Phenolic compounds as antiangiogenic CMG2 inhibitors from costa rican endophytic fungi1

Autor: Jon Clardy, Giselle Tamayo-Castillo, Michael S. Rogers, Shugeng Cao, Catalina Patricia Morales Murillo, Kaiane A. Habeshian, Lorna M. Cryan
Rok vydání: 2012
Předmět:
Zdroj: Bioorganic & Medicinal Chemistry Letters. 22:5885-5888
ISSN: 0960-894X
DOI: 10.1016/j.bmcl.2012.07.075
Popis: Anthrax Protective Antigen (PA) binds to two cell surface receptors, CMG2 and Tumor Endothelial Marker 8 (TEM8), which are responsible for allowing entry of anthrax toxin into host cells. PA and PASSSR, a mutated form of protective antigen, are anti-angiogenic, likely as a result of inhibition of CMG2-ECM (extracellular matrix) interactions.2 Thus, small molecules that bind to CMG2 and TEM8 have potential applications as both anthrax toxin antidotes and as agents for angiogenic diseases.2 Because PA is believed to use the natural ligand binding site on CMG2, compounds that inhibit the interaction of PASSSR with CMG2 are likely to inhibit tumor angiogenesis. In the course of identifying small molecules that can inhibit the interaction of PASSSR with the anthrax toxin receptor CMG2, we screened our sample libraries at Harvard Medical School’s high throughput screening facility (ICCB-L, Institute of Chemistry and Cell Biology at Longwood) using a high-throughput FRET assay. Our focus on natural products was based on an observation in a pilot screen that small molecule natural products, including those from endophytic fungi collected at Costa Rica, were frequently active for inhibition of the CMG2/PASSSR interaction. The crude extracts of the fungi CR252M3 and CR1207B3 inhibited the interaction of PASSSR with CMG2 and were selected for bioassay-guided separation. CR252M was first fractionated over a C18 SPE column, and fraction II was further separated by phenyl-hexyl prep-HPLC and then C8 semi-prep-HPLC to yield compounds 1 and 2.4 Compound 3 was obtained from CR1207B, after C18 SPE and C18 HPLC separation.4 Compound 1 was obtained as a yellow solid. Its UV absorptions in MeOH, with λmax (log e) 213 (3.16), 227 (3.24), 277 (3.28), 287 (3.31), 315 (sh), 390 (2.54) nm, indicated the presence of an extended coumarin moiety similar to that of neolambertellin.5,6 The IR spectroscopic data of compound 1, which showed absorption at 1700 cm−1, confirmed the existence of the C=O functional group. The positive ion HREIMS of 1 revealed a quasimolecular ion peak at m/z 257.0813 [M + H]+ corresponding to C15H13O4, (calculated for C15H13O4: 257.0814). The proton NMR spectrum of 1 broad singlet (δH 7.52, br s, H-4), one aromatic broad singlet (δH 6.76, br s, H-5), an ABC spin system (δH 8.14, d, J = 8.4 Hz, H-10; 7.49, t, J = 8.4 Hz, H-9; 6.97, d, J = 8.4 Hz, H-8), one methoxy (δH 4.10, s, 6-OMe), and one methyl singlet (δH 2.26, s, 3-Me). In the HMBC (Figure 1) spectrum, the 3-methyl group (δH 2.26) exhibited correlations to C-2 (δC 164.1, a lactone carbonyl), C-3 (δC 126.9), and C-4 (δC 141.4). The olefinic proton H-4 (δH 7.52) correlated to C-2, C-10b (δC 143.6), C-5 (δC 106.1), and 3-CH3 (δC 17.1); while H-5 (δH 6.76) demonstrated a 2J HMBC correlation to C-6 (δC 152.0, an oxygenated aromatic carbon) and 3J HMBC correlations to C-4, C-10b, and C-6a (δC 117.6), indicating a coumarin moiety with a methyl group at 3-position and a hydroxyl group at 6-position. Besides, the following HMBC correlations were also observed: from the 7-methoxy (δH 4.10) to C-7 (δC 157.2, an oxygenated aromatic carbon), H-8 (δH 6.97) to C-6a and C-10 (δC 115.8), H-9 (δH 7.49) to C-7 and C-10a (δC 126.4), and H-10 (δH 8.14) to C-6a, C-10b, and C-8 (δC 108.0), which indicated that the second aromatic ring was connected to the coumarin at 6a- and 10a-positions. Hence, the structure of 1 (7-O methyl neolambertellin) was determined as shown.7 Figure 1 Key HMBC correlation of 1 Compound 2 was also obtained as a yellow solid, and had a molecular formula of C16H14O4, which was 14 mass units more than 1. The proton NMR spectrum of 2 was similar to that of 1 except for the presence of the 6-O methyl group. The proton signals included one olefinic broad singlet (δH 7.92, br s, H-4), an aromatic singlet (δH 7.02, br s, H-5), an ABC spin system (δH 7.14, d, J = 7.8 Hz, H-8; 7.59, t, J = 7.8 Hz, H-9; 7.86, d, J = 7.8 Hz, H-10), two methoxy groups (δH 3.87, s, 6-OMe; 3.88, s, 7-OMe), and one methyl singlet (δH 2.14, s). Hence, it was readily deducted that 2 was the methylated product of compound 1. The ROESY (Figure 2) correlations between the 3-methyl (δH, 2.14, s) and H-4 (δH 7.92), H-4 and H-5 (δH 7.02), the 6-methoxy (δH 3.87) and H-5, and the 7-methoxy (δH 3.88) and H-8 (δH 7.14) conformed the structure of 2 as shown.8 Figure 2 Key ROESY correlations of 2 Compound 3 from CR1207B was identified to be 6,8-dihydroxy-3-methyl-1H-isochromen-1-one.9–11 Both 1 and 2 are new compounds, and 1 and 3 were active in the CMG2 FRET assay12,17 with IC50 values of 0.5 and 0.6 µM, respectively, while compound 2 was inactive. Compound 2 did not inhibit CMG2 in the FRET assay, even though it was structurally similar to compound 1, which did inhibit. Compound 1, at 39 and 117 µM, did not inhibit endothelial cell migration,2,13,17 but significantly inhibited endothelial cell proliferation.2,14 On the other hand, compound 3 did not inhibit endothelial cell proliferation at 52 and 156 µM, but inhibited endothelial cell migration by ~96.0% and ~93.1%, respectively (Figure 3). Upregulation of CMG2 during endothelial tubule formation in collagen gels suggests it may have an important functional role in the angiogenic process.15 Reeves et al have demonstrated that expression of CMG2 regulates the capacity of macrovascular endothelial cells to proliferate and form capillary networks in vitro.16 PASSSR has been shown to inhibit the migration of endothelial cells at CMG2-inhibitory concentrations, with minimal inhibition of the second anthrax receptor, TEM8.2 Endothelial cells overexpressing CMG2 migrate much faster than the regular endothelial cells.2 Inhibition of their migration means inhibition of angiogenesis. Since endothelial cell migration is a commonly used predictor of antiangiogenic activity, it is interesting that compound 3 inhibited not only CMG2 but also endothelial cell migration without any obvious anti-proliferative activity. Further studies of its effect on angiogenesis in animals may confirm it as a candidate for therapy of angiogenic diseases. Figure 3 Effect of 1 and 3 on endothelial cell migration (top panel) and cell proliferation (bottom panel). For the migration assay panel, the positive control is full serum media with vehicle (DMSO) and the negative control is serum free media with 0.1% BSA, ...
Databáze: OpenAIRE
Externí odkaz: