Chemical Synthesis and Biological Activities of Amaryllidaceae Alkaloid Norbelladine Derivatives and Precursors

Abstract

Amaryllidaceae alkaloids (AAs) are a structurally diverse family of alkaloids recognized for their many therapeutic properties, such as antiviral, anti-cholinesterase, and anticancer properties. Norbelladine and its derivatives, whose biological properties are poorly studied, are key intermediates required for the biosynthesis of all ~650 reported AAs. To gain insight into their therapeutic potential, we synthesized a series of O-methylated norbelladine-type alkaloids and evaluated their cytotoxic effects on two types of cancer cell lines, their antiviral effects against the dengue virus (DENV) and the human immunodeficiency virus 1 (HIV-1), and their anti-Alzheimer’s disease (anti-cholinesterase and -prolyl oligopeptidase) properties. In monocytic leukemia cells, norcraugsodine was highly cytotoxic (CC50 = 27.0 μM), while norbelladine was the most cytotoxic to hepatocarcinoma cells (CC50 = 72.6 μM). HIV-1 infection was impaired only at cytotoxic concentrations of the compounds. The 3,4-dihydroxybenzaldehyde (selectivity index (SI) = 7.2), 3′,4′-O-dimethylnorbelladine (SI = 4.8), 4′-O-methylnorbelladine (SI > 4.9), 3′-O-methylnorbelladine (SI > 4.5), and norcraugsodine (SI = 3.2) reduced the number of DENV-infected cells with EC50 values ranging from 24.1 to 44.9 μM. The O-methylation of norcraugsodine abolished its anti-DENV potential. Norbelladine and its O-methylated forms also displayed butyrylcholinesterase-inhibition properties (IC50 values ranging from 26.1 to 91.6 μM). Altogether, the results provided hints of the structure−activity relationship of norbelladine-type alkaloids, which is important knowledge for the development of new inhibitors of DENV and butyrylcholinesterase.

Keywords: Alzheimer’s disease; Amaryllidaceae alkaloid; O-methylation; anti-cholinesterase; antiviral; dengue virus; galanthamine; norbelladine; specialized metabolism.

Figure A1

Antiretroviral effects of four concentrations (25, 50, 100, and 200 µM) of norbelladine precursors and derivatives on HIV-1_GFP in THP1 cells. Representative dot plots are presented. DMSO (200 µM) and raltegravir (10 µM) were included as the negative and positive controls, respectively.

Figure A2

Antiflaviviral effects of norbelladine precursors and derivatives on DENV_GFP. The antiviral activity against DENV_GFP was evaluated 72 h post-infection using Huh7 cells via flow cytometry at four concentrations (25, 50, 100, and 200 µM) of norbelladine precursors and derivatives. Lycorine (5 µM) was included as the positive control and DMSO (200 µM) as the negative control. Representative dot plots are presented.

Figure 1

Chemical structures of the molecules used in this study. Amaryllidaceae alkaloid precursors (3,4-dihydroxybenzaldehyde and tyramine), intermediates (norcraugsodine and norbelladine), their corresponding O-methylated derivatives (3′-O-methylnorcraugsodine, 3′-O-methylnorbelladine, 4′-O-methylnorcraugsodine, 4′-O-methylnorbelladine, 3′,4-’O-dimethylnorcraugsodine, and 3′,4′-O-dimethylnorbelladine), and the well-known AAs galanthamine and lycorine.

Figure 2

Cytotoxic effects of norbelladine precursors and their derivatives on Huh7 and THP-1 cells. To assess the cell viability, the cellular ATP levels were measured on (a) THP-1 and (b) Huh7 cells 72 h after alkaloid addition at concentrations of 6.25 µM to 200 µM. Lycorine was utilized as a positive control at concentrations of 0.3 µM to 40 µM. Results were normalized to equivalent concentrations of DMSO and the x-axis is displayed in log₁₀. DMSO: dimethylsulfoxide; ATP: adenosine triphosphate.

Figure 3

Antiretroviral effect of norbelladine precursors and derivatives on HIV-1_GFP. The antiviral activities against HIV-1_GFP of norbelladine precursors and derivatives were evaluated 72 h post-infection using THP-1 cells via flow cytometry at concentrations ranging from 6.25 µM to 200 µM. Infections were performed with non-propagative HIV-1_GFP virus at a multiplicity of infection (MOI) of 0.1. Raltegravir served as a positive control and DMSO as a negative control at concentrations equivalent to the tested alkaloids. Results were normalized to the value with HIV-1_GFP infection without treatment and the x-axis is displayed as log₁₀.

Figure 4

Antiflaviviral effects of norbelladine precursors and derivatives on DENV_GFP. The antiviral activities against DENV_GFP of norbelladine precursors and derivatives were evaluated 72 h post-infection using Huh7 cells via flow cytometry at concentrations ranging from 6.25 µM to 200 µM. Infections were performed with propagative DENV_GFP virus at an MOI of 0.025. Lycorine was used as a positive control and DMSO as a negative control at concentrations equivalent to the tested alkaloids. Results were normalized to the value of DENV_GFP infection without treatment and the x-axis is displayed as log₁₀.

Figure 5

Anti-Alzheimer’s disease properties. (a) Prolyl oligopeptidase inhibition by norcraugsodine, 3′-O-methylnorcraugsodine, and 3′-O-methylnorbelladine. (b) Acetylcholinesterase inhibition of 3′,4′-O-methylnorbelladine using acetylthiocholine as the substrate. (c) Butyrylcholinesterase (BuChE) inhibition of norbelladine and derivatives using butyrylthiocholine (BuTCh) as the substrate. (d) Butyrylcholinesterase inhibition of norbelladine and derivatives using acetylthiocholine (ATCh) as the substrate. Galanthamine (10 μM) was used as the positive control for the AchE assays, while rivastigamine (2 mM) was used for the BuChE assays (100% inhibition not shown on the graph).

Figure 6

Prediction of norbelladine derivatives’ interactions with butyrylcholinesterase. Grey surface representation of BuChE (4BDS) active site with key subsites highlighted in different colors (catalytic triad is in yellow, pre-anionic site (PAS) is in green, anionic site is in pink, oxyanion pocket is in turquoise, and the acyl pocket is in dark grey). (a) Superimposition of docked ligands in the BuChE active site (norbelladine is in green, 3′-O-methylnorbelladine is in purple, 4′-O-methylnorbelladine is in yellow, and 3′,4′-O-dimethylnorbelladine is in turquoise). (b) Non-covalent (H-bond, hydrophobic, and P-stack) interactions of norbelladine with Trp82 from the anionic site, Tyr332 from the PAS, and Tyr440 from the binding site. (c) Non-covalent interactions of 3′-O-methylnorbelladine with W82 from the anionic site, Asp70 from the PAS, His438 from the catalytic triad, and Trp430 from the binding site. (d) Non-covalent interactions of 4′-O-methylnorbelladine with Trp82 from the anionic site. (e) Non-covalent interactions of 3′,4′-O-dimethylnorbelladine with Trp82 from the anionic site and Thr120 and Tyr440 from the binding site.