Design and Enabling Development of Hydroxyethylamine-Derived

Chapter 4

Design and Enabling Development of Hydroxyethylamine-Derived BACE1 Inhibitor Downloaded by COLUMBIA UNIV on December 12, 2016 | http://pubs.acs.org Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch004

Jason S. Tedrow*,1 and Wenge Zhong2 1Process

Development, Amgen Inc, One Amgen Center Drive, Thousand Oaks, California 91320, United States 2Discovery Research, Amgen Asia R&D Center, 99 Haike Road, 4th Floor, Building 6, Shanghai 201210, P. R. China *E-mail: [email protected].

Herein we report the hydroxyethylamine (HEA)-derived potent and orally efficacious BACE1 inhibitors as potential treatments for Alzheimer’s disease. These compounds were designed for low efflux and in vivo clearance to effect robust reduction of Aβ levels in the central nervous system (CNS). Key design strategies feature an amide masking approach for mitigating PGP-mediated efflux and the incorporation of CYP 3A4 inhibitory activity for decreased in vitro and in vivo clearance. Lead molecules demonstrated sufficient oral bioavailability and CNS penetration and were shown to be orally efficacious in pre-clinical rodent models. Collaboration between medicinal and process chemistry on the key synthetic challenges is presented including new chemistry towards challenging fragments of the HEA core structure. The new routes were designed for scalability and improved overall safety (elimination of hazardous reagents). Additionally a new, templated assembly route toward the HEA core structures was developed to overcome key challenges using traditional methods for HEA construction.

© 2016 American Chemical Society Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Downloaded by COLUMBIA UNIV on December 12, 2016 | http://pubs.acs.org Publication Date (Web): December 9, 2016 | doi: 10.1021/bk-2016-1240.ch004

Medicinal Chemistry and Discovery of HEA-Derived BACE1 Inhibitors as Potential Disease Modifying Treatments for Alzheimer’s Disease Alzheimer’s Disease (AD) is a debilitating neurodegenerative disease and the most common form of dementia. AD afflicts more than five million people in the United States (1). It is estimated that by the year 2050, the total number of AD patients will reach 16 million in the U.S. and over 50 million worldwide. There is tremendous medical and socioeconomic burden associated with the disease. Currently only symptomatic treatments are available and they provide modest temporary benefits (2). Thus finding a disease modifying treatment to slow, stop or even reverse AD represents a huge unmet medical need. One of the key characteristics of AD is the accumulation of insoluble amyloid plaques in the brain. The principal components of the amyloid plaques are the amyloid β peptides (Aβ) of various lengths, typically 38-43 amino acids (3). A large body of evidence has suggested that increased formation and/or impaired clearance of Aβ peptides are the underlying pathological mechanism for the disease (3, 4). Aβ peptides are produced in the brain via a tandem two-step proteolytic cleavages of the amyloid precursor protein (APP) by BACE1 (β-site APP cleaving enzyme or β-secretase) and γ-secretase. Thus, inhibition of secretase functions could provide a potential disease modifying approach for AD. During the last several decades, substantial effort across the pharmaceutical industry has been dedicated to the finding of viable γ-secretase inhibitors but has met with no clinical success so far (5). In more recent years, BACE1 emerged as the secretase target of focused interest across the industry. Genetic data from familial AD patients indicated that mutations in APP around the BACE1 cleavage site lead to increased processing of APP and accumulation of Aβ peptides (6). Interestingly, it was also found that a low frequency APP mutation, A673T, two amino acids after the BACE1 cleavage site, reduces APP processing by BACE1 and decreased risk of AD and cognitive decline in the aged people (7). On these genetic bases and the additional notion that the cleavage of APP by BACE1 is the rate limiting first step (8, 9), many groups committed substantial amount of efforts to the finding of effective BACE1 inhibitors as possible disease modifying agents (10, 11). It should be noted that many different BACE1 substrates other than APP have been discovered and the implications of overall inhibition of BACE1 are not well understood. It remains to be proven if BACE1 inhibitors can be effective treatments for AD with significant benefits over any potential side effects (12). Currently, two of the most advanced molecules MK-8931 (verubecestat) and AZD3293 (Figure 1) are in phase III clinical trials for mild-to-moderate and early AD, respectively (13). Amgen’s discovery research on BACE1 dates back to the cloning of the enzyme in 1999 (14). Since then, we have embarked on a long and committed journey of finding promising BACE1 inhibitors for AD. With the availability of numerous in-house crystal and co-crystal structures, we initiated a structure-based drug discovery program and started working with a chemical series that was based on the hydroxyethylene (HE) transition state isostere. Despite the fact that 138 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


single digit nM BACE1 inhibitors were quickly identified, they suffered from many drawbacks, among which most noteworthy were poor cellular activity and chemical instability due to lactone formation. For example, a representative compound in the series, 1, (Figure 2A) in the fluorescence resonance energy transfer (FRET) based BACE1 enzyme assay exhibited an IC50 of 6.1±2.3 nM, but in the cellular assay its IC50 was 500±120 nM, indicating an enzyme-to-cell shift of over 80-fold.

Figure 1. Structures of MK-8931 and AZD3293. Additionally, during the synthesis and purification of these compounds, an impurity identified as the lactone (Figure 2B) was often observed in amounts up to 15%. In order to circumvent these issues and to reduce size of the molecules, we moved on to a new series that is based on the hydroxyethylamine (HEA) transition state isostere. In this article, we describe briefly the evolution of the series leading to the highly efficacious BACE1 inhibitors and the chemistry that was developed for the large scale synthesis of these promising molecules.

