Synthesis and antiproliferative activity of (Z)-1-glycosyl-3-(5-oxo-2-thioxoimidazolidin-4-ylidene)indolin-2-ones and (Z)-3-(2-glycosylsulfanyl-4-oxo-4,5-dihydro-thiazol-5-ylidene)indolin-2-ones

Friedrich Erbena, Dirk Michalikab, Holger Feista, Dennis Kleeblatta, Martin Heina, Abdul Matinc, Jamshed Iqbald and Peter Langer*ab
aInstitut für Chemie, Universität Rostock, Albert-Einstein-Str. 3a, 18059 Rostock, Germany. E-mail: peter.langer@uni-rostock.de; Fax: +49 (381)4986412
bLeibniz-Institut für Katalyse e.V. an der Universität Rostock, Albert-Einstein-Str. 29a, 18059 Rostock, Germany
cInstitute of Biomedical and Genetic Engineering, PO Box: 2891, Sector: G-9/1, Islamabad, 44000, Pakistan
dDepartment of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad, 22060, Pakistan

Received 12th November 2013 , Accepted 27th January 2014

First published on 28th January 2014


Abstract

The reaction of N-glycosylated isatines with imidazolones and thiazolones afforded N-glycosylated (Z)-1-glycosyl-3-(5-oxo-2-thioxoimidazolidin-4-ylidene)indolin-2-ones. The reaction of S-glycosylated thiazolones with isatines gave S-glycosylated (Z)-3-(2-glycosylsulfanyl-4-oxo-4,5-dihydro-thiazol-5-ylidene)indolin-2-ones. These molecules represent hybrids of pharmacologically important moieties, including carbohydrates, isatines, 2-thiohydantoin, pseudo-thiohydantoin and rhodanine. The products show a good and selective activity against lung carcinoma cell lines H157. The best activity was obtained for 3-(5-oxo-2-thioxoimidazolidin-4-ylidene)indolin-2-ones containing a glucose moiety.


Introduction

Natural products containing a bis-indole framework, like rebeccamycin, staurosporine, K-252d and the tjipanazoles, possess a high cancerostatic activity.1,2 The akashines A, B and C, indigo-N-glycosides isolated from Streptomyces sp. GW48/1497,3 exhibit also anticancer properties, whereas unsubstituted indigo is pharmacologically inactive. Indirubin (1a) represents the red isomer of indigo (Scheme 1). Its derivatives are potent selective inhibitors of cyclin-dependent kinases (CDKs), which play an important role in cellular processes connected with growth and proliferation. Therefore, they have great potential in the treatment of cancer.4,5 For example, glycosylated indirubines are active against various cancer cell lines and are, in most cases, more active than their corresponding aglycons.6 Previous work proved that glycosylated derivatives of thiaindirubin (1b) show a remarkable influence on proliferation and viability of melanoma cells.7a Recently, the synthesis of selenaindirubins, which show activity against lung carcinoma cells, has also been reported.7b A number of N-glycosylated derivatives of isoindigo (2) have been reported by Moreau and Sassatelli and their coworkers. These compounds show a considerable activity against various human cancer cell lines.8 A prominent O-glycosylated derivative is NATURA (1-(2,3,4-tri-O-acetyl-β-D-xylopyranosyl)isoindigo).9 Recently, we reported the synthesis of a number of N,N-diglycosylated indirubin derivatives.10
image file: c3ra44362k-s1.tif
Scheme 1 Indigoid bis-indoles.

Our current interest is directed towards the synthesis of (glycosylated) indirubin or isoindigo analogues containing different 5-membered heterocycles (Scheme 1). An important strategy to synthesize bis-indoles and their analogues, such as indirubin derivatives, is based on the reaction of isatines with CH-acidic compounds. Due to the reactivity of the keto group of (glycosylated) isatines, condensation reactions with a variety of CH-acidic compounds can be carried out. In many cases, the sequence of addition of the nucleophile and subsequent elimination of water can be carried out in one step.11 In other cases the reaction can be stopped at the first step to give the hydroxylated condensation product.12 Herein we report what is, to the best of our knowledge, the first synthesis of N-gl (Z)-1-glycosyl-3-(5-oxo-2-thioxoimidazolidin-4-ylidene)indolin-2-ones and of S-glycosylated (Z)-3-(2-glycosylsulfanyl-4-oxo-4,5-dihydro-thiazol-5-ylidene)indolin-2-ones. These molecules, which represent new glycosylated indirubin analogues, exhibit an antiproliferative activity against the lung carcinoma cell line H157. The products can be regarded as hybrids of various pharmacologically highly relevant core structures, including carbohydrates, isatines, 2-thiohydantoin, pseudo-thiohydantoin and rhodanine.

Results and discussion

Synthesis

Isatine-N-glycosides 3a–d were prepared, as previously reported,10,13 by reaction of glycosylated anilines with oxalyl chloride. The reaction of isatine-N-rhamnoside 3a with 2-thiohydantoin (4), in the presence of NaOAc, AcOH and Ac2O, afforded product 5a in 54% yield (Scheme 2 and Table 1). Likewise, products 5b–d were prepared starting with 3b–d. All attempts to deprotect compounds 5a–d, using various conditions including stoichiometric or catalytic amounts of different bases (NaOMe, NaOtBu), proved to be unsuccessful (decomposition).
image file: c3ra44362k-s2.tif
Scheme 2 Synthesis of (Z)-N-(β-L-rhamnopyranosyl)-3-(5-oxo-2-thioxoimidazolidin-4-ylidene)indolin-2-one 5a. Reagents and conditions: (i) AcOH–Ac2O, NaOAc, 60 °C, 3–4 h.
Table 1 Synthesis of N-glycosylated (Z)-3-(5-oxo-2-thioxoimidazolidin-4-ylidene)indolin-2-ones 5a–d
  Yield (5)a
a Yields of isolated products.
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3a 5a, 54%
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3b 5b, 52%
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3c 5c, 57%, β[thin space (1/6-em)]:[thin space (1/6-em)]α = 5[thin space (1/6-em)]:[thin space (1/6-em)]1
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3d 5d, 26%, β[thin space (1/6-em)]:[thin space (1/6-em)]α = 20[thin space (1/6-em)]:[thin space (1/6-em)]3


The reaction of pseudo-thiohydantoin (6) with isatine-N-glycosides 3a,b, carried out under identical conditions, afforded products 7a,b (Scheme 3). During the reaction, the exocyclic nitrogen atom of pseudo-thiohydantoin was acetylated. All attempts to deprotect compounds 7a,b proved to be unsuccessful, due to decomposition.


image file: c3ra44362k-s3.tif
Scheme 3 Synthesis of 7a,b. Reagents and conditions: (i) AcOH–Ac2O, NaOAc, 60 °C, 3–4 h.

The N-glycosylated products 5a–d and 7a,b show a much better solubility in common organic solvents as compared to their non-glycosylated analogues. The better solubility is advantageous with regard to their application as potential drug candidates. However, on the other hand, all compounds had to be purified by column chromatography and proved to be slightly unstable on silica gel, which is one of the main reasons for the fairly low yields in many cases. The purification of the products by recrystallization (without chromatography) did not allow to obtain pure products. To address these issues, we turned our attention to the synthesis of products containing the carbohydrate group located at the thiazolone rather than the isatine moiety. The reaction of rhodanine (8) with glycosyl bromide 9a afforded the S-glycosylated product 10a in 47% yield (Scheme 4). The base mediated condensation of the latter with isatine (11a) afforded the (Z)-3-(2-glycosylsulfanyl-4-oxo-4,5-dihydro-thiazol-5-ylidene)indolin-2-one 12a in 62% yield. Likewise, products 12b–l were prepared in moderate to excellent yields from 8, sugars 9a–c and isatines 11a–f (Table 2). Isatines containing a hydrogen or a phenyl group located at the nitrogen atom could be successfully employed.


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Scheme 4 Synthesis of glucosylsulfanylthiazolone 10a and (Z)-3-(2-glucosylsulfanyl-4-oxo-4,5-dihydro-thiazol-5-ylidene)indolin-2-one 12a. Reagents and conditions: (i) (1) NaH, DMF, 0 °C, 15 min; (2) glycosyl bromide 9a, 20 °C, 3 h; (ii) EtOH(MeOH)–THF, cat. piperidine, 20 °C, 1–2 h.
Table 2 Synthesis of glycosylsulfanyl-thiazolones 10a–c, glucosylsulfanyl-imidazolone 10d, (Z)-3-(2-glycosylsulfanyl-4-oxo-4,5-dihydro-thiazol-5-ylidene)indolin-2-ones 12a–l and (Z)-3-(2-glucosylsulfanyl-4-oxo-4,5-dihydro-1H-imidazol-5-ylidene)indolin-2-one 12m
4, 8 9 11 Yield (10)a Yield (12)a
a Yields of isolated products.
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8 9a 11a 10a, 47% 12a, 62%
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8 9b 11a 10b, 50% 12b, 80%
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8 9c 11a 10c, 32% 12c, 49%
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8 9b 11b 10b 12d, 41%
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8 9c 11b 10c 12e, 78%
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8 9b 11c 10b 12f, 44%
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8 9b 11d 10b 12g, 67%
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8 9c 11c 10c 12h, 90%
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8 9c 11d 10c 12i, 58%
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8 9c 11e 10c 12j, 77%
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8 9b 11f 10b 12k, 80%
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8 9c 11f 10c 12l, 87%
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4 9a 11a 10d, 52% 12m, 62%


The reaction of glycosyl bromide 9a with 2-thiohydantoin (4) afforded product 10d. The reaction of the latter with isatine (11a) afforded (Z)-3-(2-glucosylsulfanyl-4-oxo-4,5-dihydro-1H-imidazol-5-ylidene)indolin-2-one (12m).

Surprisingly, the acetyl-protected S-glycosides 12a and 12m proved to be highly insoluble in various solvents. The use of pivaloyl instead of acetyl protecting groups increased the solubility. Glucose and xylose were employed as carbohydrate moieties. However, products derived from acetyl and pivaloyl protected galactose could not be prepared. In contrast to acetylated N-glycosides 5a–d and 7a,b, products 12a–m already precipitated during the reaction and could be easily isolated by filtration and recrystallization. The yields depended on the degree of solubility of the condensation products.

Due to the unstable nature of the dithioamidocarbonate unit, all attempts to carry out a base mediated deacetylation of compounds 12a–m failed. The change of the colour of the solution suggested that the chromophoric system decomposed. The employment of enzymatic techniques for the deprotection also did not meet success.

Structural studies

The Z-configuration of the double bond of products 5a–d was confirmed by the characteristic downfield shift of the H-4′ signals in the 1H NMR spectra of compounds 5a–d (8.49–8.62 ppm). In this configuration, proton H-4′ is located in the anisotropic cone of the carbonyl group of the thioxoimidazolidin moiety. Similar results were previously reported for thiaindirubin-N-glycosides7a and selenoindirubin-N-glycosides.7b For comparison, it is interesting to study the NMR data of 3-methylenindolin-2-one which can be regarded as a fragment of compounds 5a–d. In case of this compound, the signal, which is shifted most downfield, appears at 7.45 ppm.15a Furthermore, comparison of the 1H NMR data of E- and Z-configured 4,7,4′,7′-tetra-tert-butyl-1,1′-biacenaphthylenyliden-2-ones15b is interesting. In case of the Z-isomer, a significant downfield shift is observed for the proton located in the anisotropic cone of the carbonyl group, whereas the corresponding signal of the E-isomer was found in the region of the other aromatic protons (only slightly shifted downfield). Therefore, the double bond of 12a–m likely bear a Z-configuration, due to the remarkable downfield shift of the 1H NMR signals of the H-4′ protons (8.50–9.10 ppm) of the indolin-2-one moiety. However, some degree of uncertainty related to the configuration of the double bond cannot be completely excluded, because only an X-ray crystal structure analysis would provide an unambiguous proof of the structure. However, we have not yet been able to grow single crystals suitable for X-ray analysis.

For compound 12m two possible tautomeric forms exist, which differ from the proton located at nitrogen N-1 or N-3, respectively. In case of the non-glycosylated analogue of 12m, which was described in the literature,14 a tautomer was reported containing the proton located at N-1. However, the position of the proton of glycoside 12m could not be unambigiously confirmed. In the HMBC spectrum of 12m no correlation of this proton to any carbon atom of the molecule was observed.

