Synthesis of novel steroids using Mizoroki-Heck reaction, their spectroscopic analysis, anticancer activity against cervical cancer and DFT studies
Graphical abstract
Introduction
Cross coupling reactions like Heck [1], Mizoroki [2], Suzuki [3], Negishi [4], Stille [5], Sonogoshira [6] and Kumada [7] have commonly been used for the formation of new C–C bond, using palladium based catalysts. These reactions over the past few decades have revolutionized organic synthesis [8]. The Mizoroki-Heck reaction is a fundamental synthetic transformation that has been extensively used for the synthesis of substituted alkenes [9]. C-27 steroidal sapogenins, like diosgenin, are an important class of bioactive compounds having diverse pharmacological properties, including anti-oxidant, anti-inflammatory, anti-cancer and antiadipogenic activity [10] have provided interesting ’leads’ for the development of new drugs. Ester derivatives of diosgenin have also proven to have cytotoxicity, anti-adipogenic and anti-diabetic activity [11]. To the best of our knowledge not much work has been done on cross coupling reactions of steroids. In the present research work a new methodology has been adopted to synthesise novel steroidal compounds involving Steglich esterification followed by a Mizoroki-Heck reaction (Scheme 1). The method involved creation of reactive olefinic group in steroids by esterification with a subsequent Mizoroki-Heck reaction to give the cross coupling product.
All the synthesized compounds were characterized with the help of modern spectroscopic methods like 1H NMR, 13C NMR, NOESY, IR, UV–Vis spectroscopy and mass spectrometry. Single crystal X-ray analysis of (compound 2) helped in establishing the complete stereochemistry of the molecule. Quantum chemical calculations have been performed by density functional theory (DFT) using B3LYP functional and 6-31G (d, p) basis set. Nonlinear optical response of materials has wide range of applications and thus the synthesized compounds were evaluated for their non-linear optical properties. Energy gap between HOMO and LUMO characterized the chemical stability and charge transfer interaction within the molecules. Weak interactions in molecules play a vital role in deciding their biological response. For understanding the nature and strength of these weak interactions Atoms in molecules (AIM) theory was applied.
Therefore, this research paper gives a description of synthesis, anti-cancer activity, single crystal X-ray structural analysis, vibrational assignments, electronic transitions, global reactivity descriptor, non-linear optical (NLO) features and intramolecular interactions of steroidal compounds.
Section snippets
Material and measurements
2-Chloro acetic acid, crotonic acid, trans-cinnamic acid, 4-bromopropiophenone and 1-bromo-4-nitrobenzene were purchased from Sigma-Aldrich and used as received. Ethyl acetate, n-hexane, DMF and N-methyl-2-pyrrolidone purchased from Fisher Scientific Pvt. Ltd. And dried according to known procedure [12] before use. 1H NMR (300 MHz) spectra were recorded on a Bruker DRX-300 spectrometer in CDCl3 solvent and TMS as the internal standard. 13C NMR (75 MHz) spectra and NOESY (300 MHz) were recorded
Cell viability assay
The cytotoxic effect of compounds of group 1 (2, 3 and 4) and group 2 (3a and 3b) were checked on HeLa (cervical cancer) cell lines. HeLa cells at a density of 1 × 104/100 μL MEM (E) were seeded in flat bottom 96 well plates and kept in incubator at 37 °C, and 5% CO2 atmosphere. After overnight growth, cells were treated for 48 h with 1, 10, 20 and 50 μM concentrations of compounds 2, 3 and 4 and 1, 10, 20 and 50 μM conc. each of 3a and 3b prepared in fresh culture medium. Control samples
Experimental, crystal structure determination and refinement
Crystals of 3β, 25R spirost-5-en 3-yl 2-chloroacetate (Compound 2) was prepared by solvent evaporation method using mixture of hexane and ethyl acetate.
Crystals of good morphology were chosen for single crystal X-ray diffraction analysis. Intensity data was collected at 100 K using Bruker apex-II CCD diffractometer equipped with graphite-monochromatized (Mo Kα = 0.71073 Å) radiation and corrected empirically for absorption effects. The final unit cell determination, scaling of the data, and
Computational details
Quantum chemical calculations for compounds 2, 3, 3a, 3b and 4 were carried out with Gaussian 09 program package using B3LYP functional with the 6-31G (d, p) level [21,22]. The geometry optimization and vibrational frequency calculations have been carried out in gas phase, diethyl ether and DMSO. The electronic transitions and electronic properties such as HOMO-LUMO were computed with the help of time-dependent DFT (TD-DFT) method in different solvent like diethyl ether, chloroform, methanol
1H, 13C NMR, NOESY, FT-IR spectroscopy and ESIMS
In the first step of the reaction, diosgenin (compound 1) was esterified in high yield by Steglich esterification method using an ionic liquid, N-methyl-2-pyrrolidone hydrogen sulphate (NMP+HSO4) [11] to give compounds 2, 3 and 4 (reaction condition for esterification is given in Table 1). The Mizoroki-Heck reaction of compounds 1 and 2 with 4-bromopropiophenone and 4-bromonitrobenzene failed to give the desired product 6-(4-propionylphenyl)-(3β, 25 R)-spirost-5-en-3-ol (Compound 1a) and
Conclusion
Novel steroidal compounds have been synthesized using Steglich esterification and Mizoroki-Heck reactions. The structure of these novel steroid derivatives were characterized with the help of 1H, 13C, 2D NMR, FT-IR and mass spectrometry. These compounds were evaluated for anti-cancer activity against cervical cancer cell lines and it was observed that amongst them 3b showed promising activity against HeLa (cervical cancer) cell lines. X-ray analysis helped in establishing the structure and
Author contribution
Arun Sethi: Conceptualization, methodology, Writing-Review & Editing, Praveer Singh: Investigation, writing-original draft preparation. Neera Yadav: Investigation, writing-original draft preparation. Rohit Prakash: software, resources, Ranvijay Pratap Singh: Software, validation. Priyanka Yadav: Investigation, writing-original draft preparation. Monisha Banerjee: methodology, Writing-Review & Editing.
Acknowledgements
Authors are thankful to Department of Chemistry, University of Lucknow for providing Computational Research facility and spectroscopic data (1H NMR, 13C NMR, and 2D NMR), and SAIF division of Central Drugs Research Institute of Lucknow (CDRI), Lucknow for ESI-MS.
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