Figure 2. A. Structure of a Representative HE-derived Inhibitor; B. Formation of the Lactone. Compound 2 was the first promising HEA-derived BACE1 inhibitor in our program that afforded modest enzymatic activity and very small enzyme-to-cell shift (BACE1 IC50: 76±2.3 nM; Cell IC50: 210±160 nM) (Figure 3). Ring formation gave rise to a chroman derivative 3 that displayed potent BACE1 activity with an IC50 of 4.5±2.5 nM and Cell IC50 of 64±18 nM. Introduction of spirocyclobutane ring onto the chroman scaffold further improved enzymatic activities consistently, for example, 4 was identified as a 1.7±0.6 nM BACE1 inhibitor with good cellular activity (IC50: 22±4.9 nM). Based on the available structural information and modeling, the binding mode of 4 with the BACE1 enzyme is illustrated in Figure 3. We reported previously that P2′ (15) interactions with the enzyme were optimized with the introduction of a neopentyl group at this site (16). We found 139

Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


that the combination of the spirocyclobutane P1′ and neopentyl P2′ groups allowed us to truncate the left hand pyridone-derived acyl group substantially to just a simple acetyl group, rendering analogs such as 5 to retain potent activity (BACE1 IC50: 7.2±2.9 nM; Cell IC50: 81±12 nM). Ultimately, realizing the 8-position of the chroman is solvent exposed when binding to the BACE1 enzyme, we replaced chroman with 8-aza chroman which consistently preserved potent enzyme activities and minimized the enzyme-to-cell shift (6, BACE1 IC50: 5.8±6.3 nM; Cell IC50: 3.1±4.2 nM).

Figure 3. Evolution of the Early HEA-derived BACE1 Inhibitors. With potent cellular activities and chemical stability accomplished in the HEA series, we next turned our attention to achieving sufficient exposure in the central nervous system (CNS). It is well documented in the literature that major impediments for CNS penetration by small molecules are poor passive permeability and P-glycoprotein (PGP)-mediated efflux (17). Our early HEA-derived BACE1 inhibitors displayed good to excellent passive permeability as measured in the LLC-PK1 parental cell line assay, however, they suffered from high PGP-mediated efflux in the LLC-PK1 PGP-transfected cell line assay. Furthermore, these early inhibitors exhibited high clearance in rat pharmacokinetics studies, leading to poor systemic and brain exposure in vivo. In order to understand the importance of PGP-mediated efflux and in vivo clearance for achieving adequate BACE1 target coverage in the CNS for Aβ lowering efficacy, we performed co-dosing studies with a representative inhibitor 7 (Figure 4A; BACE1 IC50: 31±19 nM; Cell IC50: 34±6.8 nM). Compound 7 showed excellent passive permeability (average Papp: 26E-06 cm/s) with very high rat MDR1-mediated efflux ratio of 41. It also had a moderately high intravenous (iv) clearance in Sprague-Dawley rats at 2.6 L/h/kg. In one co-dosing study in rat, 7 was dosed at 30 mg/kg orally (i.e., per os, or p.o.) in combination with the known PGP inhibitor GF-120918 (Figure 4B; 100 mg/kg, p.o.). In another study, 7 was dosed at 30 mg/kg orally in combination with the well-known cytochrome 140



P450 3A4 CYP 3A4 inhibitor ritonavir (Figure 4C; 10 mg/kg, p.o.). Two h after dosing, cerebrospinal fluid (CSF) samples were taken, Aβ40 levels were determined, and compared to the vehicle-treated group and the 7 alone treated group (30 mg/kg, p.o.). Plasma and CSF drug levels were also measured. We should point out that in all our rodent studies, CSF Aβ lowering effects and drug levels are appropriate surrogates for brain Aβ lowering efficacy and free drug concentrations, respectively.

Figure 4. A. Structure of Compound 7; B. Structure of GF-120918; C. Structure of Ritonavir. Table 1 summarizes the results from the PGP inhibitor GF-120918 co-dosing study. When 7 was dosed alone, the plasma drug concentration ([Plasma]) and the CSF drug concentration ([CSF], an approximate indicator of free drug level in the brain) were 0.609 μM and 0.007 μM, respectively. This gave a very poor [CSF]/[Plasma] ratio of 0.011 which was suggestive of poor brain exposure, and the CSF drug level was only about 20% of the cellular IC50 of compound 7, thus no significant CSF Aβ40 reduction was observed. In contrast, when 7 was co-dosed with GF-120918, [Plasma] was modestly increased to 1.65 μM. More importantly, [CSF] was increased to 0.222 μM by a factor of greater than 30-fold. This represented a roughly ten-fold improvement in [CSF]/[Plasma] ratio to 0.134 and [CSF] was about seven-fold of the cellular IC50 for 7. As a result, a robust CSF Aβ40 reduction of 71% was achieved.

Table 1. Co-dosing Results with GF-120918 Additive

[Plasma] (μM)

[CSF] (μM)

[CSF]/[Plasma]

↓CSF Aβ40

30 mg/kg, p.o.

__

0.61

0.007

0.011

< 10%

30 mg/kg, p.o.

GF120918

1.65

0.222

0.134

71%

Compound 7

Results from the co-dosing study with CYP 3A4 inhibitor ritonavir are provided in Table 2. In this study, dosing 7 alone yielded drug levels in both plasma and CSF that were about twice as high as in the co-dosing study with GF-120918 (Table 1). This difference was presumably due to study-to-study 141 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


variations. Nevertheless, the [CSF]/[Plasma] ratio remained very low at 0.010 and CSF Aβ40 reduction was less than 10%. Co-dosing with ritonavir increased both [Plasma] and [CSF] similarly by about ten-fold with the [CSF]/[Plasma] ratio essentially unchanged. Since [CSF] reached 0.176 μM, which was five-fold of the cellular IC50 for 7, a significant CSF Aβ40 reduction of 55% was observed. Taken together, we concluded from these two co-dosing studies that reducing PGP-mediated efflux and in vivo systemic clearance for improving CNS exposure should be the main optimization strategies for identifying efficacious HEA-derived BACE1 inhibitors.