Products 3, 5, and 7, which are derived from D-mannose and L-rhamnose, exist in the form of the β-configurated anomer. In case of 3d, 5c and 5d, a small amount of the α-anomer was present. Products 10 and 12, derived from D-glucose and D-xylose, exclusively possess a β-configuration. The configuration of the anomeric carbon atom of the products was determined by 1H NMR spectroscopy, based on the coupling constants 3J1′,2′ (for 3 and 10) or 3J1′′,2′′ (for 5, 7, and 12). In case of products 10 and 12, these coupling constants are in the range of 9.0–9.4 Hz, confirming the β-configuration. In case of 3, 5 and 7, the 3J1,2 coupling constants were found to be about 1.5 Hz. The minor α-anomers of 3d (7.0 Hz), 5c (5.6 Hz) and 5d (5.4 Hz) show unexpectedly big 3J1,2 anomeric coupling constants. Usually, these 3J1,2 coupling constants are rather small for both α- and β-anomers of D-mannose and L-rhamnose derivatives. We earlier reported similar results for related D-manno and L-rhamno derivatives and extensive structure elucidations were carried out by 1H NMR spectroscopy and X-ray crystal structure analyses.6,10 As a result, we assume that a β-configuration is adopted also for the present molecules containing a small coupling constant of about 1.5 Hz. In contrast, an α-anomeric structure is presumably present in case of products containing a relatively large coupling constant. The large coupling constants in case of the α-anomers can be explained by the assumption that the pyranose ring adopts the inverted conformation in which both the protons H-1 and H-2 are located in the axial position. The β-configuration of compounds 5a, 10c, 10d and 12c was independently confirmed by NOESY experiments, which revealed correlations between protons H-1′ and H-3′/H-5′ (10c,d) and H-1′′ and H-3′′/H-5′′ (5a, 12c), respectively.

In the 1H NMR spectra of some compounds 12, signals of a second species in small amount are visible (Fig. 1). For example, in the spectrum of 12a in DMSO at 30 °C, besides the signals of the major isomer, signals of a minor isomer (17%) are present, showing exchange of signals of the major and minor isomers in a 2D EXSY experiment. In addition, in the 1H NMR spectra at higher temperature coalescence of the corresponding major and minor signals was observed. These line shape alterations could be caused by different dynamic processes, ring inversion in the sugar moiety, rotation about the C-3′–C-5 bond, and rotation hindrance about the C-2–S–C-1′′ bonding system, which is believed to be most probable. Ring inversion can be excluded, because in both species the same coupling constants were found. Rotation about the C-3′–C-5 bond is not very likely, because the H-4′ signals of both species are downfield shifted indicating that the C[double bond, length as m-dash]O in 4-position is located on the same site as the H-4′ proton.


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Fig. 1 Expansions of the 500 MHz 1H NMR spectra of 12a in DMSO-d6 recorded at different temperature (ratio major (I)–minor (II) = 5[thin space (1/6-em)]:[thin space (1/6-em)]1).

Pharmacological evaluation

The anti-proliferative activities of products 5a–d, 7a,b and 12a were studied in lung carcinoma cells (H157) and in human corneal epithelial cells (HCEC). Other derivatives were not included because of their limited solubility in water–DMSO. Assays for determination of cell density by sulforhodamine colorimetric assay revealed a dose-dependant decrease of cell density in H157 cells within 24 h by all tested derivatives, whereas the effects on HCEC cells were only weak (Table 3). At 100 μM, the percent of inhibition in H157 ranged between 20% (compound 7a) and 60% (compound 5b). Methotrexate was used as a reference. It is noteworthy that, for the molecules reported in this study, higher inhibitions were observed as compared to selenoindirubin-N-glycosides which were recently studied by us in the same assay.7b Good inhibition values were observed, despite the presence of the acetyl groups located at the carbohydrate moieties. Comparison of the pharmacological activities of derivatives 5a–d show that the type of carbohydrate moiety has a decisive influence on the activity. Derivative 5b, containing a glucosyl moiety, showed the highest inhibition. Likewise, the glucoside 7b proved to be more active than rhamnoside 7a. On the other hand, the heterocyclic moiety also plays a role which is shown by the fact that products 5 are, in general, more active than compounds 7 or 12a.
Table 3 Percentage inhibition values of tested compounds in H-157 and HCEC cells (studies carried out on a 100 μM concentration)
compound HCEC H157 H157 (blind)a
a Inhibition without presence of the tested compound.
5a 5.4 ± 0.2 46.6 ± 7.5 5.9 ± 0.6
5b 3.4 ± 1.4 60.1 ± 5.1 6.1 ± 3.6
5c 6.4 ± 2.2 44.8 ± 4.0 5.6 ± 0.6
5d 8.1 ± 4.8 43.0 ± 3.5 7.2 ± 4.4
7a 6.8 ± 3.9 20.1 ± 2.7 6.9 ± 4.6
7b 5.4 ± 0.1 33.6 ± 9.9 7.5 ± 2.3
12a 4.4 ± 1.3 42.9 ± 5.8 6.4 ± 1.3
IsTnH 7.0 ± 4.7 46.1 ± 4.8 6.6 ± 3.2
IsRh 7.2 ± 4.6 45.8 ± 10.0 3.5 ± 1.7
Methotrexate 6.5 ± 3.2 20.1 ± 1.4 4.3 ± 2.0


Conclusion

In conclusion, we reported the synthesis of N-glycosylated (Z)-1-glycosyl-3-(5-oxo-2-thioxoimidazolidin-4-ylidene)indolin-2-ones by reaction of N-glycosylated isatines with imidazolones and thiazolones. S-Glycosylated (Z)-3-(2-glycosylsulfanyl-4-oxo-4,5-dihydro-thiazol-5-ylidene)indolin-2-ones were prepared by reaction of S-glycosylated thiazolones with isatines. The products can be regarded as hybrids of various pharmacologically highly relevant core structures, including carbohydrates, isatines, 2-thiohydantoin, pseudo-thiohydantoin and rhodanine. The products show a good activity against lung carcinoma cell lines H157, while human corneal epithelial cells (HCEC) remain unattacked. A good inhibition was observed, despite the fact that the products had to be used in their acetyl protected form. These results are in line with the antiproliferative activity of NATURA (1-(2,3,4-tri-O-acetyl-β-D-xylopyranosyl)isoindigo), where the biological activity of the acetyl-protected compound is higher than that of the deprotected molecule.9

Experimental

1H NMR spectra (250.13, 300.13 and 500.13 MHz, resp.) and 13C NMR spectra (62.9, 75.5 and 125.8 MHz, resp.) were recorded on Bruker spectrometers AVANCE 250, AVANCE 300 and AVANCE 500. The chemical shifts are referenced to solvent signals (CDCl3: δ 1H = 7.26, δ 13C = 77.0; acetone-d6: δ 1H = 2.05, δ 13C = 29.8; DMSO-d6: δ 1H = 2.50, δ 13C = 39.7). The assignment of NMR signals was supported by DEPT and two-dimensional 1H, 1H COSY, 1H, 1H NOESY and 1H, 13C correlation spectra (HSQC, HMBC) using standard pulse sequences (standard Bruker software). 19F NMR spectra (282.4 MHz) were recorded on a Bruker spectrometer AVANCE 300 and are referenced to CFCl3. Mass spectra were recorded on a FINNIGAN MAT 95 XP (Thermo Electron Corporation) and a GC 6890/MS D 5973 (Agilent Technologies). Ionization was performed by electron impact (70 eV). HRMS measurements were carried out on a Time-of-flight LC/MS 6210 (Agilent Technologies). Elemental analyses were carried out on a C/H/N/S-analyzer (Thermoquest Flash EA 1112). Glycosylated isatines used as starting materials were prepared according to a previously reported procedure.13 Compounds 3a–d were prepared as previously reported.10

General procedure for the synthesis of N-glycosylated (Z)-3-(5-oxo-2-thioxoimidazolidin-4-ylidene)indolin-2-ones 5 and (Z)-3-(2-acetamido-4-oxothiazol-5-ylidene)indolin-2-one 7 (Fig. 2)

The corresponding glycosyl isatin 3, 1.2–1.5 eq. of 2-thiohydantoin (4) or pseudo-thiohydantoin (6) and 4.8–6.0 eq. of NaOAc were stirred in a mixture of acetic acid and acetic anhydride at 60 °C for 3–4 hours. Then all solvents were distilled off under reduced pressure. The residue was dissolved in EtOAc and the excess of NaOAc was filtered off. After removal of the solvent under reduced pressure the product was purified by column chromatography (heptanes–EtOAc).
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Fig. 2 Numbering of compounds 5a, 7a and related structures.

(Z)-1-(2,3,4-Tri-O-acetyl-β-L-rhamnopyranosyl)-3-(5-oxo-2-thioxoimidazolidin-4-ylidene)indolin-2-one (5a)

According to the general procedure, 1-(2,3,4-tri-O-acetyl-β-L-rhamnopyranosyl)isatine (3a) (300 mg, 0.72 mmol), 2-thiohydantoin (4) (100 mg, 0.86 mmol) and NaOAc (282 mg, 3.44 mmol) were brought to reaction in a mixture of HOAc (4 mL) and Ac2O (2 mL). Column chromatography (heptanes–EtOAc = 10[thin space (1/6-em)]:[thin space (1/6-em)]1 → 4[thin space (1/6-em)]:[thin space (1/6-em)]1) afforded compound 5a as a deep red solid (199 mg, 54%), mp 158–160 °C. 1H NMR (500 MHz, CDCl3): δ = 11.17, 9.28 (2s, 2H, NH); 8.62 (d, 3J4′,5′ = 7.9 Hz, 1H, H-4′); 7.54 (d, 3J6′,7′ = 7.9 Hz, 1H, H-7′); 7.30 (d‘t’, 3J5′,6′ = 3J6′,7′ = 7.9 Hz, 4J4′,6′ = 1.0 Hz, 1H, H-6′); 7.07 (‘t’, 3J5′,6′ = 3J4′,5′ = 7.9 Hz, 1H, H-5′); 5.93 (d, 3J1′′,2′′ = 1.5 Hz, 1H, H-1′′); 5.60 (dd, 3J2′′,3′′ = 3.3 Hz, 3J1′′,2′′ = 1.5 Hz, 1H, H-2′′); 5.32 (dd, 3J3′′,4′′ = 10.2 Hz, 3J2′′,3′′ = 3.3 Hz, 1H, H-3′′); 5.24 (‘t’, 3J3′′,4′′ = 10.2 Hz, 3J4′′,5′′ = 9.6 Hz, 1H, H-4′′); 3.82 (m, 1H, H-5′′); 2.11, 2.00, 1.82 (3s, 9H, 3 COCH3); 1.39 (d, 3J5′′,6′′ = 6.2 Hz, 3H, H-6′′). 13C NMR (125.8 MHz, CDCl3): δ = 175.6 (C-2); 170.1, 169.9, 169.8 (3 COCH3); 167.4 (C-2′); 163.6 (C-5); 141.1 (C-7a′); 132.4 (C-4); 130.5 (C-6′); 126.3 (C-4′); 123.4 (C-5′); 119.7 (C-3a′); 113.9 (C-7′); 107.9 (C-3′); 80.4 (C-1′′); 74.1 (C-5′′); 70.5 (C-3′′); 70.3 (C-2′′); 70.2 (C-4′′); 20.8, 20.7, 20.6 (3 COCH3); 17.7 (C-6′′). MS (EI, 70 eV): m/z (%) = 517 ([M]+, 29), 153 (62), 111 (53), 69 (59), 44 (53), 43 (100). HRMS (ESI/TOF-MS): calcd for C23H23N3NaO9S ([M + Na]+) 540.10472, found 540.10442.

