Antioxidant and Antitumor Activities of New Synthesized Aromatic C-Nucleoside Derivatives

The carbohydrazide 1 was used as the precursor for the synthesis of a number of new aromatic C-nucleosides containing 1,3,4-oxadiazole 7, [1,3,4]oxadiazolo[2,3-a]isoindole 10b and pyrazole units 18. On the other hand, the thiosemicarbazone 20 was used as the key intermediate for synthesis of 1,3,4-oxadiazole and 1,2,4-triazole-3-thione derivatives 21 and 23. The antioxidant activities of the prepared compounds were evaluated. The carbohydrazide 1 in particular was found to have potent antioxidant and antitumor activity.


Introduction
A number of nucleoside analogues have been found to show a broad spectrum of biological effects such as antifungal [1,2], antibacterial [1][2][3], antitumor [3][4][5], antiviral [3,4,[6][7][8][9][10][11][12][13][14] anti-inflammatory [15] and analgesic [15] activities. Moreover, 2'-deoxy-2'-fluoro-2'-C-methyl nucleoside analogues have showed promising activity against HCV replication [16]. In addition, nucleoside derivatives display inhibition of OPEN ACCESS respectively. The mass spectrum showed the expected molecular ion peak in agreement with its structure.    Condensative cyclization of carbohydrazone 3a with acetic anhydride in the presence of anhydrous sodium acetate gave a cyclized product that according to physical and chemical studies, could not be reconciled with the structure of 10a, that but rather was compatible with that of [1,3,4]oxadiazolo-[2,3-a]isoindole 10b. The infrared spectrum of this compound showed the disappearance of the carboxylic acid hydroxyl group, sugar hydroxyl groups, and CONH absorption bands. It showed instead an acetoxyl (OAc) group at γ 1,744 cm −1 and carbonyl groups at γ 1,725 and 1,712 cm −1 . Its 1 H-NMR spectrum (CDCl 3 ) showed the disappearance of signals corresponding to the sugar protons at the δ 3.00-5.00 ppm region, and only displayed the aromatic protons as a doublet at δ 7.84 (J = 7.65 Hz) for Ar-H (a) , a triplet at δ 7.65 (J = 7.65 Hz) for Ar-H (b) , a triplet at δ 7.56 (J = 7.65 Hz) for Ar-H (c) , and a doublet at δ 7.53 (J = 7.65 Hz) for Ar-H (d) , followed by a singlet attributed to CH (furan) at δ 7.00 ppm. Three singlet signals that appeared in the upper field region at δ 2.56, 2.27 and 2.14 ppm were attributed to the CH 3(furan) , COCH 3 and O-acetyl protons, respectively. It is noteworthy that the integration of the OAc protons (δ 2.14 ppm), indicated only one O-acetyl group, in accord with structure 10b. Moreover, the proposed mechanism for formation of 10b may proceed as illustrated in Schemes 3 and 4.
In addition, condensation of anhydro derivative 11 [2] with p-nitrobenzaldehyde, indoline-2,3-dione (isatin) and D-galactose in acidic medium afforded the corresponding aromatic C-nucleosides 12-14, respectively. Compounds 12 and 13 were also obtained by acid-catalyzed dehydrative cyclization of 3b and 3e, respectively. Their structures were deduced from the respective spectral data. The signals of the sugar protons of anhydro structures 12 and 13 were assigned from the characteristic chemical shifts as compared with those reported for diol derivatives [2]. Although the coupling constant value (J 1',2' = 6.85 Hz) of 12 cannot define the anomeric configuration [21], however, it could be β-in accordance with the configuration its precursor [2]. On the other hand, the anomeric configuration of 13 can be ascertained from the large observed coupling constant value (J 1',2' = 9.00 Hz) which indicates a trans arrangement of the base moiety and the 2'-hydroxyl group, i.e., β-D-configuration.
Furthermore, acetylation of 12 and 13, afforded the acetylated structures 15 and 16, in 79% and 67% yields, respectively. The infrared spectra showed OAc absorption bands at γ 1,753, 1,745 cm −1 , respectively. The 1 H-NMR spectra (CDCl 3 ) of these products revealed two singlet signals at δ 2.00-2.11 ppm attributed to two O-acetyl groups. Their mass spectra showed the expected molecular ion peaks in agreement with their proposed structures. Scheme 3. Synthesis of [1,3,4] The isopropylidene derivative 17 has been prepared from 13 in yield 88%. Its anomeric configuration was confirmed from the zero coupling constant value (J 1',2' = 0.00 Hz), as a β-D-configuration [2,21,22]. The mass spectrum showed the expected molecular ion peak in agreement with its structure (Scheme 5).
The pyrazole derivative 18 was obtained in 100% yield from the reaction of carbohydrazide 1 with pentane-2,4-dione as previously reported on other systems [23]. The infrared spectrum showed the disappearance of absorption bands corresponding to NH and NH 2 . Its 1 H-NMR spectrum (DMSO-d 6 ), revealed three singlets at δ 6.18, 2.48 and 2.16 ppm for CH (pyrazole) , CH 3(pyrazole-a) and CH 3(pyrazole-b) protons, respectively. The molecular ion peak recorded in the mass spectrum was in accordance with its molecular weight. Furthermore, the O-acetyl derivative 19 was prepared, in which the signals of the sugar protons of this product were assigned from its 2D 1 H-NMR spectrum (Scheme 6).
Moreover, condensation of 1 with phenyl isothiocyanate gave the corresponding thiosemicarbazide derivative 20 [24]. Intramolecular cyclization of this thiosemicarbazide using an improved procedure involving treatment with potassium iodide and iodine in the presence of sodium hydroxide [25] resulted in 1,3,4-oxadiazole product 21 [24] in 95% yield. The tetra-O-acetyl derivative 22 was obtained in 85% yield, the signals of the sugar protons of this product were assigned from its 2D 1 H-NMR spectrum, the mass spectrum showed the molecular ion peak at m/z 529 (M + , 19.12%), and 13 C-NMR (CDCl 3 ) spectrum confirmed the structure (Scheme 7).

