Design, Synthesis, and Evaluation of Trihalomethyl Ketone Derivatives of Neocarzilin A as Improved Antimetastatic Agents

Abstract

Vesicle Amine Transport-1 (VAT1) is a protein that is overexpressed in many cancers, including breast cancer, glioblastoma, and angiosarcoma. High VAT1 expression correlates with poor overall survival, and genetic knockout models of VAT1 indicate potent antimigratory activity, suggesting that VAT1 is a promising antimetastasis target. Recently, the natural product neocarzilin A (NCA) from Streptomyces carzinostaticus was reported to be the first validated small-molecule inhibitor of VAT1, having strong activity in metastasis models of angiosarcoma and breast cancer. While knockdown of VAT1 has no effect on cell viability, NCA has significant cytotoxicity, suggesting that NCA is not selective for VAT1. Additionally, NCA has poor aqueous solubility, making in vivo administration of NCA challenging and thus limiting its therapeutic potential. Here, we report the design, synthesis, bioactivity, and pharmacokinetics of novel NCA derivatives with improved drug-like properties. Specifically, we have developed derivatives with altered warheads, replacing chlorines on the trichloroketone with fluorines. Using a modified synthetic route, we accessed NCA derivatives with greater than 25-fold improvements in solubility and 30-fold improvements in the antimigratory to antiproliferative bioactivity ratio. The two best derivatives, along with the parent, were analyzed for oral bioavailability, with the two more soluble derivatives showing greatly improved bioavailability. Overall, these studies have resulted in the development of VAT1 inhibitors with improved properties, which will enable further study of the pharmacological inhibition of VAT1 as an antimetastatic strategy. Additionally, these studies provide insights into novel trihalomethyl ketone warheads and identify chlorodifluoroketone as a potent and selective new warhead.

Figure 1

(A) Activities previously observed or proposed for Vesicle Amine Transport-1 (VAT1). VAT1 is a member of the NADPH-dependent oxidoreductases and can reduce 9,10-phenathrene quinone. VAT1 also regulates phospholipid transport between the ER and mitochondria and regulates mitochondrial fusion through interactions with mitofusin 1 (Mfn1). VAT1 has been shown to interact with proteins critical for focal adhesion complex formation; however, it is currently unclear whether those are direct or indirect interactions. (B) Structures of FDA-approved drugs with reversible covalent warheads (red) and the natural product neocarzilin A.

Figure 2

(A) NCA (gray) was covalently docked using Schrödinger Glide with covalent attachment at the hypothesized reactive Thr175 residue (dark blue) in the ligand binding site of VAT1 (light blue; PDB: 6LHR). Other important residues, including Glu113, Phe 325, and Leu328 (dark blue), are also indicated as sticks in the image. (B) A close-up of the model shown in part A. (C) A close-up of the model of NC-3 with VAT1.

Scheme 1. Divergent Syntheses of NCA and NCA Derivatives

Figure 3

(A) Previously studied trifluoromethyl ketones and their characteristic hydration. The HDAC inhibitor 8b and the clinically evaluated human leukocyte elastase inhibitor ZD-0892 have previously been shown to equilibrate to the hydrate species in water at different ratios depending on the substituents surrounding the warhead. (B) Equilibrium of ketone to hydrate in aqueous media for NCA and the derivatives NC-7 to NC-9. n = 3; error is standard deviation. Significance was determined using a student’s t-test with each additional fluorine, resulting in a significantly higher amount of hydrate (P < 0.0001).

Figure 4

Transwell migration activity of NCA and a subset of derivatives. (A) Representative images from three independent transwell migration assays of vehicle (DMSO), NCA, and derivative-treated MDA-MB-231 cells. All treatments were performed at 1 μM. (B) Quantification of migration. Experiments were performed at least three independent times. Dots indicate results from independent experiments. Bars indicate average results, and the error shown is the standard deviation. Student’s t-test was performed to compare each treated sample to the vehicle (DMSO) control. **P < 0.01; ***P < 0.001.

Figure 5

Pharmacokinetics analysis of NCA and derivatives. (A) Compound was administered at 25 mg/kg to mice (n = 3) via oral gavage. Plasma concentration was monitored over a period of 4 h. The dotted lines indicate 10 (top) and 1 μM (bottom). (B) Due to the compound still being in the distribution phase, NC-3 and NC-4 exhibited the shortest half-life. However, the derivatives exhibited a clear improvement in the absorption and systemic concentrations of the compound compared to NCA, highlighting their potential for studying VAT1 inhibition in vivo. k_el = elimination rate constant, t_1/2 = half-life, MRT = mean retention time, t_max = time at which maximum plasma concentration was reached, and C_max = maximum plasma concentration.