(Z)-1-(2,3,4,6-Tetra-O-acetyl-β-D-mannopyranosyl)-3-(5-oxo-2-thioxoimidazolidin-4-ylidene)indolin-2-one (5b)

According to the general procedure, 1-(2,3,4,6-tetra-O-acetyl-β-D-mannopyranosyl)isatin (3b) (300 mg, 0.63 mmol), 2-thiohydantoin (4) (110 mg, 0.95 mmol) and NaOAc (247 mg, 3.01 mmol) were brought to reaction in a mixture of HOAc (3 mL) and Ac2O (3 mL). Column chromatography (heptanes–EtOAc = 4[thin space (1/6-em)]:[thin space (1/6-em)]1 → 3[thin space (1/6-em)]:[thin space (1/6-em)]2) and thin layer chromatography (heptanes–EtOAc = 3[thin space (1/6-em)]:[thin space (1/6-em)]1 → 1[thin space (1/6-em)]:[thin space (1/6-em)]2) afforded compound 5b as a red solid (187 mg, 52%), mp 168–170 °C. 1H NMR (250 MHz, CDCl3): δ = 11.10, 9.24 (2s, 2H, NH); 8.54 (dd, 3J4′,5′ = 7.8 Hz, 4J4′,6′ = 0.8 Hz, 1H, H-4′); 7.48 (br d, 3J6′,7′ = 7.9 Hz, 1H, H-7′); 7.28 (d‘t’, 3J6′,7′ = 3J5′,6′ = 7.8 Hz, 4J4′,6′ = 1.0 Hz, 1H, H-6′); 7.04 (d‘t’, 3J5′,6′ = 3J4′,5′ = 7.8 Hz, 4J5′,7′ = 0.8 Hz, 1H, H-5′); 5.96 (d, 3J1′′,2′′ = 1.5 Hz, 1H, H-1′′); 5.60 (dd, 3J1′′,2′′ = 1.5 Hz, 3J2′′,3′′ = 2.7 Hz, 1H, H-2′′); 5.48–5.37 (m, 2H, H-3′′, H-4′′); 4.41 (dd, 2J6a′′,6b′′ = 12.4 Hz, 3J5′′,6a′′ = 2.4 Hz, 1H, H-6a′′); 4.32 (dd, 2J6a′′,6b′′ = 12.4 Hz, 3J5′′,6b′′ = 5.5 Hz, 1H, H-6b′′); 4.05 (m, 1H, H-5′′); 2.12 (s, 6H), 2.00, 1.82 (2s, 6H), (4 CH3). 13C NMR (62.9 MHz, CDCl3): δ = 175.9 (C-2); 170.7, 169.8 (3) (4 COCH3); 167.1 (C-2′); 163.4 (C-5); 140.6 (C-7a′); 132.4 (C-4); 130.4 (C-6′); 126.2 (C-4′); 123.4 (C-5′); 119.6 (C-3a′); 113.7 (C-7′); 107.3 (C-3′); 80.4 (C-1′′); 75.8 (C-5′′); 70.6, 70.0, 65.5 (C-2′′, C-3′′, C-4′′); 62.4 (C-6′′); 20.9, 20.7, 20.7, 20.5 (4 CH3). MS (EI, 70 eV): m/z (%) = 575 ([M]+, 13), 169 (75), 127 (11), 109 (33), 44 (37), 43 (100). HRMS (EI): calcd for C25H25N3O11S ([M]+) 575.12043, found 575.121812. Elemental analysis: calcd for C25H25N3O11S (575.54): C, 52.17; H, 4.38; N, 7.30. Found: C, 52.02; H, 4.74; N, 7.34%.

(Z)-1-(2,3,4-Tri-O-acetyl-α,β-L-rhamnopyranosyl)-5-isopropyl-3-(5-oxo-2-thioxoimidazolidin-4-ylidene)indolin-2-one (5c)

According to the general procedure, 1-(2,3,4-tri-O-acetyl-α,β-L-rhamnopyranosyl)-5-isopropylisatin (3c) (300 mg, 0.65 mmol), 2-thiohydantoin (4) (114 mg, 0.98 mmol) and NaOAc (256 mg, 3.12 mmol) were brought to reaction in a mixture of HOAc (3 mL) and Ac2O (3 mL). Column chromatography (heptanes–EtOAc = 6[thin space (1/6-em)]:[thin space (1/6-em)]1 → 4[thin space (1/6-em)]:[thin space (1/6-em)]1) afforded compound 5c as a red solid (207 mg, 57%, α,β-anomeric mixture in a ratio of β[thin space (1/6-em)]:[thin space (1/6-em)]α = 5[thin space (1/6-em)]:[thin space (1/6-em)]1), mp 203–205 °C. 1H NMR (300 MHz, CDCl3): (β-anomer) δ = 11.20, 9.23 (2s, 2H, NH); 8.55 (d, 4J4′,6′ = 1.8 Hz, 1H, H-4′); 7.44 (d, 3J6′,7′ = 8.3 Hz, 1H, H-7′); 7.18 (dd, 3J6′,7′ = 8.3 Hz, 4J4′,6′ = 1.8 Hz, 1H, H-6′); 5.91 (d, 3J1′′,2′′ = 1.5 Hz, 1H, H-1′′); 5.59 (dd, 3J2′′,3′′ = 3.3 Hz, 3J1′′,2′′ = 1.5 Hz, 1H, H-2′′); 5.31 (dd, 3J3′′,4′′ = 10.2 Hz, 3J2′′,3′′ = 3.3 Hz, 1H, H-3′′); 5.24 (‘t’, 3J3′′,4′′ = 10.2 Hz, 3J4′′,5′′ = 9.2 Hz, 1H, H-4′′); 3.80 (m, 1H, H-5′′); 2.92 (m, 1H, CH(CH3)2); 2.11, 2.00, 1.82 (3s, 9H, 3 COCH3); 1.38 (d, 3J5′′,6′′ = 6.1 Hz, 3H, H-6′′); 1.26 (d, 3J = 6.9 Hz, 3H), 1.25 (d, 3J = 6.9 Hz, 3H), (CH(CH3)2). (α-Anomer) δ = 11.29, 9.30 (2s, 2H, NH); 8.62 (d, 4J4′,6′ = 1.6 Hz, 1H, H-4′); 7.24 (dd, 3J6′,7′ = 8.3 Hz, 4J4′,6′ = 1.8 Hz, 1H, H-6′); 7.15 (d, 3J6′,7′ = 8.3 Hz, 1H, H-7′); 6.05 (dd, 3J1′′,2′′ = 5.6 Hz, 3J2′′,3′′ = 3.9 Hz, 1H, H-2′′); 5.81 (dd, 3J3′′,4′′ = 6.4 Hz, 3J2′′,3′′ = 3.9 Hz, 1H, H-3′′); 5.72 (d, 3J1′′,2′′ = 5.6 Hz, 1H, H-1′′); 5.04 (‘t’, 3J3′′,4′′ = 3J4′′,5′′ = 6.4 Hz, 1H, H-4′′); 4.06 (m, 1H, H-5′′); 2.92 (m, 1H, CH(CH3)2); 2.15, 2.15, 2.06 (3s, 9H, 3 COCH3); 1.40 (d, 3J5′′,6′′ = 6.1 Hz, 3H, H-6′′); 1.27 (d, 3J = 6.9 Hz, 3H), 1.27 (d, 3J = 6.9 Hz, 3H), (CH(CH3)2). 13C NMR (62.9 MHz, CDCl3): (β-anomer) δ = 175.7 (C-2); 170.1, 169.9, 169.8 (3 COCH3); 167.5 (C-2′); 163.9 (C-5); 144.1 (C-5′); 139.1 (C-7a′); 132.2 (C-4); 128.8, 124.3 (C-4′, C-6′); 119.7 (C-3a′); 113.7 (C-7′); 108.4 (C-3′); 80.3 (C-1′′); 74.0 (C-5′′); 70.5, 70.3, 70.2 (C-2′′, C-3′′, C-4′′); 33.7 (CH(CH3)2); 24.0, 24.0 (CH(CH3)2); 20.8, 20.8, 20.5 (3 COCH3); 17.7 (C-6′′). (α-Anomer) δ = 175.7 (C-2); 169.9, 169.8, 169.6 (3 COCH3); 168.6 (C-2′); 164.0 (C-5); 144.7 (C-5′); 139.4 (C-7a′); 132.2 (C-4); 129.5, 124.7 (C-4′, C-6′); 119.8 (C-3a′); 113.7 (C-7′); 108.8 (C-3′); 71.5, 70.9, 69.2, 66.6 (C-2′′, C-3′′, C-4′′, C-5′′); 33.8 (CH(CH3)2); 24.0, 24.0 (CH(CH3)2); 20.9, 20.8, 20.8 (3 COCH3); 16.5 (C-6′′); (C-1′′ not given). MS (EI, 70 eV): m/z (%) = 559 ([M]+, 12), 273 (14), 153 (53), 111 (51), 83 (14), 43 (100). HRMS (ESI-TOF/MS): calcd for C26H29N3NaO9S ([M + Na]+) 582.15167, found 582.15186. Elemental analysis: calcd for C26H29N3O9S (559.59): C, 55.81; H, 5.22; N, 7.51; S, 5.73. Found: C, 55.87; H, 5.55; N, 7.41; S, 5.81%.

(Z)-1-(2,3,4,6-Tetra-O-acetyl-β-D-mannopyranosyl)-5-isopropyl-3-(5-oxo-2-thioxoimidazolidin-4-ylidene)indolin-2-one (5d)

According to the general procedure, 1-(2,3,4,6-tetra-O-acetyl-α,β-L-mannopyranosyl)-5-isopropylisatin (3d) (300 mg, 0.58 mmol), 2-thiohydantoin (4) (100 mg, 0.86 mmol) and NaOAc (227 mg, 2.77 mmol) were brought to reaction in a mixture of HOAc (3 mL) and Ac2O (3 mL). Column chromatography (heptanes–EtOAc = 4[thin space (1/6-em)]:[thin space (1/6-em)]1 → 2[thin space (1/6-em)]:[thin space (1/6-em)]1) afforded compound 5d as a red solid (91 mg, 26%, α,β-anomeric mixture in a ratio of β[thin space (1/6-em)]:[thin space (1/6-em)]α = 20[thin space (1/6-em)]:[thin space (1/6-em)]3), mp 172–174 °C. 1H NMR (300 MHz, CDCl3): (β-anomer) δ = 11.11, 9.58 (2s, 2H, NH); 8.49 (d, 4J4′,6′ = 1.5 Hz, 1H, H-4′); 7.39 (d, 3J6′,7′ = 8.4 Hz, 1H, H-7′); 7.14 (dd, 3J6′,7′ = 8.4 Hz, 4J4′,6′ = 1.5 Hz, 1H, H-6′); 5.96 (d, 3J1′′,2′′ = 1.6 Hz, 1H, H-1′′); 5.60 (dd, 3J2′′,3′′ = 2.8 Hz, 3J1′′,2′′ = 1.6 Hz, 1H, H-2′′); 5.47–5.36 (m, 2H, H-3′′, H-4′′); 4.40–4.01 (m, 3H, H-5′′, H-6a′′, H-6b′′); 2.89 (m, 1H, CH(CH3)2); 2.12, 2.11, 2.00, 1.82 (4s, 12H, 4 COCH3); 1.26–1.22 (m, 6H, CH(CH3)2). (α-Anomer) δ = 11.23, 9.48 (2s, 2H, NH); 8.61 (d, 4J4′,6′ = 1.5 Hz, 1H, H-4′); 7.40 (d, 3J6′,7′ = 8.3 Hz, 1H, H-7′); 7.23 (dd, 3J6′,7′ = 8.3 Hz, 4J4′,6′ = 1.5 Hz, 1H, H-6′); 6.08 (dd, 3J1′′,2′′ = 5.4 Hz, 3J2′′,3′′ = 3.8 Hz, 1H, H-2′′); 5.85 (dd, 3J3′′,4′′ = 6.8 Hz, 3J2′′,3′′ = 3.8 Hz, 1H, H-3′′); 5.74 (d, 3J1′′,2′′ = 5.4 Hz, 1H, H-1′′); 5.21 (‘t’, 3J3′′,4′′ = 6.8 Hz, 3J4′′,5′′ = 6.1 Hz, 1H, H-4′′); 4.58 (dd, 2J6a′′,6b′′ = 12.9 Hz, 3J5′′,6a′′ = 7.7 Hz, 1H, H-6a′′); 4.19–4.11 (m, 2H, H-5′′, H-6b′′); 2.88 (m, 1H, CH(CH3)2); 2.16, 2.15, 2.07, 2.02 (4s, 12H, 4 COCH3); 1.28–1.25 (m, 6H, CH(CH3)2). 13C NMR (62.9 MHz, CDCl3): (β-anomer) δ = 175.7 (C-2); 170.7, 169.8 (3) (4 COCH3); 167.3 (C-2′); 163.5 (C-5); 144.1 (C-5′); 138.7 (C-7a′); 132.1 (C-4); 128.7 (C-6′); 124.2 (C-4′); 119.6 (C-3a′); 113.6 (C-7′); 108.0 (C-3′); 80.3 (C-1′′); 75.7 (C-5′′); 70.0, 70.6, 65.5 (C-2′′, C-3′′, C-4′′); 62.4 (C-6′′); 33.7 (CH(CH3)2); 24.0 (2) (CH(CH3)2); 20.8, 20.7, 20.7, 20.5 (4 COCH3). (α-Anomer) δ = 175.9 (C-2); 170.8, 169.9, 169.8, 168.6 (4 COCH3); 168.6 (C-2′); 163.8 (C-5); 144.1 (C-5′); 139.2 (C-7a′); 132.1 (C-4); 129.4 (C-6′); 124.7 (C-4′); 113.6 (C-7′); 108.6 (C-3′); 78.1 (C-1′′); 72.7 (C-5′′); 68.8 (C-3′′); 67.4 (C-4′′); 66.5 (C-2′′); 61.1 (C-6′′); 33.7 (CH(CH3)2); 24.0 (2) (CH(CH3)2); 20.8, 20.7 (2), 20.5 (4 COCH3); (C-3a′) not given. MS (EI, 70 eV): m/z (%) = 617 ([M]+, 18), 287 (16), 169 (100), 109 (41), 44 (18), 43 (94). HRMS (EI): calcd for C28H31N3O11S ([M]+) 617.16738, found 617.167979. Elemental analysis: calcd for C28H31N3O11S (617.62): C, 54.45; H, 5.06; N, 6.80. Found: C, 54.09; H, 5.30; N, 6.88%.

