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Chapter 3
Discovery and Chemical Development of Verubecestat, a BACE1 Inhibitor for the Treatment of Alzheimer’s Disease David A. Thaisrivongs,1,* William J. Morris,1 and Jack D. Scott2 1Process
Research and Development, Merck & Co., Inc., Rahway, New Jersey 07065, United States 2Discovery Chemistry, Merck & Co., Inc., Kenilworth, New Jersey 07033, United States *E-mail:
[email protected] This chapter highlights the discovery and chemical development of verubecestat, a potent and selective beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor that has been evaluated in late-stage clinical studies as a potential disease-modifying therapy for the treatment of Alzheimer’s disease. The medicinal chemistry effort that culminated in the invention of verubecestat focused on the design, synthesis, and evaluation of various iminoheterocyclic BACE1 inhibitors in order to discover a compound with a profile that would enable clinical progression to evaluate the safety and efficacy of chronic administration in patients. Once verubecestat was identified as a suitable clinical candidate, a synthetic route was developed to supply multikilogram quantities of the compound to support preclinical safety studies and the early clinical program. As verubecestat progressed into Phase III studies, a short, efficient, and robust manufacturing process was developed.
Amyloid Hypothesis in Alzheimer’s Disease Alzheimer’s disease is a progressive neurodegenerative disease that accounts for 60–80% of the estimated 47 million cases of dementia worldwide; overall costs of this illness are expected to approach $1 trillion in 2018 (1, 2). It © 2018 American Chemical Society
has been predicted that by 2050, without additional health care intervention, there will be over 130 million people afflicted with dementia globally (1). The currently available therapies provide only modest symptomatic improvement for Alzheimer’s disease patients and do not target the underlying disease etiology. In the face of such a global human health crisis, the identification of a disease-modifying therapy to slow this neurodegeneration has become an urgent unmet medical need. According to the amyloid hypothesis, the buildup of neurotoxic amyloid beta (Aβ) peptides in the brain over a period of years, possibly decades, leads to the formation of the amyloid plaques and neuronal death that characterize Alzheimer’s disease (3). The formation of Aβ peptides arises from the sequential cleavage of amyloid precursor protein (APP), first by the membrane-bound aspartyl protease beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and then followed by the γ-secretase enzyme complex. This process provides Aβ peptides of varying lengths with Aβ40 predominating (4, 5). These Aβ peptides, especially Aβ42, have been shown to oligomerize, eventually leading to neurotoxicity (5). There are a number of other substrates processed by BACE1, and the biological understanding and implications of chronic inhibition of this enzyme continue to be areas of intense study (6–9). In addition, there is a structurally related aspartyl protease, BACE2, which is largely expressed in the periphery and whose function has also been extensively studied in the context of beta cell function and pigmentation (10–12). Genetic validation for the role of BACE1 in disease progression includes the identification of mutations in APP that lead to enhanced cleavage by BACE1, which account for a portion of familial Alzheimer’s disease cases (9). More recently, a study reported the identification of a mutation in APP (A673T) 2 residues toward the N-terminus of the BACE1 cleavage site that correlates with a protective effect against Alzheimer’s disease and a deceleration in cognitive decline. This mutation was shown in vitro to reduce BACE1 APP processing, leading to a 40% reduction of Aβ40 and reduced aggregation (13). On the basis of this evidence, among other compelling data, it has been hypothesized that BACE1 inhibition, which decreases the production of Aβ peptides, has the potential to be a disease-modifying therapy for Alzheimer’s disease.
Discovery of Verubecestat Verubecestat (1, Figure 1) represents the culmination of a multiyear research campaign at Merck & Co., Inc. (Kenilworth, NJ, United States) to invent potent, selective inhibitors of BACE1 for evaluation as a potential disease-modifying treatment for Alzheimer’s disease (14–19). The structure of verubecestat arose from a multifaceted discovery effort that began with the identification of weakly binding hits from an NMR-based fragment screen undertaken in parallel to more traditional approaches toward aspartyl protease inhibitors (19, 20). Enabled by an X-ray co-crystal structure of 2 bound to BACE1 and a robust collaboration with our structural biology and modeling colleagues, we designed several novel iminoheterocyclic aspartyl protease binding cores, including iminohydantoins 54
(e.g., 3), iminopyrimidinones (e.g., 4 and 5), and iminothiadiazine dioxides (e.g., 6 and verubecestat). With these cores, we explored a wide variety of substituents, including biaryls (e.g., 3, 4, and 6) and diaryl amides (e.g., 5 and verubecestat). Due to the high homology of BACE1 and BACE2 around their active sites, selectivity for BACE1 over BACE2 was difficult to achieve during the course of our discovery efforts. While the biological understanding of dual inhibition of BACE1 and BACE2 continues to evolve, the reported phenotypes did not preclude compound progression (10–12, 21).
Figure 1. Selected preclinical data of the isothiourea fragment hit and lead iminoheterocyclic BACE1 inhibitors.
Among the extensive characterization of our BACE1 inhibitors, two assays in particular provided important data to differentiate compounds during lead optimization. The first was an in vitro assay for Cathepsin D (CatD) inhibition. CatD is a widely expressed aspartyl protease with an important role in the lysosomal degradation of proteins (22). Loss of function of this enzyme through inhibition or by genetic knockout in preclinical species is not well tolerated (23, 24), and thus selectivity over CatD was assessed very early in our evaluation of 55
compounds (19). The second assay of note was an in vivo assessment of the pharmacodynamics of Aβ40 lowering in both the cerebral spinal fluid (CSF) and cortex of rats. This assay enabled assessment of the ability of an orally dosed BACE1 inhibitor to modulate the levels of Aβ40, a clinically relevant biomarker, in the central compartment of rats. Ultimately, the combination of our unprecedented iminothiadiazine dioxide core with a diaryl amide substituent distinctly provided the best overall combination of BACE1 potency and selectivity, as well as the pharmacokinetic, pharmacodynamic, and preclinical safety profile required to enable long-term clinical dosing in patients and thus test the amyloid hypothesis with a BACE1 inhibitor (18, 21). Verubecestat progressed into Phase I studies, where it was found to be generally well tolerated in both normal healthy volunteers and Alzheimer’s disease patients. A significant reduction in the levels of Aβ40 and Aβ42 in the CSF of both normal healthy volunteers and patients was observed over 14 and 7 days of dosing, respectively (21). Subsequently, verubecestat became the first BACE1 inhibitor to progress into Phase II/III studies. The first trial, EPOCH, evaluated the efficacy and safety of verubecestat in patients with mild to moderate Alzheimer’s disease. In the Phase II portion of EPOCH, the safety of verubecestat was evaluated over 3 months in 200 patients at doses of 12, 40, and 60 mg. The subsequent Phase III trial studied the safety and efficacy of verubecestat in approximately 1800 randomized patients over 78 weeks at doses of 12 and 40 mg (25). In the second Phase III trial, APECS, the safety and efficacy of verubecestat was evaluated in approximately 1500 patients with prodromal Alzheimer’s disease over 2 years at doses of 12 and 40 mg (21).
