atom of the C=S moiety in the 13 C NMR spectra. In other words, the 13 C NMR spectra of the N-glycosides 8-10 revealed signals at δ c 176.10-177.40 ppm. Anomeric carbons in both types were observed at δ 83.20-84.00 ppm.
Thermal rearrangement of the S-glycosides 5-7 under solventfree and atmospheric conditions afforded the corresponding 3-N-glycosides 8-10. The conversion was achieved in a few minutes with good to excellent yields (60-90%). Therefore, the thermal rearrangement from Sto N-glycosides may also serve as a rapid and economic (free of solvents) purification step for crude mixtures of Sand 3-N-glycosides obtained from glycosylations mediated by either Et 3 N or K 2 CO 3 . Additional experiments on crude mixtures of Sand 3-N-glycosides successfully afforded pure N-glycosides.
The mechanism of this rearrangement is presumably proceeding by an ionization-recombination pathway in which a thermally induced heterolysis of the thioglycosidic bond results in a tight ion pair generated upon ionization of this bond. An intramolecular reaction of the tight ion pair results in migration of the glycosyl moiety from sulfur to nitrogen, which proceeds with complete retention of configuration. As a result, a tight-ion-pair mechanism in which the migrating group retains chirality is suggested (Scheme 2). the mass spectra of the products 11-13 are higher by seventeen atomic mass units than would be those of the deacetylated products 14-16. Second, the IR spectra of 11-13 show new amide absorption bands that do not appear in the IR spectra of their precursors. Finally, the 13 C NMR spectra of 11-13 show signals for NC=O groups at δ C 159.40-162.90 ppm in addition to the NC=S groups at δ C 183.10-184.50 ppm. Deacetylation of 10 is only confined to the O-acetyl groups, but the N-acetyl group survived under these conditions. This was confirmed by an extra 13 C NMR signal at δ C 172.50 ppm for the NHCOCH 3 group of 13. The presence of 1 H NMR signals at δ H 6.46-6.55 ppm as doublets with coupling constants of J 1,2 = 8.5-9.0 Hz indicated the stability of the pyranose ring and its β-anomeric configuration under these conditions. Aminolysis of the S-glycosides 5-7 (Scheme 4) under the same conditions (ammonia in aqueous methanolic solution) generally led to splitting of the thioglycosyl moiety as a result of hydrolysis of the bond between the oxadiazole (C 2 ) and the glycosidic sulfur atom. Hence, the indolyloxadiazolone 20 was formed [33] whereas the expected deacetylated glycosides 17-19 were not obtained. Structure elucidation of 20 yielded a melting point of 271-273 °C [33], while literature reports give a value of 102 °C [41] or 285 °C [42]. Therefore, compound 20 was prepared in another reaction sequence [33] to prove its structure and the correctness of the physical and structural data obtained. Hence, the indol-2-carbohydrazide (21) was reacted with methyl chloroformate followed by cyclization of the resulting ester 22. As a result, 20 was obtained in high yield, and its structural analytical data were identical with those of the aminolysis product (Scheme 4).
For ultimate confirmation of the structures of Sand N-glycosides 5-10, the benzylation of the indole was chosen, because these derivatives can serve to prove the glycosyl rearrangement and glycosyl TSC formation. Moreover, crystalline glycosides are obtained that are suitable for X-ray analysis. In the presence of

X-ray analysis
Single-crystal X-ray diffraction experiments yielded unambiguous confirmations of the structural assignments of the S-glycoside 23, the 2-N-(glycosyl)thiosemicarbazide 29, and the galactosyltriazole 30. Single crystals were slowly grown in EtOH. 23 crystallized in the monoclinic space group C 2 with the following unit cell parameters: a = 25.7078 Å, α = 90º, b = 7.1500 Å, β = 105.2576º, c = 17.8411 Å, γ = 90º and V = 3163.81 Å 3 . The crystallographic data of 23 are shown in Table 1. The whole molecule is nonplanar; the phenyl group is located perpendicular to the plane of the indole ring by making a torsion angle of C(8)N(1)C(1)C(2) = 95.9º while the oxadiazole ring is located in the plane of the indole ring by making    The crystallographic analysis revealed that the sugar molecule has the glucopyranose form and has 4 C 1 conformation. The anomeric β-configuration is derived from the bond lengths of O(2)-C (18) and O(2)-C (19), which are 1.416 and 1.442 Å, and all substituents have equatorial orientation. Moreover, the crystal data revealed that the S(1)-C(17) bond length is 1.743 Å suggesting a certain degree of conjugation with the oxadiazole ring, whereas the S(1)-C(18) bond length is 1.816 Å, which is typical for single bonds of this kind [43]. The crystal structure and molecular conformation is stabilized by three intramolecular C-H···N hydrogen bonds, three intramolecular C-H···O hydrogen bonds, and five intermolecular C-H···O hydrogen bonds in the crystal network ( Figure 1, Figure 2 and Table 1).  Compound 29 (Figure 3, Figure 4 and Table 2) crystallized as a cyclic dimer and contains two independent molecules in the unit cell. The dimer is stabilized by an intermolecular hydrogen bond N (8) those found in 23), respectively, which shows that the sugar moiety still has the glucopyranose form with 4 C 1 anomeric β-configuration. In addition, the crystal structure of 29 shows that the whole molecule is nonplanar. The phenyl group makes a dihedral angle of C(1)N(1)C(9)C(10) = −100.3°, which means that it is perpendicular to the indole ring.     in 23 and 29), which shows that, first, the compound still has the cyclic galactopyranose structure, and second, the sugar moiety is stable, even if the oxadiazole moiety has opened and the TSC formed has cyclized to form a triazole. The whole structure is nonplanar and the phenyl group still oriented perpendicular to the indole ring making

Conclusion
In conclusion, 2-N-(glycosyl)thiosemicarbazides of type II (from the four glycosylthiosemicarbazide structural isomers I-IV shown in Scheme 1) were synthesized from 3-N-(glycosyl)oxadiazolinethiones, which were accessed by new regioselective glycosylations. Additionally, 3-N-(glycosyl)oxadiazolinethiones may be prepared by a mild solvent-free thermal S→N migration of the glycosyl moiety in glycosylsulfanyloxadiazoles. (Benzylindolyl)glycosylsulfanyl-1,3,4-oxadiazoles could be thermally rearranged into the corresponding N-glycosides. These may either be converted into the corresponding (benzylindolyl)-2-N-(glycosyl)thiosemicarbazides (of type II) or into the galactosyl triazolinethione from the galactosyl oxadiazolinethione, as confirmed by X-ray single-crystal analysis and from further common structural analytical data.

Supporting Information
Complete crystallographic data of the structural analysis of compounds 23, 29