(Z)-3-(2-Acetamido-4-oxo-4,5-dihydro-thiazol-5-ylidene)-1-(2,3,4-tri-O-acetyl-β-L-rhamnopyranosyl)indolin-2-one (7a)

According to the general procedure, 1-(2,3,4-tri-O-acetyl-β-L-rhamnopyranosyl)isatin (3a) (300 mg, 0.72 mmol), pseudo-thiohydantoin (6) (125 mg, 1.08 mmol) and NaOAc (352 mg, 4.29 mmol) were brought to reaction in a mixture of HOAc (3 mL) and Ac2O (3 mL). After column chromatography (heptanes–EtOAc = 4[thin space (1/6-em)]:[thin space (1/6-em)]1 → 1[thin space (1/6-em)]:[thin space (1/6-em)]1) the crude product was dissolved in EtOAc and shaken with a saturated solution of NaHCO3. Compound 7a was isolated as an orange brown oil (195 mg, 49%). 1H NMR (300 MHz, CDCl3): δ = 12.66 (s, 1H, NH); 9.09 (dd, 3J4′,5′ = 8.0 Hz, 4J4′,6′ = 1.4 Hz, 1H, H-4′); 7.57 (dd, 3J6′,7′ = 8.0 Hz, 4J5′,7′ = 1.1 Hz, 1H, H-7′); 7.38 (d‘t’, 3J6′,7′ = 8.0 Hz, 3J5′,6′ = 7.8 Hz, 4J4′,6′ = 1.4 Hz, 1H, H-6′); 7.12 (d‘t’, 3J4′,5′ = 8.0 Hz, 3J5′,6′ = 7.8 Hz, 4J5′,7′ = 1.1 Hz, 1H, H-5′); 5.97 (d, 3J1′′,2′′ = 1.5 Hz, 1H, H-1′′); 5.62 (dd, 3J2′′,3′′ = 3.0 Hz, 3J1′′,2′′ = 1.5 Hz, 1H, H-2′′); 5.31-5.19 (m, 2H, H-3′′, H-4′′); 3.79 (m, 1H, H-5′′); 2.63 (s, 3H, NHCOCH3); 2.10, 1.98, 1.85 (3s, 9H, 3 COCH3); 1.37 (d, 3J5′′,6′′ = 6.2 Hz, 3H, H-6′′). 13C NMR (75.5 MHz, CDCl3): δ = 180.3 (C-2); 178.6 (NHCOCH3); 172.0, 170.0, 169.7, 169.6, 166.9 (3 COCH3, 2 CO); 142.7 (C-7a′); 135.0 (Cq); 132.2 (C-6′); 129.3 (Cq); 128.6 (C-4′); 123.1 (C-5′); 120.5 (C-3a′); 113.8 (C-7′); 80.1 (C-1′′); 73.8 (C-5′′); 70.5, 70.4, 70.1 (C-2′′, C-3′′, C-4′′); 24.3 (NHCOCH3); 20.8, 20.7, 20.5 (3 COCH3); 17.6 (C-6′′). MS (EI, 70 eV): m/z (%) = 559 ([M]+, 23), 273 (53), 153 (77), 111 (74), 83 (39), 43 (100). HRMS (ESI-TOF/MS): calcd for C25H26N3O10S ([M + H]+) 560.13334, found 560.13334.

(Z)-3-(2-Acetamido-4-oxo-4,5-dihydro-thiazol-5-ylidene)-1-(2,3,4,6-tetra-O-acetyl-β-D-mannopyranosyl)indolin-2-one (7b)

According to the general procedure, 1-(2,3,4-tri-O-acetyl-α,β-D-mannopyranosyl)isatin (3b) (342 mg, 0.72 mmol), pseudo-thiohydantoin (6) (125 mg, 1.08 mmol) and NaOAc (352 mg, 4.29 mmol) were brought to reaction in a mixture of HOAc (3 mL) and Ac2O (3 mL). After column chromatography (heptanes–EtOAc = 3[thin space (1/6-em)]:[thin space (1/6-em)]1 → 1[thin space (1/6-em)]:[thin space (1/6-em)]1), the crude product was dissolved in EtOAc and shaken with a saturated solution of NaHCO3. Compound 7b was isolated as an orange brown foam (177 mg, 40%). 1H NMR (250 MHz, CDCl3): δ = 12.63 (s, 1H, NH); 9.09 (dd, 3J4′,5′ = 8.0 Hz, 4J4′,6′ = 1.3 Hz, 1H, H-4′); 7.56 (dd, 3J6′,7′ = 8.0 Hz, 4J5′,7′ = 1.0 Hz, 1H, H-7′); 7.37 (d‘t’, 3J6′,7′ = 8.0 Hz, 3J5′,6′ = 7.6 Hz, 4J4′,6′ = 1.3 Hz, 1H, H-6′); 7.13 (d‘t’, 3J4′,5′ = 8.0 Hz, 3J5′,6′ = 7.6 Hz, 4J5′,7′ = 1.0 Hz, 1H, H-5′); 6.05 (d, 3J1′′,2′′ = 1.5 Hz, 1H, H-1′′); 5.70 (dd, 3J2′′,3′′ = 2.8 Hz, 3J1′′,2′′ = 1.5 Hz, 1H, H-2′′); 5.50–5.34 (m, 2H, H-3′′, H-4′′); 4.38–4.21 (m, 2H, H-6′′); 3.98 (m, 1H, H-5′′); 2.60 (s, 3H, NHCOCH3); 2.12, 2.11, 1.99, 1.88 (4s, 12H, 4 COCH3). 13C NMR (62.9 MHz, CDCl3): δ = 180.3 (C-2); 178.7 (NHCOCH3); 171.9, 170.5, 169.8, 169.8, 169.6, 166.9 (4 COCH3, 2 CO); 142.7 (C-7a′); 135.1 (Cq); 132.1 (C-6′); 129.3 (Cq); 128.6 (C-4′); 123.1 (C-5′); 120.5 (C-3a′); 114.0 (C-7′); 80.6 (C-1′′); 75.3 (C-5′′); 70.7, 70.3, 65.3 (C-2′′, C-3′′, C-4′′); 62.3 (C-6′′); 24.4 (NHCOCH3); 20.8, 20.7, 20.7, 20.6 (4 COCH3). MS (EI, 70 eV): m/z (%) = 617 ([M]+, 38), 331 (78), 169 (99), 127 (49), 109 (86), 43 (100). HRMS (ESI-TOF/MS): calcd for C27H27N3NaO12S ([M + Na]+) 640.12077, found 640.12062.

General procedure for the synthesis of glycosylsulfanylthiazolones 10a–c and glycosylsulfanylimidazolone 10d (Fig. 3)

A solution of the corresponding heterocycle (8 or 4) in DMF was cooled to 0 °C, then 1.1 eq. of NaH were added. After stirring for 15 min, 1.1 eq. of glycosyl bromide 9 were added. The ice-bath was removed and the reaction mixture was stirred for 3 hours at 20 °C. Then water was added and the resulting solution was extracted several times with EtOAc. The combined organic layers were washed with water and dried over Na2SO4. The solvent was removed under reduced pressure and the residue was purified by column chromatography (heptanes–EtOAc).
image file: c3ra44362k-f3.tif
Fig. 3 Numbering of compounds 10a–c (X = S) and compound 10d (X = NH).

2-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosylsulfanyl)-4,5-dihydro-thiazol-4-one (10a)

According to the general procedure rhodanine (8) (100 mg, 0.75 mmol), sodium hydride (33 mg, 0.83 mmol; 60% dispersion in mineral oil) and 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (9a) (339 mg, 0.83 mmol) were brought to reaction in 5 mL of dry DMF. Compound 10a was isolated by column chromatography (heptanes–EtOAc = 6[thin space (1/6-em)]:[thin space (1/6-em)]1 → 3[thin space (1/6-em)]:[thin space (1/6-em)]1) and subsequent crystallization (heptanes–EtOAc) as colourless crystals (162 mg, 47%), mp 167–168 °C. 1H NMR (500 MHz, CDCl3): δ = 6.15 (d, 3J1′,2′ = 9.3 Hz, 1H, H-1′); 5.90 (‘t’, 3J1′,2′ = 3J2′,3′ = 9.3 Hz, 1H, H-2′); 5.35 (‘t’, 3J3′,4′ = 9.6 Hz, 3J2′,3′ = 9.3 Hz, 1H, H-3′); 5.20 (dd, 3J4′,5′ = 10.0 Hz, 3J3′,4′ = 9.6 Hz, 1H, H-4′); 4.25–4.16 (m, 2H, H-6′); 3.90–3.80 (m, 3H, H-5′, H-5); 2.07, 2.03, 2.01, 1.94 (4s, 12H, 4 CH3). 13C NMR (125.8 MHz, CDCl3): δ = 201.5 (C-2); 172.0 (C-4); 170.5, 170.0, 169.9, 169.3 (4 COCH3); 82.4 (C-1′); 74.8 (C-5′); 72.9 (C-3′); 67.8 (C-2′); 67.7 (C-4′); 61.5 (C-6′); 33.6 (C-5); 20.7, 20.5, 20.5, 20.3 (4 CH3). HRMS (ESI-TOF/MS): calcd for C17H22NO10S2 ([M + H]+) 464.068, found 464.0682. Elemental analysis: calcd for C17H21NO10S2 (463.48): C, 44.05; H, 4.57; N, 3.02. Found: C, 44.10; H, 4.63; N, 3.09%.

2-(2,3,4,6-Tetra-O-pivaloyl-β-D-glucopyranosylsulfanyl)-4,5-dihydro-thiazol-4-one (10b)

According to the general procedure, rhodanine (8) (300 mg, 2.25 mmol), sodium hydride (100 mg, 2.50 mmol; 60% dispersion in mineral oil) and 2,3,4,6-tetra-O-pivaloyl-α-D-glucopyranosyl bromide (9b) (1436 mg, 2.57 mmol) were brought to reaction in a mixture of 15 mL of dry DMF and 2 mL of dry THF. Compound 10b was isolated by column chromatography (heptanes–EtOAc = 100[thin space (1/6-em)]:[thin space (1/6-em)]1 → 12[thin space (1/6-em)]:[thin space (1/6-em)]1) as colourless crystals (710 mg, 50%), mp 154–155 °C. 1H NMR (250 MHz, CDCl3): δ = 6.18 (d, 3J1′,2′ = 9.3 Hz, 1H, H-1′); 5.95 (‘t’, 3J1′,2′ = 3J2′,3′ = 9.3 Hz, 1H, H-2′); 5.48 (‘t’, 3J3′,4′ = 9.8 Hz, 3J2′,3′ = 9.3 Hz, 1H, H-3′); 5.30 (‘t’, 3J3′,4′ = 3J4′,5′ = 9.8 Hz, 1H, H-4′); 4.28 (dd, 2J6a′,6b′ = 12.6 Hz, 3J5′,6a′ = 1.9 Hz, 1H, H-6a′); 4.05 (dd, 2J6a′,6b′ = 12.6 Hz, 3J5′,6b′ = 4.4 Hz, 1H, H-6b′); 3.94–3.78 (m, 3H, H-5, H-5′); 1.21, 1.16, 1.12, 1.07 (4s, 36H, 4C(CH3)3). 13C NMR (62.9 MHz, CDCl3): δ = 201.4 (C-2); 178.0, 177.7, 177.0, 176.2 (4 COC(CH3)3); 171.9 (C-4); 82.5 (C-1′); 75.2 (C-5′); 72.3 (C-3′); 67.8 (C-2′); 66.9 (C-4′); 61.0 (C-6′); 38.9, 38.9, 38.8, 38.7 (4 C(CH3)3); 33.4 (C-5); 27.1, 27.0 (3) (4C(CH3)3). HRMS (ESI-TOF/MS): calcd for C29H46NO10S2 ([M + H]+) 632.2558, found 632.2549. Elemental analysis: calcd for C29H45NO10S2 (631.80): C, 55.13; H, 7.18; N, 2.22. Found: C, 55.01; H, 7.42; N, 2.16%.