Table 2. Co-dosing Results with Ritonavir Compound 7

Additive

[Plasma] (μM)

[CSF] (μM)

[CSF]/[Plasma]

↓CSF Aβ40

30 mg/kg, p.o.

__

1.47

0.015

0.010

< 10%

30 mg/kg, p.o.

Ritonavir

14.2

0.176

0.012

55%

Studies have shown that the total number of H-bond donors (HBD’s) in a molecule is a critical physicochemical parameter and typically less than a total of three HBD’s is desirable for achieving good brain exposure (18). Since the HEA core already possesses two HBD’s that are necessary for direct interactions with the BAC1 enzyme catalytic residues at the active site, we sought to reduce the apparent total number of HBD’s by masking the amide N-H via a possible intramolecular H-bond. We contemplated that such a masking strategy could retain all key interactions with the enzyme and good passive permeability but significantly improve efflux. This indeed proved to be one of the most fruitful approaches we employed to mitigate PGP-mediated efflux for the HEA derivatives. For example, we introduced a suitably-positioned methoxy group on the acyl moiety to be capable of forming an interamolecular H-bond with, and mask the amide N-H bond (Figure 5). Similar modification of 8 thus provided analog 9 (Figure 5B). In order to augment the possibility of forming an intramolecular H-bond, we further introduced an additional methyl group to afford analog 10. To our gratification, we found that 8 and 9 displayed essentially identical enzyme and cell activities and 10 was just about two-fold less potent in both the enzyme and cell assays. The average passive permeabilities for the three compounds were comparable. Perhaps most importantly, the efflux ratios were dramatically improved, suggesting that PGP-mediated efflux was significantly reduced. In the rat MDR1- transfected LLC-PK1 cell assay, the efflux ratios for 8, 9 and 10 were 43, 18, and 7.7, respectively. When these compounds were dosed orally at 30 mg/kg in rat, 8 reduced CSF Aβ40 levels by less than 10%. In contrast, 9 and 10 reduced CSF Aβ40 levels by 39% and 50%, respectively. 142



Figure 5. Masking the Amide N-H via an Intramolecular H-bond.

Having established methods for improving efflux, we then turned our attention to focus on improve in vivo systemic clearance. Towards this end, we designed and prepared a large number of analogs that were very stable in the liver microsomes. However, these analogs continued to suffer from moderate to high in vivo clearance in rodents that led to limited exposures in plasma and in the brain for significant Aβ lowering efficacy (unpublished data). As detailed in our previous report (18), we committed to a strategy to build a CYP 3A4 inhibitory property into the HEA-derived molecules. After surveying the effects of various CYP 3A4 inhibitory functional groups around the molecule, we prioritized our effort on modifying the P1 aryl group. Among these, the benzodioxole P1 derived analogs designated as ‘699 and ‘359 emerged as the leading molecules in our program (Figure 6). While ‘699 appeared to be three-fold more potent than ‘359 in the BACE1 enzyme assay, they exhibited comparable cellular activity in the low double digit nM range (Cell IC50 for ‘699: 17 ± 5.0 nM: for ‘359: 26± 11 nM). Both compounds displayed excellent passive permeability with average Papp > 15E-06 cm/s. The efflux ratios as measured in the human and rat MDR1-transfected LLC-PK1 cell lines for ‘699 were still in the high range (hMDR1 efflux ratio: 16; rMDR1 efflux ratio: 27), however, those in the same assays for ‘359 were in the low range (hMDR1 efflux ratio: 4.0; rMDR1 efflux ratio: 6.0). As expected from the incorporation of benzodioxole group, both ‘699 and ‘359 showed potent 3A4 inhibitory activities (human 3A4 IC50 for ‘699: < 0.1 μM; for ‘359: 0.1 μM), resulting in low to moderate in vitro clearance in the human and rat microsome incubation experiments. We further profiled ‘699 and ‘359 in rat pharmacokinetics studies. When ‘699 was dosed at 2.0 mg/kg intravenously in fed male Sprague-Dawley rats, the observed clearance was 1.3 L/h/kg, volume distribution (Vss) was 5.3 L/kg, and t1/2 was 8.5 h, respectively. Its oral bioavailability was estimated to be greater than 100% from another oral pharmacokinetics study in fasted Sprague-Dawley 143



rats. We observed a similar rat pharmacokinetics profile for ‘359 and its dog and monkey pharmacokinetics profiles were also very favorable. The results were detailed in our earlier publication (18). Owing to their overall properties of potency, reduced PGP-mediated efflux, and improved in vivo clearance and systemic exposure, ‘699 and ‘359 showed robust in vivo CSF Aβ lowering of 52% and 57%, respectively (dose: 30 mg/kg as a solution in 1% Tween/2%HPMC; sampling at 4 h after dosing). With these data in hand, the team selected both ‘699 and ‘359 for further advancement.

Figure 6. Structures of Lead Molecules ‘699 and ‘359. During the course of the SAR development, the medicinal chemistry team implemented several lines of chemistry to quickly access these P1 analogs. Most noteworthy is the chiral sulfinylimine chemistry that was published by our group (15, 19). With the identification of lead molecules ‘699 and ‘359, Amgen’s medicinal chemistry and process chemistry teams started to work together. In the next section, we describe in detail the medicinal chemistry routes and process development for preparing the advanced HEA-derived BACE 1 inhibitors.

