Synthesis of marmycin A and investigation into its cellular activity

Abstract

Anthracyclines such as doxorubicin are used extensively in the treatment of cancers. Anthraquinone-related angucyclines also exhibit antiproliferative properties and have been proposed to operate via similar mechanisms, including direct genome targeting. Here, we report the chemical synthesis of marmycin A and the study of its cellular activity. The aromatic core was constructed by means of a one-pot multistep reaction comprising a regioselective Diels-Alder cycloaddition, and the complex sugar backbone was introduced through a copper-catalysed Ullmann cross-coupling, followed by a challenging Friedel-Crafts cyclization. Remarkably, fluorescence microscopy revealed that marmycin A does not target the nucleus but instead accumulates in lysosomes, thereby promoting cell death independently of genome targeting. Furthermore, a synthetic dimer of marmycin A and the lysosome-targeting agent artesunate exhibited a synergistic activity against the invasive MDA-MB-231 cancer cell line. These findings shed light on the elusive pathways through which anthraquinone derivatives act in cells, pointing towards unanticipated biological and therapeutic applications.

Figure 1. Anthraquinone-containing natural products.

a, Molecular structures of marmycin A (1) and B (2), jadomycin B (3), hedamycin (4), the clinically approved anthracycline doxorubicin (DXR, 5) and landomycin E (6). b, Retrosynthetic analysis towards marmycin A precursors featuring a stepwise fusion of the aromatic and chiral sugar moieties and a regioselective cycloaddition.

Figure 2. Chemical synthesis of the marmycins.

a, Synthesis of precursors of 1. (i) 9, 10, toluene, 100 °C, 16 h, then concentrated to dryness; MeOH, rt, 4 h, then K₂CO₃, rt, 1 h, 33%. (ii) Tf₂O, Et₃N, CH₂Cl₂, –78 °C, 6 h, 78%. (iii) Benzaldehyde dimethylacetal, PTSA, dimethylformamide (DMF), 95 °C (reduced pressure), 12 h, 98%. (iv) (ⁱPr)₂EtN, MOMCl, CH₂Cl₂, rt, 72 h, 91%. (v) sec-Buthyllithium (1.6 M, hexane), THF, –40 °C, 90 min, 61%. (vi) O-benzyl hydroxylamine HCl salt, NaOAc, EtOH, rt, 2 h, 90%. (vii) Anhydrous CeCl₃, methyllithium (3 M, diethoxymethane), THF, –78 °C, 1 h; then 16, –78 °C to rt, 1 h, 72%. (viii) H₂, Pd/C (10 mol%), MeOH, rt, 12 h, 88%. (ix) 7, 8, CuI (20 mol%), K₂CO₃, toluene, 160 °C, sealed tube, 72 h, 33%. b, Assembly of aromatic and sugar moieties. (x) BF₃·Et₂O, CH₂Cl₂, –78 °C to rt, 24 h, 42%. (xi) NaH, DMF, 0 °C, 2 h, 75%. (xii) 50% wt/wt aq. HBF₄, MeCN, 82 °C, 8 h, 13%. (xiii) NCS, C₂H₄Cl₂, 84 °C, 24 h, 61%. c, ORTEP drawing of the X-ray crystal structure of 1 showing top and side views. rt, room temperature.

Figure 3. Marmycin A kills human cancer cells independently of genome targeting.

a, Percentage of viable U2OS cells after 72 h treatment with DXR (dashed line) or 1 (black). n = 3. Error bars, s.d. b, Confocal microscopy images showing accumulation of DAPI (pan-nuclear blue), DXR (pan-nuclear red) and 1 (red puncta) in U2OS cells. Yellow boxes indicate the area of magnification of the main images. Zoomed images are ×5. Scale bar, 10 μm. c, Western blot analysis showing levels of γH2AX, p-p53 and p21 in U2OS cells treated with dimethylsulfoxide (DMSO), DXR or 1 (50 μM, 24 h) including a co-treatment with Z-DEVD-FMK.

Figure 4. Marmycin A accumulates in the lysosomes and triggers programmed cell death.

a, Live imaging of U2OS cells co-treated with 1 and DND-22. The yellow box indicates the area of magnification of the main image. The zoomed image is ×3. White arrowheads indicate sites of co-localization. The white box represents a further ×3 magnification. b, Confocal microscopy images of U2OS cells expressing GFP-Lamp1 treated with 1. Yellow boxes indicate the area of magnification of the main images. Zoomed images are ×5. White arrowheads indicate sites of co-localization. The white box shows a further ×3 magnification. Scale bars, 10 μm. c, WB analysis of p62 and LC3 in U2OS cells treated with DMSO, CQ or 1 (50 µM, 24 h) including a co-treatment with NAC. d, Western blot analysis of Bid in U2OS cells treated with DMSO or 1 (10 µM, 96 h) including a co-treatment with CB or CT inhibitors. n.i., no inhibitor. e, Molecular structures of 20 and 21. f, Percentage of viable MDA-MB-231 cells after 72 h treatment with DXR (dashed line), 1 (black), 20 (cyan), 21 (red) and an equimolar mixture of 1 and 20 (blue). n = 3; error bars, s.d.

Figure 5. Antiproliferative mechanisms of anthraquinone-containing drugs.

Black arrows indicate previously established mechanisms. Red arrows highlight the cellular activity of marmycin A. Black arrows with question marks indicate putative mechanisms of action of other anthraquinone-containing small molecules.