2-(2,3,4-Tri-O-pivaloyl-β-D-xylopyranosylsulfanyl)-4,5-dihydro-thiazol-4-one (10c)

According to the general procedure, rhodanine (8) (400 mg, 3.00 mmol), sodium hydride (120 mg, 5.00 mmol; 60% dispersion in mineral oil) and 2,3,4-tri-O-pivaloyl-α-D-xylopyranosyl bromide (9c) (1162 mg, 2.50 mmol) were brought to reaction in a mixture of 14 mL of dry DMF and 2 mL of dry THF. Compound 10c was isolated by column chromatography (heptanes–EtOAc = 100[thin space (1/6-em)]:[thin space (1/6-em)]1 → 12[thin space (1/6-em)]:[thin space (1/6-em)]1) as fine white needles (417 mg, 32%), mp 187–189 °C. 1H NMR (500 MHz, CDCl3): δ = 6.11 (d, 3J1′,2′ = 9.4 Hz, 1H, H-1′); 5.90 (‘t’, 3J1′,2′ = 3J2′,3′ = 9.4 Hz, 1H, H-2′); 5.49 (‘t’, 3J3′,4′ = 9.6 Hz, 3J2′,3′ = 9.4 Hz, 1H, H-3′); 5.15 (ddd, 3J4′,5b′ = 10.4 Hz, 3J3′,4′ = 9.6 Hz, 3J4′,5a′ = 5.6 Hz, 1H, H-4′); 4.20 (dd, 2J5a′,5b′ = 11.4 Hz, 3J4′,5a′ = 5.6 Hz, 1H, H-5a′); 3.85 (center of AB, 2J = 18.0 Hz, 2H, H-5); 3.45 (dd, 2J5a′,5b′ = 11.4 Hz, 3J4′,5b′ = 10.4 Hz, 1H, H-5b′); 1.15, 1.14, 1.07 (3s, 27H, 3C(CH3)3). 13C NMR (125.8 MHz, CDCl3): δ = 201.3 (C-2); 177.8, 177.1, 176.9 (3 COC(CH3)3); 172.3 (C-4); 83.1 (C-1′); 71.9 (C-3′); 68.3 (C-4′); 67.7 (C-2′); 65.6 (C-5′); 38.9, 38.8, 38.7 (3 C(CH3)3); 33.4 (C-5); 27.1, 27.1, 27.0 (3C(CH3)3). HRMS (ESI-TOF/MS): calcd for C23H35NNaO8S2 ([M + Na]+) 540.1696, found 540.1703. Elemental analysis: calcd for C23H35NO8S2 (517.66): C, 53.36; H, 6.81; N, 2.71. Found: C, 53. 58; H, 6.94; N, 2.81%.

2-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosylsulfanyl)-4,5-dihydro-1H-imidazol-4-one (10d)

According to the general procedure, 2-thiohydantoin (4) (150 mg, 1.29 mmol), sodium hydride (57 mg, 1.32 mmol; 60% dispersion in mineral oil) and 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (9a) (584 mg, 1.32 mmol) were brought to reaction in 15 mL dry DMF. Compound 10d was isolated by column chromatography (heptanes–EtOAc = 3[thin space (1/6-em)]:[thin space (1/6-em)]1 → 1[thin space (1/6-em)]:[thin space (1/6-em)]1) as a colourless oil (297 mg, 52%). Due to its instability, the obtained product was brought subsequently to reaction. 1H NMR (500 MHz, acetone-d6): δ = 9.20 (s, 1H, NH); 6.01 (d, 3J1′,2′ = 9.4 Hz, 1H, H-1′); 5.94 (‘t’, 3J1′,2′ = 3J2′.3′ = 9.4 Hz, 1H, H-2′); 5.43 (‘t’, 3J2′,3′ = 9.4 Hz, 3J3′,4′ = 9.5 Hz, 1H, H-3′); 5.11 (‘t’, 3J4′,5′ = 9.8 Hz, 3J3′,4′ = 9.5, 1H, H-4′); 4.27–4.00 (m, 5H, H-5, H-5′, H-6′); 2.02, 2.00, 1.97, 1.90 (4s, 12H, 4 CH3). 13C NMR (125.8 MHz, acetone-d6): δ = 184.4 (C-2); 171.5, 170.6, 170.2, 170.0, 170.0 (4 COCH3, C-4); 82.0 (C-1′); 74.8 (C-5′); 73.9 (C-3′); 68.8 (C-4′); 68.7 (C-2′); 62.6 (C-6′); 48.4 (C-5); 20.6, 20.6, 20.5, 20.4 (4 CH3). HRMS (ESI-TOF/MS): calcd for C17H22N2NaO10S ([M + Na]+) 469.0887, found 469.0890.

General procedure for the synthesis of (Z)-3-(2-glycosysulfanyl-4-oxo-4,5-dihydro-thiazol-5-ylidene)indolin-2-ones 12a-l (Fig. 4) and (Z)-3-(2-glycosysulfanyl-4-oxo-4,5-dihydro-1H-imidazol-5-ylidene)indolin-2-one 12m (Fig. 5)

The corresponding S-glycoside 10 and 1.0–1.2 eq. of isatin 11 were dissolved in a mixture of ethanol or methanol and THF. Then catalytic amounts of piperidine were added. Soon the solution turned dark and a precipitate was formed. After 1–2 hours of stirring at 20 °C all solvents were distilled off in vacuo and the product was obtained by recrystallization or the precipitate.
image file: c3ra44362k-f4.tif
Fig. 4 Numbering of compounds 12a-l.

image file: c3ra44362k-f5.tif
Fig. 5 Numbering of compound 12m.

(Z)-3-[2-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosylsulfanyl)-4-oxo-4,5-dihydro-thiazol-5-ylidene]indolin-2-one (12a)

According to the general procedure, S-glycoside 10a (100 mg, 0.22 mmol) and isatin (11a) (35 mg, 0.24 mmol) were brought to reaction in a mixture of 4 mL of dry MeOH and 2 mL of dry THF. The resulting precipitate was filtered off, washed with diethyl ether and dried in vacuo. Compound 12a was obtained as orange fine needles (80 mg, = 62%), mp 328–329 °C. 1H NMR (500 MHz, DMSO-d6) (ratio major–minor = 5[thin space (1/6-em)]:[thin space (1/6-em)]1) (major): δ = 11.30 (s, 1H, NH); 8.73 (d, 3J4′,5′ = 7.9 Hz, 1H, H-4′); 7.46 (‘t’, 3J5′,6′ = 3J6′,7′ = 7.9 Hz, 1H, H-6′); 7.13 (‘t’, 3J4′,5′ = 3J5′,6′ = 7.9 Hz, 1H, H-5′); 6.98 (d, 3J6′,7′ = 7.9 Hz, 1H, H-7′); 6.58 (d, 3J1′′,2′′ = 9.3 Hz, 1H, H-1′′); 5.92 (‘t’, 3J2′′,3′′ = 9.5 Hz, 3J1′′,2′′ = 9.3 Hz, 1H, H-2′′); 5.63 (‘t’, 3J2′′,3′′ = 3J3′′,4′′ = 9.5 Hz, 1H, H-3′′); 5.07 (‘t’, 3J4′′,5′′ = 9.8 Hz, 3J3′′,4′′ = 9.5 Hz, 1H, H-4′′); 4.39 (m, 1H, H-5′′); 4.14 (m, 2H, H-6a′′, H-6b′′); 2.04, 2.01, 1.99, 1.87 (4s, 12H, 4 COCH3); (minor): δ = 11.30 (s, 1H, NH); 8.73 (d, 3J4′,5′ = 7.9 Hz, 1H, H-4′); 7.44 (‘t’, 3J5′,6′ = 3J6′,7′ = 7.9 Hz, 1H, H-6′); 7.11 (‘t’, 3J4′,5′ = 3J5′,6′ = 7.9 Hz, 1H, H-5′); 6.98 (d, 3J6′,7′ = 7.9 Hz, 1H, H-7′); 6.40 (‘t’, 3J2′′,3′′ = 9.5 Hz, 3J1′′,2′′ = 9.3 Hz, 1H, H-2′′); 6.23 (d, 3J1′′,2′′ = 9.3 Hz, 1H, H-1′′); 5.57 (‘t’, 3J2′′,3′′ = 3J3′′,4′′ = 9.5 Hz, 1H, H-3′′); 5.01 (‘t’, 3J4′′,5′′ = 9.8 Hz, 3J3′′,4′′ = 9.5 Hz, 1H, H-4′′); 4.37 (m, 1H, H-5′′); 4.19–4.03 (m, 2H, H-6a′′, H-6b′′); 2.04, 1.99, 1.96, 1.87 (4s, 12H, 4 COCH3). 13C NMR (125.8 MHz, DMSO-d6) (major): δ = 198.5 (C-2); 170.1, 169.9, 169.6, 169.6 (4 COCH3); 168.0, 165.7 (C-4, C-2′); 145.2 (C-7a′); 133.8 (C-6′); 128.4 (C-5); 128.3 (C-4′); 126.4 (C-3′); 122.6 (C-5′); 119.8 (C-3a′); 111.1 (C-7′); 81.5 (C-1′′); 73.2 (C-5′′); 72.1 (C-3′′); 67.9, 67.5 (C-4′′, C-2′′); 61.6 (C-6′′); 20.7, 20.5, 20.4, 20.2 (4 COCH3). MS (ESI-TOF/MS): 615.0715 ([M + Na]+, 2), 292.9333 (2), 240.9874 (64), 162.9735 (100), 101.0035 (30). HRMS (ESI-TOF/MS): calcd for C25H24N2NaO11S2 ([M + Na]+) 615.0714, found 615.0716. Elemental analysis: calcd for C25H24N2O11S2 (592.59): C, 50.67; H, 4.08; N, 4.73; S, 10.82. Found: C, 50.62; H, 4.34; N, 4.95; S, 10.99%.

Alternatively, the reaction was carried out in boiling dry ethanol. The resulting precipitate was dissolved in hot dioxane to obtain larger crystals in order to extrude traces of solvents. However, the result was a decrease of the yield to 56%.

(Z)-3-[4-Oxo-2-(2,3,4,6-tetra-O-pivaloyl-β-D-glucopyranosylsulfanyl)-4,5-dihydro-thiazol-5-ylidene]indolin-2-one (12b)

According to the general procedure S-glycoside 10b (100 mg, 0.16 mmol) and isatin (11a) (23 mg, 0.16 mmol) were brought to reaction in a mixture of 3 mL of dry EtOH and 2 mL of dry THF. After recrystallization from acetone–EtOH, compound 12b was obtained as a dark red fine powder (96 mg, 80%), mp 321 °C. 1H NMR (250 MHz, CDCl3): δ = 1H NMR (250 MHz, CDCl3): δ = 8.86 (d, 3J4′,5′ = 7.9 Hz, 1H, H-4′); 8.53 (s, 1H, NH); 7.31 (‘t’, 3J5′,6′ = 3J6′,7′ = 7.7 Hz, 1H, H-6′); 7.02 (‘t’, 3J4′,5′ = 7.9 Hz, 3J5′,6′ = 7.7 Hz, 1H, H-5′); 6.86 (d, 3J6′,7′ = 7.7 Hz, 1H, H-7′); 6.41 (d, 3J1′′,2′′ = 9.3 Hz, 1H, H-1′′); 6.20 (‘t’, 3J1′′,2′′ = 9.3 Hz, 3J2′′,3′′ = 9.2 Hz, 1H, H-2′′); 5.55 (‘t’, 3J3′′,4′′ = 9.6 Hz, 3J2′′,3′′ = 9.2 Hz, 1H, H-3′′); 5.42 (‘t’, 3J4′′,5′′ = 9.8 Hz, 3J3′′,4′′ = 9.6 Hz, 1H, H-4′′); 4.39 (br d, 2J6a′′,6b′′ = 12.4 Hz, 1H, H-6a′′); 4.04 (dd, 2J6a′′,6b′′ = 12.4 Hz, 3J5′′,6b′′ = 4.0 Hz, 1H, H-6b′′); 4.00–3.91 (m, 1H, H-5′′); 1.25, 1.19, 1.15, 1.04 (4s, 36H, 4C(CH3)3). 13C NMR (62.9 MHz, CDCl3): δ = 197.7 (C-2); 178.1, 177.9, 177.0, 176.3 (4 COC(CH3)3); 168.1 (C-2′); 165.4 (C-4); 143.3 (C-7a′); 132.9 (C-6′); 129.5 (C-5); 129.3 (C-4′); 126.0 (C-3′); 123.2 (C-5′); 120.2 (C-3a′); 110.3 (C-7′); 82.0 (C-1′′); 75.2 (C-5′′); 72.5 (C-3′′); 68.0 (C-2′′); 66.9 (C-4′′); 61.1 (C-6′′); 38.9, 38.8 (3) (4 C(CH3)3); 27.2, 27.1 (3) (4C(CH3)3). HRMS (ESI-TOF/MS): calcd for C37H48N2NaO11S2 ([M + Na]+) 783.2592, found 783.2592. Elemental analysis: calcd for C37H48N2O11S2 (760.91): C, 58.40; H, 6.36; N, 3.68; S, 8.43. Found: C, 58.33; H, 6.02; N, 3.75; S, 8.32%.

