Selective synthesis of A-ring Е-arylidene derivatives from β-sitosterol and their activity

Abstract A series of 24-ethylcholest-4-ene-3,6-dione 2E-arylidene-derivatives has been synthesized by a Claisen-Schmidt reaction from a natural phytosterol β-sitosterol with yields of 80–85%. The structure of the obtained compounds was confirmed by NMR spectroscopy, including two-dimensional correlation experiments. The synthesized compounds were evaluated for their in vitro cytotoxicity and α-glucosidase inhibitory activity. It was established that compound 3 with pyridin-3-ylmethylene moiety exhibited a selective cytotoxic effect against the U251 cancer cell line with 99.31% inhibition of cancer cell growth. Compounds with pyridin-4-ylmethylene 4 and furan-2-ylmethylene-5 fragments were the most active inhibitors of α-glucosidase with IC50 64.00 and 38.95 µM, being 3- and 5-times more active than acarbose. Binding mode to α-glucosidase and ADMET characteristics for the lead molecule 5 were proposed computationally. To sum up, an efficient approach to the derivatives with promising antidiabetic activity based on available natural product β-sitosterol is suggested. Graphical Abstract


Introduction
Diabetes is a group of metabolic disorders corresponding with prolonged high blood sugar level.In recent updates on the treatment of diabetes mellitus, a-glucosidase inhibitors (AGIs) from various plant sources are trending for their ability to decrease sugar level by reduction of hydrolytic cleavage of non-reducing ends of dietary oligosaccharides and diminished release of a-glucose (Kumar et al. 2011), that retard carbohydrate digestion and absorption of glucose in small intestine.This mechanism of action plays an important role in controlling postprandial hyperglycemia, which is one of the modern therapeutic approach towards stabilizing blood glucose level in diabetic patients especially in T2D (Ghani 2015).Anti-diabetic drugs having a-glucosidase inhibiting properties such as acarbose, voglibose, miglitol and emiglitate are now commercially available for controlling postprandial hyperglycemia, but they still have high IC 50 value (from 180 to 350 in different assays).
Steroidal compounds exhibit a variety of biological functions and play an important role in life.For example, stigmasterol (IC 50 91.08mM) have antidiabetic potential being in 3.8 fold more active than acarbose.Also, steroids are highly lipophilic and readily enter most cells to interact with intracellular enzymes and receptors, and consequently, steroids demonstrate potential activity against various cancer forms including multi-drug resistant cancers.In addition, steriods also possess diverse virtues such as low cytotoxicity and high bioavailability, and several steroid-based anticancer agents such as abiraterone with N-containing substitute (Figure 1) have already been approved or under clinical evaluations for the cancer therapy, demonstrating that steroids are of great importance for the development of novel anticancer agents (Dembitsky 2020).
The literature presented a large number of publications on modifications of steroids, including the latest trend directions for the introduction of nitrogen-containing fragments by 1,3-dipolar cycloaddition (Kacprzak et al. 2016), Beckmann rearrangement (Cui et al. 2014), cyanoethylation (Khusnutdinova et al. 2017).The Claisen-Schmidt reaction is one of the simple and convenient methods to introduce this type of substituent into the molecule.By this reaction it is possible to input a chalcone moiety, that is open-chain precursors of flavonoids and isoflavonoids, presented by two aromatic rings linked by a three-carbon a,b-unsaturated carbonyl system.This reaction is especially well represented on arylidene triterpenoids with anticancer and antidiabetic activity (Wu et al. 2017;Zhong et al. 2019;Giniyatullina et al. 2021;Kazakova et al. 2021;Khusnutdinova, Petrova, et al. 2021;Khusnutdinova, Ha, et al. 2021).For example, ursolic acid arylidene-hybrid compounds inhibits glioma cell growth, induces apoptosis and arrest cell cycle through metabolic pathway downregulation (Fan et al. 2019).Ursolic acid arylidene-hydrazide derivative with chloro-substituent was found as the most effective antimicrobial compound against Staphylococcus aureus [S ¸enol et al. 2022].Abietane type semisynthetic diterpenoids modified at the carboxyl and carbonyl groups demonstrated antimicrobial activity against Mycobacterium tuberculosis H37Rv with MIC 1.25 mM [Smirnova et al. 2021].
Steroidal arylidenes have also been reported to act as antimicrobial (Banday et al. 2011;Kakati et al. 2013), antineoplastic (Shan et al. 2009), and anticancer agents (Fan et al. 2017).However, in contrast to triterpenoids, steroid-type compounds were mostly synthesized as E-ring substituted, and to the best of our knowledge, there was no data about the A-ring arylidene derivatives.
b-Sitosterol is one of the promising active phytosterols which has been reported as useful in the treatment of hypercholesterolemia, cancer, diabetes and other (Matsuoka et al. 2008;Saeidnia et al. 2014) (Figure 2).However, the examples of modification of b-sitosterol by Claisen-Schmidt reaction were not yet reported.Taking into account our data of high a-glucosidase inhibitory activity of tetrazolyl-b-sitosterol (Figure 2) (Petrova et al. 2021) we report herein the synthesis of arylidene derivatives and screening of their activity.

Chemistry
The synthetic route to b-sitosterol derivatives 3-7 is outlined in Scheme 1. 24-Ethylcholest-4-ene-3,6-dione 2 was obtained by oxidation of 1 by PCC in CH 2 Cl 2 with yield 85% according to previously reported method (Cui et al. 2008).The Claisen-Schmidt condensation of compound 2 with the corresponding aromatic aldehyde or furfural (2 eq.) in EtOH afforded 2-mono-substituted arylidene derivatives 3-7 with yields of 80-85%.To get bis-substituted compounds by enhancing the quantity of reagent, change of temperature conditions and the solvent medium was not successful, which allowed us to conclude a chemoselectivity of the reaction process.These results are in line with the previous studies concerning a selective reduction or oximination in A-ring of steroid type 3,6-diketones (Cui et al. 2008(Cui et al. , 2009)).
The structure of compounds 3-7 was determined by 1 H and 13 C NMR spectroscopy data.Thus, the formation of monosubstituted arylidene derivatives 3-7 was confirmed According to NOESY spectra and analysis of long-range J HH binding constants for compounds 3-7, the 2-30 double bond is represented in the E-configuration.In the 1 H NMR spectrum of compound 7, the H-30 proton signal is observed as a doublet-doublet with splitting 4 J 30-1b ¼ 3.3 and 4 J 30-1b ¼ 1.5 Hz with methylene protons in position C1, which indicates the E-configuration of the benzylidene fragment.
For methylene protons in the C1 position, assignment to the aor b-orientation was taken based on the NOESY cross-peaks: H a -1/H-9 (d H 2.58/1.49ppm) and H b -1/H-19 (d H 3.31/1.02ppm).The E-configuration of the double bond is also supported by NOESY data on interactions of H-32(36) aromatic protons with H-19 methyl and H b -1 methylene proton.

Biological assay
Compounds 2-7 were selected by the National Cancer Institute (NCI) Developmental Therapeutics Program (https://dtp.cancer.gov)for the in vitro cell line screening to investigate their anticancer activity against full panel of 60 human tumor cell lines representing nine different types of human cancers: leukemia, melanoma, lung, colon, central nervous system (CNS), ovarian, renal, prostate and breast cancers.Anticancer assays were performed according to the US NCI protocol, which was described elsewhere (Grever et al. 1992;Weinstein et al. 1997).Synthetic agents for the cancer screen with a known molecular weight are prepared in DMSO:glycerol 9:1 at a concentration of 4 mM.The solution was diluted 1:400, giving a High Test concentration of Scheme 1. Reagents and conditions: i. 2 eq. of the corresponding aldehyde, 40% KOH in EtOH, EtOH, 12 h, 25 C 10 mM.(https://dtp.cancer.gov/discovery_development/nci-60/handling.htm)Among tested compounds only pyridin-3-ylmethylene derivative 3 a selective cytotoxic effect against U251 cancer cell line (99.31%inhibition of cancer cell growth).
All compounds were screened for the enzyme a-glucosidase (from S. saccharomyces) inhibitory activity.The assay was performed following the modified method of Pistia Brueggeman and Hollingsworth based on the hydrolysis reaction of 4-nitrophenyl-a-D-glucopyranoside with a-glucosidase to form yellow colored 4-nitrophenol (for more details see Supporting Information file §1.4).As showed in Table S1 (see supporting information), derivative with pyridin-4-ylmethylene-4 and furan-2-ylmethylene-5 fragments were the most active with IC 50 64.00and 38.95 mM, being 3-and 5times more active than acarbose.Despite the fact that compounds 4 and 5 showed lower activity compared with the previously reported tetrazolyl-derivative (Petrova et al. 2021), a new series of b-sitosterol arylidenes including modification of the C6oxo-group for SAR analysis would be realized by our group.By these results, we show how to convert an available natural product b-sitosterol into simple derivatives with promising antidiabetic activity.

