Introduction

One of the main goals of organic and medicinal chemistry is to design and synthesize scaffolds with biological properties. Since they serve as a crucial scaffold in a variety of biologically active substances, heterocycles including nitrogen are targeted molecules in the synthetic and medicinal chemistry [1,2,3]. Benzoquinoline and its derivatives are one of the most substantial heterocyclic molecules and are found in many pharmaceuticals and natural products [4,5,6,7,8,9,10,11]. Thus, benzoquinoline compounds have a wide range of known biological characteristics and they have been utilized as templates for the synthesis of many drugs [12,13,14]. Furthermore, the biological significance of triazole, thiazolidinone, and thiazole compounds is well known due to their pharmacological effectiveness. Therefore, and in continuation of our work [15,16,17,18,19,20,21,22,23,24], it has been of significant interest to synthesize some benzo[f]quinoline bearing heterocycles through the interaction of consistent thiosemicarbazone 3 with various carbon-centered electrophiles and investigated for their antioxidant activity.

Results and discussion

Chemistry

Noteworthy, 3-chlorobenzo[f]quinoline-2-carbaldehyde (1) [25] may suffer from the nucleophilic attack at two positions: the carbonyl-carbon atom (red color) and the imino-carbon atom (blue color) (cf. Figure 1). The former attack led to the elimination of water molecule, while the later led to the removal of hydrogen chloride molecule. Thus, conduction of the aldehyde 1 with thiosemicarbazide 2 in boiling ethanol including glacial acetic acid occurred via the elimination of water molecule to provide 2-((3-chlorobenzo[f]quinolin-2-yl)methylene) hydrazine-1-carbothioamide (3) in a good yield as white crystals (Scheme 1).

Fig. 1
figure 1

Nucleophilic-attacking sides in 3-chlorobenzo[f]quinoline-2-carbaldehyde (1)

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Scheme 1
scheme 1

Synthesis of thiosemicarbazone derivative 3

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Spectroscopically, speculation of the IR spectrum of 3 lacked the carbonyl absorption but disclosed absorption bands for NH2, NH, and C=S moieties at ν 3426, 3254, 3148, and 1296 cm−1, respectively. Further, its 1H NMR spectrum offered two exchangeable singlet signals for NH and NH2 protons at δ 11.84 and 8.56 ppm, respectively.

The preferential formation of the structure of thiosemicarbazone 3 (via attack on carbonyl-carbon atom followed by elimination of water molecule) instead of attacking at imino-carbon atom may be interpreted by the charge-density calculations of the electrophilic-attacking sites which excluded the addition on imino-carbon atom and confirm the higher electron-deficiency of carbonyl group than C2-quinoline position (Figs. 2 and 3). The optimized structure, HOMO and LUMO of the aldehyde 1 are outlined in Fig. 2. Thus, it was noticed that the charge density on C=O was + 3486, and on the C2-quinoline was found + 0.3412, which excluded the addition reaction on imino-carbon atom and confirm the higher electron-deficiency of carbonyl group than C2-quinoline position, and therefore, it becomes more vulnerable to attack by the nucleophilic reagent, thiosemicarbazide (cf. Fig. 3).

Fig. 2
figure 2

Optimized structures (left), HOMO (middle), and LUMO (right) for compound 3. Atom color index: White H, Gray C, Blue N, Red O, and Green Cl

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Fig. 3
figure 3

The EHOMO of nucleophile, thiosemicarbazide 2 toward ELUMO of the starting aldehyde derivative 1