(Z)-3-[4-Oxo-2-(2,3,4-tri-O-pivaloyl-β-D-xylopyranosylsulfanyl)-4,5-dihydro-thiazol-5-ylidene]indolin-2-one (12c)

According to the general procedure, S-glycoside 10c (132 mg, 0.25 mmol) and isatin (11a) (38 mg, 0.25 mmol) were brought to reaction in a mixture of 3 mL of dry MeOH and 1 mL of dry THF. After recrystallization from acetone–EtOH, compound 12c was obtained as red brown shiny needles (103 mg, 62%), mp 294–296 °C. 1H NMR (500 MHz, CDCl3): δ = 8.88 (d, 3J4′,5′ = 8.0 Hz, 1H, H-4′); 8.44 (s, 1H, NH); 7.32 (d‘t’, 3J5′,6′ = 3J6′,7′ = 7.8 Hz, 4J4′,6′ = 1.0 Hz, 1H, H-6′); 7.04 (‘t’, 3J4′,5′ = 8.0 Hz, 3J5′,6′ = 7.8 Hz, 1H, H-5′); 6.87 (d, 3J6′,7′ = 7.8 Hz, 1H, H-7′); 6.34 (d, 3J1′′,2′′ = 9.4 Hz, 1H, H-1′′); 6.14 (‘t’, 3J1′′,2′′ = 3J2′′,3′′ = 9.4 Hz, 1H, H-2′′); 5.55 (‘t’, 3J3′′,4′′ = 9.8 Hz, 3J2′′,3′′ = 9.4 Hz, 1H, H-3′′); 5.26 (ddd, 3J4′′,5b′′ = 10.5 Hz, 3J3′′,4′′ = 9.8 Hz, 3J4′′,5a′′ = 9.6 Hz, 1H, H-4′′); 4.28 (dd, 2J5a′′,5b′′ = 11.5 Hz, 3J4′′,5a′′ = 5.6 Hz, 1H, H-5a′′); 3.53 (dd, 2J5a′′,5b′′ = 11.5 Hz, 3J4′′,5b′′ = 10.5 Hz, 1H, H-5b′′); 1.18, 1.16, 1.04 (3s, 27H, 3C(CH3)3). 13C NMR (125.8 MHz, CDCl3): δ = 197.6 (C-2); 177.9, 177.1, 177.0 (3 COC(CH3)3); 168.2 (C-2′); 165.7 (C-4); 143.2 (C-7a′); 133.0 (C-6′); 129.6 (C-5); 129.5 (C-4′); 126.0 (C-3′); 123.3 (C-5′); 120.2 (C-3a′); 110.3 (C-7′); 82.8 (C-1′′); 72.1 (C-3′′); 68.4 (C-4′′); 67.9 (2′′); 65.8 (C-5′′); 38.9, 38.8, 38.8 (3 C(CH3)3); 27.1, 27.07 (2) (3C(CH3)3). MS (EI, 70 eV): m/z (%) = 646 ([M+], 1), 97 (11), 85 (27), 57 (100), 44 (14), 41 (17). HRMS (ESI-TOF/MS): calcd for C31H38N2NaO9S2 ([M + Na]+) 669.1911, found 669.1916.

(Z)-1-Phenyl-3-[4-oxo-2-(2,3,4,6-tetra-O-pivaloyl-β-D-glucopyranosylsulfanyl)-4,5-dihydro-thiazol-5-ylidene]indolin-2-one (12d)

According to the general procedure, S-glycoside 10b (175 mg, 0.28 mmol) and N-phenylisatin (11b) (68 mg, 0.30 mmol) were brought to reaction in a mixture of 4 mL of dry EtOH and 3 mL of dry THF. After a few hours, a part of the solvents was evaporated under reduced pressure and the product was crystallized by cooling the mixture in a fridge. After filtration, washing with ethanol, several recrystallizations and drying in vacuo, compound 12d was obtained as a dark brown powder (96 mg, 41%), mp 219 °C. 1H NMR (300 MHz, CDCl3) δ = 9.09 (d, 3J4′,5′ = 7.8 Hz, 1H, H-4′); 7.59–7.32 (m, 6H, Ph, H-6′); 7.18 (‘t’, 3J4′,5′ = 3J5′,6′ = 7.8 Hz, 1H, H-5′); 6.85 (d, 3J6′,7′ = 7.9 Hz, 1H, H-7′); 6.41 (d, 3J1′′,2′′ = 9.2 Hz, 1H, H-1′′); 6.21 (‘t’, 3J1′′,2′′ = 3J2′′,3′′ = 9.2 Hz, 1H, H-2′′); 5.54 (‘t’, 3J3′′,4′′ = 9.6 Hz, 3J2′′,3′′ = 9.2 Hz, 1H, H-3′′); 5.43 (‘t’, 3J4′′,5′′ = 9.9 Hz, 3J3′′,4′′ = 9.6 Hz, 1H, H-4′′); 4.35 (br d, 2J6a′′,6b′′ = 12.4 Hz, 1H, H-6a′′); 4.07 (dd, 3J5′′,6b′′ = 4.2 Hz, 2J6a′′,6b′′ = 12.4 Hz, 1H, H-6b′′); 3.99–3.90 (m, 1H, H-5′′); 1.24, 1.18, 1.14, 1.02 (4s, 36H, 4C(CH3)3). 13C NMR (75.5 MHz, CDCl3) δ = 197.7 (C-2); 177.9, 177.3, 177.0, 176.3 (4 COC(CH3)3); 166.6, 165.4 (C-4, C-2′); 145.7 (C-7a′); 133.4 (i-Ph); 132.8 (C-6′); 130.6 (C-5); 129.8 (m-Ph); 129.4 (C-4′); 128.6 (p-Ph); 126.5 (o-Ph); 125.5 (C-3′); 123.8 (C-5′); 119.9 (C-3a′); 109.8 (C-7′); 82.1 (C-1′′); 75.3 (C-5′′); 72.5 (C-3′′); 67.7 (C-2′′); 67.0 (C-4′′); 61.1 (C-6′′); 38.9, 38.9, 38.8, 38.8 (4 C(CH3)3); 27.1 (2), 27.1 (2) (4C(CH3)3). MS (EI, 70 eV): m/z (%) = 836 ([M]+, 8), 531 (20), 251 (19), 211 (21), 85 (33), 57 (100). HRMS (ESI-TOF/MS): calcd for C43H53N2O11S2 ([M + H]+) 837.30853, found 837.30889. Elemental analysis: calcd for C43H52N2O11S2 (837.01): C, 61.70; H, 6.26; N, 3.35. Found: C, 61.64; H, 6.52; N, 3.49%.

(Z)-1-Phenyl-3-[4-oxo-2-(2,3,4-tri-O-pivaloyl-β-D-xylopyranosylsulfanyl)-4,5-dihydro-thiazol-5-ylidene]indolin-2-one (12e)

According to the general procedure, S-glycoside 10c (274 mg, 0.53 mmol) and N-phenylisatin (11b) (130 mg, 0.58 mmol) were brought to reaction in a mixture of 4 mL of dry EtOH and 2 mL of dry THF. The precipitated product was filtrated and washed with ethanol. After recrystallization (chloroform–ethanol) and drying in vacuo, compound 12e was obtained as red brown fine needles (299 mg, 78%), mp 288 °C. 1H NMR (300 MHz, CDCl3) δ = 9.10 (d, 3J4′,5′ = 7.8 Hz, 1H, H-4′); 7.58–7.40 (m, 5H, Ph); 7.36 (d‘t’, 3J4′,5′ = 3J5′,6′ = 7.8 Hz, 4J5′,7′ = 1.0 Hz, 1H, H-6′); 7.19 (d‘t’, 3J6′,7′ = 7.9 Hz, 3J5′,6′ = 7.8 Hz, 1H, H-5′); 6.85 (d, 3J6′,7′ = 7.9 Hz, 1H, H-7′); 6.35 (d, 3J1′′,2′′ = 9.3 Hz, 1H, H-1′′); 6.14 (‘t’, 3J2′′,3′′ = 9.4 Hz, 3J1′′,2′′ = 9.3 Hz, 1H, H-2′′); 5.55 (‘t’, 3J3′′,4′′ = 9.5 Hz, 3J2′′,3′′ = 9.4 Hz, 1H, H-3′′); 5.32–5.21 (m, 1H, H-4′′); 4.29 (dd, 2J5a′′,5b′′ = 11.3 Hz, 3J4′′,5a′′ = 5.6 Hz, 1H, H-5a′′); 3.53 (m, 1H, H-5b′′); 1.18, 1.16, 1.04 (3s, 27H, 3C(CH3)3). 13C NMR (62.9 MHz, CDCl3) δ = 197.6 (C-2); 177.3, 177.1, 176.9 (3 COC(CH3)3); 166.5, 165.7 (C-4, C-2′); 145.6 (C-7a′); 133.3 (i-Ph); 132.8 (C-6′); 130.4 (C-5); 129.7 (m-Ph); 129.5 (C-4′); 128.6 (p-Ph); 126.4 (o-Ph); 125.5 (C-3′); 123.9 (C-5′); 119.8 (C-3a′); 109.8 (C-7′); 82.8 (C-1′′); 72.1 (C-3′′); 68.4 (C-4′′); 67.6 (C-2′′); 65.8 (C-5′′); 38.8, 38.8, 38.7 (3 C(CH3)3); 27.1, 27.0 (2) (3C(CH3)3). MS (EI, 70 eV): m/z (%) = 722 ([M]+, 16), 518 (36), 417 (28), 199 (46), 85 (75), 57 (100). HRMS (ESI-TOF/MS): calcd for C37H42N2NaO9S2 ([M + Na]+) 745.22239, found 745.22205. Elemental analysis: calcd for C37H42N2O9S2 (722.87): C, 61.48; H, 5.86; N, 3.88. Found: C, 61.49; H, 6.02; N, 3.96%.

(Z)-5-Fluoro-3-[4-oxo-2-(2,3,4,6-tetra-O-pivaloyl-β-D-glucopyranosylsulfanyl)-4,5-dihydro-thiazol-5-ylidene]indolin-2-one (12f)

According to the general procedure, S-glycoside 10b (120 mg, 0.19 mmol) and 5-fluoroisatin (11c) (32 mg, 0.19 mmol) were brought to reaction in a mixture of 3 mL of dry MeOH and 1 mL of dry THF. After filtration, washing with ethanol and drying in vacuo, compound 12f was obtained as a brown powder (65 mg, 44%), mp 299–300 °C.1H NMR (500 MHz, CDCl3): δ = 8.57 (br, 1H, NH); 8.58 (dd, 3J4′,F = 9.6 Hz, 4J4′,6′ = 2.5 Hz, 1H, H-4′); 7.04 (d‘t’, 3J6′,7′ = 3J6′,F = 8.5 Hz, 4J4′,6′ = 2.5 Hz, 1H, H-6′); 6.80 (dd, 3J6′,7′ = 8.5 Hz, 4J7′,F = 4.3 Hz, 1H, H-7′); 6.40 (d, 3J1′′,2′′ = 9.3 Hz, 1H, H-1′′); 6.17 (‘t’, 3J2′′,3′′ = 9.4 Hz, 3J1′′,2′′ = 9.3 Hz, 1H, H-2′′); 5.56 (dd, 3J3′′,4′′ = 9.8 Hz, 3J2′′,3′′ = 9.4 Hz, 1H, H-3′′); 5.44 (‘t’, 3J3′′,4′′ = 3J4′′,5′′ = 9.8 Hz, 1H, H-4′′); 4.38 (dd, 2J6a′′,6b′′ = 12.6 Hz, 3J5′′,6a′′ = 1.9 Hz, 1H, H-6a′′); 4.04 (dd, 2J6a′′,6b′′ = 12.6 Hz, 3J5′′,6b′′ = 3.8 Hz, 1H, H-6b′′); 3.94 (ddd, 3J4′′,5′′ = 9.8 Hz, 3J5′′,6b′′ = 3.8 Hz, 3J5′′,6a′′ = 1.9 Hz, 1H, H-5′′); 1.26, 1.19, 1.17, 1.07 (4s, 36H, 4C(CH3)3). 13C NMR (125.8 MHz, CDCl3): δ = 197.3 (C-2); 178.5, 178.2, 177.1, 176.2 (4 COC(CH3)3); 167.9 (C-2′); 165.5 (C-4); 158.7 (d, 1JC,F = 240 Hz, C-5′); 139.5 (C-7a′); 131.2 (C-5); 125.2 (d, 4JC,F = 1.3 Hz, C-3′); 120.7 (d, 3JC,F = 11.3 Hz, C-3a′); 119.3 (d, 2JC,F = 24.6 Hz, C-6′); 116.3 (d, 2JC,F = 27.6 Hz, C-4′); 110.8 (d, 3JC,F = 8.1 Hz, C-7′); 82.0 (C-1′′); 75.2 (C-5′′); 72.3 (C-3′′); 68.2 (C-2′′); 66.8 (C-4′′); 60.8 (C-6′′); 39.0, 39.0, 38.8 (2) (4 C(CH3)3); 27.2, 27.1, 27.1, 27.1 (4C(CH3)3). 19F NMR (282.4 MHz, CDCl3): δ = −119.1. HRMS (ESI-TOF/MS): calcd for C37H47FN2NaO11S2 ([M + Na]+) 801.2498, found 801.2494. Elemental analysis: calcd for C37H47FN2O11S2 (778.90): C, 57.05; H, 6.08; N, 3.60. Found: C, 56.65; H, 6.10; N, 3.66%.