Process Research Toward Synthesis of the Hydroxyethylamine BACE Inhibitor Class: A New Templated Approach to the HEA Core Due to the inherent synthetic complexity and challenges to prepare gram amounts of the lead HEA molecules for the BACE program, the process chemistry team embarked on an early engagement strategy with our discovery group. The goals of the collaboration were to support and/or accelerate lead candidate selection and develop a fit-for-purpose synthetic route to enable early development trials. Process research concentrated on enabling support for the BACE program with focus on elimination of any key technological road block(s) and early process development for kilogram production with built-in flexibility to pivot on emerging SAR. Key to this was to build in technology to intercept intermediates common to the medicinal chemistry synthesis where applicable. 144



While medicinal chemistry was continuing to optimize the final candidate, the lead series contained most of the key synthetic challenges and thus we focused our enabling route selection efforts toward targeting ‘699 and ‘359 as prototypical of the ultimate compound. The medicinal chemistry route to the final molecule, while ideally positioned for maximal flexibility, presented a number of difficulties from a process chemistry perspective (Schemes 1-3). Outlined from the start, some of the key challenges toward scalability of the route was the long synthetic sequence (29 steps in total), three non-contiguous stereocenters which are set independently from one another and a challenging fragment coupling with an epimerization prone α-alkoxy aldehyde 12. Additionally the use of protecting groups and heavy reliance on chromatographic purification to control quality attributes presented substantial hurdles toward future scale-up.

Scheme 1. Discovery Assembly Route to ‘359 and ‘699

On its surface, the discovery disconnection around the central dialkyl amine (Scheme 1) was attractive and we sought to exploit similar tactics in hopes of maintaining overall convergence of the route. This approach then distilled the process chemistry challenges to definition of the disconnection strategy which in turn defines the structure of the fragments to join. 145 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Scheme 2. Discovery Synthesis of P1 Alcohol 11

Two alternate reactivity modes to build the target were considered as alternatives to the previous P1 aldehyde reductive amination (Figure 7). Route 1 relied on a fully elaborated azachromyl amine 14 reacting with an electrophilic P1 epoxide, building upon precedent from the HEA literature surrounding commercially available / late stage clinical candidates (vide infra). Multiple routes were known and likely applicable to our epoxide fragments, so technical feasibility regarding the epoxide aminolysis was the key question. Route 2 had limited precedent in the literature regarding a stereoselective reductive amination of a P1 amine fragment and an azachromyl ketone 13. Each of these pathways require redefinition of both of the key fragments and thus their synthetic routes. As the majority of the SAR optimization was focused in on the P1 fragment of the molecules, initial process development focused in on synthesis of the amine and ketone fragments to enable both synthetic approaches in Figure 7. 146



Scheme 3. Discovery Synthesis of Azachromyl Amine 14

Process Research Targeting a Scalable Synthesis of Azachromanone 13 and Azachromylamine 14 The discovery synthesis of the azachromylamine fragment (Scheme 3) presented several inherent throughput difficulties which led us toward rethinking the entire assembly strategy for the intermediate. The key transformations were built around an intramolecular SnAr of the tertiary cyclobutyl alcohol 30 into a fluoropyridine to construct the azachroman structure (Scheme 4). Intermediate 30 is built from the pyridyl Grignard addition to a functionalized cyclobutane aldehyde, which is four steps from commercially available cyclobutane. Basic concerns with this sequence surrounded the instability of the pyridyl organometallic, overall length in synthesis of the cyclobutane linker and lack of robust crystalline intermediates to control purity outside of chromatography. In rethinking the overall strategy to synthesize the target azachromylamine, a need for access to both the neopentyl and the bromoazachromyl amine systems was required by medicinal chemistry to further SAR at the 6-position. The target then became developing a synthesis to ketone 37 (Scheme 5). 147



Figure 7. Retrosynthetic Strategy Targeting Penultimate 15.

Scheme 4. Discovery Approach to Azachromanone 13

A prominent approach to access 2-aryl substituted chromanones involves intramolecular conjugate addition of a phenol to an enone (20, 21). In the case of the 2-aryl chromanones, the precursor enone is easily accessed via aldol condensation of the arylmethylketone and an aryl aldehyde. This type of method was viewed to be a challenge to target the cyclobutyl enone, however similar systems have been generated via olefination reactions of the Wittig (22, 23), Horner-Wadsworth-Emmons (HWE) (24, 25), and Peterson (26) type. Of the aforementioned transformations, the HWE approach was particularly attractive as the requisite ketophosphonates may be accessed from the corresponding esters (27–29). 148 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Scheme 5. Alternate Retrosynthesis to Azachroman Structures 38 & 13 Bromination of the readily available 2-methoxynicotinic acid 41 in the biphasic CH2Cl2/water (30) mixture afforded 5-bromo-2-methoxynicotinic acid. The product was precipitated upon antisolvent induced crystallization and was isolated directly by filtration. Esterification (refluxing H2SO4/MeOH) (31) gave the methyl ester 40 in 97% for the combined two steps. Ketophosphonate 42 was prepared by the reaction of bromonicotinic ester 40 and methyl dimethylphosphonate using previously optimized conditions (32) in 82% yield (Scheme 6).