Medicinal Chemistry Synthesis of Verubecestat The route used by the medicinal chemistry team to prepare verubecestat (1) started by adding the lithium anion of methyl sulfonamide 10 to Ellman ketimine 9 to provide sulfinyl amine 11 in moderate yield and diastereoselectivity (Scheme 1); the major isomer could be readily isolated using silica chromatography (18). The chiral auxiliary was cleaved using hydrogen chloride to produce amine 12, and the para-methoxybenzyl group was subsequently removed upon treatment with trifluoroacetic acid in the presence of 1,3-dimethoxybenzene as a benzyl cation scavenger to afford the aminosulfonamide 13. We initially attempted a three-step method to assemble the thiadiazine core using a protocol that had been efficiently executed on an analogous substrate class (Scheme 2) (18). In that case, treatment of aminosulfonamide 14 with benzoyl isothiocyanate afforded thiourea 15. The benzoyl group was readily removed with sodium methoxide to afford thiourea 16. The thiadiazine ring was then formed via activation of the sulfur atom with iodomethane and subsequent intramolecular cyclization to furnish 17 in an overall 81% yield. 56
Scheme 1. Medicinal chemistry route to aminosulfonamide 13.
Scheme 2. Thiadiazine ring formation using benzoyl isothiocyanate in an analogous substrate class.
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While the formation of analogous benzoyl isothiocyanate 18 proceeded in high conversion from aminosulfonamide 13, the subsequent benzoyl removal using sodium methoxide did not afford thiourea 19 (Scheme 3) (18). Instead, multiple side-products were detected that arose from nucleophilic aromatic substitution of the aromatic fluorine, a vulnerability that was heightened by the para-nitro group. The use of less basic sodium carbonate did, however, allow for the isolation of thiourea 19 in 70% yield. Thiadiazine ring formation using iodomethane at an elevated temperature also proved problematic given the continued susceptibility of the substrate toward unwanted nucleophilic aromatic substitution, and we were ultimately able to obtain only a modest yield of thiadiazine 20 (18). While this route did afford sufficient quantities of the target to enable initial medicinal chemistry efforts, investigations continued into alternative thiadiazine ring-closing conditions to further reduce the formation of nucleophilic aromatic substitution side-products.
Scheme 3. Initial route to thiadiazine core 20.
In short order, the use of cyanogen bromide in refluxing 1-butanol was identified as an efficient method for forming thiadiazine 20 in a single step from aminosulfonamide 13 with reasonable conversion (Scheme 4) (18). Crude 20 was treated with di-tert-butyl dicarbonate to afford protected thiadiazine 21 in moderate isolated yield over the sequence. The nitro group was then reduced under heterogeneous conditions to the corresponding aniline (22) in high yield. This functional handle allowed for extensive investigation of the structure–activity relationship of inhibitors with respect to the S3 pocket of BACE1. The medicinal chemistry synthesis of verubecestat was completed with the installation of the fluoropyridine amide and the cleavage of the thiadiazine protecting group to provide 1 in high yield over the final two steps.
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Scheme 4. Completion of the medicinal chemistry synthesis of verubecestat (1).
First Generation Process Route The first generation process route to verubecestat, which was implemented to supply both the preclinical safety work and the early clinical studies, relied substantially on the synthesis developed by the medicinal chemistry team (Scheme 5) (14, 26). This route used the same bond formation sequence as the previous one, but the discovery of two new intermediate salts were key to enabling API production on a multikilogram scale. The synthesis began with the deprotonation of N-methyl sulfonamide 10 using n-butyllithium and the diastereoselective addition of the resulting anion to sulfinyl ketimine 9 to give the corresponding sulfinyl amine 11. This intermediate was treated with excess trifluoroacetic acid to remove both the para-methoxybenzyl protecting group and the chiral auxiliary, revealing the corresponding aminosulfonamide 13. The enantiopurity of this intermediate was upgraded via the preparation and crystallization of the mandelate salt 25. This treatment provided an added advantage of avoiding the need for tedious preparative chromatography to separate the stereoisomers introduced in the formation of 11.
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Scheme 5. First generation supply route to verubecestat (1). After freebasing the mandelate salt 25, we performed the cyclization with cyanogen bromide to provide thiadiazine 20. Protection of the resulting free amine as the corresponding tert-butyl carbamate and subsequent catalytic hydrogenation of the aryl nitro group with palladium on carbon revealed aniline 22. The aniline 22 was coupled with 5-fluoro-2-picolinic acid (23) using propylphosphonic anhydride (T3P). Removal of the protecting group with para-toluenesulfonic acid allowed for the isolation of para-toluenesulfonic acid salt 26, which provided an essential point of API purity control, particularly given the ensemble of product-related impurities generated during the hydrogenation to make 22. Finally, treating salt 26 with potassium carbonate and crystallization of 1 as the freebase completed the first generation process route.
Scouting Route Alternatives An ideal manufacturing process is not only one that requires a minimum number of high-yielding chemical transformations, but one that is also 60
environmentally friendly, operationally safe, reproducibly robust, and low cost. Thus, despite the scalability and efficiency of the first generation process route, we initiated a wide-ranging exploration to identify alternative syntheses of verubecestat (1) that could form the basis of a commercial manufacturing process. The key challenge in considering how to best synthesize 1 was the enantioselective formation of the stereogenic α,α-dibranched amine. To this end, we considered a variety of retrosynthetic disconnections that offered distinct strategic approaches for targeting this central functional group (Figure 2). In particular we were highly interested in evaluating enantioselective methods for introducing the desired stereochemistry, and pursued many that incorporated C-H insertion, 1,2- and 1,4-addition, and aziridination chemistries, among others. In the end, few of these endeavors bore fruit, a reflection of the relative paucity of methods known for constructing a stereogenic α,α-dibranched amine in a highly enantioselective manner.
