(C) Viability of noncancerous MCF10A mammary epithelial cells following 72-hour treatment with FASNi in standard media. Center for Chemical Genomics using purified FASN thioesterase (FASN-TE) domain. Despite being a potent inhibitor of purified FASN-TE, compound 1 proved highly unstable in mouse plasma and only weakly cytotoxic to breast cancer (BC) cells in vitro. An iterative process of synthesis, cytotoxicity testing, and plasma stability assessment was used to identify a new lead (compound 41). This lead AP20187 is more cytotoxic against multiple BC cell lines than tetrahydro-4-methylene-27.24) or DMSO-2.49). The 13C NMR spectrum of compound 41 was recorded at 100.5 MHz in CDCl3 with CHCl3 as the internal reference (77.00). Synthesis. Synthesis of compound 1.4 from compound 1.1 was performed as shown in Scheme 1, as previously described (Solinas et al., 2008). Briefly, freshly prepared compound 1.2 was used in the reaction with AP20187 lysine derivative 1.3 to give compound 1.4 as a white or off-white solid following extraction and precipitation. This intermediate can be stored at room temperature indefinitely. Two equivalents of glycine benzyl ester hydrochloride and compound 1.5 were added to compound 1.4 in dichloromethane along with two equivalents of diisopropylethylamine, and the reaction was stirred at room temperature for 16C48 hours. The progress of each reaction was followed with thin layer chromatography. Purification by column chromatography using a gradient of hexanes and ethyl acetate afforded compound 1 as a film with 1H NMR and liquid chromatography (LC)/mass spectrometry data consistent with that previously reported for this compound (Solinas et al., 2009). Compounds 11C43 were prepared from compound 1.4 using the same approach and the corresponding amino acid or amine to replace compound 1.5. Compounds 15C24 required the synthesis of derivatives of compound 1.5 that were subsequently used in the reaction. Compounds 11C43 were purified by column chromatography using a gradient of hexanes/ethyl acetate or dichloromethane with 3% methanol (v/v). The purity and identity AP20187 of the final products were determined by LC, liquid chromatographyCtandem mass spectrometry (LC-MS/MS), and 1H NMR spectroscopy. Additional details regarding the synthesis and characterization of these derivatives are provided in the Supplemental Material. Open in a separate window Scheme 1. Synthesis of compound 1 from compound 1.1. LC and LC-MS/MS Supporting Compound Purity and Identity. Compounds were analyzed for purity and identity by LC-MS/MS using a Dionex Ultimate 3000 component LC system and Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer (LC-MS/MS) with HESI-II probe operating in positive ion mode. Solutions of the compounds were injected onto a 1.9 tests with correction for multiple comparisons (the Holm-Sidak method), where applicable, on the mean values of at least three independent in vitro experiments. Values of < 0.05 were deemed significant: *< 0.05, **< 0.01, AP20187 ***< 0.001. Error is presented as mean S.D. Plasma Stability Assay. Each FASN inhibitor (200 nM in a total volume of 50 for 20 minutes at VCL 4C. Supernatants were collected and 25 = 3C5 experiments). Here and in all ensuing figures, the IC50 values were generated using a nonlinear regression curve-fitting algorithm: log(inhibitor) vs. response-variable slope (four parameters) with Graph Pad Prism 6.0. (C) Compiled IC50 and mouse plasma stability results, represented by percentage of compound remaining after 1 hour incubation in mouse plasma at 37C (N.D., not determined). Open in a separate window Fig. 3. Structure of ester-to-amide derivatives of 1 1 along with methyl groups that add steric bulk. The Benzylamine Series. Benzylamine derivative 25 was flagged as active in the initial high-throughput screen with purified FASN-TE, and also formed the base structure of compound 13, which demonstrated improved cytotoxicity in our assays. (The original bioassay can be viewed in PubChem by searching AID 602261 under PubChem Bioassay. The SID number for compound 15 is 24833677. A direct link to the bioassay data for compound 15 is as follows: https://pubchem.ncbi.nlm.nih.gov/bioassay/602261#sid=24833677§ion=Top.) Compounds based on a substituted benzylamine (compounds 26C37) were thus prepared and tested. Substituents on the aromatic ring impacted both the cytotoxicity and the plasma stability; however, even the more potent of these compounds was not sufficiently stable to justify further investigation (Fig. 4). Open in a separate window Fig. 4. Structure, cytotoxicity, and mouse plasma stability data for compounds 25C37. (A) Benzylamine derivatives 25C37: structure, compiled IC50 values, and mouse plasma stability results, represented by percentage of compound remaining after 1-hour.