Scheme 6. Synthesis of Ketophosphonate 42 With the ketophosphonate 42 in hand, its HWE reaction with cyclobutanone was addressed. Cyclobutanone is known to undergo HWE reactions with stabilized phosphonates (33, 34), however the range of these substrates is limited and its reaction with more elaborate ketophosphonates is unknown. Screening of various conditions (35), illustrated that polar solvents and/or bases which generated a protic by-product (hexamethyldisilazine (HMDS), alcohols, water) showed diminished yields. Alkyl lithium bases with toluene as solvent showed clean reaction to the corresponding enone. Simple use of LiOMe in toluene, followed by azeotropic removal of methanol and then treatment with cyclobutanone delivered the desired enone 44 on gram scale (Scheme 7). In practice however, we found that we could isolate the lithium enolphosphonate salt and eliminate the need for extensive distillations. Additionally, the salt 42 was found to be bench stable, non-hygroscopic and served as an important crystalline holding point in the process. Formation of the lithioketophosphonate 43 was effected with a solution of LiOMe/MeOH in i-PrOH directly telescoped from the crude reaction mixture of 42. Horner-Wadsworth Emmons reaction of 43 was performed with two equivalents of cyclobutanone relative to 43 in five volumes of toluene at 90 °C. Aqueous workup and distillation 149



afford the enone 44 as a brown oil in 60% yield. Further attempts to increase the yield beyond 60% were not productive due to competing decomposition of the lithioketophosphonate under the reaction conditions (36). Demethylation and cyclization was easily effected by in situ generated iodotrimethylsilane (TMSCl/NaI in MeCN), and the product ketone could be isolated following aqueous workup in 75% yield when the purified 44 was used. However, when the crude material was subjected to the cyclization conditions, a short plug of silica gel was required for isolation of the crystalline 38. We demonstrated the robustness of the overall sequence (Scheme 7) on a ten kilogram scale and obtained 38 in 45% yield from the lithioketophosphonate 43 in two steps and in 39% yield for the overall sequence starting from 41.

Scheme 7. Optimized HWE approach to chromanone 38

With a scalable synthesis in hand of the chromanone 38, focus shifted on amine installation. From the medicinal chemistry work, the asymmetric ketone reduction / azide displacement (37) sequence constituted a dependable approach toward small-scale synthesis of azachromyl amine 46 (Scheme 8). Corey-Bakshi-Shibata (CBS) reduction of the alcohol proved unreliable and often required a significant excess of borane to achieve full conversion. Replacing the CBS reduction with a ruthenium-based transfer hydrogenation proved more robust (38). Slight modification of the alcohol reduction / azide inversion sequence could be implemented from the ketone 38 to deliver the amine tartrate 46 in 78% yield and 95% ee (Scheme 8). To circumvent the safety concerns around the use of DPPA, we chose to explore if the amine could be prepared via a diastereoselective reduction of a chiral imine which could be selectively deprotected to reveal the parent amine.

Scheme 8. Synthesis of Bromoamine 46

150 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


The chiral imine route commenced from ketone 38 already in hand. Installation of the neopentyl group using a Negishi coupling, similar to the medicinal chemistry route, revealed that the azachromanone structure 13 was prone to retrocyclization. Optimization of the coupling could only deliver a 1:2 ratio of desired cyclized to retrocyclized ketone. Fortunately, with the carbonyl functionality intact, re-cyclization could be easily achieved by treatment of the crude reaction mixture with ethanolic HCl. The neopentyl ketone 13 can be isolated in 75% yield from 38 (Scheme 9) using this recycle procedure.

Scheme 9. Negishi Route Toward Ketone 13

Investigation of imine formation of the chromanone series showed comparable propensity towards retrocyclization as with the palladium-catalyzed alkylzinc coupling (Scheme 9). Treatment of 13 with a model amine (4-fluorobenzylamine, 48) under typical imine formation conditions, revealed significant decomposition (Scheme 10). Methyl ketone 49 could be isolated in 65% yield indicating that not only retrocyclization, but retroaldolization was occurring under the conditions for imine formation. Side-product analysis by NMR and mass spectrometry led to the identification of several products along the retrocyclization/retroaldolization pathway (Figure 8). Similar results were seen with the use of other strong Brønsted acids (i.e. 4-MePhSO3H, etc) and Lewis acids.

Scheme 10. Decomposition of Ketone 13 Under Imine Formation Conditions



Figure 8. Intermediates Arising from Decomposition of 13 (Scheme 10).

Postulating that the strongly acidic conditions may be leading to the unravelling of the desired product imine / ketone, we chose to focus on imine formation under milder conditions. We obtained better results by lowering the reaction temperature and using ammonium acetate as a milder acid catalyst. Under these conditions, we achieved up to 60% conversion of 13 to the desired N-(4-fluorobenzyl)imine 50. Further analysis revealed that amine 48 was being consumed via a side reaction with acetate to generate the corresponding acetamide. Suppressing this side reaction by switching to ammonium pivolate alleviated this problem. Full conversion of the ketone to imine 50 can be achieved with 7) workup.

Scheme 11. Modified Imine Formation with Ammonium Pivolate

With suitable imine formation conditions now accessible, condensation of the 13 with S-α-methylbenzyl amine and reduction with sodium borohydride in ethanol revealed serviceable chirality transfer toward the desired stereochemistry (95:5 desired: undesired diastereoisomers). The product amine was not amenable to direct crystallization, however the bis-HCl salt is a non-hygroscopic benchstable solid and can be conveniently isolated by direct crystallization from the crude reaction mixture. Upon removal of the ethanol following the reduction, dissolving the crude product in acetone and treatment with two equivalents of HCl, the product 51 crystalized out and was isolated in 92% yield for the two steps. The crystallization served also to improve the diastereomeric purity (99:1 d.r.) of the isolated product.. Selective hydrogenolysis (39, 40) to cleave the α-methylbenzyl 152 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


group was easily accomplished with palladium on charcoal in methanol/water under 45 psig of hydrogen (Scheme 12). While the freebase of 14 product lacked suitable physical properties for a robust direct isolation, the bistosylate salt of 14 was found to be a convenient bench-stable solid and provided an upgrade in overall purity with despite variability in diastereomeric purity of the incoming stream of 51. On >100 g scale, the product could be isolated in >99% ee and 94% yield. In total (Scheme 12), the target amine was generated in 29% yield and >99% ee from 2-methoxynicotinic acid.

