Process Development and Scale-Up for the Preparation of the 1‑Methyl-quinazoline-2,4-dione Wnt Inhibitor SEN461 Matteo Betti,* ,† Eva Genesio, ‡ Alessandro Panico, § Salvatore Sanna Coccone, § and Paul Wiedenau ⊥ † Process Chemistry Unit and ‡ Compound Management & Analysis Unit, Siena Biotech SpA, 53100 Siena, Italy * S Supporting Information ABSTRACT: A practical and scalable route to the Wnt inhibitor SEN461 1 is described herein. The optimized route consists of nine chemical steps. The intermediates are solids and were isolated by filtrations. Critical reactions steps in the medicinal chemistry route were modified for an initial scale-up process, and as a result, we developed a synthetic procedure for the preparation of multihundred gram quantities of the final product. A further process development for the phase 1 clinical batch campaign is reported. ■ INTRODUCTION The Wnt signaling pathway is known to have a fundamental role in tissue development, differentiation and homeostasis; this is achieved by regulating a specific pool of genes that strictly control temporal and spatial regulation of cell growth, movement and cell survival. Chronic activation of the canonical Wnt pathway promotes uncontrolled cell growth and survival, and can consequently drive cancer formation in a range of tissues. 1-3 In the past decade a number of studies demonstrated that up-regulation of the Wnt pathway is present in various types of cancer (e.g., lung, breast, pancreatic, gastric, colorectal cancers, medulloblastoma, glioblastoma, hepatocellular carcino- ma). 4 For this reason targeting the canonical Wnt pathway can be a promising strategy in the identification and development of new anticancer agents. To date at least three Wnt-mediated intracellular signaling pathways have been identified: 5,6 the calcium-mediated and planar polarity pathways 8 and the canonical Wnt-β-catenin pathway. Siena Biotech has set up a drug discovery research program to identify new inhibitors of the β-catenin-dependent Wnt pathway for the treatment of glioblastoma multiforme. 7 After a screening campaign, a primary hit series was discovered; medicinal chemistry modifications and optimization of the original structures led to the identification of an advanced lead compound, SEN461 shown in Figure 1. ■ RESULTS AND DISCUSSIONS MedChem Synthesis. The first synthesis of SEN461 (Scheme 1), carried out in our Medicinal Chemistry laboratories, delivered gram quantities of SEN461 to support in vitro and preliminary in vivo testing. The synthesis, although long with nine steps, is convergent, but it had some important major drawbacks which necessitated its redesign: (a) the anthranilic acid 2 starting material is quite expensive; (b) the esterification of 4 with sulfuric acid and subsequent free basing gave 5 contaminated with 5-10% of its dimer a (Figure 2), which was difficult to separate in this stage and in the following stages; (c) the acylpiperazine 12 proved to be unstable, giving piperazine hydrochloride as a decom- position product and possibly contributing to the poor yield of the coupling step to give amide 13; (d) the acid 8 and its corresponding lithium salt have extremely poor solubilities and consequently, were difficult to handle; (e) N-methylation in the last step was relatively low-yielding and required chromato- graphic purification. Redesign of the Synthesis. The alternative sequence shown in Scheme 2 was designed, preserving the ring-closure step to intermediate 7 but starting from the significantly less expensive nitrobenzoic acid 14. 9 Although the sequence features the same number of steps and is more linear, it overcomes a number of problems of the original route, namely the use of the toxic and corrosive triphosgene and the use of the unstable acylpiperazine 12. Introducing the N-Me group early on (7 to 16) in the synthesis avoids the relatively low-yielding final step of the original procedure, as well as renders the intermediates, especially the acid 17, more soluble and more easy to handle. Initial Scale-Up: 50 and 500 g Campaigns. Two campaigns were carried out to synthesise SEN461 batches for preclinical studies. The first campaign furnished 50 g of the desired compound and included some route scouting and reaction optimization work, which is described in detail in the corresponding section. For the second campaign, which Received: June 3, 2013 Figure 1. Chemical structure of the Wnt inhibitor SEN461. Article pubs.acs.org/OPRD © XXXX American Chemical Society A dx.doi.org/10.1021/op400145w | Org. Process Res. Dev. XXXX, XXX, XXX-XXX