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The thiosemicarbazone 3 was utilized for the building of various heterocycles based on the benzoquinoline scaffold through reactions with some carbon electrophiles like acetic anhydride, chloroacetyl chloride, and chloroacetic acid (cf. Scheme 2). First, interaction of 3 with acetic anhydride achieved the triazolethione derivative 4 as brown powder. The IR spectrum of 4 displayed absorption bands for NH, C=O, and C=S groups. Its 1H NMR spectrum offered one exchangeable singlet signal for NH proton and two singlet signals, each of them integrated to three protons corresponding to two methyl protons. This reaction could be visualized via acetylation of the primary amino group followed by 1,5-endo-trig cyclization on C=N functionality then further acetylation (cf. Scheme 3). Second, stirring a solution of 3 with chloroacetyl chloride in dioxane under basic conditions led to the building of imidazolidinone derivative 5 as brown crystals. In the 1H NMR spectrum of 5, an exchangeable singlet signal for NH proton and singlet signal for methylene protons appeared at δ 12.56 and 4.27 ppm, respectively. This reaction was supposed to occur via tetrahedral mechanism through nucleophilic addition of NH2 of substrate 3 on the carbonyl carbon of acid chloride followed by elimination of HCl molecule (rather than attack of -SH of thiolactam form on that C=O), followed by cyclization via attack of NH on CH2Cl group (cf. Scheme 4). On the other side, cyclocondensation of 3 with chloroacetic acid in boiling dioxane containing fused sodium acetate acquired the thiazolidinone derivative 6 as yellow crystals. The IR spectrum of 6 offered NH and C=O absorptions. Also, its 1H NMR spectrum revealed NH and CH2 singlet signals at δ 12.65 and 4.71 ppm, respectively. Perhaps, this reaction took place via SN2 mechanism through nucleophilic attack of -SH (in thiolactim form) on CH2Cl group of chloroacetic acid (more electrophilic center than COOH group) followed by cyclization via attack of NH2 on the carboxylic carbon to eliminate water molecule. Probable pathways for the formation of compounds 5 and 6 are depicted in Scheme 4.

Scheme 2
scheme 2

Reactions of thiosemicarbazone 3 with acetic anhydride, chloroacetyl chloride, and chloroacetic acid

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Scheme 3
scheme 3

Suggested pathway for the formation of compound 4

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Scheme 4
scheme 4

Probable pathways for the formation of compounds 5 and 6

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It was worthy that, the interaction of thiosemicarbazone 3 with 2-bromo-1-(3-nitrophenyl)ethan-1-one (7), 2-chloro-N-phenylacetamide (9), and dimethyl but-2-ynedioate (11) was investigated (Scheme 5). Thus, the former reaction was executed in boiling dioxane including fused sodium acetate to achieve the thiazole derivative 8 as yellowish-green crystals. In turn under the same conditions, reaction of 3 with 2-chloro-N-phenylacetamide 9 afforded the triazolethione derivative 10 as faint-brown crystals. The 1H NMR spectrum of 10 revealed two exchangeable singlet signals for two NH protons and a singlet signal integrated to two protons corresponding to the methylene protons. The formation of 10 might be explained via SN2 mechanism though the nucleophilic attack of NH2 group of the substrate 3 on the CH2Cl group of the reagent 9 followed by 1,5-endo-trig cyclization on the methine carbon then dehydrogenation. Finally, boiling a methanolic solution of thiosemicarbazone 3 with dimethyl but-2-ynedioate (11) provided the thiazolidinone derivative 12 as yellow crystals. The 1H NMR spectrum of 12 lacked NH2 singlet signal but displayed one exchangeable singlet signal for NH proton and singlet signal integrated to three protons corresponding to methyl protons. The mass spectra of the synthesized compounds offered the correct molecular ion peaks as well as M + 2 peaks of the counter isotope (37Cl). A suggested pathway for the formation of thiazolidinone derivative 12 could be depicted in Scheme 6.

Scheme 5
scheme 5

Synthesis of thiazole, triazole, and thiazolidinone derivatives 8, 10, and 12

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Scheme 6
scheme 6

A probable pathway for the formation of compound 12

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Finally, hydrazinolysis of compound 3 did not proceed as expected to afford the hydrazinotriazole 13 but achieved a mixture of the bisbenzylisoquinoline azine 14 and thiosemicarbazide 2 which was separated by fractional recrystallization (Scheme 7). The chemical structure of 14 was strongly substantiated from the study of the IR and 1H NMR spectra together with chemical evidence by direct comparison with a sample prepared from reaction of the aldehyde 1 with hydrazine hydrate in refluxing ethanol (identity melting point, mixed melting point, & TLC). Probably, the formation of azine derivative 14 could be viewed via Scheme 8. The reaction time and %yields are summarized in Table 1.

Scheme 7
scheme 7

Hydrazinolysis of thiosemicarbazone 3

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Scheme 8
scheme 8

A suggested pathway for the formation of azine derivative 14

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Table 1 Reaction time and %yields of the compounds obtained
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Antioxidant activity

Antioxidants are compounds which can prevent or inhibit the oxidation of materials that can be oxidized by scavenging free radicals and assist in diminishing oxidative stress. The antioxidant activity is more fascinating since the high levels of free radicals can harm cell membranes and tissues, likewise DNA, proteins, enzymes, and lipids. To hinder free radical damage, cell antioxidants should be neutralized. The synthesized compounds were examined for antioxidant activity according to phosphomolybdenum method using ascorbic acid as standard [26]. The antioxidant activity of the sample was conveyed as the number of ascorbic acid equivalents (AAE). The results are shown in Table 2, and the values have ranged from 157.01 to 378.03 mg AAE/g sample.