Scheme 12. Optimized Chiral Auxiliary Route to 14

Epoxide Aminolysis Investigations Toward Target HEA Structure With a scalable synthesis of the azachromylamine 14 and ketone 13 in hand, we focused our attention on the strategy for the assembly of the hydroxyethylamine (HEA) core. Traditional HEA transition state isosteres are common in commercial and investigatory HIV protease inhibitors (41). Typical HEA-based inhibitors such as saquinavir (42), amprenavir (43) and palanavir (44) contain a common phenyl group in the P1/S1 region of the molecule (45). The diamino alcohol moieties are all synthesized from a key intermediate epoxide 52 via an epoxide aminolysis reaction (Figure 9). 153 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 9. Examples of Commercial / Late-stage Clinical Aspartyl Protease Inhibitors Based Upon the Hydroxyethyl Amine Transition-State Isostere. Commonly seen in a number of syntheses, the epoxide ring opening to form the intermediate for amprenavir/fosamprenavir (eq 1) is accomplished by heating the starting epoxide 52 with several equivalents of isobutyl amine (3–10 are generally used) (41). Lowering the amine stoichiometry resulted in slower reaction rates and may also result in the formation of dialkylated amine. While the complication of the dialkylation was not possible in the reaction to form saquinivir or alanavir (eq 2), the aminolysis was still low yielding (60–70%) in these cases (42, 44). These reactions often consume excess epoxide (~1.2–2 equiv) to reach full conversion, owing to competitive decomposition via anchimeric opening of the epoxide by the pendant carbamate (eq 3) (46, 47).



Initial work in this arena began with both screening a thermal aminolysis and Lewis acid-mediated epoxide openings with epoxide 52. Lewis acid mediated (48) epoxide openings with amine 14 were screened extensively with little to no avail. Desired product 56 was produced, however this was typically in less than 30% assay yield and was accompanied by several side-products. The uncatalyzed reactions of 14 with 52 in 2-propanol(IPA) (49) at 70 °C, afforded a cleaner reaction profile than the Lewis acid reactions, but could only be classified as marginally successful (assay yields of 30–40%) and useful yields (>60%) could only be obtained from using excess of the amine (50). Portionwise addition of the epoxide to a solution of the amine at elevated temperatures did not offer any improvement over the original approach and the dialkylation of the amine became a significant side-product (usually up to 20% LCAP) as the reaction progressed, resulting in lower solution yield of the desired product 57 (Scheme 13). Alkylated amine derivatives such as allyl or benzyl versions of amine 13 completely suppressed any desired product formation even under forcing conditions. Metallated (nBuLi, iPrMgCl, Et3Al) amine 14 or carbamate 14b resulted in either no reaction or complex product mixtures (Scheme 14) (48). Further attempted optimizations of thermal mediated or metallated amine derivative epoxide 50a ring-opening reactions with 13 were unsuccessful. Based upon the compilation of these results it was felt that the epoxide aminolysis approach was not viable at this juncture and an alternative disconnection needed to be explored.

Scheme 13. Epoxide 52 Aminoloysis with AzachromylAmine 14 155 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Scheme 14. Epoxide 52 Aminoloysis with AzachromylAmine 14 Derivatives


Reductive Amination Investigations Toward HEA Assembly While the epoxide route initially proved unsuccessful in generating our target HEA inhibitor, the success of the amine installation work with the azachromylamine directed us toward a possible reductive amination as a potential disconnection strategy. In this case a P1 amine moeity with the already available ketone 13. The key question would be that while methylbenzylamine was an appropriate chiral auxillary, could a P1 amine serve to successfully transfer chirality to the azachromyl center. A prototypical P1 amine was quickly generated in three steps from the commercially available epoxide (51). Condensation under the mild conditions developed for the azachromanone (Scheme 12) delivered 89% isolated yield of the target imine 58 (Scheme 15 Excess (>1.5 equiv) of the P1 amine 57 was required to achieve conversions of 12 over 80%.

Scheme 15. Imine Formation Between 59 and 13

Reduction of the imine under a variety of conditions illustrate that borohydride-type reagents showed little to no diastereoselectivity, and assay yields were moderate (53-88%; Table 3, entries 1-4). Hydrogenation with platinum on carbon in IPA showed some modest success with 100% assay yield and a ratio of 6:1 desired : undesired (Table 3, entry 5). Unfortunately, the reaction requried significant pressure of hydrogen (400 psig) to reach full conversion and while initial diastereoselectivity did show promise, further optimization of the reaction conditions did not further increase the selectivity (52). Based upon these results and the success of an alternate approach (vide infra), the imine reduction route was deprioritized. 156 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Table 3. Conditions Screen for Reduction of Imine 60 (eq 4)

Development of a Novel HEA Assembly Method: A Templated Approach While the intermolecular bond assembly strategy toward our target HEA molecules proved challenging, we chose to explore an alternate, but underutilized option which we hoped could provide improved process control. Intramolecular C-N bond formation of two tethered fragments would, in theory, mitigate reactivity issues with the epoxide aminolysis and prevent over reaction. Of additional benefit, the amine stereocenter would be controlled in the fragment amine instead of relying on the coupling chemistry to govern the selectivity. Critical to this approach we would need to select an appropriate tether which could be easily installed, facilitate the desired C-N bond formation and also be seamlessly removed (Figure 10). 157 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Initial work focused in on the use of the urethane as a tether which would cyclize to an oxazolidinone and be cleaved under basic conditions (53). Two possible disconnection strategies were conceived with this templated type of approach. The first disconnection involved acylation of an amine isocyanate with a P1 halohydrin or other such activated species (Figure 10, Path A). A similar strategy has been employed by Das and others via halohydrins (54) or other activated species (55) and simple isocyanates to form vicinal amino alcohols. No report exists of this type of strategy utilized in a complex fragment coupling such as the one we are proposing. Alternatively, an activated carbonate derivative could react with the amine to form the halourethane which upon cyclization would afford the targeted oxazolidinone intermediate (Figure 10, Path B). For purposes of testing the validity of the disconnection, path A was chosen first due to the ready availability of both coupling partners.