Figure 2. Some of the evaluated strategies for the synthesis of the core of verubecestat (1).
One notable innovation made in the course of work directed specifically at the development of an enantioselective Mannich-type reaction for the synthesis of 1 was the discovery of an enantioselective palladium-catalyzed synthesis of cyclic sulfamidates (27). In this reaction, an enantiometically pure substituted phosphinooxazoline ligand controls the stereoselectivity of a palladium-catalyzed arylation reaction between cyclic iminosulfates and arylboronic acids. Both electron-poor and ortho-substituted arylboronic acids can be engaged productively 61
in this process, including 5-bromo-2-fluorophenylboronic acid (27), which when treated with cyclic iminosulfate 28 provides cyclic sulfamidate 29 in high yield and enantioselectivity (Scheme 6). We were able to subsequently demonstrate that this intermediate can be converted in five steps to 1. Ultimately, however, this alternative route was not competitive economically with those that leveraged the diastereoselective methodology, which enabled the first synthesis of verubecestat (1) by the medicinal chemistry team.
Scheme 6. Palladium-catalyzed enantioselective synthesis of cyclic sulfamidate 29 and its application to the synthesis of verubecestat (1).
Having made the determination that a Mannich-type addition of a suitable methyl sulfonamide (10) to an Ellman sulfinyl ketimine (34) was the most efficient way of accessing the stereogenic α,α-dibranched amine of 1 (Figure 3), our retrosynthetic analysis was reduced to strategic decisions of how to order and execute the following transformations: (1) removal of both the sulfonamide protecting group and the chiral auxiliary, (2) intramolecular cyclization of the resulting diamine (30) with a source of cyanogen to provide the desired thiadiazine of 1, and (3) installation of the secondary amide. We recognized that the existing synthesis of this amide, which relied on a coupling of 5-fluoro-2-picolinic acid (23) and aniline 22 (Scheme 5), necessitated a four-step sequence that included the installation and removal of a protecting group (from 20 to 26, vide supra). From the outset of our commercial route scouting activities, we were confident that replacing this series of functional group interconversions with a single transition metal-catalyzed C–N coupling of commercially available 5-fluoro-2-picolinamide (31) with an aryl bromide (32) would substantially improve the efficiency of the overall route. All of these disconnections led back to compounds that are or can be readily prepared in one step from commercially available materials. 62
Figure 3. Retrosynthetic analysis of verubecestat (1).
Sulfinyl Ketimine Synthesis In the first generation process route to 1 (Scheme 5), exposing 1-(2-fluoro-5nitrophenyl)ethan-1-one (7) and a slight molar excess of (R)-2-methylpropane-2sulfinamide (8) to a superstoichiometric amount of the strong dehydrating agent titanium(IV) isopropoxide provided sulfinyl ketimine 7 in 67% yield and 96.7% purity on a multikilogram scale (Scheme 7). Though this process was capable of delivering suitable quantities of material to support the early clinical development program, it nevertheless required a series of very inefficient unit operations that we sought to avoid in the context of a commercial manufacturing route. Removal of the residual inorganics was achieved by adding water at the end of the reaction to precipitate them, largely as titanium dioxide. However, the physical properties of this amorphous solid made it exceedingly challenging to separate from the rest of the batch, so much so that for a pilot plant scale run, 15 separate centrifuge filtrations were necessary to recover the product solution from the paint-like mixture of inorganic salts. A subsequent carbon treatment of the filtrate at 30% w/w was still needed to further purge impurities before the product was crystallized. Even after a thorough optimization of this workup and isolation process, more than 80 volumes of solvent in total relative to 7 were required to isolate 9, and vessels used for this process required additional cleaning protocols above and beyond typical rinses between batches in a commercial manufacturing setting. This inefficiency is a general problem with the formation of such ketimines. In a recently published Organic Synthesis protocol on the same reaction using 63
acetophenone, which exemplifies the state of the art for these transformations, over 165 volumes of solvent are needed just to filter off the titanium oxide generated upon the addition of water (28).
Scheme 7. Synthesis of sulfinyl ketimine 9 in the first generation process route. There are a variety of efficient methods for the formation of sulfinyl aldimines, including dehydrating agents such as magnesium sulfate (29), copper sulfate (30), cesium carbonate (31), potassium bisulfate (32), and molecular sieves (33), strong bases such as sodium hydroxide and potassium tert-butoxide (34), and Lewis acids such as ytterbium triflate (35) and tris(2,2,2-trifluoroethyl) borate (36). The only reagent, however, that can be generally utilized in the synthesis of nearly all classes of sulfinyl ketimines is titanium(IV) isopropoxide (37). Although resins have been shown to scavenge such titanium species, an impractical loading is required since excess reagent is often necessary to drive the ketimine condensation to completion (38). Despite a wide survey of dehydrating agents, we did not identify a superior reagent for the synthesis of 36 (vide infra), both with respect to yield and bulk reagent cost. Efforts to crystallize 36 (Scheme 8) in the presence of stoichiometric metal salts were met with little success, and the addition of filtering aids like Celite did not substantially improve the characteristics of the workup. The key process development breakthrough was to simply avoid the precipitation of any inorganics altogether. This was achieved by quenching the crude reaction not with water but with a concentrated solution of excess aqueous potassium glycolate, which preferentially generated titanium glycolate over titanium oxide. Glycolic acid is a cheap, nonhazardous, and biodegradable alpha-hydroxyl acid that has low toxicity and broad metal-sequestering properties (39). Titanium glycolate is a reported inorganic complex (40) but to our knowledge has never been reported in the context of a synthetic application or as a method to simplify the aqueous workups of titanium-mediated reactions. While there are isolated reports of workup protocols for reactions employing titanium alkoxides that enable the residual metal salts to be washed away (41–43), despite the ubiquity of titanium(IV) isopropoxide-mediated formations of sulfinyl imines in the organic synthesis literature, we are not aware of such a reaction that avoids the filtration of titanium oxide. Additional optimization of the sulfinyl ketimine formation parameters revealed that at slightly elevated temperatures a much lower excess of titanium(IV) isopropoxide was required, further enhancing the robustness of the revised workup protocol. A 20% improvement in yield, an over 60% reduction in waste, and 64
the isolation of product in considerably higher purity in the commercial process compared with the first generation process route (Scheme 7) without changing a single reagent or solvent underscores how there are opportunities for development along the entire process train that can deliver substantial improvements to the overall transformation.
