Chemical Investigation of the Calcareous Marine Sponge Pericharax heteroraphis, Clathridine-A Related Derivatives Isolation, Synthesis and Osteogenic Activity

Abstract

As a result of screening a panel of marine organisms to identify lead molecules for the stimulation of endochondral bone formation, the calcareous sponge Pericharax heteroraphis was identified to exhibit significant activity during endochondral differentiation. On further molecular networking analysis, dereplication and chemical fractionation yielded the known clathridine A-related metabolites 3-6 and the homodimeric complex (clathridine A)₂ Zn²⁺ (9), together with the new unstable heterodimeric complex (clathridine A-clathridimine)Zn²⁺ (10). With the presence of the zinc complexes annotated through the LC-MS analysis of the crude extract changing due to the instability of some metabolites and complexes constituting the mixture, we combined the isolation of the predicted molecules with their synthesis in order to confirm their structure and to understand their reactivity. Interestingly, we also found a large quantity of the contaminant benzotriazoles BTZ (7) and its semi-dimer (BTZ)₂CH₂ (8), which are known to form complexes with transition metals and are used for preventing corrosion in water. All isolated 2-aminoimidazole derivatives and complexes were synthesized not only for structural confirmation and chemical understanding but to further study their bioactivity during endochondral differentiation, particularly the positively screened imidazolone derivatives. Compounds leucettamine B, clathridine A and clathridimine were found to increase type X collagen transcription and stimulate endochondral ossification in the ATDC5 micromass model.

Keywords: Pericharax heteroraphis; clathridine; clathrinida; osteoporosis; sponge; zinc complex.

Figure 1

Screening results of 47 marine sponge species revealing the osteogenic activity of the calcareous sponges framed in red.

Figure 2

t-SNE graphical representation of the selected MS/MS features from the samples constructed with MetGem. Minimal cosine score was set at 0.75. Nodes colors correspond to the relative proportions of each feature in the sample groups: Pericharax heteroraphis and Pericharax sp.

Figure 3

Structures of the 2-aminoimidazolone derivatives 1–6, the contaminants BTZ (7) and bis-BTZ (8) and Zn complexes 9 and 10.

Scheme 1

Synthesis steps from preclathridine A (13) to clathridine A (3) and clathridimine (4).

Scheme 2

Synthesis of leucettamine B (5).

Scheme 3

Preparation and structures of the three homo- and heterodimeric complexes 9, 10 and 27.

Figure 4

(A) Results of the assessment of an ATDC5-based rapid screening platform using human Col X (hCol X) proximal promoter activity for the selection of natural compounds able to stimulate endochondral differentiation. Dexamethasone can be considered in this assay as a positive control. Statistical significance is estimated by Kruskal–Wallis Dunn’s multiple comparison test (** = p < 0.005 and **** = p < 0.0001). (B) Effect of leucettamine B and clathridine A derivatives and complexes on the endochondral differentiation of ATDC5 cells. ATDC5 cells were treated in 6-well plates for 7, 14 and 21 days for Alizarin red staining (presence of calcification). (C) Enlargement by microscope of the effect of leucettamine B and clathridine A derivatives on the endochondral differentiation of ATDC5 cells. ATDC5 cells were treated for 7, 14 and 21 days for Alizarin red staining (presence of calcification) (scale bar = 200 µm). The corresponding pictures for the complexes are available in the Supplementary Materials.

Figure 5

Enlargement by microscope of the effect of clathridine A/clathridimine complexes on the endochondral differentiation of ATDC5 cells. ATDC5 cells were treated for 7, 14 and 21 days for Alizarin red staining (presence of calcification) (scale bar = 200 µm).