Figure 10. Proposed Templated Approach to HEA Core.

With limited amounts of key intermediates available at the start of this exercise, a model study was explored initially with a 4-Cl derived P1 chlorohydrin 63 (56) and commercially available S-α-methylbenzyl isocyanate 64 (Scheme 16). Treatment of chlorohydrin 63 with isocyanate 64 with 30 mol% of DABCO (57) in THF afforded a 67% isolated yield of chlorourethane 65 Further reaction with sodium tert-butoxide (NaOt-Bu) in THF was rapid (99% ee and 95% yield (Scheme 18).

Scheme 18. Enantioselective Hydrogenation Route Towards Aminoester 78 160 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Hydrolysis of ester 78 with lithium hydroxide in THF/H2O revealed the corresponding carboxylate, which upon pH adjustment and Steglich esterification with 4-nitrophenol and DCC delivered the aryl ester 77 which was crystallized from THF/n-heptane. Condensation with trimethylsulfoxionium ylide showed in minimal epimerization and after crystallization from IPA, the ylide 78 was delivered in 86% yield and >99% ee (Scheme 19).

Scheme 19. Generation of Ketoylide 80 Treatment of the ketoylide 80 with LiCl and methanesulfonic acid in THF as a source of anhydrous HCl, afforded the chloroketone 81 in 79% yield and >99% ee following a recrystallization from IPA (Scheme 20). Diastereoselective reduction using aluminum tri-isopropoxide in IPA following the protocol from Yin and coworkers (63) delivered 96:4 d.r. favoring the desired diastereoisomer. Workup with isopropyl acetate, Rochelle’s salt and crystallization from IPA resulted in 85% yield of the desired chlorohydrin 82 as essentially a single stereoisomer (>99% ee and >99:1 diastereomeric ratio). Overall the target chlorohydrin yield from piperonal 74 was 55% with seven steps and five isolations.

Scheme 20. Diastereoselective Synthesis of Chlorohydrin 82

Application and Development of the Templated Assembly Strategy toward ‘699 and ‘359 With the desired chlorohydrin 82 in hand, optimization of the final assembly route commenced by selecting the appropriate coupling mode. While the isocyanate 67 and nitrophenylcarbamate derivatives 70 of the azachromylamine were shown to synthetically deliver the desired HEA array (Schemes 16 & 17), the inherent instability of functionalized amines combined with our reluctance toward further elaboration of the already synthetically intensive azachromylamine, a more convergent mixed carbonate route was pursued. 161 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


To choose the appropriate coupling partner for the amine 14, a screen of a variety of P1 electrophiles with 14 was undertaken (Table 4). While the nitrophenylcarbonate derivative 84 showed smooth conversion to deliver the desired chlorourethane 86 (Table 4, entry 2), the corresponding phenyl carbonate 83 showed no desired product with prolonged heating up to 120 °C (Table 4, entry 1). Acyloxyimidazolide 85 (64) was contemporaneously pursued as cheaper and safer alternative to 4-nitrophenylchloroformate. Similar to 83, little to no reaction was seen with 85 under similar conditions which afforded full conversion of 84 (Table 4, entry 3).

Table 4. Electrophilic P1 Coupling with Amine 14 (eq 5)

Screening a variety of activators (65) showed that while Brønstead and Lewis acids afforded little to no conversion, common peptide coupling additives showed promise. After extensive screening N-hydroxy succinimide (HOSu) and N-hydroxypthalimide afforded >90% assay yields of the desired product 86. The loading of HOSu could be reduced to 5 mol% relative to 85 without any loss in 162 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


yield (Table 4, entry 4). Due to the time constraints and the lack of a sufficient purity control point (66) for the acyl imidazolide coupling partner, the nitrophenyl carbonate 84 was chosen over 85 as a phase-appropriate solution. Initial screen of reaction conditions coupling chlorohydrin 82 and 4-nitrophenylchloroformate in THF (67) showed that pyridine as a mild base formed the desired acylated chlorohydin 84 in nearly quantitative assay yield. While the overall solution yields in the acylation reaction were high, the reactions were contaminated with two significant impurities: bis-4-nitrophenylcarbonate 88 and 4-nitrophenol 89. The origin of both of these impurities is likely due to the hydrolysis of 4-nitrophenylchloroformate by adventitious water either before or during the reaction. Impurities 88 and 89 were generally produced in variable amounts but often >10% LCAP once the reaction has reached completion. Recrystallization of crude 84 from isopropyl acetate (IPAc) could remove the bisnitrophenylcarbonate 88 but only at considerableloss to the mother liquor (17% loss) and the isolated product still containes significant amounts of phenol 89. Screening of different solvent systems showed that a 1:1 mixture of dimethyoxyethane (DME) and water is uniquely competent to remove the two impurities with minimal loss of the desired product (100 g scale resulted in 99% wt% adjusted yield and 99% purity (Scheme 21).