Scheme 8. Synthesis of sulfinyl ketimine 36 in the commercial process route.
Synthesis of the α,α-Dibranched Amine We experimented with alternative protecting groups for methyl sulfonamide 10 (Scheme 9) in Mannich-type reactions with sulfinyl ketimine 36, including the analogous tert-butyl carbamate and tert-butyldimethylsilyl derivatives, and even evaluated the unprotected variant (which necessitated the formation of the corresponding dianion), but none proved as effective as para-methoxybenzyl. In the commercial process route, the synthesis of 10 began with the addition of para-anisaldehyde (37) to a solution of methylamine in methanol, which spontaneously formed N-methyl imine 38 (Scheme 9) (44). The subsequent reduction was accomplished by adding the crude solution of 38 to a suspension of sodium borohydride in tetrahydrofuran. The reaction was worked up with aqueous sodium hydroxide and the resulting secondary amine extracted with toluene. The solution of 39 could then be azeotropically dried efficiently, at which point triethylamine and methanesulfonyl chloride were added. Once the formation of 10 was complete, the reaction was quenched with water and the final product crystallized by adding n-heptane to the organic layer. This through-process was highly optimized, and at scale provided 10 in 92% yield over the three reactions.
Scheme 9. Synthesis of methyl sulfonamide 10 in the commercial process route. 65
The reaction of methyl sulfonamide 10 with sulfinyl ketimine 36 behaved similarly to the analogous reaction with 9 in the first generation process route (Scheme 5). Excess nucleophile was still necessary for high conversion, and the addition of the lithium anion of 10 to sulfinyl ketimine 36 had to be conducted under cryogenic conditions (–60 to –65 °C). This temperature operating window is extremely energy-intensive to achieve and maintain on a large scale. It constrains the selection of vessels and even commercial manufacturing sites for such processes. Despite a substantial effort to optimize the original reaction parameters, including an exhaustive survey of organometallic bases, additives, solvents, and protecting groups, only marginal improvements were realized, and on a pilot plant scale only a 73% assay yield of sulfone adduct 40 could be obtained (Scheme 10).
Scheme 10. Synthesis of 40 using the protocol from the first generation process route. This result is typical of such reactions with ketimines. To our knowledge, every reported diastereoselective addition of an organometallic reagent to a chiral ketimine is performed under cryogenic conditions, and with rare exception, the isolated yield is moderate (below 75%) (45). Since there were no significant sideproducts that formed during the course of this process, we attributed our modest result to the basicity of both the nucleophile and the unquenched product. When a reaction was quenched with excess deuterated acetic acid and the remaining starting materials recovered, the methyl group of sulfinyl ketimine 42 had greater than 99% incorporation of a deuterium atom (Scheme 11) (46). This result is consistent with a kinetic competition between 1,2-addition and alpha-deprotonation when the anion of methyl sulfonamide 41 reacts with sulfinyl ketimine 36. In a similar mechanistic experiment, when anion 43 (prepared by deprotonating 40 with an equimolar amount of n-hexyllithium) was added to sulfinyl ketimine 36, the methyl group of the recovered 42 had 87% deuterium incorporation (Scheme 12). Both of these side reactions irreversibly reduce the amount of 36 available for reaction, which accounts for the observed incomplete conversion even when a large excess of nucleophile is employed. We hypothesized that when this process is conducted in batch mode, the actual kinetic selectivity of the desired 1,2-addition over the competing α-deprotonation, as judged by analysis of the product distribution, is masked by the mixing rate of the reactive species (47, 48). Since we had also observed that the reaction was extremely fast even at cryogenic temperatures (i.e., complete within seconds at most), we further postulated 66
that executing the same chemistry in a continuous mode with extremely fast micromixing (49–54) should limit the exposure of unreacted sulfinyl ketimine 36 with anion 43, concomitantly reducing the amount of undesired α-deprotonation caused by the unquenched product. To evaluate the potential for a continuous process to improve the α,α-dibranched amine synthesis, we assembled a lab-scale flow reactor using four high-performance liquid chromatography pumps, stainless steel and fluoropolymer tubing, and static mixing tees (Table 1). With each of the four pumps (delivering methyl sulfonamide 10, n-hexyllithium, sulfinyl ketimine 36, and an acetic acid quench solution) set at 1 mL/min, 55% conversion to α,α-dibranched amine 40 was observed with no significant byproducts (entry 1).
Scheme 11. Deuterium quenching experiment reveals deprotonation of 36 by nucleophile 41.
