Green synthesis and biological evaluation of steroidal 2H-pyrans as anticancer and antioxidant agents

2.1 Materials and methods

All chemicals were purchased from Sigma–Aldrich (India) and Merck (India) as analytical grade. Chitosan was supplied by Sigma–Aldrich (India) as practicle grade. The solvents were purified prior to use. Melting points were determined on a Kofler apparatus in degree Celsius. The IR spectra were recorded on KBr pellets with Interspec 2020 FT-IR Spectrometer SpectroLab and values are given in cm⁻¹. ¹H and ¹³C NMR spectra were run in CDCl₃ on a JEOL Eclipse (400 MHz) instrument with TMS as internal standard and values are given in ppm (δ). Elemental analyses of all the new compounds were recorded on a Perkin Elmer 2400 CHN Elemental Analyzer. Mass spectra were recorded on a JEOL SX 102/DA- 6000 Mass Spectrometer. Thin layer chromatography (TLC) plates were coated with silica gel G and exposed to iodine vapors to check the homogeneity as well as the progress of reaction. Sodium sulfate (anhydrous) was used as a drying agent.

2.2 General method for the synthesis of steroidal 2H-pyran derivatives (4–6)

A mixture of steroidal α, β-unsaturated ketone 1, 2 or 3 (1 mmol) and ethyl acetoacetate (1 mmol) in 30 mL methanol was refluxed for 13–16 h in the presence of chitosan (20 mol%). The progress of the reaction was monitored by TLC. After completion of the reaction, chitosan was removed by filtration and the filtrate was taken in ether, washed with water, dried over anhydrous sodium sulfate and concentrated in vacuo giving a residue which was recrystallized from ethanol affording products 4, 5 or 6.

2.3 In vitro anticancer activity

2.4 Antioxidant activity

Steroidal 2H-pyran derivatives 4–6 were tested for their antioxidant property by 1,1-diphenylpicrylhydrazyl (DPPH) method (Lone et al., 2013). Drug stock solution (1 mg/mL) was diluted to final concentration of 2, 4, 6, 8, 10 and 12 mg/mL in methanol. Methanolic DPPH solution (1 mL, 0.3 mmol) was added to 3.0 mL of drug solution of different concentrations. The tube was kept at an ambient temperature for 30 min and the absorbance was measured at 517 nm. The scavenging activity was calculated by the following formula: $$[\%\text{inhibition}=[(A_{\text{Control}}-A_{\text{Sample}}/A_{\text{Control}}\times 100]$$ where A_Control is the absorbance of the l-ascorbic acid (Standard) and A_Sample is the absorbance of different compounds. The methanolic DPPH solution (1 mL, 0.3 mM) was used as control.

3.1 Chemistry

The synthesis of steroidal 2H-pyran derivatives 4–6 involves the use of 3β-acetoxy-cholest-5-en-7-one (1), 3β-chloro-cholest-5-en-7-one (2) and cholest-5-en-7-one (3), as starting materials which have been synthesized by the literature method (Dauben and Takemura, 1953). The present strategy of synthesizing compounds 4–6 by green method involves the reaction of steroidal α, β-unsaturated ketones 1–3 with ethyl acetoacetate in the presence of chitosan as an eco-friendly heterogeneous catalyst. The catalytic system is influenced by various reaction parameters, such as amount of the catalyst employed, effect of catalyst and solvent system. Therefore, in our initial investigation to establish the optimum reaction conditions 3β-acetoxy-cholest-5-en-7-one (1) and ethyl acetoacetate in methanol were selected as model substrates. We have applied a wide range of basic catalysts such as piperidine, NaOMe, pyridine, triethylamine and chitosan to improve the yield for the specific synthesis of derivatives.

As shown in Table 1, chitosan is superior to other catalysts in terms of time and yield. We then tried to screen the reaction in various organic solvents in order to optimize the solvent using chitosan as catalyst. It is noteworthy to mention that the polar solvents (methanol and ethanol) afforded better yield than the non-polar ones and the best result was obtained in methanol as we can see in Table 2.

We then tried to optimize the catalyst load for the formation of steroidal 2H-pyrans and we found that 20 mol% of the catalyst was sufficient to get optimum yields in less reaction time. Use of more than 20 mol% of the catalyst does not have any profound effect on the reaction rate as well as the yield. This result may be attributed to the coagulation of the catalyst which decreased the effective surface area of the catalyst (Bhattacharyya et al., 2012). Our study revealed that even after five cycles, the catalyst was able to carry out the reaction offering almost the same catalytic activity. In order to investigate the scope of this reaction, a variety of three steroidal compounds were subjected to this reaction and all the reactions proceeded smoothly and the reaction was completed within 13–16 h to afford the products 4–6 in good yields (70–73%). (Scheme 1).