Table 2 Total antioxidant capacity (TAC) of the investigated compounds
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The results displayed that most compounds were discovered to be effective. Also, two compounds 8 and 10 displayed the highest potent levels of activity. The higher potency of thiazole derivative 8 could be attributable to the existence of tautomeric structures and the nitrophenyl group. Likewise, the more potency of triazolethione derivative 10 might be attributed to the more tautomeric structures, hydrogen bonding, additional phenyl group, and extended conjugation (cf. Fig. 4). Three compounds 3, 5, and 6 revealed exceptional activity. In turn, moderate antioxidant activity was shown by compounds 4 and 12. This is coherent with the literature that reported good antioxidant activities by the thiazole and triazolethione derivatives [22].

Fig. 4
figure 4

SAR of the most potent compounds

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Conclusion

In summary, various benzoquinoline-based heterocycles like triazole, imidazolidine, thiazolidine, and thiazole derivatives, have been synthesized employing the 3-chlorobenzo[f]quinoline-2-carbaldehyde thiosemicarbazone as a key material. The behavior of thiosemicarbazone derivative was investigated toward 2-bromo-1-(3-nitrophenyl)ethan-1-one, N-phenyl-2-chloroacetamide, and dimethyl acetylenedicarboxylate. Finally, the hydrazinolysis of thiosemicarbazone was investigated. The antioxidant activity of the synthesized compounds revealed that thiazole and triazolethione derivatives were the most potent.

Experimental

General

Melting points (uncorrected) were measured in open capillary tubes on a MEL-TEMP II electrothermal melting point apparatus. The elemental analyses were performed on a Perkin-Elmer 2400 CHN elemental analyzer (Perkin-Elmer, Waltham, MA) at Faculty of Science, Ain Shams University. Infrared spectra (ν, cm−1) were recorded utilizing KBr wafer technique on Fourier-Transform Infrared Thermo Electron Nicolet iS10 Spectrometer (Thermo Fisher Scientific Inc. Waltham, MA) at Faculty of Science, and Faculty of Pharmacy, Ain Shams University. Mass spectra were carried out on direct probe controller inlet part to single quadrupole mass analyzer in (Thermo Scientific GC–MS) MODEL (ISQ LT) using Thermo X-CALIBUR software at regional center for mycology and biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. 1H NMR spectra (δ, ppm) were performed on BRUKER 400 MHz Spectrometer at Faculty of Pharmacy, Cairo University, with tetramethyl silane as an internal standard, using DMSO-d6 as a solvent. Thin-layer chromatography (TLC) was run utilizing TLC aluminum sheets silica gel F254.

2-((3-Chlorobenzo[f]quinolin-2-yl)methylene)hydrazine-1-carbothioamide (3)

A solution of the 3-chlorobenzo[f]quinoline-2-carbaldehyde (1) [25] (2.416 g, 0.01 mol) and thiosemicarbazide 2 (0.91 g, 0.01 mol) in absolute ethanol (20 mL) including glacial acetic acid (0.1 mL) was refluxed for 3 h. The solid deposited while hot was collected and recrystallized from ethanol/dioxane mixture (2:1) to produce white crystals, mp. 283–285 °C, yield 80%. IR (ν, cm−1): 3426, 3254, 3148 (NH,NH2), 1593 (C=N), 1296 (C=S). 1H NMR (400 MHz, δ, ppm): 11.84 (br.s, 1H, NH, exchangeable), 9.36 (s, 1H, CH=N), 8.99 (d, 1H, Ar–H, J = 7.6 Hz), 8.56 (br.s, 2H, NH2, exchangeable), 8.30 (s, 1H, C-H quinoline), 8.08 (d, 1H, Ar–H, J = 8.8 Hz), 8.03 (d, 1H, Ar–H, J = 8.8 Hz), 7.87–7.79 (m, 3H, Ar–H). EIMS (m/z, %): 316.86 (M + 2, 35), 314.61 (M+., 94), 298.83 (67), 296.12 (71), 294.11 (51), 279.66 (50), 257.21 (44), 228.75 (40), 208.30 (63), 190.01 (46), 180.83 (42), 168.86 (66), 167.22 (100), 156.66 (40), 132.44 (45.79), 117.70 (26.02), 99.18 (43), 87.13 (51), 80.49 (38). Anal. Calcd. for C15H11ClN4S (314.79): C, 57.23; H, 3.52; N, 17.80; Found: C, 57.17; H, 3.47; N, 17.84%.