Scheme 12. Deuterium quenching experiment reveals deprotonation of 36 by unquenched product 43. 67
Table 1. Proof-of-Concept for Improved Reaction Performance in Continuous over Batch Mode for the Synthesis of 40
At these relatively low flow rates, the mixing quality at the static mixing tees was poor, resulting in a significant amount of deprotonated sulfinyl ketimine 36. In stark contrast, when all other variables were kept constant and the pumping rate was increased (e.g., to 20, 32, and 40 mL/min; entries 2, 3, and 4, respectively), 86 to 88% conversion was achieved. This simple experiment provided a wealth of insight into the potential of flow chemistry to improve this process. The correlation between total flow rate and reaction conversion was consistent with our hypothesis that the quality of mixing between lithium anion 41 and sulfinyl ketimine 36 was a critical parameter governing the extent of unwanted α-deprotonation of 36 by unquenched product 43. A static micromixer was essential for making these observations; with a simple 0.05-in. inner diameter tee, the conversion at a total flow rate of 20 mL/min was only 66%. In addition, that the conversion appeared to plateau once 68
a certain mixing characteristic was achieved suggested that the balance of the material represented the unwanted kinetic competition between 1,2-addition and α-deprotonation when sulfinyl ketimine 36 reacts with 41 (vide supra). Further, the data also demonstrated that there was a substantial efficiency gain by operating in a continuous instead of a batch mode, as these superior results were all obtained at a noncryogenic temperature (-10 °C). High flow rates also significantly shortened the residence time for both the deprotonation of 10 and the reaction of 36 with 41. At the fastest flow rate (entry 4), lithium anion 41 reacted with sulfinyl ketimine 36 after only 30 ms (τ1) of its formation, and the resulting product 43 was quenched with acetic acid after only an additional 2.5 ms (τ2), yet the product was obtained in 87% conversion (entry 4). In fact, we have been unable to execute a continuous process with a residence time that is too short for both the deprotonation and sulfinyl ketimine addition to achieve completion, which suggests that as long as the mixing characteristics are sufficiently good, both reactions have reached their maximal conversion when the respective streams leave the mixing chambers. Regardless of the residence time, however, the diastereomeric ratio of the product was the same. In the first generation process route, an excess of nucleophile was necessary to achieve optimal reaction performance, with the maximum conversion achieved at 1.7 equivalents of methyl sulfonamide 10 relative to the sulfinyl ketimine. In this respect, the continuous process mirrored the batch one. An evaluation of reagent stoichiometry in flow was straightforward to perform using our lab-scale equipment by simply adjusting the relative pumping rates and collecting quenched crude reaction samples once steady state had been established at each flow rate set point. Optimal reaction conversion was observed with 1.5 equivalents of nucleophile, but to ensure robustness on a larger scale, we choose to employ 1.7 equivalents of n-hexyllithium and 1.8 equivalents of methyl sulfonamide 10 relative to sulfinyl ketimine 36. Our proof-of-concept experiments demonstrated that, unexpectedly, only moderate cooling was required to achieve significant reaction conversion (Table 1), which enabled much of our subsequent optimization work to be performed by simply submerging the heat exchangers and micromixers in a –10 °C cooling bath (Table 2, entry 1). We were unable to thoroughly evaluate reactions below –30 °C, as precipitation of both methyl sulfonamide 10 and lithium anion 41 quickly clogged the flow path at those temperatures. To our further surprise, there was no significant difference between continuous reactions conducted at –10 °C and 1 °C (entries 1 and 2). Even at ambient temperature, only a small reduction in conversion was observed (entry 3). Remarkably at 38 °C, a temperature at which the half-lives of organolithium species in ethereal solvents are on the order of mere hours (55), the reaction performance was still not substantially altered (entry 4). To our knowledge, these data represent reaction conditions that are far removed from any reported ketimine addition with a hard nucleophile and underscore the extreme conditions that are both accessible and can be uniquely effective when operating in a continuous mode (56). 69
Table 2. The Synthesis of 40 in Continuous Mode at Extreme Temperatures
Maintaining steady-state performance of an organolithium-mediated reaction in flow for many hours can be a steep challenge (57), and subsequent experiments using kilogram-scale equipment were necessary to uncover additional processing risks that were not revealed when the continuous process was conducted for relatively short periods of time. For example, although the reaction performed just as well around 0 °C as it did at -20 °C, over the course of tens of minutes we observed a gradual increase in backpressure at each of the pumps, signaling that a system clog was building. Further increasing the temperature to 20 °C hastened the decline from steady-state operation. Since rigorously dried feed solutions caused the equipment to clog at approximately the same rate as wet feed solutions, adventitious moisture did not appear to be the primary source of fouling. Neither did the issue seem to be only the insolubility of methyl sulfonamide 10 or lithium anion 41, since the effect of either should be less pronounced at higher temperatures. Instead, we hypothesized that a decomposition event at the point of mixing of methyl sulfonamide 10 and n-hexyllithium was exacerbated for prolonged periods of time at temperatures substantially above -20 °C. We measured the heat of reaction for this deprotonation as -85.2 kJ/mol with an accompanying adiabatic temperature rise of 31.2 °C, and although the heat removal in flow is generally highly efficient, the precise point along the flow 70
path at which 10 interacts with n-hexyllithium likely reaches and maintains a much warmer temperature than the surrounding cooling liquid. Further evidence that is consistent with this hypothesis was obtained when lithium anion 41 was prepared in batch at low temperature and then warmed to 20 °C; decomposition and precipitation were observed (58). When the feed solutions were all delivered at -20 °C, we could execute the continuous process on a kilogram scale for an hour without event. Nevertheless, warning signs remained that it would not be possible to perform the continuous deprotonation for the even longer periods of time that would be required for commercial-scale batches. Specifically, the deposition of unidentified insoluble material at the point of contact between methyl sulfonamide 10 and nhexyllithium led, over time, to an increase in the pressure drop through the first mixer. No data pointed to significant risks of fouling at either the sulfinyl ketimine addition or quench mixers. A way to completely avoid this issue was to generate lithium anion 41 in a batch reactor and then flow the resulting solution into the reaction mixer (Figure 4) (59). This design eliminated the fouling event associated with the continuous deprotonation, but necessarily created trade-offs. First, the feed solutions for the fully continuous process design (methyl sulfonamide 10 in tetrahydrofuran, nhexyllithium in hexanes, and sulfinyl ketimine 36 in tetrahydrofuran) were very stable at an ambient temperature, while lithium anion 41 was not. Thus it was necessary to generate and maintain 41 at a low temperature (i.e., -20 °C) over the course of the entire batch.
Figure 4. Redesigned continuous process that incorporates a batch deprotonation of 10 to improve chemical robustness.