(Z)-7-Fluoro-3-[4-oxo-2-(2,3,4,6-tetra-O-pivaloyl-β-D-glucopyranosylsulfanyl)-4,5-dihydro-thiazol-5-ylidene]indolin-2-one (12g)

According to the general procedure, S-glycoside 10b (175 mg, 0.28 mmol) and 7-fluoroisatin (11d) (50 mg, 0.30 mmol) were brought to reaction in a mixture of 3 mL of dry EtOH and 1 mL of dry THF. After filtration, washing with ethanol and drying in vacuo, compound 12g was obtained as dark red fine needles (145 mg, 67%), mp 275–277 °C. 1H NMR (300 MHz, CDCl3) δ = 8.72 (dd, 3J4′,5′ = 8.0 Hz, 4J4′,6′ = 0.8 Hz, 1H, H-4′); 8.51 (br, 1H, NH); 7.12 (br t, 3J5′,6′ = 3J6′,F = 8.5 Hz, 1H, H-6′); 7.00 (m, 1H, H-5′); 6.39 (d, 3J1′′,2′′ = 9.3 Hz, 1H, H-1′′); 6.18 (‘t’, 3J1′′,2′′ = 3J2′′,3′′ = 9.3 Hz, 1H, H-2′′); 5.55 (‘t’, 3J3′′,4′′ = 9.7 Hz, 3J2′′,3′′ = 9.3 Hz, 1H, H-3′′); 5.41 (‘t’, 3J3′′,4′′ = 3J4′′,5′′ = 9.7 Hz, 1H, H-4′′); 4.35 (dd, 2J6a′′,6b′′ = 12.5 Hz, 3J5′′,6a′′ = 1.5 Hz, 1H, H-6a′′); 4.06 (dd, 2J6a′′,6b′′ = 12.5 Hz, 3J5′′,6b′′ = 4.5 Hz, 1H, H-6b′′); 3.94 (m, 1H, H-5′′); 1.23, 1.19, 1.15, 1.03 (4s, 36H, 4C(CH3)3). 13C NMR (75.5 MHz, CDCl3) δ = 197.1 (C-2); 178.0, 177.8, 177.1, 176.3 (4 COC(CH3)3); 167.3 (C-2′); 165.2 (C-4); 146.9 (d, 1JC,F = 244 Hz, C-7′); 131.8 (C-5); 130.4 (d, 2JC,F = 13.0 Hz, C-7a′); 124.9 (d, 4JC,F = 2.7 Hz, C-4′); 124.7 (d, JC,F = 4.6 Hz), 122.6 (d, JC,F = 3.7 Hz), (C-3′, C-3a′); 123.4 (d, 3JC,F = 5.6 Hz, C-5′); 119.4 (d, 2JC,F = 16.7 Hz, C-6′); 82.0 (C-1′′); 75.3 (C-5′′); 72.4 (C-3′′); 67.9 (C-2′′); 67.0 (C-4′′); 61.1 (C-6′′); 38.9 (2), 38.8, 38.8 (4 C(CH3)3); 27.1 (2), 27.1 (2) (4C(CH3)3). 19F (282 MHz, CDCl3) δ = −133.9. MS (EI, 70 eV): m/z (%) = 778 ([M]+, 5), 574 (27), 473 (33), 387 (32), 85 (33), 57 (100). HRMS (ESI-TOF/MS): calcd for C37H47FN2NaO11S2 ([M + Na]+) 801.24975, found 801.25081. Elemental analysis: calcd for C37H47FN2O11S2 (778.9): C, 57.05; H, 6.08. Found: C, 56.91; H, 6.00%.

(Z)-5-Fluoro-3-[4-oxo-2-(2,3,4-tri-O-pivaloyl-β-D-xylopyranosylsulfanyl)-4,5-dihydro-thiazol-5-ylidene]indolin-2-one (12h)

According to the general procedure, S-glycoside 10c (200 mg, 0.39 mmol) and 5-fluoroisatin (11c) (71 mg, 0.42 mmol) were brought to reaction in a mixture of 3 mL of dry EtOH and 1 mL of dry THF. After filtration, washing with ethanol and drying in vacuo, compound 12h was obtained as a dark red brown powder (232 mg, 90%), mp 268–270 °C. 1H NMR (300 MHZ, CDCl3) δ = 8.62 (dd, 3J4′,F = 9.0 Hz, 4J4′,6′ = 2.0 Hz, 1H, H-4′); 8.45 (s, 1H, NH); 7.06 (m, 1H, H-6′); 6.81 (m, 1H, H-7′); 6.33 (d, 3J1′′,2′′ = 9.0 Hz, 1H, H-1′′); 6.12 (br ‘t’, 3J2′′,3′′ = 9.4 Hz, 3J1′′,2′′ = 9.0 Hz, 1H, H-2′′); 5.57 (‘t’, 3J2′′,3′′ = 3J3′′,4′′ = 9.4 Hz, 1H, H-3′′); 5.27 (m, 1H, H-4′′); 4.28 (dd, 2J5a′′,5b′′ = 11.5 Hz, 3J4′′,5a′′ = 5.5 Hz, 1H, H-5a′′); 3.53 (m, H-5b′′); 1.18 (s, 18H), 1.06 (s, 9H), (3C(CH3)3). 13C NMR (75.5 MHz, CDCl3) δ = 197.2 (C-2); 178.4, 177.1, 177.0 (3 COC(CH3)3); 168.0 (C-2′); 165.8 (C-4); 158.8 (d, 1JC,F = 237 Hz, C-5′); 139.4 (C-7a′); 131.2 (C-5); 125.2 (d, 4JC,F = 2.1 Hz, C-3′); 120.8 (d, 3JC,F = 10.1 Hz, C-3a′); 119.4 (d, 2JC,F = 24.2 Hz, C-6′); 116.5 (d, 2JC,F = 25.8 Hz, C-4′); 110.8 (d, 3JC,F = 7.9 Hz, C-7′); 82.8 (C-1′′); 72.0 (C-3′′); 68.4 (C-4′′); 68.0 (C-2′′); 65.8 (C-5′′); 39.0, 38.8 (2) (3 C(CH3)3); 27.2, 27.1 (2) (3C(CH3)3). 19F (282 MHz, CDCl3) δ = −118.8. MS (EI, 70 eV): m/z (%) = 664 ([M]+, 6), 460 (92), 359 (62), 199 (46), 85 (85), 57 (100). HRMS (ESI-TOF/MS): calcd for C31H36FN2O9S2 ([M − H]) 663.18517, found 663.18557. Elemental analysis: calcd for C31H37FN2O9S2 (664.76): C, 56.01; H, 5.61; N, 4.21. Gefunden: C, 55.93; H, 5.56; N, 4.47%.

(Z)-7-Fluoro-3-[4-oxo-2-(2,3,4-tri-O-pivaloyl-β-D-xylopyranosylsulfanyl)-4,5-dihydro-thiazol-5-ylidene]indolin-2-one (12i)

According to the general procedure, S-glycoside 10c (161 mg, 0.31 mmol) and 7-fluoroisatin (11d) (57 mg, 0.34 mmol) were brought to reaction in a mixture of 1 mL of dry EtOH and 1 mL of dry THF. After filtration, washing with ethanol and drying in vacuo, compound 12i was obtained as dark red powder (119 mg, 58%), mp 258 °C. 1H NMR (300 MHz, CDCl3) δ = 8.74 (d, 3J4′,5′ = 7.9 Hz, 1H, H-4′); 8.40 (br, 1H, NH); 7.13 (‘t’, 3J6′,F = 9.0 Hz, 3J5′,6′ = 8.7 Hz, 1H, H-6′); 7.03 (m, 1H, H-5′); 6.33 (d, 3J1′′,2′′ = 9.3 Hz, 1H, H-1′′); 6.11 (‘t’, 3J2′′,3′′ = 9.4 Hz, 3J1′′,2′′ = 9.3 Hz, 1H, H-2′′); 5.55 (‘t’, 3J3′′,4′′ = 9.5 Hz, 3J2′′,3′′ = 9.4 Hz, 1H, H-3′′); 5.25 (m, 1H, H-4′′); 4.27 (dd, 2J5a′′,5b′′ = 11.3 Hz, 3J4′′,5a′′ = 5.6 Hz, 1H, H-5a′′); 3.53 (m, 1H, H-5b′′); 1.18, 1.16, 1.04 (3s, 27H, 3C(CH3)3). 13C NMR (75.5 MHz, CDCl3) δ = 197.0 (C-2); 177.9, 177.2, 177.0 (3 COC(CH3)3); 167.3 (C-2′); 165.6 (C-4); 146.9 (d, 1JC,F = 243 Hz, C-7′); 131.7 (C-5); 130.3 (d, 2JC,F = 13.1 Hz, C-7a′); 125.0 (br, C-4′); 124.7 (d, JC,F = 3.7 Hz), 122.5 (d, JC,F = 3.8 Hz), (C-3′, C-3a′); 123.5 (d, 3JC,F = 5.7 Hz, C-5′); 119.5 (d, 2JC,F = 16.7 Hz, C-6′); 82.8 (C-1′′); 72.1 (C-3′′); 68.4 (C-4′′); 67.9 (C-2′′); 65.8 (C-5′′); 38.9, 38.8, 38.8 (3 C(CH3)3); 27.1, 27.1, 27.0 (3C(CH3)3). 19F (282 MHz, CDCl3) δ = −134.1. MS (EI, 70 eV): m/z (%) = 664 ([M]+, 2), 460 (39), 359 (31), 85 (46), 57 (100), 41 (30). HRMS (ESI-TOF/MS): calcd for C31H37FN2NaO9S2 ([M + Na]+) 687.18167, found 687.18235. Elemental analysis: calcd for C37H37FN2O9S2 (664.76): C, 56.01; H, 5.61; S, 9.65. Found: C, 55.68; H, 5.82; S, 9.48%.

(Z)-5-Chloro-3-[4-oxo-2-(2,3,4-tri-O-pivaloyl-β-D-xylopyranosylsulfanyl)-4,5-dihydro-thiazol-5-ylidene]indolin-2-one (12j)

According to the general procedure, S-glycoside 10c (200 mg, 0.39 mmol) and 5-chloroisatin (11e) (78 mg, 0.42 mmol) were brought to reaction in a mixture of 3 mL of dry EtOH and 1.3 mL of dry THF. After filtration, washing with ethanol and drying in vacuo, compound 12j was obtained as a dark red brown powder (203 mg, 77%), mp 285–287 °C. 1H NMR (300 MHz, CDCl3) δ = 8.72 (‘s’, 2H, H-4′, NH); 7.30 (dd, 3J6′,7′ = 8.3 Hz, 4J4′6′ = 2.0 Hz, 1H, H-6′); 6.78 (d, 3J6′,7′ = 8.3 Hz, 1H, H-7′); 6.34 (d, 3J1′′,2′′ = 9.2 Hz, 1H, H-1′′); 6.13 (‘t’, 3J2′′,3′′ = 9.4 Hz, 3J1′′,2′′ = 9.2 Hz, 1H, H-2′′); 5.58 (‘t’, 3J3′′,4′′ = 9.5 Hz, 3J2′′,3′′ = 9.4 Hz, 1H, H-3′′); 5.28 (m, 1H, H-4′′); 4.28 (dd, 2J5a′′,5b′′ = 11.3 Hz, 3J4′′,5a′′ = 5.5 Hz, 1H, H-5a′′); 3.53 (m, 1H, H-5b′′); 1.19, 1.18, 1.09 (3s, 27H, 3C(CH3)3). 13C NMR (62.9 MHz, CDCl3) δ = 197.2 (C-2); 179.3, 177.1, 177.0 (3 COC(CH3)3); 167.6 (C-2′); 165.7 (C-4); 141.8 (C-7a′); 132.4 (C-6′); 131.1 (C-5); 128.8 (C-4′); 128.2 (C-5′); 124.5 (C-3′); 120.6 (C-3a′); 111.4 (C-7′); 82.7 (C-1′′); 71.9 (C-3′′); 68.3, 68.3 (C-2′′, C-4′′); 65.8 (C-5′′); 39.1, 38.8 (2) (3 C(CH3)3); 27.2 (2), 27.1 (3C(CH3)3). MS (EI, 70 eV): m/z (%) = 682 ([M]+, [37Cl], 2), 680 ([M]+, [35Cl], 5), 476 (72), 375 (40), 199 (45), 85 (80), 57 (100). HRMS (ESI-TOF/MS): calcd for C31H38ClN2O9S2 ([M + H]+, [35Cl]) 681.17018, found 681.17038; calcd for C31H38ClN2O9S2 ([M + H]+, [37Cl]) 683.16848, found 683.16888. Elemental analysis: calcd for C31H37ClN2O9S2 (681.22): C, 54.66; H, 5.47; N, 4.11. Found: C, 54.52; H, 5.61; N, 4.43%.