Scheme 21. Synthesis of Activated Carbonate 84 The acylation of amine 14 with carbonate 84 performed well in a number of solvents to generate chlorourethane 90. Reactions in THF, 2-MeTHF, IPAc, and 2-butanol afforded 100% conversion to 90 in 18 h at 65 °C. Cyclization of 90 was easily accomplished by the treatment the crude reaction mixture with NaOt-Bu. Cyclization of crude 90 produced in either THF or 2-MeTHF afforded 88–90% assay yield, where as other solvents were found to be suboptimal due to low reactivity or competing side-reactions. In practice we chose 2-MeTHF for both 163 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


coupling and cyclization. because of the high conversion and the ease of reaction workup. On further optimization, we found potassium tert-amyloxide (1.7 M solution in toluene) to be a convenient replacement for solid sodium tert-butoxide, and could be added directly to the crude acylation reaction mixture. This change afforded the oxazolidinone 91 in 93% assay yield. Due to lack of suitable control points in 90 and 91, the choice was made to pursue a telescopic procedure through to the isolated bis-HCl salt 92. While an extensive screen was not performed, the bis-HCl salt was selected due it’s physical characteristics and ease of deprotection of the Boc group with HCl. Solvent exchange of the crude reaction tream of 91 to n-BuOH followed by treatment of 3 equiv.of anhydrous HCl at 70 °C afforded smooth Boc-deprotection. Addition of n-BuOAc as antisolvent and cooling afforded the desired bis-HCl salt 92 in 90% yield over three steps and >99% purity on >100 g scale (Scheme 22).

Scheme 22. Telescoped Process to Oxazolidinone 92

Cleavage of the oxazolidinone in 92 was most efficiently accomplished with ethanolic potassium hydroxide at elevated temperature. Treatment of an ethanol solution of the 92 with 10 equivalents of aqueous 5N KOH and heating for 18 h at 70 °C afforded full conversion and >95% assay yield of HEA 14. While the deprotection operationally simple to perform, isolation of the product was a challenge. Cooling the reaction mixture to 40 °C and charging 6 equiv of aqueous HCl (6N) led to a phase separation. Addition of toluene facilitated extraction of the diaminoalcohol 14 into the organic layer. Conveniently, the tris-HCl salt could be crystallized from a mixture of toluene and IPA. Overall on the penultimate 85 was isolated in 93% yield and 99.8% purity on >100 g scale (Scheme 23). 164 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Scheme 23. Deprotection and Isolation of Penultimate 15

Scheme 24. Summary of Demonstration Run of the Templated Route to Penultimate 15



Conclusions In summary, a new templated approach to the hydroxyethylamine core has been demonstrated on >100 g scale toward penultimate HEA 15. The new synthesis involves the use of a urethane tether to facilitate intramolecular C-N coupling of azachromylamine 14 and a P1 electrophile, followed by seamless removal and revealing the target diaminoalcohol array. Key highlights to this method include activation of chlorohydrin 82 to a stable crystalline 4-nitrophenyl carbonate 84 which occurs in excellent yield and purity (99 wt% adjusted yield). Activated carbonate 84 is readily coupled with azachromyl amine 14 to form an intermediate chlorourethane 90 which is cyclized and deprotected to afford oxazolidinone 92 in 90 wt% adjusted yield over three steps. Cleavage of the oxazolidinone tether under basic conditions affords the penultimate 15 in high yield and purity (93 wt% adjusted yield, 102 wt%). Overall 82% yield over three isolations from P1 chlorohydrin 82 (Scheme 24). In addition to the new HEA route, the early process development efforts for the BACE program were able to discover and demonstrate a new and scaleable route toward the azachromylamine 14. The route eliminated the hazardous azide chemistry, replacing it with a robust chiral auxillary route and overall shortened the sequence from 16 overall steps to 9 steps from 2-methoxy nicotinic acid. Combining the new amine synthesis with the new templated HEA route resulted in a shortening of the overall synthesis of the lead molecules from 29 steps (4% overall yield) to 19 steps. Additional process improvements resulted in of elimination of all chromatographic purifications and the overall yield was increased by nearly four-fold to 19%. The new technology described above, not only was suited for future larger scale deliveries, but also accelerated and enabled supply of several hundered grams of intermediates for molecule selection and pre-clinical toxicology work.

Acknowledgments The authors would like to acknowledge the following: Thomas A. Dineen, Matthew M. Weiss, Toni Williamson, Paul Acton, Safura Babu-Khan, Michael D. Bartberger, James Brown, Kui Chen, Yuan Chen, Martin Citron, Michael D. Chrogan, Robert T. Dunn, Joel Esmay, Russell F. Graceffa, Scott S. Harried, Dean Hickman, Stephen A. Hitchcock, Daniel B. Horne, Hongbing Huang, Ronke Imbeah-Ampiah, Ted Judd, Matthew R. Kaller, Charles R. Kreiman, Daniel S. La, Vivian Li, Patricia Lopez, Steven Louie, Holger Monenschein, Thomas T. Nguyen, Lewis D. Pennington, Tisha San Miguel, E. Allen Sickmier, Hugo M. Vargas, Robert C. Wahl, Paul H. Wen, Douglas A Whittington, Stephen Wood, Qiufen Xue, Bryant H. Yang, Vinod F. Patel, Eric Bercot, Emilio Bunel, Seb Caille, Johann Chan, Evan DiVirgilio, Jinkun Huang, Liang Huang, Anil Guram, Ken McRae, Rob Milburn, Charles Papageorgiou, Silas Wang, Filisaty Vounatsos, Jamie Zigterman, Jenny Chen, Tiffany Correll, Troy Soukup, J. Preston, Judy Ostovic, Jiemin Bao, Fang Wang, Helming Tan, Susanna Lai, Kelly Nadeau, Kevin Turney, Peter Grandsard, Margaret Faul, Mike Martinelli and Paul Reider. 166


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Design and Enabling Development of Hydroxyethylamine-Derived

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