Scheme 5.
Synthesis of aromatic C-nucleosides 12-17. 4  Alternatively, heating the thiosemicarbazide 20 with aqueous sodium hydroxide (10%) [25] gave a product 23, whose infrared spectrum showed a C=N absorption at γ 1,624 cm −1 with the disappearance of the CONH absorption. Moreover, acetylation of 23, gave 5-(5-(1',2',3',4'-tetraacetoxybutyl)-2-methylfuran-3-yl)-4-phenyl-2-N-acetyl-1,2,4-triazole-3(4H)-thione (24) in 97% yield. The 1 H-NMR spectrum (CDCl 3 ) revealed the disappearance of the NH proton and showed a singlet due to N-acetyl protons at δ 2.77 ppm, followed by three singlets at δ 2.01, 1.99, and 1.97 ppm for four O-acetyl groups. The mass spectrum showed a molecular ion peak in accordance with its molecular formula (Scheme 8).    The diphenylpicrylhydrazyl (DPPH) assay method is based on the reduction of the free radical DPPH with an odd electron which gives a maximum absorption at 517 nm. When antioxidants react with DPPH, giving DPPD-H the absorbance decreases due to decolorization with respect to the number of electrons captured. EC 50 values for each examined compound as well as standard preparations were calculated according to the method Shahwar et al. [26]. A lower EC 50 value is associated with a higher radical scavenging activity. As shown in Tables 1 and 2 Table 2.       The obtained data revealed a potential antioxidant activity of all examined compounds but with different EC 50 values compared to the standard, especially compound 20 which has a distinct thiourea group. In addition, compounds 3c and 3d revealed higher antioxidant activities as compared to the standard due to the acidic protons in the pyrrole and indole, respectively, that can be easily oxidized.

Anticancer Activity Screening (Cytotoxicity Against Three Cancer Cell Lines)
Different concentrations (50-1.56 µg/mL) of the examined compound 1 were used to screen their cytotoxicity against Human Breast Adrenocarcinoma Cells (MCF-7), Human Colon Carcinoma Cells (HCT) and Human Hepatocellular Liver Carcinoma Cells (HepG-2). Cytotoxic effects of these compounds on the cell viability of the cancer cell lines were observed, as shown in Tables 3 and 4 and Figures 7-9. The obtained data revealed that the carbohydrazide 1 has excellent cell growth inhibitory effects on HepG-2, HCT and MCF-7 with IC 50 s equal to 10.200, 8.400 and 11.700 µg, respectively compared to the IC 50 of the doxorubicin (1.200, 0.469) and vinblastine (6.100) standards used, see Table 5.

General Procedures
Melting points were determined with a Melt-temperature apparatus and are uncorrected. TLC was performed on Baker-Flex silica gel 1B-F plates and the spots were detected by UV light absorption. IR spectra were recorded on a Perkin Elmer spectrometer. 1 H-NMR and 13 C-NMR were recorded on JEOL JNM ECA 500 MHz and 300 MHz instruments using tetramethylsilane as an internal standard. Mass spectra were recorded on a GCMS DI Analysis Shimadzu Qp-2010 Plus. Solutions were evaporated under diminished pressure unless otherwise stated. ChemDraw-Ultra-8.0 has been used in generating the names of the prepared compounds.

General Procedures for the Preparation of the Aromatic C-Nucleosides 12-14
Method A. A solution of compounds 3b and 3e (2.544 mmoL) was heated with aqueous acetic acid (150 mL, 10%) under reflux for 5 h. After cooling the 3-carbohydrazones 12, 13 that separated out were filtered off, washed with water and dried.

Cytotoxicity Evaluation Using Viability Assay
For the cytotoxicity assays, cells were seeded in 96-well plate at a cell concentration of 1 × 10 4 cell per well in 100 μL of growth medium. Fresh medium containing different concentrations of the test sample was added after 24 h of seeding. The microtiter plates were incubated at 37 °C in a humidified incubator with 5% CO 2 for a period of 48 h. Three wells were used for each concentration of the tested sample. Control cells were incubated without test sample and with or without DMSO. After incubation of the cells for 24 h at 37 °C, various concentrations of the sample (50.000, 25.000, 12.500, 6.250, 3.125 and 1.560 μg) were added each separately. The incubation was continued for 48 h and viable cells yield was determined colorimetrically using 3,4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTTB). The water insoluble tetrazolium salt is converted to purple formazan by the mitochondrial dehydrogenase of viable cells. After the end of incubation period, media were aspirated and crystal violet solution (1%) was added to each well for at least 30 min. The stain was removed and plates were rinsed using tap water until all excess stain is removed. Glacial acetic acid (30%) was then added to all wells and mixed thoroughly, then the absorbance of the plates were measured after gently shaken on Microplate Reader (Tecan, Inc., city, country), at 490 nm. All results were corrected for background absorbance detected in wells without added stain. Treated sample was compared with the cell control in the absence of the tested compound. All experiments were carried out in the triplicate. The cell cytotoxic effect of the tested compound was calculated [27,28].

Conclusions
Some new aromatic C-nucleosides have been prepared from carbohydrate precursors. Their physical and chemical properties were studied, and some of the compounds showed potential antioxidant activities. One of these compounds has been screened for its antitumor activity.