(Z)-7-Bromo-3-[4-oxo-2-(2,3,4,6-tetra-O-pivaloyl-β-D-glucopyranosylsulfanyl)-4,5-dihydro-thiazol-5-ylidene]indolin-2-one (12k)

According to the general procedure, S-glycoside 10b (175 mg, 0.28 mmol) and 7-bromoisatin (11f) (69 mg, 0.30 mmol) were brought to reaction in a mixture of 3 mL of dry EtOH and 1 mL of dry THF. After filtration, washing with ethanol and drying in vacuo, 12k was obtained as fine orange needles (185 mg, 80%), mp 283–286 °C. 1H NMR (300 MHz, CDCl3) δ = 8.91 (d, 3J4′,5′ = 8.0 Hz, 1H, H-4′); 8.40 (br, 1H, NH); 7.45 (br d, 3J5′,6′ = 8.2 Hz, 1H, H-6′); 6.98 (m, 1H, H-5′); 6.39 (d, 3J1′′,2′′ = 9.3 Hz, 1H, H-1′′); 6.18 (‘t’, 3J1′′,2′′ = 3J2′′,3′′ = 9.3 Hz, 1H, H-2′′); 5.54 (‘t’, 3J3′′,4′′ = 9.8 Hz, 3J2′′,3′′ = 9.3 Hz, 1H, H-3′′); 5.40 (‘t’, 3J3′′,4′′ = 3J4′′,5′′ = 9.8 Hz, 1H, H-4′′); 4.34 (dd, 2J6a′′,6b′′ = 12.5 Hz, 3J5′′,6a′′ = 1.5 Hz, 1H, H-6a′′); 4.06 (dd, 2J6a′′,6b′′ = 12.5 Hz, 3J5′′,6b′′ = 4.5 Hz, 1H, H-6b′′); 3.94 (m, 1H, H-5′′); 1.23, 1.19, 1.14, 1.03 (4s, 36H, 4C(CH3)3). 13C NMR (62.9 MHz, CDCl3) δ = 197.1 (C-2); 177.9, 177.6, 177.0, 176.3 (4 COC(CH3)3); 167.1 (C-2′); 165.2 (C-4); 142.1 (C-7a′); 135.0 (C-6′); 132.1 (C-5); 128.0 (C-4′); 125.3 (C-3′); 124.2 (C-5′); 121.5 (C-3a′); 103.3 (C-7′); 82.1 (C-1′′); 75.3 (C-5′′); 72.4 (C-3′′); 67.9 (C-2′′); 67.0 (C-4′′); 61.1 (C-6′′); 38.9 (2), 38.8, 38.8 (4 C(CH3)3); 27.1 (3), 27.1 (4C(CH3)3). MS (EI, 70 eV): m/z (%) = 840 ([M]+, [81Br], 2), 838 ([M]+, [79Br], 2) 636 (11), 535 (12), 449 (13), 85 (19), 57 (100). HRMS (ESI-TOF/MS): calcd for C37H47BrN2NaO11S2 ([M + Na]+, [79Br]) 861.16969, found 861.16948; calcd for C37H47BrN2NaO11S2 ([M + Na]+, [81Br]) 863.16824, found 863.16803. Elemental analysis: calcd for C37H47BrN2O11S2 (839.81): C, 52.92; H, 5.64; N, 3.34. Found: C, 52.83; H, 5.99; N, 3.44%.

(Z)-7-Bromo-3-[4-oxo-2-(2,3,4-tri-O-pivaloyl-β-D-xylopyranosylsulfanyl)-4,5-dihydro-thiazol-5-ylidene]indolin-2-one (12l)

According to the general procedure, S-glycoside 10c (150 mg, 0.29 mmol) and 7-bromoisatin (11f) (72 mg, 0.32 mmol) were brought to reaction in a mixture of 2 mL of dry EtOH and 1 mL of dry THF. After filtration, washing with ethanol and drying in vacuo, compound 12l was obtained as a dark red powder (183 mg, 87%), mp 290–292 °C. 1H NMR (300 MHz, CDCl3) δ = 8.91 (d, 3J4′,5′ = 8.0 Hz, 1H, H-4′); 7.45 (d, 3J5′,6′ = 8.0 Hz, 1H, H-6′); 6.98 (‘t’, 3J4′,5′ = 3J5′,6′ = 8.0 Hz, 1H, H-5′); 6.32 (d, 3J1′′,2′′ = 9.3 Hz, 1H, H-1′′); 6.11 (‘t’, 3J2′′,3′′ = 9.5 Hz, 3J1′′,2′′ = 9.3 Hz, 1H, H-2′′); 5.55 (‘t’, 3J2′′,3′′ = 3J3′′,4′′ = 9.5 Hz, 1H, H-3′′); 5.25 (m, 1H, H-4′′); 4.27 (dd, 2J5a′′,5b′′ = 11.3 Hz, 3J4′′,5a′′ = 5.6 Hz, 1H, H-5a′′); 3.52 (‘t’, 2J5a′′,5b′′ = 11.3 Hz, 3J4′′,5b′′ = 10.5 Hz, 1H, H-5b′′); 1.17, 1.15, 1.03 (3s, 27H, 3C(CH3)3); NH not observed. 13C NMR (62.9 MHz, CDCl3) δ = 197.0 (C-2); 177.7, 177.1, 177.0 (3 COC(CH3)3); 167.1, 165.6 (C-4, C-2′); 142.1 (C-7a′); 135.1 (C-6′); 132.0 (C-5); 128.1 (C-4′); 125.3 (C-3′); 124.3 (C-5′); 121.5 (C-3a′); 103.3 (C-7′); 82.8 (C-1′′); 72.0 (C-3′′); 68.4, 67.8 (C-2′′, C-4′′); 65.8 (C-5′′); 38.9, 38.8, 38.8 (3 C(CH3)3); 27.1, 27.1, 27.0 (3C(CH3)3). MS (EI, 70 eV): m/z (%) = 726 ([M]+, [81Br], 2), 724 ([M]+, [79Br], 2), 522 (37), 199 (28), 85 (73), 57 (100), 41 (22). HRMS (ESI-TOF/MS): calcd for C31H37BrN2NaO9S2 ([M + Na]+, [79Br]) 747.10161, found 747.10202; calcd for C31H37BrN2NaO9S2 ([M + Na]+, [81Br]) 749.09996, found 749.1003.

(Z)-3-[2-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosylsulfanyl)-4-oxo-4,5-dihydro-1H-imidazol-5-ylidene]indolin-2-one (12m) (Fig. 5)

According to the general procedure, S-glycoside 10d (160 mg, 0.36 mmol) and isatin (11a) (55 mg, 0.36 mmol) were brought to reaction in a mixture of 1 mL of dry THF and 3 mL of dry MeOH. After recrystallization from acetone–EtOH, compound 12m was obtained as orange needles (101 mg, 49%), mp 331–333 °C. 1H NMR (500 MHz, DMSO-d6): δ = 11.76 (s, 1H, NH); 11.11 (s, 1H, NH); 8.50 (d, 3J4′,5′ = 7.8 Hz, 1H, H-4′); 7.36 (‘t’, 3J5′,6′ = 3J6′,7′ = 7.8 Hz, 1H, H-6′); 7.06 (‘t’, 3J4′,5′ = 3J5′,6′ = 7.8 Hz, 1H, H-5′); 6.93 (d, 3J6′,7′ = 7.8 Hz, 1H, H-7′); 6.19 (d, 3J1′′,2′′ = 9.5 Hz, 1H, H-1′′); 5.84 (‘t’, 3J1′′,2′′ = 3J2′′,3′′ = 9.5 Hz, 1H, H-2′′); 5.58 (‘t’, 3J2′′,3′′ = 3J3′′,4′′ = 9.5 Hz, 1H, H-3′′); 5.02 (‘t’, 3J4′′,5′′ = 10.0 Hz, 3J3′′,4′′ = 9.5 Hz, 1H, H-4′′); 4.35 (ddd, 3J4′′,5′′ = 10.0 Hz, 3J5′′,6a′′ = 4.6 Hz, 3J5′′,6b′′ = 2.3 Hz, 1H, H-5′′); 4.16 (dd, 2J6a′′,6b′′ = 12.8 Hz, 3J5′′,6a′′ = 4.6 Hz, 1H, H-6a′′); 4.09 (dd, 2J6a′′,6b′′ = 12.8 Hz, 3J5′′,6b′′ = 2.3 Hz, 1H, H-6b′′); 2.03, 2.00, 1.98, 1.88 (4s, 12H, 4 CH3). 13C NMR (125.8 MHz, DMSO-d6): δ = 176.2 (C-2); 170.1, 169.7, 169.7, 169.5, 169.2 (4 COCH3, C-2′); 162.2 (C-4); 143.1 (C-7a′); 131.8 (C-6′); 129.9 (C-5); 126.3 (C-4′); 122.3 (C-5′); 119.9 (C-3a′); 110.7 (C-7′); 110.4 (C-3′); 80.5 (C-1′′); 73.0 (C-5′′); 72.3 (C-3′′); 67.8 (C-2′′); 67.6 (C-4′′); 61.7 (C-6′′); 20.7, 20.5, 20.4, 20.4 (4 CH3). HRMS (ESI-TOF/MS): calcd for C25H24N3O11S ([M − H]) 574.1137, found 574.1127. Elemental analysis: calcd for C25H25N3O11S (575.54): C, 52.17; H, 4.38; N, 7.30. Found: C, 52.20; H, 4.71; N, 7.06%.

Cell lines and cell cultures

Lung carcinoma (H157) cell lines (ATCC CRL-5802) were maintained in RPMI-1640 medium [containing 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 1 mM pyruvate, penicillin (100 U mL−1) and streptomycin (100 μg mL−1)] at 37 °C in a 5% CO2 incubator, horizontally in T75 cm2 sterile tissue culture flasks. While immortalized human corneal epithelial cells (HCEC) were obtained from RIKEN Bio Resource Center, Japan and cultured routinely.16 Briefly, HCEC were grown in DMEM medium [containing 5% heat-inactivated fetal bovine serum, insulin (5 μg mL−1), epidermal growth factor human (10 ng mL−1) and DMSO (0.5%)]. Under these conditions, HCEC exhibited corneal epithelial cell-specific properties. For experiments, H157 and HCEC were grown in 96-well plates by inoculating 104 and 5 × 104 cells per 100 μL per well respectively and plates were incubated at 37 °C in a 5% CO2 incubator. At this cell density, confluent monolayer was formed within 24 h, which were subsequently used for experiments.

Analysis of cytotoxicity by sulforhodamine B (SRB) assays

Cytotoxicity assays were performed using the method of Skehan et al.17 Briefly, H157 cells were cultured in 96 well plates for overnight as described above. Next day the cell monolayer was washed with Hank's Balanced Salt Solution (HBSS) to remove non adherent cells and further incubated with various concentrations of compounds (1, 25, 50 and 100 μM final concentration) for up to 24 h at 37 °C in a 5% CO2 incubator. At the end of this incubation, cells were fixed with 50% ice cold TCA buffer and incubated further for 1 h at 4 °C. The plates were washed 5 times with HBSS to remove TCA and air dried. Fixed cells were further treated with sulforhodamine B solution (0.4%) and cells were allowed to stain for 20–30 min. After this incubation cells were rinsed with 1% acetic acid and plates were allowed to dry. Plates were treated with 10 mM Tris for 5–10 min at room temperature. Absorption was measured at 565 nm. Blank background optical density is measured in wells incubated with growth medium without cells. Control values were obtained from H157 incubated in RPMI medium alone without test compounds.

Acknowledgements

Financial support by the Deutsche Krebshilfe, by the BMBF (REMEDIS Verbundprojekt) and by the DAAD is gratefully acknowledged.

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Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/c3ra44362k

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