Molecular modeling
To elucidate a possible binding mode of the lead compound 5 to a-glucosidase protein molecular docking was performed.Since crystallographic structure of Saccharomyces cerevisiae a-glucosidase is unavailable to date homology model was used, which was obtained with SWISS-MODEL as described previously (Spasov et al. 2019).The whole enzyme molecule was targeted for molecular docking of newly identified inhibitor to take into account possible binding to allosteric site.As Figure S59 (see Supporting Information) shows, top 8 binding conformations are found in two allosteric sites close to the active center.Subsequently, ligand-protein interactions were manually analyzed for top-scoring binding pose (Figure S60 see Supporting Information).Compound 5 is predicted to accommodate favorable position inside of the mainly hydrophobic cleft.Sitosterol core forms multiple hydrophobic interactions with side chains of Tyr1005, Pro1008, Leu1413, and Val1419.In turn, C2 furfurylidene moiety is stabilized by p-cation and p-sulfur interactions with Arg1004 and Met1061, respectively.These insights can be used in future optimization efforts.
The lead molecule 5 was also computationally profiled for drug-like and ADMET properties.To obtain reliable results a consensus of four evaluations was obtained with different predictive services (Table S2, see supporting information).Not surprisingly, the compound was characterized as highly lipophilic and moderately soluble in water.The most interesting part is absorption assessment.Although the compound is anticipated to be cell-permeable and not subjected to P-glycoprotein efflux, intestinal absorption and oral bioavailability are deemed low.This is an appropriate profile for a-glucosidase inhibitor, which is intended to act in the intestine.Upon entering the systemic circulation, compound 5 is anticipated to be trapped by plasma proteins, which is supported by relatively low volume of distribution and lack blood-brain permeability.Biotransformation is likely to be catalyzed by cytochrome P450 3A4 that could facilitate rapid clearance.The compound has a good safety profile without alerts of mutagenic, cancerogenic, cardiotoxic or hepatotoxic properties (while hepatotoxicity is known issue with the marketed a-glucosidase inhibitor acarbose).

General
The spectra were recorded at the Center for the Collective Use 'Chemistry' of the Ufa Institute of Chemistry of the UFRC RAS and RCCU "Agidel" of the UFRC RAS. 1 H and 13 C NMR spectra were recorded on a "Bruker Avance-III" (Bruker, Billerica, MA, USA, 500 and 125.5 MHz respectively, d, ppm, Hz) in CDCl 3 , internal standard-tetramethylsilane.Mass spectra were obtained on a liquid chromatograph-mass spectrometer LCMS-2010 EV (Shimadzu, Kyoto, Japan).Melting points were detected on a microtable «Rapido PHMK05» (Nagema, Dresden, Germany).Optical rotations were measured on a polarimeter Perkin-Elmer 241 MC (PerkinElmer, Waltham, MA, USA) in a tube length of 1 dm.Elemental analysis was performed on a Euro EA-3000 CHNS analyzer (Eurovector, Milan, Italy), the main standard is acetanilide.Thin-layer chromatography analyzes were performed on Sorbfil plates (Sorbpolimer, Krasnodar, Russia), using the solvent system chloroform-ethyl acetate, 40:1.Substances were detected by a 10% solution of sulfuric acid solution with subsequent heating at 100-120 C for 2-3 min.All chemicals were of reagent grade (Sigma-Aldrich).b-Sitosterol 1 was commercially available from Acros Organics.24-Ethylcholest-4-ene-3,6-dione 2 was obtained as previously reported (Cui et al. 2008).

General procedure for synthesis of compounds 3-7
To a solution of compound 2 (1 mmol; 0.43 g) in ethanol (5 mL) 2 mmol of the corresponding aldehyde (furfural (0.17 mL), 3-nitrobenzaldehyde (0.30 g), 4-bromobenzaldehyde (0.37 g), 3-or 4-pyridine carboxaldehyde (0.19 mL)) and 40% KOH in ethanol (2.5 mL) were added and a mixture was stirred for 24 h at room temperature.After completing of reaction, pH was adjusted to neutral by adding an aqueous solution of 5% HCl, the mixture was poured into cold water (50 mL).The residue was filtered off, washed with water, and dried, then purified by column chromatography on SiO 2 using mixture of petroleum ether-EtOAc as eluent.All spectral and physical data are presented in the Supporting Information file.

Conclusion
A series of b-sitosterol arylidene derivatives was obtained for the first time by a Claisen-Schmidt reaction with yields of 80-85%.The biological screening revealed compounds with cytotoxic effect against U251 cancer cell line and a-glucosidase inhibitory effect.Binding mode to a-glucosidase and ADMET characteristics for lead compound 2-[(E)-furan-2-ylmethylene]-24-ethylcholest-4-ene-3,6-dione showed highly lipophilic quality and appropriate profile for anti-diabetic effect inhibitor, which is intended to act in the intestine.