The synthesis is a variation of a reported method for compound 1

The synthesis is a variation of a reported method for compound 1.32 1-(4-(Trifluoromethyl)phenyl)ethan-1-one (2) and ethyl cyanoacetate were heated in toluene to afford the ,-unsaturated ethyl ester 3 as a mixture of enzyme inhibition on a panel of eight representative MMPs. families with similar active sites. combinatorial library enumeration method implemented in the CombiGlide program (v. 3.2, Schr?dinger, LLC, NY, USA). The analysis was based on the structural template CW-069 of benzoxazinone (1), where the structural variations were introduced at the highlighted points (Figure ?Figure11A). Meanwhile, we computationally clustered snapshots from the 500 ns of the dynamics trajectory of MMP-13, which resulted in identification of a total of six conformations in which R1 and R2 pockets were created (See Supporting Information for method Akt1 details). We docked compound 1 into these structures by the use of the Glide program36 (Schr?dinger, LLC, NY, USA), and the complexes were energy-minimized. We could reproduce the crystallographic fit seen for MMP-8 in two of these conformational states within 1 ? of the root-mean-squared deviation. These two conformations were then used for screening of the 28,099-strong virtual library using Glide. The compounds were ranked for the goodness of fit, but were eliminated, CW-069 if they did not conform to Lipinskis rule of five37 and Jorgensens rule of three.38?40 With synthetic access (and availability of the starting materials) being a final filter, we selected for synthesis 19 of the high-ranking compounds (See Supporting Information for method details). Five of the target compounds are given in Table 1; an additional 14 compounds are shown in the Supporting Information (Figure S6). We highlight the five-step synthesis of compound 8 below (Scheme 1). The synthesis is a variation of a reported method for compound 1.32 1-(4-(Trifluoromethyl)phenyl)ethan-1-one (2) and ethyl cyanoacetate were heated in toluene to afford the ,-unsaturated ethyl ester 3 as a mixture of enzyme inhibition on CW-069 CW-069 a panel of eight representative MMPs. A rapid up-front screening was conducted by incubating the compounds at 10 M with the corresponding enzymes and the requisite substrates. Those compounds that showed 50% inhibition were further evaluated, and their dissociation constants (inhibition data support direct binding of the compound to the target. The pharmacokinetic properties of compound 8 were assessed in mice (= 3 mice per time point per route of administration, total 54 mice) after intravenous (iv) and oral (po) dose administration (Figure ?Figure22, and Supporting Information). After a single 1 mg/kg iv dose of 8, the plasma concentration was 4.4 1.6 M at 2 min and remained above the = 3 per time point per route of administration, total 54 mice). In conclusion, we report here an example of how crystallographically similar binding sites may be exploited for selective inhibitor design based on their dynamic nature. Our computational simulations offered insights on the flexibility of CW-069 the structurally similar binding sites of MMP-8, MMP-13, and MMP-14. Encouraged by the opportunity offered by this simulation, a virtual focused library was designed and evaluated evaluation of a subset of the library successfully identified compounds with desired activity. The kinetic data clearly document that selectivity based on the design paradigm is achievable. The compound disclosed herein has favorable pharmacokinetic properties in mice and can potentially serve as a useful tool for delineating the functions of the MMP family of enzymes. Acknowledgments We acknowledge computing resources and assistance provided by Center for Research Computing of the University of Notre Dame. Glossary AbbreviationsHATU1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-library design, virtual screening, syntheses and characterization of compounds, enzyme inhibition studies, and pharmacokinetic studies (PDF) Movie of MD simulation (AVI) Notes This work was supported in part by the Craig H. Neilsen Foundation (grant 282987 to M.C.). Notes The authors declare no competing financial interest. Supplementary Material ml7b00130_si_001.pdf(1.9M, pdf) ml7b00130_si_002.avi(2.1M, avi).