Second, temperature-controlled jacketed lines were also required to transfer lithium anion 41 from the deprotonation vessel to the reaction mixer. Critically, however, this strategy still realized the performance improvements of executing the sulfinyl ketimine addition in flow while ensuring the deprotonation of 10 was robust. Lab-scale tests of the redesigned process demonstrated that it enabled steady-state operation for multiple hours and that the changes did not diminish the overall reaction performance. 71
Nevertheless, in our first preparation of 40 in batch mode at pilot scale, we discovered a new crystalline phase of lithium anion 41 that was stable in our desired operating window (60). The resulting slurry of 41 in tetrahydrofuran was incompatible with the planned flow process, so our attention immediately focused on establishing modified reaction conditions that would solubilize this polymorph. Dilution did not appear promising, as when the concentration of 41 was halved, some solids remained. We could also not use a more polar solvent (e.g., dimethyl sulfoxide, dimethylformamide, or N-methyl-2-pyrrolidone), as these substantially reduced the reaction yield. Further, we had already demonstrated that lithium anion 41 was not sufficiently stable above -20 °C to allow us to overcome the insolubility of 41 by raising the temperature. Given the well-known tendency of organolithium species to form higher-order aggregates in solution (61), we hypothesized that additives able to perturb such species might increase the solubility of lithium anion 41. Such a group of structurally diverse and readily available additives (i.e., tetramethylethylenediamine, N,N′-dimethylpropyleneurea [DMPU], N,N′-dimethylethyleneurea, pyridine, trimethylamine, and dimethoxyethane) were tested, and DMPU was most effective in solubilizing 41 at the target concentration and temperature. Further optimization experiments demonstrated that about 1.0 equivalent of DMPU relative to lithium anion 41 was required to generate a homogeneous solution of 41 at -20 °C, which resisted crystallization even upon addition of crystalline 41 as a seed. The addition of DMPU did not significantly impact the performance of the reaction or the stability of the lithium anion feed solution. Having established that mixing efficiency was critical to high reaction conversion, one of the main objectives of our first pilot plant-scale batch was to evaluate the reaction’s performance at a range of flow rates through a commercial-scale mixer. A Y-shaped mixer was fabricated from 1/4-in. stainless steel Schedule 40 pipe with a Koflo static mixing element (62) installed in the downstream segment (Table 3). We measured the conversion and diastereoselectivity of the reaction over a range of flow rates (from 55.5 kg/h to 250.0 kg/h total solution), maintaining a constant flow rate ratio to ensure the delivery of 1.7 equivalents of lithium anion 41 relative to sulfinyl ketimine 36. Within this array of flow rates, we independently varied the temperature of the 36 feed stream (from 22 to -62 °C) while holding the temperature of the 41 feed solution between -10 and -20 °C. In all cases, the conversion monotonically improved as the total flow rate increased. This effect was most pronounced at the warmest temperature, where the conversion increased from 64.2 to 82.1% when the total flow rate was raised from 55.5 kg/h to 250.0 kg/h (entries 1 to 5). As the feed temperature of sulfinyl ketimine 36 was lowered, the overall reaction temperature was consequently reduced, leading to higher reaction conversions and a progressive decrease in the impact of the total flow rate on conversion. At the coldest temperature, the improvement in conversion increased from 80.5 to 85.6% when the total flow rate was raised from 55.5 kg/h to 250.0 kg/h (entries 16 and 20).
72
Table 3. The Impact of Flow Rate and Temperature on the Continuous Formation of 40 at Pilot Plant Scale
73
Modifications to the overall equipment train enabled rigorous temperature control at each point along the flow path. Improvements in our ability to execute the entire process rapidly enabled us to significantly shorten the length of time between the start of the n-hexyllithium charge into methyl sulfonamide 10 and the beginning of the flow reaction. Since solutions of lithium anion 41 slowly decrease in purity over time even at -20 °C, the best results are obtained with freshly prepared anion. Based on the data we had collected at pilot plant scale about the relationship between reaction conversion, diastereoselectivity, and temperature, we choose to deliver both reactants between -10 and -20 °C, a temperature range that did not necessitate cryogenic equipment. In addition, matching the feed solution temperatures enabled a single external chiller unit to supply cooling fluid to both flow heat exchangers, simplifying the engineering requirements. With these changes we were able to conduct the optimized process at steady-state conditions for hours at pilot plant scale to produce more than 100 kg of product in a single batch with consistent assay yields of 88 to 89% (Figure 5).
Figure 5. Optimized continuous process at pilot plant scale. We were only able to identify weakly crystalline polymorphs of 40, which ultimately precluded its isolation directly from the quenched reaction mixture. Instead, given our ultimate interest in cleaving the tert-butyl sulfinamide auxiliary as the next step to furnish the ideal C-N coupling substrate (vide infra), we chose to evaluate chiral acid salts of the corresponding primary amine as isolable crystalline intermediates. That substrate, amine 44, could be readily and quantitatively formed by quenching the continuous process reaction stream into an excess of aqueous hydrogen chloride (Scheme 13). We surveyed a wide array of commercially available chiral acids in the presence of 44 in a high-throughput manner; (S)-mandelic acid emerged as the best. In the optimized crystallization process, the desired salt (45) could be isolated in 98.5% yield (relative to the desired enantiomer present in the starting material), 99.9% purity, and 98.8% ee from toluene. Using a water-immiscible solvent like toluene for the crystallization after an aqueous deprotection of 43 provided a seamless connection between 74
an organic phase extraction of freebased amine 44 (the end of the continuous process) and the downstream salt isolation after freebasing.
Scheme 13. Optimized conditions for the crystallization of 45 from the sulfinyl ketimine addition reaction.
Copper-Catalyzed Amidation We initially discovered a copper-catalyzed C-N coupling between aryl bromide 40 and 5-fluoro-2-picolinamide (31), which afforded aryl amide 46 in 70% isolated yield (Scheme 14). While this reaction performed serviceably at pilot plant scale, the modest yield, very high catalyst loading, and excess of 31 required to achieve full conversion provided the impetus to further investigate the amidation.
Scheme 14. Copper-catalyzed amidation of aryl bromide 40. 75
Evaluation of alternative aryl bromide coupling partners unexpectedly revealed that the partially deprotected amine 44 also underwent efficient C–N coupling, achieving full conversion and providing the coupled adduct in 90% yield from mandelate salt 45 (Scheme 15). In addition, with this substrate we were able to reduce the catalyst loading to 20 mol%, and unlike the analogous reaction with 40, the revised C–N coupling protocol only required an equimolar amount of ligand relative to copper. The employment of aryl bromide 44 also enabled a reduction in the charge of 31 to only 1.2 equivalents.
Scheme 15. Improved amidation sequence employing aryl bromide 44.
To rationalize the difference in reaction performance between aryl bromides 40 and 44, we initially hypothesized that the free amine present in 44 coordinated to copper, favoring formation of the active catalytic species and retarding the formation of inactive cuprate complexes (63, 64). The observation that both the amounts of diamine ligand and amide 31 could be reduced in the coupling employing 44 is consistent with this proposal. In the reaction with aryl bromide 40, the excess of amide 31 was required at least in part due to a competing background hydrolysis that formed 5-fluoro-2-picolinic acid (23). Reaction monitoring by NMR revealed that this hydrolysis was promoted by copper iodide and suppressed by increasing the concentration of the diamine ligand. The availability of an additional amine from the starting material to occupy a coordination site on the metal further suppressed hydrolysis of 31, enabling the reduction of the number of equivalents required to achieve full conversion.