1,1′-(5-(3-Chlorobenzo[f]quinolin-2-yl)-3-thioxo-1,2,4-triazolidine-1,4-diyl)bis(ethan-1-one) (4)

A suspension of thiosemicarbazone 3 (3.147 g, 0.01 mol) in acetic anhydride (15 mL) was refluxed for 6 h with stirring. The precipitate formed was collected and crystallized from ethanol to furnish brown powder, mp. 302–304 °C, yield 71%. IR (ν, cm−1): 3195 (NH), 1668 (C=O), 1622 (C=N), 1286 (C=S). 1H NMR (δ, ppm): 11.88 (br.s, 1H, NH, exchangeable), 9.00 (d, 1H, Ar–H, J = 8.4 Hz), 8.17 (s, 1H, C-H quinoline), 8.09–7.80 (m, 5H, Ar–H), 7.07 (s, 1H, CH triazole), 2.37 (s, 3H, CH3), 2.03 (s, 3H, CH3). EIMS (m/z, %): 400.40 (M + 2, 24), 398.31 (M+., 65), 368.58 (42), 356.57 (42), 297.27 (18), 268.25 (50), 224.83 (39), 202.04 (37), 186.37 (91), 182.34 (100), 168.04 (51), 138.45 (82), 109.04 (41), 102.64 (23), 80.88 (72), 78.67 (27). Anal. Calcd. for C19H15ClN4O2S (398.87): C, 57.21; H, 3.79; N, 14.05; Found: C, 57.12; H, 3.70; N, 14.00%.

1-(((3-Chlorobenzo[f]quinolin-2-yl)methylene)amino)-2-thioxoimidazolidin-4-one (5)

Chloroacetyl chloride (1.42 g, 0.01 mol) was added dropwise to a stirred solution of thiosemicarbazone 3 (3.147 g, 0.01 mol) dioxane (20 mL) including triethylamine (0.1 mL) at ambient temperature and the reaction mixture was further stirred for 5 h. The precipitate formed was filtered off and crystallized from ethanol to produce brown crystals, mp. 288–290 °C, yield 75%. IR (ν, cm−1): 3263 (NH), 1736 (C=O), 1625 (C=N), 1294 (C=S). 1H NMR (δ, ppm): 12.56 (br.s, 1H, NH, exchangeable), 9.09 (s, 1H, CH=N), 9.01 (s, 1H, C-H quinoline), 8.87 (d, 1H, Ar–H, J = 8.4 Hz), 8.76 (d, 1H, Ar–H, J = 8.3 Hz), 8.34–7.67 (m, 4H, Ar–H), 4.27 (s, 2H, CH2). EIMS (m/z, %): 356.70 (M + 2, 12), 355.94 (29), 354.19 (M+., 29), 345.78 (79), 344.80 (96), 318.43 (57), 297.20 (96), 280.04 (59), 245.70 (64), 225.98 (44), 207.85 (38), 183.01 (74), 162.17 (28), 152.62 (35), 138.85 (68), 113.56 (43), 97.48 (41), 96.81 (32), 80.48 (38), 41.39 (100). Anal. Calcd. for C17H11ClN4OS (354.81): C, 57.55; H, 3.13; N, 15.79; Found: C, 57.49; H, 3.09; N, 15.76%.

2-(((3-Chlorobenzo[f]quinolin-2-yl)methylene)hydrazono)thiazolidin-4-one (6)

