Synthesis of novel steroids using Mizoroki-Heck reaction, their spectroscopic analysis, anticancer activity against cervical cancer and DFT studies

doi:10.1016/j.molstruc.2019.127512

Journal of Molecular Structure

Volume 1204, 15 March 2020, 127512

https://doi.org/10.1016/j.molstruc.2019.127512 Get rights and content

Highlights

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Diosgenin-prodrugs 3a and 3b synthesized using Mizoroki-Heck reaction.
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All the compounds were characterized by spectroscopy and 2 also by X-ray crystallography.
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Compound 3b showed higher cytotoxity.
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Recyclability of ionic liquid from the reaction mixture.
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First hyperpolarizability showed 3a (15.99 × 10-34 esu) to be a good NLO materials.

Abstract

Two novel steroids, 3β, 25R-spirost-5-en-3yl 3-(4-propionylphenyl)but-2-enoate(3a) and 3β, 25R-spirost-5-en-3yl 3-(4-nitrophenyl) but-2-enoate (3b) have been synthesized using a palladium catalyzed Mizoroki-Heck reaction in a two-step synthesis using diosgenin as a starting material. A new strategy was adopted involving conversion of diosgenin (compound 1) into compounds, 3β, 25R-spirost-5-en-3yl 2-chloroacetate (Compound 2), 3β, 25R-spirost-5-en-3yl trans-but-2-enoate (Compound 3) and 3β, 25R-spirost-5-en-3yl-trans-cinnamate (Compound 4) via Steglich esterification using an ionic liquid (NMP⁺HSO₄). The synthesized compounds were then treated with 4-bromopropiophenone and 1-bromo-4-nitrobenzene using [Pd(PPh₃)₄] as a catalyst and K₂CO₃ as a base in DMF; only compound 3 underwent reaction yielding compounds (3a) and (3b). Structure elucidation of all the synthesized compounds was done by ¹H NMR, ¹³C NMR, NOESY, IR, UV–Vis spectroscopic techniques and mass spectrometry. Stereochemistry of compound 2 was established by a single crystal X-ray structural analysis. These compounds 2, 3, 3a, 3b and 4 were investigated for cytotoxicity and apoptosis through MTT assay and Mitochondrial staining respectively, of cervical cancer cell line HeLa; the compounds showed significant anticancer activity. The molecular geometry of the compounds was optimized by density functional theory using (DFT/B3LYP) method with 6–31G (d, p) basis set and compared it with experimental data. From the low value of HOMO-LUMO energy gap, compound 3b was found to have high chemical reactivity. Nucleophilic and electrophilic sites within molecules were obtained by electrostatic potential surfaces. From global reactivity descriptors, 3b exhibited greater global electrophilicity index value (5.83eV), suggesting it to be a good electrophile. The high value of first hyperpolarizability (15.99 × 10⁻³⁰ esu) for compound 3 suggested it to be ideal for non linear optical (NLO) application. From AIM (Atoms in Molecule) approach, it is found that compound 3 was more stable in comparison to other compounds.

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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Synthesis of novel steroids using Mizoroki-Heck reaction, their spectroscopic analysis, anticancer activity against cervical cancer and DFT studies

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Material and measurements

Cell viability assay

Experimental, crystal structure determination and refinement

Computational details

1H, 13C NMR, NOESY, FT-IR spectroscopy and ESIMS

Conclusion

Author contribution

Acknowledgements

Tetrahedron Lett.

Tetrahedron Lett.

J. Mol. Struct.

J. Mol. Struct.

Tetrahedron Lett.

Adv. Quant. Chem.

J. Spectrochim. Acta A.

Mol. Struct.

Tetrahedron

J. Am. Chem. Soc.

Bull. Chem. Soc. Jpn.

J. Chem. Soc., Chem. Commun.

J. Am. Chem. Soc.

J. Am. Chem. Soc.

Chem. Rev.

J. Am. Chem. Soc.

J. Org. Chem.

Immunol. Meth.

RED Version 1.711.13

SHELXT, Acta Cryst.

¹H, ¹³C NMR, NOESY, FT-IR spectroscopy and ESIMS