Removal of the para-Methoxybenzyl Group Following the optimization of copper-catalyzed amidation, we shifted our focus toward developing robust conditions for the removal of the para-methoxybenzyl protecting group. One issue we needed to address at the 76
outset was the exceedingly poor solubility of amine 47 in nearly all organic solvents. An expansive solvent screen revealed that acetic acid provided the requisite solubility to facilitate the deprotection. Methanesulfonic acid proved to be the optimal acid to effect the cleavage of the para-methoxybenzyl group, affording clean deprotection in 2 hours at 60 °C. During the course of our lab-scale development we found that once the reaction was complete, the batch could be cooled, diluted with water, and washed with toluene before carrying out a reverse addition into concentrated ammonium hydroxide to induce the crystallization of the product. We were surprised to discover that upon performing this process on a multikilogram scale, a large, insoluble mass formed in the reactor while cooling the batch from 60 °C upon reaction completion. We analyzed this insoluble material by mass spectrometry and observed multiple species consistent with polymerization of the para-methoxybenzyl cation generated during the deprotection. To block this undesirable side reaction, we conducted the deprotection in the presence of the cation scavenger 1,3-dimethoxybenzene, which prevented the formation of these polymers by trapping the para-methoxybenzyl cation. These adducts formed during the deprotection could readily be removed during the toluene wash prior to isolation of 48 from ammonium hydroxide and provided robust conditions that could be scaled without issue (Scheme 16).
Scheme 16. Removal of the para-methoxybenzyl group.
Thiadiazine Formation and Completion of the Synthesis As in the earlier syntheses of 1 (Schemes 4 and 5), cyanogen bromide proved to be the best reagent to generate the thiadiazine heterocycle in a single chemical step. We initially observed that when amine 48 was treated with cyanogen bromide in a mixture of isopropyl acetate and acetonitrile, the hydrogen bromide salt of verubecestat (49) crystallized spontaneously (Scheme 17). This reactive crystallization provided a direct isolation of 49 in high chemical purity and was advantageous as it eliminated the need for an aqueous workup and subsequent isolation. In addition, the discovery of this reactive crystallization was fortuitous as similar thiadiazine formation conditions, which employed bases, resulted in the over-cyanation of 1 to cyanamide 50 (Scheme 18). This process was demonstrated successfully on a multikilogram scale, affording 49 in 88% yield. 77
Scheme 17. Thiadiazine formation and isolation of verubecestat (1).
Scheme 18. Over-cyanation of 1 to 50 in the presence of cyanogen bromide under basic conditions.
Surprisingly, during a pilot plant batch of this process, we observed a strikingly atypical reaction profile. When we analyzed the solid isolated from this batch, we observed a mixture of the desired product (49) along with unreacted amine 48. In addition, the supernatant contained greater than 40% of cyanamide 51, an intermediate measured to be less than 1% in the supernatant of previous batches (Figure 6).
Figure 6. Cyanamide intermediate 51 formed during the conversion of 48 to 49. The solid isolated from this batch was subjected to rigorous characterization to unambiguously determine the composition of the material. X-ray powder diffraction showed a mixture consisting of the desired crystalline phase of 49 and 78
an unknown phase (65). Scanning electron microscopy revealed that the isolated solid was composed of particles displaying two distinct morphologies. One of the morphologies was consistent with the previously characterized batches of 49, and a second, granule-like aggregate was significantly larger in size (Figure 7). The granule-like aggregates were sieved using a range of screen sizes, and the isolated solids were found to be a single pure phase by powder indexing (66).
Figure 7. Scanning electron microscope images showing two distinct morphologies in the isolated unknown solid.
Further characterization of these aggregates by high-performance liquid chromatography and inductively coupled plasma mass spectrometry revealed the purified granules were a mixture of amine 48, verubecestat (1), and two equivalents of hydrogen bromide (Table 4). This analysis demonstrated conclusively that the isolated solid from the batch was a mixture of desired product 49 and a co-crystal of the hydrogen bromide salts of both 48 and 1. This finding was also supported by single-crystal X-ray diffraction analysis of the co-crystal, which revealed a structure consistent with the component analysis via high-performance liquid chromatography (Figure 8).
Table 4. Component Analysis of the Unknown Impurity Phase Component
Fraction in impurity phase
Mol % of component
Mol % normalized to API
Amine (48)
40.1
23.6
1.01
Verubecestat (1)
42.4
23.5
1.00
Hydrogen bromide
18.6
52.8
2.25
79
Figure 8. Single-crystal X-ray diffraction of the co-crystal formed between the hydrogen bromide salts of 48 and 1. Thermal ellipsoids are shown at the 50% probability level.
The formation of the co-crystal provided insight into the mechanism of the reaction. The co-crystallization event removed hydrogen bromide from the solution phase of the reaction, which, when coupled with the observation that cyanamide remained in the supernatant, suggested that soluble hydrogen bromide was necessary for intramolecular ring closure of the thiadiazine. Indeed, when cyanamide 51 was isolated and treated with hydrogen bromide in a mixture of acetonitrile and isopropyl acetate, 49 was readily formed. The poor solubility of the co-crystal across all reaction solvents rendered the existing process untenable. Our initial efforts to modify the endgame sought to employ basic conditions in order to prevent the accumulation of hydrogen bromide salts that would lead to subsequent co-crystal formation. While this approach did generate 1, we were unable to identify conditions that suppressed the formation of the over-cyanation impurity 50, an issue that we had previously addressed using the isolation of 49 (vide supra). A more significant endgame redesign was subsequently undertaken in which we focused on a stepwise approach to initially convert amine 48 to cyanamide 51 followed by intramolecular ring closure to form the thiadiazine. This sequence would eliminate the undesired co-crystallization by preventing the formation of 1 in the presence of the amine 48 and hydrogen bromide. A stepwise approach would also address the over-cyanation issue by preventing the formation of 1 in the presence of cyanogen bromide. Our preliminary evaluation of bases revealed that amine bases weaker than N,N-diisopropylethylamine provided 51 as the exclusive product, but only in prohibitively low levels of conversion (Table 5, entries 1 to 3). Inorganic bases such as potassium phosphate monobasic or sodium bicarbonate, however, led to improved conversions across a broad range of solvents. We ultimately chose to advance sodium bicarbonate as the base and 2-methyltetrahydrofuran as the 80
solvent as that combination provided optimal conversions and streamlined the aqueous workup given its use of a water immiscible solvent.
