Structure–Activity Relationships of Withanolides as Antiproliferative Agents for Multiple Myeloma: Comparison of Activity in 2D Models and a 3D Coculture Model

Abstract

Multiple myeloma (MM) is a hematological cancer in which relapse and resistance are highly frequent. Therefore, alternatives to conventional treatments are necessary. Withaferin A, a withanolide isolated from Withania somnifera, has previously shown promising activity against various MM models. In the present study, structure–activity relationships (SARs) were evaluated using 56 withanolides. The antiproliferative activity was assessed in three MM cell lines and in a 3D MM coculture model to understand the in vitro activity of compounds in models of various complexity. While the results obtained in 2D allowed a quick and simple evaluation of cytotoxicity used for a first selection, the use of the 3D MM coculture model allowed filtering compounds that perform better in a more complex setup. This study shows the importance of the last model as a bridge between 2D and in vivo studies to select the most active compounds and ultimately lead to a reduction of animal use for more sustained in vivo studies. NF-κB inhibition was determined to evaluate if this could be one of the targeted pathways. The most active compounds, withanolide D (2) and 38, should be further evaluated in vivo.

Withaferin A: A Pleiotropic Anticancer Agent from the Indian Medicinal Plant Withania somnifera (L.) Dunal

“…The highest amount of withaferin A obtained under in vitro conditions was a concentration of 0.27 to 7.64 mg/g dry weight, while under in situ conditions the concentration was 8.06 to 36.31 mg/g dry weight [ 44 ]. The evaluation of structure–activity relationships in 56 withanolides using 2D and 3D coculture models revealed withanolide D (2) as a compound that has high antiproliferative activity against multiple myeloma [ 45 ].…”

A Homodimer of Withaferin A Formed by Base-Promoted Elimination of Acetic Acid from 27-O-Acetylwithaferin A Followed by a Diels–Alder Reaction

“…Numerous biological activities associated with withaferin A have prompted several structure–activity relationship (SAR) studies utilizing the 2,3-en-1-one moiety and the secondary OH-4 group in ring A, the 5β,6β-oxirane moiety in ring B, and the α,β-unsaturated lactone ring and the OH-27 group in ring E of the side-chain. Modifications of these structural moieties of withaferin A ( 1 ) included esterification, − silylation, ,− and oxidation − of OH-4 and OH-27, reduction of the C-1 carbonyl, reduction of the 2,3-double bond, , intermolecular 1,3-dipolar azomethine-ylide cycloaddition, and chemoselective and diastereoselective 1,3-dipolar nitrone cycloaddition to the 2,3-double bond, Michael addition of a variety of nucleophiles to the 2,3-en-1-one moiety, ,,,, 5β,6β-oxirane ring opening followed by modifications of the resulting diol, ,, and modifications of the side-chain. One of the known side-chain modification reactions, except for derivatization of the OH-27, has involved treatment of withaferin A ( 1 ) and 4,27-di- O -acetylwithaferin A ( 5 ) with a strong base (KOH/MeOH or NaOMe/MeOH) resulting in α-methylene-δ-lactone moiety in ring E and concurrent Michael addition of the base to the 2,3-en-1-one moiety of ring A affording 11 . , As the 2,3-en-1-one moiety of 1 is known to be responsible for many of its biological activities, ,, such Michael adducts are of no interest.…”

“…One of the known side-chain modification reactions, except for derivatization of the OH-27, has involved treatment of withaferin A ( 1 ) and 4,27-di- O -acetylwithaferin A ( 5 ) with a strong base (KOH/MeOH or NaOMe/MeOH) resulting in α-methylene-δ-lactone moiety in ring E and concurrent Michael addition of the base to the 2,3-en-1-one moiety of ring A affording 11 . , As the 2,3-en-1-one moiety of 1 is known to be responsible for many of its biological activities, ,, such Michael adducts are of no interest. In continuing our studies on withaferin A ( 1 ) and its analogues, ,, we were interested in modifying their side-chains without affecting the 2,3-en-1-one moiety. Herein we report that the treatment of 27- O -acetylwithaferin A ( 2 ) with the non-nucleophilic base, 1,8-diazabicyclo­[5,4,0]­undec-7-ene (DBU), under mild conditions leads to the elimination of a molecule of acetic acid from its side chain to provide 5β,6β-epoxy-4β-hydroxy-1-oxo-witha-2(3),23(24),25(27)-trienolide ( 3 ) and the intermediate 9 with an α,β -dimethylene-δ-lactone moiety, which undergoes a Diels–Alder [4 + 2] type dimerization resulting in a withaferin A homodimer ( 4 ) bearing a rare spiro­(isochromene-pyran)-dione ring system.…”