Synthesis and Evaluation of a 2,11‐Cembranoid‐Inspired Library

Abstract The 2,11‐cembranoid family of natural products has been used as inspiration for the synthesis of a structurally simplified, functionally diverse library of octahydroisobenzofuran‐based compounds designed to augment a typical medicinal chemistry library screen. Ring‐closing metathesis, lactonisation and SmI2‐mediated methods were exemplified and applied to the installation of a third ring to mimic the nine‐membered ring of the 2,11‐cembranoids. The library was assessed for aqueous solubility and permeability, with a chemical‐space analysis performed for comparison to the family of cembranoid natural products and a sample set of a screening library. Preliminary investigations in cancer cells showed that the simpler scaffolds could recapitulate the reported anti‐migratory activity of the natural products.

Trimethyl(1-phenylvinyloxy)silane (100 µL, 0.490 mmol) was then added and the reaction was warmed to RT and stirred for a further 16 h. The reaction was diluted with CH2Cl2 (20 mL), and washed with H2O (2 x 20 mL), followed by brine (20 mL). The organic layer was dried with MgSO4. The solvents were removed in vacuo and the resultant crude residue was a green oil. The product was isolated by column chromatography on silica gel (10% EtOAc:hexane) to yield 45 as a clear oil (95 mg, 78%).

SRB assay
Cells were seeded in 100μl medium into 96-well plates. After 36 hours, serial doubling dilutions of drug (or vehicle) were added to give the required dose range in sextuplicate across the plate. Cells were incubated for a further 96 hours. The medium was then removed and cells were fixed by adding 50μl of 10% (w/v) trichloroacetic acid (TCA) for one hour at room temperature. The plates were gently washed by immersion in tap water three times then air dried. 50μl of 0.4% (w/v) SRB in 1% (v/v) acetic acid was added to each well for one hour. Plates were then washed three times with 1% acetic acid and air dried. SRB was solubilised with 100ul/well 10mM Tris pH10.5. Absorbance at 570nm was measured using a SPECTRAmax 340PC plate reader.

Migration assay
Cells in standard tissue culture flasks were fluorescently labelled by incubation with 1umol/L CellTracker Green 5-chloromethylfluorescein diacetate (Invitrogen, Paisley) concomitant with serum starvation for two hours. Following trypsinisation cells were counted and added to the upper wells of 8um pore FluoroblokTM membrane inserts in 24-well companion plates (BD Biosciences, Franklin Lakes, NJ). The lower chambers were filled with 800uL medium containing 5% heat-inactivated FCS (to measure chemotaxis) or serum-free medium (to measure unstimulated random motility). The assay plates were incubated in a humidified atmosphere at 37°C in 5% CO2 in air for 16 hours. Cells which successfully migrated to the lower surface of the filter were visualised using an inverted fluorescence microscope. Figure S1. The loading scores of the Principal Components Analysis.
The loadings plot is provided in Figure S1 and can assist in interpreting the PCA scores plot of Figure 5 in the main paper. The loadings plot illustrates those molecular descriptors that are most important for explaining the variance of the molecules in those regions of the scores plot when overlaid. Descriptors that lie proximate to each other on the loadings plot are correlated, such as aromatic rings and bonds in quadrant III. Furthermore, the magnitude of deviation from the origin indicates the importance of each descriptor in that region of the plot, as seen in those molecules in quadrant III being more nitrogen-rich in their composition. Therefore, it can be readily observed that the screening collection is enriched for nitrogen-containing compounds and aromaticity, as opposed to the cembranoid-like and cembranoid libraries.   Colourless block crystals were poorly diffracting with no significant data beyond 0.84Å resolution and also non-merohedrally twinned, with a twinning ratio of 0.537:0.463. The twinned components were related by 179.8 rotation about reciprocal lattice vector 001. The data were collected on a Nonius-Kappa CCD area detector mounted at the window of an FR591 rotating anode generator with a Mo anode and equipped with an Oxford Cryosystems cryostream device. Nonius COLLECT 6 was used to record images and RigakuOD CrysAlisPro 10 was used for data integration. The structure was solved by direct methods using SHELXT 8 and refined on Fo 2 by full-matrix least squares refinement using SHELXL. 9 All non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were added at calculated positions and refined using a riding model with isotropic displacement parameters based on the equivalent isotropic displacement parameter (Ueq) of the parent atom. The structure was deposited on the Cambridge Structural Database with the deposition number CCDC 1011320. Colourless block crystals diffracted well. The data were collected on a Nonius-Kappa CCD area detector mounted at the window of an FR591 rotating anode generator with a Mo anode and equipped with an Oxford Cryosystems cryostream device. Nonius COLLECT 6 was used to record images and HKL (Denzo and Scalepack) 7 was used for for data integration. The structure was solved by direct methods using SHELXS 11 and refined on Fo 2 by full-matrix least squares refinement using SHELXL. 9 All non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were added at calculated positions and refined using a riding model with isotropic displacement parameters based on the equivalent isotropic displacement parameter (Ueq) of the parent atom. The structure was deposited on the Cambridge Structural Database with the deposition number CCDC 1435148.

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NCI cytotoxicity screening compounds Figure S2: Compounds screened at the NCI for cytotoxicity in a 60 cell line panel