A solution of chloroacetic acid (0.95 g, 0.01 mol) and thiosemicarbazone 3 (3.147 g, 0.01 mol) dioxane (20 mL) including anhydrous sodium acetate (0.82 g, 0.01 mol) was refluxed for 6 h. The formed precipitate was filtered off and crystallized from ethanol to obtain yellow crystals, mp. 288–290 °C, yield 70%. IR (ν, cm−1): 3256 (NH), 1738 (C=O), 1620 (C=N). 1H NMR (δ, ppm): 12.65 (br.s, 1H, NH, exchangeable), 9.78 (s, 1H, CH=N), 9.66 (s, 1H, C–H quinoline), 8.88–8.60 (m, 6H, Ar–H), 4.71 (s, 2H, CH2). EIMS (m/z, %): 356.70 (M + 2, 16), 355.36 (10), 354.33 (M+., 41), 334.19 (69), 298.91 (33), 296.44 (48), 268.81 (52), 234.21 (87), 221.38 (80), 209.28 (67), 194.59 (100), 162.82 (25), 118.74 (56), 99.38 (38), 84.18 (35), 71.03 (35), 67.84 (69), 49.29 (66), 48.26 (88). Anal. Calcd. for C17H11ClN4OS (354.81): C, 57.55; H, 3.13; N, 15.79; Found: C, 57.48; H, 3.08; N, 15.81%.

2-(2-((3-Chlorobenzo[f]quinolin-2-yl)methylene)hydrazinyl)-4-(3-nitrophenyl)thiazole (8)

A mixture of 2-bromo-1-(3-nitrophenyl)ethan-1-one (7) (2.42 g, 0.01 mol) and thiosemicarbazone 3 (3.147 g, 0.01 mol) in dioxane (15 mL) including anhydrous sodium acetate (0.82 g, 0.01 mol) was refluxed for 5 h. The solid obtained was collected and crystallized from ethanol to furnish yellowish-green crystals, mp. 278–280 °C, yield 72%. IR (ν, cm−1): 3299 (NH), 1530, 1345 (NO2). 1H NMR (δ, ppm): 12.68 (br.s, 1H, NH, exchangeable), 8.93 (d, 1H, Ar–H, J = 8.5 Hz), 8.78 (s, 1H, C-H quinoline), 8.63 (s, 1H, CH=N), 8.46 (s, 1H, CH nitrophenyl), 8.27 (d, 1H, Ar–H, J = 7.6 Hz), 8.10–8.03 (m, 2H, Ar–H), 7.99 (s, 1H, CH thiazole), 7.78–7.65 (m, 5H, Ar–H). EIMS (m/z, %): 461.37 (M + 2, 15), 460.56 (35), 459.96 (M+., 59), 400.36 (25), 382.32 (30), 379.69 (92), 334.25 (100), 283.80 (51), 225.55 (37), 193.69 (51), 177.48 (89), 147.70 (40), 137.96 (27), 100.93 (19), 69.65 (33). Anal. Calcd. for C23H14ClN5O2S (459.91): C, 60.07; H, 3.07; N, 15.23; Found: C, 59.08; H, 2.97; N, 15.20%.

2-(3-(3-Chlorobenzo[f]quinolin-2-yl)-5-thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl)-N-phenylacetamide (10)

A mixture of 2-chloro-N-phenylacetamide 9 (1.70 g, 0.01 mol) and thiosemicarbazone 3 (3.147 g, 0.01 mol) in dioxane (15 mL) including sodium acetate (0.82 g, 0.01 mol) was refluxed for 6 h. The solid deposited was collected and crystallized from ethanol/dioxane (2:1) to produce faint-brown crystals, mp. 274–276 °C, yield 60%. IR (ν, cm−1): 3255 (NH), 1741 (C=O), 1629 (C=N), 1318 (C=S). 1H NMR (δ, ppm): 12.15 (br.s, 1H, NHCS, exchangeable), 11.85 (br.s, 1H, NHCO, exchangeable), 9.37 (s, 1H, CH quinoline), 9.01 (d, 1H, Ar–H, J = 8.8 Hz), 8.91–7.82 (m, 10H, Ar–H), 3.95 (s, 2H, CH2). EIMS (m/z, %): 447.32 (M + 2, 28), 445.74 (M+., 76), 443.18 (36), 420.46 (48), 412.07 (65), 384.77 (74), 353.87 (47), 335.00 (50), 327.66 (70), 315.01 (73), 299.44 (100), 246.99 (40), 244.20 (52), 210.38 (50), 165.49 (58), 142.55 (52), 141.07 (97), 121.71 (39), 98.05 (26), 84.22 (60). Anal. Calcd. for C23H16ClN5OS (445.93): C, 61.95; H, 3.62; N, 15.71; Found: C, 61.82; H, 3.54; N, 15.74%.

Methyl 2-(2-(((3-chlorobenzo[f]quinolin-2-yl)methylene)hydrazono)-4-oxothiazolidin-5-ylidene)acetate (12)

Dimethyl but-2-ynedioate (11) (1.13 g, 0.01 mol) was added to a solution of thiosemicarbazone 3 (3.147 g, 0.01 mol) in methanol (20 mL), and the reaction mixture was refluxed for 5 h. The precipitated solid was collected and crystallized from methanol to produce yellow crystals, mp. 270–272 °C, yield 80%. IR (ν, cm−1): 3250 (NH), 1724 (C=O ester), 1701 (C=O thiazolidinone), 1642 (C=N). 1H NMR (δ, ppm): 13.06 (br.s, 1H, NH, exchangeable), 9.05 (s, 1H, CH=N), 9.03 (d, 1H, Ar–H, J = 8.5 Hz), 8.88 (s, 1H, C-H quinoline), 8.17–7.81 (m, 5H, Ar–H), 6.73 (s, 1H, CH =), 3.82 (s, 3H, -OCH3). EIMS (m/z, %): 426.39 (M + 2, 14), 424.81 (M+., 36), 421.78 (48), 398.75 (49), 374.36 (69), 371.94 (100), 334.38 (43), 301.04 (35), 298.91 (61), 296.29 (45), 253.90 (61), 212.70 (93), 161.47 (66), 142.37 (54), 114.37 (26), 108.14 (37), 87.18 (46). Anal. Calcd. for C20H13ClN4O3S (424.86): C, 56.54; H, 3.08; N, 13.19; Found: C, 56.40; H, 2.99; N, 13.15%.

Hydrazinolysis of thiosemicarbazone 3

A solution of thiosemicarbazone derivative 3 or the aldehyde 1 (0.01 mol) and hydrazine hydrate (0.01 mol, 80%) in absolute ethanol (20 mL) was refluxed for 4 h. The reaction mixture was concentrated and then left to cool at room temperature. In the case of using the reactant 3, the obtained colorless crystals were collected by filtration and found to be thiosemicarbazide [mp, mixed mp, TLC, & IR]. The residual part was recrystallized from ethanol and realized to be azine derivative 14. In the case of using the reactant 1, the solid obtained was collected and found to be the azine derivative 14, which was identical in all respects (IR, mp, mixed mp., & TLC).

2-Bis((3-chlorobenzo[f]quinolin-2-yl)methylene)hydrazine (14)

Yellow crystals, mp. > 300 °C, Yield: 78%. IR (ν, cm−1): 1633 (C=N). 1H NMR (δ, ppm): 8.94 (d, 2H, Ar–H, J = Hz), 8.71 (s, 2H, 2CH quinoline), 8.10 (s, 2H, 2CH=N), 8.06–7.93 (m, 6H, Ar–H), 7.77–7.59 (m, 4H, Ar–H). EIMS (m/z, %): 483.24 (15), 481.25 (M + 2, 7), 479.23 (M+., 20). EIMS, m/z (%): 482.93 (M + 4, 17), 480.30 (M + 2, 9), 478.01 (M+., 26), 453.97 (66), 449.45 (70), 448.53 (50), 414.43 (90), 387.83 (76), 341.29 (70), 320.91 (77), 278.37 (100), 224.01 (88), 199.00 (82), 163.81 (81), 154.66 (74), 114.08 (74), 100.87 (80), 81.73 (60), 78.52 (76). Anal. Calcd. for C28H16Cl2N4 (479.36): C, 70.16; H, 3.36; N, 11.69; Found: C, 70.07; H, 3.28; N, 11.64%.

Determination of total antioxidant capacity (TAC)

The antioxidant activity of each compound was recorded according to phosphomolybdenum method using ascorbic acid as standard [26]. This assay was based on the reduction of Mo (VI) to Mo (V) by the sample analyte and subsequent formation of a green colored [phosphate = Mo (V)] complex at acidic pH with a maximal absorption at 695 nm. In this method, 0.5 ml of each compound (500 µg/ml) in methanol was combined in dried vials with 5 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The vials containing the reaction mixture were capped and incubated in a thermal block at 95 °C for 90 min. After cooling the samples to room temperature, the absorbance was measured at 695 nm against a blank. The blank consisted of all reagents and solvents without the sample, and it was incubated under the same conditions. All experiments were carried out in triplicate. The antioxidant activity of the sample was expressed as the number of ascorbic acid equivalent (AAE).

Statistical analysis

All data were presented as mean ± SD using SPSS 13.0 program (SPSS Inc. USA).