Table 5. Survey of Solvents and Bases for the Conversion of 48 to 51
Once we had a solution of cyanamide 51, we quenched the residual cyanogen bromide present by adding aqueous sodium hydroxide. Interestingly, we found these conditions promoted intramolecular cyclization to form the thiadiazine, but the ring-closed adduct was once again accompanied by over-cyanation byproduct 50. We then hypothesized that a reductive workup (67, 68) would be necessary to completely destroy residual cyanogen bromide, and to that end sodium thiosulfate was identified as a suitable reducing agent. When the process stream containing 51 and residual cyanogen bromide was washed with 10% aqueous sodium thiosulfate prior to treatment with sodium hydroxide, the formation of 50 was almost completely suppressed. The resulting 2-methyltetrahydrofuran stream containing 51 was treated with aqueous sodium hydroxide to promote intramolecular cyclization to verubecestat (1), which was first isolated as para-toluenesulfonate salt 26 to ensure API purity specifications were met. A final salt break of para-toluenesulfonic acid salt 26 with potassium carbonate afforded 1 in 92% isolated yield from 48 (Scheme 19). 81
Scheme 19. Synthesis of verubecestat (1) using the revised endgame.
Emergence of a New API Polymorph Following the completion of a clinical resupply campaign, routine release testing revealed the presence of extraneous matter within a single batch of verubecestat (1), which necessitated reprocessing. The recrystallization of this particular batch was carried out using the same conditions from which the API was routinely isolated, however, X-ray powder diffraction analysis revealed a new phase of the API, labeled Form II, which had a higher melting point than the previously isolated phase (Form I). Equilibration studies conducted by slurrying Form I in a range of solvents showed conversion to Form II under all conditions evaluated. Together these data demonstrated that Form II was a more thermodynamically stable phase of verubecestat (1). The spontaneous formation of new API polymorphs in late development or even after product registration is not without precedent. The well-known example of ritonavir, which was temporarily withdrawn from the market when a new polymorph of the API was discovered, provided a cautionary tale of the potential for commercial disruption related to an unexpected API phase change (69). Based on preliminary data, we expected that our ability to robustly produce Form I would be compromised now that a more thermodynamically stable Form II had emerged. In an effort to avoid complications related to metastable polymorphs, our development team decided to target Form II in all future clinical supply deliveries and for the commercial launch. Despite its higher melting point and reduced solubility in biorelevant media, Form II was still Class I under the U.S. Food and Drug Administration guidelines for the classifications of biopharmaceutics, like Form I, and both polymorphs had a similar dissolution 82
profile across a broad pH range. This finding was significant as it assuaged concerns that the new polymorph would impact the bioavailability of verubecestat (1) and simplified the regulatory hurdles for changing the API polymorph during clinical development. The emergence of Form II forced us to reinvestigate our endgame process. First, a new API isolation that reliably produced Form II would need to be established. Second, the process that generated verubecestat prior to the formation of para-toluenesulfonic acid salt 26 (Scheme 19) would require revisions to ensure no additional complications would arise due to the reduced solubility of Form II. Given the subtle differences in solubility between the two polymorphs, we suspected the process changes would be minor. The API isolation of Form I involved concentrating an ethyl acetate solution of 1 to approximately 18 wt % and heating to 50 °C before charging 15 vol % of n-heptane and seeding with Form I. The batch was aged for 2 hours before n-heptane was added to reach 60 vol % n-heptane followed by a reduction in batch temperature to 20 °C. In order to access Form II we found that the n-heptane charge prior to seeding could be adjusted to 5 vol % and the use of Form II seeds would allow for reliable production of the Form II polymorph. Most importantly from a processing perspective, we found that Form II was equally capable of rejecting the typical impurities produced during the salt break of para-toluenesulfonic acid salt 26. We also found the processability of both forms to be similar as both reduced to similar particle size distributions during wet milling, avoiding the need for additional process development for particle size control. The endgame process, which proceeded through 1, also required only minor revisions. A solubility assessment of the newly discovered Form II in 2-methyltetrahydrofuran revealed only a subtle decrease in solubility. To prevent the undesired crystallization of 1 prior to the formation of 26, we adjusted the target volumes during distillation and were pleased to find that despite the increase in the amount of 2-methyltetrahydrofuran, we did not observe meaningful increases in product losses to the mother liquors.
Phase III Clinical Status In 2017, the EPOCH trial was stopped early following the recommendation of the external Data Monitoring Committee, which noted that while the observed safety signals did not warrant terminating the study, there was virtually no chance of finding a positive clinical effect with respect to the slowing of cognitive or functional decline (25, 70). Patients who received verubecestat did show an up to 80% reduction of Aβ peptides in their CSF compared to patients on placebo. In addition, a subset of patients had a reduction in amyloid load in their brains according to amyloid imaging using positron emission tomography (25). In February 2018, it was announced that the APECS trial would be discontinued based on the external Data Monitoring Committee recommendation that it was unlikely that a positive benefit/risk could be established if the trial continued (71). 83
Scheme 20. The commercial manufacturing process for verubecestat (1).
Summary The sum of this work constitutes the discovery of verubecestat (1) and the ultimate development of the commercial manufacturing process (Scheme 20). The overall yield of 67% from the coupling of methyl sulfonamide 10 and sulfinyl ketimine 36 through to 1 improves significantly on the first generation process route, for which the same sequence delivers a 28% overall yield. Additionally, substantial gains in efficiency across the entire process produced more than an 80% reduction in waste (72). These results were enabled by the invention of a continuous process for the addition of 41 to 36 to form the stereogenic α,αdibranched amine, the identification of a new chiral salt (45) to provide a key 84
point of stereochemical purity upgrade, a C-N coupling that takes advantage of an unexpected boost in reactivity gained by employing a substrate (44) that has been partially deprotected, and a new orchestration of the thiadiazine synthesis, which entirely circumvents the risk of co-crystallization of the starting material and product for that reaction. Finally, a revised API crystallization has been developed to deliver Form II, the recently identified API polymorph, in a robust and efficient manner.
Acknowledgments We thank the many colleagues from the Research Laboratories and the Manufacturing Division of Merck & Co., Inc., Kenilworth, NJ, United States, who contributed to this work, many of whose names are included in the references below.
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