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Scheme 1

Showing the formation of steroidal 2H-pyrans 4–6.

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The spectral studies of products were found in good agreement with the proposed structures for steroidal 2H-pyrans 4–6. In the IR spectra the absorption bands in the range 1715–1720 cm⁻¹ show the presence of CHCOCH₃ while the absorption bands at 1673–1677 cm⁻¹ confirm the presence of OCOCH. The weak absorption band in the range 1620–1622 cm⁻¹ confirms the presence of (C⚌C). In ¹H NMR study of compounds 4–6 the presence of sharp singlet at δ 5.21–5.40 was assigned to olefinic proton (C₆–H) while singlet at δ 3.10–3.31 integrated for one proton of C′₃H. In ¹³C NMR study, the signals at δ 165.1–168.3 confirm the presence of OCOCH₃ while the signals at δ 160.3–163.5 confirm the presence of OCOCH. Finally the mass spectra data of compounds showed molecular ion peaks [M⁺] at m/z: 526, 502/504 and 468 for 4, 5 and 6, respectively. All these data confirmed the formation of desired products. The stereochemical assignation of the C₅–C bond has been established on the basis of half band width (W_1/2) values of C₃-axial proton in the ¹H NMR spectra of compounds 4 and 5 which clearly suggested that A/B ring junction is trans (Shamsuzzaman et al., 2013c) and also on the basis of the fact that during the reaction, the attack of the reagent should be from (α) side which is less hindered, not from (β) side which is more hindered due to the presence of a methyl group at C₁₀, hence the C₅–C bond should be axial (α) and trans to C₁₀ methyl group (Shamsuzzaman et al., 2010). C₁₉ values in ¹³C NMR are strongly dependent on the ring fusion stereochemistry. In the synthesized compounds 4–6, C₁₉ chemical shift values were observed in the range of 16–19 ppm, which is consistent with the values, obtained for trans steroids (Iida et al., 2007). The mechanism for the formation of steroidal 2H-pyran derivatives 4–6 involves the Michael addition of active methylene of ethyl acetoacetate to conjugated ketone at the electrophilic alkene C, in nucleophilic addition type process, which undergoes subsequent intramolecular cyclization to provide the desired product (Scheme 2).

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Scheme 2

Plausible mechanism for the formation of steroidal 2H-pyrans 4–6.

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3.2 In vitro anticancer activity

Steroids with α, β-unsaturated ketone core gave the potency against human cancer cell lines (Wang et al., 2011). With this intuition, an attempt of synthesizing steroidal 2H-pyran derivatives 4–6 from steroidal α, β-unsaturated ketones 1–3 was made. The synthesized compounds were evaluated for anticancer activity toward human HeLa (cervical) and Jurkat (leukemia) cancer cells. Doxorubicin (Dox) and 5-fluorouracil (5-Fu) were used as cytotoxic drugs of reference. The conversion of the soluble yellowish MTT to the insoluble purple formazan by active mitochondrial lactate dehydrogenase of living cells has been used to develop an assay system for the measurement of cell proliferation. Dose-dependent effects of steroidal 2H-pyranes 4–6 on cell viability of HeLa, Jurkat and PBMC cell lines are given in Fig. 1.

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Figure 1

Dose-dependent effects of steroidal 2H-pyranes 4–6 on cell viability of HeLa, Jurkat and PBMC cell lines. Data shown are mean ± standard error of at least three independent experiments.

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The anticancer data given in Table 3 show that compounds 4–6 exhibited moderate to good activity while compound 6 elicited the better inhibitory activity (IC₅₀ < 19 μM) against both cell lines.

Interestingly, the IC₅₀ values of compounds 4 and 6 were close to each other, apparently indicating that the acetylation did not increase the anticancer activity. This might be due to the deacetylation of compound 4 by an esterase present indigenously in cells (Samanta et al., 2013). It is a noteworthy point that the synthesized compounds 4–6 were found to be slightly more active against HeLa. The synthesized compounds 4–6 were also tested with normal cell line (PBMC) during which they were found slightly toxic, all the compounds showed IC₅₀ > 60 μM.

3.3 In vitro antioxidant activity

The in vitro antioxidant activity and scavenging effects of steroidal 2H-pyran derivatives 4–6 were evaluated by using different reactive species assay containing DPPH radical scavenging activity. The free radical scavenging activity of the synthesized compounds was evaluated through their ability to quench the DPPH• using ascorbic acid as a reference. The potencies for the antioxidant activity of compounds 4–6 to the reference drug are shown in Table 4.

In general, all the synthesized compounds were less potent than the reference. Among the synthesized compounds, compounds 4 and 6 exhibited a slightly better antioxidant activity than compound 5.